CN110343163A - 一种与cxcr1特异性结合的肿瘤抑制肽及其用途 - Google Patents

一种与cxcr1特异性结合的肿瘤抑制肽及其用途 Download PDF

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CN110343163A
CN110343163A CN201910689947.0A CN201910689947A CN110343163A CN 110343163 A CN110343163 A CN 110343163A CN 201910689947 A CN201910689947 A CN 201910689947A CN 110343163 A CN110343163 A CN 110343163A
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cxcr1
cis
tumor suppression
suppression peptide
delivery system
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汤亭亭
韩修国
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

本发明公开了一种能与CXCR1特异性结合的肿瘤抑制肽及其用途，同时公开了一种含有该多肽的药物输送系统。该多肽可以与CXCR1直接结合并抑制CXCR1下游信号通路的激活，增强肿瘤对化疗药的敏感性，其能够单独或与其它抗肿瘤药物联合使用用于制备治疗抗肿瘤的药物。含有该多肽的药物输送系统可以通过精准靶向循环和原位的CSCs来抑制骨肉瘤的生长和转移，提高化疗敏感性。这对开发有效控制肿瘤复发和转移的方法、降低化疗药物带来的全身不良反应具有重要的作用。因此，本发明所述的抗肿瘤多肽具有要的理论和应用价值。

Description

一种与CXCR1特异性结合的肿瘤抑制肽及其用途
技术领域
本发明属于生物医学技术领域，具体涉及一种与CXCR1特异性结合的肿瘤抑制肽及其用途。
背景技术
CXCR1是IL-8的特异性结合受体，属于CXCR类趋化因子受体。研究发现CXCR1在促进包括骨肉瘤、乳腺癌、前列腺癌、肺癌、肠癌以及黑色素瘤等肿瘤的转移和化疗抵抗以及维持肿瘤干细胞特性中起到了重要作用，并且越来越多的研究证实下调CXCR1的基因表达能够明显抑制肿瘤的恶性生物学特性。因此，CXCR1可以被认为是肿瘤发生的原癌基因。
目前，对CXCR1主要通过各种抑制剂(如Reparixin)和基因工程技术来阻断CXCR1及其下游信号通路发挥作用。申请人的前期研究中证明CXCR1的基因抑制能够增强骨肉瘤对顺铂的化疗敏感性、促进骨肉瘤的失巢凋亡。但是，根据蛋白的空间结构设计多肽，从而在三维结构上抑制蛋白的功能是目前研究的热点，并且也有多个产品已经上市。基于现有技术的需求，寻求能够与CXCR1结合的抑制肽是肿瘤领域迫切的需求。
在肿瘤研究中，对CXCR1的基因抑制大多数通过基因工程相关的技术对肿瘤细胞进行基因沉默，这与临床应用还相差太远；其次，Reparixin是CXCR1/2的非竞争性变构抑制剂，作用于人体广泛的体细胞，在体内应用有不可估量的毒副作用。以上存在的问题主要有两个原因，一个是现有的基础研究实现临床转化难度较大，可能需要利用多学科的交叉融合；其次，一般的抑制剂都不具有细胞特异性，往往在实现疗效的同时会对机体产生较大的毒副作用。
发明内容
