Development of a targeted siRNA delivery system using FOL-PEG-PEI conjugate

Abstract

Receptor mediated delivery of siRNA enables silencing of target genes in specific tissues. Folate receptor (FR) is an attractive target for tumor-selective gene delivery. The focus of this study was to deliver the dihydrofolate reductase (DHFR) siRNA expressing plasmid and to silence the DHFR gene in FR positive KB cells, by complexing the plasmid with a folate-polyethylene glycol-polyethylenimine (FOL-PEG-PEI) conjugate, as a gene carrier. A DHFR siRNA sequence was cloned into a pSUPER-RNAi vector and complexed with the FOL-PEG-PEI conjugate. The complex was characterized by particle size analyzer, gel retardation and DNase protection assay. The FOL-PEG-PEI/pSUPER-siDHFR complex was transfected to FR overexpressing (KB) and FR negative (A549) cells. The transfection effiencies and gene inhibition were analyzed by fluorescence microscopy and RT-PCR. The pSUPER-siDHFR/PEI-PEG-FOL complex delivered the siRNA vector and inhibited DHFR gene in KB cells, while A549 cells were unaffected. Lipofectamine mediated transfection of pSUPER-siDHFR, delivered the vector and inhibited the DHFR gene in both KB and A549 cells. FR mediated delivery of siDHFR complexed with PEI-PEG-FOL conjugate inhibits the DHFR expression in FR positive cells alone. This strategy can be extended to deliver a wide range of drugs and post-transcriptional gene silencing therapeutics.

Polymers in Small-Interfering RNA Delivery

“…In an attempt to fabricate multifunctional delivery system which could facilitate target specific treatment of tumor, Verma group studied the folate receptor (FR)-mediated delivery of dihydrofolate reductase (DHFR) siRNA to silence the DHFR gene in FR positive KB cells (Biswal et al, 2010). A DHFR siRNA sequence was cloned into a pSUPER-RNAi vector and complexed with the folate-polyethylene glycol-PEI (FOL-PEG-PEI) conjugate.…”

Polyethylene glycol–poly(ε-benzyloxycarbonyl-L-lysine)-conjugated VEGF siRNA for antiangiogenic gene therapy in hepatocellular carcinoma

“…34 Researchers have designed various types of novel copolymers, such as pH-sensitive, temperature-sensitive, light-sensitive, magnetic-sensitive, ultrasound-sensitive, redox-sensitive, and enzyme-degraded copolymers, in order to solve the issue of low transfection efficiency of the PEGylated nanoparticles. [35][36][37][38] In this study, redox-sensitive PEG-SS-PLL was synthesized and used to deliver the novel nanoparticle complex PEG-SS-PLL/siVEGF. We investigated the transfection efficiency, cytotoxicity in vitro, tumor-inhibitory effect in vivo, and the mechanism of antitumor effect of the complex.…”

Vehicles for Small Interfering RNA transfection: Exosomes versus Synthetic Nanocarriers

“…Liposomes versatile and flexible system protection of the siRNA targeting possible membrane fusion and triggered drug release form aggregates with serum proteins immunogenic limited delivery efficiency in vivo [63] PEGylation decreased blood clearence [64] incorporation of fusogenic lipids enhanced bioavailability of siRNA [65] apolipoprotein A 95% knockdown without immunotoxicity targeted to the liver [66] pH-sensitive histidinelysine peptide inhibition of tumor growth in a pancreatic cancer model [67] peptide from Rabies Virus glycoprotein brain targeting [68] SNALPs -Stabilized Nucleic Acid Lipid Particles PEG-derivatized cationic lipid with stabilized lipid bilayer fusogenic prolonged blood circulation 80-90% efficiency after siRNA transfection into liver [34] SPANosomes sorbitan monooleate high siRNA incorporation efficiency efficient endosomal escape and cytosolic delivery 60-80% gene knockdown [69,70] Polyethylenimine -PEI toxicity of high molecular weight PEI proton sponge effect for efficient release from endosomes [21] hydrophobic lipid anchor improved cellular uptake [71] folate folate receptor mediated uptake [42] galactose and pullulan liver targeted [43,72] PEI-PEG-copolymer efficient siRNA release 75%gene knockdown efficiency suppression of tumor growth [73] bioreducible poly(amido ethylenimine) (SS-PAEI) complete release of siRNA in a reductive environment [62] Poly(dl-lactide-co-glycolide) -PLGA degraded under physiological conditions degradation rate can be controlled by composition of the polymer nucleic acid encapsulation no endosomal escape mechanism [74] cationic segments (polyamines) good binding and protections of siRNA disruption of endosomal membranes [75] poly(ethylene glycol)-poly (d,l-lactide) cationic lipic suppressed tumor growth in a breast cancer murine xenograft model [76] Chitosan biodegradable, no/low immunogenicity low siRNA transfection efficiency [77,78] salt complexes excellent cell survival high gene silencing [79] α-tocopherol succinate enhanced cellular uptake [47] thiamine pyrophosphate >70% gene silencing [80] RGD peptide targeting to cancer cells [44] guanidinylation decreased cytotoxicity and enhanced cellular internalization of siRNA enhanced gene-silencing efficiency …”

“…However, incorporation of ligands increased targeting to specific cells and tissues. PEI was modified with folate to be taken up by folate receptor expressing tumors [42], or by pullulan, a water-soluble polysaccharide, to direct it to Brought to you by | University of St Andrews Scotland Authenticated | 138.251.14.35 Download Date | 1/12/14 2:28 PM [60]. Both linear and branched PEI induced proinflammatory cytokine production and hepatic damage, although linear PEI was less toxic [61].…”