CN116421719A - 一种peg可脱落和双敏感纳米药物及其制备方法与应用 - Google Patents

一种peg可脱落和双敏感纳米药物及其制备方法与应用 Download PDF

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CN116421719A
CN116421719A CN202310226912.XA CN202310226912A CN116421719A CN 116421719 A CN116421719 A CN 116421719A CN 202310226912 A CN202310226912 A CN 202310226912A CN 116421719 A CN116421719 A CN 116421719A
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nano
drug
peg
sensitive
cap
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朱康顺
肖泽聪
谭伊童
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Second Affiliated Hospital of Guangzhou Medical University
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Second Affiliated Hospital of Guangzhou Medical University
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    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
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Abstract

本发明属于生物医药技术领域，具体涉及一种PEG可脱落和双敏感纳米药物及其制备方法与应用。该纳米药物将抗癌活性成分负载在可靶向释药的磷酸钙纳米颗粒上进行递送，并且用甲氧基‑聚(乙二醇)‑甲基马来酸酐作为屏蔽层进行保护；当纳米药物到肿瘤微环境中，可以引发相关抗癌活性成分的缓慢释放，从增强肿瘤浸润和CTL的增殖、避免肿瘤细胞逃避CTLs杀伤等多方面来协同达到显著的抗肿瘤效果。

Description

一种PEG可脱落和双敏感纳米药物及其制备方法与应用
技术领域
本发明属于生物医药技术领域。更具体地，涉及一种PEG可脱落和双敏感纳米药物及其制备方法与应用。
背景技术
肿瘤免疫治疗是近十多年来迅速发展起来的一种新型疗法，其通过激活宿主免疫系统以消灭肿瘤细胞。特别是基于免疫检查点阻断(immune checkpoint blockade，ICB)的免疫疗法，可以显著提高晚期恶性肿瘤(例如非小细胞细胞癌和晚期黑色素瘤等)患者的总生存率，为临床上的常用癌症治疗方法。但是，由于肿瘤细胞有致密的细胞外基质(extracellular matrix，ECM)构成物理屏障，阻止了细胞毒性T淋巴细胞(cytotoxic Tlymphocytes，CTLs)在大部分免疫冷肿瘤中的浸润。随着肿瘤细胞的发展，肝星状细胞(hepatic stellate cells，HSCs)等产生基质的细胞被激活后，可产生大量的ECM成分构成致密的物理屏障和分泌蛋白酶参与ECM的重塑，最终形成复杂的肿瘤免疫抑制微环境。另外的，作为ECM的关键成分，透明质酸(hyaluronan，HA)已被报道在各种肿瘤中过量表达，阻碍CTLs的浸润。
