CN115554411B - 一种酶响应的肿瘤逐级靶向药物递送系统 - Google Patents
一种酶响应的肿瘤逐级靶向药物递送系统 Download PDFInfo
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Abstract
本发明公开了一种酶响应的肿瘤逐级靶向药物递送系统,属于药物制剂技术领域。该酶响应的肿瘤逐级靶向药物递送系统包括阳离子纳米粒内核,及利用静电吸附作用在所述阳离子纳米粒表面包覆的具有酶降解特性的透明质酸外壳。由于透明质酸可有效掩盖阳离子纳米粒的正电性,并且可以与肿瘤细胞表面高表达的CD44受体进行结合,此外,在肿瘤部位高水平透明质酸酶的作用下,透明质酸外壳降解,阳离子纳米粒内核暴露,制剂恢复正电性,因此,该透明质酸包覆的阳离子纳米粒药物递送系统可以克服体内多重生物屏障,实现稳定循环、肿瘤高效靶向、深层渗透、快速摄取、溶酶体逃逸的肿瘤逐级靶向作用。
Description
技术领域
本发明属于药物制剂技术领域,具体涉及一种酶响应的肿瘤逐级靶向药物递送系统。
背景技术
近年来,纳米药物被广泛应用于肿瘤的治疗中,但是由于体内环境及肿瘤病理环境的复杂性造成的一系列生理屏障,如血液循环中网状内皮系统的消除作用、肿瘤部位的高间质压、细胞膜的屏障效应、溶酶体的降解作用等,使得纳米制剂的抗肿瘤活性受到了严重的抑制。因此,理想的肿瘤治疗用纳米药物需要具备稳定的血液循环、高效肿瘤靶向富集、肿瘤深层渗透、快速细胞摄取及溶酶体逃逸等逐级靶向特性。
阳离子纳米粒表面具有较高密度的正电荷,可有效克服细胞膜屏障快速内化入胞;阳离子纳米粒进入溶酶体后,为维持溶酶体电中性,氯离子内流入细胞,进而引起大量水分子快速进入溶酶体内,最终导致溶酶体膨胀裂解,阳离子纳米粒实现溶酶体逃逸;胞质内大量的阳离子纳米粒在转胞吞作用下可进一步实现肿瘤的深层渗透。此外,阳离子纳米粒还可通过静电作用负载多种类型药物,如基因和蛋白药物。因此,阳离子纳米粒具有较大潜力成为抗肿瘤药物的理想载体。但是阳离子纳米粒的在体毒性较大,表面较强的正电性也使其在血液循环中易被网状内皮系统吞噬清除,具有较短的半衰期,严重限制了其在抗肿瘤药物递送中的应用。此外,大量研究对肿瘤组织的高通透性和滞留效应(Enhancedpermeability and retention effect,EPR)在被动靶向抗肿瘤纳米药物递送系统中的有效性提出了质疑,因此,以被动靶向作为唯一靶向机制的抗肿瘤纳米药物靶向效率较低,在临床转化方面欠缺一定的可行性。
利用阴离子材料对阳离子纳米粒进行修饰以掩盖其正电性是降低阳离子纳米粒的毒性,并提高其半衰期和靶向性的有效方式,近年来,研究者也开发了一系列相应的药物递送系统。专利CN113577305A公开了一种白蛋白修饰的阳离子脂质体,阴离子白蛋白通过静电作用包覆在阳离子脂质体表面,屏蔽了脂质体的正电性,提高阳离子脂质体安全性的同时也延长了制剂半衰期。但是白蛋白的结合不具有可逆性,在到达肿瘤部位时,制剂难以脱去白蛋白外壳恢复阳离子脂质体正电性,导致后续细胞摄取、肿瘤渗透受到抑制,难以实现高效抗肿瘤活性。此外,修饰后的脂质体仍只具备EPR效应介导的被动靶向作用,靶向能力未得到提高。
专利CN112569369A公开了一种阳离子胶束,在其表面通过静电作用吸附有阴离子岩藻聚糖,岩藻聚糖的修饰赋予了制剂主动靶向作用,掩盖了阳离子胶束的正电性,可有效提高制剂的半衰期,但是与上述专利类似,制剂在到达靶部位时,无法恢复阳离子胶束本身的正电性。
专利CN112089704A公开了一种在阳离子白蛋白纳米粒表面包覆免疫细胞膜和肿瘤细胞膜组成的杂化细胞膜的仿生载体。细胞膜的包覆可掩盖阳离子纳米粒的表面正电荷,有效延长阳离子纳米粒半衰期,免疫细胞膜和肿瘤细胞膜的同源靶向和归巢效应可提高制剂的靶向性。但是,在全身给药时肿瘤细胞膜的存在具有较高的风险性,此外,制剂到达肿瘤部位后,无法重新恢复阳离子纳米粒的正电性,导致制剂细胞内化及肿瘤深层渗透受到抑制。
