【作者】 张继芬；

【导师】 徐晓玉；

【作者基本信息】 西南大学 ， 药用资源化学， 2011， 博士

【摘要】 背景：口服给药是临床最主要的给药途径之一。对半衰期短的难溶性药物而言,如何增强药物吸收,延长药效持续时间,以减少服药剂量和给药次数,对临床用药具有重要意义,一直是药剂学研究的热点。为此,以高分子载体为主的各种口服给药系统被不断研发。它们或具有良好的缓释性能,或能改善药物吸收,但兼而有之的给药系统却很少。脂质体和壳聚糖微球是常用的两类药物载体。两类载体各有特点：脂质体生物相容性好、可促进药物吸收,但稳定性差,尤其不宜口服给药；壳聚糖微球稳定性好,具有独特的聚合阳离子特性、亲水凝胶性和生物黏附性,可延缓药物释放、增进吸收。若能将两类载体适当结合,取长补短,或能开发出一种新的口服给药系统,既能延缓药物释放又能促进药物吸收。目的：本研究以阿魏酸(Ferulic acid.FA)为模型药物,先制备脂质体(liposome, LP).再将其包入壳聚糖微球(chitosan microsphere, CM)中,制备出骨架型的载脂质体壳聚糖微球(liposomes-in-chitosan microsphere, LICM)。重点对LICM的制备工艺、结构、体外释药机理、体内吸收等内容进行研究,旨在为LICM的制备和评价提供参考,以期最终研发出一种融合了脂质体和壳聚糖优势的新型口服给药系统。方法与结果：1.FA-CM的制备与表征首先以包封率和载药量为指标,采用单因素实验法分别研究了离子凝聚法和乳化交联法制备FA-CM的工艺,以为LICM的制备提供参考。在以三聚磷酸钠为凝聚剂的离子凝聚法中,凝聚液的pH值和用量、投药量是影响包封率和载药量的重要因素：降低凝聚液的pH值,减少用量或增大投药量均能显著提高包封率和载药量。优化工艺下3批FA-CM的平均包封率为73.77±1.89%,平均载药量为8.08±0.27%,平均粒径为1.13±0.11 mm。乳化交联法的制备工艺中,油相组成、乳化剂组成、油水相体积比、戊二醛用量、壳聚糖浓度和投药量均对包封率、载药量有一定影响。以二甲硅油为油相,浓度为2.5%的壳聚糖为水相,吐温80：司盘80(1：1)为乳化剂,适当降低油水相体积比,增大戊二醛用量和投药量,有利于提高包封率和载药量。优化工艺下3批FA-CM的平均包封率为76.33±2.07%,平均载药量为5.75±0.14%,平均粒径为88.978±12.41μm。2.FA-LP的制备与吸收随后研究了FA-LP的制备工艺和肠吸收。醋酸钙梯度法制备FA-LP。以包封率为指标,采用单因素实验法确定了主动载药的孵育条件,采用Doehlert设计对投药量、磷脂与药物的摩尔比和胆固醇与磷脂的摩尔比3个因素进行了优化。优化工艺下制备的3批FA-LP平均包封率为79.97±0.54%,平均粒径为187.6±11.9nm,zeta电位为-12.67±1.78。大鼠在体肠吸收实验结果表明,脂质体能促进阿魏酸的吸收：在十二指肠、空肠、回肠、结肠和整肠段,脂质体的ka分别为原料药的1.78、1.73、1.97、1.57和1.87倍,3h的吸收百分率分别提高了63.58%、54.52%、65.62%、64.76%和31.22%。3. FA-LICM的制备、表征及体外释放制备工艺再采用离子凝聚法和乳化交联法将FA-LP载入CM中,制备出LICM。分别研究了壳聚糖终浓度、壳聚糖溶液与脂质体的体积比、凝聚液的浓度和pH值或壳聚糖终浓度、壳聚糖溶液与脂质体的体积比、乳化剂浓度和固化剂用量4个因素对微球载药量的影响。离子凝聚法制备的载脂质体壳聚糖微球(liposomes-in-chitosan microsphere prepared by onic gelation method, LICMi),载药量在0.317-0.694%之间,凝聚液pH值和壳聚糖溶液与脂质体的体积比对载药量有较大影响：提高凝聚液pH值或增加脂质体的用量均可提高载药量。乳化交联法制备的载脂质体壳聚糖微球(liposomes-in-chitosan microsphere prepared by emulsion cross-link method, LICMe),载药量在0.426-0.974%之间,各因素对载药量均有较大影响：适当降低壳聚糖或乳化剂的浓度,提高脂质体或固化剂用量有利于提高载药量。体外释放LICM的体外释放实验结果显示,无论是离子凝聚法还是乳化交联法,脂质体自LICM中的释放速率与药物自CM中的释放速率相近：CMi中,药物2h释放了39.71%,8 h释放了79.18%,12 h释放了89.31%,符合weibull分布;各工艺的LICMi中,脂质体2h释放了15-30%左右,8h释放了55-80%,12 h释放了65-90%,均符合weibull分布。CMe的突释效应强,药物2h释放了74.7%,4h释放了88.43%,8 h即已释放了92.77%,符合weibull分布;各工艺的LICMe具有类似的强突释效应,脂质体2h释放了70%左右,4h释放了80%左右,8h释放率接近90%,也均符合weibull分布。可见,脂质体取代原料药包入CM中,并没有改变CM本身的释放性能。两种方法制备的LICM,脂质体释放后粒径均有所增大：分别由原始的115.3 nm增至330±41 nm(LICMi)和209±18nm(LICMe)。药物自LICM中的释放速率明显慢于CM。各工艺的LICMi中,2h释药5-6%,12 h释药30-40%,24h释药55-75%,大多符合weibull分布和零级动力学方程。各处方的LICMe,2h药物释放了20-30%,12 h释放了50-60%左右,24 h释放率接近65-70%,均符合weibull分布。LICMi的释药速率慢于LICMe,这与LICMi脂质体释放较慢且释放后粒径增大更多的结果一致。由此说明,LICM的药物释放分两步进行：脂质体先从LICM中释放,药物再从脂质体中释放。表征分别用扫描电镜、DSC和X射线衍射对微球进行表征。两种方法制备的微球均为球状或椭球状,离子凝聚法微球平均粒径为1.17±0.15 mm,骨架结构完整,CMi的横切面上可见到大量FA细小微晶,而LICMi的横切面上无药物微晶可见,脂质体以完整的球形分布于微球的骨架中。乳化交联法微球平均粒径为123.754±15.74μm, CMe表面较光滑、致密,LICMe表面残缺不完整,有较多孔隙,这是乳化交联法微球突释效应明显的重要原因之一。CMi的DSC图谱中FA在170℃的吸热峰明显,X射线衍射图谱中FA的结晶衍射峰明显,证实药物仍以微晶状态分布于微球中；CMe的图谱中FA的吸热峰或结晶衍射峰消失,说明药物以无定形态分布于微球中。两种方法制备的LICM中FA吸热峰或结晶衍射峰都消失。结合释放结果推知,LICM的结构为药物包封于脂质体内,脂质体再完整的分布于壳聚糖微球中。4. FA-LICM的体内药代动力学以SD大鼠为实验动物进行了体内药动学研究。大鼠灌胃给予FA原料药、FA-CMi和FA-LICMi三种制剂后定时取血,甲醇沉淀蛋白后,以香豆素为内标,甲醇-0.3%醋酸溶液(42：58)为流动相,HPLC法320 nm检测血浆中的FA含量,计算血药浓度,DAS程序计算药动学参数。FA-LICM的tmax、MRT、t1\2分别为2.5±0.354 h、7.487±0.248 h和7.818±1.161 h,较原料药的0.15±0.038 h、1.365±0.091 h和1.992±0.491 h,FA-CM的1±0.354 h、4.171±0.149 h和4.857±0.997 h都有所延长：其AUC为18.331±2.846μg·L-1·h-1,分别为原料药和FA-CM的7.08倍和2.21倍：结论：上述研究证实,LICM融合了脂质体和壳聚糖微球的双重优势,具有良好的缓释和促吸收作用,是一种有较好应用前景的新型口服给药系统。更多还原

