Abstract
RNA interference holds tremendous potential as one of the most powerful therapeutic strategies. However, the properties of short interfering RNA (siRNA), such as hydrophilicity, negative charge, and instability in serum have limited its applications; therefore, significant efforts have been undertaken to improve its cellular uptake. Cell penetrating peptides have been utilized to deliver various biologically active molecules, such as proteins, liposomes, nanoparticles, peptide nucleic acids, and recently small interfering RNAs. Here, we introduce a new cell penetrating peptide GL1(Ac-GLWRAWLWKAFLASNWRRLLRLLR-NH2) to improve the intracellular uptake of siRNA. This peptide consists of four tryptophan residues that facilitated its binding with the cell membrane, five arginine residues and one lysine residue which are positively charged at physiological pH, which induced the formation of peptide-siRNA complexes and enhanced the affinity of the peptide and cell membrane. Moreover, GL1 adopted helical secondary structure due to the altered distribution of polar and nonpolar residues in the sequence. In this study, we investigated the effect of peptide/siRNA molar ratio on the particle size, surface charge, secondary structure, and uptake efficiency. The results showed that GL1 formed stable complexes with siRNA mainly through electrostatic interaction and hydrophobic interaction, and the complexes displayed a spherical shape with the size of ~100 nm and positive surface charge. Utilizing the techniques of fluorescence microscopy and flow cytometry, the intracellular localization of Cy3-labeled GAPDH siRNA was visualized and the cellular uptake was quantified. It is worth noting that in the serum free environment, compared to Lipofectamine 2000, GL1 achieved higher cellular uptake of siRNA (~95%); in the presence of serum, GL1 retained the same level of siRNA cellular uptake (~84%) as Lipofectamine 2000. In addition, the viability of cells treated by GL1 in all studied molar ratios was >85%, which was significantly higher than that treated by Lipofectamine 2000 (~70%). Taken together, the peptide GL1 demonstrated promise as a siRNA delivery system.
Highlights
The discovery of RNA interference (RNAi) has changed the field of gene therapy
The following criteria were considered for the peptide design and its sequence contains: (1) hydrophobic residues, such as tryptophan, to facilitate membrane binding owing to its strong preference for the lipid membrane interfacial region [11]; (2) cationic residues, such as arginine and lysine, to assist the formation of peptide-short interfering RNA (siRNA) complexes and peptidemembrane interactions through electrostatic interaction and hydrogen bonding [11]; (3) altered distribution of the polar and nonpolar residues, to achieve better interaction with the cell membrane due to the formation of helical secondary structure [12]
We investigated the physicochemical characteristics of GL1 when interacting with siRNA and evaluated the effect of its physicochemical characteristics on siRNA delivery efficiency and cytotoxicity
Summary
The discovery of RNA interference (RNAi) has changed the field of gene therapy. RNAi is a post-transcriptional gene silencing process that can and potently knock down the expression of target genes both in vitro and in vivo [1, 2]. RNAi can be induced by short interfering RNA (siRNA). SiRNAs are double stranded RNA fragments with 21–23 nucleotides that are capable of inducing the cleavage of mRNAs with the complementary sequence [1–3]. The following criteria were considered for the peptide design and its sequence contains: (1) hydrophobic residues, such as tryptophan, to facilitate membrane binding owing to its strong preference for the lipid membrane interfacial region [11]; (2) cationic residues, such as arginine and lysine, to assist the formation of peptide-siRNA complexes and peptidemembrane interactions through electrostatic interaction and hydrogen bonding [11]; (3) altered distribution of the polar and nonpolar residues, to achieve better interaction with the cell membrane due to the formation of helical secondary structure [12]. We investigated the physicochemical characteristics of GL1 when interacting with siRNA and evaluated the effect of its physicochemical characteristics on siRNA delivery efficiency and cytotoxicity. CHO-K1 was used as a model system to test the biological responses for siRNA delivery in this study
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