CN112023061A - 一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物及其制备和应用 - Google Patents
一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物及其制备和应用 Download PDFInfo
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Abstract
本发明涉及一种功能化树状大分子包裹金纳米颗粒/PD‑L1 siRNA复合物及其制备和应用。该复合物为表面修饰聚乙二醇和荧光胺、内部包裹金纳米颗粒的聚酰胺‑胺树状大分子负载PD‑L1 siRNA。该方法为:G5‑PEG溶液制备,荧光胺修饰的聚乙二醇化的树状大分子包裹金纳米颗粒制备,功能化树状大分子包裹金纳米颗粒/PD‑L1 siRNA复合物制备。该复合物能够有效沉默PD‑L1的表达,实现肿瘤免疫治疗,这在肿瘤的免疫治疗方面具有良好的应用潜力。该方法简单,实验条件温和,易操作。
Description
技术领域
本发明属于多功能高分子纳米载体及其制备和应用领域,特别涉及一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物及其制备方法和应用。
背景技术
癌症是威胁人类健康的主要因素之一,传统的治疗方法有化疗、放疗和手术治疗,但存在肿瘤转移,副作用大等不良反应。而免疫疗法中的免疫检查点阻断疗法因疗效好、长期抑制肿瘤转移等成为现阶段热门的肿瘤治疗方法。其中作用机制研究较为深入的免疫检查点之一是PD-1/PD-L1,当表达在T细胞表面的PD-1与表达在肿瘤组织中的PD-L1结合时,会向T细胞传递负向调控信号,使具有免疫功能的T细胞处于休眠状态,减弱T细胞活化后期的细胞应答和攻击肿瘤细胞的免疫功能,这种现象被称之为“免疫逃逸”。而免疫检查点阻断疗法就是通过免疫相关试剂,抑制免疫细胞或肿瘤细胞表面相关受体的表达,重新激活T细胞的肿瘤识别功能,降低肿瘤细胞所造成的“免疫逃逸”,恢复T细胞的免疫应答从而杀死肿瘤细胞。
基因治疗是一种新型的有效治疗手段,通过一定的载体或手段将功能性外源基因导入受体细胞中,然后外源基因的表达产物或外源基因抑制靶基因的转录或翻译,从而介入疾病的治疗。其中siRNA(Small interfering RNA)能通过双链RNA诱导同源性的信使RNA特异性的降解,从而诱导基因沉默。因此,可以通过PD-L1 siRNA(siPD-L1)来转染诱导肿瘤组织中的PD-L1沉默,以恢复T细胞活力。然而,目前基因治疗主要的障碍就是缺乏一种安全高效的载体,非病毒载体由于其低细胞毒性、无免疫原性、高基因装载量等特点,备受研究者关注。
非病毒载体中,聚酰胺-胺树状大分子(PAMAM)是一种高度支化、结构明确的新型超支化聚合物,具有独特的单分散性和丰富的表面氨基官能团,其作为基因载体具有基因压缩包覆能力强、免疫原性低、转染效率高等突出优势。研究表明,虽然第五代PAMAM树状大分子有一定的细胞毒性,但可以通过内部包裹金纳米颗粒和表面聚乙二醇化来降低细胞毒性并提高基因转染效率(Shan et al.,Biomaterials,2012,33(10),3025-3035;Hou etal.,J.Mater.Chem.B 2016,4(17),2933-2943)。而荧光胺与第五代PAMAM树状大分子表面伯胺的反应可以使其具有蓝色荧光进而实现细胞定位评价,因此,表面荧光胺修饰的聚乙二醇化的树状大分子包裹金纳米颗粒可以广泛用于基因传递。