为了解决以上现有技术存在的问题，本发明的目的在于提供一种具有抗肿瘤活性的CXCR1靶向多肽及其应用，该抑制肽及其修饰产物对肿瘤化疗抵抗具有较好的抑制效果。并且本申请将以骨肉瘤为研究对象，在细胞和动物水平验证CXCR1靶向多肽的作用。
为了实现上述目的，本发明提供以下技术方案：
一种与CXCR1特异性结合的肿瘤抑制肽，所述的肿瘤抑制肽的氨基酸序列如SEQID NO：1所示。
进一步的，本申请所述的肿瘤抑制肽在制备药物输送系统中的用途。
进一步的，本申请所述的一种药物输送系统中，该系统包括本申请所述的肿瘤抑制肽。
进一步的，本申请所述的一种药物输送系统中，所述的药物输送系统的制备方法包括如下步骤：
(1)采用溶剂热法合成磁性Fe3O4纳米颗粒；
(2)利用Stober法制备磁性纳米二氧化硅介孔颗粒MF；并将荧光物质修饰到MF的表面得到荧光修饰的MSNs@Fe3O4；
(3)将顺铂Cis(Cisplatin)负载至步骤(2)制得的荧光MF表面得到Cis@MF，并计算载药量；
(4)采用共聚法制备氨基修饰的磁性介孔二氧化硅纳米粒Cis@MF-NH2；将羧基-PEG-氨基与本申请所述的肿瘤抑制肽进行偶联，然后再将其与Cis@MF-NH2进行接枝，得到CXCR1靶向药物纳米颗粒；
(5)通过电荷吸附将聚丙烯酸(PAA)负载到步骤(4)制得的CXCR1靶向药物纳米颗粒表面，得到靶向CXCR1的pH响应性药物输送系统。
进一步的，本申请所述的肿瘤抑制肽在制备抑制CXCR1活性的药物中的用途。
进一步的，本申请所述的肿瘤抑制肽在制备抑制癌症的药物中的用途。
一种药物制剂，所述药物制剂包含本申请所述的肿瘤抑制肽。
有益效果：本发明提供了一种与CXCR1特异性结合的肿瘤抑制肽及其用途，将该肿瘤抑制肽修饰在纳米颗粒表面制备载药纳米颗粒，并分析该纳米颗粒对骨肉瘤细胞的作用。通过测试结果得出，本发明所述的CXCR1靶向多肽能够与IL-8竞争性结合CXCR1，同时还能抑制骨肉瘤CSCs的“干细胞样”特性。通过将该CXCR1靶向多肽接枝于纳米递药系统表面，以Fe3O4磁性纳米颗粒为核(MRI显像)、荧光标记MSNs为外壳(光学成像)并负载化疗药物、PAA(pH响应性释放)/PEG表面修饰的纳米给药体系，通过精准靶向循环和原位的CSCs来抑制骨肉瘤的生长和转移，提高化疗敏感性。这对开发有效控制肿瘤复发和转移的方法、降低化疗药物带来的全身不良反应具有重要的作用。因此，本发明所述的抗肿瘤多肽具有要的理论和应用价值。
附图说明
图1为CXCR1靶向多肽与CXCR1结合的分子模拟图。
图2为本发明所述pH响应性药物输送系统的合成流程图。
图3为CXCR1靶向多肽与CXCR1结合及对骨肉瘤CSCs的抑制作用结果图。A.CXCR1靶向多肽对IL-8和CXCR1相互作用的免疫共沉淀分析图；B.表面等离子共振对CXCR1靶向多肽和CXCR1的结合力测定图；C.CXCR1靶向多肽对CSCs成球能力的影响图；D.CXCR1靶向多肽对CSCs克隆形成能力的影响图；E-F.CXCR1靶向多肽对CSCs失巢凋亡的影响图。**表示P<0.01。*表示P<0.05，所有数据结果均以均数±标准差表示，并且均来源于三次独立实验。
图4为靶向CXCR1的pH响应性药物控释纳米颗粒的表征图。A.纳米颗粒的SEM和TEM图；B.热失重实验结果图；C.氮吸附实验结果图；D.孔径检测结果图；E-F.纳米颗粒pH响应性释放药物曲线图。
