Fluorescence of Amphotericin B-Deoxycholate (Fungizone) Monomers

Fluorescence excitation and emission spectra are reported for the polyene macrolide antifungal agent Amphotericin. B formulated as micellar dispersion...
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Articles Fluorescence of Amphotericin B-Deoxycholate (Fungizone) Monomers and Aggregates and the Effect of Heat-Treatment Robin Stoodley,† Kishor M. Wasan,‡ and Dan Bizzotto*,† AdVanced Materials and Process Engineering Laboratory, Department of Chemistry, UniVersity of British Columbia, VancouVer, British Columbia, V6T 1Z1, Canada, and DiVision of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, UniVersity of British Columbia, VancouVer, British Columbia, V6T 1Z3, Canada ReceiVed March 23, 2007. In Final Form: May 23, 2007 Fluorescence excitation and emission spectra are reported for the polyene macrolide antifungal agent Amphotericin B formulated as micellar dispersion Fungizone (FZ) and its modified counterpart heat-treated Fungizone. The addition of sodium dodecyl sulfate or sodium deoxycholate surfactant to modulate the aggregation state of Amphotericin B confirms that the monomer and dimer states have different fluorescence spectra. Energy transfer from excited dimer to monomer is observed. Both FZ and heat-treated FZ (HTFZ) show expected S1 f S0 fluorescence emission as well as anti-Kasha fluorescence emission from the S2 state. The excitation and S1 f S0 emission spectra of HTFZ are similar to those of FZ, while the S2 f S0 fluorescence differs in intensity between them. The variation in the rate constant for internal conversion from S2 to S1 as the surfactant concentration is increased differs for FZ and HTFZ; we propose that this may form a new basis for examining the super-aggregated character of AmB preparations. FZ and HTFZ have a similar stability to disaggregation by added sodium dodecyl sulfate surfactant. These findings provide the groundwork for future fluorescence characterization of FZ or HTFZ interactions with cell membranes.

Introduction Amphotericin B (Figure 1; AmB) is a polyene macrolide antibiotic widely used in the treatment of systemic fungal infections. Several different AmB formulations exist, but the AmB-deoxycholate micellar dispersion commercially known as Fungizone (FZ; Squibb Canada) remains the most widely used due to its relatively low cost and good availability. AmB is thought to bind to ergosterol in the fungal membrane, forming pores and leading to leakage of intracellular components and cell death. Undesired binding to cholesterol in the host cell membrane results in dose-dependent kidney toxicity, limiting AmB’s use. Sterol binding selectivity depends on AmB’s aggregation state.1-3 Less toxic AmB formulations have been developed but are expensive. Interest in FZ has been renewed due to reports that the simple heating of AmB preparations may decrease its toxicity while maintaining efficacy by promoting the formation of so-called super-aggregates.4-6 Heat-treated FZ (HTFZ) has been shown to decrease renal cytotoxicity in human and animal models while maintaining antifungal activity.7-10 †

Department of Chemistry. Faculty of Pharmaceutical Sciences, Division of Pharmaceutics and Biopharmaceutics. ‡

(1) Bolard, J.; Legrand, P.; Heitz, F.; Cybulska, B. Biochemistry 1991, 30, 5707-5715. (2) Gruda, I.; Gauthier, E.; Elberg, S.; Brajtburg, J.; Medoff, G. Biochem. Biophys. Res. Commun. 1988, 154, 954-958. (3) Brajtburg, J.; Gruda, I.; Daigle, I.; Medoff, G. Biochim. Biophys. Acta 1989, 985, 307-312. (4) Ernst, C.; Dupont, G.; Rinnert, H.; Lematre, J. Compt. Rend. L’Acad. Sci. Paris 1978, 286, 175-178. (5) Gaboriau, F.; Cheron, M.; Leroy, L.; Bolard, J. Biophys. Chem. 1997, 66, 1-12. (6) Gaboriau, F.; Cheron, M.; Petit, C.; Bolard, J. Antimicrob. Agents Chemother. 1997, 41, 2345-2351. (7) Leon, C.; Taylor, R.; Bartlett, K. H.; Wasan, K. M. Int. J. Pharm. 2005, 298, 211-218.

Figure 1. Chemical structures of AmB and sodium deoxycholate. Numbered carbon atoms on AmB are salient to discussion of possible fluorescent contaminants.

