CN101242870B - 适用于将生物分子输入膜封闭的细胞或细胞器或从膜封闭的细胞或细胞器输出的纳米颗粒 - Google Patents
适用于将生物分子输入膜封闭的细胞或细胞器或从膜封闭的细胞或细胞器输出的纳米颗粒 Download PDFInfo
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Abstract
一种用于在体外或离体状态下适用于通过热诱导的核内体的释放而将生物分子输入到细胞中的纳米颗粒,该颗粒包括包埋在热敏性外壳中的超顺磁性核,所述热敏性外壳包括膜破裂成分和被输送的生物分子和标记物的结合位点。一种通过对携带有本发明描述的颗粒的培养细胞施加以交变磁场来引入多种所述生物分子和核内体破裂分子的释放作用的方法。
Description
技术领域
本发明涉及一种适用于通过物质的热诱导释放而将该物质输入到膜封闭的细胞或细胞器中或者将该物质从膜封闭的细胞或细胞器中输出的颗粒;本发明还涉及所述颗粒在不同方面的应用以及通过该物质的热诱导释放将物质输入到膜封闭的细胞或细胞器中或将该物质从膜封闭的细胞或细胞器中输出的方法。
背景技术
可靠的生物分子的输送方法对许多生命科学家来说是必不可少的工具。在靶细胞培养中的有效输送中关键的实例有转染、siRNA评价和各种细胞标记方法的筛选。在体外,成功的生物分子的非病毒输送方法必须克服一系列的障碍,并避免在靶细胞中产生毒性效果以及能够在细胞中有效地输送所选择的功能性生物分子。在输送过程中,第一步是生物分子到达靶细胞的表面。生物分子应当不依赖于细胞培养中的培养基组分而被输送至细胞,生物分子既能到达粘附的细胞系又能到达悬浮的细胞。一旦被输送到细胞表面,生物分子跨过细胞膜/细胞壁,或者通过内吞作用(endocytosis)进入细胞。一旦核内体中pH低,胞液和核内体内含有各种降解酶。有效的输送方法应当优选为尽可能地减少上述生物分子在细胞内的降解,无论上述的生物分子构成DNA、RNA、蛋白质、或其它分子。为了成功实现生物分子通过内吞作用进入细胞的输送过程,理想情况是促进各核内体小泡腔(endosomal compartment)尽可能多的释放生物分子至胞液中。一旦存在于胞液中,生物分子优选靶向在特异的细胞内结构如细胞核上。
目前,对于所有可利用的非病毒性生物分子的输送方法的基本技术可以分为两种方法。物理方法如电穿孔、显微注射和基因枪法,这些方法将生物分子通过细胞膜输送到胞液中。化学的/合成的方法是利用靶细胞的内吞作用。这两种方法都具有特定的特点和限制,并且都是已建立的成熟的方法。基于对干细胞、原代细胞系和培养的标准实验室细胞系进行体外研究的高通量的临床前细胞数目的增加,需要有效的、增强的和成本经济的生物分子的输送方法。
为了满足这些需求,改善化学的和合成的输送方法的输送效率是令人满意的。增加效率的方法之一是积极地促进核内体的释放。
发明内容
本发明一方面涉及一种适用于通过物质的热诱导释放而将该物质输入到膜封闭的细胞或细胞器中或者将该物质从膜封闭的细胞或细胞器中输出的颗粒,该颗粒包括:在交变磁场中具有产热能力的超顺磁性核,该核被包封在结合有所述物质与膜破裂试剂的热敏性外壳中。
本发明另一方面涉及所述颗粒在改善宿主细胞的遗传编码和/或代谢中的应用。
本发明另一方面涉及所述颗粒在从核内体细胞器或溶酶体细胞器释放所述物质中的应用。
本发明另一方面还涉及通过物质的热诱导释放以及膜破裂试剂的热诱导释放将该物质输入到膜封闭的细胞或细胞器中或者将该物质从膜封闭的细胞或细胞器中输出的方法,该方法包括:
提供含有膜封闭的细胞或细胞器和多个根据本发明的颗粒的样品;
