CN1993145A - 用于药学活性物质细胞特异性富集的基于蛋白质的纳米颗粒形式的载体系统 - Google Patents

用于药学活性物质细胞特异性富集的基于蛋白质的纳米颗粒形式的载体系统 Download PDF

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CN1993145A
CN1993145A CNA2005800073779A CN200580007377A CN1993145A CN 1993145 A CN1993145 A CN 1993145A CN A2005800073779 A CNA2005800073779 A CN A2005800073779A CN 200580007377 A CN200580007377 A CN 200580007377A CN 1993145 A CN1993145 A CN 1993145A
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扎比内·巴尔塔扎
哈根·万伯瑞森
诺贝特·迪诺尔
约尔格·克罗伊特尔
克劳斯·朗热
海德龙·沃特雷克
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LTS Lohmann Therapie Systeme AG
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Abstract

本发明涉及一种用于至少一种药理学活性物质的细胞特异性细胞内富集的基于蛋白质的纳米颗粒形式的载体系统，该系统具有通过反应基团结合的结构。所述的结构实现了纳米颗粒的细胞特异性附着和细胞吸收。本发明也涉及一种制备所述系统的方法。

Description

用于药学活性物质细胞特异性富集的基于蛋白质的纳米颗粒形式的载体系统
技术领域
本发明涉及一种用于药学活性物质的载体系统，该系统适用于药学活性物质的细胞特异性富集，并以基于蛋白质，优选基于明胶和/或血清白蛋白，特别是人血清白蛋白(HSA)的抗生物素蛋白修饰的纳米颗粒形式存在，生物素化抗体通过形成稳定的抗生物素蛋白-生物素复合物与载体系统结合，并且其中，药学活性物质对纳米颗粒的额外结合可共价或由抗生物素蛋白-生物素系统通过络合物形成以及通过掺合或吸附发生。
背景技术
纳米颗粒是粒度在10～1000nm之间的人工或天然大分子物质的颗粒，药物或其他生物活性物质可与其共价、离子或吸附结合，或所述的物质可掺合其中。
欧洲专利1 392 255公开了基于人血清白蛋白的纳米颗粒，载脂蛋白E与其共价或通过抗生物素蛋白/生物素系统结合以实现越过血脑屏障。
然而，药物疗法的特殊目的不仅是如欧洲专利1 392 255所述的实现药理学活性物质或治疗有效的药物在特定组织或器官中的特异性富集，而除此之外，也是实现在特定细胞中的特异性富集。
未修饰的纳米颗粒实现了被动“药物靶向”，其特征在于颗粒在血管内应用后被单核吞噬细胞系统(MPS)的细胞吸收。已在肝脏、脾脏、骨髓的巨噬细胞以及循环单细胞中观察到这种纳米颗粒的富集。被动“药物靶向”不同于在修饰纳米颗粒的辅助下，针对甚至在基本不可到达的身体隔室或细胞系统中定位富集活性物质的主动“药物靶向”。为此，有必要使用具有使与非靶细胞的非特异性相互作用最小化的亲水表面结构的纳米颗粒，并使其装有实现纳米颗粒的细胞特异性富集的配体。这种配体也叫做“药物靶向配体”。通过使用细胞特异性纳米颗粒作为药物载体，可能在控制条件下将药理学活性物质富集在靶细胞中，或将药理学活性物质特异性地转运到其在体内的作用位点。大多数药物没有适宜的药物形式不能实现该目的，且至多由于活性物质本身的物理化学性质表现出细胞富集或体内分布。仅施用的部分活性物质到达了所期望的目标，而剩余部分引起不需要的副作用或毒效应。因此，细胞特异性纳米颗粒有助于降低活性物质的不需要的副作用和毒性。
