中国科技论文统计源期刊 中文核心期刊
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊
日本科学技术振兴机构数据库（JST）
第七届湖北十大名刊提名奖
美国《化学文摘》《国际药学文摘》
《乌利希期刊指南》
WHO《西太平洋地区医学索引》来源期刊
日本科学技术振兴机构数据库（JST）
第七届湖北十大名刊提名奖
, 张宇佳, 魏曼, 宋辉, 郑稳生
经皮给药是一种非侵入性的给药方式,具有其他给药途径所无法比拟的优点。但由于皮肤角质层的屏障作用以及上皮细胞间的紧密连接屏障,很多药物需要通过各种促渗手段才能通过经皮给药途径吸收。细胞穿透肽作为一种高效低毒的短肽,已被证明能够促进小分子、多肽、蛋白质、脂质体、核酸等的渗透作用,其促渗机制尚不明确,但在经皮给药领域应用广泛。该文综述细胞穿透肽在经皮给药系统中的应用。
经皮给药作为一种非侵入性的给药途径,可应用于治疗全身性疾病和局部皮肤疾病。它能够避免肝脏的首关效应和胃肠道刺激,提高药物生物利用度;作为一种体表给药方式,经皮给药在实现无创给药的同时能随时中断给药,提高患者的依从性;此外,经皮给药具有缓释作用,可减少给药次数,并能维持血浆浓度长时间处于治疗窗内[1]。由于其具有很多口服给药不具备的优点,近年来已成为制剂行业的研究热点。
皮肤作为人体面积最大的器官,有多种功能,包括光保护、温度调节、激素合成、感官知觉和免疫屏障功能[2]。其中与药物吸收最相关的是免疫屏障功能。皮肤从外到内分为表皮层(包括角质层、透明层、颗粒层、棘层、基底层)、真皮层和皮下组织。经皮给药根据药物作用区域不同可以分为经皮局部给药和经皮全身性给药。经皮局部给药需要药物能够透过角质层但滞留在皮肤中,而全身性给药则需要药物透过角质层、真皮层,最终进入血液循环。药物通过经皮吸收途径进入血液循环主要有以下两种途径:通过角质层进入血液循环,或者通过皮肤附属器进入血液循环。皮肤附属器途径包括通过外泌汗腺、顶泌汗腺和毛囊以及它们相关的皮脂腺。这些附属器占全部皮肤面积的约0.1%,因此这种跨毛囊(分流)途径被认为是次要的[3]。由此可见,全身性给药中,通过角质层进入血液循环的占经皮给药途径的大部分。无论是局部给药还是全身性给药,角质层都是经皮给药系统必须通过的障碍。而上皮细胞间还存在紧密连接,这则是对经皮给药系统的另一大阻碍。对于主要穿过完整表皮角质层的药物而言,存在两种可能的微型进入途径,细胞内途径和细胞间途径。角质层富含脂质(40%脂质、40%蛋白质以及只有20%水)且含水量低,药物渗透过皮肤通过何种方法主要取决于分配系数(logk)。亲水性药物分子优先进入细胞间区域,通过角质层传递的少;而亲脂性渗透物因和角质层中细胞的细胞间脂质具有可混合性[4],则是通过细胞内通路穿过角质层而传递多一点。角质层的阻隔性使得药物若要有效穿过角质层进入真皮层必须满足以下几个条件:①相对分子质量<500;②log
CPPs也称细胞转导域(protein transduction domains,PTDs),被定义为一类由5~30 个氨基酸组成的短肽,能够溶于水。由于有多个精氨酸,其通常带正电,或具有两亲性[6-7]。该类型的多肽最早出现在1988年,人们发现I型人类免疫缺陷病毒(HIV-1)编码转录的反式激活因子(Tat)多肽,这是第一个发现能够改变细胞膜的位置并进入细胞内的多肽[8]。几年后,CPPs开始用于小的外源性多肽给药。随后发现在这些CPPs中小的结构域通常负责细胞摄取。因此与最初的Tat多肽比较,这些CPPs的序列能够被缩短至几个氨基酸残基,且不损失细胞渗透能力。从那时开始,CPPs的功能不断丰富起来,并且数量持续增长,作为具有细胞膜穿透能力的小分子多肽,它能携带生物活性物质进入细胞内,既不影响转导物质的生物活性,也不会损伤细胞,已经更多地用于多种分子物质,如小分子、小干扰RNA、载药纳米颗粒、蛋白质和多肽的体内外给药,是一种非常理想的运载工具[9]。
由于小分子药物经皮进入血液循环有多种促渗方法,用于局部给药时也往往通过常见的化学促渗剂、脂质体等方法就可有效促进小分子药物的渗透作用,因此将CPPs用于小分子药物经皮给药的研究并不多。COHEN-AVRAHAMI等[10]将一种两亲性细胞穿透肽RALA溶解在热力学稳定的反向六边形溶质液晶中介相(HⅡLC)中形成凝胶型基质,并将双氯芬酸钠溶解于其中用于经皮给药。通过小角度X射线衍射(small angle X-ray scattering,SAXS)、衰减全反射-傅里叶变换红外光谱法(attenuated total reflectance-Fourier transform infrared,ATR-FTIR)等研究方法表明,RALA导致液晶组成成分甘油单油酸酯(glycerol monooleate,GMO)有效端基面积因水合作用逐步增加,并导致液晶由六边形结构转向薄层状结构。Franz扩散池实验表明多肽促渗剂(RALA)将双氯酚酸钠从液晶中渗透入皮肤的量提高2.2倍。随后COHEN-AVRAHAMI等[11]研究比较RALA、penetratin(PEN)以及寡聚精氨酸(oligoarginine,NONA)等多种CPPs对双氯芬酸钠经皮递送的影响,并研究药物和CPPs在中介相液晶中的分布。通过差示量热扫描法(differential scanning calorimetry,DSC)和ATR-FTIR发现双氯芬酸钠分布在中介相的表面活性剂链之间的界面区(interfacial region),使得HⅡLC更加密集。亲水性的NONA插入中间相水性的柱面中引起它们的膨胀,这诱导GMO羰基合物和它们周围的物质的氢键相互作用明显增加;而两亲性的PEN则如RALA,会首先在GMO的端基区域溶解,与GMO的羟基相互作用,当GMO端基区域位点PEN达到饱和时,则分布于外部的界面区,与GMO的界面区域相互作用。在此基础上,COHEN-AVRAHAMI等[12]还比较另一种细胞穿透肽TAT对药物从立方体中介相和薄片状中介相中扩散速率的影响。研究发现立方体系统中TAT二级结构是无规则卷曲的,而当其被嵌入密堆积的薄片状系统中,则变成更有序的β-转角的紧密状态,排列在GMO的头部基团附近。而由于在薄片状中介相中TAT的结构有序性更强,更为紧密,因此TAT对药物从薄片状中介相中扩散出来的速率促进相对弱一些。TAT使得双氯芬酸钠和塞来昔布从立方体系统中扩散的量分别增加6倍和9倍,从薄片状系统中扩散的量则分别增加1.3倍和1.7倍。在CPP- HⅡLC系统中,反向六边中介相作为增溶贮库和凝胶骨架,能起到缓释的作用;而细胞穿透肽则起到促进药物经皮渗透的作用。
