Symposium OrganizersXiaoyuan (Shawn) Chen, National Institute of Biomedical Imaging and Bioengineering National Institutes of Health
Jinwoo Cheon, Yonsei University
Mansoor Amiji, Northeastern University
Shuming Nie, Emory University
VV2: Nanomedicine for Molecular Imaging and Therapy II
Tuesday PM, April 10, 2012
Marriott, Golden Gate, Salon B
2:30 AM - *VV2.1
Nanocomposite Design of Advanced Biomaterials
Jackie Y. Ying 1
1Institute of Bioengineering and Nanotechnology Singapore SingaporeShow Abstract
Nanocomposites are developed for various biomedical applications. These multicomponent materials can provide simultaneous drug delivery and bioimaging functions as theranostic systems. They can be synthesized with unique carrier materials that offer synergistic therapeutic effects with the drugs to be delivered. They can allow for the sustained delivery of growth factors to significantly enhance tissue regeneration. We have also synthesized nanocomposite scaffolds in various compositions and morphologies for the delivery of cells in tissue engineering applications.
3:00 AM - VV2.2
Spatio-temporally Concerting Gold Nanoparticle Conjugates for Highly Synergistic Cancer Therapy
Jutaek Nam 1 Wan-Geun La 2 Sekyu Hwang 1 Yeong Su Ha 3 Nokyoung Park 4 Nayoun Won 1 Sungwook Jung 5 Suk Ho Bhang 2 Yoon-Ji Ma 2 Yong-Min Cho 2 Min Jin 2 Eun Kyung Wang 3 Sang Geol Kim 6 Jong-Min Kim 4 So-Hye Cho 7 Jeongsoo Yoo 3 Byung-Soo Kim 2 Sungjee Kim 1 5
1Pohang University of Science amp; Technology Pohang Republic of Korea2Seoul National University Seoul Republic of Korea3Kyungpook National University Daegu Republic of Korea4Samsung Electronics Yongin Republic of Korea5Pohang University of Science amp; Technology Pohang Republic of Korea6Kyungpook National University Daegu Republic of Korea7Korea Institute of Science and Technology Seoul Republic of KoreaShow Abstract
Gold nanostructures can generate site-selective local heat shock, and they have great potential as an engineerable platform for thermoâ?"chemo combination cancer therapy. This combination therapy can be synergistically more effective for the treatment of cancer over the two independent treatments. However, the huge discrepancy between the sizes of the two agents limits from achieving effectively concerted actions of the two modalities, which prevent them from achieving their maximal synergistic effect. The gold nanostructures need to exploit far-red or near-infrared (NIR) light for deep tissue penetration. Properly tuning the plasmonic wavelength for photothermal therapy typically results in the nanostructures such as nanorods, nanoshells, and nanocages of which hydrodynamic sizes reach ~100 nm. Most chemo agents are small molecules; that are far smaller than gold nanostructures. This size difference inherently limits the concertion of the two agents because the size of the agents critically determines their entry to cells and their distribution in confined cellular compartments. Herein, we report a simple, spherical, 10 nm â?~smartâ?T gold nanoparticle- doxorubicin conjugates as an engineerable platform for combination cancer therapy. Upon mild acidic pH in tumor, they are designed to simultaneously release the doxorubicins and form gold nanoparticle aggregates. The gold nanoparticle aggregates served as a photothermal agent that can selectively exploit external light by their collective plasmon modes. Spatio-temporal concertion between the thermo and chemo agents can be directed at the sub-cellular, cellular, and organ levels. Both agents co-localize in the cell nuclei. The conjugates accumulate in cancer cells because of the rapid phagocytic actions of cancerous cells and effective blockage of exocytosis by the increased size of the aggregates. Similarly, the aggregates also accumulate in tumors because of their enhanced retention. The conjugates exhibit a highly synergistic effect in vitro, with their cytotoxicity enhanced by nearly an order of magnitude. They can be therapeutically effective even when they contain significantly less of each component than is required when administered alone. Using a small animal model in vivo, statistically significant tumor growth suppression is demonstrated without noticeable damage to other organs. This spatio-temporally concerting conjugate permits a new approach to combination therapy because the synergistic effect is maximized and side effects are minimized by the reduction of the dosage.
3:15 AM - VV2.3
In vivo Imaging, Cell Targeting, and Controlled Drug Release of Fe3O4 Nanospheres
Donglu Shi 1 4 Chris Huth 2 Feng Wang 2 Giovanni Pauletti 3
1Tongji University Shanghai China2University of Cincinnati Cincinnati USA3University of Cincinnati Cincinnati USA4University of Cincinnati Cincinnati USAShow Abstract
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. Thermoresponsive nanocomposites were also prepared by immobilizing a 2-3 nm thick phospholipid layer on the surface of superparamagnetic Fe3O4 nanoparticles via high-affinity avidin/biotin interactions. Hyperthermia-relevant temperatures > 40C were achieved within 10-15 min using a7-mT magnetic field alternating at a frequency of 1MHz. Loading of the surface-associated phospholipid layer with the hydrophobic dye dansylcadaverine was accomplished at an efficiency of 479 ng/mg Fe3O4. Release of this drug surrogate was temperature-dependent, resulting in a 2.5-fold greater release rate when nanoparticles were exposed to a temperature above the experimentally determined melting temperature of 39:7C. These data underline the feasibility of preparing novel, stimulus-induced drug delivery systems where payload release from a colloidimmobilized phospholipid assembly is triggered by hyperthermia.
3:30 AM - VV2.4
Exploiting the Gold Nanorod-Protein Corona Complex for Delivery and Remote Triggered Release of Small Molecular Therapeutics
James Kah 1 Christin Grabinski 2 Angel Zubieta 3 Saber Hussain 2 Kimberly Hamad-Schifferli 1 4
1Massachusetts Institute of Technology Cambridge USA2Air Force Research Laboratory, Wright Patterson AFB Dayton USA3The University of Texas at Austin Austin USA4Massachusetts Institute of Technology Cambridge USAShow Abstract
The use of nanomaterials in a biological environment often results in the formation of a protein corona on the surface of the nanomaterials due to the adsorption of proteins from a rich protein milieu commonly present in physiological fluids. Such a protein corona tends to affect the intended functionality of the nanomaterials by masking their surface properties and hence their functional characteristics, and is a cause of concern in nanomaterial design for biological applications. Despite their unintended presence, the protein corona can also be exploited as an integral part of the nanomaterial design in a way that improves their physical properties and complements the unique properties of various nanomaterials. In this study, we utilize the protein corona to stabilize cetyltrimethylammonium bromide-capped gold nanorods (CTAB-AuNR) as synthesized for biological application without complex surface ligand exchanges. We also harness the carrier properties of serum proteins in the protein corona in combination with the unique optical properties of AuNRs to effectively realize the delivery and remote triggered release of small molecular therapeutics. The protein corona was preformed on CTAB-AuNR by incubating them in horse serum in vitro before loading an antisense oligonucleotide as well as an anticancer therapeutic, doxorubicin as the payload on the horse serum-coated CTAB-AuNR (HS-AuNR). The loading of both payloads was quantified and their intracellular delivery was imaged and quantified. The release of these therapeutic payloads under laser irradiation was also characterized. The results show that the presence of a protein corona was able to effectively stabilize the CTAB-AuNR in biological media against a wide range of environmental factors. The serum proteins were also able to function as a carrier for small molecular therapeutics with an enhanced intracellular delivery of both payloads demonstrated. These payloads can be remotely released by irradiating the HS-AuNR with an ultrafast laser. Whilst the non-specific adsorption of extracellular proteins on nanomaterials could not be avoided in any biological applications, they can be embraced to provide useful functionalities as demonstrated in this study.
3:45 AM - VV2.5
Microfluidic Device for High Efficiency DNA Extraction from Single Cells
Harvey C Tian 1 2 Christopher B Wallin 2 Juraj Topolancik 2 Benjamin R Cipriany 3 Jaime J Benitez 2 David R Latulippe 2 Patrick J Murphy 4 Paul D Soloway 5 Harold G Craighead 2
1Cornell University Ithaca USA2Cornell University Ithaca USA3Cornell University Ithaca USA4Cornell University Ithaca USA5Cornell University Ithaca USAShow Abstract
We describe a polydimethylsiloxane (PDMS) microfluidic device capable of extracting, purifying, and elongating strands of DNA that are megabases in length from a single cell for on-chip analysis. The process begins with the introduction of a single cell into the microfluidic channel via hydrodynamic flow. The cell subsequently enters an array of micropillars within the channel where it becomes immobilized. The arrested cell is then lysed under continuous flow with a surfactant which degrades not only the cellâ?Ts membranes, but also denatures its proteins including the histone proteins responsible for the tight coiling behavior exhibited by DNA in its native form. Thus, as the histone proteins are stripped off by the surfactant, the DNA within the device begins to unravel. This unwinding occurs simultaneously as the DNA is flowed downstream into a spatially dense forest of micropillars where it becomes physically entangled. Here, the spacing between micropillars is nontrivial, as it serves to be a size dependent means of purification, tethering only large molecules such as strands of DNA while smaller molecules such as proteins, lipids, and RNA are washed away. The suspended DNA becomes elongated by the hydrodynamic flow and through fluorescent staining, measured visible lengths of up to 2.7cm. Finally, nucleases were used to fragment the DNA, causing its release from the microchannel. To confirm that loss of DNA was not occurring during this process, fluorescence measurements were taken of the collected extracts. However, to meet the lower sensitivity limits of current instruments, the microfluidic device was scaled up to extract DNA from a small population of less than 100 cells. Results show that this method of extraction yields over 95% of the DNA content present within the original population. Therefore, the described microfluidic device is capable of not only physically suspending the entire genomic content of a single cell without the need for chemical surface functionalization, but also high efficiency elongation, purification, staining, and collection as well.
