CN111087376B - 一种钆掺杂孟加拉玫瑰纳米点及其制备方法和应用 - Google Patents
一种钆掺杂孟加拉玫瑰纳米点及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种钆掺杂孟加拉玫瑰纳米点及其制备方法和应用,以孟加拉玫瑰红、六水合硝酸钆和乙醇为原料,通过高温高压溶剂热反应制得,其中孟加拉玫瑰红的分子中的羧基与六水合硝酸钆中的Gd(III)配位,使得Gd(III)掺杂其中。本发明选择临床光敏剂孟加拉玫瑰红(rose Bengal,简称RB),通过一步溶剂热处理制备了Gd(III)掺杂的钆掺杂孟加拉玫瑰纳米点(GRD),与游离的RB分子相比,GRD表现出高8.73倍的荧光效率以及高1.94倍的1O2生成效率,显示了更好的荧光成像和光动力治疗(PDT)功效。
Description
技术领域
本发明属于生物医用纳米材料技术领域,具体涉及一种钆掺杂孟加拉玫瑰纳米点及其制备方法和应用。
背景技术
癌症是威胁人类健康和生命的主要疾病之一,发病率日益上升。临床上常用的有外科手术切除、化疗、放疗等治疗方法,但手术切除通常会造成较大创伤而且往往病灶切除不完全,放疗所需的放射性核素会给正常组织带来不必要的辐照损伤,化疗通常具有较强的毒副作用。光动力治疗(PDT)是一种具有低毒性、高选择性的临床批准的微创手段,近年来受到广泛关注。光敏剂被合适波长的光照激发后,进而与氧气相互作用产生具有细胞毒性的单线态氧(1O2),从而杀伤癌细胞。然而,临床常用光敏剂的激发光具有有限的组织穿透深度,通常<1cm,严重限制了PDT治疗策略的临床应用,尤其是对于深层组织癌症的治疗效果不佳。而且大多数光敏剂,特别是疏水性光敏剂,在生物体系内容易发生聚集诱导猝灭(ACQ)现象,导致荧光和生成1O2效率降低。一种有效的解决方案是将光敏剂整合到纳米载体中,改善其生物相容性,提高其体内循环时间和肿瘤中的富集。另外,通过优化纳米载体并引入多种诊断/治疗剂,可以实现肿瘤诊疗一体化以及联合治疗效果。其中,联合PDT和放疗具有广阔的临床应用前景,由高质子数元素组成的纳米光敏剂可提高肿瘤的放疗敏感性的同时实现PDT疗效,而且由于放疗激发源(X射线)具有无限的组织穿透能力,可以实现对深层肿瘤的有效治疗。钆(Gd)元素是一种高质子数元素, Gd(III)容易与O、N等原子配位形成配合物,且易于掺入纳米材料中,而应用于肿瘤的磁共振成像和放射增敏。2013年,美国完成了莫特沙芬钆(NSC 695238)在患有初诊脑干神经胶质瘤的儿童的临床I期的研究(NCT00003909)。在法国,超小(<5nm)AGuIX 纳米颗粒正在用于脑转移瘤的放疗临床试验(NCT03818386和NCT04094077)。
生物医用纳米材料在肿瘤多模态诊疗一体化的基础研究已经取得了很多重大进展。但是,已经应用在临床上或正在临床试验中的研究成果还很少。
发明内容
本发明的目的在于提供一种钆掺杂孟加拉玫瑰纳米点,其由临床上最常用的显像剂和治疗剂组成的纳米诊疗剂,旨在推动其临床转化。
本发明的另一目的在于提供上述钆掺杂孟加拉玫瑰纳米点的制备方法。
本发明的再一目的在于提供上述钆掺杂孟加拉玫瑰纳米点的应用。
本发明的技术方案如下:
一种钆掺杂孟加拉玫瑰纳米点,以孟加拉玫瑰红(RB)、六水合硝酸钆和乙醇为原料,通过高温高压溶剂热反应制得,其中孟加拉玫瑰红的分子中的羧基与六水合硝酸钆中的 Gd(III)配位,使得Gd(III)掺杂其中。
在本发明的一个优选实施方案中,所述孟加拉玫瑰红和六水合硝酸钆的摩尔比为0.8-1.2∶0.8-1.2。
