Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 µM) underlines potential interest of this nanoplatform for biomedical applications.

The contribution of endocytosis to sensitization of nociceptors and synaptic transmission in nociceptive circuits.

ABSTRACT: Chronic pain involves sensitization of nociceptors and synaptic transmission of painful signals in nociceptive circuits in the dorsal horn of the spinal cord. We investigated the contribution of clathrin-dependent endocytosis to sensitization of nociceptors by G protein-coupled receptors (GPCRs) and to synaptic transmission in spinal nociceptive circuits. We determined whether therapeutic targeting of endocytosis could ameliorate pain. mRNA encoding dynamin (Dnm) 1 to 3 and adaptor-associated protein kinase 1 (AAK1), which mediate clathrin-dependent endocytosis, were localized to primary sensory neurons of dorsal root ganglia of mouse and human and to spinal neurons in the dorsal horn of the mouse spinal cord by RNAScope. When injected intrathecally to mice, Dnm and AAK1 siRNA or shRNA knocked down Dnm and AAK1 mRNA in dorsal root ganglia neurons, reversed mechanical and thermal allodynia and hyperalgesia, and normalized nonevoked behavior in preclinical models of inflammatory and neuropathic pain. Intrathecally administered inhibitors of clathrin, Dnm, and AAK1 also reversed allodynia and hyperalgesia. Disruption of clathrin, Dnm, and AAK1 did not affect normal motor functions of behaviors. Patch clamp recordings of dorsal horn neurons revealed that Dnm1 and AAK1 disruption inhibited synaptic transmission between primary sensory neurons and neurons in lamina I/II of the spinal cord dorsal horn by suppressing release of synaptic vesicles from presynaptic primary afferent neurons. Patch clamp recordings from dorsal root ganglion nociceptors indicated that Dnm siRNA prevented sustained GPCR-mediated sensitization of nociceptors. By disrupting synaptic transmission in the spinal cord and blunting sensitization of nociceptors, endocytosis inhibitors offer a therapeutic approach for pain treatment.

Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy.

We report the formulation, characterization, colloidal stability, and in vitro efficiency of Fisetin nanocrystals stabilized by poloxamer P407. Such nanocrystals present a nanometer scale (148.6 ± 1.1 nm) and a high homogeneity (polydispersity index of 0.17 ± 0.01), with a production yield of 97.0 ± 2.5%. The engineered formulations of nanocrystals suspension (pH of 7.4 ± 0.1), stabilized via steric repulsion, are stable for several days in aqueous environment (Milli Q water, NaCl 10 mM or mannitol 5% w/v), for few days in HEPES buffered saline (HBS) (20 / 150 mM) under sink conditions, and in culture medium. After freeze drying in 5% w/v mannitol, the nanocrystal formulations can be stored at -80 °C for at least 120 days. Drug release experiments displayed a 98.7 ± 5.1% cumulative release over 3 days in HBS. Compared to the free drug, the nanocrystal formulations showed an improved cytotoxicity highlighted by the decrease of the half maximal inhibitory concentration for both murine Lewis lung carcinoma (3LL) and human endothelial (EA.hy926) cell lines. In addition, after incubation with Fisetin nanosuspensions, significant changes in the cell morphology for both cell lines were observed, showing an improved anti-angiogenic effect of nanocrystals formulation compared to the free drug. Overall, Fisetin formulated as nanocrystals showed enhanced biopharmaceutical properties and in vitro activity, offering a wide range of indications for challenging applications in the clinic.

Combination of thermal ablation by focused ultrasound, pFAR4-IL-12 transfection and lipidic adjuvant provide a distal immune response.

