CN116474110A - 近红外二区长循环前药偶联物,其制备方法及与sting受体激动剂的联合应用 - Google Patents
近红外二区长循环前药偶联物,其制备方法及与sting受体激动剂的联合应用 Download PDFInfo
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Abstract
本发明涉及近红外二区长循环前药偶联物,其制备方法及与STING受体激动剂的联合应用,所述偶联物由分子探针及化疗药物偶联而成。该偶联物在体内具有长循环的特性,可以实现对实体瘤的长期成像和治疗效果监测,并减少给药次数,增加药物使用的依从性。该药物偶联物联合STING受体激动剂,调节肿瘤免疫微环境,起到化疗和免疫治疗的协同增效作用。
Description
技术领域
本发明涉及生物医学领域,具体的涉及一类具有长循环特性的近红外二区探针-化疗药物的前药偶联物,其制备方法及联合STING受体激动剂在抗肿瘤药物中的应用。
背景技术
恶性肿瘤是严重威胁人类生命健康的疾病,肿瘤的早期诊断和有效治疗是提高癌症患者生存率的重要因素。目前传统的影像技术存在灵敏度和特异性差、低时空分辨率等问题,无法对早期病变组织及时检出和有效干预。而近红外二区(NIR-II:1000-1700nm)荧光成像技术具有更高的时空分辨率、更高的信噪比及组织穿透深度,其波长较长,背景干扰低,成像质量越好,在活体成像与肿瘤治疗等方面显示出巨大的优势。
化疗是癌症治疗的一种有效且重要的治疗方法,研究表明,化疗药物除通过细胞毒性作用机制杀伤肿瘤细胞以外,还可以通过濒死肿瘤细胞途径诱导肿瘤细胞免疫原性死亡,可以刺激肿瘤抗原的呈递并引发先天性抗肿瘤免疫反应。考虑到肿瘤细胞和组织的免疫抑制特性,当采用独立的化学治疗方式时,可能会观察到反应不足,治疗效果不佳的状况。
免疫疗法是抗肿瘤领域研究热门,肿瘤免疫治疗是指通过外源干预机体免疫系统,重新启动并维持“肿瘤-免疫”循环,恢复、提高机体的抗肿瘤免疫反应,加强对肿瘤细胞的识别和杀伤能力,从而达到控制甚至特异性清除肿瘤的治疗效果,与传统治疗手段相比,肿瘤免疫治疗具有特异性强,副作用小的优点。
“冷肿瘤”的中免疫抑制微环境特征常常导致放化疗效果不佳,将“冷肿瘤”转化为“热肿瘤”,增加肿瘤微环境中的免疫浸润是肿瘤组合疗法的热点。肿瘤适应性耐药被发现与肿瘤抗原呈递与I型干扰素(IFN-I)缺乏密切关联,导致免疫反应率和客观缓解率低(<50%)。cGAS-STING通路作为胞质DNA传感通路能够激活下游IFN-I与炎症因子,引起先天免疫通路的激活,在肿瘤特异性T细胞激活和肿瘤浸润性淋巴细胞(TIL)浸润的过程中起着至关重要的作用。
发明内容
本发明的目的在于提供一种近红外二区分子探针和化疗药物相偶联的策略,该偶联物在体内具有长循环的特性,可以实现对实体瘤的长期成像和治疗效果监测,并减少给药次数,增加药物使用的依从性。
本发明解决上述技术问题所采用的方案是:
本发明提供一种偶联物(HLWP-R),所述偶联物由近红外二区分子探针及化疗药物偶联而成,分子结构为:
其中R为化疗药物。
前缀部分HLWP可使该偶联物满足在近红外二区窗口1100~1600nm范围内实现肿瘤的靶向成像应用。所述偶联物具有前药性质,在生物体内酯酶作用下,水解得到具有成像效果的NIR-II分子探针和化疗药物,实现肿瘤诊疗一体策略。
进一步地,所述化疗药物任选自紫杉醇、吉西他滨、多西他赛、奥沙利铂、苯丁酸氮芥、喜树碱衍生物。
进一步地,所述化疗药物为7-乙基-10-羟基喜树碱(SN38),其与HLWP偶联后得到的偶联物的分子结构为:
进一步地,所述化疗药物还可采用伊立替康(CPT-11),对应偶联物为HLWP-CPT-11。CPT-11为市售用药,临床用于多种肿瘤化疗,其结构为喜树碱半合成衍生物,是一种前体药物,在体内经酯酶代谢得到活性代谢产物SN38。
本发明还提供上述偶联物的制备方法,包括如下步骤:
(1)式2所示化合物与化疗药物发生酯缩合反应,得到单边取代化合物;
(2)将所得单边取代化合物与PEG2000发生酰胺缩合反应得所述偶联物。
进一步地,上述制备方法还包括将所得偶联物制备成纳米颗粒形式的步骤,具体如下:
(1)将所得偶联物溶于四氢呋喃中;
