CN116327971A - 一种靶向cd74+促炎型巨噬细胞的药物载体及其制备方法和应用 - Google Patents
一种靶向cd74+促炎型巨噬细胞的药物载体及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种靶向CD74+促炎型巨噬细胞的药物载体的制备方法，首先合成了与CD74特异性结合的MIF多肽（MIFPeptide），并将此MIF多肽偶联于PLGA‑PEG5K‑Mal，从而构建通过MIFPeptide靶向CD74+促炎型巨噬细胞的药物载体（MIFPeptide‑PLGA）。MIFPeptide‑PLGA可进一步负载抗炎药物。本发明有助于推动相关靶向抗炎药物的制备，提高骨性关节炎相关药物的疗效，并为新药物研发提供线索。
Description
技术领域
本发明涉及生物医用领域，尤其涉及一种靶向CD74+促炎型巨噬细胞的药物载体及其制备方法和应用。
背景技术
膝关节骨性关节炎（Knee Osteoarthritis，以下简称OA）是导致下肢功能障碍的主要骨科疾病之一，严重影响患者的生活质量并加剧家庭和社会的经济负担。然而目前并没有针对OA的有效药物，当病情进展到晚期时，只能接受手术治疗。
巨噬细胞与骨性关节炎（Osteoarthritis，OA）的进展密切相关。由于创伤等因素导致的膝关节内组织损伤会产生无菌性的炎性渗出，产生炎性因子，刺激单核巨噬细胞的局部浸润、增殖以及活化，活化的巨噬细胞为促炎型巨噬细胞，可分泌大量可溶性促炎因子，作用于滑膜细胞、免疫细胞等，进一步加重关节内的炎症反应，招募更多的巨噬细胞，从而形成恶性循环。
OA早期滑膜组织中即可检测到增多的巨噬细胞和促炎细胞因子，产生慢性炎症微环境，刺激滑膜细胞或软骨细胞产生MMPs或ADMTS4/5等，引起软骨的损伤，加剧OA的进展。促炎型巨噬细胞在滑膜组织炎症以及OA进展中起重要作用，及时有效的缓解巨噬细胞的炎症状态，是减轻滑膜组织炎症，延缓OA进展的有效途径。
前期的研究结果显示，在OA的进展过程中，滑膜中促炎型巨噬细胞大量增加，并高表达CD74，而CD74是巨噬细胞迁移抑制因子（Macrophage migration inhibitory factor，MIF）的特异性结合受体，因此，本发明通过构建能与CD74特异性结合的MIF多肽结构，并将此多肽结构偶联到PLGA-PEG5K-Mal支架并负载抗炎药物，可达到靶向CD74+促炎型巨噬细胞进而起到抗炎效果，从而用于制备靶向抗炎药物，减轻膝关节内炎症反应及OA进展。
发明内容
本发明的目的是提出一种靶向CD74+促炎型巨噬细胞的药物载体及其制备方法和应用，以解决现有技术中的早中期膝骨关节炎缺乏特异性抗炎药物的问题，同时提供了一种新的膝关节滑膜组织炎症的治疗靶点。
本发明的目的将通过以下技术方案得以实现：
本发明一方面提供了一种靶向CD74+促炎型巨噬细胞的药物载体的制备方法，包括以下步骤：
步骤1、合成MIF多肽：合成靶向CD74的MIF多肽结构MIFPeptide；
步骤2、偶联MIF多肽：将MIFPeptide偶联到PLGA-PEG5K-Mal上，得到靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA。
进一步的，所述MIFPeptide的氨基酸序列N’-C’为半胱氨酸-亮氨酸-半胱氨酸-甘氨酸-亮氨酸-亮氨酸-丝氨酸-天冬氨酸-精氨酸，并在其N端偶联FITC进行示踪。
本发明另一方面提供了一种上述的制备方法得到的靶向CD74+促炎型巨噬细胞的药物载体。
本发明另一方面提供了一种上述的制备方法得到的靶向CD74+促炎型巨噬细胞的药物载体在制备治疗膝关节骨性关节炎的药物或药物组合物中的应用。
进一步的，在所述靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA上偶联抗炎药物。
本发明的突出效果为：
本发明首先合成了与CD74特异性结合的MIF多肽（MIFPeptide），并将此MIF多肽偶联于PLGA-PEG5K-Mal，从而构建通过MIFPeptide靶向CD74+促炎型巨噬细胞的药物载体（MIFPeptide-PLGA）。MIFPeptide-PLGA可进一步负载抗炎药物。本发明有助于推动相关靶向抗炎药物的制备，提高骨性关节炎相关药物的疗效，并为新药物研发提供线索。
以下便结合实施例，对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
附图说明
图1A为本发明实施例1中小鼠正常及骨性关节炎滑膜组织免疫荧光染色结果图；
