【作者】 潘振华；

【导师】 张永健；

【作者基本信息】 河北医科大学 ， 药理学， 2012， 博士

【摘要】 目的：环糊精和脂质体（liposomes）作为药物的载体，可有效地提高药物的生物利用度和治疗指数。脂质体是一种具有类生物膜结构的双分子层微囊，是一种定向药物载体,属于靶向给药体系（targeting drug deliverysystem）的一种新型给药系统。其进入体内后主要被网状内皮系统吞噬而激活机体的自身免疫功能，并且通过改变被包封药物的体内分布，使药物主要在肝、脾、肺等组织器官中蓄积，并能优先聚集于肿瘤、炎症、感染等部位，从而提高药物治疗指数，减少治疗剂量并降低药物的毒性；但脂质体在包埋药物尤其是疏水性药物时往往受到药脂比等因素的限制，并且某些疏水性药物可能会干扰脂质双层的结构，影响脂质体的稳定性，使其在贮存过程发生药物泄漏。脂质体包封疏水性药物存在的这些问题在一定程度上限制了它的应用。环糊精（cyclodextrins，CYD）的特殊结构使其可与和疏水性药物形成包合物，增加难溶性药物的溶解度，提高其生物利用度。近年来的研究发现，利用环糊精与脂质体各自具备的优点，制备环糊精包合物脂质体，可以提高脂质体的稳定性,减少药物泄漏,提高载药量,改变药物的体内行为,同时使其具有缓释性和靶向性。本实验以全合成的新型小分子化合物N-[4-（4,6二甲基-2-嘧啶氧基）-3-甲基苯基]-N’-[2-（二甲氨基）]苯甲酰脲（SUD）为疏水性药物模型，研究材料组成、制备工艺对包合物-脂质体包封率的影响，考察其理化性质、体外释药情况、大鼠体内药物动力学行为及小鼠的体内分布，为这一新型给药系统的应用提供实验基础。并采用药理学试验方法进一步探讨其脂质体给药系统对人肺腺癌A549细胞体外增殖的影响，为其作为抗肿瘤药物应用于临床提供实验依据。方法：以羟丙基-β-环糊精（hydroxypropyl-β-cyclodextrin，HP-β-CD）为包合材料，分别用饱和水溶液法和研磨法制备了SUD-HP-β-CD包合物，通过对包合率及产率的评价，最后采用饱和水溶液法制备，通过四因素三水平正交实验设计对制备工艺进行优化，对包合率测定结果进行极差分析，最后确定SUD-HP-β-CD包合物的最佳处方工艺，并采用差示扫描量热法（differential scanning calorimetry, DSC）和X射线粉末衍射（X-raydiffraction, XRD）法对所得到的包合物进行鉴定。在文献和预实验的基础上初步确定SUD-HP-β-CD包合物脂质体的制备方法和处方，通过单因素考察初步确定脂质体制备的影响因素，并研究不同包封率测定方法对包封率测定结果的影响；采用薄膜分散法、注入法、逆向蒸发法、复乳法四种方法制备脂质体，利用葡聚糖凝胶柱对脂质体与未包封游离药物进行分离。测定包封率，最终确定薄膜分散法为SUD-HP-β-CD包合物脂质体的制备方法。以包封率为考察指标，通过单因素考察优化脂质体的制备工艺，并考察磷脂浓度、磷脂与胆固醇质量比、水化液类型、药物与磷脂质量比等因素对脂质体的影响。在此基础上，采用四因素三水平正交试验对各因素的水平进行筛选，对包封率测定结果进行极差分析，最后确定SUD-HP-β-CD包合物脂质体的最佳处方。考察按照优化处方制备的SUD-HP-β-CD包合物脂质体的形态外观，粒径分布及黏度等指标，考察其理化稳定性。分别绘制SUD-HP-β-CD包合物脂质体和SUD-HP-β-CD包合物的体外释药曲线并比较其体外释药过程，分别用零级、一级、Higuchi及Weibull动力学方程进行拟合，并考察相关系数。以高效液相法测定大鼠血浆及小鼠组织中SUD-HP-β-CD包合物脂质体浓度。液相条件：色谱柱：Diamonsil^TMODS C₁₈（钻石）250×4.6mm，5μm；以甲醇:水（80:20）v/v为流动相；检测波长为232nm；柱温：30℃；流速：1mL·min^-1；进样量为20μL。对结果进行处理，计算大鼠体内各种药动学参数，考察SUD-HP-β-CD包合物脂质体在小鼠体内分布，并进行靶向性评价。常规培养人肺腺癌A549细胞，采用MTT法测定SUD-HP-β-CD包合物脂质体对A549细胞增殖的抑制作用。流式细胞仪测定细胞周期分布。Western blot法测定细胞增殖核抗原（PCNA）及细胞周期蛋白（cyclin D1）蛋白表达。初步探讨SUD-HP-β-CD包合物脂质体对人肺腺癌A549细胞体外增殖的影响。结果：采用饱和水溶液法制备SUD-HP-β-CD包合物包合率较高，最佳制备工艺为SUD与HP-β-CD投料比为1:5，包合温度为50℃，包合时间为90min，搅拌速度100转/min，平均包合率为75.8%（n=3）DSC法对包合物进行鉴定，发现SUD-HP-β-CD包合物在105℃出现新的吸热峰。在XRD图中，SUD经包合后，SUD的部分特征衍射峰消失或位移,表明确实形成了包合物。通过单因素考察和正交实验，筛选出了SUD-HP-β-CD包合物脂质体的最优处方为磷脂50mg·mL^-1，磷脂与胆固醇质量比5:1，药脂比为1:30。根据优化处方制备的SUD-HP-β-CD包合物脂质体外观为均匀的乳白色混悬液，包封率为76.32%，平均粒径约为238.4nm，多分散性系数为0.258。SUD-HP-β-CD包合物溶液的体外释药最符合Weibull方程，为ln[-ln（1-Q）]=0.9045lnt^-1.4335， r²=0.9861。SUD-HP-β-环糊精包合物脂质体混悬液体外释药符合一级动力学方程，为ln（1-Q）=-0.0824t+0.0517，r2=0.9893。稳定性结果显示：脂质体在加热条件下不稳定；25℃条件下存放一个月，包封率下降33.6%，脂质体沉淀分层明显；在4℃条件下存放一个月，包封率下降1.7%，稳定性较好，说明脂质体需在冷藏条件下保存。药动学实验表明：SUD溶液和SUD-HP-β-CD包合物脂质体静脉给药后，脂质体组消除速率常数k为0.22h^-1，溶液组的k为0.41h^-1，说明制成脂质体后SUD在大鼠体内的消除减慢。脂质体组AUC为517.54μg·h·mL^-1，溶液组AUC为228.72μg·h·mL^-1，表明脂质体增加了药物在机体中的生物利用度。小鼠体内分布实验表明：SUD-HP-β-CD包合物脂质体较溶液在心、脾和肺中的药物浓度有较大增高，脂质体组中心、脾、肺的re分别为3.01、3.14、2.71，说明脂质体经静脉注射后在心、脾、肺组织有一定浓集，起到了一定的靶向目的。MTT实验结果表明，SUD-HP-β-环糊精包合物脂质体对A549细胞瘤株的生长有明显的抑制作用，随着浓度增加和作用时间的延长，对细胞增殖的抑制作用逐渐增强。流式细胞仪检测细胞周期结果表明：不同浓度含药脂质体作用48h对A549细胞周期分布均有不同程度的影响，与空白对照组相比，1、5、10μg·mL^-1SUD-HP-β-环糊精包合物脂质体组G₀/G₁期细胞百分率均明显增加（P<0.05或P<0.01）。其中5、10μg·mL^-1SUD-HP-β-环糊精包合物脂质体组S期细胞百分率明显降低（P <0.01）。10μg/mL SUD-HP-β-环糊精包合物脂质体组G₂/M期细胞百分率也明显降低（P <0.01）。