A high-throughput screening platform to identify nanocarriers for efficient delivery of RNA-based therapies.

RNA-based therapies are highly selective and powerful regulators of biological functions. Non-viral 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.

A Light-Triggerable Nanoparticle Library for the Controlled Release of Non-Coding RNAs.

RNA-based therapies offer a wide 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. Herein, we report the synthesis of a triggerable polymeric nanoparticle (NP) library composed of 160 formulations, presenting physico-chemical diversity and differential responsiveness to light. Six formulations were more efficient (up to 500 %) than commercially available lipofectamine in gene-knockdown activity. These formulations showed differential internalization by skin cells and the endosomal escape was rapid (minutes range). 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 scrambled miRNA. Light-activatable NPs offer a new strategy to topically deliver non-coding RNAs.

Induced pluripotent stem cell-derived vascular networks to screen nano-bio interactions.

The vascular bioactivity/safety of nanomaterials is typically evaluated by animal testing, which is of low throughput and does not account for biological differences between animals and humans such as ageing, metabolism and disease profiles. The development of personalized human in vitro platforms to evaluate the interaction of nanomaterials with the vascular system would be important for both therapeutic and regenerative medicine. A library of 30 nanoparticle (NP) formulations, in use in imaging, antimicrobial and pharmaceutical applications, was evaluated in a reporter zebrafish model of vasculogenesis and then tested in personalized humanized models composed of human-induced pluripotent stem cell (hiPSC)-derived endothelial cells (ECs) with "young" and "aged" phenotypes in 3 vascular network formats: 2D (in polystyrene dish), 3D (in Matrigel) and in a blood vessel on a chip. As a proof of concept, vascular toxicity was used as the main readout. The results show that the toxicity profile of NPs to hiPSC-ECs was dependent on the "age" of the endothelial cells and vascular network format. hiPSC-ECs were less susceptible to the cytotoxicity effect of NPs when cultured in flow than in static conditions, the protective effect being mediated, at least in part, by glycocalyx. Overall, the results presented here highlight the relevance of in vitro hiPSC-derived vascular systems to screen vascular nanomaterial interactions.

A light-triggerable formulation to control the stability of pro-angiogenic transcription factor hypoxia inducible factor-1α (HIF-1α).

The control of vascular remodeling mediated by transcription factor HIF-1α is critical in the treatment of several diseases including cancer, retinopathies, chronic wounds, and ischemic heart disease, among others. Gene silencing using a small interfering RNA (siRNA) is a promising therapeutic strategy to regulate HIF-1α; however, the delivery systems developed so far have limited endothelial targeting and efficiency. Herein, we have synthesized a light-triggerable polymeric nanoparticle (NP) library composed of 110 formulations which showed variable morphology, charge and disassembly rates after UV exposure. More than 35% of the formulations of the library were more efficient in gene knockdown than the siRNA delivered by a commercial transfection agent (lipofectamine RNAiMAX). The most efficient siRNA delivery formulations were tested against different cell types to identify one with preferential targeting to endothelial cells. Using a two-step methodology, we have identified a formulation that shows exquisite targeting to endothelial cells and is able to deliver more efficiently the siRNA that modulates HIF-1α than commercial transfection agents. Overall, the strategy reported here increases the specificity for tissue regulation and the efficiency for the intracellular delivery of siRNAs.

Antimicrobial peptide-gold nanoscale therapeutic formulation with high skin regenerative potential.

Chronic skin wounds affect ≈3% of persons aged >60years (Davies et al., 2007) [1]. These wounds are typically difficult to heal by conventional therapies and in many cases they get infected making even harder the regeneration process. The antimicrobial peptide (AMP) LL37 combines antimicrobial with pro-regenerative properties and thus represents a promising topical therapy to address both problems. Here, we investigated the wound healing potential of soluble and immobilized LL37 (LL37-conjugated gold nanoparticles, LL37-Au NPs), both in vitro (migration of keratinocytes) and in vivo (skin wound healing). Our results show that LL37-Au NPs, but not LL37 peptide, have the capacity to prolong the phosphorylation of EGFR and ERK1/2 and enhance the migratory properties of keratinocytes in a large in vitro wound model. We further report that both LL37 and LL37-Au NPs promote keratinocyte migration by the transactivation of EGFR, a process that seems to be initiated at the P2X7 receptor, as confirmed by chemical and genetic inhibition studies. Finally, we show in vivo that LL37-Au NPs have higher wound healing activity than LL37 peptide in a splinted mouse full thickness excisional model. Animal wounds treated by LL37-Au NPs have higher expression of collagen, IL6 and VEGF than the ones treated with LL37 peptide or NPs without LL37. Altogether, the conjugation of AMPs to NPs offers a promising platform to enhance their pro-regenerative properties.