Determination of singlet oxygen quantum yields with 1,3-diphenylisobenzofuran in model membrane systems

Journal of Biochemical and Biophysical Methods, 27 (1993) 143-149

143

© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-022X/93/$06.00

JBBM 01016

Determination of singlet oxygen quantum yields with 1,3-diphenylisobenzofuran in model membrane systems Marianne Krieg Department of Pediatrics, MACC Fund Research Center, Medical College of Wisconsin, Milwaukee, 1411(USA) (Received 19 February 1993) (Revisedversion received 13 April 1993) (Accepted 26 April 1993)

Summary The oxidation of 1,3-diphenylisobenzofuran by singlet oxygen was investigated in methanol and in two different types of liposomes. It was found that at high concentrations of scavenger 1,3-diphenylisobenzofuran, e.g., > 100 /zM in methanol, the 1:1 oxidation stoichiometry is lost and more than one scavenger molecule per molecule of singlet oxygen is consumed. In model membrane systems, where local scavenger concentrations are high due to compartmentalization, correct singlet oxygen quantum yields with 1,3-diphenylisobenzofuran are only determined if the increased oxidation is taken into account. Key words: 1,3-Diphenylisobenzofuran; Liposome; Merocyanine 540; Quantum yield; Rose bengal; Singlet oxygen

Introduction In recent years, the interest in photosensitization processes has surged mainly due to dye-mediated photodynamic effects observed in biological systems. Since singlet molecular oxygen (10 2) is believed to be a key intermediate in photodyCorrespondence address: M. Krieg, Department of Pediatrics, MACC Fund Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Abbreviations: DMPC, dimyristoyl phosphatidylcholine; DPBF, 1,3-diphenylisobenzofuran; DPPC, dipalmitoyl phosphatidylcholine; RB, rose bengal; 10 2, singlet molecular oxygen (lAg); ~-DPBF, quantum yield of 1,3-diphenylisobenzofuran consumption; ~,a, singlet oxygen quantum yield.

144

namic tumor therapy [1], the determination of singlet oxygen quantum yields (4~j) is indispensable for mechanistic investigations and the development of future photosensitizers. Due to problems associated with the detection of 10 2 in biological environments [2,3] and since primary photodamage occurs in cellular membranes [4], model membrane systems, such as micelles, vesicles and liposomes, are widely used to investigate the effect of compartmentalization on the production of 10 2.

Generally, the infrared luminescence technique is the ideal method for the determination of q~a values. However, the radiative deactivation process of IO 2 leading to the emission at 1269 nm is strongly medium dependent and, unfortunately, the rate constant for this process has its lowest value in water [3]. In aqueous and hence in biomimetic and biological media, the luminescence technique lacks the sensitivity to produce reliable quantitative data on the IO2-production of sensitizers with medium to low q~j values [5]. As a consequence, 4~j determinations in such media require the use of indirect detection methods with 1,3-diphenylisobenzofuran (DPBF) being the most frequently used I O2-scavenging agent. Recently, however, it was reported that in liposomal systems the use of DPBF may lead to the determination of erroneous (too large) q~j values [6]. It has been proposed that due to a high local concentration of DPBF in the lipid bilayer, the DPBF-endoperoxide is able to react with a second DPBF molecule to cause the consumption of two DPBF molecules per molecule of IO 2 [6,7]. On the other hand, a recent mechanistic study on the hematoporphyrin sensitized photooxidation of DPBF in small dipalmitoyl phosphatidylcholine (DPPC) liposomes suggests that interactions between excited photosensitizer and ground state DPBF may play a role as well, e.g., high concentrations of both sensitizer and DPBF enhance the probability of energy transfer from triplet photosensitizer to ground state DPBF

[81. In this work the rose bengal (RB) sensitized photooxidation of DPBF in solution and liposomes was investigated and it was found that at elevated scavenger concentrations a change in oxidation stoichiometry occurs due to intermolecular DPBF interactions. In model membrane systems, correct q~a values are only obtained if this change in oxidation stoichiometry is taken into account.

Materials and Methods

Rose bengal was from Sigma (St. Louis, MO) and purified according to literature [9]. DPBF (Aldrich, Milwaukee, WI) and merocyanine 540 (Sigma, St. Louis, MO) were used as provided. Dimyristoyl phosphatidylcholine (DMPC) and DPPC (Sigma) were of 99 + % purity. Actinochrome 475/610 was purchased from Amko (Tornesch, Germany). Unilamellar liposomes with a diameter of 70-80 nm were prepared by a modified injection method [10] using a 30 mM potassium phosphate buffer (pH 7.0 at 20°C). The incorporation of monomeric RB and DPBF was confirmed by absorption spectroscopy.

