CN113230417B - 葡萄糖和三苯基鏻修饰的脑肿瘤靶向脂质体的制备与应用 - Google Patents

葡萄糖和三苯基鏻修饰的脑肿瘤靶向脂质体的制备与应用 Download PDF

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CN113230417B
CN113230417B CN202110507676.XA CN202110507676A CN113230417B CN 113230417 B CN113230417 B CN 113230417B CN 202110507676 A CN202110507676 A CN 202110507676A CN 113230417 B CN113230417 B CN 113230417B
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吴勇
海俐
彭瑶
郭丽
李茹
卢嘉琪
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Abstract

本发明公开了葡萄糖和三苯基鏻共修饰的还原敏感型脑肿瘤靶向脂质体的制备与应用，用于实现脑肿瘤靶向药物的高效传递。所述的新型脂质体通过葡萄糖和三苯基鏻共同修饰，可分别识别血脑屏障和脑肿瘤细胞表面高表达的葡萄糖转运体GLUT1，以及与肿瘤细胞内线粒体发生静电作用，从细胞和细胞器两个层次，实现对血脑屏障、肿瘤组织以及线粒体的多重靶向功能，发挥更强的脑肿瘤靶向治疗作用。该新型脂质体可用于化疗药和作用于线粒体的化疗增敏剂的联合包载，用于协同治疗脑肿瘤，具有更精准的脑肿瘤靶向性，可提升化疗效果并减小毒副作用，拥有广阔的应用前景。

Description

葡萄糖和三苯基鏻修饰的脑肿瘤靶向脂质体的制备与应用
技术领域
本发明涉及一种新型脂质体及其在药物传递系统中的应用，具体是指以葡萄糖作为血脑屏障和脑肿瘤双重靶向配体，三苯基鏻作为线粒体靶向配体，用于共同修饰脂质体，并引入二硫键作为还原敏感键并使脂质体PEG化以实现长循环作用，将该新型多功能脂质体用于共同包载化疗药和化疗增敏剂，期望进一步提高药物对脑肿瘤的靶向治疗效果。本发明包括该材料的制备和表征，及其作为药物载体在药物传递中的应用，属于医药技术领域。
背景技术
脑胶质瘤是最常见和最致命的脑肿瘤，诊断后的预期寿命仅12-18个月。但是，传统疗法如化学疗法仍具有很多缺陷，这主要是由于脑屏障（包括血脑屏障和脑-肿瘤屏障）的存在，阻止了化疗药物到达肿瘤部位。而对肿瘤组织和药物发挥作用的细胞器缺乏选择性是脑肿瘤靶向药物开发的另一个瓶颈。为了提高疗效并减少毒副作用，药物递送系统必须具有两个重要能力：（1）克服血脑屏障并在肿瘤部位富集；（2）有控制地释放药物。
氯尼达明（Lonidamine，LND）是一种吲哚-3-羧酸的衍生物，通过使肿瘤对化学疗法、放射疗法、光动力疗法和高热疗法等敏感而具有抗肿瘤活性。据报道，作为化疗增敏剂，氯尼达明与阿霉素（DOX，最有效的广谱抗癌药物之一）协同治疗肝癌、卵巢癌及乳腺癌均取得了进展。但是，尚未报道将DOX和LND联合用于治疗比外周肿瘤复杂得多的脑肿瘤。因此，我们将设计一种有效的药物递送系统，用于阿霉素和氯尼达明联合治疗脑胶质瘤。
