Targeted therapy using engineered extracellular vesicles: principles and strategies for membrane modification

Abstract

Extracellular vesicles (EVs) are 30-150 nm membrane-bound vesicles naturally secreted by cells and play important roles in intercellular communication by delivering regulatory molecules such as proteins, lipids, nucleic acids and metabolites to recipient cells. As natural nano-carriers, EVs possess desirable properties such as high biocompatibility, biological barrier permeability, low toxicity, and low immunogenicity, making them potential therapeutic delivery vehicles. EVs derived from specific cells have inherent targeting capacity towards specific cell types, which is yet not satisfactory enough for targeted therapy development and needs to be improved. Surface modifications endow EVs with targeting abilities, significantly improving their therapeutic efficiency. Herein, we first briefly introduce the biogenesis, composition, uptake and function of EVs, and review the cargo loading approaches for EVs. Then, we summarize the recent advances in surface engineering strategies of EVs, focusing on the applications of engineered EVs for targeted therapy. Altogether, EVs hold great promise for targeted delivery of various cargos, and targeted modifications show promising effects on multiple diseases.

Keywords: Exosome; Extracellular vesicles; Surface modification; Targeted delivery.

Fig. 1

Biogenesis of EVs and the mechanisms involved in the uptake of EVs by the recipient cell. A Exosomes are generated as ILVs within MVBs and secreted after MVBs fuse with the plasma membrane. Cargo sorting and ILV formation are regulated by ESCRT-dependent, ceramide and tetraspanin pathways. the trafficking and fusion of MVBs to the plasma membrane are regulated by Rab-7, Rab-11, Rab-27a/b, SNARE, and Syntaxin. Microvesicles are generated directly through the outward budding of plasma membranes. B EVs have bilayer lipid membrane, and protein and nuclei acid contents. EVs are composed of lipid membrane and protein, nuclei acid contents. CD63, CD81, and CD9 are the common membrane proteins of EVs. C EVs can be internalized by recipient cells through micropinocytosis, phagocytosis, caveolae/raft-dependent endocytosis, direct fusion, and receptor-mediated endocytosis. LFA-1, ICAM-1, CD81 and CD9 on vesicle membranes, are important for the binding and uptake of EVs

Fig. 2

Strategies for the membrane modification and targeted delivery of EVs. Targeting EVs to specific organs or cells can be achieved by membrane proteins and lipids through genetic modification, lipid insertion, covalent ligation, metabolic modification, affinity binding, enzymatic ligation

Fig. 3

Targeted delivery of circDYM to brain by genetically engineered EVs with RVG peptide for CUS treatment [112]. A Schematic diagram of the production of RVG-decorated and circDYM-packaged EVs from HEK 293T cell. B Representative near infrared fluorescence (NIRF) images of mice brains after intravenous administration of DiR-labelled mock EVs or RVG-EVs (200 μg) at different time points. C The significantly relieved depressive-like behaviors by RVG-EVs delivered CircDYM in CUS mice as measured by the behavior tests, including SPT, FST, TST and OFT (n = 6 for each group) (^*P, ^**P, ^***P vs the Control + RVG-Vector-EVs group; ^#P, ^##P, ^###P vs the CUS + RVG-Vector-EVs group). ^*P, ^#P < 0.05; ^**P, ^##P < 0.01; ^***P, ^###P < 0.001. Two-way ANOVA followed by the Holm–Sidak test were used for the multiple comparisons (Reproduced under the terms of the CC-BY 4.0. Copyright 2022, The Authors, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles)

Fig. 4

Targeted delivery of miR-210 by chemically modified EVs for MCAO treatment [145]. A Schematic diagram of RGD modification on EV surface by EDC/NHS coupling chemistry and click chemistry. B NIRF imaging of the MCAO mouse brains 6 h after the intravenous injection of PBS, unmodified EVs with miR-210 (Exo:miR-210), scramble peptide-modified EVs with miR-210 (Scr-exo:miR-210), and RGDyK-modified EVs with miR-210 (RGD-exo:miR-210) (EVs labeled with Cy5.5). C The mRNA level of VEGF in the lesion region of MCAO mice 24 h after the intravenous injection of PBS, Scr-exo:miR-210, RGD modified EVs (RGD-exo), RGD modified EVs with controlled RNA (RGD-exo:NC), and RGD-exo:miR-210 (^**P < 0.01 vs the Sham group; ^#P < 0.05, ^##P < 0.01 vs RGD-exo:miR-210 group using one-way ANOVA followed by Tukey’s post hoc test). D Survival rate of MCAO mice after the intravenous administration of RGD-exo-NC and RGD-exo:miR-210 (Reproduced under the terms of the CC-BY 4.0. Copyright 2019, The Authors, published by BioMed Central on behalf of the Journal of Nanobiotechnology)

Fig. 5

Targeted delivery of PTX by enzymatically conjugated RBCEVs for EGFR⁺ lung cancer treatment [156]. A Schematic diagram of the conjugation between biotin modified ET-NGL peptide (bi-EL-NGL) and GL containing EV (GL-EV) by OaAEP1 ligase. B Distribution of intravenously administrated uncoated RBCEVs, Cont-coated RBCEVs and ET-coated RBCEVs (DiR labeling) in different organs from EGFR⁺ lung cancer xenografted mouse model by IVIS imaging. C H&E staining and TUNEL assay of lung sections from EGFR⁺ lung cancer xenografted mouse model with the administration of PTX, PTX delivered by uncoated RBCEVs, PTX delivered by Cont-coated RBCEVs, and PTX delivered by ET-coated RBCEVs (TUNEL, green; Cell nucleus, blue; Scale bar = 100 μm) (Reproduced under the terms of the CC-BY 4.0. Copyright 2020, The Authors, published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles)

Fig. 6

Targeted delivery of TP and miR497 by liposome-EV fused vesicels for cisplatin-resistant ovarian cancer treatment [159]. A Schematic diagram of the production of miR497/TP HENPs by membrane fusing between RGD-modified liposome and CD47-bearing EVs, and biomineraliazation for the encapsulation of miR497. B Distribution of intravenously administrated free Dir dye, Dir-labeled liposome and Dir-labeled HENPs in different organs and tumor tissue from the SKOV3-CDDP xenografted mice by the in vivo imaging apparatus. C The dissected tumor tissue and the tumor growth record curves of SKOV3-CDDP xenografted mice after the intravenous administration of miR497, miR497-HENPs, TP, TP-HENPs, and miR497/TP-HENPs (^***P < 0.001 using two-way or one-way ANOVA for independent t test analysis by GraphPad Prism software 8.0) (Reproduced under the terms of the CC-BY 4.0. Copyright 2022, The Authors, published by BioMed Central on behalf of the Journal of Nanobiotechnology)

Fig. 7

Proposed model of chemical biology approaches for surface engineering of EVs. A The exosomal membrane proteins fused with HaloTag, SNAP-tag, CLIP-tag, ybbR tag, SpyTag, and SnoopTag are expressed in eukaryotic cells. The specific ligands for each tag consisting of ligands conjugated with targeting moieties can be covalently immobilized on the exosome membrane with surface chemical reaction. B Incorporating non-canonical amino acids into EV membrane proteins can promote the azide-alkyne cycloaddition. Then an alkynyl-bearing protein is conjugated to an azide-labeled targeting moiety such as peptide/protein/antibody/nanobody etc.

Fig. 8

EVs applications in biomedicine