Study On Preparation And Targetability Of The Prodrug Folate-mediated Bovine Serum Albumin 5-fluorouracil

Objective: The prodrug folate-mediated bovine serum albumin -5-fluorouracil (F-BSA-5-FU)was synthesized,then purify,separate and optimize the synthesis conditions of it;Folate-mediated bovine serum albumin 5 - fluorouracil nanoparticles (F-BSANP-5-FU) and bovine serum albumin 5 - fluorouracil nanoparticles (BSANP-5-FU) were prepared; Tumor targeting in vivo through the preparation of loaded calcein nanoparticles and the pharmacodynamics of the conjugates nanoparticles were evaluated respectively.Methods: The synthesis of folate- bovine serum albumin was designed using orthogonal experiment;the effect factors such as the amount of folate,the molar ratio of EDC and folate,the reaction time were investigated respectively.Using the coupled rate of folate and bovine serum albumin as the evaluation of indicators, selected the optimal synthesis conditions. Using the yield as the evaluation of indicators, the 5-fluorouracil-1-acetic acid (5-FUA) and its active ester were synthesized, The synthesis of 5-FUA active ester was designed using orthogonal experiment, selected the optimal synthesis conditions,calculate yield,determine melting point of the product. The coupling of 5-FUA active ester with BSA and F-BSA were investigated,the best synthesis conditions was optimizated through the single-factor analysis.The above-mentioned products were monitored by ultraviolet,infrared,differential thermal analysis,MALDI-TOF-MS.Simultaneously the content of 5-FU in F-BSA-5-FU and BSA-5-FU were calculated by chemical methods and MALDI-TOF MS. F-BSANP-5-FU,BSANP-5-FU and loaded calcein nanoparticles were prepared by desolvation-chemical coupling method, the effect factors such as the concentration of drug,the pH of the solution,the amount of coupling agent,the coupling time were investigated. Using the average diameter and polyindex as the evaluation of indicators, selected the optimal preparation conditions, and the nanoparticles were characterized. Using tumor-bearing mice which was inoculated 7 ~ 10d for liver cancer (H22) solid tumor as animal model,the tumor targeting in vivo was determined by spectrophotofluorometer through the loaded calcein nanoparticles.The tumor inhibition information of F-BSANP-5-FU and BSANP-5-FU were evaluated by intraperitoneal injection, Original data was input to computer and was analyzed by the statistical software SPSS11.5.Inhibition rate (%)=( average tumor weight in contrast group - average tumor weight in dosage group) / average tumor weight in contrast group×100%; spleen and thymus index calculated as follows: spleen or thymus index = spleen or thymus weight (mg ) / mouse weight (g),the tumor of each group was made pathological section and the information of tumor suppression was observed .Results: The coupling ratio of about 4 of folate-bovine serum albumin was synthesized with the most optimized synthesis conditions,that is a bovine serum albumin was coupled with 4 folates; 5-FU melting point was 283℃～284℃,5-FUA melting point was 276℃～278℃, yield was 81.37%;5-FUA active ester melting point was 255℃～257℃, yield was 83.98%;The results of ultraviolet,infrared,differential thermal analysis,MALDI-TOF MS showed that compounds were synthesized successfully;The nanoparticles whose particle size was less than 200nm were prepared successfully, and the size was uniform, the shape was integrated;The tumor targeting in vivo experiments showed that the equal doses of different groups of calcein were given by intraperitoneal injection,the accumulation of the content of calcein were different in vivo (P <0.05);The content of loaded calcein the BSANP-5-FU group (A group) reached its peak at 2.0h, and then declined in tumor tissues, The content of loaded calcein of Folate-BSANP-5-FU group (B group) reached its peak at 0.5h, and then declined sharply in tumor tissues . The content of calcein of liver and kidney tissues were less than the tumor tissue, but higher than the heart,spleen,lung tissue.The peak value of B group was significantly higher than A Group; The level of animal pharmacodynamic experiments showed that the tumor growth was the slowest in the tumor-bearing mice by intraperitoneal injection of F-BSANP-5-FU high-dose group, the highest inhibition rate reached 47.19%, the inhibition rate of F-BSANP-5-FU was significantly higher than BSANP-5-FU, after adding 1 mmol ? L-1 exogenous folate, the inhibition rate of F-BSANP-5-FU was declined significantly. The inhibition rate 5-FU positive control group was higher than BSANP-5-FU.Conclusions: F-BSA-5-FU was synthesized under mild conditions, and its nanoparticles had tumor targeting, the targeting was likely to pass the folate-mediated, F-BSA-5-FU had good the inhibition of tumor,and its was a potential anti-tumor drugs that can be targeted at tumor with the high expression of folate receptor.