CUNY Advanced Science Research Center
85 St Nicholas Terrace,
New York, NY 10031
June 23rd and 24th 2016
Thursday June 24th 2016
Nano-tools and Bioinspired Tissue Engineering Approaches for the Regeneration of Different Tissues
Rui L. Reis University of Minho
This talk will describe several nano-tools and biomimetic approaches for the regeneration of different tissues. The selection of a proper material to be used as a scaffold or as a hydrogel, in many cases in combination with nanoparticles, to support, hold or encapsulate cells, as well as to control their differentiation, is both a critical and difficult choice. It will ultimately determine the success or failure of any tissue engineering and regenerative medicine (TERM) strategy.
We believe that the use of natural origin polymers is the best option for many different approaches that allow for the regeneration of different tissues. In addition to the selection of appropriate material systems it is of outmost importance the development of processing methodologies that allow for the production of adequate nano delivery systems and scaffolds/matrices.
Furthermore an adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow and adipose tissue, cells from amniotic fluids and membranes and cells obtained from umbilical cords.
The potential of each type of cells, to be used to develop novel useful regeneration therapies will be discussed. Their uses and their interactions with different nano systems and natural origin degradable scaffolds and smart hydrogels will be described. Examples of the engineering of different tissues will be presented.
Supported Biomembrane Microenvironments of Controlled Composition for Gamma-Secretase Substrate Cleavage Assays
Lane Gilchrist Memorial Sloan-Kettering Cancer Center
The intramembrane protease gamma-secretase is a current target of therapeutic intervention, with pivotal pathological functions within Alzheimer's disease and cancer. Our primary objective is to expand methods for in vitro studies of the intramembrane proteases (IMPs) with the development of a microsphere-supported (proteolipobead) and planar biomembrane platforms. IMPs are found throughout all branches of life and their functions are extremely broad. Despite extensive studies, understanding of IMP regulation and catalysis has been hampered by membrane-associated enzymology. Rhomboids and Secretases are polytopic membrane proteases that are widely conserved in all organisms. The precise reaction mechanisms of the intermembrane proteases remain to be elucidated and furthermore, rigorous analysis of the kinetics of interfacial catalysis in these systems has not yet been undertaken.
High throughput of screening of drug candidates has been carried out in bulk assay systems using cell membrane fragments, solubilized enzymes and is underway in proteoliposomes. A critical barrier to further progress in the study and HTS of gamma-secretase is that such bulk systems do not allow for the direct in situ quantification of enzyme, substrates, or inhibitors or their relative distributions within the structures under assay. We have expanded in vitro models of gamma-secretase to include supported biomembranes, enabling: 1) characterization and verification of biomembrane loading of substrates, inhibitors and protein effectors, 2) studies of co-localization, phase-partitioning and lateral mobility of membrane-bound assay constituents, and 3) the development of flow cytometry-based assays of cleavage. In this work we have characterized the structures, phase localization and compositions of gamma-secretase proteolipobead and planar systems by employing correlative optical and surface microscopy from the micro- to the nanoscale. We have probed the formation of Lo domains in situ, monitored using 3D FRET phase detection. We have probed the phase partitioning of enzyme, substrates and cleavage products in PLB systems with superresolution microscopy. A combination of biomembrane mobility methods are being used to probe the diffusivities and mobile fractions of lipid, substrates and gamma-secretase. The results of phase partioning and diffusivity measurements are being integrated into bottom-up spatial diffusion-reaction models to correlate microenvironment with enzyme function. As new drug targets in cancer and infectious disease involved in regulated intramembrane proteolysis (RIP) based cell signaling are uncovered, it is expected that other IMP enzymes can be subsequently studied and probed at high throughput with this platform
Engraftment and Function of Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells in Mice via 3D Co-aggregation and Encapsulation
Robert E. Schwartz Weill Cornell Medical College
Cellular therapies for liver diseases and in vitro models for drug testing both require functional human hepatocytes, which have unfortunately been limited due to the paucity of donor liver tissues. Human pluripotent stem cells represent a promising and potentially unlimited cell source to derive human hepatocytes. However, the hepatic functions of these human pluripotent stem cells-derived cells to date are not fully comparable to adult human hepatocytes and are more similar to fetal ones. In addition, it has been challenging to obtain functional hepatic engraftment of these cells with prior studies having been done inimmunocompromised animals. In this report, we demonstrated successful engraftment of human induced pluripotent stem cell derived hepatocyte-like cells in immunocompetent mice by pre-engineering 3D cell co-aggregates with stromal cells followed by encapsulation in recently developed biocompatible hydrogel capsules. Notably, upon transplantation, human albumin and ?1-antitrypsin (A1AT) in mouse sera secreted by encapsulated induced pluripotent stem cell derived hepatocyte-like cells/stromal cell aggregates reached a level comparable to the primary human hepatocyte/stromal cell control. Further immunohistochemistry of human albumin in retrieved cell aggregates confirmed the survival and function of iPS-H. This proof-of-concept study provides a simple yet robust approach to improve the engraftment of induced pluripotent stem cell derived hepatocyte-like cells, and may be applicable to many stem cell-based therapies.
