CN109419782A - 一种提高酶类药物稳定性的纳米制剂及其制备方法和用途 - Google Patents
一种提高酶类药物稳定性的纳米制剂及其制备方法和用途 Download PDFInfo
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Abstract
本发明属生物制药技术领域，涉及一种提高酶类药物稳定性的纳米制剂，本发明由顺式乌头酸酐(Aco)、树枝状聚左旋赖氨酸(DGL)和功能性聚乙二醇(NHS‑PEG)制成纳米粒PEG‑DGL/En‑Aco。本发明制得的纳米粒，其包封率为71.46±0.31％，纳米粒的外观圆整，形态呈球形；所述纳米粒能够减少胰蛋白酶对CAT的降解，72h内，所述PEG‑DGL/CAT‑Aco纳米粒在HL‑60细胞内的酶活性显著高于游离CAT，细胞外基质中，交联纳米粒的酶活性随时间增加而逐渐增加；制得的纳米粒能显著减少模型动物脑梗死面积；能明显抑制缺血再灌注损伤，具有潜在的神经及血管保护作用。所述的纳米粒可用于制备脑卒中治疗药物。
Description
技术领域
本发明属生物制药技术领域，涉及一种提高酶类药物稳定性的纳米制剂，具体涉及通过电荷吸附法制备的纳米制剂及其在在保护酶类药物稳定性中的用途。
背景技术
随着生物工程技术的迅猛发展，生物大分子药物已成为21世纪药物研发中最具发展前景的领域之一。现有技术公开了大分子药物如多肽和蛋白以其高度专属性，在中枢神经系统的治疗(Central nervous system，CNS)中具有明显优势，被越来越多地用于临床前及临床研究，报道显示，已有超过数百种的蛋白多肽类药物应用于临床，大分子药物已成为当前相关技术领域的研究热点，研究显示，该类药物相对于传统的化学合成药物具有药理活性高、特异性强、易被机体吸收等显著的优点。
研究表明，蛋白多肽类药物对CNS系统疾病如帕金森病、阿尔茨海默病、脑肿瘤、IS、抑郁等的治疗具有较大的临床应用前景，但由于其分子量大小、亲水性、带电性和极性等原因使之缺乏细胞膜的渗透性，难以像小分子一样穿越细胞膜而通过生理屏障，也不能透过血脑屏障(Blood Brain Barrier，BBB)；此外，该类药物的稳定性差，进入生物体内后易被酶降解，使蛋白质失活；同时由于肾小球的滤过作用及肝脏的协同作用，使得蛋白多肽类药物的体内半衰期短，等等；上述缺点都极大地限制了其应用范围，降低了其治疗效果。
树枝状大分子具有高度分支化的纳米结构，被广泛应用于蛋白质及酶类药物的载体。有研究以多聚赖氨酸(poly-L-lysines,PLL)为载体，通过静电吸附作用包载CAT后载入巨噬细胞，利用巨噬细胞在体内的趋向性实现靶向给药；其中，PLL包覆可显著提高CAT稳定性，保证其被巨噬细胞吞噬后可一定程度保持活性，并通过细胞与神经元细胞的相互作用进入神经元细胞发挥抗氧化作用，但是多聚赖氨酸来源不可控，因此其应用受到了极大的限制。
之后，有学者又开发了树枝状聚左旋赖氨酸(Dendrigraft poly-L-lysines,DGL)，是一种由赖氨酸组成的球形结构，水溶性高、热稳定性好、可完全生物降解的高分子材料，且原料已商品化、质量稳定，目前已有研究成功地将其用于基因及蛋白药物的递送，显著提高了药物稳定性’。
基于现有技术的现状，本申请的发明人拟提供一种提高酶类药物稳定性的纳米制剂及其制备方法和用途，本发明针对酶类药物体内不稳定、易被降解的特点，将DGL与酶类药物通过静电吸附制备成载药纳米粒，显著提高其体内外稳定性。为酶类药物在临床的应用提供了新的思路。
与本申请相关的技术有：
1.Fortuna,A.,G.Alves.Intranasal delivery of systemic-acting drugs:Small-molecules and biomacromolecules[J].European Journal of Pharmaceuticsand Biopharmaceutics,(2014),88(1):8-27.
