CN111954519A - 用于药物递送的脂质体系统 - Google Patents
用于药物递送的脂质体系统 Download PDFInfo
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- CN111954519A CN111954519A CN201880088777.4A CN201880088777A CN111954519A CN 111954519 A CN111954519 A CN 111954519A CN 201880088777 A CN201880088777 A CN 201880088777A CN 111954519 A CN111954519 A CN 111954519A
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Abstract
一种用于递送活性剂的脂质体系统,所述系统包含:形成脂质体的脂质组分;与所述脂质组分缔合的去稳定剂,所述去稳定剂能够形成反应性氧物质,以氧化不饱和脂质并使脂质体膜去稳定;以及活性剂;其中所述活性剂通过暴露于高能电磁辐射可从所述脂质体释放。
Description
技术领域
本技术涉及使用脂质体的药物递送系统。
相关申请
本申请基于2017年12月6日提交的澳大利亚临时专利申请号2017904916并要求其优先权,其内容通过引用以其整体并入本文。
背景
用于基因和药物递送的各种纳米材料设计的开发和应用是目前纳米医学中的关键焦点领域之一。尽管传统上病毒载体已用作基因/药物递送方法(Thomas等人2003;Zhang等人2012),但其应用受到一系列限制的阻碍,包括毒素产生、负载材料的有限大小、包装困难和重组风险(Luo,D. & Saltzman 2000)。为了克服这些限制,已经广泛地研究和开发了基于合成的纳米材料的系统。在这些纳米材料中,脂质体由于其制备的简单性和独特的特性而被很好地确立为有效的药物递送系统。脂质体由被类似于细胞膜的脂质双层包围的水性核心组成,所述脂质双层促进脂质体的细胞摄取。形成脂质体的脂质是两亲性的,因此允许包封疏水性和亲水性分子二者以及胶体颗粒。脂质体通常是生物相容的和生物可降解的,这使得它们适合于临床应用。常规脂质体,例如,商业lipofectamine 2000,不能实现按需内容物释放,这限制了它们的治疗应用,尽管它们具有高递送效率。
常规脂质体以不可控制的方式逐渐释放包封的货物,这限制了它们的治疗功效。相反,可触发的脂质体能够以更受控的方式释放基因/药物,通常更快,并且取决于触发方式,也释放至特定区域,并且这些性质有助于它们潜在更大的临床成功。先前已经采用了几种策略来设计响应性脂质体,其双层可以通过使用生理和外部刺激而去稳定。先前报道的触发方法包括pH的变化(癌症中典型) (Nahire等人,2014;Ferreira等人,2013),外部递送的热,例如经由交变磁场或红外光(Dicheva等人,2014;Kono等人,2010),酶(Sarkar等人,2005;Arouriet等人,2015)和由光照射引起的非热效应(Leung等人,2012;Puri,2013)。这些方法具有某些限制,特别是光敏脂质体的可见光触发受到光进入生物组织的相对浅(几毫米)穿透的限制(Wilson和Patterson,2008)。由于这种浅的穿透深度,可见光不能激活位于体内深处的光敏剂(PS)并产生足够量的单线态氧(1O2)或其它反应性氧物质(ROS),以释放治疗效果所需的脂质体货物。
本发明人已经开发了适于将药物或生物活性剂递送至受试者的脂质体系统。
概述
在第一方面,提供了用于递送活性剂的脂质体系统,所述系统包含:
形成脂质体的脂质组分;
与所述脂质组分缔合的去稳定剂,所述去稳定剂能够形成反应性氧物质,以氧化不饱和脂质并使脂质体膜去稳定;以及
在所述脂质体中的活性剂;
其中所述活性剂通过暴露于高能电磁辐射可从脂质体释放。
所述脂质组分可以包括形成稳定脂质体的任何合适的脂质。有用的脂质体通常由天然存在的脂质如磷脂和胆固醇形成。合适的脂质的实例包括1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)和1,2-二-(9Z-十八碳烯酰基)-3-三甲基铵-丙烷(DOTAP),或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和N-[1-(2,3-二油基氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)。
在一个实施方案中,所述脂质组分由DOPC和DOTAP形成。DOPC可负载高度疏水的分子,而DOTAP由于其正电荷可促进细胞摄取。
在一个实施方案中,所述去稳定剂为纳米颗粒。所述纳米颗粒可以是金属纳米颗粒或无机纳米颗粒。所述金属纳米颗粒可以选自金、银和铋。
在一个实施方案中,所述金属纳米颗粒为金纳米颗粒。
在一个实施方案中,所述无机纳米颗粒可以是氟化铈(CeF3)。
在一个实施方案中,所述去稳定剂为光敏剂。合适的光敏剂包括维替泊芬(VP)、玫瑰红、氨基乙酰丙酸和光敏素。应当理解,在本技术中可以使用其它光敏剂。
在一个实施方案中,所述去稳定剂为纳米颗粒和光敏剂。所述去稳定剂可以是金纳米颗粒和维替泊芬的组合。
在一个实施方案中,所述反应性氧物质是1O2 (单线态氧,是高能形式的氧)。
在一个实施方案中,所述活性剂为化疗剂、药物、医学成像剂、用于基因沉默和疗法的反义寡核苷酸和小干扰RNA (siRNA)分子、生物活性剂、抗体、抗体片段、蛋白肽或核酸。
在一个实施方案中,所述化疗剂为多柔比星、长春新碱、5-氟尿嘧啶或磷酸依托泊苷(凡毕复)。然而,应理解,本技术适用于其它适于配制脂质体的试剂。
在一个实施方案中,所述化疗剂为多柔比星。
在一个实施方案中,所述活性剂为反义寡核苷酸。
脂质体可以进一步包含引起所述脂质体摄取到受试者的靶区域或靶细胞中的材料。所述材料可以是抗原、抗体、抗体片段、肽、激素、细胞因子、叶酸、配体和受体。例如,脂质体-叶酸缀合物已经用于使脂质体成为肿瘤细胞特异性的,这是由于叶酸受体在许多癌细胞上过表达。这些叶酸缀合的脂质体将能够靶向癌细胞并通过受体介导的胞吞作用在细胞内递送其货物。
在一个实施方案中,所述高能电磁辐射为x射线辐射或γ射线辐射。所述脂质体暴露于高能电磁辐射如x射线导致在脂质组分中产生1O2,使脂质体去稳定,导致活性剂的释放。1O2的产生可以来自脂质体中的光敏剂或纳米颗粒。
在一个实施方案中,所述高能电磁辐射的能量为至少约6 MeV。
在第二方面,提供了用于递送活性剂的脂质体系统,所述系统包含:
形成脂质体的脂质组分,其包含1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)和1,2-二-(9Z-十八碳烯酰基)-3-三甲基铵-丙烷(DOTAP),或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和N-[1-(2,3-二油基氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA);
与所述脂质组分缔合的去稳定剂,其包含纳米颗粒、光敏剂或纳米颗粒和光敏剂,所述去稳定剂能够形成反应性氧物质,以氧化不饱和脂质并使脂质体膜去稳定;以及
在所述脂质体中的活性剂,其选自化疗剂或反义寡核苷酸;
其中所述活性剂通过暴露于高能电磁辐射可从所述脂质体释放。
