本文主要研究内容
作者宋义娜（2019）在《CSK和DEX修饰的粘液穿透和肠靶向性艾塞那肽纳米粒的制备与评价》一文中研究指出：蛋白和多肽类药物是药用生物活性大分子,与体内正常生理物质十分接近,因此其进行口服用药具有独特的生物活性和较少的副作用;尤其对于一些慢性疾病的治疗,但是其口服给药递送系统生物利用度低的问题亟待解决。因此,能够提高多肽类药物口服生物利用度的主动靶向纳米递药载体的研究引起了科研人员的广泛兴趣,成为现代药剂学研究的热点。艾塞那肽是一种II型糖尿病治疗药物,可以帮助患者同时控制血糖和体重。艾塞那肽与人胰高血糖素样肽（GLP）-1有53%的同源性,当血糖水平高于正常时,可降低血糖浓度。艾塞那肽通过促进胰腺β细胞增殖,抑制细胞凋亡,促进胰岛素分泌,并通过减少胰高血糖素的分泌有效控制血糖水平。目前还没有任何艾塞那肽口服制剂上市。本课题中,我们设计合成具有粘液穿透和肠靶向多功能化纳米载体材料CSK-葡聚糖-聚（乳酸-乙醇酸）（CSK-DEX-PLGA）,并以此为材料模型构建艾塞那肽-Zn2+复合物纳米载药口服递送系统,旨在解决艾塞那肽口服给药生物利用度低的问题。利用DEX的正电性中和PLGA的负电性,使纳米粒整体呈近似电中性,利于黏液层的穿透;CSK进一步对DEX-PLGA进行功能化修饰,使到达肠组织的CSK-DEX-PLGA-NPs（CDPs）易借助于CSK与杯状细胞的亲和性,提高跨膜转运效率。本研究的主要内容有艾塞那肽CDPs的制备及表征,体内外靶向性相关评价以及药代药效学相关评价,为建立高效、安全的口服多肽给药递送系统提供一定的实验和理论依据。主要研究内容与结论如下:1.嵌段聚合物CSK-DEX-PLGA的合成以CSK、DEX和PLGA为原料,利用葡聚糖末端葡萄糖分子异构化（开环）产生的醛基,与己二胺反应生成席夫碱（亚胺）并转化为稳定结构胺;然后,PLGA的-COOH端与琥珀酰亚胺发生反应,得到活化PLGA（PLGA-NHS）;最后,PLGA的-COOH与胺化DEX的-NH2反应生成酰胺键。CSK的-COOH端用己二胺活化,然后与DEX-PLGA共聚物DEX端的-OH通过酰胺键连接,制备得到CSK-DEX-PLGA。采用核磁氢谱和GPC对合成材料进行表征,并用HPLC检测其反应进程。分析结果表明所制备的聚合物为CSK-DEX-PLGA。2.纳米粒的制备与体外表征采用S/O/W复乳法制备纳米粒,以粒径、PI值、包封率、载药量为评价指标,并通过单因素考察确定最优处方及工艺为:初乳超声功率300 w,初乳超声时间30 s,外水相体积4 mL,外水相PVA浓度为3%（w/v）,聚合物浓度为20 mg/mL,DMSO与二氯甲烷的比例为1:3,理论加药量为1 mg。最优处方CDPs的粒径为136.3±8.9nm,zeta电位为0.15±0.019 mV,包封率为71.64±3.21%,载药量为4.62±1.37%。于透射电镜下观察,纳米粒呈球形,均匀分散,无黏连。分别采用含消化酶与不含消化酶的模拟胃液（SGF,pH 1.2）和模拟肠液（SIF,pH 7.4）对纳米粒的稳定性进行考察,结果表明消化酶是影响药物稳定性的重要因素,且添加Zn2+可以提高艾塞那肽的稳定性。采用不含消化酶的模拟胃肠液进行体外释放行为考察,结果显示将艾塞那肽包载于纳米粒中可以提高其稳定性,纳米粒组没有出现突释现象且具有明显的缓释效果。3.体外细胞及粘液相关评价以Caco-2细胞为模型细胞,进行体外细胞水平相关评价。采用MTT法对纳米粒的毒性进行考察,得出不同浓度DPs和CDPs对Caco-2细胞增殖影响结果无差异统计学意义,所以没有产生细胞毒性;采用流式细胞仪和激光共聚焦显微镜对香豆素-6标记纳米粒（100 ng/mL）的体外细胞摄取情况进行考察,研究结果显示CDPs的被摄取能力强于DPs,但均是通过主动运输进入细胞;摄取抑制实验结果分析表明,香豆素-6标记CDPs的被摄取涉及到网格蛋白、小窝蛋白和脂筏介导的能量依赖途径。跨膜转运实验结果表明,FITC-exenatide-CDPs的转运量明显高于FITC-exenatide-DPs;游离CSK的存在会降低FITC-exenatide-CDPs的转运量,证明CSK和CDPs与靶向结合位点的识别存在竞争关系,CDPs的转运量增加主要是由CSK肽和小肠杯状细胞的特异性亲和力所介导。采用鼠粘液平铺于Transwell小室构建粘液模型,通过细胞微孔板成像仪,定性定量的评价纳米粒的粘液穿透性。结果表明,CDPs和DPs具有相似的粘液聚集率。而CDPs因表面附有呈负电性CSK的优势,与负电性粘液的作用力稍弱。所以,CDPs粘液穿透率是DPs的2.1倍,表现出更为理想的粘液穿透性,并呈现差异显著性统计学意义。4.体内分布相关评价通过小动物活体成像系统,定性评价Dir标记CDPs在体内的靶向吸收和分布效果。活体小鼠的观察结果和离体器官（心、肝、脾、肺、肾、胃肠）的荧光强度变化表明,CDPs组的吸收和分布更快,对小肠部位具有较高的靶向性。肠上皮细胞渗透性实验和体外结扎肠环实验结果显示,十二指肠、空肠和回肠处,尼罗红标记CDPs的荧光强度强于DPs,CDPs的单位面积转运值约为DPs的1.5倍。以上结果均表明CDPs通过CSK的介导与小肠杯状细胞特异性结合,并促进其在小肠被吸收和体内循环时间的延长。5.药代动力学性质评价考察了不同剂型艾塞那肽在Sprague Dawley大鼠体内的药物代谢情况。建立体内药物含量的测定方法,采用艾塞那肽酶联免疫试剂盒测定大鼠的血药浓度,并计算药代动力学参数。