Symposium JJ: Biological Hybrid Materials for Life Sciences
Biological Hybrid Materials for Life Sciences
April 26 - 29, 2011
School of Materials Engineering
Neil Armstrong Hall of Engineering
701 W. Stadium Ave.
W. Lafayette, IN 47907-2045
Dept. of Chemistry
206 Science Center
P. O. Box 5810
Potsdam, NY 13699-5810
Universite de Perpignan
52 Ave. Paul Alduy
Perpignan Cedex, F-66860 France
Dept. of Chemistry and Institute
of Biomedical Sciences
Shanghai, 200433 China
Air Force Office of Scientific Research
National Science Foundation
Purdue University, School of Materials Engineering, School of Biomedical Engineering
Proceedings to be published in electronic-only format
(see MRS Online Proceedings Library at www.mrs.org/opl)
as volume 1355E
of the Materials Research Society
Symposium Proceedings Series.
* Invited paper
SESSION JJ1: Cell and Protein Adhesion
Chair: Ulrike Wegst
Tuesday Morning, April 26, 2011
Salons 4-6 (Marriott)
8:30 AM *JJ1.1
Regulation of Stem Cells by the Mechanics of Cell-material Interactions. Jianping Fu, Humphrey Wang, Wesley Legant and Christopher Chen; Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania.
Adhesion of cells to materials, natural or synthetic, appears to be a central regulator of cellular functions, including proliferation, differentiation, migration, and suicide. Adhesion involves not only receptor binding, but also changes in cell shape and the generation of mechanical stresses at these adhesions. Using microengineered materials as substrates for cell adhesion, we explore the relative contributions of these different aspects of adhesion to regulating cell function. In this presentation, we will present our recent progress on using microfabricated arrays of elastomeric posts as substrates for cell adhesion. The geometry of the microposts specify their rigidity, and their deflections report the forces generated by attached cells. Our studies demonstrate that mechanical forces generated either internally by the cytoskeleton or externally regulate cell structure and cell-matrix adhesions, and in so doing, modulate signals that control cell function. We show that these force are central to driving growth, multicellular patterning, and stem cell lineage commitment. We will also show some early studies on the development of a new approach to extend force measurements to the context of cells embedded within a 3D hydrogel. These studies highlight the importance of novel engineering and materials approaches to inform our basic understanding of how cells respond to their environment.
9:00 AM *JJ1.2
The Role of Surfaces in Protein Structure and Function: Amyloid Fibril Formation. Zoya Leonenko, Physics and Astronomy, University of Waterloo, Waterloo, Ontario, Canada; Biology, University of Waterloo, Waterloo, Ontario, Canada.
Many proteins are known to actively interact with biologically relevant as well as inorganic and synthetic surfaces that are widely used in nano- and bio-technology. The surfaces interact strongly with proteins and significantly affect their structure and function. Amyloid fibrils are insoluble protein aggregates in beta-sheet conformation that are implicated in at least 20 diseases for which neither the cure nor the diagnostics are currently available. The role of surfaces in amyloid fibril formation and toxicity is not well understood. Currently, the experimental data available for amyloid fibril formation both on lipid membrane and inorganic surfaces is limited. The goal of our study is to investigate how the physical properties of the surfaces affect binding of amyloid peptides and fibril formation. We use scanning probe microscopy to study amyloid beta (1-42) binding and fibril formation on model surfaces, which are functionalized with thiol monolayers with negatively or positively charged or hydrophobic functional groups. We also study interaction of amyloid peptides with model lipid membranes, which are widely used to mimic cell membrane surfaces. Effect of lipid composition, surface charge, and presence of cholesterol will be discussed. Investigation of interactions of proteins with surfaces and lipid membranes is of significant importance for the development of novel biosensing platforms.
9:30 AM JJ1.3
Dynamic Measurement of TAT Membrane Penetration. Elizabeth A. Hager-Barnard, Benjamin D. Almquist and Nicholas A. Melosh; Materials Science and Engineering, Stanford University, Stanford, California.
We present a combined experimental and theoretical study of the interactions between cell-penetrating peptides (CPPs) and lipid bilayers using dynamic AFM measurements. Understanding how CPPs can pass through cell membranes is critical for designing drug delivery agents. While CPPs like HIV-TAT have been widely studied, their ability to penetrate membranes directly, without active transport, is still a matter of considerable debate. Here, we directly measure TAT-lipid mechanics during the actual membrane translocation event using TAT-functionalized AFM probes to penetrate through a stack of lipid bilayers. Dynamic force spectroscopy revealed that both the bilayer breakthrough force and bilayer thickness depended on the TAT-bilayer contact time. The results provide a detailed view of how TAT interacts with the bilayer. Upon contact, TAT inserts into the bilayer headgroup to a depth of ~1nm in ~0.5ms. During this same timescale the bilayer thins from 4.2 to 3.2 nm, likely reflecting TAT-driven reorganization. Unexpectedly, the energy barrier for penetration actually increases 52 kT after the bilayer thins, indicating the new conformation has not weakened the bilayer and suggesting TAT cannot penetrate unassisted. Controls with polylysine or mercaptoundecanoic acid do not show this thinning behavior, implying the unique TAT-lipid interactions play a significant role. This approach can be further generalized to the interaction of a variety of peptides or membrane-active molecules with lipid bilayers. Theoretical modeling of dynamic force spectroscopy measurements shows distinct features related to the timescale and disruption of lipid membranes upon contact with a molecular species. By adapting existing AFM protocols to look for these features it will be possible to directly characterize, with microsecond resolution, the dynamics and energetics of the interactions between lipid bilayers and a wide range of peptides and molecular species.
9:45 AM JJ1.4
Amine-rich Polyelectrolyte Adhesion Layers as an Alternative to APTES for Surface Immobilization of Biomolecules. Stefan Stoianov, Jason I. Ridley and Hans D. Robinson; Virginia Polytechnic Institute and State Univerisity, Blacksburg, Virginia.
We evaluate amine enriched poly (styrene sulfonate)/poly (allylamine hydrochloride (PSS/PAH) self-assembled multilayers for the purpose of fixating freestanding or solution suspended nanostructures and biomolecules onto silicon dioxide surfaces. Unlike common strategies such as silylation of the surface with amine-containing ligands, PSS/PAH multilayers can be fabricated rapidly, without using aggressive chemicals, do not require inert processing conditions to achieve an optimal result, and can be applied to a larger variety of surfaces. PSS/PAH multilayers fabricated at pH above 8.5 are enriched in amines that promote adhesion when exposed on the surface of the film. Exposure is achieved by cycling the film to pH values below 4 and back to neutral, which causes the film to swell substantially as the amine groups are protonated. Conversely, cycling to pH 10 and back causes the amines to pack tightly into hydrophobic regions. This makes it possible to protect the surface from contamination until just before use. In addition, exposed PAH/PSS film can be selectively passivated by acetylation with acetic anhydride. The resulting surface exhibits negligible non-specific binding of negatively charged proteins. Parts of the surface covered with metal or other structures are not affected by this, making patterned adhesion easy to accomplish. We find that PSS/PAH yields at least as good adhesion of nanostructures to the surface as silylsation with 3-aminopropyltriethoxysilane (APTES). These results, along with quantitative results from ongoing investigation of biomolecule adhesion on the films will be presented.
10:15 AM *JJ1.5
Biologically Inspired Catechol Based Materials for Medicine. Phillip Messersmith, Biomedical Engineering, Northwestern University, Evanston, Illinois; Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois; Institute for Bionanotechnology in Medicine, Northwestern University, Chicago, Illinois.
Catechols are widely distributed in nature in the form of biopigments, iron sequestering compounds, neurotransmitters and adhesive protein secretions of sessile marine organisms. A plethora of chemical interactions are associated with catechols, including redox reactions, high affinity for metal ions, and strong interfacial activity. This rich landscape of potential chemical interactions confers upon catechol containing natural and synthetic compounds a large variety of potential chemical and biochemical properties. In this talk I will provide several examples of novel biomimetic materials that integrate catechols for specific functional purposes. One example is given by catecholic polymers that mimic marine wet adhesives. Marine and freshwater mussels secrete a family of specialized proteins collectively referred to as mussel adhesive proteins (MAPs). This family of proteins contain 3,4-dihydroxy-L-phenylalanine (DOPA), a catechol containing amino acid found in high concentration in MAPs. DOPA is believed to be essential for the cohesive and adhesive properties of mussel adhesive proteins. Recently we have investigated the use of catechol containing polymers for potential biomedical applications. For example, catechol-derivatized water soluble synthetic polymers have been formulated into rapid-setting liquid adhesives for tissue repair. We have investigated the use of these compounds for sealing of fetal membrane wounds, where they effectively seal fetal membrane puncture wounds and elicit a favourable tissue response in comparison to other candidate sealants. Another arena for biologically inspired catechol based materials is in surface modification, where the strong interactions of catechols towards surfaces can exploited for grafting antifouling polymers onto surfaces for control of biofouling, and for anchoring biomolecules and multifunctional coatings onto surfaces.
10:45 AM JJ1.6
Antioxidant is a Key Factor in Mussel Protein Adhesion. Jing Yu1, Wei Wei2, Eric Danner3, Jacob N. Israelachvili1 and J. Herbert Waite3; 1Chemical Engineering, University of California, Santa Barbara, Santa Barbara, California; 2Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, California; 3Department of Molecular, Cell & Developmental Biology, University of California, Santa Barbara, Santa Barbara, California.
The underwater adhesion of marine mussels relies on mussel foot proteins (mfps) rich in the catecholic amino acid 3, 4-dihydroxyphenylalanine (dopa). As a side-chain, dopa is capable of strong bidentate interactions with a variety of surfaces, but its susceptibility to oxidation often renders it unreliable for adhesion. Mussels limit dopa oxidation by imposing an acidic, reducing regime in the confined space of mfp deposition. Using the Surface Forces Apparatus (SFA) technique, we demonstrate that the adhesion of mfp-3 to mica is closely coupled with dopa redox and pH. Raising the pH from 3 to 7.5 decreases the adhesion energy of mfp-3 on mica 20-fold and appears to be driven by the pH-dependent oxidation of dopa. Addition of thiol-rich mfp-6 restores mfp-3 adhesion by coupling the oxidation of thiols to the reduction of dopaquinones. How mussels preserve adhesive dopa-containing proteins from oxidation has considerable biological and technological value.
11:00 AM JJ1.7
S-layer Crystallization on Biomimetic and Inorganic Surfaces: The Importance of Multi-stage Pathways to the Crystalline State Driven by Conformational Changes. Sungwook Chung1,2, Seong-Ho Shin1,3,4, Stephen Whitelam1,3, Babak Sanii1,3, Carolyn R. Bertozzi1,3,4 and James J. De Yoreo1,3; 1Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California; 2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; 3Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; 4Department of Chemsitry, University of California, Berkeley, Berkeley, California.
Self-assembled protein architectures exhibit a wide range of structural motifs with functions that include selective transport, structural scaffolding, mineral templating and propagation of pathogenesis. Although the primary sequences of the individual proteins define their governing interactions, their functions depend on the quaternary architecture that emerges from self-assembly. Surface layer proteins (S-layers), which form the outermost membrane of many bacteria, provide well-studied examples in which proteins assemble into 2D sheets, but the mechanisms and pathways of assembly are poorly understood. Here we report results using in situ AFM to follow 2D crystallization of S-layers on both supported lipid-bilayers (SLBs) and atomically flat mica surface at molecular-scale. In terms of the assembly process on SLBs, We show that the assembly process begins with adsorption of monomers of extended conformation that form a mobile phase on the SLBs. The proteins condense into amorphous clusters, which undergo a phase transition into 2D crystalline clusters of 2 - 15 folded tetramers. Growth of the ordered clusters proceeds by new tetramer formation from monomers on the SLB exclusively at lattice sites along the cluster edges, suggesting tetramer formation is self-catalytic. Analysis of cluster growth dynamics leads to a quantitative model in which the rate limiting parameter is the probability of tetramer creation. The estimated energy barrier of ~51 KJ/mol for this process is much less than expected from scaling laws for folding of isolated proteins, suggesting a kinetic driver for two-stage assembly. In studies of assembly on mica, we observe the emergence of two distinct phases of S-layer organization in 2D. The two phases are both composed of crystalline S-layers having the same P4 unit cell symmetry and ca. 15 nm lateral spacing between tetramers, but the domain heights are different by 2 - 3 nm. Moreover, the relative coverage of the two phases depends on growth time, temperature, and protein concentration during growth. In situ AFM reveals that the phase with the lower domain height spontaneously transforms into the taller phase, a phenomenon that may be due to a conformational change in the proteins. The results both on SLBs and mica show that kinetic constraints imposed by conformational changes lead to complex multistage pathways of protein crystallization in 2D.
11:15 AM JJ1.8
Vesicle Adsorption in Holes of a Contracted Supported Lipid Bilayer. Kimberly L. Weirich1 and Deborah K. Fygenson1,2; 1Biomolecular Science & Engineering, University of California, Santa Barbara, California; 2Physics, University of California, Santa Barbara, California.
Supported lipid bilayers (SLB)s are molecularly thin fluids that are adsorbed on a solid surface. They are commonly used as model systems in which to study biological membrane processes. One method of SLB formation involves vesicle adsorption and rupture on silica glasses. Experiments indicate that bilayer edge plays a catalytic role in SLB formation, enhancing vesicle-surface affinity and promoting rupture [K. L. Weirich, J. N. Israelachvili and D. K. Fygenson (2010) Biophys. J. 98(1):85-92]. Here we extend these investigations by controlling the surface to edge ratio. We take advantage of the large mismatch in coefficients of thermal expansion between bilayer and glass to form µm-sized holes in an SLB (regions of bare glass bounded by bilayer edge). Using fluorescence microscopy, we monitor the time-course of lipid surface coverage as vesicles adsorb and rupture within the holes as a function of hole size, lipid chain length and temperature.
11:30 AM JJ1.9
Hydroxyapaptite Growth on Nano-porous Silicon Substrates in a Hydrogels based Double Diffusion System. Jason R. Dorvee, Adele Boskey and Lara Estroff; Material Science and Engineering, Cornell University, Ithaca, New York.
Inspired by Nature’s strategy for controlling crystal growth in hydrogel-like environments, we have successfully modified a hydrogel-based double diffusion system to incorporate a functionalizable, bioactive, nano-porous silicon (pSi) substrate. The pSi substrate acts as a nucleating surface, while the hydrogel matrix acts as a crystal growth modifier for hydroxyapatite (HA). The resulting model system provides a platform with which to produce and study bioactive materials in an ECM-like environment. Using oxidized pSi allows the nucleating surface to lie normal to the precipitation front while still allowing ions to diffuse through the substrate. More traditional substrates cannot be used in the doudle-diffusion geometry. For this study, we examined the native silicon oxide as well as two physiosorbed proteins, milk casein and bovine serum albumin (BSA), on the pSi surface. Milk Casein is a known inhibitor of HA when free in a hydrogel, while BSA serves as a control, with no known effect on HA mineralization. Membranes were fabricated from highly doped P++ (100) silicon via anodic etching in 3:1 HF:ethanol solution. These surfaces were then placed in the center of a tube containing 10 w/v % gelatin. Calcium and phosphate ions were diffused into the tubes from either side to create a mineralized band of gelatin. Samples were removed at days 3 and 5, and characterized via x-ray diffraction and electron microscopy to determine the size, shape, and phase of the mineral both in the gel and on the pSi. Results show that by day 5 the mineral formed in both the hydrogel and on the pSi was HA. For all substrates, growth in the hydrogel will be compared to deposition from solution. While in the hydrogel the oxidized pSi acts as a template to adsorb organic material as seen by FE-SEM in images compared before and after placement in the hydrogel matrix, subsequently the adsorbed organic material on the substrate acts as a nucleator for the HA. This model platform, utilizing a porous membrane in a double diffusion system, facilitates the exploration of various surface functionalities and surface coatings for use on various biomedical implants.
11:45 AM JJ1.10
Cell Internalisation Pathway of Multi-walled Carbon Nanotubes into Non-phagocytic Cells . Hannah Nerl1, Alexandra E. Porter1, Khuloud T. Al-Jamal2, Karin Muller5, Hanene Ali-Boucetta2, Peter D. Haynes1, Maurizio Prato3, Alberto Bianco4 and Kostas Kostarelos2; 1Materials, Imperial College London, London, United Kingdom; 2Centre for Drug Delivery Research, The School of Pharmacy, University of London, London, WC1N 1AX, United Kingdom; 3Department of Pharmaceutical Sciences, University of Trieste, Trieste, Italy; 4CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France; 5Department of Physiology, University of Cambridge, Cambridge, United Kingdom.
Carbon nanotubes (CNTs) are being investigated for use as carriers for targeted drug delivery and diagnostic agents. Despite extensive study, little is known about the pathways which by carbon nanotubes enter cells or the intracellular distribution of CNTs after uptake. The “nanoneedle” hypothesis , of direct penetration into cells by CNTs, has been a controversial issue and its visualization has been achieved in 2D bright field imaging only   which is not sufficient as a technique to visualize the relative position of the plasma membrane with respect to the CNTs. Understanding the exact uptake mechanism is critical as a screening strategy to assess nanoparticulate materials for their suitability as drug-targeting vectors destined for specific intracellular sites. In this work we carried out short (24 h) and long-term exposures (up to 14 days) of lung epithelial cells (A549) and human macrophage cells (HMMs) to NH3+-functionalised multi-walled carbon nanotubes (f-MWNTs) to study the long-term fate of CNTs after uptake. Herein, we report, using a combination of 3-D electron tomography and energy filtered transmission electron microscopy (EFTEM) techniques, that NH3+-functionalised MWNTs (100nm length) (f-MWNTs) enter A549 via two distinct uptake mechanisms. 3-D electron tomography reconstructions confirmed that the NH3+-MWNT inserted directly into the plasma membrane of cells as “nanoneedles” and enter the cell cytoplasm. Individual NH3+-MWNTs were also found engulfed by endocytic pathways via membrane wrapping. We therefore propose a new model for the active uptake mechanism of CNTs by non-phagocytic cells. Wherein CNTs are taken up individually via membrane wrapping and then transported into the cells only to fuse with intracellular vesicles at a later stage forming large clusters of CNTs inside lysosomes. References:  C.F. Lopez et al., Proc. Natl. Acad. Sci. U. S. A 101, (2004) 4431-4434.  A.E. Porter et al., Nature Nanotech. 2 (2007) 713-717.  Q. Mu et al., Nanoletters (2009) Vol.9, No12, 4370-4375.
SESSION JJ2: Tissue Engineering
Chair: Zoya Lenonenko
Tuesday Afternoon, April 26, 2011
Salons 4-6 (Marriott)
1:45 PM *JJ2.1
Hybrid Biomaterials by Freeze Casting. Ulrike G. K. Wegst, Materials Science and Engineering, Drexel University, Philadelphia, Pennsylvania.
The functional requirements for synthetic tissue ubstitutes appear deceptively simple: they should provide a porous matrix with interconnecting porosity and surface properties that promote rapid tissue ingrowth; at the same time, they should possess sufficient stiffness, strength and toughness to prevent crushing under physiological loads until full integration and healing are reached. Despite extensive efforts and first encouraging results, current biomaterials for tissue regeneration tend to suffer common limitations: insufficient tissue-material interaction and an inherent lack of strength and toughness associated with porosity. The challenge persists to synthesize materials that mimic both structure and mechanical performance of the natural tissue and permit strong tissue-implant interfaces to be formed. In the case of bone substitute materials, for example, the goal is to engineer high-performance composites with effective properties that, similar to natural mineralized tissue, exceed by orders of magnitude the properties of its constituents. It is still difficult with current technology to emulate in synthetic biomaterials multi-level hierarchical composite structures that are thought to be the origin of the observed mechanical property amplification in biological materials. Freeze casting permits to manufacture such complex, hybrid materials through excellent control of structural and mechanical properties. As a processing technique for the manufacture of biomaterials, freeze casting therefore has great promise.
2:15 PM JJ2.2
Bone Marrow-Derived Mesenchymal Stem Cells Attachment to the 3D Scaffolds: Functionalized Vertically Aligned Carbon Nanotube Arrays. Verda Bitirim1, Gokce Kucukayan2, Donus Tuncel3, Erman Bengu3 and Can Akcali1; 1Bilkent University, Department of Molecular Biology and Genetics, Ankara, Turkey; 2Bilkent University, Material Science and Nanotechnology Graduate Program, Ankara, Turkey; 3Bilkent University, Department of Chemistry, Ankara, Turkey.
Mesenchymal Stem Cells (MSCs) have the potential to provide sources for tissue restoration in regenerative medicine and tissue engineering due to their differentiation potential into adipose, bone, cartilage, muscle, liver and nerve cells and non-immunogenic characteristics. There is no ethical concern in their usage therefore MSCs are promising tool for several cellular therapeutic approaches. A major roadblock in the use of MSCs for cell-based therapies, however, is their rareness and the long duration of their culture. Several established methods are presently available for in vitro isolation and differentiation of MSCs from bone marrow including use of nanostructured scaffolds. In this study, we aimed to mimic the extracellular matrix (ECM) environment by using functionalized vertically aligned carbon nanotube arrays (VANTA) as a 3D scaffold for bone marrow-derived MSC and to investigate the behaviors of cells on these scaffolds. VANTAs were synthesized on pre-oxidized Si (100) surfaces by chemical vapor deposition technique in different lengths using ethanol as carbon source. Synthesized flat VANTA surfaces were functionalized using the conjugate polymers with positively and negatively charged end groups. During the functionalization of VANTA surfaces, 3D cavities were formed by carbon nanotubes where some of them remained only vertically at the domain walls and some of them were completely bent parallel to the substrate surface. These modified surfaces were then seeded with rat bone marrow-derived MSCs after their characterization. We found that MSCs attached and survived according to the modification of the scaffold surfaces. Finally, our data showed that MSCs’ attachment features on VANTA surfaces decreased at the subsequent passages. Our results revealed that the modified and patterned VANTA surfaces would be a good choice as a 3D scaffold for the growth and maintenance of MSCs which may be useful tool for further applications in tissue engineering.
2:30 PM JJ2.3
Biomimetic Bone: Mineralization of Dense Collagen Matrices. Douglas E. Rodriguez1, Nadine Nassif2, Taili Thula1, Yan Wang2, Florence Babonneau2, Marie-Madeleine Giraud-Guille2 and Laurie B. Gower1; 1Materials Science & Engineering, University of Florida, Gainesville, Florida; 2Laboratoire Chimie de la Matière Condensée de Paris, Collège de France, Paris, France.
Bone is a hierarchically structured composite, which at the nanostructural level consists of an assembly of collagen fibrils that are embedded with uniaxially-aligned nanocrystals of hydroxyapatite. Our studies have revealed that this nanostructure can be reproduced in vitro using a polymer-induced liquid-precursor (PILP) mineralization process. The polymeric additive consists of acidic polypeptides (e.g. polyaspartic acid) that are a simple mimic of the non-collagenous proteins associated with bone and dentin. The high charge density of the polyanionic additive sequesters ion such that liquid-liquid phase separation occurs in the mineralization solution, forming nanodroplets of a hydrated amorphous mineral precursor. Infiltration of the nanodroplets into the collagen fibrils leads to intrafibrillar mineral, which is the foundation of bone nanostructure. Through optimization of various reaction parameters, compositions matching that of bone (over 70wt% mineral) have been achieved in porous reconstituted collagen scaffolds. Dense biogenic collagen matrices, such as rat tail tendon and demineralized bone, and dense reconstituted collagen matrices can also be mineralized by this process; however, there are currently limitations in the depth of mineral penetration that can be attained. The mineralized portion of these matrices, however, does exhibit the interpenetrating organic-inorganic nanostructure characteristic of biogenic bone. Our current studies are directed at examining the mechanism of precursor formation, because stabilization of the liquid-phase precursor may allow for greater penetration depths to be achieved. On-going work includes evaluating the mechanical properties of dense biomimetic bone as well as optimizing the mineralization conditions to create macroscale materials. The long range goal of these studies is use this as a model system to understand bone formation and pathologies, and from an applications perspective, to prepare bioresorbable load-bearing bone substitutes.
2:45 PM JJ2.4
Bio-inspired Hierarichal Vascular Network Synthesis: Electrohydrodynamic Viscous Fingering. Kristopher D. Behler and Eric D. Wetzel; Composite and Hybrid Materials Branch, U.S. Army Research Lab, Aberdeen Proving Ground, Maryland.
Vascular networks provide a physical pathway to distribute fluid throughout a system. An uninterrupted and controllable supply of liquid is optimal for many applications such as continual self-healing, drug delivery, chemical and biological agent neutralization, and thermal management. One approach to producing hierarchical vascular networks is electrohydrodynamic viscous fingering (EHVF). Hollow channels or fingers are grown from a traditional viscous fingering approach in which a low viscosity liquid, water, is pumped through a more viscous (1,000 to 100,000 cSt) fluid or polymer. Typically, only a few large fingers are formed and the resulting pattern is governed by the capillary forces which lead to breakup and isolation of the fingers and the viscous forces between the two liquids. The EHVF process deviates from traditional viscous finger by applying a high voltage, 10 - 60 kV to the injected fluid. EHVF harnesses the applied voltage to control the size of fingers in the viscous liquid or polymer thus creating many smaller features and more fingering in systems that more closely resemble natural vasculatures seen in the human circulatory system, river beds and plants. The increase in the number of fingers and the smaller size is a result of the applied voltage, which exerts a force on the system that is greater than the viscous force and capillary force. Relaxation of the fingers is seen once the applied voltage is removed from the system. Thus a method to “freeze” the grown structure is paramount. The addition of spherical particles, aligned fiber mats, random fibers and combinations of the aforementioned additives has shown to reduce the relaxation and allow the grown fingers to retain their shape for extended periods of time. The elimination of relaxation in the finger structure allows for subsequent thermal curing in systems such as poly (dimethylsiloxane), PDMS. Another approach for retaining the fingers is to use an in-situ ultra violet (UV) cure. The UV cure system can be used on both filled and un-filled systems. The subsequent curing of the polymer by either ultra violet (UV) or thermal means creates a durable material in which the grown hollow network of channels can be produced and filled and refilled with various liquids for potential self-healing and self-repairing applications.
3:30 PM *JJ2.5
Irreversible Structural Transition Induced by Mechanical Shear in a Novel Peptide-amphiphile Syst. Matthew Tirrell, Bioengineering, University of California, Berkeley, Berkeley, California.
Shear induced, self assembly has been extensively studied using model surfactant systems which form long extended micelles at a critical shear rate. These polymer based extended micelles are able to entangle, giving the system viscoelastic properties. Here we present a peptide amphiphile, a short peptide sequence attached to a fatty acid tail, which transforms into extended worm-like micelles under shear force. Peptide amphiphiles are easily synthesized and modular in design, allowing a range of bioactive peptides to be introduced into the system. The particular peptide sequence used in this study is alanine (A) rich with interspersed lysine (K) residues, which gives it a strongly alpha helical secondary structure. The peptide amphiphile initially forms spherical micelles in aqueous solution and then undergoes a switch to form long cylindrical micelles when the correct shear force is applied. The cylindrical micelles entangle and create a gel with viscoelastic mechanical properties. The physical transition from spherical to worm-like micelle coincides with a secondary structure transition from alpha helix to beta sheet which is monitored using circular dichroism. As in the surfactant system, our peptide amphiphile solution initially behaves as a Newtonian fluid and then transitions to a shear thinning gel once a critical shear rate is surpassed. The resulting gel is self healing, and stable at 4°C on the order of weeks to months. Materials properties can also be tuned to achieve the modulus of various biological tissues. The versatile nature of this system makes it an attractive material for use as an injectable tissue engineering matrix.
4:00 PM *JJ2.6
Bioactive Membranes and Cell-like Microcapsules. Samuel Stupp, Northwestern University, Evanston, Illinois.
Our laboratory has developed a superfamily of peptide modified with hydrophobic segments that are programmed to self-assemble into filamentous nanostructures. These filaments can have remarkable bioactivity as indicated by various in vivo models for the regeneration of spinal cord, cartilage, bone, enamel, and blood vessels, among others. These peptide amphiphiles are also able to co-assemble with biopolymers to create both open and closed membranes that are highly ordered and have hierarchical architectures. This process of self-assembly can occur in time scales as fast as milliseconds, but it is dynamic and can therefore lead to the growth of membrane structures over longer time scales. The integration of this hierarchical self-assembly and the bioactivity of peptide amphiphiles yields membranes and also filamentous microcapsules of high surface area that are able to signal cells. This lecture will illustrate the use of these hybrid materials to promote angiogenesis through their ability to bind and deliver proteins. The lecture will also discuss the use of the cell-like microcapsules to interact with neurons and promote neurite growth. Another possible function of the cell-like microcapsules to be described is the possibility of using them to mediate the differentiation of stem cells.
4:30 PM JJ2.7
Multiphase Anistropic Tissue Structures by Microdroplet Based Hydrogel Printing. Umut A. Gurkan1,2, Feng Xu1,2, Yuree Sung1, Banupriya Sridharan1, Ahmet S. Yavuz1 and Utkan Demirci1,2; 1Demirci Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Harvard-MIT Health Sciences and Technology, Cambrirdge, Massachusetts; 2Center for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.
Background: In this paper we present generation and preliminary assessment of multiphase anisotropic tissue structures by microdroplet based hydrogel printing method. Current cell/tissue scaffolding methods present shortcomings due to the lack of control over the spatial and temporal control over cell seeding and extracellular matrix composition. Microdroplet based hydrogel bioprinting technology can be used to engineer complex tissue anisotropies with multiple phases by producing scaffolds with controlled micro-scale spatial heterogeneity in extracellular, cellular compositions and physical properties. Therefore, we hypothesized that the microdroplet-based hydrogel bioprinting approach developed in our laboratory will successfully facilitate engineering of the complex tissue anisotropies that are composed of multiple phases. In order to test this hypothesis we printed agarose hydrogel bioinks colored with red, green and blue (RGB) high molecular weight (35-38 kDa) fluorescent dyes and assessed the phase transitions via image processing to evaluate the anisotropy of the resulting multiphase structure by measuring the RGB color intensities. Methods: Microdroplet generation process was performed with multiple ejectors in sterile laminar flow hood under controlled humidity. The inter-droplet distance was determined by the size of the droplets residing on the substrate. The prepared bio-inks (RGB colored hydrogels) were printed in a staggered configuration. The ejector was kept warm (37 degC) to minimize viscosity changes and premature gelation of the hydrogel. Printed staggered phases were gelled by incubation at 4 degC for 5 minutes. The diffusion and integration of the phases was assessed immediately after and 3 hours after printing by taking micographs and analyzing using ImageJ software. RGB color relative intensity values were used analytically to analyze the anisotropic gradient of the phases and phase transitions. Results and Discussion: The printed multiphase hydrogel structure representing an anisotropic tissue unit displayed sharp RGB boundaries between the phases immediately after printing. These sharp boundaries disappeared and smooth transitions emerged within 3 hours. These results suggest that microdroplet based hydrogel printing technology can be used to create highly anisotropic structures with smooth boundaries mimicking the complex cellular and extracellular gradients in the natural tissues. Our long term goal is to develop effective bioprinting methodologies to engineer micro-scale anisotropic complex tissue structures with multiple phases, which can be incorporated into currently available biomaterials to face the challenges of incompatibility at tissue-biomaterial interfaces.