【Abstract】 Background:Liposome and chitosan microspheres were two common drug vehicles.Both had its own characterizations.Liposome had better biocompatibility.which could promote absorption of drug.but was instabile.chitosan microspheres were normally more stable.and had unique cationic property, gel forming ability and bioadhesive property. We realized that an appropriate combination of liposome and chitosan microspheres could integrate their advantages, avoid their disadvantages.and thus lead to new applications.especially in oral administration. The new drug delivery delivery system could prompt the absorption and delay the release of insoluble drug with short half-life.Objectives:In this research. Ferulic acid was selected to be model drug to prepare liposomes-in-chitosan microsphere(LICM).Liposomes loaded Ferulic acid were prepared firstly and then were incorporated into chitosan microsoheres.The preparation technology and the structure of LICM.the releasing mechanism in vitro and absorption in vivo of drug were investigared.which could provid reference for the preparation and evaluation LICM, a new drug delivery system for oral administration which integrated advantages of liposomes and chitosan microspheres.Methods and Results:1. The preparation and characterization of FA-CMFirstly.chitosan microsphere loaded Ferulic acid(FA-CM)were prepared by onic gelation method and emulsion cross-link method. The mono-varied experimental design was used to optimize technology with as indexes. As far as onic gelation method, sodium tripolyphosphate was a proper gel agent.The entrapment efficiency and drug loading could be improved significantly by decreasing the pH and volume of sodium tripolyphosphate solution.or adding the amount of Ferulic acid.The FA-CM, whose mean entrapment efficiency was 73.77±1.89%%,mean drug loading was were 8.08±0.27 % and mean size was 1.13±0.11 mm was prepared. In another preparation technology.emulsion cross-link method, the entrapment efficiency and drug loading were affected by oil composition, emulsifier composition, phase volume ratio, amount of glutaraldehyde,concentration of chitosan and amount of Ferulic acid.To obtain higher entrapment efficiency and drug loading, dimethicone was used as oil phase,2.5% chitosan solution was used as water phase.and tween80 and span 80 mixed at a ratio of 1:1 was ued as emulsifier.Reducing phase volume ratio.raising amount of glutaraldehyde and Ferulic acid could increase entrapment efficiency and drug loading.The average entrapment efficiency of optimized FA-CM was 76.33±2.07%.average drug loading was