Safety of bovine milk derived extracellular vesicles used for delivery of RNA therapeutics in zebrafish and mice

Abstract

Extracellular 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 nanotherapeutics. 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.

Ruminant Milk-Derived Extracellular Vesicles: A Nutritional and Therapeutic Opportunity?

“…However, the presence of miRNA in milk-derived EVs suggests that they have a potential role as natural or modifiable therapeutic agents to improve or enhance human and animal health. For instance, there are studies evaluating the milk-derived EV transcriptome for use as nanotherapeutic agents [ 184 , 185 , 186 ], as disease biomarkers [ 158 , 179 , 187 ], differential mediators [ 188 , 189 ], and as a health assessment tool for lactating animals [ 175 ]. Conversely, the role of milk-derived EVs as a functional regulator has also generated concerns because continuous consumption of dairy may contribute to the pathogenesis of common Western diseases such as type 2 diabetes mellitus, allergies, and cancers [ 13 , 190 , 191 , 192 , 193 , 194 ].…”

“…The studies researched the effects of EVs and exosomes on the gut microbiota [ 201 , 223 , 225 ], on the use of a delivery vehicle [ 111 , 159 , 184 , 208 , 213 , 226 , 227 , 228 , 229 , 230 , 231 ], in the immune response [ 60 , 178 , 185 , 203 , 217 , 232 , 233 , 234 , 235 , 236 , 237 ], in diseases such as cancer, [ 188 , 189 , 209 , 228 , 231 , 236 , 238 , 239 , 240 , 241 , 242 , 243 , 244 , 245 , 246 , 247 ], and in other aspects of cell biology [ 129 , 162 , 248 , 249 , 250 , 251 , 252 , 253 ]. These studies show that milk-derived EVs can have meaningful biological effects in the model systems used, forming a basis for future research.…”

“… [ 162 ] Microvesicle/Nanovesicle Cow Suitability of nanovesicles and encapsulated siRNA as a therapeutic delivery vehicle in vivo (zebrafish) and ex vivo (C57BL/6 splenocytes). [ 184 ] Cow Demonstrated successful uptake of PKH67-labelled microvesicles in vitro (RAW 264.7 cell line). [ 203 ] The terminology used is based on the reference cited, and this division reflects older thinking and is a “pool” of EVs that are responsible for the effects (ASC, adipose-derived stem cell; DSS, dextran sulphate sodium; hPAEC: human pulmonary artery endothelial cell; MDC, monocyte-derived dendritic cell; MSC, mesenchymal stem cell; NEC, necrotising enterocolitis; NRCM, neonatal rate cardiac myocyte; PBMC, peripheral blood mononuclear cell; SCFA, short-chain fatty acid; LPS, lipopolysaccharide).…”

Overview and Update on Methods for Cargo Loading into Extracellular Vesicles

“…Milk-derived EVs can inhibit cell growth of various cell lines [128], and several studies have used milk EVs loaded with siRNA for anticancer effects. After isolating milk-derived EVs, siRNA can be loaded with transfection reagents [129][130][131][132]. Cholesterol-conjugated hydrophobic siRNA can be loaded into EVs by simple incubation [11,133].…”

Evaluation of In Vivo Toxicity of Biological Nanoparticles

“…Recent studies have identified biological nanoparticles such as extracellular vesicles as delivery vehicles for therapeutic agents. Biological nanoparticles derived from bovine milk, or MNVs, are particularly advantageous for this purpose due to the scalability and low cost of their isolation (Maji et al, 2017;Matsuda et al, 2020). MNVs can be isolated from commercially available non-fat bovine milk.…”