CN110452298A - 一种肝靶向配体及其在脂质体制剂中的应用 - Google Patents
一种肝靶向配体及其在脂质体制剂中的应用 Download PDFInfo
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Abstract
本发明公开了一种肝靶向配体及其在脂质体制剂中的应用，该配体是肝细胞生长因子经胰弹力蛋白酶水解获得的单链抗体片段，包括N末端的发夹结构和4个Kringle结构，即为NK4肝靶向配体；NK4肝靶向为NK4蛋白分子。本发明还利用配体NK4与肝细胞生长因子c‑Met受体特异性结合的原理，以HGF单链抗体片段NK4作为肝靶向配体制备NK4表面修饰脂质体制剂，介导药物主动靶向于肝脏组织；既能达到靶向识别结合肝癌细胞的目的，又能通过NK4阻断HGF/c‑Met信号通路活性，抑制肿瘤细胞生长，协同增强药物抗肿瘤效果。NK4介导的脂质体药物递送系统在肝肿瘤靶向治疗方面存在较大的应用潜力。
Description
技术领域
本发明属于医药技术领域，具体是一种新的肝靶向配体NK4及其在脂质体制剂中的应用。
背景技术
肝细胞癌（Hepatocellular carcinoma，HCC）是临床上一种常见且具有高度侵袭性的恶性癌症，发病率在全世界范围内排第5位，由于其预后差，死亡率排第3位。我国是肝癌高发地区，且发病率有上升趋势，但目前临床上缺乏安全、有效的抗肝癌药物或治疗手段。
近年来，靶向药物治疗在肿瘤治疗领域取得了重大突破，脂质体（liposome）是一种靶向药物载体，属于靶向递药系统的一种新剂型。脂质体微粒给药系统具有良好的生物相容性和生物可降解性，可以保护药物活性基团，延长药物的半衰期，经给药进入体内后，主要被网状内皮细胞所吞噬，被动靶向于肝脏等网状内皮系统。如果对脂质体表面进行修饰，如连接单克隆抗体，可使脂质体具有主动靶向性，从而进一步提高药物疗效，减少毒副作用。
肝细胞生长因子（hepatocyte growth factor，HGF）是间质细胞（如成纤维细胞、巨噬细胞等）产生的细胞因子，作用于肿瘤细胞表面的受体c-Met蛋白。HGF/c-Met信号在促进肿瘤发生、侵袭、转移和肿瘤血管生成方面发挥重要作用，因此，HGF/c-Met可以作为抗肿瘤药物设计的靶标。其中，由HGF的α链N末端和4个Kringle区域所组成的NK4蛋白是HGF的特异性拮抗剂，NK4与Met受体结合，但未激活Met受体，从而竞争性抑制HGF诱导的Met受体激活，阻断HGF/c-Met系统的信号转导，抑制肿瘤的生成、侵袭及转移，还能不依赖HGF/c-Met抑制肿瘤血管的新生，促进肿瘤细胞的凋亡。Zhu Y等人的研究发现，NK4可在体外抑制HGF介导的多种肿瘤细胞的生长发展，并可抑制裸鼠体内肿瘤的生长、侵袭和转移（Zhu Y,Cheng M, Yang Z, et al. Mesenchymal stem cell-based NK4 gene therapy in nudemice bearing gastric cancer xenografts[J]. Drug Design, Development andTherapy, 2014, 8:2449-2462.）。
发明内容
本发明的目的之一是提供一种新的肝靶向配体，该配体是肝细胞生长因子经胰弹力蛋白酶水解获得的单链抗体片段，包括N末端的发夹结构和4个Kringle结构，即为NK4肝靶向配体。
所述NK4肝靶向为NK4蛋白分子。
所述NK4肝靶向配体是抗体或抗体片段。
所述NK4肝靶向配体结合于肝细胞上的肝细胞生长因子c-Met受体，其中Kringle是NK4与c-Met受体结合的关键部位。
所述NK4肝靶向配体用于递送抗肿瘤药物靶向于肝细胞。
NK4的结构如图1所示，NK4肝靶向配体的制备方法如下：
