CN115944714B - 能够同时抗白细胞介素1和肿瘤坏死因子炎症的纳米材料及其制备方法与应用 - Google Patents
能够同时抗白细胞介素1和肿瘤坏死因子炎症的纳米材料及其制备方法与应用 Download PDFInfo
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- Pain & Pain Management (AREA)
Abstract
本发明公开了一种能够同时抗白细胞介素1和肿瘤坏死因子炎症的纳米材料及其制备方法与应用。本发明采用的纳米材料具有比表面积大,同时偶联IL‑1Ra和sTNFRI两种药物,载药能力高的优势,延长蛋白药物在体内的滞留时间,提高蛋白药物在炎症处的靶向性,并且能够增强蛋白药物的药效,在CIA(胶原诱导的类风湿性关节炎)小鼠体内取得了较好的治疗效果,这为类风湿性关节炎的治疗提供了潜在的方法和策略。
Description
技术领域
本发明涉及一种能够同时抗白细胞介素1和肿瘤坏死因子炎症的纳米材料及其制备方法与应用,属于医药技术领域。
背景技术
类风湿性关节炎(Rheumatoid arthritis,RA)属于自身免疫性疾病,是一种慢性炎症性疾病,可以引起关节发炎、滑膜增生、血管翳形成、骨骼和软骨破坏等。严重者会引发心血管、肺、心理和骨骼疾病。目前类风湿性关节炎治疗的临床用药主要有非甾体抗炎药、糖皮质激素和改善病情抗风湿药。改善病情抗风湿药包括传统的改善病情抗风湿药、免疫抑制剂和生物类改善病情抗风湿药。生物制剂以抑制类风湿性关节炎炎症进程相关的细胞因子为主,可有效抑制类风湿性关节炎发病过程中的炎症反应。
类风湿性关节炎发作过程中,关节处的白细胞介素1(IL-1)含量明显升高,并进一步引发局部的炎症反应。通过白细胞介素1受体拮抗剂(IL1Ra)抑制IL-1受体与IL-1的结合,可抑制IL-1导致的炎症反应,有效减缓病情发展。肿瘤坏死因子-α(TNF-α)是一种多功能细胞因子,在类风湿性关节炎中起关键作用。在类风湿性关节炎发展过程中,1型辅助性T细胞和巨噬细胞分泌的TNF-α激活滑膜成纤维细胞,促进表皮增生,募集炎症细胞。TNF-α抑制剂是现有证据较为充分、应用较为广泛的治疗RA的生物类DMARDs。
然而,有些患者对单一的一种生物制剂尤其是TNF、IL-1抗体治疗会产生严重的耐受性。且游离的IL-1Ra和sTNFRI在体内半衰期短,血液循环时间普遍很短,还存在在炎症关节富集量低,疗效较差的缺陷。
纳米药物一般以纳米为载体装载各种药物。纳米材料不仅便于合成,而且大部分材料在体内具有很好的可降解性。聚乙二醇(Polyethylene glycol,PEG)具有良好的水溶性和生物相容性,是目前已知的蛋白和细胞吸附水平最低的合成高分子材料。PEG经代谢会直接被排出体外,经PEG修饰的聚合物材料应用到载药微球、胶束等时,能有效避免被RES***吞噬从而实现在体内的长循环。聚乳酸-羟基乙酸共聚物(Poly Lactic-co-GlycolicAcid,PLGA)是由两种单体乳酸(LA)和羟基乙酸(GA)随机聚合而成,是一种可降解的功能高分子有机化合物。PLGA水解以后的产物是乳酸和羟基乙酸,可参与人体的新陈代谢,最终形成二氧化碳和水被排出体外。美国食品药品监督管理局(FDA)已经认定PLGA具有良好的生物相容性和安全性,被广泛应用于人类的临床医学研究。
有研究者使用聚乙二醇化可溶性肿瘤坏死因子受体I型(PEG-sTNFRI)治疗慢性炎症性疾病。并且他们的临床I/II期和早期III期数据表明PEG-sTNFRI是非免疫原性的,每周使用该药物可以缓解类风湿性关节炎患者的关节痛和关节肿胀。PEG-sTNFRI的其他临床试验正在进行中,以确定最佳剂量和时间,并进一步评估PEG-sTNFRI的疗效、安全性和潜在免疫原性。
