CN113024879A - 凝胶微球及其制备方法与应用 - Google Patents
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Abstract
本发明涉及医药技术领域,尤其涉及凝胶微球及其制备方法与应用。本发明提供了以GelMA为主要原料制备的凝胶微球,该微球具有多孔结构,且表面携带羧基,能够连接多肽药物,且在体外可通过溶胀吸附细胞并应用于组织再生工程。
Description
技术领域
本发明涉及医药技术领域,尤其涉及凝胶微球及其制备方法与应用。
背景技术
微流控技术能够同时调控多个液体流动相制备具有预设成分和稳定特殊结构的微/纳米级均质水凝胶微球,成为“精准医学”替代疗法中最具希冀的生物纳米材料候选人之一。其不仅作为生物信息传递系统表现出独特的应用前景,而且经冻干技术可加工为具有提高营养物质转运和细胞代谢产物运输功能的多孔微球。更能够以微创注射形式有效负载细胞,实现特定组织损伤后修复和再生。但是,依旧存在多孔微球负载生物因子突释、半衰期缩短以及引起局部组织水肿或炎症反应等不良因素。在此等情形下更不利于多孔微球中所搭载细胞的生物学特性,严重影响组织预后。所以如何通过微流控水凝胶微球技术长期稳定为所负载细胞搭建适宜微环境成为当下急需突破的瓶颈。
目前,微流控微球负载细胞的方法是将细胞与水凝胶材料预先混合,再交联形成载细胞微球;或先制备多孔微球而后种植细胞。后者更提高细胞生存率、增殖以及更低的细胞毒性。制备出的载细胞微球能够以微创的方式直接注射到损伤部位来促进局部组织的修复。例如:研究显示,在以局部注射载细胞或生物因子微球到动物椎间盘退变模型中,可有效补充生长因子和种子细胞,干预髓核细胞外基质合成/分解代谢失衡所引起的级联反应。但是退变椎间盘内组织细胞自噬凋亡和外周炎性细胞浸润形成恶劣炎症微环境,成为制约靶向外源性细胞因子转运和限制移植细胞存活的重要因素。
因此,改善微流控水凝胶微球长期调控病灶微环境的功能,以及提高微流控水凝胶微球负载细胞的功能,能够更高效发挥治疗作用。
发明内容
有鉴于此,本发明要解决的技术问题在于提供具有良好负载细胞功能且可修饰多肽药物的凝胶微球及其制备方法与应用。
本发明提供的凝胶微球的制备方法,包括:使分散相在连续相剪切下形成微滴,经光交联形成胶样微球,冷冻后制得凝胶微球;
所述分散相包括水、GelMA和光引发剂;
所述连续相包括油性溶剂和表面活性剂;
所述油性溶剂选自肉豆蔻酸异丙酯、硅油、矿物质油中至少一种;
所述光引发剂选自I2595、LAP中至少一种;
所述表面活性剂为span 80。
在本发明实施例中,所述分散相中包括水、5%w/v~10%w/v的GelMA和0.1%w/v~1%w/v的I2595。
一些具体实施例中,所述分散相由水和7%w/v的GelMA和0.5%w/v的I2595组成。
在本发明实施例中,所述连续相中包括肉豆蔻酸异丙酯和8%w/w~12%w/w的span 80。
一些具体实施例中,所述连续相中包括肉豆蔻酸异丙酯和10%w/w的span 80。
本发明中,所述凝胶微球采用同轴电纺的方式进行制备。
分散相通过同轴电纺喷头的内针,连续相通过同轴电纺喷头的外针。
其中,内针的直径为30G,外针的直径为22G。
在本发明中,所述分散相的流动方向与连续相的流动方向呈90°~180°角。即采用的同轴电纺喷头中,内针与外针的垂直或平行。
一些实施例中,所述分散相的流速为10~30μL/min,所述连续相的流速200~600μL/min。
