CN115487155A - 一种pH/酶双重响应的靶向细胞核的纳米载体及其制备与应用 - Google Patents
一种pH/酶双重响应的靶向细胞核的纳米载体及其制备与应用 Download PDFInfo
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Abstract
本发明涉及一种pH/酶双重响应的靶向细胞核的纳米载体及其制备与应用。该纳米载体由聚酰胺‑胺(PAMAM)、双功能的马来酰亚胺聚乙二醇活性酯(MAL‑PEG‑NHS)、吗啉‑酶响应四肽‑细胞核靶向肽(Mp‑GFLG‑PKKKRKVC)组成,以树枝状高分子聚酰胺‑胺(PAMAM)为载体核心,通过其表面氨基嫁接双功能的马来酰亚胺聚乙二醇活性酯(NHS‑PEG‑MAL),并在聚乙二醇的MAL端连接吗啉‑酶响应四肽‑细胞核靶向肽(Mp‑GFLG‑PKKKRKVC),促进纳米载体穿过细胞核膜转运到细胞核内,即可携带靶点在细胞核内的化疗药物进入细胞核,进而提高药效,减小药物的副作用。该纳米载体制备方法简单,价廉易得,在体内能够降解。可广泛应用于材料学、生物学、医学等领域,具有较好的研究价值和临床应用前景。
Description
技术领域
本发明涉及纳米生物医药材料领域,更具体的,涉及一种pH/酶双重响应的靶向细胞核的纳米载体及其制备方法与应用。该纳米递送载体制备方法简单、易于操作,且适用于抗肿瘤化疗药物的特异性靶向递送,在恶性肿瘤,如肝癌、宫颈癌、乳腺癌、胃癌、肾癌等的治疗中有潜在的应用价值。
背景技术
在现阶段,癌症仍然对人类健康存在严重的威胁。临床治疗人类肿瘤的方法包括手术、化疗和放疗。然而,大多数现有技术都存在许多副作用,如循环半衰期短、药物毒副作用大等。靶向治疗是癌症特异性破坏治疗中的一种新兴治疗方法。这种新的治疗方法可以弥补传统治疗方法的不足,提高了治疗的准确性,减少对正常组织的损伤。
亲水性高分子等纳米载体在抗癌治疗方面取得了巨大成功。然而,许多治疗药物,其靶点定位于特定的细胞器,其疗效取决于这些药物能否进入亚细胞靶点,如细胞核。然而,抗癌药物在全身给药时的高效核传递仍然是一个艰巨的挑战,这需要克服多种生理障碍,包括逃避通过肾脏排泄和单核吞噬系统(MPS)的快速清除,增强细胞摄取和核内体逃逸,并穿透双层核膜。
要克服这些生理障碍,需要药物载体具有非常不同的物理化学特性。药物载体在肿瘤部位的总体积累主要取决于它们的药代动力学特征和增强的渗透性和滞留(EPR)效应。因此,为了促进其长循环和EPR效应,全身给药的药物载体需要一定的尺寸以避免快速肾过滤,也需要相对中性的电荷以避免被MPS识别。然而,控制进入细胞核内部的核孔复合体,其功能直径仅为9nm。因此,载体的大小在很大程度上决定了其进入细胞核的效率。此外,目前的核靶向策略主要依赖于核定位序列(NLS)的偶联,NLS是一种短肽,它对底物进行“标记”,以便通过核孔复合体进行核内活性转运。然而,修饰NLS的载体需要跨越细胞质膜进入细胞质,然后才能与受体结合进入细胞核。此外,转运载体的NLS修饰可能会影响其药代动力学行为,因为该序列包含带正电荷的赖氨酸和/或精氨酸序列,可增加 MPS识别。因此,可能需要一种肿瘤环境刺激响应型递送载体来打破这些生理障碍,在纳米载体递送时实现高效的核靶向递送。
肿瘤细胞外和细胞内环境中存在的酸性pH值,被认为是肿瘤组织和/或肿瘤内吞泡(如核内体和溶酶体)中抗癌药物可控释放的适当内部触发器。与血液和健康组织的pH值(pH 7.4)比较,肿瘤细胞外pH值范围为6.0-7.2。此外,经内吞作用后,通过质子流入开始快速酸化。亚细胞器内的pH在核内体中下降到5.0-6.0,在溶酶体中下降到4.0-5.0。靶向pHe(6.5-7.2)的表面电荷逆转纳米载体被认为是一种非常有前途的策略,可以提高纳米载体在体内的稳定性,并增强其在肿瘤部位的细胞摄取。吗啉(Mp)由于胺基的脱质子-质子化作用,在血液和正常组织pH下由不带电荷转变为带正电荷。选择胺的脱质子-质子化而不是选择共价键的裂解的优点是前者是可逆的。此外,质子-脱质子化的转变比pH敏感的共价键裂解要快得多。