检索国内外相关文献和专利结果表明:以荧光胺修饰的聚乙二醇化的聚酰胺-胺树状大分子包裹金纳米颗粒为载体负载siPD-L1,应用于肿瘤的免疫治疗,尚未见报道。
发明内容
本发明所要解决的技术问题是提供一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物及其制备和应用,以填补现有技术的空白。
本发明提供一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物,所述复合物为表面修饰聚乙二醇和荧光胺、内部包裹金纳米颗粒的聚酰胺-胺树状大分子负载PD-L1 siRNA。
本发明提供一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物的制备方法,包括:
(1)将第五代聚酰胺-胺树状大分子G5.NH2和甲氧基-聚乙二醇-琥珀酰亚胺羧甲基酯M-SCM-2000分别溶于超纯水中,将得到的M-SCM-2000溶液加入到得到的G5.NH2溶液中,搅拌反应,得到G5-PEG溶液,其中G5.NH2与M-SCM-2000的摩尔比为1:18~22;
(2)将氯金酸水溶液逐滴加入到步骤(1)中的G5-PEG溶液中,混合,加入硼氢化钠溶液,搅拌反应,透析纯化,冷冻干燥,得到{G5-PEG-(Au0)25},其中,G5.NH2与氯金酸、硼氢化钠的摩尔比为1:25-27:70-80;
(3)将步骤(2)中{G5-PEG-(Au0)25}溶解于溶剂中,避光,逐滴加入荧光胺溶液,搅拌,透析纯化,冷冻干燥,得到荧光胺修饰的聚乙二醇化的树状大分子包裹金纳米颗粒{G5-PEG-(Au0)25-F},其中{G5-PEG-(Au0)25}与荧光胺的摩尔比为1:4-6;
(4)将步骤(3)中{G5-PEG-(Au0)25-F}溶于无菌水中,然后与siPD-L1溶液混合,孵育,得到功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物。
所述步骤(1)中M-SCM-2000溶液浓度为8~15mg/mL。
所述步骤(1)中G5.NH2溶液浓度为0.8~1.2mg/mL。
所述步骤(1)中搅拌反应为:室温搅拌反应3-5h。
所述步骤(2)中混合为:在冰浴搅拌的条件下,混合20~30分钟。
所述步骤(2)中搅拌反应时间为3~4h。
所述步骤(2)中透析为:选取截留分子量为5000的纤维透析袋,透析溶液为超纯水,体积为2L,透析三天,每天换水3次。
所述步骤(3)中溶剂为:pH为8的磷酸盐缓冲液。
所述步骤(3)中荧光胺溶液的浓度为3-5mg/mL,荧光胺溶液溶剂为甲醇。
所述步骤(3)中搅拌温度为35-45℃,搅拌时间为25-35分钟。
所述步骤(3)中透析:选取截留分子量为500的纤维透析袋,透析溶液为超纯水,体积为2L,透析三天,每天换水3次。
所述步骤(4)中{G5-PEG-(Au0)25-F}与siPD-L1的N/P为2:1~20:1。
所述步骤(4)中siPD-L1溶液的溶剂为DEPC水。
所述步骤(4)中孵育温度为35-37℃,孵育时间为20-30分钟。
所述步骤(4)中siPD-L1的序列为5’-CCUACUGGCAUUUGCUGAACGCAUU-3’。
本发明提供一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物在制备肿瘤免疫治疗药物中的应用。