4:45 PM JJ2.8
Nanoporous Aluminum Oxide for Tissue Engineering. Andreas Heilmann, Annika Thormann, Andreas Hoess and Andrea Friedmann; Fraunhofer Institute for Mechanics of Materials IWM, Halle (Saale), Germany.
Because of their unique highly opened and parallel pore structure, self-supporting membranes made from nanoporous anodic alumina are promising materials for cell cultivation and tissue engineering. Due to the adjustability of the membrane properties (pore diameter, membrane thickness) and additional surface modifications, the cell growth conditions can be varied to a great extent. Anodic oxidation of aluminum was performed to obtain self-supporting membranes with pore diameters between 20 and 450 nm and membrane thick¬nesses between 20 and 60 µm. By using a special mechanical stabilization con¬cept, the membrane thickness can be reduced down to 1 µm. On various porous membranes, proliferation and adhesion behavior of HepG2 hepatoma cells were investigated. The cell growth depends slightly on the pore diameter and much more on the surface properties. Scanning electron microscopy (SEM) was applied to examine cell morphology. The preparation with Focused Ion Beam (FIB) technology was used to study the cell surface interaction. Adhe¬sion points of cells to the underlying porous substrate were located. Especially cells on membranes with pore diameters larger than 220 nm developed small cell extensions, which penetrate into the pores up to 1.5 µm. In addition to the cell growth experiments a bioreactor was designed in order to use nanoporous alumina membranes as an interface between two compart¬ments. With such a concept, co-cultures of different cell types (e.g., mesen¬chymal stem cells and primary hepatocytes) under perfusion conditions were carried out successfully. This seems to be a promising approach for different applications in pharmacological research or tissue engineering.
SESSION JJ3: Poster Session: Hybrid Biological Materials I
Tuesday Evening, April 26, 2011
Exhibition Hall (Moscone West)
Hydrogel-micropatterned Nanofibers with Dual Functions as Biomimetic Scaffold for Cell Patterning and Metabolite Detection. Hyun Jong Lee, Yeol Lee, Sangphil Park, Young Ha Park and Won-Gun Koh; Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea, Republic of.
We fabricated micropatterned polymeric nanofibrous scaffold that are capable of micropatterning mammalian cells within biomimetic three-dimensional environment and detecting cellular metabolic products simultaneously. To endow micropatterned scaffold with biomimetic cell culture and biosensing capability, double layered nanofiber mats were prepared by two-step electrospinning, where bottom and upper layer of nanofiber immobilized antibody and cell adhesion proteins, respectively. Photopatterning of poly(ethylene glycol)(PEG) hydrogels on electrospun nanofibers created clearly defined hydrogel microwells incorporating PS nanofibers. The resultant micropatterned nanofibrous substrates were obtained as free-standing and bidirectionally porous sheets, where most of the nanofibers were inserted through the side walls of the hydrogel microwells. When HepG2 cells were seeded onto resultant micropatterned scaffolds, cells selectively adhered within the PS fiber microdomains and formed spheroids. Size of spheroids could be controlled by varying the size of microwells. Although primary antibodies for albumin were also positioned the entire nanofiber region, antibody microarrays that were able to detect albumin produced by micropatterned HepG2 spheroids were created using hydrogel micropatterns which block the albumin. Due to increased surface area, antibody loading in nanofibrous substrates was greater than on planar substrates, which consequently yielded a higher fluorescence signal and faster reaction rate in immunoassays than conventional well-plate systems.
Tough Hydrogel Fiber with Polypyrrole/DNA Nanoscale Tubes. Shi Hyeong Kim, Hyun-U Cho and Seon Jeong Kim; Center for Bio-Artificial Muscle, Dept. of Biomedical Engineering, Hanyang University, Seoul, Korea, Republic of.
Hydrogels, which can be used for various applications, are mechanically weak. Thereby, they have some limitations for robust material design. However, the recent investigations into the development of strong gels have shown advances in mechanical properties and suggest possibilities of applications to soft biomimetic machines. Tough gels are promising for life science applications such as artificial muscles and tissue engineering due to their high strength, high tension, and high elasticity derived from their own unique structures. The design of new strong gels would contribute to dramatic enhancement of mechanical, electrical, electrochemical performance. In this work, we have made a gel with a structure that is completely different from previous one using polypyrrole (PPy) submicron tubes and DNA fibers. The PPy/DNA gel is created from the PPy tube/DNA/water solution using a wet-spinning technique. We show the enhancement of mechanical, electrical, and electrochemical properties of the PPy/DNA tough gel compared to DNA gels.
Sequential Delivery of TAT-HSP27 and VEGF Using Microsphere/Hydrogel Combination Systems for Therapeutic Angiogenesis. Seung-Hwa Shin, Jangwook Lee, Kwang Suk Lim, Yong-Hee Kim and Kuen Yong Lee; Bioengineering, Hanyang University, Seoul, Korea, Republic of.
Ischemic vascular disease, including myocardial infarction, cardiovascular disease, and peripheral vascular disease, is the leading cause of mortality and morbidity in the world. Protein delivery systems have been extensively investigated to treat the disease in a minimally invasive manner, as surgical operation still has limitations. We previously reported a use of microsphere/hydrogel combination systems, composed of PLGA microspheres and alginate hydrogels for the controlled release of various protein drugs. In this study, we hypothesized that two different protein drugs could be released from the combination delivery systems in a sequential manner. We chose heat shock protein (HSP) and vascular endothelial factor (VEGF) as a model protein, as HSP is known to protect cells from cellular apoptosis under the hypoxia condition and VEGF is a potent angiogenic molecule that has been frequently used for therapeutic angiogenesis. TAT-HSP27 (i.e., HSP27 fused with transcriptional activator) was loaded into alginate gels and VEGF was incorporated into PLGA microspheres with various pore sizes, in order to prepare combination delivery systems. The sequential release of TAT-HSP27 and VEGF was achieved in vitro and the VEGF release rate was able to be controlled by varying the pore sizes of the microspheres. The release of TAT-HSP27 from hydrogels was completed within 3 days, while the release of VEGF from microspheres was monitored for 28 days. Combination delivery systems were next injected into the ischemic hindlimb site of a mouse model, and therapeutic angiongenesis was evaluated by histological and immunohistochemical analysis. The sequential release of TAT-HSP27 and VEGF from combination delivery systems effectively protected muscles from ischemic stress, and significantly enhanced new blood vessel formation in the ischemic site. This approach to controlling the release of various protein drugs in a sequential manner may be useful in many biomedical applications.
Sustained Drug Release from Mesoporous Geopolymers. Johan Forsgren1, Christian Pedersen1, Maria Stromme1 and Hakan Engqvist2; 1Department of Engineering Sciences, Division of Nanotechnology and Functional Materials, Uppsala, Sweden; 2Department of Engineering Sciences, Division of Applied Materials Science, Uppsala, Sweden.
A geopolymer is a type of cementitious material which typically is produced by reacting fly ash or clay-derived precursor powders (e.g. metakaolin) with an alkaline sodium silicate solution. The present study investigates the effect of Al/Si ratio on the nanostructure of geopolymers, and the geopolymers are evaluated as drug carrying vehicles for controlled release of the opioid oxycodone. Geopolymers constitute an appealing type of material in this context as they can provide mechanical strength and chemical durability to oral dosage forms, which is crucial to avoid dose dumping. Nanoparticulate precursor powders with different Al/Si-ratios (2:1, 1:1 and 1:2) are prepared by a sol-gel route, and used in the preparation of different geopolymers. It is shown that many properties of the geopolymers can be tailored by adjusting the Al/Si-ratio in the precursor powders and the curing temperature of the geopolymers. In particular, it is shown that (i) the pore sizes of the geopolymers decrease with increasing Al/Si ratio. (ii) completely mesoporous geopolymers can be synthesized at the Al/Si molar ratio 2:1. (iii) the mesoporosity is associated with a sustained and linear drug release profile (in vitro). The effect of Al/Si ratio on the nanostructure of geopolymers has never been investigated prior to this study. The easily fabricated and tunable geopolymers presented in this study constitute a novel approach in the development of controlled release formulations, not only for opioids, but whenever the clinical indication is best treated with a sustained drug release and when mechanical stability of the delivery vehicle is crucial. The geopolymers are examined with Helium-pycnometry, N2-adsorption/desorption, X-ray diffraction and ?-potential measurements. The mechanical strength of geopolymers is measured in compression mode (Autograph AGS-H universal testing machine - Shimadzu). The drug release measurements are performed in a USP-2 dissolution bath, 50rpm, 37°C (AT7 Smart, Sotax) according to the U.S. Pharmacopeia.
In-situ Polymerization of Poly (3, 4-ethylenedioxythiophene) (PEDOT) in Brain Tissue with a Local Delivery and Deposition System. Liangqi Ouyang1, Kathleen E. Feldman1, Rylie Green1,2, Michelle Dee3, Laura K. Povlich1,4, Amy L. Griffin5 and David C. Martin1; 1Department of Materials Science and Engineering, University of Delaware, Newark, Delaware; 2Australian Vision Prosthesis Group, Graduate School of Biomedical Engineering, University of New South Wales, Sydney, New South Wales, Australia; 3Department of Chemical Engineering, University of Southern California, Los Angeles, California; 4Department of Materials Science and Engineering, University of Michigan, Ann Arbor, Michigan; 5Department of Psychology, University of Delaware, Newark, Delaware.
Neural implants have shown promising therapeutic effects in the treatment of patients with sensory or motor disabilities, Parkinson’s disease and epilepsy. However, long-term implants are known to induce a chronic foreign-body reaction, which in cortex is associated with layers of activated microglia and an insulating glial scar. Here we report on a method for directly electrochemically polymerizing poly (3, 4-ethylenedioxythiophene) (PEDOT) in living brain tissue in order to effectively extend the electrodes out beyond the glial scar to maintain contact with the neurons. We have used a microcannula drive / microelectrode system to simultaneously and locally deliver monomer and facilitate in-situ electrochemical deposition. The distribution and morphology of the PEDOT and the corresponding histology of the cortical tissue itself were studied. We found that conjugated polymer filaments which extend several millimeters into the surrounding brain tissue can be grown from the working electrode tip, forming a diffuse polymer cloud within the extracellular space. The formation of the conjugated polymers was also shown to significantly decrease the impedance of the electrode as well as to increase its charge storage capacity compared to conventional metallic controls.
Lipase-resistant Poly(glycerol Sebacate) via Bulk Physical Entrapment of the Lipase Inhibitor Orlistat: Implications for Microfabricated Tissue Engineering Scaffold Degradation. Jeffrey T. Zugates, Aurelie Jean, Nafiseh Masoumi and George C. Engelmayr; Bioengineering, Penn State University, University Park, Pennsylvania.
Biodegradation is fundamental to the prototypical tissue engineering paradigm. While hydrolysis of ester bonds is a common mode of biodegradation, it is well appreciated that certain polymers degrade principally by enzymes such as lipases. Given that the principal substrate of lipase enzymes are triglycerides, it is not surprising that poly(glycerol sebacate) (PGS) is susceptible to lipase-mediated degradation. Since its introduction by [Wang et al. 2002 Nat Biotech], PGS has become one of the most promising new scaffold materials-of-construction. More recently, [Pomerantseva et al. 2009 J Biomed Mater Res A] quantified the in vitro degradation kinetics of PGS in response to lipase. Notably, while PGS degradation by surface hydrolysis is attractive in that it renders PGS more amenable to predictive modeling, excessively rapid degradation may impact its performance in applications where high concentrations of lipases naturally manifest, including small intestine, cornea (i.e., tears), and myocardium. In the cardiac context, [Engelmayr et al. 2008 Nat Mater] introduced a novel excimer laser microfabricated accordion-like honeycomb PGS scaffold that could recapitulate aspects of cardiac anisotropy. More recently, finite element simulations by [Jean and Engelmayr 2010 J Biomech] predicted microfabricated PGS scaffold anisotropy to be linked to the dimensions of the PGS struts. Thus, temporal coordination of PGS scaffold structural-mechanical evolution with tissue formation may require controlling the rate of lipase-mediated PGS degradation. By virtue of its FDA approval, low water solubility, and sterically-hindered structure, we hypothesized that the lipase inhibitor Orlistat might be suitable for inhibiting lipase-mediated PGS degradation. In the current study we are investigating whether Orlistat can: (1) be physically transported into PGS using 70% (v/v) ethanol in water as a carrier, (2) be stably entrapped within the PGS bulk upon exchange of alcohol for water, and (3) inhibit PGS degradation by lipases. In our initial experiments, control 5x5x0.25mm PGS scaffolds comprised of 50 micron-wide struts and identical scaffolds loaded by the aforementioned method with Orlistat were challenged by in vitro incubation in a lipase solution (2000 U/ml). Scaffolds were incubated at 37°C for 1.5, 2, or 3 hours and then assessed by scanning electron microscopy. While evidence of degradation was grossly apparent after 1.5 hours in the controls, little degradation was seen in the Orlistat-loaded scaffolds. By 3 hours, while Orlistat-loaded scaffolds began to exhibit modest degradation, controls had undergone rupture of structural elements in many locations. Of note, we demonstrated that at the micro-scale, the path of lipase-mediated degradation is not homogeneous, but rather appears to follow pre-existing imperfections (e.g., micro-cracks) in the PGS struts. Results will guide the design of PGS scaffolds with controllable lipase-resistance.
Recognition of Individual Carbon Nanotubes by Human Mesenchymal Stem Cells. Seon Namgung1, Ku Youn Baik1 and Seunghun Hong1,2,3; 1Physics and Astronomy, Seoul National University, Seoul, Korea, Republic of; 2Biophysics and Chemical Biology, Seoul National Univ., Seoul, Korea, Republic of; 3Interdisciplinary Program in Nano-Science and Technology, Seoul National Univ., Seoul, Korea, Republic of.
Recent studies have revealed that nanoscale cues on a solid substrate can control cell behaviors such as adhesion, proliferation, and differentiation. However, it is still unclear if cells can recognize individual nano-cues or simply respond to the bulk properties of nano-cue arrays. Herein, we demonstrated that human mesenchymal stem cells (hMSCs) could recognize individual nano-cues on a solid substrate by monitoring hMSC behaviors on a layer of carbon nanotubes (CNTs). In this work, we assembled CNTs in an aligned formation with a low or high density on solid substrates and, then, compared the adhesion and growth of hMSCs on them. The results show that the hMSCs grew along the CNT alignment directions only on CNT layers with its density low enough for the cells to recognize individual CNTs, while those on high-density CNT layers grew in a random orientation. Furthermore, the alignment of low-density CNT layers was found to affect the proliferation and differentiation of the hMSCs on them. It indicates hMSCs can recognize the individual nanoscale cues such as individual CNTs. This work provides a new insight about cell behaviors on nanostructured materials and a possibility for various applications in cell and tissue engineering.
Amine-rich Polyelectrolyte Adhesion Layers for Surface Immobilization of Biomolecules and Nanostructures. Stefan V. Stoianov, Jason I. Ridley and Hans D. Robinson; Physics, Virginia Tech, Blacksburg, Virginia.
We evaluate amine enriched poly(styrene sulfonate)/poly(allylamine hydrochloride (PSS/PAH) self-assembled multilayers for the purpose of fixating freestanding or solution suspended nanostructures and biomolecules onto glass substrates and other surfaces. Unlike common strategies such as silylation of the surface with amine-containing ligands, PSS/PAH multilayers can be fabricated rapidly, without using aggressive chemicals, do not require inert processing conditions to achieve an optimal result, and can be applied to a larger variety of surfaces. PSS/PAH multilayers fabricated at pH above 8.5 are enriched in amines that promote adhesion when exposed on the surface of the film. Exposure is achieved by cycling the film to pH values below 4 and back to neutral, which causes the film to swell substantially as the amine groups are protonated. Conversely, cycling to pH 10 and back causes deswelling as the amines pack tightly into hydrophobic regions. This makes it possible to protect the surface from contamination until just before use. In addition, the film can be selectively passivated by acetylation with acetic anhydride. The resulting surface exhibits negligible non-specific binding of negatively charged proteins. Parts of the surface covered with metal or other structures are not affected by this, making patterned adhesion easy to accomplish. We find that PSS/PAH yields at least as good adhesion of nanostructures to the surface as silylation with 3-aminopropyltriethoxysilane (APTES). We will present these results along with quantitative results from ongoing investigation of biomolecule adhesion on the films.
Nanoscale Patterning and Manipulation of Active Single-molecule DNA Structures on Surfaces. Eric A. Josephs1, Cheetar Lee2, Jingru Shao2, Gary Abel2 and Tao Ye2; 1School of Engineering, University of California, Merced, Merced, California; 2School of Natural Sciences, University of California, Merced, Merced, California.
While DNA is generally thought of solely as a biological information carrier, certain DNA motifs can be engineered to catalyze chemical reactions, bind to small-molecule ligands, or assemble into rigid or mechanically active structures. Chemical patterning of these DNA devices would allow us to obtain unprecedented control of the chemical environments around these biomolecules for both single-molecule biophysical studies and on-demand activation of biochemical reactions. Here we report our nanometer-resolution patterning, observation, and manipulation of single DNA molecules on a chemically well-defined surface by combining electrochemical atomic force microscopy and nanolithography. We are able to place single and individually-resolvable DNA oligomers consistently within select nanometer-scale patterns across micron-sized areas of a gold electrode. We find that the negatively-charged DNA molecules are covalently bound to the gold but lie flat atop the positively-charged molecules that were patterned simultaneously within a neutral monolayer. By electrochemically switching the strong resulting surface confinement of the tethered DNA we are able to modulate the activity of catalytic “DNAzymes” by controlling the electrode potential. We are currently integrating nanolithography and AFM imaging with optical microscopy to investigate both the assembly and catalytic behavior of the DNAzymes at the single-molecule level. In addition to creating a route to construct more complex biomolecular architectures, our results suggest we can combine lithographic techniques with molecular self-assembly to create dynamic biotechnological surfaces of single DNA molecules with control over biologically-relevant length-scales.
Electrochemical Control of Motor Protein Motility using Conducting Polymer on Graphene for Bio-inspired Hybrid Nanomechanical Systems. Kyung-Eun Byun1, Dong Shin Choi2, David H. Seo4, Heejun Yang4, Sunae Seo4 and Seunghun Hong1,2,3; 1Physics and Astronomy, Seoul National University, Seoul, Korea, Republic of; 2Nano Science and Engineering, Seoul National University, Seoul, Korea, Republic of; 3Biophysics and Chemical Biology, Seoul National University, Seoul, Korea, Republic of; 4Semiconductor Devices Lab., Samsung Advanced Institute of Tech., Yongin-Si, Gyeonggi-Do, Korea, Republic of.
Motor protein generates motions in a biological system by converting the chemical energy of adenosine triphosphate (ATP) into mechanical energy. It has a great potential as a key component for bio-inspired nanomechanical systems due to its small dimensions and high fuel efficiency. For such applications, it is crucial to control the motor protein motility in real time, which has been very difficult to achieve. Herein, we develop a method to control motor protein motility by electrochemically-released bioactive materials from conducting polymer structures on graphene. Graphene was functionalized to support the motility of motor protein, and it was utilized as a transparent electrode for the stimulation of bioactive materials release. This result can be a major breakthrough in the area of bio-inspired nanomechanics research and should pave the way toward advanced hybrid systems such as biomotor-based engine structures.
Investigating the Intracellular Self-assembly of Small Molecules. Yuan Gao1, Yi Kuang1, Cristina Berciu2, Daniela Nicastro2 and Bing Xu1; 1Chemistry, Brandeis University, Waltham, Massachusetts; 2Biology, Brandeis University, Waltham, Massachusetts.
Enzyme catalysis and self-assembly are two essential and ubiquitous processes during the whole span of cell life. Like cellular proteins that form fibrillary nanostructures (e.g., cytoskeletal filaments), small hydrogelator molecules self-assemble in aqueous environment to generate molecular nanofibers. While the endogenous protein filaments are crucial for normal cellular functions or highly suspectable with several human illnesses, the assembly and intracellular fate of MNFs remains unexplored. Here we choose phosphatase as the unique trigger for our enzymatic hydrogelation process. Sharing the common feature of self-assembled motif, the low molecular weight hydrogelator precursor not only serves as the substrate of phosphatase but also can easily pass the cell membrane by passive diffusion, which allows the phosphatase catalysis to regulate the formation of intracellular molecular nanofibers and subsequent hydrogelation. LC-MS confirms the gelator concentration inside cells between 2.62-9.41 mg/mL which is above the critical gelation concentration in vitro. Confocal microscopic study shows the localization of the molecular nanofibers while the electron microscopic study on the microtome section of cells reveals the molecular nanofibers at a width of 11.0±2.5 nm and its interaction with cellular organelles. This work not only provides the spatiotemporal profile of molecular nanofibers inside cells, but may lead to a new paradigm for cellular regulation based on the interactions between molecular nanofibers and organelles.
The Immunotoxicology of Mesoporous Silica Particles. Natalia Kupferschmidt1, Susanne Gabrielsson2 and Alfonso Garcia-Bennett1; 1Division of Nanotechnology and Functional Materials, Department of Engineering Sciences, The Angstrom Laboratory, Uppsala University, Uppsala, Sweden; 2Dept. of Medicine, Unit of Clinical Allergy Research, Karolinska Hospital and Institute, Stockholm, Sweden.
Mesoporous silica particles present several properties that make them highly interesting for biomedical applications. Amongst those properties the high loading capacity of highly ordered and size tuneable pores in the mesoscale range (2-50nm) enable a high number of molecules to be loaded and released (1). Their surface properties can be functionalized to tailor pharmacokinetic behaviour in a stable and biocompatible silica delivery vehicle. They offer numerous potential benefits in biomedical applications such as; the possibility of enhance oral bioavailability of drugs with limited aqueous solubility. Mesoporous materials can protect pH sensitive drugs during their passage through the stomach allowing the absorption in the intestine of the active drug form. Controlled and sustained release of drugs at an effective therapeutic level reducing the number of doses and mitigating toxicity of drugs. Targeted release to specific target cells/tissue. They can act as adjuvants by releasing minor antigen amounts and by interfering in activation and modulation of immunocompetent cells as dendritic and B cells. Mercuri et al. demonstrated that SBA-15 mesoporous material induce a better memory induction than aluminum hydroxide and a better or similar antibody response compared to incomplete Freud adjuvant (2). Our work has concentrated in this latter advantage. We first evaluate the biocompatibility of mesoporous silica particles by studying they effect on immunocompetent cells. Exposition of human macrophages to AMS-6 particles did not affect their viability or function (engulfment of target cells and cytokine secretion) (3). We shown that mesoporous silica particles induced minor size and concentration effect on human dendritic cells cytokine production (4), this could be exploited to tailor the immune response to a specific bimolecule towards the most favourable response depending on the desired effect in the development of vaccine delivery systems. 1. Hartmann, M. Chem Mater 2005, 17 (18), 4577-4593. 2. Mercuri, L. P.; Carvalho, L. V.; Lima, F. A.; Quayle, C.; Fantini, M. C.; Tanaka, G. S.; Cabrera, W. H.; Furtado, M. F.; Tambourgi, D. V.; Matos Jdo, R.; Jaroniec, M.; Sant'Anna, O. A. Small 2006, 2 (2), 254-6. 3. Witasp, E.; Kupferschmidt, N.; Bengtsson, L.; Hultenby, K.; Smedman, C.; Paulie, S.; Garcia-Bennett, A. E.; Fadeel, B. Toxicol Appl Pharmacol 2009, 239 (3), 306-19. 4. Vallhov, H.; Gabrielsson, S.; Stromme, M.; Scheynius, A.; Garcia-Bennett, A. E. Nano Lett 2007, 7 (12), 3576-82.
Binary Self-assembled Monolayers Modified Au Nanoparticles as Carriers in Biological Applications. Hsun-Yun Chang1, Yun-Wen You1, Guo-Ji Yen2, Wei-Lun Kao2, Chi-Jen Chang2 and Jing-Jong Shyue1,2; 1Research Center for Applied Science, Academia Sinica, Taipei, Taiwan; 2Material Engeneering, National Taiwan University, Taipei, Taiwan.
Nanoparticles are widely studied in cell therapies through attaching to cells and delivering medical cargos. Gold nanoparticles (AuNPs) are known as good carriers for its ease of synthesis and conjugation in biochemistry. Self-assembled monolayers (SAMs) provide a tunable system to change the interfacial properties of AuNPs, so that SAM-modified AuNPs can be used to transport biological molecules including DNA and proteins. However, these molecules are usually covalently bonded to the surface of AuNPs and required chemical cleavage inside the living cells in order to release the cargo. It might be beneficial to use electrostatic interaction to immoblilize molecules. In order to control the electrostatic interaction, AuNPs were modified with SAMs of mixed carboxylic acid and amine functional groups. It was found that a series of surface potential and the iso-electric point (IEP) of resulting AuNPs can be tailored by the chemical composition. By changing the environmental pH around the IEP, molecules could be triggered to adsorb or desorb due to the flip of surface charge. Based on different pH inside and outside of living cells, we expect that molecules can be transported by electrostatic interactions with binary SAM-modified AuNPs. In this work, the result of MTT assay revealed that binary SAM-modified AuNPs have high biocompatibility to HEK293T cells. The cellular uptake of binary SAM-modified AuNPs was examined by measuring the concentration of AuNPs remaining in the culturing medium. It was found that the degree of AuNPs ingested by cells were more obvious with pure or high loading of amine on the surface than those modified with carboxylic acid. This difference in AuNPs uptake was also observed with a scanning transmission electron microscope (STEM). The results suggested that HEK293T cells preferred amine-modified AuNPs than that modified with carboxylic acid. The difference can be rationalized by the electrostatic potential. Because most cells exhabit non-uniform negative surface charge, positively charged amine-SAM modified AuNP (+2 mV) has higher probability to attach to the cell and be ingested. For binary SAM-modified AuNPs with 1:0.33 amine to carboxylic acid ratio, they still can be ingested by cells even its surface potential (-35 mV) was close to that modified with carboxylic acid (-37 mV). This result suggested that even low concentraction of amine-modified SAM can help cells to ingest SAM-modified AuNPs of strong negative potential by decreasing the repelling of static electricity. Since binary SAM-modified AuNPs have low cytotoxicity and good affinity to cells, the ability of binary SAM-modified AuNPs as carriers were examined. The plasmid DNA containing eGFP reporter gene is chosen as cargo carried by binary SAM-modified AuNPs. It was studied whether binary SAM-modified AuNPs can carry plasmid DNA into HEK293T cells by electrostatic interactions and release it for expression.
Understanding Energy Dissipation and Thermodynamics in Biomotor-driven Self-assembly Systems. George D. Bachand1, Haiqing Liu2, Marlene Bachand3, Erik D. Spoerke4 and Bruce C. Bunker4; 1Center for Integrated Nantoechnologies, Sandia National Laboratories, Albuquerque, New Mexico; 2Surface and Interface Sciences Department, Sandia National Laboratories, Albuquerque, New Mexico; 3Nanobiology Department, Sandia National Laboratories, Albuquerque, New Mexico; 4Electronic Materials and Nanostructures Department, Sandia National Laboratories, Albuquerque, New Mexico.
Living organisms use dynamic self-assembly processes in a vast array of physiological behaviors including transcription, chromosomal segregation, and immunological synapse formation. Emergent behaviors, such as self-healing, result from the non-equilibrium nature of these assembly processes, and may be exploited to achieve "smart" behaviors in ex vivo, hybrid materials systems. The dissipation of chemical energy via ATP hydrolysis by biomolecular motors plays a crucial role in many of dynamic assembly process found in biological systems. We have recently characterized the role of energy dissipation and thermodynamics in the dynamic assembly of nanocomposite structures derived from the interaction of nanoparticles and microtubules. Several morphologically distinct nanostructures were generated by regulating the thermodynamic input and the rate of energy dissipation in this system, including (a) mobile, linear structures, (b) rotating rings, and (c) immobile, disordered aggregates. In addition, we evaluated the collective behavior of kinesin motors with respect to the rotation of ring structures. This presentation will focus on providing a fundamental understanding of how energy dissipation, thermodynamics, and mechanics may be manipulated to self-assemble structured nanomaterials that exist far-from-equilibrium. *Sandia National Laboratories is a multi-program laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin company, for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
Human Serum Albumin-mediated Formation of Silver Nanoparticles on Multi-walled Carbon Nanotubes. Andres Rodriguez-Galvan1, Flavio F. Contreras-Torres2, Victor Meza-Laguna1, Elena V. Basiuk2 and Vladimir A. Basiuk1; 1Instituto de Ciencias Nucleares, Universidad Nacional Autonoma de Mexico, Mexico-City, D. F. , Mexico; 2Centro de Ciencias Aplicadas y Desarrollo Tecnologico, Universidad Nacional Autonoma de Mexico, Mexico-City, D. F. , Mexico.
Biomedical applications of silver nanoparticles (AgNPs) based on their antibacterial activity require certain characteristics of size, shape and distribution. A number of factors, such as ready oxidation in air, tendency to agglomeration, etc., can influence the nanoparticle formation. Different methods for their in situ preparation using organic and inorganic templates, including proteins and carbon nanotubes, can be explored to optimize the process. In the present study, we developed a facile and ecologically friendly method to form AgNPs on the sidewalls of multi-walled carbon nanotubes (MWNTs) previously non-covalently functionalized with human serum albumin (HSA) protein. The deposition of AgNPs onto HSA-MWNTs was performed in situ by means of the chemical reduction of silver ions with citric acid, resulting in AgNPs/HSA-MWNTs bionanohybrid composites. The morphology and structure of AgNPs/HSA-MWNTs composites was characterized by atomic force and scanning tunneling microscopy, as well as transmission electron microscopy (TEM). Elemental chemical analysis by using TEM-coupled energy-dispersive X-ray spectroscopy (EDS) corroborated the presence of silver particles in the bionanohybrids. The following characteristics are especially important from practical point of view: (i) the protein coating is sufficiently stable to resist detachment from the nanotube surface; (ii) the bionanohybrids obtained are highly dispersible in water; (iii) the size of silver nanoparticles can be controlled by using a weak reducing agent (citric acid) in conjunction with a lowest concentration of HSA (ca. 1 µg/mL) producing AgNPs of ca. 2-nm diameter. The work was supported by the projects UNAM DGAPA-IN103009 and IN100610, CONACYT-56420, and ICyTDF-333/2009.
