STUDIES ON CELL MEMBRANE FUNCTIONALITY: AN EXPERIMENTAL SETUP FOR LIPOSOME EXPOSURE

M.D’Arienzo, A.Doria ,G.P. Gallerano, E. Giovenale A. Ramundo Orlando

ENEA C.R. Frascati - Via Enrico Fermi 45 - 00044 Frascati (Italy), INEMM-CNR -Via Del Fosso del Cavaliere, 100-00133 Roma (Italy)

In recent years, research into biological and medical effects of millimetre waves (MMW) has expanded greatly. The cell membrane is one of the site suspected to be a primary target for MMW radiation.

Several studies have indicated that millimetre wave irradiation significantly increases Ca 2+ uptake by membranes in different cells and these effects seem to be frequency dependent.

Changes of the capacitance of artificial membranes and of the transport of anionic compounds through bilayer have been also reported. Yet, in these experiments no frequency-specific effects of MMW resulted.

In this context we have chosen to study the effects induced by 80 to 140 GHz radiation on cell membrane functionality.

EXPERIMENTAL ACTIVITY

We have planned to conduct the irradiation of CA-loaded liposomes following the experimental procedure shown in the image. Small aliquots of liposomes with or without CA were placed in a 3 cm3 silica cuvette in the cell of CARY 50 spectrophotometer appositely modified to perform Free Electron Laser (FEL) irradiation in real time during the diffusion rate analysis. At the end of irradiation, saline buffer and p-NPA were added and the kinetic measurements were made.

RADIATION SOURCE

The millimetre wave source is a Free Electron Laser available at

the ENEA research centre.

FEL PROPERTIES:

Spectral Range: 80-140 GHz Bandwidth : 7 %Maximum peak power : 10 kW

THE LIPOSOMES Our cationic liposomes are composed of lipids normally found in natural membranes such as dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol), and positively charged stearylamine (SA). The soluble enzyme Carbonic Anhydrase (CA) is loaded into the aqueous space of liposomes.Once its substrate p-nitrophenyl acetate (p-NPA) is present in the external aqueous space of these liposomes basically it is slowly hydrolysed by the CA. Thus, any alteration of the lipid bilayer permeability induced by GHz radiation may be evaluated by the ability of the substrate to diffuse across the lipid bilayer. In this case it is rapidly hydrolysed by the CA entrapped inside the liposomes.

DPPC

CH2

+NH3

CH2

CH2

+N (CH3)3

CH2

O

O

O-P

OCH2

SA

CA-loaded liposome (most enzyme enclosed within

inner bilayer)

DPPC and SA polar head groups

Liposomes before being loaded with enzyme Carbonic Anhydrase (CA)

Liposomes loaded with enzyme Carbonic Anhydrase (CA)

0.06 ml 0.44 ml

0.5 ml

0.5 ml 2.4 ml 0.1 ml

ABSORPTION MEASUREMENTS

EMPTY LIPOSOMES: (50 ± 3) cm-1 FILLED LIPOSOMES: (55 ± 3) cm-1 TRIS-SALINE SOLUTION: (43 ± 8) cm-1 TRIS-SALINE SOL. With ENZYME: (48 ± 8) cm-1

0 100 200 300 400 500 600 700 800 900 1000 1100 1200

TR

AN

SM

ISS

ION

TR

AN

SM

ISS

ION

SAMPLE THICKNESS (SAMPLE THICKNESS (m)m)

1

0.1

0.01

Liposomes with enzime

Trisaline solution with enzime

“Empty” Liposome

Trisaline solution

DATA REPORTED ON THE GRAPH SHOW THE SO-CALLED “MENISCUS EFFECT” AT THE EDGE OF THE PETRI DISH, THAT RESULTS IN AN EFFECTIVE SAMPLE THICKNESS SMALLER THAN THE NOMINAL THICKNESS.

0 25 50 75 100 125 150 175 200

400

300

200

100

0

MEDIAN DIAMETER : (39 MEDIAN DIAMETER : (39 20) nm 20) nm

CC OO UN

UN

TT

VESICLE DIAMETER (nm)VESICLE DIAMETER (nm)

0 25 50 75 100 125 150 175 200

400

300

200

100

0

CC OO UN

UN

TT

VESICLE DIAMETER (nm)VESICLE DIAMETER (nm)

MEDIAN DIAMETER : (32 MEDIAN DIAMETER : (32 10) nm 10) nm

VESICLE SIZE DISTRIBUTIONFROM EM FILMS OF NEGATIVELY STAINED DPPC:CHOL:SA=5:3:2 CA-LOADED LIPOSOMES.

CONCLUSIONS

THEORETICAL APPROACH: MIE SIMULATIONS

An external shell (lipid membrane whose refractive index is the same of fat at 120 GHz 1.6-0.5i)

An inner core four times bigger (the water or ahnydrase vesicle whose refractive index can be approximated to water at 120 GHz 3.4-1.9i)

A model has been developed in which liposomes are considered a bilayer system made of two stratified spheres:

Membranen=1.6-0.5i

Water vesiclen=3.4-1.9i

EXTINCTION,SCATTERING AND ABSORPTION CROSS SECTIONS FOR LIPOSOMES:

FIGURE A-B SHOW REDUCED CROSS SECTION FOR STRATIFIED SPHERES (LIPOSOMES) IN A NON ABSORBING MEDIUM. FIGURE C SHOWS EXTINCTION CROSS SECTION FOR ENTIRE LIPOSOMES IN WATER (WITH COMPLEX REFRACTIVE INDEX AND THUS ABSORBING)

0

2

0 1

Extinction

Scattering

Absorption

REDUCED CROSS SECTIONREDUCED CROSS SECTION

MIE PARAMETERMIE PARAMETER

R2

2 Rmedium

Extinction

Scattering

Absorption

0

2

0 0.5 1

REDUCED CROSS SECTIONREDUCED CROSS SECTION

MIE PARAMETERMIE PARAMETER

R2

2 Rmedium

Reduced cross section of the core:

The extinction is owed to absorption of

radiation up to a value of the Mie parameter

0.8

Reduced cross section of the

external membrane: in the range 0-1 for the Mie parameter,

extinction of radiation is again

owed to absorption rather than scattering

In the graph the results of the irradiation of tris-saline solution and tris-saline solution with anhydrase enzime are also shown. The experimental set up and the radiation properties are exactly the same used for the irradiation experiment.

•SPECTROSCOPIC MEASUREMENTS CAN BE USED TO VERIFY THE EFFECT OF THz RADIATION ON CELL MEMBRANES

•AN IRRADIATION SYSTEM IS BEING SET-UP AT THE ENEA CENTER OF FRASCATI

•THE ENEA COMPACT FEL HAS BEEN USED TO PERFORM PRELIMINARY MEASUREMENTS ON THE OPTICAL PROPERTIES OF LIPOSOMES, THAT WILL BE USEFUL WHEN PERFORMING THE IRRADIATION EXPERIMENT

•DIFFERENCES IN ABSORPTION OF VESCICLES DEPENDING ON DIMENSIONS AND VESCICLE LOADING HAVE BEEN OBSERVED

0