对所述样品施加交变磁场,其中,所述颗粒的核的热能使所述颗粒的热敏性外壳发生分解,由此所述破裂试剂将所述封闭的细胞或细胞器的膜发生破裂,从而释放所述的物质。
附图说明
图1:为所述纳米颗粒的氧化铁核的一个例子的电镜图象;
图2:热效果
(A)为所述纳米颗粒的比吸取率(specific absorption rate)(SAR值)与施用的交变频率为10MHz的交变磁场强度的关系;(B)该曲线图说明了将100μl的样品(7mg Fe/ml,溶于0.15M NaCl中)置于交变频率为10MHz,磁场强度为2.9mT的交变磁场中,样品内温度的增加,该值通过没有颗粒的参考样品内温度的增加进行了修正,其中,采用纤维光学探针(IPITEK)对温度进行测量;
图3:为实验装置的示意性说明;
图4:为实施例5所述的颗粒处理后的细胞的高梯度磁性分离试验的结果;
图5:为表面用氨基、直链聚乙烯亚胺(PEI)和支链聚乙烯亚胺(PEI)修饰的热磁性(themagnetic)纳米颗粒的DNA结合能力的研究,不同量的颗粒与质粒DNA(phRLSV40-luc,3.7Kb)在TE缓冲液中孵育10分钟,在加入PicoGreen荧光试剂后,通过装有FITC过滤器的微孔板荧光阅读仪进行检测,荧光值降低表示存在凝缩型DNA;
图6:为实施例11所述的K562细胞的转染结果,标记为“细胞”的样品是作为对照的没有转染的细胞生成的背景。
具体实施方式
由上述可知,本发明提供了一种适用于生物分子输送的颗粒,该颗粒与核内体相结合并释放在可能输送的靶细胞中,该颗粒以高效率输送各种生物 分子。
在下文中将参照附图对本发明进行更加详细的说明,其中给出了优选的实施方式。然而,本发明可以包括多种不同的形式,不应当认为本发明限于本文中列出的说明性的实施方式。而且,提供的这些实施方式是为了使公开充分,并使本领域的技术人员更充分地理解本发明的范围。
我们以前在WO01/18168中描述了一种将生物颗粒引入生物膜封闭结构或将生物颗粒从生物膜封闭结构中提取出的方法,该方法通过向附着在膜封闭的细胞或细胞器中的超顺磁性颗粒的样品施以交变磁场,使热能增加从而对所述颗粒的超顺磁性核进行加热,同时被加热的超顺磁性核进一步导致细胞膜或细胞器的膜暂时性打开。
本发明描述了为在体外输送生物分子而设计的颗粒,该颗粒利用置于交变磁场中的超顺磁性核所释放的热能,从所述颗粒的热敏性外壳中释放出膜破裂分子如脂质、胆固醇或洗涤剂,从而以导致化学膜的破裂。在所述颗粒的超顺磁性核的加热过程中,释放出被输送的物质(下文称为货物分子)(如,生物分子)和促使细胞膜和细胞器膜如核内体膜破裂的分子。在本申请中,术语“物质”和“货物分子”可以相互交换使用。物质或货物分子通过本发明的颗粒被输送到靶细胞。物质或货物分子的实例是生物分子。
在生物医药和生物技术中,磁性分子的应用不断的增加。目前,磁性纳米颗粒被用于纯化、定量、转染、热疗(hyperthermia)、药物输送、在其它方面通过核磁共振成像。在US2003211045中描述了为了药物输送的目的将超顺磁性颗粒与前药一起包埋在脂质体中,通过对脂质体中的磁性颗粒进行加热从而将前药转变为药。而在US5441746中描述了另一种颗粒,其中,超顺磁性核被包埋在有机化合物和形成囊泡的脂质中,该颗粒通过感应加热超顺磁性核以促进杀死癌细胞。
为了与早期描述的脂质包埋的超顺磁性颗粒进行比较,本发明描述的超 顺磁性颗粒除了在交变磁场中产热能外,还被设计用于体外非病毒生物分子的输送。除了将货物分子运送到细胞表面以外,其中,在细胞表面,颗粒/货物分子复合物被细胞通过内吞作用摄入;在交变磁场中,所述颗粒还促进核内体释放货物分子。因此,所述颗粒表面并不均匀,而是具有在热脉冲下被释放或发生结构改变的热敏性的结构组合。本发明描述的大多数颗粒都具有多个货物分子的耦合和/或结合位点、一种或多种膜破裂试剂、能识别并结合到靶细胞和信号分子(如肽)的识别分子、和能使输送过程成像的荧光标记分子。
