Structural modifications of vesicular aggregates following gamma-irradiation

0146-5724/91

Radiar. Phys. Chem.Vol. 38, No. 6, pp. 513-517, 1991 Int. J. Rodiar.&I. InHum., Port C Printed in Great Britain. All rights reserved

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STRUCTURAL MODIFICATIONS OF VESICULAR AGGREGATES FOLLOWING GAMMA-IRRADIATION A. E. MANTAKA-MARKETOU~ and A. S. Domu Institute of Physical Chemistry, National Research Center, “Demokritos”, 15310Aghia Paraskevi, Attikis, Greece (Received 5 July 1990; received for publication 1 May 1991)

Abetract-The structural changes of the didodecyldimethylammonium bromide @DAB) vesicular bilayers after y-irradiation and under conditions where mainly ‘OH radicals are present are reported. Alterations of the vesicular structure, such as polarity and fluidity, were detected after a dose. of 0.65 kGy. A higher dose of _ 14kGy cause important damage to the well organized molecular structure and this is manifested by an important augmentation of the fluidity and polarity of the Stem region of the aggregates. Increased water penetration into the bilayer of the vesicle is probably the reason for these changes and electron micrographs support this hypothesis.

INTRODUCI’ION

The radiolysis products of water (OH, e,, HsO+, H) interact with biological membranes, entering into reactions with components of structural importance such as lipids or lecithin bilayers (Edwards et al., 1984; Barber and Thomas, 1978; Patterson and Hasegawa, 1978). The attack by ‘OH radicals on the bilayers of biological or synthetic membranes leads to structural damage caused by subsequent reaction of the secondary (organic) radicals. Radical-radical reactions leading to cross-linking or C-C bond breakage and could seriously affect the highly-ordered nature of the bilayer. This will be reflected in the physical properties of the bilayer such as rigidity or microviscosity (Barber and Thomas, 1978; Purohit et nl., 1980; Yoney and Kato, 1978; Yoney et al., 1979a) and polarity (Barber and Thomas, 1978; Purohit et al., 1980; Yoney et al., 1979b), owing to increased water penetration into the bilayer. Surfactant vesicles, or liposomes, have been used as biological membrane models (Fendler, 1982) and have been proposed to be drug carriers (Fendler, 1984; Romero et al., 1978). It has been reported (Paleos et al., 1982) that chemihnninescence (CL) in organized media is associated with differentiation in both quantum yields and emission spectra as compared with the same materials in a homogeneous medium. Furthermore, factors affecting the fluidity of biological membranes also affect the chemiluminescence of the lucigneninhydrogen peroxide reaction, in membrane mimetic agents such as the didodecyldimethylammonium bromide (DDAB) vesicular system of the present work (Mantaka-Marketou et al., 1985). We have tTo whom correspondence should be addressed.

investigated the y-radiation effect on the fluidity and polarity of DDAB vesicular structures by means of chemiluminescence and fluorescence probe measurements, employing the lucigenin light reaction and 17a -ethinylestradiol respectively. Finally, electron micrographs show the destructive influence of the radiation on the ordered structure of the vesicle. EXPERIMENTAL

Didodecyldimethylammonium bromide (DDAB) (Kunitake, 1977a, b) and N-methylacridone (NMA) (Akiba et al., 1978) were prepared and purified as described elsewhere. lO,lO’-Dimethyl-9,9’-biacridinium dinitrate (Lucigenin, Aldrich) was used without further purification. Vesicles were prepared by the sonication method (M.S.E. sonicator, 30min) and then extruded through 0.2pm cellulose nitrate filters (Sartorius). Irradiations were carried out in a @‘CoGammacell 220 at a dose rate of 9.99 Gymin-‘. Before irradiation the samples were deaerated by flushing with N,O, scrubbed with V*+ (to remove 0,). Chemiluminescence measurements were carried out in an Aminco “Chem-glow” photometer with the timer circuitry disconnected. To 250~1 of the sonicated, pre-irradiated solution of DDAB (lo-’ M), NaOH (30~1, 0.1 N) and H202 (30~1, 3%) were added and the light integrals Q were obtained. The same procedure was followed with non-irradiated DDAB (lo-* M) sonicated systems and values of Q, were measured. The ratio Q/Q,, is the same as the corresponding ratio of CL quantum yields. Fluorescence spectra of NMA were run on an Aminco-Bowman SPF spectrophotofluorometer. Finally, electron micrographs were taken with a Siemens Elmiscope 101 electron microscope. Both the 513

