CN116162255B - 一种电荷可转变型树状聚合物及其制备方法 - Google Patents
一种电荷可转变型树状聚合物及其制备方法 Download PDFInfo
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- CN116162255B CN116162255B CN202211628900.1A CN202211628900A CN116162255B CN 116162255 B CN116162255 B CN 116162255B CN 202211628900 A CN202211628900 A CN 202211628900A CN 116162255 B CN116162255 B CN 116162255B
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Classifications
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
本发明属于高分子材料技术领域,具体为一种电荷可转变型树状聚合物及其制备方法。本发明首先以氨基引发L‑苯丙氨酸‑N‑羧酸内酸酐开环聚合迭代地形成外围含有很多氨基的三代树状聚合物,再以其氨基引发三(苄氧羰基)‑L‑精氨酸‑N‑羧酸内酸酐和三乙二醇单甲醚功能化的L‑半胱氨酸‑N‑羧酸内酸酐聚合,脱除聚精氨酸侧链保护基后得到内核为聚苯丙氨酸、中间层为聚精氨酸、最外层为三乙二醇单甲醚功能化聚半胱氨酸的树状共聚物。所得树状聚合物具有良好的水溶性,在37℃时显电中性,在43.5℃以上时显正电性,具有良好的电荷转变能力。另外,该聚合物能有效负载疏水性药物阿霉素,可作为靶向药物的载体。
Description
技术领域
本发明属于高分子材料技术领域,具体涉及一种电荷可转变型树状聚合物及其制备方法。
背景技术
树状聚合物具有明显的空间结构,分子结构规整,表面官能团多,而且内部微孔含量非常丰富。另外,与传统的组装型纳米粒(比如:胶束和囊泡)不同,树状大分子依靠共价键结合在一起。通过调节结构和化学组份,它在水溶液中能以单分子胶束的形态存在(K.Zheng, J. Ren, J. He. Macromolecules, 2019, 52, 6780−6791),这极大地增强了这类胶束对外界环境变化(比如:温度、浓度和pH等)的耐受性。因此,其在药物运载领域具有广阔的应用前景(A. P. Dias, S. D. S. Santos, J. V. D. Silva, R. Parise-Filho, E.I. Ferreira, O. E. Seoud, J. Giarolla. Int. J. Pharmaceut., 2020, 573,118814)。目前,已经有一些树状聚合物药剂进入了临床试验和实用阶段,比如:以PAMAM为载体的Stratus® CS Acute Care TM已经在临床上用于快速检测心肌缺血。另外,树状聚赖氨酸(dendrigraft poly(L-lysine), DGL)也已经商品化,在基因治疗领域展示出了良好的应用前景(N. Hegde, V. Velingkar, B. Prabhakar. Int. J. Pept. Res. Ther.,2019, 25, 1539–1562)。
众所周知,延长血液循环时间的同时,增强其对肿瘤细胞的靶向能力是当前药物载体设计的重要原则。通常来说,在血液循环中使载药纳米粒呈电中性或电负性,待其凭高通透性和滞留效应(Enhanced permeability and retention effect, EPR effect)到达肿瘤组织后,再让它迅速地转变为正电性,可同时赋予药物血液长循环和肿瘤细胞靶向能力(H. X. Wang, Z. Q. Zuo, J. Z. Du, Y. C. Wang, R. Sun, Z. T. Cao, X. D. Ye,J. L. Wang, K. W. Leong, J. Wang. Nano Today, 2016, 11, 133–144)。但是,在现在还没有关于电荷可转变型树状聚合物的报道。因此,以目前报道的这些树状聚合物作为药物载体时,往往需要添加靶向基团(W. Cao, J. Zhou, A. Mann, Y. Wang, L. Zhu.Biomacromolecules 2011, 12, 2697–2707),仅凭载体难以同时提高药物的血液长循环时间和肿瘤细胞靶向性,这增加了载体制备的步骤,不利于树状聚合物在载药领域的应用。