Aim: Gene-based immunotherapy against cancer is limited by low gene transfer efficiency. In the literature, interleukin-12 (IL-12) encoding plasmid associated with sonoporation has been shown to enhance antitumoral activity. Moreover, non-viral carriers and high-frequency ultrasound have both been shown to promote immune response activation. Here, IL-12 encoding plasmid, non-viral carrier stimulating the immune response and focused ultrasound were combined in order to improve the antitumoral efficiency. Methods: In order to enhance a gene-based antitumoral immune response, home-made lipids Toll-like receptor 2 (TLR2) agonists and plasmid free of antibiotic resistance version 4 (pFAR4), a mini-plasmid, encoding the IL-12 cytokine were combined with high-intensity focused ultrasound (HIFU). The lipid composition and the combination conditions were selected following in vitro and in vivo preliminary studies. The expression of IL-12 from our plasmid construct was measured in vitro and in vivo. The combination strategy was evaluated in mice bearing colon carcinoma cells (CT26) tumors following their weight, tumor volume, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) levels in the serum and produced by splenocytes exposed to CT26 tumor cells. Results: Lipid-mediated cell transfection and intratumoral injection into CT26 tumor mice using pFAR4-IL-12 led to the secretion of the IL-12 cytokine into cell supernatant and mice sera, respectively. Conditions of thermal deposition using HIFU were optimized. The plasmid encoding pFAR4-IL-12 or TLR2 agonist alone had no impact on tumor growth compared with control mice, whereas the complete treatment consisting of pFAR4-IL-12, TLR2 lipid agonist, and HIFU limited tumor growth. Moreover, only the complete treatment increased significantly mice survival and provided an abscopal effect on a metastatic CT26 model. Conclusions: The HIFU condition was highly efficient to stop tumor growth. The combined therapy was the most efficient in terms of IL-12 and IFN-γ production and mice survival. The study showed the feasibility and the limits of this combined therapy which has the potential to be improved.

Spheroplexes: Hybrid PLGA-cationic lipid nanoparticles, for in vitro and oral delivery of siRNA.

Vectorized small interfering RNAs (siRNAs) are widely used to induce gene silencing. Among the delivery systems used, lipid-based particles are the most effective. Our objective was the development of novel lipid-polymer hybrid nanoparticles, from lipoplexes (complexes of cationic lipid and siRNAs), and poly (lactic-co-glycolic acid) (PLGA), using a simple modified nanoprecipitation method. Due to their morphology, we called these hybrid nanoparticles Spheroplexes. We elucidated their structure using several physico-chemical techniques and showed that they are composed of a hydrophobic PLGA matrix, surrounded by a lipid envelope adopting a lamellar structure, in which the siRNA is complexed, and they retain surface characteristics identical to the starting nanoparticles, i.e. lipoplexes siRNA. We analyzed the composition of the particle population and determined the final percentage of spheroplexes within this population, 80 to 85% depending on the preparation conditions, using fluorescent markers and the ability of flow cytometry to detect nanometric particles (approximately 200 nm). Finally, we showed that spheroplexes are very stable particles and more efficient than siRNA lipoplexes for the delivery of siRNA to cultured cells. We administered spheroplexes contain siRNAs targeting TNF-α to mice with ulcerative colitis induced by dextran sulfate and our results indicate a disease regression effect with a response probably mediated by their uptake by macrophages / monocytes at the level of lamina propria of the colon. The efficacy of decreased level of TNF-α in vivo seemed to be an association of spheroplexes polymer-lipid composition and the specific siRNA. These results demonstrate that spheroplexes are a promising hybrid nanoparticle for the oral delivery of siRNA to the colon.

Fate of Tableted Freeze-Dried siRNA Lipoplexes in Gastrointestinal Environment.

The incorporation of siRNA into nanocarriers is mandatory to facilitate its intracellular delivery, as siRNA itself cannot enter cells. However, the incorporation of these nanocarriers into oral, solid dosage forms and their fate in the gastrointestinal environment is yet to be explored. In the present work, the fate of, (i) naked siRNA, (ii) freshly prepared siRNA lipoplexes, and (iii) tableted siRNA lipoplexes, in simulated gastric and intestinal fluids was studied. The siRNA, either released from or protected within the lipoplexes, was quantified by gel electrophoresis and siRNA efficacy was assessed in cell transfection. The freshly prepared lipoplexes kept their siRNA load and transfection efficiency totally preserved during 1 h of incubation in simulated gastric fluid at 37 °C. However, in simulated intestinal fluid, despite no release of siRNA from lipoplexes after 6 h of incubation, gene silencing efficacy was dramatically decreased even after 1 h of exposure. The lipoplexes obtained from tablets efficiently protected siRNA in simulated gastric fluid, thus preserving the gene silencing efficacy, whereas their incubation in simulated intestinal fluid resulted in a marked siRNA release and decreased gene silencing efficacy. These results provided a detailed explanation for understanding the fate of siRNA in gastrointestinal conditions, when simply loaded in lipoplexes or formulated in the form of tablets.

P-selectin targeted RAGE-shRNA lipoplexes alleviate atherosclerosis-associated inflammation.

