CN117567557A - 一种整合环脂肽的制备及抗肿瘤作用 - Google Patents
一种整合环脂肽的制备及抗肿瘤作用 Download PDFInfo
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Abstract
本发明涉及一种抗癌环脂肽C8H15O‑c[Gly‑Phe‑Tyr‑Ala‑Pro]的制备及其在抗肿瘤治疗中的应用,属于生物医药领域。所述抗癌环脂肽基于膜裂解肽的关键生物学特征(正电荷、两亲性)制备。本发明提供了所述抗癌环脂肽的合成方法。本抗癌环脂肽具有溶血活性低以及血清稳定性好的特点,能有效弥补大多数抗癌肽高溶血毒性的缺陷,解决肽类药物体内稳定性差的问题。体外抗癌实验及体内抗肿瘤实验证明本抗癌环脂肽具有良好的抗肿瘤效果,有很好的应用前景。
Description
技术领域
本发明属于生物医药领域,具体涉及一种人工合成的具有良好抗癌活性的抗癌环脂肽的制备及其在肿瘤治疗中的应用。
背景技术
癌症也叫做恶性肿瘤,是严重威胁生命的常见病,化疗是临床治疗癌症的主要手段之一。化疗药物在杀死癌细胞的同时,也会破坏大量的正常细胞,同时还具有依从性差、毒副作用、转移控制不良等不良后果。因此,肿瘤的药物治疗期待着新的突破。抗癌肽(ACPs)是具有抗肿瘤活性的生物活性肽,由于其具有广谱的抗癌活性,能迅速杀灭癌细胞,不受传统化疗耐药突变的影响,同时与传统抗肿瘤药物具有良好的协同效应,且有一定的选择性以及突出的组织渗透性等特点,使其具备开发成为高选择性、低毒性、高组织渗透性及高肿瘤渗透性的新一代抗肿瘤药物的潜质。抗癌肽中净电荷数,疏水氨基酸的位置及数量,形成的二级螺旋结构会影响抗癌肽的作用机制和抗癌活性。对于大多数的阳离子肽,细胞膜是主要的靶点,α-螺旋结构的疏水面与细胞膜疏水中心之间的疏水相互作用在其发挥生物活性中起到关键作用,在多肽的非极性面增加疏水性会增强多肽的螺旋度和自组装能力,具有高疏水性的多肽会进入癌细胞膜疏水中心的内部,通过形成孔或通道从而发挥更强的抗癌活性,然而抗癌活性的提高往往伴随着溶血活性的提高。
大多数天然肽及其衍生物为线性肽,由于线性肽的结构灵活性,可以使其进行非选择性受体结合,但由于其游离氨基和羧基末端在体内不稳定,导致其生物利用度低和代谢快缺点,限制了其药效的发挥。环化是改善线性肽药物药理学性质的最常见策略之一。环脂肽通常由5至14个氨基酸组成,分子量约为5500到10000Da。环脂肽的结构有助于抵抗血液中蛋白酶的降解,从而提高其血清稳定性。并且肽的环化有助于其通过细胞膜,靶向细胞内靶标,扩大环脂肽的潜在用途。环状肽与线性肽相比,具有更高的选择性、效力、生物利用度和代谢稳定性。因此,直链肽的环化具有重要的研究价值。
发明内容
本发明的目的是通过固相合成技术合成一种具有优异抗癌活性的抗癌环脂肽并将这种抗癌环脂肽应用于抗肿瘤治疗。
为了实现本发明的目的,提供了如下技术方案:
本发明抗癌环脂肽的氨基酸序列为:C8H15O-c[Gly-Phe-Tyr-Ala-Pro],其技术方案如下:
(1)抗癌环脂肽的设计:根据抗癌肽的阳离子性和两亲性结构特点,整合成含噁二唑结构的环脂肽,同时在酪氨酸残基上用正辛酸(C8H16O2)修饰,有利于提高药物的脂溶性和稳定性,能够促进癌细胞对药物的吸收。
(2)抗癌环脂肽的合成:通过固相合成法进行制备得到抗癌环脂肽,其制备方案为,首先将偶联在树脂上的带有Fmoc保护基和侧链保护基的氨基酸脱除Fmoc保护基,再使用催化剂催化各氨基酸依次从C端到N端逐个偶联在Wang Resin上,用切割液(TFA:H2O:Tis=95:2.5:2.5)切割树脂,并脱除侧链保护基团得到线性肽,线性肽与(N-异氰基氨基)三苯基磷烷(PINC)和醛反应得到抗癌环肽,最后在酪氨酸残基上偶联正辛酸,最终得到经修饰的抗癌环脂肽。本发明抗癌环脂肽结构式如下:
(3)抗癌环脂肽的体外抗癌活性研究:通过MTT法测试抗癌环脂肽对HepG2细胞的毒性;
(4)抗癌环脂肽的破膜活性研究:将抗癌环脂肽与HepG2细胞分别共培养5min和30min后,通过PI/Hoechst 33342染色,显微镜下观察抗癌环脂肽对细胞膜形态的影响;
(5)抗癌环脂肽的血清稳定性和溶血性研究:预先用胎牛血清(FBS)和抗癌环脂肽共培养,然后用MTT检测经FBS预处理后抗癌活性变化来评估其稳定性;通过抗癌环脂肽对小鼠红细胞形态变化的影响评估其溶血活性;
(6)抗癌环脂肽的体内抗肿瘤活性研究:选用植瘤小鼠作为实验模型,隔天尾静脉注射抗癌环脂肽,并对小鼠体重及肿瘤体积进行测量,将小鼠肿瘤组织解剖并进行组织切片和HE染色来检测抗癌环脂肽的体内抗肿瘤活性。
附图说明
图1:MTT法检测抗癌环脂肽对HepG2细胞的抗癌活性。将不同浓度的抗癌环脂肽与HepG2细胞共培养24h的相对细胞存活率。
图2:抗癌环脂肽的破膜活性。环脂肽与HepG2细胞分别共孵育5min(a-b)、30min(c-d)后PI/Hoechst 33342染色,显微镜下观察抗癌环脂肽对细胞膜形态的影响。
图3:抗癌环脂肽的溶血活性和血清稳定性。小鼠尾静脉注射生理盐水(a)和2.25mg/mL抗癌环脂肽(b)2h后取血,显微镜下观察(400×)红细胞形态;(c)预先将抗癌环脂肽分别与含10%血清和不含血清的PBS在37℃下孵育24h后通过MTT法检测抗癌环脂肽对HepG2细胞的抗癌活性变化图。
图4:抗癌环脂肽的体内抗肿瘤活性。(a)植瘤小鼠隔天尾静脉注射NS和抗癌环脂肽后相对肿瘤体积随时间变化图;(b)植瘤小鼠隔天尾静脉注射NS和抗癌环脂肽后体重随时间变化图;(c)隔天注射生理盐水11天后肿瘤组织切片(200×);(d)隔天注射抗癌环脂肽11天后肿瘤组织切片(200×)。
具体实施方式
以下给出本发明的具体实施方式,用来对本发明的构成做进一步的说明,但并不认为本发明仅局限于下述的实施方式。
实施例1:抗癌环脂肽的制备方法
本发明抗癌环脂肽的合成基于Fmoc保护的固相多肽合成法进行。抗癌环脂肽的序列为C8H15O-c[Gly-Phe-Tyr-Ala-Pro],具体合成步骤如下:
(1)Gly-Phe-Tyr-Ala-Pro-NH2的合成;
通过固相合成法合成了Gly-Phe-Tyr-Ala-Pro-NH2样品;使用DMF对Wang resin-Gly-Fmoc进行溶胀,溶胀30min;取少量树脂进行茚三酮检测,若没有颜色变化,则加入含有20%哌啶的DMF溶液反应30min脱去Fmoc保护基;依次用DMF、DCM、DMF对树脂进行冲洗,冲洗完成后再用无水DMF对树脂进行溶胀,时间为30min;溶胀完成后,加入2倍当量的氨基酸和2.6倍当量的DCC、HOBT、DIEA进行反应,反应时间为48h以上;透析完成后,取少量树脂进行茚三酮检测,若没有颜色变化,则说明相应的氨基酸已被连接;然后,依次用DMF、DCM、DMF对树脂进行冲洗,用透析袋(MW8000-14000)置于95%乙醇中进行透析,透析20次以上,每次透析时间为30min;重复上述步骤,直至合成Wang resin-Gly-Phe-Tyr(OtBu)-Ala-Pro-Fmoc样品。将样品进行冷冻干燥,加入切割液(TFA:H2O:Tis=95:2.5:2.5)切割掉侧链保护基团和树脂,得到目标直链五肽Gly-Phe-Tyr-Ala-Pro-NH2。
(2)c[Gly-Phe-Tyr-Ala-Pro]的合成;
