Study On The Mechanism Of Tumor-targeted CTD Polymer Micelles

"Through poisoning" is one of the important means for Chinese medicine to treat malignant tumors.Traditional Chinese medicine clinical practice has proved that cantharidin?CTD?has a unique anti-tumor effect.The anti-tumor active ingredient is also a toxic component,so it has a narrow dose range of therapeutic,improper application can cause poisoning and even threat life.mPEG2000-PLGA2000polymer micelles can encapsulate CTD in its hydrophobic core,which can reduce the toxic side effects of drugs on normal tissues,increase its enrichment in tumor tissues,and prolong the circulation time in vivo.Our previous studies have constructed mPEG-PLGA-CTD polymer micelles.In vivo anti-tumor experiments show that mPEG-PLGA-CTD polymer micelles can alleviated the toxic damage of CTD to mouse kidney.Based on the previous research,this study will deeply explore the safe dosage and synergistic attenuation mechanism of mPEG-PLGA-CTD polymer micelles,and provide important clinical guidance for the safe use of CTD.Acute toxicity study of CTD polymer micelles in mice:?1?Acute toxicity test in normal mice:144clean-grade Kunming mice,male and female,were randomly divided into normal group,blank micelle group,original drug group and micelle group,with 12 groups in total,12 in each group.The tail vein was administered by injection?molecule solution in terms of CTD,0.20 mL/10 g?.The mice were closely observed for body weight,toxicity,and death within 24 hours,and observed continuously for 14 days.The LD50 of the median lethal dose was calculated by the Bliss method using the acute toxicity test method.?2?Acute toxicity test of tumor-bearing mice:156 clean-grade Kunming mice,male and female,were established to establish a subcutaneous transplantation tumor model of mouse liver cancer H22,which were randomly divided into model group,original drug group and micelle group.13 groups,12 in each group,were administered by tail vein injection?micelle solution in CTD,0.20 mL/10 g?.The mice were closely observed for body weight,toxicity,and death within 24 hours,and observed continuously for 14 days.The LD50 of the median lethal dose was calculated by the Bliss method using the acute toxicity test method.?3?Effects of micelles on tissues and organs of tumor-bearing mice:36 clean-grade Kunming mice,half male and half female,were established to establish a subcutaneous transplantation tumor model of mouse liver cancer H22,which were randomly divided into model group and original drug group.Three groups of micelles,12 in each group,were administered at a dose of 600?g/kg,administered intravenously every other day?micelle solution in CTD,0.20 mL/10 g?,and urine was collected.4°C refrigerator spare.After14 days of administration,blood was collected from the undead mice by eyeball extraction.The collected blood was placed in a refrigerator at 4°C for use.The mice were sacrificed by cervical dislocation and the tumor,heart and liver were dissected and removed.The spleen,kidney and thymus tissue were weighed.By calculating the organ coefficient of mice,the pathological changes of organs in various tissues were observed by HE staining,and alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,alkaline phosphatase?ALP?,creatinine?Cr?in serum and urine were measured.),urea nitrogen?BUN?,uric acid?UA?levels,comparing the toxic side effects of mPEG-PLGA-CTD micelles and CTD original drugs on mice.The results showed that the LD₅₀ value of the normal mouse micelle group was 2.29 mg/kg,the LD₅₀value of the original drug group was 1.96 mg/kg,the micelle group was 1.17 times of the original drug group.The mouse micelle group LD₅₀ of the tumor-bearing mice was 2.55 mg/kg,the LD₅₀ value of the original drug group was 1.66 mg/kg,and the micelle group was 1.54 times that of the original drug group.Compared with the model group,the liver coefficient showed a significant decrease in the original drug group?P<0.01?,and the spleen index decreased significantly?P<0.05?.Compared with the model group,the micelle group has no significant difference.The pathological sections showed that the renal tissue section of the original drug group and the micelle group became larger,the glomerular volume increased,and the renal tubule edge was blurred,but the change of the original drug group was more obvious than that of the micelle group.Significant inflammatory cell infiltration occurred in the liver tissue sections of the original drug group and the micelle group,but the change of the original drug group was more obviously than that of the micelle group.Compared with the model group,the heart tissue sections of the original drug group and the micelle group showed myocardial necrosis?nuclear lysis?accompanied by hemorrhage and inflammatory cell infiltration,but the changes in the original drug group were more obviously than those in the micelle group,necrotic cells and The number of inflammatory cells is large.The alveolar wall of the original drug group and the micelle group had a little widened and thickened.There were obvious inflammatory cell infiltration in the two groups and no significant difference between the micelle group and the model group.The ALT in serum of the mice in the original drug group and the micelle group were significantly higher than that in the model group?P<0.05?,and the change in the original drug group was more obviously than that in the micelle group.There were no significant difference between AST and ALP in the serum of the mice in the original drug group.The UA in serum of the mice in the original drug group were significantly higher than that in the model group?P<0.05?,the micelle group was not significantly different from that of the model group.The Cr and BUN in urine of the mice in the original drug group and the micelle group were not significant difference compared with the model group.The experimental results showed that mPEG-PLGA-CTD micelles can reduce the toxicity of CTD in mice,and the attenuation effect on tumor-bearing mice is more obvious.Pharmacokinetic study of CTD polymer micelles in tumor-bearing mice:A total of 228 female Kunming mice were used to establish a