Bartłomiej Kozik

Novel nanostructural photosensitizers for photodynamic therapy: In vitro studies

Photosensitizing properties of 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) functionalized by covalent attachment of one chain of poly(ethylene glycol) (PEG) with a molecular weight of 350, 2000, or 5000 Da (p-THPP-PEG(350), p-THPP-PEG(2000), p-THPP-PEG(5000)) were studied in vitro. Dark and photo cytotoxicity of these photosensitizers delivered in solution or embedded in liposomes were evaluated on two cell lines: a human colorectal carcinoma cell line (HCT 116) and a prostate cancer cell line (DU 145), and compared with these treated with free p-THPP. The attachment of PEG chains results in the pronounced reduction of the dark cytotoxicity of the parent porphyrin. Cell viability tests have demonstrated that the phototoxicity of pegylated porphyrins is dependent on the length of PEG chain and p-THPP-PEG(2000) exhibited the highest photodynamic efficacy for both cell lines. The encapsulation into liposomes did not improve the PDT effect. However, the liposomal formulation of p-THPP-PEG(2000) showed a greater tendency to induce apoptosis in both cell lines than the parent or pegylated porphyrin delivered in solution. The colocalization of p-THPP, p-THPP-PEG(2000) and p-THPP-PEG(2000) enclosed in liposomes with fluorescent markers for lysosomes, mitochondria, endoplasmatic reticulum (ER) and Golgi apparatus (GA) was determined in the HCT 116 line. The p-THPP exhibited ubiquitous intracellular distribution with a preference for membranes: mitochondria, ER, GA, lysosomes and plasma membrane. Fluorescence of p-THPP-PEG(2000) was observed within the cytoplasm, with a stronger signal detected in membranous organelle: mitochondria, ER, GA and lysosomes. In contrast, p-THPP-PEG(2000) delivered in liposomes gave a distinct lysosomal pattern of localization.

Properties of polyethylene glycol supported tetraarylporphyrin in aqueous solution and its interaction with liposomal membranes.

5,10,15,20-Tetrakis(4-hydroxyphenyl)porphyrin was functionalized by covalent attachment of poly(ethylene glycol) (PEG) chains of various molecular weights, 350, 2000, and 5000 Da. The properties of PEG-functionalized tetraarylporphyrins in aqueous solution and their interactions with liposomes have been studied. Electronic absorption spectroscopy, dynamic light scattering, atomic force microscopy, and fluorescence quenching were used to monitor aggregation of porphyrin chromophores and behavior of the attached PEG chains in the aqueous solution. The tendency for aggregation of porphyrin chromophores in aqueous solution and the efficiency of fluorescence quenching by KI decrease with increasing length of PEG chain linked to the porphyrin ring. The experimental results indicate that polymer clusters are present in aqueous solution of all pegylated porphyrins. The interactions between the pegylated porphyrins and phosphatidylcholine liposomes in the aqueous solution were studied using the fluorescence methods. The apparent binding constants of porphyrin chromophores to liposomes were determined. The degree of binding was found to be dependent on the molecular weight of the attached polymer.

Mechanistic basis for the Enantioselectivity of the Anaerobic Hydroxylation of Alkylaromatic compounds by Ethylbenzene Dehydrogenase.

The enantioselectivity of reactions catalyzed by ethylbenzene dehydrogenase, a molybdenum enzyme that catalyzes the oxygen-independent hydroxylation of many alkylaromatic and alkylheterocyclic compounds to secondary alcohols, was studied by chiral chromatography and theoretical modeling. Chromatographic analyses of 22 substrates revealed that this enzyme exhibits remarkably high reaction enantioselectivity toward (S)-secondary alcohols (18 substrates converted with >99% ee). Theoretical QM:MM modeling was used to elucidate the structure of the catalytically active form of the enzyme and to study the reaction mechanism and factors determining its high degree of enantioselectivity. This analysis showed that the enzyme imposes strong stereoselectivity on the reaction by discriminating the hydrogen atom abstracted from the substrate. Activation of the pro(S) hydrogen atom was calculated to be 500 times faster than of the pro(R) hydrogen atom. The actual hydroxylation step (i.e., hydroxyl group rebound reaction to a carbocation intermediate) does not appear to be enantioselective enough to explain the experimental data (the calculated rate ratios were in the range of only 2-50 for pro(S): pro(R)-oriented OH rebound).

LIF excitation spectra of 2,6-dicyanoaniline

Abstract 2,6-Dicyanoaniline (DCA) reveals remarkably different physicochemical and spectral properties than other aniline derivatives. The infrared and Raman spectra for DCA in solid state and laser induced fluorescence (LIF) excitation spectra of vacuum jet cooled molecules have been investigated. The ground S 0 and the S 1 (B 2 ) excited state geometries, charge distributions and vibrational frequencies of DCA have been evaluated by ab initio calculations. The Dushinsky matrices have been applied for the mode correlation and assignments of the transition frequencies in LIF excitation spectra of DCA(NH 2 ) and DCA(ND 2 ). The intensity distributions in LIF excitation spectra were modeled. The dominant vibronic bands in the excitation spectra are connected with motions of the adjacent cyano- and amino groups. The transitions involving inversion motion of the amino group have low intensities.