首先将Gly-Phe-Tyr-Ala-Pro-NH2进行冻干处理;将Gly-Phe-Tyr-Ala-Pro-NH2悬浮在二氯乙烷和乙腈的1∶1混合溶液中(DCE∶MeCN,200mL)将其进行超声处理。将丙醛(0.075mmol)加入悬浮液中,然后加入(N-异氰酸氨基)三苯基磷烷(0.05mmol),在50℃搅拌24小时。反应结束后进行旋蒸浓缩、冷冻干燥得粗品环脂肽。然后进行中性氧化铝柱纯化操作,流动相为无水乙醇:水(2:1),最终得到c[Gly-Phe-Tyr-Ala-Pro]。
(3)C8H15O-c[Gly-Phe-Tyr-Ala-Pro]的合成;
首先将c[Gly-Phe-Tyr-Ala-Pro]进行冻干处理;将2倍当量的DMAP、EDC、DIPEA、正辛酸放在无水DCM中活化2h,再加入冻干后的c[Gly-Phe-Tyr-Ala-Pro]反应48h;反应完成后,将粗品C8H15O-c[Gly-Phe-Tyr-Ala-Pro]装于透析袋内(MW500),进行透析,透析20次以上,每次透析时间为30min;然后进行中性氧化铝柱纯化操作,流动相为石油醚:乙酸乙酯(1:1),最终得到目标环脂肽C8H15O-c[Gly-Phe-Tyr-Ala-Pro]。
实施例2:抗癌环脂肽的体外抗癌活性测定
通过MTT法测试抗癌环脂肽的体外抗癌活性。选用处于对数生长期的HepG2细胞,将HepG2细胞接种到96孔板中,然后放入5%CO2、37℃的培养箱中进行培养,用培养基把环脂肽配制成不同的浓度,浓度分别设置为0μg/mL,20μg/mL,40μg/mL,60μg/mL,100μg/mL,120μg/mL,140μg/mL,160μg/mL,180μg/mL,200μg/mL。每个孔里边加入100μL含有药物的培养基,每个浓度设置5个复孔,以减小实验偶然性带来的误差影响。将细胞和药物共培养24h后,在每个孔再加入20μL(5mg·mL-1)的MTT溶液,放置在细胞培养箱中培养4h。4h后将上清液弃掉,在每个孔中加入150μL的DMSO溶液,将96孔板避光处理后放置在振荡器上,170r·min-1下振荡7min,孔内的晶体充分溶解后用酶标仪检测其OD值,酶标仪的波长设置为485nm,测量吸光度以计算癌细胞的相对存活率,评价环脂肽的体外抗癌活性。
实施例3:抗癌环脂肽对癌细胞的破膜活性研究
为研究抗癌环脂肽的破膜活性,将抗癌环脂肽与HepG2细胞分别共培养5min和30min,通过PI/Hoechst 33342染色,显微镜下观察抗癌环脂肽对细胞膜形态的影响。PI是一种对DNA染色的细胞核染色试剂,其在嵌入双链DNA后可以释放出红色荧光。PI不能进入活细胞膜进行染色,但是能穿过破碎的细胞膜从而对核进行染色。
实施例4:抗癌环脂肽的血清稳定性和溶血活性测试
通过测定在有无血清的环境下,环脂肽对HepG2细胞的杀伤能力变化来判定该环脂肽是否具有血清稳定性。同时设置对照组和实验组,对照组为PBS溶解的环脂肽,实验组为含有10%血清的PBS溶解的环脂肽。将两组置于37℃细胞培养箱孵育24h,进行MTT实验研究,计算HepG2细胞的相对存活率。
在溶血活性实验中,随机选择两只雌性小鼠,将生理盐水和2.25mg/mL(15mg·kg-1)的环脂肽通过尾静脉注射的方法注入小鼠体内,2h后通过眼球取血的方式取血,加肝素抗凝后用生理盐水稀释,荧光显微镜下观察红细胞形态。
实施例5:抗癌环脂肽的体内抗肿瘤活性
