CN115624631A - 一种蟾毒灵前药自组装纳米粒及其制备方法与应用 - Google Patents
一种蟾毒灵前药自组装纳米粒及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种蟾毒灵前药自组装纳米粒及其制备方法与应用。蟾毒灵前药自组装纳米粒通过二硫醇二羟基乙酸作为连接剂,通过简单酯化反应将蟾毒灵和脂肪酸乙二醇酯偶联形成前体药物,通过纳米沉淀法可自组装形成纳米粒。该纳米粒粒径范围在50‑250纳米之间,分散性良好,具有谷胱甘肽感应性释放特性,为提高其稳定性和体内循环时间,可与PEG化剂共组装形成PEG化前药自组装纳米粒。该蟾毒灵前药纳米粒制备工艺简单,载药量高。体外细胞实验证明可明显提高蟾毒灵对肿瘤细胞的选择性,降低对正常细胞的毒性。体内动物实验证明可显著提高蟾毒灵的半数致死量,增加蟾毒灵的安全性,有望解决由于安全性低而制约其应用于临床的关键问题。
Description
技术领域
本发明涉及抗肿瘤药物领域,具体涉及一种蟾毒灵前药自组装纳米粒及其制备方法与应用。
背景技术
在中医治疗恶心肿瘤的各种理论中,“以毒攻毒”治疗方法一直受到历代中医学家的重视,至今仍在肿瘤临床中发挥积极作用。随着中药现代化的发展,多名学者从现代医学理论水平证实毒性药材中的活性成分在抗肿瘤治疗中确有显著效果,如砒霜中的三氧化二砷、蟾蜍中的蟾毒灵等。但又因为其毒性特性导致其临床应用受到束缚(如给药剂量限制、需时时监测等)。通过高效传递的肿瘤靶向载体,将先进的纳米传递技术引入中药抗癌活性成分的应用基础研究,有利于促进中药活性成分在临床的应用。
蟾毒灵属于甲型强心苷类,是从中药蟾酥提取得到的抗肿瘤活性成分,为拓扑异构酶II抑制剂,具有抑制肿瘤细胞增殖、阻止肿瘤细胞分裂和诱导肿瘤细胞凋亡等药理作用,对各种恶性肿瘤如胃癌、胰腺癌、肝癌、白血病、肺癌、肠癌等均有明显的抑制和逆转药物耐受性的效果[参见:Wang J, Xia Y, Zuo Q, et al. Molecular and ClinicalOncology. 2018, 8(5): 631-636.]。蟾毒灵是一种有效的多靶点抗肿瘤活性成分,且能通过多方面机制来逆转不同肿瘤细胞耐药性,与其他临床化疗药物(顺铂、紫杉醇、索拉非尼、吉非替尼)联合给药具有良好的协同治疗效果(Wang H, Zhang C, Chi H, et al.International journal of oncology. 2018, 52(6): 2051-2060;Huang H, Lin X, LinY, et al. Indian Journal of Hematology and Blood Transfusion. 2018, 34(2):268-272;Liu F, Tong D, Li H, et al. Oncotarget. 2016, 7(8): 8896)。蟾毒灵能部分通过抑制紫杉醇诱导的AKT活化和上调p38的活性而增强紫杉醇诱导乳腺癌MCF -7细胞凋亡的活性[闫顺朝,焦昕,邹华伟,等. 现代肿瘤医学. 2016, 24(30): 3173-3176.],另一方面,蟾毒灵通过抑制整合素α2/FAK信号抑制AKT1的活性及激活GSK3β的表达,GSK3β的激活可以提高NK细胞对肿瘤细胞的杀伤作用(Jin F,Wu Z, Hu X, et al. Cancer BiolMed. 2019, 16(1): 38-53.)。
