氨基糖类衍生物智能水凝胶作为药物载体的研究

【作者】 滕大勇；

【导师】 李朝兴；

【作者基本信息】 南开大学 ， 高分子化学与物理， 2009， 博士

【摘要】 水凝胶由于具有丰富的交联网络结构和良好的可膨胀性,被广泛用作缓释药物的载体。本文利用具有生物相容性的氨基葡萄糖单糖衍生物、具有温敏性的异丙基丙烯酰胺(NIPAM)及具有pH敏感性的丙烯酸(AAc)共聚得到了全新的微凝胶,新型微凝胶具有良好的生物相容性、组织靶向性和对温度、pH的刺激响应性。利用天然的氨基多糖衍生物和端基活化的聚乙二醇在生理条件下,自组装得到了大孔凝胶,这种天然氨基多糖凝胶也具备某种温敏的特征。两种凝胶负载胰岛素和牛血清白蛋白(BSA)等生物大分子药物的实验均显示了良好的智能释药性能,有望成为新型的智能给药系统。一、凝胶的制备1.氨基葡萄糖单糖衍生物微凝胶的制备通过使用水溶性缩合剂联合诱导酰胺化反应合成了氨基葡萄糖单糖衍生物—丙烯酰氨基葡萄糖(AADG)。将AADG,NIPAM及AAc作为反应单体,用自由基沉淀聚合法制备了微凝胶。2.天然氨基多糖衍生物大孔凝胶的制备合成了四种具有可活化巯基型端基的聚乙二醇(PEG)交联剂,分别是:丙烯酰化、溴化、碘化及马来酸酐化端基的PEG交联剂,这些交联剂可通过端基与天然氨基多糖衍生物—巯基壳聚糖(CS-NAC)上的巯基在生理条件下自然发生迈克尔加成反应,进而形成凝胶。二、凝胶的表征1.粒径及分布实验结果表明,干态的微凝胶具有近乎球形的形态,粒径为50～60 nm左右,湿态的微凝胶粒径为100 nm左右,且分布比较均一。2.孔径通过扫描电子显微镜可以看到凝胶内部呈现多孔的类似海绵内部的结构,孔径在10μm左右,浸泡24h后可以发现网络结构发生了降解,孔径变大,达到20μm左右,浸泡7d后凝胶进一步降解,孔径增大到40～50μm。3.热稳定性通过热重实验表明壳聚糖-PEG凝胶的热失重过程主要包括两个阶段,分别对应于壳聚糖碳链的分解及PEG碳链的分解,并且由于壳聚糖刚性链对PEG结晶行为的抑制,改变了PEG失重特征峰的位置,而PEG并未对壳聚糖的失重特征峰产生影响。4.机械强度对凝胶进行了振荡应力扫描及频率扫描,结果表明,凝胶内部的交联密度越大,对应的弹性及机械强度也就越大,因此可以在制备凝胶时调整原料的类别和用量来控制凝胶的机械强度。5.溶胀及降解凝胶在37℃时快速溶胀,半个小时内就达到了最大的溶胀平衡,而在25℃时,需4h才达到。随后的24h内凝胶都快速的降解,在这之后降解逐渐缓慢下来,在6周后凝胶还未完全降解。