Engineered mRNA Delivery Systems for Biomedical Applications.

Messenger RNA (mRNA)-based therapeutic strategies have shown remarkable promise in preventing and treating a staggering range of diseases. Optimizing the structure and delivery system of engineered mRNA has greatly improved its stability, immunogenicity, and protein expression levels, which has led to a wider range of uses for mRNA therapeutics. Herein, a thorough analysis of the optimization strategies used in the structure of mRNA is first provided and delivery systems are described in great detail. Furthermore, the latest advancements in biomedical engineering for mRNA technology, including its applications in combatting infectious diseases, treating cancer, providing protein replacement therapy, conducting gene editing, and more, are summarized. Lastly, a perspective on forthcoming challenges and prospects concerning the advancement of mRNA therapeutics is offered. Despite these challenges, mRNA-based therapeutics remain promising, with the potential to revolutionize disease treatment and contribute to significant advancements in the biomedical field.

CXCR4-Expressing Mesenchymal Stem Cells Derived Nanovesicles for Rheumatoid Arthritis Treatment.

Cell membrane camouflage technology, which a demonstrated value for the bionic replication of natural cell membrane properties, is an active area of ongoing research readily applicable to nanomedicine. How to realize immune evasion, slow down the clearance from the body, and improve targeting are still worth great efforts for this technology. Herein, novel cell membrane-mimicked nanovesicles from genetically engineered mesenchymal stem cells (MSCs) are presented as a potential anti-inflammatory platform for rheumatoid arthritis (RA) management. Utilizing the synthetic biology approach, the biomimetic nanoparticles are constructed by fusing C-X-C motif chemokine receptor4 (CXCR4)-anchored MSC membranes onto drug-loaded polymeric cores (MCPNs), which make them ideal decoys of stromal cell-derived factor-1 (SDF-1)-targeted arthritis. These resulting nanocomplexes function to escape from the immune system and enhance accumulation in the established inflamed joints via the CXCR4/SDF-1 chemotactic signal axis, thereby achieving an affinity to activated macrophages and synovial fibroblasts. It is further demonstrated that the MCPNs can significantly suppress synovial inflammation and relieve pathological conditions with favorable safety properties in collagen-induced arthritis mice. These findings indicate the clinical value of MCPNs as biomimetic nanodrugs for RA therapy and related diseases.

A Biomimetic Cardiac Fibrosis-on-a-Chip as a Visible Disease Model for Evaluating Mesenchymal Stem Cell-Derived Exosome Therapy.

Cardiac fibrosis acts as a serious worldwide health issue due to its prevalence in numerous forms of cardiac disease and its essential link to cardiac failure. Considering the efficiency of stem cell therapy for cardiac fibrosis, great efforts have been dedicated to developing accurate models for investigating their underlying therapeutic mechanisms. Herein we present an elaborate biomimetic cardiac fibrosis-on-a-chip based on Janus structural color film (SCF) to provide microphysiological visuals for stem cell therapeutic studies. By coculturing cardiomyocytes (CMs) and cardiac fibroblasts (FBs) on Janus SCF with fibrosis induction, the chip can recreate physiological intercellular crosstalk within the fibrotic microenvironment, elucidating the physiological alterations of fibrotic hearts. In particular, the Janus structural color film possesses superior perceptual capabilities for capturing and responding to a weak cardiac force, demonstrating synchronized structural color shifts. Based on these features, we have not only explored the dynamic relationship between color mapping and the evaluated disease phenotype but also demonstrated the self-reporting capacity of the cardiac fibrosis-on-a-chip for the assessment of mesenchymal stem cell-derived exosome therapy. These features suggest that such a chip can potentially facilitate the evolution of precision medicine strategies and create a protocol for preclinical cardiac drug screening.

Biomimetic liposomes hybrid with erythrocyte membrane modulate dendritic cells to ameliorate systemic lupus erythematosus.

Dendritic cells (DCs)-based immunotherapy has shown immense promise in systemic lupus erythematosus (SLE) treatment. However, existing carrier strategies such as polymers, liposomes, and polypeptides, are difficult to achieve active targeting to DCs due to their intricate interaction with biological systems. Since DCs represent a class of phagocytes responsible for the removal of senescent or damaged erythrocytes, we hypothesize that hybrid vesicles containing erythrocytes membrane components could be presented to be potent drug carriers to target DCs specifically. Herein, inspired by the cell membrane fusion technique, we develop hybrid biomimetic liposomes (R-Lipo) by fusing natural erythrocyte membrane vesicles and artificial liposomes for DCs-targeted SLE therapy. The resultant R-Lipo exhibited excellent biocompatibility and was shown to be effectively internalized by DCs both in vitro and in vivo. Using an immunosuppressant, mycophenolic acid (MPA), as the model drug, MPA-loaded R-Lipo powerfully suppressed DCs maturation and efficiently controlled the duration of lupus nephritis without apparent side effects. Our findings provide a safe, effective, and easy-to-prepare biomimetic vesicle platform for the treatment of SLE and other DC-associated diseases.

