adhesion to Caco-2 cells
血清对Hela细胞整合素αvβ3受体内吞循环的影响
血清对Hela细胞整合素αvβ3受体内吞循环的影响张璐琳;刘文琦;蔡荣荣;沈皖珠;崔亚男;管华【摘要】目的探讨血清对Hela细胞整合素αvβ3受体内吞循环的影响.方法培养Hela细胞,激光共聚焦显微镜观测其表面αvβ3受体的表达情况.通过对细胞表面蛋白进行生物素标记,利用酶联免疫吸附法,考察含血清培养基培养条件下和不含血清的培养条件下,细胞表面αvβ3受体的内吞循环情况.结果αvβ3受体在Hela细胞表面具有高表达.与不含血清培养条件相比,含血清培养组αvβ3受体的入胞量在5min和10min时显著减少,差异具有统计学意义(P<0.05).结论含血清培养基培养的条件下,Hela细胞中的αvβ3受体可能更快地循环返回至细胞表面.【期刊名称】《济宁医学院学报》【年(卷),期】2019(042)003【总页数】4页(P162-165)【关键词】整合素αvβ3受体;细胞内吞;血清;人宫颈癌细胞;酶联免疫吸附法【作者】张璐琳;刘文琦;蔡荣荣;沈皖珠;崔亚男;管华【作者单位】济宁医学院药学院,日照276826;济宁医学院药学院,日照276826;济宁医学院药学院,日照276826;济宁医学院药学院,日照276826;济宁医学院药学院,日照276826;济宁医学院药学院,日照276826【正文语种】中文【中图分类】R730.1随着纳米医学以及分子生物学的发展，以肿瘤细胞中异常表达的蛋白或受体为靶标，通过对药物或载体进行特异性修饰，将药物分子选择性地递送到肿瘤细胞甚至其细胞器中，是药学工作者长期以来的研究方向[1-4]。
相应的，对于这类药物的入胞循环分子机制的探索，亦成为近年来的研究热点之一[5-6]。
目前，在给药系统对象的细胞内吞循环机制的研究方面，大多数实验采用的是无血清培养条件，对于血清的添加与否目前尚缺乏共识[7-9]。
有文献报道，细胞表面受体的内吞循环机制与培养基中添加的成分密切相关[10]。
血清作为培养基中模拟细胞体内生长环境所必需的营养成分，其成分复杂。
鼠伤寒沙门氏菌对肠道屏障的破坏作用
鼠伤寒沙门氏菌对肠道屏障的破坏作用吴姚平;武晓丽;徐锋;付金衡【摘要】为了研究鼠伤寒沙门氏菌对肠道屏障的破坏作用,应用小鼠结肠病理组织切片技术、蓝色葡聚糖2000渗透量检测并结合transwell细胞层模型和荧光定量PCR方法探寻鼠伤寒沙门氏菌破坏肠道屏障的可能机制.病理切片结果显示:鼠伤寒沙门氏菌感染可导致肠上皮细胞受损,Babl/c小鼠小肠外蓝色葡聚糖2000渗透量在2、4和6h升高(P＜0.05),昆明小鼠小肠外其渗透量在2和4h时显著升高(P＜0.005);细胞跨膜电阻值降低(P＜0.05);细胞间紧密连接蛋白ZO-1、Claudin-1基因mRNA转录水平下调(P＜0.005).结果表明,鼠伤寒沙门氏菌能够通过下词肠上皮细胞紧密连接蛋白的表达来破坏细胞间的紧密连接结构,从而达到破坏肠道屏障的作用.补充阐明了鼠伤寒沙门氏菌感染对肠道屏障的影响,对揭示研究鼠伤寒沙门氏菌的致病机制提供一定依据.%In order to investigate the break of intestinal barrier after treated with Salmonella typhimurium,the intestinal histopathologic sections and dextran blue 2000 concentration,together with the transwell tight junction structure model and RT-qPCR technology,were used to seek the mechanism.The results of histologic section were showed that the intestinal epithelial cell and intestinal barrier were broken after treated with Salmonella typhimurium,and the relative concentration of dextran blue 2000 was increased in Babl/c(P＜0.05) and Kunming mice(P＜0.005) respectively after treated in 2 h and 4 h.The relative TEER value was decreased in Caco-2 model(P＜0.05) and the mRNA expressing (ZO-1,Claudin-1) was down-regulated(P＜0.001).All those above demonstrate that Salmonella typhimurium can make thegenes of tight junction protein related genes down-regulate expression,and then intestinal epithelial cell tight junction structure and intestinal harrier were broken.This research demonstrated the effect of intestinal tract treated by Salmonella typhimurium,and it is a significant for the deep study on pathogenicity of Salmonella typhimurium.【期刊名称】《南昌大学学报（理科版）》【年(卷),期】2017(041)003【总页数】5页(P265-269)【关键词】鼠伤寒沙门氏菌;Caco-2;紧密连接;肠道屏障【作者】吴姚平;武晓丽;徐锋;付金衡【作者单位】南昌大学生命科学学院,江西南昌 330031;江西中医药大学基础医学部,江西南昌 330004;南昌大学食品科学与技术国家重点实验室,江西南昌 330047;南昌大学食品科学与技术国家重点实验室,江西南昌 330047【正文语种】中文【中图分类】Q939.9肠道，既是营养物质吸收的主要器官，也是与肠道内细菌直接接触和作用的关键部位[1-2]。
乳酸菌的研究进展
乳酸菌的研究进展
广东省佛山市三水区乐平镇动物防疫检疫站 叶 红 曾 敏
［摘 要］ 乳酸菌是应用最早、 最广泛的饲用微生态制剂， 具有多种益生作用。本文从乳酸菌的粘附、 活性物质、 免疫赋活作用、 抗肿 降低胆固醇和降血压作用进行综述， 为开发新型绿色的饲用乳酸菌奠定扎实的基础。 瘤作用、 ［关键词］ 乳酸菌 活性物质 可溶性肽 1. 竞争性排斥病原菌的粘附 乳酸菌能与肠粘膜上皮细胞结合，占据有害菌肠粘膜上皮细胞结 从人体分离的嗜酸乳杆 合位点， 对有害菌起屏障作用。Conway [1]报道， 菌 (L. acidophilus ADH)对人的回肠上皮细胞和猪的回肠上皮细胞都具 有较好的粘附性，且粘附率显著高于从乳制品中分离的保加利亚乳杆 菌(L. bulgaricus)和嗜热链球菌(S. thermophilus)， 对猪的结肠和盲肠上皮 细胞的粘附率也以嗜酸乳杆菌最高， 嗜热链球菌显著低于两株乳杆菌。 Conway 同时还指出实验中的乳酸菌的粘附都是非特异性的 。Gopal 报 道三株饲用微生态制剂菌株， 鼠李糖乳杆菌 (L. rhamnosus DR20)， 嗜酸 乳杆菌(L. acidophilus HN017)和乳酸双歧杆菌(B. lactisDR10)对人肠道上 皮细胞系 HT- 29， Caco- 2 和 HT29- MTX 有极强的粘附力，而且三株乳 H7 对肠细胞的侵袭能力和细胞结合能力 。 酸菌都能降低 E.coli O157： Pascual 发现用浓度为 l05CFU. g- 1 的唾液乳杆菌(L. salivanus)饲喂新生 雏鸡可有效预防肠道中沙门氏菌的定植。 Coconnier [2]报道人源嗜酸乳杆 从而抑制幽门螺杆菌对肠 菌 LB 菌株能吸附于结肠细胞系 HT29- MTX， 细胞的吸附。 2. 产生多种活性物质 2.1 酸性物质 乳酸菌发酵糖类产生大量的酸， 如乳酸 、 乙酸 、 丙酸等， 降低肠道 pH 值， 抑制致病菌的生长。 乳酸菌产生的有机酸使肠内 pH 及 Eh 下降， 对肠道致病菌如痢疾杆菌、 沙门氏菌、 金黄色葡萄球菌、 大肠埃希氏菌、 艰难梭菌等有拮抗作用。低 pH 及 Eh 能促进肠道蠕动， 调整肠道的菌 短链脂肪酸和乳酸能抑制腐败细菌的生长， 从而减少了这些物 群失调。 质产生的毒胺 、 靛基质 、 吲哚 、 氨、 粪臭素 、 硫化氢 、 致癌物质和其它毒 短链脂 物， 还能减少氨的吸收及尿素的生成而对肾功能不全者有疗效。 铁、 镁的吸收和代谢， 短链脂肪酸被吸 肪酸能改善一些金属离子如钙、 收后， 它的代谢产物在各种器官中被利用。 例如丁酸盐可被结肠表皮细 丙酸盐和部分醋酸盐可被肝脏所利用， 部分醋酸盐 胞所利用， 乳酸盐、 可被肌肉和周围组织所利用，所以短链脂肪酸可能在调节内源性代谢 方面起重要作用。 醋酸盐、 丙酸盐和乳酸盐也可能对降低血中极低密度 脂蛋白和胆固醇起一定的作用。 据 Sorrels 报道， 明串珠菌 (Leuconostoc citrovorum)代谢产生的乙酸 和乳酸对沙门氏菌有抑菌效果，其中乙酸起了主要作用。Michinaga O gawa 实验证明乳杆菌对 STEC(Shiga toxin- producing Escherichia coli)起 从仔猪 抑制作用的主要是乳杆菌产生的乳酸和 pH 值的降低。经报道， 小肠分离的 6 株乳杆菌对 ETEC （K88、 K99、 987P ） 有抑制作用， 且发挥 作用的抑菌物质是有机酸， 而不是细菌素或过氧化氢。 乳酸菌产生的过氧化氢， 对致病菌有抑菌作用。嗜酸乳杆菌、 乳酸乳 杆菌(L- lactis)和保加利亚乳杆菌、 戊糖片球菌(Pediococcuspentosaceus)、 乳酸片球菌(P- acidilactici)和植物乳杆菌(L- plantarum)产生过氧化氢， 对 金黄色葡萄球菌、 假单胞杆菌等致病菌起抑制作用。 2.2 可溶性肽 乳酸菌素是具有生物活性的蛋白质，它对其它相近种类的细菌具 有抑制作用， 乳酸菌素一般是带正电荷的小分子蛋白(30～60 个氨基酸 具有高等电点和亲水特性。根据化学结构， 可将乳酸菌素大致分 残基)， 为四类。 第一类是 Lantibiotics， 分子量小于 5kDa， 具有羊毛硫氨酸(Lanthionine)或 β- 甲基羊毛硫氨酸(β- methyllanthionine)基团。如 Nisin。 第二类是肽类细菌素， 分子量小于 10kDa， 具有膜活性的肽， 如 pediocin P A- 1。这类细菌素又可分为三个亚类： ①N- 末端氨基酸序列为 - Thr- Gly- Asn- Gly- Val- Xaa- Cys， 具有抗 Listeria 的活性； ②孔道复合物 由两个不同的肽的寡聚体形成； ③能被硫醇激活， 活性基团要求有还原 性半胱氨酸残基。 第三类是蛋白质细菌素，分子量大于 10kDa，热不稳定性的蛋白 质。 第四类是复合型细菌素， 除蛋白质外， 还含有碳水化合物或类脂基 团。 第二、 三、 四类细菌素不含 lanthionine 基团， 因此被称为非 lanthionine 细菌素(non- lanthionine- containing- bacteriocin)。 乳酸菌素(Lactobacillin)一般对近缘的革兰氏阳性细菌的生长具有 抑制作用， 可以防止某些腐败菌和病原菌的生长 。由于乳酸菌素能在 人体消化道内被消化液中的酶所分解 。 因此， 被认为是一种高效、 安全 的天然代谢产物。 根据研究报道乳酸菌素包括乳酸链球菌素 （Nisin ） ， 双 球菌素 （Diploccins ） ， 乳链菌素 （Lactostrepcins ） ， 乳球菌素 （Lactococcins ） ， 足球菌素 （Pediocins ）及 乳 菌 素 （Lacticin 3147 ） ， 此外， 还 有 Lactocins、 Lactacins、 Entercin ll46、 Helveticin J、 Sakacin A、 Mesentericin Y105 及 Plantaricin 423 等。这些细菌素有抑制沙门氏菌、 大肠埃希氏菌、 志贺氏 菌的作用[3]。 大多数已知的细菌素只对相同革兰氏染色的细菌有抑菌作用， 比 对多种革兰氏阳性菌如葡萄球 如某些乳球菌产生的多肽细菌素(Nisin)， 菌、 芽抱杆菌和乳杆菌有很强的抑制作用， 但对革兰氏阴性菌无效。但 随着研究的深入， 发现许多细菌素作用范围很广。 那淑敏[4]报道， 嗜酸乳 杆菌产生一种广谱抗菌肽 AP311，对致病性的革兰氏阳性菌和革兰氏 阴性菌均有抑制作用。Bernet- Camard 报道， 粘附于人类肠道的嗜酸乳 杆菌 LA1 分泌的一种抑菌物质 LA I- SCS 对革兰氏阳性菌和革兰氏阴 性菌也都有抑制作用，但这种活性物质是否应归入抗生素类物质还在 争议之中。 3. 免疫赋活作用 多种乳酸菌如嗜酸乳杆菌、双歧杆菌和酸奶中常用的保加利亚乳 杆菌 、 嗜热链球菌均在体外实验中被证明有显著的免疫调节作用 。 Donnet 等[5]实验证明含 107 个 /mL 保加利亚乳杆菌的酸奶引起人体血 液中单核细胞的吞噬活性和白细胞呼吸爆发作用的显著增加 。 Tejada- simon 等发现用 109 个 /mL 嗜酸乳杆菌给小鼠灌胃 8h，取其腹膜巨 噬细胞培养， 所产生的 INF- α 明显上升。 Pollman 给无菌仔猪口服嗜酸 乳杆菌后， 可引起白细胞数量和血清总蛋白 (主要是球蛋白 )浓度的提 高。Herich 报道， 饲喂干酪乳杆菌后仔猪白细胞总数和嗜中性细胞数显 著增加。Shiffrin 等报道了来源于母乳喂养的婴儿粪便中的双歧杆菌热 致死菌体对 IgA 产生有增强作用。嗜酸乳杆菌、 干酪乳杆菌、 植物乳杆 经 菌等菌体食用后可活化单核巨噬细胞的活性， 增强全身性免疫应答。 IgE， T 细胞和巨噬细胞的产生， 从 常食用酸奶可促使免疫球蛋白 IgG， 而有免疫增强效果， 主要是酸奶中的乳酸菌起作用。 4. 抗肿瘤作用 乳酸菌抗肿瘤作用是由于肠道菌群的改善，抑制了致癌物质的产 生， 一般认为， 乳酸菌及其代谢产物增强了机体免疫功能， 抑制了癌细 胞的增殖。乳酸菌的抗癌特性可归纳为四类。 4.1 对肿瘤细胞抑制 通过吸附、 抑制、 去除等途径来减少致癌物质和致癌物前体。 4.2 降低肠道内 pH pH 值降低，可使与致癌作用有关的酶活性及胆汁酸水解活性降 低， 因为这些酶能催化致癌前体物质转化为致癌物质。 pH 值降低， 可缩 短肠内容物在肠道的滞留时间，使变异原如 β 一葡萄糖苷酶、 β 一葡 硝基还原酶、 偶氮还原酶和 7α 一脱羟基酶等及早排出体 萄糖苷酸酶、 外。 4.3 破坏致癌物 抑制产生致癌物质的细菌增殖，抑制硝胺产生或抑制涉及硝胺合 成的硝基还原酶， 如干酪乳杆菌能抑制乳酪中硝酸盐转化为硝酸胺， 嗜 酸乳杆菌可显著降低高肉食大鼠粪便硝酸基和氮基还原酶的活性。 4.4 抗突变活性 致癌作用是通过致癌物诱导动物细胞开始的，一些乳酸菌能抑制 通过乳酸菌可以使变异原诱导染色体异常减少 80%。 动物细胞的突变。 如 Hosono 报道， 保加利亚乳杆菌 、 乳酸乳杆菌和嗜热链球菌的发酵乳 对各种变异原性物质诱导的细胞突变有抑制作用。 5. 降低胆固醇 吸收方式， 降解胆固 乳酸菌有抑制胆固醇合成的作用。通过消化、 嗜酸乳杆菌在体外试验中有降 醇和胆酸， 减少动物体内胆固醇的含量。 低胆固醇的作用，动物试验同样证明了嗜酸乳杆菌具有降低猪血清胆 固醇的功能。Taranto 等给实验鼠吃乳杆菌， 其总胆固醇下降 20%， 三甘 油脂下降 33 %， LDH/HDL 降低 17%。 乳酸菌能降低胆固醇机制目前还不清楚。大量研究认为乳酸菌可 产生 3－羟基戊－3－甲基二酸 (HMG)， 从而抑制了胆固醇合成时所必 需的羟基戊二酰辅酶 A 还原酶活性；发酵乳中的乳清酸衍生的代谢产 物可降低胆固醇浓度；乳酸菌可将胆固醇有效的同化或将其捕住使之 不能进入血液； 由于乳酸菌可使胆汁酸盐脱抱合而变得难于吸收。 6. 降血压作用 乳酸菌具有降血压作用， 这在几种乳酸菌中已有报道， 并且起作用 的物质也各不相同。高血压患者口服干酪乳杆菌的菌体自溶物的热水 每天两次， 每次 400mg， 连续 3 个月后血压明显下降： 粪肠 抽提液(LEx)， 球菌 FK- 23 热水抽提液也具有降低血压的效果，且对正常的血压完全 没有影响，进一步分析结果证实起作用的物质是分子量在 10000 ～ 70000Da 的 RNA。 瑞士乳杆菌发酵的酸奶也具有降低血压的功能， 有效 物质是两个小肽 Val- Pro- Pro(VPP)和 Ile- Pro- Pro(IPP)。 研究认为这两个 小肽形成的空间结构和降血压药物络活喜主成份结构很相似。 此外， 乳酸菌还可以通过束缚抗原、 破坏抗原而减轻过敏反应症状 ） （下转第 402 页 和提高机体抗氧化能力而有助于延缓衰老。
穿心莲内酯纳米混悬剂的制备及评价
穿心莲内酯纳米混悬剂的制备及评价王静娴;李文;陈婷;李俊松【摘要】OBJECTIVE To established the andrographolide nanosuspensions (ADG-NS) with improved its dissolution and membranepermeability.METHODS ADG-NS were prepared by wet media milling technique.The quality of the nanosuspensions was characterized including the particle size and distribution, Zeta potential, particle morphology and crystal physical state.In vitro dissolution test and Caco-2 permeability test were used to preliminarily evaluate the ADG-NS.RESULTS The particle size, polydispersity index (PDI) and Zeta potential of ADG-NS were (215.6 ± 3.3) nm, 0.165 ± 0.020 and (-36.9 ± 2.8) mV, respectively.SEM analysis showed that there was no adhesion and aggregation between particles, the particles were rod or block, and the size distribution was uniform.DSC and XRPD results showed that the nanoparticles were present in the amorphous form.The dissolution rate of ADG-NS were significantly enhanced in different pH buffer solutions compared to ADG and commercial dripping pills.Caco-2 permeability test revealed that ADG-NS showed a significant increase in the membrane permeability (Papp) compared to the group of ADG (P<0.01).CONCLUSION The ADG-NS prepared by wet media milling technique can improve the in vitro dissolution rate and membrane permeability of andrographolide (ADG).%目的制备穿心莲内酯纳米混悬剂(ADG-NS),并对其进行表征和评价.方法采用介质研磨法制备ADG-NS,对其粒径及分布、Zeta电位、粒子形态、结晶状态进行表征,采用体外溶出度试验和Caco-2细胞转运试验对ADG-NS进行初步评价.结果ADG-NS的粒径、多分散性系数、Zeta电位分别为(215.6 ± 3.3) nm、0.165 ± 0.020和(-36.9 ± 2.8) mV.扫描电子显微镜(SEM)分析结果显示,粒子多呈棒状或块状,大小分布较均匀.差示扫描量热(DSC)和x射线粉末衍射(XRPD)分析结果显示,纳米混悬剂中的药物以无定型态存在.与原料药及穿心莲内酯滴丸比较,ADG-NS在不同pH值溶出介质中,溶出速率显著提高.Caco-2细胞单层细胞渗透试验表明,与原料药相比,ADG-NS显示较高的膜渗透性.结论 ADG-NS可以提高穿心莲内酯的体外溶出度与膜渗透性.【期刊名称】《南京中医药大学学报》【年(卷),期】2017(033)003【总页数】4页(P313-316)【关键词】穿心莲内酯;纳米混悬剂;介质研磨;体外溶出;Caco-2细胞【作者】王静娴;李文;陈婷;李俊松【作者单位】南京中医药大学药学院,江苏南京 210023;南京中医药大学药学院,江苏南京 210023;南京中医药大学药学院,江苏南京 210023;南京中医药大学药学院,江苏南京 210023【正文语种】中文【中图分类】R283.6穿心莲内酯(ADG)为二萜类内酯化合物，是中药穿心莲代表性药效成分之一，拥有“天然抗生素”的美称，具有较好的抗菌、抗炎、抗肿瘤及保肝作用[1-4]，是一个极具发展潜力的药物。