近几十年来，已经开发出利用受体、转运蛋白或吸附介导的药物递送系统，以促进药物透过血脑屏障进入脑肿瘤。大脑是一个强耗能的器官，几乎完全依赖葡萄糖作为能量来源。此外，由于肿瘤的快速增殖和独特的代谢方式，肿瘤也是高度消耗葡萄糖的组织。葡萄糖主要通过GLUT1转运透过BBB或进入癌细胞，GLUT1在脑毛细血管内皮细胞和肿瘤细胞的表面均过度表达，使得葡萄糖可作为靶向血脑屏障及肿瘤的双重靶向配体。
此外，药物进入肿瘤部位后的命运仍然值得关注。与靶向组织或细胞相比，基于药物作用的分子机制和发生损伤的确切位点，细胞器已被越来越多地成为下一代药物递送的更精确的靶标。据报道，LND通过抑制糖酵解和线粒体呼吸等复杂机制来调节常规化疗药物的活性。但是，由于氯尼达明水溶性差，几乎没有LND可以进入肿瘤组织，尤其是肿瘤细胞的线粒体，导致氯尼达明的临床应用受到限制。肿瘤细胞的线粒体膜电位比正常细胞具有更高的负电荷（-160~-180 mV），这意味着亲脂性阳离子如三苯基鏻（TPP）可以选择性积聚在肿瘤细胞的线粒体中。
然而，三苯基鏻修饰的脂质体将带有很强的正电荷，无论对正常细胞还是肿瘤细胞都会增加毒性，并易于被网状内皮系统清除。用于掩盖正电荷的有效策略是利用刺激敏感键作为连接基团，连接聚乙二醇（PEG）使聚乙二醇包裹在脂质体表面从而降低脂质体的正电荷。在肿瘤细胞中，谷胱甘肽（GSH）的浓度比正常组织中的浓度高得多（高于100倍），而二硫键作为还原敏感键以响应氧化还原的肿瘤内部环境，已得到了广泛应用。
基于以上考虑，我们试图探索葡萄糖和三苯基鏻共修饰的还原敏感型多功能脂质体（Lip-SPG）用于DOX和LND协同治疗胶质瘤的有效性。 如附图1所示，该脂质体通过以下几个过程实现对脑肿瘤的协同治疗：1）通过葡萄糖与血脑屏障过表达的葡萄糖转运蛋白GLUT1的识别与转运，使脂质体透过血脑屏障；2）再一次通过葡萄糖与脑肿瘤细胞表面过表达的GLUT1的识别与转运，使脂质体识别并进入肿瘤细胞，提升在肿瘤组织中的药物浓度；3）通过PEG的屏蔽作用，使脂质体在血液循环及正常组织中带弱正电，以提升脂质体在血液循环中的稳定性及减小对正常组织的毒副作用；而在肿瘤组织的高浓度谷胱甘肽的作用下，二硫键断裂，暴露出三苯基鏻可通过静电作用靶向线粒体；4）释放出的阿霉素作用于细胞核；5）剩余的脂质体部分作用于线粒体。
发明内容
本发明提供一种新型的葡萄糖和三苯基鏻共修饰的还原敏感型脑肿瘤靶向脂质体（Lip-SPG），脂质体Lip-SPG及其配体Chol-SPG、Chol-TPP，结构如下所示：
Figure 100002_DEST_PATH_IMAGE001
Figure 100002_DEST_PATH_IMAGE002
配体Chol-SPG的具体制备方法如下所示：
Figure 100002_DEST_PATH_IMAGE003
配体Chol-TPP的具体制备方法如下所示：
Figure DEST_PATH_IMAGE004
本发明所述的新型脂质体用于共同包载化疗药和化疗增敏剂，实现对脑肿瘤的协同治疗。
附图说明
图1脂质体Lip-SPG治疗脑肿瘤的过程示意图
图2 CFPE标记的脂质体 Lip-0 和 Lip-SPG 在 bEnd.3 细胞和 C6细胞中的摄取
图3 CFPE标记的脂质体 Lip-0 and Lip-SPG 在 bEnd.3 细胞线粒体和 C6细胞线粒体中的摄取
图4 Lip-0和Lip-SPG以及游离DOX + LND对C6细胞的细胞毒性评价
图5（A）荷C6细胞的原位脑胶质瘤模型小鼠给与不同制剂后的生存曲线图，箭头表示给药时间；（B）荷C6细胞的原位脑胶质瘤模型小鼠给与不同制剂后的中位生存期统计表。
具体实施方法