一些具体实施例中,所述分散相的流速为20μL/min,所述连续相的流速200μL/min、300μL/min、450μL/min或600μL/min。
在本发明中,所述光交联的条件包括:紫外光365nm,6.9mW·cm-2,照射5min。
在本发明中,所述冷冻的温度为-10~-30℃,时间为6~10h。
一些实施例中,所述冷冻的温度为-20℃,时间为8h。
在本发明中,在所述冷冻前还包括清洗的步骤,在所述冷冻后还包括冷冻干燥的步骤;所述清洗包括以异丙醇和体积分数为75%乙醇水溶液洗涤后,以PBS缓冲液浸泡;所述冷冻干燥的时间为40~60h。其中,以PBS缓冲液浸泡的次数为6次,每次4h。每次浸泡后更换新鲜的PBS缓冲液。所述冷冻干燥的时间为48小时。冷冻干燥的条件为-60℃,真空气压为0.10mbar。
本发明所述制备方法制得的凝胶微球。
本发明所述制备方法制得的凝胶微球直径为100~400μm。
分散相流速为20μL/min连续相流速为200μL/min,制备所得凝胶微球的平均直径为400μm。
分散相流速为20μL/min连续相流速为300μL/min,制备所得凝胶微球的平均直径为300μm。
分散相流速为20μL/min连续相流速为450μL/min,制备所得凝胶微球的平均直径为200μm。
分散相流速为20μL/min连续相流速为600μL/min,制备所得凝胶微球的平均直径为100μm。
GelMA由明胶和甲基丙烯酸酐(MA)合成,采用本发明提供方法制备的凝胶微球,表面携带有-COOH,可以与多肽携带的氨基反应,从而将多肽连接在凝胶微球上。因此,本发明提供的凝胶微球可以作为多肽的载体。并且,在冷冻过程中凝胶内部产生冰晶,冻干或融化后,冰晶被抽去,因此经过冷冻的凝胶微球会形成多孔结构。因此本发明提供的凝胶微球还可以作为细胞载体。作为细胞载体的凝胶微球可以修饰多肽,也可以不修饰多肽。
本发明还提供了一种多肽-凝胶微球,其特征在于,为修饰多肽的凝胶微球,所述凝胶微球为本发明所述的凝胶微球。
在本发明实施例中,所述多肽选自细胞因子、动物毒素多肽、人源内生多肽中至少一种。
在一些具体实施例中,所述多肽为APETx2多肽。
本发明所述多肽-凝胶微球的制备方法,包括:
采用碳二亚胺法使多肽与本发明所述的凝胶微球连接,制得所述的多肽-凝胶微球。
在所述多肽-凝胶微球的制备方法中,所述多肽与所述的凝胶微球的质量比为(1~10):105。一些实施例中,所述多肽与所述的凝胶微球的质量比为5:105。
所述碳二亚胺法包括,以EDC和NHS活化所述凝胶微球,然后与多肽反应。所述活化或反应的缓冲液为MES缓冲液,其pH值为6。所述活化的条件包括37℃摇床活化15min;所述反应的条件包括:37℃摇床孵育6h。所述凝胶微球与EDC和NHS的质量比为100:8:12。所述反应后,还包括离心取沉淀、洗涤、干燥的步骤。
本发明所述的凝胶微球,或所述的多肽-凝胶微球,在制备细胞载体中的应用。
本发明还提供了一种细胞-凝胶微球,其由所述的凝胶微球吸附细胞制得。
本发明还提供了一种多肽-细胞-凝胶微球,其由所述的多肽-凝胶微球吸附细胞制得。
一些实施例中,所述多肽-细胞-凝胶微球中,所述多肽为APETx2多肽,所述细胞为髓核细胞。
一些具体实施例中,所述多肽-细胞-凝胶微球中,每100mg微球(冻干)溶胀后吸附1×106个髓核细胞。
本发明所述的细胞-凝胶微球、所述的多肽-细胞-凝胶微球在制备药物中的应用。