乳酸菌胞外多糖肠道黏附及免疫调节作用研究进展
乳酸菌胞外多糖肠道黏附及免疫调节作用研究进展李 超，王春凤，杨桂连*（吉林农业大学动物科学技术学院，吉林省动物微生态制剂工程研究中心，吉林 长春13 0118）摘 要：乳酸菌作为公认的食品级益生菌，在医学、食品科学及人类健康中发挥重要作用。
乳酸菌的胞外多糖作为其主要的细胞外分泌物与其相应生物学 功能的发 挥密切相关。
本文旨在从乳酸菌胞外多糖的理化特性和分 子结构方面入手探究 其调节免疫的机制，并总结乳酸菌胞外多糖在调节胃肠道功能和进入体内后的 免疫调节等方面的特性。
以期为乳酸菌胞外多糖的研究和应用奠定理论基础。
关键词：乳酸菌；胞外多糖；黏附；免疫调节Progress in Intestinal Adhension and Immunoregulatory Effect of Extracellular Polysaccharides of Lactic Acid BacteriaLI Chao, W ANG Chun-feng, Y ANG Gui-lian *(Jilin Provincial Engineering Research Center of Animal Probiotics, College of Animal Science and Technology,Jilin Agricultural University, Changchun 130118, China)Abstract: Lactic acid bacteria are acknowledged as one kind of food-grade probiotics, and play an important role in medical science, food science and many other fields of life science. Extracellular polysaccharide as the extracellular secretion of lactic acid bacteria is closely related to its biological characteristics. This article reviews the immune regulation mechanism, physical and chemical properties, and molecular structure of extracellular polysaccharides from lactic acid bacteria, and summarizes their gastrointestinal and immune regulatory effects. We expect that this study will provide the theoretical basis for researching and applying exopolysaccharides from lactic acid bacteria.Key words: lactic acid bacteria; extracellular polysaccharide; adhesion; immunoregulation 中图分类号：Q939.117 文献标志码：A 文章编号：1002-6630（2014）11-0314-05doi:10.7506/spkx1002-6630-201411061收稿日期：2013-06-17基金项目：国家高技术研究发展计划（863计划）项目（2013AA102806；2011AA10A215）；国家自然科学基金面上项目（31272541；31272552；81170358）；教育部“新世纪优秀人才支持计划”项目（NCET -10-0175）；吉林省科技发展计划项目（20111816；20080104）作者简介：李超（1987—），男，硕士，研究方向为动物微生态与分子生物学。
1-s2.0-S0142961214010850-main
Enhanced transport of nanocage stabilized pure nanodrug across intestinal epithelial barrier mimicking Listeria monocytogenes Dengning Xia a,1,Jinsong Tao a,b,1,Yuan He a,c,Quanlei Zhu a,Dan Chen a,Miaorong Yu a, Fude Cui d,Yong Gan a,*a Shanghai Institute of Materia Medica,Chinese Academy of Sciences,501Haike Road,Shanghai201203,Chinab Department of Pharmacy,Medical College of Nanchang University,461Bayi Road,Nanchang330066,Chinac Department of Pharmaceutics,School of Pharmacy,Chongqing Medical University,Chongqing400016,Chinad Department of Pharmaceutics,School of Pharmaceutical Science,Shenyang Pharmaceutical University,Shenyang110016,Chinaa r t i c l e i n f oArticle history:Received16August2014 Accepted2October2014 Available online25October2014Keywords:NanocrystalsNanocageEpithelial barrierWheat germ agglutininGoblet cells a b s t r a c tLigand grafted nanoparticles have been shown to enhance drug transport across epithelium barrier and are expected to improve drug delivery.However,grafting of these ligands to the surface of pure nano-drug,i.e.,nanocrystals(NCs),is a critical challenge due to the shedding of ligands along with the sta-bilizer upon high dilution or dissolving of the drug.Herein,a non-sheddable nanocage-like stabilizer was designed by covalent cross-linking of poly(acrylic acid)-b-poly(methyl acrylate)on drug nanocrystal surface,and a ligand,wheat germ agglutinin(WGA),was successfully anchored to the surface of itra-conazole(ITZ)NCs by covalent conjugation to the nanocage(WGA-cage-NCs).The cellular study showed that large amount of WGA-cage-NCs were adhered to Caco-2cell membrane,and invaded into cells, resulting in a higher drug uptake than that of ordinary NCs(ONCs).After oral administration to rats, WGA-cage-NC were largely accumulated on the apical side of epithelium cells,facilitating drug diffusing across epithelium barrier.Interestingly,WGA-cage-NCs were capable of invading rat intestinal villi and reaching to lamina propria by transcytosis across goblet cells,which behaved like a foodborne pathogen, Listeria monocytogenes.The WGA-cage-NCs showed an improved oral bioavailability,which was17.5-and 2.41-folds higher than that of coarse crystals and ONCs,respectively.To our best knowledge,this may represent thefirst report that a functional ligand was successfully anchored to the surface of pure nanodrug by using a cage-like stabilizer,showing unique biological functions in gastrointestinal tract and having an important significance in oral drug delivery.©2014Elsevier Ltd.All rights reserved.1.IntroductionIn pharmacotherapy,oral administration of drugs can signifi-cantly improve patient's compliance and quality of life.However, oral drug delivery remains a major challenge and improved tech-nologies are urgently needed.Recently,nanotechnology offers new approaches to improving the efficacy and safety of oral drug de-livery[1,2].For instance,loading drugs into nanoparticles such as solid lipid nanoparticles[3],liposomes[4],and polymeric nano-particles[5]can significantly improve drug transport across the gastrointestinal(GI)tract[6,7].The pure nanodrug,i.e.,nanocrystal (NC),is a promising nanoparticle drug delivery system and has been used in formulating of water-insoluble drugs[8e10].NC possesses almost a100%drug loading,significantly higher than other matrix nanoparticles[3].The high drug loading makes it more efficient in delivering drug into,or across cells,reaching a significantly high therapeutic concentration at drug target site.It is generally accepted that the significantly improved drug dissolution in GI endowed by NCs accounts for the enhanced oral absorption of water insoluble drugs[8].However,efficient drug absorption is still difficult to achieve when NCs encounter mucus and epithelium barrier in GI[11e13].The thick mucus layer covering the intestinal epitheliums and the intestinal epithelium layer consisting of different cells such as columnar epitheliums (CE),goblet cells(GC),and M cells,greatly hamper the oral ab-sorption of drugs.After oral administration,the drug NCs arefirstly dissolved in GI lumen;the drug molecules then diffuse across thick mucus layer and transport across the intestinal epithelial layer,and finally reach the blood capillary in lamina propria.Due to the low*Corresponding author.Tel.:þ8621202310001424;fax:þ8621202310001425.E-mail address:simm2122@(Y.Gan).1These authors contributed equally to thiswork.Contents lists available at ScienceDirect Biomaterialsjournal h omepage:/locate/biomaterials/10.1016/j.biomaterials.2014.10.0380142-9612/©2014Elsevier Ltd.All rights reserved.Biomaterials37(2015)320e332water-solubility of the drug,the diffusion process across above obstacles is greatly restricted[6].Additionally,the transport of NCs across gastrointestinal epithelia is almost impossible,due to the low binding affinity of the drug NCs to epithelia.The low cellular transport of NCs results in a limited delivery of drug,although the NCs possess an extremely high drug loading capacity.Thus far, there have been few reports on efficient cellular uptake or trans-cytosis of drug NCs after oral administration[14],and little is known about the exactly physiological pathways that are respon-sible for the cellular trafficking of NCs in GI.Recently,the concept of bioadhesion,generally using ligand grafted polymeric nanoparticles to enhance drug transport across epithelium barrier,is expected to improve drug delivery,and has gained much attention in the development of drug delivery tech-nologies[15].For example,lectins and other ligands with adhesion ability may be useful[12,16];these molecules can specifically recognize receptor-like structures on the cell membrane and bind directly to the epithelial cells,a process called‘‘cytoadhesion’’. Conjugation of these adhesion molecules to the polymeric nano-particles can enhance the bioadhesion of nanoparticles to GI epithelia,facilitating the accumulation of particles at their sites of absorption instead of being rapidly cleared in the constantly moving environment of the gut[17].Furthermore,when bioadhesion is receptor-mediated,it is not only restricted to mere binding,but may subsequently trigger the active transport of drug nanocarriers by receptor-mediated endocytosis or transcytosis,including clathrin-dependent or caveolin-dependent process[16,18,19].However,to our best knowledge,there is no literature on grafting the ligands to NCs for efficient drug delivery.The present study was designed to test the hypothesis that pure drug NCs packaged with cytoadhesion molecules could successfully traverse mucus layer and specifically target GI epithelia,resulting in improved cellular uptake and trans-port of drug across GI epithelium barrier.Grafting these ligands to NC surface faces several technical challenges due to the deficiency of functional groups on the surface of NCs,such as carboxyl groups,which can be used to anchor the ligands by covalent conjugation,and this is different from polymeric nanoparticles.To prepare NCs,efficient stabilizers hav-ing surface activity are required to prevent aggregation between the high-energy crystal surfaces produced during the size-reduction process[20,21].The stabilizers may include surfactants, e.g.,Poloxamer188,Tween80,or polymers,e.g.,polyvinyl alcohol (PVA),hydroxypropyl methyl cellulose(HPMC),and hydroxypropyl cellulose(HPC)[21].In preparing ligand grafted NCs,the stabilizers also need to provide a potential way of anchoring ligands to the NC surface.However,a critical challenge for this is that the ligands may shed along with the stabilizer upon high