羟丙基-β-环糊精修饰聚氰基丙烯酸正丁酯纳米粒的研究
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摘要
本课题采用现代靶向制剂新技术,选用羟基喜树碱为模型药物,以纳米粒为载体,研制羟丙基-β-环糊精修饰聚氰基丙烯酸正丁酯纳米粒新型靶向给药系统并进行动物体内分布及药动学研究,探索抗肿瘤药物靶向给药治疗的新方法、新思路、新技术,是药剂学前沿领域的研究热点,具有较高的科学技术价值和临床应用前景。
羟基喜树碱(Hydroxycamptothecin,HCPT)是从我国特有珙桐科旱莲属落叶植物喜树(Camptotheca acuminata)中提取出来的一种生物碱,具有显著的抗肿瘤活性及较宽的抗癌谱,且与其他常用的抗肿瘤药无交叉耐药性,可用于治疗多种恶性肿瘤。HCPT作为抗肿瘤药在临床上被广泛应用,对腹水性肝癌、头颈癌、胃癌、膀胱癌等具有明显疗效,为我国最常使用的抗肿瘤植物药之一。HCPT为水不溶性、脂难溶性药物,临床常用剂型为HCPT钠盐注射剂,但由于HCPT在体内代谢很快(t_(1/2)=5min),组织分布不理想以及内酯环不稳定性均会阻碍其充分发挥抗肿瘤作用,限制了临床应用。因此采用靶向制剂技术,将HCPT制成纳米制剂有望克服以上缺点,为HCPT的临床应用开辟更广阔的前景。
本课题以生物可降解型高分子材料氰基丙烯酸正丁酯为载体材料,研制羟丙基-β-环糊精修饰的羟基喜树碱聚氰基丙烯酸正丁酯纳米粒(HCPT-PBCA-NP)。在单因素考察的基础上,通过均匀设计优选处方及工艺,采用乳化聚合法制备纳米粒,筛选最优冻干保护剂进行冷冻干燥,采用MTT法考察体外抗肿瘤活性,建立可靠的含量测定方法及体内外质量评价指标,研究纳米粒在家兔体内的药物动力学特征及小鼠体内组织分布特征,并就羟丙基-β-环糊精用量对纳米粒性质的影响进行考察。主要研究结果如下:
1.以HCPT-PBCA-NP的形态和包封率为指标,固定处方中HCPT及羟丙基-β-环糊精用量,通过单因素试验考察表面活性剂及稳定剂种类、温度、搅拌速度、搅拌时间、pH值、氰基丙烯酸正丁酯(BCA)用量及盐类附加剂对纳米粒性质的影响。根据单因素考察结果,选择对纳米粒性质影响最显著的五个因素作为考察对象,即单体BCA用量、稳定剂Dxtran-70用量、乳化剂Pluronic F-68用量、附加剂NaHSO_3用量及pH值,以包封率为指标,依照均匀试验设计优化、筛选处方与制备工艺。得最佳处方与工艺为:BCA用量为70μL,Dxtran-70用量为15mg,Pluronic F-68用量为15mg,NaHSO_3用量为120mg,pH值为3.0,搅拌时间为3h,温度为25℃。
2.对优化所得HCPT-PBCA-NP的理化性质及稳定性进行综合评价,所制备的纳米粒在透射电镜下观察分散性好、圆整、均匀而不粘连,平均粒径为137.4nm,Zeta电位为-20.37±0.92mV,包封率为(78.56±2.17)%,载药量为(7.40±0.529)%,胶体pH值为4.96±0.079。HCPT-PBCA-NP胶体溶液在低温(4℃左右)条件下稳定性较好,而室温(25℃左右)条件下稳定性较差,长时间放置易出现沉淀,粒径和包封率改变等不稳定现象,因此需避光冷藏保存。
3.为进一步增加HCPT-PBCA-NP胶体溶液的物理及化学稳定性,延长贮存期,选择以8%甘露醇和4%海藻糖合用作冻干保护剂,制备冻干制剂,并以其微观形态,以及再分散后的粒径大小、表面电位、包封率及载药量为指标,对冻干效果进行评价。结果表明,与冻干前的HCPT-PBCA-NP胶体溶液相比,冻干复溶后粒子分布比较均匀,分散性较好,但稍有粘连;平均粒径略有增大,由137.4nm增至169.7nm;Zeta电位由-20.37 mV改变至-22.59 mV;包封率和载药量均略有降低,分别由(78.56±2.17)%及(7.40±0.529)%降低至(72.83±3.42)%及(6.27±0.368)%。