4:30 AM - VV2.6
Cholesterol-containing Polymeric Micellar Nanoparticles for Targeted Delivery of Hydrophobic Anticancer Drugs with Rigid Structures
Ashlynn Lee 1 Shrinivas Venkataraman 1 Syamilah Sirat 1 Shujun Gao 1 James L Hedrick 2 Yi-Yan Yang 1
1Institute of Bioengineering and Nanotechnology Singapore Singapore2IBM Almaden Research Center San Jose USAShow Abstract
As many anticancer agents are hydrophobic, clinical administration of these drugs typically requires dissolution using carriers. Polymeric micelles can significantly enhance the solubility of hydrophobic drugs to improve bioavailability and they can also serve as drug reservoirs to allow sustain release to prevent or decrease tissue damage upon accidental extravasations. Paclitaxel (PTX) is a hydrophobic anticancer drug with a rigid chemical structure having 11 stereocenters (4 R and 7 S), and has a strong tendency to self-assemble into long fibers. As a result, sonication is often required to prevent the self-assembly/precipitation of PTX during encapsulation and maintain the micelles within the nanosize range. Despite such arduous procedures, the drug loading content of such delivery systems is typically low. In this study, we introduce cholesterol into micellar core so that it can efficiently sequester PTX into micelles through hydrophobic interactions and stereocomplexation. The amphiphilic block copolymers with controlled composition and relatively low polydispersity index were synthesized from monomethoxy polyethylene glycol (PEG) via organocatalytic ring opening polymerization of aliphatic cyclic carbonate monomers - trimethylene carbonate (TMC) or cholesteryl 2-(5-methyl-2-oxo-1,3-dioxane-5-carboxyloyloxy)ethyl carbamate (MTC-Chol) or a copolymer of both the monomers (TMC and MTC-Chol): mPEG113-b-PTMC67, mPEG113-b-P(MTC-Chol11) and mPEG113-b-P(MTC-Cholx-co-TMCy)x+y. The role of molecular weight and composition of the hydrophobic block of the polymers in loading PTX was studied. The PTX-loaded micelles were fabricated by simple self-assembly without sonication or homogenization procedures. The results demonstrated that the presence of both MTC-Chol and TMC in the hydrophobic block significantly increased PTX loading levels, and the micelles formed from the polymer with the optimized composition were in nanosize (36 nm) with narrow size distribution (PDI: 0.07) and high PTX loading capacity (15 wt.%). PTX-loaded micelles killed human liver carcinoma HepG2 cells much more effectively than free PTX, whereas blank micelles were not cytotoxic. In addition, these nanocarriers also possessed exceptional kinetic stability. The results from non-invasive near-infrared fluorescence (NIRF) imaging studies showed that these micelles allowed effective passive targeting, and were preferably accumulated in tumor tissue with limited distribution to healthy organs. Therefore, these nanoparticles are promising candidates as carriers for hydrophobic drugs with high molecular rigidity.
4:45 AM - VV2.7
Direct Translocation of Cell-Penetrating Peptides (CPPs) across Lipid Bilayers
Andrew Wang 1 Nicholas Melosh 1
1Stanford Unviversity Palo Alto USAShow Abstract
Delivery of drugs and siRNA to cells is the key step for increasing drug efficacy, decreasing side effects, and unlocking the power of siRNA. The short, charged, amphipathic and sometimes structured cell-penetrating peptides (CPPs) have been shown promising carriers for delivering protein, RNA, DNA, and even nanoparticles into cells. Yet most CPPs have been identified based on serendipitous discovery (e.g., Tat) and trial and error attempts. The mechanistic understanding of how CPPs cross the cell membrane and what biophysical property is critical for efficient penetration is largely unknown, mostly due to the lack of proper experimental tools to measure the actual translocation event as these peptides pass through the bilayers. This fundamental problem prevents rational designs of new CPPs. We have developed a new technique that directly measures the translocation dynamics for CPPS using AFM, which can discern different mechanistic pathways and the relative efficacy of different CPPs. To gain insights in the universality of CPP translocation, we selected six peptides from three different classes of proposed mechanisms: (1) Tat and poly-arginine as structurally â?odisorderedâ?, cationic peptides that most likely enter the cell via endocytosis; (2) penetratin and pVEC as beta-stranded amphipathic peptides shown capable of directly translocating through the membrane; and (3) TP10 and modeled amphipathic peptide (MAP) as alpha-helical amphipathic peptides also being able to penetrate directly. CPPs were attached via thiol-gold linkage to Au-coated AFM probes and brought into contact with lipid bilayers. The displacement-time trajectory of CPPs inside the bilayers was directly measured during bilayer breakthroughs under different force loading rates. Biophysical characteristics, such as potential energy barrier of bilayer failure, dynamics of penetration pathways, and bilayer structural rearrangement, were derived and compared among the six peptides, which will lead to rational designs of new CPPs sharing critical biophysical properties.
5:00 AM - VV2.8
A Novel Biocompatible Nanoparticle Platform for Simultaneous Delivery of Hydrophobic, Hydrophilic Chemotherapeutic Drugs, siRNA and Gold Clusters for Cancer Therapy and Imaging
Hai Wang 1 Yan Wu 1 Guangjun Nie 1
1National Center for Nanoscience and Technology, China Beijing ChinaShow Abstract
Highly Active Antiretroviral Therapy (HAART), a strategy combining three or more anti-HIV drugs, is the most famous example of combination therapy and has become optimum strategy for treatment of HIV. In addition, combination therapy is also the main strategy to treat with malaria, multiple sclerosis and cancer. For treatment of cancer, combination therapy also has been successfully used in clinical applications, simply by administered multiple drugs for cancer patients. Due to varieties of biodistribution, pharmacokinetics, circulation time and membrane transport properties among different drug molecules, it is very challenging for physicians to choose a optimal drug regimen. Additionally, combination therapy may also cause serious side effects. Taking into account of these shortcomings, we have developed a simple method to prepare multi-drugs loaded nanoparticles, in which doxorubicin, paclitaxel, survivin siRNA and gold clusters with strong fluorescent emission have been successfully packaged into single nanoparticle with mPEG-PLGA and Îµ-Polylysine by improved double emulsion method. Both mPEG-PLGA and Îµ-Polylysine have been approved by FDA for human use, and Îµ-Polylysine have been reported with antibacterial properties, an important feature for the preservation of cationic nanoparticles. Cationic nanoparticles have been showed to absorb bacteria resulting in acute bacterial inflammation. The mean particles size were about 200 nm with the polydispersity being 0.12, and zeta potential were +32 mV. Both in vitro and in vivo experiments showed that those multi-drugs loaded nanoparticles were effective in inhibition of tumor growth, especially for in vivo study, in which we have constructed a very high degree malignancy tumor model (melanoma tumor model). Within seventeen days, the mean tumor volume in control group increased to 8438.5 mm3, but for multi-drugs loaded nanoparticles treated group, the mean tumor volume was only 469 mm3, only about one-twentieth of control group. These results strongly indicated that as-made multi-drugs loaded nanoparticles could be effective in treatment of melanoma tumor model. We have also demonstrated the protein-stabilized Au clusters could be used for fluorescent tumor imaging. Therefore, we have developed a simple strategy to effectively integrate multiple therapeutic agents and imaging probe for treatment of melanoma model both in vitro and in vivo. And this theranostic hybrid system is a promising tool to enhance the efficacy of tumor therapy.
5:15 AM - VV2.9
Superheated Nanoemulsions as Stimulus-responsive Ultrasound Contrast Agents for Cancer Detection and Imaging
Andrew P. Goodwin 1 Alan C Toledo 1 Zhe C Wu 2 Matthew A Nakatsuka 1 Jennifer N Cha 1 Sadik C Esener 1
1University of California, San Diego La Jolla USA2University of California, San Diego San Diego USAShow Abstract
Early detection of cancer correlates strongly with patient prognosis, and many are searching for a means to sense for tumor malignancy using inexpensive, non-invasive methodologies. Ultrasound imaging satisfies these latter criteria but lacks sensitivity for diseased tissue. Contrast may be enhanced significantly through the administration of colloids with physical properties dissimilar to water. Microbubbles in particular provide excellent signal-to-noise enhancement due to their ability to generate selectively detectable acoustic modes at harmonic frequencies. However, the microbubblesâ?T size (1-5 Âµm) prevents them from exiting the bloodstream and accumulating in extravascular targets like solid tumors, hindering their potential for non-invasive, early detection of cancer. An alternate approach for ultrasound imaging of cancer is the administration of superheated nanoemulsions that can respond to the tumor microenvironment. Superheated emulsions consist of liquid with a bulk boiling point below physiological temperature but, due to stabilization on the nanoscale with amphiphiles, remain stable at tens of degrees greater than normal. They can, however, be converted in situ into high-contrast microbubbles in response to incident ultrasound. In this research, it was found that the intensity of ultrasound required to initiate this transition depended upon the surface chemistry of the stabilizing amphiphiles. In their native state, the emulsions were formulated with a charged polymer shell that reduced their ability to transition into bubbles. When the polymer was removed in response to a chemical trigger, the intensity required to convert the emulsions decreased. Thus, emulsions that had responded to their chemical environment could be selectively converted into microbubbles, allowing for contrast-enhanced imaging of specific biochemical environments. Future activities include adapting this technology to sense for cancer biomarkers and studies in a mouse cancer model.
VV1: Nanomedicine for Molecular Imaging and Therapy I
Tuesday AM, April 10, 2012
Marriott, Golden Gate, Salon B
9:15 AM - VV1.1
A Closer Look at Two-photon Absorption in Colloidal Gold Nanorods
Joanna Olesiak Banska 1 Radoslaw Kolkowski 1 Marta Gordel 1 Katarzyna Matczyszyn 1 Marek Samoc 1
1Wroclaw University of Technology Wroclaw PolandShow Abstract
Among all shapes of gold nanoparticles, nanorods (NRs) have gained great interest due to particular characteristics of their surface plasmon resonances (SPRs). Anisotropic shape of the NRs introduces two modes of SPR oscillations, the longitudinal and the transverse one, resulting in strong light polarization and frequency dependence and very large field enhancements due to the â?olighting rod effectâ?. In comparison to nanospheres and nanoshells, NRs provide better tunability of SPR bands in a wider range of optical frequencies, keeping the small overall size of the nanoparticles. Gold NRs are finding increasingly important applications in linear and nonlinear optical microscopy and medical therapies (e.g. photothermal therapy) . In order to obtain specific functionalities, optical properties of NRs need to be controlled and systematic and quantitative description of light-NRs interactions in a wide range of wavelengths is required. This contribution reports on the investigations of nonlinear optical properties of gold NRs of the aspect ratio ~3 and the longitudinal SPR at Î»(Absmax)= 850nm . The NRs were synthesized using the seed-grow procedure and their surface was additionally stabilized, as was described in [3-4]. Then, the Z-scan technique was applied to determine the real and imaginary part of the cubic hyperpolarizability Ï?(3)(-Ï?;Ï?,-Ï?,Ï?) in the range from 550nm to 1600nm . The dispersion of the two-photon absorption coefficient Î±2 and two-photon absorption cross-section Ïf2 were determined taking into account the observed saturable absorption effects . The influence of plasmon resonance on the nonlinear properties of gold nanorods as well as the behaviour of gold NRs in the high intensity fs laser beam are discussed. Acknowledgement The authors acknowledge financial support from the Foundation for Polish Science â?oWelcomeâ? program.  X. Huang, S. Neretina, M. A. El-Sayed, Adv. Mater. 21, 4880 (2009)  Cheng, J. X. et al. Photochem Photobiol 85, 21 (2009)  Olesiak-Banska, J. et al. submitted (2011)  a) Sau, T. K. et al. Langmuir 20, 6414 (2004), b) Zweifel, D. A. et al. Chem. Mat. 17, 4256 (2005)  Sheik-Bahae, M. et al. IEEE J Quantum Elect 26, 760 (1990)  Samoc, M. et al. Opt Lett 23, 1295 (1998)
9:30 AM - VV1.2
Engineering Fluorescent Gold Nanoparticles for In vivo Pharmacokinetic Profiling and Tumor Imaging
Leo Chou 1 2 Warren Chan 1 2
1University of Toronto Toronto Canada2University of Toronto Toronto CanadaShow Abstract
Combining the multifunctionality of nanomaterials with their rational design for lesion-specific delivery is expected to significantly improve cancer detection and treatment. Rational design of nanoparticles builds upon systematic analyses of how nanomaterials size, shape, and surface chemistry impact their interactions with cells and tissues in vitro and in vivo. Because of their synthetic versatility and well-defined surface chemistries, gold nanoparticles are currently one of the few types of nanomaterials that can be used for this purpose. However, gold lacks the appropriate contrast for real-time detection in vivo, and rely on destructive techniques for visualization. Here we have developed and characterized a series of fluorescently-labelled GNPs, and used them to study the size-dependent kinetics of nanoparticle uptake to solid tumours. Using fluorescence imaging analysis, we also mapped the pharmacokinetics and biodistribution of nanoparticles as a function of their size. Finally, we demonstrate that multispectral imaging could be used to compare the in vivo distribution of multiple nanoparticles simultaneously within a single animal, which would eliminate data variability caused by heterogeneities in the tumour model. These results describe a novel platform that could significantly speed up translational nanoparticle research, and provide guidelines for the design of future nanomaterials.
