FIGURE 4.

Exosome engineering strategies. Two generalized approaches are employed to engineer exosomes to improve cell‐specific targeting. The first approach (a–c) relies on modifying the exosome biogenesis and assembly process in producing cells. The second approach (d–f) involves direct modification of isolated exosomes. Producing cell modification: (a) Fusing EV membrane proteins with cell type‐specific binding moieties; (b) Insertion of chimeric VSVG/cell type‐specific targeting moieties into exosome membrane; (c) Metabolic reprogramming of producing cells. Culturing producer cells in growth media supplemented with modified amino acid analogues (e.g., ADA) and/or azide‐containing saccharides introduces active sites for bioconjugation to target cell‐specific moieties via azide‐involved biorthogonal reactions, that is, click chemistry. Direct exosome modification: (d) Introduction of alkyl groups to exosome membrane proteins via modification by NHS, EDC, and 4‐pentanoic acid allows for bioconjugation to azide‐containing cell type‐specific cargo moieties mediated by CuSO₄ and bathophenanthroline; (e) Binding biotinylated cell type‐specific targeting moieties to streptavidin‐conjugated glycolipids that insert into the lipid bilayer exosome envelope; (f) Insertion of targeting moiety/anchoring peptides that recognize and bind to exosomal membrane proteins. AHA, L‐azidohomoalanine; CuSO₄, cupric sulphate; EDC,5‐ethynl‐2‐deoxycytidine; EV, extracellular vesicles; ManNAz, tetraacetylated N‐acetylazido‐D mannosamine; MVB, multivesicular body; NHS, N‐hydroxysuccinimide; scFv, single chain variable fragments.