内部交联密度的大小决定了凝胶的最大平衡溶胀度及降解速度。三、凝胶的智能释药与生物相容性1.温敏特性随着温度的升高,微凝胶的粒径有了显著的增大,表现在药物释放上,同一pH下,41℃时有明显的突释现象,2h释放了88%的药物,而在37℃时释放缓慢,2d后才释放了79%的药物。大孔凝胶对温度的敏感性也很显著,凝胶在25℃时的最大平衡溶胀度比37℃时降低了约四分之一,表现在药物释放上,37℃时3d之内药物已经释放完全,而在25℃时3d才释放不到60%的药物。2.pH敏特性当pH值小于5.0时,微凝胶的有效粒径几乎不变,而当pH值大于5.0时,粒径随着pH值的增大而增大,表现在药物释放上,同一温度下,pH 6.9时,2h释放了88%的药物,而pH 7.4时,3 h释放了79%的药物。微凝胶的pH敏特性并不如温敏特性显著,是因为在高的pH值下羧基的离子化使相变的温度范围变宽。3.组织靶向性微凝胶在肿瘤环境条件下呈现两个阶段的药物释放,初始的突释及其后的缓慢释放;而在正常生理条件下则始终是平缓的释放过程,这说明微凝胶在肿瘤环境条件下具有更快速的初始释放速率。大孔凝胶的形成时间受巯基壳聚糖的巯基含量、浓度,交联剂的端基类型、分子量、用量,以及反应的温度影响。凝胶固化时间的可控也使宏观形态的凝胶具有了“靶向性”,可以通过不同作用部位来选用不同固化时间的凝胶,确保凝胶在到达作用部位后立即固化。4.生物相容性细胞毒性实验表明两种凝胶都具有良好的细胞增殖能力,其中大孔凝胶的细胞培养实验说明了细胞可以通过凝胶的孔道而迁移至凝胶的内部生长,并且细胞的生长状态良好,说明这种新型凝胶除作为药物载体外,还有作为细胞支架潜力,可进一步应用于组织工程中。圆二色及凝胶电泳实验表明了两种凝胶都很好的保持了所包载的蛋白质药物的活性。四、智能释药机理初步探讨1.收缩微凝胶初始的突释可能是由于水分子与NIPAM中亲水的酰胺基团形成的氢键断开造成的,同时NIPAM中疏水的异丙基基团开始互相吸引,微凝胶表面疏水性增强,进而发生相转变并快速收缩使药物急剧释出,这形成了一种类似快速扩散的行为。2.溶胀使用半经验的数学模型拟合药物释放过程,证明凝胶的药物释放机理属于Fick扩散机理,并且推断出药物释放属于溶胀支配型药物释放,因此凝胶内部结构的紧密程度决定了不同凝胶药物释放的快慢。3.降解凝胶的降解并未对释放产生明显的影响,这可能是因为药物的尺寸(BSA,d_h=7.2 nm)远远小于凝胶的孔径(10μm)。更多还原

