CN114209652B - 一种微环境nk细胞免疫调控递送系统及其制备方法和应用 - Google Patents
一种微环境nk细胞免疫调控递送系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种微环境NK细胞免疫调控递送系统及其制备方法和应用,包括:外壳为在接触胎盘组织间液高表达的酶的作用下靶向崩解的酶底物多肽‑PEG修饰的脂质双分子膜;内核为胎盘自然杀伤细胞表面特异性高表达的标志物抗体修饰的药物载体;药物载体中负载超顺磁性四氧化三铁SPIO纳米粒子、调控胎盘自然杀伤细胞功能的小分子药物、治疗基因或其组合。本发明的微环境NK细胞免疫调控递送系统可有效避免母体和胎儿非特异性药物吸收,进而实现对胎盘内自然杀伤细胞特异性药物的递送和免疫功能调控。
Description
技术领域
本发明涉及化学、生物医学工程领域,具体涉及一种微环境NK细胞免疫调控递送系统及其制备方法和应用。
背景技术
妊娠期糖尿病(Gestational diabetes mellitus,GDM)是指在妊娠期间首次发生或者发现的糖代谢异常。随着生活水平的提高、饮食结构的改变和产妇年龄的增加,GDM的发病率呈逐年增加趋势,目前约20%的孕妇诊断为GDM。GDM对产妇和胎儿均具有较大损害【Nat Rev Dis Primers.2019Jul 11;5(1):47.doi:10.1038/s41572-019-0098-8.】。易发生感染、羊水过多、巨大儿、难产、产道损伤、产后出血、剖宫产率上升、胎儿畸形等。新生儿易发生新生儿呼吸窘迫综合症、低血糖、高胆红素血症等。而且,GMD胎儿成年后的糖尿病发生率远高于正常,GDM产妇再次妊娠后高血糖的发病率成倍增加。更为严重的是,GDM产妇将来发生2型糖尿病的可能性是血糖正常孕妇的7倍。现有临床研究证明,GDM如果得到有效控制,产后糖尿病的风险会明显降低【Nat Rev Endocrinol.2012Nov;8(11):639-49.doi:10.1038/nrendo.2012.96.】。
既往按照普通糖尿病的发病机制对GDM的发病进行解释,认为其主要与患者全身的胰岛素抵抗、糖耐量异常有关,进而设计疗法。但是,所设计的口服二甲双胍等普通血糖控制药物的治疗方式,并不能有效解决GDM的临床治疗问题。这是因为,该理论指导下的治疗方法,无法从胎盘甚至胎盘免疫细胞层面解决GDM的根本发病因素,所以治疗效果普遍不佳。但是随着近年来对胎盘解剖学和病理生理学研究的深入,发现GDM的发病与胎盘内NK细胞为代表的多种免疫细胞功能的失调,及其与胎盘内滋养细胞的互作有密切关系【NatMed.2006Sep;12(9):1065-74.】。特别需要引起注意的是胎盘的NK细胞【Diabetes.2011Mar;60(3):909-17.】。NK细胞是母胎免疫环境中最丰富的细胞之一,在妊娠早期占蜕膜白细胞总量的70%【Front Immunol.2019Jun 28;10:1397.】。进一步的研究发现,在GDM孕妇的发病过程中,胎盘中NK细胞数量增多、炎症因子分泌增多,引发胎盘内免疫反应过度激活【Diabetes.2011Mar;60(3):909-17.】。这一过程中的关键步骤就是未成熟NK细胞向成熟NK细胞转变的激活过程,如果能够减少NK细胞的激活,就可以有效抑制诱发GDM的胎盘免疫反应【Am J Reprod Immunol.2016May;75(5):529-38.】。
如何实现胎盘内肿瘤中NK细胞向成熟NK的激活,甚至逆转这一过程?我们在前期研究中发现,NK细胞的激活依赖一系列重要转录因子的作用,例如NFIL3(Nuclear Factor,Interleukin 3Regulated,又称E4BP4),T-bet,PU-1,STAT5,GATA3,Krf1,Ets1,Helios,Blimp-1,ID3,Eomes等等【J Recept Signal Transduct Res.2012Oct;32(5):238-49】以及IL-17、IL-15等细胞因子的作用【PNAS August 27,2019 116(35)17409-17418】。我们前期研究证实,如果可以抑制这些NK细胞活化的关键转录因子活性,可以抑制其向成熟表型转化。Neoruscogenin是甾体sapogenin家族的成员,是与发育和免疫密切相关的核受体NR1F1的调控剂。我们已经在体外研究中发现,其与NFIL3的siRNA同时作用,可以产生协同作用,有效影响NK细胞激活,降低其免疫毒性反应。所以,如果能够在不影响体内胎盘外NK细胞作用的前提下,针对胎盘内的NK细胞联合应用Neoruscogenin和NFIL3-siRNA,抑制胎盘内NK激活,促进胎盘内的未成熟NK细胞比例增加,进而降低胎盘炎症,就有望有效改善GDM无法免疫治疗的困境。