因此,如何实现阳离子纳米粒正电性的可逆掩蔽,即在血液循环中正电性被掩盖,到达肿瘤部位时重新恢复正电性,并提高其靶向效率是肿瘤逐级靶向递药系统开发领域的研究难点和热点。
发明内容
本发明的目的之一是提供一种酶响应的肿瘤逐级靶向药物递送系统。该递送系统包括阳离子纳米粒内核,及利用静电吸附作用在阳离子纳米粒内核表面包覆的具有酶降解特性的透明质酸外壳。
在本发明中,所述阳离子纳米粒内核选自阳离子脂质体、阳离子胶束、阳离子白蛋白纳米粒或者阳离子聚合物-药物结合物纳米粒。
进一步地,所述阳离子脂质体由阳离子脂质、助磷脂和胆固醇组成。
更进一步地,所述阳离子脂质选自氯化三甲基-2,3-二油烯氧基丙基铵、溴化三甲基-2,3-二油酰氧基丙基铵、三氟乙酸二甲基-2,3-二油烯氧基丙基-2-(2-精胺甲酰氨基)乙基铵、溴化三甲基十二烷基铵、溴化三甲基十四烷基铵、溴化三甲基十六烷基铵、溴化二甲基双十八烷基铵、溴化二甲基-2-羟乙基-2,3-二油酰氧基丙基铵、溴化二甲基-2-羟乙基-2,3-二油烯氧基丙基铵、溴化二甲基-3-羟丙基-2,3-二油烯氧基丙基铵、溴化二甲基-4-羟丁基-2,3-二油烯氧基丙基铵、溴化二甲基-5-羟戊基-2,3-二油烯氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十六烷氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十八烷氧基丙基铵、溴化二甲基-2-羟乙基-2,3-双十四烷氧基丙基铵、N-(2-精胺甲酰基)-N’,N’-双十八烷基甘氨酰胺、1,2-二油酰-3-琥珀酰-sn-甘油胆碱酯、脂质多聚-L-赖氨酸中的一种或多种的组合。在本发明的一个具体实施例中,优选溴化三甲基-2,3-二油酰氧基丙基铵。
所述助磷脂选自大豆卵磷脂、蛋黄卵磷脂、二硬脂酰磷脂酰胆碱、二油酰磷脂酰乙醇胺中的一种或多种的组合。在本发明的一个具体实施例中,优选大豆卵磷脂。
进一步地,阳离子胶束的单体为接枝共聚物。接枝共聚物的亲水性骨架选自聚乙烯亚胺、壳聚糖、聚精氨酸、聚赖氨酸中的一种或多种的组合;在本发明的一个具体实施例中,优选聚乙烯亚胺。接枝共聚物的疏水性嵌段选自硬脂酸、油酸、聚乳酸、聚乳酸-羟基乙酸共聚物、聚己内酯中的一种或多种的组合;在本发明的一个具体实施例中,本发明优选硬脂酸。
进一步地,阳离子白蛋白纳米粒由阳离子化合物对白蛋白进行修饰后制备得到。其中阳离子化合物选自乙二胺、三乙胺、赖氨酸、精氨酸、寡聚赖氨酸、寡聚精氨酸中的一种或多种的组合;在本发明的一个具体实施例中,优选乙二胺。白蛋白选自人血清白蛋白、牛血清白蛋白、卵白蛋白、重组人白蛋白中的一种或多种的组合,在本发明的一个具体实施例中,优选牛血清白蛋白。
进一步地,阳离子聚合物-药物结合物纳米粒的阳离子聚合物,选自聚乙烯亚胺、壳聚糖、聚精氨酸或聚赖氨酸中的一种或多种的组合,在本发明的一个具体实施例中,优选聚乙烯亚胺;药物为疏水性药物,选自紫杉醇、阿霉素、多西他赛、喜树碱、雷帕霉素、万古霉素或其衍生物中的一种或多种的组合,在本发明的一个具体实施例中,优选紫杉醇。
在本发明中,透明质酸粘均分子量为3000-1000000,优选为10000-200000,更优选为10000-20000。
本发明的另一目的是提供所述酶响应的肿瘤逐级靶向药物递送系统的制备方法。具体是先制备阳离子纳米粒,然后取适量阳离子纳米粒溶液与透明质酸溶液进行搅拌即得。该方法操作简单、绿色经济。
