Mariusz Kepczynski

Interaction of curcumin with lipid monolayers and liposomal bilayers

Curcumin shows huge potential as an anticancer and anti-inflammatory agent. However, to achieve a satisfactory bioavailability and stability of this compound, its liposomal form is preferable. Our detailed studies on the curcumin interaction with lipid membranes are aimed to obtain better understanding of the mechanism and eventually to improve the efficiency of curcumin delivery to cells. Egg yolk phosphatidylcholine (EYPC) one-component monolayers and bilayers, as well as mixed systems containing additionally dihexadecyl phosphate (DHP) and cholesterol, were studied. Curcumin binding constant to EYPC liposomes was determined based on two different methods: UV/Vis absorption and fluorescence measurements to be 4.26×10(4)M(-1) and 3.79×10(4)M(-1), respectively. The fluorescence quenching experiment revealed that curcumin locates in the hydrophobic region of EYPC liposomal bilayer. It was shown that curcumin impacts the size and stability of the liposomal carriers significantly. Loaded into the EYPC/DPH/cholesterol liposomal bilayer curcumin stabilizes the system proportionally to its content, while the EYPC/DPH system is destabilized upon drug loading. The three-component lipid composition of the liposome seems to be the most promising system for curcumin delivery. An interaction of free and liposomal curcumin with EYPC and mixed monolayers was also studied using Langmuir balance measurements. Monolayer systems were treated as a simple model of cell membrane. Condensing effect of curcumin on EYPC and EYPC/DHP monolayers and loosening influence on EYPC/DHP/chol ones were observed. It was also demonstrated that curcumin-loaded EYPC liposomes are more stable upon interaction with the model lipid membrane than the unloaded ones.

Polymeric photosensitizers 2. Photosensitized oxidation of phenol in aqueous solution

Poly(sodium styrenesulfonate-co-vinylbenzyl chloride) with rose bengal chromophores covalently attached to the polymer chain (PSSS-VBC/RB) has been successfully used as a photosensitizer for oxidation of phenol in an aqueous solution. p-Benzoquinone was identified as a primary photochemical product. The quantum yields of photooxidation of phenol was found to be strongly dependent on pH of aqueous solution.

Decomposition of microcystin-LR by Fenton oxidation

A novel and promising method of microcystin-LR (mcyst-LR) degradation is reported. The decomposition of this cyanobacterial toxin using Fenton reagent has been observed with very low initial concentrations of H2O2 and Fe2+ (Fe3+) in the reaction mixture. Mcyst-LR was isolated from a laboratory culture of Microcystis aeruginosa PCC 7813. The initial concentration of the toxin used exceeded by several orders of magnitude those occurring naturally in lakes and drinking water. Even so, the decomposition of the toxin was complete after 30 min.

Which physical and structural factors of liposome carriers control their drug-loading efficiency?

The correlation between structural and physical properties of lipid membrane and its drug-loading efficiency were studied. The properties of bilayer were altered by incorporation of several lipidic modifiers: cholesterol, oleic acid, methyl oleate, and pegylated lipid. By using the molecular probe technique it was demonstrated that the membrane properties, such as micropolarity, microviscosity and free volume were considerably changed by incorporation of the modifiers. The partitioning of two different porphyrins between the bulk aqueous phase and the modified liposomes was studied using the fluorescence methods, and liposome-binding constants were determined. It was found that cholesterol reduced the partitioning of both porphyrins into liposomal bilayer. On the contrary, the incorporation of methyl oleate and pegylated lipid causes a pronounced increase in the value of the binding constants of both porphyrins. It was concluded that the free volume rather than hydrophobicity of bilayer is a governing factor in the solute partitioning into lipid bilayers.

Comparison of photodynamic efficacy of tetraarylporphyrin pegylated or encapsulated in liposomes: In vitro studies

Two photosensitizing systems: (1) tetrakis(4-hydroxyphenyl)porphyrin (p-THPP) encapsulated in sterically stabilized liposomes (SSL) and (2) p-THPP functionalized by covalent attachment of poly(ethylene glycol) (p-THPP-PEG(2000)) were studied in vitro. The dark and photo cytotoxicity of these systems were evaluated on two cell lines: HCT 116, a human colorectal carcinoma cell line, and DU 145, a prostate cancer cell line and compared with these determined for free p-THPP. It was demonstrated that both encapsulation in liposomes as well as attachment of PEG chain result in pronounced reduction of the dark cytotoxicity of the parent porphyrin. The liposomal formulation showed higher than p-THPP-PEG(2000) photocytotoxicity towards both cell lines used in the studies.