A. E. MANTAKA-MARKETOU and A. S. D~MASOU

514

10 -

irradiated (13.5 kGy) and non-irradiated samples were coloured with an aqueous 2% uranyl acetate solution. RESULTSAND I.

DISCUSSION

Fluorescence measurements 02 -

The fluorescent probe technique is commonly used to follow structural changes of an organized vesicular or micellar system. In our case, the estrogen hormone, 17cc-ethinylestradiol was used as a fluorescent probe in the DDAB vesicular system. Its fluorescence spectrum is sensitive to the solvent polarity, a red shift being detected on increasing solvent polarity (Tables 1 and 2) and a drop of the fluorescence intensity at the maximum wavelength being observed on increasing the polarity of the solvent (Fig. 1). From Table 1 we can conclude that the hormone is bound in a micro-environment of about the same polarity as that of ethanol. Therefore, 17crethinylestradiol is an appropriate probe to follow the changes in the polar region of the vesicle-water interface, where it is bound. Vesicular systems of DDAB, N,O saturated, were irradiated with various doses of y-rays To irradiated and to non-irradiated samples an appropriate quantity of hormone was added to a concentration of 1 x 10m4M; the fluorescence intensities at the maximum wavelength (I) and (I,) respectively, were measured and the results are shown in Fig. 2(a). In the same figure (curve b) the shift of the fluorescence maximum as a function of the irradiation dose is given. Under the irradiation conditions, and in the presence of N,O, the reductive solvated electrons (e,) are converted into oxidative ‘OH radicals according to: N,O+e;q--+N,+‘OH+OH-

(1)

Questions arise about any effects of the presence of Br- counterions of the aggregates. Indeed, the ‘OH radicals can react very rapidly with Br- as follows: Br-+OH+Br’+OH-

(2)

I 20

I

I

40 60 Ethanol

80

I

I loo

Fig. 1. Variation of the intensity ratio (I/I,) of the fluorescence maximum of 17a-ethinylestradiol (1 x 10m4M) in mixtures of ethanol-water (I) and in pure ethanol (f,), as a function of % ethanol (excitation 1, = 29&294nm).

Br’ + Br- --t Br,S

(3)

Br; + Br; -+ Br, + 2Br-

(4)

where k Br + OH= 1.2 x lo* M-’ SK’ (Matheson et al., 1966). The radical-ion Br; is adsorbed on the surface of the vesicle or micelle (Thomas, 1987; Proske and Henglein, 1978; Frank et al., 1976) and the probability of bromine production, after disproportionation, depends upon the concentration of the amphiphile. In our case, however, neither Brnor Br, quench the fluorescence of 17a-ethinylestradiol (the

4” 2

0.5

Dose ( kGy

324

I

Table 1. Fluorescence wavelengths of 17aethinylestradiol in various solvents Solvent

L (Fluorescence, nm)

H,O’ (neutral pH)

322 317 312 317

Ethanol Hexene DDAB

Table 2. Fluorescence wavelengths of 17~ethinvlestradiol in ethanol-H,0 mixtures % Ethanol 0

20 30 40 60 80 100

1 (Fluorescence. nm) 322 321 320 319 318 318 317

; 320 c x

316 0

5 Dose

10 ( kGy )

15

Fig. 2. Curve (a). Variation of the intensity ratio (I/I,,) at the fluorenscence maximum of 17a -ethinylestradiol (1 x 10e4 M) in N,O-saturated pre-irradiated (I) and nonirradiated (I,) vesicular DDAB systems, (at neutral pH), as a function of dose (excitation 1 = 29k294). Curve (b). Corresponding variation of the fluorescence maximum as a

function of dose.

