CN112656944B - 一种齐墩果酸纳米凝胶的制备方法及其应用 - Google Patents
一种齐墩果酸纳米凝胶的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种齐墩果酸纳米凝胶的制备方法及其应用。该纳米凝胶由齐墩果酸在乙醇/DMSO和水的混合溶剂中自组装而构成。随着乙醇/DMSO和齐墩果酸浓度的改变,可得到不同形式的组装体,分别为:纳米粒,纳米凝胶和凝胶。相比于游离药物,该纳米凝胶具有更好的抗肿瘤活性,可改善肿瘤的纤维化程度,促进免疫细胞的浸润,用于肿瘤的化疗和免疫治疗。该纳米凝胶既可作为药物本身,也可包载光声敏化剂,得到齐墩果酸/敏化剂纳米药物,用于肿瘤的光声/化学治疗。该纳米凝胶在药物递送、组织工程和化学品等领域有很大的应用潜力。
Description
技术领域
本发明属于生物医药领域,涉及一种齐墩果酸纳米凝胶的制备方法及其应用。
背景技术
作为药物发展的起源,天然药物在药物化学中占有重要地位。存在于天然植物中的五环三萜烯齐墩果酸(OA)具有多种生物活性,例如抗肿瘤作用、抗氧化作用、增强免疫功能、肝保护作用和心脏保护作用,具有广阔的应用前景。2012年Bag等人发现齐墩果酸具有手性刚性骨架,易于在溶液中聚集,通过分子间氢键、π-π堆积作用及三萜骨架的聚集性质组装成纤维结构,然后形成超分子有机凝胶(B.G. Bag, K. Paul, Vesicular andFibrillar Gels by Self-Assembly of Nanosized Oleanolic Acid, Asian Journal ofOrganic Chemistry, 1 (2012) 150-154.)。该团队虽利用齐墩果酸有机凝胶包载阿霉素,但并未对其治疗效果进行评价,并且有机凝胶毒性大,一般应用于药物载体及医用材料的超分子凝胶主要为水凝胶。
国家癌症中心最新统计数据显示,乳腺癌位居女性恶性肿瘤发病率的首位,已经成为全球的主要公共卫生问题。在各种类型的人类乳腺癌中,三阴性乳腺癌(TNBC)是最具侵略性的类型。肿瘤微环境(TEM)由细胞外基质(ECM)、肿瘤相关成纤维细胞(CAFs)、免疫细胞和血管***组成,他们围绕着肿瘤细胞,保护肿瘤细胞免受侵犯。由成纤维细胞产生的胶原形成物理屏障,阻断药物的传递和细胞毒性T细胞的浸润。并且,CAFs通过重塑ECM支持肿瘤的发展,TNBC的预后与肿瘤的中心纤维化有关,纤维化程度越高,肿瘤向远处转移的倾向越大(Takai K , Le A , Weaver V M , et al. Targeting the cancer-associatedfibroblasts as a treatment in triple-negative breast cancer[J]. Oncotarget,2016, 7(50):82889-82901.)。因此,靶向CAFs和重构ECM可能是应对复杂的癌症病因学的有效策略。
转化生长因子-β(TGF-β)是重塑ECM和控制CAF激活的关键调节剂,可促进增生的积累和肿瘤的生长速度。TGF-β通过TGF-β/ Smad信号传导途径驱动ECM中胶原蛋白的表达。研究表明,通过阻断TGF-β,降低胶原蛋白的产生,促使血管正常化来改善药物在乳腺癌中的分布,增加肿瘤部位的免疫细胞浸润,逆转肿瘤的免疫抑制环境。齐墩果酸已被证明可以通过增加Nrf2的核转运来减轻C57BL / 6小鼠的肾纤维化和胶原蛋白沉积(Zhao D , LuanZ . Oleanolic Acid Attenuates Renal Fibrosis through TGF- β /Smad Pathway ina Rat Model of Unilateral Ureteral Obstruction[J]. 2020, 2020:1-8.)。另外,研究人员发现齐墩果酸与TGF-β1受体具有很强的结合能力,可以作为TGF-β1的拮抗剂。这表明OA通过TGF-β/ Smad信号传导减弱纤维化是有潜力的(Yoshimura H , Sugawara K ,Saito M , et al. In vitro TGF-β1 Antagonistic Activity of Ursolic andOleanolic Acids Isolated from Clerodendranthus spicatus[J]. Planta Medica,2003, 69(7):673-675.)。
