Phosphorescence Kinetics of Singlet Oxygen Produced by

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Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nano-Particles. Part II: The Case of Hypericin Loaded LDL Particles Shubhashis Datta, Andrej Hovan, Annamária Jutkova, Sergei G. Kruglik, Daniel Jancura, Pavol Miskovsky, and Gregor Bano J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00659 • Publication Date (Web): 30 Apr 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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The Journal of Physical Chemistry

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nano-Particles. Part II: The Case of Hypericin Loaded LDL Particles

Shubhashis Datta1, Andrej Hovan2, Annamária Jutková2, Sergei G. Kruglik3, Daniel Jancura1,2, Pavol Miskovsky1,2, Gregor Bánó1,2*

1

Center for Interdisciplinary Biosciences, Technology and Innovation Park, P. J. Šafárik

University, Jesenná 5, 041 54 Košice, Slovak Republic 2

Department of Biophysics, Faculty of Science, P. J. Šafárik University, Jesenná 5, 041 54 Košice,

Slovak Republic. 3

Laboratoire Jean Perrin, Sorbonne Universités, UPMC Univ. Paris 6, CNRS UMR 8237, 4 place

Jussieu, 75005 Paris, France

*corresponding author

e-mail: [email protected],

1 ACS Paragon Plus Environment

phone: +421 55 2342253

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

The phosphorescence kinetics of singlet oxygen produced by photosensitized hypericin (Hyp) molecules inside low-density lipoprotein (LDL) particles was studied experimentally and by means of numerical and analytical modelling. The phosphorescence signal was measured after short laser pulse irradiation of aqueous Hyp/LDL solutions. The Hyp triplet state lifetime determined by a laser flash-photolysis measurement was 5.3×10-6 s. The numerical and the analytical model described in Part-I of the present work were used to analyze the observed phosphorescence kinetics of singlet oxygen. It was shown that singlet oxygen diffuses out of LDL particles on a time-scale shorter than 0.1 μs. The total (integrated) concentration of singlet oxygen inside LDL is more than an order of magnitude smaller than the total singlet oxygen concentration in the solvent. The time-course of singlet oxygen concentrations inside and outside the particles were calculated using simplified representations of the LDL internal structure. The experimental phosphorescence data were fitted by a linear combination of these concentrations, using the emission factor E (the ratio of the radiative singlet oxygen depopulation rate constants inside and outside LDL) as a fitting parameter. The emission factor was determined to be E=6.7±2.5. Control measurements were carried out by adding sodium azide, a strong singlet oxygen quencher, to the solution.


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Introduction Photodynamic therapy (PDT) utilizes highly reactive oxygen species (mostly singlet oxygen) to induce cell death in cancerous tissues1-4. During the (type-II) photodynamic action, singlet oxygen is produced by energy transfer from photo-excited photosensitizer molecules to molecular oxygen. The presence of singlet oxygen either in solutions or inside cells can be detected through its phosphorescence emission at 1270 nm5-8. Various nano-carrier systems are investigated extensively to facilitate targeted transport of photosensitizer molecules in the host body9. Production of singlet oxygen inside nano-particles is

usually

studied

through

time-resolved

phosphorescence

measurements.

The

phosphorescence kinetics of singlet oxygen produced in nano-particle solutions following short (nano or sub-nanosecond range) laser pulses is affected by the diffusion of singlet oxygen in the heterogeneous environment. Due to this fact, as it was shown by several authors10-12, special care must be taken when interpreting the experimental phosphorescence kinetics data. The lifetime of singlet oxygen inside the particles may differ from that in the particle exterior, i.e. in the surrounding solvent. Moreover, the rate of singlet oxygen radiative deactivation depends on the local environment. To calculate the time-course of the overall phosphorescence intensity detected in a time-resolved experiment, the amount of singlet oxygen both inside and outside the particles needs to be known. Two theoretical models (a numerical and an analytical one) describing the phosphorescence kinetics of singlet oxygen produced in nano-particles were presented in Part I of this work. The second part is focused on a particular application of the presented models. Namely, the numerical simulation procedure is used to explain the experimentally observed time-course of singlet oxygen phosphorescence measured after 3 ACS Paragon Plus Environment


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irradiation of hypericin-loaded low-density lipoprotein (LDL) particles. Besides that, a set of LDLrelated parameters - to be used in the analytical model - is derived, too. Hypericin (Hyp) is a natural photosensitizer extracted from the plants of the genus Hypericum. The possibilities of Hyp-based photodynamic therapy have been summarized in several review articles13-17. Hyp forms biologically inactive non-fluorescent aggregates in water18,

