Bioengineered milk-derived extracellular vesicles implementing high drug loading and membrane integrity for efficient oral drug delivery

Abstract

Milk-derived extracellular vesicles (EVs) are promising for oral drug delivery, yet different loading methods exhibit distinct impacts on drug encapsulation and membrane integrity. This study demonstrated that sonication method achieved high drug encapsulation in commercial milk-derived EVs (S-CM EVs), but impaired EV structure, compromising transcytosis. Incubation method (I-CM EVs) preserved EVs delivery ability, but had low drug loading. Further proteomic and transmembrane studies showed that sonication greatly damaged membrane proteins involved in trans-epithelial transportation, especially endoplasmic reticulum-Golgi pathway. To overcome this dilemma, we generated a hybrid CM EVs (H-CM EVs) by fusing I-CM EVs and S-CM EVs. H-CM EVs demonstrated comparable drug encapsulation to S-CM EVs (56.14%), significantly higher than I-CM EVs (11.92%). Importantly, H-CM EVs could maintain efficient drug delivery capability by restoring membrane fluidity, repairing damaged proteins, and enhancing enzyme resistance of S-CM EVs. H-CM EVs exhibited excellent absorption characteristics with 1.85-fold higher of area under the curve and 2.50-fold higher of max plasma concentration than those of S-CM EVs. On type Ⅰ diabetic mice, orally delivery of insulin loaded H-CM EVs and I-CM EVs showed improved hypoglycemic effects with pharmacological availabilities of 5.15% and 5.31%, which was 1.7-fold higher than that of S-CM EVs (3.00%). This H-CM EVs platform not only achieved high drug loading and maintained functionality for effective oral delivery but also highlighted the significant translational potential for improved clinical outcomes.

Keywords

Oral drug delivery / Milk-derived EVS / Incubation / Sonication / Fusion / Encapsulation efficiency

Conflicts of interest

The authors declare no competing interests.

Acknowledgments

The authors acknowledge financial support from the the Regional Innovation and Development Joint Fund of National Natural Science Foundation of China (grant numbers: U22A20356); the National Key R&D Program of China (No. 2021YFE0115200) and the National Natural Science Foundation of China (No. 81872818).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.ajps.2025.101093. The figures and tables with "S" before the serial number are included in the Supplementary material.

References

[1] Ebrahimi SB, Samanta D. Engineering protein-based therapeutics through structural and chemical design. Nat Commun 2023; 14:2411.

[2] Rahban M, Ahmad F, Piatyszek MA, Haertlé T, Saso L, Saboury AA. Stabilization challenges and aggregation in protein-based therapeutics in the pharmaceutical industry. RSC Adv 2023; 13(51):35947-63.

[3] Kumar V, Barwal A, Sharma N, Mir DS, Kumar P, Kumar V. Therapeutic proteins: developments, progress, challenges, and future perspectives. 3 Biotech 2024; 14:112.

[4] Xie X, Yu T, Li X, Zhang N, Foster LJ, Peng C, et al. Recent advances in targeting the "undruggable" proteins: from drug discovery to clinical trials. Signal Transduct. Target Ther 2023; 8:1-71.

[5] Ibraheem D, Elaissari A, Fessi H. Administration strategies for proteins and peptides. Int J Pharm 2014; 477:578-89.

[6] Wu J, Sahoo JK, Li Y, Xu Q, Kaplan DL. Challenges in delivering therapeutic peptides and proteins: a silk-based solution. J Control Release 2022; 345:176-89.

[7] Ganesh AN, Heusser C, Garad S, Sánchez-Félix MV. Patient-centric design for peptide delivery: trends in routes of administration and advancement in drug delivery technologies. Med Drug Discov 2021; 9:100079.

[8] Brown TD, Whitehead KA, Mitragotri S. Materials for oral delivery of proteins and peptides. Nat Rev Mater 2020; 5:127-48.

[9] Ren A, Hu J, Qin C, Xia N, Yu M, Xu X, et al. Oral administration microrobots for drug delivery. Bioact Mater 2024; 39:163-90.

[10] Su FY, Lin KJ, Sonaje K, Wey SP, Yen TC, Ho YC, et al. Protease inhibition and absorption enhancement by functional nanoparticles for effective oral insulin delivery. Biomaterials 2012; 33:2801-11.

[11] Nag S, Mitra O, Maturi B, Kaur SP, Saini A, Nama M, et al. Autophagy and mitophagy as potential therapeutic targets in diabetic heart condition: Harnessing the power of nanotheranostics. Asian J Pharm Sci 2024; 19(3):100927.

[12] Wu L, Bai Y, Wang L, Liu X, Zhou R, Li L, et al. Promoting apical-to-basolateral unidirectional transport of nanoformulations by manipulating the nutrient-absorption pathway. J Control Release 2020; 323:151-60.