此外,CPPs也被用于某些小分子物质的局部经皮给药,除促进药物进入角质层之外,还具有使药物滞留在皮肤内的效果。KUMAR等[13]将SPACE多肽联合皮质类固醇使用。皮质类固醇自身可以通过皮肤进入体循环,而该类药物通常用于治疗局部炎症等疾病,进入体循环后会带来较多的系统不良反应。KUMAR等将其与SPACE多肽联合使用,加强其在表皮层的富集,同时进一步加大通过角质层进入皮肤的量,抑制药物进入体循环,同时也能降低药物用量。该研究可为CPPs的使用方法提供一种新的思路。
目前对CPPs用于大分子的经皮给药的研究主要集中在局部给药。由于某些治疗皮肤病和改善皮肤性能的大分子物质的重要性,人们更多研究这些多肽或蛋白质的局部给药。但大分子如多肽、蛋白质和核酸类物质的真皮给药或经皮给药依然是一个重大的挑战。研究者们已经尝试使用多种工具,如化学促渗剂、给药载体和不同渗透方法来发展大分子的局部制剂。但促进大分子通过皮肤渗透的化学和物理方法都有其局限性,包括化学促渗剂在高浓度时的皮肤毒性、在家使用促渗电气设备的不便性以及复杂给药系统的高成本。因此,研究者们考虑将有效、低毒的CPPs用于大分子的局部经皮给药,希望能够用来治疗某些皮肤病或其他局部疾病,改善皮肤状况[9]。STOUT等[14]演示一种细胞穿透肽RMR-丝聚合蛋白(filaggrin)复合体(RMR-FLG)的成功运用,将小鼠丝聚合蛋白基因的简单重复片段与一种细胞穿透肽(RMR)基序共价结合,并转入原核培养系统用于表达蛋白。所得RMR-FLG能很好被HEK-293T细胞摄取,重组人表皮(reconstructed human epidermis,RHE)模型的免疫染色实验表明RMR-FLG能够有效渗透入表皮组织,并且缺乏丝聚合蛋白的Flaky-Tail小鼠在局部使用后出现病理学特征上的恢复状态,表明该复合体对于过敏性皮肤炎的患者可能有治疗效果。在全身性给药研究方面,MANOSROI等[15]将鲑鱼降钙素(sCT)和细胞穿透肽Tat以摩尔比1:1在室温下共同孵育1 h,所得sCT -CPPs复合物能够使sCT更多渗透过皮肤,Franz扩散池实验中,6 h后,经过大鼠皮肤进入接收池的sCT最大值约为总药量的(58.36±12.33)%,是游离sCT渗透数量的3.5倍。且在4,25和45 ℃下储存1个月后,Tat-sCT的稳定性也比游离鲑降钙素的要好。HOU等[6]使用一种无毒的富含精氨酸的细胞内传递(AID)多肽,以一种非融合蛋白和非共价结合依赖性的方式,促进多种蛋白质快速穿过皮肤组织,且在使用化学促渗剂之后渗透量会再次增加。并考察AID多肽促进蛋白渗透的机制,使用胞饮抑制剂EIPA和纤维状肌动蛋白阻聚剂CytD处理人A549细胞后发现蛋白转导量显著降低,这表明AID介导的入胞和经皮传递涉及巨胞饮和肌动蛋白重组。虽然目前机制尚不确定,但为疫苗、新药和蛋白质类药品提供一种简单有效的给药途径。随后,AHMAD等[17]采用相同的简单混合的方法,制备Pep-1/弹性蛋白复合物。并使用光子相关光谱和等温变性法考察分别在-20,4和25 ℃下储存的Pep-1/弹性蛋白复合物的生物物理学特性,使用荧光显微法和柯达FX Pro活体成像系统考察该复合物转导入细胞和皮肤组织的能力,发现Pep-1和弹性蛋白相互作用形成球状纳米颗粒。将该复合物用于小鼠皮肤1和3 h,并与皮下注射结果进行对比,发现两者渗透入皮肤的方式相似,随时间延长,渗透量增加,而弹性蛋白自身不能穿透皮肤,有力证明了CPPs的促渗效果。GENNARI等[18]筛选出一种合成七肽DRTTLTN,证明其能促进普通肝素(unfractionated heparin,UFN)透过皮肤的量。但与其他研究中将CPPs与物质简单混合不同,该实验发现简单混合不能促进UFN的渗透,而是需要亚胺盐酸盐和
除了以上的应用之外,还有很多研究者使用CPPs,通过静电作用或共价结合等方式对脂质体、囊泡等载体进行修饰后包载大分子蛋白、多肽类药物或中药提取物等,共同增进药物的透皮效果,加强药物的局部疗效。KWON等[19]采用硫醇-马来酰亚胺反应将细胞穿透肽结合在DOPC脂质体上,以改善不溶性萹蓄提取物的经皮效果,达到抗皱目的。若要药物能够对皮肤起到抗皱效果,药物应穿过角质层进入皮肤的真皮层。DOPC脂质体和CPP-DOPC脂质体的粒径均约为120 nm,包封率为83%;两者Zeta电位却显著不同,DOPC脂质体为-45 mV,而由于CPP上带有很多带正电的精氨酸,CPP-DOPC脂质体的Zeta电位为42 mV。用罗丹明B和异硫氰酸荧光素对所制备的脂质体进行标记,使用共聚焦激光扫描显微镜(CLSM)研究脂质体在Franz扩散池中通过雌性裸鼠背部皮肤(去毛去脂肪层)的扩散情况。采用两种不同极性的染料发现,随着时间的推移,亲脂性染料异硫氰酸荧光素分散在角质层细胞间脂质中,而CPP-DOPC脂质体处理过的皮肤组中,进入亲水核的亲水性染料罗丹明B则通过角质层,进入更深层的皮肤,证明CPP-DOPC脂质体能够促进药物穿透角质层,进入深层皮肤,从而达到药效。体内实验首先比较未使用UV照射小鼠(对照组)和使用UV照射小鼠背部的皱纹情况,证明UV照射能使小鼠背部皱纹数量增多加深。随后比较在同等UV照射下,使用溶剂溶解萹蓄提取物处理的小鼠、使用DOPC脂质体包载萹蓄提取物处理的小鼠和使用CPP-DOPC脂质体包载萹蓄提取物的小鼠背部皮肤皱纹数量、深度,皮肤的胶原蛋白、弹性蛋白、金属蛋白酶的数量,证明CPP-DOPC脂质体能够有效促进萹蓄提取物的经皮效果。PATLOLLA等[20]使用纳米液晶纳米粒(nano lipid crystal nanoparticles)包载荧光染料(DID-oil),将山嵛酸甘油酯、辛酸葵酸三酰甘油和DOGS-NTA-Ni脂质通过热熔均质法制备成脂质纳米粒,并在表面覆盖TAT多肽或对照YKA多肽。应用于小鼠皮肤上24 h后,在毛囊和上皮120 μm深处出现荧光,且荧光主要出现在毛囊中。共焦显微拉曼光谱法显示在80和120 μm处,TAT修饰的纳米粒荧光强度高于YKA多肽修饰的纳米粒。将荧光染料替换成塞来昔布,出现相似的情况,TAT纳米粒包载塞来昔布的促渗效果分别是YKA纳米粒包载塞来昔布和无修饰纳米粒包载的3倍和6倍。
