Project description:The advent of novel therapeutics in recent years has urged the need for a safe, non-immunogenic drug delivery vector capable of delivering therapeutic payloads specifically to diseased cells, thereby increasing therapeutic efficacy and reducing side effects. Extracellular vesicles (EVs) have garnered attention in recent years as a potentially ideal vector for drug delivery, taking into account their intrinsic ability to transfer bioactive cargo to recipient cells and their biocompatible nature. However, natural EVs are limited in their therapeutic potential and many challenges need to be overcome before engineered EVs satisfy the levels of efficiency, stability, safety and biocompatibility required for therapeutic use. Here, we demonstrate that an enzyme-mediated surface functionalization method in combination with streptavidin-mediated conjugation results in efficient surface functionalization of EVs. Surface functionalization using the above methods permits the stable and biocompatible conjugation of peptides, single domain antibodies and monoclonal antibodies at high copy number on the EV surface. Functionalized EVs demonstrated increased accumulation in target cells expressing common cancer associated markers such as CXCR4, EGFR and EpCAM both in vitro and in vivo. The functionality of this approach was further highlighted by the ability of targeting EVs to specifically deliver therapeutic antisense oligonucleotides to a metastatic breast tumor model, resulting in increased knockdown of a targeted oncogenic microRNA and improved metastasis suppression. The method was also used to equip EVs with a bifunctional peptide that targets EVs to leukemia cells and induces apoptosis, leading to leukemia suppression. Moreover, we conducted extensive testing to verify the biocompatibility, and safety of engineered EVs for therapeutic use, suggesting that surface modified EVs can be used for repeated dose treatment with no detectable adverse effects. This modular, biocompatible method of EV engineering offers a promising avenue for the targeted delivery of a range of therapeutics while addressing some of the safety concerns associated with EV-based drug delivery.
Project description:Aims: Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we aimed to investigate the differences between NDTRs and NDMVs. Methods: The generation of neutrophil-derived EVs were visualized by live-cell fluorescence images and the physical characteristics were further analyzed using nanotracking analysis assay, scanning electron microscopic analysis, and marker expressions. Functional characteristics of neutrophil-derived EVs were analyzed using assays for bactericidal activity, monocyte chemotaxis, phenotype polarization of macrophages, and miRNA sequencing. Finally, the effects of neutrophil-derived EVs on the acute and chronic inflammation were examined in vivo. Results: Both EVs share similar characteristics including stimulators, surface marker expression, bactericidal activity, and chemoattractive effect on monocytes via MCP-1. However, the integrin-mediated physical interaction was required for generation of NDTRs whereas NDMV generation was dependent on PI3K pathway. Interestingly, NDTRs contained proinflammatory miRNAs such as miR-1260, miR-1285, miR-4454, and miR-7975, while NDMVs contained anti-inflammatory miRNAs such as miR-126, miR-150, and miR-451a. Although both EVs were easily uptaken by monocytes, NDTRs enhanced proinflammatory macrophage polarization whereas NDMVs induced anti-inflammatory macrophage polarization. Moreover, NDTRs showed protective effects against lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. Conclusion: These results suggest that NDTR is a proinflammatory subtype of neutrophil-derived EVs distinguished from NDMV.
Project description:Exosome is a subgroup of extracellular vesicles, which has been serving as an efficient therapeutic tool for various diseases. Engineered exosomes are the sort of exosomes modified with surface decoration and internal therapeutic molecules. After appropriate modification, engineered exosomes are able to deliver antitumor drugs to tumor sites efficiently and precisely with fewer treatment-related adverse effects. However, there still exist many challenges for the clinical translation of engineered exosomes. For instance, what sources and modification strategies could endow exosomes with the most efficient antitumor activity is still poorly understood. Additionally, how to choose appropriately engineered exosomes in different antitumor therapies is another unresolved problem. In this review, we summarized the characteristics of engineered exosomes, especially the spatial and temporal properties. Additionally, we concluded the recent advances in engineered exosomes in the cancer fields, including the sources, isolation technologies, modification strategies, and labeling and imaging methods of engineered exosomes. Furthermore, the applications of engineered exosomes in different antitumor therapies were summarized, such as photodynamic therapy, gene therapy, and immunotherapy. Consequently, the above provides the cancer researchers in this community with the latest ideas on engineered exosome modification and new direction of new drug development, which is prospective to accelerate the clinical translation of engineered exosomes for cancer-targeted therapy.