HTFZ also has been shown to be potent toward the malariacausing parasite Plasmodium falciparum.11 FZ and HTFZ are thought to differ only in supramolecular structure.12 Heattreatment has been shown to increase the AmB aggregate mass by several 100-fold over the aggregate size in FZ.4 In addition, (8) Sivak, O.; Bartlett, K.; Wasan, K. M. Pharmaceut. Res. 2004, 21, 15641566. (9) Kwong, E. H.; Ramaswamy, M.; Bauer, E. A.; Hartsel, S. C.; Wasan, K. M. Antimicrob. Agents Chemother. 2001, 45, 2060-2063. (10) Bartlett, K.; Yau, E.; Hartsel, S. C.; Hamer, A.; Tsai, G.; Bizzotto, D.; Wasan, K. M. Antimicrob. Agents Chemother. 2004, 48, 333-336. (11) Hatabu, T.; Takada, T.; Taguchi, N.; Suzuki, M.; Sato, K.; Kano, S. Antimicrob. Agents Chemother. 2005, 49, 493-496. (12) Baas, B.; Kindt, K.; Scott, A.; Scott, J.; Mikulecky, P.; Hartsel, S. C. AAPS PharmSci 1999, 1, 10.

10.1021/la7008573 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/18/2007

Fluorescence of Amphotericin B-Deoxycholate

HTFZ aggregates under cryo-transmission electron microscopy are larger and shaped differently than those of FZ. The heattreatment of AmB in the absence of sodium deoxycholate induced a UV-vis absorption spectra blue-shift in the aggregated AmB absorbance peak.5 AmB’s mechanism of action remains poorly understood despite much study in vivo and in vitro. Atomic force and electron microscopies, monolayer compression, differential scanning calorimetry, electrochemistry, UV-vis absorbance, circular dichroism, nuclear magnetic resonance, electron spin, and X-ray, Raman, and fluorescence spectroscopies have all been used to examine AmB’s behavior.13-27 The study of the mechanism is complicated by AmB’s amphipathic nature. Its rod-like structure with a hydrophilic poly hydroxylated moiety opposing a hydrophobic heptaene backbone leads to aggregation behavior in most solvents. AmB is known to exist as monomer, aggregate, and super-aggregate forms.5 The equilibrium between forms is sensitive to concentration, solvent, temperature, ionic strength, and ionic character.24,28-30 Numerous studies on the structure of AmB aggregates exist; the AmB dimer is a common motif.22,23,27,31-35 Gruszecki et al.’s27 work characterizing the fluorescence of the purified AmB monomer and dimer suggests new possibilities for further study. Many well-founded fluorescence techniques exist to study drug-cell and drug-model membrane interactions.36-39 Despite AmB’s low quantum yield,27 the high sensitivity of fluorescence methods should allow for fruitful study. Interest in AmB’s aggregation state has been primarly due to its influence on pharmacological behavior, but similar questions (13) Gruszecki, W. I.; Gagos, M.; Kernen, P. FEBS Lett. 2002, 524, 92-96. (14) Milhaud, J.; Ponsinet, V.; Takashi, M.; Michels, B. Biochim. Biophys. Acta 2002, 1558, 95-108. (15) Minones, J., Jr.; Dynarowicz-Latka, P.; Conde, O.; Iribarnegaray, E.; Casas, M. Prog. Colloid Polym. Sci. 2004, 126, 55-59. (16) Minones, J., Jr.; Carrera, C.; Dynarowicz-Latka, P.; Minones, J.; Conde, O.; Seoane, R.; Rodriguez Patino, J. M. Langmuir 2001, 17, 1477-1482. (17) Wojtowicz, K.; Gruszecki, W. I.; Walicka, M.; Barwicz, J. Biochim. Biophys. Acta 1998, 1373, 220-226. (18) Fournier, I.; Barwicz, J.; Tancrede, P. Biochim. Biophys. Acta 1998, 1373, 76-86. (19) Stoodley, R.; Shepherd, J.; Wasan, K. M.; Bizzotto, D. Biochim. Biophys. Acta 2002, 1564, 289-297. (20) Mazerski, J.; Bolard, J.; Borowski, E. Biochim. Biophys. Acta 1982, 719, 11-17. (21) Rinnert, H.; Thirion, C.; Dupont, G.; Lematre, J. Biopolymers 1977, 16, 2419-2427. (22) Ernst, C.; Grange, J. Biopolymers 1981, 20, 1575-1588. (23) Balakrishnan, A. R.; Easwaran, K. R. K. Biochim. Biophys. Acta 1993, 1148, 269-277. (24) Lamy-Freund, M. T.; Ferreira, V. F. N.; Schreier, S. Biochim. Biophys. Acta 1989, 981, 207-212. (25) Janoff, A. S.; Boni, L. T.; Popescu, M. C.; Minchey, S. R.; Cullis, P. R.; Madden, T. D.; Taraschi, T.; Gruner, S. M.; Shyamsunder, E. et al. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 6122-6126. (26) Ridente, Y.; Bolard, J.; Levi, G.; Aubard, J. In Spectroscopy of Biological Molecules: New Directions, European Conference on the Spectroscopy of Biological Molecules, Enschede, Netherlands, August 29 to September 2, 1999; Greve, J., Puppels, G. J., Otto, C., Eds.; Kluwer Academic Publishers: Dordrecht, 1999; pp 497-498. (27) Gruszecki, W. I.; Gagos, M.; Herec, M. J. Photochem. Photobiol., B 2003, 69, 49-57. (28) Schreier, S.; Lamy-Freund, M. T. Quim. NoVa 1993, 16, 343-349. (29) Lamy-Freund, M. T.; Schreier, S.; Peitzsch, R. M.; Reed, W. F. J. Pharmaceut. Sci. 1991, 80, 262-266. (30) Grijalba, M. T.; Cheron, M.; Borowski, E.; Bolard, J.; Schreier, S. Biochim. Biophys. Acta 2006, 1760, 973-979. (31) Anachi, R. B.; Bansal, M.; Easwaran, K. R. K.; Namboodri, K.; Gaber, B. P. J. Biomol. Struct. Dyn. 1995, 12, 957-970. (32) Hemenger, R. P.; Kaplan, T.; Gray, L. J. Biopolymers 1983, 22, 911918. (33) Barwicz, J.; Gruszecki, W. I.; Gruda, I. J. Colloid Interface Sci. 1993, 158, 71-76. (34) Mazerski, J.; Grzybowska, J.; Borowski, E. Eur. Biophys. J. 1990, 18, 159-164. (35) Mazerski, J.; Borowski, E. Biophys. Chem. 1996, 57, 205-217. (36) Bryl, K.; Langner, M. In Fluorescence Spectroscopy in Biology; Hof, M., Hutterer, R., Fidler, V., Eds.; Springer: New York, 2005; Vol. 3, pp 229-242.