采用密码子优化软件MaxCodonTM Optimization Program (V13)对提供的NK4 蛋白氨基酸序列进行优化，采用全基因合成并通过限制性酶切位点NdeI和HindIII将NK4基因插入到表达载体pET30a中，并通过酶切法和测序确认最终表达载体的准确性，最终分别转到Top10克隆菌株和BL21(DE3)表达菌株中，通过IPTG诱导表达NK4 蛋白，之后通过亲和层析（Ni-IDA树脂）纯化NK4 蛋白。
表达载体转化及诱导表达：将构建好的含有NK4 基因的质粒转化到BL21(DE3)感受态细胞中，然后均匀涂布到LB平板上（含50 μg/mL的硫酸卡那霉素），之后倒置于37℃培养箱过夜。
SDS-PAGE分析鉴定诱导表达结果：从转化的平板中挑选单克隆，接种到4 mL的LB培养基中（含50 μg/mL的硫酸卡那霉素），待培养至OD600为0.5-0.8，向试管培养液中加入终浓度0.2 mM IPTG，之后分别置于15℃、37℃诱导表达。
取诱导后的培养液12000 rpm离心5 min，去除上清液，加入PBS液重悬沉淀，最后加入SDS-PAGE上样缓冲液于100℃下加热样品10 min，然后离心取上清电泳。160 V稳压电泳至溴酚蓝带迁移至离凝胶底部1 cm，取出凝胶利用蛋白凝胶快速处理系统染脱色。
NK4 蛋白放大培养：培养4 L的表达菌，生长至OD600=0.8时，加终浓度0.2 mMIPTG，15℃诱导16 h后收集菌体。
上清中亲和层析纯化NK4 蛋白（整个纯化过程在低温下操作）：全菌采用50 mMTris(pH8.0)，300 mM NaCl，20 mM Imidazole含1% Triton X-100，1 mM DTT，1 mM PMSF超声裂解，同时以50 mM Tris(pH8.0)，300 mM NaCl，20 mM Imidazole缓冲液平衡Ni-IDA亲和层析柱，之后用不同浓度咪唑的平衡缓冲液洗脱目标蛋白，并收集每个洗脱组分进行SDS-PAGE分析检测。
包涵体通过亲和层析纯化NK4 蛋白：包涵体采用50 mM Tris(pH8.0)，300 mMNaCl含1% Triton X-100，2 mM EDTA，5 mM DTT洗涤后，以50 mM Tris(pH8.0)，300 mMNaCl，8 M Urea，20 mM Imidazole缓冲液溶解包涵体同时平衡Ni-IDA柱，最后用不同浓度咪唑的平衡缓冲液洗脱目标蛋白，并收集每个洗脱组分进行SDS-PAGE分析检测，检测情况如图2-5所示。
经Ni-IDA亲和层析纯化分析，收集纯度相对较高的Lane 4-11，下一步进行分子筛纯化。
分子筛层析（SEC）纯化NK4 蛋白：
SEC柱型号：XK 16/100
SEC填料型号： Superdex 200 prep grade
流速：1.0 mL/min
纯化缓冲液：50 mM Tris(pH8.0)，300 mM NaCl
合并纯度相对高的Lane5-7，将其加入到处理后的透析袋中，4℃环境下，透析到缓冲液［50 mM Tris(pH8.5)，300 mM NaCl，1M Urea］中复性，复性后NK4蛋白最终透析于储存液50 mM Tris(pH8.5)，300 mM NaCl溶液约6-8 h。透析结束后用0.22 μm滤器过滤，并分装冻干得NK4。
本发明另一个目的是提供了一种NK4修饰脂质体制剂，将NK4肝靶向配体修饰于脂质体表面；脂质体制剂由药物、卵磷脂、胆固醇、磷脂酰乙醇胺-聚乙二醇-马来酰亚胺、NK4组成，其中各组分比例为：药物与卵磷脂质量比为1∶5~30；胆固醇与卵磷脂质量比为1∶3~7；磷脂酰乙醇胺-聚乙二醇-马来酰亚胺与卵磷脂质量比为1∶100~300；NK4与卵磷脂质量比为1∶100~1000。
制备的NK4修饰脂质体制剂，该制剂载体可用于包载各种不同的抗肿瘤药物，以提高药物的肝靶向抗肿瘤效果。
所述NK4修饰脂质体制剂的制备方法，包括如下步骤：
（1）按脂质体制剂各组分的质量比称取药物、卵磷脂、胆固醇、磷脂酰乙醇胺-聚乙二醇-马来酰亚胺、NK4备用；
（2）取药物、胆固醇、卵磷脂，将三者混合后加入氯仿溶解药物、胆固醇与卵磷脂，得类脂溶液；氯仿的加入量以能完全溶解药物、胆固醇与卵磷脂为准；
