Development of Cationic Nanosystems Based on Poly (Styrene-Alt-Maleic Anhydride) for Combinatorial Delivery of Drug and Nucleic Acid in Cancer Therapy

Abstract

Polymeric nanosystems have been extensively investigated for delivering active agents in cancer therapy. Clinical efficacy of conventional active ingredients in cancer treatment can be significantly enhanced using nanocarriers. Nanocarriers can passively target the cancer cells by enhanced permeation and retention (EPR) effect, prolong circulation time of the loaded active agents, minimize toxic effects to healthy cells and overcome multidrug resistance. In addition, enhanced bioavailability and sustained release of the incorporated active ingredients can be achieved by nanocarriers mediated delivery. Co-delivery of drugs and nucleic acids like plasmid DNA (pDNA) vectors, double stranded RNAs (dsRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), small interfering RNAs (siRNAs) etc. has been shown to overcome various drawbacks of chemotherapeutics. Most of the genetic materials utilize RNA interference mechanism to knock down the expression of specific genes responsible for cancer cell proliferation. Dose of toxic chemotherapeutic drugs needed for clinical efficiency can be substantially reduced using the combinatorial approach, thereby minimizing the side effects of chemotherapeutics. The genetic materials cannot be administered as such in to systemic circulation for the serum nucleases will facilitate their degradation. Further, cellular entry of these nucleic acids is extremely restricted due to their poly anionic nature, high molecular weight and inherent instability. Hence it is essential to develop a suitable formulation which can dispose the loaded cargos viz. drugs and nucleic acids, by overcoming the systemic and cellular barriers. The main objective of present research work was to develop cationic polymeric nanosystems for intracellular delivery of drugs and nucleic acids. The study involves synthesis and characterization of cationic amphiphilic graft co-polymers of low molecular weight PSMA, and evaluation of their intracellular gene delivery efficacy. Various cationic moieties were selected ix from different chemical families to synthesize graft co-polymers. These cationic structures included isonicotinic acid, 2-aminoethyl piperazine, glycidyl trimethyl ammonium chloride, Larginine and spermine. Nanocarriers of the synthesized cationic graft co-polymers were formulated, characterized and evaluated for their biocompatibility, haemocompatibility, endosomal rupturing property and transfection efficiency. Highest endosomal rupturing potential was depicted by quaternized isonicotinic acid grafted polymer followed by spermine and Larginine grafts. Spermine and L-arginine grafted PSMA derivatives exhibited better transfection efficiency in comparison to the other polymeric grafts. The cationic nanocarriers of PSMA depicted core shell type morphology, which indicated the feasibility to load hydrophobic anti cancer drugs in them. Since combinatorial delivery of anti cancer drugs and siRNAs is a promising strategy to combat cancer, PSMA was suitably modified to co-deliver both drug and siRNA. Doxorubicin and PLK-1 siRNA were selected as model drug and siRNA, respectively, for the study. A cationic nanosystem was developed by grafting isonicotinic acid and arginine-acetyl lysine conjugate to the PSMA backbone using 4, 7, 10-trioxa- 1,13-decanediamine as linker molecule. The cationic polymer could self-assemble in to smooth, spherical micelles with a CMC of ~3 μg ̷ mL and particle size ranging from ~14 ˗ 30 nm. Doxorubicin was loaded in the micelles with an optimum loading content of ~ 9% w ̷ w. Efficient complexation of siRNA occured above polymer ̷ siRNA weight ratio of 10. Doxorubicin and fluorescent labeled siRNA bearing micelles depicted excellent co localization within the cytoplasm of MCF˗7 cells. MTT cell viability assay showed synergistic cytotoxic effect of the co delivered active agents. Co-delivery of drug and siRNA in Ehrlich ascites tumor (EAT) bearing Swiss albino mice, using the cationic micelles, significantly enhanced the antitumor efficacy. A stimuli sensitive polymeric nanosystem based on PSMA, which could enhance tumor specific delivery of the loaded active agents, was developed by grafting multiple disulfide linkages to the polymeric backbone. Anhydride units of the parent polymer were ring opened with methoxy polyethyleneglycol. Arginine and histidine molecules, possessing respective cell penetrating and endosomal buffering properties, were grafted to the polymer through disulfide linkages. The polymeric derivative could easily self-assemble in water to form smooth, spherical micellar structures, with a CMC of ~7 μg ̷ mL. Doxorubicin was loaded in the micelles using nanoprecipitation method, with an optimum loading content of ~ 8.6 % w ̷ w. Efficient binding and condensation of siRNA occurred above polymer ̷ siRNA weight ratio of 40, and stability of the polyplexes was achieved by coating them with bovine serum albumin (BSA). In vitro cell culture studies and in vivo tumor regression study have shown synergistic effect of the drug loaded micelleplexes, in arresting cancer cell proliferation. The thesis is divided into five chapters. First chapter include literature review on the significance of nanotechnology in cancer with specific reference to combinatorial delivery of drugs and nucleic acids. Second chapter details synthesis of cationically modified amphiphilic graft co polymers of poly (styrene-alt-maleic anhydride) (PSMA) and evaluation of their intracellular gene delivery efficacy, in vitro. Third chapter discusses development of amphiphilic graft co polymeric micelles of poly (styrene-alt-maleic anhydride), for combinatorial delivery of drug and siRNA, and evaluation of the nanosystem, in vitro and in vivo. Fourth chapter focuses on the development and optimization of stimuli sensitive nanocarriers based on poly (styrene-alt-maleic anhydride) and evaluation of their efficacy in co delivering drug and siRNA, in vitro and in vivo. Fifth chapter highlights summary, conclusion and future directions of the outcome of research works performed.