CN111973758B - 一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 - Google Patents
一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统及其制备方法。该智能递药系统是由胞内还原性谷胱甘肽响应的PTX前药纳米粒为核,可被金属基质蛋白酶剪切释放并串联穿膜肽的DNase Ⅰ接枝聚赖氨酸为壳,组成的“核‑壳”型DNase Ⅰ/PTX智能共递送载体。PTX前药纳米粒是利用前药分子PTX‑SS‑C18自组装形成,再对该纳米粒用聚赖氨酸进行包被;以MMP‑9底物肽串联穿膜肽为连接分子,将DNase Ⅰ蛋白接枝在包被于PTX前药纳米粒表面的聚赖氨酸氨基侧链上,构建智能药物递送系统,达到肿瘤微环境NETs调控与肿瘤细胞靶向的策略,联合提高抗恶性肿瘤效果。
Description
技术领域
本发明属于肿瘤及其微环境靶向与缓释给药系统技术领域,涉及一种肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统,具体涉及一种新型脱氧核糖核酸酶联合紫杉醇纳米粒的恶性肿瘤智能靶向纳米递释系统及其制备方法。
背景技术
随着科学水平和医疗技术的发展,非传染性疾病成为困扰人类健康的主要病种。其中,恶性肿瘤已在全球范围内成为仅次于心血管疾病的第二大致死病因。据世界卫生组织预测,2030年全球恶性肿瘤病人死亡人数将达到1200万,其中2/3将发生在中低收入国家。在我国,恶性肿瘤的发病率约为200/10万,每年新发病例在250万左右。恶性肿瘤给人类健康和生命造成极大威胁,给家庭及社会带来沉重的负担。因此,开发安全有效的抗肿瘤药物具有重大的社会意义和临床价值。
肿瘤微环境是在肿瘤进展过程中,由肿瘤细胞,基质细胞(成纤维细胞、炎性细胞、免疫细胞、周细胞、血管内皮细胞等),细胞外基质等共同组成的局部稳定环境,呈现出组织间液压升高与局部缺氧等病理性特征,为肿瘤的发生、发展、侵袭及转移等过程提供了必要的物质基础。一直以来,科学界对肿瘤靶向递药系统的研究,主要集中于肿瘤细胞靶向的开发。然而,由于肿瘤细胞的高度异质性,非常容易产生基因突变和表观遗传变化,导致耐药,临床获益非常有限。由于肿瘤微环境相对稳定,不容易耐药,引起了肿瘤靶向治疗研究者的高度关注。所以,靶向肿瘤细胞与调控肿瘤微环境相结合的治疗策略,成为肿瘤靶向研究领域新的热点。
肿瘤微环境被认为是“永不愈合伤口”与持续炎症状态,肿瘤组织通过释放趋化因子、细胞因子、过氧化氢等信号分子,将外周血中的中性粒细胞招募到肿瘤部位。肿瘤相关的中性粒细胞在肿瘤坏死因子α(TNF-α)、白介素8(IL-8)等细胞因子刺激下,形成NETs。NETs,即中性粒细胞胞外诱捕网(Neutrophil Extracellular Traps) 是中性粒细胞受到外界刺激活化时释放到胞外的DNA骨架纤维网,同时包含组蛋白、髓过氧化物酶(MPO)、中性粒细胞弹性蛋白酶(NE)、组织蛋白酶G(CG)、以及基质金属蛋白酶9(MMP-9)等颗粒蛋白。研究表明:NETs作为肿瘤微环境的重要组成部分,在促进肿瘤细胞增殖、远处转移以及血管生成和阻碍肿瘤治疗中发挥着关键作用。
研究发现,脱氧核糖核酸酶Ⅰ(DNaseⅠ)可以通过降解NETs抑制恶性肿瘤的增殖并阻止向远处器官的转移,可以用于恶性肿瘤的治疗。然而,提高DNase I的体内循环寿命,增强其向肿瘤组织的渗透与滞留作用,是提高DNase I体内治疗肿瘤效果的关键所在。紫杉醇(Paclitaxel,PTX)是一线广谱抗肿瘤药物,疗效显著,具有很强的微管稳定功能,在临床上已经广泛用于乳腺癌、卵巢癌、肺癌和部分头颈癌的治疗。所以,将DNase I和PTX联合用药:DNaseⅠ可以高效降解肿瘤微环境NETs,从而抑制肿瘤细胞增殖,降低肿瘤转移和减少肿瘤血管新生,还可以疏通对肿瘤细胞的机械性屏障,促进PTX制剂向肿瘤内部的渗透;PTX直接作用于肿瘤细胞,通过稳定肿瘤细胞微管蛋白而杀死肿瘤细胞。二者相得益彰,同时靶向肿瘤的“土壤”和“种子”,发挥调控与重塑肿瘤微环境和直接杀死肿瘤细胞的协同治疗作用,最大限度提高肿瘤的治疗效果。
