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聚合物胶束是最早被应用的聚合物自组装体之一,这是因为两亲性嵌段聚合物在水溶液中能够自组装形成内核疏水外壳亲水的聚合物胶束[1-4]。过去几十年里,聚合物胶束作为药物载体在生物医药领域受到了极大的关注。比起传统药物载体聚合物胶束能够增强药物溶解性,延长药物血液循环时间,改善EPR效应(Enhanced Permeability and Retention Effect)引起的药代动力学和药效等[5-14]。EPR效应是指肿瘤的高通透性和滞留效应,由于实体瘤的血管结构缺陷而造成,使得大分子药物可以有效转运到肿瘤组织中,提高了药物作用效率。不仅如此,通过改变聚合物单体,聚合度,体系结构以及其他一些参数还能够方便的调节聚合物胶束的物理化学性质。
为了实现药物的靶向输送和可控释放,各种能够响应环境刺激如pH[15,16],温度[17,18],光[19,20],氧化还原电位[21,22],超声波[23,24]、酶[25]等的刺激响应型聚合物胶束被不断发展起来。由于肿瘤细胞的恶性增殖,营养缺乏导致糖酵解和乳酸积累,肿瘤部位具有不同于正常组织的pH值,如相比于正常的组织环境(pH 7.2-7.4),肿瘤组织的pH值为6.4左右,并且肿瘤细胞内从早期核内体(pH 6-6.5),晚期核内体(pH 5-6)到溶酶体(pH 4.5-5)也存在着pH梯度变化,所以pH刺激常被作为设计刺激响应型药物载体的内源刺激[26-28]。聚甲基丙烯酸二异丙胺基乙酯(PDPA)就是能够响应pH刺激的聚合物,在不同的pH环境下能够发生质子化或去质子化,从而使亲疏水性发生改变。光是一种非接触式的外源刺激,波长和强度都能够被远程控制,也常被应用于药物载体的设计中。偶氮苯(AZO)是光响应型的官能团,偶氮苯分子存在顺式和反式两种异构体,在UV或可见光照射下能够发生可逆的光异构化过程,从而引起空间结构或亲疏水性的变化[29-32]。
多重刺激响应型聚合物能够对两种或多种刺激敏感,能够适应肿瘤部位复杂的生理环境,在癌症的药物递送和治疗中显示出了巨大的潜能。本文设计、合成得到了三种不同聚合度pH和光双重敏感的聚合物C10-AZO-C10-PDPAn-PEG45(n=30,50,80)。探究聚合物的双重敏感性,聚合物形成的胶束的基本性质,进而筛选出适合肿瘤部位药物递送的聚合物C10-AZO-C10-PDPA30-PEG45,研究此聚合物包载抗癌药物阿霉素的体外释放行为以及其生物相容性。
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甲基丙烯酸二异丙胺基乙酯(DPA,质量分数98%,百灵威科技有限公司);聚乙二醇单甲醚(mPEG-2000, 质量分数98%,百灵威科技有限公司);2-溴代异丁酰溴(质量分数98%,百灵威科技有限公司); N,N,N-N,N″-五甲基二亚乙基三胺(PMDETA, 分析纯,上海化成工业发展有限公司);CuBr(分析纯,经冰醋酸、乙醇洗涤三次,干燥后待用);芘(质量分数99%,上海阿拉丁生化科技股份有限公司); 盐酸型阿霉素(DOX • HCl,质量分数98%,百灵威科技有限公司);四氢呋喃(THF,分析纯,上海泰坦试剂有限公司,使用前重蒸除水);甲醇(分析纯,国药集团化学试剂有限公司);二氯甲烷(分析纯, 上海泰坦试剂有限公司,使用前经氢化钙处理);中性三氧化二铝(200-300目(75~48 μm),上海莲花有限公司),碱性三氧化二铝(200~300目(75~48 μm),上海莲花有限公司);人类肝癌细胞株(Huh7细胞,美国菌种保藏ATCC),细胞毒性试剂盒(CCK-8, DOJINDO. 日本)、无血清培养基(DMEM, Gibco, 美国)、热灭活胎牛血清(FBS,gibco, 美国)。
动态光散射(Zetasizer Nano ZS90,英国马尔文公司);紫外-可见分光光度计(UV-2450,日本岛津公司);荧光分光光度计(F-4500,日本);凝胶渗透色谱(GPC,PL-GPC50,英国马尔文公司);透射电子显微镜(TEM,JEM-1400,日本电子株式会社);核磁Bruker-400 MHz测定(AVANCEⅢ 500,德国);倒置显微镜(EVOS x1. AMG);荧光显微镜(EVOS FL. AMG);微孔板分光光度计(xMark Microplate Spectrophotometer. Bio-Rad)。
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在干燥的单口烧瓶中加入0.421 1 g C10-AZO-C10-OH(8.6×10-4 mol),用14 mL二氯甲烷溶解,在冰水浴搅拌的条件下用注射器逐滴加入300 μL三乙胺和300 μl 2-溴异丁酰溴,停止冰水浴,避光室温反应30 h。反应结束后,向反应液中加入少量活性炭,搅拌30 min,进行抽滤,滤饼用二氯甲烷冲洗3遍,取滤液,用旋蒸法除去其中的二氯甲烷溶剂,得到黄色粘稠状产物。
