Skimmed Bovine Milk-Derived Extracellular Vesicles Isolated via "Salting-Out": Characterizations and Potential Functions as Nanocarriers

Xiao-Hui Tan; Dong Fang; Yong-De Xu; Tie-Gui Nan; Wen-Peng Song; Yang-Yang Gu; Sheng-Ji Gu; Yi-Ming Yuan; Zhong-Cheng Xin; Li-Qun Zhou; Rui-Li Guan; Xue-Song Li

doi:10.3389/fnut.2021.769223

Skimmed Bovine Milk-Derived Extracellular Vesicles Isolated via "Salting-Out": Characterizations and Potential Functions as Nanocarriers

Front Nutr. 2021 Oct 28:8:769223. doi: 10.3389/fnut.2021.769223. eCollection 2021.

Authors

Xiao-Hui Tan^{1

2

3}, Dong Fang^{1

2

3}, Yong-De Xu⁴, Tie-Gui Nan⁵, Wen-Peng Song^{2

3

6}, Yang-Yang Gu^{2

3

7}, Sheng-Ji Gu^{1

2

3}, Yi-Ming Yuan^{1

2

3}, Zhong-Cheng Xin^{1

2

3}, Li-Qun Zhou^{1

2

3}, Rui-Li Guan^{1

2

3}, Xue-Song Li^{1

2

3}

Affiliations

¹ Department of Urology, Peking University First Hospital, Beijing, China.
² Institute of Urology, Peking University, Beijing, China.
³ Beijing Key Laboratory of Urogenital Diseases (male) Molecular Diagnosis and Treatment Center, Beijing, China.
⁴ Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, China.
⁵ State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
⁶ Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China.
⁷ Department of Radiation Medicine, Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China.

Abstract

Bovine milk-derived extracellular vesicles (BM-EVs) are recognized as promising nanoscale delivery vectors owing to their large availability. However, few isolation methods can achieve high purity and yield simultaneously. Therefore, we developed a novel and cost-effective procedure to separate BM-EVs via "salting-out." First, BM-EVs were isolated from skimmed milk using ammonium sulfate. The majority of BM-EVs were precipitated between 30 and 40% saturation and 34% had a relatively augmented purity. The separated BM-EVs showed a spherical shape with a diameter of 60-150 nm and expressed the marker proteins CD63, TSG101, and Hsp70. The purity and yield were comparable to the BM-EVs isolated via ultracentrifugation while ExoQuick failed to separate a relatively pure fraction of BM-EVs. The uptake of BM-EVs into endothelial cells was dose- and time-dependent without significant cytotoxicity. The levels of endothelial nitric oxide syntheses were regulated by BM-EVs loaded with icariside II and miRNA-155-5p, suggesting their functions as delivery vehicles. These findings have demonstrated that it is an efficient procedure to isolate BM-EVs via "salting-out," holding great promise toward therapeutic applications.

Keywords: RNA-seq; ammonium sulfate; drug delivery systems; extracellular vesicles; milk; proteome.