19

. By

contrast, Hyp has a high affinity to lipid membrane structures, where it is primarily dissolved in the monomer (fluorescent), biologically active form20-24. Only the monomer form of Hyp can produce singlet oxygen25. LDL particles were used previously as carrier of several anti-cancer drugs26-28. The interaction of Hyp with LDL was investigated in our previous studies25, 29-33. It was shown that Hyp is dissolved as a monomer in LDL particles up to a Hyp/LDL concentration ratio of 30:131. Higher concentrations of Hyp inside the LDL particles leads to Hyp aggregation and dynamic selfquenching of Hyp fluorescence. Phosphorescence studies of singlet oxygen produced by photosensitized Hyp inside LDL particles were published25. These earlier results obtained with Hyp/LDL complexes are updated and completed in this work by laser-flash photolysis measurements of triplet state Hyp lifetimes. To get a better insight into the phosphorescence kinetics of singlet oxygen present in the particle interior and exterior, the measurements were carried out also in the presence of sodium azide, a strong singlet oxygen quencher that is preferentially localized in the aqueous phase. The numerical model helped us to shed light on the details of the observed singlet oxygen phosphorescence kinetics. The analytical description, when combined with the LDL-related modelling parameters presented in this work, can be used for fast analysis of other LDL-based systems in aqueous solutions. 4 ACS Paragon Plus Environment

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Experimental methods Dimethyl-sulfoxide (DMSO) (HPLC, ≥99 %) and hypericin (HPLC, ≥95 %) were purchased from Sigma-Aldrich, Germany. LDL (SDS-PAGE, ≥95 %) was purchased from Calbiochem, USA. Stock solution of 2.0×10-3 M Hyp in DMSO was prepared. Hyp loaded LDL particles were prepared in PBS buffer (pH=7.4) setting the Hyp/LDL ratio to 30:1. The concentration of Hyp in the sample was 4.0×10-6 M. The percentage of DMSO in the solution did not exceed 1%. The samples prepared with sodium azide contained 10 mM of the quencher. The laser excitation system consisted of a pulsed OPO (GWU basiScan-M) pumped with the third harmonic of a Nd:YAG laser (Spectra-Physics, Quanta-Ray, INDI-HG-10S). The repetition rate of the 5-7 ns long laser pulses was set to 10 Hz. The OPO wavelength was tuned to 598 nm matching the absorption maximum of Hyp. 2.5 ml of the sample was placed to a 10x10x40 mm quartz cuvette equipped with an overhead-type glass stirrer, and was kept at room temperature (cca 298 K). In order to minimize sample bleaching, the average laser power was set to approx. 0.15 mW. The singlet oxygen phosphorescence signal passed through a 12501300 nm band-pass filter and was detected with a photomultiplier tube (Hamamatsu H10330A75) operated in photon counting mode. The time-course of the phosphorescence was acquired with a multichannel scaler PCI card (Becker&Hickl, MSA-300). Typically, the signal of 30.000 laser pulses was integrated at each experimental condition. To suppress the background signal originating from the optical components and the possible phosphorescence of Hyp, the emission signal was measured with two additional band-pass filters in the 1200-1250 nm and



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the 1300-1350 nm spectral range. It was assumed that the background signal (as detected by the present setup) had a slowly varying wavelength dependence in the covered spectral range. The time-course of singlet oxygen phosphorescence was calculated by subtracting the average of the two auxiliary measurements (acquired in the adjacent spectral regions) from the signal measured in the 1250-1300 nm range. This way the background phosphorescence was efficiently suppressed. The optical setup was equipped with an additional 532 nm cw laser (Cobolt, Samba), used to monitor the Hyp triplet state lifetime in a flash-photolysis experiment34, 35. The laser beam was passing through the sample area excited with the pulsed laser. The polarization of the cw laser was oriented at the magic angle with respect to the excitation beam polarization. Timeresolved absorption (at 532 nm) was measured with an avalanche photo-diode (Thorlabs, APD110A2) connected to a digitizing oscilloscope (Tektronix, DPO 7254).