在初步实验中，采用通过甲基丙烯酸羟乙酯、甲基丙烯酸和甲基丙烯酸甲酯的共聚合制备的亲水性乳胶颗粒。对这些颗粒结合兔γ-球蛋白抗体。与未修饰的颗粒相比，观察到抗体修饰的制剂与预先以兔源抗血清培养的淋巴细胞结合。
然后，采用额外结合有离子氧化物、基于聚丙烯酸酯的相应颗粒系统以进行淋巴细胞和红细胞的磁性分离。
以该基础工作为基础，然后单克隆抗CD3抗体通过C7间隔区(spacer)结构与聚丙烯酸酯纳米颗粒结合，并在细胞培养条件下检测。然而，这些工作的问题是，细胞与亚群的结合以及由此观察到的颗粒与相应亚群的结合是完全在显微镜下观察进行的，因此不是毋庸置疑的。
也检测了单克隆抗体与聚氰基丙烯酸己酯纳米颗粒的吸附结合。一方面，可观察到抗体与颗粒表面的有效吸附；另一方面，另外的血清组分的加入导致抗体由颗粒表面被竞争性取代。在该范围内，配体的吸附结合不适于生物系统中的细胞特异性药物靶向。
所述细胞特异性纳米颗粒系统的另一缺陷在于它们是基于不可生物降解的如乳胶和聚丙烯酸酯的聚合物材料。
已进行抗体对基于血清白蛋白的纳米颗粒表面的蛋白质化学结合的初步试验。在这些试验中，采用戊二醛反应通过白蛋白和抗体的初级氨基偶联抗体。作为配体，使用了路易斯肺肿瘤单克隆抗体，以及作为比较的非特异性IgG抗体。尽管游离特异性抗体在细胞培养条件下和对试验动物静脉内应用后在靶细胞中表现出清晰的富集，然而与纳米颗粒偶联后，在体内条件下肿瘤中仅检测到很低的颗粒富集。发现所施用纳米颗粒的主要部分在肝脏和肾脏中。偶联非特异性IgG抗体的纳米颗粒在肿瘤组织中未表现出任何富集。在所选实验条件下，仅可能实现基于人血清白蛋白的偶联纳米颗粒的低特异性。该颗粒系统的主要部分表现出对于被动药物靶向典型的非特异性体内分布。然而，由于所用结合纳米颗粒仅具有不充分的抗体结合特性，所以仍不清楚缺乏特异性是否是由不充分的抗体结合导致的。总之，至今仍未产生纳米颗粒在同时包围有非靶细胞的靶细胞中特异性和受体介导的吸收的证据。
发明内容
因此，本发明目的为提供没有上述纳米颗粒系统的缺陷，而即使应用于生物系统中也表现出高细胞特异性以特异性地实现药理学活性物质在选定靶细胞中富集，且基于生物可降解材料的纳米颗粒。
惊人地，通过生物素化抗体与其结合形成稳定的抗生物素蛋白-生物素复合物的抗生物素蛋白修饰的基于蛋白质的纳米颗粒形式的载体系统实现了该目的。优选，明胶和/或血清白蛋白，特别优选人血清白蛋白用作蛋白质。采用这些修饰过的纳米颗粒，药理学活性物质对纳米颗粒的额外结合能够共价、由抗生物素蛋白-生物素系统通过络合物形成、以及通过掺合或吸附的方式发生。
附图说明
图1说明通过抗生物素蛋白-生物素复合物的方式结合有抗体的基于明胶或HSA的抗生物素蛋白修饰的纳米颗粒的结构。
图2为说明通过FACS分析测定的多种乳癌细胞系中抗体(Trastazumab)修饰的明胶A纳米颗粒的细胞吸收的柱形图。在相同培养条件下，比较了各情况下的抗体修饰的纳米颗粒与未修饰的纳米颗粒。未处理的细胞作为对照。
具体实施方式
