CN114588274B - 一种负载cRGD和小分子抗肿瘤药物的复合外泌体及其制备方法和应用 - Google Patents
一种负载cRGD和小分子抗肿瘤药物的复合外泌体及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及药物制剂技术领域,具体是一种负载多肽和小分子抗肿瘤药物的复合外泌体及其制备方法和应用。本发明的复合外泌体给药系统利用人脐带间充质干细胞来源的外泌体(Exosome,EXO)作为载体,在其膜表面嵌合环状多肽cRGD,以靶向递送至高表达αvβ3整合素受体的黑色素瘤细胞,并在外泌体膜内包载小分子抗肿瘤药物,构建复合外泌体给药系统cRGD‑EXO/小分子抗肿瘤药物。体内外实验表明,该给药系统具有良好的肿瘤靶向性,在降低药物毒副作用的同时,表现出显著的抑瘤效果。本发明为αvβ3整合素受体高表达的肿瘤治疗提供了一种靶向性好、生物安全性高、抑瘤效果确切的小分子药物递送系统。
Description
技术领域
本发明涉及药物制剂技术领域,具体地说,是一种负载cRGD和小分子抗肿瘤药物的复合外泌体及其制备方法和应用。
背景技术
整合素是一类位于大多数细胞膜上的糖蛋白受体,能够介导细胞之间及细胞与细胞外基质间的黏附作用、细胞与细胞外基质间的信号传导。αvβ3整合素是24种整合素中最为重要的一员,其在肿瘤的转移、侵袭和血管的生成方面起着重要的作用。αvβ3整合素受体在活化的内皮细胞和某些肿瘤细胞(例如人恶性黑色素瘤、前列腺癌、胶质母细胞瘤和乳腺癌)上特异性表达,但在静止的内皮细胞上不特异性表达或表达很少。因此,可以利用该特征靶向于肿瘤部位或者肿瘤血管。
恶性黑色素瘤具有高度转移性,是目前最致命的皮肤恶性肿。目前,早期黑色素瘤可以通过手术进行切除,晚期黑色素瘤主要通过化疗的方法治疗。但是,黑色素瘤一旦从其起源病灶处扩散或转移到其他皮肤或皮下组织后,对治疗的反应率下降了约5-20%。此外,恶性黑色素瘤还具有耐药性,因此单一化学疗法在恶性黑色素瘤患者的治疗中效果较差,并且以全身毒性为代价。因此,寻找更有效,毒性更小的新方法对黑色素瘤的治疗具有重要意义。
雷公藤甲素(Triptolide,TPL)是从中药雷公藤(Triptygium wilfordii Hook F)根及根茎中分离的环氧二萜内酯化合物,具有抗炎、镇痛、免疫调节及抗肿瘤等作用。近年来,研究发现雷公藤甲素可用于抑制黑色素瘤、乳腺癌、肺癌、胰腺癌等肿瘤的生长。但是雷公藤甲素溶解性差,治疗窗窄,肝肾毒性强等缺点,严重限制了其临床应用。因此,需要采用合适的药物载体克服上述缺点。
外泌体(Exosome,EXO)是目前备受瞩目的天然的药物载体。外泌体是细胞主动分泌的脂质双层膜囊泡,可由多种细胞类型产生,并广泛存在于体液中,其直径分布为40-150nm。由于其毒性和免疫原性低、生物相容性好、血液循环时间长,并具有组织渗透性,甚至可以穿过血脑屏障、肿瘤部位的EPR效应(enhanced permeability and retentioneffect,EPR)以及“货物”保护能力等,被认为是目前最有潜力的药物递送载体。此外,外泌体具有内在的组织或细胞靶向特性,这归因于它们的特殊表面结构,如四跨膜蛋白,整联蛋白,粘附蛋白等。而且,还可以对其进行人工修饰,使其表达特定分子或获得更好的靶向能力。
发明内容
本发明的目的在于提供一种膜表面携带多肽cRGD,膜内包载小分子抗肿瘤药物的复合外泌体及其制备方法和应用,本发明涉及的给药系统具有肿瘤靶向性好、安全性高、抑瘤效果确切的特点。化疗药物的安全性和有效性是肿瘤化疗的关键的因素,因此,本发明以体内外靶向性和肿瘤药效为指标,制备膜表面携带cRGD,膜内包载小分子抗肿瘤药物的复合外泌体。