要实现DNaseⅠ与PTX两种药物的联合使用,选择合适的共递送载体显得尤为重要。共递送载体是药物递送领域的重要研究方向,目前共递送技术主要是将多种药物一起担载于脂质体、胶束、纳米粒等药物载体中,利用纳米载体将共载药物递送至同一作用部位,增强多种药物联用的治疗效果。然而,由于DNaseⅠ的靶点为肿瘤微环境中的NETs,而PTX的靶点为肿瘤细胞内的微管,二者靶部位不同导致现有共递释载体无法递送这两种药物至各自靶点发挥药效。
发明内容
鉴于目前仅针对肿瘤细胞的单一制剂疗效不理想,易引起耐药等缺陷,提供一种既靶向肿瘤细胞又能调控肿瘤微环境NETs的联合治疗方法,提高恶性肿瘤的治疗效果。
本发明的目的还在于,针对目前游离DNase I和PTX在治疗恶性肿瘤时存在的缺陷,提供一种智能靶向纳米药物递送载体及其制备方法,延长药物体内的循环时间,增加药物在肿瘤部位的蓄积,提高抗肿瘤疗效,降低毒副作用,达到靶向联合治疗增效减毒目的。
本发明的目的是,利用肿瘤微环境NETs中高表达MMP-9,以及肿瘤细胞内GSH 浓度高的特殊条件,设计构建一种在肿瘤部位响应性治疗的智能载体。该智能载体能够能靶向至肿瘤微环境调控NETs,同时暴露的穿膜肽增加PTX前药纳米粒的细胞内吞作用,在肿瘤细胞内,以GSH为智能释药开关,响应病灶部位GSH介导的降解作用,使前药纳米粒在肿瘤细胞内发生裂解释放出PTX杀死肿瘤细胞,达到NETs 降解与肿瘤细胞杀伤并策,联合提高恶性肿瘤的治疗效果。
本发明的目的通过以下技术方案实现的:
一种肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统,是由对GSH响应的PTX前药纳米粒、PTX前药纳米粒表面接枝的聚赖氨酸、琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮(NHS-PEG3500-N3)、MMP-9酶底物肽段与细胞穿膜肽串联氨基酸序列、4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐 (Sulfo-SMCC)以及脱氧核糖核酸酶Ⅰ(DNaseⅠ)组成;其中,所述的PTX前药纳米粒和前药纳米粒表面接枝的聚赖氨酸通过电荷吸附作用实现包裹融合。
优选上述PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇(C18-OH)化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒NP/PTX,并将NP/PTX表面涂层上阳离子聚赖氨酸,PTX-SS-C18与聚赖氨酸的质量比为1:1~5,聚赖氨酸的分子量为70000~150000。
优选上述琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮(NHS-PEG3500-N3)中的聚乙二醇分子量为500-3500,MMP-9酶底物肽段与细胞穿膜肽串联后的氨基酸序列为GRKKRRQRRRPQPLGLAGGC,所述的DNaseⅠ是通过Sulfo-SMCC为连接分子化学接枝在串联的MMP-9酶底物肽段与细胞穿膜肽上。
优选上述PTX-SS-C18与DNaseⅠ的质量比为1:0.5~3。
具体的:本发明智能递药系统是由胞内还原性谷胱甘肽(GSH)响应的PTX前药纳米粒为核,可被金属基质蛋白酶(MMP-9)剪切释放并串联穿膜肽的DNaseⅠ接枝聚赖氨酸为壳,组成的“核-壳”型DNaseⅠ/PTX智能共递送载体。所述PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇(C18-OH)化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒,对该纳米粒用聚赖氨酸进行包被;以MMP-9底物肽串联穿膜肽(GRKKRRQRRRPQPLGLAGGC)为连接分子,将DNaseⅠ蛋白接枝在包被于PTX前药纳米粒表面的聚赖氨酸氨基侧链上,构建一种肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统。通过EPR效应,将DNase I递送至肿瘤微环境降解NETs,再将PTX高效递送至肿瘤细胞内杀死肿瘤细胞,达到肿瘤微环境NETs调控与肿瘤细胞靶向的策略,联合提高抗恶性肿瘤效果。
