Engineered nanovesicles from activated neutrophils with enriched bactericidal proteins have molecular debridement ability and promote infectious wound healing.

Background: Bacterial infections pose a considerable threat to skin wounds, particularly in the case of challenging-to-treat diabetic wounds. Systemic antibiotics often struggle to penetrate deep wound tissues and topically applied antibiotics may lead to sensitization, necessitating the development of novel approaches for effectively treating germs in deep wound tissues. Neutrophils, the predominant immune cells in the bloodstream, rapidly release an abundance of molecules via degranulation upon activation, which possess the ability to directly eliminate pathogens. This study was designed to develop novel neutrophil cell engineered nanovesicles (NVs) with high production and explore their bactericidal properties and application in promoting infectious wound healing. Methods: Neutrophils were isolated from peripheral blood and activated in vitro via phorbol myristate acetate (PMA) stimulation. Engineered NVs were prepared by sequentially extruding activated neutrophils followed by ultracentrifugation and were compared with neutrophil-derived exosomes in terms of morphology, size distribution and protein contents. The bactericidal effect of NVs in vitro was evaluated using the spread plate technique, LIVE/DEAD backlight bacteria assay and observation of bacterial morphology. The therapeutic effects of NVs in vivo were evaluated using wound contraction area measurements, histopathological examinations, assessments of inflammatory factors and immunochemical staining. Results: Activated neutrophils stimulated with PMA in vitro promptly release a substantial amount of bactericidal proteins. NVs are similar to exosomes in terms of morphology and particle size, but they exhibit a significantly higher enrichment of bactericidal proteins. In vitro, NVs demonstrated a significant bactericidal effect, presumably mediated by the enrichment of bactericidal proteins such as lysozyme. These NVs significantly accelerated wound healing, leading to a marked reduction in bacterial load, downregulation of inflammatory factors and enhanced collagen deposition in a full-thickness infectious skin defect model. Conclusions: We developed engineered NVs derived from activated neutrophils to serve as a novel debridement method targeting bacteria in deep tissues, ultimately promoting infectious wound healing.

Cell-engineered virus-mimetic nanovesicles for vaccination against enveloped viruses.

Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID-19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell-engineered virus-mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane-derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VNVs expressing the SARS-CoV-2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID-19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S-specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.

Adaptive Design of Nanovesicles Overcoming Immunotherapeutic Limitations of Chemotherapeutic Drugs through Poliovirus Receptor Blockade.

Chemotherapy is currently a widely used treatment for cancer in clinical settings. Some chemotherapeutic drugs such as oxaliplatin (OXA) can cause tumor immunogenic cell death (ICD), activate immunity, and realize chemoimmunotherapy for tumors. However, the low degree of accumulation and immunosuppressive microenvironment in tumors limit the immunotherapeutic efficacy of these drugs. T cell immunoreceptor with Ig and ITIM domains (TIGIT)/poliovirus receptor (PVR) is an inhibitory immune checkpoint pathway involved in mediating natural killer (NK) cell and T cell exhaustion in tumors. TIGIT expression is up-regulated in NK cells and CD8+ T cells during tumor development. Moreover, we first found that tumors upregulated PVR expression after OXA treatment in previous work. Here, we systematically analyzed the effects of OXA on the TIGIT/PVR pathway, further proving the effectiveness of the combination of OXA and TIGIT/PVR blocking combination. We developed engineered TIGIT-expressing cell membrane nanovesicles loaded with OXA (OXA@TIGIT MVs) for synergistic cancer therapy. OXA@TIGIT showed good efficacy in several cancer models, leading to tumor regression, effectively inhibiting tumor growth and prolonging mouse survival. Furthermore, the OXA@TIGIT MVs activate a strong tumor-specific immune response in the body, providing long-term (more than 2 months) protection from tumor reactivation in the B16F10 melanoma rechallenge mouse model.

Inhalable hybrid nanovaccines with virus-biomimetic structure boost protective immune responses against SARS-CoV-2 variants.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with different antigenic variants, has posed a significant threat to public health. It is urgent to develop inhalable vaccines, instead of injectable vaccines, to elicit mucosal immunity against respiratory viral infections. METHODS: We reported an inhalable hybrid nanovaccine (NVRBD-MLipo) to boost protective immunity against SARS-CoV-2 infection. Nanovesicles derived from genetically engineered 293T cells expressing RBD (NVRBD) were fused with pulmonary surfactant (PS)-biomimetic liposomes containing MPLA (MLipo) to yield NVRBD-MLipo, which possessed virus-biomimetic structure, inherited RBD expression and versatile properties. RESULTS: In contrast to subcutaneous vaccination, NVRBD-MLipo, via inhalable vaccination, could efficiently enter the alveolar macrophages (AMs) to elicit AMs activation through MPLA-activated TLR4/NF-κB signaling pathway. Moreover, NVRBD-MLipo induced T and B cells activation, and high level of RBD-specific IgG and secretory IgA (sIgA), thus elevating protective mucosal and systemic immune responses, while reducing side effects. NVRBD-MLipo also demonstrated broad-spectrum neutralization activity against SARS-CoV-2 (WT, Delta, Omicron) pseudovirus, and protected immunized mice against WT pseudovirus infection. CONCLUSIONS: This inhalable NVRBD-MLipo, as an effective and safe nanovaccine, holds huge potential to provoke robust mucosal immunity, and might be a promising vaccine candidate to combat respiratory infectious diseases, including COVID-19 and influenza.

