Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes
Contributed by Michael L. Klein, January 22, 2016 (sent for review December 26, 2015; reviewed by Ling Peng and Donald Tomalia)
Significance
Cell surface determinants such as glycans, receptors, and adhesion molecules govern cell sociology in a complex manner. By forming cell-like hybrids of chemically programmable (glyco)dendrimersomes with bacterial membrane vesicles, evidence is obtained for the feasibility of combining chemical and biological surface design in one entity. Such tunable cell-like hybrids with custom-made combinations of surface epitopes and active receptors will likely find utility in dissecting the functionality of individual entities in complex networks and ultimately enable novel biomedical applications.
Abstract
A library of amphiphilic Janus dendrimers including two that are fluorescent and one glycodendrimer presenting lactose were used to construct giant dendrimersomes and glycodendrimersomes. Coassembly with the components of bacterial membrane vesicles by a dehydration–rehydration process generated giant cell-like hybrid vesicles, whereas the injection of their ethanol solution into PBS produced monodisperse nanometer size assemblies. These hybrid vesicles contain transmembrane proteins including a small membrane protein, MgrB, tagged with a red fluorescent protein, lipopolysaccharides, and glycoproteins from the bacterium Escherichia coli. Incorporation of two colored fluorescent probes in each of the components allowed fluorescence microscopy to visualize and demonstrate coassembly and the incorporation of functional membrane channels. Importantly, the hybrid vesicles bind a human galectin, consistent with the display of sugar moieties from lipopolysaccharides or possibly glycosylated membrane proteins. The present coassembly method is likely to create cell-like hybrids from any biological membrane including human cells and thus may enable practical application in nanomedicine.
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Naturally occurring (1), chemically modified (2, 3), and synthetic (4, 5) lipids, amphiphilic block copolymers (6, 7), polypeptides (8), Janus dendrimers (JDs) (9), and Janus glycodendrimers (JGDs) (10, 11) self-assemble into vesicles denoted as liposomes, polymersomes, dendrimersomes (DSs), and glycodendrimersomes (GDSs), respectively. These vesicles provide models for primitive (12) and contemporary (13, 14) cell membranes and drug-delivery devices (15–17). Recently, hybrid vesicles coassembled from naturally occurring phospholipids and amphiphilic block copolymers (18–20) have been described; these vesicles eliminated some of the deficiencies of liposomes, such as limited stability under oxidative conditions and general instability over time, and the deficiencies of polymersomes, which possess wide membrane thickness [8–50 nm (20)], exhibit toxicity, and can be tedious to synthesize. These hybrid vesicles combined the desirable feature of liposomes—specifically, their biologically suitable membrane thickness of 4 nm—with that of polymersomes, which are known for their stability. In addition, transmembrane proteins (21–23) could be incorporated into the phospholipid fragments of planar membranes derived from these assemblies. However, the variability in the extent of miscibility between the hydrophobic fragments of the phospholipid and the block copolymer (20) generates a complex morphology of the hybrid membrane that requires further characterization to enable practical applications both as drug-delivery devices and cell membrane models. Here, we report the coassembly of the components of DSs and GDSs with those of the bacterial membrane vesicles (BMVs) to generate functional hybrid vesicles. DSs, GDSs, and liposomes have hydrophobic fragments with similar chemical structures and similar membrane thickness (4.5–4.9 nm) (24). Therefore, the bacterial membranes with their intact native components are expected to be transferred to the hybrid vesicles, providing a new and simple method for the generation of bioactive cell-like hybrids of interest as critical nanoscale design parameters (25).
Results and Discussion
Coassembly of Giant DSs with BMVs.