在本发明的一个实施方式中,所述超顺磁性核的尺寸为1-100nm,优选为8-15纳米。
在本发明的另一个实施方式中,所述超顺磁性核包括磁铁矿石、磁赤铁矿石、氧化铁水合物、和通式为MeOxFe2O3的铁酸盐,其中,Me是选自由Co、Ni、Mn、Be、Mg、Ca、Ba、Sr、Cu、Zn、Pt、Al、Cr、Bi、及它们的组合所组成的组中的二价金属。
在本发明的另一个实施方式中,所述热敏性外壳包括与所述核直接接触的类胶束结构,其中,所述类胶束结构由至少一种疏水分子和至少一种既含有疏水部分又含有亲水部分的分子构成。所述热敏性外壳与所述物质和膜破裂试剂通过一个或多个二硫键、过氧键或偶氮键、或它们的组合相连接。
在本发明的一个实施方式中,所述亲水部分结构含有聚合物、脂质、合成分子、蛋白质、或它们的组合。
在本发明的另一个实施方式中,所述热敏性外壳还结合有至少一种识别分子,其中,所述识别分子选自由抗体、抗体片段和凝集素所组成的组中。
在本发明的另一个实施方式中,所述膜破裂试剂选自由洗涤剂、胆固醇、疏水蛋白质、表面活性剂、及它们的混合物所组成的组中。
在本发明的另一个实施方式中,所述物质或货物分子选自由DNA分子、RNA分子、蛋白质、肽、脂质、化学制剂、有机化合物、洗涤剂、合成聚合物、及它们的组合所组成的组中。
在本发明的方法的一个实施方式中,所述磁场具有频率为0.1-5MHz的交变磁场方向。所述磁场的强度为1-100mT。
在本发明的另一个实施方式中,所述磁场被反复地暴露,从而作为一系列随时间变化的脉冲,每次脉冲持续的时间优选为1-600秒,更优选为1-45秒。所述磁场可以具有交变磁场,所述交变磁场是非均匀的,且具有交变梯度磁场方向,所述交变磁场的方向由两个线圈产生,并且所述样品被插入到该两个线圈之间。所述线圈被提供有交流电源的正极或负极部分。
本发明构建了被包埋在外壳层中的超顺磁性核,其中,内层直接与超顺磁性核接触,参看实施例1和2。所述颗粒的超顺磁性核具有三个令人满意的特征。它可以用于核磁共振(MRI)跟踪;用于通过磁性分离技术对所述颗粒停留的细胞进行的分选,参考实施例2和5;最重要的是它可以被诱导产热。参考实施例3,由于所述颗粒和该颗粒中的磁性要素都旋转,变化的磁场导致了每一个颗粒的磁性核内产热。这些现象导致了热损失。其中之一控制了一种取决于颗粒大小、磁场频率和颗粒外壳的特定的运动。所述颗粒的核的尺寸优选为8-15纳米,因为在此范围内,核可以使用交变磁场的较低的MHz范围的频率,从而避免对靶细胞及其周围的培养基进行旋转加热。所述颗粒的外壳应当尽可能的薄,以确保颗粒的整体尺寸优选低于50纳米。小颗粒提高了表面积并有益地促进了与各种细胞表面的结合动力,参考实施例10。如果所述颗粒足够小,不会由于重力作用沉淀到测试管中,那么该颗粒既能与悬浮的细胞相互反应又能与粘附的细胞相互反应。
所述颗粒的外壳对于在稳定的胶状溶液中保持颗粒之间很好地分离是非常重要的。各种缓冲液和细胞培养基的稳定性对于纳米颗粒在通过各种生物化学耦合技术进行表面修饰的有用性和有效性以及对活细胞的效果方面的是很关键的。对于货物分子和/或细胞识别分子在颗粒表面的化学耦合,通过NHS(N-羟基磺酸琥珀酰亚胺酯)、SPDP(N-琥珀酰亚胺基3-(2-吡啶二硫基)丙酸酯)、戊二醛(glutaric aldehyde)结合和其它生物结合技术,氨基聚乙二醇(PEG)可以被引入到能够进行化合物耦合的所述颗粒的表面。要在体外或离体状态下(ex vivo)被输送至细胞的货物分子可以通过共价相互作用、静电相互作用或范德华力相互作用结合到外壳上。如果货物分子是DNA或RNA分子,那么DNA或RNA分子的负性骨架和正电荷分子如DOTAB之间的静电作用使它们在所述的外壳的外层成为一个整体;或者在所述热敏外壳的外层表面很容易地利用正电荷分子如聚赖氨酸、PEI(聚氮丙啶)、鱼精蛋白、富含精氨酸或赖氨酸的肽共价结合到氨基聚乙二醇分子上。其它的分子如荧光标记分子或靶细胞识别分子通过与货物分子相同的方法被结合。