然而,现有可能具有免疫功能调控作用的药物,均不可避免地在母体全身各器官分布、广泛诱导免疫反应,进而产生大量的免疫并发症;并通过胎盘,向胎儿分布,杀伤胎儿。这些药物在免疫功能调控的同时,会产生对母体和胎儿的毒性。目前包括急救药品在内的孕妇用药均存在极多的禁忌,孕妇的药物使用和新药开发中首先面临的问题是,需要考虑对药物在母体和胎儿的分布和毒性问题。绝大多数的药物可以通过胎盘,分布进入胎儿侧,影响胎儿发育。因此,目前在体外实验中实现NK细胞功能调控的药物,无法真正在保证用药安全的前提下,实现选择性针对胎盘内NK细胞的功能调控。因此,如何避免对母体和胎儿的毒性,实现NK细胞功能调控药物,对胎盘中NK细胞的精准递送,是解决NK细胞功能失调所致疾病的关键科学问题。
目前研究者尝试过的可能促进自然杀伤细胞特异性纳米药物递送的手段有2种,一种是通过加大纳米药物粒径,使其不能通过胎膜屏障,滞留于胎盘产生药物递送效果;另一种是针对自然杀伤细胞膜标志物,进行抗体修饰纳米载体的特异性递送。
加大纳米药物粒径,促进胎盘内药物分布的原理在于,实验研究发现,<300nm的纳米药物无法滞留于胎盘,容易通过胎盘进入胎儿。所以研究者尝试合成粒径>300nm的纳米药物,使其滞留于胎盘,产生对胎盘内的多种细胞的功能调控。但是,过大的药物粒径(>100nm)不利于药物的体内分布。此类>300nm的纳米药物在母体循环中多数被网状内皮系统捕获,在全身各处产生副作用,能够到达胎盘中,实现特异性分布的比例也较低。所以,需要采用其他方式实现纳米药物在胎盘中的滞留和对胎盘内自然杀伤细胞的靶向。
纳米药物可采用纳米药物链接抗体,靶向识别目的细胞的细胞膜标志,实现对目的细胞的特异性递送。自然杀伤细胞具有一些确定的,区别于胎盘组织中与其他胎盘基质细胞的表面标志如CD11b(Cluster of differentiation 11b)可供与胎盘中其他细胞进行区别。但是,全身多器官组织表达量分析发现,这种表面标志在胎盘外其他部位的部分细胞也有表达。在少量高表达细胞表面的表达丰度,与自然杀伤细胞差异不显著。如果在纳米药物载体表面连接CD11b的抗体,直接静脉注射、体内应用,将造成对体内其他表达标志物CD11b的细胞的副作用。因此,只有药物进入胎盘前,在血液循环中屏蔽纳米载体的自然杀伤细胞识别抗体,才可以避免其分布于胎盘外NK细胞和其他表达CD11b的细胞,保证其选择性分布于胎盘内的自然杀伤细胞。
综上所述,目前缺乏可有效避免母体和胎儿非特异性药物吸收,进而实现对胎盘内自然杀伤细胞选择性药物递送和功能调控的纳米载体系统。
发明内容
为了克服现有技术的缺点与不足,本发明的首要目的在于提供一种微环境NK细胞免疫调控递送系统,该系统利用胎盘微环境靶向,减少药物进入胎盘前在母体器官组织分布,利用自然杀伤细胞膜标志物靶向,减少药物通过胎盘后在胎儿器官组织分布,可有效避免母体和胎儿非特异性药物吸收,进而实现对胎盘内自然杀伤细胞特异性的药物递送和功能调控。
本发明的另一目的在于提供上述微环境NK细胞免疫调控递送系统的制备方法。
本发明是通过以下技术方案实现的:
一种微环境NK细胞免疫调控递送系统,其特征在于:
(1)外壳为在接触胎盘组织间液高表达的酶的作用下靶向崩解的酶底物多肽-PEG修饰的脂质双分子膜,所述胎盘组织间液高表达的酶为钙/钙调蛋白依赖的蛋白激酶II、溶菌酶、激肽酶、组胺酶、催产素酶或基质金属蛋白酶中的一种或几种;
(2)内核为胎盘自然杀伤细胞表面特异性高表达的标志物抗体修饰的药物载体,所述药物载体为聚乙二醇修饰的聚阳离子载体与疏水性可降解聚酯形成的共聚物,所述NK细胞可能的细胞表面标志物抗体优选为CD11b抗体的Fab段;
(3)药物载体中负载超顺磁性四氧化三铁SPIO纳米粒子、调控胎盘自然杀伤细胞功能的小分子药物、治疗基因或其组合。
