Engineered Extracellular Vesicles for Drug Delivery in Therapy of Stroke

Abstract

Extracellular vesicles (EVs) are promising therapeutic modalities for treating neurological conditions. EVs facilitate intercellular communication among brain cells under normal and abnormal physiological conditions. The potential capability of EVs to pass through the blood-brain barrier (BBB) makes them highly promising as nanocarrier contenders for managing stroke. EVs possess several potential advantages compared to existing drug-delivery vehicles. These advantages include their capacity to surpass natural barriers, target specific cells, and stability within the circulatory system. This review explores the trafficking and cellular uptake of EVs and evaluates recent findings in the field of EVs research. Additionally, an overview is provided of the techniques researchers utilize to bioengineer EVs for stroke therapy, new results on EV-BBB interactions, and the limitations and prospects of clinically using EVs for brain therapies. The primary objective of this study is to provide a comprehensive analysis of the advantages and challenges related to engineered EVs drug delivery, specifically focusing on their application in the treatment of stroke.

Keywords: blood-brain-barrier; drug delivery; engineering; extracellular vesicles; stroke.

Figure 1

Composition and Structure of EVs. The structure of EVs consists of a phospholipid bilayer that encloses proteins (membrane protein and cargo protein) and nucleic acids. Membrane proteins encompass a variety of molecules, such as tetraspanins (including CD9, CD63, and CD81, among others), adhesion molecules (such as integrins, EpCAM, and Ephrin), the major histocompatibility complex (MHC), and receptors. Nucleic acids encompass DNA and RNA, which consist of various types of RNA molecules, such as messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA). The phospholipid bilayer confers protection to the contents enclosed within. Created with BioRender.com (https://app.biorender.com/illustrations/649b3cd0422a45d3d7cc3c29, accessed date: 7 July 2023).

Figure 2

Schematic of EV Subtypes. (A) Exosomes and their formation. (B) Microvesicles and their formation. (C) Apoptotic bodies and their formation. Created with BioRender.com (https://app.biorender.com/illustrations/64d0be423f71706d207a7881, accessed date: 7 August 2023).

Figure 3

Isolation and quantification techniques for EVs. The diagram illustrates frequently employed methods for analyzing EVs. EVs can be quantified in tissue homogenates and natural fluids, such as urine, saliva, and blood. The isolation of plasma or serum from blood is utilized in this context as an illustrative case. Nanoflow cytometry enables direct labeling and quantifying EVs in various fluid samples. In contrast, EVs have the potential to be separated and subsequently utilized for further analysis. Immunoprecipitation is a technique that can be employed to enhance the specificity of EV populations. EVs can be observed using electron microscopy (EM) or alternative high-resolution microscopy methodologies. Lipidomics and proteomics methodologies can also be utilized to analyze and describe the composition of EVs populations. Ultimately, EV concentration and size measurement can now be achieved through dynamic light scattering and nanoparticle tracking analysis. Additionally, EV products can be measured by employing susceptible protein or RNA assays. Created with BioRender.com (https://app.biorender.com/illustrations/649b163862468f1db106b519, Accessed date: 7 August 2023).

Figure 4

Choices and obstacles while using EVs. The application of EVs originating from diverse cellular origins is commonly observed in managing stroke. The properties of EVs associated with each cell type may have varying levels of tropism for brain vasculature or neuronal cells, which could impact their ability to target the brain effectively. However, a complete evaluation of these properties has yet to be conducted. Crossing the blood–brain barrier (BBB) poses a significant challenge. The method of administration affects the biodistribution and clearance of EVs and can also impact the effect’s nature, i.e., whether it is localized or systematic. Lastly, the dosing schedule can be single or repeated, affecting accumulation and efficacy. Created with BioRender.com (https://app.biorender.com/illustrations/647df1821b2f09af295c29aa, accessed Date: 5 June 2023).

Figure 5

The role of natural EVs in the pathophysiology of stroke. The management of stroke has shown benefits through direct local effects in the brain, such as neuroprotection, neurogenesis, angiogenesis, antioxidant and anti-inflammatory properties, and systemic effects by modulating peripheral immune system responses. These effects may create a favorable environment for cerebral regeneration. Created with BioRender.com (https://app.biorender.com/illustrations/647df0bfe74d4f82a5bc4ade, accessed: 11 June 2023).

Figure 6

Natural EV modes of action in stroke. The transfer of MiR-133b through EVs originating from mesenchymal stem cells (MSCs) has been found to facilitate the growth of neurites. This effect is achieved through the targeting of the converting protein RhoA. Additionally, miR-124 is linked to increased neuronal viability by targeting USP-14, a ubiquitin-specific protease. Systemically, it has been demonstrated that EVs from NSC reduce pro-inflammatory Th17 cells while improving immunosuppressive Treg cells. Created with BioRender.com (https://app.biorender.com/illustrations/647ca600a95ee7a5e7fd6757, accessed: 11 June 2023).

Figure 7

EV/NP. Transport Neural Remodeling. Poly(3,4-ethylenedioxythiophene) modified with tetrapeptide was administered utilizing a biocompatible chitin scaffold. In an in vivo model, myelin thickness increased, indicating nerve regeneration. At the injury’s site, Schwann cell adhesiveness and angiogenesis increased. Created with BioRender.com (https://app.biorender.com/illustrations/64a80816d2fc5a3659e189a9, accessed: 7 July 2023).

Figure 8

Engineering strategies for modifying EV content. Cellular engineering techniques can regulate EVs, including altering primitive cells or directly loading them via various post-isolation methods. (A) Cell engineering uses genetic manipulation techniques, such as plasmid transfection or enriching cells with miRNAs or small compounds, to load a parent cell indirectly. (B) Electroporation, sonication, freeze–thaw cycles, and chemical agents modulate isolated EVs post-isolation. Therapeutic substances and loading efficiency determine the optimum EV modulation strategy. EVs cargo manipulation can treat stroke by acting on cargo type. (C) Proteins. (D) Small molecules. (E) miRNAs. Created with BioRender.com (https://app.biorender.com/illustrations/647dfe244f0c62bb59ac8cb8, accessed: 6 June 2023).

Figure 9

Surface modulation of EVs. The modulation of EV surfaces can be accomplished by genetically modifying the cells that produce them. (A) Protein plasmids or (B) Protein-residues (C) directly conjugated to lipids that are then integrated into EV membranes. (D) Bio-orthogonal chemistry identifies extracellular vesicle functional groups. Created with BioRender.com (https://app.biorender.com/illustrations/6480cf6ae6947f82704ea72e, accessed: 11 June 2023).