CN113599531B - Pcm多肽组合kala多肽的红细胞仿生纳米材料的应用及其制备方法 - Google Patents
Pcm多肽组合kala多肽的红细胞仿生纳米材料的应用及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种PCM多肽组合KALA多肽的红细胞仿生纳米材料在治疗心血管疾病中的应用,还公开了该红细胞仿生纳米材料的制备方法,该方法如下:获取适量PCM结合KALA并融合成一条融合肽;将该融合肽进行保护氨基酸操作处理,获得PCM/KALA多肽;将PCM/KALA多肽结合在红细胞膜(RBCM)表面;将结合红细胞膜的PCM/KALA多肽利用Extrusion方法通过400nm孔径的聚碳酸酯膜,构建RBCM‑PCM/KALA红细胞仿生纳米颗粒。通过本发明的方法制得的红细胞仿生纳米材料能够不依赖浓度的方式高效且快速的导入到细胞内,相比现有技术还具备心脏富集功能。
Description
技术领域
本发明涉及生物医药技术领域,具体为PCM多肽组合KALA多肽的红细胞仿生纳米材料的应用及其制备方法。
背景技术
心血管疾病是人类疾病的头号杀手。近年来,纳米技术的发展为心血管相关疾病的诊疗提供新的契机。通过纳米技术,药物能实现在心脏部位的可持续缓慢释放,达到长时间治疗目的。因此,如何将纳米药物有效递送至心肌细胞是关键。
PCM多肽(序列为WLSEAGPVVTVRALRGTGSW)具有一定的心脏靶向功能。然而,PCM的靶向效率受限于两点:1)PCM不能介导纳米材料被心肌细胞快速吞噬,仅提供与心肌细胞的亲和力,故在血液循环剪切力作用下容易重新进入循环系统;2)即使进入心肌细胞,PCM不能介导纳米材料快速从溶酶体中逃逸,导致包封的药物无法有效释放到心肌细胞,药物疗效受到制约。
现有技术,作者为王欣,于2017公开的《PCM和TAT双修饰脂质体心肌靶向传递系统的研究》论文,公开了PCM(WLSEAGPVVTVRALRGTGSW)是通过噬菌体展示技术筛选出的由20个氨基酸组成的肽段,是一种心肌细胞特异性靶向肽。TAT是近年来广泛应用于修饰药物和药物载体的一种细胞穿膜肽,它由11个氨基酸(YGRKKRRQRRR)组成,可将与之相连的药物或药物载体以一种浓度依赖的方式高效,快速地导入细胞内。该方案公开了借助靶向功能的PCM结合细胞穿模肽进行但是这种结合方式仅能利用PCM来实现靶向效果,而需要借助药物的浓度依赖将药物导入细胞。
发明内容
针对现有技术存在的不足,本发明的目的在于提供一种PCM多肽组合KALA多肽的红细胞仿生纳米材料的应用及其制备方法,通过制备方法获得的放生纳米材料能够不依赖浓度的方式高效且快速的导入到细胞内。
为实现上述目的,本发明提供了如下技术方案:PCM多肽组合KALA多肽的红细胞仿生纳米材料在治疗心血管疾病中的应用。
还提供了上述红细胞仿生纳米材料的制备方法,该制备方法包括如下步骤:
获取适量PCM结合KALA并融合成一条融合肽;
将该融合肽进行保护氨基酸操作处理,获得PCM/KALA多肽;
将PCM/KALA多肽结合在红细胞膜(RBCM)表面;
将结合红细胞膜的PCM/KALA多肽利用Extrusion方法通过400nm孔径的聚碳酸酯膜,构建RBCM-PCM/KALA红细胞仿生纳米颗粒。
作为本发明的进一步改进,PCM/KALA多肽与红细胞膜结合前先将异官能团聚乙二醇(DSPE-PEG2000-MAL)中的MAL与PCM/KALA多肽末端的SH反应,再将DSPE插入到红细胞膜表面。
作为本发明的进一步改进,该制备方法中各个组分比例PCM/KALA∶DSPE-PEG2000-MAL∶红细胞膜=2∶1∶40(w/w/w)。
作为本发明的进一步改进,保护氨基酸操作处理后,还对获得的PCM/KALA多肽进行切割处理。
作为本发明的进一步改进,DSPE-PEG2000-MAL与PCM/KALA多肽的反应条件为37℃孵育30-60min。
作为本发明的进一步改进,DSPE插入到红细胞膜表面的条件为37℃孵育30-60min。
本发明的有益效果,
