CN110368494A - 靶向内质网的纳米载体及其使用方法 - Google Patents

靶向内质网的纳米载体及其使用方法 Download PDF

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CN110368494A
CN110368494A CN201910194401.8A CN201910194401A CN110368494A CN 110368494 A CN110368494 A CN 110368494A CN 201910194401 A CN201910194401 A CN 201910194401A CN 110368494 A CN110368494 A CN 110368494A
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poly
curcumin
disease
cell
inhibitor
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谢奇璋
颜嘉良
谢达斌
蒋思澈
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Abstract

一种用囊封于聚合物纳米粒子中的肌质/内质网钙离子ATP酶泵抑制剂治疗由内质网(ER)中的蛋白质驻留所引起的疾病的方法。该聚合物纳米粒子经表面修饰以使得其靶向ER。该抑制剂减少ER中的蛋白质驻留，并且相较于施用未经囊封的抑制剂，囊封会减少该抑制剂的副作用，例如细胞毒性。还公开了一种可以用于实施该方法的药物组合物。进一步提供一种在其基因组中携带编码H338Y突变型gp91phox蛋白质的异源核酸的转基因小鼠。该转基因小鼠可以充当人类慢性肉芽肿病的模型。

Description

靶向内质网的纳米载体及其使用方法
相关申请的交叉引用
本申请要求2018年4月4日提交的美国临时专利申请号62/652,502 的优先权。
背景技术
慢性肉芽肿病(CGD)是由白血球中烟酰胺腺嘌呤二核苷酸磷酸氧化酶 2(NOX2)中的缺陷所引起的原发性免疫缺陷疾病。参见例如，Holland 2013,Hematol.Oncol.Clin.North Am.27:89-99；O'Neill等人2015,Redox Biol. 6:135-156；以及Roos 2016,Br.Med.Bull.118:50-63(Roos)。NOX2由膜结合亚单元gp91phox和p22phox，以及细胞溶质组分p40phox、p47phox、p67phox和小GTP酶Rac1/2构成。参见Roos。携带编码NOX2组分蛋白质中的一者的基因中的突变的CGD患者由于缺乏由活化白血球产生的NOX2介导的活性氧(ROS)而易受不同感染和某些自体免疫疾病影响。参见Huang等人2016,Inflamm.BowelDis.22:2794-280；Huang等人2015,Antioxid. Redox Signal.23:973-984；以及Nguyen等人2017,Front.Cell Infect. Microbiol.7:373。
已鉴别CGD患者在编码gp91phox的Cybb基因中具有点突变。参见Lin 等人2002,Biochim.Biophys.Acta 1586:275-286(Lin)。突变Cybb基因 CybbC1024T产生含有单一氨基酸变化的突变型gp91phox蛋白，其称为 H338Y-gp91phox。H338Y-gp91phox以不成熟形式存在，其保留在内质网(ER) 中并且随后通过ER中的蛋白质质量控制机制降解。参见上文。
先前已显示，肌质/内质网钙离子ATP酶(SERCA)抑制剂，即，毒胡萝卜素(thapsigargin)会挽救CGD白血球中H338Y-gp91phox的ER驻留并且恢复白血球功能。参见Huang等人2009,Free Radical Biol.Med. 47:932-940。已显示，姜黄素(也是一种SERCA抑制剂)会在囊肿性纤维化中部分校正异常ER蛋白质驻留。参见Egan等人2004,Science 304:600-602。然而，姜黄素具有细胞毒性副作用，包括ER压力和细胞凋亡。参见Gupta等人2013,Mol.Nutr.Food Res.57:1510-1528；Liu等人 2016,Evid.BasedComplement.Alternat.Med.2016:7831282；以及Reuter 等人2008,BiochemicalPharmacology 76:1340-1351。
需要研发具有较高功效和较少副作用的用于治疗ER驻留的疾病的药物组合物及方法。
发明内容
为了满足上文所论述的需要，公开了一种用于治疗由内质网中的蛋白质驻留所引起的疾病的方法。该方法是通过获得囊封于聚合物纳米粒子中的肌质/内质网钙离子ATP酶(SERCA)泵抑制剂并且向患有疾病的受试者施用有效量的抑制剂来实施。