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通过ATRP聚合的方法接枝聚合物,以C10-AZO-C10-OBr作为引发剂,PMDETA作配体,CuBr作催化剂,用MgSO4处理过的CH2Cl2作溶剂,按照摩尔比n(DPA):n(C10-AZO-C10-OBr):n(CuBr):n(PMDETA)=m:1:1:1.1的比例将药品分别放入Schlenk瓶中(m=30、50、80,合成三种不同PDPA链长的聚合物),加6 mL溶剂CH2Cl2。经过三次液氮冷冻-抽真空-溶解,在冷冻、氮气保护的条件下加催化剂CuBr,于室温避光条件下反应12 h。反应结束后,通入空气,无水无氧体系被破坏,反应停止,此时溶液呈深绿色。将反应液通过装有中性氧化铝的柱子,用THF作洗脱液除CuBr。通过旋蒸、在冷冻的无水甲醇中沉降的方法除去溶剂以及未反应的单体DPA、配体PMDETA,收集产物,得黄色粘稠状聚合物,通过1H-NMR以及 GPC检测产物纯度。
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首先将1.00 g mPEG45溶解在10 mL新蒸的THF中,在冰水浴条件下,加入0.04 gNaH,冰水浴条件下反应3 h,活化PEG中的羟基,再加入新蒸的THF 溶解的C10-AZO-C10-PDPAn-Br,室温避光反应2.5 h。反应完全后旋蒸,无水甲醇沉降三次,得到黄色粘稠状产物。用1H-NMR确定产物的结构,用GPC检测聚合物的相对分子质量(Mn)和分布系数(PDI)
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配置浓度为0.3 mg/mL,pH3.0的酸性聚合物溶液C10-AZO-C10-PDPA30-mPEG45(n=30,50,80)。用一定浓度的NaOH溶液调节溶液的pH,用DLS和UV-2450测定溶液在不同pH下的电位、透射率的变化,以电位或透射率值为纵坐标,pH值为横坐标作图。
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配置浓度为0.3 mg/mL,pH3.0的酸性聚合物溶液C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)。置于紫外分光光度计的比色皿中,用365 nm的紫外光每隔5 s或10 s照射(至曲线不再变化),然后再用470 nm的可见光照射至曲线不再发生变化,在此过程中实时检测溶液的紫外吸收值。
以芘作为荧光探针测定聚合物在水溶液中的CMC值。首先配置芘在水中的饱和溶液,然后把一定量的C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的THF溶液置于玻璃试管中,用吹风机使THF挥干,然后加入2 mL 芘的饱和溶液,使聚合物芘溶液的初始质量浓度为0.2 mg/mL,接下来用芘饱和溶液不断稀释,在此过程中,设定荧光分光光度计的激发波长为334 nm,检测不同浓度聚合物芘溶液在373 nm以及384 nm处的荧光强度,以荧光强度比值(I373/I384)为纵坐标,聚合物芘溶液浓度的对数值为横坐标作图,其突变点即为CMC值。
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使用溶剂挥发法制备聚合物胶束,首先配置6 mg/mL的聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的THF溶液,取100 μL到棕色小瓶中,在搅拌下逐滴加入2 mL pH 7.4 PBS缓冲溶液(1 mmol/L),继续搅拌12 h,使溶液中的THF挥干,得到质量浓度为0.3 mg/mL的胶束溶液。对于载药的聚合物胶束,在把聚合物的THF溶液转移到棕色小瓶后,加入盐酸型阿霉素(DOX·HCl)、适量三乙胺,搅拌1 h,使亲水性盐酸型阿霉素转变为疏水的DOX,以便包载在胶束的疏水核中,再逐滴加入pH 7.4 PBS缓冲溶液,使药物的最终质量浓度为0.5 mg/mL,继续搅拌12 h,用分子截留量为14 000 g/mol透析袋透析除去未被包载的DOX。所形成聚合物胶束的粒径、电位用DLS表征,形貌特征用TEM表征。DOX的包载量(DLC)及包封率(DLE)通过检测包裹在胶束中的DOX在480 nm处的吸收值得到。公式如下:
其中B1为胶束中的药物质量,C1为总胶束质量;C2为总药物质量
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把8 mL载DOX的C10-AZO-C10-PDPA30-mPEG45 胶束溶液装入6个透析袋中,每个透析袋装1 mL胶束溶液,分别放入装有25 mLpH=5.0、6.4、7.4的PBS缓冲液的两组离心管中,其中一组预先用365 nm的紫外光照射60 s,之后把两组分别装有不同pH缓冲液的离心管一起放入恒温振荡箱中振荡,每隔一定时间用UV-2450测量离心管中缓冲液在480 nm的紫外吸收值,从而确定药物释放量,以上剩余的2 mL胶束加HCl溶液解离,用以测量胶束中包载的总药量。DOX累计释放量(DAR)公式如下:
其中A1为在不同时间点胶束中释放出DOX的紫外吸收强度,A2为胶束包载的总DOX的紫外吸收强度。
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人肝癌细胞系Huh7和人肾上皮细胞系HEK-293T均来自于美国ATCC细胞库,这两款细胞都养在DMEM培养液中,同时培养液中加入100 U/mL青霉素G/硫酸链霉素和10%(体积比)FBS,并将细胞放在37 ℃,含5%二氧化碳的培养箱中培养。
