CN114767656A - 靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法及应用 - Google Patents
靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法及应用,本发明的纳米药物由马来酰亚胺修饰的聚乙二醇和硝酸酯环碳酸酯单体通过开环聚合得到含一氧化氮供体的聚碳酸酯嵌段共聚物,然后与含巯基或修饰后含巯基的靶头化合物进行端基修饰,修饰后的共聚物与他汀类小分子药物溶解在有机溶剂中,通过自组装得到载药纳米粒子;本发明实现一氧化氮与他汀类药物协同治疗动脉粥样硬化的增效作用,改善了临床中他汀类药物生物利用度低、无靶向性等问题,具有毒副作用小、靶向性强、载药能力好、在正常生理环境下稳定性好、生物利用度高等优势。
Description
技术领域
本发明为生物医用高分子材料学和药物制剂领域,涉及一种靶向动脉粥样硬化斑块的一氧化氮供体纳米药物其制备方法及应用。
背景技术
动脉粥样硬化(atherosclerosis,AS)是动脉壁的一种慢性疾病,导致动脉增厚和硬化,由不良的炎症反应、脂质代谢失调和斑块积聚引起。这种积聚会使动脉变窄,使血液更难流过,从而导致高血压。如果形成血凝块,它会阻止血液流动,导致致命的临床终点,例如心肌梗塞和心力衰竭,以及位于大脑内的动脉缺血性中风。AS起始于动脉内膜的内皮细胞的激活,并引发单核细胞的募集和积累,其分化成巨噬细胞并吞噬脂质,变成泡沫细胞。单核细胞通过清道夫受体(SR)依赖性摄取氧化低密度脂蛋白(oxLDL),以及SR非依赖性摄取天然和修饰的oxLDL,转化成泡沫细胞。oxLDL明显破坏了内皮型一氧化氮合酶(eNOS)/诱导型一氧化氮合酶(iNOS)的调节机制。另一方面,增加的oxLDL导致清道夫受体LOX-1的持续活化,并随后导致NF-κB活化,进而增加iNOS,从而导致内皮细胞氧化应激。泡沫细胞产生高水平的促炎细胞因子,趋化因子和生长因子,引起平滑肌细胞增殖和迁移到内膜中,导致斑块生长和纤维化。
他汀类药物治疗动脉粥样硬化已大规模使用,其副作用与有效性的平衡还远未达到完美。然而,在全身给药的临床条件下,他汀类药物的疗效有限,这可能是由于药物清除迅速,且在动脉损伤部位蓄积不令人满意造成的。
一氧化氮是一种重要的炎症反应介质,一氧化氮的产生与多种疾病有关,包括动脉粥样硬化。血管系统中的大多数NO由eNOS产生。内皮衍生的一氧化氮是一种多功能信号分子,其作为有效的内源性血管扩张剂,能抑制血管损伤形成中的关键过程。NO对相邻平滑肌细胞以及血液中血小板和白细胞的作用是维持血管稳态的重要因素。NO可以使蛋白质硝基化,各种靶蛋白的硝基化可以调节细胞增殖、凋亡、出胞、通道活性、血液流动和氧气输送等活动。此外,NO还具有抑制血小板粘附和聚集,抑制粘附分子和趋化因子表达,减少炎症细胞浸润和平滑肌细胞迁移和增殖的作用。NO信号的药理刺激可能被证明可用于预防或治疗心血管疾病,但NO信号的治疗性调节具有挑战性,因为效果必须在正确的位置、时间和数量上产生。(Nature Reviews Drug Discovery,2015,14(9):623-641;Nature ReviewsDrug Discovery,2021,20(8):589-610;Pharmaceutics,2020,12(11):1056;AdvancedHealthcare Materials,2021,10(3):2001550)因此,在利用他汀类药物和一氧化氮治疗动脉粥样硬化时,需要克服他汀类药物的生物利用度与吸收问题,以及一氧化氮体内半衰期短、无法缓慢长效释放的问题。
发明内容
发明目的:本发明针对现有技术中存在的动脉粥样硬化治疗药物吸收度低、生物利用度低且半衰期短、无法缓慢长效释放的问题,提供一种靶向动脉粥样硬化斑块的一氧化氮供体纳米药物制备方法及应用。
