CN113041232A - 靶向肠道爆破的口服结肠靶向水凝胶及其制备方法与应用 - Google Patents
靶向肠道爆破的口服结肠靶向水凝胶及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种靶向肠道爆破的口服结肠靶向水凝胶及其制备方法与应用。该口服结肠靶向水凝胶的制备方法包括:(1)利用巯基化的透明质酸与银离子交联,采用气流控技术制备得到含钙离子的HA‑SH‑Ag水凝胶微球;(2)通过钙离子扩散交联获得以海藻酸钙为壳和以HA‑SH‑Ag为核的核壳结构水凝胶微球,即得靶向肠道爆破的口服结肠靶向水凝胶微球。本发明利用气流控技术和离子扩散法制备了一种可靶向结肠爆破的核壳结构水凝胶微球,其被口服后可准确的在结肠部位“爆破”、释放水凝胶,通过黏附在肠壁局部调节肠道巨噬细胞极化、诱导良性肠道菌群组成治疗IBD,是一种安全、多效、具有临床转化意义的肠炎治疗药物。
Description
技术领域
本发明属于靶向给药技术领域,涉及一种口服结肠靶向水凝胶微球,具体涉及一种具有靶向肠道“爆破”的口服结肠靶向水凝胶微球及其制备方法与应用。
背景技术
肠道是人体最大的免疫器官,由于不断暴露在一系列的外来抗原中,当肠道免疫系统错误识别了无害的抗原,其强大的保护性机制会引起慢性肠道炎症的发生发展。随着炎症细胞的募集和浸润,肠道组内炎症因子和ROS等促炎介质逐渐累积,导致以上皮损伤、溃疡形成为基本特征的炎症性肠病(IBD)的发生。IBD近年来的发病率正稳步提升,其腹痛、腹泻、便血等IBD活动期症状及并发症会严重影响患者生活质量、加重经济负担。
然而目前而言,针对IBD的治疗手段却不尽如人意,5-氨基水杨酸、糖皮质激素属于一线抗炎治疗药物,但其非特异性抗炎特性可导致全身性副作用;新型生物制剂如TNF-α的单克隆抗体Infliximab不但价格昂贵,而且存在30%的无应答率和每年20%的耐药率。近年来,具有炎症调节作用的生物材料正逐渐走入人们的视野,研究者们发明了一系列如靶向消除ROS的纳米药、携带炎症因子siRNA和修饰有抗炎基团的纳米药物,通过对炎症介质的调控减轻肠炎小鼠的炎症水平。尽管这些免疫治疗在模式动物体内获得了初步成功,但是肠道免疫稳态的维持离不开一个健康的肠道菌群结构。因此,肠道菌群研究成为目前的研究热点。
肠道内的外来抗原主要来自于细菌群落,这些数量巨大和种类丰富的细菌群落不单单影响肠道免疫的稳态,而且参与机体免疫塑造。Microbiome-wide微生物组学研究揭示了特定微生物与一系列疾病间的相关性(J.M.Blander,R.S.Longman,I.D.Iliev,G.F.Sonnenberg,D.Artis,Nat Immunol.2017,18,851-860.),包括IBD、自身免疫性疾病、肿瘤、代谢和神经退行性疾病等,因此肠菌调节对于IBD治疗乃至人体各系统稳态至关重要。近年来,粪菌移植治疗肠道感染是菌群调节的一个新尝试,但内在机制的错综复杂、临床标准的缺乏和不良事件的频发使其难以被推广。应用抗生素对于活动性IBD有一定疗效,但易导致细菌耐药、严重副反应和有益菌减少。Nguyen et al的研究指出抗生素的使用与新发IBD风险呈剂量依赖性的正相关(L.H.Nguyen,A.K.Y.Cao,T.G.Simon,B.Roelstraete,M.Song,A.D.Joshi,K.Staller,A.T.Chan,H.Khalili,O.Olén,J.F.Ludvigsson,The lancet.Gastroenterology&hepatology.2020,5,986-995.)。传统医学对肠道菌群的干预手段的缺陷使得对安全有效调控手段的需求日益增加。虽然寻找可安全有效调节肠道菌群、调控肠道免疫稳态的药物是目前IBD治疗的主要研究方向,但是如何通过最便捷的口服给药方式将药物定点运送到肠道,使药物在局部调控免疫-肠菌稳态从而达到预期体内疗效,是实现临床转化的关键。
