CN112843241B - 可生物响应的一氧化氮供体型聚合物前药及其制备方法 - Google Patents
可生物响应的一氧化氮供体型聚合物前药及其制备方法 Download PDFInfo
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Abstract
本发明公开了可生物响应的一氧化氮供体型聚合物前药及其制备方法,该聚合物前药由含羟基化疗药物与一氧化氮供体型聚碳酸酯类大分子通过肿瘤微环境酸响应性缩醛键连接得到,然后再通过自组装得到聚合物前药纳米药物。本发明通过对化疗药物进行修饰,并在聚合物载体材料上引入一氧化氮供体,制备出高载药量、高稳定性的可生物响应的一氧化氮供体型聚合物前药,实现药物增敏和协同治疗效果,在高效抗肿瘤方面有着广泛的应用前景。
Description
技术领域
本发明涉及生物医用高分子材料学和药物制剂及其制备方法,特别涉及可生物响应的一氧化氮供体型聚合物前药及其制备方法。
背景技术
一氧化氮(nitric oxide)是一种具有多功能、剂量依赖性得到细胞信号分子,素有“明星分子”的美誉,其可在哺乳动物体内由一氧化氮合成酶(nitric oxide synthase,NOS)催化L-精氨酸氧化生成。由于NO的良好脂溶性特征,它易于穿过磷脂双分子层而作用于细胞内部,从而发挥血管扩张、神经递送、创伤愈合、肿瘤杀伤等作用。NO是一把“双刃剑”,研究结果表明,当NO浓度低于50nM 时,NO通过扩血管作用使血管的通透性会增加,从而更有利于化疗药物递送至肿瘤组织内部。随着体内NO浓度的增加(>1000nM),外源和内源性的NO与凋亡相关基因相互作用,发挥对肿瘤细胞的促凋亡作用。
常用的小分子抗肿瘤药物
化疗药物的抗肿瘤效果通常依赖肿瘤组织中的氧化作用,肿瘤组织内的乏氧可能会产生某种压力促进瘤细胞对化疗药物的抵抗。大量研究证明NO可以通过抗氧化应激、抑制P-糖蛋白外排、激活cGMP信号通路等作用作为放疗、化疗或免疫治疗的增敏剂,显著提高紫杉醇(Taxol)、环磷酰胺(Cyclophosphamide)、顺铂(Cisplatin)、阿霉素(Doxorubicin)和米托蒽醌(Mitoxantrone)等化疗药物对肿瘤细胞的杀伤作用。此外,由于大多数化疗药物母体多为多芳环结构,其具有水溶性差、血液清除快、药物靶向性差、对健康组织毒副作用较大等问题,从而限制了这些小分子化疗药物的应用。
聚合物前药是指将聚合物大分子与小分子药物进行偶联修饰得到的一种大分子前药,其化学性质与小分子前药相同,在体外无活性或活性较小,在体内经酶或非酶的转化释放出活性药物,从而发挥药效的化合物。通过对疏水性小分子抗癌药物的前药化修饰,并制备成纳米前药药物,该纳米药物可同时结合前药和纳米载体的优点,有效延长药物的血液循环时间,降低毒副作用,提高药物的利用率并发挥载体材料的协同治疗作用。理想的纳米药物体系在正常生理条件下应具有足够的稳定性,到达肿瘤部位后可以有效释放药物,其对特定的肿瘤内环境刺激具有响应性。这些刺激作用包括:pH值、光照、离子浓度、氧化还原介质等。利用肿瘤细胞的微环境特点,设计具有响应性结构的纳米药物体系,能够得到更广泛的应用。
