CN112972678A - 一种基于五角纳米金载体、制备方法及用途 - Google Patents
一种基于五角纳米金载体、制备方法及用途 Download PDFInfo
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Abstract
本发明涉及生物医药领域,特别是涉及一种基于五角纳米金载体、制备方法及用途,一种纳米载体,所述纳米载体包括五角纳米金颗粒和包覆在所述五角纳米金颗粒表面的碳酸钙层,一种纳米探针,所述纳米探针包括所述纳米载体和光敏剂,所述光敏剂与所述纳米载体的质量比为1~2:10,所述光敏剂被包覆在所述碳酸钙层内,一种诊疗剂,所述诊疗剂包括所述纳米载体、光敏剂和抗癌药,所述光敏剂与所述纳米载体的质量比为1~2:10,所述抗癌药与所述纳米载体的质量比为1~2:10,所述光敏剂和所述抗癌药均被包覆在所述碳酸钙层内。首次合成出了基于五角纳米金颗粒的多功能诊疗剂,克服了现有技术中纳米颗粒生物医药使用中的种种缺点,具有高度的临床转化价值。
Description
技术领域
本发明涉及生物医药领域,特别是涉及一种基于五角纳米金载体、制备方法及用途。
背景技术
前列腺癌是男性最常见的恶性肿瘤之一,近年来发病率呈现逐年上升的趋势。常规的去雄激素治疗对于早期前列腺癌能够取得一定的治疗效果,但是疾病一旦发展为去势抵抗性前列腺癌,去雄治疗就难以发挥作用。研究已经证实去势抵抗性前列腺癌与肿瘤的疾病进展、肿瘤转移以及放化疗治疗抵抗等密切相关。常规的放疗、化疗等治疗方式对去势抵抗性前列腺癌无法取得令人满意的效果,因此聚焦于探索针对去势抵抗性前列腺癌新的药物或诊疗方式仍然显得十分必要。随着纳米材料技术的应用越来越广泛,研究者已经关注于将纳米材料应用于肿瘤的诊疗过程中。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供一种基于五角纳米金载体、制备方法及用途,用于解决现有技术中的问题。
本发明第一方面,提供一种纳米载体,所述纳米载体包括五角纳米金颗粒和包覆在所述五角纳米金颗粒表面的碳酸钙层。
本发明第二方面,提供一种制备第一方面所述纳米载体的方法,其特征在于,所述方法至少包括以下步骤:
1)将西曲氯铵、碘化钾、氯金酸和抗坏血酸在pH为7.4~9.4溶液中混合,反应后取沉淀和再纯化,获得五角纳米金颗粒。
2)将所述五角纳米金颗粒、CaCl2和Na2CO3混合,反应后取沉淀,获得沉淀物。
3)将所述沉淀物与活化透明质酸混合,再通过离心取沉淀,获得纳米载体。
本发明第三方面,提供一种纳米探针,所述纳米探针包括第一方面所述纳米载体和光敏剂,所述光敏剂与所述纳米载体的质量比为1~2:10,所述光敏剂被包覆在所述碳酸钙层内。
本发明第四方面,提供一种诊疗剂,所述诊疗剂包括第一方面所述纳米载体、光敏剂和抗癌药,所述光敏剂与所述纳米载体的质量比为1~2:10,所述抗癌药与所述纳米载体的质量比为1~2:10,所述光敏剂和所述抗癌药均被包覆在所述碳酸钙层内。
本发明第五方面,提供制备第四方面所述诊疗剂的方法,所述制备诊疗剂方法至少包括以下步骤:
1)将所述五角纳米金颗粒、光敏剂、抗癌药CaCl2和Na2CO3混合,反应完成后通过离心去上清获得初级诊疗剂;
2)将所述初级诊疗剂与活化透明质酸混合,再通过离心取沉淀,获得诊疗剂。