dilution or dissolving of the drug,because the stabilizers are present on the crystal surface generally via physical adsorption[22,23].To achieve a high density of ligands on the particle surface,a covalent conjugation of ligand on the particle surfaces is almost mandatory[24].However,the chemistries that can be used and the chemical properties of crystal surface and the ligand are not necessarily easy to match[16].The shedding of stabilizers from the NC surface not only greatly affects the stability of NCs,but also loss the targeting ability to the cells, particularly for oral drug delivery,due to the harsh pH and enzyme environment of the GI tract[16].To resolve the aforementioned problems,here,we have suc-cessfully created a new method that anchored a ligand,wheat germ agglutinin(WGA),to NC surface by designing a“nanocage”using a crosslinkable polymer,poly(acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA)as the stabilizer.Itraconazole(ITZ),an antifungal agent with extremely low solubility[25],was used as the model drug in the present ing in vitro Caco-2cell and in vivo rat models,the transport of non-sheddable nanocage stabilized NCs across the intestinal epithelium barrier and the oral absorption of NCs was studied.We hypothesized that the designed functional NCs could significantly promote the transport of NCs across mucus and intestinal epithelium barrier and improve the oral absorption of the drug as illustrated in Scheme1.To our best knowledge,there has been no literature reporting that a functional ligand was suc-cessfully anchored to the surface of pure nanodrug by using this kind of cage-like stabilizer,showing unique biological functions in GI that will have an important significance in oral drugdelivery. Scheme1.Illustration of non-sheddable nanocage stabilized pure nanodrug and their transport across the intestinal epithelial barrier resulting in an enhanced oral absorption.D.Xia et al./Biomaterials37(2015)320e3323212.Materials and methods 2.1.MaterialsITZ were obtained from Shouguang Fukang Pharmaceutical Co.,Ltd (China).The N-(3-Dimethylaminopropyl)-N 0-ethyl carbodiimide hydrochloride (EDAC),2,20-(Ethylenedioxy)bis(ethylamine)(EbE),ethyl2-bromopropionate (2-EBP;99%),N,N,N 0,N 00,N 00-pentamethyldiethylenetriamine (PMDETA;99%),and 5-amino fluorescein were purchased from Sigma e Aldrich (St.Louis,MO,USA);Wheat germ agglutinin (WGA)and fluorescein labeled UlexEuropaeus Agglutinin I (UEA-I-FITC)were obtained from Vector laboratories (Burlingame,CA,USA),WGA-Alexa Fluor 647conjugates was obtained from Invitrogen Life technologies (Shanghai,China);Ethyl rhodamine B was obtained from Sinopharm Chemical Re-agent Co,Ltd.(Shanghai,China);and Methyl acrylate (99%),tert-butyl acrylate (99%)and Copper(I)bromide were obtained from Aladdin reagent (Shanghai,China).All other chemicals and reagents were of analytical grade.2.2.Preparation of NC suspensionsA newly synthesized polymer,poly(acrylic acid)-block-poly(methyl acrylate)(PAA-b-PMA),was used as the stabilizer for the production of NCs.The synthesis procedure was reported previously by Q.G.Ma.et al.[26],and the 1H Nuclear Magnetic Resonance (NMR)and Fourier Transform Infrared Spectroscopy (FT-IR)spectra of PAA-b-PMA are presented in Supplementary Information .The ITZ NCs were prepared using the anti-solvent precipitation-high pressure homogenization method [27].Brie fly,200mg of ITZ coarse crystals and 80mg of PAA-b-PMA were dissolved in 2.5ml of dioxane to form an organic solution.Then,the solution was rapidly injected into 40ml of pH 6.8PBS at 2 C under magnetic stirring (500rpm)and probe sonication (SCIENTZ-IID,Ningbo,China)for 10s at 200W to obtain a coarse suspension.After the anti-solvent precipitation,the coarse suspension was placed into a high pressure homogenizer (ATS Engineering Inc,Shanghai,China)equipped with a cooling device which was connected to a circulating water bath (Julabo F12,Seelbach,Germany)at 2 C.The homogenization was firstly performed at 500bar for 10circles,then,1000bar for 20circles to obtain a milky state ITZ NC suspension.The NC suspension was concentrated by centrifugation at 50,000g for 10min (Beckman Coulter Allegra 64R centrifuge,USA).The supernatant was removed,and the sediment was redispersed in 20ml of pH 6.8PBS containing 0.2%PAA-b-PMA to adjust drug concentration to 10mg/ml.To label the NCs with fluo-rescein,the hybrid NCs containing ethyl rhodamineB incorporated in crystal lattice were prepared according to the method reported by Zhao et al.,[28]with a slight modi fication.Brie fly,20mg of ethyl rhodamine,200mg of ITZ coarse crystals and 80mg of PAA-b-PMA were dissolved in 2.5ml of dioxane to form an organic solution (w/w),and the following process was the same as the preparation of ITZ nano-suspension as aforementioned.2.3.Conjugation of WGA and crosslinking of stabilizer on NC surfaceWGA was covalently conjugated to NC surface using the carbodiimide method [29].Five mg of WGA was dissolved in 0.2ml of pure water,and added in drop-wise to 15ml of ITZ NC suspension.The suspension was then stirred for 1h,using magnetic stirring at room temperature (25 C).To this suspension was added a solution of EDAC (50mg)in 0.5ml of water dropwise over 2min,and allowed to stir overnight under ambient conditions.To crosslink the stabilizer onto NC surfaces,the solution of 2,20-(ethylenedioxy)bis(ethylamine)(12.5mg in 200m l water)was added to 10ml of the aforementioned WGA conjugated NC suspension dropwise over a 2-min period.The suspension was allowed to stir for 1h at room temperature.To this suspension was added a solution of EDAC (32mg in 0.2ml water)dropwise over 2min,and allowed to stir overnight under ambient conditions.The suspensions were centrifugated at 50,000g for 10min;the supernatant was removed;and the sediment was washed thrice with pure water to remove contaminants,including unreacted EDAC,PAA-b-PMA and unbound WGA.The schematic procedure for producing WGA nanocage stabilized NCs is illustrated in Fig.1.2.4.Characterization of NCs2.4.1.Particle size and zeta potentialsThe particle sizes and zeta potentials of ITZ NCs were determined using Zetasizer (Nano e ZS,Malvern instruments,United Kingdom).The measurements of particle sizes were performed at 25 C with a scattering angle of 173 .The ITZ nano-suspensions were diluted 5times using Mili-Q water before determination.Each sample was measured in triplicate.2.4.2.Surface morphologyThe morphology of NCs was examined by transmission electron microscopy (TEM,JEM-1230,Tokyo,Japan).After diluting the samples 50-fold with puri fied water,a drop of the suspension was spread on a copper grid,and the excess liquid was removed with filter paper.A drop of 2%aqueous solution of uranyl acetate was then dropped onto the grid for negative staining for 4min.The samples were dried under room temperature for 10min,and observed under TEM.2.4.3.1H NMR and powder X-ray diffraction (XRPD)The PAA-b-PMA and nanocage were dissolved in CDCl 3and D 2O,respectively.1H NMR spectra were recorded on a Bruker Av400spectrometer (Bruker BioSpin,F €allanden,Switzerland)operating at 400MHz for protons.The crystalline form of ITZ NCs was examined using XRPD.The samples were freezing dried before analysis.Bruker AXS D8Advance diffractometer was used to analyze the samples.The sam-ples were placed on zero e background silicon plates and measured at ambient conditions in a re flection mode.A continuous 2q scan was performed in the range of 5 e 45 with a step size of 0.02 using Cu K a radiation (l ¼1.540629Å),with the scanning speed of 0.067 2q /s.The voltage and current applied were 40kV and 40mA,respectively.The data were analyzed using DIFFRAC.SUITE ™Software (Bruker).2.5.Analyses of shedding of stabilizer and WGA from NC surfaceTo determine the shedding of stabilizer from NC surface,the stabilizer was conjugated to 5-amino fluorescein using carbodiimide chemistry [30].The amount of reacted carboxyl of PAA-b-PMA was 5%as compared with theoretically calculated total amount of carboxyl.To remove the EDAC and unbounded 5-amino fluorescein,the NC suspensions (10ml)were centrifugated at 50,000g for 10min.Then,the supernatant was removed and the sediments were redispersed in 10ml of pH 6.8PBS by sonication for about 5min till they were well dispersed in PBS.The samples were washed 5times with 20ml PBS each by aforementioned cen-trifugation e redispersion method.At each time,the fluoresce intensity of nano-suspensions was determined using a microplate reader (SYNERGYH1,BioTeck,Winooski,VT,USA).To determine the WGA level on NC surface after each washing,0.3ml of nanosuspension was withdrawn,and dissolved in 3ml of methanol,the samples were vortex mixed for 1min and then centrifugated at 50,000g for 10min.The supernatant containing dissolved drug was removed,and the sediments containing precipitated WGA and stabilizer were re-dissolved in 60m l of pH 6.8PBS.The concentration of WGA was determined with a bicinchoninic acid (BCA)protein assay kit according to the manufacturer's instruction (Beyotime,Shanghai,China).The WGA levels on NC surface were expressed as amount of WGA per milligram of drug.2.6.Drug dissolution testDissolution experiments were performed according to the USP35-NF30paddle method (Symphony 7100,Distek,Inc.,USA).Water containing pH 6.8PBS and 0.2%SDS was used as dissolution medium.The temperature was maintained