title: Polyplex formation between PEGylated linear cationic block copolymers and DNA: equilibrium and kinetic studies.
abstract: The basic requirement for understanding the nonviral gene delivery pathway is a thorough biophysical characterization of DNA polyplexes. In this work, we have studied the interactions between calf-thymus DNA (ctDNA)and a new series of linear cationic block copolymers (BCPs). The BCPs were synthesized via controlled radical polymerization using [3-(methacryloylamino)propyl] -trimethylammonium chloride (MAPTAC) and poly(ethyleneglycol) methyl ether (PEGMe) as comonomers. UV−visible spectroscopy, ethidium bromide dye exclusion, and gel electrophoresis study revealed that these cationic BCPs were capable of efficiently binding with DNA. Steady-state fluorescence, UV melting, gel electrophoresis, and circular dichroism results suggested increased binding for BCPs containing higher PEG. Hydrophobic interactions between the PEG and the DNA base pairs became significant at close proximity of the two macromolecules, thereby influencing the binding trend. DLS studies showed a decrease in the size of DNA molecules at lower charge ratio (the ratio of “+” charge of the polymer to “−” charge of DNA) due to compaction, whereas the size increased at higher charge ratio due to aggregation among the polyplexes. Additionally, we have conducted kinetic studies of the binding process using the stop-flow fluorescence method. All the results of BCP−DNA binding studies suggested a two-step reaction mechanism--a rapid electrostatic binding between the cationic blocks and DNA, followed by a conformational change of the polyplexes in the subsequent step that led to DNA condensation. The relative rate constant(k'(1)) of the first step was much higher compared to that of the second step (k'(2)), and both were found to increase with an increase in BCP concentration. The charge ratios as well as the PEG content in the BCPs had a marked effect on the kinetics of the DNA−BCP polyplex formation. Introduction of a desired PEG chain length in the synthesized cationic blocks renders them potentially useful as nonviral gene delivery agents.
authors.affiliation: Department of Chemistry, Indian Institute of Technology Kharagpur , West Bengal 721302, India.
authors.forename: Debabrata
authors.initials: D
authors.lastname: Dey
authors.forename: Santosh
authors.initials: S
authors.lastname: Kumar
authors.forename: Rakesh
authors.initials: R
authors.lastname: Banerjee
authors.forename: Souvik
authors.initials: S
authors.lastname: Maiti
authors.forename: Dibakar
authors.initials: D
authors.lastname: Dhara
mesh_terms: D000643:Ammonium Chloride / Q000737:chemistry; D000818:Animals; D002412:Cations / Q000737:chemistry; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry; D007700:Kinetics; D008689:Methacrylates / Q000737:chemistry; D044367:Phase Transition; D011092:Polyethylene Glycols / Q000737:chemistry*; D058105:Polymerization; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D002412:Cations; D008689:Methacrylates; D011108:Polymers; D000643:Ammonium Chloride; D011092:Polyethylene Glycols; D004247:DNA; C427203:calf thymus DNA; C400723:MAPTAC; D004996:Ethidium