本发明以树状大分子作为纳米平台,表面修饰聚乙二醇及荧光胺、内部包裹金纳米颗粒,形成具有高转染效率以及免疫治疗效果的纳米载体。本发明利用树状大分子表面丰富的氨基,首先将聚乙二醇(M-SCM-2000)连接到G5.NH2的外围,进一步包裹金纳米颗粒,再在其表面共价修饰荧光胺。聚乙二醇(M-SCM-2000)的修饰不仅可以降低G5.NH2的细胞毒性,而且能够延长G5.NH2在体内的血液循环时间,包裹的金纳米颗粒不仅可以降低G5.NH2的细胞毒性、还可以显著增强载体的转染效率,荧光胺的修饰可以使G5.NH2具有蓝色荧光从而能在体外实验中进行示踪。
本发明以功能化的树状大分子作为纳米载体,负载siPD-L1,以黑色素瘤为研究对象,通过siRNA转染诱导肿瘤内PD-L1沉默。通过核磁共振氢谱(1H-NMR)对修饰在树状大分子上的聚乙二醇以及荧光胺的数量进行了表征;紫外可见光谱(UV-vis)对Au的成功包裹进行了表征;通过透射电镜(TEM)分析纳米金的尺寸大小;通过凝胶阻滞实验和电势粒径检测对载体负载siPD-L1的能力进行了表征;通过CCK-8实验对载体、载体/siNC(乱序的非特异性对照siRNA)及载体/siPD-L1复合物的细胞毒性进行表征;通过流式细胞仪对载体负载siPD-L1后的转染能力进行了定量分析;通过Western blot测试分析了载体/siPD-L1复合物对肿瘤细胞中PD-L1的沉默效率;最后建立黑鼠皮下肿瘤模型进行抗肿瘤实验。
有益效果
(1)本发明简单,实验条件温和,易操作;
(2)本发明制备得到的功能化树状大分子包裹金纳米颗粒,具有良好的水溶性、生物相容性、良好的基因转染能力;
(3)本发明的功能化树状大分子包裹金纳米颗粒/siPD-L1能够有效沉默PD-L1的表达,实现肿瘤免疫治疗,这在肿瘤的免疫治疗方面具有良好的应用潜力。
附图说明
图1为本发明制备的{G5-PEG-(Au0)25-F}及{G5-PEG-(Au0)25-F}/siPD-L1复合物的合成过程示意图;
图2为本发明制备的{G5-PEG-(Au0)25-F}的核磁共振氢谱图;
图3为本发明制备的{G5-PEG-(Au0)25}的UV-vis图谱;
图4为本发明制备的载体{G5-PEG-(Au0)25-F}透射电子显微镜图和粒径分布直方图;
图5为本发明制备的载体/siPD-L1复合物的凝胶阻滞试验电泳图。其中2~8分别代表N/P比值为0.125、0.25、0.5、1、2、3和4的载体/siPD-L1复合物,1代表裸siPD-L1;
图6为本发明制备的载体及载体/siPD-L1在不同氮磷比下的表面电势图(a)和水动力学直径图(b);
图7为本发明制备的{G5-PEG-(Au0)25-F}、{G5-PEG-(Au0)25-F}/siPD-L1复合物、及{G5-PEG-(Au0)25-F}/siNC复合物对B16的细胞毒性结果图;
图8为本发明制备的{G5-PEG-(Au0)25-F}/siPD-L1复合物对B16细胞摄取能力测试结果图;
图9为本发明制备的载体/siPD-L1复合物对B16细胞的胞内定位实验图;
图10为本发明制备的载体/siPD-L1复合物对B16细胞中PD-L1蛋白的Westernblot试验结果图,其中(a)为Western blot的蛋白条带图,(b)为蛋白条带的灰度定量图;
图11为本发明经瘤内注射PBS、载体/siNC、PD-L1抗体和载体/siPD-L1治疗肿瘤,记录14天内的肿瘤体积变化(a)和体重变化(b)图;
图12为本发明经瘤内注射PBS、载体/siNC、PD-L1抗体和载体/siPD-L1治疗肿瘤后第14天肿瘤组织的CD4+T细胞与CD8+T细胞的流式细胞分析图(a);图(b)为CD4+T细胞的荧光强度分析图,图(c)为CD8+T细胞的荧光强度分析图。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