近年来,酶敏感给药由于反应条件温和、特异性高、对身体组织损伤程度低,已成为一种非常受欢迎的策略。更重要的是,许多类型的酶在肿瘤组织和癌细胞中表现出高水平的表达,但在健康组织或正常细胞中没有或相对低水平的表达。例如,组织蛋白酶B是一种溶酶体中可特异性水解GFLG序列蛋白酶,在多种肿瘤中过表达。
以阿霉素(DOX)作为模型药物,我们制备了一种pH/酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM),可将DOX有效的传递至肿瘤细胞核内,显著提高抗肿瘤功效,减少药物的副作用。
发明内容
本发明的第一个目的是提供一种pH/酶双重响应的靶向细胞核的纳米载体。首先,该纳米递送载体具有良好的生物相容性、体内可降解性。其次,该纳米递送载体具有特异性靶向细胞核的功能,这一方面得益于该纳米递送载体能够通过EPR效应被动靶向到肿瘤部位;另一方面得益于该纳米递送载体能够对pH和酶做出响应,促进肿瘤细胞对pH和酶双重响应的靶向细胞核的纳米载体的摄取以及该纳米载体的进入肿瘤细胞后的核内活性转运。
本发明的第二个目的是提供所述pH/酶双重响应的靶向细胞核的纳米载体的制备方法。本发明制备方法简单,材料易得,价格低廉,且反应条件温和,易于操作,具有产业化实施的前景。
本发明的第三个目的是提供所述pH/酶双重响应的靶向细胞核的纳米载体的应用。通过物理包埋的方法负载化疗药物阿霉素,在特异性配体(Mp-GFLG-PKKKRKVC-PEG)的介导下,可有效增加携带的药物在肿瘤部位的富集,维持肿瘤细胞内高药物浓度的同时,降低药物在全身的分布,从而提高抗肿瘤功效,降低全身毒副作用。这一应用所具有的优势为肿瘤的临床治疗提供了新的思路。
本发明的上述目的是通过以下方案予以实现的:
其技术构思是:一种pH/酶双重响应的靶向细胞核的纳米载体,由聚酰胺-胺(PAMAM)、双功能的马来酰亚胺聚乙二醇活性酯(MAL-PEG-NHS)、吗啉-酶响应四肽-细胞核靶向肽(Mp-GFLG-PKKKRKVC)组成。
各部分结构式如下:
PAMAM:(结构示意图见图1)
MAL-PEG-NHS:
MP-GFLFPKKKRKVC:
一种pH/酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM):(结构示意图见图2)。
1.一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其步骤是:
(1)聚乙二醇-聚酰胺胺(PEG-PAMAM)的合成:20%W/V的PAMAM甲醇溶液用旋转蒸发器蒸干后,在pH 8.0的PBS溶液中溶解、超声,以1:10-15的摩尔比与NHS-PEG- MAL在室温下摇床反应4h,经透析、冷冻干燥后得到PEG-PAMAM。在这一阶段, PAMAM伯胺组只与双功能PEG的NHS末端反应。
(2)吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG- PAMAM)(图2)的合成:步骤(1)中所得到的PEG-PAMAM结合物重新溶解到PBS (pH 7.4)中,以1:5-10的摩尔比与Mp-GFLG-PKKKRKVC在室温下摇床反应24h。此时,PAMAM-PEG的末端MAL基团只与Mp-GFLG-PKKKRKVC的巯基反应。
所述步骤(2)中Mp-GFLF-PKKKRKVC,其中Mp为pH敏感小分子,GFLG为酶响应肽段,PKKKRKV为细胞核靶向肽。
所述步骤(1)中溶液的溶剂为pH为8的磷酸缓冲液(PBS)。
所述步骤(1)中PAMAM的分子量为14215g/mol;双功能的聚乙二醇MAL-PEG-NHS 的分子量为2000g/mol。
所述步骤(1)中PAMAM溶液的浓度为15-25mg/mL;双功能的聚乙二醇MAL-PEG- NHS溶液浓度为10-20mg/mL。
所述步骤(1)中NHS-PEG-MAL与PAMAM反应时需避光。
所述步骤(1)-(2)中透析条件为:透析膜为生物技术再生纤维(RC)膜,其截留分子量为7000Da,在去离子水中透析1-3天,每隔5-10h更换一次透析液。
