Bioactive cell-like hybrids from dendrimersomes with a human cell membrane and its components

Abstract

Cell-like hybrids from natural and synthetic amphiphiles provide a platform to engineer functions of synthetic cells and protocells. Cell membranes and vesicles prepared from human cell membranes are relatively unstable in vitro and therefore are difficult to study. The thicknesses of biological membranes and vesicles self-assembled from amphiphilic Janus dendrimers, known as dendrimersomes, are comparable. This feature facilitated the coassembly of functional cell-like hybrid vesicles from giant dendrimersomes and bacterial membrane vesicles generated from the very stable bacterial Escherichia coli cell after enzymatic degradation of its outer membrane. Human cells are fragile and require only mild centrifugation to be dismantled and subsequently reconstituted into vesicles. Here we report the coassembly of human membrane vesicles with dendrimersomes. The resulting giant hybrid vesicles containing human cell membranes, their components, and Janus dendrimers are stable for at least 1 y. To demonstrate the utility of cell-like hybrid vesicles, hybrids from dendrimersomes and bacterial membrane vesicles containing YadA, a bacterial adhesin protein, were prepared. The latter cell-like hybrids were recognized by human cells, allowing for adhesion and entry of the hybrid bacterial vesicles into human cells in vitro.

Keywords: bacterial adhesin; bacterial membrane; coassembly; hybrid vesicles; mammalian cell.

Fig. 1.

Schematic illustration of (A) the preparation of giant DSs, (B) the preparation of HMV from human kidney cells 293 (HEK293), and (C) coassembly of giant hybrid vesicles from giant DSs, and HMV from HEK293 labeled with GFP.

Fig. 2.

Coassembly of giant hybrid vesicles from DS-RhB and HMV obtained from GFP labeled HEK293. (A) JD with 1% of JD-RhB coassembles into DS-RhB. Representative microscopy images of giant hybrid vesicles containing (B) CAAX-GFP HEK293 and (C) HEK293 without CAAX-GFP. A phase-contrast image was first acquired, followed by the fluorescence image by successive exposures on the same vesicle. Scale is identical in B and C.

Fig. 3.

Illustration of (A) the preparation of giant DSs, (B) the preparation of BMV expressing YadA bacterial adhesin protein, and (C) coassembly of giant hybrid vesicles from giant DSs and E. coli BMV expressing YadA bacterial adhesin protein.

Fig. 4.

Giant hybrid vesicles coassembled from DS-Coumarin with BMV or BMV-YadA. BMV-YadA represents BMV containing the YadA bacterial adhesion protein. (A) Structure of JD-Coumarin, which self-assembles into DS-Coumarin. (B) Encapsulation of giant hybrid vesicles (DS-Coumarin + BMV-YadA) into HeLa cells. (C) Lack of encapsulation of giant hybrid vesicles (DS-Coumarin + BMV) into HeLa cells due to the absence of YadA, as the control experiment for B. Scale is identical in B and C.

Fig. 5.

Crystal violet cytotoxicity assay. HEK293 cells were treated with either giant hybrid vesicles (DS-Coumarin + BMV-YadA, DS-Coumarin + BMV only), or DS-Coumarin only. Relative cell viability was measured by crystal violet quantification. Data represent averages and SDs of six independent measurements.

Fig. 6.

Representative confocal microscopy images of giant hybrid vesicles (DS-Coumarin + BMV-YadA) localizing to the cytoplasm in HeLa cells. HeLa cell membrane was stained with red FM4-64.

Fig. 7.

Representative confocal microscopy images of giant hybrid vesicles (DS-Coumarin + BMV-YadA) localizing to the cytoplasm in HEK293 cells. The lysosomes within cell cytoplasm were stained with LysoTracker Red DND-99 dye.

Fig. 8.

Representative confocal microscopy images of E. coli incubated with (A and B) giant hybrid vesicle (DS-Coumarin + HMV) or (C and D) giant vesicle (DS-Coumarin) E. coli cells expressing YadA (E. coli-YadA⁺, in A and C) or control E. coli cells without YadA (E. coli-YadA⁻, in B and D). HMVs were prepared by HEK293 human cells. E. coli cells and vesicles were monitored in bright-field and fluorescence images, respectively.