Insights into the Mechanism of Quantum Dot-Sensitized Singlet

Article pubs.acs.org/JPCC

Insights into the Mechanism of Quantum Dot-Sensitized Singlet Oxygen Production for Photodynamic Therapy Gael̈ le Charron,†,‡ Tanya Stuchinskaya,‡ Dylan R. Edwards,§ David A. Russell,*,‡ and Thomas Nann*,∥ †

Université Paris Diderot, Sorbonne Paris Cite, ITODYS, UMR 7086, CNRS, 7-75205 Paris, France School of Chemistry, University of East Anglia, Norwich NR4 7TJ, U.K. § School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. ∥ Ian Wark Research Institute, University of South Australia, Adelaide, SA 5095, Australia ‡

ABSTRACT: Semiconductor nanoparticles or quantum dots (QDs) have been proposed as potential vehicles for photodynamic therapy (PDT) since 2003. Some studies using cadmium-based QDs have shown promising results when coupled to molecular photosensitizers. However, the toxicity of such QDs and the low overall efficiency of these hybrids are still problematic. We have coupled two types (sizes) of lesstoxic InP/ZnS QDs to the photosensitizer chlorin e6. The spectroscopic properties of these hybrids have been studied in detail. Spectroscopic methods have been applied to elucidate the energy transfer pathways and kinetics and the rate of singlet oxygen production of all components. Additionally, the PDT efficacy of the QD/chlorin e6 hybrids has been assessed against a breast cancer (MDA-MB-231) cell line using a colorimetric 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We have found that the energy transfer between QDs and the molecular photosensitizer is the ratedetermining step for the production of singlet oxygen and that the cell viabilities of the hybrid and free photosensitizer are comparable. These systematic findings show that the energy transfer between QDs and photosensitizers is a “bottleneck”, which suggests that a better chemical design of the QD/photosensitizer hybrids in future embodiments is essential.

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INTRODUCTION Photodynamic therapy (PDT) is a relatively new method for cancer treatment, where tumor cells are destroyed by lightinduced, local production of a reactive oxygen species (ROS), such as singlet oxygen (1O2).1−4 The ROS is generated by a photosensitizer, which has to be brought in close proximity to the tumor cells and is usually administered systemically. The major advantages of PDT are that it is relatively inexpensive, is noninvasive, and can be applied locally and that cumulative toxicity is not observed. Quantum dots (QDs) are semiconducting nanoparticles with size dimensions in the lower nanometer size range. Since QDs are inorganic nanocrystals, their optical properties are superior to organic fluorophores in regard to their absorption cross section, chemical and optical stability, and tunability.5 Therefore, QDs are potentially interesting candidates as photosensitizers or “light antennas” for PDT. Several groups have found that QDs by themselves do not produce a detectable amount of singlet oxygen, while others reported that QDs produce singlet oxygen with low quantum yields of up to 0.05.6,7 However, when mixed with classical photosensitizers such as methylene blue, trifluoperazine, or sulfonated aluminum phthalocyanine, a QD-mediated production of ROS has been observed.6−10 To obtain fast energy transfer from QDs to the photosensitizerand therefore increase the production of © 2012 American Chemical Society

ROSthe distance between the acceptor and donor has to be minimal (since the rate of resonance energy transfer scales inversely with the sixth power of the distance) and the overlap between electron clouds maximal.11 The easiest and most straightforward method to achieve this is to adsorb the energy acceptor molecules, i.e., the photosensitizer, directly onto the QD surface or to couple the photosensitizer by a short, adsorbed linker. Several authors have realized this type of system by chemisorption of different photosensitizers onto cadmium-based QDs.6,7,9,10,12−16 The adsorption of photosensitizers onto QD surfaces does have a significant disadvantage: the chemisorbed species are prone to desorption, and therefore, the whole system has a limited lifetime. On the other hand, covalent bonding requires a suitable “anchor” to the surface of the QDs, which is not available on as-synthesized particles. This covalent anchor can be provided by coating the QDs with a cross-linked polymeric, inorganic, or mixed shell. Covalent bonding increases the chemical stability of the donor/acceptor complex but also increases the distance between these units. Here we describe the synthesis of a QD/chlorin e6 photosensitizer system, where Received: February 2, 2012 Revised: April 3, 2012 Published: April 4, 2012 9334

dx.doi.org/10.1021/jp301103f | J. Phys. Chem. C 2012, 116, 9334−9342

The Journal of Physical Chemistry C

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the QDs are comprised of two types (sizes) of InP/ZnS about 2 and 3 nm diameterand are therefore less toxic than cadmium-based systems. The QDs were coated with a crosslinked organo-silica shell to which chlorin e6 photosensitizer molecules were covalently bound. This hybrid system has been studied systematically in terms of spectroscopic properties and singlet oxygen production and applied to a PDT model breast cancer cell line. We found that our chemically more robust and less toxic system produced singlet oxygen in a fashion similar to that reported for cadmium-based systems. More importantly, the kinetic constants have been derived and evidence that the energy transfer from the QD to the chlorin e6 photosensitizer limits the rate of singlet oxygen production.

