Prashanth Asuri

Directed Evolution of Adeno-associated Virus for Enhanced Gene Delivery and Gene Targeting in Human Pluripotent Stem Cells

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.

Three‐dimensional matrix stiffness and adhesive ligands affect cancer cell response to toxins

There is an immediate need to develop highly predictive in vitro cell‐based assays that provide reliable information on cancer drug efficacy and toxicity. Development of biomaterial‐based three‐dimensional (3D) cell culture models as drug screening platforms has recently gained much scientific interest as 3D cultures of cancer cells have been shown to more adequately mimic the in vivo tumor conditions. Moreover, it has been recognized that the biophysical and biochemical properties of the 3D microenvironment can play key roles in regulating various cancer cell fates, including their response to chemicals. In this study, we employed alginate‐based scaffolds of varying mechanical stiffness and adhesive ligand presentation to further explore the role of 3D microenvironmental cues on glioblastoma cell response to cytotoxic compounds. Our experiments suggested the ability of both matrix stiffness and cell‐matrix adhesions to strongly influence cell responses to toxins. Cells were found to be more susceptible to the toxins when cultured in softer matrices that emulated the stiffness of brain tissue. Furthermore, the effect of matrix stiffness on differential cell responses to toxins was negated by the presence of the adhesive ligand RGD, but regained when integrin‐based cell‐matrix interactions were inhibited. This study therefore indicates that both 3D matrix stiffness and cell‐matrix adhesions are important parameters in the design of more predictive in vitro platforms for drug development and toxicity screening. Biotechnol. Bioeng. 2016;113: 443–452.

Experimental Investigation of Mechanical and Thermal Properties of Silica Nanoparticle-Reinforced Poly(acrylamide) Nanocomposite Hydrogels

Current studies investigating properties of nanoparticle-reinforced polymers have shown that nanocomposites often exhibit improved properties compared to neat polymers. However, over two decades of research, using both experimental studies and modeling analyses, has not fully elucidated the mechanistic underpinnings behind these enhancements. Moreover, few studies have focused on developing an understanding among two or more polymer properties affected by incorporation of nanomaterials. In our study, we investigated the elastic and thermal properties of poly(acrylamide) hydrogels containing silica nanoparticles. Both nanoparticle concentration and size affected hydrogel properties, with similar trends in enhancements observed for elastic modulus and thermal diffusivity. We also observed significantly lower swellability for hydrogel nanocomposites relative to neat hydrogels, consistent with previous work suggesting that nanoparticles can mediate pseudo crosslinking within polymer networks. Collectively, these results indicate the ability to develop next-generation composite materials with enhanced mechanical and thermal properties by increasing the average crosslinking density using nanoparticles.

Function, Structure, and Stability of Enzymes Confined in Agarose Gels

Research over the past few decades has attempted to answer how proteins behave in molecularly confined or crowded environments when compared to dilute buffer solutions. This information is vital to understanding in vivo protein behavior, as the average spacing between macromolecules in the cell cytosol is much smaller than the size of the macromolecules themselves. In our study, we attempt to address this question using three structurally and functionally different model enzymes encapsulated in agarose gels of different porosities. Our studies reveal that under standard buffer conditions, the initial reaction rates of the agarose-encapsulated enzymes are lower than that of the solution phase enzymes. However, the encapsulated enzymes retain a higher percentage of their activity in the presence of denaturants. Moreover, the concentration of agarose used for encapsulation had a significant effect on the enzyme functional stability; enzymes encapsulated in higher percentages of agarose were more stable than the enzymes encapsulated in lower percentages of agarose. Similar results were observed through structural measurements of enzyme denaturation using an 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Our work demonstrates the utility of hydrogels to study protein behavior in highly confined environments similar to those present in vivo; furthermore, the enhanced stability of gel-encapsulated enzymes may find use in the delivery of therapeutic proteins, as well as the design of novel strategies for biohybrid medical devices.

Droplet Microfluidic Platform for the Determination of Single-Cell Lactate Release

Cancer cells release high levels of lactate that has been correlated to increased metastasis and tumor recurrence. Single-cell measurements of lactate release can identify malignant cells and help decipher metabolic cancer pathways. We present here a novel droplet microfluidic method that allows the fast and quantitative determination of lactate release in many single cells. Using passive forces, droplets encapsulated cells are positioned in an array. The single-cell lactate release rate is determined from the increase in droplet fluorescence as the lactate is enzymatically converted to a fluorescent product. The method is used to measure the cell-to-cell variance of lactate release in K562 leukemia and U87 glioblastoma cancer cell lines and under the chemical inhibition of lactate efflux. The technique can be used in the study of cancer biology, but more broadly in cell biology, to capture the full range of stochastic variations in glycolysis activity in heterogeneous cell populations in a repeatable and high-throughput manner.