Mesenchymal Stem Cell Exosomes Encapsulated Oral Microcapsules for Acute Colitis Treatment.

Mesenchymal stem cells derived exosomes (MSC-exos) exhibit an intrinsic and directed efficiency for multiple diseases, while their versatile and effective delivery to the target site is still a challenge. Herein, inspired by the acids and enzymes resistant property of sealing gelatin capsules, novel MSC-exo-encapsulated oral microcapsules are presented for colitis treatment. Based on a microfluidic electrospray technique, MSC-exos are first encapsulated in sodium alginate (SA) hydrogel microspheres with sustainable bioactivity. The resultant SA microspheres are then coated with a middle gelatin layer to protect MSC-exos from degradation. Especially, with an enteric coating-Eudragit FS30D on the outer layer, the resistance of the microcapsules in gastric juice is further enhanced. The prepared microcapsules maintain the stability and bioactivity of the MSC-exos during storage, protect them from the harsh conditions in the gastrointestinal tract, and enable the release of actives in the suitable sites for exerting their biological functions. In addition, these MSC-exos encapsulated microcapsules reduce the proinflammatory cytokines levels of inflammatory macrophages and impaired colonic epithelial cells, which exhibit superior damage repair ability in injured colon sites. Thus, it is believed that the proposed oral MSC-exos encapsulated microcapsules are valuable for many practically clinical treatments.

Transforming the spleen into a liver-like organ in vivo.

Regenerating human organs remains an unmet medical challenge. Suitable transplants are scarce, while engineered tissues have a long way to go toward clinical use. Here, we demonstrate a different strategy that successfully transformed an existing, functionally dispensable organ to regenerate another functionally vital one in the body. Specifically, we injected a tumor extract into the mouse spleen to remodel its tissue structure into an immunosuppressive and proregenerative microenvironment. We implanted autologous, allogeneic, or xenogeneic liver cells (either primary or immortalized), which survived and proliferated in the remodeled spleen, without exerting adverse responses. Notably, the allografted primary liver cells exerted typical hepatic functions to rescue the host mice from severe liver damages including 90% hepatectomy. Our approach shows its competence in overcoming the key challenges in tissue regeneration, including insufficient transplants, immune rejection, and poor vascularization. It may be ready for translation into new therapies to regenerate large, complex human tissue/organs.

Accelerated wound healing in diabetes by reprogramming the macrophages with particle-induced clustering of the mannose receptors.

The rate-limiting step in cutaneous wound healing, namely, the transition from inflammation to cell proliferation, depends on the high plasticity of macrophages to prevent inflammation in the wound tissues in a timely manner. Thus, strategies that reprogram inflammatory macrophages may improve the healing of poor wounds, particularly in the aged skin of individuals with diabetes or other chronic diseases. As shown in our previous study, KGM-modified SiO2 nanoparticles (KSiNPs) effectively activate macrophages to differentiate into the M2-type phenotype by inducing mannose receptor (MR) clustering on the cell surface. Here, we assess whether KSiNPs accelerate wound healing following acute or chronic skin injury. Using a full-thickness excision model in either diabetic mice or healthy mice, the wounds treated with KSiNPs displayed a dramatically increased closure rate and collagen production, along with decreased inflammation and increased angiogenesis in the regenerating tissues. Furthermore, KSiNPs induced the formation of M2-like macrophages by clustering MR on the cells. Accordingly, the cytokines produced by the KSiNP-treated macrophages were capable of inducing fibroblast proliferation and subsequent secretion of extracellular matrix (ECM). Based on these results, KSiNPs display great potential as an effective therapeutic approach for cutaneous wounds by effectively suppressing excessive or persistent inflammation and fibrosis.

Growing Trans-Species Islets in Tumor Extract-Remodeled Testicles.

