CN114209850B - 一种负载阿霉素靶向碳点的制备及应用 - Google Patents

一种负载阿霉素靶向碳点的制备及应用 Download PDF

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CN114209850B
CN114209850B CN202111548961.2A CN202111548961A CN114209850B CN 114209850 B CN114209850 B CN 114209850B CN 202111548961 A CN202111548961 A CN 202111548961A CN 114209850 B CN114209850 B CN 114209850B
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王建华
刘旖
汪少芸
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Abstract

本发明公开了一种负载阿霉素靶向碳点的制备及应用。本发明以荧光碳点为载体、阿霉素为抗肿瘤药物、多巴胺为支架、叶酸为靶向剂,构建了一种负载阿霉素靶向碳点。本发明所制得的碳点具有良好的荧光成像能力,并能有效释放阿霉素,对肿瘤细胞具有显著的杀伤作用。此外,肿瘤靶向分子聚乙二醇叶酸的应用可使本碳点靶向结合肿瘤细胞,从而有效实现其对肿瘤组织的生物荧光成像与靶向药物治疗的目的。

Description

一种负载阿霉素靶向碳点的制备及应用
技术领域
本发明属于生物医学领域,具体涉及一种负载阿霉素靶向碳点的制备及应用。
背景技术
荧光碳量子点(Carbon dots,CDs)又称碳点,通常被定义为尺寸小于10 nm、表面经过钝化并拥有明亮荧光的碳纳米粒子,是一种性能优良的碳纳米材料,具有良好的发光特性、生物相容性、易修饰性及可绿色合成等特性。碳点独特的光物理和化学性质促进了其在生物成像探针、高性能纳米传感器和多重纳米复合材料领域的发展,其优异的生物相容性和体内清除性也满足了体内应用要求。
叶酸(FA) 是叶酸受体(FR) 的理想配体。FR在多种肿瘤细胞上过度表达,包括卵巢、乳腺、肺、肾、脑、前列腺和喉咙的恶性肿瘤细胞。FR可主动结合叶酸,进而通过内吞作用使FA修饰的材料进入癌细胞。在肿瘤诊疗时,通过连接FA,并通过FA与肿瘤受体特异性结合,进而将抗肿瘤药物聚集于肿瘤部位同时降低对正常细胞的毒副作用,具有重要的临床意义。
恶性肿瘤是威胁人类健康的一大杀手,也是现代医学领域面临的巨大挑战。靶向性药物传递是一种既可提高治疗靶向性又较容易实现的治疗途径,主要依靠药物分子通过靶向特异性到达病变部位,杀灭致病病毒、修复受损组织或消除疾病症状。近年来,碳点在生物成像和药物递送方面的应用成为了研究的热点。碳点载药系统不仅可以特异性地递送药物到癌细胞部位,而且能够通过荧光成像来观察细胞状态和药物递送过程。然而,碳点载药体系对癌细胞的靶向性不高,不能有效区分癌细胞和正常细胞,特别是化疗及一些蛋白质药物具有很高的细胞毒性,在治疗的同时也对正常机体器官造成很大损伤。此外,基于碳点载药体系特异性探针的报道极少,这极大地限制了碳点在生物成像和药物递送领域的进一步研究。因此,合成一种靶向性载药碳点体系十分必要,也对癌症的治疗、 监测和药物释放过程的追踪具有重要意义。
发明内容
本发明目的是提供一种负载阿霉素靶向碳点的制备方法及其应用。本发明以碳点为载体、阿霉素为抗肿瘤药物、多巴胺为支架、叶酸为靶向剂构建了一种叶酸靶向负载阿霉素碳点,该碳点可用于肿瘤组织生物荧光成像以及肿瘤靶向药物治疗。
为实现上述目的,本发明采用如下技术方案:
一种负载阿霉素靶向碳点的制备方法,其以苹果酸作为碳源材料、尿素作为氮源合成荧光碳点作为载体,再以阿霉素为抗肿瘤药物、多巴胺为支架、叶酸为靶向剂,组装成一种叶酸靶向负载阿霉素碳点。
上述一种负载阿霉素靶向碳点的制备方法,具体包括如下步骤:
(1)将苹果酸与尿素混合溶于溶剂,将混合溶液转移至反应釜中进行高温反应,反应结束后将所得反应液过滤,滤液经透析和冻干处理,制得初始碳点(CDs);
