- Email: [email protected]
Interaction of Pheophorbide a molecules covalently linked to DAB dendrimers
Optics Communications 248 (2005) 295–306 www.elsevier.com/locate/optcom
Interaction of Pheophorbide a molecules covalently linked to DAB dendrimers S. Hackbarth, E.A. Ermilov, B. Ro¨der
*
Department of Photobiophysics, Institute of Physics, Humboldt-University, Newtonstr. 15, 12489 Berlin, Germany Received 29 September 2004; received in revised form 21 November 2004; accepted 23 November 2004
Abstract The photophysical properties of DAB–dendrimers from first to fourth generation as well as Diaminohexane – all substituted with the in maximum achievable quantity of Pheophorbide a (Pheo) molecules are compared with those of Pheo in ethanolic solution. The high local concentration of the linked dye molecules on the dendrimer surface yields in interactions between the chromophores within each complex. The investigation of these interactions is the aim of this paper. It was found, that a larger number of chromophores in one dendrimer–dye-complex increases the stacking probability of Pheo moieties accompanied by excitonic interaction leading to a fast non-radiative deactivation after excitation. The efficiency of deactivation depends on the relative position of the chromophores one to each other. Since the Fo¨rster radius for energy transfer between Pheo molecules is bigger than the diameter of the whole complex, energy is jumping very rapidly between the chromophores within the complex. Just one pair of excitonic interacting chromophores can act as energy trap for the whole complex. Ó 2004 Elsevier B.V. All rights reserved. PACS: 31.70.Hq; 33.50.j; 71.35.y; 78.47.+p Keywords: Pheophorbide a; Dendrimers; Fo¨rster energy transfer; Excitonic interaction; Singlet oxygen; Modular carrier system; Photodynamic therapy
1. Introduction Already years ago [1] photodynamic therapy was proposed to be a powerful treatment against *
Corresponding author. Tel.: +49 30 2093 7625; fax: +49 30 2093 7666. E-mail address: [email protected] (B. Ro¨der).
cancer and other diseases. But still today one of the crucial problems is, that no sensitizer is known, which actively and selectively accumulates in target tissues [2]. Several carrier systems have been tested in the past to overcome this problem [3] but it turned out, that only antibodies or their fragments show a sufficiently high selectivity for this purpose [2]. Due to the fact, that such antibodies can carry
0030-4018/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.optcom.2004.11.088
296
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
only a few dye molecules covalently linked without losing their bioactivity [4], it was proposed to use linker-molecules (multipliers), which can carry a number of photosensitizing molecules [5] and are linked via one chemical bond to the antibody. Based on this the modular carrier system was proposed [2] consisting of an addressator, a multiplier and lots of photosensitizers. For such systems the use of dendrimers as multiplying units was proposed [6]. Dendrimers became of rising interest in the past decade in different fields of biophysical research due to their highly branched and well-defined architecture, which consist of a high number of functional end groups [7,8]. Already in 1991, Tomalia and co-workers [9] reported the possibility to couple dendrimers to antibodies and recently Patri et al. [10] reported about antibody–dendrimer complexes carrying fluorescence markers, where the antibodies still are active and selective. During former studies with a Pheophorbide a (Pheo) substituted DAB–dendrimer of third generation [11] an efficient interaction between the covalently linked Pheo moieties was observed. The chromophores in the complex undergo Fo¨rster energy transfer along the surface of the dendrimer as well as partly excitonic interaction. These interactions result in a strong reduction of photoactivity of the Pheo molecules, especially their fluorescence and singlet oxygen generation, as long as they are linked to the dendrimer. As it was shown [11] illumination of the Pheo– DAB–dendrimer, especially in the presence of oxygen, leads to a destruction of the dendrimer backbone into fragments, that carry only one or a small number of dye molecules. Because of that for PDT relevant photophysical parameters, like fluorescence and singlet oxygen quantum yield, increase reaching nearly the values of free Pheo in solution [11]. On the other hand, in recent years lots of papers deal with the use of dendrimers to mimic light harvesting complexes [12–18]. But these systems are all based on directed energy transfer between donors and acceptors of different kind only and no other interaction between donor chromophores was reported. Additionally, a comparison with these results is impossible, since no data
are given about the photostability of these complexes. Besides our former work [11] only in one paper the existence of a stronger interaction between identical dye-molecules in dendrimers was proposed [19]. Yeow et al. only observed this effect in fifth generation of porphyrin–DAB–dendrimers and no further investigation was carried out. Nevertheless, all results shown in [19] are in good agreement with the general model developed in the present paper. In a very recent work, Hasobe et al. [18] observed a reduction of electron transfer from the same porphyrin–dendrimer-complexes to C60-fullerene with increasing generation of the dendrimers. They explained this effect with steric hindering only, but since they worked with clusters of dendrimers and C60-fullerene we can assume that excitonic deactivation of the first excited singlet state of the porphyrin moieties is a major part of this effect. Very recently, we submitted a paper [20] on pyropheophorbide a on fullerene-based dendrimers were also excitonic interaction was reported between the coupled dye-molecules, which also fits into our model. For plain DAB dendrimers especially of higher generation a high cell toxicity was reported [21]. This is strongly reduced after loading with Pheo, because the amine groups, which mainly cause the toxicity, form peptide bonds with the dye molecules. This statement is supported by cell experiments that proved Pheo loaded dendrimers to have no dark toxicity [22]. On the other side, the phototoxicity on Jurkat cells was comparable or even higher than that of Pheo administered in solution [22]. After incubation of cells in vitro with the Pheo dendrimer complexes the photophysical parameters of the complexes became similar to those of free monomeric Pheo in cells. Since a good photosensitizer comprises despite high activity in the target tissue also a low activity during transport, the intact dendrimer–dye-complexes are subject of this work. The influence of the local concentration of linked chromophores in one complex on the photophysical behaviour of the chromophores and the complex photostability was investigated for the DAB–dendrimer–Pheo system. The local Pheo concentration increases with dendrimer
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
drimers of generation 0 (Diaminohexane) to 4, respectively. Their structural formula are given in Fig. 1. Due to steric hindering for dendrimers starting with P8 it becomes more and more complicated to reach a complete loading of the DAB–dendrimers with Pheo. Steric hindering and the tendency of the coupled dye molecule to fold back, as will be shown later, inhibit in principle a complete loading. For P16, for example, a maximum loading of 13 chromophores per dendrimer was achieved [11]. All investigations were done in ethanol (Pheo– DAB was dissolved in chloroform, which was diluted with ethanol in a ratio of 1:1000) at dye concentrations of 106–107 M to reach an OD of 0.2 at the excitation wavelength.
generation due to the exponential increase of available dendrimer end groups but linear increase of dendrimer surface. Molecular modelling was carried out to verify the results.
2. Materials Pheo was isolated from stinging nettles (urtica urens) according to Willsta¨tter and Stoll [23] and activated with N-hydroxy succinimide purchased from Aldrich. All solvents were of spectroscopic grade. All dendrimers have been synthesized as described in [11] with small modifications [24]. The samples are named P2, P4, P8, P16 and P32 according to the number of end groups of the den-
P2
R N
H
H
R N
P4 N
HN R
R
N H
H N
R HN R N H
N N R
N
H
N N
HR R HR H R H RN N R R N H N NH H R R HN NH N N R R N N N N H H R R N N N NH N N RH R P32 N N H H N N R R N N N N N N H H N N H H N N N N N N R R N N H H N N RH R N N H N N N N R R H H N N N N R R N N HN NH RH R H N N R N NH R H R R HR RH
R = H or pheophorbide-amoiety
H N R
H
N
N
N R
P16
H N R
H NR
N
R HN
N
N
N
N N
R
N H
NH R
R H N
H RN
N
R
N H
R HN
H NR
P8
R
297
N
N
N
N
RN H
N
N
N
HN R
N
H NR
N R H
pheophorbide-a (Pheo):
N
N H N
H N
O HO
Fig. 1. Structural formula of the investigated samples.
O
O
R NH
O
H N R R N H NH R
NR H
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
Due to the light sensitivity of the complexes they have been prepared freshly for every experiment and the quality was checked using steadystate fluorescence setup to show no significant changes. A destruction of the dendrimer backbone would lead to an increase of the fluorescence intensity [11].
