CN115073726B - 一种靶向m2型巨噬细胞甘露糖受体的超声分子探针及其制备方法与应用 - Google Patents
一种靶向m2型巨噬细胞甘露糖受体的超声分子探针及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种靶向M2型巨噬细胞甘露糖受体的超声分子探针及其制备方法与应用,属于医学诊断技术领域。该超声分子探针的制备过程包括以下步骤:(1)合成二硬脂酰磷脂酰乙醇胺‑聚乙二醇‑甘露糖;(2)选用二硬脂酰磷脂酰乙醇胺‑聚乙二醇‑甘露糖与二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺‑聚乙二醇2000制备超声分子探针。该探针对M2型巨噬细胞具有良好的亲和力,可将其应用于心脏移植慢性排斥反应的早期诊断,且具有高敏感性、无创、无辐射、实时动态监测的优点,能够实现慢性排斥反应的早期诊断。
Description
技术领域
本发明属于医学诊断技术领域,尤其涉及一种靶向M2型巨噬细胞甘露糖受体的超声分子探针及其制备方法与应用。
背景技术
临床上常用的诊断心脏移植物血管病变(Cardiac allograft vasculopathy,CAV)的技术主要包括冠脉造影及血管内超声,皆为有创性技术,其费用昂贵且并发症的风险较高。常规超声心动图、冠状动脉造影及血管内超声等技术提示CAV时,病变已发展为中晚期,对早期病变缺乏敏感性,限制了临床应用。超声靶向分子成像在器官移植领域主要应用于监测器官移植术后排斥反应及血管栓塞等情况,评价移植物的炎症反应并进行靶向治疗,具有无创、无辐射、早期诊断、实时动态监测等优势。目前已有大量研究证实了巨噬细胞在慢性排斥反应中对器官移植物的重要作用,并且有研究发现,M2型巨噬细胞与心脏移植术后CAV发生冠脉微循环病变及心肌纤维化密切相关。因此,通过靶向示踪M2型巨噬细胞来监测心脏移植术后CAV的发生与发展,具有良好的应用前景。甘露糖受体是一种在单核/巨噬细胞细胞质内表达的特异性抗原,可用于定位M2型巨噬细胞,为无创靶向监测这一免疫反应过程提供可能。目前尚未有针对甘露糖受体的超声分子探针应用于心脏移植慢性排斥反应的早期诊断。
发明内容
本发明提供了一种靶向M2型巨噬细胞甘露糖受体的超声分子探针的制备方法,所述制备方法包括以下步骤:
(1)合成二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖
将4-异硫氢酸苯基-a-D-甘露糖苷与二硬脂酰基磷脂酰乙醇胺聚乙二醇氨基混合,溶解于无水二氯甲烷中,并加入无水三乙胺进行催化,抽真空后通入氮气保护,室温下反应6-8h;
旋转蒸发仪去除多余的无水二氯甲烷,加入适量的超纯水溶解产物,并透析,将透析后的液体冻干后即获得产物二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖,其结构式如下:
(2)制备超声分子探针
将二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖与二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000分别溶于三氯甲烷,获得浓度分别为18-22mg/ml的磷脂溶液;
将二硬脂酰基磷脂酰胆碱溶液、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶液、二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖溶液按摩尔比9:0.5:0.5移入试管中,混匀不同的磷脂成分,在氮气流的作用下使氯仿挥发掉,直至在试管壁上形成一层薄薄的磷脂薄膜,抽真空去除多余的三氯甲烷;
在试管中加入水化液,将得到的磷脂溶液加热至55-60℃,使溶液分散至透明,即获得超声分子探针;
所述水化液含有10%(体积比)甘油、10%(体积比)1,2丙二醇和80%(体积比)的0.1M的tris溶液,用盐酸调其PH值到7.4。
优选地,所述步骤(1)中4-异硫氢酸苯基-a-D-甘露糖苷与二硬脂酰基磷脂酰乙醇胺聚乙二醇氨基的摩尔比为1:2-1:10。
更优选地,所述步骤(1)中无水三乙胺的加入量为10μl。
更优选地,所述步骤(2)中二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖与二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000分别溶于三氯甲烷,获得浓度分别为20mg/ml的磷脂溶液。