1.PCM、KALA、红细胞膜三者的结合能够减少被免疫细胞清除提高血液半衰期。
2.相比现有技术来说,本方案具备靶向递送和高效且快速的导入物质,能够实现更有效的心脏富集功能,让作用效果更加集中、高效和具有针对性。
3.相比现有技术来说,本方案对浓度没有依赖。
附图说明
图1为本发明的PCM/KALA多肽合成表征;
图2为本发明的红细胞膜(RBCM)制备的效果图;
图3为本发明的RBCM-PCM/KALA或RBCM-PCM红细胞仿生纳米颗粒对心肌细胞的靶向性比较图;
图4为本发明的共聚焦激光扫描显微镜(CLSM)研究RBCM-PCM/KALA的溶酶体逃逸功能示意图;
图5为本发明的RBCM-PCM/KALA对C57BL/6鼠心脏的靶向性效果示意图。
具体实施方式
下面将结合附图所给出的实施例对本发明做进一步的详述。
参照图1-5所示,
实施例1
组合物为PCM多肽与KALA多肽组合并且插入到红细胞膜中构成的纳米材料在治疗心血管疾病中的应用。
该应用中,PCM具有心脏靶向功能,能够携带KALA多肽到达心血管疾病的目标位置,KALA多肽在pH=7.4环境中维持二级结构,与PCM相配合构成稳定的递送物质,KALA还具有蜷缩的疏水性氨基酸和暴露的氨基酸,暴露的氨基酸能够介导KALA被细胞快速吞噬,相较于现有技术中的TAT来说,不依赖浓度,吞噬效率更高。在KALA进入到溶酶体后,随着pH的下降,KALA多肽的二级结构被破坏,KALA的疏水性氨基酸暴露并与溶酶体膜融合,此时介导KALA多肽从溶酶体中逃逸,因此KALA能够介导细胞快速吞噬以及溶酶体逃逸。KALA在结合PCM之后能够实现稳定的靶向递送和快速释放KALA携带的治疗物质。连接红细胞膜能够让治疗物质携带更加稳定,三者的结合能够减少被免疫细胞清除提高血液半衰期,同时具备靶向递送和高效且快速的导入物质,能够实现更有效的心脏富集功能,让作用效果更加集中、高效和具有针对性。
实施例2
PCM/KALA多肽合成
(1)原料保护氨基酸Fmoc-Glu(otbu)-OH、Fmoc-Cys(trt)-OH、Fmoc-Ala-oh、Fmoc-Lys(boc)-oh、Fmoc-Leu-oh、Fmoc-His(trt)-oh、Fmoc-Trp(boc)-oh、Fmoc-Ser(tbu)-oh、Fmoc-Gly-oh、Fmoc-Pro-oh、Fmoc-Val-oh、Fmoc-Thr(tbu)-oh、Fmoc-Arg(pbf)-oh
起始树脂:fmoc-Ala-wang Resin
缩合剂及有机碱:HBTU,NMM
溶剂:DMF,DCM,甲醇,六氢吡啶。
(2)设备及仪器:
玻璃反应柱,真空水泵,干式加热器,低俗大容量离心机,恒温摇床,真空干燥皿
(3)各种试剂的配制:
Kaiser test试剂的配制
A液:80%的苯酚+20%的无水乙醇B液:重蒸吡啶C液:5克茚三酮+100ML无水乙醇
脱保护溶液的配制
20%的六氢吡啶+80%的DMF
多肽切割液的配制
87.5%TFA+5%苯甲硫醚+2.5%苯酚+2.5%EDT+2.5%H2O
(4)多肽的合成:
a.树脂溶胀:称fmoc-Met-wang Resin树脂倒入反应柱中,加入DCM浸泡30分钟,抽干。
b.脱保护:向反应柱中加入适量脱保护溶液,通氮气搅拌鼓动30分钟,抽干。
c.称料:量取树脂X倍摩尔量的氨基酸fmoc-Glu(otbu),再称取X倍摩尔量的HBTU,备用
d.脱保护洗涤:向反应柱中加入适量DMF,氮气鼓动2分钟,抽干,重复操作6次。
e投料:将已称好的氨基酸、保护氨基酸和HBTU加入反应柱中,再加入树脂X倍摩尔量的NMM,氮气搅拌鼓动30分钟。
f.反应后洗涤:将反应柱中的溶液抽干,加入适量DMF洗涤,氮气鼓动2分钟,抽干,重复操作3次。
