Signpost Open Access Journal of

16 downloads 0 Views 483KB Size Report
May 19, 2013 - Journal Website: http://signpostejournals.com ... quenched due to the transfer of the absorbed light energy to the ZnPcs. We discuss the ...
E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

REVIEW ARTICLE 

 

 

 

 

 

 

 

                             Open Access 

Signpost Open Access Journal of NanoPhotoBioSciences Journal Website: http://signpostejournals.com

Zinc phthalocyanines and quantum dots conjugates: physical properties and photodynamic activity E.G. Maksimov1‫כ‬, F.-J. Schmitt2, M.G. Strakhovskaya1, D.A. Gvozdev1, T. Friedrich2, V.Z. Paschenko1 and A.B. Rubin1 1 2

Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow State University, 119992, Moscow, Russia Institute of Chemistry, Biophysical Chemistry, Berlin Institute of Technology, 10623 Berlin, Germany

*Corresponding author: E.G. Maksimov Copyright: Dr. Franz-Josef Schmitt Received: May 8, 2013 Accepted: May 19, 2013

Abstract It was shown that semiconductor nanocrystals (quantum dots, QD) can be used to increase the effective absorption cross section of Zn-phthalocyanines (ZnPcs). ZnPcs and QDs form stable hybrid complexes due to electrostatic interactions in aqueous solution. The fluorescence of the QDs in such hybrid complexes is strongly quenched due to the transfer of the absorbed light energy to the ZnPcs. We discuss the mechsnism of Förster resonance energy transfer as a possible explanation for the energy migration in donor–acceptor pairs. Calculations based on the experimental data show a transient enhancement of the ZnPcs fluorescence by up to 140 % due to efficient excitation energy transfer (EET) from QDs. This enhanced fluorescence decays with biphasic exponential dynamics indicating ongoing reactions in the QD-ZnPc hybrid structures. The possible mechanism of increasing the yield of the reactive oxygen species production due to improved spectral characteristics of hybrid systems is discussed. Keywords Zinc phthalocyanines, Quantum dots, Fluorescence lifetime, Reactive oxygen species 47

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

List of Abbreviations – – FWHM – TCSPC – QD600p – QD600n – ZnPc8+ – ZnPc8- –

fluorescence lifetime fluorescence quantum yield full width at half maximum time-correlated single photon counting positively charged CdSeCdTe/ZnS quantum dot negatively charged CdTe quantum dot positively charged octakis(pyridinemethyl)-phthalocyanine negatively charged octacarboxy phthalocyanine

TEMPOL -

4-hydroxy-2,2,6,6-tetramethylpiperidin1-oxyl

DPIBF -

1,3-diphenylisobenzofuran

1. Introduction Due to their unique physical and chemical properties, phthalocyanines (Pcs) have found practical application not only as dyes [1], but as new functional materials in nonlinear optics [2], as gas sensors [3], catalysts [4,5], in artificial photosynthesis [6, 7, 8] and various other applications. One of the most rapidly developing application fields of phthalocyanines and their metal complexes (MPc) is the photodynamic therapy (PDT) of cancer and other diseases [9-11]. In addition to anticancer PDT, the photosensitizing properties of MPcs are of increasing interest for photodynamic inactivation of drug-resistant strains of pathogenic microorganisms for therapeutic purposes [12, 13] as well for water desinfection [14]. Photodynamic inactivation leads to damage of undesirable cells by reactive oxygen species generated by sensitizers in photoexcited states. Visible light activates triplet oxygen (3 Σg− O2) present in the medium to highly reactive singlet oxygen (3 Σg− O2), which in turn oxidizes biomolecules leading to the loss of the vital functions of cells and or apopthosis. MPcs are socalled type II sensitizers forming 1ΔgO2 according to the following scheme [11]: hν ⎯→ MPc ⎯⎯

1

isc 3 ⎯ MPc* ⎯⎯→ MPc*,

48

3

MPc* + 3 Σg− O2 → MPc + 1ΔgO2,

1

ΔgO2 + biomolecule → oxidation products

where 1MPc* and 3MPc* denote Pc in the singlet or triplet excited state, respectively, isc describes the inter-system crossing transition from the singlet to the triplet state. MPcs have a high quantum yield of triplet state formation and singlet oxygen generation, which determines their high phototoxic effect [12-14]. However, in aqueous solutions, phthalocyanines tend to form photochemically inactive aggregates. In the aggregates, excited states are quickly decaying via nonradiative channels, resulting in a decrease of the quantum yield of singlet oxygen generation. Since water is a universal biological solvent, the search of sensitizers for photodynamic therapy based on phthalocyanines, being in the monomeric state in aqueous media, is medically relevant [15]. Inexpensive broadband lamps are favorable sources to activate photosensitizers used for desinfection or treatment of skin infections. However, the light absorption properties of MPcs are characterized by intensive Q-bands in the far-red spectral region with a maximum near 680 nm and rather low absorption of other visible light wavelengths [15]. Thus, the possibilities to potentiate MPcs’ photoactivation by wider range of wavelengths are likely to be of great interest for biological and medical MPcs applications. This may be achieved by increasing the effective absorption cross section of MPcs through energy transfer from additional light-absorbing structures. Modern nanotechnology allows to produce semiconductor nanocrystals, or so-called quantum dots (QD), which absorb light in a broad optical range from ultraviolet to near infrared. The fluorescence spectrum of QDs is rather narrow, have a Gaussian shape, and the position of their fluorescence emission maximum can be precisely adjusted by the diameter of the nanocrystal particles [16-18]. While being slightly worse than the best fluorescent labels in terms of the magnitude of fluorescence quantum yield (up to ~70% at room temperature), quantum dots exceed the latter by several orders of magnitude in the light absorption

