CN114848854B - 一种131i-hsa-icg纳米颗粒及其制备方法和应用 - Google Patents
一种131i-hsa-icg纳米颗粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种131I‑HSA‑ICG纳米颗粒及其制备方法和应用,属于纳米药物制备技术领域。本申请以HSA为载体通过氯胺T法在中性环境下标记放射性131I,再通过搅拌反应将ICG非共价结合到131I‑HSA纳米颗粒上,从而制备得到131I‑HSA‑ICG纳米颗粒。本申请制备得到的131I‑HSA‑ICG纳米颗粒不仅可用于局部肿瘤双模态显像,还可对人未分化型甲状腺癌同时进行光热治疗与放射性核素治疗。本申请131I‑HSA‑ICG纳米颗粒合成方法简单易重复,毒性较低,治疗效果有效且显著,为未分化型甲状腺癌的治疗研究提供了新思路。
Description
技术领域
本发明涉及纳米药物制备技术领域,特别涉及一种131I-HSA-ICG纳米颗粒及其制备方法和应用。
背景技术
近年来,甲状腺癌的患病率在世界范围内逐年上升,成为影响人们健康的一个主要疾病之一。对于分化型甲状腺癌(DTC),外科手术联合放射性碘治疗后再行左甲状腺素替代治疗是公认的治疗程序。碘是滤泡细胞在正常甲状腺组织中合成甲状腺激素的重要成分。DTC中碘化钠转运体(NIS)在细胞基底膜上表达,为碘离子主动转运到甲状腺滤泡细胞提供生理基础。DTC细胞可以保持与滤泡细胞类似的功能,如碘吸收和碘化,未分化型的甲状腺癌细胞则丧失了摄碘能力。近年来的大量研究发现部分DTC的脱分化和各类基因突变(BRAF,NRAS,HRAS,RET/PTC等基因以及成纤维细胞生长因子受体、血小板衍生的生长因子受体改变等)可影响NIS的表达,从而导致放射性碘治疗晚期DTC的疗效。针对这类碘难治性甲状腺癌患者,科研人员不断开发治疗方法试图恢复NIS的表达以改善甲状腺癌细胞的摄碘能力并延缓肿瘤的进展。常见的手段如适当促甲状腺激素抑制下的观察等待、局部治疗方法和系统治疗(分子靶向治疗、再分化治疗、基因治疗、肿瘤免疫治疗)等。但是,严重的全身毒副作用、部分治疗理论和技术不完善等限制了上述方法的应用,进一步开展新的癌症治疗手段十分必要。
光热疗法(PTT)是近年来新兴的广泛应用于生物学领域的癌症治疗方式之一。PTT是指将光热纳米材料集中在肿瘤组织,通过外源性光照射引起光热杀伤作用。研究显示轻微的升温可以增加肿瘤的血管通透性,启动某些生物分子或细胞的招募,并促进光热剂在肿瘤中的扩散。40-45℃的温度可增强肿瘤的血流量和氧合能力。血管通透性的增加促进光热剂渗漏进入肿瘤组织。此外,较高的温度可以诱发肿瘤损伤,使某些生物分子或细胞进入肿瘤。常见的光热剂如金属、石墨烯、碳纳米管、等二维材料(如黑磷、氮化硼等)及花菁素类染料等均可达到良好的光热肿瘤消融效果。然而,上述有机或无机纳米材料均需要合适的纳米载体以便进入并滞留在肿瘤细胞内,从而达到最终治疗目的。
人血清白蛋白(HSA)因其优异的血液保留活性、非免疫原性、生物相容性、高水溶性、生物可降解性、化学稳定性和肾小球滤过性而广受人们青睐。HSA分子中含有丰富的结合位点,可以通过疏水作用与多种小分子药物进行物理交联,从而有助于传递抗癌药物进入肿瘤组织。除了化疗药物,功能性近红外(NIR)药物(如花菁素染料)、金纳米团簇、碳纳米管、和氧化石墨烯等均可负载在HSA上,以促进基于白蛋白载体的治疗性纳米药物的疗效。而吲哚菁绿(ICG)作为美国食品药品监督管理局批准用于临床的近红外花菁素类染料,可通过单波长近红外光激发产生强荧光用于显像及PTT。单纯的ICG因其在近红外光的照射下易分解、体循环周期短且缺乏肿瘤靶向功能等而被限制其实际应用。而HSA对ICG具有良好的亲和力,二者可通过非共价结合构建一个生物相容性良好的适用于显像及治疗的纳米材料。既往研究发现,在乳腺癌、前列腺癌、结直肠癌、肝癌等癌症治疗研究领域,可见有利用HSA-ICG纳米颗粒与其他化疗药物结合以治疗肿瘤的研究,但131I-HSA-ICG纳米颗粒用于人未分化型甲状腺癌诊疗一体化的研究尚未见报道。
