CN114601938A - 一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针 - Google Patents
一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针 Download PDFInfo
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Abstract
一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针,涉及医学技术领域。所述荧光/核磁探针为靶向VEGF‑A的GdDTPA‑HSA@ICG‑贝伐单抗近红外二区荧光/磁共振双模态分子探针。制备方法:1)制备GdDTPA‑HSA@ICG纳米颗粒;2)制备GdDTPA‑HSA@ICG‑贝伐单抗近红外二区荧光/磁共振双模态探针。所述探针作为造影剂用于实现靶向乳腺癌术前行MRI成像,评估肿瘤性质并制定保乳术方案,为术中保乳切缘评估提供重要的术前精准影像学参考;作为分子影像探针,在术中行近红外二区荧光手术导航,结合术前MRI评估,以实现术中精准示踪乳腺肿瘤边界。
Description
技术领域
本发明涉及医学技术领域,尤其是涉及基于近红外二区荧光、核磁双模态成像引导的一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针。
背景技术
乳腺癌是全球女性最常见的恶性肿瘤,保乳术已成为早期乳腺癌的标准手术方式。保乳术成功的关键在于肿瘤的完整切除,并能兼顾术后美观。当前,术中冰冻病理是较为成熟的评价肿瘤切缘的病理技术手段,但是术中冰冻病理为非连续取材,容易导致漏检且耗时长。导致保乳手术切缘阳性的另一主要原因是术中医生仅能通过“眼看、手触”等主观感受评估切缘状态,因此,如何精准、实时判定保乳术切缘状况是避免二次手术的关键。
目前临床上主要采用术中钼靶进行辅助切缘判定,该方法容易遗漏非钙化病灶、敏感性较低;术中超声对深部肿物诊断符合率低,且对微钙化灶检测不敏感。因此亟需在保乳术中切缘判定中引入一种精准、实时的检测新技术以准确判断切缘状况。光学分子影像技术具有高灵敏度、高特异性、无电离辐射等优点,特别是因其能够直观实时、可视化病灶,目前已开始应用到肿瘤外科术中实时手术导航领域。申请人与荷兰团队前期已基于贝伐单抗-IRDye800CW分子探针(靶向VEGF-A),完成在体、实时判定保乳切缘状态的II期临床研究,但由于NIR-I荧光成像下易受激发光和背景自发荧光的干扰,其成像分辨率低、特异性低且成像穿透深度浅。此外,单一光学模态的分子探针无法满足保乳术前需行MRI精准评估的需求,因而亟需探索构建融合NIR-II/MRI双模态分子影像探针,以期提高肿瘤成像分辨率、特异性及穿透深度。
发明内容
本发明的目的在于突破保乳术仅凭术者的经验以“眼看、手触”决定保乳切缘边界,无法精准评估切缘状态的临床局限,提供一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针,设计并制备NIR-II/MRI双模态分子探针,利用术前MRI评估乳腺肿瘤性质、制定手术计划,供保乳术中参考,而在术中利用NIR-II分子影像技术引导保乳切缘精准判定。利用双模态成像技术引导保乳切缘的评估这一新理念,将解决单一光学模态成像技术用于手术导航的敏感性及特异性不足的问题,本发明有望提高保乳术切缘评估的精准性。
本发明所述荧光/核磁探针为靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针,所述荧光/核磁探针包括GdDTPA-HSA纳米颗粒、包裹在GdDTPA-HSA纳米颗粒表层的ICG、脂质体NHS-PEG2000-COOH、贝伐单抗,名称为GdDTPA-HSA@ICG-贝伐单抗(简称NPs-Bev)。
所述荧光/核磁探针的粒径约为5~10nm。
所述荧光/核磁探针的制备方法包括以下步骤:
1)制备GdDTPA-HSA@ICG纳米颗粒:
将人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)混合,在pH=8.0~10.0的条件下连接,后调pH=6.0~7.0加入0.5~0.6倍当量GdCl3得到GdDTPA-HSA纳米颗粒,用质谱验证Gd连接效率,利用人血清白蛋白(HSA)的疏水口袋装入吲哚青绿(ICG),利用光谱验证ICG包裹成功,利用透射电镜验证纳米颗粒的合成;