聚合物纳米粒子经表面修饰以使得其靶向内质网(ER)。此外，有效量的抑制剂减少ER中的蛋白质驻留，并且相较于施用未经囊封的抑制剂，囊封会减少抑制剂的副作用，例如细胞毒性。
还公开了一种可以用于实施以上方法的药物组合物。该药物组合物含有囊封于聚合物纳米粒子中的SERCA泵抑制剂以及药用载体。如上文所提及，聚合物纳米粒子经表面修饰以使得其靶向内质网。
此外，提供一种在其基因组中携带编码H338Y突变型gp91phox蛋白质的异源核酸的转基因小鼠。突变型gp91phox表达于转基因小鼠的细胞中，并且野生型gp91phox未表达于转基因小鼠的细胞中。转基因小鼠可以充当人类慢性肉芽肿病的模型。
本发明的一个或多个实施方案的详情阐述于以下描述中。本发明的其他特征、目标及优点根据说明书、附图和权利要求将是显而易见的。
附图简要说明
以下描述参考附图，其中：
图1A为显示通过从未处理小鼠(-)、用游离姜黄素处理的小鼠或用所指示的负载姜黄素的PLGA纳米粒子处理的小鼠分离的嗜中性粒细胞的蛋白质印迹(Western Blot)分析测定的，具有Cybb-/-背景的CybbC1024T转基因小鼠(“TG小鼠”)中的gp91phox的糖基化形式和糖基化不足形式之间的比率的条形图(n＝4；*：p<0.05)；
图1B为显示从用游离姜黄素或用靶向ER的负载姜黄素的PLGA纳米粒子处理的TG小鼠分离的嗜中性粒细胞中的活性氧(ROS)产生的条形图(n＝4；**：p<0.01)；
图1C为显示未处理(-)及如所指示加以处理的TG小鼠中的呈菌落形成单位(CFU)形式的细菌负荷的条形图。(n＝4；*：p<0.05；**：p<0.01)；
图2为相较于非靶向的负载姜黄素的PLGA纳米粒子，对靶向ER的姜黄素PLGA纳米粒子随时间推移共定位至ER进行定量的条形图(*： p<0.05；**：p<0.01；***：p<0.001)；
图3为显示如所指示加以处理的分化HL60细胞中的钙离子通量的条形图，该钙离子通量根据胞内钙离子释放速率相对于时间的图计算为曲线下面积(AUC)(n＝4；*：p<0.05；**：p<0.01；***：p<0.001)；
图4为显示如所指示加以处理的分化HL60细胞中的钙离子通量的条形图，该钙离子通量根据胞外钙离子吸收速率相对于时间的图计算为曲线下面积(AUC)(n＝3；*：p<0.05)；
图5A为如所指示加以处理的分化HL60细胞中的用荧光染料测定的粒线体膜电位的条形图(n＝4；*：p<0.05；**：p<0.01)；
图5B为通过如所指示加以处理的分化HL60细胞群的流式细胞术测定的磷脂结合蛋白V(annexin V)阳性细胞的百分比的条形图(n＝4；*： p<0.05；**：p<0.01)；
图5C显示由从如所指示加以处理的分化HL60细胞提取的蛋白质的蛋白质印迹分析所测量的，Bip(左图)、磷酸化ERK(中图)和裂解的胱天蛋白酶-3(caspase-3)(右图)的相对强度的条形图(n＝4；*：p<0.05)；
图6A为显示通过未处理(-)及如所指示加以处理的人类外周血造血细胞(HSC)的流式细胞术测定的，呈未处理的百分比形式的CD71低CD235a 高细胞的数目的条形图(n＝3；*：p<0.05；**：p<0.01)；以及
图6B为通过如所指示加以处理的HSC的流式细胞术测定的，磷脂结合蛋白V阳性细胞的百分比的条形图(n＝3；**：p<0.01；***：p<0.001)。
发明详述
如上文所提及的，公开了一种用SERCA泵抑制剂治疗由内质网中的蛋白质驻留所引起的疾病的方法。所述抑制剂为膳食多酚化合物。化合物可为(但不限于)姜黄素、毒胡萝卜素、环匹阿尼酸(cyclopiazonic acid)、2,5- 二叔丁基氢醌、姜辣素(gingerol)、CGP37157、叔丁基氢醌、蕈青霉素 (paxilline)和柴胡皂苷d(saikosaponin-d)。也可以使用这些化合物中的一种或多种的组合。在一个特定方法中，膳食多酚化合物为姜黄素。
SERCA泵抑制剂囊封于聚合物纳米粒子中。可以用于形成纳米粒子的示例性聚合物包括聚(乳酸-共-乙醇酸)、聚(交酯)、聚(氨基酸)、聚(ε-己内酯)、聚氰基丙烯酸烷酯(polyalkylcyanoacrylate)、藻酸盐、明胶、卵磷脂 -壳聚糖、白蛋白和壳聚糖。也可以使用这些聚合物的混合物。在一个具体方法中，聚合物由聚(乳酸-共-乙醇酸)形成。
上述聚合物纳米粒子经表面修饰以使其靶向ER。表面修饰可以是与聚合物缀合的靶向ER的肽。一种或多种靶向ER的肽，例如KDEL(SEQ ID NO:1)、KKXX(SEQ ID NO:2)或基于精氨酸的RXR型ER驻留/回收 (retrieval)信号肽，可以与聚合物缀合。使用KDEL作为示例性方法中的靶向ER的肽。