[13] Han X, Lu Y, Xie J, Zhang E, Zhu H, Du H, et al. Zwitterionic micelles efficiently deliver oral insulin without opening tight junctions. Nat Nanotechnol 2020; 15:605-14.

[14] Zhu W, Chao Y, Jin Q, Chen L, Shen J, Zhu J, et al. Oral delivery of therapeutic antibodies with a transmucosal polymeric carrier. ACS Nano 2023; 17:4373-86.

[15] Xiao X, Zhang L, Ni M, Liu X, Xing L, Wu L, et al. Enhanced oral and pulmonary delivery of biomacromolecules via amplified transporter targeting. J Control Release 2024; 370:152-67.

[16] Ren C, Zhong D, Qi Y, Liu C, Liu X, Chen S, et al. Bioinspired pH- Responsive microalgal hydrogels for oral insulin delivery with both hypoglycemic and insulin sensitizing effects. ACS Nano 2023; 17:14161-75.

[17] Wang A, Fan W, Yang T, He S, Yang Y, Yu M, et al. Liver-target and glucose-responsive polymersomes toward mimicking endogenous insulin secretion with improved hepatic glucose utilization. Adv Funct Mater 2020; 30:1910168.

[18] Ma G. Microencapsulation of protein drugs for drug delivery: strategy, preparation, and applications. J Control Release 2014; 193:324-40.

[19] Sun S, Liang N, Gong X, An W, Kawashima Y, Cui F, et al. Multifunctional composite microcapsules for oral delivery of insulin. Int J Mol Sci 2017; 18:54.

[20] Drucker DJ. Advances in oral peptide therapeutics. Nat Rev Drug Discov 2020; 19:277-89.

[21] Wang L, Wang N, Zhang W, Cheng X, Yan Z, Shao G, et al. Therapeutic peptides: current applications and future directions. Signal Transduct Target Ther 2022; 7:1-27.

[22] Haddadzadegan S, Dorkoosh F, Bernkop-Schnürch A. Oral delivery of therapeutic peptides and proteins: technology landscape of lipid-based nanocarriers. Adv Drug Deliv Rev 2022; 182:114097.

[23] Renukuntla J, Vadlapudi AD, Patel A, Boddu SHS, Mitra AK. Approaches for enhancing oral bioavailability of peptides and proteins. Int J Pharm 2013; 447:75-93.

[24] Dai J, Su Y, Zhong S, Cong L, Liu B, Yang J, et al. Exosomes: key players in cancer and potential therapeutic strategy. Signal Transduct Target Ther 2020; 5:1-10.

[25] Kalluri R, McAndrews KM. The role of extracellular vesicles in cancer. Cell 2023; 186:1610-26.

[26] Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020;367:eaau6977.

[27] Chen YF, Luh F, Ho YS, Yen Y. diagnostic and Exosomes: a review of biologic function, targeted therapy applications, and clinical trials. J Biomed Sci 2024; 31:67.

[28] Samuel M, Fonseka P, Sanwlani R, Gangoda L, Chee SH, Keerthikumar S, et al. Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis. Nat Commun 2021; 12:3950.

[29] Reniker LN, Frazer LC, Good M. Key biologically active components of breast milk and their beneficial effects. Semin Pediatr Surg 2023; 32:151306.

[30] Shome S, Jernigan RL, Beitz DC, Clark S, Testroet ED. Non-coding RNA in raw and commercially processed milk and putative targets related to growth and immune-response. BMC Genomics 2021; 22:749.

[31] Tian MY, Hao DX, Liu Y, He J, Zhao ZH, Guo TY, et al. Milk exosomes: an oral drug delivery system with great application potential. Food Funct 2023; 14:1320-37.

[32] Salehi M, Negahdari B, Mehryab F, Shekari F. Milk-derived extracellular vesicles: biomedical applications, current challenges, and future perspectives. J Agric Food Chem 2024; 72:8304-31.

[33] Sanwlani R, Fonseka P, Chitti SV, Mathivanan S. Milk-derived extracellular vesicles in inter-organism, cross-species communication and drug delivery. Proteomes 2020; 8:11.

[34] Zhong J, Xia B, Shan S, Zheng A, Zhang S, Chen J, et al. High-quality milk exosomes as oral drug delivery system. Biomaterials 2021; 277:121126.

[35] Warren M R, Zhang C, Vedadghavami A, Bokvist K, Dhal P K, Bajpayee A G. Milk exosomes with enhanced mucus penetrability for oral delivery of siRNA. Biomater Sci 2021; 9:4260-77.

[36] Wu L, Wang L, Liu X, Bai Y, Wu R, Li X, et al. Milk-derived exosomes exhibit versatile effects for improved oral drug delivery. Acta Pharm Sin B 2022; 12:2029-42.

[37] Tian J, Han Z, Song D, Peng Y, Xiong M, Chen Z, et al. Engineered exosome for drug delivery: recent development and clinical applications. Int J Nanomed 2023.

