ISSN 0030-400X, Optics and Spectroscopy, 2015, Vol. 119, No. 5, pp. 733–737. © Pleiades Publishing, Ltd., 2015. Original Russian Text © A.K. Visheratina, I.V. Alisova, E.V. Kundelev, A.O. Orlova, V.G. Maslov, A.V. Fedorov, A.V. Baranov, 2015, published in Optika i Spektroskopiya, 2015, Vol. 119, No. 5, pp. 707–711.

BASIC PROBLEMS OF OPTICS

Complexes of CdSe/ZnS Quantum Dots with Chlorin E6 in Nonaqueous Media1 A. K. Visheratina, I. V. Alisova, E. V. Kundelev, A. O. Orlova, V. G. Maslov, A. V. Fedorov, and A. V. Baranov ITMO University, St. Petersburg, 197101 Russia e-mail: [email protected] Received March 23, 2015

Abstract—Complexes of semiconductor CdSe/ZnS quantum dots and molecules of chlorin e6 in dimethyl sulfoxide have been formed. Conditions of formation and spectral and luminescent properties of the complexes have been studied. The quantum yield of luminescence of chlorin e6 in the complexes has been found to correspond to that of its monomer form. It has been shown that the complexes have energy transfer from the quantum dots to the molecules of chlorin e6 being observed, with its effectiveness being about 25%. DOI: 10.1134/S0030400X15110259

INTRODUCTION Photodynamic therapy (PDT) is a relatively new method of treatment of cancer diseases [1]. This method assumes destruction of cancer cells using production of reactive oxygen species, such as singlet oxygen and free radicals, under the effect of irradiation. PDT is based on the photodynamic effect, which is a combined action of special molecules, photosensitizers, and irradiation of cancer cells. Photosensitizers have important properties: upon introduction into an organism, they are capable of selective accumulation in tumor tissues and under the effect of radiation they can produce singlet oxygen, which, in turn, destroys surrounding cancer cells [2]. A good representative of photosensitizers is chlorin e6 (Ce6), which is widely used in clinical practice as one of the main components of the drug for PDT, Fotoditazin [3]. Unfortunately, photosensitizers have a number of side effects, e.g., phototoxicity and slow excretion from the organism. Thus, searching for and making new drugs for PDT, which are able to reduce these side effects by decreasing therapeutic doses of the drugs, remain urgent. Upon last decades, there have been a lot of studies directed toward creation of complexes based on different nanomaterials with tetrapyrrole molecules [4–6]. Quantum dots (QD) represent semiconductor nanocrystals with a size of 2–10 nm in all three dimensions. Unique optical and chemical properties of QD [7] make it possible to form new hybrid structures, where those molecules are involved, which are required for certain tasks. In complexes with tetrapyrrole compounds, QD are used as a universal donor of 1 VIII

International Conference “Basic Problems of Optics” (BPO-2014, October 20–24, 2014, St. Petersburg).

photoexcitation energy, making it possible to increase singlet oxygen production by tetrapyrroles as compared to their free form [8]. There are a great number of studies devoted to formation and investigation of complexes of QDs with tetrapyrrole molecules in aqueous solutions, which report that there is a sharp decrease in the effectiveness of intracomplex energy transfer and quantum yield of luminescence of tetrapyrrole upon an increase in its relative concentration in the solution [9–11]. In the systems described, formation of the complexes occurs due to electrostatic interaction or covalent binding. The presence in an aqueous solution on the surface of QDs of charged groups of molecules of the solubilizer and charged groups at tetrapyrrole upon formation of the complexes leads to a change in the charge surroundings of both the QDs and tetrapyrrole. In turn, this may result in appearance of the concentration dependences of photophysical properties of the complexes. Therefore, it seems to be necessary to study conditions of formation and photophysical properties of the complexes in aprotic solvents, e.g., dimethyl sulfoxide (DMSO). In this case, the surface of the QDs and tetrapyrrole molecules is not charged and Ce6 molecules are in a monomer form [12]. It should be noted that DMSO could be used in experiments on living cells in vitro, since it is highly permeable in biological tissues and can be added to the growth medium of cells up to 10% in with respect to volume [13]. The data on the spectral and luminescent properties of complexes of CdSe/ZnS QDs with Ce6 molecules obtained in the study indicate their creation in DMSO being promising, since they have the effective transfer of photoexcitation energy from the QDs to