根据本发明为了制备纳米颗粒，通过双重去溶剂化过程将明胶水溶液转化为纳米颗粒，然后通过交联使后者稳定。位于这些纳米颗粒表面的官能团(氨基、羧基、羟基)通过适宜的试剂可转化为反应性的巯基。通过对氨基和自由巯基均反应的双功能间隔区分子，功能蛋白可结合于这些巯基修饰的纳米颗粒。这些功能蛋白质包括，尤其是，抗生物素蛋白衍生物或细胞特异性抗体。
当制备下文所述的用于细胞培养试验的纳米颗粒时，颗粒表面的初级氨基与2-亚氨硫醇(2-iminothiolane)反应，导致在颗粒表面引入自由巯基。以双功能间隔区磺基-MBS(m-马来酰亚胺基苯甲酰基)-N-羟基磺基琥珀酰亚胺酯(m-maleimidobenzoyl-N-hydroxysulfosuccinimideester)活化抗生物素蛋白衍生物NeutrAvidinTM的氨基，并在该活化中间阶段的柱层析纯化后，加入巯基化的明胶纳米颗粒。该抗生物素蛋白修饰的纳米颗粒的中间产物代表了用于可通过形成抗生物素蛋白-生物素络合物结合多种生物素化物质的通用载体系统。
对于抗体结合，优选单克隆抗体，抗体或者以生物素化的形式购买，或者通过用NHS生物素(N-羟基琥珀酰亚胺生物素)转化的方式生物素化，并加入抗生物素蛋白修饰的纳米颗粒。因此，通过上述抗生物素蛋白-生物素络合物形成得到了基于明胶的抗体修饰的纳米颗粒(图1)。然而，相应抗体修饰纳米颗粒也可在血清白蛋白的基础上制备，优选人血清白蛋白。
因此本发明包括一种用于至少一种药理学活性物质的细胞特异性细胞内富集的载体系统，该载体系统以基于蛋白质的纳米颗粒形式存在并包括通过反应基团结合的结构，所述的结构实现了纳米颗粒的细胞特异性附着和细胞吸附。优选考虑明胶和/或血清白蛋白，特别优选人血清白蛋白作为蛋白质基础。优选反应基团为氨基、巯基、羧基或抗生物素蛋白衍生物，且结合结构为抗体，特别优选单克隆抗体。
本发明也包括另外含有至少一种通过反应基团由吸附、掺合或共价键或配位键与载体系统或纳米颗粒结合的药学活性物质的相应载体系统。
本发明进一步包括根据本发明的载体系统用于制备药学活性物质富集到或进入特异性细胞的药剂的应用。
本发明进一步包括一种制备用于至少一种药理学活性物质的细胞特异性富集、基于蛋白质的纳米颗粒形式的载体系统的方法，该方法包括以下步骤：
-对蛋白质水溶液进行去溶剂化，
-通过交联稳定由去溶剂化形成的纳米颗粒，
-将稳定纳米颗粒表面上的官能团的一部分转化为反应性的巯基，
-通过双功能间隔区分子共价连接功能蛋白质，优选抗生物素蛋白，
-如果需要，对抗体进行生物素化
-把生物素化抗体加载在抗生物素蛋白修饰的纳米颗粒上，
-把生物素化和药学或生物活性物质加载在抗生物素蛋白修饰的纳米颗粒上。
采用根据本发明的方法，特别优选采用明胶和/或血清白蛋白，特别是人源血清白蛋白。
优选，通过搅拌和加入与水混溶的蛋白质非溶剂或盐析进行去溶剂化。所述的与水混溶的蛋白质非溶剂优选选自包括乙醇、甲醇、异丙醇和丙酮的组。
为了稳定纳米颗粒，优选采用热处理或双功能醛，特别是戊二醛或甲醛。
作为巯基修饰试剂，优选采用选自包括2-亚氨硫醇、1-乙基-3-(3-二甲基氨丙基)碳化二亚胺(1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide)和半胱氨酸的组合、或1-乙基-3-(3-二甲基氨丙基)碳化二亚胺和二氯化胱胺(cystaminium dichloride)的组合以及二硫苏糖醇的组的一种物质。
