Sequence modification of heptapeptide selected by phage display as homing device for HT-29 colon cancer cells to improve the anti-tumour activity of drug delivery systems.

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-NH2 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.

Oligo- and polypeptide conjugates of cationic porphyrins: binding, cellular uptake, and cellular localization.

Recently, we have characterized the DNA and nucleoprotein (NP) binding of bis(4-N-methylpyridyl)-15,20-di(4-carboxyphenyl)porphyrin (BMPCP) and meso-tri(4-N-methylpyridyl)-mono(4-carboxyphenyl)porphyrin (TMPCP) and their tetrapeptide conjugates (BMPCP-4P2 and TMPCP-4P, respectively). In this work, we investigated the interaction of TMPCP conjugated to the tetrapeptide 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-4P2 accumulated the most, and porphyrins with two positive charges (BMPCP and BMPCP-4P2) 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 tetrapeptide conjugated derivatives. The subcellular 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 was co-localized with nuclear marker. Instead, these derivatives showed co-localization with lysosomal and mitochondrial fluorescent probes. TMPCP-AK conjugate had different localization patterns appearing mainly in mitochondria and cytoplasmic vesicles. Our results may contribute to the further design of DNA-targeting porphyrin-based constructs.

Cellular Uptake Mechanism of Cationic Branched Polypeptides with Poly[l-Lys] Backbone.

Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes, or nucleic acids. Polylysine-based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study, we report our findings on the cellular uptake characteristics of nine structurally related polylysine-based polypeptides with cationic side chains composed of (i) single amino acid (poly[Lys(Xi)], XiK) or (ii) oligo[dl-alanine] (poly[Lys(dl-Alam)], AK) or (iii) oligo[dl-alanine] with an additional amino acid (X) at the terminal position (poly[Lys(Xi-dl-Alam)] (XAK)) or (iv) at the position next to the polylysine backbone (poly[Lys(dl-Alam-Xi)] (AXK)). In vitro cytotoxicity and cellular uptake were characterized on HT-29 human colon carcinoma and HepG2 human hepatocarcinoma cell lines. Data indicate that the polycationic polypeptides studied are essentially nontoxic in the concentration range studied, and their uptake is very much dependent on the side chain structure (length, identity of amino acid X, and distance between the terminal positive charges) and also on the cell lines. Our findings in uptake inhibition studies suggest that predominantly macropinocytosis and caveole/lipid raft mediated endocytosis are involved. The efficacy of their internalization is markedly influenced by the hydrophobicity and charge properties of the amino acid X. Interestingly, the uptake properties of the these polypeptides show certain similarities to the entry pathways of several cell penetrating peptides.

Binding of new cationic porphyrin-tetrapeptide conjugates to nucleoprotein complexes.

Ongoing research on DNA binding of cationic porphyrin derivatives and their conjugates is a subject of growing interest because of their possible DNA binding and demonstrated biological properties. In this study nucleoprotein binding of tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin (TMPCP) and tetrapeptides conjugated TMPCP (TMPCP-4P) and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin (BMPCP-4P2) was investigated with comprehensive spectroscopic methods. The key observation is that tetrapeptide-conjugates of cationic porphyrins with two or three positive charges bind to encapsidated DNA in T7 phage nucleoprotein complex. The binding modes were analyzed by fluorescent energy transfer, fluorescent life time and CD measurements. Intercalative binding is most feasible when tricationic ligands complex with DNA, especially when it is in close connection with protein capsid. It was found that larger ligand BMPCP-4P2 binds externally to encapsidated T7 DNA, and complex externally as well as by intercalation when the DNA accommodate to relaxed B-conformation. In the case of TMPCP and TMPCP-4P the intercalation is the predominant binding form both in nucleoprotein (NP) and preheated complexes. Further, melting experiments revealed that bound porphyrins do not influence the capsid stability or protein-DNA interactions, but efficiently stabilize the double helical structure of DNA without respect to binding form. A good correlation was found between porphyrin/base pair ration and DNA strand separation temperature.