CN115093459A - 一种可在外泌体表面进行轴突靶向修饰的递送复合物及其对外泌体的修饰方法 - Google Patents
一种可在外泌体表面进行轴突靶向修饰的递送复合物及其对外泌体的修饰方法 Download PDFInfo
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Abstract
本发明提供一种可在外泌体表面进行轴突靶向修饰的递送复合物及其对外泌体的修饰方法,所述递送复合物由TAxI多肽和DSPE‑PEG2000‑NHS制成;所述TAxI多肽由SEQ ID NO:1所示序列的两端各加入一个三赖氨酸嵌段并在两个半胱氨酸间形成二硫键制成;所述TAxI多肽在C端赖氨酸接Biotin,所述TAxI多肽在N端赖氨酸与DSPE‑PEG2000‑NHS接枝。所述靶向轴突的外泌体由上述递送复合物对干细胞外泌体进行表面修饰而成;所述递送复合物预先制成DSPE‑PEG2000‑TAxI‑Biotin胶束。本发明的靶向轴突的外泌体具有良好的血液稳定性、隐身性、靶向性,可有效识别中枢神经***轴突组织。
Description
技术领域
本发明属于生物技术领域,具体涉及一种可在外泌体表面进行轴突靶向修饰的递送复合物及其制备方法以及该递送复合物修饰外泌体的方法。
背景技术
神经***退行性疾病(Neurodegenerative Disease,NDD)是由神经元和神经髓鞘的丧失所致,随着时间的推移而恶化,出现功能障碍,主要有脑萎缩、阿尔茨海默病、帕金森综合征、多发性硬化症及肌肉萎缩侧索硬化等。随着老龄化人口增多,该疾病对患者的寿命和生存质量的影响也越来越严重。
靶向性轴突导入(Targeted Axonal Import ,TAxI)多肽已经被科学家通过噬菌体展示技术证实,有着良好的中枢神经***轴突靶向性,但其在血液中稳定性极低,并且在轴突的溶解度有待提高。DSPE-PEG2000(1,2-二硬脂酰基-sn-甘油-3-磷酸乙醇胺-聚乙二醇2000)是一种生物相容性高、可生物降解的两亲性材料,可用于多种生物大分子(如,功能性多肽与蛋白)功能化以实现特定功能,在药物递送***应用中广泛使用的活化脂-磷脂-聚乙二醇共轭物。
外泌体是有150nm左右的纳米级粒径,具有穿越血脑屏障的能力和良好的免疫调节功能,已有许多文章表明,干细胞外泌体在治疗神经退行性***疾病时有着较于传统药物更好的疗效。外泌体适用于静脉内递送,但其临床转化进程受阻的主要原因是外泌体的生物利用率过低,进入体内后由于脉管***的渗透性和保留(EPR)效应,大部分被肝、脾等器官降解吸收。
鉴于此,急需解决外泌体的靶向性问题,提高外泌体的生物利用率,使其应用到神经***疾病的治疗中,对于解决临床神经***退行性疾病尤为重要。
发明内容
针对现有技术的不足,本申请发明人对TAxI多肽的原序列片段进行优化,使其有更好的溶解度、血液稳定性以及便于体内示踪,并通过优化序列上的赖氨酸的氨基DSPE-PEG2000-NHS发生取代反应来接合到一起,获得靶向轴突的递送复合物,再通过控制温度和超声频率来使靶向轴突的递送复合物整合到外泌体膜表面,使被修饰外泌体具有轴突靶向功能。为实现本发明目的所使用的技术方案为:
一种可在外泌体表面进行轴突靶向修饰的递送复合物,所述递送复合物由TAxI多肽和DSPE-PEG2000-NHS制成;所述TAxI多肽由SEQ ID NO:1所示序列的两端各加入一个三赖氨酸嵌段并在两个半胱氨酸间形成二硫键制成;所述TAxI多肽在C端赖氨酸接Biotin,所述TAxI多肽在N端赖氨酸与DSPE-PEG2000-NHS接枝。
本发明中靶向轴突的TAxI多肽片段的原序列为(SACQSQSQMRCGGG)(SEQ ID NO:1),为保证其溶解性,将在序列的两端各加三赖氨酸嵌段,同时在N端赖氨酸上的氨基也将作为TAxI多肽的接枝点与DSPE-PEG2000-NHS的NHS活性酯进行酰胺反应完成接枝,C端赖氨酸接生物素(Biotin)用于示踪;另外,为了保证多肽的血液稳定性,将在多肽序列中两个半胱氨酸的巯基之间形成二硫键使多肽形成环化结构。最后用反相高效液相色谱法(RP-HPLC)对脱盐后的多肽溶液进行浓缩和纯化,获得(KKKSACQSQSQMRCGGGKKK(Biotin)-OH 二硫键6-14)(SEQ ID NO:2)多肽片段,并通过高效液相色谱法(HPLC)和质谱法(MS)对其进行鉴定。