口服给药是慢性胃肠道疾病的首选给药方式,具有简便、安全和患者依从性好的优点,且可对消化道黏膜局部产生治疗部位作用。然而这种给药方式有如下缺陷:(1)大多数口服药物难以耐受胃酸、消化酶的破坏,而腹泻症状使得常规剂型被迅速清除;(2)非靶向药物给药后的系统暴露率高和生物利用率低,为达到目的疗效而增加剂量和反复给药又可加重药物副作用。尽管许多药物在实验研究中表现出显著的治疗潜能,人体内应用时效果却不尽人意,透明质酸(HA)就是一个很好的例子。HA来源广泛、无免疫原性,不但具有良好的生物相容性,而且有研究报道HA可结合免疫细胞表面CD44受体、影响巨噬细胞的活化和分化,具有抗炎和免疫抑制的作用,具有治疗IBD的潜能(CN105749296A)。但是,其本身结构易扩散和在消化液中易降解的缺陷严重影响了肠道内抗炎和促修复的治疗效果,限制了HA在口服给药中的应用。
因此,如何提供一种新的可口服结肠靶向的生物材料,实现减少HA的过早被吸收或破坏,更好的发挥HA调节肠菌或肠黏膜溃疡处的炎症组织的作用,以打破上述应用困境,成为现阶段利用HA来实现肠道内抗炎和促进IBD的修复治疗中亟待解决的技术问题。
发明内容
本发明的目的就是为了解决上述技术问题,而提供一种具有靶向肠道“爆破”的口服结肠靶向水凝胶微球及其制备方法与应用。本发明利用气流控技术和离子扩散法制备了一种外壳可靶向结肠爆破的核壳结构水凝胶微球,该水凝胶微球被口服后可准确的在结肠部位“爆破”、释放水凝胶,通过黏附在肠壁局部调节肠道巨噬细胞极化、诱导良性肠道菌群组成治疗IBD,是一种安全、多效、具有临床转化意义的肠炎治疗药物。
本发明的目的之一是提供一种靶向肠道爆破的口服结肠靶向水凝胶微球的制备方法,其包括以下步骤:
(1)利用巯基化的透明质酸(HA-SH)可与银离子交联的原理,采用气流控技术制备得到含钙离子的HA-SH-Ag水凝胶微球;
(2)通过钙离子扩散交联获得以海藻酸钙为壳和以HA-SH-Ag为核的可口服核壳结构水凝胶微球,即得靶向肠道爆破的口服结肠靶向水凝胶微球。
本发明是基于透明质酸(HA)强大的免疫调节功能,通过合成巯基化的透明质酸(HA-SH)与抗菌银离子交联构建得到HA-SH-Ag水凝胶,并通过新型气流控技术制备含钙离子的HA-SH-Ag水凝胶微球,再通过钙离子扩散交联获得外壳为海藻酸钙和内核为HA-SH-Ag的核壳结构口服水凝胶微球。由于海藻酸钙具有在胃里不溶解和在肠道溶解的特性,本发明通过利用海藻酸独特的pH敏感性、强大的交联和降解能力等特点,通过在HA-SH-Ag水凝胶表面包裹一层海藻酸钙水凝胶的保护性外壳,赋予了该水凝胶微球以结肠靶向爆破的能力。经实验验证,该水凝胶微球被口服后可准确的在结肠部位“爆破”、释放HA-SH-Ag水凝胶,通过黏附在肠壁局部调节肠道巨噬细胞极化、诱导良性肠道菌群组成来发挥有效治疗IBD的作用。
本发明通过选择具有大规模微球制备潜能的气流控技术制备HA-SH-Ag水凝胶微球,这种微球制备方法相对于微流控法具有以下显著的优势:气流控制备法用气流代替油相来对流动的溶液进行切割,去除了微球与油相分离的步骤,一步即可完成微球制备;该方法还可通过提高溶液的流速来进行高效的大规模生产。
本发明提供的上述方法可制备大小高度均一的肠道靶向“爆破”的水凝胶微球,并可稳定量产,有利于临床转化。微球壳层在结肠部位“爆破”后,内部HA-SH-Ag水凝胶通过剩余的巯基与肠道黏蛋白形成二硫键粘附于肠黏膜,实现更长的肠道驻留时间。该口服结肠靶向水凝胶微球可以作为一种安全、多效、具有临床转化意义的肠炎治疗药物。
进一步的是,步骤(1)中所述HA-SH-Ag水凝胶的制备步骤为:利用微量注射泵将质量百分浓度为4%的HA-SH水溶液匀速注入同轴针,利用0.4L/min的氮气所产生的剪切力将溶液切割成均匀的液滴。液滴落入下方含有0.1mol/L Ca(NO3)2和0.2mol/LAgNO3水溶液的收集皿内,HA-SH液滴与Ag+交联形成HA-SH-Ag水凝胶微球。