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RESULTS AND DISCUSSION Synthesis of QD/Chlorin e6 Hybrids. The InP/ZnS QDs have been synthesized according to a previously published procedure and were dispersed in toluene.17,18 The covalent coupling of the chlorin e6 onto the surface of the QDs was achieved in two steps. First, the QDs were coated with a crosslinked, functional shell which enabled phase transfer. Second, the chlorin e6 photosensitizer was coupled to the functional groups on the outer surface of the QDs. For the first step, we adapted the coating procedure published by Jana et al.19 to our InP/ZnS QDs. Briefly, the InP/ZnS QDs were dispersed in toluene and mixed with mercaptopropyltrimethoxysilane and aminopropyltrimethoxysilane. The thiol groups are believed to displace the original QD surface ligands and act as a template for the building of a thin silica shell from the aminopropyl siloxane derivative. The thickness of the silica shell was estimated to be 7 to 10 nm from one synthesis to the other through measurement of the hydrodynamic diameter by Dynamic Light Scattering (see Experimental Section). The resulting water-soluble QDs bore pendant primary amine groups that were coupled to the carboxyl moieties of the chlorin e6 through N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide/N-hydroxysuccinimide (EDC/NHS) activated amide bond formation. According to Nakajima and Ikada,20 EDC couplings in water work best (i) under mild acidic catalysis, (ii) when carboxyl groups are deprotonated, and (iii) when amino groups are in the neutral form. Basic conditions are undesirable since hydroxyl groups are the best nucleophiles and compete with the amine groups for hydrolysis of the activated ester. For coupling on nanoparticles, one should also expect a faster reaction at a pH where the nanoparticles are well dispersed. Since primary amines generally have a pKa around 9−10, obviously not all of these conditions can be fulfilled at once. We found that the best compromise was obtained when performing the reaction in phosphate-buffered saline (PBS) at pH 7.2. At this pH, carboxyl groups are deprotonated. There is still about 10−7 M protons in solution to catalyze the addition of carboxylates on EDC and not sufficient hydroxyl groups to quickly quench the activated esters. Moreover, the pKa of surface amines on the nanoparticles is expected to be shifted by several units toward lower pH compared to that of free amines. This effect has been previously demonstrated on self-assembled monolayer-protected nanoparticles and is likely to arise from surface charge interaction.21,22 Hence, at pH 7.2, there should be a substantial amount of neutral surface amine groups. The absorption spectrum of the resulting hybrid material shows successful covalent attachment of chlorin e6 molecules through the superimposition of the characteristic Soret band of chlorin e6 on the absorption band of the QDs (cf. Figure 1).

Figure 1. Absorption spectra of chlorin e6 and the QD/chlorin e6 hybrid in DMF.

Comparison of the Soret band area in the hybrid and the pure chlorin e6 leads to an estimation of the amount of chlorin within the sample. Assuming a molar extinction coefficient for QDs of a few 105 M−1 cm−1, one can estimate the number of chlorin e6 molecules per QD to be within 5 to 20. Spectroscopic Investigation of Energy Transfer Rates. Having achieved the covalent attachment of the chlorin e6 onto the QDs, we then studied the energy transfer kinetics by means of optical spectroscopy. Figure 2 shows a diagram of the whole system with the different pathways for energy transfer indicated. Equations 1−8 describe the photochemical processes involved and 9−11 the quantum yields relevant for investigation of the kinetics. To estimate the rate of energy transfer from QDs to chlorin e6, the luminescence lifetimes of all preparations, i.e., the QDs, the chlorin e6, and the “hybrid”, were measured separately. hν

QD → QD*

(1)

k QD,l

QD* ⎯⎯⎯→ QD

(2)

k QD,nr

QD* ⎯⎯⎯⎯⎯→ QD

(3)

hν

chlorin e6 → chlorin e6* ke6,l

chlorin e6* ⎯⎯→ chlorin e6 ke6,nr

chlorin e6* ⎯⎯⎯→ chlorin e6 kET

QD* ⎯→ ⎯ chlorin e6* k SOP

chlorin e6* + 3O2 ⎯⎯⎯→ chlorin e6 + 1O2 QYe6,l = QYSOP = 9335

(4) (5) (6) (7) (8)

ke6,1 ke6,1 + ke6,nr + k SOP,a

(9)

k SOP,a ke6,1 + ke6,nr + k SOP,a

(10)



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Figure 2. Energy transfer pathways in QD/chlorin e6 hybrids. 1. No energy transfer (QD luminescence). 2. Energy transfer from QD to chlorin e6 (chlorin e6 luminescence). 3. Energy transfer from QD to chlorin e6 to oxygen. kET denotes the rate constant for the energy transfer from QD to chlorin e6, kSOP from chlorin e6 to oxygen, and kQD,1 and ke6,1 the luminescence of QD and chlorin e6.

