Nanoparticles with Embedded Porphyrin Photosensitizers for

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Nanoparticles with Embedded Porphyrin Photosensitizers for Photooxidation Reactions and Continuous Oxygen Sensing Pavel Kubát, Petr Henke, Veronika Berzediová, Miroslav Stepanek, Kamil Lang, and Jiri Mosinger ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12009 • Publication Date (Web): 28 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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ACS Applied Materials & Interfaces

Nanoparticles with Embedded Porphyrin Photosensitizers for Photooxidation Reactions and Continuous Oxygen Sensing

Pavel Kubát,† Petr Henke,‡ Veronika Berzediová, ‡ Miroslav Štěpánek, ‡ Kamil Lang$ and Jiří Mosinger‡$* †

J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, v.v.i., Dolejškova 3, 182 23 Prague 8, Czech Republic

‡

$

Faculty of Science, Charles University, 2030 Hlavova, 128 43 Prague 2, Czech Republic

Institute of Inorganic Chemistry of the Czech Academy of Sciences, v.v.i., Husinec-Řež 1001, 250 68 Řež, Czech Republic

KEYWORDS: singlet oxygen-sensitized delayed fluorescence, polystyrene nanoparticles, porphyrins, singlet oxygen, photooxidation.

ABSTRACT. We report the synthesis and characterization of sulfonated polystyrene nanoparticles (average diameter 30 ± 14 nm) with encapsulated 5,10,15,20-tetraphenylporphyrin or ionically entangled tetracationic 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, their photooxidation properties and the application of singlet oxygen-sensitized delayed fluorescence

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(SODF) in oxygen sensing. Both types of nanoparticles effectively photogenerated singlet oxygen, O2(1∆g). The O2(1∆g) phosphorescence, transient absorption of the porphyrin triplet states and SODF signals were monitored using time-resolved spectroscopic techniques. The SODF intensity depended on the concentration of the porphyrin photosensitizer and dissolved oxygen, and on the temperature. After an initial period (a few µs), the kinetics of the SODF process can be approximated as a monoexponential function, and the apparent SODF lifetimes can be correlated with the oxygen concentration. The oxygen sensing based on SODF allowed measurement of the dissolved oxygen in aqueous media in the broad range of oxygen concentrations (0.2 - 38 mg l-1). The ability of both types of nanoparticles to photooxidize external substrates was predicted by the SODF measurements and proven by chemical tests. The relative photooxidation efficacy was highest at a low porphyrin concentration, as indicated by the highest fluorescence quantum yield (ΦF), and it corresponds with negligible inner filter and selfquenching effects. The photooxidation abilities were sensitive to the influence of temperature on the diffusion and solubility of oxygen in both polystyrene and water media and to the rate constant of the O2(1∆g) reaction with a substrate. Due to their efficient photogeneration of cytotoxic O2(1∆g) at physiological temperatures and their oxygen sensing via SODF, both types of nanoparticles are promising candidates for biomedical applications.

1. INTRODUCTION Recently, significant progress in nanomaterials has led to the development of technologies that allow for the production of photoactive nanomaterials that generate singlet oxygen, O2(1∆g) with broad application potential.1,2,3 Among these materials, polymer nanofiber materials, which


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contain photosensitizers (typically porphyrins or phthalocyanines) can be applied in medicine4 or in water treatment.5 The excitation of porphyrins encapsulated in electrospun nanofiber membranes or attached to their surfaces leads to the formation of porphyrin triplet states and O2(1∆g) (Figure 1), which can efficiently kill bacteria1,6,7,8,9 and viruses.10

Figure 1. Mechanisms of prompt fluorescence, the formation of O2(1∆g) and SODF; the schematic structure of the sulfonated NPs with encapsulated TPP (TPP-NPs) and with externally bound TMPyP (TMPyP-NPs); and the structures of both porphyrin photosensitizers. There are fundamental limitations in the applications of photosensitizers in/on nanofiber membranes for O2(1∆g) photooxidation of a chemical/biological substrate, including the following: (i) the photooxidation of a substrate can occur only on the surface and/or in the close proximity of the membranes due to the short lifetime of O2(1∆g) (~3.5 ns in water) and from this


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following a limited diffusion length (tens to several hundreds of nm); (ii) the dimensions of nanofiber membranes hinder their applications in nano/micro-compartments, such as inside cells; (iii) non-negligible scattering of incident light and limited oxygen diffusion to a substrate can limit the function of photosensitizers encapsulated in polymer membranes. To overcome these limitations, we focused on the following photostable porphyrin photosensitizers with high quantum yields of O2(1∆g) formation (Φ∆) for applications in aqueous media: encapsulated 5,10,15,20-tetraphenylporphyrin, TPP (Φ∆ = 0.62 in CCl4),11 and externally bound 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, TMPyP (Φ∆ = 0.74 in H2O),11 on polystyrene nanoparticles (NPs) (Figure 1). The main advantage of NPs is their small diameter, which minimizes the diffusion length of oxygen to a porphyrin molecule. NPs with a negatively charged surface, which prevents aggregation in aqueous environments, allows O2(1∆g) to get in close proximity to chemical/biological substrates. The use of polystyrene NPs with a high oxygen permeability (P(O2) = 1.9×10-13 cm2 s-1 Pa-1),7 i.e., the parameter comprising the diffusion coefficient D(O2) and the solubility of oxygen S(O2) in polymer (P(O2) = S(O2)×D(O2)), high surface-to-volume ratio, high concentration of binding sites and good transparency to light with minimum scattering allows for easy access of oxygen to effectively quench the triplet states of the photosensitizer and form O2(1∆g) efficiently. There is a major advantage if the same material that is used for photooxidation can also be used as a sensor. It is known that the common features of polymeric materials with porphyrins not only include efficient generation of O2(1∆g) but also singlet oxygen-sensitized delayed fluorescence (SODF).6 The SODF process is a relatively rare type of delayed fluorescence12 observed for some fluorescent photosensitizers.13 The SODF is generated from repopulation of the S1 excited states of the photosensitizer from its long-lived triplet state by nearby O2(1∆g) that


