| Esophageal squamous cell carcinoma(ESCC)is a malignant tumor of digestive tract.The traditional treatment strategies,mainly including endoscopic treatment,esophagectomy,radiotherapy and chemotherapy,have various shortcomings,which limit their clinical treatment effects.Photodynamic therapy(PDT)is an emerging treatment strategy for the treatment of ESCC,which uses photosensitizers to produce reactive oxygen species(ROS)to kill tumor cells under laser irradiation.Due to the advantages of low invasion,repeatability and fewer side effects,PDT has exhibited excellent therapeutic effects in some tumor types.However,the clinical application of PDT in ESCC is limited by poor targeting of photosensitizers,tumor tissue hypoxia,and high intracellular glutathione(GSH)levels in tumor cells.To resolve these problems,we designed a multifunctional biomimetic nanomedicine TK-EC@PLGA-GA(TEPG)to enhance the efficacy of PDT for ESCC.The prepared TEPG has the long circulation time in vivo,and could utilize the homologous adhesion molecules on the tumor cell membrane to achieve efficiently enrichment at the tumor site.The catalase on the thylakoid(TK)membrane could catalyze the excess hydrogen peroxide in tumor to produce oxygen,which alleviated tumor hypoxia.Under near-infrared laser irradiation,the natural photosensitizer chlorophyll on TK further converted oxygen into ROS for PDT.At the same time,the released chemotherapeutic drug gambogic acid(GA)effectively reduced the GSH levels in tumor cells and kill tumor cells,to achieve a highly effective combination of anti-tumor effect.In this work,the physicochemical properties,anti-tumor effects in vitro,distribution,and pharmacodynamics in vivo were systematically evaluated.The details are as follows:1.Preparation and pharmaceutics of multifunctional biomimetic nanomedicine TEPG.In this study,Poly(lactic-co-glycolic acid)(PLGA)was used as the carrier to deliver the drug GA to construct the nano-core,noted as PLGA-GA.Then the TK membrane extracted from spinach leaves and the tumor cell membrane EC extracted from EC109 cells were ultrasounded to obtain composite membrane TK-EC.Then the TK-EC was successfully coated on PLGA-GA by ultrasonic and co-extrusion to prepare the final multi-functional biomimetic nanomedicine TEPG.The morphology,size,and surface charge of TEPG were characterized.The results showed that the TEPG had a typical core-shell structure,with the diameter of 208.9 ± 6.7 nm,the surface potential of-31.8 ± 0.8 m V.The results of UV-vis absorption spectra and fluorescence emission spectra showed that TEPG retained the optical properties of the TK and GA.The obvious characteristics of fluorescence co-localization of the two membranes fusion were observed by fluorescence microscope.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and western blotting experiments showed that TEPG retained the characteristic proteins of two kinds of membrane.The oxygen production and ROS generation experiments in vitro showed that TEPG could catalyze hydrogen peroxide to produce oxygen and a large number of ROS under laser irradiation.The results of drug release showed that 61.7 % of GA could be released from TEPG at p H 6.8 in vitro.Besides,the stability test showed that TEPG had good serum stability and storage stability in vitro.2.In vitro anti-tumor activity study of multifunctional biomimetic nanomedicine TEPG.Human esophageal squamous cell carcinoma cell line EC109 was selected as the experimental cell line to evaluate the anti-tumor activity of nanomedicine TEPG in vitro.The results of cell viability test showed that TEPG could kill EC109 cells efficiently under near infrared laser irradiation.Cell targeting experiments showed that TEPG could be effectively uptaken by EC109 cells,indicating its good homologous targeting ability.Based on the detection of intracellular ROS level,it was confirmed that TEPG could produce a large amount of ROS in cells under near-infrared laser irradiation,showing an excellent photodynamic effect.By detecting the content of GSH in EC109 cells,it was confirmed that TEPG could significantly reduce the level of intracellular GSH.Finally,western blotting experiments were carried out to investigate the effect of TEPG on the content of apoptosis-related proteins in EC109 cells.The results showed that TEPG could kill tumor by inducing apoptosis.3.In vivo distribution and pharmacodynamics study of multifunctional biomimetic nanomedicine TEPG.The EC109 tumor-bearing nude mice were used as animal models.With indocyanine green(ICG)labeled the nanomedicine,blood half-life and biodistribution were observed by small animal fluorescence imaging.The results showed that compared with the free ICG group and TK coated nanomedicine group,TEPG could prolong the half-life in blood,and had a higher accumulation efficiency at the tumor site.In the anti-tumor study in vivo,the tumor volume and weight,H&E and TUNEL staining of tumor tissue sections were measured and analyzed to investigate the inhibitory effects of different nanomedicine on tumors.The results showed that TEPG could significantly induce tumor cell apoptosis and inhibit the growth of ESCC.The immunohistochemical results of hypoxia inducible factor HIF-1α showed that TEPG could down-regulate the expression of HIF-1α and relieve tumor hypoxia.In addition,the safety of nanomedicine in vivo were detected by monitoring the changes of body weight of tumor-bearing mice during treatment,serum biochemical indicators,and main organs H&E staining.The safety evaluation confirmed that TEPG had no obvious toxic and side effects.Based on the above results,the multifunctional biomimetic nanomedicine TEPG prepared in this study had the homologous targeting by the decoration of the cancer cell membrane,to obtain efficient enrichment of tumor sites.TEPG achieved the enhanced photodynamic therapy effect of ESCC by relieving tumor tissue hypoxia and reducing the level of GSH in tumor cells,which provides a new idea for the clinical treatment of ESCC. |
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