Figure 3. Luminescence lifetime traces of chlorin e6, QDs, and the QD/chlorin e6 hybrid structure. Excitation at 370 nm, luminescence traces recorded at 660 nm for the chlorin e6, and the maxima of the QD emission bands. Red and black traces refer to different QD sizes.

E=

kET kET + k QD,nr + k QD,1

The luminescence lifetime of the chlorin e6 (τc) was measured to be approximately 50 ns (black trace in Figure 3, left). When coupled to QDs, this lifetime increased to 80−90 ns. Moreover, luminescence intensity measurements revealed that the luminescence of the chlorin e6 was strongly quenched when located within the hybrid. This is not surprising since QDs are competing with chlorin e6 for light absorption. Thus, at a given irradiation power, the luminescence intensity of chlorin e6 in the hybrid is expected to be weaker than that of the pure chlorin e6 as less light is directly absorbed. This diminishing of the luminescence intensity can be quantified by comparison of the luminescence QY of the free chlorin e6 (QYl,e6) and the apparent luminescence QY of chlorin e6 within the hybrid (QYl,e6,ahybrid), which was experimentally measured by treating the hybrid as a simple fluorophore. QYl,e6 was measured to be 0.11, and its apparent counterpart in the hybrid was estimated to lie below 0.01 (see Experimental Section). One can express the QYl,e6,ahybrid as a function of the true luminescence quantum yield of chlorin e6 in the hybrid

(11)

with kSOP,a = kSOP [3O2]. Figure 3 shows the luminescence lifetimes of both components (QDs and chlorin e6) and the hybrid structures on a logarithmic scale for two colors (red and yellow) of QDs (we are aware that the luminescence decay of the QDs does not follow a single exponential rate law; however, for the sake of comparison, this simplification is justified). The luminescence lifetime τQD of the unmodified QDs was typically of the order of 100−150 ns (116 and 145 ns for red and yellow QDs, respectively), which is in agreement with earlier measurements.23 When coupled to the chlorin e6, the QD luminescence was quenched but still slightly visible with the naked eye. Lifetimes in the range of 1−2 μs were recorded for QDs coupled to chlorin e6. An increase in luminescence lifetime in such a situation is characteristic for an alternative path of de-excitation in a given system. For example, short-lived excitons are quenched by energy transfer, and long-lived excitons (probably trapped ones) remain. 9336



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Figure 4. Theoretical luminescence lifetime profiles of chlorin e6 in the hybrid (green trace) compared to that of pure chlorin e6 (black trace) when energy transfer is negligible compared to direct excitation of chlorin e6 (left) and when energy transfer is non-negligible (right). hybrid QY hybrid × xe6 l,e6,q = QY l,e6

the luminescent state through energy transfer and de-excitation by luminescence (Figure 4, right). This convolution is responsible for the apparent slowing down of the luminescence decay in the hybrid as compared to the free chlorin e6, as observed in our system. The fact that the chlorin e6 luminescence slows down in the hybrid compared to the free compound indicates that the energy transfer actually slows down the overall luminescence process. This means that the energy transfer is the rate-determining step for the deexcitation of chlorin e6 and that the apparent chlorin e6 luminescence rate is in fact the energy transfer rate. On the basis of this qualitative reasoning, we can conclude that kET is exactly 12.5 × 106 s−1 if excitation occurs only by energy transfer or 12.5 × 106 s−1 at most in a more realistic picture in which excitation occurs by both direct photon absorption by chlorin e6 and energy transfer from the QD. In the following, we will derive these conclusions from quantitative analysis of the luminescence data: The exact rate of energy transfer between a donor and an acceptor (here, QD and chlorin e6, respectively) could in principle be derived from the determination of the efficiency of energy transfer using luminescence lifetime measurements according to eqs 15−17. As the luminescence rate of QD short-lived excitons in the QD/chlorin e6 hybrid was hindered by remaining long-lived excitons, the efficiency of the energy transfer could not be estimated quantitatively. However, previous studies have shown that energy transfer efficiencies fall within 10−20% for a QD to dye distance of roughly 7 nm.24−26 Assuming this is again the case in the present system, kET can be calculated to be between 6 × 105 and 14 × 105 s−1.