向患有ER驻留介导的疾病的受试者以有效治疗该疾病的量施用囊封于聚合物纳米粒子中的SERCA泵抑制剂。有效量的抑制剂减少ER中的蛋白质驻留，并且相较于施用未经囊封的抑制剂，囊封会减少抑制剂的副作用。经减少的副作用可以是细胞毒性，例如ER压力和细胞凋亡。
有效量的SERCA泵抑制剂可以促进受试者中的白血球产生活性氧，并且促进由白血球介导的腹膜细菌清除。
如上文所阐述的，本文所述的方法用于治疗由内质网中的蛋白质驻留所引起的疾病。这样的疾病包括(但不限于)慢性肉芽肿病、囊肿性纤维化、遗传性肺气肿、遗传性血色素沉着症、眼皮肤白化病、蛋白质C缺乏症、 I型遗传性血管性水肿、法布里病(Fabrydisease)、泰-萨二氏病(Tay-Sachs disease)、先天性蔗糖酶-异麦芽糖酶缺乏症、2型克果纳杰氏病 (Crigler-Najjar type 2)、糖尿病、莱伦氏综合征(Laron syndrome)、原发性甲状腺功能低下、先天性长QT综合征、甲状腺素结合球蛋白缺乏症、β-地中海贫血/Hb E病、家族性高胆固醇血症、家族性乳糜微粒血症、无β脂蛋白血症(abeta-lipoproteinemia)、先天性甲状腺功能低下、成骨不全、骨质石化病、遗传性低纤维蛋白原血症、肾源性尿崩症、垂体神经部尿崩症、夏-马-图三氏病(Charcot-Marie-Tooth disease)、家族性脑中叶硬化症(Pelizaeus-Merzbacher disease)、阿尔茨海默病、IIA型冯威利布兰德病(vonWillebrand disease)、丹吉尔病(Tangier disease)和伴随ALK1误义突变的2 型遗传性出血性毛细管扩张症(HHT)。在一个具体方法中，疾病为慢性肉芽肿病。
在又一个特定方法中，疾病为慢性肉芽肿病，膳食多酚化合物为姜黄素，聚合物为聚(乳酸-共-乙醇酸)，并且表面修饰包括与聚合物缀合的靶向 ER的肽KDEL。
为了实施以上方法，提供一种药物组合物。该药物组合物具有以上所提及的组分以及药用载体。更具体地，该组合物包括囊封于聚合物纳米粒子中的SERCA泵抑制剂，其中聚合物纳米粒子经表面修饰以使得其靶向内质网。
此外，聚合物纳米粒子由聚(乳酸-共-乙醇酸)、聚(交酯)、聚(氨基酸)、聚(ε-己内酯)、聚氰基丙烯酸烷酯、藻酸盐、明胶、卵磷脂-壳聚糖、白蛋白或壳聚糖中的一种或多种形成。SERCA泵抑制剂为选自由以下组成的组的膳食多酚化合物：姜黄素、毒胡萝卜素、环匹阿尼酸、2,5-二叔丁基氢醌、姜辣素、CGP37157、叔丁基氢醌、蕈青霉素和柴胡皂苷d或其组合。表面修饰包括与聚合物缀合的靶向ER的肽，并且靶向ER的肽为 KDEL、KKXX或基于精氨酸的RXR型ER驻留/回收信号。
一种示例性药物组合物含有囊封于经KDEL表面修饰的聚(乳酸-共- 乙醇酸)纳米粒子中的姜黄素以及药用载体。
如上文所提及的，转基因小鼠落入本发明的范围内。在其基因组中包括编码H338Y突变型gp91phox蛋白质的异源核酸的转基因小鼠在其细胞中表达突变型gp91phox。在一个实例中，表达H338Y突变型gp91phox蛋白质的细胞为白血球。转基因小鼠在腹膜细菌清除方面有缺陷。特别地，相较于野生型小鼠，转基因小鼠中的腹膜细菌性清除有所减少。
以下特定实例仅解释为示例性的，并且不以任何方式限制本发明的其余部分。无需进一步详细描述，据信本领域技术人员可以基于本文中的描述最大程度地利用本发明。本文中所引用的所有出版物均以全文引用的方式并入本文中。
实施例
实施例1：CGD转基因小鼠的产生
如下在Cybb基因敲除背景下建立由H338Y突变型gp91phox所引起的慢性肉芽肿病(CGD)的转基因小鼠模型。含有鼠类Cybb基因的细菌人工染色体(BAC)(克隆编号RP23-210P9)从奥克兰儿童医院及研究机构 (Children's Hospital Oakland ResearchInstitute)(美国加利福尼亚州奥克兰 (Oakland,CA USA))的BACPAC资源中心获得。使用反选择BAC修饰试剂盒(Counter Selection BAC Modification Kit)(Gene Bridges有限公司)， BAC经修饰而含有突变型CybbC1024T。使用标准技术将所得CybbC1024T BAC 转基因经由原核注射引入至从靶向Cybb的突变型B6.129S-Cybbtm1Din/J小鼠(杰克逊实验室(JacksonLabs)目录号002365)分离的卵子中。通过PCR 自尾部DNA且通过直接DNA测序鉴别携带BAC转基因的原代及后代。通过蛋白质印迹分析确认全长突变型CybbC1024T的表达。转基因小鼠进一步与Cybb基因敲除小鼠回交以产生具有Cybb基因敲除背景的CybbC1024T转基因小鼠。
C57BL/6小鼠用作野生型(WT)小鼠并且购自中国台湾台南市的成功大学(NCKU)的动物中心(Animal Center)。所有小鼠均常规地回交至 C57BL/6背景并且经历全基因组基因分型以确认遗传学背景。将小鼠圈养于NCKU的实验室动物中心(Laboratory AnimalCenter)的动物设施中。所有程序均经NCKU的实验动物照护及使用委员会(InstitutionalAnimal Care and Use Committee)批准。
