Cancer Letters

Cancer Letters

Volume 371, Issue 1, 1 February 2016, Pages 48-61
Cancer Letters

Original Articles
Bovine milk-derived exosomes for drug delivery

https://doi.org/10.1016/j.canlet.2015.10.020Get rights and content

Highlights

  • Bovine milk is a biocompatible and scalable source of exosomes.

  • Milk exosomes can serve as a vehicle to deliver both hydrophilic and lipophilic small drug molecules.

  • Exosomal formulations provide enhanced biological efficacy.

  • Milk exosomes can be functionalized with ligands such as folic acid to achieve tumor targeting.

  • Milk exosomes per se exhibit protective effects presumably due to the endogenous cargo of macromolecules.

Abstract

Exosomes are biological nanovesicles that are involved in cell–cell communication via the functionally-active cargo (such as miRNA, mRNA, DNA and proteins). Because of their nanosize, exosomes are explored as nanodevices for the development of new therapeutic applications. However, bulk, safe and cost-effective production of exosomes is not available. Here, we show that bovine milk can serve as a scalable source of exosomes that can act as a carrier for chemotherapeutic/chemopreventive agents. Drug-loaded exosomes showed significantly higher efficacy compared to free drug in cell culture studies and against lung tumor xenografts in vivo. Moreover, tumor targeting ligands such as folate increased cancer-cell targeting of the exosomes resulting in enhanced tumor reduction. Milk exosomes exhibited cross-species tolerance with no adverse immune and inflammatory response. Thus, we show the versatility of milk exosomes with respect to the cargo it can carry and ability to achieve tumor targetability. This is the first report to identify a biocompatible and cost-effective means of exosomes to enhance oral bioavailability, improve efficacy and safety of drugs.

Introduction

Over the last three decades, a number of nanoparticle-delivery systems have been developed for cancer therapy, including natural and synthetic polymer-based, lipid-based, and organic and inorganic materials [1], [2]. However, due to inherent limitations, only a handful of them have been studied in clinics. The encapsulation of doxorubicin in liposomes (DoxilR) and paclitaxel in protein-based nanoparticles (AbraxaneR) represents two of the successful applications [3], [4], [5]. Factors that have stalled the clinical introduction of other nanoparticles include high cost, difficulty in reproducibly synthesizing them in sufficient quantities, and/or toxicity issues [2], [6]. The development of ideal nanoparticles with attributes such as long circulation time, evasion of the host immune system, and ability to target specific cells, minimum off site toxicity, and ability to carry versatile therapeutics remains elusive [7], [8].

Nature-derived nanoparticles could potentially overcome some of the limitations of synthetic liposomes. Among the different secreted membrane vesicles, exosomes intrinsically possess many attributes of a drug delivery vehicle [9], [10], such as they: (i) are well tolerated in the body, as evidenced by their wide distribution in various biological fluids (including milk) [11], [12], [13], (ii) have longer circulating half-life, (iii) are internalized by other cells, (iv) carry a cargo of macromolecules such as miRNA, mRNA, DNA and proteins that make these vesicles as delivery vehicles of therapeutics [14], [15], [16], and (v) are amenable to ligand attachment for tumor targetability.

Although the field of exosome-based therapeutics is in its infancy, the ability to engineer exosomes to display proteins, incorporate specific nucleic acid and protein cargos, load therapeutic agents, its targeted uptake and tolerance in vivo has been demonstrated to some extent [14], [15]. However, before exosomes are accepted as a delivery vehicle in clinics, the development of biocompatible, economically-viable source and methods for harvesting exosomes, which are effective and well-tolerated in vivo, must be demonstrated.

We report here the suitability of bovine milk as a potentially scalable source of exosomes that could serve as a drug delivery vehicle. Bovine milk consumption is generally considered to be safe and to provide important nutritional benefits [17]. Thus, availability, cost and toxicity considerations make bovine milk a suitable natural source for large-scale production of exosomes. We demonstrate that milk-derived exosomes can serve as a vehicle to deliver both hydrophilic and lipophilic small molecules, including chemotherapeutic (chemo) drugs. Using in vitro and in vivo models, we show enhanced biological efficacy of the exosomal formulations. This effect was further increased by the addition of tumor-targeting ligand, folic acid (FA). Therefore, milk exosomes represent a scalable, biocompatible and cost-effective means to potentially enhance oral bioavailability, improve efficacy and safety of drugs.

Section snippets

Isolation of exosomes

Milk from pasture-fed Holstein and Jersey cows during the mid-lactation period was obtained from a local dairy; colostrum was from 1–2 days postpartum. Exosomes were isolated by differential centrifugation. Briefly, milk was centrifuged at 13,000 × g in 250 mL centrifuge bottles (Nalgene, Thermofisher Scientific, Holtsville, NY) using TA-10.250 rotor and Allegra 25R centrifuge (Beckman Coulter Inc, Fullerton, CA) at 4 °C for 30 min to remove fat globules, casein aggregates and other debris. The

Isolation and biological characterization of milk-derived exosomes

We isolated milk exosomes from bovine raw milk by differential centrifugation (Fig. 1A, Fig. S1). The average yield of the exosomes was 335 ± 48 mg per liter of the milk prepared at different times. These exosomes were largely 40–100 nm in diameter, as measured by NanoSight (Fig. 1B), and exhibited a polydispersity index (PDI) of 0.22 ± 0.06, calculated at 37 °C using Zetasizer (Fig. 1C). The exosome size was confirmed to be <80 nm by atomic force microscopy (AFM) and scanning electron

Discussion

The focus of this study was to develop a biocompatible, exosome-based drug delivery technology. Secreted membrane vesicles such as exosomes inherently possess many characteristics of a drug delivery vehicle and could be accepted in clinics [30]. Here, we show bovine milk as a viable source for isolation of exosomes. Exosomes have been purified from biological fluids and cell culture media using a variety of strategies such as ultracentrifugation, density-based separation (sucrose and

Conclusions

In summary, we demonstrated that raw mature bovine milk can serve as a biocompatible and cost-effective source for harvesting bulk quantities of exosomes and that milk exosomes have tremendous potential as a drug carrier for hydrophilic and lipophilic agents, including chemo drugs. This nanodevice technology can overcome the limitations associated with the poor oral bioavailability of chemopreventives and chemotherapeutics and lower the total administered dose, thus minimizing or eliminating

Conflict of interest

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by the USPHS grants CA-118114, CA-125152 and R41-CA-189517, Kentucky Lung Cancer Research Program, Agnes Brown Duggan Endowment, and Helmsley Funds. Drs. Tereza Paronyan, Sham Kakar and Douglas D. Taylor are gratefully acknowledged for their assistance and use of SEM and AFM, Zetasizer and Nanosight, respectively. and Ms. Sarah Wilcher for drug gavage treatment. We thank Drs. Manicka Vadhanam and Wendy Spencer for useful discussions. We also thank Charles Dudley Tapp of

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    Authors contributed equally to this work.

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