本发明中二棕榈酰磷脂酰乙醇胺-聚乙二醇-琥珀酰亚胺酯(DSPE-PEG2000-NHS),分子式为C139H271N4O57P,PEG分子量:2000,整体分子量:2900。结构式为:
DSPE-PEG2000-NHS是一种线性异双功能聚乙二醇化试剂,含有DSPE磷脂和活化的羧酸NHS酯。它是一种自组装试剂,用于制备聚乙二醇化脂质体或胶束,同时还提供NHS基团用于与含胺分子共轭,可提供隐身性,延长循环半衰期,减少经其修饰的外泌体与非特异性蛋白质结合或细胞粘附。将TAxI多肽片段的N端赖氨酸上的氨基作为接枝点,通过赖氨酸上的氨基与DSPE-PEG2000-NHS上的NHS基团之间的共价共轭作用生成了酰胺键,最终合成了DSPE-PEG2000-TAxI-biotin线性靶向轴突的递送复合物,用于外泌体的膜表面靶向修饰。
本发明还提供一种轴突靶向递送复合物修饰外泌体的方法,利用上述递送复合物对干细胞外泌体进行表面修饰而成;所述递送复合物预先制成DSPE-PEG2000-TAxI-Biotin胶束;所述DSPE-PEG2000-TAxI-Biotin胶束和外泌体的质量比为1:1 (按µg蛋白:µg蛋白)。
优选地,所述外泌体为人脐带间充质干细胞外泌体、血液外泌体、唾液外泌体或尿液外泌体的任意一种。
外泌体膜表面的DSPE-PEG2000-TAxI***修过程,发明人将DSPE-PEG2000-TAxI-biotin与外泌体通过一系列温度变化和超声振荡条件下的共孵育来进行。另外,通过DiR染料对轴突靶向递送复合物修饰的外泌体进行膜标记,也可以通过染料标记(如,HRP、FITC等)的链霉亲和素蛋白嵌合体与DSPE-PEG2000-TAxI-Biotin中的生物素偶联组成生物反应放大***来对DSPE-PEG2000-TAxI-biotin进行标记。
优选地,具体制备步骤如下:
(1)合成TAxI多肽;
(2)将AxI多肽和DSPE-PEG2000-NHS的接合,获得递送复合DSPE-PEG2000-TAxI-Biotin;
(3)将步骤(2)合成的递送复合物制成胶束,再***外泌体脂双层膜中。
优选地,所述合成TAxI多肽的步骤是将SEQ ID NO:1序列C-端氨基酸的羧基以共价键的形式与不可溶的高分子树脂相接,再以这一氨基酸的氨基作为多肽合成的起始点,同其他的氨基酸已活化的羧基反应生成肽键,持续重复该过程,获得多肽后,再在多肽片段的两个半胱氨酸的巯基间形成二硫键进行环化修饰,最后用高效液相色谱进行纯化,即得TAxI多肽。
优选地,步骤(2)中,DSPE-PEG2000-NHS与TAxI多肽的物质的量比为2:1。
优选地,步骤(3)中,递送复合物预先在60℃形成胶束后冷却到室温,再与外泌体条件下共孵育***外泌体脂双层膜中。
本发明的有益效果是:
相对于自然外泌体,DSPE-PEG2000-TAxI-biotin修饰后的外泌体具有良好的血液稳定性、隐身性、靶向性,可有效识别中枢神经***轴突组织,达到良好的神经退行性疾病的治疗效果。
附图说明
图1是本发明实施例1 TAxI多肽的高效液相色谱图,在出样的第8min收集到目标多肽,纯度为96.06%。
图2是本发明实施例1 TAxI多肽的质谱图,结果显示该多肽在质荷比(m/z)941.649处出现峰值[M+2H]2+,相对分子质量为2453.0。
图3是本发明实施例2的递送复合物的核磁共振氢谱图。
图4是本发明实施例2的递送复合物的红外光谱图。
图5是本发明实施例3轴突靶向递送复合物修饰外泌体膜表面的过程图。
图6是本发明实施例3的轴突靶向递送复合物修饰的外泌体的HT7700透射电镜结果,标尺为200nm。
图7是本发明实施例3的轴突靶向递送复合物修饰的外泌体的ZetaView粒径分析仪结果,经修饰外泌体与自然外泌体相比,粒径约增大25nm。
图8是本发明实施例4轴突靶向递送复合物修饰的外泌体在小动物体内的示踪结果,给药组与对照组相比有着良好的轴突靶向性。