进一步的是,步骤(2)中所述核壳结构水凝胶微球的制备步骤为:收集HA-SH-Ag水凝胶微球并洗涤两次以去除表面残留的离子后,将其加入到质量百分浓度为0.5%的海藻酸钠溶液中,微球内的钙离子逐渐扩散到微球表面并交联海藻酸钠形成海藻酸钙水凝胶外壳,从而得到核壳结构水凝胶微球。
进一步的是,步骤(1)中所述巯基化的透明质酸的制备步骤为:将5g透明质酸粉末通过机械搅拌溶解于250mL的2-(N-吗啉代)乙磺酸缓冲液(MES,10.0mM,pH=5.5)中,然后加入3,3'-二硫代双(丙酸二酰肼)溶液(626mg溶解在10mL DMSO中)和二甲氧基-1,3,5-三嗪-2-基-4-甲基吗啉氯化物溶液(2.91g溶解在10mLMES溶液中)在室温下继续搅拌12h得到凝胶态化合物。然后将三(2-羧乙基)膦盐酸盐(3g)溶解在20mL去离子水中并滴加到上述凝胶体系中并继续搅拌3h以获得均匀溶液。最后,在溶液中加入NaCl(10g)并搅拌均匀以破坏溶液中强的物理作用,再用含有0.1M NaCl和稀HCl(pH=4.5)通过透析袋(MWCO 7000,)透析两天,再用稀HCl(pH=4.5)溶液继续透析两天,得到纯化的HA-SH溶液后冻干。
进一步的是,步骤(1)中所述HA-SH-Ag水凝胶微球的大小为264.64±13.98μm。
本发明的目的之二是提供一种靶向肠道爆破的口服结肠靶向水凝胶微球,其是由如上所述的方法制备而成。
进一步的是,该靶向肠道爆破的口服结肠靶向水凝胶微球经口服后到达结肠并释放HA-SH-Ag水凝胶。
进一步的是,所述释放的HA-SH-Ag水凝胶黏附于肠道黏膜,并发挥抗炎和菌群双调节的作用。
本发明的目的之三是提供如上所述的靶向肠道爆破的口服结肠靶向水凝胶微球在制备炎症性肠病治疗的药物中的应用,该口服结肠靶向水凝胶微球可以作为一种安全、多效、具有临床转化意义的肠炎治疗药物,在IBD治疗上是一个理想的候选者。
与现有技术相比,本发明的有益效果如下:
本发明制备了一种外壳可靶向结肠爆破的核壳结构水凝胶微球,该核壳结构水凝胶微球可很好用于肠炎治疗,其内部HA-SH-Ag水凝胶可黏附肠道黏膜,并发挥抗炎和菌群双调节的作用。体外研究验证了本发明的水凝胶微球能够具有调节巨噬细胞分化和炎症因子TNF-a和IL-6等分泌的作用。通过小鼠模型验证了微球可显著缓解DSS诱导的结肠炎;通过对小鼠粪便的16s rRNA测序发现微球可优化肠菌组成结构,上调益生菌如双歧杆菌和乳酸杆菌的丰度,有利于肠炎缓解和修复。总之,本发明提供的水凝胶微球一种安全、多效、具有临床转化意义的肠炎治疗药物,在IBD治疗上是一个理想的候选者。
附图说明
图1为:(a)靶向肠道爆破的口服结肠靶向水凝胶微球的制备流程示意图;(b)靶向肠道爆破的口服结肠靶向水凝胶微球应用于肠炎的治疗示意图。
图2为HA-SH的合成路线图。
图3为HA-SH的红外表征图谱。
图4为:(a)HA-SH冻干样的照片(左)和核磁共振波谱(1H NMR)(右);(b)采用气体剪切装置将HA-SH水溶液制备成HA-SH微球;(c)获得的HA-SH微球的光学图像(左)和微球的尺寸分布(右),比例尺50μm;(d)HMs的扫描电子显微镜(SEM)图像和元素分析;(e)HAMs的SEM图像和元素分析;(f)HMs在海藻酸钠水溶液中的停留时间(0~60min)与壳层厚度的变化曲线;(g)不同外壳厚度的HAMs浸入不同pH模拟消化液中降解曲线图;(h)HAMs的壳层(海藻酸钙水凝胶)在模拟消化液中降解拍照图,比例尺100μm;(i)在透明质酸酶的存在下,HAMs核(HA-SH水凝胶微球)发生缓慢降解,比例尺200μm。
