CN115364240A - 一种基于聚多肽的门控纳米粒及其制备方法和应用 - Google Patents
一种基于聚多肽的门控纳米粒及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于聚多肽的“门控”纳米粒及其制备方法和应用,属于聚多肽技术领域。本发明以中空介孔二氧化硅为核心,表面修饰聚多肽,通过调控聚多肽电荷的转变、二级结构的变化和聚合物的柔顺性,进而控制药物的释放;本发明中的聚多肽纳米载体具有粘液层和生物被膜渗透能力以及高效抗菌能力并且实现药物的有效释放,在抗炎、抗肿瘤以及抗菌治疗中具有极大潜力。
Description
技术领域
本发明属于聚多肽技术领域,尤其涉及一种基于聚多肽的门控纳米粒及其制备方法和应用。
背景技术
蛋白质作为最重要的生物大分子之一,参与细胞内生化反应催化、调节细胞内信号和控制细胞内物质运输等过程。其中,不同的二级结构(α螺旋、折叠、无规卷曲)对于蛋白质发挥其功能至关重要。例如,载体蛋白是一类重要的跨膜蛋白分子,能够通过自身的构象变化控制葡萄糖、氨基酸、核苷酸或离子等的运输。螺旋结构参与小分子的结合并为小分子跨膜运输提供通道。当螺旋结构被破坏后,蛋白质的物质转运效率显著降低;细胞膜上的水通道是一个高度特异性的亲水通道,由4个亚基组成的四聚体,每个亚甲基由6个α螺旋组成。螺旋的刚性结构与侧链的有序排列允许水分子从水势较高的地方向水势较低的地方扩散。
聚多肽是一类通过化学合成的并具有良好的生物相容性、生物降解性以及可调二级结构的聚合物。侧链的相互作用影响主链氢键的有序排列,从而改变其二级结构。这些相互作用包括静电相互作用、极性以及氢键。通过调节聚多肽侧链作用力,从而实现二级结构的转换并发挥聚多肽结构的独特优势为聚多肽在药物递送、抗菌、抗炎以及组织工程等应用中提供了可靠的手段。例如,Yin等设计了一种侧链含阳离子和碱性磷酸酶响应的聚多肽。磷酸基团在被碱性磷酸酶水解后脱落后,实现二级结构由α螺旋到无规线团的转变,从而激活聚多肽的抗菌性。
除此之外,中空介孔二氧化硅纳米粒具有尺寸和孔隙率可调、比表面积大、孔容大以及良好的生物相容性等特点,在药物递送领域中发挥重要作用。智能中空介孔二氧化硅递送体系能够对内源性或外源性刺激做出反应,在建立下一代精确化医学中具有巨大潜力。传统的智能载体通过在中空介孔二氧化硅表面偶连、物理包覆或静电吸附刺激响应材料等方法制备。然而,降解性较差、合成成本相对较高、表面修饰过程复杂等缺点限制了其进一步的应用。
发明内容
为解决上述技术问题,本发明公开了一种基于聚多肽的门控纳米粒及其制备方法和应用,并合理地利用了不同二级结构柔顺性的差异,通过调控聚多肽的二级结构,调控中空介孔二氧化硅表面聚多肽由“躺”到“站”,进而控制药物的释放。在聚多肽侧链引入合适的响应基团,设计了“门控”聚多肽纳米材料,从而实现了孔的关闭与打开,为构建智能纳米递送系统提供了一个有效的技术手段。
本发明是通过以下技术方案实现的:
本发明的第一个目的在于提供一种“门控”聚多肽纳米粒,所述“门控”聚多肽纳米粒为核壳结构,其中由多肽主链、与聚多肽主链键合的阳离子基团和阴离子基团构成外壳,介孔材料构成内核;所述内核与外核通过化学键相连,所述“门控”聚多肽纳米粒结构式如(I)-(Ⅲ)任一项所示:
其中,R4为阳离子基团;R5为阴离子基团;R6为介孔材料;
n为10-1000的任一整数,x为0.01-0.99,m为1-20的任一整数。
在本发明的一个实施例中,聚多肽主链选自天然氨基酸序列或通过人工合成的氨基酸序列。通常,所述聚多肽主链包括10个-1000个氨基酸的残基,优选20个-500个氨基酸的残基,更优选20个-200个氨基酸的残基,最优选50个-200个氨基酸的残基。
在本发明的一个实施例中,所述氨基酸是天然存在的氨基酸及其衍生物,天然存在的氨基酸包括但不限于谷氨酸、酪氨酸、丝氨酸、高丝氨酸、赖氨酸、半胱氨酸、组氨酸、缬氨酸、精氨酸、谷氨酰胺、甘氨酸、亮氨酸、色氨酸等,氨基酸衍生物包括但不限于谷氨酸酯、丝氨酸脂以及同型半胱氨酸等。
在本发明的一个实施例中,聚多肽主链包括一种或多种氨基酸或其衍生物的残基。如包括两种氨基酸或其衍生物的残基、三种或四种氨基酸或其衍生物的残基。
在本发明的一个实施例中,所述聚多肽主链可为聚(γ-炔丙基-L-谷氨酸苄酯)、聚(L-酪氨酸)、聚(L-赖氨酸)、聚(L-半胱氨酸)、聚(L-谷氨酸-γ-炔丙酯)、聚(γ-3-氯丙基-L-谷氨酸酯)或聚(γ-3-氯己基-L-谷氨酸酯)等。
