Study of cell penetrating peptide and Europium(III) and Terbium(III) Schiff base complexes interaction

During the last decade newly developed potential therapeutic drugs such as proteins, nucleic acids, and new types of hydrophilic drugs are being reported. However, these drugs mostly have limitations due to their inability to reach the appropriate intracellular targets as a consequence of poor possibility to penetrate through cell membranes or deactivation by resistance mechanisms that transport these compounds out of the cell, both limiting their interaction with intracellular targets [1,2]. It has been also reported that poor cell specificity and normal cell cytotoxicity are common in application of standard technique for drug delivery systems, such as microinjection, electroporation, liposomal formulation and use of viral vectors [3-5]. In the search for new anticancer agents and drug delivery systems, cell penetrating peptides (CPP) attracted attention due to their possibilities for intracellular delivery of a wide range of macromolecules. CPP are short peptides consisting of less than 30 amino acids. CPP structures are mostly composed of positively charged amino acids (e.g. Arg, Lys and His) providing them possibility to translocate through the cell membrane by various mechanism, including endocytosis, and easily deliver various cell-impermeable covalently or noncovalently conjugated bioactive cargo such as proteins [6], nucleic acid [7], siRNA [8], peptide nucleic acid [9] and quantum dots [10]. CPPs as delivery agents were in focus of many investigations with the aim to increase stability and efficiency of cargo delivery avoiding the problem of cytotoxicity effect, lack of cell specificity and unexpected side effects. However, it has been shown that side effects on normal cells during cancer therapy or antibacterial application are minimized [11-13]. Peptides as drug carriers offer some advantages over other carriers as they are relatively easy to modify with various organic or inorganic materials, especially with compounds that have fluorescent properties, enabling easy tracking of drugs after application and for better understanding of the structure and functions of biological systems [14]. Many of luminescent materials such as organic fluorophores [15], recombinant proteins [16], semiconductor nanoparticles [17], and emissive metal complexes [18] are being used for peptide labeling. Application of organic dyes has many limitations associated with poor extinction coefficient or quantum yield and low stability against bleaching. However, on the other hand, metal complexes, especially rare earth metal-based materials show excellent optical properties since the f−f emission lines of Pr(III), Sm(III), Eu(III), Tb(III), Dy(III) and Tm(III) ions are in the visible range [19,20]. Especially great attention attracts luminescent rare-earth metal complexes with Eu(III) and Tb(III) as the excited states of these ions are less sensitive to vibrational quenching by intra or intermolecular energy transfer to adjacent high-energy vibrators such as hydroxyl groups [21]. Comparing metal-rare complex with quantum dots (QDs), they have long life time fluorescence and the fluorescence wavelength of Ln(III) ions is not sensitive to particle size, where the study of the function and properties of these compounds is simplified in comparison to QDs [22]. However, Ln(III) complexes are mostly used for study of their magnetic properties and imagine purpose, only few scientific reports are dealing with application of the complexes in biological applications [23-25]. Based on this consideration, we were interested in preparation of Schiff bases 2-[(E)-2-pyridylmethyleneamino]-N-[2-[(E)-2-pyridylmethyleneamino]ethyl]ethanamine (S-5) and 2-[(E)-2-pyridylmethyleneamino]-N,N-bis[2-[(E)-2-pyridylmethyleneamino]ethyl]ethanamine (S-6) and their europium(III) and terbium(III) complexes Eu(III)-S-5 and Tb(III)-S-6 with luminescent properties in order to study their interaction with cell penetrating peptide and possible biological applications. The compounds were evaluated against several bacterial species with respect to their toxicity.

Figure 1: (a) Absorption and fluorescence spectra of cell penetrating peptide, Tb(III)-S-5 and Tb(III)-S-6 Schiff base complex. (b) Absorption and fluorescence spectra of cell penetrating peptide, Eu(III)-S-5 and Eu(III)-S-6 Schiff base complex.

Figure 2: (a,b) Mass spectra of peptide. (c) Mass spectrum of peptide interaction with Eu(III)-S-5 complex, (d) Tb(III)-S-5 complex, (e) Eu(III)-S-6 complex, (f) Tb(III)-S-6 complex.

Figure 3: Spectrophotometric determination of growth curves obtained by treatment with 0, 0.125, 0.312, 0.625, 1, 2.5 and 5 mM concentration of Eu(III) and Tb(III) Schiff base complex conjugate with cell penetrating peptide of: (a) E. coli after application of Eu(III)-S-5 conjugate with CPP. (b) E. coli after application of Eu(III)-S-6 conjugate with CPP. (c) E. coli after application of Tb(III)-S-5 conjugate with CPP. (d) E. coli after application of Tb(III)-S-6 conjugate with CPP.

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