| Objective:Oral squamous cell carcinoma(OSCC)is the most common malignant tumor of head and neck.Despite many advanced therapies,the 5-year survival rate of OSCC patients still stagnate at 40-50%.Oral squamous cell carcinoma(OSCC)is the most common malignant tumor in the head and neck.Highly expressed tumor receptors can be used as targets for diagnosis and treatment of tumors.Studies have shown that gastrin-releasing peptide receptors are highly expressed on the surface of many tumors,including head and neck squamous cell carcinoma.Therefore,the purpose of this study is to:1.Construction of a tagert against OSCC probes of NGO-ant BBN-AF750 and DOX @ NGO-ant BBN-AF7502.In vitro study of the feasibility of molecular probes for the integration of OSCC diagnosis and treatment.Methods:1.Immunohistochemical fluorescence was used to investigate the targeted binding of ant BBN to GRPR in OSCC tissues.2.The ultraviolet fluorescence spectrophotometer was used to analyze the effect of NGO on fluorescence quenching to determine the optimal NGO concentration.Nano-graphene oxide(NGO)and ant BBN-AF750 are coupled via hydrogen bonds and π-π bonds.The molecular probe was metered by Fourier infrared spectroscopy,TEM and UV fluorescence spectrophotometer.3.HSC-3 cell line with high GRPR expression was used in vitro.Flow cytometry and confocal laser microscopy were used for cell binding,uptake and internalization.4.The tumor microenvironment was simulated with different pH to study the release of the anti-tumor drug DOX from the DOX @ NGO-ant BBN-AF750 complex under different pH conditions.CCK8 was used to determine the effect of free DOX on the proliferation of HSC-3 cells.Prism 6 was used to calculate its half maximal inhibitory concentration(IC50).The toxicity of NGO-antBBN-AF750,DOX @ NGO-ant BBN-AF750,and free DOX to HSC-3 cells was further determined.Results:1.Immune tissue fluorescence showed high GRPR expression in OSCC and low expression in normal oral mucosa.2.Fluorescence quenching and recovery experiments showed that: 0.1mg/ml NGO was selected for in vitro analysis,and ant BBN-AF750 could be detached from the probe,which played a targeting role when the probe encountered GRPR.3.Cell binding showed that the HSC-3 cells in the uptake group where cells were incubated with ant BBN-AF750 displayed strong NIR fluorescence signals,while the HSC-3 cells in the blocking groups where cells were incubated with excessive BBN(1-14)peptide for 10 minutes prior to incubation with ant BBN-AF750 had no fluorescence signal.The results of uptaking and internalization showed that the uptake of the probes by HSC-3 was time-dependent.With increase of incubation time,cells uptake increased,and at the first 15 min,we observed a rapid binding of up to 80%.Whereas,after 15 min,the fluorescence showed little increase with time.The cell uptake was 83 ± 15% and 89 ± 29%(ant BBN-AF750)and 85 ± 15% and 96 ± 20%(NGO-ant BBN-AF750)at 30 min and 60 min,respectively.ant BBN-AF750 was observed on the membrane of HSC-3,agreeing with its GRPR antagonist’s property on cell surface.However,stronger fluorescence signals were appeared overlapping with the nucleus of HSC-3 cells in the NGO-ant BBN-AF750 group,indicating a cell internalization activity by NGO-ant BBN-AF750.No fluorescence was observed in HOK cells which was selected as a negative control as they do not express GRPR.4.Different pH response studies showed that during the whole drug release process,at pH 5.6 the drug release rate is faster than the other two(pH 6.6 and 7.4).And within the initial 9 h,the DOX is released very fast.After 36 h,the drug release rates tend to be flat.We observed that cumulative release amount of drugs in PBS solution were about19.8%,17.7% and 15.3% after 24 h,at pH 5.6,6.6 and 7.4.CCK8 assayed illustrated that with the increase of DOX concentration,the activity of HSC-3 cells decreased,and the IC50 value was 5ug/ml(prism 6).there is no cytotoxicity of the GO within the study concentration range.For free DOX and DOX@NGO-ant BBN-AF750.Compared with the free DOX groups,the cell viability of DOX@NGO-ant BBN-AF750 groups increased with the same concentration of DOX(5,10,20ug/ml).And as the pH of the culture medium decreased,the cell viability of the NGO-ant BBN-AF750 group decreased.Conclusion:1.This study successfully synthesized fluorescent targeting probes NGO-ant BBN-AF750 and DOX @ NGO-ant BBN-AF750.2.In vitro cell uptake,binding and internalization experiments confirm that NGO-ant BBN-AF750 targets oral squamous cell carcinoma GRPR and can be applied to OSSS fluorescence imaging.3.Experimental confirmation of pH response,cell internalization and proliferation in vitro DOX@NGO-ant BBN-AF750 has inhibitory effect on HSC-3. |
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