发明内容
本发明的目的在于提供一种具有电荷转变功能的树状聚合物及其制备方法,该树状聚合物在37℃时显电中性,而在高于43.5℃时显正电性。
本发明提供的具有电荷转变功能的树状聚合物的制备方法,具体步骤为:
(1)以L-苯丙氨酸和三光气为原料,在蒎烯作用下合成L-苯丙氨酸-N-羧酸内酸酐,记为L-Phe NCA;
(2)以三(苄氧羰基)-L-精氨酸和二氯甲基甲醚为原料,在二氯甲烷溶液中合成三(苄氧羰基)-L-精氨酸-N-羧酸内酸酐,记为Z2-Arg NCA;
(3)以三乙二醇单甲醚和甲基丙烯酰氯为原料,在二氯甲烷溶液中合成甲基丙烯酰三乙二醇单甲醚酯,记为EG3MA;以EG3MA与L-半胱氨酸反应制备功能性半胱氨酸,记为EG3MA-C;其在三光气和蒎烯作用下生成三乙二醇单甲醚功能化的L-半胱氨酸-N-羧酸内酸酐,记为EG3MA-C NCA;
(4)以N-(叔丁氧基羰基)-L-半胱氨酸甲酯与磺酰氯反应,所得产物同Boc-L-半胱氨酸反应制备N, N'-双(叔丁氧羰基)-L-胱氨酸单甲醚,记为N, N’-diBoc-L-CME;
(5)在密闭受氮气保护的玻璃容器中,以胱胺为引发剂引发L-Phe NCA聚合,形成分子链中间含有一个二硫键,两端均为氨基的聚苯丙氨酸,记为G0;该分子链末端的氨基与N, N’-diBoc-L-CME的羧基发生缩合反应,经TFA脱除Boc保护基团后,得到分子链起、始端各含两个氨基的聚苯丙氨酸,记为G0-4NH2;
(6)以G0-4NH2为引发剂,引发L-Phe NCA聚合后,分子链末端的氨基再与N, N’-diBoc-L-CME的羧基发生反应,并用TFA脱除Boc保护基团,得到分子链末端含有8个氨基的第一代树状聚合物,记为G1-8NH2;
(7)以G1-8NH2为引发剂,引发L-Phe NCA聚合后,分子链末端的氨基再与N, N’-diBoc-L-CME的羧基发生反应,并用TFA脱除Boc保护基团,得到分子链末端含有16个氨基的第二代树状聚合物,记为G2-16NH2;
(8)以G2-16NH2为引发剂,引发L-Phe NCA聚合后,分子链末端的氨基再与N, N’-diBoc-L-CME的羧基发生反应,并用TFA脱除Boc保护基团,得到分子链末端含有32个氨基的第三代树状聚合物,记为G3-32NH2;
(9)以G3-32NH2中的氨基引发Z2-Arg NCA和EG3MA-C NCA连续聚合,得到内核为聚苯丙氨酸、中层为含保护基聚精氨酸、最外层三乙二醇单甲醚改性聚半胱氨酸的树状聚合物(dendrimer-like poly(amido acid)s),记为,DLPAA。
本发明步骤(1)中,优选地,L-苯丙氨酸与三光气和蒎烯的摩尔比为1:(2.0-3.0):(2.0-3.0)。
本发明步骤(2)中,优选地,三(苄氧羰基)-L-精氨酸与二氯甲基甲醚的摩尔比为1:(1.4-1.6)。
本发明步骤(3)中,优选地, 三乙二醇单甲醚与甲基丙烯酰氯的摩尔比为1:(1.1-1.2),半胱氨酸与EG3MA的摩尔比为1:(1.4-1.6);EG3MA-C与三光气和蒎烯的摩尔比为1:(2.0-3.0):(2.0-3.0)。
本发明步骤(4)中,优选地,N-(叔丁氧基羰基)-L-半胱氨酸甲酯与磺酰氯的摩尔比为1:(1.1-1.2);其产物与Boc-L-半胱氨酸反应的摩尔比为1:(1.0-1.05)。
本发明步骤(5)中,优选地,胱胺与L-Phe NCA的摩尔比为1:(8-10);氨基与N, N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上。
本发明步骤(6)中,优选地,G0-4NH2中氨基的量与L-Phe NCA的摩尔比为1:(15-20);
聚合物与N'-diBoc-L-CME的羧基发生反应时,其所含氨基与N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上。