Optimisation of the Material and Culture Conditions for the Formation of a Hard/soft Tissue Interface. Jennifer Z. Paxton and Liam M. Grover; School of Chemical Engineering, University of Birmingham, Birmingham, United Kingdom.
The anterior cruciate ligament (ACL) is the most common musculoskeletal injury in the developed world . Due to the insufficient quality of ligament repair and the low long-term success of synthetic grafts, the demand for tissue-engineered ligament tissues for implantation is increasing. However, in vivo bone-ligament healing is often inadequate, with a failure to generate the graded mineral and mechanical properties of the natural interface . It is therefore pertinent to establish improved regeneration at the hard/soft tissue interface or to engineer this complex structure in vitro if engineered tissues are to become a realistic clinical possibility. We have developed a system whereby artificial bone-ligament-bone constructs can be engineered using a ceramic and cell-seeded hydrogel to produce a ceramic-hydrogel-ceramic composite. Our previous work showed that an in vitro hard/soft tissue interface could be formed using brushite cement and cell-seeded fibrin hydrogel and importantly, that varying the shape of the ceramic (bone) and the collagen content of the fibrin (ligament) could affect both interface strength and longevity between the two materials . Our current work is focussed on three main areas; 1) the assessment and comparison of the ability of various other materials (e.g. hydroxyapatite, calcium pyrophosphate, magnesium oxychloride, calcium silicate, calcium carbonate, alumina) to form the in vitro hard-soft tissue interface; 2) augmenting the collagen deposition profile of tendon fibroblasts seeded within our fibrin hydrogel by chemical conditioning with ascorbic acid and growth factors (e.g. transforming growth factor, TGFß, fibroblast growth factor, FGF-4 and growth and differentiation factor, GDF-7) and 3) mechanical conditioning of the constructs using specially-designed bioreactors to generate functional tissues. Examination of these variables is essential to determine the optimal material, collagen content and mechanical properties of the hard/soft tissue interface to allow the production of clinically relevant bone-to-bone ligaments for future implantation.  Spalazzi, JP. et al., 2006 Tissue Eng. 12(12):3497-508  Fu, FH. et al., 1999 Am J Sports Med 27(6): 821-30.  Paxton, JZ et al., 2010 Ann Biomed Eng. 38(6):2155-66.
Cell Attachment and Proliferation Study on a High Pressure Torsion (HPT) ``Gum Metals”. Kelvin Y. Xie1, Yanbo Wang2, Babak Sarafpour3, Hans Zoellner3, Simon P. Ringer1 and Xiaozhou Liao2; 1The Australian Centre for Microscopy and Microanalysis, The University of Sydney, Sydney, New South Wales, Australia; 2School of Aeronautical, Mechanical and Machatronics Engineering, The University of Syendy, Sydney, New South Wales, Australia; 3Faculty of Dentistry, The University of Syendy, Sydney, New South Wales, Australia.
Gum metal is a type of specially designed multifunctional beta-type titanium alloy. It has high strength and excellent deformability because it deforms via giant fault instead of ordinary dislocation flows. It possesses ultra-low Young’s modulus which reduces the problems associated with stress shielding introduced by conventional bone implants. These attractive properties make gum metal great potential material for biomedical applications. When gum metal is processed with high pressure torsion (HPT), apparent grain refinement from micron level to ~10 nm was observed, accompanied with substantial increment in hardness. Cells were seeded directly on both as-received and HPT gum metals using human gingival fibroblasts (passage number 6-7). Cells were confluent on all samples and no obvious differences in cells number density was observed between as-received and HPT gum metal 4 days after cells seeding. However, enhanced cell attachment was noticed 90 min after cell seeding on HPT gum metal in comparison to the as-received ones. Even better than conventional gum metal, HPT gum metal seems to have the potential to become an alternative to current Ti-based materials for dental implants as it exhibited superior mechanical properties. Furthermore, enhanced cell attachment can be beneficial especially for the initial healing process after implantation.
SESSION JJ4: Hybrid Materials for Biosensing
Chair: Matthew Maye
Wednesday Morning, April 27, 2011
Salons 4-6 (Marriott)
8:30 AM *JJ4.1
Application of Biohybrid Material in Biosensing Devices. Jean-Louis Marty, IMAGES EA 4218, Universite de Perpignan Via Domitia, Perpignan, France.
A biohybrid material can be defined as a material that includes two moieties blended on the molecular scale. Commonly one of these compounds is inorganic and the other one organic in nature The rapid increase of interest in the field of biological hybrid materials that exhibit improved structural and functional properties is attracting more and more researchers from life science. Combining advanced materials with biology has become one of the most innovative research fields. We focus our talk on the biohybrid materials specifically designed for biosensor application developed in our laboratory. A biosensor responds to the presence of a biological agent by combining a sensitive biological element with a physical device. The living systems are able to perform a series of complex and attractive catalytic reactions. However, cells, isolated from their native environment, are generally fragile or unsuitable in the development of technologies. In the last years, abundant researches have proved that encapsulation of biomolecules; enzymes and microorganisms into various artificial matrices can be very attractive to create new functional bio-composites. We developed various approaches - Encapsulation of biomolecules, enzymes and fragile biological species (thylakoids, chloroplasts) into various artificial matrices can be very attractive to create new functional bio-composites - Immobilization of enzymes, antibodies, DNA and aptamers on functionalized magnetic beads. This material provides a platform to integrate different steps in the sensing process (separation, reaction and immobilization) and open the path to the realization of lab-on-a-chip system. - Biomolecule-nanoparticles (NP) hybrid systems with gold nanoparticles, SWCNT or MWCNT allow the fabrication of many novel high-sensitivity biosensors. - Use of porous materials, macroporous ordered silica foams (MOSF) and carbon mesoporous materials (CMM) The final objective of this science is to develop hybrid material to fabricate a biosensor, as a physical device out of the biological element itself.
9:00 AM JJ4.2
CdSe/ZnS QDs-G-quadruplex Hybrids as Optical Sensors for DNA and Telomerase Activity. Ronit Freeman, Etery Sharon and Itamar Willner; Institute of Chemistry, The Center for Nanoscience and Nanotechnology,, The Hebrew University of Jerusalem, Jerusalem, Israel.
Quantum dots (QDs) attract substantial recent research interest as optical labels for sensing and biosensing events, due to their unique size-controlled luminescence properties. Also, the horseradish peroxidase (HRP) mimicking DNAzyme, a hemin/G-quadruplex conjugate was, recently, extensively used as a catalytic amplifier for biorecognition events. The modification of the CdSe/ZnS QDs with DNA G-quadruplex/hemin conjugates and the interactions (electron transfer quenching) between metal-porphyrin/DNA structures and quantum dots enabled the analysis of DNA and aptamer-substrate complexes. Also, the luminescence quenching of functionalized quantum dots by Gquadruplex/ hemin complexes generated by telomeric chains was used to monitor telomerase activity. The structure, composition, and operation of the different systems will be presented.
9:15 AM JJ4.3
Electrodeposited Iridium Oxide Electrodes for the Detection of Glutamate in vitro. Danielle R. Rand, Silke Musa and Wolfgang Eberle; imec, Leuven, Belgium.
Iridium oxide (IrO2) is an attractive electrode material due to its biocompatibility, wide pH response range, and excellent electrical conductivity. Our approach investigates the use of IrO2 electrodes for real-time monitoring of the release of neurotransmitters from neuronal networks in vitro. Here, we describe the incorporation of neurotransmitter receptors into electrodeposited IrO2 films to create a biomimetic environment that ensures neuronal survival. A three-electrode configuration was used for the electrochemical deposition and measurements. Microfabricated Si devices with TiN electrodes 50 µm in diameter served as working electrodes. Prior to deposition, various electrode pre-treatments were examined, including potential cycling in H2SO4. This proved important in controlling the surface roughness and thus increasing the surface area of the TiN electrode. Atomic force microscopy determined that a low rate of cycling (1.55 V/s) in H2SO4 led to a root mean square roughness value of rms = 25.1 nm, while a fast rate (8.55 V/s) led to rms = 3.72 nm for 129 potential cycles. IrO2 was electrodeposited on these TiN electrodes using various methods, including constant anodic potential. Cyclic voltammetry suggest that longer deposition times and higher deposition potentials up to 0.6 V result in optimal films. The pH response of these IrO2 electrodes was then tested in phosphate buffer (PB) by varying the pH and measuring the potential change. For an average pH change of 0.19 ± 0.2, a potential change of 18.9 ± 2.2 mV was measured in the pH range of 6-8, with very little hysteresis. The IrO2 electrodes were also used for the detection of the neurotransmitter glutamate. This was achieved by exploiting the enzyme glutamate oxidase (GLOD), which uses glutamate as a substrate and releases NH3 as a product, thus creating a detectable local pH change in the solution. The immobilization procedure of GLOD onto IrO2 electrode surfaces involves the electrodeposition of IrO2, followed by immersion into 2 mg/mL GLOD solution. This process was repeated at least 5 times. After immobilization, the enzyme remained active, as monitored by an assay that correlates absorbance with GLOD activity. The assay showed that 0.093 µg/mL GLOD was incorporated into the IrO2 films when the immobilization procedure was repeated 5 times, while almost a doubling of 0.141 µg/mL GLOD incorporation was achieved with 10 procedure repeats. Cyclic voltammograms show that the incorporation of GLOD does not cause crack formation and, thus, does not induce additional stress in the IrO2 layer. Furthermore, the IrO2/GLOD layer remains stable for at least 16 h after deposition. Glutamate was detected in PB using the IrO2/GLOD electrodes by small additions of 1 M GLOD solution. The measured potential change was 55 mV ± 21 mV, thus proving that electrodeposited IrO2 electrodes can anchor glutamate-sensitive enzymes and be used for the detection of glutamate in vitro.
9:30 AM JJ4.4
Chemically Modified M-13 Bacteriophages Amplifiable Colorimetric Antigen Detection. Ju Hun Lee and Jennifer N. Cha; Department of Nanoengineering and Materials Science and Engineering Program, University of california, San Diego, La Jolla, California.
For early disease detection, biomarker detection with extremely high sensitivities are critical. However, in order to meet the demand for diagnostics, advanced technical platforms that are reliable, rapid, low-cost, high throughput are needed. Additionally, in order for these sensors to have true global applicability, the devices must not be depended on additional equipment, such as high end freezers or spectrophotometers. Therefore, the ideal diagnostic would be one that is solution based and can provide a rapid colorimetric response with extremely high specificities and sensitivities. As a means to address these challenges, we show here that M13 bacteriophages can act as amplifiable platforms to induce color changes when mixed with metal nanocrystals in direct response to an antigen in solution. The advantages of using M13 bacteriophages for sensor technologies include their ease of production, their stability in most environments as compared to enzymes, their antigen recognition and the ability to modify their majority coat protein that can serve as the amplifiable platform for sensor diagnostics. We demonstrate here a complete biosensor based on thiol modified M13 bacteriophage that were screened using phage display to bind specifically to rabbit immunoglobulins. As a means to drive gold nanocrystal flocculation, we modified the major coat protein of the viruses with thiol groups using cysteamine and carbodiimide chemistry. Any antigen in solution were detected then by reacting with the thiolated M13 bacteriophages which were then captured using magnetic microparticles. The bound phage were then released into solution under acidic conditions and were detected in solution by adding gold nanocrystals and looking for colorimetric changes due to changes in plasmon resonance. We demonstrate that the phage based sensors could show visible color changes with femtomolar sensitivities lending to the notion of providing low-cost diagnostics that can be used in any location around the world.
9:45 AM JJ4.5
Regenerative Immobilization of Biomolecules on Label-free Optical Biosensors. Heather K. Hunt1, Carol Soteropulos2 and Andrea M. Armani1,3; 1Mork Family Department of Chemical Engineering and Materials Science, Univeristy of Southern California, Los Angeles, California; 2Department of Biomedical Engineering, University of Southern California, Los Angeles, California; 3Ming Hsieh Department of Electrical Engineering-Electrophysics, University of Southern California, Los Angeles, California.
The rational design of drug-based therapeutics to detect and combat antigens, such as viral infections and cancer cells, requires a fundamental understanding of the relationship between structure and function of biological macromolecules. To fully characterize the effects of the molecules’ activity, conformation, binding sites, and local environment on intermolecular interactions, it is necessary to study macromolecular binding, both at the ensemble and single molecule level. Typically, single molecule binding events are investigated using fluorescence imaging techniques, which require a label to detect the binding event of interest, suffer from fluorophore instability, and can adversely affect the active centers. Recently, a new class of label-free, high-performance optical devices, based on whispering gallery microcavities, has demonstrated unique capabilities as biosensors. The very low optical loss of these devices, characterized quantitatively by the quality factor (Q) of the cavity, enables “optical amplification” of otherwise undetectable signals to occur within the structure, improving the signal to noise ratio. For example, using ultra-high-Q (Q > 10^8) silica microcavity devices, researchers have shown label-free detection of protein conformation changes and bacteria, as well as label-free detection of viruses and molecules at the single molecule concentration level. While the low optical loss of these devices leads to high sensitivities, the performance of the optical platform may be further improved by the addition of specificity towards biomolecules of interest. However, no general methods exist for the covalent bioconjugation of these devices, primarily because of the difficulty of functionalization without device degradation. Here, we demonstrate a facile method to impart specificity to optical microcavities without adversely impacting their optical performance, using ultra-high-Q, silica microtoroids and microspheres as test platforms. In this approach, the sensors are selectively functionalized with biotin via a 3-step grafting process. The as-fabricated and surface-modified devices are characterized by XPS, fluorescence, optical, and scanning electron microscopy, as well as microcavity analysis techniques, to determine the impact of the surface functionalization on the device sensitivity (Q factor). Additionally, the resulting biotinylated device is used to detect streptavidin as a proof of concept. The functionalized sensors have a uniform probe coverage, low optical absorption, specificity to the target ligand, high sensitivity, and are stable to long-term storage. Lastly, the sensor surface may be renewed via O2 plasma etching without sensor or binding site degradation. This work represents one of the first examples of non-physisorption-based bioconjugation of optical microtoroid resonators for the label-free detection of biomolecules in real time.
10:30 AM JJ4.6
Layer by Layer Deposition of Polymer Spheres for Biosensors. Alexandra Snyder and Lia Stanciu; Materials Engineering, Purdue University, West Lafayette, Indiana.
Latex microspheres were used as a template for layer by layer deposition of polyelectrolytes with opposite surface charges. The latex was coated with three alternating layers of polyacrylic acid (PAA) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy and zeta potential measurements verified the deposition of the polyelectrolyte layers. The enzymes acetylcholinesterase (AChE), and horseradish peroxidase (HRP) were separately immobilized onto the surface of the polymer spheres by electrostatic forces and hydrogen bonding. Screen printed electrodes were coated with the immobilized enzymes in order to measure current response and stability of the enzyme. Enzyme concentration, pH, and applied voltage were optimized for each system. Higher current response was observed when AChE was immobilized onto polymer spheres with a terminal PAA layer, despite the fact that both the polyelectrolyte and the enzyme are negatively charged. This can be attributed to positively charged PAH molecules blocking the negatively charged active site of the enzyme during immobilization. Inhibition tests will be done to measure the paraoxon detection limit of these biosensors and long term stability of the electrodes will be observed. The electrodes coated with immobilized HRP exhibited hydrogen peroxide detection limits that ranged from 0.8 to 8 µM H2O2. Stability measurements confirm that the biosensor retains 87% of its initial current response after 10 weeks of storage.
10:45 AM JJ4.7
Chromatic Lipid/Polymer Vesicles Encapsulated within Sol-gel Matrixes for Diagnostic Applications. Raz Jelinek, Sofiya Kolusheva and Angel Porgador; Ben Gurion University, Beer Sheva, Israel.
Non-invasive, early detection of lethal diseases, such as cancer, is considered the single most effective factor in treatment and survival. We describe a new disease diagnostic approach, denoted “reactomics”, based upon reactions between blood sera and an array of gel-encapsulated vesicular aggregates comprising lipids and polydiacetylene (PDA), a chromatic polymer. The lipid/PDA vesicles in the array constitute an effective biosensing platform, as the lipid surface attracts lipophilic molecules in serum, and the PDA subsequently reports upon lipid interactions through both colorimetric and fluorescence transformations. We show that reactions between sera and the lipid/PDA vesicle array produce chromatic patterns which depend both upon the sera composition as well as the specific lipid constituents within the vesicles. The chromatic patterns were processed through machine-learning algorithms, and the bioinformatics analysis could distinguish both between disease-bearing and healthy patients, respectively, as well between different types of disease. This chromatic reactomics concept is generic, robust, and does not require a priori knowledge upon specific disease markers in sera.
11:00 AM JJ4.8
Plasmonic Nanopore Arrays for Label-free Kinetic Biosensing of IgM Antibody Binding to Lipid Membranes. Hyungsoon Im1, Nathan J. Wittenberg1, Brent R. Wright2, Arthur E. Warrington2, Moses Rodriguez2 and Sang-Hyun Oh1; 1Electrical and Computer Engineering, University of Minnesota, Minneapolis, Minnesota; 2Neurology, Mayo Clinic, Rochester, Minnesota.
We integrated periodic nanopore arrays in a thin gold film with supported lipid membranes for surface plasmon resonance (SPR) biosensing inside microfluidic channels. Surface plasmon-enhanced light transmission through the periodic nanopore arrays enables real-time label-free sensing of molecular binding on the lipid membrane surface. To facilitate formation of a lipid bilayer, the nanopore surface was encapsulated by a 12-nm-thick silica layer deposited by atomic layer deposition, which makes the surface hydrophilic. A supported lipid bilayer is formed over the nanopore arrays by vesicle rupture in microfluidic channels and the continuity and fluidity of the supported lipid bilayer was characterized by fluorescence recovery after photobleaching (FRAP). We investigated binding kinetics of immunoglobulin M (IgM) O1 and O4 antibodies, which are mouse anti-galactocerebroside and anti-sulfatide antibodies, respectively, using the surface plasmon-enhanced light transmission through the nanopores. These antibodies bind to oligodendrocytes in vitro and IgM O4 has been shown to promote myelin regrowth in a mouse model of multiple sclerosis. We were able to detect specific binding of IgM O1 and IgM O4 to galactocerebroside and sulfatide containing lipid membranes, respectively. Negative controls showed that the presence of galactocerebroside and sulfatide are essential for binding of IgM O1 and IgM O4 onto the membranes, respectively, and there is little cross-binding between the antibodies and membranes. This work shows the potential use of the nanopore arrays for real-time label-free sensing with cell membranes and eventually characterization of therapeutic antibodies for new drug discovery.
11:15 AM JJ4.9
Fabrication of Nanostructured Multi-catalyst System Entrapping Fe3O4 Magnetic Nanoparticles and Oxidase in Large Pore Sized Mesoporous Materials for Colorimetric Biosensing and Electrochemical Biosensing. Jinwoo Lee1, Moon Il Kim2, Jongmin Shim1, Youngjin Ye1 and Hyun Gyu Park2; 1POSTECH, Pohang, Korea, Republic of; 2Chemical Engineering, KAIST, Daejon, Korea, Republic of.
Large cellular porous silica materials were synthesized by the self-assembly of commercially available block copolymers, P123. One-pot multi-catalyst system, so called “nanofactory”, was developed entrapping magnetic nanoparticles (Fe3O4) and oxidases in large cellular mesoporous silica with high loadings of simultaneously above 40 wt% MNPs and 20 wt% enzymes. Inorganic magnetic nanoparticle was used as peroxidase mimetics. This multi-catalyst system can be employed to achieve nano-scale colorimetric sensing. The results of the investigations demonstrate that the multi-catalyst system, incorporating GOx or ChOx, have high selectivities and sensitivities for the detection of the corresponding target molecules along with excellent reusabilities and highly enhanced stabilities. Our approach provided highly loaded MNP system and any highly loaded enzymes with superior activity, stability, and reusability, thereby making further applications as versatile sensors for detecting DNA, protein, and cell highly promising. We also demonstrated a novel strategy for developing efficient and robust electrochemical biosensing platform by incorporating magnetic nanoparticles as mimetic peroxidase and glucose oxidase in a conductive mesoporous carbon
11:30 AM JJ4.10
Modulation of Electron Transfer at the Enzyme-electrode Interface for Ultrasensitive Bio-detection. Siu-Tung Yau and Yongki Choi; Cleveland State University, Cleveland, Ohio.
We have demonstrated that, by applying an external gating voltage to the working electrode of an amperometric enzymatic detector, the electron transfer at the enzyme-electrode interface could be modulated, resulting in ultrasensitive bio-detection . The modulated electron transfer gave rise to voltage-controlled amplification of the detector’s amperometric signal. The signal amplification has allowed us to push the system’s detection limit from the milli-molar (10-3 M) range into the pico-molar (10-12 M) range with pico-molar detection resolution. We have demonstrated this detection technique with two important and well-known enzymatic systems: the glucose-glucose oxidase system and the ethanol-alcohol dehydrogenase system. The amplification could be reversibly controlled by the gating voltage while the enzyme’s bio-specificity was preserved. In particular, the selectivity of the enzyme was preserved in the presence of interfering substances, whose concentrations are 103 times higher than that of the analyte. The modulation of electron transfer is believed to be the result of modulating the enzyme-electrode interfacial electronic structure using the electric field induced at the interface by the gating voltage. To show the versatility of the detection technique, we have applied it to amperometric immuno-sensing systems. In this regard, we achieved the detection of protein cancer biomarkers with a detection limit of 1 unit/mL. Our results indicate the applicability of the technique to point-of-care diagnosis.  Y. Choi Y and S.-T. Yau, Analytical Chemistry 81 (2009) 7123-7126.
11:45 AM JJ4.11
Electrochemical Sensors for Neurodegenerative Disease Detection. Savindra Branigampala2, Gaurav Chatterjee1, Krishna Vattipalli2, Michael Sierks3 and Shalini Prasad2,1; 1School of Electrical, Computer and Energy Engineering, Arizona State University, Tempe, Arizona; 2Electrical Engineering and Computer Science, Wichita State University, Wichita, Kansas; 3School of Mechanical, Aerospace, Chemical and Materials Engineering, Arizona State University, Tempe, Arizona.
Neurodegenerative diseases (dementias) such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) will affect an increasing number of people as our population ages. For AD alone, over 5 million Americans currently are living with the disease, with nearly half a million new cases expected each year with total yearly economic costs of nearly $150 billion. In early stages of dementias, patients are classified as having Mild-Cognitive Impairment (MCI), a more general evolving term used to classify early, non-disabling cognitive disorders. Although MCI represents a transitional state between normal aging and dementia, not all MCI case progress to dementia. Pathological changes associated with these different dementias have been shown to occur long before symptoms are evident, suggesting that biomarkers have promise to facilitate early diagnoses of these different diseases. We have designed and developed a sensitive label-free biosensor technology operating using electrochemical impedance spectroscopy that is ideally suited to identify protein biomarkers in clinical samples.
SESSION JJ5: Bionanomaterials
Chair: Jean Louis Marty
Wednesday Afternoon, April 27, 2011
Salons 4-6 (Marriott)
1:30 PM *JJ5.1
Bio-inspired, Kinetically Controlled, Low-temperature Nanofabrication of Semiconductor and Ferroelectric Thin Films and Nanoparticles: Unique Advantages for High-performance Li-ion Batteries. Daniel E. Morse and Hong-Li Zhang; Institute for Collaborative Biotechnology, University of California, Santa Barbara, Santa Barbara, California.
Biological systems fabricate high-performance materials at low temperatures and near-neutral pH with a precision of nanostructural control that exceeds the capabilities of present human engineering. We discovered the mechanism governing nanofabrication of silica in a marine sponge, and translated this mechanism to a generic new, "biologically inspired" low-temperature method for the kinetically controlled catalytic synthesis of a wide range of nanostructured semiconductor thin films and nanoparticles without organic templates. Employing gentle catalysis at low temperature, this method preserves the intermetallic organization of bimetallic precursors that are thus incorporated into crystalline solids without phase segregation. Results include the first low-temperature synthesis of 6 nm barium titanate nanoparticles with low polydispersity, good electronic properties and no organic contaminants. A wide range of other materials made by this low-temperature process offers unique combinations of structures and properties not readily attainable by conventional high-temperature processes; these exhibit potential advantages now under investigation for improved solar energy, high power-density and fireproof batteries, ferroelectric random access memory, infrared and piezoelectric detectors and optoelectronic devices. Especially promising applications have been discovered for high-performance Li ion and Li batteries. Anodes made by this method consist of tin or silicon nanoparticles uniformly dispersed by synthesis in situ within the pores of highly compliant and conductive microparticulate graphite; these composites exhibit electrochemical capacity and power density significantly greater than present commercial materials and exceptionally stable cyclability. Cathodes made by this method also exhibit superior cyclability, with capacities greater than present commercial levels.
2:00 PM JJ5.2
Effect of Structure - Property Relationship on the Photoresponse Functionality of Hematite Based Hybrid Integrated System With Light Harvesting Protein. Debajeet K. Bora1,2, Artur Braun1, Elena Rozhkova3, Thomas Graule1 and Edwin C. Constable2; 1Laboratory for High Performance Ceramics, Empa.Swiss Federal Laboratory for Material Science and Technology, Dubendorf, Switzerland; 2Department of Chemistry, University of Basel, Basel, Switzerland; 3Center for Nanoscale Materials, Argonne National Laboratory, Chicago, Illinois.
The integration of light harvesting protein and other photosynthetic molecular machinery with semiconductor surface plays an important role in improving its performance as a solar cell active material and also to acheive the action of “ Photosynthesis Process” by artificial biomimetic route. The motivation of this study is to gain knowledge about the effect of structure - property relationship on the photoresponse of an integrated system consisting of hematite - and light harvesting protein such as phycocyanin. The integrated system was fabricated by surface functionalization of nanostuctured hematite surface which was synthesized by dip coating of iron fatty acid complex on FTO substrate. The structural and optical property of the system has been studied with XRD and UV-Vis spectroscopy. The morphology, surface structure and the composition of the integrated system was studied with FESEM, EDX and AFM. Raman spectroscopy has been applied for further validating the structure of the chromophore inside the protein molecule . On comparing the photocurrent obtained from the finally conjugated phycocyanin hematite system with respect to pristine hematite film, 2 fold increase in photocurrent value was obtained. This increase was attributed due to the higher absorption value of the pristine hematite film after integration with protein molecule as evident from the UV-Vis spectra .This signified the harvesting of more photon due to the presence of phycocyanin which results in enhanced photocurrent. Next, from the long term stability measurement of the phycocyanin adsorbed on FTO substrate, we have found a promising increase in photocurrent which deciphers its role in showing the enhanced photocurrent of hematite during the present study.
2:15 PM JJ5.3
Engineering Electrical Connections in Living Cells. Heather M. Jensen1,2, Jay T. Groves1 and Caroline M. Ajo-Franklin2; 1UC Berkeley, Berkeley, California; 2Lawrence Berkeley National Lab, Berkeley, California.
Hybrid living-non-living systems have the potential to combine the specialties of the technological world with those of the living world, such as sensing, synthesizing, and energy harvesting. The challenge in creating bioelectronic interfaces is competing with the naturally insulating cell membranes that inherently create an electronic barrier between intracellular processes and inorganic materials. Additionally, while the toolbox for engineering inorganic materials seems infinite, the toolbox for engineering cells as a material without disrupting cell viability is rather limited. To overcome these barriers, we used synthetic biology to ‘grow’ electrical connections in living cells by engineering the cell to construct a well-defined electron pathway. The dissimilatory metal-reducing microbe, Shewanella oneidensis MR-1, inspired our approach: it has the unusual ability to transport electrons to extracellular minerals via a trans-membrane electron transport pathway. We seek to generalize this ability to grow electrical contacts between living microbes and inorganic materials, and thus have genetically re-engineered a portion of the Shewanella electron transport pathway into the model microbe, Escherichia coli. Native E. coli proteins complete the partial electron pathway by supplying electrons to the functionally expressed Shewanella proteins. These 'electrified' strains exhibit ~8x and ~4x faster the metal reducing efficiency with aqueous Fe(III) citrate and insoluble Fe2O3, respectively, than wild-type E. coli. The ‘electrified’ strains are also capable of generating a current at an electrode surface. This engineered electron conduit is unique in that it requires no experimenter intervention because it is self-maintained and -repaired by the microbe. In addition, the genetic nature of this strategy makes the electron conduit transferable to other microbes with different applications (ex: photosynthetic bacteria). These experiments not only expand the toolbox for engineering cells as a material for living electrical devices but they also provide the first steps towards growing microbial-electrical connections. Reference: H.M.Jensen, et.al. (2010) PNAS
2:30 PM JJ5.4
Biocompatible Luminescent Nanoparticles on the Basis of Calcium Phosphate. Sofia Dembski1, Carsten Gellermann1, Torsten Klockenbring2, Marcel Dyrba3 and Stefan Schweizer3,4; 1Fraunhofer ISC, Wuerzburg, Germany; 2Fraunhofer IME, Aachen, Germany; 3Centre for Innovation Competence SiLi-nano®, Martin Luther University of Halle-Wittenberg, Halle (Saale), Germany; 4Fraunhofer CSP, Halle (Saale), Germany.
Calcium phosphate (CP) is the inorganic component of many biological hard tissues. Materials based on CP have been used extensively in medicine due to their chemical composition identical to natural tissue, their excellent biocompatibility and osteoconductivity. Moreover, current studies have shown the possibility to use nanoparticles (NPs) on the basis of CP as non-viral carriers in gene delivery or as luminescent labels. One advantage of CP NPs is their high capacity to accept various ionic substitutions such as lanthanide ions. Furthermore, they exhibit superior structural and optical properties and high photostability. Here, we present our recent activities in the research field of medical diagnostics concerning the development of novel biocompatible luminescent NPs. The focus of our work is on the wet-chemical synthesis and surface functionalization of luminescent NPs on the basis of calcium phosphate with mean particle sizes < 50 nm. For example, luminescent SiO2/CP core/shell structured NPs with mean diameters in the range of 40 - 50 nm can be synthesized via a modified Pechini process and doped with various rare-earth ions. The resulting NPs exhibit an amorphous SiO2 core and a crystalline luminescent hydroxyapatite shell. The structure, size, and composition of NPs can easily be controlled to tailor their chemical and physical properties. Like quantum dots, luminescent CP NPs with mean diameters of 5 - 10 nm can also be obtained by high temperature thermolysis of metal precursors in the presence of stabilizing agents. These organic molecules ensure the slow particle growth and stabilize CP NPs in non-polar solvents. The characterization of CP-based NPs is done by conventional methods such as dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), inductively coupled plasma optical emission spectrometry (ICP-OES), and photoluminescence (PL) spectroscopy. We have further demonstrated a subsequent surface modification of the resulting NPs with amine and carboxyl functionalities for a later attachment of biomolecules to enable their use as luminescent markers in biological or medical diagnostics. The presence of functional groups was determined by ?-potential measurements as a function of pH and Fourier transform infrared (FT-IR) spectroscopy. Additionally, the newly developed NPs were proven to be non-toxic, e.g. as per ISO 10993 standards.
