CN113425860A - 砷荧光量子点及其制备方法与应用 - Google Patents
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Abstract
本发明提供了一种砷荧光量子点及其制备方法与应用，该量子点分子式为As4S4@HA QDs，其中，As4S4和HA分别为雄黄和透明质酸，该砷荧光量子点以透明质酸为稳定剂，在其表面形成稳定的HA水化膜；其制备方法为：将雄黄溶解于乙二胺中，以透明质酸为稳定剂，并加入盐酸调节pH，形成形态结构稳定的砷荧光量子点。本发明量子点制备方法简单，结构稳定，可通过自发强荧光的方式实时示踪胞内递送，并通过透明质酸配体特异性识别肿瘤细胞，实现肿瘤靶向递送，选择性杀伤肿瘤细胞，并降低潜在毒副作用，具备广阔的应用前景。
Description
技术领域
本发明涉及纳米材料和纳米生物技术领域，具体涉及一种砷荧光量子点及其制备方法与应用。
背景技术
雄黄(realgar，As4S4)是代表性的中药砷化物，具有广泛的抗癌谱，其肿瘤治疗机制主要包括：诱导肿瘤细胞凋亡、抑制肿瘤增殖、促进肿瘤细胞分化和抗肿瘤血管生成等。然而，雄黄在抗肿瘤应用中，存在诸多亟待解决的问题。雄黄是高晶格能的晶体，难溶于水和其它常见的试剂，其制剂成型具有局限性，往往通过口服途径大剂量给药，但胃肠道吸收差，生物利用度低，降低了治疗效果，长期使用潜在毒性较大，同时也是一种资源的浪费。因此，克服上述的困难，有助于拓宽雄黄的治疗范围。
纳米技术是研究结构尺寸在0.1-100nm范围内的材料的性质和应用的一种技术，目前已经广泛应用于各领域。研究表明，将难溶性、生物利用度低和强毒性的药物与纳米技术结合，制成纳米晶体药物或纳米载体药物可以改善药物的物理化学特性。纳米药物能克服生理屏障，提高生物利用度，增强药物的靶向性和疗效，降低用药剂量，减轻毒副作用，显著优于传统的药物。目前，雄黄纳米粒的制备方法则有物理法和化学法两大类，物理法包括高能球磨法和气流粉碎法等，具体方法就是利用强外力或其它优势条件对固体雄黄进行粉碎研磨，并在体系中加入不同表面活性剂如聚乙二醇、吐温-80、羧甲基纤维素钠和聚乙烯吡咯烷酮等，以提高纳米晶的分散性，但物理法制备的纳米雄黄存在粒径不均一、稳定性差等诸多问题，化学法则包括溶剂接力法、共沉淀法和化学配位法，制备的纳米雄黄粒径小、水溶性好、生物利用度更高。
常见的纳米递药系统虽然疗效显著，但难以对药物体内行为进行实时有效地监测，因此研究者常常在纳米体系中引入了荧光物质(如荧光染料、量子点等)，用以观测药物在体内分布和浓度。量子点(quantum dots，QDs)是一种能够接受激发光而产生荧光的半导体纳米晶粒，表现出独特的光电特性。量子点与荧光染料相比，具有显著优势，如发射光谱可通过调节尺寸而改变，荧光强度更高、光稳定性更好、生物相容性好，因此具有广阔的发展空间，在生命科学领域可用作荧光探针。然而，药物与荧光物质结合的纳米递药系统也同样存在缺陷，药物和荧光物质可能在体内分离，丧失了示踪功能。因此，开发成像和治疗效应整合一体化的纳米药物是大势所趋，有利于为个体化用药提供便利。
发明内容
为解决上述技术问题，本发明提出了一种砷荧光量子点及其制备方法与应用，其目的是构建一种自发荧光的砷纳米制剂，实现药物递送的可视化实时定位，并通过透明质酸对雄黄纳米粒子进行表面包裹和修饰，既能实现雄黄的肿瘤靶向递送，又能实现肿瘤的可视化高效低毒治疗。所述砷荧光量子点制备方法简单，具有简单且稳定的结构，粒子大小均匀度高度一致，有较高荧光下效率，在肿瘤诊疗一体化中具备广阔的应用前景。
为了实现上述目的，本发明首先提供了一种砷荧光量子点，所述量子点分子式为As4S4@HA QDs，其中，As4S4和HA分别为雄黄和透明质酸，所述砷荧光量子点以透明质酸为稳定剂，在其表面形成稳定的HA水化膜。
作为优选，所述砷荧光量子点中，As4S4和HA的质量浓度比为5:1～1:5。
