CN114010798A - 一种酶控自组装与解组装的负载姜黄素药物的水凝胶前体及其应用 - Google Patents
一种酶控自组装与解组装的负载姜黄素药物的水凝胶前体及其应用 Download PDFInfo
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Abstract
本发明公开了一种酶控自组装与解组装的负载姜黄素药物的水凝胶前体及其应用，其中负载姜黄素药物的水凝胶前体简记为Cur‑FFEYp，其结构式如下所示：
本发明水凝胶前体Cur‑FFEYp通过对姜黄素进行修饰，极大增加了姜黄素的溶解性，提高了其生物利用度。其中，Cur‑FFEYp可以被肠炎细胞中过度表达的ALP特异性识别剪切，生成的Cur‑FFEY通过π‑π堆积自组装形成纳米纤维，富集在肠炎细胞中，增强滞留时间；随后，Cur‑FFEY被肠炎细胞中富含的酯酶缓慢水解，原位释放出姜黄素。这种酶控制的自组装富集与解组装缓慢释放的过程提高了Cur治疗炎症性肠病的效果，具有良好的应用前景。
Description
技术领域
本发明涉及一种酶控自组装与解组装的负载姜黄素药物的水凝胶前体及其应用，实现炎症性肠病的靶向给药及治疗。
背景技术
近年来，随着国内生活方式的多样化，炎症性肠病(Infiammatory BowelDisease，IBD)的发病趋势为发病率越来越高，越来越年轻化，且极易反复，尤其是在北京、上海、深圳等发达城市。IBD包括两种类型：溃疡性结肠炎(Ulcerative colitis，UC)和克罗恩病(Crohn’s disease，CD)。其区别在于，溃疡性结肠炎发生在结肠，是一种连续性炎症疾病，随着疾病的加深，发病部位会由直肠蔓延到整个结肠。克罗恩病则发生在整个消化道，是一种非连续性的炎症疾病，发病部位会分布在消化道的各个部位。目前，IBD的治疗方式以抗炎药物及免疫治疗药物为主，虽然这些药物已经被证明可以有效地抑制炎症，但在进一步的临床使用过程中，会出现长期治疗效果不佳及严重的副作用。因此急需开发一种新的递药系统用于炎症性肠病的治疗。
姜黄素(Cur)是一种从姜黄根茎中提取出来的疏水性多酚化合物。已知Cur具有抗氧化、抗癌、抗炎等多种药理作用，对健康细胞的内在毒性很小甚至没有。已有研究表明，Cur可以通过降低IL-7、IL-23、TNF-α等炎症因子的表达，进而减轻炎症反应达到抗炎效果。有学者发现，Cur可以预防和抑制多种类型的癌细胞的生成、转化、增殖和转移，例如乳腺癌，结肠癌，宫颈癌，胃癌，胰腺癌和肝癌。但是，由于其水溶性极差(≈11ng/ml)和低生物利用度，Cur的应用仍然非常有限。
为了解决上述问题，现有学者通过使用生物材料增加Cur的水溶性，提高其生物利用率。例如，Yang等人开发了一种基于Cur的凝胶因子Cur-FFE-ss-ERGD，该凝胶因子通过二硫键还原形成的水凝胶可以通过酯键水解持续释放出Cur。Feng等人将Cur封装到MPEG-PCL共聚物中，以提供载有Cur的胶束，其药物封装效率达到93.57％，明显增强Cur的水溶性，其制备的超分子水凝胶Cur-H显著提高了Cur的抗皮炎活性且抗炎效果明显高于地塞米松软膏。在碱性条件下，Cur苯环上的羟基以氧负离子形式存在，其给电子能力增强，致使碳链的亲电反应活性大大增强，故Cur在碱性条件下极其不稳定。因此，增强Cur水溶性及稳定性，提高其生物利用率有很大的发展前景。
发明内容
本发明针对上述现有技术所存在的问题，提供了一种酶控自组装与解组装的负载姜黄素药物的水凝胶前体及其应用。本发明通过对Cur结构进行修饰，获得了负载Cur药物的水凝胶前体，具有良好的水溶性及稳定性，提高了姜黄素的生物利用度。
本发明首先提供了一种负载姜黄素药物的水凝胶前体，简记为Cur-FFEYp，结构式如下所示：
本发明负载姜黄素药物的水凝胶前体的制备方法，是以姜黄素衍生物和多肽分子为原料，发生活化酯反应得到Cur-FFE(OtBu)Yp，随后以50％的三氟乙酸脱除谷氨酸的羧氨基保护基OtBu得到目标化合物Cur-FFEYp。
所述姜黄素衍生物是由姜黄素与戊二酸酐反应获得，以酯键形式连接，延伸出羧基，用于酰胺键的形成。具体结构以及反应路线如下所示：
