pubs.acs.org/Langmuir © 2009 American Chemical Society
Inhibition of Aggregation of [Pd]-Bacteriochlorophyllides in Mesoporous Silica J€org Dandler and Hugo Scheer* Department Biologie I - Botanik, Universit€ at M€ unchen, Menzinger Strasse 67, 80638 M€ unchen, Germany Received July 28, 2009. Revised Manuscript Received September 9, 2009 Aggregation is a major factor affecting the photophysical properties of chlorophylls. For two [Pd]-bacteriochlorophyll derivatives that are currently under clinical testing as sensitizers for photodynamic therapy, aggregation control in aqueous solution has been studied with folded-sheet mesoporous silica (FSM) of different pore sizes (20, 45, 83 A˚) and with detergent (Triton X-100). With both the moderately polar WST09 and the highly polar WST11, no pigment oligomers were formed in FSM, and the monomer-dimer equilibrium was shifted toward the monomer with decreasing pore diameter.
Introduction Porphyrins and, in particular, chlorophylls show complex aggregation in polar as well as in nonpolar solvents.1-4 These aggregates often exhibit dramatically changed photophysics; research into the understanding and control of the aggregation behavior has therefore been substantial not only for photosynthesis research but also in applying chlorophylls as sensitizers in photodynamic therapy (PDT). Aggregation, besides inducing band shifts of the major optical transitions and the reduction of excited-state lifetimes, is therefore deleterious for both photosynthesis, where the efficiency of light-harvesting is proportional to the 1S lifetime, and PDT, where long 1S lifetimes allow for increased intersystem crossing and, consequently, the increased generation of reactive oxygen species.5-10 Aggregation can be controlled by the choice of solvent: examples are disaggregation by the addition of ligating solvents2 or amphiphiles.1,3,11-13 A second approach to aggregation control is chemical modification by introducing functional groups *Corresponding author. E-mail:
[email protected]. Phone: þ49-8917861-295. Fax: þ49-89-81099334.
(1) Katz, J. J.; Bowman, M. K.; Michalski, T. J.; Worcester, D. L. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; pp 211-236. (2) Katz, J. J.; Shipman, L. L.; Cotton, T. M.; Janson, T. R. In The Porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; pp 402-458. (3) Scherz, A.; Rosenbach-Belkin, V.; Michalski, T. J.; Worcester D. L. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; pp 237-268. (4) Cosma, P.; Longobardi, F.; Agostiano, A. J. Electroanal. Chem. 2004, 564, 35–43. (5) Damoiseau, X.; Tfibel, F.; Hoebeke, M.; Fontaine-Aupart, M.-P. Photochem. Photobiol. 2002, 76, 480–485. (6) Damoiseau, X.; Schuitmaker, H. J.; Lagerberg, J. W.; Hoebeke, M. J. Photochem. Photobiol. B 2001, 60, 50–60. (7) Hoebeke, M.; Damoiseau, X.; Schuitmaker, H. J.; Van de Vorst, A. Biochim. Biophys. Acta 1999, 1420, 73–85. (8) Borisov, S. M.; Blinova, I. A.; Vasil0 ev, V. V. High Energy Chem. 2002, 36, 189–192. (9) Tanielian, C.; Schweitzer, C.; Mechin, R.; Wolff, C. Free Radical Biol. Med. 2001, 30, 208–212. (10) Tanielian, C.; Heinrich, G. Photochem. Photobiol. 1995, 61, 131–135. (11) Gottstein, J.; Scheer, H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 2231–2234. (12) Dentuto, P. L.; Catucci, L.; Cosma, P.; Fini, P.; Agostiano, A.; Hackbarth, S.; Rancan, F.; Roeder, B. Bioelectrochemistry 2007, 70, 39–43. (13) Gottstein, J.; Scherz, A.; Scheer, H. Biochim. Biophys. Acta 1993, 1183, 413– 416. (14) Oba, T.; Tamiaki, H. Photochem. Photobiol. 1998, 67, 295–303. (15) Balaban, T. S.Linke-Schaetzel, M.Bhise, A. D.Vanthuyne, N. a. R. C. Eur. J. Org. Chem. 2004, 3919-3930. (16) Tamiaki, H. Photochem. Photobiol. Sci. 2005, 4, 675–680.