本发明还提供了一种药物,其包括药学上可接受的辅料和所述的细胞-凝胶微球、或所述的多肽-细胞-凝胶微球。
本发明还提供了一种治疗椎间盘退变的药物,其包括所述的多肽-细胞-凝胶微球,其中述多肽为APETx2多肽,所述细胞为髓核细胞。
一个具体实施例中,提供了一种多肽-细胞-凝胶微球,其包括本发明所述的凝胶微球、APETx2和髓核细胞;
所述APETx2与凝胶微球通过酰胺键连接,所述髓核细胞吸附于凝胶微球的孔洞中;所述APETx2与所述的凝胶微球的质量比为(1~10):105;
每100mg凝胶微球(冻干后的质量)吸附1×106个髓核细胞。
本发明提供了以GelMA为主要原料制备的凝胶微球,该微球具有多孔结构,且表面携带羧基,能够多肽药物,且在体外可通过溶胀吸附细胞并应用于组织再生工程。
附图说明
图1示调节局部“炎症过激”的可注射“多肽-细胞-水凝胶”微球促进髓核再生的原理图;
图2示GelMA微球的物理表征,(a)油相中微球分布;(b)水相中微球分布;(c,d)不同流速下微球的分布;(e)微球的弹性模量;(f)微球通过微量注射器注射图;
图3示在微球上共价接枝APETx2的表征,(a,b)微球接枝多肽前后的SEM结果;(c-f)微球接枝多肽前后的XPS分析;(g)微球接枝荧光多肽前后的荧光图;(h)多肽微球在不同pH中的释放曲线;
图4示微球中的髓核细胞的生存能力和增殖能力,(a)活死染色;(b)CCK-8结果;(c)细胞骨架染色和细胞SEM图;(d)单个载细胞微球的细胞增殖图;
图5示在体外酸性诱导7天后,在mRNA水平多肽微球调节炎症过激和代谢平衡的作用;
图6示“多肽-细胞-水凝胶”微球应用的手术示意图;
图7示动物实验的影像学资料,(a)穿刺示意图,(b)大鼠尾椎X线图片,(c-e)椎间盘高度指数DHI(%),(f)MRI图片,(g)椎间盘MRI分级;
图8示动物实验的组织学评价,(a)H&E染色图;(b)S-O染色图;(c)COL2免疫组化染色图;(d-f)椎间盘组织学评分。
具体实施方式
本发明提供了凝胶微球及其制备方法与应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明针对传统微流控技术中装置昂贵,重复利用率低等缺点,使用同轴电纺可拆卸喷头制备GelMA凝胶微球。同轴电纺可拆卸喷头方便购买,拆卸清理容易,能够重复利。在微量控制器的控制下,通过改变分散相和连续相的流速,能够制备出理想大小的微球。此外,装置出口处的紫外同步交联盘,能够保障所有微球的微球处在均一的交联时间,避免了过度交联或者交联不完全。
本发明中,所述凝胶微球是指以GelMA为主要原料经同轴电纺、光交联、冷冻后形成的多孔微球材料,其中,同轴电纺步骤由流动相切割含有GelMA的分散相使其形成微球形态,然后在光固化的作用下交联、凝固形成胶样微球。胶样微球不存在孔洞结构,在冷冻过程中胶样微球中产生冰晶冻干或融化后,冰晶被抽去,因此经过冷冻的凝胶微球会形成多孔结构。产生孔洞的微球在冻干后再次溶胀,能够恢复多孔状态。
制备凝胶微球的原料可以是多样的,采用GelMA作为主要原料更有利于形成多孔结构。在本发明中,所述光引发剂又称为光敏剂或光固化剂,其可以在紫外光的作用下,促进GelMA交联固化。在本发明一些具体实施例中,所述光引发剂采用2-羟基-4'-(2-羟乙氧基)-2-甲基苯丙酮,即I2595。在本发明中,所述GelMA和光引发剂的质量比为(5~10):(0.1~1)。一些具体实施例中,所述GelMA和I2595的质量比为7:0.5。
