CN114588277B - 装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法 - Google Patents

装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法 Download PDF

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CN114588277B
CN114588277B CN202210362680.6A CN202210362680A CN114588277B CN 114588277 B CN114588277 B CN 114588277B CN 202210362680 A CN202210362680 A CN 202210362680A CN 114588277 B CN114588277 B CN 114588277B
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吴浩
魏民
张恒柱
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Abstract

本发明公开了一种装载有替莫唑胺和Pep‑1的聚多巴胺纳米颗粒的构建方法,属于纳米载药颗粒的制备领域,所述的构建方法具体为:步骤一、PDA的合成;步骤二、替莫唑胺‑聚多巴胺,即TMZA‑PDA的合成:TMZA是TMZ在体内的活性代谢物具有与TMZ相同的细胞毒性,替莫唑胺酸TMZA的羧基与二胺连接臂缩合,其中TMZA首先与NH2‑PEG‑NH2进行反应,使得TMZA末端拥有的氨基‑NH2能够与PDA反应,同时加入DCC及DAMP进行催化,将替莫唑胺TMZA修饰到聚多巴胺PDA上;步骤三、Pep‑1‑TMZA‑PDA的合成;本发明的Pep‑1‑TMZ‑PDA的纳米载药颗粒,基于PDA纳米递药系统对于能够提高临床一线化疗药物TMZ肿瘤靶向性、增强其抗肿瘤活性、改善其理化性质和生物利用度的有效工具。

Description

装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法
技术领域
本发明属于纳米载药颗粒的制备领域,具体是一种修饰联有替莫唑胺和Pep-1的PDA纳米颗粒的构建方法。
背景技术
在实际的临床工作中,众所周知,多形性胶质母细胞瘤(GBM)是一种起源于脑胶质瘤的恶性肿瘤,占大脑恶性肿瘤的47.10%,从过去到现在,全球原发性脑恶性肿瘤的发病率依旧逐年上升,根据CBTRUS最新数据显示在中枢神经系统原发的恶性肿瘤中胶质母细胞瘤仍是比例最高,5年的相对生存率为23.5%,非恶性脑及其他中枢神经系统肿瘤的5年相对生存率为82.4%,严重威胁着人类的健康,迄今为止手术切除依然是主要的治疗方式,但是要做到对于肿瘤的彻底切除,却存在着很大的难度,因此结合术后的放化疗是GBM的标准治疗方式。
但事实上放化疗也因胶质瘤生长部位的特殊性、不同病理类型的生物学特性的差异性等很难确定有效的治疗方案。化疗是继胶质瘤手术切除之后的诸多治疗环节中至关重要的一环,其成败直接影响着患者的生活质量以及预后。替莫唑胺(TMZ)是经美国食品和药物管理局批准(FDA)的治疗GBM的一线临床治疗药物,可通过口服或静脉注射的途径给药,通过诱导凋亡杀死胶质瘤细胞。然而,TMZ也因MGMT及各种分子信号转导机制等诸多因素的存在而肿瘤靶向性差、毒副作用明显、产生耐药性等诸多缺点,对于GBM治疗效果并不理想,经积极治疗后依然存在复发或者进一步恶化的可能,长期困扰着临床工作,并且不能改善患者术后的五年生存率,截至目前对于胶质母细胞瘤的治疗既是难点又是热点,近年各类的纳米载药系统对于术后胶质瘤放化疗的基础研究及临床试验的研究更是焦点。但是对于临床一线药物替莫唑胺纳米载药的研究相对甚少。
发明内容