建立体内荷瘤小鼠模型检验环脂肽的体内抗肿瘤活性。实验所用小鼠均为(24~27g)雌性昆明小白鼠,并严格按照“动物实验的护理和使用指南”饲养。将1×106个HepG2细胞皮下注射接种到小鼠的左前肢腋下,当肿瘤长至150-220mm3时进行抗肿瘤活性实验。将小鼠随机分为生理盐水组和环脂肽组,每组5只。对上述2组小鼠进行4次给药,每次注射剂量为0.2mL,浓度为2.25mg·mL-1,每组注射给药的间隔时间为1天。实验为期9天,通过颈椎脱臼的方法处死小鼠。在每次注射给药前以及在最终解剖前都要记录小鼠的体重和肿瘤的体积,将各组小鼠肿瘤解剖后浸泡在4%的多聚甲醛溶液中固定24h以上,最后对肿瘤组织切片并进行HE染色。
本发明制备抗癌环脂肽,具有良好的生物医学性能:
(1)本抗癌环脂肽具有优异的体外抗癌活性
通过MTT法检测抗癌环脂肽的体外抗癌活性。由图1所示,随着环脂肽浓度的增加,HepG2细胞的相对细胞存活率逐渐降低,且在200μg/mL时相对细胞存活率为49.1%,说明环脂肽具有较强的体外抗癌效果,且对癌细胞的杀伤具有浓度依赖性。
(2)本抗癌环脂肽具有强效的穿膜能力
PI是一种对DNA染色的细胞核染色试剂,其在嵌入双链DNA后可以释放红色荧光,不能穿透活细胞膜进行染色,但是能穿过破碎的细胞膜从而对核进行染色。从图2a中可以看到,红色荧光非常少,表明在5min时,HepG2细胞膜仍然较为完整。从图2c中可以看到,红色荧光较图2a有了明显增加,表明在30min时,抗癌环脂肽已经能够发挥穿膜作用,HepG2细胞膜因此发生了变化,其通透性显著增加。荧光染料Hoechst 33342本身能少许地进入正常的细胞膜,使其染上低蓝色,所以从图2b可以观察到在5min时,因为Hoechst 33342的进入,已经出现蓝色荧光。从图2d中可以观察到蓝色荧光增强,这是因为凋亡细胞的膜通透性增强,因此进入凋亡细胞中的Hoechst 33342比正常细胞的多,荧光强度要比正常细胞中要高。此外,凋亡细胞的染色体DNA的结构发生了改变从而使该染料能更有效地与DNA结合,并且凋亡细胞膜上的p-糖蛋白泵功能受到损伤,不能有效地将Hoechst 33342排出到细胞外,使之在细胞内积累,蓝色荧光增强。
(3)本抗癌环脂肽具有较高的血清稳定性和较低的溶血毒性
通过抗癌环脂肽对小鼠红细胞形态的影响来评估抗癌环脂肽的溶血活性。如图3a-b所示,生理盐水组与抗癌环脂肽组小鼠红细胞形态变化不大,均呈正常的双凹盘形状,因此本抗癌环脂肽溶血毒性较小,具备较高的生物安全性。
很大一部分抗癌肽之所以不能很好地发挥体内抗肿瘤作用,是因为其在体内的生物半衰期很短,生物稳定性较低,体内的蛋白酶很容易就能将其水解失活。胚胎牛血清(FBS)是一种人们在牛胎中采集到的血液成分,其中含有多种蛋白水解酶,多肽会因为蛋白酶的水解而减弱了对肿瘤的抑制活性。因此,首先分别通过PBS和含有10%胎牛血清(FBS)的PBS溶液预先处理环脂肽,通过MTT法检测环脂肽的抗癌活性是否受到影响。图3c为将抗癌环脂肽单独以及和血清共培养24h后对HepG2细胞的杀伤能力对比,可以看到经血清共培养后的环脂肽对癌细胞的杀伤能力略有降低,但仍然具有很强的杀伤能力,表明环脂肽具有良好的血清稳定性。
(4)本抗癌环脂肽具有良好的体内抗肿瘤效果
为了研究该环脂肽的体内抗肿瘤活性,建立了荷瘤小鼠模型,在小鼠左侧腋下皮下注射大约1×106个H22细胞,待肿瘤体积长到150-220mm3时,开始注射环脂肽药物,并测量肿瘤体积和称重。实验结束后通过颈椎脱臼的方法处死小鼠,并将小鼠的肿瘤组织进行解剖,再经4%多聚甲醛固定后进行组织切片和HE染色处理。如图4a所示,抗癌环脂肽组的小鼠相对肿瘤体积明显小于生理盐水组的小鼠相对肿瘤体积。如图4b所示,抗癌环脂肽组荷瘤小鼠的体重随着给药次数的增加变化不大,说明抗癌环脂肽毒副作用较小,可以在不影响小鼠体重的情况下发挥抗肿瘤作用。为了能够更加直接地了解环脂肽的抗癌效果,对肿瘤组织进行了HE染色并置于显微镜下观察。如图4c-d所示,与NS组(图4c)肿瘤组织相比,抗癌环脂肽组(图4d)肿瘤细胞密度有了明显降低,可以看出本抗癌环脂肽具有良好的体内抗肿瘤效果。
