| Background and Purpose:Oral Squamous Cell Carcinoma(OSCC)is the most common malignant tumor of the maxillofacial area,chemotherapy is a commonly used treatment for patients with advanced cancer,Doxorubicin(Dox)as an anthracycline chemotherapy drug,can effectively treat a variety of cancers,but because of its serious cardiotoxicity and liver,kidney toxicity,the clinical application of Dox is limited.Photodynamic Therapy(PDT)has the advantages of non-invasive and few side effects,and is widely used in clinical cancer treatment.Traditional photosensitizers have problems such as poor water solubility,short half-life,short excitation wavelength,and weak tissue penetration.Therefore,980 nm near-infrared excited Up-converting Nanoparticles(UCNP)were used to solve the problem of weak tissue penetration of traditional PDT.A nanodrug carrier system combining chemotherapy and photodynamic therapy was constructed by using the red blood cell membrane biomimetic system,which significantly extended the circulation time of the drug in vivo and greatly reduced the toxic side effects of Dox.The application of nanocarriers realizes the combined application of chemotherapy,PDT and tumor immunotherapy,effectively inhibits tumor growth,and provides new ideas for the treatment of oral squamous cell carcinoma.Experimental methods:1.We put DSPE-PEG2000,DSPE-PEG2000-ce6,DSPE-PEG2000-up-conversion according to a certain percentage.The conversion and mixing form nano carriers through selfassembly,and use the nano coprecipitation method to prepare nano particles Dox/ce6/UCNP.The erythrocyte membrane was extracted by hypotonic permeation,and the erythrocyte membrane and nanoparticles were mixed according to a certain proportion.The nano drug loading system Dox/ce6/UCNP@R with erythrocyte membrane camouflage was obtained by extrusion method.Dynamic Light Scattering(DLS)detects particle size,Transmission Electron Microscope(TEM)observes the morphology of nano-drug carriers,ultramicrospectrophotometer measures the UV pattern and drug inclusion rate of each component drug,steady/transient fluorescence spectrometer detects the fluorescence spectrum of UCNP.Western blotting assays detect changes in major functional membrane proteins of nanodrugloaded systems,and in vitro release of Dox is detected using dialysis bags.2.The inhibitory ability of each component of nanodrugs on cal27 and scc7 tumor cells was detected by CCK-8 method,the release of ROS was detected by DCFH-DA fluorescent probe,and the endosomal escape was detected by the colocalization experiment between nanomedicine and lysosome.Calreticulin(CRT)valgus and Adenosine Triphosphate(ATP)release in vitro were detected to verify the immunogenic death of tumor cells.3.To detect the intensity of Dox and ce6 in the blood of mice within a certain period of time,to verify the in vivo circulation time of free Dox+ce6,Dox/ce6/UCNP and Dox/ce6/UCNP@R,a unilateral tumor model of C3H mice was constructed by scc7,the fluorescence image of tumor tissue in mice at 2、4、6、8、12 and 24 h was detected by the small animal living 3D imaging system,and tumor,heart,liver,spleen,lung,kidney and muscle tissue were collected after the sacrifice of mice.Fluorescence images were taken to detect the distribution of nanodrugs in vivo,bilateral tumor models of C3H mice were constructed by scc7,nanodrugs were injected through tail veins,bilateral tumor volume and mouse weight changes were monitored,mice were sacrificed on day 14,bilateral tumor tissues and major tissues and organs were dissected and collected,and in situ tumor tissues were immunofluorescence stained for CRT and HMGB1.Experimental results:1.The particle size of Dox/ce6/UCNP@R detected by dynamic light scattering was 151.3±12.01 nm,and the Zeta potential was-13.9±1.9 mV,and transmission electron microscopy showed that the spherical nanoparticles were wrapped with a layer of vesicles,showing a typical shell-core structure.The ultra-microspectrophotometer measured the UV spectrum of each component and used 478 nm as the detection wavelength of Dox and 403 nm as the detection wavelength of ce6.The drug loading rates of the nanoparticles Dox and ce6 prepared by us were calculated to be 14.8±0.57%and 8.3±0.5%,respectively.The fluorescence spectrum of UCNP at 980 nm indicates that it emits narrow spectral bands at 645-675 nm.Western blot experiments show that the main functional membrane proteins CD47 and CD44 on the surface of red blood cell membranes are basically not reduced after the erythrocyte membrane is coated with nanoparticles,and the main functions of cell membrane surface proteins are retained,which also shows the successful preparation of nano-drug delivery systems.In vitro drug release experiments showed that the nanodrug-loaded system could slowly release Dox for a long time,and the release rate and total release of Dox increased in acidic environment(PH=4.5).2.The results of CCK-8 experimental results showed that all component drugs could effectively inhibit the proliferation of cal27 and scc7 cells,and showed concentrationdependence,while the cell inhibition rate in the laser treatment group was significantly higher than that in the non-laser treatment group,and the higher the concentration of Dox,the more obvious the difference,indicating that the tumor inhibition effect of chemotherapy and photodynamic therapy was significantly better than chemotherapy alone.The ROS fluorescence results showed that the green fluorescence of Dox/ce6/UCNP@R was significantly enhanced after laser irradiation,indicating that Dox/ce6/UCNP@R showed good photodynamic therapeutic effect.Endosomal escape experiments show that Dox/ce6/UCNP@R can be effectively taken up by tumor cells and escape from lysosomes,better killing tumor cells.The increase of CRT valgus and ATP release in the Dox/ce6/UCNP@R group after laser irradiation proved that PDT could induce significant ICD in tumor cells.3.In vivo circulation experiments in mice,it was found that the fluorescence decay rate of Dox and ce6 in the Dox/ce6/UCNP@R group was significantly lower than that in the Dox/ce6/UCNP and free Dox+ce6 groups,indicating that the coating of erythrocyte membrane significantly prolonged the internal circulation time of the drug.In vivo distribution experiments found that nanoparticles could effectively accumulate into tumor tissues,reducing the ineffective distribution in normal tissues.Tumor suppression experiments have proved that Dox/ce6/UCNP@R(+)can effectively inhibit the growth of bilateral tumors without obvious toxic side effects on normal tissues.Tissue immunofluorescence staining showed that obvious ICDs occurred in situ tumor tissues,and the growth inhibition of distant tumors proved that PDT-induced ICDs could enhance anti-tumor immune responses in vivo.Conclusion:In summary,the nano-drug loading system we built has good biological safety,prolongs the circulation time in the body,and can effectively inhibit tumor growth.By inducing the ICD of tumor cells,the nanoparticle can regulate the body’s antitumor immune response,and provide new ideas for enhancing tumor immunotherapy. |
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