在连续相中,所述油性溶剂是指与水不能互溶的液体,并且其不与GelMA或光引发剂发生反应。利用油性溶剂与水不能互溶的特性,通过控制分散相和流动相的流速,以连续相切割分散相,形成油包水(water in oil)的形式的液滴,进入交联盘。在一些具体实施例中,所述油性溶剂采用肉豆蔻酸异丙酯。为了更好的形成液滴连续相中添加表面活性稳定剂,一些具体实施例中,采用Span80作为表面活性稳定剂。
分散相和连续相通过同轴电纺喷头的直径和流速与形成液滴的粒径密切相关。多孔微球孔洞的大小与GelMA的浓度,交联时间,冷冻的条件密切相关。粒径过小则无法形成完整孔洞,而粒径过大则无法实现注射,只能采用体外移植的方式使用。孔洞直径过小无法吸附细胞、但孔洞直径过大也不利于细胞的定植。本发明实施例中,提供的参数下形成的多孔微球粒径和孔洞大小最适宜用于担载多肽药物和细胞。
采用GelMA作为主要原料,其携带的羧基可以与氨基缩合,从而将凝胶微球与多肽偶联。与多肽偶联的凝胶微球在本发明中记为“多肽-凝胶微球”,其中多肽可选自细胞因子、动物毒素多肽、人源内生多肽中至少一种。所述细胞因子选自白细胞介素、集落刺激因子、干扰素、肿瘤坏死因子、转化生长因子-β家族、生长因子、趋化因子家族中至少一种。所述动物毒素多肽来自蛇毒、唾液酸、蜂毒、蛙毒、蝎毒、水蛭素、海葵毒素、竽螺毒素或苍蝇杀菌肽。所述人源内生多肽包括脑啡肽、胸腺肽、胰脏多肽中至少一种。
冻干的凝胶微球表面通过碳二亚胺法接枝功能化的多肽,能够赋予多孔微球调节局部炎症过激的能力。不仅如此,凝胶微球在体外可通过溶胀能够吸附细胞并应用于组织再生工程。通过溶胀负载细胞不仅无副作用,还能够作为3D培养支架促进细胞的存活和增殖。
利用本发明所述的凝胶微球,直接溶胀后吸附细胞形成的生物材料在本发明中记为“细胞-凝胶微球”。在本发明中,所述“多肽-凝胶微球”溶胀后并吸附细胞形成的生物材料在本发明中记为“多肽-细胞-凝胶微球”。可担载的细胞根据治疗目的进行选择,例如,可选自表皮细胞、粘膜细胞、被覆细胞、淋巴细胞、造血细胞、间皮细胞、平滑肌细胞、软骨细胞、间叶细胞、上皮细胞、神经细胞、骨骼肌细胞、心肌细胞、间充质干细胞、造血干细胞、iPS细胞或髓核细胞。
本研究中,受椎间盘退变局部病理炎症微环境启发,设计构建了兼具炎症微环境调控和促进椎间盘组织再生潜能的仿生微流控水凝胶多孔微球。我们先通过微流控技术制备GelMA水凝胶多孔微球,之后再通过碳二亚胺法在微球表面共价修饰APETx2活性肽,长效下调ASIC-3表达,抑制局部髓核细胞自噬作用,减少炎症因子分泌,提高负载髓核细胞增殖活力,增强细胞外基质沉积作用,达到抑制椎间盘退变的同时促进髓核组织再生疗效。在表征相关实验中,首先,通过光镜、电镜、药物释放曲线等对该复合多孔微球的理化特性进行相关评价。随后,体外模拟椎间盘退变酸性微环境实验中,通过CCK-8、活死染色、PCR和Western blot等对其免疫炎症调控和抑制髓核细胞自噬生物学性能进行充分表征。最后,注射入大鼠椎间盘退变模型,通过X线、MRI、组织学评分、免疫组化对抑制炎症反应和促髓核组织再生功能进行系统性评估。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1
(1)GelMA凝胶微球的制备:
将同轴电纺喷头通过两个硅胶管(0.5×2mm)连接到两个单独的微流控注射泵上。同轴电纺喷头的内针和外针的直径分别是30G和22G。以外针为连续相,即外针的溶液是10%w/w span 80的肉豆蔻酸异丙酯溶液。以内针作为分散相,即内针的溶液是7%w/vGelMA水溶液和0.5%w/v的光引发剂(I2595);启动注射泵,通过调节外相流速为600μl/min,内向流速为20μl/min,凝胶在连续相液体剪切应力作用下形成了100μm大小的微滴,并从出口输送到交联盘。在紫外光(365nm,6.9mW·cm-2)的照射下,水凝胶液滴通过光胶联形成固体的胶样微球。收集到的微球用异丙醇和75%乙醇反复洗涤,除去表面活性剂和矿物油,并且在新鲜PBS中每4h浸泡一次微球,重复6次,对未交联的水凝胶单体和光交联剂进行清洗。纯化后的微球经-20℃冷冻8h,再经冷冻干燥48h得到多孔的凝胶微球。
(2)“多肽-凝胶微球“的制备:
步骤(1)制备的100mg的“凝胶微球”(GM)分散在1ml的MES缓冲溶液中(pH=6),之后依次加入8mg的EDC,12mg的NHS在37℃摇床活化15min,然后再加入5ug的APETx2在37℃摇床孵育6h。然后通过快速离心分离得到共价接接枝的多肽-凝胶微球(GM-APETx2),离心后的GM-APETx2在洗涤三遍后,冷冻干燥48h。通过XPS分析多肽-微球的元素以和化学键,以及通过接枝荧光多肽的方法在荧光显微镜下观察,共同证明了多肽通过酰胺键共价接枝到了微球表面。
(3)“多肽-细胞-凝胶微球“的制备:
步骤(2)制备的“多肽-凝胶微球”在辐照杀菌后,将1ml浓缩了1×106细胞的悬液滴加入100mg的微球中,放置培养箱中培养30min,待细胞被充分吸入微球后,使用培养基重悬这些载细胞微球并接种到24孔板中等待进一步应用。
实施例2凝胶微球的物理特性
(1)凝胶微球的形貌特征SEM
使用SEM(SEM,S-4800;Hitachi,Tokyo,Japan)观察实施例1步骤(1)制备的凝胶微球的大小及表面孔径。冻干后的微球进行喷金处理(SC7620,Quorum Technologies,UK),SEM图像设定放大倍率为3000倍。
(2)实施例1步骤(1)制备的凝胶微球的弹性模量。
使用AFM测量了微球表面杨氏模量,将赫兹接触模型应用于拉伸力位移曲线。
(3)多肽-凝胶微球的释放率。
反应后的上清和洗涤后的上清收集在一起作为总的上清。为了测定APETx2在“多肽-凝胶微球”上的载药率,通过高效液相色谱(HPLC)测定了总上清中的APETx2含量,并通过以下公式计算了载药率:
载药率=(APETx2的总质量-残留的APETx2的质量)/APETx2的总质量×100%。
等量的GM-APETx2分别放置在pH7.4和pH6.5的1mlPBS溶液中,分别在(1,3,5,14和28天)收集1ml上清并储存在-80℃,同时分别加入1ml新的pH7.4和pH6.5的PBS溶液。所收集的上清中APETx2的含量最后通过HPLC测定,并且计算每个时间点的释放量和累计释放率。
(4)微球的降解率。
为了评估GM接枝多肽后的降解能力,分别将100mg的GM和GA加入到1ml含有0.2%二型胶原酶的PBS中,放置在37℃,120rpm的摇床中持续一周。每天更换新的含有二型胶原酶的PBS来维持酶的活性,并在预设的时间点收取微球,用去离子水洗涤两边后冻干称重,并用以下的公式计算降解率:
W0代表最初冻干的样品质量;Wt代表时间t后冻干的样品质量。
实施例3细胞在微球上的粘附生长
(1)微球对细胞的毒性实验LIVE/DEAD
