壳聚糖肺部缓释微球的研制及其药效学研究
|
摘要
支气管哮喘是一种呼吸系统严重危害人民健康的常见病,在过去的20年中,其发病率和病死率在世界范围内呈上升趋势,这不仅引起了呼吸学界的普遍关注,也得到了WHO和许多国家政府的关注,如何有效控制哮喘成为各国的公共卫生问题。氨茶碱是治疗支气管哮喘的常用药物,具有松弛呼吸道平滑肌的作用,尚有一定的抗炎和免疫调节作用。由于治疗指数小(10～20μg/ml),普通片的半衰期短(约6 h),需频繁给药才能达到治疗浓度。口服给药后容易出现血药浓度峰谷现象及引起对胃肠道和心脏的不良反应。制成缓控释制剂虽然可以提供较平稳的血药浓度,但不能避免长期口服的副作用。肺部吸入缓释制剂药物可维持局部治疗浓度,减少全身副作用,延长药物作用时间,提高患者的顺应性。因此,运用生物降解材料及控释技术与呼吸临床治疗技术的紧密结合,探索一种新的控释技术和剂型,成为目前研究的热点。
壳聚糖是一种聚阳离子多糖,因具有良好的物理化学特性、生物相容性和生物可降解性,来源丰富,无毒,成为药剂学研究的热点而被广泛应用于药物控释体系中。壳聚糖作为药物载体可以控制药物释放,提高难溶性药物的溶出。已有大量文献报道用壳聚糖作为缓释药物载体如珠、膜、海绵、凝胶、微球等递药系统。壳聚糖阳离子聚合物的性质能与粘膜的负电荷相互作用,提高粘附性和降低清除率。最近研究显示,壳聚糖能打开细胞连接,提高生物利用度5～10倍,而且对粘膜功能的影响是可逆的,尤其适用于肺部给药系统。本文采用壳聚糖和β-环糊精为原料,以氨茶碱为模型药物,采用喷雾干燥法制备不同药物/载体比例的茶碱/壳聚糖/β-环糊精微球,旨在研究其理化性质及粉末特性,生物相容性、降解性,并对其药效进行评价。
利用喷雾干燥法成功地制备了三种不同药物/载体比例的茶碱/壳聚糖/β-环糊精微球,通过扫描电镜、激光粒度测定仪、红外光谱仪、紫外分光光度计对微球的物理化学特性进行观察。结果微球外观圆整,表面光滑或有皱折,粒径分布范围窄,符合肺部粒子吸入的要求。微球具有较高的产率、载药量和包封率;红外光谱结果显示茶碱与壳聚糖和β-环糊精高分子间发生氢键反应;体外释放结果证实茶碱/壳聚糖/β-环糊精微球具有缓释作用,并且与释放介质、载药量、溶胀性及药物性质有关,茶碱/壳聚糖/β-环糊精微球B具有良好的缓释效果。稳定性实验表明,在实验条件下茶碱/壳聚糖/β-环糊精微球载药量和形态无明显的变化,药物较易透过粘膜。茶碱/壳聚糖/β-环糊精微球B吸湿速率为0.3071 mg/h,固密度为0.44 g/cm3。为克服壳聚糖酸溶性的不足,利用喷雾干燥法、以壳聚糖衍生物羧甲基壳聚糖
为主要载体,成功地制备了三种不同药物/载体比例的茶碱/羧甲基壳聚糖/β-环糊精微球,并对其物理化学特性进行了研究。结果得到的微球外观圆整,表面光滑或有皱折,理论空气动力学直径小5μm。茶碱/羧甲基壳聚糖/β-环糊精微球微球具有高的较高的产率、载药量和包封率;红外光谱结果显示茶碱与羧甲基壳聚糖高分子间发生了氢键反应。茶碱/羧甲基壳聚糖/β-环糊精微球在实验条件下保存稳定,其载药量和形态无明显的变化。微球具有较高的药物透过粘膜透过率(>14.7%),较低的固密度(<0.44 g/cm3),吸湿速率在0.0538～0.1864 mg/h之间,因而具有良好的粉末特性;体外释放结果证实茶碱/羧甲基壳聚糖/β-环糊精微球在pH6.8介质中的释放快于pH1.2,并且与载药量有关,微球F(pH1.2)具有良好的缓释效果。
通过细胞毒性实验、溶血实验、微核实验、肺泡灌洗液乳酸脱氢酶及肌肉植入实验等对茶碱/壳聚糖微球、茶碱/壳聚糖/β-环糊精及茶碱/羧甲基壳聚糖/β-环糊精微球等进行了一系列生物学性能的表征。结果表明,茶碱/壳聚糖/β-环糊精及茶碱/羧甲基壳聚糖/β-环糊精微球无毒,不引起溶血,试验材料组微核率为0.99‰,小于3‰,骨髓微核实验呈阴性。茶碱/壳聚糖、茶碱/壳聚糖/β-环糊精微球及茶碱/羧甲基壳聚糖/β-环糊精微球的纤毛持续运动时间分别为493.00、512.33和514.33 min,纤毛运动频率为92.4%、96.0%和96.4%,对蟾蜍口腔上腭纤毛无明显毒性;气管给药后总蛋白含量增加,LDH活性与正常组无差异显著性;肌肉埋植实验证明,肌肉植入后各个时期伤口无红肿、化脓、坏死等现象,对造血组织和血红蛋白的合成没有影响,对肝肾功能无损伤;病理结果显示埋植组织的反应与缝合丝线相比无差别,具有良好生物相容性,能够作为缓释药物的安全载体用于肺部给药。
微球的体外降解结果表明,微球具有良好的降解性,体外酶解快于体外水解。微球A的降解快于微球B、C、和空白微球;电镜结果表明体外酶解和水解至12周,微球形貌完全改变,表面不再光滑且粘连成一体,有微孔出现;体内降解至8周,茶碱/壳聚糖/β-环糊精微球多数已降解成为不规则、片状、多孔结构,直径小于5μm,茶碱/壳聚糖/β-环糊精微球和壳聚糖/β-环糊精微球体内降解无明显差异。
通过建立大鼠哮喘病模型、高血压及哮喘合并高血压大鼠模型,开展茶碱/壳聚糖/β-环糊精微球疗效的实验研究,并与茶碱溶液雾化的疗效进行比较。通过全自动生化分析仪测定BALF中LDH、ALT、AST和Cr,计数BALF中白细胞计数及分类、全自动血液分析仪测定血液中白细胞计数及分类,并HE染色观察肺组织水肿及Eos浸润情况,探讨茶碱/壳聚糖/β-环糊精微球对气道炎症、LDH及血压、肝肾功能的影响。结果显示,哮喘模型组与正常对照组相比,BALF中白细胞总数、Eos计数均明显增加(p < 0.05),血液中白细胞总数、Eos计数也具有显著差异(p < 0.05);地塞米松治疗组与哮喘模型组相比,具有显著差异(p < 0.05),BALF和血液中白细胞总数、中性粒细胞、Eos均明显下降;茶碱溶液雾化治疗组、茶碱/壳聚糖/β-环糊精微球肺部给药治疗组、茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘口服茶碱/壳聚糖/β-环糊精微球口服治疗组经治疗后BALF和血液中白细胞总数、Eos数与哮喘模型组相比明显下降(p <0.05);而茶碱/壳聚糖/β-环糊精微球肺部给药治疗组、茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘茶碱/壳聚糖/β-环糊精微球口服治疗组相互比较,差异无显著性(p﹥0.05)。肺组织病理可见哮喘组大鼠气道粘膜、粘膜下组织大量Eos浸润,粘膜水肿,腔内可见脱落的上皮细胞、渗出的炎性细胞,而正常组大鼠无上述改变;经地塞米松治疗后病理可见粘膜水肿减轻,炎性细胞浸润明显减少,肺组织结构基本正常;茶碱溶液雾化治疗组病理可见肺泡间隔断裂,水肿,细支气管周围有少量炎性细胞浸润,肺组织结构基本正常,证明茶碱对中支气管哮喘有明显的治疗作用;茶碱/壳聚糖/β-环糊精微球肺部给药治疗组有少量炎性细胞浸润,可见Eos,肺组织结构基本正常;茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘口服茶碱/壳聚糖/β-环糊精微球口服治疗组病理表现与哮喘组比较炎症明显减轻,细支气管周围有少量炎性细胞浸润,粘膜断裂,肺间质水肿,肺组织结构基本正常。茶碱/壳聚糖/β-环糊精微球肺部给药对BALF中LDH、蛋白含量及肝、肾功能皆无影响,口服也具有同样的规律;正常大鼠诱发哮喘后没有引起血压升高,高血压大鼠诱发哮喘亦未致血压升高;肺部和口服给药后对血压皆亦没有影响,提示肺部给药有可能在支气管哮喘的治疗中发挥作用。
Asthma is a serious commonly chronic airway disease which do harm to health of people, in the past two decades, the prevalence and the mortality rates of asthma are increasing in all countries throughout the world, which has been paid much more attention not only by the specialist of respiratory, but also by the WHO and the government of many countries, so how to manage asthma effectively has become a public health problem. Theophylline (TH) is one of the most important drugs for treatment of asthma and recent studies indicate that it has anti-inflammatory effects. Thus, great attentions have been shown due to the new effect on treatment of asthma. Unfortunately, since its short half-life (6 h), conventional dosage forms have to be administered 3-4 times a day in order to avoid large fluctuations in plasma concentrations, which lead to poor patient compliance. The large fluctuations of plasma TH concentration lead to gastrointestinal and cardiovascular adverse effects. Moreover, its therapeutic index is narrow (10-20μg/ml). The therapeutic effects of TH require plasma concentration of TH at least 5-10μg/ml and toxic effects are frequent above 20μg/ml. Sustained release dosage forms can overcome these drawbacks, but long-term oral therapy can’t avoid the side effects of gastrointestinal, cardiovascular and central nervous system’s disturbances because its elimination half-life varies widely between patients. Hence a long-term TH therapy also leads to serious management problems in asthmatics. Despite the occurrence of numerous new sustained release products of TH, an optimal therapeutic use continues to evolve. Inhalation of aerosolized sustaining release of drugs in the lung can prolong drug action, reduce side effects and improve patient compliance. Therefore new sustained microparticles have been paid much attention by applying biomaterial and the techniques of pharmaceutics and clinical respiratory.
Chitosan (CTS) is a cationic natural copolymer of glucosamine, which is abundant biodegradable biomaterial. It has been widely used in several pharmaceutical formulations as sustained release carrier systems such as beads, gels, films, sponges and microspheres for its many unique properties such as low toxicity, biocompatibility, biodegradability and good physicochemical properties. Furthermore, CTS has been used as a drug carrier to attain the desirable drug release profile and enhance dissolution rate of low water soluble drugs. Recently, CTS has become a good candidate for using in pulmonary delivery because its effect on the lung epithelium is reversable. It can bind with mucosal surfaces due to its cationic nature, which leads to bioadhesion and a reduced mucociliary clearance. In addition, CTS has another dramatic effect in terms of improving drug absorption (5～10 fold) by opening the intercellular tight junctions of the lung epithelium. In this paper, TH is used as model drug, while CTS andβ-cyclodextrin (β-CD) are used as drug carriers. The main aim is to study the characterization of TH/CTS/β-CD micropheres with different formulations made by spray drying as pulmonary sustained drug delivery systems. The microspheres are characterized by a series of physicochemical and powder properties, biocompability, degradability and pharmacodynamic effect.
The TH/CTS/β-CD microspheres are prepared by spray drying method. The characteristics of TH/CTS/β-CD microspheres are carried out by Scanning Electron Microscopy (SEM), Laser diffraction, Fourier Transform Infrared Spectroscope (FT-IR) and UV-Vis Spectrophotometer. The result of SEM shows spherical microspheres with smooth or slightly wrinkled surfaces. The microspheres B have a narrower particle size distribution with the diameter between l and 10μm. FT-IR spectroscopy reveals that hydrogen bonds are formed between TH and CTS orβ-CD. The drug entrapments significantly increase from 13.33% to 35.70% with an increase of the ratio of drug/polymer. The encapsulation efficiencies are from 85.16% to 91.40%. The in vitro release of TH from microspheres is slower at pH 6.8 than that at pH 1.2 and also relate to the swelling ability, especially to the ratio of drug/polymer. The microspheres B have a prolonged release pattern with the release rate of 60.20% (pH 6.8) within 8 h. The results of permeation test reveale that the microspheres are easy to penetrate the membrane.The microspheres are stability under the experimental condition. The microspheres B have the low rate of moisture uptake (0.3071 mg/h) and low tap density (0.44 g/cm3).