Project description:Exosomes are nanosized membranous vesicles secreted by a variety of cells. Due to their unique and pharmacologically important properties, cell-derived exosome nanoparticles have drawn significant interest for drug development. By genetically modifying exosomes with two distinct types of surface-displayed monoclonal antibodies, we have developed an exosome platform termed synthetic multivalent antibodies retargeted exosome (SMART-Exo) for controlling cellular immunity. Here, we apply this approach to human epidermal growth factor receptor 2 (HER2)-expressing breast cancer by engineering exosomes through genetic display of both anti-human CD3 and anti-human HER2 antibodies, resulting in SMART-Exos dually targeting T cell CD3 and breast cancer-associated HER2 receptors. By redirecting and activating cytotoxic T cells toward attacking HER2-expressing breast cancer cells, the designed SMART-Exos exhibited highly potent and specific anti-tumor activity both in vitro and in vivo. This work demonstrates preclinical feasibility of utilizing endogenous exosomes for targeted breast cancer immunotherapy and the SMART-Exos as a broadly applicable platform technology for the development of next-generation immuno-nanomedicines.
Project description:The complex pathogenesis of osteoporosis includes excessive bone resorption, insufficient bone formation and inadequate vascularization, a combination which is difficult to completely address with conventional therapies. Engineered exosomes carrying curative molecules show promise as alternative osteoporosis therapies, but depend on specifically-functionalized vesicles and appropriate engineering strategies. Here, we developed an exosome delivery system based on exosomes secreted by mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (iPSCs). The engineered exosomes BT-Exo-siShn3, took advantage of the intrinsic anti-osteoporosis function of these special MSC-derived exosomes and collaborated with the loaded siRNA of the Shn3 gene to enhance the therapeutic effects. Modification of a bone-targeting peptide endowed the BT-Exo-siShn3 an ability to deliver siRNA to osteoblasts specifically. Silencing of the osteoblastic Shn3 gene enhanced osteogenic differentiation, decreased autologous RANKL expression and thereby inhibited osteoclast formation. Furthermore, Shn3 gene silencing increased production of SLIT3 and consequently facilitated vascularization, especially formation of type H vessels. Our study demonstrated that BT-Exo-siShn3 could serve as a promising therapy to kill three birds with one stone and implement comprehensive anti-osteoporosis effects.
Project description:Extracellular vesicles (EVs) are nanoscale membrane-formed compartments naturally secreted from cells, which are intercellular mediators regulating physiology and pathogenesis, therefore they could be a novel therapeutic carrier for targeted delivery. However, the translation of EVs is hindered by the heterogeneous composition, low yield, inefficient drug loading and unlikely scalability. Here we report a strategy to generate EVs using nitrogen cavitation (NC-EVs) that instantly disrupts neutrophils to form nanosized membrane vesicles. NC-EVs are similar to naturally secreted EVs (NS-EVs), but contain less subcellular organelles and nuclear acids. The production of NC-EVs was increased by 16 folds and is easy to scale up for clinical use compared to NS-EVs. To examine the usefulness of NC-EVs as a drug delivery platform, piceatannol (an anti-inflammation drug) was remotely loaded in NC-EVs via the pH gradient. We found that piceatannol-loaded NC-EVs dramatically alleviated acute lung inflammation/injury and sepsis induced by lipopolysaccharide (LPS). Our studies reveal that nitrogen cavitation is a novel approach to efficiently generate EVs from any cell type and could be exploited for personalized nanomedicine.