(12)

where xe6 stands for the fraction of incident energy directly absorbed by chlorin e6 (for the sake of simplicity, the contribution of energy transfer from QDs is neglected here). Comparison of the absorption spectra of pure chlorin e6 and hybrid indicates that the fraction of energy directly absorbed by chlorin e6 is roughly 57%. Hence, if the true quantum yield of luminescence of the chlorin e6 in the hybrid is identical to that of the pure molecule, one should expect an apparent quantum yield of 0.57 × 0.11 = 0.06. The experimental value is much less and therefore indicates quenching of the luminescence of chlorin e6 when supported on QDs. The true luminescence QY of chlorin e6 within the hybrid is below 0.01/0.57−0.02. The increase in the lifetime of chlorin e6 luminescence when coupled to the QDs leads to two conclusions, which we derive in the following sections. First, excitation of chlorin e6 in the hybrid is triggered for a substantial part by energy transfer from the excited QDs. Second, the energy transfer process is the ratedetermining step for all subsequent de-excitation processes. For the sake of simplicity, we first qualitatively demonstrate these assertions assuming the de-excitation pathways of the chlorin e6 in the hybrid are the same as in the free molecule and are characterized by the same kinetic constants. The rate laws of de-excitation of chlorin e6* can be written by taking eqs 5 to 8 into account −

d[e6*] = (ke6,l + ke6,nr + k SOP,a)[e6*] dt

−

d[e6*] = (ke6,l + ke6,nr + k SOP,a)[e6*] − kET[QD*] dt

(13)

E=1−

(14)

Equation 13 represents the rate law for de-excitation of the free chlorin e6 and eq 14 for a hybrid structure. In the hybrid system, the excited luminescent state is populated by both direct absorption of light by chlorin e6 and energy transfer from the QDs (the term kET[QD*] in eq 14also cf. to Figure 1). The contribution of energy transfer to the excited state of chlorin e6 in the hybrid leads to a superimposition of the chlorin photosensitizer decay, in a similar manner to the free molecule, and to a rise term through the contribution of excited QDs according to eq 14. Figure 4 shows a schematic of this situation. The time-scale of energy transfer from a QD to an absorbed dye has been reported to be in the order of magnitude of a few hundred nanoseconds for distances of the order of 6−7 nm.24 Therefore, the feeding of the excited luminescent state of chlorin e6 extends long after the laser pulse is over. The observed decay then results from the convolution of feeding of

E=

hybrid τQD

τQD

(15)

kET −1 τQD

+ kET

(16)

E −1 × τQD (17) 1−E The rate constant kSOP,a of chlorin e6 can be expressed as a function of the quantum yields of singlet oxygen production and fluorescence and of the rate of fluorescence kET =

k SOP,a =

QYSOPke6,l QYe6,l

(18)

For the free chlorin e6 molecule, the luminescence quantum yield was measured to be 0.11. Moreover, the luminescence rate of chlorin can be derived from its lifetime state and its fluorescence quantum yield according to eqs 9 and 19. 9337



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Figure 5. Bleaching of ADPA by chlorin e6 (left) and the QD/chlorin e6 hybrid (right) under illumination with 350 nm.

τF =

1 ke6,1 + ke6,nr + k SOP,a

chlorin e6. Second, the luminescence quantum yield of the chlorin e6 in the hybrid is at least five and a half times less than in the free chlorin e6 (less than 0.02 vs 0.11). This suggests that the luminescence de-excitation pathway of chlorin e6 is shut down in the hybrid, to the advantage of another de-excitation path through increasing of its respective rate. This observation led us to consider two possible scenarios. In the worst case scenario, the portion of energy lost for the luminescence path is gained by the nonradiative paths other than singlet oxygen production (QYe6,nr = 1 − 0.02 − 0.65 = 0.33). Hence, the corresponding apparent singlet oxygen production rate is calculated to be 6.14 × 107 s−1 using QYe6,1 = 0.02, QYSOP = 0.65, and ke6,1 = 1.89 × 106 s−1, which is at least 44 times faster than the ET rate calculated above. In the best case scenario, the portion of energy lost by the luminescence path is gained by the singlet oxygen production path (QYSOP = 1 − 0.25 − 0.02 = 0.73), which leads to an apparent singlet oxygen production rate of 6.90 × 107 s−1, again roughly 50 times faster than the highest reasonable ET rate calculated above. These findings confirm our qualitative result according to which the energy transfer from QD to chlorin e6 acts as a bottleneck for the singlet oxygen production. The spectroscopic results indicate that the energy transfer kinetics actually slows the excitation of the photosensitizer down. Nevertheless, there are some factors that might still render the hybrid system advantageous compared with the free chlorin e6 photosensitizer alone. For example, the large absorption cross section of the QDs might increase the efficiency of light absorption and thus the overall performance.31 Furthermore, dimerization of chlorin e6 is a prime cause for lowering its singlet oxygen conversion rate. It can be assumed that anchoring chlorin e6 onto the surface of QDs using short linkers might prevent dimerization and thus increase its performance. Measurement of Singlet Oxygen Production. We have quantified the singlet oxygen production activities of chlorin e6, water-soluble QDs, and the QD/chlorin e6 hybrid by colorimetrically measuring the rate of conversion of the singlet oxygen probe anthracene dipropionic acid (ADPA) to its endoperoxide.

