OXIDATIVE STRESS STUDIES ON LIPID MODEL MEMBRANES

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1 OXIDATIVE STRESS STUDIES ON LIPID MODEL MEMBRANES MARCELA ELISABETA BARBINTA-PATRASCU *, LAURA TUGULEA * * Faculty of Physics, University of Bucharest, Romania Received December 21, 2004 The liposomes with chlorophyll a (Chla) are excellent models for biomembranes and are successfully used to study the influence of different agents on the lipid bilayer structures at molecular level. The spectral features of Chla allow its use as a sensor to monitor the modifications in the lipid membrane. Key words: liposomes, oxidative stress, chlorophyll a, procaine, vitamin E, VIS spectra. 1. INTRODUCTION The living organisms are known to be often exposed to oxidative stress due to the action of most of environmental factors: the exposure to athmospheric oxygen (O 2 ), the pollution, the ozone and the ultraviolet radiation level increasing. The consequence of this fact is the biomolecules (proteins, nucleic acids and lipids) damaging. The oxidative stress mainly affects the membranous systems of aerobic cells and the consequently oxidative damage is associated with the peroxidation reactions in membrane lipids. Thus, the use of the liposomes as cell model membranes is an important tool for oxidative stress studies. The liposomes with chlorophyll a (Chla) are excellent models for biomembranes and are successfully used to study the interactions of different agents inside the bilayer structures. The chlorophyll is an important photopigment, but insufficient exploited in biotechnological applications. The strong visible absorption and fluorescence of Chla allow its use as a sensor for the interactions at molecular level and as a fluorescence marker; it reflects certain aspects of the supramolecular structure of the lipid phase: fluidity, lipid and liposomes aggregation [1]. It is known that the chlorophyll has antioxidant properties. Derivatives of chlorophyll a were found to be more effective radical quenchers than those of chlorophyll b [2]. This is why we utilised in our studies the Chla. Paper presented at the 5th International Balkan Workshop on Applied Physics, 5 7 July 2004, Constanþa, Romania. Rom. Journ. Phys., Vol. 50, Nos. 9 10, P , Bucharest, 2005

2 1172 M. E. Barbinta-Patrascu, Laura Tugulea 2 Since liposomes can undergo many different changes with the time passage, one of the ways to overcome this problem is an antioxidant including as a component of the lipid membrane. In order to improve the liposomes stability, vitamin E (α-tocopherol acetate) was used as an antioxidant (scavenger for oxygen free radicals, lipid peroxide radicals and singlet oxygen) and as a membrane stabilizer (influencing the membrane lipid organization and restricting the molecular mobility). The present work was aimed at using the liposomes with Chla as membrane models to study the interaction of different agents at the molecular level. 2. EXPERIMENTAL A reproducible protocol for Chla-SUVs (small unilamellar vesicles) preparation was developed. Chlorophyll a extraction. Chla was extracted from fresh spinach leaves by a chromatographic method using different organic solvents (petroleum ether, n-propanol and ethanol) as mobile phase. The purity of the collected chl a samples was monitored by VIS absorption spectra recorded with a computer assisted PERKIN-ELMER LAMBDA 2S spectrophotometer. Liposomes preparation. Liposomes were prepared using the thin-film hydration method. The lipids used: β-stearoyl-γ-oleoyl L-α-phosphatidylcholine (SOPC) and dimyristoyl phosphatidylcholine (DMPC) were dissolved in chloroform together with Chla (Chla/lipid molar ratio = 1/100) in a rotary evaporator BIOBLOCK SCIENTIFIC Heildolph 94200, 120 rpm. Multilamellar vesicles were obtained by the thin-films hydration with a phosphate buffer solution (NaH2PO4-K2HPO4), ph = Small unilamellar vesicles (SUVs) were obtained from multilamellar vesicles by mechanical dispersion (VIBRAX stirrer, 200 rpm) and bath sonication (BRANSON 1210). The size and morphology of liposomes as determined from spectral data were correlated with negative stain electron microscopy [3]. The α-tocopherol acetate was incorporated (2 mol% of phospholipids final concentration) into liposomal membrane during the lipid thin film preparation together with Chla to increase liposomes stability. Different types of liposomes were obtained: 1) Chla/ DMPC liposomes; 2) Chla/ DMPC/ α-tocopherol acetate liposomes; 3) Chla/ SOPC liposomes; 4) Chla/ SOPC/ α-tocopherol acetate liposomes. 3. RESULTS AND DISCUSSION Chlorophyll a incorporated in the lipid bilayer of liposomes was used to monitor the oxidative effects on liposomes. A connection between lipid