Nanoparticle Based Analysis of Biomolecules, Cells and Tissue
Duncan Graham University of Strathclyde
Metallic nanoparticles offer many opportunities in terms of detection including light scattering, surface plasmon resonance and surface enhanced Raman scattering (SERS). We are interested in the optical properties of metal nanoparticles and their potential application in a range of different biological studies. We can make use of the optical properties of nanoparticles in two ways.
1. The nanoparticle can act as an extrinsic label for a specific biomolecular target in the same way as a fluorescent label is used. The advantage of using the nanoparticle is its optical brightness (typically several orders of magnitude more than fluorophores) and the lack of background vibrational signals. Functionalisation of the nanoparticle with a specific targeting species such as an antibody or peptide aptamer allows this approach to be used in a wide range of studies including cell, tissue and in vivo analysis.
2. Nanoparticles can be designed to contain a specific recognition probe designed to cause a change in the aggregation status of the nanoparticles resulting in a discernible optical change when it interacts with its biomolecular target. This allows separation free analysis of specific biomolecular interactions and can be applied to a range of different probe/target interactions such as DNA-DNA, peptide-protein and sugar-protein.
We have been making use of nanoparticles in both of these approaches in conjunction with SERS which is an advanced vibrational spectroscopy. To demonstrate the applicability of the two different approaches examples will be given on the use of nanoparticles for cell imaging in two and three-dimensions, imaging of nanoparticles in tissue and also their ability to report on biological molecules in vitro and in vivo.
Michelle S. Bradbury Memorial Sloan Kettering Cancer Center
Despite recent advances in imaging probe development for biomedicine, the translation of targeted diagnostic platforms remains challenging. Nanomaterials platforms currently under evaluation in oncology clinical trials are largely non-targeted drug delivery vehicles or devices to thermally treat tissue; these are typically not surface modified for targeted detection by clinical imaging tools. New tumor-selective platforms need to satisfy critical safety benchmarks, in addition to assaying targeted interactions with the microenvironment and their effects on biological systems. Coupled with metabolic imaging and analysis tools, such as PET, complete and quantitatively accurate data sets for whole body distributions, targeting kinetics, and clearance profiles of new diagnostic platforms undergoing preclinical testing or transitioning into early-phase clinical trials can be acquired.
In the operating theatre, there is also an urgent need for implementing new image-directed visualization tools that can enhance surgical vision, facilitate minimally invasive surgical procedures, and dramatically alter surgical outcomes of oncological patients. The lack of clear surgical vision impacts the ability of the operating surgeon to accurately and specifically identify the extent of malignancy, microscopic tumor burden, or remnant disease. Collectively, these factors affect therapeutic outcome, prognosis, and treatment management. Newer molecular imaging probe designs coupled with state-of-the-art device technologies, may enhance cancer care, provide real-time imaging guidance, and lead to new, more efficient approaches for early-stage detection and treatment.
Advances in nanotechnology have also fueled a paradigm shift in targeting and safely delivering drugs in conjunction with image-directed approaches. The size, architecture, and chemical composition of particle-based drug delivery vehicles can be fine-tuned to achieve properties optimal for loading and controlled release of therapeutic agents, patient safety/compliance, favorable kinetic profiles, and reducing unwanted side effects. By combining therapeutic particle tracer preparations with quantitative bioimaging approaches, drug delivery, lesion localization, and the extraction of key tumor biologic properties can be achieved for individualizing treatment planning. In turn, dosage regimens needed to achieve therapeutic efficacy might be estimated based on knowledge of drug specific activity and dose, uptake kinetics, and IC50 values.
