Design and Drug Carrier Application of a Photo-Responsive and pH-Sensitive Azobenzene Polymer Molecule
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摘要: 采用原子转移自由基聚合（ATRP）法合成了一系列具有pH/光双敏感的两亲性嵌段聚合物C10-偶氮苯（AZO）-C10-聚甲基丙烯酸二异丙胺基乙酯（PDPAn）-聚乙二醇（PEG45）（n=30,50,80），用核磁、凝胶渗透色谱（GPC）对合成的聚合物进行表征，用动态光散射（DLS）测定聚合物自组装形成的胶束的粒径及电位，用紫外分光光度计测定聚合物的光敏感性以及聚合物胶束的pH敏感点，采用荧光分光光度计测定聚合物胶束的临界胶束浓度（CMC），模拟体外阿霉素释药。结果表明，合成的聚合物具有良好的pH敏感性和紫外可见光敏感性，并可以自组装得到粒径约为140~200 nm的均一、稳定的球状胶束，pH敏感点在6.3～7.2之间，并且在波长为365 nm和470 nm的两种光线下发生顺反结构的转变。选择pH敏感点在6.3左右的聚合物胶束运载抗癌药物阿霉素，后续体外释药实验表明聚合物胶束在pH或光刺激下可以很好地实现可控性释药，同时利用细胞毒性实验验证了聚合物胶束具有很低的细胞毒性，该聚合物有望成为一种潜在的pH/光响应型靶向药物载体。Abstract: A series of amphiphilic block copolymers C10-AZO-C10-PDPAn-PEG45(C10-azobenzene-C10-poly[2-(diisopropylamino)ethyl methacrylate]-polyethylene glycol) (n=30,50,80) were designed and synthesized to improve stimuli-responsibility of pH/photo dual-responsive drug delivery system. In this paper, we used three steps to synthesize the copolymers. The data of proton nuclear magnetic resonance spectroscopy (1H-NMR) and gel permeation chromatography (GPC) showed that copolymers were successfully synthesized. The florescence intensity ratio (I373/I384) of copolymeric micelles indicated that CMC (critical micelles concentrations) was negative correlation with PDPA polymerization degree. The image of transmission electron microscope showed that the copolymers were self-assembled to obtain uniform stable spherical micelle with particle size of 140—200 nm (0.3 mg/mL). Dynamic light scattering measurements were used to determine the diameters and Zeta potentials of the self-assembled copolymer micelles. Results showed that the copolymeric micelles were well dispersed in water. The structure of micelles was sensitively correlation with pH change. The pH-triggering points of copolymers were in the range of 6.3—7.2. And then, the reversible photo responsibility was investigated via UV-visible spectrophotometer. It was found that the absorption value of each copolymer micelle solution decreased at 350 nm (characteristic absorption peak of trans-AZO) and increased slightly at 440 nm (characteristic absorption peak of cis-AZO) when they were irradiated with 365 nm ultraviolet light, which indicated that the trans-structure was changed into cis-structure. After the solutions were irradiated with visible light of 470 nm, the cis-structures were found to convert into trans-structures again. Herein, we chose C10-AZO-C10-PDPA30-PEG45 for further experiment as its pH-triggering points fitted the pH environment of normal and cancer cells. In vitro release kinetics of copolymer micelles were studied using fluorescence spectrophotometer under different conditions. The cumulative drug release amount at pH 7.4 was apparently lower than those at pH 6.4 and 5.0. And the release amounts of micelles irradiated by ultraviolet rays were higher than those of non-irradiated samples. These results showed that the environmental stimuli responsibility of the copolymer micelles could control drug release by pH and light stimulations. Finally, we transfected the blank micelles to Hela cell. Cytotoxicity assay indicated that micelles had low cytotoxicity. Therefore, this copolymer-based drug carrier could be expected to achieve controllable drug release in response to different conditions.
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Key words:
- pH/photo dual-responsive /
- copolymeric micelle /
- drug carrier /
- cytotoxicity /
- controlled release
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图 2 (Ⅰ) C10-AZO-C10-OBr、(Ⅱ) C10-AZO-C10-PDPAn 和(Ⅲ) C10-AZO-C10-PDPAn-mPEG45在CDCl3中的1H-NMR谱图
Figure 2. 1H-NMR spectra of (Ⅰ) C10-AZO-C10-OBr, (Ⅱ) C10-AZO-C10-PDPAn and (Ⅲ) C10-AZO-C10-PDPAn-mPEG45 in CDCl3
图 3 不同pH环境下聚合物胶束溶液的（a）透射率及（b）Zeta电位的变化（聚合物胶束溶液质量浓度0.1 mg/mL）
Figure 3. (a) Transmittance and (b) Zeta potential of polymeric micelle solution in different pH environments(Mass concentration of copolymeric micelle solution: 0.1 mg/mL)
图 4 反式 (a) C10-AZO-C10-PDPA30-mPEG45, (c) C10-AZO-C10-PDPA50-mPEG45, (e) C10–AZO-C10-PDPA80-mPEG45受到不同时间紫外光（365 nm）照射后的紫外吸收谱图; 顺式 (b) C10-AZO-C10-PDPA30-mPEG45, (d) C10-AZO-C10-PDPA50-mPEG45, (f) C10-AZO-C10-PDPA80-mPEG45受不同时间可见光（470 nm）照射后的紫外吸收谱图
Figure 4. Ultraviolet absorption spectra after irradiation of ultraviolet (365 nm) after different time intervals of trans-(a) C10-AZO-C10-PDPA30-mPEG45, (c) C10-AZO-C10-PDPA50-mPEG45, (e) C10-AZO-C10-PDPA80-mPEG45; Ultraviolet absorption spectra after irradiation of visible light (470 nm) after different time intervals of cis-(b) C10-AZO-C10-PDPA30-mPEG45, (d) C10-AZO-C10-PDPA50-mPEG45, (f) C10-AZO-C10-PDPA80-mPEG45
图 5 (a)聚合物C10-AZO-C10-PDPAn-mPEG45（n=30,50,80）的临界胶束浓度随荧光强度比值I373/I384变化曲线（pH=7.4）；(b)聚合物胶束的粒径分布图（pH=7.4）；(c) 聚合物胶束的透射电镜图（pH=7.4）
Figure 5. (a) Florescence intensity ratio (I373/I384) versus logarithm of critical micelles concentrations（C10-AZO-C10-PDPAn-mPEG45（n=30,50,80））; (b) Size distribution of the copolymer micelles; (c) Transmission electron micrographs of copolymer micelles (pH=7.4)
图 6 聚合物C10-AZO-C10-PDPA30-mPEG45在不同pH和紫外光照射条件下的累计释放曲线
Figure 6. Accumulated release curves of C10-AZO-C10-PDPA30-mPEG45 in different pH under irradiation of UV light
图 7 不同质量浓度的聚合物空白胶束对Huh7和 HEK-293T的细胞毒性
Figure 7. Cytotoxicity to Huh7 and HEK-293T cells at different massconcentrations of copolymer micelle
表 1 C10-AZO-C10-PDPAn-mPEG45共聚物的分子量和分散系数
Table 1. Molecular weight and dispersity index(PDI) of C10-AZO-C10-PDPAn-mPEG45 copolymers
Copolymer Mn,NMR Mn,GPC PDI C10-AZO-C10-PDPA30-mPEG45 8894 9748 1.73 C10-AZO-C10-PDPA50-mPEG45 13160 11355 1.82 C10-AZO-C10-PDPA80-mPEG45 19560 22524 1.63 
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