技术方案:本发明的所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:由功能性聚乙二醇和硝酸酯环碳酸酯单体通过开环聚合得到含一氧化氮供体的聚碳酸酯嵌段共聚物,然后与含巯基或修饰后含巯基的靶头化合物进行端基修饰,修饰后的共聚物与小分子药物溶解在有机溶剂中得到载药纳米粒子。
优选的,所述功能性聚乙二醇为马来酰亚胺修饰聚乙二醇;所述功能性聚乙二醇分子量为1000-20000g/mol,所述功能性聚乙二醇与硝酸酯环碳酸酯单体分子量大小比例为1:0.5-1。
优选的,所述的含巯基或修饰后含巯基的靶头化合物为巯基衍生物为S2P肽(CRTLTVRKC)、巯基修饰透明质酸或Cys-RGD肽中的一种。
所述靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法具体为:
(1)含一氧化氮供体的聚碳酸酯嵌段共聚物的合成:在惰性气体保护下,将硝酸酯环碳酸酯单体溶于有机溶剂,然后加入功能性聚乙二醇作为引发剂,加入催化剂,形成混合溶液A,通过开环聚合反应制备得到含一氧化氮供体的聚碳酸酯嵌段共聚物Mal-PEG-PNTC;合成路线通式如下:
(2)靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物的合成:含巯基或修饰后含巯基的靶头化合物与Mal-PEG-PNTC溶解在DMF中,加入三乙胺形成混合溶液B,室温反应制备得到靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物;合成路线如下:
所制得的靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物的结构通式如下所示;
(3)靶向动脉粥样硬化斑块的载药纳米粒子的制备:将靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物与小分子药物溶解在DMF溶剂中,反应后透析得到靶向动脉粥样硬化斑块的聚碳酸酯类载药纳米粒子。
优选的,所述小分子药物为他汀类药物。
优选的,步骤(1)中,所述有机溶剂为二氯甲烷,所述催化剂为双(双三甲基硅基)胺锌,混合溶液A中,硝酸酯环碳酸酯单体、功能性聚乙二醇、有机溶剂和催化剂的质量比为0.5-1:1:25:1。
优选的,步骤(2)中,所述混合溶液B中含巯基或修饰后含巯基的靶头化合物、Mal-PEG-PNTC、DMF和三乙胺的质量比为1:5:25:0.1。
优选的,步骤(3)中,所述靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物、小分子药物、DMF溶剂的质量比为1:0.02-0.3:50,所述透析采用的介质为去离子水或PB缓冲液。
本发明还公开了所述靶向动脉粥样硬化斑块的一氧化氮供体纳米药物在制备治疗动脉粥样硬化药物中的应用。
本发明的一氧化氮供体纳米药物具有显著的抗动脉粥样硬化作用,通过在动脉粥样硬化斑块部位释放一氧化氮,抑制平滑肌细胞的迁移,与他汀类药物联合治疗,通过抑制NF-kb信号通路,降低斑块炎症,同时抑制诱导性一氧化氮合酶活性,促进M1型巨噬细胞极化为M2型巨噬细胞,达到治疗动脉粥样硬化的效果。
有益效果:与现有技术相比,本发明具有如下显著优点:
(1)本发明的一氧化氮供体纳米药物具有良好的载药性能,并且将聚乙二醇良好的生物学相容性和一氧化氮的抗动脉粥样硬化作用有机结合于一体,克服了他汀类药物的生物利用度与吸收问题,一氧化氮体内半衰期短、无法缓慢长效释放的问题,又实现一氧化氮与他汀类药物协同治疗动脉粥样硬化的目的;
(2)本发明的一氧化氮供体纳米药物具有显著的动脉粥样硬化斑块靶向性,可以通过主动靶向,利用碳酸酯键的还原响应性来实现在动脉粥样硬化斑块内的响应性药物释放,解决了他汀类药物的无靶向性问题,提高了药物在斑块部位的富集,降低了药物的毒副作用。
附图说明
图1为实施例1中功能性Mal-PEG-PNTC的核磁图谱;
图2为实施例2中靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物的核磁图谱;
