CN111840229A - 抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法 - Google Patents
抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法 Download PDFInfo
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Abstract
本发明公开了一种抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，涉及药物化学的合成领域，更具体地涉及从合成线粒体靶向磷脂、合成载药脂质体等各步的具体方法。抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法具体包括以下步骤：（1）利用酰胺反应合成线粒体靶向磷脂（2）利用薄膜超声法合成抑制氨基酸合成的声动力线粒体靶向纳米脂质体。所得到的复合纳米药物递送系统优化合成方式，具有较好的声动力性能,较高的载药量以及良好的生物相容性等优点。
Description
技术领域
本发明涉及化学药物领域，具体涉及具有声动力疗法以及氨基酸合成抑制的联合疗法的靶向线粒体脂质体纳米粒子的制备与用途。
背景技术
癌细胞与正常分化细胞相比表现出不寻常的代谢活动，往往成为癌症治疗的靶点。特别是，靶向氨基酸代谢在癌症治疗中是开发新型治疗药物的一种很有前途的策略，其中线粒体内的谷氨酸脱氢酶1通过氧化脱氨作用将谷氨酸转化为α-酮戊二酸，α-酮戊二酸是三羧酸循环内一种产物。R162是谷氨酸脱氢酶1特异性抑制剂，降低了细胞α-酮戊二酸水平，影响三羧酸循环正常运行，造成细胞内延胡索酸浓度降低，也导致癌细胞谷胱甘肽过氧化物酶活性减弱造成癌细胞活性氧清除能力降低。因此，谷氨酸脱氢酶1处于细胞活性氧调节的关键位点，细胞内谷氨酸代谢明显影响氧化还原稳态。然而R162抑制效果通常不够令人满意，以及药物自身没有线粒体靶向作用，需要其他辅助治疗来促进的抗肿瘤效果。
声动力疗法作为一种新兴但具有代表性的基于超声的癌症无创治疗方法，具有较高的组织穿透能力、最小的侵袭性、高可控性和低成本。超声波是一种机械波和非辐射刺激，超声波到达非表面物体，并集中靶向组织，激活像IR780这样的超声增敏剂来诱导活性氧产生造成局部细胞毒性，同时尽量减少对周围正常组织的不良损伤。声敏剂IR780激活后产生活性氧，通过凋亡或坏死途径诱导癌细胞死亡。结合R162药物治疗作用，癌细胞清除活性氧能力被抑制，进一步加强声动力对癌细胞的杀伤作用。
脂质体是由两性分子如磷脂和鞘脂分散于水相时，分子的疏水尾部倾向于聚集在一起，避开水相，而亲水头部暴露在水相，形成具有双分子层结构的封闭囊泡。脂质体常用于转基因或药物载体。利用脂质体可以和细胞膜融合的特点，将药物送入细胞内部，生物安全性较高。通过修饰线粒体靶向的嵌段，脂质体能线粒体靶向递送药物，增强R162抑制效果。
发明内容
由于目前用于治疗肿瘤的脂质体无靶向效果，而且近红外光对于组织穿透深度有限，超声穿透较深组织层面。因此本发明旨在合成一种结合声动力以及氨基酸合成抑制疗法的靶向线粒体新型药物递送纳米平台，具有较高的生物相容性，较长的体内循环时间。
本发明的技术方案具体如下：
抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于，包含以下步骤：
（1）将三苯基膦PPh3Br–(CH2)4–COOH加入pH 5.4的2-(N-吗啉)乙磺酸缓冲液MES中，然后磁力搅拌至三苯基膦完全溶解，得到混合溶液；
（2）向步骤（1）得到的混合溶液中加入N-羟基丁二酰亚胺NHS、1-(3-二甲基胺丙基)-3-乙基碳二亚胺EDC后，室温下磁力搅拌20 min，得到混合溶液；
（3）将步骤（2）得到的混合溶液中加入溶于pH7.2的磷酸盐缓冲液PBS的氨基聚乙二醇化二硬脂酰基磷脂酰乙醇胺DSPE-PEG2000-NH2，室温下磁力搅拌24 h，得到混合溶液；
（4）向步骤（3）得到的混合溶液转移至分子量2000的透析袋中，在去离子水环境中透析纯化48 h后冷冻干燥，得到线粒体靶向的磷脂冻干粉末；