当所述颗粒是与用于基因转染的目的货物分子如DNA或RNA分子相结合的复合物时,所述颗粒应当充当一个稳定的非毒性的载体。此外,所述颗粒应当优选保护它们的货物分子不被降解,并且促进该分子在细胞内靶位点的释放。通过将货物分子结合到所述颗粒的表面的附近,对降解酶来说,这样引入了一个位阻,从而抑制了货物分子被酶降解。
除了携带货物分子外,所述纳米颗粒还携带信号分子(如肽)和荧光标记分子。本发明的颗粒除了能携带不同的货物分子外,还能诱导纳米颗粒表面的热释放作用。热诱导释放的意义是为了增加分子在颗粒附近的扩散速率以及促进结合到颗粒表面的分子的释放。
由两层分子组成外壳。内层包括疏水分子,例如,脂肪酸如油酸;或疏水分子的混合物。第二层包括具有一个疏水部分和一个亲水部分的分子的混合物。所述疏水部分形成了类胶束结构的双层,在第一层中,亲水部分朝外进入环境介质中使该颗粒在水基缓冲液和生长培养基中保持稳定。还利用该亲水部分结合到货物分子和其它任何结合到颗粒外壳表面的识别分子或信号分子。
当所述外壳被暴露在外壳脂质的相变温度时,上述外壳的内层分子发生重排或由于外壳从颗粒中泄漏导致外壳的破裂,这又进一步导致了结合在所述外壳上的物质的释放。所述相变温度定义为能导致脂质的物理状态从有序的胶质态转变为无序的液体晶态的温度,其中,所述的有序的胶质态是烃链充分伸展并堆积的状态;所述无序的液体晶态是烃链无规定向并且是流动的状态。有许多因素直接影响相变温度,包括烃链长度、不饱和度、电荷和终端基团(head group)的种类。
由于所述颗粒的超顺磁性核在交变磁场中可以被诱导而产热,因此加热可以精确到所述颗粒附近的区域。所述靶细胞作为一个整体而不被加热。
通过使外部脂质层包括多种分子来构建多功能的热释放纳米颗粒。已知的辅助分子如胆固醇、DOPE(二油酰基磷脂酰乙醇胺)脂质是作为表面外层的部分/成员被输送的。洗涤剂如Triton X和SDS(十二烷基磺酸钠)及类似的分子也可以作为外壳外层的部分/成员。当这些洗涤剂被整合到颗粒中时,它们对靶细胞的毒性很小,但当从颗粒中被释放时,它们能促进核内体膜的破裂,参见实施例11。此外,通过对外壳外层的分子与货物分子之间的选择温度敏感的共价桥进行选择,可以更有利地促进货物分子从颗粒中的释放,并随后离开核内体,参见实施例4和6。
在本发明的一个实施方式中描述了一种方法,其中,使用本发明的颗粒在体外或离体状态下将物质输送到培养的细胞中。所需要的一种或多种物质通过静电作用、共价作用或范德华力作用结合到所述颗粒的表面而作为货物分子。将颗粒/货物分子的复合物与细胞培养基进行混合。所述颗粒表面可以携带能结合到在靶细胞表面特定的靶位点的亲和分子,或者所述颗粒表面具有能促进颗粒/货物复合物与靶细胞带负电的表面区域相互作用的净正电荷。所述颗粒可以通过内吞快速地被靶细胞摄取,早期的核内体随后能被输送到 细胞中并随后被细胞加工成晚期核内体,然后被运送到细胞中的高尔基体和内质网细胞器中。然后施加磁场,磁场脉冲的时间为几秒到几分钟,脉冲的间隔时间与脉冲的时间相同。可以施加两个到几百个脉冲,反复进行加热从而导致颗粒外壳分子的泄漏,进而释放出与外壳连接的货物分子和膜破裂分子。所述膜破裂试剂插入到核内体膜中,从而导致了核内体的破裂。如果货物分子被共价的结合到颗粒外壳上,当加热时,热敏共价键如二硫桥能够进一步促进释放的效果。在暴露于磁场前,细胞与颗粒的孵育时间将影响输送的过程,因此,对于特定的细胞系需要分别优化孵育时间。此外,施加的磁场可以多于一次,以改善随时间推移的输送过程,在每次处理的孵育时间小于1小时至几个小时。
