Eva Kočišová

Hypocrellin A Photosensitization Involves an Intracellular pH Decrease in 3T3 Cells

Abstract— The fluorescent pH probe carboxy‐seminaphtorhoda‐fluor‐1 (C‐Snarf‐1) has been used for laser microspectrofluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al, J. Am. Chem. Soc. 118, 9484‐9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 μM hypocrellin A after 90 s of exposure to 0.1 μW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A‐treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C‐Snarf‐1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C‐Snarf‐1 that results in an increase of C‐Snarf‐1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.

SERS Microspectroscopy of Biomolecules on Dried Ag Colloidal Drops

We report the application of dried Ag hydroxylamine-reduced colloidal drops to surface-enhanced (resonance) Raman scattering (SE(R)RS) study of biomolecules using Raman microspectroscopy. 5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin (TMPyP), amino acid tryptophan, and phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) served as testing biomolecules. Ag colloid/biomolecule drop dried on glass support forms a ring in the edge part of the drop in which almost all nanoparticles are clustered. This specific drying process promotes adsorption of the studied biomolecules in highly enhancing sites (“hot spots”) as well as concentrates them in the ring. We were able to obtain SE(R)RS spectra from the ring that cannot be acquired directly from Ag colloidal solutions (SERRS spectrum of 1×10−10 M TMPyP by 1 s accumulation time, SERS spectrum of 2×10−7 M DSPC). Despite the spectral irreproducibility and problems with spurious bands in some cases, SERS microspectroscopy of studied biomolecules using dried Ag colloid/adsorbate systems improves SERS applicability and sensitivity in comparison to measurements directly from Ag colloidal solution.

Drop coating deposition Raman spectroscopy of liposomes: role of cholesterol.

Drop coating deposition Raman (DCDR) spectroscopy was used to study liposomes (DPPC and asolectin) with growing proportion of cholesterol. Deposited samples of both liposomes on special hydrophobic surface formed a dried drop with a circular shape with a ring of concentrated liposomes at the outer edge. The presence of cholesterol in liposome causes a diminishing of the drop size and an increasing in diameter of the ring, but DPPC with 20% of cholesterol forms the compact drop without the ring. Raman spectra contain characteristics of both lipids and cholesterol, liposomes do not change their initial phase state after drying. Spectral mapping shows that maximum Raman intensity originated from the inner part of the ring. Our results suggest that DCDR spectroscopy can be used for studying lipids containing cholesterol in situ.

Advanced Microfluorescence Methods in Monitoring Intracellular Uptake of “Antisense” Oligonucleotides

Antisense strategy represents a promising molecular tool for efficient and selective chemotherapeutic action. It belongs among oligonucleotide strategies that employ specific single-stranded sequences of deoxyriboand ribonucleotides or their synthetic analogs to block or suppress expression of a pathogen in its early stage. This approach is also promising for studies of the biological function of the gene. However, the routine use of modified oligonucleotides in practice is complicated by non-ideal properties of currently available oligonucleotide analogs. A successful medical treatment requires not only proper binding of the modified oligonucleotide to its cellular target but also its efficient cellular uptake, stability and appropriate distribution in the intracellular environment. The latter processes can be effectively studied by various microfluorescence techniques. The paper reviews the current situation in the application of advanced microfluorescence methods in this field and gives a brief description of the oligonucleotide strategy and possibilities to support the cellular uptake, theoretical and technical basics of current fluorescence microimaging and fluorescence microspectroscopy including time-resolved measurements. Second part of the paper describes experiment preparation, surveys the most interesting studies published so far and outlines the perspectives.

Antimicrobial Peptide from the Eusocial Bee Halictus sexcinctus Interacting with Model Membranes

Halictine-1 (Hal-1)—a linear antibacterial dodecapeptide isolated from the venom of the eusocial bee Halictus sexcinctus—has been subjected to a detailed spectroscopic study including circular dichroism, fluorescence, and vibrational spectroscopy. We investigated Hal-1 ability to adopt an amphipathic α-helical structure upon interaction with model lipid-based bacterial membranes (phosphatidylcholine/phosphatidylglycerol-based large unilamellar vesicles and sodium dodecylsulfate micelles) and helix inducing components (trifluoroethanol). It was found that Hal-1 responds sensitively to the composition of the membrane model and to the peptide/lipid ratio. The amphipathic nature of the helical Hal-1 seems to favour flat charged surfaces of the model lipid particles over the nondirectional interaction with trifluoroethanol. Increasing fraction of polyproline II type conformation was detected at low peptide/lipid ratios.