3:15 PM *JJ5.5
A Modular Phase Transfer and Biomimetic Assembly Protocol for Quantum Dots. Mathew M. Maye, Department of Chemistry, Syracuse University, Syracuse, New York.
The use of bio-inspired approaches for nanoparticle self-assembly, bioimaging, and drug-delivery takes advantage of the natural tunability and addressability of encoded biomolecular interactions. Integrating such bio-encoding with semiconductive quantum dot (qdot) photoluminescence has led to a number of powerful sensing technologies and imaging probes. However, a number of challenges need to be overcome for the full potential to be achieved, which include: ease and stability of biofunctionalization, tailorable biomaterial coverage, and minimum hydrodynamic radius increase. To help address this, we have developed a new phase transfer protocol using the simple amino acids and small molecules that allow not only for rapid phase transfer, but also the preservation of quantum yields. In addition, the modified qdots readily undergo further ligand exchange or biofunctionalization in aqueous buffers, greatly improving utility. The resulting qdots can then be assembled into large 3D assemblies, as well as molecule like clusters, with defined interparticle distances and energy transfer.
3:45 PM JJ5.6
Identification of Protein-nanoparticle Interaction Site. Luigi Calzolai, Fabio Franchini, Douglas Gilliland and Francois Rossi; JRC IHCP, Ispra, Italy.
The interaction of nanoparticles with proteins is a key parameter in nanomedicine and nanotoxicology. When nanoparticles (NP) interact with proteins, they might alter protein conformation, expose new epitopes on the protein surface or perturb the normal protein function, which could induce unexpected biological reactions and lead to toxicity. Here we show that is possible to characterize the interaction of gold nanoparticles to proteins at atomic level resolution. For the first time, by using state of the art Nuclear Magnetic Resonance techniques, it has been possible to identify the amino acids of the ubiquitin protein (shown in red in the figure) that bind to gold nanoparticles . Using NMR, chemical shift perturbation analysis, and dynamic light scattering we have identified a specific domain of human ubiquitin that interacts with gold nanoparticles.. The ubiquitin proteins interact with the gold surface via a limited number of amino acids that form a well-defined Au-binding area on the protein surface. These results open up the possibility of characterizing at atomic level resolution a large variety of nanoparticles-protein complexes in physiological conditions with a great potential impact in the nanotoxicology field and in nanomedicine.
4:00 PM JJ5.7
Selective Bio-functionalization of Nanopatterned Surfaces: DNA Nanoarrays for Protein-DNA Interaction Studies and Nanostructures Assembly. Matteo Palma1,2, Erika Penzo1, Justin Abramson2, Risheng Wang1, Alon Gorodetsky3, Mike P. Sheetz4, Colin Nuckolls3, James Hone2 and Shalom J. Wind1; 1Applied Physics and Applied Mathematics, Columbia University, New York, New York; 2Mechanical Engineering, Columbia Univeristy, New York, New York; 3Chemistry, Columbia Univeristy, New York, New York; 4Biological Sciences, Columbia Univeristy, New York, New York.
The ability to control biomolecules on surfaces with nanometer resolution is of great interest in the field on nanoscience and nanotechnology. Nanopatterned arrays of biomolecules can offer unmatched sensitivity, and smaller test sample volumes, in molecular diagnostics. DNA nanoarrays, in particular, are of interest in the study of DNA-protein interactions, for biodiagnostic investigations and as a tool to drive self-organization of nanomoieties on surfaces. In this context, achieving a highly specific nanoscale assembly of oligonulceotides at surfaces is critical. Here we describe different strategies to control the immobilization of single- and double-stranded DNA, as well as DNA nanostructures (DNA “origami”), on nanopatterned surfaces, with features down to the sub-10nm regime. Using electron-beam and nanoimprint lithography we fabricated sub-10nm metal dots arranged in multiple configurations on Si and glass substrates. We have developed bio-chemical strategies for the selective bio-functionalization of these patterns, at the single nanodot level: each step of the biochemical functionalization has been monitored by Fluorescence Microscopy. The bio-functionalization approach used allowed for the formation of non-sterically hindered DNA nanodomains where the dsDNA attached to the nanodots is accessible for interaction with DNA-binding proteins and maintains its native conformation, as confirmed by restriction enzymes studies. This allowed us, moreover, to follow the activity (at surfaces) of a restriction enzyme in real time and with single-molecule resolution: the monitoring of protein-DNA interactions with such biological nanoarrays will be discussed. We will highlight the broader utility and application of such nanopatterned surfaces for the self-organization of DNA nanostructures. In-situ hybridization between the complementary strands on DNA nanostructures and on functionalized nanodots has been achieved, resulting in the ordered placement of the origami on the dot patterns, as demonstrated by Atomic Force Microscopy (AFM) imaging, both in liquid and in air.
4:15 PM JJ5.8
Immobilization of Oligonucleotide-functionalized Magnetic Nanobeads in DNA Coils Studied by Electron Microscopy and Atomic Force Microscopy. Mattias Stroemberg1, Sultan Akhtar1, Klas Gunnarsson1, Teresa Zardan Gomez de la Torre1, Camilla Russell2, David Herthnek2, Jenny Goeransson1,2,3, Peter Svedlindh1, Mats Nilsson2, Klaus Leifer1 and Maria Stroemme1; 1Engineering Sciences, Uppsala University, Uppsala, Sweden; 2Genetics and Pathology, Uppsala University, Uppsala, Sweden; 3Q-linea AB, Uppsala, Sweden.
Immobilization of oligonucleotide-functionalized magnetic nanobeads by hybridization in rolling circle amplified DNA coils has been investigated using electron microscopy and atomic force microscopy (AFM). This particular immobilization process of magnetic beads is the basis of a newly invented substrate-free and lab-on-a-bead magnetic biosensor principle denoted the volume-amplified magnetic nanobead detection assay (VAM-NDA) [Strömberg et al., Nano Lett. 8 (2008) 816]. The VAM-NDA has up to now been demonstrated for the detection of synthetic single-stranded DNA target molecules [Strömberg et al., Nano Lett. 8 (2008) 816] but also for detection of real bacterial genomes [Göransson et al., Anal. Chem. (2010) DOI: 10.1021/ac102133e]. In the former case, the target is recognized by hybridization to a DNA padlock probe followed by ligation giving a circular DNA molecule. Addition of DNA polymerase enzymatically amplifies each DNA circle to a macromolecular DNA coil (typically one micrometer in diameter). The DNA coils are then detected by adding oligonucleotide-functionalized magnetic nanobeads (typically 40 to 250 nm in nominal diameter) where the oligonucleotide is complementary to the DNA coil sequence. Therefore, the beads will hybridize to the DNA coils and experience a dramatic increase in hydrodynamic size which can be measured by recording the dynamic magnetization of the sample. In absence of target molecules, no DNA circles are formed and hence no DNA coils. The main aim of these microscopy studies is to obtain increased understanding of how the magnetic beads interact with the DNA coils. Dry samples of non-stained DNA coils (not labeled with heavy metal stains or other contrast increasing substances) with beads of two different sizes (40 nm and 130 nm) were studied by transmission electron microscopy (TEM) [Akhtar et al., J. Phys. Chem. B 114 (2010) 13255]. The bead-coil complexes were appearing as salt residues of the DNA coils with beads. A quantitative analysis of the number of beads per salt-DNA residue and a qualitative analysis of the location of beads within the salt residues were performed. The average number of beads per salt-DNA residue obtained from TEM micrographs were found to be in very good agreement with magnetic measurements on similar samples, thereby confirming the bead counting reliability of the TEM method. The observed location of beads inside the salt-DNA residues was consistent with earlier findings [Zardán Gómez de la Torre et al., J. Phys. Chem. B 114 (2010) 3707] that small beads tend to more easily diffuse into and hybridize inside DNA coils than larger beads. As a complement to the electron microscopy, AFM was conducted on dry samples of DNA coils with and without magnetic beads. This technique provided direct visualization of the DNA coils as thread-like objects. DNA coils with immobilized beads typically appeared as a collection of beads with thread-like features going out from the boundary.
4:30 PM JJ5.9
Fluorescent Silver and Gold Nanoclusters: Design, Structure and Function. Jennifer S. Martinez, Hsin-Chih Yeh, Jaswinder Sharma and James H. Werner; Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico.
Fluorescent metal nanoclusters (<1 nm) are collections of several to tens of atoms of gold or silver. While for gold nanoclusters, the fluorescence emission is thought to scale as a function of the number of atoms within the cluster, for silver nanoclusters, the relation of atom number to fluorescence emission is less clear, as is the nature of the cluster. Nevertheless, fluorescent metal nanoclusters are gaining much interest because of their desirable photophysical properties, smaller size than quantum dots, and inherent biocompatibility. As a compliment to quantum dots and molecular fluorophores, fluorescent metal nanoclusters have been produced using templates of dendrimers and polymers, small molecular ligands, or within biological materials of interest, such as DNA. Recently, we have synthesized and photophysically characterized Ag-nanoclusters (AgNCs), which were templated on DNA under one reaction condition (near physiological pH), with distinct, and narrow, excitation and emission profiles tuned to common laser lines. We have taken those clusters and developed an intrinsically fluorescent recogonition ligand based on a DNA aptamer-templated AgNC for specific and sensitive protein detection. Our intrinsically fluorescent recognition ligand combines the strong fluorescence of oligonucleotide templated AgNCs with the specificity and strong binding affinity of DNA aptamers for their target proteins, to develop a new strategy for detection of specific proteins. Here the AgNC-aptamer assembly serves as both a fluorescent label and a specific binding ligand. Our in situ method is a single-step that requires no covalent attachment of aptamer, nor protein, to fluorophore, and is thus simple, inexpensive, and a facile detection method. Additionally, we find that the red fluorescence of oligonucleotide-templated silver nanoclusters can be enhanced 500 fold using a proximity technique. Based upon this newly observed phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that “lights up” upon target binding. In a separation-free assay, a signal-to-background ratio of 175 was demonstrated for the detection of H1N1 In addition to eliminating the need to purify DNA nanocluster probes that do not bind targets, there is no need to remove the silver nanocluster precursors used during nanocluster formation. The synthesis, design, and physical characterization will be discussed.
4:45 PM JJ5.10
A Bio-inspired Approach to Tip-based Nanofabrication. Sungwook Chung1,2, Jonathan Felts4, Delan H. Tai1, Iva Urbanova1,3, Frantisek Svec1,3, William P. King4 and James J. De Yoreo1,3; 1Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California; 2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; 3Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; 4Department of Mechanical Science and Engineering, University of Illinois Urbana-Champaign, Urbana, Illinois.
Developing generic platforms to organize discrete molecular elements and nanostructures into deterministic patterns at surfaces is one of the central challenges in the field of nanotechnology. To address this challenge, we are developing a tip-based approach to fabrication of nanowires and quantum dots that relies on patterning of peptides selected for their ability to induce material-specific formation of inorganic solids under mild reaction conditions. These peptides are linked to the substrate either through a base self-assembled monolayer (SAM) or a thiol functionalized polyethylene (PE) polymer patterned via thermal dip-pen nanolithography (tDPN) and/or nanografting. For the case of SAM, the region surrounding the active patterns are passivated with a hydrophobic SAM such as octadecane thiol or an hydroxy-terminated polyethylene glycol thiol. When using the polymers, which provide a means for patterning on insulating substrates, linkages to the peptides are established through UV activated grafting of glycidyl methacrylate followed by the reaction with cysteamine to produce the exposed sulfhydryl groups. The dimensions of the pattern are further controlled by generating a steep temperature gradient away from the tip through the use of heated AFM probes. When peptides selected by phage display for their ability to nucleate Au nanoparticles from an auric acid solution are linked to either the base SAM or thiol functionalized PE polymer, films consisting of Au nanoparticles grow on the patterns. We find that the areal density and vertical thickness of the films, as well as the average gold particle size depend strongly on the choice of solution conditions. Introduction of silver ion in aqueous solution enhances the nucleation density, producing nearly continuous gold films when tested on patterns formed via microstamping. Results of in situ AFM studies on the formation of the Au nanoparticles as well as the effects of solution composition, peptide sequence and peptide polarity on film morphology will be reviewed.
SESSION JJ6: Poster Session: Hybrid Biological Materials II
Wednesday Evening, April 27, 2011
Salons 7-9 (Marriott)
The Use of Diamond as a Flexible Surface for the Attachment of Different Bioreceptors in the Development of Impedimetric Biosensors. Natalie Vanden Bon1, Veronique Vermeeren1, Bart Van Grinsven2, Lars Grieten2, Stoffel D. Janssens2, Dinh T. Tran3, Jeroen Lammertyn3, Marcel Ameloot1, Ken Haenen2,4, Ward De Ceuninck2,4, Patrick Wagner2,4 and Luc Michiels1; 1Biomedical Research Institute, Hasselt University, Diepenbeek, Belgium; 2Institute for Materials Research, Hasselt University, Diepenbeek, Belgium; 3Catholic University Leuven, Leuven, Belgium; 4Division IMOMEC, IMEC vzw, Diepenbeek, Belgium.
In healthcare the demand for point-of-care diagnostics is growing. Point-of-care diagnostics entails the detection of certain disease markers on site, e.g. at the GP’s office or in the patient’s home. Essential features are fast result analysis, cheap production cost, reliability and sensitivity. To supply for this demand research on biosensor development is increasing rapidly. Biosensors that use electronic read-out techniques, such as Electrochemical Impedance Spectroscopy (EIS), are favored for point-of-care applications, because they are cheap to produce and they can generate a fast and real-time response and don’t require the targets of interest to be labeled. These electronic biosensors are based on biological receptor molecules which are bound to a semiconductive surface, the transducer. To obtain a sensitive and specific biosensor, the layer of biological receptor molecules has to be dense, stable and functionally active. Several popular transducer materials, such as Si and Ge, have been used in biosensor development, although these materials are receptive to hydrolysis. This causes gradual loss of bioreceptors from the surface and instability of the sensor platform, leading to a decrease in sensivity and specificity. Therefore, our research focuses on diamond, which exceeds Si and Ge on many levels. Diamond is chemically inert and it can be made semiconductive by doping. In addition, diamond is biocompatible, because it only consists of carbon (C) atoms. For this reason it’s also able to form strong C-C bonds with biomolecules, leading to a stable functional layer on the diamond surface. We developed a real-time, and hence fast, label-free biosensor platform using chemically inert nanocrystalline diamond (NCD) as a transducer material and EIS as an electronic signal read-out technique. By now we successfully functionalized this generic sensing platform with several biological receptor molecules, such as aptamers, ssDNA, and antibodies, to develop impedimetric NCD-based aptamer-, DNA- and immunosensors, respectively, allowing real-time DNA mutation and protein detection. Within 30 minutes, the prototypes of the apta- and immunosensors can detect IgE and CRP, respectively, in biological serum samples in real-time with clinically relevant sensitivity. The real-time DNA-sensor can distinguish between DNA with and without a single base mismatch during the denaturation phase, and hence displays single nucleotide polymorphism (SNP) sensitivity, in only 5 minutes. This principle is based on the difference in stability of a perfect DNA duplex and a DNA duplex with a SNP. We are exploring the use of this principle in EIS for mutation identification. As a proof of application patient mutations in the gene for Phenylalanine Hydrolylase (PAH) leading to Phenylketonuria (PKU) are analyzed.
Rational Design of Responsive Self-assembling Peptide Hydrogels Derived from Native Sequences. Hongzhou Huang1, Jishu Shi2, Ziyan Liu2, David S. McVey3 and Xiuzhi S. Sun1; 1Grain Science and Industry, Kansas State University, Manhattan, Kansas; 2College of Veterinary Medicine, Kansas State University, Manhattan, Kansas; 3Veterinary Diagnostic Center, Veterinary and Biomedical Sciences University of Nebraska, Lincoln, Nebraska.
Using peptide hydrogels as injectable materials for drug delivery systems and tissue engineering applications has been an important discovery made over the past few decades. Most published hydrogel-forming sequences have been achieved by designing model peptides with alternating-charged and non-charged amino acids or blocks of hydrophobic and hydrophilic copolymers. Because the physical properties of hydrogel are greatly dependent on the highly complex self-assembly pathway, modifying the proper residue of the peptide primary structure is a common way to change hydrogel characteristics. Here, we report a novel-designed peptide FLIVIGSIIGPGGDGPGGD (h9e), which was designed by rationally combining two native sequences from an elastic segment of spider silk and a trans-membrane segment of human muscle L-type calcium channel. The h9e peptide formed two distinct hydrogels at water content greater than 99.5%, without primary structure modification, induced by adding Ca2+ and adjusting pH respectively. The h9e Ca2+ hydrogel has shear-thinning, rapid-strength-recovering properties, which indicated a high potential for injectable materials for medical applications.
Biocompatibility of Novel Nano-engineering Cellulose-polypyrrole Composite. Natalia Ferraz1, Leif Nyholm2, Maria Stromme1 and Albert Mihranyan1; 1Nanotechnology and Functional Materials, Uppsala University, Uppsala, Sweden; 2Materials Chemistry, Uppsala University, Uppsala, Sweden.
In the present work we studied the biocompatibility of a novel cellulose-polypyrrole composite. Polypyrrole (PPy) and its derivatives are probably one of the most promising of currently known conductive polymers useful in a broad range of applications. Due to properties such as biocompatibility and the ability to entrap and controllably release biomolecules, the use of PPy in biological applications has increased exponentially. PPy has been described as a promising candidate for electroactive matrix material to stimulate electrochemically responsive cells such as bone, cardiac and nerve cells . We have developed a novel composite material consisting of nanofibrous cellulose coated with a homogeneous 30-50 nm thick layer of PPy . The composite is prepared by chemical polymerization of pyrrole on cellulose from Cladophora sp.algae using iron chloride as the oxidant agent. The material in question presents large surface area (60 m2/g) and extensive porosity, combined with excellent potential-controlled ion-exchange properties. These distinct properties could be exploited in the field of biomedical applications where electro stimulus can enhance tissue regeneration, promote cell differentiation or controlled release drugs. The nanofibrous structure of the Cladophora sp. algae cellulose and its extensive porosity could be further exploited to enhance the functional properties of the composite. Tissue and cell reactions to PPy-based materials are likely to be modulated by a variety of factors, including synthesis conditions, dopant, and washing steps. Moreover, results may differ depending on the cell type considered. Therefore, to evaluate the feasibility of using the novel PPy composite as a biomaterial the new synthesized composites need to be tested for their biocompatibility. In this study biocompatibility was measured in terms of cellular response in an indirect culture assay using fibroblasts and monocyte cell lines. The effect of extractables on the cell lines was studied by evaluating cell proliferation, viability and morphology. Results indicate that the cellulose-PPy composite presents no toxicity, with cell viability and proliferation comparable to the results obtained with extracts from the tissue culture substrate thermanox. However, we found that after chemical synthesis extensive washing is required and 48 h extraction is needed to ensure the release of potential toxic substances such as oligomers from the synthesis step. The cytotoxicity due to residual pyrrole and its oligomers can therefore be overcome by appropriate washing and extraction procedures. These promising results push forward the investigation of the biocompatibility of the novel cellulose-PPy composite. Next steps will include blood compatibility studies and acute toxicity tests.  Guimard et al. Prog Polym Sci, 2007;32:876-921  Mihranyan et al. J Phys Chem B. 2008; 112: 12249-12255
Creation of Biocompatible Hemodialysis Membrane by Surface Modification. Yoshiyuki Ueno, Masaki Fujita, Hiroyuki Sugaya and Ichiro Itagaki; Toray Industries, Inc., Siga, Japan.
The polysulfone (PSf) membrane is now becoming the mainstream in the hemodialysis treatment because PSf membrane has a high membrane performance. Besides, most of PSf membranes hydrophilized by blending polyvinylpyrrolidone (PVP) are well known to have biocompatibility in clinical use. However, in recent years, the improvement in biocompatibility of the hemodialysis membrane is needed. Especially, when blood components adhere to the membrane, it is pointed out to generate the oxidative stress. Then, it aimed at creation of the hemodialysis membrane that did not adhere blood components based on the PSf membrane. We found that the amount of the platelet adhesion was decreased by increasing the amount of the surface PVP on the model surface of the PSf membrane. However, some platelets were admitted to adhere. Then, we used a new hydrophilic polymer instead of PVP to the PSf membrane by taking note of the adsorbed water on it. Now, we found that the platelet adhesion was decreased by rising mobility of the absorbed water by using several hydrophilic polymers. In addition, mobility of the absorbed water correlates with the relaxation time of that. Therefore, We examined the relaxation time of absorbed water using the dielectric measurements. The mobility of the absorbed water on the new hydrophilic polymer we used was faster than that of PVP. As the result, platelets hardly adhered at the surface covered the new hydrophilic polymer in a monolayer. Furthermore, we invented the new surface modification technology by the new polymer was arranged on the hemodialysis membrane in monolayer order. Applying this technology, we created the biocompatible hemodialysis membrane which had a high membrane performance, retained low elution and dramatically prevented the fibrinogen and the platelet from adsorbing to the surface.
Multifunctional Superparamagnetic MnO@SiO2 Core/Shell Nanoparticles and Their Application for Optical and Magnetic Resonance Imaging. Thomas D. Schladt1,2, Kerstin Schneider1, Steve Pruefer3, Heiko Bauer1, Filipe Natalio1, Renugan Raidoo1, Weber Stefan4, Laura M. Schreiber4, Hansjoerg Schild3 and Wolfgang Tremel1; 1Institute for Inorganic and Analytical Chemistry, Johannes Gutenberg University Mainz, Mainz, RLP, Germany; 2SpinAps, IBM Almaden Research Center, San Jose, California; 3Institute for Immunology, Johannes gutenberg University, Mainz, RLP, Germany; 4Institute for Medical Physics, Johannes gutenberg University, Mainz, RLP, Germany.
Highly biocompatible multifunctional nanocomposites consisting of monodisperse manganese oxide nanoparticles with luminescent silica shells were synthesized by a novel approach, involving a combination of w/o-microemulsion techniques and common sol-gel procedures. The nanoparticles were characterized by TEM analysis, powder XRD, SQUID magnetometry, FT-IR-, UV/Vis- and fluorescence spectroscopy and dynamic light scattering. Due to the presence of hydrophilic poly(ethylene glycol) (PEG) chains on the SiO2 surface, the nanocomposites are highly soluble and stable in various aqueous solutions, including physiological saline, buffer solutions and human blood serum. As the quantification of the particle bound target molecules is crucial for their drug loading capacity the number of surface amino groups available for ligand binding on each particle were determined using a colorometric assay with fluorescein isothiocyanate (FITC). MnO@SiO2 nanoparticles were less prone to Mn-leaching compared to nanoparticles coated with a conventional bi-functional dopamine-PEG ligand. Interestingly, the presence of a silica shell did not change the magnetic properties significantly, and therefore, the MnO@SiO2 nanocomposite particles showed a T1 contrast with relaxivity values comparable to those of PEGylated MnO nanoparticles.
A Polarimetric Glucose Sensor Based on a Double Pin a-SiC:H Photodetector for Selective Wavelength Measurements. Joao Costa1,2, Miguel Fernandes1,2, Paula Louro1,2, Yuri Vygranenko2 and Manuela Vieira1,2,3; 1Electronics Telecommunications and Computer Engineering, ISEL, Lisbon, Portugal; 2CTS, UNINOVA, Lisbon, Portugal; 3DEE, FCT-UNL, Lisbon, Portugal.
Glucose concentrations can be estimated indirectly by measuring the rotation of the polarization state of light after propagating through a sample containing glucose. In fact, optical polarimetric sensors which work by probing aqueous humor glucose concentrations are a potential alternative for noninvasive diabetic glucose monitoring. The novelty of the polarimeter proposed in this study is in the fact that it is designed for multiple wavelength measurements using a tailor made amorphous silicon photodetector. The advantage of making measurements at multiple wavelengths is in the reduction of noise, in particular the artifacts caused by time-variant birefringence due to sample motion. The photodetector was produced by Plasma Enhanced Chemical Vapour Deposition on a glass substrate and consists of a double pi’n/pin a-SiC:H heterostructure (p(a-SiC:H)- í'(a-SiC:H)-n(a-SiC:H)-p(a-SiC:H)-i(a-Si:H)-n(a-Si:H). Due to the stacked architecture wavelength selectivity can be tunned in the visible range by adjusting the bias voltage. Closed-loop control is used to bring the noise down and enable measurements at physiological levels. In this paper we present an experimental and theoretical characterization of the photodetector, in terms of the wavelength selectivity and its dependence on the bias voltage, and demonstrate its applicability in the real-time closed loop polarimetric sensor proposed.
NAD+/NADH-Sensitive Quantum Dots: Applications to Probe NAD+- Dependent Enzymes and to Sense the RDX Explosive. Ronit Freeman and Itamar Willner; Institute of Chemistry, The Center for Nanoscience and Nanotechnology,, The Hebrew University of Jerusalem, Jerusalem, Israel.
Quantum Dots (QDs) exhibit unique size-controlled optical properties. While QDs were extensively applied as fluorescence labels, the use of QDs to follow dynamic processes, such as recognition or biocatalysed processes, by fluorescence resonance energy transfer (FRET), or electron transfer (ET), attracts only recent research efforts. We have developed different approaches to implement QDs for the FRET-induced or ET-activated analysis of biomolecular processes or for the construction of new paradigms of QDs-based chemosensors. The biomolecular-based CdSe/ZnS QDs sensors will be exemplified with the development of 1,4-dihydronicotinamide (NADH)-sensitive QDs and their use to follow NAD+-dependent enzymes, and to probe intracellular metabolic pathways. The ET different features of quenching of the QDs by NAD+ or NADH were used to develop an ultrasensitive biosensor for the RDX explosive.
Folate Functionalized Hollow Silica Nanoshells and Their Selective Targeting of HeLa Cervical Cancer Cells. Alexander Liberman1, Sergio Sandoval2, Jian Yang3, Jesus G. Alfaro4, Sharraya Aschemyer3, David Martin5, Milan Makale6, Kristana K. Mitchell3, Andrew C. Kummel3 and William C. Trogler3; 1Materials Science and Engineering, UCSD, La Jolla, California; 2Bioengineering, UCSD, La Jolla, California; 3Chemistry and Biochemistry, UCSD, La Jolla, California; 4Nanoengineering, UCSD, La Jolla, California; 5Electrical and Computer Engineering, UCSD, La Jolla, California; 6Moores Cancer Center, UCSD, La Jolla, California.
An important area of biomedical nanotechnology is based on the interaction of living systems with inorganic and organic materials at the nanoscale. Silica nanoshells (NS) are attractive biomaterials because of their advantages as readily functionalized transport and imaging devices: the porous amorphous structure of silica colloid allows small molecule storage; the surface of silica can be modified easily with trimethoxysilyl reagents; silica has low biotoxicity and good biocompatibility. Silica nanoshells potentially have multiple biomedical applications as imaging agents, targeted drug delivery agents or gene transferring motherships. A simple method to fabricate hollow silica NS with 100 nm or 200 nm diameters has been developed and tested. Amino polystyrene beads were used as templates and a 5-10 nm thick silica gel coating was formed by the sol-gel reaction. After removing template by calcinations, porous dehydrated silica gel nanoshells of uniform size were obtained. The porous structure of silica shell wall was characterized by transmission electron microscopy measurements, while particle size and zetapotentials of the particles suspended in aqueous solution were characterized by dynamic light scattering. The surfaces of the NS have been functionalized with folic acid in order to specifically target cancer cells. Folic acid, also known as vitamin B9 or Folate, is essential for the synthesis of nucleotide bases and binds with high affinity to Folate receptors, which are frequently over-expressed in tumor cells such as ovarian carcinomas. With the use of confocal and two-photon microscopy, it was found that as the amount of folate on the surface of the NS was increased, a higher amount of NS endocytose into HeLa cancer cells, a cervical cancer cell line. Cytotoxicity studies will quantify the effectiveness of using folate coated silica shells for enhancing endocytosis of chemotherapy drugs in cell lines and in animal studies.
Single Particle Tracking Reveals Corralling of a Transmembrane Protein in a Double Cushioned Lipid Bilayer Bio-mimetic Assembly. Kumud R. Poudel1, David J. Keller2 and James A. Brozik1; 1Materials science and engineering, Washington State University, Pullman, Washington; 2Department of Chemistry and Biological Chemistry, University of New Mexico, Albuquerque, New Mexico.
A predominate question associated with supported bio-mimetic bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT3A, has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT3A receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bio-mimetic bilayer assemblies include: a simple POPC supported planar bilayer, a “single-cushion” POPC bilayer with a PEG layer between membrane and support, and a “double-cushion” POPC bilayer with both a PEG layer and a layer of BSA. Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble Fluorescence Recovery After Photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT3A molecules shows that individual proteins in the double-cushion system have quite high local mobilities, but are spatially confined within small corralling domains ( nm). Comparisons with the simple POPC membrane and the single-cushion POPC-PEG membrane reveal that BSA serves to both minimize interactions with the solid support and creates the corrals that reduce the long range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion bio-mimetic assemblies, proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.
Particle Transport through Silica Diatom Nanopores Immobilized on Silicon Substrates. Michael Goryll1, Kai-Chun Lin2, Xiaofeng Wang1, Shankar Ramakrishnan1, Kaushal Rege2, Sandwip Dey2 and B. L. Ramakrishna2; 1Electrical, Computer and Energy Engineering, Arizona State University, Tempe, Arizona; 2School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, Arizona.