孕妇胎盘中富含多种促进胎盘发育和胎儿营养的酶类,本发明所述胎盘组织间液高表达的的酶为钙/钙调蛋白依赖的蛋白激酶II(Calcium/Calmodulin DependentProtein Kinase II,CaMKII)、溶菌酶、激肽酶、组胺酶、催产素酶或基质金属蛋白酶中的一种或几种,其中CaMKII在胎盘和组织液中表达量极高,正常人体血液和组织液中几乎不表达,因此,优选钙/钙调蛋白依赖的蛋白激酶II。
CaMKII底物多肽可以选择Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu,分子量:1527.77Da。
本发明的药物载体为聚乙二醇修饰的聚阳离子载体与疏水性可降解聚酯形成的共聚物,所述共聚物为聚乙二醇-聚乙烯亚胺-聚己内酯PEG-PEI-PCL、聚乙二醇-聚乙烯亚胺-聚乳酸PEG-PEI-PLA或聚乙二醇-聚乙烯亚胺-聚乳酸-羟基乙酸PEG-PEI-PLGA中的一种或几种,优选聚乙二醇-聚乙烯亚胺-聚己内酯PEG-PEI-PCL。
本发明所述的共聚物可通过现有技术合成,如先将PEG通过化学反应与聚阳离子载体形成共聚物,然后利用聚阳离子的活性基团与活化后的聚酯段反应形成共聚物。
本发明所述的共聚物也可通过市购得到。
本发明药物载体中负载超顺磁性四氧化三铁SPIO纳米粒子、调控胎盘自然杀伤细胞功能的小分子药物、治疗基因或其组合。所述小分子药物为Neoruscogenin,所述治疗基因为抑制NFIL3基因表达的siRNA。
所述胎盘自然杀伤细胞表面特异性高表达的标志物为CD11b、CD16、CD56、CD122、CD244、CD3ζ链、CD57、CD59、CD11b、CD94、CD34、CD133、NKG2D、NKG2A、NKP80、CD161、KIR、CD335、CD337、CD45RA、CD117和LAK-1等。优选的,所述的胎盘自然杀伤细胞表面特异性高表达的标志物抗体为CD11b抗体的Fab段。
本发明所述微环境NK细胞免疫调控递送系统的平均粒径为80nm-300nm,优选为100nm-205nm,粒径太大不利于体内循环,粒径太小制备难度增大而且不利于负载药物和基因。
本发明还提供了上述微环境NK细胞免疫调控递送系统的制备方法,包括以下步骤:
S1、将超顺磁性四氧化三铁SPIO纳米粒子、调控胎盘自然杀伤细胞功能的小分子药物和/或基因负载到共聚物,得到复合纳米粒子;
S2、将胎盘自然杀伤细胞表面标志物抗体链接到复合纳米粒子;
S3、将胎盘中高表达的酶的底物多肽链接PEG,得到多肽-PEG;
S4、将多肽-PEG和脂质体混合形成多肽-PEG修饰的脂质双分子膜脂质双分子膜;
S5、将多肽-PEG修饰的脂质双分子膜与复合纳米粒子组装成微环境NK细胞免疫调控递送系统。
优选的,步骤S1中,共聚物和超顺磁性四氧化三铁SPIO纳米粒子的质量比为5-15:1。
本发明通过CaMKII底物多肽-PEG修饰的脂质双分子膜作为外壳,保证了纳米传输体系在进入胎盘酶环境前,在无酶的血液中的分布稳定,减少药物泄露,减少或避免胎盘外NK细胞以及其他细胞吞噬。从而保证了母体胎盘外其他组织和器官的安全性;CaMKII酶敏感外壳在胎盘母体侧含酶的微环境中崩解,释放药物,可以保证药物在母体胎盘中的高效释放和分布;由于酶敏感外壳的引入,无需通过采用大粒径纳米载体结构即可保证对胎盘的分布效率,有效降低纳米载体粒径,保证了药物在进入胎盘前,循环分布的稳定,且保证了胎盘外的网状内皮系统不会大量吞噬载体引起疗效降低和副作用增大。
本发明采用胎盘自然杀伤细胞表面标志物抗体修饰的药物载体作为内核,药物由自然杀伤细胞表面标志物抗体修饰,可在释放后,确切锚定胎盘中的自然杀伤细胞膜,保证了在复杂的胎盘环境中,对自然杀伤细胞的特异性给药,同时避免胎盘中其他细胞服药,产生不必要的胎盘功能损伤;
绝大多数进入胎盘的药物通过抗体靶向,确切锚定于自然杀伤细胞,保证了药物极少渗漏通过胎盘屏障,进入胎儿侧,保证了胎儿的安全;药物被锚定于自然杀伤细胞膜后,促进治疗药物和治疗基因内吞入自然杀伤细胞,实现功能调控,保证了确切的自然杀伤细胞功能调控。
本发明还提供了上述微环境NK细胞免疫调控递送系统在制备调控胎盘自然杀伤细胞功能失调疾病药物中的应用,所述调控胎盘自然杀伤细胞功能失调疾病为妊娠期糖尿病(妊娠期高血糖)。