在本发明的一个优选实施方案中,所述孟加拉玫瑰红和乙醇的比例为0.2-0.3mol∶12-17mL。
在本发明的一个优选实施方案中,所述孟加拉玫瑰红、六水合硝酸钆和乙醇的比例为 0.2-0.3mol∶0.2-0.3mol:12-17mL。
上述钆掺杂孟加拉玫瑰纳米点的制备方法,包括如下步骤:
(1)将六水合硝酸钆和孟加拉玫瑰红溶于乙醇中,超声分散20-30min;
(2)将步骤(1)所得的物料转移至聚四氟乙烯内衬的高压釜中,加热至170-185℃进行所述高温高压溶剂热反应10-14h;
(3)将步骤(2)所得的物料自然冷却至室温后,加入超纯水分散均匀;
(4)将步骤(3)所得的物料经7000-9000rpm离心8-15min,获得上清液;
(5)将上述上清液用截留分子量为0.8-1.2kDa的透析袋在超纯水中透析,即得。
上述钆掺杂孟加拉玫瑰纳米点在制备肿瘤诊断药物中的应用。
上述钆掺杂孟加拉玫瑰纳米点在制备肿瘤治疗药物中的应用。
在本发明的一个优选实施方案中,所述肿瘤治疗药物为光动力治疗药物和放疗药物。
本发明的有益效果是:
1、本发明选择临床光敏剂孟加拉玫瑰红(RB),通过一步溶剂热处理制备了Gd(III) 掺杂的的钆掺杂孟加拉玫瑰纳米点(GRD),与游离的RB分子相比,GRD表现出高 8.73倍的荧光效率以及高1.94倍的1O2生成效率,显示了更好的荧光成像和PDT功效。
2、本发明中的Gd(III)的掺杂带来良好的T1磁共振成像(MRI)性质和放射增敏性质,显示了本发明可以在MRI和增强放射治疗中的应用。
3、通过磁共振/荧光双模态成像实时监测,本发明的GRD可通过EPR效应有效地被4T1肿瘤细胞摄取,在肿瘤最佳富集时间点给予放射线和激发光照射后,GRD显示出优异的抗肿瘤性能,且对机体没有产生长期毒副作用。
附图说明
图1为本发明实施例1制得的GRDs的具体表征图,其中,a为GRDs的TEM图像及其溶液外观(橙色);b为GRDs的高分辨TEM图像;c为GRDs的STEM-mapping元素分布。
图2为本发明实施例1制得的GRDs的水合粒径分布图。
图3为本发明实施例1制得的GRDs的XRD谱图。
图4为本发明实施例1制得的GRDs的EDS能谱以及各元素含量。
图5为利用ICP-MS测得的浓度逐渐增加的本发明实施例1制得的GRDs中RB和 Gd的含量变化图。
图6为本发明实施例1制得的GRDs的XPS分析图。
图7为本发明实施例1制得的GRDs的FTIR光谱图。
图8为RB和本发明实施例1制得的GRDs的表面电位对比图。
图9为本发明实施例1制得的GRDs在9.4T场强下的MRI的T1造影信号图。
图10为本发明实施例1制得的GRDs在不同分散介质中的胶体稳定性以及荧光稳定性图。
图11为本发明实施例1制得的GRDs在紫外灯照射不同时间后的荧光发光强度图。
图12为RB和本发明实施例1制得的GRDs的荧光发光强度以及荧光量子产率对比图。
图13为浓度逐渐增加的RB和含有相应浓度RB的GRDs在白光和紫外光照射下的发光情况图。
图14为本发明实施例1制得的GRDs的光谱分析图(吸收光谱和荧光光谱)。
图15为以ABDA为探针检测溶液中生成的单线态氧,浓度相同、相同激发条件下,RB和GRDs的单线态氧产生情况图。
图16为本发明实施例2中4T1肿瘤细胞对RB和GRDs的摄取情况图。
图17为本发明实施例2中的4T1肿瘤细胞杀伤效果评价图,其中,a为4T1肿瘤细胞用含不同浓度RB的RB或GRDs孵育24h后,经过激光照射处理前后的存活率。b和 c为经过不同治疗方式处理后,4T1肿瘤细胞的细胞活力(b)以及活细胞(绿色荧光)/ 死细胞(红色荧光)共染色荧光图像(c)。
