Proper cell-cell communication through physical contact is crucial for a range of fundamental biological processes including, cell proliferation, migration, differentiation, and apoptosis and for the correct function of organs and other multi-cellular tissues. The spatial and temporal arrangements of these cellular interactions in vivo are dynamic and lead to higher-order function that is extremely difficult to recapitulate in vitro. The development of 3-dimensional (3D), in vitro model systems to investigate these complex, in vivo interconnectivities would generate novel methods to study the biochemical signaling of these processes, as well as provide platforms for tissue engineering technologies. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts in 3D through chemoselective cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. This strategy uses liposome fusion for the delivery of ketone or oxyamine groups to different populations of cells for subsequent cell assembly via oxime ligation. We demonstrate how this method can be used for several applications including, the delivery of reagents to cells for fluorescent labeling, the formation of small, 3D spheroid cell assemblies, and the generation of large and dense, 3D multi-layered tissue-like structures.

A great effort is being carried out to successfully establish drug delivery systems (DDS) since, especially for cancer treatment, DDS hold tremendous potential to overcome and/or attenuate undesired side effects from drugs while enhancing their therapeutic effectiveness. As a matter of fact, as much as ~60% of new chemical entities fail clinical trials not because of a lack of biological activities per se but due to poor pharmacokinetics, undesirable metabolic properties, and toxicity.¹ Despite the large amount of different DDS and drugs with antitumor activity, the in vitro approach to test them has remained relatively undeveloped in the past decades and mainly 96-well plates with pre-cultured cells at static conditions are employed, and in vivo experiments are often the only way out. However, due to high costs and ethical reasons, a better in vitro pre-screen approach would be highly beneficial, since any DDS administered intravenously will get in contact with the circulatory system of the human body, and static experimental systems fail to consider major physical factors of the blood stream such as the shear stress (tau;).
Since liposomes (L) are a dominant class of DDS² and polyethylene glycol (PEG) coated L result in long circulating particles able to escape from the Reticulo-Endothelial (RES) or Mononuclear Phagocytic (MPS) Systems, herein we present a new simple and flexible method to coat L for stabilization and functionalization purposes via poly(dopamine) (PDA) “self-“ polymerization.³ Since PDA coated L can be subsequently PEGylated in one step (L_PEG), we evaluated the association with myoblast cells of L and L_PEG in the absence or presence of tau;. Our results show a significant higher association with cells for L when compared to L_PEG under the effect of tau; when compared to static conditions (tau;₀), indicating that the low-fouling properties of PEG are also valid under the influence of tau;, thus confirming the potential of L_PEG as a stealth DDS under flow conditions.
To assess if the presence of tau; can enhance the therapeutic response of the cells, we encapsulated Thiocoraline, a potent anticancer drug, in L and assessed the cell viability (CV) after 24h. The reduction in CV was significantly higher when comparing tau; vs tau;₀, and, importantly, for unloaded L, the CV of the myoblasts was not affected by the tau; and the pristine L.
Furthermore, since gene therapy also holds great potential for cancer treatment, we delivered a dsRNA oligomer together with Lipofectamine designed for use in RNAi analysis as an indicator of the transfection efficiency (TE) under flow conditions and observed a significant increase in both TE and amount of transfected cells in the presence of tau; as compared to tau;₀.
1.Wang, Drug Delivery: Principles and Applications. 2005.
2.Farokhzad, ACS Nano 2009.
3.van der Westen, Biointerphases 2012.

Due to its potential for detecting ultra-low levels of biomolecules in solution, methods for developing surface-enhanced Raman spectroscopy (SERS) diagnostics has been studied extensively in recent years. The largest amplification in Raman signal is obtained when a Raman active molecule is placed directly within a gap, or “hotspot,” between two metal nanoparticles spaced a few nanometers apart. To take advantage of this, we have designed a conformationally switchable nanoparticle-based “nanodumbbell” probe that undergoes significant changes in interparticle distance before and after addition of an analyte. The nanodumbbell (NDB) probe is composed of two Au@Ag core-shell nanoparticles joined together by a strand of Cy3-labeled, dithiolated DNA containing a hairpin sequence. The NDB is first fully extended to ~10nm after hybridization with an ATP aptamer. After ATP is added to the NDB, the ATP-aptamer interaction causes full dehybridization from the NDB. This leaves the single stranded DNA-NP strand to form a hairpin, thereby reducing interparticle spacing to ~2nm. This dramatic decrease in interparticle spacing caused significant enhancement in Raman signal from the Cy3 molecule in direct response to ATP binding. Control experiments using CTP, GTP, and no ATP showed no increase in Raman signals. The detection limits thus far are in the pmole range with the potential for higher sensitivities. The ability to create switchable discrete Au@Ag core-shell nanoparticle conjugates that respond to a single binding event with a biomolecular analyte paired with the high sensitivity of SERS will have great potential for the next generation of biosensors.

The ability to regulate cellular activities in a controlled manner is one of the most challenging issues in field ranging from cell biology to biomedicine. In contrast to conventional biochemical ligands, magnetic nanoparticles have the potential of becoming useful tools for controlling cell signaling pathways in a space and time selective fashion. Because these particles can be coupled with magnetic field to produce enough force to initiate the actuation of biological objects, this magnetic stimulation system can switch the cellular activity on and off. In addition, the nanoscale dimensions of nanoparticles make them beneficial for probing cellular sensory structures and functions at the molecular level and for inducing specific cellular activation process. In this study, we demonstrate that magnetic triggering of membrane receptor clustering-mediated cellular signaling is possible in cellular system.

Circulating Tumor Cells (CTCs) are released into the blood through primary and metastatic tumors. By capturing and analyzing CTCs from the blood of a patient, clinicians could obtain insight into the nature of the cancer as well as the efficacy of various treatments for that cancer [1]. The challenges for CTC capture arise from the low population of CTCs in blood relative to red and white blood cells. In this work, we describe a new approach for CTC capture using beads that are functionalized to enable binding to CTCs. Using a microfluidic tubing device, we generate monodisperse beads of the biopolymer, chitosan. The tubing device uses an oil co-flow to generate aqueous droplets of chitosan, which are collected in a solution of the cross-linker glutaraldehyde to form uniform solid polymer beads [2]. The beads are then functionalized with streptavidin, allowing for selective binding to cells that have been previously treated with a biotinylated antibody. To enable CTC capture, the above beads are packed into an array within a microfluidic device and a cell suspension is flowed across this array. CTCs that are captured by the beads can be easily visualized by fluorescence microscopy. We show that the above approach is a simpler and cheaper alternative to existing devices for CTC capture and is thus likely to be adaptable for clinical use.
[1] J. den Toonder, "Circulating tumor cells: the Grand Challenge," Lab on a Chip, 11, 375, 2011.
[2] K. Jiang, et al., "Microfluidics: A New Approach to In-Situ “Micromanufacturing”: Microfluidic Fabrication of Magnetic and Fluorescent Chains Using Chitosan Microparticles as Building Blocks (Small 17/2011)," Small, 7, 2469, 2011.

The National Cancer Institute (NCI) is exploring new and innovative approaches to better understand and control cancer. By partnering with scientists from various non-biology disciplines, NCI envisions novel approaches to help generate answers to some of the major questions and barriers in cancer research. The Alliance for Nanotechnology in Cancer (ANC) has demonstrated that interfacing materials science and nanotechnology with oncology has brought significant advancements and potentially novel breakthroughs in the way we clinically diagnose and treat cancer. Another NCI initiative is the Physical Sciences in Oncology which attempts to better understand the physical laws and principles that shape and govern the emergence and behavior of cancer through a physical sciences perspective (e.g., physics, material science, etc.). In this talk, examples of blending materials science and nanotechnology with a physical sciences perspective on oncology will be presented to illustrate that fostering the development of innovative ideas and new fields of study could lead to a paradigm shift in the way we understand and ultimately and treat cancer.

With recent advances in stem cell therapies and oncological research, prolonged tracking of single cells in vivo is critical to understanding their unusual biology. To tag these cells, nanocrystal-based probes are highly desirable since their composition can be tailored for specific imaging techniques, such as magnetic resonance imaging or whole-body fluorescence imaging. We will a robust strategy to incorporate nanocrystals to the cytosol of living cells for tracking using a unique class of core-shell colloidal polymer vectors decorated with both amine and guanidine functionalities. These materials exhibit unusual dual responsive character to both pH and temperature that can be leveraged to translocate nanocrystal/polymer assemblies to the cytosol in less than an hour. By avoiding prolonged exposure to the acidic microenvironment of late endosomes, the intact nanocrystals no longer pose a problem to cell health. Such a rapid trajectory for these mixed-component assemblies into live cells is unprecedented should make practical a wider array of nanoparticle-based tracking schemes in biological systems than is currently possible. We will also describe the synthetic development of new nanocrystal probes that when coupled to these delivery vectors, enable prolonged imaging schemes in cells via MRI.

Sensitive, efficient and easy to use platforms are required for the detection of circulating tumor biomarkers for early diagnosis of cancer and cancer risk assessment. To this aim, we are developing two classes of innovative, nanotechnology-based intrinsically high throughput platforms to detect and quantify (circulating) cancer biomarkers.
On one side, ELISA-like nanoimmuno assays suitable for proteomics/interactomics studies in low sample volumes. We exploit the approach of DNA microarray technologies applied to proteomics [1], in combination with atomic force microscopy (AFM) to generate DNA/RNA nanoarrays [2] and functional protein nanoarrays [3,4]: semisynthetic DNA-protein conjugates are immobilized by bioaffinity within a nanoarray of complementary ssDNA oligomers produced by AFM nanografting (NG). A nanoarray of different antibodies or synthetic molecular binders can be generated in a single operation, on a DNA nanoarray with optimized packing density to achieve optimum bio-recognition. Each DNA nanoarray can be copied in multiple copies (few tens) by means of Supramolecular Stamping [5]. A nanoliter dispenser pipette, is being produced in our lab, to dispense small volumes of analytes on the array surface. As a preliminary result of this technique, we will show the detection by accurate AFM topography measurements, of GFAP (Gliar Fibrillar Acidic Protein) in the pM range (few tens of pM) in a cell lysate mixed to synthetic GFAP and in lysate of GFAP-expressing cells. Also, we will present results on the detection sensitivity of miRNAs.
The second platform consists of capacitance immunosensors based on the measurement of the double layer capacitance forming at the electrode-electrolyte interface, in microfluidic channels. When a low potential (about 10 mV) is applied at variable frequency between two micro-electrodes in the channel, charge distribution variations at the electrodes occurring when a molecule of interest (e.g. an antigen) binds to the metal active surface, will lead to a low-frequency measurable change in the double layer capacitance. We will show preliminary results obtained with such devices on the detection of DNA hybridization and antigen-antibody recognition. We will also discuss the use of this class of cheap, real-time lab-on-chip sensors, in the context of therapeutic drug monitoring.
[1] C.M. Niemeyer, in Nanobiotechnology (Weinheim: Wiley-VCH (2004))
[2] E. Mirmomtaz, M. Castronovo, C. Grunwald, F. Bano, D. Scaini, A.A. Ensafi, G. Scoles, L. Casalis, Nano Lett (2008) 8 (12) pp. 4134-9
[3] F. Bano, L. Fruk, B. Sanavio, M Glettenberg, L. Casalis, C.M. Niemeyer, G. Scoles, Nano Lett (2009) 9 (7) pp. 2614-8
[4] B. Sanavio, D. Scaini, G. Grunwald, G. Legname, G. Scoles, L. Casalis, ACS Nano (2010) 4 (11) pp. 6607-16
[5] O. Akbulut, J.-M. Jung , R. D. Bennett , Y. Hu , H.-T. Jung , R. E. Cohen , A. M. Mayes , F. Stellacci , Nano Lett. (2007) 11, pp. 3493-8

In order to enable protein biomarker based diagnostics for diseases like cancer, sensitive platforms are necessary which can assay proteins in high throughput multiplex format. One of the main difficulties arising in assaying blood for low abundance proteins in multiplex format is due to the high level of background arising from the presence of blood cells and also highly abundant proteins (~1-1000mu;M) like albumin, IgG, IgA, transferrin, haptoglobin and α-1-antitrypsin making up 90% of the protein mass. The dynamic range of proteins in blood plasma alone spans nine orders of magnitude resulting in difficulties for quantification. Depletion of cells from blood requires centrifugation which is bulky and unsuitable for point of care diagnostics. We present an on-chip platform for depletion of cells and highly abundant serum proteins in blood. Our platform consists of two components, the first of which is a microfluidic mixer which mixes beads containing antibodies against the highly abundant proteins with the whole blood. For microfluidic mixing we use an adapted herringbone structure (Whitesides et. al) to induce chaotic mixing of beads with proteins in the channel. Using the adapted herringbone serpentine channel we achieve depletion of Immunoglobin G all the way down to 35 nanoMolar in concentration. This complex mixture (consiting of beads, cells, and serum proteins) is then injected into the second component of our microfluidic platform which consists of a filter trench to capture all of the cells and the beads. In order to significantly enhance the trapping of the cells and beads into the filter trench we have integrated a pair of interdigitated electrodes at the top of the channel above the trench in order to use negative dielectrophoresis in order to push all of the micron sized particles (cells and beads which have captured the highly abundant proteins) down into the trench allowing the serum proteins of lower abundance to flow through. In general dielectrophoresis is incapable of producing forces beyond the low piconewton range. However using atomic layer deposition to deposit a 10 nm pin-hole free layer of SiO2 on top of the electrodes protecting them from corrosion, we were able to demonstrate DEP forces in the nanonewton range. Using the enhanced negative DEP electrodes in combination with the filter trench, we were able to demonstrate 100% depletion of all micron sized particles in the mixture.

The drug gradients, which occur in tumor microenvironment, are likely to play an essential role for the evolution of drug resistance. We have developed microfluidic structures which allow drug gradients to be engineered into a cell culture region with near zero flow rate, which enables the long term (16 days) culture of breast cancer. Although either cancer chemotaxis or viability inhibition by drug has been studied separately using microfluidic gradient approaches, an integration of both migration and growth response in the drug gradient is still less complete.
In this talk, we examine the population growth versus time of MDA-MB-231, an invasive breast cancer cell line, in a gradient of 0 to 200nM doxorubicin in a 1 mm culture region. We observe a population gradient in response to the drug gradient, so that there were fewer cells in the regions with higher drug concentration.
We further observe substantial population growth in both the low and high drug regions of the culture chamber. To examine underlying mechanisms, we tracked positions of individual cells over 3 days in the culture with different drug concentrations. We find that there was no substantial net migration (chemotaxis) of cells from the high to low drug region or vice versa. Furthermore, the mean net displacement of individual cells was around 70 microns. Thus, cells were localized in similar drug concentration. Finally, we will discuss possibility of cell-cell interaction from low to high drug regions and how it may affect cell dynamics.

Since the discovery of RNA interference (RNAi), there has been an explosion of interest and knowledge in using this technology for clinical applications. Although small molecule inhibitors and monoclonal antibodies have led to many successful therapies for cancer, many important cancer therapy targets are difficult to inhibit using these strategies. Use of short interfering RNA (siRNA) as a method of gene silencing has rapidly become a powerful tool in protein function delineation, gene discovery, and drug development. The promise of specific RNA degradation has also generated much excitement as a possible therapeutic modality, but in vivo siRNA delivery has proven difficult. Moreover, many physiological obstacles stand in the way of successful and efficient delivery. To overcome these limitations, we have developed a number of biocompatible nanoparticle strategies for highly efficient delivery of siRNA. We have used a novel complex of siRNA with a neutral nanoliposome (1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine - DOPC) for in vivo siRNA delivery. This delivery platform has shown substantial efficacy with regard to target modulation and anti-tumor effects in many different tumor models. To target the tumor microenvironment, we have also developed delivery methods (e.g., chitosan nanoparticles) that allow highly efficient delivery of siRNA into both tumor cells as well as tumor associated endothelial cells. Therefore, the chitosan nanoparticles allow silencing of genes that play a functional role in tumor angiogenesis. In addition, we have also utilized these nanoparticles for targeted delivery by attaching peptides to the nanoparticles, which permits increased delivery into the tumor microenvironment. Collectively, these approaches offer new opportunities for therapeutic gene silencing and are being developed toward clinical testing.