(2)超声状态下将偶联物与四氢呋喃的混合溶液逐滴加入一级水中,至完全溶解成透明溶液,室温搅拌过夜;
(3)除去其中的有机溶剂,超滤浓缩,得到所述偶联物纳米颗粒。
进一步地,制备所述HLWP-SN38偶联物的过程如下:
本发明还提供一种药物组合,所述药物组合包括上述的偶联物及STING通路受体激动剂;或者,所述药物组合物包括上述制备方法所得偶联物及STING通路受体激动剂。
进一步地,所述STING通路受体激动剂任选自环状核苷酸类激动剂ADU-S100、MK-1454,或者任选自非环状核苷酸类STING激动剂diABZI、SR717、MSA-2、DMXAA。
本发明该提供上述偶联物或者药物组合在制备治疗或预防或监测肿瘤的药物中的应用。
进一步地,所述偶联物为水溶性固体或纳米颗粒形式。纳米颗粒形式在用于生物体内成像及靶向治疗方面可能更有优势。所述纳米粒子可通过自组装形成。
本发明有益效果在于:
1,本发明提供一种近红外二区窗口的化疗前药,可用于近红外二区肿瘤成像和治疗,可实现很好的时间和空间分辨率,具有很好的应用前景。
2,本发明提供的前药HLWP-SN38具有较好的长循环特征,其半衰期为30h,可有效维持药物血药溶度,减少给药次数,提高患者给药的依从性,具有良好的应用前景。
3,本发明提供一种化学疗法联合免疫治疗的协同治疗方法,具体来讲涉及近红外二区化疗前药HLWP-SN38和STING受体激动剂的联合治疗策略,对于逆转肿瘤免疫抑制微环境,增加肿瘤化疗疗效具有较大的使用价值和开发前景。
附图说明
图1为实施例2所得HLWP-SN38纳米粒子的透射电镜和动态光散射结果;
图2为HLWP-SN38纳米粒子对4T1、CT26、HepG2、U87细胞的细胞毒性结果;
图3为HLWP-SN38在E0771细胞水平的48h细胞摄取实验结果;
图4为不同时间下CT26细胞的FITC-AnnexV/PI凋亡流式图,上图为24小时的细胞凋亡结果,下图为48小时的细胞凋亡结果;
图5为不同时间点下CT26皮下肿瘤模型的近红外二区成像及肿瘤区荧光强度随时间变化的散点图;其中:(a)不同时间点下肿瘤区域荧光强度变化散点图;(b)不同时间点下小鼠各组织及器官的离体荧光强度分布图;(c)不同时期小鼠肿瘤活体荧光成像图;
图6为不同治疗组小鼠肿瘤及体重变化情况,其中左上为肿瘤体积变化曲线,右上为治疗过程各组小鼠体重变化曲线,左下为治疗结束后各小组小鼠肿瘤立体图片,右下为各组小鼠离体肿瘤重量;
图7为治疗过程中各小鼠活体NIR-II成像检测结果;
图8为流式细胞术实验分析不同治疗组小鼠脾脏DC细胞的成熟状况;
图9为不同治疗组小鼠血浆中IL-6和IFN-β分泌水平研究,左图为IL-6的分泌水平,右图为IFN-β的分泌水平;
图10为不同实验组中小鼠主要器官(心、肝、脾、肺、肾)的H&E染色结果。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。以下实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行,使用的方法如无特别说明,均为本领域公知的常规方法,使用的耗材和试剂如无特别说明,均为市场购得。除非另有说明,本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
实施例1HLWP-SN38的合成
本实施例提供的偶联物HLWP-SN38结构式如下图所示:
合成路线概括如下:
化合物1经Witting反应制得化合物2;化合物2经过Pd/H2还原制得化合物3;化合物3经酯水解反应得化合物4;化合物4经过酯缩合得到TMS保护的化合物5;化合物5经过NBS溴代和双频哪醇硼酸酯反应得化合物6;化合物6发生Suzuki偶联得化合物8;化合物8在TFA条件下脱去TMS保护得化合物9,;化合物9与SN38发生酯缩合反应得到单边取代的化合物10;化合物10与PEG2000发生酰胺缩合反应得化合物HLWP-SN38。
由化合物1制备HLWP-SN38的具体步骤如下:
步骤1:称取计算量化合物1(5g,18.3mmol),(三苯基膦烯)乙酸乙酯(7.65g,22mmol)于圆底烧瓶中,30ml无水THF溶解,反应液室温下搅拌24h,至TLC监测反应结束,反应液旋干,PE/EA=60/1为洗脱液经柱层析法纯化,得到浅黄色油状化合物2。1H NMR(400MHz,CDCl3)δ7.66(d,J=15.9Hz,1H),7.40(d,J=8.6Hz,2H),7.36–7.28(m,4H),7.19–7.08(m,6H),7.04(d,J=8.6Hz,2H),6.33(d,J=15.9Hz,1H),4.29(q,J=7.1Hz,2H),1.37(t,J=7.1Hz,3H).13C NMR(101MHz,CDCl3)δ167.4,149.8,146.9,144.2,129.5,129.2,127.5,125.3,123.9,121.7,115.4,60.3,14.4.
步骤2:称取计算量化合物2(6g,17.5mmol)和10% Pd/C(0.037g)于50ml乙酸乙酯溶剂中,H2置换,室温下搅拌24h至TLC监测反应结束。反应液硅藻土过滤,有机相旋干柱层析纯化,得无色油状化合物3。1H NMR(400MHz,CDCl3)δ7.23–7.17(m,4H),7.09–7.03(m,6H),7.02–6.94(m,4H),4.13(q,J=7.3Hz,2H),2.90(t,J=7.8Hz,2H),2.60(t,J=7.8Hz,2H),1.24(t,J=7.1Hz,3H).13C NMR(101MHz,CDCl3)δ173.2,148.1,146.2,135.3,129.4,129.3,124.7,124.1,122.7,60.6,36.2,30.6,14.5.
步骤3:称取计算量(5g,14.5mmol)化合物3于30ml THF中,冰浴下滴加LiOH(0.42g,17.4mmol)水溶液并搅拌1h,反应液缓慢恢复至室温,继续搅拌14h至反应结束,2MHCl调节反应液pH=3,乙酸乙酯萃取,无水Mg2SO4干燥旋干,得产物化合物4,无须纯化可直接用于下一步反应。
步骤4:称取计算量化合物4(3g,9.5mmol)于DCM中,反应液中加入DMAP(0.23g,1.2mmol)、EDCI(5.46g,28.5mmol)和2-三甲基硅乙醇(2.25g,19mmol),反应液室温下搅拌16h。TLC监测反应结束,DCM萃取,有机相干燥旋干,柱层析法纯化得无色油状化合物5。1HNMR(400MHz,CDCl3)δ7.31(t,J=7.9Hz,4H),7.17(t,J=7.4Hz,6H),7.13–7.04(m,4H),4.35–4.22(m,2H),3.00(t,J=7.8Hz,2H),2.70(t,J=7.8Hz,2H),1.14–1.03(m,2H),0.15(s,9H).13C NMR(101MHz,CDCl3)δ173.3,148.1,146.2,135.3,129.4,129.3,124.7,124.1,122.7,62.9,36.4,30.6,17.5,-1.1.
步骤5:称取计算量化合物5(2g,4.8mmol)于三颈瓶中,加入计算量醋酸铵(0.74g,9.6mmol),在0-5℃下冰浴5min,分批加入NBS(1.03g,5.76mmol),反应液恢复至室温搅拌1h,反应液DCM萃取,有机相干燥旋干,所得粗品直接用于下一步反应;称取计算量双(三苯基膦)二氯化钯(II)(0.4g,0.48mmol),KOAc(1.4g,14.4mmol)和双频哪醇硼酸酯(1.46g,5.76mmol)于DMF中,加入上述粗产物,Ar2保护,85℃加热反应5h,TLC监测反应结束,反应液乙酸乙酯萃取,合并有机相,干燥后旋干,柱层析色谱法纯化得无色油状物6。1H NMR(400MHz,CDCl3)δ7.70(t,J=7.6Hz,2H),7.28(dd,J=9.6,6.2Hz,2H),7.17–7.10(m,4H),7.09–7.01(m,5H),4.26–4.18(m,2H),2.95(t,J=7.8Hz,2H),2.64(t,J=7.8Hz,2H),1.38(d,J=7.6Hz,12H),1.08–0.98(m,2H),0.11–0.05(m,9H).13C NMR(101MHz,CDCl3)δ173.1,150.6,147.4,145.5,135.8,135.8,129.3,129.2,125.2,124.8,123.2,121.5,83.5,62.7,36.1,30.4,24.9,17.3,1.0,-1.4.