很多研究者都研究过CPPs协助传递核酸药物进入细胞内。在这之前,由于寡核苷酸的大小和高度带负电荷的性质,核酸通过细胞膜的有效传递有些障碍,病毒载体是唯一克服寡核苷酸传递的细胞膜阻碍的工具,但它具有严重的致癌性和免疫原性。因此,带正电或两亲性的CPPs是寡核苷酸如siRNA全身性或局部给药的病毒载体的一种有效且毒性更小的替代选择[9,21]。但CPPs主要还是用于穿透肿瘤细胞或设计成靶向载体进入肿瘤细胞等方向。在经皮给药中运用得并不是很多,往往是被用于局部给药治疗某些皮肤疾病如过敏性皮炎。siRNA是过敏性皮炎的一种潜在疗法,因为它们能特异沉默过敏性皮炎基因表达的一种相关因子。虽然对于过敏性皮炎患者的皮肤来说,角质层已经被破坏,不足以维持正常的屏障作用,但表皮层中细胞之间的紧密连接对于siRNA的经皮给药来说依然是一个巨大的障碍。为了克服紧密连接的限制,UCHIDA等[22]使用一种合成的六肽AT1002联合Tat共同促进siRNA的经皮给药。其中,AT1002是一种紧密连接调节剂,能够可逆促进分子通过细胞间转运穿过表皮屏障。通过对PAM212细胞中RelA的mRNA沉默效应的考察,证明相比于无修饰的siRelA,Tat/siRelA复合物能够进入细胞。使用荧光免疫分析法分析AT1002对小鼠皮肤上一种紧密蛋白ZO-1结构的影响,证明AT1002能导致ZO-1蛋白发生络氨酸磷酸化,并且该反应可能是可逆的。将AT1002与Tat联合使用,发现siRNA抗核糖核酸酶的能力大大提升。且由于AT1002和Tat的共同作用,siRNA通过角质层细胞旁路途径从皮肤表面或者毛囊通道广泛地分布在皮肤中。随后,UCHIDA等[23]使用AT1002/Tat联合一种抗RelA的siRNA(小鼠皮肤上核因子-jB家族的一员)涂抹在患有AD的NC/Nga小鼠耳朵上,最终发现小鼠的耳朵厚度、皮炎症状、局部细胞因子水平,以及血清IgE产物量均有所改善。KANAZAWA等[24]使用一种功能性细胞穿透硬脂酰-寡肽OK-102(functional cell-penetrating stearoyl-oligopeptide OK-102),作为一种细胞质应答纳米载体递送新型治疗AD的RNA干扰剂(RNAi)——nkRNA和PnkRNA。相比于无包载的nkRNA和PnkRNA,该复合物能有效沉默巨噬细胞中的RelA mRNA,并在小鼠AD模型中表现出明显的治疗效果。VIJ等[25]在恒速的涡流下,通过向一种两亲性多肽Mgpe9溶液中逐滴加入质粒DNA的方法通过静电相互作用制备稳定的Mgpe9/质粒纳米复合物,并发现Mgpe9可能是通过短暂改变皮肤脂质分布、可逆破坏细胞间紧密连接的方式促进纳米复合物透过角质层进入上皮细胞中。并与市场上销售的促渗载体如Lipofectamine 2000TM进行比较,证明Mgpe9/质粒纳米复合物对皮肤和细胞的伤害比Lipofectamine 2000TM更小。
目前,关于细胞穿透肽如何穿透细胞膜进入细胞并没有确定的解释,但现在研究者们普遍对多数CPPs的细胞易位机制比较认同[26]。CPPs透过细胞膜主要有以下几个特点:①渗透量具有细胞系种类的依赖性,但是否渗透不具有细胞特异性;②与温度、时间、CPPs浓度相关;③CPP 与小分子的结合物的入胞机制是静电作用或易位、跨膜转导作用;而CPP 与大分子的结合物是通过能量依赖的内吞作用进入细胞等。马冬旭等[27]研究不同细胞系对细胞穿透肽摄取的不同效果,并比较它们的穿透机制。采用异硫氰酸荧光素标记CPPs,发现CPPs穿透细胞没有选择性,但摄取量与细胞种类有关。在采用的4 种肿瘤细胞系(MCF-7、MDA-MB-231、C6 和B16F10)中,MCF-7对CPPs的摄取量最大。而随细胞的孵化时间的延长,细胞内的荧光强度逐渐增强;且当增加CPPs的浓度时,细胞内的荧光强度也会明显增高。
但细胞穿透肽通过皮肤角质层的机制似乎与穿过细胞膜的机制并不完全相同。因为角质层中的细胞是死细胞,因此这些细胞只有代谢活性环境,而不会有内吞作用。只有某些由于皮炎而导致角质层缺损的经皮研究中,可以忽略角质层带来的促渗机制上的差异。除此之外,角质层和普通细胞膜在细胞间的脂域、脂质成分、含水量和脂质/蛋白质比率等方面也有很多不同点[9]。因此,可能代谢活性、角质层的非活性细胞和脂质和PTDs之间的相互作用在CPPs通过角质层运中起到重要作用[28-29]。HOU等[16]的研究已表明巨胞饮和肌动蛋白重组都涉及到CPPs促进药物经皮给药。此外,多项研究表明[22,25],CPPs能够可逆地作用于上皮细胞间的紧密连接蛋白,使得皮肤结构发生短暂变化,从而促进药物渗透。近期,WANG等[30]发现,上皮细胞上的Na+/K+-ATP酶β亚基(ATP1B1)对TD1(一种11个氨基酸残基构成的短肽)调节的药物促渗有重要作用。通过ELISA和免疫沉淀法验证在酵母和哺乳动物细胞中TD1与ATP1B1能特异性结合。TD1主要与ATP1B1的羧基端相互作用,不仅能够影响ATP1B1的表达和分布,还影响上皮的结构。该相互作用可被外源性ATP1B1竞争性拮抗,或被哇巴因(一种可以特异性抑制ATP1B1的抑制剂)抑制,这会导致大分子药物通过皮肤的递送量降低。该研究将CPPs促渗机制锁定到某一个分子的级别,对未来CPPs促渗研究有着重要的影响。但至今CPPs通过皮肤层的准确机制还未彻底阐明,需要进一步的研究来确认CPPs介导从非活性至活性皮肤层的转运机制。
CPPs作为一种短序列多肽,是具有生物活性的分子细胞内转运工具,转导效率高、低毒、使用方法简单、成本相对较低,对于通过皮肤的局部给药和全身性给药都有一定的促进作用,具有很多促渗方法所不具备的优点。但是由于目前还没有统一关于CPPs命名和质量控制的确定,因此目前众多的CPPs的经皮促渗效果参差不齐,没有形成一个比较完善的体系。且CPPs不具有细胞特异性,需通过其他手段辅助才能实现靶向性给药。此外,近年来发现,细胞穿透肽的应用面临着药物释放率、代谢降解、细胞系的分化状态和Rho-GTPases活性的依赖性等问题[31],这些都阻碍CPPs的发展。因此,研究者们还应加强对其机制和性能的研究,扩大CPPs在蛋白质、多肽、核酸以及各种载体中的经皮应用。
The authors have declared that no competing interests exist.
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[本文引用:1]
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ABSTRACT:68 The development of the human skin from intrauterine to extrauterine life is a balletic interplay of maturing layers and interlocking structures. We discuss this transition and then branch out to touch on issues of premature infant as well as neonatal skin care. Disruption of the barrier function due to toxins and development errors are expounded upon. Staph scalded skin syndrome, collodion membrane, bullous congenital ichthyosiform erythroderma, autosomal recessive ichthyosis (lamellar and congenital ichthyosiform erythroderma), and harlequin fetus are used as examples of these disruptions. Discussion of therapy with the authors’ experience highlights each disease.
