Shreyas Shah

Design, Synthesis, and Characterization of Graphene–Nanoparticle Hybrid Materials for Bioapplications

Graphene is composed of single-atom thick sheets of sp2 bonded carbon atoms that are arranged in a perfect two-dimensional (2D) honeycomb lattice. Because of this structure, graphene is characterized by a number of unique and exceptional structural, optical, and electronic properties.1 Specifically, these extraordinary properties include, but are not limited to, a high planar surface area that is calculated to be 2630 m2 g−1,2 superior mechanical strength with a Young’s modulus of 1100 GPa,3 unparalleled thermal conductivity (5000 W m−1 K−1),4 remarkable electronic properties (e.g., high carrier mobility [10 000 cm2 V−1 s−1] and capacity),5 and alluring optical characteristics (e.g., high opacity [~97.7%] and the ability to quench fluorescence).6 As such, it should come as no surprise that graphene is currently, without any doubt, the most intensively studied material for a wide range of applications that include electronic, energy, and sensing outlets.1c Moreover, because of these unique chemical and physical properties, graphene and graphene-based nanomaterials have attracted increasing interest, and, arguably, hold the greatest promise for implementation into a wide array of bioapplications.7 In the last several years, numerous studies have utilized graphene in bioapplications ranging from the delivery of chemotherapeutics for the treatment of cancer8 to biosensing applications for a host of medical conditions9 and even for the differentiation and imaging of stem cells.10 While promising and exciting, recent reports have demonstrated that the combination of graphene with nanomaterials such as nanoparticles, thereby forming graphene–nanoparticle hybrid structures, offers a number of additional unique physicochemical properties and functions that are both highly desirable and markedly advantageous for bioapplications when compared to the use of either material alone (Figure 1).11 These graphene–nanoparticle hybrid structures are especially alluring because not only do they display the individual properties of the nanoparticles, which can already possess beneficial optical, electronic, magnetic, and structural properties that are unavailable in bulk materials, and of graphene, but they also exhibit additional advantageous and often synergistic properties that greatly augment their potential for bioapplications. Open in a separate window Figure 1 Graphene nanoparticle hybrids exist in two forms, as graphene–nanoparticle composites and graphene-encapsulated nanoparticles, and can be used for various bioapplications including biosensors, photothermal therapies, stem cell/tissue engineering, drug/gene delivery, and bioimaging. Panel (A) reprinted with permission from ref 110. Copyright 2012 Wiley. Panel (B) reprinted with permission from ref 211. Copyright 2013 Elsevier. Panel (C) reprinted with permission from ref 244. Copyright 2013 Wiley.

Guiding Stem Cell Differentiation into Oligodendrocytes Using Graphene‐Nanofiber Hybrid Scaffolds

Damage to the central nervous system (CNS) from degenerative diseases or traumatic injuries is particularly devastating due the limited regenerative capabilities of the CNS. Among the current approaches, stem cell-based regenerative medicine has shown great promise in achieving significant functional recovery by taking advantage of the self-renewal and differentiation capabilities of stem cells, which include pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs).[1] However, the low survival rate upon transplantation has been a longstanding barrier for scientists and clinicians to overcome.[2] To this end, numerous types of natural and synthetic biomaterial scaffolds have been developed, the two main classes being hydrogels and nanofibers, in an attempt to mimic the cellular microenvironment, support cellular growth and improve cellular viability.[3] Yet, designing scaffolds with defined properties to selectively guide stem cell differentiation towards a specific neural cell lineage is still an ongoing challenge.

Controlling Differentiation of Adipose-Derived Stem Cells Using Combinatorial Graphene Hybrid-Pattern Arrays

Control of stem cell fate by modulating biophysical cues (e.g., micropatterns, nanopatterns, elasticity and porosity of the substrates) has emerged as an attractive approach in stem cell-based research. Here, we report a method for fabricating combinatorial patterns of graphene oxide (GO) to effectively control the differentiation of human adipose-derived mesenchymal stem cells (hADMSCs). In particular, GO line patterns were highly effective for modulating the morphology of hADMSCs, resulting in enhanced differentiation of hADMSCs into osteoblasts. Moreover, by generating GO grid patterns, we demonstrate the highly efficient conversion of mesodermal stem cells to ectodermal neuronal cells (conversion efficiency = 30%), due to the ability of the grid patterns to mimic interconnected/elongated neuronal networks. This work provides an early demonstration of developing combinatorial graphene hybrid-pattern arrays for the control of stem cell differentiation, which can potentially lead to more effective stem cell-based treatment of incurable diseases/disorders.