3. Methods The absorption spectra were recorded using a commercial spectrophotometer Shimadzu UV160A. Time resolved fluorescence and fluorescence anisotropy measurements were carried out using time-correlated single photon counting (TCSPC), containing of an SPC 300 plug-in card (Becker Hickl, Berlin) and a multi-channel plate (Hamamatsu) described in [25]. For data analysis the decay curves were deconvoluted and fitted using the least square method based on Newton algorithm. For mono exponential fits v2-values less than 1.1 were reached. For fittings with more than one decay times, still smaller v2-values close to 1.0 were obtained. The device for steady-state luminescence measurements is based on an L-setup with different light sources (xenon lamp, Yb+ Disc-YAG pumped dye laser) and has been described previously [26]. The singlet oxygen quantum yield (UD) of all samples was determined twice using steady state (nitrogen cooled InGaAs-diode, Laser Components) and time resolved (nitrogen cooled Ge-diode, Northcoast and recording oscilloscope HP) luminescence detection as described in [27,28] to exclude the influence of different possible non-radiative deactivation pathways. Using these two different methods identical quantum yields (UD ± 0.05) for all samples were obtained. For this reason in the results section only one value of UD is given for each sample. Transient absorption measurements were performed using the pump–probe-setup described in [29]. Molecular mechanical calculations have been carried out using the program Hyperchem with forcefield MM+ in vacuum at room temperature.
4. Results 4.1. Absorption In the visible range the absorption spectra of all samples are similar to that of Pheo in ethanolic solution. With higher dendrimer generation the Soret band is broadened and the lowest Q-band is bathochromic shifted between 3 nm for P2 and 7 nm for P32 (Fig. 2 and Table 1). The extinction coefficients of the covalently linked dye are strongly reduced. For P4 to P32 the absorption is reduced by a factor of about 3 related to equal amounts of Pheo molecules in solution. A more exact determination is not possible due to statistical loading of the dendrimers. 4.2. Steady-state fluorescence For all dendrimer–dye-complexes the fluorescence spectra are shifted bathochromic (Fig. 3 and Table 1). The shift of the fluorescence maximum relative to that of Pheo increases from P2 over P4 to P8. For P16 the shift is less than for P8 and the maximum of emission of P32 is shifted only 2 nm relative to Pheo in solution. The fluorescence quantum yield is reduced for all dendrimer–dye-complexes relative to that of Pheo. Already for P2 the fluorescence intensity is reduced to 22%. It further decreases for P4 and P8 and nearly reaches zero for P16 and P32 (Table 1).
absorption (normalized)
298
1 0.8 0.6 640
0.4
660
680
700
0.2 0 300
400
500
600
700
800
wavelength [nm] Fig. 2. Absorption spectra of Pheo (thin line), P2 (black), P4 (grey), P8 (light grey), P16 (black dotted) and P32 (grey dotted) in ethanol.
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
299
Table 1 Maxima of absorption and emission of the samples under investigation, their fluorescence and singlet oxygen quantum yields relative to Pheo (UD = 0.52 ± 0.05) [27] as well as steady-state anisotropy r0 in ethanolic glass kmax (nm) (Q-absorption)
kmax (nm) (fluorescence)
UFl relative to Pheo ± 0.005
UD relative to Pheo ± 0.03
r0 at 110 K ± 0.02
Pheo
666
674
1.00
1.00
P2 P4 P8 P16 P32
669 669 670 672 673
675 680 683 680 676
0.22 0.070 0.065 0.021 0.005
0.33 0.29 0.25 0.12 0.08
0.30 0.27 0.16 0.07 0.04 0.03 0.02
(675 (700 (700 (700 (700 (700 (700
nm) nm) nm) nm) nm) nm) nm)
fluorescence (normalized)
1 0.8 0.6 0.4 0.2 0 625 650 675 700 725 750 775 800 825 850 875 900
(a)
wavelength [nm]
fluorescence (normalized)
1 0.8 0.6 0.4 0.2 0 625 650 675 700 725 750 775 800 825 850 875 900 (b)
wavelength [nm]
Fig. 3. Fluorescence spectra of (a) P2 (black), P4 (grey), and P8 (light grey); (b) P8 (black), P16 (grey) and P32 (light grey). For comparison the fluorescence of Pheo in ethanol is shown as thin line.
The initial fluorescence anisotropy has been measured in ethanolic glass at 110 K excited at 633 nm. For Pheo a value of 0.3 was obtained at
the emission wavelength at 675 nm. At 700 nm this value is decreased slightly only. For all other samples a much stronger reduction of the initial
300
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
anisotropy was observed at 700 nm and longer wavelengths – from 0.17 for P2 down to nearly zero for P32 (Table 1). At shorter wavelengths (around 675 nm – not shown) this reduction was much weaker.
0.7 ns (decay time 3) can be detected at both wavelengths. Starting with P4 a fourth, very fast decay with 0.03–0.06 ns (decay time 4) becomes dominant (Table 2). Its relative amplitude increases with dendrimer generation number.
4.3. Time-resolved fluorescence
4.4. Transient picosecond absorption
Based on the results of anisotropy measurements, it was supposed that besides monomeric Pheo a second emitter must exist in the complexes with much lower initial fluorescence anisotropy. Since the influence of this additional emitter is most pronounced at 700 nm and higher, the fluorescence decay time was determined at 675 and 700 nm. For Pheo both decay curves could be fitted mono exponential to a value of 5.7 ns, which is in a good agreement with former publications [11,30]. For all other samples at both wavelengths a slightly shorter decay time (4.4–4.7 ns) was observed (decay time 1). Such shortened lifetime has been already reported for Pheo in inhomogeneous environments [26,30]. A second faster decay with 2.0–2.6 ns (decay time 2) appears at 675 nm for P4 to P16. At 700 nm this decay time was also observed for P2 and P32. The relative amplitude of this decay time reaches its maximum for P8. For all dendrimers a third shorter decay time of 0.4–
Investigating the samples using picosecond transient absorption spectroscopy mainly three decay times were obtained. Two of them are similar to decay times 3 and 4 obtained with TCSPC. The latter one has much smaller relative amplitude than it was found in fluorescence measurements for P4 to P32 and does not exist for P2. The third decay time of ground state depletion lies between fluorescence decay time 1 and 2. It is probably a mixture of both times which cannot be resolved due to the signal/noise ratio (values not shown). For illustration the decays of the ground state depletion of P2 and P32 are shown in Fig. 4. 4.5. Singlet oxygen In general the singlet oxygen luminescence is also decreasing with rising dendrimer generation. The well-known singlet oxygen quantum yield of Pheo, 0.52 [27] reduces to 0.17 for P2 and it continues decreasing down to 0.04 for P32 (Table 1). Interestingly, for all samples the quantum yield
Table 2 Fluorescence decay times and relative amplitudes of all samples at 675 and 700 nm s1 (ns) ±0.2
A1 (rel.) ±0.3
s2 (ns) ±0.3
675 nm Pheo P2 P4 P8 P16 P32
5.72 4.52 4.48 4.61 4.68 4.75
5.0 0.5 <2 <2 <2
– – 2.55 2.46 2.14 –
700 nm Pheo P2 P4 P8 P16 P32
5.72 4.71 4.43 4.45 4.68 4.60
1.6 0.35 <2 <2 <2
– 2.63 2.20 2.16 2.14 1.96
A2 (rel.) ±0.3
1.5 2.5 1.6
1.6 1.4 2.0 1.7 0.4
s3 (ns) ±0.1
A3 (rel.) Ref.
s4 (ns) ±0.015
A4 (rel.) ±0.5
– 0.661 0.50 0.68 0.52 0.63
1 1 1 1 1
– – 0.034 0.063 0.054 0.044
1.5 3.3 3.6 3.8
– 0.66 0.48 0.70 0.52 0.42
1 1 1 1 1
– – 0.032 0.060 0.054 0.032
1.0 3.0 3.5 5.3
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306 ground state depletion
0.35
ground state depletion
0.3 0.25 0.2
301
0.33 0.31 0.29 0.27 0.25 0.23 0.21 0.19 0.17 0
0.15
500
1000
1500
2000
time [ps]
0.1 0.05 0 0
(a)
10000
15000
time [ps] ground state depletion
0.2 0.18 ground state depletion
5000
0.16 0.14 0.12 0.1
0.19 0.17 0.15 0.13 0.11 0.09 0.07 0.05 0
500
0.08
1000
1500
2000
time [ps]
0.06 0.04 0.02 0
(b)
0
5000
10000
15000
time [ps]
Fig. 4. Ground state depletion and transient emission of P2 (a) and P32 (b) as determined with TAS.
of singlet oxygen is less reduced relative to that of Pheo than that of fluorescence. For P32 the fluorescence intensity is reduced by a factor of about 200 relative to that of Pheo in ethanol, the corresponding singlet oxygen quantum yield is reduced only by a factor of 13. 4.6. Molecular modelling In Fig. 5, energetically optimized conformations of P4 and P16 with 4 and 12 chromophores, respectively, are shown. The calculations were carried out in vacuum at room temperature. These
pictures show each just one possible conformation, but they visualize an effect, that was obtained from all calculations for all samples. The covalently linked Pheo molecules show a strong tendency to fold back to the dendrimer backbone and to stack with each other. In case of higher generation dendrimers, the back folded chromophores cover some of the not substituted end groups of the dendrimers, which probably inhibit further binding at these positions. The nearest-neighbour centre-to-centre distances for the chromophores in all complexes ˚ , while the where found to be smaller than 20 A
302
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
Fig. 5. Energy optimised conformations of P4 and P16 in vacuum at room temperature. The pictures show each just one possible conformation typical for all achieved results. The dendrimers backbone is light, the Pheo moieties are dark painted.
