Transfection Efficiency Of Chitosan Research Articles

Relationship between phosphorylamine-modification and molecular weight on transfection efficiency of chitosan

The aim of this study is both to design the chitosan (Chi) nanoparticles with different Mw containing the phosphoester bonds and increase their amino function for the transfection. The phosphorylamine-modification of Chi and depolymerized Chi (DChi) was realized using o-phosphorylethanolamine (o-PEA) and characterized, for the first time. The nanoparticles (nMChi and nMDChi) were prepared by ionic gelation and their particle size, polydispersity index (PDI), zeta potential, stability, gene binding capacity and cytotoxicity were examined. The effects of the Mw of Chi on the cytotoxicity, gene binding capacity, and in vitro transfection efficiency of the nanoparticles on Human Embryonic Kidney 293 (HEK293) cells were also examined. Green Fluorescent Protein Circular Plasmid DNA (pEGFN1) loaded nanoparticles (gnMChi and gnMDChi) were used in the transfection. This study showed that the Mw of phosphorylamine-modified Chi significantly affected the characteristics, cytotoxicity, gene binding capacity and transfection efficiency of the nanoparticles.

Transfection efficiency of chitosan and thiolated chitosan in retinal pigment epithelium cells: A comparative study

OBJECTIVE:Gene therapy relies on efficient vector for a therapeutic effect. Efficient non-viral vectors are sought as an alternative to viral vectors. Chitosan, a cationic polymer, has been studied for its gene delivery potential. In this work, disulfide bond containing groups were covalently added to chitosan to improve the transfection efficiency. These bonds can be cleaved by cytoplasmic glutathione, thus, releasing the DNA load more efficiently.MATERIALS AND METHODS:Chitosan and thiolated chitosan nanoparticles (NPs) were prepared in order to obtain a NH3+:PO4− ratio of 5:1 and characterized for plasmid DNA complexation and release efficiency. Cytotoxicity and gene delivery studies were carried out on retinal pigment epithelial cells.RESULTS:In this work, we show that chitosan was effectively modified to incorporate a disulfide bond. The transfection efficiency of chitosan and thiolated chitosan varied according to the cell line used, however, thiolation did not seem to significantly improve transfection efficiency.CONCLUSION:The apparent lack of improvement in transfection efficiency of the thiolated chitosan NPs is most likely due to its size increase and charge inversion relatively to chitosan. Therefore, for retinal cells, thiolated chitosan does not seem to constitute an efficient strategy for gene delivery.

Improved transfection efficiency of chitosan‐DNA complexes employing reverse transfection

AbstractThe present study was designed to systematically compare the conventional and reverse transfection methodologies for chitosan/DNA complexes using a low molecular weight (MW) chitosan. The hydrodynamic diameter of the complexes, measured by Dynamic Light Scattering (DLS) was found to be ∼ 216 nm and TEM investigations showed spherical and compact complexes with an average size of 200 nm. The transfection efficiency of chitosan using the two methodologies was assessed by employing reporter gene coding for green fluorescent protein (GFP) and luciferase. More than 50% of HEK 293 cells were transfected when transfection done using reverse transfection strategy at pH 6.5 with 10% serum for 24 h followed by media replenishment with pH 7.4 with 10% serum for an additional 24 h period. Also, the cytotoxicity of chitosan/DNA complexes was also considerably lower than the commercially available transfection reagent lipofectamine. Our investigation concludes that maximal transgene expression levels could be achieved using reverse transfection where the chitosan/DNA complexes are pre‐incubated on the plate surface followed by plating of cells at pH 6.5 with 10% serum for 24h and media resupplemented with pH 7.4 with 10% serum for an additional 24 h period. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Transfection efficiency of depolymerized chitosan and epidermal growth factor conjugated to chitosan–DNA polyplexes

