CN108948413B - 波聚合制备胸腺五肽分子印迹水凝胶的方法 - Google Patents
波聚合制备胸腺五肽分子印迹水凝胶的方法 Download PDFInfo
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Abstract
本发明涉及一种波聚合制备胸腺五肽分子印迹水凝胶的方法。它的原料的质量组成:胸腺五肽4.33%‑11.94%;N‑异丙基丙烯酰胺10.00%‑36.44%;丙烯酸5.88%‑21.86%;N,N’‑亚甲基双丙烯酰胺0.36%‑0.74%;氯化胆碱9.92%‑33.58%;乙二醇30.12%‑53.68%。本发明采用波聚合制备方法,以生物活性分子胸腺五肽为模板,以绿色溶剂DES体系为致孔剂,制备了分子印迹水凝胶缓释材料,其作为药物载体物理和化学性质稳定,印迹效果明显;载药量大,对模板分子胸腺五肽有明显的缓控释效果;相比热聚合印迹水凝胶,可延长药物体内代谢时间,提高药物生物利用度。
Description
技术领域
本发明涉及一种波聚合制备胸腺五肽分子印迹水凝胶的方法。
背景技术
胸腺五肽(Arg-Lys-Asp-Va1-Tyr,TP-5)是一种人工合成的五肽,对应于胸腺生成素Ⅱ的活性位点。自发现以来,TP-5一直被临床用于治疗免疫缺陷病如类风湿性关节炎,癌症,乙型肝炎病毒感染和获得性免疫缺陷综合征(AIDS)。然而,由于酶促降解的广泛性和膜通透性差,TP-5在人血浆中的半衰期很短,为30s,因此其临床应用受到很大限制。目前临床上使用的多为TP-5肌肉或静脉注射,对TP-5口服给药的研究却很少见,原因是多肽类药物在胃肠道中稳定很差、吸收率低,所以制备一种可以保护胸腺五肽不被胃肠道破坏的载药装置延长治疗效果很有必要。
水凝胶是介于固体和液体之间的能够溶胀于水但在水中并不能溶解的亲水聚合物凝胶,它由聚合物链组成的三维网络或互穿网络结构,并在水中溶胀至热力学平衡。鉴于水凝胶的生物相容性和无毒性,它们作为药物递送中的生物材料是极好的选择。亲水性药物可以溶解在水凝胶内部含水的三维网络状结构中,得到了保护,水凝胶可以帮助药物抵抗生物体内相对比较恶劣的环境,从而使药物可以在体内发挥更好的疗效。但是水凝胶作为药物载体仍存在一些缺陷,如水凝胶机械强度差,在体内药物释放过程不可控等,导致其实际应用受限。
分子印迹技术(Molecularly Imprinting Technique,MIT)是通过聚合过程在合成材料中对模板分子产生人工识别位点的技术,用此技术合成的分子印迹聚合物(Molecular imprinted polymers,MIPs)对模板分子具有较强的选择性,能够识别生物和化学分子。在印迹体系中,许多互补或空间定位的功能单体与模板药物相互作用,印迹孔穴内较强的相互作用减缓或延缓了药物从聚合物网络的释放,因此MIPs由于高选择性和亲和性作为药物载体控制给药受到了广泛关注。
MIPs具有特异性识别能力,水凝胶具有很好的溶胀性能,将这两种技术结合起来制备的分子印迹智能水凝胶可以兼顾两者优势,保护胸腺五肽不被胃肠道破坏,有效延长胸腺五肽的作用时间。传统的MIP为了提高对模板分子的高选择性,往往会增大交联剂的使用量来提高聚合物的机械性能,降低其在溶液中的溶胀,来形成稳定的三维立体结构。然而吸水溶胀是水凝胶的特性,分子印迹水凝胶必须采用低交联来保证溶胀性能。与传统的具有刚性基质的传统MIP相比,低交联度的分子印迹水凝胶韧性和机械强度差,特别是处于水溶胀状态时过于柔软,印迹孔穴不稳定,所以迫切需要改进分子印迹水凝胶的制备工艺。
