A Targeted and pH-Responsive Nano-Graphene Oxide Nanoparticle Loaded with Doxorubicin for Synergetic Chemo-Photothermal Therapy of Oral Squamous Cell Carcinoma

Abstract

Purpose: Oral squamous cell carcinoma (OSCC) is a malignant disease with serious impacts on human health and quality of life worldwide. This disease is traditionally treated through a combination of surgery, radiotherapy, and chemotherapy. However, the efficacy of traditional treatments is hindered by systemic toxicity, limited therapeutic effects, and drug resistance. Fibroblast activation protein (FAP) is a membrane-bound protease. Although FAP has limited expression in normal adult tissues, it is highly expressed in the tumor microenvironment of many solid cancers - a characteristic that makes it an ideal target for anticancer therapy. In this study, we constructed a nano-drug delivery system (NPF@DOX) targeting FAP to increase the therapeutic efficiency of synergistic chemo-photothermal therapy against OSCC.

Methods: We utilized PEGylated nano-graphene oxide (NGO) to link doxorubicin (DOX) and fluorescently-labeled, FAP-targeted peptide chains via hydrogen bonding and π-π bonding to enhance the targeting capability of NPF@DOX. The synthesis of NPF@DOX was analyzed using UV-Vis and FT-IR spectroscopy and its morphology using transmission electron microscopy (TEM). Additionally, the drug uptake efficiency in vitro, photo-thermal properties, release performance, and anti-tumor effects of NPF@DOX were evaluated and further demonstrated in vivo.

Results: Data derived from FT-IR, UV-Vis, and TEM implied successful construction of the NPF@DOX nano-drug delivery system. Confocal laser scanning microscopy images and in vivo experiments demonstrated the targeting effects of FAP on OSCC. Furthermore, NPF@DOX exhibited a high photothermal conversion efficiency (52.48%) under near-infrared radiation. The thermogenic effect of NPF@DOX simultaneously promoted local release of DOX and apoptosis based on a pH-stimulated effect. Importantly, FAP-targeted NPF@DOX in combination with PTT showed better tumor suppression performance in vivo and in vitro than did either therapy individually.

Conclusion: NPF@DOX can precisely target OSCC, and combined treatment with chemical and photothermal therapy can improve the therapeutic outcomes of OSCC. This method serves as an efficient therapeutic strategy for the development of synergistic anti-tumor research.

Keywords: fibroblast activation protein; nano-graphene oxide; oral squamous cell carcinoma; photothermal therapy; targeted combination therapy.

Figure 1

The synthetic route and function of the NPF@DOX nanocarriers. The release of DOX was accelerated in the low-pH microenvironment, and the amount of DOX released increased with temperature.

Figure 2

Nanocarrier preparation, characterization, and measurements of photothermal effects and stability. (A) TEM images of NGO, NP, NPF, and NPF@DOX nanocarriers. Scale bar: 200 µm. (B) Photothermic heating curves of NPF@DOX under 808 nm irradiation (1.0 W/cm²) followed by cooling to room temperature. (C) Photothermal properties of NPF@DOX throughout five cycles of laser irradiation at 808 nm (1.0 W/cm²). (D) Drug release curves of nanoparticles under pH 7.4 or 5.5 conditions with or without 808 nm NIR.

Figure 3

FAP is highly expressed in OSCC. (A) Representative immunohistochemistry images of FAP staining in normal and OSCC tissues. Scale bar: 100 µm. (B) Representative immunohistochemistry images of FAP staining in HOK and CAL-27 cells. Scale bar: 50 µm.

Figure 4

In vitro cellular uptake and drug release. CLSM images of CAL-27 cells treated with NP@DOX or NPF@DOX with or without 808 nm NIR irradiation (1.0 W/cm²) to evaluate cellular uptake. Scale bar: 20 µm.

Figure 5

Cytotoxicity and in vitro combination therapy. (A) Viability of CAL-27 cells after treatment with control, NPF, NPF-NIR, NP@DOX, NPF@DOX, NP@DOX-NIR, and NPF@DOX-NIR. Data are presented as means ± SD, n=3 (***P<0.001). (B) CAL-27 cells stained with calcein AM (green) and propidium iodide (red) following various treatments were analyzed for live/dead status. Scale bar: 50 µm.

Figure 6

Tumor-specific infiltration using nanocarrier fluorescence. (A) In vivo imaging of CAL-27 tumor-bearing mice 1, 3, 6, 9, 12, and 24 h after intravenous injection of NPF-Cy5.5 or NS. (B) Ex vivo fluorescence images of NPF in the tumors and major organs/tissues after intravenous injection.

Figure 7

Continued.

Figure 7

In vivo antitumor efficacy of NPF@DOX in BALB/c mice and biosafety analysis. (A) Representative images of tumor-bearing mice taken 12 d post-treatment. The arrows in the images points to the location of the tumor. (B) Tumor volume growth after various treatments. Data are presented as means ± SD, n=3 (***P<0.001) (C) Body weight comparisons of mice following intravenous administration of different treatments in tumor-bearing BALB/c nude mice (mean ± SD) with 3 mice in each group. (D) H&E staining images of excised tumors at the end of treatment. Scale bar: 50 µm. (E) H&E staining images of hearts, livers, spleens, lungs, and kidneys of mice in each group after tail vein injection of nanocarriers. Scale bar: 50 μm.