图9是本发明实施例5轴突靶向递送复合物修饰的外泌体在与BV2细胞共孵育的观察结果,修饰外泌体组与对照组相比有着良好的细胞结合能力与贮留能力。
具体实施方式
为了使本发明更加清楚、完整,下面结合实施例对本发明方案做进一步详细描述,显然,所描述的实施例仅是本发明所选择的一部分,而不是全部的实施例。基于发明中的实施例,本领域普通技术人员在没有作出创造性劳动及试剂改进的前提下,所获得的所有其他实施例,都属于本发明保护的范围。
各个实施例中技术方案和实际操作的步骤顺序可以根据实际情况进行相应的调整,但是必须是以本领域普通技术人员能够实现为基础,当该调整与本发明技术方案的结合出现相互矛盾或无法实现与实际操作相对应的检测结果时,应当认为这种技术方案的调整与结合不存在,即不在本发明的保护范围之内。
需要说明,本发明实施例中所附图结果均仅为某一步骤所得粗产物在既定仪器及参数条件下的结果,如果该仪器和参数条件中其一发生改变时,则该结果也会随之相应的改变。
实施例1 合成TAxI多肽
在本实施例中,先将所需合成的目标多肽(SACQSQSQMRCGGG)(SEQ ID NO:1)的C-端氨基酸的羧基以共价键形式与不可溶的高分子树脂相接,然后以这一氨基酸的氨基做为多肽合成的起始点,同其他的氨基酸早已活化的羧基功效产生肽键,持续重复这一过程,用高效液相色谱进行纯化,就可以获得靶向多肽的序列为(KKKSACQSQSQMRCGGGKKK(biotin)-OH 二硫键6-14)(SEQ ID NO:2)。
在实际操作中,根据Fmoc化学合成步骤,
一、树脂溶涨,称取0.6g的取代度为0.4mmol/g的2-Chlorotrityl ChlorideResin(2-CTC Resin)树脂,将树脂放入反应管中,加入二氯甲烷15ml(15ml/g),振荡30min;
二、接第一个氨基酸, 通过沙芯抽滤掉溶剂,加入3倍摩尔过量的Fmoc-Lys(biotin)--OH氨基酸,再加入10倍摩尔过量的N,N-二异丙基乙胺(DIPEA),最后加入少量DMF溶解,振荡1h,用N,N-二甲基甲酰胺(DMF)和二氯甲烷(DCM)交替清洗6次;
三、脱保护,加15ml的20%哌啶DMF溶液(15ml/g),5min,去掉再加15ml的20%哌啶DMF溶液(15ml/g),15min;
四、检测,使用DMF洗涤6次,取少量树脂(大约10颗树脂即可)进行颜色反应。抽掉DMF溶液,取少量树脂,用乙醇洗三次,进行定性颜色反应茚三酮显色法(Kaiser法)加入6%茚三酮乙醇溶液、2% 0.001M KCN的吡啶溶液、80%苯酚乙醇溶液各一滴,105℃-110℃加热5min,变深蓝色为阳性反应;
五、置换溶剂,DMF(10ml/g)两次,甲醇(10ml/g)两次,DMF(10ml/g)两次;
六、缩合,保护氨基酸(Fmoc-L-Gly-OH)三倍过量,苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)三倍过量,用少量DMF溶解,加入反应管,之后立刻加入N-甲基***啉(NMM)十倍过量,反应30min,重复步骤五;
七、重复步骤二至六,从右到左依次连接序列中的氨基酸;
八、脱保护,最后一个氨基酸连接后洗树脂,DMF(10ml/g)两次,甲醇(10ml/g)两次,DMF(10ml/g)两次,DCM(10ml/g)两次,抽干10min;
九、从树脂上切割多肽,配制切割液(10ml/g): TFA 94.5%,水2.5%,EDT 2.5%,TIS1%,将干燥树脂装入烧瓶中,树脂和切割液比例按照10ml/g,恒温震荡2h;
十、抽滤沉淀洗涤,将切割液用氮气尽量吹干,用冰***与滤液6:1混合均匀,3000rpm/min*10min,洗三次,真空抽干,所得粗品多肽可以-20℃保存;
十一、将上述粗品多肽用HPLC纯化多肽。
对上述合成的多肽片段在两个半胱氨酸的巯基间形成二硫键以进行环化修饰。
在实际操作中,将脱保护的游离出半胱氨酸巯基和处于还原态的环化TAxI-biotin多肽溶于浓度为20%二甲基亚砜(DMSO)水溶液中,多肽片段中两个半胱氨酸的硫原子之间脱氢氧化形成二硫键来将线性多肽环化,这个过程在pH 8的条件下进行氧化,反应进行3-4小时。