4.采用荧光分光光度法进行生物样品的测定。本方法灵敏度较高,重现性较好,线性范围广,且操作简单,快速,准确,用此法测定家兔血浆、小鼠血浆及各组织脏器中HCPT浓度,计算药动学参数,取得良好结果。为保证测定结果的准确性,在生物样品处理过程中,应先加入冰醋酸避光酸化,使开环的HCPT闭合成内酯形式,再用甲醇-乙腈(1:1)混合液沉淀蛋白提取药物后测定荧光吸收强度F。血浆及各组织器官药物浓度线性关系良好,回收率均在90%～110%内,日内精密度小于6%,日间精密度小于8%,且内源性物质不干扰HCPT的测定,可满足生物样品测定的要求。
5.家兔静脉注射HCPT注射液和HCPT-PBCA-NP后,血药浓度测定结果用DAS 2.0药物动力学程序进行处理,两者体内药动学过程均符合双室模型特征,动力学方程分别为:C=0.582e~(-1.747t)+0.097e~(-0.306t)和C=0.204e~(-0.464t)+0.115e~(-0.044t)。通过对两种制剂静脉注射给药后的药动学参数比较可知,纳米粒组AUC值(2.805μg/mL ~*h)和t_(1/2β)(15.712h)明显高于注射液组AUC值(0.699μg/mL~*h)和t_(1/2β)(2.265h);纳米粒组药物清除率CL(0.006 L/h/kg)小于注射液组药物清除率CL(0.026 L/h/kg)。证明HCPT纳米粒静脉注射给药后,可显著延长药物在体内的存留时间及药物与靶组织器官的接触时间,具有缓释长效作用。
6.小鼠组织分布试验表明HCPT制成纳米粒后,与HCPT注射液相比,药物在各组织器官的分布发生明显改变,结果显示:肝、脾的相对摄取率r_e分别为25.000和19.889;肝的总靶向效率T_e由9.21%提高到49.32%(5.36倍),脾的总靶向效率T_e由6.33%提高到26.98%(4.26倍),而肾的总靶向效率则由35.03%降至12.04%;肝脏内峰浓度提高至2.693倍,脾脏内峰浓度提高至4.816倍。表明该纳米粒具有显著的肝靶向性分布及较低的肾毒性。
7.通过考察羟丙基-β-环糊精用量对纳米粒形态和粒径分布、表面电位、包封率和载药量、体外释药动力学及体外抗肿瘤活性等性质的影响,发现通过羟丙基-β-环糊精对纳米粒进行表面修饰后可起到减小粒径,降低表面负电性,提高包封率及载药量,延缓药物释放速率,增强体外抗肿瘤活性的作用,且上述影响与羟丙基-β-环糊精用量呈现出一定的剂量依赖关系。以此为依据可进一步改善药物疗效、作用时间及组织器官靶向性。
8.体外释放试验结果表明,游离HCPT的体外释药过程可用一级动力学方程描述,4h药物已全部释放出来;对于HCPT-PBCA-NP,羟丙基-β-环糊精用量越大,纳米粒的缓释长效作用越明显。HCPT纳米粒的体外释放可用双相动力学方程拟合,前期为快速释药,后期缓慢释药。这种释药特点比较符合抗肿瘤药物的用药原则,即给药后到达靶部位可快速释放药物,以达到较高药物浓度发挥治疗作用;慢速释药可不断补充药物到靶部位以维持有效药物浓度,使制剂在较长时间发挥治疗作用。
9.采用MTT比色法研究并比较HCPT-PBCA-NP及HCPT注射液对人肝癌细胞HepG-2体外生长及增殖的影响,评价体外抗肿瘤活性。结果表明,与HCPT注射液对照组相比,HCPT纳米粒在体外可有效抑制人肝癌细胞HepG-2的生长增殖,并具有一定的剂量依赖性;随着羟丙基-β-环糊精用量的增加,HCPT纳米粒的体外抗肿瘤活性也表现出不断增强的趋势。
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,NaHSO_3 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;NaHSO_3 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.