Marine diatoms feature silica shells with interesting structural properties. These biomineralized structures have hierarchical pore architecture with the smallest pore diameters being 40 nm. The hierarchical support structure makes these nanomembranes exceptionally mechanically stable, while providing direct access to the nanopores. Moreover, the nanopores are homogeneous in size and have a low aspect ratio, enabling fast diffusion-driven transport. With the shells growing up to 200 µm in diameter, they provide a large area of nanopores. These features make the biomineralized silica shells an interesting low-cost alternative to microfabricated nanoporous membranes. We will report how these biomineralized nanoporous membranes can be immobilized on silicon substrates, enabling studies of particle transport through the pores. Through-wafer via holes with diameters between 5 µm and 30 µm were etched using a dry etching technique to allow free access to the bottom of the immobilized diatom shell. Two pathways towards immobilization of the diatom shells were used, one being a chemical linkage approach using poly-L-lysine and the other involving UV-polymerizable epoxy. Controlled etching of the silica structure has been employed to manipulate both the dimensions of the nanopores as well as the pore hierarchy. Studies of transport phenomena were carried out after mounting the silicon chip in a fluidic chamber with reservoirs on either side, holding up to 3 ml of aqueous solution. Electrical contact was made via Ag/AgCl electrodes, connected to a transimpedance amplifier. Results on our transport studies based on size and chemical considerations will be presented for ions, such as potassium and sodium, present in physiologically relevant isotonic buffers, polystyrene nanobeads and gold nanoparticles. Upon application of a transmembrane bias, current fluctuations were observed in the presence of nanoparticles, corresponding to translocation events. Analysis of the amplitude of the current reduction correlates with the size of the particles, making this setup an interesting candidate for a nanoparticle analysis system. We will further use this nanopore-based sensor with electrochemical signal transduction for the detection of biologically molecular targets, such as DNA and proteins.
DNA-Directed Self-Assembly of Asymmetric Nanoclusters Hang Xing1, Zidong Wang2 and Yi Lu1,2; 1Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois; 2Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois.
Asymmetric nanoassembly is of particular interest because of the unique anisotropic properties which are difficult to attain with their symmetric counterparts. However, controlled fabrication of asymmetric structure in nanoscale remains a challenge. Herein, we report the DNA-directed approach for the assembly of asymmetric nanoclusters by using Janus nanoparticles as starting building blocks. The results demonstrate the essential role of DNA in controlling the self-assembly process and the novel optical properties of obtained nanoclusters. Furthermore, the approach of incorporating DNA strands with Janus nanoparticles is modular and programmable. A series of asymmetric nanostructures have been rationally designed and assembled through programmable base-paring interactions to systematically study the size-dependent assembly process.
Biosensors for Liver Function Detection Utilizing Low-cost Optical System. Meng-Che Tu and Tri-Rung Yew; Materials Science and Engineering, National Tsing Hua University, Hsinchu, Taiwan.
This work shows a sensitive biosensor with a simple structure and a low-cost optical system for targeting human serum albumin (HSA), an important biomarker of liver function. The optical biosensing system including light source and photodetector was used for rapid and quantitative detection of HSA. The photo-electrical signal at various HSA concentrations was measured by photodetector directly and quantified. To improve biocompatibility, amino groups were utilized for the modification of sensing region on the glass substrate. The surface morphology of sensing area was observed before and after antibody-modification by scanning electron microscopy (SEM), atomic force microscopy (AFM), and fluorescence microscopy. The amount of bound antibodies was also characterized by enzyme-linked immunosorbent assay (ELISA). The functional groups of surface treatment were also characterized by Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS).
Study of Disinfection Mechanism of Nanoparticles. Sajid Bashir1,4, Karthik Chamakura2, Rafael Perez-Ballestero2, Zhiping Luo3 and Jingbo L. Liu4,5,1; 1Chemical Biology Research Group, Texas A&M University-Kingsville, Kingsville, Texas; 2Department of Biological and Health Sciences, Texas A&M University-Kingsville, Kingsville, Texas; 3Microscopy and Imaging Center, Texas A&M University, College Station, Texas; 4Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas; 5Department of Chemistry, Texas A&M University, College Station, Texas.
Comparison of silver nanoparticles to chemical disinfectants confirms that silver nanoparticles exhibit both bactericidal and biocidal activities with much greater persistence than chemical disinfectants, which lose their potency after 4 hours. In comparison with nanoparticle, this effect was not observed. The nanocomposite of AgTiO2 exhibited similar bactericidal activity as silver with Escherichia coli, however with Staphylococcus aureus; the composite was slightly superior in measured minimal bacterial concentration (MBC). This discrepancy might be attributed to variable clustering (composite cluster might be larger than 12 nm) of the nanocomposite, whereas the AgNP were more monodispersed, therefore yielding a more fixed surface area to volume ratio to the microbe. Both nanoparticles exhibited lower MBCs for with both microbes when compared with chemical disinfectants (bleach), although the lowest MBC was observed with Escherichia coli. The mode of action as also investigated, whereas silver nanoparticles exhibited efficacy under either light or dark conditions, the composite was ineffective in the dark, suggesting the light activation was necessary. Previous tests with TiO2 under either dark or light conditions were ineffective support literature observations of disinfection of water with UV activated TiO2. Collectively, our studies indicate that reduction/oxidation of the composite to generate hydrogen peroxide require visible light energy in the first instances in a similar manner to production of H2O with TiO2 where UV activation is required.
Bactericidal Activity of Ag-TiO2NP in Two Different Environments and with Two Different Cell or Types. Sajid Bashir4,1, Karthik Chamakura2, Rafael Perez-Ballestero2, Zhiping Luo3 and Jingbo L. Liu1,5,4; 1Department of Chemistry, Texas A&M University-Kingsville, Kingsville, Texas; 2Department of Biological and Health Sciences, Texas A&M University-Kingsville, Kingsville, Texas; 3Microscopy and Imaging Center, Texas A&M University, College Station, Texas; 4Chemical Biology Research Group, Texas A&M University-Kingsville, Kingsville, Texas; 5Department of Chemistry, Texas A&M University, College Station, Texas.
The Ag-TiO2 nanocomposite bactericidal activity on Escherichia coli was previously determined using visible light to promote photo catalytic activity, however the effect of environmental conditions on the interaction between nanocomposite and bacteria was unknown, which is the subject of the current study. The minimum bacteria concentration (MBC) of the composite under iso-osmotic and hypo-osmotic conditions was investigated and found to be the same, suggesting that osmotic conditions have an negligible effect on the measured MBC on Escherichia coli. The relationship between addition of bulk metal (e.g. wire) with chemical disinfectant (e.g., hypochlorite) and measured MBC was investigated and compared to Ag-TiO2. With chemical disinfectant, higher doses were required in the presence of metal salts to achieve the same MBC as when no metals were added. This was not the case with silver nanoparticles, where the measured MBC appeared to be independent of external metal addition. Lastly, addition of both (uncoated) silver and silver titania on NIH/3T3 cell line (somatic mouse immortalized fibroblast) was investigated, to compare and contrast with MBC determined with prokaryotic cells (Escherichia coli, Staphylococcus aureus). The NIH/3T3 cell were susceptible vulnerable to toxicity when incubated with either silver or silver titania nanoparticles at a concentration range of 10-40 parts-per-million (ppm). The slight difference in toxicity in Ag versus Ag-TiO2 may be due to slower uptake of Ag-TiO2 due to their relative higher size when compared to the smaller diameter of silver clusters. These results indicate that silver titania nanoparticles are more rugged in usage when compared to chemical disinfectants under different environmental conditions and against different microbes or cell lines.
Effect of the Cross-linking with Calcium Ions to Improve the Thermo-mechanical Properties of Alginate Films. Turner K. Vidal-Urquiza1, Oscar Perales Perez2, Boris Renteria2 and Marcelo Suarez2; 1Department of Mechanical Engineering, University Of Puerto Rico at Mayaguez, Mayaguez, Puerto Rico; 2Department of Engineering Science & Materials, Univesity of Puerto Rico at Mayaguez, Mayaguez, Puerto Rico.
The development of coatings based on Calcium alginate is becoming very attractive because of the biocompatibility and mechanical strength exhibited by this polymeric material. On this basis, the synthesis of Calcium alginate films has been attempted via an immersion method where precursor Na-alginate films are contacted with a CaCl2 aqueous solution to promote the ionic cross-linking in the resulting polymeric material. The effect of Ca2+-concentration (0.1M-0.2M), temperature (10oC-60oC) and immersion time in the 0.1-24 hours range on the structural and thermo-mechanical properties of produced films were investigated. X-ray diffraction, Raman and FT-IR spectroscopy analyses verified the incorporation of the Ca2+ ions into alginate structure and the corresponding cross-linking in the films. Raman spectroscopy revealed the systematic shift of the vibration modes corresponding to carboxylic group from 1415cm-1(in pure Na-alginate film) to around 1429cm-1 (for cross-linked Ca-alginate films). Thermo-gravimetric analyses, under nitrogen atmosphere, of the films synthesized at different conditions revealed a moderate mass loss (10%-25%) between 78°C and 230°C, which was attributed to molecular water within the films. The thermal decomposition of the films started at 300°C. Micro-hardness measurements coupled with Thermo-mechanical (TMA) and Dynamic Mechanical Analyses (DMA) revealed the dependence of the mechanical properties with the immersion time and Ca ions concentration in the starting aqueous solutions.
Development of Porous Nanoparticle-supported Lipid Bilayers (Protocells) for Targeted Delivery of Plasmid DNA to Hepatocellular Carcinoma. David P. Padilla1, Carlee Ashley1, Eric Carnes1, Katie Epler1, Robert Castillo1, Caroline Bouvie1, Dan Wilkinson1, Natalie Johannes1 and C. Jeffery Brinker1,2; 1Center for Micro-Engineered Materials, University of New Mexico, Albuquerque, New Mexico; 2Self-Assembled Materials Division, Sandia National Laboratories, Albuquerque, New Mexico.
The porous nanoparticle-supported lipid bilayer (protocell), formed via fusion of liposomes to nanoporous silica particles, is a novel type of nanocarrier that addresses multiple challenges associated with targeted delivery of cancer therapeutics and diagnostics. Like liposomes, protocells are biocompatible, biodegradable, and non-immunogenic, but their nanoporous silica core confers drastically enhanced cargo capacity and prolonged bilayer stability when compared to similarly-sized liposomal delivery agents. The porosity and surface chemistry of the core can, furthermore, be modulated to promote encapsulation of a wide variety of therapeutic agents, such as drugs, nucleic acids, and protein toxins. The rate of cargo release can be controlled by pore size and the overall degree of silica condensation, making protocells useful in applications requiring either burst or controlled release profiles. Finally, the protocell’s supported lipid bilayer (SLB) can be modified with ligands to promote selective delivery and with PEG to extend circulation times. Here we report the use of peptide-targeted protocells to achieve highly specific delivery of a plasmid that encodes small hairpin RNA (shRNA), which induces growth arrest and apoptosis of transfected cells by silencing cyclin B1. We synthesized silica nanoparticles with pores large enough to accommodate histone-packaged plasmids using a dual surfactant approach. A non-ionic surfactant (Pluronic® F-127), when employed in conjunction with a swelling agent (1,3,5-trimethylbenzene) served as the template for large pores, while a fluorocarbon surfactant (FC-4) promoted growth of the silica core. Resulting particles had diameters ranging from 100-nm to 300-nm and contained an ordered network of 20-nm pores with 17.3-nm entrances. Supercoiled plasmid DNA was packaged with histones, and the resulting complex (~15-nm in diameter) was modified with a nuclear localization sequence (NLS) prior to being loaded into the silica core. Fusion of liposomes to the nanoporous core promoted long-term retention (> 1 month) of encapsulated DNA upon exposure to simulated body fluids at 37°C. Using phage display, we identified a targeting peptide with nanomolar affinity for hepatocyte growth factor receptor (c-Met), which is known to be overexpressed by various types of hepatocellular carcinoma (HCC). Protocells loaded with the DNA-histone-NLS complex and modified with ~240 copies each of the targeting peptide and a fusogenic peptide that promotes endosomal escape of protocells and encapsulated DNA were capable of transfecting both dividing and non-dividing HCC. Furthermore, targeted protocells effectively induced G2/M arrest and apoptosis of HCC (LC90 = 25 nM) without affecting the viability of non-cancerous cells, including hepatocytes, endothelial cells, and immune cells (PBMCs, B cells, and T cells).
Structurally Supported Porous Silicon Membranes for Biomedical Applications. Rachel Lowe, Hilkka Ylinara, Torin Yeager, Shuvo Roy and Tejal Desai; UCSF, San Francisco, California.
Nanostructured membranes are increasingly being developed for molecular separation applications. The introduction of both micro and nanofabrication techniques has allowed more precise structural control of pore size, porosity and membrane thickness. In particular, silicon membranes offer great potential due to their biocompatibility. However, fabrication of membranes with high porosity has been limited by structural fragility. In this work, we present a simple fabrication process for structurally supported porous silicon membranes and demonstrate the membranes capability to withstand pressure driven flow. The fabrication process utilizes photolithography to pattern the mask to create etch windows for an initial buffered oxide etch and a subsequent potassium hydroxide etch on the backside of the silicon wafer. The final stage is to then electrochemically etch the remaining silicon on the top-side to form discrete porous silicon membrane regions. The multi-dimensional tuning capabilities of porous silicon allow the fabrication of pore dimensions for size selective separations. Another favourable property of porous silicon is the ease of which it can be chemically modified with various functionalities. While fabrication of membranes with pore sizes in the nanometer range should be sufficient to prevent passage of cells and large proteins, small proteins may still pass through. Efforts to further prevent the passage of these macromolecules will be presented. The ability to introduce surface modification also plays a significant role in reducing degradation and biofouling, which is essential for biomedical applications. Here, we present the hydraulic permeability and permselectivity of structurally supported porous silicon membranes.
SESSION JJ7: Drug Delivery Systems
Chair: Laurie Gower
Thursday Morning, April 28, 2011
Salons 4-6 (Marriott)
8:30 AM *JJ7.1
Double Emulsions: Versatile Core-shell Structures for Encapsulation. Carlos Martinez, Purdue University, West Lafayette, Indiana.
Double emulsions are suspensions consisting of small droplets dispersed within larger drops, which are themselves dispersed in another fluid. Their use in advanced applications such as bio-encapsulation and targeted drug delivery has been limited since their traditional fabrication by means of a two-step emulsification offers little control over their structure. Recently, microcapillary devices have emerged as powerful platform for double emulsion fabrication. It is now possible to controllably form monodisperse double emulsions at frequencies ranging from 100 to 10,000 Hz. It is also possible to use these monodisperse double emulsions not only as fixed core-shell structures but also as sacrificial templates within which we can perform polymerization reactions such as particle synthesis. In this talk, I'll highlight my group efforts to use microcapillary devices to fabricate monodisperse functional core-shell structures for applications in colloidal crystal fabrication, cell encapsulation and the formation of cellulose nanocrystal (CNC) capsules. I'll describe the use of the double emulsion droplets as flexible semipermeable sacs that permit the osmotically driven exchange of water between the inner drop and the continuous phase. This water exchange is used to form colloidal crystals in the inner drop of the double emulsion droplet. I'll also describe a technique to encapsulate cells in monodispersed alginate hydrogels. The encapsulation begins by generating monodispersed double emulsions where the inner phase is an alginate/cells suspension, the middle phase is mineral oil, and the continuous phase is a glycerol-water mixture; we collect the double emulsions in a 100 mM CaCl2 solution. The mineral oil temporarily shields the encapsulated alginate suspension from the contact with the CaCl2 solution, preventing uncontrollable gelation. However, by tuning the metastability of these emulsions, the alginate solution controllably coalesces with the continuous phase, thereby forming monodispersed hydrogels. Finally, a technique to generate capsules composed of cellulose nanocrystals will be discussed.
9:00 AM JJ7.2
Simultaneous Gene Delivery and Tracking of Cells Using Fluorescent Upconversion Nanoparticles for Cell Therapy. Muthu Kumara Gnanasammandhan Jayakumar1 and Zhang Yong1,2; 1Division of Bioengineering, National University of SIngapore, Singapore, Singapore; 2Nanoscience and Nanotechnology Initiative, Singapore, Singapore.
Cell therapy has attained new heights with the current developments in the field of stem cell therapy and is often done in combination with gene therapy. Much research has gone into developing safe vectors to deliver genes to the cells used for cell therapy and also in tracking the transplanted cells and determining their fate. Even though viral vectors are very efficient in gene delivery, there are major safety concerns related to it. Herein, we report the use of novel fluorescent upconversion nanoparticles (UCNs) for simultaneous gene delivery and tracking of cells for cell-based therapies. Plasmid encoding Vascular endothelial growth fac-tor (VEGF) was loaded on to the mesoporous silica coating of the nanoparticles and used for transfecting mouse skeletal myoblasts. The inherent nature of these nanoparticles to absorb NIR light and emit in the UV-Visible region is exploited to track the gene delivery process and also to track the chimeric cells in-vivo after transplantation. Our results prove that UCNs are very efficient in gene encapsulation and delivery and provide excellent imaging capabilities too with minimal autofluorescence and high signal-to-noise ratio in deep tissues.
9:15 AM JJ7.3
Active Scaffolds for On-demand Drug and Cell Delivery. Xuanhe Zhao1 and David J. Mooney2; 1Mechanical Engineering, Duke University, Durham, North Carolina; 2School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts.
For over two decades, porous biomaterials fabricated from natural and synthetic polymers, metals, ceramics and glasses have been intensively studied and widely used as drug delivery vehicles and scaffolds for tissue regeneration. For example, collagen sponges that release bone morphogenetic proteins are widely used in spine surgery, with an approximately billion-dollar market. However, the porous scaffolds currently used are mostly passive in that they deliver biological agents mainly through mechanisms involving molecular diffusion, material degradation, and cell migration, which do not allow for dynamic external regulations. Here, we present a new active porous scaffold that can be remotely controlled by a magnetic field to deliver various biological agents on demand. The active porous scaffold, in the form of a macroporous ferrogel, gives a large deformation and volume change of over 70% under a moderate magnetic field. The deformation and volume variation allows a new mechanism to trigger and enhance the release of various drugs including mitoxantrone, plasmid DNA, and a chemokine from the scaffold. The porous scaffold can also act as a depot of various cells, whose release can be controlled by external magnetic fields. The controlled releases of various drugs and cells were demonstrated in vitro and in vivo.
9:30 AM JJ7.4
PLGA/rhBMP2 Mesoporous Silicon Microparticles for Orthopedic Disc Regeneration. Christopher Loo1,2, Dongmei Fan1,2, Iman Yazdi1,2, Ennio Tasciotti2, Mauro Ferrari2 and Bradley K. Weiner2,3; 1Nanomedicine and Biomedical Engineering, The Methodist Hospital Research Institute, Houston, Texas; 2Nanomedicine and Biomedical Engineering, The University of Texas Health Science Center, Brown Foundation Institute of Molecular Medicine, Houston, Texas; 3Orthopedic Surgery, The Methodist Hospital, Houston, Texas.
Low back pain caused by degenerative disc disease remains a significant cause of morbidity and mortality. We describe a novel drug delivery system utilizing PLGA-coated mesoporous silica microparticles for the delivery of rhBMP2 for the treatment of degenerative disc disease in bovine nucleus pulposus (NP) cells. Mesoporous silicon microparticles were fabricated with a mean diameter of 3.2±0.2 µm, pore diameters 6.0±2.1 nm. Heavily doped p++ type (100) silicon wafers were used as the base source and by deposition of 200-nm layer of silicon nitride followed by standard photolithography was used to pattern it using an EVG 620 contact aligner. A current density of 320 mAcm-2 was applied. Particle surface was further oxidized using H2O2. The suspension was heated to 100-110C, washed, and resuspended in isopropyl alcohol. PLGA-coating was prepared by a modified solid in oil in water (S/O/W) emulsion method. rhBMP2 loaded particles were suspended in 10% PLGA solution. The organic phase was mixed with 2.5% w/v PVA. NP cells were cultured in complete media at 37C, 5% CO2. Encapsulation of NP cells into alginate beads was accomplished by the addition of cells into a 1.2% alginate solution. Beads were cultured in chondrogenic media and were allowed to equilibrate for 72 hrs prior to rhBMP2 treatment. Cells were treated with 10 ng/ml rhBMP2, 1x106 empty PLGA/Psi microparticles, chondrogenic media, or 1x106 BMP2-loaded PLGA/Psi microparticles. After 14 days, beads were collected and dissolved. Alginate-cultured NP cells were recovered on 24-well plates, and lysed for proteoglycan analysis. MTT assay was used to assess cellular viability and proliferation. Proteoglycan expression was measured using the dimethylmethylene blue (DMMB) assay. Bovine trachea provided with the kit was used as a standard. DNA picogreen assay was used to normalize values. Alcian blue and Safranin-O staining were used to stain for cartilage expression. Images were taken with an inverted Zeiss Axio microscope. Histogram analysis was performed to quantitate staining differences. P-values<0.05 were determined to be statistically significant. No significant differences in proliferation or cytotoxicity were observed after 3 days. After 14 days of treatment, the greatest amount of proteoglycan expression was measured in cells treated with BMP2-loaded PLGA/Psi microparticles (p=0.005) and cells treated with 10 ng/ml of BMP2 (p=0.002). This effect was significant compared to untreated cells and the unloaded microparticle treatment group (p<0.01). Significantly greater collagen staining was observed in cells treated after 10 days. Image analysis demonstrated that cells treated with BMP2-loaded PLGA/Psi showed the greatest increase in cartilage staining (37.54±12.09%, p<0.001). Comparison of phenotypic expression of cartilage in the BMP2-loaded microparticle group with controls were statistically significant (p<0.05).
9:45 AM JJ7.5
New Drug Nanocarriers Based on Porous Iron Carboxylates MOFS. Patricia Horcajada1, Tamim Chalati2, Tarek Baati1, Ruxandra Gref2, Patrick Couvreur2 and Christian Serre1; 1CNRS. Universite de Versailles, Versailles, France; 2CNRS. Universite Paris Sud, Chatenay Malabry, France.
The need of molecules of very high molecular weight and/or with a low aqueous solubility in chemotherapy makes indispensable the development of new drug carriers. Actual delivery systems are not able to satisfy the critical requirement for a large number of molecules of high therapeutic interest such as Busulfan (antitumoral agent)(1) and Azidothimidine triphosphate (antiretroviral drug), which show important problems such as a poor stability(2), toxicity effects(3) or a low bioavailability(4). A new alternative to encapsulate drugs, never tried before, is to use nanoparticles of porous Metal-Organic-Frameworks (MOFs). These solids combine a high pore volume and a regular porosity, as well as the presence of organic groups easily tuneable within the framework(5). Thus, very high drug storage capacities, up to 1.4 g of Ibuprofen/g of MOF, with a complete drug controlled release under physiological conditions from 3 to 6 days were achieved using rigid MOFs(6). In addition, the use of flexible porous MOFs led to an unsually long drug release(3 weeks) with a loading capacity of 20% (wt/wt).(7) This work reports the use for the first time of biocompatible and non toxic nanoparticles of porous iron (III) carboxylates as new drug delivery systems(8). The encapsulation and release of different antitumoral and retroviral drugs into these porous hybrids solids were studied. Exceptionally high drug loadings, up to 60 and 4 times more effective than in liposomes and the best systems polymer-based respectively, were achieved with a controlled release of the active form of drug. In addition, the design of porous hybrid solids, playing with the wide range of compositions and topologies, will allow adapting these porous hybrid matrices to the guest molecule, according to its structure and its dosage requirements. 1. Nashyap A. et al. Biol. Blood Marrow Transplant., 2002, 8, 493 2. Li X. et al., Adv. Drug Deliv. Rev. 39,1999, 81 3. Baron F. et al. Haematologica 1997, 82, 718 4. Hillaireau H. et al. J. Control. Release 116, 2006, 346 5. Serre C. et al. Science, 2007, 315, 1828; Férey G. et al. Science, 2005, 309, 2040. 6. Horcajada P. et al. Angew. Chem. Int. Ed., 2006, 45, 5974. 7. Horcajada P. et al. J. Am. Chem. Soc.,2008, 130, 6774. 8. Ghermani N.E. et al. Pharmac. Research, 2004, 21, 598. 9. Horcajada P.,et al, Nature Mater., 2010, 9, 172.
10:30 AM JJ7.6
In Vitro Evaluation of Layer by Layer Assembled Lectin Mediated Polystyrene and Paclitaxel Nanoparticles on Caco-2 Cell Lines. Xiao Yu and Michael V. Pishko; Texas A&M University, College Station, Texas.
In this study, hydrophobic properties of anticancer drug paclitaxel (PTX-NPs) and polystyrene nanoparticles (PS NPs) were modified by chitosan and dextran polyelectrolytes using a layer by layer self-assembled method (lbl)1. To facilitate material biocompatibility and cell targeting, functional groups poly (ethylene glycol) (PEG) and fluorescent labeled wheat germ agglutinin (F-WGA) were conjugated onto particle surfaces (PTX/PS-lbl-PEG-WGA) 2. PEG is a hydrophilic polymer to improve material biocompatibility2a. In this study, viability of Caco-2 cells was analyzed by fluorescence microscopy. Less than 10 % of Caco-2 cells were injured/killed after contacting with PS-lbl-PEG NPs (20 hrs, 37 degree), which was almost the same as controls (PS NPs, PS-lbl NPs). In vitro evaluation of WGA conjugated nanoparticles’ binding and uptaken behavior with Caco-2 cells was performed by flow cytometry (PS-lbl-PEG-WGA NPs 1.5 - 15 µg/mL). 61.3 % of Caco-2 cells were fluorescent labeled at 15 µg/mL of WGA (21 hrs at 4 degree) towards binding saturation. Efficacy of WGA as a targeting moiety was studied by comparing the percentage of PS-lbl-PEG-WGA NPs bound Caco-2 cells, with that of PS-lbl-PEG / PS NPs bound cells (1.5 µg/mL of WGA). 35 % of Caco-2 cells were fluorescent labeled by PS-lbl-PEG-WGA NPs versus less than 10 %/5 % of cells by PS-lbl-PEG NPs/ PS NPs, proving that WGA was an effective targeting group for colon cancer Caco-2 cells. Further study involved replacing PS NPs by PTX NPs, and efficacy of this drug delivery system could be tested by contacting PTX-lbl-PEG-WGA NPs with Caco-2 cells. To summarize, the layer by layer self-assembly coatings, PEG and WGA modifications onto PTX NPs surfaces provided a potential way to achieve controllable releases of drug from a biocompatible and targeting system.
10:45 AM JJ7.7
Rapid Delivery of Nanocrystals into the Cytosol of Live Cells with Responsive Polymer Colloids. Brett A. Helms, Andrea R. Bayles, Dev S. Chahal, Harvind S. Chahal and Emma N. Rudie; The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California.
Luminescent nanocrystals are increasingly sophisticated, bright, photostable probes that show great promise in bioimaging. Nevertheless, researchers often find them difficult to employ in live cell imaging. Nanocrystals, when applied to a plate of cells, are taken up in vesicles known as endosomes. Due to their sequestration in endosomes, they are unable to reach subcellular targets. We will describe our recent results in achieving efficient cytosolic delivery of nanocrystals to live cells, free from vesicular entrapment using a small library of polymeric nanomaterials with membrane disrupting character. We will discuss the role of surface chemistry, particle size, and other design parameters in the delivery capabilities to different cell types.
11:00 AM JJ7.8
Cellulose-nanocrystalline Hydroxyapatite Composite Meshes for the Retention and Delivery of Recombinant Protein Therapeutics. Tera Filion1,2, Artem Kutikov1,2 and Jie Song1,2; 1Department of Orthopedics & Physical Rehabilitation, University of Massachusetts Medical School, Worcester, Massachusetts; 2Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts.
Two-dimensional polymer fiber meshes with a combination of cytocompatibility, good physical handling characteristics, and the ability to retain/release protein therapeutics are of significant value to regenerative medicine applications. To achieve stable absorption and sustained release of proteins, a polymer scaffold may be integrated with other structural components capable of absorbing proteins. Taking advantage of the innate high affinity of hydroxyapaptite (HA), the major inorganic component of bone, for a wide range of biomolecules, we aim to mineralize cellulose fiber meshes with nanocrystalline hydroxyapaptite (nHA) as a protein therapeutics delivery platform. Cellulose fiber meshes, obtained by hydrolyzing electrospun cellulose acetate meshes, were annealed using a hot hydraulic press and mineralized with nHA using a urea-mediated heterogeneous mineralization technique developed in our lab. The impact of thermal/mechanical annealing and nHA-mineralization on the mechanical properties of the fiber mesh was investigated using a dynamic mechanical analyzer at dry and hydrated states. Annealing at 90 °C under a 3750-psi compressive loading significantly improved the elastic moduli and ultimate tensile strengths of the fiber meshes by 100-200% without compromising their ultimate tensile strains (7-9%). Mineralization of the annealed fiber meshes, on the other hand, reduced both elastic moduli and ultimate tensile strengths while maintaining ultimate strains of the meshes. An optimal annealing and mineralization condition was identified to prepare mineralized meshes with good tensile compliances for potential surgical manipulations. To evaluate the ability of the mineralized meshes to retain/release protein therapeutics in a sustained manner, human recombinant proteins BMP-2, IGF-1 and VEGF165 were absorbed to the meshes and their release kinetics in PBS at 37 °C were monitored using ELISA. No burst release of the growth factors from the mineralized fiber meshes was observed, with 65-90% of the proteins retained on the meshes after a 1-week incubation. Further, using a BMP-induced osteogenic trans-differentiation of myoblast C2C12 cell culture model, we show that the BMP-2 retained on the mineralized mesh after the 1-week incubation remained biologically active, enabling the trans-differentiation of C2C12 cells attached to the mesh into osteoblasts. Overall, we show that the combination of attractive mechanical properties and protein retention/release profile of nHA-cellulose composite meshes can be obtained. Synergistic delivery of multiple growth factors using this platform for tissue engineering applications will be explored.
11:15 AM JJ7.9
Folate Receptor Targeting Drug Loaded PLGA-lipid Hybrid Nanoparticles and Their Selectivity for HeLa Cervical Cancer Cells. Sergio Sandoval1, Alex Liberman2, Jian Yang3, Sharraya Aschemeyer3, Jesus G. Alfaro4, Liangfang Zhang5, William C. Trogler3 and Andrew C. Kummel3; 1Bioengineering, University of California, San Diego, La Jolla, California; 2Material Science Engineering, University of California, San Diego, La Jolla, California; 3Chemistry and Biochemistry, University of California, San Diego, La Jolla, California; 4Chemical Engineering, University of California, San Diego, La Jolla, California; 5Nanoengineering, University of California, San Diego, La Jolla, California.