更优选地,所述步骤(2)中水化液的加入量为4-6ml。
更优选地,所述步骤(2)中二硬脂酰基磷脂酰胆碱溶液、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶液、二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖溶液的摩尔比为9:0.5:0.5。
本发明的另一方面提供了一种诊断试剂,所述诊断试剂包括上述靶向M2型巨噬细胞甘露糖受体的超声分子探针。
本发明的另一方面提供了靶向M2型巨噬细胞甘露糖受体的超声分子探针在制备心脏移植慢性排斥反应诊断试剂中的应用。
本发明的另一方面提供了上述超声分子探针制备方法在制备心脏移植慢性排斥反应诊断试剂中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明制备了一种靶向甘露糖受体的超声分子探针,其对M2型巨噬细胞具有良好的亲和力,可将其应用于心脏移植慢性排斥反应的早期诊断,且具有高敏感性、无创、无辐射、实时动态监测的优点,能够实现慢性排斥反应的早期诊断。
附图说明
图1为实施例1中超声分子探针的结构示意图。
图2为试验例1中微泡体外成像效果图。
图3为试验例2中M2型巨噬细胞流式鉴定图。
图4为试验例2中靶向微泡与M2型巨噬细胞粘附效果图。
图5为试验例3中分别注射普通微泡(MB)和甘露糖靶向微泡(Man-MB)在大鼠移植心脏的成像效果及对比图。
图6为试验例3中分别注射MB和Man-MB的大鼠移植心脏成像信号强度图。
图7为试验例3中大鼠心脏CAV模型心脏切片EVG染色图,其中左侧为成功制备的大鼠腹腔异位心脏移植模型,右侧为发生慢性排斥反应的移植心脏EVG染色。
实施例1
本实施例提供一种靶向M2型巨噬细胞甘露糖受体的超声分子探针,其制备方法包括以下步骤:
(1)合成二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖
将9mg 4-异硫氢酸苯基-a-D-甘露糖苷(CAS号为96345-79-8)与8mg二硬脂酰基磷脂酰乙醇胺聚乙二醇氨基(CAS号为474922-26-4)混合,溶解于20ml无水二氯甲烷中,并加入10μl无水三乙胺进行催化,抽真空后通入氮气保护,室温下反应6-8h;旋转蒸发仪去除多余的无水二氯甲烷,加入适量的超纯水溶解产物,超纯水作为透析外液透析24h;将透析后的液体冻干24h后即获得产物二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖。
(2)制备超声分子探针
将二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖与二硬脂酰基磷脂酰胆碱(CAS号为816-94-4)、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(CAS号为147867-65-0)分别溶于三氯甲烷,获得浓度分别为20mg/ml的磷脂溶液;
将二硬脂酰基磷脂酰胆碱溶液、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶液、二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖溶液按摩尔比9:0.5:0.5移入试管中,用涡旋振荡器形成涡旋混匀不同的磷脂成分,在氮气流的作用下使氯仿挥发掉,直至在试管壁上形成一种薄薄的一层磷脂薄膜,抽真空24h,去除多余的三氯甲烷;
在试管中加入5.5ml水化液(含有10%(体积比)甘油、10%(体积比)1,2丙二醇和80%(体积比)的0.1M的tris溶液(用购买的tris粉溶于超纯水中所获得的溶液),用盐酸调其PH值到7.4),将得到的磷脂溶液在超声清洗机水浴5min(提前将超声清洗机中的水加热至相变温度55-60℃)使溶液分散至透明,1ml/瓶分装入西林瓶,用橡胶塞及铝塑盖封口;
将上述封口的西林瓶置于换气装置上,抽真空30min以上抽尽瓶中的空气,随后通入全氟丙烷气体,做好标记,放入4℃冰箱储存备用。
本实施例制备的超声分子探针的结构示意图如图1所示。
试验例1