根据上述现有技术中存在的技术问题,本发明公开了一种修饰联有替莫唑胺和Pep-1的PDA纳米颗粒的构建方法,本发明根据纳米载体的特征,公开了一种可以载药替莫唑胺,并且具有较高的生物利用度、较好的组织相容性、可观的载药率及包封率,较轻的毒副作用的纳米载药颗粒,本发明的Pep-1-TMZ-PDA的纳米载药颗粒,基于PDA纳米递药系统对于能够提高临床一线化疗药物TMZ肿瘤靶向性、增强其抗肿瘤活性、改善其理化性质和生物利用度的有效工具。
本发明是这样实现的:
一种装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法,其特征在于,所述的构建方法具体为:
步骤一、PDA的合成:
将5mg的多巴胺加入到10ml的Tris-HCI缓冲液中,在常温下振荡8h,然后通过超速离心机得到PDA;
步骤二、替莫唑胺-聚多巴胺,即TMZA-PDA的合成:替莫唑胺酸TMZA的羧基与二胺连接臂缩合,其中TMZA首先与NH2-PEG-NH2进行反应,使得TMZA末端拥有的氨基-NH2,能够与PDA反应,同时加入DCC及DAMP进行催化,通过形成席夫碱、加成等反应,将替莫唑胺TMZA修饰到聚多巴胺PDA上;具体的过程为:
2.1,在20mL的Tris-HCl缓冲液中溶解连有连接臂的TMZA,并添加20mg PDA;
2.2,在25℃下搅拌6h后,以14000rpm离心15分钟,得到PDA-TMZA;盐酸替莫唑胺酸TMZ与替莫唑胺TMZA具有相同的细胞毒性TMZA与NH2-PEG-NH2反应,使得TMZ具有了氨基,进一步利用PDA中的活性基团可与氨基发生迈克尔加成反应、席夫碱反应的原理,使化疗药物功能性装载到PDA的得到PDA-TMZA;
步骤三、Pep-1-TMZA-PDA的合成:
Pep-1的合成参数:NH2-CGEMGWVRC-SH;氨基酸残基数:9个;序列:Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys;分子量:1040.26g/mol;等电点:6.2PH=7时静电荷为-0.1;平均亲水性:-0.2;亲水残基比例:22%;
利用Pep-1结构中半胱氨酸残基侧链的巯基,按2.2的步骤将Pep-1装载到PDA表面,得到Pep-1-TMZ-PDA。
本发明的方法主要是:如何把药物替莫唑胺酸能够修饰到PDA上,替莫唑胺酸与临床用药的替莫唑胺具有相同的毒性,所以我们选用替莫唑胺酸,我们首先利用了TMZA与NH2-PEG-NH2反应,使得TMZA的分子式里面多了一个氨基NH2,因为只有TMZA分子式具有了NH2,才能使得TMZA能够修饰到PDA上面,虽然PDA具有穿透血脑屏障的能力及肿瘤屏障的能力,但是为了更高效的穿透血脑屏障及肿瘤屏障,并且精准进入肿瘤细胞,我们选用了Pep-1作为导航器,因为Pep-1不仅能加速进入肿瘤细胞,并且能够更精准的进入肿瘤细胞,因为Pep-1能够与肿瘤细胞高表达的IL-13Ra2具有高的亲和力,这样子一来,按照本发明装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法制备的纳米颗粒可以更精准,更高效的进入肿瘤细胞,将TMZA药物运载到肿瘤细胞,对其进行起到化疗的作用。PEP-1的特征:①序列为KETWWETWWTEWSQPKKKRKV。②起源于富含色氨酸的簇/SV40T抗原NLS。③属于亲水和疏水的两性分子,同时带有正电荷。④水溶性好⑤具有直接渗透作用⑥长度24。本发明中利用PDA纳米颗粒所修饰的TMZA的化疗作用及PDA的光热作用来联合治疗胶质瘤做出。
进一步,本发明中采用10mM、pH=8.5的Tris-HCI缓冲液。
进一步,所述的超速离心机设置为12000rpm,20min。
本发明与现有技术相比的有益效果在于:
本发明中的Pep-1能够有效透过血脑屏障及能够与肿瘤表面过度表达的白细胞介素-13受体α2(IL-13Rα2)具有高度的亲和力,并且可以特异性结合的特点,同时我们秉着利用简单的方法去解决复杂的问题,以便于更好的推广和使用,研究对于胶质母细胞瘤术后TMZ的化疗作用,同时结合PDA多方面的生物价值及较强的光热转换作用,以PDA为核,Pep-1为导航器,运载化疗药物TMZ的纳米颗粒Pep-1-TMZ-PDA通过内化机制精准到达胶质瘤细胞,双重联合打击肿瘤细胞治疗胶质母细胞瘤,以期达到治疗目的的最优化;