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| [3] |
Abstract There is considerable interest in the skin as a site of drug application both for local and systemic effect. However, the skin, in particular the stratum corneum, poses a formidable barrier to drug penetration thereby limiting topical and transdermal bioavailability. Skin penetration enhancement techniques have been developed to improve bioavailability and increase the range of drugs for which topical and transdermal delivery is a viable option. This review describes enhancement techniques based on drug/vehicle optimisation such as drug selection, prodrugs and ion-pairs, supersaturated drug solutions, eutectic systems, complexation, liposomes, vesicles and particles. Enhancement via modification of the stratum corneum by hydration, chemical enhancers acting on the structure of the stratum corneum lipids and keratin, partitioning and solubility effects are also discussed. The mechanism of action of penetration enhancers and retarders and their potential for clinical application is described.
[本文引用:1]
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| [4] |
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| [5] |
Abstract The selective permeability of the plasma membrane prohibits most exogenous agents from gaining cellular access. Since many therapeutics and reporter molecules must be internalized for activity, crossing the plasma membrane is essential. A very effective class of transporters harnessed for this purpose are cell penetrating peptides (CPPs), a group of short cationic sequences with a remarkable capacity for membrane translocation. Since their discovery in 1988, CPPs have been employed for the delivery of a wide variety of cargo including small molecules, nucleic acids, antibodies and nanoparticles. This review describes recent advances in the use of CPPs for biological and therapeutic applications. In particular, an emphasis is placed on novel systems and insights acquired since 2006. Basic research on CPPs has recently yielded techniques that provide further information on the controversial mechanism of CPP uptake and has also resulted in the development of new model membrane systems to evaluate these mechanisms. In addition, recent use of CPPs for the development of new cellular imaging tools, biosensors, or biomolecular delivery systems have been highlighted. Lastly, novel peptide delivery vectors, designed to tackle some of the drawbacks of CPPs and enhance their versatility, will be described. This review will illustrate the diverse applications for which CPPs have been harnessed and also demonstrate the remarkable advancements these peptides have facilitated in cell biology.