作为优选，所述HA的质量浓度为30-40mg/mL。
作为优选，所述砷荧光量子点的粒径为1-3nm。
作为优选，所述砷荧光量子点在350-410nm激发波长下具有强荧光信号。
基于一个总的发明构思，本发明还提供了一种所述砷荧光量子点的制备方法，包括以下步骤：
S1、将雄黄溶于乙二胺中，超声、离心，取上清，配置雄黄溶液；
S2、将雄黄溶液分散于水中，加入HA水溶液，搅拌，加入HCl调节pH，搅拌，70-80℃油浴孵育48-72h，得到粗品。
S3、通过离心去除沉淀，并经葡聚糖凝胶柱层析分离得到纯化的砷荧光量子点。
作为优选，所述S1中超声时间为10-20min，10000-15000rpm离心5-10min。
作为优选，所述S2中HCl的浓度为3-5mol/L，所述PH值小于或等于8。
作为优选，所述S2中油浴温度为75℃，油浴孵育时间为72h。
基于一个总的发明构思，本发明还提供了上述砷荧光量子点在制备治疗肿瘤药物中的应用和在制备肿瘤示踪药物中的应用。本发明可用于构建一种自发荧光的砷纳米制剂，以实现药物递送的可视化实时定位，并通过表面的透明质酸修饰，实现雄黄的肿瘤靶向递送，实现肿瘤的可视化高效低毒治疗。
本发明的上述方案有如下的有益效果：
1、本发明提供了一种砷荧光量子点，该砷荧光量子点以透明质酸(HA)为稳定剂，让量子点长在HA上，在其表面形成稳定的HA水化膜，稳定量子点，增强荧光效率。
2、本发明构建了一种自发荧光的砷纳米制剂，该砷荧光量子点能通过自发荧光的方式示踪药物递送，实现药物递送的可视化；通过砷荧光量子点表面包裹的HA水化膜，靶向CD44高表达的肿瘤细胞，将药物选择性输送至治疗部位，增加药物在肿瘤部位的累积量，降低对正常组织的毒副作用，实现疗效最大化。
3、本发明提供了一种砷荧光量子点的制备方法，其制备过程简单可控，制备得到的砷荧光量子点既可作为产生治疗效应的纳米药物，又能自身标记成像，这为创新型的诊疗一体化纳米递送平台的构建提供了理论依据。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1中As4S4和HA不同质量浓度比所制备的As4S4@HA QDs在日光及紫外光下图片。
图2为本发明实施例2中不同油浴时间下制备As4S4@HA QDs的荧光强度图。
图3为本发明实施例3中As4S4@HA QDs在350-410nm激发光下的荧光光谱图。
图4为本发明实施例4中在加或不加HA条件下油浴加热不同时间制备As4S4@HAQDs，在日光及紫外激发光下的图片。
图5为本发明实施例5中As4S4@HA QDs的透射电镜图，在50nm下观察平均粒径及形态，在高分辨率(2nm)下观察晶体结构。
图6为本发明实施例6中As4S4@HA QDs在HEK293及MDA-MB-231细胞中的摄取图。
图7为本发明实施例7中As4S4@HA QDs对HEK293及MDA-MB-231细胞杀伤的浓度依赖性图；其中图(a)为HEK293和MDA-MB-231两种细胞在不同浓度As4S4@HA QDs条件下的细胞毒性，图(b)为HEK293和MDA-MB-231两种细胞的IC50情况对比。
图8为本发明实施例8中As4S4@HA QDs对HEK293及MDA-MB-231细胞杀伤的IC50结果,其中图(a)为对照组，图(b)为HEK293细胞凋亡情况，图(c)为MDA-MB-231细胞凋亡情况。
图9为本发明实施例9中为As4S4@HA QDs诱导HEK293及MDA-MB-231细胞凋亡情况图，其中图(a)为20内三组不同给药条件下肿瘤体积变化曲线，图(b)为20天后三组不同给药条件下肿瘤体重。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段；若未特别指明，实施例中所用试剂均为市售。
实施例1
砷荧光量子点的制备：