图18为本发明实施例2中经过不同方式处理后,利用SOSG检测4T1肿瘤细胞内单线态氧的产生情况(激光共聚焦荧光成像;绿色荧光信号由单线态氧与SOSG作用产生)。
图19为本发明实施例2中单细胞电泳实验(彗星试验)测定经过不同治疗方式处理后4T1肿瘤细胞的DNA损伤程度照片。
图20为本发明实施例2中4T1肿瘤细胞通过不同辐照治疗方式处理后的细胞增殖能力曲线图。
图21为本发明实施例3中的实验鼠尾静脉注射PBS(control)或GRDs(10mg·kg-1)后第3天和第7天的血清生化分析图。
图22为本发明实施例3中的实验鼠尾静脉注射PBS(control)或GRDs(10mg·kg-1)后第3天和第7天的血细胞计数图。
图23为本发明实施例3中的尾静脉注射GRDs不同时间点(0.5、1、2、4、6、10 和24h)后,GRDs在血液中的相对含量图(a)以及主要器官(脑、心脏、肝脏、脾脏、肺、肾脏)和肿瘤中的分布图(b)。(n=5)
图24为本发明实施例3中尾静脉注射GRDs的1、2、4、6、10和24h后,荷瘤实验鼠的MRI成像图(a)、对应的肿瘤部位的T1信号强度图(b)、体内荧光成像图(c)、体外组织荧光成像图(d)以及对应的肿瘤部位的荧光信号图(e)。黑色虚线圈所示是肿瘤部位。
图25为本发明实施例3中经过不同方式治疗期间,荷瘤实验鼠的肿瘤生长曲线图(a, ***,P<0.001)、治疗结束后各实验组小鼠肿瘤照片(b)、在治疗开始(第0天)以及第3、9、15天,各实验组的小鼠照片(c)以及治疗结束后各实验组肿瘤切片的H&E染色图(d)。(n=5)
图26为本发明实施例3中经过不同方式治疗后,实验鼠的主要脏器(脑、心脏、肝脏、脾脏、肺和肾脏)切片的H&E染色分析图。(标尺:100μm)。
图27为本发明实施例3中经过不同方式治疗期间,荷瘤实验鼠的体重变化曲线图。(n=5)
具体实施方式
以下通过具体实施方式结合附图对本发明的技术方案进行进一步的说明和描述。
下述各实施例的所有数据均表示为平均值±标准偏差。用Student′s t检验进行数据比较(*,P<0.05;**,P<0.01;***,P<0.001)。
实施例1
(1)将六水合硝酸钆(100mg,0.22mmol)和孟加拉玫瑰红(220mg,0.22mmol) 溶于15mL乙醇中,超声分散30min;
(2)将步骤(1)所得的物料转移至聚四氟乙烯内衬的高压釜中,加热至180℃进行所述高温高压溶剂热反应12h;
(3)将步骤(2)所得的物料自然冷却至室温后,加入35mL超纯水分散均匀;
(4)将步骤(3)所得的物料经8000rpm离心10min,获得上清液;
(5)将上述上清液用截留分子量为1kDa的透析袋在超纯水中透析3天,即得本发明的钆掺杂孟加拉玫瑰纳米点,简称GRDs。
利用JEM-2100透射电子显微镜测定GRDs的TEM图像。使用Zeta-sizer纳米颗粒分析仪(Malvern Ltd.)测量GRDs的水合粒径(DLS)和表面Zeta电位。使用UltimaIVX 射线粉末衍射仪(Rigaku Co.,Japan)测定GRDs的XRD谱图。使用K-A驷ha+X射线光电子能谱仪(单色Al Kα源)(6mA,12kV)进行XPS分析。使用UV-2100分光光度计测定紫外可见吸收光谱。使用F-7000荧光光度计测定荧光光谱。使用IVIS Lumina II荧光成像设备和小动物9.4T MRI成像设备测定材料的荧光和MRI信号。荧光成像参数为: Ex,470nm;Em,535nm。MRI成像(使用T1反转恢复快速自旋回波序列),参数为: TR=5000ms;TE=12ms;ETL=8;反转时间=5,10,30,50,80,200,500,700,900, 1200,3000ms;256×256矩阵。