研究结果显示,与皮下注射组相比,口服exenatide-Zn2+-CDPs的生物利用度为9.2%;与艾塞那肽溶液口服给药组相比,生物利用度提高了4倍,而且比exenatide-Zn2+-DPs的生物利用度高1.61倍。实验结果表明,CSK能促进CDPs与小肠杯状细胞的靶向识别,提高艾塞那肽的口服给药生物利用度。6.体内药效学评价采用db/db小鼠作为二型糖尿病模型,在动物水平考察不同剂型艾塞那肽的药理降血糖效果。实验结果表明,CSK修饰的CDPs在单次给药和多次给药中均表现出较明显和持续的降血糖作用。酶联免疫组化分析研究结果显示,CDPs组的胰岛素染色面积相比于口服艾塞那肽溶液组和DPs组是更高的,表明口服载艾塞那肽的CDPs可以改善胰腺β细胞的功能,并且促进β细胞分泌胰岛素。本研究成功的构建载艾塞那肽CDPs,细胞和动物水平实验均充分证明功能化外源性CDPs具备提高粘液穿透率和肠道靶向性功能,提高了艾塞那肽的口服生物利用度,具有较明显的降血糖效果。为艾塞那肽制备成治疗二型糖尿病的口服制剂提供了理论依据。
Abstract
dan bai he duo tai lei yao wu shi yao yong sheng wu huo xing da fen zi ,yu ti nei zheng chang sheng li wu zhi shi fen jie jin ,yin ci ji jin hang kou fu yong yao ju you du te de sheng wu huo xing he jiao shao de fu zuo yong ;you ji dui yu yi xie man xing ji bing de zhi liao ,dan shi ji kou fu gei yao di song ji tong sheng wu li yong du di de wen ti ji dai jie jue 。yin ci ,neng gou di gao duo tai lei yao wu kou fu sheng wu li yong du de zhu dong ba xiang na mi di yao zai ti de yan jiu yin qi le ke yan ren yuan de an fan xing qu ,cheng wei xian dai yao ji xue yan jiu de re dian 。ai sai na tai shi yi chong IIxing tang niao bing zhi liao yao wu ,ke yi bang zhu huan zhe tong shi kong zhi xie tang he ti chong 。ai sai na tai yu ren yi gao xie tang su yang tai （GLP）-1you 53%de tong yuan xing ,dang xie tang shui ping gao yu zheng chang shi ,ke jiang di xie tang nong du 。ai sai na tai tong guo cu jin yi xian βxi bao zeng shi ,yi zhi xi bao diao wang ,cu jin yi dao su fen bi ,bing tong guo jian shao yi gao xie tang su de fen bi you xiao kong zhi xie tang shui ping 。mu qian hai mei you ren he ai sai na tai kou fu zhi ji shang shi 。ben ke ti zhong ,wo men she ji ge cheng ju you nian ye chuan tou he chang ba xiang duo gong neng hua na mi zai ti cai liao CSK-pu ju tang -ju （ru suan -yi chun suan ）（CSK-DEX-PLGA）,bing yi ci wei cai liao mo xing gou jian ai sai na tai -Zn2+fu ge wu na mi zai yao kou fu di song ji tong ,zhi zai jie jue ai sai na tai kou fu gei yao sheng wu li yong du di de wen ti 。li yong DEXde zheng dian xing zhong he PLGAde fu dian xing ,shi na mi li zheng ti cheng jin shi dian zhong xing ,li yu nian ye ceng de chuan tou ;CSKjin yi bu dui DEX-PLGAjin hang gong neng hua xiu shi ,shi dao da chang zu zhi de CSK-DEX-PLGA-NPs（CDPs）yi jie zhu yu CSKyu bei zhuang xi bao de qin he xing ,di gao kua mo zhuai yun xiao lv 。