发明内容
本发明为了解决上述技术问题,提供了一种131I-HSA-ICG纳米颗粒及其制备方法和应用。
第一方面,本发明提供一种131I-HSA-ICG纳米颗粒,是通过以下技术方案得以实现的。
一种131I-HSA-ICG纳米颗粒,所述纳米颗粒为在人血清白蛋白上负载吲哚菁绿并标记放射性核素131I制备得到的粒径为25-45nm的颗粒。
第二方面,本发明提供一种131I-HSA-ICG纳米颗粒的制备方法,是通过以下技术方案得以实现的。
一种上述131I-HSA-ICG纳米颗粒的制备方法,包括以下步骤:
S1.将HSA、氯胺T和偏重亚硫酸钠分别溶于缓冲液中;
S2.抽取一定单位计数的放射性Na131I溶液:根据细胞或者动物实验需求计算用量,131I的标记率在90%以上,每次需在计算量基础上多取1mCi用于损耗;
S3.将HSA、氯胺T、Na131I溶液混匀,震荡反应1-2min后,加入偏重亚硫酸钠溶液终止反应,再将得到的溶液进行超滤离心;
S4.将步骤S3得到的放射性131I-HSA溶液与ICG溶液按照摩尔比为HSA:ICG=1:1的比例混合,反应1-1.5h,再将得到的溶液进行超滤离心,即得。
进一步的,步骤S1中,HSA的浓度为20-60mg/mL。优选的,HSA的浓度为20mg/mL。
进一步的,步骤S3中,溶液超滤离心后,测定滤液放射性标记率≥90%。
进一步的,步骤S3和S4中,超滤离心的处理条件为截留分子量30kDa,转速6000-8000r/min,8-10min,冲洗2-3次。优选的,超滤离心的处理条件为截留分子量30kDa,转速6000r/min,10min,冲洗3次。
第三方面,本发明提供一种131I-HSA-ICG纳米颗粒在制备肿瘤诊断试剂中的应用,是通过以下技术方案得以实现的。
一种上述131I-HSA-ICG纳米颗粒在制备肿瘤显像剂中的应用。
第四方面,本发明提供一种131I-HSA-ICG纳米颗粒在制备抗肿瘤药物中的应用,是通过以下技术方案得以实现的。
一种上述131I-HSA-ICG纳米颗粒在制备治疗未分化型甲状腺癌药物中的应用。
本申请具有以下有益效果。
本申请制备得到的131I-HSA-ICG纳米颗粒不仅可用于局部肿瘤治疗时及治疗后显像(放射性核素显像和荧光成像),而且其对肿瘤进行光热治疗与放射性核素治疗双重叠加的效果优于单一治疗组的治疗效果,可构建一个用于人未分化型甲状腺癌诊疗一体化的体系。而且131I-HSA-ICG纳米颗粒中的HSA为天然载体,具有优异的血液保留活性、非免疫原性、生物相容性、高水溶性、生物可降解性、化学稳定性和肾小球滤过性等优势,HSA分子中含有丰富的结合位点,可以通过疏水作用与多种小分子药物进行物理交联,从而有助于传递放射性核素进入肿瘤组织;ICG对蛋白质有很高的亲和力,可非共价结合HSA,结合后的纳米颗粒比游离ICG具有更高的荧光效率和稳定性。本申请充分利用HSA、ICG和131I的优势,可利用ICG准确勾勒肿瘤范围,减少显像时131I的放射性伪影导致的范围预估误差,同时结合光热治疗与放射性核素治疗,避免单一治疗效果不显著或者过度治疗带来的损伤和局限性。本申请131I-HSA-ICG纳米颗粒合成方法简单易重复,毒性较低,在肿瘤内的滞留时间更长,稳定且不易分解,治疗效果有效且显著,可以同时进行双模态显像和治疗,为未分化型甲状腺癌的治疗研究提出新思路。
附图说明
图1是本发明131I-HSA-ICG纳米颗粒用于人未分化型甲状腺癌双模态显像与治疗的实验方案示意图;