处理后的细胞被自动标记,并可核磁共振进行跟踪;此外,可将荧光标记分子加入到颗粒外壳中以便可以在荧光显微镜下跟踪输送的过程。此外,可以对细胞进行洗涤,并在任何时候用高梯度磁场分离方法对细胞进行分选,在显微镜下可以观察到当细胞处于运动的永久磁铁环境中的细胞的运动。
实施例1
羧酸铁的热分解
采用下述方法制备11±1nm的单分散性颗粒。将360mg的氧化铁(III)研磨成很细的颗粒,并加入到5g的油酸和11g的1-十八烯中。将溶液混合并转移到三颈圆底烧瓶中,将该烧瓶置于加热壁炉架上,用磁力搅拌转子搅拌,将溶液加热到至少320℃,此时1-十八烯开始沸腾。在不低于上述温度的条件下反应一个小时。一小时后,将溶液降至室温。用乙醇、甲醇、氯仿或其混合物对颗粒进行反复洗涤,以从过量的反应物中纯化出颗粒,最后将颗粒重悬在氯仿或己烷中。
实施例2:
聚乙二醇-脂质表面外壳
简单地说,将颗粒悬浮在氯仿中,随后进行真空干燥。将氯仿溶液加入到干燥的颗粒中,使颗粒返回到溶液中,该氯仿溶液含有30-60mol%的磷脂(如,1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱或化学结构类似的其它物质)和40mol%或更高的聚乙二醇脂质(如,1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇)-2000]、1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺-N-[氨基(聚乙二醇)-2000]或类似的聚乙二醇脂质,或化学结构类似的其它物质)。蒸发氯仿,并对颗粒进一步干燥1-4小时。然后用水、NaCl或普通的生理缓冲液对颗粒进行水合,并进行温和地超声处理,获得清澈的溶液,该溶液可以通过100nm的滤器进行无菌过滤。通过高梯度的磁性分离将颗粒从脂质和PEG脂质中纯化出,然后在4℃下进行保存。然而,可以在室温下将颗粒保存数周,并且没有观察到颗粒性质的变化。在生理盐浓度的普通缓冲液中,颗粒的胶体稳定性可保持数周。颗粒甚至在含有10%的FCS(胎牛血清)的复杂生长培养基中保持稳定的时间超过3天,尽管没有测定时间的上限,但颗粒在溶液中可以保存更长的时间。
颗粒悬浮液可以通过磁性柱(磁性高梯度分离柱)或通过尺寸排阻层析而轻易地从结合的化合物和/或盐中纯化出。
磁性高梯度分离柱
将颗粒悬浮液加入到含有磁性基质的柱中,所述磁性基质置于在1.2T的高永久磁场中。只要基质置于高永久磁场中,颗粒就能够保持在磁性基质中。当除去磁场后,颗粒可以被水或期望的缓冲溶液洗脱。还可以通过用少量体积的洗脱液在磁性柱中对铁磁流体进行浓缩。
尺寸排阻层析
将颗粒悬浮液加入装有SephacrylTM S-500高分辩胶的柱中。通过尺寸排 阻将铁磁流体从结合的化合物和/或盐中分离出。或用期望的缓冲溶液对铁磁流体进行洗脱。
尺寸和稳定性
通过TEM(反式电子显微镜)的检测,颗粒的氧化铁核的平均直径为11±1nm,见图1。整个颗粒的直径取决于脂质表面层的组成和载液的特性。磁性纳米颗粒可以很容易地被过滤除菌,并在在普通的生理缓冲液中保持胶体稳定性,因此在制备颗粒和进一步修饰颗粒的过程中,可以使用尺寸排阻层析、透析、和高磁场分离。可以通过Fluram在标准的方法下,对颗粒表面的氨基进行分析,每个颗粒上的氨基的数量可以为几百到几千,而不影响颗粒的稳定性。此外,氨基易于通过普通的已建立完好的结合化学方法来实现共价结合,以加入其它的功能基团或功能分子。