所述步骤(1)中超声条件为20-200W,超声时间为5-15min。
所述步骤(2)中Mp-GFLG-PKKKRKVC的分子量为1501.38g/mol。
所述步骤(2)中Mp-GFLG-PKKKRKVC溶液浓度为10-15mg/mL。
2.将步骤(2)中所制备的pH/酶双重响应的靶向细胞核的纳米载体经透射电镜观察、动态光散射分析,发现相较Mp-GFLG-PKKKRKV-PEG-PAMAM,经木瓜蛋白酶处理的Mp-GFLG-PKKKRKV-PEG-PAMAM的粒径明显减小,这更有利于该纳米载体进入细胞核。(图 4,图5)
3.将步骤(2)中制备的一种pH和酶双重响应的靶向细胞核的纳米载体在pH为6.8和 7.4的条件下分别与HepG2肿瘤细胞共培养,检测不同浓度的该载体对细胞活度的影响。发现其对肿瘤细胞均无明显毒性,细胞存活率高,证明这种pH和酶双重响应的靶向细胞核的纳米载体具有优良的生物安全性。(图6)
一种pH和酶双重响应的靶向细胞核的纳米载体在制备治疗或预防肿瘤的局部化疗药物中的应用,步骤如下:
1.聚乙二醇-聚酰胺胺(PEG-PAMAM)的合成:吸取90μL的20%W/V的PAMAM 甲醇溶液至圆底烧瓶中,旋蒸后,在烧瓶中加入1mL pH 8.0的PBS溶液,待溶解后超声10 min。然后在避光条件下称取28mg NHS-PEG-MAL并将其溶解于1mLpH为8的PBS溶液中,溶解后将其逐滴滴入到含有PAMAM的烧瓶中,溶液呈现淡淡的粉色,在室温下摇床反应4h。将反应后的混合物转移到透析袋中(分子截留量为7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM伯胺组只与双功能PEG的末端NHS反应。
2.吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG-PAMAM)的合成:步骤(1)中所得到的PEG-PAMAM结合物重新溶解到PBS(pH 7.4) 中,然后,称取10.56mg Mp-GFLG-PKKKRKVC溶于1mL PBS PEG-PAMAM在PBS(pH 7.4)中,逐滴加入上述反应体系,室温下摇床反应24h(pH 7.0)中以1:5-10的摩尔比与 Mp-GFLG-PKKKRKVC在室温下摇床反应24h。将反应后的混合物转移到透析袋中(分子截留量为7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM-PEG的末端MAL基团只与Mp-GFLG-PKKKRKVC的巯基反应。
3.载有化疗药物阿霉素(DOX)的pH和酶双重响应的靶向细胞核的纳米体系的合成:
称取10mg的DOX,溶于5mL的去离子水,转移至含有20mg Mp-GFLG-PKKKRKV- PEG-PAMAM的2mL去离子水溶液中,室温下避光摇床反应24h。反应结束后,将产物转移至透析袋中(透析袋截留分子量为7000Da),避光透析12h,冷冻干燥后,得到负载化疗药物阿霉素的一种pH和酶双重响应的细胞核靶向纳米体系,称重。(图3)
4.将步骤3中获得的一种负载化疗药物阿霉素的pH/酶双重响应的靶向细胞核的纳米体系利用紫外分光光度法检测药物的载药量和包封率。
5.将步骤3中获得的一种负载化疗药物阿霉素的pH/酶双重响应的细胞核靶向纳米体系与肿瘤细胞共培养,利用荧光显微镜、流式细胞术等手段检测肿瘤细胞摄取效率,发现载药纳米体系可有效的被肿瘤细胞内吞并穿过细胞核膜将上载药物DOX传递至细胞核中。
6.将游离DOX、PAMAM/DOX、PEG-PAMAM/DOX、PKKKRKV-PEG- PAMAM/DOX、及步骤3中获得的一种负载化疗药物阿霉素的pH/酶双重响应的靶向细胞核的纳米体系分别经尾静脉注射入移植瘤小鼠模型体内,每天监测小鼠的体重及肿瘤生长变化,结果发现经步骤3获得的纳米体系治疗的小鼠,肿瘤体积增长缓慢,小鼠体重缓慢上升。治疗结束后,安乐处死小鼠,取出肿瘤并称重,发现步骤3中获得的纳米体系在上述各个组中具有最高的抑瘤率。
