Bovine mRNAs in small extracellular vesicles from cow’s milk are not bioavailable in mice and translation products are not detectable in reticulocyte lysates and human U937 cells

Jiang Shu; Camila Pereira Braga; Juan Cui; Jiri Adamec; Janos Zempleni

doi:10.55092/exrna20240013

Article

Open Access

Bovine mRNAs in small extracellular vesicles from cow’s milk are not bioavailable in mice and translation products are not detectable in reticulocyte lysates and human U937 cells

Jiang Shu¹Camila Pereira Braga²Juan Cui¹Jiri Adamec²Janos Zempleni³^,∗

¹ School of Computing, University of Nebraska-Lincoln, Lincoln, NE 68583, USA

² Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68583, USA

³ Department of Nutrition and Health Science, University of Nebraska-sLincoln, Lincoln, NE 68583, USA

* jzempleni2@unl.edu

Volume
Volume 6 Issue 3, 2024
Citation
Shu J, Braga CP, Cui J, Adamec J, Zempleni J. Bovine mRNAs in small extracellular vesicles from cow’s milk are not bioavailable in mice and translation products are not detectable in reticulocyte lysates and human U937 cells. ExRNA 2024(3):0013, https://doi.org/10.55092/exrna20240013.
DOI
10.55092/exrna20240013
Copyright
Copyright2024 by the authors. Published by ELSP.

Abstract

Aim: Small extracellular vesicles from bovine milk (BEVs) have garnered attention as vehicles for delivering therapeutics to pathological tissues. Theoretically, mRNAs in BEVs might be translated into proteins, thereby eliciting immune responses in patients. The objectives of this study were to provide a comprehensive analysis of mRNAs in BEVs, assess mRNA bioavailability, and determine whether mRNAs are translated into proteins. Methods: BEVs were purified from raw cow’s milk (RM) by ultracentrifugation and treated with RNase to remove mRNA adsorbed to BEVs. BEVs were also isolated from store-bought milk (SBM) and analyzed with (SBM+) and without (SBM–) RNase treatment. mRNAs were analyzed using the Illumina HiSeq2500 platform. Bioavailability was assessed by administering BEVs loaded with IRDye-labeled bovine Casein Kappa (CSN3) mRNA by oral gavage in Balb/c mice. Translation was assessed using a rabbit reticulocyte lysate system (using RNA purified from BEVs) and human U937 monocytes (cultured with BEVs). Results: We identified transcripts of 4,858, 2,680, and 4,554 genes in BEVs from RM, SBM+, and SBM–, respectively. IRDye-labeled CSN3 mRNA, encapsulated in BEVs and delivered by oral gavage, was not detectable in murine tissues. Upon incubation with RNA and BEVs, bovine proteins were not detected in rabbit reticulocyte lysates and U937 monocytes, respectively. Conclusion: We conclude that bovine CSN3 mRNA, encapsulated in BEVs and delivered by oral gavage, is not bioavailable in mice, and translation products of RNA are not detectable in reticulocyte lysates and human U937 cells.