g.检测:取适量(10-20颗)树脂于小试管中,加入A,B,C液各两滴。放入干式加热器中加热3分钟(110摄氏度)。取出后若溶液显蓝色,树脂有杂色不透明,则反应没有完全,需要重新再反应一次;若溶液颜色微黄,树脂无色透明则为反应完全,可以连接下一个氨基酸,具体步骤重复以上b-f这五个步骤,直到连完主链上最后一个氨基酸fmoc-Trp(boc)-oh。在此基础上,脱去最后的fmoc,再连接boc制备boc-WEAKLAKALAKALA-K(DDE)-HLAKALAKALKACEA-wangresin树脂多肽。最后,脱去K侧链的DDE将直链C端第一个氨基酸Fmoc-Trp(boc)连在K的侧链,然后按照b-f继续往下连支链第二个氨基酸Fmoc-Leu-oh,一直连到支链最后一个氨基酸Fmoc-Trp(boc)结束,并脱除其Fmoc。h.合成完毕后的洗涤和干燥:上述多肽,抽干,向反应柱中加入适量甲醇,氮气鼓动2分钟,抽干,再加入适量DCM,氮气鼓动2分钟,抽干重复操作3次。最后反应釜中加入适量甲醇,氮气鼓动2分钟,抽干,重复操作2次,将树脂装入合适器皿中置于真空干燥器内真空干燥12小时待切割。
(5)多肽的切割
切割:将干燥后的树脂装入合适的圆底烧瓶中,加入适量配好的切割液(1g/10m1),放置于恒温摇床中25C恒温振荡2小时。
过滤:用50ml的砂芯漏斗过滤掉树脂颗粒,然后将滤液倒入100ml的离心管内,加入6-8倍体积量的无水乙醚,边加边搅拌,析出的白色固体为即所需多肽粗品。
洗涤:将离心管密封后放入离心机中以4000转每分钟的转速离心3分钟,取出,将上层清液倒掉,再加入乙醚,用玻璃棒搅拌均匀,再次离心;如此重复操作洗涤5次。
干燥:将以洗涤5次的后的多肽放入真空干燥器中真空干燥24小时。最后的所得白色粉末即为所需多肽的粗品,称量,待纯化。
(5)多肽的纯化
取少量样品进行超声溶解,溶解完成后取20微升样品。放分析型高效液相色谱仪分析,梯度为10-100,时间是0~25分钟,A泵是100%乙腈加0.1%TFA,B泵100%水加0.1%TFA,进样分析,搜取目标峰后把质谱确定是否正确,确定目标峰后给出相应的梯度进行制备,制备型液相色谱仪的流动相和分析的相同时间也同样在目标峰出现后进行质谱确认,确认好质谱后给出相应的梯度进行分析,等分析合格后冻干。
多肽的表征如图1所示。
实施例3
红细胞膜(RBCM)的制备
红细胞来源于C57BL/6鼠(B6鼠),过程如下:收集B6鼠全血,3000-5000rpm离心15min去除白细胞、血小板和血清,获得红细胞。将250μL的红细胞加入950μL超纯水中,冰浴30-60min,加入20x PBS调渗透压至lx,混匀后,14000rpm离心10min,弃上清,加950μL超纯水重悬,再次冰浴,调渗透压,离心,以此循环直至上清无血红蛋白,并收集沉淀(图2)。收集的沉淀即为红细胞膜(Red Blood Cell Membrane,RBCM)。通过BCA法测定RBCM中膜蛋白的浓度。
实施例4
DSPE-PEG2000-PCM/KALA、DSPE-PEG2000-PCM的合成
准确称取DSPE-PEG2000-MAL、PCM/KALA以及PCM分别溶于超纯水中,随后分别按照DSPE-PEG2000-MAL∶PCM/KALA为1∶2(mol/mol)、DSPE-PEG2000-Mal∶PCM为1∶2(mol/mol)混合,37℃孵育30-60min分别合成DSPE-PEG2000-PCM/KALA、DSPE-PEG2000-PCM待用。
实施例5
DSPE-PEG2000-PCM/KALA或DSPE-PEG2000-PCM与RBCM结合
将RBCM分别与DSPE-PEG2000-PCM/KALA或DSPE-PEG2000-PCM按照DSPE-PEG2000-MAL与RBCM质量(以膜蛋白质量标定)比l∶20(w/w)混合,37℃孵育30-60min。完成反应后将上述反应物置于离心机中,14000rpm离心10min,去除多余DSPE-PEG2000-PCM/KALA或DSPE-PEG2000-PCM,制备RBCM-PCM/KALA或RBCM-PCM。
实施例6
RBCM-PCM/KALA或RBCM-PCM红细胞仿生纳米颗粒对心肌细胞的靶向性比较