Fo¨rster radius for the dipole-dipole resonant energy transfer between Pheo molecules was calcu˚. lated to be 62 A
5. Discussion 5.1. Interactions of chromophores in one dendrimer– dye complex The strong reduction of the fluorescence quantum yield (Section 4.2) and the appearance of up to three additional decay times (Section 4.3) for the dendrimer–dye complexes are a prove for an intense interaction between chromophores in one dendrimer–dye-complex. The reduced initial fluorescence anisotropy of the dendrimer–dye-complexes (Section 4.2) indicates, that Fo¨rster transfer occurs. But if we as-
sume Fo¨rster transfer to be the only interaction, the excitation energy can hop from one dye molecule to the next, but the fluorescence intensity should be reduced not significantly. The extremely high amplitudes of the short decay times of Pheo in the higher generation dendrimers indicate, that another interaction should exist yielding a non- or less radiative deactivation process. Pheo is known to undergo such deactivation in dimers [31]. So the reason for the non-radiative deactivation is likely to be an excitonic interaction between stacked chromophores. This conclusion is supported by the red shift and slight broadening of the S0,0 ! S1,0 absorption of the dendrimer–dyecomplexes compared to that of Pheo (Fig. 2). The possibility of dimer formation by chromophores in one dendrimer–dye-complex was proofed using molecular modelling. The calculations showed that such dimer formation can occur
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
in all dendrimer–dye-complexes (Section 4.6). Moreover, the stacking of Pheo molecules linked to the same dendrimer became dominant in all calculations. We suggest that these calculations show the general tendency of the linked Pheo molecules to fold back and to stack with each other. Since the calculations have been carried out in vacuum, in solution these effects should be reduced. Otherwise, already for P4 a nearly complete quenching of fluorescence, much stronger than observed, should occur. Due to the fact, that the calculated Fo¨rster radius for Pheo is much larger than the whole complex (Section 4.6), the stacking of just one pair of chromophores leading to excitonic interaction (trap) is enough for a very effective quenching of the whole complex. Even a nearest ˚ makes energy transfer neighbour distance of 20 A about 900 times more probable than any other processes. That means for example, in case of P4, that in the presence of one trap every energy hop originating from one of the remaining chromophores has a 50% probability to end in the trap. Nearly no fluorescence should be obtained due to the fast deactivation of the trap. Consequently, the real conformation can not be as tight as calculated in vacuum. That means also that there exist also dendrimer–dye-complexes without any trap or with non-perfect traps. Non-perfect means, that their deactivation is not that fast resulting in a certain probability of radiative deactivation or back transfer to a single chromophore in the complex. Nevertheless, the tendency of the linked Pheo molecules to stack with each other must be very strong, since already for P2 the fluorescence intensity is reduced to 0.22 of that of Pheo in solution. Because of just two chromophores in each complex no other reason for the strong reduction of the fluorescence and appearance of additional decay time is possible. With rising generation number and hence rising number of covalently linked dye molecules the probability of trap formation increases and as a result the fluorescence quantum yield decreases. 5.2. General model Several dimers of reduced porphyrins, among them pyropheophorbide a-dimers have been re-
303
ported to have a bathochromic shifted emission maximum [32–35]. This stands in a good agreement with our former findings, that Pheo molecules densely packed in Langmuir Blodgett Films also form dimers/aggregates which show a bathochromic shifted absorption and remaining emission. Such an aggregation was also observed for Pheo in mixed solutions of ethanol and water [31]. The high local concentration of Pheo moieties at the dendrimers surface yields in dimer (trap) formation and hence the emission spectrum of the dendrimer–dye-complexes is a superposition originating from single chromophores and from traps. While the fluorescence of single chromophores will be more pronounced at 675 nm the remaining trap emission will be visible more intense at 700 nm. For this reason the time-resolved fluorescence was measured at these two wavelengths. 5.2.1. Decay time 1 This decay time was observable at both registration wavelengths for all complexes. For P2 at 675 nm it is the strongest decay component. This decay time can be assigned to chromophores in the complexes where no energy traps were formed. Since the distances between the chromophores in one complex are very small, energy transfer processes should occur very fast. But, as already pointed out in Section 5.1, Fo¨rster transfer between identical chromophores without traps does not influence the fluorescence decay time. 5.2.2. Decay time 4 In the presence of one or more traps in a complex an additional decay time arises, which represents the probability of energy transfer to the trap. For a very rough estimation of this additional decay time a random walk of the energy between a set of 20 chromophores was analyzed. If two of these molecules form a trap, they are very close to each other and can be approximated as one acceptor. For simplicity it is assumed, that every of the remaining 18 chromophores as well as the trap have five nearest neighbours, all at equal distances. Due to the r6 dependency Fo¨rster transfer practically happens only to these nearest neighbours. The probability of the trap to be among
304
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
the five neighbours is about 1/3. If we approximate, that the transfer probabilities to a monomer and the trap are similar [36], than the probability pTrap of every energy hop to go to the trap is 1/15. Once caught by the trap a back transfer is very unlikely due to the red-shifted emission of the trap, its low fluorescence quantum yield and the reduced fluorescence lifetime due to non-radiative deactivation. In general the additional decay time s caused by transfer to a trap is given by s ¼ i sHop , where sHop is the time for one energy hop, which in this simple approximation is just 1/5 of the Fo¨rster transfer time sFT between two of these chromophores [37]. Here, i represents the number of transfer hops for which the probability of not being caught by the trap is 1/e 1 i ¼ ð1 pTrap Þ : e For a set of 20 chromophores with one trap we get approximately 33 hops and a decay time of s = 6.6 Æ sFT. ˚ centre to centre distance of two Pheo For a 20 A molecules the Fo¨rster transfer time is about 0.006 ns giving a value of 0.04 ns for the additional decay time caused by energy transfer to the trap in this approximation. These simple calculations already demonstrate that the fourth decay time describes the energy transfer process to the trap. This explains, why this decay time is only much weaker visible in transient absorption (Section 4.4). It does not represent a transfer to the ground state but a transfer to chromophores with lower extinction. It also explains, why it does not appear in P2, because such transfer process is impossible. The two chromophores in P2 can form a trap or behave like monomers. 5.2.3. Decay times 2 and 3 Basing on the absence of an energy transfer to traps in P2 the decay times 2 and 3 assign to the traps themselves. It is known [32–35], that the fluorescence decay times of dimers can strongly differ due to the relative orientation of the dipole mo-
ments of the two chromophores. Because of this fact we suggest that the shorter decay time 3 originates from the trap (trap 1) with more parallel oriented, while decay time 2 represents a more or less tilted trap (trap 2). The big angle between the dipole moments of the two partners in such a tilted trap prevents a more intense interaction yielding in a longer decay time. So the fluorescence of trap 2 is much stronger compared with the one of trap 1. Carrying out molecular mechanical calculations pairs of chromophores with more or less parallel as well as nearly perpendicular relative orientation of the dipole moments have been found (Fig. 5). The relative amplitude of decay time 2 is decreased in P32. In our model this fact can be explained with the higher probability of multiple stacking and the higher probability of a parallel orientation of chromophores yielding in formation of trap 1. At 675 nm this decay time is not observed due to the lower intensity of trap emission and stronger emission of single chromophores at this wavelength. This also fits with the picture found for P2. Here, the probability of non-interacting dye molecules is much higher and so decay time 1 is much more pronounced than the quite similar decay time 2, which then can not be distinguished during fitting procedure. 5.2.4. Shift of steady-state fluorescence Since the emission from trap 2 is much stronger than that from trap 1, it is not surprising, that the steady-state emission of P4, P8, and P16 where both types of traps are present is more bathochromic shifted than for P32, where the formed traps are mainly of type 1. The emission from trap 1 is so weak, that even the short time of energy transfer from the remaining single chromophores to the traps enables sufficient monomer emission to override the trap emission. 5.2.5. Anisotropy reduction The higher the number of linked dye molecules in a complex the stronger the reduction of anisotropy due to the higher probability of energy transfer (Section 4.2 and Table 1) should be. The anisotropy of P2 is slightly more than half of that of Pheo in solution at 675 nm. Such reduction is expected for a random orientation of two linked
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
Pheo molecules in presence of Fo¨rster transfer. The probability of emission from the originally excited molecule is nearly the same as for the second molecule. With rising number of chromophores in the complex the probability for an emission from any randomly oriented chromophore increases. The majority of the energy is caught by the traps and since they can have any orientation relative to the excited chromophore, the anisotropy is reduced especially at wavelengths, where the traps emit. At shorter wavelengths the fluorescence mainly originates from single chromophores and their number is reduced due to trap formation. Hence at these wavelengths the anisotropy is not reduced that strong. In Fig. 6, a schematic sketch of the transfer processes in a dendrimer–dye-complex is given. 5.2.6. Singlet oxygen As it was pointed out above, the majority of the absorbed energy in the dendrimer–dye complexes is caught by traps yielding mainly in an efficient non-radiative deactivation. But all samples, even P32 generate singlet oxygen. For all complexes the fluorescence quantum yield has been reduced stronger than that of singlet oxygen. As a consequence not only the single chromophores but also the traps should undergo intersystem crossing to allow further energy transfer to molecular oxygen. But, as it was found using transient absorption spectroscopy (Fig. 4) the ISC quantum yield of the complexes is much smaller
Fig. 6. Schematic sketch of a dendrimer–dye-complex to illustrate the proposed energy transfer processes between dye molecules in one complex.