波聚合(Frontal polymerization,FP)是一种单体在局部区域反应,然后靠自身反应放热产生聚合波沿着反应区域轴向传播,将未反应区域的单体转化成聚合物的反应模式,也称为前端聚合。与常规的聚合方法相比,其反应时间大大缩短,避免了出现聚合溶液发生两相分离的情况,因此该法制得的水凝胶结构更为均一。此外,在使用两种单体共聚时,波聚合的高温会减少两种单体竞聚率的差异,得到的共聚物分子链结构均匀,接近理想共聚。因此,用波聚合方法制备分子印迹水凝胶工艺简单、反应迅速、转化率高,并且制备的水凝胶的分子链结构均匀,有望形成更加稳定均一的印迹孔穴,进而提高MIP的分子识别能力。
波聚合制备技术相比于常规制备方法具有很大优势,但是由于其反应温度很高,使用水或者其他挥发性溶剂作为溶剂时,聚合波前沿溶剂温度不稳定甚至沸腾,聚合过程会产生间歇性气泡,气泡生长时,压力增大抑制聚合波的蔓延,造成聚合波失稳甚至熄灭,极大地限制了它的应用,以往是使用惰性填料或具有高沸点的溶剂避免这些问题。
低共熔溶剂(DESs)是一种新型绿色溶剂,由氢键供体(羧酸、多元醇、尿素等)和氢键受体(季铵盐)组成的,具有无毒性、高粘度、高度的热稳定性和化学稳定性、高溶解度等优势。DESs用于波聚合反应溶剂可以提高聚合波温度的稳定性,聚合过程中不会产生气泡,避免发生聚合波熄灭的情况,从而维持聚合波快速稳定的传播。综上所述,波聚合制备技术在DESs环境下反应有望制备高性能的MIP水凝胶。
中国专利CN201310241426.1公开了一种胸腺五肽分子印迹磁性微球的制备方法,具体是制备油酸改性的Fe3O4磁性纳米粒子,然后在十二烷基硫酸钠做表面活性剂下,以过硫酸钾为引发剂,二甲基丙烯酸乙二醇酯为交联剂,利用甲基丙烯酸甲酯和甲基丙烯酸制备羧基功能化的Fe3O4磁性纳米粒子。最后以胸腺五肽为模板分子、丙烯酰胺为功能单体、二甲基丙烯酸乙二醇酯为交联剂、羧基功能化的Fe3O4为磁性载体,制备出胸腺五肽分子印迹磁性微球。该制备方法将分子印迹聚合物的专一识别特性和磁性微球易在外界磁场作用下分离的特性合二为一,可应用在分离、富集、检测、药物靶向治疗等。目前尚未发现采用波聚合制备胸腺五肽分子印迹水凝胶的方法。
发明内容
本发明的目的在于提供一种波聚合制备胸腺五肽分子印迹水凝胶的方法。该发明是以人工合成的胸腺五肽为原料,使用波聚合法制备得到胸腺五肽分子印迹水凝胶,解决了胸腺五肽半衰期短的问题,本发明具有良好的生物相容性,可以延长药物在体内作用时间,波聚合制备方法,其反应可在20min内快速完成,在提高聚合速率的基础上又可提高水凝胶的均一性,使用高沸点的绿色溶剂DESs作为波聚合反应溶剂,提高了波聚合反应的稳定性;相比于常规制备方法的分子印迹水凝胶,波聚合制备的分子印迹水凝胶对模板的选择性更好,同时延长了在大鼠体内的代谢时间。本发明广泛用于治疗免疫缺陷病。
本发明提供的胸腺五肽分子印迹水凝胶的原料的质量百分数组成:
胸腺五肽4.33%-11.94%
N-异丙基丙烯酰胺10.00%-36.44%
丙烯酸5.88%-21.86%
N,N’-亚甲基双丙烯酰胺0.36%-0.74%
氯化胆碱9.92%-33.58%
乙二醇30.12%-53.68%
上述的各原料的质量组成之和为100%。
本发明提供的上述一种胸腺五肽分子印迹水凝胶的制备方法,采用波聚合和热聚合两种制备方法,制备方法分别如下:
波聚合制备:
DES体系的制备:按计量将氯化胆碱和乙二醇混合,在80℃油浴锅中磁力搅拌,直到得到澄清、均一的溶液(氯化胆碱和乙二醇组成的DES,在室温下为无色透明液体),室温保存,备用。氯化胆碱和乙二醇的质量比为1:3。
1)按计量分别将胸腺五肽、N-异丙基丙烯酰胺、丙烯酸、N,N’-亚甲基双丙烯酰胺混合,加入致孔剂步骤1)DES体系或DMSO,超声溶解,使之均匀、澄清。加入引发剂过硫酸铵,超声溶解,通入氮气以除去液体中氧气,然后将反应液倒入试管中。
2)上述装有反应液的试管底端浸入到90℃的油浴锅中来引发反应,底部反应液开始反应后立即将试管从油浴锅中取出,然后通过反应液自身反应产生的热量向上传播到整个试管,进而维持剩余反应液的反应,整个反应完成后,将试管冷却至室温。
3)上述得到的分子印迹水凝胶切成小块并使用水-乙酸混合物(90:10,v/v)洗脱三天,每隔12h更换一次洗脱液,接着使用甲醇洗脱一天,直到模板以及未反应的单体不能再通过紫外分光光度计检测出来,然后将水凝胶干燥。
非印迹水凝胶的合成除不加模板分子胸腺五肽外,其余步骤同上。
热聚合制备:除将步骤3)油浴反应改为60℃水浴中反应30min外,其余步骤同上。
本发明提供了一种波聚合制备胸腺五肽分子印迹水凝胶的方法,首次合成了加入低共熔溶剂DES体系的低交联胸腺五肽分子印迹水凝胶药物载体材料,波聚合制备过程简单,反应时间短,节能降耗。利用场发射扫描电镜可以看出分子印迹水凝胶材料表面光滑,具有明显的三维网络大孔结构。根据分子印迹水凝胶对胸腺五肽的平衡吸附试验表明,相比于热聚合制备的分子印迹水凝胶,其对胸腺五肽的选择性和印迹效果更好。通过MTT实验测得细胞在水凝胶浸提液中培养的相对增值率均在75%以上,证明其具有很好的生物相容性。由大鼠体内药代动力学研究表明,分子印迹水凝胶作为胸腺五肽载体,与商品药相比大大延长了药物作用时间,与热聚合分子印迹水凝胶材料相比,相对生物利用度有了很大提高。由一系列实验结果表明本发明物理和化学性质稳定,作为一种特殊的药物载体材料适用于缓控释缓释材料。
总之,本发明采用波聚合方法制备了胸腺五肽分子印迹水凝胶缓释材料,作为药物载体物理和化学性质稳定,印迹效果明显,载药量大,细胞毒性小,对模板分子胸腺五肽在大鼠体内有明显的缓控释效果;相比热聚合印迹水凝胶,其印迹效果更好,并且提高了药物生物利用度。本发明广泛用于治疗免疫缺陷病如类风湿性关节炎,癌症,乙型肝炎病毒感染和获得性免疫缺陷综合征(AIDS)。
附图说明
图1为本发明制备的水凝胶缓释材料的场发射扫描电镜图。
图2为本发明制备的水凝胶缓释材料对胸腺五肽的平衡吸附图。
图3为细胞在本发明制备的水凝胶缓释材料浸提液上培养不同时间后的细胞相对增值率图。
图4为本发明制备的水凝胶缓释材料作为药物载体时体内血药浓度随时间变化的药时曲线图。
具体实施方式
下面结合具体实施例,进一步详细阐述本发明。实施例中未注明具体条件的实验方法,通常按照常规条件以及手册中所述的条件,或按照制造厂商所建议的条件;所用的通用设备、材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
为了更明确的考察胸腺五肽分子印迹水凝胶的表面形态结构,我们分别使用波聚合制备分子印迹水凝胶(FP-MIP)和非印迹水凝胶(FP-NIP),热聚合制备分子印迹水凝胶(BP-MIP)和非印迹水凝胶(BP-NIP)。并对水凝胶材料进行了场发射扫描电镜分析表征水凝胶的结构特征。具体操作步骤如下:
a.波聚合制备胸腺五肽分子印迹水凝胶药物载体材料:
1)DES体系的制备:按计量将氯化胆碱(4g)和乙二醇(10.8mL)混合,在80℃油浴锅中磁力搅拌,直到得到澄清、均一的溶液,室温保存备用。
2)按计量分别将胸腺五肽(326mg)、N-异丙基丙烯酰胺(670mg)、丙烯酸(0.4mL)、N,N’-亚甲基双丙烯酰胺(25mg)混合,加入致孔剂步骤1)DES体系(2.5mL)或DMSO(2.5mL),超声溶解(超声条件:40KHz,20min),使之均匀、澄清。加入引发剂过硫酸铵(10mg),超声溶解5min,通入氮气以除去液体中氧气,然后将反应液倒入100mm长的试管(直径10mm)中。
3)上述装有反应液的试管底端(1cm)浸入到90℃的油浴锅中来引发反应,底部反应液开始反应后立即将试管从油浴锅中取出,然后通过反应液自身反应产生的热量向上传播到整个试管,进而维持剩余反应液的反应,整个反应完成后,将试管冷却至室温。
4)上述得到的分子印迹水凝胶切成小块并使用水-乙酸混合物(90:10,v/v)洗脱三天,每隔12h更换一次洗脱液,接着使用甲醇洗脱一天,直到模板以及未反应的单体不能再通过紫外分光光度计检测出来,然后将水凝胶干燥。
b.波聚合制备非印迹水凝胶药物载体材料:
波聚合非印迹水凝胶的合成除不加模板分子胸腺五肽外,其余步骤同上述方法(实施例1a)。
c.热聚合制备胸腺五肽分子印迹水凝胶药物载体材料:
热聚合分子印迹水凝胶的合成除将步骤3)90℃油浴反应改为60℃水浴中反应30min外,其余步骤同上述方法(实施例1,a)。
d.热聚合制备非印迹水凝胶药物载体材料:
热聚合非印迹水凝胶的合成除不加模板分子胸腺五肽外,其余步骤同上(实施例1c)。
e.场发射扫描电镜
测试前将干燥的水凝胶在蒸馏水中浸泡三天达到溶胀平衡,然后用冷冻干燥机干燥,将冻干后的水凝胶切成薄片,并在表面喷上一层薄薄的金,通过高分辨率场发射扫描电子显微镜(FE-SEM;FEI Nova NanoSEM450,荷兰)观察水凝胶的表面形态。
通过场发射扫描电镜图可以看出FP-MIP、FP-NIP、BP-MIP和BP-NIP表面光滑,具有明显的三维网络大孔结构(见图1)。FP-NIP网络结构中只有大量直径在50μm左右的大孔,然而BP-MIP网络结构中不仅有直径在50μm左右的大孔,还有大量直径在10μm左右的小孔,说明模板的加入对水凝胶孔径影响很大。
实施例2
胸腺五肽平衡吸附实验用来研究分子印迹水凝胶对模板分子胸腺五肽的特异性吸附性能,为了考察印迹水凝胶的对胸腺五肽的特异性识别能力,测定了胸腺五肽分子印迹水凝胶及非印迹水凝胶在0.5-5.0mmol/L范围内的吸附等温线。具体操作步骤如下:
a. 同上述方法(实施例1)合成波聚合分子印迹水凝胶(FP-MIP)、波聚合非印迹水凝胶(FP-NIP)、热聚合分子印迹水凝胶(BP-MIP)、热聚合非印迹水凝胶(BP-NIP)。
b. 分别称取干燥的水凝胶材料10.0mg放入5ml离心管之中。然后配制0.5-5.0mmol/L TP-5的水溶液,将上述离心管中分别加入不同浓度的TP-5溶液3.0mL,于37℃水浴锅中浸泡24h,然后将离心管放入高速离心机中以10000r/min的转速离心5min,取上清液600μL用蒸馏水稀释至10mL,使用紫外分光光度计在231nm波长处检测胸腺五肽的平衡浓度。
