【作者】 孟丽娜；

【导师】 张广宇；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 内科学， 2013， 博士

【摘要】 随着结核病在世界范围内的流行日趋严峻，耐药结核病的发病率亦呈上升趋势，我国是结核病高负担国家，耐药结核疫情不容乐观。在抗结核新药匮乏的条件下，着眼于现有抗结核药物剂型和给药途径的改良，已成为当前肺结核治疗的研究热点，肺部靶向给药又因其优势独特而日益受到重视。支气管镜下介入给药，因其操作技术成熟且较为安全，注入药物剂量准确可控，并且可经气管镜直视病灶部位注入抗结核药物，目前在临床上应用广泛，特别是对于支气管结核、耐多药肺结核及空洞型肺结核其靶向性更强。但目前支气管镜局部注药剂型多为口服或静脉药物溶解于真溶液或混悬于凝胶中配制而成，药物及凝胶的滞留效果差，无法长时间在病灶局部维持有效药物浓度，凝胶基质人体不能吸收，且无法在局部降解，影响治疗效果。因此本课题意在研究一种支气管镜局部介入治疗专属的，有较好的组织相容性、留滞性和粘附性的，并可自身降解且安全性高的缓释抗结核药物系统。本研究以壳聚糖作为药物载体，环丙沙星作为研究药物，以羟乙基纤维素[1-3]作为缓释微球的辅料，应用乳化交联法制备环丙沙星壳聚糖缓释微球，并对其粒径、粒径分布、载药量、包封率、通针性、降解度、肺部滞留性、缓释性、安全性等方面进行评价，进而评估其经支气管镜介入给药的可行性，以期达到环丙沙星肺部缓慢持续释放，减少给药次数，提高患者依从性的目的。第一部分环丙沙星壳聚糖缓释微球的制备目的：制备可用于支气管镜肺部介入治疗的环丙沙星壳聚糖缓释微球，采用紫外分光光度法测定环丙沙星含量，并通过考察环丙沙星壳聚糖缓释微球制备过程中的多种影响因素，对其表征及理化性质进行评价，筛选出适合支气管镜介入治疗的处方工艺。方法：以壳聚糖为药物载体，环丙沙星为模型药物，以羟乙基纤维素[1-3]作为缓释微球的辅料，以戊二醛作为交联剂，采用乳化交联法制备环丙沙星壳聚糖缓释微球，选取载药量和包封率作为评价指标，正交实验设计筛选处方工艺，并应用扫描电镜、UV等检测手段对其形态、粒径、载药量、包封率及恒温条件下体外释放度等指标进行测定。测试环丙沙星壳聚糖缓释微球能否以10ml注射器7号针头顺利吸取，并能否通过支气管镜导管打出。结果：环丙沙星壳聚糖缓释微球的优化处方工艺条件：壳聚糖质量分数为2%，环丙沙星与壳聚糖质量比为1:3，戊二醛质量分数为6%，HEC质量分数3%。以优化处方制备的环丙沙星壳聚糖微球球型完整，球体光滑，有少量皱褶，粒度分布均匀符合正态性分布，平均粒径（142.31±7.85）μm，平均载药量为（36.23±0.42）%，平均包封率（78.54±1.58）%，平均Zeta电位值为（51.68±2.3）mV。该微球具有较好的分散性及通针性，能顺利通过7号针头，可经支气管镜管道顺畅注射。结论：成功制备环丙沙星壳聚糖缓释微球，含药微球的形态表征、载药量及包封率较为理想。第二部分环丙沙星壳聚糖缓释微球的体外评价目的：考察环丙沙星壳聚糖缓释微球制备过程中主要处方因素对含药微球体外释放情况的影响，及以优化处方工艺制备的环丙沙星壳聚糖缓释微球的体外释放效果。方法：采用紫外分光光度法进行环丙沙星壳聚糖缓释微球的体外释放试验，精密称取一定量载药微球，加入到装有1ml PH7.4的PBS的透析袋中，然后置于具塞试剂瓶中，PH7.4的PBS100ml作为释放介质，将装置放在转速100rpm、温度(37±0.5)℃的恒温搅拌器上，定时取样5ml，并补足等体积释放介质，第一天于2h、4h、6h、8h、12h、16h、20h、24h、36h、48h、60h、72h取样，样品经过微孔（0.45μm）滤膜滤过，检测其吸光度并计算药物浓度。结果：当壳聚糖溶液、戊二醛、HEC质量分数升高，环丙沙星与壳聚糖质量比降低时，环丙沙星壳聚糖缓释微球的体外释药速率减低。以优化处方工艺制备的环丙沙星壳聚糖缓释微球的体外释放时间可达6d左右，前2d释放较快，第2d累计释放率达49.3%，2d至6d缓慢平稳释放，第6d时累计释放率为95%。结论：环丙沙星壳聚糖缓释微球具有较好的体外缓释效果。第三部分环丙沙星壳聚糖缓释微球肺内应用的安全性评价目的：利用新西兰白兔及比格犬模型观察环丙沙星壳聚糖缓释微球的溶解性、渗透性以及吸收降解情况，对环丙沙星壳聚糖缓释微球肺内应用的可行性和安全性做出初步评价。方法：将60只新西兰白兔随机分为3组，每组20只，分别为：对照组，于左肺注入生理盐水2ml。空白微球组，于左肺注入空白微20mg加生理盐水，总体积为2ml。含药微球组，于左肺注入环丙沙星壳聚糖缓释微球20mg加生理盐水，总体积为2ml。将60只比格犬随机分为3组，每组20只，分别为：对照组，于左肺注入生理盐水5ml。空白微球组，于左肺注入空白微球300mg加生理盐水，总体积为5ml。含药微球组，于左肺注入环丙沙星壳聚糖缓释微球300mg加生理盐水，总体积为5ml。各组新西兰白兔和比格犬在进行试验操作前均测量体温、体重，试验操作后每日测量体温，观察呼吸、饮食、活动性、反应性、二便情况等，每周测量体重；均于注入前、注入后1d、7d、14d采集静脉血进行血常规、T细胞亚群、IL-1β、IL-6、IL-8、IL-10、TNF-α含量检测；注入后14d处死解剖，观察各组试验动物胸腔及腹腔脏器位置、形态、颜色是否正常，重点观察左右肺组织有无无差异，左右肺对比主支气管是否通畅，颜色是否正常，有无出血、水肿等，选取注入局部肺组织进行病理学HE染色，以免疫组化法检测肺组织中IL-1β、IL-6、IL-8、IL-10、TNF-α表达情况。此外，尚在各组比格犬中进行以下试验：以呼吸生理记录仪描记注入前至注入后60min时比格犬的呼吸频率、最大呼气峰压及最小吸气压；经支气管镜观察注入3d、7d后支气管粘膜的变化及微球留滞情况；于比格犬注入前、注入后1d、7d、14d采集动脉血进行血气分析；选取比格犬注入局部肺组织进行扫描电镜、透射电镜观察。