图5为靶向CXCR1的pH响应性药物控释纳米颗粒在体内的靶向抑制作用图。A.CSCs和纳米颗粒的体内实时显像图；B-C.体内纳米颗粒对CSCs在肺中存活的实时监测图；D.纳米颗粒治疗4周后原位肿瘤取材后大体照片及肺转移HE染色图；E.肿瘤大小统计图；F.肺转移率统计图。***表示P<0.001，**表示P<0.01所有数据结果均以均数±标准差表示。
具体实施方式
下面结合具体实施例来进一步描述本发明，但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
下面结合具体的实施例对本发明做进一步地说明，以更好地理解本发明。
一、本申请所述的肿瘤抑制肽的设计思路申请人发现IL-8与CXCR1的结合是分两步完成的。首先，IL-8的N端存在一个环状的Loop(N-loop)序列，IL-8的N-loop与CXCR1受体的N端结合。这种最初的结合诱导了IL-8发生了结构的动态变化，尤其是30s N-loop序列和N-端残基，使得IL-8发生空间构象的改变，这种改变可以使IL-8的N-loop序列的ELR氨基酸残基准确的与CXCR1受体N端结合。随后CXCR1受体发生构象变化，导致下游信号的激活。所以，IL-8能够与CXCR1结合主要是通过N-Loop序列，而随后的空间构象的变化是激活CXCR1信号通路的关键步骤。申请人进一步对N-Loop全长序列中与CXCR1特异性结合的氨基酸进行了分析。综合文献报道，申请人选取了N-loop的序列中N端的第1到第20位。此段氨基酸包含能与CXCR1特异性结合的E4L5R6(谷氨酸4-亮氨酸5-精氨酸6)序列、1-15位的丝氨酸-丙氨酸-赖氨酸-谷氨酸-亮氨酸-精氨酸-半胱氨酸-谷氨酰胺-异亮氨酸-赖氨酸-苏氨酸-酪氨酸-丝氨酸-赖氨酸(SAKELRCQCIKTYSK)序列以及K15、H18、K20(15位赖氨酸，18位组氨酸，20位赖氨酸)三个氨基酸。因此，需要合成和研究的多肽序列全长如下：丝氨酸-丙氨酸-赖氨酸-谷氨酸-亮氨酸-精氨酸-半胱氨酸-谷氨酰胺-异亮氨酸-赖氨酸-苏氨酸-酪氨酸-丝氨酸-赖氨酸-脯氨酸-苯丙氨酸-组氨酸-脯氨酸-赖氨酸(SAKELRCQCIKTYSKPFHPK)。如图1所示，分子模拟结果显示该多肽序列能够特异性结合CXCR1。
二、靶向CXCR1的pH响应性药物输送系统的制备以下所述制备过程如图2所示：
1.磁性Fe3O4纳米颗粒的合成：根据文献采用溶剂热法合成Fe3O4纳米微球。利用强磁铁收集产物分别用无水乙醇和去离子水洗涤三次后，置于40℃干燥箱中干燥24h。
2.磁性纳米二氧化硅介孔颗粒(MSNs@Fe3O4，MF)的合成：根据文献采用Stober法制备二氧化硅纳米颗粒包裹磁性Fe3O4纳米颗粒，60℃真空干燥得最终制得MSNs@Fe3O4(MF)样品。
3.Cy7-MF的合成：根据文献通过EDC/NHS偶联法合成用于生物显像的荧光纳米粒Cy7-MF。纳米粒离心分离后，用超纯水离心洗涤3次，冷冻干燥即得Cy7-MF产物。
4.顺铂(Cis)负载至MF：称取顺铂100mg，置于10mL容量瓶中，用PBS(pH 7.4)溶液溶解并定容至25mg/mL。取上述顺铂溶液4mL，置于西林瓶中，加入10mg冻干纳米粒(Cy7-MF)，常温下搅拌24h。离心去除未负载的游离药物，PBS洗涤两遍，冻干。本步骤的中间产物为Cis@MF。
5.氨基化表面修饰Cis@MF：通过共聚法制备氨基修饰的磁性介孔二氧化硅纳米粒(Cis@MF-NH2)。取100mg的Cis@MF纳米颗粒分散在10mL超纯水中，加入0.4mL二乙烯三胺基丙基三甲氧基硅烷(AEPTMS)。反应于40℃搅拌24h后r/min离心收集产物。反应产物反复多次离心洗涤后，冷冻干燥即得Cis@MF-NH2产物。
