Cellular uptake of DNA-chitosan nanoparticles: the role of clathrin- and caveolae-mediated pathways.

The success of gene therapy depends on efficient delivery of DNA and requires a vector. A promising non-viral vector is chitosan. We tailored chitosan to optimize it for transfection by synthesizing self-branched and trisaccharide-substituted chitosan oligomers (SBTCO), which show superior transfection efficacy compared with linear chitosan (LCO). The aim of the work was to compare the cellular uptake and endocytic pathways of polyplexes formed by LCO and SBTCO. Both polyplexes were taken up by the majority of the cells, but the uptake of LCO was lower than SBTCO polyplexes. LCO polyplexes were internalized through both clathrin-dependent and clathrin-independent pathways, whereas SBTCO polyplexes were primarily taken up by clathrin-independent endocytosis. The different level of cellular uptake and the distinct endocytic pathways, may explain the difference in transfection efficacy. This was supported by the observation that photochemical internalization increased the transfection by LCO polyplexes considerably, whereas no effect on transfection was found for SBTCO polyplexes.

Intravital microscopy in window chambers: a unique tool to study tumor angiogenesis and delivery of nanoparticles.

Solid tumor growth is heavily dependant on angiogenesis. Tumor angiogenesis is the result of a complex interplay between tumor cells, endothelial cells, and other stromal cells. It has been found to be under strict control of a plethora of molecular factors that function as angiogenic up- and down-regulators; nevertheless, the identification of molecular and cellular players and their roles in angiogenesis is still ongoing. The microvasculature resulting from tumor angiogenesis lacks hierarchy and has a high permeability for macromolecules and nanoparticles, which offers significant potential for nanoparticulate tumor imaging and drug delivery platforms. However, improvements in the delivery to poorly vascularized regions and the distribution throughout the tumor interstitium are critical for nanoparticles to become more effective in the battle against cancer. A tool that has proven extremely valuable in both unraveling angiogenic pathways and characterizing in vivo nanoparticle behavior in solid tumors is intravital microscopy of tumors grown in window chamber preparations. In this review this technique is explained, several exciting examples illustrating its value in elucidating tumor angiogenesis are presented and the study of nanoparticle behavior in solid tumors using this approach is described. We conclude with a discussion of the potential value of intravital microscopy in window chambers in multimodality studies of tumor pathophysiology and nanoparticle dynamics.

Molecular design of chitosan gene delivery systems with an optimized balance between polyplex stability and polyplex unpacking.

Chitosan is an attractive gene delivery vehicle, but the criteria and strategies for the design of efficient chitosan gene delivery systems remain unclear. The purpose of this work was to investigate how the strength of the charge-based interaction between chitosan and DNA determines the gene expression levels and to design chitosan vectors with an optimized balance between polyplex stability and polyplex unpacking. Using 21 formulations based on low molecular weight chitosans with constant charge density and a number-average degree of polymerization (DPn) in the range of 21-88 (M(w) 4.7-33kDa), we studied the relationship between the chain length and the formulation properties, cellular uptake of polyplexes and gene transfer efficacy. We were able to identify a narrow interval of DPn31-42 that mediated the maximum level of transgene expression. An increase in chain length and/or the amino-phosphate (A/P) ratio reduced and delayed transgene expression. Compared to DPn31, transfection with the same amount of DPn72 or DPn88 resulted in 10-fold-lower expression levels. The gene transfer pattern correlated with the ability of heparin to release DNA from the polyplexes. As a tool to facilitate the unpacking of the polyplexes, we substituted the chitosans with uncharged oligosaccharides that reduced the interaction with DNA. The substitution of chitosans that originally yielded too stable polyplexes, such as DPn72 and DPn88 resulted in a 5-10-fold enhancement of the expression levels. However, the substitution of chitosans shorter than DP28 completely abolished transfection. Tailoring of the chain length and the substitution of chitosan were shown to be feasible tools to modulate the electrostatic interactions between the chitosan and DNA and to design chitosans with an optimized balance between polyplex stability and polyplex unpacking.

Characterizing DNA condensation by structurally different chitosans of variable gene transfer efficacy.

Chitosan can be used as a nonviral gene delivery vector for which DNA condensation and transfection efficacy strongly depend on structural parameters. In this study, we characterized the condensation of DNA by three molecularly tailored chitosans, including linear, trisaccharide substituted-, and self-branched trisaccharide substituted chitosan oligomers. No significant differences could be detected in the hydrodynamic diameters formed by the various chitosans as analyzed by dynamic light scattering. However, atomic force microscopy revealed that self-branched chitosan formed complexes with a higher ratio of globules to rods, and the heights of both globules and rods were larger than for complexes formed by the other chitosans. Using an amino/phosphate ratio of 10, fluorescence correlation spectroscopy measurements showed that self-branched chitosan exhibited a lower fraction (30%) of bound chitosan than the other chitosans. YOYO-1 was a superior fluorescent DNA-label compared to Cy5 and PicoGreen, since labeling with YOYO-1 had least effect on the size and structure of the complexes.