临床上尚未有蟾毒灵单体制剂。经大量文献调研与市场咨询,蟾毒灵应用受限的原因主要有三点:问题一,蟾毒灵体内代谢快,蛋白结合率高。虽然蟾毒灵体外显示出优越的抗癌活性,但体内给药后,97%以上与蛋白结合,游离药物也会被快速代谢,导致作用于肿瘤组织的含量非常低。问题二,蟾毒灵的毒性大,治疗窗窄。与紫杉醇相比,蟾毒灵毒性更大。蟾毒灵属于强心甾类固醇(cardiotonic steroids,CTS)中的六元内酯环强心苷类(如蟾蜍甾类、海葱类)化合物,具有明显的心脏毒性。蟾毒灵的耐受量也非常的低,治疗窗窄,给药剂量受限,甚至很难达到治疗效果。问题三,肿瘤递送率低。有关蟾毒灵的组织分布研究结果显示,蟾毒灵在肿瘤组织中的分布相对较少。过去解决上述问题的方法主要有药物结构修饰(前药)及纳米载体递送。前药系指化学改性的、生物惰性的小分子药物,其能在体内转化成有药理活性的药物。前药提供了一种提高药物药理活性的手段,即通过简单化学修饰就可以提高药物药理活性。纳米载体是一种新型的药物传递载体,特别适合包载疏水性药物,通过包裹药物于内部,再利用纳米粒的小尺寸效应、比表面效应和界面效应等性质将药物传递到靶部位,从而达到药物治疗的最终目的。国内外已有报道脂质体、无机和聚合物纳米粒对蟾毒灵进行纳米包裹,并表现出一定的优势,但亦存在很多不足之处,如药物突释及释放缓慢等,特别是载药量和载体材料本身安全性的限制。
中国专利申请CN201510023486.5 《一种基于聚乙二醇的水溶性蟾毒灵大分子前药的制备方法》,是关于通过PEG化改善蟾毒灵的水溶性和系统性毒性,但原料药很难从酯类前药中完全释放出来,不具备肿瘤环境感应释放特性。需要进一步改性处理,提高其应用前景。
发明内容
针对现有技术的不足,本发明提供了一种蟾毒灵前药自组装纳米粒及其制备方法与应用,该自组装纳米粒稳定性强,药效好,且安全性高,制备方法简单,通过体外抗肿瘤活性实验可知,适合应用于肝癌疾病的治疗。
一种蟾毒灵前药自组装纳米粒,以二硫醇二羟基乙酸作为连接剂,将蟾毒灵和脂肪酸乙二醇酯进行偶联形成前体药物,所述前体药物通过纳米沉淀法制备得到蟾毒灵前药自组装纳米粒。
作为改进的是,所述脂肪酸乙二醇酯中脂肪酸酸为亚油酸、油酸、或亚麻酸中的一种。
上述蟾毒灵前药自组装纳米粒的制备方法,包括以下步骤:步骤1,按照摩尔比1:2~10称取脂肪酸和无水乙二醇,加入无水二氯甲烷溶解,再加二环己基碳二亚胺(DCC)和4-二甲氨基吡啶(DMAP),所述二环己基碳二亚胺(DCC)与脂肪酸的摩尔比为1~1.5:1,所述4-二甲氨基吡啶(DMAP)与脂肪酸的摩尔比为0.1~1:1,在密封条件下,连续搅拌反应2~14小时,经过萃取和柱色谱纯化后,得到目标产物脂肪酸乙二醇酯,记为FA-CH2-CH2-OH;
步骤2,将二硫醇二羟基乙酸加入乙酸酐中,在30℃的密封条件下,连续搅拌反应2-4小时后,除掉乙酸酐后,加入无水二氯甲烷溶解油状液体,再加入步骤1的FA-CH2-CH2-OH和4-二甲氨基吡啶,所述FA-CH2-CH2-OH与二硫醇二羟基乙酸的摩尔比为1:4~10, 4-二甲氨基吡啶与二硫醇二羟基乙酸的摩尔比为0.1~1:1,连续搅拌反应2-4小时,经过萃取和柱色谱纯化后,得到目标产物脂肪酸乙二醇二硫醇二羟基乙酸,记为FA-ss-COOH;
步骤3,称取上述步骤2 的产物FA-ss-COOH和蟾毒灵,加入无水二氯甲烷溶解后,再加入DCC和DMAP,所述FA-ss-COOH、蟾毒灵、DCC和DMAP的摩尔比为1︰0.8~1.2︰1~1.5︰0.1~1,常温下连续搅拌反应2-24小时,经过萃取和柱色谱纯化后,得到目标产物脂肪酸-乙二醇-二硫醇二羟基乙酸-蟾毒灵,记为BFL-ss-FA;
步骤4,称取步骤3产物BFL-ss-FA,配制浓度为10 mg/mL~40 mg/mL的有机溶剂溶液,在搅拌条件下,慢慢滴加入双蒸水中,有机溶剂与双蒸水的体积比为1:4~10,旋干除去有机溶剂,自组装得蟾毒灵前药自组装纳米粒,记为BFL-ss-FA-NPs。
作为改进的是,步骤4中有机溶剂为无水乙醇、 DMSO、乙腈、丙酮中的一种或者多种混合溶剂。
作为改进的是,步骤1和步骤2所述的纯化处理的具体形式为硅胶柱纯化、C8、C18填料柱层析或液相制备纯化。
作为改进的是,步骤4中还包括在有机溶剂中添加PEG化剂,且PEG化剂与BFL-ss-FA的质量比为1:1~10。
进一步改进的是,所述PEG化剂为二棕榈酰磷脂酰乙醇胺-聚乙二醇(DSPE2k)、维生素E聚乙二醇琥珀酸酯(TPGS)、或亚油酸-聚乙二醇(LA-PEG2k)。