结果：新西兰白兔及比格犬肺内注入空白微球及环丙沙星壳聚糖缓释微球后，与对照组相比：均未引起窒息、咳嗽及呛咽表现，活力及进食情况无异常；体温、体重、红细胞数、白细胞数、血红蛋白量、单核细胞百分比、CD3、CD4、CD8百分比均无显著性差异（P>0.05）；处死解剖后观察胸腔及腹腔脏器形态、颜色、位置均正常，两侧主支气管均通畅，颜色正常，无出血、水肿、硬结、坏死，左右肺组织正常无差异，左肺底细支气管无空白微球存留，右肺未见有空白微球扩散进入；病理学HE染色光镜下可见支气管粘膜各层结构清晰，内膜细胞形态完整，肌层平滑肌结构清晰；肺泡结构完整，肺泡内未见细胞脱落及红细胞，肺泡间隔未见增厚；注入处局部肺组织IL-1β、IL-6、IL-8、IL-10、TNF-α表达比较无显著性差异（P>0.05）。兔血清中细胞因子检测发现，空白微球组、含药微球组及对照组注入后1d较注入前IL-1β、IL-6、IL-8、IL-10、TNF-α含量均升高，且空白微球组、含药微球组较对照组的含量要高（P<0.05）。注入后7d，对照组IL-1β、IL-6、IL-10、TNF-α含量与注入前相比已无差异（P>0.05），空白微球组、含药微球组IL-8、IL-10含量也恢复至注入前水平且与对照组无差异（P>0.05），但空白微球组、含药微球组IL-1β、IL-6、TNF-α含量虽较注入后1d有所下降但仍比注入前高，亦高于对照组（P<0.05）。注入后14d，含药微球组IL-1β、IL-6、TNF-α含量降至注入前水平且与对照组无差异（P>0.05），空白微球组IL-6、TNF-α含量降至注入前水平且与对照组无差异（P>0.05），但IL-1β虽较注入后7d有所下降但仍略高于注入前水平，与对照组及载药微球组相比无差异（P>0.05）。犬血清中细胞因子检测结果呈现出与兔检测结果大致相同的变化规律。比格犬空白微球组、含药微球组与对照组的呼吸频率、最大呼气峰压、最小吸气谷压绝对值注入后即刻比较无显著性差异（P>0.05）；注入后10min比较呼吸频率、最小吸气谷压绝对值有显著性差异（P<0.05）；注入后30min比较均无显著性差异（P>0.05）；恢复正常呼吸所需时间比较有显著性差异（P<0.05）。比格犬空白微球、含药微球注入后3d经支气管镜观察可见，微球粘附于注入部位支气管粘膜，分散较为均匀，可见含药微球颜色变浅；空白微球、含药微球注入后7d经支气管镜观察，含药微球已不可见，镜下对比观察发现，支气管粘膜外观未见异常，无充血、水肿、硬结及坏死。比格犬空白微球组、含药微球组与对照组在注入前、注入后1d、7d、14d的动脉血pH值、动脉血氧分压、动脉血二氧化碳分压及血氧饱和度结果比较均无显著性差异（P>0.05）。比格犬注入局部肺组织扫描电镜观察可见，对照组肺泡壁光滑，结构完整，间隔无增大，肺泡内无纤维渗出物，支气管粘膜纤毛排列有序，无倒伏，透射电镜观察可见，对照组肺泡上皮细胞形态结构完整，肺泡上皮细胞之间细胞连接结构完整，胞浆均匀，线粒体等细胞器无损伤，空白微球组及含药微球组与对照组比较，均未见异常。结论：环丙沙星壳聚糖缓释微球肺内应用具有较好的留滞性和粘附性，并可降解吸收，不会影响试验动物的正常呼吸功能，对支气管粘膜无刺激性，不会造成肺组织病理性改变，可行性和安全性得到初步证实。更多还原

【Abstract】 With the prevalence of tuberculosis in the world is worsening, the incidence ofMDR tuberculosis was also uptrend.China is high burden countries with TB, MDRtuberculosis epidemic is not optimistic. In the conditions of the lack of new drug fortuberculosis, modified of existing dosage form and administration route has beenpresent research focus,lung targeting drug delivery get more attention. Interventionaladministration with bronchoscopy is a safety and mature technology, and canaccurately control dosage, dugs is delivered to local focus of pneumonophthisis withbronchoscopy, especially for bronchial tuberculosis, MDR and chronic fibrocavernouspulmonary tuberculosis.Currently,effective concentration is short, drug-loadedsubstrate can’t be absorbed and implanted to distal bronchus. This article is intendedto develop a new dosage form that will be a exclusive drug for Bronchoscopy therapy,can degrade and be absorbed, and is safety for pulmonary delivery.In this study, drug carrier is chitosan, model drug is Ciprofloxacin, hydroxyethylcellulose[1-3]as a sustained-release microsphere excipients,glutaraldehyde crosslinkedmicrospheres are prepared for sustained release of Ciprofloxacin using chitosan.Microspheres were characterized by scanning electron