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

energy to native photosynthetic pigment–protein complexes [24-28].

cross section [19]. In addition, the organic coating by bi- or trifunctional polymers provides water solubility and electrostatic interaction due to polar groups bound to the surface [20-22]. Functional groups of organic coating are also available for conjugation, which makes it possible to create artificial light-harvesting complexes based on quantum dots, which can serve as highly effective energy donors for photosynthetic pigments and pigment–protein complexes [23-28].

In the present study, we investigate the possibility of creating hybrid structures based on zinc Pcs, bearing 8 anionic or cationic substituents, and differently charged QDs. We mainly investigated the enhancement of the effective absorption cross section of ZnPc by efficient energy transfer from the QDs. Probably, such structures can be used in photodynamic therapy because QDs can greatly expand the action spectrum of the ZnPcs and reduce their concentration and additionally allow the creation of switchable PDT dyes, since the coupling between QDs and organic structures critically depends on external parameters like temperature [25-26].

Recently, the possibility of energy transfer from QDs to MPcs photosensitizers was demonstrated in [29-38]. However, absorption characteristics of the obtained conjugates differed with the charge of MPcs used in the study. Compared with almost minor changes in the absorption of anionic MPc in combination with QDs [30] the significant changes were observed for combination cationic MPc-QD [35]. Moreover, the number of charged substituents in MPc molecules may influence their aggregation capacity and thus photophysical and photochemical characteristics [15].

2. Materials Two types of ZnPcs were used. Negatively charged octacarboxy phthalocyanine (ZnPc8- as shown in Figure 1) and positively charged octakis(pyridinemethyl)-phthalocyanine (ZnPc8+) synthesized in the Organic Intermediates and Dyes Institute (Moscow).

Previously, we experimentally established that quantum dots, used as additional artificial light collectors, efficiently absorb light in the ultraviolet and visible regions of the spectrum and transmit    

 

  Figure 1. Structures of Zn-phthalocyanines used in this study. 49

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages 1,4 1,2 1,0

O.D./Fluorescence

0,8

QD600n abs QD600n em 8+ ZnPc abs 8+ ZnPc em

A

QD600p abs QD600p em 8ZnPc abs 8ZnPc em

B

0,6 0,4 0,2 0,0 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 300

350

400

450

500

550

600

650

700

750

800

Wavelength (nm)

Figure 2. Normalized absorption (abs) and emission (em) spectra of quantum dots and zinc phthalocyanines at room temperature. Two types of quantum dots with an emission maximum at 600 nm were used. One with a core of CdSeCdTe/ZnS (further denoted as QD600p), with positively charged polymer shell "poly T-APS", and another with a core of CdTe (termed QD600n), with negatively charged carboxyl groups. Both types of QD were synthesized by “NanotechDubna”, Russia. Concentrations of quantum dots were calculated as described in [39]. Normalized absorption and fluorescence spectra of QDs and ZnPcs are presented in Figure 2AB. 3. Methods Fluorescence measurements were performed by time- and wavelength-correlated single photon counting with the equipment described in [40-41]. The setup consists of a system with a Hamamatsu R5900 16-channel multi-anode photomultiplier tube with 16 separate output (anode) elements and a common cathode and dynode system (PML-16, Becker&Hickl, Berlin, Germany). The polychromator was equipped with 600 grooves/mm grating resulting in a spectral bandwidth of the PML-16 of about 200 nm (resolution of 12.5 nm/channel). Excitation was performed with a pulsed 405 nm laser diode (IOS, Saint Petersburg, 50

Russia) delivering 30 ps FWHM pulses, driven at a repetition rate of 50 MHz. To study the dynamics of fluorescence quenching we measured the changes of initial fluorescence of the donor after the addition of the acceptor and vice versa. The signal was recorded form of 64 cycles in time (f(t,T) mode of B&H SPC [40]) with a duration of 3 seconds signal accumulation for each cycle with count rates up to 400.000 photons per second. This setup allowed us to record the dynamics of the transition of the samples from the initial to quenched state, by simultaneous measurement of the fluorescence intensity and the fluorescence decay time. Thus, each experiment resulted in 1024 fluorescence decay curves (16 spectral sections for each of 64 time windows). For time-integrated measurements we used Fluoromax 4 (Horiba Jobin Yvon, France) and a USB-connected fluorometer system with CCD array USB4000 (Ocean Optics, USA). During the processes of quenching measuring, the fluorescence signal was accumulated for 1 s, this procedure was repeated 100 times (i.e., 100 fluorescence spectra were recorded in steps of 1 s). Changes in the fluorescence intensity were analyzed in the bands of the QD (600 nm) and the ZnPc (700 nm) fluorescence spectrum.