在本发明的一个实施例中,所述介孔材料选自中空介孔二氧化硅和/或介孔二氧化硅。
本发明的第二个目的在于提供一种“门控”聚多肽纳米粒的制备方法,包括以下步骤:
S1:将两种或两种以上式Ⅳ所示的化合物在引发剂的作用下开环聚合,制备得到聚多肽主链;
S2:通过在聚多肽侧链上接枝阳离子基团和阴离子基团,得到所述“门控”聚多肽纳米粒;
其中,A为氨基酸或氨基酸衍生物。
在本发明的一个实施例中,以介孔二氧化硅为引发剂,引发两种或两种以上式Ⅳ所示的氨基酸及衍生物的N-羧酸酐单体(NCA)无规共聚,得到侧基可功能化的无规共聚多肽,再通过季铵盐化反应在所述无规共聚多肽侧链接枝阳离子基团;通过脱保护反应和酰胺化反应等在所述无规共聚多肽侧链接枝阴离子基团,得到所述聚多肽纳米开关。
无规共聚时,溶剂为N,N-二甲基甲酰胺、二氯甲烷或二氯甲烷和水的混合溶液。
在本发明的一个实施例中,步骤S1中,所述引发剂选自氨基修饰的介孔二氧化硅和/或中空介孔二氧化硅。
在本发明的一个实施例中,所述中空介孔二氧化硅或介孔二氧化硅的粒径为50nm-200nm。
在本发明的一个实施例中,介孔二氧化硅内核可包载任何疏水或亲水药物以及任何荧光剂或光敏剂。
本发明的第三个目的在于提供所述的“门控”聚多肽纳米粒在制备外源性响应纳米开关或内源性响应纳米开关中的应用。
在本发明的一个实施例中,所述响应纳米开关具有磷酸酯酶响应、pH响应或活性氧响应中的一种或多种。
本发明的第四个目的在于提供所述的“门控”聚多肽纳米粒在制备跨粘液层材料、跨生物被膜材料、抗肿瘤药物递送材料、光动力试剂递送材料、抗炎剂递送材料或抗菌剂递送材料中的应用。
在本发明的一个实施例中,在内源性或外源性的刺激下,聚多肽抗菌性被特异性激活,载体包括上述聚多肽或“门控”聚多肽纳米粒。
本发明所述的聚多肽纳米开关具有磷酸酯酶响应、pH响应和活性氧 (ROS)响应等,可实现磷酸酯酶、pH或ROS浓度变化等触发的构象转变以及药物释放。具体来说,将阴离子基团为羧酸基的“门控”聚多肽用PBS(pH 6.0)处理后,相对于中性PBS(pH 7.4),药物释放量大大增多。
本发明主要基于正常细胞和肿瘤细胞中pH地差异,实现聚多肽“门控”药物释放的功能。以DOX为例,在正常情况下,聚多肽为柔性、负电的无规线团结构,此时由于聚多肽“躺”在介孔二氧化硅表面,将DOX堵在孔内。在肿瘤细胞的微酸条件下,侧链羧酸基团水解脱去,聚多肽转变为螺旋结构,“站”在介孔二氧化硅表面,孔打开并释放DOX。
pH响应的“门控”聚多肽可以实现抗菌的特异性激活。“门控”聚多肽侧链疏水基团的引入增强了聚多肽与细菌细胞膜的结合能力,导致膜破裂。具体来说,在细菌微酸性条件下,“门控”聚多肽螺旋结构和正电性恢复并插入到细菌细胞膜内,导致细菌因内容物泄露而死亡。
本发明的技术方案相比现有技术具有以下优点:
本发明利用了聚多肽不同构象间柔顺性的差异,设计了一种构象可调的聚多肽,用于控制核内药物释放,为聚多肽的构效关系研究以及智能纳米材料的设计提供了指导。
本发明以中空介孔二氧化硅为核心,表面修饰聚多肽,通过调控聚多肽电荷的转变,调控二级结构的变化,调控聚合物的柔顺性,进而控制药物的释放;本申请中的聚多肽纳米载体具有粘液层和生物被膜渗透能力以及高效抗菌能力并且实现药物的有效释放,在抗炎、抗肿瘤以及抗菌治疗中具有极大潜力。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中
图1是本发明实施例1中MCEBK的核磁共振氢谱图;
图2是本发明实施例2中MEBK的核磁共振氢谱图;
图3是本发明实施例3中MEK的核磁共振氢谱图;
图4是本发明实施例4中MEKCA的核磁共振氢谱图;
图5是本发明实施例4中MEKCA在pH 6.0和7.4的电位、粒径图和圆二色谱图;其中图5-A为MEKCA在pH 6.0和7.4的电位图;图5-B为MEKCA在pH 6.0和pH 7.4的粒径图;图5-C为MEKCA在pH 6.0和7.4的圆二色谱图;
图6是本发明一种基于聚多肽的“门控”纳米粒的机理图;
图7是本发明测试例1中MEKCA/CAZ纳米粒在pH 6.0和pH 7.4的CAZ 积累释放曲线;
图8是本发明测试例2中不同浓度MEKCA的细胞毒性测试图;