In order to overcome the disadvantage of acid soluble of chitosan, three kinds of carboxymethyl chitosan(CMCS)/β-CD microspheres loaded TH are successfully made by spray drying method and the characteristics are investigated. The microspheres obtained after spray drying are found to be spherical shaped with smooth or wrinkled surface. The mean particle size is between 3.39 and 6.06μm. The micropheres demonstrate high product yield (43.7-50.2%), high drug loading (13.7-38.1%), and high encapsulation efficiency (86.9-92.8%). The results of FT-IR indicate that there are interactions of TH with CMCS matrix. The TH/CMCS/β-CD microspheres are stability under the experimental condition which have the low rate of moisture uptake (0.0538～0.1864 mg/h) and tap density (<0.44 g/cm3). The in vitro release of TH from microspheres is slower at pH 1.2 than that at pH 6.8 and also related to the ratio of drug/polymer. The microsphere F has a prolonged release pattern (pH1.2).
To study the biocompability of the microspheres, the tests such as the cell toxicity experiment, hemolytic test, acute cell toxicity experiment (LDH and protein in BALF), micronucleus test and muscle implantation test have been investigated. The results demonstrate that the microspheres have no toxicity and don’t cause hemolysis. The micronucleus ratio of the microspheres is 0.99‰, being less than 3‰. The result of medullary micronucleus test is reported negative. The ciliary movement time of TH/CTS microspheres, TH/CTS/β-CD microspheres, and TH/CMCS/β-CD microspheres are 493.00、512.33 and 514.33 min,and the ciliary beat frequencies are 92.4%、96.0% and 96.4%.This result shows that the microspheres have no significant toxicity to palate mucosa. This result suggests that the microspheres can effectively reduce the ciliotoxicity and possess better adaptability. The total contents of protein and LDH in BALF of pulmonary delivery group are not significantly different from that of normal group. The wounds are free from suppuration and necrosis after muscle implantation in all periods. The results of implantation show that the microspheres have no effect on the hematopoisis, hemoglobin and have no toxicity in the liver and kidney. The inflammations of muscle tissue are not significantly different from that of operative suture, therefore, the TH/CTS/β-CD microspheres possess good biocompability and can be applied as pulmonary sustained release systems.
In vitro degradation was performed in PBS buffer solution and with enzyme solution. Microspheres are found to be degraded by enzymatic hydrolysis and buffer solution, which indicate that enzymatic hydrolysis is faster than that in buffer solution. The degradation rate of MS A is faster than that of other microspheres (MS B、MS C and blank microspheres). Morphology of SEM of microparticles with PBS and enzymic solution develope rough surfaces and become irregularly shaped and porous after 12 weeks. In addition, microparticle aggregation is also observed both in PBS and enzyme solution. Microparticles degrade faster in vivo and show similar morphology after 8 weeks, which the TH/CTS/β-CD microspheres also degrade into irregular, sheet, porous shape, and the diameters are smaller than 5μm. The degradation rate of TH/CTS/β-CD microspheres has no different from that of the TH/CTS microspheres.
The objective of this experiment is to study the influence of TH/CTS/β-CD microspheres on the allergen induced asthmatic rat model and the asthma-hypertension rat model, which also compare with the effect of TH solution. The quantity of WBC and its classification in blood is detected by automatic blood analyzer and in BALF is smeared sedimentation of the cell before stained with Wright-Giemsa and count differential leukocyte number. The LDH in BALF, ALT、AST and Cr in blood is detected by automatic clinical analyzer. The contents of protein in BALF are measured by protein quantification kit-rapid. To investigate the change of inflammation in the lung tissue and the blood pressure in asthma–hypertension rat model, the middle lobe of right lung is fixed in 10% formalin, then stained with hematoxylin-eosin and the blood pressure is measured. The total of WBC and Eos in blood and BALF of asthmatic group are higher than that of normal group (p < 0.05). Compared with the asthmatic group, the total of WBC and Eos in blood and BALF from DXM group is significantly different from asthmatic group (p < 0.05), the decrease tendency is found. The total of WBC and Eos in blood and BALF results of TH solution group, the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po) are all significantly different from asthmatic group (p < 0.05), which are lower than that of asthmatic group, furthermore, there are no significant difference between these groups (p>0.05). The result of histological changes in asthmatic model reveal obvious allergic inflammation such as dissepiments of bronchioles and small vessels becoming thicker, epithelial damage and the tissue edema compared to normal group. The lung tissue of DXM group seem normal except a few inflammatory cell infiltration. The lung tissue of TH solution group seem normal too except a few inflammatory cell and Eos infiltration. In the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po), the lung tissue also seem normal, but still a few inflammatory cell infiltration and tissue edema, which are signicantly different from asthmatic group. The routes of pulmonary delivery and po have no damages to the liver and kindney, and also have no effect on the blood pressure. There is no effect on the LDH and contents of protein in BALF. In short, the TH/CTS/β-CD microspheres can significantly reduce the quantity of Eos and the total of inflammatory cell in blood and in BALF and can inhibit airway inflammation, whose efficiency is similar to dexamethasone. This study indicates that the TH/CTS/β-CD theophylline/chitosan/β-cyclodextrin microspheres applied as pulmonary delivery is feasible.
壳聚糖是一种聚阳离子多糖,因具有良好的物理化学特性、生物相容性和生物可降解性,来源丰富,无毒,成为药剂学研究的热点而被广泛应用于药物控释体系中。壳聚糖作为药物载体可以控制药物释放,提高难溶性药物的溶出。已有大量文献报道用壳聚糖作为缓释药物载体如珠、膜、海绵、凝胶、微球等递药系统。壳聚糖阳离子聚合物的性质能与粘膜的负电荷相互作用,提高粘附性和降低清除率。最近研究显示,壳聚糖能打开细胞连接,提高生物利用度5～10倍,而且对粘膜功能的影响是可逆的,尤其适用于肺部给药系统。本文采用壳聚糖和β-环糊精为原料,以氨茶碱为模型药物,采用喷雾干燥法制备不同药物/载体比例的茶碱/壳聚糖/β-环糊精微球,旨在研究其理化性质及粉末特性,生物相容性、降解性,并对其药效进行评价。
利用喷雾干燥法成功地制备了三种不同药物/载体比例的茶碱/壳聚糖/β-环糊精微球,通过扫描电镜、激光粒度测定仪、红外光谱仪、紫外分光光度计对微球的物理化学特性进行观察。结果微球外观圆整,表面光滑或有皱折,粒径分布范围窄,符合肺部粒子吸入的要求。微球具有较高的产率、载药量和包封率;红外光谱结果显示茶碱与壳聚糖和β-环糊精高分子间发生氢键反应;体外释放结果证实茶碱/壳聚糖/β-环糊精微球具有缓释作用,并且与释放介质、载药量、溶胀性及药物性质有关,茶碱/壳聚糖/β-环糊精微球B具有良好的缓释效果。稳定性实验表明,在实验条件下茶碱/壳聚糖/β-环糊精微球载药量和形态无明显的变化,药物较易透过粘膜。茶碱/壳聚糖/β-环糊精微球B吸湿速率为0.3071 mg/h,固密度为0.44 g/cm3。为克服壳聚糖酸溶性的不足,利用喷雾干燥法、以壳聚糖衍生物羧甲基壳聚糖
为主要载体,成功地制备了三种不同药物/载体比例的茶碱/羧甲基壳聚糖/β-环糊精微球,并对其物理化学特性进行了研究。结果得到的微球外观圆整,表面光滑或有皱折,理论空气动力学直径小5μm。茶碱/羧甲基壳聚糖/β-环糊精微球微球具有高的较高的产率、载药量和包封率;红外光谱结果显示茶碱与羧甲基壳聚糖高分子间发生了氢键反应。茶碱/羧甲基壳聚糖/β-环糊精微球在实验条件下保存稳定,其载药量和形态无明显的变化。微球具有较高的药物透过粘膜透过率(>14.7%),较低的固密度(<0.44 g/cm3),吸湿速率在0.0538～0.1864 mg/h之间,因而具有良好的粉末特性;体外释放结果证实茶碱/羧甲基壳聚糖/β-环糊精微球在pH6.8介质中的释放快于pH1.2,并且与载药量有关,微球F(pH1.2)具有良好的缓释效果。
通过细胞毒性实验、溶血实验、微核实验、肺泡灌洗液乳酸脱氢酶及肌肉植入实验等对茶碱/壳聚糖微球、茶碱/壳聚糖/β-环糊精及茶碱/羧甲基壳聚糖/β-环糊精微球等进行了一系列生物学性能的表征。结果表明,茶碱/壳聚糖/β-环糊精及茶碱/羧甲基壳聚糖/β-环糊精微球无毒,不引起溶血,试验材料组微核率为0.99‰,小于3‰,骨髓微核实验呈阴性。茶碱/壳聚糖、茶碱/壳聚糖/β-环糊精微球及茶碱/羧甲基壳聚糖/β-环糊精微球的纤毛持续运动时间分别为493.00、512.33和514.33 min,纤毛运动频率为92.4%、96.0%和96.4%,对蟾蜍口腔上腭纤毛无明显毒性;气管给药后总蛋白含量增加,LDH活性与正常组无差异显著性;肌肉埋植实验证明,肌肉植入后各个时期伤口无红肿、化脓、坏死等现象,对造血组织和血红蛋白的合成没有影响,对肝肾功能无损伤;病理结果显示埋植组织的反应与缝合丝线相比无差别,具有良好生物相容性,能够作为缓释药物的安全载体用于肺部给药。
微球的体外降解结果表明,微球具有良好的降解性,体外酶解快于体外水解。微球A的降解快于微球B、C、和空白微球;电镜结果表明体外酶解和水解至12周,微球形貌完全改变,表面不再光滑且粘连成一体,有微孔出现;体内降解至8周,茶碱/壳聚糖/β-环糊精微球多数已降解成为不规则、片状、多孔结构,直径小于5μm,茶碱/壳聚糖/β-环糊精微球和壳聚糖/β-环糊精微球体内降解无明显差异。
通过建立大鼠哮喘病模型、高血压及哮喘合并高血压大鼠模型,开展茶碱/壳聚糖/β-环糊精微球疗效的实验研究,并与茶碱溶液雾化的疗效进行比较。通过全自动生化分析仪测定BALF中LDH、ALT、AST和Cr,计数BALF中白细胞计数及分类、全自动血液分析仪测定血液中白细胞计数及分类,并HE染色观察肺组织水肿及Eos浸润情况,探讨茶碱/壳聚糖/β-环糊精微球对气道炎症、LDH及血压、肝肾功能的影响。结果显示,哮喘模型组与正常对照组相比,BALF中白细胞总数、Eos计数均明显增加(p < 0.05),血液中白细胞总数、Eos计数也具有显著差异(p < 0.05);地塞米松治疗组与哮喘模型组相比,具有显著差异(p < 0.05),BALF和血液中白细胞总数、中性粒细胞、Eos均明显下降;茶碱溶液雾化治疗组、茶碱/壳聚糖/β-环糊精微球肺部给药治疗组、茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘口服茶碱/壳聚糖/β-环糊精微球口服治疗组经治疗后BALF和血液中白细胞总数、Eos数与哮喘模型组相比明显下降(p <0.05);而茶碱/壳聚糖/β-环糊精微球肺部给药治疗组、茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘茶碱/壳聚糖/β-环糊精微球口服治疗组相互比较,差异无显著性(p﹥0.05)。肺组织病理可见哮喘组大鼠气道粘膜、粘膜下组织大量Eos浸润,粘膜水肿,腔内可见脱落的上皮细胞、渗出的炎性细胞,而正常组大鼠无上述改变;经地塞米松治疗后病理可见粘膜水肿减轻,炎性细胞浸润明显减少,肺组织结构基本正常;茶碱溶液雾化治疗组病理可见肺泡间隔断裂,水肿,细支气管周围有少量炎性细胞浸润,肺组织结构基本正常,证明茶碱对中支气管哮喘有明显的治疗作用;茶碱/壳聚糖/β-环糊精微球肺部给药治疗组有少量炎性细胞浸润,可见Eos,肺组织结构基本正常;茶碱/壳聚糖/β-环糊精微球口服治疗组和高血压合并哮喘口服茶碱/壳聚糖/β-环糊精微球口服治疗组病理表现与哮喘组比较炎症明显减轻,细支气管周围有少量炎性细胞浸润,粘膜断裂,肺间质水肿,肺组织结构基本正常。茶碱/壳聚糖/β-环糊精微球肺部给药对BALF中LDH、蛋白含量及肝、肾功能皆无影响,口服也具有同样的规律;正常大鼠诱发哮喘后没有引起血压升高,高血压大鼠诱发哮喘亦未致血压升高;肺部和口服给药后对血压皆亦没有影响,提示肺部给药有可能在支气管哮喘的治疗中发挥作用。
Asthma is a serious commonly chronic airway disease which do harm to health of people, in the past two decades, the prevalence and the mortality rates of asthma are increasing in all countries throughout the world, which has been paid much more attention not only by the specialist of respiratory, but also by the WHO and the government of many countries, so how to manage asthma effectively has become a public health problem. Theophylline (TH) is one of the most important drugs for treatment of asthma and recent studies indicate that it has anti-inflammatory effects. Thus, great attentions have been shown due to the new effect on treatment of asthma. Unfortunately, since its short half-life (6 h), conventional dosage forms have to be administered 3-4 times a day in order to avoid large fluctuations in plasma concentrations, which lead to poor patient compliance. The large fluctuations of plasma TH concentration lead to gastrointestinal and cardiovascular adverse effects. Moreover, its therapeutic index is narrow (10-20μg/ml). The therapeutic effects of TH require plasma concentration of TH at least 5-10μg/ml and toxic effects are frequent above 20μg/ml. Sustained release dosage forms can overcome these drawbacks, but long-term oral therapy can’t avoid the side effects of gastrointestinal, cardiovascular and central nervous system’s disturbances because its elimination half-life varies widely between patients. Hence a long-term TH therapy also leads to serious management problems in asthmatics. Despite the occurrence of numerous new sustained release products of TH, an optimal therapeutic use continues to evolve. Inhalation of aerosolized sustaining release of drugs in the lung can prolong drug action, reduce side effects and improve patient compliance. Therefore new sustained microparticles have been paid much attention by applying biomaterial and the techniques of pharmaceutics and clinical respiratory.