图5为:(a)平板涂布法实验流程示意图;(b)通过平板涂布法测定HAMs的抗菌活性;(c)大肠杆菌和鼠柠檬酸杆菌的菌落计数;(d)在模拟消化液中银离子释放的检测;(e)calcein-AM/PI细胞活力测定法检测了HAMs对Caco-2和iBMDM细胞株中的细胞毒性,标尺500μm;(f)qPCR检测促炎症细胞因子TNF-α、IL-1β和IL-6的mRNA水平,实验重复3次;(g)qPCR检测M2型巨噬细胞相关基因IL-10、Arginase1、Fizz1、YM1的mRNA水平,实验重复3次;(h)健康小鼠和DSS诱导结肠炎的小鼠给与ICG-HAMs灌胃后6小时,在IVIS下成像;(i)健康小鼠和DSS诱导结肠炎的小鼠给与ICG-HAMs灌胃后,取其肠段用PBS冲洗,在IVIS下分别对冲洗前后的肠段进行成像。
图6为HAMs的抑菌圈实物图及抑菌圈直径统计结果。
图7为:通过cck8试剂盒检测细胞活性来反映生物安全性。(a)HAMs与肠上皮细胞系Caco-2共培养后的细胞活性曲线;(b)HAMs与巨噬细胞系iBMDM共培养的细胞活性曲线。
图8为:LPS刺激下巯基化透明质酸对TNF-α、IL-1β和IL-6促炎症因子的mRNA分泌的抑制能力。
图9为:(a)造模设计:前两组给予正常饮水,其余组给予含3%w/vDSS的饮用,其他处理皆通过灌胃进行,每天一次;(b)各组代表性的结肠图片,n=5;(c)各组结肠长度的测量和分析,n=5;(d)各组代表性的脾脏图片,n=5;(e)各组脾脏重量的测量和分析,n=5;(f)每日体重的变化和分析,n=5;(g)疾病活动指数分析,n=5;(h)代表性的结肠组织苏木精伊红(HE)染色图像;(i)通过各组HE染色统计并分析结肠损伤评分;
采用Mann-Whitney U-test进行分析,ns不显著,*P<0.05,**P<0.01,***P<0.001,****P<0.0001。
图10为各组小鼠的处理方式。
图11为:(a)代表性的MPO免疫组化染色,标尺100μm;(b)MPO免疫组化的定量分析;(c)代表性的PCNA免疫组化染色,标尺100μm;(d)PCNA免疫组化的定量分析;(e)westernblot分析小鼠结肠组织中inos和Arginase1水平的代表性图像;(f)通过ImageJ软件量化inos和Arginase1的蛋白水平;(g)免疫荧光分析M1型巨噬细胞(DAPI蓝色、CD68绿色、iNOS红色))和M2型巨噬细胞(DAPI蓝色、CD68绿色、CD163红色),通过共焦显微镜观察,比例尺100μm;(h)qPCR检测结肠组织TNF-α、IL-1β、IL-6、TGF-β等细胞因子的mRNA表达水平;(i)ELISA检测血清TNF-α、IL-1β、IL-6、TGF-β等细胞因子的浓度;
采用Mann-Whitney U-test进行分析,ns不显著,*P<0.05,**P<0.01,***P<0.001。
图12为HAMs灌胃的小鼠的心脏、肝脏、肺、肾脏等主要器官的组织HE染色图。
图13为16s rRNA测序技术对三组小鼠(健康对照组、DSS组、DSS+HAMs组)的粪便菌群在群落丰富度的分析结果。
图14为16S rRNA测序分析结果:(a)代表肠道微生物群落α-多样性的Shannon和Simpson指数;(b)代表肠道微生物群落β-多样性的PCOA分析,每个点代表一只老鼠,n=5;(c)群落直方图显示门水平上的肠道菌群组成;(d)Heatmap显示在科水平上的肠道菌群的相对丰度;(e)LEfSe分析对从门到属不同分类单元下丰度有差异的群体进行识别;(f)线性判别分析(LDA);(g)小鼠门水平上的肠菌组成中厚壁菌门、放线菌门和变形菌门的丰度分析;(h)小鼠科水平上的肠菌组成中乳杆菌科、双歧杆菌科和肠杆菌科的丰度分析;
统计分析采用Kruskal-Wallis检验。ns不显著,*P<0.05,**P<0.01。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例对本发明进行具体描述,有必要指出的是,以下实施例仅仅用于对本发明进行解释和说明,并不用于限定本发明。本领域技术人员根据上述发明内容所做出的一些非本质的改进和调整,仍属于本发明的保护范围。
实施例1