microscopy(SEM)、UV andother detection means to evaluate its morphology, particle size distribution,encapsulation efficiency, drug-loaded, pulmonary retention, and accumulated release.To assess the feasibility of its bronchoscopic intervention therapy, in order to achievethe purpose of targeted therapy, improves anti-tuberculosis drugs ciprofloxacin releasetime in lung, reduces the frequency of administration and improves patients’compliance.PART ONE Preparation of Ciprofloxacin citosan microspheres Objective: To prepare sustained release Ciprofloxacin chitosan microspheres,whichcould be used in lung, and establish determination methond of Ciprofloxacin with UVspectrophotometry, and investigate influence factors in preparation process, andoptimize formulation and technology.Methods: drug carrier is chitosan,model drug is Ciprofloxacin,hydroxyethyl cellulose[1-3]as a sustained-release microsphere excipients,glutaraldehyde crosslinkedmicrospheres are prepared for sustained release of Ciprofloxacin using chitosan.Select drug loading and encapsulation efficiency as the evaluation index, orthogonalexperimental design Filter prescription process.Microspheres were characterized byscanning electron microscopy(SEM)、 UV and other detection means to evaluate itsmorphology, particle size distribution, encapsulation efficiency, drug-loaded,pulmonary retention, and accumulated release. Testing that Ciprofloxacin chitosanmicrospheres whether can be aspirated or injected by7＃needle or catheter.Result: The optimum formulation and technology is that chitosan mass fraction of2%,Cciprofloxacin and chitosan mass ratio of1:3, glutaraldehyde6%, the HEC massfraction of3%.Complete preparation of Ciprofloxacin chitosan microspheres Spheretype, smooth sphere, a small amount of folds, uniform particle size distribution in linewith the normal distribution, the average particle size (142.31±7.85) μm, the averagedrug loading was (36.23±0.42)%, with an average encapsulation efficiency (78.54±1.58)%, the average zeta potential value (51.68±2.3) mV.Microspheres can beinjected by7＃needle or catheter.Conclusion: Ciprofloxacin citosan microspheres is successfully prepared,characterization characteristics, Entrapment efficiency and drug loading are accordedwith requirement.PART TWO In vitro evaluation of Ciprofloxacin chitosan microspheresObjective:To investigate sustained release effect of Ciprofloxacin chitosanmicrospheres in vitro.Methods: By UV spectrophotometry method Ciprofloxacin chitosan microspheres invitro release test accurately weighed amount of drug-loaded microspheres into areactor