作为双功能间隔区分子，优选采用选自包括m-马来酰亚胺基苯甲酰基-N-羟基磺基琥珀酰亚胺酯、磺基琥珀酰亚胺-4-[N-马来酰亚胺基-甲基]环己烷-1-羧酸酯(sulfosuccinimidyl-4-[N-maleimido-methyl]cyclohexane-1-carboxylate)、磺基琥珀酰亚胺基-2-[m-叠氮基-o-硝基苯甲酰胺基]-乙基-1，3′二硫代丙酸酯(sulfosuccinimidyl-2-[m-azido-o-nitrobenzamido]-ethyl-1，3′dithiopropionate)，二甲基-3，3′-二硫代双丙酰亚胺-二盐酸化物(dimethyl-3，3′-dithiobispropionimidate-dihydrochloride)和3，3′-二硫代双[磺基琥珀酰亚胺基丙酸酯](3，3′-dithiobis[sulfosuccinimidyl propionate])的组的一种物质。
实施例
为了制备蛋白质纳米颗粒，加热同时将500mg明胶A溶于10.0ml净化水，并通过加入10.0ml丙酮沉淀为沉淀物。分离沉淀的明胶，加热同时再溶于10.0ml水，并将溶液的pH值调至pH2.5。通过滴加30ml丙酮(去溶剂化过程)由该溶液制得纳米颗粒。
通过加入625μl8％的戊二醛并搅拌过夜稳定纳米颗粒。通过离心和超声处理的再分散的5次循环方式在2.0ml等分部分中纯化纳米颗粒。为了颗粒表面的巯基化，将2.5ml 30mg 2-亚氨硫醇(Traut’s试剂)于Tris缓冲液(pH8.5)中的溶液加入1.0ml纳米颗粒悬浮液(20mg/ml)，并搅拌24小时。巯基化后，重复上述纯化。
抗生物素蛋白衍生物FITC-NeutrAvidinTM通过双功能间隔区磺基-MBS(m-马来酰亚胺基苯甲酰基-N-羟基磺基琥珀酰亚胺酯)与巯基化的纳米颗粒结合。为了活化抗生物素蛋白衍生物，将0.75mg磺基-MBS加入2.5mg FITC-NeutrAvidinTM于500μl PBS缓冲液(pH7.0)的溶液中，并在室温下搅拌1小时。通过分子排阻色谱分离未反应的磺基-MBS与活化的NeutrAvidinTM。将那些通过分光光度检测在280nm检测到的NeutrAvidinTM部分结合，加入巯基化纳米颗粒的悬浮液，并在室温下搅拌12小时。如上所述进行现在的共价FITC-NeutrAvidinTM修饰的纳米颗粒的进一步纯化。光度检测由颗粒纯化得到的上清液中未结合的NeutrAvidinTM，并由其计算共价结合NeutrAvidinTM的部分。通过采用生物素-4-荧光素滴定试验，测定表达为每抗生物素蛋白分子生物素结合位点数目的结合NeutrAvidinTM的官能度。表明理论上存在于抗生物素蛋白分子中的4个生物素结合位点中的2.4个在与纳米颗粒偶联后仍然具有功能性。为了加载载体，将500μl生物素化的抗体(25μg/ml)加入到150μl NeutrAvidinTM修饰的纳米颗粒(20mg/ml)中，随后在10℃下培养90分钟。
培养后，通过离心和再分散再次纯化颗粒。所得的颗粒上清液通过蛋白质印记分析检测未结合的抗体。表明多于80％的所用抗体结合于颗粒系统存在。
在所述颗粒系统的辅助下，在不同细胞培养试验中发现了在携带被抗体识别的表面抗原的靶细胞中细胞特异性颗粒的富集。采用了以下细胞培养模型：
1.具有CD3表面抗原的淋巴靶细胞(Jurkat T细胞)。
以生物素化抗CD3抗体加载纳米颗粒。
2.具有HER2表面抗原表达的人乳癌细胞系(SK-Br-3-，MCF-7-，BT474细胞)。
以预先生物素化的安全抗体Trastuzumab(Herceptin)加载纳米颗粒。