进一步地,阳离子纳米粒与透明质酸的质量比为1:20~20:1,搅拌速度为100~2000rpm,温度为4~60℃,搅拌时间为5~240min。
本发明的第三个目的是提供上述酶响应的肿瘤逐级靶向药物递送系统在制备肿瘤治疗药物的应用。
本发明利用透明质酸通过静电作用结合在阳离子纳米粒表面,可实现阳离子纳米粒正电荷的可逆掩蔽,从而具有稳定血液循环、高效肿瘤靶向、肿瘤深层渗透、高效细胞摄取、快速溶酶体逃逸等优势,此外,本发明还提供了可应用于逐级靶向系统透明质酸的最佳分子量范围。
与现有技术相比,本发明提出的酶响应的肿瘤逐级靶向药物递送系统具有以下有益效果:
(1)透明质酸通过静电作用结合在阳离子纳米粒表面后可有效掩盖阳离子纳米粒的正电荷,此外,透明质酸具有较高的生物相容性,因此制剂在体循环中具有较高的稳定性和安全性;
(2)透明质酸仅通过静电作用与阳离子纳米粒结合,充分保留了其主动靶向特性,与EPR效应介导的被动靶向共同实现肿瘤部位的高效特异性富集;
(3)制剂到达肿瘤部位时,透明质酸被肿瘤微环境高表达的透明质酸酶降解,阳离子纳米粒暴露,制剂恢复正电荷,可有效克服细胞膜屏障快速内化入胞;
(4)阳离子纳米粒快速入胞进入溶酶体后,可通过质子海绵效应实现溶酶体逃逸,实现胞质靶向;
(5)胞质内大量的阳离子纳米粒在转胞吞作用下可进一步实现肿瘤的深层渗透。
附图说明
图1为实施例1中阳离子聚合物-药物结合物纳米粒及不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒的电镜图;
图2为实施例1中不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒在透明质酸酶孵育条件下的电位变化情况;
图3为实施例1中聚合物-药物结合物纳米粒、不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒在体内的靶向分布情况;
图4为实施例1中聚合物-药物结合物纳米粒、不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒在肿瘤球中的渗透情况;
图5为实施例1中不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒在透明质酸酶孵育条件下的细胞摄取情况;
图6为实施例1中阳离子聚合物-药物结合物纳米粒及不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒的溶酶体逃逸情况;
图7为实施例1中游离药物、阳离子聚合物-药物结合物纳米粒及不同分子量透明质酸包覆的阳离子聚合物-药物结合物纳米粒的体内抗肿瘤药效情况。
具体实施方式
透明质酸(Hyaluronic acid,HA)是一种天然的阴离子大分子多糖,具有良好的安全性和生物相容性,可通过静电作用包覆在阳离子纳米粒表面以掩盖其正电性,从而提高阳离子纳米粒半衰期,并降低全身给药带来的毒性;许多肿瘤细胞表面过度表达HA受体CD44,透明质酸的包裹还赋予了制剂主动靶向特性,与EPR效应共同实现肿瘤部位高效富集;此外,HA还可被肿瘤区域高水平的透明质酸酶(Hyal)降解,因此在制剂到达肿瘤部位时,透明质酸外壳会被降解,暴露阳离子纳米粒,制剂成功恢复正电性,从而实现阳离子纳米粒正电性的可逆掩蔽。
