Nucleic acids complexation with cationic elastin-like polypeptides: Stoichiometry and stability of nano-assemblies

Abstract

Positively charged elastin-like polypeptides (ELPs) were synthesized for the compaction of genetic material. A recombinant ELP (VPGXG) 40 with X=V,M (3:1) was post-modified in two steps to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. Positively charged ELPs were characterized by SDS-PAGE, size exclusion chromatography, 1 H NMR, potentiometric titrations and dynamic light scattering to assess their purity and determine their degree of functionalization, molecular weight, isoelectric point and thermo-responsive behaviour. Electrostatic complexation between the different ELP derivatives and nucleic acids was studied to determine the stoichiometry of ELP S /nucleic acids complex formation, and to find optimal conditions leading to stable nanoparticles with controlled size and surface potential. The stability of these complexes was investigated in the presence of salts at physiological concentrations and in the presence of surfactant. This study revealed that two regimes of stable nanoparticles in terms of size and charge can be obtained from the electrostatic complexation between the primary amine containing ELP derivative, ELP(-NH 2 ), and plasmid DNA. Resulting complexes were found to be stable to dissociation for charge ratios up to 2.5 under physiological salt concentrations (154 mM NaCl), showing that plasmid DNA was completely condensed by the polycationic ELP and protected against electrolyte-mediated dissociation.

Coupling of RAFT polymerization and chemoselective post-modifications of elastin-like polypeptides for the synthesis of gene delivery hybrid vectors

“…Slow dropwise mixing of cationic hybrid ELPs to a negatively charged DNA solution was selected as complex formation method. 71,[76][77][78] The stabilization of pDNA/Cationic hybrid ELPs complexes was reached after 5 min. of each cationic ELP addition.…”

“…The native recombinant ELP[M 1 V 3 -40] corresponding to the sequence (VPGXG) 40 , where X=V/M (3:1), here simply referred to as ELP (Fig. 3a) 34,71 , was chemoselectively modified onto methionine residues in order to graft in a second step the cationic PTMAEMA oligomers. Fig.…”

“…5.27. 71,91 Alkyne-modified ELP presented an IP of 7.9. The IP of the cationic hybrid ELPs were found to be around 10.4, 11.7 and 11.8, for ELP-g-P(TMAEMA) 6 , ELP-g-P(TMAEMA) 9 and ELP-g-P(TMAEMA) 15 , respectively.…”

“…64,70 In a previous contribution, we have described the synthesis of positively charged ELPs through a two steps post-modification of a recombinant ELP, (VPGXG) 40 with X = V,M (3:1), to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. 71 These ELPs were found to be able to interact with plasmid DNA and to form stable complexes for gene delivery. Furthermore, preliminary in vitro testing on HEK 293 cells viability showed that the modified ELPs complexed with DNA were non toxic to these cells.…”

“…The CuAAC reaction was achieved using similar conditions to those described by Bravo-Anaya et al for the modification of alkyne-functionalized ELPs with amino groups (i.e., EtOH 75%, Cu(II)SO 4 , sodium ascorbate, PMDETA). 71 A reaction time of 72 h and excess reagents of 1.5 equiv. azido-oligomers per Met residue was necessary to maximize the coupling onto ELP(-CCH).…”