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To develop a chitosan-based nonviral gene carrier capable of delivering genes specifically into hepatoma cells, a bifunctional peptide composed of the TAT (transactivator of transcription) peptide and luteinizing hormone-releasing hormone (LHRH) was conjugated with low molecular weight chitosan, resulting in a TAT-LHRH-chitosan conjugate (TLC). TLC/DNA nanoparticles (TLCDNPs) were characterized by agarose gel retardation, atomic force microscopy, and dynamic light scattering analysis. In vitro targeting specificity and transfection efficiency were analyzed with a GE IN Cell Analyzer 2000 High-Content Cellular Analysis System. The results demonstrated that TLC had stronger DNA condensing power than unmodified chitosan, and that TLCDNPs were of roughly round shape with average diameter of 70-85 nm and zeta potential of +30 mV and were relatively stable in solution. The in vitro study demonstrated TLC was highly selective for hepatoma cells and essentially nontoxic.

Previous reports showed that arginine-rich peptides and oligoarginines facilitated the cellular internalization of DNA and proteins. A number of studies demonstrated that arginine-conjugated chitosan (CS)/DNA nanoparticles (ACGN) mediated significantly higher expression of the transgenes when compared with CS/DNA nanoparticles (CGN). However, the underlying mechanisms remain poorly understood. In the current study, the endocytic pathways through which cells internalize ACGN and CGN were explored by incubating the fluorescent CGN or ACGN with A10 cells in the presence of a variety of inhibitors of different endocytic pathways. The data accumulated in the current study revealed that conjugation of arginine moieties onto CS molecules enhanced the cellular uptake of the polymer/DNA nanoparticles and their transgenic efficacy, probably due to changes in the endocytic pathways, which led to the preference of internalization of ACGN by the caveolin-mediated endocytosis in comparison with that of CGN. These findings provide further support to the previous observations that ACGN mediated much higher expression of the transgenes when compared with CGN., (Copyright © 2011 Wiley Periodicals, Inc.)

Previous studies showed that arginine-conjugated chitosan (ACS)/DNA nanoparticles (ACGN) mediated significantly higher expression of the transgenes when compared with chitosan (CS)/DNA nanoparticles (CGN). However, the interactions between ACGN and immune cells still remain poorly understood. The present study investigated whether ACGN affected the initial differentiation direction of human naive CD4(+) T cells, either directly or indirectly. It was demonstrated that both ACGN and CGN induced slightly higher production of IL-12 by THP-1 cells in the order of ACGN > CGN. However, this macrophage stimulating activity was much less significant when compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4(+) T cells separated from the nanoparticles and THP-1 cells by a 0.1-μm diameter polycarbonate semipermeable membrane, which allows the pass through of macromolecules including IL-12. It also demonstrated that, when directly exposed to naive CD4(+) T cells, none of the nanoparticles induced either the activation of the naive CD4(+) T cells in the absence of recombinant human IL-4 (rhIL-4) or IFN-γ (rhIFN-γ) that induce naive CD4(+) T cell polarization or any changes in the differentiation direction of the naive CD4(+) T cells in the presence of the corresponding cytokines., (Copyright © 2010 Wiley Periodicals, Inc.)

Arginine-chitosan conjugate (Arg-CS) was demonstrated to mediate significantly elevated expression of the transgenes in comparison to chitosan (CS). However, little is known about the hemocompatibility of Arg-CS/DNA nanoparticles (ACGN). This study evaluated the coagulation properties of the ACGN and CS/gene nanoparticles (CGN) by looking at the impact of the nanoparticles on the activated partial thrombin time (APTT), prothrombin time (PT), thrombin time (TT), and hemolysis ratio (HR). It demonstrated that both ACGN and CGN, at the concentration containing 50 μg/mL of DNA, led to moderate increases of APTT but no obvious change in PT and TT in vitro. Intravenous injection of CGN or ACGN (125 μg of DNA content/100 g of body weight) into rat did not result in any significant changes in PT but moderately increased APTT and TT. ACGN was demonstrated to be a slightly stronger inhibitor of blood coagulation as compared to CGN. No obvious hemolysis was induced by the nanoparticles. Taken together, the results in the present study indicated that introduction of arginine moieties into chitosan enhanced its anticoagulant property, which would be beneficial for applications in direct contact with blood., (© 2010 Wiley Periodicals, Inc.)