获得了足量的环化靶向多肽片段,并利用高效液相色谱进行纯化,如附图1。同时利用质谱进行分子量的鉴定,结果如附图2。
实施例2 合成递送复合物
在本实施例中,用DSPE-PEG2000-NHS来装载实施例1合成的环化多肽片段,合成最终的靶向轴突的递送复合物。
在实际操作中,将溶解于适量水中的DSPE-PEG2000-NHS加入到溶解在PBS (0.1M磷酸盐,pH 7.4)中的环化的TAxI多肽中,物质的量2:1,pH 8-8.5,室温搅拌过夜。之后用酸进行中和至pH7.0左右,并用蒸馏水进行透析,最后用反相高效液相色谱法(RP-HPLC)对脱盐后的多肽溶液进行浓缩和纯化。
然后进行表征检测,FITC和NH2-PEG2000-DSPE在DMF中溶解2h,通过共价共轭合成FITC-PEG2000-DSPE。采用茚三酮试验确定无游离氨基存在,表明DSPE-PEG2000-NH2均与FITC连锁。对上述混合物进行蒸馏水透析(MWCO 3500Da)去除过量的环化TAxI多肽及试剂,然后将溶液真空冻干,用1HNMR (Varian 400MHz, Palo Alto, USA)对功能材料进行表征。
如附图3,在DSPE-PEG2000-NHS的核磁氢谱中,NHS基团在3.7ppm处出现了一个峰,而在DSPE-PEG2000-TAxI-Biotin的氢谱中该峰完全消失,说明NHS基团与TAxI赖氨酸上的氨基发生了反应。另外,在DSPE-PEG2000-TAxI-Biotin的红外光谱中,在1640-1820cm^-1区域内产生强吸收峰证明有羰基的存在,同时在3500 cm^-1附近有中等强度吸收峰证明酰胺键的存在,如附图4。
实施例3 合成轴突靶向递送复合物修饰的外泌体
如附图5,将实施例2合成的DSPE-PEG2000-TAxI-Biotin与自然外泌体进行一系列条件下共孵育,即可完成轴突靶向递送复合物对外泌体的修饰,选用的是人脐带间充质干细胞外泌体。
在实际操作中,将 DSPE-PEG2000-TAxI-Biotin溶解于100µM的HEPES缓冲溶液中(pH7.4) 60℃下溶解15 min,形成胶束,完成后冷却到室温。外泌体与DSPE-PEG2000-TAxI-Biotin胶束按1:1比例混合(µg蛋白:µg蛋白),总体积为100-150µl,在PCR仪中40°C孵育2小时。完成孵育后,将混合物冷却到4°C,通过排阻色谱法立即从游离胶束中纯化修饰的外泌体。对靶向递送复合物所修饰的外泌体进行透射电镜下观察和粒径分析。
如附图6所示,在电镜下观察,经靶向递送复合物修饰的外泌体膜表面暗区阴影加深,证明靶向递送复合物已有效的修饰到外泌体膜表面。附图7的历经分析结果佐证了附图6的电镜结果,经靶向复合物修饰的外泌体,粒径明显增大,证明修饰成功。
实施例4 轴突靶向递送复合物修饰的外泌体的动物实验
使用DiR染料对轴突靶向递送复合物修饰的外泌体进行表面标记,选用C57BL/6小鼠用作体内示踪实验动物来进行DiR染料标记外泌体的小动物活体成像实验。
在实际操作中,配制2µM的 DiR工作液,向工作液中加入靶向修饰外泌体到DiR工作液中终浓度为1x10^8- 1x10^9 particles/ml,37℃水浴20min,用100KDa超滤管置换溶剂为PBS缓冲液,终浓度为10µg/µl。选用C57BL/6小鼠用作体内示踪实验动物,每组小鼠静脉给药100µg DiR染色的靶向修饰外泌体,并在IVIS Spectrum小动物活体成像仪的720nm激发光下检测,如附图8。
结果显示,与自然外泌体相比,经靶向递送复合物修饰的外泌体有更好的血液稳定性,可在脑部有效驻留48h。
实施例5 轴突靶向递送复合物修饰的外泌体的细胞结合能力和贮留能力实验
在本实施实例中,使用BV2细胞(一种小鼠神经小胶质细胞系)作为对象进行靶向递送复合物的细胞结合能力与贮留能力。使用DiR染料对轴突靶向递送复合物修饰的外泌体进行表面标记,使用CFSE染料对BV2细胞进行标记,最后在激光共聚焦显微镜下进行观察。
