CN113842470B - p-HA和三苯基鏻修饰的pH敏感靶向脂质体的制备与应用 - Google Patents

p-HA和三苯基鏻修饰的pH敏感靶向脂质体的制备与应用 Download PDF

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CN113842470B
CN113842470B CN202111165237.1A CN202111165237A CN113842470B CN 113842470 B CN113842470 B CN 113842470B CN 202111165237 A CN202111165237 A CN 202111165237A CN 113842470 B CN113842470 B CN 113842470B
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吴勇
海俐
郭丽
彭瑶
李茹
卢嘉琪
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Abstract

本发明公开了对羟基苯甲酸和三苯基鏻共修饰的pH敏感型的多靶向脂质体的制备与应用，用于实现化疗药物对脑肿瘤的靶向传递。所述的多靶点脂质体通过对羟基苯甲酸的修饰，识别血脑屏障上的多巴胺受体和sigma受体，并进一步识别脑肿瘤细胞上的sigma受体从而进入脑组织，实现对肿瘤细胞的靶向；再通过三苯基鏻与游离氨基的正电性与肿瘤细胞内线粒体发生静电作用，实现细胞器靶向。该脂质体可同时包载化疗药和化疗增敏剂，通过多重靶向性对脑肿瘤发挥高效的协同作用，不仅显著提升治疗效果，抑制肿瘤迁移和侵袭，并减小了毒副作用，拥有巨大的应用潜力。

Description

p-HA和三苯基鏻修饰的pH敏感靶向脂质体的制备与应用
技术领域
本发明涉及一种脂质体及其在药物传递系统中的应用，具体是指利用对羟基苯甲酸靶向血脑屏障和脑肿瘤，三苯基鏻和正电性氨基靶向线粒体，并引入酰胺键作为pH敏感键，使脂质体PEG化实现长循环效果，将该多功能脂质体用于化疗药和化疗增敏剂的联合包载，以提高药物对脑肿瘤的治疗效果。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，属于医药化学领域。
背景技术
脑胶质瘤由于其常见又具有侵袭性的特征，目前的治疗仍存在很多问题：（1）血脑屏障阻止治疗药物到达病变区域；（2）脑胶质瘤的高度异质性和浸润性，使传统疗法不可避免地损害正常组织；（3）脑胶质瘤的侵袭性和浸润性，不仅影响治疗效果，而且导致肿瘤的频繁复发和不良预后。因此，有效的治疗手段必须具有以下特点：能够跨越血脑屏障、选择性靶向肿瘤细胞；能够抑制脑胶质瘤细胞的迁移和侵袭；毒副作用小。
传统的单药疗法不能满足脑胶质瘤治疗的复杂要求，有报道称阿霉素（DOX）和氯尼达明（LND）相结合起来能够协同治疗脑胶质瘤，而有效的给药系统可以提高它们的协同效率，因此，基于靶向纳米技术的药物传递系统越来越普遍，并在克服脑癌方面显示出巨大的前景。DOX是一种对多种癌症都有效的化疗药物，但由于严重的副作用，在临床应用中受到限制。LND是一种化疗增敏剂，它通过抑制肿瘤细胞线粒体的有氧呼吸，使肿瘤细胞对各种治疗方法敏感而具有抗癌活性。
对羟基苯甲酸（p-HA）是一种对多巴胺受体和sigma受体均具有高亲和力的小分子配体，已被用于治疗中枢神经系统疾病。多巴胺受体和sigma受体分布在中枢神经系统的大部分部位，如血脑屏障。另外，sigma受体在包括脑肿瘤在内的各种肿瘤中也过表达，表明p-HA拥有作为靶向血脑屏障和脑肿瘤的配体的能力。
为了更好地发挥LND的作用，在递药系统中引入了三苯基鏻（TPP），不仅可以提高LND的溶解度，还可以利用三苯基鏻所带的正电荷以提高线粒体选择性。脂质体进入肿瘤细胞后，在肿瘤的酸性微环境下，酸敏感键断裂暴露出正电性的游离氨基，并在TPP的亲脂性和静电吸附作用下，选择性地靶向膜电位为−160~−180mV的线粒体，从而实现了脂质体对肿瘤细胞以及细胞器的双重靶向。