at 37±0.5 C,and the paddle speed used was 100rpm.Accurately weighed samples containing the equivalent of 4mg drug were dispersed in the 500ml of dissolution medium,and the samples (4ml each)were withdrawn at different times and passed through a 0.1-m m syringe filter (Millipore Filters,USA).Quantitative analyses of the samples were done using a UV spectrophotometer (Mode TU-1901,Purkinje Gen-eral,China)at 263nm.Fig.1.Schematic illustration of producing WGA-cage-NC.A non-sheddable nanocage stabilizer formed by cross-linking the PAA-b-PMA on crystal surface was designed to anchor WGA to the surface of ITZ NCs.D.Xia et al./Biomaterials 37(2015)320e 3323222.7.Cellular uptake of NCsCaco-2cells were seeded in12-well plates at a density of1Â105cells per well and grown in an incubator at37 C with a controlled atmosphere containing5%CO2 and90%relative humidity for4days with feeding every2days.The cellular uptake experiments with Caco-2cells were performed4days after reaching confluence.The fluorescent labeled NCs were dispersed in Hank's Balanced Salt Solution(HBSS)to form test suspensions with thefinal drug concentration of1mg/ml.Cells were pre-incubated with blank HBSS at37 C for30min.After removing the blank HBSS,the cells were exposed to the0.5ml of the test suspensions at37 C for2h.Then,the test suspensions were removed,and the cell layer were washed thrice by2ml HBSS each.The cells werefixed with4%buffered paraformaldehyde for10min,and stained with diamidino-phenyl-indole(DAPI)(5m g/ml,Beyotime,Shanghai),and finally imaged under confocal laser scanning microscopy(CLSM)(FluoView FV10i, Olympus,Japan).In a separate experiment,the cell layers which had been incubated with the test suspensions for2h were trypsinized after washing thrice with2ml HBSS each.The cells were centrifuged and then washed with ice-cold medium.The cells were then re-suspended in ice-cold medium.And the samples were analyzed by FACS Calibur(Becton e Dickinson,USA).To distinguish between extracellularly and intracellularly localized NCs,the samples were incubated in a0.2%trypan blue solution in HBSS for5min at room temperature to quench extracellularfluorescence [31].The amount of intracellularfluorescence was also quantified using FACS Cal-ibur.All experiments were performed in thrice and a total of10,000cells per sample were analyzed for each experiment.2.8.Animal studies2.8.1.Histological examination of the intestine after oral administration of NCsMale Sprague e Dawley rats(weighing250e280g)were provided by SLAC Lab Animal Ltd.(Shanghai,China).The experiments were performed under the guide-lines approved by the Institutional Animal Care and Use Committee of the Shanghai Institute of Materia Medica,Chinese Academy of Sciences,Shanghai,China(IACUC, 2013-07-GY-12).The rats were kept in a12h/12h light/dark cycle at the animal care facility,given daily fresh diet with free access to water,and acclimatized for at least5 days prior to the experiments.The rats were fasted for12h prior to the experiments but had free access to water.Thefluorescence-labeled NC suspensions were administered to the rats at 30mg/kg of itraconazole(ITZ)by oral gavage.At2h after dosing,the animals were anesthetized by intraperitoneal injection of25%urethane(1g/kg).A3-cm midline incision was made on the abdomen.The intestinal segments of duodenum,jejunum and ileum of small intestine were identified.The proximal jejunum was located by identifying the ligament of Trietz;the distal jejunum and the beginning of the proximal ileum were located by visualizing an increase in the density of gut-associated lymphatic tissue(GALT);and the distal ileum was identified by locating the cecum.The intestinal segments of duodenum,jejunum and ileum were removed,and washed extensively with large volumes of cold saline.These tissues were carefully everted,and embedded in Tissue-Tek®O.C.T™Compound(Sakura Finetek,USA)atÀ20 C and sectioned at20m m(Leica CM1950,Germany).The sections werefixed with4%buffered paraformaldehyde,stained with40,6-diamidino-2-phenylindole(DAPI)(5m g/ml,Sigma),and then imaged under LCSM.A standard lectin staining procedure was also used for the identification of cell types. After thefixation,the tissue sections were blocked with3%PBS-BSA(Sigma e Aldrich)by dropping40m l of3%PBS-BSA on the sample and incubated for1h.The lectin-labeling experiment was performed withfluorescein labeled UEA-I and WGA-Alexa Fluor647conjugate at a concentration of20m g/ml in3%PBS-BSA for2h[32].2.8.2.Ex vivo imaging of NCs transportation through GI tractThefluorescein labeled NC suspensions were administered to the rats at30mg/ kg of ITZ by oral gavage.At different times after oral dosing(0.5,1,2,and5h),the rats were anesthetized with1ml of25%(w/v)urethane,and the GI tract of the rats (stomach and small intestine)were removed.The spectralfluorescence images of the GI tract were obtained by the Caliper IVIS®Spectrum in vivo Imaging System (PerkinElmer,USA).An internal laser of570nm was used for excitation light of ethyl rhodamine.Tunablefilter automatically stepped in20-nm increments from520to 720nm was employed for light emission,whereas the camera captured images at each wavelength interval with constant exposure.Spectral unmixing algorithms were applied to create unmixed images offluorescein and auto-fluorescence.All images were analyzed using the living image®4.3.1software provided by Caliper Life Sciences.2.8.3.Pharmacokinetic study in ratsFour groups of SD rats(5or6rats per group)were orally administrated with4 formulations,namely coarse ITZ crystal suspension,ordinary NC suspension without WGA(ONC),WGA functionalized NCs by physical absorption(WGA-NC),and WGA functionalized NCs by nanocage(WGA-cage-NC)at an equivalent dose of30mg/kg. The rats were allowed to access to food4h after oral drug administration.Blood samples(400m l)were collected via the orbital venous plexus under isoflurane anesthesia at1,2,3,4,5,6,8,12,25,and36h after dosing,and transferred into heparinized tubes.Plasma sample was obtained by centrifugation at8000rpm for 5min and was kept atÀ20 C until analysis.The ITZ concentrations in blood were quantified by HPLC[33,34].The samples for HPLC analysis were prepared by liquid e liquid extraction according to reported procedures with minor modification[33,34].Briefly,30m l of internal standard so-lution(fenofibrate,8m g/mL in acetonitrile)were added to0.15ml of rat plasma,and vortex-mixed for5s.Then,0.8ml heptane e isoamyl alcohol(95:5,v/v)mixture was added and vortex-mixed for2min to extract ITZ.The extraction was repeated twice. The organic layer was separated by centrifugation at8000rpm for5min,and transferred to another clean tube.The pooled organic layer was evaporated under a stream of nitrogen at40 C,and the residue was reconstituted in50m l of acetonitrile. The drug concentration was analyzed by HPLC Agilent1100series(Agilent Tech-nologies,Santa Clara,California)with an Agilent Extend C18column(3.5cm, 250Â5mm2;Agilent Technologies,Santa Clara,CA,USA).The mobile phase was composed of acetonitrile and water(78/22,v/v),with aflow rate of1ml/min.UV-detection was performed at263nm.Quantification of ITZ in the plasma samples was based on linear regression between the HPLC peak area ratio of ITZ to the in-ternal standard.Linearity was observed in the concentration range of0.05e4m g/ml with correlation coefficients of over0.99.Pharmacokinetic parameters were derived from drug concentration-time data by a drug and statistics software(Phoenix®WinNonlin6.3,Pharsight Corporation, USA)using a non-compartmental analysis model.The parameters included the maximal blood concentration of drug(C max),the time taken to reach maximum blood concentration(T max),and the area under the blood drug concentration-time curve(AUC0e24h).2.8.4.Statistical analysisThe experimental data were analyzed using the Excel2013(Microsoft,USA). Student's t-test was used to determine the significance of differences between2 groups.The difference was considered to be statistically significant if the probability value was less than0.05(p<0.05),and to be very significant if p<0.01.All data were expressed as mean±S.D.unless otherwise indicated.3.Results and discussion3.1.Design,preparation and characterization of WGA-nanocage stabilized drug NCsTo design a non-sheddable“nanocage”for anchoring a lectin, wheat germ agglutinin(WGA),to NC surface,a crosslinkable poly-mer,poly(acrylic acid)-b-poly(methyl acrylate)(PAA-b-PMA)was used as the stabilizer.The polymer was synthesized by atom transfer radical polymerization method and is presented in Materials and Methods[26].The structure of the PAA-b-PMA and the prepara-tion procedure of the nanocage-stabilized NCs are illustrated in Fig.1. The molecule weight of PAA-b-PMA and the number of carboxyl per molecule were determined as2800g/mol and26,respectively,via 1H NMR end group analysis[35].The stabilizing copolymer contains hydrophobic polymer segments,poly(methyl acrylate),and hydro-philic polymer segments,poly(acrylic acid),which can prevent NC from agglomeration by masking their high-energy surfaces during preparation,and the hydrophilic domain extends to the water environment,providing steric barriers for the NCs,which is benefit for the stabilization of NCs.And most importantly,the carboxyl groups of the polymer open the opportunity for future conjugation of the hydrophilic corona