在步骤1)中,所述人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA )的质量比可为1︰1至1︰1.5;优选在pH=8.2的条件下进行连接,后调pH=6.5加入GdCl3得到GdDTPA-HSA纳米颗粒。
2)制备GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态探针:
选择NHS-PEG2000-COOH将GdDTPA-HSA@ICG纳米颗粒与靶向元件贝伐单抗(Bev)连接,利用催化剂量的1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化,得到所述靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针。
在步骤2)中,所述GdDTPA-HSA@ICG纳米颗粒与靶向元件贝伐单抗(Bev)的质量比可为1︰2至1︰3。
所制备的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态探针可在引导乳腺癌保乳切缘评估中应用。作为造影剂可实现靶向乳腺癌术前行MRI成像,评估肿瘤性质并制定保乳术方案,为术中保乳切缘评估提供重要的术前精准影像学参考;作为分子影像探针,在术中行近红外二区荧光手术导航,结合术前MRI评估,实现术中精准示踪乳腺肿瘤边界,并完成精准切除。
本发明建立NIR-II/MRI双模态分子影像引导的乳腺肿瘤精准切除的手术导航体系。与现有技术相比,本发明的有益效果在于:
本发明将进一步制备、表面改性及优化靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗的NIR-II/MRI双模态分子探针,将实现术中可视化保乳边界,旨在建立NIR-II/MRI双模态分子影像引导的乳腺肿瘤精准切除的手术导航体系。
实验表明,本发明的分子探针具有生物相容性高,靶向乳腺癌高表达受体VEGF-A靶向性好,近红外二区荧光性能稳定、成像效果好的优点。相对于临床常用的钆喷酸葡胺具有更好的MRI成像效果,信号背景比钆喷酸葡胺组超过2倍,相对于白光手术(直视下),本发明导引下的乳腺癌切除术的局部复发率降低60%,总生存率达到100%。
附图说明
图1为NPs-Bev的电镜图。
图2为NPs-Bev的光学表征图。其中:左图为吸收波;中图为发射波;右图为近红外二区拖尾信号。
图3为NPs-Bev的磁学表征图。
图4为NPs-Bev细胞荧光靶向性图实验的效果图。
图5为NPs-Bev探针在MDA-MB-231荷瘤小鼠动物靶向性实验的效果图。
图6为NPs-Bev探针在MDA-MB-231荷瘤小鼠中的靶向性实验的效果图。
图7为NPs-Bev引导下的乳腺癌手术精准切除实验示意图。
图8为NPs-Bev引导下的乳腺癌病理和荧光切片匹配图。
图9为NPs-Bev引导下的乳腺癌手术术后复发情况实验参考图。
图10为NPs-Bev探针合成模式图。
具体实施方式
为了使本发明创造的目的及优点更加清楚明白,以下结合附图及实施例,对本发明创造进行进一步详细说明。
本发明实施例所述荧光/核磁探针为靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针,所述荧光/核磁探针包括GdDTPA-HSA纳米颗粒、包裹在GdDTPA-HSA纳米颗粒表层的ICG、脂质体NHS-PEG2000-COOH、贝伐单抗,名称为GdDTPA-HSA@ICG-贝伐单抗(简称NPs-Bev)。所述荧光/核磁探针的粒径约为5~10nm。
所述靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针的合成包括以下步骤:
1)制备GdDTPA-HSA@ICG纳米颗粒:将人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)按质量比1︰1至1︰1.5混合,在pH=8.2的条件下进行连接,后调pH=6.5加入GdCl3得到GdDTPA-HSA纳米颗粒,用质谱验证Gd连接效率,利用HSA的疏水口袋装入ICG,利用光谱验证ICG包裹成功,利用透射电镜验证纳米颗粒的合成。