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Ce6 molecules observed, their quantum yield of luminescence corresponding to that of their monomer form. These facts allow the hypothesis that the production of singlet oxygen by these complexes and, correspondingly, photodynamic effect may be significantly higher than those for a monomer form of Ce6. MATERIALS AND METHODS In the study, CdSe/ZnS QDs with an average core diameter of 2.5 nm and extinction coefficient in the first absorption band of 6.2 × 10 4 M–1 cm–1 were used. These QDs were synthesized according to the method described in [14]. To form complexes of QDs with Се6 molecules in DMSO, CdSe/ZnS QDs stabilized with cysteamine molecules were used. Cysteamine was purchased from Aldrich and used without further purification. The QDs stabilized with cysteamine used in the study had the quantum yield of luminescence in an aqueous solution about 8%. To obtain colloidal solutions of the QDs in DMSO, microquantities of a concentrated solution of the QDs were added to DMSO and, then, thoroughly mixed. Chlorin e6 (Fronter Scientific), the main component of a Fotoditazin drug, represented a tetrapyrrole compound. To investigate the conditions of formation and optical properties of the complexes of the QDs and Се6, the QD solution in DMSO (C ~ 10–6 mol/L) was supplemented with a Се6 solution (C ~ 2 × 10‒7 mol/L) in DMSO so that the mixture had the ratio of the molar concentrations of the QDs and Ce6 ~ 1 : 0.5, respectively. To record the absorption spectra of the samples, a UV-Probe 3600 spectrophotometer (Shimadzu) was used, the luminescence spectra of the samples were obtained using a Cary Eclipse spectrofluorimeter (Varian), and the kinetics of the luminescence of the solutions was recorded using a MicroTime100 confocal microscope (PicoQuant). RESULTS AND DISCUSSION As was shown in [15], the complexes of QDs and tetrapyrrole have energy transfer from the QD to tetrapyrrole occurring according to FRET with its main conditions: (1) an overlap between the luminescence spectra of a donor (QD) and the absorption spectra of an acceptor (Ce6), (2) the distance between the donor and acceptor should be sufficiently small and, in the case of the complexes of QD and tetrapyrrole, it should constitute no more than 10 nm. As could be seen from Fig. 1, the absorption spectrum of Ce6 has a good overlap with the luminescence spectrum of the QDs; therefore, in the case of formation of the complexes of the QDs and Ce6, effective FRET from the QDs to the Ce6 molecules can be implemented. Due to the relative arrangement of the absorption spectrum

Luminescence intensity, rel. units

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2 1 3

Optical density

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4

300

400

500 600 Wavelength, nm

700

Fig. 1. (1, 3) Absorption and (2, 4) luminescence spectra of (1, 2) CdSe/ZnS QD solutions and (3, 4) Ce6 molecules in DMSO. The shaded area shows the region of overlap of the absorption spectrum of Ce6 and luminescence spectrum of the QDs.

of the QD and luminescence spectrum of Ce6, back transfer of energy is almost impossible. The complexes of the QDs and Ce6 were obtained as a result of microaddition of the concentrated solution of Ce6 to that of the QDs in DMSO. Figure 2 presents (a) absorption and (b) luminescence spectra of the individual components and mixed solution of the QDs and Ce6. Analysis of the absorption spectra presented in Fig. 2a revealed that the absorption spectrum of Ce6 in the mixture with QDs almost did not differ from that of its free form. Upon addition of the solution of Ce6 to that of QDs, effective quenching of the luminescence of the quantum dots and appearance of the luminescence of Ce6, the position and shape of the luminescence band of which (maximum at 670 nm) are almost identical to the luminescence spectra of its free form (curves 2 and 3), were observed. It could be seen from Fig. 2b that, upon excitation of the mixed solution with light at a wavelength of 460 nm, the intensity of the luminescence of Се6 appears to be significantly higher than that of Ce6 at the same concentration. Hence, it could be concluded that these complexes have effective energy transfer from the QD to Ce6 molecules implemented. Thus, quenching of the luminescence of the QDs and appearance of the sensitized luminescence of Ce6 observed are indirect evidences of the formation of complexes of the QDs and Ce6 with effective energy transfer from the QDs to Ce6. In the absence of the charge on the surface of the QDs and at Ce6, the formation of the complexes as a result of the electrostatic interaction of the amino group of cysteamine on the surface of the QDs with the carboxyl group of Ce6 is unlikely, although electrostatic interaction of ionized groups formed during proton transfer cannot be excluded. It is suggested to clarify the binding mecha-

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nism of the QDs and Ce6 in DMSO into the complex in further studies.

Optical density 0.3

(а)

2

Direct manifestation of the intracomplex energy transfer from the QDs to the Ce6 molecules is a contribution of the absorption band of the QDs to the excitation spectrum of the luminescence of Ce6 (Fig. 3), which is clearly seen in the region from 300 to 550 nm.

1 0.2

Earlier, it was found that, in similar systems of QDs and Ce6 in aqueous solutions, a decrease in the quantum yield of luminescence of Ce6 associated in the complex with the QDs was observed [16]. One reasons for the dependence observed may be that Ce6 molecules in an aqueous solution are in a dimeric form [17].