实施例2：CGD转基因小鼠的分析
使用抗gp91phox抗体通过SDS-PAGE及蛋白质印迹法检查来自 CybbC1024T转基因Cybb-/-小鼠的嗜中性粒细胞中的H338Y-gp91phox蛋白质的表达。为了分离嗜中性粒细胞，用3ml的3％巯基乙酸盐对小鼠进行腹膜内注射，以引出腹膜嗜中性粒细胞。四小时之后，处死小鼠，并且用5ml 的具有2mM乙二醇四乙酸的汉克斯缓冲盐溶液(Hanks buffered saltsolution；HBSS)洗涤腹腔两次以收集引出的腹膜嗜中性粒细胞。随后在使用之前用PBS洗涤腹膜嗜中性粒细胞。
结果表明，从CybbC1024T转基因Cybb-/-小鼠分离的嗜中性粒细胞产生 H338Y-gp91phox的糖基化不足形式。相较于野生型小鼠，CybbC1024T转基因 Cybb-/-小鼠中的H338Y-gp91phox蛋白质的糖基化形式的水平显著较低。如所预期，Cybb-/-小鼠中未检测到gp91phox表达。这些结果表明，成功地建立携带CybbC1024T突变并且展现模拟见于人类CGD患者中的迁移缺陷性 (trafficking-defective)H338Y-gp91phox蛋白质表达的蛋白质表达的鼠类CGD 模型。
实施例3：负载姜黄素的纳米粒子的产生
用经改良的双重乳化法(W1/O/W2乳液；参见Zhong等人2010,J.Nanobiotechnology 8:6)制备负载姜黄素的聚(乳酸-共-乙醇酸)(PLGA)纳米粒子。简言之，将姜黄素溶解于500μL醇(W1)中，同时将PLGA(PLA:PGA ＝50:50，MW＝60,000)分散于二氯甲烷(O)中。将W1添加至O中并且用探针型超声振动器进行混合，形成第一乳液。将第一乳液即W1/O添加至 1％聚乙烯醇(PVA)水溶液(W2)中并且加以搅拌，形成含有PLGA纳米粒子的第二乳液(W1/O/W2)。随后使第二乳液在真空下脱气以移除有机溶剂，并且通过离心移除任何游离姜黄素及剩余PVA。通过使用NanoDrop 1000TM(美国NanoDrop Technologies有限责任公司)测量435nm处的光密度来分析PLGA纳米粒子的平均负载量。
实施例4：靶向ER的负载姜黄素的纳米粒子的产生
如下将如前述段落中所述的所产生的负载姜黄素的PLGA纳米粒子与靶向ER的肽缀合。将负载姜黄素的PLGA纳米粒子在乙基二甲基氨基丙基碳二亚胺/N-羟基丁二酰亚胺溶液中温育1小时，并且随后从溶液移除。将负载姜黄素的PLGA纳米粒子以及10倍摩尔过量的靶向ER的肽KDEL 悬浮于H2O中并且温育18小时，其后通过离心移除过量的肽。将经标记的纳米粒子再分散于磷酸盐缓冲盐水(PBS)溶液中。
实施例5：用负载姜黄素的纳米粒子处理CybbC1024T转基因Cybb-/-小鼠
测试含有或不含靶向ER的KDEL肽的负载姜黄素的PLGA纳米粒子对CybbC1024T转基因Cybb-/-小鼠中的H338Y-gp91phox蛋白质的表达和成熟的影响。简言之，小鼠未经处理或经游离姜黄素、负载姜黄素的PLGA纳米粒子或负载姜黄素的靶向ER的PLGA纳米粒子以相当于20mg/kg姜黄素的量单独地处理24小时。如上文所述，自小鼠引出嗜中性粒细胞，加以分离，并且分析H338Y-gp91phox蛋白质的表达。
用Prism软件(格拉夫帕德软件公司(GraphPad Software))进行统计学分析。各组的平均值呈现为平均值±SEM并且通过使用以下方式进行比较：单向方差分析(ANOVA)，之后是事后最小显著差异测试；或双向ANOVA，之后是邦弗朗尼事后测试(Bonferroni posthoc test)。在所有实验中，低于 0.05的p值视为统计学上显著的。
结果显示，所有经处理及未处理的CybbC1024T转基因Cybb-/-小鼠均表达H338Y-gp91phox的糖基化不足形式。如与未处理的小鼠相比，用游离姜黄素处理的小鼠在所表达的糖基化H338Y-gp91phox的量方面未显示出任何变化。另一方面，如与未处理的小鼠相比，用负载姜黄素的PLGA纳米粒子处理的小鼠显示出糖基化H338Y-gp91phox的表达增加1.3倍。此外，如与未处理的小鼠相比，用负载姜黄素的靶向ER的PLGA纳米粒子处理的小鼠表达出高1.7倍的糖基化H338Y-gp91phox。分析相同样本中的糖基化对糖基化不足H338Y-gp91phox的比率后，获得类似结果。参见图1A。
这些数据显示，经由靶向ER的PLGA纳米粒子将姜黄素递送至 CybbC1024T转基因Cybb-/-小鼠中的ER有助于嗜中性粒细胞中的突变型 H338Y-gp91phox蛋白质的成熟。
实施例6：CybbC1024T转基因Cybb-/-小鼠中的活性氧的产生