空胶束的细胞毒性:在96孔板上接种人肝癌细胞系Huh7或人肾上皮细胞系HEK-293T细胞(104细胞/孔),培养过夜。然后加入不同浓度的空胶束(7.5~225 μg/mL),共孵育8 h后换成新鲜培养液再继续培养16 h,在每个孔中加入10 μl CCK-8试剂,将培养板置于培养箱中孵育1 h,然后用酶标仪检测在450 nm处的吸光度。
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本实验的合成路线如示意图1所示,首先用C10-AZO-C10-OH和2-溴异丁酰溴反应合成C10-AZO-C10-OBr,将其作为引发剂,用原子转移自由基聚合(ATRP)法,合成聚合物C10-AZO-C10-PDPAn,最后接枝亲水性的聚乙二醇mPEG,得到具有pH和光双重敏感的两亲性聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)。
每一步合成产物的1H-NMR如图1所示,图中(Ⅰ) (Ⅱ)(Ⅲ) 分别为C10-AZO-C10-Br,C10-AZO-C10-PDPAn和C10-AZO-C10-PDPAn-mPEG45的1H NMR谱图。(Ⅰ)中a处对应于AZO上苯环的特征峰,b(δ=1.96)处的峰归属于Br-C-(CH3)2上的质子。(Ⅱ) 中 c(δ=1.01)处的强峰归属于异丙基的甲基质子,是PDPAn的特征峰,表明了PDPAn的成功连接。(Ⅲ)中d处出现了mPEG45中的重复单元-CH2CH2O-上质子的特征峰,说明mPEG45的成功修饰。综上,说明成功制备了嵌段聚合物。
图 1 The 1H NMR spectrum of (Ⅰ) C10-AZO-C10-Br (Ⅱ) C10-AZO-C10-PDPAn 和(Ⅲ) C10-AZO-C10-PDPAn-mPEG45 in CDCl3
同时,GPC检测结果进一步证明了嵌段聚合物的成功合成,表1列出了实验中合成聚合物的组成、数均分子量和分布系数。从表中可以看到,随着PDPA聚合度的增加,数均分子量增大,聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的PDI值偏大,有可能是合成聚合物时,存在少量未反应C10-AZO-C10-PDPAn-Br,且不溶于无水甲醇中,导致PDI偏大。
Copolymers 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 表 1 各聚合物的数均分子量和分散系数
Table 1. Molecular weight and copolymer dispersity index (PDI) of the C10-AZO-C10-PDPAn-mPEG45 copolymers
通过测定嵌段聚合物在不同pH环境下的透射率和电位的变化来确定pH敏感点。当pH<5.5时,嵌段聚合物溶液澄清透明,透过率近乎100%,随着NaOH溶液的滴加,PDPA逐渐开始脱质子化,由亲水性变成疏水性,溶液逐渐变得浑浊,从而确定pH敏感点,结果如图2(a),pH敏感点为6.3~7.2。且随着PDPA聚合度的增加,嵌段聚合物的pH敏感点略有增大。这可能是因为PDPA链段越长,其从亲水性转变为疏水性更为困难,需要更多的OH-用以脱质子化。另一方面,随着pH的升高Zeta电位开始下降,这是由于在pH<5.5时,大量H+存在,因而溶液为正电性,随着NaOH的量逐渐增多电位由正转为负。如图2(b)所示,Zeta电位的结果和透过率法测出的嵌段聚合物pH敏感点基本一致。
图 2 嵌段聚合物在不同pH环境下溶液的透射率(a)及Zeta电位的变化(b) (聚合物胶束溶液浓度0.1 mg/mL)
Figure 2. Transmittance (a) and Zeta potential (b) of copolymer micelles in different pH environments(concentration of copolymer: 0.1 mg/mL)
嵌段聚合物的光敏感性可以通过测定聚合物溶液在不同光照下的紫外吸收值来确定。其中偶氮苯以反式Trans-构型存在的在350 nm处有一强的特征吸收峰,顺式Cis-偶氮苯在440 nm处有较强的吸收峰。从图3(a,c,e)的实验结果看出,当用365 nm的紫外光照射0.3 mg/mL的各聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80; pH=3)溶液时,随着光照时间的增加,350 nm处的紫外吸收值逐渐减小,440 nm处的峰值略有增高,Trans-构型的AZO逐步向Cis-构型转变,当光照时间约为20 s以上紫外吸收曲线均不再发生变化。说明反式偶氮苯向顺式偶氮苯转化已达到平衡。之后再用470 nm的可见光照射,结果如图3(b, d, f)所示,在350 nm左右处的吸收峰强度逐渐增强,440 nm处的吸收峰强度逐渐减弱,Cis-构型的AZO逐步向Trans-构型转变。实验发现三种聚合物分别在可见光照5、10、35 s之后,顺式又向反式偶氮苯转化并且达到平衡,其中,C10-AZO-C10-PDPA30-mPEG45反异构化速度最快,表明连接较大尺寸的聚合物可能会阻碍反异构化的进行。同时,与紫外光照之前相比,350 nm处的吸收值有所降低,440 nm处的吸收值略有升高,表明偶氮苯不能完全回复到紫外光照前的状态,这可能是由于PDPA嵌段对AZO基团的异构化具有一定的阻碍作用[33]。上结果说明聚合物具有可逆的光敏感性。
