Fig. 1: Bioengineered nanovesicles with cancer cell recognition and enzyme-controlled membrane fusion functions for cytosolic siRNA delivery.

a Scheme shows engineering siRNA@eS-BNVs by genetically programming HEK293T cells via ACE2 receptor knock out and S protein knock in plasmids for obtaining eS-HEK293T^ACE2- cell membranes to encapsulate siRNA/PEI complexes. b Scheme shows siRNA@eS-BNVs for intracellular siRNA delivery by recognizing ACE2 receptors on cancer cells and enzyme-controlled membrane fusion via Thr-triggered S protein conformation change. c Structures and sequences of S and S^WT proteins. Left: simulated structural alignments of S (pink) and S^WT (blue, PDB: 6VXX), and S2’ (dark blue) in the S protein. Right: S protein and its cleavage site. S^WT is a transmembrane (TM) protein with S1 and S2 domains. S1 contains the native RBD domain for recognition. S2 contains S2’ linker, FP, HR1 and HR2 domains. FP domain is exposed by digestion of S2’ site, then HR1 and HR2 form six helical bundles, which are the main structures for membrane fusion. d Western blot analysis of ACE2 and S expressed on HEK293T and eS-HEK293T^ACE2- cells. β-actin was an internal reference. e Representative flow cytometry results show higher expression of S protein on eS-HEK293T^ACE2- cells (blue) than HEK293T^ACE2- cells (gray). f Diameter of eS-BNVs. g Representative TEM image of eS-BNVs. Scale bar,100 nm. h SDS-PAGE and western blot analysis of S protein in eS-BNVs and No-BNVs nanovesicles. PEI, polyethylenimine; FP, fusion peptide; HR1, heptad repeat 1‌; HR2, heptad repeat 2; RBD, receptor binding domain; S1, S1 Subunit; S2, S1 Subunit; S1/S2, S1/S2 cleavage site; S2’, S2’ cleavage site; S^WT, wild-type spike protein; S, mutant spike protein; Thr, Thrombin enzyme; EGFR, epidermal growth factor receptor; RISC, RNA-induced silencing complex. M, marker. Experiments in (d, g and h) were repeated three times independently with similar results. Source data are provided in a Source Data file.