本发明步骤(7)中,优选地,G1-8NH2中氨基的量与L-Phe NCA的摩尔比为1:(15-20);聚合物与N'-diBoc-L-CME的羧基发生反应时,其所含氨基与N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上。
本发明步骤(8)中,优选地,G2-16NH2中氨基的量与L-Phe NCA的摩尔比为1:(15-20);聚合物与N'-diBoc-L-CME的羧基发生反应时,其所含氨基与N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上。
本发明步骤(9)中,优选地,G3-32NH2中氨基的量与Z2-Arg NCA和EG3MA-C NCA的摩尔比为1:(40-60):(2-3.5);HBr的摩尔量是保护基团的3倍以上。
本发明中,所制备的树状聚合物可以负载疏水药物,比如:阿霉素、紫杉醇、索拉菲尼等。其中:
负载阿霉素的具体流程为:
首先,准确称取10 mg的树状聚合物于50 mL圆底烧瓶中,加入8-10 mL DMSO使其完全溶解,再加入150-300 mg的阿霉素(DOX);
其次,把上述溶液逐滴加入到剧烈搅拌的磷酸盐缓冲溶液中(pH: 6.8, 100 mL),用透析的方法,除去溶液中的DMSO和未被包裹的DOX;
最后,用冷冻干燥的方法,得到载药纳米粒。
负载紫杉醇的具体流程为:
首先,准确称取10 mg的树状聚合物于50 mL圆底烧瓶中,加入8-10 mL DMSO使其完全溶解,再加入100-200 mg的紫杉醇;
其次,把上述溶液逐滴加入到剧烈搅拌的磷酸盐缓冲溶液中(pH: 6.8, 100 mL),用透析的方法,除去溶液中的DMSO和未被包裹的紫杉醇;
最后,用冷冻干燥的方法,得到载药纳米粒。
负载索拉菲尼的具体流程为:
首先,准确称取10 mg的树状聚合物于50 mL圆底烧瓶中,加入8-10 mL DMSO使其完全溶解,再加入200-300 mg的索拉菲尼;
其次,把上述溶液逐滴加入到剧烈搅拌的磷酸盐缓冲溶液中(pH: 6.8, 100 mL),用透析的方法,除去溶液中的DMSO和未被包裹的索拉菲尼;
最后,用冷冻干燥的方法,得到载药纳米粒。
本发明中,所用到的有机溶剂都是用氢化钙除水后再通过减压蒸馏得到,以便除去溶剂中的水分。
本发明制备的聚合物用如下方法进行表征:
NMR:AVANCE III HD 400MHz型(Bruker公司),测试温度为25℃;
DMF流动相凝胶色谱仪:流动相为含有LiBr的DMF(1.6 g/L),洗脱液流速为1.0mL/min,色谱柱温度为50℃,用PMMA标定色谱柱标准曲线,测定相对分子量(M n)。系统上也连接了美国公司生产的多角度光散射系统用于测定聚合物样品的绝对分子量(M w, MALLS);
ALV动静一体光散射仪:用互相关模式,在90º散射角检测样品的流体力学半径(R H);
Cyro-TEM:荷兰FEI公司生产的Tecnai G20型,加速电压200 kV,点分辨率0.27nm,线分辨率0.14 nm。使用Vitrobot冷冻制样设备和Gatan 626冷冻转移系统。样品的水溶液浓度为0.5 mg/mL。
本发明以氨基酸为原料,首先合成氨基酸-N-羧酸内酸酐,再利用开环聚合和发散的方法合成树状聚合物,所得聚合物分子链为聚氨基酸,这有助于提高载体与生物体的相容性;聚合物接枝点处含有二硫键,有助于其再体内降解。
本发明合成树状聚合物,其内核是聚苯丙氨酸,中间层是聚精氨酸,最外层为少量三乙二醇单甲醚改性的聚半胱氨酸。其最外层不带电、亲水且具有温敏性,中间层显正电性,内核为疏水性结构,其特殊的结构不仅能负载疏水性化合物,而且阻碍了分子团聚,在37℃水溶液中以单分子胶束的形成存在,具有良好的水溶性,且整个胶束显电中性。温度升高至43.5℃以上时,整个分子显正电性,有良好的电荷转变能力。在水溶液中的流体力学半径为(R H)为19.6 nm,绝对分子量为956.51×103 g/mol。
本发明合成的树状聚合物之所以具有电荷转变能力,是因为在高温下,最外层分子中三乙二醇单甲醚与水的氢键被破坏,从而使其转变为疏水状态并向内折叠。此时,中间层的聚精氨具有亲水性,更容易向外突出,从而使整个粒子显正电性。
本发明合成的树状聚合物,其形成的单分子胶束可以负载阿霉素有效地进入肝癌细胞,起到了载药进入癌细胞的功效。