图9是本发明测试例3中MEKCA和MEK的渗透系数测定图与粘蛋白吸附效果图;其中图9-A为MEKCA和MEK的渗透系数测定图;图9-B为 MEKCA和MEK的黏蛋白吸附效果图;
图10是本发明测试例4中材料的生物膜渗透效果图;
图11是本发明测视例5中CAZ、MEKCA、MEKCA/CAZ对金黄色葡萄球菌、大肠杆菌、铜绿假单胞菌生物膜的抑制效果图;
图12是本发明测试例6中不同材料对不同细菌的最低抑菌浓度值与 MEKCA与CAZ联用后对不同细菌的协同抑菌指数;
图13是本发明测试例7中小鼠体内材料的抗氧化效果图;
图14是本发明测试例7中小鼠体内材料抗炎效果图;
图15是本发明测试例8中小鼠动脉血的血气分析图;
图16是本发明测试例9中雾化给药后,小鼠主要脏器的H&E染色切片图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例中,氨基修饰的中空介孔二氧化硅(hollow mesoporous silicananoparticles,HMSN)购买于先锋纳米(氨基含量为1.68mmol/g,南京,中国);碘化钠、正丁胺、L-谷氨酸、L-谷氨酸-γ-苄酯、18-冠-6和乌头酸酐购买于安耐吉化学(上海,中国);γ-6-氯己基-N-羧酸酐单体(CH-NCA)、L-叔丁氧羰基-N-赖氨酸羧酸酐单体(Boc-lys-NCA)、γ-(4-炔丙氧基苄基)-L-谷氨酸-N- 羧酸酐单体(POBLG-NCA)均根据文献报道的方法合成(Nano Lett,2020,20: 1738–1746;PNAS,2015,112,43,13159;Angew,2017,56,10826–10829); ELISA试剂盒以及商业化试剂盒均购买于碧云天(上海,中国)。所有使用的玻璃仪器购买于欣维尔。
分析天平购买于Sartorius(型号:BSA224S)。磁力搅拌器购买于IKA(型号:RHdigital)。离心机购买于Thermo SCIENTIFIC(型号:MULTIFUGE X1R)。旋转蒸发仪购买于IKA(型号:RV10)。圆二色谱仪(CD,型号:J-900)。荧光光谱仪(FLUOROMAX-4)。凝胶渗透色谱仪(GPC,1260Infinity)。激光共聚焦显微镜(CLSM,TCS-SP5)。核磁共振氢谱仪(1HNMR,400MHz)。多功能酶标仪(A-2082)。
在本文中,如果没有特别的说明,百分数(%)都指相对于组合物的摩尔百分数;所涉及的各组分或其优选组分可以相互组合形成新的技术方案;所提到的所有实施方式以及优选实施方式可以相互组合形成新的技术方案;所提到的所有技术特征以及优选特征可以相互组合形成新的技术方案;组合物中各组分的含量之和为100%;组合物中各组分的份数之和可以为100摩尔份;数值范围表示a到b之间的任意实数组合的缩略表示,其中a和b都是实数。例如数值范围“0-20”表示本文中已经全部列出了“0-20”之间的全部实数,“0-20”只是这些数值组合的缩略表示;整数数值范围“a-b”表示a到b之间的任意整数组合的缩略表示,其中a和b都是整数。例如整数数值范围“1-N”表示 1、2……N,其中N是整数;“其组合”表示所述各元件的多组分混合物,例如两种、三种、四种以及直到最大可能的多组分混合物;所用的术语“一种”指“至少一种”;所述的百分数(包括摩尔百分数)的基准都是所述组合物的总物质的量。
本文所公开的“范围”以下限和上限的形式。可以分别为一个或多个下限,和一个或多个上限。给定范围是通过选定一个下限和一个上限进行限定的。选定的下限和上限限定了特别范围的边界。所有可以这种方式进行限定的范围是包含和可组合的,即任何下限可以与任何上限组合形成一个范围。例如,针对特定参数列出了60-120和80-110的范围,理解为60-110和80-120的范围也是预料到的。此外,如果列出的最小范围值1和2,和如果列出了最大范围值3,4和5,则下面的范围可全部预料到:1-3、1-4、1-5、2-3、2-4、和2-5;所述的“残基”表示化合物或单体在反应之后形成的产物(例如聚合物) 中的相应部分;“氨基酸”一般包括氨基酸及其衍生物。
