Cell-penetrating peptides (CPPs) have attracted great interest as delivery vehicles in medicine, with potential for the development of novel therapeutic agents or cosmetic products. Biological membranes are typically waterproof to almost all compounds with a molecular weight greater than 500 Da.

In a recent study, Mareike Horn and Ines Neundorf covalently coupled to wound-healing promoting sequence (Tylotoin) with a cell-penetrating peptide to improve covalently coupled delivery of the drug across cell membranes. Cellular uptake of these novel peptide conjugates into keratinocytes was observed with internalization studies to show significantly improved delivery with good tolerability.

The promising novel peptide chimera (Tylotoin-sC18 *) was investigated in the study in a scratch wound closure assay to understand its wound healing capacity. The results indicated that the fusion of Tylotoin with a peptide did not cause a loss of its activity. In addition, its proliferative impact on keratinocytes was observed as an important step in the process of wound healing.

Tylotoin-sC18 *

Tylotoin-sC18 * exhibited strong antimicrobial activities to prevent bacterial infection in the wound. As a result of Their multiple functions, the authors assume the novel peptide can be used Chimera to treat infected wounds as an agent in the future. The results are now published on  Scientific Reports.

Tremendous efforts have been made in recent years on the use of CPPs as novel transdermal delivery systems to provide a safer alternative in the cosmetic industry. For example, in the treatment of inflammatory skin diseases, cyclosporine A was attached to polyarginine-7 to form R7-CsA. More recently, fusion proteins were designed with four structural classes of  CPPs with the human epidermal growth factor (hEGF) as a wound healing agent.

In the present study, Horn and Neundorf report on the fusion of a well-characterized cell-penetrating peptide called sC18 to a wound healing peptide sequence known as Tylotoin. The short peptide Tylotoin was recently identified and isolated from the skin of the salamander, Tylototriton verrucosus. The novel conjugates were evaluated for their cytotoxicity to keratinocytes and in a cell line in the study, to observe cellular internalization behavior and the impact of peptides on wound healing in vitro.

To eliminate cytotoxicity of the potential wound healing agents, the authors next investigated cell viability of the peptides on human immortalized keratinocytes (HaCaT). The results showed no cytotoxic effects on keratinocytes for Tylotoin-sC18, sC18 * alone and with Tylotoin-sC18 *. The internalization of peptides into keratinocytes was visualized using CF-labelled peptides. All peptides were internalized, and chimeric peptides notably demonstrated a strong distribution in the cytosol and nucleoli.

CF-labeled Tylotoin-sC18 * ???????

In the next level of experiments, the authors investigated the initial step of cell entry on a molecular level using artificial membrane systems known as giant unilamellar vesicles (GUVs). The GUVs were composed of zwitterionic lipids to ensure better cell compatibility. In the study, the authors used CF-labeled Tylotoin-sC18 * alongside the fluorescently labeled CF-Tylotoin and CF-sC18 * as controls for incubation at 90 minutes with neutral GUVs and anionic GUVs.

To detect the effect of the chimeric Tylotoin-sC18* on wound healing, the authors used a scratch wound healing assay and observed keratinocyte migration. In the presence of Tylotoin-sC18*, the migration of keratinocytes into the wounded area distinctly increased 30 hours after scratching. Similar results were also observed with the human epidermal growth factor (hEGF). The novel chimeric peptide conjugate designed in the study had comparable migration efficacy to Tylotoin and hEGF.

Anticancer activity 

The behavior of the novel peptide conjugate was also investigated in HeLa cells to understand its impact on a cancer cell line. The CF-labelled Tylotoin-sC18* HeLa cells were characterized by an extremely high uptake of the dye in comparison to the control peptides. The study also interestingly showed decreased viability in Tylotoin-sC18* induced cancer cells, significant even at lower concentrations hinting at potential anticancer activity of the peptide.

The study also evaluated the inhibition of endocytosis and its effect on cellular uptake of Tylotoin-sC18* in to HaCaT cells. To establish conditions of endocytosis inhibition and constrain the energy-dependent pathway, HaCaT cells were incubated with Tylotoin-sC18* at 4 degrees C. The authors nevertheless observed the peptide's ability to enter the cells, with notable localization in the nucleoli, leading to the assumption that the peptide chimera can internalize via direct translocation.

In addition, the fusion of the peptide sC18 * to Tylotoin did not impair the inherent healing capacity of Tylotoin. As observed, the chimeric peptide increased healing capacity to promote in vitro wound closure by inducing migration and proliferation of keratinocytes. Coupled with antimicrobial properties, the novel cell-penetrating chimeric peptide Tylotoin-sC18 * can be used as a topical agent for infected wound treatment.