本发明的第六方面,提供第五方面所述一种诊疗剂用于制备诊断和治疗肿瘤产品用途。
如上所述,本发明的一种诊疗剂,具有以下有益效果:
本发明首次合成了五角纳米金,本发明的通过化学合成碳酸钙的方法巧妙地将光敏剂药物IR820以及化疗药物多烯紫杉醇包覆入内,使得诊疗剂可以在肿瘤偏酸的微环境中响应性降解表面碳酸钙层从而靶向性释放抗肿瘤药物。并且我们利用活化的透明质酸对碳酸钙表面进行生物有机修饰,使其增加肿瘤靶向性,促进诊疗剂被肿瘤细胞内吞摄取,极大的提升了多功能诊疗剂的诊疗能力。
附图说明
图1为本发明制备的五角纳米金的透射电镜照片;
图2为本发明制备的五角纳米金的高分辨透射电镜照片;
图3为本发明制备的纳米载体的元素构成照片;
图4为本发明制备的五角纳米金的光热转换效率实验结果;
图5为本发明制备的诊疗剂的细胞指数评估;
图6为本发明制备的诊疗剂的细胞摄取TEM照片;
图7为本发明制备的诊疗剂的细胞凋亡;
图8为本发明制备的诊疗剂的体内荧光照片;
图9为本发明制备的诊疗剂的HE和TUNEL照片。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
请参阅图1至图9。需要说明的是,本实施例中所提供的图示仅以示意方式说明本发明的基本构想,遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制,其实际实施时各组件的型态、数量及比例可为一种随意的改变,且其组件布局型态也可能更为复杂。
本发明第一方面,提供一种纳米载体,所述纳米载体包括五角纳米金颗粒和包覆在所述五角纳米金颗粒表面的碳酸钙层。本发明中的五角纳米金颗粒具有良好的光热效果和稳定性,光热效率可达59.43%。
本发明中位于五角纳米金颗粒表面的所述碳酸钙层可用包覆载药剂,例如光敏剂和抗癌药等,所述碳酸钙层在偏酸的微环境中响应性降解,释放包覆的药剂,可实现靶向性释放。
在一实施例中,所述碳酸钙层的厚度为1~5nm。
实验人员根据药剂负载量需要选择碳酸钙层的厚度,例如可以是1~2nm、2~3nm、3~4nm或4~5nm。
优选的,所述碳酸钙层的厚度为5nm,此厚度下碳酸钙层可包覆更多的药剂。
在一实施例中,所述五角纳米金颗粒的粒径为50~65nm。
实验人员根据需求选择所述五角纳米金颗粒的粒径,例如可以是50~60nm或60~65nm。
在一实施例中,所述碳酸钙层表面附着有透明质酸。此种设计有利于促进纳米载体被肿瘤细胞内吞摄取,增加了肿瘤靶向性。
本发明第二方面,提供一种制备第一方面所述纳米载体的方法,所述方法至少包括以下步骤:
1)将西曲氯铵、碘化钾、氯金酸和抗坏血酸在pH为7.4~9.4溶液中混合,反应后取沉淀和再纯化,获得五角纳米金颗粒。
2)将所述五角纳米金颗粒、CaCl2和Na2CO3混合,反应后取沉淀,获得沉淀物。
3)将所述沉淀物与活化透明质酸混合,再通过离心取沉淀,获得纳米载体。
在步骤1)中,称取一定量的西曲氯铵、碘化钾、氯金酸和抗坏血酸加入到水溶液内,再加入碱性试剂调节在pH为7.4~9.4,静置过程中西曲氯铵、碘化钾、氯金酸和抗坏血酸进行反应1~2h,待反应完成后,通过高速离心并利用透析膜进行纯化可获取金五角纳米颗粒。
在步骤2)中,将步骤1)获得的所述五角纳米金颗粒、CaCl2和Na2CO3进行搅拌混合,反应一段时间,反应完成后通过离心机进行高速离心并去除上层清液,获得沉淀物。