Structural modifications of vesicular aggregates

515

influences of Br- in chemiluminescence measurements is discussed below). Taking into account the results of Figs 1 and 2 and those of Table 2, we can draw some conclusions regarding the polarity changes of the microenvironment of the probe as a function of irradiation dose: (a) For doses up to 0.65 kGy the polarity of the region, where the hormone is bound in the vesicle, remains unaltered and its value is the same as in ethanol. (b) From 0.65 and up to 6.80 kGy the polarity increases becoming the same as that of a mixture of ethanol:water (30:70) (c) From 6.80 to 13.00 kGy there is a “plateau” and the fluorescence intensity and the maximum wavelength remain roughly stable, indicating that the polarity does not change. (d) Finally, for doses > 13.00 kGy another increase of the polarity is detected reaching roughly that of aqueous solutions of the hormone. The plateau of curves (a) and (b) of Fig. 2 correspond to the same ratio of concentrations of the alcohol:water solutions. II. Chemilurninescence

measurements

The lucigenin-NaOH-H,Oz light reaction leads to electronically excited N-methylacridone (NMA). De-excitation of this primary emitter results in light emission with &,,, ca. 430 nm plus energy transfer to other species (lucigenin included) with subsequent emission at ca. 5OOnm. The light integrals of the chemiluminescent reaction were measured in DDAB vesicular systems, after NzO saturation and y-irradiation with various doses and were compared with those in non-irradiated systems. The possible role of H,Or-the molecular product of water radiolysis-was investigated. The calculated maximum amount of Hz02 produced with the highest given dose was of the order of 1.19 x lo-’ M and it was found that such a concentration of Hz02 had no effect on the light integrals.

I 0

I IO

5 Dose

(kGy

I 15

1

Fig. 4. Variation of fluorescence intensities of added NMA in N,O saturated, pre-irradiated (Z) and non-irradiated (Z,) DDAB vesicular systems as a function of dose.

The effect of the presence of Br- counterions was also considered in association with the CL measurements, since Br, is possibly produced according to reactions (2)-(4). In particular, using constant concentrations of DDAB and the emitter NMA and varying the concentration on added Br, we obtained the results given in Fig. 3, where an important quenching of the NMA fluorescence by Br, was detected. Using the same technique we found that Br- had a minor quenching effect. Furthermore, in N,O-saturated DDAB vesicular system, irradiated with doses up to 16.5 kGy, appropriate quantities of NMA were added and the fluorescence intensity of NMA was measured. The same procedure was followed in non-irradiated vesicular systems. The results are shown in Fig. 4, where no additional quenching on the NMA fluorescence in the irradiated vesicular systems was detected. It seems, therefore, that there is no significant accumulation of Br, in these systems. Effects on the quantum yield of the light reaction (expressed as Q/Q,, where Q is the measured light integral of irradiated and Q,, of non-irradiated samples) are shown in Fig. 5, as a function of dose. Experiments in the absence [curve (a)] and in the presence [curve (b)] of 17a!-ethinylestradiol were performed by adding the appropriate quantities of

1.0

P ,I2

0

0.0

0

t-

2 0.6

0.4

L

I

I

J

0

5

10

15

DosetkGy) 1

CBr,l

xlC4

M

Fig. 3. Variation of fluorescence intensities of NMA in the absence (I,) and in the presence (Z) of Br, as a function of [BrJ in DDAB vesicles.

Fig. 5. Quantum yield ratios of the lucigenin light reaction in N,O saturated pre-irradiated (Q) and non-irradiated (Qo) DDAB vesicular systems as a function of the dose: (a) in the absence of 17aethinylestradio~ (b) in the presence of 17a-ethinylestradiol.

