阳离子环肽对胰岛素经皮和跨膜促渗作用研究
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摘要
经皮给药系统具有维持药物血药浓度稳定,避免口服给药引起的肝脏首过效应,给药方便等优点。亲水性大分子药物很难透过皮肤产生药效,目前主要以注射的方式给药,导致患者适应性较差。因此亲水性大分子经皮肤的有效跨膜转运研究具有重要意义。
文献报道环肽TD-1(ACSSSPSKHCG)可以促进胰岛素的经皮吸收。由于富含阳离子氨基酸的细胞穿膜肽具有跨膜转运能力,因此本研究以TD-1为骨架采用阳离子氨基酸精氨酸或赖氨酸扫描的方法设计合成了系列阳离子环肽,发现在设计的系列环肽中,N-5位是阳离子环肽的结构改造位点,其中N-5和N-6位双赖氨酸取代的阳离子环肽TD-34(ACSSKKSKHCG)表现出最好的经皮和跨膜促渗活性。5μmol/mL TD-34和21IU/mL胰岛素的混合溶液分别进行了在体经皮输送和跨膜转运实验,经皮给药8h后,糖尿病大鼠血糖浓度下降为初始值的26%；跨膜转运2h后,胰岛素跨Caco-2细胞单层膜(BL→AP)的Papp值从0.27×10-6cm/s增加到0.69×10-6cm/s。对系列环肽的经皮和跨膜促渗能力比较,发现不同环肽作用下胰岛素在Caco-2细胞单层膜外排侧(BL→AP)的转运能力与在体经皮吸收能力具有较好的相关性(R2=0.73),因此Caco-2细胞单层膜的外排侧可以用于阳离子环肽对胰岛素经皮促渗活性的评价。
采用原子力显微镜(AFM)轻敲模式观察大气环境下Caco-2细胞膜的微观形态变化,发现阳离子环肽TD-34(ACSSKKSKHCG)作用于Caco-2细胞膜后,Caco-2细胞膜表面失去原有的平整性和光滑性,细胞膜表面粗糙度较未处理组提高91%,推测该变化可能与TD-34内吞进入细胞过程有关。在跨膜转运实验中,与未处理组比较胞吞抑制剂处理组TD-34跨膜转运量下降约49%,进一步证明了TD-34主要以内吞的方式跨膜转运。
分别进行了三组胰岛素跨膜转运实验(对照组,胞吞抑制剂组,4℃组),空白对照组胰岛素跨细胞膜两侧转运的Papp值比值(PDR)明显高于1.5,说明胰岛素跨膜转运存在主动转运过程,同时这一转运过程并没有随着实验温度由37℃下降到4℃或胞吞抑制剂(氧化苯砷)的作用而完全被抑制,因此推测胰岛素跨Caco-2细胞单层膜的转运包括了被动扩散在内的多种转运方式。此外,采用促渗肽TD-34处理的Caco-2细胞单层膜阻值呈迅速下降趋势,去除TD-34后Caco-2细胞单层膜的阻值逐渐恢复,整个过程中的Caco-2细胞单层膜电阻值呈现可逆性的变化。免疫组化研究表明Caco-2细胞单层膜电阻值的变化与紧密连接蛋白Claudin-1的荧光的消失与重现具有密切相关性,该结果有力的证明了TD-34通过可逆地松弛Caco-2细胞单层膜间的紧密连接促进了胰岛素的细胞旁路转运。
综合以上研究得到如下结论：在阳离子环肽中阳离子基团的含量和位置决定了环肽对胰岛素经皮和跨膜促渗的能力,Caco-2细胞单层膜外排侧可用于预测环肽对胰岛素的经皮和跨膜促渗能力,机理研究证明TD-34通过可逆的松弛紧密连接蛋白增加胰岛素的细胞旁路转运。该结论为今后研究生物大分子经皮输送促渗技术提供了重要的理论依据。
The advantages of transdermal delivery system are as follows:It can provide constant blood levels in the plasma; It can provide first-pass metabolism in the gastrointestinal tract and liver; It is suitable for patients who are unconscious or vomiting and rely on self-administration and so on. The biomolecules are usually taken as an injected way, so effective delivery of therapeutic biomolecules across skin and biomembranes is a challenging topic.
Transdermal peptide (TD-1) has exhibited enhancement activity on insulin transdermal delivery. As cell penetrating peptides were rich in cationic amino acids, a series of cationic cyclopeptides based on the sequence of TD-1(ACSSSPSKHCG) were designed by partial arginine or lysine scan method. Among these peptides, TD-34(ACSSKKSKHCG) with bis-substituted lysine in N-5and N-6showed the best transdermal and transmembrane enhancement activity, the blood glucose level lower to about26%of initial after transdermal administrating21IU/mL insulin with5μmol/mL TD-34for8h to diabetic rats in vivo, and the Papp value of insulin in basolateral side of Caco-2cell monolayers was increased from0.27×10-6cm/s to0.69×10-6cm/s. In addition, the transmembrane permeability in Caco-2cell monolayers (BL→AP) exhibited preferable correlation with percutaneous absorption of insulin (R2=0.73), it is suggested that Caco-2cell monolayers can be used in assessing the transdermal enhancement activity of cationic cyclopeptides.