9:45 AM - *VV1.3
Designed Synthesis of Uniform-sized Iron Oxide Nanoparticles for New MRI Contrast Agents
Taeghwan Hyeon 1 Nohyun Lee 1 Byung Hyo Kim 1 Mihyun Park 1 Daishun Ling 1 Soo Hong Yi 1 Youjin Lee 1
1Seoul National University Seoul Republic of KoreaShow Abstract
Superparamagnetic iron oxide nanoparticles of 5 nm ~ 20 nm have been extensively used as MR contrast agents due to its unique magnetic property and biocompatibility. We developed a generalized synthetic procedure, called heat-up process, to produce uniform-sized iron oxide nanocrystals with controlled sizes ranging from 1.5 nm to 100 nm. Since the magnetic property of iron oxide nanoparticles is dependent on their size, smaller and/or larger nanoparticles will extend the application of MR contrast agents. Extremely small iron oxide nanoparticles (ESION) below 5 nm exhibit very small magnetization because most of their magnetic spins are canted. On the other hand, ferrimagnetic iron oxide nanoparticles (FION) of > 20 nm exhibit extremely large magnetization and coercivity. The 3 nm-sized ESION exhibited a high r1 relaxivity of 4.78 mM-1s-1 and non-toxic. Intravenously administered ESIONs showed much longer circulation time than the clinically available T1 contrast agent, DOTAREM, and such long circulating nanoparticles enable high-resolution MR imaging. Various blood vessels with sizes down to 0.2 mm were clearly observed after injection of ESIONs. The ferrimagnetic iron oxide nanoparticles (FION) coated with PEG-phospholipid exhibited a very high r2 relaxivity of 324 mM-1s-1. The cells labeled with FIONs showed so enormous T2 contrast effect that even single cells were readily image using 9.4 T MR scanner. The uptake of FIONs by islets was so fast that single islets were readily imaged using 1.5 T MR scanner as early as 2 hr after the incubation. The viability and functionality of the labeled islets did not vary compared with the unlabeled islets. After transplantation of syngeneic islets, the diabetic rats became euglycemic, and the transplanted islets were observed up to 150 days. Immune rejection was also observed as decrease in number of dark spots representing transplanted islets after transplantation of allogeneic islets.
10:15 AM - VV1.4
Conjugated Polymer Nanoparticles for Small Interfering RNA Delivery
Joongho Moon 1 Eladio Mendez 1 Youri Lee 1
1Florida International Univ Miami USAShow Abstract
Positively charged conjugated polymers (CPs) are promising fluorescent tracers useful for delivery of small interfering RNA (siRNA). Structurally, CPs contain rigid hydrophobic backbones and flexible charged side chains. While side chains of CPs can attract negatively charged nucleic acids, hydrophobic backbones can also stabilize or help cellular uptake of the complexes through hydrophobic interaction. The photophysical robustness of CPs offers visualization of delivery events using microscopic imaging techniques. Previously, we demonstrated that conjugated polymer nanoparticles (CPNs) fabricated by acetic acid treatment of an amine-containing poly(phenylene ethynylene) (PPE-NH2) were taken up by live cells without inhibiting cell viability. Herein, we demonstrate that the CPNs can be a nontoxic and efficient siRNA carrier, with the delivery event easily visualized by fluorescent microscopic imaging. Loosely aggregated CPNs formed stable complexes with siRNA, and the complexes were successfully localized inside live cells. The CPNs delivered biologically active siRNA for the actin B gene into HeLa cells, and significant down-regulation of the target protein was observed without measurable toxic effects.
11:15 AM - *VV1.6
Multifunctional Mesoporous Silica Nanoparticles for Targeting, Imaging and Drug Delivery
Jeffrey I Zink 1
1UCLA Los Angeles USAShow Abstract
Mesoporous silica nanoparticles (particle diameter ~ 100 nm, pore diameter ~ 2 nm) are derivatized with molecules designed to induce multiple functionality. The most important functionality is the ability to trap molecules in the pores and release them in response to specific stimuli and/or on external command by using molecular machines. Other functions highlighted in this talk include incorporation of smaller metal nanocrystals (for antimicrobial activity and/or magnetic manipulation), targeting molecules (towards specific cells or microorganisms), and fluorescence (for imaging). Two types of molecular machines that are based on molecules that undergo large amplitude motion when attached to mesoporous silica are described: impellers and valves. Derivatized azobenzene molecules, attached to the interior pore walls function as impellers that can move other molecules through the pores. Nanoparticles containing toxic molecules in the mesopores are taken up by cancer cells, and stimulation of the impellers drives out the toxic molecules and kills the cells. Nanovalves consisting of rotaxanes and pseudorotaxanes, placed at pore entrances, can trap and release molecules from the pores in response to stimuli. Two methods of activation that have been demonstrated for in vitro studies will be discussed: pH changes and oscillating magnetic fields. Lysosomal acidification causes self-opening of the valves, and externally applied magnetic fields afford external control. Activation by both of these in living cancer cells will be discussed.
11:45 AM - VV1.7
Nanoparticle Size and PEG Density Controls Serum Protein Adsorption and Macrophage Uptake
Carl D Walkey 1 4 Jonathan B Olsen 2 3 4 Hongbo Guo 2 3 4 Andrew Emili 2 3 4 Warren Chan 1 4
1University of Toronto Toronto Canada2University of Toronto Toronto Canada3University of Toronto Toronto Canada4University of Toronto Toronto CanadaShow Abstract
Nanomaterials hold tremendous promise as efficient and selective diagnostic and therapeutic agents. However, the inability to control the interaction of nanomaterials with complex physiological systems has prevented realization of their potential. When a nanomaterial is injected intravenously, its surface is rapidly modified by the adsorption of serum proteins, giving it a â?~biological identityâ?T that is distinct from its â?~synthetic identityâ?T. Adsorbed serum proteins promote efficient recognition and clearance of nanomaterials by macrophages of the reticuloendothelial system (RES), leading to preferential accumulation in the liver and spleen. On top of lowering the diagnostic and therapeutic efficiency of nanomaterials, macrophage uptake increases the potential for toxic side-effects. To suppress serum protein adsorption, the surface of a nanomaterial is typically modified with the highly hydrophilic, charge-neutral polymer poly(ethylene glycol) (PEG). Despite its ubiquitous application, little is known about how the design of PEG-modified nanomaterials influences their interaction with serum proteins and macrophages. Here, using a gold nanoparticle model system, we show that a combination of nanoparticle size and PEG surface density controls the adsorption of over 80 different proteins from serum to the nanoparticle surface. Each protein adsorbs preferentially to nanoparticles grafted with PEG at a characteristic density. By modulating the composition of the adsorbed serum protein layer, nanoparticle size and PEG density also control the interaction of nanoparticles with macrophages in an in vitro model. At low PEG densities, nanoparticles are rapidly internalized and accumulate in intracellular vesicles. As PEG density is increased, the efficiency of macrophage uptake decreases. Above ~0.5 PEG/nm^2, macrophage uptake is minimized and is independent of adsorbed serum protein, regardless of nanoparticle size. However, â?~serum-independentâ?T macrophage uptake is more efficient for larger nanoparticles, suggesting that at high densities, PEG preferentially suppresses macrophage uptake of smaller nanoparticles. Ultimately, this study establishes principles for the design of nanomaterials with controlled serum protein adsorption and macrophage uptake, and serves as a framework to evaluate alternatives to PEG with improved performance.
12:00 PM - VV1.8
Gold Nanoparticle-based Assay for Acetylcholinesterase in Cerebrospinal Fluid of Transgenic Mice with Alzheimerrsquo;s Disease
Dingbin Liu 1 Xingyu Jiang 1 Zhuo Wang 1
1National Center for Nanoscience and Technology, China Beijing ChinaShow Abstract
Gold nanoparticle (AuNP)-based assays still face challenges in real samples due to their moderate sensitivity and selectivity. This study improves AuNP-based assay for acetylcholinesterase (AChE) by monitoring the fluorescence of Rhodamine B detached from Au surfaces and the simultaneous color change of AuNPs solutions. The detection limit (0.1 mU/mL) is much lower than existing reported probes for AChE. Such high sensitivity allows the measurement of AChE in the cerebrospinal fluid of transgenic mice even after being diluted over two thousands folds, where tiny amounts of analyte could still be effectively detected while the interferents can be avoided. These measurements show that the level of AChE in transgenic mice that suffer from Alzheimerâ?Ts disease (AD) is lower than that from healthy mice. This assay also allows the monitoring of AChE in transgenic mice treated with different doses of drugs for AD, which provides information on the progression of AD and the effects of treatment. We hope this RB-AuNP-based assay could be useful in monitoring AChE in human CSF for early diagnostics and prognostics of AD especially in combination with other currently existing neuroimaging and CSF biomarkers as well as other settings such as lab-on-a-chip systems.
