Targeting Inflammatory Vasculature by Extracellular Vesicles

Abstract

Extracellular vesicles (EVs) are cell membrane-derived compartments that regulate physiology and pathology in the body. Naturally secreted EVs have been well studied in their biogenesis and have been exploited in targeted drug delivery. Due to the limitations on production of EVs, nitrogen cavitation has been utilized to efficiently generate EV-like drug delivery systems used in treating inflammatory disorders. In this short review, we will discuss the production and purification of EVs, and we will summarize what technologies are needed to improve their production for translation. We describe the drug-loading processes in EVs and their applications as drug delivery systems for inflammatory therapies, focusing on a new type of EVs made from neutrophil membrane using nitrogen cavitation.

Keywords: drug delivery; extracellular vesicles; inflamed endothelium; neutrophils.

Figure 1.

Flow chart of purification of EVs based on differential centrifugation. There are two major steps: 1) centrifugations at low speeds are used to separate cells/dead cells from EVs; 2) high speed ultra-centrifugations are used to isolate and purify EVs from soluble proteins and lipids.

Figure 2.

A. Scheme of a preparation process of cell membrane-formed nanovesicles by nitrogen cavitation and a series of centrifugations. B. The yield of EVs generated using nitrogen cavitation (NC-EVs) and EVs made from natural secretion in culture media (NS-EVs), determined based on the same quantity of cells. [Reprinted by permission from Elsevier, copyright 2016 and 2017, (42, 43)].

Figure 3.

Overview of drug loading strategies in EVs. Left: Loading small RNA or small molecules in EVs before EVs are generated. Therapeutics are introduced into cells via transfection or incubation, and thus, EVs contain loaded therapeutics when they are released from cells. Right: Different approaches for loading therapeutics in preformed EVs, including electroporation, sonication, incubation at room temperature (RT incubation), freeze/thaw, and extrusion.

Figure 4.

Schematic representation of targeting EVs to inflamed endothelium. Neutrophil membrane-derived nanovesicles possess integrin β₂ that binds ICAM-1 highly expressed on inflamed endothelium.

Figure 5.

Neutrophil (HL60 cells) membrane-derived nanovesicles mitigate acute lung inflammation. (A) Western blot of Integrin β₂ on HL-60 and erythrocyte cells (as control) and their nanovesicles. (B) Adhesion of DiO-labeled vesicles (green) to a cremaster venule labeled with anti-CD31 antibody (pink) in a live mouse after treatment with TNF-α (0.5 μg). The image was taken at 488 nm and 640 nm using a confocal microscope. TNF-α (C) and IL-6 (D) in BALF 10 h after administration of HBSS, TPCA-1 solution, EV-TPCA-1 (erythrocyte vesicles loaded with TPCA-1) and HV-TPCA-1 (HL60 vesicles loaded with TPCA-1) in an LPS-induced lung inflammation mouse model. All data are expressed as the mean ± SD (3–4 mice per group). * and ** represent p value<0.05 and 0.01, respectively. [Reprinted by permission from Elsevier, copyright 2016, (42)].

Figure 6.

(A) Scheme of remote loading of piceatannol into NC-EVs. (B) Leukocyte numbers in BALF (bronchoalveolar lavage fluid) in an LPS-induced lung inflammation mouse model. Vehicle, piceatannol and pic-NC-EVs were injected into mice 2 h after LPS challenge at a dose of 10 mg/kg, and BALF was examined 12 h after LPS challenge (C) Mouse survival rates in a LPS-induced sepsis model. The model was established by intraperitoneal administration LPS at a dose of 22 mg/kg. All data expressed as the mean ± SD (3–4 mice per group). ** and *** represent p value<0.01 and 0.001, respectively. [Reprinted by permission from Elsevier, copyright 2017, (43)].