说明SUD-HP-β-环糊精包合物脂质体可使A549细胞停滞在G₀/G₁期，从而抑制细胞增殖。Western blot结果显示，与空白对照组相比，空白脂质体对PCNA、cyclin D1蛋白表达均没有明显影响。而5μg·mL^-1SUD原药以及1、5、10μg·mL^-1SUD-HP-β-环糊精包合物脂质体作用于A549细胞48h均不同程度地下调了PCNA和cyclin D1的蛋白表达（P<0.05或P<0.01）。结论：以HP-β-CD为包合材料，以饱和水溶液法制备SUD-HP-β-CD包合物；以磷脂、胆固醇为主要膜材，以薄膜分散法制备SUD-HP-β-CD包合物脂质体，制备工艺简单，质量可控，重现性好，可提高药物靶向性，从而降低毒副作用，有望开发成为有良好应用前景的新剂型。SUD-HP-β-CD包合物脂质体给药系统对人肺腺癌A549细胞体外增殖有明显的抑制作用，使细胞周期停滞于G₀/G₁期，还明显下调了PCNA和cyclin D1的蛋白表达，为其作为抗肿瘤药物应用于临床提供实验依据。更多还原

【Abstract】 Objective: Cyclodextrins （CYD） and liposomes have been used in recentyears as drug delivery vehicles, improving the bioavailability and therapeuticindex of many poorly water soluble drugs. Liposome is a kind of ultra-fineform globular carrier preparation and encapsulates the drug in the thin filmformed by lipid bilayer. Liposome is analog to cellular structure, and has thecharacteristics and function of biological membrane. Liposomes have beenextensively used as drug carriers that can enhance the therapeutic index of anumber of drugs and reduce the toxicity. Liposomes can accumulate greateramounts of drug in some tissues such as liver, spleen and lung comparing freedrug administration.However,incorporation of lipophilic drugs into lipidbilayers of liposomes is often limited in terms of drug-to-lipid mass ration anddrug choice，whitch can interfere with bilayer formation and its stability. CYDcan accommodate water insoluble drugs in their cavities to form inclusioncomplexes. The formed inclusion complex has increased solubility, stabilityand pharmacological activity. Entrapping water-soluble drug/CYD inclusioncomplexes into the aqueous phase of liposomes combines the advantages ofcertain properties of cyclodextrins and liposomes into a single system tocircumvent problems associated with both systems. In our present study, SUDwas used as lipophilic drug model. SUD-HP-β-CD liposome was prepared toincrease the bioavailability and cut down toxicity to achieve the effect ofprolonged action. Meanwhile, some pharmacological experiments wereapplied to explore the effect of SUD-HP-β-CD liposome on A549cellproliferation and the related mechanisms, which would be expected to provideexperimental evidence in future clinical application of SUD-HP-β-CD liposome as an anti-tumor drug.Methods: Inclusion complexes of SUD with hydropropyl-β-cyclodextrin（HP-β-CD） were prepared by the saturation solution method and ultrasionmethod，respectively. However，the saturation solution method was the betterthan the ultrasion method on the basis of the load efficiency. L49（3） orthogonaldesign of the preparation technology of HP-β-CD was made to screen thedosage of adjuvant. The optimized prescription was decided according to theresult of encapsulated ratio determination by range analysis. Inclusioncomplexes were verified by differential scanning calorimetry （DSC） and X-raydiffraction （XRD）.Preparation technique and prescription of liposomal of SUD-HP-β-CDwere initially determined on the basis of literature and preliminary test, andinfluential factors were