将实施例1制备得到的分子探针(微泡)用磷酸盐缓冲溶液分别稀释至1×106个/ml、5×105个/ml、1×105个/ml,将各个浓度的微泡移入制备好的1.5%(质量百分数)琼脂糖仿体孔中,用Philips EPic7超声仪器检测不同浓度下超声造影剂成像效果,获取超声造影信号,结果如图2所示,由图2可知实施例1制备得到的分子探针(微泡)在不同浓度下都具有良好的成像性能。
试验例2
为获取M2型巨噬细胞,将Raw264.7细胞培养贴壁后,加入细胞因子IL-4+IL-13各20ng/ml诱导48h,与CD206抗体孵育后进行流式检测,以验证M2型巨噬细胞是否诱导成功。对照组为未加细胞因子的Raw264.7细胞,结果如图3所示,由图3可知,诱导后的细胞CD206表达量高达39.1%,与对照组相比明显增加,表明M2型巨噬细胞诱导成功。
二硬脂酰基磷脂酰胆碱溶液与二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶液以摩尔比9:1并按照实施例1的方法制备普通微泡MB。
将Raw264.7细胞接种于六孔板中,每孔1×106个细胞,加入IL4+IL-13各20ng/ml诱导48h后,分别加入3×107个有靶向作用的Man-MB(实施例1中制备的分子探针)和无靶向作用的普通微泡(MB)孵育30min,用磷酸盐缓冲液清洗三遍,洗去多余的微泡,显微镜下观察微泡与细胞粘附情况,结果如图4所示,由图4可知,细胞表面有较多的靶向微泡粘附,而非靶向微泡未见明显粘附现象,证实了靶向微泡对M2型巨噬细胞的靶向性。
试验例3
构建大鼠心脏移植模型,皮下注射FK506(1mg/kg)14天预防急性排斥反应,后对移植心进行切片并行EVG染色,结果如图7所示,可见血管发生弥漫性狭窄,表明CAV模型制备成功。4W后对发生慢性排斥反应的模型鼠进行超声造影,每只大鼠以3.5%异氟烷诱导麻醉后,以浓度为2%异氟烷维持麻醉,麻醉后取仰卧位固定四肢,腹部备皮脱毛。应用GE LOGIQE9超声诊断仪进行成像,将MB、Man-MB(实施例1制备的分子探针)分别稀释至1×109个/ml,各取300μl通过尾静脉注射入同一大鼠体内,并进行超声分子成像,两种微泡注射间隔时间30min。通过击破-再灌注的方法定量局部粘附的超声造影剂的信号强度(GrayscaleIntensity),以dB值表示。通过二维图像确定心肌组织边界,勾画心肌,分析时间-强度曲线(time-intensity curve,TIC),通过TIC曲线读出击破之前和击破之后信号的平均值,两者相减,即可计算出局部粘附的微泡产生的超声信号,结果如图5和图6所示,发生慢性排斥反应的移植心的心肌中粘附的Man-MB信号明显高于MB(P<0.05),表明制备的Man-MB可用于心脏移植慢性排斥反应的早期诊断。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种靶向M2型巨噬细胞甘露糖受体的超声分子探针的制备方法,所述制备方法包括以下步骤:
(1)合成二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖
将4-异硫氢酸苯基-a-D-甘露糖苷与二硬脂酰基磷脂酰乙醇胺聚乙二醇氨基混合,溶解于无水二氯甲烷中,并加入无水三乙胺进行催化,抽真空后通入氮气保护,室温下反应6-8h;
旋转蒸发仪去除多余的无水二氯甲烷,加入适量的超纯水溶解产物,并透析,将透析后的液体冻干后即获得产物二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖;
(2)制备超声分子探针
将二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖与二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000分别溶于三氯甲烷,获得浓度分别为18-22mg/ml的磷脂溶液;
将二硬脂酰基磷脂酰胆碱溶液、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶液、二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖溶液按摩尔比(9.5-n):0.5:n,移入试管中,混匀不同的磷脂成分,在氮气流的作用下使氯仿挥发掉,直至在试管壁上形成一层薄薄的磷脂薄膜,抽真空去除多余的三氯甲烷,所述n的取值为0.1~1;
在试管中加入水化液,将得到的磷脂溶液加热至55-60℃,使溶液分散至透明,即获得超声分子探针;