12:15 PM - VV1.9
Metal Chelating Crosslinkers and Their Application in Hydrogels and Hydrogel Nanoparticles for Non-invasive Imaging
Minnie Chan 1 Adah Almutairi 1
1University of California San Diego San Diego USAShow Abstract
Hydrogels have been widely used as biomaterial. They are usually spatially monitored by the inclusion of fluorescent dyes either entrapped in or conjugated to the gel matrix. Our group is interested in non-invasive imaging of delivery systems in vivo, such as by Magnetic Resonance Imaging (MRI). Towards this aim, we have developed metal chelating crosslinkers that are able to form both bulk hydrogels as well as hydrogel nanoparticles. When chelate with Gadolinium (Gd), these gels become active MRI contrast agents, which enjoy a high relaxivity that can be altered on gel density. Gd and Eu (Europium), a luminescent lanthanide, can also be concurrently chelated, giving these gels dual modality imaging properties. Hydrogel nanoparticles can also function as theranostic delivery vehicles when formulated to contain biomolecules. Incorporation of an acid-degradable crosslinker allows for release of these contained biomolecules. We are investigating the in vivo applications of these gel systems as trackable imaging systems and non-invasive monitoring of gel integrity.
12:30 PM - VV1.10
Hybrid Lipid-Polymer Nanoparticles for Safe and Effective Delivery of Small Interfering RNA
Jinjun Shi 1 2 Alexander R Votruba 1 Xiaoyang Xu 1 2 Zeyu Xiao 1 2 Robert Langer 2 Bruce R Zetter 3 Omid C Farokhzad 1
1Brigham and Women's Hospital, Harvard Medical School Boston USA2Massachusetts Institute of Technology Cambridge USA3Childrenrsquo;s Hospital Boston, Harvard Medical School Boston USAShow Abstract
Since the discovery of RNA interference (RNAi), by which the activity of specific genes can be efficiently suppressed, synthetic small interfering RNA (siRNA)-mediated RNAi has been of substantial interest for various disease treatments. However, the safe and effective delivery of siRNA to target cells remains a major hurdle for its widespread clinical applications. Herein, we will present a novel nanoparticle (NP) platform to tackle the challenges associated with siRNA delivery, using biodegradable and biocompatible polymers and lipids. The hybrid lipid-polymer NP has a differentially charged hollow core/shell nanostructure which provides the delivery system with three distinct functional features: (i) a positively charged inner hollow core for dense loading of siRNA; (ii) a middle hydrophobic polymer layer for sustained siRNA release; and (iii) a relatively neutral lipid-polyethylene glycol (PEG) surface to keep the NP stable and prolong its systematic circulation. Moreover, the hybrid NPs can be formulated in a simple and robust way that may facilitate future scale-up. By screening various factors, such as inner cationic lipids, outer PEG chain length, and formulation parameters, the hybrid NPs show excellent in vitro knockdown efficacy at low doses of siRNA, with greater than 80% luciferase silencing at an siRNA dose of 1.0 pmol (10 nM) and over 95% silencing at a dose of 4.0 pmol (40 nM). In addition, these hybrid NPs demonstrate promising in vivo results for delivering siRNA to luciferase-expressing xenograft tumors. Furthermore, we are applying this NP platform to co-deliver siRNA (e.g., anti-MDR1) and chemotherapeutic drugs (e.g., taxanes) carried within the hydrophobic polymer layer for the treatment of multidrug resistant cancers.
Symposium OrganizersXiaoyuan (Shawn) Chen, National Institute of Biomedical Imaging and Bioengineering National Institutes of Health
Jinwoo Cheon, Yonsei University
Mansoor Amiji, Northeastern University
Shuming Nie, Emory University
VV4: Nanomedicine for Molecular Imaging and Therapy IV
Wednesday PM, April 11, 2012
Marriott, Golden Gate, Salon B
2:30 AM - *VV4.1
Use of High Throughput Discovery at the Nano/Bio Interface to Improve Nanotherapeutics and Nanosafety
Andre Nel 1
1UCLA David Geffen School of Medicine Los Angeles USAShow Abstract
In both the design of safe and improved nanocarrier systems for delivery of drugs as well as safety assessment of commercial nanoparticles such as metals, metal oxides and carbon nanotubes, it is required that we understand how the physicochemical characteristics of the engineered nanomaterials relate to biological responses such as cellular uptake, biodistribution, bioavailability and the catalysis of potentially useful or hazardous biological responses the nano/bio interface. While this is a potentially rewarding platform for discovery, the number of perturbations at the nano-bio interface is potentially overwhelming and requires the use of high content and high throughput screening approaches to perform modeling and predictions. My talk will delineate the implementation of high throughput methodology for cells and zebrafish and describe how the systems can be used for the safety assessment of nanomaterials in general as well as the improvement of nanoparticles that can be used for drug and siRNA delivery. I will describe how the use of compositional and combinatorial nanomaterial libraries are being used to elucidate the material properties that drive biological injury response pathways as well as how to use a multifunctional mesoporous silica nanoparticle drug delivery system to improve safety, biodistribution and therapeutic efficacy through redesign of size, shape, and surface functionalization. I will also show how in silico data transformation and decision-making tools can help to speed up the rate of discovery.
3:00 AM - VV4.2
Cancer Cell Targeting by a Novel Coating Material for Carbon Nanotubes
Magdalena Swierczewska 1 2 Ki Young Choi 2 Seulki Lee 2 Xiaoyuan Chen 2
1Stony Brook University Stony Brook USA2NIBIB/NIH Bethesda USAShow Abstract
Although single-walled carbon nanotubes (SWCNTs) have biomedical potential as drug carriers, imaging agents, and photothermal therapeutics, they are greatly limited in their application due to insolubility and lack of targetability. We developed a unique and facile SWCNT coating by noncovalent absorption that simultaneously offers: a) effective solubility in physiological conditions, b) cancer cell targetability and uptake, and c) intracellular cancer biomarker detection capabilities, with the use of a hydrophobically modified, FDA-approved hyaluronic acid (HA). The coating, hyaluronic acid-5Î²-cholanic acid (HACA), interacts hydrophobically between the graphene wall of the nanotube and the steroid, cholanic acid, conjugated on HA. The coating is performed by simple sonication with HACA and SWCNTs, without the need for additional chemical modification. HACA is biocompatible, cell-permeable, non-immunogenic and specifically binds to cancer cells that overexpress CD44, a cell-surface glycoprotein responsible for tumor metastasis. In addition, it can be degraded by hyaluronidase (HYAL), a prostate, bladder, head and neck cancer biomarker. Since CNTs efficiently quench fluorophores from the visible to near infrared region, dyes labeled onto HACA coated SWCNTs (HACA-SWCNTs) can activate fluorescence upon HA degradation by HYAL. Thus, HACA-SWCNTs serve as platforms for intracellular activatable probes for cancer cell imaging. First, HACA-SWCNTs were characterized by TEM, AFM, Raman spectroscopy and for stability in physiological conditions. Next, HACA-SWCNTs were tested for cell uptake and cancer cell imaging capabilities. The fluorescent dye, FITC, was conjugated onto HACA and wrapped onto the SWCNT surface. FITC fluorescence was quenched when in contact with the strong absorbing nanotube. After HA degradation by HYAL, the FITC is released from the nanotube and fluorescence is recovered. By fluorescence imaging and Raman mapping, significant uptake of FITC-HACA-SWCNTs in CD44 positive, cancer cell lines is seen over CD44 negative, non-cancerous cells. After confirming cancer cell targetability and cell uptake, FITC fluorescence recovery time curves were studied at various concentrations of enzyme and HACA-SWCNTs. Specificity for HYAL was examined by fluorescence activation with other extracellular degrading enzymes, like matrix metalloproteinases. HACA-SWCNTs exhibit high activation and specificity for HYAL. In conclusion, FITC-HACA coating simultaneously offers the CNT solubility, cancer cell targeting, high cancer cell uptake, and finally, specific fluorescence activation upon exposure to a cancer biomarker. Work is underway to use SWCNTsâ?T high absorption properties and efficient transfer of absorbed energy into photothermal energy as a photothermal therapy agent. In this way, a true theranostic material can be engineered that targets and images cancer cells, induces localized apoptosis by photothermal therapy, and monitors its therapeutic efficacy.
3:15 AM - VV4.3
SERS Biotags (SBTs) for the Quantitative Ratiometric Discrimination between Noncancerous and Cancerous Prostate Cells
Alessia Pallaoro 1 Gary Braun 2 Martin Moskovits 1
1University of California Santa Barbara Santa Barbara USA2Sanford Burnham Medical Research Institute - UCSB Santa Barbara USAShow Abstract
Early and rapid identification of malignant cells is a central goal in cancer research. Normal Raman signals from cells and their infrared (FTIR) absorbance patterns have been previously used to differentiate between cancer and normal cells, while surface-enhanced Raman spectroscopy (SERS) has been used as an alternative immunohistochemistry tool for the detection of biomarkers in biological fluids or in vivo, and for cancer detection from blood. SERS biotags (SBTs) can be routinely synthesized and simultaneously excited with a single, very low intensity laser source, making the determination of the relative contribution of the individual SBTs to the overall spectrum tractable. Importantly for biomedical applications, SERS employs tissue-penetrating lasers in the red to near-infrared range resulting in low autofluorescence. Here, we describe a multiplexed, ratiometric method that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro based on neuropilin-1 (NRP-1) overexpression. SERS biotags are made from polymer-encapsulated silver nanoparticle dimers infused with unique Raman reporter molecules, and carry peptides as recognition moieties. Two sets of SBTs were used: One targets the NRP-1 receptors of cancer cells and the other functions as a positive control (PC). Point-by-point 2D Raman ratio maps were constructed with subcellular resolution from cells simultaneously incubated with the two sets of SBTs while in suspension, thus simulating the cellsâ?T capture from blood. We show that using SBTs ratiometrically can provide highly statistically significant, quantitative measures of receptor overexpression insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, and turbidity.