本发明的有益效果体现在:
1.本发明以小粒径的树枝状高分子聚酰胺-胺(PAMAM)为内核,并通过其表面氨基嫁接双功能的马来酰亚胺聚乙二醇活性酯(NHS-PEG-MAL),形成聚乙二醇-聚酰胺按 (PEG-PAMAM)。一方面屏蔽PAMAM本身的正电荷,降低纳米颗粒的毒性,延长其循环时间。另一方面,增大纳米颗粒的粒径使其能够更好的通过EPR效应被动靶向肿瘤组织,进而增加纳米载体在肿瘤部位的积累。
2.进一步地,本发明在聚乙二醇-聚酰胺按(PEG-PAMAM)中PEG的MAL末端连接吗啉-酶响应四肽-细胞核靶向肽(Mp-GFLG-PKKKRKVC),形成吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG-PAMAM)。一方面Mp对酸性pH敏感,在正常组织及血液的pH条件下去质子化,Mp呈现中性,在肿瘤微环境的pH条件下被质子化,Mp携带正电荷,增强肿瘤细胞对纳米载体的摄取。另一方面,Mp-GFLG可对PKKKRKV表面的正电荷产生干扰,减小对PKKKRKV的MPS识别,但当纳米载体被肿瘤细胞内吞后,溶酶体中的组织蛋白酶B可使GFLG断裂,Mp-GFLG从Mp-GFLG- PKKKRKV-PEG-PAMAM上掉落,细胞核靶向肽PKKKRKV完全暴露在纳米载体外围, PKKKRKV与受体结合后,纳米载体通过核孔复合体穿过核膜进入细胞核。
3.此外,该纳米载体合成原料廉价易得,合成过程简单易操作,合成条件温和,在体内能够被降解,是一种优良的pH/酶双重响应的靶向细胞核的纳米载体。
附图说明
图1是树枝状聚酰胺-胺纳米载体(PAMAM)的结构示意图。其中:NH2代表氨基,64代表纳米颗粒表面有64个氨基。
图2是一种pH/酶双重响应的细胞核靶向纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM)的结构示意图。由聚酰胺-胺(PAMAM)、双功能的马来酰亚胺聚乙二醇活性酯(MAL-PEG-NHS)、吗啉-酶响应四肽-细胞核靶向肽(Mp-GFLG-PKKKRKVC)组成。
图3是一种载有化疗药物阿霉素的pH/酶双重响应的靶向细胞核的纳米载体的结构示意图。通过物理吸附的方法上载化疗药物阿霉素(DOX)。
图4是一种pH/酶双重响应的靶向细胞核的纳米载体有无经木瓜蛋白酶(Papain)处理的粒径分布图:A:Mp-GFLG-PKKKRKV-PEG-PAMAM,B:Mp-GFLG-PKKKRKV-PEG- PAMAM+Papain。
将pH/酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM)以及经木瓜蛋白酶处理的pH/酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV- PEG-PAMAM+Papain)分别进行动态光散射实验,检测处理前后的纳米载体的粒径变化。结果发现,经木瓜蛋白酶处理后,纳米载体的粒径从67.19nm减小到35.94nm,这表明 GFLG对木瓜蛋白酶对敏感,Mp-GFLG从Mp-GFLG-PKKKRKV-PEG-PAMAM上掉落,细胞核靶向肽PKKKRKVC完全暴露在纳米载体外围且纳米载体的粒径减小,细胞核靶向肽 PKKKRKVC更好的发挥靶向细胞核的功能,使得纳米载体穿过核膜进入细胞核。
图5是一种pH/酶双重响应的靶向细胞核的纳米载体在有无经木瓜蛋白酶(Papain)处理的透射电镜图。A:Mp-GFLG-PKKKRKV-PEG-PAMAM,B:Mp-GFLG-PKKKRKV-PEG-PAMAM+Papain。
将pH/酶双重响应的细胞核靶向纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM)以及经木瓜蛋白酶处理的pH/酶双重响应的细胞核靶向纳米载体(Mp-GFLG-PKKKRKV-PEG- PAMAM+Papain)分别进行透射电镜实验,检测纳米颗粒的形貌、粒径。结果发现,有无经木瓜蛋白酶处理的纳米颗粒均呈球形,且粒径减小,这与动态光散射的检测结果一致。