2)制备GdDTPA-HSA@ICG-贝伐单抗荧光/磁共振双模态探针:选择NHS-PEG2000-COOH将GdDTPA-HSA@ICG纳米颗粒按质量比1︰2至1︰3混合,随后与靶向元件贝伐单抗(Bev)连接,利用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化,得到靶向肿瘤的贝伐单抗荧光/磁共振双模态探针。另外,用IgG构建对照探针GdDTPA-HSA@ICG-IgG。
以下结合附图给出具体实施例。
1.设计并制备靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针(NPs-Bev探针的合成模式图参见图10)
1.1制备GdDTPA-HSA@ICG纳米颗粒:将一定量的人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)在pH=8.2的条件下进行连接,后调pH=6.5加入GdCl3得到GdDTPA-HSA纳米颗粒,用质谱验证Gd连接效率,利用HSA的疏水口袋装入ICG,利用光谱验证ICG包裹成功,利用透射电镜验证纳米颗粒的合成。
1.2制备GdDTPA-HSA@ICG-贝伐单抗荧光/磁共振双模态探针:选择NHS-PEG2000-COOH将GdDTPA-HSA@ICG纳米颗粒与靶向元件贝伐单抗(Bev)连接,利用1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化,得到靶向肿瘤的贝伐单抗荧光/磁共振双模态探针。另外,用IgG构建对照探针GdDTPA-HSA@ICG-IgG。
2.体外和在体水平评估分别评估荧光/磁共振双模态分子探针的安全性以及靶向性
2.1体外水平研究:
1)亚细胞水平分布:利用荧光显微镜检测探针在亚细胞水平的分布;2)探针对细胞性能的影响:在细胞水平设置探针孵育时间、浓度梯度,对分子探针进行体外安全性及适用性评价;3)靶向性研究:探针与不同VEGF-A表达水平的乳腺癌细胞共孵育后,用荧光成像、流式等方法进行多角度分析,验证探针的VEGF-A靶向性;
2.2在体水平研究:
1)评估分子探针的在体安全性:根据探针在小鼠体内各个脏器的探针影像信号强度判断其代谢途径和生物分布,最后根据探针对小鼠体重及各个脏器组织学水平的影响初步评价在体毒性;2)靶向性研究:取乳腺癌皮下移植瘤模型鼠15只,随机分为GdDTPA-HSA@ICG组、GdDTPA-HSA@ICG-贝伐单抗组和GdDTPA-HSA@ICG-贝伐单抗+贝伐单抗阻断组,每组各5只,分别经尾静脉注射最佳剂量的相应探针,贝伐单抗阻断组每只小鼠尾静脉同时注入最佳剂量GdDTPA-HSA@ICG-贝伐单抗和适量贝伐单抗,在最佳观测时间置于Edmund filtersNIR-Ⅱ荧光成像系统采集肿瘤荧光信号,分析对比三组间的信号背景比。
3.在小动物活体水平评估GdDTPA-HSA@ICG-贝伐单抗NIR-II/MRI双模态分子探针对乳腺癌切缘精准评估的有效性
3.1构建乳腺癌移植瘤小鼠模型:1)将处于对数生长期的MDA-MB-231-Luc细胞消化离心制备浓度为5×107个/ml细胞悬液。2)常规消毒后,在BALB/c-Nude雌性小鼠左侧第四对乳房下方脂肪垫部位,注射100μl体积的细胞悬浮液,细胞总数为5×106个细胞。3)术后每隔3天用D-萤光素钾盐(Beyotime,#ST196)进行生物发光监测肿瘤的生长。
3.2术前核磁共振及术中NIR-II荧光成像系统引导的小鼠原位乳腺移植瘤手术导航:1)取乳腺癌原位移植瘤模型小鼠20只,尾静脉注射最佳浓度的探针,在探针最佳观察时间,麻醉;2)术前磁共振成像评估肿瘤病灶情况:对麻醉好的小鼠进行T1磁共振成像(参数同上)评估肿瘤解剖位置、边界及微卫星灶等情况;3)术中荧光成像引导乳腺癌精准切除:将上述完成磁共振成像的小鼠立即仰卧位固定于黑纸板上,常规消毒、切开皮肤,暴露小鼠左侧第4对乳房,置于Edmund filters NIR-Ⅱ荧光成像系统下检测癌灶边缘并进行标记,而后对有荧光信号的组织进行完整切除,再次检测癌床是否有荧光信号残留,将残留荧光信号的组织继续切除,并取下一块正常乳腺组织,术后常规消毒切口、缝合、镇痛。4)比较在体及离体影像学信息、石蜡块及切片近红外荧光图像、病理学诊断和免疫组化染色分析,观察原位乳腺癌组织中荧光分布、病理信息、分子靶标是否具有一致性。最后通过生物发光的方式随访小鼠肿瘤局部残留情况,并对比各组小鼠生存情况。