2.Yi,X.,D.S.Manickam.Agile delivery of protein therapeutics to CNS[J].J Control Release,(2014),190:637-663.
3.Brasnjevic,I.,H.W.Steinbusch.Delivery of peptide and protein drugsover the blood-brain barrier[J].Prog Neurobiol,(2009),87(4):212-251.
4.Neuwelt,E.,N.J.Abbott.Strategies to advance translational researchinto brain barriers[J].The Lancet Neurology,(2008),7(1):84-96.
5.Tomlinson,I.M.Next-generation protein drugs[J].NatureBiotechnology,(2004),22(5):521-522.
6.Zhao,Y.,M.J.Haney.Polyelectrolyte complex optimization formacrophage delivery of redox enzyme nanoparticles[J].Nanomedicine(Lond),(2011),6(1):25-42.
7.Sideratou,Z.,N.Sterioti.Arginine end-functionalized poly(L-lysine)dendrigrafts for the stabilization and controlled release of insulin[J].JColloid Interface Sci,(2010),351(2):433-441.
8.An,S.,Y.Kuang.Brain-targeting delivery for RNAi neuroprotectionagainst cerebral ischemia reperfusion injury[J].Biomaterials,(2013),34(35):8949-8959.。
发明内容
本发明的目的是针对现有技术中有关酶类药物体内不稳定、易被降解的缺点，提供一种提高酶类药物体内外稳定性的纳米制剂及其制备方法和应用。
本发明的可提高酶类药物体内外稳定性的纳米制剂，由顺式乌头酸酐(cis-Aconitic anhydride,Aco)、树枝状聚左旋赖氨酸(Dendrigraft poly-L-lysines,DGL)、功能性聚乙二醇(NHS-PEG)制成；
本发明提供了所述的可提高酶类药物体内外稳定性的纳米制剂的制备方法，其包括：采用功能性聚乙二醇(PEG-NHS)与树枝状聚左旋赖氨酸(Dendrigraft poly-L-lysines,DGL)发生偶合反应，生成PEG-DGL，然后采用交联剂N-琥珀酰亚胺3-(2-吡啶二硫基)(N-succinimidyl 3-(2-pyridyldithio)-propionate，SPDP)表面的NHS发生特异性反应，DGL表面的部分氨基被PDP基团取代，得到PEG-DGL-PDP，之后采用顺式乌头酸酐(cis-Aconitic anhydride,Aco)修饰酶类药物(Enzyme，En)，反应生成En-Aco，增加酶类药物负电荷密度；最后通过电荷吸附法将PEG-DGL和En-Aco交联成纳米粒PEG-DGL/En-Aco。
本发明中，DGL表面的氨基与功能PEG一端的NHS发生特异性反应，其中，按DGL:PEG＝1:5～1:8(mol/mol)的比例称量，将NHS-PEG溶于PBS 7.4缓冲溶液中，然后逐滴加入搅拌的DGL溶液中(5mg/ml,PBS,pH＝7.4)，室温搅拌反应1h，转移至5kDa的超滤管内，加入PBS，6000rpm离心20min，重复两次，纯化得到PEG-DGL；