如图1a所示,纯化后的GRDs呈透明状,均匀分散在水中,呈橙色,粒径为3.3±0.8nm,粒径分布较窄,水合粒径为7.7±1.4nm(图2)。高分辨电镜图像(图1b)显示,GRDs 的结晶度较弱,层间距为0.21nm,归因于石墨烯的(100)晶格间距(图3)。根据能量色散X射线能谱(EDS)和ICP-MS分析,测得钆的掺杂率可以分别达到21.3wt%和30.0 wt%(图4,图5),同时,含碘元素的量高达28.5%(wt%),高于RB分子内碘的含量,富含高质子数元素(钆和碘)有助于实现放疗增敏。如图1c元素分布测定结果显示,C、 O、Cl、I、Gd元素在GRDs中均匀分布。
如图6,RB和GRDs的XPS能谱显示,RB分子和Gd(III)混合物经过溶剂热处理后,在形成的GRDs中,Na 1s信号消失,C、O、Cl、I、Gd信号仍然存在。C 1s的高分辨率XPS谱图显示了sp3/sp2碳(C-C/C=C)和氧合碳(C=O和C-O),O 1s带由两个峰组成,分别归属于C=O和C-O,同时在GRDs中,存在Gd 3d3/2、Gd 3d5/2、Gd 4d3/2、Gd 4d5/2的特征峰,证明Gd(III)的存在。RB和GRD的傅里叶变换红外光谱(FTIR)(图 7),进一步证实了组成和官能团的变化。苯环C=C(1545,1454,1340cm-1)和羰基C=O (1614cm-1)的特征峰。GRDs中730、850cm-1处出现新的C=C峰,1750cm-1处出现新的羧基C=O峰。这些结果表明,RB的羧基和酚羟基中的氧与Gd(III)发生配位反应。这些变化使得材料的表面电位由RB的-28.87mV变为GRD的+0.96mV(图8)。
对GRDs的光学和磁学性质进行了表征,发现GRD的最佳激发/发射峰为530/560nm(图9)。GRDs的紫外-可见吸收光谱在450-550nm处呈现宽而强的吸收带。与RB相比, GRD的吸收发生了轻微的蓝移,这可能是纳米点形成后芳香结构中电子共轭减少的结果。此外,在RB浓度相同的情况下,GRDs的荧光强度高于RB,且随着其中Gd(III)和 RB浓度的增大,GRDs的荧光发生轻微的红移(图10)。以RB(ΦRB=0.11)为标准,测定GRD的荧光量子产率为0.97(图11)。经过紫外灯照射6h后,GRDs的荧光强度几乎没有减弱,表明GRDs的光稳定性高于许多商用有机染料和量子点(图12)。GRDs在不同生物介质(包括水、PBS(pH 7.4)、FBS(10%)、DMEM)中放置10天后,仍然表现出较好的胶体稳定性和光稳定性(图13)。此外,由于掺杂了大量Gd(III),GRDs显示出明显的MRI-T1信号(图14a),r1弛豫率为9.6mM-1·s-1,是Gd-DTPA的3.1倍(3.1 mM-1·s-1)(图14b)。
光学表征结果表明,GRDs在450-550nm处有较强的吸收,因此,本实施例使用了532nm的激光作为激发源,以9,10-二亚甲基二丙二酸(ABDA)作为单线态氧检测探针,通过测定溶液中单线态氧的生成来评价PDT性能。以相同浓度的RB作为对照,利用532 nm的激光(30mW·cm-2)照射,测定378nm处的吸光度的下降值(0、10、20min时间点)(图15)。计算单线态氧量子产率,结果表明GRDs的单线态氧量子产率是RB的1.94 倍。