图2是本发明I-HSA-ICG纳米颗粒的表征图(其中,a.透射电镜下I-HSA-ICG纳米颗粒的形貌和尺寸图;b.不同浓度HSA-ICG、I-HSA-ICG在纯水及PB缓冲液中的Zeta电势对比图;c.I-HSA-ICG纳米颗粒在纯水中的水合直径图;d.HSA、HSA-ICG、I-HSA-ICG纳米颗粒的FT-IR光谱图);
图3是本发明HSA-ICG纳米颗粒的UV-Vvis-NIR吸收光谱图以及吸收光谱拟合曲线图(其中,a.按照不同比例合成HSA-ICG纳米颗粒的UV-Vvis-NIR吸收光谱图;b.按照不同比例合成的HSA-ICG纳米颗粒在808nm处的UV-Vvis-NIR吸收光谱拟合曲线图;c.不同浓度的HSA-ICG纳米颗粒的UV-Vvis-NIR吸收光谱图;d.不同浓度HSA-ICG纳米颗粒在808nm处的UV-Vvis-NIR吸收光谱拟合曲线图);
图4是本发明I-HSA-ICG纳米颗粒的长期静置稳定性结果图(每一幅图中,从左至右的溶剂依次为超纯水、PB缓冲液(10mM,pH=7.4)、FBS、Gibco 1640培养基);
图5是本发明I-HSA-ICG纳米颗粒光热性能评估结果图(其中,a.在2.0W/cm2的808nm激光照射下,I-HSA-ICG纳米颗粒(CICG=0.1mg/mL)的光热升温效果图;b.I-HSA-ICG纳米颗粒的热传导时间常数,表示为900s冷却期后的线性时间数值与温度自然对数的负数;c.在2.0W/cm2的808nm激光照射下,水及不同浓度I-HSA-ICG纳米颗粒的光热升温曲线图;d.在2.0W/cm2的808nm激光照射下,水及不同浓度I-HSA-ICG纳米颗粒的红外热值图像;e.水、纯ICG、HSA-ICG、I-HSA-ICG纳米颗粒的光热升温曲线图;
图6是本发明131I-HSA-ICG纳米颗粒的体外细胞毒性实验图(其中,a.不同浓度HSA-ICG处理FRO、4T1细胞的细胞存活率图;b.不同浓度HSA-ICG、I-HSA-ICG处理ARO细胞的细胞存活率图;
图7是本发明131I-HSA-ICG纳米颗粒体外细胞放射性核素杀伤实验及光热杀伤实验结果图(其中,a.不同放射剂量药物处理后ARO细胞的细胞存活率图;b.不同浓度药物在不同功率下对ARO细胞的杀伤结果图;c.不同ICG浓度的131I-HSA-ICG处理后的ARO细胞在不同功率下的存活率图);
图8是本发明放射性核素治疗联合PTT的体外肿瘤细胞杀伤实验结果图(其中,a.不同处理组ARO细胞的细胞存活率图;b.不同处理组的死活细胞染色图);
图9是本发明I-HSA-ICG纳米颗粒的动物活体毒理实验结果图(其中,a.对照组及实验组小鼠体重变化图;b.对照组及实验组小鼠血生化指标对比图;c.对照组及实验组各个重要脏器HE染色图);
图10是本发明I-HSA-ICG纳米颗粒活体成像图(其中,a.不同处理组小鼠在不同时间点的活体荧光成像图;b.不同处理组小鼠在不同时间点的SPECT断层显像图);
图11是本发明131I-HSA-ICG纳米颗粒的活体治疗实验结果图(其中,a.808nm激光照射过程中各组肿瘤表面温度变化红外热值图像;b.808nm激光照射过程中各组肿瘤表面温度变化曲线图;c.不同处理组小鼠肿瘤体积变化趋势图;d.17天后不同处理组小鼠肿瘤重量与体重之比结果图);
图12是本发明不同处理组治疗后的活体观察图。
具体实施方式
下面结合附图和实施例对本发明进行进一步说明。
本申请以HSA为载体通过氯胺T法在中性环境下标记放射性131I,然后通过简单的搅拌反应将ICG非共价结合到131I-HSA纳米颗粒上,以制备131I-HSA-ICG纳米颗粒。为了避免放射性辐射的影响,本申请首先进行非放射性碘标记,利用I-HSA-ICG纳米颗粒进行一系列表征。通过MTT法分别考察I-HSA-ICG、131I-HSA-ICG纳米颗粒的细胞毒性、放射性杀伤作用。通过尾静脉注射药物,进行组织病理切片染色来从活体水平评估药物的安全性。通过溶液升温实验、细胞/活体水平的肿瘤消融实验来证实131I-HSA-ICG纳米颗粒的光热转换效能及放射性杀伤作用。实验过程参见图1。具体实验方法如下:
本申请所有试剂均为分析级。吲哚菁绿(Indocyanine green,ICG)购买自阿拉丁生化科技股份有限公司(中国上海);人血清白蛋白(HSA)购自鼎国生物技术有限公司(中国北京);放射性131碘(Na131I)购买自天津原子高科股份有限公司;氯胺T购买自希恩思生化科技有限公司(中国天津);偏重亚硫酸钠购买自中国医药公司北京采购供应站;碘化钠(NaI)购买自毕得医药科技有限公司(中国上海);氢氧化钠(NaOH)、磷酸二氢钠(NaH2PO4-2H2O)、磷酸氢二钠(Na2HPO4-12H2O)购买自天津市光复科技发展有限公司;噻唑蓝溴化四唑(methyl thiazolyl tetrazolium,MTT)购买自SDN公司;二甲基亚砜(dimethyl sμLfoxide,DMSO)购买自索莱宝科技有限公司(中国北京);胎牛血清(fetal bovine serum,FBS)购买自M&C有限公司;Gibco DMEM、1640培养基和胰蛋白酶消化液(ethlene diaminetetraacetic acid,EDTA)购买自海克隆生物化学有限公司(美国);钙黄绿素乙酰氧基甲酯(Calcein-AM)和碘化丙啶(PI)购买自东仁化学科技有限公司(中国上海);生理盐水购买自中国大冢制药有限公司;5%水合三氯乙醛购买自默克Sigma-Aldrich公司;超纯水购买自娃哈哈集团有限公司(中国杭州)。
本申请通过马尔文纳米粒度分析仪(Nano系列ZS,英国)测定I-HSA-ICG纳米颗粒的水合直径和zeta电势;利用透射电子显微镜(TEM)(日立HT7700,日本)来观察I-HSA-ICG纳米颗粒的形貌和尺寸;以纯溴化钾为背景,通过傅里叶变换-红外光谱仪(尼高力AVATAR-360,美国)记录I-HSA-ICG纳米颗粒的傅立叶变换-红外(FT-IR)光谱(400-4000cm-1);通过UV-3600plus分光光度计(日立,日本)测定I-HSA-ICG纳米颗粒的紫外-可见光-近红外吸收光谱;利用红外热值相机(菲力尔E75系列,美国)拍摄所需红外热值照片;808nm激光器购自长春新兴工业光电技术有限公司(中国);SPECT Discovery 670购自GE(通用电气)公司;离心机购自湘仪离心机仪器有限公司(TGL-16M,中国长沙);酶标仪购买自芬兰(labsystemsmμLtiskan MS)公司;显微镜购自奥林巴斯有限公司(TH4-200,日本);磁力搅拌器购买自司乐仪器有限公司(中国上海);电子天平购买自梅特勒-托利多仪器有限公司(中国上海)。
制备例1
HSA-ICG纳米颗粒的制备
按照摩尔比为HSA:ICG=1:1的比例,称取HSA 20mg溶于4mL H2O,对应ICG0.234mg溶于1mL H2O,反应体系5mL,可取2mg/mL ICG溶液0.117mL加水混匀至1mL,与4mLHSA溶液搅拌反应1h后超滤离心(截留分子量30kDa,转速6000r/min,10min,冲洗3次),将超滤后的溶液扩容至5mL,置于培养皿中避光冻存(4℃冰箱30min,-20℃冰箱2h,-80℃冰箱过夜)。利用冻干机将提纯后的HSA-ICG纳米颗粒冻干,避光保存于4℃冰箱以备后续实验使用。
实施例1
I-HSA-ICG纳米颗粒的制备
(1)配置0.01M的PB缓冲液:分别称取7.16g Na2HPO4-12H2O及3.12g NaH2PO4-2H2O分别溶于100ml水溶液,取19mL 0.2mol/mL的Na2HPO4-12H2O溶液,81mL 0.2mol/mL的Na2HPO4-12H2O溶液,混匀(0.2M,pH=7.4)后取50mL,加水稀释至1000mL备用。