通过从胸腺五肽初始浓度减去平衡时上清液中的胸腺五肽浓度计算水凝胶吸附胸腺五肽的吸附量(Q e ,mmol/L),计算公式为:
其中C o 、C e (mmol/L)分别为TP-5溶液的初始浓度和平衡浓度,V(L)是溶液的体积,M(g)是水凝胶的质量。实验结果平行测定3次,取平均值。
以Q e 对C e 作图,得到水凝胶对加替沙星的吸附等温线。
结果表明,随着吸附母液C o 浓度的增大,水凝胶材料对胸腺五肽的吸附量随之增大。FP-MIP对TP-5的吸附量大于FP-NIP,但是BP-MIP与BP-NIP的吸附量却无明显区别(见图2)。说明制备方法会影响MIP对模板的吸附能力和印迹效果,波聚合制备得到的分子印迹水凝胶对模板的选择性更高,印迹效果更好。
实施例3
MTT实验研究水凝胶材料的细胞毒性。为了考察水凝胶材料的细胞相容性,测定细胞在水凝胶材料浸提液上培养不同时间后的细胞相对增值率。具体操作步骤如下:
a. 同上述方法(实施例1)合成波聚合分子印迹水凝胶(FP-MIP)、波聚合非印迹水凝胶(FP-NIP)、热聚合分子印迹水凝胶(BP-MIP)、热聚合非印迹水凝胶(BP-NIP)。
b. 取对数期生长的小鼠成纤维细胞,经胰酶消化后,用含10%胎牛血清(FBS)的DMEM培养基稀释成1×104浓度,加入96孔板,每孔加入细胞悬浮液100μL,然后将细胞置于细胞培养箱(37℃,5%CO2)中培24h,弃去原来培养液,在96孔板内加入水凝胶浸提液,每个样品需做三组平行孔。不加水凝胶浸提液,用10%FBS的DMEM培养基培养细胞的孔板作为阴性对照组,只含10%FBS的DMEM培养基不含细胞的孔板作为空白对照组。
c. 分别在细胞培养24h、48h、72h后,在倒置显微镜下观察细胞形态。吸出孔板内原有培养基,加入150μL培养基和20μLMTT,然后放回细胞培养中培养5h,吸出孔板内液体,每孔加入200μL DMSO显色,将孔板置于37℃恒温振荡器上震荡5min,最后使用酶标仪在波长490nm处检测吸光度OD值。细胞相对增值率(Relative growth ratio,RGR)计算公式如下:
RGB%=(OD test -OD 0 )/(OD control -OD 0 )*100%
其中,OD test 为加入水凝胶浸提液的实验组的吸光度值,OD control 为阴性对照组的吸光度值,OD 0 为空白对照组的吸光度值。
图3为小鼠成纤维细胞在水凝胶浸提液中培养1天、2天和3天后的细胞相对增值率。结果表明,在水凝胶浸提液中细胞培养不同时间后,与只加培养液的对照组相比细胞数量相差不大,细胞相对增值率均在75%以上,并且随着培养时间的延长,细胞的相对增值率不断增大。综上所述,我们制备的分子印迹水凝胶材料无细胞毒性,可以用于生物体内研究。
实施例4
药物体内释放实验研究载有胸腺五肽的水凝胶药物载体在大鼠胃内的缓释作用。为了考察药物在大鼠体内缓释效果,测定了载药后的水凝胶材料和对照组胸腺五肽原料药一定时间内在大鼠体内的血药浓度。具体操作步骤如下:
a. 同上述方法(实施例1)合成波聚合分子印迹水凝胶(FP-MIP)、波聚合非印迹水凝胶(FP-NIP)、热聚合分子印迹水凝胶(BP-MIP)、热聚合非印迹水凝胶(BP-NIP)。
b. 配制浓度为1500μg/mL TP-5的水溶液,秤取40mg水凝胶浸泡于10ml上述浓度的TP-5溶液中,25℃下浸泡三天,离心取上清液,用紫外分光光度计231nm波长处测得上清液浓度计算得到载药量,最后将载药后的水凝胶在烘箱中40℃下干燥72h。