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of molecular aggregation are often considered in relation to dyes for photographic, photovoltaic, and photosynthesis processes.40,41 Surfactants are commonly used to control the aggregation state while such systems are probed with fluorescence and absorbance measurements.42-44 The separate hydrophobic and hydrophilic regions of surfactants often leads to self-association behavior such as micelle formation at the critical micelle concentration (CMC). The anionic surfactant sodium dodecyl sulfate (SDS) is widely used in dye aggregation studies as well as being fully characterized by its widespread use to influence protein structure. Dye-surfactant interactions are generally governed by the interplay of hydrophobic and electrostatic contributions. Gruda et al. used light-scattering and absorbance measurements to characterize AmB aggregation in the presence of sodium deoxycholate or lauryl sucrose in the course of studying the effect of aggregation state on sterol binding and efficacy.2,3,45 Lamy-Freund and co-workers used static and dynamic lightscattering experiments to study AmB-deoxycholate aggregates and their stability at different deoxycholate (DOC; Figure 1) concentrations,29 while Shervani examined the self-association of AmB in the presence of non-ionic surfactant Triton X-100.46 AmB formulated as HTFZ has not yet been examined, and additionally, the use of fluorescence spectroscopy to characterize AmB is in its infancy. Our interest lies in probing the interaction of FZ with model cell membrane systems and comparing results of the heated and unheated forms. AmB fluorescence may prove to be an ideal way to monitor its behavior in such systems. Presented here are the results of fluorescence characterization of FZ and HTFZ. To better understand our results in the context of separate AmB monomer and aggregate fluorescence, we have introduced varying concentrations of SDS or DOC to modulate the AmB aggregation state. We also compare the relative stability of FZ and HTFZ to disaggregation by SDS. Materials and Methods A stock solution of AmB (1.45 × 10-4 M) in the form of commercial micellar dispersion FZ was prepared in Milli-Q water. FZ was supplied as a powder with each vial containing 50 mg of AmB, 41 mg of DOC, and 20.2 mg of sodium phosphate. The 2.50 mL aliquots of stock solution (unheated or heated at 70 °C for 20 min) were diluted to a 7.00 mL volume with Milli-Q water. Additions of 0.6635 M SDS (Sigma L3771) or 0.5672 M DOC (Sigma D5670) stock solutions varied between 0 and 109 µL volume, resulting in a maximum dilution of AmB solution by 1.6%. DOC, sodium phosphate (Fisher ACS grade), and Milli-Q water were added to Nystatin (Sigma N3503) to prepare a solution analogous to FZ. All solutions were made the same day as the measurement and were protected from exposure to light. Fluorescence spectra were collected on ISS K2 fluorometer equipped with a 300 W Xe arc lamp and 0.1 mm entrance and exit (37) Gumbleton, M.; Stephens, D. J. AdV. Drug DeliVery ReV. 2004, 57, 5-15. (38) White, N. S.; Errington, R. J. AdV. Drug DeliVery ReV. 2004, 57, 17-42. (39) Porcar, I.; Gomez, C. M.; Codoner, A.; Abad, C.; Campos, A. Recent Res. DeVelop. Polym. Sci. 1998, 2, 431-446. (40) Chen, H.; Farahat, M. S.; Law, K.-Y.; Whitten, D. G. J. Am. Chem. Soc. 1996, 118, 2584-2594. (41) Chibisov, A. K.; Prokhorenko, V. I.; Gorner, H. Chem. Phys. 1999, 250, 47-60. (42) Pereira, R. V.; Gehlen, M. H. Spectrochim. Acta, Part A 2005, 61, 29262932. (43) Brichkin, S. B.; Kurandina, M. A.; Nikolaeva, T. M.; Razumov, V. F. High Energy Chem. 2004, 38, 373-381. (44) Micheau, J. C.; Zakharova, G. V.; Chibisov, A. K. Phys. Chem. Chem. Phys. 2004, 6, 2420-2425. (45) Tancrede, P.; Barwicz, J.; Jutras, S.; Gruda, I. Biochim. Biophys. Acta 1990, 1030, 289-295. (46) Shervani, Z.; Etori, H.; Taga, K.; Yoshida, T.; Okabayashi, H. Colloids Surf., B 1996, 7, 31-38.