516

A. E. MANTAKA-MARKETOU and A. S. D~MASW.

Fig. 6. Vesicular

DDAB

aggregates

NaOH, (lucinigen) and H,O,--as described in the experimental-to a pre-irradiated and non-irradiated vesicular system. The two curves show the same trend of change with dose; the lower ratio of the +/& for

Fig. 7. N20

saturated

vesicular

DDAB

in non-irradiated

solution

curve (b) is due to the ctrect of estrogen on the vesicular structure. which increases the fluidity of the system. Also, in this latter case. there is a pronounced effect of irradiation on the organired system.

aggregates

irradiated

with

13.50 kGy

Structural modifications of vesicular aggregates III. Efict of y-irradiation on the morphology of DDAB vesicles

Electron microscopy was employed, as described in the experimental section, to demonstrate the damage to the vesicle-water interface; after irradiation the difference of the appearance of the interface before irradiation (Fig. 6) and after irradiation (Fig. 7) is remarkable. During irradiation attack by the strongly oxidative ‘OH radicals has resulted in the observed grooves of the vesicular-water interface. Apparently here, primary ‘OH radical attack and, to a minor extent, H-atom attack, results in hydrogen abstraction from a-methylene groups and to C-C bond breakage of the carbohydrate chains followed by cross-linking with the model membrane or by removal of chain fragments. All of these events result in the perturbation of the water-vesicle interface and the penetration of an appreciable quantity of water. The appearance of the surface grooves of the aggregates in the electron micrographs and the alteration of the chemiluminescence and fluroescence data after irradiation support this hypothesis. The alterations of the micro-environment of the amphiphile depend upon dose. Hence, doses up to 0.65 kGy do not affect the well-organized vesicular structure and parameters such as fluidity and polarity remain unchanged. On increasing the dose (up to 6.50 kGy) the number of the structural molecular units attacked increases and becomes important. The intermolecular forces of cohesion are disturbed and the probability of C-C bond breakage is higher, resulting in an important increase of the polarity and fluidity of the Stem region micro-environment. The observed “plateau” for higher doses implies a steady-state situation, in which the vesicular structure can confront the damage by a repair process. The conclusion that the vesicular structure is quite resistant to irradiation can be drawn from Fig. 7

517

where 13.50 kGy does not completely destroy the structure. On the other hand, structural changes are important and a significant water penetration alters the polarity and fluidity of the Stern region. Such phenomena have also been reported, after irradiation, for lecithin liposomes and erythrocyte membranes. REFERENCES Akiba K., Ishikawa K. and Inamoto N. (1978) Bull. Chem. Sot. Jpn. 51, 674. Barber D. J. W. and Thomas J. K. (1978) Radial. Res. 74, 51. Edwards J. C., Chapman W. A., Cramp W. A. and Yalvin M. B. (1984) Prog. Biophys. Biol. 43, 71. Fendler J. H. (1982) Membrane Mimetic Chemistry. Wiley, New York. Fendler J. H. (1984) Chem. Brit. 1096. Frank A. J., Gtitzel M. and Kozak J. J. (1976) J. Am. Chem. Sot. 98, 3317. Kunitake T. (1977a) J. Macromot. Sci. A13, 587. Kunitake T., Okahata Y., Tamaki K., Kumamura F. and Takayanagi M. (1977b) Chem. LQU. 387. Mantaka-Marketou A. E., Vassilopoulos G. and Nikokavouras J. (1985) Monatsh. Chem. 116, 973 (and references cited th&ein). Matheson M. S.. Mulac W. A.. Weeks J. L. and Rabani J. (1966) J. Phy.k Chem. 70, 2092. Paleos C. M., Vassilopoulos G. and Nikokavouras J. (1982) J. Phorochem. 18, 327 (and references cited therein). Patterson L. K. and Hasegawa K. (1978) Ber. Bunsenges. Phys. Chem. 82, 95 1. Proske T. and Hengfein A. (1978) Ber. Bunseges. Phys. Chem. 82, 711. Purohit S. C., Bisby R. H. and Cundall R. B. (1980) In?. J. Radial. Biol. 30, 147. Romero A., Tran C. D., Klahn P. L. and Fendler J. H. (1978) Life Sci. 22, 1447. Thomas J. K. (1987) Radiation Chemistry: Principles and Applications. VGH Publishers. Yon& S., Todo T. and Kato M. (1979a) Rod. Res. 80,484. Yonei S.. Todo T. and Kato M. (1979b) ht. J. Radial. Biol. . 35, 16i. Yoney S. and Kato M. (1978) Rad. Res. 75, 31. I