The ability to flexibly adapt the formulation of clinically-promising drugs to improve their physicochemical and/or biological properties, in combination with metabolic imaging tools, will be important to quantify and establish suitable clinical trial endpoints. Issues relating to solubility, transport, barrier penetration, time-dependent changes in drug uptake, and intratumoral distribution are additional considerations. These properties are often not generally evaluated in the context of drug delivery due to the complexity of the biological systems involved and the inability to serially monitor this process non-invasively in the absence of drug labeling. The future success of molecular medicine will, in part, rest upon our ability to offer improved clinical trial designs addressing the foregoing issues. In conclusion, the adoption of such an approach for image-directed drug delivery in clinical settings will have far-reaching implications for personalizing cancer care in terms of treatment planning, stratification to appropriate trial arms, and response monitoring.
Traceable and Thermoresponsive Multifunctional Engineered Protein Drug Delivery Agents for Metastatic Breast Cancer
Joseph A. Frezzo NYU Tandon School of Engineering
Theranostics is the field whereby both drug delivery and medical imaging are merged to promote more effective therapies. This is especially important in the delivery of small molecule cancer therapeutics such as doxorubicin (DOX) where clinicians must strike a balance between administering an effective dose while limiting off-target cardiotoxicity risks. In this work, a fluorinated protein is investigated for theranostic use as a chemotherapeutic carrier and 19F MRI agent. The fluorinated protein, CE2-RGD-TFL, is comprised of two functional domains: 1) a coiled-coil domain (C), flanked by two integrin targeting domains, capable of encapsulating small hydrophobic drugs and; 2) two elastin-like peptide domains (E) that impart concentration-dependent thermoresponsiveness. Fluorination imparts interesting thermoresponsive properties upon CE2-RGD. While the circular dichroism analysis reveals that CE2-RGD-TFL is less structured than the wild-type variant, CE2-RGD-TFL coacervates in the physiological range for hyperthermic treatment (39-42oC). Furthermore, fluorination permits greater doxorubicin binding as CE2-RGD-TFL exhibits 49.1% loading of drug while CE2-RGD possesses 17.8% loading. Inversion recovery experiments show little change in R1 values, regardless of temperature or concentration. Car-Purcell-Meiboom-Gill echo train experiments reveal a remarkable dependence in R2 values as a function of concentrations and temperatures. Based on r2/r1 as a function of concentration, there is support for imaging the protein using ultra-short echo time (UTE) pulse sequences. Interestingly, the protein provides an excellent image using UTE at relatively low fluorine concentrations and a short time.
Influence of Surface Chemistry on the Response of Stem Cells via Distinct Fibronectin Adsorption
Ricardo A. Pires University of Minho
Glycosaminoglycans (GAGs) are main building elements of the extracellular matrix where they act in synergism with proteins and are equally critical for the development, growth, function or survival of an organism . GAGs are anionic linear polysaccharides made of repeating disaccharide units, whose negative charge is due to the presence of sulfate groups. These charged units have a crucial role in GAGs interactions with proteins and therefore in key biochemical/signalling processes related to cell functionality and survival. In a previous work we have developed a GAG mimicking platform that is based on self-assembled monolayers (SAM) and allows precise control of the surface exposed hydroxyl (-OH) and sulfonic groups (-SO3H) . We proved that those surfaces are reliable tools to study GAGs interactions with proteins (FGF-2) or cells [2, 3]. Herein, we used the same platform to study the influence of -OH or -SO3H functionalities on fibronectin (FN) adsorption/conformation and the impact of these events on the adhesion and morphology of human adipose derived stem cells (ASCs).
QCM-D data showed that similar amounts of FN were adsorbed on the -OH and -SO3H functionalized surfaces. However, the adsorbed protein underwent different organization on these surfaces and induced distinct ASCs response: it accelerates the cell adhesion on the -OH surfaces and slows down this process on the -SO3H ones. Interestingly, single cell force spectroscopy (SCFS) experiments revealed similar maximum cell adhesion forces for -OH surfaces with or without adsorbed FN, while significantly stronger adhesion was determined for FN-coated -SO3H surfaces, when compared with the non-coated ones. Finally, we have also evaluated the influence of the FN adsorption and organization on the integrin expression of the ASCs cultured on these surfaces. In the case of -SO3H surfaces, the FN coating provoked an overexpression of the ?V?3 integrin and no significant variation of the α5β1 that interacts with the RGD epitotes of FN but also with its synergy domain . On the other hand, FN adsorbed on the -OH surfaces caused underexpression of αVβ3 integrin that recognizes and interact specifically with the RGD domains of FN .