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

Absorption spectra were recorded using a USB2000 spectrometer with a DT-MINI-2-GS deuterium tungsten halogen light source (Ocean Optics, USA), in standard 10 mm quartz cuvette. In all experiments, for mixing the solutions during the measurements, a magnetic stirrer was used. The fluorescence decay kinetics were approximated by the sum of the exponential functions used to fit the experimental data. To compare different kinetic patterns, we calculated the average decay time according to the expression: , where is the lifetime of the i-th component and is the fraction of the amplitude of the i-th component of the fluorescence decay normalized to . To obtain the time-integrated fluorescence spectra, the number of photons in each spectral channel was summed up. All calculations were performed using Origin 8.0 (OriginLab Corporation, USA) and SPCImage (Becker&Hickl, Germany) software packages. To determine the rate of reactive oxygen species, we analyzed the changes in absorption spectra of 1,3-diphenylisobenzofuran (DPIBF, Sigma Aldrich) as described in [42] as well as changes in the electronic paramagnetic resonance spectra of 4hydroxy-2,2,6,6tetramethylpiperidin-1-oxyl (TEMPOL) as described in [15, 42]. To determine the bactericidal activity of Zn-phthalocyanines alone and hybrid systems we used a bioluminescent bacterial test system based on a genetically engineered strain E. coli K-12 TG1, which emits bioluminescence due to complete lux-operon (commercially available biosensor ECOLUM, Russia). The method is based on the correlation between photosensitized bioluminescence quenching and inactivation of bacterial colony forming units and is suitable for studying photodynamic effects [43-44]. Bioluminescence intensity was recorded with a luminometer Sirius Smart Line TL (Titertek, USA). Each experiment was repeated at least five times. 51

4. Results and Discussion The absorption and fluorescence spectra of individual solutions of ZnPcs and quantum dots (Figure 2AB) were used to calculate the corresponding overlap integrals according to the formula [45]: , where is the normalized fluorescence spectrum of the donor, denotes the absorption spectrum of the acceptor, is the light wavelength. The Förster radius was calculated as: , where is the quantum yield of the donor in absence of acceptor, is the refractive index of the surrounding medium, denotes the orientation factor between the transition dipole moments of the donor and acceptor. The value of the Förster radius was calculated to be about 60 Å for the QD600p and 62 Å for the QD600n (as the fluorescence spectra of the latter is a bit broader, Figure 2) for all ZnPc acceptors, due to the high similarity of the absorption spectra of all studied compounds. Titrations were performed to estimate the quenching of the QDs by Pcs. Initially, an aqueous solution of the QD (donor of energy) was prepared and then gradually increasing concentrations of ZnPc (acceptor of energy) were added. After the addition of each new concentration of acceptor, the solution was incubated for 5 minutes with intense stirring. The steady-state fluorescence spectra of QD and ZnPc were recorded to analyze the changes of the related quantum yield. To determine the enhancement of the acceptor fluorescence, the same sequence of experiments was carried out with ZnPc in the absence of quantum dots. The results of the titration are shown in Figure 3. It clearly shows that in pairs of similarly charged donor and acceptor of energy, the quenching of donor is not as strong as in the case of opposite charged pairs. After mixing

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages 9

9

A

8-

ZnPc QD600n 8ZnPc + QD

Fluorescence (a.u.)

8

8+

ZnPc QD600n 8+ ZnPc + QD

B

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0 0

20

40 8-

60

80

0

10

-8

20

30

8+

ZnPc (10 M)

40

-8

ZnPc (10 M)

12 11

8-

ZnPc QD600p 8ZnPc + QD

10 9

Fluorescence (a.u.)

8

C

8+

ZnPc QD600p 8+ ZnPc + QD

D

12 11 10 9

8

8

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0 0

20

40 8-

60

80

-8

0

10

20 8+

ZnPc (10 M)

30

40

-8

ZnPc (10 M)

 

  Figure 3. Relative quantum yields of fluorescence of quantum dots and zinc phthalocyanines during the titration. Fluorescence excitation at 405 nm, CW mode. Red curves – QDs, black – ZnPcs without QDs, blue – ZnPcs in presence of quantum dots. Initial concentrations of QD600p and QD600n were 7·10-9 M and 2·10-8 M, respectively. QD600n and ZnPc8+ as well as in solution of QD600p and ZnPc8-, significant quenching of both, donor and acceptor, was observed at high acceptor concentrations. The same procedure of titration was studied with time-resolved fluorescence spectroscopy (data not shown), the changes of the overall photon numbers in the spectral channels corresponding to the QD and ZnPc fluorescence 52

were in a good agreement with the results presented in Figure 3. Those series of experiments were used to estimate the efficiency of energy transfer ( ) from QDs to ZnPc as a result of changes of the fluorescence lifetime ( ) of the donor (QD):