(2)称取HSA 20mg、氯胺T 5mg、偏重亚硫酸钠5mg分别溶于1mL PB缓冲液,NaI·2H2O 930mg溶于1mL水。将HSA、氯胺T、NaI·2H2O溶液混匀,利用震荡器震荡反应1min后,加入偏重亚硫酸钠溶液(5mg/mL,1mL)终止反应(反应1min)。将上述反应后的溶液超滤(截留分子量30kDa,转速6000r/min,10min,冲洗3次),然后加高纯水扩容至4mL。在避光条件下,按照摩尔比HSA:ICG=1:1计算对应浓度的ICG,即取2mg/mL ICG溶液0.117mL,加水混匀至1mL,与标过碘的4mL HSA溶液搅拌反应1h后超滤离心(截留分子量30kDa,转速6000r/min,10min,冲洗3次),将超滤后的溶液扩容至5mL,置于培养皿中避光冻存(4℃冰箱30min,-20℃冰箱2h,-80℃冰箱过夜)。利用冻干机将提纯后的I-HSA-ICG纳米颗粒冻干,避光保存于4℃冰箱以备后续实验使用。
实施例2
131I-HSA-ICG纳米颗粒的制备
根据实验所需,穿好铅衣,在通风橱中抽取一定单位计数的放射性Na131I溶液(体积小于1mL,所取计数大于目标计数至少1个单位),根据氯胺T法标记HSA并超滤离心(截留分子量30kDa,转速6000r/min,10min,冲洗3次),重新测滤液的放射性计数(标记率在90%以上),然后将放射性131I-HSA溶液与配好的ICG溶液反应(步骤同上),将反应后的溶液超滤离心至250μL,以备使用。
实验结果参见图2,本申请在中性环境中成功制备得到131I-HSA-ICG纳米颗粒,其具有合适的纳米尺寸(直径25-45nm)和形貌(类球形),使其由于EPR效应长时间滞留在瘤体内,避免其向周围正常组织渗漏。
性能检测
1.长期静置稳定性
固定HSA的浓度不变,取不同浓度的ICG按照不同摩尔比合成I-HSA-ICG溶液(HSA:ICG=1:0.01、1:0.02、1:0.04、1:0.06、1:0.08、1:0.1、1:0.2、1:0.3)(图3a、b);称取适量I-HSA-ICG并配置成1mg/mL溶液,按照一定比例稀释成不同浓度(0.1-1.0mg/mL)(图3c、d),通过UV-3600plus分光光度计(日立,日本)测定上述I-HSA-ICG纳米颗粒的紫外-可见光-近红外吸收光谱,并作出808nm对应的紫外吸光度拟合曲线。
根据紫外吸光度结果推算ICG在HSA上的负载率,计算出4mg/mL HSA-ICG溶液所对应的纯ICG浓度。然后,以超纯水、PB缓冲液(10mM,pH=7.4)、FBS、Gibco 1640培养基作为溶剂分别溶解纯ICG、I-HSA-ICG纳米颗粒,静置观察,在为期14天的不同时间点(0h,0.5h,1h,2h,4h,6h,12h,24h,2d,3d,5d,7d,14d)跟踪拍摄照片。
实验结果参见图4,表明本申请制备的I-HSA-ICG可以在不同溶液介质中保持优异的稳定性。
2.光热性能评估
(1)根据紫外结果计算I-HSA-ICG中ICG的负载率,按照ICG浓度为1mg/mL分别配置纯ICG、HSA-ICG、I-HSA-ICG溶液,使用808nm激光器分别照射超纯水、纯ICG溶液、HSA-ICG溶液、I-HSA-ICG溶液(1mL)5min;再分别照射不同浓度(1mg/mL、2mg/mL、4mg/mL)I-HSA-ICG溶液和超纯水(1mL)5min。在光照液体升温过程中,使用红外热值相机记录超纯水、纯ICG、HSA-ICG和I-HSA-ICG溶液的温度变化热值图像,并每隔30s记录一次温度(图5c-e);光照期间使用磁力搅拌确保样品中热量的均匀分布。
(2)光热转换效率计算
利用808nm激光(2W/cm2)分别照射I-HSA-ICG溶液(取配置的梯度浓度中升温效果较佳的浓度,即4mg/mL,1mL)和超纯水(1mL),待溶液温度不再上升后停止照射,自然冷却至最初环境温度,期间每半分钟记录一次温度(图5a、5b)。通过下述公式计算I-HSA-ICG纳米颗粒的光热转换效率(η):