11988 DOI: 10.1021/la902767x
that enhance solute-solute interaction14-16 or by introducing polar groups that enhance solubility in water.17,18 A third approach to aggregation control is spatial confinement; this is the case in most chlorophyll proteins, where aggregation is limited and controlled by the protein and additional cofactors.19 Chlorophylls and their derivatives can also only be incorporated in their monomeric state in heme proteins such as myoglobin.20,21 An alternative to biopolymers in spatial control are synthetic structures that contain cavities of defined size. Chlorophylls form cyclodextrin complexes, but in this case, the pigment does not fit into the cavity.12 Larger cavities are present in mesoporous materials such as folded-sheet mesopores silica (FSM): they contain hexagonal tubes of a well-defined constant diameter that cross the FSM from one side to the other.22-25 The pore diameter of FSM can be adjusted during manufacturing in the range of 20-100 A˚; the materials have high specific surface areas (e1500 m2/g) and pore volumes (e1.5 mL/g).26 The aggregation of chlorophylls is complex: in hydrophobic environments, it is dominated by interactions of peripheral groups of one molecule with peripheral substituents (mainly CdO groups) of the binding partner. In hydrophilic environments, such interactions are less dominant, as shown by the aggregation of pheophorbides27 and Pd complexes28 in Triton (17) Brandis, A.; Salomon, Y.; Scherz, A. In Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics, Functions and Applications; Grimm, B., Porra, R., R€udiger, W., Scheer, H., Eds.; Springer: Dordrecht, The Netherlands, 2006; pp 485-494. (18) Pandey, R. K.; Goswami, L. N.; Chen, Y.; Gryshuk, A.; Missert, J. R.; Oseroff, A.; Dougherty, T. J. Lasers Surg. Med. 2006, 38, 445–467. (19) Green, B., Parson, W. Light-Harvesting Antennas in Photosynthesis; Kluwer: Dordrecht, The Netherlands, 2003. (20) Kuki, A.; Boxer, S. G. Biochemistry 1983, 22, 2923–2933. (21) Pr€oll, S.; Wilhelm, B.; Robert, B.; Scheer, H. Biochim. Biophys. Acta 2006, 1757, 750–763. (22) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.; Schmitt, K. D.; Chu, C. T. W.; Olson, D. H.; Sheppard, E. W.; et al. J. Am. Chem. Soc. 1992, 114, 10834–10843. (23) Inagaki, S., Fukushima, Y., Kuroda, K. J. Chem. Soc., Chem. Commun. 1993, 680-682. (24) Yang, P.; Zhao, D.; Margolese, D. I.; Chmelka, B. F.; Stucky, G. D. Nature 1998, 396, 152–155. (25) Inagaki, S.; Guan, S.; Ohsuna, T.; Terasaki, O. Nature 2002, 416, 304–307. (26) Fukushima, Y.; Kajino, T.; Itoh, T. Curr. Nanosci. 2006, 2, 211–218. (27) Scheer, H.; Paulke, B.; Gottstein, J. In Optical Properties and Structure of Tetrapyrroles; Blauer, G., Sund, H., Eds.; De Gruyter: London, 1985; pp 507-521. (28) Limantara, L.; Koehler, P.; Wilhelm, B.; Porra, R. J.; Scheer, H. Photochem. Photobiol. 2006, 82, 770–780.
Published on Web 09/22/2009
Langmuir 2009, 25(20), 11988–11992
Dandler and Scheer
Letter
Figure 1. Molecular structures of WST09 and WST11. WST09 is generally present as a 132-epimer mixture; only the 132R epimer is shown, which is present in large excess over the 132S epimer.