图1B为本发明实施例1中小鼠正常及骨性关节炎滑膜组织细胞流式分析结果图；
图1C为本发明实施例1中临床正常及滑膜炎患者的滑膜组织免疫荧光染色结果图；
图2A为本发明实施例2中Raw 264.7细胞经LPS处理后细胞流式分析结果图；
图2B为图2A的统计分析；
图2C为本发明实施例2中正常以及OA患者滑膜组织炎症性巨噬细胞标志物CD86以及CD74的共染色；
图3为本发明实施例3中靶向CD74的MIFPeptide氨基酸序列示意图；
图4为本发明实施例4中靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA-PEG-MAL的结构示意图；
图5为本发明实施例5的免疫荧光染色结果图；
图6为本发明实施例6的靶向CD74+促炎型巨噬细胞的抗炎药物MIFPeptide-PLGA-Luteolin的结构示意图；
图7A为本发明实施例7中软骨组织番红O/快绿染色；
图7B为本发明实施例7中膝关节HE染色，评估滑膜炎症水平；
图7C为本发明实施例7中骨关节炎严重程度的Mankin score，分值越高，软骨损伤越严重；
图7D为本发明实施例7中滑膜炎严重程度的Synovitis score，分值越高，滑膜组织炎症越严重；
实施方式
为使本发明的目的、技术方案及效果更加清楚、明确，以下参照实施例对本发明作进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限本发明。
本发明上下文中所使用的的试剂，均通过市售获得。本发明PLGA-PEG5K-Mal购买自Ruixibio，Luteolin购买自MedChemExpress。本发明所使用的实验方法，如分子克隆、细胞实验及动物实验等均为本领域的常规方法和技术。对于动物实验，相关程序及方法符合医学伦理要求。本发明所使用的实验方法均为本领域的常规方法和技术。
实施例1：CD74+巨噬细胞在骨性关节炎滑膜组织中表达增加
将小鼠正常滑膜组织（Sham组）及骨性关节炎滑膜组织（OA组）进行免疫荧光染色，结果如图1A所示，相比于Sham组，OA组中F4/80+CD74+的巨噬细胞明显增加。提取小鼠正常滑膜组织（Sham组）及骨性关节炎滑膜组织（OA组），通过流式细胞术分析F4/80+CD74+巨噬细胞数量，结果如图1B所示，相比于Sham组，OA组中F4/80+CD74+的巨噬细胞增加了3.32倍。将临床正常的滑膜组织（Control组）和滑膜炎患者的滑膜组织（OA组）进行免疫荧光染色，结果如图1C所示，相比于Control组，OA组中F4/80+CD74+的巨噬细胞也明显增加。
实施例2：CD74+巨噬细胞为促炎型巨噬细胞
利用LPS（50 ng/ml）刺激小鼠Raw 264.7巨噬细胞系24 h后，细胞流式分析结果如图2A和2B所示，F4/80+CD74+的巨噬细胞显著增加。免疫荧光染色显示，相比于Control组，OA组的CD74表达明显增加，并且CD74+细胞同样特异性高表达炎症性巨噬细胞标志物CD86，（图2C），故称此群巨噬细胞为CD74+的促炎型巨噬细胞，与骨性关节炎滑膜组织的炎症反应密切相关，可能为OA的潜在治疗靶点。
实施例3：合成靶向CD74的MIF多肽结构
通过常用的Fmoc固相合成法合成MIF多肽（MIFPeptide）。包括1）树脂的选择及氨基酸的固定；2）氨基、羧基、侧链的保护及脱除；3）成肽反应；4）裂解及合成肽链的纯化步骤。首先选择固相合成多肽的载体羧基树脂（如王氏树脂），将所要合成肽链的羟末端氨基酸的羟基以共价键的结构同王氏树脂相连，然后以此结合在王氏树脂上的氨基酸作为氨基组份，利用碱性溶剂piperidine去除Fmoc保护柱和单体氨基的保护基团，以便于后续肽链的合成，经过脱去氨基保护基并同过量的活化羧基组分反应，氨基酸的羧基与游离的氨基反应交联，形成肽键；重复操作，接长肽链，达到所要合成的肽链长度。最后多肽从柱上洗脱下来，其保护基团被脱保护剂TFA洗脱和脱保护，最终合成氨基酸序列（N’-C’）为“半胱氨酸-亮氨酸-半胱氨酸-甘氨酸-亮氨酸-亮氨酸-丝氨酸-天冬氨酸-精氨酸”的肽段（图3），并在其N端增加FITC进行示踪。
实施例4：构建一种靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA
为了将MIFPeptide偶联到PLGA-PEG-Mal聚合物上，选择马来酰亚胺-硫醇化学反应，将存在于MIF肽C端半胱氨酸中的巯基(SH)共价连接到PLGA-PEG-Mal聚合物的Mal分子上。这个半胱氨酸不是原来的MIF肽氨基酸序列的一部分，它是在商业肽合成过程中插入于一侧。具体的合成步骤如下：在加入PLGA-PEG-MAL纳米颗粒之前，为了保证游离SH基团的存在，将TCEP添加到MIFPeptide (摩尔比率：1:1)中，在室温下孵育1小时后加入PLGA-PEG-MAL纳米颗粒。为了使PLGA-PEG-Mal末端与MIF多肽C端巯基键形成共价键，将MIFPeptide/TCEP溶液与PLGA-PEG-Mal溶液(摩尔比率：1.5:1)混合在一起, 溶于适量中性PBS溶液中,避光搅拌过夜后4℃保存。同时，将没有加入MIFPeptide 的聚合物作为对照。为了去除非共轭肽，聚合物用冷乙醚沉淀，3200 g离心5分钟，收集上清，在每次聚合物沉淀之间用超声波重复一次(3分钟)。此外，聚合物用超纯水清洗三次，最终得到靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA-PEG-MAL（简写为MIFPeptide-PLGA）最后，将MIFPeptide-PLGA聚合物冷冻干燥并在- 20°C保存。（图4）。
实施例5：MIFPeptide-PLGA靶向小鼠滑膜炎症组织中CD74+巨噬细胞
利用内侧半月板失稳手术构建OA模型，通过关节腔注射技术将MIFPeptide-PLGA（FITC标记）输送到小鼠关节腔内，使其作用于膝关节局部组织，24 h后收集小鼠小鼠膝关节标本并制作切片，免疫荧光染色显示，结果如图5所示，MIFPeptide-PLGA（FITC）与CD74存在明显的共定位现象，表明MIFPeptide-PLGA可以靶向CD74+巨噬细胞。
实施例6：构建靶向CD74+促炎型巨噬细胞的靶向抗炎药物MIFPeptide-PLGA-Luteolin
采用PLGA和PLGA-PEG-MAL共混物制备了聚合物纳米颗粒。将PLGA和PLGA-PEG-MAL聚合物(w/w=4:1)溶于乙酸乙酯和二氯甲烷组成的溶液中(v/v = 3/7)，Luteolin溶于二氯甲烷，与MIFPeptide-PLGA溶液充分混合，在冰水浴中进行超声处理(功率：180W, 3 s开，1 s关，5分钟)。接下来，将得到的乳剂在超声处理下滴入3%聚乙烯醇(PVA)溶液中。最后，利用旋转蒸发器对有机溶剂进行挥发干燥，得到MIFPeptide-PLGA-Luteolin（其结构如图6所示）。
实施例7：MIFPeptide-PLGA-Luteolin延缓小鼠骨性关节炎的进程
通过DMM手术构建小鼠OA模型后，立即开始关节腔注射PBS，Luteolin及MIFPeptide-PLGA-Luteolin（30 μM），每3周注射一次，共持续12周。番红O/快绿染色显示，如图7A所示，PBS组呈现明显的软骨破坏，软骨表层及中间层缺失，肥大软骨细胞增多，番红O染色减弱，骨性关节炎评分（Mankin Score）达到8.6；单纯Luteolin组也呈现明显的软骨损伤，结构缺失，染色减弱，骨性关节炎评分为7.6；而MIFPeptide-PLGA-Luteolin组的软骨结构基本完整，只出现表层的纤维化，番红O染色效果良好，骨性关节炎评分降低到2.4（图7C）；并且相比于PBS组以及单纯Luteolin组，MIFPeptide-PLGA-Luteolin组的滑膜炎评分明显降低（图7B和D），表明相比于单纯的Luteolin，MIFPeptide-PLGA-Luteolin能够更加有效延缓小鼠骨性关节炎的进程。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (5)
1.一种靶向CD74+促炎型巨噬细胞的药物载体的制备方法，其特征在于包括以下步骤：
步骤1、合成MIF多肽：合成靶向CD74的MIF多肽结构MIFPeptide；
步骤2、偶联MIF多肽：将MIFPeptide偶联到PLGA-PEG5K-Mal上，得到靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA。
2.根据权利要求1所述的制备方法，其特征在于：
所述MIFPeptide的氨基酸序列N’-C’为半胱氨酸-亮氨酸-半胱氨酸-甘氨酸-亮氨酸-亮氨酸-丝氨酸-天冬氨酸-精氨酸，并在其N端偶联FITC进行示踪。
3.根据权利要求1-2任一项所述的制备方法得到的靶向CD74+促炎型巨噬细胞的药物载体。
4.根据权利要求1-2任一项所述的制备方法得到的靶向CD74+促炎型巨噬细胞的药物载体在制备治疗膝关节骨性关节炎的药物或药物组合物中的应用。
5.根据权利要求4所述的应用，其特征在于：在所述靶向CD74+促炎型巨噬细胞的药物载体MIFPeptide-PLGA上偶联抗炎药物。
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