光引发的光热治疗(PTT)和光动力学治疗(PDT)具有高特异性,即给药后外加光源照射肿瘤部位使其产生热或活性氧(ROS),对其他组织的副作用小。但是,PTT和PDT的缺点是光穿透深度有限,因此单独使用PTT或PDT可能无法有效抑制肿瘤。超声作为一种机械波,在生物体内的良好的穿透能力,能够到达深层的肿瘤,可用于肿瘤的诊断和治疗。声敏剂在超声刺激下,产生ROS,促使细胞内蛋白或DNA的氧化损伤,导致细胞死亡(Zhu, Piao,Chen, et al. Nanoenzyme-Augmented Cancer Sonodynamic Therapy by CatalyticTumor Oxygenation[J]. Acs Nano, 2018,12:3780-3795.)。
因此,我们通过改变乙醇比例和齐墩果酸浓度,得到一种纳米凝胶。该纳米凝胶能有效抑制乳腺癌细胞的增殖,通过降低肿瘤的纤维化程度,重塑肿瘤微环境,提高免疫细胞在肿瘤部位的比例,可用于肿瘤的化疗和免疫治疗。该纳米凝胶可用于包载光声敏化剂,得到OA/敏化剂纳米药物,用于肿瘤的光声/化学治疗。
发明内容
本发明的目的在于提供一种齐墩果酸纳米凝胶的制备方法及应用。本发明以齐墩果酸为原料,构建了一种具有调控肿瘤微环境功能的纳米凝胶,可用于肿瘤的化疗和免疫治疗。该纳米凝胶可包载光声敏化剂,得到OA/敏化剂纳米药物,用于肿瘤的光声/化学治疗。
为达到上述目的,本发明采取了如下技术方案:
一种齐墩果酸纳米凝胶的制备方法,包括如下步骤
(1)将齐墩果酸溶解在乙醇或DMSO中;
(2) 将步骤(1)得到的溶液在涡旋状态下滴加到水溶液中,室温静置0.5~24 h,得到齐墩果酸纳米凝胶。
所述的水溶液选自水、含药物分子的水溶液、生理盐水溶液、磷酸盐缓冲液、细胞培养基或其混合溶液。
所述纳米凝胶中齐墩果酸的浓度为0.1~5mg/mL。
所述的纳米凝胶中乙醇或DMSO的体积浓度为10%~100%。
齐墩果酸纳米凝胶在制备抗肿瘤药物中的应用。
齐墩果酸纳米凝胶在抗纤维化方面的应用。
齐墩果酸纳米凝胶在免疫治疗中的应用。
OA/敏化剂纳米药物在光声/化学治疗中的应用。
本发明的有益效果是:
第一,本发明通过改变乙醇比例和齐墩果酸浓度,得到具有不同构造的组装体;
第二,本发明得到的纳米凝胶,相比于游离药物和齐墩果酸纳米粒,抗肿瘤效果大大提升。
第三,本发明得到的纳米凝胶,可以调控肿瘤的微环境,减弱肿瘤的纤维化程度,促进毒性T细胞的浸润,可用于肿瘤的化疗和免疫治疗。
第四,本发明得到的OA/敏化剂纳米药物,可用于肿瘤的光声/化学治疗,抑制肿瘤的增长。
附图说明
图1为实施例1 制备的齐墩果酸凝胶。
图2为实施例1 制备的齐墩果酸凝胶在频率扫描下的流变学结果。
图3为实施例2 制备的OA-NG。
图4为实施例2制备的OA-NG的透射电镜图。
图5为实施例3 制备的OA-NP的透射电镜图。
图6为实施例5制备的ORM的紫外光谱图。
图7为实施例6中OA-NG对4T1细胞毒性的影响。
图8为实施例7中ORM对4T1细胞毒性的影响。
图9为实施例8中OA-NG对4T1荷瘤小鼠肿瘤体积的影响。
图10为实施例8中OA-NG对肿瘤纤维化的影响。
图11为实施例9中OA-NG对4T1荷瘤小鼠肿瘤部位免疫细胞的影响。
具体实施方式
下面结合具体实施例,对本发明进行进一步说明,有助于本领域的普通技术人员进一步理解本发明,但不以任何形式限制本发明。
实施例1