专利CN109550057A将透明质酸通过电荷作用和化学键结合作用包覆于阳离子纳米粒表面,以降低阳离子纳米粒毒性并提高其靶向性,但是,化学键的形成会消耗透明质酸上游离的羧基,而羧基则是透明质酸实现靶向性的关键功能性基团,因此,与本发明提出的仅通过静电作用实现透明质酸修饰阳离子纳米粒的给药系统相比,该专利公开的给药系统的主动靶向性受到了一定的限制。此外,该专利对于透明质酸分子量的说明较为模糊,而透明质酸酶对透明质酸的降解效应与透明质酸分子量有密切关系,其说明书中也未记载所使用透明质酸的分子量,同时未证明透明质酸酶可以对所使用的透明质酸进行降解。专利CN112999363A将透明质酸通过静电作用包覆与阳离子胶束表面,专利CN112999159A将透明质酸通过静电作用包覆与阳离子脂质体表面,但二者仅利用了透明质酸对阳离子纳米粒的电荷掩蔽作用及主动靶向性,均未明确说明所使用透明质酸的分子量,也未证明透明质酸酶可以有效降解制剂外层透明质酸,恢复阳离子纳米粒的正电性。
为实现阳离子纳米粒正电性的可逆掩蔽,并提高靶向效率,本发明提供了一种酶响应的肿瘤逐级靶向药物递送系统,该递送系统包括阳离子纳米粒内核,及利用静电吸附作用在所述阳离子纳米粒表面包覆的具有酶降解特性的透明质酸。
由于透明质酸分子量对其生物学功能(如靶向性、酶降解特性)具有显著影响(AdvDrug Deliv Rev,2016,97:204-36;J Control Release,2010,141:2-12.),本发明通过对比不同分子量透明质酸对制剂性能的影响,成功筛选出可应用于逐级靶向系统透明质酸的最佳分子量范围。
下面结合附图和具体实施例对本发明作进一步详细说明,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照本领域常规条件。
实施例1
透明质酸包覆的阳离子聚合物-药物结合物纳米粒的制备与表征
1.阳离子聚合物-药物(聚乙烯亚胺-紫杉醇)结合物的合成
称取紫杉醇(PTX)100mg(0.117mmol),溶于5mL N,N-二甲基甲酰胺,在搅拌条件下依次加入14.30mg 4-二甲氨基吡啶(0.117mmol)、17.60mg琥珀酸酐(0.176mmol)和25μL三乙胺(0.179mmol),继续室温搅拌反应12h。反应结束后,置于旋转蒸发仪上旋至近干,加入适量0.01M稀盐酸沉淀产物,8000rpm冷冻离心10min,弃去上清,重复上述步骤三次。之后加入适量纯水洗涤沉淀,冷冻离心,弃去上清,重复三次,将最后一次离心所得沉淀真空干燥48h,得到白色固体,即为羧基化紫杉醇(PTX-COOH)。
称取25mg PTX-COOH(0.0262mmol),溶于2.5mL甲醇,在搅拌条件下加入20.10mg1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(0.105mmol)及12.06mg N-羟基琥珀酰亚胺(0.105mmol),室温持续搅拌活化30min。之后加入25mg聚乙烯亚胺(PEI,0.0025mmol),在室温下继续搅拌24h。反应结束后将反应液转移至透析袋(MWCO:7000Da)中,于甲醇中透析24h,接着转移至纯水中透析24h,透析结束后取出透析袋内液体冷冻干燥48h,可得白色疏松固体,即为聚乙烯亚胺-紫杉醇结合物。
2.聚乙烯亚胺-紫杉醇阳离子纳米粒的制备
称取冻干后的聚乙烯亚胺-紫杉醇结合物溶解在水溶液中(浓度为2mg/mL),即自组装成阳离子纳米粒PgP。
3.透明质酸包覆的聚乙烯亚胺-紫杉醇阳离子纳米粒(HAPs)的制备
粒径和电位作为纳米药物的关键质量属性对制剂性能有较大影响,HA与PgP的质量比以及HA的浓度是影响粒径和电位的关键因素,经过发明人前期的预实验得知,由于电荷相互作用,当HA与PgP的质量比超过一定范围时会导致体系不稳定,产生沉淀。因此,需要选择通过筛选PgP与HA的质量比以及HA的浓度得到稳定性良好且粒径和电位接近的系列HAPs,进而保证不同HAPs的体内外性能差异仅由HA分子量的不同导致。