在现有技术中,已有一些关于生物响应性的前药的报道,PHPMA-PTX (PNU166945)和PGlu-PTX(CT-2103,Xyotax)已分别进入了临床一期和三期试验中。但是,作为一个具有临床应用前景的聚合物前药还应具备以下特点:(1) 聚合物前药能够在水溶液中形成稳定的纳米粒子,且粒径分布均一;(2)聚合物前药纳米粒子拥有稳定的结构,避免在体内循环过程中过早释放载药,但是在到达靶部位可根据生物环境响应性降解并释放载药;(3)聚合物前药纳米粒子通过主动或被动靶向作用到达肿瘤细胞时,聚合物前药的壳层结构既能提高聚合物前药的跨膜效率,又有利于聚合物前药纳米粒子通过内吞作用快速进入细胞内,提高药物的利用率;(4)聚合物材料不仅单一的作为抗肿瘤药物的输送载体,还可以携带具有协同治疗作用的信号分子,在肿瘤细胞内特定的生物条件(如酸性 pH值,高GSH浓度等)刺激下,聚合物前药纳米粒子结构响应性解体,释放出抗肿瘤药物和协同治疗分子,从而达到协同抑制肿瘤细胞增殖的目的。
发明内容
发明目的:针对上述问题,本发明提供了可生物响应的一氧化氮供体型聚合物前药及其制备方法,该聚合物前药可显著提高多羟基类化疗药物的水溶性和抗肿瘤活性。
技术方案:本发明提供的可生物响应的一氧化氮供体型聚合物前药,首先由含羟基化疗药物与一氧化氮供体型聚碳酸酯类大分子通过肿瘤微环境酸响应性缩醛键连接得到聚合物前药,然后再通过自组装得到前药纳米药物。
进一步地,含羟基化疗药物与连接臂形成小分子酸降解型前药,再与一氧化氮供体型聚碳酸酯类大分子反应,实现一氧化氮供体型聚碳酸酯类大分子与含羟基类化疗药物的连接,所述连接臂是以肿瘤微环境酸响应性缩醛键相连。
进一步地,所述一氧化氮供体型聚碳酸酯类大分子由硝酸酯环状碳酸酯单体利用含羟基类引发剂通过开环聚合,或与环状酯类单体衍生物开环共聚得到生物可降解聚碳酸酯类大分子。
进一步地,所述生物可降解聚碳酸酯类大分子用作一氧化氮供体或作为药物载体。
进一步地,所述环状酯类单体衍生物为三亚甲基碳酸酯(TMC)单体、丙烯酸环碳酸酯(AC)单体以及叠氮环碳酸酯(AEC)、丙交酯(LA)、乙交酯(GA)、己内酯(CL)。
进一步地,所述含羟基化疗药物选自雌二醇、紫杉醇、多西紫杉醇、羟基喜树碱、长春新碱、多柔比星、柔红霉素、米托蒽醌、依托泊苷或替尼泊苷。
进一步地,聚合物前药中环碳酸酯单元在聚合物链上的质量百分数为5%~100%;聚合物的分子量为1000~20000。
进一步地,所述的生物可降解的聚碳酸酯类大分子作为一氧化氮供体或作为药物载体的应用。
进一步地,所述一氧化氮供体可在肿瘤微环境的刺激下响应性释放出一氧化氮气体分子,并作为化疗药物的增敏剂。涉及的增敏作用为抗氧化应激、抑制 P-糖蛋白外排、激活cGMP信号通路,增强EPR效应,激活肿瘤细胞免疫应答等。
所述的可生物响应的一氧化氮供体型聚合物前药的制备方法,包括如下步骤:
(1)酸响应型缩醛键修饰的小分子含羟基化疗药物的合成:
将小分子含羟基化疗药物溶于有机溶剂中,再加入小分子连接臂,以对甲苯磺酸为催化剂,进行亲核化学反应得到酸降解型缩醛键修饰的小分子含羟基化疗药物前药;
(2)官能化生物响应性一氧化氮供体型聚碳酸酯类大分子的合成:
在惰性气体保护下,将环状碳酸酯单体衍生物溶于有机溶剂,然后加入引发剂,通过开环聚合反应得到聚碳酸酯类聚合物大分子,再通过化学偶联反应得到末端炔基化的聚碳酸酯类共聚物;
(3)一氧化氮供体型聚合物前药的制备:
将步骤(1)中制备得到的酸响应型含羟基类前药溶于有机溶剂中,再加入 (2)中制备的炔基修饰的功能化聚碳酸酯类共聚物,通过无铜点击化学反应得到酸降解型聚合物前药;
(4)生物响应型聚合物前药胶束的制备:
将(3)中制备得到的酸降解型聚合物前药溶于有机溶剂中,通过溶剂交换法制备得到聚合物前药胶束,经纯水透析,即可。
具体来说,以生物响应性缩醛键为连接臂,首先通过亲核反应得到缩醛键连接的羟基类化疗药物的前药小分子,再以环碳酸酯单体作为反应物,异丙醇、苄醇或单甲氧基PEG等分子为引发剂,通过开环聚合反应得到聚乙二醇-聚碳酸酯大分子聚合物,最后通过偶联反应将大分子聚合物与羟基类前药小分子连接在一起,合成了具有肿瘤微环境响应释药特性的聚合物前药。该聚合物前药通过在溶液进行自组装得到了生物响应性聚合物前药纳米药物,可有效改善多醇类化疗药物的水溶性,降低毒副作用,提高聚合物前药纳米药物的体内循环稳定性,有效防止载药的突释情况。
技术效果:本发明相对于现有技术具有如下效益:
1、本发明聚合物前药,具有水溶性好、毒副作用小、在正常生理环境下稳定好等优点。有效解决了临床使用中雌二醇、阿霉素等难溶性化疗药物水溶性差,毒副作用大等问题。此外,该大分子前药可在肿瘤细胞内酸性环境的刺激下快速释放出雌二醇等多羟基类化疗药物,且该一氧化氮供体型聚乙二醇-聚碳酸酯类高分子载体可在肿瘤细胞内高GSH浓度情况下,响应性释放NO,并逐渐被降解,体现出良好的生物相容性和生物可降解性,制备方法简便易行,便于操作推广。
2、本发明聚合物前药具有显著的肿瘤靶向性。大分子前药复合物粒径为纳米级别,可通过EPR滞留效应实现被动靶向;用缩醛键的pH敏感性实现肿瘤细胞内的快速响应性释药,克服了普通大分子前药入肿瘤细胞后释药速率缓慢的缺陷。该设计实现了从生理环境到肿瘤组织再到肿瘤细胞的层层靶向释药特性,有效提高多羟基类化疗药物在肿瘤部位的蓄积,并降低其肝肾毒性。
3、本发明聚合物前药具有逆转肿瘤多药耐药特性。包含一氧化氮供体的硝酸酯类聚碳酸酯大分子前药复合物,在肿瘤部位释放一氧化氮信号分子,显著改善肿瘤微环境,提高肿瘤细胞对化疗药物的敏感性,并逆转肿瘤细胞的多药耐药性;同时,结合纳米粒子的内吞机制及一氧化氮介导的多药耐药逆转功能,大分子前药复合物有望高效克服多羟基类小分子化疗药物在临床治疗中的耐药障碍。
4、本发明聚合物前药具有可控释药特性。通过调节大分子前药复合物中聚乙二醇和聚碳酸酯大分子的聚合度,不仅可以灵活调整聚碳酸酯类高分子与多羟基类化疗药物组分的剂量比,也可调节大分子前药复合物中多羟基类化疗药物的释药速率。该方法与物理包载药物型的纳米载体相比,制备工艺简单,操作简便,可控性极高。
5、本发明聚合物前药具有循环稳定性。可降解型高分子聚合物前药胶束具有很强的稳定性,可以显著增强高分子前药复合物体系的循环稳定性,防止多羟基类化疗药在复杂的生理环境中发生突释现象。
6、本发明聚合物前药具有高度的安全性。本发明使用的高分子载体材料为聚乙二醇-聚碳酸酯类高分子,均为高安全性、无毒环保、可生物降解的药用辅料。
附图说明
图1为小分子2-氮杂乙基乙烯基醚(AzVE)的核磁氢谱图;
图2为小分子前药雌二醇-2-氮杂乙基乙烯基醚复合物(Estradiol-AzVE)的核磁氢谱图;
图3为酸敏感性聚合物前药聚碳酸酯-2-氮杂乙基乙烯基醚-雌二醇复合物 (PEG-PNTC-co-Estradiol)的核磁氢谱图;