Keywords

drug delivery; extracellular vesicle; milk; mRNA; translation

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References

[1] O'Brien K, Breyne K, Ughetto S, Laurent LC, Breakefield XO. RNA delivery by extracellular vesicles in mammalian cells and its applications. Nat. Rev. Mol. Cell Biol. 2020, 21(10):585–606.
[2] Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 2007, 9(6):654–659.
[3] Purushothaman A, Bandari SK, Liu J, Mobley JA, Brown EE, et al. Fibronectin on the surface of myeloma cell-derived exosomes mediates exosome-cell interactions. J. Biol. Chem. 2016, 291(4):1652–166
[4] Keerthikumar S, Chisanga D, Ariyaratne D, Al Saffar H, Anand S, et al. ExoCarta: A web-based compendium of exosomal cargo. J. Mol. Biol. 2016, 428(4):688–692.
[5] Zhao Z, Zlokovic BV. Remote control of BBB: A tale of exosomes and microRNA. Cell Res. 2017, 27(7):849–850.
[6] Andras IE, Toborek M. Extracellular vesicles of the blood-brain barrier. Tissue barriers 2016, 4(1):e1131804.
[7] Escrevente C, Keller S, Altevogt P, Costa J. Interaction and uptake of exosomes by ovarian cancer cells. BMC Cancer 2011, 11:1–10.
[8] Alvarez-Erviti L, Seow Y, Yin H, Betts C, Lakhal S, et al. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 2011, 29(4):341–345.
[9] Liu Y, Li D, Liu Z, Zhou Y, Chu D, et al. Targeted exosome-mediated delivery of opioid receptor Mu siRNA for the treatment of morphine relapse. Sci. Rep. 2015, 5(1):17543.
[10] Zhou Y, Yuan Y, Liu M, Hu X, Ouan Y, et al. Tumor-specific delivery of KRAS siRNA with iRGD-exoxomes efficiently inhibits tumor growth. ExRNA 2019, 1(1):1–7.
[11] Mendt M, Kamerkar S, Sugimoto H, McAndrews KM, Wu CC, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 2018, 3(8):e99263.
[12] Sukreet S, Pereira Braga C, An TT, Adamec J, Cui J, et al. Isolation of extracellular vesicles from byproducts of cheese making by tangential flow filtration yields heterogeneous fractions of nanoparticles. J. Dairy Sci. 2021, 104(9):9478–9493.
[13] Milk produced per cow in the United States from 1999 to 2020 (in pounds). 2021 Available: https://www.statista.com/statistics/194935/quantity-of-milk-produced-per-cow-in-the-us-since-1999/ (accessed on 24 May 2021).
[14] Izumi H, Kosaka N, Shimizu T, Sekine K, Ochiya T, et al. Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions. J. Dairy Sci. 2012, 95(9):4831–4841.
[15] Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J. MicroRNAs are absorbed in biologically meaningful amounts from nutritionally relevant doses of cow’s milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. J. Nutr. 2014, 144(10):1495–1500.
[16] Wolf T, Baier SR, Zempleni J. The intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma caco-2 cells and rat small intestinal IEC-6 cells. J. Nutr. 2015; 145(10):2201–2206.
[17] Kusuma RJ, Manca S, Friemel T, Sukreet S, Nguyen C, et al. Human vascular endothelial cells transport foreign exosomes from cow’s milk by endocytosis. Am. J. Physiol. Cell Physiol. 2016, 310(10):C800–C807.
[18] Manca S, Upadhyaya B, Mutai E, Desaulniers AT, Cederberg RA, et al. Milk exosomes are bioavailable and distinct microRNA cargos have unique tissue distribution patterns. Sci. Rep. 2018, 8(1):11321.
[19] Sadri M, Shu J, Kachman SD, Cui J, Zempleni J. Milk exosomes and microRNAs cross the placenta and promote embryo survival in mice. Reprod. 2020, 160(4):501–509.
[20] Zhou F, Ebea P, Mutai E, Wang H, Sukreet S, et al. Small extracellular vesicles in milk cross the blood-brain barrier in murine cerebral cortex endothelial cells and promote dendritic complexity in the hippocampus and brain function in C57BL/6J mice. Front. Nutr. 2022, 9:838543.
[21] Khanam A, Ngu A, Zempleni J. Bioavailability of orally administered small extracellular vesicles from bovine milk in C57BL/6J mice. Int. J. Pharm. 2023, 639:122974.
[22] Agrawal AK, Aqil F, Jeyabalan J, Spencer WA, Beck J, et al. Milk-derived exosomes for oral delivery of paclitaxel. Nanomed. 2017, 13(5):1627–1636.
[23] Aqil F, Munagala R, Jeyabalan J, Agrawal AK, Kyakulaga AH, et al. Milk exosomes - Natural nanoparticles for siRNA delivery. Cancer Lett. 2019, 449:186–195.
[24] Munagala R, Aqil F, Jeyabalan J, Gupta RC. Bovine milk-derived exosomes for drug delivery. Cancer Lett. 2016, 371(1):48–61.