本发明的第一方面,提供一种负载多肽cRGD和小分子抗肿瘤药物的复合外泌体,所述复合外泌体为cRGD-EXO/小分子抗肿瘤药物,所述的多肽cRGD嵌合于外泌体的膜表面可靶向肿瘤细胞高表达的整合素αvβ3受体,所述的小分子抗肿瘤药物包载于cRGD-EXO膜内。
进一步的,所述的复合外泌体是先从细胞培养上清中提取细胞分泌的外泌体(EXO)。
进一步的,通过优选实验,确定EXO和cRGDfk的比例,将环状多肽cRGD嵌入EXO膜中,即得cRGD-EXO药物靶向递送载体。
进一步的,再将小分子抗肿瘤药物载入cRGD-EXO膜中制得靶向药物递送系统cRGD-EXO/小分子抗肿瘤药物。
进一步的,所述的外泌体来源的细胞选自巨噬细胞、NK细胞、T细胞、间充质干细胞、及肿瘤细胞中的一种或两种以上。
进一步的,所述的多肽cRGD为甘氨酸-天冬氨酸-丝氨酸(cyclic Arginine-Glycine-Aspartate),在制备过程中优选的cRGD为经过二硬脂酰磷脂酰乙醇胺(Distearoylphosphatidylethanolamine,DSPE)-聚乙二醇2000(polyethylene glycol-2000,PEG-2000)修饰的多肽,即硬脂酰磷脂酰乙醇胺改性多肽DSPE-PEG2000-(cRGDfk),购自西安瑞禧生物科技有限公司。化学结构如式(I)所示:
进一步的,所述的外泌体具有典型的“茶托”样结构,表面有CD9、CD63、TSG101特征蛋白的表达。
进一步的,所述的小分子抗肿瘤药物,来源于天然药物,如雷公藤甲素TPL;分子靶向药物,如曲美替尼;化疗药物,如阿霉素。
本发明的第二方面,提供一种如上所述的负载cRGD和小分子抗肿瘤药物的复合外泌体的制备方法,包括以下步骤:
S1.细胞培养上清中提取细胞分泌的外泌体的方法为梯度离心法,从细胞培养上清中提取细胞分泌的外泌体,标记为EXO;
S2.采用后插入法,将cRGD多肽嵌入EXO,即cRGD-EXO;
S3.将小分子抗肿瘤药物载入cRGD-EXO,得到负载cRGD和小分子抗肿瘤药物的复合外泌体,即cRGD-EXO&小分子抗肿瘤药物;所述复合外泌体的膜表面携带cRGD多肽,膜内包裹小分子抗肿瘤药物。
进一步的,步骤S1具体包括以下步骤:
取对数生长期的细胞,当细胞融合达到70%-80%时,弃去培养液,PBS清洗2-3遍后,更换至无血清的培养基进行饥饿培养;培养12h后,收集细胞上清液,采用梯度超速离心法分离外泌体。在4℃下,将细胞上清液300g离心10min除去细胞,2000g离心10min去除死细胞,10000g离心30min除去细胞碎片,120000g超高速离心70min,收集沉淀,PBS重悬后再次进行120000g离心70min,收集沉淀,并用PBS重悬后,保存在-80℃。
进一步的,所述的外泌体来源的细胞为人脐带来源的间充质干细胞。
进一步的,步骤S2具体包括以下步骤:
A)将DSPE-PEG2000-c(RGDfk)于4-(2-羟乙基)-1-哌嗪乙烷磺酸缓冲液(HEPES)中处理15min,从而形成胶束;
B)将胶束超声处理2×5s,以减小胶束的大小从而促进其与EXO的分离;
C)将EXO悬浮液与上述悬浮液在40℃下混合2h;
D)立即冷却至4℃,超速离心70min纯化外泌体,得到cRGD-EXO。
优选的,EXO与DSPE-PEG2000-cRGDfk的质量比为1:1。
进一步的,步骤S3中所述的载药方法为超声法、电穿孔法或共孵育法。
优选的,载药方法为共孵育法。
进一步的,步骤S3具体包括以下步骤:
将cRGD-EXO与药物按质量比10:1-1:10(μg蛋白:μg)混合均匀,200rpm条件下孵育90min。以药物包封率和载药量为评价标准,进行单因素考察。通过超速离心纯化载有药物的RGD-EXO,即得到纯化的RGD-EXO/小分子药物。通过高效液相色谱法检测药物的含量。
优选的,所述的小分子抗肿瘤药物为雷公藤甲素TPL。
优选的,cRGD-EXO和雷公藤甲素的质量比为1:1。
本发明的第三方面,提供一种如上所述的负载cRGD和小分子抗肿瘤药物的复合外泌体在制备肿瘤治疗药物中的应用。
进一步的,所述的肿瘤为αvβ3整合素受体高表达的肿瘤细胞,如人恶性黑色素瘤、前列腺癌、胶质母细胞瘤和乳腺癌。
进一步的,所述的负载cRGD和小分子抗肿瘤药物的复合外泌体在制备靶向黑色素瘤部位药物中的应用。
本发明优点在于:
1、本发明采用外泌体作为药物载体,具有靶向性好、毒性小、生物相容性高以及本身固有的靶向归巢作用等特点。将多肽cRGD与外泌体相结合能够特异性靶向于整合素高表达的内皮细胞和某些肿瘤细胞,增强靶向性。
2、本发明中的膜表面携带多肽cRGD,膜内包载小分子抗肿瘤药物的复合外泌体,外泌体分泌量多、制备方法简单、外泌体结构特征稳定。
3、经过优化的外泌体载药系统表现出优异的理化性质,具有粒径小和较高的载药量、包封率,表现出高效的抗黑色素瘤的能力,同时增强外泌体的靶向性、降低雷公藤甲素的毒副作用,为肿瘤治疗提供了一种新思路。
4、本发明研究开发了一种有效的基于外泌体的肿瘤治疗方法,并改善了外泌体在药物递送中的普适性,为肿瘤药物递送载体的研究提供了理论依据和新思路。
附图说明
图1.EXO的透射电镜(A)和粒径(B)。
图2.DSPE-PEG2000-c(RGDfk)与不同含量EXO的结合比例筛选:(A)流式细胞仪检测图;(B)流式细胞仪检测平均荧光强度。
图3.cRGD-EXO/TPL的透射电镜(A)和粒径(B)。
图4.cRGD-EXO/TPL表面蛋白CD9,CD63及TSG101的Western blot分析。
图5.cRGD-EXO/TPL对黑色素瘤细胞A375的细胞增殖影响。
图6.cRGD-EXO/TPL对黑色素瘤细胞A375的细胞迁移和侵袭抑制效果:(A)不同处理组对细胞迁移的影响;(B)不同处理组迁移的细胞数;(C)不同处理组对细胞侵袭的影响;(D)不同处理组侵袭的细胞数。
图7.cRGD-EXO/TPL对黑色素瘤细胞A375的细胞凋亡影响:(A)不同处理组的细胞凋亡流式图;(B)不同处理组的细胞凋亡率。
图8.cRGD-EXO/TPL的体内分布评价:(A)荷瘤裸鼠尾静脉给药后1、2、4、6、12和24h的荧光分布;(B)荷瘤裸鼠尾静脉给药24h的离体组织荧光分布;(C)荷瘤裸鼠尾静脉给药24h后离体器官荧光强度值。
图9.cRGD-EXO/TPL体内抗肿瘤效果评价;(A)给药周期结束后各组裸鼠肿瘤形态;(B)治疗期间裸鼠的肿瘤体积变化;(C)治疗周期结束后各组裸鼠的瘤重;(D)各组裸鼠生存曲线。
图10.注射cRGD-EXO/TPL后的肿瘤组织HE染色和Tunel染色。
图11.cRGD-EXO/TPL的安全性考察:(A)各器官组织的HE染色;(B)治疗过程中裸鼠的体重变化。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。
实施例1:EXO的制备及表征
1.外泌体提取:取对数生长期的细胞,当细胞融合达到70%-80%时,弃去培养液,PBS清洗2-3遍后,更换至无血清的培养基进行饥饿培养。培养12h后,收集细胞上清液,采用梯度超速离心法分离外泌体:在4℃下,将细胞上清液300g离心10min除去细胞;2000g离心10min去除死细胞;10000g离心30min除去细胞碎片;120000g超高速离心70min,收集沉淀,PBS重悬;再次进行120000g离心70min,收集沉淀,并用PBS重悬后,保存在-80℃,用于后续实验。