本发明中通过多巴胺合成聚多巴胺(PDA)再进一步利用TMZA的羧基与二胺连接臂缩合、末端的氨基与PDA反应,通过席夫碱将TMZA修饰到PDA上得到TMZ-PDA,利用同样的方法,得到Pep-1-TMZ-PDA的纳米颗粒,通过Pep-1能够高效穿透血脑屏障、直接渗透/内吞的机制到达肿瘤细胞的特性,进入后与IL-13Ra2高度亲和,使得化疗药物进入肿瘤细胞,并结合PDA的光热作用实现对胶质瘤进行化疗及光热的治疗。
附图说明
图1为本发明Pep-1-TMZA-PDA纳米颗粒合成过程;
图2为本发明PDA由DA与Tris-HCI在常温下合成过程;
图3为本发明PDA-TMZA-Pep-1的合成模式图
图4为本发明TMZA与NH2-PEG-NH2反应图;
图5为本发明实施例中Pep-1的分子式;
图6为本发明实施例中PDA纳米颗粒的TEM及SEM表征结果图;
图7为本发明实施例中TMZA、Pep-1、及Pep-1-TMZA-PDA的傅立叶红外光谱图;
图8为本发明实施中Pep-1-TMZA-PDA的粒径大小;
具体实施方式
为使本发明的目的、技术方案及效果更加清楚,明确,以下列举实例对本发明进一步详细说明。应当指出此处所描述的具体实施仅用以解释本发明,并不用于限定本发明。
1.纳米递药系统的构建和表征,合成过程以及合成模式如图1/3所示;
步骤1:PDA的合成:将5mg的多巴胺(DA)加入到10ml的Tris-HCI中(10mM,PH8.5),在常温下振荡8h,然后通过超速离心机(SIGMA3-30KS,12000rpm,20min)得到PDA,如图2所示。
步骤2:TMZ-PDA的合成:TMZA是TMZ在体内的活性代谢物具有与TMZ相同的细胞毒性。TMZA的羧基与二胺连接臂缩合,末端的氨基与PDA反应,通过形成席夫碱将TMZA修饰到PDA上。在20mL的10mM Tris-HCl缓冲液(pH 8.5)中溶解连有连接臂的TMZA,并添加PDA(20mg)。在25℃下搅拌6h后,以14000rpm离心15分钟,得到TMZ-PDA。
步骤3:利用Pep-1结构中半胱氨酸残基侧链的巯基,按关键技术2的步骤将Pep-1修饰到PDA表面,得到Pep-1-TMZA-PDA,如图4所示,反应后使得TMZA拥有一个基团NH2,使得其能够与PDA发生加成反应,其中反应的过程中需要DCC及DMAP催化。DCC:N,N’-dicyclohexylcarbodiimide;DMAP:4-dimethylaminopyridine。
步骤4:Pep-1的合成参数:NH2-CGEMGWVRC-SH;氨基酸残基数:9个;序列:Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys;分子量:1040.26g/mol;等电点:6.2PH=7时静电荷为-0.1;平均亲水性:-0.2;亲水残基比例:22%,如图5所示。Pep-1含有丰富的功能基团,其中有巯基-SH,可以与PDA发生加成等一些列反应。
步骤5:通过核磁共振氢谱和傅立叶红外光谱(FT-IR)等表征手段。通过比较TMZA、Pep-1和Pep-1-TMZA-PDA的特征核磁信号和FT-IR信号确定TMZA和Pep-1被成功修饰到PDA表面。在合成PDA-TMZA时,收集上清液,通过HPLC测定上清液中TMZA的量,利用下面公示计算TMZA的载药率(DL%)和包封率(EE%)。紫外-可见光光度分析可测试。
Figure BDA0003584594350000051
Figure BDA0003584594350000052
步骤6:材料稳定性的验证:为了再次验证材料的溶解性和稳定性,将其直接溶解在水溶液和胎牛血清(100%FBS)中,观察其在水中和FBS中的透明度及澄清的均匀性,如果溶液均保持澄清透明,证明其良好的溶解性和稳定性。并且进行紫外可见吸收光谱测试,绘制吸收光谱曲线图进行比对,行进一步验证。预期结论:通过验证颜色透明,清亮,表明稳定性良好。PDA纳米颗粒的TEM及SEM表征结果图,以及本发明实施例中TMZA、Pep-1、及Pep-1-TMZA-PDA的傅立叶红外光谱图如图6~7所示,Pep-1-TMZA-PDA的粒径大小如图8所示。。