[本文引用:1]
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| [6] |
Abstract The development of short-interfering RNA (siRNA) offers new strategies for manipulating specific genes responsible for pathological disorders. Myriad cationic polymer and lipid formulations have been explored, but an effective, non-toxic carrier remains a major barrier to clinical translation. Among the emerging candidates for siRNA carriers are cell penetrating peptides (CPPs), which can traverse the plasma membrane and facilitate the intracellular delivery of siRNA. Previously, a highly efficient and non-cytotoxic means of gene delivery was designed by complexing plasmid DNA with CPPs, then condensing with calcium. Here, the CPP TAT and a longer, 'double' TAT (dTAT) were investigated as potential carriers for siRNA. Various N/P ratios and calcium concentrations were used to optimize siRNA complexes in vitro. Upon addition of calcium, 'loose' siRNA/CPP complexes were condensed into small nanoparticles. Knockdown of luciferase expression in the human epithelial lung cell line A549-luc-C8 was high (up to 93%) with no evidence of cytotoxicity. Selected formulations of the dTAT complexes were dosed intravenously up to 1000 mg/kg with minimal toxicity. Biodistribution studies revealed high levels of gene knockdown in the lung and muscle tissue suggesting these simple vectors may offer a translatable approach to siRNA delivery. Published by Elsevier B.V.
[本文引用:2]
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| [7] |
Cell penetrating peptides hold considerable potential for academic and pharmaceutical remits with an interest in delivering macromolecules to the insides of cells. Hundreds of sequences now fall within the cell penetrating peptide classification and HIV-Tat, penetratin, transportan, and octaarginine represent extensively studied variants. The process by which membrane translocation is achieved has received significant interest in an aim to exploit new mechanistic knowledge to gain higher efficiency of penetration. There is evidence that many of the most well studied peptides are able to deliver themselves, relatively small cargo and possibly large macromolecular structures directly across the plasma membrane but there is also support for the involvement of an endocytic pathway or pathways. This review focuses on recent findings relating to experimental protocols and cell penetrating peptide modifications or extensions that yield significant effects on penetration capability. Relatively small changes in extracellular peptide concentrations, the inclusion or absence of serum from the incubation medium and the in vitro model exemplify variables that significantly influence the capacity of CPPs to penetrate membranes. Attachment of any type of cargo to these entities has the potential to affect their interaction with cells. There is increasing evidence to suggest that this is true for relatively small molecules such as fluorescent probes and hydrophobic adducts such as lipids and short peptide sequences designed as peptide therapeutics. Information gained from these findings will improve our knowledge of, and capacity to study the interactions of CPPs with cells, and this will accelerate their translation as efficient vectors from the in vitro setting into the clinical arena.
[本文引用:1]
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| [8] |
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| [9] |
In the last decade, almost one-third of the newly discovered drugs approved by the US FDA were biomolecules and biologics. Effective delivery of therapeutic biomolecules to their target is a challenging issue. Innovations in drug delivery systems have improved the efficiency of many of new biopharmaceuticals. Designing of novel transdermal delivery systems has been one of the most important pharmaceutical innovations, which offers a number of advantages. The cell-penetrating peptides have been increasingly used to mediate delivery of bimolecular cargoes such as small molecules, small interfering RNA nucleotides, drug-loaded nanoparticles, proteins, and peptides, both in vitro and in vivo, without using any receptors and without causing any significant membrane damage. Among several different drug delivery routes, application of cell-penetrating peptides in the topical and transdermal delivery systems has recently garnered tremendous attention in both cosmeceutical and pharmaceutical research and industries. In this review, we discuss history of cell-penetrating peptides, cell-penetrating peptide/cargo complex formation, and their mechanisms of cell and skin transduction.
[本文引用:4]
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| [10] |
The combined H II LC-CPE systems can serve as high potential vehicles for a variety of drugs, as they can easily be modified by varying the composition and temperature, according to the required dose and delivery features.
[本文引用:1]
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Abstract Glycerol monooleate (GMO)-based mesophases offer extensive prospects for incorporation of various bioactive molecules. This work deals with the solubilization of selected cell-penetrating peptides (CPPs) together with sodium diclofenac (Na-DFC) within the H(II) mesophase for transdermal applications. The effect of CPPs such as RALA (an amphipatic CPP), penetratin (PEN), and oligoarginine (NONA) on Na-DFC skin permeation kinetics to provide controlled release and tune the drug transdermal diffusion was studied. The location of the drug and the CPPs within the mesophase was probed by DSC and FTIR. Na-DFC was found to be located at the interfacial region between the surfactant chains, leading to denser H(II) mesophase. The hydrophilic NONA was intercalated into the aqueous cylinders and caused their swelling. It induced a significant decrease in the hydrogen binding between the GMO carbonyls and their surrounding. The amphiphilic PEN was entrapped within two different regions, depending on its concentration. PEN and NONA improved Na-DFC permeation by 100%, whereas RALA enhanced permeation by 50%. When estimating Na-DFC migration rate out of the mesophase toward surrounding aqueous media, it appeared to be slower with the CPPs. The peptides were not involved at this diffusion-controlled step. It seems that their effect on skin permeation is based on their specific interaction with the skin. 2011 American Chemical Society
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| [12] |
Sodium diclofenac (Na-DFC) and celecoxib (CLXB) are common nonsteroidal anti-inflammatory (NSAID) drugs which suffer from poor bioavailability and severe side effects when consumed orally, and their transdermal delivery might present important advantages. In this study, the drugs were solubilized in cubic and lamellar mesophases as transdermal delivery vehicles, and a cell-penetrating peptide, HIV-TAT (TAT), was examined as a skin penetration enhancer. SD-NMR, ATR-FTIR, and EPR measurements revealed that, in the cubic mesophase (which is rich in water content), TAT populates the aqueous cores and binds water, while in the dense lamellar system (with the lower water content) TAT is bound also to the glycerol monooleate (GMO) and increases the microviscosity and the order degree. TAT secondary structure in the cubic system was found to be a random coil while once it was embedded in the closely packed lamellar system it transforms to a more ordered compact state of 尾-turns arranged around the GMO headgroups. TAT remarkably increased the diffusion of Na-DFC and CLXB from the cubic systems by 6- and 9-fold enhancement, respectively. TAT effect on drug diffusion from the lamellar systems was limited to an increase of 1.3- and 1.7-fold, respectively. The dense packing and strong binding in the lamellar phase led to slow diffusion rates and slower drug release in controlled pattern. These effects of the chemical composition and vehicle geometry on drug diffusion are demonstrated with the impacts of TAT which can be specifically utilized for controlling skin delivery of drugs as required.
