Cell‐penetrating peptides (CPPs) are still an interesting and viable alternative for drug delivery applications. CPPs contain considerably high amounts of positively charged amino acids, imparting them with cationic character. Tumor cells are characterized by an enhanced anionic nature of their membrane surface, a property that could be used by CPPs to target these cells. We recently identified a branched CPP that displays a high internalization capacity while exhibiting selectivity for certain tumor cell types. In this study we elucidated this observation in greater detail by investigating the underlying mechanism behind the cellular uptake of this peptide. An additional cytotoxicity screen against several cancer cell lines indeed demonstrates high cytotoxic activity against cancer cells over normal fibroblasts. Furthermore, we show that this feature can be used for delivering the anticancer drug actinomycin D with high efficiency in the MCF‐7 cancer cell line.

Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety for drug delivery systems. The anti-tumor activity of the previously developed GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] conjugate and the novel synthesized GnRH-III-[2ΔHis,3d-Tic,4Lys(Bu),8Lys(Dau=Aoa)] conjugate, containing the anti-cancer drug daunorubicin, were evaluated. Here, we demonstrate that both GnRH-III-Dau conjugates possess an efficient growth inhibitory effect on more than 20 cancer cell lines, whereby the biological activity is strongly connected to the expression of gonadotropin-releasing hormone receptors (GnRH-R). The novel conjugate showed a higher in vitro anti-proliferative activity and a higher uptake capacity. Moreover, the treatment with GnRH-III-Dau conjugates cause a significant in vivo tumor growth and metastases inhibitory effect in three different orthotopic models, including 4T1 mice and MDA-MB-231 human breast carcinoma, as well as HT-29 human colorectal cancer bearing BALB/s and SCID mice, while toxic side-effects were substantially reduced in comparison to the treatment with the free drug. These findings illustrate that our novel lead compound is a highly promising candidate for targeted tumor therapy in both colon cancer and metastatic breast cancer.

The Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most aggressive and dangerous cancerous diseases, leading to a high rate of mortality. Therefore, the development of new, more efficient treatment approaches is necessary to cure this illness. Peptide-based drug targeting provides a new tool for this purpose. Previously, a hexapeptide Cys-Lys-Ala-Ala-Lys-Asn (CKAAKN) was applied efficiently as the homing device for drug-loaded nanostructures in PDAC cells. In this research, Cys was replaced by Ser in the sequence and this new SKAAKN targeting moiety was used in conjugates containing daunomycin (Dau). Five different structures were developed and tested. The results indicated that linear versions with one Dau were not effective on PANC-1 cells in vitro; however, branched conjugates with two Dau molecules showed significant antitumor activity. Differences in the antitumor effect of the conjugates could be explained with the different cellular uptake and lysosomal degradation. The most efficient conjugate was Dau=Aoa-GFLG-K(Dau=Aoa)SKAAKN-OH (conjugate 4) that also showed significant tumor growth inhibition on s.c. implanted PANC-1 tumor-bearing mice with negligible side effects. Our novel results suggest that peptide-based drug delivery systems could be a promising tool for the treatment of pancreatic cancers.

Development of peptide-based conjugates for targeted tumour therapy is a current research topic providing new possibilities in cancer treatment. In this study, VHLGYAT heptapeptide selected by phage display technique for HT-29 human colon cancer was investigated as homing peptide for drug delivery. Daunomycin was conjugated to the N-terminus of the peptide directly or through Cathepsin B cleavable spacers. Conjugates showed moderate in vitro cytostatic effect. Therefore, sequence modifications were performed by Ala-scan and positional scanning resulting in conjugates with much higher bioactivity. Conjugates in which Gly was replaced by amino acids with bulky apolaric side chains provided the best efficacy. The influence of the cellular uptake, stability and drug release on the anti-tumour activity was investigated. It was found that mainly the difference in the cellular uptake of the conjugates generated the distinct effect on cell viability. One of the most efficient conjugate Dau=Aoa-LRRY-VHLFYAT-NH 2 showed tumour growth inhibition on orthotopically developed HT-29 colon cancer in mice with negligible toxic side effect compared to the free drug. We also indicate that this sequence is not specific to HT-29 cells, but it has a remarkable effect on many other cancer cells. Nevertheless, the Phe-containing conjugate was more active in all cases compared to the conjugate with the parent sequence. The literature data suggested that this sequence is highly overlapped with peptides that recognize Hsp70 membrane bound protein overexpressed in many types of tumours.

The first representatives of fluorescent boro-β-carbolines were applied for labeling trastuzumab. The antibody fluorophore conjugate was confirmed as a useful tool for labeling and imaging tumour cells in confocal and two-photon microscopy.