Daniel Jancura

On the Diffusion of Hypericin in Dimethylsulfoxide/Water Mixtures— The Effect of Aggregation

Hypericin (Hyp) is a natural photosensitizing pigment with a possible application in the photodynamic therapy of cancer. Hyp is readily dissolved in dimethylsulfoxide (DMSO) but forms nonsoluble aggregates in an aqueous environment. Fluorescence spectroscopy and diffusion coefficient measurements are used to investigate the self-association of Hyp molecules in DMSO/water mixtures. Fluorescence measurements reveal that Hyp remains in its monomeric form in DMSO/water mixtures containing up to ∼20-30 wt % water. At higher water concentration, Hyp starts to form nonfluorescent aggregates. To determine the size of the aggregates, the diffusion coefficient of Hyp is determined for different DMSO/water mixtures both experimentally and theoretically. Our data indicate that the size of the aggregates increases as more water is added into DMSO. At 50 wt % water content, the effective diffusion coefficient is about 30% smaller than the calculated value for the stacked Hyp tetramer. The results indicate that in an aqueous environment, Hyp presumably produces large molecular weight stacked H-aggregates. We have also confirmed that in an aqueous environment at alkaline pH, molecules of Hyp remain in the monomeric state.

High Level of Low-density Lipoprotein Receptors Enhance Hypericin Uptake by U-87 MG Cells in the Presence of LDL

The dependence of the uptake of hypericin (Hyp) by human glioma U‐87 MG cells on the level of expression of low‐density lipoprotein (LDL) receptors has been studied in this work. A special role of the LDL receptor‐pathway for Hyp delivery to U‐87 MG cells in the presence of LDL was revealed by the substantial increase of Hyp uptake in the situation, when the number of LDL receptors on the cell surface was elevated. Moreover, the colocalization experiments showed the lysosomal localization of Hyp following the uptake and that the concentration of Hyp in these organelles was enhanced in the cells with elevated number of LDL receptors when the incubation medium contained LDL. Both these findings suggest that LDL and LDL receptor‐pathway play an important role in the delivery and accumulation of Hyp into the cells.

Fluorescence Spectroscopic Study of Hypericin‐photosensitized Oxidation of Low‐density Lipoproteins

Abstract By means of UV-VIS absorption and fluorescence spectroscopy, we demonstrate that the photosensitizer hypericin (Hyp) interacts nonspecifically with low-density lipoproteins (LDL), most probably with the lipid fraction of LDL. The molar ratio of monomeric Hyp binding to nonoxidized LDL and mildly oxidized LDL is 30:1. Increasing the Hyp concentration further leads to the formation of Hyp aggregates inside the LDL molecule. We also demonstrate that photoactivated Hyp oxidizes LDL in a light dose and excitation wavelength dependent manner. The level of oxidation of LDL depends on the amount of Hyp inside the LDL molecule. The maximum of the photosensitized oxidation of the LDL by Hyp is achieved for a 30:1 molar ratio, which corresponds to the maximum concentration of monomeric form of Hyp in LDL.

Interaction dynamics of hypericin with low-density lipoproteins and U87-MG cells.

The natural photosensitizer hypericin exhibits potent properties for tumor diagnosis and photodynamic therapy. Fluorescent properties of hypericin along with various technical approaches have been used for dynamic studies of its interaction with low-density lipoprotein and U87 glioma cells. Evidences for hypericin release from low-density lipoprotein towards cells plasmatic membrane are addressed. Subsequent subcellular bulk flow redistribution leading to non-specific staining of intracellular membranes compartment were observed within cells. It was shown, that monomers of hypericin are the only redistributive forms. Increasing concentration of hypericin leads to the formation of non-fluorescent aggregates within low-density lipoprotein as well as within the U87 cells, and can preclude its photosensitizing activities. However, the aggregation process can only account for a part of the observed emission decrease. As shown by the excited state lifetime measurements, this fluorescence quenching actually results from a combination of aggregation process and energy transfer from monomers to aggregates. In all experiments, hydrophobic character of hypericin appears as the driving force of its redistribution process.

Time-resolved Luminescence and Singlet Oxygen Formation After Illumination of the Hypericin–Low-density Lipoprotein Complex

Time‐resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low‐density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self‐quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime (∼0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime (∼8 μs) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.