High yield engineered nanovesicles from ADSC with enriched miR-21-5p promote angiogenesis in adipose tissue regeneration.

BACKGROUND: Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been found to have a great potential for soft tissue repair due to various biological functions, including pro-angiogenesis and low immunogenicity. However, the low yield and heterogeneity of MSC-EVs limited their clinical transformation. This study was designed to develop a novel adipose-derived stem cell engineered nanovesicles (ADSC-NVs) with high production and explore its pro-angiogenetic effect and application in adipose tissue regeneration. METHODS: Adipose-derived stem cell-derived extracellular vesicles (ADSC-EVs) were isolated from an EVs-free culture medium for human ADSCs (hADSCs). ADSC-NVs were prepared by sequentially extruding ADSCs followed by iodixanol density gradient ultracentrifugation and were compared with ADSC-EVs in morphology, size distribution, protein contents and yield. The pro-angiogenetic effect of ADSC-NVs in different doses (0, 5, 20 and 80 µg/mL) in vitro was determined using transwell assay, tube formation assay, western blot and qRT-PCR. In vivo, BALB/c nude mice were administered injection of a mixture of fat granules and different dose of ADSC-NVs and grafts were harvested at 12 weeks post-transplantation for further analysis. By analyzing the weight and volume of grafts and histological evaluation, we investigated the effect of ADSC-NVs in vessel formation and adipose tissue regeneration. RESULTS: Our results showed yield of purified ADSC-NVs was approximately 20 times more than that of ADSC-EVs secreted by the same number of ADSCs. In vitro, both ADSC-NVs and ADSC-EVs exhibited a dose-dependent pro-angiogenetic effect, despite their distinct miRNA profiles. These effects of ADSC-NVs may be mediated by enriched miR-21-5p via PTEN inhibition and PI3K/p-Akt signaling activation. Furthermore, after a mixed injection of ADSC-NVs, vessel formation and adipose regeneration were observed in vivo in fat implants. CONCLUSIONS: Our study developed a potent alternative of ADSC-EVs. ADSC-NVs have a high pro-angiogenesis potential and can be used as cell-free therapeutic biomaterials in soft tissue regeneration.

Engineered Nanovesicles from Fibroblasts Modulate Dermal Papillae Cells In Vitro and Promote Human Hair Follicle Growth Ex Vivo.

Alopecia is a common medical condition affecting both sexes. Dermal papilla (DP) cells are the primary source of hair regeneration in alopecia patients. Therapeutic applications of extracellular vesicles (EVs) are restricted by low yields, high costs, and their time-consuming collection process. Thus, engineered nanovesicles (eNVs) have emerged as suitable therapeutic biomaterials in translational medicine. We isolated eNVs by the serial extrusion of fibroblasts (FBs) using polycarbonate membrane filters and serial and ultracentrifugation. We studied the internalization, proliferation, and migration of human DP cells in the presence and absence of FB-eNVs. The therapeutic potential of FB-eNVs was studied on ex vivo organ cultures of human hair follicles (HFs) from three human participants. FB-eNVs (2.5, 5, 7.5, and 10 µg/mL) significantly enhanced DP cell proliferation, with the maximum effect observed at 7.5 µg/mL. FB-eNVs (5 and 10 µg/mL) significantly enhanced the migration of DP cells at 36 h. Western blotting results suggested that FB-eNVs contain vascular endothelial growth factor (VEGF)-a. FB-eNV treatment increased the levels of PCNA, pAKT, pERK, and VEGF-receptor-2 (VEGFR2) in DP cells. Moreover, FB-eNVs increased the human HF shaft size in a short duration ex vivo. Altogether, FB-eNVs are promising therapeutic candidates for alopecia.

Revisiting the Role of Exosomes in Colorectal Cancer: Where Are We Now?

Exosomes (Exos) are nano-sized extracellular vesicles constitutively released by both prokaryotic and eukaryotic cells. Their role as inter-cellular messengers involved in both physiological and pathological processes has overwhelmingly come to light in the last decade, and their contribution to cancerogenesis and tumor metastasis is under intensive investigation. Here we review the most recent information concerning Exos in colorectal cancer (CRC) and focus on their effects on tumor microenvironment and the immune system, as well as unravel their role in the formation of the pre-metastatic niche and in drug resistance. Such a recent knowledge on Exos depicts their potential translations into the clinical arena, either as an alternative tool of "liquid biopsy" or novel therapeutic approaches for CRC. However, due to the limited data available from clinical trials, they need further validations before addressing their putative application in oncology.

Cell-Engineered Nanovesicle as a Surrogate Inducer of Contact-Dependent Stimuli.

Heterotypic interactions between cells are crucial in various biological phenomena. Particularly, stimuli that regulate embryonic stem cell (ESC) fate are often provided from neighboring cells. However, except for feeder cultures, no practical methods are identified that can provide ESCs with contact-dependent cell stimuli. To induce contact-dependent cell stimuli in the absence of living cells, a novel method that utilizes cell-engineered nanovesicles (CNVs) that are made by extruding living cells through microporous membranes is described. Protein compositions of CNVs are similar to their originating cells, as well as freely diffusible and precisely scalable. Treatment of CNVs produced from three different stromal cells successfully induces the same effect as feeder cultures. The results suggest that the effects of CNVs are mainly mediated by membrane-associated components. The use of CNVs might constitute a novel and efficient tool for ESC research.