Design and Synthesis of New Cell Penetrating Peptides: Diffusion and Distribution Inside the Cornea

Abstract

The role of cell penetrating peptides (CPPs) has been challenged in recent years for drug delivery to ocular tissues for the targeting of both anterior and posterior segments. The enhancement of trans-corneal transport for anterior segment targeting is a very important issue possibly leading to important outcomes on efficacy and to the opportunity of topical administration of molecules with unfavorable penetration properties. The aim of the present work was the design and synthesis of new CPPs, deriving from the structure of PEP-1 peptide. Synthesized peptides were labeled with 5-carboxyfluorescein (5-FAM), and their diffusion behavior and distribution inside the cornea were evaluated by a validated ex vivo model and a confocal microscopy approach. Newly synthesized peptides showed similar corneal permeation profiles as PEP-1 (P = 0.75 ± 0.56 × 10 cm/s), about 2.6-fold higher than 5-FAM (P = 0.29 ± 0.08 × 10 cm/s) despite the higher molecular weight. Confocal microscopy experiments highlighted the tendency of PEP-1 and its derived peptides to localize in the intercellular space and/or in the plasma membrane. Noteworthy, using penetratin as positive control, a higher trans-corneal permeation (P = 6.18 ± 1.46 × 10 cm/s) was evidenced together with a diffusion by intracellular route and a different accumulation between wings and basal epithelial cells, probably depending on the stage of cell development. Finally, PEP-1 and pep-7 proved to be safe and well tolerated when tested on human conjuctival cell line.

ABCA1 Regulates IOP by Modulating Cav1/eNOS/NO Signaling Pathway

“…5A; Supplementary Table S1). The LogP of GW3965 is 5.9, 22,23 which is much higher than the maximum optimal LogP for cornea permeation, that is, between 1 and 3. Topical application of 100 μM GW3965 did not change the IOP (n = 4, P > 0.05) ( Supplementary Fig.…”

Synthesis and Ex Vivo Trans-Corneal Permeation of Penetratin Analogues as Ophthalmic Carriers: Preliminary Results

“…Finally, peptides were purified and characterized by RP-HPLC and mass spectrometry as previously reported [ 6 ]. Analytical data are shown in Supplementary Materials (Table S1 and Figures S1–S15) .…”

“…Nevertheless, once applied ex vivo to porcine cornea, all PEP-1 analogues and PEP-1 itself, were not able to match PNT, chosen as reference CPP, demonstrating its superiority in both permeation across and distribution within corneal layers. Moreover the synthesized peptides and PEP-1 itself showed a preferential paracellular penetration route in comparison with the intracellular penetration route of PNT [ 6 ]. Therefore, according to these evidences, the aim of the present paper is the design and synthesis of new PNT analogues by the mimotopic approach and the study of their capability to cross porcine cornea ex vivo.…”

Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization

“…3 Positively charged functional groups, particularly guanidinium groups, are essential for total cellular uptake for most CPPs, and this property is also related to cytosolic localization. [13][14][15][16][17][18][19][20][21][22][23] However, amphipathic, hydrophobic, or even anionic CPPs have also been described. 24,25 Although there has been quite an effort to clarify the mechanism(s) by which CPPs translocate to the inside of a cell, it remains elusive how exactly a particular peptide or peptide-cargo complex crosses the membrane.…”