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is formed via a photosensitized reaction (Figure 1). The close proximity of the encapsulated/bounded photosensitizer triplet states and the O2(1∆g) in/on polystyrene NPs could significantly contribute to the efficient repopulation of the S1 fluorescent states. The SODF signal strongly depends on the concentration of dissolved oxygen. In this paper, we tested the use of SODF for in situ monitoring of the oxygen concentration using NPs with porphyrins. Previously published data showed that SODF of endogenous protoporphyrin IX was successfully used for oxygen sensing in cells.14,15

2. EXPERIMENTAL SECTION Chemicals. 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetrakis(N-methylpyridinium4-yl)porphyrin

tetra-p-toluensulfonate

(TMPyP),

uric

acid

(UA),

cyclohexanone,

tetraethylammonium bromide (TEAB), potassium iodide, and other inorganic salts were purchased from Sigma-Aldrich. The polystyrene, Synthos PS GP 137, was purchased from Synthos Kralupy, Czech Republic. Tetrahydrofuran and sulfuric acid were purchased from LachNer, Czech Republic. Tetrahydrofuran (THF, HPLC-grade) was dried with a PureSolv MD5 solvent purification system (Innovative Technology). All other chemicals were used as delivered. Electrospinning. A mixture of 0.07 wt % TEAB and 99.93 wt % polystyrene was dissolved in cyclohexanone to prepare a 17 % solution for the fabrication of the polystyrene nanofiber material. The polystyrene nanofiber membranes were produced using the modified NanospiderTM electrospinning industrial technology by the simultaneous formation of charged liquid jets on the surface of a thin wire electrode, where the number and location of the jets were set to their optimal positions.1 The nanofiber diameters were measured using the NIS Elements 4.0 image analysis software (Laboratory Imaging, Czech Republic).


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Preparation of sulfonated NPs. The electrospun polystyrene nanofiber membranes (250 cm2, typically 150 mg) fixed on quartz substrates were treated by immersion in 96 % sulfuric acid at room temperature for 54 hours. The materials were washed with deionized water until a neutral pH was reached and then stored in deionized water. Typically, a wet sulfonated nanofiber membrane was immersed in 16 ml of dry THF for 60 s with stirring. For the preparation of TPP encapsulated in NPs (TPP-NPs), dry THF was enriched by 0.07 mg of TPP to obtain 0.05 wt% TPP in NPs. The same method was used for the preparation of NPs with 0.005, 0.5, 5 and 10 wt% of encapsulated TPP. Then, deionized water (80 ml) was added and THF was removed by evaporation under vacuum. The resulting dispersion of NPs in water was centrifuged for 10 min at 4700 g to remove the microparticles. The dispersion of NPs was then dialyzed using a FloatA-Lyzer® G2 membrane with a molecular weight cut-off of 50 kD for 18 hours in deionized water at room temperature to remove traces of sulfuric acid and THF. The final volume of NPs stock solution was 45 ml. The required number of NPs was obtained by dilution of the stock solutions by H2O. Ion exchange capacity (IEC). The IEC of the sulfonated NPs was determined by titration. Approximately 20 ml (~ 3 × 1013 NPs ml-1) of dialyzed NPs was treated with 10 ml of a 0.2 M NaOH solution for 1 hour to completely replace H+ with Na+. The remaining NaOH was titrated potentiometrically with 0.1 M HCl. The concentration of HCl was determined using a primary standard of sodium tetraborate. The IEC values were related to the mass of the dried NPs. Gravimetric analysis. Twenty milliliter samples of NPs were dried at 50 °C to a constant weight. The weight was determined using a GR-200 analytical balance (A&D Instruments Ltd., Japan). The use of 154 mg of nanofiber membrane led to approximately 140 mg of dry NPs (Table S1).


