Alla Synytsya

Novel Porphyrin Conjugates with a Potent Photodynamic Antitumor Effect: Differential Efficacy of Mono- and Bis-β-cyclodextrin Derivatives In Vitro and In Vivo

Abstract In the present study we investigated the photosensitizing properties of two novel mono- and bis-cyclodextrin tetrakis (pentafluorophenyl) porphyrin derivatives in several tumor cell lines and in BALB/c mice bearing subcutaneously transplanted syngeneic mouse mammary carcinoma 4T1. Both studied sensitizers were localized mainly in lysosomes and were found to induce cell death by triggering apoptosis in human leukemic cells HL-60. In 4T1 and other cell lines both apoptotic and necrotic modes of cell death occurred depending on drug and light doses. Mono-cyclodextrin porphyrin derivative P(β-CD)1 exhibited stronger in vitro phototoxic effect than bis-cyclodextrin derivative P(β-CD)2. However, in vivo P(β-CD)2 displayed faster tumor uptake with maximal accumulation 6 h after application, leading to complete and prolonged elimination of subcutaneous tumors within 3 days after irradiation (100 J cm−2). In contrast, P(β-CD)1 uptake was slower (48 h) and the reduction of tumor mass was only transient, reaching the maximum at the 12 h interval when a favorable tumor-to-skin ratio appeared. Thus, P(β-CD)2 represents a new photosensitizing drug displaying fast and selective tumor uptake, strong antitumor activity and fast elimination from the body.

pH-Controlled Self-Assembling of meso-Tetrakis(4-sulfonatophenyl)porphyrin−Chitosan Complexes

Solid meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS(4))-chitosan supramolecular complexes were prepared by addition of porphyrin to an aqueous solution of chitosan at pH values. The precipitates obtained were assigned as 1 (pH 6.8) and 2 (pH 2.5) and characterized by spectroscopic, thermal, and microscopic methods. Spectroscopic investigation confirmed the presence of TPPS(4) and chitosan in both products and that the porphyrin is highly self-associated. H-type (stacked) of TPPS(4) aggregation was proposed for 1 and J-type (tilted) for 2. Thermal analysis revealed different pyrolysis routes of the complexes depending on their structural diversity. Light microscopic analysis indicated fibrous and lamellar microstructures, respectively, for 1 and 2. SEM and AFM analysis showed that both complexes consist of compact nanostructures; their size and interconnection is different for 1 and 2. Based on structural inferences, self-assembling hierarchy models were proposed for both of the TPPS(4)-chitosan supramolecular complexes.

Raman spectroscopy of tissue samples irradiated by protons

Purpose: Since the number of cancer patients treated by proton irradiation has increased in the last few years, it seems appropriate to study dose‐dependent effects of proton irradiation on mammalian tissues in more detail. Materials and methods: Tissue samples of normal skin of mouse and swine, of a human tumour model xenograph, and of normal skin and a skin tumour (basal cell carcinoma) of a human patient of about 1 mm thickness were irradiated by 24 MeV protons (uniform delivered doses of 1, 7 and 50 Gy: skin of mouse and a human tumour model xenograph, and 0.5, 5 and 50 Gy: swine and human skin). Raman spectra of non‐irradiated and irradiated samples were recorded and analysed. Results: Amide I, P=O and C‐O bond vibrations and aromatics were sensitive to the proton irradiation dose. In the C‐H stretching region, the irradiation‐mediated change of Raman spectra was significant only in the case of the skin tumour. Conclusions: It has been shown that Raman spectroscopy is suited to assess the radiation damage done to biological samples by protons. Proteins of the human skin tumour seem to be more sensitive to proton irradiation than proteins of normal human skin.

Mekabu fucoidan: Structural complexity and defensive effects against avian influenza A viruses

Fucoidan from the sporophyll (Mekabu) of brown seaweed Undaria pinnatifida (wakame) is interesting due to its various biological activities. Mekabu fucoidan (Mw ∼ 9 kDa) of this study (MF) was previously isolated and characterized by chemical and separation methods including GPC and methylation analysis (Lee, Hayashi, Hashimoto, Nakano, & Hayashi, 2004). It was found that this fucoidan composed of partially sulphated (DS ∼ 0.72) fucose and galactose at approximately equal amounts. Methylation analyses revealed complex structure of MF. However, it has been still unclear about the linkages between units and substitution patterns. To solve these structural tasks, spectroscopic methods (FTIR, FT Raman and NMR) were used in the analysis of native MF and its deesterified derivatives. According to obtained results, this polysaccharide was defined as O-acetylated sulphated fucogalactan. The defensive effects of MF were evaluated on mice infected with avian influenza A viruses (H5N3 and H7N2 subtypes); its efficacy was determined in reducing viral replication and increasing antibody production. Oral administration of MF resulted in suppressing virus yields. In addition, the production of neutralizing antibodies and mucosal IgA in the animals inoculated with the avian influenza A viruses was significantly increased. These results suggested that MF could be used for the prevention of viral infection.

Analysis of human blood plasma and hen egg white by chiroptical spectroscopic methods (ECD, VCD, ROA)

AbstractChiroptical methods are widely used in structural and conformational analyses of biopolymers. The application of these methods to investigations of biofluids would provide new avenues for the molecular diagnosis of protein-misfolding diseases. In this work, samples of human blood plasma and hen egg white were analyzed using a combination of conventional and chiroptical methods: ultraviolet absorption/electronic circular dichroism (UV/ECD), Fourier transform infrared absorption/vibrational circular dichroism (FTIR/VCD), and Raman scattering/Raman optical activity (Raman/ROA). For comparison, the main components of these substances—human serum albumin (HSA) and ovalbumin (Ova)—were also analyzed by these methods. The ultraviolet region of the ECD spectrum was analyzed using the CDNN CD software package to evaluate the secondary structures of the proteins. The UV/ECD, FTIR/VCD, and Raman/ROA spectra of the substances were quite similar to those of the corresponding major proteins, while some differences were also detected and explained. The conclusions drawn from the FTIR/VCD and Raman/ROA data were in good agreement with the secondary structures calculated from ECD. The results obtained in this work demonstrate that the chiroptical methods used here can be applied to analyze not only pure protein solutions but also more complex systems, such as biological fluids. FigureAnalysis of human blood plasma and hen egg white by ECD, VCD and ROA.