Synthesis of strong polycations with improved biological properties

Poly(allylamine hydrochloride) (PAH) has found many applications both in biotechnology and biomedical fields. However, its high toxicity toward various mammalian cells significantly limits its effective usage. This study focuses on improving the biological properties of PAH by its modification to strong polyelectrolytes. The strong polycations were prepared by the direct quaternization of PAH amino groups or by the attachment of glycidyltrimethylammonium chloride to these groups. The biological properties, such as cytotoxicity toward human skin fibroblasts (HSFs), proliferation and migration of the cells on a polymeric surface, and antibacterial activities against two pathogenic bacteria, Staphylococcus aureus and Escherichia coli, were determined. All the modified polyelectrolytes are considerably less toxic to HSFs as compared to PAH. Moreover, the directly quaternized polycations are stronger biocides against S. aureus than the parent polymer. Contrary to PAH, thin films of the modified polyelectrolytes improve or do not affect HSFs proliferation and can stimulate cell migration into the wound, as was demonstrated using an in vitro model. The relationship between the structure of the modified polymers (amount and localization of the quaternary ammonium groups) and the biological activity is discussed. Due to the improved biological properties, the obtained polycations may be potentially useful for a variety of biotechnological and biomedical applications.

Novel synthesis of α-arylnaphthalenes from diphenylacetaldehydes and 1,1-diphenylacetones

Abstract A two-step synthesis of 1-amino-4-arylnaphthalene-2-carbonitriles from diphenylacetaldehydes and 1,1-diphenylacetones involves condensation of the carbonyl compounds with malonodinitrile and cyclization of the aryl-ylidenemalonodinitriles obtained in concentrated sulfuric acid. The benzannulation reaction is accompanied with a quasi-aromatic rearrangement. Some of synthesized aminonitriles reveal considerable biological activity against phytopathogenic fungi.

Behavior of 2,6-bis(decyloxy)naphthalene inside lipid bilayer.

Interactions between small organic molecules and lipid or cell membranes are important because of their role in the distribution of biologically active substances inside the membrane and their permeation through the cell membranes. In the current paper, we have explored the effect of the attachment of long hydrocarbon tails on the behavior of small organic molecule inside the lipid membrane. Naphthalene with two decyloxy groups attached at the opposite sites of the ring (2,6-bis(decyloxy)naphthalene, 3) was synthesized and incorporated into phosphatidylcholine (PC) vesicles. Fluorescence methods as well as molecular dynamic (MD) simulations were used to estimate the position, orientation, and migration of compound 3 in PC bilayer. It was found that the naphthalene ring of compound 3 resides in the upper acyl chain region of the bilayer and the hydrocarbon tails are directed to the center of the bilayer. As was shown with cryotransmission electron microscopy (cryo-TEM), such lipidlike conformation enables compound 3 to be incorporated into liposomes at a very high content without their disintegration. Moreover, compound 3 can migrate from one leaflet to other. The mechanism of this process is, however, different from that characteristic of the flip-flop event of lipid molecules in the membrane. Finally, the possible application of compound 3 as a rotational molecular probe for monitoring fluidity of liposomal membrane in the acyl side chain region was checked by studies of the effect of cholesterol on the fluorescence anisotropy of 3.

Growth and motility of human skin fibroblasts on multilayer strong polyelectrolyte films

Polyelectrolyte multilayers (PEMs) have found application in modifying material surfaces to make them adhesive or non-adhesive for animal cells. However, PEMs made of strong polyelectrolytes are not fully recognized in the literature. This study focuses on the interplay between the properties of PEM assembled from strong polyelectrolytes and cell adhesion and motility. Strong polycations (with quaternary ammonium groups) and a polyanion (with sulfonate groups) were obtained by modification of poly(allylamine hydrochloride) (PAH). Two types of multilayer films were assembled from these PAH derivatives and used to investigate the behavior of human skin fibroblasts (HSFs). The effect of surface charge, hydrophobicity, and film thickness on adhesion of HSFs in a serum-containing medium was studied with immunofluorescence microscopy. The results showed that adhesion of HSFs was strongly depended on the chemical functions of the terminal layer, whereas the wettability was not important. The surface of PEM can be strongly cytophobic (the quaternary ammonium terminal groups) or strongly cytophilic (the sulfonate terminal groups). Finally, the motile activity of HSFs seeded on glass coated with a varying number of polymer layers was investigated. It was demonstrated using an in vitro model that coating the substrate with only two polymer layers can considerably increase the average speed of HSFs movement and stimulate cell migration into the wound.