与现有技术相比,本发明具有如下有益效果:
(1)本发明以可以在接触胎盘组织间液高表达的特定酶的作用下靶向崩解的酶底物多肽-PEG修饰的脂质双分子膜作为外壳;以胎盘自然杀伤细胞表面特异性高表达的标志物抗体修饰的药物载体作为内核;合成双层结构的微环境NK细胞免疫调控递送系统。这种双层结构能保证纳米药物在孕妇血液循环中,脂质体外壳结构稳定,保持稳定循环,不容易被网状内皮系统在内的其他组织和细胞捕获,降低在孕妇体内影响胎盘之外的其他组织(包括胎盘外的NK细胞)分布和释放,降低毒副作用;
(2)在传输体系随血液循环进入胎盘后,其外壳中的酶底物被胎盘组织中高表达的对应的酶分解,保护性脂质双分子外壳在胎盘中迅速崩解,释放出可锚定自然杀伤细胞膜表面标志的、抗体修饰的纳米药物。该纳米药物避免被母体胎盘中其他组织细胞吸收,特异性锚定于胎盘中的自然杀伤细胞膜,进而被自然杀伤细胞特异性内吞,产生免疫功能调控作用,保证对胎盘疾病进行确切的治疗;
(3)通过确切的“抗原-抗体反应”,使脂质双分子外壳崩解后药物滞留于富含自然杀伤细胞的胎盘,减少药物渗漏通过胎盘屏障,减少了对胎儿的毒副作用;也避免影响胎盘中血管内皮细胞、免疫细胞和其他基质细胞。
附图说明
图1为本发明实施例1制备得到的微环境NK细胞免疫调控递送系统的结构示意图。
具体实施方式
下面通过具体实施方式来进一步说明本发明,以下实施例为本发明较佳的实施方式,但本发明的实施方式并不受下述实施例的限制。
本发明的原料来源如下:
Fe含量测定方法:
用原子吸收分光光度计法测纳米药物体系中的Fe含量,用于衡量纳米药物的剂量。取一定量制备好的药物溶液(例如步骤三中溶液取1mL)冻干称重,再将其溶解到1molL-1的HCl溶液中,放置24小时使SPIO中Fe充分离子化,用原子吸收分光光度计检测Fe原子在248.3nm处的吸光度,代入用Fe标准溶液做出的标准曲线中算出Fe的浓度,再反算出冻干前药物溶液中的Fe含量。
粒径测试方法:
用Zeta-Plus电位粒径仪(Brooken Haven)测试样品的粒径,入射激光波长λ=532nm,入射角θ=90°,温度为25℃;取三次测量值的平均值。
实施例1:
S1、聚乙酰亚胺接枝聚乙二醇(PEG-PEI)的合成
采用两步法合成聚乙烯亚胺接枝聚乙二醇(PEG-PEI),先用羰基二咪唑将单甲基醚聚二醇的端羟基活化,再与聚乙烯亚胺的氨基反应生成PEG-PEI。具体操作如下:称取单甲基醚乙二醇(8.0g,Mn=2kDa)于反应瓶中,80℃真空干燥6h,在氩气氛围下加入THF(60mL)将其溶解。称取羰基二咪唑(CDI,6.4g)与另一反应瓶中,再将溶有mPEG-OH的THF用恒压滴液漏斗缓慢滴加到CDI瓶中,室温下搅拌反应过夜。加入蒸馏水(0.648mL)使过量的CDI失活,继续搅拌30min。将溶液沉淀到大量的冷乙醚中,过滤后真空干燥得白色粉末状固体mPEG-CDI;
称取PEI(4.4g,MW=1.8kDa)加入到两口瓶(50mL)中,加入三氯甲烷(20mL)使其溶解加入PEG-CDI(3.2g),室温下搅拌反应24h,将溶液装到透析袋(MWCO=3.5kDa)中,用三氯甲烷中透析24h,将透析袋中溶液减压浓缩,然后沉淀在大量冷乙醚中,过滤干燥得白色粉末装产物mPEG-PEI;
S2、聚乙酰亚胺接枝聚乙二醇接枝聚己内酯(PEG-PEI-PCL)的合成
首先合成PCL-OH,15g干燥的十二醇加入两口瓶中,70℃下真空干燥8h,加2mlSn(Oct)2继续干燥0.5h,然后加入400mL干燥的的ε-己内酯,在105℃搅拌反应24h;冷却后加入100mL乙醇溶解未反应的ε-己内酯,过滤,粗产品溶于250mL四氢呋喃,沉淀在大量无水乙醚中,过滤后干燥得白色粉末状产物,产率96%;
然后合成PCL-CDI,10g PCL-OH(Mn=5000)加入两口瓶中,50℃下真空干燥8h,溶于50mL四氢呋喃后加入7.2g(10eq.)的羰基二咪唑(CDI),氩气保护,室温反应24h,沉淀在大量无水乙醚中,过滤,室温真空干燥,得白色粉末状产物,产率90%;
最后将PCL-CDI与PEG-PEI反应制得PEG-PEI-PCL,1.6g PEG-PEI加入到50mL两口瓶中,加入30mL三氯甲烷使其溶解,然后缓慢滴入10mL含200mg PCL-CDI的三氯甲烷溶液,在室温下搅拌反应24h,用透析袋(MWCO=5kDa)在1000mL三氯甲烷中透析24h,减压除去部分三氯甲烷,然后沉淀在无水乙醚中,过滤干燥得白色粉末产物,产率86%;
