CN107441499A - 一种改性壳聚糖的核酸靶向递送载体及其制备方法和应用 - Google Patents
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Abstract
本发明涉及一种改性壳聚糖的核酸靶向递送载体及其制备方法和应用。三甲基化壳聚糖与琥珀酰亚胺乙酸酯‑聚乙二醇‑邻二硫吡啶进行反应,得到三甲基化壳聚糖‑g‑聚乙二醇‑邻二硫吡啶;在常温条件下,将反应产物与短肽缬氨酸‑丙氨酸‑脯氨酸‑甘氨酸‑半胱氨酸(Val‑Ala‑Pro‑Gly‑Cys,VAPG‑Cys)反应,制得三甲基化壳聚糖‑g‑聚乙二醇‑VAPG。本发明的三甲基化壳聚糖‑g‑聚乙二醇‑VAPG带有正电荷,能够与带有负电荷的microRNA‑145通过静电作用复合得到纳米粒子,纳米粒子复合物在水中分散性好,靶向血管平滑肌细胞,转染效率高,能够调节血管平滑肌细胞的表型,可用于医药基因治疗领域。
Description
技术领域
本发明属于载体材料技术领域,具体涉及一种基于改性壳聚糖的靶向血管平滑肌细胞的microRNAs递送载体及其制备方法和应用。
背景技术
血管一般由血管内皮细胞层、血管平滑肌细胞层和成纤维细胞层构成,其中,血管平滑肌细胞(VSMC)存在收缩型和合成型两种表型。正常情况下,VSMC为收缩表型,有利于对抗血管张力并维持血管壁稳态,但是当VSMC受到病理和生理刺激后会发生表型改变,由收缩型转变为合成型,同时VSMC过度增殖导致内膜增生,造成血管狭窄,因此需要对VSMC表型进行有效调控,抑制其过度增殖。MicroRNAs(miRNAs)是一种长度为20~24nt的非编码核糖核酸,在血管再生中具有重要作用(Peng B,Chen Y,Leong KW.MicroRNA delivery forregenerative medicine.Advanced Drug Delivery Reviews,2015,88:108-122)。其中,miRNA-145可在血管平滑肌细胞内高度表达,对血管平滑肌细胞的增殖和表型具有调控作用(Hergenreider E,Heydt S,Tréguer K,et al.Atheroprotective communicationbetween endothelial cells and smooth muscle cells through miRNAs.Nature CellBiology,2012,14:249-256)。但是,由于miRNAs带有负电荷,难于通过内吞作用进入细胞,并且裸露的miRNAs在血液中容易降解,因此,设计有效的miRNAs靶向递送体系是很必要的(Guzman-Villanueva D,El-Sherbiny IM,Herrera-Ruiz D,et al.Formulationapproaches to short interfering RNA and microRNA:Challenges andimplications.Journal of Pharmaceutical Sciences,2012,101:4046-4066)。
三甲基化壳聚糖(TMC)是对壳聚糖的氨基进行甲基化处理后的产物,具有良好的生物相容性,以聚乙二醇(PEG)接枝TMC,可降低其细胞毒性,增强其在水中的溶解性。选择不同的多肽接枝在壳聚糖上,利用其选择特异性,对提高载体的靶向能力具有重要意义。本实验室前期研究表明,通过双官能团的PEG,可将三甲基化壳聚糖和具有靶向功能的精氨酸-谷氨酸-天冬氨酸-缬氨酸(Arg-Glu-Asp-Val,REDV)连接,能够特异性地识别血管内皮细胞(Zhou F,Jia X,Yang Q,et al.Targeted delivery of microRNA-126to vascularendothelial cells via REDV peptide modified PEG-trimethylchitosan.Biomaterials Science,2016,4:849-856)。Gobin AS发现来源于缬氨酸-甘氨酸-缬氨酸-丙氨酸-脯氨酸-甘氨酸(VGVAPG)的短肽缬氨酸-丙氨酸-脯氨酸-甘氨酸(Val-Ala-Pro-Gly,VAPG),可以作为血管平滑肌细胞的生物特异性粘附配体(Gobin AS,WestJL.Val-ala-pro-gly,an elastin-derived non-integrin ligand:Smooth muscle celladhesion and specificity.Journal of Biomedical Materials Research,2003,67A:255-259)。
本发明进一步扩展了三甲基化壳聚糖-g-聚乙二醇-短肽的应用范围,它是一种带有正电荷的壳聚糖与聚乙二醇和短肽构成的载体,具有生物可降解性。与miRNAs复合后得到纳米粒子,通过调节壳聚糖的分子量得以控制纳米粒子的直径,并有利于进入血管平滑肌细胞内部,同时,接枝适量的亲水性PEG和短肽,在体内可以屏蔽多余的正电荷,具有较低的毒副作用,有效促进miRNAs在细胞内中产生作用,提高转染效率。