305
(UISC < 0.2 already for P2) than that of the dye in solution (0.6 ± 0.05). For this reason the singlet oxygen quantum yield is reduced in comparison to Pheo in solution, but not as strong as the fluorescence quantum yield.
6. Conclusions Pheo molecules covalently linked to DAB dendrimers undergo very efficient energy transfer processes, Fo¨rster-transfer as well as partly excitonic interaction. These processes already occur in the case of Diaminohexane coupled Pheo where only two chromophores are within each complex. The covalently linked Pheo molecules show a tendency to fold back to the dendrimer and to stack with neighbouring molecules. Such close contact gives rise to excitonic interaction yielding in non-radiative deactivation of such stacked chromophores. The shortening of decay time caused by this interaction depends on the position of the involved chromophores relative to each other. While those with parallel oriented dipole moments are very short lived, other pairs of Pheo molecules with no parallel orientation of the dipole moments have about half the decay time of isolated chromophores in the complexes and a stronger remaining fluorescence. Since the Fo¨rster radius of energy transfer between Pheo molecules is longer than the overall diameter of the complex every pair of excitonic interacting chromophores acts like an energy trap for the whole complex. Due to the small nearest neighbour distances the transfer velocity of energy to the traps is about two orders of magnitude higher than that of radiative relaxation of the chromophores. As a consequence the quantum yields of fluorescence, inter system crossing and singlet oxygen are strongly reduced in all complexes. The probability of trap formation increases with the number of coupled dyes and hence with generation number of the dendrimer and so does the effect of these traps. This yields in a nearly complete quenching of fluorescence in case of P32. Nevertheless, the traps should undergo inter system crossing and so some singlet oxygen is generated even for highest generation.
306
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
References [1] D. Dolphin, Can. J. Chem. 72 (1994) 1005. [2] B. Ro¨der, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry: Instrumentation and Applications, Wiley, Chinchester, 2000. [3] Y.N. Konan, R. Gurny, E. Alle´mann, J. Photochem. Photoboil. B 66 (2002) 89. [4] C. Dressler, U. Mo¨ller, T. Lewald, H.-P. Berlien, B. Ro¨der, H.-J. Risse, Laser Med. Sci. 7 (1992) 174. [5] J.G. Moser, A. Heuermann, P. Oehr, H. Scheer, A. Vervoorts, S. Andrees, SPIE Proc. 2325 (1994) 92. [6] B. Ro¨der, S. Hackbarth, G. Wo¨hlecke, US-Patent, NDN 172-0037-2622-7. Publication Nr. 00108704 WO, German Patent Nr. 199 36 997.6 02.08.1999. [7] G.J. Dykes, Chem. Technol. Biotechnol. 76 (2001) 903. [8] M. Fischer, F. Vo¨gtle, Angew. Chem. Int. Ed. 38 (1999) 884. [9] J.C. Roberts, Y.A. Adams, D. Tomalia, J.A. MercerSmith, D.K. Lavalee, Bioconjugate Chem. 1 (1990) 305. [10] A.K. Patri, T. Thomas, J.R. Baker, N.H. Bander, Polym. Mater. Sci. Eng. 86 (2002) 130. [11] S. Hackbarth, V. Horneffer, A. Wiehe, F. Hillenkamp, B. Ro¨der, Chem. Phys. 269 (2001) 339. [12] V. Balzani, P. Ceroni, M. Maestri, V. Vicinelli, Curr. Opin. Chem. Biol. 7 (2003) 657. [13] M. Cotlet, R. Gronheid, S. Habuchi, A. Stefan, A. Barbafina, K. Mu¨llen, J. Hofkens, F.C. De Schryver, JACS 125 (2003) 13609. [14] B.L. Davis, J.S. Melinger, D. McMorrow, Z. Peng, Y. Pan, J. Lumin. 106 (2004) 301. [15] D. Rana, G. Gangopadhyay, J. Chem. Phys. 118 (2003) 434. [16] P.R. Hania, D.J. Heijs, T. Bowden, A. Pugzlys, J. van Esch, J. Knoester, K. Duppen, J. Phys. Chem. B 108 (2004) 71. [17] M.R. Shortreed, S.F. Swallen, Z.-Y. Shi, W. Tan, Z. Xu, C. Devadoss, J.S. Moore, R. Kopelmann, J. Phys. Chem. B 101 (1997) 6318. [18] T. Hasobe, Y. Kashiwagi, M.A. Absalom, J. Sly, K. Hosomizu, M.J. Crossley, H. Imahori, P.V. Kamat, S. Fukuzumi, Adv. Mater. 16 (2004) 975. [19] E.K.L. Yeow, K.P. Ghiggino, J.N.H. Reek, M.J. Crossley, A.W. Bosman, A.P.H.J. Schenning, E.W. Meijer, J. Phys. Chem. B 104 (2000) 2596.
[20] E. Ermilov, S. Hackbarth, S. al Omari, M. Helmreich, N. Jux, A. Hirsch, B. Ro¨der, Opt. Commun. (2004) submitted. [21] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, E.W. Meijer, W. Paulus, R. Duncan, J. Control. Release 65 (2000) 133. [22] A. Paul, S. Hackbarth, A. Mo¨lich, C. Luban, S. Oelckers, F. Bo¨hm, B. Ro¨der, Laser Phys. 13 (2003) 22. [23] A. Willsta¨tter, R. Stoll, Untersuchungen u¨ber Chlorophyll, Springer, Berlin, 1913. [24] All the dendrimers have been synthesized by adding 200 mg Pheophorbide a succinimide ester to the unmodified DAB dendrimer (DSM) in 20 ml DCM in a molar ratio of 6:1. A small amount (5 drops) of triethylamine has been added as well as 100 mg dicyclocarbodiimide for deprotonation of the dendrimer and catalization. After stirring for 4 days at room temperature in the dark the product was dried an rinsed several times with acetone and supersonic. [25] J. Zimmermann, J.v. Gersdorff, H. Kurreck, B. Ro¨der, J. Photochem. Photobiol. B 40 (1997) 209. [26] O. Korth, T. Hanke, I. Ru¨ckmann, B. Ro¨der, Exp. Tech. Phys. 41 (1995) 25. [27] W. Spiller, H. Kliesch, D. Wo¨hrle, St. Hackbarth, B. Ro¨der, J. Porphyr. Phthalocynaines 2 (1998) 145. [28] St. Oelckers, M. Sczepan, Th. Hanke, B. Ro¨der, J. Photochem. Photobiol. B 39 (1997) 219. [29] A. Zeug, I. Ru¨ckmann, B. Ro¨der, J. Opt. B 3 (2001) 251. [30] B. Ro¨der, T. Hanke., S. Oelckers, S. Hackbarth, C. Symietz, J. Porphyr. Phthalocynaines 4 (2000) 37. [31] I. Eichwurzel, H. Stiel, B. Ro¨der, J. Photochem. Photobiol. B 54 (2000) 124. [32] A. Osuka, Y. Wada, S. Shinoda, Tetrahedron 52 (1996) 4311. [33] A. Osuka, K. Maruyama, JACS 110 (1988) 4454. [34] E.I. Zenkevich, V.N. Knyukshto, A.M. Shulga, V.A. Kuzmitsky, V.I. Gael, E.G. Levinson, A.F. Mironov, J. Lumin. 75 (1997) 229. [35] A.N. Kozyrev, V. Suresh, S. Das, M.O. Senge, M. Shibata, T.J. Dougherty, R.K. Pandey, Tetrahedron 56 (2000) 3353. [36] Due to the better overlap of the bathochromic shifted trap absorption with the monomer fluorescence spectrum the transfer probability is increased, but on the other hand decreased due to the lower extinction of the traps. [37] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic Publishers, New York, 1999.