由氨基酸形成α-螺旋聚多肽主链并对其二级结构进行调控的方法是本领域已知的。例如可参见Chem Soc Rev,2018,47:7401–7425;J Mater Chem B, 2020,8:6530–6547;Angew Chem Int Ed,2017,56:10826–10829;Proc Natl Acad Sci U SA,2017,114:12675–12680。具体来说,以六甲基二硅氮烷为引发剂,以N,N-二甲基甲酰胺为溶剂,通过开环聚合,引发基于两种氨基酸及衍生物的N-羧酸酐单体(NCA)无规共聚,并在聚多肽侧链接枝阳离子季铵盐基团和响应性阴离子磷酸根或羧酸根,从而制备二级结构可调节的聚多肽,通过磷酸酯酶或pH响应对其二级结构进行调节。
在下述实施例中,根据聚多肽的侧基种类以及接枝率不同,将该聚多肽简称为MEKCA(x,n)、MEQYP(x,n)或MAEQYP(x,n),其中M代表中空介孔二氧化硅;E代表谷氨酸及衍生物;Y代表酪氨酸;K代表赖氨酸;P 代表磷酸根;n代表聚多肽的聚合度;x代表聚多肽中阳离子的接枝率。
以下反应流程列举了部分本发明所述聚多肽纳米开关的制备:
实施例1 MCEBK的制备
通过调节聚合度和赖氨酸含量,合成中空介孔二氧化硅引发的无规共聚多肽MCEBK(m,n)。以合成聚合度为100、谷氨酸酯含量为80%的MCEBK 为例。将CH-NCA(500mg,1.71mmol)和Boc-lys-NCA(116.68mg,0.43 mmol)溶于无水DCM(1.5mL)。混合物分别用PBS(0.5mL×4)和Tris 缓冲液(0.5mL×1,pH 9.0)洗涤。加入18-冠-6(6mg,0.02mmol)以及HMSN/H2O悬浮液(0.3mL,42.53mg/mL,氨基含量为0.07M),室温剧烈搅拌,通过FTIR监测NCA单体特征峰(1793cm-1)的变化。当单体转化率大于99%(通常在15分钟内)时,混合物用乙醚/正己烷(50mL,v/v=1/1) 沉淀,离心(5000rpm,10分钟)收集沉淀,真空干燥,得MCEBK(434.4 mg,产率80%)。所得MCEBK的核磁共振氢谱图如图1所示。
实施例2通过季铵盐化合成MEBK
将MCEBK(100mg,氯原子的物质的量为0.4mmol)溶于DMF(1mL),加入N,N-二甲基-1-辛胺(189mg,1.2mmol)和NaI/ACE溶液(1mL,180 mg/mL),80℃搅拌48小时。反应结束后,加入NaCl溶液(3mL,1M),室温搅拌3小时以进行离子交换。粗产物在去离子水中透析3天(MWCO= 3500Da),冻干,得白色固体(127.2mg,产率73%)。所得MEBK的核磁共振氢谱图如图2所示。
实施例3 MEK的制备
将MEBK(100mg,Boc基团的物质的量为0.06mmol)和TFA/DCM (2mL,v/v=1/3)混合,室温搅拌3小时。反应结束后,真空除去溶剂,得黄色固体。为了进一步除去残留的TFA,粗产物在去离子水中透析3天 (MWCO=3500Da),冻干,得白色固体(64.7mg,产率67%)。所得MEK 的核磁共振氢谱图如图3所示。
实施例4 MEKCA的制备
将MEK(100mg,氨基的物质的量为0.06mmol)溶于去离子水(2mL),加入乌头酸酐(cis-aconitic anhydride,CA,27.1mg,0.17mmol),NaOH 溶液(0.2M)调节体系pH至9.0,室温搅拌12小时。反应结束后,混合物在去离子水(pH=9.0)中透析3天(MWCO=3500Da),冻干,得白色固体(60mg,产率55%)。所得MEKCA的核磁共振氢谱图如图4所示。通过 DLS和CD分析了微酸触发MEKCA的电位、粒径以及二级结构转变。如图 5所示,MEK纳米粒为荷正电的α-螺旋结构,其zeta电位为+43mV,尺寸为140nm。引入CA后,由于侧链羧基中和了聚多肽的正电荷,导致所得 MEKCA纳米粒的zeta电位转变为负值(-28mV,图5-A)。同时,由于侧链静电相互吸引,聚多肽主链α-螺旋结构被破坏,MEKCA转变为无规线团构象,并使纳米粒的粒径减小至120nm(图5-B和5-C)。微酸条件下(pH 6.5), MEKCA侧链的羧基水解并脱落,纳米粒重新恢复为正电性(+34mV,图 5-A);同时由于聚多肽侧链间静电相互吸引作用消除,其二级结构由柔性的无规线团转变为刚性、棒状的α-螺旋,纳米粒的粒径恢复至140nm(图5-B 和5-C)。
实施例5 MEKCA/CAZ的制备