步骤6:称取计算量化合物6(1.0g,1.85mmol),化合物7(0.5g,0.62mmol)和Pd(PPh3)4(0.144g,0.124mmol)于THF中,加入2ml 14wt%NaHCO3,混合物N2保护,反应液在75℃反应2h,TLC监测至反应结束,硅胶柱层析纯化,得到绿色固体化合物8。1H NMR(400MHz,CDCl3)δ7.51(d,J=8.4Hz,5H),7.29(dd,J=8.8,5.7Hz,4H),7.23(d,J=3.8Hz,2H),7.14(d,J=6.3Hz,8H),7.07(dd,J=8.1,4.9Hz,9H),4.29–4.16(m,4H),2.95(t,J=7.7Hz,4H),2.64(t,J=7.8Hz,4H),1.07–0.95(m,4H),0.15–0.03(m,18H).13C NMR(101MHz,CDCl3)δ173.1,148.3,147.6,147.6,147.3,145.7,145.4,135.8,129.3,129.2,126.3,124.9,124.7,124.5,124.2,123.1,123.1,62.7,36.1,30.4,17.3,-1.4.MALDI-TOF-MS Calcdfor:C50H64N6O4S4([M+H]+):1136.29,found:1137.3271.
步骤7:化合物8(0.6g,0.41mmol)于0℃溶于TFA/DCM(1/2)的混合溶液中,缓慢恢复至室温,继续搅拌8h,TLC监测至反应结束,反应液乙酸乙酯萃取,合并有机相,干燥旋干得粗产物9,绿色固体,无需经过纯化直接用于下一步反应。
步骤8:称取计算量化合物9(0.4g,0.31mmol),EDCI(0.18g,0.94mmol)、DMAP(0.0076g,0.062mmol)和SN38(0.146g,0.372mmol),适量DCM溶解,室温下搅拌24h,TLC监测至反应结束;旋干反应液,EA萃取,合并有机相,干燥旋干,柱层析色谱法纯化得单边SN38取代的化合物10,绿色固体。
步骤9:称取计算量化合物10(0.1g,0.06mmol),HATU(0.068g,0.18mmol)、DIPEA(0.039g,0.3mmol)和PEG2K-NH2(0.24g,0.12mmol),适量DCM溶解,室温下搅拌24h,TLC监测至反应结束;旋干反应液,EA萃取,合并有机相,干燥旋干,柱层析色谱法纯化得目标化合物HLWP-SN38。MALDI-TOF-MS Calcd for:C100H106N7O7S4([M+H]+):1645.7070,found:1647.6362。
实施例2HLWP-SN38自组装纳米粒子的制备
使用实施例1所得化合物产物HLWP-SN38制备HLWP-SN38纳米粒子,具体步骤如下:
称取HLWP-SN38 5mg溶于1mL四氢呋喃中,超声下至完全溶解,接着在超声破碎仪强烈超声状态下逐滴加入至10mL一级水中,至完全溶解成透明溶液,室温搅拌过夜除去里面的有机溶剂,然后再将上述溶液用50mL超滤离心管(30KDa)超滤浓缩至1mL。即得到自组装纳米粒子HLWP-SN38@NPs。
对所得纳米粒子进行透射电镜和动态光散射测试,结果如图1所示,可知所得纳米粒子粒径约为20~40nm。
实施例3化合物HLWP-SN38细胞毒性研究
