Cell-Penetrating Peptides Enhance the Activity of Human Fibroblast Growth Factor 2 by Prolonging the Retention Time: A New Vision for Drug-Delivery Systems

Abstract

Cell-penetrating peptides (CPPs) are defined by their ability to deliver cargo into cells and have been studied and developed as a promising drug-delivery system (DDS). However, the issue of whether the CPPs that have already entered the cells can be re-released or reused has not been studied. The purpose of this research was to construct CPP-conjugated human fibroblast growth factor 2 (hFGF2) and investigate whether they can be re-released from the cell membrane for reuse. This study combined hFGF2 with Tat or Ara27, a newly developed CPP derived from the zinc knuckle (CCHC-type) family protein of Arabidopsis. Human dermal fibroblast (HDF) was treated with Tat-conjugated hFGF2 (tFGF2) and Ara27-conjugated hFGF2 (NR-FGF2) for both long and short durations, and the effects on cell growth were compared. Furthermore, tFGF2 and NR-FGF2 re-released from the cells were quantified and the effects were evaluated by culturing HDF in a conditioned medium. Interestingly, the proliferation of HDF increased only when NR-FGF2 was treated for 1 h in endocytosis-independent manner. After 1 h, NR-FGF2 was significantly re-released, reaching a maximum concentration at 5 h. Furthermore, increased proliferation of HDF cultured in the conditioned medium containing re-released NR-FGF2 was discovered. While previous studies have focused on the delivery of cargo and its associated applications, this study has revealed that combinations of superior CPPs and therapeutics can be expected to prolong both the retention time and the cell-penetrating capacity, even in the presence of external factors. Therefore, CPPs can be applied in the context of topical drugs and cosmetics as a new DDS approach.

Cellular internalization effect of Ara27 in various cell lines

“…In our previous study, Ara27-conjugated human fibroblast growth factor 2 penetrated HDF and re-released from the cells after 1 h treatment and an HDF condition medium increased the proliferation of HDF (Lee et al, 2020). CPP can also deliver nucleic acid such as DNA, mRNA and siRNA (Meade et al, 2007;Srimanee et al, 2018;Yokoo et al, 2021;Mahjoubin-Tehran et al, 2022) and can mediate anti-cancer effects (Klimpel et al, 2019).…”

Photothermally Modulated Intracellular Delivery of Therapeutic Proteins Using Gold-Collagen Hybrid Nanoparticles

“…Among these, CPPs are small peptide sequences consisting of fewer than 30 amino acids that have been extensively applied in preclinical research for disease diagnosis and therapy. , Introducing CPPs to the cargo can achieve effective cellular delivery because they can enter the cells in a noninvasive manner via various endocytosis pathways. − Moreover, CPPs do not usually destroy the plasma membrane structure. In previous studies, the conjugation of Ara27 CPP, an amphipathic CPP, to FGF2 significantly increased cell permeability. , However, the peptide alone can degrade and lose its function under delivery conditions, owing to its relatively poor stability.…”

“…In previous studies, the conjugation of Ara27 CPP, an amphipathic CPP, to FGF2 significantly increased cell permeability. 19,20 However, the peptide alone can degrade and lose its function under delivery conditions, owing to its relatively poor stability.…”