Giant DSs ranging in diameter from 2 to 50 μm were prepared by hydration of JD films with ultrapure water or PBS (Fig. 1) (9, 24, 26). This protocol was previously used for the encapsulation of hydrophilic and hydrophobic dyes and drugs within giant DSs. Giant DSs prepared by this method were used for coassembly with BMVs via a dehydration–rehydration technique (27) (Fig. 1). In addition to a library of giant DSs, giant GDSs (10, 11, 28–31) self-assembled from sugar-presenting JGDs were also used for coassembly with BMVs. This process was expected to maintain the integrity of the components of bacterial membranes. The envelope of Gram-negative bacteria such as Escherichia coli contains an inner phospholipid bilayer membrane and an outer membrane consisting of an asymmetric bilayer with phospholipids in the inner leaflet and lipopolysaccharides (LPSs) in the outer leaflet (32–34). The inner and outer membranes contain various peripheral and integral membrane proteins that perform diverse functions such as membrane receptors, transporters, channels, and enzymes. To label BMVs, a small integral membrane protein, MgrB, which localizes to the inner membrane, was tagged with mCherry and expressed in E. coli (35). These BMVs enriched with red fluorescent mCherry–MgrB and provide a simple means for following the incorporation of an integral membrane protein by fluorescence microscopy, allowing the self-assembly and coassembly processes to be monitored.
Fig. 1.
Coassembly of Monodisperse Nanohybrids.
The coassembly of JD-3 (3,5)12G1-PE-(3,4,5)-3EO-G1-(OCH3)6 (9, 24) with the components of BMV was investigated by the injection of ethanol solubilized JD-3 and BMVs into PBS. Control experiments for the self-assembly of JD-3 and BMV components under identical conditions were also performed. All assemblies were studied by dynamic light scattering (DLS). This method produced monodisperse hybrid vesicles with dimensions of about 140 nm (Fig. S1). These values are in the range of DS-3 prepared by injection under similar conditions. The initially prepared BMV vesicles exhibit a bimodal molecular weight distribution, whereas BMV vesicles reassembled by ethanol injection into PBS buffer under identical conditions as DS-3 also exhibited bimodal molar mass distribution and dimensions ranging from 340 and 430 nm. These experiments suggest that the size and molar mass distribution of the hybrid vesicles coassembled by the injection method is determined by the contribution of the JD component. Monodisperse nanohybrids obtained by the injection method open numerous potential applications in nanomedicine.
Fig. S1.
Visualization of BMVs, Giant DSs, and of Their Coassembly.
Four JDs were selected from 19 libraries containing 144 compounds (Fig. 2) (9, 24, 26, 36). JD-1, JD-2, and JD-3 are twin–twin molecules (9, 24) containing twin-hydrophobic and twin-hydrophilic dendrons, whereas JD-4 is a single–single compound (26) based on single-hydrophobic and single-hydrophilic dendrons. These JDs were characterized by a combination of complementary methods including DLS, cryogenic transmission electron microscopy (cryo-TEM), differential interference contrast (DIC) microscopy, X-ray diffraction, confocal fluorescence microscopy, and micropipette aspiration (9, 24, 26). JDs possess a bilayer thickness of ∼4.5–4.9 nm (24), which is comparable to that of the phospholipid bilayer of biological membranes (4 nm), unlike most polymersomes, which are generally thicker (8–50 nm). The giant hybrid vesicles coassembled from DSs with BMVs containing mCherry–MgrB by the dehydration–rehydration protocol are comparable in size to giant DSs. These DS vesicles showed a strong red fluorescence signal along the boundary, indicating that these vesicles consist of both dendrimers and bacterial membrane components. It must be mentioned that BMVs alone do not form giant vesicles (Fig. S2) and instead only form small vesicles ranging from 20 to 200 nm (37).
Fig. 2.
Fig. S2.
Dual-Color Imaging of Coassembled Hybrid DS Vesicles.
To demonstrate the coassembly of the DSs with BMVs, DSs labeled with a suitable fluorophore compatible with mCherry were synthesized. Two modified JDs based on the structure of JD-3 were elaborated (Fig. 3). The pentaerythritol (PE) from the center of the JD was changed to Tris(hydroxymethyl)aminomethane (Tris) and the small hydrophobic fluorescent dye coumarin was incorporated to generate the fluorescent FL-JD-5 and FL-JD-6 (Figs. S3 and S4). These two molecules are constitutional isomers with the same ratio of hydrophobic and hydrophilic dendrons and differ only in their connectivity. FL-JD-6 is easier to prepare and purify because the hydrophobic fragments were introduced in the last step of the synthesis, dramatically changing the polarity from that of their precursors (Fig. S4). Both FL-JD-5 and FL-JD-6 self-assembled into identical giant DSs upon film hydration and showed intense cyan fluorescence (Fig. 3 A and B and Fig. S5). When the giant fluorescent DSs (FL-DSs) were coassembled with BMVs enriched with mCherry–MgrB, both cyan and red fluorescence along the boundary of the vesicles were observed, demonstrating that the assemblies were hybrid vesicles (Fig. 3 C and D). Together with their 3D intensity plots, this dual-color design proved that the coassembly of giant FL-DSs and BMVs was achieved.