ben yan jiu de zhu yao nei rong you ai sai na tai CDPsde zhi bei ji biao zheng ,ti nei wai ba xiang xing xiang guan ping jia yi ji yao dai yao xiao xue xiang guan ping jia ,wei jian li gao xiao 、an quan de kou fu duo tai gei yao di song ji tong di gong yi ding de shi yan he li lun yi ju 。zhu yao yan jiu nei rong yu jie lun ru xia :1.qian duan ju ge wu CSK-DEX-PLGAde ge cheng yi CSK、DEXhe PLGAwei yuan liao ,li yong pu ju tang mo duan pu tao tang fen zi yi gou hua （kai huan ）chan sheng de quan ji ,yu ji er an fan ying sheng cheng xi fu jian （ya an ）bing zhuai hua wei wen ding jie gou an ;ran hou ,PLGAde -COOHduan yu hu po xian ya an fa sheng fan ying ,de dao huo hua PLGA（PLGA-NHS）;zui hou ,PLGAde -COOHyu an hua DEXde -NH2fan ying sheng cheng xian an jian 。CSKde -COOHduan yong ji er an huo hua ,ran hou yu DEX-PLGAgong ju wu DEXduan de -OHtong guo xian an jian lian jie ,zhi bei de dao CSK-DEX-PLGA。cai yong he ci qing pu he GPCdui ge cheng cai liao jin hang biao zheng ,bing yong HPLCjian ce ji fan ying jin cheng 。fen xi jie guo biao ming suo zhi bei de ju ge wu wei CSK-DEX-PLGA。2.na mi li de zhi bei yu ti wai biao zheng cai yong S/O/Wfu ru fa zhi bei na mi li ,yi li jing 、PIzhi 、bao feng lv 、zai yao liang wei ping jia zhi biao ,bing tong guo chan yin su kao cha que ding zui you chu fang ji gong yi wei :chu ru chao sheng gong lv 300 w,chu ru chao sheng shi jian 30 s,wai shui xiang ti ji 4 mL,wai shui xiang PVAnong du wei 3%（w/v）,ju ge wu nong du wei 20 mg/mL,DMSOyu er lv jia wan de bi li wei 1:3,li lun jia yao liang wei 1 mg。zui you chu fang CDPsde li jing wei 136.3±8.9nm,zetadian wei wei 0.15±0.019 mV,bao feng lv wei 71.64±3.21%,zai yao liang wei 4.62±1.37%。yu tou she dian jing xia guan cha ,na mi li cheng qiu xing ,jun yun fen san ,mo nian lian 。fen bie cai yong han xiao hua mei yu bu han xiao hua mei de mo ni wei ye （SGF,pH 1.2）he mo ni chang ye （SIF,pH 7.4）dui na mi li de wen ding xing jin hang kao cha ,jie guo biao ming xiao hua mei shi ying xiang yao wu wen ding xing de chong yao yin su ,ju tian jia Zn2+ke yi di gao ai sai na tai de wen ding xing 。cai yong bu han xiao hua mei de mo ni wei chang ye jin hang ti wai shi fang hang wei kao cha ,jie guo xian shi jiang ai sai na tai bao zai yu na mi li zhong ke yi di gao ji wen ding xing ,na mi li zu mei you chu xian tu shi xian xiang ju ju you ming xian de huan shi xiao guo 。