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| [13] |
The balance of efficacy and safety of topical corticosteroids (TCs) depends on their ability to penetrate into and be retained within the skin. Here, we evaluated the ability of SPACE64 peptide to enhance epidermal delivery and localization of three model TCs. In vitro and in vivo skin penetration studies were performed to evaluate penetration of TCs into and across the skin in the presence of various formulations of SPACE64 peptide. Topical formulations of corticosterone containing free SPACE64 peptide produced significantly enhanced epidermal penetration and localization. Ratio of drug deposition in the skin and receiver (efficacy/safety, indicative of ratio of local to systemic uptake) exhibited higher values for SPACE64 peptide-based formulation as compared to aqueous and hydroethanolic solutions and Cortizone64 cream. Mass spectrometry analysis showed that SPACE64 peptide associates with corticosterone, which may explain its enhanced retention effect. SPACE64 peptide also enhanced dermal retention of two more TCs (hydrocortisone and triamcinolone acetonide) compared to the vehicle control. An in vivo study in mice further established the ability of SPACE64 peptide to enhance skin retention of hydrocortisone without producing elevated blood concentrations. These results show that SPACE64 peptide is an effective additive to the formulation for enhanced skin localization of topical steroids.
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| [14] |
This study was designed to engineer a functional filaggrin (FLG) monomer linked to a cell-penetrating peptide (RMR) and to test the ability of this peptide to penetrate epidermal tissue as a therapeutic strategy for genetically determined atopic dermatitis (AD). A single repeat of the murine filaggrin gene ( Flg ) was covalently linked to a RMR motif and cloned into a bacterial expression system for protein production. Purified FLG+RMR (mFLG+RMR) was applied in vitro to HEK-293T cells and a reconstructed human epidermis (RHE) tissue model. Immunochemistry demonstrated RMR-dependent cellular uptake of FLG+RMR in a dose- and time-dependent manner in HEK cells. Immunohistochemical staining of the RHE model identified penetration of FLG+RMR to the stratum granulosum, the epidermal layer at which FLG deficiency is thought to be pathologically relevant. In vivo application of FLG+RMR to FLG-deficient flaky tail ( ft / ft ) mice skin demonstrated internalization and processing of recombinant FLG+RMR to restore the normal phenotype. These results suggest that topically applied RMR-linked FLG monomers are able to penetrate epidermal tissue, be internalized into the appropriate cell type, and be processed to a size similar to wild-type functional barrier peptides to restore necessary barrier function, and prove to be therapeutic for patients with AD.
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| [15] |
Context: Highly organized structure of stratum corneum (SC) is the major barrier of the delivery of macromolecules such as proteins and peptides across the skin. Recently, cell penetrating peptides (CPPs) such as HIV1-trans-activating transcriptional (Tat) have been used to enhance the topical delivery of proteins and peptides. Objective: This study aimed to enhance the transdermal absorption and chemical stability of salmon calcitonin (sCT) by co-incubation with Tat. Materials and methods: Tat-sCT mixture at 1: 1 molar ratio was prepared. Transdermal absorption and chemical stability of the mixture was evaluated in comparing with free sCT. Results: Tat-sCT mixture gave higher cumulative amounts and fluxes of sCT than free sCT. The maximum percentage of sCT of 58.36 +/- 12.33% permeated into the receiving chamber was found in Tat-sCT mixture at 6 h which was 3.50 folds of free sCT. Tat-sCT mixture demonstrated better sCT stability than sCT solution after 1 month storage at 4 C, 25 degrees C and 45 degrees C. Discussion: The positively-charged arginine groups in Tat might be responsible for the binding of peptide complexes to negatively charged cell surfaces by electrostatic interactions and also the translocation of sCT through the excised skin. Conclusion: This study demonstrated the enhancements of transdermal absorption and stability of sCT by Tat peptide with potential for further application in transdermal delivery of other therapeutic peptides.
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| [16] |
Abstract Plasma membranes of animal cells are generally impermeable to macromolecules. Protein transduction mediated by protein transduction domains (PTDs) covalently cross-linked to cargoes for cellular internalization has previously been demonstrated. Peptides with PTDs could be an effective way to deliver proteins into living cells or tissues in vitro. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to facilitate the delivery of proteins into animal cells and to penetrate skin tissues rapidly. This cellular internalization and transdermal delivery of proteins is mediated by non-toxic AID peptides in a non-fusion protein and non-conjugation dependent manner. The efficiency of intracellular transport is further increased in the presence of chemical enhancer oleic acid. The mechanism of the AID-mediated cellular entry may involve macropinocytosis and actin rearrangement. Thus, we confirm that direct delivery of bioactive proteins into living cells and tissues mediated by non-covalent actions of AID peptides represents a useful strategy in pharmaceutics, therapeutics and cosmetics.
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| [17] |
Transdermal drug delivery of proteins is challenging because the skin acts as a natural and protective barrier. Several techniques including using the cell-penetrating peptides have been studied to increase the penetration of therapeutic proteins into and through the skin. Cell-penetrating peptides facilitate and improve the transduction of large and hydrophilic cargo molecules through plasma membrane. We have recently reported an efficient skin delivery of elastin protein in complex with a cell-penetrating peptide called Pep-1. As the biophysical characteristics of cell-penetrating peptide/protein complexes have been linked with their biological responses, in this study, we investigated biophysical properties of Pep-1/elastin complexes (ratio 10:1) stored in three temperatures (-20C, 4C and 25C) by photon correlation spectroscopy, circular dichroism and isothermal denaturation. We also evaluated the ability of transduction of this complex into cells and skin tissue using both fluorescence microscopy and Kodak In-Vivo FX Pro Imaging System.