AFM was used to observe the microstructure of Caco-2cell membrane treated with a cationic cyclopeptide (TD-34) at air condition and tapping mode using AFM. Results showed that the surface of Caco-2cell membrane treated with TD-34became rough, the mean square roughness increased91%, which implied the increased roughness of Caco-2cell membrane might be correlated with endocytosis process of TD-34. Transportation of TD-34decreased49%when Caco-2cell monolayers were treated with an endocytosis inhibitor. This result further testified TD-34transported across Caco-2cell monolayers by an endocytosis route.
Transport studies (control group, endocytosis inhibitor group,4℃group) of insulin were did. PDR of insulin was large than1.5in control group which indicated insulin transported across Caco-2cell monoayers by an active process. But when an endocytosis inhibitor (PAO) was added or temperature was decreased from37℃to4℃, Papp value of insulin was not totally inhibited. This result indicated that insulin transported across Caco-2cell monolayers by mutiple routes including passive diffusion. In addition, transepithelial electrical resistance (TEER) value was monitored for24h immediately after the beginning of transport experiments. Moreover, the tight junction protein (Claudin-1) was localized by confocal immunofluorescence microscopy. Results showed the transport of insulin alone across biomembranes was attributable to multiple routes including passive diffusion. When TD-34was treated on Caco-2cell mono layers, TEER values decreased reversibly, and it was correlated with the reappearance of tight junction proteins by immunostaining assay. This result testified TD-34enhanced insulin delivery by loosening tight junction in a reversible way.
It is concluded that appropriate content and position of cationic group in cyclopeptides may improve percutaneous absorption and transmembrane ability of insulin, and Caco-2cell monolayers (BL→AP) might be applied to predict the percutaneous absorption of insulin chaperoned by a cyclopeptide in vivo. The mechanism studies testified that the cationic cyclopeptide (TD-34) had the potential to enhance paracellular delivery of insulin across Caco-2cell monolayers by loosening tight junction in a reversible way. This work provided an important theoretically basis for researching the transdermal or transmembrane enhancement techniques for biomacromolecules.
文献报道环肽TD-1(ACSSSPSKHCG)可以促进胰岛素的经皮吸收。由于富含阳离子氨基酸的细胞穿膜肽具有跨膜转运能力,因此本研究以TD-1为骨架采用阳离子氨基酸精氨酸或赖氨酸扫描的方法设计合成了系列阳离子环肽,发现在设计的系列环肽中,N-5位是阳离子环肽的结构改造位点,其中N-5和N-6位双赖氨酸取代的阳离子环肽TD-34(ACSSKKSKHCG)表现出最好的经皮和跨膜促渗活性。5μmol/mL TD-34和21IU/mL胰岛素的混合溶液分别进行了在体经皮输送和跨膜转运实验,经皮给药8h后,糖尿病大鼠血糖浓度下降为初始值的26%；跨膜转运2h后,胰岛素跨Caco-2细胞单层膜(BL→AP)的Papp值从0.27×10-6cm/s增加到0.69×10-6cm/s。对系列环肽的经皮和跨膜促渗能力比较,发现不同环肽作用下胰岛素在Caco-2细胞单层膜外排侧(BL→AP)的转运能力与在体经皮吸收能力具有较好的相关性(R2=0.73),因此Caco-2细胞单层膜的外排侧可以用于阳离子环肽对胰岛素经皮促渗活性的评价。
采用原子力显微镜(AFM)轻敲模式观察大气环境下Caco-2细胞膜的微观形态变化,发现阳离子环肽TD-34(ACSSKKSKHCG)作用于Caco-2细胞膜后,Caco-2细胞膜表面失去原有的平整性和光滑性,细胞膜表面粗糙度较未处理组提高91%,推测该变化可能与TD-34内吞进入细胞过程有关。在跨膜转运实验中,与未处理组比较胞吞抑制剂处理组TD-34跨膜转运量下降约49%,进一步证明了TD-34主要以内吞的方式跨膜转运。
分别进行了三组胰岛素跨膜转运实验(对照组,胞吞抑制剂组,4℃组),空白对照组胰岛素跨细胞膜两侧转运的Papp值比值(PDR)明显高于1.5,说明胰岛素跨膜转运存在主动转运过程,同时这一转运过程并没有随着实验温度由37℃下降到4℃或胞吞抑制剂(氧化苯砷)的作用而完全被抑制,因此推测胰岛素跨Caco-2细胞单层膜的转运包括了被动扩散在内的多种转运方式。此外,采用促渗肽TD-34处理的Caco-2细胞单层膜阻值呈迅速下降趋势,去除TD-34后Caco-2细胞单层膜的阻值逐渐恢复,整个过程中的Caco-2细胞单层膜电阻值呈现可逆性的变化。免疫组化研究表明Caco-2细胞单层膜电阻值的变化与紧密连接蛋白Claudin-1的荧光的消失与重现具有密切相关性,该结果有力的证明了TD-34通过可逆地松弛Caco-2细胞单层膜间的紧密连接促进了胰岛素的细胞旁路转运。
综合以上研究得到如下结论：在阳离子环肽中阳离子基团的含量和位置决定了环肽对胰岛素经皮和跨膜促渗的能力,Caco-2细胞单层膜外排侧可用于预测环肽对胰岛素的经皮和跨膜促渗能力,机理研究证明TD-34通过可逆的松弛紧密连接蛋白增加胰岛素的细胞旁路转运。该结论为今后研究生物大分子经皮输送促渗技术提供了重要的理论依据。
