ELECTRO-MANIPULATION OF THE BIOLOGICAL CELLS basIcs and applIcatIos

ELECTRO-MANIPULATION OF THE BIOLOGICAL CELLS basIcs and applIcatIos

The accumulation of the mobile charges at the interfaces between media with different electrical properties (interfacial polarization) induces the occurrence of an electric dipole into a cell exposed to the action of an external electric field. The consequences depend on the electric field features (amplitude, frequency, spatial distribution) and on the cell electrical characteristics (conductivity and permittivity of the media which compound the cell). Some of the mechanical effects produced by the interaction between the electric field and the induced dipole led the so called “electro-manipulation” of the biological cells. On the other hand, the electrical potential induced across the cellular membranes is able to produce local reversible increase of the permeability (electroporation) allowing to the exogenous chemical species to diffuse into the cell.

The electro-mechanical techniques (dielectrophoresis, electro-rotation and electro-orientation) will be reviewed addressing the mechanisms, the theoretical models and the applications. The multi-shell model and its use to the description of the yeast cells mechanical behavior in an external electric field will be discussed. Also other techniques rely on the cell electro-mechanical behavior will be described.

The electroporation of the artificial and natural membrane will be analyzed in connection with the mechanism of the phenomenon reversibility. The diffusion of the different kind of molecules will be discussed.

The combination of these effects with other techniques, as laser trapping of the cell, produced other complex noninvasive methods for cells investigation and manipulation that will be briefly presented.

OCTOBER 14, 2003 – WEDNESDAY

HALL B

ORAL PRESENTATION 1

IMMUNOLIPOSOMES DIRECTED TOWARD VCAM-1: VEHICLES FOR SPECIFIC DRUG DELIVERY TO ACTIVATED ENDOTHELIAL CELLS

Manuela VOINEA¹, Elena DRAGOMIR¹, Ileana MANDUTEANU¹, Monica CAPRARU², Dorin CAPRARU², Maya SIMIONESCU<sup>1

1</sup>Institute of Cellular Biology and Pathology “Nicolae Simionescu", Bucharest, ROMANIA, ² Millitary Hospital "D.Gerota", Bucharest, ROMANIA

mvoinea@simionescu.instcellbiopath.ro

Introduction: The use of liposomes as carriers for selective targeting of drugs and genes to endothelial cells is an attractive strategy in the treatment of cardiovascular diseases. A potential molecular target is vascular cell adhesion molecule-1 (VCAM-1) which is over-expressed in vivo by activated endothelial cells (EC) covering the developing atheromatous plaque and has an significant role in leukocyte adhesion to these cells. Taking advantage that VCAM-1 can be induced in cultured EC in the presence of inflamatory cytokines and endotoxins, we searched for the mechanisms of interaction between activated EC and liposomes targeted to VCAM-1 expressed on the cell surface.

Materials and methods: Human EC line (EAhy 926) activated with TNF- were exposed to small unilamellar liposomes, plain or coupled with anti-VCAM-1 (L-VCAM-1) or with irrelevant IgG. For binding studies the cells were incubated with fluorescently labelled liposomes for 2h at 4^oC. To follow the fate of liposomes after binding to the cell's surface we analyzed the uptake and the transmigration of liposomes and the subsequently induced intracellular changes using radioactively labelled liposomes. As methods, flow cytometry, liquid scintilation counting, fluorescence microscopy and fluorimetry were employed.

Results: The data showed that: (i) liposome coupled to anti-VCAM-1 bind selectively to activated EC; (ii) the immunoliposomes are taken up by specific(e.g. receptor-mediated endocytosis) and unspecific mechanisms; (iii) binding of L-VCAM-1 to EC surface induced rearrangements of actin filaments and rises in intracellular calcium concentration; (iv) a small percent of liposomes transmigrate into subendothelial space.

Conclusion: The data suggest that VCAM-1 may be an appropriate molecular target for specific delivery of drugs to activated EC using immunoliposomes.

This work was supported by the Ministry of Education and Research, National Program VIASAN (Grant nr. 031/2001).

DEVELOPMENT OF TWO AMPEROMETRIC BIOSENSORS BASED ON CATALASE IMMOBILIZED IN GELATIN-ALGINATE AND GELATIN--CARRAGEENAN FOR ALCOHOL DETERMINATION

Erol AKYILMAZ and Erhan DİNÇKAYA

Ege University, Faculty of Science, Biochemistry Department, 35100 Bornova-İzmir/ TURKEY

akyilmaz@sci.ege.edu.tr

Ethanol is a toxic material which is very often in great need of being determined in forensic medicine and clinical toxicology especially as the alcoholic drinks are widely consumed.

Biosensors are defined as an analytical device incorporating a biological sensing element such as enzyme, tissue, microorganism, cell, and DNA with a suitable transducer.

In this study, two different amperometric biosensors based on catalase immobilized in gelatin-alginate and gelatin--carrageenan on a dissolved oxygen (DO) probe covered with a oxygen sensitive teflon membrane, were developed for the alcohol determination. Measurements were made by standard curves which were obtained by the determination of consumed oxygen level related to alcohol concentration according to two reactions catalyzed by catalase given below;

Catalase

H₂O₂ + H₂O₂ O₂ + 2H₂O (1)

CH₃CH₂OH + H₂O₂ CH₃COH + 2H₂O (2)

The response of the both of two biosensors depended linearly on a alcohol concentration range of 0.05 – 0.8 mM with a response time of two min. For the biosensors developed in order to optimize working conditions some optimization studies such as determination of optimum pH, temperature, the most suitable buffer system and concentration were done. In the characterization studies of the biosensors substrate specificity, reproducibility, determination of interference effects of some substances, operational and storage stability experiments were done. Results obtained in the optimization and the characterization studies for both two biosensors were also compared eachother.

ORAL PRESENTATION 3