实施例2细胞实验
以鼠源乳腺癌4T1细胞为研究模型,进一步探究了GRDs纳米材料用于多模态成像指导的肿瘤的联合治疗。
(1)细胞摄取试验
将4T1肿瘤细胞与含有RB或GRDs的培养基共孵育24h,PBS洗涤,补充新鲜培养基,通过细胞荧光成像实验测得细胞摄取情况,使用倒置荧光显微镜(Ex/Em:547/572nm) 获取材料在4T1肿瘤细胞中的荧光图像。结果显示,与RB分子相比,纳米点GRDs更容易被4T1肿瘤细胞摄取,从而显示出更强的细胞质内荧光信号(图16)。
(2)细胞毒性测定
将4T1肿瘤细胞接种在96孔板(每孔104个细胞),实验前在培养箱中培养24h;利用含有不同浓度(0、2.5、5、10、20、40μg·mL-1)纳米材料GRDs的新鲜培养基,在避光条件下,继续与4T1肿瘤细胞共孵育24h;PBS洗涤,并补充新鲜DMEM培养基后,使用标准MTT试验测定细胞存活率:利用酶标仪测定570nm处的吸光度,以空白对照组的细胞存活率为百分之百作为对照,根据各个材料实验组与空白对照组的吸光度比值计算得到各实验组的细胞活力。如图17a显示,GRDs纳米材料没有表现出明显的细胞毒性。
(3)4T1肿瘤细胞杀伤效果评价
A.利用MTT试验评价了对4T1肿瘤细胞的杀伤效果。将4T1肿瘤细胞与不同浓度的RB或GRDs(含有的RB的浓度分别为:0、1.5、3.0、6.0、12、24μg·mL-1)共孵育 24h;PBS洗涤;给予532nm激光照射(30mW·cm-2,10min);继续培养24h后,使用标准MTT试验测定细胞存活率。如图17a所示,与GRDs孵育24h后经过532nm激光照射激发的PDT效应导致了更多的肿瘤细胞的凋亡,4T1细胞活力显著降低;而相同条件下,与RB进行孵育或者只是与GRDs进行孵育并没有给予激光照射的4T1细胞,其存活率较高。说明RB与GRDs具有良好的生物相容性,且GRDs比RB的光动力疗效更好。经过532nm激光(30mW·cm-2)照射10min后,GRDs导致63.5%的细胞死亡,而RB 仅引起36.4%的细胞杀伤。
B.此外,由于钆和碘这两种高质子数元素起到一定的辐射增敏作用,因此在X射线辐照下可以产生一定的增强放疗效果。为了比较不同治疗方法对4T1肿瘤细胞的杀伤效果,对4T1肿瘤细胞进行不同处理:PBS、PBS+L、PBS+X、PBS+L+X,RB、RB+L、 RB+X、RB+L+X,GRD、GRD+L、GRD+X、GRD+L+X,L:532nm激光照射(30mW·cm-2, 5min),X:X射线(1Gy);继续培养24h后,使用标准MTT试验测定细胞存活率。如图17b显示,GRDs可以联合PDT(532nm激光,30mW·cm-2,5min)和放疗(X射线, 1Gy),产生更强的肿瘤细胞杀伤效应(细胞活力为37.4±1.3%)。使用Calcein-AM/PI双染色试剂盒进行活/死细胞染色直观检测肿瘤细胞杀伤情况。加入Calcein-AM/PI双染色试剂,在37℃下染色30min;PBS洗涤后,利用倒置荧光显微镜测定活细胞(Ex/Em:490/515 nm)和死细胞(Ex/Em:535/617nm)的荧光。如图17c所示,活/死细胞染色分析结果与MTT试验结果一致,GRD+L+X组的肿瘤细胞具有最明显的受损现象,坏死细胞的细胞核被PI(碘化吡啶)染色所呈现的红色荧光更明显。进一步表明,在激光和高能射线的作用下,GRDs纳米点可以起到对4T1肿瘤细胞有效的杀伤作用,而且联合治疗比单一治疗方法具有更好的疗效。
(4)4T1肿瘤细胞杀伤机制