将MEK纳米粒(10mg,表面含5.8μmol氨基)超声分散于磷酸盐缓冲溶液(phosphatebuffered saline,PBS,2mL,pH=7.4),加入头孢他啶 (ceftazidime,CAZ,5mg),避光搅拌12小时。将混合液转移至超滤管(MWCO =3500Da),超滤(4000rpm,10分钟),PBS洗涤3次,收集负载CAZ的 MEK(MEK/CAZ)纳米粒。将MEK/CAZ纳米粒重悬于去离子水(2mL),加入CA(2.7mg,17.4μmol),用NaOH溶液(0.2M)调节pH至9.0,室温搅拌2小时。混合液在去离子水(pH 9)中透析12小时(MWCO=3500Da),冻干,得MEKCA/CAZ纳米粒。
实施例6叠氮小分子的制备
将N,N-二甲基氯丙胺盐酸盐(1.0g,6.33mmol)溶于去离子水(10mL),加入叠氮化钠(0.82g,12.65mmol),70℃反应12小时。反应结束后,将反应液冷却至室温,用氢氧化钠溶液(0.1mol/L)调pH至12,无水乙醚(30mL ×5)萃取。合并有机相,有机相用无水硫酸钠干燥,过滤,真空除去溶剂,得N,N-二甲基叠氮丙胺。
将N,N-二甲基叠氮丙胺(1.12g,8.74mmol)用无水乙醚(5mL)溶解,缓慢加入碘甲烷(1.86g,13.11mmol),室温反应12小时。反应结束后,离心除去溶剂,将白色固体用无水乙醚洗涤(30mL×5),旋蒸除去溶剂,得叠氮季铵盐小分子。
实施例7碱性磷酸酶响应“门控”聚多肽制备
将L-酪氨酸(1.0g,5.52mmol)和三光气(0.71g,2.39mmol)溶于无水四氢呋喃(25mL),50℃反应6小时。反应结束后,过滤除去不溶物,真空除去四氢呋喃以及残余的三光气,得淡黄色固体。随后加入四氢呋喃(5mL) 将粗产物溶解,加入正己烷(60mL)将产物沉淀出,反复三次,得Tyr-NCA。
以合成聚合度为100、谷氨酸含量为80%的PEP为例。在手套箱中,将 POBLG-NCA(100mg,0.315mmol,结构式如下所示)和Tyr-NCA(22mg, 0.105mmol)溶于无水N,N-二甲基甲酰胺(4mL),加入含中空介孔二氧化硅纳米粒的N,N-二甲基甲酰胺(0.5mL,20mg/mL,氨基物质的量为3×10-3 mmol),室温反应三天。通过傅里叶变换红外光谱监测反应进程,直至单体转化率大于99%。反应结束后,将其逐滴滴入去离子水中(60mL),得粗产物,用去离子水洗涤三次,以除去残余杂质,真空干燥,得到白色固体MOBEY。
在手套箱中,将MOBEY(146,0.75)(30mg,炔基物质的量为0.08mmol) 和叠氮季铵盐小分子(60mg,0.22mmol)溶于无水N,N-二甲基甲酰胺(3mL),加入五甲基二亚乙基三胺(34μL)和溴化亚铜(18mg),室温反应24小时。反应结束后,将混合物暴露于空气中使过量的溴化亚铜氧化,加入盐酸(4mL, 1mol/L),粗产物在去离子水中透析三天(MWCO=3500Da),冻干,得白色固体MEQY(146,75%)。
通过磷酸化反应合成MEQYP(n,x)。将MEQY(146,0.75)(30mg) 分散于无水N-甲基吡咯烷酮(3mL),冰浴下缓慢滴加无水三乙胺(200μL) 和磷酰氯(200μL),避光、室温反应12小时。反应结束后,向反应液中加入饱和碳酸氢钠溶液(7-8mL),室温继续反应2小时。粗产物在去离子水中透析三天(MWCO=3500Da),冻干,得白色固体MEQYP(146,75%)。
实施例8
将γ-炔丙基-L-谷氨酸酯(1.0g,5.4mmol)和三光气(0.8g,2.7mmol) 溶于无水四氢呋喃(25mL),室温反应24小时。反应结束后,真空除去四氢呋喃以及残余的三光气,得淡黄色液体。随后加入乙酸乙酯(50mL)将粗产物溶解,分别用饱和碳酸氢钠(50mL×3)和饱和氯化钠(50mL×3)洗涤,无水硫酸钠干燥,过滤,真空除去溶剂,得无色粘稠液体PLG-NCA。
通过调节聚合度、酪氨酸含量以及介孔二氧化硅种类,合成中空介孔二氧化硅引发的无规共聚多肽(n,x)。以聚合度为100、谷氨酸酯含量为75%的MOBEY(146,0.75)的合成为例,在手套箱中,将PLG-NCA(100mg, 0.315mmol)和Tyr-NCA(20mg,0.105mmol)溶于无水N,N-二甲基甲酰胺 (4mL),加入含中空介孔二氧化硅的N,N-二甲基甲酰胺(0.5mL,20mg/mL,氨基物质的量为3×10-3mmol),室温反应三天。通过傅里叶变换红外光谱监测反应进程,直至单体转化率大于99%。反应结束后,将其逐滴滴入去离子水中(60mL),得粗产物,用去离子水洗涤三次,以除去残余杂质,真空干燥,得到白色固体MAEY。