S3、聚乙二醇-聚乙烯亚胺-聚己内酯负载SPIO纳米粒子、药物(PEG-PEI-PCL-SPIO/drug)的制备
SPIO(超顺磁性四氧化三铁)按文献【S.H.Sun,H.Zeng,D.B.Robinson,S.Raoux,P.M.Rice,S.X.Wang,G.X Li.Monodisperse MFe2O4(M=Fe,Co,Mn)Nanoparticles.J.Am.Chem.Soc.2004,126,273-279】报道的方法合成,乙酰丙酮铁Fe(acac)31.4126g(4mmol),1,2-十六烷二醇5.16g(20mmol),油酸3.8ml(12mmol),油胺3.8ml(12mmol),加入到200ml三口瓶中,然后在氮气保护下加入40ml二苄醚搅拌溶解,在沙浴中加热到200℃回流搅拌2h,然后加热到300℃回流1h,反应体系由暗红色慢慢的变成黑色;在空气中自然冷却,沉淀在150ml乙醇中,在10000rpm转速下离心5分钟,弃去上层清夜,下层沉淀溶解在分别加有4滴油酸和油胺的70ml正己烷中,然后再在10000rpm转速下离心10min去掉不溶解部分,溶液再沉淀在200ml乙醇中,再在10000rpm转速下离心10min,下层沉淀溶解在60ml正己烷中,通入氩气保护,置于4℃下保存备用;
将SPIO的正己烷溶液吹干称重收集5mg的SPIO纳米粒子于血清瓶(10mL)中,称取50mg PEG-PEI-PCL聚合物、Neoruscogenin5mg,用三氯甲烷(3mL)将它们溶解混合均匀,把上述溶液在超声分散下逐滴加入20mL蒸馏水中,挥发除去三氯甲烷,用12000r/mim的转速离心,收集沉淀,弃去上清液。再用水将沉淀溶解,超声分散,重复离心操作,最后将制备的PEG-PEI-PCL-SPIO/drug纳米粒子超声分散到水中,用孔径为220nm针头过滤器过滤率,加入纯净水,定容调整PEG-PEI-PCL-SPIO/drug纳米粒子浓度至Fe含量为0.145mg/mL,产物置于4℃下保存备用;
S4、抗体靶向的聚乙二醇-聚乙烯亚胺-聚己内酯负载SPIO纳米粒子/药物(Fab-PEG-PEI-PCL-SPIO/drug)的制备
先采用现有文献中的方法裂解CD11b抗体,获得CD11b的Fab段,并提纯。然后将CD11b-Fab链接到mal-PEG-COOH上,再用酰胺化反应将连有抗体的PEG与PEG-PEI-PCL-SPIO纳米粒子上的氨基反应制备出Fab-PEG-PEI-PCL-SPIO;
具体操作如下:称取10mg的CD11b抗体,在0.5mg·ml-1的木瓜蛋白酶、10mmol·L-1的半胱氨酸、2mmol·L-1的EDTA,pH7.6的条件下酶解4h。酶解产物经ProteinA亲和色谱法分离,穿透峰经DEAE阴离子交换色谱法进一步纯化,透析除盐冻干后得到纯度较高的CD11b的Fab片段;
称取1mg的CD11b的Fab片段(Mn=45kDa),用EDTA溶液(500μL 0.5M)在4℃预处理15min。加入5ml的PBS溶液溶解,加入二硫苏糖醇1mg,25℃反应30min。用截留分子量为1k的离心超滤管离心去除二硫苏糖醇后,加入5ml的PBS溶液溶解,再加入mal-PEG-COOH(2mg,Mn=4k)混和均匀,在4℃放置过夜。再用截留分子量为5k的离心超滤管离心去除过量的mal-PEG-COOH。用EDC和NHS各500μg活化Fab-PEG-COOH中的羧基15min,后加入步骤3制备好的PEG-PEI-PCL-SPIO/drug 16mL,4℃反应过夜,最后超滤离心除去过量的EDC、NHS的小分子杂质,12000r/min离心除去未连接上的抗体,收集固体溶液,超声分散到蒸馏水中,定容调整Fab-PEG-PEI-PCL-SPIO/drug纳米粒子浓度至Fe含量为0.145mg/mL备用;
S5、治疗基因复合纳米粒子的制备