将雄黄溶于乙二胺中，超声15min，15000rpm离心5min，取上清。将1.2mL上清分散于8mL水中，搅拌5min，加入2.5mL HA水溶液(40mg/mL)，搅拌5min，加入4.8mL 4M HCl，搅拌10min，然后将混合溶液置于250mL三口烧瓶中，75℃油浴72h，得到粗品。最后收集离心上清，经葡聚糖凝胶柱层析分离得到纯化的砷荧光量子点。
实施例2
考察实施例1中不同油浴时间形成的砷荧光量子点的荧光强度。
在实施例1制备方法中，75℃油浴由12h逐渐延长至72h，由图2可知，随着油浴时间的延长，荧光强度逐渐增强，直至72h达到平衡。
实施例3
考察不同激发波长下砷荧光量子点的荧光强度。
取As4S4@HA QDs在不同激发波长上进行荧光发射光谱扫描，得到荧光光谱图，由图3可知，As4S4@HA QDs在350nm-410nm激发波长下均具有较强荧光。
实施例4
考察加和不加HA情况下荧光量子点的荧光强度。
按实施例1的方法制备砷荧光量子点，对照组为不加入HA，油浴加热反应不同时间，对所制备的荧光量子点在日光灯及紫外灯下观察，结果表明，HA存在下所制备的荧光量子点荧光显著增强，表明以HA为稳定剂的量子点具有更强的量子产率，其主要原因是HA稳定下形成粒径更均匀，尺寸更小的量子点。
实施例5
考察砷荧光量子点形态结构。
取As4S4@HA QDs在透射电镜及高分辨率投射电镜下观察。在50nm标尺下观察荧光量子点平均粒径分布，发现量子点为平均粒径3nm的近球形纳米粒；插图为在高分辨率透射电镜下(标尺为2nm)下观察到量子点的晶格结构，表明量子点具有典型砷晶体结构。
实施例6
考察砷荧光量子点对于肿瘤细胞的特异性选择和示踪。
用人乳腺癌细胞(MDA-MB-231)和人胚胎肾细胞293(HEK293)两种细胞进行培养与实验。首先，将两种细胞分别复苏后接种到细胞培养瓶中用RPMI1640细胞培养基(含10％FBS和1％双抗)进行培养。两种细胞的培养条件均为37℃，5％CO2的潮湿环境。细胞培养过程中隔天进行培养基更换，以确保细胞生长状态良好，以备后续实验。
选用处于对数生长期的MDA-MB-231及HEK293细胞，加入As4S4@HA QDs孵育4h后，在荧光显微镜下观察摄取情况。
由图6可知，As4S4@HA QDs在MDA-MB-231细胞中的荧光信号明显强于HEK293细胞，表明As4S4@HA QDs的自发荧光可用于示踪其细胞内转运，且量子点可选择性进入肿瘤细胞，具有特异性。
实施例7
用MTT实验评价As4S4@HA QDs的细胞毒性。
选用处于对数生长期的MDA-MB-231及HEK293细胞进行实验，去掉细胞培养基，用PBS洗涤细胞，胰酶消化使其游离，终止消化后接种到96孔无菌细胞培养板中(5×103个细胞/孔)，37℃，5％CO2环境中培养过夜，待细胞完全贴壁且生长状态良好时去掉培养基，将含有不同浓度As4S4@HA QDs(0-5μg/mL)的新鲜培养基加入培养孔中与细胞孵育，每孔加入的体积为100μL。以没有经As4S4@HA QDs处理的细胞为空白对照。将处理的细胞继续在培养箱中培养24h，然后向每孔中加入20μL用PBS溶解的MTT溶液(5mg/mL)。将细胞置于培养箱中继续培养4h，然后弃去上清细胞培养基。每孔加入100μL的二甲基亚砜，避光震荡10min，待蓝紫色的甲瓒结晶完全溶解后，用酶标仪测定490nm波长处的紫外吸收，用于定量分析砷荧光量子点对细胞的毒性。
图7(a)可知，As4S4@HA QDs对两种细胞均具有肿瘤浓度依赖性杀伤；由图7(b)可知，As4S4@HA QDs对MDA-MB-231具有更强的细胞毒性(IC50值更低)，具有肿瘤细胞选择性细胞毒。
实施例8
As4S4@HA QDs对MDA-MB-231及HEK293的细胞凋亡实验：
(1)取对数生长的细胞，计数，适量IMDM培养基稀释至106cells/mL的细胞悬液，每孔1mL接种于6孔板，总共接种5个孔。