（3）将步骤（2）的类脂溶液转移至茄形瓶中，减压蒸发至有机溶剂完全蒸发，得类脂膜；
（4）在蒸发得到的类脂膜中加入pH为6.8的HEPES缓冲液水合反应，得脂质体溶液；HEPES缓冲液的加入量达到卵磷脂浓度为30 mg/mL即可；
（5）取磷脂酰乙醇胺-聚乙二醇-马来酰亚胺溶于HEPES缓冲液，配制0.5 mg/mL的磷脂酰乙醇胺-聚乙二醇-马来酰亚胺溶液，按组分比例加入到步骤（4）脂质体溶液于60℃下孵育1 h；
（6）按组分比例将NK4加入到步骤（5）含磷脂酰乙醇胺-聚乙二醇-马来酰亚胺的脂质体溶液中，4℃下偶联反应24 h；
（7）将脂质体溶液用脂质体挤出仪经200 nm 聚碳酸酯膜来回挤出，制备得NK4配体修饰脂质体制剂。
所述磷脂酰乙醇胺-聚乙二醇-马来酰亚胺-NK4配体偶联，合成方法如下：
。
本发明NK4修饰脂质体制剂，利用配体（NK4）与肝细胞生长因子受体（c-Met）特异性结合的原理，以HGF单链抗体片段NK4作为肝靶向配体制备NK4表面修饰脂质体制剂，介导药物主动靶向于肝脏组织；既能达到靶向识别结合肝癌细胞的目的，又能通过NK4阻断HGF/c-Met信号通路活性，抑制肿瘤细胞生长，协同增强药物抗肿瘤效果。NK4介导的脂质体药物递送系统在肝肿瘤靶向治疗方面存在较大的应用潜力。
附图说明
图1为NK4蛋白结构图；
图2为SDS-PAGE分析NK4 蛋白于BL21(DE3)表达情况图；
图3为SDS-PAGE分析NK4 蛋白上清纯化图；
图4为 SDS-PAGE分析包涵体中NK4 蛋白纯化图；
图5为 SDS-PAGE分析SEC纯化图；
图6为NK4修饰羟基喜树碱脂质体（NK4-HCPT-Lip）的示意图；
图7为NK4-HCPT-Lip透射电镜图；
图8为NK4-HCPT-Lip粒径分布图；
图9为HCPT溶液和NK4-HCPT-Lip体外释放图。
具体实施方式
下面结合具体实施例和附图对本发明内容作进一步的说明，只是举例说明，不应当构成对本发明的限定。
实施例
制备NK4修饰脂质体制剂，包括如下步骤：
（1）按脂质体制剂各组分的质量比称取药物、卵磷脂、胆固醇、磷脂酰乙醇胺-聚乙二醇-马来酰亚胺、NK4备用：
药物与卵磷脂质量比为1∶5~30；胆固醇与卵磷脂质量比为1∶3~7；磷脂酰乙醇胺-聚乙二醇-马来酰亚胺与卵磷脂质量比为1∶100~300；NK4与卵磷脂质量比为1∶100~1000；
（2）本实施例的药物为羟基喜树碱（HCPT），取羟基喜树碱6 mg、胆固醇30 mg、卵磷脂120 mg，将三者混合后再加入10 mL氯仿溶解药物、胆固醇与卵磷脂得类脂溶液；
（3）类脂溶液转移至250 mL茄形瓶中，减压蒸发至有机溶剂完全蒸发；
（4）在蒸发得到的类脂膜中加入 4 mL、 pH为6.8的HEPES缓冲液，水合1.5 h，得脂质体溶液；
（5）将磷脂酰乙醇胺-聚乙二醇-马来酰亚胺 0.5 mg 溶于1 mL HEPES缓冲液，并与羟基喜树碱脂质体溶液于60 ℃下孵育1 h；
（6）随后，将1 mg NK4加入到含磷脂酰乙醇胺-聚乙二醇-马来酰亚胺的羟基喜树碱脂质体溶液中，4 ℃下偶联反应24 h；
（7）将脂质体溶液用脂质体挤出仪经200 nm 聚碳酸酯膜来回挤20-30次，制备得NK4配体修饰的羟基喜树碱脂质体（NK4-HCPT-Lip），如图6所示。
脂质体包封率与载药量的测定：采用葡聚糖凝胶柱层析法，取脂质体混悬液0.5mL，上Sephadex G-50葡聚糖凝胶柱，用HEPES缓冲液（pH6.8）洗脱收集，分离脂质体与游离药物，加甲醇对脂质体破乳，高效液相色谱法（HPLC）测定含量，计算脂质体包封率与载药量。
包封率（%）=（包入脂质体中的HCPT量/HCPT投入量）×100%
载药量（%）=（包入脂质体中的HCPT量/脂质体总质量）×100%
体外释放度的考察：