3.ti wai xi bao ji nian ye xiang guan ping jia yi Caco-2xi bao wei mo xing xi bao ,jin hang ti wai xi bao shui ping xiang guan ping jia 。cai yong MTTfa dui na mi li de du xing jin hang kao cha ,de chu bu tong nong du DPshe CDPsdui Caco-2xi bao zeng shi ying xiang jie guo mo cha yi tong ji xue yi yi ,suo yi mei you chan sheng xi bao du xing ;cai yong liu shi xi bao yi he ji guang gong ju jiao xian wei jing dui xiang dou su -6biao ji na mi li （100 ng/mL）de ti wai xi bao she qu qing kuang jin hang kao cha ,yan jiu jie guo xian shi CDPsde bei she qu neng li jiang yu DPs,dan jun shi tong guo zhu dong yun shu jin ru xi bao ;she qu yi zhi shi yan jie guo fen xi biao ming ,xiang dou su -6biao ji CDPsde bei she qu she ji dao wang ge dan bai 、xiao wo dan bai he zhi fa jie dao de neng liang yi lai tu jing 。kua mo zhuai yun shi yan jie guo biao ming ,FITC-exenatide-CDPsde zhuai yun liang ming xian gao yu FITC-exenatide-DPs;you li CSKde cun zai hui jiang di FITC-exenatide-CDPsde zhuai yun liang ,zheng ming CSKhe CDPsyu ba xiang jie ge wei dian de shi bie cun zai jing zheng guan ji ,CDPsde zhuai yun liang zeng jia zhu yao shi you CSKtai he xiao chang bei zhuang xi bao de te yi xing qin he li suo jie dao 。cai yong shu nian ye ping pu yu Transwellxiao shi gou jian nian ye mo xing ,tong guo xi bao wei kong ban cheng xiang yi ,ding xing ding liang de ping jia na mi li de nian ye chuan tou xing 。jie guo biao ming ,CDPshe DPsju you xiang shi de nian ye ju ji lv 。er CDPsyin biao mian fu you cheng fu dian xing CSKde you shi ,yu fu dian xing nian ye de zuo yong li shao ruo 。suo yi ,CDPsnian ye chuan tou lv shi DPsde 2.1bei ,biao xian chu geng wei li xiang de nian ye chuan tou xing ,bing cheng xian cha yi xian zhe xing tong ji xue yi yi 。4.ti nei fen bu xiang guan ping jia tong guo xiao dong wu huo ti cheng xiang ji tong ,ding xing ping jia Dirbiao ji CDPszai ti nei de ba xiang xi shou he fen bu xiao guo 。huo ti xiao shu de guan cha jie guo he li ti qi guan （xin 、gan 、pi 、fei 、shen 、wei chang ）de ying guang jiang du bian hua biao ming ,CDPszu de xi shou he fen bu geng kuai ,dui xiao chang bu wei ju you jiao gao de ba xiang xing 。chang shang pi xi bao shen tou xing shi yan he ti wai jie za chang huan shi yan jie guo xian shi ,shi er zhi chang 、kong chang he hui chang chu ,ni luo gong biao ji CDPsde ying guang jiang du jiang yu DPs,CDPsde chan wei mian ji zhuai yun zhi yao wei DPsde 1.5bei 。