CN111995745B - 一种双锁型聚合物及其制备方法和应用 - Google Patents
一种双锁型聚合物及其制备方法和应用 Download PDFInfo
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- CN111995745B CN111995745B CN202010861678.4A CN202010861678A CN111995745B CN 111995745 B CN111995745 B CN 111995745B CN 202010861678 A CN202010861678 A CN 202010861678A CN 111995745 B CN111995745 B CN 111995745B
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Abstract
Description
技术领域
本发明涉及聚合物荧光探针技术领域,具体涉及一种双锁型聚合物及其制备方法和应用。
背景技术
光学成像是以发光探针的发光强度为检测信号的可视化成像技术,主要包括生物发光技术和荧光技术,已经广泛应用于癌症的早期、准确诊断。激活型荧光探针可以对肿瘤微环境发出响应信号,具有较低的背景噪声和较高的信号-背景比,能对肿瘤进行灵敏、准确的诊断,效果要显著优于“常开”光学探针。
近年来,随着前体药物和纳米技术在药物传递领域的广泛应用,极大地丰富了抗肿瘤药物的传递策略,而基于小分子前药的自组装纳米递送系统结合了前体药物策略和纳米技术的优点,载药量高、稳定性好、毒副作用小,成为了研究的热点。无论是前药还是纳米给药系统,智能地触发药物在靶点的选择性释放对制剂的有效性和安全性都非常重要。与正常细胞相比,肿瘤细胞特殊的微环境已被广泛应用于设计刺激反应药物传递系统。
目前,大多数用于癌症成像和治疗的纳米材料都可能对健康组织造成非特异性背景和毒性,并引起严重的副作用。为了提高特异性,许多对癌症特异性生物标志物(如低pH值、过表达酶或谷胱甘肽)有反应的刺激反应纳米材料已经被开发用于癌症成像和治疗,但大多数刺激反应型纳米材料仅对单一刺激产生反应,可能并不足以区分肿瘤组织,并会导致成像的高背景信号和治疗的副作用。为了改善位点的特异性转运和降低非靶标细胞的毒性,开发能够对多种癌症相关生物标志物共存做出反应的智能纳米材料是非常可取的。然而,开发能够同时对正交刺激做出反应,并具有集成的成像和治疗功能的纳米探针仍是一项挑战。
发明内容
本发明的目的在于提供一种双锁型聚合物及其制备方法和应用。
本发明所采取的技术方案是:
一种双锁型聚合物,结构式为:
式中,x为110~117的自然数,y为70~80的自然数,z为1~3的自然数。
上述双锁型聚合物的制备方法,包括以下步骤:
1)进行二异丙基乙醇胺和甲基丙烯酰氯的反应,得到2-(二异丙基氨基)甲基丙烯酸乙酯;
优选的,步骤1)所述二异丙基乙醇胺、甲基丙烯酰氯的摩尔比为1:(1.0~1.2)。
优选的,步骤1)所述反应的时间为20~30h。
优选的,步骤2)所述甲氧基聚乙二醇、4-氰基-4-(硫代苯甲硫基)戊酸的摩尔比为1:(4~5)。
优选的,步骤2)所述酯化反应的时间为40~70h。
优选的,步骤3)所述PEG-CPDB、2-(二异丙基氨基)甲基丙烯酸乙酯、甲基丙烯酸的摩尔比为1:(80~90):(3~4)。
优选的,步骤3)所述聚合反应的时间为10~20h。
优选的,步骤4)所述反应的时间为10~20h。
优选的,步骤5)所述反应的时间为15~25h。
优选的,步骤7)所述反应的时间为20~30h。
本发明的有益效果是:本发明的双锁型聚合物具有良好的生物相容性和可降解性,由其自组装形成的纳米颗粒可以用于肿瘤成像与治疗。
具体来说:
本发明的双锁型聚合物自组装形成的纳米颗粒,在肿瘤细胞内的内涵体/溶酶体酸性环境中会通过质子化使颗粒内核由疏水性向亲水性转变,颗粒崩解,使小分子肽和荧光分子连接键暴露出来,并且在溶酶体内组织蛋白酶B的作用下,连接键断裂,荧光分子Cy-NH2释放出来,小分子Cy-NH2不仅可以荧光成像,而且能够靶向至肿瘤细胞线粒体,使线粒体活性氧上升,ATP下降,从而杀死肿瘤细胞,达到诊疗一体的目的,具有巨大的临床应用潜能。
附图说明
图1为实施例中的S-PPDPA-VC-Cy的合成路线图。
图2为实施例中的PEG-CPDB的核磁共振氢谱图。
图3为实施例中的PEG-b-(PDPA-co-MAA)的核磁共振氢谱图。
图4为实施例中的Fmoc-Val-Cit-NH-Cy的核磁共振氢谱图。
图5为实施例中的S-PPDPA-VC-Cy的核磁共振氢谱图。
图6为对比例中的inS-PPDPA-VC-Cy的合成路线图。
图7为对比例中的inS-PPDPA-VC-Cy的核磁共振氢谱图。
图8为纳米颗粒在水溶液中的粒径及粒径分布图。
图9为纳米颗粒在酸性条件下和中性条件下酶作用后的荧光光谱图。
图10为S-NP在酸性条件下酶作用不同时间后的荧光光谱图。
图11为S-NP和inS-NP在不同pH环境下酶作用后的荧光光谱图。
图12为S-NP和inS-NP对4T1细胞杀伤效果实验图。
图13为流式检测细胞摄取两种纳米颗粒后细胞凋亡情况图。
图14为激光共聚焦观察4T1细胞和MEF细胞摄取纳米颗粒后的荧光恢复情况图。
图15为激光共聚焦观察肿瘤细胞摄取纳米颗粒后荧光分子的胞内分布情况图。
图16为激光共聚焦观察肿瘤细胞摄取纳米颗粒后胞内ROS和ATP水平情况图。
图17为小动物活体成像情况图。
具体实施方式
下面结合具体实施例对本发明做进一步的解释和说明。
Cy-NH2:参照“Activity-Based Near-Infrared Fluorogenic Probe forEnabling in Vitroand in Vivo Profiling of Neutrophil Elastase,Anal.Chem.2019,91,3877-3884”制备。
Fmoc-Val-NHS:参照“Cathepsin B-Labile Dipeptide Linkers for LysosomalRelease of Doxorubicin from Internalizing Immunoconjugates:Model Studies ofEnzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity,Bioconjugate Chem.2002,13,855-869”制备。
实施例:
一种双锁型聚合物S-PPDPA-VC-Cy(合成路线图见图1),其制备方法包括以下步骤:
1)将0.1mol的二异丙基乙醇胺、0.1mol的三乙胺和0.001mol的对苯二酚分散在100mL的四氢呋喃中,再滴加0.1mol的甲基丙烯酰氯,滴加完后在氩气气氛下室温搅拌反应24h,过滤除去沉淀的三乙胺盐酸盐,旋转蒸发除去四氢呋喃,85℃真空(0.05mmHg)蒸馏,得到2-(二异丙基氨基)甲基丙烯酸乙酯(无色液体);
2)将0.4mmol的mPEG114分散在20mL的无水二氯甲烷中,再加入1.6mmol的4-氰基-4-(硫代苯甲硫基)戊酸和0.2mmol的4-二甲基氨基吡啶,再在冰浴下滴加10mL的二环己基碳二亚胺的二氯甲烷溶液(含二环己基碳二亚胺1.6mmol),滴加完后室温搅拌反应48h,减压浓缩,用乙醚沉淀3次,得到(PEG-CPDB,核磁共振氢谱如图2所示);
3)将0.1mmol的PEG-CPDB、8mmol的2-(二异丙基氨基)甲基丙烯酸乙酯(DPA)和0.02mmol的偶氮二异丁腈分散在5mL的1,4-二氧六环中,60℃反应12h,再加入0.3mmol的甲基丙烯酸,继续反应12h,将产物先后用二甲基甲酰胺和纯水透析,再冷冻干燥,得到(PEG-b-(PDPA-co-MAA),核磁共振氢谱如图3所示);