分别取对数生长期4T1、CT26、HepG2、U87细胞接种于96孔板中,每孔180μL,待细胞贴壁生长至70%~80%。将待测化合物溶解于适量DMSO中,配制为母液,并分别用适量培养基稀释。每孔加入待测化合物稀释液20μL,使每个孔终体积为200μL,DMSO含量为0.1%,待测化合物终浓度为相当于0.01μg/mL、0.1μg/mL、1μg/mL、10μg/mL、20μg/mL的SN38单药的浓度。在37℃、5%CO2条件下培养24h,每孔加入10mL CCK-8溶液,在37℃、5%CO2条件下继续孵育2h,使用Tecan Infinite M1000多功能酶标仪测量在490nm波长的吸光度,计算细胞存活率。每个浓度均设置5个复孔,其中以临床药物SN38作为阳性对照药物,结果如图2所示:化合物HLWP-SN38对四种肿瘤细胞均具有较好的增值抑制效果,其中对于CT26细胞抑制效果最佳,在药物浓度大于0.1μg/mL时其细胞毒性显著优于SN38阳性对照药。上述结果表明化合物HLWP-SN38对多种癌细胞都有一定的药物毒性。
实施例4E0771细胞对HLWP-SN38的药物摄取试验
取对数生长期的E0771细胞置于6孔板中,当细胞密度达到70~80%,将含HLWP-SN38(10μM)的DMEM培养基2mL加入至每个复孔,其中一个孔加入空白培养基做对照,然后置于孵育箱中培育48h,接着细胞消化,离心,细胞沉淀用PBS洗涤三次,最后加入200μL PBS,混合均匀,置于Cytoflex S流式细胞仪中分析。图3为不同孵育时间下E0771细胞悬液的平均荧光强度。由图可知,随着孵育时间的增长,E0771细胞悬浮液的荧光强度增加,呈现出对HLWP-SN38良好的摄取能力。
实施例5流式细胞术分析HLWP-SN38纳米粒子处理细胞后的凋亡情况
取对数期生长的CT26小鼠结肠癌细胞培养于12孔板中,每孔加入含有细胞数约1×105RPMI-1640培养基1mL。在37℃,含5% CO2的细胞孵育箱中培养过夜使细胞贴壁,第二天吸去旧的培养基,并分别向孔中加入空白培养基、含HLWP-SN38以及含SN38的RPMI-1640培养基各1mL,接着培养24小时后,收集每孔上层培养基,下层细胞沉淀用0.25%胰酶(500μL)消化2min,加入培养基(500μL),小心吹打均匀,使细胞沉淀悬浮,然后与每孔上层培养基收集一起,1000g离心5min,移去上层清液,下层细胞沉淀用PBS洗涤3次,1000g离心5min,移去上层清液,加入细胞凋亡Annexin V-FITC/PI工作液(索莱宝生物试剂),轻轻混匀,避光染色15min,接着对空白组、药物组、阳性对照组进行流式细胞凋亡分析。图4为不同时间条件下CT26细胞的FITC-AnnexV/PI凋亡流式图。由图可知,在24h时HLWP-SN38的细胞凋亡率略高于PBS组,但与阳性对照组SN38差距较大,然而在48h时HLWP-SN38组的细胞凋亡率显著提高,且大于阳性对照SN38。结果证明HLWP-SN38能够显著诱导CT26细胞的凋亡,且具有长效缓释的作用。
实施例6HLWP-SN38对乳腺癌皮下肿瘤模型的近红外二区荧光成像研究
对小鼠右后肢进行脱毛处理,取对数期CT26小鼠结肠癌细胞于1.5mL无血清的RPMI-1640培养基配成细胞悬浮液,浓度约为1×107个细胞/毫升。将上述细胞悬液接种至小鼠右后肢,每只100μL的体积,约3-4周后,肿瘤体积长至100~200mm3,将肿瘤模型鼠通过腹腔注射125μL戊巴比妥钠(1mg/mL)麻醉,将小鼠置于近红外二区成像系统采集不同时间点的荧光图像,研究其肿瘤组织对HLWP-SN38的摄取规律,小动物活体成像仪所用相机为InGaAs检测器,激发光源为808nm波长光纤耦合激光器,成像激光功率密度约为0.1W/cm2,滤光片选择1000nm长通滤光片。图5为不同时间点下CT26皮下肿瘤模型的近红外二区成像及肿瘤区荧光强度随时间变化的散点图。
实施例7化合物HLWP联合STING激动剂的三阴性乳腺癌皮下肿瘤模型的诊疗一体化研究。
选取30只C57BL/6J小鼠作为实验动物,接种前,对小鼠右后肢进行脱毛处理。取对数生长期E0771小鼠乳腺癌细胞,于无血清的DMEM培养基中,配成浓度约为1×107个细胞/毫升的细胞悬浮液。将上述细胞悬液接种至C57小鼠右后肢,每只接种体积为100μL,待肿瘤体积长至100mm3时,计为第0天,上述30只老鼠随机分为5组(空白对照组PBS、阳性对照组CPT-11、单药组(单独使用HLWP-SN38)、联合治疗组(HLWP-SN38+STING激动剂SR717),用于小鼠肿瘤成像及治疗实验。