在进一步的临床试验中，并非所有患者都能从基于ICB的免疫疗法中获益。据研究报道，随着肝细胞癌(hepatocellular carcinoma，HCC)发展过程中，ECM被过度产生，不仅促进肿瘤的生长和转移，还增加了液体压力和增强了物理屏障的形成，阻碍了免疫细胞的浸润。为了解决上述问题，中国专利申请CN108884159A公开了一种用于癌症治疗的包含肿瘤抑制基因治疗和免疫检查点阻断的组合物，将免疫检查点抑制剂和p53和/或MDA-7基因治疗联合使用，并结合细胞外基质降解蛋白来增强抗肿瘤效果。但是上述蛋白类药物在体内非常容易被酶水解，导致生物半衰期短，并且，全身用药也导致了严重的毒副作用，存在一定的安全隐患。因此，迫切需要提供一种保护蛋白类药物不被水解延长生物半衰期，在ECM环境中具有较好浸润性，具有靶向肿瘤释药效果的纳米药物。
发明内容
本发明要解决的技术问题是克服现有抗肿瘤蛋白药物易被水解，用量大毒副作用大的缺陷和不足，提供一种保护蛋白类药物不被水解延长生物半衰期，在ECM环境中具有较好浸润性，具有靶向肿瘤释药效果的PEG可脱落和双敏感纳米药物。
本发明的目的是提供所述PEG可脱落和双敏感纳米药物的制备方法。
本发明另一目的是提供所述PEG可脱落和双敏感纳米药物的应用。
本发明上述目的通过以下技术方案实现：
本发明的思路是消除ECM的影响，通过促进免疫细胞的浸润来提高基于ICB的免疫疗法对肿瘤的有效性。研究表明，透明质酸酶(hyaluronidases，HAase)对清除ECM，提高药物渗透和CTLs在肿瘤中的浸润有较大的临床作用。可以利用透明质酸酶进行辅助治疗，协助基于ICB的免疫疗法促进更好的抗肿瘤疗效。另一方面，白细胞介素-12(IL-12)等免疫因子在抗肿瘤免疫中发挥着重要作用，其中IL-12可以促进CD8+T细胞增值并增加其细胞毒性活性，同时促进巨噬细胞极化为M1样的表型，以杀死肿瘤细胞，并促进肿瘤抗原的呈现。发明人尝试将HAase与IL-12等免疫因子还有ICB结合，通过促进CTLs的浸润和增殖来增加肿瘤组织中CTLs的丰度，提高ICB对HCC的抗肿瘤效果。但是，这类蛋白质类药物，如HAase和IL-12在体内容易被酶水解，导致生物半衰期短；并且HAase和IL-12等免疫因子的全身给药也导致了严重的毒性，例如，IL-12的全身给药容易引起细胞因子风暴综合征，而HAase的全身给药可使正常组织和器官中的透明质酸降解，引起一系列不良反应，ICB也有诱发免疫相关不良事件(immune-related adverse events，irAEs)的风险，包括肝炎、肺炎和心肌炎。
为了解决上述问题，本发明提供了一种PEG可脱落和双敏感纳米药物，所述PEG可脱落和双敏感纳米药物将透明质酸酶和免疫因子包载于磷酸钙纳米颗粒中，通过二苯并环辛烯-聚乙二醇-聚天冬氨酸(DBCO-PEG-PAsp)共聚物来控制磷酸钙纳米颗粒的形成，将MMP敏感肽修饰的aPD-L1抗体连接在磷酸钙纳米颗粒的表面，甲氧基-聚(乙二醇)-甲基马来酸酐与aPD-L1抗体的N端氨基连接形成屏蔽层。
