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.

Recently we have characterized the DNA and nucleoprotein (NP) binding of bis(4-Nmethylpyridyl)-15,20-di(4-carboxyphenyl)porphyrin (BMPCP) and meso-tri(4-Nmethylpyridyl)-mono(4-carboxyphenyl)porphyrin (TMPCP) and their tetra-peptide conjugates (BMPCP-4P 2 and TMPCP-4P, respectively). In this work we investigated the interaction of TMPCP conjugated to the tetra-peptide branches of branched chain polymeric polypeptide with poly-L-lysine backbone (AK) with DNA or NP using spectroscopic methods.Analysis of absorption spectra revealed the external binding, but no intercalation of TMPCP-AK to DNA. There was no evidence for the interaction between TMPCP-AK and encapsidated DNA. Furthermore, we examined the cellular uptake of BMPCP and TMPCP and their tetra-or polypeptide conjugates by flow cytometry and analyzed how charge, size and structure of the compounds affect their incorporation. In comparison, liposomal association constants of these derivatives were determined. BMPCP-4P 2 accumulated the most, and porphyrins with two positive charges (BMPCP and BMPCP-4P 2 ) showed better accumulation than the tri-cationic TMPCP or TMPCP-4P.Cellular uptake of polycationic TMPCP-AK was significantly lower than that of the free or tetra-peptide conjugated derivatives. The sub-cellular localization of all the five compounds was investigated in co-localization studies by confocal microscopy with special attention to their nuclear localization. Neither free nor conjugated BMPCP or TMPCP were co-localized with nuclear marker. Instead, these derivatives showed co-localization with lysosomal and mitochondrial fluorescent probes. TMPCP-AK conjugate had different localization pattern appearing mainly in mitochondria and cytoplasmic vesicles.Our results may contribute to the further design of DNA targeting porphyrin based constructs.

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