Theoretical Methods The numerical model The numerical model employed to simulate the concentration ∆ of singlet oxygen produced by photosensitized Hyp-loaded LDL particles was described in Part I of this work. Shortly, the spatio-temporal distribution of singlet oxygen was calculated by solving the diffusion equation complemented with source and depletion terms for singlet oxygen: ∆

= ∇ ∆ + −

∆ ∆

− q azide∆.


(1)

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D and ∆ are the singlet oxygen diffusion coefficient and lifetime, respectively, and S is the production rate of singlet oxygen. The last term in Equation 1 stands for singlet oxygen quenching by sodium azide, where kq is the quenching rate constant. The above equation was solved numerically on a radial grid of equal spacing. The details of the numerical scheme are given in Part I of the present paper.

Figure 1. The schematic view of LDL particles and the approximations used in the numerical and the analytical models. The internal structure of LDL particles is depicted in Figure 1. LDL consists of a hydrophobic core predominantly formed of cholesteryl esters and triglycerides, and an outer layer of phospholipids, cholesterol and the ApoB-100 protein36. The applied numerical model was derived for spherically symmetric particles due to which a simplified LDL representation was introduced in the simulations. The particles were divided into two concentric regions, the core and the ApoB-100 protein-containing (3 nm thick) outer shell37, assuming homogeneous local environment in both of them (see Figure 1). The outer diameter of LDL was set to 21 nm. The radial distribution of singlet oxygen concentration inside and outside the particle was calculated as a function of time and was used to predict the time-course of the experimental phosphorescence signal. The time step used in the simulations was 0.25×10-12 s.



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The lifetime of singlet oxygen is influenced by the environment through the local unimolecular (pseudo-first-order) rate constants of singlet oxygen radiative and non-radiative deactivation: 1/τΔ = kr + knr. The phosphorescence emitted by singlet oxygen molecules at a certain position is proportional to the product of the radiative deactivation rate constant kr and the local concentration of singlet oxygen. The rate constant of the radiative deactivation may be different in the particle core, the shell and the surrounding solvent38, 39. The physico-chemical mechanisms of singlet oxygen interaction with the environment and the effect of the environment on the singlet oxygen radiative (and non-radiative) deactivation rate constants have been published40, 41. For the sake of simplicity a single emission factor = rIN /r ! was assigned to the entire LDL particle in the present work, including both the core and shell regions. With this simplification, the phosphorescence signal P(t) is proportional to the linear combination of the total amount of singlet oxygen inside and outside the particle (∆IN tot and ∆OUT tot ) and can be expressed as follows (see equations 7 and 8 in Part-I): OUT '( = )∆IN tot + ∆tot ,

(2)

A is a proportionality factor, which is specific for the given experimental arrangement. There is an indication that Hyp dissolved in LDL particles is preferentially located close to the particle surface29, 30. To account for this fact, non-zero source of singlet oxygen was only used in the outer shell of the particle. The time course of the source term was approximated by an exponential decay as measured for the Hyp triplet state concentration in the laser flashphotolysis experiment. As the absolute value of the singlet oxygen source term S (see


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Equation 1) is not known, only the relative concentration of singlet oxygen is calculated by the model. The three compartments (the core, the shell and the solvent) of the simulated system were characterized by their own set of physical parameters (diffusion coefficients and singlet oxygen lifetime values). Besides that, partitioning of singlet oxygen between the LDL particle and the aqueous surroundings was taken into account. Only a single effective partition coefficient K was assigned to the whole particle interior. In principle, all the mentioned parameters can be determined by fitting the computational results to the experimental data. However, one needs to be aware of the limitations of this approach, mostly originating from the quality (signal to noise ratio and time-resolution) of the available experimental data. The philosophy of the present work was to present a description of the studied Hyp/LDL system in two steps. First, the diffusion coefficient, lifetime and partition coefficient values were estimated based on the data available in the literature and only the emission factor E was varied to fit the computational results to the experimental phosphorescence curve. In the next step, the effect of the assumed modelling parameters on the obtained emission factor was investigated by running the simulations with modified parameter sets (see the Results and Discussion section). Table 1. The Primary Set of LDL-Related Parameters. D -9

τΔ 2 -1

-6

[x 10 m s ]

[ x 10 s ]

LDL core

0.7

20

LDL shell

0.49

10

τt

K

-6

[ x 10 s ]

5.3 ± 0.1 5.1 ± 0.1 (with azide)


3


solvent

2.0

3.5 0.23 (with azide)