以下实施例旨在说明本发明而不是对本发明的进一步限定。下面参照实施例进一步详细阐述本发明，但本发明并不限于这些实施例以及使用的制备方法。而且，本领域技术人员根据本发明的描述可以对本发明进行等同替换、组合、改良或修饰，但这些都将包括在本发明的范围内。
本发明通过以下技术方案实现上述目的：
所述的新型脂质体具体由以下步骤制备：
脂质体配体的合成
以下实施例1-4以分子量为1000的聚乙二醇为例，说明配体Chol-SPG的合成方法。
实施例1
化合物3的制备
Figure DEST_PATH_IMAGE005
-5 ℃下，向3,3'-二硫代二丙酸（4.054 g，19.28 mmol）CH2Cl2（30 mL）溶液中加入EDCI（4.75g，24.1mmol），DMAP（2.95 g，24.1 mmol），和DIPEA（3.74g，28.92mmol），搅拌30分钟。然后，向反应液中逐滴加入化合物2（本实验室自制，5.00g，9.64mmol）的CH2Cl2（30mL）溶液，并在室温下反应过夜。将反应溶液依次用HCl水溶液（1N，100mL×2）和饱和NaCl（100mL×3）洗涤。有机相进一步用无水Na2SO4干燥，并除去溶剂。最后，通过硅胶柱层析（CH2Cl2/CH3OH＝300/1）纯化，得浅黄色半固体（5.067 g），收率74.06％。1H-NMR (400 MHz, CDCl3,ppm) δ: 0.67 (s, 3H), 0.86 (dd, 6H, J 1 = 6.6 Hz, J 2 =1.6 Hz), 0.91 (d, 3H, J=6.4 Hz), 0.99 (s, 3H), 0.85-2.39 (remaining cholesterol protons), 2.75-2.79(m, 4H), 2.93 -2.97 (m, 4H), 3.15-3.24 (m, 1H), 3.64-3.68 (m, 8H), 3.72-3.74(m, 2H), 4.26-4.28 (m, 2H), 5.34 (s, 1H). HRMS (ESI) calculated forC39H66O7S2Na+[M+Na]+-733.4142, found 733.4135.
实施例2
化合物4的制备
Figure DEST_PATH_IMAGE006
-5 ℃下，于化合物3（738 mg，1.04 mmol）的CH2Cl2（10 mL）溶液中，加DCC（257 mg，1.247 mmol）和DMAP（25 mg，0.208 mmol）搅拌30 分钟。然后，向反应液中逐滴加入PEG（1.039 g，1.04 mmol）的CH2Cl2（10mL）溶液，室温搅拌过夜。滤出副产物（DCU）后，将反应溶液浓缩。最后，通过硅胶柱层析（CH2Cl2/CH3OH＝70/1）纯化，得到浅黄色油状物（898 mg），收率51.05％。1H-NMR (400 MHz, CDCl3, ppm)δ: 0.67 (s, 3H), 0.86 (dd, 6H, J 1 = 6.8Hz, J 2 =1.2 Hz), 0.91 (d, 3H, J= 6.4 Hz), 0.99 (s, 3H), 0.85-2.38 (remainingcholesterol protons), 2.75-2.79 (m, 4H), 2.91 -2.94 (m, 4H), 3.10-3.22 (m,2H), 3.47-3.83 (m, 85 H), 4.25-4.27 (m, 4H), 5.34 (s, 1H).