羟基喜树碱(Hydroxycamptothecin,HCPT)是从我国特有珙桐科旱莲属落叶植物喜树(Camptotheca acuminata)中提取出来的一种生物碱,具有显著的抗肿瘤活性及较宽的抗癌谱,且与其他常用的抗肿瘤药无交叉耐药性,可用于治疗多种恶性肿瘤。HCPT作为抗肿瘤药在临床上被广泛应用,对腹水性肝癌、头颈癌、胃癌、膀胱癌等具有明显疗效,为我国最常使用的抗肿瘤植物药之一。HCPT为水不溶性、脂难溶性药物,临床常用剂型为HCPT钠盐注射剂,但由于HCPT在体内代谢很快(t_(1/2)=5min),组织分布不理想以及内酯环不稳定性均会阻碍其充分发挥抗肿瘤作用,限制了临床应用。因此采用靶向制剂技术,将HCPT制成纳米制剂有望克服以上缺点,为HCPT的临床应用开辟更广阔的前景。
本课题以生物可降解型高分子材料氰基丙烯酸正丁酯为载体材料,研制羟丙基-β-环糊精修饰的羟基喜树碱聚氰基丙烯酸正丁酯纳米粒(HCPT-PBCA-NP)。在单因素考察的基础上,通过均匀设计优选处方及工艺,采用乳化聚合法制备纳米粒,筛选最优冻干保护剂进行冷冻干燥,采用MTT法考察体外抗肿瘤活性,建立可靠的含量测定方法及体内外质量评价指标,研究纳米粒在家兔体内的药物动力学特征及小鼠体内组织分布特征,并就羟丙基-β-环糊精用量对纳米粒性质的影响进行考察。主要研究结果如下:
1.以HCPT-PBCA-NP的形态和包封率为指标,固定处方中HCPT及羟丙基-β-环糊精用量,通过单因素试验考察表面活性剂及稳定剂种类、温度、搅拌速度、搅拌时间、pH值、氰基丙烯酸正丁酯(BCA)用量及盐类附加剂对纳米粒性质的影响。根据单因素考察结果,选择对纳米粒性质影响最显著的五个因素作为考察对象,即单体BCA用量、稳定剂Dxtran-70用量、乳化剂Pluronic F-68用量、附加剂NaHSO_3用量及pH值,以包封率为指标,依照均匀试验设计优化、筛选处方与制备工艺。得最佳处方与工艺为:BCA用量为70μL,Dxtran-70用量为15mg,Pluronic F-68用量为15mg,NaHSO_3用量为120mg,pH值为3.0,搅拌时间为3h,温度为25℃。
2.对优化所得HCPT-PBCA-NP的理化性质及稳定性进行综合评价,所制备的纳米粒在透射电镜下观察分散性好、圆整、均匀而不粘连,平均粒径为137.4nm,Zeta电位为-20.37±0.92mV,包封率为(78.56±2.17)%,载药量为(7.40±0.529)%,胶体pH值为4.96±0.079。HCPT-PBCA-NP胶体溶液在低温(4℃左右)条件下稳定性较好,而室温(25℃左右)条件下稳定性较差,长时间放置易出现沉淀,粒径和包封率改变等不稳定现象,因此需避光冷藏保存。
3.为进一步增加HCPT-PBCA-NP胶体溶液的物理及化学稳定性,延长贮存期,选择以8%甘露醇和4%海藻糖合用作冻干保护剂,制备冻干制剂,并以其微观形态,以及再分散后的粒径大小、表面电位、包封率及载药量为指标,对冻干效果进行评价。结果表明,与冻干前的HCPT-PBCA-NP胶体溶液相比,冻干复溶后粒子分布比较均匀,分散性较好,但稍有粘连;平均粒径略有增大,由137.4nm增至169.7nm;Zeta电位由-20.37 mV改变至-22.59 mV;包封率和载药量均略有降低,分别由(78.56±2.17)%及(7.40±0.529)%降低至(72.83±3.42)%及(6.27±0.368)%。
