Ran Pan

Serum stability and physicochemical characterization of a novel amphipathic peptide C6M1 for siRNA delivery.

The efficient delivery of nucleic acids as therapeutic agents is a major challenge in gene therapy. Peptides have recently emerged as a novel carrier for delivery of drugs and genes. C6M1 is a designed amphipathic peptide with the ability to form stable complexes with short interfering RNA (siRNA). The peptide showed a combination of random coil and helical structure in water but mainly adopted a helical conformation in the presence of anions or siRNA. Revealed by dynamic light scattering (DLS) and microscopy techniques, the interaction of C6M1 and siRNA in water and HEPES led to complexes of ∼70 and ∼155 nm in size, respectively, but showed aggregates as large as ∼500 nm in PBS. The time-dependent aggregation of the complex in PBS was studied by DLS and fluorescence spectroscopy. At molar ratio of 15∶1, C6M1 was able to completely encapsulate siRNA; however, higher molar ratios were required to obtain stable complexes. Naked siRNA was completely degraded in 4 h in the solution of 50% serum; however C6M1 protected siRNA against serum RNase over the period of 24 h. Western blotting experiment showed ∼72% decrease in GAPDH protein level of the cells treated with C6M1-siRNA complexes while no significant knockdown was observed for the cells treated with naked siRNA.

Uptake Mechanism and Direct Translocation of a New CPP for siRNA Delivery

Since their development, cell-penetrating peptides (CPPs) have been used as delivery vehicles for various genetic or therapeutic agents; however, the uptake mechanisms of CPPs and the delivery details are still unclear. Understanding the mechanisms of cellular internalization of CPPs facilitate their development of CPPs as gene delivery vectors. In the present study, we evaluated the internalization process of a previously designed CPP, STR-KV, complexed with small interference RNA (siRNA) targeting at the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Using heparin treatment and chemical endocytic inhibitors, we elucidated that the electrostatic interaction of STR-KV/siRNA complex with heparin sulfate proteoglycans at the cell membrane surface triggered the energy-independent uptake of the majority of the complexes, which most likely through a direct translocation pathway. The intracellular trafficking and internalization kinetics observed by confocal microscopy also confirmed that the complex was uptaken through a nonendocytic pathway.

A novel peptide for efficient siRNA delivery in vitro and therapeutics in vivo

Small interfering RNA (siRNA) shows great therapeutic potential due to its ability to regulate gene expression in a highly selective manner. However, its application has been limited by ineffective cellular uptake of siRNAs. To achieve successful gene-silencing efficiency, a safe and effective delivery vector is generally required. In this study, we designed a series of amphipathic peptides that comprised a variant of a nuclear localization sequence, 0-6 histidine residues and an optional stearic acid group. Among these candidates, STR-HK exhibited good characteristics as a safe and efficient siRNA delivery vector, facilitating efficient siRNA delivery to mammalian cells without causing cytotoxicity. Moreover, the intratumoral injection of STR-HK/siRNA complexes achieved high anti-tumor activity through the downregulation of the Bcl-2 protein in mice, with an inhibition rate of 62.8%. Our data demonstrate that STR-HK is a highly promising siRNA delivery vector for therapeutic applications.

Design and Evaluation of Endosomolytic Biocompatible Peptides as Carriers for siRNA Delivery

Gene therapy using RNA interference (RNAi) technology has been explored to treat cancers, by regulating the expression of oncogene. However, even though small interfering RNA (siRNA), which triggers RNAi, may have great therapeutic potential, efforts at using them in vivo have been hampered by the difficulty of effective and safe delivery into cells of interest. In this study, to develop a safe and efficient carrier for in vitro and in vivo siRNA delivery, we designed a peptide library. These peptides are improved variants of a known peptide based siRNA carrier C6. All the modifications improved the transfection efficiency of C6 to some degree. After completing prescreening for activity, several promising candidates were used for further evaluation. Selected peptides C6M3 and C6M6 could form stable complexes with siRNA. These complexes could be greatly uptaken by cells and showed a punctate perinuclear distribution. Moreover, peptide/siRNA complexes achieved high transfection efficiency in vitro without inducing substantial cytotoxicity. We have validated the therapeutic potential of this strategy for cancer treatment by targeting Bcl-2 gene in mouse tumor models, and demonstrated that tumor growth was inhibited. In order to address possible immune side effects of these peptide carriers, biocompatibility study in terms of complement activation and cytokine activation assay were carried out, whereas none of the peptides induced such effects. In conclusion, these results support the potential of these peptides as therapeutic siRNA carrier.

Effective small interfering RNA delivery in vitro via a new stearylated cationic peptide.