图3为实施例2中靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物的红外图谱;
图4为实施例3中载药胶束的粒径图;
图5为实施例4中聚合物胶束在GSH(10mM,pH 7.4)条件下一氧化氮释放结果;
图6为实施例5中载药胶束在GSH(10mM,pH 7.4)条件下阿托伐他汀释放结果;
图7为实施例6中细胞存活率测定的结果图;
图8为实施例7中细胞内iNOS蛋白含量测定的结果图;
图9为实施例8中细胞内P-P65和P65蛋白含量测定的结果图。
具体实施方式
下面结合附图对本发明的技术方案作进一步说明。
实施例1
功能性Mal-PEG-PNTC的合成:
在手套箱中称取0.1g NTC单体和0.1g Mal-PEG-OH于密闭反应器中,加无水二氯甲烷溶解,然后加入双(双三甲基硅基)胺锌催化剂3滴,将反应器密封,转移出手套箱,在30-50℃油浴条件下反应48h,然后用2滴冰乙酸终止反应,在冰乙醚中沉淀,过滤真空干燥得到产物。核磁结果表明其分子量为5000-3200g/mol。核磁表征见附图1。
实施例2
靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物的制备:
将50mg Mal-PEG-PNTC共聚物和10mg S2P肽溶解在2mL DMF中,加入1mg三乙胺催化剂,室温反应24h,然后透析去除有机试剂和游离肽,随后通过冷冻干燥得到产物,核磁结果显示马来酰亚胺双键消失,红外结果显示S2P肽的肽键峰出现。核磁表征见附图2,红外表征见附图3。
实施例3
靶向动脉粥样硬化斑块的载药纳米粒子的制备:
通过溶剂交换法制备载阿托伐他汀纳米胶束,取0.1mL一氧化氮供体嵌段共聚物S2P-PEG-PNTC的DMF溶液(10mg/mL)及10μL阿托伐他汀的DMF溶液(10mg/mL)混合均匀,随后超声条件下将PB缓冲液缓慢滴入含药物和聚合物的DMF溶液中,混合液继续室温超声半小时,随后用PB缓冲液透析除去有机溶剂和未被包裹的阿托伐他汀。胶束包裹阿托伐他汀的含量通过高效液相色谱法测量。载药量(DLC)和包封率(DLE)通过下面的公式计算:
载药量(%)=(装载药物质量/聚合物与药物总质量)×100%
包封率(%)=(装载药物质量/药物总投入量)×100%
S2P-PEG-PNTC聚合物胶束对阿托伐他汀的理论载药量分别为5%、10%、15%和20%时,其实际包封率如表1所列,当载药量提高时,包封率有所下降,实际载药量达到15.1%时,包封率达到75.6%,表明材料具有良好的载药效率。
表1.包裹阿托伐他汀的聚合物胶束的表征
通过动态光散射分析仪,测得样品的尺寸,实验结果显示载药胶束粒径良好,不同载药量对胶束粒径影响不大,PDI都在0.16-0.21之间,表明胶束粒径均一,实验结果见附图4。
实施例4
聚合物胶束体外NO释放实验:
NO的释放量用Griess试剂测定。NO的体外释放实验在37℃下进行,取两种不同介质:(1)磷酸缓冲液,pH 7.4;(2)磷酸缓冲液中含有10mM GSH。取制备好的胶束(每份NO为100μM),转移到透析袋,置于相应的缓冲液中,然后放入37℃恒温摇床,在指定时间点,从释放体系中取释放介质,并补充相同体积的介质,取出的释放介质与Griess试剂混合后,用酶标仪在UV=540nm下测定。该释放实验重复三次。数据显示,一氧化氮可以稳定持续的释放,在48h,可释放6umol左右一氧化氮,属于对心血管有益的浓度。实验结果见附图5。
实施例5
载药聚合物胶束体外阿托伐他汀释放实验:
阿托伐他汀的体外释放实验在37℃下进行,取两种不同介质:(1)磷酸缓冲液,pH7.4;(2)磷酸缓冲液中含有10mM GSH。取制备好的载药胶束,转移到透析袋,置于相应的缓冲液中,然后放入37℃恒温摇床,在指定时间点,从释放体系中取释放介质,并补充相同体积的介质,用高效液相色谱测定阿托伐他汀含量。该释放实验重复三次。数据显示,胶束药物释放表现出预期的GSH响应释放,在36h可以释放70%左右的药物,而PB缓冲液中药物释放缓慢,响应性的特点可以防止药物未达到靶点部位突释,并且胶束对药物释放起到缓释作用,有利于延长药物半衰期,增加药物生物利用度。实验结果见附图6。