Chitosan (CTS) is a cationic natural copolymer of glucosamine, which is abundant biodegradable biomaterial. It has been widely used in several pharmaceutical formulations as sustained release carrier systems such as beads, gels, films, sponges and microspheres for its many unique properties such as low toxicity, biocompatibility, biodegradability and good physicochemical properties. Furthermore, CTS has been used as a drug carrier to attain the desirable drug release profile and enhance dissolution rate of low water soluble drugs. Recently, CTS has become a good candidate for using in pulmonary delivery because its effect on the lung epithelium is reversable. It can bind with mucosal surfaces due to its cationic nature, which leads to bioadhesion and a reduced mucociliary clearance. In addition, CTS has another dramatic effect in terms of improving drug absorption (5～10 fold) by opening the intercellular tight junctions of the lung epithelium. In this paper, TH is used as model drug, while CTS andβ-cyclodextrin (β-CD) are used as drug carriers. The main aim is to study the characterization of TH/CTS/β-CD micropheres with different formulations made by spray drying as pulmonary sustained drug delivery systems. The microspheres are characterized by a series of physicochemical and powder properties, biocompability, degradability and pharmacodynamic effect.
The TH/CTS/β-CD microspheres are prepared by spray drying method. The characteristics of TH/CTS/β-CD microspheres are carried out by Scanning Electron Microscopy (SEM), Laser diffraction, Fourier Transform Infrared Spectroscope (FT-IR) and UV-Vis Spectrophotometer. The result of SEM shows spherical microspheres with smooth or slightly wrinkled surfaces. The microspheres B have a narrower particle size distribution with the diameter between l and 10μm. FT-IR spectroscopy reveals that hydrogen bonds are formed between TH and CTS orβ-CD. The drug entrapments significantly increase from 13.33% to 35.70% with an increase of the ratio of drug/polymer. The encapsulation efficiencies are from 85.16% to 91.40%. The in vitro release of TH from microspheres is slower at pH 6.8 than that at pH 1.2 and also relate to the swelling ability, especially to the ratio of drug/polymer. The microspheres B have a prolonged release pattern with the release rate of 60.20% (pH 6.8) within 8 h. The results of permeation test reveale that the microspheres are easy to penetrate the membrane.The microspheres are stability under the experimental condition. The microspheres B have the low rate of moisture uptake (0.3071 mg/h) and low tap density (0.44 g/cm3).
In order to overcome the disadvantage of acid soluble of chitosan, three kinds of carboxymethyl chitosan(CMCS)/β-CD microspheres loaded TH are successfully made by spray drying method and the characteristics are investigated. The microspheres obtained after spray drying are found to be spherical shaped with smooth or wrinkled surface. The mean particle size is between 3.39 and 6.06μm. The micropheres demonstrate high product yield (43.7-50.2%), high drug loading (13.7-38.1%), and high encapsulation efficiency (86.9-92.8%). The results of FT-IR indicate that there are interactions of TH with CMCS matrix. The TH/CMCS/β-CD microspheres are stability under the experimental condition which have the low rate of moisture uptake (0.0538～0.1864 mg/h) and tap density (<0.44 g/cm3). The in vitro release of TH from microspheres is slower at pH 1.2 than that at pH 6.8 and also related to the ratio of drug/polymer. The microsphere F has a prolonged release pattern (pH1.2).
To study the biocompability of the microspheres, the tests such as the cell toxicity experiment, hemolytic test, acute cell toxicity experiment (LDH and protein in BALF), micronucleus test and muscle implantation test have been investigated. The results demonstrate that the microspheres have no toxicity and don’t cause hemolysis. The micronucleus ratio of the microspheres is 0.99‰, being less than 3‰. The result of medullary micronucleus test is reported negative. The ciliary movement time of TH/CTS microspheres, TH/CTS/β-CD microspheres, and TH/CMCS/β-CD microspheres are 493.00、512.33 and 514.33 min,and the ciliary beat frequencies are 92.4%、96.0% and 96.4%.This result shows that the microspheres have no significant toxicity to palate mucosa. This result suggests that the microspheres can effectively reduce the ciliotoxicity and possess better adaptability. The total contents of protein and LDH in BALF of pulmonary delivery group are not significantly different from that of normal group. The wounds are free from suppuration and necrosis after muscle implantation in all periods. The results of implantation show that the microspheres have no effect on the hematopoisis, hemoglobin and have no toxicity in the liver and kidney. The inflammations of muscle tissue are not significantly different from that of operative suture, therefore, the TH/CTS/β-CD microspheres possess good biocompability and can be applied as pulmonary sustained release systems.
In vitro degradation was performed in PBS buffer solution and with enzyme solution. Microspheres are found to be degraded by enzymatic hydrolysis and buffer solution, which indicate that enzymatic hydrolysis is faster than that in buffer solution. The degradation rate of MS A is faster than that of other microspheres (MS B、MS C and blank microspheres). Morphology of SEM of microparticles with PBS and enzymic solution develope rough surfaces and become irregularly shaped and porous after 12 weeks. In addition, microparticle aggregation is also observed both in PBS and enzyme solution. Microparticles degrade faster in vivo and show similar morphology after 8 weeks, which the TH/CTS/β-CD microspheres also degrade into irregular, sheet, porous shape, and the diameters are smaller than 5μm. The degradation rate of TH/CTS/β-CD microspheres has no different from that of the TH/CTS microspheres.
The objective of this experiment is to study the influence of TH/CTS/β-CD microspheres on the allergen induced asthmatic rat model and the asthma-hypertension rat model, which also compare with the effect of TH solution. The quantity of WBC and its classification in blood is detected by automatic blood analyzer and in BALF is smeared sedimentation of the cell before stained with Wright-Giemsa and count differential leukocyte number. The LDH in BALF, ALT、AST and Cr in blood is detected by automatic clinical analyzer. The contents of protein in BALF are measured by protein quantification kit-rapid. To investigate the change of inflammation in the lung tissue and the blood pressure in asthma–hypertension rat model, the middle lobe of right lung is fixed in 10% formalin, then stained with hematoxylin-eosin and the blood pressure is measured. The total of WBC and Eos in blood and BALF of asthmatic group are higher than that of normal group (p < 0.05). Compared with the asthmatic group, the total of WBC and Eos in blood and BALF from DXM group is significantly different from asthmatic group (p < 0.05), the decrease tendency is found. The total of WBC and Eos in blood and BALF results of TH solution group, the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po) are all significantly different from asthmatic group (p < 0.05), which are lower than that of asthmatic group, furthermore, there are no significant difference between these groups (p>0.05). The result of histological changes in asthmatic model reveal obvious allergic inflammation such as dissepiments of bronchioles and small vessels becoming thicker, epithelial damage and the tissue edema compared to normal group. The lung tissue of DXM group seem normal except a few inflammatory cell infiltration. The lung tissue of TH solution group seem normal too except a few inflammatory cell and Eos infiltration. In the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po), the lung tissue also seem normal, but still a few inflammatory cell infiltration and tissue edema, which are signicantly different from asthmatic group. The routes of pulmonary delivery and po have no damages to the liver and kindney, and also have no effect on the blood pressure. There is no effect on the LDH and contents of protein in BALF. In short, the TH/CTS/β-CD microspheres can significantly reduce the quantity of Eos and the total of inflammatory cell in blood and in BALF and can inhibit airway inflammation, whose efficiency is similar to dexamethasone. This study indicates that the TH/CTS/β-CD theophylline/chitosan/β-cyclodextrin microspheres applied as pulmonary delivery is feasible.
引文
Aiba S. Studies on chitosan lysozymic hydrolysis of partially N-acetylated chitosan. Int J Bio Macromol, 1992, 14: 225-225.
Aiedeh K, Taha MO. Synthesis of iron-crosslinked chitosan succinate and iron-crosslinked hydroxamated chitosan succinate and their in vitro evaluation as potential matrix materials for oral theophylline sustained-release beads. Eur J Pharm Sci, 2001, 13: 159-168.
Akbuga J. The effect of the physiochemical properties of a drug on its release from chitosonium malate matrix tablets. Int J Pharm, 1993, 100: 257-261.
Alexakis T, Boadi DK, Quong D, et al. Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application. Appl Biochem Biotechnol, 1995, 50(1): 93-106.
Alpar HO, Somavarapu S, Atuah KN, et al. Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery. Adv Drug Deliv Rev, 2005, 57: 11-430.
Altiere RJ, Thompson DC. Physiology and pharmacology of the airways. In: Hickey AJ, (Eds). Inhalation aerosols. New York: Marcel Dekker, 1996, 133-272.
Anil KA, Willem FS, Carmen RL. Ionotropic cross-linked chitosan microspheres for controlled release of ampicillin. Int J Pharm, 2006, 312: 166-173.
Artursson P, Lindmark T, Davis SS, et al. Effect of chitosan on the permeability of intestinal epithelial cells (Caco-2). Pharm Res, 1994, 11(9): 1358-1361.
Asada M, Takahashi H, Okamoto H, et al. Theophylline particle design using chitosan by the spray drying. Int J Pharm, 2004, 270: 167-174.
Aspden TJ, Adler J, Davis SS, et al. Chitosan as a nasal delivery system: evaluation of the effect of chitosan on mucociliary clearance rate in the frog palate model. Int J Pharm, 1995, 122(1/2): 69-78.
Autian J. Biological model systems for the testing of the toxicity of biomaterials. In: Kronentheal RI, Oser Z, (Eds). Polymer science and technology, vol. 8: polymers in medicine and surgery. New York, 1975, Plenum Press 181.
Barnes PJ. Mechanisms of action of glucocorticoids in asthma. AM J Respir Crit Care Med, 1996, 154: 21-27.
Batycky R, Deaver D, Dwivedi S, et al. The development of large porous particles for inhalation drug deliver, In: M.J. rathbene, J. Hadgraft, M.S. Roberts (Eds), Modified-Release Drug Delivery Technology, 1st ed, Marcel Dekker, New York, 2003, 891-902.
Beaulac C, Sachetelli S, Lagace J. Aerosolization of low phase transition temperature liposomal tobramycin as a dry powder in an animal model of chronic pulmonary infection caused by pseudomonas aeruginosa. J Drug Target, 1999, 7(1): 33-41.
Ben-Jebria A, Chen D, Eskew ML, et al. Large porous particles for sustained protection from carbachol-induced bronchoconstriction in guinea pigs. Pharm Res, 1999, 16: 551-561.
Bentley AM, Hamid Q, Robinson DS, et al. Prednisolone treatment in asthma: reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa. AM J Respir Crit Care Med, 1996, 153: 551-556.
Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microshperes as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs J Control Release , 1996, 39: 17-25.
Bosquillon C, Lombry C, Préat V, et al. Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance. J Control Release, 2001, 70: 329-339.
Bugamelli F, Raggi MA, Orienti I, et al. Controlled insulin release from chitosan microspheres. Arch Pharm, 1998, 331(4):133-138.
Calvo P, Vila-Jato JL, Alonso MJ. Effect of lysozyme on the stability of polyester nanocapsules and nanoparticles: stabilization approaches. Biomaterials, 1997, 18: 1305-1310.
Carbral Marques HM, Hadgraft J, Kellaway IW, et al. Studies of cyclodextrin inclusion complexes: molecular modeling and 1H-NMR evidence for the sabutamol-β-cyclodextrin complex. Int J Pharm, 1990, 63: 267-264.
Cerchiara T, Luppi B, Bigucci F, et al. Chitosan and poly (methyl vinyl ether-co-maleic anhydride) micropaticles as nasal sustained delivery systems. Int J Pharm, 2003, 258: 209-215.
Chan, HK, Chew NYK, Novel alternative methods for the delivery of drugs for the treatment of asthma. Adv Drug Deliv Rev, 2003, 55: 793-805.
Chandler MJ, Zeiss CR, Leach CI, et al. Levels and specificity of antibody in bronchoalveolar lavage(BAL) and serum in an animal model of trimellitic anhydride-induced lung injury. J Allergy Clin Immunol, 1987, 80(2): 223-229.
Chen LY, Du YM, Zeng XQ, Relationships between the molecular structure and moisture-absorption and moisture-retention abilities of carboxymethyl chitosan II. Effect of degree of deacetylation and carboxymethylation. Carbohyd Res, 2003, 338: 333-340.
Chen SC, Wu YC, Mi FL, et al. A novel pH-sensitive hydrogel composed of N, O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J Control Release, 2004, 96: 285-300.
Chen XG, Zheng L, Wang Z, et al. Molecular affinity and permeability of different molecular weight chitosan membranes. J Agric Food Chem, 2002, 50: 5915-5918.
Chew NYK, Chan HK. Use of solid corrugated particles to enhance powder aerosol performance. Pharm Res, 2001, 18: 1570-1577.
Chobot V, Kremenak J, Opletal L. Phytotherapeutic aspects of diseases of the circulatory system. 4. Chitin and chitosan. Ceska Slov Farm, 1995, 44: 190-195.
Clarke MJ, Tobyn MJ, Staniforth JN. The formulation of powder inhalation systems containing a high mass of nedocromil sodium trihydrate. J Pharm Sci, 2001, 90(2): 213-223.
Cook RO, Pannu RK, Kellaway IW. Novel sustained release microspheres for pulmonary drug delivery. J Controll Release, 2005, 104: 79-90.
Corrigan DO, Healy AM, Corrigan OI. Preparation and release of salbutamol from chitosan and chitosan co-spray dried compacts and multiparticulates. Eur J Pharm Biopharm, 2006, 62: 295-305.
David P, Manssur Y, Mark S. Unusual susceptibility of chitosan to enzymic hydrolysis. Carbohydr Res, 1992, 237: 325-332.
Davide G, Andreas BS. In vitro evaluation of polymeic excipients protecting calcitonin against degradation by intestinal serine proteases. Int J Pharm, 2003, 252: 187-196.
Davis SS. Delivery of peptide and non-peptide drugs through the respiratory tract. Pharm Sci Technol Today, 1999, 2(11): 450-456.
Di Benedetto G, Lopez-Vidriero MT, Carratu L, et al. Effect of amiloride on human bronchial ciliary activity in vitro. Respiration, 1990, 57: 37-39.
Edwards DA, Hanes J, Caponetti G, et al. Large porous particles for pulmonary drug delivery. Science,1997, 276: 1868-1871.
El-Baseir MM, Kellaway IW. Poly (L-lacticacid) microspheres for pulmonary drug delivery: release kinetics and aerosolization studies. Int J pharm, 1998, 175: 135-145.
Erjefalt I, Persson CG. Anti-asthma drugs attenuate inflammation leakage of plasma into airway lumen. Acta Physiol Scand, 1986, 128: 653-654.
Evora C, Soriano I, Rogers RA, et al. Relating the phagocytosis of microparticles by alverolar macrophages to surface chemistry: the effect of 1, 2 dipalmitoyl phosphatidylcholine. J Control Release, 1998, 51: 143-152.
Faul J, Tormey V, Leonard C, et al. Lung immunopathology in cases of sudden asthma death. Eur Respir J, 1997, 10: 301-307.
Fernandes CM; Veiga FJB. Effect of the hydrophobic nature of triacetyl-β-cyclodextrin on the complexation with nicardipine hydrochloride: physicochemical and dissolution properties of the kneaded and spray-dried complexes. Chem Pharm Bull, 2002, 50, 1597-1602.
Fiebrig I, Harding SE, Rowe AJ, et al. Transmission electron microscopy on pig gastric mucin and its interaction with chitosan. Carbohydr Polym, 1995, 28: 239-244.
Fiegel J, Fu J, Hanes J. Poly (ether-anhydride) dry powder aerosols for sustained drug delivery in the lungs. J Control Release, 2004, 96: 411-423.
Filipovic-Grcic J, Becirevic-Lacan M, ?kalko N, et al. Chitosan microspheres of nifedipine and nifedipine-cyclodextrin inclusion complexes. Int J Pharm, 1996, 135: 183-190.
Finnerty JP, Lee C, Wilson S, et al. Effects of theophylline on inflammatory cells and cytokines in asthmatic subjects: a placebo-controlled parallel group study. Eur Respir J, 1996, 9: 1672-1677.
Folkesson HG, Westrom BR, Karlsson BW. Permeability of the respiratory tract to different-sized macromolecules after intratracheal instillation in young and adult rats. Acta Physol Scand, 1990, 139: 347-354.
Fukaya H, Iimura A, Hoshiko K, et al. A cyclosporine A/maltosyl-alpha-cyclodextrin complex for inhalation therapy of asthma. Respir J, 2003, 22: 213-219.
Ganguly S, Dash AK. A novel in situ gel for sustained drug delivery and targeting. Int J Pharm, 2004, 276: 83-92.
Ganza-González A, Anguiano-Igea S, Otero-Espinar FJ, et al. Chitosan and chondroitin microspheres for oral-administration controlled release of metoclopramide. Eur J Pharm Biopharm, 1999, 48:149-155.
Gupta KC, Ravi Kumar MNV. Drug release behavior of beads and microgranules of chitosan. Biomaterials, 2000, 20: 115-119.
Hardy JG, Chadwick TS. Sustained release drug delivery to the lungs. Clin Pharmacokinet, 2000, 39: 1-4.
Hayes ER. N, O-carboxymethyl chitosan and preparative method therefore, U.S. Patent 4, 619, 995, October 28, 1986.
He P, Davis SS, Illum L. Chitosan microspheres prepared by spray drying. Int J Pharm, 1999, 187: 53-65.
Henderson RF, Damon EG, Henderson TR. Early damage indicators in the lung. I. Lactate dehydrogenase activity in the airways. Toxicol Appl Pharmacol, 1978, 44: 291-297.
Henderson RF. Use of bronchoalveolar lavage to detect lung damage. Environ Health Perspect, 1984, 56, 115-129.
Herrmann G, Aynesworth MB. Succesful treatment of persistent extreme dyspnea status asthmatics: use of theophylline ethylene diamine intravenously. J Lab Clin Med, 1937, 23: 135-148.
Huang YC, Yeh MK, Chiang CH. Formulation factors in preparing BTM-chitosan microsperes by spray drying method. Int J Pharm, 2002, 242: 239-242.
Jameela SR, Kumary TV, Lal AV, et al. Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled deliver system. J Control Release, 1998, 52(1-2): 17-24.
Jrudi JA, Julian DT, Nicholas SJ, etal. Chitosan as a nasal Delivery system: the effect of chitosan solutions on in vitro and In vivo mucociliary transportrate sin human turbinates and volunteers. J Pharm Sci, 1997, 86(4): 509-513.
Jumaa M, Furkert FH, Muller BW. A new lipid emulsion formulation with high antimicrobial efficacy using chitosan. Eur J Pharm Biopharm, 2002, 53: 115-123.
Karlson L, Thuresson K, Lindman B. A rheological investigation of the complex formation between hydrophobically modified ethyl (hydroxyl ethyl) cellulose and cyclodextrin, Carbohyd Polym, 2002, 50: 219-226.
Kawashima Y, Yamamoto H, Takeuchi H, et al. Pulmonary delivery of insulin with nebulized DL-lactidePglycolidecopolymer (PLGA) nanospheres to prolong hypoglycemic effect. J Control Release, 1999, 62: 279-287.
Kennedy R, Costain DJ, MeAlister VC, et al. Prevention of experimental postoperative peritoneal adhesions by N, O-carboxymethyl chitosan. Surgery, 1996, 120: 866-870.
Kenneth H, Hiroaki N, Eli Y. Expression, hormonal regulation, and cyclic variation of chemokines in the rat ovary: key determinants of the intraovarian resistance of representatives of the white blood cell series. Endocrinology, 2002, 143(3): 784-791.
Kidney J, Domingulez M, Taylor PM, et al. Immunomodulation by theophylline in asthma: demontration with withdrawal of therapy. Am J Respir Crit Care Med, 1995, 151: 1907-1914.
Kimelberg HK, Mayhew EG. Properties and biological effects of liposomes and their uses in pharmacology and toxicology. CRC Crit Rev Toxicol, 1978, 6: 25-79.
Kinnarinen T, Jarho P, J?rvinen K, et al. Pulmonary deposition of a budesonide/γ-cyclodextrin complex in vitro. J Control Release, 2003, 90: 197-205.
Kockisch S, Rees GD, Young SA, et al. In situ evaluation of drug-loaded microspheres on a mucosal surface under dynamic test conditions. Int J Pharm, 2004, 276(1-2): 51-58.
Konstan MW, Chen PW, Sherman JM, et al. Human lung lysozyme: sources and properties. Am Rev Respir Dis, 1981, 123: 120-124.
Koping-Hoggard M, Tubulekas I, Guan H, et al. Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo. Gene Ther, 2001, 8(14): 1108-1121.
Koshkina NV, Gilbert BE, Waldrep JC, et al. Distribution of camptothecin after delivery as liposome aerosol or following intramuscular injection in mice. Cancer Chemother Pharmacol, 1999, 44(3): 187-192.
Kumar M, Behera AK, Lockey RF, et al. Intranasal gene transfer by chitosan-DNA nanospheres protects BALB/ c mice against acute respiratory syncytial virus infection. Hum Gene Ther, 2002, 13(17): 1415-1425.
Kumar M, Kong X, Behera A K, et al. Chitosan IFN-gamma-pDNA nanoparticle (CIN) therapy for allergic asthma. GenetVaccines Ther, 2003, 1: 3-13.
Kwon JH, Lee BH, Lee JJ, et al. Insulin microcrystal suspension as a long-acting formulation for pulmonary delivery. Eur J Pharm Sci, 2004, 22: 107–116.
Leffler CC, Muller BW. Influence of the acid type on physical and drug liberation properties of chitosan gelatin sponge. Int J Pharm, 2000, 194: 229-237.
Leu?en HL, de Leeuw BJ, Langemeyer WME, et al. Mucoadhesive polymers in peroral peptide drug delivery. IV. Polycarbophil and chitosan are potent enhancers of peptide transport across intestinal mucosae in vitro. J Control Release 1997, 45: 15-23.
Lim S, Tomita K, Carramori G, et al. Low dose theophylline reduces eosinophilic inflammation but not exhaled nitric oxide in mild asthma. Am J Respir Crit Care Med, 2001, 164: 273-276.
Lim ST, Mrtin GP, Berry DJ, et al. Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. J Control Release, 2000, 66: 281-292.
Lin YH, Liang HF, Chung ChK, et al. Physically crosslinked alginate/N, O-Carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs. Biomaterials, 2005, 26: 2105-2113.
Liu FY, Shao Z, Kildsig DO, et al. Pulmonary delivery of free and liposomal insulin. Pharm Res, 1993, 10: 228-232.
Liu LS, Liu SQ, Steven YN, et al. Controlled release of interleukin-2 for tumor immunotherapy using alginate/chitosan porous microspheres. J Control Release, 1997, 43: 65-74.
