Bacterial Outer Membrane Vesicle (OMV)-Encapsulated TiO2 Nanoparticles: A Dual-Action Strategy for Enhanced Radiotherapy and Immunomodulation in Oral Cancer Treatment

Abstract

Oral squamous-cell carcinoma (OSCC) poses significant treatment challenges due to its high recurrence rates and the limitations of current therapies. Titanium dioxide (TiO₂) nanoparticles are promising radiosensitizers, while bacterial outer membrane vesicles (OMVs) are known for their immunomodulatory properties. This study investigates the potential of OMV-encapsulated TiO₂ nanoparticles (TiO₂@OMV) to combine these effects for improved OSCC treatment. TiO₂ nanoparticles were synthesized using a hydrothermal method and encapsulated within OMVs derived from Escherichia coli. The TiO₂@OMV carriers were evaluated for their ability to enhance radiosensitivity and stimulate immune responses in OSCC cell lines. Reactive oxygen species (ROS) production, macrophage recruitment, and selective cytotoxicity toward cancer cells were assessed. TiO₂@OMV demonstrated significant radiosensitization and immune activation compared to unencapsulated TiO₂ nanoparticles. The system selectively induced cytotoxicity in OSCC cells, sparing normal cells, and enhanced ROS generation and macrophage-mediated antitumor responses. This study highlights TiO₂@OMV as a dual-action therapeutic platform that synergizes radiotherapy and immunomodulation, offering a targeted and effective strategy for OSCC treatment. The approach could improve therapeutic outcomes and reduce the adverse effects associated with conventional therapies.

Keywords: TiO2 nanoparticles; oral cancer; outer membrane vesicles; radiotherapy.

Figure 1

(a) TEM images of the TiO₂ NPs synthesized by TB/OA/OM = 1:6:4 molar ratio, the formation of spherical particles of TiO₂ NPs with an average size of 2–5 nm. (b) SEM images of the TiO₂ NPs with an aggregated spherical structure. (c) EDX of the TiO₂ NPs, showing the composition of carbon (C), oxygen (O), and titanium (Ti) elements. (d) XRD of the TiO₂ NPs.

Figure 2

Preparation and characterization of DH5-α outer membrane-coated nanoparticles (TiO₂@OMV NPs). TEM images of (a) OMV NPs, (b) bare TiO₂, and (c) TiO₂@OMV NPs. (a,c) Samples were negatively stained with phosphotungstic acid before imaging.

Figure 3

(a) Cell viability using PrestoBlue Cell Viability Reagent for CAL27 and L929 cells exposed to OMV at different concentrations (0, 0.1, 2, 5, 10 μg/mL) at 24 h. (b) Cell viability using PrestoBlue Cell Viability Reagent for CAL27 and L929 cells exposed to TiO₂@OMV nanoparticles at different concentrations (0, 0.1, 2, 5,10 μg/mL) at 24 h. The results are expressed as mean ± standard deviation (SD) of n = 3 biologically independent samples. Statistical analysis was performed using two-way ANOVA.

Figure 4

Fluorescence microscopic images of the localization of TiO₂@OMV nanoparticle uptake in (a) CAL27 cells and (b) L929 cells treated with TiO₂@OMV nanoparticle for 0, 3, 12, and 24 h. TiO₂@OMV nanoparticle concentration: 10 μg/mL (TiO₂@OMV NPs were labeled with vibrant DiD (red) fluorescence). Nucleuses were labeled with vibrant DAPI (blue) fluorescence. Actin was labeled with phalloidin (green) fluorescence. (c) The statistics of cell uptake with MetaMorphsoftware 7.10.4. Representative images were taken by a Leica fluorescence microscope at a magnification of 20× (Scale bar = 50 μm).

Figure 5

The effects of TiO₂@OMV nanoparticles on cell cycle distribution and apoptosis. (a) CAL27 and L929 cells were treated with media containing 0, 5, 7.5, and 10 μg/mL TiO₂@OMV nanoparticles. After 24 h, the cells were harvested and fixed in ice alcohol. Then, the cells were incubated in PBS containing 40 μg/mL propidium iodide and 100 μg/mL RNase A. Propidiumiodide-labeled nuclei were analyzed by flow cytometry. (b) The statistics of CAL27 cell cycle. (c) The statistics of CAL27 apoptosis. (d) The statistics of L929 cell cycle. (e) The statistics of L929 apoptosis.

Figure 6

The generation of ROS from TiO₂@OMV, detected by DCFDA after being exposed to a 6 MeV X-ray beam at radiation doses of 2 Gy (excitation: 485 nm and emission: 535 nm). The results are expressed as mean ± standard deviation (SD) of n = 3 biologically independent samples. Statistical analysis was performed using two-way ANOVA.

Figure 7

Cell viability of (a) CAL27 oral cancer cells, and (b) L929 normal cells incubated with TiO₂@OMV nanoparticles (various concentrations = 0, 0.1, 2, 5, 10 μg/mL) for 12 h and subjected to X-ray irradiation (6 MV, 2 Gy) or no irradiation. The results are expressed as mean ± standard deviation (SD) of n = 3 biologically independent samples. Statistical analysis was performed using two-way ANOVA **** p < 0.0001).

Figure 8

(a) Macrophage migration was analyzed 24 h after counterstaining cells in the lower chamber with DAPI. Representative images were taken by a Leica fluorescence microscope at a magnification of 20× (Scale bar = 50 μm). (b) The statistics of red fluorescent macrophages seeded to bottom chambers. (c) The presentation of the macrophage–cancer cell co-culture set up in transwell plates (the 3 μm microporous membrane allows for the migration of macrophages). DiD-labeled macrophages were seeded onto the insert and CAL27 cancer cells were seeded onto the bottom chamber. Cancer cells treated with OMVs (5 ug/mL) and TiO₂@OMV (5 ug/mL) for 12 h and were subjected to X-ray irradiation (6 MV, 2 Gy) or no irradiation.

Figure 9

The cell viability of the macrophage–cancer cell co-culture set up in transwell plates (0.4 μm microporous membrane). Macrophages were seeded onto the insert and CAL27 cancer cells were seeded onto the bottom chamber. Cancer cells treated with OMVs (5 ug/mL) and TiO₂@OMV (5 ug/mL) for 12 h and were subjected to X-ray irradiation (6 MV, 2 Gy) or no irradiation. The results are expressed as mean ± standard deviation (SD) of n = 3 biologically independent samples. Statistical analysis was performed using two-way ANOVA.

Figure 10

Cytokine levels of CAL27 cells medium. Macrophage–cancer cell co-culture and cancer cells treated with OMVs (5 ug/mL) and TiO₂@OMV (5 ug/mL) for 12 h and were subjected to X-ray irradiation (6 MV, 2 Gy) or no irradiation. After two days, the ELISA kit analysis (a) of TNF-α Cytokine levels. (b) IL-6 Cytokine levels.