实施例6
细胞存活率的测定,细胞培养及分组:
细胞培养:小鼠单核巨噬细胞白血病细胞(RAW264.7)以含10%胎牛血清的DMEM培养基(含10%胎牛血清、100IU/mL青霉素和100IU/mL链霉素)在37℃、5%CO2条件下培养,根据细胞生长情况,每隔1-2天换液一次。
实验分组:AT(0.1mg/mL)组,S2P-PEG-PNTC(2mg/mL)组,S2P-PEG-PNTC@AT(2mg/mL,0.1mg/mL)组。
按照实验分组:AT(0.1mg/mL)组,S2P-PEG-PNTC(2mg/mL)组,S2P-PEG-PNTC@AT(2mg/mL,0.1mg/mL)组,各组干预24小时后,进行检测。首先将细胞的DMEM悬浮液铺于96孔培养板中,并置于37℃,5%二氧化碳条件下培养12h使单层细胞的覆盖率达到70%-80%。然后向每孔中加入10μL不同组的药物溶液。共孵育培养24h后,向每孔中加入10μL 3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)的PBS溶液(5mg/mL),并放入培养箱继续培养4h使MTT与活细胞作用。随后移除含有MTT的培养液,向每孔中加入150μL DMSO以溶解活细胞与MTT产生的紫色甲瓒结晶,并使用酶标仪测定每个吸收孔在570nm处的吸收,实验数据平行三组进行。按下式计算各实验组细胞活力。
细胞活力(%)=(OD570样品/OD570对照)×100%
实验结果见附图7,由图7的结果可知,共聚物S2P-PEG-PNTC浓度低于1mg/mL时,对细胞基本没有明显毒性,随着共聚物S2P-PEG-PNTC材料含量增加到2mg/mL,RAW264.7的细胞活力降低。将NO与阿托伐他汀联合使用后,对于RAW264.7的细胞活力的影响更大。
实施例7
Western Blot法检测RAW264.7细胞iNOS的蛋白表达情况:
实验分组:空白对照组,阳性对照组,AT(25ug/mL)组,S2P-PEG-PNTC(1mg/mL)组,S2P-PEG-PNTC@AT(1mg/mL,25ug/mL)组。
按适量数量将RAW264.7细胞接种于6孔板内,培养箱中孵育12小时贴壁,更换为分别含有上述不同复合物的细胞培养基,除空白对照组,其余组别加脂多糖(1ug/mL)刺激12h,药物孵育24小时;提取RAW264.7细胞的总蛋白,BCA蛋白定量,检测、拟合蛋白含量的标准曲线,将样品的蛋白浓度调至一致;用8%的SDS-PAGE凝胶分离RAW264.7细胞蛋白;电泳完成后,将蛋白转移到PVDF膜上,用兔抗鼠iNOS作为抗体进行检测。实验结果见附图8。
如图8,空载的S2P-PEG-PNTC聚合物胶束和单独阿托伐他汀表现出显著的iNOS抑制,S2P-PEG-PNTC载药胶束对于iNOS的抑制活性比单独阿托伐他汀和空载的聚合物胶束更加明显,说明NO供体和阿托伐他汀联用达到增效的作用。
实施例8
Western Blot法检测RAW264.7细胞P-P65和P65的蛋白表达情况:
实验分组:空白对照组,阳性对照组,AT(25ug/mL)组,S2P-PEG-PNTC(1mg/mL)组,S2P-PEG-PNTC@AT(1mg/mL,25ug/mL)组。
按适量数量将RAW264.7细胞接种于6孔板内,培养箱中孵育12小时贴壁,更换为分别含有上述不同复合物的细胞培养基,除空白对照组,其余组别加脂多糖(1ug/mL)刺激12h,药物孵育24小时;提取RAW264.7细胞的总蛋白,BCA蛋白定量,检测、拟合蛋白含量的标准曲线,将样品的蛋白浓度调至一致;用8%的SDS-PAGE凝胶分离RAW264.7细胞蛋白;电泳完成后,将蛋白转移到PVDF膜上,用兔抗鼠P-P65和P65作为抗体进行检测。实验结果见附图9。
如图9,空载的S2P-PEG-PNTC聚合物胶束和单独阿托伐他汀表现出显著的P-P65抑制,S2P-PEG-PNTC载药胶束也表现出对于P-P65的高抑制活性,说明NO供体和阿托伐他汀都能对RAW264.7细胞的NF-kb信号通路进行抑制,从而抑制动脉粥样硬化。