with ligand by a simple carbodiimide method[36].To prevent the shedding of the polymer and anchored ligand,a cross-linker,2,20-(Ethylenedioxy)bis(ethylamine)(EbE), was used to crosslink PAA-b-PMAs on surface of the crystals by the carbodiimide method between carboxylic acid groups of PAA and the amine functionalities of EbE[36].The NCs were prepared by the anti-solvent precipitation e high pressure homogenization method[27].The particle sizes and Zeta potential values of ordinary nanocrystals(ONC),NCs functionalizedTable1Particle size,zeta potential and WGA level of nanocrystals after washing four times. Nanocrystals Size(nm)Zeta potential(mV)WGA level(m g/mg)ONC227±7nmÀ45.2±1.30WGA-NC229±6nmÀ43.1±2.1 3.2±1.6WGA-cage-NC243±5nmÀ31.4±5.010.2±2.0D.Xia et al./Biomaterials37(2015)320e332323with WGA by sheddable stabilizer(WGA-NC),and NCs functionalized with WGA by non-sheddable nanocage(WGA-cage-NC)are shown in Table1.The particle sizes of WGA-NC and ONC were similar,while the particle size of WGA-cage-NC was slightly increased.During the preparation of NCs,amorphous ITZ nanoparticles with particle size about180nm were obtained by anti-solvent precipitation at2 C in this experiment.Although,with the stabilization of stabilizer,the amorphous nanoparticle suspension was still extremely unstable under ambient conditions,and an amorphous to crystalline trans-formation processed with increased particle size and change in polymorphism in few days[37,38].The high pressure homogeniza-tion could convert materials from thermodynamically unstable amorphous particles into a more stable crystalline nanoparticles [39,40].As a result,the combination use of anti-solvent precipitation and high pressure homogenization method,we prepared the ITZ NCs with reproducible particle size(~220nm),and a uniform particle size distribution(PDI~0.05),which was the smallest size achievable with the other experimental setup such as wet-milling[41]and high pressure homogenization[34].The prepared NCs are reproducible and stable for at least six months at4 C.The Zeta potentials of ONC and WGA-NC were similar.The negative charge of ONC and WGA-NC was mainly contributed by the dissociation of carboxylic acid groups of stabilizer,PAA-b-PMA, which presented on the surface of NCs by physical absorption.The chemical crosslinking of stabilizer,which occurred between the carboxylic acid groups of PAA-b-PMAs and the amine groups of EbE within the shell layer of the crystals,resulted in a significantly increased Zeta-potential of WGA-cage-NC.As shown in Fig.2a,under a transmission electron microscope (TEM),the NCs showed an irregular shape(Fig.2a,left panel); WGA-cage-NC showed a clear core e shell structure(Fig.2a.middle panel).After removing of crystal core by dissolution of WGA-cage-NC,the intact polymeric coatings were observed(Fig.2a,right panel).1H NMR spectra of the polymeric coatings(Fig.2b)showed that the ethylenedioxy(O e CH2e CH2e O)gave signals at 3.1e3.3ppm,a proof for intermolecular cross-linking of stabilizers. These results also demonstrated that the nanocage remained structurally intact even following complete removal of the drug. This system was reminiscent of hollow cage-like structures,as prepared by Turner and Wooley,which were obtained bycoreFig.2.Characterization of NCs.(a)TEM images of ONC,WGA-cage-NC and core removed shell(an intact polymeric coatings were observed after dissolution of the drug from WGA-cage-NC);(b)1H NMR spectra of PAA-b-PMA(400MHz,CDCl3)and nanocage(400MHz,D2O)shows the cross-linking of PAA-b-PMA with EbE;(c)XRPD patterns of coarse crystals, ONC and WGA-cage-NC.D.Xia et al./Biomaterials37(2015)320e332324。
通脉方中异黄酮类化合物在人源肠Caco2细胞模型的吸收转运
通脉方中异黄酮类化合物在人源肠Caco2细胞模型的吸收转运通脈方是由葛根、丹参和川芎3味药按质量1∶1∶1组成的复方。
该文研究通脉方中异黄酮类化合物大豆苷元、芒柄花素、5羟基芒柄花苷、芒柄花苷、大豆苷、3′甲氧基葛根素、染料木苷、葛根素、芒柄花素8CβD呋喃芹糖基（1→6）OβD吡喃葡萄糖苷、芒柄花素7OβD呋喃芹糖基（1→6）OβD吡喃葡萄糖苷、澳白檀苷、葛花宁、大豆苷元7，4′二OβD吡喃葡萄糖苷、泰国野葛根素、3′羟基葛根素、3′甲氧基大豆苷、芒柄花素8CβD吡喃木糖基（1→6）OβD吡喃葡萄糖苷、染料木素8CβD呋喃芹糖基（1→6）OβD吡喃葡萄糖苷、染料木素7OβD 呋喃芹糖基（1→6）OβD吡喃葡萄糖苷、3′羟基泰国野葛根素、6″OβD木糖基葛根素、鹰嘴豆芽素A8CβD呋喃芹糖基（1→6）OβD吡喃葡萄糖苷、3′甲氧基大豆苷元7，4′二OβD吡喃葡萄糖苷、大豆苷元7OβD吡喃葡萄糖基（1→4）OβD 吡喃葡萄糖苷和大豆苷元7OαD吡喃葡萄糖基（1→4）OβD吡喃葡萄糖苷在肠的吸收转运。
采用人源肠Caco2细胞单层模型，研究通脉方中上述25个异黄酮类化合物由绒毛面（AP侧）到基底面（BL侧）或从BL侧到AP侧2个方向的转运过程。
应用高效液相色谱法分离、紫外检测法对化合物进行定量分析，计算表观渗透系数（Papp），并与阳性对照药普萘洛尔和阿替洛尔比较。
大豆苷元和芒柄花素由AP侧到BL侧的Papp分别为（255±003）×10-5，（306±001）×10-5 cm·s-1；由BL侧到AP侧的Papp分别为（262±0）×10-5，（265±011）×10-5 cm·s-1。
与本试验中在Caco2细胞单层模型上呈良好吸收的阳性对照药普萘洛尔的Papp （266±032）×10-5 cm·s-1和呈难吸收的阳性对照药阿替洛尔的Papp（234±010）×10-7 cm·s-1比较，大豆苷元和芒柄花素与普萘洛尔在同一数量级；其他化合物与阿替洛尔在同一数量级。
醋氯芬酸贴剂
RESEARCH ARTICLEFormulation and biopharmaceutical evaluation of a transdermal patch containing aceclofenacYun-Seok Rhee •Thuong Nguyen •Eun-Seok Park •Sang-Cheol ChiReceived:6January 2013/Accepted:4February 2013/Published online:11March 2013ÓThe Pharmaceutical Society of Korea 2013Abstract To reduce the adverse effects of aceclofenac that accompanied with oral administration of this drug,trans-dermal patches in the form of drug-in-adhesive (DIA)pat-ches,containing aceclofenac,were formulated.The effect of formulation factors on the skin permeation of the drug and physical properties of the patch were evaluated using excised rat skins.The optimized patch contained 12%aceclofenac and 20%lauryl alcohol in DT-2852as a pressure-sensitive adhesive.The pharmacokinetic characteristics of the DIA patch were determined after application of the transdermal patches to human volunteers.The calculated relative bio-availability of the aceclofenac DIA patch was 18.2%com-pared to oral administration of the drug.The findings of this study suggest that transdermal application of aceclofenac can substitute for oral administration of the drug.Keywords Aceclofenac ÁTransdermal patch ÁSkin permeation ÁPharmacokinetics IntroductionAceclofenac,one of potent non-steroidal anti-inflammatory drugs,has been used for the treatment of rheumatoid arthritis and its related conditions (Brogden and Wiseman 1996).It blocks prostaglandin E 2production following its intracellular conversion into cyclooxygenase inhibitors (Yamazaki et al.1997).However,some adverse effects such as gastrointestinalirritation,abdominal pain,diarrhea,or constipation are accompanied with oral administration of the drug.To reduce the adverse effects of aceclofenac,transdermal application may be the dosing route of choice.However,the large daily dose of 100mg makes it difficult to formulate a transdermal dosage form of this drug because usually only drugs required in doses of \20mg per day are candidates for transdermal patch application.Nevertheless,the fact that rheumatoid arthritis occurs near the body surface indicates that transder-mal application of aceclofenac may provide a ‘‘site of action’’potential thereby allowing the drug to more effectively reach its target areas.There have been some reports about trans-dermal dosage forms of aceclofenac such as cream (Tessari et al.1995),gel (Thorat and Rane 2010)or microemulsion (Yang et al.2002);however,there has not been any previous report on the formulation of aceclofenac as a transdermal patch using pressure-sensitive adhesive (PSA).The aim of this study was to formulate drug-in-adhesive (DIA)patches containing aceclofenac using PSA.There are many formulation factors for such a patch including PSA,penetration enhancer,loading amount of the drug,and the physical properties of the patch.This study examined the effects of these factors on the skin perme-ation of aceclofenac using excised rat skin.The pharma-cokinetic characteristics of an optimized DIA patch containing aceclofenac were determined after its transder-mal application to healthy volunteers.Materials and methods MaterialsThe following reagents were used as purchased without further purification:aceclofenac (Saniver,Hongkong),Y.-S.RheeCollege of Pharmacy and Research Institute of Pharmaceutical Sciences,Gyeongsang National University,Jinju 660-701,Korea T.Nguyen ÁE.-S.Park ÁS.-C.Chi (&)Sungkyunkwan University,Suwon 440-746,Korea e-mail:scchi@Arch.Pharm.Res.