(2)将初始碳点(CDs)溶于去离子水中,向其中加入阿霉素,随后调节混合溶液pH至7.2,室温避光搅拌过夜后,将所得反应液过滤过滤,滤液经透析和冻干处理,制得负载阿霉素碳点(CDs-DOX);
(3)将负载阿霉素碳点(CDs-DOX)溶于Tris缓冲液中,向其中加入多巴胺盐酸盐,随后调节混合溶液pH至8.5,室温下避光搅拌反应8-12 h,将所得反应液过滤,滤液经透析和冻干处理,制得载阿霉素的聚多巴胺碳点(CDs-DOX-PDA);
(4)将载阿霉素的聚多巴胺碳点(CDs-DOX-PDA)溶于弱碱性溶液, 随后依次向其中加入三(2-羧乙基)膦和巯基-聚乙二醇-叶酸(SH-PEG 1000-FA),室温避光搅拌反应3~12h后,将所得反应液过滤,滤液经透析和冻干处理,制得所述负载阿霉素靶向碳点(CDs-DOX-PDA-FA)。
进一步的,上述制备方法的步骤(1)中,所述苹果酸的终浓度为 0.07 g/mL-0.15g/mL;所述尿素的终浓度为 0.14 g/mL-0.3 g/mL;所述溶剂为超纯水、Tris缓冲液(浓度为0.05 mol/L,pH为8.5)、二甲基亚砜、N ,N-二甲基甲酰胺中的任意一种;所述高温反应温度为120~200℃,反应时间为6~10 h;所述反应釜的内衬为聚四氟乙烯。
进一步的,上述制备方法的步骤(2)中,所述CDs的终浓度为1 mg/mL;所述阿霉素的终浓度为1 mg/mL。
进一步的,上述制备方法的步骤(3)中,所述CDs-DOX的终浓度为0.25 mg/mL;所述Tris缓冲液的浓度为0.05 mol/L,pH为8.5;所述多巴胺盐酸盐的终浓度为0.125 mg/mL-1.25 mg/mL。
进一步的,上述制备方法的步骤(4)中,所述CDs-DOX-PDA的终浓度为0.5 mg/mL;所述弱碱性溶液为PBS缓冲液或Tris缓冲液中的任意一种,其pH为8.5~10,浓度为0.01-0.05 mol/L;所述三(2-羧乙基)膦的终浓度为0.05 mg/mL;所述巯基-聚乙二醇-叶酸的终浓度为0.1~5 mg/mL。
一种利用上述制备方法制得的负载阿霉素靶向碳点。
上述一种负载阿霉素靶向碳点在细胞成像中的应用。
上述一种负载阿霉素靶向碳点在制备抗肿瘤药物中的应用
本发明与原理如下:
多巴胺在负载阿霉素碳点(CDs-DOX)表面聚合形成聚多巴胺(polydopamine,PDA),可利用PDA表面官能团与肿瘤靶向剂叶酸(FA)的化学反应以及强黏附性制备所述负载阿霉素靶向碳点。载阿霉素靶向碳点在肿瘤诊疗时,通过FA与肿瘤受体特异结合,进而将抗肿瘤药物阿霉素聚集于肿瘤部位同时减少对正常细胞的毒性,不仅可以特异性地递送药物到癌细胞部位,而且能够通过荧光成像来观察细胞状态和药物递送过程。
与现有技术相比,本发明具有以下有益效果:
本发明所述一种负载阿霉素靶向碳点的制备方法具有工艺简单、绿色无污染、成本低廉的优点。同时,所制得的负载阿霉素靶向碳点具有大斯托克斯位移(Stokes shift),在细胞成像实验中表现出了良好的荧光生物成像能力,在荧光生物成像方面有巨大潜力,在抗肿瘤治疗时,本发明所得到的负载阿霉素靶向碳点能与肿瘤细胞靶向结合,并有效释放抗癌药DOX,对肿瘤细胞具有显著的杀伤作用,从而实现了其对肿瘤组织的生物荧光成像与靶向药物治疗目的。
附图说明
图1为实施例1中初始碳点的透射电子显微镜(TEM)图。
图2为实施例1中初始碳点的粒径分布图。
图3为实施例2中CDs、CDs-DOX、CDs-DOX-PDA-PEGFA的红外光谱图。
图4为实施例2中CDs、CDs-DOX、CDs-DOX-PDA-PEGFA的紫外-可见吸收光谱。
图5为实施例3中CDs-DOX-PDA-PEGFA的激发光谱图和发射光谱图。
图6为应用实施例1中DOX和CDs-DOX-PDA-PEGFA对Hela细胞的细胞毒性作用图。
图7为应用实施例2中488 nm波长激发下Hela细胞与CDs-DOX-PDA-PEGFA分别共孵育6 h和24 h的荧光成像图。
具体实施方式
下面结合具体实施例对本发明作进一步阐述,但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径得到。
实施例1
本实施例提供了一种负载阿霉素靶向碳点的制备方法,所述方法包括如下步骤:
(1)称取3 g苹果酸与6 g尿素,放入同一烧杯中,向其中加入20 mL超纯水,室温搅拌溶解后将溶液转移至反应釜中,在200℃下反应8 h,冷却至室温后取出,用0.22μm滤膜过滤,滤液用截留分子量为200 Da的透析袋在超纯水中透析2 d,最后收集透析袋中的液体,冷冻干燥后即得到初始碳点(CDs)。
(2)按1 mg:1 mL的比例将步骤(1)得到的初始碳点(CDs)溶于去离子水中,向其中加入终浓度为1 mg/mL的阿霉素,随后用浓度为0.01 mol/L,pH为7.4的PBS缓冲液调节混合溶液pH值至7.2,室温避光搅拌过夜后,用0.22μm滤膜过滤,滤液用截留分子量为200 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到负载阿霉素碳点(CDs-DOX)。
(3)称取5 mg步骤(2)得到的的负载阿霉素碳点(CDs-DOX),将其溶于20 mL 浓度为0.01 mol/L,pH为8.5的Tris缓冲液中,随后向其中加入终浓度为0.5 mg/mL的多巴胺盐酸盐(购自上海麦克林生化科技有限公司),再用0.1 mol/L NaOH溶液调节混合溶液pH至8.5,室温下避光搅拌10 h后,用0.22μm滤膜过滤,滤液用截留分子量为500 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到载阿霉素的聚多巴胺碳点(CDs-DOX-PDA)。
(4)称取10 mg步骤(3)得到的载阿霉素的聚多巴胺碳点(CDs-DOX-PDA ),将其溶于20 mL pH 8.5浓度为0.01 mol/L的Tris缓冲液中,随后向其中加入1 mg还原剂三(2-羧乙基)膦,再加入5 mg巯基-聚乙二醇-叶酸(SH-PEG1000-FA,购自上海芃硕生物科技有限公司),室温避光搅拌12 h后,用0.22μm滤膜过滤,滤液用截留分子量为500 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到所述负载阿霉素靶向碳点(CDs-DOX-PDA-FA)。
利用透射电子显微镜(TEM)对本实施例所得到的初始碳点(CDs)进行表征,结果如图1所示,由图1可以看出,CDs为分散的近球形颗粒,黑色,无明显团聚现象。
图2为本实施例所得初始碳点(CDs)的粒度分布。由图2可以看出,CDs的粒径主要集中在1.3-1.6 nm,平均粒径为1.475 nm。
实施例2
本实施例提供了一种负载阿霉素靶向碳点的制备方法,所述方法包括如下步骤:
(1)称取5 g苹果酸与10 g尿素,放入同一烧杯中,向其中加入70 mL N ,N-二甲基甲酰胺,室温搅拌溶解后将溶液转移至反应釜中,在180℃下反应8 h,冷却至室温后取出,用0.22μm滤膜过滤,滤液用截留分子量为200 Da的透析袋在超纯水中透析48 h,最后收集透析袋中的液体,冷冻干燥后即得到初始碳点(CDs)。
(2)按1 mg:1 mL的比例将步骤(1)得到的初始碳点(CDs)溶于去离子水中,向其中加入终浓度为1 mg/mL的阿霉素,随后用浓度为0.01 mol/L,pH为7.4的PBS缓冲液调节溶液pH至7.2,室温避光搅拌过夜后,用0.22μm滤膜过滤,滤液用截留分子量为200 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到负载阿霉素碳点(CDs-DOX)。
(3)称取5 mg步骤(2)得到的的负载阿霉素碳点(CDs-DOX),将其溶于20 mL浓度为0.05 mol/L,pH 8.5的Tris缓冲液中,随后向其中加入终浓度为0.5 mg/mL的多巴胺盐酸盐(购自上海麦克林生化科技有限公司),再用0.1 mol/L NaOH溶液调节混合溶液pH值至8.5,室温下避光搅拌10 h后,用0.22μm滤膜过滤,滤液用截留分子量为500 Da的透析袋透析24h,最后收集透析袋中的液体,冷冻干燥后即得到载阿霉素的聚多巴胺碳点(CDs-DOX-PDA)。
(4)称取10 mg步骤(3)得到的载阿霉素的聚多巴胺碳点(CDs-DOX-PDA),将其溶于20 mL pH 8.5浓度为0.01mol/L的PBS缓冲液中,随后向其中加入1 mg还原剂三(2-羧乙基)膦,再加入5 mg巯基-聚乙二醇-叶酸(SH-PEG1000-FA,购自上海芃硕生物科技有限公司),室温避光搅拌12 h后,用0.22μm滤膜过滤,滤液用截留分子量为500 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到所述负载阿霉素靶向碳点(CDs-DOX-PDA-FA)。
