Study On The Effect Of Hydroxyethyl Starch-epirubicin Prodrug On Bladder Cancer

Objective In China,bladder cancer is the malignant tumor of the urinary system with the highest incidence.In recent years,the incidence of bladder cancer is increasing year by year.Epirubicin is commonly used in bladder cancer perfusion therapy,but it has strong irritation,high tissue affinity and permeability,and there is a risk of bladder epithelial stimulation and plasma exposure,which produces side effects and affects clinical treatment.The p H-sensitive hydroxyethyl starch-epidoxorubicin（HES-EPI）and folic acid-targeted hydroxyethyl starch-epidoxorubicin（FA-HES-EPI）prodrug micelles were prepared and evaluated in vitro and in vivo.Methods In order to increase the concentration of drugs in the tumor site and improve the therapeutic effect,hydroxyethyl starch-epidoxorubicin prodrug micelles with p H-sensitive and folic acid-targeting function were constructed.EPI was combined with aldehyde hydroxyethyl starch（HES-CHO）by amine or hydrazone bond,folic acid（FA）was combined with aldehyde hydroxyethyl starch by ester bond,and amphiphilic prodrugs were self-assembled by nanoprecipitation method to form micelles.The particle size,morphology and in vitro release behavior were investigated.Construction of orthotopic bladder tumor model in SD rats N-methyl-N-nitrosourea（MNU）bladder perfusion method was used to establish an animal model.In vivo irritation,plasma exposure,tissue distribution,anti-tumor effect,histopathological analysis and evaluation were performed,and cell experiments were performed to verify the anti-bladder cancer effect of the constructed hydroxyethyl starch epirubicin prodrug micelles.Results The particle size and potential of drug-loaded micelles were determined by dynamic light scattering method.The average particle size of HES-EPI imide-bonded micelles was 124.30±0.30 nm,PDI was 0.255±0.008,and the average potential was-25.80±2.40 m V.The average particle size of HES-EPI micelles was 163.30±0.50 nm,PDI was 0.202±0.012,and the average potential was-0.33±0.16 m V.The average particle size of FA-HES-EPI micelles was136.90±0.89 nm,PDI was 0.264±0.097,and the average potential was-9.07±0.67 m V.The average particle size of FA-HES-EPI micelles was 136.40±0.95 nm,the PDI was 0.183±0.023,and the average potential was-0.24±0.26 m V.The morphology of prodrug micelles was observed by transmission electron microscopy（TEM）to be spherical.By monitoring the changes of micelle size and drug loading at room temperature,it was considered that the physical stability of prodrug micelles was good.The release behavior of prodrug micelles in vitro showed slow release and certain acid sensitivity.The HE staining results showed that the prodrug micelles had lower irritation than the prototype drug.The C_max of EPI·HCl group was 98.40±4.13 ng·m L^-1,AUC_0-t was 70.56 ng·m L^-1·h^-1,the C_max of HES-EPI imine group was 72.27±4.92ng·m L^-1,AUC_0-t was 20.09 ng·m L^-1·h^-1,the C_max of HES-EPI hydrazone group was 44.07±6.25ng·m L^-1,AUC_0-t was 8.48 ng·m L^-1·h^-1.It showed that the plasma drug concentration of the prototype drug group was significantly higher than that of the prodrug micelle group after perfusion and drug excretion in vitro,which confirmed that the prodrug micelle had lower plasma exposure than the prototype drug.Using EPI spontaneous fluorescence,in vitro tumor fluorescence imaging results showed that prodrug micelles in tumor tissue specific accumulation;in addition,the drug content in tumor tissues and normal tissues of bladder in EPI HCl group,HES-EPI hydrazone group and FA-HES-EPI hydrazone group was measured,indicating that the active targeting system significantly improved the tissue distribution of drugs in vivo,and realized the increase of drug concentration in tumor sites and the decrease of healthy organs.The excellent antitumor effect of prodrug micelles was confirmed by evaluating the tumor growth inhibition on the inner and outer surfaces of bladder.The bladder tissue of FA-HES-EPI micelles group showed no obvious tumor,smooth surface,normal size and normal morphology,and the lowest tumor mass was 0.26 mg.By measuring body weight,it was found that at the end of treatment,the body weight of rats in the free EPI·HCl group decreased by 22.9%compared with that before treatment.During the whole chemotherapy process,the HES-EPI imide bond and hydrazone bond prodrug micelles group did not show significant weight loss.The weight of the experimental animals in the folic acid active targeting FA-HES-EPI imide bond and hydrazone bond prodrug micelles group increased steadily during treatment.After treatment,the weight increased by 0.45%compared with that at the beginning of treatment,indicating that its systemic toxicity was negligible.The reduced systemic toxicity indicates that the prodrug micelles have reliable biological safety.The results of histopathological HE section were consistent with those of anti-tumor in vivo.Compared with the normal bladder tissue cells in the control group,a large number of cancer cells were observed in the free drug group.The structure was disordered,and the nuclear heteromorphism was obvious.There were spherical or fusiform nuclei and enlarged nuclei or heterogeneous binuclei,indicating that there were still bladder tumor tissues.Compared with the free drug group,the number of tumor cells in all prodrug micelles treatment groups was significantly reduced.Among them,the number of tumor cells in FA-HES-EPI prodrug micelles actively targeting folic acid was significantly less than that in HES-EPI prodrug micelles.At the same time,compared with the imide-bonded prodrug micelles group,the hydrazone-bonded prodrug micelles group showed fewer tumor cells with enlarged nuclei or heterogeneous dinuclei.At the same time,TUNEL method was used to detect apoptotic DNA fragments.The number of green particles was the largest in FA-HES-EPI prodrug micelles group,indicating that the degree of apoptosis was the largest.The ability of confocal laser scanning microscope（CLSM）and flow cytometry（FCM）to detect cell uptake was always stronger in the targeted FA-HES-EPI group than in the non-targeted HES-EPI group,and this gap gradually increased with the increase of folic acid ratio.It indicates that the increase of folic acid content is expected to increase the uptake ability of tumor cells to drugs,thereby enhancing the targeted therapeutic effect of folic acid bonded prodrug micelles on bladder cancer.The above results reveal that folic acid active targeting prodrug micelles have better inhibitory effect on tumor growth.Conclusion Nanomicelles were self-assembled by coupling hydroxyethyl starch（HES）and epirubicin（EPI）via p H-sensitive imine or hydrazone bonds.Improve drug stability,delay drug release,increase drug concentration in tumor site,reduce toxicity,improve drug treatment index.In order to further increase the anti-tumor effect,folic acid was modified on the surface of micelles to give it active targeting ability.Since the high expression of folic acid-specific receptors on tumors promotes drug accumulation,compared with unmodified prodrug micelles,folic acid modification further increases the tumor targeting of micelles,enhances the killing ability of cancer cells,and reduces the toxicity to normal tissues.To sum up,the micelles based on targeted delivery,accurate drug release strategy,is expected to improve the clinical value of epirubicin in the treatment of bladder cancer.This paper has 24 figures,9 tables and 74 references.