Boosting Hydroxyl Radical Generation with Nitrogen Vacancy-Modified Carbon Nitride for Triggering Dual Damage of Cancer Nucleus DNA-Mitochondria against Hypoxic Tumors

Abstract

Introduction: Oral squamous cell carcinoma (OSCC) is a prevalent and deadly cancer, with over 350,000 new cases yearly. A hypoxic tumor microenvironment is the bottleneck of photodynamic therapy (PDT) and significantly weakens overall therapeutic efficacy.

Methods: In this study, we introduce nitrogen vacancy-modified PCN (N_V-PCN), a novel metal-free and O₂-independent photosensitizer designed for PDT. N_V-PCN targets Cal-27-induced OSCC by reducing highly expressed H₂O₂ in tumors to highly reactive •OH. This innovative approach aims to overcome the limitations posed by the hypoxic environment and enhance the effectiveness of PDT in treating OSCC.

Results: The introduction of N_V not only further improves the cell accessibility of PCN by increasing the content of -NH₂ but also provides more reactive sites for H₂O₂ reduction and facilitates carrier separation. Under illumination, N_V-PCN generates a burst of •OH around the nuclei and mitochondria of Cal-27 cells, which effectively kills the cells via synchronously leading to DNA damage and mitochondrial dysfunction. Compared to the conventional photosensitizer chlorin e6, N_V-PCN-based PDT exhibits excellent anticancer performance in vitro and in vivo, highlighting its potential as a next-generation therapeutic agent.

Conclusion: Collectively, the high •OH-generation efficiency, strong anticancer activity, and overall safety of the O₂-independent nanoparticle opens up new avenues for in-depth study on carbon nitride-based cancer PDT strategies. This work offers new hope for the effective treatment of OSCC and other challenging cancers.

Keywords: DNA-damage repair; hydroxyl radical; nitrogen vacancy; photodynamic therapy; polymeric carbon nitride.

Figure 1

Schematic illustration of (a) the preparation process and (b) the therapeutic process of N_V-PCN.

Figure 2

Characterization of as-prepared N_V-PCN. (a) TEM image of N_V-PCN. (b) XRD patterns, (c) FTIR spectra, and (d) N1s XPS signals of PCN and N_V-PCN. (e) Structure diagram of N_V-PCN. (f) UV-vis diffuse reflectance spectra. Inset: Tauc plots. (g) Band structures of PCN and N_V-PCN. h) In situ DRIFT spectra of H₂O₂ on PCN and N_V-PCN under illumination for 10 min at 5-min intervals. (i) In situ EPR signals of H₂O₂ over PCN and N_V-PCN under illumination.

Figure 3

PDT definitely promoted tumoral •OH expression in vitro. (a)Depicts a schematic diagram of detecting intracellular •OH in Cal-27 cells that have been pre-exposed to light, utilizing a •OH fluorescent probe. (b) Flow-cytometry analysis of •OH production in Cal-27 cells. (c) CLSM images of Cal-27 cells exposed to various conditions, where green fluorescence reflects •OH expression.

Figure 4

(a) Optimal concentrations of N_V-PCN acting on cells under light and dark conditions. **P<0.01, ****P<0.0001 compared to 0 μg/mL N_V-PCN + light. (b) Hemolysis values of various samples collected from the supernatants. ****P<0.0001 compared to the other groups.

Figure 5

N_V-PCN as a photosensitizer induces dual damage to cancer nuclear DNA and mitochondria. (a) Relative viability of Cal-27 cells incubated with PCN and N_V-PCN at a concentration of 1 mg·cm⁻¹ for 24 h with white LED light illumination for 30 min. (b) Cellular uptake evaluation of Cal-27 cells treated with N_V-PCN for 0.5–6 h using CLSM images. (c) Immunofluorescence images of γH₂AX foci (green) in Cal-27 cells treated withcontrol, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. Cell nuclei were stained with DAPI (blue). (d) Confocal microscopy images of the JC-1 probe in Cal-27 cells. (e) Flow cytometry of total ROS generation in Cal-27 cells under different treatments using DCFH-DA as intracellular total ROS indicator. Significance calculated by one-way ANOVA: *P<0.05, ****P<0.0001 compared to the control group.

Figure 6

N_V-PCN-mediated PDT promoted Cal-27 cell apoptosis. (a) Intracellular ATP levels of Cal-27 cells after various treatments. (b) Expression of 53BP1 and GADD45A in Cal-27 cells examined by fluorescence microscopy. (c) Expression levels of γH2AX, GADD45A, 53BP1, pro-caspase 3, cleaved caspase 3, and β-actin proteins in cells treated for 24 h in different groups detected by Western blot experiments. β-actin protein was used as the internal control. (d) EDU assay of Cal-27 cells with control, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. (e) Transwell assay of Cal-27 cells with control, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. The concentration of N_V-PCN was 1 mg·mL⁻¹. Significance calculated by one-way ANOVA: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 7

(a) Qualitative flow-cytometry data plot indicating the increase in apoptosis of Cal-27 cells after different treatments for 24 h. (b) Immunofluorescence images of cleaved caspase 3 (green) in Cal-27 cells treated with control, N_V-PCN, light, Ce6 + light, and N_V-PCN + light. Cell nuclei were stained with DAPI (blue).

Figure 8

In vivo antitumor effect of N_V-PCN-mediated PDT. (a) Schematic of the therapeutic process for cancer-bearing nude mice. (b) Fluorescence images of Cal-27 cancer-bearing mice and ex vitro fluorescence images of major organs and tumor tissue after intratumoral injection of Cy5.5–N_V-PCN at different time points. (c) Time-dependent surveillance of body weight for mice with different treatments over 22 days (n=4). (d) Time-dependent surveillance of tumor volume for mice with different treatments over 22 days (n=4). (e) Cancer images of each group derived from BALB/c mice at day 22 posttreatment. (f) H&E and immunohistochemical staining of tumor tissue of mice after various treatments. Significance calculated by one-way ANOVA: ****P<0.0001.

Figure 9

In vivo toxicity and safety assessment of N_V-PCN. (a) Hematoxylin and eosin–stained tissue sections from the mice to monitor histological changes in heart, liver, spleen, lung, and kidney 22 days after intratumoral injection of the N_V-PCN solution. (b) Blood biochemistry analysis of the mice treated with Ce6 and N_V-PCN. (c) Blood hematology analyses of mice on the last day.