2.外泌体表征:透射电镜实验:用100μL 2%多聚甲醛重悬EXO,取5μL EXO悬液滴到有碳膜的200目铜网上,室温干燥20min,将铜网置于100μL PBS液滴上清洗1min,用1%戊二醛室温固定5min,再将铜网用ddH2O洗涤2min,重复洗涤8次,将铜网放在50μL醋酸双氧铀液滴上负染5min,再置于甲基纤维素液滴上10min以形成一层薄甲基纤维素膜,室温干燥10min,用透射电镜(TEM)在80kV加速电压下观察拍照。
粒径分析实验:取适量EXO用PBS重悬稀释,置于比色皿中。使用马尔文激光粒度仪在室温下捕获并分析EXO的平均粒径。
Western blot验证外泌体蛋白标记物CD9、CD63、TSG101:将超速离心得到的EXO用50μL RIPA裂解液裂解,在使用前数分钟内加入蛋白酶抑制剂,在振荡器上充分震荡数次。充分裂解后,12000rpm离心10min,取上清。所提取的蛋白在使用前先用BCA法测定蛋白的浓度,根据BCA说明书利用标准品绘制标准曲线,并计算蛋白样品的浓度。配制8%的分离胶和5%的浓缩胶,依次灌胶。待胶凝固后,置于电泳槽中,取EXO的蛋白提取液,调整蛋白浓度,和等体积2×蛋白上样缓冲液混合,煮沸变性15min,将蛋白样本加入电泳孔中,恒压75V电泳至溴酚蓝刚跑出即可终止。将甲醇活化的PVDF膜盖于胶上,300mA恒流转膜30min。将转好的膜在摇床上用5%脱脂奶粉封闭1h。每组分别加入CD9(Abcam 1:1000),CD63(Abcam 1:1000),TSG101(Abcam1:1000),4℃孵育过夜。用TBST在摇床上洗膜5min×3次。按照一抗种属分别加入相对应的辣根过氧化物酶(HRP)标记的山羊抗兔二抗(Abcam 1:3000)室温孵育2h。用TBST在摇床上洗10min×3次。用ECL试剂盒曝光,显影、定影。
通过透射电镜观察到,EXO具有明显的“茶托”样结构;马尔文激光粒径仪结果表明外泌体的平均粒径为82.82nm;在EXO样品中均可明显地检测到外泌体特异性蛋白CD9、CD63和TSG101的表达(图1)。
实施例2:cRGD-EXO的制备
1.制备:将DSPE-PEG2000-c(RGDfk)(以下简称cRGDfk)在60℃下于4-(2-羟乙基)-1-哌嗪乙烷磺酸缓冲液(HEPES)中处理15min,从而形成胶束。
将胶束以10μm的振幅超声处理2×5s,以减小胶束的大小从而促进其与EXO的分离。将EXO悬浮液与上述悬浮液在40℃下混合2h。立即冷却至4℃,以120,000×g离心70min纯化外泌体,得到cRGD-EXO。
2.EXO和cRGDfk质量比优化:以细胞摄取的平均荧光强度为指标,将EXO和cRGDfk的质量比设置为5:1、1:1、1:5、1:10、1:15(μg蛋白质:μg),采用流式细胞仪进行单因素优化。流式细胞仪的具体操作步骤如下:将A375细胞以5×105cells/mL的密度接种于6孔板中,在37℃,5%CO2培养箱中培养24h。待细胞贴壁后,弃去培养液,用PBS清洗2遍,分别加入PKH67标记的cRGD-EXO(cRGDfk与EXO的质量比分别为5:1、1:1、1:5、1:10、1:15)和EXO(100μg/mL蛋白浓度),继续培养6h。吸去上清,PBS洗3次,消化离心后用PBS重悬至400μL,涡旋后利用流式细胞仪检测,记录流式图谱,并定量评价A375细胞对EXO和不同比例的cRGD-EXO的摄取情况和细胞的平均荧光强度。