上述公式中,热传导系数表示为h,容器的表面积表示为s。在稳定的环境温度中所测得的I-HSA-ICG纳米颗粒溶液的温度变化最大值表示为Tmax-Tsurr。容器和溶剂所吸收的光辐射热量表示为Q0。激光辐射的功率密度以I表示,I-HSA-ICG纳米颗粒在808nm处的吸光度则表示为A。
体外升温实验证实131I-HSA-ICG纳米颗粒的近红外区吸光度优良,体外升温效果显著,可升温25℃左右,同比水温未见明显上升,其光热转换效率可达24.25%。
3.体外细胞毒性实验
首先评估HSA-ICG、I-HSA-ICG纳米颗粒对未分化的人未分化甲状腺癌细胞(AROcell)的毒性。另单独评估HSA-ICG纳米颗粒对小鼠乳腺癌细胞(4T1 cell)、低分化的人甲状腺癌细胞(FRO cell)的毒性。将10%FBS加入含有1%链霉素-青霉素的Gibco 1640,配置成ARO、FRO的细胞培养基;4T1细胞的培养基则是将10%FBS加入含有1%链霉素-青霉素的Gibco DMEM中混合而成。将细胞均放在90mm的培养皿中并置于含有5%CO2的37℃恒温孵箱中培养。按照1×104/孔的密度将ARO细胞接种在96孔板内,放入孵箱培养24h后,使用PBS缓冲液(10mM,pH=7.4)从侧壁轻轻冲洗每个孔,洗掉未贴壁的死细胞。取冻干后的HSA-ICG粉末,用培养基溶解并配成3.2、1.6、1.4、1.2、1.0、0.8、0.6、0.4、0.2、0mg/mL(现配现用),用于评估该纳米颗粒对4T1、FRO细胞的毒性;取冻干后的HSA-ICG、I-HSA-ICG粉末用培养基分别配成1.0、0.8、0.6、0.4、0.2、0mg/mL(现配现用),用于评估它们对ARO细胞的毒性。按每个孔200μL的体积向96孔板内加入上述溶液,并继续培养24h。次日继续使用PBS冲洗1次,每个孔内加入10μL MTT(5mg/mL)溶液及190μL新鲜培养基,3-4h后轻轻从侧壁吸走含有MTT的培养基,不要破坏孔底部的紫色甲臜结晶,每孔加入150μL DMSO,低速震荡10min以上,待紫色结晶溶解后利用酶标仪测定每个孔在492nm处的吸光度(OD)。细胞存活率由下述公式计算得到。
Cell viability=ODeg/ODcg×100%
其中ODeg代表不同浓度的纳米颗粒处理过的实验组细胞在492nm处的吸光度值,ODcg代表空白对照组细胞在492nm处的吸光度值。
实验结果如图6所示,从图6a、6b可以看出,HSA-ICG纳米颗粒对ARO、FRO、4T1三种癌细胞系均无明显毒副作用,I-HSA-ICG纳米颗粒处理后的ARO细胞可存活70%以上。
4.体外细胞放射性核素杀伤实验
在96孔板内按照3×103/孔的密度接种ARO细胞,置于含有5%CO2的37℃恒温孵箱中培养24h后,分为两组,每组两个板,分别加入不同放射性梯度计数的131INa、131I-HSA-ICG溶液(放射性计数0、7.81、15.63、31.25、62.5、125、250、500μCi,CICG=10ppm),溶剂均为配置好的新鲜培养基,培养24h后使用标准MTT法分别测定其细胞存活率(图7a)。
5.131I-HSA-ICG纳米颗粒对体外肿瘤细胞的光热杀伤实验
按照5×103/孔的密度在96孔板中隔孔接种ARO细胞,将96孔板放入37℃恒温孵箱(含5%CO2)中培养24h后,用PBS冲洗每个孔以除去死细胞,每孔分别加入200μL不同浓度的I-HSA-ICG溶液(0、0.5、1.5mg/mL),培养24h后,使用808nm激光(0、1.0、1.5、2.5W/cm2)照射每个孔5min,使用标准MTT法测定各组细胞存活率(图7b)。再取一个96孔板,按照上述方法种板,每孔分别加入200μL PBS溶液及不同ICG浓度的131I-HSA-ICG溶液(CICG=5、10ppm),培养24h后,808nm激光(0、1.5、2.5W/cm2)照射每个孔5min,使用标准MTT法测定各组细胞存活率(图7c)。