（5）将步骤（4）得到的线粒体靶向的磷脂冻干粉末与二棕榈酰磷脂酰胆碱DPPC、胆固醇和IR780以一定比例混合溶解于三氯乙烯，将含有混合溶液的圆底烧瓶转移至旋转蒸发仪干燥混合溶液直到形成一层薄膜；
（6）将步骤（5）得到的含有薄膜的圆底烧瓶内加入含有R162的PBS溶液水化30 min，超声10 min后离心分离得到抑制氨基酸合成的声动力线粒体靶向纳米脂质体。
进一步，所述步骤（1）中的PPh3Br–(CH2)4–COOH的浓度范围为6×10-4 mol/L~8×10-4 mol/L，所述步骤（1）中的混合溶液的MES范围浓度是1×10-3 mol/L~1×10-2 mol/L。
进一步，所述步骤（2）中的混合溶液中的NHS的浓度范围为2×10-2 mol/L~3×10-2 mol/L， EDC的浓度范围为1×10-2 mol/L ~ 2×10-2 mol/L。
进一步，所述步骤（3）中的DSPE-PEG2000-NH2的浓度范围为4×10-4 mol/L~5×10-4mol/L。
进一步，所述步骤（5）中的磷酸磷脂冻干粉末、DPPC、胆固醇和IR780的质量比为5:10:3:4。
进一步，所述步骤（6）中的R162的浓度范围为1×10-5 mol/L~2×10-5 mol/L；所述步骤（6）中的超声功率为100 W，超声脉冲持续时间4 s以及超声脉冲暂停时间5 s；所述步骤（6）中的离心的转速为6000 rpm，离心的时间为15 min。
本发明主要优点有：
针对目前纳米药物载体存在的不足，本项目创造性地提出能够实现多种治疗方法协同治疗同时又具有较好生物相容性的纳米药物递送平台。本项目创新性地设计制备了一种载药脂质体纳米粒子，将声动力和氨基酸合成抑制治疗巧妙结合用于乳腺癌治疗。相较于近红外光，超声波组织穿透性强，声动力治疗深层次肿瘤效果更佳。同时，脂质体纳米粒子具有靶向线粒体能力，更高效递送谷氨酸脱氢酶1抑制剂R162进入线粒体，提高R162的抑制效果。谷氨酸脱氢酶1被抑制后进一步影响三羧酸循环，从而影响谷胱甘肽过氧化物酶活性，细胞清除活性氧能力受限，进一步加强声动力效果。脂质体表面修饰的PEG2000延长体内循环时间，提高生物相容性。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图：
图1为本发明实施例1中抑制氨基酸合成的声动力线粒体靶向纳米脂质体的治疗流程图。
图2为本发明实施例1中抑制氨基酸合成的声动力线粒体靶向纳米脂质体的体外性能特征图。
图3为本发明实施例1中抑制氨基酸合成的声动力线粒体靶向纳米脂质体对4T1癌细胞的体外毒性图。
图4为本发明实施例1中抑制氨基酸合成的声动力线粒体靶向纳米脂质体在小鼠体内血常规结果图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1 制备抑制氨基酸合成的声动力线粒体靶向纳米脂质体
抑制氨基酸合成的声动力线粒体靶向纳米脂质体的作用原理图如图1所示，包括如下合成步骤：
（1）线粒体靶向磷脂的制备：首先将7.58×10-4 mol/L浓度的三苯基膦加入4 mL 1×10-2 mol/L浓度的2-(N-吗啉)乙磺酸缓冲液中，常温下磁力搅拌均匀，使其完全溶解。在上述体系中加入2.7×10-2 mol/L 浓度的N-羟基丁二酰亚胺、1.68×10-2 mol/L 浓度的1-(3-二甲基胺丙基)-3-乙基碳二亚胺后，室温下磁力搅拌20 min，得到混合溶液。在上述混合溶液中加入溶于pH7.2的1×10-2 mol/L磷酸盐缓冲液的氨基聚乙二醇化二硬脂酰基磷脂酰乙醇胺浓度为4.56×10-4 mol/L室温下磁力搅拌24 h，得到的混合溶液转移至分子量2000的透析袋中，在去离子水环境中透析纯化48 h后冷冻干燥，得到线粒体靶向的磷脂冻干粉末。