The response rate of breast cancer to first line chemotherapies is encouraging, but 20-30% of patients develop chemoresistance to these drugs, and consequently, have a cancer reoccurrence 7-10 months after their last treatment. Chemoresistance is believed to be due to drug efflux proteins responsible for the removal of many commonly used anti-neoplastic agents. One possible way to overcome these drug efflux pumps is to give higher doses of chemotherapy, but high doses of such agents commonly lead to chemocytotoxicity. Targeted PLGA-Lipid hybrid nanoparticle (NP) drug delivery systems have been developed that can deliver high doses of chemotherapy agents specifically to breast cancer cells. A practical cancer targeting drug delivery system will reduce the overall amount of chemotherapy agents given to patients for a given amount of targeted NPs endocytosed by cancer cells. Biodegradable NPs were synthesized using a novel nano-precipitation lipid-polymer hybrid platform which also allows for the encapsulation of hydrophobic chemotherapy drugs within the NPs. Using this method, drug free NPs have been shown to have an average diameter size of 81.78 nm (PDI: 0.25), while single loaded NPs, with Paclitaxel or Doxorubicin, show an average size between 72.33 to 89.64 nm (PDI: 0.242 to 0.339), signifying that the synthesis technique creates consistent sub 100nm particles. The majority of all NPs show a zeta-potential value of > -30 mV consistent with the NPs having sufficient repulsive interaction to be mono-dispersed in solution under physiological conditions. Folate receptors are often over expressed on the surfaces of cancer cells; therefore, folic acid was incorporated to the surface of these NPs as a cancer targeting ligand. Previous studies have shown that HeLa cells, a cervical cancer cell line, over expresses folate receptors. Immunofluorescence studies show that folic acid coated PLGA-Lipid hybrid NPs are readily endocytosed by HeLa cells compared to non-targeted NPs. Cytoxicity studies will determine the increased effectiveness of drug delivery with targeted PLGA-Lipid hybrid NPs vs. untargeted PLGA-Lipid hybrid NP in cell lines and in animal models.
11:30 AM JJ7.10
Fluorescent, Superparamagnetic Nanospheres for Drug Storage, Targeting, and Imaging. Donglu Shi1, Hoon Sung Cho1, Zhongyun Dong2, Giovanni M. Pauletti3, Rodney C. Ewing4, Hongchen Gu5, Hong Xu5 and Jie Lian6; 1Chemical and Materials Engineering, University of Cincinnati, Cincinnati, Ohio; 2Internal Medicine, University of Cincinnati, Cincinnati, Ohio; 3James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, Ohio; 4Geological Sciences, University of Michigan, Ann Arbor, Michigan; 5Med-X Institute, Shanghai Jiao Tong University, Shanghai, China; 6Mechanical, Aerospace & Nuclear Engineering, Rensselaer Polytechnic Institute, Troy, New York.
For early cancer diagnosis and treatment, a nano carrier system is designed and developed with key components uniquely structured at nano scale according to medical requirements. For imaging, quantum dots with emissions near infrared range (~800 nm) are conjugated onto the surface of a nano composite consisting of a spherical polystyrene matrix (~150 nm) and the internally embedded, high fraction of superparamagnetic Fe3O4 nanoparticles (~10 nm). For drug storage, the chemotherapeutic agent paclitaxel (PTX) is loaded onto the surfaces of these composite multifunctional nano-carriers by using a layer of biodegradable poly(lactic-co-glycolic acid) (PLGA). A cell-based cytotoxicity assay is employed to verify successful loading of pharmacologically active drug. Cell viability of human, metastatic PC3mm2 prostate cancer cells is assessed in the presence and absence of various multifunctional nano-carrier populations using the MTT assay. PTX loaded composite nano-carriers are synthesized by conjugating anti-Prostate Specific Membrane Antigen (anti-PSMA) for targeting. Specific detection studies of anti-PSMA-conjugated nano carrier binding activity in LNCaP prostate cancer cells are carried out. LNCaP cells are targeted successfully in vitro by the conjugation of anti-PSMA on the nano carrier surfaces. To further explore targeting, the nano carriers conjugated with anti-PSMA are intravenously injected into tumor-bearing nude mice. Substantial differences in fluorescent signals are observed ex vivo between tumor regions treated with the targeted nano-carrier system and the non-targeted nano-carrier system, indicating considerable targeting effects due to anti-PSMA functionalization of the nano carriers.
11:45 AM JJ7.11
Hydrogel-based Drug Delivery Systems for the Controlled Release of Hydrophobic Drugs and Proteins. Itsuro Tomatsu, Ke Peng and Alexander Kros; LIC, Leiden University, Leiden, Netherlands.
Hydrogel based biomaterials are of interest because of their excellent biocompatibilities and the similarity between their highly hydrated three-dimensional polymeric networks and hydrated body tissues . Particularly, hydrogel based drug delivery systems have recently been paid much attention as promising drug carriers [2,3]. To advance applications of hydrogels in drug delivery systems, careful design of the hydrogel materials is critically important in order to tune its drug release characteristics. In this contribution, we will present our recent development in hydrogel based drug delivery systems using maleimide modified dextran (Dex-mal) and various thiol functionalized compounds. In the first example [4,5], we have used thiol-functionalized cyclodextrins (CD) to modify the hydrogel in order to increase the affinity of hydrophobic drugs with the hydrogel matrix. This is necessary because most hydrogels are typically hydrophilic, which limits their successful use as a drug delivery system for hydrophobic drugs due to rapid and uncontrolled drug release in the initial phase. In the second example, we have used the inclusion complex formation between trans-azobenzene and CD as a photo-switchable crosslinker to develop a photo-responsive supramolecular hydrogel resulting in a light controlled protein release system. This system can facilitate an efficient protein delivery since it can transport the physically entrapped protein to the target site and the release is induced by light. References:  N.A. Peppas, J.Z. Hilt, A. Khademhosseini, R. Langer, Hydrogels in biology and medicine: From molecular principles to bionanotechnology, Adv. Mater. 18 (2006) 1345-1360.  C. Hiemstra, Z.Y. Zhong, M.J. van Steenbergen, W.E. Hennink, J. Feijen, Release of model proteins and basic fibroblast growth factor from in situ forming degradable dextran hydrogels, J. Control. Release 122 (2007) 71-78.  F. van de Manakker, K. Braeckmans, N. el Morabit, S.C. De Smedt, C.F. van Nostrum, W.E. Hennink, Protein-Release Behavior of Self-Assembled PEG-beta-Cyclodextrin/PEG-Cholesterol Hydrogels, Adv. Funct. Mater. 19 (2009) 2992-3001.  K. Peng, I. Tomatsu, A.V. Korobko, A. Kros, Cyclodextrin-dextran based in situ hydrogel formation: a carrier for hydrophobic drugs, Soft Matter 6 (2010) 85-87.  K. Peng, C. Cui, I. Tomatsu, F. Porta, A.H. Meijer, H.P. Spaink, A. Kros, Cyclodextrin/dextran based drug carriers for a controlled release of hydrophobic drugs in zebrafish embryos, Soft Matter 6 (2010) 3778-3783.  K. Peng, I. Tomatsu, A. Kros, Light controlled protein release from a supramolecular hydrogel, Chem. Commun. 46 (2010) 4094-4096.
SESSION JJ8: Hybrid Materials for Implant Applications
Chair: Carlos Martinez
Thursday Afternoon, April 28, 2011
Salons 4-6 (Marriott)
1:30 PM *JJ8.1
Remineralization of Partially Demineralized Human Dentin via the Polymer-induced Liquid-irecursor (PILP) Process. Taili Thula-Mata1, Anora Burwell2, Laurie Gower1, Stefan Habelitz2 and Grayson Marshall2; 1Materials Science & Engineering, University of Florida, Gainesville, Florida; 2Department of Preventive and Restorative Dental Sciences, University of California, San Francisco, California.
Dental caries represents a major problem in dentistry despite the significant advances in preventive care. Repair of caries using resin-composite fillings is the preferred treatment. However, bonding between dentin and fillings created by adhesives fails with time due to in vivo degradation of demineralized collagen. A common experimental approach to improve the durability of these bonds is the remineralization of dentin in situ. Our group has been able to achieve intrafibrillar mineralization of diverse collagen matrices using a novel method known as the polymer-induced liquid-precursor (PILP) mineralization process. The PILP process consists of the addition of anionic polymers to a supersaturated mineralization solution in order to induce a fluidic amorphous calcium phosphate precursor. To determine if the PILP process might be effective in the remineralization of dentin caries, an in vitro model system was used based on acid-etched dentin samples with a 100-150 µm thick zone of demineralized dentin on top of a mineralized dentin base. This biogenic collagen-based matrix was then remineralized via the PILP process using poly-L-aspartic acid as the polymeric process-directing agent. The artificial lesions remineralized by the PILP process presented a surface morphology very similar to the intact mineralized dentin’s architecture, in contrast to samples mineralized via the conventional nucleation and growth method, where no polymer additive was present. In the latter, hydroxyapatite (HA) spherulites were observed on the surface of the dentin samples. Energy dispersive x-ray spectroscopy analysis of the PILP-mineralized samples showed the presence of calcium and phosphate ions at high levels. Since no HA clusters were observed on the surface of the PILP-mineralized samples, we could conclude the signal was produced from the mineral embedded within the collagen fibrils. TEM of thin sections prepared by focused ion beam (FIB) milling showed a microstructure very similar to native dentin. The remineralized lesions were also analyzed by micro-XCT with respect to their mineral content, where the PILP- remineralized specimen recovered about 80% of the mineral lost within seven days of treatment. Within the following seven day treatment, mineral content recovered to normal dentin tissue values. While mineral content appears normal after remineralization, nanomechanical properties examined by AFM nanoindentation were not fully restored within that time-frame. Thus current efforts are focused on methods to improve the functional restoration of the dentin lesions. More recently, we have also been examining remineralization of natural caries, which present a more heterogeneous and challenging microenvironment.
2:00 PM JJ8.2
Composite PDLLA-Bioglass Biodegradable Scaffolds for Bone Tissue Engineering: Characterization of Bioglass Transformation and Integration within the Scaffold. Sara Abdollahi and Marta Cerruti; Mining & Materials Engineering, McGill University, Montreal, Quebec, Canada.
When bone is damaged such as in an injury, essentially four options exist to restore its function: replace the damaged part with (i) an inert, biocompatible implant, (ii) a bone graft from another species (xenograft), (iii) a graft from a human source (allograft), or (iv) bone from the patient’s own skeleton (autograft). Although attractive, the first option is problematic in that an inert implant does not allow vascularization and is unable to interact with its surrounding environment to accommodate changes in load and physiological conditions. The second and third options are possible sources of disease transmission. The fourth alternative requires a second surgical site, not to mention the quantity limitations when taking grafts from the patient and risks associated with donor-site complications. The ideal choice would be to incite bone around the damaged area to repair itself. This is possible by using a biodegradable, bioactive scaffold, with a porous structure similar to bone, which is biomechanically stable and can temporarily replace the bone in the site of injury. A scaffold that is biodegradable resorbs into the body generating non-toxic degradation products while new tissue reforms. On the other hand, a bioactive scaffold encourages the surrounding tissue to regenerate and gradually fill in the missing bone area. In the present study, we make composite scaffolds using poly-DL-lactide (PDLLA), a biodegradable polymer, as the matrix, and incorporate 45S5 Bioglass (BG) to stimulate scaffold bioactivity. BG is a silicate-based glass that when in contact with body fluids, dissolves and generates silicate, phosphate, calcium and sodium ions. Calcium and phosphate ions reprecipitate on the BG surface and form a layer of hydroxycarbonate apatite. The precipitation of hydroxycarbonate apatite is most welcome because it is similar to the inorganic component of bone. After it is formed, it integrates with surrounding tissue fibrillar collagen, the organic component of bone, to form a matrix that attracts osteoblasts and incite bone tissue regrowth. The composite scaffold is synthesized by the solvent casting and particulate leaching technique, which stands out for its simplicity. This method involves the insertion of a porogen in a polymer solution that later leaches out to create a porous structure. In order to assess the scaffold’s bioactivity, it is of utmost importance to understand the fate of BG throughout the scaffold’s processing, and its integration within the polymeric matrix. Specifically, this study focuses on the transformations of BG during the leaching step and on the BG-scaffold interface. BG dissolution in the leachate is detected with inductively coupled plasma atomic emission spectroscopy. The BG-scaffold interface is characterized with infrared and Raman spectroscopy, as well as scanning electron microscopy, to understand the chemical and morphological transformations occurred in BG during the scaffold’s synthesis.
2:15 PM JJ8.3
Evolution of Fibril Orientations and Composite Mechanical Properties in Collagen Gel-infiltrated Accordion-like Honeycomb Poly(glycerol Sebacate) Scaffolds: Implications for Tissue Engineering Functionally Anisotropic Cardiac Muscle. Aurelie Jean, Sarah R. Bass and George C. Engelmayr; Bioengineering, Penn State University, University Park, Pennsylvania.
Tissue engineering cardiac muscle is both a clinically relevant and intellectually stimulating pursuit. In aiming to overcome structural-mechanical limitations of previous scaffolds, Engelmayr et al. recently developed a novel accordion-like honeycomb poly(glycerol sebacate) (PGS) scaffold pore geometry that was found to be intrinsically capable of promoting the alignment of seeded neonatal heart cells in a manner similar, albeit suboptimal, to native myocardium [Engelmayr et al. 2008 Nat Mater]. Toward better delineating the roles of scaffold microstructure and extracellular matrix properties in mediating local, intra-pore cardiomyocyte elongation, phenotype and deformation, in related work we are investigating a model system comprised of neonatal rat cardiomyocytes sparsely distributed within a collagen gel infiltrating the pores of the accordion-like honeycomb PGS scaffold. In the current study we are particularly focusing on the structural-mechanical coupling between the PGS struts comprising the accordion-like honeycomb scaffold and the nascent fibrils of collagen gelled in situ within the accordion-shaped pores. For this purpose, serial dilutions of acid-solubilized bovine dermal collagen solution (1.5, 3, 4.5, and 6 mg/ml; Sigma) are being studied to vary the collagen gel effective stiffness in the initial stress-strain regime, as well as different pore geometries to elucidate the explicit contribution of the pore geometry on the evolution of collagen fibril orientations and different PGS stiffnesses (by varying the curing conditions). Following collagen solution infiltration and in situ fibrillogenesis within the scaffold (25x5x0.25mm) pores, the effective stiffnesses of the gel-infiltrated scaffolds will be measured by uniaxial tensile testing of specimens oriented in the two orthogonal material directions defined with respect to the pore long axis. Then, the effective initial modulus of the collagen gel (under isotropic and transverse isotropic assumptions) will be predicted by an inverse method using finite element simulations of the composite gel-infiltrated scaffold in conjunction with homogenization theory. A periodic finite element model, previously utilized to characterize the mechanical behavior of the accordion-like honeycomb scaffold itself [Jean and Engelmayr, 2010 J Biomech] will be utilized. Further, in addition to mechanical testing and finite element simulation, the collagen gel-infiltrated scaffolds are currently being evaluated by confocal reflectance microscopy to image the collagen fibrils. Specifically, the influence of fibril position relative to a coordinate system defined by PGS struts will be assessed and orientation distributions will be integrated into the finite element simulations. Collectively, the results of these studies will aid in the design of scaffold pore geometries suitable for generating functionally anisotropic tissue engineered cardiac muscle.
2:30 PM JJ8.4
Viscoelastic Rheological Model for Reconstructed Tissue In-vitro. Leslie Laquieze1,2, Mohamed Ben Tkaya1, Cyril Pailler-Mattei1, Geraldine Aimond2, Pascal Sommer2 and Hassan Zahouani1; 1LTDS Ecole Centrale de Lyon, Ecully cedex, France; 2Institut de Biologie et Chimie des Protéines, Lyon, France.
Over the last years, several models of reconstructed skin have been developed to contribute large burns and chronic wounds treatment. In the same way, in agreement with European regulation, skin equivalents have been developed to provide alternative methods for innocuousness and effectiveness validation of dermo-cosmetic products. Skin equivalents are presenting several limits for a rigorous mechanical characterization. Indeed, skin equivalent can be presented as a visco-elastic, adhesive, heterogeneous and multi-layered material. Several applications of the contact problems have given rise in last decades to special studies in the range of isotropic elasticity and elstoplastic theory. Indentation is frequently used to characterize the elastic property of the material. The assumption made is that the material is elastoplastic. However many many material exhibit a visco-elastic behavior in one hand. In other hand biological material is adhesive. The indentation and relaxation experiments have been conducted for different reconstructed skin samples to provide data to predict mechanical property of is models. To emphasize our study the indentation of standard viscoelastic solids has been investigated theoretically. This work shows how conventional spring and dashpot element can be used to model the relaxation and indentation behavior of a different reconstructed skin. The results show a good agreement between experimental load-displacement data and theoretical model during relaxation. This approach is used to identify rheological parameters of reconstructed skins in the aim to extend this approach to the study of human skin in-vivo.
2:45 PM JJ8.5
Microelectrode Arrays Employing Collagen for Improved Neuronal Recording. Alfred Miller1, Matthew Taylor2, Patrick Goerrn1, Lynn Enquist2, Naveen Verma1, Sigurd Wagner1 and Simon Archibald3; 1Electrical Engineering, Princeton University, Princeton, New Jersey; 2Molecular Biology, Princeton University, Princeton, New Jersey; 3Integra Life Sciences, Plainsboro, New Jersey.
Bridging the gap between electronics and biology will be the key to solving many critical and compelling questions in biology. In particular, neuroscience is a branch brimming with questions that await the collaborative effort of neuroscientists and electrical engineers. Neuroscientists would like to develop brain-machine interfaces. One major obstacle to designing chronic brain-machine interfaces is the inability to record signals from within the brain over long time periods. Current microelectrodes are ill-suited for recording chronic neural signals because of their diminishing performance over time and eventual failure. The next generation of implantable microelectrode arrays must possess qualities that are ideal from the perspective of both biology and electronics. This project makes such a union of biology and electronics a reality. It aims to develop a new type of implantable microelectrode array for chronic neural recording. We approach such a device by combining materials used for flexible electronics (i.e., thin-film electronics on polyimide substrates) with materials used in regenerative medicine (i.e., collagen). A device able to reliably record chronic neural signals will have an immense impact. The project is forging connections between molecular biologists, neuroscientists, and engineers. Moreover, our novel approach of combining electronics and biology can be applied more broadly to biological research, such as on tissue morphodynamics. Fabrication of our prototype microelectrode array starts with the use of photolithography to pattern thin film gold traces on a polyimide backplane. Biocompatible tungsten wire electrodes are threaded through and bonded to the backplane using conductive paint and PDMS encapsulation. A collagen matrix, which engulfs the tungsten wire electrodes, is then added. The collagen layer is essential since it provides an environment conducive to homeostatic biochemistry. We record neural signals in vitro and eventually will record in vivo. At present we are explanting superior cervical ganglion cells onto the microelectrode arrays and perform electrophysiology. We will report device fabrication, cell explantation, and the results of neural recording.
3:30 PM JJ8.6
Noncollagenous Structures - The Springs Within the Human Dentin. Luiz E. Bertassoni and Michael V. Swain; Biomaterials, University of Sydney, Sydney, New South Wales, Australia.
It has become increasingly evident that structural interactions at different length scales play an important role in increasing resistance to deformation in load-bearing mineralized tissues, such as dentin and bone. A great deal is known about the role of the mineral, the collagen fibrils and interfibrillar molecules on the dissipation of mechanical energy during tissue strain, particularly during fracture. However, the structural origins of the outstanding durability of teeth and its complex relationship to damage accumulation and ability to recover remain poorly understood. Here we report that dentin contains noncollagenous components that both hamper deformation during compression and enable recovery after compression is ceased. Dentin specimens were cut, polished, half masked and demineralized with 10 vol% citric acid for 2 min. Both the normal and demineralized sides of all specimens were treated with 1 mg/mL Trypsin (pH 7.9) for 48 h at 37°C, unselectively targeting all noncollagenous structures in the matrix. Specimens were indented in water using an UMIS system and imaged with an FE-SEM. Data was analysed with student’s t-test. Creep response was determined using a holding time of 30 sec at a maximum load (5 mN), and recovery was recorded during another 30 sec at a minimum load (1 mN), without incremental loading or unloading segments. Results revealed that removal of noncollagenous structures yielded a decrease in hardness of nearly 23% (p<0.001) for normal and about 27% for demineralized dentin. The normalized creep deformation of normal dentin nearly doubled from 15.50±2.12 nm to 31.67±3.79 nm (p<0.01) after removal of noncollagenous structures. Similarly, demineralized dentin had an increase in creep deformation from 26.67±2.31 nm to 52.00±5.66 nm following trypsin digestion (p<0.001). To our surprise, the tissue nearly lost its ability to recover from loading once noncollagenous structures were removed. Normal dentin recovered approximately 83% of the creep deformation before removal of noncollagenous structures, after digestion the recovery represented only ~16% (p<0.05). Similarly, demineralized dentin recovered nearly 62% of the creep deformation, whereas the digested tissue recovered only about 11% (p<0.05). In SEM images the collagenous matrix appeared as a much more exposed network after removal of noncollagenous structures. High resolution images of digested dentin suggested that removal of noncollagenous structures consistently unraveled 20 nm thick collagen subfibrils, consistent with untwisting of the 100-200 nm collagen fibrils initially seen, putatively due to loss of surface proteoglycans, thus revealing a hierarchical level that has never been reported for mineralized tissues. In summary, we found that noncollagenous structures, such as proteoglycans and glycosaminoglycans, although a minor component of the tissue’s matrix, may be a critical element responsible for the high durability of teeth.
3:45 PM JJ8.7
Comparison of Properties and Microstructure of Dense Hydroxyapatite-carbon Nanotube Composites Prepared by Spark Plasma Sintering and Pressureless Sintering. Ashley A. White1, Fawad Inam2, Sebastian W. Pattinson1, Michael J. Reece2, Alan H. Windle1 and Serena M. Best1; 1Dept. of Materials Science & Metallurgy, University of Cambridge, Cambridge, United Kingdom; 2Nanoforce Technology Ltd. and School of Engineering and Materials Science, Queen Mary, University of London, London, United Kingdom.
The bioactivity of hydroxyapatite (HA) and the strength and stiffness of carbon nanotubes (CNTs) suggest that composites of these two materials may be mechanically-superior as a bone replacement material. This work is the first direct comparison of spark plasma sintering (SPS) and pressureless sintering (PLS) to prepare dense composites of HA and CNTs. Previous studies of HA-CNT composites have typically used one or the other of these techniques, but since the exact powder properties, including phase purity, particle size distribution, and CNT morphology, can affect sinterability, it is difficult to compare the results of different authors. It is thus necessary to use the same powder to make a direct comparison of the two sintering techniques. The sintering conditions for HA-CNT composites are crucial. The high temperatures necessary for sintering can oxidize CNTs, while the presence of water vapour or high pressure is necessary to retain HA’s phase purity and hydroxylation. SPS offers the advantages of fast processing times (minutes) and lower sintering temperatures, but requires expensive equipment and limited geometry. PLS, on the other hand, involves cheap, readily-available equipment, but requires much longer processing times (hours) and higher sintering temperatures. The atmosphere must also be controlled carefully to balance CNT retention and preservation of HA phase purity and hydroxylation. HA-CNT powders (CNT loadings between 0 - 5 wt.%) were prepared by co-precipitation of HA in the presence of multi-walled CNTs. The material was then dried, ground, and pressed into tablets. SPS tablets were sintered at 1050°C for 3 min. at 70 MPa. PLS tablets were sintered in an atmosphere of carbon monoxide and hydrogen (optimized in a previous study for density and CNT retention) at 1200°C for 1 hr. Powder from the same batch was used for both techniques. The sintered material was characterized for phase purity (XRD), hydroxylation (FTIR), % porosity, CNT retention, microhardness, microstructure (SEM), and HA-CNT interface (TEM). Tablets produced by both techniques were found to be phase pure and hydroxylated. The SPS tablets were more dense (97-99% vs. 74-94%, depending on CNT loading) and had far superior CNT retention (99+% vs. up to 50%). In correlation with density, the microhardness of the SPS materials was up to 500% greater compared with their PLS counterparts. SEM and TEM analysis showed a much smaller grain size for SPS materials and better coating of the CNTs with HA. PLS can offer an affordable method to prepare phase-pure HA-CNT composites but at the expense of density and mechanical properties. SPS, on the other hand, produces fully dense materials with no CNT loss, enhanced coating of CNTs with HA, and increased microhardness.
4:00 PM JJ8.8
Evaluation of Elastic Modulus of Hydroxyapatite-nanocomposites for Bone Implants Using Finite Element Method. Kristopher Doll and Ani Ural; Villanova University, Villanova, Pennsylvania.
Bone replacement using artificial materials have been widely used to allow patients to regain mobility compromised by aging, disease or trauma. These artificial materials have traditionally been biologically inactive and do not entirely mimic the mechanical behavior of the bone. Recently, bioactive and biomimetic materials such as hydroxyapatite (HA) have been proposed as new bone implant materials. HA is a biologically active calcium phosphate ceramic that promotes bone growth on its surface and forms a strong interface with the bone. However, HA has low tensile strength and fracture toughness compared to that of bone, therefore, a second phase reinforcing material needs to be added to monolithic HA to improve its mechanical properties. Among the possible reinforcements for HA, nanoscale inclusions such as graphene and carbon nanotubes provide promising alternatives to conventional reinforcements due to their exceptionally high strength and the large interface area that they form with the matrix material. In this study, we investigate how the addition of graphene nanosheets and carbon nanotubes affect the elastic modulus of HA using finite element modeling (FEM) to provide insight into the mechanical behavior of HA-nanocomposites as candidate materials for bone implant applications. The FEM was chosen to enable the controlled evaluation of the effects of a single parameter on the mechanical properties of the HA-nanocomposites. Random distributions of nonintersecting nanofillers within a given volume are generated with a MATLAB program for varying aspect ratio, area, alignment and volume fraction of graphene and carbon nanotubes. Following this step, the position data of each particle is imported into the finite element program. In the FE models, HA is represented by a solid block of material, graphene sheets are modeled as planar rectangular particles embedded in the HA matrix and carbon nanotubes are represented by beam elements of a tubular cross-section. In order to determine the elastic modulus of the material along each direction, loading is applied along the three principal axes of the composite. The results indicate that nanocomposites reinforced by graphene increase the elastic modulus of HA several times more than that of carbon nanotubes at the same volume fraction. Assuming perfect bonding, the results predict a linear relationship between the elastic modulus and volume fraction, whereas particle aspect ratio and area have negligible effect on the composite’s modulus. When most of the graphene nanosheets are aligned towards a given direction the composite behaves like a transversely isotropic material and has a much greater stiffness in the plane of the graphene sheets than in the normal direction. In summary, this study provides additional insight into the effect of nanofillers on mechanical properties of HA-nanocomposites as bone implant materials and demonstrates an effective computational tool for evaluating their properties.
4:15 PM JJ8.9
Layer by Layer Assembled Nanostructures for Neural Interface. Huanan Zhang1 and Nicholas A. Kotov1,2,3; 1Chemical Engineering, University of Michigan, Ann Arbor, Michigan; 2Biomedical Engineering, University of Michigan, Ann Arbor, Michigan; 3Material Science and Engineering, University of Michigan, Ann Arbor, Michigan.
Electrical stimulation is a technique for diagnosis and therapy of many neurological diseases. We often utilize a neural prosthetic device (NPD) to deliver the electrical stimulation.[1, 2] The common clinical NPDs have several problems, such as low charge storage capacity, large size , chronic inflammatory response, and mismatch of mechanical properties with brain tissue. In order to improve the current design of NPDs, we need to design nanocomposite materials that exhibit superior properties and functionalities than current materials. Layer by layer (LBL) assembled gold nanoparticle (Au NP) multilayers are composite materials made from gold nanoparticles and polyelectrolyte polymers. Nanocomposite materials made from combining nanoparticles and LBL assembly technique have superior electrochemical properties, long term chemical stability,and tunable mechanical properties. These remarkable properties are very attractive for chronically implanted NPDs. The versatility of the LBL assembly enables us to easily modify current clinical NPDs to create multifunctional devices. As a coating material, LBL assembled Au NP nanocomposite can potentially improve the performance of implantable NPD by increasing its charge injection capacity and decrease electrical impedance. In addition, we could also create freestanding LBL assembled Au NP film to directly fabricate flexible implantable electrodes. Reference: 1. Merrill, D.R., M. Bikson, and J.G. Jefferys, Electrical stimulation of excitable tissue: design of efficacious and safe protocols. J Neurosci Methods, 2005. 141(2): p. 171-98. 2. Benabid, A.L., Deep brain stimulation for Parkinson's disease. Curr Opin Neurobiol, 2003. 13(6): p. 696-706. 3. Kotov, N.A., et al., Nanomaterials for Neural Interfaces. Advanced Materials, 2009. 21(40): p. 3970-4004. 4. Cogan, S.F., Neural stimulation and recording electrodes. Annu Rev Biomed Eng, 2008. 10: p. 275-309.
4:30 PM JJ8.10
Biomineralization of Nano-rough Titanium Surface Functionalized with Statherin-inspired Recombinant Biopolymers. Yuping Li1, Jose C. Rodriguez-Cabello2 and Conrado Aparicio1; 1Restorative Sciences, Minnesota Dental Research Center for Biomaterials and Biomechanics,University of Minnesota, School of Dentistry, Minneapolis, Minnesota; 2University of Valladolid, Valladolid, Spain.
Modification of Ti surfaces to mimic unique chemical and topographical features of bone plays an important role on the clinical success of dental and orthopedic implants. In that sense, bioactive implant surfaces with ability to regenerate a calcium-phosphate layer either biomimetically in vitro or naturally in vivo are preferential candidates to speed-up implant osseointegration. In this work we aimed to a) covalently-functionalize nano-rough Ti surface with the synthetic elastin-like recombinant biopolymers (pHAP), and b) biomineralize the functionalized surface with biominerals using a biomimetic route. We hypothesize that pHAP has potential to induce controlled hydroxyapatite formation. This is because the polymer includes a peptide sequence derived from a saliva protein, statherin, with strong affinity for hydroxyapatite on tooth enamel. Ti discs were prepared as follows: ultrasonic solvent-cleaning (Ti), etched with 5M-NaOH solution (eTi); silanizated with 3-(chloropropyl)-triethoxysilane; and final immobilized with the recombinant biopolymers in aqueous solution (Ti-pHAP). Control surfaces: Ti, eTi, and eTi immobilized with biopolymers with same-residues but no bioactivity (Ti-pREF). The mineralization was conducted in osteogenic media (OM) supplemented with 25mM of CaCl2 at 37oC. Characterization before and after biomineralization was carried out by Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFT), X-Ray Photoelectron Spectroscopy (XPS), Field-Emission Scanning Electron Microscopy (FE-SEM), and Transmission Electron Microscopy with Selected Area Electron Diffraction (TEM-SAED). FE-SEM revealed that eTi, Ti-pREF, and Ti-pHAP surfaces before mineralization had nano-rough topography. The attachment of pHAP and pREF on Ti was confirmed: amide bands from the peptides were detected by DRIFT; and strong N1s peaks were observed by XPS. Isolated mineral nuclei with 10-40 nm in diameter on Ti-pHAP surfaces were observed after 3 days of mineralization. A homogeneous layer of minerals covered on nano-rough Ti-pHAP surface was achieved after 7 days of mineralization. The minerals were carbonated calcium phosphates as shown in representative DRIFT spectrum: broad phosphate band at 1062 cm-1 and carbonate-?2 at 871 cm-1. TEM-SAED analysis indicated the presence of minerals were an amorphous phase. On control experiments, we found that plain Ti surfaces did not induce mineral formation, while eTi and Ti-pREF surfaces formed a layer of minerals overgrowing on top of the surface with disappearance of the nano-rough topography. This suggests that the biopolymers containing the statherin-derived peptide can control mineral formation. We have proved to obtain titanium surfaces combining topographical and bioactive chemical features through a biomimetic route. This unique nano-rough implant-surface covered with a calcium-phosphate layer will be further tested in vitro for promising synergic stimuli for osteoblasts differentiation.