附图说明
图1为L-Phe NCA的合成路线。
图2为Z2-Arg NCA的合成路线。
图3为EG3MA-C NCA的合成路线。
图4为N, N'-diBoc-L-CME的合成路线。
图5为氨基酸型树状聚合物(DLPAA)的合成路线。
图6为G0, G1-8NH2、G2-16NH2、G3-32NH2、DLPAA的凝胶渗透色谱(GPC)检测结果(以DMF为流动相)。
图7为DLPAA在水溶液中的流体力学半径分布曲线(PDI代表粒径分布)。
图8为DLPAA在水溶液中的冷冻透射电镜图片。
图9为树状聚合物DLPAA的Zeta电位随温度变化的曲线。
图10 激光共聚焦显微镜观察DLPAA载药纳米粒在不同温度下进入肝癌细胞能力的结果(孵育时间为1 h)。
图11为L-胱氨酸-N-NCA合成路线图。
图12为实施例2中超支化型树状聚合物HBPAA的合成路线图。
图13为HBPAA合成过程的GPC测试图。
图14超支化型树状聚合物HBPAA的Zeta电位随温度变化的曲线。
图15为树状聚合物电荷转变的原理图。
具体实施方式
下面通过具体实施例进一步介绍本发明。
实施例1:
(1)合成L-Phe NCA(参见图1)
① 在氩气保护下,往干燥的三口烧瓶中加入10 g L-苯丙氨酸(60.5 mmol)、18 g三光气(60.5 mmol)和120 mL干燥的THF,磁力搅拌10 min;
② 往上述反应瓶中加入19.2 mL (+)-alpha-pinene(121 mmol),并逐步升温至40℃;在氩气保护下,反应5 h;
③ 在干燥箱中,利用减压蒸馏技术,蒸干反应液中的THF;往瓶中加入50 mL干燥的THF,待固体物完全溶解,再蒸干THF,以除去反应过程中产生的HCl;最后,加入50 mL干燥的THF,使固体物完全溶解;
④ 在干燥箱中,把产物的THF溶液滴加入剧烈搅拌的干燥正己烷(500 mL)中,减压抽滤;
⑤ 把产物装入史莱克瓶中,在真空线上抽真空干燥后,充入氩气,密封;放入干燥器中后,在-20℃冰箱中保存。
(2)合成Z2-Arg NCA(参见图2)
① 在氩气保护下,往装有球形冷凝管的三口烧瓶中加入 2 g 三(苄氧羰基)-L-精氨酸(3.5 mmol)和100 mL干燥的CH2Cl2;在磁力搅拌下,使三(苄氧羰基)-L-精氨酸溶解后,往反应瓶中加入0.5 mL二氯甲基甲醚(5.5 mmol);
② 油浴锅逐步升温至48℃,反应36 h;
③ 在干燥箱中,用蒸馏装置除去反应瓶中的CH2Cl2,再往反应瓶中加入20 mL干燥的THF,使产物完全溶解;
④ 在干燥箱中,把产物的THF溶液滴加入剧烈搅拌的干燥正己烷(200 mL)中,减压抽滤;
⑤ 把产物装入史莱克瓶中,在真空线上抽真空干燥后,充入氩气,密封;放入干燥器中后,在-20℃冰箱中保存。
(3)合成EG3MA-C NCA(参见图3)
① 往500 mL 三口烧瓶中加入120 mL干燥CH2Cl2、40 mL三乙胺(249.6 mmol)三乙二醇单甲醚、34.6 mL(249.6 mmol)干燥三乙胺和1.52 g DMAP(12.5 mmol),置于冰水混合物中搅拌20 min;
② 配制含有24.08 mL(249.6 mmol)甲基丙烯酰氯和40 mL干燥CH2Cl2溶液,并缓慢滴加入上述三口烧瓶中;滴加结束后,在室温条件下,搅拌反应过夜;
③ 所得反应液用25 mL 1 mol/L HCl水溶液洗2次后,再用饱和NaHCO3水溶液洗2次,最后用饱和食盐水洗2次;用无水硫酸镁干燥后,过滤除去硫酸镁;用旋转蒸法仪除去溶剂后,用1H NMR表征该产物(EG3MA);
④ 往100 mL圆底烧瓶中加入5 g L-cysteine(31.7 mmol)和50 mL去离子水,在磁力搅拌作用下,用1 mol/L NaOH水溶液调节其pH至7.5;
⑤ 往上述圆底烧瓶中加入8.1 g EG3MA(34.9 mmol),在室温环境中搅拌过夜;
⑥ 反应液用乙酸乙酯洗三次(50 mL/次),除去多余的EG3MA,收集水溶液;用旋转蒸发仪把水溶液浓缩至20 mL后,用冷冻干燥机干燥;
⑦ 往三口烧瓶中加入2 g EG3MA-C(5.7 mmol)、3.4 g三光气(11.4 mmol)和40mL干燥THF,磁力搅拌10 min;