除非特殊说明,否则所有化学试剂都是可商购的,无需进一步纯化即可直接使用。G5.NH2购自美国Dendritech公司。氯金酸购自国药集团化学试剂有限公司(中国上海)。甲氧基-聚乙二醇-琥珀酰亚胺基(M-SCM-2000)购自上海拓旸生物有限公司(上海,中国)。荧光胺购自上海叶源生物公司(中国上海)。B16细胞(鼠类黑色素瘤细胞系)来自中国科学院生物化学与细胞生物学研究所。RPMI-1640培养基(1640培养基,GIBCO,Invitrogen,Carlsbad,CA),胎牛血清(FBS,GIBCO),青霉素-链霉素(HyClone,Thermo Scientific,Logan,UT)和胰蛋白酶0.25%溶液(HyClone)购自杭州吉诺生物医学技术有限公司(中国杭州)。Cell Counting Kit-8(CCK-8)来自7Sea Biotech Co.,Ltd.(中国上海)。从上海Slac实验动物中心(中国上海)购买约4-6周大的小黑鼠。所有实验中使用的电阻率高于18.2MΩ.cm的水均通过实验室水净化系统(Cascada I,PALL,北京,中国)进行净化。
实施例1
(1)称取第五代聚酰胺-胺树状大分子(PAMAM)50mg溶于50mL超纯水中,称取M-SCM-2000(78mg)溶于7.8mL超纯水中,超声15分钟后快速加入到G5.NH2溶液中,室温搅拌反应4小时,得到G5-PEG粗产品。
(2)在上述得到的G5-PEG中逐滴加入687μL HAuCl4(30mg/mL),冰浴搅拌混合20分钟后,加入过量预冷的NaBH4溶液(5.67mg,1mg/mL)继续反应4小时,得到{G5-PEG-(Au0)25}粗产品。
(3)将上述得到的{G5-PEG-(Au0)25}溶解在pH为8的磷酸盐缓冲液(25mL)中,然后逐滴滴加680μL荧光胺溶液(4mg/mL,甲醇溶解),在避光、40℃水浴并搅拌反应30分钟,然后使用截留分子量5000的透析袋对水溶液透析3天(2L/次,3次/天),透析液经冷冻干燥后得到载体{G5-PEG-(Au0)25-F}。
实施例2
取6mg的{G5-PEG-(Au0)25-F}溶解于600μL的D2O中,对溶液超声10分钟,然后使用核磁共振波谱仪测定材料的核磁氢谱。核磁结果如图2所示,在化学位移2.25-3.4ppm处的质子峰代表G5.NH2的亚甲基质子峰,3.63ppm是(M-SCM-2000)重复单元-CH2CH2-的质子峰,根据它们积分面积之比,计算出每个G5.NH2上连接了16.8个M-SCM-2000分子,接近其投料摩尔比G5.NH2:M-SCM-2000(1:20)。处于7.2-7.6ppm的质子峰,其归属于荧光胺苯环上的质子,8.63ppm处的质子峰归属于荧光胺与树状大分子的氨基(-NH2)反应后生成的-COOH上的质子,根据它们积分面积之比,计算出每个G5.NH2上连接了3.8个荧光胺分子,接近其投料摩尔比G5.NH2:荧光胺(1:5)。
实施例3
将{G5-PEG-(Au0)25}溶解于超纯水中,配制成浓度为200μg/mL溶液,利用紫外-可见分光光度检测在300nm-800nm波段范围内的紫外吸收值。紫外-可见分光光度检测结果如图3所示,{G5-PEG-(Au0)25}在520nm左右有吸收峰,说明金纳米颗粒的成功包裹。
实施例4
将得到的功能化树状大分子包裹金纳米颗粒{G5-PEG-(Au0)25-F}溶解于超纯水中,配制成浓度为1mg/mL的溶液,滴到有碳膜的铜网表面,置于室温下干燥,制得样品。然后采用JEM-2010F型透射电子显微镜在200kV下检测样品(图4)。采用Image J软件进行测量并计算出样品尺寸,样品的直径约为1.9nm,尺寸分布较窄,具有良好的分散性。