在步骤3)中,将步骤2)中获得的所述沉淀物与经过活化的透明质酸进行混合,并搅拌一段时间,通过离心机进行高速离心取离心后的沉淀,获得纳米载体。
在一实施例中,在步骤1)中,所述西曲氯铵、所述碘化钾、所述氯金酸和所述抗坏血酸在溶液中的摩尔比为15~40:0.08~0.2:0.2~0.35:0.48~0.8。
在一实施例中,步骤1)中,采用氢氧化钠调节所述溶液的pH。
在一实施例中,步骤1)中,所述离心的转速为10000-12000rpm。
在一实施例中,步骤1)中,采用透析膜进行透析。
在本实施例中,所述透析膜的型号:MWCO:3500-5000,所述透析膜为市场常规产品,具体可从spectrumlabs公司购买。
在一实施例中,步骤2)中,所述五角纳米金、所述CaCl2和所述Na2CO的质量比为10:2~3:2~3。
实验人员根据碳酸钙层的厚度需求选择所述五角纳米金、所述CaCl2和所述Na2CO的质量比,例如可以是10:2~2.5:2~2.5、10:2.5~3:2.5~3、10:2:2或10:3:3。
在一实施例中,步骤2)中,所述反应时间为12~36h。
实验人员需要选择所述反应时间,例如可以是12~24h或24~36h。
在一实施例中,所述沉淀物与所述活化透明质酸的质量比为1~3:3.3~4.5。
实验人员需要选择所述沉淀物与所述活化透明质酸的质量比,例如可以是1~2:3.3~4.5、2~3:3.3~4.5、1~3:3.3~4或1~3:4~4.5。
在一实施例中,所述沉淀物与所述活化透明质酸混合的时间为18~24h。
实验人员需要选择所述沉淀物与所述活化透明质酸混合的时间,例如可以是18~20h、20~22h或22~24h。
在一实施例中,所述活化透明质酸由透明质酸、1-乙基-(3-二甲基氨基丙基)碳二亚胺和羟基琥珀酰亚胺在磷酸盐缓冲液中混合获得。
优先地,所述透明质酸、1-乙基-(3-二甲基氨基丙基)碳二亚胺和羟基琥珀酰亚胺在磷酸的质量比为0.8~1:1.5~2:1~1.5。
实验人员根据需要选择所述透明质酸、1-乙基-(3-二甲基氨基丙基)碳二亚胺和羟基琥珀酰亚胺在磷酸的质量比,例如可以是0.8~0.9:1.5~2:1~1.5或0.9~1:1.5~2:1~1.5。
本发明第三方面,提供一种纳米探针,所述纳米探针包括第一方面所述纳米载体和光敏剂,所述光敏剂与所述纳米载体的质量比为1~2:10,所述光敏剂被包覆在所述碳酸钙层内。
在本实施例中,实验人员可以选择所述光敏剂与所述纳米载体的质量比,例如可以是1~1.5:10或1.5~2:10。
在本发明中,所述光敏剂被包覆在所述纳米载体的碳酸钙层内,所述纳米载体可定向于肿瘤部位释放光敏剂,提高了光敏剂的使用效率,减少光敏剂对正常组织伤害。
优选地,所所述光敏剂为IR820的发射波长在近红外区,生物自身荧光干扰较少。
本发明第四方面,提供一种诊疗剂,所述诊疗剂包括第一方面所述纳米载体、光敏剂和抗癌药,所述光敏剂与所述纳米载体的质量比为1~2:10,所述抗癌药与所述纳米载体的质量比为1~2:10,所述光敏剂和所述抗癌药均被包覆在所述碳酸钙层内。
在本实施例中,实验人员可以选择所述光敏剂与所述纳米载体的质量比,例如可以是1~1.5:10或1.5~2:10,还可以选择所述抗癌药与所述纳米载体的质量比为,例如可以是1~1.5:10或1.5~2:10。