Interaction of Pheophorbide a molecules covalently linked to DAB dendrimers S. Hackbarth, E.A. Ermilov, B. Ro¨der
*
Department of Photobiophysics, Institute of Physics, Humboldt-University, Newtonstr. 15, 12489 Berlin, Germany Received 29 September 2004; received in revised form 21 November 2004; accepted 23 November 2004
Abstract The photophysical properties of DAB–dendrimers from first to fourth generation as well as Diaminohexane – all substituted with the in maximum achievable quantity of Pheophorbide a (Pheo) molecules are compared with those of Pheo in ethanolic solution. The high local concentration of the linked dye molecules on the dendrimer surface yields in interactions between the chromophores within each complex. The investigation of these interactions is the aim of this paper. It was found, that a larger number of chromophores in one dendrimer–dye-complex increases the stacking probability of Pheo moieties accompanied by excitonic interaction leading to a fast non-radiative deactivation after excitation. The efficiency of deactivation depends on the relative position of the chromophores one to each other. Since the Fo¨rster radius for energy transfer between Pheo molecules is bigger than the diameter of the whole complex, energy is jumping very rapidly between the chromophores within the complex. Just one pair of excitonic interacting chromophores can act as energy trap for the whole complex. Ó 2004 Elsevier B.V. All rights reserved. PACS: 31.70.Hq; 33.50.j; 71.35.y; 78.47.+p Keywords: Pheophorbide a; Dendrimers; Fo¨rster energy transfer; Excitonic interaction; Singlet oxygen; Modular carrier system; Photodynamic therapy
1. Introduction Already years ago [1] photodynamic therapy was proposed to be a powerful treatment against *
Corresponding author. Tel.: +49 30 2093 7625; fax: +49 30 2093 7666. E-mail address: [email protected] (B. Ro¨der).
cancer and other diseases. But still today one of the crucial problems is, that no sensitizer is known, which actively and selectively accumulates in target tissues [2]. Several carrier systems have been tested in the past to overcome this problem [3] but it turned out, that only antibodies or their fragments show a sufficiently high selectivity for this purpose [2]. Due to the fact, that such antibodies can carry
0030-4018/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.optcom.2004.11.088
296
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
only a few dye molecules covalently linked without losing their bioactivity [4], it was proposed to use linker-molecules (multipliers), which can carry a number of photosensitizing molecules [5] and are linked via one chemical bond to the antibody. Based on this the modular carrier system was proposed [2] consisting of an addressator, a multiplier and lots of photosensitizers. For such systems the use of dendrimers as multiplying units was proposed [6]. Dendrimers became of rising interest in the past decade in different fields of biophysical research due to their highly branched and well-defined architecture, which consist of a high number of functional end groups [7,8]. Already in 1991, Tomalia and co-workers [9] reported the possibility to couple dendrimers to antibodies and recently Patri et al. [10] reported about antibody–dendrimer complexes carrying fluorescence markers, where the antibodies still are active and selective. During former studies with a Pheophorbide a (Pheo) substituted DAB–dendrimer of third generation [11] an efficient interaction between the covalently linked Pheo moieties was observed. The chromophores in the complex undergo Fo¨rster energy transfer along the surface of the dendrimer as well as partly excitonic interaction. These interactions result in a strong reduction of photoactivity of the Pheo molecules, especially their fluorescence and singlet oxygen generation, as long as they are linked to the dendrimer. As it was shown [11] illumination of the Pheo– DAB–dendrimer, especially in the presence of oxygen, leads to a destruction of the dendrimer backbone into fragments, that carry only one or a small number of dye molecules. Because of that for PDT relevant photophysical parameters, like fluorescence and singlet oxygen quantum yield, increase reaching nearly the values of free Pheo in solution [11]. On the other hand, in recent years lots of papers deal with the use of dendrimers to mimic light harvesting complexes [12–18]. But these systems are all based on directed energy transfer between donors and acceptors of different kind only and no other interaction between donor chromophores was reported. Additionally, a comparison with these results is impossible, since no data
are given about the photostability of these complexes. Besides our former work [11] only in one paper the existence of a stronger interaction between identical dye-molecules in dendrimers was proposed [19]. Yeow et al. only observed this effect in fifth generation of porphyrin–DAB–dendrimers and no further investigation was carried out. Nevertheless, all results shown in [19] are in good agreement with the general model developed in the present paper. In a very recent work, Hasobe et al. [18] observed a reduction of electron transfer from the same porphyrin–dendrimer-complexes to C60-fullerene with increasing generation of the dendrimers. They explained this effect with steric hindering only, but since they worked with clusters of dendrimers and C60-fullerene we can assume that excitonic deactivation of the first excited singlet state of the porphyrin moieties is a major part of this effect. Very recently, we submitted a paper [20] on pyropheophorbide a on fullerene-based dendrimers were also excitonic interaction was reported between the coupled dye-molecules, which also fits into our model. For plain DAB dendrimers especially of higher generation a high cell toxicity was reported [21]. This is strongly reduced after loading with Pheo, because the amine groups, which mainly cause the toxicity, form peptide bonds with the dye molecules. This statement is supported by cell experiments that proved Pheo loaded dendrimers to have no dark toxicity [22]. On the other side, the phototoxicity on Jurkat cells was comparable or even higher than that of Pheo administered in solution [22]. After incubation of cells in vitro with the Pheo dendrimer complexes the photophysical parameters of the complexes became similar to those of free monomeric Pheo in cells. Since a good photosensitizer comprises despite high activity in the target tissue also a low activity during transport, the intact dendrimer–dye-complexes are subject of this work. The influence of the local concentration of linked chromophores in one complex on the photophysical behaviour of the chromophores and the complex photostability was investigated for the DAB–dendrimer–Pheo system. The local Pheo concentration increases with dendrimer
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
drimers of generation 0 (Diaminohexane) to 4, respectively. Their structural formula are given in Fig. 1. Due to steric hindering for dendrimers starting with P8 it becomes more and more complicated to reach a complete loading of the DAB–dendrimers with Pheo. Steric hindering and the tendency of the coupled dye molecule to fold back, as will be shown later, inhibit in principle a complete loading. For P16, for example, a maximum loading of 13 chromophores per dendrimer was achieved [11]. All investigations were done in ethanol (Pheo– DAB was dissolved in chloroform, which was diluted with ethanol in a ratio of 1:1000) at dye concentrations of 106–107 M to reach an OD of 0.2 at the excitation wavelength.
generation due to the exponential increase of available dendrimer end groups but linear increase of dendrimer surface. Molecular modelling was carried out to verify the results.
2. Materials Pheo was isolated from stinging nettles (urtica urens) according to Willsta¨tter and Stoll [23] and activated with N-hydroxy succinimide purchased from Aldrich. All solvents were of spectroscopic grade. All dendrimers have been synthesized as described in [11] with small modifications [24]. The samples are named P2, P4, P8, P16 and P32 according to the number of end groups of the den-
P2
R N
H
H
R N
P4 N
HN R
R
N H
H N
R HN R N H
N N R
N
H
N N
HR R HR H R H RN N R R N H N NH H R R HN NH N N R R N N N N H H R R N N N NH N N RH R P32 N N H H N N R R N N N N N N H H N N H H N N N N N N R R N N H H N N RH R N N H N N N N R R H H N N N N R R N N HN NH RH R H N N R N NH R H R R HR RH
R = H or pheophorbide-amoiety
H N R
H
N
N
N R
P16
H N R
H NR
N
R HN
N
N
N
N N
R
N H
NH R
R H N
H RN
N
R
N H
R HN
H NR
P8
R
297
N
N
N
N
RN H
N
N
N
HN R
N
H NR
N R H
pheophorbide-a (Pheo):
N
N H N
H N
O HO
Fig. 1. Structural formula of the investigated samples.
O
O
R NH
O
H N R R N H NH R
NR H
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
Due to the light sensitivity of the complexes they have been prepared freshly for every experiment and the quality was checked using steadystate fluorescence setup to show no significant changes. A destruction of the dendrimer backbone would lead to an increase of the fluorescence intensity [11].
3. Methods The absorption spectra were recorded using a commercial spectrophotometer Shimadzu UV160A. Time resolved fluorescence and fluorescence anisotropy measurements were carried out using time-correlated single photon counting (TCSPC), containing of an SPC 300 plug-in card (Becker Hickl, Berlin) and a multi-channel plate (Hamamatsu) described in [25]. For data analysis the decay curves were deconvoluted and fitted using the least square method based on Newton algorithm. For mono exponential fits v2-values less than 1.1 were reached. For fittings with more than one decay times, still smaller v2-values close to 1.0 were obtained. The device for steady-state luminescence measurements is based on an L-setup with different light sources (xenon lamp, Yb+ Disc-YAG pumped dye laser) and has been described previously [26]. The singlet oxygen quantum yield (UD) of all samples was determined twice using steady state (nitrogen cooled InGaAs-diode, Laser Components) and time resolved (nitrogen cooled Ge-diode, Northcoast and recording oscilloscope HP) luminescence detection as described in [27,28] to exclude the influence of different possible non-radiative deactivation pathways. Using these two different methods identical quantum yields (UD ± 0.05) for all samples were obtained. For this reason in the results section only one value of UD is given for each sample. Transient absorption measurements were performed using the pump–probe-setup described in [29]. Molecular mechanical calculations have been carried out using the program Hyperchem with forcefield MM+ in vacuum at room temperature.