将上述比例的外泌体进行染色,用于后续流式细胞仪的检测。用1mL稀释液Diluent C稀释外泌体溶液,混匀。取4μL PKH67染色液加入1mL Diluent C稀释液,按体积比1:1将稀释后的外泌体和染色液混合,避光放置2min。用2mL 1%BSA终止染色1min,120000g超速离心70min去除游离的染料,用PBS重悬外泌体沉淀,即得PKH67荧光探针标记的外泌体溶液。
图2显示,相比于EXO(81.80±0.56),当EXO与cRGDfk的质量比为1:1时,cRGD-EXO(231±3.61)组中细胞内的平均荧光强度更强,因此选用该比例进行后续实验。
实施例3:cRGD-EXO/TPL的制备及表征
1.cRGD-EXO/TPL的制备及载药量优化:在室温下,将cRGD-EXO与TPL混合均匀,摇床孵育90min,转速200rpm。为了选择最优的cRGD-EXO和TPL的比例,以药物包封率和载药量为评价标准,将cRGD-EXO和TPL的质量比设置为10:1、5:1、1:1、1:5、1:10(μg蛋白:μg),进行单因素考察。最后,通过超速离心纯化载有TPL的cRGD-EXO,即得到纯化的cRGD-EXO/TPL。通过高效液相色谱法检测cRGD-EXO/TPL中的TPL含量。
2.cRGD-EXO/TPL表征:透射电镜实验:cRGD-EXO/TPL分别经固定、干燥、清洗、负染、自然风干后,在TEM上检测,设加速电压为80kV,观察并拍照。
粒径分析实验:取适量cRGD-EXO/TPL用PBS重悬稀释,置于比色皿中。使用马尔文激光粒度仪在室温下捕获并分析cRGD-EXO/TPL的平均粒径。
Western blot验证该外泌体给药系统表面蛋白标记物CD9、CD63、TSG101,按照实施例1中的Western blot实验步骤对cRGD-EXO/TPL外泌体蛋白标记物CD9、CD63、TSG101进行检测。
图3表明,透射电镜结果显示,cRGD-EXO/TPL的结构完整,与EXO的外观结构无明显差异。粒径分析结果显示,与EXO相比,cRGD-EXO/TPL的粒径略有增加,约为160nm,并且经过cRGD多肽修饰以及TPL的包载不影响外泌体表面蛋白的表达(图4)。
实施例4:cRGD-EXO/TPL对的细胞增殖影响检测
A375细胞培养至80%融合状态时,胰酶消化并计数,以8×103cells/孔的密度铺于96孔板,37℃,5%CO2培养24h。弃上清,加入PBS洗3次,加入不同浓度TPL(0、20、40、60、80、100ng/mL),计算TPL的半数抑制浓度(IC50)。计算IC50之后,重新铺板,分别加入EXO、cRGD-EXO、TPL、EXO/TPL、cRGD-EXO/TPL(外泌体蛋白浓度为70μg/mL,TPL浓度为70ng/mL),对照组只加培养基,每组设置6个复孔。同时,为了防止边缘液体蒸发带来的误差,孔板边缘的孔作为空白孔,加入100μL的DMEM。37℃,5%CO2培养24h。弃去培养基,每孔加入10%体积的CCK-8试剂,培养箱中培养1h。取出培养板,使用酶标仪检测450nm处的吸光度。
图5表明,TPL对A375细胞的IC50为69.69ng/mL。图5显示出在相同浓度的TPL(70ng/mL)下用EXO、cRGD-EXO、TPL、EXO/TPL、cRGD-EXO/TPL处理24h后的A375细胞的活力。cRGD-EXO/TPL组比其他EXO组或游离药物组具有更低的细胞生存力。
实施例5:cRGD-EXO/TPL对细胞迁移和侵袭抑制的影响
1.细胞迁移实验
(1)细胞悬液的制备