(2013)36:602–607DOI10.1007/s12272-013-0073-yflurbiprofen(Sigma Chemical Co.,USA),lauryl alcohol (Kanto Chemical Co.,Japan),oleyl alcohol(Yakuri Pure Chemicals Co.,Japan),and HPLC-grade acetonitrile and methanol(J.T.Baker Co.,USA).Duro-TakÒ(DT)adhe-sives were obtained from National Starch and Chemical Co.,USA and Bio-PSAÒadhesives from Dow Corning, USA.All other chemicals were of reagent grade.Water was purified by reverse osmosis andfiltered in-house.Preparation of DIA patches containing aceclofenacThe DIA patches containing aceclofenac were prepared with various PSA and penetration enhancers.A laboratory-coating unit(Labcoater LTE-S,Mathis,Switzerland)was used to prepare the DIA patches.Appropriate amounts of aceclofenac and penetration enhancer,when needed,were added to the PSA solution and mixed homogenously with a mechanical stirrer.The solution was coated onto afluoro-polymer-treated polyester release liner(ScotchPakÒ1022, 3M,USA).The coating thickness wasfixed at100l m. After the solvent had been removed,it was laminated with a polyester backingfilm(ScotchPakÒ9732,3M,USA). Determination of skin permeation of aceclofenacSkin permeation rates of aceclofenac from DIA patches of different formulations were measured using Franz diffusion cellsfitted with excised rat skins.The rat skins were obtained from male Sprague-Dawley rats weighing 230±20g after the hair had been removed with electric clippers(900,TGC Inc.,Japan).A393cm patch of skin was excised from the dorsal region and the adhering fats and other visceral tissues were removed carefully.The excised rat skins were stored at-20°C and used within 1week after the skin harvest.The receptor compartment of the Franz diffusion cell was11.5ml and the effective diffusion area was1.77cm2. The receptor medium was pH7.4phosphate buffer (0.03M).Its temperature was maintained at37±0.5°C using a thermostatic water pump(WBC1520,Jeio Tech Co.,Korea)and stirred at a constant rate of600rpm during the experiment.At2,4,6,8,12,16,and24h after transdermal application of the patch containing aceclofenac on the excised rat skin,200l l of the receptor medium was withdrawn and replaced with an equal volume of freshly prepared medium.The amount of aceclofenac that per-meated through the skin into the receptor medium was determined using a validated HPLC method.Data analysisThe cumulative amounts of aceclofenac permeated through the skin were plotted as a function of time.From these graphs,the skin permeation rate of the drug was calculated from the slope of the linear portion of each plot. Evaluation of physical properties of DIA patchesThe prepared patches were cut into strips2.54cm wide and conditioned for24h at23±2°C and50±5%RH.The samples were applied to an adherent plate made of stainless steel,smoothened with a4.5pound rollerfive times,and pulled from the substrate at a180°angle at a rate of 300mm/min.Adhesion/release tester(AR-1000,ChemIn-strument,USA)was used to determine peel adhesion force. For the determination of tackiness,the prepared patches were cut into strips1091.5cm and conditioned for24h at23±2°C and50±5%RH,and mounted on loop tack tester(LT-100,ChemInstrument,USA). Biopharmaceutical evaluation of DIA patches containing aceclofenacThe pharmacokinetic characteristics of the DIA patch containing aceclofenac were evaluated following its transdermal application to healthy volunteers using the oral administration of a tablet containing the drug as a refer-ence.This experiment was carried out after approval of the protocol by the ethics committee of our school.The vol-unteers were25–47years old and weighed between55and 87kg.A total of12volunteers were allocated randomly into two groups,one group for the transdermal application and the other for the oral administration.DIA patches with the optimized formula of696cm,equivalent to100mg of aceclofenac,were applied to the backs of volunteers and removed at24h post-dose.For oral administration of aceclofenac,Airtal(Dae Woong Co.,Korea),which con-tains100mg of aceclofenac,was used as a reference.Blood(10ml)was collected from the forearm vein of volunteers at0,1,2,3,4,6,8,10,12,24,27,30,and33h after transdermal application of the patches,and at0,0.5, 1,1.5,2,3,4,5,6,9,12,and15h after oral administration of the aceclofenac tablet.The blood was transferred immediately into glass tubes containing a small amount of sodium heparin and centrifuged at3,000rpm for10min. The plasma collected was stored at-20°C until analysis.The maximum plasma concentration(C max)and the time to reach C max after dosing(T max)were directly determined from the plasma concentration of aceclofenac versus time profiles.The area under the plasma concentration–time curve(AUC)up to the last sampling time was calculated using the linear trapezoidal rule.The relative bioavail-ability of the aceclofenac patch compared to the oral administration of the aceclofenac tablet was calculated using the AUC values obtained.Transdermal patch containing aceclofenac603HPLC determination of aceclofenacConcentrations of aceclofenac in the receptor medium or human plasma were determined using a validated HPLC method with a slight modification(Bae et al.1999).The HPLC system consisted of an isocratic pump(L-7100, Hitachi,Japan),a UV detector(L-7400,Hitachi,Japan),an automatic injector(L-7200,Hitachi,Japan),and an inte-grator(L7000,Hitachi,Japan).The column used was a Luna C18column(4.69250mm,5l m particle size, Phenomenex,USA).The mobile phase was a mixture of acetonitrile,methanol,and pH7.4phosphate buffer (0.03M)(18:41:41).Theflow rate was1ml/min and the detection wavelength was275nm.For the permeation study,samples were appropriately diluted with the mobile phase,and100l l was injected onto the column.For the pharmacokinetic study,50l l of an internal standard solution(flurbiprofen,5l g/ml),2ml of2.5N phosphoric acid,and3ml of an extraction solvent of n-hexane:iso-propanol(9:1)were added to500l l of plasma and shaken for30min.After centrifugation at2,500rpm for10min, the organic phase was taken and evaporated to dryness at40°C under a gentle stream of nitrogen.The residue was reconstituted with500l l of the mobile phase and100l l of thefinal solution was injected onto the column.StatisticsEach experiment was repeated at least three times.The mean values and standard errors are presented.The phar-macokinetic parameters were compared using Student’s t test with the level of significance set at P\0.05. Results and discussionEffect of PSAs on the skin permeation of aceclofenacThree types of PSAs are commonly used in transdermal drug delivery devices:polyisobutylenes,silicones,and acrylate copolymers.These are hypoallergenic,synthetic, biologically inert,non-irritating PSAs and have no sys-temic toxicity(Venkatraman and Gale1998).The selection of an appropriate PSA is the most important factor in designing a transdermal drug delivery system(Tan and Pfister1999).Physicochemical properties of a PSA can affect the skin permeation rate of a drug from the PSA matrix.Some parameters such as glass transition temper-ature,PSA functional group,interaction between the drug and PSA,adhesive force,and many other factors can influence the skin permeation rate of a drug.To investigate the effect of PSAs on skin permeation of aceclofenac,DIA patches were prepared using various PSAs.The concentration of the drug in the patch wasfixed at12%.The skin permeation profiles of aceclofenac from the prepared patches through excised rat skins were obtained and the skin permeation rates,calculated from these profiles,are shown in Table1.Among PSAs studied, acrylate PSAs resulted in pronounced skin permeation of aceclofenac,while polyisobutylene and silicone PSAs showed negligible skin permeation of the drug.Among the PSAs,DT-2852showed the highest skin permeation rate of aceclofenac;1.19l g/cm2/h.Acrylate PSAs with hydroxyl groups also showed relatively high skin permeation rates. However,DT-2051and DT-2052showed low skin per-meation rates,despite their similarity to DT-2852as acrylic PSAs containing carboxylic groups.Other studies have also reported that different functional groups in acrylate PSAs can result in different permeation rates of drugs (Guyot and Fawaz2000;Chedgzoy et al.2002).Based on these results,DT-2852was chosen as the adhesive of choice for the aceclofenac DIA patch.Effect of loading amount of aceclofenac on skin permeation of aceclofenacTo determine the maximum skin permeation rate of ace-clofenac from patches prepared with DT-2852,the effect of the loading amount of aceclofenac in the DIA patch was evaluated using excised rat skins.The amount of ace-clofenac in the patch was tested at concentrations of1,3,6, 12,18,and24%in DT-2852.Figure1shows the perme-ation rates of aceclofenac through excised rat skins from the patches as a function of the loading amount of Table1Permeation rates of aceclofenac through excised rat skins from transdermal patches prepared with different PSAsPSA Type ofPSAFunctionalgroupPermeation rate(l g/cm2/h)DT-2852A–COOH 1.19±0.01a DT-2525AV–OH0.69±0.30 DT-2510A–OH0.62±0.32 DT-2287AV–OH0.57±0.21 DT-9301A None0.31±0.14 DT-4098AV None0.29±0.18 DT-2052AV–COOH0.26±0.12 DT-2051A–COOH0.18±0.00 DT-6432P None0.09±0.00 DT-6430B P None0.03±0.00 Bio-PSAÒ7-4101S None0.00±0.00 Bio-PSAÒ7-4201S None0.00±0.00 Bio-PSAÒ7-4302S None0.13±0.00A acrylate,AV acrylate-vinyl acetate,P polyisobutylene,S silicone a Mean±SE;n=3604Y.-S.Rhee et al.aceclofenac.The skin permeation rate of the drug increasedup to12%aceclofenac and then stayed almost constant beyond this concentration,indicating that12%may be the saturation solubility of the drug in DT-2852.Effect of fatty alcohols on skin permeationof aceclofenacTo develop a DIA patch for a drug,an appropriate pene-tration enhancer is often needed to enhance the permeation of the drug through the skin.According to preliminary studies,fatty alcohols showed good penetration enhancing effects for the skin permeation of aceclofenac.Fatty alco-hols are effective penetration enhancers used widely in percutaneous absorption of many drugs(Aungst et al. 1986;Cho et al.2001;Rhee et al.2007).Fatty alcohols, classified as lipid disrupting agents,fluidize the stratum corneum lipids and reduce its barrier properties,thereby increasing drug transport through the skin(Lee et al.2006).The effect of fatty alcohols on the skin permeation of aceclofenac was evaluated using patches containing lauryl alcohol,a saturated fatty alcohol,and oleyl alcohol,an unsaturated fatty alcohol.The adhesive used was DT-2852. The concentration of the drug wasfixed at12%,while the concentrations of fatty alcohols were varied from0to25%.The skin permeation rates of aceclofenac were calculated from the skin permeation profiles of the drug from the prepared patches containing different amounts of lauryl alcohol;these rates are presented in Fig.2.The skin per-meation rate of aceclofenac increased up to8.08l g/cm2/h when lauryl alcohol was added to the patch.The increase of the skin permeation rate of the drug was almost proportional to the concentration of lauryl alcohol,up to20%,in the patch.It is well known that the effects of penetration enhancers are often dependent upon their concentrations (Takayama et al.1999;Narishetty and Panchagnula2004). However,beyond this concentration of lauryl alcohol,the skin permeation of aceclofenac did not continue to increase. Aungst et al.