were 5.75±0.14% and average size was 88.978±12.41μm.2. The preparation and absorption of FA-LPLiposomes loaed Ferulic acid(FA-LP) were prepared by calcium acetate gradient method. The mono-varied experimental design was used to study the incubation condition of drug loading and Doehlert design was used to optimize prescription, which included the amount of Ferulic acid.the mole ratio of phospholipid to drug and the mole ratio of phospholipid to cholesterol. The average entrapment efficiency of optimized liposome was 79.97=0.54%.the average size was 187.6±11.9nm and the Zeta potential was-12.67±1.78. The absorption of FA-LP and Ferulic acid solution were compared by rat intestines recirculation experiments.The results demonstrated that liposome could promote absorption of Ferulic acid. The absorption rate of liposome in duodenum, jejunum, ileum. colon and complete intestine were as 1.78、1.73、1.97、1.57 and 1.87 times as Ferulic acid solution respectively. The absorption amount at 3h of liposome in duodenum, jejunum, ileum. colon and complete intestine were higher 63.58%、54.52%、65.62%、64.76%and 31.22% than Ferulic acid solution respectively.3. The preparation, characterization and release of FA-LICMThen FA-LP was incorporated into CM by onic gelation method and emulsion cross-link method。The effect of the concentration of chitosan. the volume ratio of chitosan solution and liposome, the concentration and pH of sodium tripolyphosphate solution or the concentration of chitosan. the volume ratio of chitosan solution and liposome. the concentration of emulsifier and the amount of glutaraldehyde on drug loading were investigated respectively.The drug loading of liposomes-in-chitosan microsphere prepared by onic gelation method(LICMi) were between 0.317% and 0.694% and increasing the pH of sodium tripolyphosphate solution or adding the amount of liposome could raise drug. loading. The drug loading of liposomes-in-chitosan microsphere prepared by emulsion cross-link method(LICMe) were between 0.426% and 0.974% and could be improved if the phase volume ratiooremulsifier concentration were reduced properly or the amount of liposome or glutaraldehyde were raised.A law was found through the release experiments in vitro.that was the release rate of liposome from LICM was near to that of drug from CM.Ferulic acid was released 39.71% at 2h.79.18% at 8h and 89.31% at 12h from CMi.Liposome was released 15-30% at 2h.55-80% at 8h and 65-90% at 12h from various LICMi.There was a remarkable burst effect in CMe and LICMe.74.7% Ferulic acid and about 70% liposome were released from CMe and LICM at 2h respectively.All these release were in accordance with weibull distribution.These verified that the release properity of CM was not changed when liposome was iocoporated into CM insead of Ferulic acid.The sizes of liposome released were raised from 115.3 mn to 330±41 nm (LICMi) or 209±18nm (LICMe).The release rate of drug from LICM was much slower than from