图6是纳米递送载体在空载与载药情况下对HepG2细胞活度的影响。其中VPP/DOX:PKKKRKV-PEG-PAMAM/DOX,MGPP/DOX:Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX。
将空载的pH/酶双重响应的细胞核靶向纳米载体以及载有药物DOX的各载药纳米体系分别与肝癌HepG2细胞孵育24h,孵育结束后采用酶标仪检测490nm下每孔的吸光值(OD值)。结果发现,一种pH/酶双重响应的靶向细胞核的纳米载体(Mp-GFLG- PKKKRKV-PEG-PAMAM)对肿瘤细胞存活无明显影响(A)。而载药后的各组中,pH和酶双重响应的靶向细胞核的载药纳米体系(Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX)(pH 6.8)具有最高的细胞毒性(B)。综合以上结果,一种pH/酶双重响应的细胞核靶向纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM)具有优良的生物安全性,上载药物后,该载药纳米体系具有较高的细胞毒性,能有效抑制肿瘤细胞的生长。
图7是HepG2细胞对修饰不同配体的载药纳米体系的摄取能力。其中VPP/DOX:PKKKRKV-PEG-PAMAM/DOX,MGPP/DOX:Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX。
各载药纳米体系分别与肝癌HepG2细胞孵育4h,采用Hoechst33432染料对孵育后的细胞核进行染色,利用荧光显微镜观察HepG2细胞对各载药纳米体系的摄入情况。结果发现,与PEG-PAMAM/DOX、Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX(pH 7.4)相比,细胞对Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX(pH 6.8)的摄入量最高,除此之外, PKKKRKV-PEG-PAMAM/DOX也具有相对较高的细胞摄入量,这表明在正常组织(pH 7.4)中,Mp呈现中性,且Mp-GFLG可在一定程度上遮蔽PKKKRKV所带的正电荷。在肿瘤组织(pH 6.8)中,Mp质子化,发生电荷反转,从pH 7.4条件下的不带电荷转变为pH 6.8条件下的带正电荷,促进肿瘤细胞对纳米体系的摄取。当纳米体系被肿瘤细胞内吞后,溶酶体中的组织蛋白酶B可使GFLG断裂,Mp-GFLG从Mp-GFLG-PKKKRKV-PEG- PAMAM/DOX上掉落,细胞核靶向肽PKKKRKVC完全暴露在纳米载体外围,细胞核靶向肽PKKKRKVC更好的发挥靶向细胞核的功能,使得纳米载体更有效的穿过核膜进入细胞核。值得注意的是,细胞对游离DOX的摄取量高于Mp-GFLG-PKKKRKV-PEG- PAMAM/DOX(pH 6.8),这主要是由于小分子DOX通过自由扩散的方式进入细胞。
图8是各载药纳米体系的体内抗肿瘤功效。其中VPP/DOX:PKKKRKV-PEG- PAMAM/DOX,MGPP/DOX:Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX。
将磷酸盐缓冲液(PBS)、游离DOX、上载阿霉素的PAMAM(PAMAM/DOX)、嫁接聚乙二醇的载药纳米体系(PEG-PAMAM/DOX)、修饰细胞核靶向肽的载药纳米体系 (PKKKRKV-PEG-PAMAM/DOX)以及pH/酶双重响应的靶向细胞核的载药纳米体系(Mp- GFLG-PKKKRKV-PEG-PAMAM/DOX)分别经尾静脉注射入肝癌移植瘤小鼠模型体内,隔天注射1次,共注射7次,治疗时间共计14天。在治疗期间,每天测量小鼠的体重及肿瘤大小(肿瘤最长径×肿瘤最短经2/2),治疗结束后,将小鼠安乐处死,取出肿瘤,拍照并称重。结果发现,pH/酶双重响应的靶向细胞核的纳米体系(Mp-GFLG-PKKKRKV-PEG- PAMAM/DOX)抗肿瘤生长效果最好,抑瘤率最高,并且小鼠体重一直呈现缓慢增长的趋势。