（2）优化抑制氨基酸合成的声动力线粒体靶向纳米脂质体的制备条件：线粒体靶向的磷脂冻干粉末与二棕榈酰磷脂酰胆碱DPPC、胆固醇和IR780以质量比为5:10:3:4混合溶解于三氯乙烯，将含有混合溶液的圆底烧瓶转移至旋转蒸发仪干燥混合溶液直到形成一层薄膜。含有薄膜的圆底烧瓶内加入含有浓度为1×10-5 mol/L的R162的10 mL PBS溶液水化30 min，超声10 min后离心分离得到抑制氨基酸合成的声动力线粒体靶向纳米脂质体。其肿瘤治疗原理模式图见图1。其纳米脂质体形貌见图2，从图中可以看出，脂质体水合粒径（2a）约为151 nm，TEM形貌图（2b）。图3是所得纳米颗粒对4T1癌细胞体外毒性情况。从图中可以看出，12 h（3a）与24 h（3b）HUVEC 细胞毒性较低，而4T1癌细胞的细胞毒性明显较高，且与药物浓度成正比。不同处理组下，施加超声的线粒体靶向纳米颗粒的细胞毒性最高（3c），并且该组处理的细胞内产生活性氧浓度最高（3d），4T1癌细胞内α-酮戊二酸含量（3e）、延胡索酸含量（3f）和谷胱甘肽过氧化物酶活性（3g）结果表示，抑制剂R162影响谷氨酸脱氢酶1活性，减少细胞内α-酮戊二酸、延胡索酸含量，降低谷胱甘肽过氧化物酶活性，削弱细胞清除活性氧能力，对4T1肿瘤细胞的毒性表现出一个很好的声动力与氨基酸合成抑制疗法的联合效果。图4是所得纳米颗粒对BALB/c小鼠生物相容性检测结果图，从图中可以看出静脉注射纳米颗粒后，不同时间点的血液指标与空白组相比并无明显差距，证明纳米颗粒具有较高的生物相容性。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其做出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
Claims (6)
1.抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于：包含以下步骤：
（1）将三苯基膦PPh3Br–(CH2)4–COOH加入pH 5.4的2-(N-吗啉)乙磺酸缓冲液MES中，然后磁力搅拌至三苯基膦完全溶解，得到混合溶液；
（2）向步骤（1）得到的混合溶液中加入N-羟基丁二酰亚胺NHS、1-(3-二甲基胺丙基)-3-乙基碳二亚胺EDC后，室温下磁力搅拌20 min，得到混合溶液；
（3）将步骤（2）得到的混合溶液中加入溶于pH7.2的磷酸盐缓冲液PBS的氨基聚乙二醇化二硬脂酰基磷脂酰乙醇胺DSPE-PEG2000-NH2，室温下磁力搅拌24 h，得到混合溶液；
（4）向步骤（3）得到的混合溶液转移至分子量2000的透析袋中，在去离子水环境中透析纯化48 h后冷冻干燥，得到线粒体靶向的磷酸磷脂冻干粉末；
（5）将步骤（4）得到的线粒体靶向的磷酸磷脂冻干粉末与二棕榈酰磷脂酰胆碱DPPC、胆固醇和IR780以一定比例混合溶解于三氯乙烯，将含有混合溶液的圆底烧瓶转移至旋转蒸发仪干燥混合溶液直到形成一层薄膜；
（6）将步骤（5）得到的含有薄膜的圆底烧瓶内加入含有R162的PBS溶液水化30 min，超声10 min后离心分离得到抑制氨基酸合成的声动力线粒体靶向纳米脂质体。
2. 根据权利要求1所述的抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于：所述步骤（1）中的PPh3Br–(CH2)4–COOH的浓度范围为6×10-4 mol/L~8×10-4mol/L，所述步骤（1）中的混合溶液的MES范围浓度是1×10-3 mol/L~1×10-2 mol/L。
3. 根据权利要求1所述的抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于：所述步骤（2）中的混合溶液中的NHS的浓度范围为2×10-2 mol/L~3×10-2 mol/L， EDC的浓度范围为1×10-2 mol/L ~ 2×10-2 mol/L。
4. 根据权利要求1所述的抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于：所述步骤（3）中的DSPE-PEG2000-NH2的浓度范围为4×10-4 mol/L~5×10-4 mol/L。
5.根据权利要求1所述的抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于：所述步骤（5）中的磷酸磷脂冻干粉末、DPPC、胆固醇和IR780的质量比为5:10:3:4。