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| [18] |
Abstract This study aimed to identify a new skin penetrating peptide (SPP) able to enhance unfractionated heparin (UFH) permeation through human epidermis by screening a phage display peptide library. The effects of the synthesized heptapeptide (DRTTLTN) on human stratum corneum organization were investigated by ATR-FTIR spectroscopy and molecular dynamics simulation. The DRTTLTN penetration within the human epidermis caused both a fluidization of the stratum corneum lipids and the extension of keratins due to the increase of the contribution of -helices. The coadministration of DRTTLTN with UFH resulted ineffective in increasing skin penetration due to UFH affinity for keratins. The conjugation of DRTTLTN to UFH by N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride and sodium N-hydroxysulfosuccinimide led to an increase of the flux of 24-36-fold with respect to raw UFH, depending on the adopted synthetic procedure. The new compounds showed a decrease of the antifactor Xa activity of about 4-5 times. DRTTLTN also permitted to increase the fluxes of small model molecules. In conclusion, these data support the use of SPP to enhance the skin penetration of poorly absorbed compounds even in the case of macromolecules as polysaccharides.
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| [19] |
Abstract In this study, Polygonum aviculare L. extract, which has superior antioxidative and cellular membrane protective activity, was loaded onto cell penetrating peptide (CPP) conjugated liposomes to enhance transdermal delivery. The physical characteristics of typical liposomes and CPP-conjugated liposomes containing P. aviculare extract were evaluated. The particle sizes of both liposomes were approximately 150 nm. Whereas the zeta potential of typical liposomes was -45 mV, that of CPP-conjugated liposomes was +42 mV. The loading efficiency of P. aviculare extract in both liposomes was calculated to be about 83%. Fluorescent-labeled liposomes were prepared to evaluate cellular uptake and skin permeation efficiency. Using flow cytometry, we found that CPP-conjugated liposomes improved cellular uptake of the fluorescent dye as compared with the typical liposomes. In addition, the skin permeation of CPP-conjugated liposomes was proved higher than that of typical liposomes by confocal laser scanning microscopy studies and Franz diffusion cell experiments. The improved cellular uptake and skin permeation of the CPP-conjugated liposomes were due to the cationic arginine-rich peptide. In vivo studies also determined that the CPP-conjugated liposomes were more effective in depigmentation and anti-wrinkle studies than typical liposomes. These results indicate that the CPP-conjugated liposomes could be effective for transdermal drug delivery of antioxidant and anti-aging therapeutics. Copyright 2015 Elsevier B.V. All rights reserved.
[本文引用:1]
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| [20] |
The objective of the current study was to evaluate the ability of cell penetrating peptides (CPP) to translocate the lipid payload into the skin layers. Fluorescent dye (DID-oil) encapsulated nano lipid crystal nanoparticles (FNLCN) were prepared using Compritol, Miglyol and DOGS-NTA-Ni lipids by hot melt homogenization technique. The FNLCN surface was coated with TAT peptide (FNLCNT) or control YKA peptide (FNLCNY) and in vitro rat skin permeation studies were performed using Franz diffusion cells. Observation of lateral skin sections obtained using cryotome with a confocal microscope demonstrated that skin permeation of FNLCNT was time dependent and after 2402h, fluorescence was observed upto a depth of 12002μm which was localized in the hair follicles and epidermis. In case of FNLCN and FNLCNY formulations fluorescence was mainly observed in the hair follicles. This observation was further supported by confocal Raman spectroscopy where higher fluorescence signal intensity was observed at 80 and 12002μm depth with FNLCNT treated skin and intensity of fluorescence peaks was in the ratio of 2:1:1 and 5:3:1 for FNLCNT, FNLCN, and FNLCNY treated skin sections, respectively. Furthermore, replacement of DID-oil with celecoxib (Cxb), a model lipophilic drug showed similar results and after 2402h, the CXBNT formulation increased the Cxb concentration in SC by 3 and 6 fold and in epidermis by 2 and 3 fold as compared to CXBN and CXBNY formulations respectively. Our results strongly suggest that CPP can translocate nanoparticles with their payloads into deeper skin layers.
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| [21] |
Efficient delivery of therapeutic and diagnostic molecules to the cells and tissues is a difficult challenge. The cellular membrane is very effective in its role as a selectively permeable barrier. While it is essential for cell survival and function, also presents a major barrier for intracellular delivery of cargo such as therapeutic and diagnostic agents. In recent years, cell-penetrating peptides (CPPs), that are relatively short cationic and/or amphipathic peptides, received great attention as efficient cellular delivery vectors due to their intrinsic ability to enter cells and mediate uptake of a wide range of macromolecular cargo such as plasmid DNA (pDNA), small interfering RNA (siRNAs), drugs, and nanoparticulate pharmaceutical carriers. This review discusses the various uptake mechanisms of these peptides. Furthermore, we discuss recent advances in the use of CPP for the efficient delivery of nanoparticles, nanocarriers, DNA, siRNA, and anticancer drugs to the cells. In addition, we have been highlighting new results for improving endosomal escape of CPP-cargo molecules. Finally, pH-responsive and activable CPPs for tumor-targeting therapy have been described.
