Investigation Of Nanoparticles Combined Polybutylcyanoacrylate And Hydroxypropyl-β-cyclodextrin

In the present study,hydroxycamptothecin(HCPT)was taken as the model drug to prepare targeted-oriented hydroxycamptothecin nanoparticles drug system combined polybutylcyanoacrylate and hydroxypropyl-β-cyclodextrin (HCPT-PBCA-NP)by modern targeted drug delivery technique.The characteristics of distribution and pharmacokinetics in vivo were examined at the same time.The accomplishment of this project is a great achievement of nanoparticles drug system and develop a new idea,new method and new technique for targeted therapy of antitumor drugs.Hydroxycamptothecin(HCPT)is a kind of microamount alkaloids extracted from Camptotheca acuminata.The in vitro and vivo experiments were all revealed that HCPT had distinguished anti-tumour effect,wide anti-tumour pattern and achiasmate drug fast with other antineoplastic agents,it can be used to cure many kinds of malignancy.Now HCPT is generally used as an anti-tumour drug to cure liver cancer,stomach cancer,leukemia and many malignant tumors.However,its clinical applications are confined by its special physico-chemical properties(for example,it is insolubilize in water and difficultly dissolves in lipids,the lactone band is also instable),so its curative effects decrease and the intracorporal half life (t_1/2=5min)is too short.In the present study,HCPT was taken as the model drug to prepare hydroxycamptothecin nanoparticles combined polybutylcyanoacrylate and hydroxypropyl-β-cyclodextrin(HCPT-PBCA-NP)by modern targeted drug delivery technique.The emulsification evaporation method was used to prepare HCPT-PBCA-NP.According to the results of the effects of different variables, the uniform experimental design was applied to optimize the prescription and technology of preparation.The effects of the quantity of hydroxypropyl-β-cyclodextrin on the nanoparticles' characters were investigated and the nanoparticles were developed to improve the stabllitiy and extend the storage time using freeze-drying technology;The effects of HCPT-PBCA-NP on the growth of hepatoma carcinoma cell HepG-2 in vitro were investigated using MTT colorimetric method to evaluate the antineoplasmic activity;To establish the quality control standard for HCPT-PBCA-NP in vivo and in vitro,pharmacokinetics and tissue distribution in rabbits and mouse were investigated to evaluate its targeting efficacy.The main results were as follows:1.The particle shape and entrapment efficiency were taken as criterions to evaluate the effects of surfactant,temperature,mixing rate,mixing time,pH,the quantity of BCA and supplemental agents on the properties of HCPT-PBCA-NP after fixing the quantity of HCPT and hydroxypropyl-β-cyclodextrin.As a result,the quantity of BCA,Dxtran-70,PluronicF-68,NaHSO₃ and pH were proved to have great effect on the properties of HCPT-PBCA-NP.The uniform experimental design was applied to optimize the prescription and technology based on the entrapment efficiency of HCPT-PBCA-NP.The optimization is as follows:BCA was 70μL;Dxtran-70 was 15mg;PluronicF-68 was 15mg;NaHSO₃ was 120mg;pH was 3.0;mixing time was 3h;temperature was 25℃.2.To evaluate the physico-chemical properties and stabilities of nanoparticles which were prepared using the optimized formulation.From the transmission electron microscope observation,the nanoparticles were spherically shaped, ranged in uniformity size and had good dispersibility.The particle size was 137.4nm,the entrapment efficiency and drug loading were(78.56±2.17)%and (7.40±0.529)%;The zeta potential was-20.37±0.92mV,which attained the predicted objective.HCPT-PBCA-NP showed unstable under room temperature(about 25℃) for storing a long time;it showed preferable stability under cold storage,which indicated that nanoparticles needed to be stored under cold conditions(about 4℃) away from light.3.The nanoparticles were developed to improve stabllitiy and extend storage time using freeze-drying technology,8%manicol and 4%trehalose were choosen as freeze-drying