将DiD染料分别与RBCM或RBCM-PCM/KALA或RBCM-PCM按照DiD与RBCM质量(以膜蛋白质量标定)比6∶1000(w/w)混合,37℃孵育30min。随后,14000rpm离心10min去除上清,并利用PBS清洗3次,确保完全去除游离的DiD染料。然后,利用超纯水重悬上述制备的三种荧光染料标记的纳米颗粒,最后利用Extrusion方法分别制备标记DiD染料的RBCM-PCM/KALA或RBCM-PCM或RBCM红细胞仿生纳米颗粒待用。
实验组分:NC组(Negative Control,无任何处理组);RBCM组(仅加入DiD标记的DNA&Pro@RBCM);3.PCM组(仅加入DiD标记的DNA&Pro@RBCM-PCM);4.PCM拮抗组(利用freePCM预处理4h后,再加入DiD标记的DNA&Pro@RBCM-PCM);5.PCM/KALA组(仅加入DiD标记的DNA&Pro@RBCM-PCM/KALA);6.PCM/KALA拮抗组(利用free PCM/KALA预处理4h后,再加入DiD标记的DNA&Pro@RBCM-PCM/KALA)。大致实验流程:在6孔板中培养H9C2细胞(大鼠来源的心肌细胞),待其汇合率至80%后分别加入上述不同实验组,每个组RBCM膜蛋白统一浓度为100μg/mL。2h后,去除上清,再利用PBS清洗3次。随后,利用胰酶消化细胞成单细胞悬液,再通过流式细胞术检测细胞内DiD荧光信号强弱。流式细胞术实验结果统计见图3。结果表明,PCM组和PCM/KALA组较单独RBCM组的心肌细胞靶向效率分别提高约2倍和10倍。在PCM组中,PCM仅提高颗粒与H9C2细胞之间的亲和力,并不能快速介导颗粒被H9C2细胞吞噬,因此靶向效果仅为RBCM组的2倍。对于PCM/KALA组,PCM在增加颗粒与H9C2细胞亲和力的同时,KALA能快速介导颗粒被H9C2细胞吞噬,故靶向效果达到RBCM组的10倍。尽管KALA在介导H9C2细胞快速吞噬颗粒的过程中起到关键作用,但PCM的靶向作用是前提。从拮抗实验结果可以得出,若利用free PCM/KALA预处理H9C2细胞(PCM/KALA拮抗组),使H9C2细胞表面PCM受体提前封闭,该细胞不再有效吞噬PCM/KALA组颗粒,其靶向效果约为RBCM组的2倍。同样,经过free PCM预处理的PCM拮抗组也能显著降低颗粒被H9C2细胞吞噬。综上所述,DNA&Pro@RBCM-PCM/KALA先通过PCM靶向结合到H9C2细胞表面,同时再利用KALA实现快速的细胞吞噬。
实施例7
通过共聚焦激光扫描显微镜(CLSM)研究RBCM-PCM/KALA的溶酶体逃逸功能
将DiD染料和已制备的RBCM-PCM/KALA、RBCM-PCM及RBCM按照一定的质量比混合(RBCM∶DiD=500∶3w/w),37℃孵育30min。随后,14000rpm离心10min去除游离DiD染料,再用PBS洗3次,然后用超纯水重悬,最后利用Extrusion方法制备DiD荧光标记的RBCM-PCM/KALA、RBCM-PCM及RBCM红细胞仿生纳米颗粒。同时,在共聚焦细胞培养皿内铺入2*104个心肌细胞,待其汇合率到达约50%时加入DiD荧光标记的不同实验组红细胞仿生纳米颗粒。共培养2h,除去上清,并用PBS洗3次,然后加入新鲜的完全培养基,继续培养至24h。24h时间点时,取出细胞,加入DAPI染细胞核。CLSM结果说明(见图4),RBCM-PCM/KALA组中溶酶体荧光信号(第二列)与纳米颗粒荧光信号(第一列)几乎不重叠,说明颗粒已从溶酶体中逃逸。相反的,在相同时间点,RBCM-PCM或RBCM组中溶酶体荧光信号(第二列)与颗粒荧光信号(第一列)大部分重叠,说明颗粒仍处于溶酶体内。以上结果表明,KALA多肽的修饰能够有效介导颗粒穿透溶酶体进入细胞质。
实施例8
RBCM-PCM/KALA对C57BL/6鼠心脏的靶向性研究
按上述方法分别制备带有DiD荧光标记的RBCM-PCM/KALA或RBCM-PCM或RBCM红细胞仿生纳米颗粒。将三种颗粒以RBCM剂量(膜蛋白为标定)为50mg/kg通过尾静脉分别注射至C57BL/6鼠体内,24h后解剖小鼠,利用小动物活体成像仪检测不同实验组小鼠心脏内颗粒的富集情况(见图5)。结果表明,RBCM组几乎不会被心肌细胞摄取,而RBCM-PCM组由于PCM对心肌细胞的亲和力,能够介导一部分颗粒进入心肌细胞。对于RBCM-PCM/KALA,PCM能提高颗粒与心肌细胞的亲和力,同时KALA能够快速介导颗粒被心肌细胞吞噬,实现更有效的心脏富集功能。