本发明中，DGL表面的氨基与交联剂N-琥珀酰亚胺3-(2-吡啶二硫基)(N-succinimidyl 3-(2-pyridyldithio)-propionate，SPDP)表面的NHS发生特异性反应，其中，SPDP与PEG-DGL(DGL:SPDP＝1:2～1:5mol/mol)混合于PBS溶液中(100mM Na3PO4,1m MEDTA,pH＝7.4)，室温搅拌反应1～3h。DGL表面的部分氨基被PDP基团取代，得到PEG-DGL-PDP；
本发明中，顺式乌头酸酐(cis-Aconitic anhydride,Aco)与荷正电酶类药物(Enzyme，En)表面的氨基反应增加其表面的负电荷，其中，首先，按照En:Aco＝3:1～6:1的质量比，称取En和Aco，然后，将Aco溶于DMSO中，加入Na2CO3(pH＝7.4～8.5)溶解的En溶液中，再加入2μl N，N-二异丙基乙胺，室温搅拌2h，反应生成En-Aco；
精密称取En-Aco(En，1～2mg/ml)和PEG-DGL-PDP(DGL 2～10mg/ml)，分别溶于10mM Tris(pH 7.4)溶液中，水浴超声2min。定量称取二硫苏糖醇(1,4-Dithiothreitol，DTT)，溶于10mM Tris(pH 7.4)缓冲液中，使其浓度为1～2mg/ml，按照1:40～1:50的体积比(DTT:DGL)加入PEG-DGL-PDP溶液中，迅速涡旋15min，将PDP的二硫键还原成巯基，并逐滴加入搅拌的等体积En-Aco溶液中(DGL:En＝2:1～10:1wt/wt)，室温持续搅拌20min。用5L10mM Tris(pH＝7.4)溶液透析1h，移除DTT和释放的硫代吡啶酮。透析过程中，氧化DGL赖氨酸侧链引入的巯基使其形成二硫键，制得交联的纳米粒PEG-DGL/En-Aco。
本发明制得的纳米粒，其包封率为71.46±0.31％，纳米粒的外观圆整，形态呈球形；经稳定性考察，结果显示所述纳米粒能够减少胰蛋白酶对CAT的降解，72h内，所述PEG-DGL/CAT-Aco纳米粒在HL-60细胞内的酶活性显著高于游离CAT，细胞外基质中，交联纳米粒的酶活性随时间增加而逐渐增加。
本发明进一步进行了包载CAT纳米粒对缺血再灌模型动物脑梗死面积及其血氧饱和度的影响实验，结果显示，制得的交联纳米粒能够显著减少模型动物脑梗死面积；所述的交联纳米粒实验组两侧脑半球血氧饱和度比值显著高于对照组，实验结果标明，所述的PEG-DGL/CAT-Aco纳米粒能明显抑制缺血再灌注损伤，具有潜在的神经及血管保护作用。
本发明制得的纳米粒可用于制备脑卒中治疗药物。
下列附图和具体的实施方案仅作为本发明的解释，但绝不是对本发明的限制。
附图说明
图1PEG-DGL/CAT-Aco纳米粒透射电镜照片。
图2过氧化氢标准曲线。
图3制备纳米粒后CAT活性测定。
图4制备纳米粒后CAT体外稳定性测定。
图5细胞内外CAT活性结果n＝3,**P＜0.01，***P＜0.001,与CAT组比较。
图6载CAT纳米粒对模型动物脑梗死面积的影响及结果分析。
图7载CAT纳米粒对模型动物脑血氧饱和度的影响及结果分析。
具体实施方式
实施例1
合成PEG-DGL
DGL表面的氨基与功能PEG一端的NHS发生特异性反应。按照DGL:PEG＝1:6(mol/mol)的比例称量，将NHS-PEG溶于PBS 7.4缓冲溶液中，然后逐滴加入搅拌的DGL溶液中(5mg/ml,PBS,pH＝7.4)，室温搅拌反应1h。转移至5kDa的超滤管内，加入PBS，6000rpm离心20min，重复两次，纯化得到PEG-DGL；
制备PEG-DGL-PDP
DGL表面的氨基与交联剂N-琥珀酰亚胺3-(2-吡啶二硫基)(N-succinimidyl 3-(2-pyridyldithio)-propionate，SPDP)表面的NHS发生特异性反应。SPDP与PEG-DGL(DGL:SPDP＝1:2mol/mol)混合于PBS溶液中(100mM Na3PO4,1mM EDTA,pH＝7.4)，室温搅拌反应1～3h。DGL表面的部分氨基被PDP基团取代，得到PEG-DGL-PDP；
制备CAT-Aco
顺式乌头酸酐(cis-Aconitic anhydride,Aco)与过氧化氢酶(Catalase，CAT)表面的氨基反应增加其表面的负电荷。首先，按照CAT:Aco＝3:1的质量比，称取CAT和Aco。然后，将Aco溶于DMSO中，加入Na2CO3(pH＝7.4～8.5)溶解的CAT溶液中，再加入2μl N，N-二异丙基乙胺，室温搅拌2h，反应生成CAT-Aco；