c. 体内药代动力学实验是在雄性Wistar大鼠(体重180-200g,8-10周)体内进行的。将载药后的水凝胶(大鼠灌胃量均为1mg kg-1TP-5)分散在2.0mL生理盐水溶液中。大鼠灌胃前禁食12h,然后将上述配制好的样品以灌胃的方式喂食大鼠。以0.5,1,1.5,2,3,4,5,6和8h给药后的指定时间间隔从麻醉大鼠的眼睛内眦取血(约100μL),取血后立即将血液样品置于加有肝素化的试管中,以4500rpm离心5min。
d. 取50μL上清液,加入150μL乙腈混合30s,在13000rpm下离心5min,取上清液有机层在温和的氮气流下干燥,待样品干燥后加入HPLC流动相,在超声仪超声20min使样品充分溶解在流动相中,用滤膜过滤,进样量为20μL,用HPLC测定药物释放量。
以血药浓度对时间作图,绘制药物的药时曲线(见图4)。可以看出,商品药TP-5达峰时间T max 为0.16h,在大鼠体内代谢时间为2h。FP-MIP达峰时间T max 为0.16h,在大鼠体内代谢时间为2h。结果说明分子印迹水凝胶作为胸腺五肽载体可以延长其在大鼠体内的作用时间,具有一定的缓控释效果。
本发明提供了一种波聚合制备胸腺五肽分子印迹水凝胶材料的方法。通过场发射扫描电镜可以看出水凝胶材料表面光滑,具有明显的三维网络孔结构;波聚合制备得到的印迹水凝胶可以在水中实现对胸腺五肽的特异性识别作用,与非印迹水凝胶和常规制备方法得到的水凝胶相比,对模板的吸附能力更高,印迹效果更好;细胞毒性试验测得水凝胶材料具有很好的生物相容性;以水凝胶为TP-5载体口服给药大鼠,与商品药相比大大延长了药物作用时间。这些特性能使得波聚合制备的印迹水凝胶可作为一种新型缓释材料。
Claims (3)
1.一种胸腺五肽分子印迹水凝胶,其特征在于它的原料的质量组成:
胸腺五肽4.33%-11.94%
N-异丙基丙烯酰胺10.00%-36.44%
丙烯酸5.88%-21.86%
N,N’-亚甲基双丙烯酰胺0.36%-0.74%
氯化胆碱9.92%-33.58%
乙二醇30.12%-53.68%
上述的各原料的质量组成之和为100%。
2.权利要求1所述的胸腺五肽分子印迹水凝胶的制备方法,其特征在于经过下列步骤:
1)DES体系的制备:按计量将氯化胆碱和乙二醇混合,在80℃油浴锅中磁力搅拌,直到得到澄清、均一的溶液,室温保存备用;
2)按计量分别将胸腺五肽、N-异丙基丙烯酰胺、丙烯酸、N,N’-亚甲基双丙烯酰胺混合,加入致孔剂步骤1)DES体系,超声溶解,使之均匀、澄清;加入引发剂过硫酸铵,超声溶解,通入氮气以除去液体中氧气,然后将反应液倒入试管中;
3)上述装有反应液的试管底端浸入到90℃的油浴锅中来引发反应,底部反应液开始反应后立即将试管从油浴锅中取出,然后通过反应液自身反应产生的热量向上传播到整个试管,进而维持剩余反应液的反应,整个反应完成后,将试管冷却至室温;
4)上述得到的分子印迹水凝胶切成小块并使用水-乙酸混合物,体积比为90:10,v/v,洗脱三天,每隔12h更换一次洗脱液,接着使用甲醇洗脱一天,直到模板以及未反应的单体不能再通过紫外分光光度计检测出来,然后将水凝胶干燥。
3.按照权利要求2所述的制备方法,其特征在于氯化胆碱和乙二醇的质量比为1:3。
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