上述肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统的制备方法,将聚赖氨酸借助电荷吸附包被在PTX前药纳米粒表面,得到P-NP/PTX;利用Sulfo-SMCC为linker,将DNase I与N端炔基化的 (Pro)GRKKRRQRRRPQPLGLAGGC肽进行接枝,得到多肽-蛋白偶联 (Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,再将该偶联物通过NHS-PEG3500-N3为 linker,利用NHS端与P-NP/PTX表面游离氨基反应,另一端的叠氮基团与 (Pro)GRKKRRQRRRPQPLGLAGGC-DNase I偶联物N端的炔基发生点击化学反应,将(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I偶联物锚定在P-NP/PTX表面即组装成智能药物递送系统。
PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒NP/PTX;其中,PTX-SS-C18制备方法包括以下步骤:将1.0g二硫代甘醇酸与15mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应3h后,旋转蒸发去除乙酸和多余的乙酸酐,旋蒸后的产物溶于二氯甲烷,并加入1.49g的十八醇和65mg的DMAP,室温下搅拌反应15h后用1%HAc 终止,有机层用无水硫酸钠干燥;粗产物经硅胶柱层析纯化后得到中间体 HOOC-CH2-SS-CH2-COOC18;将中间体和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入200mgPTX,在避光条件下,继续室温反应10h;待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥;粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。
所述前药分子自组装形成PTX前药纳米粒NP/PTX的方法为采用乙醇注入法制备GSH响应的PTX前药纳米粒,具体为:称取5mgPTX-SS-C18溶于0.5mL无水乙醇,室温下将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得NP/PTX。
将制备好的PTX前药纳米粒与5mg/mL的聚赖氨酸溶液混合,借助电荷吸附,将聚赖氨酸包被在PTX前药纳米粒表面,多余的聚赖氨酸通过高速离心法去除,得到P-NP/PTX,PTX-SS-C18与聚赖氨酸的质量比为1:1~5。
上述智能药物递送系统组装过程具体如下:(1)首先,将P-NP/PTX用HEPES(pH7.4)重新分散,加入浓度为5mg/mL的NHS-PEG3500-N3溶液,室温搅拌2小时后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用;(2)将4mgDNase I溶解于PBS(pH 7.4)中,加入0.5mg的Sulfo-SMCC,室温氮气保护下反应1小时;将5mg(Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES缓冲溶液中,加入上述DNase I溶液,室温氮气保护下搅拌反应4小时,得到(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物;(3)表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时后冷冻高速离心,水洗,即得。
本发明构建一种具有程序化释药能力的智能共递送载体,首先靶向肿瘤微环境NETs,到NETs部位后,释放出DNaseⅠ,降解NETs,然后智能载体再携载PTX渗透进入肿瘤细胞,在胞内再释放出PTX,是亟待解决的关键问题。MMP-9是NETs 的重要组成部分,即NETs部位过表达MMP-9。所以,可以利用MMP-9对底物的酶切作用,触发DNase I在NETs部位的智能释放。还原性谷胱甘肽(GSH)在肿瘤细胞内的浓度(~2-10mM)是胞外和血浆中的(~2-10μM)1000倍。因此,GSH成为抗肿瘤药物胞内释放的“智能开关”。通过二硫键构建纳米递药载体(胶束、聚合物纳米粒、高分子结合物等),在模拟血液生理环境下载体稳定,而在模拟肿瘤细胞内 GSH浓度下,载体解聚,释放出药物,显示出对GSH高度的响应性,有利于抗肿瘤药物在肿瘤细胞内的智能释放。
