Synthesis and characterization of variable-architecture thermosensitive polymers for complexation with DNA.
Copolymers of N-isopropylacrylamide with a fluorescent probe monomer were grafted to branched poly(ethyleneimine) to generate polycations that exhibited lower critical solution temperature (LCST) behavior. The structures of these polymers were confirmed by spectroscopy, and their phase transitions before and after complexation with DNA were followed using ultraviolet and fluorescence spectroscopy and light scattering. Interactions with DNA were investigated by ethidium bromide displacement assays, while temperature-induced changes in structure of both polymers and polymer-DNA complexes were evaluated by fluorescence spectroscopy, dynamic light scattering, laser Doppler anemometry, and atomic force microscopy (AFM) in water and buffer solutions. The results showed that changes in polymer architecture were mirrored by variations in the architectures of the complexes and that the overall effect of the temperature-mediated changes was dependent on the graft polymer architecture and content, as well as the solvent medium, concentrations, and stoichiometries of the complexes. Furthermore, AFM indicated subtle changes in polymer-DNA complexes at the microstructural level that could not be detected by light scattering techniques. Uniquely, variable-temperature aqueous-phase AFM was able to show that changes in the structures of these complexes were not uniform across a population of polymer-DNA condensates, with isolated complexes compacting above LCST even though the sample as a whole showed a tendency for aggregation of complexes above LCST over time. These results indicate that sample heterogeneities can be accentuated in responsive polymer--DNA complexes through LCST-mediated changes--a factor that is likely to be important in cellular uptake and nucleic acid transport.
Pennadam
Sivanand S
SS
The School of Pharmacy, Boots Science Building, University of Nottingham, University Park, Nottingham NG7 2RD, UK.
Ellis
James S
JS
Lavigne
Matthieu D
MD
Górecki
Dariusz C
DC
Davies
Martyn C
MC
Alexander
Cameron
C
D000178:Acrylamides / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006358:Hot Temperature; D011094:Polyethyleneimine / Q000737:chemistry*; D013329:Structure-Activity Relationship
D000178:Acrylamides; D005456:Fluorescent Dyes; D011094:Polyethyleneimine; D004247:DNA; C067295:N-isopropylacrylamide