X-100: the former lack the central Mg2þ, and in the latter, it is replaced by Pd2þ that is only four-coordinate and binds no extra ligands in [Pd]-chlorophylls and [Pd]-bacteriochlorophylls. [Pd]-(bacterio)chlorophylls (abbreviations and structures, see Figure 1) are currently under investigation for applications in photodynamic therapy and diagnostics. Because they are chemically and photochemically more stable than the parent pigments, they are also of interest for solar energy conversion; in all cases, it is relevant to control aggregation. During pharmacokinetic studies, we observed aggregation in human blood plasma.29 We now report that the aggregation of such pigments can be controlled by incorporation into FSM-type silica with pore diameters of 20, 45, and 83 A˚.
Materials and Methods Chemicals were analytical grade or better and purchased from Sigma-Aldrich (Taufkirchen, DE), Roth (Karlsruhe, DE), or Merck (Darmstadt, DE). Doubly demineralized water was used for all aqueous solutions. WST09 and WST11 were gifts from Negma-Lerads (Magny-Les-Hameaux, FR) and A. Scherz (Weizmann Institute of Science, Rehovot, Israel), respectively. They were stored dry, in the dark and under argon at -20 °C. All experiments were carried out under green light (95%) was in the monomeric state (λmax = 764 nm).29 Aggregates in Aqueous Solutions. The three aggregation states of WST09 were also attained in an aqueous detergent system; the relative concentrations varied depending on the amount of detergent Triton X-100 used (Supporting Information, Figure S1). By stepwise increments in the detergent concentration, the WST09 oligo-/polymers (Qy = 913 nm) were first converted to WST09 dimers (Qy = 820 nm) and then to WST09 monomers (Qy =761 nm). The disaggregation of the pigment dimers started just above the critical micelle concentration (cmc) of Triton X-100 (0.016% v/v).30 The Qx absorption band showed pronounced hypochromism upon pigment aggregation. Populations with comparable spectra and assigned to the same aggregation states were reported for BChl a in aqueous solutions of Triton X-100.11,27,31 Unlike chlorophyll aggregation in organic solvents,2 aggregation in aqueous systems does not require coordination to the central metal; therefore, it also occurs with Pd complexes that do not bind extra ligands.32 (30) Mohr, A.; Talbiersky, P.; Korth, H.-G.; Sustmann, R.; Boese, R.; Blaeser, D.; Rehage, H. J. Phys. Chem. B 2007, 111, 12985–12992. (31) Scherz, A.; Parson, W. W. Biochim. Biophys. Acta 1984, 766, 653–665. (32) Hartwich, G.; Fiedor, L.; Simonin, I.; Cmiel, E.; Schaefer, W.; Noy, D.; Scherz, A.; Scheer, H. J. Am. Chem. Soc. 1998, 120, 3675–3683.
DOI: 10.1021/la902767x
11989
Letter
Figure 3. Absorption spectra of WST09 adsorbed to FSM with pore diameters of (a) 20, (b) 45, and (c) 83 A˚. The spectra were recorded 0, 1, and 24 h after the dried WST09-FSM conjugates had been suspended in 50% (v/v) aqueous glycerol. The absorption spectrum of WST09 in toluene is given for comparison in plot a. Deconvolution yields the following monomer/dimer ratios: 20 A˚: 26.0 f 17.2 f 6.0; 45 A˚: 0.44 f 0.34 f 0.26; 83 A˚: 0.079 f 0.074 f 0.067 after 0 f 1 f 24 h of incubation.