结合实施例7和实施例8,对于iNOS和P-P65的抑制活性,一氧化氮虽然能达到抑制P-P65的治疗效果,但在治疗动脉粥样硬化时,单一的材料能发挥的作用毕竟是有限的,需要配合药物联合治疗才能起到更好的治疗效果,在实施例7的iNOS的抑制实验中,体现了本发明所述的一氧化氮供体纳米药物与药物协同的增效作用。
Claims (10)
1.一种靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:由功能性聚乙二醇和硝酸酯环碳酸酯单体通过开环聚合得到含一氧化氮供体的聚碳酸酯嵌段共聚物,然后与含巯基或修饰后含巯基的靶头化合物进行端基修饰,修饰后的共聚物与小分子药物溶解在有机溶剂中得到载药纳米粒子。
2.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:所述功能性聚乙二醇为马来酰亚胺修饰聚乙二醇。
3.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:所述功能性聚乙二醇分子量为1000-20000g/mol,所述功能性聚乙二醇与硝酸酯环碳酸酯单体分子量大小比例为1:0.5-1。
4.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:所述的含巯基或修饰后含巯基的靶头化合物为巯基衍生物为S2P肽(CRTLTVRKC)、巯基修饰透明质酸或Cys-RGD肽中的一种。
5.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于,所述制备方法具体为:
(1)含一氧化氮供体的聚碳酸酯嵌段共聚物的合成:在惰性气体保护下,将硝酸酯环碳酸酯单体溶于干燥的有机溶剂中,然后加入功能性聚乙二醇作为引发剂,加入催化剂,形成混合溶液A,通过开环聚合反应制备得到含一氧化氮供体的聚碳酸酯嵌段共聚物Mal-PEG-PNTC;
(2)靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物的合成:含巯基或修饰后含巯基的靶头化合物与Mal-PEG-PNTC溶解在N,N-二甲基甲酰胺(DMF)中,加入三乙胺形成混合溶液B,室温反应制备得到靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物;
(3)靶向动脉粥样硬化斑块的载药纳米粒子的制备:将靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物与小分子药物溶解在DMF溶剂中,反应后透析得到靶向动脉粥样硬化斑块的聚碳酸酯类载药纳米粒子。
6.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:所述小分子药物为他汀类药物。
7.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:步骤(1)中,混合溶液A中,硝酸酯环碳酸酯单体、功能性聚乙二醇、有机溶剂和催化剂的质量比为0.5-1:1:25:1。
8.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:步骤(3)中,所述靶向动脉粥样硬化斑块的一氧化氮供体嵌段共聚物、小分子药物、DMF溶剂的质量比为1:0.02-0.3:50。
9.根据权利要求1所述的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物的制备方法,其特征在于:步骤(3)中,所述透析采用的介质为去离子水或PB缓冲液。
10.权利要求1-9任一项制备的靶向动脉粥样硬化斑块的一氧化氮供体纳米药物在治疗动脉粥样硬化中的应用。
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