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Light scattering (LS). The LS setup (ALV, Langen, Germany) consisted of a 22 mW He-Ne laser (λ = 632.8 nm), an ALV CGS/8F goniometer, an ALV High QE APD detector and an ALV 5004 multibit, multitau autocorrelator. The measurements were carried out at 25 °C with aqueous NPs dispersions (mass concentration, c, = ~0.15 mg ml-1). For details on the LS data evaluation, see the Supporting Information, Figure S1. Scanning electron microscopy (SEM). The nanofiber morphology was studied using a scanning electron Quanta 200 FEG microscope (FEI, Czech Republic). Transmission electron microscopy (TEM). TEM micrographs were obtained with a Tecnai G2 Spirit Twin12 microscope (FEI, Czech Republic) at an acceleration voltage of 120 kV. A 2 µl aliquot of the 3 mg ml-1 NPs dispersion was dropped onto a copper TEM grid coated with electron-transparent carbon film. After 1 min, the solution was removed by touching the bottom of the grid with filter paper. UV/Vis and fluorescence spectroscopy. UV/Vis absorption spectra were recorded on Unicam 340 and Varian 4000 spectrometers equipped with an integration sphere. The spectra were recorded in transmission mode. Steady-state fluorescence spectra were monitored using an FLS 980 (Edinburgh Instruments, UK) spectrofluorimeter. The absolute photoluminescence quantum yields were measured using a Quantaurus QY C11347-1 spectrometer (Hamamatsu). The dispersions of NPs were excited at the Soret and Q-regions, as indicated in Table S2. Phosphorescence of O2(1∆g). The time-resolved near-infrared phosphorescence of O2(1∆g) at 1270 nm was recorded at a right angle to the excitation light (a Lambda Physik FL3002 laser, λexc = 425 nm, pulse length ~28 ns) using a homemade detector unit (interference filters, Ge diode). The temporal profiles of O2(1∆g) phosphorescence were averaged and calculated as the


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difference between the signals in oxygen-/air- and argon-saturated water. The details are given in Supporting Information. Singlet oxygen-sensitized delayed fluorescence (SODF). The SODF signals were measured on an LKS 20 kinetic spectrometer (Applied Photophysics) and calculated as the difference between the signal in a solution with a given concentration of oxygen and the detector response in an argon-saturated solution or after the addition of NaN3 (i.e., singlet oxygen quencher). The initial parts of signals are obscured by light scattering and the strong prompt porphyrin fluorescence and are omitted in all figures. The temperature-dependence measurements were performed in a 10 mm quartz cell placed inside a thermostatically controlled holder that was heated or cooled from 10 to 80 °C using the Peltier effect with a temperature stability within 0.1 °C. Dissolved oxygen sensing. For the simultaneous measurement of the SODF and the dissolved oxygen concentration, a luminescence flow cell equipped with an InPro 6880i oxygen sensor (Mettler Toledo), a peristaltic pump and a connection to argon and oxygen gas bottles was used. The cell contained 40 ml of a TPP-NPs dispersion (~ 1.5 × 1013 NPs ml-1 in water). Different dissolved oxygen concentrations were maintained by bubbling inert gas (argon) or oxygen. Photooxidation properties. A 1.5 ml aliquot of the NPs was placed in a thermostated 10 mm quartz cell (7 - 60 °C) that contained either 2×10-4 mol l-1 uric acid in 0.02 M phosphate buffer (pH = 7.0) or a 0.1 mol l-1 iodide detection solution. The cell was irradiated with visible light using a stabilized xenon lamp (500 W, Newport) equipped with a longpass filter (λ ≥ 400 nm, Newport). The UV/Vis absorbance changes at 291 nm, attributed to the photodegradation of uric acid,16 and at 287 or 351 nm, attributed to the formation of I3- in the iodide test,17 were recorded


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at regular intervals and compared to a blank solution of the same composition that was stored in the dark.

3. RESULTS AND DISCUSSION 3.1 Preparation and characterization of the nanomaterials. Stable polystyrene NPs free of photosensitizers and TPP-NPs with different amounts of encapsulated TPP were prepared by a slightly modified top-down nanoprecipitation method using nanofiber membranes (Figure 2).18 The membranes were prepared by electrospinning, and they served as a precursor for the preparation of NPs after extensive sulfonation. The nanofiber character of the membranes (Figure 3) and the long-term sulfonation (54 hours) yielded NPs with an average IEC of ~ 3×10-4 mol g-1. Instead of using sulfonated polystyrene nanofiber membranes with encapsulated TPP, we added appropriate amount of TPP to THF during nanoprecipitation. The modified method allows for deliberate tuning the amount of the encapsulated compound from 0.005 wt% to 10 wt% TPP distributed over the various number of NPs (from 3×1013 to 1.5×1010 NPs ml-1). The well-defined concentration series of NPs enables to analyze how their spectral, photophysical and photochemical properties are affected by the varying content of TPP using the same number of NPs or by the packing of TPP in NPs, investigated by keeping the same overall amount of encapsulated TPP over the series (i.e., the varying number of NPs by dilution). We also prepared photoactive NPs with various amounts of TMPyP, TMPyP-NPs, by adsorption of the tetracationic photosensitizer on the surfaces of the NPs (up to TMPyP/IES = 0.1). For example, TMPyP-NPs with a TMPyP/IEC ratio of 0.001 were prepared by mixing 100 µl 1×10-4 mol/l TMPyP with 10 ml of NPs stock solution (3.2 mg ml-1).


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The adsorption was monitored by UV-Vis absorption spectroscopy. Based on gravimetric analysis of the NPs, the average concentration of the prepared NPs (stock dispersion) was ~ 3 × 1013 NPs ml-1 (Supporting Information, Table S1). Note that the NPs can be simply removed from the aqueous environment by filtering through the original nanofiber membranes.18

Figure 2. Schematic diagram of the preparation protocol of photoactive NPs. 3.2. Morphology, size and stability of the NPs. The structure of the original electrospun polystyrene nanofiber materials was visualized by SEM (Figure 3 A, B). The typical nanofiber diameter was in the range of 150–400 nm. The modified nanoprecipitation method led to typically spherical NPs (Figure 3C) with a broad size distribution (Figure 3D); the average radius of the particles was 15±7 nm. The presence of encapsulated or attached porphyrins at all concentrations used had no influence on the morphology or size of the NPs.