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

spectrum indicate the formation of dimers of ZnPc8+, as characterized by low fluorescence quantum yield and the low yield of singlet oxygen [44, 46]. Therefore, it is assumed that two (or more) molecules of ZnPc8+ can simultaneously bind to a single QD of this type. We can assume that this explains the significant decrease of the fluorescence intensity of ZnPc8+ in the presence of QD600n. On the contrary, the absorption spectrum of the hybrid system QD600p-ZnPc8- in the red region corresponds well to the absorption spectrum of an aqueous solution of the ZnPc8-. Thus, from the point of view of the possible increase in the photodynamic effect, the most interesting hybrid structure is QD600p-ZnPc8- hybrid system, since it indicates a highly efficient transfer of energy, and the ratio of QD to ZnPC in the hybrid system is probably 1 to 1, despite a much larger number of phthalocyanine molecules in solution.

where is the fluorescence lifetime of the donor in presence of the acceptor and denotes the fluorescence lifetime of the donor in absence of the acceptor. Despite of a significant reduction in the fluorescence intensity of quantum dots in pairs of QD600n-ZnPc8and QD600p-ZnPc8+, the efficiency of energy migration does not exceed the values of 0.1 and 0.5, respectively, and might be even lower due to radiationless dynamic quenching of the donor by the acceptor opening concurring decay channels that lead to reduction of the lifetime without real “transfer” of energy. In contrast, in pairs of oppositely charged QD and ZnPc, the efficiency of energy migration due to the reduction of lifetime of the donor exceeded 0.9, corresponding to ~ 30 Å distance between the donor and the acceptor. Thus, the quantum dots can interact with oppositely charged ZnPc due to electrostatic interactions and form hybrid structures with highly efficient energy transfer.It is also important to note that the absorption spectrum of QD600n-ZnPc8+ hybrid system cannot be obtained by simple summing the optical densities of the individual solutions (see Figure 4B). Significant changes in the red region of the absorption

It was found that the fluorescence intensity of the donor and the acceptor species is strongly timedependent after the mixing, and relative quantum yields can change dramatically in a few minutes. This indicates that a slow chemical reaction

1,0

1,0

A

QD600p 8ZnPc Hybrid system 0,8

O.D. (units)

B

QD600n 8+ ZnPc Hybrid system

0,8

0,6

0,6

0,4

0,4

0,2

0,2

0,0

0,0 300

400

500

600

700

Wavelength (nm)

800

300

400

500

600

700

800

Wavelength (nm)

Figure 4. Absorption spectra of quantum dots and Zn-phthalocyanines and their hybrid systems at room temperature. The concentration ratio was selected from Figure 3, corresponding 1/133 and 1/500 for QD600p/ZnPc8- and QD600n/ZnPc8+, respectively, showing the most efficient quenching of the QDs. 53

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

proceeds (in contact of QDs and ZnPCs) when QDs and ZnPcs are in contact. To analyze such a slowly proceeding chemical reaction by continuous imaging of the EET efficiency, time-resolved spectra were taken with highest possible time resolution for the single decay curves. Since the relative fluorescence quantum yield may change due to static and dynamic fluorescence quenching [45] that develops on a macroscopic time scale of up to minutes, the dynamics of the quenching process was investigated by measuring the timeresolved fluorescence in steps of 3 seconds. For these experiments, we prepared a solution with a certain concentration of the acceptor. Simultaneously, the registration of the fluorescence decay kinetics and the steady-state fluorescence spectra was started with an increment of 3 seconds. Then, the donor was added to the measured volume. The concentration ratio for the dynamic experiments was selected based on the results of the titration (Figure 3). Of greatest interest are the points with the largest difference between the relative quantum yields of the individual acceptor and the acceptor in the presence of the donor (black and blue curves in Figure 3). However, to obtain a high count rates (up to 400,000 photons per second) by the single photon-counting method, the concentrations of acceptor and donor were increased 10-fold and were exactly the same as in Figure 4 (1/133 and 1/500 for QD600p/ZnPc8- and QD600n/ZnPc8+, respectively). The most interesting results are presented in Figure 5. It was found that the fluorescence intensity and lifetime of QD600p are sharply reduced after the injection into a solution of ZnPc8-, and this decrease continues with a typical biphasic behavior comprising characteristic time constants of about 8 s (major component) and 45 s (minor component). The efficiency of energy migration, calculated as a change of fluorescence lifetime of QD600p, reaches 0.94. Monitoring the ZnPc8- fluorescence shows that after injection QD600p, there is a fast increase of fluorescence intensity of ZnPc8- by almost 140% compared to the initial level of fluorescence. The enhancement factor gradually decreases with characteristic time constants of about 8 s (major 54