Based on these results, we propose a model according to which the RGD epitotes of FN interact with the -OH surfaces reducing their availability to participate in integrin-mediated cell binding. In the case of -SO3H surfaces, the FN interaction with the surface is non-RGD mediated, leaving the RGD epitopes available to interact with the ASCs and to promote the αVβ3-mediated cell binding.
 Pashkuleva I and Reis RL, J Mat Chem 2010, 20: 8803-8818;  Soares da Costa D et al., J Mat Chem 2012, 22: 7172-7178;  Amorim S et al., Langmuir 2013, 29: 7983-7992.;  Danen EHJ et al., J Cell Biol 2002, 159, 6: 1071-1086.
We acknowledge the EC H2020 (grant number H2020-TWINN-2015-692333 Chem2Nature) and the European Research Council (grant number ERC-2012-ADG 20120216-321266 ComplexiTE).
Myocardial Delivery of Lipidoid Nanoparticle Carrying modRNA Induces Rapid and Transient Expression
Kevin D Costa Icahn School of Medicine at Mount Sinai
Nanoparticle-based delivery of nucleotides offers an alternative to viral vectors for gene therapy. We report highly ef?cient in vivo delivery of modi?ed mRNA (modRNA) to rat and pig myocardium using formulated lipidoid nanoparticles (FLNP). Direct myocardial injec-tion of FLNP containing 1-10 μg eGFPmodRNA in the rat (n = 3 per group) showed dose-dependent enhanced green ?uorescent protein (eGFP) mRNA levels in heart tissue 20 hours after injection, over 60-fold higher than for naked modRNA. Off-target expression, including lung, liver, and spleen, was <10% of that in heart. Expres-sion kinetics after injecting 5 μg FLNP/eGFPmodRNA showed robust expression at 6 hours that reduced by half at 48 hours and was barely detectable at 2 weeks. Intra-coronary administration of 10 μg FLNP/eGFPmodRNA also proved successful, although cardiac expression of eGFP mRNA at 20 hours was lower than direct injec-tion, and off-target expression was correspondingly higher. Findings were cofirmed in a pilot study in pigs using direct myocardial injection as well as percutane-ous intracoronary delivery, in healthy and myocardial infarction models, achieving expression throughout the ventricular wall. Fluorescence microscopy revealed GFP-positive cardiomyocytes in treated hearts. This nanopar-ticle-enabled approach for highly effcient, rapid and short-term mRNA expression in the heart offers new opportunities to optimize gene therapies for enhancing cardiac function and regeneration.
A New Microscope Objective for Imaging Large Biomedical Specimens with Sub-cellular 3D Resolution
Gail McConnell University of Strathclyde
Optical lenses reached the limit of resolution set by the wavelength of light more than a century ago. However, no attempt was made to achieve the maximum resolution in the case of low-magnification lenses, probably because the visual image would then have contained detail too fine to be perceived by the human eye. Currently available lenses of less than 10x magnification are of simple construction and their numerical apertures (which determine their resolving power) are 0.2 or less, as compared with 1.3 or more in high-power lenses. They are perfectly adequate for the eye or a standard camera, but in the 1980s, confocal microscopy and improvements in camera resolution revealed a need for better low-power lenses: many researchers found that thin confocal optical sections could not be obtained at 4x magnification.
We have developed a novel lens system called the Mesolens, which achieves an N.A. of nearly 0.5 at a magnification of only 4x. When compared with a standard 4x objective, its lateral resolution is 2.5 to 5 times better and its depth resolution (which is vital for confocal or multi-photon microscopy) is 10x better. This unusually large objective lens provides, for the first time, good optical sectioning of specimens as large as entire 12.5-day mouse embryos (>5 mm long) with sub-cellular detail in every developing organ. We are creating at the University of Strathclyde a facility for the development and application of Mesolenses in biomedical science. Progress in the creation of the facility will be presented, together with recent super-wide-field and confocal mesoscopy datasets.
