CN116421741A - 一种工程化血小板及其制备方法和应用 - Google Patents
一种工程化血小板及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种工程化血小板及其制备方法和应用。所述工程化血小板通过将经过含有***素的缓冲液冲洗后的血小板与磷脂‑聚乙二醇‑配体混合孵育而获得的,其中磷脂‑聚乙二醇‑配体通过磷脂嵌插锚定在血小板的细胞膜上。所述工程化血小板通过对肿瘤和损伤部位的归巢能力以及葡萄糖和葡萄糖转运蛋白介导的亲和作用,实现对肿瘤的精准识别靶向和药物的精准递送,进而对肿瘤的未来治疗具有重要意义。
Description
技术领域
本发明涉及医用生物材料技术领域,特别涉及一种工程化血小板及其制备方法和应用。
背景技术
恶性肿瘤严重威胁到了人们的生命健康,给患者和社会都带来了沉重的压力和负担。
目前临床上的肿瘤治疗方法有很多,传统的肿瘤治疗方式有手术、化学疗法、放射疗法,这三种方法仍然是实体瘤最常见的治疗选择。随着医疗技术的不断提升,免疫治疗、靶向治疗、热疗、干细胞移植等技术也逐渐应用于临床,这些治疗方式虽然可以达到抑制肿瘤成长的效果,但是都存在着一定的局限。
化疗是肿瘤治疗的重要方法之一,在很多方面有着不可替代的作用。传统化疗的局限性推动了药物递送***的发展,极大改善了抗肿瘤药物的递送效率和疗效。细胞药物是一种基于活细胞的药物递送***,可以有效防止药物泄露,避免被机体清除,延长体内循环时间,跨越生物屏障,精准靶向聚集在病变部位,降低药物在非靶向部位的蓄积,具有更优良的生物安全性以及更低的免疫原性。
血小板是一种无核血细胞,参与肿瘤的生长和转移,并对肿瘤微环境具有归巢效应,可自发富集在肿瘤部位。正常生理状态下,循环血小板处于平衡稳态,但当血管内皮出现损伤,或肿瘤微环境刺激可被迅速激活,经历形态和功能的系列变化。由于血小板独特的生物学功能,其已被应用于肿瘤药物载体的开发。
葡萄糖、透明质酸、叶酸等在多种肿瘤细胞表面的受体(如葡萄糖转运蛋白GLUTs、CD44、叶酸受体等)表达量高于正常细胞,以满足快速增殖的肿瘤细胞对能量摄入、迁移和信号传导等生理需求,因此,这些受体成为肿瘤药物靶向递送的理想靶点。
发明内容
本发明提供了一种工程化血小板,通过将“磷脂-聚乙二醇-配体”嵌***血小板细胞膜,利用血小板的肿瘤组织归巢效应和肿瘤细胞表面受体介导的亲和反应,实现携带药物对肿瘤细胞的精准靶向递送,提高抗肿瘤疗效。
为了实现本发明的目的,拟采用以下技术方案:
本发明提供了一种工程化血小板,包括血小板和磷脂-聚乙二醇-配体;所述磷脂-聚乙二醇-配体通过磷脂嵌插锚定在血小板的细胞膜上。
进一步的,所述磷脂-聚乙二醇-配体中的磷脂为二硬脂酰磷脂酰乙醇胺、二月桂酰磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、二肉豆蔻酰磷脂酰乙醇胺、二棕榈酰磷脂酰乙醇胺、二亚油酰磷脂酰乙醇胺、二亚麻酰磷脂酰乙醇胺中的至少一种。
进一步的,所述磷脂-聚乙二醇-配体中的配体为葡萄糖、透明质酸、叶酸中的至少一种。
优选的,所述磷脂-聚乙二醇-配体为二硬脂酰磷脂酰乙醇胺-聚乙二醇-葡萄糖。
本发明还提供了所述的工程化血小板的制备方法,其特征在于,包括以下步骤:
(1)取血液离心取血小板沉淀,加入含有***素的缓冲液中冲洗,得到血小板;
(2)将步骤(1)的血小板与磷脂-聚乙二醇-配体混合孵育,离心后得到细胞膜中嵌插磷脂-聚乙二醇-配体的血小板;
(3)使用含有***素的缓冲液冲洗步骤(2)中的血小板,得到工程化血小板。
进一步的,所述步骤(1)和(3)中缓冲液中的***素浓度为1μM-2μM,其中还含有2mM-4mM的EDTA。
进一步的,所述步骤(2)中血小板与磷脂-聚乙二醇-配体的添加比例为:1×107-108个血小板加入1mg磷脂-聚乙二醇-配体。
进一步的,所述步骤(2)中的孵育温度为22℃~37℃,时间为0.5h~2h。
本发明还提供了所述的工程化血小板在用于制备抗肿瘤药物中的应用。