As a noninvasive technology, fluorescence should offer ultrasensitive detections in biomolecular mechanisms of cancer biology. However, practical applications encounter many challenges, including optical interferences, strong autofluorescence, and the limitation of the wave properties of light. One key to solving these problems is to introduce a frequency component on top of the fluorescence intensity. In other words, photoswitchable molecular probes produce such a desired frequency by alternating between bright-and-dark states in response to external light stimulation. For example, modulation of nanoparticles targeted to breast cancer cells switches their fluorescence on and off. This feature enables super-resolution, which enhances resolution by an order of magnitude greater than the longstanding diffraction-limit barrier. The reversible modulation of such molecular probes at a particular frequency significantly amplifies this frequency-bearing target signal while suppressing interferences and autofluorescence. Thus, these new approaches offer unambiguous detection of superbrightness when data are processed in frequency domain. Additionally, photoswitching can be carried out in near infrared (NIR) to avoid physiological damages typically associated with UV-irradiation. In this presentation, we present a series of biological applications to demonstrate the power of photoswitching-enabled fluorescence imaging and detections.

Superparamagnetic iron oxide nanoparticles have received great attention owing to their potential applications as contrast agents for magnetic resonance imaging (MRI). Through the thermal decomposition approach high quality magnetic iron oxide nanocrystals can facilely be obtained. Nevertheless, acids, alcohols, and amines bearing long hydrophobic chain are commonly adopted as stabilizing agents. Consequently the direct products of the thermal decomposition syntheses are typically hydrophobic. Only through further surface engineering, they become usable as MRI contrast agents.
We recently developed an alternative approach, by using carboxylated PEG as surface capping agent, to achieve water soluble and biocompatible iron oxide nanocrystals and meanwhile inherit the advantages of the thermal decomposition method, which greatly simplifies the procedures for producing high quality Fe3O4-based MRI contrast agents.
In this presentation, we will report our recent results on the preparations of biocompatible Fe3O4 nanocrystals and their bioapplications, especially in in vivo imaging of tumors by MRI. In addtion, tumor imaging results based on paramagnetic NaGdF4 particles will also be presented.

References
1 NaGdF4 Nanoparticle-based Molecular Probes for Magnetic Resonance Imaging of Intraperitoneal Tumor Xenografts in vivo, Yi Hou, Ruirui Qiao, Fang Fang, Xuxia Wang, Chengyan Dong, Kan Liu, Chunyan Liu, Zhaofei Liu, Hao Lei,* Fan Wang, and Mingyuan Gao*, ACS Nano, 2012, accepted
2 Receptor-Mediated Delivery of Magnetic Nanoparticles across the Blood-Brain Barrier, R. R. Qiao, Q. Jia, S. Hüwel, R. Xia, T. Liu, F. Gao, H.-J. Galla, and M. Y. Gao, ACS Nano, 2012, 6, 3304.
3 Gelification: An Effective Measure for Achieving Differently Sized Biocompatible Fe3O4 Nanocrystals through a Single Preparation Recipe, Q. Jia, J. Zeng, R. R. Qiao, L. H. Jing, L. Peng, F. L. Gu, and M. Y. Gao, J. Am. Chem. Soc., 2011, 133, 19512.
4 Facile synthesis of ultrasmall PEGylated iron oxide nanoparticles for dual-contrast T1-and T2-weighted magnetic resonance imaging, F. Q. Hu*, Q. Jia, Y. L. Li and M. Y. Gao, Nanotechnology, 2011, 245604.
5 Investigations on the interactions between plasma proteins and magnetic iron oxide nanoparticles with different surface modifications, S. Liu, Y. Han, R. Qiao, J. Zeng, Q. Jia, Y. Wang, and M. Y. Gao, J. Phys. Chem. C, 2010, 114, 21270.
6 A novel type of dual-modality molecular probe for MR and nuclear imaging of tumor: Preparation, characterization and in vivo application, S. Liu, B. Jia, R. Qiao, Z. Yang, Z. Yu, Z. Liu, K. Liu, H. Ouyang, F. Wang, and M. Y. Gao, Mol. Pharm., 2009, 6, 1074.
7 Detection of toxoplasmic lesions in mouse brain by USPIO-enhanced magnetic resonance imaging, L. Wei, G. Zhou, Z. Li, L. He, M. Y. Gao, J. Tan, and H. Lei, Magnetic Resonance Imaging, 2007, 25, 1442.
8 Preparations of biocompatible magnetite nanocrystals for in vivo magnetic resonance detection of cancer, F. Hu, L. Wei, Z. Zhou, Y. Ran, Z. Li, M. Y. Gao, Adv. Mater., 2006, 18, 2553.
9 Preparation of water-soluble magnetite nanocrystals by refluxing ferric hydrated salts in 2-pyrrolidone: Mechanism leading to Fe3O4, Z. Li, Q. Sun, and M. Y. Gao, Angew. Chem. Int. Ed., 2005, 44, 123.
10 One-pot reaction to synthesize biocompatible magnetite nanoparticles, Z. Li, L. Wei, M. Y. Gao, and H. Lei, Adv. Mater., 2005, 17, 1001.
11 One-pot reaction to synthesize water-soluble magnetite nanocrystals, Z. Li, H. Chen, H. Bao, and M. Y. Gao, Chem. Mater., 2004, 16, 1391.

Molecular imaging is that field of imaging in which imaging agents are often utilized to target specific cellular and molecular events to interrogate biology in vivo. We are developing novel imaging agents and instruments that leverage on advances in nanotechnology in order to achieve advances in early disease detection and improved disease management. I will highlight our efforts in photoacoustic and Raman imaging that are being advanced by novel nanoparticles with an emphasis on cancer. The goals of increasing sensitivity and multiplexing will be highlighted. The need for novel nanoparticles will be discussed and specific applications provided.

In the last decade self-assembled lipidic nanoparticles have been increasingly explored as intravenously injectable agents for biomedical purposes. They can serve as drug delivery vehicles and/or molecular imaging probes. Particularly, lipid-coated inorganic nanocrystals are of great interest as such nanoparticles exhibit unprecedented possibilities with respect to their multifunctionality, potential for derivatization and biocompatibility.[1] Although many lipidic nanoparticles have been developed and applied in vivo for cancer diagnosis and therapy, most studies failed to consider two essential questions: Do these self-assembled particles retain their original composition, and how do they disassemble at and clear from the targeted site? Therefore, an improved understanding of the in vivo dynamics of the lipidic nanoparticles and subsequent trafficking of the different nanoparticle components would allow an improved tailoring of nanoparticle design to application.
To this aim, we here introduce a PEG-lipid stabilized nanoparticle that is composed of a near infrared (NIR) quantum dot (QD) core and a NIR dye-lipid corona label (QD-Cy7-PEG).[2] This nanoparticle allowed us to investigate the dynamics of nanoparticle accumulation and dissociation in a tumor mouse model. Since both the QD core and Cy7-lipid emit in the NIR range we were able to trace the QD and Cy7-lipid simultaneously using an NIR fluorescence imaging system in vivo. Moreover, Förster resonance energy transfer (FRET) between the QD core and the Cy7-lipid allowed us to sensitively monitor nanoparticle dissociation. Upon intravenous administration of the nanoparticles, the disassociation process was observed through a decrease of FRET signal in the tumors in vivo. Ex vivo organ imaging revealed the QD nanocrystal core and the lipid coating to follow different clearance pathways. In vivo imaging experiments with mice that received peritumoral injections of QD-Cy7-PEG revealed its trafficking to sentinel lymph nodes. Through fast protein liquid chromatography analyses of blood plasma, we found the nanoparticle&’s lipid coating vividly exchanged with plasma proteins as well as lipoproteins.
In conclusion, our study allowed in vivo imaging of the accumulation, disassociation and trafficking of lipid-coated nanocrystals. Our technology helps optimizing and tailoring design criteria when using lipid-coated nanocrystals for biomedical purposes in general, and our results warrant caution in the interpretation of imaging data when using them as diagnostic agents specifically.
1. T. Skajaa, Y. Zhao et al. Nano Lett. 2010, 10,5131
2. Y. Zhao, I. van Rooy et al. unpublished

In this talk, a spectroscopic device suitable for the investigation of ligand-receptor interactions will be presented. In particular, the research focused on optical waveguides constituted by an attenuated total internal reflection (ATR) element, transparent in the infrared and whose surfaces can be activated in view of covalently binding a receptor. Silicon and germanium ATR elements have been considered. The original method is based on the grafting of bifunctional spacer molecules directly at the surface of the germanium crystal, avoiding the deposition of an intermediate metal layer. This tool has been successfully used for the detection of proteins or of small molecules.
A specific example will be described dealing with Verrucarin A (VerA) which is a toxic trichotecene mycotoxin that can be produced indoors at very low level by moulds contaminating dwellings and may be associated with several human health problems. We will describe our biosensor for VerA which makes use of a new anti-VerA rat monoclonal antibody (mAb). This antibody has been directly grafted at the surface of the infrared element. Competitive ELISA and FTIR-ATR techniques were compared for detection of VerA in buffer and in complex dust samples obtained from dwellings. After optimization, the competitive ELISA showed a sensitivity of 7.43 ng/ml of VerA in PBS and a dynamic range below one order of magnitude. The FTIR technique improved the detection of the VerA by three orders of magnitude (2.5 pg/ ml in buffer and 6 pg/ml when spiked in dust samples). The dynamic range for its detection extended over four orders of magnitude.
Our results clearly show that this antibody-based spectroscopic biosensor allows a better detection of VerA as compared to classical immunoassays and can be very efficiently used in the field of indoor mycotoxin detection.

The microtubule, a rigid cellular biopolymer, is an important target for anticancer chemotherapies due to its important role in cell division. Drugs such as epothilones and taxanes act by stabilizing microtubules against depolymerization, which therefore inhibits cell division and tumor growth. While such drugs are effective in reducing solid tumor masses, they unfortunately can also cause painful and debilitating dose-dependent neurological effects. Microtubules also play an important role in axonal transport in neuronal cells, and the onset of transport defects has been observed before the onset of pain. This suggests a possible link between transport defects and neurological symptoms, and further, suggests that early interventions may be possible, if the detailed molecular mechanisms of drug action were known.
In this study, we use a suite of biophysical tools to assess how the mechanical, structural, and functional properties of microtubules are influenced by the chemotherapeutic compounds epothilone-A, epothilone-B, and taxol. In each case, we find that chemically stabilized microtubules can support kinesin motion in vitro. To characterize kinesin function, we couple small ensembles of proteins to quantum dots, and observe them using a custom-built total internal reflection fluorescence (TIRF) microscope. We find that kinesin speed is sensitive not only to the type of small molecule stabilizer used, but also to the presence of the essential microtubule-associated protein tau. Additionally, using a novel spectral analysis method to measure microtubule rigidity in situ, we find that epothilone-stabilized microtubules are substantially less stiff than taxol-stabilized microtubules. The addition of tau proteins to microtubules stabilized by either epothilone compound or taxol further reduces stiffness. Taken together, these results suggest that small molecule stabilizers do not simply stabilize a ‘native&’ microtubule structure, but rather they modulate the structure, function, and mechanics of the microtubules they bind. This may have important consequences to the therapeutic use of these agents in cancer chemotherapies.

Cancer of the head and neck is the sixth most common cancer worldwide, accounting for more than 300,000 deaths per year. Current treatment strategies include surgical removal combined with radiotherapy and/or chemotherapy, but the prognosis is poor and the 5-year survival rate is only 50%. A critical issue is that there are no methods for tracking the effectiveness of the treatment in real-time. Since high expression of epidermal growth factor receptor (EGFR) in tumor cell membranes is a signature of this type of cancer, we aimed to develop a liposomal probe bearing anti-EGFR antibodies as well as gadolinium (Gd), a positive (image-brightening) contrast agent for magnetic resonance imaging (MRI). Here, we report a simple self-assembly-based strategy to synthesize such liposomal probes, which involves combining biocompatible phospholipids, a chelated-Gd lipid, and a biotinylated lipid, followed by attachment of biotinylated antibodies to the liposomes using avidin as linker. Our results demonstrate that these probes preferentially bind to head and neck cancer cells compared to low-EGFR-expressing cells. Moreover, we show that probe-targeted head and neck cancer cells can be easily tracked using MRI. Such probes can potentially provide real-time feedback during cancer treatment and allow physicians to adjust their therapeutic strategies, thus improving the overall efficacy of the treatment.

Effective diagnosis and treatment are essential for reducing the high mortality of pancreatic cancer (PaCa). The overexpression of Neutrophil gelatinase-associated lipocalin (NGAL), a small 25kDa secreted acute phase protein, has been implicated in early stage PaCa. We report a magneto-fluorescent theranostic nanocomplex for the imaging and therapy of PaCa. Silica core-gold shell nanoshells (GNS) resonant at 810 nm were encapsulated in silica epilayers doped with iron oxide and the NIR dye ICG, resulting in theranostic gold nanoshells (TGNS), which were subsequently conjugated with antibodies targeting NGAL. Human PaCa cells AsPC-1 were employed for in vitro and in vivo studies in nude mice models. Animals were imaged at varying time points post-injection using both NIR and T2 weighted MR imaging. Biodistribution of the nanocomplexes was studied by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). The results clearly show that AntiNGAL-conjugated TGNS specifically targeted PaCa cells in vitro and in vivo , providing contrast for both NIR fluorescence and T2 weighted MR imaging, with higher tumor contrast than can be obtained using long-circulating but non-targeted PEGylated nanoparticles. The complexes also enabled highly specific cancer cell death via NIR photothermal therapy in vitro. We also demonstrated that NIR therapy and the nanocomplexes themselves are noncytotoxic in vitro. The ICP-MS based gold quantification confirmed the favorable biodistribution of NGAL-targeted nanocomplexes compared to controls.

An ongoing challenge in cancer therapy remains site-specific delivery to solid tumors. In this work, we demonstrate that tumor hyperthermia, delivered via gold nanorod-mediated plasmonic photothermal therapy, can be utilized to enhance the delivery of polymer therapeutics. Gold nanorods were delivered to the tumor mass following intravenous administration via the enhanced permeability and retention (EPR) effect. Localized tumor hyperthermia was then induced via laser radiation of the gold nanorods, resulting in a targetable heat shock response. Subsequent administration of heat shock targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates resulted in enhanced delivery. Specifically, the effect of heat shock protein expression on cellular uptake and cytotoxicity of targeted HPMA copolymers was evaluated in vitro followed by in vivo evaluation of their tumor accumulation and organ biodistribution. While increases in tumor delivery were observed using targeting and hyperthermia strategies alone, substantial increases in tumor delivery were achieved using a combination of the two. Spatial distribution within the tumor was visualized by magnetic resonance imaging of Gd labeled HPMA copolymers, where increased delivery to both the tumor periphery and tumor center was observed. Heat shock protein targeted HPMA copolymer-drug conjugates bearing the anticancer agents aminohexylgeldanamycin, docetaxel, or cisplatin were then evaluated in vitro in combination with hyperthermia to evaluate for synergistic effects. Heat shock targeted HPMA copolymer-aminohexylgeldanamcyin and -docetaxel conjugates showed marked synergism with hyperthermia and docetaxel conjugates were evaluated for efficacy in vivo by tumor regression in human prostate cancer bearing mice. In combination with tumor hyperthermia, delivered via gold nanorod-mediated plasmonic photothermal therapy, treatment with heat shock targeted HPMA copolymer-docetaxel conjugates resulted in tumor regression over 30 days following a single IV administration at 10 mg/kg docetaxel equivalent. These results demonstrate the potential for enhanced delivery and efficacy of targeted polymer therapeutics using gold nanorod-mediated plasmonic photothermal therapy.