培养细胞以100～1000μg/ml浓度的纳米颗粒系统培养，且4小时的培养时间后，通过清洗细胞分离未结合的纳米颗粒。通过流式细胞仪(FACS)以及共聚焦显微术(CLSM)检测细胞的纳米颗粒吸收。
对于在淋巴细胞中生物素化-抗CD3抗体修饰的纳米颗粒的细胞特异性吸收试验，将Jurkat-T细胞以1×106个细胞/孔的密度散布在24孔微孔板上并在RPMI培养基中培养。培养基加有10％(vol/vol)胎牛血清(FCS)，2％L-谷氨酸和1％青霉素/链霉素。以抗体修饰的纳米颗粒以1000μg/ml的浓度与细胞培养4小时。为了证实通过T细胞受体的特异性细胞吸收，进行了不同的对照试验。一方面，使用加载有非特异性IgG抗体的纳米颗粒代替特异性抗CD3抗体。另外，以每1×106个细胞用2.5μg游离IgG或抗CD3抗体预培养30分钟的Jurkat T细胞进行试验。此后，加入加载有抗CD3抗体的纳米颗粒。另一方面，比较试验采用不具有CD3表面抗原的MCF-7细胞进行。通过共聚焦显微术以及流式细胞仪对细胞吸收进行了定量评价。
对于在乳癌细胞中生物素化-抗HER2抗体修饰的纳米颗粒的细胞特异性吸收试验，将HER2过表达细胞(BT474和SK-Br-3)以每孔分别为2×105、1×105个细胞的密度散布在24孔微孔板上并分别在RPMI培养基和McCoy’s5A中培养。BT474的培养基加有20％(vol/vol)胎牛血清(FCS)、2％L-谷氨酸、1％青霉素/链霉素和100U胰岛素。SK-Br-3的培养基加有10％(vol/vol)胎牛血清(FCS)、2％L-谷氨酸和1％青霉素/链霉素。抗体修饰的纳米颗粒以100μg/ml的浓度与细胞培养3小时。为了证实通过HER2受体的特异性细胞吸收，进行了不同的比较试验。一方面，使用未加载特异性抗体的纳米颗粒。另一方面，采用MCF-7细胞(正常HER2表达)进行试验。另外，对照试验采用以每2×105个细胞2.5μg/ml游离抗HER2抗体(Trastuzumab)预培养30分钟的SK-Br-3细胞。此后，加入加载有抗HER2抗体的纳米颗粒。通过共聚焦显微术以及流式细胞仪对细胞吸收进行定量评价。
结果
淋巴靶细胞(Jurkat T细胞)
FACS和CLSM均表明以细胞特异性抗CD3抗体修饰形式使用的纳米颗粒被细胞吸收。在加入颗粒前以游离特异性抗体处理的细胞可避免细胞吸收。然而，以游离非特异性IgG抗体预处理没有显示出对颗粒吸收的任何影响。以非特异性IgG抗体代替特异性抗CD3抗体修饰的纳米颗粒同样不会导致靶细胞中的吸收。进一步以没有CD3表面抗原的乳癌细胞(MCF-7细胞)进行了对照试验。在这些对照试验中，在任何所选条件下都没有观察到纳米颗粒制剂的吸收。
人乳癌细胞系(SK-Br-3-、MCF-7-、BT474细胞)
所用细胞在不同程度上表现出用作抗体修饰的纳米颗粒的细胞吸收攻击点的HER2表面抗原的表达。在以纳米颗粒培养前，细胞的表达通过蛋白质印记分析测定(表1)。
细胞系   表达HER2[％]
BT474MCF-7SK-Br-3   311100366
表1：通过蛋白质印记分析测定的不同肿瘤细胞表面上HER2表面抗原的表达。
          表达相对“正常表达”MCF-7细胞值计算。
FACS和CLSM均表明以细胞特异性抗体Trastuzumab修饰的形式使用的纳米颗粒被细胞吸收(图2)。在加入颗粒前以游离特异性抗体处理的细胞可避免特异性纳米颗粒的细胞吸收。不使用以生物素化抗体形式修饰的同批次的纳米颗粒在所选条件下仅表现出低细胞富集。抗体修饰的纳米颗粒的细胞吸收程度可与HER2表面抗原的表达程度相关。
