Peptide-siRNA Supramolecular Particles for Neural Cell Transfection (original) (raw)
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Small (Weinheim an der Bergstrasse, Germany), 2015
Lipopolymer 49, a solid-phase synthesized T-shaped peptide-like oligoamide containing two central oleic acids, 20 aminoethane, and two terminal cysteine units, is identified as very potent and biocompatible small interfering RNA (siRNA) carrier for gene silencing in glioma cells. This carrier is combined with a novel targeting polymer 727, containing a precise sequence of Angiopep 2 targeting peptide, linked with 28 monomer units of ethylene glycol, 40 aminoethane, and two terminal cysteines in siRNA complex formation. Angiopep-polyethylene glycol (PEG)/siRNA polyplexes exhibit good nanoparticle features, effective glioma-targeting siRNA delivery, and intracellular siRNA release, resulting in an outstanding gene downregulation both in glioma cells and upon intravenous delivery in glioma model nude mice without significant biotoxicity. Therefore, this novel siRNA delivery system is expected to be a promising strategy for targeted and safe glioma therapy.
Journal of pharmaceutical sciences, 2013
The application of gene and RNAi-based therapies to the central nervous system (CNS), for neurological and neurodegenerative disease, offers immense potential. The issue of delivery to the target site remains the single greatest barrier to achieving this. There are challenges to gene and siRNA (small interfering RNA) delivery which are specific to the CNS, including the post-mitotic nature of neurons, their resistance to transfection and the blood–brain barrier. Viral vectors are highly efficient and have been used extensively in pre-clinical studies for CNS diseases. However, non-viral delivery offers an exciting alternative. In this review, we will discuss the extracellular and intracellular barriers to gene and siRNA delivery in the CNS. Our focus will be directed towards various non-viral strategies used to overcome these barriers. In this regard, we describe selected non-viral vectors and categorise them according to the barriers that they overcome by their formulation and targeting strategies. Some of the difficulties associated with non-viral vectors such as toxicity, large-scale manufacture and route of administration are discussed. We provide examples of optimised formulation approaches and discuss regulatory hurdles to clinical validation. Finally, we outline the components of an “ideal” formulation, based on a critical analysis of the approaches highlighted throughout the review. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
Nucleic Acids Research, 2011
While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/ siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.
Identification and Characterization of Receptor-Specific Peptides for siRNA Delivery
ACS nano, 2012
Tumor-targeted delivery of siRNA remains a major barrier in fully realizing the therapeutic potential of RNA interference. While cell-penetrating peptides (CPP) are promising siRNA carrier candidates, they are universal internalizers that lack cell-type specificity. Herein, we design and screen a library of tandem tumor-targeting and cell-penetrating peptides that condense siRNA into stable nanocomplexes for cell type-specific siRNA delivery. Through physiochemical and biological characterization, we identify a subset of the nanocomplex library of that are taken up by cells via endocytosis, trigger endosomal escape and unpacking of the carrier, and ultimately deliver siRNA to the cytosol in a receptor-specific fashion. To better understand the structureÀactivity relationships that govern receptor-specific siRNA delivery, we employ computational regression analysis and identify a set of key convergent structural properties, namely the valence of the targeting ligand and the charge of the peptide, that help transform ubiquitously internalizing cell-penetrating peptides into cell type-specific siRNA delivery systems.
Hydrocarbon-Stapled Peptide Based-Nanoparticles for siRNA Delivery
Nanomaterials, 2020
Small interfering RNAs (siRNAs) are promising molecules for developing new therapies based on gene silencing; however, their delivery into cells remains an issue. In this study, we took advantage of stapled peptide technology that has emerged as a valuable strategy to render natural peptides more structured, resistant to protease degradation and more bioavailable, to develop short carriers for siRNA delivery. From the pool of stapled peptides that we have designed and synthesized, we identified non-toxic vectors that were able to efficiently encapsulate siRNA, transport them into the cell and induce gene silencing. Remarkably, the most efficient stapled peptide (JMV6582), is composed of only eight amino-acids and contains only two cationic charges.
Tf-lipoplexes for neuronal siRNA delivery: A promising system to mediate gene silencing in the CNS
Journal of Controlled Release, 2008
Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both in vitro and in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tflipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both in vitro an in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.
Receptor-targeted liposome-peptide nanocomplexes for siRNA delivery
Biomaterials, 2011
a b s t r a c t RNA interference induced by double-stranded, small interfering RNA (siRNA) molecules has attracted great attention as a genetic therapeutic approach. Despite major advances in this field, new nanoparticle formulations are required for in vivo delivery of siRNA, particularly for tissue-specific delivery of siRNA reagents. We have developed and optimized LYR nanocomplex formulations for siRNA delivery that consist of a liposome (DOTMA/DOPE; L) and a targeting peptide (K 16 GACYGLPHKFCG; Y) which selfassemble on mixing at optimal ratios with siRNA (R). Biophysical measurements indicated that LYR nanocomplexes were strongly cationic, mainly spherical particles of less than 100 nm. These formulations packaged and protected siRNA on incubation with RNAseA with >90% intact siRNA recovery. In addition, intact siRNA was recovered from LYRs upon heparin treatment. A critical synergy was observed between the lipid and peptide components for LYR particle stability and transfection efficiency. To evaluate targeting, transfections were compared with non-targeted formulations containing K 16 with no targeting ligand. Gene knockdown efficiencies with targeted formulations were more than two-fold better in all cell lines tested (p < 0.01). LYR formulations with liposomes containing DOTMA, which has an 18-carbon (C18) alkyl tail, were significantly better in silencing than formulations containing cationic lipids with shorter alkyl tails. LYRs with siRNA against endogenous luciferase and GAPDH were successful in silencing these genes in 3 cell lines (1HAEo-human airway epithelial, B104 rat neuroblastoma, Neuro2A-Luc mouse neuroblastoma) in vitro with 80% efficiency, similar in efficiency to Lipofectamine 2000. Confocal microscopy analysis with LYRs containing fluorescently labelled siRNA (Cy3) showed that the siRNA was located in the perinuclear region of the cytoplasm, where the RNA-induced silencing complex (RISC) is likely to be found. The LYR formulations may have applications for the further development of siRNA-based therapeutics.