然而，TPP的修饰会使脂质体带强烈的正电荷，使其容易在血液循环中被内皮网状系统清除，因此，通过对脂质体进行PEG化，掩蔽TPP导致的正电荷，可以实现脂质体在体内的长循环。另外，酰胺键作为酸敏感键修饰脂质体，在肿瘤的酸性微环境下断裂后，暴露出正电性的游离氨基，并与TPP的正电荷共同作用下，可以通过质子海绵效应使其具有溶酶体逃逸能力，以便药物能够进一步进入细胞器发挥治疗作用。
发明内容
本发明提供一种对羟基苯甲酸和三苯基鏻共修饰的pH敏感的多靶向脂质体（Lip-CTPP），用于高效递送DOX和LND以协同治疗脑胶质瘤。Lip-CTPP通过以下步骤有效发挥脑胶质瘤治疗作用：（1）聚乙二醇化的Lip-CTPP在血液循环中保持稳定；（2）利用p-HA对多巴胺受体和sigma受体的亲和力，Lip-CTPP穿透血脑屏障；（3）通过sigma受体介导的内吞作用，Lip-CTPP进入肿瘤细胞；（4）酸敏感的酰胺键在溶酶体中被裂解，暴露TPP和游离氨基，使脂质体带正电；（5）Lip-CTPP通过质子海绵效应从溶酶体中逃逸；（6）DOX从脂质体中释放并进入细胞核；（7）剩余的脂质体部分进入线粒体。
Figure DEST_PATH_IMAGE001
Figure DEST_PATH_IMAGE002
本发明所述的脂质体用于化疗药和化疗增敏剂的联合包载，以协同治疗脑肿瘤。
附图说明
图1. Lip-0、Lip-CTPP 在 bEnd.3和C6细胞中的摄取；
图2. Lip-0、Lip-CTPP在bEnd.3和C6细胞线粒体中的摄取；
图3. Free DOX + LND、Lip-0和Lip-CTPP对C6细胞的细胞毒性评价；
图4. Free DOX + LND、Lip-0和Lip-CTPP对C6细胞的凋亡能力评价；
图5. Free DOX + LND、Lip-0和Lip-CTPP抑制C6细胞迁移侵袭的能力评价；
图6. DOX（A）和LND（B）在血浆中的药-时曲线；DOX（C）和LND（D）在脑中的药-时曲线；
图7. (A) 脑胶质瘤小鼠尾静脉注射不同制剂后的生存曲线图，箭头表示给药时间；(B) 脑胶质瘤小鼠尾静脉注射不同制剂后的中位生存期统计表。
本发明通过以下技术方案实现上述目的。
具体实施方式
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。
所述的脂质体具体由以下步骤制备：
脂质体配体的合成
实施例1
化合物3的制备
Figure DEST_PATH_IMAGE003
将叔丁基羰酰基6-氨基己酸（2.23 g, 9.64 mmol）溶解于二氯甲烷 （30 mL），于-10 ℃下加入DCC（3.97 g, 19.28 mmol）和DMAP（235 mg, 1.93 mmol），搅拌30分钟。将化合物2（5.00 g, 9.64 mmol）用二氯甲烷 (20 mL) 溶解，缓慢滴加至上述反应液中，并移至室温搅拌反应过夜。将反应液过滤，滤液经减压蒸馏除去溶剂后，经硅胶柱层析纯化（石油醚/乙酸乙酯=50/1），得无色油状物5.11 g，收率72.6%。1H-NMR (400 MHz, CDCl3, ppm) δ:0.67 (s, 3H), 0.86 (dd, 6H, J 1 = 1.6 Hz, J 2 = 6.4 Hz), 0.91 (d, 3H, J= 6.4 Hz),0.99 (s, 3H), 1.44 (s, 9H), 0.86-2.24 (remaining cholesterol protons), 2.34(t, 2H, J= 7.2 Hz), 3.10-3.22 (m, 3H), 3.63-3.66 (m, 8H), 3.70 (t, 2H, J= 4.8Hz), 4.23 (t, 2H, J= 4.8 Hz), 5.34 (s, 1H)。
实施例2
化合物4的制备