3:30 AM - VV4.4
Study of Cellular Reponse to Functional Silicon Nanowires Using Nonlinear Optical Imaging
Chen Yang 1 Weixia Zhang 1 Ji-xin Cheng 2
1Purdue University West Lafayette USA2Purdue University West Lafayette USAShow Abstract
Interfacing nanostructures with biology has shown great promise for imaging and treatment of diseases. However, the translation of nanomedicine to clinical setting has been hampered by the limited fundamental understanding of the interactions between nanomaterials and cells. To establish this understanding, and thus enable rational design of nanomedicine, we aims at understanding the role of nanostructure in regulating cellular activities, as well as evaluating the potential of bio-conjugated nanostructures in therapeutic applications. In this work, we first performed the non-linear optical imaging study of silicon nanowires. This intensive and stable intrinsic optical signal from nanowires, together with depth resolution offered by non-linear optical imaging, enabled intravital imaging of nanowires for the first time. We demonstrated intravital imaging of nanowires circulating in the peripheral blood of a mouse and mapping of nanowires accumulated in organs. Using intensive and intrinsic nonlinear optical signal of SiNWs, we visualized the interaction between the folate and amine group functionalized SiNWs and cells by monitoring the cellular uptake and intracellular distribution of SiNWs in real time. We found strong specific ligand-receptor interaction between folate on NWs and folate receptors on cell membranes greatly expedited agglomeration of folate modified SiNWs on cells and internalization of NWs. Weaker non-specific charge-charge attraction led to longer time required for amino group modified SiNWs to be bound on cell membrane. No effective accumulation was noticed for unmodified SiNW with native oxidized surface layer, which had the weakest dipole-charge interaction. Length effect of NWs on cellular response was also investigated. Our investigation suggested that silicon nanowire system provides an excellent system to study bio-interactions with 1-d nanomaterials, with its unparalleled capability in size and shape control, its intensive intrinsic nonlinear optical signal for imaging, and the flexible chemistry on silicon surface.
3:45 AM - VV4.5
Bacteriophage MS2 Virus-Like Particles (VLPs) - A Combinatorial Platform for Affinity Selection and Targeted Delivery of Therapeutics
Mekensey D Buley 1 Katharine Epler 5 6 Carlee Ashley 2 8 Cheryl Willman 8 9 Stephan Grupp 10 Jeffrey Brinker 4 3 8 Walker Wharton 9 David Peabody 7 David Padilla 5
1University of New Mexico Albuquerque USA2Sandia National Labs Livermore USA3Sandia National Labs Albuquerque USA4University of New Mexico Albuquerque USA5University of New Mexico Albuquerque USA6University of New Mexico Albuquerque USA7University of New Mexico Health Sciences Center Albuquerque USA8University of New Mexico Health Sciences Center Albuquerque USA9University of New Mexico Health Sciences Center Albuquerque USA10The Childrens' Hospital of Philadelphia Philadelphia USAShow Abstract
Over the past decade, targeted delivery of chemotherapeutic drugs using nanoparticle carriers has become a major investment area in cancer research. Phage display is commonly employed to identify peptides that can direct the cell-specific binding and internalization of targeted nanocarriers. After characterization and sequencing of affinity-selected phage clones, targeting peptides are typically produced synthetically and conjugated to the nanocarrier surface. Transfer of the peptide to a different structural environment frequently results in reduced affinity for the target receptor, however. To address this problem, we have developed virus-like particles (VLPs) of bacteriophage MS2 as both a drug delivery vehicle (see the cover article of the July 26, 2011 edition of ACS Nano) and a platform for affinity selection of targeting peptides from random sequence libraries. Thus, the structural context present during affinity optimization of a targeting peptide is strictly preserved during delivery. Here we present the development and optimization of MS2 VLPs that target a component of the thymic stromal lymphopoietin receptor (CRLF2), which is over-expressed by high-risk pediatric acute lymphoblastic leukemia (ALL). We performed affinity selections against a recombinant form of human CRLF2, as well as a murine leukemic cell line (BaF3) that was transfected to over-express CRLF2. By including counter-selections against parental BaF3 cells, we favored selection of CRLF2-specific clones. After five rounds of selection, we recovered twelve heptapeptides that have a 50- to 1000-fold higher affinity for BaF/CRLF2 cells than for parental BaF3 cells; the three highest affinity peptides (Kd values = 10-50 nM) were selected for further analysis. We employed flow cytometry and confocal fluorescence microscopy to characterize the binding and internalization properties of VLPs that display 90 copies of CRLF2-specific peptides. Our results demonstrate that CRLF2-targeted VLPs selectively bind to BaF3/CRLF2 cells, as well CRLF2-positive human leukemic cells, and that PEGylation of the VLP capsid reduces non-specific interactions. We are currently investigating the biodistribution of CRLF2-targeted and wild-type VLPs within NOD/SCID mice engrafted with CRLF2-positive and CRLF2-negative human leukemic cells. If preliminary in vivo results demonstrate that CRLF2-targeted VLPs co-localize with CRLF2-positive cells (transfected to express click beetle green luciferase for imaging purposes), we will encapsidate and deliver siRNA that silences expression of cyclin B1 in order to induce G2/M arrest and apoptosis of CRLF2-positive ALL. This work is supported by the Excellence in Engineering Research Fellowship and the Laboratory Directed Research and Development Program at Sandia National Laboratories.
4:30 AM - *VV4.6
Supramolecular Structures Self-assembled from Engineered Block Copolymers for Theranostics Nanodevice
Kazunori Kataoka 1
1University of Tokyo Tokyo JapanShow Abstract
Nanotechnology-based medicine (Nanomedicine) has received progressive interest for the treatment of intractable diseases, such as cancer, as well as for the non-invasive diagnosis through various imaging modalities. Engineered polymeric nanodevices with smart functions play a key role in Nanomedicine, including drug carriers, gene vectors, and imaging probes. This presentation focuses present status and future trend of the development of polymeric nanodevices particularly for drug and gene delivery. Polymeric nanodevices with 10 to 100 nm in size can be prepared by programmed self-assembly of block copolymers in aqueous entity. Most typical example is polymeric micelles with distinctive core-shell architecture. Several micellar formulations of antitumor drugs have been intensively studied in preclinical and clinical trials, and their utility has been demonstrated. Compared with conventional formulations, such as liposomes, polymeric micelles have several advantages, including controlled drug release, tissue penetrating ability and reduced toxicity. Critical features of the polymeric micelles as drug carriers, including particle size, stability, and loading capacity and release kinetics of drugs, can be modulated by the structures and physicochemical properties of the constituent block copolymers. The development of smart polymeric micelles that dynamically change their properties due to sensitivity to chemical or physical stimuli is the most promising trend, directing to the targeting therapy with high efficacy and ensured safety. Notable anti-tumor efficacy against intractable cancer, including pancreatic cancer, of DachPt-incorporated polymeric micelles with pH-responding property was demonstrated to emphasize a promising utility of Nanomedicine for the cancer treatment. Indeed, five different formulations of polymeric micelles loaded with anti-cancer drugs have already been in clinical trial worldwide, including Japan, Taiwan, Singapore, UK, France, and USA, featuring high utility of polymer-based therapeutics.
5:00 AM - VV4.7
Biodegradable Polymeric Nanoparticles for Gene Delivery: The Effect of Polymer Structures on Nanoparticle Stability and Transfection Efficiency
Zhe Zhang 3 Michael Keeney 1 Fan Yang 1 2
1Stanford University Stanford USA2Stanford University Stanford USA3Tsinghua University Beijing ChinaShow Abstract
Introduction Gene therapy offers a promising approach for promoting stem cell lineage-specific differentiation by up-regulating inductive genes via DNA delivery, and/or down-regulating inhibitory genes via RNA interference delivery. Cationic polymers can complex plasmid DNA into nanoparticles (NPs) and are potentially safer than viral based approach. We have previously reported that freshly prepared biodegradable nanoparticles using poly(Î²-amino)esters (PBAEs) can deliver DNA into stem cells with high transfection efficiency and low toxicity. To facilitate regulating cell fate in situ, NPs released from scaffold overtime that maintains its ability to transfect cells would be highly desirable. The goal of this study is to characterize the effects of polymer chemical structure on NP stability and the ability of NPs incubated over different time to transfect cells. We hypothesized that small changes in the polymer structure may enhance the transfection efficiency of NPs over time by controlling the degradation rate of polymeric nanoparticles. Materials and Methods To evaluate the effects of polymer structures on NP stability, a subset of PBAEs with increasing hydrophobicity were synthesized. Nanoparticles were formed by mixing PBAEs and plasmid DNA at the optimized ratios as we previously reported. To examine NP stability, NPs were then incubated for different time (2 hr-5 days) before analysis. Freshly prepared NPs were included as a control. NPs stability was evaluated using electrophoresis (for NP integrity) and dynamic light scattering (for particle size and surface charge) using a ZetaSizer Nano ZS. To quantify the amount of free DNA dissociated from complexed NPs over time, NPs were treated with or without DNase and PicoGreen assay was conducted to measure the amount of free DNA. To examine the ability of NPs to transfect cells, human Embryonic Kidney Cells (HEK293) were transfected using NPs formed using different PBAE polymers and GFP plasmid. Outcome was analyzed using fluorescence imaging. Results and Discussion PBAEs with varying hydrophobicity all formed NPs of comparable size upon mixing with DNA plasmids. NPs formed using PBAE with the least hydrophobicity led to the fastest NP dissociation as early as 6 hours, and free DNA could be detected by PicoGreen assay. Dynamic light scattering also demonstrated a decrease in particle size as the time of incubation increased. NPs incubated over 12 hours before transfection showed a significant decrease in their transfection efficiency. PBAEs with increasing hydrophobicity demonstrated enhanced NP stability, with significant transfection detected using NPs incubated over 96 hours prior to transfection. Our results suggest that NP stability may be controlled by controlling the hydrophobicity of the cationic polymer, which would be of great use for designing optimal polymers for sustained gene delivery to regulate cell fate in situ.
5:15 AM - VV4.8
Selective Release from Gold Nanorods for Applications in Nanomedicine
Kimberly Hamad-Schifferli 1
1MIT Cambridge USAShow Abstract
The synergistic combination of nanotechnology and biology has resulted in numerous of innovative approaches for using biomolecules as machines, new therapies for diseases, and biological and biomolecular sensors. One of the most exciting prospects of nanotechnology is that nanoparticles can act as a â?ohandleâ? by which one can control nanoscale processes, particularly biological ones. We use laser excitation of gold nanorods to control the release of multiple species independently. Ultrafast laser excitation at the nanorod longitudinal surface plasmon resonance (SPR) heats the nanorod to a high local temperature, inducing melting, which can release biomolecules conjugated to the nanorod. Because the SPR is tunable by changing nanorod aspect ratio, nanorods with different aspect ratios can be excited independently at different wavelengths. We exploit this property for selective and mutually exclusive release of two distinct DNA oligonucleotides, and show that the released DNA is still functional. Furthermore, one of the biggest barriers for effective use of nanoparticles in biology is non-specific adsorption, where proteins and DNA non-covalently stick to nanoparticles. Typically, non-specific adsorption is viewed as a major hindrance to nanobiotechnology. We have developed methods to quantify interface effects, and are also exploiting the unique properties of the nano-bio interface to improve utility of nanoparticles for a variety of applications in nanomedicine.
