Sivakumar Vijayaraghavalu

3-D Tumor Model for In Vitro Evaluation of Anticancer Drugs

The efficacy of potential anticancer drugs during preclinical development is generally tested in vitro using cancer cells grown in monolayer; however, a significant discrepancy in their efficacy is observed when these drugs are evaluated in vivo. This discrepancy, in part, could be due to the three-dimensional (3-D) nature of tumors as compared to the two-dimensional (2-D) nature of monolayer cultures. Therefore, there is a need for an in vitro model that would mimic the 3-D nature of tumors. With this objective, we have developed surface-engineered, large and porous biodegradable polymeric microparticles as a scaffold for 3-D growth of cancer cells. Using the MCF-7 cell line as model breast cancer cells, we evaluated the antiproliferative effect of three anticancer drugs: doxorubicin, paclitaxel and tamoxifen in 3-D model vs in 2-D monolayer. With optimized composition of microparticles and cell culture conditions, a density of 4.5 x 10 (6) MCF-7 cells/mg of microparticles, which is an 18-fold increase from the seeding density, was achieved in six days of culture. Cells were observed to have grown in clumps on the microparticle surface as well as in their interior matrix structure. The antiproliferative effect of the drugs in 3-D model was significantly lower than in 2-D monolayer, which was evident from the 12- to 23-fold differences in their IC 50 values. Using doxorubicin, the flow cytometry data demonstrated approximately 2.6-fold lower drug accumulation in the cells grown in 3-D model than in the cells grown as 2-D monolayer. Further, only 26% of the cells in 3-D model had the same concentration of drug as the cells in monolayer, thus explaining the reduced activity of the drugs in 3-D model. The collagen content of the cells grown in 3-D model was 2-fold greater than that of the cells grown in 2-D, suggesting greater synthesis of extracellular matrix in 3-D model, which acted as a barrier to drug diffusion. The microarray analysis showed changes in several genes in cells grown in 3-D, which could also influence the drug effect. In conclusion, the cells grown in 3-D are more resistant to chemotherapy than those grown in 2-D culture, suggesting the significant roles of cellular architecture, phenotypic variations, and extracellular matrix barrier to drug transport in drug efficacy. We propose that our model provides a better assessment of drug efficacy than the currently used 2-D monolayer as many of its characteristic features are similar to an actual tumor. A well-characterized 3-D model can particularly be useful for rapid screening of a large number of therapeutics for their efficacy during the drug discovery phase.

Biophysics of Cell Membrane Lipids in Cancer Drug Resistance: Implications for Drug Transport and Drug Delivery with Nanoparticles

In this review, we focus on the biophysics of cell membrane lipids, particularly when cancers develop acquired drug resistance, and how biophysical changes in resistant cell membrane influence drug transport and nanoparticle-mediated drug delivery. Recent advances in membrane lipid research show the varied roles of lipids in regulating membrane P-glycoprotein function, membrane trafficking, apoptotic pathways, drug transport, and endocytic functions, particularly endocytosis, the primary mechanism of cellular uptake of nanoparticle-based drug delivery systems. Since acquired drug resistance alters lipid biosynthesis, understanding the role of lipids in cell membrane biophysics and its effect on drug transport is critical for developing effective therapeutic and drug delivery approaches to overcome drug resistance. Here we discuss novel strategies for (a) modulating the biophysical properties of membrane lipids of resistant cells to facilitate drug transport and regain endocytic function and (b) developing effective nanoparticles based on their biophysical interactions with membrane lipids to enhance drug delivery and overcome drug resistance.

Drug Resistance in Breast Cancer Cells: Biophysical Characterization of and Doxorubicin Interactions with Membrane Lipids

Understanding the role of lipids in drug transport is critical in cancer chemotherapy to overcome drug resistance. In this study, we isolated lipids from doxorubicin-sensitive (MCF-7) and -resistant (MCF-7/ADR) breast cancer cells to characterize the biophysical properties of membrane lipids (particularly lipid packing and membrane fluidity) and to understand the role of the interaction of cell membrane lipids with drug/nanocarrier on drug uptake and efficacy. Resistant cell membrane lipids showed significantly different composition and formed more condensed, less fluid monolayers than did lipids from sensitive cells. Doxorubicin, used as a model anticancer agent, showed a strong hydrophobic interaction with resistant cell membrane lipids but significantly less interaction, as well as a different pattern of interaction (i.e., ionic), with sensitive ones. The threshold intracellular doxorubicin concentration required to produce an antiproliferative effect was similar for both sensitive and resistant cell lines, suggesting that drug transport is a major barrier in determining drug efficacy in resistant cells. In addition to the biophysical characteristics of resistant cell membrane lipids, lipid-doxorubicin interactions appear to decrease intracellular drug transport via diffusion as the drug is trapped in the lipid bilayer. The rigid nature of resistant cell membranes also seems to influence endosomal functions that inhibit drug uptake when a liposomal formulation of doxorubicin is used. In conclusion, biophysical properties of resistant cell membrane lipids significantly influence drug transport, and hence drug efficacy. A better understanding of the mechanisms of cancer drug resistance is vital to developing more effective therapeutic interventions. In this regard, biophysical interaction studies with cell membrane lipids might be helpful to improve drug transport and efficacy through drug discovery and/or drug delivery approaches by overcoming the lipid barrier in resistant cells.