在第三方面,提供了将活性剂给予受试者的方法,所述方法包括:
向受试者提供根据第一方面或第二方面所述的脂质体系统;以及
将所述受试者暴露于高能电磁辐射,以从所述脂质体释放所述活性剂,以治疗所述受试者。
脂质体系统可以通过任何合适的途径提供给受试者,例如口服、静脉内、局部和肠内。
所述方法可以进一步包括在将所述受试者暴露于高能电磁辐射之前,允许所述脂质体被所述受试者的部位中的细胞吸收。该部位可以是肿瘤、感染、伤口、器官或其区域,例如骨区域、皮肤和血管,包括在眼睛中。
高能电磁辐射可以通过将患者暴露于x射线或γ射线辐射来提供。高能电磁辐射可以是定点暴露或整个受试者暴露。
在第四方面,提供了根据第一方面或第二方面所述的脂质体系统在将活性剂给予受试者中的用途。
在第四方面,提供了根据第一方面或第二方面所述的脂质体系统在制备将活性剂给予受试者的药物中的用途。
所述受试者可以是任何动物,例如人。
在整个本说明书中,除非上下文另有要求,否则词语"包含(comprise)"或变型如"包含(comprises)"或"包含(comprising)"将被理解为暗示包括所述的要素、整数或步骤,或要素、整数或步骤的组,但不排除任何其它要素、整数或步骤,或要素、整数或步骤的组。
对本说明书中包括的文件、法案、材料、装置、制品等的任何讨论仅用于提供本发明的语境的目的。不应承认任何或所有这些内容因为其存在于本说明书的每个权利要求的优先权日之前而形成现有技术基础的一部分或为本发明相关领域中的公知常识。
为了可以更清楚地理解本技术,将参考以下附图和实例来描述优选的实施方案。
附图简述
图1是由X射线辐射触发的掺有维替泊芬和金纳米颗粒的脂质体递送平台的基因沉默和癌细胞杀伤作用的示意图。将两种类型的货物,反义寡核苷酸和多柔比星(Dox)分别包埋在脂质体中间腔内,用于证明体外基因和药物递送。还通过使用掺有Dox并由X射线触发的脂质体进行体内证实。
图2显示脂质体的物理和光学性质。(a)和(b)含有金纳米颗粒的脂质体和纯脂质体的透射电子显微术(TEM)图像。(c)通过动态光散射确定的尺寸分布。(d)脂质体、纯维替泊芬(VP)和纯金纳米颗粒的吸收光谱。
图3显示(a)在360 nm照射下在不同时间点,和(b)在不同剂量的X射线辐射下,不同脂质体样品中单线态氧传感器绿色(SOSG)荧光强度的增加百分比。
图4显示(a)在360 nm LED照射下和(b) X射线照射下钙黄绿素从不同脂质体样品中释放的动力学。
图5(a)显示PAC1R在处理后不同时间点的间接免疫荧光染色的代表性共焦图像。(a)上子图:用X射线和脂质体处理的细胞。(a)下子图:用脂质体单独处理的细胞。由在不同时间点(b)用X射线辐射和(c)不用X射线辐射从脂质体释放的反义寡核苷酸诱导的PAC1R基因沉默的定量评价。降低的PAC1R表示为对照的百分比。与细胞一起孵育的脂质体的浓度为25 μM。比例尺为75 μm。
图6显示在各个时间点(0h、2h、4h和24h),(a)用4 Gy的X射线辐射和(b)不用4 Gy的X射线辐射,LipoDox对HCT116的细胞杀伤作用。将细胞在96孔板中培养。Dox的浓度为6、20、60和160 ng/孔。存活率表示为相对于对照细胞的平均百分比和标准偏差(n=4)。(c)显示在4 Gy的X射线辐射后24小时,LipoETP对HCT116的细胞杀伤作用。ETP的浓度为33、100、300和900 ng/孔。存活率表示为相对于对照细胞的平均百分比和标准偏差(n=4)。
图7(a)显示在孵育后24小时和48小时,负载有VP (Lipo-VP)和金纳米颗粒(Lipo-VP-金)的脂质体对PC12细胞的体外毒性测定。(b)处理后24小时和48小时,4 Gy的X射线对PC12、HCT116和CCD 841 CoN细胞的毒性。(c)孵育后24小时,脂质体配制的Dox对CCD 841CoN细胞的毒性。存活率表示为相对于对照细胞的平均百分比和标准偏差(n=4)。(d)在以不同剂量进行X射线暴露后,反义寡核苷酸(10 μg/mL)以及寡核苷酸与VP (10 μg/mL寡核苷酸和32 μg/mL维替泊芬)的混合物的琼脂糖凝胶电泳。从左至右泳道:未经处理的对照样品、1 Gy、2 Gy和4 Gy。
图8显示在UV照射下,负载有VP和金纳米颗粒的脂质体以及仅负载有VP的脂质体的1O2产生的定量。(a) SOSG强度作为UV照射时间的函数。(b)这些样品的吸收光谱。
图9 (a)显示由仅负载有VP的脂质体吸收的UV光子数量作为时间的函数;(b)产生的单线态氧数量相对于吸收的UV光子数量。
图10 (a)显示对于负载有VP和金纳米颗粒的脂质体,SOSG强度作为X射线剂量的函数;(b)对应于各X射线产生的单线态氧数量。
图11显示在含有不同浓度FBS的PBS (pH 7.4)中孵育0 h、2 h、4 h、18 h、24 h和48 h后,从(a)常规脂质体和(b)聚乙二醇化脂质体中释放的Dox的百分比。(c)从在含有10%的FBS的PBS (pH 7.4、6.0和5.0)中孵育的聚乙二醇化脂质体样品中释放的Dox的百分比。
图12显示用负载有荧光寡核苷酸的脂质体纳米颗粒(25 μM)孵育的PC12细胞的共焦激光扫描显微术图像。比例尺为75 μm。
图13显示X射线触发的LipoDox在结肠直肠癌的异种移植模型中的抗肿瘤活性。(a和c)在各种所示处理后,小鼠的肿瘤和体重变化。黑色箭头指示处理给药的时间。使用t检验分析平均肿瘤体积。*P<0.05,**P<0.01,***P<0.001。(b)在终点分离的肿瘤的照片。
定义
在整个本说明书中,除非上下文明确另有要求,否则词语"包含(comprise)"或变型如"包含(comprises)"或"包含(comprising)"将被理解为暗示包括所述的要素、整数或步骤,或要素、整数或步骤的组,但不排除任何其它要素、整数或步骤,或要素、整数或步骤的组。
在整个本说明书中,术语"由……组成"是指仅由……组成。
对本说明书中包括的文件、法案、材料、装置、制品等的任何讨论仅用于提供本技术的语境的目的。不应承认任何或所有这些内容因为其存在于本说明书的每个权利要求的优先权日之前而形成现有技术基础的一部分或为本技术相关领域中的公知常识。
除非上下文另有要求或相反地特别说明,否则本文中作为单数的整数、步骤或要素叙述的技术的整数、步骤或要素明确包括所叙述的整数、步骤或要素的单数和复数形式二者。
在本说明书的上下文中,术语"a"和"an"用于指该冠词的一个或多于一个(即,至少一个)语法对象。例如,提及"一个要素"是指一个要素或多于一个要素。
在本说明书的上下文中,术语"约"是指对数字或值的提及不应被视为绝对数字或值,而是包括在该数字或值之上或之下的变化的容限,其与技术人员根据本领域的理解一致,包括在误差或仪器限制的典型容限内。换句话说,术语"约"的使用被理解为是指本领域技术人员在实现相同功能或结果的情况下将认为与所叙述的值等同的范围或近似值。
本领域技术人员将理解,本文所述的技术易于进行除具体描述的那些以外的变化和修改。应当理解,本技术包括所有这些变化和修改。为了避免疑惑,该技术还包括在本说明书中单独或共同提及或指出的所有步骤、特征和化合物,以及所述步骤、特征和化合物中的任何两者或更多者的任何和所有组合。
实施方案的描述