145 Steady-state irradiation experiments were performed at 24 + I°C as previously described [11] by using a GG475 cut-off filter, a 552 + 10 nm interference filter (Schott, Mainz, Germany) and various calibrated neutral density filters (Oriel, Stratford, CT). The quantum yield of DPBF consumption (~-DPBF) was determined by following the bleaching of DPBF with irradiation time by absorption spectroscopy at 410 nm (solution) or 418 nm (liposomes) and by performing actinometry with Actinochrome 475/610 [12]. Only data points with < 15% bleaching of DPBF were evaluated. For each determination, three to five runs were carried out and results were always independent of the sensitizer concentration. Overall concentrations of RB were in the range of 2-15 ~ M in methanol and 5-21 /~M in liposomes (lipid : dye = 70 : 1-340 : 1).

Results and Discussion

As previously described [6,13], q~a values can be determined with DPBF by using Eqn. 1:

1

1( 1+

t~-DPBF ~A

(1)

where/3 represents the ratio k d / k r with k d being the rate constant for the natural decay of lO 2 to its ground state and k r the rate constant for the chemical reaction between 10 2 and DPBF. Plotting experimentally obtained values of 1/(b.DPBF VS. I / [ D P B F ] results in a linear relationship and the reciprocal of the intercept produces ~a. When scavenging experiments are performed in methanol with DPBF concentrations of 7-40/xM, a linear relationship according to Eqn. 1 is observed and the intercept produces a q~a = 0.69 + 0.12 for RB. This qbz is in good agreement with a published value of 0.76 in methanol [14]. When higher DPBF concentrations are used, i.e., > 90/zM, a linear behavior is still observed, but the reciprocal of the intercept now produces a value of 1.02 + 0.10 which is approx. 1.5-times larger than the real ~ . Similarly, when DPBF scavenging experiments are performed in liposomes, the linear relationship is maintained, but the reciprocal of the intercepts (Eqn. 1) produces values which are larger than one, i.e., 3.03 + 0.36 in DMPC- and 1.46 + 0.24 in DPPC-liposomes. In comparison, RB has a q~z = 0.75 in all types of liposomes [15,16]. Although overall DPBF concentrations in liposomes were 7 - 5 0 / z M , true concentrations were considerably higher due to incorporation of the scavenger into the lipid bilayer: with a radius of 39 nm for DMPC- and 35.5 nm for DPPC-liposomes [10], an aggregation number of 34292 and 29837 for DMPC and DPPC [10], respectively, and assuming a spherical shape with a bilayer thickness of 4.5 nm [17], true concentrations are 505 (DMPC) and 475 (DPPC)-times larger than overall concentrations, i.e., liposomal DPBF concentrations were 3000-25 000/xM.

146 These results in homogeneous and biomimetic systems suggest that at elevated scavenger concentrations Eqn. 1 indeed is no longer valid and hence erroneous q~z values are obtained. Although high DPBF concentrations play a role, these experiments do not allow to determine if scavenger-scavenger a n d / o r photosensitizer-scavenger interactions are involved. In order to probe the existence of photosensitizer-DBPF interactions, scavenging experiments in liposomes were carried out with D P B F and RB locally separated by solubilizing the substrates in separate liposomes. In DMPC-, as well as in DPPC-liposomes, experimental results are not affected by this separation. Consequently, it must be concluded that at elevated DPBF concentrations a change in the generally observed 1:1 oxidation stoichiometry [18] occurs and that photosensitizer-DPBF interactions [8] do not play a role. The oxidation of more than one DPBF molecule per molecule of 10 2 leads to an increase in ~DPBF values which results in the observed determination of e r r o n e o u s / t o o large q~a values. A recent mechanistic investigation of the methylene blue sensitized oxidation of 2,5-diphenylfuran in acetonitrile has shown that the oxidation yield increases due to the onset of a furan induced chain reaction at high scavenger concentrations [19]. The following expression was developed by taking the chain reaction into account [19]:

~b oaaF

~ba

1+

~

~ a+[DPBF]