如上文所提及，由于ER中的gp91phox发生螯合(sequestration)，CGD 患者中对细菌清除至关重要的利用嗜中性粒细胞中的NOX2活性产生活性氧(ROS)受损。通过测量用游离姜黄素处理或用负载姜黄素的靶向ER 的PLGA纳米粒子处理的这些小鼠的嗜中性粒细胞中的ROS产生来检查 CybbC1024T转基因Cybb-/-小鼠中的NOX2功能。
ROS产生分析
如上文所述，引出并且分离CybbC1024T转基因Cybb-/-小鼠的腹膜嗜中性粒细胞。用1×HBSS洗涤嗜中性粒细胞并且用5％FBS洗涤三次，并且随后在37℃下与10μM 2',7'-二氯二氢荧光素二乙酸酯一起温育30分钟。随后在37℃下将细胞与100ng/ml佛波醇肉豆蔻酸酯13-乙酸酯(西格玛 (Sigma))一起温育15分钟，以诱导产生ROS。通过流式细胞术使用FACSCalibur仪器(BD生物科学(BD Biosciences))检测经PMA处理的细胞中的荧光，并且用Flowing Software 2软件分析数据。结果展示于图1B中。
由从用负载姜黄素的靶向ER的PLGA纳米粒子处理的CybbC1024T转基因Cybb-/-小鼠分离的嗜中性粒细胞产生的ROS明显高于从经游离姜黄素处理的动物分离的那些。这些数据显示，经由靶向ER的PLGA纳米粒子将姜黄素递送至CybbC1024T转基因Cybb-/-小鼠中的ER会恢复嗜中性粒细胞中的NOX2功能，并且与ER递送的姜黄素的能力相关，以促进嗜中性粒细胞中的突变型H338Y-gp91phox蛋白质的成熟。
实施例7：腹膜细菌清除测试
NOX2功能对清除细菌感染也是必要的。通过将细菌即金黄色葡萄球菌(S.aureus)腹膜内注射至未处理的CybbC1024T转基因Cybb-/-小鼠、用姜黄素处理的转基因小鼠和用经PLGA囊封的姜黄素处理的转基因小鼠中来检查细菌清除。
细菌制备
在37℃下在具有10％绵羊血液的胰蛋白酶大豆琼脂板上过夜培养金黄色葡萄球菌(ATCC第25923号)。从该板挑取单一菌落并且在37℃下在鲁利亚-贝尔塔尼(Luria-Bertani；LB)培养基中继代培养16小时。用5mL LB 以1:50稀释细菌培养物并且在37℃下温育6小时。通过将10μl培养物液滴铺展于TSA板上，将该板温育过夜并且计算菌落数来测定金黄色葡萄球菌培养物中的菌落形成单位(CFU)。
用金黄色葡萄球菌处理小鼠
CybbC1024T转基因Cybb-/-小鼠不加处理或用20mg/kg如上文所阐述的游离姜黄素、负载姜黄素的PLGA纳米粒子或负载姜黄素的靶向ER的 PLGA纳米粒子处理24小时。用100μlPBS中的1×107CFU的金黄色葡萄球菌对小鼠进行腹膜内注射。4小时之后，处死小鼠并且经由用5ml PBS 灌洗来收集腹膜细胞。通过将50μl灌洗流体的液滴涂铺于TSA板上并且计算过夜温育之后生长的金黄色葡萄球菌菌落来测定灌洗流体中的金黄色葡萄球菌的CFU。
展示于图1C中的结果表明，用负载姜黄素的靶向ER的PLGA纳米粒子处理的小鼠中的细菌负荷明显低于未处理的小鼠中的细菌负荷，并且也低于用游离姜黄素或用非靶向的负载姜黄素的PLGA纳米粒子处理的小鼠中的细菌负荷。
在以上程序之后，据测定，从用游离姜黄素、负载姜黄素的PLGA纳米粒子或负载姜黄素的靶向ER的PLGA纳米粒子处理的小鼠分离的腹膜泌出物中的经巯基乙酸盐引出的嗜中性粒细胞的百分比不存在显著差异。
这些发现一起显示，经由靶向ER的PLGA纳米粒子将姜黄素递送至 CybbC1024T转基因Cybb-/-小鼠中的ER会恢复嗜中性粒细胞的抗菌功能。
实施例8：PLGA纳米粒子的ER靶向的定量
通过在具有嗜中性粒细胞的多种特征的分化HL-60细胞中进行免疫染色和共定位，测量用含有或不含靶向ER的肽装饰的负载姜黄素的PLGA 纳米粒子处理的细胞中的胞内分布。
HL-60细胞的分化
在37℃下在补充有10％热灭能胎牛血清的RPMI 1640中培养HL-60 细胞。用1.3％二甲亚砜(西格玛-奥德里奇(Sigma-Aldrich))处理密度为3×105个细胞/毫升的细胞以诱导分化。温育4天之后，收集经分化细胞，并且经由聚蔗糖-泛影葡胺(Ficoll-Hypaque)通过离心移除死细胞。
纳米粒子的亚细胞定位的测定
将分化HL-60细胞接种于8孔载片上，并且随后用ER-Tracker(一种定位至ER的细胞渗透性荧光染料)标记。随后用10μM负载姜黄素的 PLGA纳米粒子或用10μM负载姜黄素的靶向ER的PLGA纳米粒子处理经标记细胞。通过共聚焦显微镜使用488nm和374nm的激发波长(即分别刺激姜黄素和ER-Tracker染料的荧光的波长)获取延时荧光影像。通过皮尔森相关性方法(Pearson's correlation method)根据延时影像计算 ER-tracker染料与姜黄素纳米粒子的共定位程度。结果展示于图2中。