Aggregates in FSM. WST09 has four narrow absorption bands in toluene at 333 (By), 387 (Bx), 534 (Qx), and 764 nm (Qy) (Figure 3a). After incubation with FSM, most of the pigment was bound to the particles. After centrifugation, the supernatant (toluene) contained no pigment in the case of 20 and 45 A˚ FSM and only ∼1% of the original pigment content in the case of 83 A˚ FSM. The absorption spectra of the respective WST09-FSM conjugates were quite different in the NIR region for these three preparations (Figure 3). In 20 A˚ FSM, the absorption maxima of WST09 (By = 333, Bx = 379, Qx = 533, and Qy = 761 nm) were shifted only slightly relative to their positions in toluene (Figure 3a), indicating an 11990 DOI: 10.1021/la902767x
Dandler and Scheer
environment of similar polarity. The somewhat larger apparent hypsochromic shift of the Bx band is due to its loss of intensity, and deconvolution results in the same value (390 ( 1 nm) for this band in WST09 in all FSM samples and at 387 nm in toluene solution. All absorption bands were considerably broadened, however, compared to those of a toluene solution. The fwhh of the Qy absorption band, for example, tripled from 29 nm (497 cm-1) in toluene to 88 nm (1525 cm-1) in 20 A˚ FSM. Broadening is also obvious in the Soret region; assuming only monomers at early times, Bx increases from 28 nm (1872 cm-1) in toluene to 56 nm (3675 cm-1) in 20 A˚ FSM, and By increases from 35 nm (3165 cm-1) to 55 nm (5150 cm-1). A satisfactory deconvolution of mixed populations was not possible in this region because of the overlap of the Bx and By bands. Upon standing for 24 h at 4 °C in the dark, the bandwidth increased further, for example, to 98 nm (1690 cm-1) for the Qy band, and this additional broadening was most pronounced on the longwavelength side, whereas the positions of all absorption bands remained nearly constant. Deconvolution of the Qy bands resulted in a decrease of the monomer/dimer ratio from 26.0 to 6.0 within 24 h; during this time, the aggregation states were equilibrated. When WST09 was incorporated into 45 A˚ FSM, the positions of the By and Qx bands were nearly unchanged as compared to the conjugates with 20 A˚ FSM (Figure 3b). The Bx band was very weak so that only a small shoulder at ∼385 nm remained, but its position upon deconvolution was unchanged. The only significant shift was that of the Qy band from λmax =761 to 771 nm, but this is due to changes in the monomer/dimer ratio, and the position of the deconvoluted band remains at 761 nm. There was again an increase in the quantity of dimers (λmax,app = 814 nm, λmax,deconv = 825 nm) upon standing: a red shoulder is already clearly visible at t = 0, which rises within 24 h to a distinct absorption band. An isosbestic point (∼785 nm) in the Qy region suggests a direct interconversion of monomers and dimers; the maxima of the other absorption bands remained unchanged. The absorption of WST09 in 83 A˚ FSM immediately after preparation is similar to that in 45 A˚ FSM after 24 h, indicating mainly the presence of dimers; the Qy band was slightly redshifted to 821 nm within the next 24 h (Figure 3c). In none of the WST09-FSM preparations were there any absorption bands indicative of oligo-/polymers (λmax ∼915 nm, see above); this was supported by spectral deconvolution that never required such a band. Furthermore, spectral deconvolution revealed the absorption maxima of monomers and dimers at ∼760 and ∼825 nm, respectively, and similar bandwidths for all conjugates. WST11 has four absorption bands in aqueous solution at 330 (By), 380 (Bx), 521 (Qx), and 747 nm (Qy) (Supporting Information, Figure S2). The bands remained stable over 24 h, except that a small fraction was oxidized to the corresponding chlorin derivative (Qy = 641 nm), despite storing the solution at 4 °C in the dark; this probably relates to the lower oxidation potential of this pigment.33 The binding of WST11 to FSM was much weaker than that of WST09, and a considerable fraction remained in the supernatants, with absorptions that were nearly identical to that in an FSM-free aqueous solution (Figure 4a). In the presence of 20 and 45 A˚ FSM, there were only minor absorption decreases (∼7 and ∼14%, respectively) during the first 3 h; in contrast, in the presence of 83 A˚ FSM a major absorption decrease of ∼45% occurred within this time. There (33) Ashur, I.; Goldschmidt, R.; Pinkas, I.; Salomon, Y.; Szewczyk, G.; Sarna, T.; Scherz, A. J. Phys. Chem. 2009, 113, 8827–8837.