DCDR Spectroscopy as Efficient Tool for Liposome Studies: Aspect of Preparation Procedure Parameters

Drop-coating deposition Raman (DCDR) spectroscopy was employed to study liposome suspensions. The method is based on a specific drying process on the hydrophobic surface that efficiently accumulates the macromolecular sample in a ring of the edge of the dried drop. We studied liposome suspensions purchased from two sources (Avanti Polar Lipids, Inc. and Sigma-Aldrich, Co.) and prepared under different conditions. Structure of the dried drop substantially depends on the lipid concentration, lipid composition of the sample, and used solvent. Optimal lipid concentration is about 0.3 mg/ml in all cases, asolectin and DSPC suspensions form compact dried drops when dissolved in water and phosphate buffer, respectively. Drying process of the sample drop does not influence the initial phase state (gel or liquid-crystalline) of the studied liposomes excepting DSPC from Sigma-Aldrich, Co.

Interaction of porphyrin/oligonucleotide complex with liposomes studied by drop coating deposition Raman spectroscopy

Drop coating deposition Raman (DCDR) microspectroscopy was used to investigate interaction of the complexed cationic copper 5,10,15,20-tetrakis(1-methyl-4-pyridyl) porphyrin (CuP) and phosphorothioate analog of dT15 oligonucleotide with liposomes, the lipid composition of which imitated the natural plasmatic membrane. Great advantage of dried drops on DCDR plates over a solution sample is that the specific drying process on the special hydrophobic surface efficiently separates liposomes from small species in the solvent. In our case, liposomes with bound CuP/oligonucleotide complexes formed a ring at the edge part of the dried drop while dried solution of this complex remained inside this ring. High quality spectra measured from the ring by using Raman confocal microspectrometer revealed unperturbed arrangement of lipid chains by the drying process, partial binding of the CuP/oligonucleotide complexes to liposomes, and a certain reorientation of lipid chains as a consequence of this interaction.

Time-resolved Microspectrofluorometry and Fluorescence Imaging Techniques: Study of Porphyrin-mediated Cellular Uptake of Oligonucleotides

Time‐resolved confocal microspectrofluorometry and fluorescence microscopy imaging were applied to monitor the cellular uptake of fluorescent‐labeled oligonucleotides (ONs) delivered by a porphyrin molecule. The fate of porphyrin–ON complexes inside living cells has also been monitored. Due to intrinsic fluorescence of the porphyrin and sensitivity of its characteristics to microenvironment, multicomponent analysis of time‐resolved fluorescence provides unique information about stability of the porphyrin–ON complexes, ON interactions with their target sequences, and ON and porphyrin distributions after delivery inside the cells. Time‐resolved confocal microspectrofluorometry indeed delivers additional information compared with fluorescence confocal microscopy imaging widely employed to study ON uptake.

Monitoring of labeled antisense oligonucleotides within living cells by using a multifrequency phase/modulation approach for fluorescence lifetime measurements

A multifrequency phase/modulation method has been developed for our UV confocal laser microspectrofluorimeter (modulation frequency 1 ‐ 200 MHz) for fluorescence lifetime measurements. This technique enables excited state lifetimes of mixed fluorescent components to be resolved and the fluorescence spectral contribution of each species to be determined without using any model spectra. This approach is very efficient for analyzing intracellular multicomponent fluorescence signals. Our effort is focused on the elucidation of the intracellular behavior of synthetic modified oligonucleotides - potential drugs for antisense and/or antigene strategies of curing viral and malignant diseases. A novel type single stranded dT15 oligomer analogue containing isopolar, non-isosteric, phosphonate-based internucleotide linkages (3 0 -O‐P‐CH 2‐ O-5 0 ), labeled with tetramethylrhodamine dye at the 3 0 -end, has been utilized. This method, along with fluorescence micro-imaging, was used to monitor uptake, distribution and stability of our modified oligonucleotide inside living cells. Binding to Escorte vector leads to an homogeneous intracellular distribution of fluorescent labeled oligonucleotide, including nucleus staining, while point distribution only is achieved for its free form. q 2003 Elsevier Science B.V. All rights reserved.

Intracellular Monitoring of AS1411 Aptamer by Time-Resolved Microspectrofluorimetry and Fluorescence Imaging

Time-resolved microspectrofluorimetry and fluorescence microscopy imaging—two complementary fluorescence techniques—provide important information about the intracellular distribution, level of uptake and binding/interactions inside living cell of the labeled molecule of interest. They were employed to monitor the “fate” of AS1411 aptamer labeled by ATTO 425 in human living cells. Confocal microspectrofluorimeter adapted for time-resolved intracellular fluorescence measurements by using a phase-modulation principle with homodyne data acquisition was employed to obtain emission spectra and to determine fluorescence lifetimes in U-87 MG tumor brain cells and Hs68 non–tumor foreskin cells. Acquired spectra from both the intracellular space and the reference solutions were treated to observe the aptamer localization and its interaction with biological structures inside the living cell. The emission spectra and the maximum emission wavelengths coming from the cells are practically identical, however significant lifetime lengthening was observed for tumor cell line in comparison to non–tumor one.