髓核细胞在两种pH条件下,在“凝胶微球”和“多肽-凝胶微球”上培养3天和7天后,去除培养基,用PBS冲洗三遍,然后将配好的活死染色溶液(将5μl钙黄绿素和20μl溴乙非啶豪莫二聚体,加入到10ml PBS中)加入到样品中,每个样品200μl,室温孵育30min,去除染色溶液,PBS清洗3次,拍照。
(2)细胞在微球上的增殖实验CCK-8
使用cell counting kit-8(CCK-8)实验测试髓核细胞在两种pH条件下,在“凝胶微球”和“多肽-凝胶微球”上1、3、7天后的增殖情况,培养基去除后PBS清洗三遍,300μL培养液加30μL的CCK-8试剂,放入37℃培养箱2小时。然后将100μl的孵育介质转移到另一个96孔板中。最后用酶标仪在450nm波长下读出吸光度的数值。
(3)细胞在微球上的形态
将髓核细胞吸附于微球上,培养24小时后去除培养液,PBS冲洗三遍,使用4%多聚甲醛固定半小时后PBS冲洗三遍,接着使用逐步增加浓度的酒精溶液进行梯度脱水,在干燥容器中进行干燥,喷金后进行SEM观察。
将髓核细胞吸附于微球上培养24小时后去除培养基,4%多聚甲醛固定半小时,PBS冲洗三遍,使用0.3%的Triton X-100进行通透处理20分钟,PBS再次冲洗后4%牛血清白蛋白阻断45分钟,最后使用50mg/ml的鬼笔环肽避光40分钟进行细胞骨架荧光染色,细胞核使用DAPI染色,PBS冲洗三遍后荧光显微镜观察细胞的荧光影像。
(4)多肽-凝胶微球对酸性条件下退变髓核的影响
在“多肽-细胞-凝胶微球”通过pH7.4和pH6.5培养基干预7天后,通过实时荧光定量PCR分析检测了ASIC-3,IL-1β,IL-6,TNF-α,MMP-3,ADAMTS-5,COL2,AGGRECAN的基因的表达量,最后通过相对定量分析结果。
结果表明,(1)在pH7.4培养下,各组间细胞存活率无差异。但是,在pH6.5培养下,“多肽-凝胶微球”组的细胞存活率高于“凝胶微球”组。(2)在pH7.4培养下,各组间细胞7天的增殖情况无差异。但是,在pH6.5培养下,“多肽-凝胶微球”组在7天的细胞增殖率高于“凝胶微球”组和空白组。(3)细胞骨架荧光染色和SEM的结果均表明细胞能够在微球上得到良好的黏附。(4)在pH7.4培养下,各组间细胞的各个基因表达无差异。但是,在pH6.5培养下,“多肽-凝胶微球”组的ASIC-3表达量明显低于“凝胶微球”组和单纯酸干预组。同时,炎症相关基因和基质分解的表达量在“多肽-凝胶微球”组明显降低,而基质合成基因的表达量明显高于其他两组。
实施例4可注射“多肽-细胞-水凝胶”微球对椎间盘的修复作用
前期试验表明,将多肽与髓核细胞联用直接给与动物效果非常有限。并且,由于多肽的半衰期极短,且细胞缺少赖以生存的基质,存活率会明显降低,因此直接给药的方式需要多次给药,而每次注射给药也会对椎间盘造成的损伤。因此,考虑采用本发明提供的方案给药:
30只雄性SD大鼠被随机分为5组:未处理组为the normal control group(NC),单纯穿刺组为the degradation control group(DC),注射“凝胶微球”+髓核细胞组为GN组,注射“多肽-凝胶微球”组为GA组,注射“多肽-细胞-凝胶微球”组为GNA组。在退变的椎间盘内注射10μl各组材料,评价椎间盘内的再生情况,进行组织学检测。结果如图所示,最终GNA组的修复效果均好于其他3组,并与未处理组相似。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (19)