上述蟾毒灵前药自组装纳米粒在制备抗肿瘤药物中的应用。
一种药物组合物,其有效成分为蟾毒灵前药自组装纳米粒。
作为改进的是,所述药物组合还包括至少一种药学上可接受载体。
有益效果:
与现有技术相比,本发明一种蟾毒灵前药自组装纳米粒及其制备方法与应用,具有如下优势:
1、本发明合成蟾毒灵前药自组装纳米粒,通过纳米沉淀法可制备得到自组装纳米粒,该纳米粒具有谷胱甘肽感应性释放特性,显著提高蟾毒灵对肿瘤细胞的选择性;
2、另外,本发明还可以加入PEG化,所制备得到蟾毒灵前药自组装纳米粒经PEG化后,稳定性提高,半数致死量显著高于原型药,提高蟾毒灵的给药安全性。
附图说明
图1为目标产物(BFL-ss-LA)的1H-NMR图谱;
图2为BFL-ss-LA-NPs和BFL-ss-LA/TPGS-NPs的透射电镜图;
图3为BFL-ss-LA-NPs和BFL-ss-LA/TPGS-NPs在水介质(A)和pH=7.4的PBS(B)介质中的稳定性考察;
图 4为体外释放实验:BFL-ss-LA/TPGS-NPs在含GSH的四种介质(1 mM和10 μMGSH)中的BFL累计释放率;
图5为BFL-ss-LA/TPGS-NPs对HepG2肿瘤细胞的杀伤作用。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
以下实施例中用到的技术均为本领域常规技术手段,所用材料均为现有市售材料,无需特殊说明。
实施例1. 亚油酸-蟾毒灵前药的制备
第一步:分别称取4.28 mmol的亚油酸1.2 g、32.3 mmol的无水乙二醇2 g、5.24mmol的DCC 1.08g和1.11 mmol的DMAP 0.13g在圆底烧瓶里,再加入50 mL的无水二氯甲烷,在30℃水浴条件下,600 rpm连续搅拌反应24小时。滤膜过滤反应液,胶头滴管除去上层乙二醇溶液,现配饱和NaHCO3溶液洗三遍反应液,采用无水硫酸钠干燥二氯甲烷层。最后,采用硅胶柱纯化,用体积比为石油醚:乙酸乙酯=10:1,且乙酸占其百分之二的洗脱剂,湿法装柱,加压,收集流出液,通过薄层色谱法确定目标溶液。再利用旋转真空蒸发仪进行旋转蒸发后得到白色油状物——亚油酸乙二醇酯(LA-CH2-CH2-OH),收集目标产物。经1H-NMR鉴证,1H-NMR (400 MHz, CDCl3),δ5.59-5.03 (4H, m), 4.38-4.13 (2H, t), 3.67 (2H, t),2.84-2.55 (2H, m), 2.36 (2H, t), 2.06 (4H, m), 1.64 (2H, m), 0.95 (3H, t)。
第二步,称取4.40 mmol的二硫醇二羟基乙酸0.8 g在圆底烧瓶中,再加入10 mL的乙酸酐,在30℃的密封条件下,连续搅拌反应2小时。反应完成后,在反应液中加入200mL甲苯,利用旋转蒸发仪加压旋干甲苯,重复4次加甲苯和旋干,观察得到,圆底烧瓶中留有油状液体,往烧瓶里加入10 mL无水二氯甲烷溶解油状液体,加入第一步中1.23 mmol的LA-CH2-CH2-OH 0.4 g和0.1 g DMAP,在30℃水浴条件下,600rpm连续搅拌反应2小时。最后,采用硅胶柱纯化,用正己烷:乙酸乙酯:乙酸体积比为10:1:1的洗脱剂,湿法装柱,每50mL洗脱液收集一瓶,通过薄层板确定目标产物(第7和8瓶),展开剂为正己烷:乙酸乙酯:乙酸体积比为=10:1:1,显色剂为高锰酸钾显色剂,Rf值为0.24。再利用旋转真空蒸发仪进行旋转蒸发后得产物LA-ss-COOH,收集目标产物。经1H-NMR鉴证,1H-NMR (400 MHz, CDCl3),δ H (400 MHz,CDCl3): 5.59 (4 H, m), 4.35 (4H, m), 3.64 (4 H, d), 2.88 (2H, d), 2.36 (2 H,t), 0.90(3H, t).