Fig. 3.
Fig. S3.
Fig. S4.
Fig. S5.
Bioactive Hybrid Vesicles Containing Functional Channels.
E. coli is a Gram-negative bacterium with a complex cell envelope containing diverse functional components such as channel proteins, transporters, receptors, enzymes, and LPSs in its membranes (32–34) (Fig. 1). Among the transmembrane proteins, nonspecific porins such as OmpF and OmpC located on the outer membrane are particularly abundant (33, 38). These β-barrel proteins allow passive diffusion of small molecules up to 600 Da in size. The coassembly of BMVs with giant DSs was likely to incorporate functional porins in the hybrid vesicles, rendering the membrane permeable to low molecular weight compounds. To test for membrane permeability, the fluorescent dye rhodamine 6G (Rho) (479 Da), which is smaller than the porin size cutoff, was encapsulated into the giant DS (Fig. 4) to confirm the membrane impermeability demonstrated previously (9). As expected, the giant DS display intense yellow fluorescence in the center of the vesicle (Fig. 4C). Because of the wide emission spectrum of Rho (Fig. S6), a fluorescence signal was also detected in the red wavelength region. When Rho was coassembled with dendrimers and bacterial membranes to make hybrid vesicles, on the other hand, the yellow fluorescence was not detectable, whereas the red fluorescence of the mCherry–MgrB from bacterial membranes was visible at the boundary of the vesicle, as expected. These results are consistent with the low-molecular-weight dye diffusing through porin channels in the hybrid vesicles. In addition to Rho, a green fluorescent dye, calcein (622 Da), which is close in size to the porin diffusion limit was used. Calcein was encapsulated in both the giant DSs (Fig. 5B) and in the hybrid vesicles (Fig. 5C) without any leakage being observed, as expected for membrane permeability mediated by porins in the hybrid vesicles. The fluorescence of calcein is strongly quenched by cations such as Co2+ at near neutral pH (39). If there are functional channel proteins within the hybrid vesicles, Co2+ should quench the fluorescence of calcein. To test this possibility, Co2+ (1 mM) was added to calcein-loaded giant DS (Fig. 5D) as well as to the hybrid vesicles (Fig. 5E). Subsequently, samples were studied under the microscope less than 5 min after the addition of Co2+. The fluorescence inside the giant DSs (Fig. 5D) remained unquenched, whereas the calcein fluorescence from the hybrid vesicles (Fig. 5 A and E) was quenched. Interestingly, closer examination of a number of hybrid vesicles in this experiment revealed variable levels of calcein fluorescence quenching (Fig. S7). This result may be attributable to a heterogeneous distribution of porins within each individual hybrid vesicle that depends on the amount of BMVs stochastically incorporated into each hybrid vesicle. It is also possible that some of these vesicles are multilamellar, with interior layers possibly lacking porin channels. These results indicate that small molecules like rhodamine 6G (Fig. 4) and cations like Co2+ (Fig. 5) can pass through the bacterial membrane porin channels, whereas larger molecules such as calcein, which are closer to the size of the porin cutoff (Fig. 5), are not able to pass through.
Fig. 4.
Fig. 5.
Fig. S6.
Fig. S7.
Human Galectin-8 Binds the LPSs from the Surface of Hybrid Vesicles.