A.将4T1肿瘤细胞接种在玻璃底细胞培养皿培养24h后,分别与PBS、RB、GRD共孵育24h;PBS洗涤三次后,加入SOSG(1μM)继续孵育30min,PBS洗涤;辐照组细胞给予532nm激光照射(30mW·cm-2,10min);使用O1ympus FV1200激光共聚焦显微镜(FITC滤光器)检测单线态氧绿(SOSG)和细胞内单线态氧反应所产生的绿色荧光。如图18所示,虽然RB+L组4T1细胞内显示出单线态氧绿绿色荧光,但是 GRD+L组明显产生更多的细胞内单线态氧,即使是正在进行分裂增殖中的细胞内也清晰可见绿色荧光信号。说明在532nm激光照射下,GRDs可以在4T1肿瘤细胞内产生大量单线态氧,也即GRDs可以有效地介导4T1肿瘤细胞内PDT效应。
B.将4T1肿瘤细胞接种在6孔板中,培养24h,分为三组:PBS(空白对照组), PBS+X(放疗组),GRD+X(放疗增敏组),其中X射线辐照剂量为1Gy;PBS洗涤,培养24h后收集细胞;使用彗星分析试剂盒试剂对细胞进行预处理;进行单细胞凝胶电泳 (21V,45min);避光条件下用DAPI染色30min,利用倒置荧光显微镜测定细胞核的荧光。如图19所示,空白对照组的细胞核保持完整形态;当受到X射线辐照后的放疗组的4T1细胞核,呈现明显的彗星拖尾状,说明高能辐照导致DNA双链断裂,在电泳条件下,断裂的片段核酸从类核中迁移出来;与空白组和放疗组相比,GRD+X射线组处理后的4T1细胞,其类细胞核呈现出更加明显的长而强烈的彗尾拖尾,说明摄取了GRDs的 4T1肿瘤细胞在受到辐照后,其细胞核内DNA双链断裂更为严重,断裂的核酸碎片在电泳条件下“喷涌”而出。GRDs纳米材料具有放疗增敏效应。
C.将4T1肿瘤细胞接种在6孔板中,培养24h,分为两组(n=3):PBS+X(放疗组) 和GRD+X(放疗增敏组),其中X射线照射剂量为0、1、2、4、6、8Gy;PBS洗涤,继续培养24h后,收集细胞并取1000个活细胞重新接种到新的6孔板中,继续培养10 天;福尔马林固定细胞,用0.5%龙胆紫进行细胞染色;计数含有50个以上的着色细胞的集落,计算存活分数(以不施加辐照组的细胞存活率作为100%进行计算)。结果显示(图 20),当用高剂量X射线照射(>4Gy)后,与PBS+X射线处理组相比,GRDs+X射线处理后的4T1细胞,其长期增殖能力也明显减弱更多。这一结果也表明GRDs纳米点具有放射增敏性质,这种增强的放疗效应主要归因于GRDs纳米材料中含有相对较多的钆和碘原子。
实施例3动物实验
实验动物模型:由厦门大学实验动物中心自上海斯莱克实验动物中心代为购买的雌性 BALB/c小鼠(体重为16-18g)。
肿瘤模型:通过皮下注射4T1肿瘤细胞在实验鼠右后臀部,接种皮下4T1肿瘤;当肿瘤体积达到80~100mm3时,荷4T1皮下瘤的BALB/c小鼠用于动物实验。
所有动物实验都符合厦门大学实验动物使用委员会和实验动物伦理委员会批准的动物保护条例。
(1)纳米材料GRDs的生物毒性
通过尾静脉内向实验鼠(n=3)体内注射PBS或GRDs(10mg·kg-1);分别在注射后第3天和第7天,采集血清和全血样品,用于生化分析和的血细胞分析。使用全自动生化分析仪BS-220测量实验鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶 (ALP)、血清白蛋白(ALB)、总胆红素(T-Bil-V)、血尿素氮(UREA)、总胆汁酸(TBA)、胆碱酯酶(CHE)、肌酸酐(CREA-S)的浓度或含量。