Although pancreatic islet transplantation holds promise for the treatment of type I diabetes, its application has been significantly hampered by transplant rejection. Here, an approach is demonstrated to support trans-species islet beta cells from a rat to grow and function in the body of a mouse host while overcoming graft rejection. This approach, which builds on remodeling of the mouse testicle by local injection of a tumor homogenate, establishes an immunosuppressive and proregenerative niche in the testicle. This remodeling proves necessary and effective in shaping the testicle into a unique site to accommodate xenograft cells. Rat pancreatic beta cells-from both the insulinoma (cancer cells) and pancreatic islet (normal tissue)-survive, grow, and form a desirable morphology in the remodeled mouse testicle. Notably, when hyperglycemia is induced in the host body, these xenografts secrete insulin to regulate the blood glucose level in mice for as long as 72 days. Furthermore, no graft rejection, acute inflammation, or safety risks are observed throughout the study. In summary, it is demonstrated that the growth of xenogeneic insulinoma cells in a mouse testicle might serve as an alternative approach for islet transplantation.

Producing anti-inflammatory macrophages by nanoparticle-triggered clustering of mannose receptors.

Macrophages are highly plastic cells that can either mediate or suppress inflammation, depending on their cellular phenotype and cytokine secretion. Inducing macrophages from an inflammatory ('M1') to anti-inflammatory ('M2') phenotype has significant implications for the treatment of inflammatory diseases and regeneration of injured tissues. Although certain cytokines, such as interleukin-4 and -13, are known to induce this phenotypic switch, their therapeutic use in vivo has both safety and efficacy concerns. Here, we demonstrate an alternative approach to change macrophage phenotype from M1 to M2, through inducing the clustering of mannose receptors (MR) on the cell surface, by using carbohydrate-presenting substrates. We prepared and screened glucomannan-decorated silicon oxide of different sizes ranging from 10 to 1000 nm, and identified one type (KSiNP30) that could potently induce MR clustering on macrophages and thereby stimulated the cells into an M2 phenotype - as an unexpected consequence of MR activation. Further administration of KSiNP30 in a murine model of inflammatory bowel disease efficiently alleviated the colitis symptoms, indicating the translational potential of our finding for therapeutic applications. In summary, we report for the first time an approach to modulate cellular immune responses by manipulating the assembly of cell-surface receptors, without the aid of cytokines. Our approach may provide insights for the development of new anti-inflammatory therapies.

Probiotics protect mice from CoCrMo particles-induced osteolysis.

Wear particle-induced inflammatory osteolysis is the primary cause of aseptic loosening, which is the most common reason for total hip arthroplasty (THA) failure in the med- and long term. Recent studies have suggested an important role of gut microbiota (GM) in modulating the host metabolism and immune system, leading to alterations in bone mass. Probiotic bacteria administered in adequate amounts can alter the composition of GM and confer health benefits to the host. Given the inflammatory osteolysis that occurs in wear debris-induced prosthesis loosening, we examined whether the probiotic Lactobacillus casei could reduce osteolysis in a mouse calvarial resorption model. In this study, L. casei markedly protected mice from CoCrMo particles (CoPs)-induced osteolysis. Osteoclast gene markers and the number of osteoclasts were significantly decreased in L. casei-treated mice. Probiotic treatment decreased the M1-like macrophage phenotype indicated by downregulation of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and inducible nitric oxide synthase (iNOS) and increased the M2-like macrophage phenotype indicated by upregulation of IL-4, IL-10 and arginase. Collectively, these results indicated that the L. casei treatment modulated the immune status and suppressed wear particle-induced osteolysis in vivo. Thus, probiotic treatment may represent a potential preventive and therapeutic approach to reduced wear debris-induced osteolysis.

Expression of XBP1s in fibroblasts is critical for TiAl6 V4 particle-induced RANKL expression and osteolysis.

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the expression of Receptor activation of nuclear factor (NF)-kB (RANKL) by fibroblasts in periprosthetic membrane played a crucial role in wear particle-induced osteolysis. However, the underlying mechanism of RANKL expression remains largely unknown. In the present study, we investigated the effect of TiAl6 V4 particle (TiPs)-induced XBP1s (spliced form of X-box binding protein 1) on RANKL expression and osteoclastogenesis both in vitro and in vivo. The levels of XBP1s in peri-implant membrane, animal models, and TiPs-stimulated fibroblasts were determined by western blots. To assess the effect of XBP1s on RANKL expression, fibroblasts were treated with both a small interfering RNA (siRNA) and an inhibitor of XBP1 prior to exposure to TiPs. The effect of XBP1s on osteoclasts formation was determined by tartrate-resistant acid phosphatase (TRAP) staining in vitro osteoclastogenesis assay and in animal models. The resorption of bone was assessed by micro-computed tomography (micro-CT) with three-dimensional reconstruction. Our results demonstrated that XBP1s was activated in periprosthetic membrane, mouse calvaria models, and TiPs-stimulated human synovial fibroblasts. Further, inhibition of XBP1s decreased the expression of RANKL and osteoclasts formation in vitro. In mouse calvaria models, both of the osteoclastogenesis and osteolysis were inhibited XBP1s inhibitor. Our results suggested that XBP1s mediated TiPs-induced of RANKL expression in fibroblasts, and down regulating XBP1s may represent a potential therapy for wear particle-induced osteolysis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:752-759, 2017.