component) and 100 s (minor component). It should be noted that after addition of the QD600p the fluorescence spectrum of the ZnPc8- is red-shifted by approximately 5 nm. Simultaneous measurements of the fluorescence lifetime of the acceptor shows that upon the addition of QD, the fluorescence lifetimes increases by 7% and essentially does not change with time. This phenomenon indicating that there is a significant EET from the quantum dots that leads to appearance of the fluorescence rise kinetic and therefore to a virtual prolongation of the fluorescence decay time. That means that even after a long time, hybrid structures with efficient EET can be detected in the measured volume. Thus, QD600p form stable hybrid complexes with ZnPc8-. Due to a highly efficient energy transfer, the effective absorption cross section of ZnPc can be strongly increased. In other investigated pairs of QDs and ZnPcs, the increase of the acceptor fluorescence was insignificant, probably due to weak electrostatic interactions, radiationless quenching, or the formation of dimers (Figure 4). Since the ZnPc8- in the excited state can generate singlet oxygen, it is reasonable to assume that the energetic interaction between the positively charged QD600p and the negatively charged ZnPc8- should lead to increased amount of 1ΔgO2 generation, which also might cause the degradation of the Znphthalocyanine molecules and reducing the measured fluorescence amplitude. Such photodegradation would lead to the gradual decay of the increased fluorescence intensity of ZnPc without influence on the fluorescence decay time (Figure 5). However, the question remained to be answered, if quantum dots, when used as additional light-harvesting antennas, can increase the rate of 1 ΔgO2 generation by ZnPc. Therefore, standard methods to detect reactive oxygen were used [42]. The presence of 5·10-6 M ZnPcs caused characteristic changes of the DPIBF absorption spectrum and light-induced oxidation of the TEMPOL spin label was registered. The addition of a solution of sodium azide, which is known to bind singlet oxygen, caused a sharp decrease in the amplitude of the EPR signal.

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages 3200 2,4

1,0

3150

2,2 0,8

0,6

I/I0 QD600p τ/τ0 QD600p

0,4

0,2

3100

1,8

Enhancement coef. 8τ ZnPc

1,6

3050

3000

1,4

1,2

Lifetime (ps)

Enhancement (units)

I/I0, τ/τ0 (units)

2,0

2950

1,0 0,0

A 0

30

60

90

120

150

180

B

0,8

0

30

Time (s)

60

90

120

150

2900

180

Time (s)

Figure 5. (A) Changes in the relative quantum yield and the fluorescence lifetime of the QD600p (compared to individual QD aqueous solution) when adding it to the solution of ZnPc8-. At zero time, fluorescence lifetime and intensity of QD correspond to those in case of donor in the absence of acceptor. (B) The enhancement of ZnPc8- fluorescence (comparing to initial level) and changes of the fluorescence lifetime as a result of the addition of quantum dots with positive surface charges. The ratio of donor and acceptor is exactly the same as in Figure 4A. Fluorescence excitation - 405 nm, 30 ps, 50 MHz. A magnetic stirrer was used with constant stirring rate. Arrows indicate the time of QD injection.

8-

8-

Intensity (a.u.)

ZnPc Hybrid system

ZnPc Hybrid system

3000

3000

2000

2000

1000

1000

0

0

-1000

-1000

-2000

-2000

-3000

A

B Magnetic Field

-3000

Magnetic Field

Figure 6. Left (A) - EPR signal of TEMPOL after 5 minutes of white light illumination of solution of ZnPc8(black) and QD600p/ZnPc8- (red). Right (B) - EPR spectrum of ZnPc8- before (black) and after addition of QD600p (red). 55

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

These results clearly show that ZnPcs exposed to light are able to generate singlet oxygen. However, no significant increase in the rate of generation of singlet oxygen could be registered with the described methods after additionally adding quantum dots to ZnPcs. Conversely, in the presence of hybrid systems, changes of the optical density of DPIBF were not longer detected, and the amplitude of the EPR signal of TEMPOL (and ZnPcs themselves) decreased dramatically (see Figure 6), wich points out the reduction of TEMPOL. Moreover, it was shown in control experiments that the quantum dots themselves are able to interact with DPIBF and TEMPOL, probably causing their reduction. Thus, we assume that these methods of singlet oxygen detection do not give adequate results, since QDs can interact directly with DPIBF and TEMPOL (this mechanism is interesting by itself), and, obviously, other methods of testing our hypothesis are required. The most interesting test would be to measure the direct interaction between bacteria and QD-ZnPc hybrid complexes. For that purpose, we investigated the photoinactivation of the special strain E. coli K-12 TG1 after addition of ZnPcs, QDs and hybrid structures. The level of the K-12 TG1

bioluminescence was measured immediately after the addition of ZnPcs and QDs to the cell suspension, and then at regular intervals after irradiation with white light. The main results are presented in Figure 7. It is seen from Figure 6, not only the zinc phthalocyanines [15, 46], but also the aqueous solution of quantum dots display bactericidal activity [47-50]. Treatment of the cell suspension by a solution of hybrid structures leads to a more efficient photoinactivation. The effect of bacteria inactivation by ZnPc-QD hybrids, however, is smaller compared to the summarized effect in the presence of both, QDs and ZnPC. Probably, positively charged QDs bind to negatively charged bacterial cell walls and protect cells from singlet oxygen generated in solution by the anionic photosensitizer. Therefore, it is assumed that our investigated structure does not release more 1ΔgO2 than ZnPc or QD only. Thus, the effect of quantum dots on the ability of zinc phthalocyanine to generate singlet oxygen is unclear and requires alternative research methods.

100

Bioluminescence, %

80

60

40

control QD600p 8ZnPc Hybrid

20

0 0

2

4

6

8

10

12

Dose of white light, J/cm

14

16

18

20

2

Figure7. Level of E. coli K-12 TG1 bioluminescence comparing to initial level ( 56

).