3 3 Studies on lipid model membranes 1173 oxidation and Chla oxidation exists. Antioxidant effect of α-tocopherol acetate was studied by exploiting the spectral properties (absorption in VIS) of Chla. It is known that the liposomes scatter light. The over-estimated absorbances values were corrected for the contribution of light scattering. Fig. 1. Time evolution of the DMPC/vitamin E/Chla liposomes. Fig. 2. Time evolution of the DMPC/Chla liposomes

4 1174 M. E. Barbinta-Patrascu, Laura Tugulea 4 Figs. 1 4 show the evolution in time of the liposomes. During the time passage, a modification in the Soret band and at the main red peak is observed. Fig. 3. Time evolution of the SOPC/vitamin E/Chla liposomes. Fig. 4. Time evolution of the SOPC/Chla liposomes.

5 5 Studies on lipid model membranes 1175 To monitor the Chla oxidation in the presence of the atmospheric oxygen and the degradation with the liposomes ageing, several parameters were calculated: the absorbance ratio E437nm/E419nm and the relative variation of the absorbances ratio % = (ΔR/R0)*100, where R = E437nm/E419nm; Ro = the absorbance ratio in the 1th day; Ri = the absorbance ratio at several times; ΔR = R0 Ri. The Chla bleaching results in a spectral modification in the Soret band, in a great decrease of the absorbance ratio E437nm/E419nm, therefore in during the time passage (see Fig. 5). Fig. 5. Relative decrease in the Soret band absorbance ratio during the time passage. Great values for the relative variation of the absorbances ratio indicate a degradation of the Chla incorporated in liposomes. Thus, in the Chla-vitamin E-liposomes, the values of this parameter are less than in the Chla-liposomes; this fact suggests that the liposomes with vitamin E incorporated are more stable in time. For the SOPC liposomes, the values of this parameter are greater than in DMPC liposomes, because the SOPC system contains unsaturated fatty acids which are more susceptible to oxidation than the DMPC system which contains only saturated compounds. This fact suggests a connection between lipid oxidation and Chla oxidation. In order to study the influence of different agents on the bilayer structures at molecular level, different concentrations of procaine were added to liposomal suspensions.

6 1178 M. E. Barbinta-Patrascu, Laura Tugulea 8 Procaine is a local anesthetic; their potency is determined mainly by lipid solubility. The procaine molecule consists of a lipophilic portion containing an aromatic ring, an intermediate chain, and a hydrophilic amine. The Figs. 6 9 show the procaine effects on lipid membrane of the liposomes. There is a competition between Chla and procaine in the lipid bilayer evidentiated in the case of the Chla/Vitamin E/ SUVs. Because their smaller dimensions, Chla is excluded in the aqueous environment, thus explaining the peak at the 740 nm characteristic for the Chla-water adduct (Chla H 2 O)n. 4. CONCLUSIONS Chlorophyll a incorporated in the liposomal lipid bilayer was used as a sensor to monitor the oxidative effects on liposomes and to study the influence of different agents on the lipid bilayer. By incorporating vitamin E into membrane bilayer, the Chla oxidation, as monitored by modification of absorption spectra in VIS, was reduced in this case. Chla and procaine proved to be competitors in the lipid bilayer. A future task is also to improve the liposome stability by incorporating different antioxidants with synergistic actions and also modifying the bilayer composition. REFERENCES 1. L. Tugulea, S. Pascanu, Chlorophyll a -molecular sensor in model lipid membranes, Romanian J. Biophys., vol. 6, nos. 1 2, M. G. Ferruzzi et al., Antioxidant and Antimutagenic Activity of Dietary Chlorophyll derivatives Determined by Radical Scavenging and Bacterial Reverse Mutagenesis Assays, J. of Food Sci., Vol. 67, No. 7, M. E. Barbinta-Patrascu, M. Voinea, L. Tugulea, Antioxidant effects on liposomes with chlorophyll a, Ann. Bucharest Univ., p , 2003.

7 Fig. 6. Procaine effect on the DMPC/vitamin E/Chla liposomes. Fig. 7. Procaine effect on the SOPC/vitamin E/Chla liposomes.

8 Fig. 8. Procaine effect on the DMPC/Chla liposomes. Fig. 9. Procaine effect on the SOPC/Chla liposomes.