(1986)also reported that the enhancing effect of lauryl alcohol on naloxone penetration depended on the concentration of the enhancer.In their study,however,they found that higher concentrations of lauryl alcohol decreased skin permeation of naloxone,possibly because the partition coefficient of the drug was reduced.As shown in Fig.2,oleyl alcohol also enhanced skin per-meation of aceclofenac,although the penetration enhancing effect of oleyl alcohol was not as pronounced as the effect of lauryl alcohol.The maximum skin permeation rate for ace-clofenac was obtained at15%oleyl alcohol and the rate remained almost unchanged beyond this concentration. However,unlike lauryl alcohol,oleyl alcohol reduced the skin permeation rate of aceclofenac at its low concentrations, possibly due to the high solubilizing capacity of oleyl alcohol. Effect of lauryl alcohol on physical propertiesof the patchTo investigate the effect of lauryl alcohol on the adhesion properties of the prepared patches,physical characteristicsTransdermal patch containing aceclofenac605of the patches were measured.Peel force and tackiness of the patches containing aceclofenac and different amounts of lauryl alcohol are presented in Fig.3.The peel force of the patch decreased almost proportionally to increasing con-centrations of the enhancer.The tackiness also decreased gradually as the concentration of lauryl alcohol was increased.Nevertheless,the peel force obtained with 25%lauryl alcohol,123g/inch,is still high enough to be con-sidered good adhesion.Likewise,the tackiness obtained with 25%lauryl alcohol (555g)is also high enough for good adhesion.Based on the above results,the optimal concentration of lauryl alcohol in the patch is considered to be 20%.Pharmacokinetic evaluationTo evaluate the bioavailability of aceclofenac via a trans-dermal patch,the patch was applied transdermally at a dose of 100mg aceclofenac.The aceclofenac patch used con-tained 12%aceclofenac and 20%lauryl alcohol in DT-2852.The oral administration of the drug was used as the reference.The obtained plasma concentration–time profiles of aceclofenac are shown in Fig.4.After oral administration of an aceclofenac tablet,the drug showed typical oral pharmacokinetic characteristics (Table 2)similar to previous reports (Lee et al.2000;Kang and Kim 2008;Ghosh and Barik 2010).The plasma concentration–time profile,obtained after transdermal application of a DIA patch containing aceclofenac at a dose of 100mg,is also shown in Fig.4.The lag time was 4h after the transdermal application of the patch.The plateau concen-tration reached at 10h post-dose and the averageconcentration of aceclofenac in plasma was 0.15l g/ml.Based on these results,the calculated relative bioavail-ability of aceclofenac in the DIA patch was 18.2%com-pared to the oral administration of the drug.Even though the relative bioavailability of the drug after transdermal application is low compared to that of oral administration,transdermal application of aceclofenac may provide a larger benefit than would be anticipated from its measured bioavailability.The disease sites of arthritis are usually located near the body surface,therefore application of the drug via the skin above the disease sites could result in locally high concentrations of the drug at the disease site,while the concentration of aceclofenac in the systemic blood would remain low.The low systemic blood con-centration could reduce the adverse systemic effects thatTable 2Pharmacokinetic parameters of aceclofenac obtained after transdermal application of a DIA patch and oral administration to healthy volunteers at a dose of 100mg ParametersAdministration route TransdermalOral C max (l g/ml)0.19±0.03a,*7.46±0.90T max (h)22.0±1.8* 2.2±0.3AUC (l g*h/ml)3.59±0.52*19.68±1.67*Significantly different compared to the oral administration (P \0.05)aMean ±SE,n =6606Y.-S.Rhee et al.occur as a result of high concentrations of the drug in the blood.Therefore,in the case of anti-inflammatory or anti-arthritis drugs,the systemic bioavailability does not need to be equivalent to the oral administration of the drug.Rather, it is better to have low bioavailability with high local concentration and low blood concentration of the drug. Future testing in an animal model is required to determine whether the concentration of the drug,as delivered via the DIA patch,is high enough to have a pharmacological effect.Acknowledgments This work was supported by the Korean Health Technology R&D Project,Ministry for Health,Welfare&Family Affairs(#A092018).ReferencesAungst,B.J.,N.J.Rogers,and E.Shefter.1986.Enhancement of naloxone penetration through human skin in vitro using fatty acids,fatty alcohols,surfactants,sulfoxides and amides.The International Journal of Pharmaceutics33:225–234.Bae,J.H.,K.E.Choi,S.-C.Chi,and E.-S.Park.1999.Bioequivalence evaluation of aceclofenac tablets.Korean Journal of Clinical Pharmacy9:44–48.Brogden,R.N.,and L.R.Wiseman.1996.Aceclofenac.A review of its pharmacodynamic properties and therapeutic potential in the treatment of rheumatic disorders and in pain management.Drugs 52:113–124.Chedgzoy,P.,G.Winckle,and C.M.Heard.2002.Triclosan:Release from transdermal adhesive formulations and in vitro permeation across human epidermal membranes.The International Journal of Pharmaceutics235:229–236.Cho,S.J.,D.R.You,and K.S.Kim.2001.The effects of enhancers on the transdermal absorption of ketoprofen packs.Yakche Hak-hoechi31:107–112.Ghosh,S.,and B.B.Barik.2010.A comparative study on the pharmacokinetics of conventional and sustained-release tablet formulations of aceclofenac in healthy male subjects.Tropical Journal of Pharmaceutical Research9:395–399.Guyot,M.,and F.Fawaz.2000.Design and in vitro evaluation of adhesive matrix for transdermal delivery of propranolol.The International Journal of Pharmaceutics204:171–182.Kang,W.,and E.-Y.Kim.2008.Simultaneous determination of aceclofenac and its three metabolites in plasma using liquid chromatography-tandem mass spectrometry.Journal of Phar-maceutical and Biomedical Analysis46:587–591.Lee,P.J.,N.Ahmad,nger,S.Mitragotri,and V.P.Shastri.2006.Evaluation of chemical enhancers in the transdermal delivery of lidocaine.The International Journal of Pharmaceutics308: 33–39.Lee,S.L.,C.K.Jeong,S.J.Choi,S.B.Kim,M.H.Lee,G.I.Ko,andD.H.Sohn.2000.Simultaneous determination of aceclofenacand diclofenac in human plasma by narrowbore HPLC using column-switching.Journal of Pharmaceutical and Biomedical Analysis23:775–781.Narishetty,S.T.K.,and R.Panchagnula.2004.Transdermal delivery of zidovudine:Effect of terpenes and their mechanism of action.The Journal of Controlled Release95:367–379.Rhee,Y.-S.,J.-Y.Huh,C.-W.Park,T.-Y.Nam,K.-R.Yoon,S.-C.Chi, and E.-S.Park.2007.Effects of vehicles and enhancers on transdermal delivery of clebopride.Archives of Pharmacal Research30:1155–1161.Takayama,K.,J.Takahara,M.Fujikawa,H.Ichikawa,and T.Nagai.1999.Formula optimization based on artificial neural networks in transdermal delivery.The Journal of Controlled Release62: 161–170.Tan,H.S.,and W.R.Pfister.1999.Pressure-sensitive adhesives for transdermal drug delivery systems.Pharmaceutical Science& Technology Today2:60–69.Tessari,L.,L.Ceciliani,A.Belluati,G.Letizia,U.Martorana,L.Pagliara, A.Pognani,G.Thovez, A.Siclari,G.Torri,L.Solimeno,and E.Montull.1995.Aceclofenac cream versus piroxicam cream in the treatment of patients with minor traumas and phlogistic affections of soft tissues:A double-blind study.Current Therapeutic Research56:702–712.Thorat,S.P.,and S.I.Rane.2010.Formulation and in vitro evaluation of lecithin(soya and egg)aceclofenac organogels.Journal of Pharmacy Research3:1438–1441.Venkatraman,S.,and R.Gale.1998.Skin adhesives and skin adhesion1.Transdermal drug delivery systems.Biomaterials19: 1119–1136.Yamazaki,R.,S.Kawai,T.Matsuzaki,N.Kaneda,S.Hashimoto, T.Yokokura,R.Okamoto,T.Koshino,and Y.Mizushima.1997.Aceclofenac blocks prostaglandin E2production following its intracellular conversion into cyclooxygenase inhibitors.The European Journal of Pharmacology329:181–187.Yang,J.-H.,Y.-I.Kim,and K.-M.Kim.2002.Preparation and evaluation of aceclofenac microemulsion for transdermal deliv-ery system.Archives of Pharmacal Research25:534–540.Transdermal patch containing aceclofenac607。
01-硅烷基础知识培训
Silane
R-NCO + H2N-Polymer
O
Si
O - Si-CH2CH2CH2-NH-C-NH
C
O
O
Si
O - Si-CH2CH2CH2-NH-C-NH
C
O
Adhesive
O
Si
O - Si-CH2CH2CH2-NH-C-NH
C
Silicone Basic Course
玻璃纤维填充聚氨酯的界面
Silicone Basic Course
Si (OC2H5)3
Alkyl Silane : A-137
O O
Si (OCH3)3
Epoxy Silane : A-187
硅烷的偶联和交联作用
Y
Si – (OR)3
Organo-functional Group
Linker
Silicon atom
Hydrolysable group
水性涂料的耐水性，耐化学品，耐擦洗
Silicone Basic Course
硅烷的偶联作用 在有机-无机界面形成化学键接. 防止湿气沿着界面渗透. 防止界面破坏. 将强度由填料传递到树脂. 提高填料的分散性或降低材料的粘度.