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

5. Conclusion Creation of hybrid structures in solution due to electrostatic interactions, without the use of additional reagents for the formation of covalent bonds, opens up a number of promising new areas of research and corresponding applications. It was shown that in a mixture of ZnPcs and QDs, stable hybrid complexes can be formed due to electrostatic interactions. The fluorescence of QDs in such hybrid complexes is strongly quenched due to quenching of the absorbed energy by ZnPcs. In the framework of FRET, the distance between the donor and the acceptor of energy in hybrid complexes should not exceed ~ 30 Å. According to steady-state and time-resolved spectral measurements, quantum dots can transfer the excitation energy to the zinc phthalocyanine, increasing the effective absorption cross section of ZnPc and the number of excited states. Especially in ZnPc8- /QD-600p complexes, a characteristic fluorescence rise (prolongation of the acceptor fluorescence) was observed indicating a strong EET. Calculations based on the experimental data show that the enhancement of ZnPcs fluorescence can reach a factor of 2.4 due to efficient energy migration from QDs. Interestingly, this EET is transient and seems to occur concomitant to strong degradation of ZnPc, possibly due to the interaction with 1 gO2. This degradation might be caused by the generation of reactive oxygen species, which can reduce the concentration of ZnPcs and QDs itself. However, the rate of reactive oxygen species generation through improved spectral characteristics of hybrid systems is unclear. It was shown that quantum dots can interact with DPIBF and TEMPOL making them insensitive to singlet oxygen, so alternative methods are necessary to study this process. Additionally the strong photodegradation of ZnPc might occur due to the interaction with localized 1 ΔgO2 therefore preventing the release of 1ΔgO2 into the solution. This finding is supported by the observation that the biocidal effect on bacteria is not significantly enhanced in the case of hybrid complexes compared to the effects of ZnPc or QDs 57

only. It is important to note that a very strong bactericidal action by QDs alone was shown. Some additional questions should be studied in the future works. For example, what is the nature of the dynamics of the amplitude decrease and virtual destruction of the acceptor? What is the exact mechanism of donor quenching? The role of diffusion should be analyzed in future studies by the means of temperature-dependent investigation. The exact dependency of the interaction between donors and acceptors in the QD-ZnPc hybrid structures on the light intensity and the ionic strength of the buffer medium will be targeted by future studies, as well as investigations of the stability of the complexes. Acknowledgements Authors are grateful to Dr. K.N. Timofeev for assistance with the registration of the EPR spectra. Financial support by BMBF bilateral cooperation funds RUS 10/026 and RUS 11/014 is gratefully acknowledged. V.Z. Paschenko and E.G.Maksimov also thank the Russian Foundation for Basic Research (project no. 11-04-01617 and no. 12-0431100). References 1. 2. 3. 4.

5.

Moser F.H., Thomas A.L. (1983) The Phthalocyanines. Vol.1,2, CRC Press, Boca Raton, Fla Torre G, Vazquez P, Agullo-Lopez F, Torres T 2004 Phthalocyanines. Properties and Applications Chem Rev. 104, 3723 Dogo S., Germain J.P., Maleysson C., Pauly A. (1992) Thin Solid Films, 219, 251 Kaliya O.L., Lukyanets E.A., Vorozhtsov G.N. (1999) Catalysis and photocatalysis by phthalocyanines for technology, ecology and medicine, J. Porphyrins Phthalocyanines, 3, 592 Wohrle D., Suvorova O., Gerdes R., Bartels O., Lapok L., Baziakina N., Makarov S., Slodek A. (2004) Efficient Oxidations and Photooxidations with Molecular Oxygen

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

6.

7.

8. 9.

10.

11.

12. 13.

14.

15.

using Metal Phthalocyanines as Catalysts and Photocatalysts, J. Porphyrins Phthalocyanines, 8, 1020 Carraro M., Sartorel A., Toma F., Puntoriero F., Scandola F., Campagna S., Prato M., Bonchio M., (2011) Artificial Photosynthesis Challenges: Water Oxidation at Nanostructured Interfaces. Topics in Current Chemistry, 303, 121–50. doi:10.1007/128_2011_136 Walter M.G., Rudine A.B., Wamser C.C. (2010) Porphyrins and phthalocyanines in solar photovoltaic cells, J. Porphyrins Phthalocyanines, 14: 759–92, doi: 10.1142/S1088424610002689 McConnell I., Li G., Brudvig G.W. (2010) Energy Conversion in Natural and Artificial Photosynthesis Chem. Biol. 17(5), 434-47 Cook M.J., Chambrier I., Cracknell S.J., Mayes D.A., Russell D.A. (1995) Octa-alkyl zinc phthalocyanines: potential photosensitizers for use in the photodynamic therapy of cancer. Photochem. Photobiol. 62. 542-5 Ochsner M. (1997) Photophysical and photobiological processes in the photodynamic therapy of tumours, J Photochem. Photobiol. B. 39, 1-18 Sekkat N., van den Bergh H., Nyokong T., Lange N. (2011) Like a bolt from the blue: phthalocyanines in biomedical optics. Molecules. 17, 98-144 Jori G., Brown S. (2004) Photosensitised inactivation of microorganisms, Photochem. Photobiol. Sci. 3, 403-5 Hamblin M., Hasan T. (2004) Photodynamic therapy: a new antimicrobial approach to infectious diseases? Photochem. Photobiol. Sci. 3, 436-50 Kuznetsova N.A., Yuzhakova O.A., Strakhovskaya M.G., Shumarina A.O., Kozlov A.S., Krasnovsky A.A., Kaliya O.L. (2011) New heterogeneous photosensitizers with phthalocyanine molecules covalently linked to aminopropyl silica gel, J. Porphyrins and Phthalocyanines. 15, 718-26 Makarov D.A., Kuznetsova N.A., 58

16.

