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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

The azide anion is often used as a physical quencher of singlet oxygen, the important active intermediate in photosensitized oxidation. An observed effect of azide on the rate of a reaction is considered an indication to the involvement of singlet oxygen. In most biological photosensitizations, the light-absorbing sensitizer is located in a membrane or in an intracellular organelle, whereas azide is water soluble. The quenching it causes relies on a physical encounter with singlet oxygen during the latter's short lifetime. This can happen either if azide penetrates into the membrane's lipid phase or if singlet oxygen is intercepted when diffusing in the aqueous phase. We demonstrate in this article the difference, in liposomes’ suspension, between the effect of azide when using a water-soluble and membrane-bound chemical targets of singlet oxygen, whereas this difference does not exist when micelles are used. We explain the difference on the population of sensitizer and target in the liposome vs micelle. We also show the effect that exists on azide quenching of singlet oxygen by electrically charged lipids in liposomes. This is a result of the accumulation or dilution of azide in the debye layer near the membranes’ surface, due to the surface Gouy–Chapman potential.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Biological photosensitization entails the use of a photosensitizer molecule that, upon the absorption of light, undergoes a series of energy transduction processes. At the end of these transitions, an oxidizing species is generated, which can induce a photochemical reaction or, when employed in living cells, is cytotoxic and causes cell death. Biological photosensitization has been used very widely for photodynamic therapy (PDT) of cancer and bacterial and viral infections [1-5]. PDT is currently employed as an experimental procedure for various types of solid tumors and several photosensitizer molecules were approved for PDT by regulatory agencies.

Several molecular species were implicated as intermediate agents that are capable of carrying out the chemical oxidation reaction, which serves as the damaging step in biology. These include various oxygenic radicals, but the main culprit appears to be singlet molecular oxygen (1Δg). Monitoring singlet oxygen in samples that undergo photosensitization reactions is possible by direct observation of its weak luminescence, centered near 1270 nm [6]. Indirect methods employ chemical targets that upon oxidation or peroxidation exhibit a marked change in their ESR signal [7], or even more simply, by monitoring changes in absorption or fluorescence spectra of the trap. These targets include chemicals such as 9,10-dimethylanthracene (DMA) or 1,3-diphenylisobenzofuran, which have very high reaction rate constants with 1Δg (ca 109 m−2 s−1 ;[8]) and their fluorescence disappears upon oxidation [9-11]. 9,10 disubstituted anthracenes, such as DMA, interact with singlet oxygen almost exclusively and with high yield, to form the nonfluorescent endoperoxide [8, 12, 13]. Newly reported detector molecules include Singlet Oxygen Sensor Green® and fluorescein derivatives [14, 15].

In all indirect methodologies, which rely on the use of chemical molecular traps, there is a need to establish the identity of the reactive species that the target responds to and reacts with, as the specificity to singlet oxygen needs proof. A common approach is to employ singlet oxygen quenchers to establish whether they do indeed diminish the rate of the reaction that is being studied. Such quenchers that have a very high intrinsic quenching rate of singlet oxygen include β-carotene, 1,4-diazabicyclo[2.2.2]octane and azide anion. β-carotene is highly hydrophobic hence it cannot be used in aqueous media and its exogenous insertion into cellular membranes is not easy.

Sodium azide is an extensively used 1Δg quencher. It was shown to be a purely physical quencher [16], with a reported rate constants for the quenching reaction of 1Δg in the range of [5-15].108 m−1 s−1 [8]. The azide anion is therefore a very efficient quencher, and aqueous concentrations of 1–50 mm are usually used. Sodium azide was employed, however, also in cases when the sensitizer was bound to a liposomal or biological membrane [17-24]. In such cases, the quenching action of the azide anion might happen while singlet oxygen is diffusing in the aqueous solution, following its generation by a membrane- or cellular-bound photosensitizer. Azide might also quench singlet oxygen within the membrane's lipid bilayer, if azide penetrates momentarily into the membrane phase. This might be enabled in spite of the azide being charged because it has a low charge-to-volume ratio. This is similar to the way an iodide anion, also having a low charge-to-volume ratio, is capable of shooting into a membrane's lipid phase and quenching fluorophores that are buried deep inside a membrane [25, 26]. Naturally, the quenching of singlet oxygen by azide in a system containing micelles, liposomes, vesicles or cells could be a combination of both mechanisms.