Insights into Molecular Mechanisms of Heart Diseases: Cryo-electron Microscopy Uncovers the Atomic Structure of the Ryanodine Receptor and its Gating Mechanism
Amedee des Georges CUNY Advanced Science Research Center
Cryo-electron microscopy has revolutionized structural biology with its ability to obtain 3D structures of macromolecular complexes without crystals. In particular, it has opened an avenue to the structure of membrane proteins, which are notoriously difficult to crystallize and study by X-ray diffraction. This work sheds light on how RyR1 may be regulated and how known mutations affect its function, a first step towards designing drugs to cure their associated heart and muscular diseases.
Imaging Molecular Structure in Living Animals
Hyungsik Lim Hunter College, The City University of New York.
I will present two stories in this talk. First, I will describe second-harmonic generation for capturing the dynamics of protein conformation in vivo. The significance of our work in neurodegenerative disorders such as glaucoma will be discussed. Second, I will briefly explain our endeavor to interrogate transcription at the single molecule level in living brain.
Fibrous Protein Biomaterials - Designs for Function
David L. Kaplan Tufts University
The study of biomaterials underpins drug delivery systems, medical devices and tissue engineering-regenerative medicine scaffolding. Fibrous proteins, including collagens, elastins and silks provide a useful suite of structural templates upon which to build insight into design-function relationships for biomaterials, as well as useful material systems for the above applications. Traditional trial and error strategies, while useful, extend time frames for discovery and utility. Thus improved experimental strategies to shorten the path from design to utility are needed. Strategies being implemented towards this goal, attained by marrying modeling with experimental platforms, will be discussed. The goal is to develop predictive tools for biomaterial designs, with a current focus on hierarchical features and mechanics.
Engineering with Biomolecular Motors
Jacek Wychowaniec University of Manchester
In the last two decades, significant efforts have been made to develop soft materials exploiting the self-assembly of short peptides for biomedical applications. So-called β-sheet forming peptides are very attractive for the design of biomaterials, in particular hydrogels. Due to the 'simplicity' of the structure formed at the molecular level, the relative robustness of the β-sheet assembly and the ease of functionalization, very stable functional hydrogels with tailored properties, in particular mechanical, can be designed which mimic the cell niche with potential application in a range of fields from tissue engineering and cell culture [2,3] to drug delivery [4,5].
One particular aspect which has attracted significant interest is the ability to use these systems in-vivo. Their low immunogenicity, cell-retention ability and biodegradability make them ideal 3D injectable delivery systems for cell-based therapies. In this context, understanding the shear-thinning and recovery mechanisms of peptide hydrogels is key to their design. The actual structural shear-thinning mechanism is still a matter of debate  as two mechanisms are possible: 1. breaking of self-assembled fibres under shear resulting in the breaking of the hydrogels into small isotropic disconnected aggregates (junction strength > fibre cohesive strength) or 2. deformation of the fibrillar network under shear resulting in sliding, disentanglement and alignment of the fibres (junction strength < fibre cohesive strength).
In this project we have explored the shear thinning and recovery properties of series of β-sheet forming peptides with varying hydrophilicities which design is based on the alternation of hydrophobic and hydrophilic residues originally developed by Zhang and co-workers [7,8]. The phase behaviour and shear thinning properties of these peptides was investigated using a range of techniques including oscillatory shear rheology and shear polarised light imaging microscopy. Our results show that the shear thinning mechanism depends on the aggregation propensity of the peptide fibres and the strength of the fibre-fibre interactions which is controlled by the peptide design and overall peptide concentration of the hydrogel. These results give an insight into the deformation mechanism which is important to understand the level of shear cells are subjected to when injected with these materials.