实施例5
实施例1制备得到的载体对siRNA的压缩能力采用琼脂糖凝胶阻滞实验进行测定。按照氮磷比(树状大分子的伯氨基与siRNA骨架上磷酸基团的摩尔比)为0.125、0.25、0.5、1、2、3或4,siRNA量为1μg/孔,制备载体/siPD-L1复合物,孵育20分钟。配制8孔含有4SGreen plus nucleic acid stain的琼脂糖凝胶(1.0%w/v),将载体与siPD-L1复合物与上样缓冲液混匀后分别加到琼脂糖凝胶的孔中,电压90V,时间30分钟。电泳结束后用凝胶成像仪拍照分析siPD-L1在凝胶中的迁移情况。图5是{G5-PEG-(Au0)25-F}/siPD-L1复合物的琼脂糖凝胶电泳图,图中显示材料和siPD-L1复合物在氮磷比为1时,siPD-L1被材料完全压缩,说明载体具有较强的基因包裹能力。
实施例6
根据实施例1制备的{G5-PEG-(Au0)25-F}纳米颗粒溶解于超纯水中,按照N/P分别为2:1、5:1、10:1、20:1与5μg siPD-L1制备成不同N/P的载体/siPD-L1复合物,室温下孵育20分钟,然后加入超纯水,使其终体积为1mL。采用马尔文激光粒度仪(Malvern,MK)在633nm激光波长处测定复合物的水动力学粒径和表面电势。结果如图6所示:载体/siPD-L1复合物的电势均在15mV以下,水合粒径均在200nm左右。因此,制备的{G5-PEG-(Au0)25-F}纳米颗粒适合用于基因传递。
实施例7
以B16细胞为模型细胞来检测实施例1所制备的载体以及载体/siPD-L1复合物的细胞毒性。以1×104细胞每孔的密度将B16细胞接种于96孔板中,所用培养基为添加了100U/mL青霉素、100U/mL链霉素和10%FBS的1640完全培养基,在37℃、5%CO2浓度下过夜培养。然后将培养基换为载体浓度分别为0、500、1000、2000、3000nM的含载体、载体/1μgsiPD-L1复合物或载体/1μg siNC复合物的完全培养基,在37℃、5%CO2浓度下共培养24h后,倒掉培养基,加入含10%的CCK-8(10μL)的1640培养基(100μL/孔),在培养箱继续培养3h后,最后放置于多功能酶标仪中在波长为450nm处测试每孔的吸光度,以PBS处理的细胞作为空白对照,细胞活力记为100%。结果如图7所示,与对照组(材料浓度为0,PBS溶液)相比,随着载体浓度的增加,单独的载体逐渐显示出细胞毒性,表现为细胞活力降低;而载体/siPD-L1复合物与载体/siNC复合物几乎没有细胞毒性,这可能是由于负载基因消耗了纳米颗粒表面正电荷从而增强了材料的细胞相容性。
实施例8
以B16细胞为细胞模型来评价细胞对载体/Cy3-siPD-L1的吞噬能力。按照1.5×105细胞每孔的密度接种在12孔板的细胞培养板上,所用培养基为添加了100U/mL青霉素、100U/mL链霉素和10%FBS的1640完全培养基,置于5%CO2、37℃条件下孵育12h。然后将培养基换成含有载体/Cy3-siPD-L1在不同氮磷比(N/P=2、5、8、10或20)的复合物的培养基,与细胞在5%CO2、37℃条件下共培养4h。随后将孔板中的培养基和材料弃去,并用PBS清洗2遍,用胰酶消化细胞,离心收集细胞,用流式细胞仪检测细胞样品的荧光强度以表征细胞对载体/siPD-L1复合物的细胞摄取情况。由图8可以看出,载体/Cy3-siPD-L1复合物经过细胞转染4小时后,在不同氮磷比下都能够成功被细胞内吞,随着氮磷比的增加,荧光强度呈现出先增强后减弱的趋势,并且当N/P=10时,检测到最强的荧光强度,表明在该N/P下载体/siPD-L1复合物最易被细胞摄取,内吞效率最高。