Figure DEST_PATH_IMAGE004
将化合物3（5.00 g, 6.83 mmol）用二氯甲烷（20 mL）和三氟乙酸（10 mL）溶解，室温搅拌30分钟。将反应液浓缩，残留物用二氯甲烷 (200 mL) 重新溶解，依次用饱和NaHCO3（100 mL × 2）、饱和NaCl（100 mL × 2）洗涤，有机相用无水Na2SO4干燥，过滤，浓缩，得黄色油状物4.21 g，收率98.0 %。
实施例3
化合物5的制备
Figure DEST_PATH_IMAGE005
氩气保护下，将化合物5（5.00 g, 7.91 mmol）和顺-4-环己烯-1,2-二羧酸酐（1.81 g, 11.87 mmol）用无水二氯甲烷（50 mL）溶解，加入2 mL三乙胺，室温下搅拌2小时。将反应液用饱和NaCl（100 mL × 3）洗涤，有机相用无水Na2SO4干燥，过滤，浓缩。经硅胶柱层析纯化（石油醚/乙酸乙酯=1/1），得黄色油状物4.58 g，收率73.8 %。1H-NMR (400 MHz,CDCl3, ppm) δ: 0.67 (s, 3H), 0.86 (dd, 6H, J 1 = 1.6 Hz, J 2 = 6.4 Hz), 0.91 (d,3H, J= 6.4 Hz), 0.99 (s, 3H), 0.86-2.28 (remaining cholesterol protons), 2.35(t, 2H, J= 7.2 Hz), 2.51-2.64 (m, 6H), 2.97-3.00 (m, 6H), 3.16-3.22 (m, 1H),3.24-3.30 (m, 2H), 3.62-3.66 (m, 8H), 3.69-3.72 (m, 2H), 4.22-4.24 (m, 2H),5.34 (s, 1H), 7.25 (br, 1H). HRMS (ESI) calculated for C47H77NO8Na+[M+Na]+-806.5541, found 806.5558。
实施例4
化合物6的制备
Figure DEST_PATH_IMAGE006
将化合物5（1.00 g, 1.28 mmol）用二氯甲烷（10 mL）溶解，于 -10 ℃下加入HBTU（0.97 g, 2.55 mmol），HOBT（0.34 g, 2.55 mmol），DIPEA（0.53 mL, 3.19 mmol）搅拌30分钟。将PEG2000-NH2（实验室自制，3.84 g, 1.92 mmol）用二氯甲烷（30 mL）溶解后，缓慢滴加至上述反应液中，并移至室温搅拌过夜。将反应液用饱和NaCl（20 mL × 2）洗涤，有机相用无水Na2SO4干燥，过滤，浓缩，经硅胶柱层析纯化（二氯甲烷/甲醇=50/1），得淡黄色固体2.13g，收率60.3 %。熔点：48.5-50.2 ℃。1H-NMR (400 MHz, CDCl3, ppm) δ: 0.68 (s, 3H),0.86 (d, 6H, J= 6.4 Hz), 0.91 (d, 3H, J= 6.4 Hz), 0.99 (s, 3H), 0.86-2.27(remaining cholesterol protons), 2.31-2.35 (m, 4H), 2.88-2.94 (m, 3H), 3.16-3.19 (m, 1H), 3.39-3.47 (m, 8H), 3.62-3.67 (m, 197H), 3.79-3.82 (m, 2H), 4.22(t, 2H, J= 4.8 Hz), 5.34 (s, 1H), 5.71-5.78 (m, 2H)。
实施例5
化合物7的制备
Figure DEST_PATH_IMAGE007