本发明的有益效果:该智能共递送药物载体集长循环、被动靶向、肿瘤微环境NETs部位MMP-9响应性释药、“原位暴露”式肿瘤细胞靶向、肿瘤胞内GSH响应性释药等多功能于一体。本发明构建的智能共递送载体,通过EPR效应聚集在肿瘤部位,有效延长药物在体内的半衰期,对NETs的降解效果显著提高,抑制了肿瘤细胞的增殖和远处转移。在NETs部位高表达的MMP-9对多肽响应性剪切,释放DNase I 发挥效果,同时暴露的穿膜肽增加PTX前药纳米粒的细胞内吞作用,以GSH为智能释药开关,响应肿瘤细胞内GSH介导的降解作用,使前药纳米粒在肿瘤细胞内发生裂解,释放出PTX杀死肿瘤细胞。达到肿瘤微环境调控与肿瘤细胞杀伤并策,联合提高恶性肿瘤的治疗效果。
对本发明制备的用于治疗肿瘤的靶向纳米递药系统进行了体内外评价:
对本发明实施例方法制备的肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统进行了细胞毒性实验、细胞摄取实验、细胞迁移实验、体内药效学研究,结果表明,将DNase I锚定在纳米粒上后,显著增加了DNase I在肿瘤部位的蓄积,达到预期目的。而MMP-9响应性剪切之后,暴露出细胞穿膜肽,大大增加肿瘤细胞对药物的摄取,明显提高PTX的抗肿瘤效果,具体如下:
1)细胞摄取
前药纳米粒通过香豆素-6荧光标记后考察载体系统的细胞定性摄取情况,结果如图2所示,细胞对纳米粒的摄取具有浓度依赖性和时间依赖性,1μg/mL组的荧光强度明显强于0.5μg/mL组,2h组的荧光强度明显强于1h组。
2)细胞毒性实验
将体外诱导生成的NETs与A549细胞共培养在96孔板中,采用MTT法测定 P-NP/PTX、游离DNase I和对MMP-9响应的纳米粒mP-NPS-DNase/PTX、对MMP-9 不响应的纳米粒nP-NPS-DNase/PTX以及P-NP/PTX和游离DNase I共同给药对 A549细胞的体外抑制情况,结果如图3所示,mP-NPS-DNase/PTX纳米粒组对A549 的细胞毒作用与共同给药组没有显著的统计学差异,且明显强于其它对照组。
3)细胞迁移实验
将体外诱导生成的NETs与4T1细胞共培养于Transwell的上室中,分别给药 P-NP/PTX、游离DNase I和mP-NPS-DNase/PTX、nP-NPS-DNase/PTX以及P-NP/PTX 和游离DNase I共同给药,结果如图4所示,mP-NPS-DNase/PTX纳米粒组显著减少 4T1细胞迁移至小室下层,抑制作用与共同给药组没有显著的统计学差异。
4)体内靶向性评价
皮下瘤小鼠分别尾静脉注射罗丹明-NHS标记的游离DNase I和 mP-NPS-DNase/PTX,12h后的肿瘤组织切片荧光结果显示(图5),mP-NPS-DNase/PTX 在肿瘤部位的荧光强度明显强于游离组,该结果表明将DNase I接枝在纳米粒上后能增加递药系统在肿瘤部位的主动靶向与蓄积。
5)体内抗肿瘤药效学评价
对BALB/c鼠乳房垫接瘤后分别给药mP-NPS-DNase/PTX、nP-NPS-DNase/PTX、P-NP/PTX、DNase I、P-NP/PTX与DNase I共同给药和生理盐水,通过肿瘤大小以及肺转移情况评价纳米载体的治疗效果。结果如图6所示,给药组小鼠体重与生理盐水组无明显差异,表明纳米载体生物相容性较好,无明显的毒副作用。通过图7可以发现,mP-NPS-DNase/PTX组的肿瘤明显小于其它对照组,对原发肿瘤的生长有较好的抑制作用。如图8所示,使用mP-NPS-DNase/PTX治疗后,肺结节数明显少于其它对照组,证明该纳米载体有效抑制了肿瘤的肺转移。
附图说明
图1为本发明共递送给药系统的透射电镜图,纳米粒呈规整的类球形。其中,图1A:NP/PTX,图1B:mP-NPS-DNase/PTX。