本发明人通过将光敏剂和/或金纳米颗粒(3-5 nm)共包埋在脂质双层内部,已设计了触发的脂质体。在这项工作中选择金,这是因为由于其高原子序数,它与X射线辐射强烈地相互作用,例如通过金纳米颗粒诱导的生物组织内的辐射增强所示。尽管在设计中光敏剂可以是反应性氧物质(ROS)的主要来源,以氧化不饱和脂质和使脂质体膜去稳定,但是暴露于X射线的金纳米颗粒也产生一定水平的ROS。更复杂的作用也是可能的,例如在X射线与金纳米颗粒相互作用期间产生的二次电子可以从金转移到光敏剂并导致PS诱导的1O2 (单线态氧是高能量形式的氧)或其它ROS的产生。选择维替泊芬(VP)作为光敏剂来证明该技术,其在临床上被批准用于年龄相关性黄斑变性的光动力疗法(PDT)。选择1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)和1,2-二-(9Z-十八碳烯酰基)-3-三甲基铵-丙烷(DOTAP)作为脂质体制剂中的脂质组分,这是因为DOPC可以负载高度疏水的分子,并且DOTAP由于其正电荷可以促进细胞摄取。通过分别使用单线态氧绿色传感器(SOSG)和钙黄绿素释放测定,评价了在365nm LED照射下以不同时间点(2、4、6、8和10分钟)和以不同剂量(1、2和4 Gy)的X射线辐射从不同脂质体样品的1O2产生和通过1O2的脂质双层去稳定。SOSG是一种常用的、高度特异的荧光探针,用于检测1O2产生。其被鉴定为与蒽部分共价结合的荧光素。钙黄绿素是在高浓度下自猝灭的荧光染料,这使得可以通过监测X射线辐射时钙黄绿素荧光强度的增加来检测其从脂质体到周围环境的释放。另外,还使用本发明人以前开发的方法,基于实验数据计算在UV光照射下的1O2量子产率和作为X射线辐射结果产生的1O2数量。
X射线触发释放脂质体货物通过以下来验证:(a)证明X射线触发的基因敲减的效率和(b)基于该脂质体递送系统的化疗的有效性增加(图1)。对于基因沉默,将与特定的垂体腺苷酸环化酶激活多肽(PACAP)受体PAC1R互补的反义寡核苷酸包封在脂质体内部。在脂质体被大鼠PC12细胞吸收后,以4 Gy剂量施加X射线辐射。由于暴露于电离辐射,在脂双层中产生的1O2使脂质体去稳定,导致反义寡核苷酸的释放。然后该反义核苷酸能够通过阻断翻译起始复合物而阻止PAC1R mRNA的翻译。通过观察X射线照射后细胞中PAC1R间接免疫荧光染色的荧光强度的降低来监测基因敲减。对于X射线触发的化疗,将抗肿瘤药物多柔比星(Dox)负载到脂质体中。脂质体被人结肠直肠癌HCT116细胞吸收并施用X射线。随后通过MTS测定检查脂质体配制的Dox的体外癌细胞杀伤功效。为了比较,还在除了省略X射线辐射之外的相同实验条件下进行对照实验。
通过在脂质体双层内部引入金纳米颗粒和光敏剂维替泊芬,开发了X射线可触发的脂质体。对于6 MeV的X射线辐射,定量单线态氧产生量子产率,其中发现4 Gy的剂量产生约7250个单线态氧分子/脂质体。单线态氧分子使脂质体膜去稳定,导致从脂质体腔释放货物(基因沉默剂和/或药物)。这通过在大鼠PC12细胞中X射线触发的垂体腺苷酸环化酶激活多肽(PACAP)受体之一PAC1R的基因敲减来证明。负载有化疗药物多柔比星的相同的X射线触发的脂质体比没有X射线触发下更有效地杀死人结肠直肠癌HCT116细胞。这表明在标准放疗过程中,6 MeV的X射线与通过X射线触发的脂质体递送的化疗组合的协同作用的可能性。重要的是,X射线介导的脂质体释放策略通过消除其深度限制为深层组织光动力疗法提供了新的前景。因此,与放疗、化疗或基因疗法组合的新型脂质体可提供新的癌症治疗选择。
材料
脂质体
脂质组分可以包括形成稳定脂质体的任何合适的脂质。有用的脂质体通常由天然存在的脂质如磷脂和胆固醇形成(Miranda和Lovell,2016)。
脂质体通常由溶解的脂质分子自组装形成,每个脂质分子含有亲水性头基和疏水性尾部。这些脂质采取产生熵有利状态的低自由能的缔合,在一些情况下,形成双分子脂质小叶。这样的小叶的特征在于疏水性烃尾部彼此面对,而亲水性头基面向外部以与水溶液缔合。此时,双层形成仍然是能量不利的,因为分子的疏水部分仍然与水接触,该问题通过形成双层膜在其自身上弯曲以形成具有闭合边缘的囊泡而克服。这种自由能驱动的自组装是稳定的,并且已经被用作特异性地针对给定系统的需要而工程化脂质体的有力机制。脂质体中使用的脂质分子是保守实体,其具有通过骨架连接基如甘油连接的头基和疏水性烃尾部。阳离子脂质通常通过存在于极性头基中的一个或多个胺获得正电荷。带正电荷的胺的存在促进与阴离子如在DNA中发现的那些阴离子的结合。由此形成的脂质体是通过与DNA的静电结合和范德华力的能量贡献的结果,这部分决定脂质体形状。由于DNA的多阴离子性质,阳离子(和中性)脂质通常用于基因递送,而阴离子脂质体的使用已相当局限于递送其它治疗性大分子(Balazs和Godbey,2011)。
用于阳离子脂质转染的充分表征且广泛使用的商业试剂包括N-[1-(2,3-二油基氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)、[1,2-双(油酰基氧基)-3-(三甲铵基)丙烷](DOTAP)、3β[N-(N',N-二甲氨基乙烷)-氨基甲酰基]胆固醇(DC-Chol)和双十八烷基酰氨基甘氨酰精胺(DOGS)。中性脂质二油酰基磷脂酰乙醇胺(DOPE)可以与阳离子脂质结合使用,这是因为其在低pH下具有膜去稳定化作用,这有助于内溶酶体逃逸。
脂质双层可以包括形成稳定脂质体的任何合适的脂质。合适的脂质的实例包括1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)和1,2-二-(9Z-十八碳烯酰基)-3-三甲基铵-丙烷(DOTAP),或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和N-[1-(2,3-二油基氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)。
由DOPC和DOTAP形成的脂双层适合用作本技术的脂质体。DOPC可负载高度疏水的分子,DOTAP由于其正电荷可促进细胞摄取。与DOPC和DOTAP的组合类似,DOTMA可以与DOPE以1/1摩尔比偶联以形成脂质体,其中阳离子DOTMA可以增强细胞摄取,而DOPE在外部触发下具有膜去稳定作用(Farhood,1995)。
去稳定剂
纳米颗粒
纳米颗粒可以是金属纳米颗粒或无机纳米颗粒。合适的金属纳米颗粒包括金、银和铋,其可以增强X射线或γ射线辐射以及来自X射线或γ射线辐射的能量传递(Su等人,2014)。
无机纳米颗粒可以选自氟化铈(CeF3)。无机纳米颗粒可以用作有效的闪烁剂,其在X射线或γ射线激发时产生可见光以触发ROS产生(Clement等人,2016)。
光敏剂
合适的光敏剂包括维替泊芬(VP)、玫瑰红、氨基乙酰丙酸和光敏素。VP (商品名Visudyne)是苯并卟啉衍生物,其传统上用作光动力疗法的光敏剂,以消除与病症例如湿性黄斑变性相关的眼中异常血管。
纳米颗粒和光敏剂的组合也可用于本技术。例如,在下面的实例中金纳米颗粒和VP已经显示良好起作用。
活性剂
活性剂可以是可掺入脂质体中的任何合适的试剂。活性剂可以是化疗剂、药物、医学成像剂、用于基因沉默和疗法的反义寡核苷酸和小干扰RNA (siRNA)分子、生物活性剂、抗体、抗体片段、蛋白肽或核酸。
在一个实施方案中,化疗剂为多柔比星、长春新碱、5-氟尿嘧啶或磷酸依托泊苷(凡毕复)。然而,应理解,本技术适用于其它适于为脂质体配制的试剂。
在一个实施方案中,化疗剂为多柔比星。
活性剂可以是反义寡核苷酸。
多柔比星和反义寡核苷酸
高能电磁辐射
可以使用高能电磁辐射。能量高于约5 keV的电磁辐射,特别是能量为至少约6 MeV的X射线或γ射线辐射可用于本技术。