Liu LS, Thompson AY, Heidaran MA et al. An osteoeonducdve collagen/hyaluronate matrix for bone regenerate. Biomaterials, 1999, 20(12): 1097-1108.
Louey MD, Mulvaney P, Stewart PJ. Characterisation of adhesional properties of lactose carriers using atomic force microscopy. J Pharm Biomed Anal, 2001, 25(3-4): 559-567.
Maa Y, Nguyen P, Sweeney T, et al. Protein inhalation powder: spray drying vs spray freeze drying. Pharm Res, 1999, 16(2): 249-254.
Maestrelli F, Zerrouk N, Chemtob C, et al. Influence of chitosan and its glutamate and hydrochloride salts on naproxen dissolution rate and permeation across Caco-2 cells. Int J Pharm, 2004, 271: 257-267.
Majeti NV, Ravi K. A review of chitin and chitosan applications. React Funct Polym, 2000, 46(1): 1-27.
Malcolmson RJ, Embleton JK. Dry powder formulations for pulmonary delivery. Pharm Sci Tech Today, 1998, 1(9): 394-398.
Mao HQ, Roy K. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control release, 2001, 70: 399-421.
McConville JT, Patel N , Ditchburn N, et al. Use of a novel modified TSI for the evaluation ofcontrolled-release aerosol formulations. Drug Dev Ind Pharm, 2000, 26(11): 1191-1198.
Mellstrand T, Svedmyr N, Fagerstorm PO. Absorption of theophylline from conventional and sustained release tablets. Eur J Respir Dis Suppl, 1980, 109: 54-61.
Merkus FWHM, Verhoef JC, Marttin E, et al. Cyclodextrins in nasal drug delivery. Adv Drug Deliv Rev, 1999, 36: 41-57.
Mi FL, Shyu SS, Chen CT, et al. Porous chitosan microsphere for controlling the antigen release of newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release. Biomaterials, 1999, 20: 1603-1612.
Mi Fl, Tan YC, Liang HF, et al. In vivo biocompability and degradability of a novel injectable-chitosan-based implant. Bioaterials, 2002, 23(1): 181-191.
Mitra S, Gaur U, Ghosh PC, et al. Tumour targeted delivery of encapsulated dextran doxorubicin conjugate using chitosan nanoparticles as carrier. J Control Release, 2001, 74(1-3): 317-323.
Miyazaki S, Yamaguchi H, Yokouchi C, Takada M, How WM. Sustained-release and intragastric-floating granules of indomethacin using chitosan in rabbits. Chem Pharm Bull, 1988, 36: 4033-4038.
Mooren FC, Berthold A, Domschke W, et al . Influence of chitosan microspheres on the transport of prednisolone sodium phosphate across HT229 cell monolayers. Pharm Res, 1998, 15(1): 58-65.
Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controllrd drug delivery–a review of the state of the art. Eur J Pharm Biopharm, 2000, 50(1): 161-177.
Muzzarelli RA. Biochemical significance of exogenous chitins, chitosan in animals, patients. Carbohydr Polym, 1993, 20: 7-16.
Muzzarelli RAA, Mattioli-Belmonte M, Pugnaloni A, et al. Biochemistry, histology and clinical uses of chitins and chitosans in wound healing, In: P. Jolles, R.A.A. Muzzarelli (Eds.), Chitin and chitinases. Basel: Birkhauser Verlag; 1999, 260-261.
Muzzarelli RAA. Human enzymatic activities related to the therapeutic administration of chitin derivatives. Cell Mol Life Sci, 1997, 53, 131-140.
Newman SP, Holling Worth A, Clark AR, et al. Effect of defferent modes of inhalation on drug delivery from a dry powder inhaler. Int J Pharm, 1994, 102(2): 127-132.
Niven RW. Delivery of biotherapeutics by inhalation aerosol. Crit Rev Ther Drug Carrier Syst. 1995, 12: 151-231.
Okamoto H, Nishida S, Todo H, et al. Pulmonary gene delivery by chitosan-pDNA complex powder prepared by a supercritical carbon dioxide process. J Pharm Sci, 2003, 92(2): 371-380.
Oneda F, RéMI. The effect of formulation variables on the dissolution and physical properties of spray-dried microspheres containing organic salts. Powder Technol, 2003, 130: 377-384.
Oungbho K, Müller BW. Chitosan sponges as sustained release drug carriers. Int J Pharm, 1997, 156: 229-237.
Patel VR, Amiji MM. Preparation and characterization of freeze-dried chitosan-poly (ethylene oxide) hydrogels for site-specific antibiotic delivery in the stomach. Pharm Res, 1996, 13: 588-593.
Patton JS, Trinchero P, Platz R. Bioavailability of pulmonary delivered peptides and proteins: α-interferon, calcitonins and parathyroid hormones. J Control Release, 1994, 28: 79-85.
Pelletier A, Lemire, I, Syguch, J, Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization. Biotech Bioeng, 1990, 36: 310-315.
Philip VA, Mehta RC, DeLuca PP. In vitro and in vivo respirable fractions of isopropanol treated PLGA microspheres using a dry powder inhaler. Int J Pharm, 1997, 151: 175-182.
Portero A, Remunan-Lopez C, Vila-Jato JL. Effect of chitosan glutamate enhancing the dissolution properties of the poorly water soluble drug nifedipine. Int J Pharm, 1998, 175: 75-84.
Poznansky MJ, Juliano RL. Biological approaches to the controlled delivery of drugs: a critical review. Pharmacol Rev, 1984, 36: 277-336.
Puttipipatkhachorn S, Nunthanid J, Yamamoto K, et al. Drug physical state and drug polymer interaction on drug release from chitosan matrix films. J Control Release, 2001, 75: 143-153.
Richardson SCW, Kolbe HVJ, Duncan R. Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int J Pharm, 1999, 178(2): 231-243.
Saari M, Vidgren MT, Koskinen MO, et al. Pulmonary distribution and clearance of two beclomethasone liposome formulations in healthy volunteers. Int J Pharm, 1999, 181: 1-9.
Saks SR, Gardner LB. The pharmacoeconomic value of controlled release dosage forms. J Control Release, 1997, 48: 237-242.
Sashiwa H, Saimoto H, Shigemasa, Ogawa R, et al. Lysozyme susceptibility of partially deacetylated chitin. Int J Biol Macromol, 1990, 12: 295-296.
Sawayanagi Y, Nambu N, Nagai T. Use of chitosan for sustained-release preparations of water-soluble drugs. Chem Pharm Bull, 1982, 30: 4213-4215.
Shahiwala A, Misra A. A preliminary pharmacokinetic study of liposomal leuprolide dry powder inhaler: A technical note. AAPS Pharm Sci Tech, 2005, 6(3): 482-486.
Shen Z, Zhang Q, Wei S, et al. Proteolytic enzymes as a limitation for pulmonary absorption of insulin:in vitro and in vivo investigations. Int J Pharm, 1999, 192: 115-121.
Shigemasa Y, Saito K, Sashiwa H, et al. Enzymatic degradation of chitins and partially deacetylated chitins. Int J Biol Macromol, 1994, 16(1): 43-49.
Shiraishi S, Arahira M, Imai T, Otagiri M. Enhancement of dissolution rates of several drugs by low-molecular weight chitosan and alginate. Chem Pharm Bull, 1990, 38: 185-187. Smith SJ, Bernstein JA. Therapeutic used of long aerosols. In: AJ Hickey (Eds), Inhalation aerosols Physical and Biological Basis for Therapy, Marcel Dekker Inc., New York, 1996, 233-269.
Sugibayashi K, Morimoto Y, Nadai T, et al. Drug-carrier property of albumin microspheres in chemotherapy II. Preparation and tissue distribution in mice of microspher-entrapped 5-fluorouracil. Chem Pharm Bull, 1979, 27(1): 204-209.
Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterialsin cartilage tissue engineering: a review. Biomaterials, 2000, 21(24): 2589-2598.
Sullivan P, Bekir S, Jaffar Z, et al. Anti-inflammatory effects of low-dose oral theophylline in atopic asthma. Lancet, 1994, 343: 1006-1008.
Sun LP, Du YM, Fan LH, et al. Preparation, characterization and antimicrobial activity of quaternized carboxymethyl chitosan and application as pulp-cap. Polymer, 2006, 47: 1796-1804.
Szoka F, Papahadjopoulos D. Liposomes: preparation and characterization. In: Knight CG, ed. Liposomes: From Physical Structure to Therapeutic Applications. New York, NY: Elsevier/North-Holland Biomedical Press; 1981: 51-82.
Takeuchi H, Yamamoto H, Kawashima Y. Mucoadhesive nanoparticle systems for peptide drug delivery. Adv Drug Deliv Rev, 2001, 47(1): 39-54.
Takeuchi H, Yamamoto H, Niwa T, Hino T, Kawashima Y. Enteral absorption of insulin in rats from mucoadhesive chitosan-coated liposomes. Pharm Res, 1996, 13: 896–900.
Takeuchi H, Yasuji T, Hino T, et al. Spray-dried composite particles of lactose and sodium alginate fordirect tabletting and controlled releasing. Int J Pharm, 1998, 174: 91-100.
Tarayer JP, Aliaga M, Barbara M, et al. Theophylline reduces pulmonary eosinophilia after various types of active anaphylactic shock in guinea-pigs. J Pharm Pharmacol, 1991, 43: 877-879.
Thomas C, Sharma P. Chitosan as a biomaterial. Biomater Artif Cells Artif Org, 1990, 18: 1-24.
Todo H, Okamoto H, Iida K, et al. Effect of additives on insulin absorption from intratracheally administered dry powders in rats. Int J Pharm, 2001, 220: 101-110.
Tohda Y, Muraki M, Iwanaga T, et al. The effect of theophylline on blood and sputum eosinophils and ECP in patients with bronchial asthma. Int Immunopharmaco, 1998, 20: 173-181.
Tomihata K, Ikada Y. In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials, 1997, 18: 567-573.
Tomkiewicz RP, App EM, De Sanetis GT, et al. A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine:airway epithelial function and mucus change in dog. Pulm Pharmacol, 1995, 8: 259-265.
USP 24 Phisical tests, 1999.
Vanbever R, Mintzes JD, Wang J, et al. Formulation and physical characterization of large porous particles for inhalation. Pharm Res, 1999, 16: 1735-1742.
Waldrep JC, Gilbert BE, Knight CM, et al. Pulmonary delivery of beclomethasone liposome aerosol in volunteers. Tolerance and safety. Chest. 1997, 111: 316-323.
Wall DA. Pulmonary absorption of peptides and proteins. Drug Deliv, 1995, 2: 1-20.
Waserman S, Xu LJ, Olivenstein R, et al. Association between late allergic bronchoconstriction in the rat and allergen-stimulated lymphocyte proliferation in vitro. Am J Respir Crit Care Med, 1995, 151: 470-474.
Watanabe A, Hayashi H. Allergen-induced biphasic bronchoconstriction in rats. Int Arch Allergy Appl Immunol, 1990, 93(1): 26-34.
Weiberger M, Hendeler L. Theophylline in asthma. New Eng J Med, 1996, 334 (21): 1380-1382.
Williams RO, Barron MK, Alonso MJ, et al. Investigation of a pMDI system containing chitosan microspheres and P134a. Int J Pharm, 1998, 174: 209-222.
Williams RO, Liu J. Formulation of a protein with propellant HFA 134a for aerosol delivery. Eur J Pharm Sci, 1998, 7: 137-144.
Yamamoto H, Kuno Y, Sugimoto S, et al. Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions.J Control Release, 2005, 102: 373-381.
Li YP, Ashim KM. Effects of phospholipid chain length concentration, charge, and vesicle size on pulmonary insulin absorption. Pharm Res, 1996, 16: 76-79.
Zeng XM, Martin GP, Marriott C. The controlled delivery of drugs to the lung. Int J Pharm, 1995, 124: 149-164.
Zhang Q, Shen ZC, Nagai T. Prolonged hypoglycemic effect of insulin-loaded polybutylcyanoacrylate nanoparticles after pulmonaryadministration to normal rats. Int J Pharm, 2001, 218: 75–80.
Zhou HY, Zhang YL, Biggs DL, et al. Microparticle-based lung delivery of INH decreases INH metabolism and targets alveolar macrophages. J Control Release, 2005, 107: 288–299
陈萍,林小平,冻娟,等.过敏性哮喘血清嗜酸性粒细胞阳离子蛋白水平在丙酸倍氯米松治疗前后的变化.中国实用内科杂志, 1988, 5: 277-281.