通过点击化学反应合成阳离子聚多肽MAEQY(n,x)。以聚合度为146、谷氨酸酯含量为75%的MEQY(146,75%)为例。在手套箱中,将MAEY(146, 0.75)(30mg,炔基物质的量为0.08mmol)和叠氮季铵盐小分子(60mg,0.22 mmol)溶于N,N-二甲基甲酰胺,加入五甲基二亚乙基三胺(34μL)和溴化亚铜(18mg),室温反应24小时。反应结束后,将混合物暴露于空气中使过量的溴化亚铜氧化,加入盐酸(4mL,1mol/L),粗产物在去离子水中透析三天 (MWCO=3500Da),冻干,得白色固体MAEQY(100,0.75)。
通过和实施例7相同的方法合成磷酸化的MAEQY。将MAEQY(146,0.75) (30mg)分散于无水N-甲基吡咯烷酮(3mL),冰浴下缓慢滴加无水三乙胺 (200μL)和磷酰氯(200μL),避光、室温反应12小时。反应结束后,向反应液中加入饱和碳酸氢钠溶液(7-8mL),室温继续反应2小时。粗产物在去离子水中透析三天(MWCO=3500Da),冻干,得白色固体MAEQYP(146,0.75)。
测试例
(1)构象调节可调控药物释放速率的测试
基于无规线团和α-螺旋构象柔顺性差异,“门控”聚多肽纳米粒MEKCA可有效包载药物,实现微酸触发的药物可控释放。MEK纳米粒中聚多肽为α-螺旋结构,此时HMSN的“孔”打开,可将CAZ装载进HMSN的空腔中。CA修饰后,聚多肽转变为无规线团结构,HMSN的“孔”关闭,CAZ保留在空腔内,从而制备了“门控”聚多肽载药纳米粒MEKCA/CAZ(图6)。将MEKCA/CAZ纳米粒(6.7mg,含有0.8mg CAZ)重悬于PBS(5mL,pH 7.4或6.0),混合液加入透析袋(MWCO=3500Da)。具体而言,将透析袋浸没于PBS(45mL, pH 7.4或6.0)中,37℃恒温摇床孵育。在不同时间点(0小时、0.5小时、1 小时、2小时、4小时、8小时、10小时、12小时和24小时),从透析袋外取出释放介质(2mL),并加入等体积的新鲜介质。使用紫外-可见分光光度计测定释放介质在255nm处的吸光度,计算释放介质中CAZ的浓度,从而计算出CAZ的释放量。如图7所示,pH 7.4时,24小时内仅24%的CAZ被释放,表明由于柔性聚多肽链的紧密堆叠,“孔”被堵住,因此CAZ难以有效释放。而pH 6.0时,12小时内CAZ的释放量可达80%。这主要是由于无规线团到α-螺旋的转变致使聚多肽由“平躺”状态转变为“站立”状态,从而打开 HMSN的“孔”并加速药物释放。
(2)细胞毒性测试
将HLF1细胞接种至96孔板(1×104个细胞/孔),培养24小时。将MEKCA纳米粒按15.6μg/mL、31.3μg/mL、62.5μg/mL、125μg/mL、250μg/mL和500 μg/mL的终浓度添加到孔中,37℃孵育24小时,MTT法测定细胞存活率。以未经纳米粒处理的细胞的存活率作为100%,计算纳米粒处理后细胞的相对存活率。如图8所示,MEKCA(100,0.80)“门控”聚多肽对HLF1细胞具有较低的细胞毒性。
(3)粘液层渗透测试
将Calu-3细胞(5×105细胞/cm2)接种至Transwell(0.33cm2,孔径3.0μm,Corning,NY),下室加入培养基(300μL)。细胞接种后,每2天更换上室和下室中的培养基。在第4天吸出上室和下室培养基,仅在下室中加入新鲜培养基。37℃孵育,每2天更换下室培养基,使上室暴露在空气中。在第7天到第14天内,每天测定跨膜电阻(transepithelialelectrical resistance,TEER),通常14天内TEER值可达700Ω·cm2。14天后,上室用PBS洗3次,下室加入含1%BSA的Hank’s平衡盐缓冲液(Hank’s balanced salt solution,HBSS,500μL),将Cy5MEKCA纳米粒或Cy5MEK纳米粒(20μL,0.25mg/mL)分别与含1%BSA的HBSS(200μL)混合并加入至上室,37℃孵育6小时。收集下室培养基,多功能酶标仪测定荧光强度(λex=649nm,λem=670nm)并计算纳米粒浓度。按公式Papp=Q/Act计算纳米粒的表观渗透系数(Papp),其中 Q表示下室中纳米粒的总量(ng),A表示细胞单层的面积(cm2),c表示上室中纳米粒的初始浓度(μg/cm3),t表示渗透时间(s)。如图9-A所示,与荷正电的MEK纳米粒相比,荷负电的MEKCA纳米粒的Papp提高了5倍,表明负电修饰显著增强了纳米粒在粘液层中的渗透。
