Hydroxyl versus permethylated glycopolymers as gene carriers.

The main parameters that contribute to non-viral gene delivery are chemical structure and charge distribution. Indeed, saccharide units have been reported to have specific interactions with proteins located in the outer leaflet of the plasma cell membrane that facilitate the cellular internalization of plasmid-DNA vector complexes. In this work, glycopolymers based on statistical copolymers were synthesized through radical copolymerization of a cationic unit, N-ethyl pyrrolidine methacrylamide (EPA), with two styrenic monomers derived from the hydroxylated and permethylated forms of α-glucose. These copolymers were evaluated as possible non-viral gene carriers, and their ability to complex DNA was evaluated. The transfection efficiency and cytocompatibility of the polyplexes, in both fibroblastic and tumoral murine cell lines, was evaluated. Systems derived from α-glucose (GLCSt), over a monomer concentration range of 5-70mol%, exhibited high toxicity and low transfection efficiency, and were not able to significantly improve on results obtained from positive poly-EPA (PEPA) and polyethyleneimine (PEI) controls. However, systems derived from the permethylated form of α-glucose (MGLCSt), formed stable complexes with DNA or polyplexes, which showed improved transfection efficiency and cytocompatibility in comparison to positive controls. The high transfection efficiency can be clearly attributed to their cytocompatibility, which was notably found to be different for Swiss fibroblasts and B16 melanoma cells, high for Swiss and low for B16. As such, we present permethylated MCLCSt copolymers as good candidates for the possible development of therapies against melanoma.

Cell Adhesion and Proliferation on Sulfonated and Non-Modified Chitosan Films.

Three types of chitosan-based films have been prepared and evaluated: a non-modified chitosan film bearing cationizable aliphatic amines and two films made of N-sulfopropyl chitosan derivatives bearing both aliphatic amines and negative sulfonate groups at different ratios. Cell adhesion and proliferation on chitosan films of C2C12 pre-myoblastic cells and B16 cells as tumoral model have been tested. A differential cell behavior has been observed on chitosan films due to their different surface modification. B16 cells have shown lower vinculin expression when cultured on sulfonated chitosan films. This study shows how the interaction among cells and material surface can be modulated by physicochemical characteristics of the biomaterial surface, altering tumoral cell adhesion and proliferation processes.

Polymeric Gene Carriers Bearing Pendant ß-Cyclodextrin: The Relevance of Glycoside Permethylation on the "In Vitro" Cell Response.

The incorporation of cyclodextrins (CDs) to nonviral cationic polymer vectors is very attractive due to recent studies that report a clear improvement of their cytocompatibility and transfection efficiency. However, a systematic study on the influence of the CD derivatization is still lacking. In this work, the relevance of ß-CD permethylation has been addressed by preparing and evaluating two series of copolymers of the cationic N-ethyl pyrrolidine methacrylamide (EPA) and styrenic units bearing pendant hydroxylated and permethylated ß-CDs (HCDSt and MeCDSt, respectively). For both cell lines, CDs permethylation shows a strong influence on plasmid DNA complexation, "in vitro" cytocompatibility and transfection efficiency of the resulting copolymers over two murine cell lines. While the incorporation of the hydroxylated CD moiety increased the cytotoxicity of the copolymers in comparison with their homopolycationic counterpart, the permethylated copolymers have shown full cytocompatibility as well as superior transfection efficiency than the controls. This behavior has been related to the different chemical nature of both units and tentatively to a different distribution of units along the polymeric chains. Cellular internalization analysis with fluorescent copo-lymers supports this behavior.

Multivalent Glycosylation of Fluorescent Gold Nanoclusters Promotes Increased Human Dendritic Cell Targeting via Multiple Endocytic Pathways.

We report the synthesis and characterization of gold nanoclusters (Au NCs) stabilized by a mixture of zwitterionic and multivalent mannose ligands. Characterization of this carbohydrated nanosystem confirms its small size (∼2 nm), intense red-NIR fluorescence, relatively high affinity to lectin (ConA), and stability in physiological media. Cell studies performed using human-monocyte-derived dendritic cells (DCs) show that Au NC uptake efficiency is greatly enhanced by the presence of surface carbohydrate (>250% compared to noncarbohydrated Au NCs), allowing their detection in cells by fluorescence following incubation with concentrations as low as 1 µg mL(-1). Investigation using electron microscopy and pharmacological inhibitors indicates that Au NC uptake is mediated by multiple endocytic pathways involving the engulfment of Au NCs into endosomes and partial transport to lysosomes. Results show that clathrin- and F-actin-dependent pathways play major roles in Au NC uptake by DCs, regardless of whether or not they are coated with carbohydrates. In contrast, a specific C-lectin inhibitor induces a 60% decrease in DC particle uptake only for the carbohydrate-coated Au NCs. This study demonstrates that the combination of ultrasmall gold NCs and functionalization with multivalent mannose ligands results in greatly enhanced human DC targeting, presumably due to increased diffusion and target cell binding, respectively.

Preparation of surface-attached polymer layers by thermal or photochemical activation of α-diazoester moieties.

We report on the attachment of polymer monolayers or surface-attached, polymer networks onto SiO2 and/or polymer surfaces using thermo- and photoreactive α-diazoester groups. In the prior case, the α-diazoester groups are introduced into the system in the form of self-assembled monolayers of appropriately functionalized silanes. The monolayer decorated substrates are coated by polymer films and the α-diazoester groups in the monolayer are activated by heat or irradiation with UV-light. Upon activation, they cleave off nitrogen and the resulting carbene intermediates insert into C-H bonds of neighboring polymer chains. As a result of this binding process, surface-attached monolayers of the deposited polymer are obtained. When the polymers themselves carry such reactive moieties, the photo- or thermal activation leads to cross-linking of the polymers and thin surface-attached polymer networks result from the same process. The formation of the surface-attached layer is studied as a function of activation conditions, especially the temperature and the wavelength of the light used in the process.