yi shang jie guo jun biao ming CDPstong guo CSKde jie dao yu xiao chang bei zhuang xi bao te yi xing jie ge ,bing cu jin ji zai xiao chang bei xi shou he ti nei xun huan shi jian de yan chang 。5.yao dai dong li xue xing zhi ping jia kao cha le bu tong ji xing ai sai na tai zai Sprague Dawleyda shu ti nei de yao wu dai xie qing kuang 。jian li ti nei yao wu han liang de ce ding fang fa ,cai yong ai sai na tai mei lian mian yi shi ji he ce ding da shu de xie yao nong du ,bing ji suan yao dai dong li xue can shu 。yan jiu jie guo xian shi ,yu pi xia zhu she zu xiang bi ,kou fu exenatide-Zn2+-CDPsde sheng wu li yong du wei 9.2%;yu ai sai na tai rong ye kou fu gei yao zu xiang bi ,sheng wu li yong du di gao le 4bei ,er ju bi exenatide-Zn2+-DPsde sheng wu li yong du gao 1.61bei 。shi yan jie guo biao ming ,CSKneng cu jin CDPsyu xiao chang bei zhuang xi bao de ba xiang shi bie ,di gao ai sai na tai de kou fu gei yao sheng wu li yong du 。6.ti nei yao xiao xue ping jia cai yong db/dbxiao shu zuo wei er xing tang niao bing mo xing ,zai dong wu shui ping kao cha bu tong ji xing ai sai na tai de yao li jiang xie tang xiao guo 。shi yan jie guo biao ming ,CSKxiu shi de CDPszai chan ci gei yao he duo ci gei yao zhong jun biao xian chu jiao ming xian he chi xu de jiang xie tang zuo yong 。mei lian mian yi zu hua fen xi yan jiu jie guo xian shi ,CDPszu de yi dao su ran se mian ji xiang bi yu kou fu ai sai na tai rong ye zu he DPszu shi geng gao de ,biao ming kou fu zai ai sai na tai de CDPske yi gai shan yi xian βxi bao de gong neng ,bing ju cu jin βxi bao fen bi yi dao su 。ben yan jiu cheng gong de gou jian zai ai sai na tai CDPs,xi bao he dong wu shui ping shi yan jun chong fen zheng ming gong neng hua wai yuan xing CDPsju bei di gao nian ye chuan tou lv he chang dao ba xiang xing gong neng ,di gao le ai sai na tai de kou fu sheng wu li yong du ,ju you jiao ming xian de jiang xie tang xiao guo 。wei ai sai na tai zhi bei cheng zhi liao er xing tang niao bing de kou fu zhi ji di gong le li lun yi ju 。
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论文作者分别是来自烟台大学的宋义娜，发表于刊物烟台大学2019-08-29论文，是一篇关于艾塞那肽论文,肠靶向论文,粘液穿透论文,口服递送论文,烟台大学2019-08-29论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自烟台大学2019-08-29论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：艾塞那肽论文; 肠靶向论文; 粘液穿透论文; 口服递送论文; 烟台大学2019-08-29论文;