可生物降解两亲性聚天冬氨酸衍生物的合成及其pH敏感性研究

【作者】 王勇；

【导师】 马建标；

【作者基本信息】 南开大学 ， 高分子化学与物理， 2009， 博士

【摘要】 环境刺激响应高分子材料因能够对外界刺激(如pH、温度、光、电信号的变化)进行识别和响应,而在许多领域显示了良好的应用前景。如果环境刺激响应高分子同时具有亲水-疏水两亲性,则可以在选择性溶剂中形成纳米尺度的高分子胶束。以往研究的具有两亲性环境刺激响应高分子,多数为不可生物降解的高分子化合物,如聚(N-异丙基丙烯酰胺)、聚丙烯酸、聚4-乙烯基吡啶等,作为生物医用材料在体内使用时会受到很多限制。如果能够将环境刺激响应性能、两亲性、生物降解性能赋予一种高分子中,就可以得到适于在体内应用的新型高分子材料。这类材料形成的纳米级胶束,可以用作具有定位释放功能的纳米药物制剂的高分子载体;此类高分子也可以在特定组织中原位形成高分子水凝胶,用作诱导组织再生的支架材料(组织修复材料)。聚氨基酸由于具有良好的生物相容性,一般对生物体无毒、无副作用,并可以通过体内的水解或酶解反应最终降解为小分子氨基酸,被人体吸收或排出体外。有望广泛用于医学领域,比如作为药物控释载体等。聚(α,β-L-天冬氨酸)(α,β-L-PAsp)具有良好的生物相容性和生物降解性,而且合成简便,成本较低,作为药物载体和组织工程支架材料已多有研究。如果对聚(α,β-L-天冬氨酸)进行部分酯化,则聚(α,β-L-天冬氨酸)的侧链上含有疏水性的酯基和亲水性的羧基,部分酯化的聚(α,β-L-天冬氨酸)在一定条件下会呈现出两亲性。由于侧链羧基可以电离,在不同pH介质环境中有不同的电离度,因此部分酯化的聚(α,β-L-天冬氨酸)会表现出pH敏感性。不论聚酰胺主链还是酯基侧链,在生理条件下都是可以生物降解的。因此,部分酯化的聚(α,β-L-天冬氨酸)同时具有环境刺激响应性能、两亲性、生物降解性能,本实验室以该类高分子化合物为例,研究一类具有环境刺激响应性能的生物降解高分子纳米材料。(1)以L-天冬氨酸为起始物,采用热缩聚法制备得到聚琥珀酰亚胺(PSI),聚琥珀酰亚胺,开环水解得到聚(α,β-L-天冬氨酸)的钠盐。为调节聚合物的亲疏水平衡,赋予聚合物两亲性,对侧链羧基进行部分酯化,得到共聚物:聚(α,β-L-天冬氨酸盐)/聚(α,β-L-天冬氨酸丙酯)(PAsp-Na/PAsp-P)。利用FTIR,~1H-NMR,DSC,TG等方法对各阶段得到的聚合物中间体和两亲性目标聚合物进行了结构与性能表征。(2)对PAsp-Na/PAsp-P共聚物在水溶液中的pH诱导胶束化行为进行了系统研究。诱导胶束形成的临界pH值在3附近,由于低pH溶液环境中,PAsp-Na链段侧链羧基处于质子化状态,PAsp-Na链段和PAsp-P链段均表现为疏水性,聚合物PAsp-Na/PAsp-P在强酸性环境中会产生沉淀;pH升高,羧基去质子化,PAsp-Na链段表现为亲水性,整个PAsp-Na/PAsp-P共聚物表现为两亲性,可自组装形成胶束。对pH=4时的胶束形态进行了SEM,TEM和AFM观察,并结合动态/静态光散射验证了pH敏感核-壳胶束的存在,证明聚合物形成了以PAsp-Na为亲水性壳层,PAsp-P为疏水性核的核.壳胶束结构。而pH=6条件下,核-壳结构由于环境pH的改变而遭到破坏,形成空心曲面结构。同时利用ζ电位仪测定聚合物表面电荷,证明胶束解离与形成的驱动力的确是聚合物侧链羧基的质子化与去质子化。同时胶束形态变化具有可逆性,随pH变化,聚集体会在沉淀,核-壳胶束和空心曲面结构间相互转变。(3)为得到聚(α-L-天冬氨酸)片段含量较高的聚(α,β-L-天冬氨酸)衍生物,采用另一种反应路线制备两亲性聚天冬氨酸酯(PAsp-Na/PAsp-R)。以聚琥珀酰亚胺为研究对象,选用不同的醇钠做为亲核取代反应进攻试剂,使聚琥珀酰亚胺开环制备聚(α,β-L-天冬氨酸酯)(PAsp-R)。研究发现,不同的亲核试剂进攻聚酰亚胺环时,得到共聚物中聚α-氨基酸与聚β-氨基酸的比例不同,空间位阻和电子效应共同影响反应结果。具有较长烷基链的亲核试剂会优先选择进攻空间位阻较小的β-位羰基碳原子,从而得到聚α-氨基酸占优势的聚(α,β-L-天冬氨酸酯)。由于与α-位碳原子相连羰基碳具有更强的电正性,电负性较大的亲核试剂会优先进攻α-位羰基碳而得到聚β-氨基酸占优势的聚(α,β-L-天冬氨酸酯)。PAsp-R部分水解,得到侧链既有亲水性羧基又有疏水性酯基的两亲性聚合物。以聚(α,β-L-天冬氨酸丁酯)为例,利用UV、~1H NMR等方法研究聚合物的水解降解行为。通过~1H NMR中侧链酯基质子信号峰的消失和主链游离氨基在紫外光谱中吸光度的变化,证明水解降解过程包括侧链酯键的水解和主链酰胺键的裂解两部分,水解过程受环境温度和缓冲溶液pH值的共同影响。水解速率具有温度依赖性,环境温度越高水解速率越快(37℃时的水解速率＞25℃时的水解速率)。溶液pH对聚合物降解速率的影响体现为:pH 12＞pH 4＞pH7。实验结果证实所合成的聚(α,β-L-天冬氨酸酯)是一类性能优良且可生物降解的聚合物。更多还原