本发明含有五角纳米金颗粒因其无毒性、较好的生物相容性以及高效光热转化能力等特性,以五角纳米金颗粒为基础,通过荷载上抗肿瘤药物和光敏剂可以达到成像和治疗的目的。考虑到偏酸性是肿瘤微环境一个重要的特征,通过化学合成碳酸钙的方法巧妙地将光敏剂以及抗癌药包覆入内,使得诊疗剂可以在肿瘤偏酸的微环境中响应性降解表面碳酸钙层从而靶向性释放光敏剂和抗癌药。并且我们利用活化的透明质酸对碳酸钙层进行生物有机修饰,使其增加肿瘤靶向性,促进诊疗剂被肿瘤细胞内吞摄取,极大的提升了诊疗剂的诊疗能力。
优选地,所述抗癌药为多烯紫杉醇。
本发明第五方面,提供制备第四方面所述诊疗剂的方法,所述制备诊疗剂方法至少包括以下步骤:
1)将所述五角纳米金、光敏剂、抗癌药CaCl2和Na2CO3混合,反应完成后通过离心去上清获得初级诊疗剂;
2)将所述初级诊疗剂与活化透明质酸混合,再通过离心取沉淀,获得诊疗剂。
在步骤1)中,分别称取一定量的五角纳米金颗粒、光敏剂、抗癌药CaCl2和Na2CO3混合进行混合,并搅拌12~36h,待反应完成后,使用离心机用过离心去除上清液获得初级诊疗剂。
在步骤2)中,步骤1)中获得的所述初级诊疗剂与经过活化的透明质酸进行混合,并搅拌一段时间,通过离心机进行高速离心,取离心后的沉淀,获得诊疗剂。
本发明的第六方面,提供第五方面所述一种诊疗剂用于制备诊断和治疗肿瘤产品用途。
本发明中的诊疗剂,诊疗剂的基底材料为角纳米金颗粒可用于肿瘤的光热治疗,通过化学合成碳酸钙的方法将光敏剂以及抗癌药包覆在碳酸钙层内,碳酸钙层在肿瘤环境下发生降解,释放空腔内负载的光敏剂和抗癌药,可实现靶向性释放,实现了化疗,光热和荧光成像联合治疗肿瘤。
实施例1
(1)将1.5ml的西曲氯铵(CTAC,试剂浓度为0.1M),用9ml的超纯水稀释,再加入80-100μl的碘化钾(KI,试剂浓度为0.01)以及80μl氯金酸(HAuCl4,浓度为25mM),轻度摇匀。接着加入20μl的氢氧化钠(NaOH,浓度为0.1M),充分摇匀。再加入80-100μl的抗坏血酸(AA,浓度为60mM),充分摇匀。最后再次滴加10μl的氢氧化钠(NaOH,浓度为0.1M),充分摇匀后静置1-2h。待反应完成后,通过高速离心(10000-12000rpm)并利用透析膜纯化可获取五角纳米金颗粒。
(2)利用上述步骤获得的五角纳米颗粒,加入含有光敏剂药物IR820(500mg/ml)的CaCl2(10mg/ml)溶液中,充分搅拌18-24h;将上述溶液通过高速离心后获取的沉淀物加入含有化疗药物DTX(多烯紫杉醇)(500mg/ml)的Na2CO3(10mg/ml)溶液中,再次充分搅拌24h;待反应完成后,通过高速离心获取沉淀物。
(3)将800mg的透明质酸加入40ml磷酸盐缓冲液中,充分混匀搅拌,再加入1.5g的1-乙基-(3-二甲基氨基丙基)碳二亚胺以及1g的羟基琥珀酰亚胺进行活化,充分混匀搅拌20-40min,获得活化的透明质酸溶液。
(4)将步骤3中获取的沉淀物用步骤4中获取的活化透明质酸溶液充分摇匀18-24h,再通过高速离心(10000-12000rpm)后可获得诊疗剂。
实施例2