本发明所提供的PEG可脱落和双敏感纳米药物，其中的活性成分透明质酸酶(HAase)、IL-12等免疫因子和aPD-L1抗体使用pH值和MMP双敏感聚合物磷酸钙(CaP)纳米颗粒载体进行联合递送。外围的甲氧基-聚(乙二醇)-甲基马来酸酐(mPEG20k-CDM)作为屏蔽层，保护其中的蛋白质类药物不被体内的酶水解，当纳米药物到达肿瘤细胞附近时，肿瘤细胞偏酸(pH 6.5)的微环境条件下，引发CaP的溶解从而促进IL-12等免疫因子和负责ECM消化的HAase的释放，增强了肿瘤浸润和CTL的增殖；与此同时，肿瘤微环境中过度表达的MMP酶会引发纳米药物中aPD-L1的释放，防止了肿瘤细胞逃避CTLs的杀伤作用。这种组合策略诱发了强大的抗肿瘤免疫力，能显著抑制肿瘤细胞的生长。
进一步地，所述免疫因子选自白细胞介素-12、白介素IL-2、白介素IL-6中的一种或多种。
更进一步地，所述MMP敏感肽为末端修饰了叠氮的MMP敏感肽。优选地，所述MMP敏感肽为末端修饰了叠氮的MMP-2敏感肽(N3-MMP-2敏感肽)或末端修饰了叠氮的MMP-9敏感肽(N3-MMP-9敏感肽)。
进一步地，所述纳米药物还可以负载荧光试剂、造影剂、蛋白多肽类药物、小分子药物中的一种或多种，来达到相应的效果。
另外的，本发明还提供了所述PEG可脱落和双敏感纳米药物的制备方法，具体包括以下步骤：
S1、将可溶性钙盐溶于水中，0～5℃加入透明质酸酶、免疫因子和DBCO-PEG-PAsp共聚物，混合均匀，滴加磷酸盐溶液，充分反应后离心，收集沉淀，洗涤，得负载透明质酸酶和免疫因子的磷酸钙纳米颗粒；
S2、将步骤S1所得磷酸钙纳米颗粒与MMP-2敏感肽修饰的aPD-L1抗体和甲氧基-聚(乙二醇)-甲基马来酸酐在水中充分混合，再加入甲氧基-聚(乙二醇)-甲基马来酸酐，反应完全，后处理，即得。
进一步地，步骤S1中，所述可溶性钙盐选自硝酸钙、氯化钙，磷酸钙中的一种或多种。
更进一步地，步骤S1中，所述磷酸盐为磷酸氢二钠。
另外的，本发明还要求保护所述PEG可脱落和双敏感纳米药物在制备抗癌药物中的应用。
进一步地，所述癌包括肝细胞癌、乳腺癌、胰腺癌、结直肠癌。
另外的，本发明还要求保护所述PEG可脱落和双敏感纳米药物在制备诊断制剂中的应用。
本发明具有以下有益效果：
本发明提供了一种PEG可脱落和双敏感纳米药物，将抗癌活性成分负载在可靶向释药的磷酸钙纳米颗粒上进行递送，并且用甲氧基-聚(乙二醇)-甲基马来酸酐作为屏蔽层进行保护；当纳米药物到肿瘤微环境中，可以引发相关抗癌活性成分的缓慢释放，从增强肿瘤浸润和CTL的增殖、避免肿瘤细胞逃避CTLs杀伤等多方面来协同达到显著的抗肿瘤效果。
附图说明
图1为本申请实施例1纳米药物的表征特征图，其中，图1中的(a)为DBCO-PEG-PAsp的1H NMR谱图；图1中的(b)为P-αPD-L1-CaP@H/I纳米药物的制备流程示意图；图1中的(c)为αPD-L1-CaP@H/I纳米药物动态光散射(DLS)测量颗粒大小数据统计图；图1中的(d)P-αPD-L1-CaP@H/I纳米药物动态光散射(DLS)测量颗粒大小数据统计图；图1中的(e)为DLS测定αPD-L1-CaP@H/I纳米药物和P-αPD-L1-CaP@H/I纳米药物的zeta电位数据统计图；图1中的(f)为αPD-L1-CaP@H/I纳米药物和P-αPD-L1-CaP@H/I纳米药物在不同条件下(pH 7.4和pH 6.5+MMP-2；MMP-2的浓度，10nM)的方案和透射电子显微镜(TEM)图像；图1中的(g)为在pH7.4和pH6.5+MMP-2下，P-αPD-L1-CaP@H/I纳米药物的HAase体外释放量数据图；图1中的(h)为在pH7.4和pH 6.5+MMP-2条件下，体外从P-αPD-L1-CaP@H/I纳米药物中释放aPD-L1抗体数据图。