探针形貌和尺寸检测:将少量样品稀释至浅绿色。吸取10μl溶液滴加在超薄碳膜上,室温晾干。探针稀释于PBS,取部分悬浮液滴于铜网,自然干燥,并采用透射电子显微镜,观察样品的形貌、分布和大小,选取清晰、无污染的视野拍照保存。如图1所示,探针在电镜下粒径较为均一,分散性好,大小约为5~10nm。
探针光谱检测:取适量探针溶于不同溶剂中,将样品装进比色皿,置于样品仓中。利用酶标仪检测400~900nm探针吸光度。利用荧光光谱仪检测其荧光发射光谱(近红外1区:激发:720nm发射:750~900nm;近红外2区:激发:808nm发射:1000~1400nm)实验结果参见图2:如图2(左)的吸收光谱所示,探针在780nm左右有最大吸收峰,说明NPs-Bev的合成未改变ICG的荧光性能。如图2(中)的近红外一区荧光光谱所示,探针在800nm左右有最大的发射强度。如图2(右)的近红外二区荧光光谱所示,该探针在808nm的激发光下,在近红外二区有较强的荧光拖尾信号,说明该探针可用于近红外二区荧光成像。
磁学表征:取适量探针在PBS中稀释稀释至不同浓度,利用去离子水作为对照,利用台式磁共振弛豫测量系统测量纵向弛豫时间(T1),并利用1/T1与Gd浓度的曲线计算样品的纵向弛豫率(γ1)。所有样本采用T1加权成像。实验结果参见图3:不同的Gd3+离子浓度具有不同T1磁共振信号,利用1/T1与Gd浓度的曲线计算样品的纵向弛豫率(γ1)为7.53。说明该探针有较好的T1弛豫信号,可用于磁共振成像。
图4给出NPs-Bev细胞荧光靶向性图。探针共孵育法定量分析内源性VEGF-A不同表达水平的乳腺癌细胞对纳米探针的摄入能力,通过12孔板进行细胞爬片,待细胞贴壁后加入4μg/ml浓度纳米探针,进行探针孵育,孵育4h。从图4可以看出,所合成的探针用于孵育不同VEGF-A的乳腺癌细胞(MDA-MB-231细胞为高表达VEGF-A),证明在细胞水平探针具有特异性靶向VEGF-A的能力。
图5给出NPs-Bev探针在MDA-MB-231荷瘤小鼠动物靶向性实验结果图。MDA-MB-231乳腺癌细胞进行皮下种植,共得移植瘤裸鼠6只,随机分为2组。每组小鼠通过尾静脉分别注射NPs-Bev和NPs-IgG对照探针进行多个时间点的荧光成像。实验结果显示,种植MDA-MB-231细胞的荷瘤小鼠尾静脉注射NPs-Bev探针后会在肿瘤部位特异性聚集,而NPs-IgG对照组探针未见显著聚集,二组间近红外荧光信号具有差异性,并在36h达到最佳信号背景比的差别,证明该探针在小动物体内具有特异靶向VEGF-A的能力。
图6给出NPs-Bev探针在MDA-MB-231荷瘤小鼠中的靶向性实验结果图。MDA-MB-231乳腺癌细胞进行皮下种植,共得移植瘤裸鼠9只,随机分为9组,每组小鼠通过尾静脉分别注射NPs-Bev,NPs-IgG对照探针和Gd-DTPA对照造影剂,分别于注射前及注射后多个时间点使用9.4T小动物磁共振成像设备采集肿瘤磁共振图像,通过软件分析各组在不同时间点的信号强度差异。从图6可以看出,核磁共振成像显示种植MDA-MB-231细胞的荷瘤小鼠尾静脉注射NPs-Bev探针后会在肿瘤部位特异性聚集,而NPs-IgG,Gd-DTPA对照组探针不会。磁共振可检测其信号差异,并在12h达到最佳信号背景比的差别,证明该探针在小动物体内具有特异靶向VEGF-A的能力。
图7给出NPs-Bev引导下的乳腺癌手术精准切除实验示意图。MDA-MB-231乳腺癌细胞进行皮下种植,共得移植瘤裸鼠16只,随机分为2组。每组小鼠通过尾静脉分别注射NPs-Bev和NPs-IgG对照探针,最佳成像时间36小时的时候,进行荧光成像并在近红外二区手术导航仪进行手术切除移植瘤。实验结果显示,注射NPs-Bev探针在近红外荧光二区手术导航仪可有效引导下的乳腺癌手术精准切除,直至肿瘤瘤腔未见荧光信号,并标记切除癌组织为(T1/T2/T3),证明该探针在小动物有效导引乳腺癌移植瘤切除术。
图8给出NPs-Bev引导下的乳腺癌病理和荧光切片匹配图。MDA-MB-231乳腺癌细胞进行皮下种植,共得移植瘤裸鼠16只,随机分为2组。每组小鼠通过尾静脉分别注射NPs-Bev和NPs-IgG对照探针,最佳成像时间36小时的时候,进行荧光成像并在近红外二区手术导航仪进行手术切除移植瘤,切除切除癌组织为(T1/T2/T3),随后进行病理HE染色及荧光切片扫描。实验结果显示,注射NPs-Bev探针在近红外荧光二区手术导航仪可有效引导下的乳腺癌手术精准切除,直至肿瘤瘤腔未见荧光信号,并标记切除癌组织为(T1/T2/T3),切片的病理(HE)与荧光信号相互匹配,证明该探针在小动物水平可有效靶向乳腺癌移植瘤。
