Successful intracellular delivery of genes requires an efficient carrier, as genes by themselves cannot diffuse across cell membranes. Because of the toxicity and immunogenicity of viral vectors, nonviral vectors are gaining tremendous interest in research. In this work, we have investigated the temperature-dependent DNA condensation efficiency of various compositions of a thermosensitive block copolymer viz., poly(N-isopropylacrylamide)-b-poly(2-(diethylamino)ethyl methacrylate) (PNIPA-b-PDMAEMA). Three different copolymer compositions of varying molecular weights were successfully synthesized via the RAFT polymerization technique. Steady-state fluorescence and circular dichroism (CD) spectroscopies, dynamic light scattering (DLS) and zeta potential measurements, agarose gel electrophoresis, and atomic force microscopy techniques were utilized to study the interaction of the copolymers with DNA at temperatures above and below the critical aggregation temperature (CAT). All these experiments revealed that, above the CAT, there was formation of highly stable and tight polymer–DNA complexes (polyplexes). The size of polyplexes was dependent on the temperature up to a certain charge ratio, as determined by the DLS results. The results obtained from temperature-dependent fluorescence spectroscopy, CD, and gel electrophoresis indicated that the DNA molecules were shielded more from aqueous exposure above the CAT because of the formation of relatively more compact complexes. The polyplexes also exhibited changes in the particle morphology below and above the CAT, with particles generated above CAT being more spherical in morphology. These results suggested at the possibility of modulating the complex formation by temperature modification. The present biophysical studies would provide new physical insight into the design of novel gene carriers.

Cationic polymeric micelles that are capable of coreleasing drugs and DNA into cells have attracted considerable interest as combination chemotherapy in cancer treatment. To this effect, we have presently developed a cationic fluorescent amphiphilic copolymer, poly(N,N′-dimethylaminoethylmethacrylate)-b-(poly(2-(methacryloyl)oxyethyl-2′-hydroxyethyl disulfidecholate)-r-2-(methacryloyloxy)ethyl-1-pyrenebutyrate) [PDMAEMA-b-(PMAODCA-r-PPBA)], having pendent cholate moiety linked through a redox-responsive disulfide bond. The amphiphilic nature of the copolymer facilitated the formation of cationic micellar nanoparticles in aqueous medium. The self-assembly of the copolymer to form micelles and subsequent destabilization of the micelles in the presence of glutathione (GSH) was monitored by the change in the fluorescence characteristic of the attached pyrene resulting from alteration in the hydrophobicity of its neighborhood. These micellar nanoparticles were subsequently utilized in encapsulating hydrophobic anticancer drug, doxorubicin (DOX), in the core of the micelles, whereas the cationic shell of the micelles was used for complexation with oppositely charged DNA to form micelleplexes. Gel retardation assays, ethidium bromide (EB) exclusion assay, and DLS and AFM studies confirmed the successful binding of the cationic micelles with DNA. The binding capability of the micelles was higher than corresponding cationic linear PDMAEMA. The kinetics of the simultaneous release of encapsulated DOX and complexed DNA in the presence of glutathione was thoroughly studied using various techniques. All the experiments showed fast and efficient release of DOX and DNA from DOX-loaded micelleplexes. The study implies that these redox-responsive cationic micelles may open up new opportunities toward co-delivery of DNA and anticancer drugs in combinatorial therapy.

Copper-free click reaction for synthesis of redox-responsive water-soluble polymer based core cross-linked nanoparticles for cancer therapy.

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