图4为聚合物前药胶束(PEG-PNTC-co-Estradiol)的粒径表征图;
图5为聚合物前药胶束(PEG-PNTC-co-Estradiol)在不同pH条件下放置不同时间的粒径图;
图6为聚合物前药胶束在体外模拟生理条件下的一氧化氮释放结果图;
图7为聚碳酸酯大分子材料(PEG-PNTC)对PC-3细胞的细胞毒性;
图8为聚合物前药胶束(PEG-PNTC-co-Estradiol)对PC-3细胞的细胞毒性结果图。
具体实施方式
实施例1
(1)合成小分子2-氮杂乙基乙烯基醚(AzVE)
小分子AzVE的合成路线如下:
称取2-氯乙基乙烯基醚(29.5mL,0.290mol)溶于175mL无水N,N-二甲基甲酰胺中,在N2保护条件下,缓慢加入叠氮化钠(21.2g,0.326mol),80℃反应5h后,经减压蒸馏,得到2-氮杂乙基乙烯基醚(AzVE),为无色透明液体,产率72%,核磁氢谱如图1所示。
(2)合成小分子前药雌二醇-2-氮杂乙基乙烯基醚复合物(Estradiol-AzVE)
小分子Estradiol-AzVE的合成路线如下:
分别取2-氮杂乙基乙烯基醚(0.5g,4.4mmol)和雌二醇(120mg,0.44mmol) 于反应瓶中,加入适量二氯甲烷溶剂,,于氮气保护下加入对甲苯磺酸(PTSA) 20mg,室温搅拌反应3小时。待反应完全后,加入300mL二氯甲烷溶解产物并用水萃取三次,有机相用无水硫酸镁干燥,过滤,减压浓缩。采用硅胶柱层析法分离出雌二醇-2-氮杂乙基乙烯基醚复合物,得到淡黄色粘稠状雌二醇-2-氮杂乙基乙烯基醚复合物,产率:39.6%,核磁氢谱如图2所示。
(3)合成酸敏感性聚合物前药聚碳酸酯-2-氮杂乙基乙烯基醚-雌二醇复合物(PEG-PNTC-co-Estradiol)
在N2保护下,将0.8g环碳酸酯单体溶于8mL二氯甲烷,依次加入引发剂聚乙二醇5k1.0g,催化剂磷酸二苯酯0.5g,40℃加热反应,反应结束后,三乙胺终止反应,冰乙醚沉淀,得到聚碳酸酯大分子m-PEG-PNTC,核磁氢谱如图3 所示。另称取1.0g聚碳酸酯大分子mPEG-PNTC溶于30mL无水二氯甲烷,依次加入吡啶59mg,0.75mmol,对硝基氯甲酸苯酯125mg,0.625mmol,室温搅拌反应24h,冰乙醚沉淀,得到对硝基氯甲酸苯酯活化的聚碳酸酯大分子。将该产物溶于20mL无水二氯甲烷,再加入乙二胺10mg,室温搅拌5h,冰乙醚沉淀,得到胺基修饰的聚碳酸酯大分子。最后将胺基修饰的聚碳酸酯大分子溶于 10ml无水二氯甲烷,依次加入对硝基氯甲酸苯酯活化的环辛炔,三乙胺,室温搅拌12h,冰乙醚沉淀得到0.75g末端炔基化的聚乙二醇-聚碳酸酯类聚合物,最后加入1.0倍量酸敏感性羟基小分子前药,室温反应过夜,甲醇透析得到酸敏感性聚合物前复合物(PEG-PNTC-co-Estradiol)。
大分子酸敏感聚合物前药PEG-PNTC-co-Estradiol的合成路线如下:
实施例2可生物响应的一氧化氮供体型聚合物雌二醇前药胶束 (PEG-PNTC-co-Estradiol)的制备
