Browsing by Author "Asthana, Vishwaratn"
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Item A mechanistic investigation exploring the differential transfection efficiencies between the easy-to-transfect SK-BR3 and difficult-to-transfect CT26 cell lines(BioMed Central, 2017) Figueroa, Elizabeth; Bugga, Pallavi; Asthana, Vishwaratn; Chen, Allen L.; Stephen Yan, J.; Evans, Emily R.; Drezek, Rebekah A.Abstract Background Gold–polyamidoamine (AuPAMAM) has previously been shown to successfully transfect cells with high efficiency. However, we have observed that certain cell types are more amenable to Au–PAMAM transfection than others. Here we utilized two representative cell lines—a “difficult to transfect” CT26 cell line and an “easy to transfect” SK-BR3 cell line—and attempted to determine the underlying mechanism for differential transfection in both cell types. Using a commonly established poly-cationic polymer similar to PAMAM (polyethyleneimine, or PEI), we additionally sought to quantify the relative transfection efficiencies of each vector in CT26 and SK-BR3 cells, in the hopes of elucidating any mechanistic differences that may exist between the two transfection vectors. Results A comparative time course analysis of green fluorescent protein reporter-gene expression and DNA uptake was conducted to quantitatively compare PEI- and AuPAMAM-mediated transfection in CT26 and SK-BR3, while flow cytometry and confocal microscopy were used to determine the contribution of cellular uptake, endosomal escape, and cytoplasmic transport to the overall gene delivery process. Results from the time course analysis and flow cytometry studies revealed that initial complex uptake and cytoplasmic trafficking to the nucleus are likely the two main factors limiting CT26 transfectability. Conclusions The cell type-dependent uptake and intracellular transport mechanisms impacting gene therapy remain largely unexplored and present a major hurdle in the application-specific design and efficiency of gene delivery vectors. This systematic investigation offers insights into the intracellular mechanistic processes that may account for cell-to-cell differences, as well as vector-to-vector differences, in gene transfectability.Item An inexpensive, customizable microscopy system for the automated quantification and characterization of multiple adherent cell types(PeerJ, 2018) Asthana, Vishwaratn; Tang, Yuqi; Ferguson, Adam; Bugga, Pallavi; Asthana, Anantratn; Evans, Emily R.; Chen, Allen L.; Stern, Brett S.; Drezek, Rebekah A.Cell quantification assays are essential components of most biological and clinical labs. However, many currently available quantification assays, including flow cytometry and commercial cell counting systems, suffer from unique drawbacks that limit their overall efficacy. In order to address the shortcomings of traditional quantification assays, we have designed a robust, low-cost, automated microscopy-based cytometer that quantifies individual cells in a multiwell plate using tools readily available in most labs. Plating and subsequent quantification of various dilution series using the automated microscopy-based cytometer demonstrates the single-cell sensitivity, near-perfect R2 accuracy, and greater than 5-log dynamic range of our system. Further, the microscopy-based cytometer is capable of obtaining absolute counts of multiple cell types in one well as part of a co-culture setup. To demonstrate this ability, we recreated an experiment that assesses the tumoricidal properties of primed macrophages on co-cultured tumor cells as a proof-of-principle test. The results of the experiment reveal that primed macrophages display enhanced cytotoxicity toward tumor cells while simultaneously losing the ability to proliferate, an example of a dynamic interplay between two cell populations that our microscopy-based cytometer is successfully able to elucidate.Item Changes in Optical Properties of Plasmonic Nanoparticles in Cellular Environments are Modulated by Nanoparticle PEGylation and Serum Conditions(Springer, 2016) Chen, Allen L.; Jackson, Meredith A.; Lin, Adam Y.; Figueroa, Elizabeth R.; Hu, Ying S.; Evans, Emily R.; Asthana, Vishwaratn; Young, Joseph K.; Drezek, Rebekah A.When plasmonic nanoparticles (NPs) are internalized by cells and agglomerate within intracellular vesicles, their optical spectra can shift and broaden as a result of plasmonic coupling of NPs in close proximity to one another. For such optical changes to be accounted for in the design of plasmonic NPs for light-based biomedical applications, quantitative design relationships between designable factors and spectral shifts need to be established. Here we begin building such a framework by investigating