Abstract
BackgroundThe successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers.ResultsFor better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model.ConclusionThe designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.
Highlights
The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape
Design of elastin-like polypeptide (ELP) and physical characterization For targeted gene delivery, tumor specific highly intracellular penetrable ELPs were constructed by combining coding sequence of Tat and IL-4 receptor specific targeting ligand (AP1) along with ELP sequence (Fig. 1a and Additional file 1: Figure S1)
ELP sequences with pentapeptide repeat (Val–Pro–Gly–Xaa–Gly), in which the Xaa residue was phenylalanine (F) was used to obtain ELP of lower molecular weight with transition temperatures (Tt) near body temperature
Summary
The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Yi et al J Nanobiotechnol (2020) 18:15 target mRNA complementary to the single strand RNA so that it inhibits the pathogenic protein translation at the first place [13]. For these reasons, siRNA delivery systems that protect siRNA from degradation by serum proteins and enable the specific uptake or release of siRNA in cells or tissues with substantially reduce risks of systemic toxicity and immunogenicity are being activity investigated. Arginine-rich cell penetrating peptides (CPPs) have been shown to encapsulate siRNA efficiently due to electrostatic interactions, but these increase risks of collateral cytotoxicity [25,26,27]
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