带正电的PEG-PEI-SPIO(或Fab-PEG-PEI-SPIO)纳米粒子与带负电的NFIL3-siRNA可以通过静电作用复合制成纳米复合物。具体操作如下:将400μg的NFIL3-siRNA用PBS稀释至终体积1.5mL,振荡均匀。取步骤3制备好的PEG-PEI-SPIO 1.5mL,(或步骤4制备好的Fab-PEG-PEI-SPIO净重1.6mL)纳米粒子超声分散均匀,将NFIL3-siRNA稀释溶液与PEG-PEI-SPIO(或Fab-PEG-PEI-SPIO)纳米粒子溶液混匀,将复合物体系定容至Fe含量为0.061mg/mL,吹打混匀并静置30分钟,制得均匀复合物;
S6、PEG-多肽的合成
将0.05mmol钙/钙调蛋白依赖的蛋白激酶II(Calcium/Calmodulin DependentProtein Kinase II,CaMKII)敏感的多肽(Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu,分子量:1527.77Da)、5mmol的EDC和5mmol的DMAP溶于10mL乙腈水溶液(乙腈:水=1:1),N2保护下于冰水浴上,500rpm下磁力搅拌2h,以活化Peptide。2h后加入0.5mmol的PEG-NHS(分子量3000Da),继续反应72h。反应结束后,将反应液置于透析袋(MWCO=3.5kDa)中,透析72h,冷冻干燥,得到产物PEG-多肽;
S7、PEG-多肽修饰的脂质体壳@治疗基因复合纳米粒子的制备
PEG-多肽和胆固醇(重量各20mg)溶于5mL的二氯甲烷中,用真空旋转真发器将二氯甲烷旋干,使PEG-多肽和胆固醇在圆底烧瓶壁上形成一层脂质体薄膜。缓慢的搅拌下将步骤5制备的治疗基因复合纳米粒子2mL以0.5mL/min的速度滴加到上述PEG-多肽和胆固醇形成的脂质体薄膜中。滴加完成后再继续搅拌30min,使脂质体和治疗基因复合纳米粒子充分组装,最后用强磁铁将载有治疗基因复合纳米粒子的脂质体和空脂质体分开。最后加入2mL生理盐水(0.9%NaCl)溶液将PEG-多肽修饰的脂质体壳@治疗基因复合纳米粒子溶解,孔径为220nm针头过滤器过滤率,定容至Fe含量为0.061mg/mL,4℃下保存,备用。
制备得到的微环境NK细胞免疫调控递送系统的具体结构示意图如图1所示。
实施例2-4,对比例1-6:
同实施例1相比,改变步骤S3中聚合物、药物和SPIO的投料量或省略步骤S3、S4、S5、S6、S7中某个步骤可以制备出实施例2-4或对比例1-6,具体见下表1:
表1:实施例和对比例
功能评价实验
1.核磁共振(MRI)实验,评价药物在妊娠期糖尿病模型的胎盘特异性递送功能
模型建立和体重检测:
8周龄SPF级C57BL/6小鼠(购自广东省医学实验动物中心),小鼠被安置在SPF光控饲养环境中,在22℃±2℃的恒温和60%的湿度下进行12小时的光/暗循环,自由获取食物和饮用水,饲养在代谢笼中。雌性小鼠与雄性小鼠2:1于发情期合笼交配,第二天雌鼠阴道分泌物涂片行帕帕尼科拉乌染色,光学显微镜下观察标本阴道精子阳性者诊断为妊娠,标记为妊娠第0天(D0)。孕4天晚8:00禁食12h至孕5天后,于模型组尾静脉快速注射50mg/kgSTZ(以pH=4.2~4.5的柠檬酸-柠檬酸钠缓冲液新鲜配制为0.1mol/L溶液)。等量溶剂注射小鼠为对照组。
空腹血糖测定:提前禁食12h后,于孕10天早8:00,剪尾采血检测孕鼠空腹血糖,将孕鼠空腹血糖≥11.1mmol/L者视为造模成功,建立妊娠期糖尿病模型。
同前法于提前禁食12h后,孕17天早8:00剪尾采血测量空腹孕鼠空腹血糖。
MRI影像学检测药物的胎盘分布:
妊娠期糖尿病模型动物于第11天,水合氯醛麻醉后,行MRIT2序列在药物注射前(0h)和注射后2小时(2h)时间点扫描,观察含有SPIO的纳米药物体内分布。尾静脉注射纳米药物的剂量为:(治疗剂量0.31mg/Kg铁当量药物,或等体积的生理盐水);
使用Philips Intera 1.5T MRI扫描仪,及其动物专用线圈进行C57BL/6j小鼠子宫MRI成像。在MRIbTFE序列上观察小鼠体内子宫和胚胎区域的信号强度演变,并采用T2map成像技术,测量随着药物中SPIO在体内子宫、胎盘、胚胎和其他器官分布引起的T2弛豫时间改变,计算0h时和2h时分别的驰豫率R2。计算药物注射后2h时R2的相对增加比率(RSI(Relative Signal Intensity)%=R22h/R20h),结果见表2。