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| [22] |
Abstract Topical use of small interfering RNA (siRNA) as a therapeutic nucleic acid is increasingly studied for the treatment of skin diseases and for the improvement of skin properties. However, naked siRNA transdermal delivery is limited by its low stability in the body and low permeability into target cells. This is due to various skin barriers such as the stratum corneum that has multiple lipid bilayers and epidermal layers that have tight junctions. In this study, we investigate non-invasive transdermal siRNA delivery using two functional peptides: AT1002, which is a tight junction modulator and 6-mer synthetic peptide belonging to a novel class of compounds that reversibly increases paracellular transport of molecules across the epithelial barrier; and Tat, which is a cell-penetrating peptide applicable as a transdermal siRNA delivery enhancer. We examined whether expression of the tight junction protein zonula occludens protein 1 (ZO-1) was detected in mouse skin applied with AT1002. Additionally, siRNA stabilities for RNaseA using Tat and AT1002 were assessed. We also determined the intradermal delivery efficiency of siRNA using functional peptides by confocal laser microscopy of fluorescently labeled siRNA in mouse skin. We found that the Tat analog and AT1002 strongly increased siRNA stability against RNaseA. In addition, ZO-1 disappeared from the skin after treatment with AT1002, yet recovered with time after washing. Finally, we also found that Tat and AT1002 peptides accelerate transdermal siRNA delivery both widely and effectively. Thus, combination of Tat and AT1002 is expected to be a transdermal delivery enhancer of siRNA. 2011 Pharmaceutical Society of Japan
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| [23] |
Abstract Atopic dermatitis (AD) has high morbidity and poor prognosis because safe and effective treatments are scarce. Recently, short interfering RNA (siRNA) has shown promise as an effective treatment for targeting specific aberrantly expressed genes. However, naked siRNAs are too inefficient because of various enzymatic, membrane, and cellular barriers. We previously reported that a Tat analog acting as a cell-penetrating peptide, combined with AT1002, which reversibly increases paracellular transport of molecules across the epidermal barrier in epidermis-disrupted mice and enhances the skin permeation of water-soluble siRNA. In the present study, to develop a novel treatment for AD, we determined the intradermal permeation of siRNAs and the antiallergic effects of a siRNA that silences RelA, a member of the nuclear factor-B family, using Tat and AT1002 peptides in an AD mouse model. We first showed that the Tat analog and AT1002 delivered siRNA into the skin of ICR mice and, upon topical application to the AD-induced ears of NC/Nga mice, changed zonula occludens protein 1 expression. In addition, the silencing effects on the mRNA of RelA in JAWS II cells transfected with siRNA oligonucleotides for mouse RelA, complexed with Tat, were as effective as a commercial vector. Furthermore, the ear thickness, clinical skin severity, topical cytokine levels, and serum IgE production in AD model mice treated with anti-RelA siRNA with Tat and AT1002 were improved.
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| [24] |
Small interfering RNAs (siRNAs) are a potential treatment of atopic dermatitis (AD) because they can specifically silence the gene expression of AD-related factors. However, siRNA alone cannot exert a sufficiently strong therapeutic effect due to low delivery efficiency to the target tissues and cells; simply increasing the amount used is not possible due to the possibility of off-target effects. We previously reported a novel class of therapeutic RNA interference (RNAi) agents called nkRNA 03; and PnkRNA 03; , which have been shown to be effective in several disease models, have greater resistance to nuclease degradation than canonical siRNAs, and do not induce any immunotoxicity. In the present study, we describe a non-invasive and effective transdermal RNAi therapeutic system for atopic dermatitis that uses the functional cell-penetrating stearoyl-oligopeptide OK-102 as a cytoplasm-responsive nanocarrier for nkRNA 03; and PnkRNA 03; . The two RNAi agents were targeted against RelA, a subclass of NF-κB (nuclear factor kappa B), and, as part of OK-102 complexes, they strongly silenced RelA mRNA in macrophage cells and demonstrated a significant therapeutic effect in a mouse model of AD. It was shown that OK-102-complexed RNAi agents were an efficient therapeutic system for AD and caused no adverse reactions.
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| [25] |
Topical delivery to skin is an essential step in non-invasive application of nucleic acid therapeutics for cutaneous disorders. The barrier posed by different layers of the skin – stratum corneum on top followed by the viable epidermis below – makes it extremely challenging for large hydrophilic molecules like nucleic acids to efficiently enter the uncompromised skin. We report an amphipathic peptide Mgpe9 (CRRLRHLRHHYRRRWHRFRC) that can penetrate the uncompromised skin, enter skin cells and deliver plasmid DNA efficiently as nanocomplexes in vitro and in vivo without any additional physical or chemical interventions prevalent currently. We observe efficient gene expression up to the highly proliferating basal layer of the skin without observable adverse reactions or toxic effects after delivery of reporter plasmids. The entry mechanism of nanocomplexes possibly involves reversible modulation of junction proteins accompanied by transient changes in skin structure. This peptide holds potential to be used as an efficient transporter of therapeutic nucleic acids to the skin.
[本文引用:2]
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| [26] |
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| [27] |
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| [28] |
ABSTRACT Topical or transdermal drug delivery is challenging because the skin acts as a natural and protective barrier. Therefore, several methods have been examined to increase the permeation of therapeutic molecules into and through the skin. One approach is to use the nanoparticulate delivery system. Starting with liposomes and other vesicular systems, several other types of nanosized drug carriers have been developed such as solid lipid nanoparticles, nanostructured lipid carriers, polymer-based nanoparticles and magnetic nanoparticles for dermatological applications. This review article discusses how different particulate systems can interact and penetrate into the skin barrier. In this review, the effectiveness of nanoparticles, as well as possible mode of actions of nanoparticles, is presented. In addition to nanoparticles, cell-penetrating peptide (CPP)-mediated drug delivery into the skin and the possible mechanism of CPP-derived delivery into the skin is discussed. Lastly, the effectiveness and possible mechanism of CPP-modified nanocarriers into the skin are addressed.
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| [29] |
Abstract Many systemically effective drugs such as cyclosporin A are ineffective topically because of their poor penetration into skin. To surmount this problem, we conjugated a heptamer of arginine to cyclosporin A through a pH-sensitive linker to produce R7-CsA. In contrast to unmodified cyclosporin A, which fails to penetrate skin, topically applied R7-CsA was efficiently transported into cells in mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation. These data establish a general strategy for enhancing delivery of poorly absorbed drugs across tissue barriers and provide a new topical approach to the treatment of inflammatory skin disorders.
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| [30] |
DOI:10.1021/mp500789h
URL
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