聚合物前药胶束(PEG-PNTC-co-Estradiol)采用溶剂交换法制备。在超声条件下将0.1mL聚合物前药PEG-PNTC-co-Estradiol的N,N-二甲基甲酰胺溶液(20 mg/mL)缓慢加入到1mL的高纯水中,所得混合溶液继续超声半小时,最后在高纯水中透析2h。图4是聚合物前药胶束(PEG-PNTC-co-Estradiol)的粒径表征图。结果显示,前药胶束的平均粒径为103nm,PDI为0.21。
实施例3生物响应性聚合物雌二醇前药胶束(PEG-PNTC-co-Estradiol)体外降解实验
准备两份聚合物前药胶束(PEG-PNTC-co-Estradiol,1mL,1mg/mL)并加入到玻璃样品池中,向其中一个样品池中加入一定量盐酸溶液使胶束溶液最终 pH为5.0,另一个样品池中加入等量pH 7.4磷酸盐缓冲溶液使胶束溶液最终pH 为7.4,然后玻璃样品池用橡胶塞封住,摇晃均匀,置于37℃恒温摇床(200rpm) 中,在选定时间、37℃下,通过动态激光光散射(DLS)来跟踪测定颗粒的粒径变化。图5为聚合物前药胶束(PEG-PNTC-co-Estradiol)在不同pH条件下放置不同时间的粒径图。结果表明,在pH 7.4条件下,前药胶束在放置24小时后粒径没有发生明显变化,而在pH 5.0,20mM DTT条件下放置24小时前药胶束粒径明显变大,达到186nm,说明前药纳米胶束疏水端硝酸酯在还原条件下释放出一氧化氮后转变为亲水性羟基,使胶束内部发生溶胀。
实施例4生物响应性聚合物雌二醇前药胶束(PEG-PNTC-co-Estradiol)体外一氧化氮释放实验
NO-M在PB中NO的释放量用Griss试剂测定。PB中NO的释放实验在37℃下,两种不同介质:(i)PB,pH 7.4;(ii)PB含有10mM GSH,pH 7.4中测定。这两种介质的浓度均是10mM。制备好的聚合物胶束样品分成三份,转移到透析袋(Spectra/Pore,MWCO 12000-14000)中,将透析袋置于相应缓冲液中,然后放入37℃恒温摇床。在指定的时间点,从释放体系中取出50μL释放介质,取出的释放介质与Griss试剂混合后室温放置10分钟,然后,用酶标仪在UV=540 nm下测定。该释放实验重复三次。图6是聚合物前药胶束在体外模拟生理条件下的一氧化氮释放结果,从图中看出,在没有GSH存在的情况下,长达70小时仅有不超过2μM的一氧化氮释放出来,而在10mM GSH存在的情况下,有高达 25μM的一氧化氮被释放出来,结果显示出了显著的生物响应性,这也为一氧化氮供体型聚合物材料递送药物提供了必要条件。
实施例5聚合物前药胶束(PEG-PNTC-co-Estradiol)对前列腺癌细胞PC-3 的毒性测试(MTT)
聚合物前药胶束(PEG-PNTC-co-Estradiol)在PC-3细胞中的毒性通过MTT 法测定。首先将100μL细胞的FL2K悬浮液(FL2K培养基中含10%胎牛血清、 100IU/mL青霉素和100μg/mL链霉素)铺于96孔培养板中,并置于37℃,5%二氧化碳条件下培养24h使单层细胞的覆盖率达到70~80%。然后向每孔中加入 10μL不同浓度的自由雌二醇以及聚合物前药胶束(PEG-PNTC-co-Estradiol)的 PB溶液,使药物在细胞孔中的最终浓度为0.5、1、2、5、10和20μg/mL。待继续培养48h后,向每孔中加入10μL3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐 (MTT)的PBS溶液(5mg/mL),并放入培养箱继续培养4h以使MTT与活细胞作用。随后移除含有MTT的培养液,向每孔中加入150μL DMSO以溶解活细胞与MTT产生的紫色甲瓒结晶,并使用酶标仪(SpectraMax i3x)测定每个孔在570 nm处的吸收。细胞相对存活率通过与只有空白细胞的对照孔在570nm处的吸收相比得到。实验数据均是平行四组进行的。