SESSION JJ9: Poster Session: Hybrid Biological Materials III
Thursday Evening, April 28, 2011
Salons 7-9 (Marriott)
Development of Deformable Ca-Si-Zn Bone Restoration Complexes with Collagen. Ren-Jei Chung, Huan-Yu Wu and Kai-Shiang Chen; Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei, Taiwan.
This study aimed to develop deformable Ca-Si-Zn bone restoration complex, and focused on these analyses including mechanical properties, crystalline phases, surface properties, thermal analysis, ionic releasing and in vitro tests. This bone restoration complex is a type of “inorganic-organic” composite. The inorganic composition is Zn0.075Ca2.925SiO5, which was calcined at 1400oC by tricalcium silicate (Ca3SiO5) doped with ZnO. The organic composition is commercial deimmunized collagen. Tricalcium silicate(C3S) is one of the main composition of Portland cement. It provides the property of self-setting, after completely setting, deformation is unable. According to the previous studies, few ZnO doping in C3S could inhibit the formation of free CaO during preparation. ZnO not only stabilizes the structure of C3S but also provides antibiotic ability. In addition, collagen could increase plasticity of the composites and absorb tissue fluid and blood during wound healing. Zn0.075Ca2.925SiO5 was prepared through a solid-state reaction and then mixed with collagen and NaH2PO4-2H2O solution as buffer to obtain the complex. This bone restoration complex had a long working time, high plasticity and high compressive strength. So that, the bone restoration complexes are able to be shaped in advance before application and served in tissue engineering as scaffolds. For in vitro study, we used the mouse fibroblast cell line (L929) and human osteoblast cell line (MG63). Investigations including cell attachment observation, PI staining, cell toxicity test (MTT), DNA content evaluation, alkaline phosphatase test (ALP) and flowcytometry detection were carried out to evaluate the biocompatibility of the materials and soaking mediums. The research results showed that the cell numbers increased with cultivation time through OM observation and PI staining. According to cell toxicity test and aptosis analysis using flowcytometry, soaking mediums of different amounts of materials didn’t affect cell growth harmfully. Through alkaline phosphatase test, results showed that the soaking mediums would stimulate the osteoblast and trigger the biomineralization processes leading to higher secretion of ALP during cultivation. In conclusion, these in vitro studies insured the biocompatibility of our hybrid complex for future applications.
Hybridization State Detection of DNA Functionalized Gold Nanoparticles Using Hyperspectral Imaging. Richard C. Murdock1,2, Omar A. Khan2,3, Nancy Kelley-Loughnane1, Thomas J. Lamkin1 and Saber M. Hussain2; 1711 HPW/RHPC, Wright Patterson Air Force Base, Dayton, Ohio; 2711 HPW/RHPB, Wright Patterson Air Force Base, Dayton, Ohio; 3Neuroscience and Behavioral Biology Department, Emory University, Atlanta, Georgia.
The objective of this investigation was to further explore the ability noble metal nanoparticles to produce unique local surface plasmon resonance (LSPR) responses, in order to detect the binding states of particles for sensor applications. Hyperspectral imaging has the unique ability to capture spectral data at multiple wavelengths in each pixel image. With this imaging technique, one is able to distinguish, with certainty, different nanomaterials as well as discriminate between nanomaterials and biological materials. In this study, 4 nm and 13 nm gold nanoparticles (Au NPs) were synthesized, functionalized with complimentary oligonucleotides, and hybridized to form large networks of similar particles. Reflected spectra were collected from each sample (unfunctionalized, functionalized, and hybridized) and evaluated. The spectra showed unique peaks for both sizes of Au NPs, as well as exhibited narrowing and an increase in intensity of the spectra as the NPs were functionalized and subsequently hybridized. This change in the reflected spectrum is different from normal aggregation effects where the absorption peak is red-shifted and broadens, while the reflected spectrum usually is broadened as well. Rather, the DNA linkage of the Au NPs intensifies the LSPR and appears to be dependent on the interparticle distance through oligonucleotide length, which is also explored through the incorporation of a poly-A spacer into the sequence. Also, the hybridized Au NPs were exposed to cells with no adverse affects and still retained their unique spectral signatures. With the ability to distinguish between nearly individual NP binding states, hyperspectral imaging can provide a method of tracking the intracellular actions of nanomaterials as well as have potential biosensing applications.
A Soluble Strontium Carbonate Implant Coating for Local and Targeted Cell Stimulation. Johan Forsgren, Andreas Hoess, Marjam Ott, Maria Strømme and Hakan Engqvist; Engineering Sciences, Uppsala University, Uppsala, Sweden.
A concept for local delivery of strontium ions from prosthetic implants has been developed in order to stimulate and improve the bone formation after implantation. Strontium acts by increasing the differentiation and bone forming activity of osteoblasts while also reducing the bone-resorbing activity of osteoclasts. This is believed to have a positive effect on the bone healing process, reduce convalesce time after surgery and improve implant fixation. In the present work, a coating of strontium carbonate was deposited on titanium substrates via a biomimetic mineralization process where chemically treated titanium was immersed in a strontium acetate solution. Due to electrostatic interactions, ions in the solution accumulate and bind to the surface and eventually form a nanocrystalline film of SrCO3 on the substrate. The final coating has a nanoporous morphology and is soluble in aqueous solutions. The dissolution rate is slow and easily adjusted by simple thermal treatment of the coating, which enables a controlled and sustained effect from the strontium. When immersed in simulated body fluid, the SrCO3 coating interacts with ions in the solution and bone like apatite is formed on the surface. In vitro cell studies with human osteosarcoma osteoblast-like cells demonstrated that the SrCO3 coating promoted cell adhesion and proliferation. In this study, cells were cultured on heat-treated SrCO3 coatings with relatively low solubility for 10 days. No signs of cytotoxicity were observed and the cell viability was equal to that of cells seeded on conventional tissue culture plastics. In addition, alkaline phosphatase (ALP) activity in the cells was increased extensively during the first three days of cultivation on the SrCO3 coated surfaces. At this point the ALP levels in these cells were twice as high as in the control group. The elevation in ALP activity correlated precisely with the amount of strontium released from the surface. The release of strontium ions declined after a couple of days, which resulted in a decreased ALP activity. However, the ALP levels were at all times higher in the cells seeded on SrCO3. Scanning electron microscopy studies confirmed that the cells were thriving on the SrCO3 coating; the cells were stretching out over the porous surface and exhibited well-developed filopodia. The findings from this study are encouraging. Strontium carbonate has to our knowledge never been considered for biomedical applications before. Here it was formed via a biomimetic mineralization method on titanium, which allowed the formation of a nanoporous coating. The coating was evaluated in cell culture experiments where the release of strontium ions increased the activity of osteoblast-like cells. The positive effect on the cells lasted for at least 3 days. By increasing the solubility of the coating through altering the thermal treatment, it is proposed that an even more sustained effect can be achieved.
T Cell-specific Delivery of HDAC Inhibitor Targeting Latent HIV Using Antibody-conjugated PLGA Nanoparticles. Jangwook Lee, Sunmi Choi, Sang Kyung Lee and Kuen Yong Lee; Hanyang University, Seoul, Korea, Republic of.
Drug delivery systems based on nanoparticles, with consideration of excellent pharmaco-kinetics and dynamics as well as targeting effects, have found potential for many biomedical applications. We have developed a novel delivery system using PLGA-based nanoparticles for delivering histone deacetylase inhibitor (HDACi) to human T cells, the major reservoirs for latent HIV. Latent HIV can be activated through inhibition of histone deacetylase (HDAC) using HDACi. However, pleiotropic effects of HDACi include reactivation of other latent viruses due to lack of selectivity, leading to systemic toxicity. In order to restrict the effects of HDACi to human T cells we conjugated a single chain fragment antibody targeting the T cell-specific CD7 molecule (scFvCD7) to the surface of PLGA nanoparticles encasing the HDACi, valproic acid (VPA). These nanoparticles displayed a sustained VPA release over 3 days and a significant association with human T cells with minimal cytotoxicity. Importantly, VPA-loaded scFvCD7-conjugated PLGA nanoparticles successfully activated latent HIV in human T cells in vitro. This approach suggests a new method geared to eradicate HIV through the elimination of HIV reservoirs in supportive HIV therapy.
Tree-dimension Imaging of Fibroblast Cells by Scanning Transmission Electron Microscopy with Various Tilting Angles. Yun-Wen You1, Hsun-Yun Chang1, Wei-Lun Kao2, Guo-Ji Yen2, Chi-Jen Chang2 and Jing-Jong Shyue1,2; 1Academia Sinica, Taipei, Taiwan; 2National Taiwan University, Taipei, Taiwan.
Three-dimension (3D) structure information is important in biological research. For the well studied internal structure of organism, the human embryonic kidney cell-line 293T (HEK293T) fibroblast cell is selected in this work for developing 3D imaging techniques by scanning transmission electron microscopy (STEM) and electron tomography (ET). The STEM used here is based on a scanning electron microscope (SEM) operated at 20 kV with home-made specimen holder and a multi-angle solid-state detector behind the sample. Because of the low acceleration voltage, stronger electron-atom scattering yields stronger contrast in the resulting image. While the higher scattering probability leads to diffused background in traditional TEM image and diminish the contrast, the incoherent STEM imaging allowed the observation of thick specimens. In this work, 2D STEM images of 950 nm thick of cell slices with projection angles between ±25° are collected and the 3D volume structure was reconstructed using ART model with the TomoJ plugin in ImageJ. Although the tilting angle is restrained and limits the image resolution, slicing the reconstructed volume generated depth profile of the thick specimen and cross-sectional structure can also be obtained using ImageJ. Comparing with existing systems capable of doing ET, the method presented here yields better contrast on thick biological specimens and SEM-based instrument costs less than a TEM. Using this technique, cellular uptake of self-assembled monolayer modified Au nanoparticles with different surface charge is examined and the final position of these nanoparticles inside the cell is imaged.
Carbon Highways in High Surface Area Conducting Paper Membranes for Efficient DNA Separation. Aamir Razaq1, Maria Stromme1, Leif Nyholm2 and Albert Mihranyan1; 1Engineering Science, Uppsala University, Uppsala, Sweden; 2Material Chemistry, Uppsala University, Uppsala, Sweden.
The ability to separate and purify biomolecules, e.g. DNA, from complex biological samples at larger scale is always in demand for biotechnology applications. The interaction of DNA with polypyrrole (PPy) films coated on different substrates has been investigated, and irreversible adsorption of DNA onto PPy has commonly been observed [Misoska et al., Synth. Metals 123 (2001) 279; Pande et al., Biomater 19 (1998) 1657]. It could be hypothesized that this was due to two different phenomena: i) slow diffusion of DNA during the reduction as opposed to migration in an electric field during PPy oxidation; and ii) the PPy films becoming non-conductive during reduction which hinders the electron transport needed for an efficient release of the trapped DNA molecules. To verify these hypotheses, we chemically polymerized a thin ~30-50 nm PPy layer on a high surface area Cladophora-cellulose substrate [Mihranyan et al, J. Phys. Chem. B 112 (2008) 12249]. The resulting composite membrane had the appearance of a black flexible paper sheets with good mechanical strength, large surface area (80 m2/g) and high electron conductivity (~2 S/cm). The latter material was previously utilized for efficient (and fully reversible) extraction of organic and inorganic anions [Razaq et al, J. Phys. Chem. B 113 (2009) 426; Gelin et al, Electrochimica Acta 54 (2009) 3394]. The oxidation of these PPy conducting membranes resulted in the inclusion of [dT]6 hexamer as a counter ion into the film even in the presence of other competitor anions [Rubino et, al J. Phys. Chem. B 114 (2010) 13644]. The goal of the present study was to study the possibilities of obtaining a complete release of [dT]6 hexamers. By exploiting 30-50 nm thick PPy layers, firstly we observed a partially reversible release of [dT]6 hexamers accounting for ~ 30% of the DNA absorbed. In order to increase the conductivity of the membrane in the reduced state, we incorporated chopped carbon fibers (CCFs) into the composite at a 1:1 wt. ratio during the synthesis. A 20% increase in the electrical conductivity of the membrane with CCFs was observed which resulted in a release efficiency of ~ 50%. It was concluded that CCFs acted as conducting highways which facilitated the electron transport in the reduced state and made it possible to access a larger portion of electroactive PPy sites during the reduction. The results, thus, suggest that improved conductivity in the reduced state significantly improves the efficiency of the DNA release. The effects of an application of an external electric field as a driving force for the DNA release will also be discussed as a way of developing a new technology platform for novel electrochemically-controlled separation techniques in biotechnology, nanoanalytical systems, paper-based diagnostic devices, and immunosensors.
Culturing Cells in a 3D Ordered Cellular Solid. Keng-hui Lin1,5, Jing-ying Lin5, Wan-jung Lin2, Wei-jung Lin2, Wei-chun Hong1, Hsiang-haw Ning2, Stephanie H. Nowotarski3, Susana M. Gouveia4 and Ines Cristo6; 1Physics, Academia Sinica, Taipei, Taiwan; 2Physics, National Taiwan University, Taipei, Taiwan; 3Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; 4Gulbenkian de Ciencia, Oeiras, Portugal; 5Biophysics, National Central University, Chungli, Taiwan; 6Science and Technology Austria, Vienna, Austria.
We demonstrated a high throughput method to fabricate gelatin-based ordered cellular solids with tunable pore size and solid fraction. The process involved generating high air fraction and monodisperse liquid foam by a cross-flow microfluidic device. The monodisperse liquid foam was further processed into open-cell solid foam. The solid foams were used as tissue engineering scaffolds where cells were cultured inside. Three different cell types were chosen and they showed physiological morphology and functions. Epithelial cells formed cyst-like structures and were polarized inside pores. Myoblasts formed tubular structure and fused into myotubes. Fibroblasts exhibited wide varieties of morphologies depending on their locations in scaffolds. This ordered cellular solids open doors to study the effect of pore size on cells and the mechanical properties of microscopic foam structures.
Behaviour of Two Titanium Alloys in Simulated Body Fluid. Julia Mirza Rosca1, Domingo Herrera1, Agustin Santana1, Agurtzane Martinez3, Jose-Antonio Garcia3 and David Gonzalez2; 1University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain; 2Technological Institute of Canarias, Las Palmas de Gran Canaria, Spain; 3Advanced Surface Engineering Center AIN, Pamplona, Spain.
Titanium possesses an excellent corrosion resistance in biological environments because the titanium dioxide formed on its surface is extremely stable. When aluminium and vanadium are added to titanium in small quantities, the alloy achieves considerably higher tensile properties than of pure titanium and this alloy is used in high stress-bearing situations. But these metals may also influence the chemostatic mechanisms that are involved in the attraction of biocells. V presence can be associated with potential cytotoxic effects and adverse tissue reactions. The alloys with aluminium and iron or with aluminium and niobium occur to be more suitable for implant applications: it possesses similar corrosion resistance and mechanical properties to those of titanium-aluminium-vanadium alloy; moreover, these alloys have no toxicity. In this paper, pure Ti, Ti-6Al-7Nb and Ti-6Al-4Fe with a nanostructured surface were studied. Data about mechanical behaviour are presented. The mechanical behaviour was determined using optical metallography, tensile strength and Vickers microhardness. For the electrochemical measurements a conventional three-electrode cell with a Pt grid as counter electrode and saturated calomel electrod (SCE) as reference electrode was used. AC impedance data were obtained at open circuit potential using a PAR 263 A potentiostat connected with a PAR 5210 lock-in amplifier. The ESEM and EDAX observations were carried out with an environmental scanning electron microscope Fei XL30 ESEM with LaB6-cathode attached with an energy-dispersive electron probe X-ray analyzer (EDAX Sapphire). After 3 days of immersion in simulated body fluid the nucleation of the bone growth was observed on the implant surface. It resulted that the tested oxide films presented passivation tendency and a very good stability and no form of local corrosion was detected. The mechanical data confirm the presence of an outer porous passive layer and an inner compact and protective passive layer. EIS confirms the mechanical results. The thicknesses of these layers were measured. SEM photographs of the surface and EDX profiles for the samples illustrate the appearance of a microporous layer made up of an alkaline titanate hydrogel. The apatite-forming ability of the metal is attributed to the amorphous sodium titanate that is formed on the metal during the surface treatment. The results emphasised that the surface treatment increases the passive layer adhesion to the metal surface and improves the biocompatibility of the biomedical devices inducing the bone growth on the implant surface.
Development of Novel Nanoparticle/Protein Conjugates for Applications in Nanomedicine. Valeria Marangoni, Ana I. Camargo, Leila M. Beltramini and Valtencir Zucolotto; University of Sao Paulo, Sao Carlos, Brazil.
Nanobiocomposites have presented a high potential for biomedical applications due to their ability of combining the recognition properties of biomaterials with the unique electronic, photonic, and catalytic features of nanoparticles. In this study we describe a new strategy for conjugating gold nanoparticles and the centrin protein (BeCen1), a member of the calcium-binding EF-hand protein superfamily, usually located at microtubule-organizing centers. BeCen1 also exhibits the ability to form filaments via a nucleation-controlled polymerization. The nanoparticles are formed in the presence of the proteins using diluted acid formic as reducing agent. The protein-nanoparticle binding was confirmed by size exclusion chromatography and Transmission Electron Microscopy (TEM) images. Circular dichroism analyses (CD) revealed that the protein maintained its secondary structure upon conjugation with the nanoparticles. Furthermore, we observed that AuNP/BeCen1 conjugated kept the polymerization ability. The BeCen/AuNPs nanoconjugates exhibit high potential for biotechnological applications including biosensors and catalysis.
Direct Oligonucleotide Quantification by Sequence-specific Gold Nanoparticle Assay. Prayanka Rajendran, Janos Voros and Marcy Zenobi-Wong; Laboratory of Biosensors and Bioelectronics, Institut f. Biomedizinische Technik, ETH Zurich, Zurich, Switzerland.
A sensitive and sequence-based direct detection of oligonucleotides is essential for quantifying the amount of DNA/RNA in single cell. In general, direct oligonucleotide quantification assays are attractive detection systems since they offer the potential to monitor single cell RNA levels without the usage of amplification steps. This method reduces the risk of contamination and eliminates time-consuming intermediate steps. Based on these advantages a spectral based oligonucleotide detection system with thiol-functionalized oligonucleotide-modified 50 nm gold probes is studied. In this system, oligonucleotide-modified gold nanoparticles are dispersed into the microwells and the target analyte is spotted on the coverslip. The coverslip is placed on the microarray structures which contains the gold nanoparticles. The gold nanoparticles then form aggregated polymeric networks via hybridization events with complementary target oligonucleotides that were spotted onto a coverslip. The hybridization events induce aggregation which leads to concomitant change in the extinction spectra. The distinct light scattering properties of the gold nanoparticles can be utilized for the detection and quantification of DNA. The binding of the analyte (target DNA molecule) to the gold nanoparticles during the assay brings the plasmonic nanoparticles in close vicinity to each other. Due to this proximal effect they become optically coupled, creating a more enhanced field and giving a strong resonance depending on the coupling strength or interparticle distance. As a result there is a second extinction peak towards the red region. The assay made in a microwell array structures makes it possible to detect DNA in naturally occurring quantities. The 50 nm gold colloids (7 × 1011 colloids ml-1) (GC50, British Biocell, UK) were tagged with thiolated-DNA (Probe1 and Probe2) (Eurogentec, Belgium). The glass substrate with microwell structures is rendered hydrophilic property and then filled with the mixture of Probe 1 and 2. This is then covered with a coverslip on which the target analyte is dried. The spectral measurements were conducted by a custom built microscope (Axiovert 200, Zeiss, Germany) with a spectrometer (SpectraPro 2150, PIXIS 400, Princeton Instruments, US). The online data analysis and the control of the spectrometer were performed by a custom made program. The microwell array system filled with all components for a biochemical detection assay is advantageous compared to the earlier methods firstly because the dimensions of the microwells define the total volume of the assay so that sample dilution and the reagent consumption is minimized and secondly all the assay components are preloaded to the microwells which makes fluidic components unnecessary. The future directions will be to quantify the RNA from single cells without the need for amplification.
Electroactive Peptides via Phage Display for Biosensor Applications. Ya-Wen Yeh1, Chih-Wei Liao2, Seonhoo Kim2, David Norton2 and Laurie Gower2; 1Department of Biomedical Engineering, University of Florida, Gainesville, Florida; 2Department of Materials Science and Engineering, University of Florida, Gainesville, Florida.
For biosensor devices, functionalizing the surface with covalent linkers is usually employed. However, it is difficult to avoid the loss of activity following the bioreceptor-analyte binding event, which limits the lifetime of the device. The goal of our group is to use phage display to biopan for inorganic binding peptides that are reversible upon applying of an electric field. This can provide dynamic functionalization of surfaces, with applications such as self cleaning devices. For example, when the bioreceptor becomes clogged, the peptides may be released by triggering an electric field to generate a non-binding state. A fresh surface of bioreceptors can then be applied via a flow-through setup. Our group has been panning for peptides that bind strongly to indium zinc oxide (IZO), a transparent semiconducting oxide which makes it an attractive electrode for biochemical sensors. An electro-releasing device is being used to collect the strong binding peptides that are subsequently released. In an alternative method, because a reversible peptide may not necessarily be a strong binding peptide, our group has also developed a novel phage display biopanning protocol with an electro-elution process instead of the regular chemical elution. Our recent works has shown that M13 phage can be electroeluted and the eluted phage that were collected have shown good binding to indium zinc oxide surface. On-going work examines and compares these two approaches for the goal for developing self-cleaning devices and other potential applications of electroactive peptides.
Scattering under Shear: Alignment of a Disordered Bicontinuous Mesophase. Annela Seddon1 and Adam Squires2; 1Physics, University of Bristol, Bristol, United Kingdom; 2Chemistry, University of Reading, Reading, United Kingdom.
In this work we present evidence that biological lipids can form types of disordered bicontinuous nanostructured phases, known as sponge phases, which until now have been more commonly observed in surfactant / brine systems. By using a combination of small angle x-ray scattering under shear and rheology, we are able to identify these phases and obtain information upon the dynamics of their formation. Understanding the self-assembly behaviour of biological amphiphiles, their rheological properties and their structural response to shear can provide a foundation for the design of nanostructured materials for biological applications. Biological amphiphiles such as lipids can adopt a wide range of mesophases upon self-assembly in water. These mesophases can be classified in terms of their desire for surface curvature and range from flat phases such as the fluid lamellar phase to highly curved phases such as the inverse hexagonal phase. One such phase which is of interest both as a mimic for cell membranes and as a template for nanostructured materials is the L3, or sponge phase. This consists of a series of interconnected lipid bilayers forming a random bicontinuous network of tubules. The inherent long range disorder of this phase has meant that it is more challenging to study with techniques such as small angle x-ray scattering (SAXS) than the more ordered bicontinuous cubic phases. Previous work on surfactants in brine which form sponge phases has shown that the application of shear to the sponge phase leads to the formation of highly orientated lamellar phases. In this work we show that the application of shear to a sponge phase formed by lipids causes the mesophase to form a highly orientated lamellar phase which returns to the sponge phase on cessation of shear. By combining SAXS under shear with rheological measurements, it is possible to identify sponge phase regions for a range of lipid systems under biologically relevant conditions.
Enhanced Detection of Cardiovascular Biomarker Proteins: A Detailed Study of Nanoconfinement in Nanoporous Membranes. Savindra Brandigampala2, Paige Feikert1, Krishna Vattipalli1,2 and Shalini Prasad1,2; 1Bioengineering Program, Wichita State University, Wichita, Kansas; 2Electrical Engineering and Computer Science, Wichita State University, Wichita, Kansas.
The goal of this work is to understand the role of nano confinement in designing biosensors. We have been investigating silicon based micro devices incorporated with nanoporous membranes in designing sensors. We have observed that nanoporous membranes enable nanoscale size based confinement of biomolecules such as proteins onto micro platforms. This in turn enhances the concentration of the biomolecules which in turn enhances the sensitivity in detecting biomolecules. It is critical that ultralow detection of biomolecules be achieved as they have significant impact in designing diagnostics platforms for early disease diagnosis. Commercially available nano-porous membranes made out of anodized alumina and polycarbonate are evaluated for their role in nano-confinement and enhancing sensitivity of detection. In this biosensor configuration sandwich assay, an electrical double layer is formed between a test protein (C-reactive protein) and the gold surface underneath the porous membrane. Using electrical impedance spectroscopy, the capacitance changes in the electrical double layer, translating to the sensitivity and the linear dose response over a large dynamic range will be analyzed for each of the physical characteristic of the porous membrane - pore densities, height of the pore and diameter of pore.
Zinc Oxide Nanorod Films for Electrochemical Urea Biosensor. Netza Palomera2, Marcia Balaguera3, Maharaj S. Tomar4, Jaime E. Ramírez-Vick1, Sunil Arya5 and Surinder P. Singh1; 1Engineering Science and Materials, University of Puerto Rico Mayaguez, Mayaguez; 2Mechanical Engineer, University of Puerto Rico, Mayaguez; 3Chemistry, University of Puerto Rico, Mayaguez; 4Physics, University of Puerto Rico, Mayaguez; 5Electrical Engineer, University of South Florida, Tampa, Florida.
Metal oxide nanostructures have shown significant promise for biosensors, gas sensors, photocatalyst and other biomedical applications in recent past. Among these, Zinc oxide (ZnO) nanostructures, exhibiting interesting properties such as high catalytic activity, biocompatibility, high iso-electric point, large surface to volume ratio, make them good candidate for biosensing applications. Here we report the synthesis of ZnO nanorods (ZnONR) on ITO film in aqueous phase and its application in Urea biosensor fabrication. ZnONR have been synthesized by two step method, first seed growth of ZnO by sputtering on ITO films followed by decomposition of zinc nitrate hexahydrate / hexamethylenetetramine (HMT) in aqueous phase. Using high isoelectric point of ZnO, Urs/ZnONR/ITO bioelectrode has been fabricated by physical binding of Urease (Urs) enzyme onto ZnONRs. XRD, FE-SEM, and cyclic voltammetry (CV) has been used to characterize ZnONR and Urs/ZnONR/ITO bioelectrode. The FE-SEM measurements confirm the formation of ZnO nanorods. CV measurements on Urs/ZnONR/ITO biolectrode reveal linearity of 10 - 125 mg/dL with high sensitivity of 10 µAdL/mg (1.66 µA/mM) and relatively low Michaelis-Menten constant (Km) of 2.21 mM for urea. The results indicate the potential of ZnO NR films for fabrication of commercial biosensors.
Transferred to JJ10.7
Cytotoxicity of Heterobifunctional Crosslinkers used for Conjugation and Functionalization of Biomaterials. Nihar Shah1, Jill Covert1, Rishabh Jain2, Ankit Agarwal2, Nicholas L. Abbott2 and Christopher J. Murphy1; 1Vet Med Surgical and Radiological Sciences, University of California, Davis, California; 2Chemical and Biological Engineering, University of Wisconsin, Madison, Wisconsin.
Heterobifunctional crosslinkers are ubiquitously used in multiple life science fields for a variety of applications, including: biosensors, protein- and cell-surface interaction analysis, protein-protein and DNA/RNA-protein conjugation, immunogen preparation, and biomaterials surface modification. Specifically for tissue- and cell based applications, crosslinkers are used to functionalize material surfaces, devices, and scaffolds, with bioactive molecules that can influence cell behavior. Despite the wide-ranging possibilities for cellular applications, few published reports on the comparative cytotoxicity of crosslinkers exist. Furthermore, studies have been limited to one or two commonly used crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and often within too narrow a concentration range to evaluate their reaction efficiency and relative cytotoxicity. In the development of engineered therapeutic delivery systems, it is critical to assess the potential cellular toxicity of these essential crosslinking reagents. We have determined the relative cytotoxicity of a wide panel of heterobifunctional crosslinkers selected based on multiple criteria including popularity for existing applications, their reactive moieties and functional linking groups. We restricted our selection to water-soluble crosslinkers that could be delivered to cells and tissues in buffered solutions. Specifically, we tested two main families of crosslinkers, commercially available synthetic heterobifunctional crosslinkers and self-reactive catecholamines that were first identified in mussel adhesives. We tested these chemicals immediately after preparation as well as after hydrolysis (crosslinkers) or aggregation (catecholamines). Concentrations spanned from 1 nM up to 10 mM. Cells (primary human dermal microvasculature endothelial cells (HMVECs) and immortalized human keratinocyte cells (HaCaT) were exposed for 1, 4, or 24 hours. Significant findings include: differential responses based on concentration, crosslinker state, treatment time, and cell type. For most crosslinkers tested, cells remain viable from 1 nM up to 100 µM. This large concentration window will provide flexibility for conjugation reaction optimization within cellular viability limits. In some cases, differences were observed between the fresh and modified forms which resulted in either increased (e.g. aggregated catecholamines) or decreased (e.g. hydrolyzed crosslinkers) toxicity. Further segregation of toxicity was delineated by temporal assays, wherein longer treatment durations shifted the onset of toxicity towards lower concentrations. Additionally, for some of the chemicals tested, primary cells (HMVECs) showed greater sensitivity at lower concentrations than immortalized HaCaT cells. The data provided informs the rational selection of crosslinking reagents for cell and tissue applications.
Nucleation Kinetics of Calcium Phosphate on Collagen. Jinhui Tao1, Jim De Yoreo1 and George Nancollas2; 1Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California; 2Department of Chemistry, University at Buffalo, Buffalo, New York.