称取粘均分子量10000、20000、50000、100000、200000的HA各20mg,配制成相应浓度溶液。分别吸取不同分子量的HA溶液适量置于西林瓶中,在1000rpm搅拌条件下向西林瓶中逐滴加入适量PgP溶液,滴加完毕继续室温搅拌30min,得到HAP10、HAP20、HAP50、HAP100及HAP200。通过筛选,采用表1所示条件制备HAP10、HAP20、HAP50、HAP100及HAP200。由表1粒径和电位结果可知,不同分子量HA的修饰均可有效掩盖阳离子聚合物-药物结合物纳米粒正电荷,粒径接近,均适用于全身给药。
表1
制剂 | HA与PgP质量比 | HA浓度(mg/mL) | 粒径(nm) | PDI | 电位(mV) |
PgP | / | / | 154.0±6.1 | 0.250±0.011 | 49.5±0.5 |
HAP10 | 20:1 | 1 | 179.4±4.4 | 0.150±0.034 | -30.1±0.6 |
HAP20 | 10:1 | 1 | 184.9±4.8 | 0.167±0.046 | -33.6±1.3 |
HAP50 | 2:1 | 2 | 187.8±2.4 | 0.179±0.005 | -30.2±1.2 |
HAP100 | 2:1 | 1 | 165.8±3.4 | 0.173±0.034 | -31.4±0.6 |
HAP200 | 2:1 | 1 | 176.7±3.1 | 0.226±0.006 | -32.3±0.3 |
此外,发明人还利用3000分子量的HA制备了HAPs,制备过程中发现HA与PgP的质量比在一定范围内时,PgP的正电荷并未得到完全掩蔽,制剂仍呈现正电性,推测原因可能是由于HA分子量较小,链长较短,不能与PgP充分结合,大部分HA以游离形式存在。然而随着HA用量的进一步增加,尽管PgP的正电荷得到了一定程度的掩蔽,但是溶液稳定性随之变差,产生沉淀。因此,3000分子量的HA无法在保证制剂稳定性的前提下实现阳离子纳米粒正电荷的成功掩蔽,与其他分子量的HA相比具有明显劣势。
4.制剂形态观察
吸取HAP10、HAP20、HAP50、HAP100及HAP200溶液约10μL滴加至表面膜化处理的铜网上,静置5min,使样品充分沉积至铜网,用滤纸吸干周围多余的样品溶液。随后,向铜网上滴加1%磷钨酸溶液20μL,静置染色5min,然后用滤纸小心吸干磷钨酸溶液,置于红外灯下烤干,转移至透射电子显微镜中观察各组制剂的外观形态,结果如图1所示,各制剂均呈规整的球形或类球形,另外,在经HA包覆后,可在纳米粒表面明显地观察到一圈透明质酸层,也说明了HA外壳的成功形成。
5.透明质酸酶(Hyal)降解性质考察
取HAP10、HAP20、HAP50、HAP100及HAP200溶液适量,向其中加入适量Hyal(2mg/mL)后,置于恒温振荡器上振摇(37℃,100rpm),于不同时间点(0h、1h、2h、4h、6h)取出适量,测定电位。由图2可知,在孵育1h后,HAPs的电位均显著减小,逐渐向正电荷翻转,而当孵育时间延长至2h时,HAPs的电位均发生由负向正的翻转,说明HAPs具有透明质酸酶响应特性,能够被肿瘤组织高表达的Hyal识别并脱去外层HA,暴露出内核,重新恢复阳离子纳米粒正电性。此外,HAP10及HAP20在不同时间点的电位均显著高HAP50、HAP100及HAP200,表明与其它HAPs相比,HAP10及HAP20的酶响应速度最快。
6.体内靶向研究