(19)

Rearrangement of eqs 9 and 19 gives the following expression for the luminescence rate ke6,1 =

QYe6,1 τF

(20)

with a measured chlorin e6 luminescence QY of 0.11 and a measured lifetime of 53 ns, and eq 20 leads to a luminescence rate of 1.89 × 106 s−1. Measurement of the SOP quantum yield by luminescence means would have required the determination of the luminescence quantum yield and lifetime of singlet oxygen. However, such a study involves the monitoring of the singlet to triplet oxygen radiative de-excitation at 1275 nm with an optical instrumentation suited for the NIR range that was not available to us. Nevertheless, Redmond and Gamlin compiled quantum yields for chlorin e6 in various solvents and irradiation conditions.27 In particular, the quantum yield of several salts of chlorin e6 in ethanol under irradiation at 347 nm were determined to be on average 0.65. This value was used as an estimation of the singlet oxygen quantum yield (ΦΔ or QYSOP) of chlorin e6 under 350 nm irradiation. Substitution of the luminescence QY, luminescence rate, and singlet oxygen QY leads to an apparent rate of singlet oxygen production of 1.1 × 107 s−1 for the free chlorin e6. However, for the chlorin e6 within the hybrid, the singlet oxygen production rate can be foreseen to be faster than this value for two reasons. First, chlorins tend to dimerize in aqueous solution because of strong π-stacking, a phenomenon that has been reported to weaken the singlet oxygen quantum yields. 28 The effect on the singlet oxygen production performance can be understood in terms of the number of accessible sites on the chlorin e6 for the oxygen. As dimers, chlorins display only two accessible faces for dioxygen adsorption through noncovalent interaction.29,30 When supported onto QDs, it is likely that dimers are broken. Each supported chlorin e6 displays two accessible interaction sites by itself. From a statistical point of view, the energy transfer rate through intersystem crossing from chlorin e6 to triplet oxygen should therefore double in the hybrid as compared to the free 9338



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the following the consequences of these two situations on the rate of ADPA consumption. If the consumption of ADPA is much faster than every other step involving singlet oxygen, then the production of singlet oxygen is the rate-determining step for ADPA consumption. One can then apply the steady state approximation to 1O2.

d[1O2 ] =0 dt k SOP,a[e6*] = (kADPA[ADPA] − k H2O[H 2O]

The various samples were irradiated at 350 nm over incremental 10 min periods. After each 10 min irradiation, the visible absorbance spectrum was recorded, and the change in ADPA was monitored via the decrease of the characteristic absorption band at 400 nm. The diminution of the ADPA concentration is an indirect reflection of the production of singlet oxygen in the medium. Figure 5 shows four absorption spectra of an ADPA−chlorin e6 and ADPA−QD/chlorin e6 hybrid recorded following irradiation. The “bleaching” of the ADPA as the singlet oxygen was produced can be observed clearly. For the QDs on their own, the amount of ADPA endoperoxide species produced was negligible. Although representative of the presence of singlet oxygen in solution, the degradation of ADPA does not necessarily mirror the total amount of singlet oxygen produced by the photosensitizer. Indeed, conversion of ADPA into its endoperoxide species through reaction with singlet oxygen may be slower than the singlet oxygen production itself or slower than other competitive singlet oxygen de-excitation processes, namely, luminescence or nonradiative deactivation by the solvent (in our case, DMF/water 1:10). Careful examination of the data is therefore needed to probe the meaning of ADPA degradation in terms of the amount of singlet oxygen produced. The kinetics of the reaction at play in the presence of ADPA is described by the following equations k SOP

chlorin e6* + 3O2 ⎯⎯⎯→ chlorin e6* + 1O2 kADPA

1

O2 + ADPA ⎯⎯⎯⎯→ endoperoxide k H2O

1

O2 + H 2O ⎯⎯⎯→ 3O2 + H 2O + heat kDMF

1

O2 + DMF ⎯⎯⎯⎯→ 3O2 + DMF + heat

1

k1O2,1

O2 ⎯⎯⎯⎯→ 3O2

− kDMF[DMF] − kl,1O2)[1O2 ] ≈kADPA[ADPA][1O2 ]

−

d[ADPA] = kADPA[ADPA][1O2 ] dt

d[ADPA] Δ[ADPA] ≈− = k SOP,a[e6*] dt Δt

(31)

and Δn(1O2 )produced = Δn(ADPA)consumed

(32)

We examine now the possibility of an ADPA consumption rate in the same order of magnitude or smaller than the other processes. In this case, the steady state approximation cannot be applied to [1O2]. It then follows that the ADPA consumption does not mirror the total production of singlet oxygen and depends on the concentration of ADPA −

d[ADPA] Δ[ADPA] ≈− = kADPA[ADPA][1O2 ] dt Δt

(33)