Theranostics are nanodevices with diagnostic, therapeutic and possibly treatment-monitoring functions for treating cancers. Combining magnetic and plasmonic properties of nanoparticles (NPs) may provide new, high performance theranostics for millions of cancer patients and hence has provided a strong impetus for developing novel, multifunctional NPs. Fe3O4 NPs, owing to their magnetic property, have found medical applications such as magnetic resonance imaging and hyperthermia therapy for cancers. Au NPs are also attractive due to their high surface-enhanced Raman scattering (SERS), which can be used for high-sensitivity biodetection and photothermal ablation of cancer cells. Therefore, synthesizing hybrid NPs of Au and Fe3O4 can provide new platforms for developing novel theranostics. In the present study, a facile approach was developed to fabricate core-shell structured Fe3O4@Au NPs, which had a high efficiency for producing Fe3O4@Au NPs with desired size and shape in order to achieve the optimal optical property arising from the Au shell and the magnetic property due to the Fe3O4 core. After synthesizing Fe3O4@Au NPs, a coating of thermo-sensitive polymer poly(N-isopropylacrylamide) (pNIPAm) which was incorporated with anti-cancer drug 5-FU and the Raman reporter 4-MBA was produced on the NPs, forming multifunctional Fe3O4@Au@pNIPAm nanoparticles as a new type of theranostics. Owing to SERS, Fe3O4@Au@pNIPAm NPs could provide greatly amplified Raman signals, making them as highly desirable SERS tags in medical imaging for cancer detection. HRTEM images showed the formation of Au shell on Fe3O4 core. STEM mapping of Au and Fe and EDX analysis of Fe3O4@Au@pNIPAm NPs revealed the existence of Au and Fe in NPs. UV-Vis spectroscopy exhibited an SPR peak at ~530 nm for Fe3O4@Au@pNIPAm NPs, suggesting the NPs had an equivalent Au NP diameter of ~10 nm. DSC analysis of NPs indicated a lower critical solution temperature (LCST) of 32.3°C owing to the pNIPAm coating, and TGA results showed that the NPs contained 42.7 wt.% of pNIPAm. In vitro drug release studies were also conducted. At 30°C (below LCST), only a small amount (6.4%) of loaded 5-FU was released from Fe3O4@Au@pNIPAm NPs within 48 hr. At 42 °C (above LCST), 5-FU was released quickly from the NPs in the initial 6 hr and the release reached 83.1% within 48 hr. Fe3O4@Au@pNIPAm NPs with the attached anti-HER2 antibody could target SK-BR-3 cancer cells. In the presence of a magnetic field or NIR irradiation, there was a significant decrease in cell viability for SK-BR-3 cells incubated with 5-FU-loaded NPs because of combined hyperthermia therapy and photothermally guided drug delivery. Drug-loaded Fe3O4@Au@pNIPAm NPs are therefore multifunctional and have emerged as a novel theranostics.

In this talk, I will discuss integrated molecular diagnostic systems (iMDx) that do not require any external connections, tethers, or tubing to deliver and analyze a raw whole-blood sample. iMDx only requires the user to place a droplet of whole-blood at the inlet port of the device, whereupon the stand-alone iMDx performs on-chip removal of red and white cells, without external pumping mechanisms, followed by analyte detection in platelet-containing plasma. Red and white blood cells are removed by trapping them in an integral trench structure. Powered by pre-evacuation of its polymeric substrate, the guiding design principle of iMDx is the integration of the minimal number of functional components without sacrificing effectiveness in performing rapid complete bioassays including detection systems and automated wireless communication, a critical step towards point-of-care molecular diagnostics. In summary, I will discus the convergence of science, engineering, and medicine to find the solutions for translational personalized medicine and low-cost global healthcare systems.

Multifunctional probes for simultaneous magnetic resonance imaging (MRI) and drug delivery have attracted intensive interest due to their promising potential applications in the early-stage diagnosis and therapy of the diseases. In this study, hollow manganese phosphate nanoparticles (HMP NPs) with an average diameter of 18 nm were synthesized and aminated through silanization, which enabled the covalent conjugation of biocompatible PEG on their surfaces. Contact NPs in physiological condition (pH=7.4) resulted in low MRI T1 contrast (r1=1.19 mM-1 s-1), whereas high T1 enhancement (r1=5.22 mM-1 s-1) was achieved after dissolving them in endosome/lysosome mimetic condition (pH=5.4). This is due to the fact that those NPs were easily eroded and then resulted in the releasing of Mn2+ at low pH condition. To use this interesting phenomenon for targeting drug delivery by conjugating targeted ligand folic acid (FA) on HMP NPs, we further investigated their drug delivery capacity and cytotoxicity to cell lines expressed different amount of folate receptor (FR). KB cells showed more significant cellular uptake than HeLa cells and A549 cells, confirmed by confocal laser scanning microscopy (CLSM), flow cytometry and cellular T1-weighted MRI. Furthermore, the drug-loaded HMP NPs exhibited greater cytotoxicity in KB cells. Our results suggested that functionalized HMP NPs can act as an effective multifunctional probe for selective diagnosing with MRI as well as efficient targeted drug delivery.

The reported positive margin rate from wire localized excisions of breast cancers is approximately 20-50%; however, by preoperatively injecting a radio-active seed into the tumor under CT guidance, the excision rate is halved because the surgeon can constantly reorient the dissection to place the seed in the center of the specimen. Unfortunately, radioactive seed localization has several safety challenges, only single focus can be localized, and incisions are required to implant the seeds, so it is rarely employed. As a safe alternative, gas-filled hollow Fe-doped silica particles have been developed, which can be used for ultrasound-guided surgery even for multiple foci. The function of the Fe doping is to render the silica shells biodegradable. The particles are synthesized through a sol-gel method on a polystyrene template, and calcined to create hollow, rigid nanoshells. The Fe-doped silica shell is derived from tetramethoxy orthosilicate (TMOS) and iron ethoxide, which forms a rigid, mesoporous shell upon calcination. The nanoshells are filled with perfluoropentane (PFP) vapor. The flourous phase is contained within the porous shell due to its extremely low solubility in water. Quantification of particle functionality, signal persistence and acoustical properties have been performed using various phantoms including ultrasound gel, chicken breast, and excised human mastectomy tissue. In vitro studies have shown that continuous particle imaging time is up to approximately 45 minutes. Additional in vivo particle injection longevity studies have been performed in rabbit and mouse models which are consistent with in vitro data showing signal presence even five days post injection. These silica shells break under acoustic excitation to release uncovered gas pockets which increase acoustic energy absorption and reduce acoustic cavitation threshold locally. Therefore they may also be employed as a sensitizing agent in high intensity focused ultrasound (HIFU) therapy. Traditional ultrasound agents pose several potential drawbacks such as poor in vivo persistence (minutes) and high risk during continuous perfusion. Preliminary in vitro and in vivo HIFU ablation studies show that very few particles are needed in order to develop a sensitizing effect to HIFU thereby substantially reduce the amount of HIFU exposure time necessary to achieve an ablative effect. When HIFU alone was focused on a rabbit liver using a continuous 800KHz pure tone waveform with a peak negative pressure at 3MPa, no measurable response could be detected with less than a 30 second exposure. However, after giving a rabbit a dose of nanoshells at 0.5 mg/kg and allowing for a 15 minute circulation time, a measureable response could be detected with as little as 2 seconds of HIFU exposure. This may potentially allow for a larger area to be ablated in less time with less power. This would potentially reduce unwanted thermal or mechanical damage to the surrounding healthy tissues.

Photodynamic therapy (PDT) is a selective and promising targeted cancer treatment modality, especially because the chances of multi-drug resistance are minimal and patients suffer little cosmetic scarrings after treatment. In PDT, non-toxic photosensitizers (PS) generate reactive oxygen species (ROS) and/or highly reactive cytotoxic singlet (1O2) after absorption at a specific wavelength. The most commonly used agents for clinical application are haematoporphyrin derivatives such as Photofrin® [1]. Despite its apparent success, it still presents important disadvantages, such as skin photosensitivity, and a weak absorption peak around 600 nm, used for PDT treatment. The lack of hydrosolubility could also be a major problem for in vivo intravenous PDT drug bioavailability. In order to overcome the shortcomings of PS, the search for new photosensitizers remains an important goal. In this area, stable ruthenium(II) complexes (such as polypyridyl compounds [2]) have shown considerable promise as anticancer agents [3]. Due both to their absorption in the visible, and their long lived triplet metal-to-ligand charge transfer states (3MLCT) [4], tris-(bipyridyl)ruthenium(II) complexes proved to be efficient singlet oxygen photosensitizers for PDT[5]. Encapsulation of such Ru(II) complexes in PLGA-based nanoparticles (NPs) provides numerous advantages over the common treatments regarding the properties of the complex - third order nonlinear optical properties can be used in a two-photon absorption process, allowing the irradiation in the biological spectral window - and the properties of the NPs - their small sizes provide an effective diffusion into the cell improving targeting.
In this study, we use a correlative microscopy (CM) approach to evaluate the efficiency of the NPs. By combining Two-Photon Microscopy (TPM), Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM), we have characterized the Ru(II)-NPs and their effects on C6 tumoral cells. The results suggest internal concentration of the NPs in C6 tumoral cells and a strong improvement under irradiation of the efficiency of the NPs compared to the “free” complex. The results of the microscopy studies clearly demonstrate that encapsulation facilitates the O2 access to the photosensitizer, and the drug delivery to cancer cells which makes the NPs suitable candidates for PDT and two-photon excited PDT (TPE-PDT) [7].
[1] Sharman, W. M. et al., Drug Discoverv. Today. 1999, 4, 507; [2] Erkkila, K. E. et al., Chem. Rev., 1999, 99, 2777; [3] Ang, W. H. et al., Eur. J. Inorg. Chem. 2006, 4003 ; [4] Juris, A. et al., Coord. Chem. Rev. 1988, 84, 85, 277. ; [5] Schmitt, F. et al., J. Med. Chem., 2008, 51, 1811 ; [6] Girardot, C. et al., Tetrahedron Lett., 2008, 49, 1753. ; [7] Ogawa, K. et al., J. Med. Chem., 2006, 49, 2276.

In recent years external stimuli-responsive inorganic-organic core-shell nanoparticles have received great attention due to their potential applications in the effective encapsulation of therapeutics and controlled release systems. Such hybrid materials can provide multifunctionality and triggered drug release mechanisms to ascertain specific drug delivery, especially in cancer therapy. Specifically, mesoporous silica nanoparticles have attracted great interest as host system.[1] Due to their high loading capacity and the possibility of multifunctional modification, they provide precise control of spatial and time-resolved drug release. The delivery of anti-cancer therapeutics into cancer cells by employing nanoparticle carriers has made significant progress. However, a higher targeting efficiency and improved release of the drug from the endosome into the cytosol is essential to increase the efficacy of the drug.[2]
Here we report on colloidal mesoporous silica nanoparticles as a multifunctional platform for different stimuli-responsive trigger systems for escaping from the endosomal entrapment. Controlled endosomal escape via photo-activated endosomal membrane rupture as well as proton sponge effects based on covalently linked polymer brushes will be discussed. Coating of nanoparticles with different organic shells improves biocompatibility, facilitates attachment of targeting ligands for specific cellular recognition, and can be utilized for effective encapsulation of cancer therapeutics. We analyze the in vitro behavior of the colloidal particles with live-cell imaging, thus demonstrating high targeting specificity and efficient delivery. Achieving these properties simultaneously is a main challenge for targeted nanocarriers. We anticipate that the integration of all these strategies into one multifunctional drug delivery vehicle as well as the efficient synthesis and modification of the nanoparticles for multiple cancer cell types will hold promise for wide-ranging applications in cancer therapy.
Acknowledgement
The authors are grateful for funding from the DFG through the SFB 749 and the NIM Cluster (LMU Munich).
[1] V. Cauda, A. Schlossbauer, J. Kecht, A. Zuerner, T. Bein, JACS2009, 131, 11361.
[2] A. M. Sauer, A. Schlossbauer, N. Ruthardt, V. Cauda, T. Bein, C. Braeuchle, Nano Lett.2012, 10, 3684.

Combined cancer treatment via co-delivery of small interfering RNAs (siRNAs) and an anticancer drug can be a promising strategy due to the synergistic effect of simultaneously minimizing drug/gene administration and associated side effects while maximizing anticancer efficacy. In this study, Bcl-xL siRNA and doxorubicin (DOX) were encapsulated into newly designed methoxypoly(ethylene glycol)-b-poly(D,L-lactic acid) (mPEG-b-PLA) block copolymer polymersomes (PSomes). PSomes prepared by the thin film hydration method consisted of a hydrophilic core and hydrophobic shell to load Bcl-xL siRNA and DOX respectively and provided significant storage capacity. These PSomes were highly efficient for loading of hydrophilic Bcl-xL siRNA and hydrophobic drug. A study of time and pH resolved gene/drug release kinetics revealed distinctly different release profiles that were attributed to the degradation of mPEG-b-PLA copolymers and the relative locations of the siRNA and DOX within the PSomes. Moreover, a study of the cytotoxicity of Bcl-xL siRNA and DOX co-encapsulated PSomes (CPSomes) against MKN-45 and MKN-28 human gastric cancer cell lines demonstrated that CPSomes showed superior transfection efficiency than LipofectamineTM, resulting in higher inhibition of proliferation compared with single treatment with gene- or drug- loaded PSomes. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) revealed that CPSomes successfully silenced the targeted Bcl-xL and activated Bax mRNA expression levels. Consequently, these results demonstrated that co-delivery of gene and drug using PSomes resulted in a synergistic efficacy for cancer treatment and indicated the potential of PSomes as efficient nanocarriers for combined gene therapy and chemotherapy.

We have developed a smart nanovector consisting of hyaluronic acid (HA) and poly-L-lysine-graft-imidazole (PLI)-based polyplexes containing Bcl-xL-specific shRNA (HA/PLI/shRNA) for CD44 targeted gastric cancer therapy. The prepared ternary polyplexes have a negative surface charge of -24 mV and a size of approximately 100 nm at an N/P ratio of 5 with HA/PLI molar ratio of 0.03. Gel electrophoresis and cell viability experiments demonstrated that the ternary polyplexes showed high stability and no cytotoxicity due to the anchored HA molecules on the surface of PLI/pDNA binary polyplexes. Selective cancer cell death was achieved by CD44-mediated gene delivery and the internalized gene was effectively escaped from endosomes due to the buffering capacity of imidazole groups in an acidic environment. These novel nanovectors may be highly efficient gene delivery tools that allow the selective destruction of metastatic gastric cancer cells.

Nanoparticles are widely studied in cancer therapies through attaching to cells and delivering medical cargos. AuNPs are known as good carriers for its ease of synthesis and conjugation in biochemistry. Self-assembled monolayers (SAMs) provide a tunable system to change the interfacial properties of AuNPs, so that SAM-modified AuNPs can be used to transport biological molecules by electrostatic interaction. In order to control the electrostatic interaction, AuNPs were modified with SAMs of homogeneously mixed carboxylic acid and amine functional groups. It was found that a series of surface potential and the iso-electric point (IEP) of resulting AuNPs can be tailored by the chemical composition. By changing the environmental pH around the IEP, molecules could be triggered to adsorb or desorb due to the flip of surface charge. Based on different pH inside and outside of living cells, we expect that molecules can be transported by electrostatic interactions with binary SAM-modified AuNPs. In this work, the result of MTT assay revealed that binary SAM-modified AuNPs have high biocompatibility to HEK293T cells. The cellular uptake of binary SAM-modified AuNPs was examined by measuring the concentration of AuNPs remaining in the culturing medium. It was found that the degree of AuNPs ingested by cells were more obvious with pure or high loading of amine on the surface than those dominated by carboxylic acid. This difference in AuNPs uptake was also confirmed with STEM. The results suggested that HEK293T cells preferred amine-modified AuNPs than that modified with carboxylic acid. The difference can be rationalized by the electrostatic potential. Because most cells exhibit non-uniform negative surface charge, positively charged amine-SAM modified AuNP (+2 mV) has higher probability to attach to the cell and be ingested. For binary SAM-modified AuNPs with 20%:80% amine to carboxylic acid ratio, they still can be ingested by cells even its surface potential (-35 mV) was close to that modified with 100% carboxylic acid (-37 mV). This result suggested that even low concentraction of amine-modified SAM can help cells to ingest SAM-modified AuNPs of strong negative potential by decreasing the repelling of static electricity. Since binary SAM-modified AuNPs have low cytotoxicity and good affinity to cells, the ability of binary SAM-modified AuNPs as carriers were examined. The plasmid DNA containing eGFP reporter gene is chosen as cargo carried by binary SAM-modified AuNPs. By using confocal microscopy, it was found that AuNPs modified with 20~100% amine groups (complementary carboxylate group) can carry plasmid DNA into HEK293T cells by electrostatic interactions and release it for expression under 4 mu;g plasmid and 100 mu;M AuNPs. Compared to lipofetamine 2000, the transfection efficiency of binary SAM-modified AuNPs is weaker. Nevertheless, the effect of surface chemical composition and surface potential to the transfection efficiency can be studied.