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Figure 3. SEM micrographs of the polystyrene nanofiber membrane before (A) and after (B) sulfonation and the TEM micrograph (C) with the corresponding distribution of NPs radii (D) of the NPs prepared by the nanoprecipitation method. The combined static and dynamic LS measurement confirmed the spherical shape of the particles, providing gyration and hydrodynamic radii of the NPs of Rg = 42 nm and RH = 52 nm, respectively, and thus, the Rg/RH ratio of 0.81 is close to the value for homogeneous spheres, ρsph = (3/5)1/2. The values correspond to the radii of the largest particles observed by TEM because their contribution to the overall scattering is dominant (the scattering cross section of homogeneous spheres is proportional to R6). The molar mass of NPs from the LS measurement was Mw = 6.8×107 g mol-1, which corresponds to a NP density of d = 3Mw/4πRH3NA = ~0.2 mg ml-1, suggesting that nanoprecipitation leads to swollen NPs rather than compact NPs, with the density close to that of bulk polystyrene (dPS = 1.05 mg ml-1).24


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In our previous study, we found that long-time sulfonation (54 hours) during the preparation of NPs increased their stability, even in environments with high ionic strengths, due to the high number of sulfonic groups per NP.18 Using DLS measurements, we observed long-time stability even in the case of the TMPyP-NPs, where no change in the size was observed in the stock solution of the NPs after a month of storage. 3.3. UV/Vis absorption/emission spectra and photostability of the NPs. The UV/Vis absorption spectra of the TPP-NPs with the same overall content of TPP distributed over different number of NPs show differences (Supporting Information, Figure S2). The spectra of TPP-NPs with 5 and 10 wt% TPP are nearly identical to that of TPP in nonpolar solvents, such as toluene (Soret band at 421 nm and four Q bands), documenting that TPP molecules are located in nonpolar polystyrene matrix predominantly in the monomeric form whereas the sulfonic substituents are oriented towards the solvent. In contrast, TPP-NPs with the lower content of TPP contain a mixture of the non-protonated and protonated forms of TPP (the Soret band at 442 nm and the Q band at 650 nm), even after thorough washing by dialysis and/or by dispersing the NPs in neutral buffers. The protonation of the nitrogen atoms in the porphyrin macrocycle is not expected to affect photophysical properties considerably because this process does not result in an appreciable change of the singlet oxygen quantum yields.19 Extensive aggregation of TPP, which is characterized by broadening of the Soret band or hypochromicity, was not observed for either sample. The absorption spectra of the TMPyP-NPs (Supporting Information, Figure S3) with various TMPyP loadings are characterized by a Soret band at 430 nm and four Q bands. The Soret band of the TMPyP-NPs was significantly red-shifted (8 nm) compared with that of TMPyP in water. Spectral shifts for water-soluble porphyrins in the presence of aromatic compounds have been


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well documented and are proportional to the energy of association of the aromatics with the porphyrin. The fluorescence emission spectra of the TPP-NPs (Supporting Information, Figure S4) indicated that most of the porphyrin molecules were in a monomeric state, well encapsulated in the polystyrene bulk, but were influenced, to some extent, by protonation and re-absorption effects. The two bands at 654-680 nm and 716-718 nm are typical of TPP in a hydrophobic enviroment.6 The fluorescence spectra of TMPyP-NPs revealed the monomeric state of TMPyP, but no protonation or re-absorption effects occurred (Supporting Information, Figure S5). The two bands at 655-659 nm and 719-720 nm, which are in agreement with previously published data,20 were slightly red-shifted at high TMPyP loadings. The measurement of the fluorescence quantum yields (ΦF) of TPP-NPs dispersions revealed that ΦF decreased with increasing porphyrin loading due to inner filter and self-quenching effects (Supporting Information, Table S2). To test the photostability of the prepared TPP-NPs and TMPyP-NPs, a water dispersion of the corresponding NPs was irradiated with intense visible light. We found only a negligible photobleaching effect, as the absorbance of the Soret band of TPP in the TPP-NPs decreased by 6 % and that of the TMPyP-NPs decreased by 1 % after 2.5 hours of irradiation with a 500 W Xe-lamp with a longpass filter (λ ≥ 400 nm)) (Supporting Information, Figure S6). 3.4. Singlet oxygen-sensitized delayed fluorescence (SODF). The photogeneration of O2(1∆g) by both TPP-NPs and TMPyP-NPs was confirmed by direct measurements of the phosphorescence of O2(1∆g) at 1270 nm (Supporting Information, Figure S7) in aqueous media. These phosphorescence measurements are complicated by the very low