The chosen primary set of input parameters is shown in Table 1. Detailed justification of the used diffusion coefficient, singlet oxygen lifetime and partition coefficient values is given in the Supporting Information. The analytical model The analytical model introduced in Part I of the present work approximates the nanoparticles by homogeneous spheres. Singlet oxygen is created uniformly in the entire volume of the particle. The diffusion of singlet oxygen out of the particle is characterized by a single effective diffusion time Deff , which can be calculated by the semi-empirical formula (see Part-I) when the singlet oxygen diffusion coefficients DIN and DOUT, the lifetime of singlet oxygen in the solvent τΔIN, the particle radius R and the partition coefficient between the particle interior and exterior K are known. The model describes the time-course of singlet oxygen concentration inside and outside the particle in a form of analytical formulae. The capability of the simplified analytical description to reproduce the numerical results was tested in this work for LDL particles. The following LDL-related input parameters were used in the analytical model: DIN=0.6×10-9 m2s-1, τΔIN=20×10-6 s. The remaining parameters were identical to the ones presented in Table 1. The calculated effective diffusion time Deff was 0.066×10-6 s and 0.053×10-6 s for the solvent without and with sodium azide, respectively.


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Results and Discussion The results of Hyp triplet state transient absorption measurements are shown in Figure 2. The intensity of the 532 nm laser beam detected by the avalanche photodiode (following short pulse excitation) is shown as a function of time for the Hyp/LDL solutions without sodium azide (Fig. 2a) and in the presence sodium azide (Fig. 2b). The signals were fitted with single exponential curves. The obtained lifetimes of the Hyp triplet state were (5.3±0.1)×10-6 s and (5.1±0.1)×10-6 s for the samples without and in the presence of sodium azide, respectively. The shorter Hyp triplet state lifetime measured in the presence of azide indicates a minor effect of azide on Hyp molecules embedded in LDL particles. This observation can be rationalized by the fact that Hyp molecules dissolved in LDL particles are preferentially located close to the particle surface29, 30. The numerical simulations were run for the parameter set of Table 1. The source term S (see Equation 1) was set to be proportional to the exponentially decaying Hyp triplet state concentration detected in the laser flash-photolysis experiment (Figure 2). The obtained kinetics of singlet oxygen total concentration inside and outside the LDL particles – as calculated by the numerical and the analytical model – is shown in Figure 3. In the case of pure Hyp/LDL solution without azide (Fig. 3a), the calculated total concentration in the particle exterior is significantly larger than the concentration inside LDL, except for the very first 100 ns, when singlet oxygen is predominantly located in the particle interior (see the inset in Fig. 3a). This clearly shows that singlet oxygen diffuses out of LDL very fast. Indeed, the duration of the fast concentration increase inside the particle (during the first 400 ns) is limited by singlet oxygen diffusion out of LDL. The difference between the numerical and analytical results is 11 ACS Paragon Plus Environment


most pronounced in this initial time period. In the numerical model, the source of singlet oxygen is located close to the particle surface, which may be the reason for the faster concentration increase outside the particle, as compared to the analytical data.

Figure 2. The kinetics of hypericin triplet state decay measured by flash-photolysis on Hyploaded LDL particles in PBS buffer without a) and with b) sodium azide. The acquired raw data are indicated by the open symbols. The solid circles represent the smoothed signal. The solid lines are single exponential fits to the original data points.


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Figure 3. The kinetics of singlet oxygen concentration calculated by the numerical and analytical models for Hyp/LDL solutions without sodium azide a) and in the presence of sodium azide b). The open symbols represent the total (integrated) singlet oxygen concentration in the solvent and inside LDL, as calculated by the numerical model. The parameters of Table 1 were used for the calculations. The results of the analytical model are plotted by solid lines. The first 500 ns period is zoomed-in in the inset of panel a). The exponential late-time decay of singlet oxygen concentration inside the particle is determined by the Hyp triplet state lifetime. The concentration of singlet oxygen outside the particle reaches its maximum at about 4 μs. In general, there is a good match between the analytical and numerical results in the late-time period. It is interesting to note that because of the short diffusion time, the concentration kinetics of singlet oxygen in the solvent can be well



