Shambhavi Pandey

Synergistic effects of nanotopography and co-culture with endothelial cells on osteogenesis of mesenchymal stem cells.

Inspired by the aligned nanostructures and co-existence of vascular cells and stem cells in human cancellous bone, we quantitatively investigated the relative contributions of nanotopography and co-culture with human umbilical endothelial cells (HUVECs) to the osteogenesis of human mesenchymal stem cells (hMSCs). Although both nanotopography and co-culture independently enhanced the osteogenesis of hMSCs, osteogenesis was further enhanced by the two factors in combination, indicating the importance of synergistic cues in stem cell engineering. Interestingly, nanotopography provided a larger relative contribution to the osteogenesis of hMSCs than did co-culture with HUVECs. Furthermore, the osteogenesis of hMSCs was also affected by the density of parallel nanogrooves, exhibiting a maximum at a 1:3 spacing ratio, as defined as the ratio of ridge width to groove width. Analysis of (i) biochemical soluble factors, (ii) hMSC-substrate interaction and (iii) hMSC-HUVEC interaction suggests that (ii) and (iii) play a crucial role in mediating osteogenic phenotypes.

Nucleotide biosynthesis arrest by silencing SHMT1 function via vitamin B6-coupled vector and effects on tumor growth inhibition.

Serine hydroxymethyltransferase isoforms (SHMT1 & SHMT2α), which serve as scaffold protein for the formation of a multi-enzyme complex and generate one-carbon unit for the de novo thymidylate biosynthesis pathway during DNA synthesis, are vitamin B6 (VB6)-dependent enzyme. Cancer cells with high proliferation intensity need increased SHMT activation which enforces the facilitated-diffusion of VB6 for the continuous functioning of thymidylate synthase cycle. Therefore, SHMT knockdown presents an alternative approach to prevent DNA synthesis in cancer cells; however, its potential to inhibit cancer growth remains unknown so far. Here we demonstrated that VB6 coupled to poly(ester amine) (VBPEA) enforces a high level of VTC (VB6-transporting membrane carriers)-mediated endocytosis of the complexed SHMT1 siRNA (siSHMT1) to interrupt the thymidylate biosynthesis pathway of cancer cells. The detrimental effect of SHMT1 knockdown on the disintegration of multi-enzyme complex resulted in cell cycle arrest and a decrease in cells genomic DNA content, leading to enhanced apoptotic events in cancer cells. A reduction in tumor size was observed with constant SHMT1 suppression in xenograft mice. This study illustrates how silencing the SHMT1 expression inhibits cancer growth and the increased VB6 channeling for sustenance of cancer cells promotes VB6-coupled vector to elicit enhanced delivery of siSHMT1.

Enhanced chitosan–DNA interaction by 2-acrylamido-2-methylpropane coupling for an efficient transfection in cancer cells

Gene therapy is the treatment of human disorders by the introduction of genetic material to specific target cells of a patient. Chitosan and its derivatives show excellent biological properties including biocompatibility, biodegradability and nonallergenicity. Primary amines of chitosan are responsible for its cationic nature and hence binding and protection of DNA for intracellular delivery. But the transfection efficiency of chitosan based gene transporters is severely hampered by its poor physical properties such as low water solubility and high viscosity. In this study, primary amines of low molecular weight (LMW) chitosan were coupled with 2-acrylamido-2-methylpropane sulphonic acid (AMP) making it water soluble for its application in gene delivery. AMP modified chitosan (CSAMP) showed an enhanced interaction with DNA and a higher buffering capacity due to AMP amines leading to a higher transfection efficiency in cancer cells (A549, HeLa and HepG2) compared to native chitosan and Lipofectamine®. In vivo studies in Balb/c through intravenous injection demonstrated a higher luciferase expression compared to LMW chitosan.

Triphenylamine coupled chitosan with high buffering capacity and low viscosity for enhanced transfection in mammalian cells, in vitro and in vivo

Chitosan and its derivatives show excellent biological properties, including biocompatibility, biodegradability and non-allergenicity. The primary amines of chitosan are responsible for its cationic nature, which confer its electrostatic binding with anionic DNA and protects from DNA degradation during intracellular delivery. However, its poor physical properties, such as low water solubility and high viscosity, severely hamper the transfection efficiency and in vivo applicability of chitosan based gene transporters. In this study, highly soluble triphenylamine coupled chitosan (TPAC) was synthesized by coupling triphenylamine (TPA) with primary amines of low molecular weight (LMW) chitosan, offering lower viscosity at biological pH and at the concentrations required for in vivo gene delivery. TPAC inherits a higher buffering capacity due to the tertiary amines of TPA leading to enhanced endosomal escape compared to native LMW chitosan. Intracellular fate and co-localization studies of TPAC showed decreased co-localization of polyplexes with lysosomes, demonstrating an increased availability of delivered plasmid DNA to the nucleus. Low viscosity and smaller pGL3/TPAC polyplex size enabled in vivo studies in Balb/c mice through intravenous injection. The in vitro transfection and in vivo biodistribution of the pGL3/TPAC nanoplexes showed higher luciferase expression compared to chitosan, polyethyleneimine (PEI 25K) and lipofectamine®. Physicochemical characterization, cell viability assays, and degradation studies demonstrated that TPAC meets the standards of a good transfection agent.