脂质体摄取材料
脂质体可以进一步包含引起脂质体摄取到受试者的靶区域或靶细胞中的材料。所述材料可以是抗原、抗体、抗体片段、肽、激素、细胞因子、配体和受体。例如,由于叶酸受体在许多癌细胞上过表达,脂质体-叶酸缀合物已经用于制备肿瘤细胞特异性的脂质体(Low等人,2007)。可以使用以下方法合成缀合(Gabizon等人,1999)。简言之,将过量的叶酸溶解在二甲基亚砜中。将聚乙二醇化的脂质体和吡啶加入到叶酸中,随后加入二环己基碳二亚胺。在室温下继续反应4小时,随后在旋转蒸发下从反应混合物中除去吡啶。在向混合物中加入水后,将其离心,以除去痕量的不溶性物质。上清液用盐水和水透析,以除去二甲基亚砜和未缀合的反应物。这些叶酸缀合的脂质体将能够靶向癌细胞并通过受体介导的胞吞作用在细胞内递送其货物(Kularatne和Low,2010)。
方法
负载有金纳米颗粒和维替泊芬的脂质体的制备
将溶解在氯仿(100 mg/mL,Sigma-Aldrich,编号288306-1L)中的350 μL的DOTAP(Avanti Polar Lipids,编号890890P)与溶解在氯仿(100 mg/mL)中的370 μL的DOPC(Avanti Polar Lipids,编号850375P)混合,随后加入溶解在二甲基亚砜(DMSO,2.3 mg/mL,Sigma-Aldrich,编号472301-500ML)中的40 μL金纳米颗粒悬浮液(Nanocomposix,Inc)和50 μL的VP (Sigma-Aldrich,编号SML0534-5MG)。为了合成空脂质体、仅含VP的脂质体、仅含金纳米颗粒的脂质体,省略了其它成分。使用氯仿将混合物稀释至1.0 mL总体积并轻轻涡旋10分钟。用氩气流蒸发掉氯仿,并在冷冻干燥下蒸发剩余的DMSO,这在冷冻干燥机(Alpha 1-4 LDplus, John Morris Scientific Pty Ltd)中进行。通过向玻璃试管中加入1.0 mL的去离子水,随后剧烈搅拌直至悬浮液均质化,使脂质膜水合。将水合的脂质悬浮液放置过夜,以允许脂质体的最大溶胀。然后在具有两个1.0 mL玻璃注射器的挤出机(AvantiPolar Lipids,Inc)中挤出该悬浮液十一次。聚碳酸酯膜(Avanti Polar Lipids,Inc)的孔径为200 nm。将所得悬浮液在氩气下在4℃下储存。为了在脂质体内部包封钙黄绿素,使用1.0 mL钙黄绿素溶液(100 mM,Sigma-Aldrich,编号C0875-5G)作为脂质水合溶液,代替去离子水。为了包封寡核苷酸,使用1.0 mL含有针对PAC1R基因的具有3' FAM标记的反义寡核苷酸(10 μM,5'-TGGTGCTTCCCAGCCACTAT-3') (Integrated DNA Technologies Pte.Ltd.)的PBS (pH 7.4)溶液来水合脂质膜,随后进行上述水合程序。为了除去水合后存在于上清液中的钙黄绿素和寡核苷酸,然后按照制造商的说明书,通过使用Pall Nanosep离心装置(Sigma-Aldrich)将脂质体在14000×g下离心10分钟。
脂质体配制的多柔比星(LipoDox)的合成
按照公开的方案,使用具有较小修改的梯度交换法(Li等人,2009),进行脂质体内部多柔比星的包封。将1 mL硫酸铵(250 mM,Sigma-Aldrich,编号A4418-100G)加入蒸发有机溶剂后形成脂质膜的玻璃试管中,随后进行上述水合程序。通过在PBS溶液(pH 7.4)中透析除去游离的硫酸铵,缓冲液交换重复四次。随后将Dox溶液(10mg/mL,Sigma-Aldrich,编号D1515-10MG)加入到水合脂质体悬浮液中,其中药物与脂质的质量比为1:10,随后在60℃下孵育1小时。通过在PBS溶液(pH 7.4)中透析,除去未负载的Dox,其中缓冲液交换四次。
掺入依托泊苷(ETP)、VP和金纳米颗粒的脂质体(LipoETP)的制备
通过一定改良的薄膜水合(Sengupta等人,2000)制备掺入ETP、VP和金纳米颗粒的脂质体。简言之,将100 μL的DOTAP (50 mg/mL于氯仿中)与54 μL的DOPC (100 mg/mL于氯仿中)混合,随后加入6 μL金纳米颗粒悬浮液、7 μL的VP (2.3 mg/mL于DMSO中)和83.5 μL的ETP(Sigma-Aldrich,编号E1383-25MG,1 mg/mL于氯仿和乙醇(1:1,V/V)中)。蒸发有机溶剂后,用1 mL的PBS (pH 7.4)使脂质膜水合。水合和挤出程序与上述相同。通过在PBS溶液(pH7.4)中透析,除去未负载的依托泊苷,其中缓冲液交换重复四次。
叶酸缀合的脂质体的制备
通过将DSPE-PEG2000-叶酸胶束后插入预先形成的脂质体中,具有轻微改良(Ishida等人1999;Yoshino等人2012),制备叶酸缀合的脂质体。简言之,将1 mg的DSPE-PEG2000-叶酸(Avanti Polar Lipids,编号880124)溶解于320 μL的DMSO中,随后与3.1 mL蒸馏水水合,产生100 μM胶束悬浮液。然后将悬浮液在10000 MWCO透析管中对1L水透析三次,以除去DMSO。此后,将40 μL胶束加入到1 mL预先形成的硫酸铵(250 mM)中的脂质体悬浮液中,并在60℃下加热1小时,以产生叶酸拴系的脂质体。通过透析除去泄漏的硫酸铵和未掺入的胶束。为了确定与脂质体缀合的叶酸含量,在缀合程序中使用裸脂质体代替负载VP的脂质体。制备后,通过用0.1%的Triton X-100裂解脂质体后,在285 nm下测定UV吸光度,并与已知浓度的叶酸的标准曲线相比较,确定叶酸的量。
脂质体的表征
用分光光度计(Cary 5000 UV-Vis-NIR, Varian Inc.)测定负载金纳米颗粒和VP、单独的VP以及单独的金纳米颗粒的脂质体的消光光谱。脂质体的尺寸分布和ζ电势用得自Malvern Instruments的Zetasizer Nano Series测定。使用透射电子显微术(TEM)记录脂质体的形态。对于TEM成像,通过将一滴悬浮液置于铜格栅上并风干制备脂质体样品,随后用一滴2%乙酸双氧铀水溶液进行负染色以增强对比度。然后使用PHILIPS CM10系统在100KV的加速电压下对经空气干燥的样品成像。用Olympus Megaview G10照相机和iTEM软件捕获图像。为了确定脂质体内部负载的寡核苷酸和Dox的包封效率,将Triton X-100 (0.1%,Sigma-Aldrich,编号T8787-50ML)加入到原样制备的脂质体溶液中,导致荧光寡核苷酸和Dox的释放。在Fluorolog-Tau-3系统(Jobin Yvon-Horiba,US)上记录FAM荧光(Ex/Em:494nm/520 nm)和Dox荧光(Ex/Em 485/590 nm),并分别与相应的寡核苷酸和Dox标准曲线相比较。
用光和X射线外部触发的单线态氧产生测试
对于光照,使用365nm的LED照射样品。将16 μL的SOSG (0.5 mM,Thermo FisherScientific Inc,编号S36002)与3 mL脂质体悬浮液混合,然后将混合物置于比色杯中,随后在365nm的LED下照射(2.5 mW/cm2,照射10分钟)。在照射后,使用荧光分光光度计记录488 nm激发时在525 nm的SOSG荧光。对于X射线辐射,使用线性加速器(6 MeV LINAC,Elekta AB, Sweden)向样品递送不同剂量(1 Gy、2 Gy和4 Gy)。将每孔中具有200 μL脂质体悬浮液和2 μL的SOSG (0.5 mM)的96孔板暴露于X射线辐射。使用来自前和后定向辐射场的6 MeV的X射线光子进行样品的照射。照射后,用微板读数器(PHERAstar FS system, BMGLABTECH, Germany)记录SOSG荧光。