Silicone Basic Course
硅烷的偶联作用
Glass (Substrate)
T alc
1% polyethersilane
No silane
ZnO
Yellow Iron Molybdate
Oxide
Orange
Silicone Basic Course
硅烷用于提高湿态 附着力
Silquest A-187 用于丙烯酸乳液胶粘剂中
基于网络药理学的宣肺止嗽合剂止咳作用机制研究
基于网络药理学的宣肺止嗽合剂止咳作用机制研究∗孙丽丽，白海英，郑文惠，杨志刚△兰州大学，甘肃兰州730000［摘要］运用网络药理学的方法探究宣肺止嗽合剂止咳的作用机制。
从中药系统药理学数据库及分析平台（traditional Chinese medicine systems pharmacology database and analysis platform，TCMSP）中检索宣肺止嗽合剂中的化学成分，以口服生物利用度（OB＞30%）、Caco-2细胞通透性（Caco-2＞0.4）和药物相似性（DL>0.18）为标准筛选潜在的活性成分。
利用STITCH数据库检索宣肺止嗽合剂中活性成分的作用靶点，以“cough”为检索词在OMIM、TTD、CTD数据库中检索疾病靶点库。
利用STRING数据库构建PPI网络，根据度中心性筛选关键靶点，并用LeDock做分子对接验证。
运用DAVID平台对关键靶点进行通路分析，结合Omic-share平台注释和可视化分析。
从宣肺止嗽合剂中筛选出34个活性成分，涉及到50个关键靶点，这些靶点主要集中在肿瘤坏死因子（tumor necrosis factor，TNF）、哮喘（asthma）、丝裂原活化蛋白激酶（mitogen-acti-vated protein kinase3，MAPK3）、甲型流感（influenza A）等多条信号通路上，分子对接显示罂粟碱能稳定的对接到AKT1的活性口袋中。
宣肺止嗽合剂通过作用于AKT1、TNF、MAPK3等关键靶点，干预了多个与抗炎、免疫调节和呼吸道疾病相关的生理过程，初步揭示了宣肺止嗽合剂止咳的分子机制。
［关键词］宣肺止嗽合剂；网络药理学；止咳；作用靶点；分子对接；信号通路［中图分类号］R285［文献标识码］A［文章编号］1004-6852（2020）12-0007-10Research on Antitussive Mechanism of Xuanfei Zhisou Mixture Basedon Network PharmacologySUN Lili,BAI Haiying,ZHENG Wenhui,YANG Zhigang△Lanzhou University,Lanzhou730000,ChinaAbstract The antitussive mechanism of Xuanfei Zhisou mixture is discussed using the method of network pharmacology.Chemical ingredients of Xuanfei Zhisou mixture were searched in TCMSP,and the potential active ingredients were screened according to oral bioavailability(OB>30%),Caco-2cellular permeability(Caco-2>0.4) and drug similarity(DL>0.18).The targets of active ingredients in Xuanfei Zhisou mixture were retrieved from STITCH,"cough"was chosen as the key words in disease target library from OMIM,TTD and CTD.PPI network was constructed using STRING database,the key targets were selected according to centrality,molecular docking verification was performed using LeDock.The signals of key targets were analyzed using DAVID platform, annotation and visual analysis were performed combined with Omicshare platform.All34active ingredients were selected from Xuanfei Zhisou mixture,involved50key targets,and these targets mainly focused on multiple signaling pathways of TNF,asthma,MAPK3,influenza A and others,molecular docking displayed that papaverine could dock into the active pocket of AKT1stably.Xuanfei Zhisou mixture intervened physiological process related to resisting inflammation,immunoregulation and respiratory disease,primarily revealed the molecular mechanism of antitussive effects of Xuanfei Zhisou mixture.Keywords Xuanfei Zhisou mixture;network pharmacology;antitussive;target;molecular docking;signaling pathway宣肺止嗽合剂是《中华人民共和国药典》所收载的非处方中成药，由荆芥、前胡、桔梗、蜜百部、蜜紫菀、陈皮、鱼腥草、薄荷、蜜罂粟壳、蜜甘草等10味中药组成。
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Received 5 January 2005; accepted 26 April 2005 Available online 9 June 2005
Abstract Probiotic bacteria play an important role in protecting the host from intestinal colonization of pathogenic bacteria. We have developed a new analytical approach based on a real-time PCR technique for quantifying Bifidobacterium adhesion to intestinal epithelial cells. Real-time PCR analysis showed that adhesion to enterocyte-like Caco-2 cells represented a variable phenotype in the genus Bifidobacterium, enabling classification of three adhesion behaviors: high adhesiveness (>40 bifidobacterial cells/Caco-2 cell); adhesiveness (5–40 bifidobacterial cells/Caco-2 cell); no adhesiveness (<5 bifidobacterial cells/Caco-2 cell). This molecular methodology was successfully used in competition studies in enteropathogens. All bifidobacterial strains examined evidenced displacement activity towards important enteropathogens (S. typhimurium, Y. enterocolitica and E. coli EPEC). Real-time PCR is a rapid, accurate and sensitive method for detecting and quantifying different bacterial genera and species simultaneously adhering to a epithelial cell monolayer. 2005 Elsevier SAS. All rights reserved.
* Corresponding author.
E-mail address: patrizia.brigidi@unibo.it (P. Brigidi). 0923-2508/$ – see front matter 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2005.04.006
Marco Candela a , Gerd Seibold b , Beatrice Vitali a , Sabrina Lachenmaier b , Bernhard J. Eikmanns b , Patrizia Brigidi a,∗
a Department of Pharmaceutical Sciences, CIRB—Center for Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy b Department of Microbiology and Biotechnology, University of Ulm, 89069 Ulm, Germany
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M. Candela et al. / Research in Microbiology 156 (2005) 887–895
concept of modulating the human microbiota by administration of probiotic bacteria has been established [4,6,16, 20,22,23]. Probiotics are defined as “live microbial feed supplements which beneficially affect the host animal by improving its intestinal balance” [6]. Bifidobacterium represents such a probiotic bacterial genus and therefore is widely used in food and pharmaceutical probiotic preparations [12,24–26]. Bifidobacteria represent about 8–10% of the normal adult fecal flora and constitute one of the most important human intestinal microbial groups [2,9,19]. The presence of bifidobacteria in the gastrointestinal tract has been associated with several health-promoting effects such as prevention of diarrheas, amelioration of lactose intolerance and immunomodulation [18,24,25]. Furthermore, a role for Bifidobacterium in host infection resistance has been proposed, as in vitro and in vivo studies suggested that some bifidobacterial strains exert an antagonistic activity toward enteropathogens such as Escherichia coli EPEC, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis [1,8]. However, although the genome of B. longum has recently been sequenced and annotated [21], there is only incomplete information about bifidobacterial physiology and ecology, and very little is known about the specific mechanisms of direct interactions between bifidobacteria and the host. In this study we propose a new analytical approach, based on real-time PCR and genus- species-specific primers, for the in vitro evaluation of bacterial adhesion to epithelial cells. In comparison with traditional techniques available (viable counts, radiolabeled bacteria, light and electron microscopy) [32], the analytical approach proposed here is rapid, sensitive and particularly useful for bacterial competition studies. This new molecular approach has been used to study the competition between different bifidobacterial strains belonging to B. lactis, B. bifidum and B. longum species, and three important enteropathogens (S. enterica serovar Typhimurium, Y. enterocolitica and E. coli EPEC) for adhesion to monolayers of enterocyte-like Caco2 cells.
colonization of enteric pathogens [15]. From the host point of view, such a barrier effect can be considered as one of the main functions exerted by the gut microbiota. Three mechanisms of action are involved in the barrier effect played by the intestinal microflora: (i) prevention of enteropathogenic adhesion to the host enterocytes; (ii) favorable competition with exogenous pathogens for nutrient availability in the gastrointestinal ecological niches; (iii) inhibition of growth of pathogenic bacteria by bacteriocin production and lowering of the pH. Intestinal disorders, antibiotic treatment, stress, and changes in diet influence the individual microbiota, resulting in their depletion and imbalance [7]. The reduction in the normal microflora has negative effects on human wellbeing and can be frequently associated with greater host susceptibility to enteropathogenic bacterial infections. In order to overcome problems associated with microflora imbalance, or to generally improve the health of the host, the