CN109106954B - 光热治疗与基因治疗协同增效作用的碳纳米管复合载体及其制备方法与应用 - Google Patents

光热治疗与基因治疗协同增效作用的碳纳米管复合载体及其制备方法与应用 Download PDF

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CN109106954B
CN109106954B CN201811314938.5A CN201811314938A CN109106954B CN 109106954 B CN109106954 B CN 109106954B CN 201811314938 A CN201811314938 A CN 201811314938A CN 109106954 B CN109106954 B CN 109106954B
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张树彪
赵轶男
曹英楠
周泉
刘一宁
吴琼
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Dalian Minzu University
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Abstract

本发明提供了一种光热治疗和基因治疗协同增效作用的碳纳米管复合载体及其制备方法与应用。由载体部分和基因组成，所述载体部分包括碳纳米管、肽脂质和/或助剂。采用自组装的方法将修饰剂固定于碳纳米管，制得可携带和转运基因的复合载体。该复合载体克服了单纯碳纳米管水溶性差、生物相容性低、携带和转运基因效率不佳的问题，同时具备更高的光热转换性能和基因转运效率，并降低了碳纳米管的细胞毒性，缓解了碳纳米管局部蓄积的问题。利用光热治疗与基因治疗协同增效的作用，解决了肿瘤治疗中单一治疗方法疗效差的问题。

