Multifunctional, self-assembling anionic peptide-lipid nanocomplexes for targeted siRNA delivery

Abstract

Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5' RACE-PCR and Western blot analysis of protein.

Practical Considerations of Convection Enhanced Delivery for Novel Therapies Treating Spinal Cord Injury Related Neuropathic Pain

“…The utility of using anionic, siRNA nanoparticles developed from PEGylated liposomes, contemplated of therapeutic significance for treating neuronal diseases as compared to cationic Nano complexes use [53].They evaluated administration of siRNA to BACE1 in the rat brain showed both the silencing of BACE1 and substrate-enzyme specificity and was achieved in vivo following a single CED injection of anionic nanoparticles.…”

A method for concentrating lipid peptide DNA and siRNA nanocomplexes that retains their structure and transfection efficiency

“…The anionic liposomes that were made were: DOPG:DOPE:DOPE PEG2000 (L AP 1); and DOPG:DOPE:DPPE PEG2000 (L AP 2) at a molar ratio of 47.5:47.5:5 mol%, respectively. 16,21 Anionic nanocomplexes were prepared in water at a 4:3:1 molar charge ratio 16 …”

“…Adult male Wistar rats (Charles River Laboratories International, Inc., Wilmington, MA, USA) (225-275 g) were used for convection-enhanced delivery (CED), as described previously. 15,21 A total volume of 5 μL (6 μg of siRNA or 0.024 mg/kg) of anionic PRL nanoparticles (with peptide RVG-9R) in 5% dextrose was delivered to the striatum at an infusion rate of 2.5 μL/minute. Rats were euthanized 48 hours later, and the striata were placed in RNAlater (Thermo Fisher Scientific).…”

“…21 Briefly, total protein was extracted from mouse brain using the Precellys ® Homo genizer (Stretton Scientific Ltd, Derbyshire, UK). Forty micrograms of protein were loaded and separated using NuPAGE ® Precast gels (10% Bis-Tris; Thermo Fisher Scientific) and then transferred electrophoretically to a polyvinylidene fluoride membrane (Thermo Fisher Scientific).…”

“…Empirically optimized ratios of these components are required to produce monodisperse nanoparticles with peak transfection efficiency. The formulation used in this study comprises a mixture of liposomes and targeting peptides that self-assemble on mixing with DNA [11][12][13][14][15][16][17][18] or siRNA [19][20][21] to form LPD or LPR receptor-targeted nanocomplexes (RTNs). Based on this platform, we have described both cationic and anionic RTN formulations and shown their in vivo potential by delivery The formulation of RTNs is more complicated than simple lipoplex or polyplexes and becomes increasingly problematic in our experience at higher nucleic acid concentrations of more than 0.5 mg/mL with large aggregates and precipitates forming, which leads to poor biodistribution, systemic toxicity, and low transfection efficiency in vivo.…”