Bacterial outer membrane vesicle nanorobot

Abstract

Autonomous nanorobots represent an advanced tool for precision therapy to improve therapeutic efficacy. However, current nanorobotic designs primarily rely on inorganic materials with compromised biocompatibility and limited biological functions. Here, we introduce enzyme-powered bacterial outer membrane vesicle (OMV) nanorobots. The immobilized urease on the OMV membrane catalyzes the decomposition of bioavailable urea, generating effective propulsion for nanorobots. This OMV nanorobot preserves the unique features of OMVs, including intrinsic biocompatibility, immunogenicity, versatile surface bioengineering for desired biofunctionalities, capability of cargo loading and protection. We present OMV-based nanorobots designed for effective tumor therapy by leveraging the membrane properties of OMVs. These involve surface bioengineering of robotic body with cell-penetrating peptide for tumor targeting and penetration, which is further enhanced by active propulsion of nanorobots. Additionally, OMV nanorobots can effectively safeguard the loaded gene silencing tool, small interfering RNA (siRNA), from enzymatic degradation. Through systematic in vitro and in vivo studies using a rodent model, we demonstrate that these OMV nanorobots substantially enhanced siRNA delivery and immune stimulation, resulting in the utmost effectiveness in tumor suppression when juxtaposed with static groups, particularly evident in the orthotopic bladder tumor model. This OMV nanorobot opens an inspiring avenue to design advanced medical robots with expanded versatility and adaptability, broadening their operation scope in practical biomedical domains.

Keywords: bacterial outer membrane vesicle; enzyme propulsion; nanorobots; surface bioengineering.

Fig. 1.

Fabrication and characterization of OMV-siR robots. Schematic of (A) the fabrication process of OMV-siR robots with surface-bioengineered CPP capable of tumor-targeted binding and penetration, and (B) motion-enhanced gene silencing and immune stimulation for bladder cancer therapy. Fluorescence images showing the loaded siRNA (C) or modified urease (D) of OMV-siR robots in two independent experiments. siRNA or urease were labeled with Cy5 (red), respectively. The OMV body was stained with Dio (green). (Scale bar, 1.5 μm.) (E) TEM images of the siR-OMV and OMV-siR robot. (Scale bar, 100 nm.) (F) Hydrodynamic diameters and zeta potential of unmodified OMVs, siR-OMVs, and OMV-siR robots (n = 3; means ± SD). (G) Gel electrophoresis analysis of proteins presented on unmodified OMVs and OMV-siR robots. The samples were run at equal protein content and stained with Coomassie blue. Gel electrophoresis analysis representing siRNA degradation of free siRNA or OMV-siR robots after exposure to RNase solution (H) or serum-containing medium (I) for various durations.

Fig. 2.

Motion performance of OMV-siR robots. (A) Typical motion trajectories (over 25 s), (B) MSD, (C) diffusion coefficient (D_eff), and (D) speed of OMV-siR robots in PBS solution with various urea concentrations (n = 15; means ± SEM). (E) Typical motion trajectories (over 25 s), (F) MSD, (G) Deff, and (H) speed of unmodified OMVs and OMV-siR robots in real urine, collected from mice bearing the orthotopic bladder tumor (n = 15; means ± SEM).

Fig. 3.

Drug loading and release profiles, and in vitro anticancer effect of OMV-siR robots. (A) Loading efficiency of OMV-siR robots upon different siRNA inputs (n = 3; means ± SD). (B) Cumulative siRNA release from OMV-siR robots at pH 5.0 or 7.4 over 12 h (n = 3; means ± SD). (C) Fluorescence images of MBT-2 cells after incubation with siR-OMVs or OMV-siR robots for 2 h in the presence of 100 mM urea. Cells and siRNA were labeled by Hoechst 33342 (blue) and Cy5 (red), respectively. (Scale bar, 10 μm.) (D) Viability of MBT-2 cells after first incubation with various solutions for 2 h at 100 mM urea, including PBS, siRNA, OMVs, siR-OMVs, and OMV-siR robots, then separation for another 22 or 46-h incubation (n = 3; means ± SD). (E) Relative survivin mRNA expression in MBT-2 cells after incubation with nanorobots and other control groups for 2 h at 100 mM urea, followed by isolation for another 22-h culture (n = 3, mean ± SD). (F) Western blot for survivin in MBT-2 cells after culture with nanorobots and other constructs for 2 h at 100 mM urea, then separated to incubate for 46 h; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. *P < 0.05; one-way ANOVA.

