Vitamin B6-Coupled Poly(ester amine) Mediated DNA and siRNA Delivery via Modulation of Intracellular Uptake

Abstract

The innovative methodologies in the broad field of cancer gene therapy promises a number of potential benefits for diagnosis and treatment that are likely to become important in preventing deaths from cancer. DNA synthesis mechanism underlying cancer cell proliferation and mutations can be selected for therapeutic attack to cause cell death or slow the growth of the cancer, which is one of the objectives of the research study. Elevated serine hydroxymethyltransferase (SHMT, one of the components of DNA synthesis machinery) activity has been shown to be coupled with the increased demand for DNA synthesis in rapidly proliferating cancer cells. It is also found that complete knockout of SHMT leads to glycine auxotrophy which leads to impairment in the synthesis of purines and pyrimidines. Therefore the central role of SHMT in nucleotide biosynthesis makes it a suitable anticancer target which can be selectively silenced by delivering siRNA to shut down the whole DNA synthesis machinery in cancer cells. However, the immense potential of RNAi in anti-cancer therapy is impeded by the non-availability of a suitable delivery agent, off-target effects and induction of innate immune response. Therefore, engineering a non-viral vector which can efficiently deliver the siRNA against SHMT involved in DNA biosynthesis with higher affinity to cancer cells becomes an interesting research area. Vitamin B6 (VB6) plays an essential role as a coenzyme in various cellular metabolic functions, including DNA biosynthesis for cellular growth and proliferation. VB6 is taken up by cells through facilitated diffusion via VB6 transporting membrane carrier (VTC). In this study, it was demonstrated that the VB6-coupled poly(ester amine) (VBPEA) gene transporter utilizes this uptake mechanism, leading to enhanced vector transport inside the rapidly proliferating cancer cells with relatively high affinity. Physicochemical characterization, cell viability assays, and transfection studies showed VBPEA to meet the standards of a good transfection agent. Competitive inhibition of VBPEA uptake by its structural analogue 4-deoxypyridoxine hydrochloride revealed the involvement of VB6 specific transporting membrane carrier in VBPEA internalization in tumor cells. VBPEA elicit higher transfection levels in lung cancer cells (A549) than in normal lung cells (16HBE), indicating that cancer cells which have a high demand for VB6, have a higher affinity for VB6-coupled vector. VB6 coupling to the gene transporter is important to enforce a high level of VTC-mediated endocytosis compared to VB6 alone. This system exemplified how understanding of the VB6 membrane transporter specificity allowed for the design of a VB6-coupled gene transporter with accelerated transfection activity in cancer cells owing to an advanced mode of internalization. The advancement in study was done by examining its therapeutic application in cancer tissue using RNAi technology. Serine hydroxymethyltransferase isoforms (SHMT1 & SHMT2α), which serve as scaffold protein for the formation of a multienzyme complex and generate one-carbon unit for the de novo thymidylate biosynthesis pathway during DNA synthesis, are vitamin B6-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 it was demonstrated that VB6 coupled to poly(ester amine) enforces a high level of VTC-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 multienzyme 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.