虽然目前已经成功设计出许多运输蛋白的策略,但也经常受到蛋白活性降低的限制,因此在设计新疗法时应仔细考虑生物材料融合对受体结合亲和力的影响。目前,国内外均无专利或文献报道将IL-1Ra和sTNFRI进行整合的产品和方法。
发明内容
为解决上述技术问题,本发明提供一种同时连接IL-1Ra和sTNFRI的纳米材料,增强其药效,减少患者的耐受性。
本发明的第一个目的是提供一种能够同时抗白细胞介素1和肿瘤坏死因子炎症的纳米材料,所述的纳米材料是将人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI和人白介素1受体拮抗剂hs-IL1Ra连接至PLGA-PEG-NHS纳米颗粒上得到。
进一步地,所述的人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI和人白介素1受体拮抗剂hs-IL1Ra通过酯交联反应与PLGA-PEG-NHS纳米颗粒连接。
进一步地,所述的人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI的氨基酸序列如SEQ ID NO.1所示。
LVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTT
进一步地,所述的人白介素1受体拮抗剂hs-IL1Ra的氨基酸序列如SEQ ID NO.2所示。RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE
本发明的第二个目的是提供所述的纳米材料的制备方法,包括如下步骤:
S1、将PLGA-PEG-NHS粉末溶解在有机溶剂中,在缓冲液中进行透析,透析后去除有机溶剂,得到PLGA-PEG-NHS纳米颗粒溶液;
S2、向S1步骤得到的PLGA-PEG-NHS纳米颗粒溶液中加入人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI和人白介素1受体拮抗剂hs-IL1Ra进行酯交联反应,反应后进行超滤离心去除未结合的蛋白,得到所述的纳米材料。
进一步地,在S2步骤中,PLGA-PEG-NHS纳米颗粒溶液中PLGA-PEG-NHS纳米颗粒的浓度为10~20mg/mL。
进一步地,在S2步骤中,人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI按照终浓度为1~2mg/mL加入,人白介素1受体拮抗剂hs-IL1Ra按照终浓度为1~2mg/mL加入。
进一步地,所述的有机溶剂为四氢呋喃、DMSO、二氯甲烷、丙酮中的一种或多种。
进一步地,所述的缓冲液为HEPES缓冲液、磷酸盐、碳酸盐-碳酸氢盐或硼酸盐缓冲液。
进一步地,所述的酯交联反应是在0~30℃下,在pH 7.2~8.5的缓冲液中反应0.5~4小时。
本发明的第三个目的是提供所述的纳米材料在制备治疗类风湿性关节炎的药物中的应用。
本发明的有益效果是:
本发明采用的纳米材料具有比表面积大,同时偶联IL-1Ra和sTNFRI两种药物,载药能力高的优势,延长蛋白药物在体内的滞留时间,提高蛋白药物在炎症处的靶向性,并且能够增强蛋白药物的药效,减少患者的耐受性。通过细胞实验证明本发明中涉及到的交联反应没有破坏蛋白sTNFRI和IL1Ra的活性,因此我们制备的纳米颗粒既能维持sTNFRI和TNF-α结合的生物学活性,也能维持IL1Ra和IL-1β结合的生物学活性。
附图说明
图1为人白细胞介素1受体拮抗剂(hs-IL1Ra)基因表达载体的构建;A.目的基因hs-IL1Ra的扩增;B.重组质粒酶切鉴定;M:DNA marker;
图2为人可溶性肿瘤坏死因子受体I胞外段(hs-sTNFRI)基因表达载体的构建;A.目的基因hs-sTNFRI的扩增;B.重组质粒酶切鉴定;M:DNA marker;
图3为重组蛋白诱导表达后电泳图;A.重组蛋白hs-IL1Ra诱导表达后电泳图;B.重组蛋白hs-sTNFRI诱导表达后电泳图;1,3:未诱导重组菌破碎上清;2,4:诱导后重组菌破碎上清;M:蛋白分子标准marker;