determined by single factor investigation. The effect ofdifferent assay methods on encapsulated ratio was studied. Four methods suchas film dispersion method, injection method, reverse-phase method, doubleemulsion method were used to prepare liposomes, and sephadex column wasused to separate liposome with free drug. The encapsulated ratio of theliposomes was determined, which showed that the film dispersion method wasthe best preparation method. Encapsulated ratio as index, the preparationtechnology of liposomes of SUD-HP-β-CD was optimized by single factorinvestigation, Consequently, L9（34） orthogonal design was made to screen thedosage of adjuvant. The optimized prescription was decided by rangeanalysis to the encapsulated ratio determination result.The shape,appearance,viscosity,particle size distribution、and physical andchemical stability were observed. The accumulative release curve ofSUD-HP-β-CD liposomes and SUD-HP-β-CD solution was protractedrespectively and compared. Zero order, first order, Higuchi, and Weibullkinetic equations were used to fit the release process, and coefficientcorrelation were calculated.A simple HPLC method was developed for the determination of SUD inrat plasma and mice tissues. HPLC conditions: column: Diamonsil C18column （200×4.6mm,5μm）; methanol: water （80:20） v/v as mobile phase; detectionwavelength was232nm; column temperature:30℃; flow rate1mL·min^-1;injection volume was20μL. The pharmacokinetic parameters were calculatedand the drug distribution in tissues of mice was investigated to evaluatetargeting in tissues.A549cells were cultured. The proliferation of A549cells was evaluatedby MTT assay. Cell-cycle distribution was determined by flow cytometryassay. The expression of PCNA and Cyclin D1was evaluated by western blotanalysis.Results: SUD-HP-β-CD prepared by saturated aqueous solution has ahigher inclusion rate, the optimal feed ratio of SUD and HP-β-CD is1:5; theinclusion temperature is50℃,the inclusion time is90min, the stirring speedis100rev/min and the average inclusion rate is75.8%（n=3）. The inclusioncompound was identified by DSC method and it has been found that newendothermic peak appeared in SUD-HP-β-CD inclusion compound at105℃.Whereas, in the XRD patterns, part of the characteristic diffraction peaks ofSUD disappeared or displaced after SUD was inclused, which indicated thatthe inclusion complex had been formated indeed.The optimized prescription of SUD-HP-β-CD inclusion compoundliposomes was: phospholipid50mg·mL^-1, phospholipid to cholesterol massratio of5:1, drug to lipid ratio of1:30. Liposomes were made by filmdispersion method and the encapsulation was determined by sephadex column.The appearance of SUD-HP-β-CD inclusion compound liposomes wasuniform milky suspension, the encapsulated ratio was76.32%, the averageparticle size of about238.4nm, and the polydispersity coefficient was0.258.The release process in vitro of SUD-HP-β-CD correspond to the Weibullequation, ln[-ln（1-Q）]=0.9045lnt^-1.433, R²=0.9861. In vitro release ofliposome-encapsulated