4. Results 4.1. Absorption In the visible range the absorption spectra of all samples are similar to that of Pheo in ethanolic solution. With higher dendrimer generation the Soret band is broadened and the lowest Q-band is bathochromic shifted between 3 nm for P2 and 7 nm for P32 (Fig. 2 and Table 1). The extinction coefficients of the covalently linked dye are strongly reduced. For P4 to P32 the absorption is reduced by a factor of about 3 related to equal amounts of Pheo molecules in solution. A more exact determination is not possible due to statistical loading of the dendrimers. 4.2. Steady-state fluorescence For all dendrimer–dye-complexes the fluorescence spectra are shifted bathochromic (Fig. 3 and Table 1). The shift of the fluorescence maximum relative to that of Pheo increases from P2 over P4 to P8. For P16 the shift is less than for P8 and the maximum of emission of P32 is shifted only 2 nm relative to Pheo in solution. The fluorescence quantum yield is reduced for all dendrimer–dye-complexes relative to that of Pheo. Already for P2 the fluorescence intensity is reduced to 22%. It further decreases for P4 and P8 and nearly reaches zero for P16 and P32 (Table 1).
absorption (normalized)
298
1 0.8 0.6 640
0.4
660
680
700
0.2 0 300
400
500
600
700
800
wavelength [nm] Fig. 2. Absorption spectra of Pheo (thin line), P2 (black), P4 (grey), P8 (light grey), P16 (black dotted) and P32 (grey dotted) in ethanol.
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
299
Table 1 Maxima of absorption and emission of the samples under investigation, their fluorescence and singlet oxygen quantum yields relative to Pheo (UD = 0.52 ± 0.05) [27] as well as steady-state anisotropy r0 in ethanolic glass kmax (nm) (Q-absorption)
kmax (nm) (fluorescence)
UFl relative to Pheo ± 0.005
UD relative to Pheo ± 0.03
r0 at 110 K ± 0.02
Pheo
666
674
1.00
1.00
P2 P4 P8 P16 P32
669 669 670 672 673
675 680 683 680 676
0.22 0.070 0.065 0.021 0.005
0.33 0.29 0.25 0.12 0.08
0.30 0.27 0.16 0.07 0.04 0.03 0.02
(675 (700 (700 (700 (700 (700 (700
nm) nm) nm) nm) nm) nm) nm)
fluorescence (normalized)
1 0.8 0.6 0.4 0.2 0 625 650 675 700 725 750 775 800 825 850 875 900
(a)
wavelength [nm]
fluorescence (normalized)
1 0.8 0.6 0.4 0.2 0 625 650 675 700 725 750 775 800 825 850 875 900 (b)
wavelength [nm]
Fig. 3. Fluorescence spectra of (a) P2 (black), P4 (grey), and P8 (light grey); (b) P8 (black), P16 (grey) and P32 (light grey). For comparison the fluorescence of Pheo in ethanol is shown as thin line.
The initial fluorescence anisotropy has been measured in ethanolic glass at 110 K excited at 633 nm. For Pheo a value of 0.3 was obtained at
the emission wavelength at 675 nm. At 700 nm this value is decreased slightly only. For all other samples a much stronger reduction of the initial
300
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
anisotropy was observed at 700 nm and longer wavelengths – from 0.17 for P2 down to nearly zero for P32 (Table 1). At shorter wavelengths (around 675 nm – not shown) this reduction was much weaker.
0.7 ns (decay time 3) can be detected at both wavelengths. Starting with P4 a fourth, very fast decay with 0.03–0.06 ns (decay time 4) becomes dominant (Table 2). Its relative amplitude increases with dendrimer generation number.
4.3. Time-resolved fluorescence
4.4. Transient picosecond absorption
Based on the results of anisotropy measurements, it was supposed that besides monomeric Pheo a second emitter must exist in the complexes with much lower initial fluorescence anisotropy. Since the influence of this additional emitter is most pronounced at 700 nm and higher, the fluorescence decay time was determined at 675 and 700 nm. For Pheo both decay curves could be fitted mono exponential to a value of 5.7 ns, which is in a good agreement with former publications [11,30]. For all other samples at both wavelengths a slightly shorter decay time (4.4–4.7 ns) was observed (decay time 1). Such shortened lifetime has been already reported for Pheo in inhomogeneous environments [26,30]. A second faster decay with 2.0–2.6 ns (decay time 2) appears at 675 nm for P4 to P16. At 700 nm this decay time was also observed for P2 and P32. The relative amplitude of this decay time reaches its maximum for P8. For all dendrimers a third shorter decay time of 0.4–
Investigating the samples using picosecond transient absorption spectroscopy mainly three decay times were obtained. Two of them are similar to decay times 3 and 4 obtained with TCSPC. The latter one has much smaller relative amplitude than it was found in fluorescence measurements for P4 to P32 and does not exist for P2. The third decay time of ground state depletion lies between fluorescence decay time 1 and 2. It is probably a mixture of both times which cannot be resolved due to the signal/noise ratio (values not shown). For illustration the decays of the ground state depletion of P2 and P32 are shown in Fig. 4. 4.5. Singlet oxygen In general the singlet oxygen luminescence is also decreasing with rising dendrimer generation. The well-known singlet oxygen quantum yield of Pheo, 0.52 [27] reduces to 0.17 for P2 and it continues decreasing down to 0.04 for P32 (Table 1). Interestingly, for all samples the quantum yield
Table 2 Fluorescence decay times and relative amplitudes of all samples at 675 and 700 nm s1 (ns) ±0.2
A1 (rel.) ±0.3
s2 (ns) ±0.3
675 nm Pheo P2 P4 P8 P16 P32
5.72 4.52 4.48 4.61 4.68 4.75
5.0 0.5 <2 <2 <2
– – 2.55 2.46 2.14 –
700 nm Pheo P2 P4 P8 P16 P32
5.72 4.71 4.43 4.45 4.68 4.60
1.6 0.35 <2 <2 <2
– 2.63 2.20 2.16 2.14 1.96
A2 (rel.) ±0.3
1.5 2.5 1.6
1.6 1.4 2.0 1.7 0.4
s3 (ns) ±0.1
A3 (rel.) Ref.
s4 (ns) ±0.015
A4 (rel.) ±0.5
– 0.661 0.50 0.68 0.52 0.63
1 1 1 1 1
– – 0.034 0.063 0.054 0.044
1.5 3.3 3.6 3.8
– 0.66 0.48 0.70 0.52 0.42
1 1 1 1 1
– – 0.032 0.060 0.054 0.032
1.0 3.0 3.5 5.3
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306 ground state depletion
0.35
ground state depletion
0.3 0.25 0.2
301
0.33 0.31 0.29 0.27 0.25 0.23 0.21 0.19 0.17 0
0.15
500
1000
1500
2000
time [ps]
0.1 0.05 0 0
(a)
10000
15000
time [ps] ground state depletion
0.2 0.18 ground state depletion
5000
0.16 0.14 0.12 0.1
0.19 0.17 0.15 0.13 0.11 0.09 0.07 0.05 0
500
0.08
1000
1500
2000
time [ps]
0.06 0.04 0.02 0
(b)
0
5000
10000
15000
time [ps]
Fig. 4. Ground state depletion and transient emission of P2 (a) and P32 (b) as determined with TAS.
of singlet oxygen is less reduced relative to that of Pheo than that of fluorescence. For P32 the fluorescence intensity is reduced by a factor of about 200 relative to that of Pheo in ethanol, the corresponding singlet oxygen quantum yield is reduced only by a factor of 13. 4.6. Molecular modelling In Fig. 5, energetically optimized conformations of P4 and P16 with 4 and 12 chromophores, respectively, are shown. The calculations were carried out in vacuum at room temperature. These
pictures show each just one possible conformation, but they visualize an effect, that was obtained from all calculations for all samples. The covalently linked Pheo molecules show a strong tendency to fold back to the dendrimer backbone and to stack with each other. In case of higher generation dendrimers, the back folded chromophores cover some of the not substituted end groups of the dendrimers, which probably inhibit further binding at these positions. The nearest-neighbour centre-to-centre distances for the chromophores in all complexes ˚ , while the where found to be smaller than 20 A
302
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
Fig. 5. Energy optimised conformations of P4 and P16 in vacuum at room temperature. The pictures show each just one possible conformation typical for all achieved results. The dendrimers backbone is light, the Pheo moieties are dark painted.