所述多肽分子是由Fmoc-Phe-OH、Fmoc-Phe-OH、Fmoc-Glu-OH、Fmoc-Tyr(H2PO3)-OH通过固相合成得到，合成路线如下所示：
本发明负载姜黄素药物的水凝胶前体的制备方法，具体包括如下步骤：
以吡啶为溶剂，向溶剂中加入Cur和戊二酸酐，在室温下反应6小时，得到姜黄素衍生物Cur-GA-COOH；将Cur-GA-COOH充分分散在DMF中，然后加入1-乙基-(3-(二甲氨基)丙基)-碳二亚胺盐酸盐和N-羟基琥珀酰亚胺，搅拌8-12h以进行活化酯反应；随后加入固相合成的多肽分子FFEYp和N,N-二异丙基乙胺，在室温下搅拌反应8-12h，得到Cur-FFE(OtBu)Yp，随后以50％的三氟乙酸脱除谷氨酸的羧氨基保护基OtBu得到目标化合物Cur-FFEYp。每一步反应用HPLC分离纯化，确认纯度，用1H NMR，13C NMR和MS确认分子结构。
本发明通过姜黄素与戊二酸酐反应，封住了姜黄素一边的酚羟基，抑制了其在碱性条件下的给电子能力，阻止了其碳链上亲电反应活性的增大，从而增强了姜黄素在碱性条件下的稳定性；通过增加H2PO3基团，极大增强了姜黄素的溶解性。
本发明负载姜黄素药物的水凝胶前体的合成路线如下所示：
本发明负载姜黄素药物的水凝胶前体采用亲水性溶剂(比如水)，分散后获得溶液。
本发明负载姜黄素药物的水凝胶前体的应用，是作为原位富集以及缓慢释放Cur的药物递送系统，实现酶控自组装与解组装，用于体内炎症性肠病的治疗。
本发明负载姜黄素药物的水凝胶前体，可以被肠炎细胞中过度表达的碱性磷酸酶(ALP)特异性识别剪切，利用磷酸基团与碱性磷酸酶的相互作用，Cur-FFEY(kcat/kM＝1.45×104M-1s-1)通过π-π堆积自组装形成纳米纤维，富集在肠炎细胞中，增强滞留时间；随后，Cur-FFEY被肠炎细胞中富含的酯酶(Esterase)缓慢水解(kcat/kM＝2.23×102M-1s-1)，原位释放出姜黄素。这种酶控制的自组装富集与解组装缓慢释放的过程提高了Cur治疗炎症性肠病的效果，具有良好的应用前景。
具体地，通过Esterase水解Cur与戊二酸酐连接的酯键，将富集在肠炎细胞中的纳米纤维解组装，使得Cur在发病部位以单分子形式释放，被粘膜吸收，从而发挥有效的抗炎作用，不仅从根本上解决了Cur水溶性与稳定性差的问题，还进一步解决了Cur无法实现靶向给药的问题。
实验结果证明，本发明的负载Cur药物的水凝胶前体可制备得冻干粉末制剂，可以良好的溶于水溶液。通过体内外实验证明，该水凝胶前体可被ALP有效识别并剪切(kcat/kM＝1.45×104M-1s-1)，通过π-π堆积自组装形成平均宽度为4.287±0.495nm的水凝胶纳米纤维。随后通过Esterase水解(kcat/kM＝2.23×102M-1s-1)缓慢释放Cur单分子，达到治疗炎症性肠病的目的，具有良好的医疗前景。
附图说明
图1为本发明Cur-FFEYp结构式以及治疗炎症性肠病的示意图。
图2为本发明Cur-FFEYp的氢谱(1H NMR)分析结果图。
图3为本发明Cur-FFEYp的碳谱(13C NMR)分析结果图。
图4为本发明Cur-FFEYp的质谱(MS)分析结果图。
图5为本发明使用紫外光谱法测定的临界胶束浓度结果图。
图6为本发明使用流变仪检测Cur-FFEY自组装后所形成的水凝胶随频率变化储能模量(G’)和耗能模量(G”)和曲线。
图7为本发明使用反相液相色谱(HPLC)实时监测ALP与Esterase酶切Cur-FFEYp的动态过程。
图8为本发明Cur-FFEYp经ALP酶切后自组装及Esterase酶切后解组装的冷冻电镜图。
图9为本发明Cur-FFEYp与Cur在不同pH及H2O2存在条件下的稳定性比较。
图10为本发明Cur-FFEYp的酶促动力学测定。
图11为本发明Cur-FFEYp用于治疗IBD小鼠模型的实验过程图。
图12为使用本发明Cur-FFEYp治疗IBD小鼠模型的体重变化结果图。
图13为本发明Cur-FFEYp治疗IBD小鼠模型的结肠长度变化结果图。
具体实施方式
以下将结合图例对实验方案进行详细阐述，本发明所提供的思路仅为说明本发明的设计方案而非限制，故尽管在此我们对实验技术进行了详细说明，但本领域的普通技术人员应当理解，若对本方案进行优化或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的权利要求范围内。