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Racemic Hydrogels from Enantiomeric Peptides: Predictions from Linus Pauling
Joel Schneider National Cancer Institute, National Institutes of Health
We have reported that hydrogel materials can be prepared from self-assembling beta-hairpin peptides. For example, the 20-residue peptide MAX1 rapidly self-assembles into a hydrogel network of monomorphic fibrils whose molecular structure was recently determined by solid state NMR. The enantiomer of MAX1, namely DMAX1 assembles affording a hydrogel of identical crosslink density, mesh size, and mechanical rigidity to the MAX1 gel. Surprisingly, the gelation of a 1 wt % equimolar solution of peptide enantiomers occurs more rapidly resulting in a racemic hydrogel network whose mechanical rigidity is over four-fold greater than gels prepared from either pure enantiomer. Keeping in mind that the total amount of peptide in the racemic gel is equal to that of either pure enantiomeric gel, this observation is truly unexpected and suggests that biomolecular chirality, at the level of the monomer, is directly influencing the mechanical properties of the self-assembled hydrogel. We interrogated the self-assembly process and resulting fibrillar and network morphologies of the racemic gel employing CD spectroscopy, isotope-edited FTIR, transmission electron microscopy labeling experiments, small angle neutron scattering, diffusing wave spectroscopy, solid state NMR and molecular modeling to uncover the molecular basis for this behavior. In light of the NMR structure of pure MAX1 fibrils, the mechanism of enantiomeric assembly and their molecular arrangement in the solid state will be presented. The mode of molecular assembly uncovered in our studies was predicted by Linus Pauling in 1953 in the course of deriving models of the pleated β-sheet, a fold ubiquitous in protein structure.
Friday June 24th 2016
Towards Dynamic Hybrid Architectures: Or Can We Make Materials Adaptive?
Joanna Aizenberg Harvard University
Dynamic structures that respond reversibly to changes in their environment are central to self-regulating thermal and lighting systems, targeted drug delivery, sensors, and self-propelled locomotion. Since an adaptive change requires energy input, an ideal strategy would be to design materials that harvest energy directly from the environment and use it to drive an appropriate response. New synthetic approaches that would lead to such adaptive materials present a real challenge for materials chemistry in the 21st century. To address this challenge, I will describe the design of a novel class of reconfigurable materials that, similar to skeletomuscular systems, use a hybrid architecture to interconvert energy between different forms and scales. To specify the materials' functions, we use surfaces bearing arrays of nanostructures put in motion by environment-responsive gels. Their unique topography can be designed to confer a wide range of adaptive optical, wetting, adhesive, anti-bacterial, motion-generating, and other behaviors, similar to their natural counterparts used by lotus leaves to shed water, geckos to stick to surfaces, cephalopods to change color, echinoderms to keep their skin clean, and fish to sense flow. Using both experimental and modeling approaches as well as new fabrication methods, we are developing our ability to take full advantage of the immense potential for energy coupling within these hybrids to create a generation of sustainable, self-reporting, self-adapting materials.
Designing Biomimetics and Smart-materials for Recognition and Capture of Biologics
Cecilia Roque Universidade NOVA de Lisboa
Over the past 40 years monoclonal antibodies and derived structures became the standard binding proteins representing powerful tools in biotechnology and biosensing. Other synthetic and protein scaffolds, with the robustness and versatility required, are being explored by our group to enable the selective capture and oriented immobilization of biomolecules onto surfaces. We employed biological and chemical combinatorial libraries supported by computational design tools to develop robust peptidomimetics against several targets, namely tagged recombinant proteins (1), GFP fusion proteins (2), and virus-like particles (3). Our research has also focused on the development of strategies for the surface coating of magnetic nanoparticles with natural (4) and synthetic polymers and subsequent decoration with the novel affinity reagents (5). These modified magnetically-driven materials are applied to biotechnological and biomedical areas which will be discussed in the seminar.
(1) Pina et al., 2014, ChemBioChem, 15 (10), 1423-1435. (2) Pina et al., 2015, Journal of Chromatography A, 1418, 83-93. (3) Fernandes et al., 2016, Journal of Chromatography A, 1438, 160-170. (4) Palma et al., 2015, Nanoscale (7), 14272-14283. (5) Barroso et al., 2014, Advanced Functional Materials, 24(28), 4528-4541.
Cancer Carbohydrate Nanotechnology: Understanding and Targeting Cell Surface Glycosylation in Disease Therapy and Diagnosis
Adam B. Braunschweig University of Miami
Every eukaryotic cell is coated with a layer of carbohydrates 'termed the glycocalyx' whose glycosylation pattern is cell specific. Because of their rapid division, cancer cells promote immature glycans to the cell surface, and as such display distinct glycosylation patterns that could be exploited for diagnosis or drug delivery. Currently, however, cell-surface carbohydrates are considered 'undruggable targets'. attractive and validated cancer targets that remain outside of the reach of pharmacological regulation, because of (1) the difficulty associated with selectively binding in water saccharides with subtle structural differences, and (2) the primitive understanding of how surface effects contribute to molecular recognition at biological interfaces. This talk will present recent efforts from the Braunschweig group to develop selective molecular receptors for binding non-glucosidic saccharides in water, and developing new printing methods including a platform for 4D organic nanolithography for creating complex cell-surface models.[2-4] These examples demonstrate how emerging tools from supramolecular chemistry and nanoscience can be leveraged to develop new strategies for addressing cancer and understanding how information is communicated within molecular systems and biological networks.
