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

Abstract

Nonviral gene and small interfering RNA (siRNA) delivery formulations are extensively used for biological and therapeutic research in cell culture experiments, but less so in in vivo and clinical research. Difficulties with formulating the nanoparticles for uniformity and stability at concentrations required for in vivo and clinical use are limiting their progression in these areas. Here, we report a simple but effective method of formulating monodisperse nanocomplexes from a ternary formulation of lipids, targeting peptides, and nucleic acids at a low starting concentration of 0.2 mg/mL of DNA, and we then increase their concentration up to 4.5 mg/mL by reverse dialysis against a concentrated polymer solution at room temperature. The nanocomplexes did not aggregate and they had maintained their biophysical properties, but, importantly, they also mediated DNA transfection and siRNA silencing in cultured cells. Moreover, concentrated anionic nanocomplexes administered by convection-enhanced delivery in the striatum showed efficient silencing of the β-secretase gene BACE1 . This method of preparing nanocomplexes could probably be used to concentrate other nonviral formulations and may enable more widespread use of nanoparticles in vivo.

Peptide and nucleic acid-directed self-assembly of cationic nanovehicles through giant unilamellar vesicle modification: Targetable nanocomplexes for in vivo nucleic acid delivery

“…In this study, we replaced the liposomal component of lipopolyplexes, which was derived through sonication of multi-lamellar vesicles [8,[15][16][17][18]45], with GUVs as initial templates for vector assembly (nanovesicles and nanovesicle complexes). Following further processing, these preparations were characterized and compared with sonicated liposomes.…”

“…Furthermore, it has been shown that shape is an important factor in cellular uptake and that rods may enter cells more readily than spheres under static conditions and particularly from a side-on mode of contact with plasma membrane [52,53]. Indeed, nanovesicle particles were mostly spherical in shape, whereas lipopolyplexes contained a high number of rods and torroids [9,16,45,54] as well as particle clusters, which could explain their higher uptake. Our results further showed that the nanocomplexes that were made with nanovesicles after prolonged storage had larger size and reduced cell uptake.…”

Silencing E3 Ubiqutin ligase ITCH as a potential therapy to enhance chemotherapy efficacy in p53 mutant neuroblastoma cells

“…In our experiments, we used nanoparticles as a carrier system to deliver the ITCH siRNA in vivo. This was based on our previous findings that the receptor-targeted liposome-peptide-siRNA nanoparticle is an efficient delivery system for ex vivo and in vivo applications [49][50][51][52][53] . The formulation of the nanoparticles contains peptide ME27 which can mediate receptor-specific targeting via its interaction with integrin αvβ3 and αvβ5 on cells.…”

Receptor-targeted liposome-peptide-siRNA nanoparticles represent an efficient delivery system for MRTF silencing in conjunctival fibrosis

“…Peptide Y closely resembles part of a targeting protein expressed by the intracellular pathogen Legionella pneumophila 5 22 but the identity of the receptor is still unknown 55 . However, we have shown that peptide Y mediates the targeted delivery of siRNA in nanocomplexes to cells of neuronal origin 5 24 , lung cells 6 26 , primary vascular cells and rabbit aorta 22 23 , and is thus a peptide that could be used for different target tissues.…”

“…The liposome-peptide-siRNA nanoparticles with their synergistic lipid and peptide components can effectively package the siRNAs and protect them from enzymatic cleavage, can be dissociated by heparin, and are localised in the cytoplasm following transfection 5 24 . We have also developed PEGylated formulations to further increase the receptor-targeted specificity and transfection efficiency in cells and to enable better biocompatibility of the nanocomplexes 25 26 .…”