An efficient non-viral gene delivery for varieties of cells has been considered essential for gene therapy and tissue engineering. This study evaluated transfection efficiency of chitosan (HW) with molecular weights (Mw) at 470 and degree of deacetylation (DDA) 80% and its depolymerization product (LW) with Mw at 16 kDa and DDA 54%, as well as epidermal growth factor (EGF) conjugated to chitosan-DNA microparticles of both HW and LW by using either disulfide linkage or NHS-PEO(4)-Maleimide as a cross linker. The results revealed that the depolymerized LW at chitosan/DNA charge ratio 56:1 and pH 6.9 gave high transfection efficiency in both KB, a cancer cell line, and fibroblast cells at about the same level of Lipofectamine, but the EGF-conjugated chitosan-DNA polyplexes from these methods did not improve transfection efficiency, which may come from the aggregation and fusing of the complexes as shown in scanning electron microscopy. However, this depolymerized LW chitosan showed the potential for further development as a safe and cost-effective non-viral gene delivery vehicle.

Enhancing Polysaccharide-Mediated Delivery of Nucleic Acids Through Functionalization with Secondary and Tertiary Amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.

Effects of alginate inclusion on the vector properties of chitosan-based nanoparticles

Chitosan nanoparticles have shown considerable promise as gene vectors but do not mediate transfection with satisfactory efficiency. To improve upon the transfection efficiency of chitosan, we approached the development of alginate–chitosan nanoparticles with the goals of maintaining low toxicity and biocompatibility. Through ionic gelation, particles were formed with a mean Z-average diameter of 157 nm and a zeta potential of + 32 mV. Competition binding assays indicated that the presence of alginate reduces the strength of interaction between chitosan and DNA, contributing to improved transfection. Cell viability assays indicated that nanoparticles exhibit the same low toxicity as chitosan, and significantly reduced toxicity compared to a commercial liposome formulation. As well, complexation with nanoparticles maintained DNA integrity and protected it from nuclease degradation better than chitosan alone. Alginate–chitosan nanoparticles were able to mediate transfection of 293T cells four times that achieved by chitosan nanoparticles; at 48 h, the transfection efficiency was as high as with Lipofectamine™, with significantly reduced cytotoxicity. Overall, alginate inclusion improved the vector properties of chitosan-based nanoparticles, demonstrating superior transfection ability while maintaining biocompatibility and low toxicity.

Efficient gene delivery by urocanic acid-modified chitosan

Nonviral delivery systems for gene therapy have been increasingly proposed as safer alternatives to viral vectors. Chitosan is considered to be a good candidate for the gene delivery system since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic charge potential. However, the use of chitosan for gene delivery is limited due to low transfection efficiency. To enhance the transfection efficiency, water-soluble chitosan (WSC) was coupled with urocanic acid (UA) bearing imidazole ring which can play the crucial role in endosomal rupture through proton sponge mechanism. The urocanic acid-modified chitosan (UAC) was complexed with DNA, and UAC/DNA complexes were characterized. The sizes of UAC/DNA complexes under physiological condition (109–342 nm) were almost same as those of chitosan–DNA complexes. UAC also showed good DNA binding ability, high protection of DNA from nuclease attack, and low cytotoxicity. The transfection efficiency of chitosan into 293T cells was much enhanced after coupling with UA and increased with an increase of UA contents in the UAC.

Efficient gene delivery by urocanic acid-modified chitosan

Nonviral delivery systems for gene therapy have been increasingly proposed as safer alternatives to viral vectors. Chitosan is considered to be a good candidate for the gene delivery system since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic charge potential. However, the use of chitosan for gene delivery is limited due to low transfection efficiency. To enhance the transfection efficiency, water-soluble chitosan (WSC) was coupled with urocanic acid (UA) bearing imidazole ring which can play the crucial role in endosomal rupture through proton sponge mechanism. The urocanic acid-modified chitosan (UAC) was complexed with DNA, and UAC/DNA complexes were characterized. The sizes of UAC/DNA complexes under physiological condition (109–342 nm) were almost same as those of chitosan–DNA complexes. UAC also showed good DNA binding ability, high protection of DNA from nuclease attack, and low cytotoxicity. The transfection efficiency of chitosan into 293T cells was much enhanced after coupling with UA and increased with an increase of UA contents in the UAC.