实施例9
以B16细胞为模型细胞,利用胞内定位实验定性评价载体/Cy3-siPD-L1复合物的内吞效率。以2×105/孔的密度将B16细胞种于共聚焦专用培养皿,所用培养基为添加了100U/mL青霉素、100U/mL链霉素和10%FBS的1640培养基,随后在37℃、5%CO2浓度下过夜培养。按每孔1μg/mL、N/P=10制备载体/Cy3-siPD-L1复合物,以单独的siPD-L1为对照。将上述三种材料加入皿中与细胞共培养,待转染细胞4h后,用激光共聚焦显微镜观察拍摄。胞内定位结果如图9所示,单独的siPD-L1几乎无法进入细胞,而在N/P=10时,载体/siPD-L1复合物可成功进入细胞。
实施例10
以B16细胞为细胞模型来评价载体/siPD-L1对PD-L1的沉默效率。按照每孔2×105B16细胞种植于6孔板中,所用培养基为添加了100U/mL青霉素、100U/mL链霉素和10%FBS的1640培养基,置于5%CO2、37℃条件下孵育24h,然后将培养基换成PBS、裸PD-L1siRNA、载体/siPD-L1复合物(N/P=10)和载体/siNC复合物(N/P=10)与细胞共培养4小时。然后更换为含10%FBS的培养基,继续培养24小时。利用胰酶消化并收集细胞,将细胞在冰上裂解,并于4℃,12000rpm离心5分钟,收集上清蛋白液。随后依次进行SDS-PAGE电泳、转膜、免疫反应、ECL化学显影剂定影实验,并对凝胶图像进行分析。实验结果如图10(a-b)所示,裸siRNA和载体/siNC几乎没有基因沉默现象,而载体/siPD-L1具有明显的基因沉默现象。说明载体/siPD-L1能够有效的沉默B16细胞表面PD-L1的表达。
实施例11
所有动物实验均严格按照动物保护协会标准进行。实验用的5周雌性C57BL/6小黑鼠购自上海斯莱克实验动物中心(中国,上海)。将2×106个B16细胞接种到小鼠的右腿,待肿瘤体积达到约100mm3左右时,将小鼠随机分为4组(每组6只)。分组如下:对照组(PBS,100μL);载体/siNC(N/P=10,siNC=0.31mg kg-1,100μL);PD-L1抗体组(a-PD-L1,100μL);载体/siPD-L1(N/P=10,siPD-L1=0.31mg kg-1,100μL),将各组材料通过瘤内注射到小鼠体内。开始治疗的当天记为第0天,每4天治疗一次,每隔2天记录老鼠体重和肿瘤尺寸。从图11(a)可以看出,相对于PBS组和载体/siNC组,a-PD-L1组和载体/siPD-L1均表现出一定程度的抑制肿瘤效果。相比较于a-PD-L1组,载体/siPD-L1肿瘤体积更小,可能是由于载体/siPD-L1组中PD-L1 siRNA从内源上抑制了黑色素瘤细胞表面的PD-L1表达,因而更多的减少PD-1与PD-L1的结合,从而恢复T细胞的免疫应答功能进而杀死肿瘤细胞。从图11(b)可以看出,各组体重变化不明显,说明材料的生物相容性良好。
实施例12
取实施例12中治疗最后一天各组小鼠一只,无菌条件下取出其肿瘤组织,剪碎研磨经400目滤网过滤,得到细胞悬液,再通过各种动物肿瘤浸润组织淋巴细胞分离试剂盒分离出淋巴细胞,最后通过尼龙毛柱获取T淋巴细胞悬液,将上述获取的T细胞分别用CD4/CD8抗体进行标记,通过流式细胞仪对肿瘤组织中CD4+T细胞与CD8+T细胞进行定量分析。结果如图12(a-c)所示,PBS组和载体/siNC组肿瘤组织中的CD4/CD8的表达都低于a-PD-L1组和载体/siPD-L1组,而载体/siPD-L1组中CD4/CD8的表达量最高是由于载体/siPD-L1组从内源上阻止了PD-L1的表达。结果说明a-PD-L1组和载体/siPD-L1组在不同程度上恢复了T细胞的免疫应答功能,而载体/siPD-L1组效果更加明显。