傅继华,陈宇飞,张琪.肾性高血压大鼠实验模型制作改进.中国药科大学学报, 1995, 26(6): 383-384.
黄英,张雷,刘国祥.Wistar大鼠支气管哮喘模型的建立.中华名医论坛,2003, 11: 58-50
蒋新国,崔景斌,方晓玲,等.药物的鼻黏膜纤毛毒性及评价方法.药学学报,1995, 30(11): 848-853.
金方,谢保源,施丽西,等.无载体色甘酸钠粉雾剂的研究Ⅱ.评价疗效的体外试验方法的研究.中国医药工业杂志,1998, 29(1): 19-22.
金方,谢保源,施丽西,等.无载体色甘酸钠粉雾剂的研究-处方设计及粉体性质研究.中国医药工业杂志,1996, 27(9): 404-407.
金方.呼吸道给药的新剂型-粉雾剂.国外医学药学分册,1996, 23: 1-6.
李凤前,胡晋红,朱全刚,等.干粉末吸入环丙沙星缓释微球的制备及其体外释药调控.中国药学杂志.2002, 37(2): 110-113.
李惠萍,何国钧,易祥华.氨茶碱雾化吸入对正常及哮喘豚鼠气道粘膜的影响.上海医学,2000, 23(8): 465-469.
李明华,殷凯生,朱栓立.哮喘病学.北京:人民卫生出版社,1998, 346-348.
林芳,孙铭,肖琅,等.二丙酸倍氯米松缓释微囊在豚鼠体内释药浓度的测定.生物医学工程与临床,2002, 6(4): 191-202.
刘海弟,陈运法,曾冬冬,等.用于肺部给药的壳聚糖空心微球的制备.功能材料,2005, 36(4): 616-618.
刘利萍,李苹,吴泽志,等.肺靶向利福平壳聚糖磁微球的构建.化学世界, 2003, 44(4): 200-202.
陆彬.药物新剂型与新技术.北京:人民卫生出版社,1998.
梅兴国.生物技术药物制剂—基础与应用.北京:化学工业出版社,2004, 221-222.
沈赞聪,张强,魏树礼.胰岛素毫微球肺部给药对正常大鼠的降血糖作用.北京医科大学学报,1999, 31: 436-438.
施新猷.现代医学实验动物学,人民军医出版社, 2000, 448-449.
史志诚.小鼠骨髓微核实验方法,第三篇毒物检验与毒理实验.北京:中国农业出版社,2001.
唐翠,印春华.肺部给药系统的研究进展.中国医药工业杂志, 2001, 32(12): 560-564.
王伟,王志宏.吸入型胰岛素的研究进展.武警医学院学报,2004, 13(7): 340-342.
吴婉莹,李云谷.金雀异黄素壳聚糖微球胶囊的体内药动学研究.中药材, 2002, 25(4): 282-284.
肖琅,孙铭,林芳,等.吸入丙酸倍氯米PLGA纳米缓释微囊在哮喘治疗中的作.中国临床医学, 2002, 9(4): 334-342.
辛嘉英,张复升.高分子药物缓释用壳聚糖微球的制备.天然药物研究与开发, 1996, 8(3): 91-95.
熊莲洁,朱家壁.鲑降钙素吸入粉雾剂的制备及其药剂学性质研究.药学学报,2003, 38(3): 218-222.
杨兴海,钱京萍,柳蔚.氯氨地平伍用VitE对肾性高血压大鼠左室肥厚的逆转作用.实用医学进修杂志, 2002, 30 (3): 155-157.
姚康德,成国祥.智能材料.北京:化学工业出版社,2002.
尹承慧,侯春林,徐皓,等.羧甲基壳聚糖/环丙沙星植入缓释微球防治局部感染的实验研究.第二军医大学学报,2005, 26 (1): 72-74.
张建湘,蔡克勤,马卫东,等.壳聚糖棒材的组织相容性和安全性评价.生物医学工程杂志等,1996, 13(4): 293-297
张珠.利福平壳聚糖蛋白微球的制备及性能研究.武汉理工大学学报,2001, 23(1): 21-23.
赵磊,姜斌,王铭远,等.壳聚糖微球的纤毛毒性和粘附力的考察.中国医院药学杂志,2005, 25(8): 697-699.
郑爱萍,于少云,赵莹,等鼻用氟尿嘧啶壳聚糖微球的制备及其特性研究北京大学学报(医学版),2004, 36 (3): 300-306.
朱慧,朱家壁.胰岛素吸入粉雾剂粉体性质的研究.中国药科大学学报,2004, 35(5): 424-428.
朱元珏,陈文彬.呼吸病学.北京:人民卫生出版社,2003, 634-635.
Aiedeh K, Taha MO. Synthesis of iron-crosslinked chitosan succinate and iron-crosslinked hydroxamated chitosan succinate and their in vitro evaluation as potential matrix materials for oral theophylline sustained-release beads. Eur J Pharm Sci, 2001, 13: 159-168.
Akbuga J. The effect of the physiochemical properties of a drug on its release from chitosonium malate matrix tablets. Int J Pharm, 1993, 100: 257-261.
Alexakis T, Boadi DK, Quong D, et al. Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application. Appl Biochem Biotechnol, 1995, 50(1): 93-106.
Alpar HO, Somavarapu S, Atuah KN, et al. Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery. Adv Drug Deliv Rev, 2005, 57: 11-430.
Altiere RJ, Thompson DC. Physiology and pharmacology of the airways. In: Hickey AJ, (Eds). Inhalation aerosols. New York: Marcel Dekker, 1996, 133-272.
Anil KA, Willem FS, Carmen RL. Ionotropic cross-linked chitosan microspheres for controlled release of ampicillin. Int J Pharm, 2006, 312: 166-173.
Artursson P, Lindmark T, Davis SS, et al. Effect of chitosan on the permeability of intestinal epithelial cells (Caco-2). Pharm Res, 1994, 11(9): 1358-1361.
Asada M, Takahashi H, Okamoto H, et al. Theophylline particle design using chitosan by the spray drying. Int J Pharm, 2004, 270: 167-174.
Aspden TJ, Adler J, Davis SS, et al. Chitosan as a nasal delivery system: evaluation of the effect of chitosan on mucociliary clearance rate in the frog palate model. Int J Pharm, 1995, 122(1/2): 69-78.
Autian J. Biological model systems for the testing of the toxicity of biomaterials. In: Kronentheal RI, Oser Z, (Eds). Polymer science and technology, vol. 8: polymers in medicine and surgery. New York, 1975, Plenum Press 181.
Barnes PJ. Mechanisms of action of glucocorticoids in asthma. AM J Respir Crit Care Med, 1996, 154: 21-27.
Batycky R, Deaver D, Dwivedi S, et al. The development of large porous particles for inhalation drug deliver, In: M.J. rathbene, J. Hadgraft, M.S. Roberts (Eds), Modified-Release Drug Delivery Technology, 1st ed, Marcel Dekker, New York, 2003, 891-902.
Beaulac C, Sachetelli S, Lagace J. Aerosolization of low phase transition temperature liposomal tobramycin as a dry powder in an animal model of chronic pulmonary infection caused by pseudomonas aeruginosa. J Drug Target, 1999, 7(1): 33-41.
Ben-Jebria A, Chen D, Eskew ML, et al. Large porous particles for sustained protection from carbachol-induced bronchoconstriction in guinea pigs. Pharm Res, 1999, 16: 551-561.
Bentley AM, Hamid Q, Robinson DS, et al. Prednisolone treatment in asthma: reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa. AM J Respir Crit Care Med, 1996, 153: 551-556.
Berthold A, Cremer K, Kreuter J. Preparation and characterization of chitosan microshperes as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs J Control Release , 1996, 39: 17-25.
Bosquillon C, Lombry C, Préat V, et al. Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance. J Control Release, 2001, 70: 329-339.
Bugamelli F, Raggi MA, Orienti I, et al. Controlled insulin release from chitosan microspheres. Arch Pharm, 1998, 331(4):133-138.
Calvo P, Vila-Jato JL, Alonso MJ. Effect of lysozyme on the stability of polyester nanocapsules and nanoparticles: stabilization approaches. Biomaterials, 1997, 18: 1305-1310.
Carbral Marques HM, Hadgraft J, Kellaway IW, et al. Studies of cyclodextrin inclusion complexes: molecular modeling and 1H-NMR evidence for the sabutamol-β-cyclodextrin complex. Int J Pharm, 1990, 63: 267-264.
Cerchiara T, Luppi B, Bigucci F, et al. Chitosan and poly (methyl vinyl ether-co-maleic anhydride) micropaticles as nasal sustained delivery systems. Int J Pharm, 2003, 258: 209-215.
Chan, HK, Chew NYK, Novel alternative methods for the delivery of drugs for the treatment of asthma. Adv Drug Deliv Rev, 2003, 55: 793-805.
Chandler MJ, Zeiss CR, Leach CI, et al. Levels and specificity of antibody in bronchoalveolar lavage(BAL) and serum in an animal model of trimellitic anhydride-induced lung injury. J Allergy Clin Immunol, 1987, 80(2): 223-229.
Chen LY, Du YM, Zeng XQ, Relationships between the molecular structure and moisture-absorption and moisture-retention abilities of carboxymethyl chitosan II. Effect of degree of deacetylation and carboxymethylation. Carbohyd Res, 2003, 338: 333-340.
Chen SC, Wu YC, Mi FL, et al. A novel pH-sensitive hydrogel composed of N, O-carboxymethyl chitosan and alginate cross-linked by genipin for protein drug delivery. J Control Release, 2004, 96: 285-300.
Chen XG, Zheng L, Wang Z, et al. Molecular affinity and permeability of different molecular weight chitosan membranes. J Agric Food Chem, 2002, 50: 5915-5918.
Chew NYK, Chan HK. Use of solid corrugated particles to enhance powder aerosol performance. Pharm Res, 2001, 18: 1570-1577.
Chobot V, Kremenak J, Opletal L. Phytotherapeutic aspects of diseases of the circulatory system. 4. Chitin and chitosan. Ceska Slov Farm, 1995, 44: 190-195.
Clarke MJ, Tobyn MJ, Staniforth JN. The formulation of powder inhalation systems containing a high mass of nedocromil sodium trihydrate. J Pharm Sci, 2001, 90(2): 213-223.
Cook RO, Pannu RK, Kellaway IW. Novel sustained release microspheres for pulmonary drug delivery. J Controll Release, 2005, 104: 79-90.
Corrigan DO, Healy AM, Corrigan OI. Preparation and release of salbutamol from chitosan and chitosan co-spray dried compacts and multiparticulates. Eur J Pharm Biopharm, 2006, 62: 295-305.
David P, Manssur Y, Mark S. Unusual susceptibility of chitosan to enzymic hydrolysis. Carbohydr Res, 1992, 237: 325-332.
Davide G, Andreas BS. In vitro evaluation of polymeic excipients protecting calcitonin against degradation by intestinal serine proteases. Int J Pharm, 2003, 252: 187-196.
Davis SS. Delivery of peptide and non-peptide drugs through the respiratory tract. Pharm Sci Technol Today, 1999, 2(11): 450-456.
Di Benedetto G, Lopez-Vidriero MT, Carratu L, et al. Effect of amiloride on human bronchial ciliary activity in vitro. Respiration, 1990, 57: 37-39.
Edwards DA, Hanes J, Caponetti G, et al. Large porous particles for pulmonary drug delivery. Science,1997, 276: 1868-1871.
El-Baseir MM, Kellaway IW. Poly (L-lacticacid) microspheres for pulmonary drug delivery: release kinetics and aerosolization studies. Int J pharm, 1998, 175: 135-145.
Erjefalt I, Persson CG. Anti-asthma drugs attenuate inflammation leakage of plasma into airway lumen. Acta Physiol Scand, 1986, 128: 653-654.
Evora C, Soriano I, Rogers RA, et al. Relating the phagocytosis of microparticles by alverolar macrophages to surface chemistry: the effect of 1, 2 dipalmitoyl phosphatidylcholine. J Control Release, 1998, 51: 143-152.
Faul J, Tormey V, Leonard C, et al. Lung immunopathology in cases of sudden asthma death. Eur Respir J, 1997, 10: 301-307.
Fernandes CM; Veiga FJB. Effect of the hydrophobic nature of triacetyl-β-cyclodextrin on the complexation with nicardipine hydrochloride: physicochemical and dissolution properties of the kneaded and spray-dried complexes. Chem Pharm Bull, 2002, 50, 1597-1602.
