| Aims: Vitexin,a natural flavonoid extracted from licorice,has potential therapeutic effect on cancer.However,drug resistance is common occurrence during long-term chemotherapy.In this study,Indocyanine green(ICG),a photosensitized agent with high biosafety,was chosen for photothermal and photodynamic synergistic therapy based on drug therapy.This approach not only shortened the treatment cycle of cancer,but also significantly improved the therapeutic effect.Given the poor solubility of vitexin,this study utilized liposome as a carrier to investigate their antitumor effects and mechanisms in combination with ICG.To precisely control the particle size and ensure particle homogeneity,a microfluidic(MF)chip was employed for liposomes preparation,aiming to save costs and enable continuous production of formulations.Finally,a 3D tumor cell model that is closer to human physiological environment was constructed on the basis of 2D tumor cell model to further study the mechanism of vitexin liposome synergistic photothermal and photodynamic treatment of tumor,providing new insights for cancer treatment.Methods:(1)An in vitro analysis method of vitexin was established using high performance liquid chromatography(HPLC)to lay the foundation for the subsequent vitexin liposome preparation.(2)The Corel DRAW2020 software was used to design the channel diagram of the MF chip,followed by the selection of chip preparation materials,and the preparation process of the chip was investigated and optimized by a laser cutter.(3)The single-factor experimental design and orthogonal experimental design were used to screen and optimize the formulation of vitexin liposome.The total flow rate and flow rate ratio was used to optimize the preparation process of vitexin liposome,and the particle size,polydispersity index(PDI),and zeta potential of vitexin liposome were investigated by a laser particle size analyzer.The appearance of vitexin liposome was observed using transmission electron microscope(TEM).In addition,the encapsulation rate and drug loading of vitexin liposome were detected using the dextran gel column chromatography.The drug release ability of vitexin liposome in different release media and storage stability in aqueous solution were investigated and evaluated.(4)Based on 2D cell model,suitable drug concentration and near-infrared irradiation time were screened by MTT method,suitable drug concentrations and near-infrared light exposure times were screened using the MTT assay.The effect of vitexin liposomes on tumor cell migration was investigated using the scratch assay,and the drug-induced apoptosis of tumor cells was analyzed using tunel kit and flow cytometry.Finally,western blot technology was used to explore the mechanism of drug-induced tumor cells apoptosis.(5)By screening the culture methods of 3D tumor spheroids,3D tumor cell model was constructed,and the difference in treatment effectiveness between 2D and 3D tumor cell models was analyzed and compared for vitexin liposome-mediated photothermal and photodynamic therapy.Results:(1)HPLC technology was used to establish vitro analysis method of vitexin.The method validation results demonstrated that the method was good specificity,precision,method recovery and stability.(2)Polydimethylsiloxane(PDMS)was selected as the material for the chip,and polymethyl methacrylate(PMMA)was used for the cover and bottom plate.A T-shaped flow focusing MF chip was successfully prepared and assembled,and the sealing of the chip was detected.(3)The optimization of the vitexin liposome formulation was investigated using a singlefactor and orthogonal experimental design.The final vitexin liposome formulation contained 22 mg of lecithin,4 mg of cholesterol,2 mg of sodium cholate,and 5 mg of Isopropyl myristate(IPM).The optimization of the preparation process resulted a total flow rate(TFR)of 3 m L/min,with a water phase and organic phase flow rate ratio(FRR)of 10:1.Characterization and evaluation of the prepared vitexin liposomes showed that the free vitexin was almost insoluble in several release media.However,after preparing it into liposomes,the cumulative drug release rate of vitexin was increased by approximately 40%,and the encapsulation rate was 94.40 ± 0.23%,with a drug loading rate was 13.49 ± 0.027%.The average particle size of the prepared vitexin liposomes was107.33 ± 1.03 nm,PDI of 0.220 ± 0.010 and zeta potential of-11.67 ± 0.14 m V.The TEM results showed that the surface of vitexin liposomes was smooth and spherical,with a distinct bilayer structure.The stability test results over 35 days indicated that the prepared liposomes were stable.(4)To investigate the synergistic effect of vitexin liposome on tumor in photothermal and photodynamic therapy,2D and 3D tumor models were constructed.After using MTT assay to screen vitexin concentrations(40,50,60 μg/m L),it was found that vitexin formulation had an excellent anti-tumor effect.Optical treatment results showed that after the tumor cells completely absorbed vitexin,5 minutes of near-infrared light irradiation produced a large amount of heat,killing tumor cells,and inducing the production of a large amount of reactive oxygen species to promote tumor cell apoptosis.The scratch experiment results showed that vitexin could effectively inhibit the migration of tumor cells.At 40 μg/m L of vitexin,the cell invasion assay showed that,and the cell wound closure rate of vitexin liposome group decreased from 20.37 ± 1.17% to 12.32± 1.16%.Cell wound closure rate decreased from 23.83 ± 0.36% to 15.42 ± 0.14% in free vitexin group and from 3.67 ± 1.54.18 ± 0.41% in near infrared irradiation group.When the concentration of vitexin increased from 40 μg/m L to 60 μg/m L,the results showed that the apoptotic rate of U251 in vitexin liposome group was 33.20 ± 2.19%,49.78 ± 2.45% and 57.67 ± 1.02%,respectively.The results of cell apoptosis experiment showed that the percentage apoptosis tumor cell in the near infrared irradiation group was 82.73 ± 0.81 %,86.48 ± 1.26 % and 94.96 ± 1.40 %,which were higher than those in the free vitexin group(16.18 ± 1.96%,32.35 ± 2.40% and 43.65± 2.87%).The inhibition and apoptosis effects on tumor cells were observed to be dose-dependent.Western blot analysis demonstrated that vitexin promoted apoptosis in tumor cells by increasing the expression of bax protein and decreasing the expression of bcl-2 protein.To investigate the therapeutic potential of vitexin in a more physiologically environment,an ultra-low adhesion plate was prepared with 1% Polyvinyl alcohol 1799(PVA1799),and a 3D tumor spheroid model was successfully constructed using the ultra-low adhesion plate culture method.Results from the cell apoptosis experiment and Western blot analysis were consistent with those obtained from the 2D tumor model.Compared with free vitexin,vitexin liposome was found to be more efficient in penetration the cell membrane and entering the tumor cells.Moreover,the levels of apoptosisrelated proteins were significantly increased in both the inner and outer layers of tumor spheroids.Near-infrared rays were also found to penetrate the outer cells and induce the cells inside the tumor spheroids,resulting in the death of a large amount of reactive oxygen species,and thus improving the therapeutic efficacy of vitexin.Conclusion: In this study,the vitexin /ICG liposome prepared by MF technology can effectively improve the solubility of vitexin and increase the bioavailability of drugs.The combination of photothermal and photodynamic synergistic therapy had been found to significantly enhance the therapeutic effect against tumors.The application of 2D and 3D tumor models has enabled drug concentration screening and mechanistic investigation into the anti-tumor effect of the treatment.The findings of this study provide a promising and straightforward to the clinical application of vitexin /ICG liposome. |
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