实施例3
化合物5的制备
Figure DEST_PATH_IMAGE007
-5 ℃下，于1,2,3,4-四-O-三甲基甲硅烷基-6-琥珀酰-D-吡喃葡萄糖（本实验室自制，386 mg，0.23 mmol）的CH2Cl2（10 mL）溶液中，加入DCC（84 mg，0.41 mmol）、DMAP（8mg，0.065mmol）搅拌30分钟。然后，向混合物中逐滴加入化合物4（200 mg，0.34 mmol）的CH2Cl2（10 mL），室温搅拌过夜。滤出DCU后，浓缩反应溶液。最后，通过硅胶柱层析（CH2Cl2/CH3OH＝100/1）纯化，得浅黄色油状物（158 mg），收率61.24％。1H-NMR (400 MHz, CDCl3, ppm)δ:0.14-0.15 (m, 36H), 0.68 (s, 3H), 0.86 (d, 6H, J= 6.6 Hz), 0.91 (d, 3H, J=6.0 Hz), 0.99 (s, 3H), 0.85-2.39 (remaining cholesterol protons), 2.66-2.67(m, 4H), 2.75-2.79 (m, 4H), 2.91-2.94 (m, 4H), 3.10-3.21 (m, 2H), 3.64-3.92(m, 95 H), 4.03-4.07 (m, 2H) 4.25-4.27 (m, 6H), 4.35-4.38 (m, 1H), 4.42-4.50(m, 1H), 5.00 (d, 1H, J=2.4 Hz), 5.34 (s, 1H).
实施例4
化合物6（配体Chol-SPG）的制备
Figure DEST_PATH_IMAGE008
将三氟乙酸（CF3COOH，2 mL）加入到化合物5（222 mg，0.10 mmol）的CH2Cl2（4 mL）溶液中，室温反应2小时。浓缩反应物，并通过硅胶柱色谱法（CH2Cl2/CH3OH＝ 30/1）纯化，得浅黄色半固体（123 mg），收率63.73％。1H-NMR (400 MHz, DMSO-d6, ppm)δ: 0.65 (s,3H), 0.84 (dd, 6H, J 1 = 6.4 Hz, J 2 = 1.6 Hz), 0.89 (d, 3H, J= 6.4 Hz), 0.94 (s,3H), 0.83-2.33 (remaining cholesterol protons), 2.56-2.57 (m, 4H), 2.70-2.73(m, 4H), 2.89-2.93 (m, 4H), 3.00-3.15 (m, 4H), 3.41-3.62 (m, 110 H), 3.67-3.69 (m, 1H), 3.76-3.80 (m, 1H), 3.94-4.02 (m, 1H), 4.11-4.16 (m, 6H), 4.28-4.31 (m, 1H), 4.90-4.91 (m, 1H), 5.31 (s, 1H). HRMS (ESI) calculated forC95H172O38S2Na+[M+Na]+-2008.0860, found 2008.0702.
实施例5
化合物7（配体Chol-TPP）的制备
Figure DEST_PATH_IMAGE009
-5 ℃下，于3-羧丙基三苯基溴化鏻（124 mg，0.29 mmol）的CH2Cl2（5 mL）溶液中加入DCC（79 mg，0.38 mmol）和DMAP（4.7 mg，0.0385 mmol）搅拌30分钟。 然后，向混合物中逐滴加入化合物2（100 mg，0.193 mmol）的CH2Cl2（5 mL）溶液，室温搅拌过夜。浓缩反应物，并通过硅胶柱层析（CH2Cl2/CH3OH＝ 50/1）纯化，得白色泡沫固体（143 mg），收率84.60％。1H-NMR (400 MHz, CDCl3, ppm)δ: 0.67 (s, 3H), 0.86 (dd, 6H, J 1 = 6.6 Hz, J 2 =1.6Hz), 0.91 (d, 3H, J= 6.4 Hz), 0.98 (s, 3H), 0.85-2.37 (remaining cholesterolprotons), 2.88-2.96 (m, 2H), 3.13-3.21 (m, 1H), 3.61-3.63 (m, 8H), 3.67-3.69(m, 2H), 4.00-4.07 (m, 2H), 4.19-4.21 (m, 2H), 5.33 (s, 1H), 7.68-7.73 (m,6H), 7.78-7.82 (m, 3H), 7.86-7.91 (m, 6H). HRMS (ESI) calculated for C55H78O5P+[M-Br]+-849.5587, found 849.5577.