表2胎盘特异性递送功能评价结果
由上述结果可知,对比例1中未链接胎盘自然杀伤细胞表面标志物抗体,多肽--PEG修饰的脂质双分子层崩解后,内容的药物无法锚定于自然杀伤细胞获得胎盘滞留,大量漏过胎盘屏障,检测到胎盘RSI较低;药物在胚胎聚集,导致胚胎RSI较高;药物无法锚定于自然杀伤细胞获得胎盘滞留,也导致部分药物脱离胎盘,全身分布,导致肝脏RSI较高。
对比例2的递送系统不含有多肽-PEG修饰的脂质双分子膜做外壳,无法实现针对胎盘微环境的靶向释放;另外,CD11b抗体靶向包括自然杀伤细胞在内的体内其他多种细胞膜表达CD11b的细胞,胎盘靶向性不强;所以,检测到胎盘RSI较低,肝脏RSI较低;没有脂质膜的药物粒径较小,进入胎盘者,以较大比例通过胎盘屏障,检测到胚胎RSI较高。
对比例3和4的递送系统,不含CD11b抗体,进入胎盘的药物无法靶向锚定于自然杀伤细胞,无法获得胎盘滞留,大量漏过胎盘屏障,检测到胎盘RSI较低;药物在胚胎聚集,导致胚胎RSI较高。同时,对比例3的脂质双分子膜外壳没有酶敏感多肽修饰,在胎盘的分布降低,也导致胎盘RSI较低,肝脏的RSI较高。对比例4没有脂质双分子膜外壳,相比对比例3,胎盘的RSI更低,肝脏的RSI更高。
对比例5的粒径过大,导致其体内循环分布效果极差,药物主要被肝脏的网状内皮系统大量吞噬,导致肝脏的RSI明显较高,胎盘RSI明显较低;但是其大粒径阻滞其漏过母胎屏障,所以胚胎RSI较低。对比例6的粒径远大于对比例5,循环更差,所以其肝脏RSI高于对比例5;其粒径更大,更不容易漏过母胎屏障,所以胎盘RSI低于对比例5。
实施例1-4中采用CaMKII的底物多肽-PEG修饰的脂质双分子膜作为外壳,以胎盘自然杀伤细胞表面标志物抗体修饰的药物载体作为内核,合成双层结构的微环境NK细胞免疫调控递送系统,粒径范围为80-205nm。其100nm左右的粒径,和外层的负电脂质双分子膜,便于避免被网状内皮系统大量吞噬,实现体内循环时间延长,实现体内的有效循环。其底物多肽-PEG修饰的脂质双分子膜外壳在体内其他组织器官的循环中稳定,到达特异性高表达CaMKII的胎盘微环境中,随着多肽的降解而崩解,实现在胎盘组织中药物特异性分布。药物外壳在胎盘微环境崩解后,显露含有CD11b抗体片段的药物内核。CD11b抗体片段在胎盘中可以锚定于细胞膜特异性高表达CD11b的自然杀伤细胞,促进药物被自然杀伤细胞特异性内吞后实现自然杀伤细胞功能调控,并减少在胎盘其他细胞中的分布,减少对胎盘功能的影响。CD11b抗体使胎盘中的药物锚定于自然杀伤细胞,也有效减少了药物漏过母胎屏障,减少了药物到达胚胎。
2.建立妊娠期糖尿病动物模型评价治疗效果
于D3、D6、D9、D12、D15注射药物治疗(治疗剂量0.31mg/Kg铁当量药物,或等体积的生理盐水),并于D17进行系列检测,检测结果如表3所示:
胎盘和和胎仔检查:胎盘组织:处死孕鼠,打开腹腔,剖开子宫,依次取出胎仔和胎盘,记录存活的胎仔数量。剔除胎盘上的胎膜及脐带,沿脐带根部将胎仔端脐带剪除,将胎盘及胎仔分别置于无菌纱布上吸干表面羊水,分析天平称重胎盘和胎仔。剪取胎盘组织放入液氮中,-80℃保存。
表3妊娠期糖尿病动物模型评价治疗效果
由上述结果可知,对比例1中未链接胎盘自然杀伤细胞表面标志物抗体,多肽---PEG修饰的脂质双分子层崩解后,内容的药物无法锚定于自然杀伤细胞获得胎盘滞留,大量漏过胎盘屏障,检测到治疗效果较差,血糖较高,胎仔重量较低,产仔数较低;同时,药物在胚胎聚集,导致胚胎毒性,也导致胎仔重量较低,产仔数较低。
对比例2的递送系统不含有多肽-PEG修饰的脂质双分子膜做外壳,针对胎盘微环境的靶向释放无法实现;CD11b抗体靶向包括胎盘外的自然杀伤细胞在内的体内表达CD11b细胞,胎盘靶向性不强,检测到治疗效果较差,血糖较高,胎仔重量较低,产仔数较低。同时,没有脂质膜的药物粒径较小,进入胎盘者,以较大比例通过胎盘屏障,也导致胎仔重量较低,产仔数较低。
对比例3和4的递送系统,不含CD11b抗体,进入胎盘的药物无法靶向锚定于自然杀伤细胞,无法获得胎盘滞留,导致大量漏过胎盘屏障,检测到治疗效果较差,血糖较高,胎仔重量较低,产仔数较低。同时,对比例3的脂质双分子膜外壳没有酶敏感多肽修饰,在胎盘的分布降低,检测到治疗效果较差,血糖较高,胎仔重量较低,产仔数较低。对比例4没有脂质双分子膜外壳,相比对比例3,治疗效果更差。
