Researches On Cationic Cyclopeptides For Enhancing Insulin Transdermal And Transmembrane Delivery

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 (BLâ†'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 (BLâ†'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.