CM. Ferulic acid was released 5-6% at 2h.30-40% at 12h and 55-75% at 24h from various LICMi.which fitted weibull distribution and zero-order kinetics.The release rates of Ferulic acid from various LICMe were 20-30%at 2h.50-60% at 12h and 65-70% at 24h, which also fitted weibull distribution. The drug releaseof LICMi was slower than LICMe.and this was in line with the fact that the size of liposome released from LICMi was larger than from LICMe. Therefore, it could be concluded that the drug release from LICM should be devided into two steps:liposome was released from LICM firstly, and then drug was released from liposome.The structure of LICM was demonstrated by scanning electron microspheres. differential scanning calorimetry and X-ray diffractometry. All microspheres were sphericity or spheroidicity with a dimater of 1.17±0.15mm for LICMi or 123.754±15.74μm for LICMe. There was much microcrystal of Ferulic acid in the transverse section of CMi,while no microcrystal was founded in the transverse section of LICMi.Liposomes were distributed in microspheres intactly.The surface of CMe was smooth and compact while the surface of LICMe was incomplete and had many pores. There was a remarkable heat absorption peak at170℃in the DSC curves of CMi or crystallization diffraction peaks in the X-ray diffractometry pattern, which demonstrated that Ferulic acid was distributed in CMi in microcrystal form. The heat absorption peak or crystallization diffraction peaks of Ferulic acid disappeared in corresponding patterns of CMe.which verified that Ferulic acid was distributed in CMi in amorphous form. The heat absorption peak or crystallization diffraction peaks of Ferulic acid also disappeared in LICMi and LICMe.Considering the release results of liposome and drug, it could be concluded that the structure of LICM was that Ferulic acid was incorporated into liposomes.and liposomes were distributed in CM intactly.4. The pharmacokinetics of FA-LICMThe pharmacokinetics was investigated by Sprague-Dawley rats. Three group of rats were admintted orally Ferulic acid. FA-CM and FA-LICM respectively. At determined time. the blood was obtained and plasma protein was precipited by methanol. The concentration of Ferulic acid in blood was analyzed by HPLC method using coumarin as internal standard, methol-0.3% acetic acid (42:58) as mobile phase at 320 nm. The data were analysed by DAS program. The tmax.MRT and t1/2βwere as follows: FA-LICM.2.5±0.354h.7.487±0.248h and 7.818±1.161h respectively;Ferulic acid.0.15±0.038h.1.365±0.091h and 1.992±0.491h:FA-CM.1±0.354h.4.171=0.149h and 4.857±0.997h. The AUC of FA-LICM was 18.331±2.846μg·L-1·h-1,which was asr 76.08 times as Ferulic acid and 2.21 times as FA-CM.Conclusion:All these studies verified that LICM integrated advantages of liposomes and chitosan microspheres, so could enhance absorption and delay drug release.which was a new drug delivery system for oral administration with good application prospersity.更多还原