Highly efficient gene transfection by a hyperosmotic polymannitol based gene tranporter through regulation of caveolae and COX-2 induced endocytosis

The regulation of cellular uptake to cross the cell membrane is one of the key strategies of importance for efficient gene transfection of non-viral vectors. Hyperosmotic activity of polyplexes may facilitate crossing of the membrane barrier by elevating the osmolarity of the extracellular matrix. In this study, we demonstrated that a polymannitol based gene transporter (PMGT) utilizes the hyperosmoticity contributed by the polymannitol backbone leading to accelerated cellular uptake and enhanced gene transfection. Mannitol dimethacrylate (MDM) monomer was synthesized by esterification of mannitol and methacryloyl chloride. The prepared MDM was then cross-linked with low molecular weight (LMW) branched polyethyleneimine (bPEI) by Michael addition reaction to produce PMGT. PMGT provided polyplex stability in serum, low cytotoxicity, and degradability due to the ester linkages present in the polymannitol backbone. Elevated transfection activity and efficiency, both in vitro and in vivo, were achieved by modulating the mode of cellular uptake due to the effect of the hyperosmotic properties of PMGT. Cyclooxygenase-2 (COX-2) inhibition by SC58236 revealed the up-regulation of this osmoprotectant molecule against the hyperosmotic activity of polymannitol, inducing rapid endocytosis of PMGT in order to re-balance the hyperosmotic environment. Various inhibition studies of endocytosis showed caveolae-mediated endocytosis to be the main route of cellular internalization to account for the enhanced transgene expression.

Development of a bio-electrospray system for cell and non-viral gene delivery

Bio-electrospray technology is a very attractive tool for preparing scaffolds and depositing desired solutions on various targets by electric force. In this study, we focused on the application of a bio-electrospray (BES) technique to spray cells on the target and to simultaneously deliver genetic constructs into the cells, called non-viral gene delivery-based bio-electrospray (NVG-BES). Using this method, we tried to harvest the electric charge produced during electrospray for the cellular internalization of cationic polymer/DNA nanoparticles as well as the delivery of living cells on the desired substrate. Furthermore, we optimized the voltage, culture medium and polymeric cationic charges for high transfection efficiency and cell viability during NVG-BES. As a result, the solutions used during the NVG-BES process played an important role in improving transfection efficiency. We determined that a voltage of 10 kV with PBS as the spraying solution showed high transfection efficiency, probably due to the facilitation of cationic polymer/DNA nanocomplexes in cellular internalization and their subsequent expression. In conclusion, NVG-BES, as a novel method, is expected to deliver genes to cells and simultaneously deliver transfected cells to any substrate or scaffold.

Correction: Development of a bio-electrospray system for cell and non-viral gene delivery

Correction for ‘Development of a bio-electrospray system for cell and non-viral gene delivery’ by Myung Chul Lee et al., RSC Adv., 2018, 8, 6452–6459.

JNK2 silencing and caspase-9 activation by hyperosmotic polymer inhibits tumor progression

c-Jun N-terminal kinase 2 (JNK2) is primarily responsible for the oncogenic transformation of the transcription factor c-Jun. Expression of the proto-oncogene c-Jun progresses the cell cycle from G1 to S phase, but when its expression becomes awry it leads to uncontrolled proliferation and angiogenesis. Delivering a JNK2 siRNA (siJNK2) in tumor tissue was anticipated to reverse the condition with subsequent onset of apoptosis which predominantly requires an efficient delivering system capable of penetrating through the compact tumor mass. In the present study, it was demonstrated that polymannitol-based vector (PMGT) with inherent hyperosmotic properties was able to penetrate through and deliver the siJNK2 in the subcutaneous tumor of xenograft mice. Hyperosmotic activity of polymannitol was shown to account for the enhanced therapeutic delivery both in vitro and in vivo because of the induction of cyclooxygenase-2 (COX-2) which stimulates caveolin-1 for caveolae-mediated endocytosis of the polyplexes. Further suppression of JNK2 and hence c-Jun expression led to the activation of caspase-9 to induce apoptosis and inhibition of tumor growth in xenograft mice model. The study exemplifies PMGT as an efficient vector for delivering therapeutic molecules in compact tumor tissue and suppression of JNK2 introduces a strategy to inhibit tumor progression.

Synergistic effects of hyperosmotic polymannitol based non-viral vectors and nanotopographical cues for enhanced gene delivery

Here, we report the synergistic effects of hyperosmotic and nanotopographical cues designed using non-viral vectors and nanopatterned matrices for gene delivery. We show that efficiency of gene delivery can be further enhanced by two factors in combination, indicating the importance of synergistic cues in designing non-viral gene delivery platforms and strategies for gene therapy.