图 3 反式C10-AZO-C10-PDPA30-mPEG45(a)/C10-AZO-C10-PDPA50-mPEG45(c)/C10–AZO-C10-PDPA80-mPE G45 (e) 受到不同时间紫外光照射后紫外吸收谱图;顺式C10-AZO-C10-PDPA30-mPEG45(b)/ C10-AZO-C10-PDPA50-mPEG45(d)/ C10-AZO-C10-PDPA80-mPEG45(f)受不同时间可见光照射的紫外吸收谱图(pH=3)
Figure 3. Ultraviolet absorption spectrum after irradiation of ultraviolet after different times intervals of C10-AZO-C10-PDPA30-mPEG45(a), C10-AZO-C10-PDPA50-mPEG45(c), C10-AZO-C10-PDPA80-mPEG45(e). Ultraviolet absorption spectrum after irradiation of visible light after different intervals of cis C10-AZO-C10-PDPA30-mPEG45(b), C10-AZO-C10-PDPA50-mPEG45(d), C10-AZO-C10-PDPA80-mPEG45(f)(pH=3)
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CMC(临界胶束浓度)是嵌段聚合物形成胶束最基本的物性参数,本实验利用芘作为荧光探针,测得各聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的CMC值分别在0.1、1×10−2、1×10−2 mg/mL(图4(a)),且随着PDPA聚合度的增加而减小,这可能是由于PDPA链长越长,形成胶束时疏水聚集能力越强,越容易形成胶束,因此CMC值越小。通过DLS,TEM表征所形成胶束的粒径以及形貌,结果如图4(a)、(c)所示,各聚合物形成球状、粒径分布均匀的胶束,其粒径分别在140、200、220 nm左右。在pH=7.4 的PBS(1 mmol/L)缓冲液中,嵌段聚合物所形成胶束的粒径随着PDPA聚合度的增加而增大。原因可能在于PDPA疏水嵌段越长,所形成胶束疏水内核越大,粒径就越大。TEM所观察到的粒径和DLS的结果基本一致。
图 4 (a)聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的临界胶束浓度(I372/I383)变化曲线;(b)聚合物胶束的粒径分布图;(c) 聚合物胶束的透射电镜图pH=7.4
Figure 4. (a) The florescence intensity ratio (I372/I383) 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
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人体正常生理环境的pH约为7.4,肿瘤微环境为弱酸性约为6.5,而肿瘤细胞内部环境的pH更低约为5.5-6.5[34]。本实验选取了pH敏感点为6.3左右的嵌段聚合物C10-AZO-C10-PDPA30-mPEG45制备胶束,作为抗癌药物DOX的载体进行体外模拟释药。载药量为12.1%±1%。
我们选取pH5.0、6.4、7.4三种pH环境进行药物释放研究。DOX的释药结果如图5所示,累计释药量随着环境pH值的降低而增大。在pH=7.4时,25 h后的累计释药量约为11.3±4%,说明在人体正常生理环境下,聚合物胶束能够有效的包载DOX,药物泄漏量少。在pH=6.4和pH=5.0条件下,相应的DOX最终平衡累计释药量约为59.6%±5%,81.0±5%。这是因为溶液酸性越强,聚合物PDPA嵌段越容易发生质子化,从疏水性逐渐变成亲水性嵌段,引起胶束结构动摇从而释放包裹在胶束疏水层内的DOX。
图 5 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 5. Accumulated release curves of C10-AZO-C10-PDPA30-mPEG45 in different pH under irradiation of UV light
同时,我们还考察了紫外光刺激后的释药效果。在开始释药前,用365 nm的紫外光照射各个pH下载药样品,发现各个pH下的释药率都增加了约10%左右。这是因为部分疏水性DOX包裹在C10-AZO-C10嵌段所形成的疏水层,紫外光照使AZO由反式构象变为顺式构象,导致胶束的疏水层发生扰动,促进了DOX的释放。
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为了检测聚合物C10-AZO-C10-PDPA30-mPEG45胶束本身的生物相容性,我们分别用肝癌细胞Huh7和人正常细胞HEK-293T与不同浓度的C10-AZO-C10-PDPA30-mPEG45胶束共孵育8 h,然后换成新鲜培养液继续孵育16hr,用CCK-8方法检测细胞的存活率。实验结果如图6所示,与不同浓度的空胶束作用后这两个细胞系的细胞存活率依然很高,即使空胶束浓度高达225 μg/mL,Huh7和HEK-293T细胞的存活率仍保持在90%以上,这说明聚合物C10-AZO-C10-PDPA30-mPEG45胶束无论对肝癌细胞还是正常细胞的毒性都极小。表明此聚合物胶束具有良好的生物相容性,适合作为药物载体。
