Introduction: It is well known that gene therapy can cure serious diseases, such as cancer, infection, innate immunodeficiency, cystic fibrosis, and cardiovascular diseases. However, gene delivery within one's own body decreases transfection efficiency because the gene is degraded by enzymes in body[1]. Among the polymer-based non-viral gene carriers, chitosan is a good candidate to interact with DNA or RNA. Therefore the psiRNA-bcl2 gene and (KLA)4 peptide coated by LMWSC with targeting ligand was delivered by receptor-mediated endocytosis. The delivered compounds were denoted ternary complexes. These complexes effectively inhibited cancer cell growth via the co-activity of psiRNA-bcl2 and (KLA)4 peptide.
(KLA)4 peptide of cationic, induced apoptosis by disrupting mitochondrial membrane, is fused to deliver with gene by electrostatic interaction. In addition, polyplexed genes and peptides can be degraded by enzymes in-vivo[2]. Therefore, low molecular weight-water soluble chitosan (LMWSC) as a non-viral vector which is a linear cationic polysaccharide, has good biocompatibility, antibacterial activity, solubility, and stability. In addition, LMWSC can act as a gene carrier because it has many free amine-groups and LMWSC used in this study was modified with PEGylated transferrin.
In this study, a combination therapy with peptides and genes effectively inhibited cancer growth via mitochondrial apoptosis and gene silencing. To deliver the peptide and gene, ternary complexes with targeting ligand-grafted LMWSC were prepared. The ternary complexes were then investigated to confirm the ability to condense with genes and undergo transfection, as well as for cytotoxicity and anticancer activity.
Experimental Methods: DNA/(KLA)4/LMWSC-TfP ternary complexes were prepared by electrostatic charge interaction in aqueous reactions. To prepare DNA/(KLA)4 polyplexes, (KLA)4 was dissolved in distilled water. The solution of (KLA)4 peptide was then added to the pDNA solution and incubated for 30 min at room temperature. Finally, the LMWSC-TfP solution was added to the polyplex solution and incubated for 30 min at room temperature.
Binding affinity, stability from Dnase, and releasing assay of ternary complexes was conducted by gelretardation assay. Also, transfection efficacy of ternary complexes was accomplished in PC3 cell lines. In addition, cytotoxicity and antitumor activity of that was confirmed by MTT assay in L929 and PC3 cell lines.
Results and Discussion: To deliver effectively gene and peptide in cancer cell, targeting-ligand grafted ternary complex was prepared by electrostatic interaction. The delivered gene and (KLA)4 peptide showed that psiRNA exerted RNA-silencing action in the nucleus, while the (KLA)4 peptide inhibited cancer growth through mitochondrial apoptosis.
Gene transfection of ternary complex showed that targeting-ligand grafted ternary complex was transfected more effectively than that without targeting-ligand at all ratio (1:1:64, 1:2:64, 1:4:64) because cellular uptake of it was increased by targeting-ligand. Also, antitumor activity of ternary complex was indicated that the more amount of psiRNA was increased, the more cell viability was decreased. It means that ternary complex was entered into PC3 cells and tumor growth was inhibited by gene scilencing of psiRNA and mitochondrial apoptosis of KLA peptide, respectively.
Conclusion: In this study, we prepared ternary complex with targeting-ligand grafted LMWSC by electrostatic interaction. The transfection of ternary complex was increased by targeting-ligand. Moreover, antitumor activity showed that was increased by gene and peptide co-action. These results suggest that ternary complex can be effectively inhibiting tumor growth by delivered gene and peptide.
This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning. (No. NRF-2014R1A2A1A10053027).
References:
[1] G.Y. Wu et al., Biochemistry, 27(1988), 887-892.
[2] S. Hyun et al., Biomacromolecules, 15(2014), 3746-3752