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

Tan, Xiao-Hui; Fang, Dong; Xu, Yongde; Nan, Tie-Gui; Song, Wenpeng; Gu, Yi; Gu, Sheng-Ji; Yuan, Yiming; Xin, Zhongcheng; Zhou, Liqun; Guan, Ruili; Li, Xuesong

doi:10.3389/fnut.2021.769223

Cited by 12 publications

(8 citation statements)

References 40 publications

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“…In such cases, some published studies use precipitation‐based methods, such as ExoQuick ® and ExoEasy ® (Bickmore & Miklavcic, 2020 ; Kupsco et al., 2021 ; Soares Martins et al., 2018 ). Although these methods are efficient, they can result in co‐isolation of non‐EV proteins (Lobb et al., 2015 ; Tan et al., 2021 ; Wijenayake et al., 2021 ; Zlotogorski‐Hurvitz et al., 2015 ). Additionally, EV fractions obtained from ExoQuick ® may contain traces of biopolymers that can interfere with mass spectrometry (Taylor & Shah, 2015 ).…”

Section: Discussionmentioning

confidence: 99%

Neonatal enteroids absorb extracellular vesicles from human milk‐fed infant digestive fluid

Yung,

Zhang,

Kuhn

et al. 2024

J of Extracellular Vesicle

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Human milk contains extracellular vesicles (HMEVs). Pre‐clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density‐gradient ultracentrifugation following two‐step skimming, acid precipitation of caseins, and multi‐step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super‐resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β‐casein and lactalbumin. HMEV and some dEV fractions contain mammary gland‐derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin‐mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.

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Section: Discussionmentioning

confidence: 99%

Neonatal enteroids absorb extracellular vesicles from human milk‐fed infant digestive fluid

Yung,

Zhang,

Kuhn

et al. 2024

J of Extracellular Vesicle

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show abstract

“…In such cases, some published studies use precipitation-based methods, such as ExoQuick ® and ExoEasy ® (82)(83)(84). Although these methods are efficient, they can result in co-isolation of non-EV proteins (85)(86)(87)(88). Additionally, EV fractions obtained from ExoQuick ® may contain traces of biopolymers that can interfere with mass spectrometry (89).…”

Section: Discussionmentioning

confidence: 99%

Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid

Yung,

Zhang,

Kuhn

et al. 2023

Preprint

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Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, nanoparticle tracking analysis, and western blotting. EV uptake and effects were tested in human neonatal enteroids. HMEVs and digesta EVs (dHMEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEVs and dHMEVs contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dHMEVs in part via clathrin-mediated endocytosis. Our data suggest that HMEVs survive to the duodenum and can be absorbed by IECs.

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“…However, several obstacles restrict its further clinical translation, including poor aqueous solubility, low membrane permeability, and obvious efflux from cells. Bovine milk-derived extracellular vesicles are recognized as promising nanoscale delivery to resolve this problem ( Tan et al, 2021 ). In conclusion, we demonstrated that ICA Ⅱ has protective effects and can rescue HUVECs from injury and dysfunction caused by PA in vitro via the SRPK1-Akt-eNOS signaling pathway.…”

Section: Discussionmentioning

confidence: 99%

Icariside Ⅱ Attenuates Palmitic Acid-Induced Endothelial Dysfunction Through SRPK1-Akt-eNOS Signaling Pathway

Tan

Song

et al. 2022

Front. Pharmacol.

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Background: Endothelial dysfunction is commonly accompanied by a reduced capacity for nitric oxide (NO) production and decreased NO sensitivity, playing a central role in numerous vascular diseases. Saturated free fatty acids are known to reduce NO production and then induce endothelial dysfunction. Alternative splicing participates in the regulation of cellular and tissular homeostasis and is highly regulated by serine-arginine protein kinase (SRPK1). The role of SRPK1 in the biology of endothelial cells remains elusive. Icariside Ⅱ (ICA Ⅱ) has been reported to have protective effects on endothelial function. However, the specific molecular mechanisms are still unknown. The purpose of this study is to explore the role of SRPK1 in the biology of endothelial cells and the underlying mechanism of ICA Ⅱ on palmitic acid (PA) induced endothelial dysfunction.Methods: Endothelial dysfunction was induced using PA in human umbilical vein endothelial cells (HUVECs). The expression and phosphorylation of related proteins in the SRPK1-Akt-eNOS signaling pathway were detected by Western Blot. Cell Counting Kit-8 assay and Ki-67 immunofluorescence were used to estimate cell viability. Endothelial cell function was assessed by detecting NO production using DAF-FM DA. Interaction between ICA Ⅱ and SRPK1 was demonstrated by a biotinylated protein interaction pull-down assay.Results: The expressions of eNOS, Akt, and SRPK1 were down-regulated in the endothelial dysfunction stimulated by PA. SRPK1 inhibitor SPHINX31 restrained endothelial cell viability in a dose-dependent manner. Moreover, inhibition of SRPK1 using SPHINX31 and knockdown of SRPK1 by shRNA also showed a down-regulation of the proteins associated with the SRPK1-Akt-eNOS signaling pathway. Biotinylated protein interaction pull-down assay revealed that ICA Ⅱ could be directly bound with SRPK1. On the other hand, ICA Ⅱ could attenuate the PA-induced endothelial dysfunction and restore cell viability through the SRPK1-Akt-eNOS pathway.Conclusions: ICA Ⅱ, bound with SRPK1, could attenuate the endothelial dysfunction induced by the PA in HUVECs via the SRPK1-Akt-eNOS signaling pathway.

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

Cited by 12 publications

References 40 publications

Neonatal enteroids absorb extracellular vesicles from human milk‐fed infant digestive fluid

Neonatal enteroids absorb extracellular vesicles from human milk‐fed infant digestive fluid

Neonatal enteroids absorb extracellular vesicles from human milk-fed infant digestive fluid

Icariside Ⅱ Attenuates Palmitic Acid-Induced Endothelial Dysfunction Through SRPK1-Akt-eNOS Signaling Pathway

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