实施例1：水凝胶前体Cur-FFEYp的制备
以吡啶为溶剂，向溶剂中加入Cur(368mg,1mmol)和戊二酸酐(GA)(125mg,1.1mmol)，在室温下反应6小时，得到姜黄素衍生物Cur-GA-COOH；将Cur-GA-COOH(184.8mg,0.1mmol)充分溶解在DMF(1mL)中，然后加入1-乙基-(3-(二甲氨基)丙基)-碳二亚胺盐酸盐(EDC·HCl,98.15mg,0.15mmol)和N-羟基琥珀酰亚胺(NHS,52.9mg,0.12mmol)，进一步搅拌过夜以进行活化酯反应；随后加入固相合成的多肽分子FFEYp(88mg,0.12mmol)和N,N-二异丙基乙胺(DIPEA,60μL)，在室温下搅拌过夜，得到Cur-FFE(OtBu)Yp，随后以50％的三氟乙酸脱除谷氨酸的羧氨基保护基OtBu得到目标化合物Cur-FFEYp。每一步反应用HPLC分离纯化，确认纯度，用1H NMR，13C NMR和MS确认分子结构。
Cur-FFEYp的1H NMR分析结果：1H NMR(400MHz,d6-DMSO)δ(ppm)：8.23(d,J＝6.9Hz,1H),8.18–7.99(m,3H),7.68–7.54(m,1H),7.52–7.42(m,1H),7.29(d,J＝38.3Hz,4H),7.20(d,J＝13.6Hz,5H),7.17–6.95(m,8H),6.89–6.75(m,2H),6.64(dd,J＝23.0,8.5Hz,1H),4.51(s,2H),4.40(d,J＝5.5Hz,1H),4.32(d,J＝6.3Hz,1H),3.85–3.77(m,6H),3.75–3.71(m,3H),2.98(dd,J＝28.1,13.2Hz,4H),2.36(s,2H),2.18(s,2H),2.12(s,2H),1.85(s,2H),1.73(d,J＝24.1Hz,4H),1.35–1.12(m,2H).
Cur-FFEYp的13C NMR分析结果：13C NMR(101MHz,d6-DMSO)δ(ppm)：176.42,174.52(2C),173.16(2C),171.88,171.72,171.53,171.26,151.61,150.70,147.86,145.17,138.46(2C),138.17(2C),134.23,133.24,131.94,130.56(4C),129.65(4C),128.50(4C),126.72,123.73,122.92,121.62,120.75,120.33(3C),115.76,112.92,101.55,56.11(3C),54.11(2C),52.01,42.13,37.93,37.66,36.26,34.39,32.89,30.41,28.23,20.99.
Cur-FFEYp的MS分析结果：理论分子量为[(M+H)+]:1149.37；实际分子量为[(M+H)+]:1149.25。
实施例2：水凝胶前体Cur-FFEYp的性质表征
在磷酸盐缓冲溶液中，将目标化合物Cur-FFEYp与商业上可购买到的ALP及Esterase分别及组合孵育，用HPLC实时监测去磷酸根以及Cur药物释放的过程，找到稳定形成纳米纤维以及纳米纤维解组装的时间点。在这个时间点下，用透射电子显微镜(TEM)表征酶切后自组装形成纳米结构及解组装后释放Cur的形貌和尺寸。通过TEM图可清晰看到Cur-FFEYp经ALP酶切后通过π-π堆积自组装形成平均宽度为4.287±0.495nm的纳米纤维。
由于Cur稳定性较差，所以我们利用HPLC初步观察了探针Cur-FFEYp及Cur在不同pH下的稳定性。由于Cur在体内的不稳定性可能源于酶的氧化，我们比较了Cur和Cur-FFEYp在中性pH(7.4)下在过氧化氢(H2O2)存在不同时间的稳定性。由图10可明显看到，通过对Cur进行修饰，极大提高了Cur耐酸耐碱以及耐氧化的能力。
配置不同浓度Cur-FFEYp，经ALP酶切24h后使用紫外光谱法测定纳米胶束的临界胶束浓度；