本实施例提供了一种靶向肠道爆破的口服结肠靶向水凝胶微球的制备方法,其包括以下步骤:
(1)利用巯基化的透明质酸(HA-SH)可与银离子(Ag+)交联的原理,采用气流控技术制备得到含钙离子的HA-SH-Ag水凝胶微球;
(2)通过钙离子扩散交联获得以海藻酸钙为壳和以HA-SH-Ag为核的可口服核壳结构水凝胶微球,即得靶向肠道爆破的口服结肠靶向水凝胶微球。
具体的,本实施例中巯基化的透明质酸的制备步骤为:将5g透明质酸粉末通过机械搅拌溶解于250mL的2-(N-吗啉代)乙磺酸缓冲液(MES,10.0mM,pH=5.5)中,然后加入3,3'-二硫代双(丙酸二酰肼)溶液(626mg溶解在10mL DMSO中)和二甲氧基-1,3,5-三嗪-2-基-4-甲基吗啉氯化物溶液(2.91g溶解在10mLMES溶液中)在室温下继续搅拌12h得到凝胶态化合物;然后将三(2-羧乙基)膦盐酸盐(3g)溶解在20mL去离子水中并滴加到上述凝胶体系中并继续搅拌3h以获得均匀溶液;最后,在溶液中加入NaCl(10g)并搅拌均匀以破坏溶液中强的物理作用,再用含有0.1M NaCl和稀HCl(pH=4.5)通过透析袋(MWCO 7000,)透析两天,再用稀HCl(pH=4.5)溶液继续透析两天,得到纯化的HA-SH溶液后冻干。
HA-SH-Ag水凝胶微球的制备步骤为:利用微量注射泵将质量百分浓度为4%的HA-SH水溶液匀速注入同轴针,利用0.4L/min的氮气所产生的剪切力将溶液切割成均匀的液滴,液滴落入下方含有0.1mol/L Ca(NO3)2和0.2mol/LAgNO3水溶液的收集皿内,HA-SH液滴与Ag+交联形成HA-SH-Ag水凝胶微球。
核壳结构水凝胶微球的制备步骤为:收集HA-SH-Ag水凝胶微球并洗涤两次以去除表面残留的离子后,将其加入到质量百分浓度为0.5%的海藻酸钠溶液中,微球内的钙离子逐渐扩散到微球表面并交联海藻酸钠形成海藻酸钙水凝胶外壳,从而得到核壳结构水凝胶微球。
如图1所示为发明的靶向肠道爆破的口服结肠靶向水凝胶微球的制备步骤及应用示意图。图1(a)中的流程示意了采用气流控技术制备得到大小均一的HA-SH-Ag水凝胶微球(HA-SH-Ag hydrogel microsphere,简记为HMs),接着钙离子从微球内部向外扩散并在微球表面交联海藻酸,从而在HMs表面形成海藻酸钙水凝胶外壳,得到具有核壳结构的水凝胶微球(HA-SH-Alg hydrogel microsphere,简记为HAMs)。图1(b)中的流程示意了靶向结肠爆破的水凝胶微球口服后到达结肠并释放HA-SH-Ag水凝胶,HA-SH-Ag水凝胶在炎症的结肠黏膜中积累,通过抑制促炎细胞因子的分泌和诱导M2型巨噬细胞分化调节肠道炎症,并通过上调益生菌的丰度和抑制有害菌群优化了肠道菌群组成。后续实验例将详细阐述该机理。
实验例1
实施例1中HA-SH的合成步骤如图2所示,为了验证HA-SH的成功合成,通过1HNMR和FITR进行了表征(图3,图4a)。如图4b、4c所示,通过气流控法制备的HA-SH-Ag微球(HMs),大小相对均一(264.64±13.98μm),产率约为100mg/min。
在扫描电子显微镜(scanning electron microscope,SEM)下,可观察到HMs和HAMs的外表面是光滑的,通过能谱仪(EDS)检测两种微球表面的元素分布验证了其结构(图4d、图4e)。
为了确保HAMs成功被递送到结肠并快速释放核心微球,发明人通过对海藻酸钠水凝胶外壳厚度进行微调。结果发现,随着HMs在海藻酸钠水溶液中停留时间(5~40min)的增加,壳体不断变厚(0~12μm)(图4f),但交联时间超过40min后,由于Ca2+的耗尽壳层厚度停止增加。依次将不同外壳厚度的HAMs浸入模拟消化道各部位pH值的模拟消化液中,发现8μm的壳层厚度可使HAMs相对完整地通过模拟胃液和模拟小肠液,但在模拟结肠液中仅10分钟就会完全降解(图4g、4h)。另外,在微球壳层降解后,发现HAMs中的水凝胶核可在1至2天内逐渐膨胀并缓慢降解(图4i),该缓慢降解的过程有利于体内药物的持续释放和作用。