【Abstract】 Stimuli-responsive polymers have been extensively investigated and used as smart biomaterials such as drug delivery systems（DDS）.The phase transition or phase separation of polymer is induced by a change in various conditions such as temperature,pH,electric field and solvent composition.However,the application of polymers to DDS is difficult because most stimuli-responsive polymers are not biodegradable and some are toxic.Polymeric biodegradability and biocompatibility are the most important factors for the application of polymers to drug delivery systems.Poly（amino acid）s is a kind of biodegradable materials in vivo.They are useful in pharmaceutical and biomedical areas.The precursor polymer,polysuccinimide（PSI）,was synthesized by the thermal polycondensation of L-aspartic acid.Poly（α,β-L-aspartate）（PAsp） was prepared by hydrolysis of PSI.And PAsp was partially esterified to afford an amphiphilic biodegradable polyanion,poly（sodium aspartate-co-propyl aspartate）（PAsp-Na/ PAsp-P）.The synthesized polyanion could be assembled into the nano-scaled aggregates in aqueous medium.The aggregate morphologies were studied by scanning electron microscopy（SEM） and transmission electron microscopy（TEM） as a function of pH.It was demonstrated that micellization of this random copolymer occurred with stimulus of pH changes to form various morphological micelles.The copolymer existed as precipitate at low pH（pH＜2）.When pH increased to 4,the polymers were associated into spherical micelles with the core of poly（propyl aspartate）（PAsp-P） hydrophobic units and shell of some negatively charged poly（sodium aspartate）（PAsp-Na） units.At higher pH（pH＞5）,toroidal nanostructures of the micelles were formed because rigid polyamide chains directly assemble into the large hollow spheres.The circular dichroism（CD） study showed that the conformation underwent a transition betweenα-helix and random coil at pH 3-7.The cooperative transitions were regulated by the degree of ionization of carboxylic side chains.When they were protonated（neutralized）,the molecular backbone was in favor of the regular helical structure;when deprotonated（ionized）, the electrostatic repulsions among side chains destabilized the intramolecular hydrogen bonds,thus randomizing the regular conformation.Some kinds of novel biodegradable polymers,poly（α,β-alkoxy-L-aspartates）, were successfully synthesized by nucleophilic substitution in polysuccinimide. Biocompatible polysuccinimide derivatives conjugated with alkoxy side chains were obtained by using sodium alkoxide（n=1-4）,sodium isopropoxide and sodium tertbutoxide as nucleophilic agents.Due to the steric hindrance effect and electronegative effect of the nucleophilic agent,poly（alkoxy-L-aspartate） with differentαandβ-amide unit compositions could be obtained.The contents ofα- andβ-amide unit were determined by ¹H NMR spectroscopy and ¹³C NMR spectroscopy.The highest content ofα-polyaspartate was 60%,obtained in poly（α,β-butoxy-L- aspartate）.The lowest content ofα-polyaspartate was 30%, obtained in poly（α,β-tertbutoxy-L-aspartate） and poly（sodium aspartate） （PAsp-Na）.Both steric and electronegative contributions were important factors that determine over all reactivity via either pathway.On ring-opening modes of the site selectivity,the stronger nucleophilicity of the nucleophilic agents,compared among methoxy,ethoxy,isopropoxy and tert-butoxy anions,would induce higherβ-amide content in the polymer.The steric hindrance effect of the nucleophilic agents such as methoxy,ethoxy,n-propoxy and n-butoxy anions leads to an increase in the extent ofαpeptide bonds in the reaction product.Amphiphilic polymer PAsp-Na/PAsp-R can be obtained by partially hydrolysis of poly（α,β-alkoxy-L-aspartates）.Hydrolysis process of poly（α,β-butoxy-L-aspartates） （PAsp-B） was studied by UV spectroscopy and ¹H NMR.The results showed that the degradation of PAsp-B includes the rapid elimination of butyl side chains and the moderate cleavage ofpeptide linkages in backbone as well as that the hydrolytic properties of PAsp-B were controlled by hydrolytic environments such as temperature and pH value.The hydrolysis rate of PAsp-B in aqueous solutions decreased in the order of pH 12＞pH 4＞pH 7 and increase with increasing the environmental temperature（for example,the hydrolysis rate of PAsp-B is higher at 37℃than that at 25℃）.更多还原