图4为重组蛋白hs-IL1Ra梯度洗脱液的蛋白电泳结果以及免疫印迹分析;M:蛋白分子标准marker;S:细胞裂解上清;FF:穿过液;
图5为重组蛋白hs-sTNFRI梯度洗脱液的蛋白电泳结果以及免疫印迹分析;M:蛋白分子标准marker;S:细胞裂解上清;FF:穿过液;
图6为肠激酶切割融合蛋白以及内毒素检测标准曲线;A:4℃条件下用1.5U肠激酶切割0.5mg蛋白hs-IL1Ra;B:4℃条件下用1.5U肠激酶切割0.5mg蛋白hs-sTNFRI;1,3:蛋白酶切前收样电泳;2,4:蛋白酶切后48h收样电泳;C:内毒素标准曲线;
图7为纯化的蛋白在体外生物学活性的检测;A:cck-8检测胸腺细胞的增殖水平以反映hs-IL1Ra的生物学活性;B:cck-8检测L929的增殖水平以反映hs-sTNFRI的生物学活性;
图8为纳米粒子PLGA-PEG-NHS的表征以及生物相容性的检测;A:DLS测定的粒径及粒径分布;B:PLGA-PEG-NHS的TEM图;C:不同浓度纳米颗粒与L929孵育后的细胞活性实验;D:不同时间对纳米颗粒粒径的检测;
图9为装载hs-IL1Ra和hs-sTNFRI纳米颗粒的生物学活性研究;A:不同浓度的纳米颗粒和蛋白的连接效率;B:检测hs-IL1Ra纳米颗粒的生物学活性;C:检测hs-sTNFRI纳米颗粒的生物学活性;
图10为游离蛋白或蛋白纳米复合物在体内滞留实验的研究;A:小鼠关节腔注射Cy5.5标记的游离蛋白或蛋白纳米复合物后的腿部荧光成像;B:A图的荧光半定量分析;
图11为游离蛋白或蛋白纳米复合物在体内滞留实验的研究;A:小鼠尾静脉注射药物后不同时间点荧光成像图;B:A图的荧光半定量分析;
图12为蛋白纳米复合物治疗关节炎小鼠的初步探索;A:实验流程图;B:治疗后不同组小鼠后爪代表性图片;C:小鼠爪子关节炎临床评分。
具体实施方式
下面结合具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1:构建人可溶性肿瘤坏死因子受体Ⅰ胞外段(hs-sTNFRI)基因表达载体和人白介素1受体拮抗剂(hs-IL1Ra)基因表达载体
1.1引物设计及PCR
人肿瘤坏死因子受体胞外端蛋白(hs-sTNFRI)在UNIPROT查到胞外端区域为(22AA-211AA),计算后对应NCBI数据库的碱基序列为:
CTGGTCCCTCACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGCACTGAGGACTCAGGCACCACA(SEQ ID NO.3)
长度为546bp。选取质粒载体pET-32a上的EcoRⅠ和HindⅢ为限制性内切酶的酶切位点,设计的引物序列为:
F:5’-CGGAATTCGACGACGACGACAAGCGCCCTTCTGGGAAAAGACC-3’;
R:5’-CCCAAGCTTCTATTGGTCTTCCTGGAAGTAG-3’。
人白介素1受体拮抗剂(hs-IL1Ra)在NCBI数据库查得的碱基序列,CDS区除信号肽的序列为目的序列,NCBI数据库对应的碱基序列为:
CGACCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAAAGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTCCAGGAGGACGAGTAG(SEQ IDNO.4)
全长459bp。选取质粒载体pET-28a上的EcoRⅠ和HindⅢ为限制性内切酶的酶切位点,设计的引物序列为:
F:5’-CGGAATTCGACGACGACGACAAGCGACCCTCTGGGAGAAAATCCA-3’;
R:5’-CCCAAGCTTCTACTCGTCCTCCTGGAAGTAG-3’。