实验例2
HMs在缓慢降解过程中释放Ag+和HA-SH单体,其中银长久以来被作为抗菌剂使用,而大量的证据表明肠道菌群在IBD发病中发挥了重要作用。因此,本实验例中对HAMs的抗菌作用进行了体外验证。
首先,通过平板涂布法(图5a)和Kindy-Bauer法在体外利用E.coli和一种鼠类致病菌——鼠柠檬酸杆菌探究HAMs的抗菌能力(参考S.Wang,H.Zheng,L.Zhou,F.Cheng,Z.Liu,H.Zhang,L.Wang,Q.Zhang,Nano Lett.2020,20,5149-5158.)。如图5所示,图5b、5c的菌落计数和以及图6的抑菌圈直径统计结果表明:HAMs以及其浸提液对E.coli和鼠柠檬酸杆菌有一定的抑制作用。
另一方面,众所周知Ag+具有细胞毒性,但文献表明微量的Ag+对微生物的毒性远大于对人体的毒性。于是测定了HAMs在模拟消化液中Ag+的释放,并将HAMs与肠上皮细胞系Caco-2和巨噬细胞系iBMDM进行共培养,通过检测细胞生存和增殖能力来评估HAMs生物兼容性。从图5d、5e,图7可以看到,微量的Ag+在HAMs的缓慢降解过程中被缓慢释放,另外与HAMs共培养的细胞表现出良好的活力,这表明HAMs具有良好的生物相容性。
接着,在体外对HAMs的生物特性进行了检测。如背景技术中所述,虽然HA具有抗炎能力,但尚不确定巯基基团的修饰是否会改变其抗炎特性。因此,通过检测LPS刺激下iBMDM细胞内细胞因子的mRNA表达和分泌,体外评估HA-SH的抗炎能力。结果发现HA-SH对于TNF-α、IL-1β和IL-6等促炎症因子的mRNA表达和分泌的抑制能力不弱于HA,表明经巯基修饰后,HA-SH具备相当甚至更强的抗炎能力(图5f、图8)。
由于有文献报道关于IBD患者的炎症结肠组织中M1型巨噬细胞广泛聚集,而该类细胞可大量分泌上述促炎症细胞因子。激活的巨噬细胞可分化为两型,即具有完全相反功能的促炎M1型和抗炎促愈合的M2型巨噬细胞。因此,为了了解HA-SH是否可通过诱导巨噬细胞分化发挥其抗炎能力,发明人检测了Arginase1、Fizz1、YM1等M2相关基因的mRNA水平变化(图5g),发现HA-SH可显著上调M2相关基因表达,提示HA-SH可调控巨噬细胞分化,但仍需要体内实验验证。
为了在体内获得更好的生物利用度,需要赋予药物以靶向性并促使其在局部富集。HAMs的壳层水凝胶靶向结肠爆破后释放的HA-SH-Ag水凝胶,其内部剩余的巯基在理论上可与肠黏膜表面黏液中富含半胱氨酸的糖蛋白形成二硫键,可延长其在肠黏膜的停留时间。此外,HA-SH因其丰富的羧基而带负电荷,而炎症组织富含如转铁蛋白和抗菌肽等带正电荷的物质,这有也助于其在炎症的结肠组织中滞留。为了验证HAMs的体内黏附性,发明人制备了ICG修饰HA-SH制备的HAMs,对小鼠进行灌胃并进行了IVIS成像。在图5h中可见,DSS诱导结肠炎小鼠体内的荧光信号较健康小鼠更强,且图5i中可见PBS冲洗肠段前后的IVIS成像提示DSS诱导结肠炎小鼠的肠道荧光物质的滞留的健康小鼠多。
实验例3
本实验例中,通过构建DSS诱导结肠炎小鼠模型来检测HAMs在体内的治疗效果。DSS诱导结肠炎是最常用的一种IBD动物模型,DSS溶解于饮用水中被小鼠饮用后可损伤上皮细胞、破坏肠道屏障,致使肠道微生物群的入侵随后引起炎症。
实验方法如下:
将小鼠随机分为对照组、HAMs组、DSS组、DSS+HAMs组、DSS+HA-SH组、DSS+5-ASA组。前两组给予正常饮水,其余组给予含3%w/v DSS的饮用,其他处理皆通过灌胃进行,每天一次(图9a)。结果发现给与HAMs灌胃处理可显著维持DSS诱导结肠炎小鼠的体重水平(P<0.0001,图9b),疾病活动指数(P<0.0001,图9c、图10)反映出小鼠的一般健康状况也好于对照组。解剖小鼠后,发现DSS+HAMs组结肠长度显著长于DSS组(P<0.0001,图9d、9e)、脾脏重量也显著轻于DSS组(P<0.001,图9f、9g)。从图9h和9i的HE染色结肠损伤评分(P<0.001)可以发现,HAMs在治疗DSS诱导的结肠炎过程中,对维持结肠上皮的完整性、减少黏膜炎症细胞的浸润方面均有一定作用。以上证据表明,HAMs通过口服可缓解小鼠的肠道炎症。
此外,虽然5-ASA是治疗IBD的一线药物,但有复发风险和多重不良反应,因此发明人还比较了HAMs和5-ASA对于DSS诱导肠炎的治疗效果。从图9的结果中可发现,HAMs治疗效果在从体重、结肠长度、脾脏重量、结肠损伤评分等多个方面均较5-ASA显著。
实验例3
为了揭示HAMs保护小鼠免受DSS诱导的结肠炎具体机制,通过对小鼠结肠组织的免疫组化分析,发现髓过氧化物酶(myeloperoxidase,MPO)阳性的中性粒细胞数在DSS诱导结肠炎的小鼠结肠组织中显著增加,而HAMs治疗显著降低了该细胞的比例(P<0.001,图11a、11b),这进一步验证了HAMs能够缓解结肠炎症。进一步还发现在HAMs或HA-SH处理组,肠道组织PCNA(细胞增殖的重要组织标记物)的表达水平显著高于对照组(P<0.001,图11c、11d),表明HA-SH也可以促进组织修复。这也解释了前文观察到接受HAMs或HA-SH治疗的小鼠结肠上皮相对完整,但是HA-SH主要调节免疫反应,而DSS主要损伤上皮。
由于在体外发现HA-SH可促进M2巨噬细胞分化,而M2的特点是抗炎和组织修复。因此,通过Westernblot检测结肠组织中inos和arginase 1(M1和M2标记物)的蛋白水平。结果发现,HAMs治疗组小鼠结肠组织arginase 1显著上调,inos表达下调(图11e、11f)。结肠组织的免疫荧光分析也表明,HAMs治疗明显增加了组织内M2巨噬细胞的水平、减少了M1型巨噬细胞(图11g)。
此外,对结肠组织细胞因子mRNA表达和血清中细胞因子水平的分析结果与前文体内外发现的结果一致,即HAMs治疗上调了组织修复相关的细胞因子TGF-β的表达和分泌,并下调了促炎症细胞因子TNF-α、IL-1β和IL-6的表达和分泌(图11h、11i),体内外结果一致。另外,除了追求治疗效果,体内用药的安全性也至关重要,从图12中可以看出,给与HAMs灌胃的小鼠的心脏、肝脏、肺、肾脏等主要器官的组织HE染色与其他组相似,没有异常炎症反应或病变,提示HAMs在体内具有良好的生物相容性。
实验例5
众所周知,肠道微生物群组成和丰度的异常波动与IBD密切相关,而参与水凝胶交联过程的银离子具有杀菌作用。虽然Ag+具有广泛的抗菌谱,但多项证据表明这种杀菌剂具有选择性,而肠道生物膜中也有一部分是益生菌,对微环境的健康有重要意义。综上,本实施例中探究了HAMs影响是否以及如何影响肠道微生物群的组成或丰度。通过采用先进的16s rRNA测序技术对三组小鼠(健康对照组、DSS组、DSS+HAMs组)的粪便菌群进行深入分析,发现三组小鼠的粪便菌群在群落丰富度和alpha多样性上都没有显著差异(图13、图14a),这可能是由于建模周期较短,菌群在整体水平上的变化不显著所致。但是PCOA分析表明,HAMs处理确实改变了结肠炎小鼠的菌群组成(图14b)。
进一步深入分析了肠菌的具体变化,每个样品在门、科水平上的肠杆菌总体组成分别用柱形图和热图表示(图14c、14d)。线性判别分析效应大小分析(Lineardiscriminant analysis Effect Size,LEfSe)对从门到属不同分类单元下丰度有差异的群体进行识别(图14e、14f),发现HAMs治疗组肠菌组成在门水平上与健康对照组接近,而DSS组相对于这两组厚壁菌门和放线菌门数量减少,变形菌门丰度增加(图14g),DSS组的肠菌变化文献报道的IBD患者的菌群特征类似。
