FAP-targeted Nano-drug Delivery System Combined With Photothermal Therapy For Oral Squamous-cell Carcinoma

Objective: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.The combination of targeted drug chemotherapy and photothermal therapy(PTT)provides a new therapeutic strategy for OSCC.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.The aim of this study was to construct a nano-drug delivery system targeting FAP,NGO-PEG-FAP targeting peptide@DOX,to improve the efficacy of chemotherapy-photothermal therapy for OSCC,and to investigate its therapeutic feasibility through in vitro and in vivo.Methods:1.Preparation and characterization of NPF@DOXNano-graphene oxide(NGO)was linked to chitosan-Polyethylene glycol(PEG)and targeted short peptides that can be specifically recognized and sheared by FAP.NPF@DOX nano-drug carrier was prepared by linking DOX with NGO via hydrogen bond and π-π bond.The morphology of NPF@DOX was observed by transmission electron microscopy(TEM),and the NPF@DOX was analyzed by UV-Vis spectroscopy and FT-IR.2.In vitro performance evaluationThe physicochemical analysis and photothermal properties and therapeutic effects of the nano-drug delivery system at the solution and cellular levels were investigated.The absorbance values of DOX were recorded by UV spectrophotometer and analyzed by substituting into the regression equation to verify the high drug loading rate of the NGO.PBS solution corresponding to the p H of tumor and normal tissue was set up in vitro to observe the drug release of the drug delivery system in it.The NPF@DOX was illuminated with an 808 nm laser emitter,observed by infrared thermography,and evaluated for its photothermal performance.This experiment used oral squamous cell carcinoma CAL-27 cells as a cell model to test the fluorescence imaging and therapeutic effects of the nano-drug delivery system,and the cytotoxicity of the nano-drug delivery system was evaluated by CCK-8 and apoptosis assay.3.In vivo performance evaluationThe tumor models were established by of Balb/c nude mice.NPF@DOX was injected into the tail vein and FL imaging was performed the distribution of the drug in vivo.The effect of photothermal therapy after drug injection was also investigated in vivo.The biological safety of NPF@DOX was studied by H&E staining and pathological analysis.Results:1.TEM images show that NPF@DOX nano-drug delivery system was successfully prepared.The distance between the layers of the NGO increases,resulting in wrinkles and curls due to PEG modification.Small particles as well as porous structures appear on the surface,which is made approximately porous by FAP-targeting peptides and π-πstacking bonds between the aromatic ring of DOX and the graphene oxide nanoparticles.The characteristic peaks in Fourier IR and UV spectra indicate that DOX and FAP target peptides successfully bind to NGO.2.In the drug loading experiment,the high drug loading rate of NPF@DOX system was 44.5 ± 2.1%.The results of drug release experiments at different p H showed that the cumulative drug release at p H 5.5 was 33% higher than that at p H 7.4.The photothermal properties of NPF@DOX were evaluated at 808 nm near infrared.The experimental results showed that the temperature of NPF@DOX solution rose sharply to about 50 ℃ with the increase of time and concentration,its temperature is enough to make the deep tissue achieve the effective photothermal treatment effect.In addition,the results of five heating and cooling cycles showed that NPF@DOX had no obvious attenuation in the rise of temperature,which indicated that the NPF@DOX drug-loading system had excellent photothermal stability.The results of CCK-8 and confocal experiments showed that NPF@DOX not only showed significant tumor targeting performance and high cell uptake,but also showed a significant increase in tumor uptake after further irradiation with 808 nm laser,the survival rate of CAL-27 cells significantly decreased to 42%,indicating that NPF@DOX combined with photothermal therapy can effectively induce apoptosis.3.Balb/c nude mice(subcutaneous CAL-27 tumor-bearing mice)were injected with NPF@DOX by tail vein.In vivo fluorescence imaging revealed that the activatable NPF probe fluoresces rapidly and specifically to the tumor,which helps distinguish tumor areas from normal tissues.At the tumor area,the FL signal was gradually enhanced and peaked at 6 hours after injection.Moreover,NPF@DOX combined with photothermal therapy showed almost complete tumor ablation,and the therapeutic effect was significantly higher than that of other groups.Conclusion:In this study,we successfully constructed a dual-stimulus response tumor-targeted NPF@DOX drug delivery system.The NPF exhibits good tumor aggregation through the EPR effect,where the targeted peptides are specifically cleaved by FAP to release DOX.After tumor cell uptake,the anti-tumor ablation effect of NPF@DOX in NIR-treated groups were significantly higher than that in other groups in vitro and in vivo,suggesting that the therapeutic effect of synergistic therapy was enhanced.NPF@DOX provides a simple therapeutic strategy for promoting synergistic anti-tumor research and has a broad clinical application prospect for oral squamous cell carcinoma.