建立原位乳腺癌(4T1)小鼠模型,待肿瘤体积长至200mm3,对小鼠尾静脉注射DiR荧光标记的PgP及HAPs,分别于给药后1h、3h、6h及24h将小鼠麻醉并置于小动物活体成像仪中荧光成像。给药24h时将各组小鼠处死,并取出主要脏器(心、肝、脾、肺和肾)及肿瘤,对各组织荧光成像评价各制剂的体内分布情况,活体成像及组织成像结果如图3所示。在进入体内后,游离DiR快速被机体消除,基本无肿瘤蓄积。DiR/PgP在体内的分布情况与游离DiR相似,主要分布在肝脏和肾脏部位,说明进入体内后快速被肝肾代谢清除,具有较短半衰期。而DiR/HAP10、DiR/HAP20、DiR/HAP50、DiR/HAP100和DiR/HAP200注射入体内后迅速分布至全身,在3h时开始出现肿瘤部位的聚集,6h时肿瘤部位的荧光强度显著增加,待给药24h后取出心、肝、脾、肺、肾和肿瘤进行荧光成像,发现DiR/HAP10、DiR/HAP20、DiR/HAP50、DiR/HAP100和DiR/HAP200组在肿瘤部位的荧光强度逐步降低,表明HA分子量对HAPs的靶向性具有一定的影响,10000分子量HA的修饰具有最强的靶向能力。
7.肿瘤深层渗透实验
体外建立4T1乳腺癌3D肿瘤球模型,将香豆素6(C6)标记的PgP及HAPs与肿瘤球共孵育12小时,然后利用激光共聚焦显微镜观察不同制剂渗透情况,结果如图4所示,C6/PgP、C6/HAP10、C6/HAP20、C6/HAP50、C6/HAP100及C6/HAP200均能够渗透到80μm左右的深度,而C6/HAP100及C6/HAP200在80μm深度时已经开始出现肿瘤球边缘较中心区域亮度更强的现象,表明这两组制剂的渗透能力不及C6/PgP、C6/HAP10、C6/HAP20、C6/HAP50。另外,从绿色荧光强度的变化可以看出,C6/PgP组的荧光强度最高,显然,这是由于C6/PgP表面具有高正电荷密度,因此渗透的效果最好。C6/HAP10、C6/HAP20、C6/HAP50渗透强度依次减小主要是由于透明质酸分子量越低,被透明质酸酶降解的速度就越快,从而加快电荷翻转,暴露正电性内核,进一步实现更佳的深层渗透功效。
8.摄取实验
预先将C6/HAPs与Hyal在37℃条件下孵育1h,随后与4T1细胞共孵育4小时,孵育结束后收集细胞进行流式检测,考察Hyal对C6/HAP10、C6/HAP20、C6/HAP50、C6/HAP100及C6/HAP200摄取量的影响。由图5可知,与未经处理的C6/HAPs相比,各组C6/HAPs加透明质酸酶处理后细胞摄取率均有不同程度的增加,且C6/HAP10、C6/HAP20、C6/HAP50、C6/HAP100及C6/HAP200的增幅呈现依次降低的趋势,C6/HAP10和C6/HAP20组的摄取量均增大为原来的2倍左右,而C6/HAP100和C6/HAP200组的摄取量仅增大为原来的1.3倍左右,造成该现象的原因可能是各组HAPs对Hyal的响应速度存在差异,低分子量的HA更容易发生降解,因此更快暴露出正电内核增加摄取量。
9.溶酶体逃逸实验
利用C6(绿色)标记PgP及HAPs,溶酶体探针(红色)标记溶酶体,Hoechst(蓝色)标记细胞核,制剂与细胞共孵育1h、3h、6h时进行荧光成像,考察各组制剂溶酶体逃逸情况。结果如图6所示,在1h时,各组制剂均散在分布一些黄色荧光,即绿色荧光与红色荧光发生重叠,表明有少部分制剂已经进入溶酶体中。3h时各组出现大量较强的黄色荧光,表明大部分制剂与溶酶体发生共定位。6h时,各组制剂的黄色荧光均较3h时发生显著减少,即大量制剂从溶酶体成功逃出,其中C6/PgP、C6/HAP10和C6/HAP20组基本看不到黄色荧光点,而C6/HAP100及C6/HAP200组的黄色荧光仍相对较多,说明这两组的逃逸能力不及其它几组。C6/HAP10及C6/HAP20组发生大量制剂逃逸的原因可能在于溶酶体中高表达的Hyal能够快速降解C6/HAP10及C6/HAP20的外层HA分子,暴露出由PEI构成的PgP内核,产生质子海绵效应,促进制剂快速逃出溶酶体到细胞质中发挥作用。而C6/HAP100及C6/HAP200组由于对Hyal的响应能力不及C6/HAP10及C6/HAP20组(如细胞摄取结果所示),所以在6h后,仍有相对较多制剂在溶酶体中。
