Oligonucleotide-decorated carboxyl-modified PVP nanogels: a new platform for diagnosis and gene-therapy

Abstract

Nanogels, or nanoscalar chemically crosslinked polymer networks, were generated by simultaneous crosslinking of Poly(N-vinyl pyrrolidone) (PVP) and grafting of acrylic acid (AA) monomer through e-beam irradiation of their aqueous solutions. The yield of the process is very high and no separation or complex purification procedures are required since no recourse to surfactants or organic solvents is made. The administered irradiation dose (40 kGy) imparts also sterility to the obtained nanogels in the form of aqueous dispersions. Control of particle size and size-distribution can be achieved by proper selection of the irradiation conditions. In particular, we obtained nanogel variants with average hydrodynamic diameters in the range 30 – 100 nm with PDI lower than 0.3 and negative surface charge density. Confirmation of the molecular architecture and detection of the surface composition have been sought through complementary spectroscopic techniques, such as FTIR and XPS. Preliminarily to any biological evaluation or bio-conjugation, absence of cytotoxicity was tested through MTT assay. Studies on nanogel viability thought cell compartments, carried out with fluorescent PVP nanogels variants incubated for different times in cell cultures and observed by confocal microscopy, showed cytoplasmic distribution of nanogels and preferential localization of around the nuclei during the first 7 hours and progressive disappearance during the following 17 hours. Nanogels secretion as waste products could be hypothesized. The availability of accessible carboxyl groups for subsequent facile bio-conjugation reactions makes this family of functional nanogels very promising for decoration with oligonucleotides. As a proof of concept, a fluorescent oligonucleotide semi-helix was conjugated through an amino group attached to the terminal 3’. The recognition ability of the conjugated forward semi-helix toward the reverse semi-helix was proved by spectrofluorimetric readings using a reverse oligo carrying a fluorescence quencher.