17.

18. 19.

20.

21.

22.

23.

Yuzhakova O.A., Savvina L.P., Kaliya O.L., Lukyanets E.A., Negrimovskii V.M., Strakhovskaya M.G. (2009) Effects of the degree of substitution on the physicochemical properties and photodynamic activity of zinc and aluminum phthalocyanine polycations, Russian Journal of Physical Chemistry A. 83, 1044-50 Sukhanova A., Baranov A.V., Klinov D., Oleinikov V., Berwick K., Cohen J.H.M., Pluot M., Nabiev I. (2006) Self-assembly of charged microclusters of CdSe/ZnS core/shell nanodots and nanorods into hierarchically ordered colloidal arrays, Nanotechnology, 17, 4223-8 Bawendi M.G., Carroll P.J., Wilson W.L., Brus L.E. (1992) Luminescence properties of CdSe quantum crystallites: Resonance between interior and surface localized states’, J. Chem. Phys. 96, 2, 946 Bergmann L., Schäfer C., Niedrig H. (2004) Optik, Lehrbuch der Experimentalphysik Band 3, 9. Auflage, de Gruyter, Berlin Leatherdale C.A., Woo W.-K., Mikulec F.V., Bawendi M.G. (2002) On the absorption cross section of CdSe nanocrystal quantum dots. J. Phys. Chem. B, 106 (31), 7619-22 Sukhanova A., Artemyev M., Sharapov O., Baranov A., Jardillier J.C., Nabiev I. (2001) European, Eurasian and USA patents EP1366347, US2004105973, WO02073155. Oleinikov V.A., Sukhanova A.V., Nabiev I.R. (2007) Fluorescent Semiconductor Crystals in Biology and Medicine, Ros. Nanotekhnol., 2, 160-73 Medintz I.L., Mattoussi H. (2009) Quantum dot-based resonance energy transfer and its growing application in biology, Phys. Chem. Chem. Phys., 11, 17-45 Nabiev I., Rakovich A., Sukhanova A., Lukashev E., Zagidullin V., Pachenko V., Rakovich Y.P., Donegan J.F., Rubin A.B., Govorov A.O. (2010) Fluorescent Quantum Dots as Artificial Antennas for Enhanced Light Harvesting and Energy Transfer to Photosynthetic Reaction Centers, Angewandte Chemie, 49, 7217-21

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

24.

25.

26.

27.

28.

29.

30.

31.

Maksimov E.G., Gostev T.S., Kuzminov F.I., Sluchanko N.N., Stadnichuk I.N., Paschenko V.Z., Rubin A.B. (2010) Hybrid systems of quantum dots mixed with the photosensitive protein phycoerythrin, Ros. Nanotekhnol., 5, 531-7 Schmitt F.-J., Maksimov E.G., Suedmeyer H., Jeyasangar V., Theiss C., Paschenko V.Z., Eichler H.J., Renger G. (2010) Time resolved temperature switchable excitation energy transfer processes between CdSe/ZnS nanocrystals and phycobiliprotein antenna from Acaryochloris marina, Photon Nanostruct: Fundam Appl, 9 (2), 190-5 Schmitt F.-J., Maksimov E.G., Hätti P., Weißenborn J., Jeyasangar V., Razjivin A.P., Paschenko V.Z., Friedrich T., Renger G. (2012) Coupling of different isolated photosynthetic light harvesting complexes and CdSe/ZnS nanocrystals via Förster resonance energy transfer, Biochim Biophys Acta. 1817 (8), 1461-70 Maksimov E.G., Kurashov V.N., Mamedov M.D., Paschenko V.Z. (2012) Hybrid system based on quantum dots and photosystem 2 core complex. Biochemistry (Moscow), Biokhimiya, 77(6), 624–30 Borissevitch I.E., Parra G.G., Zagidullin V.E., Lukashev E.P., Paschenko V.Z., Knox P.P., Rubin A.B. (2013) Cooperative effects in quenching of cdse/zns-pegoh quantum dot luminescence by water soluble porphyrins. Journal of Luminescence, 134, 83–7 Idowu M., Chen Ji –Yao, Nyokong T. (2008) Photoinduced energy transfer between watersoluble CdTe quantum dots and aluminium tetrasulfonated phthalocyanine. New J. Chem., 32, 290–6 Britton J., Antunes E., Nyokong T. (2009) Fluorescence studies of quantum dots and zinc tetraaminophthalocyanine conjugates. Inorganic chemistry communications, 12, 828–31 Britton J., Antunes E., Nyokong T. (2010) Fluorescence quenching and energy transfer in conjugates of quantum dots with zinc and indium tetraamino phthalocyanines. J. 59

32.