图2为纳米药物体外免疫调节效果，其中，图2中的(a)为用流式细胞仪测量各种处理后的CD8+T细胞的增殖情况图；图2中的(b)为各种处理后增殖的CD8+T细胞百分比的量化分析图；图2中的(c)为流式细胞仪分析接受不同治疗后的骨髓来源巨噬细胞(BMDMs)的M1样(CD80+)和M2样(CD206+)的高分型；图2中的(d)为各种处理后M1样(CD80+)巨噬细胞的百分比的量化分析图；图2中的(e)为M2样(CD206+)巨噬细胞的百分比的量化分析图。
图3为纳米药物体内抗肿瘤活性，其中，图3中的(a)为体内抗肿瘤研究的流程示意图；图3中的(b)为分别用PBS,P-αPD-L1-CaP@I,P-αPD-L1-CaP@H,αPD-L1-CaP@H/I,P-iso-CaP@H/I,和P-αPD-L1-CaP@H/I处理的Hepa1-6肿瘤小鼠的肿瘤生长曲线图；图3中的(c)为21天治疗后从不同组收集的皮下肿瘤的典型图像；图3中的(d)为用不同配方治疗的Hepa1-6肿瘤小鼠的肿瘤重量统计图；图3中的(e)为接受不同治疗的Hepa1-6肿瘤携带小鼠的生存曲线图。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
其中，所述DBCO-PEG-PAsp可通过购买也可以DBCO-PEG-NH2为引发剂，采用BLA-NCA开环聚合法自行制备，具体制备方法如下：
在氩气保护下，将DBCO-PEG-NH2(0.20g，2200g/mol，0.1mmol)溶解在无水二氯甲烷(20mL)中，置于50mL干燥的schlenk烧瓶；将BLA-NCA(1.00g，249.22g/mol,，4.0mmol)溶解于无水DMF(2mL)中，加入上述schlenk烧瓶中，封闭烧瓶，35℃反应48h；将溶液沉淀到300mL冷却乙醚中，离心收集产物，用乙醚洗涤三次，真空干燥得到DBCO-PEG-PBLA，收益率:89％。
将所得DBCO-PEG-PBLA(1.45g，14500g/mol，0.10mmol)溶解在DMSO(20mL)中，在N2保护下搅拌加入乙酰氯(Ac-Cl)(0.0157g，78.5g/mol，0.20mmol，2eq.)，在室温下反应4h后，搅拌加入溴化氢(HBr，33wt.％醋酸溶液)(65μL，12mmol，2等量苄基的DBCO-PEG-PBLA)和三氟乙酸(92μL，114.02g/mol，12mmol，2等量苄基)，在35℃反应4h后，用去离子水透析(MWCO:1kDa)48h，冷冻干燥得到DBCO-PEG-PAsp，收益率:87％。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1一种PEG可脱落和双敏感纳米药物的制备
所述PEG可脱落和双敏感纳米药物的制备，具体包括以下步骤：
S1、负载HAase和IL-12的CaP纳米颗粒的制备：
将29.5mg Ca(NO3)2·4H2O溶于50mL蒸馏水中，在冰浴条件下加入1mL蛋白质HAase(1mg mL-1)和IL-12(5μg mL-1)并搅拌均匀，加入0.5gDBCO-PEG-PAsp至上述溶液中并搅拌15分钟，得到Ca2+混合物溶液；将21.2mg Na2HPO4溶于50ml蒸馏水并充分搅拌，滴加到上述Ca2+混合物溶液中，搅拌30分钟后以10000rpm离心30分钟，用蒸馏水洗涤三次后，收集沉淀物，为负载HAase和IL-12的CaP纳米颗粒(CaP@H/I)。
S2、P-αPD-L1-CaP@H/I纳米颗粒的制备：