Separation of circulating tumor cells (CTCs) from blood stream is most important step for the early diagnosis of cancer and cancer therapies. The optical and immunofluorescence techniques are the most common tools to detect CTCs using CTC-specific anti-Ep-CAM antibodies. However, the amount of cells captured by anti-Ep-CAM antibodies was found to be insufficient for the sensitive detection/separation of CTCs. Therefore, a new detection system is needed that enables the capture of various kinds of CTCs and the sensitive detection of biomarkers expressed by CTCs, simultaneously.
In this study, we report a new chip-based detection system capable of simultaneous detection and characterization of CTCs based on surface-enhanced Raman spectroscopy (SERS). A simple electro-deposition method was used to fabricate gold nano-patterned ITO substrate used for platform of CTC chip. A multi-functional nanoparticle composed of gold nanoparticles (GNP), antibody, and thiolated double strand DNA (dsDNA) was fabricated to detect and capture CTCs efficiently. Different kinds of Raman reports that matches with antibodies specific for different targets were further conjugated for SERS detection. The structural characteristics of the fabricated nanoparticles were confirmed by both transmission electron microscopy and surface-enhanced Raman spectroscopy. Thereafter, different kinds of nanoparticles were treated on various subtypes of human breast cancer cells that induced the attachment of CTCs on the chip surface through Au-S bond. Rationalizing membrane protein overexpression of CTC was successfully monitored by SERS method which was validated by conventional immunofluorescence analysis. The fabricated CTCs chip combined with multi-functional nanoparticles can be used as an effective tool for the cancer diagnosis, development of anticancer drugs and prognosis monitoring.
Acknowledgments: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2012-0000163), and by the Nano/Bio Science & Technology Program (M10536090001-05N3609-00110) of the Ministry of Education, Science, and Technology (MEST), and by the Ministry of Knowledge Economy (MKE) and Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology.

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 stability, effective endosomal escape, and a high capacity for disparate cargos. To this end, we have developed mesoporous silica nanoparticle-supported lipid bilayers (‘protocells&’, see the May 2011 cover of Nature Materials, the March 2012 cover of ACS Nano, and the May 2012 cover of Advanced Healthcare Materials), which synergistically combine properties of liposomes and mesoporous silica nanoparticles (MSNs). Protocells can be loaded with combinations of therapeutic (drug cocktails, small interfering RNA, plasmids, protein toxins) and diagnostic (quantum dots, iron oxide nanoparticles) agents and modified with PEG to enhance stability and with targeting and endosomolytic peptides to promote cell-specific binding and endosomal escape, respectively. We have previously reported that the high capacity of the MSN core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer (SLB) 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 MSNs. Liposome fusion to cargo-loaded cores results in a 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 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 ex ovo avian embryos and murine xenografts. We are, additionally, determining whether protocells can simultaneously deliver a combination of pCB1, the hydrophobic drug, paclitaxel, and siRNA that silences expression of the anti-apoptosis protein, Bcl-2, to SNU-398, a highly paclitaxel-resistant form of HCC.

Targeted delivery of therapeutic agents to tumor sites increases efficacy and limits off-target toxicity. Nanoparticles are an emerging class of targeted drug delivery systems. Commonly, nanoparticles are coated with poly(ethylene glycol) (PEG) to reduce off-target uptake by cells of the mononuclear phagocyte system (MPS) and a targeting moiety to promote uptake at the desired location. This approach holds great promise, but such constructs still predominantly accumulate in the liver. Here we demonstrate a different approach to tumor targeting using nanoparticles functionalized with a PEG coating that is shed in the presence of matrix metalloproteinase-2 (MMP-2), which is overexpressed in many tumor microenvironments. There was very little uptake of intact particles by human breast adenocarcinoma cells. Whereas, when the same cells were treated with particles in the presence of MMP-2 the resulting denuded particles were rapidly taken up by the cells. This selective targeting mechanism for nanoparticles may be useful for improved imaging and targeted drug delivery for solid tumors.

We present results in the development of a new ion source for imaging mass spectrometry (IMS) based on a novel approach to electrospray ionization (ESI). The ion source, called the Scanning Mass Spectrometry (SMS) Probe, allows for imaging of transient events at submerged interfaces in solution. It thus provides a route to previously inaccessible information in proteomics and metabolomics research.
Transient sampling from solution is made possible by novel implementation of nano-electrospray ionization concepts. [1] By minimizing the length of the liquid flow path from sampling point to spray point, we provide the shortest response time possible, making transient imaging experiments practical.[2] The ability to sample from a cell-sized region of liquid eliminates some obstacles to current proteomic and metabolomic based efforts for biomarker discovery: (1) dilution and mixing with non-disease-cell-specific biochemicals; and, (2) detection of non-secreted proteins and metabolites. Coupling the SMS probe with a time of flight mass spectrometer, we have demonstrated the ability to detect proteins, peptides and metabolites in transient biochemical reactions with sensitivities in the mu;M range, and with spatial and temporal resolutions of 10 mu;m and 1 s, respectively. [2]
This ambient ionization approach faces technical challenges, in that most relevant biochemistry occurs in aqueous solutions with high salt content, which makes successful identification of analytes via ESI-MS difficult. In order to apply the SMS probe to cancer research we have developed the capability to remove salts in the short distance between the sample and ionization points using on-line microdialysis. As initially reported [3] this requires increased transit length, which negatively impacts both spatial and temporal resolution. Here we report results of efforts to mitigate the negative impact of microdialysis on SMS probe resolution. We present results of simulations, analysis and experiments on synergistic application of ion source microfabrication and dialysis enhancement via exploitation of electrokinetic separations. Furthermore, we describe the path forward, including obstacles and enabling strategies to overcome, to application of the SMS probe as a biomarker discovery tool.
The work described was supported by Grant Number R21GM103539 from the National Institute of General Medical Science (NIGMS), a component of the National Institutes of Health (NIH). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.
1. Fedorov AG, Degertekin FL. 2006. Electron Lett 42: 793-5.
2. Kottke PA, Degertekin FL., Fedorov AG. 2010. Anal Chem 82: 19-22.
3. Olivero D, LaPlaca M, Kottke PA, 2012 Anal Chem 84: 2072-5.

In order to selectively detect hydrogen peroxide (H2O2) produced to mediate the angiogenic signaling pathways during angiogenesis in human umbilical vein endothelial cells (HUVEC), we functionalized single-walled carbon nanotubes (SWNT) emitting near-infrared (nIR) fluorescence with a collagen protein in non-covalent manner. We utilized the SWNT/collagen sensor array to measure the single molecule efflux of H2O2 from HUVEC in response to angiogenic stimulation. Two different types of angiogenic agents were investigated: the first one was the pro-angiogenic cytokine, vascular endothelial growth factor A (VEGF-A) and the second one was the recently identified inorganic pro-angiogenic factor, europium (III) hydroxide nanorods. According to the experimental results, it was found that the production of H2O2 following VEGF stimulation was significantly elevated outside of the cells as well as inside, but it was mainly increased inside the cytoplasm of HUVEC for stimulation via inorganic nanorods, which suggests two distinct signaling pathways.

Well-designed nanoparticle-mediated, image-guided cancer therapy has attracted interest for increasing the efficacy of cancer treatment. A new class of smart theragnostic nanoprobes employing cetuximab (CET)-conjugated polyethylene glycol (PEG)ylated gold nanorods (CET-PGNRs) is presented; these nanoprobes target epithelial cancer cells using near-infrared light. The cetyltrimethylammonium bromide bilayer on GNRs is replaced with heterobifunctional PEG (COOH-PEG-SH) to serve as a biocompatible stabilizer and to increase specificity. The carboxylated GNRs are further functionalized with CET using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC-NHS) chemistry. To assess the potential of such GNRs, their optical properties, biocompatibility, colloidal stability, in vitro/in vivo binding affinities for cancer cells, absorption imaging, and photothermal therapy effects are investigated. CET-PGNRs exhibit excellent tumor targeting ability and strong potential for simultaneous absorption imaging and photothermal ablation of epithelial cancer cells.

Well-designed photothermal nanomaterials have attracted much research interest to accurately diagnose cancer and further assess the treatment due to their photothermal ablation ability guided by near-infrared (NIR) laser without damage of normal human tissues.
Polyaniline, biocompatible and widely used as an electroactive material, exhibits transition states from emeraldine base (EB, neutral) to emeraldine salt (ES, acidic) by proton doping process that induces the movement of electrons and reduces the excitation energy level. Thus, the optical absorbance peak of polyaniline is red-shifted toward the NIR region due to its transition from EB to ES state which generates a substantial amount of heat energy sufficient for cancer cell ablation
Herein, we synthesized hollow walnut-shape polyaniline nanoparticles (HWPANPs) for ablation of cancer cells. First, polystyrene (PS) nanospheres were prepared as a core template and then their surface was covered with ammonium persulfate as an oxidant for anchoring the polyaniline nanofiber. Subsequently, polyaniline over-layer was formed on the PS surface through dropwised addition of aniline in acidic environment (pH2) at room temperature. Moreover, the thickness and length of polyaniline nanofiber were efficiently controlled by adjusting the amount ratios of aniline (5.256 × 10-4 mole) to oxidant (1.314 × 10-5 mole) tested with various types of surfactants (CTAB, CDAB, dextran, tween 40 and tween 80). Finally, HWPANPs were obtained after dissolution of PS core template using tetrahydrofuran (THF). Well engineered formation of HWPANPs were verified by scanning electron microscopy (SEM) showing their overall size of sim;400 nm with 30 nm in thichkness and 700 nm in length of polyaniline nanofibers using dynamic light scattering (DLS). In addition, FT-IR and UV-vis data strongly demonstrated successful synthesis of the desired HWPANPs. We evaluate their functional use in photothermal ablation and the results showed that our HWPANPs exhibited excellent NIR absorption characteristics with sufficient heat generation capability and sufficient cancer cell killing efficacy without cytotoxicity in vitro. Furthermore, the hollow space of HWPANPs can provide an additional cargo for another payload such as gene or drug delivery which may be highly potent multifunctional nanoparticles in biomedical applications.

Several mineral fibers are both fibrogenic and carcinogenic to lungs. Among the mineral fibers, asbestos is well known for its cytotoxicity. In asbestosis lungs, asbestos bodies are frequently recognized in the pulmonary interstitium, a medium of lung fibrosis and microenvironment for carcinogenesis. Although cytotoxic effects on pulmonary alveolar cells, the first target of inhaled fibers, have been studied, the etiology of pathological effects on the microenvironment of lung fibrosis is yet to be elucidated. In the present work, we present our preliminary study on the different types of asbestos fibers. Fibers from different origins were classified by examining the experimental X-ray diffraction patterns against the known database files. A real-time cell analyzer (xCELLigence, Roche diagnostics) was utilized to observe the cellular reactions to lung fibroblasts (IMR-90), which was considered the microenvironment of lung fibrosis. Fibers of serpentine (chrysotile) and amphibole (amosite and crocidolite) minerals were studied in concentrations of 10, 50, and 100 mu;g/mL. The investigated fibers showed diverse cytotoxic effects on IMR-90. Chrysotile showed the cytostatic effect in all concentrations, crocidolite caused steady cell death, and amosite had an effect similar to the mitosis interference. In conclusion, all types of asbestos fibers showed cytotoxic effects on the lung fibroblasts in vitro, however, in different manners. Further investigations are necessary to fully understand the underlying mechanisms of the cellular interactions and their ramifications in vivo.

We developed a novel engineered viral particle that can be used for therapeutic nanoparticles with improved cancer targeting capability through genetic engineering of M13 bacteriophage (phage). M13 phage possesses multiple advantageous structural features to develop novel improved drug delivery vehicles. M13 can easily express foreign peptides on their major and minor coat proteins. It has no tropism for eukaryotic cells. It can be easily amplified through bacterial amplification in a large scale. Utilizing these features, we developed novel engineered phage for cancer target application with improved internalization efficiency. Previously, M13 was engineered with an integrin binding peptide, RGD (Arg-Gly-Asp), and extensively studied. However, in the homing ability for cancerous tissue, this system usually relies on the short ligands expressed on the pIII minor coat protein, which is placed at one end of phage particle with only five copies. Although the major coat protein of the M13 phage was engineered with the RGD-peptide for the better targeting for the cancer cell, the improvement of the function was limited. Here, we developed novel engineered phage that can express dual peptides on its major coat proteins. We have engineered additional major pVIII coat protein gene with Tac promoter on the M13 phage genome and expressed two different types of the major coat proteins on the phage body. The newly engineered phage can also endure large foreign peptide expression. By expressing cyclic form of RGD-peptide on the newly incorporated pVIII protein, we could enhance the targeting ability for the cancer cells with improved internalization efficiency. As a demonstration of concept, we could deliver streptavidin- tagged fluorescent dyes into the target cell by incorporating streptavidin-binding peptide (His-Pro-Gln) on the pVIII major coat protein. By replacing the fluorescent dye to different drug molecules, this newly developed phage could be used as safe and efficient drug delivery vehicle.

Compared with synthesized nanomaterials, natural nanomaterials have many advantages for drug delivery applications, including possible evasion of the immune system, excellent biocompatibility, and increased biodegradability. The development of the naturally occurring nanoparticles for use as a new class of drug delivery carrier is a rapidly emerging research area. As such, we have investigated the use of nanoparticles previously reported to form in green tea for cancer therapy. Due to the diverse phytochemicals in the beverage and excellent biocompatibility, we suppose through this study that these nanoparticles will have many uses in drug delivery. In this study, the ability of nanoparticles formed from green tea infusions, to be used as a vehicle for a drug delivery system was evaluated using Doxorubicin (DOX) as the model drug. First, the nanoparticles from the green tea infusion were isolated by dialysis and characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), and dynamic light scattering (DLS). AFM and SEM revealed that the nanoparticles were spherical with diameters ranging from 50-200 nm with a zeta potential of -20 to -30 mV. After isolation of the TNPs, the nanoparticles were complexed with DOX to develop a novel nanoparticle-based drug delivery vehicle. The DOX was effectively loaded into TNPs using physical adsorption via electrostatic and hydrophobic interactions. The loading and releasing of DOX from TNPs were characterized under various pH conditions, and the drug loading and release from TNPs was pH dependent. More importantly, the results from flow cytometry and confocal microscopy showed that the use of DOX-loaded TNPs increased up to 1.42 fold for the cellular uptake of DOX, and, thus had higher cytotoxicity in human lung cancer A549 and mouse melanoma B16BL6 cells when compared to free DOX, indicating the potential for TNPs as a natural nanocarrier for cancer chemotherapy.

Naturally occurring nanoparticles (NONPs) have drawn significant interests from scientific communities, due to their unique properties and promising biocompatibility. However, most studies on NONPs have focused on higher organisms, and few studies have been conducted for producing NONPs from the microbial world. Given the earth&’s rich biological diversity, it is reasonable to believe that NONPs, with various forms and functions, may be produced from a variety of organisms, ranging from microbes to metazoans. Arthrobotrys oligospora, a representative flesh eater in the fungal kingdom, can change from a saprophyte into a predatory stage in the presence of nematodes or proteinaceous substances, characterized by the formation of 3D adhesive trapping networks that can capture, penetrate and digest free-living nematodes in diverse environments. Due to the presence of specialized 3D adhesive traps, the fungus was often used for parasitic nematodes control in both plants and animals in the past. Several biopolymers isolated from the 3D-trapping networks have also been reported to be involved in the adhesion process. Thus, we hypothesize that A. oligospora is a potential source for natural-based biomaterials due to the unique biological interactions between the fungus and its prey, nematodes. The purpose of this study is to discover novel nanomaterials that occur naturally in A. oligospora and to exploit its potential applications in cancer therapy. For this purpose, a new culture method, fungal sitting drop culture method, was established to monitor the growth of A. oligospora in situ and produce the nanoparticles without interfering or contamination from the solid media. Abundant spherical nanoparticles secreted from the fungus were revealed by scanning electron microscopy and atomic force microscopy. They had an average size of 360-370 nm, with a zeta potential of -33 mV at pH 6.0. Further analyses revealed that there were ~28 µg of glycosaminoglycan and ~550 µg of protein per mg of nanoparticles. Interestingly, the NONPs from A. oligospora have demonstrated potential capability as an immunostimulatory and antitumor agent. The nanoparticles significantly induced TNF-α secretion in RAW264.7 mouse macrophages, indicating a potential immunostimulatory effect. The nanoparticles themselves were also found slightly cytotoxic to mouse melanoma B16BL6 and human lung cancer A549 cells, and showed a synergistic cytotoxic effect upon conjugation with doxorubicin against both cells. Therefore, they may play multifunctional roles as a promising candidate for potential tumor immunochemotherapy. In this respect, the synergistic antitumor efficacy for chemotherapy, drug delivery and immune regulation are expected in vivo. This study has proposed a new approach for producing organic nanoparticles from microorganisms. It may open a new avenue for controlling the synthesis of organic nanoparticles using synthetic biology.