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3D Cell Culture in Anchored Hydrogel Droplets for High Throughput Drug Screening
Alan M. Lyons ARL Designs LLC
Current drug-discovery approaches are highly inefficient, as <10% of new drugs entering the pipeline receive FDA approval. The tools used today may explain this low success rate as researchers typically study cells cultured in microplates; a non-natural 2D environment. Studying the response of cells cultured in a more physiologically relevant 3D environment has recently shown promise for increasing the drug-discovery success rate. Similarly, isolated lymphocytes, cultured in 3D, are an important source of new biological drugs. Available tools for culturing cells in 3D are challenging to use and difficult to automate, especially for long-term culture studies. The lack of appropriate platforms for handling cells in 3D severely limits the development of 3D cell assays for new drug discovery.
We have developed a new technology, composed of arrays of specially designed surface structures, on which droplets of hydrogels containing cells can be anchored. These structures are built into the AIM lid which is compatible with standard 96 and 384 well microplates. Hydrogel droplets containing cells are dispensed on the AIM lid by standard pipetting or by immersing the surface features into a solution of hydrogel. To form tissues, different types of cells can be juxtaposed by dispensing successive layers of solutions containing different cell types or hydrogel chemistries. After cure, the AIM lid is placed on a standard microplate, immersing the hydrogel droplets into the culture medium or any other solution contained in the microwells. The cells remain within the gel, but nutrients and gases can rapidly diffuse through the nano-porous gels. Medium changes, washing steps, staining and drug introduction are achieved by moving the lid to a new microplate containing fresh solution.
The long term culturing of cells (> 10 days) as well as the diffusion of peptides and proteins in anchored 3D gel droplets was studied as a function of hydrogel composition and cross-link density using optical and high resolution (63x) florescence microscopy. Techniques for dispensing nanoliter droplets of cells in high density arrays (>500 droplets/cm2) will also be described.
P-selectin is a Nanotherapeutic Delivery Target to the Tumor Microenvironment
Yosi Shamay Memorial Sloan Kettering Cancer Center
Disseminated tumors are poorly accessible to nanoscale drug delivery systems due to the vascular barrier which attenuates extravasation at the tumor site. We investigated P-selectin, a molecule expressed on activated vasculature that facilitates metastasis by arresting tumor cells at the endothelium, for its potential to target metastases using this property. We found unexpectedly that P-selectin is expressed on cancer cells in many human tumors. To develop a targeted drug delivery platform, we employed a fucosylated polysaccharide with nanomolar affinity to P-selectin. The nanoparticles targeted tumor vasculature to localize chemotherapeutics and a targeted MEK inhibitor at tumor sites in both primary and metastatic models, resulting in superior anti-tumor efficacy. On non-expressing tumors, we found that ionizing radiation guided the nanoparticles to the disease site by inducing P-selectin expression. Radiation concomitantly produced an abscopal-like phenomenon in unirradiated tumors, suggesting a potential strategy to target disparate drug classes to almost any tumor.
Microfluidics for 3D Tissue Engineering and Personal Health Diagnostics
Samuel K. Sia Columbia University
I will discuss the use of microfluidic techniques for two different applications: controlling 3D microenvironments of cells and tissues, and for developing low-cost point-of-care diagnostics for use in U.S. and in developing countries.
1) A number of microfluidic techniques have been developed in our group for controlling the 3D microenvironments of cells and tissues to high resolution. These techniques are useful for studying microvascularization in a number of organ systems, and for engineering implantable devices.
2) In the second half of the talk, I will discuss the development of lab-on-a-chip devices for personal health in the U.S., and for diagnosing diseases for global health. I will discuss our lab's current efforts, in conjunction with partners in industry, public health, and local governments, to develop new rapid diagnostic tests for use in sub-Saharan Africa.