10.药效试验
建立原位乳腺癌(4T1)小鼠模型,待肿瘤体积生长至约50mm3时,将小鼠随机分为8组,每组5只。按照10.0mg/kg PTX的给药剂量尾静脉注射生理盐水、PTX、PgP、HAP10、HAP20、HAP50、HAP100及HAP200,每3天给药一次,共给药4次。至给药第19天时,将小鼠脱颈处死,取出肿瘤块拍照记录,并称取瘤重。如图7所示,与PTX和PgP相比,HAPs显著抑制了肿瘤生长,说明HA的包覆可以显著提高阳离子纳米粒的抗肿瘤活性,并且随着HA分子量的减小,抑瘤效果越佳。
综上,HA的包覆可实现阳离子聚合物-药物结合物纳米粒正电荷的可逆掩蔽,从而实现稳定血液循环、高效肿瘤靶向、肿瘤深层渗透、高效细胞摄取、快速溶酶体逃逸等效果,最终实现高效的抗肿瘤活性,此外,还证明了10000和20000的低分子量HA具有更优异的靶向性和酶降解特性。
实施例2
透明质酸包覆的阳离子脂质体的制备与表征
1.载药阳离子脂质体的制备
精密称取大豆卵磷脂10mg、溴化三甲基-2,3-二油酰氧基丙基铵30mg、胆固醇10mg、紫杉醇4mg于离心管中,加入三氯甲烷5mL溶解后转移至茄型瓶中,旋转蒸发仪设置温度40℃,旋蒸时间2h,除去三氯甲烷,在茄型瓶瓶壁形成一层薄膜,加入生理盐水5mL于制备条件下继续旋转15min,使得薄膜充分水化,将水化得来的初级脂质体于冰浴下探头超声,超声结束后的溶液依次过0.45μm滤膜和0.22μm滤膜,得到具有乳光的阳离子脂质体溶液(Lip@PTX)。
2.透明质酸包覆的阳离子脂质体的制备
称取10000分子量的HA 5mg溶于5mL水中,取含有约2.5mg Lip@PTX的脂质体溶液,800rpm搅拌条件下逐滴加入HA溶液中,滴加完毕后继续搅拌1h,即得HA包覆的阳离子脂质体(HA/Lip@PTX),采用动态光散射法测定Lip@PTX及HA/Lip@PTX电位及粒径,结果如表2所示。由结果可知,HA包覆后,阳离子脂质体粒径增大,并且实现了正电荷的掩蔽。
表2
制剂 | 粒径(nm) | PDI | 电位(mV) |
Lip@PTX | 124.0±3.0 | 0.110±0.014 | 44.9±2.3 |
HA/Lip@PTX | 157.1±6.2 | 0.160±0.035 | -27.1±1.3 |
3.透明质酸酶降解性质考察
取HA/Lip@PTX溶液适量,向其中加入Hyal(2mg/ml)后,置于恒温振荡器上振摇(37℃,100rpm),6h后取出适量,测定电位,电位为13.8±1.4mV,说明HA包覆的阳离子脂质体在到达肿瘤部位时,可以有效脱去外壳,制剂成功实现电荷翻转,进而具备后续肿瘤深层渗透、高效摄取、溶酶体逃逸等性能。
实施例3
透明质酸修饰的阳离子胶束的制备与表征
1.载药阳离子胶束的制备
称取10mg硬脂酸(SA)、13.48mg 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐,8.1mg N-羟基琥珀酰亚胺于茄形瓶中,加入10mL无水乙醇溶解,室温搅拌活化1h,加入100mg分子量10000的PEI,室温反应24h,反应能结束后,依次用无水乙醇和超纯水透析24h,透析结束后冻干,得到胶束单体PEI-SA(PS)。
称取10mg胶束单体PS、1mg PTX于离心管中,加入无水乙醇5mL溶解后转移至茄型瓶中,旋转蒸发仪设置温度45℃,旋蒸时间1h,除去无水乙醇,在茄型瓶瓶壁形成一层薄膜,加入生理盐水2mL于制备条件下继续旋转15min,使得薄膜充分水化,即得载药阳离子胶束PS@PTX。
2.透明质酸包覆的阳离子胶束的制备