由于目的片段连接在质粒载体中,而基因转录所用的的启动子和蛋白纯化所用的6×His标签均在目的片段上游的质粒上,所以除了目的基因外尚有质粒部分连在目的片段前面,因此在设计上游引物时预设了肠激酶酶切位点(GACGACGACGACAAG)以便后期纯化时切除多余部分。
1.2目的片段和载体双酶切以及重组质粒的连接、转化和验证
1)对重组质粒和目的片段进行双酶切。
2)将酶切好的目的片段的质粒连接,转化到大肠杆菌DH5α,抽提质粒得到所需重组质粒。双酶切重组质粒后将显示酶切成功的质粒送去公司进行序列测序鉴定。公司测序结果比对,显示测序正确。表明重组质粒构建成功。结果如图1、2所示。
实施例2:重组蛋白hs-IL1Ra和hs-sTNFRI的诱导表达及纯化
1)将测序正确的重组表达载体转化至E.coli BL21(DE3)。
2)挑取单克隆进行诱导表达,IPTG终浓度为0.1mM。表达结果如图3所示。原核表达提供了大量生产的可能性。从图中可以看出,成功表达了重组蛋白hs-IL1Ra和hs-sTNFRI。
3)超声破裂细菌后使用Ni柱进行蛋白亲和层析,通过分步洗脱的方式,得到较纯的重组蛋白。亲和纯化SDS-PAGE电泳图和蛋白Western如图4、5所示。
实施例3:重组蛋白hs-IL1Ra和hs-sTNFRI的肠激酶酶切以及内毒素含量的检测
1)使用肠激酶酶切除重组蛋白多余部分,为了尽量保持蛋白活性,选择在4℃条件下用1.5U肠激酶切割0.5mg目的蛋白48小时。然后再次使用Ni柱进行蛋白亲和层析得到目的蛋白。酶切结果如图6中A、B所示。
2)表达纯化之后的蛋白在进行后续细胞实验之前,需要先检测内毒素含量。使用重组C因子内毒素检测试剂盒。根据内毒素标准品作出标准曲线,将待测的重组蛋白(1μg/ml)的吸光度代入标准曲线(图6中C),计算得到纯化的两种蛋白的内毒素含量分别为0.58EU/μg,0.675EU/μg。检测结果符合细胞实验的内毒素标准。
实施例4:目的蛋白hs-IL1Ra和hs-sTNFRI在体外生物学活性的检测
测定hs-IL1Ra体外活性的方法:ConA刺激小鼠胸腺细胞测定hs-IL1Ra生物学活性。小鼠胸腺细胞在刺激下表达IL-1R,在有IL-1β条件下,IL-1β协同ConA促进T细胞的增殖作用,而hs-IL1Ra竞争性与IL-1R结合,从而拮抗细胞的增殖作用,根据给药后细胞的增殖水平反映IL-1检测hs-IL1Ra的生物学活性。测定hs-sTNFRI体外活性的方法:以L929细胞为靶细胞,检测hs-sTNFRI对TNF-α杀伤细胞的抑制作用。实验结果如图7中A所示,纯化蛋白hs-IL1Ra能明显抑制胸腺细胞的增殖作用,图7中B显示纯化蛋白hs-sTNFRI能明显挽救TNF-α对L929的杀伤。
实施例5:纳米粒子PLGA-PEG-NHS的制备与表征以及生物相容性的检测
纳米颗粒PLGA-PEG-NHS连接蛋白的原理:NHS酯是通过羧酸盐分子的碳二亚胺活化形成的反应基团。NHS酯活化的交联剂在生理至弱碱性条件下(pH 7.2至9)与伯胺反应,形成稳定的酰胺键。反应释放出N-羟基琥珀酰亚胺(NHS)。由于PLGA具有不溶于水,易溶于有机溶剂的特性,因此,称取公司购买的PLGA-PEG-NHS冻干粉20mg溶在200μL四氢呋喃里,使用磁力搅拌器充分溶解约1小时后逐滴滴入800μL双蒸水中稀释。由于NHS-酯交联反应最常在室温或4℃下于pH 7.2-8.5的磷酸盐、碳酸盐-碳酸氢盐、HEPES或硼酸盐缓冲液中进行,因此需再将纳米溶液装入透析袋中置于HEPES缓冲液中透析过夜,最终得到的纳米颗粒浓度为20mg/mL。