将步骤S1所得CaP@H/I与N3-MMP-2敏感肽结合的aPD-L1抗体在水中混合，并涡旋8小时，得到αPD-L1-CaP@H/I纳米药物；再加入PEG20k-CDM(聚乙二醇(Mn＝20kDa)-2，3，二甲基马来酰胺键)并在室温下涡旋4小时，通过透析除去未反应的N3-MMP-2敏感肽结合的aPD-L1抗体和PEG20k-CDM，从而得到pH/MMP-2敏感的纳米药物(P-αPD-L1-CaP@H/I)。
HAase和IL-12装载到pH敏感的磷酸钙(CaP)纳米颗粒；同时，应用DBCO-PEG-Pasp共聚物来控制CaP纳米颗粒的形成(DBCO-PEG-CaP@HAase/IL-12)；进一步通过点击反应将用MMP-2敏感肽修饰的αPD-L1抗体连接在纳米粒子的表面，甲氧基-聚(乙二醇)-(mPEG20K-CDM)与抗体的N端氨基反应，形成屏蔽层。流程示意图参见图1中的(b)。
实施例2一种PEG可脱落和双敏感纳米药物的制备
与实施例1不同之处在于，实施例2中，将蛋白质HAase替换为吲哚青绿(ICG)，其他参数及操作参考实施例1。可制备得到应用于生物分布研究的纳米粒子。
实施例3一种PEG可脱落和双敏感纳米药物的制备
与实施例1不同之处在于，实施例3中，将Ca(NO3)2·4H2O替换为Ca(NO3)2·4H2O和MnCl2质量比为1:1的混合物，其他参数及操作参考实施例1。可制备得到应用于磁共振成像实验的纳米粒子。
实施例1～3效果相似，以下以实施例1为例进行实验。
实验例1纳米药物的表征
测定DBCO-PEG-PAsp的1H NMR图，参见图1中的(a)，结果证实了DBCO-PEG-PAsp的成功合成。
测定αPD-L1-CaP@H/I和P-αPD-L1-CaP@H/I的动态光散射(DLS)，结果参见图1中的(c)和(d)，由图可见，αPD-L1-CaP@H/I纳米药物拥有151.5nm的流体力学直径。然而，P-αPD-L1-CaP@H/I的流体动力学直径略微增加到168.2nm，可能是由于纳米药物上装饰的mPEG20k，形成了一个屏蔽层。
测定αPD-L1-CaP@H/I和P-αPD-L1-CaP@H/I的zeta电位，结果参见图1中的(e)，由图可见，αPD-L1-CaP@H/I和P-αPD-L1-CaP@H/I纳米药物的zeta电位表现相似，分别为-2.3和-1.3。
使用透射电子显微镜(TEM)研究不同条件下αPD-L1-CaP@H/I和P-αPD-L1-CaP@H/I的形态。如图1中的(f)所示，αPD-L1-CaP@H/I纳米药物呈现为球形纳米颗粒。在pH 7.4时，P-αPD-L1-CaP@H/I显示出核壳结构，即CaP的深色核心和PEG20k的灰色外壳。同时，还观察到纳米药物的直径略有增加。在pH 6.5和MMP-2酶的作用下，P-αPD-L1-CaP@H/I纳米药物被完全分解。在pH 7.4和pH 6.5+MMP-2条件下，测量了P-αPD-L1-CaP@H/I的体外HAase和aPD-L1释放曲线。如图1中的(g)和(h)所示，HAase在pH 7.4时释放非常缓慢，但在pH 6.5+MMP-2时则加速释放，这是因为纳米药物在酸性肿瘤微环境中崩解。同样，aPD-L1抗体在pH 7.4时几乎没有释放，但在pH 6.5+MMP-2时迅速释放，由于MMP-2敏感肽的裂解，仅12小时就释放了80.3％。上述结果表明，pH/MMP-2双敏感纳米药物可以在模拟肿瘤微环境的条件下有效释放药物。
实验例2纳米药物的体外免疫调节效果
1、不同pH条件下纳米药物对CD8+T细胞增殖的影响