Magnetic nanoparticles can be one of the most effective tools for hyperthermia which is the thermal treatment of cancer under an AC magnetic field. The non-invasive nature and the ability of selective heating at targeted areas without any penetration depth limit make magnetic hyperthermia a promising technique in cancer research. The use of magnetic hyperthermia, however, has been limited so far owing to its poor treatment efficacy caused by the development of thermal tolerance.
In this study, we introduce a new approach to efficient apoptotic hyperthermia at low temperature by utilizing double-effector nanoparticles. Reactive oxygen species (ROS) induced by the double-effector nanoparticles render cancer cells more vulnerable to subsequent magnetic hyperthermia. High potency is seen in vitro and, remarkably, in vivo. Xenograft tumors (100 mm³, MDA-MB-231) in mice are completely eliminated after a single magnetic hyperthermia treatment using the double-effector nanoparticles at 43 ^oC.

In the past decades, there has been a great improvement in the domain of the imaging systems. But commercialised techniques are still very expensive and potentially harmful. Thus optical imaging, in which photons are the information source, is a rapidly expanding field. We have recently developed inorganic luminescent nanoparticles (NPs) suitable for in vivo imaging and that can master the difficulties due to the biological environment. The NPs are first excited by a UV light for a few minutes outside the animal, then injected to the animal, where they emit visible light for hours after the injection. Different families of materials have been considered as host lattices for doping with transition metal and lanthanide ions: silicates, gadolinium oxysulfides (Gd2O2S) and calcium phosphates. Our starting material composition for the silicate compounds was Ca0.2Zn0.9Mg0.9Si2O6. These new long luminescent NPs emit in the red-near infrared range and the emission can last for several hours. In the aim of improving their luminescent properties, we studied the influence of the chemical composition on the luminescence: Ca/Mg/Si molar ratio, TEOS and the doping elements and ratio. We studied Gd2O2S: Eu2+, Ti4+, Mg2+ which are interesting bimodal sensors (luminescence and magnetism). Gadolinium compounds are already used in clinical medicine as MRI contrast agents (visualization of blood vessels). Gd2O2S NPs are synthesized by hydrothermal method. The final objective of this work is to obtain biocompatible and/or biodegradable NPs with the highest luminescence intensity and time, therefore we want to develop NPs of doped calcium phosphates, which are the most important inorganic constituents of biological hard tissues.
All these compounds were characterized by TEM, XRD, NMR, FTIR and their luminescent properties were studied with a CCD camera coupled with a spectrometer for spectral analysis.

We have recently reported on the development of arrays of high performance micro-magnet (NdFeB, SmCo) on the one hand using a laser-based technique (Thermo-Magnetic Patterning) [1] and on the other hand using a lithography-based process (Topographic Patterning) [2]. The extrinsic magnetic properties (remanence, coercivity) of the µ-magnets are comparable to high quality commercial bulk magnets. A combination of Scanning Hall Probe Microscopy and simulations indicate that these µ-magnets produce stray magnetic field gradients as high as 10^6 T/m [3].
Based on the high quality and on the autonomous nature of these µ-magnets, they are very well adapted to use in point-of-care devices to trap magnetic carriers and very small nanoparticles (down to the sub-10 nm size).
To study the trapping efficiency of µ-magnet-based systems, different magnetic structures were investigated ex-vivo [4, 5] in microfluidic devices [6] and fluid circulation with different micro/nano magnetic particles.
Based on these results, we have developed a specific magnetic tool, the “MAGPIE” (MAGnetic Probe for In-vivo Experiments) to trap magnetic carriers circulating in the human body (blood, cerebrospinal fluid).
In this communication, we will show results on the ex-vivo and in-vivo trapping of magnetic particles using such a tool.
[1] F. DUMAS-BOUCHIAT et al,
Applied Physics letters 96 (2010).
[2] A. WALTHER et al,
Journal of magnetism and magnetic material, 321 (2009).
[3] M. KUSTOV et al,
Journal of Applied Physics 108 (2010).
[4] L.F. ZANINI et al,
Journal of Applied Physics, 111 (2012).
[5] O. OSMAN et al.
Biomedical Microdevices, 14 (2012).
[6] L.F. ZANINI et al,
Applied Physics Letters, 99 (2011).

The basic pharmacokinetic behavior of a therapeutic agent (i.e. absorption into the blood, distribution throughout the body, metabolism, and excretion) is primarily determined by its chemical properties, regardless of its pharmacologic classification. High capacity loading of therapeutic agents within porous nanoparticles serves to mask their chemical properties by improving their aqueous solubility and mitigating their charge, thus allowing the pharmacokinetics of a particular drug to be tailored. Mesoporous silica nanoparticle-supported lipid bilayers (or ‘protocells&’) are a robust nanocarrier that synergistically combines the advantages of both mesoporous nanoparticles and liposomes, while simultaneously addressing the limitations of each. Protocells are formed by electrostatic fusion of liposomes to spherical, high-surface-area mesoporous silica nanoparticles. The overall diameter, pore size, pore volume, surface chemistry, and surface charge are all tailorable, allowing for precise control of core properties. Additionally, the composition of the supported lipid bilayer (SLB) can also be precisely controlled to control its fluidity and stability. Collectively, the protocells possesses unique biophysical and biochemical properties, making it amenable to high capacity loading with a wide variety of cargos. Here we demonstrate that the mesoporous silica nanoparticle core can be functionalized to facilitate high capacity loading of various therapeutic agents, including chemotherapeutics, non-steroidal anti-inflammatory drugs (NSAIDS), and anti-fungals and that the resulting pharmacokinetic behavior of these drugs is controlled by the protocell rather than the drug&’s inherent chemical properties. Some of the chemotherapeutic drugs that are involved include Paclitaxel, Carboplatin, Gemcitibine, and Doxorubicin. Using various techniques that optimize the particle-liposome relationship and the drug-particle dual solubility, high-capacity drug loading can be achieved. The applications of this particle and liposomal relationship also include effectively increasing the solubility of certain hydrophobic drugs in aqueous solutions. Paclitaxel's aqueous solubility can effectively be increased by a factor of 10 in a 0.5x PBS solution when loaded into protocells. The kinetics of release and stability can also be effectively controlled by this relationship. The release of drug can be controlled to have characteristics of a burst release when added to a pH=5 solution, which results in full drug release in less than two days. Also, the addition of these protocells to pseudo-bodily conditions resulted in a long lasting stability of over 10 days. Effectively, this particle-liposome relationship allows a shorter burst release in pH=5 stomach conditions and long period of stability and circulation in pH=7.4 conditions.

Research in the area of cancer treatments often focuses on ways of targeting and killing cancer, but with brain tumors and other neurological diseases, there is one more obstacle that must first be overcome. The blood brain barrier (BBB) is a complex barrier which helps to maintain the brain&’s unique physiology as compared to the rest of the human body. Unfortunately, in cancer treatment, it also inhibits the delivery of drugs, making treatments ineffective. Drugs like Manitol have been used to increase permeability, though once administered it continues uncontrolled action until removed, and if repeated treatments are needed, they have diminished effects. In this research, magnetic nanowires were incubated with the arachnoid cells of BBB in vitro, and a magnetic field was applied to stress the cells such that the tight junction between cells was stretched. Specifically, 100nm diameter, 5-20um long Ni nanowires were incubated for 24 hours with a confluent layer of arachnoid cells, during which time the nanowires were uptaken into the cells (as shown by TEM). The cells appeared unaffected by the presence of the wires over the course of days. Interestingly, microscopy of the cells themselves did not show statistically significant morphological changes, as the cells were observed with and without nanowires, and with and without a magnetic field. Next, the permeability of the confluent layer was measured using trans epithelial electrical resistance (TEER) measurements. After uptake of nanowires, the confluent layer&’s permeability appeared not to change until the application of an external magnetic field which led to changes of a few ohms in the TEER resistance.This appears to have caused an increase in permeability of the barrier through a simple the flip of a switch. In addition, cells were detached from the membrane when the fields were increased beyond a few kOe due to magnetic forces on the nanowires. Initial toxicity measurements showed that cells in nanowire-containing media doubled at robust rates of 2-3 days which is typical of this cell line. In further studies, unattached cells were manipulated by external fields and separated from the assay. In this talk, we will discuss the permeability of the BBB and how it can be enhanced for delivery of cancer therapeutics through the use of magnetic nanowires which could yield switchable permeability for delivery of cancer therapies and brain perfusion for other conditions.

Nanoscale materials are a promising platform for novel approaches to disease diagnostics and therapeutics due to their unique optical, magnetic, electronic and biological properties. Magnetic nanoparticles are especially attractive for biological and medical applications since they can be detected and manipulated remotely which allows for utilizing them in vivo. Chemically synthesized superparamagnetic iron oxide nanoparticles are currently being studied for a variety of purposes including magnetic resonance imaging, molecular sensing, drug delivery, cell sorting, magnetofection of living cells, and hyperthermia. The majority of biological applications of magnetic materials developed to date utilize chemically synthesized superparamagnetic particles on the order of tens of nanometers. While these particles do not interact with each other, their typically small magnitude of magnetization calls for very strong magnetic fields required for their detection and manipulation. Ferromagnetic particles fabricated out of soft 3d transition metals offer the advantage of high magnetization saturation which translates to high responsivity of the particles to even weak external magnetic fields [1-2]. Furthermore, tailoring the geometry of ferromagnetic particles allows for modulating their magnetic properties. Our group has recently developed disk-shaped ferromagnetic particles with zero net magnetization which do not agglomerate in the absence of external magnetic field. In this talk, we will discuss the applications of these microdisks both in high frequency and in low frequency magnetic field for targeted control of cancer cells. First, we will consider the hyperthermic treatment based on cell heating with ferromagnetic microdisks. It will be shown that these particles are more efficient for hyperthermia compared to superparamagnetic ones. Second, we will discuss the magnetomechanical stimulation of cancer cells which triggers apoptosis as a result of disks oscillations on the cell membrane in low frequency a.c. magnetic field.
1. D.-H. Kim, E.A. Rozhkova, I.V. Ulasov, S. D. Bader, T. Rajh, M. S. Lesniak, V. Novosad, Biofunctionalized magnetic-vortex microdiscs for targeted cancer-cell destruction. Nature Materials 2009, 9, (2), 165-171.
2. E. A. Vitol, V. Novosad, E. A. Rozhkova, Microfabricated magnetic structures for future medicine: from sensors to cell actuators, Nanomedicine, 2012, 7(10), 1611-1624.

Due to their unique porous structure, tunable dimensions, ease of surface functionalization and overall versatility, mesoporous silica nanoparticles (MSNs) have emerged as the carrier of choice for delivering various payloads to cells in a controlled manner. In addition to spontaneous diffusion of the bio-molecules loaded within the pores of MSNs, it is also possible to control/trigger their release by apt use of molecular valves which respond to various chemical and physical stimuli such as light, pH, redox potential, enzyme and temperature. While there have been numerous reports on the development of stimuli-sensitive MSNs for drug/gene delivery and subsequent intracellular release, monitoring the release of drugs from these MSNs in real-time is still not explored, thereby necessitating the development of real-time monitoring systems incorporated within the biomolecule-loaded MSNs.
To address the afore-mentioned challenges, we demonstrate redox-sensitive fluorescent MSNP-based drug delivery system (FMSNs), which enabled FRET-based real-time monitoring of the drug release from the pores of MSNPs in the reducing environment found inside cells. The FMSNs were labeled with coumarin on the outer surface of their pores and were subsequently capped with FITC-conjugated β-cyclodextrin (FITC-β-CD) via a disulfide bond. This endowed the FMSNs with redox-responsive capabilities, wherein the FITC-β-CD would act as a molecular gate to entrap the cargo inside the pores in non-reducing conditions, while releasing it only in the presence of stimuli such as glutathione. Additionally, under non-reducing conditions, the intact disulfide bond results in formation of a FRET complex - a donor-acceptor model between the coumarin-labeled FMSN and the FITC-β-CD molecular gate. At this stage (FRET ON), coumarin acts as the donor and FITC acts as an acceptor, thereby resulting in emission at 520 nm (which corresponds to FITC), when the FMSNs are excited at 405 nm. However, in the presence of reducing environment, such as that encountered by the FMSNs in the cytoplasm of cancer cells, the disulfide bond is cleaved which results in the release of FITC-β-CD, thereby unlocking the pores and release of encapsulated drug. At this stage (FRET OFF), the donor-acceptor interaction between coumarin and FITC is abolished and hence the NPs show the emission at 450 nm, characteristic of coumarin, when excited at 405 nm. Since the modulation of FRET was incorporated into the unlocking event, the triggered release of the in target location will be reported and the drug dosing can be estimated from the FRET signal. Thus, the proposed drug delivery system provides a unique as well as universal strategy for real-time monitoring of drug delivery which can be extended to a wide array of biological applications.

Deterministic lateral displacement (DLD) arrays have been used to concentrate circulating tumor cells (CTCs) in diluted whole blood at flow rates as high as 10 mL/min with capture efficiencies exceeding 85% (K. Loutherback et al., AIP Advances, 2012). However, the equivalent volume of undiluted whole blood that can be processed is limited to 2 mL due to clogging of the array. Since the concentration of CTCs can be as low as 1 - 10 cells/mL in clinical samples, increasing the volume that can be processed with DLD arrays is important in order to allow collection of sufficient numbers of CTCs for biological experiments. Furthermore, by bumping the CTCs into a buffer stream, DLD arrays can be used to harvest the CTCs free of the background of leukocytes, erythrocytes, platelets, and plasma present in blood, resulting in a highly enriched product (J.A. Davis et al., PNAS, 2006). Reducing clogging of the array is even more important for this application since clogging of the array will cause contamination of the buffer stream by leukocytes and erythrocytes, thus reducing the enrichment.
In this talk, we (i) identify the key fundamental components of blood that lead to clogging and (ii) demonstrate experimental approaches that can increase the throughput before clogging by well over an order of magnitude. Clogging in the DLD array is not due to large clots that have formed before blood enters the chip but rather happens dynamically at the beginning of the array of micro-posts. By experimentally removing or adding different blood components before the sample enters the chip, we present the relative contributions of different components of blood (leukocytes, erythrocytes, platelets, and plasma) to clogging and identify platelets as the primary cause of clogging. Further, we hypothesize that clogging is due to a clotting-type process involving activation of the platelets by the micro-post array. We seek to avoid clogging without the use of agents such as ficoll or lysis buffer that are necessary for other cell separation techniques. We find that an increased blood dilution and increased flow rate reduce clogging for the equivalent volume of undiluted whole blood processed, combining to increase the equivalent volume of undiluted whole blood processed by 10x. Finally, we show how the post shape and geometry can be used to further reduce clogging. Based on the results of these experiments, we discuss the possible dominant platelet activation mechanisms leading to clogging.

Protein sensing is an issue with great interest in medical and biological applications. One possible approach to protein detection takes advantage of measuring changes in fluorescence resonance energy transfer (FRET) between a fluorescent donor and an acceptor within a protein which undergoes induced changes in conformation. This technique has grown in popularity due to the emergence of various fluorescent proteins with shifted spectral properties. To accomplish this process it is necessary thee detection of low intensity fluorescent signals in the visible spectrum.
In this paper we analyzed the emission spectrum obtained from fluorescent labels attached to a protein which changes its conformation in the presence of glucose using a commercial spectrofluorometer. Different glucose nanosensors were used to measure the output spectra with fluorescent signals located at visible bands of the spectrum.
A new device is presented based on multilayered a-SiC:H heterostructures to detect identical transient visible signals. The transducer consists of a p-i'(a-SiC:H)-n/p-i(a-Si:H)-n heterostructure optimized for the detection of the fluorescence resonance energy transfer between fluorophores with excitation in the violet (400 nm) and emissions in the visible range of the spectrum. The device was characterized through transmittance and spectral response measurements (400-800 nm), under reverse electrical bias (-10VResults show that the device photocurrent signal measured under reverse bias and using appropriate steady state optical bias, allows the separate detection of the fluorescence signals.
An electrical model, supported by a numerical simulation, gives insight into the transduction mechanism.

