Cross-linked polyethylenimine-tripolyphosphate nanoparticles for gene delivery.

The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site-enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery.

Effect of CD95 on inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVE: Many CD95-expressing cells don't always undergo apoptosis after stimulation with CD95 ligation. The purpose of this paper is to investigate the role of expression of CD95 (Fas/Apo1) on inflammatory response in fibroblast-like synoviocytes (FLS) obtained from rheumatoid arthritis (RA) and to evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB or Akt) pathways within this process. METHODS: The expression levels of CD95 were monitored by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Apoptotic cells were detected by in situ apoptosis detection (TUNEL) assay. The RA-FLS were treated with agonistic anti-CD95 antibody or CD95 siRNA. Then the proliferation was detected by CCK-8, and mRNA level of inflammatory cytokines was detected by RT-PCR. After the RA-FLS were treated with agonistic anti-CD95 antibody, the total Akt and pAkt protein expression was analyzed by Western blot, and the changes mentioned above were observed while pre-incubated with the PI3K inhibitor LY294002. RESULTS: A significant increase of CD95 antigen was found in RA compared with osteoarthritis (OA) samples, while apoptosis in RA synovial tissue was not obvious. Low concentrations of agonistic anti-CD95 antibody could promote RA-FLS growth and interleukin-6 (IL-6) mRNA expression, while high concentrations could induce apoptosis. And both of these phenomena could be inhibited by CD95 siRNA. Agonistic anti-CD95 antibody could stimulate the expression of pAkt, and PI3K specific inhibitor LY294002 could induce opposite changes. CONCLUSION: Stimulation of CD95 could promote RA-FLS proliferation and inflammation, and activation of the PI3K/Akt signaling pathway might be the possible mechanism.