分别于第0天、第7天通过尾静脉注射前药HLWP,在第1-第5天通过腹腔注射STING激动剂SR717,阳性药CPT-11分别于0、4、8、12天通过尾静脉注射,治疗过程中隔天监测记录小鼠体重及肿瘤体积变化。
肿瘤体积计算公式如下:
体积=(长×宽×宽)/2。
实施例8化合物HLWP联合STING激动剂的细胞因子水平表征(STING信号通路体内激动表征)
上述HLWP-STING联合治疗组治疗周期结束后,于取样前给予最后一次STING激动剂施用,于给药后的4h通过眼眶取血,所得血浆样品于-80℃保存。用ELISA试剂盒表征待测血浆样品中IL-6、IFN-β的分泌水平,依据标准曲线进行定量分析。结果如图9所示:SN38和HLWP-SN38均能一定程度促进IL-6和IFN-β的分泌,但其效果较弱,而HLWP-SN38+SR717联合治疗组的分泌水平则显著增加;取治疗周期第14天处死各组小鼠,取脾脏制备单细胞悬液,通过流式细胞术分析体内DC细胞成熟状态。
实施例9各器官HE染色及肿瘤组织免疫组化分析
取实施例8中各治疗组中C57BL/6J小鼠,每只小鼠进行离体取心、肝、脾、肺、肾、肿瘤,并用组织固定液固定,用于组织切片及免疫组化分析。图10为不同实验组中小鼠主要器官(心、肝、脾、肺、肾)的H&E染色,说明HLWP-SN38及其HLWP-SN38+SR717的联合治疗组具有良好的生物相容性,不会引起大的毒副作用。
以上所述是本发明的优选实施方式而已,当然不能以此来限定本发明之权利范围,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和变动,这些改进和变动也视为本发明的保护范围。
Claims (10)
1.一种偶联物,其特征在于,所述偶联物由近红外二区分子探针及化疗药物偶联而成,分子结构为:
其中R为化疗药物。
2.根据权利要求1所述的偶联物,其特征在于,所述化疗药物任选自紫杉醇、吉西他滨、多西他赛、奥沙利铂、苯丁酸氮芥、喜树碱衍生物。
3.根据权利要求1所述的偶联物,其特征在于,所述化疗药物为7-乙基-10-羟基喜树碱或伊立替康。
4.权利要求1~3任一项所述偶联物的制备方法,其特征在于,包括如下步骤:
(1)式2所示化合物与化疗药物发生酯缩合反应,得到单边取代化合物;
(2)将所得单边取代化合物与PEG2000发生酰胺缩合反应得所述偶联物。
5.根据权利要求4所述的制备方法,其特征在于,还包括将所得偶联物制备成纳米颗粒形式,包括如下步骤:
(1)将所得偶联物溶于四氢呋喃中;
(2)超声状态下将偶联物与四氢呋喃的混合溶液逐滴加入一级水中,至完全溶解成透明溶液,室温搅拌过夜;
(3)除去其中的有机溶剂,超滤浓缩,得到所述偶联物纳米颗粒。
6.根据权利要求4所述的制备方法,其特征在于,反应过程如下:
7.一种药物组合,其特征在于,包括权利要求1~3任一项所述的偶联物及STING通路受体激动剂;或者,所述药物组合物包括权利要求4~6任一项所述制备方法所得偶联物及STING通路受体激动剂。
8.根据权利要求7所述的药物组合物,其特征在于,所述STING通路受体激动剂任选自环状核苷酸类激动剂ADU-S100、MK-1454,或者任选自非环状核苷酸类STING激动剂diABZI、SR717、MSA-2、DMXAA。
9.如权利要求1~3任一项所述的偶联物或者权利要求7~8任一项所述的药物组合在制备治疗或预防或监测肿瘤的药物中的应用。
10.如权利要求9所述的应用,其特征在于,所述偶联物为水溶性固体或纳米颗粒形式。
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