Project description:Despite significant advances in diagnostic methods and treatment strategies, the prognosis for patients with advanced colon cancer remains poor, and mortality rates are often high due to metastasis. Increasing evidence showed that it is of significant importance to investigate how the tumor microenvironment participates in the development of colorectal cancer (CRC). In this manuscript, neutrophils were sequentially stimulated with all-trans retinoic acid and transforming growth factor-β in turn to induce the neutrophil polarization. Differentially expressed miRNA in neutrophil exosomes have been sequenced by microarray profile, and the effect of N2-like neutrophil-derived exosomal miR-4780 on epithelial-mesenchymal transition (EMT) and angiogenesis was investigated. In our results, we found that neutrophils were enriched in CRC tumor tissue and that CD11b expression correlated with tumor site and serous membrane invasion. At the same time, we demonstrated that internalization of N2 exosomes exacerbated the viability, migration, and invasion of CRC cell lines and inhibited apoptosis. To further investigate the molecular mechanism, we analyzed the miRNA expression profile in the N2-like neutrophils, which led to the selection of hsa-miR-4780 for the subsequent experiment. The overexpression of miR-4780 from N2-like neutrophil-derived exosomes exacerbated EMT and angiogenesis. Moreover, miR-4780 can regulate its target gene SOX11 to effect EMT and angiogenesis in CRC cell lines. CRC with liver metastasis model also validated that aberrant expression of miR-4780 in N2-like neutrophil exosomes exacerbated tumor metastasis and development of tumor via EMT and angiogenesis. In conclusion, our current findings reveal an important mechanism by which mR-4780 from N2-like neutrophil exosomes exacerbates tumor metastasis and progression via EMT and angiogenesis.
Project description:Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil-platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.
Project description:Cancer-targeted drug delivery systems (DDS) based on carbon nanostructures have shown great promise in cancer therapy due to their ability to selectively recognize specific receptors overexpressed in cancer cells. In this paper, we have explored a green route to synthesize nanobiochar (NBC) endowed with graphene structure from the hydrothermal carbonization (HTC) of orange peels and evaluated the suitability of this nanomaterial as a nanoplatform for cancer therapy. In order to compare the cancer-targeting ability of different widely used targeting ligands (TL), we have conjugated NBC with biotin, riboflavin, folic acid and hyaluronic acid and have tested, in vitro, their biocompatibility and uptake ability towards a human alveolar cancer cell line (A549 cells). The nanosystems which showed the best biological performances-namely, the biotin- and riboflavin- conjugated systems-have been loaded with the poorly water-soluble drug DHF (5,5-dimethyl-6a-phenyl-3-(trimethylsilyl)-6,6a-dihydrofuro[3,2-b]furan-2(5H)-one) and tested for their anticancer activity. The in vitro biological tests demonstrated the ability of both systems to internalize the drug in A549 cells. In particular, the biotin-functionalized NBC caused cell death percentages to more than double with respect to the drug alone. The reported results also highlight the positive effect of the presence of oxygen-containing functional groups, present on the NBC surface, to improve the water dispersion stability of the DDS and thus make the approach of using this nanomaterial as nanocarrier for poorly water-soluble drugs effective.
Project description:Small RNAs (microRNAs [miRNAs] or small interfering RNAs [siRNAs]) are effective tools for cancer therapy, but many of the existing carriers for their delivery are limited by low bioavailability, insufficient loading, impaired transport across biological barriers, and low delivery into the tumor microenvironment. Extracellular vesicle (EV)-based communication in mammalian and plant systems is important for many physiological and pathological processes, and EVs show promise as carriers for RNA interference molecules. However, some fundamental issues limit their use, such as insufficient cargo loading and low potential for scaling production. Plant-derived vesicles (PDVs) are membrane-coated vesicles released in the apoplastic fluid of plants that contain biomolecules that play a role in several biological mechanisms. Here, we developed an alternative approach to deliver miRNA for cancer therapy using PDVs. We isolated vesicles from watermelon and formulated a hybrid, exosomal, polymeric system in which PDVs were combined with a dendrimer bound to miRNA146 mimic. Third generation PAMAM was chosen due to its high branching structure and versatility for loading molecules of interest. We performed several in vivo experiments to demonstrate the therapeutic efficacy of our compound and explored in vitro biological mechanisms underlying the anti-tumor effects of miRNA146, which are mostly related to its anti-angiogenic activity.