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probability of the O2(1∆g)→O2(3Σg-) radiative transition21 and the short lifetime of O2(1∆g) (~ 3.5 µs in H2O).22 The SODF signal is several orders of magnitude stronger than the phosphorescence at 1270 nm, and therefore requires the averaging of fewer traces to achieve a comparable S/N ratio. In contrast to porphyrins in solutions,12 the delayed fluorescence of the NPs with porphyrins consisted of SODF only because the delayed fluorescence of the NPs in argon-saturated H2O disappeared (Figure 4, panel A). The absence of the delayed fluorescence in the µs/ms time scale indicates that the contribution of other mechanisms, e.g., thermally activated delayed fluorescence or triplet-triplet annihilation, depending on the local concentration of the porphyrin triplet states (the triplet states have a lifetime of a few hundreds of µs in oxygen-free conditions), is negligible. This observation corresponds with a high singlet-triplet energy gap in porphyrins and considerable repulsion forces between negatively charged NPs and also with results published for nanofiber materials.23 A strong SODF signal occurred for the reaction of the triplet porphyrin states with O2(1∆g) (Figure 1). The relatively long lasting signal of prompt fluorescence is due to long excitation pulse (pulse length ~28 ns).

Figure 4. Normalized fluorescence of the NPs with 0.5 wt% TPP in oxygen- (a), air- (b) and argon- (c) saturated H2O (panel A). SODF of the TPP-NPs with 0.5 wt% TPP in oxygen- (d) and air-(e) saturated H2O calculated as the difference between the raw data in oxygen/air (a, b) and


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the prompt fluorescence (fluorescence in argon-saturated H2O, c) (panel B); SODF of the TMPyP-NPs in oxygen- (f) and air- (g) saturated H2O (panel C). Red lines are single exponential fits to the experimental data. In the polystyrene matrix, the lifetimes of both porphyrin triplet states and O2(1∆g) are several times longer than the corresponding lifetimes in H2O (Supporting Information, Figures S8-S10). The diffusion of O2(1∆g) in the TPP-NPs occurred mainly through polystyrene with less efficient quenching (τ∆ =15-30 µs) in comparison with H2O (τ∆ = 3.5 µs). In the TMPyP-NPs, the porphyrin molecules are localized on the NPs surface, and the TMPyP triplet states are in close contact with water molecules; thus, the diffusion of O2(1∆g) in the water environment prevailed. Taking into account the different lifetimes of O2(1∆g) and the oxygen diffusion coefficients, the SODF signal of the TPP-NPs decays on a longer time scale than that of the TMPyP-NPs. This function was tested for in situ oxygen sensing in H2O over a broad range of oxygen concentrations. It is known that the decay of the porphyrin triplet state is a monoexponential process (equation 1) and that the kinetics of O2(1∆g) can be described by equation 2 (it is monoexponential function for τ∆>> τT):23 [3P]=[3P]0exp(-t/τT),

(1)

[O2(1∆g)]=ASO(τ∆/(τT-τ∆))(exp(-t/τT)-exp(-t/τ∆)),

(2)

where ASO is a parameter, τT is the lifetime of the triplet states, τ∆ is the lifetime of O2(1∆g), [O2(1∆g)] denotes the concentration of O2(1∆g) at time t, and [3P]0 and [3P] are the initial concentrations of the porphyrin (TPP or TMPyP) triplet states at time t0 (immediately after excitation) and at time t, respectively.


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The SODF kinetics is quite complex, calculating τ∆ and τT values from the SODF traces using a simplified model (equation 3) based on equations 1, 2 and Figure 124,25 for the heterogeneous system (i.e. polymeric NPs and H2O) may provide different values than the corresponding values obtained from phosphorescence or transient absorption measurements of the porphyrin triplet states. ISODF = A

τ∆ τT − τ ∆

exp(-t/τT) × (exp(-t/τT)-exp(-t/τ∆)),

(3)

In this equation, A is the parameter and ISODF is the intensity of SODF. In general, the kinetics of SODF depends on other parameters (e.g., excitation energy) and cannot be expressed by a simple equation. The experimental results showed that the SODF kinetics of the TPP-NPs and TMPyP-NPs can be approximated by a single exponential kinetics after elimination of the initial data points (usually up to 5 µs), and it was strongly dependent on the oxygen concentration (Figure 4). The apparent lifetime of SODF, τSODF changed from 3.5 to 12.6 µs in the 0.5 wt% TPP-NPs (Figure 4, panel B) and from 1.2 to 2.1 µs in the TMPyP-NPs (Figure 4, panel C) in oxygen- and airsaturated H2O. The value of τSODF is lower than the lifetimes of both porphyrin triplet states and the apparent lifetime of O2(1∆g) (Supporting Information, Figure S11). Due to the strong dependence of SODF on the concentration of dissolved oxygen and the relatively low τSODF in air-saturated H2O, the TMPyP-NPs are suitable for sensing low concentrations of oxygen, where τSODF achieves a value of several tens of microseconds and the SODF signal can be well separated from the prompt fluorescence. In contrast, the higher value of τSODF predisposes the TPP-NPs to oxygen sensing in a broad range of oxygen concentrations from anaerobic to oxygen-saturated media. Moreover, the TPP-NPs were much less sensitive to NPs environment than the TMPyP-NPs, where TMPyP may be in direct contact with species


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diffused toward NPs. It is evident that the presence of NaN3, an O2(1∆g) quencher,26 with free access to TMPyP in the TMPyP-NPs and close proximity to O2(1∆g), significantly reduced the SODF signal due to the quenching of O2(1∆g) (Figure 5). In contrast, the encapsulated TPP molecules in the TPP-NPs are well protected by the polystyrene core, and the corresponding SODF signals are only slightly reduced by the presence of NaN3 (see also Supporting Information, Figure S12). This result indicates that only a negligible portion of the O2(1∆g) that is released from TPP-NPs into the aqueous solution diffuses back to the NPs to be quenched by the TPP triplet states and contributes to the SODF signal.