图2本发明中A549细胞对纳米粒的摄取荧光图,其中,图2 A和图2 C纳米粒浓度为0.5μg/mL,图2 B和图2 D纳米粒浓度为1μg/mL,图2 A、图2 B是A549细胞与P-NP/PTX 共孵育1h的摄取情况,图2 C、图2 D是A549细胞与P-NP/PTX共孵育2h的摄取情况。
图3为本发明中A549细胞存活率柱状图。
图4为本发明中4T1细胞迁移率柱状图。
图5为本发明中游离DNase I和mP-NPS-DNase/PTX分别在肿瘤部位的蓄积。
图6为本发明中纳米粒给药组与生理盐水组小鼠体重随时间变化。
图7为本发明中各个给药组小鼠肿瘤大小比较。
图8为本发明中各个给药组肿瘤肺转移后形成结节的数量比较。
具体实施方式
下面结合具体实施例对本发明作进一步的阐述,具体实施例是在本发明的优选条件下进行。所述方法如无特别说明均为常规方法,所述原材料如无特别说明均能从公开商业途径而得。
肿瘤微环境中性粒细胞胞外诱捕网(NETs)调控的智能药物递送系统制备的步骤:
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入 (Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的 (Pro)GRKKRRQRRRPQPLGLAGGC-DNaseI,水洗2遍后,即得mP-NPS-DNase/PTX。实施例1:纳米粒理化性能表征及细胞迁移实验所用mP-NPS-DNase/PTX纳米粒的合成
将1.0g二硫代甘醇酸与15mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应 3h。待反应完全后,旋转蒸发去除乙酸和多余的乙酸酐。旋蒸后的产物溶于二氯甲烷,并加入1.49g的十八醇及65mgDMAP,室温下搅拌反应15h后用1%HAc终止,有机层用无水硫酸钠干燥。粗产物经硅胶柱层析纯化后得到中间体 HOOC-CH2-SS-CH2-COOC18。将HOOC-CH2-SS-CH2-COOC18和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入200mgPTX,在避光条件下,继续室温反应10h。待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥。粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。采用乙醇注入法制备GSH响应的PTX前药纳米粒。称取5mgPTX-SS-C18溶于0.5mL无水乙醇。室温下,将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得PTX前药纳米粒。
纳米粒溶液外观澄清,有明显的蓝色乳光。激光粒度分析表明,所得纳米粒子以106nm为有效直径呈正态分布,多分散性为0.113。扫描电镜下观察该纳米粒具有规整的球形外观,在溶液中分散良好,并具有较好的稳定性。
将制备好的PTX前药纳米粒与聚赖氨酸溶液混合,借助电荷吸附,将聚赖氨酸 (分子量为70000~150000)包被在PTX前药纳米粒表面,多余的聚赖氨酸(分子量为70000~150000)通过高速离心法去除,得到P-NP/PTX,PTX-SS-C18与聚赖氨酸的质量比为1:1~5。将P-NP/PTX用HEPES(pH 7.4)重新分散,加入NHS-PEG3500-N3 (购自于北京键凯科技有限公司),室温搅拌2小时后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用。PTX-SS-C18与NHS-PEG3500-N3(聚乙二醇分子量为500-3500)的质量比为1:2。
DNaseⅠ溶解于PBS(pH 7.4)中,加入Sulfo-SMCC,室温氮气保护下反应1小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的Sulfo-SMCC。Sulfo-SMCC与DNase Ⅰ的质量比为1:1.5~7.5。将(Pro)GRKKRRQRRRPQPLGLAGGC多肽(购自于吉尔生化(上海)有限公司)溶解于HEPES(pH 7.0)缓冲溶液中,加入上述DNaseⅠ溶液,室温氮气保护下搅拌反应4小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的(Pro)GRKKRRQRRRPQPLGLAGGC,得到 (Pro)GRKKRRQRRRPQPLGLAGGC-DNaseⅠ多肽-蛋白偶联物,用HEPES(pH 7.4)缓冲液重新分散后,备用。Sulfo-SMCC与(Pro)GRKKRRQRRRPQPLGLAGGC的质量比为1:2.5。PTX-SS-C18与DNaseⅠ的质量比为1:3。