⑧ 往上述反应瓶中加入1.8 mL (+)-alpha-pinene(11.4 mmol),并逐步升温至50℃;在氩气保护下,反应5 h;
⑨ 在干燥箱中,利用减压蒸馏技术,蒸干反应液中的THF;往瓶中加入10 mL干燥的THF,待固体物完全溶解,再蒸干THF,以除去反应过程中产生的HCl;最后,加入10 mL干燥的THF,使固体物完全溶解;
⑩ 在干燥箱中,把产物的THF溶液滴加入剧烈搅拌的干燥正己烷(100 mL)中,减压抽滤;
⑪ 把产物装入史莱克瓶中,在真空线上抽真空干燥后,充入氩气,密封;放入干燥器中后,在-20℃冰箱中保存。
(4)合成N, N'-diBoc-L-CME(参见图4)
① 往史莱克瓶中加入7 g N-(叔丁氧基羰基)-L-半胱氨酸甲酯(30 mmol)和30mL干燥的CH2Cl2,冻融脱气3次后,充入氩气,置于冰水混合物中;
② 用注射器往反应瓶中逐滴加入4.5 g 磺酰氯(33 mmol)后,磁力搅拌反应3 h;
③ 配制含有6.6 g Boc-L-半胱氨酸(30 mmol)、5 g NaHCO3(60 mmol)和150 mL的甲醇悬浮液,冻融脱气三次;然后用注射器把该悬浮液加入到上述反应液中,在磁力搅拌下反应2 h;
④ 往反应瓶中再加入100 mL甲醇,先用减压过滤的方法除去主要的不溶物,再用0.22 μm滤头过滤除去残存的微量不溶物;
⑤ 用旋转蒸发仪除去甲醇,所得样品用乙醚洗三次,最后置于真空干燥箱中干燥。
(5)合成树状聚合物DLPAA(参见图5)
采用发散法,逐步地合成氨基酸类树状聚合物;具体步骤如下:
① 往连有双排管的体系中加入17.20 g (90 mmol) L-PheNCA,抽真空充氮气三次,使整个体系在氮气的保护氛围中;然后,往反应瓶中加入80 mL干燥的四氢呋喃;在磁力搅拌作用下使L-Phe NCA完全溶解,加入1.52 g(10 mmol)胱胺;开启油浴加热至30℃,搅拌反应12 h;
② 把反应液滴加入400 mL 正己烷中,离心后再用正己烷洗三次,放入真空干燥箱中30℃烘干,记为G0(M w, MALLS = 1.31×103 g/mol,GPC表征结果参见图6);
③ 取10g G0(约含有15.37 mmol氨基)和22.71 g (50 mmol)N, N'-diBoc-L-CME于250 mL圆底烧瓶中,加入100 mL四氢呋喃使其完全溶解后,再加入5.75 g(50 mmol)N-羟基丁二酰亚胺(NHS)和7.76 g(50 mmol)1-(3-二甲基氨丙基)-3-乙基碳酰亚胺(EDC);在50℃条件下磁力搅拌12 h后,把反应液滴加入300 mL正己烷中沉淀,离心分离后,在30℃真空干燥箱中烘干;把所得样品加入到100 mL圆底烧瓶中,用50 mL二氯甲烷溶解后,再加入10mL三氟乙酸,在室温下磁力搅拌3 h;用旋转蒸发仪除去二氯甲烷后,再用20 mL四氢呋喃溶解;把聚合物的四氢呋喃溶液滴加入100 mL正己烷中后,离心分离后,所得样品(G0-4NH2,M w, MALLS = 1.50×103 g/mol)放入真空烘箱中干燥;
④ 准确称量45.73 g L-PheNCA(239.40 mmol)于500 mL连有双排管的烧瓶中,抽真空充氮气三次后加入300毫升干燥的THF,然后再加入5 g G0-4NH2(约含有13.33 mmol氨基),开启油浴加热至30℃,搅拌反应24 h;把反应液滴加入1200 mL 正己烷中,减压抽滤后,所得聚合物(M w, MALLS =9.16×103 g/mol)放入真空干燥箱中30℃烘干; 取10 g干燥聚合物(约含有4.40 mmol氨基)和6.81 g(15 mmol)N, N'-diBoc-L-CME于250 mL圆底烧瓶中,加入60 mL四氢呋喃使其完全溶解后,再加入1.73 g(15 mmol)N-羟基丁二酰亚胺(NHS)和2.38 g(15 mmol)1-(3-二甲基氨丙基)-3-乙基碳酰亚胺(EDC);在50℃条件下磁力搅拌12 h后,把反应液滴加入200 mL正己烷中沉淀,离心分离后,在30℃真空干燥箱中烘干;把所得样品加入到100 mL圆底烧瓶中,用50 mL二氯甲烷溶解后,再加入10 mL三氟乙酸,在室温下磁力搅拌3 h;用旋转蒸发仪除去二氯甲烷后,再用20 mL四氢呋喃溶解;把聚合物的四氢呋喃溶液滴加入100 mL正己烷中后,离心分离;所得样品(G1-8NH2, M w, MALLS = 9.33×103 g/mol,GPC表征结果在图6中展示)放入真空烘箱中干燥;