The advantages of transdermal delivery system are as follows:It can provide constant blood levels in the plasma; It can provide first-pass metabolism in the gastrointestinal tract and liver; It is suitable for patients who are unconscious or vomiting and rely on self-administration and so on. The biomolecules are usually taken as an injected way, so effective delivery of therapeutic biomolecules across skin and biomembranes is a challenging topic.
Transdermal peptide (TD-1) has exhibited enhancement activity on insulin transdermal delivery. As cell penetrating peptides were rich in cationic amino acids, a series of cationic cyclopeptides based on the sequence of TD-1(ACSSSPSKHCG) were designed by partial arginine or lysine scan method. Among these peptides, TD-34(ACSSKKSKHCG) with bis-substituted lysine in N-5and N-6showed the best transdermal and transmembrane enhancement activity, the blood glucose level lower to about26%of initial after transdermal administrating21IU/mL insulin with5μmol/mL TD-34for8h to diabetic rats in vivo, and the Papp value of insulin in basolateral side of Caco-2cell monolayers was increased from0.27×10-6cm/s to0.69×10-6cm/s. In addition, the transmembrane permeability in Caco-2cell monolayers (BL→AP) exhibited preferable correlation with percutaneous absorption of insulin (R2=0.73), it is suggested that Caco-2cell monolayers can be used in assessing the transdermal enhancement activity of cationic cyclopeptides.
AFM was used to observe the microstructure of Caco-2cell membrane treated with a cationic cyclopeptide (TD-34) at air condition and tapping mode using AFM. Results showed that the surface of Caco-2cell membrane treated with TD-34became rough, the mean square roughness increased91%, which implied the increased roughness of Caco-2cell membrane might be correlated with endocytosis process of TD-34. Transportation of TD-34decreased49%when Caco-2cell monolayers were treated with an endocytosis inhibitor. This result further testified TD-34transported across Caco-2cell monolayers by an endocytosis route.
Transport studies (control group, endocytosis inhibitor group,4℃group) of insulin were did. PDR of insulin was large than1.5in control group which indicated insulin transported across Caco-2cell monoayers by an active process. But when an endocytosis inhibitor (PAO) was added or temperature was decreased from37℃to4℃, Papp value of insulin was not totally inhibited. This result indicated that insulin transported across Caco-2cell monolayers by mutiple routes including passive diffusion. In addition, transepithelial electrical resistance (TEER) value was monitored for24h immediately after the beginning of transport experiments. Moreover, the tight junction protein (Claudin-1) was localized by confocal immunofluorescence microscopy. Results showed the transport of insulin alone across biomembranes was attributable to multiple routes including passive diffusion. When TD-34was treated on Caco-2cell mono layers, TEER values decreased reversibly, and it was correlated with the reappearance of tight junction proteins by immunostaining assay. This result testified TD-34enhanced insulin delivery by loosening tight junction in a reversible way.
It is concluded that appropriate content and position of cationic group in cyclopeptides may improve percutaneous absorption and transmembrane ability of insulin, and Caco-2cell monolayers (BL→AP) might be applied to predict the percutaneous absorption of insulin chaperoned by a cyclopeptide in vivo. The mechanism studies testified that the cationic cyclopeptide (TD-34) had the potential to enhance paracellular delivery of insulin across Caco-2cell monolayers by loosening tight junction in a reversible way. This work provided an important theoretically basis for researching the transdermal or transmembrane enhancement techniques for biomacromolecules.
引文
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