在实际操作中,DiR工作液标记外泌体的操作如上所述,配制5µM的CFSE工作液,向工作液中加入重悬的BV2细胞到终浓度为1x10^6个/ml,37℃水浴30min,加入5倍体积的冷培养基终止染色并孵育5min,1000rpm/min * 5min离心沉淀细胞并用培养基洗涤三次,最后收集细胞重新接种到皿里与浓度为1x10^8 particles/ml经DiR染色的经靶向递送复合物修饰的外泌体孵育24h后,在激光共聚焦显微镜下用488nm激发光和750nm激发光进行观察,如附图9。
结果显示,与自然外泌体相比,经靶向递送复合物修饰的外泌体有更好的与BV2细胞的结合能力与贮留能力。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内所作的任何修改、等同替换、改进等,均应包含在本发明的包含范围之内。
序列表
<110> 广西大学
<120> 一种可在外泌体表面进行轴突靶向修饰的递送复合物及其对外泌体的修饰方法
<141> 2022-05-26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14
<212> PRT
<213> 人工序列(Artificial sequence Latin)
<400> 1
Ser Ala Cys Gln Ser Gln Ser Gln Met Arg Cys Gly Gly Gly
1 5 10
<210> 2
<211> 20
<212> PRT
<213> 人工序列(Artificial sequence Latin)
<400> 2
Lys Lys Lys Ser Ala Cys Gln Ser Gln Ser Gln Met Arg Cys Gly Gly
1 5 10 15
Gly Lys Lys Lys
20
Claims (10)
1.一种可在外泌体表面进行轴突靶向修饰的递送复合物,其特征在于,所述递送复合物
由TAxI多肽和DSPE-PEG2000-NHS制成;
所述TAxI多肽由SEQ ID NO:1所示序列的两端各加入一个三赖氨酸嵌段制成;
所述TAxI多肽在C端赖氨酸接Biotin,所述TAxI多肽在N端赖氨酸与DSPE-PEG2000-NHS接枝。
2.根据权利要求1所述的可在外泌体表面进行轴突靶向修饰的递送复合物,其特征在于,所述TAxI多肽的序列如SEQ ID NO:2所示。
3.根据权利要求1或2所述的可在外泌体表面进行轴突靶向修饰的递送复合物,其特征在于,所述TAxI多肽的两个半胱氨酸间形成二硫键使多肽环化。
5.权利要求4所述的可在外泌体表面进行轴突靶向修饰的递送复合物,其特征在于,所述DSPE-PEG2000-NHS的分子式为C139H271N4O57P,分子量为2900。
6.一种轴突靶向递送复合物修饰外泌体的方法,其特征在于,由权利要求1的递送复合物对干细胞外泌体进行表面修饰而成;所述递送复合物预先制成DSPE-PEG2000-TAxI-Biotin胶束;
所述DSPE-PEG2000-TAxI-Biotin胶束和外泌体的质量比为1:1 (按µg蛋白:µg蛋白)。
7.根据权利要求6所述轴突靶向递送复合物修饰外泌体的方法,其特征在于,所述外泌体为人脐带间充质干细胞外泌体、血液外泌体、唾液外泌体或尿液外泌体的任意一种。
8.根据权利要求6所述轴突靶向递送复合物修饰外泌体的方法,其特征在于,具体制备步骤如下:
(1)合成TAxI多肽;
(2)将AxI多肽与DSPE-PEG2000-NHS接合,获得递送复合物;
(3)将步骤(2)合成的递送复合物制成胶束,再***外泌体脂双层膜中。
9.根据权利要求8所述轴突靶向递送复合物修饰外泌体的方法,其特征在于,所述合成TAxI多肽的步骤是将SEQ ID NO:1序列C端氨基酸的羧基以共价键的形式与不可溶的高分子树脂相接,再以这一氨基酸的氨基作为多肽合成的起始点,同其他的氨基酸已活化的羧基反应生成肽键,持续重复该过程,获得多肽后,再在多肽片段的两个半胱氨酸的巯基间形成二硫键进行环化修饰,最后用高效液相色谱进行纯化,即得TAxI多肽。
10.根据权利要求8所述轴突靶向递送复合物修饰外泌体的方法,其特征在于,步骤(2)中,DSPE-PEG2000-NHS与TAxI多肽的物质的量比为2:1。
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