Deciphering the thermodynamic and structural controls underlying nucleation of calcium phosphate on collagen is critical for understanding the organic-inorganic interface of biominerals such as bone and dentine. In bone, apatite crystals are arranged with their  axis parallel to the long axis of collagen and the nucleation of apatite (AP) is believed to be controlled by the collagen fibers. However, the influence of collagen on the thermodynamics and kinetics of the initial stages of apatite nucleation is still unclear. To determine the interfacial energy of calcium phosphate on collagen and the underlying mechanisms of nucleation, in-situ atomic force microscopy (AFM) was used to monitor the nucleation process of calcium phosphate in the presence of collagen. The experiment was divided into two steps. In the first step collagen was physically adsorbed onto mica surfaces in the presence of potassium ions over a range of pH values. The organization of collagen evolved from random fibers to co-aligned fibers to ordered bundles to D-banded micro-ribbons as the K+ ion concentration and pH were varied. In the second step, nucleation experiments were performed at pH 7.40 for a series of supersaturations (S) between 3.08 and 3.47 with respect to AP and the number density of calcium phosphate nuclei as a function of time was measured. High-resolution TEM was used to determine the initial phase of the calcium phosphate nuclei. We found that the initially formed phase was highly dependent on S, with AP forming directly for S = 3.31 and amorphous calcium phosphate (ACP) forming first for S = 3.36 before transforming to octacalcium phosphate (OCP) and then AP. Using classical nucleation theory to analyze the data, the interfacial energies of apatite and ACP were estimated to be 91±1 and 54±0.4 mJ/m2, respectively. With these energies, at the smallest S (S=3.36) for which ACP first forms, the free energy barrier (Gc) for ACP formation should be about 200 times that for AP formation and equal to over 1800kT. Consequently, ACP should not form under these conditions. The failure of the classical expressions for both the barrier and the nucleation rate may be a consequence of the presence of pre-nucleation clusters reported elsewhere. Assuming that these clusters have excess free energy relative to the solution, if nucleation occurs through their aggregation the free energy barrier decreases to several kT. Moreover, while this thermodynamic barrier should still favor AP nucleation, its low value suggests that the unexpected appearance of ACP is a result of significant kinetic barriers to AP formation, such as those associated with the structural rearrangements required to transform the clusters into AP.
Analysis of Amelogenin Assembly at the Oil-water Interface: The Role of Hydrophilic C-terminus. Olga M. Martinez-Avila1, Shenping Wu2, Yifan Cheng2 and Stefan Habelitz1; 1Department of Preventive and Restorative Dental Sciences, School of Dentistry, UCSF, San Francisco, California; 2Department of Biochemistry & Biophysics, UCSF, San Francisco, California.
Self-assembly of amelogenin proteins plays a key role in controlling enamel biomineralization. Full length amelogenin protein self-assembles into nanospheres of 20-40 nm under certain conditions and these nanospheres are thought to regulate the growth and organization of nanofibrous apatite crystals through a mechanism still unknown. However, the amphiphilic nature of the bipolar full-length protein provide the characteristics that might allow assembly into supramolecular structures of high order similar to surfactant molecules. Our group recently reported on the use of water-in-oil metastable emulsions to induce the formation of amelogenin nanoribbons that are formed from reverse micelles at the oil-water interface. The hydrophilic C-terminus of the protein may play an essential role in the assembly. In this system, the interactions between the hydrophobic tails of human recombinant full length amelogenin rH174 occur and prevent the formation of amelogenin nanospheres. The purpose of this study was to elucidate the role of the hydrophilic C-terminus by studying the self-assembly of two recombinant MMP-20 proteolytic products, rH163 and rH146 in the water-in-oil system. The effects on protein self-assembly and crystal formation as a function of calcium and phosphate concentration, protein concentration, pH, water-oil ratio and incubation time for rH174, rH163 and rH146 were studied. The gel-matrix was analyzed using Atomic force microscopy, Transmission and Scanning electron microscopy, Energy Dispersive X-ray analysis, Dynamic Light Scattering, Circular Dichroism and helical reconstruction modeling. rH163, lacking the hydrophilic C-terminus, self assemble into regular nanospheres, which do not vary with pH, ion concentration or time, suggesting a clear role of the C-terminus in stabilization of the water-in-oil emulsion and for the generation of reverse micelles that initiates amelogenin self assembly into nanoribbons as shown for full length protein rH174. Surprisingly, rH146, self-assembly into a mixture of helical architectures. Width of rH146 helical structures is similar to untwisted amelogenin nanoribbons formed by rH174 suggesting the twisting of regular nanostrings that may be due to hydrophobic repulsions. Co-existence of few isolated regular nanostrings together with abundant helical structures of amelogenin rH146 highlight the enhanced contribution of the central domain in amelogenin assembly at the oil water-interface Funded by NIH-NIDCR R01-DE017529.
The Effects of Hydroxyapatite Nanoparticles on Breast Cancer Bone Metastasis in 3-D Scaffolds. Debra D. Lin1, Siddharth P. Pathi2, Jason R. Dorvee1, Lara A. Estroff1 and Claudia Fischbach-Teschl2; 1Materials Science & Engineering, Cornell University, Ithaca, New York; 2Biomedical Engineering, Cornell University, Ithaca, New York.
Breast cancer metastasis to bone often leads to the formation of osteolytic lesions, areas with severe mineral density loss. However, the role of cell-material interactions, between cancer cells and the bone mineral hydroxyapatite HA, Ca10(PO4)6(OH)2, in promoting metastatic and osteolytic activity remains unclear. To examine the effects of mineral properties on cancer cell activity, we have developed a method to synthesize a series of well-defined HA particles that vary in size, crystallinity, and composition. Monodispersed HA particles were obtained via a 2-step process of a wet chemical precipitation followed by hydrothermal aging of the precipitate for variable lengths of time to obtain specific particle sizes. The size, crystallinity, and composition of the HA nanoparticles were characterized with X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR). The particles were then incorporated into 3-D porous poly(lactide-co-glycolide) (PLG)-scaffolds via a gas-foaming salt-leach process. The pore morphology and particle distribution in the scaffolds were characterized via TEM and SEM. The solubility of particles in the presence of media was assessed by analyzed by Inductively Coupled Plasma (ICP). Scaffolds were then seeded with a bone-specific breast cancer cell line, MDA-MB231. Protein adsorption and cell activity were examined for the different scaffolds. Comparing HA-containing scaffolds with control scaffolds, marked increase in cell adhesion and proliferation were observed for scaffolds with HA. Secretion of interleukin-8 (IL-8), an osteolytic and pro-angiogenic cytokine, was also enhanced in HA-containing scaffolds. Scaffolds containing different HA particles exhibited enhanced protein adsorption with decreasing HA particle size and crystallinity, while IL-8 was upregulated with increasing HA particle size and crystallinity. Our results suggest that the nanoscale properties of HA in the bone matrix play a role in regulating metastatic breast cancer cell behavior. These mineralized scaffolds may provide innovative platforms for modeling the interaction of cells with the bone microenvironment.
SESSION JJ10: Biopolymers and Tissue Engineering
Chair: Carlos Martinez
Friday Morning, April 29, 2011
Salons 4-6 (Marriott)
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8:00 AM *JJ10.1
Sea Urchin Inspired Photonic Solids. Chekesha Liddell Watson, Department of Materials Science and Engineering, Cornell University, Ithaca, New York.
Anisotropic shape in biological and natural systems continues to inspire the development of structural complexity in synthetic materials. For instance, during gastrulation the shape of sea urchin embryos undergoes a dramatic systematic change that has been compared to particle models. Controlled deformation morphologies arise from the evacuation of swelling solvent and unreacted monomer through a mildly cross-linked skin layer as colloids contract during seeded polymerization. Deformation is driven by the pressure gradient across the porous shell surface. Isotropic shrinkage transitions to regimes where the inhomogeneous membrane shell flattens, curvature reversal occurs forming a depression, an invagination ensues and its tip deepens until tearing connects to a hollow or solid central core region. Here, to incrementally capture the morphology development a geometric model joining a hemisphere to a torus with varied inner and outer hemi-toroid radii is defined. The consequence of packing and transformation of colloidal building blocks from hemisphere through 'mushroom cap' shaped particles to spheres for the optical properties of photonic crystals will be presented. Self-organizaiton of such nonspherical particles into photonic solids under confinement conditions will also be discussed.
8:30 AM JJ10.2
Investigation of the Nest Cell Lining of Colletes Inaequalis. Shannon Taylor, Rebecca Belisle, Debbie Chachra and Christopher Morse; Olin College of Engineering, Needham, Massachusetts.
As we look to create new hybrid polymers from renewable resources instead of petroleum, we find inspiration in natural materials. The nest cell lining of Colletes inaequalis, a species of solitary bee native to New England, is a particularly interesting naturally-occurring polymer composite. Waterproof, antibacterial, antifungal, and resistant to degradation, it is a promising case study for a robust, bio-derived polymer. Bees of the genus Colletes create nest cells in soil, lined with a cellophane-like material produced in their enlarged Dufour’s gland. Researchers have confirmed that the nest cell lining is a polyester, composed of polymerized lactones from the Dufour’s gland [1-3]. The bees have been observed to use their brushlike bilobed tongues to spread the gland secretions on the soil walls of their nest cell cavities to form the linings [2, 3]. However, the polymerization mechanism is still unknown, and the mode of construction of the nest cells has not been fully described [2, 4]. We have begun to characterize this thermally and chemically stable polymer and consider its potential for bio-inspiration. We have shown that these cell linings are extremely robust, insoluble, and resistant to hydrolysis by methanolic hydrochloric acid, with high thermal stability (60% to 70% of the material by mass remained intact at 360°C). This work suggests that the polyester characterized by Hefetz et al.  is not the only component of the nest cell lining material. To further characterize the cell lining composition and structure, confocal and scanning electron microscopy were used to investigate the linings of Colletes inaequalis nest cells excavated from a site in Acton, MA. In the images, both a continuous matrix and distinct fibers were observed. This suggests that the linings are a composite material. The fiber diameters range from 1 µm to 10 µm. Combustion analysis of the linings indicated the presence of nitrogen, consistent with a protein content of approximately 9%. Amino acid analysis confirmed the presence of protein, with a composition similar to silks (including 10% serine). Fourier-transform spectrometry with a focal plane array was used to localize the presence of amide bonds in the fibers, suggesting the material is indeed a polyester with a silk supporting structure. Finally, staining with Sypro Red confirmed a higher concentration of protein in the fibers. As a silk-polyester hybrid, this biological material can serve as inspiration for a novel class of biomimetic composites. Further research on the microstructure and the chemical pathways that underlie this unique polymer may lead to the development of innovative processes to create plastics that are both bioderived and robust. 1. A Hefetz et al. Science 204, 415, (1979) 2. SWT Batra. J Kansas Entomol. Soc. 62:121-4 (1980) 3. JH Cane. J Chem Ecology, 7:403-410. (1981) 4. PF Torchio et al. Ann Entomol. Soc. Am. 81(4):606-625 (1988)
8:45 AM JJ10.3
Engineered Biomimetic Polymers as Tunable Agents for Controlling Rates of Crystal Growth and Tissue Mineralization. Chun-Long Chen, Jiahui Qi, Ronald N. Zuckermann and James J. DeYoreo; The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California.
In nature, proteins are known to a play significant role in the control of mineral nucleation and growth, both to create functional tissues and prevent pathological mineralization. The research described here addresses the challenge of developing a class of compounds that mimic the mineralizing functions of proteins for use in the synthesis of functional crystalline materials and as therapeutic agents. Peptoids, or poly-N-substituted glycines, are a novel class of non-natural polymers recently developed to mimic both structures and functionalities of peptides and proteins, and bridge the gap between biopolymers and bulk polymers. As with peptides, sequence-specific peptoids can be efficiently synthesized by using automated solid-phase synthesis starting from a large number of chemically diverse amine building blocks. Moreover, peptoids exhibit much higher protease stability and thermal stability than peptides or proteins. Inspired by recent research that showed low concentrations of acidic peptides and proteins can significantly accelerate calcite growth, we designed and synthesized a suite of anionic peptoids and screened them for control over calcite morphology and growth rate. Results to date demonstrate both a high degree of morphological control and extreme levels of acceleration, with both characteristics observed to be highly dependent on peptoid hydrophobicity, number of carboxylic acid groups, peptoid sequence and concentration. At high concentrations (50µM), calcite crystals formed in the presence of peptoid exhibit various unique shapes ranging from elongated spindles and twisted paddles to crosses and spheres. At low concentrations (<250nM), a number of the peptoids increased calcite growth rates by as much as a factor of 25, with the most effective being strongly amphiphilic. Based on previous research into peptide-induced acceleration, the energetic source of acceleration is likely to be a reduction in the activation barrier that controls the rate of solute addition at atomic steps on the calcite surface. Here we estimate the magnitude of the barrier and present a number of structural scenarios that could result in its reduction, including enhanced cation desolvation rates, disruption of the near-surface solution layer, and displacement of the waters believed to be strongly bound to calcite surfaces.
9:00 AM JJ10.4
Morphology of Cholesterol-incorporated Poly(ethylene glycol) Polymer Networks via Small Angle X-ray Scattering. Kristin Engberg1, Dale Waters1, Rachel Parke-Houben1, Laura Hartmann1, Michael F. Toney2 and Curtis W. Frank1; 1Chemical Engineering, Stanford University, Stanford, California; 2Stanford Synchrotron Radiation Lightsource, Menlo Park, California.
Cholesterol molecules were incorporated into photopolymerized poly(ethylene glycol) (PEG) hydrogel networks, thus altering the structure and properties of a well-characterized and commonly used biomaterial. Addition of a hydrophobic biomolecule like cholesterol could enhance the functionality of a PEG hydrogel by allowing for attachment of drug-delivering liposomes or by simply altering the properties of the material to allow for greater diffusion of nutrients or shifted mechanical strength. Cholesterol-PEG-acrylamide (Chol-PEG-AAm) molecules were synthesized and added, in molar ratios ranging from 10% to 50% Chol-PEG-AAm, to precursor solutions of PEG-diacrylate macromonomers in both chloroform and deionized water. Photopolymerization resulted in end-linked PEG-co-(PEG-chol) polymer networks that were subsequently washed and swollen in both organic and aqueous solvents. Small angle X-ray scattering (SAXS) was used as a technique to determine the molecular level morphology of the PEG-co-(PEG-chol) gels in order to relate the network structure to the macroscopic properties. Control samples of PEG networks without cholesterol exhibited a peak in the scattering curve regardless of the solvent used during network formation or in swelling afterwards. The observed peak represents an ordered spacing within the network structure and has been shown in previous work to correlate to the spacing of large, hydrophobic crosslink junctions with high functionality. Scattering curves of PEG-co-(PEG-chol) networks polymerized in organic solvent reveal a collapse of the cholesterol into the crosslink junctions upon transference of the networks from an organic to aqueous swelling solvent. This collapse led to a greater spacing between crosslink junctions with increased amounts of incorporated cholesterol. In situ polymerization of PEG-co-(PEG-chol) networks in water using the SAXS X-ray beam was performed. Scattering observed during polymerization demonstrated the presence of micelle-like structures formed by the Chol-PEG-AAm macromonomers in aqueous solution (20% to 50% molar ratios) and showed that they remained present during network formation. Preliminary results of protein diffusion through PEG-co-(PEG-chol) networks showed that the hydrogel network morphology changes caused by the cholesterol incorporation may improve diffusive properties. A solid understanding of the relationship between the nano-scale and macro-scale features of these polymer networks will allow us to design and tune the materials for specific biomedical applications.
9:15 AM JJ10.5
Design of Polyethylene Glycol-based Multidentate and Multifunctional Oligomers for Biocompatible Semiconductor and Gold Nanocrystals. Goutam Palui, Hyon Bin Na and Hedi Mattoussi; Department of Chemistry and Biochemistry, Chemical Science Laboratories, Florida State University, Tallahassee, Florida.
There has been a great interest in developing inorganic nanocrystals as novel probes and platforms for use in bio-inspired applications. They range from tunable fluorescent platforms such as semiconductor quantum dots (QDs), plasmonic probes (including Au nanoparticles, AuNPs) to magnetic nanoparticles. The development of effective and reproducible strategies for making aggregate-free (monodispersed) water-soluble luminescent QDs and AuNPs that are stable and compatible with commonly used biological coupling chemistries is highly sought, since such platforms promise great advances in understanding a variety of biological processes. High quality QDs are generally synthesized using high temperature solution reaction of organometallic precursors, and as-prepared they are dispersible in hydrophobic (organic) solvents. AuNPs are often made via citrate-reduction but require subsequent surface-functionalization to make them biocompatible. Thus, post-synthetic surface modification is required to render these nanocrystals stable in aqueous media and biologically compatible. We have developed a new set of compact multifunctional ligands that contain each an oligomer coupled to several copies of a short poly(ethylene glycol) (PEG)-appended thioctic acid (TA). Reduction of TA (e.g., in the presence of NaBH4) produces dihydrolipoic acid (DHLA) appended-oligomer ligands. Here the insertion of several PEG segments in the ligand structure promotes water solubility, while TA and DHLA groups provide multidentate anchoring onto Au and ZnS-overcoated semiconductor QDs, respectively. Dispersion of QDs and AuNPs that exhibit remarkable colloidal stability over a broad range of pHs and in the presence of added electrolytes and reducing agents have been prepared. Moreover, introducing different functional groups (such as COOH, N3 and NH2) into the oligomers opens the opportunity for effective orthogonal coupling of these platforms to target biomolecules such as proteins and peptides. We will discuss the ligand design, preparation, capping of the nanocrystals, coupling to target biomolecules and use in specific biological investigations such as sensor design.
9:30 AM JJ10.6
Ocular Drug Delivery Devices Utilizing Nanostructures for Controlled Release. Daniel A. Bernards1, Mark R. Steedman1, Paula Wynn2, On-tat Lee2, Robert B. Bhisitkul2 and Tejal A. Desai1; 1Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, California; 2Ophthalmology, University of California, San Francisco, San Francisco, California.
There is need for sustained drug release devices that are highly efficacious for delivery of protein-based therapeutics ocularly. Many contemporary delivery routes, such as injections, are hampered by patient compliance, discomfort, risk, and inconvenience, as well as limited bioavailability and frequent administration. Alternatively, a therapeutic device capable of delivering a constant rate of drug over time can circumvent these issues. One attractive platform for such a device is the use of nanoporous materials. In porous materials, constant-rate diffusion is possible when the size of a diffusing species is comparable to material pore size. A process often referred to as “single-file” diffusion, this situation can lead to zero-order constant-rate release. For macromolecule delivery this requires pores on the order of a few tens of nanometers. To this end, nanoporous biodegradable polymers have been fabricated for delivery of protein therapeutics within the eye. Utilizing biodegradable poly(caprolactone) (PCL) thin films with transport-controlling nanostructures and structural microstructures, we have fabricated devices for the delivery of protein-based therapeutics. PCL is an excellent candidate material since it biodegrades yet maintains its structural integrity during the majority of the degradation time course: this allows structural degradation to follow the effective therapeutic lifetime of the device. To validate these devices, we characterize the release of Lucentis, an age-related macular degeneration therapeutic, from nanoporous PCL thin film devices over extended periods. Furthermore, PCL films and devices have been implanted or injected into rabbit eyes to establish the long-term biocompatibility, structural integrity, and functionality of these devices.
10:15 AM JJ10.7
Self-assembly and Mineralization of Organics on a Template. Elaine DiMasi, Brookhaven National Laboratory, Upton, New York.
Biomineralization depends upon an appropriate template consisting of organic material - proteins, polysaccharides, or similar - being available to nucleate mineral and control crystal growth. The organics in turn require the correct conditions to self-assemble into the appropriate structures. For proteins tertiary structures such as fibrils may be required; for surfactants such as membrane lipids, conditions must permit the formation of sheets or vesicles. When model systems are assembled to study biomimetic mineralization, a properly chosen experimental substrate - gels, hard wafer surfaces, etc - can be crucial. It has been demonstrated across a variety of experimental systems that better molecular order of organic templates can be achieved on surfaces bearing net negative charge. We will review these results and present new observations in surfactant and extracellular matrix protein systems, as well as cultured cell systems, studied with scanning probe and synchrotron x-ray microscopies.
10:30 AM JJ10.8
Hybrid Nanowire Arrays for Multifunctional Adhesive Surfaces. Hyunhyub Ko1,2,3, Zhenxing Zhang2,3,4, Rehan Kapadia2,3,4 and Ali Javey2,3,4; 1Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan Metropolitan City, Korea, Republic of; 2Electrical Engineering and Computer Sciences, University of California at Berkeley, Berkeley, California; 3Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California; 4Berkeley Sensor and Actuator Center, University of California at Berkeley, Berkeley, California.
The biological systems with functional micro- and nanostructured surfaces have inspired the fabrication of functional surfaces with unique optical, chemical and mechanical properties. Inspired by the micro- and nanofibrillar structures found in various biological adhesive systems, here, we report the fabrication of hybrid, inorganic/organic nanowire (NW) arrays for multifunctional adhesive surfaces. In particular, inspired by the adhesion systems found in dragonfly and gecko lizard, we demonstrate the first connector (i.e., fasteners to bind things together) concept that utilizes van der Waals interactions rather than mechanical or magnetic interactions as the main binding mechanism. In addition, we demonstrate that hybrid NW connectors can be easily tuned to provide underwater binding operation, self-cleaning superhydrophobic properties, anisotropic adhesion, thermo-responsive smart adhesion, and electrically conductive connection by manipulating the composition of hybrid nanostructures. Since many biological systems have evolved into multi-scale, hierarchical structures with sophisticated and smart functions, we also introduce a simple method for fabricating hierarchical fibrillar arrays based on polymer micropillar arrays (µPLR) decorated with ZnO nanowires on mechanically flexible substrates. The hierarchical µPLR/NW arrays show superhydrophobic surface properties, with the contact angle higher than that of planar surfaces and µPLR arrays without nanostructures. The fabrication strategy suggested here may be potentially extended to fabricate other organic/inorganic hierarchical systems for different applications in biomedical devices, micro-robotics, and in any device that require reversible assembly of components. . Hyunhyub Ko, Zhenxing Zhang, Kuniharu Takei, and Ali Javey, “Hierarchical polymer micropillar arrays decorated with inorganic nanowires”, Nanotechnology, 2010, 21, 295305. . Hyunhyub Ko, Yu-Lun Chueh, Zhenxing Zhang, and Ali Javey, Thermo-responsive smart nanowire connectors, Angew. Chem. Int. Ed., 2010, 49, 616-619. . Hyunhyub Ko, Zhenxing Zhang, Johnny C. Ho, Kuniharu Takei, Rehan Kapadia, Yu-Lun Chueh, Weizhen Cao, Brett A. Cruden, and Ali Javey, Flexible carbon nanofiber connectors with anisotropic adhesion properties, Small, 2010, 6, 22-26. . Hyunhyub Ko, Zhenxing Zhang, Yu-Lun Chueh, Johnny C. Ho, Jongho Lee, Ronald S. Fearing, and Ali Javey, Wet and dry adhesion properties of self-selective, nanowire connectors, Adv. Funct. Mater. 2009, 19, 3098-3102. . Rehan Kapadia, Hyunhyub Ko, Yu-Lun Chueh, Johnny Ho, Toshitake Takahashi, Zhenxing Zhang, and Ali Javey, Hybrid core-multi-shell nanowire forests for electrical connector applications, Appl. Phys. Lett. 2009, 94, 263110. . Hyunhyub Ko, Jongho Lee, Bryan E. Schubert, Yu-Lun Chueh, Paul W. Leu, Ronald S. Fearing and Ali Javey, Hybrid Core-Shell Nanowire Forests as Self-Selective Chemical Connectors, Nano Lett. 2009, 9, 2054-2058.
10:45 AM JJ10.9
A pH-sensitive Cross-linker based on a Hyperbranched Acetal-polymer. Hongliang Cao, Network of Excellence for Functional Biomaterials, National University of Ireland, Galway, Galway, Ireland.
There are numerous pH gradients that exist in both normal and pathophysiological states in the human body. For example, tumor and inflammed tissue have a pH slightly more acidic than the blood and normal tissue. In addition, the intracellular vesicles of cells involved in the endocytosis mechanism, i.e. endosomes and lysosomes are also acidic. Therefore, pH-responsive systems can potentially be used in a broad range of tissue engineering and drug delivery applications. Acetals compounds and polymer are promising candidates for the development of acid-sensitive systems because the hydrolysis of acetal group is strongly dependant on pH value. In this study, a new hyperbranched polymer with acid-cleavable acetal groups was developed as a pH sensitive cross-linker for protein. This technique involves the formation of covalent bonds between proteins by using multifunctional reagents containing reactive end groups, e.g. N-hydroxysuccinimide (NHS) that react with functional groups—such as primary amines and sulfhydryls—of amino acid residues from protein. The acetal-NHS co-polymer was synthesized by the ATRP method, The 1H NMR spectrum illustrated the double bonds and characteristic acetal proton peak within the acetal-NHS co-polymer. These peaks also provide the integral values required to calculate the final polymer composition, which consist of: 81.8% molar percentage of PEG-A, 10.1% acetal units, 8.0% NHS units, 4.0% vinyl groups and 6.1% branching degree. The pH-dependent hydrolysis of the acetal crosslinker was investigated at pH 3.0, 5.0, and 7.4. At pH 3.0, the polymer was completely hydrolyzed in 24h. At pH 5.0, the polymer was found to be hydrolyzed with half-life of approx. 50 hrs. At neutral pH the hyperbranched polymer remained relatively stable over the same period. It will be hydrolyzed approx. 25% after incubated at room temperature for 120 hrs. Mouse 3T3 fibroblast cells were utilized for the cytotoxicity assessment of the polymer. The results showed the synthesized polymer did not exhibit reduction in cellular metabolic activity at 0.5mg/ml and 1mg/ml concentration. The acetal-NHS hyperbranched polymer was used as a crosslinker for proteinaceous scaffold (collagen type I). Hydrogels made from collagen and the crosslinker were prepared in 96 well plates for 2D and 3D culture. The results of cell viability showed that the hydrogel made from acetal-NHS polymer was not significantly different from star-PEG as cross-linker and cell alone by ADSC and 3T3 cell culture. Acknowledgements. The authors would like to thank the European Commission under the DISC REGENERATION project (NMP3-LA-2008-213904) financial support of this research.
11:00 AM JJ10.10
Design and Synthesis of Self-oscillatory Hydrogels. Ye Zhang1, Jorge Delgado1, Anna C. Balazs2, Irving R. Epstein1 and Bing Xu1; 1Chemistry, Brandeis University, Waltham, Massachusetts; 2Chemical Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania.
Inspired by the natural materials that convert chemical energy into mechanical movement, we designed and fabricated chemomechanical hydrogels. By using Belousov-Zhabotinsky (BZ) oscillating chemical reaction as theoretical modeling, we built the catalyst of BZ reaction-ruthenium complex into a copolymer or a supramolecular gelator to form hydrogels. The periodic oxidation and reduction of the anchored ruthenium ion in BZ reaction induce the corresponded hydrating and dehydrating effects in the hydrogels that finally result into its oscillatory volume change. All these efforts make the hydrogels self-oscillate without external stimuli. The overall aim of our research is to explore the possibility of utilizing hererogeneous chemoresponsive gels to produce multifunctional materials with the ability to convert the chemical energy of an oscillating chemical reaction into controllable mechanical forces.
11:15 AM JJ10.11
Bio-inspired Hierarchical Vascular Network Synthesis: Electrical Treeing. Kristopher D. Behler, Zachary R. Melrose and Eric D. Wetzel; Composite and Hybrid Materials Branch, U.S. Army Research Lab, Aberdeen Proving Ground, Maryland.
Vascular networks provide a physical pathway to distribute fluid throughout a system. An uninterrupted and controllable supply of liquid is optimal for many applications such as continual self-healing, drug delivery, chemical and biological agent neutralization, and thermal management. One approach to producing hierarchical vascular networks is electrical treeing (ET). Hollow channels or “trees” are grown from a series of step-wise, partial discharges in a dielectric material resulting from a high local electric field that is greater than the dielectric breakdown strength of the material. Continual breakdown leads to hollow tubules which from an intricate network of branches, ranging in diameter from sub micron to less than 100 microns, within a polymer or epoxy system. Electrical trees have been grown using voltages of 20 kV (AC) and up to 60 kV (DC) resulting in “bush-like”, dense, networks of hollow tubules and less dense “tree-like” structures, respectively. Filling of these structures has been visualized using a dissolved UV sensitive dye pumped into the trees via a syringe pump.
11:30 AM JJ10.12
Bone-Inspired Multicomponent Bionanocomposites with a Simple Drop-cast Processing Strategy. Abhijit Biswas1, He Zhao2, Ilker Bayer3 and Alexandru S. Biris4; 1Electrical Engineering, Center for Nanoscience and Technology, University of Notre Dame, Notre Dame, Indiana; 2Aerospace and Mechanical Engineering, Center for Nanoscience and Technology, University of Notre Dame, Notre Dame, Indiana; 3Smart Materials Group, Italian Institute of Technology, Arnesano, Italy; 4Nanotechnology Center, University of Arkansas at Little Rock, Little Rock, Arkansas.
Bone is a specialized form of connective tissue that forms the skeleton of the body and is built at the nano and micro levels as a multicomponent composite material consisting of a hard inorganic phase (minerals) in an elastic, dense organic network. Mimicking bone structure presents an important frontier in the fields of materials science and bone tissue engineering. An ideal bonelike or bone-mimetic biomaterial would replicate the predominant coalignment of the organic and mineral phases of the actual bone tissue architecture. This essentially involves nano- to micro-scale features of both the organization of collagen fibers in a characteristic three dimensional architecture and the coalignment of important mineral such as hydroxyapatite (HAP) crystals within the collagen fibers. It is a significant challenge to achieve such complex and special three dimensional multicomponent bonelike features. We describe an innovative and simple drop-cast processing strategy to create bonelike multicomponent bionanocomposite materials that consist of an organic poly(e-caprolactone) (PCL) matrix, minerals such as hydroxyapatite (HAP) and CaCO3, and collagen fibers. The process allows morphological and structural control to achieve the desired nanostructure of the bone mimics. The synthesis process involves adding inorganic and organic components sequentially followed by controlling the growth conditions and composition. This enables organization of collagen nanofibers (~ 100 nm) into scaffolds while simultaneously allowing nucleation and co-alignment of hydroxyapatite spheres (~ 100 - 500 nm) within aligned, thermally stable collagen fibers in the porous PCL matrix. We achieved high calcium (26%) and oxygen (17%) within the bioscaffold and adequate phosphorous compositions comparable to the levels of bone tissues. These breakthroughs provide the viable approach to help maintain the required bone mineral density and revascularization for nutrient and compensate for the loss of oxygen delivered to the bone cells. It suggests the huge potential of these bone-inspired materials for bone grafting technologies.