进一步还发现HAMs治疗组小鼠肠道内乳杆菌和双歧杆菌丰度显著高于DSS组,而在炎症性肠病的患者体内丰度相对高的肠杆菌科在HAMs治疗组肠道内丰度减少(图14h)。益生菌可通过竞争性抑制有害微生物定植和调节肠道免疫反应来维持一个健康的肠道微环境,因此HAMs干预后这些菌群的变化有利于构建健康肠道菌群以及肠炎缓解。综上所述,HAMs可以优化小鼠肠道菌群的组成,可减少应用抗生素导致的副作用和预防耐药细菌导致的严重感染,因此HAMs是治疗炎症性肠病理想的候选药物。
Claims (10)
1.一种靶向肠道爆破的口服结肠靶向水凝胶微球的制备方法,其特征在于,包括以下步骤:
(1)利用巯基化的透明质酸与银离子交联,采用气流控技术制备得到含钙离子的HA-SH-Ag水凝胶微球;
(2)通过钙离子扩散交联获得以海藻酸钙为壳和以HA-SH-Ag为核的核壳结构水凝胶微球,即得靶向肠道爆破的口服结肠靶向水凝胶微球。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述HA-SH-Ag水凝胶微球的制备步骤为:利用微量注射泵将质量百分浓度为4%的巯基化的透明质酸水溶液匀速注入同轴针,利用0.4L/min的氮气所产生的剪切力将溶液切割成均匀的液滴,液滴落入下方含有0.1mol/L Ca(NO3)2和0.2mol/LAgNO3水溶液的收集皿内,巯基化的透明质酸液滴与Ag+交联形成HA-SH-Ag水凝胶微球。
3.根据权利要求1所述的制备方法,其特征在于,步骤(2)中所述核壳结构水凝胶微球的制备步骤为:收集HA-SH-Ag水凝胶微球并洗涤两次以去除表面残留的离子后,将其加入到质量百分浓度为0.5%的海藻酸钠溶液中,微球内的钙离子逐渐扩散到微球表面并交联海藻酸钠形成海藻酸钙水凝胶外壳,从而得到核壳结构水凝胶微球。
4.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述巯基化的透明质酸的制备步骤为:将5g透明质酸粉末溶解于250mL的2-(N-吗啉代)乙磺酸缓冲液,然后加入3,3'-二硫代双(丙酸二酰肼)溶液和二甲氧基-1,3,5-三嗪-2-基-4-甲基吗啉氯化物溶液在室温下继续搅拌12h得到凝胶态化合物;然后将3g三(2-羧乙基)膦盐酸盐溶解在20mL去离子水中并滴加到上述凝胶体系中并继续搅拌3h以获得均匀溶液;最后,在溶液中加入10gNaCl并搅拌均匀以破坏溶液中强的物理作用。接着用含有0.1M NaCl和pH=4.5的稀HCl通过透析袋透析两天,再用pH=4.5的稀HCl溶液继续透析两天,得到纯化的HA-SH溶液后冻干,即得。
5.根据权利要求4所述的制备方法,其特征在于,所述2-(N-吗啉代)乙磺酸缓冲液的浓度为10.0mM,pH=5.5;所述3,3'-二硫代双(丙酸二酰肼)溶液是将626mg 3,3'-二硫代双(丙酸二酰肼)溶解在10mL DMSO中得到;所述二甲氧基-1,3,5-三嗪-2-基-4-甲基吗啉氯化物溶液是将2.91g原料溶解在10mL 2-(N-吗啉代)乙磺酸缓冲液中而得。
6.根据权利要求1所述的制备方法,其特征在于,步骤(1)中所述HA-SH-Ag水凝胶微球的大小为264.64±13.98μm。
7.一种靶向肠道爆破的口服结肠靶向水凝胶微球,其特征在于,是由权利要求1-6任一项所述方法制备而成。
8.根据权利要求7所述的靶向肠道爆破的口服结肠靶向水凝胶微球,其特征在于,所述靶向肠道爆破的口服结肠靶向水凝胶微球经口服后到达结肠并释放HA-SH-Ag水凝胶。
9.根据权利要求8所述的靶向肠道爆破的口服结肠靶向水凝胶微球,其特征在于,所述释放的HA-SH-Ag水凝胶黏附于肠道黏膜,并发挥抗炎和菌群双调节的作用。
10.如权利要求1-6任一项所述方法制备的或权利要求7-9任一项所述的靶向肠道爆破的口服结肠靶向水凝胶微球在制备炎症性肠病治疗的药物中的应用。
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