上述细胞培养试验的结果清楚地表明基于明胶的抗体修饰的纳米颗粒实现了靶细胞中的特异性富集。在对比条件下，颗粒系统仅在相应靶细胞中被吸收，但没有在对照细胞中被吸收。以游离特异性抗体预处理明显表明颗粒吸收通过受体介导的内吞过程发生。因此，如果这些靶细胞的表面性质不同于健康细胞，则已开发的纳米颗粒药物载体系统提供了将药物特异性传递至疾病细胞的可能。
采用根据本发明的基于明胶的抗体修饰的纳米颗粒，提供了一种性能良好的颗粒载体系统，该系统通过所述载体系统表面上携带的功能药物靶向配体实现了甚至通过吸附、掺合或通过共价键或配位键结合的这种药学活性物质的细胞特异性吸收和富集。

Claims (14)

1、用于至少一种药理学活性物质的细胞特异性细胞内富集的载体系统，其特征在于，所述的载体系统以基于蛋白质，优选基于明胶和/或血清白蛋白，特别优选基于人血清白蛋白的纳米颗粒形式存在，且其具有通过反应基团结合的结构，所述的结构实现纳米颗粒的细胞特异性附着和细胞吸收。
2、根据权利要求1的载体系统，其特征在于，所述反应基团为氨基、巯基、羧基或抗生物素蛋白衍生物。
3、根据权利要求1或2的载体系统，其特征在于，所述结合结构为抗体。
4、根据权利要求3的载体系统，其特征在于，所述抗体为单克隆抗体。
5、根据上述任一权利要求的载体系统，其特征在于，其另外包括通过吸附、掺合或共价键或配位键由反应基团与载体系统结合的药学活性物质。
6、根据上述任一权利要求的载体系统用于制备药学活性物质至/在特异性细胞中富集的药剂的应用。
7、制备用于至少一种药理学活性物质的细胞特异性富集的基于蛋白质的纳米颗粒形式的载体系统的方法，其特征在于，该方法包括以下步骤：
-对蛋白质水溶液进行去溶剂化，
-通过交联稳定由去溶剂化形成的纳米颗粒，
-将稳定纳米颗粒表面上的官能团的一部分转化为反应性的巯基，
-通过双功能间隔区分子共价连接功能蛋白质，优选抗生物素蛋白，
-如果需要，对抗体进行生物素化，
-把生物素化抗体加载在抗生物素蛋白修饰的纳米颗粒上，
-把生物素化和药学或生物活性物质加载在抗生物素蛋白修饰的纳米颗粒上。
8、根据权利要求7的方法，其特征在于，蛋白质基础为明胶和/或血清白蛋白，优选人血清白蛋白。
9、根据权利要求7或8的方法，其特征在于，通过搅拌和加入与水混溶的蛋白质非溶剂或通过盐析进行去溶剂化。
10、根据权利要求9的方法，其特征在于，与水混溶的蛋白质非溶剂选自包括乙醇、甲醇、异丙醇和丙酮的组。
11、根据权利要求7～10任一项的方法，其特征在于，采用热处理或双功能醛或甲醛稳定纳米颗粒。
12、根据权利要求11的方法，其特征在于，戊二醛用作双功能醛。
13、根据权利要求7～12任一项所述的方法，其特征在于，采用选自包括2-亚氨硫醇、1-乙基-3-(3-二甲基氨丙基)碳化二亚胺和半胱氨酸的组合、或1-乙基-3-(3-二甲基氨丙基)碳化二亚胺和二氯化胱胺的组合以及二硫苏糖醇的组的一种物质作为巯基修饰剂。
14、根据权利要求7～13任一项所述的方法，其特征在于，采用选自包括m-马来酰亚胺基苯甲酰基-N-羟基磺基琥珀酰亚胺酯、磺基琥珀酰亚胺基-4-[N-马来酰亚胺基-甲基]环己烷-1-羧酸酯、磺基琥珀酰亚胺基-2-[m-叠氮基-o-硝基苯甲酰胺基]-乙基-1，3′二硫代丙酸酯、二甲基-3，3′-二硫代双丙酰亚胺-二盐酸化物和3，3′-二硫代双[磺基琥珀酰亚胺基丙酸酯]的组的一种物质作为双功能间隔区分子。
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