Fo¨rster radius for the dipole-dipole resonant energy transfer between Pheo molecules was calcu˚. lated to be 62 A
5. Discussion 5.1. Interactions of chromophores in one dendrimer– dye complex The strong reduction of the fluorescence quantum yield (Section 4.2) and the appearance of up to three additional decay times (Section 4.3) for the dendrimer–dye complexes are a prove for an intense interaction between chromophores in one dendrimer–dye-complex. The reduced initial fluorescence anisotropy of the dendrimer–dye-complexes (Section 4.2) indicates, that Fo¨rster transfer occurs. But if we as-
sume Fo¨rster transfer to be the only interaction, the excitation energy can hop from one dye molecule to the next, but the fluorescence intensity should be reduced not significantly. The extremely high amplitudes of the short decay times of Pheo in the higher generation dendrimers indicate, that another interaction should exist yielding a non- or less radiative deactivation process. Pheo is known to undergo such deactivation in dimers [31]. So the reason for the non-radiative deactivation is likely to be an excitonic interaction between stacked chromophores. This conclusion is supported by the red shift and slight broadening of the S0,0 ! S1,0 absorption of the dendrimer–dyecomplexes compared to that of Pheo (Fig. 2). The possibility of dimer formation by chromophores in one dendrimer–dye-complex was proofed using molecular modelling. The calculations showed that such dimer formation can occur
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
in all dendrimer–dye-complexes (Section 4.6). Moreover, the stacking of Pheo molecules linked to the same dendrimer became dominant in all calculations. We suggest that these calculations show the general tendency of the linked Pheo molecules to fold back and to stack with each other. Since the calculations have been carried out in vacuum, in solution these effects should be reduced. Otherwise, already for P4 a nearly complete quenching of fluorescence, much stronger than observed, should occur. Due to the fact, that the calculated Fo¨rster radius for Pheo is much larger than the whole complex (Section 4.6), the stacking of just one pair of chromophores leading to excitonic interaction (trap) is enough for a very effective quenching of the whole complex. Even a nearest ˚ makes energy transfer neighbour distance of 20 A about 900 times more probable than any other processes. That means for example, in case of P4, that in the presence of one trap every energy hop originating from one of the remaining chromophores has a 50% probability to end in the trap. Nearly no fluorescence should be obtained due to the fast deactivation of the trap. Consequently, the real conformation can not be as tight as calculated in vacuum. That means also that there exist also dendrimer–dye-complexes without any trap or with non-perfect traps. Non-perfect means, that their deactivation is not that fast resulting in a certain probability of radiative deactivation or back transfer to a single chromophore in the complex. Nevertheless, the tendency of the linked Pheo molecules to stack with each other must be very strong, since already for P2 the fluorescence intensity is reduced to 0.22 of that of Pheo in solution. Because of just two chromophores in each complex no other reason for the strong reduction of the fluorescence and appearance of additional decay time is possible. With rising generation number and hence rising number of covalently linked dye molecules the probability of trap formation increases and as a result the fluorescence quantum yield decreases. 5.2. General model Several dimers of reduced porphyrins, among them pyropheophorbide a-dimers have been re-
303
ported to have a bathochromic shifted emission maximum [32–35]. This stands in a good agreement with our former findings, that Pheo molecules densely packed in Langmuir Blodgett Films also form dimers/aggregates which show a bathochromic shifted absorption and remaining emission. Such an aggregation was also observed for Pheo in mixed solutions of ethanol and water [31]. The high local concentration of Pheo moieties at the dendrimers surface yields in dimer (trap) formation and hence the emission spectrum of the dendrimer–dye-complexes is a superposition originating from single chromophores and from traps. While the fluorescence of single chromophores will be more pronounced at 675 nm the remaining trap emission will be visible more intense at 700 nm. For this reason the time-resolved fluorescence was measured at these two wavelengths. 5.2.1. Decay time 1 This decay time was observable at both registration wavelengths for all complexes. For P2 at 675 nm it is the strongest decay component. This decay time can be assigned to chromophores in the complexes where no energy traps were formed. Since the distances between the chromophores in one complex are very small, energy transfer processes should occur very fast. But, as already pointed out in Section 5.1, Fo¨rster transfer between identical chromophores without traps does not influence the fluorescence decay time. 5.2.2. Decay time 4 In the presence of one or more traps in a complex an additional decay time arises, which represents the probability of energy transfer to the trap. For a very rough estimation of this additional decay time a random walk of the energy between a set of 20 chromophores was analyzed. If two of these molecules form a trap, they are very close to each other and can be approximated as one acceptor. For simplicity it is assumed, that every of the remaining 18 chromophores as well as the trap have five nearest neighbours, all at equal distances. Due to the r6 dependency Fo¨rster transfer practically happens only to these nearest neighbours. The probability of the trap to be among
304
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
the five neighbours is about 1/3. If we approximate, that the transfer probabilities to a monomer and the trap are similar [36], than the probability pTrap of every energy hop to go to the trap is 1/15. Once caught by the trap a back transfer is very unlikely due to the red-shifted emission of the trap, its low fluorescence quantum yield and the reduced fluorescence lifetime due to non-radiative deactivation. In general the additional decay time s caused by transfer to a trap is given by s ¼ i sHop , where sHop is the time for one energy hop, which in this simple approximation is just 1/5 of the Fo¨rster transfer time sFT between two of these chromophores [37]. Here, i represents the number of transfer hops for which the probability of not being caught by the trap is 1/e 1 i ¼ ð1 pTrap Þ : e For a set of 20 chromophores with one trap we get approximately 33 hops and a decay time of s = 6.6 Æ sFT. ˚ centre to centre distance of two Pheo For a 20 A molecules the Fo¨rster transfer time is about 0.006 ns giving a value of 0.04 ns for the additional decay time caused by energy transfer to the trap in this approximation. These simple calculations already demonstrate that the fourth decay time describes the energy transfer process to the trap. This explains, why this decay time is only much weaker visible in transient absorption (Section 4.4). It does not represent a transfer to the ground state but a transfer to chromophores with lower extinction. It also explains, why it does not appear in P2, because such transfer process is impossible. The two chromophores in P2 can form a trap or behave like monomers. 5.2.3. Decay times 2 and 3 Basing on the absence of an energy transfer to traps in P2 the decay times 2 and 3 assign to the traps themselves. It is known [32–35], that the fluorescence decay times of dimers can strongly differ due to the relative orientation of the dipole mo-
ments of the two chromophores. Because of this fact we suggest that the shorter decay time 3 originates from the trap (trap 1) with more parallel oriented, while decay time 2 represents a more or less tilted trap (trap 2). The big angle between the dipole moments of the two partners in such a tilted trap prevents a more intense interaction yielding in a longer decay time. So the fluorescence of trap 2 is much stronger compared with the one of trap 1. Carrying out molecular mechanical calculations pairs of chromophores with more or less parallel as well as nearly perpendicular relative orientation of the dipole moments have been found (Fig. 5). The relative amplitude of decay time 2 is decreased in P32. In our model this fact can be explained with the higher probability of multiple stacking and the higher probability of a parallel orientation of chromophores yielding in formation of trap 1. At 675 nm this decay time is not observed due to the lower intensity of trap emission and stronger emission of single chromophores at this wavelength. This also fits with the picture found for P2. Here, the probability of non-interacting dye molecules is much higher and so decay time 1 is much more pronounced than the quite similar decay time 2, which then can not be distinguished during fitting procedure. 5.2.4. Shift of steady-state fluorescence Since the emission from trap 2 is much stronger than that from trap 1, it is not surprising, that the steady-state emission of P4, P8, and P16 where both types of traps are present is more bathochromic shifted than for P32, where the formed traps are mainly of type 1. The emission from trap 1 is so weak, that even the short time of energy transfer from the remaining single chromophores to the traps enables sufficient monomer emission to override the trap emission. 5.2.5. Anisotropy reduction The higher the number of linked dye molecules in a complex the stronger the reduction of anisotropy due to the higher probability of energy transfer (Section 4.2 and Table 1) should be. The anisotropy of P2 is slightly more than half of that of Pheo in solution at 675 nm. Such reduction is expected for a random orientation of two linked
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
Pheo molecules in presence of Fo¨rster transfer. The probability of emission from the originally excited molecule is nearly the same as for the second molecule. With rising number of chromophores in the complex the probability for an emission from any randomly oriented chromophore increases. The majority of the energy is caught by the traps and since they can have any orientation relative to the excited chromophore, the anisotropy is reduced especially at wavelengths, where the traps emit. At shorter wavelengths the fluorescence mainly originates from single chromophores and their number is reduced due to trap formation. Hence at these wavelengths the anisotropy is not reduced that strong. In Fig. 6, a schematic sketch of the transfer processes in a dendrimer–dye-complex is given. 5.2.6. Singlet oxygen As it was pointed out above, the majority of the absorbed energy in the dendrimer–dye complexes is caught by traps yielding mainly in an efficient non-radiative deactivation. But all samples, even P32 generate singlet oxygen. For all complexes the fluorescence quantum yield has been reduced stronger than that of singlet oxygen. As a consequence not only the single chromophores but also the traps should undergo intersystem crossing to allow further energy transfer to molecular oxygen. But, as it was found using transient absorption spectroscopy (Fig. 4) the ISC quantum yield of the complexes is much smaller
Fig. 6. Schematic sketch of a dendrimer–dye-complex to illustrate the proposed energy transfer processes between dye molecules in one complex.