SUD-HP-β-CD inclusion compound correspond tofirst order kinetic equation, Ln （1-Q）=-0.0824t+0.0517, r²=0.9893. Thestability results showed that the liposome was unstable under heatedconditions. When stored at25℃for one month, the encapsulation efficiency decreased by33.6%, liposome deposited obviously; stored for one month at4℃, the encapsulation efficiency decreased by1.7%, indicating that theliposomes need to be stored under refrigeration.Pharmacokinetic experiments showed that the liposome elimination rateconstant k of free SUD was0.22h^-1and the k value of SUD-HP-β-CDinclusion compound liposome was0.41h^-1, indicating that the elimination ofSUD slowerd after liposome formation. AUC was517.54μg·h·mL^-1and228.72μg·h·mL^-1in liposome group and SUD group, indicating that theliposome increased the bioavailability of SUD in the body. Theconcentration of the drug in heart, spleen and lung of mice in SUD-HP-β-CDinclusion compound liposomes group was much higher than that in SUDgroup. The revalue was3.01,3.14and2.71, respectively, which indicated thatliposomes played a certain targeted purpose in heart, spleen and lung afterintravenous injection.The results of MTT assay suggested that SUD-HP-β-CD inclusioncompound liposomes significantly inhibited A549cell proliferation in aconcentration-and time-dependent manner. The results of flow cytometryassay suggested that liposome-encapsulated SUD-HP-β-CD inclusioncompound1,5and10μg·mL^-1increased percentage of cells in G0/G1phase（P<0.05or P<0.01）， liposome-encapsulated SUD-HP-β-CD inclusioncompound5and10μg·mL^-1decreased the percentage of cells in S phase（P<0.01）, and the percentage of cells in G₂/M phase was also decreased byliposome-encapsulated SUD-HP-β-CD inclusion compound10μg·mL^-1（P<0.01）. Western blot analysis indicated that the expression of PCNA andCyclin D1was not effected by control liposomes. Whereas, in5μg·mL^-1SUDand1,5,10μg·mL^-1SUD-HP-β-CD inclusion compound liposomes groups,the level of PCNA and Cyclin D1was markedly decreased compared withcontrol group （P<0.05or P<0.01）.Conclusion: HP-β-CD was used as the inclusion material, and theSUD-HP-β-CD inclusion compound was prepared by method of the saturatedaqueous solution; the SUD-HP-β-CD inclusion compound liposome, with the characteristics of simple preparation, controllable quality and goodreproducibility, was prepared by film dispersion method, and phospholipidsand cholesterol were used as the main membrane phospholipids. Theliposome-encapsulated SUD-HP-β-CD inclusion complex can improve drugtargeting and reduce toxicity, which would be expected to be developed into anew prospective dosage form. SUD-HP-β-CD inclusion compound liposomessignifacantly inhibited A549cell proliferation in vitro, and obviously arrestedthe cell-cycle progression in the G₀/G₁phase. Meanwhile, the proteinexpression of PCNA and Cyclin D1were markedly decreased bySUD-HP-β-CD inclusion compound liposomes, which would be expected toprovide experimental evidence in future clinical application of SUD as ananti-tumor drug.更多还原