称取10000分子量的HA 5mg溶于5毫升水中,取含有约2mg PS@PTX的胶束溶液,1500rpm搅拌条件下逐滴加入HA溶液中,滴加完毕后继续搅拌2h,即得HA包覆的阳离子胶束(HA/PS@PTX),采用动态光散射法测定PS@PTX及HA/PS@PTX电位及粒径,结果如表3所示。由结果可知,透明质酸包覆后,阳离子胶束粒径增大,并且实现了正电荷的掩蔽。
表3
制剂 | 粒径(nm) | PDI | 电位(mV) |
PS@PTX | 151.4±5.4 | 0.220±0.008 | 31.7±3.3 |
HA/PS@PTX | 185.5±6.2 | 0.262±0.014 | -28.4±1.9 |
3.透明质酸酶降解性质考察
取HA/PS@PTX溶液适量,向其中加入Hyal(2mg/ml)后,置于恒温振荡器上振摇(37℃,100rpm),6h后取出适量,测定电位,电位为9.0±1.2mV,说明HA包覆的阳离子胶束在肿瘤部位高表达Hyal的作用下可以有效脱去外壳,成功实现电荷翻转。
实施例4
透明质酸修饰的阳离子白蛋白纳米粒的制备与表征
1.载药阳离子白蛋白纳米粒制备
称取500mg牛血清白蛋白(BSA)溶于5mL超纯水中,随后加入圆底烧瓶中,搅拌条件下缓慢滴加乙二胺溶液(取4mL乙二胺溶液用超纯水稀释至40mL,在用0.1M盐酸调节pH至4.75),称取30mL 1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐缓慢加入上述溶液中,继续搅拌2h,加入400μL pH 4.75的醋酸缓冲液(4mol/L)终止反应,随后超纯水透析48小时,冻干得到阳离子白蛋白cBSA。
称取48mg cBSA与茄形瓶中,加入4.8mL超纯水溶解,搅拌条件下缓慢加入0.333mL紫杉醇乙醇溶液(12mg/ml),室温继续搅拌4h,100W功率探头超声10min(2s on,5s off),即得载药阳离子白蛋白纳米粒cBSA@PTX。
2.透明质酸包覆的阳离子白蛋白纳米粒的制备
称取10000分子量的HA 5mg溶于5mL水中,取含有约5mg cBSA@PTX的阳离子白蛋白纳米粒溶液,1000rpm搅拌条件下逐滴加入HA溶液中,滴加完毕后继续搅拌4h,即得HA包覆的阳离子白蛋白纳米粒(HA/cBSA@PTX),采用动态光散射法测定cBSA@PTX及HA/cBSA@PTX电位及粒径,结果如表4所示。由结果可知,HA包覆后,阳离子白蛋白纳米粒粒径增大,并且实现了正电荷的掩蔽。
表4
制剂 | 粒径(nm) | PDI | 电位(mV) |
cBSA@PTX | 130.6±6.6 | 0.104±0.016 | 35.0±1.9 |
HA/cBSA@PTX | 164.4±5.1 | 0.247±0.014 | -29.6±1.0 |
3.透明质酸酶降解性质考察
取HA/cBSA@PTX溶液适量,向其中加入Hyal(2mg/ml)后,置于恒温振荡器上振摇(37℃,100rpm),6h后取出适量,测定电位,电位为11.3±1.4mV,说明HA包覆的阳离子白蛋白纳米粒在肿瘤部位高表达Hyal的作用下可以有效脱去外壳,成功实现电荷翻转。
Claims (2)
1.药物递送系统在制备肿瘤治疗药物的应用,其特征在于,所述药物递送系统包括阳离子纳米粒内核以及包覆在阳离子纳米粒内核表面的透明质酸外壳;所述阳离子纳米粒为聚乙烯亚胺-紫杉醇阳离子纳米粒;所述透明质酸的粘均分子量为10000-20000;所述阳离子纳米粒与透明质酸的质量比为1:20~20:1。
2. 根据权利要求1 所述的应用,其特征在于,所述药物递送系统的制备方法是先制备得到阳离子纳米粒,然后取阳离子纳米粒溶液与透明质酸溶液进行搅拌即得。
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