0.1 3 0 400

500

600

700

Investigation of the stability of the spectral characteristics of the mixed solutions of the QDs and Ce6 in DMSO demonstrated that the process of formation of the complex in this solvent at concentration regions of the QDs and Ce6 chosen could last during several hours. Figure 4 gives the time dependences of the change in the luminescence intensity of the QDs sens. (IQDs) and sensitized luminescence of Ce6 (I Се6 ), which are normalized to the corresponding intensity of the luminescence at 0 min, i.e., correspond to the values in a freshly prepared solution of the QD and Ce6. The intensity of the sensitized luminescence of Ce6 was found according to

Luminescence intensity, rel. units 80 (b) 2

60

20

1 10

40

2

3 0

20

650

700

3

0 500

735

550 600 650 700 Wavelength, nm

sens rec intr I Се6 = I Се6 − I Се6 ,

750

rec where I Се6 is the luminescence intensity of Се6 in the mixture (the wavelength of the excitation light is intr 460 nm) and I Се6 is the luminescence intensity of Ce6 caused by the intrinsic absorption of Ce6 at a wavelength of the excitation light of 460 nm.

Fig. 2. (a) Absorption and (b) luminescence spectra upon excitation with light at a wavelength of 460 nm of (1) CdSe/ZnS QDs, (2) mixture of the QDs and Ce6, and (3) Ce6 in DMSO.

200 Optical density

Luminescence intensity, rel. units

0.2

0.1 100 1

2 3 0 300

400 500 Wavelength, nm

600

0

Fig. 3. (1) Spectra of absorption of QDs, (2) excitation of luminescence of complexes of the QDs with Ce6, and (3) excitation of luminescence of Ce6. The luminescence intensity was recorded at λrec = 680 nm. OPTICS AND SPECTROSCOPY

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1.0 1

1.4

0.9

1.3 0.8 1.2 0.7

1.1

2 0.6

1.0 0

20

40

Intensity of sensitized luminescence of Ce6, norm

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Intensity of luminescence of QD, norm

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60 80 100 120 140 160 Time, min

Fig. 4. Time evolution of luminescence intensity of QDs and sensitized luminescence of Ce6.

Herewith, as has been shown in Fig. 4, a gradual decrease in the luminescence intensity of the QD is observed, which is accompanied by an increase in the sensitized luminescence of Ce6. The luminescence lifetime of the QDs does not depend on the concentration of Ce6. This means that, similarly to the situation in aqueous solutions [18, 19], binding of the QD with one molecule of Ce6 leads to quenching of the luminescence of this QD. Using the degree of luminescence quenching of the QD in the mixture with ce6, this makes it possible to determine the part of the QDs associated in the complex as

F =1− I , I0

E =

sens

a

Iλ Dλ intr d , I λ Dλ F

(3)

where I λsens is the intensity of the acceptor luminescence sensitized by the donor at the wavelength λ, I λintr is the luminescence intensity of the acceptor caused by its intrinsic absorption at the wavelength λ, Dλa and Dλd are the optical densities of the acceptor and donor at wavelength λ, and F is the part of the donor in the complex with the acceptor.

FRET efficiency 0.24

0.22

0.20

0.18 25

Since, for this system, the contribution of the absorption spectrum of the QDs to the luminescence spectrum of Ce6 is obvious (Fig. 3, curves 1 and 2) and the absorption spectrum of Ce6 upon association in the complex almost does not change, the FRET efficiency can be assessed by analyzing the luminescence spectra of free аnd bound Се6:

(2)

where I and I0 are the luminescence intensities of the QDs in the presence and absence of molecules of the

0

quenching agent, respectively, at a wavelength of the excitation light of 460 nm.

50

75

100

125 150 Time, min

Fig. 5. The time dependence of FRET efficiency (E) from the QDs to Ce6 in complexes of QDs and Ce6 calculated according to (3).

Figure 5 presents the time dependence of FRET efficiency from the QD to Ce6 in the complexes of the QDs and Ce6 calculated according to (3). It could be seen that, 1 h after the preparation of the samples, the FRET efficiency reaches 23% and achieves saturation. The lower value of the efficiency of the energy transfer (18%) in the freshly prepared solution is most likely connected with the presence in it of free Ce6, which, according to (3), leads to a lower value. Comparison of the dependences demonstrated in Figs. 4 and 5 has revealed that 3 h is sufficient for the equilibrium concentration of the complexes of the QDs and Ce6 in the samples to be established within the concentration range chosen, with most complexes being formed within the first 60–70 min. OPTICS AND SPECTROSCOPY