实施例3
交变磁场中的热损失
颗粒核是超顺磁性的,当该颗粒被置于交变磁场中时,热被损失到环境介质中。检测决定颗粒的比吸收率(SRA)的频率,并进行评价。在频率为10MHz以及磁场强度为2.8mT时,获得的SRA值超过500watt/g,如图2的说明。
实施例4
热对分子从颗粒表面的释放的影响
将含有携带磷脂和氨基的通过Fluram荧光标记的氨基-PEG-脂质的颗粒的样品置于10MHz,2.9mT的磁场中处理5分钟,然后通过磁性分离,得到热对颗粒表面和在磁场中可能的热释放的第一个指标。在对比检测中,用温度为40-95℃的常规水浴处理样品5分钟。通过颗粒流过磁性分离器,来检测释放的通过 标记的氨基。
结果概括如下:
1、通过加热,2-6%的氨基从颗粒上释放出。
2、在温度为50-60℃时,释放的氨基的量最多,高温时释放量的减少。
3、在磁场中处理样品5分钟,2-3%的氨基的被释放。
在温度提高的程度相对较小的情况下,氨基-聚乙二醇-nn即开始离开颗粒。这一特性使颗粒适于缓慢的释放。如果分子被共价的结合到氨基上,那么通过交变磁场进行反复处理,可以实现缓慢并反复的释放。
实施例5
为了确定氨基-聚乙二醇-nn和磷脂是否在相同的温度下并以相同的速率进行释放,采用温度为40-95℃的常规水浴来处理含有携带通过FITC标记的磷脂的颗粒样品5分钟。
结果概括如下:
1、用温度高于80℃处理5分钟,高达25%的FITC标记的磷脂从颗粒中释放出。
2、在高于80℃的条件下处理样品,处理后发生絮凝,表明样品的胶体稳定性差。
该实施例说明,与聚乙二醇-脂质相比,磷脂更不易于离开氧化铁核,并且在超过80℃进行加热时,将对颗粒悬浮液的胶体稳定性产生严重的影响。
实施例6
按照如上所述的方法对共价结合到颗粒表面的氨基上的含有携带FITC标记的HIV TAT肽的颗粒样品进行处理。设计该实施例是为了确定通过在氨 基和其它分子之间构建的热敏性共价键是否能够提高释放作用。以SPDP作为介体,将肽结合到颗粒上,并在颗粒与TAT肽之间形成二硫桥。已知二硫桥为相对热敏性的。
结果概括如下:
1、通过加热,3-13%的ATA肽从颗粒中释放出。
2、释放作用随温度的增加而提高。与实施例2获得的结果相比,提高的释放作用可能是由于热而导致二硫桥的破裂。
3、通过磁场对样品进行处理,3-5%的TAT肽的被释放出。
实施例7
细胞的生长
粘附细胞系COS-7生长在含有10%的FCS和5%的链霉素/青霉素的DMEM中。悬浮细胞系K562生长在含有10%的FCS的RPMI中,其中,CO2的含量为5%。细胞每周传代两次。
增值
收获COS-7和K562细胞并将它们分别接种到每孔含有100μl的DMEM和RPMI完全培养基的96孔板中,细胞的接种密度为105个细胞/孔。为了获得细胞的增值标准曲线,在每孔中接种的细胞为104-105,平行实验三组。用适当的缓冲液稀释具有氨基、直链PEI或支链PEI的颗,使Fe的终浓度为每个细胞对应于50-250pg的Fe,平行实验四组。此外,COS-7细胞的增值试验是通过将颗粒/细胞复合物置于交变频率为10MHz,磁场强度为2.9mT的交变磁场中来实现的,该实验是在如图3所示的实验装置中进行的。用水浴对线圈进行冷却,并维持样品在预期的温度。用不受射频(RF)磁场影响的光纤维温度计连续检测样品中的温度。