The receptor for advanced glycation end products (RAGE) plays a central role in the chronic inflammatory process associated with atherosclerosis development. We aimed to develop lipoplexes carrying RAGE-short hairpin (sh) RNA, targeted to the adhesion molecule P-selectin, selectively expressed on the surface of activated endothelium (Psel-lipo/shRAGE) to down-regulate RAGE expression as a therapeutic strategy for atherosclerosis. In vitro, Psel-lipo/shRAGE lipoplexes were efficiently taken up by activated endothelial cells (EC), decreased the expression of RAGE protein, and proved to be functional by reducing the monocyte adhesion to activated EC. In ApoE-deficient mice, the targeted lipoplexes accumulated specifically and efficiently transfected the aorta. The repeated administration of Psel-lipo/shRAGE lipoplexes, twice per week for one month: i) reduced the expression of RAGE protein in the aorta by decreasing the expression of NF-kB and TNF-α; ii) diminished the plasma levels of TNF-α, IL6, IL-1ß, and MCP-1; iii) inhibited the atherosclerotic plaque development and iv) had no significant adverse effects. In conclusion, the newly developed Psel-lipo/shRAGE lipoplexes reduce the inflammatory processes associated with RAGE signaling and the progression of atherosclerosis in ApoE-deficient mice. Downregulation of RAGE employing these lipoplexes may represent a promising new targeted therapy to block atherosclerosis progression.

Neuroprotective Effect of siRNA Entrapped in Hyaluronic Acid-Coated Lipoplexes by Intravitreal Administration.

Since the possibility of silencing specific genes linked to retinal degeneration has become a reality with the use of small interfering RNAs (siRNAs), this technology has been widely studied to promote the treatment of several ocular diseases. Despite recent advances, the clinical success of gene silencing in the retina is significantly reduced by inherent anatomical and physiological ocular barriers, and new strategies are required to achieve intraocular therapeutic effectiveness. In this study, we developed lipoplexes, prepared with sodium alginate as an adjuvant and strategically coated with hyaluronic acid (HA-LIP), and investigated the potential neuroprotective effect of these systems in a retinal light damage model. Successful functionalization of the lipoplexes with hyaluronic acid was indicated in the dynamic light scattering and transmission electron microscopy results. Moreover, these HA-LIP nanoparticles were able to protect and deliver siRNA molecules targeting caspase-3 into the retina. After retinal degeneration induced by high light exposure, in vitro and in vivo quantitative reverse transcription-PCR (RT-qPCR) assays demonstrated significant inhibition of caspase-3 expression by HA-LIP. Furthermore, these systems were shown to be safe, as no evidence of retinal toxicity was observed by electroretinography, clinical evaluation or histology.

Compression of Vectors for Small Interfering RNAs Delivery: Toward Oral Administration of siRNA Lipoplexes in Tablet Forms.

Currently, most nonviral nucleic acid vectors are in the form of colloidal suspensions administered primarily parenterally. This type of formulation and the mode of administration impose strong constraints such as the size of the administered vectors or the production of sterile preparations. The tablet form provides access to easy oral administration, well accepted by patients; As regards nucleic acid vectors, a dry form represents an advance in terms of stability. Using an optimized lipid-based small interfering RNA-delivery system, we studied the tabletability of a liquid suspension of these vectors. We optimized the conditions of freeze-drying by choosing excipients and process, allowing for the conservation of both the gene-silencing efficacy of the formulated siRNAs and the supramolecular structure of the lipid particulate system. Gene-silencing efficacy was assayed on luciferase-expressing cells and the structure of the siRNA vector in freeze-dried and tablet forms was examined using small-angle X-ray scattering (SAXS) synchrotron radiation. The freeze-dried powders were then mixed with excipients necessary for the good progress of the compression by allowing for a regular supply of the matrix and the reduction of friction. The compression was carried out using a rotary press simulator that allows for complete monitoring of the compression conditions. After compression, formulated siRNAs retained more than 60% of their gene-silencing efficacy. Within the tablets, a specific SAXS signal was detectable and the lamellar and cubic phases of the initial liquid suspension were restored after resuspension of siRNA vectors by disintegration of the tablets. These results show that the bilayer lipid structures of the particles were preserved despite the mechanical constraints imposed by the compression. If such a result could be expected after the freeze-drying step, it was never shown, to our knowledge, that siRNA-delivery systems could retain their efficacy and structure after mechanical stress such as compression. This opens promising perspectives to oral administration of siRNA as an alternative to parenteral administration.

Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis.

Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a "quiescent" to an "activated" phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

Targeting endothelial thioredoxin-interacting protein (TXNIP) protects from metabolic disorder-related impairment of vascular function and post-ischemic revascularisation.