使用空白培养基饥饿培养A375细胞12h,去除血清对细胞的影响。A375细胞培养至80%融合状态时,PBS清洗3次,胰酶消化,终止消化后离心,去除上清,PBS清洗2次,用无血清培养基重悬细胞,A375细胞的密度调整为1×104cells/孔。
(2)接种细胞
以1×104cells/孔的密度铺于8μm Transwell上室,分别加入100μL EXO、cRGD-EXO、TPL、EXO/TPL、cRGD-EXO/TPL。下室加入800μL含有10%FBS的DMEM作为趋化因子,37℃,5%CO2培养24h。用无菌棉签擦拭上室表面上的细胞,将迁移至下室的细胞用4%多聚甲醛固定15min,细胞用0.1%结晶紫染色10min,并用PBS洗涤3次,在倒置显微镜下随机选择五个视野来计数迁移细胞并拍照。
2.细胞侵袭实验
将Matrigel以1:8的比例进行稀释,取稀释液50μL,均匀覆盖在Transwell小室的上室面,37℃干燥2h,使Matrigel胶凝固。之后的步骤与上述的细胞迁移实验一致。
图6显示,细胞迁移和侵袭实验中,EXO组穿过膜的细胞数量与对照组相比无显著性差异(P>0.05);cRGD-EXO、TPL、EXO/TPL、cRGD-EXO/TPL组穿过膜的细胞数量依次降低。而且与其他组相比,cRGD-EXO/TPL组细胞形态发生了变化,A375细胞的迁移和细胞侵袭数量的组间差异具有统计学意义(P<0.001)。结果表明,cRGD-EXO/TPL能够有效抑制A375细胞的迁移和侵袭。
实施例6:cRGD-EXO/TPL对A375细胞凋亡实验
A375细胞培养至80%融合状态时,胰酶消化并计数,以3×105cells/孔的密度铺于12孔板,37℃,5%CO2培养24h。吸出上清,PBS洗涤3遍,分别加入EXO、cRGD-EXO、TPL、EXO/TPL和cRGD-EXO/TPL,收集旧培养基,PBS洗一遍,使用不含EDTA的胰酶消化,将消化后的细胞悬液和之前收集的旧培养基一起离心,可以残余约50μL左右的培养液,避免吸取细胞。加入1mL4℃预冷的PBS清洗一次,用1×Binding buffer重悬细胞沉淀,调整浓度为106-107cells/mL。向100μL的细胞悬液中加入5μL Annexin V-APC和10μL7-AAD,混匀,室温避光孵育15min。无需洗涤,每管加入385μL 1×Binding buffer,混匀,尽快用流式细胞仪进行检测。
图7所示,对照组与EXO和cRGD-EXO组之间无显著差异(6.17%±1.33%,11.03%±0.86%和12.63%±1.05%),表明EXO、cRGD-EXO不会诱导细胞凋亡。cRGD-EXO/TPL诱导的细胞凋亡率为42.85%±2.34%,明显高于TPL(22.10%±1.66%)和EXO/TPL组(37.99%±0.40%)的细胞凋亡率。这些结果表明cRGD-EXO/TPL可以增加TPL对A375细胞中的凋亡作用。
实施例7:cRGD-EXO/TPL的体内分布
构建黑色素瘤裸鼠移植瘤模型:四周龄的雄性无胸腺裸鼠适应新环境2-3天。裸鼠右肢腋下皮下注射A375细胞悬液(106cells/mL)。继续饲养裸鼠,当裸鼠的肿瘤体积生长至100mm3时,进行后续试验。
我们使用黑色素瘤裸鼠模型评估了cRGD-EXO/TPL在体内的肿瘤靶向作用。用近红外荧光染料DiR标记EXO,PBS洗涤3次,并使用超高速离心进行纯化。裸鼠(n=3)尾静脉注射200μL DiR标记的EXO,cRGD-EXO。使用IVIS Spectrum于注射后1、2、4、6、12和24h在750/780nm波长处扫描裸鼠。最后一次拍照后处死裸鼠,并记录每只裸鼠心脏,肝脏,脾脏,肺,肾,血液,肿瘤处的荧光强度。使用LivingImage软件分析所有数据。