在培养24小时后进行增值检测。可以推出的是,如果将携带有相对较 高浓度的颗粒细胞置于交变磁场中,则该细胞的增值降低。此外,通过PEI(聚氮丙啶)修饰的颗粒对细胞的毒性更大,K562对颗粒的敏感性比COS-7更高。
实施例8
细胞的分离和洗涤
将K562细胞接种在24孔培养板中,每孔中的培养基为1ml。将具有DOTAB/DOPE/磷脂混合物的外壳的颗粒以下列浓度加入到细胞中:
样品1:1皮克的Fe/细胞,孵育2小时;
样品2:2皮克的Fe/细胞,孵育2小时;
样品3:2皮克的Fe/细胞,孵育2小时,在分离柱中未使用磁铁;
样品4:2皮克的Fe/细胞,孵育24小时;
样品5:2皮克的Fe/细胞,孵育48小时;
样品6:2皮克的Fe/细胞,孵育72小时;
样品7:0皮克的Fe/细胞,孵育2小时;
将细胞从培养皿中移出,并用缓冲液(PBS+BSA)通过短时间离心进行洗涤。洗涤后用钙黄绿素AM对活细胞进行标记,然后加入到高梯度磁性的分离柱中(Milteyi)。分别将每一个样品置于不同的柱中进行处理,分离样品nr 3时,不施加额外静磁场。用三倍于柱体积的缓冲液对细胞进行洗涤,并按照供用商的使用手册将柱从永久磁铁中释放,以洗脱细胞。检测不同洗脱液部分的钙黄绿素荧光的强度。
流通(FT)
洗涤1、2、3(W1、W2、W3)
洗脱(E)
从图4所示的结果可以清楚的看出,加入2皮克的FE/细胞能够为处理 的细胞提供充足的磁性材料,以利用高梯度磁场进行分离和洗涤,并且这种效果可以持续72小时。
实施例9
颗粒的细胞吸取及其在细胞内的分布
用荧光显微镜对同超顺磁性纳米颗粒孵育了1小时和24小时的COS-7细胞进行检测,以跟踪颗粒在细胞内的运输。可以看出颗粒能够容易地且相对较快地被细胞吸取,并均匀地分布在整个细胞中。
在另一个实验中,将COS-7细胞与用德克萨斯红标记的、被通过FITC标记的HIV-1 TAT肽共价修饰的磁性纳米颗粒(100pg铁/细胞)一起孵育12小时。该肽能够以天然的状态标靶并进入细胞核。德克萨斯红标记物(标记磷脂)和FITC标记的HIV-1 TAT肽共同定位在细胞质中。
实施例10
质粒DNA的结合能力
将与直链PEI(聚氮丙啶)或支链PEI共价结合于颗粒表面的氨基PGE的纳米颗粒、和无直链PEI(聚氮丙啶)或支链PEI共价结合于颗粒表面的氨基PGE的纳米颗粒通过缓冲液稀释至相同的颗粒浓度。颗粒与相同量的DNA(100ng)以不同的比例(0.1-500)进行混合。制备DNA的标准曲线(10-500ng/ml)。将溶液在室温下孵育10分钟,然后加入PIco 并检测荧光强度(Ex484/Em520)。结果如图5表示,可以推出,所有的颗粒都具有与DNA结合的能力,并且表面具有支链PEI的颗粒能更有效地与DNA相结合。
实施例11
携带SDS、DOPE和DOTAB混合物的颗粒的转染效率与用仅携带PEG-NH2的颗粒的转染效率的比较
将细胞(K562)和具有携带德克萨斯红和编码荧光素酶(phRL-SV40)的质粒的含有DOTAB/DOPE/SDS的外壳的颗粒一起孵育3小时;将作为对照的细胞与具有携带编码荧光素酶(phRL-SV40)的质粒的含有氨基-PEG外壳的颗粒以相同的方法进行孵育。孵育后,通过AMF(交变磁场)对细胞进行反复处理,其中,交变磁场的脉冲时间为30秒,且每两个脉冲之间的静息时间也为30秒。
在用AMF处理细胞后,将细胞接种到新的培养皿中,后处理48小时后,读取增值的荧光值。从图6可以看出,携带洗涤剂和辅助分子脂质(DOTAB/DOPE/SDS)的颗粒表现出相当高的表达率。
Claims (24)