第三步,分别称取0.123 mmol的LA-ss-COOH 60 mg和0.112 mmol的BFL (购于宝鸡市辰光生物科技有限公司)44 mg在圆底烧瓶中,再加入6 mL的无水二氯甲烷溶解后,还需加入0.14 mmol的DCC 29 mg和0.15 mmol的DMAP 15mg,在30℃水浴条件下,600rpm搅拌反应2小时。反应结束后滤膜过滤反应液,除去二环己基脲(DCU)。最后,采用硅胶柱纯化,用石油醚:乙酸乙酯体积比为2:1的洗脱剂,湿法装柱,每20mL洗脱液收集一瓶,通过薄层板确定目标产物(第8和9瓶),展开剂为石油醚:乙酸乙酯体积比为2:1,高锰酸钾显色,Rf值为0.25。再利用旋转真空蒸发仪进行旋转蒸发后得产物BFL-ss-LA,收集目标产物。上述目标产物采用红外和1H-NMR(图1)进行表征。IR (KBr) υmax (m-1):2926、2854、1738、1730和421cm-1。
结合图1可知,δ7.79(1H, dd)、7.16(1H, d)和6.20(1H, d)位移蟾毒灵上六元不饱和内酯环上三个氢原子峰。δ5.24-5.26(4H, m)是亚油酸上不饱和质子峰。δ4.30-4.40(4H, m)是乙二醇上的四个质子峰。δ3.60(4H, d)是二硫醇二羟基乙醇酸上的四个质子峰。δ0.96(3H, s, H-19)、0.89 (3H, t, LA-CH3)和0.71(3H, s, H-18)为蟾毒灵和亚油酸的三个甲基峰。
实施例2 BFL-ss-LA纳米粒(BFL-ss-LA-NPs)的制备
首先,配制混合浓度20 mg/mL的BFL-ss-LA无水乙醇溶液。其次,在800 rpm搅拌条件下,移液枪吸取200 μL的上述无水乙醇溶液平稳缓缓滴加到体积大概在2 mL的超高纯度蒸馏水(超纯水)中。最后,利用旋转真空蒸发仪进行旋转蒸发旋干除去无水乙醇,从而得BFL-ss-LA纳米粒(BFL-ss-LA-NPs)。BFL-ss-LA/TPGS-NPs的制法同上,只是在BFL-ss-LA无水乙醇中加入TPGS(最终浓度为5 mg/L)。
实施例3 亚油酸-蟾毒灵前体药物纳米的表征
①采用纳米粒度仪(Bruke粒径与Zeta电位分析仪)利用动态光散射法原理测量纳米粒的粒径与Zeta电位。步骤流程:首先,将制备的BFL-ss-LA-NPs用超纯水稀释至适宜浓度(0.5 mg/mL),放入纳米粒度仪测量光强分布图,测定结果:BFL-ss-LA-NPs粒径大小为107.4±0.9 nm,PDI为0.153±0.014,Zeta为-39.4±0.5 mV。BFL-ss-LA/TPGS-NPs粒径大小为126.4±1.3 nm,PDI为0.193±0.03,Zeta为-32.5±1.59 mV;
②采用透射电镜仪观察纳米粒的大小和表面。步骤:在铜网上,滴加适量稀释后的BFL-ss-LA-NPs或者BFL-ss-LA/TPGS-NPs(0.5 mg/mL),红外干燥后。将BFL-ss-LA-NPs或者BFL-ss-LA/TPGS-NPs放置在透射电镜下仔细观察其外观形态并拍照,结果见图2,BFL-ss-LA-NPs和BFL-ss-LA/TPGS-NPs形态规整,分散均匀。
实施例4稳定性试验
制备BFL-ss-LA-NPs或者BFL-ss-LA/TPGS-NPs,分别分散在水、0.01 M PBS(pH=7.4)的介质中,常温放置,在时间点第1、2、3、5、10、15、20、25、30天时测量粒径,评价其稳定性。
如图3所示,BFL-ss-LA/TPGS-NPs在PBS(pH=7.4)中的稳定性明显高于BFL-ss-LA-NPs。
实施例6体外释放