EXO和cRGD-EXO分别用DiR标记。从图8(A)可知,EXO主要分布于肝脏部位,并且随着时间的延长,荧光信号降低迅速;cRGD-EXO组在尾静脉注射后开始逐渐向肿瘤部位聚集,于第4h肿瘤部位的荧光最强,之后逐渐降低。cRGD-EXO组的肿瘤区域中的荧光强度显著高于EXO组,这表明,相比EXO组,cRGD-EXO组能够在肿瘤部位积聚更多的外泌体。
如图8(B)所示,给药24h后,进一步检测裸鼠心,肝,脾,肺,肾,血液和肿瘤中离体荧光信号。cRGD-EXO组的肿瘤中的荧光强于EXO组。而且EXO组的荧光主要分布在肝脏和脾脏中。cRGD-EXO组的荧光强度总体上大于EXO组,这可能是由于cRGD多肽上携带的PEG2000的长循环作用。我们的结果表明,cRGD-EXO比EXO能够更有效地积聚在肿瘤组织中,证明了cRGD-EXO的体内肿瘤靶向能力。
实施例8:cRGD-EXO/TPL的体内抗肿瘤作用
将黑色素瘤裸鼠模型随机分为5组,每组6只。每隔一天通过尾静脉注射0.9%生理盐水、cRGD-EXO、TPL、EXO/TPL和cRGD-EXO/TPL。给药第一天记为第0天,每次给药前测量裸鼠的重量,测量皮下肿瘤的最长直径(L)和最短直径(W),并通过公式(L×W2)/2计算肿瘤体积。以时间为横坐标,绘制相应的肿瘤的生长曲线和裸鼠体重随时间变化的曲线。给药周期结束后对裸鼠实施安乐死,取出肿瘤组织、重要器官并称重,与心、肝、脾、肺、肾各个器官用多聚甲醛固定,用于后续的HE染色和凋亡情况考察。为考察不同给药组对裸鼠生存期的影响,需要另外准备30只肿瘤体积为100mm3的人黑色素瘤皮下移植瘤裸鼠,同上分组,给药,记录各组小鼠的生存情况,并绘制生存期曲线。
对组织进行切片染色,可以直观的观察到组织的病理变化。本研究中,通过肿瘤组织的HE染色和Tunel染色考察药物对肿瘤细胞的作用。
体内抗肿瘤实验表明,以给药的时间作为横坐标,裸鼠肿瘤的体积数据作为纵坐标,对每组裸鼠肿瘤的生长抑制曲线进行绘制,如图9(B)所示,对照组与cRGD-EXO组肿瘤体积无明显差异,肿瘤生长几乎没有受到抑制。而TPL、EXO/TPL和cRGD-EXO/TPL组肿瘤生长受到不同程度的抑制。cRGD-EXO/TPL组可以显著抑制肿瘤的生长(P<0.05),第14天时,EXO/TPL、TPL和cRGD-EXO组的肿瘤体积分别是cRGD-EXO/TPL的2.07、2.62和3.01倍。图9(C)肿瘤重量结果显示,对照组与cRGD-EXO组平均肿瘤重量没有显著性差异(P>0.05)。第14天时,EXO/TPL、TPL和cRGD-EXO组的肿瘤重量分别是cRGD-EXO/TPL的1.61、2.23和2.74倍,与上述肿瘤体积结果一致。
为了评估cRGD-EXO/TPL的长期效果,在黑色素瘤模型中记录了总生存期。图9(D)显示了各个组的裸鼠的生存期。对照组中裸鼠的平均存活时间为25.5天,而cRGD-EXO、TPL、EXO/TPL和cRGD-EXO/TPL组的平均存活时间分别为26.5、29.5、38和44.5天。与其他组相比,cRGD-EXO/TPL组的裸鼠的存活期显著延长(P<0.001),这表明cRGD-EXO/TPL组具有更强的治疗效果,并增加了荷瘤小鼠的存活率。
在肿瘤组织中(图10),对照组与cRGD-EXO组中,细胞核较大、深染,胞浆较少,表现出肿瘤组织的病理学特征。而经TPL、EXO/TPL和cRGD-EXO/TPL组治疗后,肿瘤细胞间的空隙增大,细胞核缩小,胞浆增多,cRGD-EXO/TPL组这一现象更为显著,说明cRGD-EXO/TPL的治疗效果最优。在Tunel染色结果中,TPL、EXO/TPL和cRGD-EXO/TPL组的肿瘤组织出现不同程度的凋亡,其中cRGD-EXO/TPL组最为明显,说明cRGD-EXO/TPL对肿瘤细胞的凋亡作用最强。与上述的体内的药效实验结果一致。