以上所述仅为本发明的优选实例,并不用于限制于上述事实。凡在本发明基础上所作出的非本质的改动、修改、替换等,均应包含在本发明的保护范围内。
Claims (3)
1.一种抗癌环脂肽,其特征在于,其从C端到N端的序列为C8H15O-c[Gly-Phe-Tyr-Ala-Pro],其中Gly为甘氨酸,Phe为苯丙氨酸,Tyr为酪氨酸,Ala为丙氨酸,Pro为脯氨酸,C8H15O表示正辛酸,抗癌环肽结构式如下:
2.抗癌环脂肽C8H15O-c[Gly-Phe-Tyr-Ala-Pro]的制备方法,其特征在于,包括以下步骤:(1)Gly-Phe-Tyr-Ala-Pro-NH2的合成;
通过固相合成法合成了Gly-Phe-Tyr-Ala-Pro-NH2样品;使用DMF对Wang resin-Gly-Fmoc进行溶胀,溶胀30min;取少量树脂进行茚三酮检测,若没有颜色变化,则加入含有20%哌啶的DMF溶液反应30min脱去Fmoc保护基;依次用DMF、DCM、DMF对树脂进行冲洗,冲洗完成后再用无水DMF对树脂进行溶胀,时间为30min;溶胀完成后,加入2倍当量的氨基酸和2.6倍当量的DCC、HOBT、DIEA进行反应,反应时间为48h以上;透析完成后,取少量树脂进行茚三酮检测,若没有颜色变化,则说明相应的氨基酸已被连接;然后,依次用DMF、DCM、DMF对树脂进行冲洗,用透析袋(MW8000-14000)置于95%乙醇中进行透析,透析20次以上,每次透析时间为30min;重复上述步骤,直至合成Wang resin-Gly-Phe-Tyr(OtBu)-Ala-Pro-Fmoc样品;将样品进行冷冻干燥,加入切割液(TFA:H2O:Tis=95:2.5:2.5)切割掉侧链保护基团和树脂,得到目标直链五肽Gly-Phe-Tyr-Ala-Pro-NH2。(2)c[Gly-Phe-Tyr-Ala-Pro]的合成;
首先将Gly-Phe-Tyr-Ala-Pro-NH2进行冻干处理;将Gly-Phe-Tyr-Ala-Pro-NH2悬浮在二氯乙烷和乙腈的1∶1混合溶液中(DCE∶MeCN,200mL)将其进行超声处理;将丙醛(0.075mmol)加入悬浮液中,然后加入(N-异氰酸氨基)三苯基磷烷(0.05mmol),在50℃搅拌24小时;反应结束后进行旋蒸浓缩、冷冻干燥得粗品环肽,然后进行中性氧化铝柱纯化操作,流动相为无水乙醇:水(2:1),最终得到c[Gly-Phe-Tyr-Ala-Pro]。
(3)C8H15O-c[Gly-Phe-Tyr-Ala-Pro]的合成;
首先将c[Gly-Phe-Tyr-Ala-Pro]进行冻干处理;将2倍当量的DMAP、EDC、DIPEA、正辛酸放在无水DCM中活化2h,再加入冻干后的c[Gly-Phe-Tyr-Ala-Pro]反应48h;反应完成后,将粗品C8H15O-c[Gly-Phe-Tyr-Ala-Pro]装于透析袋内(MW500),进行透析,透析20次以上,每次透析时间为30min;然后进行中性氧化铝柱纯化操作,流动相为石油醚:乙酸乙酯(1:1),最终得到目标环脂肽C8H15O-c[Gly-Phe-Tyr-Ala-Pro]。
3.权利要求1所述抗癌环脂肽C8H15O-c[Gly-Phe-Tyr-Ala-Pro]在制备抗肿瘤药物中的应用。
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