图3为本实施例所得到的CDs、CDs-DOX、CDs-DOX-PDA-PEGFA的红外光谱图。从图3中可以看出,CDs在3419 cm-1处有吸收峰,归因于O-H的伸缩振动;与CDs红外光谱相比,CDs-DOX在1286 cm-1处出现了新吸收峰,归因于DOX的C-O-C伸缩振动,这表明DOX与CDs结合成功;与CDs-DOX红外光谱相比,CDs-DOX-PDA-PEGFA在1106 cm-1处出现了一个显著的吸收峰,这归因于PEGFA叔醇结构的C-O伸缩振动。上述结果验证了CD-DOX-PDA-PEGFA的成功合成。
图4为本实施例所得到的CDs、CDs-DOX、CDs-DOX-PDA-PEGFA的紫外-可见吸收光谱。从图4中可以看出,CDs在317 nm处有吸收峰,源自于CDs表面态的跃迁,对应C=N的n-π*跃迁;DOX在488 nm处有特征峰,归因于蒽醌的特征吸收;CDs与DOX结合后,CDs-DOX的紫外-可见吸收光谱既包含488 nm处出现的明显吸收峰,又包含317 nm处出现的微弱吸收峰;PEGFA在283 nm处有吸收峰,借助PDA负载PEGFA后,虽然CDs-DOX-PDA-PEGFA在283 nm处未出现明显吸收峰,但吸光度比相同浓度单纯CDs-DOX明显要大,表明CDs-DOX-PDA-PEGFA在283 nm处吸光度是CDs-DOX和PEGFA共同贡献的,PDA成功起到连接作用。以上结果证明了CDs-DOX-PDA-PEGFA制备成功。
实施例3
本实施例提供了一种负载阿霉素靶向碳点的制备方法,所述方法包括如下步骤:
(1)称取3 g苹果酸与6 g尿素,放入同一烧杯中,向其中加入40 mL N ,N-二甲基甲酰胺,室温搅拌溶解后将溶液转移至反应釜中,在180℃下反应8 h,冷却至室温后取出,用0.22μm滤膜过滤,滤液用截留分子量为200 Da的透析袋在超纯水中透析2 d,最后收集透析袋中的液体,冷冻干燥后即得到初始碳点(CDs)。
(2)按1mg:1mL比例将步骤(1)得到的初始碳点(CDs)溶于去离子水中,向其中加入终浓度为1 mg/mL的阿霉素,随后用浓度为0.01 mol/L,pH为7.4的PBS 缓冲液调节混合溶液pH值至7.2,室温避光搅拌过夜后,用0.22μm滤膜过滤,滤液用截留分子量为200 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即到得负载阿霉素碳点(CDs-DOX)。
(3)称取5 mg步骤(2)得到的负载阿霉素碳点(CDs-DOX),将其溶于20 mL 浓度为0.05 mol/L,pH8.5的Tris缓冲液中,随后向其中加入终浓度为0.2 mg/mL的多巴胺盐酸盐,再用0.1 mol/L NaOH溶液调节混合溶液pH至8.5,室温下避光搅拌10 h后,用0.22μm滤膜过滤,滤液用截留分子量为500 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到载阿霉素的聚多巴胺碳点(CDs-DOX-PDA)。
(4)称取10 mg步骤(3)得到的载阿霉素的聚多巴胺碳点(CDs-DOX-PDA),将其溶于20 mL浓度为0.05 mol/L,pH8.5的Tris缓冲液中,随后向其中加入1 mg还原剂三(2-羧乙基)膦,再加入10 mg巯基-聚乙二醇-叶酸(SH-PEG1000-FA,购自上海芃硕生物科技有限公司),室温避光搅拌过夜后,用0.22μm滤膜过滤,滤液用截留分子量为500 Da的透析袋在超纯水中透析24 h,最后收集透析袋中的液体,冷冻干燥后即得到所述负载阿霉素靶向碳点(CDs-DOX-PDA-FA)。