(2)每孔加入1ml 2μg/ml As4S4@HA QDs，重复3个复孔，孵育48h。
(3)用EP管收集细胞，1200rpm 5min离心，用PBS洗两次，离心除去培基。
(4)用200μl 1*的Binding Buffer重悬细胞,每管加入5μl Annexin V-FITC和5μlPI，室温，避光，孵育15min，加入Buffer至300μL，轻轻混匀
(5)最后在1h内使用用流式细胞仪检测。
由图8可知，处理48h后两种细胞均出现凋亡，表明As4S4@HA QDs通过诱导细胞凋亡来杀死肿瘤细胞，同时As4S4@HA QDs表现出肿瘤细胞特异性。
实施例9
As4S4@HA QDs的体内抗肿瘤活性研究：
(1)荷瘤裸鼠造模：裸鼠由中南大学实验动物学部统一在符合动物实验要求的条件下购买并饲养。所有的动物护理和处理均由当地伦理委员会批准，并按照中国相关动物保护法的指导方针实施。在裸鼠右前侧进行皮下接种事先准备好的含有MDA-MB-231细胞、PBS和基质胶的混悬液，每只裸鼠的接种量约为5x106个细胞，然后正常饲养接种后的裸鼠，待肿瘤体积生长到合适大小后再用于后续的动物实验。
(2)体内抗肿瘤实验：按照上述方法建立皮下荷MDA-MB-231异体移植的肿瘤裸鼠模型，待肿瘤长到约60mm3时，荷瘤裸鼠被随机分为三组(n＝3)：
A、PBS组；
B、As4S4@HA QDs 2.5mg/kg组；
C、As4S4@HA QDs 5mg/kg组；
接下来对每组荷瘤裸鼠进行标记、体重称量和瘤径测定，然后根据设置的组别，对每只荷瘤裸鼠进行尾静脉给药。给药方式：隔天给药，共8次，同时在治疗期间内隔天进行体重称量和瘤径测定。治疗20天后，把荷瘤裸鼠安乐处死，取出肿瘤称重。
图9结果显示As4S4@HA QDs呈现剂量依赖性抑制肿瘤组织的生长，在生物医学领域具有重大的应用价值。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种砷荧光量子点，其特征在于，所述砷荧光量子点分子式为As4S4@HA QDs，其中，As4S4和HA分别为雄黄和透明质酸，所述砷荧光量子点以透明质酸为稳定剂，在其表面形成稳定的HA水化膜。
2.根据权利要求1所述的砷荧光量子点，其特征在于，所述砷荧光量子点中As4S4和HA的质量浓度比为5:1～1:5。
3.根据权利要求1所述的砷荧光量子点，其特征在于，所述砷荧光量子点的粒径为1-3nm。
4.根据权利要求1所述的砷荧光量子点，其特征在于，所述砷荧光量子点在350-410nm激发波长下具有强荧光信号。
5.根据权利要求1-4任一项所述砷荧光量子点的制备方法，其特征在于，包括以下步骤：
S1、将雄黄溶于乙二胺中，超声、离心，取上清，配置雄黄溶液；
S2、将雄黄溶液分散于水中，加入HA水溶液，搅拌，加入HCl调节pH，搅拌，70-80℃油浴孵育48-72h，得到粗品；
S3、通过离心去除沉淀，并经葡聚糖凝胶柱层析分离得到纯化的砷荧光量子点。
6.根据权利要求5所述的制备方法，其特征在于，所述S1中超声时间为10-20min，10000-15000rpm离心5-10min。
7.根据权利要求5所述的制备方法，其特征在于，所述S2中HCl的浓度为3-5mol/L，所述pH值小于或等于8。
8.根据权利要求5所述的制备方法，其特征在于，所述S2中油浴温度为75℃，油浴孵育时间为72h。
9.根据权利要求1-4任一项所述的砷荧光量子点或权利要求5-8任一项所述制备方法制备得到的砷荧光量子点在制备治疗肿瘤药物中的应用。
10.根据权利要求1-4任一项所述的砷荧光量子点或权利要求5-8任一项所述制备方法制备得到的砷荧光量子点在制备肿瘤细胞示踪药物中的应用。
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