⑤ 准确称量45.73 g(239.40 mmol)于500 mL连有双排管的烧瓶中,抽真空充氮气三次后加入300毫升干燥的THF,然后再加入15.51 g G1-8NH2(约含有13.33 mmol氨基),开启油浴加热至30℃,搅拌反应24 h;把反应液滴加入1200 mL 正己烷中,减压抽滤后,所得聚合物(M w, MALLS =24.61×103 g/mol)放入真空干燥箱中30℃烘干; 用与第④步相同的方法和添加比例使其与N, N'-diBoc-L-CME反应,并用10 mL TFA脱去保护基得到G2-16NH2(M w, MALLS =24.90×103 g/mol,GPC表征结果在图6中展示);
⑥ 重复第⑤步反应得到G3-32NH2(M w, MALLS= 488.96×103 g/mol,GPC表征结果在图6中展示);
⑦ 准确称量7.73 g(16.50 mmol)Z2-Arg NCA于100 mL连有双排管的烧瓶中,抽真空充氮气三次后加入50毫升干燥的DMF ,然后再加入5 g G3-32NH2(约含有0.33 mmol氨基);磁力搅拌反应72 h后,滴入500 mL甲基叔丁基醚中沉淀;减压过滤后,所得样品(M w, MALLS= 938.43×103 g/mol)放入真空烘箱干燥;
⑧ 准确称量5 g第⑦步所得样品(约含有0.17 mmol氨基)于100 mL连有双排管的烧瓶中,抽真空充氮气三次后加入30毫升干燥的DMF,然后再加入0.19 g (0.51 mmol)EG3MA-C NCA,磁力搅拌反应12 h后,滴入300 mL甲基叔丁基醚中沉淀;减压过滤后,所得样品(Mw, MALLS= 956.62×103 g/mol)放入真空烘箱干燥;把所得样品加入100 mL圆底烧瓶,加入50 mL二氯甲烷使其完全溶解,然后再加入10 mL HBr的冰醋酸溶液(33%),磁力搅拌2 h后,装入透析袋(截留分子量为3500 g/mol)对水透析三次;所得聚合物水溶液冷冻干燥后,放入冰箱保存, 记为DLPAA(Mw, MALLS= 956.51×103 g/mol,GPC表征结果在图6中展示);
所得树状聚合物DLPAA在水中能完全溶解,其粒径分布如图7所示,从中可以看出其流体力学半径为19.7 nm;图8为DLPAA的冷冻电镜图片,图中分散着一些球状粒子,半径约为18 nm,粒径大小与动态光散射测试结果(图7)相符;图9展示了DLPAA随温度变化,电位发生的变化;从图中可以看出,在低于42.5℃时,其电位值几乎为0,而温度高于43.5℃时,展示出了约10 mV的正电性,这说明随着温度的转变,粒子表面由电中性转变为了正电性。
(6)载药纳米粒的制备以及其进入肝癌细胞能力的实验
以负载阿霉素为例:
首先,准确称取适量的本发明制备的树状聚合物于不同的圆底烧瓶中,加入10 mLDMSO使其完全溶解,再加入适量的阿霉素(DOX),(聚合物与DOX的质量比为1:20);其次,把上述溶液逐滴加入到剧烈搅拌的PBS(pH = 6.8, 100 mL)溶液中,用透析的方法,除去溶液中的DMSO和未被包裹的DOX;最后,用冷冻干燥的方法,得到载药纳米粒;
将肝癌细胞以5×105个/孔的密度接种于5个6孔板中,培养24 h;分别往不同的6孔板中加入浓度为50 µg/mL的载药纳米粒子水溶液;随后,在37℃和43℃条件下,分别孵育0.25、0.5和 1 h,并用共聚焦显微镜观察;从图10可以看出,在37℃时,DLPAA负载的DOX几乎不能进入肝癌细胞,而在43.5℃时,该载药纳米粒可以有效地进入肝癌细胞。
按上述类似操作,同样可以负载紫杉醇、索拉菲尼,并有同样的效果。具体操作不重复。
实施例2:
(1)合成L-胱氨酸-N-羧酸内酸酐(图11)
准确称量10.0 g(41.6 mmol)L-胱氨酸和43.5 g(145.6 mmol)三光气于500 mL干燥的圆底烧瓶中,置换成氩气气氛后,加入300 mL干燥四氢呋喃,剧烈搅拌形成悬浮液;放入60℃油浴中加热并磁力搅拌3 h,反应液变为澄清透明;待反应液降至室温后,逐滴加入到1 L干燥的正己烷中,减压过滤后用THF作为良溶剂,正己烷作为不良溶剂重结晶三次;减压过滤后,放入30℃真空烘箱中干燥,所得L-胱氨酸-N-羧酸内酸酐记为L-Cystine NCA。