制备纳米粒
精密称取CAT-Aco(CAT，1mg/ml)和PEG-DGL-PDP(DGL 1mg/ml)，分别溶于10mMTris(pH 7.4)溶液中，水浴超声2min。定量称取二硫苏糖醇(1,4-Dithiothreitol，DTT)，溶于10mM Tris(pH 7.4)缓冲液中，使其浓度为1～2mg/ml，按照1:40～1:50的体积比(DTT:DGL)加入PEG-DGL-PDP溶液中，迅速涡旋15min，将PDP的二硫键还原成巯基，并逐滴加入搅拌的等体积CAT-Aco溶液中(DGL:CAT＝2:1wt/wt)，室温持续搅拌20min，用5L 10mM Tris(pH＝7.4)溶液透析1h，移除DTT和释放的硫代吡啶酮。透析过程中，氧化DGL赖氨酸侧链引入的巯基使其形成二硫键，得到交联的纳米粒PEG-DGL/CAT-Aco。
测定纳米粒的包封率
精密称量CAT-Aco，用10mM Tris(pH＝7.4)稀释成浓度分别为1.60、0.80、0.40、0.20、0.10、0.05、0.04mg/ml的溶液，紫外分光光度计法在404nm处测定吸光度，绘制标准曲线。吸取制备纳米粒的CAT-Aco投药溶液，通过紫外分光光度计检测在404nm下吸光度值，得到纳米粒的总含药量(Ct,mg/mL)。另取纳米粒的混悬液于超速离心机上，在4℃条件下，3000000g离心30min，将游离CAT-Aco与纳米粒分离。吸取上清液，通过紫外分光光度计检测在404nm下吸光度值，得上清液中游离CAT-Aco的浓度(Ce，mg/ml)，按以下公式计算包封率：
超速离心法测得的包封率结果显示：PEG-DGL/CAT-Aco纳米粒的包封率为71.46±0.31％；
PEG-DGL/CAT-Aco纳米粒的粒径、电位及形态考察
制备PEG-DGL/CAT-Aco纳米粒后，加入10mM Tris(pH＝7.4)缓冲液稀释到适当浓度，采用粒度/zeta电位测定仪测定纳米粒粒径分布及zeta电位，结果纳米粒的平均粒径约为100nm，zeta电位为5.0mV左右。
将纳米粒混悬液与1％的醋酸铀溶液等体积混合，滴加到透射电镜专用的铜网上，灯下照射3min后，用滤纸吸取多余的溶液，透射电镜观察纳米粒的形貌。透射电镜结果见图1所示，纳米粒的外观圆整，形态呈球形。
纳米粒中CAT活性测定
取适量30％过氧化氢，用10mM Tris(pH＝7.4)稀释100倍，测定A240，根据公式：过氧化氢浓度(mM＝22.94×A240)计算过氧化氢的实际浓度，并根据实际测定的浓度绘制标准曲线。用10mM Tris(pH＝7.4)稀释成浓度分别为18.6、15.5、12.4、9.3、6.2mM的溶液，紫外分光光度计法在240nm处测定吸光度，绘制标准曲线(如图2所示)，以浓度为横坐标，A240为纵坐标进行线性回归，得回归方程A240＝0.0437C+0.021，表明A240在0.3-0.8范围，线性关系良好；
配制过氧化氢溶液(A240≈0.8)用于PEG-DGL/CAT-Aco纳米粒(DGL与CAT质量比为1.25,2.50,5.00,7.50,10.00)中CAT活性的测定，取一定量新制备的纳米粒(含过氧化氢酶1μg)加入3ml过氧化氢溶液中，通过紫外分光光度计检测在240nm下不同时间的变化，考察其活性；
DGL和CAT按不同质量比制备纳米粒，并检测其对过氧化氢的降解能力，考察不同DGL与CAT复合比例对酶活性的影响，实验结果如图3所示，DGL与CAT质量比为1.25时，与裸酶相比较，纳米粒的活性显著减低，当质量比≥2.5时，纳米粒的活性与裸酶并没有明显的差别。
实施例2纳米粒中CAT体外稳定性考察实验
将新制备的PEG-DGL/CAT-Aco纳米粒(0.5mg/ml CAT)加胰蛋白酶(10-5M)混合，37℃共孵育3小时，以CAT-Aco作为对照，依据上述方法检测PEG-DGL/CAT-Aco纳米粒(质量比为1.25,2.50,5.00,7.50,10.00)被胰蛋白酶降解后残存的CAT活性，考察纳米粒的稳定性；