LPS is a major component of the outer leaflet of the outer membrane of E. coli and other Gram-negative bacteria. LPS is important for membrane stability and defense against antimicrobial compounds. Galectins are a class of secreted sugar-binding proteins that have specificity for β-galactosides. Galectin-8 (Gal-8), a tandem-repeat type galectin (Fig. 6A) (40) regulates cell–matrix interaction and is important for animal cell growth. Gal-8 also plays a significant role in autophagy, enabling mammalian host defense against bacterial invasion (41). E. coli LPS contains numerous sugar moieties. In addition, some E. coli membrane proteins may be glycosylated (42). To test whether the surface of E. coli MG1655 and membrane vesicles derived from this strain can be labeled with Gal-8, a fluorescein isothiocyanate (FITC)-labeled Gal-8 (Gal-8–FITC) was used to perform binding experiments with both live E. coli cells (Fig. 6B) and BMVs (Fig. 6C) enriched with mCherry–MgrB. Green fluorescence from FITC was detected in both samples, confirming that the lectin binds the E. coli cell surface as well as BMVs. As a control experiment, the giant DS-3 was tested for binding with Gal-8–FITC. No fluorescence was detected in the control experiment (Fig. 6D). However, when hybrid vesicles coassembled from giant DS-3s with BMVs were incubated with Gal-8–FITC, both green fluorescence from Gal-8–FITC (although a weak signal) and red fluorescence from mCherry–MgrB were observed along the boundary of the vesicle (Fig. 6E). Giant GDSs (10, 11, 28–31) were assembled from the JGD JGD-7 (Fig. 6 F and G) using the protocol used for giant DSs and subsequently were tested for Gal-8–FITC binding. As shown in Fig. 6E, Gal-8–FITC is strongly targeted to the giant GDSs because of the very high surface density of lactose [β-d-galactopyranosyl-(1→4)-d-glucose], which is a β-galactoside sugar (β-galactose + glucose) (11, 30, 43). Hybrid vesicles coassembled from giant GDS-7 and BMVs enriched with mCherry–MgrB also clearly exhibited both strong green and red fluorescence on the membrane (Fig. 6H).
Fig. 6.
Conclusions
A simple method to create hybrid vesicles from the coassembly of DSs or GDSs with vesicles generated from bacterial membranes is demonstrated. This method transfers many of the components of the bacterial inner and outer membranes, including transmembrane proteins such as MgrB and porins, and possibly LPSs, into the structure of the hybrid vesicle. These cell-like hybrid structures provide a platform that should be readily applicable to the coassembly of other biological membranes—including those from mammalian cells—with DSs and GDSs. Through standard molecular genetic methods, specific membrane components of interest can be engineered (such as the membrane protein mCherry–MgrB shown in this work) and incorporated in the hybrid vesicles. Our attempts to coassemble polymersomes with BMVs by a similar method did not result in hybrid vesicles, likely because of the large thickness of polymersome membranes. Artificial cell-like hybrid assemblies such as those described here have potential to impact diverse fields, such as targeted drug delivery, detection by fluorescence, vaccines, and other areas of nanomedicine (44–49). Future experiments have the potential to alter the membranes of living cells directly by DSs or GDSs into their membranes without the need for genetic manipulation.
Methods
Giant DSs and GDSs were prepared by the following general method. A solution of JD or JGD (10–40 mg⋅mL–1, 200 μL) in tetrahydrofuran (THF) was deposited on the top surface of a roughened Teflon sheet (1 cm2), placed in a flat-bottom vial, and followed by evaporation of the solvent for 2 h. The Teflon sheet was dried in vacuo for additional 12 h. Milli-Q water (2 mL) was added to merge the dendrimer film on Teflon sheet, and the vial was placed in a 60 °C oven for 12 h for hydration. The sample was then mixed using a vortex mixer for 30 s with a final concentration of 1–4 mg⋅mL–1.
Strain AML20 (35), which is derived from E. coli K-12 strain MG1655, contains a gene deletion mgrB. AML20 cells were transformed with plasmid pAL22 expressing the mCherry–MgrB fusion protein, which is inserted into the bacterial inner membrane. Strain AML20/pAL22 was streaked out from the –80 °C freezer stock on LB (Lysogeny broth; Fisher Scientific) agar (1.5% wt/vol) and incubated overnight at 37 °C to obtain isolated colonies. A single colony was then inoculated in LB medium for overnight growth at 37 °C on a roller drum for aeration. A saturated culture of AML20/pAL22 was diluted 1:200 in 50 mL of LB medium supplemented with ampicillin (100 μg⋅mL–1) in a 250-mL culture flask. Following growth for 1.5 h at 37 °C with aeration, mCherry–MgrB expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM. Protein induction was carried out for 3.5 h, and then cells were harvested at 3,500 × g for 10 min using a Beckman table-top centrifuge at 4 °C. The cell pellets were washed in Tris(hydroxymethyl)aminomethane⋅HCl (Tris⋅HCl) buffer (50 mM, pH 8.0) and then frozen at –80 °C.