将Cy5MEKCA纳米粒或Cy5MEK纳米粒(20μL,0.25mg/mL)与粘蛋白溶液(0.3%或0.5%,w/v,2mL)混合,涡旋10秒,37℃孵育30分钟。离心 (1500rpm,2分钟)收集沉淀,PBS洗涤2次,随后加入NaOH溶液(200μL, 5M),室温孵育15分钟,通过酶标仪测定溶液的荧光强度(λex=649nm,λem=670nm)。如图9-B所示,与0.3%粘蛋白孵育后,Cy5MEK纳米粒的荧光强度比Cy5MEKCA纳米粒高40倍,并且在0.5%粘蛋白中也观察到类似结果。表明荷负电纳米粒可通过减少粘蛋白吸附增强其在粘液中的运动能力。
(4)生物被膜渗透测试
将P.aeruginosa悬浮液(100μL,1×108CFU/mL)接种至玻璃培养皿, 37℃培养2天,每天更换新鲜的LB培养基。去除培养基,PBS洗3次,得 P.aeruginosa生物膜。分别加入Cy5MEKCA纳米粒、Cy5MEK纳米粒或Cy5MEKSA纳米粒(10μL,2μg/mL)溶液,37℃孵育2小时。PBS洗3次,SYTO 9(100 μL,2μM,用于生物膜染色)染色15分钟,CLSM观察并拍照,通过Image J软件测定Cy5的荧光强度。如图10所示,经电荷可反转的Cy5MEKCA纳米粒处理后的生物膜能检测出大量红色荧光(Cy5)信号,而荷正电的Cy5MEK 纳米粒只能检测出少量红色荧光信号,非响应型荷负电的Cy5MEKSA纳米粒几乎检测不到荧光信号。这主要归因于电荷可反转纳米粒能在生物膜中同时实现渗透与滞留。
(5)抗生物膜效果测试
将P.aeruginosa、E.coli或S.aureus悬浮液(200μL,pH 6.5,1×108 CFU/mL)分别与CAZ、MEKCA纳米粒或MEKCA/CAZ纳米粒(浓度为MIC) 混合,孵育24小时。除去培养基,PBS洗3次,结晶紫(crystal violet,CV) 溶液(200μL,1%,w/w)染色20分钟,PBS洗3次,加入乙酸(200μL,33%,v/v)溶液,摇床震荡(100rpm)15分钟以溶解染色的生物膜。通过酶标仪测定570nm处的吸光值(OD570)。如图11所示,经CAZ或MEKCA纳米粒处理后,生物膜的形成均减少了1-2倍,而经MEKCA/CAZ纳米粒处理后,生物膜的形成减少了4-5倍。表明与CAZ或MEKCA纳米粒单独处理相比, CAZ联合MEKCA可有效抑制生物膜的形成。
(6)抗菌效果测试
将E.coli、P.aeruginosa或S.aureus在溶菌肉汤(lysogenybroth,LB) 培养基中培养24小时。CAZ、MEKCA纳米粒和MEKCA/CAZ纳米粒用LB培养基(pH 6.0)梯度稀释,将不同浓度的纳米粒(200μL)和细菌悬浮液(2μL, 1×108CFU/mL)混合后加入96孔板中,孵育24小时。通过酶标仪测定600 nm处的吸光值(OD600),以OD600<0.09对应的药物/纳米粒的最低浓度作为其最低抑菌浓度(minimum inhibitory concentration,MIC)。随后,利用棋盘法评估CAZ和MEKCA纳米粒的协同抗菌作用。将CAZ和MEKCA纳米粒用 LB培养基(pH 6.0)梯度稀释,随后将CAZ溶液用不同浓度的MEKCA溶液稀释,配制一系列不同浓度梯度的溶液。将其加入至96孔板(100μL/孔),并加入细菌悬浮液(100μL/孔,1×106CFU/mL),孵育24小时。通过酶标仪测定OD600,并计算协同抑菌系数。
由图12可知,当MEKCA纳米粒联合CAZ对细菌进行处理后,CAZ对S. aureus、E.coli和P.aeruginosa的MIC分别降了26、80和45倍,同时MEKCA纳米粒的MIC分别下降了14、4和11倍。这主要归因于MEKCA纳米粒与CAZ 的协同作用能够相互增效,大幅度减少了MEKCA纳米粒与CAZ的用量。随后,通过测定了MEKCA纳米粒联合CAZ的FICI进一步验证了该结论。结果显示,MEKCA纳米粒联合CAZ的FICI分别为0.19、0.38和0.13,均小于0.5,表明MEKCA纳米粒和CAZ之间有较强的协同作用。