Cancer is an enormous global health problem, accounting for 1 in 8 deaths worldwide with over 1.6 million new cases predicted for 20121. The financial cost of a disease of this magnitude is also staggering, so there is a definite need for more efficient, less invasive and cheaper treatments that eliminate the severe symptoms of those currently available. Recently, much progress has been made in the use of nanoparticles (NPs) as early detection and treatment options for many different types of cancer. Human serum albumin (HSA), the most abundant human blood protein, has been heavily studied as an extremely promising cancer detection and targeted-treatment colloidal carrier system.
A desolvation and cross-linking creation method was employed to encase pre-formed gold nano-shells (AuNSs) and solid gold nanoparticles (AuNPs), into HSANPs. These relatively non-toxic, metallic NPs offer combinational therapy via imaging and photo-thermal ablation capabilities, due to their unique surface plasmon resonance properties. This encasement strategy will facilitate a perfectly sized HSA shell for efficient internal enclosure of AuNPs, as well as drugs, dyes or other biomarker molecules. The strategy also enables maximum external surface area for the functionalization of target-specific ligands.
HSANPs, formed via a desolvation and cross linking method have had major success, with the size of the HSANPs having crucial importance for loading and in vivo performance. Therefore, studies have focused on controlling size by manipulating the parameters of the preparation process. However, the HSANP sizes these studies report seem to vary somewhat in comparison with one another, even when equal preparation methods have been used; leaving the exact preparation for a particular particle size still rather illusive.
This research reports 1) an efficient method for preparing and characterizing HSANPs by varying preparation parameters and, notably, further improving the washing and purification step; 2) determination of efficient loading of a drug analog, rhodamine 6G (Rh6G) dye, into HSANPs and 3) development of an encasement strategy for loading AuNP/NSs into HSANPs.

We recently developed a new class of nanocarriers that synergistically combine features of mesoporous silica nanoparticles and liposomes. Fusion of liposomes to a spherical, high-surface-area, mesoporous silica core followed by modification of the resulting supported lipid bilayer (SLB) with multiple copies of a targeting peptide, an endosomolytic peptide, and PEG results in a nanocarrier construct (the ‘protocell&’) that, compared with liposomes improves on capacity, selectivity, and stability and enables targeted delivery and controlled release of high concentrations of multicomponent cargos (chemotherapeutic drugs, siRNA, etc.) within the cytosol or nucleus of cancer cells. Specifically, owing to its high surface area, the mesoporous silica core possesses a higher capacity for therapeutic and diagnostic agents than similarly sized liposomes. The fluid but stable SLB allows multivalent interactions with the target cell at very low targeting peptide densities, features crucial to maximizing specific binding, minimizing nonspecific binding, reducing dosage, and mitigating immunogenicity. Here using an in vivo egg embryo model, we image dynamic protocell/cellular interactions at the individual nanoparticle scale and benchmark protocells against alternative nanocarriers.

Pancreatic cancer has very high mortality rates and often has a poor prognosis, even when diagnosed early. Efficient delivery of anti-cancer drugs to the tumor in the pancreas in combination with other treatment strategies will significantly improve outcome for patients suffering from this deadly disease. The use of multifunctional nanoparticles for theranostics - therapy as well as diagnostics/prognosis -holds great promise for pancreatic cancer therapy. In this study we report the synthesis, characterization and in vitro application of layered double hydroxide nanoparticles [LDH NPs, based on the Hydrotalcite (Mg₆Al₂(OH)₁₆CO₃#9679;4H₂O) structure] for pancreatic cancer therapy. Various parameters critical for the synthesis of LDH NPs (~300 nm in diameter) as well as intercalation with anions and anti-cancer drugs were optimized. These parameters included pH, reactant concentration, heat treatment, solvent for ion exchange and intercalation temperature. The phase purity, particle size, morphology and chemical composition of the resulting LDH NPs were characterized by XRD, dynamic light scattering, scanning electron microscopy, and inductive coupled plasmon optical emission spectrometer. To demonstrate the ability of the newly synthesized LDH NPs for drug delivery, a known inhibitor of the proliferation of pancreatic cancer cells - valproic acid - was intercalated into the NPs. When cultured pancreatic cancer BXPC3 cells were treated with the valproic acid containing LDH NPs a significant reduction in cell viability was observed. These results indicate that LDH NPs, synthesized by the methods optimized in this study, have enormous potential of for pancreatic cancer therapy.

Polyesters have a strong and increasing importance in material science due to their biocompatibility and biodegradability that make these materials attractive for biological uses, nowadays mainly devoted to drug delivery applications, such as nanoparticles (NPs) production. In this work biocompatible and biodegradable NPs, with tunable hydrophobicity and biodegradation kinetics, were synthesized using novel Comb-Like Polymers (CLB). The synthesis and characterization of both polymers and NPs based on polylactic acid (PLA), polycaprolactone (PCL) and polylactic-co-glycolic acid (PLGA) is detailed. The proposed NPs synthesis allows obtaining small and better controlled particle sizes than current synthetic methods such as nanoprecipitation which, in addition, involves the use of an organic solvent. Moreover using this process it is relatively easy to tune final nanoparticles features: influence of parameters such as emulsifier type, feeding mode and macromonomer chain length has been investigated. In addition, comb-like polymer obtained through the copolymerization of these novel macromonomers with pegylated hydroxyethylmethacrylate monomers (HEMA-PEG) have been produced through a surfactant free polymerization process.
All the novel NPs produced satisfy requirements for biological purpose for intravenous drug delivery in terms of size, between 20 and 300 nm, particle size distribution, biodegradability and biocompatibility. The synthesis and development of these NPs is part of an ambitious project aimed to elaborate new diagnostic/prognostic tools and novel therapeutic strategies, in particular to treat triple-negative breast cancer. As a results, a number of obtained results for imaging (in vitro, ex vivo and in vivo), cell tracking (stem cells), cellular uptake studies, drug loading (antitumorals), toxicity studies, degradation and release studies are reported.

Nanoparticles have attracted great interest as therapeutic vehicles for potential treatment of disease. DNA nanostructures offer several advantages over conventional carriers such as liposomes and polymeric particles. These include low immunogenicity, a simple fabrication process with a uniform particle size and controllable spatial location of ligands. We have previously shown that DNA nanostructures are resistant to nuclease degradation and can be used to deliver antisense DNA to cells. However, these and other carriers generally lack target specificity and exhibit limited cell uptake. In this work, we design and demonstrate that self-assembled DNA nanostructures bearing multivalent AS1411 aptamers can selectively target cancer cells and induce efficient intracellular uptake. The improved uptake relative to naked DNA nanostructures and linear DNA relies on the aptamer multivalency and aptamer concentration. In addition, the AS1411 aptamer is by itself being studied in clinical trials as a potential therapeutic agent for cancer. The bioactivity of AS1411 is due to a reduction in the level of Bcl-2, which is a well-known apoptosis regulator. We are currently exploring the co-presentation of Bcl-2 antisense and AS1411 aptamers on our DNA nanostructures to further promote the degradation of Bcl-2 mRNA and inhibit Bcl-2 protein expression. Overall, our in vitro results demonstrate the potential synergy between AS1411 aptamers and Bcl-2 antisense and suggest that such multifunctional DNA nanostructures could be used as therapeutic delivery vehicles in cancer.

Mussel-inspired chemistry has aroused great interest in planar surface coating on various materials, including metals, metal oxides, glasses, ceramics and polymers [1]. We report the one-step assembly of catechol-based dopamine and polymer-dopamine conjugates onto spherical templates, followed by removal of these templates to form robust capsules [2-4]. Several templates including silica particles [2], polystyrene particles, and dimethyldiethoxysilane emulsion droplets [3] have been used for capsule fabrication. Mussel-inspired catecholic chemistry allows for the preparation of monodisperse capsules with tuneable diameters (0.3minus;5 µm), shell thicknesses (10minus;140 nm), and enzymatic degradability. Several functional substances, such as hydrophobic (nanoparticles and anticancer drug) and hydrophilic (enzyme) cargos, have been easily encapsulated in capsules. Polydopamine capsules have been used to immobilize polymer-drug conjugates based on thiol-polydopamine reaction for drug delivery into cancer cells [5]. The drug-loaded capsules show higher toxicity to cancer cells compared with same equivalent of free drugs. In addition, PDA capsules without cargos were shown to exhibit negligible toxicity toward LIM1215 and HeLa cells, which is important in biomedical applications. The reported technique is versatile, relatively quick and low-cost, using commonly available and naturally occurring compounds, which provides new avenues for the preparation of polymer capsules with defined properties and for encapsulation and drug delivery systems.
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[4] C. J. Ochs, T. Hong, G. K. Such, J. Cui, A. Postma, F. Caruso Chem. Mater. 2011, 23, 3141.
[5] J. Cui, Y. Yan, G. K. Such, K. Liang, C. J. Ochs, A. Postma, F. Caruso Biomacromolecules 2012, 13, 2225.

The use of nanoparticles for cancer detection requires their detailed characterization within biological systems. Owing to their small size, the use of advanced high resolution imaging and analysis techniques is necessary to locate and view individual nanoparticles and their position within larger biological structures like the liver or a tumor. High resolution scanning electron microscopy (SEM), aberration corrected transmission electron microscopy (TEM), and high resolution secondary ion mass spectrometry (NanoSIMS) are three powerful tools for carrying out such analyses in complex systems.
In a recent study, scanning transmission electron microscopy (STEM) was used to investigate the accumulation of gold core SERS nanoparticles injected either intravenously or intrarectally in mice[1]. Liver tissue from select mice was analyzed for nanoparticle accumulation. The results indicated that the nanoparticles injected intrarectally did not pass through the colon wall and into the blood stream whereas those injected intravenously accumulated in the liver. This analysis required the examination of extremely large volumes of tissue for TEM, which the STEM approach enabled. In another study, SEM was utilized correlatively with light microscopy to locate nanoparticles within brain tissue[2]. Both backscattered and secondary electron imaging in the SEM demonstrated that the nanoparticles accumulated in a brain tumor and not in the surrounding healthy tissue and that the nanoparticles normally accumulate in clusters. These two examples demonstrate the capabilities of advanced characterization techniques to provide insight into the interactions of nanoparticles with biological systems.
NanoSIMS is increasingly being used in biological applications due to the combination of medium spatial and compositional resolution. We performed a comparative study between NanoSIMS and SEM to determine the advantages and disadvantages of this technique. For our purposes the SEM approach is often adequate. Aberration corrected monochromated TEM provides even more information about the structure, local chemistry and bonding states of nanoparticles in different environments. This technique has been utilized to study the effect of size on surface plasmon resonance in silver nanoparticles down to 2 nanometers[3].
We will present strategies for the use of these instruments to obtain the best possible results and will describe examples of their successful application to complex and pressing problems in cancer detection.
1. Thakor, A.S., et al., The Fate and Toxicity of Raman-Active Silica-Gold Nanoparticles in Mice. Science Translational Medicine, 2011. 3(79): p. 79ra33.
2. Kircher, M.F., et al., A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nat Med, 2012.
3. Scholl, J.A., A.L. Koh, and J.A. Dionne, Quantum plasmon resonances of individual metallic nanoparticles. Nature, 2012. 483(7390): p. 421-427.

Many triggered release systems based on the presence of physiological stimuli, such as pH, enzymes and redox-potential, have emerged to the forefront for advanced therapeutic delivery in biomedical applications. We have recently demonstrated that several independent release mechanisms can be brought together and synergistically function to tune cargo release profile. Dual-responsive polymer capsules were assembled using alkyne-modified poly(2-diisopropylaminoethyl methacrylate) (PDPAAlk) via layer by layer assembly and cross-linked by click chemistry using a biazide cross-linker containing a central disulfide moiety, and hence can degrade under reducing conditions due to the presence of intracellular thiol species such as glutathione (GSH).¹ These capsules synergistically respond to variations in pH and redox potential, thus facilitating cargo release at ultralow concentrations of GSH. This mechanism is based on the capsules exhibiting reversible size and charge variation, in response to pH changes that occur as a result of endocytosis. Therefore, at physiological pH (pH 7.4) the capsules shrink and remain stable for long periods of time; however, under acidic conditions (pH 6.0) they swell, allowing degradation of the disulfide moieties. This provides the capsules with the ability to exploit the environmental complexity that exists within cells. More importantly, we have also demonstrated a versatile way of controlling the intracellular degradation rate of these capsules by tailoring their degree of cross-linking.² The degree of cross-linking was tuned from 65% to 98% by adjusting the amount of cross-linker used to stabilize the polymer films. We showed that the pH responsiveness of the capsules was maintained, regardless of the degree of cross-linking. Cellular studies showed that the rate of intracellular degradation of the capsules could be controlled between 0 and 6 h by altering the degree of cross-linking in the polymer capsules. These studies also demonstrated that the cellular degradation of highly cross-linked capsules (>90%) was significantly retarded compared to degradation in simulated cellular conditions. This suggests that the naturally occurring cellular reducing environment is rapidly depleted, and there is a significant delay before the cells can replenish the reducing environment. The modular and versatile nature of this approach lends itself to application to a wide range of polymer carriers and thus offers significant potential for the design of polymer-based systems for drug and gene delivery.
[1] Liang, K.; Such, G. K.; Zhu, Z.; Yan, Y.; Lomas, H.; Caruso, F., Charge-Shifting Click Capsules with Dual-Responsive Cargo Release Mechanisms. Adv. Mater. 2011, 23, H273-H277.
[2] Liang, K.; Such, G. K.; Zhu, Z.; Dodds, S., J.; Johnston, A., P., R.; Cui, J., Ejima, H.; Caruso, F., Engineering Cellular Degradation of Multilayered Capsules through Controlled Cross-Linking. ACS Nano. DOI: 10.1021/nn3039353.

Over the past ten years, nanosized mesoporous silica (MS) particles have been extensively investigated in biomedical applications and shown the ability to deliver therapeutic cargos and incorporate imaging modalities. Recent reports [1-2] have demonstrated that the biodistrbution and toxicity of MS nanoparticles are greatly influenced by particle geometry. However, the in vitro or in vivo study of shape effect of MS nanoparticles is only performed on spherical and rod-shaped particles because of limited morphology-controllable syntheses.
In this work, we developed a novel synthetic approach to transform ellipsoid-shaped MS particles to torus using a heat treatment. The morphology of MS nanoparticles (from ellipsoid to biconcave to torus shape) can be easily controlled based on varying hydrothermal temperatures. These unique shaped MS nanoparticles were characterized by transmission electron microscopy (TEM), scanning electron microscopy, X-ray diffraction, nitrogen adsorption-desorption measurements, infrared and Raman spectroscopy. A possible formation mechanism of torus MS nanoparticles is proposed based on a time-course TEM study of shape transformation of MS ellipsoids (after different heat-treated times). In addition, the doxorubicin (DOX) and protein loading capacity of the ellipsoidal, biconcave-, and torus-shaped MS is investigated. The results show that torus-shaped MS nanoparticles have the greater DOX loading capacity compared to ellipsoidal and biconcave-shaped particles. Finally, the comparison of in vivo cell-particle interactions and blood circulation time of fluorescence-labelled MS ellipsoid and torus nanoparticles are studied in a ex ovo chick embryo model using intravital microscopy.
References:
[1] Huang, X.; Li, L.; Hao, N.; Liu, H.; Chen, D.; Tang, F. ACS Nano, 2011, 5, 5390-5399.
[2] Yu, T.; Greish, K.; McGill, L. D.; Ray, A.; Ghandehari, H. ACS Nano, 2012, 6, 2289-2301.

