【作者】 李晓丹；

【导师】 赵燕军；

【作者基本信息】 天津大学 ， 药学（专业学位）， 2017， 硕士

【摘要】 纳米粒子是光动力疗法（PDT）治疗癌症的重要载体,临床效果往往受限于单线态氧（¹O₂）的极短寿命和有限的的扩散半径。为解决此难题,本课题假设¹O₂响应型纳米胶束载体,光触发后粒径膨胀,则可实现光敏剂的按需释放。咪唑,一种众所周知的¹O₂清除剂,通过胺解法连接于两亲性共聚物胶束的疏水内核,同时和具有生物相容的锌离子Zn²⁺进行配位络合,以此来提高载体的稳定性。模型光敏剂,二氢卟吩e6（Ce6）,物理包载于两亲性共聚物胶束的疏水内核。包载光敏剂的胶束对光照高度敏感,光触发产生¹O₂引发载体由咪唑向亲水性尿素的转化,进而引起载体粒径的增大,导致Ce6的迅速释放以及胞内分布。载体聚合物mPEG-PAsp-IM和mPEG-PBLA的成功合成通过核磁共振氢谱（¹H NMR）得以验证。载体的临界胶束浓度（CMC）使用芘荧光探针法测定,结果为11.7±0.4μg/mL。对各载体胶束的粒径和形貌使用马尔文粒度仪（DLS）和透射电子显微镜（TEM）进行考察,结果显示,实验组胶束mPEG-PAsp-IM-Zn²⁺/Ce6,30 min内随着激光照射时间的延长,胶束粒径逐渐增大,30 min至2 h粒径基本不再变化,而对照组胶束mPEG-PBLA/Ce6,其粒径在光照前后几乎没有变化。TEM结果与DLS结果一致。胶束的载药量利用高效液相色谱法进行测定,实验组为3.3%,对照组为0.6%。体外释放实验中,在pH 7.4条件下,对实验组和对照组分别进行光照和不光照处理,实验组未经光照的胶束36 h的累计释放量只有20.8%,光照的胶束释放了56.7%,差别较明显;而对照组在相同条件下未经光照和光照的胶束释放量分别为19.4%和21.2%,差别较微弱。说明载体mPEG-PAsp-IM-Zn²⁺中的咪唑与激光照射产生的¹O₂发生了反应,导致载体结构变化,粒径随之变化,因而加快Ce6释放;载体mPEG-PBLA不含咪唑环,¹O₂不与之发生反应,粒径无明显变化,Ce6释放速率基本一致。在细胞和动物实验中,与游离Ce6对照相比,这种粒径可膨胀的纳米系统基本上将更多的Ce6递送至肿瘤部位,并且在4T1肿瘤小鼠中显示出改善的体内抗肿瘤功效,使得PDT具有整合¹O₂清除剂和光敏剂的前所未有的级联反应。综上所述,本课题构建的¹O₂敏感纳米载体有望用于高效递送不同类型光敏分子,具有较高临床转化价值。更多还原

【Abstract】 Photodynamic therapy（PDT）efficacy is limited by very short half-life and limited diffusion radius of singlet oxygen（¹O₂）.We report ¹O₂-responsive micellar nanoplatform subject to considerable size-expansion upon light triggering to facilitate on-demand release of photosensitizer.Imidazole,a well-known ¹O₂ scavenger,was incorporated in the hydrophobic core of amphiphilic copolymer micelle,and was used to coordinate with biocompatible Zn²⁺and encapsulate Chlorin e6（Ce6）,a photosensitizer.The micelles were highly sensitive to the light irradiation;¹O₂triggering induced dramatic particle size expansion due to the conversion of imidazole to hydrophilic urea,resulting in controlled prompt release of Ce6 and rapid intracellular distribution.The synthesis of mPEG-PAsp-IM and mPEG-PBLA was confirmed by ¹H NMR.The critical micelle concentration（CMC）of the carrier was 11.7±0.4μg/m L by pyrene fluorescence probe method.The particle size and morphology of each micelle were investigated by using Malvern particle size analyzer（DLS）and transmission electron microscopy（TEM）.The results showed that the particle size of the experimental group mPEG-PAsp-IM-Zn²⁺/Ce6 increased gradually with the laser irradiation time in 30 min,and almost unchanged from 30 min to 2 h.While the particle size of the control group mPEG-PBLA/Ce6 was almost unchanged before and after laser irradiation.The TEM results were consistent with the DLS results.The drug loading of the micelles was determined by high performance liquid chromatography（HPLC）,3.3%in the experimental group and 0.6%in the control group.In the in vitro release experiment,under the condition of pH 7.4,the experimental group and the control group were irradiated with light and not irradiated respectively.The cumulative release of the experimental group without the light was only 20.8%,and with the light was increased to 56.7%,the difference was obvious;and the control group under the same conditions,without the light the cumulative release was 19.4%and with the light was 21.2%,nearly no difference.It was shown that the imidazole in the carrier mPEG-PAsp-IM-Zn²⁺could be reacted with the ¹O₂,which led to the change of the carrier structure and the change of the particle size,thus accelerating the release of Ce6.The carrier mPEG-PBLA does not contain imidazole ring,it could not be reacted with ¹O₂,so there was no significant change in particle size,Ce6 release rate was nearly the same.In cell and animal experiments,this size-expandable nanosystem delivered substantially more Ce6 to tumor sites as compared to free Ce6 control,and exhibited improved antitumor efficacy in vivo in 4T1 tumor-bearing mice,enabling PDT with the unprecedented,cascade responses of the integrated ¹O₂ scavenger and photosensitizer.The current work presents a novel ¹O₂-sensitive nanoplatform for efficient photosensitizer delivery with high potential clinical value.更多还原