如图4所示，当纳米粒的质量比(DGL:CAT)在1.25-10.00范围内，将CAT-Aco嵌入PEG-DGL中显著增加酶的稳定性，随着DGL与CAT质量比的增加，PEG-DGL/CAT-Aco纳米粒的稳定性逐渐增大，当达到一定程度后，纳米粒稳定性保持在一定范围内，表明纳米粒能够减少胰蛋白酶对CAT的降解，且不同质量比的PEG-DGL/CAT-Aco纳米粒之间无显著性差异。
实施例3纳米粒中CAT在细胞内稳定性的考察实验
HL-60细胞分别与过氧化氢酶及不同种类纳米粒在无血清培养液中混匀，37℃共孵育1h。收集细胞悬液，2000rpm离心5min。加入HBSS重悬，2000rpm离心5min，重复两遍，并加入新鲜培养液，置于30℃孵育箱中培养。不同时间点，收集细胞及培养液，使用过氧化氢酶检测试剂盒，按照说明书操作对细胞内过氧化氢酶活性及释放出HL-60细胞的酶活性进行测定；
对于不同时间点细胞内过氧化氢酶活性的测定，需将收集的细胞加入一定量的细胞裂解液(含1％蛋白酶抑制剂)，冰上孵育15min后，用超声粉碎仪粉碎细胞(功率100w，超声3s)，145000rpm离心15min，取上清。一部分用于过氧化氢酶活性的测定，另一部分用于蛋白浓度的测定(BCA试剂盒)，酶活性用蛋白浓度进行标准化处理，测定酶活性；
不同纳米粒抵抗HL-60细胞内的降解，保护CAT活性的能力如图5所示，HL-60细胞与过氧化氢酶及不同纳米粒共孵育1h后，加入PBS洗两遍，然后加入新鲜培养液。在不同时间，利用过氧化氢分解率对释放到基质中及保留在细胞内的酶活性进行评价，结果标明：72h内，PEG-DGL/CAT-Aco纳米粒在HL-60细胞内的酶活性最高，显著高于游离CAT。细胞外基质中，交联纳米粒的酶活性随着时间的增加而逐渐增加。纳米粒在细胞内对CAT保护作用的原因，可能是表面存在大量的氨基引发质子海绵效应，进而减弱内涵体酸化。
实施例4包载CAT纳米粒对缺血再灌模型动物脑梗死面积的影响实验
TTC与活细胞线粒体内的琥珀算脱氢酶反应，产生红色的产物用于鉴定细胞活力[103]。因此，利用TTC染色评价缺血再灌注后的神经损伤，如图6所示，I/R模型大鼠，再灌后3h给予PBS、CAT-Aco和PEG-DGL/CAT-Aco纳米粒，给药后48h，交联纳米粒组与PBS组相比，能够显著减少梗死面积，而CAT-Aco组与PBS组无显著区别(P＜0.05)。
实施例5
包载CAT纳米粒对缺血再灌注模型动物脑梗死部位血氧饱和度的影响实验
利用光声成像(Photoacoustic Imaging,PAI)测量脑缺血区域血液中血氧饱和度(SaO2)，考察载CAT纳米粒对缺血再灌注损伤的作用。光声成像系统具有光声(PA)和超声(ultrasound,US)双模式，共定位结果如图7所示，I/R模型小鼠，再灌后3h进行光声成像，左侧缺血半球的血氧饱和度比左右正常脑半球的血氧饱和度明显降低，占80％左右，说明缺血再灌注损伤能够显著降低血氧饱和度含量。随后立即静注给予PBS及纳米粒，再灌后24h，光声成像检测血氧饱和度，结果显示PBS组、PEG-DGL/CAT-Aco组两侧脑半球血氧饱和度比值继续降低。再灌后48h，PEG-DGL/CAT-Aco组两侧脑半球血氧饱和度比值显著高于PBS组。实验结果标明，PEG-DGL/CAT-Aco纳米粒能够明显抑制缺血再灌注损伤。而这一现象的发生，可能是CAT降解过氧化氢释放氧气，增加血液中氧气的含量，亦或是减少活性氧(ROS)对缺氧易感器官造成的氧化应激损伤，发挥潜在的神经及血管保护作用。
Claims (7)
1.一种提高酶类药物体内外稳定性的纳米制剂，其特征在于，由顺式乌头酸酐(Aco)、树枝状聚左旋赖氨酸(DGL)和功能性聚乙二醇(NHS-PEG)制成。
2.按权利要求1所述的提高酶类药物体内外稳定性的纳米制剂的制备方法，其特征在于，其包括：采用功能性聚乙二醇(PEG-NHS)与树枝状聚左旋赖氨酸(DGL)发生偶合反应，生成PEG-DGL，然后采用交联剂N-琥珀酰亚胺3-(2-吡啶二硫基)(SPDP)表面的NHS发生特异性反应，DGL表面的部分氨基被PDP基团取代，得PEG-DGL-PDP，之后采用顺式乌头酸酐(Aco)修饰酶类药物(En)，反应生成En-Aco，增加酶类药物负电荷密度；最后通过电荷吸附法将PEG-DGL和En-Aco交联制成纳米粒PEG-DGL/En-Aco。