将对羟基苯甲酸（0.10 g, 0.72 mmol）用DMF（10 mL）溶解，于 -10 ℃下加入HBTU（0.27 g, 0.72 mmol），HOBT（0.10 g, 0.72 mmol），DIPEA（0.15 mL, 0.90 mmol）搅拌30分钟。将化合物6（1.00 g, 0.36 mmol）用DMF（10 mL）溶解后，缓慢滴加至上述反应液中，并移至室温搅拌过夜。将反应液浓缩，残留物用二氯甲烷（200 mL）溶解，用饱和NaCl（100 mL ×2）洗涤，有机相用无水Na2SO4干燥，过滤，浓缩。经硅胶柱层析纯化（二氯甲烷/甲醇=15/1），得白色固体0.65 g，收率62.3 %。熔点：39.7-41.1 ℃。1H-NMR (400 MHz, CDCl3, ppm) δ:0.67 (s, 3H), 0.86 (dd, 6H, J 1 = 1.6 Hz, J 2 = 6.4 Hz), 0.91 (d, 3H, J= 6.4 Hz),0.99 (s, 3H), 0.86-2.23 (remaining cholesterol protons), 2.31-2.34 (m, 4H),2.88-2.98 (m, 3H), 3.38-3.47 (m, 8H), 3.59-3.69 (m, 197H), 3.80-3.81 (m, 2H),4.22 (t, 2H, J= 4.8 Hz), 5.34 (s, 1H), 5.70-5.77 (m, 2H), 6.90 (d, 2H, J= 8.4Hz), 7.73 (d, 2H, J= 8.4 Hz). HRMS (ESI) calculated for C143H263N3O52Na+[M+Na]+-2598.6068, found 2598.5601。
以下实施例6-13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治疗作用。
实施例6
脂质体的制备
脂质体的制备处方为：脂质材料/DOX/LND＝40/1/1（质量比），其中Chol-CTPP的脂质材料处方为：Chol/SPC/Chol-CTPP/Chol-TPP= 34:60:3:3（摩尔比）；Lip-0 的脂质材料处方为SPC/Chol/=60/40（摩尔比）。将脂质材料和LND溶解在CHCl3/CH3OH（v/v=2/1）的混合溶剂中，并在38 ℃下旋蒸以形成均匀的薄膜。然后，加入DOX水溶液在50 ℃下水化1小时。最后，间歇超声（80W，5S，5S）3分钟后得到脂质体。荧光标记的脂质体通过将上述的LND替换为荧光标记的磷脂（CFPE），并将DOX水溶液替换为PBS缓冲液即可。
实施例7
脂质体的表征
将超声后的脂质体加入超滤管（10 kd）中并在10000 rpm下离心30分钟，上层即得到包有药物的脂质体。通过HPLC分析DOX和LND的含量。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、PDI和Zeta电位，结果如表1所示。两种脂质体均分散均匀，带微弱正电荷，且对DOX和LND的包封率均大于80%。
表1：脂质体 Lip-0 和Lip-CTPP的表征
Figure DEST_PATH_IMAGE008
体外活性评价
实施例8
细胞摄取
将C6和bEnd.3细胞在12孔板中培养24小时，再和CFPE标记的Lip-0、Lip-CTPP孵育4小时后，将细胞收集，洗涤，并用流式细胞仪测定荧光强度。如图1所示，相比于Lip-0，C6和bEnd.3细胞对Lip-CTPP的摄取能力显著提升。
实施例9
线粒体摄取
将C6和bEnd.3细胞在12孔板中培养24小时后，用CFPE标记的Lip-0、Lip-CTPP孵育12小时，然后收集，洗涤细胞，用线粒体提取试剂盒提取出线粒体并用流式细胞仪测定其荧光强度。如图2所示，相比于Lip-0，C6和bEnd.3细胞的线粒体对Lip-CTPP的摄取能力大大提升。
体外抗肿瘤活性评价
实施例10
细胞毒性评价
将C6细胞在96孔板中培养24小时，然后用20, 10, 5, 2.5, 0.5, 0.1 µg/mL药物浓度的Free DOX+LND、Lip-0、Lip-CTPP孵育24小时。用MTT法检测细胞毒性。如图3所示，不同制剂对C6细胞增殖的抑制能力为Lip-CTPP>Lip-0>Free DOX+LND，IC50分别为0.675、2.112、2.720 µg/mL。
