| Tongue squamous cell carcinoma（TSCC）is a malignant tumor with high morbidity and mortality in the oral cavity.In recent years,with the continuous improvement of tumor early screening and treatment,the survival rate of TSCC patients has improved significantly compared with the past decades,but its associated mortality rate has not decreased significantly.Currently,TSCC is mainly treated by surgery,which is often supplemented by chemotherapy and radiotherapy in order to prevent recurrence.However,these modalities still have certain limitations and side effects,such as high trauma,low efficacy,and damage to normal tissues.The use of targeted drug therapy can improve drug utilization and reduce the impact of drugs on normal tissues,which is expected to increase the survival rate and improve the quality of life of patients with TSCC.Our previous research found that Evodiamine（Evo）can inhibit the proliferation of TSCC cells.However,the poor aqueous solubility of EVO,low targeting efficiency and inability to be delivered to tumor tissues in free form have hindered its clinical application.How to improve the aqueous solubility and bioavailability of EVO is a key scientific issue in this research field.Objective: EVO-loaded nano-drug delivery system designed by targeting molecular folic acid（FA）-modified graphene quantum dots（GQDs）can effectively improve its bioavailability while overcoming the low bioavailability of EVO.The effect of treating TSCC opens up a new way for the treatment of TSCC and the targeted delivery of the anticancer drug EVO.Research methods:1.Use Molecular Dynamics（MD）simulation calculations to gain an in-depth understanding of the nature of the interaction between EVO and GQDs.GQDs were used as the carrier of EVO,and FA was used as the targeting molecule of tongue squamous cell carcinoma cells.The GQDs-FA-EVO nanocomposite system was synthesized by self-assembly method.2.The synthesized GQDs-FA-EVO nanocomposite systems were characterized by transmission electron microscopy（TEM）,ultraviolet-visible absorption spectroscopy（UV-vis）,fluorescence spectroscopy（FL）,Fourier infrared spectroscopy（FTIR）,dynamic light diffraction（DLS）and zeta potential for morphology,material composition and structure,fluorescence properties at different excitation wavelengths,various The GQDs-FA-EVO nanocomposite systems were characterized in terms of morphology,composition and structure,fluorescence properties at different excitation wavelengths,various chemical bonds and different chemical groups,particle size distribution,and surface charge.3.The effects of GQDs-FA-EVO nanocomposite system on the uptake,activity and apoptosis of tongue squamous cell carcinoma cells were evaluated by laser confocal microscopy（CSCM）imaging,CCK-8 assay,clone formation assay,CalceinAM/PI and Annexin V-FITC/PI double staining assay.4.The transplantation tumor model was established by subcutaneous injection of tongue squamous carcinoma cells into the back of nude mice.After successful modeling,Saline,GQDs,GQDs-FA,EVO and GQDs-FA-EVO were injected into the tail vein,observing the growth,tumor volume changes of nude mice;the effect of GQDs-FA-EVO nanocomposite system in targeting tumor cells was observed by small animal live imaging system;the morphology of tumor cells was observed by HE staining;Western blot and immunohistochemistry were performed to detect the expression of Ki67,Caspase-9 and Caspase-3 in transplanted tumor tissues;the histological changes of heart,liver,spleen,lung and kidney tissues of nude mice were observed by HE staining to evaluate the biosafety of GQDs-FA-EVO nanocomposite system.Research results:1.The binding form of three substances was confirmed by theoretical simulations at the atomic scale to achieve effective synthesis between substances and to obtain the binding behavior and steady-state structure at the energy steady state.In the GQDs-based system,EVO forms strong π-π interactions with the surface of GQDs,indicating that EVO will spontaneously move toward GQDs and form stable complexes.2.The results of TEM,UV-vis,FL,FTIR,DLS and zeta potential characterization showed that the GQDs-FA-EVO nanocomposite system with good physicochemical properties was prepared.3.The results of CLSM imaging showed that GQDs-FA-EVO nanocomposite system could label tongue squamous cell carcinoma cells;the results of CCK-8 assay,clone formation assay,Calcein-AM/PI and Annexin V-FITC/PI double-staining assay showed that GQDs-FA-EVO nanocomposite system inhibited the activity of tongue squamous cell carcinoma cells and promoted their apoptosis（P<0.01）.4.Successfully established a subcutaneous transplantation tumor model in nude mice,and after 18 d of treatment,the GQDs-FA-EVO nanocomposite system significantly reduced the tumor volume by 19% compared with the only EVO group（P<0.05）;the results of small animal live imaging showed that the GQDs-FAEVO nanocomposite system could target to the tumor site.The HE results showed that the EVO,GQDs-FA-EVO group had smaller nuclei,wider cell-to-cell gaps,and abundant apoptotic/necrotic areas,while the control group（Saline,GQDs,GQDs-FA）had variable tumor cell morphology,obvious heterogeneity,poor differentiation,irregular morphology.Western blot experiments and immunohistochemistry results showed that Ki67 expression was lower in the EVO and GQDs-FA-EVO groups than in the control group,and its expression in the GQDs-FA-EVO group was significantly lower than that in the EVO group（P<0.05）;caspase-9 and caspase-3 expression in the GQDs-FA-EVO group was significantly higher than that in the EVO group（P<0.05）,and their expression in the control group was not significantly different.The HE results confirmed that the cellular structures of the vital organs（heart,liver,spleen,lung and kidney）in the GQDs-FA-EVO and EVO groups and the control group were clear and showed no signs of necrosis,and no morphological abnormalities or obvious pathological changes were observed.Research conclusions: The GQDs-FA-EVO nanocomposite system not only exhibited excellent physicochemical properties and biosafety,but also had good antitumor effects,and its mechanism of action might be related to the inhibition of Ki67 expression and promotion of Caspase-9 and Caspase-3 expression. |
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