Biodistribution Assessment of a Lutetium(III) Texaphyrin Analogue in Tumor-bearing Mice Using NIR Fourier-transform Raman Spectroscopy¶

The use of near‐infrared (NIR)‐excited Fourier‐transform (FT) Raman spectroscopy as a technique for evaluating the extent of photosensitizer localization in tumor (human pancreatic adenocarcinomas)‐bearing mice has been tested using lutetium(III) texaphyrin analogue Ln‐T2B2Tex. The complex was injected subcutaneously in the form of three injections given during the course of 3 days. The kinetics of biodistribution were then followed over a time scale of 1–6 days. The NIR‐FT‐Raman spectra of tissue samples obtained from the xenographic tumor, muscle, heart, brain, liver, spleen, kidney and blood were recorded and used to identify the presence of Lu‐T2B2Tex in these tissues. Five Raman sensitizer markers were used to estimate the relative content of Lu‐T2B2Tex in tumor at various postinjection times. UV‐Visible (Vis) absorption spectroscopic detection of this sensitizer in tissue extracts was applied as a conventional method. Both spectroscopic methods were in good agreement with each other and confirm that Lu‐T2B2Tex localizes well in tumor tissue. Maximal drug content was observed 3 days after the final injection. This time delay seems to be optimal for tumor irradiation in photodynamic therapy.

In vitro interaction of macrocyclic photosensitizers with intact mitochondria: a spectroscopic study.

Six water-soluble macrocyclic photosensitizers, the members of two groups of expanded porphyrins (metallotexaphyrins and free-base sapphyrins) containing hydrophilic substituents and meso-tetra(4-sulfonatophenyl)-porphyrin, were tested by UV-Vis absorption and resonance Raman spectroscopy in the in vitro binding experiments with intact mitochondria isolated from swine liver. Studied macrocycles showed markedly different affinity to mitochondria. The highest uptake was observed for sapphyrin-sugar conjugate and metallotexaphyrins. Sapphyrin-polyamine conjugates exhibit something less affinity to mitochondria, while the porphyrin of anionic character showed very low mitochondrial uptake. Obtained spectroscopic results confirm that the binding process altered the self-aggregation degree of expanded porphyrins.

Resonance Raman and UV-Visible Spectroscopic Studies of Water-Soluble Sapphyrin Derivative: Drug Localization in Tumor and Normal Mice Tissues

Resonance Raman (RR) and UV-visible (UV-vis) spectroscopy have been applied to the study of sapphyrin localization in tumor human pancreatic adenocarcinoma-bearing mice. The photosensitizer was subcutaneously injected, and the kinetics of drug biodistribution have been followed in a time scale from 1 to 9 days. RR spectra of tumor and tissue samples and UV-vis absorption spectra of tissue extracts in water/methanol (1:4 v/v) mixture were measured. Spectral data were used to identify the presence and to estimate the relative content of sapphyrin in various tissues at various times after the last injection. Results showed a preferential localization of sapphyrin in tumor tissue. Maximal content of drug in tumor tissue was observed at 6 days after the last injection. Results obtained by RR and UV-vis spectroscopy were in good agreement with each other.

The effect of proton-irradiation on the Raman spectroscopy of tissue samples.

Tumor and healthy tissue samples were irradiated by 24 MeV protons. The samples were exposed to doses from 0 to 50 Gy and subsequently examined by Raman spectroscopy. The analysis of the intensity of characteristic peaks as a function of radiation dose exhibits different trends for the two types of tissue.

Interaction of metallotexaphyrins with mono- and polysaccharides

The interactions of two water-soluble metallotexaphyrins, containing coordinated lutetium(III) and gadolinium(III) cations, with uronic acids (D-galacturonic and D-glucuronic acids), neutral (amylose, galactan) and anionic (pectate, alginate) polysaccharides were studied using UV–VIS titrations. In the case of polyuronides, strong red shifts in the Soret (9–12 nm) and Q-like (4–9 nm) transitions were observed when solutions of the metallotexaphyrin were subject to titration with increasing ligand concentrations. The interaction with neutral polysaccharides leads to significantly smaller bathochromic shifts in these same bands (1–2 nm). No shift of these bands was observed in the case of interaction with uronic acids. If pectate is replaced by partially C-6 methylated pectinates, the extent of the bathochromic shift was seen to decrease as the extent of methylation increased, becoming minimal in the case of methyl pectate (0.5–1 nm). The origin and magnitude of the observed red shifts is rationalised in terms of the conversion of aggregated forms of the metallotexaphyrin into less aggregated (e.g., monomeric) texaphyrin–polysaccharide species. UV–VIS titrations support the conclusion that polyuronides interact with metallotexaphyrins, presumably by acting as polydentate carboxylic ligands for the Lewis acidic lanthanide(III) metallotexaphyrin centres. In the case of the polyuronides, where near complete conversion to monomers is observed, this decrease in aggregation is thought to reflect binding to carboxylate sites. Such binding interactions are not possible in the case of neutral polysaccharides and methyl pectate and the macrocycles remain highly aggregated. Interaction with uronic acids, however, also does not lead to deaggregation.