equipped1ml PH7.4PBS dialysis bag, then placedwith stopper reagent bottle, PBS100ml of pH7.4as the release medium, the device on the rotational speed of100rpm, temperature (37±0.5)°C thermostat stirrer, regular sampling5ml, and iscomplementary to an equal volume of release medium, the first day of2h,4h,6h,8h,12h,16h,20h,24h,36h,48h,60h,72h sampling, sample after microporous (0.45μm)membrane filtration, detected by absorbance and calculate the concentration of thedrug.Result: As mass fraction of chitosan、glutaraldehyde and HEC increased, mass ratioof Ciprofloxacin and chitosan decreased, drug release rate was slower. Release time ofCiprofloxacin chitosan microspheres is6d in vitro. Accumulative release rate was49.3%in2d. Drug release was slowly stationary in2d-6d. Accumulative release ratewas95%in6d.Conclusion: Ciprofloxacin chitosan microspheres have better sustained release effectin vitro.PART THREE Safety evaluation of Ciprofloxacin chitosan microspheres usedin lungObjective: To observe solubility, permeability and absorption of Ciprofloxacinchitosan microspheres used in lung of new zealand white rabbits and beagle dogs.Methods：60rabbits are randomly divided into3groups,10in each group, controlgroup injected2ml physiological saline into left lung, blank microspheres groupinjected2ml blank microspheres(20mg) plus physiological saline into left lung, drugmicrospheres group injected2ml drug microspheres(20mg) plus physiological salineinto left lung.60dogs are randomly divided into3groups,20in each group, controlgroup injected5ml physiological saline into left lung, blank microspheres groupinjected5ml blank microspheres(300mg) plus physiological saline into left lung, drugmicrospheres group injected5ml drug microspheres(300mg) plus physiological salineinto left lung. For rabbits and dogs, body temperature and weight were detected;general conditions were observed; blood routine, T lymphocyte subsets and cytokine(IL-1β, IL-6, IL-8, IL-10, TNF-α) were detected after injecting1d,7d,14d; allanimals were sacrificed after injecting14d, and observed lung gross anatomy. Lungtissue of local injecting were evaluated by HE staining and immunohistochemistry (IL-1β, IL-6, IL-8, IL-10, TNF-α). For dogs bronchial mucosa was observed bybronchoscopy after injecting3d and7d. Respiratory rate, maximal expiratory pressureand minimal inspiratory pressure were observed by respiratory record instrumentbefore injecting to after injecting60min. Blood gas analysis were done after injecting1d,7d,14d. Lung tissue of local injecting were observed by SEM andTEM(transmission electron