protective agents.The freeze-drying effect of HCPT-PBCA-NP was examined using the qualities such as the particle size,particle distribution,zeta potential,entrapment efficiency and drug loading,and the results showed that HCPT-PBCA-NP freeze-drying preparation had good uniformity and dispersibility, but adhered slightly,compared with the colloidal solution,it had a little larger mean diameter(137.4nm to169.7nm),lower zeta potential(-20.37 mV to -22.59 mV), lower entrapment efficiency((78.56±2.17)%to(72.83±3.42)%)and drug loading ((7.40±0.529)to(6.27±0.368)%).4.Fluorospectrophotometry was established to investigate pharmacokinetic and drug distribution in rabbits and mouse.This method had a higher sensibility, better reproducibility and wide linear range,simple,fast and accurate.And during the treating process of these biological specimens,glacial acetic acid should be added earlier away from light for 4h to turn seco-HCPT to lactone pattern,then determined the fluorescence intensity(F)after precipitating proteinum with methanol- acetonitrile(1:1).The results satisfied the testing requests of biological specimens.5.The pharmacokinetic experiment of HCPT solution and HCPT nanoparticles was carded out on rabbits,and DAS 2.0 pharmacokinetics procedure was applied to calculate pharmacokinetic parameters and chose the best compartment method.The HCPT solution and HCPT-PBCA-NP were all fitted two compartment model after i.v.administrations.Compared with HCPT solution,the AUC of HCPT-PBCA-NP were increased and the retention time in blood were also prolonged, while CL were significantly reduced.As a result,the circulating time of the drug in blood was remarkedly prolonged which helped to increase the concentration of the drug in target organs for enhancing its curative effect.6.The tissue distribution of HCPT solution and HCPT-PBCA-NP was investigated after i.v.administrations into mouse.Compared with HCPT solution, HCPT-PBCA-NP injection distinctly changed the distribution of HCPT in vivo and increased the concentration of drug in target organs greatly.The relative taking efficiency(r_e)of liver and spleen were 25.000 and 19.889;the maximum concentrations(C_max)of liver and spleen were enhanced by 2.693 times and 4.816 times;and the targeting efficiency(T_e)of liver and spleen were also improved from 9.21%to 49.32%(5.36 times)and 6.33%to 26.98%(4.26 times)respectivly, while T_e of kidney was cut down from 35.03%to 12.04%.In conclusion, HCPT-PBCA-NP could greatly increase the distribution of HCPT in liver and spleen with lower nephrotoxicity.7.Through the investigation about effects of hydroxypropyl-β-cyclodextrin on particle size,zeta potential,entrapment efficiency,drug loading,the release pharmacokinetics and antineoplasmic activity in vitro,we found that the nanoparticles' entrapment efficiency and drug loading could be raised,the superficial electronegativity became lower and the particle diameter became smaller combined with hydroxypropyl-β-cyclodextrin,which attained the predicted objective for delaying release rate,target distribution and improving the curative effect.8.The drug release behavior from HCPT solution and HCPT nanoparticles in vitro was in accord with first order kinetics model and double phase kinetics model respectivly.The HCPT-PBCA-NP release behavior showed sustained-release effect, HCPT released quickly during protophase and released slowly during anaphase,this characteristic consistented with the medication principle of antitumor drug,and the release rate became more and more slower with the increasing quantity of hydroxypropyl-β-cyclodextrin.9.The effects of HCPT solution and HCPT-PBCA-NP on the growth of hepatoma carcinoma cell HepG-2 in vitro were investigated using MTT colorimetric method to evaluate the antineoplasmic activity in vitro.As a result,compared with HCPT solution,HCPT-PBCA-NP had a active suppression on the growth of HepG-2 with dose dependent,and the antineoplasmic activity could be enhanced greatly with the increasing quantity of hydroxypropyl-β-cyclodextrin.