进一步的,将所述工程化血小板作为运送体系负载药物,进而靶向贴附于肿瘤细胞表面和/或聚集于出血损伤部位,达到精准靶向递药,实现抗肿瘤的作用。
进一步的,所述工程化血小板与药物的混合比例为:3×108-109个工程化血小板中加入0.01mmol-0.5mmol药物。
与现有的技术相比,本发明的有益效果和优点是:
(1)本发明制备的工程化血小板是将具有生物活性的血小板作为药物载体,通过简单温和的方法制备,工程化血小板仍保留静息状态,保留了血小板结构和功能完整性。
(2)本发明制备的工程化血小板通过对肿瘤和损伤部位的归巢能力以及葡萄糖和葡萄糖转运蛋白介导的亲和作用,实现对肿瘤的精准识别靶向和药物的精准递送,有利于肿瘤药物的开发和利用。
附图说明
图1为实施例1中DPG-PL@DOX的扫描电镜图。
图2为实施例1中不同DOX初始浓度下PL和DPG-PL的药物装载量。
图3为本发明实施例1中不同DOX初始浓度下PL和DPG-PL的药物载药率。
图4为实施例1中载DOX的DPG-PL和凝血酶激活的血小板的药物累积释放量图。
图5为实施例1中不同血小板的总蛋白质含量定量图。
图6为实施例1中不同血小板的LDH释放量。
图7为实施例1中不同血小板细胞膜表面CD62P表达量。
图8为实施例1中血小板及工程化血小板对不同肿瘤细胞吸附能力评价的激光共聚焦荧光显微观察图。
图9为实施例2中大鼠出血点荧光成像图。
图10为实施例2中B16F10荷瘤小鼠出血点荧光成像图。
图11为实施例2中第11天收集的肿瘤照片图。
图12为实施例2中不同治疗组小鼠的存活率图。
具体实施方式
为了进一步理解本发明,下面将结合本发明实施例,对发明实施例中的技术方案进行描述。如无特殊说明,本发明实施例中所涉及的试剂均为市售产品。
实施例1
1、载阿霉素工程化血小板的制备
称取100~300mg DSPE-PEG-COOH,溶于4~10mL超纯水中,然后加入200~400mgN-N’环己基碳二亚胺(EDC)活化羧基,室温搅拌20~60min,加入100~300mg N-羟基琥珀酰亚胺(NHS),室温搅拌2~5h后,加入40~100mg氨基葡萄糖,室温搅拌18~36h,将反应溶液充分透析,冻干得DSPE-PEG-Glu固体粉末。
在室温下将健康个体的新鲜全血利用枸橼酸钠抗凝后,以200g离心15min,用移液枪小心吸取上层浅黄色富血小板血浆(PRP),然后以800g离心10min,得到血小板沉淀。弃上清后,将血小板重悬于含有1μM***素E1(PGE1)和2mM EDTA的PBS缓冲液中以防止血小板的激活,并用PBS(1μM PGE1,2mM EDTA)冲洗两遍得到洗涤血小板,并对提取出的血小板进行计数,调整血小板浓度,在5mL(2×107个/mL)血小板中加入5mg二硬脂酰磷脂酰乙醇胺-聚乙二醇-葡萄糖(DSPE-PEG-Glu)。在37℃,100rpm条件下振荡孵育1h,然后800g离心10min,去除游离的DSPE-PEG-Glu,得到细胞沉淀。用含1μMPGE1和2mM EDTA的PBS缓冲液重悬细胞沉淀,得到DSPE-PEG-Glu修饰的血小板(DPG-PL)悬液。将阿霉素溶液(DOX,50μM)与血小板悬液(3×109个/mL)混合,轻轻吹打至混合均匀。将混合物在37℃避光条件下100rpm振荡反应1小时,然后经800g离心去除未负载的药物,重悬后得到双靶向载阿霉素血小板(DPG-PL@DOX)悬液。
2、多项检测
(1)将50μM的DOX分别与血小板悬液(3×109个/mL)混合,轻轻吹打至混合均匀,将混合物在37℃避光条件下100rpm振荡反应1小时,然后经800g离心10min取沉淀,使用含1μM***素E1(PGE1)和2mM EDTA的PBS缓冲液重悬,得DPG-PL@DOX悬液,将滴加至细胞爬片上,静置30min,用2.5%的戊二醛溶液固定30min。将固定后的样品放置于乙醇中进行超临界干燥,然后对样品进行喷金处理,通过扫描电子显微镜进行观察。
观察结果如图1所示,工程化血小板呈椭圆形或圆盘形,结构完整,未出现放射状的突起,处于静息状态。