结合上述实施例,本方案主要为如下步骤,首先将PCM和KALA的关键序列按照预先设计的氨基酸序列,通过一定的排列方式融合成一条融合肽,并通过保护氨基酸的一系列反应(如树脂溶胀、脱保护、称料、脱保护洗涤、投料、反应后洗涤、检测和最后的切割)完成PCM/KALA多肽的合成。随后,将异官能团聚乙二醇(DSPE-PEG2000-MAL)中的MAL与PCM/KALA末端的SH反应,再利用DSPE插入到预先制备的红细胞膜(RBCM)表面,最后通过Extrusion方法构建RBCM-PCM/KALA红细胞仿生纳米颗粒。通过各个组分比例优化,获得最佳的比例为:PCM/KALA∶DSPE-PEG2000-MAL∶红细胞膜=2∶1∶40(w/w/w)。
综上所述,将PCM多肽与KALA多肽的关键序列按照一定的排列方式进行组合能保留两者的生物学功能,并且结合在一起能够具有稳定高效的递送效果,实现靶向心肌细胞以及被心肌细胞快速吞噬和溶酶体逃逸的目的。该多肽与红细胞膜结合能够制备一种靶向心肌细胞的红细胞仿生纳米药物递送体系,有望用于靶向治疗心肌细胞功能失调导致的多种心血管疾病。
上述主要名词的译名解释如下:
DCM 二氯甲烷
DMF N,N-二甲基甲酰胺
NMM N-甲基吗啉
HBTU 苯并三氮唑-四甲基脲六氟磷酸盐
Kaiser test 茚三酮检测
EDT 1,2-乙二硫醇
TFA 三氟乙酸
DiD 细胞膜红色荧光探针
CLSM 共聚焦扫描显微镜
以上所述仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。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前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.PCM多肽组合KALA多肽的红细胞仿生纳米材料的制备方法,其特征在于,包括如下步骤:
获取适量PCM结合KALA并融合成一条融合肽;
将该融合肽进行保护氨基酸操作处理,获得PCM/KALA多肽,其中PCM/KALA多肽的结构为WEAKLAKALAKALAK(NHCO-WLSEAGPVVTVRALLRGTGSW)HLAKALAKALKACEA;
将PCM/KALA多肽结合在红细胞膜RBCM表面,PCM/KALA多肽与红细胞膜结合前先将异官能团聚乙二醇DSPE-PEG2000-MAL中的MAL与PCM/KALA多肽末端的SH反应,获得DSPE-PEG2000-PCM/KALA,再将DSPE-PEG2000-PCM/KALA插入到红细胞膜表面,获得RBCM-PCM/KALA;
将RBCM-PCM/KALA利用Extrusion方法通过400 nm孔径的聚碳酸酯膜,构建RBCM-PCM/KALA红细胞仿生纳米颗粒;
DSPE-PEG2000-MAL:PCM/KALA为1:2mol/mol,反应条件为37℃孵育30-60 min;
DSPE-PEG2000-PCM/KALA与RBCM以膜蛋白质量标定质量比1:20w/w混合,37℃孵育30-60min。
2.根据权利要求1所述的制备方法,其特征在于,保护氨基酸操作处理后,还对获得的PCM/KALA多肽进行切割处理。
3. PCM多肽组合KALA多肽的红细胞仿生纳米材料在制备治疗心血管疾病的药物中的应用,其特征在于,其中PCM多肽组合KALA多肽的结构为WEAKLAKALAKALAK(NHCO-WLSEAGPVVTVRALLRGTGSW)HLAKALAKALKACEA,且心血管疾病由心肌细胞功能失调导致,且PCM多肽组合KALA多肽的红细胞仿生纳米材料由权利要求1或2的PCM多肽组合KALA多肽的红细胞仿生纳米材料的制备方法制备获得。
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