SEQUENCE LISTING
<110> 东华大学
<120> 一种功能化树状大分子包裹金纳米颗粒/PD-L1
siRNA复合物及其制备和应用
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> 人工序列
<400> 1
ccuacuggca uuugcugaac gcauu 25
Claims (10)
1.一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物,其特征在于,所述复合物为表面修饰聚乙二醇和荧光胺、内部包裹金纳米颗粒的聚酰胺-胺树状大分子负载PD-L1siRNA。
2.一种功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物的制备方法,包括:
(1)将第五代聚酰胺-胺树状大分子G5.NH2和甲氧基-聚乙二醇-琥珀酰亚胺羧甲基酯M-SCM-2000分别溶于超纯水中,将得到的M-SCM-2000溶液加入到得到的G5.NH2溶液中,搅拌反应,得到G5-PEG溶液,其中G5.NH2与M-SCM-2000的摩尔比为1:18~22;
(2)将氯金酸水溶液逐滴加入到步骤(1)中的G5-PEG溶液中,混合,加入硼氢化钠溶液,搅拌反应,透析纯化,冷冻干燥,得到{G5-PEG-(Au0)25},其中,步骤(1)中G5.NH2与氯金酸、硼氢化钠的摩尔比为1:25-27:70-80;
(3)将步骤(2)中{G5-PEG-(Au0)25}溶解于溶剂中,避光,逐滴加入荧光胺溶液,搅拌,透析纯化,冷冻干燥,得到荧光胺修饰的聚乙二醇化的树状大分子包裹金纳米颗粒{G5-PEG-(Au0)25-F},其中{G5-PEG-(Au0)25}与荧光胺的摩尔比为1:4-6;
(4)将步骤(3)中{G5-PEG-(Au0)25-F}溶于无菌水中,然后与siPD-L1溶液混合,孵育,得到功能化树状大分子包裹金纳米颗粒/PD-L1 siRNA复合物。
3.根据权利要求2所述方法,其特征在于,所述步骤(1)中M-SCM-2000溶液浓度为8~15mg/mL;G5.NH2溶液浓度为0.8~1.2mg/mL。
4.根据权利要求2所述方法,其特征在于,所述步骤(1)中搅拌反应为:室温搅拌反应3-5h。
5.根据权利要求2所述方法,其特征在于,所述步骤(2)中混合为:在冰浴搅拌的条件下,混合20~30分钟;搅拌反应时间为3~4h。
6.根据权利要求2所述方法,其特征在于,所述步骤(3)中溶剂为:pH为8的磷酸盐缓冲液;荧光胺溶液的浓度为3-5mg/mL,荧光胺溶液溶剂为甲醇。
7.根据权利要求2所述方法,其特征在于,所述步骤(3)中搅拌温度为35-45℃,搅拌时间为25-35分钟。
8.根据权利要求2所述方法,其特征在于,所述步骤(4)中siPD-L1溶液的溶剂为DEPC水;孵育温度为35-37℃,孵育时间为20-30分钟。
9.根据权利要求2所述方法,其特征在于,所述步骤(4)中{G5-PEG-(Au0)25-F}与siPD-L1的N/P为2:1~20:1;siPD-L1的序列为5’-CCUACUGGCAUUUGCUGAACGCAUU-3’。
10.一种如权利要求1所述复合物在制备肿瘤免疫治疗药物中的应用。
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