Two kinds of chitosan of different molecular weight (50 kDa and 400 kDa) were employed to form nanoparticles with 32P-labeled plasmid DNA at different N/P ratios by complex coacervation method. The characteristics of chitosan gene nanoparticles (CGN) were measured. The cellular uptake of DNA nanoparticles was evaluated by A10 and K562 cells. The in vitro cytotoxicity of DNA nanoparticles was determined by the MTT assays. Cellular uptake of the DNA nanoparticles increased with increasing chitosan molecular weight and N/P ratio. It also correlated with the zeta potential of the DNA nanoparticles. Chitosan-DNA nanoparticles were much less cytotoxic when compared with Lipofectamine 2000-DNA nanoparticles.

The preparation and cell transfection of chitosan-DNA nanoparticles were studied. The TFPI (tissue factor pathway inhibitor) or EGFP (enhanced green fluorescent protein) plasmid DNA was encapsulated with chitosan to form gene nanoparticles. The results with TEM showed that the nanoparticles were of sphere shape. The mean diameter of the nanoparticles was 149 nm and the diameter ranged from 80-250 nm, which were measured by the photo related spectrometry (PCS). The encapsulation efficiency of DNA was 96% +/- 1.38% and the DNA content in the nanoparticles was 37% +/- 3.0%. The encapsulated DNA could be protected from the degradation by DNase I. The transfection efficiency of chitosan nanoparticles were about equivalent to that of the LipofectAMINETM reagent. Our results also showed that chitosan nanoparticles were nontoxic to cultured cells.

Chitosan (CS) is one of the most widely studied polymers in non-viral gene delivery since it is a cationic polysaccharide that forms nanoparticles with DNA and hence protects the DNA against digestion by DNase. However, the impact of CS/DNA nanoparticle on the immune system still remains poorly understood. Previous investigations did not found CS/DNA nanoparticles had any significant impact on the function of human and murine macrophages. To date, little is known about the interaction between CS/DNA nanoparticles and naive CD4 T cells. This study was designed to investigate whether CS/DNA nanoparticles affect the initial differentiation direction of human naive CD4 T cells. The indirect impact of CS/DNA nanoparticles on naive CD4 T cell differentiation was investigated by incubating the nanoparticles with human macrophage THP-1 cells in one chamber of a transwell co-incubation system, with the enriched human naive CD4 T cells being placed in the other chamber of the transwell. The nanoparticles were also co-incubated with the naive CD4 T cells to explore their direct impact on naive CD4 T cell differentiation by measuring the release of IL-4 and IFN-γ from the cells. It was demonstrated that CS/DNA nanoparticles induced slightly elevated production of IL-12 by THP-1 cells, possibly owing to the presence of CpG motifs in the plasmid. However, this macrophage stimulating activity was much less significant as compared with lipopolysaccharide and did not impact on the differentiation of the naive CD4 T cells. It was also demonstrated that, when directly exposed to the naive CD4 T cells, the nanoparticles induced neither the activation of the naive CD4 T cells in the absence of recombinant cytokines (recombinant human IL-4 or IFN-γ) that induce naive CD4 T cell polarization, nor any changes in the differentiation direction of naive CD4 T cells in the presence of the corresponding cytokines. [ABSTRACT FROM AUTHOR]

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan (Arg-CS). Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CSIDNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and 13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering (DLS) and atomic force microscopy (AFM). The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection. [ABSTRACT FROM AUTHOR]