HR9: An Important Cell Penetrating Peptide for Delivery of HCV NS3 DNA into HEK-293T Cells.

BACKGROUND: The delivery of exogenous genes into cells for functional expression is required for development of DNA vaccine and gene therapy in medicine and pharmacology. Cell Penetrating Peptides (CPPs) were considered to mediate gene and drug delivery into living cells. In this study, an attempt was made to evaluate the efficiency of an arginine-rich CPP, HR9, in HCV NS3 gene delivery compared to TurboFect cationic polymer and supercharged +36 GFP into HEK-293T cells. METHODS: The recombinant pEGFP-NS3 was constructed and their accuracy was confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into HEK-293T cells by TurboFect, +36 GFP and HR9 gene delivery systems. The expression of NS3 protein was assessed by fluorescent microscopy, flow cytometry and western blotting. RESULTS: Our data indicated that HR9 peptide was able to form stable complexes with plasmid DNA and increased its delivery into HEK-293T cells in a non-covalent manner. Furthermore, treatment of cells with HR9 and HR9/DNA complexes resulted in a viability of 90-95% indicating this CPP was not cytotoxic. The analysis of zeta potential and size showed the importance of interactions between positively-charged HR9/pEGFP-NS3 complexes and negatively-charged plasma membranes. CONCLUSION: The non-toxic HR9 CPP can be considered an effective carrier for delivering plasmid DNA harboring Hepatitis C virus (HCV) gene in therapeutic vaccine design.

Simultaneous use of natural adjuvants and cell penetrating peptides improves HCV NS3 antigen-specific immune responses.

To improve an effective hepatitis C virus (HCV) therapeutic vaccine, induction of a strong and long term HCV antigen-specific immune response is an important parameter. HCV non-structural protein 3 (NS3) has antigenic properties and plays a major role in viral clearance. In this study, DNA constructs encoding HCV NS3 and heat shock protein 27 (Hsp27)-NS3 genes, and the recombinant (r) NS3 and rHsp27-NS3 proteins complexed with HR9 and Cady-2 cell penetrating peptides (CPPs) were utilized to evaluate antibody, cytokine and Granzyme B secretion in mice. Herein, the formation of NS3 and Hsp27-NS3 DNA/ HR9 CPP complexes were revealed by gel retardation assay and protection against DNase and protease. Cady-2 peptide was used to form the nanoparticles with rNS3 and rHsp27-NS3 proteins. The size and charge of the nanoparticles were confirmed by SEM and Zetasizer instruments. Next, in vitro transfection of the nanoparticles was assessed by flow cytometry and western blotting. Finally, humoral and cellular immune responses were evaluated using different modalities in mice. Our data showed that HR9 and Cady-2 could form stable nanoparticles with DNA and proteins, respectively and enhance their delivery into HEK-293 T cells in a non-covalent approach. Furthermore, the heterologous Hsp27-NS3 DNA + HR9 prime/rHsp27-NS3+Cady-2 protein boost elicited a higher Th1 cellular immune response with a predominant IgG2a, IgG2b, IFN-γ profile and strong Granzyme B secretion than those induced by other groups. Briefly, the combination of a natural adjuvant (Hsp27) and CPPs (HR9 and Cady-2) could significantly stimulate effective immune responses as a promising approach for development of HCV therapeutic vaccines.

Evaluation of AgClNPs@SBA-15/IL nanoparticle-induced oxidative stress and DNA mutation in Escherichia coli.

The bactericidal effects of silver nanoparticles have been demonstrated in the past years. Recently, the new antimicrobial compounds of silver nanoparticles with different formulations have been developed. In this work, AgClNPs@SBA-15/IL as a new compound of Ag nanoparticles, was synthesized and characterized by XRD, TEM, SEM, FTIR, and EDX. The antibacterial activity and the molecular mechanism effects of AgClNPs@SBA-15/IL nanoparticles (SNPs) on Escherichia coli DH5α cells were investigated by analyzing the growth inhibitory, H2O2 level, catalase activity, DNA mutation, and plasmid copy number following treatment with AgClNPs@SBA-15/IL nanoparticles. In experimental results, the minimum inhibitory concentration (MIC) was observed in 75 µg/ml and the antibacterial efficacy (ABE) in CFU analysis was estimated 95.3 %. In bacterial cells treated with 75 and 100 µg/ml, H2O2 level significantly increased and catalase activity decreased compared with control. The random amplified polymorphic DNA (RAPD) was used to evaluate the effect of AgClNPs@SBA-15/IL nanoparticles in DNA damages and mutation in E. coli genome. RADP-PCR results indicated different banding patterns including appearance or disappearance of bands and differences in their intensity. Cluster analysis of the RAPD-PCR results based on genetic similarity showed genetic difference between E. coli cells treated with AgClNPs@SBA-15/IL nanoparticles, and control and phylogenetic tree were divided to two clusters. Plasmid copy number analysis indicated that after 8 h incubation of E. coli cells with 50, 75, and 100 µg/ml AgClNPs@SBA-15/IL nanoparticles, copy number of pET21a (+) significantly decreased compared with control which indicating DNA replication inhibition by Ag nanoparticles. In conclusion, the results of this study indicated that AgClNPs@SBA-15/IL nanoparticles can be used as an effective bactericidal agent against bacterial cells.