用光和X射线照射的钙黄绿素释放测定
通过使用用10 mM Tris/HCl平衡的Pall Nanosep®离心装置(Sigma-Aldrich),将负载钙黄绿素的脂质体与游离钙黄绿素分子分离。然后分别通过光照和电离辐射活化脂质体。除了省略SOSG之外,实验过程与本文所述的相同。该释放诱导了先前包含在脂质体中的钙黄绿素的释放和随后的稀释,导致钙黄绿素荧光的增加。在485 nm激发时,在510 nm记录钙黄绿素荧光信号。如下计算在不同照射时间点或X射线剂量的钙黄绿素释放百分比(R c (%)):
其中Ft和F0分别表示在不同照射时间点和没有照射下的钙黄绿素的荧光强度。Fmax是指在通过加入0.1%的Triton X-100破坏脂质体后钙黄绿素的总荧光强度。对于X射线辐射,Fd是在各种辐射剂量d下钙黄绿素的荧光强度。
LipoDox的血清稳定性
在含有不同浓度(0%、10%、25%和50%)胎牛血清(FBS)的PBS (pH 7.4)中稀释200 μL的LipoDox。将所有样品在37℃下针对PBS (pH 7.4)透析48小时。在各个时间点(0h、2h、4h、18h、24h和48h),取PBS的等分试样用于释放的Dox的荧光表征。通过用0.1%的Triton X-100破坏脂质体,测定总Dox荧光。通过使用与应用于钙黄绿素释放测定的公式相同的公式,计算在各个时间点的Dox释放百分比。在pH触发的药物释放研究中,将200 μL负载Dox的聚乙二醇化脂质体悬浮液与pH分别调节至7.4 (对照)、6.0和5.0的PBS (含有10%的FBS)一起孵育,随后进行上述相同的透析程序和荧光测定。
细胞制备和细胞的电离辐射处理
大鼠PC12细胞、人结肠腺癌细胞(HCT116)和正常人结肠上皮细胞(CCD 841 CoN)购自American Type Culture Collection (Rockville, MD)。将PC12细胞在Dulbecco改良的Eagle培养基(DMEM)中培养;将HCT116细胞在McCoy's 5A (改良)培养基中培养;将CCD 841CoN细胞在Eagle's Minimum Essential Medium (EMEM)中培养。所有培养基都补充有10%胎牛血清和1%抗生素-抗真菌药。将瓶维持在具有5% CO2加湿空气的37℃培养箱中。用胰蛋白酶分离细胞,并以适当的稀释度转移到用于细胞存活率测定的96孔板或用于细胞成像的玻璃底培养皿中。对于X射线辐射实验,通过使用与上述相同的加速器辐射细胞。
脂质体的细胞摄取的成像和定量分析
将PC12细胞(3×104/mL)附着于玻璃底培养皿,并在37℃下孵育24小时。除去培养基后,在补充10%的FBS的培养基中,用脂质体悬浮液(25 μM)将细胞孵育1小时、4小时和10小时。然后用PBS (1×,PH 7.4)洗涤细胞三次,以除去游离的脂质体。为了评价脂质体纳米颗粒的摄取,将细胞用2.5%多聚甲醛在室温下固定10分钟,用PBS (1×,PH 7.4)洗涤两次,并在成像前在室温下用Hoechst 33342 (5 μg/ml)染色10分钟。使用Leica SP2共焦激光扫描显微系统对细胞成像。405 nm的紫色激光和496 nm的氩激光分别用于激发包埋在脂质体内部的VP和FAM标记的寡核苷酸。如上所述,也进行FA靶向脂质体在HCT116细胞和CCD 841CoN细胞中的摄取活性的成像。为了定量分析,采用荧光标记的DOTAP (Avanti PolarLipids,编号810890P)代替标准DOTAP,以制备荧光脂质体。将PC12细胞(1×104/mL)在培养皿中在37℃下培养24小时。除去旧的培养基后,向培养皿中加入1 mL含有10 μL荧光标记的脂质体(0.5 mg/mL)的新鲜培养基,并将细胞在37℃下再孵育4小时。孵育后,用新鲜培养基洗涤细胞三次,以除去游离的脂质体,用胰蛋白酶从培养皿中分离,并用细胞计数器(得自Thermo Scientific的Countess II FL自动细胞计数器)计数。随后将100μL的NaOH (1M)和100μL的Triton X-100 (1% v/v)加入到800μL细胞悬浮液中。在恒定摇动下于室温下裂解细胞2小时。细胞裂解后,在Fluorolog-Tau-3系统上记录荧光(Ex/Em:460/535 nm),并与游离荧光DOTAP溶液的标准曲线相比较。每个细胞的脂质体数量的详细计算描述如下。
PAC1R的间接免疫荧光染色
PC12细胞用2.5%多聚甲醛固定10分钟,并用0.1%的Triton X-100在室温下透化另外10分钟,随后用5%牛血清白蛋白封闭30分钟。然后将细胞与山羊抗PAC1R一抗(1:50稀释,Santa Cruz Biotechnology,编号sc-15964)一起室温孵育90分钟,并和与FITC缀合的驴抗山羊IgG二抗(1:100稀释,Santa Cruz Biotechnology,编号sc-2024)在室温下孵育30分钟。
X射线辐射后LipoDox对HCT116细胞的细胞毒性测定
使用MTS测试评价X射线触发的LipoDox的体外抗肿瘤作用。处理前,HCT116细胞(2x104/mL)在96孔板中在含有10%的FBS的培养基中生长24小时。除去旧培养基后,将细胞与在含有10%的FBS的培养基中稀释的一系列LipoDox样品一起孵育4小时。孵育后,除去旧培养基,向细胞中加入新鲜培养基,随后用4Gy的X射线辐射。根据制造商的说明书,通过MTS测试(Promega Co., WI, USA,编号G3582)确定X射线触发的LipoDox在HCT116细胞中在各个时间点(0小时、2小时、4小时和24小时)的细胞毒性,并与未经任何处理的对照细胞进行比较。然后将细胞存活率计算为未经处理的对照样品的吸光度的百分比。后者被设定为100%。为了比较的目的,在相同的实验条件下也评价了单独用LipoDox处理的细胞的存活率。
脂质体、LipoDox和X射线辐射的毒性
PC12、HCT116和CCD 841 CoN细胞(1-4×104/mL)分别在96孔板中在含有10%的FBS的培养基中生长24小时。对于脂质体和LipoDox处理实验,PC12细胞和CCD 841 CoN细胞分别与不同的脂质体和LipoDox样品孵育4小时,随后在新鲜培养基中再孵育24小时。对于X射线暴露实验,所有三种类型的细胞用4 Gy辐射,随后在新鲜培养基中再孵育24小时和48小时。通过使用与上述相同的方法评价细胞存活率。对于纯DNA分子以及DNA和维替泊芬的混合物的X射线处理,分别将50 μL反义寡核苷酸溶液(10 μg/mL)和50 μL混合物溶液(10 μg/mL的DNA和32 μg/mL维替泊芬)暴露于不同剂量(1、2和4 Gy)的X射线辐射。处理后,在1.2%琼脂糖凝胶中在Tris-乙酸盐-EDTA (TAE)缓冲液中在95V下进行凝胶电泳45分钟。用SYBR SafeDNA Gel Stain (Thermo Fisher)对凝胶染色,并在UV光下使用Bio-Rad成像系统照相。
通过X射线触发的药物释放的体内抗肿瘤功效