subcutaneous transplantation tumor model of mouse liver cancer H22.They were randomly divided into 19 groups,12 in each group,and injected intravenously?the micelle solution was calculated by CTD,0.20 mL/10 g?.).Blood was taken from the eyelids at 5 min,15 min,30min,1 h,2 h,4 h,8 h,12 h,24 h after administration,and centrifuged?4°C,3000 r/min,10 min?,separation Upper serum.The sample was treated by ethyl acetate extraction,and clofibrate was selected as an internal standard by GC-MS.DAS 2.0 software calculates pharmacokinetic parameters and investigated the pharmacokinetic properties of mice.The experimental results show that the GC-MS method has high specificity,plasma CTD has a good linear relationship in the range of 4¹000ng/mL,The regression equation is Y=0.0081x+0.0695?R²=0.9979?,the minimum detection limit is 2 ng/mL,precision and stability are both Meet the requirements.The plasma concentrations of the CTD solution and the mPEG-PLGA-CTD micelle solution after tail vein administration were consistent with the three-compartment model.The area under the curve of CTD and mPEG-PLGA-CTD micelles in mice was619.99 and 3771.56?g/L*h,respectively;the peak concentrations of Cmax were 348.21 and 638.78?g/L,respectively;The MRT prolongation time was 2.25 and 9.82 h,respectively;the elimination half-life t_1/2?was 6.45 and 17.69 h,respectively;the clearance rates were 0.97 and 0.14 L/h/kg,respectively.The resesuts showed that the mPEG-PLGA-CTD micelle can accelerate the drug absorption after intravenous injection in mice,prolong the circulation of the drug in the blood effectively,and AUC_{0-24 h}-24 h is 608.32%of the original drug.Dynamic tissue distribution and tumor targeting of CTD polymer micelles in tumor-bearing mice:mice were divided into groups and administered with pharmacokinetic studies.The mice were sacrificed by cervical dislocation at each time point after administration,and the heart,liver,spleen,lung,kidney and tumor were dissected,and each tissue was treated by protein precipitation.The sample was treated with ethyl acetate extraction.The content of the drug in the tissue was determined by using clofibrate as the internal standard and GC-MS method.Using Origin 8.5 software to process the data,to investigate the characteristics of dynamic tissue distribution and conduct targeted evaluation in mice.The experimental results showed that the GC-MS method has a high specificity,and the CTD in tissue has a good linear relationship in the range of 4⁶40 ng/mL,The regression equations of CTD in mouse heart,liver and kidney tissues were Y=0.0081x+0.0983,Y=0.0078x+0.1006,and Y=0.0075x+0.0969?R²is greater than 0.99 in each tissue?.and the minimum detection limitmation is 2 ng/mL.Precision and stability are in line with the requirements.After the tail vein administration of CTD solution and mPEG-PLGA-CTD micelle solution,CTD was distributed in all tissues.The comparison method showed that mPEG-PLGA-CTD micelles were in tissues compared with the original drug variously.The drug distribution was low;the AUC of micelles and the original drug in the tumor were 3276.63,1907.15?g/L*h,and the micelle was 1.72 times than that of the original drug.The drug was in heart,liver,spleen,lung and kidney.The targeting indices?TI?in tumors were 0.75,0.88,0.71,0.49,0.39,and 1.71,and the overall targeting efficiencies?TE?were12.88,31.73,9.71,2.20,3.11,and 19.18,respectively.The efficiency?RTE?was-29.27,-1.702,-33.63,-54.07,-63.45,and 59.70%,respectively.It can be seen that the TE and TI of micelles in tumor tissues are higher than other tissues,and the organs are relative tumor overall efficiency is only positive for tumors.The results show that mPEG-PLGA-CTD micelles can not only reduce the toxicity of drugs to normal tissues,but also improve the targeting of drugs to tumors,so that CTD can be better enriched in diseased tissues,thus improving its treatment of tumors.effect.Permeability study of CTD polymer micelles in tumor tissues:Permeability experiments of CTD in tumor tissues were performed by using clean-grade female Kunming mice.and mice were divided into groups and administered with pharmacokinetic studies.At each time point after administration,the mice were sacrificed by cervical dislocation,the tumor tissue was dissected,and the intact tumor tissue was equally cut into three different levels of tissue samples at the inner,middle and outer levels,and processed by protein precipitation method.It was treated by extraction with ethyl acetate.The content of the drug in the tissue was determined by using clofibrate as the internal standard and single quadrupole GC-MS method?chromatography and mass spectrometry conditions and pharmacokinetic studies?.The data was processed using Origin 8.5 software to investigate the degree of penetration of micelles in solid tumors.The experimental results show that CTD can be detected at all levels of tumor tissue after saturate CTD solution and mPEG-PLGA-CTD micelle solution.The AUC of the inner,middle and outer layers of the micelle group are 2373.47,2947.06,3813.03?g/L*h,the AUC of the inner,middle and outer layers of the original drug group were 1235.04,2289.94,and 2752.68?g/L*h,respectively.The overall targeting efficiencies of the drugs in the tumor,in the middle and outer layers were respectively It is 19.70,24.47,31.66.The comparison method showed that the distribution of mPEG-PLGA-CTD micelles in the CTD group had a certain degree of increasing in the distribution of tumors,and the distribution of the outermost layer of tumors was the highest,followed by the middle layer,and finally inner layer.The results showed that mPEG-PLGA-CTD micelles can enhance the permeability of drugs in tumor tissues,enhance the enrichment of drugs in tumor sites,thereby reducing the distribution of drugs in other parts,reducing their toxic side effects and improving anti-tumor efficacy.In summary,mPEG-PLGA-CTD micelles can increase the accumulation of drugs in the tumor site,improve the pharmacokinetic characteristics of CTD,prolong the half-life of the drug,increase the retention time of the drug in the blood circulation,and reduce its toxicity to the body.Side effects improve anti-tumor efficacy.