5:30 AM - VV4.9
Improved Cellular Uptake of Drug-containing Polymer Nanoparticles through Surface Charge-Stabilization
Sai Archana Krovi 1 Elden P Swindell 1 Thomas V Orsquo;Halloran 1 SonBinh T Nguyen 1
1Northwestern University Evanston USAShow Abstract
Polymer nanoparticles (PNPs) have emerged as promising polymer-based drug delivery vehicles to cancer cells due to their high drug loading, narrow particle size distributions, and tailorable surface functionality for covalent attachment of targeting ligands, imaging agents, and other biologically active species. Previously, we have demonstrated that drug-containing amphiphilic block copolymers undergo directed assembly into therapeutically active core-shell PNPs of tunable diameter (130-1600 nm) and up to 50% of particle weight as drug. However, these drug-loaded PNPs suffer from poor stability in physiological conditions, limiting their application as an in vivo therapeutic delivery platform. In this presentation, we discuss the stabilization of PNPs in biological buffers through amine functionalization post-nanoparticle formation. Surface amine quantification studies indicated that a minimum density of 1.59x10-4 amino groups per nm2 was required to prevent PNP aggregation in buffers. Exposing human breast cancer cell lines to the amine-functionalized PNPs results in greatly inhibited cell proliferation compared to a non-functionalized PNP control.
5:45 AM - VV4.10
Targeted Delivery of Therapeutic RNA to Virally-infected Host Cells via Nanoporous Particle-supported Lipid Bilayers (Protocells)
Carlee Ashley 1 Katharine Epler 2 Oscar Negrete 1 Eric Carnes 2 3
1Sandia National Labs Livermore USA2University of New Mexico Albuquerque USA3University of New Mexico Albuquerque USAShow Abstract
Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family, has been classified as a BSL-4 select agent due to its numerous routes of transmission and the high mortality rates associated with infection. Despite recent advances in understanding the cellular tropism of NiV, treatment remains primarily supportive. To this end, we have developed nanoporous particle-supported lipid bilayers (â?~protocellsâ?T - see Nature Materials (2011) 10: 389-397) that specifically deliver high concentrations of therapeutic RNA to host cells stably transfected with NiV genes. To generate peptide-targeted, siRNA-loaded protocells, we first soaked porous silica nanoparticles (100-nm in diameter with 10-nm pores) in a solution of siRNA that silences expression of NiV nucleocapsid protein (NiV-N); due to its high surface area, the nanoporous silica core can be loaded with high concentrations (50-65 wt%) of siRNA. Liposome fusion to siRNA-loaded cores results in a supported lipid bilayer (SLB) that promotes long-term (>3 months) cargo retention and provides a fluid interface for ligand display. To generate targeted protocells, we employed phage display to identify peptides that bind to ephrin B2 (EB2), a protein expressed by human endothelial cells and neurons that acts as the primary receptor for NiV entry via macropinocytosis. TGAILHP (specific clone-1, or SC1) was the predominant sequence after five rounds of affinity selection against CHO-K1 cells stably transfected with human EB2. Using flow cytometry, we found that SC1-targeted protocells have a nanomolar affinity (Kd = 0.25-1.5 nM) for EB2-positive cells (CHO-K1/EB2 and HEK 293) at both high (1.50 wt%) and low (0.015 wt%) valencies. Importantly, protocells modified with 0.015 wt% of SC1 and 15 wt% PEG-2000, which promotes colloidal stability and reduces non-specific interactions, have a 1000-fold higher affinity for EB2-positive cells than for EB2-negative cells (parental CHO-K1). Using confocal fluorescence microscopy, we determined that SC1-targeted protocells are rapidly internalized by both CHO-K1/EB2 and HEK 293 and that pre-treatment of cells with various macropinocytosis inhibitors reduces uptake by 60-80%. To promote macropinosome escape, we further modified the SLB with a histidine-rich fusogenic peptide (H5WYG), which disrupts macropinosomal membranes via the proton-sponge mechanism and promotes cytosolic distribution of siRNA. We have found that SC1-targeted, siRNA-loaded protocells silence 98% of NiV-N mRNA and protein expression in EB2-positive cells without affecting NiV-N levels in EB2-negative cells. Furthermore, 4 mg/kg of SC1-targeted protocells silence ~90% of GFP expression in mice pre-exposed to a NiV pseudovirus that transfects host cells with GFP upon infection. Due to their enormous cargo capacity, as well as their stability and specificity, protocells show promise as delivery vehicles for therapeutic agents capable of preventing viral replication and transmission.
VV5: Poster Session
Wednesday PM, April 11, 2012
Marriott, Yerba Buena, Salons 8-9
9:00 AM - VV5.10
Targeted Delivery of Therapeutic Agents to Hepatocellular Carcinoma via Nanoporous Particle-supported Lipid Bilayers
Katharine Elizabeth Epler 3 5 Dan C Wilkinson 3 Robert E Castillo 3 David P Padilla 3 Trevin Heisey 3 Brian S Wilkinson 3 Natalie Johannes 3 Cameron Burgard 3 Jason Townson 3 Jeffrey Brinker 2 4 6 Carlee Ashley 1 4 Eric C Carnes 2 4
1Sandia National Laboratories Livermore USA2University of New Mexico Albuquerque USA3University of New Mexico Albuquerque USA4University of New Mexico Health Sciences Center Albuquerque USA5Sandia National Laboratories Albuquerque USA6Sandia National Laboratories Albuquerque USAShow Abstract
Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate the side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including high specificity, enhanced colloidal stability, extended circulation times, effective endosomal escape, improved intracellular targeting, and a high capacity for disparate cargos. To this end, we have developed nanoporous particle-supported lipid bilayers (â?~protocellsâ?T) that synergistically combine properties of liposomes and porous silica nanoparticles. Protocells can be loaded with combinations of therapeutic (drugs and drug cocktails, small interfering RNA, plasmids, protein-based toxins) and diagnostic (quantum dots, iron oxide nanoparticles) agents and modified with targeting and endosomolytic peptides to promote cell-specific binding and endosomal escape, respectively, and with PEG to enhance stability. As we reported in the May 2011 cover article of Nature Materials, the enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma (HCC) cell. We have also successfully used peptide-targeted protocells to deliver a plasmid (pCB1) that encodes a cyclin B1-specific small hairpin RNA to dividing and non-dividing HCC cells with nearly 100% efficiency. We use histones to package pCB1 into ~18-nm particles that are subsequently modified with a nuclear localization sequence and incorporated within the pores of mesoporous silica nanoparticles. Liposome fusion to cargo-loaded cores results in a supported lipid bilayer (SLB) that we modify with PEG, an endosomolytic peptide, and a targeting peptide (MC40) that binds to hepatocyte growth factor receptor, a protein known to be over-expressed by many types of HCC. MC40-targeted protocells have a 1000-fold higher affinity for HCC (Hep3B) than for untransformed hepatocytes and are selectively internalized by Hep3B via receptor-mediated endocytosis. MC40-targeted, pCB1-loaded protocells, furthermore, induce a dose and time-dependent decrease in the expression of cyclin B1 mRNA and protein when exposed to Hep3B in vitro. Effective silencing of cyclin B1 results in rapid G2/M arrest and apoptosis of Hep3B at pCB1 concentrations < 5 pM. We are currently assessing the biodistribution and therapeutic efficacy of MC40-targeted, pCB1-loaded protocells in orthotopic mouse models of HCC. We are, additionally, determining whether protocells can simultaneously deliver a combination of pCB1, the hydrophobic drug, taxol, and siRNA that silences expression of the anti-apoptosis protein, Bcl-2, to mice engrafted with SNU-398, a highly taxol-resistant form of HCC.
9:00 AM - VV5.11
Glutathione-Responsive Silica Nanocarriers with Cyclodextrin Gatekeeper
Chulhee Kim 1 Jeonghun Lee 1 Minji Kim 1 Hyunjeong Kim 1 Hyemi Lee 2 Heon Joo Park 2
1Inha University Incheon Republic of Korea2Inha University Incheon Republic of KoreaShow Abstract
The development of multifunctional nanomaterials with advanced features for combination of therapeutic application has been one of key issues in nanomedicinal research. The multifunctional nanomaterials encompass a wide range of organic and inorganic nanoparticles such as liposomes, dendrimers, polymers, quantum dots, inorganic oxides etc. In particular, silica NPs recently have attracted great interests as a delivery platform for drug molecules or imaging agents due to low cytotoxicity, high chemical stability, structural diversity, and facile loading of guest drugs in the nanochannel. We report on the stimulus-responsive Si-MPs with cyclodextrin (CD) gatekeepers covalently connected onto the particle surface via disulfide unit and their glutathione (GSH)-induced controlled release characteristics of guest molecules from the pore. In particular, we also report a GSH-stimulated release of doxorubicin (DOX), an anticancer drug, from Si-MPs in the GSH-rich cancer cell, A549. The Si-MPs are composed of the surface CD gatekeepers, disulfide stalks as GSH-induced cleavable linker, and DOX or calcein as guests within the porous channels. In addition, for the release experiment in the cell line, the surface of Si-MPs was PEGylated for enhanced solubility in aqueous media. For in vitro experiment, DOX was loaded in the pore of Si-MPs, and its release from Si-MPs was investigated. We have demonstrated that the tethering of the CD gatekeepers linked via disulfide unit to the surface of Si-MPs is a very efficient approach not only to entrap the guest anticancer drug in the pore reservoir but also to release the guest in response to GSH which can remove the gatekeeper by cleavage of the disulfide stalk moiety. In addition, the GSH induced release of DOX from the CD-capped Si-MPs was investigated in the cancer cells. The results indicated that DOX was released from the Si-MP internalized in the cell, which was induced by GSH that cleaved the disulfide unit in the stalk of the CD gatekeeper.
9:00 AM - VV5.12
Effectiveness of microRNA-21 Inhibitor Gene Therapy Combined with Temozolomide for Glioblastoma Depend on the Time and Sequence Influence
Yu Ren 1 Xiaomin Qian 2 Zhendong Shi 3 Xuan Zhou 4 Lixia Long 2 Xubo Yuan 2 Zhenduo Cui 2 Chunsheng Kang 3 Chaoyong Liu 1
1Tianjin Medical University Tianjin China2Tianjin University Tianjin China3Tianjin Medical University General Hospital Tianjin China4Tianjin Cancer Hospital amp; Institute Tianjin ChinaShow Abstract
Substantial studies have shown that down-regulation microRNA-21 (miR-21) can inhibit cancer cell proliferation and induce cell apoptosis in human cancer cell lines. Temozolomide (TMZ) is considered the first-class chemotherapeutics in glioblastoma. In this study, we design to explore the most effective schedule of combination treatment by using PAMAM dendrimer as vector of miR21 inhibitor. Three brain tumor cell lines U251, LN229 and U87 were subjected to miR-21 inhibitor (miR-21i) and TMZ for varying lengths of time and combination schedule (miR-21i before TMZ 4h,8h; TMZ before miR-21i 4h, 8h; and concurrent exposure) to evaluate the anti-tumor effect in vitro. A synergistic anti-proliferative and proapoptotic activity was only obtained when chemotherapy was 4h followed by treatment with miR-21i in both U251 and U87 cells, while the best anti-tumor effect is achieved by using concomitant treatment in LN229 cells. These effects were accompanied by the arrest of the surviving cancer cells in the G2/M phase and by a maximum inhibition in the activated, phosphorylated forms of STAT3. The results show that the time and sequence of administration is a determining factor in glioma combination therapy.