数据显示，相较于非靶向的纳米粒子，靶向ER的纳米粒子在添加至细胞中4分钟内显著定位至ER。参见图2。在添加纳米粒子之后20分钟时段内，该显著定位差异增加。结果证实，靶向ER的纳米粒子的确优先定位于活细胞中的ER。
实施例9：经姜黄素诱导的胞内钙离子释放的测定
诸如姜黄素的SERCA抑制剂引起胞内钙离子自ER的释放增加。将姜黄素诱导钙离子释放的能力与经PLGA纳米粒子囊封的姜黄素的能力进行比较。
将分化HL-60细胞接种于无钙离子的培养基中的8孔载片上，并且负载有胞内钙离子敏感指示染料Fluo-4AM。用毒胡萝卜素处理(阳性对照) 或用0.5、1或10μM姜黄素、负载姜黄素的PLGA纳米粒子或负载姜黄素的靶向ER的PLGA纳米粒子处理细胞。在经处理的细胞中通过共聚焦显微镜测量并且使用ImageJ软件分析随时间推移的荧光强度。相对于时间绘制随时间推移的荧光强度的变化除以添加钙离子之前的基线荧光强度(ΔF/F0)。
相较于用0.5μM及1μM游离姜黄素处理，用10μM游离姜黄素处理细胞诱导明显较高水平的钙离子以较快速率释放。更具体地，用10μM游离姜黄素处理后40、50和60秒，细胞中的钙离子水平明显高于同一时间点时用0.5μM或1μM游离姜黄素处理的细胞中的钙离子水平。相较于用较低浓度的游离姜黄素处理的细胞，用10μM游离姜黄素处理的细胞中的胞内钙离子增长速率(其由荧光对时间曲线推导得出)也是最高的。
还在用负载姜黄素的PLGA纳米粒子和负载姜黄素的靶向ER的 PLGA纳米粒子处理的细胞中测量钙离子释放。数据显示，用0.5μM和1 μM的这些纳米粒子中的任一者处理分化HL-60细胞诱导胞内钙离子水平升高和与由10μM游离姜黄素处理诱导的增长速率相似的增长速率。指示为曲线下面积(AUC)的总体钙离子通量展示于图3中。
结果显示，0.5μM的负载姜黄素的PLGA纳米粒子和靶向ER的负载姜黄素的PLGA纳米粒子能够将胞内钙离子水平提高至毒胡萝卜素处理的相同水平。姜黄素纳米粒子浓度增加至10μM并未进一步增加经处理的细胞中的胞内钙离子水平的峰值水平或增长速率。
实施例10：经姜黄素诱导的胞外钙离子流入的测定
在如下用游离姜黄素处理或用负载姜黄素的纳米粒子处理的细胞中测量胞外钙离子流入，其通过ER钙离子储存消耗触发。
在37℃下用游离姜黄素(10μM)、负载姜黄素的PLGA纳米粒子(0.5 μM)和负载姜黄素的靶向ER的PLGA纳米粒子(0.5μM)过夜预处理分化 HL-60细胞。用无钙离子HBSS洗涤细胞，将其以1×106个细胞/毫升的密度接种于经聚赖氨酸涂布的8孔载片(德国普拉内格的易必迪(ibidi, Planegg,Germany))上，并且在37℃下温育30分钟。在37℃下用2μM钙离子敏感荧光染料Fluo-4AM和1μM ER-tracker标记细胞30分钟。用1.25 mM CaCl2处理经标记细胞。
添加CaCl2之后，通过共聚焦显微镜每10秒获取一次荧光影像，持续 5分钟。使用ImageJ软件分析这些影像中的荧光强度。相对于时间绘制随时间推移的荧光强度的变化除以添加钙离子之前的基线荧光强度(ΔF/F0)，并且从该图推导出钙离子水平增长速率(ΔF/F/s)。总体钙离子通量计算为ΔF/F/s对时间曲线的曲线下面积(AUC)。结果展示于图4中。
添加1.25mM胞外钙离子并未在未处理的细胞中诱导明显的钙离子流入。毒胡萝卜素(一种用作阳性对照的SERCA抑制剂)在添加之后10秒内诱导钙离子流入。
10μM的游离姜黄素较早诱导胞外钙离子流入，其类似于由毒胡萝卜素诱导的流入。相比之下，仅在用这些纳米粒子处理后60秒检测到由0.5 μM负载姜黄素的PLGA纳米粒子和0.5μM负载姜黄素的靶向ER的 PLGA纳米粒子诱导的胞外钙离子进入。
荧光强度增长速率的测量显示，相较于负载姜黄素的纳米粒子，用游离姜黄素处理细胞在较早时间点诱导较显著的钙离子流入。特别地，用10 μM游离姜黄素处理后20秒的钙离子流入速率明显高于用0.5μM负载姜黄素的PLGA纳米粒子或用0.5μM负载姜黄素的靶向ER的PLGA纳米粒子处理的细胞中的速率。
与由游离姜黄素诱导的胞外钙离子流入相比，由经PLGA纳米粒子囊封的姜黄素诱导的胞外钙离子进入的延迟降低总体钙离子流入。参见图4。鉴于相较于PLGA纳米粒子中所负载的姜黄素，游离姜黄素诱导相似的 ER钙离子消耗水平的事实，此结果为未预期的。如以上所指出，ER钙离子的消耗触发胞外钙离子流入。
实施例11：姜黄素对ER压力和细胞存活的影响
如上文所提及，已显示，由于姜黄素会引发ER压力和细胞凋亡，因此其具有细胞毒性。通过在经处理的细胞中测量粒线体膜电位(一种细胞毒性的量度)、细胞凋亡标志物和ER压力相关蛋白质表达，对游离姜黄素的细胞毒性与经囊封的姜黄素的细胞毒性进行比较。
粒线体膜电位的测量
用0.5μM或10μM游离姜黄素、负载姜黄素的PLGA纳米粒子或负载姜黄素的靶向ER的PLGA纳米粒子处理分化HL-60细胞达24小时。在37℃下用250nM粒线体膜电位指示染料四甲基若丹明(TMRM)标记经处理的细胞达30分钟。通过流式细胞术分析经TMRM标记的细胞，并且使用Flowing Software 2软件包对其粒线体膜电位进行定量。
