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

Abstract

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.

Nucleic Acids Based Polyelectrolyte Complexes: Their Complexation Mechanism, Morphology, and Stability

“…The cationic block includes many of the synthetic cationic polymers discussed in the previous section. Some common examples include PLL, ,, PDMAEMA, , and poly­(vinyl benzyl trimethyl­ammonium chloride) (PVBTMAC). , The neutral block can be either hydrophobic or hydrophilic. Poly­(ethylene glycol) (PEG) is the most commonly employed hydrophilic neutral block. ,, For ease of block copolymer synthesis, poly­(oligo­(ethylene glycol) methyl ether methacrylate) (POEGMA) is also used .…”

“…The cationic block includes many of the synthetic cationic polymers discussed in the previous section. Some common examples include PLL, 36,37,118 PDMAEMA, 44,119 and poly(vinyl benzyl trimethylammonium chloride) (PVBTMAC). 9,38 The neutral block can be either hydrophobic or hydrophilic.…”

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

“…Steady-state as well as time-resolved fluorescence spectral studies to evaluate the DNA binding ability of different cationic DNA carriers are usually done using the EB dye. ,,− However, because of the limited ability of EB to augment fluorescence intensity on binding with DNA, all these fluorescence studies using EB were carried out at very high concentrations of DNA. For example, Dey et al showed that the fluorescence intensity of EB increased only around 10 times from free EB to EB–DNA complexes using the concentration of calf thymus DNA at 25 μM (in terms of phosphate groups present in the DNA backbone) and the ratio of the concentrations of EB and the nucleotide was maintained as 1:15.…”

“…Steady-state fluorescence spectroscopy of DNA-intercalating dyes such as ethidium bromide (EB) is extensively carried out in dye displacement assays to probe the DNA-binding ability of various gene carriers. − Nevertheless, this approach cannot be considered suitable for estimating the binding capability of gene carriers with DNA quantitatively because of overlapping and broad spectra of excluded and DNA-intercalated dyes as well as the conformational change of DNA in polyplexes. , This is why there arises a necessity for developing new techniques to study polyplex formation at various N/P ratios where maximum DNA condensation or gene transfection capability is exhibited.…”

“…20−22 Nevertheless, this approach cannot be considered suitable for estimating the binding capability of gene carriers with DNA quantitatively because of overlapping and broad spectra of excluded and DNA-intercalated dyes as well as the conformational change of DNA in polyplexes. 20,21 This is why there arises a necessity for developing new techniques to study polyplex formation at various N/P ratios where maximum DNA condensation or gene transfection capability is exhibited. Nowadays, the time-resolved fluorescence spectroscopy method has been an interesting as well as useful tool to investigate the state of DNA inside the polyplexes.…”