所有程序都是在Macquarie University Animal Ethics Committee的批准下(动物伦理学批准号2017/001)进行的。将6-7周龄BALB/c nu/nu雌性小鼠(The Animal ResourcesCentre, Perth, Australia)皮下注射悬浮于无FBS的McCoy's 5A (改良)培养基中的5×106个HCT116细胞至胁腹。每两天用卡尺测量肿瘤,并通过使用下式计算体积(V):
其中L和W是肿瘤的大直径和短直径。
当肿瘤体积达到约100 mm3时,将小鼠随机分成4组(每组n= 4)用于不同的处理:A组通过瘤内注射PBS (20 μL)处理;B组通过瘤内注射脂质体悬浮液(20 μL,10mg/kg)处理;C组用X射线辐射(4 Gy,单次)处理;以及D组通过瘤内注射脂质体悬浮液(20μL,10mg/kg)和X射线辐射(4 Gy,单次)处理。然后将小鼠再维持2周。每隔一天测量体重和肿瘤体积。两周后,处死小鼠并取出肿瘤,照相并用10%中性缓冲福尔马林固定,用于组织学分析。
结果
脂质体的表征
图2(a)说明含有金纳米颗粒和VP的脂质体的典型TEM图像。由于与脂质相比金属金的电子密度更高,因此容易观察到金纳米颗粒簇。通过动态光散射确定脂质体的平均尺寸为约165 nm,ζ电势为37.3±4 mV (图2(b))。图2(c)显示不同脂质体样品的吸收光谱,其中观察到来自金纳米颗粒和VP二者的特征吸收峰。估计负载在脂质体内部的寡核苷酸和Dox的包封效率,发现分别为约37.5%和44%。
通过使用两种外部刺激(光和X射线)的单线态氧产生测试
单线态氧的产生是不饱和脂质氧化的一个因素,导致脂质体结构的破坏。通过使用SOSG并监测在488 nm激发时荧光强度的增强来证实1O2产生。1O2与SOSG反应以产生内过氧化物,该内过氧化物在488 nm激发下在525 nm处具有强荧光信号,而在没有1O2存在时具有弱荧光。已知金纳米颗粒和VP分子在525 nm的激发波长下都不发荧光。在这些情况下,在525 nm处测定的荧光强度主要与由VP分子产生的单线态氧的量有关。SOSG荧光强度增强分别作为光照时间和X射线剂量的函数绘制于图3。图3(a)显示负载金纳米颗粒和VP的脂质体比其它样品产生更多的单线态氧,照射10分钟后增加约102%。计算该样品(负载金纳米颗粒和VP的脂质体)的单线态氧量子产率(SOQY)为0.75±0.18,表明与仅负载VP的脂质体相比,增强因子为1.42。下面解释该计算的细节。从VP产生1O2的增强归因于由金纳米颗粒诱导的电磁场的近场增强。类似地,在X射线辐射实验中,在负载金纳米颗粒和VP的脂质体中也观察到1O2产生的增强,但程度较小。如图3(b)所示,在相同的实验条件下,掺杂有金纳米颗粒和VP分子的脂质体产生最大量的1O2,在用4 Gy的X射线辐射下增加的百分比为约79%,而仅含有金纳米颗粒的脂质体和仅含有VP的样品产生有限量的1O2,增加的百分比分别为约48%和40%。在4 Gy的X射线辐射下,从负载VP和金纳米颗粒的脂质体产生的单线态氧的数量计算为7250/单个脂质体。下面提供计算。
在金纳米颗粒存在下观察到的X射线诱导的单线态氧产生的增强可以通过以下机理解释。金是一种与X射线强烈相互作用的重金属元素,当用这种射线照射时,其导致生物组织中的能量沉积显著增加。因此,金纳米颗粒是公知的能够放大肿瘤组织中的辐射剂量的辐射敏化剂。此外,金纳米颗粒可以选择性地散射和(或)吸收高能X射线辐射,导致从X射线到光敏剂的增强的能量传递。由于这些机制的贡献,与金纳米颗粒紧密接近的VP分子能够比VP本身更强烈地与电离辐射相互作用,导致增强的1O2产生。
在两种外部刺激下的钙黄绿素释放测定
使用两种刺激方式已经证实了从脂质体内部包埋的VP产生1O2,通过使用钙黄绿素释放测定评价脂质体内容物的释放,其基于荧光自猝灭原理。图4显示分别在UV照射和X射线暴露下从不同的脂质体样品中释放的钙黄绿素的比例。从掺杂有金纳米颗粒和VP的脂质体中释放的钙黄绿素的量分别在光照10分钟后达到44%的最大值(图4(a))和在用4 Gy的X射线辐射后达到19%的最大值(图4(b))。然而,在对照(仅掺杂有VP的脂质体)中观察到较低的渗漏,在相同的实验条件下仅有31%和13%的钙黄绿素被释放。与1O2产生的这些结果相似,发现表明在UV照射和X射线辐射二者下,与仅含VP分子的样品相比,在脂质体内部引入金纳米颗粒有助于增加包埋的钙黄绿素的释放。
脂质体纳米颗粒的细胞摄取
为了研究脂质体的细胞摄取,PC12细胞用脂质体处理1小时、4小时和10小时。与处理1小时的细胞相比,在4小时孵育后观察到来自VP的更高的红色荧光信号。确定与PC12细胞一起孵育4小时后,脂质体的细胞摄取的详细特征。此外,在4小时孵育后也清楚地观察到来自FAM标记的寡核苷酸的绿色荧光(图12)。与脂质体一起孵育10小时后,细胞被大的红色簇包围,表明大量负载VP的脂质体被细胞内化。然而,由于非特异性结合,在其它区域也观察到一些簇。因此,在本研究中选择4小时孵育时间。基于被细胞内化的荧光脂质的浓度,估计2550±89个脂质体被每个HCT116细胞内化。基于ICP-MS数据,每个脂质体的金纳米颗粒的数量估计为156±24。因此,在本研究中,由每个HCT116细胞内化的金纳米颗粒的数量估计为3.98×105。下面提供每个细胞的脂质体数量和每个脂质体的金纳米颗粒数量的详细计算。
叶酸缀合的脂质体的细胞摄取活性
叶酸受体(FR)在许多类型的癌细胞中显著表达,而其在大多数正常组织中的表达通常低。叶酸(FA)对FR具有非常高的亲和力,即使在与其它纳米材料缀合后对其结合能力的影响也最小。因此,FA可显著增强基于纳米颗粒的递送系统靶向癌细胞的能力。在该研究中,我们用叶酸修饰脂质体表面,并基于样品中叶酸和脂质体的总量确定每个脂质体中叶酸分子的平均数量,估计其为约480。为了评价叶酸靶向的脂质体对肿瘤细胞的靶向特异性,将结肠直肠癌HCT116细胞对脂质体的摄取活性与正常人结肠上皮CCD 841细胞的摄取进行比较。用叶酸缀合的脂质体纳米颗粒处理的癌细胞在1小时孵育后清楚地显示细胞质中来自VP的红色信号。相反,在相同的实验条件下,显示CCD 841 CoN细胞的脂质体摄取水平相当低。这些结果表明FA诱导了与在HCT116细胞表面上表达的叶酸受体的特异性结合,导致与正常CCD 841细胞相比,靶向脂质体具有高得多的内化率。
在365 nm波长照射后,从负载VP和金纳米颗粒的脂质体确定单线态氧量子产率
单线态氧量子产率(φ)是光敏剂(PS)分子吸收的光子数量与产生的单线态氧数量的比率。参考方法是计算φ的最常用方法(Lin,H等人,2013)。PS的单线态氧量子产率(φ PS )可使用以下等式(Clement等人,2016a),基于具有已知量子产率(φ REF )的参比PS计算:
其中r PS 和r REF 是荧光检测探针与分别由PS和参比PS产生的单线态氧的反应速率。T PS 和T REF 表示在照射波长下PS和参比PS的透射率。
在这种情况下,通过采用作为参比PS的单独VP的φ (0.53±0.06),在365 nm下测定负载VP和金纳米颗粒的脂质体的单线态氧量子产率φ。图8(a)显示对于只负载VP的脂质体以及负载VP和金纳米颗粒的脂质体,在525nm处SOSG强度作为UV照射时间的函数的变化。这些纳米复合材料的吸收光谱示于图8(b)中。基于其吸收光谱,从单独的VP和负载VP和金纳米颗粒的脂质体的吸光度计算365 nm处的透射率值。用等式(1)和从图8获得的反应速率和吸光度值,本工作中获得的负载VP和金纳米颗粒的脂质体的单线态氧量子产率φ估计为0.75±0.18。这个结果表明,与只负载VP的脂质体相比,负载VP和金纳米颗粒的脂质体的量子产率值增强到1.42倍。这种增强暂时归因于存在于负载金的脂质体中的金纳米颗粒周围的电场增强。
