Preparation And Application Of Nanofibrous Membrane With Triple Combined Anti-tumor Effect

Cancer is one of the most dangerous diseases that seriously threaten human health,and its treatment remains a major challenge in biomedicine to date.Tumor drug resistance is the main reason for the failure of chemotherapy.Therefore,it is often difficult to effectively eradicate cancer with a single drug.Photothermal therapy（PTT）is a method to inhibit and kill cancer cells by using the thermal effect generated by light,which has the advantages of being less invasive and simple to operate,and has been a hot research topic in recent years.Photodynamic therapy（PDT）can kill bacteria and cancer cells by reactive oxygen species and free radicals produced by photosensitizers under aerobic conditions,and its low systemic toxicity,suitability for local treatment and minimal invasiveness have also received significant attention from researchers.DOX is a commonly used chemotherapeutic agent with a broad-spectrum anticancer effect by inhibiting the synthesis of RNA and DNA in cancer cells and killing tumor cells of all growth cycles.Serine fibroin（SF）is a natural protein polymer with excellent biocompatibility and biodegradability,which is widely used in the research of biomaterials.Polylactic acid glycolic acid（PLGA）is a biodegradable polymer with good mechanical properties and has a wide range of applications in the field of biomaterials,but materials prepared only from PLGA have defects such as poor hydrophilicity and poor cell affinity.Composites prepared using synthetic and natural polymers can complement each other and perform better.In view of the complexity,diversity and heterogeneity of tumors and the current treatment status of a single chemotherapeutic modality which is difficult to achieve the desired effect.In this study,SF and PLGA hybrid electrostatic spinning were used as nanofiber membrane matrix,loaded with photosensitizer ICG and chemotherapeutic drug DOX,to prepare nanofiber membrane which can have triple synergistic therapeutic effects of chemotherapy,photothermal and photodynamic therapy at the same time,and applied to the study of cancer treatment.The fluorescence signal of ICG and DOX can monitor the distribution of drugs in vivo in real time,while the controlled release of drugs can be achieved by the light power to ensure the effect in cancer treatment.This study can provide new avenues for the application research of drug-loaded silk fibroin/polylactic acid glycolic acid nanofiber membranes,and provide richer options for the research and application of tumor multi-treatment.The main results of this study are as follows.1.Preparation and performance study of indocyanine green silk fibroin/polylactic acid glycolic acid nanofibrous membraneSilk fibroin,polylactic acid glycolic acid and indocyanine green solution were co-blended to prepare indocyanine green silk protein/polylactic acid glycolic acid nanofiber membrane by electrostatic spinning technique.The WVTR value of SF/PLGA/20ICG was 3040.49±157.11 g·m^-2day^-1,showing good moisture permeability and hydrophilicity.The seven-day cumulative ICG release of SF/PLGA/20ICG was 54.97%,which can continuously act on the wound or cancer site.The photothermal performance test results showed that the maximum temperature of SF/PLGA/20ICG could be increased by 44.4°C in the dry state,and the photothermal effect of the ICG-loaded nanofiber membrane could be controlled by adjusting the laser power.The photothermal effect of SF/PLGA/20ICG can inhibit Staphylococcus aureus and Escherichia coli under near-infrared radiation with 91.53%and 87.95%inhibition rates,respectively.Cellular experiments showed that the photothermal effect of SF/PLGA/20ICG had a significant inhibitory effect on the growth of tumor cells.2.Preparation and performance study of dual drug-loaded silk protein/polylactic acid glycolic acid nanofiber membraneThe dual drug-loaded silk protein/polylactic acid glycolic acid nanofiber membranes were prepared by electrostatic spinning technique after the co-blending of silk protein,polylactic acid glycolic acid,indocyanine green and adriamycin hydrochloride solution.The semi-inhibitory concentration test showed that the IC50 of DOX was 2.761μg/m L and 3.092μg/m L for Hep G2 and B16 cells,respectively,and the contact angle test showed that SF/PLGA/20ICG/DOX had good hydrophilicity,which was beneficial to the adhesion of tissue cells.The photothermal performance test results showed that the loading of DOX did not affect the photothermal performance of ICG,and the maximum temperature of SF/PLGA/20ICG/DOX in the dry state increased by 43.9°C.The photothermal effect of the nanofiber membrane could be well controlled by adjusting the laser power and time.The drug release helps to ensure a long-lasting effect on the wound or cancer site,and the slow release test showed that after cycling the 808 nm IR laser a total of 4 times in 12 hours,the cumulative release of DOX from the three groups of nanofiber membranes（0,0.6,and 1.2 W/cm² light）was 39.49%,53.63%,and 64.39%,respectively;0,0.6,and 1.2 W/cm² NIR illumination,the cumulative release of DOX from nanofiber membranes for seven days（one light per day）was 49.69%,61.52%,and 68.93%,respectively.The results of slow release provide a basis for light-controlled drug release.In vitro cancer inhibition experiments showed that the SF/PLGA/20ICG/DOX nanofiber membranes achieved74.75%and 82.97%cancer inhibition against Hep G2 and B16,respectively,after NIR light exposure.3.Tumor therapeutic performance of dual drug-laden silk protein/polylactic acid glycolic acid nanofiber membranesA tumorigenic model of B16 mouse melanoma cells was established to study the therapeutic effects of the dual drug-loaded filipin/polylactic acid glycolic acid nanofiber membrane on tumors and the in vivo distribution of the drug.The results showed that the light-irradiated biphasic serine protein/polylactic acid glycolic acid acid nanofiber membranes could inhibit the growth of tumors.After fourteen days of treatment,the tumor volume was 1055.89±167.44 mm³ in the light group and1454.76±267.28 mm³ in the control group.the nanofiber membrane still had a strong photothermal conversion effect in the tumor tissue of mice,and the warming curve and thermal imaging pictures proved the excellent photothermal effect after 20 minutes of near-infrared light exposure to 60.6℃.The photothermal conversion property of the photothermal agent at the tumor converts light energy into heat energy thus increasing the temperature at the tumor and achieving the effect of ablating tumor cells at high temperature.A fluorescence imaging test was performed to determine the in vivo distribution of the drug.The results showed that the fluorescence of ICG at the 1st and6th hours was very high in the tumor,while intravenous ICG and DOX showed a completely different biodistribution pattern.From 0 to 12 h after drug administration,the fluorescence of the drug was mainly concentrated in the liver rather than in the tumor.The advantages of local treatment with nanofibrous membranes were demonstrated,avoiding the toxicity of systemic drug administration.In histopathological tests,H&E,TUNEL and ki67 staining demonstrated the good cancer inhibitory effect of SF/PLGA/20ICG/DOX-L.In summary,SF/PLGA/20ICG and SF/PLGA/20ICG/DOX nanofiber membranes possess good physicochemical properties and biological properties,and ICG generates ROS and photothermal effects under NIR light illumination to exert photothermal and photodynamic therapy for cancer.At the same time,the slow release of DOX is controlled based on the photothermal effect,and the DNA and RNA of cancer cells are destroyed by the chemotherapeutic effect of DOX to achieve a triple synergistic therapeutic effect on tumors together with photothermal and photodynamic therapy to suppress cancer.SF/PLGA/20ICG nanofiber membrane has potential applications in the field of trauma dressing and cancer therapy,and SF/PLGA/20ICG/DOX nanofiber membranes have promising applications in the field of cancer therapy.