实施例9:cRGD-EXO/TPL的安全性考察
cRGD-EXO/TPL系统给药的生物安全性主要通过两个方面进行表征,一是裸鼠的体重在治疗期间的变化情况,二是通过HE染色结果观察TPL对裸鼠心、肝、脾、肺、肾组织的毒性情况。
结果表明,如图11(A)所示,通过心、肝、脾、肺、肾的HE染色结果可以看出cRGD-EXO/TPL组的生物相容性较好,各个脏器没有出现明显毒性。TPL组可以看出肝脏组织和肾脏组织出现不同程度的损伤。可见cRGD-EXO/TPL可降低TPL的系统毒性,可能是因为其具有缓释效果并且能够减少药物在非靶向器官的分布。
如图11(B)所示,TPL组裸鼠体重出现下降的趋势,最终体重为19.81±0.95g,这可能是由于累计用药产生了系统毒性。其余组裸鼠体重均呈不同程度的波动上升趋势。TPL组裸鼠体重与cRGD-EXO/TPL组(23.08±1.91g)、EXO/TPL(22.60±2.21)、cRGD-EXO(22.85±0.76)和对照组(23.13±1.85g)相比,无显著性差异(P>0.05),说明单纯TPL给药的系统毒性较大,外泌体的包载能够有效降低TPL的毒性。
以上已对本发明创造的较佳实施例进行了具体说明,但本发明创造并不限于所述实施例,熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换,这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Claims (5)
1.一种负载由甘氨酸-天冬氨酸-丝氨酸组成的cRGD和抗肿瘤药物雷公藤甲素TPL的复合外泌体,其特征在于,所述的外泌体的膜表面携带靶向于整合素αvβ3受体的环状多肽cRGD,膜内包载抗肿瘤药物雷公藤甲素TPL;所述的复合外泌体是先从细胞培养上清中提取细胞分泌的外泌体EXO;再通过后插入法将环状多肽cRGD嵌入EXO膜中,得cRGD-EXO;再将抗肿瘤药物雷公藤甲素TPL载入cRGD-EXO膜中制得,即cRGD-EXO/TPL;其中多肽cRGD和EXO的质量比1:1,cRGD-EXO和雷公藤甲素TPL的质量比1:1。
2.根据权利要求1所述的负载由甘氨酸-天冬氨酸-丝氨酸组成的cRGD和抗肿瘤药物雷公藤甲素TPL的复合外泌体,其特征在于,所述的外泌体来源的细胞选自间充质干细胞、巨噬细胞、NK细胞、T细胞、及肿瘤细胞中的一种或两种以上。
3.一种如权利要求1或2所述的负载由甘氨酸-天冬氨酸-丝氨酸组成的cRGD和抗肿瘤药物雷公藤甲素TPL的复合外泌体的制备方法,其特征在于,包括以下步骤:
S1.采用梯度超速离心法,从细胞培养上清中提取细胞分泌的外泌体,标记为EXO;
S2.通过后插入法,以多肽cRGD和EXO按1:1质量比,将cRGD嵌入EXO,即cRGD-EXO;
S3.以cRGD-EXO和抗肿瘤药物TPL按1:1的质量比,将TPL载入cRGD-EXO,得到负载多肽cRGD和抗肿瘤药物TPL的复合外泌体,即cRGD-EXO/TPL。
4.根据权利要求3所述的复合外泌体的制备方法,其特征在于,步骤S3中载药方法为超声法、电穿孔法或共孵育法。
5.根据权利要求1或2所述的负载由甘氨酸-天冬氨酸-丝氨酸组成的cRGD和抗肿瘤药物雷公藤甲素TPL的复合外泌体在制备肿瘤治疗药物中的应用,其特征在于,所述的肿瘤为整合素αvβ3受体高表达的人恶性黑色素瘤。
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