| Objective: In this study,a Balb/c mouse tumor model was made by subcutaneously implanting H22 hepatoma cells,and the TAMS aggregation of tumor tissues was observed by immunohistochemistry.The mannosylated chitosan nanoparticles that have been found in previous studies have the ability to specifically bind to mannose receptors.In this study,a preliminary understanding of the safety dose of nano-drugs in mice was performed to evaluate the tissue distribution of the nano-drugs in the model mice and the inhibitory effect on the model mice.To explore the mechanism of the anti-tumor effects of the nano-particles at the cellular level.Methods: 1.Production of model mice: H22 hepatoma cells were injected into the abdominal cavity of balb/c mice,centrifuged in ascites and resuspended with saline 1×107/ml,and transplanted into the mouse's left groin subcutaneously with 0.2 ml each.The mouse that forms a tumor under the skin is a model mouse.2.Macrophage aggregation of tumor tissue in mouse model: Using immunohistochemical staining,tumor-associated macrophages(TAMS,M2 macrophages)were labeled with CD68 antibody and CD163 antibody,respectively.Light microscopy was used to observe the accumulation of positive staining cells in the tumor tissue.3.Safe dose test of nano-drugs: Three gradient nano-drug experimental groups were designed.Nano-drug concentrations at the tail vein were 1 g/kg,500 mg/kg,and 200 mg/kg,respectively,and the saline group was the control group.The presence or absence of death in mice was recorded within 7 days.The changes in diet,activity,and body weight of the mice were observed,safety was evaluated,and the amount of the drug used in thesubsequent experiment was found.4.Tissue distribution of nano-drugs in model mice: Heart,liver,spleen,lung,kidney,and tumor were dissected 3hours,24 hours,and 72 hours after nano-drugs were injected into the tail vein of model mice.The microscope observes the fluorescence intensity in the tissue and records the fluorescence value.5.Anti-tumor experiments of nano-drugs:experimentally designed high,medium and low concentration nano drug experimental group(MICE),saline group and blank nano group as negative control group,interferon group as positive control group.On the second day after transplantation,different drugs were treated,and the volume change of tumor growth in each group of mice was recorded within 21 days after the administration.At the end of the experiment,tumors of the remaining mice in each group were necropsied,weighed and measured,and the tumor volume inhibition rate and tumor mass inhibition rate of each group of mice were calculated.The experiment was repeated and the number of mice survived within 30 days after administration was recorded.6.Effects of nano-drugs on immune cells in cancer nests: The nano-drug group,interferon group,blank nanoparticle group,and saline group were experimentally designed.On the second day after transplantation,different drugs were administered,and the experiment was completed 15 days after the subcutaneous implant model.Blood was collected from the fundus veins and the tumor tissue was dissected.The tumor tissues were mechanically prepared as individual cells.The proportions of CTL,Treg,M1,and M2 macrophages in venous blood and tumor tissues were examined by flow cytometry.Results: 1.On the 8th day after the subcutaneous implantation,the mice developed neoplastic lesions,indicating successful establishment of the model.2.Results of tumor immunohistochemistry: TAMS and M2 macrophages were aggregated in both tumor parenchyma and mesenchyme,and tumor interstitial aggregates of TAMS and M2 macrophages were more common than tumor parenchyma.3.Drug Safe Dose Test Results for Animals: In the 7-day observation period,thegeneral conditions of the three concentration gradient nanomedicine experimental groups were,and no obvious drug toxicity was observed.There was no significant difference in the color and morphology of the vital organs and organs of the necropsy group between the experimental group and the control group.The safety of the medicine was better,suggesting that the dose of the later experiment was within 1g/kg.4.Results of tissue distribution of nano-drugs in model mice: The fluorescence values of the hearts at 3 hours,24 hours,and 72 hours after the administration were similar to those of the blank control group,with no statistical significance.The average fluorescence values of liver,spleen,lung,kidney,and tumor at 3 hours after treatment were greater than those of the corresponding blank control group(P<0.01).At 24 hours after administration,only the liver,kidney,and tumor had higher average fluorescence values than the control group(P<0.01).At 72 hours after administration,only the average fluorescence value of the tumor was higher than that of the control group(P<0.01).5.Results of anti-tumor experiments of nano-drugs: The tumor formation time of the high-concentration nanometer group was later than that of the other five groups.After the tumor formation,the tumor volume change of the mice in each group was significantly different.The tumors in the high concentration nanometer group had a slow growth,and the other five groups had a faster growth rate.At the end of the experiment,the tumor volume in the high-concentration group was significantly smaller than the other five groups(P<0.01).In the high concentration nano group,tumor volume inhibition rate and tumor mass inhibition rate were higher,which could reach more than 80%;the other five groups had little difference in tumor volume inhibition rate and tumor quality inhibition rate,and were all lower than30%.During the experiment,the high-concentration nanogroup died zero,and deaths of model mice occurred in other groups,suggesting that the high-concentration nano-drugs have a significant inhibitory effect on tumors.6.Flow cytometry results: Tumor tissue test results: Compared with the salinegroup,the proportion of immunosuppressive cells M2 and Treg in the nano-drug group decreased,and the proportion of immuno-enhanced cells M1 and CTL cells increased.The proportion of Treg and M2 in the immunosuppressive cells of the interferon group increased,the proportion of immune-enhanced cell CTL cells increased,and M1 decreased;the proportion of immune cells in the blank nanogroup and saline was similar.Venous blood test results: Compared with the saline group,the immunosuppressive cell Treg ratio in the nano-drug group was increased,M2 was similar,and the proportion of immuno-enhanced cells M1 and CTL increased;In the interferon group,the proportion of Treg in immunosuppressive cells was increased,M2 was similar,and the proportion of immune-enhanced cells M1 and CTL increased;The percentage of immunoregulatory Treg cells in the blank nanogroup was higher,M2 was similar,the proportion of immune-enhanced cells was increased,and M1 was decreased.Conclusions: 1.A large number of TAMS and M2 macrophages were accumulated in the tumor tissue of the mouse model,which provided an animal model for tumor immunotherapy.2.The safe dose of nanomedicines should be at least within 1g/kg;3,Nano-drugs can be targeted to accumulate in tumor tissue,and the maintenance time is longer;4.High concentrations of nano-drugs(500 mg/kg)have a significant inhibitory effect on tumor growth;5.The mechanism of nano-drugs exerting an anti-tumor effect may be related to the decrease of the proportion of M2 macrophages and Treg cells in immune cells and the increase in the proportion of CTL and M1 macrophages. |
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