1.凝胶微球的制备方法,包括:使分散相在连续相剪切下形成微滴,经光交联形成胶样微球,冷冻后制得凝胶微球;
所述分散相包括水、GelMA和光引发剂;
所述连续相包括油性溶剂和表面活性剂;
所述油性溶剂选自肉豆蔻酸异丙酯、硅油、矿物质油中至少一种;
所述光引发剂选自I2595、LAP中至少一种;
所述表面活性剂为span 80。
2.根据权利要求1所述的制备方法,其特征在于,
所述分散相中包括水、5%w/v~10%w/v的GelMA和0.1%w/v~1%w/v的I2595;
所述连续相中包括肉豆蔻酸异丙酯和8%w/w~12%w/w的span 80。
3.根据权利要求1或2所述的制备方法,其特征在于,
所述分散相的流动方向与连续相的流动方向呈90°~180°角;
所述分散相的流速为10~30μL/min,所述连续相的流速200~600μL/min;
所述光交联的条件包括:紫外光365nm,6.9mW·cm-2,照射5min;
所述冷冻的温度为-10~-30℃,时间为6~10h。
4.根据权利要求1~3任一项所述的制备方法,其特征在于,
在所述冷冻前还包括清洗的步骤,在所述冷冻后还包括冷冻干燥的步骤;所述清洗包括以异丙醇和体积分数为75%乙醇水溶液洗涤后,以PBS缓冲液浸泡;所述冷冻干燥的时间为40~60h。
5.权利要求1~4任一项所述制备方法制得的凝胶微球。
6.根据权利要求5所述的凝胶微球,其特征在于,其直径为100~400μm。
7.多肽-凝胶微球,其特征在于,为修饰多肽的凝胶微球,所述凝胶微球为权利要求5或6所述的凝胶微球。
8.根据权利要求7所述的多肽-凝胶微球,其特征在于,所述多肽选自细胞因子、动物毒素多肽、人源内生多肽中至少一种。
9.根据权利要求7所述的多肽-凝胶微球,其特征在于,所述多肽为APETx2多肽。
10.权利要求7~9任一项所述的多肽-凝胶微球的制备方法,包括:
采用碳二亚胺法使多肽与权利要求5或6所述的凝胶微球连接,制得所述的多肽-凝胶微球。
11.根据权利要求10所述的制备方法,其特征在于,所述多肽与权利要求5或6所述的凝胶微球的质量比为(1~10):105。
12.权利要求5或6所述的凝胶微球,或权利要求7~9任一项所述的多肽-凝胶微球,在制备细胞载体中的应用。
13.细胞-凝胶微球,其由权利要求5或6所述的凝胶微球吸附细胞制得。
14.多肽-细胞-凝胶微球,其由权利要求7~9任一项所述的多肽-凝胶微球吸附细胞制得。
15.根据权利要求14所述的多肽-细胞-凝胶微球,其特征在于,所述多肽为APETx2多肽,所述细胞为髓核细胞。
16.权利要求13所述的细胞-凝胶微球、权利要求14或15所述的多肽-细胞-凝胶微球在制备药物中的应用。
17.一种药物,其包括药学上可接受的辅料和权利要求13所述的细胞-凝胶微球、权利要求14或15所述的多肽-细胞-凝胶微球。
18.多肽-细胞-凝胶微球,其包括权利要求5或6所述的凝胶微球、APETx2和髓核细胞;
所述APETx2与凝胶微球通过酰胺键连接,所述髓核细胞吸附于凝胶微球的孔洞中;
所述APETx2与权利要求5或6所述的凝胶微球的质量比为(1~10):105;
每100mg凝胶微球吸附1×106个髓核细胞。
19.一种治疗椎间盘退变的药物,其特征在于,包括权利要求18所述的多肽-细胞-凝胶微球。
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