实验方法：将C57BL/6j小鼠脾脏中分离的淋巴细胞悬浮在含有0.1％ FBS的PBS中，密度为每毫升5×107个细胞；在黑暗条件下用荧光染料CFSE(10μM)染色10分钟后，加入5倍体积的含40％ FBS的PBS，并在冰上孵育10分钟；CFSE预染色的淋巴细胞用1640培养基洗3次，再用所得CFSE预染色的淋巴细胞以每毫升1×106个细胞的密度在12孔板中进行铺板，并接受不同的处理(pH7.4培养基(Control)；pH 7.4培养基包含P-αPD-L1-CaP@H/Inanoparticles(pH 7.4+NPs)；pH 6.5培养基(pH 6.5)；pH 6.5培养基包含P-αPD-L1-CaP@H/I纳米颗粒(pH 6.5+NPs)和pH 7.4培养基包含IL-12(IL-12))。培养72小时后，在4℃下用氟色素结合的CD3和CD8抗体对细胞进行染色30分钟，然后，收集细胞并通过流式细胞仪进行分析。结果参见图2中的(a)和(b)。
实验结果分析：由图可见，与接受PBS处理的CD8+T细胞相比，P-αPD-L1-CaP@H/I纳米药物在pH 7.4孵育24小时后，用CFSE标记的增殖CD8+T细胞的百分比几乎没有增加；相反，在pH 6.5可以检测到T细胞增殖的明显增加(22.79％)。上述结果表明纳米药物在pH6.5条件下可以释放的IL-12诱导CD8+T细胞增殖。
2、不同pH条件下纳米药物对BMDMs细胞的影响
实验方法：将C57BL/6j小鼠骨髓来源的单细胞(BMDMs)在含有小鼠M-CSF(巨噬细胞集落刺激因子，PeproTech)的完全DMEM培养基中培养，在第三天，更换一半体积的细胞悬液；分化6天后，BMDMs被分化成M0型巨噬细胞。将粘附的M0型巨噬细胞以每毫升1x 105个细胞的密度接种在6孔板中，并接受不同的处理(pH 7.4培养基(Control)；pH 7.4培养基包含P-αPD-L1-CaP@H/I纳米颗粒(pH 7.4+NPs)；pH 6.5培养基(pH 6.5)；pH 6.5培养基包含P-αPD-L1-CaP@H/I纳米颗粒(pH 6.5+NPs)和pH 7.4培养基包含IL-12(IL-12))，培养48小时后，离心收集细胞。所得细胞在4℃下用氟色素结合的F4/80、CD11b和CD86的抗体染色30分钟后，用含有1％ Triton X-100的4％多聚甲醛固定细胞，并在37℃下用氟色素结合的CD206抗体染色30分钟。通过离心法收获细胞，用流式细胞仪进行分析。结果参见图2中的(c)、(d)和图(e)。
实验结果分析：由图2中的(a)可见，与接受pH 7.4培养基处理的BMDMs相比，接受P-αPD-L1-CaP@H/I处理的细胞在pH 7.4时，M1巨噬细胞和M2巨噬细胞的百分比没有明显差异；然而，当pH值调整到6.5时，M1巨噬细胞的百分比明显增加，同时M2巨噬细胞则减少。
考虑到HAase可以降解致密ECM中的透明质酸，消除阻止淋巴细胞浸润的物理障碍；IL-12可以诱导CD8+T细胞增殖，并在体外增加M1巨噬细胞的极化，结合上述结果可以说明，纳米药物中封装的HAase和IL-12纳米药物在pH6.5条件下可以释放出来，提高肿瘤中CD8+T细胞和M1巨噬细胞的丰度，从而扭转HCC的"寒冷"免疫微环境。
实验例3纳米药物的体内抗肿瘤活性
实验方法：
6周大的雄性C57BL/6j小鼠皮下注射Hepa1-6细胞，以建立动物肿瘤模型。当肿瘤达到约100mm3，将小鼠随机分为六组(PBS，P-αPD-L1-CaP@I，P-αPD-L1-CaP@H，αPD-L1-CaP@H/I，P-iso-CaP@H/I和P-αPD-L1-CaP@H/I)。每三天通过尾静脉注射PBS和纳米药物；每三天用游标卡尺测量肿瘤体积，并使用以下公式计算：肿瘤体积＝长×宽2×0.5。此外，每三天记录一次身体的重量，同时每天记录生存率。结果参见图3。
