| Background Osteosarcoma(OS)is the most common primary malignant bone tumor in children,its high degree of malignancy,strong invasiveness,early distant transmission can occur,the current clinical treatment effect is poor,serious threat to the health of children and adolescents.Clinically,new treatments are urgently needed to inhibit OS progression and improve treatment effectiveness,thereby improving patient prognosis.Objective By injecting K7M2 cells into the scrotum cavity to establish a mouse localization transplant model,we explore the role and mechanisms of loaded antiviral uranium(Melittin,MLT)nanopolymer tape LC09-MLT@F127 in targeting intra-and external OS and inhibiting tumor progression in order to provide new strategies for the treatment of OS.Methods (1)MLT@F127 is prepared by thin-film hydration and optimized for comparison,and then combined with the adapter LC09 for the specificity combination of K7M2 cells by static absorption to prepare nanoparticles LC09-MLT@F127,which can specifically target the K7 M2 cell,to characterize its shape and particle diameter,zeta power level and other basic properties using a nanoparticle power positioner and a transmitted electron microscope;Lowry method to detect LC09-MLT@F127 encapsulation rate and drug load rate;polar stability and critical cloth concentration of LC09-MLT@F127 to be detected by uranium fluorescent probe method;(2)Taking red blood cells from mice to detect intramuscular hemorrhoidal activity of LC09-MLT@F127;dialysis to detect extracellular discharge of LC09-MLT@F127,and in vitro cultivation of K7M2 cells to use MTS to detect the destructive effects of Free MLT,Rd-MLT@F127 and LC09-MLT@F127,in different concentrations on K7 M2 celles.(3)Preparation of LC09-MLT@F127 and co-incubation with K7M2 cells using the same method to load near-infrared dye Di R-BOA to evaluate the targeted effect of LC09-MLT@F127 by in vitro immune fluorescence and fluid cellography;construction of OS-locally transplanted mouse models and observation of the bio-distribution of LC09 MLT@f127 in hormone mice by small animal live imaging and plasma organ imaging;(4)Infrared cultivation of K7M2 cells,using immune fluorescence to observe the expression of CRT,HMGB1 in PBS,Free MLT and LC09-MLT@F127 cells after treatment;extracting BMDC cells using free MLT containing different concentrations of MLT,Rd-MLT@F127 and LC09-MLT@f127 processed on-cell fluid with BMDC incubated for 24 hours,through fluid cytological testing of CD80+CD86+cell ratio,to assess the maturity of tree mutant cells(Dendritic Cell,DC)cells;(5)Injecting K7M2 osteoarthritis cells into the bone marrow of 4-6 w Bal B/C mice to build a locally transplanted OS mouse model,and modeled the successful mice randomly divided into PBS control group,F127 treatment group,Rd-MLT@F127 therapy group and LC09-MLT@F127-treatment group to perform four tail intravenous injection therapy,observe the general condition of mice and tumor growth,after the end of the treatment cycle to the tumor tissue for pathology H&E coloring,TUNEL coloring observed the death of various groups of tumour tissue,immunofluorescence observed tumor,the expression of CRT,HMGB1,CD4~+CD8~+T cell immersion.The relevant biochemical indexes were detected by serum,and the heart,liver,spleen,lung and kidney tissues were taken for histopathological H&E staining to evaluate the toxic side effects of each treatment group.Results (1)The average particle diameter of LC09-MLT@F127 prepared by the co-drying method was 32.05±7.24 nm,the average PDI was 0.29±0.07,while TEM was seen as the single-dispersed nanoparticles of LC09-MLT@F127,distributed evenly;by comparison optimization,we determined that adding 2 mg of MLT per 100 mg of F127 had a higher drug efficiency,its encapsulation rate was calculated at 57.2%,and the CMC of LC09-MLT@F127,detected by the helium fluorescent spectrum,was approximately 80.36μg/m L,indicating its polar stability.(2)Intra-organic hemorrhoidal activity analysis shows that when the MLT concentration of LC09-MLT@F127 is loaded up to 25μM,it results in only approximately 13.9%hemorrhage rate,indicating that it significantly improves the hemorrhagicity of Free MLT;the release curve of LC09-MLT@F127 appears at 72 h with a drug release rate of about 68.6%with a certain buffering effect;and the MTS results show a relatively untargeted K7M2 cell-destruction effect of Rd-MLT@F127,LC09-MLT@F127.(3)The quantitative results of the FCM showed that in K7M2 cells in the LC09-MLT@F127 processing group,Di R-BOA’s mean fluorescence intensity(MFI)was significantly higher than in the Rd-MLT@F127 group,while the CLSM results showed strong Di R-BOA fluorescent signals in the inkubated K7M2 celles in LC09-MLT@F127.(4)In-body anti-tumor experiments show that compared to PBS,F127,Rd-MLT@F127 and LC09-MLT@F127 groups show good inhibition of tumor proliferation,while the active targeted LC09-MLT@F127 anti-tumor effects are better,the difference is statistically significant.Pathology H&E coloring can be observed in the LC09-MLT@F127 treatment group tumor tissue can be seen a large number of nucleic solidification,nucleic rupture and a wide range of dead areas;TUNEL coloring is visible in LC09-MLT@F127 group tumour tissue visible a large amount of fluorescent signals,indicating that LC09-MLT@f127 can effectively induce the death of cells in the tumor body;H&E coloring observed the major organ damage,the treatment group heart,liver,spleen,lungs,kidney and other tissues did not show significant heterogeneous changes;serological testing showed the treatment of different groups of mice glutathione transaminase(ALT),grain transaminases(AST)and hemoglobin(CRE)levels.(5)The results of IF showed that compared to the PBS control group,free MLT and LC09-MLT@F127 processed 12 h of K7M2 cells visible CRT fluorescent surface expression,the drug processed 24 h of cell visible in-core expression HMGB1 significantly released to the outer cells;the flow results show that,compared with PBS group,the average fluorescencyintensityofCD86andCD80issignificantally increased BMDC which treatment with LC09-MLT@F127,indicating that LC09-MLT@F129 can effectively promote DC cell maturity.(6)The results of IHC-Fr showed visible surface CRT expression in the tumor tissue of hormonal mice treated with Rd-MLT@F127and LC09-MLT@F127,a significant decrease in in-nuclear HMGB1 expression and a more pronounced CD4+CD8+fluorescence signal compared to the PBS and F127 treatment groups.Conclusion (1)Successfully prepared the NPs and named LC09-MLT@F127,which has the characteristics of small size,good polarity stability,and certain slow-release,which can effectively improve the hemolytic activity of free MLT while payload MLT,which meets the requirements of further experiments.(2)LC09-MLT@F127 can demonstrate a good active targeting effect through EPR effect and adapter-specific identification of K7M2 cells,effectively accumulating within the OS tumor group.(3)LC09-MLT@F127 can play a good tumor suppression role by inducing tumor cell ICD,promoting DC maturity and further activating the body’s adaptive immune response. |
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