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的 (Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,水洗2遍后,即得mP-NPS-DNase/PTX。
制得的mP-NPS-DNase/PTX外观呈规整的类球形,如图1B所示。该纳米粒对肿瘤细胞的生长有较好的抑制作用,如图3所示。通过细胞迁移实验,可发现该纳米粒能有效抑制肿瘤细胞的迁移,结果如图4所示。
实施例2:细胞定性摄取实验所用mP-NPS-DNase/PTX纳米粒的合成
PTX-SS-C18的合成步骤同实施例1。称取香豆素-6和5mg PTX-SS-C18溶于0.5mL 无水乙醇。室温下,将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得香豆素-6 标记的PTX前药纳米粒。
后续步骤同实施例1,得标记了香豆素-6的mP-NPS-DNase/PTX纳米粒。该纳米粒可被肿瘤细胞摄取,且摄取的量具有时间依赖性和浓度依赖性,如图2所示。
实施例3:体内药效学所用mP-NPS-DNase/PTX纳米粒的合成
将5.0g二硫代甘醇酸与75mL无水乙酸酐混合,在氮气保护下,35℃搅拌反应 3h。待反应完全后,旋转蒸发去除乙酸和多余的乙酸酐。旋蒸后的产物溶于二氯甲烷,并加入7.45g的十八醇及325mg DMAP,室温下搅拌反应15h后用1%HAc终止,有机层用无水硫酸钠干燥。粗产物经硅胶柱层析纯化后得到中间体 HOOC-CH2-SS-CH2-COOC18。将HOOC-CH2-SS-CH2-COOC18和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N,N-二异丙基乙胺,反应30min后加入1.0g PTX,在避光条件下,继续室温反应10h。待反应完全后,混合物先后用1%HAc和纯水洗涤,并用无水硫酸钠干燥。粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。采用乙醇注入法制备GSH响应的PTX前药纳米粒。称取48mg PTX-SS-C18溶于5mL无水乙醇。室温下,将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5 min,旋转蒸发除去乙醇,最后经0.22μm微孔滤膜过滤,即得PTX前药纳米粒。
将制备好的PTX前药纳米粒与144mg聚赖氨酸混合,借助电荷吸附,将聚赖氨酸包被在PTX前药纳米粒表面,多余的聚赖氨酸通过高速离心法去除,得到P-NP/PTX。将P-NP/PTX用HEPES(pH 7.4)重新分散,加入NHS-PEG3500-N3,室温搅拌2h后冷冻高速离心,水洗2遍后,得到表面聚乙二醇叠氮化P-NP/PTX,用HEPES缓冲液重新分散,备用。PTX-SS-C18与NHS-PEG3500-N3的质量比为1:2。
将7.2mg DNaseⅠ溶解于PBS(pH 7.4)中,加入Sulfo-SMCC,室温氮气保护下避光反应1小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的Sulfo-SMCC。 Sulfo-SMCC与DNaseI的质量比为1:1.5。将(Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES(pH 7.0)缓冲溶液中,加入上述DNaseⅠ溶液,室温氮气保护下搅拌反应4小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的 (Pro)GRKKRRQRRRPQPLGLAGGC,得到 (Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物,用HEPES(pH 7.4)缓冲液重新分散后,备用。Sulfo-SMCC与(Pro)GRKKRRQRRRPQPLGLAGGC的质量比为1:2。
