Moirangthem, Anuradha

Extracellular vesicles (EVs) show promise for targeted drug delivery but face production challenges with low yields. Cell-derived nanovesicles (CDNVs) made by reconstituting cell membranes could serve as EV substitutes. In this study, CDNVs were generated from mesenchymal stem cells by extrusion. Their proteomic composition, in vitro and in vivo toxicity, and capacity for loading RNA or proteins were assessed. Compared with EVs, CDNVs were produced at higher yields, were comprised of a broader range of proteins, and showed no detrimental effects on cell proliferation, DNA damage, or nitric oxide production in vitro or on developmental toxicity in vivo. CDNVs could be efficiently loaded with RNA and engineered to modify surface proteins. The feasibility of generating immunomodulatory CDNVs was demonstrated by preparing CDNVs with enhanced surface expression of PD1, which could bind to PD-L1 expressing tumor cells, enhance NK and T cell degranulation, and increase immune-mediated tumor cell death. These findings demonstrate the adaptability and therapeutic promise of CDNVs as promising substitutes for natural EVs that can be engineered to enhance immunomodulation.

AbstractBiologically derived nanoparticles such as extracellular vesicles are promising candidates for therapeutic applications. In vivo toxicity of biological nanoparticles can result in tissue or organ damage, immunological perturbations, or developmental effects but cannot be readily predicted from in vitro studies. Therefore, an essential component of the preclinical assessment of these particles for their use as therapeutics requires screening for adverse effects and detailed characterization of their toxicity in vivo. However, there are no standardized, comprehensive methods to evaluate the toxicity profile of nanoparticle treatment in a preclinical model. Here, we first describe a method to prepare bovine milk‐derived nanovesicles (MNVs). These MNVs are inexpensive to isolate, have a scalable production platform, and can be modified to achieve a desired biological effect. We also describe two vertebrate animal models, mice and zebrafish, that can be employed to evaluate the toxicity profile of biologically derived nanoparticles, using MNVs as an example. Treatment‐induced organ toxicity and immunological effects can be assessed in mice receiving systemic injections of MNVs, and developmental toxicity can be assessed in zebrafish embryos exposed to MNVs in embryo water. Utilizing these animal models provides opportunities to analyze the toxicity profiles of therapeutic extracellular vesicles in vivo. © 2021 Wiley Periodicals LLC.Basic Protocol 1: Preparation of milk‐derived nanovesiclesBasic Protocol 2: In vivo screening for organ toxicity and immune cell profiling using miceBasic Protocol 3: In vivo developmental toxicity screening using zebrafish

AbstractExtracellular vesicles are endogenous biological nanoparticles that have potential for use as therapeutic nanoparticles or as delivery vehicles for therapeutic agents. Milk nanovesicles (MNV) are extracellular vesicles isolated from bovine milk that have been explored for use as delivery vehicles for RNA therapeutics such as small interfering RNA (siRNA). We performed in vivo toxicological studies of MNV or therapeutic MNV (tMNV) loaded with siRNA as a prelude to their clinical use. Development toxicity was assessed in zebrafish embryos. Acute toxicity was assessed in both mice and zebrafish whereas safety, biochemical, histological and immune effects after multiple dosing were assessed in mice. Zebrafish embryo hatching was accelerated with MNV and tMNV. While acute toxicity or effects on mortality were not observed in zebrafish, developmental effects were observed at high concentrations of MNV. There was a lack of discernable toxicity, mortality and systemic inflammatory or immunological responses in mice following administration of either MNVs or tMNVs. The tolerability and lack of discernable developmental or systemic in vivo toxicity support their use as biological nano‐therapeutics. Adoption of a standardized protocol for systematic analysis of in vivo safety and toxicity will facilitate preclinical assessment of EV based formulations for therapeutic use.