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CONCLUSIONS In the study, the complexes of the CdSe/ZnS quantum dots with the molecules of chlorin Ce6 have been formed in DMSO for the first time. It has been demonstrated that, within the concentration range chosen, at room temperature 3 h is necessary for the equilibrium concentration of the complexes to be established. Analysis of the spectral and luminescent properties of the samples has revealed that the spectral form and quantum yield of the luminescence of Ce6 in the complexes with the CdSe/ZnS QDs almost do not differ from these characteristics for its monomer form in DMSO. This fact together with the highly effective intra-complex energy transfer from the QD to Ce6 (~25%) indicates that studying complexes of semiconductor quantum dots with tetrapyrrole molecules in DMSO is promising, since it gives grounds to expect a noticeable increase in the production of singlet oxygen by these complexes, when compared to free tetrapyrrole. In addition, it is suggested to study concentration dependences of photophysical properties of the complexes and photophysical properties of the complexes of the QD and Ce6 formed in DMSO and aqueous and biological media. ACKNOWLEDGMENTS This study was supported by the Ministry of Education and Science of the Russian Federation, project no. 14.B25.31.0002 and state contract no. 3.17.2014/K. REFERENCES 1. H. Kashtan, R. Haddad, and Y. Skornick, International Symposium on Biomedical Optics Europe’94 (Int. Soc. Opt. Photon., 1995), pp. 290–296. 2. R. R. Allison, G. H. Downie, R. Cuenca, X.-H. Hu, C. J. H. Childs, and C. H. Sibata, Photodiagn. Photodyn. Therapy 1 (1), 27 (2004). 3. T. G. Rudenko, A. B. Shekhter, A. E. Guller, N. A. Aksenova, N. N. Glagolev, A. V. Ivanov, R. K. Aboyants, S. L. Kotova, and A. B. Solovieva, Photochem. Photobiol. 90 (6), 1413 (2014). 4. C. Constantin and M. Neagu, Nanomedicine 5 (2), 307 (2010).

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5. E. I. Zenkevich, E. I. Sagun, V. N. Knyukshto, A. S. Stasheuski, V. A. Galievsky, A. P. Stupak, T. Blaudeck, and C. Borczyskowski, J. Phys. Chem. C 115 (44), 21535 (2011). 6. V. V. Danilov, A. V. Baranov, G. K. El’Yashevich, A. O. Orlova, G. G. Khokhlov, and A. I. Khrebtov, Opt. Spectrosc. 108 (6), 941 (2010). 7. A. M. Smith, H. Duan, M. N. Rhyner, G. Ruan, and S. Nie, Phys. Chem. Chem. Phys. 8 (33), 3895 (2006). 8. V. G. Maslov, A. O. Orlova, and A. V. Baranov, Photosensitizers in Medicine, Environment, and Security (Springer, 2012). 9. A. O. Orlova, M. S. Gubanova, V. G. Maslov, G. N. Vinogradova, A. V. Baranov, A. V. Fedorov, and I. Gounko, Opt. Spectrosc 108 (6), 927 (2010). 10. I. V. Martynenko, A. O. Orlova, V. G. Maslov, A. V. Baranov, A. V. Fedorov, and M. Artemyev, Beilstein J. Nanotechnol. 4 (1), 895 (2013). 11. A. Skripka, J. Valanciunaite, G. Dauderis, V. Poderys, R. Kubiliute, and R. Rotomskis, J. Biomed. Optics 18 (7), 078002 (2013). 12. D. A. Schwartz and D. R. Gamelin, Proc. SPIE—Int. Soc. Opt. Eng. 5224, 1 (2003). 13. T. M. Rippin, V. J. Bykov, S. M. Freund, G. Selivanova, K. Wiman, and A. R. Fersht, Oncogene 21 (14), 2119 (2002). 14. B. O. Dabbousi, J. Rodriguez-Viejo, F. V. Mikulec, J. R. Heine, H. Mattoussi, R. Ober, K. F. Jensen, and M. G. Bawendi, J. Phys. Chem. B 101 (46), 9463 (1997). 15. E. I. Zenkevich, A. Shulga, T. Blaudeck, F. Cichos, and C. von Borczyskowski, Proc. SPIE—Int. Soc. Opt. Eng. 6002, 60020 (2005). 16. J. Valanciunaite, A. Skripka, G. Streckyte, and R. Rotomskis, Proc. SPIE—Int. Soc. Opt. Eng. 7376, 737607 (2010). 17. L. Kelbauskas and W. Dietel, Photochem. Photobiol. 76 (6), 686 (2002). 18. A. K. Visheratina, I. V. Martynenko, A. O. Orlova, V. G. Maslov, Yu. K. Gun’ko, A. V. Fedorov, and A. V. Baranov, J. Opt. Technol. 81 (8), 444 (2014). 19. A. O. Orlova, V. G. Maslov, A. A. Stepanov, I. Gounko, and A. V. Baranov, Opt. Spectrosc. 105 (6), 889 (2008).

Translated by E. Berezhnaya