(1)将2.0ml的西曲氯铵(CTAC,试剂浓度为0.2M),用12ml的超纯水稀释,再加入100μl的碘化钾(KI,试剂浓度为0.02M)以及100μl氯金酸(HAuCl4,浓度为35mM),轻度摇匀。接着加入40μl的氢氧化钠(NaOH,浓度为0.2M),充分摇匀。再加入100μl的抗坏血酸(AA,浓度为80mM),充分摇匀。最后再次滴加20μl的氢氧化钠(NaOH,浓度为0.2M),充分摇匀后静置1-2h。待反应完成后,通过高速离心(10000-12000rpm)并利用透析膜纯化可获取五角纳米金颗粒。
(2)利用上述步骤获得的五角纳米金颗粒,加入含有光敏剂药物IR820(800mg/ml)的CaCl2(20mg/ml)溶液中,充分搅拌18-24h;将上述溶液通过高速离心后获取的沉淀物加入含有化疗药物DTX(多烯紫杉醇)(800mg/ml)的Na2CO3(20mg/ml)溶液中,再次充分搅拌24h;待反应完成后,通过高速离心获取沉淀物。
(3)将1000mg的透明质酸加入60ml磷酸盐缓冲液中,充分混匀搅拌,再加入2.0g的1-乙基-(3-二甲基氨基丙基)碳二亚胺以及1.5g的羟基琥珀酰亚胺进行活化,充分混匀搅拌20-40min,获得活化的透明质酸溶液。
(4)将步骤3中获取的沉淀物用步骤4中获取的活化透明质酸溶液充分摇匀18-24h,再通过高速离心(10000-12000rpm)后可获得诊疗剂。
利用型号为JEM-2100F(JEOL,Japan)的透射电镜对我们合成的多功能诊疗探针进行EDS元素能谱分析,金元素为Au,元素Ca代表被覆的碳酸钙层,元素N代表探针表面有机透明质酸层。
结果如图1至图3所示,通过透射电镜发现五角纳米金颗粒呈五角星状,发现碳酸钙层包覆在五角纳米金颗粒表面,通过EDS能谱分析,发现碳酸钙均匀地包覆在五角纳米金颗粒表面。
实施例3
(1)测量样品浓度为50μg/ml,溶剂为超纯水,加入石英比色皿中。
(2)设置808nm激光发射器功率为2W,激光光线头距离样品2cm,并放置传感器检测输出功率约为100mW。
(3)使用热红外成像仪观察样品温度,链接电脑将数据实时导入FLIR Tools软件中分析计算。
如图4所示,五角纳米金颗粒的光热性能好,光热转换效率为59.43%。
实施例4
实验方法:将PC-3细胞按照1×104个孔的密度种植于细胞培养E板上后,进行干预处理加入诊疗剂,放入细胞分析仪后,机器记录对应增殖参数生成生长曲线。利用实时细胞分析仪(RTCA分析仪)连续监测PC-3细胞的增殖情况。实验结果如图5所示,约26小时后随着碳酸钙层的降解,化疗药物DTX释放出来,抑制了PC-3细胞的增殖。
图中的金五角多功能探针为本发明的诊疗剂,下述实施例中的金五角多功能探针也为本发明的诊疗剂。
实施例5
将PC-3细胞与多功能诊疗剂共孵育过夜后,消化离心用戊二醛固定,再按照TEM检测样品制备程序制备观测。
实验结果如图6所示,TEM检测观察PC-3细胞对诊疗剂的摄取方式与能力评估。可发现PC-3细胞可以有效的以巨胞饮的形式摄取我们构建的多功能诊疗剂。
实施例6
分别将不同浓度的诊疗剂与PC-3细胞一同培养作为实验组,生理盐水与PC3细胞一同培养的实验组作为对照组,在细胞生长的环境下共同孵育24h。
实验结果如图7所示,诊疗剂与细胞共孵育24h后,近红外激光(1w/cm2)照射3min后,用凋亡试剂盒检测发现与对照组相比,诊疗剂孵育组能够显著的增加肿瘤细胞凋亡。
实施例7