结果显示，相较于对照未处理细胞，用10μM游离姜黄素处理的细胞显示粒线体膜电位显著降低。参见图5A。相比之下，用10μM负载姜黄素的PLGA纳米粒子或10μM负载姜黄素的靶向ER的PLGA纳米粒子处理的细胞中的粒线体膜电位并未与对照未处理细胞不同，并且当与经游离姜黄素处理的细胞相比时显著较高。参见上文。
凋亡细胞的检测
用冰冷磷酸盐缓冲盐水洗涤如上用游离或经囊封的姜黄素处理的细胞，并且在室温下用藻红素-磷脂结合蛋白V和7氨基-放线菌素D(7-AAD) 染色15分钟。磷脂结合蛋白V结合至经历细胞凋亡的细胞表面，并且 7-AAD仅进入和染色凋亡细胞。对染色细胞进行流式细胞术分析，并且使用Flowing Software 2分析软件包分析结果。
结果显示，如通过结合至细胞表面的磷脂结合蛋白V所测量，用10μM 游离姜黄素处理后，凋亡细胞的数目显著增加。参见图5B。用相同浓度的负载姜黄素的PLGA纳米粒子或负载姜黄素的靶向ER的PLGA纳米粒子处理的细胞显示明显较低数目的凋亡细胞。参见上文。
ER压力反应
为了测定ER压力反应的活化与经姜黄素诱导的细胞凋亡有关，通过用10μM姜黄素或经囊封的姜黄素处理的细胞的蛋白质印迹分析对ER压力标志物Bip和磷酸化ERK的水平，以及裂解的胱天蛋白酶-3(一种细胞凋亡的标志物)的水平进行定量。
简言之，使经处理的细胞在100μl裂解缓冲液(1％Triton-X100、150 mM NaCl、10mM Tris碱、1mM EDTA、1mM EGTA、50×蛋白酶抑制剂混合液，pH 7.4)中裂解。对裂解样本进行10％SDS-PAGE，并且转移至膜以供使用结合至Bip、磷酸化ERK、总ERK、裂解的胱天蛋白酶-3和β- 肌动蛋白的抗体进行蛋白质印迹分析。使用市售的加强型化学发光试剂观测这些抗体的结合。根据影像使用ImageJ软件对化学发光信号进行定量。
数据显示，相较于未处理的对照细胞，用游离姜黄素和负载姜黄素的 PLGA纳米粒子处理细胞会增加Bip表达和ERK磷酸化。出乎意料地，用负载姜黄素的靶向ER的PLGA纳米粒子处理的细胞中的Bip表达和ERK 磷酸化明显低于经游离姜黄素处理的细胞中的Bip表达和ERK磷酸化。参见图5C的左图和中图。
胱天蛋白酶-3裂解的测量显示，相较于用游离姜黄素进行处理，经 PLGA纳米粒子囊封的姜黄素诱导明显较少胱天蛋白酶-3裂解。参见图 5C，右图。
总而言之，前述实例中所阐述的发现表明，处于可以抑制SERCA的浓度(即10μM)下的游离姜黄素会诱导快速钙离子流入，引起粒线体受损， ER压力并且诱导细胞凋亡。具有相等SERCA抑制活性的经PLGA纳米粒子囊封的姜黄素诱导延迟的钙离子流入并且保护细胞免受粒线体受损、 ER压力和细胞凋亡。
此外，负载姜黄素的靶向ER的PLGA纳米粒子，通过抑制SERCA，成功地释放螯合于ER中的H338Y-gp91phox，由此恢复NOX2功能而不会过度损害细胞活力。
实施例12：姜黄素对成红细胞分化的影响
如下在用游离姜黄素处理或用负载姜黄素的纳米粒子处理的人类外周血造血细胞(HSC)中测量成红细胞分化。
在含有15％热灭活胎牛血清、2U/ml重组人类红细胞生成素、20ng/ml 干细胞因子和10ng/ml白细胞介素-3(IL-3)的伊氏经改良的Dulbecco培养基中培养人类CD34+HSC。在第4天，用不含IL-3的新制培养基置换该培养基并且将HSC再培养5天。
通过用流式细胞术检测CD71和CD235a的表面表达来分析成红细胞的成熟。结果展示于图6A中。数据显示，游离姜黄素和靶向ER的负载姜黄素的PLGA纳米粒子显著提升经处理的HSC中的较成熟CD71低、 CD235高细胞的百分比。
磷脂结合蛋白V阳性细胞的测量显示，相较于用游离姜黄素进行处理，经靶向ER的PLGA纳米粒子囊封的姜黄素在经处理的HSC中诱导明显较少细胞凋亡。参见图6B。
总而言之，用靶向ER的负载姜黄素的PLGA纳米粒子处理HSC促进成红细胞成熟，并且同时防止发生经姜黄素诱导的细胞凋亡。
其他实施方案
本说明书中公开的所有特征可以以任何组合形式组合。本说明书中公开的各特征可以经用作相同、等效或类似目的的替代性特征置换。除非另外明确说明，否则所公开的各特征仅为一系列等效或类似通用特征的一个实例。
根据以上描述，本领域技术人员可以容易地确定本发明的基本特征，并且在不脱离本发明的精神和范围的情况下可以对本发明作出各种改变和修改以使其适应各种用途和条件。因此，其他实施方案也在以下权利要求的范围内。

Claims (21)

1.一种用于治疗由内质网中的蛋白质驻留所引起的疾病的方法，所述方法包括：
获得囊封于聚合物纳米粒子中的肌质/内质网钙离子ATP酶(SERCA)泵抑制剂，以及
向需要其的受试者施用有效量的所述抑制剂，
