Alternatives to efficient viral vectors in gene therapy are desired because of their poor safety profiles. Chitosan is a promising non-viral nucleotide delivery vector because of its biocompatibility, biodegradability, low immunogenicity and ease of manufacturing. Since the transfection efficiency of chitosan polyplexes is relatively low compared to viral counterparts, there is an impetus to gain a better understanding of the structure-performance relationship. Recent progress in preparation and characterisation has enabled coupling analysis of chitosans structural parameters that has led to increased TE by tailoring of chitosan's structure. In this review, we summarize the recent advances that have lead to a more rational design of chitosan polyplexes. We present an integrated review of all major areas of chitosan-based transfection, including preparation, chitosan and polyplexes physicochemical characterisation, in vitro and in vivo assessment. In each, we present the obstacles to efficient transfection and the strategies adopted over time to surmount these impediments.
The transfection efficiency (TE) of chitosan-plasmid DNA (pDNA) polyplexes can be critically modulated by the polymer degree of deacetylation (DDA) and molecular weight (MW). This study was performed to test the hypothesis that the TE dependence on chitosan MW and DDA is related to the polyplex stability, hence their intracellular decondensation/unpacking kinetics. Major barriers to nonviral gene transfer were studied by image-based quantification. Although uptake increased with increased DDA, it did not appear to be a structure-dependent process affecting TE, nor was nuclear entry. Colocalization analysis showed that all chitosans trafficked through lysosomes with similar kinetics. Fluorescent resonant energy transfer (FRET) analysis revealed a distinct relationship between TE and polyplex dissociation rate. The most efficient chitosans showed an intermediate stability and a kinetics of dissociation, which occurred in synchrony with lysosomal escape. In contrast, a rapid dissociation before lysosomal escape was found for the inefficient low DDA chitosan whereas the highly stable and inefficient complex formed by a high MW and high DDA chitosan did not dissociate even after 24 hours. This study identified that the kinetics of decondensation in relation to lysosomal escape was a most critical structure-dependent process affecting the TE of chitosan polyplexes.
This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan–DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8–24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24–40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine™. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression.
Mechanical loading of articular cartilage can produce catabolic and anabolic changes in tissue metabolism. Most previous studies in this area have focussed on aggrecan. Little information concerning load-induced collagen modifications has been obtained. We have therefore conducted studies where mechanical loads are applied in vitro to full thickness cartilage explants retaining a thin layer of bone, in order to investigate mechanically induced collagen breakdown and consequent turnover, in addition to aggrecan changes and mechanical property alterations. Tissue explant disks were subjected to unconfined compression and either immediately frozen or kept in static culture for 10 days. Mechanical tests of the disks immediately prior to and just after the cyclic loading period were also performed. They showed a weakening of the collagen network and an increased hydraulic permeability due to the cyclic loading. Load-induced alterations of the extracellular matrix was then clearly evidenced by an increase in denatured collagen in the disks frozen immediately after loading compared to unloaded controls. Loaded disks maintained in culture for 10 additional days following cyclic loading no longer expressed this increase in denatured collagen suggesting that mechanically denatured collagen I1 had undergone a removal process which could represent turnover or repair, or the beginning of progressive degradation. Indeed matrix fragments of collagen I1 and glycosaminoglycans were found to be released to post-loading culture medium in increased quantities compared to unloaded controls. Our data further demonstrates the ability of mechanical load of articular cartilage to modulate turnover and metabolism of collagen and proteoglycan in a complex and multifactorial manner that may be of particular significance in the pathogenesis of osteoarthritis and in the development of pharmacological agents to modulate its progression.
Polycations having a high buffering capacity in the endosomal pH range, such as polyethylenimine (PEI), are known to be efficient at delivering nucleic acids by overcoming lysosomal sequestration possibly through the proton sponge effect, although other mechanisms such as membrane disruption arising from an interaction between the polycation and the endosome/lysosome membrane, have been proposed. Chitosan is an efficient delivery vehicle for nucleic acids, yet its buffering capacity has been thought to be significantly lower than that of PEI, suggesting that the molecular mechanism responsible for endolysosomal escape was not proton sponge based. However, previous comparisons of PEI and chitosan buffering capacity were performed on a mass concentration basis instead of a charge concentration basis, the latter being the most relevant comparison basis because polycation-DNA complexes form at ratios of charge groups (amine to phosphate), rather than according to mass. We hypothesized that chitosan has a high buffering capacity when compared to PEI on a molar basis and could therefore possibly mediate endolysosomal release through the proton sponge effect. In this study, we examined the ionization behavior of chitosan and chitosan-DNA complexes and compared to that of PEI and polylysine on a charge concentration basis. A mean field theory based on the use of the Poisson-Boltzmann equation and an Ising model were also applied to model ionization behavior of chitosan and PEI, respectively. We found that chitosan has a higher buffering capacity than PEI in the endolysosomal pH range, while the formation of chitosan-DNA complexes reduces chitosan buffering capacity because of the negative electrostatic environment of nucleic acids that facilitates chitosan ionization. These data suggest that chitosans have a similar capacity as PEI to mediate endosomal escape through the proton sponge effect, possibly in a manner which depends on the presence of excess chitosan.
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