Development of a new LDL-based transport system for hydrophobic/amphiphilic drug delivery to cancer cells

Low-density lipoproteins (LDL), a natural in vivo carrier of cholesterol in the vascular system, play a key role in the delivery of hydrophobic/amphiphilic photosensitizers to tumor cells in photodynamic therapy of cancer. To make this delivery system even more efficient, we have constructed a nano-delivery system by coating of LDL surface by dextran. Fluorescence spectroscopy, confocal fluorescence imaging, stopped-flow experiments and flow-cytometry were used to characterize redistribution of hypericin (Hyp), a natural occurring potent photosensitizer, loaded in LDL/dextran complex to free LDL molecules as well as to monitor cellular uptake of Hyp by U87-MG cells. It is shown that the redistribution process of Hyp between LDL molecules is significantly suppressed by dextran coating of LDL surface. The modification of LDL molecules by dextran does not inhibit their recognition by cellular LDL receptors and U-87 MG cellular uptake of Hyp loaded in LDL/dextran complex appears to be similar to that one observed for Hyp transported by unmodified LDL particles. Thus, it is proposed that dextran modified LDL molecules could be used as a basis for construction of a drug transport system for targeted delivery of hydrophobic/amphiphilic drugs to cancer cells expressing high level of LDL receptors.

Characterization of the interaction of hypericin with protein kinase C in U-87 MG human glioma cells.

Abstract A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of Kds (∼100 nM) suggest that Hyp binds with high affinity to PKC. Finally, molecular modeling was used to compare structural models of the interaction of C1B domain of PKC (PKC isoforms α, δ, γ) with Hyp and our previously published model of the (C1B domain PKCγ)/PMA complex. The influence of Hyp on PKC translocation in U-87 MG cells in comparison with PMA, colocalization fluorescence pattern of Hyp and PKC, the higher binding affinity of Hyp to PKC in comparison with known binding constants of phorbol esters, as well as the binding mode of Hyp and PMA to the C1B domain of PKC suggested by molecular modeling, support the idea that Hyp and PMA might competitively bind to the regulatory domain of PKC.

Kinetics of Hypericin Association With Low-Density Lipoproteins

Steady‐state and time‐resolved fluorescence spectroscopy have been used for the study of the incorporation kinetics of hypericin (Hyp) into low‐density lipoproteins (LDL). Biphasic kinetics of Hyp association with LDL was observed when solutions of Hyp and LDL were mixed at various concentration ratios. The rapid phase of Hyp incorporation is completed within seconds, while the slow phase lasts several minutes. The relative contributions of the individual phases show that a higher amount of Hyp molecules (65%) are incorporated into LDL in the second phase. The kinetics of the incorporation of Hyp into LDL particles preloaded with Hyp (Hyp/LDL = 25:1) was also investigated. The decreased intensity of Hyp fluorescence is a sign of the formation of Hyp aggregates after penetration of additional Hyp molecules into Hyp/LDL = 25:1 complex. The time dependence of Hyp fluorescence was measured after mixing the complex Hyp/LDL = 200:1 with appropriate amounts of free LDL molecules. For each final Hyp/LDL ratio, an increase in the intensity and lifetime of Hyp fluorescence was observed, suggesting a monomerization of Hyp aggregates. The half‐time of Hyp transfer from Hyp/LDL complex to LDL particles is similar to the half‐time of the slow phase of Hyp incorporation into free LDL particles.

Interaction of antiviral and antitumor photoactive drug hypocrellin A with human serum albumin.

Absorption, resonance Raman, surface-enhanced Raman spectroscopy and differential scanning microcalorimetry were employed to study the interaction of hypocrellin A with human serum albumin. The identification of the binding place for hypocrellin A as well as the model for the albumin-hypocrellin A complex are proposed. In this model hypocrellin A interacts with albumin through more than one binding site placed on the protein surface. This model of non-specific interaction could explain why the absorption spectrum of hypocrellin A does not change in the presence of albumin and why the presence of the drug does not change significantly the thermodynamic parameters of the protein, while the Raman spectra show evident changes concerning both the protein and the drug structure. Even if hypocrellin A does not interact with an interior binding site, it can affect deeply the general albumin structure.

Spatial orientation and electric-field-driven transport of hypericin inside of bilayer lipid membranes.

Fluorescence experiments were carried out to investigate the interaction of hypericin (Hyp), a natural hydrophobic photosensitizer, with artificial bilayer lipid membranes. The spatial orientation of Hyp monomers incorporated in diphytanoyl phosphatidylcholine (DPhPC) membranes was determined by measuring the dependence of the Hyp fluorescence intensity on the angle of incidence of p- and s-polarized excitation laser beams. Inside of the membrane, Hyp monomers are preferentially located in the layers near the membrane/water interface and are oriented with the S(1) ← S(0) transition dipole moments perpendicular to the membrane surface. Transport of Hyp anions between the two opposite sides of the lipid bilayer was induced by applying rectangular electric field pulses to the membrane. The characteristic time for Hyp transport through the membrane center was evaluated by the analysis of the Hyp fluorescence signal during the voltage pulses. In the zero-voltage limit, the transport time approached 70 ms and gradually decreased with higher voltage applied to the membrane. In addition, our measurements indicated an apparent pK(a) constant of 8 for Hyp deprotonation in the membrane.