Description

光热治疗与基因治疗协同增效作用的碳纳米管复合载体及其 制备方法与应用
技术领域
本发明属于肿瘤治疗领域新型药物制剂领域，具体涉及一种可进行光热和基因联合治疗的碳纳米管复合载体的制备和应用。
背景技术
随着科学技术的发展，出现一些治疗恶性肿瘤的新方法和新技术，其中光热治疗技术和基因治疗技术受到研究者广泛关注。光热治疗(Photothermal therapy，PTT)是用具有较高光热转换效率的材料，将其注射入生物体内，利用靶向识别技术聚集在肿瘤组织附近，并在外部近红外光的照射下将光能转化为热能，提高肿瘤部位的温度，利用局部过热引起的热杀伤作用及其继发效应来治疗肿瘤。其机制为，高温将细胞质中Bid蛋白剪切，形成活性分子tBid并进入线粒体，导致细胞色素C从线粒体释放，放大凋亡信号，激活Caspase9和Caspase3蛋白，从而诱导肿瘤细胞凋亡。这种方法能够通过近红外光靶向照射肿瘤部位杀伤肿瘤细胞而避免损伤正常组织细胞，因而成为一种比较有效的治疗肿瘤的新手段。但是，在治疗过程中热能不仅会杀伤肿瘤组织，也会波及周围正常的组织细胞，并且在治疗过后伴随的炎症问题也是该治疗手段的缺陷之一。
光热治疗的关键是光热材料的性能且能否在病灶富集，随着研究的不断深入多种光热转换材料应运而生，碳纳米管作为一种较为可靠的光热材料在光热治疗领域始终占有一席之地。碳纳米管是一种管状的碳分子，碳纳米管的管径小的不到一纳米，粗的有几十纳米，管长在几纳米在半米之间。碳纳米管碳原子的SP2杂化和碳—碳σ键的排列使其在医学上有着广泛应用，它能够吸附多种药物并较为容易的穿透细胞膜，从而达到携带和转运药物提升药效的目的，但在实际应用中仍会出现体内团聚与蓄积的问题。
基因治疗是将外缘基因导入患者细胞纠正缺陷基因而达到治疗的目的，基因治疗的关键之一是基因转运载体的构建，非病毒基因载体以其安全低毒的特点受到人们的广泛关注。肽脂质是一种头部带有正电荷的表面活性剂，由于头部亲水尾部疏水的结构一直被用来通过自组装的方式制备成阳离子脂质体，能够有效地运载药物和基因。但肽脂质载体的运载效率低一直是人们亟需解决的问题。无论是单纯的基因治疗还是单纯的光热治疗其治疗效果都很难达到优秀的治疗效果，多种治疗手段同时进行是目前人们研究的趋势。
发明内容
为提高现有转染试剂转运基因的能力和增强碳纳米管的生物相容性，本发明提供一种用于癌症肿瘤治疗的新型碳纳米管复合载体。
本发明的发明构思是：由肽脂质等修饰剂通过对碳纳米管进行非共价修饰，使其具有负载基因的能力进行基因治疗，在特定性能上得到提升，同时利用碳纳米管的光热转换效率进行光热联合治疗，实现基因与光热联合治疗的效果。
本发明的目的是通过以下技术方案实现的：一种碳纳米管复合基因载体，由载体部分和基因组成，所述载体部分包括碳纳米管、肽脂质和/或助剂。载体部分与基因的N/P质量比为0.5:1—8:1。优选的N/P比为2:1—3:1。所述肽脂质与助剂用量的摩尔比为1:0.2—1:10。所述的肽脂质与碳纳米管的质量比为1:0.1—1:100，优选的比为1:0.5—1:5。
所述助剂为地高辛(Digoxin)、塞来考昔(celecoxib)、槲皮素、白藜芦醇、蔗糖酯中的一种或一种以上。
所述基因为质粒DNA，小干扰RNA或信使mRNA。
所述的碳纳米管为多壁碳纳米管、单壁碳纳米管、羧基化的多壁碳纳米管、羧基化的单壁碳纳米管、氨基化的多壁碳纳米管、氨基化的单壁碳纳米管、羟基化的多壁碳纳米管、羟基化的单壁碳纳米管中的一种或一种以上。
本发明利用肽脂质所带有的正电性和碳纳米管表面的化学键易与基因结合，可以增强与基因的结合效率；地高辛、塞来考昔、槲皮素、白藜芦醇可以增强载体对肿瘤的治疗。本发明还提供了碳纳米管复合载体的制备方法，首先利用碳管表面的π键和疏水作用将肽脂质、地高辛(Digoxin)、塞来考昔(celecoxib)、槲皮素、白藜芦醇、蔗糖酯充分吸附在碳纳米管表面形成碳管水性分散液，再将DNA或RNA稀释液缓慢滴加到碳管分散液中，利用静电作用制备成碳纳米管复合载体。
上述复合基因载体的制备方法具体为：将肽脂质与助剂地高辛(Digoxin)、塞来考昔(celecoxib)、槲皮素、白藜芦醇、蔗糖酯中一种或一种以上溶解于有机试剂中，利用薄膜分散法将肽脂质和助剂均匀分散于容器表面，真空干燥12-36h，优选时间为24h，然后将碳纳米管水分散液缓慢滴入，同时50-60℃下超声振荡，再利用高速离心的方法去除未结合和结合较少的碳纳米管，取上清，按载体部分与基因的N/P质量比为0.5:1—8:1将载体部分与基因稀释液混合，通过静电复合的方法制备出复合基因载体可用于光热治疗和基因治疗的制剂。
所述的有机溶剂为甲醇、氯仿中的一种或一种以上。
本发明另一个目的请求保护碳纳米管复合基因载体用于制备肿瘤光热和基因联合治疗药物或制剂中的应用。
本发明提供的复合载体可注射到生物体内，利用载体部分的被动靶向富集在病灶周围，通过近红外光的照射使病灶周围的温度升高，利用肿瘤细胞的热敏感性杀伤肿瘤细胞，携带的地高辛、塞来考昔、槲皮素、白藜芦醇、蔗糖酯会阻止肿瘤细胞受到热损伤后启动的自我修复机制，配合光热治疗。本发明提供的碳纳米管复合基因载体体外实验表明具有一定压缩、携带DNA、RNA的能力，复合载体富集在肿瘤细胞周围后，可以穿越细胞膜进入细胞质中释放携带的DNA或RNA进行基因治疗，实现光热与基因联合治疗的作用。该载体系统的构建为肿瘤的治疗提供了新思路，具有极大的临床应用价值。该复合载体克服了单纯碳纳米管水溶性差、生物相容性低、携带和转运基因效率不佳的问题，同时具备更高的光热转换性能和基因转运效率，并降低了碳纳米管的细胞毒性，缓解了碳纳米管局部蓄积的问题。利用光热治疗与基因治疗协同增效的作用，解决了肿瘤治疗中单一治疗方法疗效差的问题。
附图说明
图1为制备的碳纳米管复合基因载体的透射电镜图。