Fiebrig I, Harding SE, Rowe AJ, et al. Transmission electron microscopy on pig gastric mucin and its interaction with chitosan. Carbohydr Polym, 1995, 28: 239-244.
Fiegel J, Fu J, Hanes J. Poly (ether-anhydride) dry powder aerosols for sustained drug delivery in the lungs. J Control Release, 2004, 96: 411-423.
Filipovic-Grcic J, Becirevic-Lacan M, ?kalko N, et al. Chitosan microspheres of nifedipine and nifedipine-cyclodextrin inclusion complexes. Int J Pharm, 1996, 135: 183-190.
Finnerty JP, Lee C, Wilson S, et al. Effects of theophylline on inflammatory cells and cytokines in asthmatic subjects: a placebo-controlled parallel group study. Eur Respir J, 1996, 9: 1672-1677.
Folkesson HG, Westrom BR, Karlsson BW. Permeability of the respiratory tract to different-sized macromolecules after intratracheal instillation in young and adult rats. Acta Physol Scand, 1990, 139: 347-354.
Fukaya H, Iimura A, Hoshiko K, et al. A cyclosporine A/maltosyl-alpha-cyclodextrin complex for inhalation therapy of asthma. Respir J, 2003, 22: 213-219.
Ganguly S, Dash AK. A novel in situ gel for sustained drug delivery and targeting. Int J Pharm, 2004, 276: 83-92.
Ganza-González A, Anguiano-Igea S, Otero-Espinar FJ, et al. Chitosan and chondroitin microspheres for oral-administration controlled release of metoclopramide. Eur J Pharm Biopharm, 1999, 48:149-155.
Gupta KC, Ravi Kumar MNV. Drug release behavior of beads and microgranules of chitosan. Biomaterials, 2000, 20: 115-119.
Hardy JG, Chadwick TS. Sustained release drug delivery to the lungs. Clin Pharmacokinet, 2000, 39: 1-4.
Hayes ER. N, O-carboxymethyl chitosan and preparative method therefore, U.S. Patent 4, 619, 995, October 28, 1986.
He P, Davis SS, Illum L. Chitosan microspheres prepared by spray drying. Int J Pharm, 1999, 187: 53-65.
Henderson RF, Damon EG, Henderson TR. Early damage indicators in the lung. I. Lactate dehydrogenase activity in the airways. Toxicol Appl Pharmacol, 1978, 44: 291-297.
Henderson RF. Use of bronchoalveolar lavage to detect lung damage. Environ Health Perspect, 1984, 56, 115-129.
Herrmann G, Aynesworth MB. Succesful treatment of persistent extreme dyspnea status asthmatics: use of theophylline ethylene diamine intravenously. J Lab Clin Med, 1937, 23: 135-148.
Huang YC, Yeh MK, Chiang CH. Formulation factors in preparing BTM-chitosan microsperes by spray drying method. Int J Pharm, 2002, 242: 239-242.
Jameela SR, Kumary TV, Lal AV, et al. Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled deliver system. J Control Release, 1998, 52(1-2): 17-24.
Jrudi JA, Julian DT, Nicholas SJ, etal. Chitosan as a nasal Delivery system: the effect of chitosan solutions on in vitro and In vivo mucociliary transportrate sin human turbinates and volunteers. J Pharm Sci, 1997, 86(4): 509-513.
Jumaa M, Furkert FH, Muller BW. A new lipid emulsion formulation with high antimicrobial efficacy using chitosan. Eur J Pharm Biopharm, 2002, 53: 115-123.
Karlson L, Thuresson K, Lindman B. A rheological investigation of the complex formation between hydrophobically modified ethyl (hydroxyl ethyl) cellulose and cyclodextrin, Carbohyd Polym, 2002, 50: 219-226.
Kawashima Y, Yamamoto H, Takeuchi H, et al. Pulmonary delivery of insulin with nebulized DL-lactidePglycolidecopolymer (PLGA) nanospheres to prolong hypoglycemic effect. J Control Release, 1999, 62: 279-287.
Kennedy R, Costain DJ, MeAlister VC, et al. Prevention of experimental postoperative peritoneal adhesions by N, O-carboxymethyl chitosan. Surgery, 1996, 120: 866-870.
Kenneth H, Hiroaki N, Eli Y. Expression, hormonal regulation, and cyclic variation of chemokines in the rat ovary: key determinants of the intraovarian resistance of representatives of the white blood cell series. Endocrinology, 2002, 143(3): 784-791.
Kidney J, Domingulez M, Taylor PM, et al. Immunomodulation by theophylline in asthma: demontration with withdrawal of therapy. Am J Respir Crit Care Med, 1995, 151: 1907-1914.
Kimelberg HK, Mayhew EG. Properties and biological effects of liposomes and their uses in pharmacology and toxicology. CRC Crit Rev Toxicol, 1978, 6: 25-79.
Kinnarinen T, Jarho P, J?rvinen K, et al. Pulmonary deposition of a budesonide/γ-cyclodextrin complex in vitro. J Control Release, 2003, 90: 197-205.
Kockisch S, Rees GD, Young SA, et al. In situ evaluation of drug-loaded microspheres on a mucosal surface under dynamic test conditions. Int J Pharm, 2004, 276(1-2): 51-58.
Konstan MW, Chen PW, Sherman JM, et al. Human lung lysozyme: sources and properties. Am Rev Respir Dis, 1981, 123: 120-124.
Koping-Hoggard M, Tubulekas I, Guan H, et al. Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo. Gene Ther, 2001, 8(14): 1108-1121.
Koshkina NV, Gilbert BE, Waldrep JC, et al. Distribution of camptothecin after delivery as liposome aerosol or following intramuscular injection in mice. Cancer Chemother Pharmacol, 1999, 44(3): 187-192.
Kumar M, Behera AK, Lockey RF, et al. Intranasal gene transfer by chitosan-DNA nanospheres protects BALB/ c mice against acute respiratory syncytial virus infection. Hum Gene Ther, 2002, 13(17): 1415-1425.
Kumar M, Kong X, Behera A K, et al. Chitosan IFN-gamma-pDNA nanoparticle (CIN) therapy for allergic asthma. GenetVaccines Ther, 2003, 1: 3-13.
Kwon JH, Lee BH, Lee JJ, et al. Insulin microcrystal suspension as a long-acting formulation for pulmonary delivery. Eur J Pharm Sci, 2004, 22: 107–116.
Leffler CC, Muller BW. Influence of the acid type on physical and drug liberation properties of chitosan gelatin sponge. Int J Pharm, 2000, 194: 229-237.
Leu?en HL, de Leeuw BJ, Langemeyer WME, et al. Mucoadhesive polymers in peroral peptide drug delivery. IV. Polycarbophil and chitosan are potent enhancers of peptide transport across intestinal mucosae in vitro. J Control Release 1997, 45: 15-23.
Lim S, Tomita K, Carramori G, et al. Low dose theophylline reduces eosinophilic inflammation but not exhaled nitric oxide in mild asthma. Am J Respir Crit Care Med, 2001, 164: 273-276.
Lim ST, Mrtin GP, Berry DJ, et al. Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. J Control Release, 2000, 66: 281-292.
Lin YH, Liang HF, Chung ChK, et al. Physically crosslinked alginate/N, O-Carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs. Biomaterials, 2005, 26: 2105-2113.
Liu FY, Shao Z, Kildsig DO, et al. Pulmonary delivery of free and liposomal insulin. Pharm Res, 1993, 10: 228-232.
Liu LS, Liu SQ, Steven YN, et al. Controlled release of interleukin-2 for tumor immunotherapy using alginate/chitosan porous microspheres. J Control Release, 1997, 43: 65-74.
Liu LS, Thompson AY, Heidaran MA et al. An osteoeonducdve collagen/hyaluronate matrix for bone regenerate. Biomaterials, 1999, 20(12): 1097-1108.
Louey MD, Mulvaney P, Stewart PJ. Characterisation of adhesional properties of lactose carriers using atomic force microscopy. J Pharm Biomed Anal, 2001, 25(3-4): 559-567.
Maa Y, Nguyen P, Sweeney T, et al. Protein inhalation powder: spray drying vs spray freeze drying. Pharm Res, 1999, 16(2): 249-254.
Maestrelli F, Zerrouk N, Chemtob C, et al. Influence of chitosan and its glutamate and hydrochloride salts on naproxen dissolution rate and permeation across Caco-2 cells. Int J Pharm, 2004, 271: 257-267.
Majeti NV, Ravi K. A review of chitin and chitosan applications. React Funct Polym, 2000, 46(1): 1-27.
Malcolmson RJ, Embleton JK. Dry powder formulations for pulmonary delivery. Pharm Sci Tech Today, 1998, 1(9): 394-398.
Mao HQ, Roy K. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. J Control release, 2001, 70: 399-421.
McConville JT, Patel N , Ditchburn N, et al. Use of a novel modified TSI for the evaluation ofcontrolled-release aerosol formulations. Drug Dev Ind Pharm, 2000, 26(11): 1191-1198.
Mellstrand T, Svedmyr N, Fagerstorm PO. Absorption of theophylline from conventional and sustained release tablets. Eur J Respir Dis Suppl, 1980, 109: 54-61.
Merkus FWHM, Verhoef JC, Marttin E, et al. Cyclodextrins in nasal drug delivery. Adv Drug Deliv Rev, 1999, 36: 41-57.
Mi FL, Shyu SS, Chen CT, et al. Porous chitosan microsphere for controlling the antigen release of newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release. Biomaterials, 1999, 20: 1603-1612.
Mi Fl, Tan YC, Liang HF, et al. In vivo biocompability and degradability of a novel injectable-chitosan-based implant. Bioaterials, 2002, 23(1): 181-191.
Mitra S, Gaur U, Ghosh PC, et al. Tumour targeted delivery of encapsulated dextran doxorubicin conjugate using chitosan nanoparticles as carrier. J Control Release, 2001, 74(1-3): 317-323.
Miyazaki S, Yamaguchi H, Yokouchi C, Takada M, How WM. Sustained-release and intragastric-floating granules of indomethacin using chitosan in rabbits. Chem Pharm Bull, 1988, 36: 4033-4038.
Mooren FC, Berthold A, Domschke W, et al . Influence of chitosan microspheres on the transport of prednisolone sodium phosphate across HT229 cell monolayers. Pharm Res, 1998, 15(1): 58-65.
Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controllrd drug delivery–a review of the state of the art. Eur J Pharm Biopharm, 2000, 50(1): 161-177.
Muzzarelli RA. Biochemical significance of exogenous chitins, chitosan in animals, patients. Carbohydr Polym, 1993, 20: 7-16.
Muzzarelli RAA, Mattioli-Belmonte M, Pugnaloni A, et al. Biochemistry, histology and clinical uses of chitins and chitosans in wound healing, In: P. Jolles, R.A.A. Muzzarelli (Eds.), Chitin and chitinases. Basel: Birkhauser Verlag; 1999, 260-261.
Muzzarelli RAA. Human enzymatic activities related to the therapeutic administration of chitin derivatives. Cell Mol Life Sci, 1997, 53, 131-140.
Newman SP, Holling Worth A, Clark AR, et al. Effect of defferent modes of inhalation on drug delivery from a dry powder inhaler. Int J Pharm, 1994, 102(2): 127-132.
Niven RW. Delivery of biotherapeutics by inhalation aerosol. Crit Rev Ther Drug Carrier Syst. 1995, 12: 151-231.
Okamoto H, Nishida S, Todo H, et al. Pulmonary gene delivery by chitosan-pDNA complex powder prepared by a supercritical carbon dioxide process. J Pharm Sci, 2003, 92(2): 371-380.
Oneda F, RéMI. The effect of formulation variables on the dissolution and physical properties of spray-dried microspheres containing organic salts. Powder Technol, 2003, 130: 377-384.
Oungbho K, Müller BW. Chitosan sponges as sustained release drug carriers. Int J Pharm, 1997, 156: 229-237.
Patel VR, Amiji MM. Preparation and characterization of freeze-dried chitosan-poly (ethylene oxide) hydrogels for site-specific antibiotic delivery in the stomach. Pharm Res, 1996, 13: 588-593.
Patton JS, Trinchero P, Platz R. Bioavailability of pulmonary delivered peptides and proteins: α-interferon, calcitonins and parathyroid hormones. J Control Release, 1994, 28: 79-85.
Pelletier A, Lemire, I, Syguch, J, Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization. Biotech Bioeng, 1990, 36: 310-315.
Philip VA, Mehta RC, DeLuca PP. In vitro and in vivo respirable fractions of isopropanol treated PLGA microspheres using a dry powder inhaler. Int J Pharm, 1997, 151: 175-182.