6. 根据权利要求1所述的抑制氨基酸合成的声动力线粒体靶向纳米脂质体制备方法，其特征在于：所述步骤（6）中的R162的浓度范围为1×10-5 mol/L~2×10-5 mol/L；所述步骤（6）中的超声功率为100 W，超声脉冲持续时间4 s以及超声脉冲暂停时间5 s；所述步骤（6）中的离心的转速为6000 rpm，离心的时间为15 min。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160228466A1 (en) * | 2015-02-06 | 2016-08-11 | Emory University | Glutamate Dehydrogenase 1 Inhibitors and Methods of Treating Cancer |
| CN109646676A (zh) * | 2019-01-30 | 2019-04-19 | 重庆医科大学 | 线粒体靶向的声响应性的多层次肿瘤深穿透声敏剂的制备方法 |
| US20200206138A1 (en) * | 2017-06-16 | 2020-07-02 | Consiglio Nazionale Delle Ricerche | Nanoparticles as delivery vehicles of active ingredients and methods for the production thereof |
-
2020
- 2020-07-30 CN CN202010753021.6A patent/CN111840229A/zh active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160228466A1 (en) * | 2015-02-06 | 2016-08-11 | Emory University | Glutamate Dehydrogenase 1 Inhibitors and Methods of Treating Cancer |
| US20200206138A1 (en) * | 2017-06-16 | 2020-07-02 | Consiglio Nazionale Delle Ricerche | Nanoparticles as delivery vehicles of active ingredients and methods for the production thereof |
| CN109646676A (zh) * | 2019-01-30 | 2019-04-19 | 重庆医科大学 | 线粒体靶向的声响应性的多层次肿瘤深穿透声敏剂的制备方法 |
Non-Patent Citations (5)
| Title |
|---|
| CAIXIA YUE 等: "Mitochondria-targeting near-infrared light-triggered thermosensitive liposomes for localized photothermal and photodynamic ablation of tumors combined with chemotherapy", 《NANOSCALE》 * |
| CAIXIA YUE 等: "Mitochondria-targeting near-infrared light-triggered thermosensitive liposomes for localized photothermal and photodynamic ablation of tumors combined with chemotherapy", 《NANOSCALE》, vol. 10, no. 31, 4 July 2017 (2017-07-04), pages 1110, XP055614429, DOI: 10.1039/C7NR02193C * |
| 吴正红 等: "《药剂学》", 30 April 2020, 中国医药科技出版社, pages: 361 * |
| 易衡静 等: "靶向肿瘤细胞的相变型纳米粒多模态显像及其声动力治疗的体外实验研究", 《解放军医学杂志》 * |
| 易衡静 等: "靶向肿瘤细胞的相变型纳米粒多模态显像及其声动力治疗的体外实验研究", 《解放军医学杂志》, vol. 43, no. 10, 1 October 2018 (2018-10-01), pages 823 * |
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