9:00 AM - VV5.13
A New TEM Imaging Technique for Liposomes
Sarah Hoppe 1 Darryl Sasaki 2 David Nackashi 3 John Damiano 3 Khalid Hattar 1
1Sandia National Laboratories Albuquerque USA2Sandia National Laboratories Livermore USA3Protochips, Inc. Raleigh USAShow Abstract
Liposomes have become increasingly important in nanomedicine because of their potential as drug carriers. They can be targeted to bind specific proteins or carry cytotoxic species to specific cells. While the synthesis of these hollow vesicles is well understood, it is more difficult to study their structure-behavior relationship. Imaging liposomes by transmission electron microscopy provides the greatest resolution, but requires special treatment like heavy element staining or cryo-TEM because biologoical samples are difficult to see in the instrument. These techniques can alter the sampleâ?Ts structure and may not be the best representation of functional lipsomes. In this work, recent advancements in microfluidic TEM stages will be utilized to investigate liposome structure in solution. Recently, beta testing has begun on a microfluidic TEM stage from Protochips Inc. that allows for in-situ solution studies with results recorded in real time. This allows for the liposomes to be imaged in their native solution environment without any post-treatment, which gives the most accurate images of the liposomes and their behavior. The ability to do in-situ solution experiments is being applied to watch the liposomes bind to proteins, and opens up a new realm of nano-biomaterial characterization; the current status of this research will be presented. Furthermore, the stage technology as well as initial results involving POPC liposomes will be discussed. Procurement of the microfluidic stage was supported by the Department of Energyâ?Ts Office of Basic Energy Sciences. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.
9:00 AM - VV5.14
Luminescent Silicon Carbide Quantum Dots Prepared by Reactive Bonding and Subsequent Wet Chemical Etching: Characterization and Potential Applications
David Beke 1 Zsolt Szekrenyes 1 Istvan Balogh 1 Adam Gali 1 2
1Research Institute for Solid State Physics and Optics Budapest Hungary2Budapest University of Technology and Economics Budapest HungaryShow Abstract
Visual analysis of biomolecules is an integral avenue of basic and applied biological research. Quantum dots (QDs) are semiconductor inorganic nanoparticles that are emerging as alternative or complementary tools to the organic fluorescent dyes currently used in bioimaging. In comparison with traditional organic fluorophores, QDs have a number of advantages including broad excitation and narrow emission spectra. QDs are more resistant to photobleaching than their organic counterparts, making QDs as a superior alternative of bio-imaging and further its applications in basic and applied biology. QDs are often made from of group II and VI elements (e.g. CdSe and CdTe) or group III and V elements (e.g. InP and InAs). Although these QDs have great potential as probes for bioimaging, certain limitations may restrict their applications. These QDs were found to be cytotoxic through the release of free metallic ions. Therefore, a protective shell must be systematically added. However, no protective shell can guarantee an efficient chemical isolation of the extremely toxic elements from the living cell environment. Cytotoxicity strongly influencing is one of the major limiting factors for the application of II-VI QDs in efficient in vivo imaging. We propose silicon carbide (SiC) QDs for bioimaging in order to eliminate numerous disadvantages of traditional QDs. SiC is a stable, chemically inert wide band gap indirect semiconductor. SiC QDs, with about 3 eV excitation energy, were successfully fabricated in many ways. The typical diameter is often less than 5 nm. Small size is also of importance in living cell applications for clearance. In addition, biocompatibility of bulk SiC and SiC QDs has been proven by several research teams. We developed a two-step experimental routine of producing SiC QDs. First, microcrystalline SiC (SiC MCs) is formed by reactive bonding method which, principally, allows us to produce highly doped SiC MCs in order to modulate the optical properties of the prepared SiC QDs made from them. SiC QDs form by electroless wet chemical etching of the SiC MCs. Under the etching, highly porous layer is created on the SiC MCs by using an acid mixture of HNO3 and HF. SiC QDs are obtained by sonication of the porous SiC MCs in a specific solvent. SiC QDs make stable suspension in water without any surfactant added or any surface modification thanks to the surface termination that was studied by attenuated total internal reflection infrared spectroscopy (ATR IR). We found clear evidence of surface related IR bands characteristic of Si-O-Si, C-O-C, CH as well as COOH and OH. These polar groups on the surface give hydrophilic characteristic of the SiC QDs and give a chance to functionalize these QDs for sensing selected biomolecules and for therapy.
9:00 AM - VV5.15
Multifunctional Nanoparticles as Imaging Probes and Cooperative Therapeutic Agents in Tumor Cells
Prasad Subramaniam 1 Birju P Shah 1 Ki-Bum Lee 1
1Rutgers University Piscataway USAShow Abstract
This presentation focuses on i) the synthesis of multifunctional nanomaterials for the delivery of various biomolecules (antibodies, siRNA etc.) to cancer cells and, ii) utilizing the functional nanomaterials as multimodal imaging and therapeutic agents against malignant tumors like glioblastoma multiforme (GBM). More specifically, we have synthesized graphitic-carbon protected FeCo MNPs (FeCo/C NPs) using a novel hydrothermal approach, for the targeted delivery of siRNA for sensitizing tumor cells towards hyperthermia and for the selective knockdown of a key oncogene (EGFRvIII) expressed and implicated in brain cancer cells. Furthermore, the FeCo/C NPs could be used as multimodal imaging (MRI and Raman) agents, both in vitro and in vivo. This poster will also enlist our efforts towards the generation of a library of non-toxic ZnS-AgInS2 quantum dots (ZAIS QDs) using a rapid sonochemical synthetic methodology and their application as imaging agents in cancer and stem cells. The ZAIS QDs exhibit excellent biocompatibility for cellular imaging/labeling in mesenchymal stem cells and cancer cells with negligible QD-induced cytotoxicity, thereby making them an attractive alternative over conventional QD-based molecular imaging probes. Collectively, our endeavors would facilitate the development of novel therapeutic and imaging systems aimed at the successful diagnosis and therapy of malignant cancers such as brain and breast tumors.
9:00 AM - VV5.16
Multifunctional Polypeptide-based Nanocarriers for Targeted Drug and Gene Delivery to Tumor Cells
Prasad Subramaniam 1 Dipti N Barman 1 Cheoljin Kim 1 Ki-Bum Lee 1
1Rutgers University Piscataway USAShow Abstract
Even though the survival rate of cancer patients has increased significantly over the last few decades due to advances in the diagnosis and in-depth knowledge of cancer biology, brain cancer remains virtually incurable and the median survival is very short. This has been mainly attributed to the scant availability of delivery platforms capable of simultaneously translocating multiple payloads to the target cells. Hence, an effective treatment against GBM requires the development of novel methods of intracellular delivery that can efficiently deliver anticancer drugs and biomolecules to brain tumor cells at high concentrations in order to activate and inhibit key cellular functions and induce apoptosis of the tumor cells. This talk focuses on the development of a polypeptide-based nanocarrier that can integrate multiple functions in one system, including the capability to accommodate a combination of therapeutic modalities (i.e., nucleic acids and anticancer drugs), cancer cell targeting and controlled drug release. The polypeptide-based nanocarrier synthesized using a facile N-carboxy anhydride (NCA) chemistry, allowed for the attachment of multiple anticancer drugs (SAHA and Doxorubicin) using acid labile or enzymatically degradable linkages, thereby enabling the controlled and sustained release of the drugs in target brain tumor cells. Furthermore, the polypeptides also helped in solubilizing these hydrophobic drugs, thereby increasing their bioavailability and leading to a decrease in the IC50 values. In addition, the presence of multiple lysine residues on the polypeptide was shown to effectively condense and transfect small interfering RNA (siRNA) into brain tumor cells, thereby demonstrating the potential for the co-delivery of nucleic acids (siRNA, miRNA and plasmid DNA) and anticancer drugs for obtaining a synergistic response. When delivered to brain tumor cells, it was observed that the combined delivery of multiple anticancer drugs using our polypeptide-nanocarrier led to a remarkable increase in cell death as compared to the free drugs. The nanocarrier surface could be further modified with antibodies and dyes for cell-specific delivery and imaging. Collectively, the use of our biodegradable polypeptide-based nanocarrier could be used for the simultaneously delivery of multiple payloads in an efficient and target-specific manner to glioblastoma cells. Our synthetic techniques afforded facile manipulation of the polypeptide structure to achieve efficient transfection with minimal nanocarrier-mediated cytotoxicity. This would open new avenues for the treatment of malignant GBM by reducing the systemic toxicity of the traditional anticancer drugs and effectively circumventing drug resistance in brain tumors.