(2)合成超支化型树状聚合物(HBPAA)(图12)
① 准确称量0.5 g(1.34 mmol)EG3MA-C NCA于连有双排管的玻璃瓶中,抽真空充氮气三次,把整个体系置换为氮气气氛后,往其中加入50 mL干燥DMF和58.75 μL(0.45mmol)正己胺;在30℃油浴中磁力搅拌6 h后,所得聚合物记为Poly(EG3MA-C),取出0.1 mL进行分子量分析(M w, MALLS= 0.65×103 g/mol,GPC测试结果在图13中);再往反应瓶中加入10.54 g(22.50 mmol)Z2-Arg NCA,继续搅拌反应48 h;所得反应液滴入200 mL甲基叔丁基醚中沉淀;减压过滤后,所得聚合物(记为poly(EG3MA-C)-b-poly(Z2-Arg),M w, MALLS=34.26×103 g/mol,GPC测试结果在图13中)放入30℃真空烘箱中干燥12 h;
② 往连接有双排管的玻璃瓶中加入2 g(10.47 mmol)L-PheNCA,抽真空充氮气三次,把整个体系置换为氮气气氛后,往其中加入20 mL干燥DMF、5g poly(EG3MA-C)-b-poly(Z2-Arg)(约含有0.20 mmol氨基)和0.5 g(1.71 mmol)L-Cystine NCA;在30℃油浴中磁力搅拌24 h后,所得反应液滴入60 mL甲基叔丁基醚中沉淀;减压过滤后,所得聚合物放入30℃真空烘箱中干燥12 h;把所得样品加入100 mL圆底烧瓶,加入20 mL二氯甲烷使其完全溶解,然后再加入5 mL HBr的冰醋酸溶液(33%),磁力搅拌2 h后,装入透析袋(截留分子量为3500 g/mol)对水透析三次;所得聚合物水溶液冷冻干燥后,放入冰箱保存,所得样品记为HBPAA(M w, MALLS=396.69×103 g/mol,GPC测试结果在图13中);所得超支化型树状聚合物HBPAA随温度的变化也会出现电转现象,其结果如图14所示;从中可以看出,在低温下其几乎显电中性,而随着温度的升高会表面电位会升高到7 mV左右,展示出了电荷转变能力。
(3)载药纳米粒的制备
首先,准确称取适量的本发明制备的树状聚合物于不同的圆底烧瓶中,加入10 mLDMSO使其完全溶解,再加入适量的阿霉素(DOX),(聚合物与DOX的质量比为1:20);其次,把上述溶液逐滴加入到剧烈搅拌的PBS(pH = 6.8, 100 mL)溶液中,用透析的方法,除去溶液中的DMSO和未被包裹的DOX;最后,用冷冻干燥的方法,得到载药纳米粒。
Claims (8)
1.一种电荷可转变型树状聚合物的制备方法,其特征在于,具体步骤为:
(1)以L-苯丙氨酸和三光气为原料,在蒎烯作用下合成L-苯丙氨酸-N-羧酸内酸酐,记为L-Phe NCA;
(2)以三(苄氧羰基)-L-精氨酸和二氯甲基甲醚为原料,在二氯甲烷溶液中合成三(苄氧羰基)-L-精氨酸-N-羧酸内酸酐,记为Z2-Arg NCA;
(3)以三乙二醇单甲醚和甲基丙烯酰氯为原料,在二氯甲烷溶液中合成甲基丙烯酰三乙二醇单甲醚酯,记为EG3MA;以EG3MA与L-半胱氨酸反应制备功能性半胱氨酸,记为EG3MA-C;将EG3MA-C在三光气作用下生成三乙二醇单甲醚功能化的L-半胱氨酸-N-羧酸内酸酐,记为EG3MA-C NCA;
(4)以N-(叔丁氧基羰基)-L-半胱氨酸甲酯与磺酰氯反应,所得产物同Boc-L-半胱氨酸反应制备得N, N'-双(叔丁氧羰基)-L-胱氨酸单甲醚,记为N,N'-diBoc-L-CME;
(5)在密闭受氮气保护的玻璃容器中,以胱胺为引发剂引发L-Phe NCA聚合,形成分子链中间含有一个二硫键,两端均为氨基的聚苯丙氨酸,记为G0;该分子链末端的氨基与N, N'-diBoc-L-CME的羧基发生缩合反应,经TFA脱除Boc保护基团后,得到分子链起、始端各含两个氨基的聚苯丙氨酸,记为G0-4NH2;
(6)以G0-4NH2为引发剂,引发L-Phe NCA聚合后,分子链末端的氨基再与N,N'-diBoc-L-CME的羧基发生反应,并用TFA脱除Boc保护基团,得到分子链末端含有8个氨基的第一代树状聚合物,记为G1-8NH2;