The rate of quenching of singlet oxygen by pure water can be calculated from the phosphorescence quantum yield and lifetime (kH2O,a = kH2O[H2O] = 3 × 105 s−1).32 Similarly, the rate of quenching of singlet oxygen by pure DMF is calculated to be kDMF,a = kDMF[DMF] = 5 × 104 s−1. The total apparent quenching rate due to the solvent is then 2.8 × 105 s−1. The rate constant of ADPA endoperoxo formation has been reported to be kADPA = 108 M−1 s−1.33 Using a concentration in ADPA of roughly 100 μM leads to an apparent quenching rate of singlet oxygen by ADPA of kADPA,a = kADPA[ADPA] = 104 s−1, which is roughly 30 times slower than the apparent quenching rate due to the solvent. According to these figures, ADPA consumption is always a minor deactivation path compared to quenching by the solvent, regardless of the rate of singlet oxygen production, and therefore the second mechanistic hypothesis holds. This was confirmed by the experimental observation of the dependence of the rate of ADPA consumption on the concentration of ADPA, all chlorin e6 quantities being equal. Hence, the monitoring of ADPA consumption gives access to a quantity that is proportional but not equal to the amount of singlet oxygen produced during the irradiation period, which we will hereafter refer to as the singlet oxygen production activity Ω

(22) (23) (24) (25) (26)

d[1O2 ] = k SOP,a[e6*] − (kADPA[ADPA] − k H2O[H 2O] dt

−

(30)

Therefore, it follows that the rate of ADPA consumption fully mirrors the rate of singlet oxygen production and is independent of the ADPA concentration in the medium

and the rates of the singlet oxygen formation and ADPA consumption are, respectively, given by

− kDMF[DMF] − k 1O2,1)[1O2 ]

(29)

(27)

(28)

Ω=

To interpret the singlet oxygen production from the ADPA degradation, two possibilities need to be considered. The rate of ADPA is either (i) much higher or (ii) of the same magnitude or smaller than the rate of singlet oxygen production and all other singlet oxygen de-excitation paths. We examine in

Δn[ADPA] 1 × Δt [ADPA]

= kADPA[1O2 ] × V = k[1O2 ] 9339

(34) dx.doi.org/10.1021/jp301103f | J. Phys. Chem. C 2012, 116, 9334−9342


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with V as the reaction volume and Δn[ADPA] as the number of mole of ADPA consumed during the reaction. To examine the production of singlet oxygen of the hybrid system through Ω, one can adopt two perspectives. In the first one, the singlet oxygen production rates are compared in terms of the efficiency of each chlorin e6 molecule (represented by Re6). For this, one needs to normalize the singlet oxygen production rates of both the hybrid and free chlorin e6 compound on the concentration of chlorin e6 (we have normalized our data on the area of the Soret band for this purpose as shown in Figure 1). The second perspective involves looking at the efficiency of each photon absorbed by a QD. In other words, it consists of following the efficiency of each hybrid moiety as a whole. If we consider a hybrid bearing on average n chlorin e6 molecules, then the rate of singlet oxygen production in terms of hybrid moieties (Rhybrid) hybrid is n*Re6. Table 1 gives values of Rhybrid for different possible values of n. Obviously, if n = 1, both possibilities are identical.

0.033 J/cm2). The cells were incubated for a further 18 h before proceeding with the MTT assay. Figure 6 shows the results of

Figure 6. Cell viability represented as percentage of viable cells after 3 h treatment with QDs, chlorin e6, and the QD/chlorin e6 hybrid following UV irradiation (0.033 J·cm−2) assessed with a MTT assay.

Table 1. Amount of singlet oxygen produced per mole of sacrificial ADPA probe for a Soret band area of 1 expressed in moles per moles of ADPA. Corresponding rates of singlet oxygen production per mole of sacrificial ADPA probe for a Soret band area of 1 expressed in moles per moles of ADPA per minute Ω [×10−7 moles per mol·L−1 of ADPA per min] 10 min 20 min chlorin e6 QD/chlorin e6 hybrid

7.6 9.0

7.3 6.4

the MTT assay in terms of chlorin e6 moieties. The cell viability decreased slightly when a concentration of 13 nM QD/ chlorin e6 hybrid was used. When the concentration of the hybrid was increased to 26 nM, the cell viability decreased by 40% as compared to nontreated control cells. A similar decrease in cell viability was observed when the chlorin e6 (24 nM) alone was used as the photosensitizer. Cells without any treatment, or treated with quantum dots alone displayed no decrease in viability following the same period and conditions of irradiation.

average Ω [×10−7 moles per mol·L−1 of ADPA per min]