Targeted delivery of let-7b to reprogramme tumor-associated macrophages and tumor infiltrating dendritic cells for tumor rejection.

Both tumor associated macrophages (TAMs) and tumor infiltrating dendritic cells (TIDCs) are important components in the tumor microenvironment that mediate tumor immunosuppression and promote cancer progression. Targeting these cells and altering their phenotypes may become a new strategy to recover their anti-tumor activities and thereby restore the local immune surveillance against tumor. In this study, we constructed a nucleic acid delivery system for the delivery of let-7b, a synthetic microRNA mimic. Our carrier has an affinity for the mannose receptors on TAMs/TIDCs and is responsive to the low-pH tumor microenvironment. The delivery of let-7b could reactivate TAMs/TIDCs by acting as a TLR-7 agonist and suppressing IL-10 production in vitro. In a breast cancer mouse model, let-7b delivered by this system efficiently reprogrammed the functions of TAMs/TIDCs, reversed the suppressive tumor microenvironment, and inhibited tumor growth. Taken together, this strategy, designed based upon TAMs/TIDCs-targeting delivery and the dual biological functions of let-7b (TLR-7 ligand and IL-10 inhibitor), may provide a new approach for cancer immunotherapy.

Autophagy mediated CoCrMo particle-induced peri-implant osteolysis by promoting osteoblast apoptosis.

Wear particle-induced osteolysis is the leading cause of aseptic loosening, which is the most common reason for THA (total hip arthroplasty) failure and revision surgery. Although existing studies suggest that osteoblast apoptosis induced by wear debris is involved in aseptic loosening, the underlying mechanism linking wear particles to osteoblast apoptosis remains almost totally unknown. In the present study, we investigated the effect of autophagy on osteoblast apoptosis induced by CoCrMo metal particles (CoPs) in vitro and in a calvarial resorption animal model. Our study demonstrated that CoPs stimulated autophagy in osteoblasts and PIO (particle-induced osteolysis) animal models. Both autophagy inhibitor 3-MA (3-methyladenine) and siRNA of Atg5 could dramatically reduce CoPs-induced apoptosis in osteoblasts. Further, inhibition of autophagy with 3-MA ameliorated the severity of osteolysis in PIO animal models. Moreover, 3-MA also prevented osteoblast apoptosis in an antiautophagic way when tested in PIO model. Collectively, these results suggest that autophagy plays a key role in CoPs-induced osteolysis and that targeting autophagy-related pathways may represent a potential therapeutic approach for treating particle-induced peri-implant osteolysis.

ER Stress Mediates TiAl6V4 Particle-Induced Peri-Implant Osteolysis by Promoting RANKL Expression in Fibroblasts.

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the synovial fibroblasts present in the periprosthetic membrane are important targets of wear debris during osteolysis. However, the interaction mechanisms between the wear debris and fibroblasts remain largely unknown. In the present study, we investigated the effect of ER (endoplasmic reticulum) stress induced by TiAl6V4 particles (TiPs) in human synovial fibroblasts and calvarial resorption animal models. The expression of ER stress markers, including IRE1-α, GRP78/Bip and CHOP, were determined by western blot in fibroblasts that had been treated with TiPs for various times and concentration. To address whether ER stress was involved in the expression of RANKL, the effects of ER stress blockers (including 4-PBA and TUDCA) on the expression of RANKL in TiPs-treated fibroblasts were examined by real-time PCR, western blot and ELISA. Osteoclastogenesis was assessed by tartrate resistant acid phosphatase (TRAP) staining. Our study demonstrated that ER stress markers were markedly upregulated in TiPs-treated fibroblasts. Blocking ER stress significantly reduced the TiPs-induced expression of RANKL both in vitro and in vivo. Moreover, the inhibition of ER stress ameliorated wear particle-induced osteolysis in animal models. Taken together, these results suggested that the expression of RANKL induced by TiPs was mediated by ER stress in fibroblasts. Therefore, down regulating the ER stress of fibroblasts represents a potential therapeutic approach for wear particle-induced periprosthetic osteolysis.