[38] da Silva TLT, Danthine S, Martini S. Influence of sonication, temperature, and agitation, on the physical properties of a palm-based fat crystallized in a continuous system. Ultrason Sonochem 2021; 74:105550.

[39] Sukreet S, Silva BVRE, Adamec J, Cui J, Zempleni J. Sonication and short-term incubation alter the content of bovine milk exosome cargos and exosome bioavailability (OR26-08-19). Curr Dev Nutr 2019;3:nzz033.

[40] Kim HI, Park J, Zhu Y, Wang X, Han Y, Zhang D. Recent advances in extracellular vesicles for therapeutic cargo delivery. Exp Mol Med 2024; 56:836-49.

[41] Mukherjee A, Bisht B, Dutta S, Paul MK. Current advances in the use of exosomes, liposomes, and bioengineered hybrid nanovesicles in cancer detection and therapy. Acta Pharmacol Sin 2022; 43:2759-76.

[42] Che Shaffi S, Hairuddin ON, Mansor SF, Syafiq TMF, Yahaya BH. Unlocking the potential of extracellular vesicles as the next generation therapy: challenges and opportunities. Tissue Eng Regen Med 2024; 21:513-27.

[43] Wang L, Wang D, Ye Z, Xu J. Engineering extracellular vesicles as delivery systems in therapeutic applications. Adv Sci 2023; 10:2300552.

[44] Somiya M, Yoshioka Y, Ochiya T. Biocompatibility of highly purified bovine milk-derived extracellular vesicles. J Extracell Vesicles 2018; 7:1440132.

[45] Li Y, Xing L, Wang L, Liu X, Wu L, Ni M, et al. Milk-derived exosomes as a promising vehicle for oral delivery of hydrophilic biomacromolecule drugs. Asian J Pharm Sci 2023; 18:100797.

[46] Piffoux M, Silva AKA, Wilhelm C, Gazeau F, Tareste D. Modification of extracellular vesicles by fusion with liposomes for the design of personalized biogenic drug delivery systems. ACS Nano 2018; 12:6830-42.

[47] Hoffmann P, Burmester M, Langeheine M, Brehm R, Empl MT, Seeger B, et al. Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells. PLoS One 2021;16: e0257824.

[48] Pochapski DJ, Carvalho dos Santos C, Leite GW, Pulcinelli SH, Santilli CV. Zeta potential and colloidal stability predictions for inorganic nanoparticle dispersions: effects of experimental conditions and electrokinetic models on the interpretation of results. Langmuir 2021; 37:13379-89.

[49] Midekessa G, Godakumara K, Ord J, Viil J, Lättekivi F, Dissanayake K, et al. Zeta potential of extracellular vesicles: toward understanding the attributes that determine colloidal stability. ACS Omega 2020; 5:16701-10.

[50] Batrakova EV, Kim MS. Using exosomes, naturally-equipped nanocarriers, for drug delivery. J Controlled Release 2015; 219:396-405.

[51] Zeng H, Guo S, Ren X, Wu Z, Liu S, Yao X. Current strategies for exosome cargo loading and targeting delivery. Cells 2023; 12:1416.

[52] Wang J, Chen D, Ho EA. Challenges in the development and establishment of exosome-based drug delivery systems. J Control Release 2021; 329:894-906.

[53] Skotland T, Sandvig K, Llorente A. Lipids in exosomes: current knowledge and the way forward. Prog Lipid Res 2017; 66:30-41.

[54] Villarroya-Beltri C, Baixauli F, Mittelbrunn M, Fernández-Delgado I, Torralba D, Moreno-Gonzalo O, et al. ISGylation controls exosome secretion by promoting lysosomal degradation of MVB proteins. Nat Commun 2016; 7:13588.

[55] Kumar S, Karmacharya M, Michael IJ, Choi Y, Kim J, Kim I, et al. Programmed exosome fusion for energy generation in living cells. Nat Catal 2021; 4:763-74.

[56] Chattopadhyay M, Krok E, Orlikowska H, Schwille P, Franquelim HG, Piatkowski L. Hydration layer of only a few molecules controls lipid mobility in biomimetic membranes. J Am Chem Soc 2021; 143:14551-62.

[57] Chernomordik LV, Kozlov MM. Mechanics of membrane fusion. Nat Struct Mol Biol 2008; 15:675-83.

[58] Mukherjee D, Paul D, Sarker S, MdN Hasan, Ghosh R, Prasad SE, et al. Polyethylene glycol-mediated fusion of extracellular vesicles with cationic liposomes for the design of hybrid delivery systems. ACS Appl Bio Mater 2021; 4:8259-66.

[59] Lin Y, Wu J, Gu W, Huang Y, Tong Z, Huang L, et al. Exosome-liposome hybrid nanoparticles deliver CRISPR/Cas 9 system in MSCs. Adv Sci 2018; 5:1700611.