对比例5和6的粒径过大,导致其体内循环分布效果极差,药物主要被肝脏的网状内皮系统大量吞噬,导致胎盘药物分布不足,治疗效果较差,血糖较高,胎仔重量较低,产仔数较低。对比例6的粒径远大于对比例5,循环分布更差,所以其治疗效果相比对比例5更差。
实施例1-4中采用CaMKII的底物多肽-PEG修饰的脂质双分子膜作为外壳,以胎盘自然杀伤细胞表面标志物抗体修饰的药物载体作为内核,合成双层结构的微环境NK细胞免疫调控递送系统,粒径范围为80-205nm。其100nm左右的粒径,和外层的负电脂质双分子膜,便于避免被网状内皮系统大量吞噬,实现体内循环时间延长,实现体内的有效循环。其底物多肽-PEG修饰的脂质双分子膜外壳在体内其他组织器官的循环中稳定,到达特异性高表达CaMKII的胎盘微环境中,随着多肽的降解而崩解,实现在胎盘组织中药物特异性分布。药物外壳在胎盘微环境崩解后,显露含有CD11b抗体片段的药物内核。CD11b抗体片段在胎盘中可以锚定于细胞膜特异性高表达CD11b的自然杀伤细胞,促进药物被自然杀伤细胞特异性内吞后实现自然杀伤细胞功能调控,并减少在胎盘其他细胞中的分布,减少对胎盘其他细胞功能的影响,通过选择性的自然杀伤细胞功能调控,实现了较好的治疗效果。CD11b抗体使胎盘中的药物锚定于自然杀伤细胞,也有效减少了药物漏过母胎屏障,减少了药物到达胚胎,对胎仔的毒性较小。
3.药物用于动物模型的毒性评价
正常对照组小鼠注射药物后72小时,尾静脉取血,检测肝功能指标谷丙转氨酶(alanine transaminase,ALT)、总胆红素(total bilirubin,TBil)和肾功能指标血尿素氮(blood urea nitrogen,BUN)和血清肌酐(serum creatinine,sCr)。检测仪器为日立7600型全自动生化分析仪,检测结果见表4。
表4毒性评价结果
由上述结果可知,本发明制备得到的微环境NK细胞免疫调控递送系统,未见对母体和胎儿发生明显的毒副作用。
Claims (5)
1.一种微环境NK细胞免疫调控递送系统,其特征在于,包括:
(1)外壳为在接触胎盘组织间液高表达的酶的作用下靶向崩解的酶底物多肽-PEG修饰的脂质双分子膜,所述胎盘组织间液高表达的酶为钙/钙调蛋白依赖的蛋白激酶II;
(2)内核为胎盘自然杀伤细胞表面特异性高表达的标志物抗体修饰的药物载体,所述药物载体为聚乙二醇修饰的聚阳离子载体与疏水性可降解聚酯形成的共聚物;所述胎盘自然杀伤细胞表面特异性高表达的标志物抗体为 CD11b抗体的Fab段; 所述共聚物为聚乙二醇-聚乙烯亚胺-聚己内酯PEG-PEI-PCL;
(3)药物载体中负载超顺磁性四氧化三铁SPIO纳米粒子、调控胎盘自然杀伤细胞功能的小分子药物和治疗基因;所述小分子药物为Neoruscogenin,所述治疗基因为抑制NFIL3基因表达的siRNA;
所述微环境NK细胞免疫调控递送系统的平均粒径为80nm-300nm。
2.根据权利要求1所述的微环境NK细胞免疫调控递送系统,其特征在于,所述微环境NK细胞免疫调控递送系统的平均粒径为100nm-205nm。
3.权利要求1-2任一项所述的微环境NK细胞免疫调控递送系统的制备方法,其特征在于,包括以下步骤:
S1、将超顺磁性四氧化三铁SPIO纳米粒子、调控胎盘自然杀伤细胞功能的小分子药物和治疗基因负载到共聚物,得到复合纳米粒子;
S2、将胎盘自然杀伤细胞表面标志物抗体链接到复合纳米粒子,得到抗体复合纳米粒子;
S3、将胎盘中高表达的酶底物多肽链接PEG,得到多肽-PEG;
S4、将多肽-PEG和脂质体混合形成多肽-PEG修饰的脂质双分子膜;
S5、将多肽-PEG修饰的脂质双分子膜与抗体复合纳米粒子组装成微环境NK细胞免疫调控递送系统。
4.权利要求3所述的微环境NK细胞免疫调控递送系统的制备方法,其特征在于,步骤S1中,共聚物和超顺磁性四氧化三铁SPIO纳米粒子的质量比为5-15:1。
5.权利要求1-2任一项所述的微环境NK细胞免疫调控递送系统在制备调控胎盘自然杀伤细胞功能失调所致疾病的药物中的应用,所述调控胎盘自然杀伤细胞功能失调所致疾病为妊娠期高血糖。
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