Controlling Differentiation of Neural Stem Cells Using Extracellular Matrix Protein Patterns

The ability of stem cells to differentiate into specialized lineages within a specific microenvironment is vital for regenerative medicine. For harnessing the full potential of stem cells for regenerative therapies, it is important to investigate and understand the function of three types of micro-environmental cues—soluble signals, cell–cell interactions, and insoluble (physical) signals—that dynamically regulate stem cell differentiation.[1] Neural stem cells (NSCs) are multipotent and differentiate into neurons and glial cells,[2] which can provide essential sources of engraftable neural cells for devastating diseases such as Alzheimer’s disease,[3] Parkinson’s disease[4] and spinal cord injury.[5] One of the major challenges involved in the differentiation of NSCs is to identify and optimize factors which result in an increased proportion of NSCs differentiating into neurons as opposed to glial cells. To this end, soluble cues such as brain-derived neurotrophic factor (BDNF),[6] sonic hedgehog (Shh),[7] retinoic acid (RA),[6c] and neuropathiazol[8] have been shown to significantly increase neuronal differentiation of NSCs in vitro. However, the research toward studying the function of the other two microenvironmental cues (cell–cell interactions and insoluble cues) during the neurodifferentiation of NSCs is limited, mainly due to the lack of availability of methods for the investigation.[9] While various aspects such as cell–cell interactions,[10] combinations of extracellular matrix (ECM) proteins,[1a,11] and physical properties of substrates have been shown to play a vital role in determining the fate of other adult stem cells such as mesenchymal stem cells (MSCs),[12] cardiac stem cells,[13] and hematopoetic stem cells,[14] little is known about the influence of such factors on the neuronal differentiation of NSCs. Therefore, there is a pressing need to develop methods for investigating the role of cell–cell interactions and insoluble signals in selectively inducing the differentiation of NSCs into specific neural cell lineages.

Generation of a Library of Non‐Toxic Quantum Dots for Cellular Imaging and siRNA Delivery

The development of non-toxic quantum dots and further investigation of their composition-dependent cytotoxicity in a high-throughput manner have been critical challenges for biomedical imaging and gene delivery. Herein, we report a rapid sonochemical synthetic methodology for generating a library of highly biocompatible ZnS-AgInS(2) (ZAIS) quantum dots for cellular imaging and siRNA delivery.

Nanotopography-mediated Reverse Uptake for siRNA Delivery into Neural Stem Cells to Enhance Neuronal Differentiation

RNA interference (RNAi) for controlling gene expression levels using siRNA or miRNA is emerging as an important tool in stem cell biology. However, the conventional methods used to deliver siRNA into stem cells result in significant cytotoxicity and undesirable side-effects. To this end, we have developed a nanotopography-mediated reverse uptake (NanoRU) delivery platform to demonstrate a simple and efficient technique for delivering siRNA into neural stem cells (NSCs). NanoRU consists of a self-assembled silica nanoparticle monolayer coated with extracellular matrix proteins and the desired siRNA. We use this technique to efficiently deliver siRNA against the transcription factor SOX9, which acts as a switch between neuronal and glial fate of NSCs. The knockdown of SOX9 enhanced the neuronal differentiation and decreased the glial differentiation of the NSCs. Our NanoRU platform demonstrates a novel application and the importance of nanotopography-mediated siRNA delivery into stem cells as an effective method for genetic manipulation.

Single vehicular delivery of siRNA and small molecules to control stem cell differentiation.