用动态激光光散射仪(DLS)测定粒径如图8中A所示。图8中B为PLGA-PEG-NHS的TEM图。由于蛋白质作为治疗药物的主要障碍是运输材料的毒性,于是制备好纳米颗粒后,我们先检测了不同浓度的纳米颗粒对L929的毒性,以选取合适的材料浓度进行后续的实验。图8中C是不同浓度PLGA-PEG-NHS对L929的毒性分析结果,从图8中C中可以看出,纳米颗粒即使用到5.4mg/mL的浓度时,细胞活性与不加材料的对照组相比没有显著差异。这一结果表明,纳米颗粒几乎没有细胞毒性,其生物相容性很好,这表明该颗粒作为蛋白运输载体是安全的。图8中D是不同时间对纳米颗粒粒径的检测,结果表明一定时间内纳米颗粒粒径没有明显差异,这表明该颗粒作为蛋白运输载体是相对稳定的。
实施例6:装载人可溶性肿瘤坏死因子受体I胞外段(hs-sTNFRI)蛋白和人白介素1蛋白受体拮抗剂(hs-IL1Ra)的纳米颗粒的生物学活性研究
为了保证一定条件下蛋白和纳米颗粒的连接效率,我们首先用不同浓度的纳米颗粒和蛋白连接,结果如图9A所示,15mg/mL的纳米颗粒和蛋白的连接率最高,后续实验所需的纳米颗粒均用此浓度。接着我们制备了装载hs-IL1Ra的纳米颗粒(hs-IL1RaNanoparticles)、装载hs-sTNFRI的纳米颗粒(hs-sTNFRI Nanoparticles)以及同时装载hs-IL1Ra和hs-sTNFRI两种蛋白的纳米颗粒(hs-IL1Ra+hs-sTNFRI Nanoparticles)。装载方法:取PLGA-PEG-NHS纳米颗粒溶液(15mg/mL)200μL于EP管中,再分别加入纯化的人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI(1.5mg/mL)100μL和人白介素1受体拮抗剂hs-IL1Ra(1.5mg/mL)100μL。用移液枪轻轻吹打混匀后放于冷库摇床反应过夜。第二天将反应产物加到含有4mL HEPES缓冲液的超滤离心管中,离心去掉未结合的蛋白。轻轻吸取超滤离心管上方的溶液约400μL保存于4℃冰箱。使用检测蛋白浓度的试剂盒检测剩余蛋白浓度,最终得到同时连接两种蛋白(350-400μg/ml)的纳米颗粒(7-7.5mg/mL)溶液。
然后进行细胞实验检测其活性。
1)测定hs-IL1Ra体外活性的方法:ConA刺激胸腺细胞测定hs-IL1Ra生物学活性。6-8周C57小鼠,取胸腺制备成细胞悬液,细胞密度1.5*106到96孔板中。加入ConA(3μg/mL),IL-1β(1ng/mL)。然后分别加入hs-IL1Ra(5μg/mL),hs-IL1Ra Nanoparticles(5μg/mL),hs-IL1Ra+hs-sTNFRI Nanoparticles(5μg/mL),BSA Nanoparticles(5μg/mL)。72小时后加入cck-8检测450nm处的吸光度。
2)测定hs-sTNFRI体外活性的方法:L929按1*104的细胞密度接种到96孔板中,24时后加入TNF-α(150ng/mL),然后分别加入hs-sTNFRI(16μg/mL),hs-sTNFRINanoparticles(16μg/mL),hs-IL1Ra+hs-sTNFRI Nanoparticles(16μg/mL),BSANanoparticles(16μg/mL)。24小时后加入cck8检测450nm处的吸光度。
实验结果如图9所示。B图显示装载hs-IL1Ra的纳米颗粒的抑制作用与hs-IL1Ra蛋白质自身相比差别不大,这表明,在形成纳米颗粒的过程中,修饰等作用对hs-IL1Ra原有的生物活性没有影响。同时,作为阴性对照,装载BSA的纳米颗粒确实不能抑制IL-1β对胸腺细胞的增殖作用。C图显示装载hs-sTNFRI的纳米颗粒虽然和hs-sTNFRI蛋白质自身相比稍低,但也能明显挽救TNF-α对L929的杀伤。同样作为阴性对照,装载BSA的纳米颗粒确实不能抑制TNF-α对L929的杀伤。综上所述,此纳米结构能够用来作为蛋白运输载体的材料。上文已经明确了两种蛋白纳米复合物的生物学活性,然后我们将两种蛋白同时连接到纳米颗粒上后同样进行了细胞实验。实验结果如图9显示,B图显示装载hs-IL1Ra和sTNFRI的纳米颗粒的抑制作用与hs-IL1Ra蛋白质自身相比差别不大。C图显示装载hs-IL1Ra和sTNFRI的纳米颗粒虽然和hs-sTNFRI蛋白相比稍低,但也能明显挽救TNF-α对L929的杀伤。综上所述,同时复合两种蛋白的纳米颗粒同样具有生物学活性。