在X射线辐射下来自负载VP和金纳米颗粒的脂质体的单线态氧的定量
为了定量在特定剂量的X射线辐射下,由负载VP和金纳米颗粒的脂质体产生的单线态氧的数量,采用与先前出版物(Clement等人,2016b)相似的方式,建立由X射线辐射产生的单线态氧分子的数量与SOSG荧光的强度之间的关系。
首先,用下列等式计算仅负载VP的脂质体吸收的UV光子数量(N uv (t))作为时间的函数:
其中P是在样品表面上检测到的光功率,E是365 nm光子的能量,且t是照射时间。F是吸收因子,并且从样品的吸收光谱计算。该N uv (t)相对于时间作图,如图9(a)所示。从上述VP的已知单线态氧量子产率φ和由图9(a)的N uv (t),计算对应于吸收的每个UV光子产生的单线态氧数量。如果将该数量与图8(a)中的SOSG强度进行比较,则获得了转换因子,其给出了SOSG信号相对于所产生的单线态氧数量的校准。
图10(a)显示SOSG强度作为应用于负载VP和金纳米颗粒的脂质体的X射线剂量的函数。使用上述估计的转换因子,计算对应于每个X射线剂量产生的单线态氧数量。在这种情况下,对于4 Gy,由负载VP和金纳米颗粒的脂质体产生的单线态氧数量为约2.9×1018。通过除以该样品中脂质体的数量,由每个脂质体产生的单线态氧数量估计为约7250。在这种情况下,由于在暴露于X射线辐射之前产生的内过氧化物的存在,考虑到SOSG显示一些背景荧光的事实。
每个细胞的脂质体数量的计算
首先按照以下等式(Güven等2009)计算每个脂质体中脂质分子的数量:
其中d是脂质体的直径,h表示对于脂质制剂计算为4.7 nm的脂质体双层的厚度(Small,1984),并且a表示平均脂质头基面积,其值根据a = a 1 N 1 + a 2 N 2 + a 3 N 3 + ···计算,其中N是每种脂质组分的摩尔分数,并且在本研究中,对于DOTAP,a为70Å (Koltover等人1999),而对于DOPC,a为72.4Å (Kučerka等人2006)。
通过使用以下等式来估计已知脂质浓度的脂质体的数量:
其中[脂质]是脂质浓度,N A 是阿伏加德罗数(6.023×1023 mol/L),并且N tot 是每个脂质体的脂质总数。
基于等式(3)和(4)获得每个细胞的脂质体数量。
每个脂质体的金纳米颗粒的数量的估计
基于ICP-MS分析和以下等式计算脂质体样品中金原子的总数(N atom ):
其中[Au 3+]是Au (III)的浓度,V代表样品体积,M表示金的原子量,并且N A 是阿伏加德罗数(6.023×1023 mol/L)。
每个金纳米颗粒的金原子的平均数(U)也通过使用以下等式(Chithrani等人,2006)计算:
其中D是指金纳米颗粒的直径,并且α是晶胞的边界,其值为4.0786 Å。因此,脂质体样品中金纳米颗粒的数量(N gold )基于以下等式计算:
最后,根据以下等式估计每个脂质体的金纳米颗粒的数量(N):
聚乙二醇化脂质体的血清和pH稳定性研究
对于血清稳定性研究,从具有和没有PEG修饰的脂质体释放的Dox的累积百分比示于图11(a)和11(b)。在48小时孵育期间,不同量的Dox从常规脂质体中释放,当在含有10%和50%的FBS的PBS中孵育时,48小时时总量大于30%和50% (图11(a))。然而,图11(b)所示的Dox释放概况表明,与没有聚乙二醇化的脂质体相比,聚乙二醇化的脂质体中释放速率大大降低。在含有10%和50%的FBS的PBS中孵育,在48小时时,脂质体仍然保留了超过其初始药物含量的90%和80%,表明脂质体表面上的PEG链将有助于改善其在血液循环中的稳定性。考虑到pH降低是肿瘤组织的主要特征,并且它可能影响药物从脂质体的释放,我们还评价了由不同pH值触发的Dox释放。这些聚乙二醇化的脂质体在不同的缓冲液pH值(7.4、6.0和5.0)下显示相似的Dox释放概况。即使在pH5.0下孵育48小时,释放的Dox的总量也小于10% (图11(c))。这些发现表明,在本研究中制备的脂质体制剂不受降低的pH值显著影响,在对肿瘤部位应用光或X射线之前,使脂质体在肿瘤微环境中的稳定性最大化。
X射线触发的体外基因沉默和化疗
X射线辐射下的PAC1R基因沉默
脂质体负载反义寡核苷酸,以通过将脂质体递送到PC12细胞并施加4 Gy的X射线辐射来进行PAC1R基因敲减。使用共焦显微术观察在各个时间点的剩余PAC1R荧光。为了比较,使用相同的成像条件,将用脂质体单独处理但不触发的细胞也成像。如图5(a)所示,在X射线暴露后24小时清楚地观察到细胞样品中荧光的降低,表明从脂质体释放的反义寡核苷酸有效地敲减PAC1R基因表达。对于用脂质体单独处理的细胞,在处理后24小时也观察到PAC1R荧光信号降低,但与用X射线辐射处理的细胞相比,该降低较不显著。基于细胞荧光图像定量分析在不同时间点的PAC1R抑制。在处理后4小时,在用X射线处理的细胞中观察到PAC1R水平降低20%,而在用脂质体处理但省略X射线辐射的细胞中在相同时间点几乎观察不到PAC1R水平的变化。自X射线暴露24小时后,PAC1R的密度降低约45%,而未暴露于X射线但接受具有反义寡核苷酸的脂质体的细胞中PAC1R的水平仅降低30% (图5(b)和图5(c))。
在X射线辐射后LipoDox在HCT116细胞中的细胞毒性
除了通过使用X射线触发的脂质体证明基因沉默外,还研究了在HCT116细胞中负载不同量的Dox的类似脂质体的体外细胞杀伤作用。图6中呈现的一系列药物稀释测定揭示,在1.6 μM包封在脂质体内并由X射线辐射触发的Dox下,实现50%的细胞杀伤(IC50)。然而,单独的LipoDox,没有X射线触发,但具有1.6 μM的相同Dox浓度,仅杀死约10%的癌细胞。这说明,并非出乎意料地,与仅LipoDox相比,使用X射线辐射时LipoDox杀死细胞的功效更高。MTS测定在X射线处理后0小时、4小时和24小时没有揭示细胞存活率的任何显著变化,而在处理后2小时观察到细胞存活率的增加。本文所述的X射线触发的LipoDox处理的结果表明,X射线触发的化疗和放疗与相同X射线的组合似乎产生协同作用,并且其产生癌细胞杀伤的改善的功效。应当提及的是,化疗和放疗都可能导致心脏毒性的发展,其发病率与包括抗肿瘤药物类型在内的不同因素有关。因此,我们评价了另一种化疗药物ETP与X射线辐射组合的细胞杀伤作用。与Dox相比,ETP引起相对较低的心脏毒性发病率。如图6c所示,与单独的LipoETP相比,在4 Gy的X射线辐射后24小时观察到LipoETP在HCT116细胞中更高的细胞毒性。
脂质体纳米颗粒、LipoDox和X射线暴露的毒性测定
评价掺杂有金纳米颗粒和VP的脂质体的毒性。与对照组相比,在我们的研究中,用至多50 μM (高于用于基因和药物递送的那些)的脂质体浓度处理的PC12细胞的存活率中没有观察到显著变化(图7(a))。在该研究中设计的脂质体配制的Dox还应该对没有X射线触发的正常细胞具有最小的毒性作用。为了验证这一点,我们通过改变Dox浓度来检查LipoDox对CCD 841 CoN细胞的毒性。如图7(b)所示,我们在与脂质体配制的Dox样品(Dox浓度:3 μg/ml和2 μg/ml)孵育后24小时没有观察到细胞存活率的显著降低(至多14%细胞死亡),表明在体外条件下,我们的具有这两种Dox浓度的LipoDox样品可能不影响CCD 841 CoN细胞的存活率。