使用血细胞分析仪BC-2600测量实验鼠注射纳米材料前后的血液中白细胞(WBC)、红细胞(RBC)、血红蛋白(HGB)、血细胞比容(HCT)、平均红细胞体积(MCV)、平均红细胞血红蛋白(MCH)、平均红细胞血红蛋白浓度(MCHC)、红细胞分布宽度(RDW-CV和RDW-SD)、血小板(PLT)、平均血小板体积(MPV)、血小板分布宽度(PDW)、血小板压积(PCT)、血小板比率 (P-LCR)的计数。如图21所示的各项生物酶浓度或含量和图22所示的血细胞计数或浓度,给药前后,实验鼠的各项生化指标和血细胞计数没有发生显著性变化,说明所使用的GRDs纳米材料对实验鼠并没有产生明显的身体功能以及血细胞的损伤,生物毒性较低,具有一定的生物应用安全性。
(2)GRDs在体内肿瘤诊断中的应用
A.荷4T1皮下瘤的BALB/c小鼠,通过尾静脉注射GRDs(10mg·kg-1)(n=3),在给药后的0.5、1、2、4、6、10、24h,断颈处死;解剖取出主要器官,包括血液(~50μL)、脑、心脏、肝脏、脾脏、肺、肾脏和肿瘤,称重;加入适量浓HNO3-H2O2(体积比1∶3) 进行消解;超纯水稀释后,通过ICP-MS测定组织样品中钆的含量;计算为每克组织中含有钆的量相对于总注射的钆的量的百分比(%ID/g)。如图23所示,GRDs纳米点显示出较长的体内循环时间(血液清除半衰期为5.5±0.8h);除肝脏和脾脏等网状内皮系统组织外,相对于大部分正常组织器官,GRDs纳米点在肿瘤中具有较高的被动靶向摄取,尾静脉注释4h后,4T1肿瘤内摄取高达13.0±1.6%ID/g。
B.荷4T1皮下瘤的BALB/c小鼠,通过尾静脉注射GRDs(10mg·kg-1),分别在给药前(0h的时间点)和给药后的1、2、4、6、10、24h的时间点,使用IVIS Lumina II 小动物荧光系统和9.4T小动物磁共振成像设备进行全身荧光成像和磁共振成像,成像参数与材料测定的设置相同。如图24所示,GRDs对4T1肿瘤具有较好的被动靶向能力,在考察的时间范围内,尾静脉给药4-6h后,具有最好的肿瘤摄取。因此,在后续的治疗实验中,本实施例选择在尾静脉给药4h后,对肿瘤部位施加激光照射和X射线辐照。
(3)4T1肿瘤的治疗效果
荷4T1皮下瘤的BALB/c小鼠随机分成8组(n=5):(1)PBS,(2)PBS+L,(3)PBS+X,(4)PBS+L+X,(5)GRD,(6)GRD+L,(7)GRD+X,(8)GRD+L+X;其中,L:532 nm激光照射(140mW·cm-2,15min),X:X射线(1Gy);尾静脉内给药GRDs(10mg·kg-1) 4h后,仅对实验鼠的肿瘤区域施加532nm激光和X射线照射,除肿瘤外的实验鼠的身体其他部位都用铅板进行遮蔽。在相应的治疗后的15天内,每隔一天记录一次实验鼠的体重和肿瘤体积。如图25a肿瘤生长曲线所示,对照组((1~5)组)的肿瘤生长迅速,单一PDT((6)组)或放疗增敏组((7)组)可以起到一定的抑制效果,肿瘤生长缓慢(第 15天的肿瘤抑制率分别是:(6)组,50.5%;(7)组,43.8%)。与之相比,联合治疗组((8) 组)有效地消除肿瘤,最终的肿瘤抑制率高达98.8%(图25b,c)(肿瘤抑制率计算公式为: (1-实验组肿瘤体积/空白对照组肿瘤体积)×100%)。苏木精和伊红(H&E)对肿瘤切片染色试验结果显示,GRDs介导的联合治疗显著破坏了4T1肿瘤的组织结构,可见明显降低的肿瘤细胞密度(图25d)。
