Abstract Lipid‐based nanocarriers have demonstrated high interest in delivering genetic material, exemplified by the success of Onpattro and COVID‐19 vaccines. While PEGylation imparts stealth properties, it hampers cellular uptake and endosomal escape, and may trigger adverse reactions like accelerated blood clearance (ABC) and hypersensitivity reactions (HSR). This work highlights the great potential of amphiphilic poly(N‐methyl‐N‐vinylacetamide) (PNMVA) derivatives as alternatives to lipid‐PEG for siRNA delivery. PNMVA compounds with different degrees of polymerization and hydrophobic segments, are synthesized. Among them, DSPE (1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine)‐PNMVA efficiently integrates into lipoplexes and LNP membranes and prevents protein corona formation around these lipid carriers, exhibiting stealth properties comparable to DSPE‐PEG. However, unlike DSPE‐PEG, DSPE‐PNMVA 24 shows no adverse impact on lipoplexes cell uptake and endosomal escape. In in vivo study with mice, DSPE‐PNMVA 24 lipoplexes demonstrate no liver accumulation, indicating good stealth properties, extended circulation time after a second dose, reduced immunological reaction, and no systemic pro‐inflammatory response. Safety of DSPE‐PNMVA 24 is confirmed at the cellular level and in animal models of zebrafish and mice. Overall, DSPE‐PNMVA is an advantageous substitute to DSPE‐PEG for siRNA delivery, offering comparable stealth and toxicity properties while improving efficacy of the lipid‐based carriers by minimizing the dilemma effect and reducing immunological reactions, meaning no ABC or HSR effects.
Hypoxia‐inducible factor‐1 (HIF‐1) is a transcription factor activated by hypoxia. The HIF‐1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF‐1 activation. We demonstrated that in human microvascular endothelial cells‐1 (HMEC‐1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia‐induced HIF‐1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF‐1α is phosphorylated in hypoxia by an ERK‐dependent pathway. These results evidence the role of mitogen‐activated protein kinase in the transcriptional response to hypoxia.
The growth and spread of cancerous tumors is possible through angiogenesis: the formation of new blood vessels from the pre-existing vasculature. These new capillaries allow a supply of oxygen and nutrients necessary for the tumor development. They also allow the spread of the tumor in the body and the formation of metastases. A family of enzymes, named histones deacetylases (HDAC), play key role in angiogenesis. Among the different HDAC members the specific inhibition of HDAC7 disturbs the angiogenic process, making it an attractive target for an anti-angiogenic therapy (1). To specifically knockdown HDAC7 expression, siRNA technology is used. This specific siRNA recognizes and leads to the degradation the mRNA encoding for HDAC7 protein. The delivery of siRNA into the cytoplasm of target cells to exert their effects remains a significant challenge. Novel nanoparticle-based approaches that enable more efficient delivery of siRNA sequences are constantly progressing towards the goal of meeting the challenge of delivery. Vectors used in this work are polyplexes formed by the self-assembly of biodegradable polycarbonate polymers and siRNA specifically targeted against HDAC7. To be effective in vivo , polyplexes must meet several physico-chemical characteristics. To get the best physico-chemical characteristics, many parameters can be modified like the polymer structure, the polyplexes formulation, the preparation method, ... (2). The main characteristics are the incorporation of the siRNA into the polyplexes (determined by agarose gel electrophoresis or by the Quant-iT™ RiboGreen® kit), the size (measured by dynamic light scattering), the charge (zeta potential measured by laser Doppler velocimetry) and the buffering capacity (measured by titration). The transfection capacity of polyplexes with good physicochemical characteristics has been examined in HeLa cells (determined by flow cytometry and microscopy). A western blot has been performed to assess the expression level of HDAC7 protein in treated cells compared to a control. Different architectures of biodegradable polycarbonate polymers at different ratios (N/P, polymer/siRNA) have been tested. Most of them show a complete incorporation of the siRNA at N/P above 10. At these N/P ratios, size measurements show an average diameter between 200 and 500 nm. Ideally, the diameter should be around 200 nm; not smaller to avoid renal excretion and not too big to escape the monomolecular phagocytic system. This scale also allows accumulation of nanoparticles in the tumor due to the enhanced permeability and retention (EPR) effect (3). Zeta potential is slightly positive, around +5mV. To interact with cell membranes, nanoparticles must have a slight positive charge, lower than 10 mV to avoid a too high toxicity. In vitro , flow cytometry shows a high transfection level for most selected polyplexes, up to 90% of transfected cells. Unfortunately, no decrease of the expression of HDAC7 has been observed by western blot. The two main hypotheses to explain the lack of efficacy of our new polyplexes are either a too high affinity between the polymer and siRNA that prevents the release of the siRNA in the cytoplasm or either the use of an endocytosis pathway without vesicle acidification. This acidification allows the endosome bursting using the buffering capacity of the polymer through the « proton sponge effect » (4). These two hypotheses will be further studied.
Muscle formation is controlled by a number of key myogenic transcriptional regulators that govern stage-specific gene expression programs and act as terminal effectors of intracellular signaling pathways. To date, the role of phosphatases in the signaling cascades instructing muscle development remains poorly understood. Here, we show that a specific PP2A-B55δ holoenzyme is necessary for skeletal myogenesis. The primary role of PP2A-B55δ is to dephosphorylate histone deacetylase 4 (HDAC4) following myocyte differentiation and ensure repression of Myocyte enhancer factor 2D (MEF2D)-dependent gene expression programs during myogenic fusion. As a crucial HDAC4/MEF2D target gene that governs myocyte fusion, we identify ArgBP2, an upstream inhibitor of Abl, which itself is a repressor of CrkII signaling. Consequently, cells lacking PP2A-B55δ show upregulation of ArgBP2 and hyperactivation of CrkII downstream effectors, including Rac1 and FAK, precluding cytoskeletal and membrane rearrangements associated with myoblast fusion. Both in vitro and in zebrafish, loss-of-function of PP2A-B55δ severely impairs fusion of myocytes and formation of multinucleated muscle fibers, without affecting myoblast differentiation. Taken together, our results establish PP2A-B55δ as the first protein phosphatase to be involved in myoblast fusion and suggest that reversible phosphorylation of HDAC4 may coordinate differentiation and fusion events during myogenesis.