For membrane preparation, frozen cell pellets were resuspended in 400 μL of solution of sucrose (20%) and Tris⋅HCl (30 mM, pH 8.0). A fresh stock solution of lysozyme (Sigma L6876) was prepared by dissolving 10 mg of lysozyme in 1 mL of EDTA (0.1 M, pH 7.3); 50 μL of lysozyme solution was added to the resuspended pellet and incubated on ice for 30 min. After this step, 5 mL of the solution containing EDTA (3 mM, pH 7.3) and 30 μL of protease inhibitor mixture (1:100 dilution final; Sigma P8849) was added to each tube. Cells were sonicated for 20 s at a low power (30% amplitude) and repeated for a total of five rounds of 20 s each with 1 min of incubation on ice between each round of sonication. Cells were spun at 4 °C for 5 min at 7,500 × g and the supernatant was transferred to a Nalgene white screw-cap centrifuge tube to isolate the membranes from the cytoplasmic fraction and cell debris. This supernatant was then spun at 4 °C for 30–45 min at 40,000 × g in a Beckman centrifuge fitted with SL-50T rotor. The bacterial membrane fraction was obtained as vesicles following the high-speed centrifugation, and the supernatant was discarded. The membrane vesicles were resuspended in 500 μL of buffer composed of Tris⋅HCl (20 mM, pH 8.0) and EDTA (0.1 mM).
The hybrid vesicle preparation protocol was adapted from the literature (27). Bacterial membrane preparation enriched with mCherry–MgrB was added at a final dilution of 1:25–1:100 to a solution of premade giant DSs or GDSs (50 μL) in a flat-bottomed glass vial. The solution was mixed and dehydrated using microcentrifuge adapters in a vacuum centrifugal evaporator (Savant SpeedVac). Dehydration was performed at room temperature for ∼30 min, until all of the liquid was removed and a dry film was formed. The glass vials were then loosely capped and transferred to a humid chamber containing excess water to create a water saturated atmosphere and incubated at 37 °C for 1 h. Sucrose (500 mM, 25 μL) solution was added to each vial, and the hybrid vesicles were allowed to swell overnight (∼16 h) at 37 °C. For vesicles encapsulated with dyes, sucrose solution (500 mM, 25 μL) prepared in Tris⋅HCl (10 mM, pH 7.5) was added during overnight swelling.
Acknowledgments
This work was supported by National Science Foundation Grants DMR-1066116 and DMR-1120901 (to V.P.), the P. Roy Vagelos Chair at the University of Pennsylvania (V.P.), the Humboldt Foundation (V.P.), National Science Foundation Grant DMR-1120901 (to M.L.K., M.G., and D.A.H.), National Institutes of Health Grant R01-GM080279 (to M.G.), and the EC Seventh Framework Programme (GLYCOPHARM) (H.-J.G.).
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Published online: February 16, 2016
Published in issue: March 1, 2016
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Acknowledgments
This work was supported by National Science Foundation Grants DMR-1066116 and DMR-1120901 (to V.P.), the P. Roy Vagelos Chair at the University of Pennsylvania (V.P.), the Humboldt Foundation (V.P.), National Science Foundation Grant DMR-1120901 (to M.L.K., M.G., and D.A.H.), National Institutes of Health Grant R01-GM080279 (to M.G.), and the EC Seventh Framework Programme (GLYCOPHARM) (H.-J.G.).
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The authors declare no conflict of interest.
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Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes, Proc. Natl. Acad. Sci. U.S.A.
113 (9) E1134-E1141,
https://doi.org/10.1073/pnas.1525589113
(2016).
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