6.CXCR1靶向多肽改性：利用1-乙基-3-(3-二甲基氨丙基)碳二亚胺(EDC)活化法将CXCR1靶向肽与羧基-PEG-氨基结合。在EDC存在的情况下，将羧基-PEG-氨基中的氨基与CXCR1靶向肽中的谷氨酸的羧基接枝。
7.CXCR1靶向药物纳米颗粒(Cisplatin@MSNs@Fe3O4-PEG-CXCR1 targetingpeptide，Cis@MFPC)的合成：反应步骤同(6)，在本步骤中是将羧基-PEG-氨基中的羧基与氨基化修饰的Cis@MF进行接枝。本步骤的中间产物为Cis@MFPC。此外，我们还合成了不接靶向多肽的对照纳米颗粒，即将未经过CXCR1靶向多肽修饰的羧基-PEG-氨基中的羧基与氨基化修饰的Cis@MF接枝，得到Cis@MFP。
8.PAA负载至Cis@MFPC和Cis@MFP：PAA可以通过电荷吸附到纳米颗粒上。反应完毕后冻干，得到最终产物Cis@MFPPC和Cis@MFPP。
随后我们对合成的纳米颗粒进行表征，包括扫描电镜(SEM)、透射电镜(TEM)、氮吸附、热重分析以及药物pH响应性释放。
实施例1免疫共沉淀(IP)检测CXCR1靶向多肽与CXCR1的结合
1.细胞裂解
(1)取对数期生长的骨肉瘤CSCs，分别加入10mL含有0、0.5、1μg/mL的细胞培养基孵育24h。吸去培养皿中的培养基，PBS缓冲液洗三遍，并吸干残留液体；
(2)将培养皿置于冰上，向培养皿中加入预冷的IP裂解液，冰上裂解30min，不时摇晃；
(3)将裂解液转移至1.5mL离心管中，13000g，4℃离心10min。
(4)将上清转移至新管中。
2.免疫复合体制备
将每组蛋白裂解液分成2份，分别检测等量IL-8在CXCR1上的结合量和等量CXCR1上结合的IL-8的量。
(1)向蛋白裂解液中加入5μg IP抗体(IL-8和CXCR1抗体)；
(2)蛋白裂解液不足500μL的用IP裂解液补足至500μL；
(3)4℃摇床孵育过夜。
3.免疫沉淀
为了保证磁珠均一性，使用前上下颠倒混匀或者轻柔涡旋。
(1)向一个1.5mL离心管中加入25μLProteinA/G磁珠；
(2)向磁珠中加入175μL IP裂解液，轻柔涡旋混匀；
(3)磁力架4℃吸附2min，弃去上清；
(4)再加入1mL IP裂解液，上下颠倒或者轻柔涡旋混匀1min。磁力架4℃吸附2min。弃去上清，磁珠保留在管中；
(5)向有预清洗的磁珠的离心管中加入蛋白裂解液/抗体复合物，4℃摇床摇动4-6小时；
(6)磁力架4℃吸附2min收集抗原/抗体/磁珠复合体，弃上清；
(7)向管中加入500μL改良IP washing buffer(TBS+0.1％NP40+5％甘油，也可用TBS+5％甘油将IP裂解液稀释十倍)；
(8)磁力架4℃吸附2min，弃上清，总共洗涤珠子三次；
(9)使用低pH洗脱，洗脱前先用超纯水洗涤珠子一次，而后向磁珠中加入100μLElutionBuffer，室温震动孵育10min。磁力架吸附，转移上清至新管，加入10μL中和缓冲液中和pH。
(10)最后加入适量上样缓冲液煮沸10min。
4.WesternBlot
电泳、转膜后封闭。CXCR1或IL-8一抗孵育过夜，一抗以1:1000用TBST配制；TBST摇床洗膜，每次7min，共洗3次；荧光二抗以1:10000比例用TBST配制，室温摇床孵育1h，避光；二抗孵完后再次加TBST洗膜，每次7min，共洗3次。随后将膜放入Odyssey红外成像系统中拍照，保存图像。