30 min

n=1

n=2

n=3

8.9 6.1

7.9 7.0

14.0

21.0

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The results of Table 1 show that the rate of production of singlet oxygen per chlorin e6 molecule (n = 1) is smaller when coupled to a QD. This finding is in line with the conclusions made above from the luminescence lifetime measurements that the energy transfer rate kET is the rate-determining step in the energy transfer cascade (cf. Figure 2). However, in reality, more chlorin e6 molecules are bound to each QD, and the rate of production of singlet oxygen indeed exceeds that of free chlorin e6 for n = 2, when comparing molecular chlorin e6 with the number of hybrid structures. Thus, one could argue that the large absorption cross section of the QDs enhances the net singlet oxygen production. In Vitro Study Performance of QD/Chlorin e6 Hybrids. To determine the suitability of the QD/chlorin e6 hybrids for PDT, we have performed in vitro studies using the MDA-MB231 breast cancer cell line. The colorimetric 3-(4,5 dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic activity assay adapted to incorporate a cell irradiation protocol34,35 was used to determine cell viability after irradiation with UV light. The QD/chlorin e6 hybrid, chlorin e6 alone, and the QDs alone were separately added in 1 μL of phosphate buffer saline to the cell line seeded in 96-well plates and incubated for 3 h. The amount of QDs was adjusted so that it matched the concentration of QDs in the hybrid solution, which was equivalent to 26 nM of chlorin e6 (final concentration in each well); Triton X 100 lysis solution was used as a positive control to induce cell death. After incubation, cells were washed thoroughly with buffer to remove the QDs that had not been internalized, and then the plate was irradiated with a UV light for 5 min (UV lamp, total irradiation dose

EXPERIMENTAL SECTION Materials. All solvents and reagents were reagent grade and purchased from Sigma-Aldrich and used without further purification unless otherwise noted. MDA-MB-231 (breast carcinoma cell line) was obtained from the Cancer Research UK bank; cell culture materials were purchased from GIBCO. InP/ZnS quantum dots have been synthesized according to a previously published procedure.17,18 Silica Coating. Stock solutions (5 mL) of 0.5 M (3mercaptopropyl)trimethoxysilane (MPS) and (3-aminopropyl)trimethoxysilane (APS) respectively in toluene were prepared in a glovebox. In a vial, 67 μL of MPS stock was mixed with 333 μL of APS stock and diluted with 100 μL of toluene. Then, 100 μL of InP/ZnS QDs stock solution (the concentration in NPs was in the 15−50 μM range) was added. The solution was stirred in an ultrasonic bath setup at 65 °C for 45 min. At the end of the reaction, the nanoparticles spontaneously precipitated. After isolation by centrifugation, the QDs were washed with 4 mL of toluene, 4 mL of chloroform, and 4 mL of dimethylformamide (DMF). Finally, the silica-coated QDs were dispersed in 5 mL of DMF. A 100 μL aliquot of the QD solution was dissolved in 1 mL of 1% acetic acid buffer for dynamic light scattering (DLS) and zeta potential measurements. The hydrodynamic diameter was estimated between 16 and 25 nm from one experiment to the other; the zeta potential was between +30 and +40 mV. Chlorin e6 Conjugation. In a vial settled in an ice bath, 7.5 mg of chlorin e6 (Frontier Scientific), 114 mg of Nhydroxysulfosuccinimide sodium salt (sulfo-NHS), and 170

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μL of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) were dissolved in 15 mL of phosphate-buffered saline (PBS). The silica-coated QDs suspended in DMF were then added under stirring. The mixture was reacted in an ice-cold ultrasonic bath for 1 h. The functionalized QDs were recovered from the reaction medium using amicon centrifuge filters with a mass cutoff of 30 kDa. Purification was achieved by washing the nanoparticles on the filters with 100 mL of PBS buffer, after which no free chlorin e6 could be detected in the filtrate. Finally, the QD/chlorin e6 hybrid was redispersed in 10 mL of PBS buffer and stored in the dark at −18 °C. Luminescence Studies. All luminescence spectra were recorded using excitation and emission slits of 5 and 5 nm, respectively. The concentrations of the solutions were adjusted so that absorbance intensity was no more than 0.3 to prevent inner filter effects. Fluorescence quantum yields were calculated by comparison with Rhodamine, which has a QY of 0.31 at 514 nm.36 For the chlorin e6, the fluorescence quantum yield was determined to be 0.11 at 350 nm excitation following emission at 660 nm. In the hybrid, the fluorescence quantum yield was determined to be zero under the same conditions (no luminescence). Time-resolved experiments were performed under excitation at 370 nm (Horiba Jobin Yvon, Fluorolog 3 Spectrophotometer). The luminescence decay of the chlorin e6, as a solution of the free chlorin e6 and in the QD/chlorin e6 hybrid, was measured using a 660 nm emission. Luminescence decays of QD, as the QD alone, and QD/chlorin e6 hybrids were measured at 550 and 600 nm emission for yellow and red QD, respectively. Measurement of Singlet Oxygen Production. UV−vis spectra were recorded on a Hitachi U-3000 UV−visible spectrophotometer. The generation of singlet oxygen by QDs, chlorin e6, and the QD/chlorin e6 hybrids was determined using the molecular probe disodium, 9,10anthracenedipropionic acid (ADPA). Singlet oxygen causes the conversion of ADPA into an endoperoxide (cf. eq 21) which can be monitored by UV−visible spectrophotometry by following the decay of the absorption band of ADPA at 400 nm.37 In a typical experiment, 3 mL of a PBS (1X) solution about 20 μM in chlorin e6 (either free or in the QD hybrid, as determined by estimation of the Soret band area) was mixed with 200 μL of a 1.5 mM ADPA solution in DMF in a fluorescence quartz cuvette. The absorption spectrum of the mixture was immediately measured between 300 and 800 nm. The mixture was oxygenated by bubbling 6 mL of air using a syringe. The solution was irradiated with 350 nm excitation wavelength for 10 min by placing the cuvette into the fluorescence spectrometer. The visible spectrum was again recorded, and 6 mL more air was bubbled in the solution. The overall procedure was repeated a further two times to achieve 30 min cumulated irradiation time. As the ADPA 400 nm absorption band is superimposed on the Soret band of the chlorin e6, it is necessary to assess the photobleaching behavior of the chlorin e6 (both free and in the QD hybrid) during the experiment in order to not overestimate the consumption of ADPA. To this goal, 3 mL of a PBS solution about 20 μM in chlorin e6 was mixed with 200 μL of DMF. The solution was irradiated, with air bubbled through the solution at 10 min intervals, while the UV−vis absorption spectra were obtained (as described above) as a control experiment.