4)将0.033mol的L-瓜氨酸和3.24mmol的NaHCO3分散在60mL的水中,再加入由0.02mol的(Fmoc-Val-NHS)、60mL的二甲醚和39mL的四氢呋喃组成的混合溶液,搅拌反应16h,再边搅拌边加入103mL质量分数15%的柠檬酸水溶液,反应混合物用2-丙醇/乙酸乙酯(100mL,2-丙醇的质量分数为10%)萃取3次,有机层用水(100mL)洗涤2次,用无水MgSO4干燥,将有机层蒸发至干,得到(Fmoc-Val-Cit,白色粉末状,收率84%);
5)将0.22mmol的Fmoc-Val-Cit和0.34mmol的2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)分散在10mL的二甲基甲酰胺中,再加入0.16mmol的N,N-二异丙基乙胺,室温搅拌反应2h,再加入5mL的(Cy-NH2)的二甲基甲酰胺溶液(含0.088mmol的Cy-NH2),室温搅拌反应16h,减压除去溶剂,再将粗产物通过硅胶柱色谱法纯化,得到(Fmoc-Val-Cit-NH-Cy,收率40%,核磁共振氢谱如图4所示);
7)将0.02mmol的PEG-b-(PDPA-co-MAA)、0.024mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和0.02mmol的N-羟基琥珀酰亚胺分散在2mL的二甲基甲酰胺中,再将得到的混合液加入步骤6)的反应液中,室温反应24h,将产物先后用二甲基甲酰胺和纯水透析,再冷冻干燥,得到(S-PPDPA-VC-Cy,核磁共振氢谱图如图5所示)。
对比例:
一种双锁型聚合物inS-PPDPA-VC-Cy(合成路线图见图6),其制备方法包括以下步骤:
性能测试:
一、纳米颗粒的制备:
通过纳米沉淀法制备纳米颗粒,具体方法如下:
将10mg的S-PPDPA-VC-Cy分散在1mL的四氢呋喃中,再边搅拌边滴加10mL的超纯水,滴加完后搅拌2h,将得到的颗粒溶液转移到透析袋(MWCO 7000)中,在超纯水中透析24h除去四氢呋喃,再将颗粒溶液用0.45μm过滤器过滤,得到的纳米颗粒标记为S-NP。同样,将S-PPDPA-VC-Cy替换成inS-PPDPA-VC-Cy,经过相同的制备过程得到的纳米颗粒标记为inS-NP。
二、双锁型聚合物S-PPDPA-VC-Cy的酸响应性:
纳米颗粒的酸响应:
将S-NP和inS-NP分别加入pH=5.0、5.5、6.0、6.5、6.8、7.4的0.2mol/L的PBS缓冲液(模拟内涵体/溶酶体酸性微环境)中,孵育培养10min,测试纳米颗粒在水溶液中的粒径及粒径分布,测试结果如图8所示。
由图8可知:随着pH的下降S-NP的尺寸减小到10nm左右,而inS-NP的尺寸变化很小。
三、双锁型聚合物S-PPDPA-VC-Cy的体外荧光光谱:
1)纳米颗粒在酸性条件下和中性条件下酶作用后的荧光光谱:
将S-NP和inS-NP分别加入pH=5.5和pH=7.4的0.2mol/L的PBS缓冲液(模拟内涵体/溶酶体酸性微环境)中,并加入木瓜蛋白酶(2μM),孵育培养12h,S-NP和inS-NP在酸性条件下酶作用后的荧光光谱图如图9所示。
由图9可知:S-NP在酸性条件下酶作用后有明显的荧光恢复,而中性条件下未有荧光恢复,并且在相同条件下inS-NP不存在荧光恢复。
2)纳米颗粒在酸性条件下酶作用不同时间后的荧光光谱:
将S-NP加入pH=5.5的0.2mol/L的PBS缓冲液(模拟内涵体/溶酶体酸性微环境)中,并加入木瓜蛋白酶(6μM),孵育培养0、0.5h、1.0h、1.5h、2.0h、2.5h、3.0h、3.5h、4.0h,S-NP在酸性环境中酶作用不同时间后的荧光光谱图如图10所示。