6.放射性核素治疗联合PTT的体外肿瘤细胞杀伤实验
按照5×103/孔的密度在96孔板内接种ARO细胞,再将96孔板放入37℃恒温孵箱(含5%CO2)中培养,24h后将其分为6组处理每孔细胞:PBS、Na131I(500μCi/孔)、131I-HSA-ICG(500μCi/孔)、PBS+光照、HSA-ICG+光照、131I-HSA-ICG+光照(HSA浓度为1mg/mL,ICG浓度为0.0117mg/mL),溶剂均为配好的新鲜1640培养基。培养24h后,使用808nm激光照射每个孔(2.5W/cm2,5min),并使用标准MTT法测定各组细胞存活率。另取一个96孔板重复上述操作,在808nm激光照射完毕后,使用PBS轻轻冲洗每个孔两遍,每个孔中加入1:1混合的碘化丙啶(PI)和钙黄绿素乙酰氧基甲酯(Calcein-AM)对细胞进行染色,将染色后的细胞放入孵箱内温育15min,利用PBS多次(2遍以上)小心冲洗每个孔,避免吸走贴壁不牢固的细胞,随后再加入200μL PBS保持细胞活性,避光环境下,通过倒置荧光显微镜获取死活细胞的荧光图像。
实验结果如图8所示,从图8a、8b可以看出,131I-HSA-ICG纳米颗粒处理组、Na131I处理组及HSA-ICG纳米颗粒+光照组的存活率低于PB组,而131I-HSA-ICG纳米颗粒+光照处理组的ARO细胞存活率最低,表明联合治疗效果优于单一治疗。
7.未分化甲状腺癌荷瘤鼠模型构建
选取若干只4-5周的雄性BALB/c裸鼠(15-18g)(中国北京华阜康生物科技股份有限公司),将ARO肿瘤细胞种植在小鼠左侧(或右侧)大腿背侧皮下,待小鼠皮下肿瘤长大至直径6mm左右时对其进行活体放射性核素联合PTT治疗。对需要注射含有131I的所有裸鼠在注射前一周需喂服含1%NaI的水溶液,注射前一天需腹腔注射含1%NaI的水溶液1mL,以封闭裸鼠甲状腺避免其在治疗过程中摄取放射性131I。
8.I-HSA-ICG的动物活体毒理实验
将4-5周的雄性昆明小鼠(15-18g)(中国北京华阜康生物科技股份有限公司)随机分成2组(A-B),每组5只,A组:尾静脉注射生理盐水100μL,B组:尾静脉注射I-HSA-ICG溶液100μL(5mg/mL),观察20天并记录不同时间点的小鼠体重变化,20天后取出小鼠心、肝、脾、肺、肾进行HE染色,取眼球血进行生化检验,观察药物对各个脏器有无毒性。
实验结果如图9所示,从图9可以看出,对照组及I-HSA-ICG纳米颗粒处理组小鼠体重均稳定上升,血生化指标均无明显异常,观察20天后的病理H&E染色显示I-HSA-ICG纳米颗粒对小鼠各个脏器均无明显毒性作用,表明该纳米颗粒生物相容性良好。
9.131I-HSA-ICG纳米颗粒肿瘤局部滞留效果评估
本申请通过放射性核素显像及小动物活体荧光成像来评估131I-HSA-ICG纳米颗粒肿瘤局部滞留效果。将荷瘤裸鼠随机分成4组(a,b,c,d),每组3只,显像前均进行腹腔麻醉(2.5%水合氯醛溶,130-150μL/只),a组:瘤内注射纯ICG溶液(CICG=0.1mg/mL)100μL,b组:瘤内注射I-HSA-ICG溶液(CICG=0.1mg/mL)100μL,c组:瘤内注射Na131I溶液(800μCi/只)100μL,d组:瘤内注射131I-HSA-ICG溶液(800μCi/只,HSA浓度4mg/mL,ICG浓度0.04mg/mL)100μL,分别在注射药物前、注射药物后8天内的不同时间点对小鼠进行显像,其中a、b组利用小动物活体荧光成像仪器进行图像采集,c、d组利用SPECT/CT进行图像采集。
实验结果如图10所示,从图10可以看出,I-HSA-ICG纳米颗粒在瘤体内可滞留较长时间,荧光滞留时间可达7天以上,具有良好的局部肿瘤荧光成像效果,而纯ICG则很快扩散出去;放射性核素SPECT/CT显像结果显示,相对于Na131I,131I-HSA-ICG纳米颗粒在瘤体内的滞留时间较长(8天左右,接近放射性131I的半衰期),有利于进行放射性核素治疗。