(2)

where F represents the degree of deactivation of the chain carrier and a a ratio of chain carrier oxidation rates. Since our data show that only interactions between DPBF molecules are the actual reason for the determination of higher ~DPBF values, the applicability of Eqn. 2 on our experimental data was investigated. The experimental data obtained in methanol were fitted to Eqn. 2 by using a @a = 0.76 for RB [14], a/3 = 7 . 1 0 -5 M in methanol [20], and an experimentally determined F value of 0.75 (extrapolated using data with [DPBF] > 90/xM). Fig. 1 shows the experimental data and the fit to a of Eqn. 2. The fit is in good agreement with the experimental data and provides an a = 1 - 1 0 -5 M in methanol. Since Eqn. 2 changes to Eqn. 1 when DPBF concentrations are low, i.e., [DPBF] << a ( F is an efficiency and < 1), this a value means that at [DPBF] > 100 /xM the chain process becomes efficient and hence Eqn. 1 is no longer valid. On the other hand, at high DPBF concentrations or for [DPBF] x F >> a, Eqn. 2 transforms into Eqn. 3:

t~ - D P B F

~zl

1+

(3)

Eqn. 1 and Eqn. 3 are in agreement with the reported results in homogeneous and biomimetic media, and are able to explain why always linear relationships but different intercepts were obtained: when scavenger concentrations become larger

147 I .00

0

,

.

0.00. 0

i

2

T

l

5 I

250

~ I

500

I

750

DPBF c o n c e n t r a t i o n (/~Y) Fig. 1. Dependence of t~DPBF on the concentration of DPBF in methanol. Experiments were performed at 24_+1°C using RB concentrations of 5 and 15 ~M. v, Experimentally obtained values; --, fit to a using Eqn. 2 with/3 = 7.10 -5 M, ~ = 0.76, and F = 0.75. than a, Eqn. 3 is activated and the factor F in the intercept becomes important (or F < 1). Eqn. 3 is also important for the determination of ~ values in model membrane systems. In these media, local scavenger concentrations wil! be always so large, e.g., > 3000 /zM, that the application of Eqn. 3 is justified. By using overall scavenger concentrations (true concentrations are not necessary) and the corresponding factor F, ~a values are obtained from the intercept of the linear relationship expressed by Eqn. 3. The factor F, however, has to be determined for each biomimetic system and type of scavenger separately. As a matter of fact, RB is an ideal standard for such determinations since its q~z is not influenced by the environment, i.e., q~z = 0.72 _+ 5% in all investigated media [14-16,21,22]. F values for methanol and liposomes are summarized in Table 1. In order to actually test Eqn. 3 in model membrane systems, ~a values of merocyanine 540 in D M P C - and DPPC-liposomes were determined and compared to literature values. By applying Eqn. 3 and using the corresponding factors F from Table 1, a q~a = 0.011 + 0.002 in DMPC- and a ~ = 0.038 + 0.005 in DPPCliposomes were measured. These values are in good agreement with literature values of 0.016 [11] and 0.02-0.05 [23] in DMPC-liposomes, and 0.037 [11] in DPPC-liposomes (literature values were obtained by using a reference). TABLE 1 Determination of 4)3 values of RB with DPBF: parameters in methanol and liposomes Medium

[DPBF] (/~M)

~,a/F (Eqn. 3)

F

methanol methanol DMPC DPPC

< 40 > 90 > 3,000 > 4,000

0.69 _+0.12 1.02+ 0.10 3.03 + 0.36 1.46+ 0.24

1.00 0.75 _+0.07 0.25 + 0.03 0.51 + 0.08

148

This study demonstrates that in model membrane systems a high local DPBF concentration promotes a change in oxidation stoichiometry. This work also shows that correct q~a values in model membrane systems are obtained if the change in oxidation stoichiometry is taken into account. The sensitized photooxidation of DPBF does not change from 1:1 to a clean 1:2 stoichiometry as suggested [6,7], but the stoichiometry and the factor F rather depend on the scavenger and the surrounding medium. These findings are in agreement with a recent observation that consumption of DPBF increases as DPPC-liposomes undergo phase transition from a gel- to liquid-like state [24]. Indeed, the determination of a smaller F value (representing a larger ~-DPBF) for DMPC-liposomes than for DPPC-liposomes reflects the higher fluidity of the DMPC-membrane at 24°C.

Acknowledgements This work was supported by NCI grant CA50733 and the MACC Fund.

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