其中所述聚合物纳米粒子经表面修饰以使得其靶向内质网(ER)，有效量的所述抑制剂减少ER中的蛋白质驻留，并且相较于施用未经囊封的所述抑制剂，囊封减少所述抑制剂的副作用。
2.根据权利要求1所述的方法，其中所述抑制剂为选自由以下组成的组的膳食多酚化合物：姜黄素、毒胡萝卜素、环匹阿尼酸、2,5-二叔丁基氢醌、姜辣素、CGP37157、叔丁基氢醌、蕈青霉素和柴胡皂苷d或其组合。
3.根据权利要求2所述的方法，其中所述膳食多酚化合物为姜黄素。
4.根据权利要求3所述的方法，其中所述副作用为ER压力或细胞凋亡。
5.根据权利要求2所述的方法，其中所述聚合物为聚(乳酸-共-乙醇酸)、聚(交酯)、聚(氨基酸)、聚(ε-己内酯)、聚氰基丙烯酸烷酯、藻酸盐、明胶、卵磷脂-壳聚糖、白蛋白或壳聚糖。
6.根据权利要求5所述的方法，其中所述膳食多酚化合物为姜黄素并且所述聚合物为聚(乳酸-共-乙醇酸)。
7.根据权利要求2所述的方法，其中所述表面修饰包括与所述聚合物缀合的靶向ER的肽，并且所述靶向ER的肽为KDEL(SEQ ID NO:1)、KKXX(SEQ ID NO:2)或基于精氨酸的RXR型ER驻留/回收信号。
8.根据权利要求7所述的方法，其中所述聚合物为聚(乳酸-共-乙醇酸)、聚(交酯)、聚(氨基酸)、聚(ε-己内酯)、聚氰基丙烯酸烷酯、藻酸盐、明胶、卵磷脂-壳聚糖、白蛋白或壳聚糖。
9.根据权利要求8所述的方法，其中所述膳食多酚化合物为姜黄素并且所述聚合物为聚(乳酸-共-乙醇酸)。
10.根据权利要求2所述的方法，其中所述疾病为慢性肉芽肿病、囊肿性纤维化、遗传性肺气肿、遗传性血色素沉着症、眼皮肤白化病、蛋白质C缺乏症、I型遗传性血管性水肿、法布里病、泰-萨二氏病、先天性蔗糖酶-异麦芽糖酶缺乏症、2型克果纳杰氏病、糖尿病、莱伦氏综合征、原发性甲状腺功能低下、先天性长QT综合征、甲状腺素结合球蛋白缺乏症、β-地中海贫血/Hb E病、家族性高胆固醇血症、家族性乳糜微粒血症、无β脂蛋白血症、先天性甲状腺功能低下、成骨不全、骨质石化病、遗传性低纤维蛋白原血症、肾源性尿崩症、垂体神经部尿崩症、夏-马-图三氏病、家族性脑中叶硬化症、阿尔茨海默病、IIA型冯威利布兰德病、丹吉尔病或伴随ALK1误义突变的2型遗传性出血性毛细管扩张症(HHT)。
11.根据权利要求10所述的方法，其中所述聚合物为聚(乳酸-共-乙醇酸)、聚(交酯)、聚(氨基酸)、聚(ε-己内酯)、聚氰基丙烯酸烷酯、藻酸盐、明胶、卵磷脂-壳聚糖、白蛋白或壳聚糖。
12.根据权利要求11所述的方法，其中所述疾病为慢性肉芽肿病，所述膳食多酚化合物为姜黄素，所述聚合物为聚(乳酸-共-乙醇酸)，并且所述表面修饰包括与所述聚合物缀合的靶向ER的肽KDEL(SEQ ID NO:1)。
13.根据权利要求1所述的方法，其中有效量的所述抑制剂促进所述受试者中的白血球产生活性氧。
14.根据权利要求13所述的方法，其中有效量的所述抑制剂促进由所述白血球介导的腹膜细菌清除。
15.一种药物组合物，所述药物组合物包含囊封于聚合物纳米粒子中的SERCA泵抑制剂以及药用载体，其中所述聚合物纳米粒子经表面修饰以使得其靶向内质网。
16.根据权利要求15所述的组合物，其中所述聚合物纳米粒子由以下中的一种或多种形成：聚(乳酸-共-乙醇酸)、聚(交酯)、聚(氨基酸)、聚(ε-己内酯)、聚氰基丙烯酸烷酯、藻酸盐、明胶、卵磷脂-壳聚糖、白蛋白或壳聚糖，并且所述SERCA泵抑制剂为选自由以下组成的组的膳食多酚化合物：姜黄素、毒胡萝卜素、环匹阿尼酸、2,5-二叔丁基氢醌、姜辣素、CGP37157、叔丁基氢醌、蕈青霉素和柴胡皂苷d或其组合。
17.根据权利要求16所述的组合物，其中所述膳食多酚化合物为姜黄素并且所述聚合物为聚(乳酸-共-乙醇酸)。
18.根据权利要求17所述的组合物，其中所述表面修饰包括与所述聚合物缀合的靶向ER的肽，并且所述靶向ER的肽为KDEL(SEQ ID NO:1)、KKXX(SEQ ID NO:2)或基于精氨酸的RXR型ER驻留/回收信号。
19.一种转基因小鼠，所述转基因小鼠在其基因组中包含编码H338Y突变型gp91phox蛋白质的异源核酸，其中所述突变型gp91phox表达于所述转基因小鼠的细胞中，并且野生型gp91phox未表达于所述转基因小鼠的细胞中。
20.根据权利要求19所述的转基因小鼠，其中所述转基因小鼠的细胞为白血球。
21.根据权利要求20所述的转基因小鼠，其中相较于野生型小鼠，所述转基因小鼠中的腹膜细菌清除减少。
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AU2019200903A1 (en) 2019-10-24
AU2019200903B2 (en) 2024-07-04
US10912843B2 (en) 2021-02-09
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