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Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection.

RNA interference has emerged as a powerful tool in biological and pharmaceutical research; however, the enzymatic degradation and polyanionic nature of short interfering RNAs (siRNAs) lead to their poor cellular uptake and eventual biological effects. Among nonviral delivery systems, cell-penetrating peptides have been recently employed to improve the siRNA delivery efficiency. Here we introduce an 18-mer amphipathic, amino-acid-pairing peptide, C6, as an siRNA delivery carrier. Peptide C6 adopted a helical structure upon coassembling with siRNA. The C6-siRNA coassembly showed a size distribution between 50 and 250 nm, confirmed by dynamic light scattering and atomic force microscopy. The C6-siRNA interaction enthalpy and stoichiometry were 8.8 kJ·mol(-1) and 6.5, respectively, obtained by isothermal titration calorimetry. A minimum C6/siRNA molar ratio of 10:1 was required to form stable coassemblies/complexes, indicated by agarose gel shift assay and fluorescence spectroscopy. Peptide C6 showed lower toxicity and higher efficiency in cellular uptake of siRNA compared with Lipofectamine 2000. Fluorescence microscopy images also confirmed the localization of C6-siRNA complexes in the cytoplasm using Cy3-labeled siRNAs. These results indicate high capabilities of C6 in forming safe and stable complexes with siRNA and enhancing its cellular uptake.

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Peptide-based nanoparticles have emerged as promising drug delivery systems for targeted cancer therapy. Yet, the biocompatibility of these nanoparticles has not been elucidated. Here, the in vitro biocompatibility and toxicity and in vivo immunocompatibility and bioactivity of the self/coassembling peptide AC8 in its nanoparticle form are evaluated. AC8 showed minimal hemolytic activity (5%) and did not cause aggregation of red blood cells. The in vitro assay revealed that AC8 did not activate the complement system via the classical or alternative pathway but did activate the lectin pathway to a small extent. However, AC8 showed no C3a and C5a anaphylotoxin activation suggesting that complement activation did not proceed to the later, inflammatory, stages. The in vivo immune response assay showed that administration of AC8 to BALB/c mice had no effect on the weight of immune organs or body weight of mice at doses less than 0.1 mg/kg. This peptide also did not have any effect on the expression of CD3+ T-cells and natural killer (NK) cells, the ratio of CD4+/CD8+ T-cell, and the proliferation of B-cells. These results suggest that AC8 can be a potential carrier candidate for drug delivery.

Amino acid pairing peptide-based nanoparticles were recently introduced as promising carriers for hydrophobic anticancer drugs. The AC8 peptide, n-FEFQFNFK-c, is based on the amino acid pairing (AAP) design with 8 amino acids and hence the designated name AAP8. The nanoparticles (NPs) AAP8 have modified either on the C-terminal or on both terminal, by conjugation with diethylene glycol (DEG) . Here, the in vitro biocompatibilities of the NPs and their modified versions were compared and the potential of these NPs as carriers for the hydrophobic anticancer drug pirarubicin was determined as well as the peptide-drug co-assembly complexes. The toxicity of the NPs, DEGylated NPs, and blended mixtures with pirarubicin, was tested against the human adenocarcinoma lung cancer cell line, A549. The amino-end DEGylated NP, (NP-I), had superior biocompatibility over the non-modified NPs or double DEGylated NPs (NP-II). NP-I had very low hemolytic activity (1%) while NP and NP-II had marginal (8%) and acceptable (5%) hemolytic activity, respectively. All three types of NPs did not activate the complement system via the classical and alternative pathways nor did they activate the anaphylotoxin C3a. However, NP-II and its drug complex effectively activate the complement terminal attack complex. The lectin pathway was not activated by NP-I and NP-II, but was to a small extent by the non-modified NPs, with no lectin activation when complexed with drug. These results indicate NP-I is the most promising peptide for use as a drug delivery system, highlighting the importance of proper modification in peptides for drug delivery systems.