图9给出NPs-Bev引导下的乳腺癌手术术后复发情况实验参考图。MDA-MB-231乳腺癌细胞进行皮下种植,共得移植瘤裸鼠16只,随机分为2组。每组小鼠通过尾静脉分别注射NPs-Bev和NPs-IgG对照探针,最佳成像时间36小时的时候,进行荧光成像并在近红外二区手术导航仪进行手术切除移植瘤,并对复发情况及生存时间进行随访记录。实验结果显示,注射NPs-Bev探针对比对照组NPs-IgG,在近红外荧光二区手术导航仪可有效引导下的乳腺癌手术精准切除,该组的术后复发低,总生存率更长。
上述实施例仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (8)
1.一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针,其特征在于其为靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针,所述荧光/核磁探针包括GdDTPA-HSA纳米颗粒、包裹在GdDTPA-HSA纳米颗粒表层的ICG、脂质体NHS-PEG2000-COOH、贝伐单抗,命名为GdDTPA-HSA@ICG-贝伐单抗,简称NPs-Bev。
2.如权利要求1所述一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针,其特征在于所述荧光/核磁探针的粒径为5~10nm。
3.如权利要求1所述一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针的制备方法,其特征在于包括以下步骤:
1)制备GdDTPA-HSA@ICG纳米颗粒:
将人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)混合,在pH=8.0~10.0的条件下连接,后调pH=6.0~7.0加入0.5~0.6倍当量GdCl3得到GdDTPA-HSA纳米颗粒,用质谱验证Gd连接效率,利用人血清白蛋白(HSA)的疏水口袋装入吲哚青绿(ICG),利用光谱验证ICG包裹成功,利用透射电镜验证纳米颗粒的合成;
2)制备GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态探针:
选择NHS-PEG2000-COOH将GdDTPA-HSA@ICG纳米颗粒与靶向元件贝伐单抗(Bev)连接,利用催化剂量的1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDC)和N-羟基琥珀酰亚胺(NHS)将羧基活化,得到所述靶向VEGF-A的GdDTPA-HSA@ICG-贝伐单抗近红外二区荧光/磁共振双模态分子探针。
4.如权利要求3所述一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针的制备方法,其特征在于在步骤1)中,所述人血清白蛋白(HSA)与二乙基三胺五乙酸(DTPA)的质量比为1︰1至1︰1.5。
5.如权利要求3所述一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针的制备方法,其特征在于在步骤1)中,在pH=8.2的条件下进行连接,后调pH=6.5加入GdCl3得到GdDTPA-HSA纳米颗粒。
6.如权利要求3所述一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针的制备方法,其特征在于在步骤2)中,所述GdDTPA-HSA@ICG纳米颗粒与靶向元件贝伐单抗(Bev)的质量比为1︰2至1︰3。
7.如权利要求1所述一种引导乳腺癌保乳切缘精准评估的荧光/核磁探针在引导乳腺癌保乳切缘评估中应用。
8.如权利要求7所述应用,其特征在于其具体包括:作为造影剂用于实现靶向乳腺癌术前行MRI成像,评估肿瘤性质并制定保乳术方案,为术中保乳切缘评估提供重要的术前精准影像学参考;作为分子影像探针,在术中行近红外二区荧光手术导航,结合术前MRI评估,以实现术中精准示踪乳腺肿瘤边界。
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