10.放射性核素治疗联合PTT的活体治疗
将荷瘤小鼠随机分成5组(A-G),每组3只,治疗前均进行腹腔麻醉(2.5%水合氯醛溶,130-150μL/只),A组:瘤内注射100μL PB缓冲液(10mM,pH=7.4)并进行808激光照射10min,B组:瘤内注射Na131I溶液100μL(800μCi/只),C组:瘤内注射131I-HSA-ICG溶液100μL(800μCi/只,HSA浓度4mg/mL,ICG浓度0.04mg/mL),D组:瘤内注射HSA-ICG溶液100μL(HSA浓度4mg/mL,ICG浓度0.04mg/mL)并使用808nm激光照射10min,E组:瘤内注射131I-HSA-ICG溶液100μL(800μCi/只,HSA浓度4mg/mL,ICG浓度0.04mg/mL)并使用808nm激光照射10min,上述光照过程中均使用热值相机进行照射部位局部升温情况记录并拍照。所有治疗结束后,在为期17天的时间里观察治疗后情况,在不同时间点记录裸鼠体重及肿瘤体积变化并拍照,17天后取出小鼠肿瘤并称重。
实验结果如图11-12所示,活体治疗实验也显示131I-HSA-ICG纳米颗粒+光照处理组的肿瘤组织明显消融,且随观察期延长,未见明显复发倾向,呈持续低水平;而HSA-ICG纳米颗粒+光照组在一周后开始出现肿瘤复发迹象,131I-HSA-ICG纳米颗粒处理组、Na131I处理组小鼠肿瘤虽然增长缓慢,其速度明显快于131I-HSA-ICG纳米颗粒+光照处理组,证实本申请131I-HSA-ICG纳米颗粒具有明显的放射性杀伤作用及光热肿瘤消融效果。
本申请的数据均表示为平均值±标准差。通过单向ANOVA分析或者双尾t检验比较组间的差异。p值小于0.05时代表其具有显著统计学差异(p<0.05)。
本具体实施方式的实施例均为本发明的较佳实施例,并非依此限制本发明的保护范围,故:凡依本发明的结构、形状、原理所做的等效变化,均应涵盖于本发明的保护范围之内。
Claims (7)
1.一种131I-HSA-ICG纳米颗粒,其特征在于:所述纳米颗粒为在人血清白蛋白上负载吲哚菁绿并标记放射性核素131I制备得到的粒径为25-45nm的颗粒。
2.一种权利要求1所述131I-HSA-ICG纳米颗粒的制备方法,其特征在于:包括以下步骤:
S1.将HSA、氯胺T和偏重亚硫酸钠分别溶于缓冲液中;
S2.抽取一定单位计数的放射性Na131I溶液:根据细胞或者动物实验需求计算用量,131I的标记率在90%以上;
S3.将HSA、氯胺T、Na131I溶液混匀,震荡反应1-2min后,加入偏重亚硫酸钠溶液终止反应,再将得到的溶液进行超滤离心;
S4.将步骤S3得到的放射性131I-HSA溶液与ICG溶液按照摩尔比为HSA:ICG=1:1的比例混合,反应1-1.5h,再将得到的溶液进行超滤离心,即得。
3.根据权利要求2所述的131I-HSA-ICG纳米颗粒的制备方法,其特征在于:步骤S1中,HSA的浓度为20-60mg/mL。
4.根据权利要求2所述的131I-HSA-ICG纳米颗粒的制备方法,其特征在于:步骤S3中,溶液超滤离心后,测定滤液放射性标记率≥90%。
5.根据权利要求2所述的131I-HSA-ICG纳米颗粒的制备方法,其特征在于:步骤S3和S4中,超滤离心的处理条件为截留分子量30kDa,转速6000-8000r/min,8-10min,冲洗2-3次。
6.一种权利要求1所述131I-HSA-ICG纳米颗粒在制备肿瘤显像剂中的应用。
7.一种权利要求1所述131I-HSA-ICG纳米颗粒在制备治疗未分化型甲状腺癌药物中的应用。
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