具体实施方式
结合实施例对本发明作进一步的说明,应该说明的是,下述说明仅是为了解释本发明,并不对其内容进行限定。
实施例1:
一种pH/酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV-PEG-PAMAM) 的制备:
1.聚乙二醇-聚酰胺胺(PEG-PAMAM)的合成:吸取90μL的20%W/V的PAMAM 甲醇溶液至圆底烧瓶中,旋蒸后,在烧瓶中加入1mLpH 8.0的PBS溶液,待溶解后超声10 min。然后在避光条件下称取28mg NHS-PEG-MAL并将其溶解于1mLpH为8的PBS溶液中,溶解后将其逐滴滴入到含有PAMAM的烧瓶中,溶液呈现淡淡的粉色,在室温下摇床反应4h。将反应后的混合物转移到透析袋中(分子截留量为7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM伯胺组只与双功能PEG的末端NHS反应。
2.吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG-PAMAM)的合成:将步骤(1)中所得到的PEG-PAMAM结合物重新溶解到PBS(pH 7.4)中。然后,称取10.56mg Mp-GFLG-PKKKRKVC溶于1mL PBS(pH 7.4),逐滴加入上述反应体系后,室温下摇床反应24h。将反应后的混合物转移到透析袋中(分子截留量为 7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM-PEG的末端MAL基团只与Mp-GFLG-PKKKRKVC的巯基反应。
实施例2:
一种pH和酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV-PEG- PAMAM)的生物安全性检测,其步骤如下:
1.聚乙二醇-聚酰胺胺(PEG-PAMAM)的合成:吸取90μL的20%W/V的PAMAM 甲醇溶液至圆底烧瓶中,旋蒸后,在烧瓶中加入1mL pH 8.0的PBS溶液,待溶解后超声10 min。然后在避光条件下称取28mg NHS-PEG-MAL并将其溶解于1mLpH为8的PBS溶液中,溶解后将其逐滴滴入到含有PAMAM的烧瓶中,溶液呈现淡淡的粉色,在室温下摇床反应4h。将反应后的混合物转移到透析袋中(分子截留量为7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM伯胺组只与双功能PEG的末端NHS反应。
2.吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG-PAMAM)的合成:将步骤(1)中所得到的PEG-PAMAM结合物重新溶解到PBS(pH 7.4)中。然后,称取10.56mg Mp-GFLG-PKKKRKVC溶于1mL PBS(pH 7.4),逐滴加入上述反应体系后,室温下摇床反应24h。将反应后的混合物转移到透析袋中(分子截留量为 7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM-PEG的末端MAL基团只与Mp-GFLG-PKKKRKVC的巯基反应。
3.将步骤2中制备的一种pH和酶双重响应的靶向细胞核的纳米载体进行透射电镜观察、动态光散射分析。结果发现,经木瓜蛋白酶处理后,纳米载体的粒径从67.19nm减小到35.94nm,这表明GFLG对木瓜蛋白酶对敏感,Mp-GFLG从Mp-GFLG-PKKKRKV-PEG- PAMAM上掉落,细胞核靶向肽PKKKRKVC完全暴露在纳米载体外围且纳米载体的粒径减小,细胞核靶向肽PKKKRKVC更好的发挥靶向细胞核的功能,使得纳米载体更有效的穿过核膜进入细胞核。(图4、图5)