In this study, we demonstrate the differences that are observed in azide's quenching efficiency of singlet oxygen when the microenvironment is composed of micelles vs liposomes; when the chemical target that reacts with singlet oxygen resides in the microenvironment or is soluble in water; and the effect on the quenching that is observed when the lipid molecules that constitute the microenvironment are neutral or charged. We show that these three classes of properties are strongly manifested in the efficiency of quenching.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
Chemicals and sample preparation

The following phospholipids were purchased from Avanti Polar Lipids (Alabaster, AL): two charged phospholipids; 1,2-dimyristoyl-s-glycero-3-phospho-l-serine (DMPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and natural egg-yolk lecithin. The surfactant sodium dodecyl sulfate (SDS) was obtained from Merck (Darmstadt, Germany).

Hematoporphyrin IX (HP) and the singlet oxygen quenchers DMA and anthracene-9,10-dipropionic acid disodium salt (ADPA) were purchased from Sigma (St. Louis, MO). We employed HP from a stock solution of 1 mm in N,N-dimethyformamide (Bio-Lab Ltd., Jerusalem, Israel). Diethyl ether (>99.8%) and sodium azide were from Fluka Chemie (Buchs, Switzerland). Chloroform was from Bio-Lab Ltd.

Preparation of liposomes

The lipid was layered at the bottom of a vial, by evaporating the ethanol solvent under a stream of nitrogen. Diethyl ether and chloroform were added and the solution was thoroughly re-evaporated to complete dryness. Phosphate buffer (5 mm, pH 7.5) was added to form a lipid concentration of 5 mg mL−1. The sample was vortexed for 1 min and was then sonicated for 20 min at 4°C, using a probe sonicator (Soniprep 150; MSE, Crawley, Sussex, UK) for clarity. Three different stock solutions were prepared by this process: one containing 100% lecithin, the second 95% lecithin-5% DMPS and the third 95% lecithin-5% DOTAP.

Spectroscopic measurements

Absorption spectra were recorded on a Shimadzu (Kyoto, Japan) UV-2501PC UV–visible spectrophotometer. Fluorescence spectra and fluorescence time-drive measurements were performed on a Perkin-Elmer LS-50B digital fluorimeter (Norwalk, CT).

Preparation of solutions and photochemical measurements

The reaction solution, containing liposomes’ suspension in buffer, membrane-bound HP, and DMA or ADPA was placed in a cuvette in the fluorimeter. It was illuminated by a diode laser (Ningbo Lasever Inc., Ningbo, China) beam at 532 nm, which is within one of the excitation bands of HP, and the singlet oxygen quantum yield (Ф) values were measured by monitoring the decrease in the target's fluorescence intensity. The sample was stirred magnetically throughout the illumination, to obtain uniform exposure of the whole content of the sample. The laser power (around 70 mW) was measured at the sample's surface with a power meter (model PD2-A; Ophir, Jerusalem, Israel), before and after each measurement, to ensure that the power remained constant during the experiment. The production rate of excited photosensitizer molecules, in molar concentration units per second, kpho is given by the following equation [27]:

  • display math(1)

where P is the laser power (in mW), abs is the optical density per cm of the photosensitizer at the laser's wavelength, L is the length of the laser beam path along the sample (in cm), E is the number of Einstein units (1 Einstein = 6.023 × 1023 photons) of light energy per second per watt of light at the illumination wavelength and V is the sample's volume (in mL). The factor 0.98 corrects for Fresnel's light reflection at the air/sample interface.

The time-drive traces of diminishing fluorescence intensity of DMA or ADPA were fitted to an exponential decay (Eq. (2)) by least-squares fitting (Microcal Software; Microcal Origin, Northampton, MA).