Fig. 4.

In vitro and in vivo evaluation of tumor targeting and penetration of OMV-siR robots. (A) Schematic, (B) fluorescence images, and (C) quantified Dil fluorescence intensity showing the binding affinity of OMV-siR robots after incubation with various cells for 2 h in the presence of 100 mM urea, including bEnd.3 mouse brain endothelial cells, SV-HUC-1 human uroepithelial cells and MBT-2 mouse bladder carcinoma cells. Cells and nanorobots were stained by Hoechst 33342 (blue) and Dil (red), respectively. (Scale bar, 10 μm.) (D) Schematic and (E) fluorescence images representing the adhesion and penetration of siR-OMV or OMV-siR robots to MBT-2 cell spheroids after coincubation for 2 h at 100 mM urea. OMV membranes and cell spheroids were stained with Dil (red) and Hoechst 33342 (blue), respectively. The fluorescence images of cell spheroids were captured at various depths. (Scale bar, 20 μm.) (F) Schematic and (G) Fluorescence images showing the distribution of siR-OMV or OMV-siR robots after intravesical instillation to the mouse bearing orthotopic bladder tumor for 2 or 12 h. OMVs and bladder tumor tissues were stained with Dil (red) and Hoechst 33342 (blue), respectively. (Scale bar, 1 mm.)

Fig. 5.

In vivo immunomodulation and gene silencing of OMV-siR robots. (A) Schematic of the treatment course of mice bearing orthotopic MBT-2 bladder tumor, where the effects of immune stimulation and gene silencing were assessed on day 15. The percentage of (B) matured DCs (gated on CD11c+ DC cells), (C) CD4+ and (D) CD8+ T cells (gated on CD3+CD45+ T cells), and (E) macrophages in resected bladder tumor tissues from mice intravesically instilled with PBS, OMVs, siR-OMVs, and OMV-siR robots (n = 3; means ± SD). Cytokine contents in excised bladder tumor tissues from mice treated with nanorobots and other control groups: (F) IFN-γ, (G) TNF-α, and (H) IL-6 (n = 3; means ± SD). (I) Western blot and (J) immunohistochemistry staining of survivin expression in resected bladder tumor tissues from mice upon intravesical administrations of nanorobots and other control groups. (Scale bar: 100 μm.)

Fig. 6.

In vivo antitumor effect of OMV-siR robots using an orthotopic bladder tumor model. (A) Schematic of the treatment course of mice bearing orthotopic MBT-2 bladder tumor. (B) Representative in vivo bioluminescence images of MBT-2 bladder tumor-bearing mice and (C) quantified bioluminescence intensity upon various intravesical administrations over the treatment course, including PBS, OMVs, siR-OMVs, and OMV-siR robots. (D) Survival rate of mice treated with nanorobots and other control groups over the treatment course (n = 5; mean ± SD). (E) Images, (F) weights and (G) hematoxylin and eosin (H&E) staining (Top: overall view; Bottom: zoom-in view) of bladder tumor tissues from mice upon intravesical instillations of nanorobots and other control groups at the end (day 32) of treatment (n = 5; mean ± SD). Scale bar, 2.5 mm for Top row and 100 μm for the Bottom row. (H) Body weights of tumor-bearing mice upon various intravesical administrations. The expression level of serum biomarkers of tumor-bearing mice after treated with nanorobots and other control groups at the end of treatment: (I) aspartate aminotransferase (AST), (J) BUN, (K) ALP, (L) LDH (n = 3; mean ± SD). ***P < 0.001; one-way ANOVA.