Figure 5. SODF signals of the TMPyP-NPs (0.1 TMPyP/IES) and 0.05 wt% TPP-NPs with (red line) and without (black line) NaN3 in H2O. For the sensing of oxygen and verification of the pseudo-monoexponential kinetics, we studied the dependence of the SODF signal from the TPP-NPs on the concentration of TPP in the NPs, the concentration of NPs, the temperature and the concentration of dissolved oxygen. Concentration of TPP in the TPP-NPs. NPs with different TPP loadings (i.e., average number of TPP per NP) were prepared to test their influence on the SODF kinetics. Using the average molar mass of the NPs (6.8×107 g mol-1), the calculated number of TPP molecules per


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NP changed from ~ 10.000 (10 wt% TPP) to ~ 5 (0.005 wt% TPP) (Table S2). The number of TPP molecules and the average diffusion length of O2(1∆g) in polystyrene (several hundreds of nm)7 allows their efficient interaction by the SODF mechanism (Figure 1). The SODF traces of TPP-NPs with high TPP loadings can be fitted by a single exponential function starting from a few µs after excitation due to a shorter rise time in comparison with TPP-NPs with low loading (Figure 6, panel A). The SODF apparent lifetimes decreased from 13.9 µs (0.005 wt% TPP-NPs) to 10.7 µs (5 wt% TPP-NPs). This observation is in accordance with a shorter average distance between TPP molecules in NPs with the high loading and from this following higher probability for O2(1∆g) to diffuse to and interact with a nearby porphyrin triplet state and to form fluorescent singlet excited state.

Figure 6. Comparison of the SODF kinetics of 5 wt% (a), 0.5 wt% (b), 0.05 wt% (c) and 0.005 wt% (d) TPP-NPs in air-saturated H2O, where the intensity of the SODF signal 2 µs after excitation was normalized to 1.0 (panel A); Normalized SODF profiles of 0.5 wt% TPP-NPs in air-saturated H2O of the original sample (e) and after 100-fold dilution (f) corrected for concentration (panel B). Red lines are the single-exponential fits of the experimental data.


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Concentration of NPs. The TPP-NPs sample (0.5 wt% TPP) was diluted by 100, and its SODF kinetics were compared with that of the undiluted dispersion (Figure 6, panel B). The diluted sample had slightly shorter SODF kinetics (τSODF = 11.1 µs, Figure 6, panel B, f) in comparison with the original sample (τSODF = 12.6 µs, Figure 6, panel B, e). It is obvious that a small portion of the O2(1∆g) in the undiluted dispersion was deactivated by diffusion to nearby NPs, where it interacted with the porphyrin triplet states to produce SODF (Figure 7). In the diluted solution where the number of NPs was 100 times lower, the majority of the O2(1∆g) released into the H2O was deactivated prior to reaching another NP.

Figure 7. Inter- and intraparticle mechanisms of SODF.

Temperature. The dependence of SODF on temperature was studied over a broad range of temperatures from 10° C (Figure 8, panel A, a) to 80 C° (Figure 8, panel A, b). An increase in the temperature led to an increase in the diffusion coefficient of the O2(1∆g) in both the polystyrene matrix27 and H2O28 and, therefore more efficient quenching of the TPP triplets (Supporting Information, Figure S13).29 These data correspond to a significant increase in the SODF signal at high temperature and a shortening of the SODF apparent lifetime (τSODF). The opposite effect leading to the decrease of the SODF signal, i.e., a lower solubility of oxygen at higher temperatures, is less effective within the studied temperature region.30


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Figure 8. SODF of 0.5 wt% TPP-NPs in air-saturated H2O at 10 °C (a) and 80 °C (b) (panel A) and the corresponding Arrhenius plot (c) (panel B). Red lines are the fits to the experimental data. The Arrhenius equation (Figure 8, panel B, c) was applied to the reaction of the TPP triplet states with O2(1∆g), which results in SODF (equation 4): kSODF = A´ exp(-Ea´/RT).

(4)

The apparent activation energy Ea´ is 15.9±0.2 kJ mol-1. Oxygen concentration. Finally, we tested the TPP-NPs for sensing of dissolved oxygen over a broad range of concentrations from 0.2 mg l-1 (2×10-3 % or 6.25 µM) to 38 mg l-1 (0.38 % or 1187 µM) at temperatures of 25 and 37 °C. Note that these oxygen concentrations comprise anaerobic, air-saturated (8.27 mg l-1, 0.083 % or 258.4 µM at 25 °C and 101 kPa) and oxygensaturated conditions. The SODF signals did not show any significant deviation from the single exponential kinetics (Figure 9, panel A, a, b). The potential for oxygen sensing was analyzed quantitatively using the apparent lifetimes of the SODF signal at the different oxygen concentrations and the Stern–Volmer equation (equation 5): 1/τSODF=1/τ0SODF + kq [O2],

(5)


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where τ0SODF and τSODF are the apparent lifetimes of SODF at [O2] = 0 and a given concentration of O2, respectively, and kq is the bimolecular quenching constant. The limiting value of τ0SODF is the lifetime of the TPP triplet states in oxygen-free conditions (τ0SODF → τT), where the SODF amplitude is zero. The Stern–Volmer plot is nearly linear, from which kq values of (6.4 ± 0.1) ×103 l mg-1s-1 at 25 °C (Figure 9, panel B, c) and (6.5 ± 0.1) ×103 l mg-1s-1 at 37 °C (Figure 9, panel B, d) were estimated.