发明内容
本发明的技术方案如下:
一种基于改性壳聚糖的miRNAs靶向递送载体,以VAPG作为特异性识别短肽,载体具体为三甲基化壳聚糖-g-聚乙二醇-VAPG(TMC-g-PEG-VAPG),其结构式为:
所述壳聚糖的重均分子量为1~50kDa;
所述壳聚糖脱乙酰度大于90%;
所述三甲基化壳聚糖的甲基化度为20~60%;
所述聚乙二醇的双端的活性基团分别为琥珀酰亚胺乙酸酯基团和邻二硫吡啶基团;聚乙二醇的数均分子量为1~5kDa;载体中聚乙二醇的接枝率为10~30%。
所述的miRNAs为miRNA-145或miRNA-143等双链模拟物,其中miRNA-145为:5′-GUCCAG UUU UCC CAG GAA UCC CU-3';5′-GGA UUC CUG GGA AAA CUG GAC UU-3′。
本发明的基于改性壳聚糖的miRNAs靶向递送载体的制备方法,包括以下步骤:
(1)制备三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶:琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶与三甲基壳聚糖反应,其质量比为1~5:1,三甲基壳聚糖的浓度为5~10mg/mL,去离子水作为溶剂,室温下反应2~10h,产物使用去离子水透析,最后冻干得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶;
(2)制备三甲基化壳聚糖-g-聚乙二醇-VAPG:三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶与缬氨酸-丙氨酸-脯氨酸-甘氨酸-半胱氨酸(VAPG-Cys)短肽的质量比为5~20:1,去离子水为溶剂,室温反应2~8h,未参加反应的短肽通过透析除去,通过冻干的方法制得三甲基化壳聚糖-g-聚乙二醇-VAPG共聚物载体(TMC-g-PEG-VAPG)。
本发明基于改性壳聚糖的miRNA-145靶向递送载体,应用于靶向血管平滑肌细胞,对血管平滑肌细胞的表型具有调控作用。
应用方法是:将制备的载体溶解于焦碳酸二乙酯处理过的水中,与microRNA-145溶液根据载体中氮原子和microRNA-145中磷原子的N/P摩尔数比例为12~20进行混合,室温静置10~30min。
载体复合miRNA-145后的纳米粒子的动态光散射测试的粒径为50~200nm,ζ电位为2~30mV。
本发明公布了三甲基化壳聚糖-g-聚乙二醇-VAPG载体的制备方法,第一步为三甲基化壳聚糖与琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶进行反应,得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶;第二步为常温条件下,将上一步反应产物与特异性短肽VAPG-Cys反应,利用邻二硫吡啶基团和短肽中巯基进行偶联反应,制得三甲基化壳聚糖-g-聚乙二醇-VAPG,该制备方法的优点是在常温下进行的反应,操作过程简单易行。本发明的三甲基化壳聚糖-g-聚乙二醇-VAPG产物带有适当量正电荷,能够与带有负电荷miRNA-145通过静电作用复合得到纳米粒子,此复合物在水溶液中分散性好,粒径较均一,整体所带电荷为正值,并且具有靶向血管平滑肌细胞和转染效率高的优点,能够调节血管平滑肌细胞的表型,可以用于医药基因治疗领域。
附图说明
图1:实施例1制备的三甲基化壳聚糖(TMC)的核磁谱图。
图2:实施例1制备的三甲基化壳聚糖-g-聚乙二醇-VAPG(TMC-g-PEG-VAPG)共聚物的核磁谱图。
图3:实施例1制备的载体与miRNAs复合的纳米粒子的原子力显微镜照片。
具体实施方式
下面通过实施案例对本发明的技术方案作进一步的描述,以下实施案例是对本发明的进一步说明,并不限制本发明的适用范围,其中所用miRNAs为microRNA-145:5′-GUCCAG UUU UCC CAG GAA UCC CU-3';5′-GGA UUC CUG GGA AAA CUG GAC UU-3′。
实施例1:
在装有磁力搅拌的三口瓶中,将1g脱乙酰度为90%以上的壳聚糖(Mw=5kDa)和2.5g碘化钠加入到5.5mL质量分数为15%的NaOH和45mL N-甲基-2-吡咯烷酮的混合溶液中,并加入6mL碘甲烷,在避光条件下,60℃回流反应45min,加入5.6mL的15%NaOH溶液和3mL碘甲烷,继续在60℃下反应45min,将反应后的体系加入40mL乙醇中终止反应,将产物离心沉淀,并使用乙醚洗涤。最后将沉淀产物溶解在40mL质量分数为10%NaCl水溶液中,搅拌3h进行离子交换,最后使用去离子水透析72h,冻干得到三甲基化壳聚糖。所得壳聚糖甲基化度为60%,所得产物1H-NMR如图1所示。