(7)抗氧化应激以及抗炎效果测试
将Balb/c小鼠每天暴露于香烟中2小时(泰山香烟,济南,中国),重复 20天(每天4支香烟)。第7天气管内雾化注射脂多糖(lipopolysaccharide, LPS)溶液(20μL,1mg/mL)。第21天气管内雾化注射P.aeruginosa悬浮液 (10μL,1×108菌落形成单位(colonyformingunits,CFU)/mL)用于模拟 COPD的急性加重期。第22、23和24天,雾化注射PBS、CAZ、MEKCA纳米粒和MEKCA/CAZ纳米粒(2.4mg CAZ/kg、12.6mg MEKCA/kg),每组6只小鼠,以未进行COPD诱导和药物治疗的小鼠作为正常组。
第3次给药24小时后,处死小鼠,收集肺组织,加入含蛋白酶抑制剂的裂解缓冲液(1mL),匀浆,离心(4℃,15000rpm,10分钟)收集上清液,通过ELISA测定上清液中炎性因子(TNF-α、IL-1β、IL-8、MMP-9和IL-6) 浓度并通过商用试剂盒测定氧化应激标志物(H2O2、SOD、Nrf2和MDA)浓度。如图13和图14所示,经过MEKCA/CAZ纳米粒治疗后,小鼠肺组织内氧化因子和炎性因子均恢复至正常值,表明MEKCA/CAZ纳米粒具有良好的抗氧化应激和抗炎功效。
(8)血气分析
将COPD小鼠随机分成5组,每组6只,连续3天气管内雾化注射PBS、 CAZ、MEKCA纳米粒或MEKCA/CAZ纳米粒(2.4mg CAZ/kg或12.6mg MEKCA/kg)。以未经COPD诱导和纳米粒治疗的正常小鼠作为对照,第3次给药24小时后,从颈动脉采集血样,使用血气分析仪(Radiometer,中国上海)测定氧分压PaO2、二氧化碳分压PaCO2和pH值。如图15所示,经MEKCA/CAZ纳米粒治疗后,小鼠动脉血中氧分压、二氧化碳分压和pH值恢复至正常值,表明小鼠肺功能逐渐恢复。
(9)生物相容性测试
正常Balb/c小鼠随机分成2组,每组3只,连续3天气管内雾化注射PBS (50μL)或MEKCA/CAZ纳米粒(2.4mg CAZ/kg)。第3次给药24小时后,收集主要器官(心、肝、脾、肺和肾),福尔马林(10%,w/v)固定,石蜡包埋,切片(厚度为8μm),H&E染色,光学显微镜观察并拍照。如图16所示,给药后主要脏器均没有发生明显变化,表明“门控”聚多肽具有良好的生物相容性。
综上,以氨基修饰的中空介孔二氧化硅为引发剂,通过引发无规共聚,经季铵盐化、脱保护反应和酰胺化反应制备了一系列具有微酸响应的“门控”聚多肽纳米粒,旨在通过二级结构变化对控制药物的释放。研究表明,正常生理环境时,由于侧链的静电相互吸引,MEKCA为柔性的无规线团结构,此时聚多肽“躺”在中空介孔二氧化硅表面。一旦纳米粒子进入细菌微酸性环境,羧酸基团水解离去,由于此时侧链缺乏静电相互作用,聚多肽转变为刚性的α-螺旋构象,使聚多肽“站”在中空介孔二氧化硅表面,释放药物,从而实现智能的药物递送。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
2.根据权利要求1所述的“门控”聚多肽纳米粒,其特征在于,所述介孔材料选自中空介孔二氧化硅和/或介孔二氧化硅。
6.根据权利要求5所述的制备方法,其特征在于,步骤S1中,所述引发剂选自氨基修饰的介孔二氧化硅和/或中空介孔二氧化硅。
7.根据权利要求6所述的制备方法,其特征在于,所述中空介孔二氧化硅或介孔二氧化硅的粒径为50nm-200nm。
8.如权利要求1所述的“门控”聚多肽纳米粒在制备外源性响应纳米开关或内源性响应纳米开关中的应用。
9.根据权利要求8所述的应用,其特征在于,所述响应纳米开关具有磷酸酯酶响应、pH响应或活性氧响应中的一种或多种。
10.如权利要求1-4任一所述的“门控”聚多肽纳米粒在制备跨粘液层材料、跨生物被膜材料、抗肿瘤药物递送材料、光动力试剂递送材料、抗炎剂递送材料或抗菌剂递送材料中的应用。
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