Dual TNF-α/IL-12p40 Interference as a Strategy to Protect Against Colitis Based on miR-16 Precursors With Macrophage Targeting Vectors.

Cytokines are central components of the mucosal inflammatory responses that take place during the development of Crohn's disease. Cell-specific combination therapies against cytokines may lead to increased efficacy and even reduced side effects. Therefore, a colonic macrophage-specific therapy using miR-16 precursors that can target both TNF-α and IL-12p40 was tested for its efficacy in experimental colitic mice. Galactosylated low molecular weight chitosan (G-LMWC) associated with miR-16 precursors were intracolonically injected into mice. The cellular localization of miR-16 precursors was determined. The therapeutic effects and possible mechanism were further studied in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitic mice. The results show that specific upregulation of miR-16 level in colonic macrophages significantly reduces TNF-α and IL-12p40 expression, which could suppress the associated mucosal inflammation and ultimately result in the relief of colitic symptoms. This strategy, based on the dual silencing of colonic macrophage-specific cytokines, represents a potential therapeutic approach that may be valuable for colitis therapy.

The fibroblast expression of RANKL in CoCrMo-particle-induced osteolysis is mediated by ER stress and XBP1s.

Particle-induced osteolysis is a major cause of aseptic loosening, which is the most common reason for total hip arthroplasty (THA) failure and revision surgery. Although existing studies suggest that synovial fibroblasts present in the interfacial membrane are important targets of wear particles during bone resorption, the interaction mechanisms between the particles and fibroblasts remains elusive. In the present study, we investigated the effect of ER stress induced by CoCrMo particles (CoPs) in fibroblasts, calvarial resorption animal models and aseptic loosening clinical samples and its role in the stimulation of the RANKL expression. Our study further demonstrated that CoPs could induce significant ER stress in fibroblasts. Blocking ER stress with a specific inhibitor dramatically reduced the particle-induced expression of RANKL in vitro and in vivo. Furthermore, in fibroblasts, downregulation of the expression of XBP1s, a signaling molecule of ER stress, significantly reduced the expression of RANKL induced by wear particles. Moreover, inhibition of ER stress or XBP1s both ameliorated the CoPs-induced osteolysis in animal models. Collectively, these results suggested that in particle-induced osteolysis, CoPs could stimulate fibroblasts to secret RANKL through ER stress and the signaling molecule XBP1s. Therefore, downregulating ER stress or the signaling molecule XBP1s of fibroblasts represents a potential therapeutic approach for treating particle-induced peri-implant osteolysis. STATEMENT OF SIGNIFICANCE: For the first time, our study demonstrated that ER stress mediated the induction of RANKL expression by CoPs in fibroblasts and promoted particle-induced osteolysis. Furthermore, the upregulation of RANKL by CoPs in fibroblasts was mediated by the ER stress signaling molecule XBP1s. Both blocking ER stress and inhibiting the protein XBP1s by specific inhibitors resulted in downregulation of the expression of RANKL and amelioration of osteolysis induced by the implanted particles. Collectively, these findings suggest a possible mechanism underlying the RANKL expression induced by wear particles in fibroblasts, and downregulating ER stress and the XBP1s expression of fibroblasts represents a potential therapeutic approach for treating aseptic loosening.

An orally administrated nucleotide-delivery vehicle targeting colonic macrophages for the treatment of inflammatory bowel disease.

Tumor necrosis factor-alpha (TNF-α) plays a central role in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF-α therapies have shown protective effects against colitis, but an efficient tool for target suppression of its secretion - ideally via oral administration - remains in urgent demand. In the colon tissue, TNF-α is mainly secreted by the colonic macrophages. Here, we report an orally-administrated microspheric vehicle that can target the colonic macrophages and suppress the local expression of TNF-α for IBD treatment. This vehicle is formed by cationic konjac glucomannan (cKGM), phytagel and an antisense oligonucleotide against TNF-α. It was given to dextran sodium sulfate (DSS) colitic mice via gastric perfusion. The unique swelling properties of cKGM enabled the spontaneous release of cKGM& antisense nucleotide (ASO) nano-complex from the phytagel scaffold into the colon lumen, where the ASO was transferred into colonic macrophages via receptor-mediated phagocytosis. The treatment significantly decreased the local level of TNF-α and alleviated the symptoms of colitis in the mice. In summary, our study demonstrates a convenient, orally-administrated drug delivery system that effectively targets colonic macrophages for suppression of TNF-α expression. It may represent a promising therapeutic approach in the treatment of IBD.

[Expression of human tumstatin in Pichia pastoris and its bioactivity].

Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.