Interactions of a hydrophobically modified polycation with zwitterionic lipid membranes.

The interactions between synthetic polycations and phospholipid bilayers play an important role in some biophysical applications such as gene delivery or antibacterial usage. Despite extensive investigation into the nature of these interactions, their physical and molecular bases remain poorly understood. In this Article, we present the results of our studies on the impact of a hydrophobically modified strong polycation on the properties of a zwitterionic bilayer used as a model of the mammalian cellular membrane. The study was carried out using a set of complementary experimental methods and molecular dynamic (MD) simulations. A new polycation, poly(allyl-N,N-dimethyl-N-hexylammonium chloride) (polymer 3), was synthesized, and its interactions with liposomes composed of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC) were examined using dynamic light scattering (DLS), zeta potential measurements, and cryo-transmission electron microscopy (cryo-TEM). Our results have shown that polymer 3 can efficiently associate with and insert into the POPC membrane. However, it does not change its lamellar structure, as was demonstrated by cryo-TEM. The influence of polymer 3 on the membrane functionality was studied by leakage experiments applying a fluorescence dye (calcein) encapsulated in the phospholipid vesicles. The MD simulations of model systems reveal that polymer 3 promotes formation of hydrophilic pores in the membrane, thus increasing considerably its permeability.

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.

Interaction of Hematoporphyrin with Lipid Membranes

Natural or synthetic porphyrins are being used as photosensitizers in photodiagnosis (PD) and photodynamic therapy (PDT) of malignancies and some other diseases. Understanding the interactions between porphyrins and cell membranes is therefore important to rationalize the uptake of photosensitizers and their passive transport through cell membranes. In this study, we consider the properties of hematoporphyrin (Hp), a well-known photosensitizer for PD and PDT, in the presence of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer that we use as a model system for protein-free cell membranes. For this purpose, we employed 200 ns atomic-scale molecular dynamics (MD) simulations for five systems containing the neutral (Hp(0)) or the dianionic form (Hp(2-)) of Hp and the POPC bilayer. MD simulations allowed one to estimate the position, orientation, and dynamics of Hp molecules inside the membrane. The dye molecules were found to reside in the phospholipid headgroup area close to the carbonyl groups of the POPC acyl chains. Their orientations were dependent on the protonation state of two propionic groups. Hp(2-) was found to have a lower affinity to enter the membrane than the neutral form. The dianions, being in the aqueous phase, formed stable dimers with a strictly determined geometry. Our results fully supported the experimental data and provide a more detailed molecular-level description of the interactions of photosensitizers with lipid membranes.

New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.

Endothelium in Spots – High-Content Imaging of Lipid Rafts Clusters in db/db Mice

Lipid rafts (LRs) are dynamic, sterol- and sphingolipid-enriched nanodomains involved in the regulation of cellular functions and signal transduction, that upon stimuli, via (e.g. association of raft proteins and lipids), may cluster into domains of submicron or micron scale. Up to date, however, lipid raft clusters were observed only under artificially promoted conditions and their formation in vivo has not been confirmed. Using non-destructive approach involving Raman and Atomic Force Microscopy imaging we demonstrated the presence of clustered lipid rafts in endothelium of the aorta of the db/db mice that represent a reliable murine model of type 2 diabetes. The raft clusters in the aorta of diabetic mice were shown to occupy a considerably larger (about 10-fold) area of endothelial cells surface as compared to the control. Observation of pathology-promoted LRs confirms that the cellular increase of lipid content results in clustering of LRs. Clustering of LRs leads to the formation of assemblies with diameters up to 3 micrometers and increased lipid character. This massive clustering of lipid rafts in diabetes may trigger a signaling cascade leading to vascular inflammation.