A Novel 2.5D Culture Platform to Investigate the Role of Stiffness Gradients on Adhesion-Independent Cell Migration

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration.

Biomolecule-Nanomaterial Interactions: Effect on Biomolecular Structure, Function, and Stability

We have characterized the influence of protein–carbon nanotube interactions on protein structure and function using various techniques such as Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and atomic force microscopy. This structure-based analysis revealed that different proteins interact with nanotubes differentially, consistent with the observed biological activity data. Furthermore, the high degree of surface curvature of the nanoscale support was found to play an important role in stabilizing proteins under denaturing conditions. Along with these fundamental studies, various applications of such highly active and stable nanotube–protein conjugates have been pursued, which include self-cleaning nanobiocomposite films, interfacial biocatalysis in a biphasic medium, and synthesis of nanotube–nanoparticle hybrids, among others.

Trehalose effectiveness as a cryoprotectant in 2D and 3D cell cultures of human embryonic kidney cells

Abstract Post cryopreservation viability of human embryonic kidney (HEK) cells under two-dimensional (2D) and three-dimensional (3D) culture conditions was studied using trehalose as the sole cryoprotective agent. An L9 (34) Taguchi design was used to optimize the cryoprotection cocktail seeding process prior to slow-freezing with the specific aim of maximizing cell viability measured 7 days post thaw, using the combinatorial cell viability and in-vitro cytotoxicity WST assay. At low (200 mM) and medium (800 mM) levels of trehalose concentration, encapsulation in alginate offered a greater protection to cryopreservation. However, at the highest trehalose concentration (1200 mM) and in the absence of the pre-incubation step, there was no statistical difference at the 95% CI (p = 0.0212) between the viability of the HEK cells under 2D and 3D culture conditions estimated to be 17.9 ± 4.6% and 14.0 ± 3.6%, respectively. A parallel comparison between cryoprotective agents conducted at the optimal levels of the L9 study, using trehalose, dimethylsulfoxide and glycerol in alginate microcapsules yielded a viability of 36.0 ± 7.4% for trehalose, in average 75% higher than the results associated with the other two cell membrane-permeating compounds. In summary, the effectiveness of trehalose has been demonstrated by the fact that 3D cell cultures can readily be equilibrated with trehalose before cryopreservation, thus mitigating the cytotoxic effects of glycerol and dimethylsulfoxide.

Effect of crosslinker length on the elastic and compression modulus of poly(acrylamide) nanocomposite hydrogels

Polymer hydrogelshave shown to exhibit improved properties upon the addition of nanoparticles; however, the mechanical underpinnings behind these enhancements have not been fully elucidated. Moreover, fewer studies have focused on developing an understanding of how polymer parameters affect the nanoparticle-mediated enhancements. In this study, we investigated the elastic properties of silica nanoparticle-reinforced poly(acrylamide) hydrogels synthesized using crosslinkers of various lengths. Crosslinker length positively affected the mechanical properties of hydrogels that were synthesized with or without nanoparticles. However the degree of nanoparticle enhancement was negatively correlated to crosslinker length. Our findings enable the understanding of the respective roles of nanoparticle and polymer properties on nanoparticle-mediated enhancement of hydrogels and thereby the development of next-generation nanocomposite materials.

Influence of Macromolecular Crowding and Confinement on Enzyme Activity and Structure under Native and Denaturing Conditions

It is now well appreciated that both crowding and confinement influence enzyme structure and function due to excluded volume effects; however, the relative efficacies of these environments on protein fates remain unclear due to a lack of direct comparison studies. In this study, we explore the use of the biopolymer alginate to develop an in vitro platform to investigate the effects of both crowding and confinement on the behavior of two model enzymes - horseradish peroxidase and β-galactosidase. Alginate, in its solution phase, can be used as a crowding agent and, in its gel phase by crosslinking using divalent cations, to encapsulate and confine proteins, thereby allowing us to use the same system to directly compare the effects of crowding and confinement. Different degrees of crowding and confinement were achieved by varying the alginate concentration, and these studies demonstrated a clear dependence of enzyme activity on the degree of crowding and confinement. Moreover, our data also suggested that protein confinement in crosslinked alginate gels led to higher enhancements in enzyme activity under denaturing conditions relative to non-crosslinked crowded environments. Results from the kinetic analyses were corroborated using structural measurements of protein denaturation using the 8-anilinonaphthalene-1-sulfonic acid fluorescence assay.