Cell-penetrating peptides (CPP) are promising tools for the transport of a broad range of compounds into cells. Since the discovery of the first members of this peptide family, many other peptides have been identified; nowadays, dozens of these peptides are known. These peptides sometimes have very different chemical–physical properties, but they have similar drawbacks; e.g., non-specific internalization, fast elimination from the body, intracellular/vesicular entrapment. Although our knowledge regarding the mechanism and structure–activity relationship of internalization is growing, the prediction and design of the cell-penetrating properties are challenging. In this review, we focus on the different modifications of well-known CPPs to avoid their drawbacks, as well as how these modifications may increase their internalization and/or change the mechanism of penetration.

Cancer of the skin is by far the most common of all cancers. Although the incidence of melanoma is relatively low among skin cancers, it can account for a high number of skin cancer deaths. Since the start of deeper insight into the mechanisms of melanoma tumorigenesis and their strong interaction with the immune system, the development of new therapeutical strategies has been continuously rising. The high number of melanoma cell mutations provides a diverse set of antigens that the immune system can recognize and use to distinguish tumor cells from normal cells. Peptide-based synthetic anti-tumor vaccines are based on tumor antigens that elicit an immune response due to antigen-presenting cells (APCs). Although targeting APCs with peptide antigens is the most important assumption for vaccine development, peptide antigens alone are poorly immunogenic. The immunogenicity of peptide antigens can be improved not only by synthetic modifications but also by the assistance of adjuvants and/or delivery systems. The current review summarizes the different chemical approaches for the development of effective peptide-based vaccines for the immunotherapeutic treatment of advanced melanoma.

Utilizing McMurry reactions of 4,4′-dihydroxybenzophenone with appropriate carbonyl compounds, a series of 4-Hydroxytamoxifen analogues were synthesized. Their cytotoxic activity was evaluated in vitro on four human malignant cell lines (MCF-7, MDA-MB 231, A2058, HT-29). It was found that some of these novel Tamoxifen analogues show marked cytotoxicity in a dose-dependent manner. The relative ROS-generating capability of the synthetized analogues was evaluated by cyclic voltammetry (CV) and DFT modeling studies. The results of cell-viability assays, CV measurements and DFT calculations suggest that the cytotoxicity of the majority of the novel compounds is mainly elicited by their interactions with cellular targets including estrogen receptors rather than triggered by redox processes. However, three novel compounds could be involved in ROS-production and subsequent formation of quinone-methide preventing proliferation and disrupting the redox balance of the treated cells. Among the cell lines studied, HT-29 proved to be the most susceptible to the treatment with compounds having ROS-generating potency.

Cell-penetrating peptides (CPPs) are commonly modified to increase their cellular uptake, alter the mechanism of penetration or enhance their endosomal release. Earlier, we described the internalization enhancement ability of the 4-((4-(dimethylamino)phenyl)azo)benzoyl (Dabcyl) group. We proved that this modification on the N-terminus of tetra- and hexaarginine enhanced their cellular uptake. The introduction of an aromatic ring 4-(aminomethyl) benzoic acid, AMBA) into the peptide backbone has a synergistic effect with Dabcyl, and the tetraarginine derivatives had outstanding cellular uptake. Based on these results, the effect of Dabcyl or Dabcyl-AMBA modification on the internalization of oligoarginines was studied. Oligoarginines were modified with these groups and their internalization was measured using flow cytometry. The concentration dependence of the cellular uptake of selected constructs was compared too. Their internalization mechanism was also examined by using different endocytosis inhibitors. While the effect of the Dabcyl group was optimal for hexaarginine, the Dabcyl-AMBA group increased the cellular uptake in the case of all oligoarginines. All derivatives, with the exception of only tetraarginine, were more effective than the octaarginine control. The internalization mechanism was dependent on the size of the oligoarginine and was independent of the modification. Our findings suggest that these modifications enhanced the internalization of oligoarginines and resulted in novel, very effective CPPs.

Delivering therapeutic agents into cells has always been a major challenge. In recent years, cyclization emerged as a tool for designing CPPs to increase their internalization and stability. Cyclic ring(s) can protect the peptide from enzymatic degradation, so cyclic peptides remain intact. Therefore they can be good carrier molecules. In this work, the preparation and investigation of efficient cyclic CPPs are described. Different oligoarginines were designed to conjugate with rigid aromatic scaffolds or form disulfide bonds. The reaction between the scaffolds and the peptides forms stable thioether bonds, constraining the peptide into a cyclic structure. The constructs presented very efficient internalization on cancerous cell lines. Our peptides use more than one endocytic pathway for cellular uptake. In this way, short peptides, which can compete with the penetration of well-known CPPs such as octaarginine (Arg₈), may be synthesized through cyclization.

Malignant melanoma is one of the most aggressive and resistant tumor types, with high metastatic properties. Because of the lack of suitable chemotherapeutic agents for treatment, the 5-year survival rate of melanoma patients with regional and distant metastases is lower than 10%. Targeted tumor therapy that provides several promising results might be a good option for the treatment of malignant melanomas. Our goal was to develop novel melanoma-specific peptide–drug conjugates for targeted tumor therapy. Melanocortin-1-receptor (MC1R) is a cell surface receptor responsible for melanogenesis and it is overexpressed on the surface of melanoma cells, providing a good target. Its native ligand, α-MSH (α-melanocyte-stimulating hormone) peptide, or its derivatives, might be potential homing devices for this purpose. Therefore, we prepared three α-MSH derivative–daunomycin (Dau) conjugates and their in vitro and in vivo antitumor activities were compared. Dau has an autofluorescence property; therefore, it is suitable for preparing conjugates for in vitro (e.g., cellular uptake) and in vivo experiments. Dau was attached to the peptides via a non-cleavable oxime linkage that was applied efficiently in our previous experiments, resulting in conjugates with high tumor growth inhibition activity. The results indicated that the most promising conjugate was the compound in which Dau was connected to the side chain of Lys (Ac-SYSNleEHFRWGK(Dau=Aoa)PV-NH₂). The highest cellular uptake by melanoma cells was demonstrated using the compound, with the highest tumor growth inhibition detected both on mouse (38.6% on B16) and human uveal melanoma (55% on OMC-1) cells. The effect of the compound was more pronounced than that of the free drug.