(2)体积为200μL,将不同浓度的DOX分别与血小板悬液(3×109个/mL)混合,其中DOX的起始浓度为10,20,30,40和50μM,轻轻吹打至混合均匀。将混合物在37℃避光条件下100rpm振荡反应1小时,然后经800g离心10min去除未负载的药物,收集上清,DOX的浓度通过酶标仪检测480nm处吸光值进行定量。药物的负载效率通过药物的负载量与加入药物总量的比值来进行计算。
结果如图2和3所示,随着DOX浓度的增加,血小板对DOX的装载量和载药量都逐渐增加,且在DOX浓度为50μM时,DPG-PL对DOX的装载量可达4.62μΜ/108个血小板,对DOX的载药率可达28%。
(3)采用动态透析法研究包载DOX血小板的体外释放行为。分别在pH 5.5和7.4的条件下测定药物的释放效率。将5mL载药血小板体系装入透析袋中,将透析袋置于20mL PBS缓冲液中。以加入凝血酶(2U/mL)激活的血小板作为对照组。各组在37℃避光条件下100rpm振动。分别在1、2、4、8、16和24h从容器中取出1mL溶液,并且补加等量新鲜PBS。通过酶标仪在480nm处测定药物浓度,并根据药物释放比绘制药物在血小板中的累积释放量。
结果如图4所示,在模拟肿瘤微环境pH=5.5时,DOX能够快速从DPG-PL@DOX中释放。
(4)将具有相同血小板浓度的工程化血小板DPG-PL和载药工程化血小板DPG-PL@DOX,超声破碎,凝血酶处理血小板组(PL+Thrombin)为阳性对照,天然血小板(PL)为阴性对照,离心收集上清,用BCA蛋白浓度试剂盒测定各组血小板的总蛋白浓度。
如图5所示,DPG-PL和DPG-PL@DOX组与空白血小板(PL)总蛋白浓度类似,DPG的锚定和DOX的负载不会导致血小板激活及损伤。
(5)将相同血小板浓度的PL、DPG-PL和DPG-PL@DOX,以凝血酶激活的血小板(PL+Thrombin)为对照,加入提取液后超声破碎各组血小板,用血小板乳酸脱氢酶(LDH)活性检测试剂盒检测各组血小板中LDH的含量。
如图6所示,DPG-PL和DPG-PL@DOX组与空白血小板(PL)没有明显LDH释放,表明血小板细胞膜完整,而被凝血酶激活的血小板(PL+Thrombin)细胞膜被破坏,导致LDH释放。
(6)将PL、DPG-PL、DPG-PL@DOX和加入凝血酶激活的血小板(PL+Thrombin)以106个/mL的浓度与5μL PE标记的CD62P(P-selectin)抗体避光孵育30min,800g离心洗涤三次后,使用流式细胞仪检测荧光强度。
结果如图7所示,CD62P是储存在静息血小板α颗粒中的一种糖蛋白,随着血小板的激活转移到质膜上,是血小板激活的标志蛋白。DPG-PL和DPG-PL@DOX组与空白血小板(PL)表面的CD62P表达没有显著变化,但被凝血酶激活的血小板(PL+Thrombin)的CD62P表达显著增强。
(7)将提取的血小板用PBS稀释,以1×107个/mL的浓度转移至无菌离心管中,加入经0.22μm无菌滤膜过滤后的DSPE-PEG-Glu溶液,37℃、100rpm条件下振荡孵育1h,800g离心10min,去除游离的DSPE-PEG-Glu,得DPG-PL悬液,分别向等浓度的PL和DPG-PL悬液加入DiI(0.01mg/mL),37℃振荡孵育15min,PBS洗涤三次,用培养基重悬血小板沉淀;取对数生长期的L929、4T1、B16F10和HepG-2,制备细胞悬液,分别用含10%胎牛血清的培养基稀释,以每孔1×105个/mL的密度接入12孔细胞培养板中,培养12h,而后弃去培养基,在L929、4T1、B16F10和HepG-2细胞培养孔中分别加入培养基重悬的PL和DPG-PL悬液,培养4h,使用含EDTA的胰酶将细胞消化,200g离心后用培养基重悬,待细胞贴壁后,用4%的多聚甲醛进行固定,并用DAPI染色,PBS冲洗三次后在激光共聚焦显微镜下观察。
如图8所示,对比正常胎鼠成纤维细胞L929,DPG-PL@DOX对细胞表面过表达GLUT1的乳腺癌细胞4T1、黑色素瘤细胞B16F10或肝癌细胞HepG-2均表现出显著的靶向黏附作用。