microscope).Result: Compared with control group in rabbits and dogs after injecting blank or drugmicrospheres, asphyxia and cough didn’t be found; general conditions were normal;body temperature and weight, blood routine and T lymphocyte didn’t showsignificantly diversity(P>0.05); lung gross anatomy didn’t show abnormal;histopathological examination showed that bronchiole didn’t obviously changed bycomparing with control group’s, and alveolus had complete structure and spacing ofalveolus was not incrassation; expression of IL-1β, IL-6, IL-8, IL-10and TNF-αdidn’t show significantly diversity in lung tissue of local injecting (P>0.05).For rabbits after injecting1d, content of IL-1β, IL-6, IL-8, IL-10and TNF-α ofcontrol group, blank microspheres and drug microspheres group were higher thanbefore injecting in blood; blank microspheres and drug microspheres were higher thancontrol group (P<0.05). After injecting7d, IL-1β, IL-6,IL-10and TNF-α of controlgroup didn’t show significantly diversity compared with before injecting(P>0.05);IL-8and IL-10of blank microspheres and drug microspheres didn’t showsignificantly diversity compared with before injecting(P>0.05); IL-1β, IL-6andTNF-α of blank microspheres and drug microspheres were higher than controlgroup(P<0.05). After injecting14d, except that IL-1β of blank microspheres groupwere higher than before injecting(P>0.05), others didn’t show significantly diversitycompared with before injecting(P>0.05). In dogs variations of cytokine were similarto that in rabbits.For dogs bronchial mucosa didn’t show abnormal, and no hyperemia, edema,induration and necrosis were found after injecting3d,7d by bronchoscopyobservation. Respiratory rate, maximal expiratory pressure and minimal inspiratorypressure didn’t show significantly diversity in control group, blank microspheres and drug microspheres group after immediate injecting(P>0.05), but Respiratory rate,minimal inspiratory pressure show significantly diversity after injecting10min(P<0.05), didn’t show significantly diversity after injecting30min(P>0.05).Blood gas analysis didn’t show significantly diversity in control group, blankmicrospheres and drug microspheres group after injecting1d,7d,14d(P>0.05). SEMand TEM didn’t show abnormal in in control group, blank microspheres and drugmicrospheres groupConclusion: Ciprofloxacin chitosan microspheres has better adhesion, and noinfluence on normal respiration, no irritation to bronchial mucosa, and can bedegraded and absorbed. Safty and feasibility of Ciprofloxacin chitosan microspheresused in lung are preliminariely confirmed.更多还原