取0.1 mL实施案例2得到的BFL-ss-LA/TPGS-NPs样品(浓度为2 mg/mL),将其放入提前准备好的透析袋(型号为MW=3000)中,接下来放至体积为20 mL的30%乙醇-0.01M PBS(pH=7.4)内,其中PBS含有GSH的浓度分别为(10μM、1mM)。完成上述操作后,置于水浴(37℃)中,以100 rpm的速度进行震荡,最后选择如下:0.5 h、1 h、3 h、6h、12 h、24 h这6个释放时间点,取用1 mL介质释放,除上述操作外,还需补加1 mL的新配介质。通过液相色谱仪进行BFL的含量测定,并根据得到的数据算出其对应的释放率。如图4所示,BFL-ss-LA/TPGS-NPs在GSH低浓度时(10 μM)的释放率明显低于高浓度(1mM)时,具有一定谷胱甘肽感应性释放特性。
实施例7 细胞毒性评价
取对数生长旺盛期的HepG2人肝癌肿瘤细胞(购于中科院上海细胞库),经胰蛋白酶消化后,1500 r/min离心5min,用10%FBS DMEM(高糖)培养基重悬细胞,将细胞浓度配置至2 × 104个/mL,再加入到96孔细胞培养板中,每孔100 μL,继续在细胞培养箱(37℃,5 %CO2)中培养24 h。吸取培养基,加入含不同浓度受试药物(蟾毒灵和BFL-ss-LA/TPGS-NPs)的10%FBS DMEM(高糖)培养基100 μL,阴性对照组加入浓度0.5% DMSO的培养基,各组均设3个复孔。加药后继续培养72 h后,每孔加入5 mg/mL MTT试剂20 μL,在37℃下放置4 h。每孔加入DMSO 100 μL,振荡混匀,在波长570 nm处测定各孔吸光度值。
计算细胞生长抑制率:抑制率(%)=(1-受试药物组吸光度/阴性组吸光度)×100%。结果如图5所示,BFL在0.1、0.2、0.4、0.8、1 μM浓度时L02的生长率分别为63%、43%、26%、13%和7%,HepG2的生长率分别为76%、44%、26%、15%和9%。BFL-ss-LA/TPGS-NPs在0.1、0.2、0.4、0.8、1 μM浓度时L02的生长率分别为96%、78%、45%、26%和9%,而HepG2的生长率分别为88%、48%、31%、17%和9%,在0.1、0.2、0.4、0.8μM浓度时,BFL-ss-LA/TPGS-NPs能降低蟾毒灵对正常肝细胞 L02 的毒性,同时尽可能地维持对HepG2 肿瘤细胞的毒性。
实施例8 从半数致死量反馈给药安全性
取健康KM小鼠60只,雌雄各半,分开饲养,待小鼠适应实验室环境一周后,随机分为6组,每组10只,雌雄各半。一组为蟾毒灵-0.05%TPGS溶液作为对照组,小鼠经静脉注射给药的剂量为0.88~5 mg/kg;另一组为BFL-ss-LA/TPGS-NPs给药组,小鼠经静脉注射给药的剂量为3.55~20 mg/kg。给药容积为0.2 mL/20 g体重,给药后禁食禁水3 h,仔细观察并详细记录给药后,即刻及14天内动物可能出现的毒性反应和死亡情况。
结果显示,BFL-ss-LA/TPGS-NPs在给药剂量为20mg/kg时,死亡率为0,结果说明半数致死量大于20 mg/kg,蟾毒灵的半数致死量为1.85 mg/kg,从中可以看出,本发明蟾毒灵前药自组装纳米粒有效提高给药安全性。
综上所述本发明进一步提供了亚油酸-蟾毒灵前药自组装纳米粒的表征。动态光散射法测定纳米粒的粒径范围为50nm-250nm纳米之间,zeta电位为-21.4 mV左右。透射电镜观察纳米粒形态呈规则球形,分散性良好。
本发明进一步提供了亚油酸-蟾毒灵前体药物自组装纳米粒的稳定性和体外释放特性研究。一个月内该纳米粒在水和PBS(pH=7.4)介质中的粒径无明显变化。该纳米粒在PBS(pH=7.4)介质中药物释放和降解速率随着谷胱甘肽浓度的升高而提高,具有一定的氧化还原感应性。
通过急性毒性评价,表明本发明纳米粒对小鼠的半数致死量明显高于原药。