以下实施例6-14以阿霉素作为化疗药、氯尼达明作为化疗增敏剂为例，说明所述的新型脑肿瘤靶向脂质体的具体制备方法及其对脑肿瘤的靶向及协同治疗作用。
实施例6
脂质体的制备
脂质体Lip-SPG的脂质处方（摩尔比）为：SPC/Chol/Chol-SPG/Chol-TPP=60/34/3/3；未修饰的脂质体Lip-0 的脂质处方（摩尔比）为SPC/Chol/=60/40。脂质材料/药物的质量比为脂质/DOX/LND＝40/1/1。将脂质材料和LND溶解在CHCl3-CH3OH混合溶剂（v/v=2/1）中，并在40 ℃下旋转蒸发以形成均匀的薄膜。然后，加入DOX水溶液在50 ℃下水化1 h。最后，通过间歇超声（80W，5S，5S）5 min后得到脂质体。荧光标记的脂质体通过将上述操作中的药物替换为荧光标记的磷脂（CFPE），并将水化液替换为PBS缓冲液即可。
实施例7
脂质体的表征
采用超滤法除去未包载的药物：将超声后的脂质体加入超滤管（10 kd）中并离心（10000 rpm，30分钟），上层为载有药物的脂质体，下层的滤液为游离药物。通过HPLC分析DOX和LND的浓度。 DOX或LND的包封率（EE）计算公式如下：
EE（％）=（1-W1 / W2）×100％
W1为未载药量，W2为超滤前脂质体溶液中的药物总量。并通过激光粒度仪测量脂质体的粒径和Zeta电位。结果如表1所示，两种脂质体包封率良好，均大于80%。粒径分散均匀；Lip-SPG带少量正电。
表1： DOX-LND 共载脂质体 Lip-0 和Lip-SPG的表征
Figure DEST_PATH_IMAGE010
体外活性评价
实施例8
细胞摄取
将bEnd.3和C6细胞用CFPE标记的脂质体（Lip-0，Lip-SPG）处理4小时。然后，将细胞消化、收集并用PBS洗涤。最后，用流式细胞仪测定细胞的荧光强度。结果如图2所示，Lip-SPG可显著提升bEnd.3细胞和C6细胞对脂质体的摄取。
实施例9
线粒体摄取
将bEnd.3和C6细胞用CFPE标记的脂质体（Lip-0，Lip-SPG）处理12小时后，将细胞消化、收集并用PBS洗涤。然后用线粒体提取试剂盒提取线粒体，最后，用流式细胞仪测定线粒体的荧光强度。结果如图3所示，Lip-SPG可显著提升bEnd.3细胞线粒体和C6细胞线粒体对脂质体的摄取。
体外抗肿瘤活性评价
实施例10
细胞毒性评价
将C6细胞用梯度药物浓度的共载DOX-LND的Lip-0和Lip-SPG以及游离DOX + LND处理24小时。然后，用MTT法检测细胞存活率。结果如图4所示，各组制剂对C6细胞增殖的抑制能力为Lip-SPG>Lip-0>Free DOX+LND，IC50分别为0.85，2.82和3.02 µg/mL。
实施例11
细胞凋亡评价
将C6细胞用DOX和LND浓度为0.5µg/mL的共载DOX-LND的Lip-0和Lip-SPG以及游离DOX + LND处理24小时。然后，用Annexin V-FITC/PI凋亡试剂盒检测细胞的凋亡情况。结果显示各组制剂诱导C6细胞凋亡的能力为Lip-SPG>Lip-0>Free DOX+LND，Lip-SPG诱导的凋亡和坏死的细胞分别为Lip-0的2.18倍，为Free DOX+LND的3.29倍。
体内活性评价
实施例12
药代动力学和体内脑靶向评价