【Abstract】 Due to plentiful crosslinking network and favorable expansibility,hydrogels have been studied extensively for application as drug delivery carrier.In the present study,a novel microgel was designed by the incorporation of temperature sensititive N-isopropylacrylamide(NIPAM) and pH sensitive acrylic acid(AAc) to copolymerize with glucosamine derivative,acrylamido-2-deoxy-glucose(AADG). The novel microgel has favorable biocompatibility,tissue-targeting and stimuli-responsibility to pH and temperature.Moreover,a novel in situ cross-linked hydrogel was prepared under physiological conditions,based on self-assembly of natural amino polysaccharide derivatives with homo-bifunctional PEG derivatives using Michael type addition,and the natural amino polysaccharide hydrogel also has some temperature sensitivity.The in vitro macromolecular drug delivery experiment suggested the favorable intelligent drug release abilities of these two gels,and they have potential to be novel intelligent drug delivery system.Ⅰ.Preparation of gels1.Preparation of glucosamine derivative microgelAcrylic acid was attached to glucosamine via an amide bond between the carboxylic group and a free primary group.The microgels of the obtained functional glucosamine-AADG with NIPAM and AAc as the comonomer were prepared by the method of a free radical precipitation polymerization.2.Preparation of natural amino polysaccharide hydrogelFour homo-bifunctional PEG derivatives were synthesized:acrylate,bromoacetate, iodoacetate and maleimide functional PEG..And they were examined as cross-linking agents for thiol-modified derivatives of chitosan.The hydrogels were prepared by Michael type addition between the bifunctional PEG derivatives and thiol-modified chitosan.Ⅱ.Characterization of gels1.Size and morphology of microgel The morphology of the resulting microgel was investigated by SEM.It is evident that microgels are well dispersed as individual particles with spherical shapes.It can be seen that the size of the microgels is around 50～60 nm in diameter in the solid state,and the microgels exhibit a narrow size distribution with an average diameter of around 100 nm in the wet state.2.Pore size and morphology of hydrogelThe SEM images of freeze-dried hydrogels exhibited a highly macroporous spongelike structure and the average mesh size is about 10μm.After being dipped in PBS for 24 h and 7 d,the chains in the network degraded and the pores of the hydrogel increased3.Thermal stability of hydrogelAll the hydrogels showed a two-stage thermal decomposition,the first-stage decomposition temperature was similar to that of chitosan,suggesting the introduction of PEG via Michael type addition did not decrease thermal stability of chitosan.And the weight loss results in the second-stage thermal decomposition were similar to the contents of PEG in hydrogels and indicate that the second-stage decomposition was caused by PEG.The second-stage degradation temperature increased when decreasing the content of PEG,which may be due to the inhibition of crystal growth of PEG caused by the stiff chitosan chain.4.RheologyThe resulting of oscillatory stress sweep and frequency sweep indicated the higher mechanical strength and elasticity of hydrogel can be achieved by increasing the cross-linking density.5.Swelling and degradationAll the hydrogels swelled rapidly and reached equilibrium within 0.5 h at 37℃, but within 4 h at 25℃.After reaching swelling equilibrium,all the hydrogels were rapidly degraded in the first 24 h,and then the degradation became slowly and hydrogels did not completely dissolve within six weeks.Ⅲ.Intelligent drug release and biocompatibility of gels1.Temperature sensitivityThe results of dynamic light scattering measurement show that the size of the microgels significantly increased with an increase in the temperature.And drug release profile represents the temperature sensitivity,under the same pH value,at 41℃,about 88%of drug was released within the initial 2 h;at 37℃,only about 79% of drug was released after 2 d.The temperature sensitivity of hydrogel is also remarkable,the equilibrium swelling ratio of hydrogel at 25℃decreased to 25%of that at 37℃.For drug release profile,at 37℃,drug was completely released within 3 d;at 25℃,only about 60%of drug was released after 3 d.2.pH sensitivityWhen pH＜5.0,the diameters of microgels were nearly invariable regardless of the change of pH,and when pH≥5.0,the diameters became larger when the pH value was increased.And drug release profile represents the pH sensitivity,under the same temperature,at pH 6.9,about 88%of drug was released within the initial 2 h;at pH 7.4,about 79%drug was released within the initial 3 h.3.Tissue-targetingThe drug release from microgel was two-stage under the tumor-surrounding environment,the initial burst release and subsequent slow release;and for the drug release under physiological conditions,the diffusing process was sustained slow and mild.Therefore,the release of drug at the tumor-surrounding environment is faster than that under normal physiological conditions.The gelation time of hydrogel was determined by the thiols contents and concentration of CS-NAC,the terminal group type,molecular weight and the amount of cross-linking agents,and the reaction temperature.The controllability of hydrogel gelation time make macro form gel have tissue-targeting ability.4.BiocompatibilityThe cell viability study showed that the use of glucosamine may improve the biocompatibility of the microgels.The cell culture results illustrate that cells can migrate into the hydrogel networks and remain viable and maintain their 3D morphology during the 3 days culture.The secondary structures of insulin and BSA as determined by CD spectra and SDS-PAGE were preserved in all gels. Ⅵ.Mechanism of intelligent drug release1.ShrinkageThe drug release from microgel has an initial burst,which was correlated to the NIPAM segment in the microgels.As the temperature increased up to a certain point, the water contained in the microgels was expelled due to the disruption of hydrogen bonding between water and the hydrophilic amide groups,then the hydrophobic isopropyl groups began to associate.And following the structural change and consequent enhanced surface hydrophobicity,the microgels shrunk and aggregated, the insulin was squeezed out.2.SwellingFitted by semi-empirical mathematical model,mechanism of drug release from hydrogel was determined for Fick diffusion,and the release type was belong to swelling dominating drug release by further inference.Therefore,the crosslinking density determined the release rate of different hydrogels.3.DegradationDegradation of hydrogel did not have significant effect on drug release,which may be due to the huge difference between hydrodynamic diameter of BSA(d_h=7.2 nm) and hydrogel pore size(10μm).更多还原