Portero A, Remunan-Lopez C, Vila-Jato JL. Effect of chitosan glutamate enhancing the dissolution properties of the poorly water soluble drug nifedipine. Int J Pharm, 1998, 175: 75-84.
Poznansky MJ, Juliano RL. Biological approaches to the controlled delivery of drugs: a critical review. Pharmacol Rev, 1984, 36: 277-336.
Puttipipatkhachorn S, Nunthanid J, Yamamoto K, et al. Drug physical state and drug polymer interaction on drug release from chitosan matrix films. J Control Release, 2001, 75: 143-153.
Richardson SCW, Kolbe HVJ, Duncan R. Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int J Pharm, 1999, 178(2): 231-243.
Saari M, Vidgren MT, Koskinen MO, et al. Pulmonary distribution and clearance of two beclomethasone liposome formulations in healthy volunteers. Int J Pharm, 1999, 181: 1-9.
Saks SR, Gardner LB. The pharmacoeconomic value of controlled release dosage forms. J Control Release, 1997, 48: 237-242.
Sashiwa H, Saimoto H, Shigemasa, Ogawa R, et al. Lysozyme susceptibility of partially deacetylated chitin. Int J Biol Macromol, 1990, 12: 295-296.
Sawayanagi Y, Nambu N, Nagai T. Use of chitosan for sustained-release preparations of water-soluble drugs. Chem Pharm Bull, 1982, 30: 4213-4215.
Shahiwala A, Misra A. A preliminary pharmacokinetic study of liposomal leuprolide dry powder inhaler: A technical note. AAPS Pharm Sci Tech, 2005, 6(3): 482-486.
Shen Z, Zhang Q, Wei S, et al. Proteolytic enzymes as a limitation for pulmonary absorption of insulin:in vitro and in vivo investigations. Int J Pharm, 1999, 192: 115-121.
Shigemasa Y, Saito K, Sashiwa H, et al. Enzymatic degradation of chitins and partially deacetylated chitins. Int J Biol Macromol, 1994, 16(1): 43-49.
Shiraishi S, Arahira M, Imai T, Otagiri M. Enhancement of dissolution rates of several drugs by low-molecular weight chitosan and alginate. Chem Pharm Bull, 1990, 38: 185-187. Smith SJ, Bernstein JA. Therapeutic used of long aerosols. In: AJ Hickey (Eds), Inhalation aerosols Physical and Biological Basis for Therapy, Marcel Dekker Inc., New York, 1996, 233-269.
Sugibayashi K, Morimoto Y, Nadai T, et al. Drug-carrier property of albumin microspheres in chemotherapy II. Preparation and tissue distribution in mice of microspher-entrapped 5-fluorouracil. Chem Pharm Bull, 1979, 27(1): 204-209.
Suh JK, Matthew HW. Application of chitosan-based polysaccharide biomaterialsin cartilage tissue engineering: a review. Biomaterials, 2000, 21(24): 2589-2598.
Sullivan P, Bekir S, Jaffar Z, et al. Anti-inflammatory effects of low-dose oral theophylline in atopic asthma. Lancet, 1994, 343: 1006-1008.
Sun LP, Du YM, Fan LH, et al. Preparation, characterization and antimicrobial activity of quaternized carboxymethyl chitosan and application as pulp-cap. Polymer, 2006, 47: 1796-1804.
Szoka F, Papahadjopoulos D. Liposomes: preparation and characterization. In: Knight CG, ed. Liposomes: From Physical Structure to Therapeutic Applications. New York, NY: Elsevier/North-Holland Biomedical Press; 1981: 51-82.
Takeuchi H, Yamamoto H, Kawashima Y. Mucoadhesive nanoparticle systems for peptide drug delivery. Adv Drug Deliv Rev, 2001, 47(1): 39-54.
Takeuchi H, Yamamoto H, Niwa T, Hino T, Kawashima Y. Enteral absorption of insulin in rats from mucoadhesive chitosan-coated liposomes. Pharm Res, 1996, 13: 896–900.
Takeuchi H, Yasuji T, Hino T, et al. Spray-dried composite particles of lactose and sodium alginate fordirect tabletting and controlled releasing. Int J Pharm, 1998, 174: 91-100.
Tarayer JP, Aliaga M, Barbara M, et al. Theophylline reduces pulmonary eosinophilia after various types of active anaphylactic shock in guinea-pigs. J Pharm Pharmacol, 1991, 43: 877-879.
Thomas C, Sharma P. Chitosan as a biomaterial. Biomater Artif Cells Artif Org, 1990, 18: 1-24.
Todo H, Okamoto H, Iida K, et al. Effect of additives on insulin absorption from intratracheally administered dry powders in rats. Int J Pharm, 2001, 220: 101-110.
Tohda Y, Muraki M, Iwanaga T, et al. The effect of theophylline on blood and sputum eosinophils and ECP in patients with bronchial asthma. Int Immunopharmaco, 1998, 20: 173-181.
Tomihata K, Ikada Y. In vitro and in vivo degradation of films of chitin and its deacetylated derivatives. Biomaterials, 1997, 18: 567-573.
Tomkiewicz RP, App EM, De Sanetis GT, et al. A comparison of a new mucolytic N-acetylcysteine L-lysinate with N-acetylcysteine:airway epithelial function and mucus change in dog. Pulm Pharmacol, 1995, 8: 259-265.
USP 24 Phisical tests, 1999.
Vanbever R, Mintzes JD, Wang J, et al. Formulation and physical characterization of large porous particles for inhalation. Pharm Res, 1999, 16: 1735-1742.
Waldrep JC, Gilbert BE, Knight CM, et al. Pulmonary delivery of beclomethasone liposome aerosol in volunteers. Tolerance and safety. Chest. 1997, 111: 316-323.
Wall DA. Pulmonary absorption of peptides and proteins. Drug Deliv, 1995, 2: 1-20.
Waserman S, Xu LJ, Olivenstein R, et al. Association between late allergic bronchoconstriction in the rat and allergen-stimulated lymphocyte proliferation in vitro. Am J Respir Crit Care Med, 1995, 151: 470-474.
Watanabe A, Hayashi H. Allergen-induced biphasic bronchoconstriction in rats. Int Arch Allergy Appl Immunol, 1990, 93(1): 26-34.
Weiberger M, Hendeler L. Theophylline in asthma. New Eng J Med, 1996, 334 (21): 1380-1382.
Williams RO, Barron MK, Alonso MJ, et al. Investigation of a pMDI system containing chitosan microspheres and P134a. Int J Pharm, 1998, 174: 209-222.
Williams RO, Liu J. Formulation of a protein with propellant HFA 134a for aerosol delivery. Eur J Pharm Sci, 1998, 7: 137-144.
Yamamoto H, Kuno Y, Sugimoto S, et al. Surface-modified PLGA nanosphere with chitosan improved pulmonary delivery of calcitonin by mucoadhesion and opening of the intercellular tight junctions.J Control Release, 2005, 102: 373-381.
Li YP, Ashim KM. Effects of phospholipid chain length concentration, charge, and vesicle size on pulmonary insulin absorption. Pharm Res, 1996, 16: 76-79.
Zeng XM, Martin GP, Marriott C. The controlled delivery of drugs to the lung. Int J Pharm, 1995, 124: 149-164.
Zhang Q, Shen ZC, Nagai T. Prolonged hypoglycemic effect of insulin-loaded polybutylcyanoacrylate nanoparticles after pulmonaryadministration to normal rats. Int J Pharm, 2001, 218: 75–80.
Zhou HY, Zhang YL, Biggs DL, et al. Microparticle-based lung delivery of INH decreases INH metabolism and targets alveolar macrophages. J Control Release, 2005, 107: 288–299
陈萍,林小平,冻娟,等.过敏性哮喘血清嗜酸性粒细胞阳离子蛋白水平在丙酸倍氯米松治疗前后的变化.中国实用内科杂志, 1988, 5: 277-281.
傅继华,陈宇飞,张琪.肾性高血压大鼠实验模型制作改进.中国药科大学学报, 1995, 26(6): 383-384.
黄英,张雷,刘国祥.Wistar大鼠支气管哮喘模型的建立.中华名医论坛,2003, 11: 58-50
蒋新国,崔景斌,方晓玲,等.药物的鼻黏膜纤毛毒性及评价方法.药学学报,1995, 30(11): 848-853.
金方,谢保源,施丽西,等.无载体色甘酸钠粉雾剂的研究Ⅱ.评价疗效的体外试验方法的研究.中国医药工业杂志,1998, 29(1): 19-22.
金方,谢保源,施丽西,等.无载体色甘酸钠粉雾剂的研究-处方设计及粉体性质研究.中国医药工业杂志,1996, 27(9): 404-407.
金方.呼吸道给药的新剂型-粉雾剂.国外医学药学分册,1996, 23: 1-6.
李凤前,胡晋红,朱全刚,等.干粉末吸入环丙沙星缓释微球的制备及其体外释药调控.中国药学杂志.2002, 37(2): 110-113.
李惠萍,何国钧,易祥华.氨茶碱雾化吸入对正常及哮喘豚鼠气道粘膜的影响.上海医学,2000, 23(8): 465-469.
李明华,殷凯生,朱栓立.哮喘病学.北京:人民卫生出版社,1998, 346-348.
林芳,孙铭,肖琅,等.二丙酸倍氯米松缓释微囊在豚鼠体内释药浓度的测定.生物医学工程与临床,2002, 6(4): 191-202.
刘海弟,陈运法,曾冬冬,等.用于肺部给药的壳聚糖空心微球的制备.功能材料,2005, 36(4): 616-618.
刘利萍,李苹,吴泽志,等.肺靶向利福平壳聚糖磁微球的构建.化学世界, 2003, 44(4): 200-202.
陆彬.药物新剂型与新技术.北京:人民卫生出版社,1998.
梅兴国.生物技术药物制剂—基础与应用.北京:化学工业出版社,2004, 221-222.
沈赞聪,张强,魏树礼.胰岛素毫微球肺部给药对正常大鼠的降血糖作用.北京医科大学学报,1999, 31: 436-438.
施新猷.现代医学实验动物学,人民军医出版社, 2000, 448-449.
史志诚.小鼠骨髓微核实验方法,第三篇毒物检验与毒理实验.北京:中国农业出版社,2001.
唐翠,印春华.肺部给药系统的研究进展.中国医药工业杂志, 2001, 32(12): 560-564.
王伟,王志宏.吸入型胰岛素的研究进展.武警医学院学报,2004, 13(7): 340-342.
吴婉莹,李云谷.金雀异黄素壳聚糖微球胶囊的体内药动学研究.中药材, 2002, 25(4): 282-284.
肖琅,孙铭,林芳,等.吸入丙酸倍氯米PLGA纳米缓释微囊在哮喘治疗中的作.中国临床医学, 2002, 9(4): 334-342.
辛嘉英,张复升.高分子药物缓释用壳聚糖微球的制备.天然药物研究与开发, 1996, 8(3): 91-95.
熊莲洁,朱家壁.鲑降钙素吸入粉雾剂的制备及其药剂学性质研究.药学学报,2003, 38(3): 218-222.
杨兴海,钱京萍,柳蔚.氯氨地平伍用VitE对肾性高血压大鼠左室肥厚的逆转作用.实用医学进修杂志, 2002, 30 (3): 155-157.
姚康德,成国祥.智能材料.北京:化学工业出版社,2002.
尹承慧,侯春林,徐皓,等.羧甲基壳聚糖/环丙沙星植入缓释微球防治局部感染的实验研究.第二军医大学学报,2005, 26 (1): 72-74.
张建湘,蔡克勤,马卫东,等.壳聚糖棒材的组织相容性和安全性评价.生物医学工程杂志等,1996, 13(4): 293-297
张珠.利福平壳聚糖蛋白微球的制备及性能研究.武汉理工大学学报,2001, 23(1): 21-23.
赵磊,姜斌,王铭远,等.壳聚糖微球的纤毛毒性和粘附力的考察.中国医院药学杂志,2005, 25(8): 697-699.
郑爱萍,于少云,赵莹,等鼻用氟尿嘧啶壳聚糖微球的制备及其特性研究北京大学学报(医学版),2004, 36 (3): 300-306.
朱慧,朱家壁.胰岛素吸入粉雾剂粉体性质的研究.中国药科大学学报,2004, 35(5): 424-428.
朱元珏,陈文彬.呼吸病学.北京:人民卫生出版社,2003, 634-635.