图5为本实施例所得到的CDs-DOX-PDA-PEGFA的激发光谱图和发射光谱图。在365nm-700 nm范围内,测量CDs-DOX-PDA-PEGFA的发射光谱,得到最佳发射波长为454 nm。如图5所示,以最佳激发波长454 nm为固定发射波长进行激发光谱的检测,得到最佳激发波长382 nm;固定382 nm的最佳激发波长测量发射光谱,得到最佳发射波长为463 nm,计算得到CDs-DOX-PDA-PEGFA的斯托克斯位移为81 nm。而CDs-DOX-PDA-PEGFA的斯托克斯位移较大,可以降低成像过程中的背景干扰以及减少组织的损伤,并具有更高的穿透性和信噪比,有利于荧光生物成像。
应用实施例1
将本发明实施例3所得到的负载阿霉素靶向碳点(CDs-DOX-PDA-FA)应用于抑制人宫颈癌Hela细胞生长,具体步骤如下:
将l×105 cells/mL的HeLa细胞按每孔100 μL接种于96孔板中,在37℃、5% CO2、饱和湿度的培养箱中恒温下孵育24 h(细胞培养液配方为:DMEM培养基+10%体积分数胎牛血清+终浓度100 μg/mL青霉素+终浓度100 μg/mL链霉素);孵育结束后,小心吸取并弃去96孔板中上清,将实施例3所得到的负载阿霉素靶向碳点(CDs-DOX-PDA-FA)用DMEM培养基分别配制成不同浓度(20、10、5、2.5、1.25 μg/mL)的分散液,按每孔10 μL加入到96孔板细胞中,37℃、5% CO2、饱和湿度的培养箱中培养24 h后,每孔中加入10 μL MTT (5.0 mg/mL)继续培养4 h,弃上清,随后每孔加入100 μL DMSO溶液,最后在550 nm波长下用酶标仪检测吸光度。将DOX用DMEM培养基配制成不同浓度(20、10、5、2.5、1.25 μg/mL)的分散液,作为对照。
图6为DOX和实施例3所得到的CDs-DOX-PDA-PEGFA对Hela细胞的细胞毒性实验结果。从图中可以看出,CDs-DOX-PDA-PEGFA对HeLa细胞具有显著的杀伤作用,且杀伤效果与同浓度的DOX相近。当浓度为1.25 μg/mL时,CDs-DOX-PDA-PEGFA表现出明显的杀伤作用,与同浓度DOX作用相近,HeLa细胞成活率显著降低。以上结果表明CDs-DOX-PDA-PEGFA能有效释放DOX,从而达到肿瘤治疗目的。
应用实施例2
将本发明实施例3所得到的负载阿霉素靶向碳点(CDs-DOX-PDA-FA)应用于人宫颈癌HeLa细胞成像,具体步骤如下:
将l×105 cells/mL的HeLa细胞按每孔100 μL接种于96孔板中,在37℃、5%CO2、饱和湿度的培养箱中恒温下孵育24 h(细胞培养液配方为:DMEM培养基+10%体积分数胎牛血清+终浓度100 μg/mL青霉素+终浓度100 μg/mL链霉素),随后向细胞培养液中加入终浓度为200 μg/mL的负载阿霉素靶向碳点(CDs-DOX-PDA-PEGFA)。将碳点与细胞在37℃下分别共孵育6 h和24h后,将HeLa细胞用4%质量分数的多聚甲醛固定30 min,再用浓度为0.01 mol/L,pH为7.4的PBS缓冲液清洗,最后用激发波长为488nm的光源激发,进行共聚焦成像,在荧光倒置显微镜下观察CDs-DOX-PDA-FA进入细胞的情况。将DOX以及实施例3所得到的初始碳点(CDs)分别用DMEM培养基配制成不同浓度(20、10、5、2.5、1.25 μg/mL)的分散液,作为对照。
结果如图7所示,在488 nm波长激发下,在蓝色通道只有CDs组HeLa细胞发出荧光;在绿色和红色通道,CDs、DOX、CDs-DOX-PDA-PEGFA组HeLa细胞在经过6 h孵育后均有明亮荧光发出,与无叶酸修饰的CDs、DOX组相比,CDs-DOX-PDA-PEGFA一组HeLa细胞显示出更强的荧光信号。这些结果表明制备的CDs-DOX-PDA-PEGFA具有明显的癌细胞靶向性。与孵育6 h相比,在孵育24 h后CDs、DOX组的荧光增强,CDs-DOX-PDA-PEGFA组荧光仍未猝灭。上述结果表明CDs-DOX-PDA-PEGFA可被Hela细胞有效内化摄取,并在细胞内具有良好的荧光生物成像能力。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。

Claims (3)

1.一种用于抑制人宫颈癌细胞生长的负载阿霉素靶向碳点的制备方法,其特征在于:其以苹果酸作为碳源材料、尿素作为氮源合成荧光碳点作为载体,再以阿霉素为抗肿瘤药物、多巴胺为支架、叶酸为靶向剂,组装成一种叶酸靶向负载阿霉素碳点;所述制备方法包括如下步骤:
1)将苹果酸与尿素混合溶于溶剂中,将混合溶液转移至反应釜中进行高温反应,反应结束后将所得反应液过滤,滤液经透析和冻干处理,制得初始碳点CDs;
2)将初始碳点CDs溶于去离子水中,向其中加入阿霉素,随后调节混合溶液pH至7.2,室温避光搅拌反应过夜,将所得反应液过滤,滤液经透析和冻干处理,制得负载阿霉素碳点CDs-DOX;
3)将负载阿霉素碳点CDs-DOX溶于Tris缓冲液中,向其中加入多巴胺盐酸盐,随后调节混合溶液pH至8.5,室温下避光搅拌反应8~12 h,将所得反应液过滤,滤液经透析和冻干处理,制得载阿霉素的聚多巴胺碳点CDs-DOX-PDA;
4)将载阿霉素的聚多巴胺碳点CDs-DOX-PDA溶于弱碱性溶液中,随后依次向其中加入三(2-羧乙基)膦和巯基-聚乙二醇-叶酸SH-PEG1000-FA,室温避光搅拌反应3~12h后,将所得反应液过滤,滤液经透析和冻干处理,即制得所述负载阿霉素靶向碳点CDs-DOX-PDA-FA;
步骤1)中,所述溶剂为超纯水、N,N-二甲基甲酰胺、二甲基亚砜、Tris缓冲液中的任意一种;所述苹果酸的终浓度为0.07g/mL~0.15g/mL,尿素的终浓度为0.14g/mL~0.3g/mL;所述高温反应温度为120~200℃,反应时间为6 ~10 h;
步骤2)中,所述CDs的终浓度为1mg/mL,阿霉素的终浓度为1mg/mL;
步骤3)中,所述CDs-DOX的终浓度为0.25mg/mL,多巴胺盐酸盐的终浓度为0.125mg/mL~1.25mg/mL;所述Tris缓冲液的浓度为0.05mol/L,pH为8.5;
步骤4)中,所述CDs-DOX-PDA的终浓度为0.5 mg/mL;所述弱碱性溶液为PBS缓冲液或Tris缓冲液中的任意一种,其pH为8.5~10,浓度为0.01~0.05mol/L;所述三(2-羧乙基)膦的终浓度为0.05 mg/mL,巯基-聚乙二醇-叶酸的终浓度为 0.1~5mg/mL。
2.一种利用如权利要求1所述的制备方法制得的负载阿霉素靶向碳点。
3.如权利要求2所述的负载阿霉素靶向碳点在制备用于抑制人宫颈癌细胞生长的药物中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106806343A (zh) * 2017-02-17 2017-06-09 清华大学深圳研究生院 一种叶酸和聚多巴胺修饰的肿瘤靶向介孔二氧化硅纳米粒及制备方法与应用
CN108163834A (zh) * 2018-01-22 2018-06-15 华南师范大学 一种碳量子点荧光材料的合成方法
CN113736456A (zh) * 2021-09-10 2021-12-03 四川大学 一种基于叶酸偶联碳量子点的肿瘤靶向纳米探针及其制备方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106806343A (zh) * 2017-02-17 2017-06-09 清华大学深圳研究生院 一种叶酸和聚多巴胺修饰的肿瘤靶向介孔二氧化硅纳米粒及制备方法与应用
CN108163834A (zh) * 2018-01-22 2018-06-15 华南师范大学 一种碳量子点荧光材料的合成方法
CN113736456A (zh) * 2021-09-10 2021-12-03 四川大学 一种基于叶酸偶联碳量子点的肿瘤靶向纳米探针及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
A portable synthesis of water-soluble carbon dots for highly sensitive and selective detection of chlorogenic acid based on inner filter effect;Huang Yang等;《Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy》;20170801;第189卷;第139-146页 *

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