实施例2:工程血小板用于肿瘤治疗功效评价
选用实施例1制备的载阿霉素工程化血小板DPG-PL@DOX,将其通过尾静脉注射至小鼠体内(DPG-PL@DOX组);尾静脉注射生理盐水作为对照组;注射游离DOX的组别为DOX组;注射未修饰血小板载阿霉素的组别为PL@DOX组,治疗后,记录小鼠生存曲线,并测量小鼠肿瘤体积大小。
选用实施例1制备的DPG-PL@DOX(2×109个/mL),采用NHS-Cy5.5标记,而后通过尾静脉注射至大鼠体内,然后分别在大鼠的眼眶、前爪、后爪以及股动脉部位制造出血伤口,小动物活体成像仪观察。
结果如图9所示,荧光染料Cy5.5标记的DPG-PL@DOX经尾静脉注射后,聚集在小鼠眼眶、前爪、后爪以及股动脉的出血处。
将B16F10细胞(1×106)移植到C57BL/6小鼠的右侧腹股沟外侧,建立小鼠黑色素肿瘤模型,通过尾静脉分别将生理盐水、NHS-Cy5.5标记的PL和DPG-PL@DOX(200μL,2×109个/mL)注射入荷瘤小鼠体内,并在肿瘤部位制造出血点,小动物活体成像仪观察。
如图10所示,构建小鼠肿瘤出血模型,对比肿瘤非出血模型,DPG-PL@DOX在肿瘤出血部位的荧光强度更强。
构建荷瘤小鼠,将小鼠随机分为四组,每组8只,通过尾静脉分别注射生理盐水、DOX、PL@DOX和DPG-PL@DOX,其中DOX的注射剂量为1mg/kg,分别于第5、7和9天进行给药,共给药治疗三次,第11天治疗结束,每组5只小鼠通过颈椎脱臼法处死,取出肿瘤,拍照观察肿瘤尺寸,每组剩余3只监测存活率。
如图11所示,经过治疗后,各治疗组小鼠的肿瘤明显减小,DPG-PL@DOX组相比于PL@DOX组和DOX组,肿瘤尺寸最小。
如图12所示,各治疗组治疗后,小鼠的生存时间明显延长,DPG-PL@DOX治疗组小鼠生存时间最长。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (10)
1.一种工程化血小板,其特征在于,所述工程化血小板包括血小板和磷脂-聚乙二醇-配体;所述磷脂-聚乙二醇-配体通过磷脂嵌插锚定在血小板的细胞膜上。
2.根据权利要求1所述的工程化血小板,其特征在于,所述磷脂-聚乙二醇-配体中的磷脂为二硬脂酰磷脂酰乙醇胺、二月桂酰磷脂酰乙醇胺、二油酰磷脂酰乙醇胺、二肉豆蔻酰磷脂酰乙醇胺、二棕榈酰磷脂酰乙醇胺、二亚油酰磷脂酰乙醇胺、二亚麻酰磷脂酰乙醇胺中的至少一种。
3.根据权利要求1所述的工程化血小板,其特征在于,所述磷脂-聚乙二醇-配体中的配体为葡萄糖、透明质酸、叶酸中的至少一种。
4.权利要求1-3任一项所述的工程化血小板的制备方法,其特征在于,包括以下步骤:
(1)取血液离心取血小板沉淀,加入含有***素的缓冲液中冲洗,得到血小板;
(2)将步骤(1)的血小板与磷脂-聚乙二醇-配体混合孵育,离心后得到细胞膜中嵌插磷脂-聚乙二醇-配体的血小板;
(3)使用含有***素的缓冲液冲洗步骤(2)中的血小板,得到工程化血小板。
5.根据权利要求4所述的制备方法,其特征在于,所述步骤(1)和(3)中缓冲液中的***素浓度为1μM-2μM,其中还含有2mM-4mM的EDTA。
6.根据权利要求4所述的制备方法,其特征在于,所述步骤(2)中血小板与磷脂-聚乙二醇-配体的添加比例为:1×107-108个血小板加入1mg磷脂-聚乙二醇-配体。
7.根据权利要求4所述的制备方法,其特征在于,所述步骤(2)中的孵育温度为22℃~37℃,时间为0.5h~2h。
8.权利要求1-3任一项所述的工程化血小板在用于制备抗肿瘤药物中的应用。
9.根据权利要求8所述的应用,其特征在于,将所述工程化血小板作为运送体系负载药物,进而靶向贴附于肿瘤细胞表面和/或聚集于出血损伤部位,达到精准靶向递药,实现抗肿瘤的作用。
10.根据权利要求9所述的应用,其特征在于,所述工程化血小板与药物的混合比例为:3×108-109个工程化血小板中加入0.01mmol-0.5mmol药物。
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