图2为制备的碳纳米管复合基因载体的扫描电镜图。
图3为碳纳米管复合基因载体与携带的RNA电泳延滞实验图，其中第1泳道均为Marker(λDNA-EcoR I+Hind III Markers)第2泳道为裸RNA，第3至第9泳道分别为复合载体/RNA复合质量比1:1、2:1、4:1、6:1、8:1、16:1)。
图4为碳纳米管复合基因载体诱导hela细胞凋亡实验结果图。
图5为碳纳米管复合基因载体对hela细胞存活率影响实验图。
图6为碳纳米管复合基因载体对A549细胞荧光素酶干扰实验结果图。
图7为碳纳米管复合基因载体在808nm的激光下光热转换图。
具体实施方式
下面通过附图和具体实施例详述本发明，但不限制本发明的保护范围。如无特殊说明，本发明所采用的实验方法均为常规方法，所用实验器材、材料、试剂等均可从化学公司购买。
实施例1
取肽脂质、地高辛、白藜芦醇各1mg溶解于氯仿中，利用氮吹仪使三种试剂均匀分布在容器表面，真空干燥24h。取直径1-2nm，长度在1-3μm之间的单壁碳纳米管1mg超声分散在1ml超纯水中，将碳纳米管水分散液缓慢滴入涂有三种试剂的容器中，在55℃下不断超声振荡30min，将形成的悬浊液8000r/min离心30min弃掉沉淀，取上层溶液。按照N/P为3/1的计量缓慢加入到RNA水溶液中静置20min得到的复合载体在4℃储存。
实施例2
取肽脂质1mg、槲皮素0.4mg、蔗糖酯0.8mg溶解于氯仿中，利用氮吹仪使三种试剂均匀分布在容器表面，真空干燥36h。取直径0.5-1nm，长度在400-800nm之间的单壁碳纳米管1mg超声分散在1ml超纯水中，将碳纳米管水分散液缓慢滴入涂有三种试剂的容器中，在55℃下不断超声振荡30min，将形成的悬浊液10000r/min离心30min弃掉沉淀，取上层溶液。按照N/P为2/1的计量缓慢加入到RNA水溶液中静置20min得到的复合载体在4℃储存。
实施例3
取肽脂质1mg溶解于在1ml超纯水中，取1.2mg直径3-5nm，长度在400-800nm之间的酸化后的多壁碳纳米管超声分散在肽脂质溶液中，在55℃下不断超声振荡30min，将形成的悬浊液10000r/min离心30min弃掉沉淀，取上层溶液。按照N/P为4/1的计量缓慢加入到RNA水溶液中静置20min得到的复合载体在4℃储存。
实施例4
取肽脂质1mg、蔗糖酯0.2mg溶解于氯仿中，利用氮吹仪使两种试剂均匀分布在容器表面，真空干燥36h。取直径0.5-1nm，长度在400-800nm之间的单壁碳纳米管1mg超声分散在1ml超纯水中，将碳纳米管水分散液缓慢滴入涂有三种试剂的容器中，在55℃下不断超声振荡30min，将形成的悬浊液10000r/min离心30min弃掉沉淀，取上层溶液。按照N/P为2/1的计量缓慢加入到RNA水溶液中静置20min得到的复合载体在4℃储存。
实施例5
取肽脂质1mg、槲皮素0.8mg、蔗糖酯1mg溶解于氯仿中，利用氮吹仪使三种试剂均匀分布在容器表面，真空干燥36h。取直径2-5nm，长度在400-600nm之间的多壁碳纳米管1mg超声分散在1ml超纯水中，将碳纳米管水分散液缓慢滴入涂有三种试剂的容器中，在55℃下不断超声振荡30min，将形成的悬浊液10000r/min离心30min弃掉沉淀，取上层溶液。按照N/P为4/1的计量缓慢加入到RNA水溶液中静置20min得到的复合载体在4℃储存。
实施例6
本发明选择宫颈癌细胞Hela为研究对象，将Hela细胞按照1×107/孔的密度接种于12孔板中，在10％浓度血清的DMEM培养液中培养24h，更换成无血清低糖的培养基，将得到的复合载体稀释在无血清低糖的培养基中，转染4-5h，移去带有复合载体的培养基，用PBS将附着在细胞表秒的复合载体清洗干净，更换成10％血清4.5g/l葡萄糖的DMEM培养液，利用808nm波长1w/cm2功率的激光器垂直照射细胞培养板产生光热转换效应，使细胞温度维持在40-43℃之间5-10min，利用高温诱导癌细胞凋亡，在诱导后24-48小时检测，细胞凋亡效率在50％左右，而在使用剂量下致死的细胞不足1％。
实施例7
将载体部分与带有荧光标记的FAM-siRNA复合，导入Hela细胞，在转染4h后利用流式细胞仪在细胞中检测出本发明碳纳米管复合基因载体，载体细胞摄入效率在80％以上。
实施例8
本发明选择肺腺癌细胞A549为研究对象，将载体部分与带有可以干扰荧光素酶表达的Luc-siRNA复合，导入A549细胞，在转染4h后利用酶标仪检测细胞中荧光素酶的表达含量，如图6所示，实验证明复合载体具有一定干扰能力。
实施例9
活细胞的线粒体中含有琉拍酸脱氢酶，而死细胞中没有，MTT能够被活细胞中的琉拍酸脱氢酶还原生成水不溶性的蓝紫色甲攒，本发明利用这一性质，用MTT对Hela细胞分别染色，用酶标仪检测，可间接反映活细胞相对于死细胞的数量，发现在正常使用计量下细胞存活率较高，说明本发明复合载体具有较低的毒性。
实施例10
取本发明复合载体经静脉注射到荷瘤小鼠体内，按10mg/kg的剂量每两天注射一次，利用808nm波长3w/cm2功率的激光设备每24h照射一次，共持续7天，每次照射5min，照射时使病变部位温度升高，利用热红外成像观察病变部位的形貌，与此同时复合载体中的小干扰RNA进行基因治疗达到治疗和辅助成像的目的。每日照射后检测小鼠肿瘤体积和体重，治疗完成后颈椎脱臼处死小鼠，取出肿瘤和其他脏器做病理方面的研究。
以上所述，仅为本发明创造较佳的具体实施方式，但本发明创造的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明创造披露的技术范围内，根据本发明创造的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明创造的保护范围之内。