在2mL去离子水中,加入合成的20mg三甲基化壳聚糖和40mg琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶(Mn=1kDa)中,室温下反应6h,反应后的溶液使用去离子水透析,冻干得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶。
在10mg三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶和0.8mgVAPG-Cys短肽中,加入1mL的去离子水并反应8h,未参加反应的短肽通过去离子水透析除去,制得三甲基化壳聚糖-g-聚乙二醇-VAPG共聚物,作为载体备用。
所制得的三甲基化壳聚糖-g-聚乙二醇-VAPG共聚物的聚乙二醇的接枝率约为30%。最终所得产物1H-NMR如图2所示。使用DEPC处理后的水将上述产物配制成5mg/mL的溶液,与miRNAs溶液根据N/P为18进行混合,室温静置30min,得到载体与核酸复合的纳米粒子。所制得纳米粒子,通过动态光散射测定的粒径为50~130nm,ζ电位约为11mV。所制得纳米粒子的原子力显微镜照片如图3所示,其中纳米粒子的粒径为112±1.9nm。
实施例2:
在装有磁力搅拌的三口瓶中,将1g脱乙酰度为90%以上的壳聚糖(Mw=20kDa)和2.5g碘化钠加入到5.5mL质量分数为15%的NaOH和45mL N-甲基-2-吡咯烷酮的混合溶液中,并加入6mL碘甲烷,在避光条件下,60℃回流反应45min,加入5.6mL的15%NaOH溶液,继续在60℃下反应45min,将反应后的体系加入40mL乙醇中终止反应,将产物离心沉淀,并使用乙醚洗涤。最后将沉淀产物溶解在40mL质量分数为10%NaCl水溶液中,搅拌3h进行离子交换,最后使用去离子水透析72h,冻干得到三甲基化壳聚糖。所得壳聚糖甲基化度为39%。
在已经制备的20mg三甲基化壳聚糖和20mg琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶(Mn=5kDa)中,加入2mL去离子水作为溶剂,室温下反应3h。反应产物使用去离子水透析,最后冻干得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶。
在10mg三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶和0.6mg VAPG-Cys短肽中,加入2mL的去离子水并反应3h。未参加反应的短肽通过离心超滤并透析除去,制得三甲基化壳聚糖-g-聚乙二醇-VAPG共聚物,作为载体备用。
所制得的产物,聚乙二醇的接枝率约为10%,使用DEPC处理后的水配制成10mg/mL的溶液,与miRNAs溶液根据N/P为14进行混合,室温静置25min,得到载体与核酸复合的纳米粒子。所制得纳米粒子通过动态光散射测定的粒径为50~150nm。ζ电位约为6mV,纳米粒子的粒径为125±1.3nm。
实施例3:
在装有磁力搅拌的三口瓶中,将1g脱乙酰度为90%以上的壳聚糖(Mw=5kDa)和2.5g碘化钠加入到4mL质量分数为15%的NaOH和45mL N-甲基-2-吡咯烷酮的混合溶液中,并加入6mL碘甲烷,在避光条件下,60℃回流反应45min,加入5.6mL的15%NaOH溶液和1mL碘甲烷,继续在60℃下反应45min,将反应后的体系加入40mL乙醇中终止反应,将产物离心沉淀,并使用乙醚洗涤。最后将沉淀产物溶解在40mL质量分数为10%NaCl水溶液中,搅拌3h进行离子交换,最后使用去离子水透析72h,冻干得到三甲基化壳聚糖。所制得的三甲基化壳聚糖其甲基化度约20%。
在2mL去离子水中,加入合成的20mg三甲基化壳聚糖和30mg琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶(Mn=2kDa)中,室温下反应6h,反应后的溶液使用去离子水透析,冻干得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶。
在10mg三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶和0.5mgVAPG-Cys短肽中,加入1mL的去离子水并反应2h,未参加反应的短肽通过去离子水透析除去,制得三甲基化壳聚糖-g-聚乙二醇-VAPG共聚物,作为载体备用。
所制得的产物的聚乙二醇的接枝率约为10%。使用DEPC处理后的水配制成5mg/mL的溶液,与miRNAs溶液根据N/P为20进行混合,室温静置30min,得到载体与核酸复合的纳米粒子。所制得纳米粒子,通过动态光散射测定的粒径为50~120nm,ζ电位约为8mV。原子力显微镜照片中纳米粒子的粒径为102±0.4nm。