Figure 9. Kinetics of SODF of the TPP-NPs at limiting concentrations of oxygen of 0.2 (a) and 38 mg l-1 (b) at 25 °C, where the red lines are the single-exponential fits to the experimental data (panel A); Stern–Volmer dependence of the SODF apparent lifetime on the oxygen concentration at 25 °C (c) and at 37 °C (d) (panel B). All independent measurements of the oxygen concentration showed very good reproducibility. Additional evaluation of the SODF data at different O2 concentrations based on the signal amplitudes or overall fluorescence intensity after a selected time (20 µs) is shown in the Supporting Information (Figure S14).


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3.5. Photooxidation of external substrates by TPP-NPs and TMPyP-NPs. The SODF signal and O2(1∆g) phosphorescence of both the TPP-NPs and TMPyP-NPs dispersions in water revealed the efficient generation of O2(1∆g). The SODF sensitivity of the TPP-NPs and TMPyP-NPs to external quenchers (Figure 5) as well as the proven ability of O2(1∆g) from one NP to reach another (Figure 7 and Figure 6B) can serve as important tools for predicting that photogenerated O2(1∆g) from both types of NPs is able to reach (and oxidize) external substrates in aqueous solutions. This assumption is not obvious in the case of the TPPNPs with an encapsulated photosensitizer. To prove this assumption, we used uric acid (UA), a known specific acceptor of O2(1∆g),16 and I-, a nonspecific but highly sensitive acceptor,17 as model substrates. We found a strong photooxidation ability for both type of NPs (TPP-NPs and TMPyP-NPs) toward both types of acceptors. As an example, irradiation of the 0.05 wt% TPP-NPs or TMPyP-NPs dispersions in a detection solution of UA led to its photobleaching, as observed by a decrease in absorbance at 291 nm, in contrast to the behavior in the dark (Figure 10, panels A and C).

Figure 10. Decrease in the UA absorbance at 291 nm with irradiation time for the 0.05 wt% TPP-NPs (panel A) and TMPyP-NPs (0.01 TMPyP/IES) (panel C), and the increase in the I3absorbance at 351 nm in an air-saturated iodide detection solution with 0.05 wt% TPP-NPs


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(panel B). Dispersions (1.5×1012 NPs ml-1) were investigated without (a) and with 0.01 M NaN3 quencher (b), saturated with N2 (c) or in the presence of 50 % D2O (d). Dispersions of NPs without TPP (e) were used as references.

No photobleaching was observed during irradiation in the presence of 0.01 mol l-1 NaN3, a known physical quencher of O2(1∆g),26 or in N2- saturated solutions or when using NPs without a photosensitizer. Similarly, the iodide test for the detection of O2(1∆g) indicated a linear increase in the I3− concentration (an increase in the absorbance bands at 287 or 351 nm) proportional to the photogenerated amount of O2(1∆g), in contrast to a blank solution stored in the dark or irradiated in the presence of NaN3 (Figure 10, panel B). In further experiments, we examined the effect of the photosensitizer concentration and temperature on the photooxidation rate of UA. Photosensitizer concentration. The photooxidation rate increased with increasing photosensitizer concentration (Supporting Information, Figure S15). The relative photooxidation efficacy (PE) of the samples was calculated as the slope of UA absorbance at 291 nm, divided by the amount of absorbed light by porphyrin, ΣI0(1-10-Ai), between 400 and 700 nm vs. irradiation time (Figure 11). The calculation clearly shows that the PE is the highest at a low concentration of photosensitizer. The highest PE corresponds to the highest ΦF (Supporting information, Table S2) and it is evidently related to negligible inner filter and self-quenching effects. The inner filter and self-quenching effects were clearly observed when we examined the photooxidation of a UA detection solution containing dispersions of TPP-NPs or TMPyP-NPs with the same overall concentration of photosensitizer (TPP (2.4×10-8 mol l-1) or TMPyP (5.0 ×10-8 mol l-1)) but a different number of NPs (Supporting Information, Figure S16). Evidently, a higher photosensitizer/NP ratio significantly decreased the photooxidation rate.


