RNA-based therapies offer aw ide range of therapeutic interventions including the treatment of skin diseases; however,the strategies to efficiently deliver these biomolecules are still limited due to obstacles related to the cellular uptake and cytoplasmic delivery.H erein, we report the synthesis of atriggerable polymeric nanoparticle (NP) library composed of 160 formulations,p resenting physico-chemical diversity and differential responsiveness to light. Six formulations were more efficient (up to 500 %) than commercially available lipofectamine in gene-knockdownactivity.These formulations showed differential internalization by skin cells and the endosomal escape was rapid (minutes range). The NPs were effective in the release of siRNAa nd miRNA. Acute skin wounds treated with the top hit NP complexed with miRNA-150-5p healed faster than wounds treated with scrambled miRNA. Lightactivatable NPs offer an ew strategy to topically deliver noncoding RNAs.
Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).
Modulation of endogenous adult stem cell niches represents a promising strategy for regeneration of tissues and to correct cell abnormalities, including cancer. Recent advances show the possibility to target endogenous stem cells or their progenies by using nanoparticles conjugated with specific biomolecules. In addition, the targeting of the stem cell niche can be accomplished by using stem cells loaded with nanoparticles. This review examines principles for the targeting of endogenous stem cells as well as factors for the modulation of stem cells.
RNA-based therapies are highly selective and powerful regulators of biological functions. Nonviral vectors such as nanoparticles (NPs) are very promising formulations for the delivery of RNA-based therapies but their cell targeting, cell internalization and endolysomal escape capacity is rather limited. Here, we present a methodology that combines high-throughput synthesis of light-triggerable NPs and a high-content imaging screening to identify NPs capable of efficiently delivering different type of RNAs. The NPs were generated using polymers synthesized by Michael type addition reactions and they were designed to: (i) efficiently complex coding (mRNAs) and non-coding (miRNAs and/or lncRNAs) RNA molecules, (ii) allow rapid cell uptake and cytoplasmic release of RNA molecules and (iii) target different cell types based on their composition. Furthermore, light-responsive domains were attached to the polymers by distinctive methods to provide diverse disassembly strategies.The most efficient formulations were identified using cell-based assays and high-content imaging analysis. This strategy allows precise delivery of RNA-based therapies and provides an effective design approach to address critical issues in non-viral gene delivery.
Messenger RNA (mRNA)-based therapies offer enhanced control over the production of therapeutic proteins for many diseases. Their clinical implementation warrants formulations capable of delivering them safely and effectively to target sites. Owing to their chemical versatility, polymeric nanoparticles can be designed by combinatorial synthesis of different ionizable, cationic, and aromatic moieties to modulate cell targeting, using inexpensive formulation steps. Herein, 152 formulations are evaluated by high-throughput screening using a reporter fibroblast model sensitive to functional delivery of mRNA encoding Cre recombinase. Using in vitro and in vivo models, a polymeric nanoformulation based on the combination of 3 specific monomers is identified to transfect fibroblasts much more effectively than other cell types populating the skin, with superior performance than lipid-based transfection agents in the delivery of Cas9 mRNA and guide RNA. This tropism can be explained by receptor-mediated endocytosis, involving CD26 and FAP, which are overexpressed in profibrotic fibroblasts. Structure-activity analysis reveals that efficient mRNA delivery required the combination of high buffering capacity and low mRNA binding affinity for rapid release upon endosomal escape. These results highlight the use of high-throughput screening to rapidly identify chemical features towards the design of highly efficient mRNA delivery systems targeting fibrotic diseases.
RNA-based therapies offer a wide range of therapeutic interventions including for the treatment of skin diseases; however, the strategies to deliver efficiently these biomolecules are still limited due to obstacles related to the cellular uptake and cytoplasmic delivery. Herein, we synthesized a triggerable polymeric nanoparticle (NP) library composed by 160 formulations, presenting physico-chemical diversity and differential responsiveness to light. Six formulations were more efficient (up to 500%) than commercial Lipofectamine in gene knockdown activity. These formulations had differential internalization by skin cells and the endosomal escape was rapid (minutes range) as shown by the recruitment of galectin-8. The NPs were effective in the release of siRNA and miRNA. Acute skin wounds treated with the top hit NP complexed with miRNA-150-5p healed faster than wounds treated with scramble miRNA. Light-activatable NPs offer a new strategy to deliver topically non-coding RNAs. The capacity to regulate intracellular gene expression with RNA-based therapeutics such as small interfering RNAs (siRNAs) or miRNAs has enormous potential for the treatment of many diseases [1]. Unfortunately, the intracellular delivery of RNA-based therapeutics is difficult because of their susceptibility to enzymatic degradation and low capacity to cross cell membrane without a vector/carrier. Several delivery
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