1.一种颗粒,该颗粒适用于通过物质的热诱导释放而将该物质输入到膜封闭的细胞或细胞器中,或者将该物质从膜封闭的细胞或细胞器中输出,该颗粒包括:在交变磁场中具有产热能力的超顺磁性核,该核被包埋在结合有所述物质和膜破裂试剂的热敏性外壳中。
2.根据权利要求1所述的颗粒,其中,所述超顺磁性核的尺寸为1-100nm。
3.根据权利要求2所述的颗粒,其中,所述超顺磁性核的尺寸为8-15nm。
4.根据权利要求1或2所述的颗粒,其中,所述超顺磁性核包括磁铁矿石、磁赤铁矿石、氧化铁水合物、和通式为MeOxFe2O3的铁酸盐,其中,Me为选自由Co、Ni、Mn、Be、Mg、Ca、Ba、Sr、Cu、Zn、Pt、Al、Cr、Bi、及它们的组合所组成的组中的二价金属。
5.根据权利要求1所述的颗粒,其中,所述热敏性外壳包括与所述核直接接触的类胶束结构。
6.根据权利要求5所述的颗粒,其中,所述类胶束结构由至少一种疏水分子和至少一种既含有疏水部分又含有亲水部分的分子构成。
7.根据权利要求5所述的颗粒,其中,所述热敏性外壳与所述物质和膜破裂试剂通过一个或多个二硫键、过氧键、偶氮键、或它们的组合相连接。
8.根据权利要求6所述的颗粒,其中,所述亲水部分的结构含有聚合物、脂质、合成分子、蛋白质、或它们的组合。
9.根据权利要求1所述的颗粒,其中,所述热敏性外壳还结合有识别分子。
10.根据权利要求9所述的颗粒,其中,所述识别分子选自由抗体、抗体片段和凝集素所组成的组中。
11.根据权利要求1所述的颗粒,其中,所述膜破裂试剂选自由洗涤剂、胆固醇、疏水蛋白质、表面活性剂、及它们的混合物所组成的组中。
12.根据权利要求1所述的颗粒,其中,所述物质选自由DNA分子、RNA分子、蛋白质、肽、脂质、化学制剂、有机化合物、洗涤剂、合成聚合物、及它们的组合所组成的组中。
13.权利要求1-12中的任意一项所述的颗粒在体外改善宿主细胞的遗传编码和/或代谢中的应用。
14.权利要求1-12中的任意一项所述的颗粒在体外从核内体细胞器或溶酶体细胞器释放所述物质中的应用。
15.一种在体外通过物质的热诱导释放以及膜破裂试剂的热诱导释放而将该物质输送到膜封闭的细胞或细胞器中或者将该物质从膜封闭的细胞或细胞器中输出的方法,该方法包括:
提供含有膜封闭的细胞或细胞器和多个根据权利要求1-12中的任意一项所述的颗粒的样品;
对所述样品施加交变磁场,其中,所述颗粒的核的热能导致所述颗粒的热敏性外壳发生分解,由此所述破裂试剂使封闭的细胞或细胞器的膜发生破裂,从而释放出所述物质。
16.根据权利要求15所述的方法,其中,所述磁场具有频率为0.1-5MHz的交变磁场方向。
17.根据权利要求15或16所述的方法,其中,所述磁场的强度为1-100mT。
18.根据权利要求17所述的方法,其中,所述磁场被反复地暴露从而作为一系列随时间变化的脉冲。
19.根据权利要求18所述的方法,其中,每次脉冲持续的时间为1-600秒。
20.根据权利要求18所述的方法,其中,每次脉冲持续的时间为1-45秒。
21.根据权利要求15所述的方法,其中,所述磁场具有交变磁场,所述交变磁场是非均匀的并且具有交变梯度的磁场方向,所述交变磁场的方向由两个线圈产生,所述样品插入到该二个线圈之间。
22.根据权利要求21所述的方法,其中,所述线圈被提供有交流电源的正极或负极部分。
23.根据权利要求15所述的方法,其中,所述物质选自由DNA分子、RNA分子、蛋白质、肽、脂质、化学制剂、有机化合物、洗涤剂、合成聚合物、及它们的组合所组成的组中。
24.根据权利要求15所述的方法,其中,所述膜破裂试剂选自由洗涤剂、胆固醇、疏水蛋白质、表面活性剂、及它们的混合物所组成的组中。
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