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Figure 11. Photooxidation efficacy (PE) for 0.005 (a), 0.05 (b), 0.5 (c), 5 (d), and 10 (e) wt% TPP-NPs (panel A) and for TMPyP-NPs with TMPyP/IEC of 0.001 (a), 0.01 (b), and 0.1 (c) (panel B) irradiated by a 500 W Xe-lamp equipped with a long pass filter (λ ≥ 400 nm). Temperature effects. In our previous studies,18,

23

we analyzed the mean radial diffusion

length of O2(1∆g), lr, during its lifetime (τ∆) inside a polystyrene matrix and in water based on equation 6: lr=(6 D(O2)τ∆)1/2 ,

(6)

where D(O2) is the oxygen diffusion coefficient. Using literature data (Supporting Information, Table S3)27,29,30,31 in the calculation using equation 6, the estimated values of lr at 20 °C were 41 and 200 nm in polystyrene and H2O, respectively. Note that the values of τ∆ and D(O2) depend on the temperature in both media.22,29 The estimated values of lr at 50 °C were 53 and 280 nm in polystyrene and H2O, respectively. The higher D(O2), together with higher reaction rate constant at higher temperatures, is responsible for the experimentally observed faster photooxidation of UA at a higher temperature (Figure 12), which is in agreement with the SODF measurement (section 3.4). The effect is substantial mainly for the TPP-NPs with TPP encapsulated in a polystyrene core (Figure 12, panel A), where the increase in D(O2) with increasing temperature


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contributes to the photooxidation rate, in contrast to the externally bound TMPyP (TMPyPNPs). The photooxidation of UA using TMPyP-NPs was less dependent on temperature (Figure 12, panel B); in fact, the photooxidation rate at 60 °C was slightly lower than at 37 °C. This result could correspond to the lower oxygen concentration in water at a higher temperature,30 which limited the accelerating effects on both diffusion and the rate constant of UA photooxidation.

Figure 12. The effect of temperature on the kinetics of UA photooxidation by 0.5 wt% TPP-NPs (TPP concentration ~ 3 ×10-7 mol l-1, panel A) or TMPyP-NPs (0.01 TMPyP/IES, TMPyP concentration ~ 1 ×10-7 mol l-1, panel B). CONCLUSIONS In this study, we reported the photooxidation properties and oxygen sensing capabilities of two types of sulfonated polystyrene nanoparticles with encapsulated hydrophobic TPP (TPP-NPs) or ionically entangled hydrophilic TMPyP (TMPyP-NPs) porphyrin photosensitizers. A simple method based on SODF permitted the in situ continuous measurement of the dissolved oxygen concentration in aqueous media in the broad region of oxygen concentrations from anaerobic conditions to oxygen-saturated media without the addition of any external oxygen sensor. The analysis of SODF can be also useful for predicting the photooxidation ability of photoactive


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materials towards external chemical/biological substrates. In addition, the measurement of the SODF signals is a very sensitive method of singlet oxygen detection compared to the direct measurement of the weak singlet oxygen phosphorescence. The short-lived SODF signal of the TMPyP-NPs could not be fully separated from the prompt fluorescence in air- or oxygen-saturated media due to the short lifetime of the TMPyP triplet states and/or O2(1∆g). In contrast, the kinetics and intensity of the long-lived SODF signal of the TPP-NPs reflect the longer lifetimes of all excited states in the polystyrene matrix over a broad range of oxygen concentrations. The amplitude of the SODF signal increased and the corresponding apparent lifetime decreased with increasing temperature and increasing concentration of dissolved oxygen. The photooxidation efficacy of both photoactive NPs strongly depended on the concentration of the corresponding photosensitizer due to the inner filter and self-quenching effects. The photooxidation rates were significantly influenced by the temperature due to changes in the oxygen diffusion and oxygen solubility, both in the polystyrene NPs and in their surrounding environment.

ASSOCIATED CONTENT Supporting Information. Experimental methods, transient absorption of the porphyrin triplets, time-resolved phosphorescence of O2(1∆g), evaluation of photophysical data, and physical properties of oxygen are provided. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author


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* E-mail address: [email protected] (J. M.) Funding Sources This work was supported by the Czech Science Foundation (16-15020S) and by the OP VVV “Excellent Research Teams”, project No. CZ.02.1.01/0.0/0.0/15_003/0000417 – CUCAM. ACKNOWLEDGMENT The authors thank Dr. Lukáš Plíštil for the preparation of the nanofiber materials via electrospinning, Dr. Miroslav Šlouf for the TEM measurements and Dr. Pavel Engst for help with the photophysical measurements. ABBREVIATIONS

TPP,

5,10,15,20-tetraphenylporphyrin;

TMPyP,

5,10,15,20-tetrakis(N-methylpyridinium-4-

yl)porphyrin; TPPS, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin; TPP-NPs, sulfonated polystyrene nanoparticles with encapsulated TPP; TMPyP-NPs, sulfonated polystyrene nanoparticles with ionically bound TMPyP; UA, uric acid; IEC, ion exchange capacity; PE, relative photooxidation efficacy.


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REFERENCES

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(28) Han, P.; Bartels, D. M. Temperature Dependence of Oxygen Diffusion in H2O and D2O. J. Phys. Chem. 1996, 100, 5597−5602. (29) Suchánek, J.; Henke, P.; Mosinger, J.; Zelinger, Z.; Kubát, P., Effect of Temperature on Photophysical Properties of Polymeric Nanofiber Materials with Porphyrin Photosensitizers. J. Phys. Chem. B 2014, 118, 6167−6174. (30) Battino, R.; Rettich, T. R.; Tominaga, T., The Solubility of Oxygen and Ozone in Liquids. J. Phys. Chem. Reference Data 1983, 12, 163−178. (31) 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.

GRAPHICAL ABSTRACT


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Nanoparticles with Embedded Porphyrin Photosensitizers for

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