  • display math(2)

where kDMA is the rate constant for the disappearance of DMA's fluorescence intensity, DMAflu. Ф is proportional to the singlet oxygen quantum yield of HP and expressed by the following Eq. (3).

  • display math(3)

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

The experimental parameter that was measured throughout this study is defined by Eq. (3). It is the rate of the photosensitized destruction of the chemical target, DMA or ADPA, normalized for the rate of light absorption by the HP photosensitizer. This parameter is proportional to the yield of singlet oxygen. For each experimental setup, we measured this parameter and we display its change upon increased concentrations of NaN3.

As a first set of data, we show in Fig. 1 the effect of azide concentration on the rate of photodestruction of DMA and ADPA in a suspension of liposomes. HP was bound to the liposomes, and from its measured binding constant [28] we evaluated that in the presence of 0.83 mg lipid mL−1, which we used in these experiments, more than 90% of the HP was in the lipid phase. DMA is also mainly membrane bound, whereas the double negatively charged ADPA is water soluble and is in the water phase. As can be seen, the quenching effect of azide on the photoperoxidation of DMA is small (less than 40%), whereas the same concentration of azide decreases the rate of ADPA's oxidation almost 10-fold. These results are explainable on the basis of the following calculations: we assume an average liposome diameter of 400 nm as we have observed by electron microscopy on lecithin vesicles that are obtained by the sonication method that we use. We take 62 Å2 as the average surface area per lipid molecule [29]. These numbers imply that a liposome having a 400 nm diameter is composed of 1.62 × 106 phospholipid molecules. At a lipid concentration of 0.83 mg mL−1 the molar concentration of liposomes is 7.31 × 10−10 m. Thus, at our HP and DMA concentrations of 4.16 × 10−6 and 1.66 × 10−6 m, respectively, which are intercalated into the lipid bilayer, each liposome contains in its lipid phase 5700 and 2300 HP and DMA molecules, respectively. DMA has, therefore, a chance to react with singlet oxygen, which is diffusing rapidly [30] in the lipid phase, before it escapes into the aqueous phase. Thus, quenching of singlet oxygen which is diffusing within the lipid phase of the liposome in which it was generated is feasible if the azide injects into the membrane. However, the chance for this is probably low, in comparison with the much better probability for singlet oxygen reacting with the abundant DMA in the membrane. Once singlet oxygen has diffused out of the membrane into the water phase, we would observe its reaction with DMA only if it diffused and entered into another liposome, where it would meet and react with DMA molecules. The average distance between the liposomes, under these experimental conditions, is ca 130 nm and based on the reported value of oxygen's diffusion constant of 4.7 × 10−5 cm2 s−1 [30], the average time to cross this distance is ca 1 μs, which is shorter than the lifetime of 1Δg in water, 3 μs. Azide can quench it during this diffusion in the water. Both mechanisms of quenching by azide are probably not very competitive with the reaction of 1Δg with DMA within the liposome where it was generated. Therefore, we observe the relatively small effect of azide as compared to when the chemical target is water-soluble ADPA. In this case, the reaction of 1Δg with ADPA occurs in the water phase, after 1Δg has diffused out of the liposome. In this case, azide is a strong competitor with ADPA, especially as the reaction rate constant for the first is higher than for the second [8] 5 × 108 m−1 s−1 or more vs 1 × 108 m−1 s−1, and because of the large difference in their concentrations.

image

Figure 1. The effect of azide concentration on the effective yield of singlet oxygen, normalized to that at no azide, that was generated by photosensitization of HP in lecithin liposomes and measured by its reaction with DMA (●) and ADPA (○). Concentrations of DMA and ADPA were 1.66 µm.