Efficacy of decitabine-loaded nanogels in overcoming cancer drug resistance is mediated via sustained DNA methyltransferase 1 (DNMT1) depletion

DNA methyltransferase 1 (DNMT1) promotes DNA methylation to maintain cancer drug resistance. The epigenetic drug, decitabine (DAC) is a potent hypomethylating agent, but its effect is transient because of its instability. We tested the efficacy of DAC-loaded nanogels in doxorubicin-resistant breast cancer cells, DAC-resistant melanoma cells, and leukemia cells. DAC in nanogel sustained DNMT1 depletion, prolonged cell arrest in the G2/M cell-cycle phase, and significantly enhanced antiproliferative effect of DAC. The efficacy of DAC-loaded nanogels was more significant in resistant than sensitive cells. Our data suggest that effective delivery of DAC and prolonged DNMT1 depletion are critical to overcoming drug resistance.

Recent advances targeting innate immunity-mediated therapies against HIV-1 infection.

Early defence mechanisms of innate immunity respond rapidly to infection against HIV‐1 in the genital mucosa. Additionally, innate immunity optimises effective adaptive immune responses against persistent HIV infection. Recent research has highlighted the intrinsic roles of apolipoprotein B mRNA‐editing, enzyme‐catalytic, polypeptide‐like 3G, tripartite motif‐containing protein 5, tetherin, sterile α‐motif and histidine/aspartic acid domain‐containing protein 1 in restricting HIV‐1 replication. Likewise, certain endogenously secreted antimicrobial peptides, namely α/β/θ‐defensins, lactoferrins, secretory leukocyte protease inhibitor, trappin‐2/elafin and macrophage inflammatory protein‐3α are reportedly protective. Whilst certain factors directly inhibit HIV, others can be permissive. Interferon‐λ3 exerts an anti‐HIV function by activating Janus kinase‐signal transducer and activator of transcription‐mediated innate responses. Morphine has been found to impair intracellular innate immunity, contributing to HIV establishment in macrophages. Interestingly, protegrin‐1 could be used therapeutically to inhibit early HIV‐1 establishment. Moreover, chloroquine inhibits plasmacytoid dendritic cell activation and improves effective T‐cell responses. This minireview summarizes the recently identified targets for innate immunity‐mediated therapies and outlines the challenges that lie ahead in improving treatment of HIV infection.

Reaching for the Stars in the Brain: Polymer-Mediated Gene Delivery to Human Astrocytes

Astrocytes, the “star-shaped” glial cells, are appealing gene-delivery targets to treat neurological diseases due to their diverse roles in brain homeostasis and disease. Cationic polymers have successfully delivered genes to mammalian cells and hence present a viable, non-immunogenic alternative to widely used viral vectors. In this study, we investigated the gene delivery potential of a series of arginine- and polyethylene glycol-modified, siloxane-based polyethylenimine analogs in primary cultured human neural cells (neurons and astrocytes) and in mice. Plasmid DNAs encoding luciferase reporter were used to measure gene expression. We hypothesized that polyplexes with arginine would help in cellular transport of the DNA, including across the blood-brain barrier; polyethylene glycol will stabilize polyethylenimine and reduce its toxicity while maintaining its DNA-condensing ability. Polyplexes were non-toxic to human neural cells and red blood cells. Cellular uptake of polyplexes and sustained gene expression were seen in human astrocytes as well as in mouse brains post-intravenous-injections. The polyplexes also delivered and expressed genes driven by astrocyte-restricted glial fibrillary acidic protein promoters, which are weaker than viral promoters. To our knowledge, the presented work validates a biocompatible and effective polymer-facilitated gene-delivery system for both human brain cells and mice for the first time.

Nanogel-mediated delivery of a cocktail of epigenetic drugs plus doxorubicin overcomes drug resistance in breast cancer cells

Epigenetic modifications (e.g., DNA methylation or histone deacetylation) are commonly implicated in cancer chemoresistance. We previously showed that pretreating resistant MCF-7/ADR breast cancer cells with a demethylating agent (5-aza-2′-deoxycytidine (DAC)) or with an inhibitor of histone deacetylase (suberoylanilide hydroxamic acid (SAHA)) sensitized resistant cells to doxorubicin (DOX) treatment. However, even with increasing doses of DOX, a fraction of resistant cells remained nonresponsive to this pretreatment (~ 25% pretreated with DAC, ~ 45% with SAHA). We hypothesized that pretreating resistant cells with a combination of epigenetic drugs (DAC + SAHA) could more effectively overcome drug resistance. We postulated that delivery of epigenetic drugs encapsulated in biodegradable nanogels (NGs) would further enhance their efficacy. MCF-7/ADR cells were first treated with a single drug vs. a combination of epigenetic drugs, either as solutions or encapsulated in NGs, then subjected to DOX, either in solution or in NGs. Antiproliferative data showed that pretreatment with epigenetic drugs in NGs, then with DOX in NGs, was most effective in overcoming resistance; this treatment inhibited cell growth by > 90%, even at low doses of DOX. Cell cycle analysis showed that a major fraction of cells treated with a cocktail of epigenetic drugs + DOX, all in NG formulations, remained in the G2/M cell cycle arrest phase for a prolonged period. The mechanism of better efficacy of epigenetic drugs in NGs could be attributed to their sustained effect. A similar strategy could be developed for other cancer cells in which drug resistance is due to epigenetic modifications.