Claims (2)

1.一种碳纳米管复合基因载体，其特征在于，由载体部分和基因组成，所述载体部分包括碳纳米管、肽脂质和助剂；载体部分与基因的N/P质量比为2:1—3:1；所述肽脂质与助剂用量的摩尔比为1:0.2—1:10；所述的肽脂质与碳纳米管的质量比为1:0.5—1:5；所述助剂为地高辛、塞来考昔、槲皮素、白藜芦醇、蔗糖酯中的一种以上；所述基因为小干扰RNA或mRNA；所述的碳纳米管为多壁碳纳米管、单壁碳纳米管、羧基化的多壁碳纳米管、羧基化的单壁碳纳米管、氨基化的多壁碳纳米管、氨基化的单壁碳纳米管、羟基化的多壁碳纳米管、羟基化的单壁碳纳米管中的一种以上；用于制备肿瘤光热和基因联合治疗药物或制剂。
2.一种如权利要求1所述的碳纳米管复合基因载体的制备方法，其特征在于，将肽脂质与助剂地高辛、塞来考昔、槲皮素、白藜芦醇、蔗糖酯中一种以上溶解于有机溶剂中，利用薄膜分散法将肽脂质和助剂均匀分散于容器表面，真空干燥12-36h，然后将碳纳米管水分散液缓慢滴入，同时50-60℃下超声振荡，再利用高速离心的方法去除未结合和结合较少的碳纳米管，取上清，按载体部分与基因的N/P质量比为2:1—3:1将载体部分与基因稀释液混合，通过静电复合的方法制备出复合基因载体；所述的有机溶剂为甲醇、氯仿中的一种以上。
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