取脂质体溶液3批，以等浓度的羟基喜树碱溶液作对照，分别吸取1 mL移入已处理好的透析袋中，将透析袋两端扎紧，置于200 mL pH7.4 的PBS中，恒温（37±1）℃、恒速（100 r/min）搅拌，分别于0.5、1、2、4、6、8、12、24、48、72 h取释放液1 mL，同时补加同体积同温度的释放介质。
样品用10 μL冰醋酸酸化2 h，将药液以0.22 μm的微孔滤膜过滤，HPLC测定含量，计算平均累积释放百分率。
如图7-9所示，结果显示：HCPT溶液体外释药迅速，2 h累积释放达78%，脂质体药物释放缓慢，72 h累积释放率为61%，与HCPT溶液相比，NK4-HCPT-Lip具有较好的缓释作用。
细胞毒性实验：选用HepG2肝癌细胞，在37℃、5％CO2培养箱中培养。取对数生长期的HepG2肝癌细胞100 μl，接种于96孔板，分别加入不同浓度（1、10、20、40、60 μl/ml ）HCPT溶液、HCPT-Lip、NK4-HCPT-Lip 各50 μl，每个浓度设5个复孔，培养箱中孵育48 h后，每孔加入20 μl MTT，继续放至培养箱中培养4 h，4 h后吸掉培养液，每孔加入150 μl DMSO，充分震荡10 min，酶标仪以490 nm波长测吸光度值。相比于HCPT-Lip，NK4-HCPT-Lip对HepG2肝癌细胞抑制作用更强，且在一定浓度范围内，具有剂量依赖性趋势。这表明NK4加强了HepG2肝癌细胞对载药脂质体的内吞作用。
Claims (8)
1.一种肝靶向配体，其特征在于：该配体是肝细胞生长因子经胰弹力蛋白酶水解获得的单链抗体片段，包括N末端的发夹结构和4个Kringle结构，即为NK4肝靶向配体。
2.根据权利要求1所述的肝靶向配体，其特征在于：所述NK4肝靶向为NK4蛋白分子。
3.根据权利要求1所述的肝靶向配体，其特征在于：所述NK4肝靶向配体是抗体或抗体片段。
4.根据权利要求1-3任一项所述的肝靶向配体，其特征在于：所述NK4肝靶向配体结合于肝细胞上的肝细胞生长因子c-Met受体，其中Kringle是NK4与c-Met受体结合的关键部位。
5.根据权利要求1-4任一项所述的肝靶向配体，其特征在于：所述NK4肝靶向配体用于递送抗肿瘤药物靶向于肝细胞。
6.一种NK4修饰脂质体制剂，其特征在于：将权利要求1所述的NK4肝靶向配体修饰于脂质体表面；
脂质体制剂由药物、卵磷脂、胆固醇、磷脂酰乙醇胺-聚乙二醇-马来酰亚胺、NK4组成，其中各组分比例为：药物与卵磷脂质量比为1∶5~30；胆固醇与卵磷脂质量比为1∶3~7；磷脂酰乙醇胺-聚乙二醇-马来酰亚胺与卵磷脂质量比为1∶100~300；NK4与卵磷脂质量比为1∶100~1000。
7.根据权利要求6所述NK4修饰脂质体制剂的制备方法，其特征在于，包括如下步骤：
（1）按脂质体制剂各组分的质量比称取药物、卵磷脂、胆固醇、磷脂酰乙醇胺-聚乙二醇-马来酰亚胺、NK4备用；
（2）取药物、胆固醇、卵磷脂，将三者混合后加入氯仿溶解药物、胆固醇与卵磷脂，得类脂溶液；氯仿的加入量以能完全溶解药物、胆固醇与卵磷脂为准；
（3）将步骤（2）的类脂溶液转移至茄形瓶中，减压蒸发至有机溶剂完全蒸发，得类脂膜；
（4）在蒸发得到的类脂膜中加入pH为6.8的HEPES缓冲液水合反应，得脂质体溶液；HEPES缓冲液的加入量达到卵磷脂浓度为30 mg/mL即可；
（5）取磷脂酰乙醇胺-聚乙二醇-马来酰亚胺溶于HEPES缓冲液，配制0.5 mg/mL的磷脂酰乙醇胺-聚乙二醇-马来酰亚胺溶液，按组分比例加入到步骤（4）脂质体溶液于60℃下孵育1 h；
（6）按组分比例将NK4加入到步骤（5）含磷脂酰乙醇胺-聚乙二醇-马来酰亚胺的脂质体溶液中，4℃下偶联反应24 h；
（7）将脂质体溶液用脂质体挤出仪，经200 nm 聚碳酸酯膜来回挤出，制备得NK4配体修饰脂质体制剂。
8.根据权利要求7所述NK4修饰脂质体制剂的制备方法，其特征在于：步骤（6）所述磷脂酰乙醇胺-聚乙二醇-马来酰亚胺-NK4配体偶联，合成方法如下：
。
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