由图10可知:S-NP在酸性条件随着酶作用后时间的延长,荧光逐渐增强。
3)纳米颗粒在不同pH条件下酶作用后的荧光光谱:
将S-NP和inS-NP分别加入pH=5.0、5.5、6.0、6.5、6.8、7.4的0.2mol/L的PBS缓冲液(模拟内涵体/溶酶体酸性微环境)中,并加入木瓜蛋白酶(2μM),孵育培养12h,S-NP和inS-NP在不同pH环境下酶作用后的荧光光谱图如图11所示。
由图11可知:S-NP在酸性条件下酶作用后有明显的荧光恢复,而中性条件下未有荧光恢复,并且在相同条件下inS-NP不存在荧光恢复。
四、双锁型聚合物的体外细胞实验:
1)S-NP和inS-NP对小鼠乳腺癌细胞(4T1)和小鼠成纤维细胞(MEF)的杀伤效果实验:
将MEF或4T1细胞接种于96孔组织培养板(1×104细胞/孔)中培养12h,然后在所需的Cy-NH2、Val-Cit-NH-Cy、S-NP或inS-NP浓度下培养细胞,培养24h后用标准的商用甲基噻唑四唑蓝(MTT)法测定细胞活性;流式细胞术分析细胞凋亡;4T1细胞(每孔1×105细胞)在24孔培养板上培养12h,然后用PBS、Cy-NH2、Cy-NH2+Vc(维生素C)、S-NP、S-NP+Vc、S-NP+inhibitor(CA-074组织蛋白酶B抑制剂)、inS-NP、inS-NP+inhibitor等剂量(Cy-NH2:6μg/mL,抑制剂:10mM,Vc:50μM)处理4T1细胞6h;使用膜联蛋白V FITC和碘化丙啶的死细胞凋亡试剂盒(KeyGEN BioTECH,中国)检测细胞凋亡水平;最后,将细胞用PBS缓冲液洗涤3次,收集并通过流式细胞术分析,S-NP和inS-NP对4T1细胞杀伤效果实验图和流式检测细胞摄取两种纳米颗粒后细胞凋亡情况图分别如图12和图13所示。
由图12和图13可知:在7个浓度梯度下,游离的Cy-NH2对4T1和MEF细胞的杀伤效果最强,但是相对于正常细胞,Cy-NH2对肿瘤细胞的杀伤效果更好,另外S-NP对肿瘤细胞的杀伤效果比inS-NP的效果更好,对正常细胞MEF的杀伤效果较弱;流式结果与MTT结果一致。
2)激光共聚焦观察肿瘤细胞摄取纳米颗粒后的荧光恢复实验:
细胞共聚焦荧光成像:将MEF和4T1细胞(每孔2×105细胞)在玻璃盖玻片上培养12h,4T1细胞用S-NP、S-NP+inhibitor、inS-NP、inS-NP+inhibitor等剂量(Cy-NH2:6μg/mL)处理6h,MEF细胞用S-NP、inS-NP等剂量(Cy-NH2:6μg/mL)处理6h;用PBS缓冲液洗涤3次,用4%甲醛溶液固定细胞;最后,用CLSM观察细胞,激光共聚焦观察肿瘤细胞摄取纳米颗粒后的荧光恢复情况图如图14所示。
由图14可知:S-NP经细胞摄取胞内有明显的荧光,而添加了组织蛋白酶抑制剂的组和inS-NP组细胞内无明显的荧光,说明在胞内溶酶体酸性环境和组织蛋白酶B的存在下,S-NP在胞内才会有荧光的恢复。
3)激光共聚焦观察肿瘤细胞摄取纳米颗粒后荧光分子的胞内分布:
将4T1细胞(3×105细胞/孔)在35mm共焦培养皿中培养12h,然后用S-NP(Cy-NH2:6μg/mL)培养4h,然后在37℃下用Lyso-Tracker Green DND-26(150nM)染色2h,线粒体在37℃下用Mito-Tracker Green FM(100nM)染色0.5h,用PBS缓冲液洗涤三次,然后置于无血清的新鲜培养基中,用CLSM观察细胞,激光共聚焦观察肿瘤细胞摄取纳米颗粒后荧光分子的胞内分布情况图如图15所示。
由图15可知:S-NP在经细胞摄取后通过酸性条件和组织蛋白酶B响应后释放出Cy-NH2,Cy-NH2会迅速靶向至线粒体,在细胞线粒体处富集。