实施例4:
在装有磁力搅拌的三口瓶中,将1g脱乙酰度为90%以上的壳聚糖(Mw=50kDa)和2.5g碘化钠加入到4mL质量分数为15%的NaOH和45mL N-甲基-2-吡咯烷酮的混合溶液中,并加入6mL碘甲烷,在避光条件下,60℃回流反应45min,加入5.6mL的15%NaOH溶液和3mL碘甲烷,继续在60℃下反应45min,将反应后的体系加入40mL乙醇中终止反应,将产物离心沉淀,并使用乙醚洗涤。最后将沉淀产物溶解在40mL质量分数为10%NaCl水溶液中,搅拌3h进行离子交换,最后使用去离子水透析72h,冻干得到三甲基化壳聚糖。所制得的三甲基化壳聚糖其甲基化度约40%。
在2mL去离子水中,加入合成的20mg三甲基化壳聚糖和40mg琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶(Mn=5kDa)中,室温下反应6h,反应后的溶液使用去离子水透析,冻干得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶。
在8mg三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶和0.7mgVAPG-Cys短肽中,加入1mL的去离子水并反应6h,未参加反应的短肽通过去离子水透析除去,制得三甲基化壳聚糖-g-聚乙二醇-VAPG共聚物,作为载体备用。
所制得的产物的聚乙二醇的接枝率约为30%。使用DEPC处理后的水配制成5mg/mL的溶液,与miRNAs溶液根据N/P为12进行混合,室温静置10min,得到载体与核酸复合的纳米粒子。所制得纳米粒子,通过动态光散射测定的粒径为50~150nm,ζ电位约为9mV。原子力显微镜照片中纳米粒子的粒径为129±2.7nm。
Claims (8)
1.一种改性壳聚糖的核酸靶向递送载体及其制备方法和应用,其特征是以短肽缬氨酸-丙氨酸-脯氨酸-甘氨酸(Val-Ala-Pro-Gly,VAPG)作为特异性识别短肽,载体具体为三甲基化壳聚糖-g-聚乙二醇-VAPG,其结构式为:
2.如权利要求1所述的载体,其特征是壳聚糖的重均分子量为1~50kDa;壳聚糖脱乙酰度大于90%。
3.如权利要求1所述的载体,其特征是三甲基化壳聚糖的甲基化度为20~60%。
4.如权利要求1所述的载体,其特征是聚乙二醇的双端的活性基团分别为琥珀酰亚胺乙酸酯基团和邻二硫吡啶基团;聚乙二醇的数均分子量为1~5kDa;载体中聚乙二醇的接枝率为10~30%。
5.权利要求1所述的基于改性壳聚糖的核酸靶向递送载体的制备方法,其特征在于包括以下步骤:
(1)制备三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶:琥珀酰亚胺乙酸酯-聚乙二醇-邻二硫吡啶与三甲基壳聚糖反应,其质量比为1~5:1,三甲基化壳聚糖的浓度为5~10mg/mL,去离子水作为溶剂,室温下反应2~10h,产物使用去离子水透析,最后冻干得到三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶;
(2)制备三甲基化壳聚糖-g-聚乙二醇-VAPG:三甲基化壳聚糖-g-聚乙二醇-邻二硫吡啶与缬氨酸-丙氨酸-脯氨酸-甘氨酸-半胱氨酸(Val-Ala-Pro-Gly-Cys,VAPG-Cys)短肽的质量比为5~20:1,去离子水为溶剂,室温反应2~8h,未参加反应的短肽通过透析除去,通过冻干的方法制得三甲基化壳聚糖-g-聚乙二醇-VAPG。
6.如权利要求1所述的基于改性壳聚糖的核酸靶向递送载体的应用,其特征是所述的核酸为microRNAs,具体为microRNA-145的双链模拟物,载体与microRNA-145复合得到纳米粒子,可靶向血管平滑肌细胞,调节血管平滑肌细胞的表型。
7.如权利要求6所述的应用,其特征是包括步骤:将制备的载体溶解于焦碳酸二乙酯处理过的水中,与microRNA-145溶液根据载体中氮原子和microRNA-145中磷原子的N/P摩尔数比例为12~20进行混合,室温静置10~30min。
8.如权利要求6所述的应用,其特征是所述的纳米粒子的动态光散射测试的粒径为50~200nm,ζ电位为2~30mV。
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CN114316086A (zh) * | 2021-12-30 | 2022-04-12 | 广西医科大学 | 改性琥珀酰壳聚糖、载药纳米颗粒及其制备靶向肝癌细胞药物中的应用 |
CN114316086B (zh) * | 2021-12-30 | 2022-08-02 | 广西医科大学 | 改性琥珀酰壳聚糖、载药纳米颗粒及其制备靶向肝癌细胞药物中的应用 |
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