取昆明小鼠（20-25克）通过尾静脉分别注射游离DOX+LND和DOX-LND共载脂质体（Lip-0，Lip-SPG）。注射后5、15、30、60、120、240、480、720和1440分钟，从眼眶取血并离心（10000 rpm，10 min）以获得血浆样品。然后处死动物剖取全脑，并用使用盐水（w/w =1：2）匀浆以制得脑匀浆。将三倍体积的乙腈加入到血浆（100 μL）或脑匀浆（300 μL）中并涡旋5分钟以提取DOX和LND。 然后，将样品离心（10,000 rpm，10分钟）并吹干上清液。最后，将残余物重新溶解，并离心（10,000 rpm，10 min），取上清液用HPLC分析。结果显示，脂质体可显著改善游离DOX的药代动力学行为，减缓阿霉素的清除速率，促进药物进入脑组织；而葡萄糖和三苯基鏻的修饰，可通过GLUT1的介导以及静电吸附作用，进一步促进脂质体进入中枢，提高药物的脑靶向性。
实施例13
体内抗肿瘤活性评价
取荷C6细胞的原位脑胶质瘤模型小鼠，随机分为4组，于建模第4、7、10、13天，按DOX和LND剂量为5 mg/kg，分别给予生理盐水、Free DOX+LND和DOX-LND共载脂质体(Lip-0、Lip-SPG)，并监测小鼠生存状况。结果如图5所示，各组制剂的小鼠存活时间为Lip-SPG>Lip-0>Free DOX+LND>生理盐水，Lip-SPG相比于生理盐水组，可将中位生存期从19天延长至39天。

Claims (5)

1.一种葡萄糖和三苯基鏻共修饰的还原敏感型脑肿瘤靶向脂质体Lip-SPG，其特征在于脂质体包含两种配体：葡萄糖修饰的胆固醇Chol-SPG和三苯基鏻修饰的胆固醇Chol-TPP；三者的结构如下式所示：
Figure DEST_PATH_IMAGE001
Figure DEST_PATH_IMAGE002
Figure DEST_PATH_IMAGE003
其中，Chol-SPG中使用的聚乙二醇分子量为1000，1500，2000，3000，4000中的一种。
2. 根据权利要求1所述的葡萄糖和三苯基鏻共修饰的还原敏感型脑肿瘤靶向脂质体，其特征在于主要由膜材与活性剂组成，膜材包含磷脂、胆固醇和配体Chol-SPG、Chol-TPP，各组分配比关系如下：胆固醇和磷脂的摩尔比为1:1~1:4，脂质体配体的摩尔含量为胆固醇和磷脂的总摩尔数的1~10%，两种配体的摩尔比为Chol-TPP /Chol-SPG =1/1~3/1；所述的活性剂为化疗药和化疗增敏剂，化疗药/化疗增敏剂的质量比=1/1~3/1；活性剂的质量占总脂质体的1%~10%。
3.根据权利要求2所述的脂质体，其中，所述脂质体中的磷脂是大豆磷脂、卵磷脂、磷脂酰乙醇胺、磷酯酰丝氨酸、磷脂酰肌醇、磷脂酰甘油、二磷脂酰甘油中的一种。
4.根据权利要求2所述的脂质体，其中，所述脂质体中的化疗药是抗肿瘤药物，为烷化剂、抗代谢物、抗肿瘤抗生素、植物生物碱、紫杉醇、拓朴异构酶抑制剂、单克隆抗体、光敏剂、激酶抑制剂和含铂化合物的一种。
5.根据权利要求2所述的脂质体，其中，所述脂质体中的化疗增敏剂是干扰线粒体功能的药物，为线粒体己糖激酶抑制剂，线粒体丙酮酸转运蛋白抑制剂，细胞增殖抑制剂，氧化磷酸化抑制剂，ATP竞争性抑制剂中的一种。
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