how functionalization of gold NPs (AuNPs) with biocompatible poly(ethylene) glycol (PEG), and the serum conditions in which the NPs are introduced to cells impact the optical changes exhibited by NPs in a cellular context. Utilizing darkfield hyperspectral imaging, we find that PEGylation decreases the spectral shifting and spectral broadening experienced by 100 nm AuNPs following uptake by Sk-Br-3 cells, but up to a 33 ± 12 nm shift in the spectral peak wavelength can still occur. The serum protein-containing biological medium also modulates the spectral changes experienced by cell-exposed NPs through the formation of a protein corona on the surface of NPs that mediates NP interactions with cells: PEGylated AuNPs exposed to cells in serum-free conditions experience greater spectral shifts than in serum-containing environments. Moreover, increased concentrations of serum (10, 25, or 50 %) result in the formation of smaller intracellular NP clusters and correspondingly reduced spectral shifts after 5 and 10 h NP-cell exposure. However, after 24 h, NP cluster size and spectral shifts are comparable and become independent of serum concentration. By elucidating the impact of PEGylation and serum concentration on the spectral changes experienced by plasmonic NPs in cells, this study provides a foundation for the optical engineering of plasmonic NPs for use in biomedical environments.Item Development and Characterization of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics(2018-08-03) Asthana, Vishwaratn; Drezek, RebekahCancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment including differentiating self from non-self, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is a profound need to develop an all encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome cancer resistance. We describe herein, a new class of programmable self-assembling dsRNA-based cancer therapeutics that uniquely targets aberrant genetic sequences, and in a functionally decoupled manner, initiates a therapeutic polymerization cascade that induces apoptosis and immune activation. We further show that a DNA protector can be used to selectively seal the therapeutic RNA and induce specific and potent killing of cells containing the target oncogenic sequence, but not wildtype.Item Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: A Proof-of-Concept Investigation(Cell Press, 2020) Asthana, Vishwaratn; Stern, Brett S.; Tang, Yuqi; Bugga, Pallavi; Li, Ang; Ferguson, Adam; Asthana, Anantratn; Bao, Gang; Drezek, Rebekah A.Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment, including differentiating self from nonself, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein a new class of programmable self-assembling double-stranded RNA (dsRNA)-based cancer therapeutics that uniquely targets aberrant genetic sequences and in a functionally decoupled manner, undergoes oncogenic RNA-activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof of concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing sarcoma cells that are end blocked with phosphorothioate bonds and additionally sealed with a 2′-deoxyuridine (2′-U)-modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence but not wild type.Item Metallic nanoparticles for cancer immunotherapy(Elsevier, 2018) Evans, Emily Reiser; Bugga, Pallavi; Asthana, Vishwaratn; Drezek, RebekahCancer immunotherapy, or the utilization of the body’s immune system to attack tumor cells, has gained prominence over the past few decades as a viable cancer treatment strategy. Recently approved immunotherapeutics have conferred remission upon patients with previously bleak outcomes and have expanded the number of tools available to treat cancer. Nanoparticles – including polymeric, liposomal, and metallic formulations – naturally traffic to the spleen and lymph organs and the relevant immune cells therein, making them good candidates for delivery of immunotherapeutic agents. Metallic nanoparticle formulations, in particular, are advantageous because of their potential for dense surface functionalizationand their capability for optical or heat-based therapeutic methods. Many research groups have investigated the potential of nanoparticle-mediated delivery platforms to improve the efficacy of immunotherapies. Despite the significant preclinical successes demonstrated by many of these platforms over the last twenty years, only a few metallic nanoparticles have successfully entered clinical trials with none achieving FDA approval for cancer therapy. In this review, we will discuss preclinical research and clinical trials involving metallic nanoparticles (MNPs) for cancer immunotherapy applications and discuss the potential for clinical translation of MNPs.