利用本发明纳米粒对HepG2细胞进行体外抗肿瘤活性实验。实验结果表明,该纳米粒抑制HepG2细胞的生长能力显著高于对正常细胞L02生长的抑制,可用于制备预防或治疗肝癌相关产品上应用。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (10)
1.一种蟾毒灵前药自组装纳米粒,其特征在于,以二硫醇二羟基乙酸作为连接剂,将蟾毒灵和脂肪酸乙二醇酯进行偶联形成前体药物,所述前体药物通过纳米沉淀法制备得到蟾毒灵前药自组装纳米粒。
2.根据权利要求1所述的一种蟾毒灵前药自组装纳米粒,其特征在于,所述脂肪酸乙二醇酯中脂肪酸酸为亚油酸、油酸、或亚麻酸中的一种。
3.基于权利要求1所述的蟾毒灵前药自组装纳米粒的制备方法,其特征在于,包括以下步骤:
步骤1,按照摩尔比1:2~10称取脂肪酸和无水乙二醇,加入无水二氯甲烷溶解,再加二环己基碳二亚胺和4-二甲氨基吡啶,所述二环己基碳二亚胺与脂肪酸的摩尔比为1~1.5:1,所述4-二甲氨基吡啶与脂肪酸的摩尔比为0.1~1:1,在密封条件下,连续搅拌反应2~14小时,经过萃取和柱色谱纯化后,得到目标产物脂肪酸乙二醇酯,记为FA-CH2-CH2-OH;
步骤2,将二硫醇二羟基乙酸加入乙酸酐中,在30℃的密封条件下,连续搅拌反应2-4小时后,除掉乙酸酐后,加入无水二氯甲烷溶解油状液体,再加入步骤1的FA-CH2-CH2-OH和4-二甲氨基吡啶,所述FA-CH2-CH2-OH与二硫醇二羟基乙酸的摩尔比为1:4~10, 4-二甲氨基吡啶与二硫醇二羟基乙酸的摩尔比为0.1~1:1;连续搅拌反应2-4小时,经过萃取和柱色谱纯化后,得到目标产物脂肪酸乙二醇二硫醇二羟基乙酸,记为FA-ss-COOH;
步骤3,称取上述步骤2 的产物FA-ss-COOH和蟾毒灵,加入无水二氯甲烷溶解后,再加入DCC和DMAP,所述FA-ss-COOH、蟾毒灵、DCC和DMAP的摩尔比为1︰0.8~1.2︰1~1.5︰0.1~1,常温下连续搅拌反应2-24小时,经过萃取和柱色谱纯化后,得到目标产物脂肪酸-乙二醇-二硫醇二羟基乙酸-蟾毒灵,记为BFL-ss-FA;
步骤4,称取步骤3产物BFL-ss-FA,配制浓度为10 mg/mL~40 mg/mL的有机溶剂溶液,在搅拌条件下,慢慢滴加入双蒸水中,有机溶剂与双蒸水的体积比为1:4-10,旋干除去有机溶剂,自组装得蟾毒灵前药自组装纳米粒,记为BFL-ss-FA-NPs。
4.根据权利要求3所述的权利要求1所述的蟾毒灵前药自组装纳米粒的制备方法,其特征在于,步骤4中有机溶剂为无水乙醇、 DMSO、乙腈、丙酮中的一种或者多种混合溶剂。
5.根据权利要求3所述的权利要求1所述的蟾毒灵前药自组装纳米粒的制备方法,其特征在于,步骤1和步骤2所述的纯化处理的具体形式为硅胶柱纯化、C8、C18填料柱层析或液相制备纯化。
6.根据权利要求3所述的权利要求1所述的蟾毒灵前药自组装纳米粒的制备方法,其特征在于,步骤4中还包括在有机溶剂中添加PEG化剂,且PEG化剂与BFL-ss-FA的质量比为1:1-10。
7.根据权利要求6所述的权利要求1所述的蟾毒灵前药自组装纳米粒的制备方法,其特征在于,所述PEG化剂为二棕榈酰磷脂酰乙醇胺-聚乙二醇、维生素E聚乙二醇琥珀酸酯、或亚油酸-聚乙二醇。
8.基于权利要求1-7中任一种蟾毒灵前药自组装纳米粒在制备抗肿瘤药物中的应用。
9.一种药物组合物,其特征在于,其有效成分为权利要求1-8中任一种蟾毒灵前药自组装纳米粒。
10.根据权利要求8所述的权利要求1所述的药物组合物,其特征在于,所述药物组合还包括至少一种药学上可接受载体。
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