实施例7:蛋白纳米复合物的体内滞留实验
实验选择C57BL/6J(8-10周龄)小鼠,用4%水合氯醛深度麻醉后将5μg Cy5.5标记的体积为20μL的游离蛋白hs-IL1Ra+sTNFRI或装载hs-IL1Ra和hs-sTNFRI的纳米颗粒注入右膝关节中。在预定的时间点使用成像***(IVIS Spectrum In Vivo Imaging system,perkinElmer,USA)对小鼠进行活体成像。
装载蛋白的纳米颗粒若要有治疗效果,不仅要保持蛋白的活性,还要延长蛋白在必要部位的停留时间。因此,我们用Cy5.5对蛋白进行标记以监测装载蛋白的纳米颗粒和蛋白分别在体内的停留情况。结果如图10所示,整个成像过程持续了8天。第一天刚注射时,结合A、B图中的Day 0我们可以看出,装载蛋白纳米颗粒组和游离蛋白组的起始荧光强度是一样的,这是该实验继续进行下去所需要满足的基本条件。一天后,游离蛋白组的荧光强度减弱,装载蛋白纳米颗粒组的荧光强度虽然也比刚注射时变弱了,但和游离蛋白组相比荧光强度明显更多(***p<0.001)。第4天和第8天时,两组间的差异依然明显。这些结果表明,蛋白进入体内后,由于体内的代谢清除作用,游离蛋白组的蛋白质被很快清除掉,而纳米颗粒组则由于纳米颗粒对蛋白质的保护作用明显延长了蛋白的半衰期,使其在体内停留更长的时间。
实施例8:酵母聚糖诱导的关节炎(ZIA)小鼠模型的建立和蛋白纳米复合物的靶向性研究
为了建立ZIA小鼠模型,首先按计划称取一定量的酵母聚糖粉末,然后用PBS配置25mg/mL的酵母聚糖溶液,加热沸腾后继续煮5-10分钟成为乳液状,再超声10分钟即可使用。在C57BL/6小鼠左腿关节腔注射20μL酵母聚糖乳液,右腿注射PBS做对照。24小时后,可见小鼠膝关节处出现明显肿胀,即建成ZIA小鼠模型用于实验。为了研究蛋白纳米复合物在ZIA小鼠炎症部位的被动靶向能力,在ZIA小鼠模型建立24小时后,将小鼠分为两组(每组4只),通过尾静脉注射200μL相同蛋白质量(5μg)的Cy5.5标记的蛋白溶液或蛋白纳米复合物,在预定时间点对小鼠活体成像。
为了研究人蛋白纳米复合物对于炎症部位的靶向性,我们在小鼠左腿关节部位建立了如上述ZIA模型。随后将Cy5.5标记的游离蛋白和蛋白纳米复合物分别经尾静脉注射到小鼠体内,通过活体荧光成像来观察药物在小鼠主要器官及炎症部位的分布随时间的变化。结果显示,蛋白药物能够在小鼠左腿RA关节处迅速富集,平均富集量在8h达到峰值,随后降低(图11中B)。观察的32小时内,游离蛋白组的左腿RA关节处荧光值显著更低,蛋白纳米复合物在RA关节的富集约是游离蛋白组的2倍,且滞留时间更长,32h仍保持高荧光强度。这说明装载蛋白hs-IL1Ra和hs-sTNFRI的纳米颗粒具有较强的体内被动靶向炎症部位的能力,为接下来进一步发挥蛋白的抗炎效果奠定了基础。
实施例9:小鼠胶原诱导性关节炎(CIA)模型的建立
小鼠胶原诱导关节炎(CIA)是临床试验期间常用模型之一,胶原诱导的关节炎构建策略:将牛二型胶原溶液与弗氏完全佐剂1:1混合制成乳化剂,DBA/1小鼠尾部皮下注射100μg胶原。21天后,再于小鼠尾部皮下注射100μg胶原与弗氏不完全佐剂1:1混合的乳化剂,随后每天观察小鼠,观察关节炎的进展情况。
CIA模型构建成功后每天观察关节炎的进展情况并按下表评分。在小鼠CIA模型中,小鼠的四个爪子均可被累及,小鼠整体的关节炎严重程度由四只爪子的评分相加得到(总分16分)。
表1
严重度评分 | 视觉病理 |
0 | 无红斑和肿胀的证据 |
1 | 红斑和轻度肿胀局限于足中段或踝关节 |