Achieving a controlled and reproducible means to direct stem cell differentiation is the single most critical concern scientists have been trying to address since the discovery of stem cells. In this regard, the use of small molecules and RNA interference offers unique advantages by targeting different cellular mechanisms. Our cyclodextrin-modified dendritic polyamine construct (termed DexAM) combines the unique properties of two distinct chemical moieties in a single delivery vehicle. DexAM is a single vehicle that not only solubilizes hydrophobic small molecules in physiological solutions but also forms complexes with siRNA molecules, making it an attractive delivery system for controlling stem cell differentiation. Herein, we report the synthesis and application of DexAM to simultaneously deliver hydrophobic small molecules and siRNA into neural stem cells to significantly enhance their neuronal differentiation.

Hybrid Upconversion Nanomaterials for Optogenetic Neuronal Control

Nanotechnology-based approaches offer the chemical control required to develop precision tools suitable for applications in neuroscience. We report a novel approach employing hybrid upconversion nanomaterials, combined with the photoresponsive ion channel channelrhodopsin-2 (ChR2), to achieve near-infrared light (NIR)-mediated optogenetic control of neuronal activity. Current optogenetic methodologies rely on using visible light (e.g. 470 nm blue light), which tends to exhibit high scattering and low tissue penetration, to activate ChR2. In contrast, our approach enables the use of 980 nm NIR light, which addresses the short-comings of visible light as an excitation source. This was facilitated by embedding upconversion nanomaterials, which can convert NIR light to blue luminescence, into polymeric scaffolds. These hybrid nanomaterial scaffolds allowed for NIR-mediated neuronal stimulation, with comparable efficiency as that of 470 nm blue light. Our platform was optimized for NIR-mediated optogenetic control by balancing multiple physicochemical properties of the nanomaterial (e.g. size, morphology, structure, emission spectra, concentration), thus providing an early demonstration of rationally-designing nanomaterial-based strategies for advanced neural applications.

Photo-triggerable hydrogel–nanoparticle hybrid scaffolds for remotely controlled drug delivery

Remotely-triggerable drug delivery systems enable the user to adjust dosing regimens on-demand based on a patients physiological response and clinical needs. However, currently reported systems are limited by the non-specific leakage of drugs in the absence of triggering and the lack of repeatability over multiple cycles of release. To this end, we have designed a unique hydrogel-nanoparticle hybrid scaffold that provides a chemically-defined, remotely-triggerable and on-demand release of small molecule drugs. Our hybrid platform consists of three distinct components: 1) a photo-triggerable chemical compound, which serves to release a covalently-bound drug upon photo-irradiation, 2) a nanoparticle, which serves to covalently bind the photo-triggerable compound, and 3) a polymeric hydrogel, which serves to hold the drug-conjugated nanoparticle. Upon photo-irradiation, the activation of the photo-triggerable compound is designed to initiate a series of intramolecular chemical rearrangements, which would cleave the covalently-bound drug and release it from the hydrogel. The combination of these distinct components in a single scaffold proved to be an effective drug delivery system, as demonstrated by the delivery of a model drug to a malignant cancer line. Our hybrid scaffold can be easily tuned for practically any biological application of interest, thus offering immense potential for clinical therapies.

Cyclophilin A promotes cell migration via the Abl-Crk signaling pathway

Summary Cyclophilin A (CypA) is over-expressed in a number of human cancer types, but the mechanisms by which CypA promotes oncogenic properties of cells are not understood. Here we demonstrate that CypA binds to and prevents the CrkII adaptor protein from switching to the inhibited state. CrkII is involved in cell motility and invasion by mediating signaling through its SH2 and SH3 domains. CrkII Tyr221 phosphorylation by the Abl or EGFR kinases induces an inhibited state of CrkII, by means of an intramolecular SH2-pTyr221 interaction, causing signaling interruption. We show that the CrkII phosphorylation site constitutes a binging site for CypA. Recruitment of CypA sterically restricts the accessibility of Tyr221 to kinases, thereby suppressing CrkII phosphorylation and promoting the active state. Structural, biophysical, and in vivo data show that CypA augments CrkII-mediated signaling. A strong stimulation of cell migration is observed in cancer cells wherein both CypA and CrkII are greatly up-regulated.