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These results can be contrasted with the very different ones obtained in SDS micelles (Fig. 2). Herein, we observed that the quenching effect exerted by azide is practically identical with both DMA and ADPA. The difference is rooted in the very large difference between the size of a liposome and a micelle, which is reflected in the results in more than one aspect. A micelle provides a much smaller nonpolar environment and singlet oxygen can diffuse out of it within a small fraction of 3 μs. In addition, the critical micelle concentration (cmc) of SDS is 8 mm [31], thus at 27 mm SDS concentration, the concentration of SDS that is micellized is 19 mm. The aggregation number of SDS micelles was reported as 60 [32]. These numbers dictate that at 27 mm SDS, the concentration of micelles, as such, is 317 μm. The probability of finding n molecules of a solute in a micelle into which it partitions well, such as HP or DMA, Pn, is given by Poisson distribution:

  • display math(4)

where <Q> is the ratio between the solute's and micelles’ concentration. With the concentrations of HP and DMA that we used here, 3.44 × 10−6 and 1.38 × 10−6 m, respectively, the <Q> values are 0.0108 and 0.0043. P1 is calculated as 0.0129 and 0.0052, respectively, and the probability of finding both a HP and DMA molecule in the same micelle is not more than 8.46 × 10−5. It remains clear, therefore, that the chance for singlet oxygen to react with DMA within the micelle in which it was generated by HP is extremely slim, and singlet oxygen will diffuse rapidly out of a micelle. However, because of the high concentration of micelles, namely 317 μm, which is several orders of magnitude higher than the concentration of liposomes that was calculated above, a singlet oxygen molecule will quickly hit a nearby micelle, and have a chance to interact with a DMA molecule that might be present there. The calculated average distance between micelles is ca 20 nm, about seven times shorter than the interliposomal distance that we encountered in liposomes above. This is the reason why one does not observe a difference in the quenching effect of azide on the reaction efficiencies of singlet oxygen between micelle-localized DMA or water resident ADPA: in both cases azide might intercept singlet oxygen mainly while it is diffusing in water.

image

Figure 2. The effect of azide concentration on the effective yield of singlet oxygen, normalized to that at no azide, that was generated by photosensitization of HP in SDS micelles and measured by its reaction with DMA (●) and ADPA (○). Concentrations of DMA and ADPA were 1.38 µm.

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To advance the understanding of azide quenching of singlet oxygen, which diffuses between liposomes, we studied the effect of surface charges on the liposomes. As azide and ADPA are anionic, it is expected that their concentrations near the surface of the liposomes will differ from the bulk concentration, and will differ from each other because ADPA is doubly charged. This should affect the probability of singlet oxygen, which diffuses out of a membrane, to encounter these anions.

We compare the effect of azide on the reactions of singlet oxygen with DMA or with ADPA in suspensions of liposomes composed of neutral and charged lipids. Positively and negatively charged liposomes were composed of 95% lecithin and 5% DOTAP or DMPS, respectively. The Gouy–Chapman electrostatic theory of surface charges enables the calculation of the surface potential, Ψ0, using the following Eq. (5) [33]:

  • display math(5)

where σ is the density of membrane surface charges, in unit charges per Å2, C is the concentration of the electrolyte, having a valence of z, in the solution, N is Avogadro's number, ∈r is the solution dielectric constant and ∈0 is vacuum permittivity. In our case, at 5% fraction of charged lipid molecules, namely charge density of one electronic charge per 1240 Å2, and at a 5 mm electrolyte concentration the surface potential is calculated as ±62.3 mV, depending on the lipid charge. This is the difference between the electric potential at the membrane's surface and at a distant point in the bulk.

The relevance of the surface potential to our case is that the potential decays exponentially from the surface into the bulk, Eq. (6) [29, 33]. This causes a varying concentration of solution cations and anions, C(x), according to a Boltzman distribution, Eq. (7):

  • display math(6)

At low values of surface potential Ψ(x) can be represented by: inline image however, in our case we used the full Eq. (6).

  • display math(7)

For a 1:2 electrolyte, such as the phosphate buffer which we used, B can be given (in nm−1) as inline image [29], where C(∞) is the bulk electrolyte's concentration. B−1 is called as the debye thickness and in our case is 2.49 nm. This is the distance from the surface to where the electric potential decreases to 1/e of its value at the surface. Anions, such as azide or ADPA will be attracted or repelled from the environment near the surface of the liposomes, which contain DOTAP or DMPS, respectively.