细胞存活率(%)=(OD570样品/OD570对照)×100%
图7聚碳酸酯大分子材料(PEG-PNTC)对PC-3细胞的细胞毒性,图8为聚合物前药胶束(PEG-PNTC-co-Estradiol)对PC-3细胞的细胞毒性结果图。结果表明:未载药的PEG-PNTC聚合物前药胶束随着浓度升高其细胞毒性也随之增强,说明该聚合物前药可以在一定程度上抑制PC-3细胞生长;而随着孵育时间的增加,细胞毒性也随之增强,说明键连在聚合物上的药物缓慢从聚合物前药胶束中释放出来。
Claims (5)
1.一种可生物响应的一氧化氮供体型聚合物前药纳米药物,其特征在于:首先由含羟基化疗药物与一氧化氮供体型聚碳酸酯类大分子通过肿瘤微环境酸响应性缩醛键连接得到聚合物前药,然后再通过自组装得到聚合物前药纳米药物;所述含羟基化疗药物为雌二醇;聚合物前药按如下方法制得:
(1)酸响应型缩醛键修饰的小分子含羟基化疗药物的合成:将小分子含羟基化疗药物溶于有机溶剂中,再加入小分子连接臂,以对甲苯磺酸为催化剂,进行亲核化学反应得到酸降解型缩醛键修饰的小分子含羟基化疗药物前药;所述小分子连接臂为2-氮杂乙基乙烯基醚;
(2)官能化生物响应性一氧化氮供体型聚碳酸酯类大分子的合成:在惰性气体保护下,将环状碳酸酯单体溶于有机溶剂,然后加入引发剂,通过开环聚合反应得到聚碳酸酯类聚合物大分子,再通过化学偶联反应得到末端炔基化的聚碳酸酯类共聚物;所述末端炔基化的聚碳酸酯类共聚物由胺基修饰的聚碳酸酯类聚合物大分子和对硝基氯甲酸苯酯活化的环辛炔反应制得;
(3)一氧化氮供体型聚合物前药的制备:将步骤(1)中制备得到的酸响应型含羟基类小分子前药溶于有机溶剂中,再加入(2)中制备的炔基修饰的功能化聚碳酸酯类聚合物,通过无铜点击化学反应得到酸降解型聚合物前药;
(4)生物响应型聚合物前药胶束的制备:将(3)中制备得到的酸降解型聚合物前药溶于有机溶剂中,通过溶剂交换法制备得到聚合物前药胶束,经纯水透析,即可。
2.根据权利要求1所述的可生物响应的一氧化氮供体型聚合物前药纳米药物,其特征在于:生物可降解的一氧化氮供体型聚碳酸酯类大分子由硝酸酯环碳酸酯(NTC)单体在含羟基类引发剂的存在下通过开环聚合反应得到。
3.根据权利要求1所述的可生物响应的一氧化氮供体型聚合物前药纳米药物,其特征在于:聚合物前药中环碳酸酯单元在聚合物链上的质量百分数为5%~100%;聚合物的分子量为1000~20000。
4.根据权利要求1所述的可生物响应的一氧化氮供体型聚合物前药纳米药物,其特征在于:所述的生物可降解的聚碳酸酯类大分子作为一氧化氮供体或作为药物载体的应用。
5.根据权利要求1所述的可生物响应的一氧化氮供体型聚合物前药纳米药物,其特征在于:所述一氧化氮供体可在肿瘤微环境的刺激下响应性释放出一氧化氮气体分子,并作为化疗药物的增敏剂。
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