INTRODUCTION: Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biological functions, the contribution of endothelial TXNIP has not been well-defined in regards to endothelial and vascular function or in post-ischemic revascularisation. We postulated that inhibition of endothelial TXNIP with siRNA or in a Cre-LoxP system could be involved in protection from high fat, high protein, low carbohydrate (HFHPLC) diet-induced oxidative stress and endothelial dysfunction, leading to vascular damage and impaired revascularisation in vivo. METHODS AND RESULTS: To investigate the role of endothelial TXNIP, the TXNIP gene was deleted in endothelial cells using anti-TXNIP siRNA treatment or the Cre-LoxP system. Murine models were fed a HFHPLC diet, known to induce metabolic disorders. Endothelial TXNIP targeting resulted in protection against metabolic disorder-related endothelial oxidative stress and endothelial dysfunction. This protective effect mitigates media cell loss induced by metabolic disorders and hampered metabolic disorder-related vascular dysfunction assessed by aortic reactivity and distensibility. In aortic ring cultures, metabolic disorders impaired vessel sprouting and this alteration was alleviated by deletion of endothelial TXNIP. When subjected to ischemia, mice fed a HFHPLC diet exhibited defective post-ischemic angiogenesis and impaired blood flow recovery in hind limb ischemia. However, reducing endothelial TXNIP rescued metabolic disorder-related impairment of ischemia-induced revascularisation. CONCLUSION: Collectively, these results show that targeting endothelial TXNIP in metabolic disorders is essential to maintaining endothelial function, vascular function and improving ischemia-induced revascularisation, making TXNIP a potential therapeutic target for therapy of vascular complications related to metabolic disorders.

Osteoblasts mineralization and collagen matrix are conserved upon specific Col1a2 silencing.

Classical osteogenesis imperfecta (OI) is an inherited rare brittle bone disease caused by dominant mutations in the COL1A1 or COL1A2 genes, encoding for the α chains of collagen type I. The definitive cure for the disease will require a gene therapy approach, aimed to correct or suppress the mutant allele. Interestingly, individuals lacking α2(I) chain and synthetizing collagen α1(I)3 homotrimers do not show bone phenotype, making appealing a bone specific COL1A2 silencing approach for OI therapy. To this aim, three different Col1a2-silencing RNAs (siRNAs), -3554, -3825 and -4125, selected at the 3'-end of the murine Col1a2 transcript were tested in vitro and in vivo. In murine embryonic fibroblasts Col1a2-siRNA-3554 was able to efficiently and specifically target the Col1a2 mRNA and to strongly reduce α2(I) chain expression. Its efficiency and specificity were also demonstrated in primary murine osteoblasts, whose mineralization was preserved. The efficiency of Col1a2-siRNA-3554 was proved also in vivo. Biphasic calcium phosphate implants loaded with murine mesenchymal stem cells were intramuscularly transplanted in nude mice and injected with Col1a2-siRNA-3554 three times a week for three weeks. Collagen α2 silencing was demonstrated both at mRNA and protein level and Masson's Trichrome staining confirmed the presence of newly formed collagen matrix. Our data pave the way for further investigation of Col1a2 silencing and siRNA delivery to the bone tissue as a possible strategy for OI therapy.

Targeted Transfection Using PEGylated Cationic Liposomes Directed Towards P-Selectin Increases siRNA Delivery into Activated Endothelial Cells.

: The progress in small-interfering RNA (siRNA) therapeutics depends on the development of suitable nanocarriers to perform specific and effective delivery to dysfunctional cells. In this paper, we questioned whether P-selectin, a cell adhesion molecule specifically expressed on the surface of activated endothelial cells (EC) could be employed as a target for nanotherapeutic intervention. To this purpose, we developed and characterized P-selectin targeted PEGylated cationic liposomes able to efficiently pack siRNA and to function as efficient vectors for siRNA delivery to tumour necrosis factor-α (TNF-α) activated EC. Targeted cationic liposomes were obtained by coupling a peptide with high affinity for P-selectin to a functionalized PEGylated phospholipid inserted in the liposomes' bilayer (Psel-lipo). As control, scrambled peptide coupled cationic liposomes (Scr-lipo) were used. The lipoplexes obtained by complexation of Psel-lipo with siRNA (Psel-lipo/siRNA) were taken up specifically and at a higher extent by TNF-α activated b.End3 endothelial cells as compared to non-targeted Scr-lipo/siRNA. The Psel-lipo/siRNA delivered with high efficiency siRNA into the cells. The lipoplexes were functional as demonstrated by the down-regulation of the selected gene (GAPDH). The results demonstrate an effective targeted delivery of siRNA into cultured activated endothelial cells using P-selectin directed PEGylated cationic liposomes, which subsequently knock-down the desired gene.