Despite significant progress in development of new chemotherapeutic agents and methods of dug delivery, the effective therapy for treatment of brain tumors remains a challenge. To overcome the blood brain barrier problem, high doses of systemic intravenous delivery of anticancer drugs are required, that can cause adverse side effects. It is becoming clear that a major unmet challenge for the field is to develop methods that allow effective and local delivery of chemotherapeutic agents in cellular level. Many 'solutions' to this problem have been published on this subject during the last decade, but we yet have to see an effective delivery technology.
Here we report a new method for local and on-demand release of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an anticancer agent, using electrical actuation of conducting polymer poly (3,4-ethylenedioxythiophene) (PEDOT). We fabricated BCNU-loaded PEDOT micro/nanospherical cups (PEDOT MNSCs) on the surface of conductive substrate. The fabrication process consists of electrochemical deposition of PEDOT around the electrosprayed BCNU-loaded poly(lactic-co-glycolic acid) (PLGA) micro/nanospheres. The electrochemical deposition was carried out with an applied current density of 0.5 mA/cm2. We were able to control the size of PLGA and thickness of PEDOT layers by adjusting electrospray and electrodeposition parameters. Scanning electron microscopy (SEM) revealed that the size of BCNU-loaded PLGA spheres and the wall thickness of PEDOT spherical cups ranged from 200 nm to 3 mu;m and 50 nm to 100 nm, respectively. BCNU was precisely triggered by electrical actuation of PEDOT MNSCs with an applied voltage of 1V.
Our preliminary results indicated a significant release of BCNU from PLGA spheres due to fast hydrolytic degradation of PLGA. Adding a PEDOT layer as a shell around the drug-loaded PLGA spheres dramatically decreased the rate of BCNU release. Finally, on-demand and precisely triggered release of BCNU was observed during electrical stimulation of the PEDOT MNSCs. This innovative drug delivery system holds a considerable promise for a highly precise control of anticancer agent release at the tumor sites to minimize the drug site effects. Future study will focus on effect of on-demand release of BCNU on brain tumor cells both in vitro and in vivo.

Magnetoelastic (ME) materials are ferromagnetic materials with magnetostrictive properties, which allow them to generate dynamic mechanical loads (vibrations) on the order of sub-micrometers when activated by an AC magnetic field. We have previously demonstrated that these mechanical loads can control cellular adhesion on an ME substrate surface [1], and local mechanical factors (i.e. matrix stiffness) have recently been shown to influence proliferation and chemotherapeutic responsiveness of hepatocellular carcinoma (HCC) cells [2]. We hypothesize that small, local mechanical loads can be used as an adjuvant therapy to improve the chemotherapeutic response of HCC cells. In this work, we will use ME materials to investigate the effect of these loads on the behavior of HCC cells. HCC cells (HepG2) were seeded on ME materials (1x10^4 cells/cm^2 cultured at 37 degrees Celsius and 5% Carbon Dioxide). After 6 or 48 hours of culture, cells were loaded with ME vibrations (150-170kHz; 0.15µm effective displacement) for 1 hour (control = cells cultured on non-vibrated ME materials; n = 3 each group). A media change containing 10µM cis-diamminedichloroplatinum(II) (cisplatin) was applied to all groups for 24 hours. Media was then renewed for an additional 24 hours (total incubation time = 96 hours) prior to staining with Calein-AM (live) and Ethidium Bromide (dead). Percent cell survival, defined as the number of live cells divided by the total number of cells, was determined through direct cell counting and analyzed using student&’s t-test, p-values less than 0.05 were considered significant. Results showed a significant reduction in cell survival for both the 6 hour (63.23±11.80) and 48 hour (70.17±8.63) loading groups in comparison to the non-vibrated control (98.73±0.40); no significant differences in cell survival were observed between loading groups. This result suggests that small, local mechanical vibrations can influence the chemotherapeutic response of HCC cells, and presents the potential of using this approach as an adjuvant therapy to improve the efficacy of current chemotherapies. This approach could be realized clinically through the use of low intensity ultrasound. Future work will focus on identifying the mechanisms responsible for these observations, which could also improve our understanding of the mechanisms by which tumors develop stiffness related resistance to particular agents.
[1] E Vlaisavljevich, LP Janka, KG Ong, RM Rajachar. Magnetoelastic materials as novel bioactive coatings for control of cell adhesion. IEEE Transactions on Biomedical Engineering. 58(3):698-704, 2011.
[2] J Schrader et al. Matrix stiffness modulates proliferation chemotherapeutic response, and dormancy in hepatocellular carcinoma cells. Hepatology. 53(4):1192-1205, 2011.

Mesoporous silica nanoparticles (particle diameter ~ 50 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 magnetic heating, resonance imaging and/or manipulation), targeting molecules (towards specific cells), 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 optical 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 affords external control. Activation by both of these methods in living cancer cells will be discussed.

Nano-structure field-effect transistor (FET) biosensors have gained much attention in recent years, and have emerged as a great candidate for point-of-care systems. In fabricating these nanobiosensors, top-down approaches better support the goal of high yield, controllability, and uniformity necessary for portable and reliable sensing than bottom-up approaches do. However, many top-down approaches require costly material or time-consuming fabrication to achieve the critical dimensions required to achieve high sensitivity. Borrowing from emerging metal oxide thin-film transistor (TFT) technology, we have fabricated In₂O₃ nanoribbon FET biosensors using a simple 2-step photolithography process. The In₂O₃ nanoribbon channels are deposited using scalable RF sputtering to create channel thicknesses ranging from 50nm to 10nm and below. The electrical properties of 50 In₂O₃ nanoribbon FETs showed good uniformity in on-state current, mobility, and threshold voltage. To verify the sensitivity of our In₂O₃ nanoribbon FETs to changes in ionic concentration, we measured changes in device conduction in solutions of varying pH. The sensors demonstrated good sensitivity to small pH changes within the physiological pH ranges and also showed stable changes in a wider pH range from 4 to 9. Furthermore, we demonstrated a negative correlation between In₂O₃ nanoribbon channel thickness and sensitivity to ions. As a proof of principle, we demonstrated that the In₂O₃ nanoribbon FETs have similar detection limits for the ovarian cancer marker CA125 when compared with In₂O₃ nanowire FET biosensors, suggesting that the gain in yield and controllability does not compromise device sensitivity. These combined characteristics give In₂O₃ nanoribbon biosensors a great advantage when integrating with other functional device groups in a point-of-care platform.

Surface enhanced Raman scattering (SERS) Au nanoparticles are designed and evaluated as optical labels for imaging of cell surface receptors. Charged Raman-active dyes are physisorbed on to the surfaces of citrate-capped Au nanoparticles, and thiol-terminated 5kDa polyethylene glycol (PEG) is used as a particle coating. PEG imparts stability to the nanoparticles in buffer solutions and functional groups in the PEG layer facilitate conjugation to monoclonal antibodies for cell targeting. Multiple characterization methods are used to investigate the particles&’ monodispersity in suspension, and functionalization with PEG and targeting antibodies. The SERS particles are targeted to multiple surface antigens of interest in B cell leukemia and lymphoma as demonstrated using primary patient chronic lymphocytic leukemia (CLL) cells and LY10 lymphoma cell line, respectively. The specificity of the SERS labels for their cell surface protein targets are assessed using non-specific control particles, and competitive binding experiments. Targeting moieties include both research grade antibodies, as well as rituximab, a therapeutic antibody that is specific for CD20. Raman spectroscopy, dark field microscopy, and flow cytometry as used to evaluate the targeting of malignant B cells by SERS nanoparticles. Triplexed detection of three cell surface molecules simultaneously on LY10 cells is resolved using Raman images derived from the results of least-squares fitting. Fluorescence flow cytometry is demonstrated as a valuable method for detecting cell labeling by SERS particles using both fluorescence secondary labeling, and competitive binding protocols. Additionally, the particles&’ SERS was directly detected using a flow cytometer. Immunophenotyping of lymphoproliferative disorders depends on the effective measurement of cell surface markers, typically relying on fluorescent labels. SERS particles have a greater potential for multiplexed detection due to narrow bandwidth spectra. Excitation of multiple SERS particle reporter types can ideally be accomplished with a single laser wavelength, decreasing the cost and complexity of diagnostic imaging equipment required.

The utilization of fluorescent and non-cytotoxic nanodiamonds as an active biolabeling agent to replace organic fluorophores and semiconductor nanocrystals is garnering extensive attention. Nanodiamonds, being almost wholly carbon, and containing photostable fluorescent defect centers (Nitrogen Vacancy Centers (NVCs)) have the advantage of being heavy-metal free, surface functionalizable, and with the world&’s most sensitive electric and magnetic field center in NVCs. Experimentally the literature has concentrated on detonation nanodiamonds, and we have found some stark contrasts in our investigation of high-pressure, high-temperature (HPHT) nanodiamonds. In this work with HPHT nanodiamonds we have several new insights concerning their crystallographic and electronic structure quality, impurity identification, and surface species identification after oxidative purification leading to aggregate free nanodiamonds with readily active NVCs. Namely, we identify their superior crystallographic and electronic structure via Raman spectroscopy in conjunction with NEXAFS and HRTEM. The major impurity in the HPHT nanodiamond was found to be amorphous carbon and not graphene nor graphite. Raman spectroscopy also revealed that dangling carbon atoms on the HPHT surface reconstruct to form localized sp2 networks referred to as Pandey chains. Also, we identify that with increasing oxidative temperature the percentage of alcohol surface groups increases quickly in comparison to ketones. Using DRIFTS, XPS and NEXAFS we conclude that the surface of oxidized HPHT NDs does not contain detectable concentrations of carboxylic acids, but instead is largely alcohols and ketones. This is in stark contrast to the literature which cites that aerobic oxidation leads to a largely carboxylic acid terminated surface, especially in the case of detonation NDs. We also find that piranha treatment followed by aerobic oxidation at 575°C can readily decrease the presence of ketones altogether, while maintaining an alcohol rich surface. Interestingly this leads to a view that chemical and aerobic oxidative treatments etch unstable carbons with 2 and 3 dangling bonds, leading to a 111 diamond surface largely terminated with alcohols (1 dangling bond). These findings unambiguously show that HPHT NDs share properties similar to bulk diamond, and thus our findings have broad implications for the successful chemical functionalization routes of HPHT NDs, and in turn result in stable NVCs that can be manipulated and optically readout for active biolabeling.

Prostate, breast, colon, and lung cancer are the most prevalent cancers in the US among men and women. Current detection methods include mammograms, self-exams, colonoscopies, digital rectal exams, CT scans, and biopsies. Most methods can be invasive, and they only detect the cancer after sizeable growths have formed. Earlier diagnosis could help with prognosis and allow for better treatment options. We have previously shown that quantum dots encoded in mesoporous silica beads conjugated with antibodies specific to breast cancer biomarkers can detect multiple biomarkers in a single assay by using a different color of quantum dot for each biomarker. While results indicate promise as a multiplex detection assay, the limited number of emission wavelengths in singly loaded silica beads does not allow for an enhanced system able to screen for a multitude of protein biomarkers. We have utilized the unique luminescent and spectral properties of CdSe/ZnS quantum dots to demonstrate the development of quantum dot-mesoporous silica composite particles encoded with quantum dots of multiple wavelengths at precisely controlled ratios. Previous methods incorporated a quantum dot with one emission color into silica beads which were then coated with a single antibody, making a probe for one biomarker. Additional beads, each with a different single color and antibody combination, were then prepared and used together for multiplex detection of up to three biomarkers. In the current approach, up to three different colors of quantum dots are loaded into each bead with controlled ratios. For example, the red, green, blue emission ratio for one bead might be 1:1:1, while another bead might be 1:3:1. Once prepared, beads of a given ratio are then labeled with a single antibody. Combinations of these beads can allow multiplex detection of multiple (>3) biomarkers. We have succeeded in controlling quantum dot loading into silica beads with different quantum dot ratios. This novel method will increase the target number of biomarkers that can be screened in a multiplex assay. Bioconjugation of these composite particles with antibodies will allow for the simultaneous screening of multiple cancers or other diseases.

Advances in nanotechnologies and nanomaterials offer numerous opportunities to develop powerful imaging probes for cancer imaging. The standard approach in the field is to design and synthesize nanoparticles in vitro and then apply them in vivo. We are exploring a different concept of developing nanoparticle probes—in celluo synthesis of nanoparticles for tumor imaging. Instead of synthesizing nanoparticles in vitro and applying them in vivo, this new approach will deliver small molecules to cells as building blocks and then synthesize nanoparticles from them inside cells.
Our strategy is based on a biocompatible chemical condensation reaction between two chemical groups -- 1,2-aminothiol and 2-cyanobenzothiazole [ref 1-3]. We have demonstrated that this condensation chemistry can lead to the formation and assembly of nanoparticles in vitro and in living cells under the control of pH, disulfide reduction and/or enzymatic cleavage. Now we have new results that suggest this in celluo nanoparticle assembly strategy is also working in living animals and compatible with several imaging modalities. Based on this strategy, we have successfully developed probes that can image drug-induced apoptosis in tumor cells in vivo with whole-body fluorescence, positron emission tomography (PET), and magnetic resonance imaging (MRI).
Ref:
1. Liang G, Ren H, Rao J. Nature Chemistry 2010, 2, 54-60.
2. Ye D, Gao L, Ma ML, Rao J. Angewandte Chemie International Edition 2011, 50, 2275-2279.
3. Liang G, Ronald J, Chen Y, Ye D, Pandit P, Ma ML, Rutt B, Rao J. Angewandte Chemie International Edition 2011, 50, 6283-6286.

We will describe a method that used chemically modified carbon nanotubes to selectively bind to diseased tissue (e.g. arterial plaques) and were imaged using several modalities (ultrasound, fluorescence and thermal imaging). The system also incorporated a sensing system that was able to detect down to picomolar levels. Other ongoing work using graphene and carbon dots will be presented for performance comparison.

It was reported that the prostate cancer is considered as the second main cause of cancer related death among men in USA [1]. In fact, this serious disease is identified as a genetical disorder occurring in the epithelial cells of the prostate which induces a high production of prostate specific antigen (PSA) proteins. A PSA concentration superior than 4 ng/ml uses to be a strong indication of the presence of cancer [2]. Nevertheless, we find in the human serum two categories of PSA. Only the proteolyticcally-active one is a useful serological marker for the early diagnosis and monitoring of prostate cancer [3]. For this reason, we build-up a very simple biosensing system based on the detection of the proteolysis activity of the PSA through the cleavage of the fixed peptide-magnetic nano-carriers and the subsequent attraction of the released magnetic beads onto the external magnet. This dramatic change on the structure and the morphology of the sensitive monolayer can be monitored easily by different physical transducers such as : the electrochemical impedance spectroscopy (EIS), surface plasmon resonance (SPR) and colorimetric assay. The results show the ability of our system to detect the PSA at pg/ml level within an improved timing (from 30 min to 5 min with the colorimetric assay). Moreover, the same construction was efficient in distinguishing different PSA substrates as well as the non specific ones which offers more possibilities to develop an advanced platform for ultra-rapid drug discovery.
[1] R. Siegel, C.A Cancer, J. Clin., 2011. 61: 212-36.
[2] H.B Carter, N. Engl. J. Med., 350 (22): 2292-4 2004.
[3] P. Wu, L. Zhu, U.H. Stenman, J. Leinonen, Clin. Chem., 2004 ; 50 (1) :125-9.

Magnetic barcode nanowires composed of gold/nickel multilayers with various surface coatings were used as agents for imaging and magnetic separation of Osteosarcoma (OSCA) cells. Magnetic beads, quantum dots and fluorescent probes are among current technologies applied in studies of cellular uptake, cell sorting and identification. However, cell identification is limited by single antibody-labeled magnetic beads or by the number of spectrally resolvable fluorophores (wavelengths). The ability of barcode nanowires to label populations of cells with unique signatures would enable identification and separation of multiple cell populations. Here, selective targeting of OSCA cells was followed by excitation of localized surface plasmon resonance or application of alternating magnetic fields to study potential therapies using the energy absorbed by phagocytosed nanowires. Importantly, the number of codes available in this technology is a function of the distinguishable materials and lengths used in the multi-layers of the barcode nanowires which can be read using reflectance microscopy and magnetic detection. Various imaging techniques will be shown to illustrate the use of the barcodes as image-contrast agents in OSCA cells, including differential interference contrast (DIC), reflectance, confocal and dark-field microscopy. While reflectance microscopy was excellent in distinguishing the multilayers in the barcodes, a combination of bright and dark field optical microscopy was found to provide comprehensive information on interactions between nanowires and cells and their response to external fields. Moreover, a combination of these techniques and SEM/TEM imaging was used to study dependence of cellular uptake on nanowire concentrations, lengths and surface coatings and also to quantify separation yields using magnetic separation. For example, the separation yield increased from 16.6% to 35.11% as the initial nanowire to cell plating ratio increased from 1:1 to 100:1. A comparative empirical study of specific and non-specific uptake of the barcodes by OSCA was used to better understand the internalization pathways. Toxicity analysis included exposing peritoneal macrophages to the barcodes nanowires and then monitoring IL-1b, TNF-α levels, and metabolic activity. Nearly no cell death was induced and minimal quantities of IL-1b and TNF-α were induced over a six hour incubation period. In short, barcode nanowires hold much promise for multiplexed diagnosis, magnetic separation and therapy.