在治疗后的第15天,对所有实验鼠断颈处死,解剖取出主要组织器官:包括脑、心脏、肝脏、脾脏、肺和肾脏,H&E染色各组织切片,进行组织学分析。如图26所示,所有实验组的主要器官没有发生病理变化,说明这些治疗方法都没有对正常器官造成明显的损伤。此外,在整个治疗监测期间,所有实验组的实验鼠未显示出明显的体重下降(图 27)。进一步证实了本实施例中所使用的GRDs和治疗手段不会造成较大的生物毒性。
综上所述,本发明以临床上已经应用的光敏剂孟加拉玫瑰红(rose Bengal,RB)作为前驱体,通过一步简单的溶剂热反应制备了Gd(III)配位掺杂的超小纳米点Gd@RBnanodots(GRDs)(<5nm)。与前驱体RB分子相比,纳米点GRDs具有独特的光学性质和增强荧光及光敏特性,显示了高出7.73倍的荧光量子产率和高出0.94倍的单线态氧量子产率,而且具有较强的光稳定性(>6h),可以更好地应用于荧光成像和PDT;通过与 RB nanodots结合有效地掺杂Gd(III),与Gd-DTPA配合物相比,纳米点GRDs显示了高出2.1倍的T1磁共振成像弛豫率。较高的单线态氧量子产率使得GRDs显示出高效的PDT 效应;较高的高质子数元素(钆和碘)含量使得GRDs具有放疗增敏效应。以鼠源乳腺癌 (4T1)为研究模型,在体外细胞实验和体内肿瘤诊疗实验中,GRDs纳米点显示了较高的4T1细胞摄取和4T1肿瘤被动靶向能力,实现了荧光/磁共振双模态成像指导下对肿瘤的PDT和放疗增敏联合治疗,有效地抑制了肿瘤生长并最终消除肿瘤。由于是利用临床诊疗剂作为前体,超小GRDs纳米点显示了优良的生物相容性和生物应用安全性,具有广阔的临床转化应用前景。
以上所述,仅为本发明的较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (8)
1.一种钆掺杂孟加拉玫瑰纳米点,其特征在于:以孟加拉玫瑰红、六水合硝酸钆和乙醇为原料,通过高温高压溶剂热反应制得,其中孟加拉玫瑰红的分子中的羧基与六水合硝酸钆中的Gd(III)配位,使得Gd(III)掺杂其中。
2.如权利要求1所述的一种钆掺杂孟加拉玫瑰纳米点,其特征在于:所述孟加拉玫瑰红和六水合硝酸钆的摩尔比为0.8-1.2∶0.8-1.2。
3.如权利要求1所述的一种钆掺杂孟加拉玫瑰纳米点,其特征在于:所述孟加拉玫瑰红和乙醇的比例为0.2-0.3mol∶12-17mL。
4.如权利要求1所述的一种钆掺杂孟加拉玫瑰纳米点,其特征在于:所述孟加拉玫瑰红、六水合硝酸钆和乙醇的比例为0.2-0.3mol∶0.2-0.3mol∶12-17mL。
5.权利要求1至4中任一权利要求所述的一种钆掺杂孟加拉玫瑰纳米点的制备方法,其特征在于:包括如下步骤:
(1)将六水合硝酸钆和孟加拉玫瑰红溶于乙醇中,超声分散20-30min;
(2)将步骤(1)所得的物料转移至聚四氟乙烯内衬的高压釜中,加热至170-185℃进行所述高温高压溶剂热反应10-14h;
(3)将步骤(2)所得的物料自然冷却至室温后,加入超纯水分散均匀;
(4)将步骤(3)所得的物料经7000-9000rpm离心8-15min,获得上清液;
(5)将上述上清液用截留分子量为0.8-1.2kDa的透析袋在超纯水中透析,即得。
6.权利要求1至4中任一权利要求所述的钆掺杂孟加拉玫瑰纳米点在制备肿瘤诊断药物中的应用。
7.权利要求1至4中任一权利要求所述的钆掺杂孟加拉玫瑰纳米点在制备肿瘤治疗药物中的应用。
8.如权利要求7所述的应用,其特征在于:所述肿瘤治疗药物为光动力治疗药物和放疗药物。
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