Modifying internal organization and surface morphology of siRNA lipoplexes by sodium alginate addition for efficient siRNA delivery.

Vectorized small interfering RNAs (siRNAs) are widely used to induce specific mRNA degradation in the intracellular compartment of eukaryotic cells. Recently, we developed efficient cationic lipid-based siRNA vectors (siRNA lipoplexes or siLex) containing sodium alginate (Nalg-siLex) with superior efficiency and stability properties than siLex. In this study, we assessed the physicochemical and some biological properties of Nalg-siLex compared to siLex. While no significant differences in size, ζ potential and siRNA compaction were detected, the addition of sodium alginate modified the particle morphology, producing smoother and heterogeneous particles characterized by transmission electron microscopy. We also noted that Nalg-siLex have surface differences observed by X-ray photoelectron spectroscopy. These differences could arise from an internal reorganization of components induced by the addition of sodium alginate, that is indicated by Small-Angle X-ray Scattering results. Moreover, Nalg-siLex did not trigger significant hepatotoxicity nor inflammatory cytokine secretion compared to siLex. Taken together these results suggest that sodium alginate played a key role by structuring and reinforcing siRNA lipoplexes, leading to more stable and efficient delivery vector.

Cationic lipids as one-component vaccine adjuvants: A promising alternative to alum.

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1ß secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants.

Liposomes as Gene Delivery Vectors for Human Placental Cells.

Nanomedicine as a therapeutic approach for pregnancy-related diseases could offer improved treatments for the mother while avoiding side effects for the fetus. In this study, we evaluated the potential of liposomes as carriers for small interfering RNAs to placental cells. Three neutral formulations carrying rhodamine-labelled siRNAs were evaluated on an in vitro model, i.e., human primary villous cytotrophoblasts. siRNA internalization rate from lipoplexes were compared to the one in the presence of the lipofectamine reagent and assessed by confocal microscopy. Results showed cellular internalization of nucleic acid with all three formulations, based on two cationic lipids, either DMAPAP or CSL-3. Moreover, incubation with DMAPAP+AA provided a rate of labelled cells as high as with lipofectamine (53 ± 15% and 44 ± 12%, respectively) while being more biocompatible. The proportion of cells which internalized siRNA were similar when using DMAPAP/DDSTU (16 ± 5%) and CSL-3 (22 ± 5%). This work highlights that liposomes could be a promising approach for gene therapy dedicated to pregnant patients.

Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIPfl/fl cdh5cre). Control (TXNIPfl/fl) and TXNIPfl/fl cdh5cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIPfl/fl and TXNIPfl/fl cdh5cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIPfl/fl cdh5cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1ß. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Delivery of miR-146a to Ly6Chigh Monocytes Inhibits Pathogenic Bone Erosion in Inflammatory Arthritis.

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.

Neurokinin 1 receptor signaling in endosomes mediates sustained nociception and is a viable therapeutic target for prolonged pain relief.

Typically considered to be cell surface sensors of extracellular signals, heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) control many pathophysiological processes and are the target of 30% of therapeutic drugs. Activated receptors redistribute to endosomes, but researchers have yet to explore whether endosomal receptors generate signals that control complex processes in vivo and are viable therapeutic targets. We report that the substance P (SP) neurokinin 1 receptor (NK1R) signals from endosomes to induce sustained excitation of spinal neurons and pain transmission and that specific antagonism of the NK1R in endosomes with membrane-anchored drug conjugates provides more effective and sustained pain relief than conventional plasma membrane-targeted antagonists. Pharmacological and genetic disruption of clathrin, dynamin, and ß-arrestin blocked SP-induced NK1R endocytosis and prevented SP-stimulated activation of cytosolic protein kinase C and nuclear extracellular signal-regulated kinase, as well as transcription. Endocytosis inhibitors prevented sustained SP-induced excitation of neurons in spinal cord slices in vitro and attenuated nociception in vivo. When conjugated to cholestanol to promote endosomal targeting, NK1R antagonists selectively inhibited endosomal signaling and sustained neuronal excitation. Cholestanol conjugation amplified and prolonged the antinociceptive actions of NK1R antagonists. These results reveal a critical role for endosomal signaling of the NK1R in the complex pathophysiology of pain and demonstrate the use of endosomally targeted GPCR antagonists.

Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers.

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.