随后，利用表面等离子共振来验证CXCR1靶向多肽与CXCR1的结合力。成球试验、克隆形成实验和失巢凋亡检测验证CXCR1靶向多肽对骨肉瘤CSC“干性”的影响。
实施例2纳米颗粒在体内对CSCs的靶向抑制作用检测
选取SPF级4周龄雌性裸鼠(BALB/c-nu/nu)，先用3.5％戊巴比妥钠麻醉，每只裸鼠腹腔注射100μL。待老鼠麻醉后，经腹部将肠系膜血管暴露，在保持裸鼠存活的前提下将肠管放在10cm培养皿中，放于荧光显微镜(OLYMPUS,CKX41)下调整好焦距，随后各纳米颗粒组先经尾静脉先后注射1×107个GFP标记的CSCs，随后按照前述剂量注射相应的纳米颗粒。随后用荧光显微镜观察肠系膜血管，寻找CSCs带有的GFP和荧光纳米颗粒带有的红色荧光，记录各自的位置。另一方面，选取SPF级4周龄雌性裸鼠(BALB/c-nu/nu)15只，经尾静脉注射1×107个GFP标记的CSCs，随后注射相应的纳米材料，分别在注射后0、0.5、1和2h进行活体成像，观察CSCs在体内的存活情况。
实施例3本申请所述肿瘤抑制肽的测试效果如下：
1、CXCR1靶向多肽特异性结合CXCR1以及其对骨肉瘤肿瘤干细胞(CSCs)生物学特性的影响将CXCR1靶向多肽作用于骨肉瘤CSCs后，免疫共沉淀结果显示，在加入相同量CXCR1进行共沉淀后，加入CXCR1靶向多肽能够抑制IL-8与CXCR1的结合量，并且具有浓度依赖性。在加入相同量的IL-8后进行共沉淀同样发现多肽能够浓度依赖性抑制CXCR1的免疫共沉淀量(附图3A)。
通过表面等离子共振技术检测了CXCR1靶向多肽与CXCR1的结合率为90％(附图3B)，这表明CXCR1靶向多肽与CXCR1具有很强的结合力。
对CXCR1靶向多肽对骨肉瘤CSCs生物学特性的影响进行研究，通过成球试验和克隆形成实验表明CXCR1靶向多肽能够抑制CSCs的成球能力和克隆形成能力(附图3C-D)。
失巢凋亡实验证实，在低粘附培养条件下，CXCR1靶向多肽能够促进CSCs的失巢凋亡(附图3E-F)。
综上所述，CXCR1靶向多肽能够通过结合CXCR1来抑制IL-8与CXCR1的结合，同时能够抑制CSCs的恶性生物学表型。
2、靶向CXCR1的pH响应性药物输送载体的表征结果
纳米颗粒(NPs)合成后，通过SEM和TEM对各组材料的形貌进行表征。从SEM图中可以发现，各组纳米颗粒的粒径在100nm左右，具有均一性。并且在进行修饰后，纳米颗粒的介孔在MF组中非常清晰，在逐步添加修饰后逐渐模糊，在Cis@MFPPC组中介孔已经模糊不清，但分散性未受影响。TEM结果除了能够显示SEM相同的表现之外，同时能够看到制备的MF纳米颗粒具有有序介孔孔道，负载顺铂后，Cis@MF的部分介孔孔道消失。将Cis@MFPP用CXCR1靶向多肽修饰后，孔道进一步消失，表面还有一层厚度约为5nm的透亮层包裹(附图4A)。
进一步对纳米颗粒的功能化进行了检测：
热失重实验表明MF、Cis@MF、Cis@MF-PEG-CXCR1靶向多肽(Cis@MFPC)和Cis@MFPPC四种纳米颗粒在800℃的热失重比例分别是8.2±0.4％、17.5±1.1％、21.4±1.4％以及24.8±1.7％，通过负载顺序我们可以推算出Cis、PEG修饰的靶向多肽和PAA的负载质量比分别为9.3±0.5％、3.9±0.3％和3.4±0.4％(附图4B)。