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的 (Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,水洗2遍后,即得mP-NPS-DNase/PTX。
制得的mP-NPS-DNase/PTX对小鼠的体重无明显影响,具有良好的安全性,如图 6所示。该纳米粒抑制了原发肿瘤的生长,并且阻止了肿瘤向肺部的转移,肺结节数明显少于其他对照组,结果如图7、8所示。
实施例4:评估纳米粒在肿瘤部位聚积所用mP-NPS-DNase/PTX纳米粒的合成
表面聚乙二醇叠氮化P-NP/PTX的合成步骤同实施例3。
将7.2mgDNaseⅠ溶解于PBS(pH 7.4)中,加入0.1mg荧光染料罗丹明-NHS,室温下避光搅拌2h得DNaseⅠ-Rhodamine。向DNaseⅠ-Rhodamine溶液中加入Sulfo-SMCC,室温氮气保护下避光反应1小时,反应液经MidiTrapTMG-25脱盐柱去除未反应的 Sulfo-SMCC。Sulfo-SMCC与DNase I的质量比为1:1.5。将 (Pro)GRKKRRQRRRPQPLGLAGGC多肽溶解于HEPES(pH7.0)缓冲溶液中,加入上述DNaseⅠ-Rhodamine溶液,室温氮气保护下搅拌反应4小时,反应液经MidiTrapTM G-25脱盐柱去除未反应的(Pro)GRKKRRQRRRPQPLGLAGGC,得到 (Pro)GRKKRRQRRRPQPLGLAGGC-DNase I-Rhodamine多肽-蛋白偶联物,用HEPES (pH 7.4)缓冲液重新分散后,备用。Sulfo-SMCC与 (Pro)GRKKRRQRRRPQPLGLAGGC的质量比为1:1.5。
表面聚乙二醇叠氮化P-NP/PTX溶液中加入硫酸铜和抗坏血酸钠后,再加入上述(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I-Rhodamine多肽-蛋白偶联物溶液,避光,氮气保护下click反应6小时,反应液经14000rpm冷冻高速离心,弃上清中未反应的(Pro)GRKKRRQRRRPQPLGLAGGC-DNase I,水洗2遍后,即得接枝了荧光染料的mP-NPS-DNase/PTX。
制得的mP-NPS-DNase/PTX通过EPR效应聚积在肿瘤部位,其荧光强度明显亮于游离DNase I组,如图5所示。
实施例5:对MMP-9不响应的纳米粒nP-NPS-DNase/PTX的合成
MMP-9不响应是通过设计(Pro)GRKKRRQRRRPQPLGLAGGC肽中底物序列 PLGLA全部用相应的D-型氨基酸取代得到。其余步骤同实施例3。
nP-NPS-DNase/PTX对肿瘤细胞的毒性以及抑制肿瘤细胞的能力均弱于 mP-NPS-DNase/PTX,且体内药效实验结果均差于MMP-9响应性纳米粒 mP-NPS-DNase/PTX。结果如图3、4、7、8所示。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征在于该智能药物递送系统是由对GSH响应的PTX前药纳米粒、PTX前药纳米粒表面接枝的聚赖氨酸、琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮、MMP-9酶底物肽段与细胞穿膜肽串联氨基酸序列、4-(N-马来酰亚胺甲基)环己烷-1-羧酸磺酸基琥珀酰亚胺酯钠盐Sulfo-SMCC以及脱氧核糖核酸酶Ⅰ制成,MMP-9酶底物肽段与细胞穿膜肽串联氨基酸序列为N端炔基化的GRKKRRQRRRPQPLGLAGGC肽,所述的智能药物递送系统的制备方法是通过以下步骤实现的:将聚赖氨酸借助电荷吸附包被在PTX前药纳米粒表面,得到P-NP/PTX;利用Sulfo-SMCC为连接分子,将DNaseⅠ与N端炔基化的GRKKRRQRRRPQPLGLAGGC肽进行接枝,得到多肽-蛋白偶联N端炔基化的GRKKRRQRRRPQPLGLAGGC-DNaseⅠ,再将该偶联物通过NHS-PEG3500-N3为连接分子,利用NHS端与P-NP/PTX表面游离氨基反应,另一端的叠氮基团与N端炔基化的GRKKRRQRRRPQPLGLAGGC-DNaseⅠ偶联物N端的炔基发生点击化学反应,将N端炔基化的GRKKRRQRRRPQPLGLAGGC-DNaseⅠ偶联物锚定在P-NP/PTX表面即组装成智能药物递送系统。