(7)以G1-8NH2为引发剂,引发L-Phe NCA聚合后,分子链末端的氨基再与N,N'-diBoc-L-CME的羧基发生反应,并用TFA脱除Boc保护基团,得到分子链末端含有16个氨基的第二代树状聚合物,记为G2-16NH2;
(8)以G2-16NH2为引发剂,引发L-Phe NCA聚合后,分子链末端的氨基再与N,N'-diBoc-L-CME的羧基发生反应,并用TFA脱除Boc保护基团,得到分子链末端含有32个氨基的第三代树状聚合物,记为G3-32NH2;
(9)以G3-32NH2中的氨基引发Z2-Arg NCA和EG3MA-C NCA连续聚合,得到内核为聚苯丙氨酸、中层为含保护基聚精氨酸、最外层三乙二醇单甲醚改性聚半胱氨酸的树状聚合物,记为,DLPAA。
2.根据权利要求1所述的制备方法,其特征在于:
步骤(1)中,L-苯丙氨酸与三光气和蒎烯的摩尔比为1:(2.0-3.0):(2.0-3.0)
步骤(2)中,三(苄氧羰基)-L-精氨酸与二氯甲基甲醚的摩尔比为1:(1.4-1.6);
步骤(3)中,三乙二醇单甲醚与甲基丙烯酰氯的摩尔比为1:(1.1-1.2),半胱氨酸与EG3MA的摩尔比为1:(1.4-1.6);EG3MA-C与三光气和蒎烯的摩尔比为1:(2.0-3.0):(2.0-3.0);
步骤(4)中,N-(叔丁氧基羰基)-L-半胱氨酸甲酯与磺酰氯的摩尔比为1:(1.1-1.2);其产物与Boc-L-半胱氨酸反应的摩尔比为1:(1.0-1.05)。
3.根据权利要求1所述的制备方法,其特征在于:
步骤(5)中,胱胺与L-Phe NCA的摩尔比为1:(8-10);氨基与N,N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上;
步骤(6)中,G0-4NH2中氨基的量与L-Phe NCA的摩尔比为1:(15-20);
聚合物与N,N'-diBoc-L-CME的羧基发生反应时,其所含氨基与N,N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上;
步骤(7)中,G1-8NH2中氨基的量与L-Phe NCA的摩尔比为1:(15-20);聚合物与N,N'-diBoc-L-CME的羧基发生反应时,其所含氨基与N,N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上;
步骤(8)中,G2-16NH2中氨基的量与L-Phe NCA的摩尔比为1:(15-20);聚合物与N,N'-diBoc-L-CME的羧基发生反应时,其所含氨基与N,N'-diBoc-L-CME的摩尔比为1:(3-4),TFA的摩尔量是保护基团的3倍以上。
4. 根据权利要求1所述的制备方法,其特征在于,步骤(9)中,G3-32NH2中氨基的量与Z2-Arg NCA和EG3MA-C NCA的摩尔比为1:(40-60):(2-3.5)。
5.一种由权利要求1-4之一所述制备方法得到的电荷可转变型树状聚合物,在水溶液中以单分子胶束的形式存在;其内核为聚苯丙氨酸,中层为正电性的聚精氨酸,最外层为中性的三乙二醇单甲醚改性的聚半胱氨酸;在37℃水溶液中显电中性,在43℃以上显正电性。
6.如权利要求5所述的电荷可转变型树状聚合物作为靶向药物的载体的应用。
7.根据权利要求6所述的电荷可转变型树状聚合物作为靶向药物的载体的应用,所述靶向药物为阿霉素、紫杉醇或索拉菲尼。
8.根据权利要求7所述的应用,其负载靶向药物的流程为:
(1)首先,称取10 mg的树状聚合物于圆底烧瓶中,加入8-10 mL DMSO使其完全溶解,再加入靶向药物;
(2)其次,把上述溶液逐滴加入到剧烈搅拌的磷酸盐缓冲溶液中,用透析的方法,除去溶液中的DMSO和未被包裹的靶向药物;
(3)最后,通过冷冻干燥,得到载药纳米粒;
流程(2)中,所述靶向药物为阿霉素,其加入量为150-300 mg;或者,所述靶向药物为紫杉醇,其加入量为100-200 mg;或者,所述靶向药物为索拉菲尼,其加入量为200-300 mg。
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