所述水化液含有体积比10%甘油、体积比10% 1,2丙二醇和体积比80%的0.1M的tris溶液,用盐酸调其pH值到7.4。
2.根据权利要求1所述的制备方法,其特征在于,所述步骤(1)中4-异硫氢酸苯基-a-D-甘露糖苷与二硬脂酰基磷脂酰乙醇胺聚乙二醇氨基的摩尔比为1:2-1:10。
3.根据权利要求2所述的制备方法,其特征在于,所述步骤(1)中无水三乙胺的加入量为10μl。
4.根据权利要求3所述的制备方法,其特征在于,所述步骤(2)中二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖与二硬脂酰基磷脂酰胆碱、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000分别溶于三氯甲烷,获得浓度分别为20mg/ml的磷脂溶液。
5.根据权利要求4所述的制备方法,其特征在于,所述步骤(2)中水化液的加入量为4-6ml。
6.根据权利要求5所述的制备方法,其特征在于,所述步骤(2)中二硬脂酰基磷脂酰胆碱溶液、二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000溶液、二硬脂酰磷脂酰乙醇胺-聚乙二醇-甘露糖溶液的摩尔比为9:0.5:0.5。
7.一种诊断试剂,其特征在于,所述诊断试剂包括权利要求1-6中任一项所述的制备方法制备得到的超声分子探针。
8.权利要求1-6中任一项所述的制备方法制备得到的超声分子探针在制备心脏移植慢性排斥反应诊断试剂中的应用。
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Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0241376A1 (fr) * | 1986-04-08 | 1987-10-14 | Centre National De La Recherche Scientifique (Cnrs) | Nouveaux liposomes à base de phosphatidylinositolmannosides, et compositions pharmaceutiques les contenant |
WO2004067732A2 (en) * | 2003-01-27 | 2004-08-12 | Ústav Makromolekulární Chemie Akademie Vedceské Republiky | Polymer carriers with bonded saccharides for immobilization of biological systems |
CN102973506A (zh) * | 2011-09-05 | 2013-03-20 | 中国科学院深圳先进技术研究院 | 阳离子脂质体及其制备方法 |
WO2014021678A1 (ko) * | 2012-08-02 | 2014-02-06 | (주)아이엠지티 | 암의 진단 및 치료를 위한 마이크로버블-나노리포좀 복합체 |
CN108578711A (zh) * | 2018-04-04 | 2018-09-28 | 哈尔滨医科大学 | 一种乙酰化糖酯-聚乙二醇-磷脂酰乙醇胺共轭物及其制备方法与应用 |
CN109745326A (zh) * | 2017-11-02 | 2019-05-14 | 中国科学院上海药物研究所 | 一种包含吉非替尼和组蛋白去乙酰酶抑制剂的药物组合物,其脂质体制剂及其制药用途 |
CN109893515A (zh) * | 2019-02-26 | 2019-06-18 | 华中科技大学 | 一种巨噬细胞载药微颗粒制剂及其制备方法 |
CN111150857A (zh) * | 2020-01-07 | 2020-05-15 | 山东大学齐鲁医院 | 一种靶向肿瘤相关巨噬细胞的脂质纳米级超声造影剂及其制备方法与应用 |
CN112870388A (zh) * | 2021-03-04 | 2021-06-01 | 新疆医科大学第一附属医院 | 纳米级amh靶向超声造影剂及其制备方法和应用 |
