Extracellular Vesicles as Precision Delivery Systems for Biopharmaceuticals: Innovations, Challenges, and Therapeutic Potential

Abstract

Unlike traditional small-molecule agents, biopharmaceuticals, like synthetic RNAs, enzymes, and monoclonal antibodies, are highly vulnerable to environmental conditions. Preservation of their functional integrity necessitates advanced delivery methods. Being biocompatible, extracellular vesicles (EVs) gained attention as a promising system for delivering biopharmaceuticals, addressing challenges related to the stability and efficacy of sensitive therapeutic molecules. Indeed, EVs can cross biological barriers like the blood-brain barrier, delivering therapeutic cargo to tissues that are traditionally difficult to reach. Recent innovations in surface modification technologies, including ligand and antibody attachment, have further enhanced EVs' targeting capabilities, making them particularly effective in personalized medicine. Here, we review the versatile suitability of EVs for being next-generation delivery vehicles of biopharmaceuticals, including current standings, practical challenges, and possible future directions of the technology.

Keywords: RNA therapeutics; biopharmaceuticals; extracellular vesicles; gene therapy.

Figure 1

Comparative overview of exosome and microvesicle biogenesis and composition. Explored on the left in the figure, exosomes originate from the inward budding of endosomal membranes forming intraluminal vesicles (ILVs) within multi-vesicle bodies (MVBs). MVBs are destined for either lysosomal degradation or exocytosis via plasma membrane fusion. Exosomes are characterized by CD9, CD63, and CD81. In contrast, microvesicles (MVs, right side of the figure), are formed directly via the outward budding and fission of the plasma membrane. MVs are enriched in proteins like integrins and proteases, and lipids like phosphatidylserine. Shared components between the two types of EVs include nucleic acids (mRNAs, miRNAs, non-coding RNAs) and proteins involved in vesicle trafficking and fusion (e.g., ALIX, TSG101). EGFR: Epidermal Growth Factor Receptor; SOD-1: Superoxide Dismutase; HSP70: Heat Shock Protein-70; ALIX: ALG-2-interacting protein-X; RAB: Ras-associated binding protein; TSG101: Tumor susceptibility gene-101; ICAM-1: Intracellular adhesion molecule-1; MDRP: Multidrug Resistant Protein. Created with BioRender.

Figure 2

Extracellular vesicle sources, yield optimization, isolation, and characterization strategies. Extracellular vesicles can be derived from mesenchymal stem cells (MSCs), neural stem cells (NSCs), macrophages, tumor cells, HEK293T cells, milk cells, and plant-derived cells (e.g., lemon EVs). These EVs can be obtained from body fluids such as saliva, urine, blood, cerebrospinal fluid (CSF), and lymph, or directly from milk and plants. To enhance EV yield prior to isolation, donor cells can undergo yield optimization strategies such as hypoxic preconditioning, cytokine stimulation (e.g., TNF-α, IL-1β), or genetic engineering targeting key EV biogenesis regulators (e.g., Rab27a/b, TSG101, ALIX, CD63). To isolate EVs, most implemented techniques include ultracentrifugation (high-speed spinning to separate EVs based on density), extrusion (forcing fluids through nanoporous membranes for size-based separation), ultrafiltration (filtering EVs through membranes of specific pore sizes), and size-exclusion chromatography (separating EVs from contaminants based on molecular size differences). Once isolated, EVs are characterized using methods such as nanoparticle tracking analysis (NTA) for size/concentration, mass spectrometry for cargo profiling, PCR for nucleic acid quantification, atomic force microscopy (AFM) and electron microscopy (EM) for morphology, and flow cytometry for surface marker profiling. The result is a purified EV population ready for use. Created with BioRender.

Figure 3

Cargo loading strategies for extracellular vesicle-mediated biopharmaceutical delivery. Pre-loading includes the natural integration of cargo into EVs during biogenesis, as well as the engineering of donor cells by transduction and transfection. Co-incubation, electroporation, sonication, extrusion, freeze–thaw, and microfluidics are post-loading techniques that include introducing cargo directly into isolated EVs. These techniques enable the loading of a variety of therapeutic molecules, such as membrane proteins (Lamp2b, Glypican-3 (GPC3) fusion protein, E7-Lamp2b fusion), siRNAs (HGF-specific siRNA, BACE1 siRNA, and Zika virus genome-specific siRNA), mRNAs (LDLR mRNA), CRISPR/Cas9 constructs, and enzymes/proteins (Neprilysin/CD10, Catalase, and monoclonal antibodies like Trastuzumab). Neurodegenerative diseases, lung cancer, hepatocellular carcinoma, cystic fibrosis, kidney cancer, gastrointestinal cancers, and inflammatory diseases like osteoarthritis may all benefit from the use of these EV-based treatments, which can be delivered intradermally, intranasally, intramuscularly, or subcutaneously. Created with BioRender.