众所周知,X射线辐射导致的水分子的辐解作用会通过产生毒性自由基而损伤DNA分子。尽管细胞修复了大多数损伤,但它们有时留下小的错修复区域,导致DNA突变,并可能导致包括癌症在内的健康问题。记住X射线辐射对遗传物质和细胞具有潜在的副作用,我们特别地通过用不同剂量的X射线照射寡核苷酸和细胞来检查X射线对基因和细胞的存活率的细胞毒性。
对于细胞实验,MTS测试未揭示PC12细胞、HCT116细胞和CCD 841 CoN细胞在X射线暴露后24小时和48小时的存活率明显降低(图7(c))。关于X射线对基因的作用,与对照相比,DNA凝胶电泳没有显示出在X射线辐射后明显的DNA条带分散,表明这种剂量的X射线辐射没有对DNA分子造成明显的损伤(图7(d))。
另外,通过用X射线照射寡核苷酸和VP的混合物溶液,发明人还检查了单线态氧对遗传物质的作用。如图7(d)所示,与对照相比,没有观察到明显的寡核苷酸损伤。单线态氧是负责PDT技术中涉及的光生物活性的主要细胞毒性剂。它可以通过与许多生物分子反应而损伤细胞,所述生物分子包括氨基酸、核酸和具有双键的不饱和脂肪酸以及含硫氨基酸。幸运的是,单线态氧的短寿命阻止其行进较大的距离,因此其主要引起定位在产生其的光敏剂处的损伤。在该研究中,由负载在脂质双层中的VP产生的单线态氧主要使不饱和脂质去稳定,并因此诱导药物/基因释放。此外,单线态氧的寿命在与脂质反应后也将显著降低。因此,单线态氧对寡核苷酸的不利作用将显著最小化。
X射线触发的脂质体在体内的治疗作用的评价
为了确定X射线触发的脂质体在体内的效力,我们检测了它们在携带HCT 1116细胞的异种移植小鼠模型中控制肿瘤生长的能力。在用不同条件处理的小鼠上的肿瘤大小示于图13(a)中。在整个期间(处理后两周),经PBS处理的、经脂质体处理的和经X射线处理的肿瘤分别3.0倍、2.9倍和3.4倍增加,表明这些处理不能延迟肿瘤进展。相反,在用X射线触发的脂质体处理的组中,肿瘤大小在相同的时间过程中逐渐缩小,与PBS对照组相比,肿瘤体积减小74%。对不同处理后小鼠的肿瘤大小也拍照并示于图13(b),与PBS对照、单独的X射线辐射和单独的脂质体相比,用X射线触发的脂质体处理的小鼠生长更慢。这些发现表明,与其它单一处理相比,组合处理可以显著抑制肿瘤生长,实现更好的处理结果。此外,在用X射线触发的脂质体处理后14天期间没有观察到死亡,并且与对照相比没有观察到经处理的小鼠的体重减轻,表明这种组合技术在目前条件下对小鼠是无毒的(图13(c))。
讨论
在本研究中,采用X射线辐射作为新的外部脂质体触发方式来激活脂质体基因/药物递送系统。通过将光敏剂、VP和金纳米颗粒包封在脂质体双层中,设计X射线可触发的脂质体。当这些脂质体暴露于X射线时,由于金纳米颗粒与入射x射线之间的相互作用,实现从VP增强的1O2产生。该1O2氧化不饱和脂质并使膜去稳定,允许从脂质体释放包埋的货物。证明这种新的释放策略具有体外基因敲减的能力,并在X射线辐射后,通过释放两种货物,即针对PAC1R基因的反义寡核苷酸和抗肿瘤药物(Dox),增强癌细胞杀伤功效。在体内实验中,X射线触发的脂质体被证明比其它单一处理条件更有效地控制结肠直肠肿瘤生长。尽管广泛认为临床上用于诊断和治疗某些医学病症的X射线和其它形式的电离辐射导致细胞水平的DNA突变,从而导致健康问题,但是与光相比,X射线可以更容易地穿透组织和身体,一旦X射线触发的脂质体到达它们的靶,就激活基因/药物释放。这一特征将为从触发的基因疗法和化疗直到目前受制于照射光(通常在UV和可见光区)的有限穿透深度的增强PDT的生物医学研究和临床医学打开许多新的机会。另外,这里描述的策略已经被设计为与未来的临床转化相容。本研究中所用的材料和方法,如VP、脂质、Dox和X射线,在临床上用于治疗肿瘤。尽管在本研究中使用的金纳米颗粒还没有被管理机构批准,但是它们的尺寸与肾清除的要求相容。这样,如果不被消除,长期的纳米颗粒毒性可能最小化。此外,当应用于靶向疗法,特别是用于肿瘤治疗时,易于用合适的连接基,例如脂质-聚乙二醇(PEG)将靶向配体与脂质体表面缀合将是额外的优点。
本领域技术人员将理解,在不背离如广泛描述的本发明的精神或范围的情况下,可以对如具体实施方案中所示的本发明进行许多变化和/或修改。因此,本发明的实施方案在所有方面都被认为是说明性的而非限制性的。
参考文献
Claims (20)
1.用于递送活性剂的脂质体系统,所述系统包含:
形成脂质体的脂质组分;
与所述脂质组分缔合的去稳定剂,所述去稳定剂能够形成反应性氧物质,以氧化不饱和脂质并使脂质体膜去稳定;以及
在所述脂质体中的活性剂;
其中所述活性剂通过暴露于高能电磁辐射可从脂质体释放。
2.根据权利要求1所述的脂质体系统,其中脂质组分是天然存在的脂质,包括磷脂和胆固醇。
3.根据权利要求2所述的脂质体系统,其中所述脂质组分包含1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)和1,2-二-(9Z-十八碳烯酰基)-3-三甲基铵-丙烷(DOTAP),或1,2-二油酰基-sn-甘油-3-磷酸乙醇胺(DOPE)和N-[1-(2,3-二油基氧基)丙基]-N,N,N-三甲基氯化铵(DOTMA)。
4.根据权利要求1-3中任一项所述的脂质体系统,其中所述去稳定剂为纳米颗粒、光敏剂或纳米颗粒和光敏剂。
5.根据权利要求4所述的脂质体系统,其中所述纳米颗粒为金属纳米颗粒或无机纳米颗粒。
6.根据权利要求5所述的脂质体系统,其中所述金属纳米颗粒选自金、银和铋,并且所述无机纳米颗粒为氟化铈(CeF3)。
7.根据权利要求4-6中任一项所述的脂质体系统,其中所述光敏剂选自维替泊芬、玫瑰红、氨基乙酰丙酸和光敏素。
8.根据权利要求7所述的脂质体系统,其中所述光敏剂为维替泊芬。
9.根据权利要求4所述的脂质体系统,所述系统包含金纳米颗粒和维替泊芬。
10.根据权利要求1-9中任一项所述的脂质体系统,其中所述活性剂为化疗剂、药物、医学成像剂、用于基因沉默和疗法的反义寡核苷酸和小干扰RNA (siRNA)分子、生物活性剂、抗体、抗体片段、蛋白肽或核酸。
11.根据权利要求10所述的脂质体系统,其中所述化疗剂为多柔比星、长春新碱、5-氟尿嘧啶或磷酸依托泊苷。
12.根据权利要求11所述的脂质体系统,其中所述化疗剂为多柔比星。
13.根据权利要求10所述的脂质体系统,其中所述活性剂为反义寡核苷酸。
14.根据权利要求1-13中任一项所述的脂质体系统,所述系统进一步包含引起所述脂质体摄取到受试者的靶区域或靶细胞中的材料。
15.根据权利要求14所述的脂质体系统,其中所述摄取材料选自抗原、抗体、抗体片段、肽、激素、细胞因子、叶酸、配体和受体。
16.根据权利要求1-15中任一项所述的脂质体系统,其中所述高能电磁辐射的光子能量为至少约6 MeV。
17.根据权利要求16所述的脂质体系统,其中所述高能电磁辐射为x射线辐射或γ射线辐射。
18.将活性剂给予受试者的方法,所述方法包括:
向受试者提供权利要求1-15中任一项所述的脂质体系统;以及
将所述受试者暴露于高能电磁辐射,以从所述脂质体释放所述活性剂,以治疗所述受试者。
19.根据权利要求18所述的方法,其中所述高能电磁辐射为x射线辐射或γ射线辐射。
20.根据权利要求1-15中任一项所述的脂质体系统用于将活性剂给予受试者的用途。
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