Injectable “nano-micron” combined gene-hydrogel microspheres for local treatment of osteoarthritis

Sustained and controllable local gene therapy is a potential method for treating osteoarthritis (OA) through the delivery of therapeutic microRNAs (miRNAs) to targeted cells. However, direct injection of crude miRNAs for local gene therapy is limited due to its inadequate transfection efficiency, easy inactivation, and short half-life. Here, a multifunctional gene vector, arginine, histidine, and phenylalanine-modified generation 5 polyamidoamine (named G5-AHP), was employed to form G5-AHP/miR-140 nanoparticles by forming a complex with microRNA-140 (miR-140). Then, the nanoparticles were entrapped in hydrogel microspheres (MSs) to construct a “nano-micron” combined gene hydrogel to alleviate the degradation of articular cartilage. Monodisperse gelatin methacryloyl hydrogel MSs were produced under ultraviolet light using one-step innovative microfluidic technology. Evenly dispersed MSs showed better injectability in sustainable and matrix metalloproteinases (MMPs)-responsive degradation methods for local gene delivery. The G5-AHP/miR-140 nanoparticles released from the MSs exhibited high gene transfection efficacy and long-term bioactivity, facilitated endocytosis, and thus maintained the metabolic balance of cartilage matrix by promoting the expression of type II collagen and inhibiting the expression of a disintegrin and metalloproteinase with thrombospondin motifs-5 and MMP13 in chondrocytes. After injection of the “nano-micron” combined gene hydrogel into the articular cavity of the OA model, the gene hydrogel increased G5-AHP/miR-140 nanoparticle retention, prevented articular cartilage degeneration, and reduced osteophyte formation in a surgically induced mouse model of OA. The present study provides a novel cell-free approach to alleviate the progression of OA that shows potential for locally injected gene delivery systems.

Hyaluronic acid hydrogel encapsulated BMP-14-modified ADSCs accelerate cartilage defect repair in rabbits

Background Cartilage defect has a limited capacity to heal. In this context, we hypothesized that hyaluronic acid (HA) hydrogel encapsulated BMP-14-modified adipose-derived mesenchymal stem cells (ADSCs) could accelerate cartilage defect repair in rabbits. Methods ADSCs were isolated and identified by flow cytometry. ADSCs were treated with adenovirus vector encoding BMP-14 (Ad-BMP-14) or adenovirus vector encoding control (Ad-ctrl). Real-time PCR (RT-PCR) and western blot assay was performed to verify the transfection efficacy and chondrogenic differentiation markers (ACAN, Collagen II and SOX9). Rabbit cartilage defect model was performed and randomly divided into following groups: control group, HA hydrogel + ADSCs, ADSCs, HA hydrogel + BMP-14 transfected ADSCs, HA hydrogel + BMP-14 transfected ADSCs. At 6, 9 and 12 weeks after surgery, scanning electron microscopy, hematoxylin–eosin, Safranin-O/Fast Green and immunohistochemical staining for Collagen II were performed to determine the role of HA hydrogel encapsulated BMP-14-modified ADSCs in cartilage repair in vivo. Results ADSCs were successfully isolated and positively expressed CD29, CD44 and CD90. Transfection efficacy of Ad-BMP-14 was verified by RT-PCR and western blot assay. Moreover, Ad-BMP-14 could significantly increased chondrogenic differentiation markers (ACAN, Collagen II and SOX9). The LV-BMP-14-ADSCs and HA hydrogel + LV-BMP-14-ADSCs groups revealed smoother surface cartilage repair that was level with the surrounding cartilage and almost complete border integration. Conclusions HA hydrogel encapsulated BMP-14-modified ADSCs accelerate cartilage defect repair in rabbits. We need to further validate the specific mechanism of action of HA hydrogel encapsulated LV-BMP-14-ADSCs involved in the repairing cartilage damage in vivo.

mRNA-Based Nanomedicinal Products to Address Corneal Inflammation by Interleukin-10 Supplementation

The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated in vitro in human corneal epithelial (HCE-2) cells. Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.

Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy

Cell-penetrating peptides (CPPs), as non-viral gene delivery vectors, are considered with lower immunogenic response, and safer and higher gene capacity than viral systems. In our previous study, a CPP peptide called RALA (arginine rich) presented desirable transfection efficacy and owns a potential clinic use. It is believed that histidine could enhance the endosome escaping ability of CPPs, yet RALA peptide contains only one histidine in each chain. In order to develop novel superior CPPs, by using RALA as a model, we designed a series of peptides named HALA (increased histidine ratio). Both plasmid DNA (pDNA) and siRNA transfection results on three cell lines revealed that the transfection efficacy is better when histidine replacements were on the C-terminal instead of on the N-terminal, and two histidine replacements are superior to three. By investigating the mechanism of endocytosis of the pDNA nanocomplexes, we discovered that there were multiple pathways that led to the process and caveolae played the main role. During the screening, we discovered a novel peptide-HALA2 of high cellular transfection efficacy, which may act as an exciting gene delivery vector for gene therapy. Our findings also bring new insights on the development of novel robust CPPs.

Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2–3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

OTME-18. Targeted CRISPR/Cas9 gene-editing regulates the brain tumor environment

Glioblastoma multiform (GBM) is the most common malignant brain tumor. Recent immunotherapy has demonstrated potential to treat GBM. However, the immune suppressive tumor environment in the brain represents a significant barrier for the treatment of GBM. Overexpression of programmed death ligand-1 (PD-L1) in GBM tumor cells and macrophages plays a key role in GBM vitality, proliferation, and migration, while also suppressing the immune system. We developed a CRISPR/Cas9 gene-editing system to delete whole cell PD-L1. Human PD-L1 targeted sgRNA were cloned into CRISPR/Cas9 plasmids with or without an HDR templet. CRISPR/Cas9 were treated to human GBM U87 cells for 15, 30, 60, 120 and 240 minutes. The intracellular concentration of CRISPR/Cas9 exhibited a time-dependent increases. A GFP tagged CRISPR/Cas9 plasmid was developed to test the transfection efficacy. Higher levels of GFP+ U87 cells were observed at day 3. CRISPR/Cas9 showed a greater PD-L1 knockout at day 3. The PD-L1 reduction limited the proliferation of U87 cells. A scratch assay showed that PD-L1 deletion inhibited the migration of U87 cells. An in vitro GBM model was developed by co-cultivation of U87 cells and macrophages. CRISPR/Cas9 treated co-cultures changed the ratios of U87 cells and macrophages and polarized tumor associated macrophages (TAM) from M2 toward M1. CRISPR/Cas9 gene-editing effectively deleted PD-L1 in U87 cells. Successful deletion of PD-L1 prevented U87 cells growth and migration, and altered the TAMs plasticity and the tumor environment.

Degradable branched polycationic systems are promising gene therapy vectors

Degradable branched polycationic systems have exhibited exceptional potential for nucleic acid delivery due to their good responsive degradability and exceptional transfection efficacy. We focused on current work on the development of ARP for nucleic acid delivery in this study. The results of numerous responsive degradable branching polycationic systems were assembled. For the synthesis of ARP, the typical amino-epoxy ring-opening procedure was primarily presented. For nucleic acid delivery, the properties of redox- and pH-responsive degradable branching systems were addressed. Despite great development, there are still a lot of barriers to overcome in this discipline. The step growth strategy is more convenient than the chain growth technique. However, the polydispersity index (PDI) of most branched polymers generated by the step growth process is relatively high, making biological investigations hard. As a result, regulating the batch-to-batch quality of branched polycations is important for the translation of ARP from laboratories to clinics. Furthermore, as diseases develop more complicated and treatment processes get more diversified, single-function nucleic acid carriers are increasingly unable to satisfy complicated needs. Nucleic acid carriers must be manufactured in a precise and multifunctional method. In nucleic acid-based therapy, branched polycations must have degradability, targeting capability, and multimodal therapeutic properties in addition to degradability. In the future, one problem will be how to develop degradable, targeted, and multifunctional branching polycations for nucleic acid delivery in a regulated manner. Degradable branching polycationic systems will have a substantial effect on nucleic acid delivery and will become key instruments for nucleic acid treatment as research improves and allied areas such as materials science and medicine improve quickly.

Effects of lyoprotectants on long-term stability and transfection efficacy of lyophilized poly(lactide-co-glycolide)-graft-polyethylenimine/plasmid DNA polyplexes

Aim: We investigated the effect of lyoprotectants on the long-term stability and transfection efficiency of lyophilized (Lyo.) polyplexes prepared from poly(lactide-co-glycolide)-graft-polyethylenimine (PgP) and plasmid DNA encoding green fluorescent protein (pGFP). Materials & methods: Lyo. PgP/pGFP polyplexes prepared with/without lyoprotectants were stored at -20°C over 6 months. Polyplex stability was analyzed by gel electrophoresis and heparin competition assay. Transfection efficiency and cytotoxicity were evaluated in rat glioma (C6) cells in medium containing 10% serum. Results: Lyo. PgP/pGFP polyplexes prepared with 5% sucrose as a lyoprotectant remained stable up to 6 months and retained transfection efficiency up to 4 months. Conclusion: Lyo. PgP-based polyplexes retain bioactivity during extended storage, potentially enabling transport to remote regions and less stable settings, increasing access to life-changing gene therapy.