2 | 红斑和轻度肿胀从踝关节蔓延至足中段 |
3 | 红斑和中度肿胀从踝关节蔓延至趾关节 |
4 | 红斑和重度肿胀包括了踝、足和趾 |
CIA小鼠在接受第一次免疫刺激后的第35天开始接受不同治疗,每只小鼠尾静脉注射200μL溶液。具体分组如下:PBS,游离蛋白(Soluble hs-IL1Ra+sTNFRI),纳米颗粒(BSANanoparticles),单独蛋白纳米复合物(Hs-IL1Ra Nanoparticles),单独蛋白纳米复合物(Hs-sTNFRI Nanoparticles),两种蛋白纳米复合物(Hs-IL1Ra+sTNFRI Nanoparticles)。从第35天开始,每隔5天给一次药,共计给药5次,流程图如图12中A所示。治疗结束后,各治疗组CIA小鼠的爪子拍照保存。实验结果如图12B所示,CIA模型建立后,小鼠踝关节、足和趾肿胀明显。经过蛋白药物治疗后,关节评分明显降低(*p<0.05),而同时复合两种蛋白的纳米颗粒治疗组的CIA小鼠关节评分和蛋白药物组相比显著降低(*p<0.05)。并且同时复合两种蛋白的纳米颗粒的治疗效果明显优于一种蛋白纳米复合物(*p<0.05)。对照PBS组和纳米组的CIA小鼠未见明显好转。上述结果初步说明,同时复合两种蛋白的纳米颗粒对CIA小鼠起到了显著的治疗效果。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (7)
1.一种能够同时抗白细胞介素1和肿瘤坏死因子炎症的纳米材料,其特征在于,所述的纳米材料是将人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI和人白介素1受体拮抗剂hs-IL1Ra连接至PLGA-PEG-NHS纳米颗粒上得到;
所述的人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI和人白介素1受体拮抗剂hs-IL1Ra通过酯交联反应与PLGA-PEG-NHS纳米颗粒连接;
所述的人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI的氨基酸序列如SEQ IDNO.1所示;
所述的人白介素1受体拮抗剂hs-IL1Ra的氨基酸序列如SEQ ID NO.2所示。
2.一种权利要求1所述的纳米材料的制备方法,其特征在于,包括如下步骤:
S1、将PLGA-PEG-NHS粉末溶解在有机溶剂中,在缓冲液中进行透析,透析后去除有机溶剂,得到PLGA-PEG-NHS纳米颗粒溶液;
S2、向S1步骤得到的PLGA-PEG-NHS纳米颗粒溶液中加入人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI和人白介素1受体拮抗剂hs-IL1Ra进行酯交联反应,反应后进行超滤离心去除未结合的蛋白,得到所述的纳米材料。
3.根据权利要求2所述的制备方法,其特征在于,在S2步骤中,PLGA-PEG-NHS纳米颗粒溶液中PLGA-PEG-NHS纳米颗粒的浓度为10~20mg/mL,人可溶性肿瘤坏死因子受体I胞外段蛋白hs-sTNFRI按照终浓度为1~2mg/mL加入,人白介素1受体拮抗剂hs-IL1Ra按照终浓度为1~2mg/mL加入。
4.根据权利要求2所述的制备方法,其特征在于,所述的有机溶剂为四氢呋喃、DMSO、二氯甲烷、丙酮中的一种或多种。
5.根据权利要求2所述的制备方法,其特征在于,所述的缓冲液为HEPES缓冲液、磷酸盐、碳酸盐-碳酸氢盐或硼酸盐缓冲液。
6.根据权利要求2所述的制备方法,其特征在于,所述的酯交联反应是在0~30℃下,在pH7.2~8.5的缓冲液中反应0.5~4小时。
7.权利要求1所述的纳米材料在制备治疗类风湿性关节炎的药物中的应用。
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