A crucial bottleneck in RNA interference-based gene therapy is the lack of safe and efficient delivery systems. Here, a novel small interfering RNA (siRNA) delivery peptide, STR-HK, was constructed by conjugating a stearyl end to the N-terminus of the peptide sequence HHHPKPKRKV, where PKPKRKV is an altered sequence of the nucleus localization signal (PKKKRKV) and contributes to the cytosol localization of STR-HK–siRNA complexes. Histidine is a linker and plays an important role in disrupting the endosomal membrane via the proton sponge effect. As expected, STR-HK formed complexes with siRNA with a particle size of 80–160 nm in diameter and efficiently delivered Cy3-labeled glyceraldehyde 3-phosphate dehydrogenase siRNA into PC-3 human prostate cancer cells. The transfection efficiency of STR-HK at molar ratio of 60/1 was comparable to that of Lipofectamine 2000, one of the most efficient commercially available transfection reagents. Furthermore, the STR-HK–siRNA complexes exhibited minimal cytotoxicity, which was significantly lower than that of Lipofectamine. Taken together, the strategy of conjugating the stearyl moiety with HHHPKPKRKV as a non-viral siRNA delivery system is advantageous.

An amphipathic lytic peptide for enhanced and selective delivery of ellipticine

Cationic lytic peptides (CLPs) have shown promise in treating bacterial infection and cancer via selective disruption of bacterial or cancer cell membranes. In this work, we used a CLP, C6, as a nanocarrier for a hydrophobic anticancer agent, ellipticine (EPT). The size of the resulting C6-EPT complex was ∼190 nm. The in vitro studies using A549 lung cancer cells showed an enhanced anticancer activity of the C6-EPT complex compared to that of C6 or the EPT control. This enhancement was found to correlate with the membrane disruption induced by C6, which facilitated the entry of EPT into cells. More importantly, the C6-EPT complex showed a higher selectivity than that of C6 towards cancer cells upon comparison of their cytotoxicities against A549 cells and NIH-3T3 fibroblast cells. The enhanced therapeutic activity was also found in in vivo studies using an A549 tumor-bearing BALB/c nude mice model. This study provides a new CLP strategy for the development of multifunctional drug delivery systems.

Supramolecular peptide amphiphile based nanocarrier for pH-triggered Dox release, overcoming drug resistance

We conjugated a self-assembling peptide amphiphile (PA), Palm-CR3, with doxorubicin (Dox) via a hydrazone linker. The Dox loaded PA system (Palm-CR3–Dox) formed fibrous particles with a pH-triggered Dox release profile. Palm-CR3–Dox enhanced the cellular uptake of Dox and showed drug resistance reversal potential. Both the membrane disruptive activity of Palm-CR3 and the conjugation of Dox with PA were suggested to contribute to the drug resistance reversal effect. This work explored the potential of the self-assembling PA system as a versatile drug delivery platform for cancer treatment.

Co-delivery of drug nanoparticles and siRNA mediated by a modified cell penetrating peptide for inhibiting cancer cell proliferation

Co-delivery of anti-cancer agent Ellipticine (EPT) and Bcl-2 siRNA was mediated by a modified cell penetrating peptide, stearylated H16R8 (STR-H16R8). The stability and efficacy of the EPT nanoparticles was improved significantly by the amphiphilic peptide. Efficacy of STR-H16R8 stabilized EPT nanoparticles was further enhanced with Bcl-2 siRNA.

In vitro and in vivo therapeutic siRNA delivery induced by a tryptophan-rich endosomolytic peptide

At the forefront of medicine, gene therapy provides an effective way to treat a range of diseases by regulating defective genes at the root of the disease. Short interfering RNAs (siRNAs) hold great promise as therapeutic agents in this domain; however, intracellular delivery remains a major obstacle to clinical applications of therapeutic siRNAs. Here we report a peptide designed to mediate siRNA delivery. This peptide, C6M1, is rationally designed to promote the endosomal escape ability of an existing peptide sequence. Formed C6M1-siRNA nanoscale complexes are able to deliver siRNA into cells and induce specific gene knockdown with low toxicity. The increased membrane disruption ability under acidic conditions of the peptide with tryptophan residue substitution may contribute to the enhanced gene silence efficacy. Intratumoral injection of the complexes results in a marked reduction of tumor growth through downregulation of antiapoptotic Bcl-2 protein in mice. In addition, the C6M1-siRNA complex was proven safe at transfection concentration by cytotoxicity assay. These results demonstrate that the C6M1-siRNA complex is a potent system for efficient gene delivery in vitro and in vivo.