33.

34.

35.

36.

37.

38.

39.

Photochem. Photobiol. A: Chem., 210, 1–7 Narband N., Mubarak M., Ready D., Parkin I.P., Nair S.P., Green M.A., Beeby A., Wilson M. (2008) Quantum dots as enhancers of the efficacy of bacterial lethal photosensitization. Nanotechnology, 19, 445102 Samia A.C.S, Dayal S., Burda C. (2006) Quantum dot-based energy transfer: perspectives and potential for applications in photodynamic therapy, Photochem. Photobiol., 82, 617–25 Suchánek J., Lang K., Novakova V., Zimcik P., Zelingera Z., Kubát P. (2013) Photophysical properties of CdSe quantum dot self-assemblies with zinc phthalocyanines and azaphthalocyanines Photochem. Photobiol. Sci., Advance Article DOI: 10.1039/C2PP25348H Idowu M., Nyokong T., (2010) Spectroscopic behavior of cationic metallophthalocyanines in the presence of anionic quantum dots, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 75(1), 411-6, doi 10.1016/j.saa.2009.10.050 Lei Li, Jin-Feng Zhao, Nayoun Won, Ho Jin, Sungjee Kim and Ji-Yao Chen (2012) Quantum Dot - Aluminum phthalocyanine Conjugates perform photodynamic reactions to kill cancer cells via fluorescence resonance energy transfer (FRET) Nanoscale Research Letters, 7, 386 doi:10.1186/1556276X-7-386 Duygu Aydın Tekdaş, Mahmut Durmuş, Hülya Yanık, Vefa Ahsen, (2012) Photodynamic therapy potential of thiolstabilized CdTe quantum dot-group 3A phthalocyanine conjugates (QD-Pc), Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 93, 313-20 Idowu M., Nyokong T. (2009) Interaction of water-soluble CdTe quantum dots with octacarboxy metallophthalocyanines: A photophysical and photochemical study, Journal of Luminescence, 129(4), 356-62 Jasieniak J., Smith L., van Embden J.,

E.G. Maksimov et al.. (2013) Signpost Open Access J. NanoPhotoBioSciences. 1, 47-60. Volume 01, Article ID 010302, 14 pages

40.

41.

42.

43.

44.

45. 46.

47.

Mulvaney P. (2009) Re-examination of the Size-Dependent Absorption Properties of CdSe Quantum Dots, J. Phys. Chem. C, 113, 19468–74 Becker & Hickl GmbH, PML-16-C 16 channel detector head for timecorrelated single photon counting, User Handbook, Berlin, 2006 http://www.beckerhickl.de/pdf/pml16c21.pdf Schmitt F.J. (2011) Picobiophotonics for the investigation of pigment–pigment and pigment–protein interactions in photosynthetic complexes, thesis, Technische Universität Berlin http://opus.kobv.de/tuberlin/volltexte/2011/3 202/pdf/schmitt_franzjosef.pdf. Nakamura K., Ishiyama K., Ikai H., Kanno T., Sasaki K., Niwano Y., Kohno M. (2011) Reevaluation of analytical methods for photogenerated singlet oxygen. J. Clin. Biochem. Nutr., 49(2), 95 Strakhovskaya M.G., Parkhomenko I.M., Rumbal Y.V., Zarubina A.P., Danilov V.S., Stranadko E.F. (2002) Photoquenching of the bioluminescence of the genetically engineered Escherichia coli TG1 (pXen7) strain in the presence of photodithazine, Microbiology (Russia). 71, 294-7 Strakhovskaya M.G., Zarubina A.P., Zhukhovitskii V.G., Mironov A.F., Rubin A.B. (2004) Bioluminescent genetically transformed bacteria as a new effective tool for testing photosensitization activity, Dokl. Biochem. Biophys. 396, 177-80 Lakowicz J.R. (1999) Principles of Fluorescence Spectroscopy, 2nd ed, Kluwer Academic/Plenum Publishers, 465. Strakhovskaya M.G., Antonenko Y.N., Pashkovskaya A.A., Kotova E.A., Kireev V., Zhukhovitsky V.G., Kuznetsova N.A., Yuzhakova O.A., Negrimovsky V.M., Rubin A.B. (2009) Electrostatic binding of substituted metal phthalocyanines to enterobacterial cells: its role in photodynamic inactivation, Biochemistry (Moscow). 74, 1305-14 Shiohara A., Hoshino A., Hanaki K., Suzuki 60

48.

49.

50.

K., Yamamoto K. (2004) On the cytotoxicity caused by quantum dots. Microbiol. Immunol., 48, 669–75 Derfus A.M., Chan W.C.W., Bhatia S.N. (2004) Probing the cytotoxicity of semiconductor quantum dots. Nano Lett., 4, 11–8. Bakalova R., Ohba H., Zhelev Z., Ishikawa M., Baba Y. (2004) Quantum dots as photosensitizers? Nature biotechnology, 22, 1360–1 Samia A.C.S., Chen, Burda C. (2003) Semiconductor quantum dots for photodynamic therapy. J. Am. Chem. Soc., 125, 15736–7.