The number of moles of singlet oxygen produced was determined as follows. A(t), Ae6(t), and AADPA(t) represent the total absorbance, the absorbance due to the chlorin e6, and the absorbance due to ADPA, respectively, at time t of the chlorin e6/ADPA mixture. Ae6(t) is determined using the control experiment for the chlorin e6/DMF mixture. A(t ) = Ae6(t ) + AADPA (t )

(35)

The number of moles of singlet oxygen produced after a cumulated irradiation time t is given by Δn(ADPA)consumed =

AADPA (0) − AADPA (t ) × n(ADPA, 0) AADPA (0)

(36)

where n(ADPA,0) refers to the initial amount of ADPA probe. The amount of ADPA endoperoxide produced was normalized to the amount of chlorin e6 by dividing each by the area of the Soret band between 350 and 450 nm. The rate of ADPA endoperoxide production of the QDs was determined in the same way using silica-coated QDs dispersed in PBS buffer. The amount of ADPA endoperoxide produced was found to be negligible. Cell Culture. Breast carcinoma MDA-MB-231 cells were routinely cultured in 75 cm3 tissue culture flasks in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% L-glutamine. The amount of cells for the cell viability assay was monitored by counting viable cells in a hemocytometer by using the trypan blue exclusion method.38 MTT Assay. The effect of the QD, chlorin e6, and QD/ chlorin e6 hybrid on cell viability was determined using the MTT assay.34,35 Cells at a density of 1 × 104 cells per well were seeded on clear 96-well plates and treated with the QD, chlorin e6, or QD/chlorin e6 hybrid as described above followed by light irradiation with a UV lamp at a fluence rate of 0.11 mW/ cm2 for 5 min. MTT (10 μL) (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay solution (5 mg/mL in PBS) was added to each well together with 200 μL of DMEM with supplements (as described above). The plates were further incubated for 4 h at 37 °C. The medium was removed, and the resultant formazan crystals were washed with 200 μL of PBS (three times) and then dissolved in 200 μL of dimethyl sulfoxide. The absorbance intensity of each well was measured at 550 nm on a Dynatech MRX plate reader. Lysis solution Triton X 100 (2 μL per 100 μL from 9% stock in DMSO) was a positive control to induce cell death. Each variable (the concentrations of the chlorin e6, the QD/chlorin e6 hybrid, and the controls) was assessed through MTT assay in triplicate.

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CONCLUSIONS We have succeeded in covalently coupling the photosensitizer chlorin e6 onto a thin silica layer surrounding InP/ZnS QDs. Spectroscopic investigations of the hybrid system showed that the energy transfer between the QDs and photosensitizer is the rate-determining step for the production of singlet oxygen. When comparing the singlet oxygen production activities of the chlorin e6 photosensitizer, it was found that the rate of production of singlet oxygen of the hybrid was slightly inferior to that of the free photosensitizer, assuming that just one chlorin e6 molecule was bound to each QD. This result was strengthened by the in vitro measurements, where a similar cell viability was found for both the free chlorin e6 and QD/chlorin 9341



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e6 hybrids. The key finding of our study is that the ratedetermining step for the production of singlet oxygen is the energy transfer from QDs to the photosensitizer molecules. Future research might focus on binding photosensitizers closer to the QD surface and/or thinning the protective shell surrounding the QDs to enhance the energy transfer rate.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Big C cancer charity (Grant Number 10-20R) and the Engineering and Physical Sciences Research Council (EPSRC − programme SOLARCAP) is gratefully acknowledged.

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Insights into the Mechanism of Quantum Dot-Sensitized Singlet

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