Imunofan—RDKVYR Peptide—Stimulates Skin Cell Proliferation and Promotes Tissue Repair

Abstract

Regeneration and wound healing are vital to tissue homeostasis and organism survival. One of the biggest challenges of today’s science and medicine is finding methods and factors to stimulate these processes in the human body. Effective solutions to promote regenerative responses will accelerate advances in tissue engineering, regenerative medicine, transplantology, and a number of other clinical specialties. In this study, we assessed the potential efficacy of a synthetic hexapeptide, RDKVYR, for the stimulation of tissue repair and wound healing. The hexapeptide is marketed under the name “Imunofan” (IM) as an immunostimulant. IM displayed stability in aqueous solutions, while in plasma it was rapidly bound by albumins. Structural analyses demonstrated the conformational flexibility of the peptide. Tests in human fibroblast and keratinocyte cell lines showed that IM exerted a statistically significant (p < 0.05) pro-proliferative activity (30–40% and 20–50% increase in proliferation of fibroblast and keratinocytes, respectively), revealed no cytotoxicity over a vast range of concentrations (p < 0.05), and had no allergic properties. IM was found to induce significant transcriptional responses, such as enhanced activity of genes involved in active DNA demethylation (p < 0.05) in fibroblasts and activation of genes involved in immune responses, migration, and chemotaxis in adipose-derived stem cells derived from surgery donors. Experiments in a model of ear pinna injury in mice indicated that IM moderately promoted tissue repair (8% in BALB/c and 36% in C57BL/6 in comparison to control).

Functionalized Peptide Fibrils as a Scaffold for Active Substances in Wound Healing

“…The aim of our project was to develop new peptide scaffolds and to test their physicochemical and biological properties in the field of skin wound regeneration. For this purpose, we designed peptide constructs that were a hybrid of three interconnected sequences, namely, a self-assembling QAGIVV fragment, named FC in this work corresponding to the steric zipper sequence from a human protein—cystatin C, with shuffled residues to ensure high fibrillization propensities [ 31 , 32 ]; AAPV, which is sensitive to neutrophil elastase; and biologically active RDKVYR [ 33 ] or KGHK or GHK [ 34 , 35 , 36 , 37 ] sequences, which show propensities toward skin wound regeneration. The individual segments were connected by linkers consisting of three glycine residues ( Figure 2 ).…”

“…The aggregation tests, ThT assay and microscopic techniques (TEM and AFM) confirmed that the FC peptide forms stable fibrils, and thus can be a scaffold for sequence modifications and further research. We then included this fibrillogenic fragment in new hybrid compounds, which also contained an elastase-specific sequence (AAPV) and three pro-regenerative sequences: (i) GHK [ 36 , 37 ]; (ii) KGHK [ 34 ] and (iii) RDKVYR [ 33 ]. Aggregation tests were also performed for these peptides.…”

“…As an in vitro model, we chose immortalized human cell lines, namely HaCaT keratinocytes and 46BR.1N fibroblasts, which constitute a reliable model for the initial assessment of peptide activity in wound healing. This model was successfully used to evaluate the activity of different peptides, e.g., IM or PDGF peptide derivatives, in our previous works [ 33 , 57 , 58 ]. The analyses performed using the LDH test did not show any cytotoxic effect for any of the tested free peptides or fibrils.…”

“…MK401) was measured to determine the cytotoxicity of the tested compounds, and the XTT assay was used to evaluate their effect on cell proliferation. Both tests were performed as previously described [ 33 , 57 ] according to manufacturers’ instructions. Briefly, cells were seeded into 96-well plates, at a density of 5000 cells per well, in DMEM supplemented with 10% FBS.…”

Short peptide sequences: current knowledge and future prospects

“…Peptidergic regulation of homeostasis is supposed to be phylogenetically the oldest form of bioregulation [2]. Being biocompatible, biodegradable, bioactive, and relatively amenable to large-scale production, peptides are currently gaining interest as potential agents in different fields of medicine [4]. Peptide drugs have obvious advantages over the non-peptide drugs such as high activity, low toxicity since they consist of endogenous amino acids, the absence of severe side effects, and lower probability of tolerance or dependence [5].…”