Histidine-Based Reduction-Sensitive Star-Polymer Inclusion Complex as a Potential DNA Carrier: Biophysical Studies Using Time-Resolved Fluorescence as an Important Tool.

An ideal DNA carrier is one that is capable of effectively condensing DNA into complexes of optimum size and shape, preventing premature decomplexation in the bloodstream and efficiently releasing the DNA into affected cells. In this context, we have developed a novel ß-cyclodextrin (ß-CD)-based four-arm star-shaped polymer inclusion complex (IC) with arms made of a poly(l-histidine)-based cationic polymer. The polymer was well characterized by gel permeation chromatography, NMR, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We have also investigated its DNA complexation and release properties. Bisadamantane containing a disulfide bond was synthesized that linked two such poly(l-histidine)-containing ß-CD units via guest-host interactions to prepare the presented IC. Besides using the conventional steady-state fluorescence spectroscopy, the ability of this IC to condense DNA to form polyplexes and their release behavior have been established by using the time-resolved fluorescence spectroscopy technique. Thiazole orange (TO) was used for the first time as a DNA-intercalating dye in the time-resolved fluorescence spectroscopic study. The superior DNA-condensing ability of the IC as compared to that of the precursor two-arm ß-CD and linear poly(l-histidine) of a comparable molecular weight, as confirmed by dynamic light scattering, zeta potential, atomic force microscopy, and gel electrophoresis studies, could be attributed to a higher charge density. The IC-DNA polyplexes were found to be stable in a medium similar to an extracellular fluid but could efficiently release DNA in the presence of 10 mM glutathione, a concentration prevalent in the intracellular fluid of cancer cells. Hence, here, we have successfully demonstrated the synthesis of a novel biocompatible star-shaped IC with the potential to carry and release DNA in cancer cells and also established the feasibility of using the time-resolved fluorescence spectroscopic technique to study the complexation behavior of the polycation and DNA using TO as a DNA-intercalating dye.

Cationic cross-linked polymers containing labile disulfide and boronic ester linkages for effective triple responsive DNA release.

Disruption of DNA carriers triggered by intracellular bio-stimulants has been broadly considered as most convenient strategy for efficient DNA delivery. In this direction, we have designed and synthesized pH, redox and ATP responsive cationic cross-linked polymers (CLPs) having disulfide and reversible boronic ester linkages. These CLPs also contain folate groups that are known for their targeting capability towards cancer cells. Biophysical studies showed that these cationic CLPs exhibited more effective DNA condensation in comparison to cationic linear polymers resulting in the formation of nano-sized polyplexes with sufficient positive zeta potentials and good colloidal stability at neutral pH (∼7.4). More interestingly, the polyplexes prepared from these CLPs have the ability to selectively release complexed DNA under conditions similar to those prevalent in cancer cells such as acidic pH, ATP rich surroundings or presence of glutathione, as revealed by ethidium bromide exclusion assay, agarose gel electrophoresis, AFM measurements, etc. Therefore, these cross-linked polymers have high potential of being effective non-viral gene delivery vehicles.

Redox-Responsive Efficient DNA and Drug Co-Release from Micelleplexes Formed from a Fluorescent Cationic Amphiphilic Polymer.

Cationic polymeric micelles that are capable of co-releasing 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.

Temperature- and Composition-Dependent DNA Condensation by Thermosensitive Block Copolymers.

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.