实施例11
细胞凋亡评价
将C6细胞在6孔板中培养24小时后，用药物浓度为0.5 µg/mL的Free DOX + LND、Lip-0和Lip-CTPP孵育24小时。然后收集细胞，用凋亡试剂盒处理细胞，并在流式细胞仪中分析细胞凋亡情况。如图4所示，不同制剂诱导C6细胞凋亡的能力为Lip-CTPP>Lip-0>FreeDOX+LND，Lip-CTPP诱导的细胞凋亡和坏死率为Lip-0的1.45倍，为Free DOX+LND的1.87倍。
实施例12
迁移侵袭能力评价
将1 mg/mL的基质胶加入到上室中孵育6小时，再在上室中加入含有不同制剂和C6细胞的培养基，下室中加入10% FBS的培养基，孵育48h。然后对上室细胞进行擦拭、固定、染色，并测定吸光度以评价细胞的侵袭能力。细胞迁移能力的评价过程，除了不在上室中加入基质胶，其余和上述一致。如图5所示，与Free DOX+LND和Lip-0相比，Lip-CTPP明显抑制了C6细胞的迁移和侵袭能力。
体内活性评价
实施例13
药代动力学和体内脑靶向性评价
将昆明鼠（20-25 g）随机分成三组，每组分别通过尾静脉注射Free DOX+LND、Lip-0、Lip-CTPP。注射后的30，60，120，240，480，720和1440分钟，对小鼠进行眼眶取血获得血浆样品，处死小鼠剖出脑子获得脑样。将血样在10000 rpm下离心10分钟收集上清液，将脑样加入生理盐水（w/w =1：2）匀浆以制得脑匀浆。在血浆（100 μL）或脑匀浆（300 μL）中加入三倍体积的乙腈，涡旋5分钟。然后，将样品在10000 rpm下离心10分钟并吹干上清液。残余物用甲醇/水=1/1的混合溶剂重新溶解，并于10000 rpm下离心10 分钟，取上清液用HPLC分析DOX和LND浓度。结果如图6所示，与游离药物相比，Lip-CTPP显著改善了DOX和LND的药代动力学行为，不仅减慢了药物在体内的清除速率，还促使药物进入脑组织，大大提高了药物的脑靶向性。
实施例14
体内抗肿瘤活性评价
用C6细胞建立原位脑胶质瘤昆明鼠模型，随机分为4组，建模后第4、7、10、13天，按5 mg/kg的DOX和LND剂量分别给每组小鼠给予生理盐水、Free DOX+LND、Lip-0和Lip-CTPP，并每天监测小鼠生存情况。结果如图7所示，各组小鼠存活时间趋势为Lip-CTPP>Lip-0>Free DOX+LND>生理盐水，相比于生理盐水组，Lip-CTPP组小鼠的中位生存期从16.5天延长至41.5天。

Claims (3)

1.一种对羟基苯甲酸和三苯基鏻共修饰的pH敏感型多靶向脂质体Lip-CTPP，其结构特征在于，含有配体对羟基苯甲酸修饰的胆固醇Chol-CTPP和三苯基鏻修饰的胆固醇Chol-TPP，结构如下所示：
Figure QLYQS_1
，
Figure QLYQS_2
。
2.根据权利要求1所述的对羟基苯甲酸和三苯基鏻共修饰的pH敏感型多靶向脂质体Lip-CTPP，其特征在于，由膜材与活性剂组成，膜材的组成是磷脂、胆固醇和两种配体Chol-CTPP、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-CTPP=1/1~3/1；所述的活性剂为治疗剂，两种活性剂的质量比为化疗药/化疗增敏剂=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所述的对羟基苯甲酸和三苯基鏻共修饰的pH敏感型多靶向脂质体Lip-CTPP，其特征在于，磷脂为大豆磷脂、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。
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