[25] Leiferman A, Shu J, Grove R, Cui J, Adamec J, et al. A diet defined by its content of bovine milk exosomes and their RNA cargos has moderate effects on gene expression, amino acid profiles and grip strength in skeletal muscle in C57BL/6 mice. J. Nutr. Biochem. 2018, 59:123–128.
[26] United States Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research. Nonclinical safety evaluation of the immunotoxic potential of drugs and biologics (draft guidance for industry). Rockville MD, Eds. In: Pharmacology/Toxicology. Food and Drug Administration 2020, pp. 1–10. Available: https://www.fda.gov/media/135312/download (accessed on 9 August 2024)
[27] Kanada M, Bachmann MH, Hardy JW, Frimannson DO, Bronsart L, et al. Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc. Natl. Acad. Sci. 2015, 112(12):E1433–E1442.
[28] Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines - a new era in vaccinology. Nat. Rev. Drug Discov. 2018, 17(4):261–279.
[29] Morgan AE. The synergistic effect of gentamicin and ceftazidime against Pseudomonas fluorescens. Biosci. Horiz. 2014, 7:hzu007.
[30] Bender AT, Sullivan BP, Lillis L, Posner JD. Enzymatic and chemical-based methods to inactivate endogenous blood ribonucleases for nucleic acid diagnostics. J. Mol. Diagn. 2020, 22(8):1030–1040.
[31] FastQC. 2017. Available: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ (accessed on 24 August 2017).
[32] Martin M. Cutadapt removes adapter aequences from high-throughput sequencing reads. EMBnetjournal. 2011. Available: http://journal.embnet.org/index.php/embnetjournal/article/view/200/0 (accessed on 9 August 2024).
[33] Rosen BD, Bickhart DM, Schnabel RD, Koren S, Elsik CG, et al. De novo assembly of the cattle reference genome with single-molecule sequencing. Gigascience 2020, 9(3):1–9.
[34] Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, et al. STAR: ultrafast universal RNA-seq aligner. Bioinf. 2013, 29(1):15–21.
[35] Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf. 2011, 12:1–16.
[36] Danecek P, Bonfield JK, Liddle J, Marshall J, Ohan V, et al. Twelve years of SAMtools and BCFtools. Gigascience 2021, 10(2):1–4.
[37] Haisma H, Coward WA, Albernaz E, Visser GH, Wells JC, et al. Breast milk and energy intake in exclusively, predominantly, and partially breast-fed infants. Eur. J. Clin. Nutr. 2003, 57(12):1633–1642.
[38] Leiferman A, Shu J, Upadhyaya B, Cui J, Zempleni J. Storage of extracellular vesicles in human milk, and microRNA profiles in human milk exosomes and infant formulas. J. Pediatr. Gastroenterol. Nutr. 2019, 69(2):235–2
[39] Chung S, Lapoint K, Martinez K, Kennedy A, Boysen Sandberg M, et al. Preadipocytes mediate lipopolysaccharide-induced inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes. Endocrinol. 2006, 147(11):5340–5351.
[40] Khanam A, Yu J, Zempleni J. Class A scavenger receptor-1/2 facilitates the uptake of bovine milk exosomes in murine bone marrow-derived macrophages and C57BL/6J mice. Am. J. Physiol. Cell Physiol. 2021, 321(3):C607–C614.
[41] Shelke GV, Lasser C, Gho YS, Lotvall J. Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum. J. Extracell. Vesicles 2014, 3(1):24783.
[42] Malheiros JM, Braga CP, Grove RA, Ribeiro FA, Calkins CR, et al. Influence of oxidative damage to proteins on meat tenderness using a proteomics approach. Meat Sci. 2019, 148:64–71.
[43] UniProt C. UniProt: the universal protein knowledgebase in 2023. Nucleic Acids Res. 2023; 51(D1):D523–D531.
[44] Hogan MJ, Pardi N. mRNA vaccines in the COVID-19 pandemic and beyond. Annu. Rev. Med. 2022, 73(1):17–39.
[45] Batagov AO, Kurochkin IV. Exosomes secreted by human cells transport largely mRNA fragments that are enriched in the 3'-untranslated regions. Biol. Direct 2013, 8:1–8.
[46] Kurosaki T, Maquat LE. Nonsense-mediated mRNA decay in humans at a glance. J. Cell Sci. 2016; 129(3):461–467.
[47] Mutai E, Ramer-Tait AE, Zempleni J. MicroRNAs in bovine milk exosomes are bioavailable in humans but do not elicit a robust pro-inflammatory cytokine response. ExRNA 2020, 2:1–9.
[48] Alsaweed M, Lai CT, Hartmann PE, Geddes DT, Kakulas F. Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk. Sci. Rep. 2016; 6(1):20680.