We have shown in a previous study that the uptake of charged photosensitizers by liposomes is modulated by their surface potential [34]. In this study, we wish to exhibit that the existence of surface charges on liposomes can affect the results of photosensitized damage to the singlet oxygen chemical targets and its quenching by azide. This is a result of two different mechanisms. (1) Azide is negatively charged. Using Eq. (7), we calculate that for a 5% negatively charged liposome, the average concentration of azide within a layer that has a thickness of 2 debye layers, namely 5 nm, is 0.43 times its bulk concentration. For a 5% positively charged liposome, this concentration is 3.14 times higher than the bulk concentration. (2) ADPA is doubly charged at neutral pH. The same calculation yields that for a 5% negatively charged liposome, the average concentration of HP within 2 debye thicknesses is 0.30 times its bulk concentration. For a 5% positively charged liposome, this concentration is 10.1 times higher than the bulk concentration. These results are summarized in Table 1.

Table 1. Average concentration of ADPA and azide in the solution near the membrane for positively and negatively charged liposomes
Average concentration of azide in the solution layer of a thickness of 1, 2 or 5 debye units, near the membraneAverage concentration of ADPA in the solution layer of a thickness of 1, 2 or 5 debye units, near the membrane  
  • display math
  • display math
Liposomes with 5% DOTAP (positive Ψ0)
  • display math
  • display math
  • display math
  • display math
  • display math
  • display math
Liposomes with 5% DMPS (negative Ψ0)
  • display math
  • display math
  • display math
  • display math

Figure 3 summarizes the results which we obtained. As can be seen, in almost all cases there is no clear large change in the trend of azide's effect between neutral, positively or negatively charged liposomes. Only in the case of ADPA did we observed a consistent trend of a weaker effect of azide, about two-fold, when the liposomes were positively charged with 5% DOTAP. Our explanation for these observations is that azide has a much higher probability for interacting and quenching singlet oxygen than DMA or ADPA, for two reasons: its higher reaction rate constant [8] and its higher concentration, namely 0–5 mm vs 3–4 μm of the chemical targets. Therefore, when the membrane carries a negative surface potential, the aqueous layer near the surface is depleted from anions. If we integrate over a thickness of 2 debye layers, ADPA is depleted to 30% the bulk value, as compared with 43% for azide. Singlet oxygen which diffuses out of a liposome will diffuse longer before encountering ADPA or inline image, but the latter has a better probability of reaction than ADPA, as said above, and azide will be removed from the nearby volume only slightly more than ADPA. With a positively charged liposome anions will accumulate near the surface at a concentration higher than their nominal bulk values, but ADPA will be present there at a more pronounced concentration, by a factor of 10.1 vs 3.14 for azide when considering a thickness of 2 debye layers. Values for average concentrations, calculated for layers of different thicknesses are shown in Table 1. Singlet oxygen is more prone to being quenched by ADPA than by azide, which leads to the difference observed in Fig. 3D.

image

Figure 3. The effect of azide concentration on the effective yield of singlet oxygen that was generated by photosensitization of HP in liposomes made purely of lecithin (●) or of 95% lecithin +5% DMPS (○ in plates A and C), or of 95% lecithin +5% DOTAP (○ in plates B and D), and measured by its reaction with DMA (plates A and B) and ADPA (plates C and D).

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In conclusion, we show that using azide ions as quenchers of singlet oxygen may have different consequences in different experimental set-ups. In the very common methodology, of using a singlet oxygen chemical trap, one observes a different extent of azide's quenching when the target is aqueously distributed vs when membrane bound, or when the nonpolar environment in which the sensitizer present is a liposome or a micelle. However, differences are observed between azide's quenching when the membrane is neutral vs surface charged. These should be taken into consideration when azide quenching is employed as a tool to point at the involvement of singlet oxygen in a photosensitized reaction in samples that contain nonpolar microenvironments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

This research was supported by a grant of the US-Israel Binational Science Foundation, by the Michael David Falk Chair in Laser Phototherapy and by the Katz Family Grant Incentive Program.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
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