将2mg Cur-FFEYp溶于200μL磷酸盐缓冲液(PB,0.5M,pH＝7.4)，得到1.0wt％的Cur-FFEYp溶液。然后在溶液中加入40U ALP，并在37℃条件下孵育24h,发现上述孵育混合物已转换为水凝胶Cur-FFEY。为了评估凝胶的粘弹性，我们首先使用动态应变扫描来确定水凝胶的动态频率扫描的适当条件。如图7所示，水凝胶Cur-FFEY的G'和G”的值表现出从0.1％到10.0％应变的弱相关性(G'主导G”)，表明样品是水凝胶。将应变幅度设置为1.0％(在应变幅度的线性响应范围内)后，我们使用动态频率扫描来研究水凝胶。在整个范围内(0.1-10Hz)，G'的值主导G”值，表明水凝胶Cur-FFEY对外力具有耐受外部剪切力。
为了验证我们的策略是水凝胶先自组装富集在肠炎部位，随后解组装原位释放Cur，我们分别获得了Cur-FFEYp的ALP催化去磷酸化反应和Esterase催化水解反应的动力学参数。我们首先获得了Cur-FFEYp和Cur在420nm处的HPLC峰面积校准曲线。将不同浓度的过量Cur-FFEYp与ALP或Esterase在37℃下在PB缓冲液(PH＝7.4)中孵育相应时间。然后根据Cur-FFEYp的初始浓度绘制初始速度并拟合Michaelis-Menten模型，使用Lineweaver-Burk分析，我们获得了ALP去磷酸化的米氏常数kcat/kM＝1.45×104M-1s-1，是脂酶催化水解的米氏常数kcat/kM＝2.23×102M-1s-1的65倍。这些结果表明，在等量的酶浓度水平下，Cur-FFEYp先进行被ALP去磷酸化自组装，然后被酯酶水解释放Cur。
实施例3：Cur-FFEYp对IBD小鼠模型的治疗作用
用含有2.5％葡聚糖硫酸钠(Dextran SulphateSodium,DSS)的水连续喂养C57BL/6小鼠(0-5天)，之后换成正常的水，建立溃疡性结肠炎模型。从1天开始，按5mg/kgCur的剂量连续腹腔注射Cur-FFEYp或Cur或PBS(1-7天)，每日监测小鼠体重，计算小鼠体重下降率。8天后，解剖取其结肠测定结肠长度。
体重结果表明，实验组(未经治疗组)的小鼠体重出现大幅下降(26％)。相比于未经治疗组，Cur治疗组小鼠体重有所改善(21％)，但还是有比较明显的下降。Cur-FFEYp治疗组的小鼠体重只有小幅度下降(6％)，与Cur治疗组有显著性差异(P＜0.001)。说明Cur-FFEYp可以有效缓解IBD导致的体重下降。
解剖出的结肠长度数据亦表明，Cur-FFEYp治疗组的结肠长度与Cur治疗组结肠长度有显著性差异。说明Cur-FFEYp可以有效缓解IBD导致的结肠溃烂。
以上数据表明，水凝胶前体Cur-FFEYp可以有效地缓解IBD，这种酶控制的自组装富集与解组装缓慢释放的过程极大提高了姜黄素的治疗效果。
Claims (6)
1.一种酶控自组装与解组装的负载姜黄素药物的水凝胶前体，其特征在于：
所述负载姜黄素药物的水凝胶前体简记为Cur-FFEYp，其结构式如下所示：
2.一种权利要求1所述的负载姜黄素药物的水凝胶前体的制备方法，其特征在于：
以姜黄素衍生物A和多肽分子B为原料，通过活化酯反应得到Cur-FFE(OtBu)Yp，随后以50％的三氟乙酸脱除谷氨酸的羧氨基保护基OtBu得到目标化合物Cur-FFEYp；
反应路线如下所示：
3.根据权利要求2所述的制备方法，其特征在于：
所述姜黄素衍生物是由姜黄素与戊二酸酐反应获得，以酯键形式连接，延伸出羧基，用于酰胺键的形成；反应路线如下所示：
4.根据权利要求2所述的制备方法，其特征在于：
所述多肽分子是由Fmoc-Phe-OH、Fmoc-Phe-OH、Fmoc-Glu-OH、Fmoc-Tyr(H2PO3)-OH通过固相合成得到。
5.一种权利要求1所述的负载姜黄素药物的水凝胶前体的应用，其特征在于：
所述负载姜黄素药物的水凝胶前体作为原位富集以及缓慢释放Cur的药物递送系统，实现酶控自组装与解组装。
6.根据权利要求5所述的应用，其特征在于：
所述负载姜黄素药物的水凝胶前体被肠炎细胞中过度表达的碱性磷酸酶ALP特异性识别剪切，利用磷酸基团与碱性磷酸酶的相互作用，通过π-π堆积自组装形成平均宽度为4.287±0.495nm的水凝胶纳米纤维，富集在肠炎细胞中，增强滞留时间；随后被肠炎细胞中富含的酯酶Esterase缓慢水解，原位释放出姜黄素。
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