305
(UISC < 0.2 already for P2) than that of the dye in solution (0.6 ± 0.05). For this reason the singlet oxygen quantum yield is reduced in comparison to Pheo in solution, but not as strong as the fluorescence quantum yield.
6. Conclusions Pheo molecules covalently linked to DAB dendrimers undergo very efficient energy transfer processes, Fo¨rster-transfer as well as partly excitonic interaction. These processes already occur in the case of Diaminohexane coupled Pheo where only two chromophores are within each complex. The covalently linked Pheo molecules show a tendency to fold back to the dendrimer and to stack with neighbouring molecules. Such close contact gives rise to excitonic interaction yielding in non-radiative deactivation of such stacked chromophores. The shortening of decay time caused by this interaction depends on the position of the involved chromophores relative to each other. While those with parallel oriented dipole moments are very short lived, other pairs of Pheo molecules with no parallel orientation of the dipole moments have about half the decay time of isolated chromophores in the complexes and a stronger remaining fluorescence. Since the Fo¨rster radius of energy transfer between Pheo molecules is longer than the overall diameter of the complex every pair of excitonic interacting chromophores acts like an energy trap for the whole complex. Due to the small nearest neighbour distances the transfer velocity of energy to the traps is about two orders of magnitude higher than that of radiative relaxation of the chromophores. As a consequence the quantum yields of fluorescence, inter system crossing and singlet oxygen are strongly reduced in all complexes. The probability of trap formation increases with the number of coupled dyes and hence with generation number of the dendrimer and so does the effect of these traps. This yields in a nearly complete quenching of fluorescence in case of P32. Nevertheless, the traps should undergo inter system crossing and so some singlet oxygen is generated even for highest generation.
306
S. Hackbarth et al. / Optics Communications 248 (2005) 295–306
References [1] D. Dolphin, Can. J. Chem. 72 (1994) 1005. [2] B. Ro¨der, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry: Instrumentation and Applications, Wiley, Chinchester, 2000. [3] Y.N. Konan, R. Gurny, E. Alle´mann, J. Photochem. Photoboil. B 66 (2002) 89. [4] C. Dressler, U. Mo¨ller, T. Lewald, H.-P. Berlien, B. Ro¨der, H.-J. Risse, Laser Med. Sci. 7 (1992) 174. [5] J.G. Moser, A. Heuermann, P. Oehr, H. Scheer, A. Vervoorts, S. Andrees, SPIE Proc. 2325 (1994) 92. [6] B. Ro¨der, S. Hackbarth, G. Wo¨hlecke, US-Patent, NDN 172-0037-2622-7. Publication Nr. 00108704 WO, German Patent Nr. 199 36 997.6 02.08.1999. [7] G.J. Dykes, Chem. Technol. Biotechnol. 76 (2001) 903. [8] M. Fischer, F. Vo¨gtle, Angew. Chem. Int. Ed. 38 (1999) 884. [9] J.C. Roberts, Y.A. Adams, D. Tomalia, J.A. MercerSmith, D.K. Lavalee, Bioconjugate Chem. 1 (1990) 305. [10] A.K. Patri, T. Thomas, J.R. Baker, N.H. Bander, Polym. Mater. Sci. Eng. 86 (2002) 130. [11] S. Hackbarth, V. Horneffer, A. Wiehe, F. Hillenkamp, B. Ro¨der, Chem. Phys. 269 (2001) 339. [12] V. Balzani, P. Ceroni, M. Maestri, V. Vicinelli, Curr. Opin. Chem. Biol. 7 (2003) 657. [13] M. Cotlet, R. Gronheid, S. Habuchi, A. Stefan, A. Barbafina, K. Mu¨llen, J. Hofkens, F.C. De Schryver, JACS 125 (2003) 13609. [14] B.L. Davis, J.S. Melinger, D. McMorrow, Z. Peng, Y. Pan, J. Lumin. 106 (2004) 301. [15] D. Rana, G. Gangopadhyay, J. Chem. Phys. 118 (2003) 434. [16] P.R. Hania, D.J. Heijs, T. Bowden, A. Pugzlys, J. van Esch, J. Knoester, K. Duppen, J. Phys. Chem. B 108 (2004) 71. [17] M.R. Shortreed, S.F. Swallen, Z.-Y. Shi, W. Tan, Z. Xu, C. Devadoss, J.S. Moore, R. Kopelmann, J. Phys. Chem. B 101 (1997) 6318. [18] T. Hasobe, Y. Kashiwagi, M.A. Absalom, J. Sly, K. Hosomizu, M.J. Crossley, H. Imahori, P.V. Kamat, S. Fukuzumi, Adv. Mater. 16 (2004) 975. [19] E.K.L. Yeow, K.P. Ghiggino, J.N.H. Reek, M.J. Crossley, A.W. Bosman, A.P.H.J. Schenning, E.W. Meijer, J. Phys. Chem. B 104 (2000) 2596.
[20] E. Ermilov, S. Hackbarth, S. al Omari, M. Helmreich, N. Jux, A. Hirsch, B. Ro¨der, Opt. Commun. (2004) submitted. [21] N. Malik, R. Wiwattanapatapee, R. Klopsch, K. Lorenz, H. Frey, J.W. Weener, E.W. Meijer, W. Paulus, R. Duncan, J. Control. Release 65 (2000) 133. [22] A. Paul, S. Hackbarth, A. Mo¨lich, C. Luban, S. Oelckers, F. Bo¨hm, B. Ro¨der, Laser Phys. 13 (2003) 22. [23] A. Willsta¨tter, R. Stoll, Untersuchungen u¨ber Chlorophyll, Springer, Berlin, 1913. [24] All the dendrimers have been synthesized by adding 200 mg Pheophorbide a succinimide ester to the unmodified DAB dendrimer (DSM) in 20 ml DCM in a molar ratio of 6:1. A small amount (5 drops) of triethylamine has been added as well as 100 mg dicyclocarbodiimide for deprotonation of the dendrimer and catalization. After stirring for 4 days at room temperature in the dark the product was dried an rinsed several times with acetone and supersonic. [25] J. Zimmermann, J.v. Gersdorff, H. Kurreck, B. Ro¨der, J. Photochem. Photobiol. B 40 (1997) 209. [26] O. Korth, T. Hanke, I. Ru¨ckmann, B. Ro¨der, Exp. Tech. Phys. 41 (1995) 25. [27] W. Spiller, H. Kliesch, D. Wo¨hrle, St. Hackbarth, B. Ro¨der, J. Porphyr. Phthalocynaines 2 (1998) 145. [28] St. Oelckers, M. Sczepan, Th. Hanke, B. Ro¨der, J. Photochem. Photobiol. B 39 (1997) 219. [29] A. Zeug, I. Ru¨ckmann, B. Ro¨der, J. Opt. B 3 (2001) 251. [30] B. Ro¨der, T. Hanke., S. Oelckers, S. Hackbarth, C. Symietz, J. Porphyr. Phthalocynaines 4 (2000) 37. [31] I. Eichwurzel, H. Stiel, B. Ro¨der, J. Photochem. Photobiol. B 54 (2000) 124. [32] A. Osuka, Y. Wada, S. Shinoda, Tetrahedron 52 (1996) 4311. [33] A. Osuka, K. Maruyama, JACS 110 (1988) 4454. [34] E.I. Zenkevich, V.N. Knyukshto, A.M. Shulga, V.A. Kuzmitsky, V.I. Gael, E.G. Levinson, A.F. Mironov, J. Lumin. 75 (1997) 229. [35] A.N. Kozyrev, V. Suresh, S. Das, M.O. Senge, M. Shibata, T.J. Dougherty, R.K. Pandey, Tetrahedron 56 (2000) 3353. [36] Due to the better overlap of the bathochromic shifted trap absorption with the monomer fluorescence spectrum the transfer probability is increased, but on the other hand decreased due to the lower extinction of the traps. [37] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic Publishers, New York, 1999.