2.根据权利要求1所述的中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征是:所述PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒,并将PTX前药纳米粒表面涂层上阳离子聚赖氨酸,聚赖氨酸的分子量为70000~150000。
3.根据权利要求1所述的中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征是:所述的琥珀酰亚胺基羧甲基酯-聚乙二醇-叠氮中的聚乙二醇分子量为500-3500,所述的DNase Ⅰ是通过Sulfo-SMCC为连接分子化学接枝在串联的MMP-9酶底物肽段与细胞穿膜肽上。
4.根据权利要求2所述的中性粒细胞胞外诱捕网调控的智能药物递送系统,其特征是:所述PTX-SS-C18与DNase Ⅰ的质量比为1:0.15~3。
5.一种权利要求1所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征是:将聚赖氨酸借助电荷吸附包被在PTX前药纳米粒表面,得到P-NP/PTX;利用Sulfo-SMCC为连接分子,将DNase Ⅰ与N端炔基化的GRKKRRQRRRPQPLGLAGGC肽进行接枝,得到多肽-蛋白偶联N端炔基化的GRKKRRQRRRPQPLGLAGGC-DNaseⅠ,再将该偶联物通过NHS-PEG3500-N3为连接分子,利用NHS端与P-NP/PTX表面游离氨基反应,另一端的叠氮基团与N端炔基化的GRKKRRQRRRPQPLGLAGGC-DNaseⅠ偶联物N端的炔基发生点击化学反应,将N端炔基化的GRKKRRQRRRPQPLGLAGGC-DNaseⅠ偶联物锚定在P-NP/PTX表面即组装成智能药物递送系统。
6.根据权利要求5所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征在于PTX前药纳米粒是利用二硫代甘醇酸将PTX与十八醇化学键合,得到PTX前药分子PTX-SS-C18,前药分子自组装形成PTX前药纳米粒NP/PTX;其中,PTX-SS-C18制备方法包括以下步骤:将1.0g二硫代甘醇酸与15mL无水乙酸酐混合,在氮气保护下,35˚C搅拌反应3h后,旋转蒸发去除乙酸和多余的乙酸酐,旋蒸后的产物溶于二氯甲烷,并加入1.49g的十八醇和65mg的DMAP,室温下搅拌反应15 h后用1%乙酸终止,有机层用无水硫酸钠干燥;粗产物经硅胶柱层析纯化后得到中间体HOOC-CH2-SS-CH2-COOC18;将中间体和HBTU在冰浴下溶于二氯甲烷,并逐滴加入N, N-二异丙基乙胺,反应30 min后加入200mgPTX,在避光条件下,继续室温反应10 h;待反应完全后,混合物先后用1%乙酸和纯水洗涤,并用无水硫酸钠干燥;粗产物经硅胶柱层析纯化,干燥后得到PTX-SS-C18。
7.根据权利要求6所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征在于所述前药分子自组装形成PTX前药纳米粒NP/PTX的方法为采用乙醇注入法制备GSH 响应的PTX 前药纳米粒,具体为:
称取5mgPTX-SS-C18溶于0.5 mL无水乙醇,室温下将无水乙醇溶液逐滴加入到不断搅拌的去离子水中,滴加完毕后继续搅拌5 min,旋转蒸发除去乙醇,最后经0.22μm 微孔滤膜过滤,即得NP/PTX。
8.根据权利要求7所述的肿瘤微环境中性粒细胞胞外诱捕网调控的智能药物递送系统的制备方法,其特征在于:将制备好的PTX前药纳米粒与5mg/mL的聚赖氨酸溶液混合,借助电荷吸附,将聚赖氨酸包被在PTX前药纳米粒表面,多余的聚赖氨酸通过高速离心法去除,得到P-NP/PTX,PTX-SS-C18与聚赖氨酸的质量比为1:1~5。
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