CN113456838A (zh) * | 2021-02-09 | 2021-10-01 | 深圳市人民医院 | 磁性黑磷微泡及其在制备超声诊断试剂和治疗乳腺癌药物中的应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104189897A (zh) * | 2014-05-21 | 2014-12-10 | 深圳先进技术研究院 | 一种树突状细胞高效负载抗原的制备方法 |
CN110522917A (zh) * | 2018-05-25 | 2019-12-03 | 成都瑞博克医药科技有限公司 | 一种甘露糖修饰的靶向纳米制剂 |
-
2022
- 2022-07-04 CN CN202210786178.8A patent/CN115073726B/zh active Active
Patent Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0241376A1 (fr) * | 1986-04-08 | 1987-10-14 | Centre National De La Recherche Scientifique (Cnrs) | Nouveaux liposomes à base de phosphatidylinositolmannosides, et compositions pharmaceutiques les contenant |
WO2004067732A2 (en) * | 2003-01-27 | 2004-08-12 | Ústav Makromolekulární Chemie Akademie Vedceské Republiky | Polymer carriers with bonded saccharides for immobilization of biological systems |
CN102973506A (zh) * | 2011-09-05 | 2013-03-20 | 中国科学院深圳先进技术研究院 | 阳离子脂质体及其制备方法 |
WO2014021678A1 (ko) * | 2012-08-02 | 2014-02-06 | (주)아이엠지티 | 암의 진단 및 치료를 위한 마이크로버블-나노리포좀 복합체 |
CN109745326A (zh) * | 2017-11-02 | 2019-05-14 | 中国科学院上海药物研究所 | 一种包含吉非替尼和组蛋白去乙酰酶抑制剂的药物组合物,其脂质体制剂及其制药用途 |
CN108578711A (zh) * | 2018-04-04 | 2018-09-28 | 哈尔滨医科大学 | 一种乙酰化糖酯-聚乙二醇-磷脂酰乙醇胺共轭物及其制备方法与应用 |
CN109893515A (zh) * | 2019-02-26 | 2019-06-18 | 华中科技大学 | 一种巨噬细胞载药微颗粒制剂及其制备方法 |
CN111150857A (zh) * | 2020-01-07 | 2020-05-15 | 山东大学齐鲁医院 | 一种靶向肿瘤相关巨噬细胞的脂质纳米级超声造影剂及其制备方法与应用 |
CN113456838A (zh) * | 2021-02-09 | 2021-10-01 | 深圳市人民医院 | 磁性黑磷微泡及其在制备超声诊断试剂和治疗乳腺癌药物中的应用 |
CN112870388A (zh) * | 2021-03-04 | 2021-06-01 | 新疆医科大学第一附属医院 | 纳米级amh靶向超声造影剂及其制备方法和应用 |
Non-Patent Citations (4)
Title |
---|
Mannosylation of budesonide palmitate nanoprodrugs for improved macrophage targeting;Ludmila Pinheiro等;《European Journal of Pharmaceutics and Biopharmaceutics》;正文第112-120页 * |
基于包裹染料木黄酮的甘露糖靶向长循环纳米脂质体抗肿瘤效应研究;杨凡;秦爱平;李;彭倩;王晨旭;洪秀琴;;激光生物学报(04);全文 * |
心脏移植慢性排斥反应机制的探讨;张建军, 罗兆榴;中国免疫学杂志(07);全文 * |
甘露糖化羧甲基壳聚糖与依替膦酸复合物纳米粒靶向结合M2型巨噬细胞的实验研究;黄燕;蒋鸥;文庆莲;刘宇;林盛;何玉;吴敬波;孟凡智;马成;;癌症进展(05);全文 * |
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