4.将不同浓度(5-125μg/mL)的步骤2中制备的一种pH和酶双重响应的靶向细胞核的纳米载体Mp-GFLG-PKKKRKV-PEG-PAMAM(pH 7.4)和Mp-GFLG-PKKKRKV-PEG- PAMAM(pH6.8)分别与肝癌HepG2细胞共培养,然后用酶标仪检测细胞的增殖和活力变化。结果发现,一种pH和酶双重响应的靶向细胞核的纳米载体(Mp-GFLG-PKKKRKV- PEG-PAMAM)对肿瘤细胞存活均无明显影响。就pH和酶双重响应的靶向细胞核的纳米载体而言,即使在125μg/mL的浓度下孵育24h,细胞存活率仍高于85%,证明这种pH和酶双重响应的靶向细胞核的纳米载体具备优良的生物安全性。(图6)
实施例3:载有化疗药物阿霉素的一种pH和酶双重响应的靶向细胞核的载药纳米体系的制备方法,其步骤如下:
1.聚乙二醇-聚酰胺胺(PEG-PAMAM)的合成:吸取90μL的20%W/V的PAMAM 甲醇溶液至圆底烧瓶中,旋蒸后,在烧瓶中加入1mLpH 8.0的PBS溶液,待溶解后超声10 min。然后在避光条件下称取28mg NHS-PEG-MAL并将其溶解于1mLpH为8的PBS溶液中,溶解后将其逐滴滴入到含有PAMAM的烧瓶中,溶液呈现淡淡的粉色,在室温下摇床反应4h。将反应后的混合物转移到透析袋中(分子截留量为7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM伯胺组只与双功能PEG的末端NHS反应。
2.吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG-PAMAM)的合成:将步骤(1)中所得到的PEG-PAMAM结合物重新溶解到PBS(pH 7.4)中。然后,称取10.56mg Mp-GFLG-PKKKRKVC溶于1mL PBS(pH 7.4),逐滴加入上述反应体系后,室温下摇床反应24h。将反应后的混合物转移到透析袋中(分子截留量为 7000Da),在500mL的去离子水体系中透析2天。透析结束后,将其转移到烧杯中冷冻干燥。在这一阶段,PAMAM-PEG的末端MAL基团只与Mp-GFLG-PKKKRKVC的巯基反应。
3.称取10mg的DOX,溶于5mL的去离子水,转移至含有20mg Mp-GFLG- PKKKRKV-PEG-PAMAM的2mL去离子水溶液中,室温下避光摇床反应24h。反应结束后,将产物转移至透析袋中(透析袋截留分子量为7000Da),避光透析12h,冷冻干燥后,得到负载化疗药物阿霉素的一种pH和酶双重响应的靶向细胞核的纳米体系,称重。
4.收集对数生长期的HepG2细胞,按照每孔4×105-5×105个细胞的密度种在六孔板中,置于5%CO2、37℃条件下培养24小时,弃掉原培养液,将游离DOX、PEG- PAMAM/DOX、PKKKRKV-PEG-PAMAM/DOX和步骤1中制备的一种载有化疗药物阿霉素的pH/酶双重响应的靶向细胞核的纳米载体系分别与肝癌HepG2细胞共培养,4h后,去除孵育药物并用PBS清洗3-5次,加入Heochst33435孵育10min后,去除多余染料并用PBS 清洗3次,对细胞核进行染色,在荧光显微镜下观察细胞中DOX的荧光信号密度。结果发现,细胞对Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX(pH 6.8)的摄入量较多,证明在特异性配体(Mp-GFLG-PKKKRKVC)的介导下,促进了肿瘤细胞对pH和酶双重响应的靶向细胞核的纳米体系的摄取以及该纳米体系的进入肿瘤细胞后的核内活性转运。值得注意的是,细胞值得注意的是,细胞对游离DOX的摄取量高于Mp-GFLG-PKKKRKV-PEG- PAMAM/DOX(pH 6.8),这主要是由于小分子DOX通过自由扩散的方式进入细胞。(图 7)
实施例4:
一种载有化疗药物阿霉素的pH/酶双重响应的靶向细胞核的纳米体系在制备治疗或预防肝癌的局部化疗药物中的应用,其步骤如下:
1.在每只体重在的20g左右的BalB/c雄性小鼠右腋下皮下注射150μL细胞悬液,构建小鼠肝癌移植瘤模型,其中细胞悬液的浓度为2×107个/mL,每天观察并测量肿瘤体积(肿瘤最长径×肿瘤最短经2/2),待肿瘤体积达到150mm3后将小鼠随机分为6组(n=6),用于后续实验。