4.采用荧光分光光度法进行生物样品的测定。本方法灵敏度较高,重现性较好,线性范围广,且操作简单,快速,准确,用此法测定家兔血浆、小鼠血浆及各组织脏器中HCPT浓度,计算药动学参数,取得良好结果。为保证测定结果的准确性,在生物样品处理过程中,应先加入冰醋酸避光酸化,使开环的HCPT闭合成内酯形式,再用甲醇-乙腈(1:1)混合液沉淀蛋白提取药物后测定荧光吸收强度F。血浆及各组织器官药物浓度线性关系良好,回收率均在90%～110%内,日内精密度小于6%,日间精密度小于8%,且内源性物质不干扰HCPT的测定,可满足生物样品测定的要求。
5.家兔静脉注射HCPT注射液和HCPT-PBCA-NP后,血药浓度测定结果用DAS 2.0药物动力学程序进行处理,两者体内药动学过程均符合双室模型特征,动力学方程分别为:C=0.582e~(-1.747t)+0.097e~(-0.306t)和C=0.204e~(-0.464t)+0.115e~(-0.044t)。通过对两种制剂静脉注射给药后的药动学参数比较可知,纳米粒组AUC值(2.805μg/mL ~*h)和t_(1/2β)(15.712h)明显高于注射液组AUC值(0.699μg/mL~*h)和t_(1/2β)(2.265h);纳米粒组药物清除率CL(0.006 L/h/kg)小于注射液组药物清除率CL(0.026 L/h/kg)。证明HCPT纳米粒静脉注射给药后,可显著延长药物在体内的存留时间及药物与靶组织器官的接触时间,具有缓释长效作用。
6.小鼠组织分布试验表明HCPT制成纳米粒后,与HCPT注射液相比,药物在各组织器官的分布发生明显改变,结果显示:肝、脾的相对摄取率r_e分别为25.000和19.889;肝的总靶向效率T_e由9.21%提高到49.32%(5.36倍),脾的总靶向效率T_e由6.33%提高到26.98%(4.26倍),而肾的总靶向效率则由35.03%降至12.04%;肝脏内峰浓度提高至2.693倍,脾脏内峰浓度提高至4.816倍。表明该纳米粒具有显著的肝靶向性分布及较低的肾毒性。
7.通过考察羟丙基-β-环糊精用量对纳米粒形态和粒径分布、表面电位、包封率和载药量、体外释药动力学及体外抗肿瘤活性等性质的影响,发现通过羟丙基-β-环糊精对纳米粒进行表面修饰后可起到减小粒径,降低表面负电性,提高包封率及载药量,延缓药物释放速率,增强体外抗肿瘤活性的作用,且上述影响与羟丙基-β-环糊精用量呈现出一定的剂量依赖关系。以此为依据可进一步改善药物疗效、作用时间及组织器官靶向性。
8.体外释放试验结果表明,游离HCPT的体外释药过程可用一级动力学方程描述,4h药物已全部释放出来;对于HCPT-PBCA-NP,羟丙基-β-环糊精用量越大,纳米粒的缓释长效作用越明显。HCPT纳米粒的体外释放可用双相动力学方程拟合,前期为快速释药,后期缓慢释药。这种释药特点比较符合抗肿瘤药物的用药原则,即给药后到达靶部位可快速释放药物,以达到较高药物浓度发挥治疗作用;慢速释药可不断补充药物到靶部位以维持有效药物浓度,使制剂在较长时间发挥治疗作用。
9.采用MTT比色法研究并比较HCPT-PBCA-NP及HCPT注射液对人肝癌细胞HepG-2体外生长及增殖的影响,评价体外抗肿瘤活性。结果表明,与HCPT注射液对照组相比,HCPT纳米粒在体外可有效抑制人肝癌细胞HepG-2的生长增殖,并具有一定的剂量依赖性;随着羟丙基-β-环糊精用量的增加,HCPT纳米粒的体外抗肿瘤活性也表现出不断增强的趋势。
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,NaHSO_3 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;NaHSO_3 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.
引文
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