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本论文利用肿瘤细胞微环境的特点,设计合成了具有pH响应和紫外光响应的嵌段共聚物,通过自组装过程制备pH/光可控释药载体。首先测得了各聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的各项物理化学性质,其CMC值分别0.1~1×10−2 mg/mL,而其粒径也在140~220 nm之间,满足EPR效应的基本要求。嵌段聚合物在不同pH环境下,表现出不同的亲疏水性,且受到聚合物嵌段长度的影响而具有不同的pH敏感点,其敏感点在6.3~7.2之间。通过对嵌段聚合物的紫外光响应性能考察,证实了聚合物上的AZO基团的顺反结构可以在350 nm和440 nm的紫外-可见光照射下发生可逆转换,照射下具有良好的光响应性能。最后我们验证了水相中聚合物具有优良的pH/光双敏感可控性,通过照射可使原先pH敏感的聚合物胶束的释药率提高10%左右,而CCK8实验表明所获得的聚合物胶束不论是在低质量浓度(75 μg/mL)下还是在较高质量浓度(225 μg/mL)下都能有较高的生物相容性,可以预见该pH/光双敏感的聚合物胶束在靶向药物载体研制领域具有良好的应用前景。
pH/光双敏感含偶氮苯聚合物分子的设计及药物载体的应用
Design and drug release 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-AZO-C10-PDPAn-PEG45(n=30,50,80),用核磁、GPC对合成的聚合物进行表征,用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聚合物胶束的CMC值、体外阿霉素模拟释药。结果表明,此聚合物具有良好的pH敏感性和紫外可见光敏感性,并可以自组装得到粒径在140~200 nm左右均一、稳定的球状胶束,pH敏感点在6.3~7.2之间,并且在波长在350 nm和440 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其在pH或光刺激下可以很好地实现可控性释药,同时利用CCK-8实验验证了其具有很低的细胞毒性。有望成为一种潜在的的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) had been designed to improve stimuli-responsibility of pH/ photo dual-responsive drug delivery system. In this paper, we used three steps to synthesize copolymers. The data of proton nuclear magnetic resonance spectroscopy (1H NMR) and gel permeation chromatography (GPC) showed that copolymers were accurately synthesized. The florescence intensity ratio (I372/I383) of copolymeric micelles determined that the CMC (critical micelles concentrations) was negative correlation with PDPA polymerization degree. The image of Transmission electron microscope (bar=100 nm) showed 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 assess the diameters and zeta potentials of self-assembled copolymer micelles. Results showed that the copolymeric micelles were fine dispersed in water environment. The structure of micelles was sensitively correlation with pH change. Their pH-triggering points of copolymers were in the range of 6.3-7.2. And then, the reversible photo responsibility had been investigated via UV-visible spectrophotometer. It was found that the absorption value of each copolymer micelle‘s solution decreased in 350 nm (characteristic absorption peak of trans-AZO) and increased slightly in 440 nm (characteristic