实验结果分析：
在携带皮下Hepa1-6肿瘤的C57BL/6j小鼠中研究了抗肿瘤活性，小鼠被随机分为六组，然后每3天接受不同的治疗(PBS，P-αPD-L1-CaP@I，P-αPD-L1-CaP@H，αPD-L1-CaP@H/I，P-iso-CaP@H/I和P-αPD-L1-CaP@H/I)，共三个周期流程参见图3中的(a)。
根据肿瘤大小和肿瘤重量来评估抗肿瘤活性，详见图3中的(b)～(d)。与对照组相比，两种药物(即HAase或IL-12)与aPD-L1抗体联合治疗都表现出一定的抗肿瘤活性，意味着这两种药物增强了aPD-L1抗体的抗肿瘤功效。尽管接受aPD-L1抗体与HAase和IL-12(αPD-L1-CaP@H/I)联合治疗的小鼠表现出更好的抗肿瘤效果，但PEG可脱落的纳米药物(P-αPD-L1-CaP@H/I)治疗表现出最好的抗肿瘤效果，应该是由于其在肿瘤积累方面的优异表现。同时，与P-iso-CaP@H/I组相比，用P-αPD-L1-CaP@H/I治疗的小鼠也表现出更好的抗肿瘤活性，纳米药物使用异型抗体替代aPD-L1抗体，表明aPD-L1抗体与HAase和IL-12的联合疗法发挥了协同抗肿瘤作用。一致的是，尽管这些纳米药物都对小鼠的存活率显示出积极的影响，但P-αPD-L1-CaP@H/I似乎是延长动物存活率最有效的一种(图3中的(e))。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

1.一种PEG可脱落和双敏感纳米药物，其特征在于，所述PEG可脱落和双敏感纳米药物将透明质酸酶和免疫因子包载于磷酸钙纳米颗粒中，通过DBCO-PEG-PAsp共聚物来控制磷酸钙纳米颗粒的形成，将MMP敏感肽修饰的aPD-L1抗体连接在磷酸钙纳米颗粒的表面，甲氧基-聚(乙二醇)-甲基马来酸酐与aPD-L1抗体的N端氨基连接形成屏蔽层。
2.根据权利要求1所述PEG可脱落和双敏感纳米药物，其特征在于，所述免疫因子选自白细胞介素-12、白介素IL-2、白介素IL-6中的一种或多种。
3.根据权利要求1所述PEG可脱落和双敏感纳米药物，其特征在于，所述MMP敏感肽为末端修饰了叠氮的MMP敏感肽。
4.根据权利要求1～3任一所述PEG可脱落和双敏感纳米药物，其特征在于，所述纳米药物还负载荧光试剂、造影剂、蛋白多肽类药物、小分子药物中的一种或多种。
5.权利要求1～4任一所述PEG可脱落和双敏感纳米药物的制备方法，其特征在于，具体包括以下步骤：
S1、将可溶性钙盐溶于水中，0～5℃加入透明质酸酶、免疫因子和DBCO-PEG-PAsp共聚物，混合均匀，滴加磷酸盐溶液，充分反应后离心，收集沉淀，洗涤，得负载透明质酸酶和免疫因子的磷酸钙纳米颗粒；
S2、将步骤S1所得磷酸钙纳米颗粒与MMP-2敏感肽修饰的aPD-L1抗体和甲氧基-聚(乙二醇)-甲基马来酸酐在水中充分混合，再加入甲氧基-聚(乙二醇)-甲基马来酸酐，反应完全，后处理，即得。
6.根据权利要求5所述制备方法，其特征在于，步骤S1中，所述可溶性钙盐选自硝酸钙、氯化钙，磷酸钙中的一种或多种。
7.根据权利要求5所述制备方法，其特征在于，步骤S1中，所述磷酸盐为磷酸氢二钠。
8.权利要求1～4任一所述PEG可脱落和双敏感纳米药物在制备抗癌药物中的应用。
9.根据权利要求8所述应用，其特征在于，所述癌包括肝细胞癌、乳腺癌、胰腺癌、结直肠癌。
10.权利要求1～4任一所述PEG可脱落和双敏感纳米药物在制备诊断制剂中的应用。
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