3.按权利要求2所述的制备方法，其特征在于，DGL表面的氨基与功能PEG一端的NHS发生特异性反应中，按DGL:PEG＝1:5～1:8mol/mol的比例称量，将NHS-PEG溶于PBS 7.4缓冲溶液中，然后逐滴加入搅拌的DGL溶液中(5mg/ml,PBS,pH＝7.4)，室温搅拌反应1h，转移至5kDa的超滤管内，加入PBS，6000rpm离心20min，重复两次，纯化得PEG-DGL。
4.按权利要求2所述的制备方法，其特征在于，DGL表面的氨基与交联剂N-琥珀酰亚胺3-(2-吡啶二硫基)SPDP)表面的NHS发生特异性反应中，SPDP与PEG-DGL(DGL:SPDP＝1:2～1:5mol/mol)混合于PBS溶液中(100mM Na3PO4,1mM EDTA,pH＝7.4)，室温搅拌反应1～3h，DGL表面的部分氨基被PDP基团取代，得PEG-DGL-PDP。
5.按权利要求2所述的制备方法，其特征在于，顺式乌头酸酐(Aco)与荷正电酶类药物(En)表面的氨基反应增加其表面的负电荷中，首先，按En:Aco＝3:1～6:1的质量比，称取En和Aco，然后，将Aco溶于DMSO中，加入Na2CO3(pH＝7.4～8.5)溶解的En溶液中，再加入2μl N，N-二异丙基乙胺，室温搅拌2h，反应生成En-Aco。
6.按权利要求2所述的制备方法，其特征在于，所述方法中，称取En-Aco其中En，1～2mg/ml和DGL 2～10mg/ml，分别溶于10mM Tris，pH 7.4，溶液中，水浴超声2min；定量称取二硫苏糖醇(DTT)，溶于10mM Tris，pH 7.4，缓冲液中，使其浓度为1～2mg/ml，按DTT:DGL体积比为1:40～1:50加入PEG-DGL-PDP溶液中，涡旋15min，将PDP的二硫键还原成巯基，并逐滴加入搅拌的等体积En-Aco溶液中(DGL:En＝2:1～10:1wt/wt)，室温持续搅拌20min，用5L10mM Tris，pH＝7.4，溶液透析1h，移除DTT和释放的硫代吡啶酮，透析过程中，氧化DGL赖氨酸侧链引入的巯基使其形成二硫键，制得交联的纳米粒PEG-DGL/En-Aco。
7.权利要求1的提高酶类药物体内外稳定性的纳米制剂在用于制备脑卒中治疗药物中的用途。
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| CN112624967A (zh) * | 2019-10-08 | 2021-04-09 | 复旦大学 | 索拉菲尼巯基衍生物及其应用 |
| CN114767839A (zh) * | 2021-10-28 | 2022-07-22 | 严然 | 一种纳米复合物及应用 |
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| CN101879313A (zh) * | 2009-05-08 | 2010-11-10 | 复旦大学 | 一种基于树枝状聚合物的抗肿瘤纳米前药系统及其制备方法 |
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| CHUN ZHANG，等: "Direct Macromolecular Drug Delivery to Cerebral Ischemia Area using Neutrophil-Mediated Nanoparticles", 《THERANOSTICS》 * |
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| CN112624967A (zh) * | 2019-10-08 | 2021-04-09 | 复旦大学 | 索拉菲尼巯基衍生物及其应用 |
| CN114767839A (zh) * | 2021-10-28 | 2022-07-22 | 严然 | 一种纳米复合物及应用 |
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