absorption peak of cis-AZO) when they were irradiated with 365 nm ultraviolet light. The result indicated that the trans structure alter to cis structure. After the solutions were irradiated with visible light of 470 nm, the cis structures were found to alter to trans structures again. Herein, we choose C10-AZO-C10-PDPA30-PEG45, whose pH-triggering points fitted the pH environment of normal and cancer cells, to use in further experiment. In vitro release kinetics of copolymer micelles were studied using fluorescence spectrophotometer under different conditions. The cumulative drug release amount in pH=7.4 was apparently lower than pH=6.4 and 5.0. And it was found that the release amount of micelles irradiated by Ultraviolet rays were higher than no-irradiated sample. These results exhibited that the environmental stimuli responsibility of the copolymer micelles could control drug release through pH and light stimulation. Finally, we transfected the blank micelles to Hela cell, the CCK8(Cell Counting Kit-8) experiments 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. -
Key words:
- pH/photo dual-responsive /
- copolymeric micelle /
- drug carrier /
- Cytotoxicity /
- Controlled release
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图 3 反式C10-AZO-C10-PDPA30-mPEG45(a)/C10-AZO-C10-PDPA50-mPEG45(c)/C10–AZO-C10-PDPA80-mPE G45 (e) 受到不同时间紫外光照射后紫外吸收谱图;顺式C10-AZO-C10-PDPA30-mPEG45(b)/ C10-AZO-C10-PDPA50-mPEG45(d)/ C10-AZO-C10-PDPA80-mPEG45(f)受不同时间可见光照射的紫外吸收谱图(pH=3)
Figure 3. Ultraviolet absorption spectrum after irradiation of ultraviolet after different times intervals of C10-AZO-C10-PDPA30-mPEG45(a), C10-AZO-C10-PDPA50-mPEG45(c), C10-AZO-C10-PDPA80-mPEG45(e). Ultraviolet absorption spectrum after irradiation of visible light after different intervals of cis C10-AZO-C10-PDPA30-mPEG45(b), C10-AZO-C10-PDPA50-mPEG45(d), C10-AZO-C10-PDPA80-mPEG45(f)(pH=3)
图 4 (a)聚合物C10-AZO-C10-PDPAn-mPEG45(n=30,50,80)的临界胶束浓度(I372/I383)变化曲线;(b)聚合物胶束的粒径分布图;(c) 聚合物胶束的透射电镜图pH=7.4
Figure 4. (a) The florescence intensity ratio (I372/I383) 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
表 1 各聚合物的数均分子量和分散系数
Table 1. Molecular weight and copolymer dispersity index (PDI) of the C10-AZO-C10-PDPAn-mPEG45 copolymers
Copolymers 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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