本发明还将一种装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法制备的纳米颗粒进行一系列的实验,具体如下:
2.体外药物释放:采用透析法考察Pep-1-TMZ-PDA(100μg mL-1)的体外释药行为。采用pH 5.0和pH 7.4的PBS缓冲液为释放介质。pH 7.4模拟生理条件的pH值,pH 5.0模拟肿瘤细胞酸性微环境。取1mL载药纳米粒溶液,加入到经预溶胀处理的透析袋内(MWCO=7000Da),扎紧两端袋口,放入装有40mL释放介质的离心管中,置于恒温摇床中(37℃、120rpm)进行体外释放实验。在每个预设的取样时间点(2、4、6、8、10、12、24、33、48、57、72h)从释放介质中取样400μL,同时立即以等量空白释放介质补充。每组纳米粒溶液体外释放平行操作3份,采用HPLC法(高效液相色谱)测定样品中TMZA浓度,计算TMZA累积释放百分率,绘制体外累积释放曲线图。预期结论:表明制备的Pep-1-TMZ-PDA具有良好的pH响应性的释药能力,在生理环境中具有较高的稳定性,不释放药物,而在肿瘤的酸性环境可以进行药物的释放,对细胞进行杀伤作用。
分组不同时间段(2、4、6、8、10、12、24、33、48、57、72h):
①.PH=5.0模拟肿瘤细胞酸性微环境:a:TMZ-PDA b:Pep-1-TMZ-PDA
②.PH=7.4模拟正常生理条件下环境:a:TMZ-PDA b:Pep-1-TMZA-PDA
备注:绘制释放曲线图:纳米材料是否对TMZA的释放起到缓释作用,是否可更好地发挥药物的作用,注意PDA-TMZA与PDA-TMZA-Pep-1在不同时间段内的累积释放量有无显著差别,是否可以表明Pep-1肽的修饰未影响纳米粒材料中TMZA的释放。
3.光热效应研究:配置不同浓度(20~100μg mL-1)的Pep-1-TMZ-PDA的PBS(pH7.4)溶液,置于石英比色皿中,用近红外光照射(808nm,1.0W cm-2),于不同时间点(0,15,30,60,90,120,180,240,300,360,420,480,540,600s)利用红外热成像仪测定溶液的温度,考察PDA-TMZA-Pep-1的光热效应。以PDA作为阳性对照(用光热成像仪FLIR E50对温度及红外热图像进行记录)。为了研究光热的稳定性,分别记录2个循环,4个循环及6个循环的激光开/关周期内的Pep-1-TMZ-PDA溶液的光热升温和自然冷却变化情况,验证其光热性能的稳定性及可利用性。
4.细胞摄取研究:
细胞株选用:
①实验组:人恶性胶质瘤细胞U87MG和C6;②对照组:非胶质瘤细胞A549和HepG2
为方便检测,利用疏水—疏水作用将荧光分子香豆素(CUM)装载于PDA-TMZA-Pep-1中,制备得①.CUM-Pep-1-TMZ-PDA②.CUM-TMZ-PDA CUM-Pep-1-TMZ-PDA的制备:通过超声处理将Pep-1-TMZ-PDA分散在去离子水(20mL)中,然后加入含CUM的乙醇(20mL)溶液。避光搅拌24h后,通过离心分离产物,并进一步用乙醇和水洗涤,以除去过量的CUM,得CUM-PDA-TMZA-Pep-1。
细胞接种:细胞接种于24孔板中,经24h培养后加入含CUM-Pep-1-TMZ-PDA和CUM-TMZ-PDA的DMEM溶液,37℃孵育1h,弃去溶液,用PBS洗涤3遍,利用激光扫描共聚焦显微镜(CLSM)观察细胞内香豆素的荧光(Ex=465nm,Em=502nm),以此考察纳米材料的细胞摄取行为。[共聚焦激光扫描显微镜(CLSM)图像的采集使用奥林巴斯FV1000MPE]
DMEM是一种含各种氨基酸和葡萄糖的培养基,是MEM培养基的基础上研制的。与MEM比较增加了各种成分用量,同时又分为高糖型(高于4500mg/L)和低糖型(低于1000mg/L)。高糖型有利于细胞停泊于一个位置生长,适于生长较快、附着较困难肿瘤细胞等。
5.细胞毒性研究:[为了验证Pep-1-TMZ-PDA的生物相容性,采用MTT测定细胞活性]细胞株选用人恶性胶质瘤细胞U87MG和C6,同时选用正常细胞HEK293和L02作为对照,考察Pep-1-TMZ-PDA的肿瘤细胞选择性。对于每一种细胞,试验分组如下:
①空白对照
②Pep-1-TMZ-PDA
③TMZ-PDA
④Pep-1-TMZ-PDA+近红外光照(化疗与光热协同治疗组)
每个给药组设定5个不同浓度(0、10、25、50、100μg mL-1)(按照TMZA计算)。细胞与不同给药组孵育48h后,利用经典的MTT法测定细胞活力。近红外光照组在给药12h后光照10min(808nm,1.0W cm-2),期间注意观察化-光协同作用的的细胞杀伤力是否效果显著明显,同时也可采用共聚焦成像的方法进一步验证,最后绘制曲线图进行比对。预期结论:Pep-1-TMZ-PDA+近红外光照组的效果更加明显。
6.体内靶向性研究:利用荧光显微镜定性观察
利用小鼠原位胶质瘤模型,通过尾静脉注射香豆素标记的
①CUM-Pep-1-TMZ-PDA
②CUM-TMZ-PDA
③CUM-Pep-1-TMZ-PDA+近红外光照
利用荧光显微镜定性与定量观察体内分布的情况:观察给药0.5h,1h,1.5h后肿瘤区域的荧光分布,观察是否疗效是否CUM-PDA-TMZA-Pep-1+近红外光照>CUM-PDA-TMZA-Pep-1>CUM-PDA-TMZA。
预期结论:CUM-Pep-1-TMZ-PDA+近红外光照>CUM-Pep-1-TMZ-PDA>CUM-TMZ-PDA。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进,这些改进也应视为本发明的保护范围。

Claims (4)

1.一种装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法,其特征在于,所述的构建方法具体为:
步骤一、PDA的合成:
将5mg的多巴胺加入到10ml的Tris-HCI缓冲液中,在常温下振荡8h,然后通过超速离心机得到PDA;
步骤二、替莫唑胺-聚多巴胺,即TMZA-PDA的合成:TMZA是TMZ在体内的活性代谢物具有与TMZ相同的细胞毒性,替莫唑胺酸TMZA的羧基与二胺连接臂缩合,其中TMZA首先与NH2-PEG-NH2进行反应,使得TMZA末端拥有的氨基-NH2,能够与PDA反应,同时加入DCC及DAMP进行催化,将替莫唑胺TMZA修饰到聚多巴胺PDA上,具体的过程为:
2.1,在20mL的Tris-HCl缓冲液中溶解连有连接臂的TMZA,并添加20mgPDA;
2.2,在25℃下搅拌6h后,以14000rpm离心15分钟,得到PDA-TMZA;盐酸替莫唑胺酸TMZ与替莫唑胺TMZA具有相同的细胞毒性,TMZA与NH2-PEG-NH2反应,使得TMZ具有了氨基,进一步利用PDA中的活性基团可与氨基发生迈克尔加成反应、席夫碱反应的原理,使化疗药物功能性装载到PDA上得到PDA-TMZA;
步骤三、Pep-1-TMZA-PDA的合成:
Pep-1的合成参数:NH2-CGEMGWVRC-SH;氨基酸残基数:9个;序列:Cys-Gly-Glu-Met-Gly-Trp-Val-Arg-Cys;分子量:1040.26g/mol;等电点:6.2PH=7时静电荷为-0.1;平均亲水性:-0.2;亲水残基比例:22%;
利用Pep-1结构中半胱氨酸残基侧链的巯基-SH,按2.2的步骤将Pep-1装载到PDA表面,得到Pep-1-TMZA-PDA。
2.根据权利要求1所述的一种装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法,其特征在于,采用10mM、pH=8.5的Tris-HCI缓冲液。
3.根据权利要求1所述的一种装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法,其特征在于,所述的超速离心机设置为12000rpm,20min。
4.一种如根据权利要求1所述的一种装载有替莫唑胺和Pep-1的聚多巴胺纳米颗粒的构建方法制备的纳米颗粒。
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