4)激光共聚焦观察肿瘤细胞摄取纳米颗粒后胞内活性氧(ROS)和5'-三磷酸腺苷(ATP)水平:
在96孔黑色组织培养板中培养4T1细胞(1×106细胞/孔),用PBS、Cy-NH2、Cy-NH2+Vc、S-NP、S-NP+Vc、S-NP+inhibitor、inS-NP、inS-NP+inhibitor处理6h,将细胞用PBS缓冲液冲洗3次,然后用2',7'-二氯荧光黄双乙酸盐(DCFH-DA)(20μM)染色,使用微孔板系统(DCF:Ex/Em=480/525nm)分析ROS水平,激光共聚焦观察肿瘤细胞摄取纳米颗粒后胞内ROS和ATP水平情况图如图16所示。
线粒体膜电位测定:使用JC-1探针评估4T1细胞中的线粒体去极化。将细胞在35mm共聚焦培养皿中培养(3×106个细胞/孔)12h,然后用PBS、Cy-NH2、Cy-NH2+Vc、S-NP、S-NP+Vc、S-NP+inhibitor处理,处理12h后,将细胞与等体积的含JC-1染料(5mg/L)的无血清培养基在37℃下孵育20min。染色结束时,将探针用PBS缓冲液洗涤3次,然后置于无血清的新鲜培养基中。最后,通过CLSM观察细胞。
细胞ATP水平的测量:将4T1细胞(每孔1×106个细胞)在24孔板上培养12h,然后用PBS、Cy-NH2、Cy-NH2+Vc、S-NP、S-NP+Vc、S-处理NP+inhibitor、inS-NP、inS-NP+inhibitor12h。之后,将细胞用PBS缓冲液洗涤3次,在37℃下用胰蛋白酶消化1min,然后加入RPMI1640培养基以终止反应和通过离心收集细胞。使用5'-三磷酸腺苷(ATP)生物发光分析试剂盒和微孔板系统评估ATP水平。
由图16可知:S-NP经细胞摄取以后能够使细胞内的ROS水平上升,胞内ATP的水平下降,通过破坏线粒体膜电位影响细胞活性,最终杀死肿瘤细胞。
五、动物水平实验:
小动物活体成像:
将荷瘤小鼠随机分组(n=3),当肿瘤体积达到200mm3时,静脉注射S-NP(Cy-NH2:1.25mg/kg,100μL),S-NP+inhibitor(Cy-NH2:1.25mg/kg,100μL,抑制剂:5mg/kg,腹腔注射),inS-NP(Cy-NH2:1.25mg/kg,100μL),inS-NP+inhibitor(Cy-NH2:1.25mg/kg,100μL,抑制剂:5mg/kg,腹腔注射)。所有小鼠分别于注射前0h、2h、4h、8h、12h、24h和36h,用Xtreme(德国布鲁克)活体荧光仪采集全身荧光图(Ex/Em=650/700nm)。给药后36h处死小鼠,采集主要脏器(肝、肾、肺、脾、心)和肿瘤组织,进行Cy-NH2分布的离体检测,小动物活体成像情况图如图17所示。
由图17可知:S-NP组可以很好的富集在肿瘤部位,并且其他对照组不能在肿瘤部位有很好的成像效果。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (9)
2.权利要求1所述的聚合物的制备方法,其特征在于,包括以下步骤:
1)进行二异丙基乙醇胺和甲基丙烯酰氯的反应,得到2-(二异丙基氨基)甲基丙烯酸乙酯;
3.根据权利要求2所述的制备方法,其特征在于:步骤1)所述二异丙基乙醇胺、甲基丙烯酰氯的摩尔比为1:(1.0~1.2)。
4.根据权利要求2所述的制备方法,其特征在于:步骤2)所述甲氧基聚乙二醇、4-氰基-4-(硫代苯甲酰)戊酸的摩尔比为1:(4~5)。
5.根据权利要求2所述的制备方法,其特征在于:步骤3)所述PEG-CPDB、2-(二异丙基氨基)甲基丙烯酸乙酯、甲基丙烯酸的摩尔比为1:(80~90):(3~4)。
9.权利要求1所述的聚合物用于制备抗肿瘤药物的应用。
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