Currently available radio-frequency (rf) hyperthermia and ablation techniques, even if minimally invasive, are non-selective, and frequently induce thermal necrosis in both normal and malignant cells surrounding the source of rf energy inserted in the tissue. It has been demonstrated that rf procedures can be non-invasive and cell-selective when an external source of rf is combined with either directly or systemically injected functionalized nanoparticles (NPs) acting as rf absorption enhancers. We describe a characterization method of the efficiency of rf heating of nanoparticles (NPs) suspended in an aqueous medium. Contrary to the common approach, where heating of magnetic NPs is assumed to be caused only by field interaction with spins, we have analyzed all rf loss components which include electric field induced losses in NPs and eddy-currents losses in NPs solution. We carried out measurements of water suspended 5 nm, 30 nm and 50 nm superparamagnetic iron-oxide NPs with dextran matrix for three different configurations of electric and magnetic rf fields. A 30 MHz high-Q resonator was designed to measure samples placed inside a parallel plate capacitor and solenoid coil with or without a specially designed electric field shield. To quantify rf heat enhancement due to the presence of NPs, their specific absorption rate (SAR) was determined by measuring the temperature change of the NP suspension using a solenoid coil. Literature reports on SAR measurements of magnetic NPs recognize only the presence of the magnetic field component in the solenoid, as a result, only the circumferential electric field component (E_phi;) has been considered responsible for eddy currents losses and axial magnetic field (H_z) for spin related losses. Surprisingly, the existence of the axial conservative electric field component (E_z) was disregarded. However, our analysis determined that the magnitude of the E_z component, in most solenoid configurations is at least comparable to the magnitude of the E_phi; component and significantly contributes to the total rf electric field (E_s = E_phi; + E_z); thus affecting the sample heating. The SAR of the NPs and solution for the all componants of electric field loss E_s was determined to be 8.5 kW/kg and 3.8 W/kg, respectively, while the SAR for magnetic field loss componant H_z was determined to be 4.1 kW/kg. Further, we have shown that if E_s is assumed negligible, as it is done in literature, all heat loss produced by the electric field components would be counted as magnetic loss, and the magnetic SAR would be 14 kW/kg. This is a very significant overestimation of the NPs SAR, compared to the more accurate value of 4.1 kW/kg obtained in this work (giving 250% systematic error).The presented methodology of SAR determination was also applied to metallic (Au) and dielectric (silica) NPs. We will show relevant rf loss components for these NPs and discuss mechanisms of heating.

Most anti-cancer drugs have limited water solubility, and short blood circulation in the body systems, leading to frequent administrations. In addition, they cause harmful side-effects due to their non-specificity. To combat these problems, liposomes, dendrimers, polymeric micelles and peptide assemblies have been reported as vehicles to deliver anti-cancer drugs. In particular, biodegradable polymeric micelles have attracted increasing attention due to their well-defined core-shell nanostructures that allow hydrophobic anti-cancer drugs to be encapsulated in the core while providing increased water solubility, prolonged blood circulation, reduced protein adsorption and recognition by the mononuclear phagocytic system, and nanosize that gives rise to passive accumulation in tumor tissues (i.e. the enhanced permeability and retention (EPR) effect).
In this study, aliphatic polycarbonates were chosen as the building units for amphiphilic copolymers because of their biodegradability and non-toxic degradation products. Four diblock copolymers of acid- and urea-functionalized polycarbonate with the same length and poly(ethylene glycol) (PEG) with different molecular weights (Mn: 5 kDa and 10 kDa) were synthesized via organocatalytic ROP and employed to prepare mixed micelles via acid-urea non-covalent interactions. An amine-containing anticancer drug, doxorubicin (DOX) was loaded into the mixed micelles via a self-assembly process. DOX-loaded 5K and 10K PEG mixed micelles had sizes below 100 nm with narrow size distribution, and high drug loading capacity due to ionic interaction between the amine group in DOX and acid groups in the micelles. DOX-loaded 5K PEG mixed micelles had greater kinetic stability. In a 4T1 mouse breast cancer model, the micelles were preferably transported to the tumor. In particular, DOX-loaded 5K PEG micelles accumulated in the tumor more rapidly to a larger extent, and inhibited tumor growth more effectively than free DOX and DOX-loaded 10K PEG micelles without causing significant toxicity. The anti-tumor efficacy of DOX-loaded micelles is based on DOX-induced apoptosis. In addition, when thioridazine, which also contains an amine group, and DOX were delivered simultaneously using the mixed micelles into MCF-7 human breast cancer cells, the population of cancer stem cells was reduced significantly as compared to individual formulations. These mixed micelles hold great potential for use as carriers for targeted delivery of anticancer drugs.

Emerging evidence from clinical studies has suggested the inversion association between cancer and Alzheimer&’s disease, but the molecular mechanisms of such inverse comorbidity remains unknown. In this study, we used a small hydrophobic molecule that self-assemble in water to form soluble fibrillar aggregates, which are morphologically and phenotypically similar to soluble oligomers of aberrant proteins (e.g., Aβs)—a plausible causal agent of Alzheimer&’s disease. According to cell-free and cell-base assays, these aggregates interact with cytoskeletal proteins to disrupt formation of microtubules, arrest cell-cycle, and inhibit the growth of several mammalian cancer cell lines while exhibiting little acute toxicity to a neuronal cell line. This work presents a new molecular mechanism that suggests the differential cytotoxicity of soluble fibrillar aggregates of hydrophobic molecules may function as a common origin of the inverse comorbidity between cancers and neurodegenerative diseases, which ultimately may contribute to the development of molecular aggregates for understanding and treating the two most devastating diseases.

Oncolytic viruses (OVs) can target multiple mechanisms of action while at the same time exploit validated genetic pathways known to be disregulated in many cancers. In particular, the oncolytic virus TAV-255 has shown viral replication attenuation in normal cells while retaining cytolytic activity in tumor cells by taking advantage of defects in the p53-tumor suppressor pathway. Despite its several advantages, the utility of OVs for cancer therapy is limited by 1) neutralization by antibodies mediated by the immune system, 2) rapid clearance by the reticuloendothelial (RE) system in the liver, and 3) the lack of expression of surface receptors (CAR) in certain cancers necessary for OV transduction. With the aim to overcome an immune response and to enhance its potential use to treat primary and metastatic tumors, a method for liposomal encapsulation of adenovirus has been developed. The encapsulation of Adenovirus in an anionic non-toxic liposome has been prepared by self-assembly of Lecithin around the viral capsid. The developed method has shown that encapsulated viruses retain their ability to infect cancer cells. Furthermore, an immunoprecipitation (IP) technique has shown to be a fast and effective method to extract non-encapsulated viruses and homogenize the liposomes remaining in solution. Extracting non-encapsulated viruses from solution may prevent an adverse immune response when used in an in vivo model and may enhance treatment for multiple administrations.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in humans. They have very poor prognoses with a median survival time of 14 months. Gene therapy could potentially improve the dismal prognosis of patients with glioma, but this treatment modality has not yet reached the bedside due to the lack of safe and effective gene delivery vehicles. For glioma gene therapy, viral vectors have been used as a delivery vehicle. However, effective gene delivery to brain tumor cells in vivo remains a main challenge for clinical application due to the inability of current delivery vehicles to overcome extra- and intra-cellular barriers in the brain. Furthermore, concerns over safety and potential side effects have slowed their advancement into the clinic. To address these limitations, various non-viral vectors have been developed to safely deliver genes. These systems have shown promise in vitro, but exhibit significantly reduced transfection efficiencies in vivo due to the lack of site specificity and limited internalization by target cells. This presentation covers development and assessment of a cancer-cell specific magnetic nanovector construct for efficient gene delivery and non-invasive monitoring through magnetic resonance imaging (MRI). The nanovector is comprised of a superparamagnetic iron oxide nanoparticle core and a polymer shell complexed with DNAs and conjugated with a tumor-targeting peptide to improve tumor specificity and potency. The strategies for surface engineering of nanovector to bypass the physiological barriers including blood brain barriers, maximize blood half-life, overcome intracellular barriers, and enable controlled drug release will be discussed. Finally, the design and characterization of several nanovectors for targeted delivery of genes and siRNAs and the nanovector with dual targeting and therapeutic functions will be presented. Our results showed that these nanovectors overcame various biological barriers and induced effective gene transfection both in vitro and in vivo.

Layer-by-layer assembly was introduced almost two decades ago as a versatile technique for the construction of thin multiple layer films composed out of polyelectrolytes. Shortly after the concept was extended from planar to spherical geometry, resulting in polyelectrolyte multilayer capsules. The semipermeable wall of the capsules (with a thickness of a few nanometers), and the cavity can be further loaded with inorganic colloidal nanoparticles (NPs) made of different materials, and with multiple cargos, respectively. The resulting multifunctional capsules are well suited for in vitro delivery of cargo inside cells. This concept has been highlighted in several recent reviews. Meanwhile technology has advanced to a point at which these capsules could be a helpful tool for controlled multifunctional in vitro delivery. Nowadays the cavity of capsules can be loaded with a large variety of cargo. While large molecules such as proteins will be readily kept inside the cavity, small molecules need to be either linked to macromolecules such as dextran,or be embedded inside micelles.The micelle approach even allows for encapsulation of small hydrophobic molecules. The materials forming the polyelectrolyte wall can be chosen such that capsules internalized by cells are not degraded and preserve their cargo over weeks. Leakage of the cargo molecule is reduced and controlled release of cargo upon external stimuli such as photothermal heating with Au NPs can be performed.

Nanoparticle based drug carriers represent a promising approach for the design of anticancer therapeutics. Careful optimization of size, stability and cargo retention is required to facilitate the translation of different nanoplatforms as clinically relevant therapeutics. In order to address the challenges in the field of nanomedicine, we have designed ultra small and highly stable micelles based on self assembly of polymer conjugates of coiled-coil three helix bundles. The present study is focused on the evaluation of the structure and biological stability of doxorubicin loaded 3-helix micelles as potential nanocarriers. Doxorubicin loaded micelles with drug content ranging from 1-8wt% were prepared by solvent evaporation method. The self assembly of amphiphiles into spherical micelles was maintained after drug incorporation and the effect of drug content on size of 3-helix micelles was minimal. Structure, stability and release kinetics of micelles with highest drug loading were investigated by light and X-ray scattering, differential scanning calorimetry, liquid chromatography and fluorescence spectroscopy. Doxorubicin loaded 3-helix micelles, ~ 15 nm in size, could be formulated in aqueous solutions at ~8wt% drug content without free drug, in a reproducible manner. Differential calorimetry indicated drug interaction with micelle core and peptide structure in micellar shell was maintained after drug incorporation. Drug loaded micelles exhibit minimal protein adsorption and exceptionally slow release in serum albumin. Localized delivery of micellar-doxorubicin in normal mice brain indicated greater extent of distribution and significantly reduced toxicity of micelle incorporated drug compared to free drug. Drug loaded 3-helix micelles with small size, extended stability and minimal drug leakage in protein rich biological environment meet some of the most critical requirements for safe and effective nanocarriers for intravenous delivery and could be potential candidates for drug carriers with stable drug incorporation in circulation before reaching the target site. Molecular properties of the amphiphile can be easily tailored for generating micelles compatible with different drugs. The present system appears to be a promising and versatile platform for the design of stable nanocarriers for biomedical applications.

Nanomedicine is a growing field that utilizes nanoscale materials to treat or cure diseases at the cellular level. Controlled release processes from porous materials and cell targeting represent challenging fields of research. For targeted drug delivery, the triggered release of bioactive compounds at specific locations, times and conditions is highly desirable. Recently we have developed strategies to incorporate molecular functionality at different locations of colloidal mesoporous silica (CMS) nanoparticles.[1,2] Here we present our recent studies on a polymer-based modular porous nanosystem that features, simultaneously, pH-sensitive release, cell targeting and long term stability.[3]
To the surface of the amino-functionalized shell of our core-shell CMS particles, poly(2-vinylpyridine) (PVP) was attached as a pH-sensitive cap system. At pH values of ~7 or higher, this polymer is hydrophobic and blocks the pores, whereas at acidic pH it is water soluble and allows the release of a drug. Furthermore, we used poly(ethylene glycol) (PEG) as solubilizer, folic acid as targeting ligand and a red light photosensitizer for triggering endosomal escape. Release in HeLa cells was studied by confocal fluorescence microscopy, whereas targeting experiments were conducted in KB cells. Long-term stability tests of our silica-polymer composite material were performed in simulated body fluid at different pH-values. Under these conditions, no degradation of the mesoporous silica host could be observed, whereas in unfunctionalized silica strong degradation occurred.
Our results show the great potential of this polymer-capped system for targeted drug delivery.
Acknowledgement
The authors are grateful for funding from DFG through the SFB 749, the NIM cluster and the “Fonds der Chemischen Industrie”.
[1] J. Kecht, A. Schlossbauer, T. Bein, Chem. Mater. 2008, 20, 7207.
[2] C. Cauda, A. Schlossbauer, J. Kecht, A. Zürner, T. Bein, J. Am. Chem. Soc. 2009, 131, 11361.
[3] S. Niedermayer, V. Weiss, A. Schmidt, C. Bräuchle, T. Bein, in preparation

Biological systems are exquisitely sensitive to the location and timing with which physiologic cues and drugs are presented. However, exploring the relationship between cue timing and tissue response in vivo remains challenging, particularly for transient signals. Polymer-based delivery systems are used extensively for attaining localized, sustained release of bioactive molecules, and release rates depend on kinetic processes such as hydrolysis and passive diffusion. We hypothesized that these processes could be transiently accelerated by applying ultrasound to hydrogels, but that permanent damage to the material would be avoided if the gel were formed by reversible crosslinks which could reform when the ultrasound was withdrawn. Consequently, release could be switched on and off, on-demand.
We first investigated the mechanical disruption of ionically crosslinked calcium-alginate hydrogels via low intensity ultrasound (18 mW/cm2). At this intensity, there was no significant change in the temperature of the saline solution bathing the hydrogel, but visual inspection and scanning electron microscopy revealed marked degradation, which was further confirmed by a substantial decrease in elastic modulus and dry mass of the material. Strikingly, however, no visual change in hydrogel appearance, microstructure, or mechanical properties was observed when hydrogels were maintained in media with physiological levels of calcium during and after ultrasound treatment; this confirms the self-healing ability of these materials. The ability to release a variety of potentially therapeutic molecules, including cytokines, oligonucleotides and small molecules on-demand was confirmed in vitro. These studies indicated the ability to exert nearly digital control over drug release, and that the rate of on-demand release could be controlled by manipulating the extent of molecular interactions between the polymer and drug molecule. The ability to elicit on-demand release from other types of hydrogels was demonstrated via ultrasound induced drug delivery from ionically crosslinked chitosan hydrogels.
Finally, this system was exploited in vivo to treat xenograft tumors formed from metastatic human MDA-MB-231 breast cancer cells. A daily application of ultrasound to mitoxantrone-laden alginate gels dramtically reduced tumor growth rate and improved animal survival, compared to treatment with either mitoxantrone eluting hydrogels without the ultrasound stimulus, or with ultrasound alone. Altogether, this study demonstrates a general ability of reversibly crosslinked hydrogels to exhibit self-healing under ultrasound, which can be exploited for on-demand drug delivery. This method of achieving digital control over drug release is likely to be applicable to a broad variety of polymers and bioactive molecules, and is a potentially useful tool for studying how the timing of factor delivery controls cell fate in vivo.