fitted by the conventional two-exponential time-dependence of singlet oxygen kinetics in homogeneous media (not shown), using the Hyp triplet state lifetime and the lifetime of singlet oxygen in the solvent. The experimental phosphorescence kinetics, however, contains the contribution from singlet oxygen both inside and outside the particles. When sodium azide is added to the solution, the kinetics of singlet oxygen concentration changes dramatically. As a result of strong quenching by azide, the calculated maximal concentration of singlet oxygen in the solvent drops by an order of magnitude. At the same time, the concentration of singlet oxygen inside LDL is not affected considerably (see Fig. 3b, note the different scales in panels a and b). At these conditions, the late-time decay of singlet oxygen is set, both outside and inside the particle, by the Hyp triplet state lifetime. The experimental time-course of singlet oxygen phosphorescence is shown in Figure 4 for Hyp/LDL solutions in the absence and presence of sodium azide. The measured data were fitted by Equation 2 (starting at 0.5 μs after the laser pulse) using the singlet oxygen concentrations calculated by the numerical model (see Figure 3). The emission factor E was set to different values between 2 and 15 and the sum of squared differences (SSD) of the measured and calculated data sets was minimized by tuning the A parameter of Equation 2. It is important to note that the two curves (with and without azide) were treated in a single fitting procedure, using the same A parameter for both. The SSD values obtained by this way are shown in the inset of Figure 4 as a function of the emission factor E. It can be seen that E=6.7 gives the best match of the calculated and measured curves. The corresponding theoretical phosphorescence time-courses of singlet oxygen are plotted by black solid lines in Figure 4. In general, the kinetics of the phosphorescence signal and the intensity ratio of the two experimental curves 14 ACS Paragon Plus Environment

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are well reproduced by the numerical model. It is noted, however, that the reliability of the emission factor determined this way is limited by the quality of the experimental data and the validity of the modelling assumptions. The obtained value of the emission factor E=6.7 falls between the one reported by the Röder group for phospholipid membranes11 E=3.25 ± 0.5 and the value derived for the protein environment inside myoglobin10 E=8.1 ± 1.3. This is in agreement with the expectations for the LDL interior composed of lipid and protein components.

Figure 4. The experimental phosphorescence kinetics of singlet oxygen produced by photosensitized Hyp/LDL complexes. The experimental curves were fitted by Equation 2 using singlet oxygen concentrations calculated by the numerical model (see Figure 3). Only the amplitude parameter A was varied during the fitting procedure. The black lines represent the theoretical phosphorescence kinetics obtained for emission factor of E=6.7 that describe the



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experimental data the best. Inset: the sum of squared differences between the experimental and theoretical curves obtained for different emission factors. The effect of the experimental and modelling uncertainties is analyzed as follows. The timecourse of the singlet oxygen concentration (inside and outside LDL, see Figure 3) can be divided into two parts. First, there is a fast increase of the concentration inside the particle during the first 400 ns period after the laser pulse. This fast increase is set by the diffusion time of singlet oxygen out of the particle, which in the case of the numerical model mostly depends on the diffusion coefficient values estimated for the LDL core and the LDL shell (Table 1). As already mentioned, the effective diffusion time of singlet oxygen Deff is explicitly included in the analytical model. In the presence of sodium azide, the concentration of singlet oxygen reaches its maximum within 1 μs after the laser pulse not only inside but also outside the particle (Fig. 3b). This fast concentration increase is reflected in the fast onset of the phosphorescence signal which is observed experimentally in the presence of azide (Figure 4). Detailed comparison between calculated and measured phosphorescence kinetics in this initial time period is hindered by the limited time resolution and the noise level of the experimental phosphorescence data. The calculated kinetics of singlet oxygen concentration after the initial 0.5 μs period can be readily correlated with the measured phosphorescence signal. The time-dependence of singlet oxygen concentration in this later period is mainly determined by the two most reliable parameters included in the model, the Hyp triplet state lifetime (measured experimentally) and the lifetime of singlet oxygen in the solvent τΔOUT=3.5×10-6 s

42

. The uncertainty of the LDL-

related parameters (Dcore, Dshell, τΔcore, τΔshell and K) is higher. These parameters, however, when 16 ACS Paragon Plus Environment

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changed in a reasonable range (keeping the relations Deff a(1)Delta(g) Radiative Transitions of O-2. J. Phys. Chem. A 1999, 103, 6091-6096.


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42. Jensen, R. L.; Arnbjerg, J.; Ogilby, P. R., Temperature Effects on the Solvent-Dependent Deactivation of Singlet Oxygen. J. Am. Chem. Soc. 2010, 132, 8098-8105.

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Phosphorescence Kinetics of Singlet Oxygen Produced by

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