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RNA interference (RNAi) is among the most potential approach for the therapy of hepatocellular carcinoma and the major barrier hindering siRNA therapeutics is the low efficiency of delivery to the desired cells. The current study aimed at developing a novel peptide for more efficient hepatoma targeted siRNA delivery, by combining luteinizing hormone-releasing hormone with hepatoma targeting specificity and MPG with cytoplasm-delivery tendency. The developed bifunctional peptide LHRH-MPG and siRNA were mixed together and resulted in LHRH-MPG/siRNA polyplexes through self-assembly. The polyplexes were characterized by agarose gel retardation and dynamic light scatting analysis. Hepatoma targeting specificity was analyzed with the GE IN Cell Analyzer 2000 High-Content Cellular Analysis System after cell transfection, and the effect of RNA interference was detected by RT-PCR. The results demonstrated that LHRH-MPG was able to assemble with siRNA to form stable and nano-sized peptide/siRNA polyplexes, which could inhibit the expression of the target gene and was essentially non-cytotoxic, as compared with the commercial transfection reagent lipofectamine 2000. [ABSTRACT FROM AUTHOR]

A simple and ultrasensitive detection of human IgG based on signal amplification using a novel bio-barcode assay and DNA chip technology was developed. The sensing platform was a sandwich system made up of antibody-modified magnetic microparticles (Ab-MMPs)/human IgG/Cy3-labeled single-stranded DNA and antibody-modified gold nanoparticles (Cy3-ssDNA-Ab-AuNPs). The MMPs (2.5 μm in diameter) modified with mouse anti-human IgG monoclonal-antibodies could capture human IgG and further be separated and enriched via a magnetic field. The AuNPs (13 nm in diameter) conjugated with goat anti-human IgG polyclonal-antibodies and Cy3-ssDNA could further combine with the human IgG/Ab-MMP complex. The Cy3-ssDNA on AuNPs was then released by TCEP to hybridize with the DNA chip, thus generating a detectable signal by the fluorescence intensity of Cy3. In order to improve detection sensitivity, a three-level cascaded signal amplification was developed: (1) The MMP enrichment as the first-level; (2) Large quantities of Cy3-ssDNA on AuNPs as the second-level; (3) The Cy3-ssDNA conjugate with DNA chip as the third-level. The highly sensitive technique showed an increased response of the fluorescence intensity to the increased concentration of human IgG through a detection range from 1 pg mL to 10 ng mL. This sensing technique could not only improve the detection sensitivity for the low concentration of human IgG but also present a robust and efficient signal amplification model. The detection method has good stability, specificity, and reproducibility and could be applied in the detection of human IgG in the real samples. [ABSTRACT FROM AUTHOR]

Tissue factor pathway inhibitor (TFPI) was reported to suppress the proliferation and migration of vascular smooth muscle cells (VSMCs) which play an important role in several vascular proliferative disorders including restenosis. Our preliminary studies demonstrated that TFPI gene induced apoptosis in human vascular smooth muscle cells (hVSMCs). The current study was designed to address the role TFPI gene plays in the cell cycle of hVSMCs. hVSMCs isolated from human umbilical artery were transfected with pIRES-TFPI plasmid which expresses TFPI in eukaryotic cells. As measured by RT-PCR, the expression of TFPI was elevated in the TFPI treated cells, leading to the arrest of the cells at G1 phase as analyzed by flow cytometry. Further study by Western blotting demonstrated that TFPI gene transfer increased the amount of p21 and p53 and decreased the amount of cyclin D and phosphorylated cdk4 and cdk6 in the cells. [ABSTRACT FROM AUTHOR]

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]