Langmuir 2009, 25(20), 11988–11992
Dandler and Scheer
Figure 4. Absorption spectra of WST11 after incubation of an aqueous solution with FSM of different pore diameters (20, 45, and 83 A˚) for 24 h. (a) Aqueous supernatants of centrifuged samples and original solution after 24 h. (b) Pellets with WST11-FSM conjugates suspended in 50% (v/v) aqueous glycerol.
was no further WST11 adsorption to the FSM over the next 21 h, and the oxidation to the chlorine derivative was similar (∼12 mOD/h) in the presence and absence of FSM. The spectra of the WST11-FSM conjugates with different pore sizes were very similar-they showed no indication of aggregate formation-and the maxima of all absorption bands were only slightly shifted relative to the respective supernatants containing monomers (Figure 4b). Quantitatively, the Qy band intensities of the spectra reflect increased binding with increasing pore size at ratios of approximately 1.0 (20 A˚ FSM) to 2.0 (45 A˚ FSM) to 6.5 (83 A˚ FSM). No pigment was adsorbed on any FSM from a methanolic solution of WST11 over a 24 h period.
Discussion Disaggregation of WST09 is induced in whole blood,34 isolated blood fractions, and liposomes.29,34,35 In all cases, the aggregation equilibrium is shifted toward the monomer (λmax ≈ 760 nm), and large aggregates (λmax > 900 nm) were absent. The formation of large aggregates is also inhibited in FSM; aggregation is restricted to the monomers (M760) and dimers (D825). Molecular modeling indicates that in a tube with a 20 A˚ inner diameter there is insufficient space for aggregation that involves stacking of the tetrapyrrole macrocycles such as found in the special pair of the (34) Weersink, R. A.; Bogaards, A.; Gertner, M.; Davidson, S. R. H.; Zhang, K.; Netchev, G.; Trachtenberg, J.; Wilson, B. C. J. Photochem. Photobiol., B 2005, 79, 211–222. (35) Vakrat-Haglili, Y.; Weiner, L.; Brumfeld, V.; Brandis, A.; Salomon, Y.; McIlroy, B.; Wilson, B. C.; Pawlak, A.; Rozanowska, M.; Sarna, T.; Scherz, A. J. Am. Chem. Soc. 2005, 127, 6487–6497.
Langmuir 2009, 25(20), 11988–11992
Letter
reaction center,36 in the light-harvesting complexes of purple bacteria,37 or in aggregates in aqueous solution.3 The diameter of a tetrapyrrole macrocycle is 12 A˚, which increases to 14 A˚ in the presence of (short) side chains.38 If one considers, in addition, the vertical extension of the π system of ∼3.5 A˚ and the noncoplanar side chains at C-7, C-8, C-132, C-17, and C-18, then a staggeredtype interaction of the two macrocycles is sterically inhibited. This is reflected by the near absence of red-shifted absorption maxima in 20 A˚ FSM pigment preparations. (According to deconvolution, the increase in bandwidth on the red side of the Qy band reflects a small amount of aggregation (see legend of Figure 3 for quantification), which can possibly occur at the outer FSM surface, at the end of the tubular pores, or with broken material.) In the FSM with larger pores, aggregation is sterically possible but still considerably reduced as compared to the situation in solution: larger aggregates (λmax >860 nm) are completely absent, and in the 45 A˚ FSM there is even a large fraction present as monomers. This is quite remarkable because the pigment concentration in the FSM pores is more than 2 orders of magnitude higher than in the applied toluene solution. On the basis of pore volumes of 0.65, 0.89, and 1.22 mL/g for the 20, 45, and 83 A˚ FSM, respectively (private communication with T. Kajino, Toyota Central R & D Laboratories, 2009), the concentration increases are 460-fold, 340-fold, and 250-fold. This is probably explained by relatively tight pigment binding to the moderately hydrophobic FSM surface26 that competes efficiently with pigment-pigment interactions that, in aqueous solution, are also largely hydrophobic.1,11,27,31 In all three cases, there is still only a low surface coverage of