将500 μL含有4 mg/mL齐墩果酸的乙醇溶液在涡旋状态下滴加到500 μL的水中,室温静置24 h,观察到果冻状凝胶,得到齐墩果酸凝胶,如图1所示。通过流变仪表征凝胶的流变学性质,如图2所示,G’比 G’’高出一个数量级,并且随着扫描频率的变化,G’基本保持恒定,满足凝胶的流变学性质。
实施例2
将150 μL含有3.33mg/mL齐墩果酸的乙醇溶液在涡旋状态下滴加到850 μL的水中,室温静置24 h,离心观察到果冻状凝胶,得到齐墩果酸纳米凝胶OA-NG,如图3所示。齐墩果酸纳米凝胶的结构纳米凝胶呈边缘模糊的球体,部分小球之间存在黏连(图4)。
实施例3
将50 μL含有10 mg/mL齐墩果酸的乙醇溶液在涡旋状态下滴加到950 μL的水中,室温静置24 h,得到齐墩果酸纳米粒OA-NP。通过透射电镜观察发现,纳米粒呈边缘明确的球体,球体均匀分布没有黏连(图5)。
实施例4
将400 μL含有5 mg/mL齐墩果酸的DMSO溶液在涡旋状态下滴加到200 μL的水中,室温静置24 h,观察到果冻状凝胶,得到齐墩果酸有机凝胶。
实施例5
将150 μL含有3.33 mg/mL齐墩果酸的乙醇溶液在涡旋状态下滴加到850 μL的含有亚甲蓝(MB)和孟加拉玫瑰红(RB)的水溶液中,室温静置24 h, 离心观察到果冻状凝胶,得到纳米凝胶ORM。测定其紫外吸收,结果如图6所示,RB在565nm处有特征吸收峰,MB在667nm处有紫外吸收峰,ORM在565nm和667nm处均有特征吸收峰,表明OA成功包载RB和MB。
实施例6
实施例2中的纳米凝胶OA-NG对鼠源乳腺癌细胞4T1的抗肿瘤活性被MTT法测定。结果如图7所示,相同OA (50 μg/mL)浓度下,OA-NP和OA-NG实验组的细胞存活率比游离的OA更低,OA-NG对4T1细胞的增殖抑制的作用比OA-NP更强。
实施例7
ORM对鼠源乳腺癌细胞4T1的抗肿瘤活性被MTT法测定。结果如图8所示,ORM的光声联合治疗优于单一治疗,抗肿瘤效果大大提高。
实施例8
4T1原位乳腺癌小鼠模型被用于验证纳米凝胶的体内抗肿瘤活性。其中一组小鼠通过尾静脉注射实施例2中的OA-NG(15 mg/kg)进行治疗,一组小鼠通过尾静脉注射实施例3中的OA-NP(15 mg/kg)进行治疗;一组小鼠通过尾静脉注射游离OA(15 mg/kg)进行治疗;另一组则通过静脉注射PBS作为对照。每隔一天测量肿瘤的体积,结果如图9所示,经过14天治疗后,OA-NG组的肿瘤的体积明显小于游离OA组和OA-NP组。对治疗后肿瘤组织进行天狼星红染色,结果如图10所示,OA-NG组胶原蛋白的表达量最少,OA-NG抑制肿瘤纤维化的能力强于OA-NP。
实施例9
将实施例2中的OA-NG(15 mg/kg)、实施例3中的OA-NP(15 mg/kg)、游离OA(15 mg/kg)分别尾静脉注射到4T1原位乳腺癌小鼠模型中,通过静脉注射PBS作为对照组,24小时后,取出肿瘤组织,通过流式细胞仪检测肿瘤中浸润的毒性T细胞的含量。结果如图11所示,小鼠经过OA-NG治疗后,肿瘤部位有14.6%的CD8+T细胞浸润,OA-NP组有6.57%的CD8+T细胞浸润,而PBS组只有1.26%的CD8+T细胞浸润。相比于OA-NP和游离OA,OA-NG的免疫治疗效果最好。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
1.一种齐墩果酸纳米凝胶的制备方法,其特征在于:所述方法步骤如下:
将150 μL含有3.33 mg/mL齐墩果酸的乙醇溶液在涡旋状态下滴加到850 μL的水中,室温静置24 h,离心观察到果冻状凝胶,得到齐墩果酸纳米凝胶。
2.一种权利要求1所述方法制备的齐墩果酸纳米凝胶在制备抗乳腺癌药物中的应用。
3.一种权利要求1所述方法制备得到的齐墩果酸纳米凝胶在制备调控肿瘤微环境药物中的应用。
4.一种权利要求1所述方法制备得到的齐墩果酸纳米凝胶在制备免疫治疗药物中的应用。
5.一种权利要求1所述方法制备得到的齐墩果酸纳米凝胶在制备药物载体中的应用。
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