成功建立PC3裸鼠荷瘤模型后,尾静脉注射生理盐水与诊疗剂(浓度为5mg/mL),注射48小时后,利用Bruker体外荧光检测系统活体观测诊疗剂在体内的分布情况。
实验结果图8所示,尾静脉注射PBS与诊疗剂,48h后利用Bruker体外荧光检测系统活体条件下观察,诊疗剂能够聚集于肿瘤部位。
实施例8
裸鼠分为两组,分别尾静脉注射诊疗剂与生理盐水。再喂食21天后对小鼠实施安乐死获取肿瘤,再进行标准H&E染色。使用显微镜记录H&E染色图像。免疫荧光染色步骤:肿瘤石蜡切片脱蜡,蛋白酶K工作液修复,滴加破膜工作液,按照TUNEL试剂盒中配置工作液孵育,BSA封闭切片,一抗孵育过夜,二抗染色,DAPI复染细胞核,封片,荧光显微镜拍摄。
实验结果图9所示,与对照组相比,诊疗剂组在受到激光照射治疗后,肿瘤组织的坏死显著增多,进一步证明了本发明制备的诊疗剂具有较好的治疗能力。TUNEL:多功能诊疗剂治疗组的凋亡坏死细胞显著多于对照组。
综上所述,本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种纳米载体,其特征在于,所述纳米载体包括五角纳米金颗粒和包覆在所述五角纳米金颗粒表面的碳酸钙层。
2.根据权利要求1所述的一种纳米载体,其特征在于:所述碳酸钙层的厚度为1~5nm。
3.根据权利要求1所述的一种纳米载体,其特征在于:所述五角纳米金颗粒的粒径为50~65nm。
4.根据权利要求1所述的一种纳米载体,其特征在于:所述碳酸钙层表面附着有透明质酸。
5.一种制备权利要求1至4任一项所述纳米载体的方法,其特征在于,所述方法至少包括以下步骤:
1)将西曲氯铵、碘化钾、氯金酸和抗坏血酸在pH为7.4~9.4溶液中混合,反应后取沉淀和再纯化,获得五角纳米金颗粒。
2)将所述五角纳米金颗粒、CaCl2和Na2CO3混合,反应后取沉淀,获得沉淀物。
3)将所述沉淀物与活化透明质酸混合,再通过离心取沉淀,获得纳米载体。
6.根据权利要求5所述的方法,其特征在于,包括以下特征中的一项或多项:
在步骤1)中,所述西曲氯铵、所述碘化钾、所述氯金酸和所述抗坏血酸在溶液中的摩尔比为15~40:0.08~0.2:0.2~0.35:0.48~0.8;
在步骤2)中,所述五角纳米金颗粒、所述CaCl2和所述Na2CO的质量比为10:2~3:2~3;
在步骤3)中,所述沉淀物与所述活化透明质酸的质量比为1~3:3.3~4.5。
7.一种纳米探针,其特征在于:所述纳米探针包括权利要求1至4任一项所述纳米载体和光敏剂,所述光敏剂与所述纳米载体的质量比为1~2:10,所述光敏剂被包覆在所述碳酸钙层内。
8.一种诊疗剂,其特征在于:所述诊疗剂包括权利要求1至4任一项所述纳米载体、光敏剂和抗癌药,所述光敏剂与所述纳米载体的质量比为1~2:10,所述抗癌药与所述纳米载体的质量比为1~2:10,所述光敏剂和所述抗癌药均被包覆在所述碳酸钙层内。
9.一种制备权利要求8所述诊疗剂的方法,其特征在于,所述制备诊疗剂方法至少包括以下步骤:
1)将所述五角纳米金颗粒、光敏剂、抗癌药、CaCl2和Na2CO3混合,反应完成后通过离心去上清获得初级诊疗剂;
2)将所述初级诊疗剂与活化透明质酸混合,再通过离心取沉淀,获得诊疗剂。
10.一种如权利要求8所述的诊疗剂用于制备诊断和治疗肿瘤产品用途。
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