氮吸附实验表明MF、Cis@MF和Cis@MFPPC的比表面积分别为456±36m2g-1、172±24m2 g-1以及67±10m2 g-1(附图4C)。进一步检测出MF、Cis@MF和Cis@MFPPC的平均介孔孔径分别为1.75±0.16cm3 g-1、0.36±0.11cm3 g-1以及0.11±0.05cm3 g-1(附图4D)。
pH响应性释放结果提示，在pH为5.0时，Cis@MFPPC在0.5h时迅速释放，随后释放逐渐放缓，12h时逐渐平稳；在pH为6.5时，Cis@MFPPC在0.5h时也有一个释放高峰，随后释放逐渐放缓，12h时逐渐平稳，但是释放量远小于pH为5.0时；在pH为7.4时，顺铂几乎不释放(附图4E-F)。因此，合成的Cis@MFPPC具有pH响应性释放化疗药物的特性，符合实验需求。
综上所述，所合成的Cis@MFPPC成功负载了顺铂、CXCR1靶向多肽和PAA，具有pH响应性释放的特性。
3、CXCR1靶向纳米颗粒在体内对CSCs的靶向抑制作用及对原位骨肉瘤肺转移的抑制作用
采用荧光显微镜在体内血液循环中观察Cis@MFPP和Cis@MFPPC两种纳米颗粒对CSCs的靶向和抑制作用。将CSCs和对应的纳米颗粒尾静脉先后注射入裸鼠体内，荧光显微镜观察肠系膜血管的实时荧光成像。结果表明，红色荧光标记的Cis@MFPPC和Cis@MFPP纳米材料和绿色荧光标记的CSCs能够在注射后1h左右观测到(附图5A)。进一步对不同纳米颗粒注射后的CSCs存活进行了实时小动物活体成像监测，发现Cis@MFPPC明显抑制CSCs的存活，Cis@MFPP的抑制作用明显比Cis@MFPPC弱(附图5B-C)。综上所述，Cis@MFPPC纳米颗粒能够精准靶向循环中的CSCs，并且对CSCs进行有效的杀灭，进一步在体内证实了合成的Cis@MFPPC纳米颗粒的靶向治疗作用。
建立裸鼠原位骨肉瘤模型，待一周成瘤后进行4周的纳米颗粒的治疗。结果说明Cis@MFPPC具有最好的抑制肿瘤生长的效果(附图5D)，主要是肿瘤体积的减小(附图5E)。最重要的是，肺组织的病理切片结果显示，Cis@MFPPC组的肺转移被明显抑制(附图5D)，转移率明显下降(附图5F)。

Claims (7)

1.一种与CXCR1特异性结合的肿瘤抑制肽，其特征在于，所述的肿瘤抑制肽的氨基酸序列如SEQ ID NO：1所示。
2.一种如权利要求1所述的肿瘤抑制肽在制备药物输送系统中的用途。
3.一种药物输送系统，其特征在于，该系统包括权利要求1所述的肿瘤抑制肽。
4.根据权利要求3所示的药物输送系统，其特征在于，所述的药物输送系统的制备方法包括如下步骤：
(1)采用溶剂热法合成磁性Fe3O4纳米颗粒；
(2)利用Stober法制备磁性纳米二氧化硅介孔颗粒MF；并将荧光物质修饰到MF的表面得到荧光修饰的MSNs@Fe3O4；
(3)将顺铂Cis负载至步骤(2)制得的荧光MF表面得到Cis@MF，并计算载药量；
(4)采用共聚法制备氨基修饰的磁性介孔二氧化硅纳米粒Cis@MF-NH2；将羧基-PEG-氨基与权利要求1所述的肿瘤抑制肽进行偶联，然后再将其与Cis@MF-NH2进行接枝，得到CXCR1靶向药物纳米颗粒；
(5)通过电荷吸附将聚丙烯酸负载到步骤(4)制得的CXCR1靶向药物纳米颗粒表面，得到靶向CXCR1的pH响应性药物输送系统。
5.权利要求1所述的肿瘤抑制肽在制备抑制CXCR1活性的药物中的用途。
6.权利要求1所述的肿瘤抑制肽在制备抑制癌症的药物中的用途。
7.一种药物制剂，其特征在于，所述药物制剂包含权利要求1所述的肿瘤抑制肽。
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