2.将6组小鼠分别通过尾静脉注射PBS、游离DOX、PEG、PEG-PAMAM/DOX、 PKKKRKV-PEG-PAMAM/DOX以及Mp-GFLG-PKKKRKV-PEG-PAMAM/DOX,DOX的用量为5mg/kg,每只小鼠注射0.2mL,注射结束后将小鼠放回笼中饲养,隔天注射1次,共注射7次,治疗时间共计14天,治疗期间每天记录小鼠的体重和肿瘤体积。
3.治疗结束后,将小鼠安乐处死,取肿瘤组织,拍照并称量肿瘤重量。
4.相比于其他各组,一种载有化疗药物阿霉素的pH和酶双重响应的靶向细胞核的纳米体系发挥了最优的抗肿瘤效果,肿瘤体积增长缓慢,抑瘤率最高,并且小鼠体重呈现缓慢上升的趋势,不存在明显的毒副作用(图8)。
Claims (11)
2.权利要求1所述的一种pH/酶双重响应的靶向细胞核的纳米载体,所述方法包括以下步骤:
(1)聚乙二醇-聚酰胺胺(PEG-PAMAM)的合成:20%W/V的PAMAM甲醇溶液用旋转蒸发器蒸干后,在pH8.0的PBS溶液中溶解、超声,以1:10-15的摩尔比与NHS-PEG-MAL在室温下摇床反应4h,经透析、冷冻干燥后得到MAL-PEG-PAMAM。在这一阶段,PAMAM伯胺组只与双功能NHS-PEG-MAL的NHS末端反应。
(2)吗啉-酶响应四肽-细胞核靶向肽-聚乙二醇-聚酰胺胺(Mp-GFLG-PKKKRKV-PEG-PAMAM)的合成:步骤(1)中所得到的MAL-PEG-PAMAM重新溶解到PBS(pH7.4)中,以1:5-10的摩尔比与Mp-GFLG-PKKKRKVC在室温下摇床反应24h。此时,PAMAM-PEG的末端MAL基团只与Mp-GFLG-PKKKRKVC的巯基反应。
3.根据权利要求1所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:在Mp-GFLG-PKKKRKVC中,Mp为pH敏感小分子,GFLG为酶响应四肽,PKKKRKVC为细胞核靶向肽。
4.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(1)中溶液的溶剂为pH为8.0的磷酸缓冲液(PBS)。
5.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(1)中PAMAM的分子量为14215g/mol;MAL-PEG-NHS的分子量为2000g/mol。
6.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(1)中PAMAM溶液的浓度为15-25mg/mL;MAL-PEG-NHS溶液浓度为10-20mg/mL。
7.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(1)中NHS-PEG-MAL与PAMAM反应时需避光。
8.根据权利要求2所述的一种pH/酶双重响应的细胞核靶向纳米载体的制备方法,其特征在于:所述步骤(1)中超声条件为20-200W,超声时间为5-15min。
9.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(2)中Mp-GFLG-PKKKRKVC的分子量为1501.38g/mol。
10.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(2)中Mp-GFLG-PKKKRKVC溶液浓度为10-15mg/mL。
11.根据权利要求2所述的一种pH/酶双重响应的靶向细胞核的纳米载体的制备方法,其特征在于:所述步骤(1)-(2)中透析条件为:透析膜为生物技术再生纤维(RC)膜,其截留分子量为7000Da,在去离子水中透析1-3天,每隔5-10h更换一次透析液。
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