9:00 AM - VV5.17
Enhancement of Gene Delivery Efficiency for Human Neural Stem Cells by Magnetically Guided Penetration
Eunmi Kim 1 Ji-Seon Oh 1 Ik-Sung Ahn 1 Kook In Park 2 Jae-Hyung Jang 1
1Yonsei University Seoul Republic of Korea2Yonsei University College of Medicine Seoul Republic of KoreaShow Abstract
Current virus mediated clinical approaches are broadly explored and gene therapy has been elucidated for identifying the molecular signals such as stem cell fates and cancerous growths, but current delivery vehicles or mechanisms still have limitations to integrate clinical trials. Here, we report that magnetically guided an adeno-associated virus (AAV) delivery system to human neural stem cells (hNSCs). To enhance gene delivery to human neural stem cells (hNSCs), we employed a magnetic force to AAV delivery system. In this system, Adeno-associated viral vector (AAV) was genetically modified to display hexa-histidine (6xHis) on the AAV2 capsid (6xHisAAV). Streptavidin-coated superparamagnetic iron oxide nanoparticles (SPIONs) were used to form complexes with 6xHisAAV vectors, and nickel ions chelated on the NTA-biotin conjugated to streptavidin-coated superparamagnetic iron oxide nanoparticles (NiStNPs). This system resulted in short delivery times on target cells induced by forced penetration of the vectors across the plasma membranes. Also, these NiStNP-6xHisAAV complexes delivery by magnetic force led to significantly enhanced transgene expressions in hNSCs, just in 2~10minutes. Using this magnetically guided AAV delivery system, we have gene delivery efficiency that is up to 3~4 times higher. Magnetically guided 6xHis AAV delivery induced rapid internalization for the significantly enhanced cellular transduction. Additionally, to investigate the energy-dependent endocytosis pathway, cellular transduction by magnetically guided 6xHisAAV vectors at 4Â°C was comparable with that at 37Â°C. Since, it suggests that direct penetration of viral vectors by magnetic forces can be a key factor in enhancing cellular transduction in non-permissive cell types, and this system is a highly efficient AAV-mediated gene delivery system. Reference : E. Kim et al. Biomaterials 32 (2011) 8654~8662
9:00 AM - VV5.18
Preparation of Dendrimer Porphyrin-containing Multilayered Hollow Nanocapsules for Combination Cancer Therapy
Won-Gun Koh 1 Kyung Jin Son 1 Tae Geuk Lim 1
1Yonsei Univ Seoul Republic of KoreaShow Abstract
Multifunctional hollow nanocapsules were prepared via layer-by-layer (LbL) self assembly of dendrimer porphyrin (DP) and poly(allylaminehydrochloride) (PAH) on polystyrene (PS) nanoparticles and the subsequent removal of PS nanoparticles to realize combined chemotherapy and photodynamic therapy (PDT). The formation of multilayer was demonstrated with not only Î¶-potential measurement but also with UV-Vis absorbance and fluorescence emission by virtue of the optical properties of DP. SEM and TEM studies further confirmed successful fabrication of hollow nanocapsules and resultant nanocapsules were stable in the wide range of pH (3-11). Since DP that served as photosensitizing agent for PDT was used as a multilayer component, the amount of DP that determines the strength of PDT effect was easily controlled by changing the number of deposited layers. The cavities of hollow nanocapsules were loaded with doxorubicin (DOX) to implement chemotherapy as well as PDT and sustained release of DOX from the hollow nanocapsules was achieved by controlling the degree of crosslinking between PAH and DP. Cell viability study revealed that combined treatment demonstrated synergetic effect, resulting in higher therapeutic efficacy than chemotherapy and PDT alone.
9:00 AM - VV5.19
Polyethylene Oxide-polystyrene Oxide Triblock Copolymers as Biological-responsive Nanocarriers
Silvia Barbosa 1 Adriana Cambon 1 Ana Rey-Rico 2 Jose Brea 3 Isabel M Loza 3 Stephen G Yeates 4 Carmen Alvarez-Lorenzo 2 Angel Concheiro 2 Pablo Taboada 1 Victor Mosquera 1
1Universidad de Santiago de Compostela Santiago de Compostela Spain2Universidad de Santiago de Compostela Santiago de Compostela Spain3Universidad de Santiago de Compostela Santiago de Compostela Spain4University of Manchester Manchester United KingdomShow Abstract
In the present work, we synthesized two triblock polyethylene oxide-polystyrene oxide triblock copolymers E33S13E33 and E38S10E38, where E denotes oxyethylene and S oxystyrene, and the subscripts the block lengths, and characterized their self-assembling properties in water. The block lengths were selected with the objective of ensuring an optimal compromise between chain solubility, micelle fomation ability and core size for enhanced drug solubilization. We evaluated the biocompatibility of the copolymers and assessed their ability to dissolve and chemically protect the anticancer drug doxorubicin, DOXO. We also analyzed the temporal stability of the drug-loaded micelles and drug release profile. Finally, we studied their efficacy as an antitumoral polymeric formulation in vitro by using a multidrug resistant ovarian tumour cell line (NCI-ADR-RES), with the special aim of analyzing the possible capability of both copolymers as potential P-glycoprotein efflux pump inhibitors to enhance DOXO accumulation in this cell line.
9:00 AM - VV5.2
Ordered Arrays of Single Elongated Chromatin Fragments: Towards the Mapping and Analysis of Multiple Epigenetic Marks
Aline Cerf 1 Harvey C Tian 1 Benjamin R Cipriany 2 Jaime J Benitez 1 Harold G Craighead 1
1Cornell University Ithaca USA2Cornell University Ithaca USAShow Abstract
Chromatin contains valuable information in epigenetic and genetic disorders such as cancer. Analyzing and identifying the placement of multiple epigenetic marks such as DNA methylation and histone modification across the entire genome would represent an important tool for early diagnosis and monitoring of treatment. But the characterization of chromatin in its compacted state hinders obtaining such information that can be more easily read from elongated molecules. Here we show how soft-lithography in combination with molecular elongation can be used to generate arrays of isolated and elongated chromatin fragments onto various supports for imaging and statistical analysis of these fragments. We have performed analysis using fluorescence microscopy, atomic force microscopy and electron microscopy, with electron microscopy providing the possibility to resolve the genomic location of epigenetic marks with single base resolution. In our process, the assembly of chromatin fragments is carried out using a system designed to elongate molecules and transfer those to even the most delicate substrates such as single-atom-thick graphene. In our approach, a controlled volume of chromatin fragments in solution is injected between a fixed glass spreader and a topographically microstructured polydimethylsiloxane stamp. The liquid contact line is then moved over the stamp at a controlled velocity. Chromatin fragments are partially trapped inside the microfeatures of the stamp and elongated. The so-assembled chromatin fragments can then be transfer-printed with high placement accuracy and yield onto a hydrophilic or a hydrophobic substrate such as cover slips for optical or scanning-probe imaging, but also, and for the first time, single-layer graphene support layers on TEM grids for electron imaging and morphological/elemental composition studies. This methodology can handle molecules functionalized in bulk with specific antibodies or energy-loss resolved labels allowing the mapping of multiple epigenetic marks simultaneously.
9:00 AM - VV5.20
Comparison and Correlation between PET and Fluorescence Dynamic Imaging by Using a Dual-labeled Cyclic RGD Analog
Lei Zhu 1 Ning Guo 1 Seulki Lee 1 Xiaoyuan Chen 1
1NIH Bethesda USAShow Abstract
Real-time dynamic imaging methods for quantitative and visual monitoring of certain target expression could provide opportunities for assessing therapeutic intervention. Dynamic PET imaging has long been used as a powerful technique providing quantitative information about dynamic physiological and biochemical processes in vivo. Recent developments in fluorescence imaging techniques have enabled real-time dynamic imaging in small animals. However, the comparison and correlation of PET and fluorescence dynamic imaging is rarely reported. To address this issue, we synthesized a probe that was dually labeled with near-infrared (NIR) dye and 64Cu-DOTA labeled RGD analog and studied its initial biodistribution and dynamic PET and fluorescence imaging results in an MDA-MB-435 tumor model. The resulting probe, NIR Dye-(64Cu-DOTA)-c(RGDyK), was synthesized and its chemical/biological properties were characterized. One hour dynamic PET and fluorescence scans for the dual-labeled probe were implemented and a two-compartment model of pharmacokinetic analysis in the tumor was applied with time activity curves (TACs) derived from the series of dynamic images. Logan plots were implemented in both the optical and PET studies. The TACs were fit linearly, and the distribution volume-ratio (DVR) (1+k3/k4) was determined as the slope of the fitted line. We quantitatively compared the dual labeled RGD peptide uptake parameters in tumor and also correlated the dynamic parameters gained from PET and fluorescence dynamic imaging. We have determined (1) the rates of change of the intensity of the probe in tumor and (2) the rate of uptake and wash-out in the animal. In addition, body compartment data provided information on the temporal distribution of the agent during the experiment and acted as input for rate-of-change or body compartment models. We found a good correlation between PET and fluorescence modalities in probe concentration and spatial signal distribution. The binding potential of dynamic fluorescence imaging was well correlated with that of PET dynamic imaging. This finding suggests that dynamic fluorescence imaging is a useful tool for pharmacokinetic modeling and can potentially be used as a surrogate for dynamic PET imaging for biodistribution studies.
9:00 AM - VV5.21
NMR Force Microscopy of Ammonium Sulfate with 12mu;m Resolution
Isaac Vicente Manzanera 1 John T Markert 1
1University of Texas Austin USAShow Abstract
We report details of the construction and use of a variable temperature nuclear magnetic resonance (NMR) force microscopy probe, as well as the development of its control system for three dimensional nanoscale imaging and spectroscopy. The probe contains two 3-axis piezo-driven slip-stick motion stages for fiber interferometer and for gradient magnet positioning. The control station is a LabView-software-based control system capable of performing signal generation, data acquisition, and analysis. Position-dependent NMR force measurements on a 25x15x7 Î¼m3 single crystal of ammonium sulfate (NH4)2SO4 were performed at room temperature in a sample-on-oscillator configuration. Force signals were detected with a signal-to-noise ratio of 6 and 12 Î¼m resolution in a one-dimensional scan. Measurements of NMR relaxation times T2* = 1.5Â±0.2 Î¼s, T2 = 44Â±2 Î¼s, and T1 = 5.6Â±0.7s were obtained, thus opening a window to dynamical 3-D imaging scans in materials research on single crystals with resolution on the micron scale at room temperature, and at sub-micron scales at lower temperatures.
9:00 AM - VV5.22
Gold-silver Alloy Nanoparticle Interaction with Optically Active Proteins
Don N Ho 1 Andy Blaeser 2 Arto Nurmikko 2 Shouheng Sun 1
1Brown University Providence USA2Brown University Provid USAShow Abstract
Surface enhanced fluorescence from nano-sized metal materials occurs through near-field coupling of the local electromagnetic field. We focus on controlling the surface plasmon resonance of AuAg alloy nanoparticles (AuAgNP) with a tunable method for transferring energy with fluorescent proteins. Our current work focuses on synthesis, surface modification, and localization of AuAgNPs to protein targets. We use fluorescein isothiocyanate as a model fluorophore to measure optimal energy transfer conditions. We also examine green fluorescent protein as a target protein fluorophore for the protein model. Determination of alloy content, distance control, and targeting abilities for enhanced fluorescence can optimize current fluorescence imaging techniques and assays with greater sensitivity. Our aim is to further use particles in vitro to examine optically active proteins and fluorophores.
9:00 AM - VV5.23
Organocatalytic Polymerization of Cyclic Esters and Carbonates for the Formation of Unimolecular Delivery Vehicles
Amanda C Engler 1 Daniel J Coady 1 Yi Yan Yang 2 James L Hedrick 1
1IBM San Jose USA2Institute of Bioengineering and Nanotechnology The Nanos SingaporeShow Abstract
In the field of nanostructured biomaterials for drug delivery, it is widely accepted that both size and shape play critical roles in the ability of particles to navigate biological barriers. A common method utilized to achieve vehicles with precise shape, size, an