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Figure 2. UV-vis absorption spectrum of 5.16 × 10-5 M AmB as FZ with varying concentrations of added SDS as labeled. SDS concentrations are given as mol ratio with respect to AmB. Corresponding data for HTFZ are given as Supporting Information. slits to give an 8 nm excitation and emission bandwidth. The measured fluorescence intensity was normalized for variations in excitation intensity and recorded at 1 nm intervals as the average photomultiplier tube output over 2.5 s. Samples were held in a Starna quartz dual path length cuvette (2 and 10 mm) with the long axis perpendicular to the excitation beam. UV-vis absorption spectra were collected with a 2 mm path length cuvette on a Varian Cary 4000 spectrometer operating with a 2 nm bandwidth, 0.5 nm interval, 0.1 s integration time, and 300 nm/min scan rate. The Cary 4000 light source was switched automatically from a tungsten halogen to a deuterium arc lamp at 345 nm. Recorded fluorescence intensities and absorbances were corrected for the dilution effect of added surfactant solution. Inner filter effect corrections were made to fluorescence intensities.47

Results and Discussion UV-vis absorption spectra for solutions of 5.16 × 10-5 M AmB as FZ with varying SDS concentrations are presented in Figure 2. SDS concentrations are expressed with respect to AmB and vary between 0 and 209 mol of SDS/mol of AmB. The UV-vis absorbance of FZ in the absence of added SDS agrees well with that given in the literature.22 The principal absorption is at 326 nm, with less intense bands at 292, 363, 386, 406, and 418 nm. In the AmB literature, there are two cases for which absorption peaks at 326 nm are observed: aggregated AmB complexed with deoxycholate48 and super-aggregates formed by heat-treating AmB alone.5 Pure AmB aggregates absorb at 340 nm; deoxycholate is thought to interact with AmB aggregates such that the monomers are moved closer to each other,33 blueshifting the absorbance to 326 nm. The FZ absorption spectrum thus principally corresponds to aggregated AmB. As expected, the addition of SDS shifts the main absorption from 326 nm of aggregated AmB to the 411 nm band diagnostic of monomeric AmB5 along with the appearance of vibrational fine structure peaks at 388, 367, and 350 nm. The UV-vis absorbance spectrum of HTFZ (provided as Supporting Information) is nearly identical and undergoes similar changes upon addition of SDS. Similarity between FZ and HTFZ absorption spectra suggests that the DOC present acts to limit the extent of exciton coupling in the aggregates because there is no further blue-shift on super-aggregation. Recently, Gruszecki et al.27 reported the detection of monomeric AmB and dimeric AmB using fluorescence spectroscopy. They reported that excitation at 350 nm excites the AmB dimer while 408 nm light stimulates the AmB monomer. The assignment of the AmB dimer as a fluorescent species was made based on two (47) Parker, C. A.; Barnes, W. J. Analyst 1957, 82, 606-618. (48) Egito, E. S. T.; Araujo, I. B.; Damasceno, B. P. G. L.; Price, J. C. J. Pharmaceut. Sci. 2002, 91, 2354-2366.

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Figure 3. Schematic diagram of AmB electronic energy levels. 11Ag, 21Ag, and 11Bu states are alternately denoted S0, S1, and S2. Arrangement of AmB transition dipoles in card-pack aggregation leads to shown exciton splitting. Energy levels for higher aggregates of AmB depend strongly on nature and size of aggregate.

principal arguments: the presence of vibrational fine structure and a van’t Hoff plot of aggregate to monomer intensity. We note that the van’t Hoff analysis fits trimer or tetramer models nearly as well as the dimer and that the presence of fine structure is indicative of the fluorescent species being a small aggregate, not specifically indicative of a dimer. Accordingly, the assignment of dimer fluorescence may be uncertain, although it seems likely that the fluorescence originates from small AmB aggregates. For clarity, we retain here the usage of the term dimer, with the understanding that various low-N aggregates might be a more accurate term. Excitation of higher aggregates has been suggested to occur at 325 nm,49 in agreement with the UV absorbance of purified AmB. Aggregation-induced spectral shifts for AmB absorbance and fluorescence may be rationalized via exciton theory.50 Briefly, variations in the packing pattern within aggregates leads to a red- or blue-shift of spectral features relative to the monomer spectrum. One simple model that accounts for the AmB aggregate blue-shift is card-pack aggregation; helical structures have also been proposed for AmB aggregates in agreement with the blue-shifted spectra.32,51 In card-pack aggregation, a parallel orientation of AmB electronic transition dipole moments in a direction perpendicular to the plane of aggregation results in splitting of the excited-state energy level as shown in Figure 3. The transition dipole for a linear polyene such as AmB is modeled to lie approximately 13° off the polyene axis.52 The transition probability depends on the square of the vector sum of the dipoles; attraction between out-of-phase dipoles lowers the energy level but results in a forbidden transition. In-phase dipolar arrangement results in an allowed transition with a higher energy level, giving the characteristic blue-shifted absorption band upon AmB aggregation. Fluorescence excitation of the AmB dimer is allowed into the high-lying exciton state, while emission results from the low-lying exciton state. Assuming strictly card-pack aggregation, the splitting between the exciton bands enlarges with an increasing number of molecules in the aggregate, doubling in separation from dimer to infinite aggregate. A collection of aggregates with varying sizes displays a range of energy levels, represented by the shaded box in Figure 3. The (49) Gruszecki, W. I., personal communication. (50) Kasha, M.; Rawls, H. R.; El-Bayoumi, M. A. Pure Appl. Chem. 1965, 2, 371-392. (51) Millie, P.; Langlet, J.; Berges, J.; Caillet, J.; Demaret, J.-P. J. Phys. Chem. B 1999, 103, 10883-10891. (52) Birge, R. R.; Zgierski, M. Z.; Serrano-Andres, L.; Hudson, B. S. J. Phys. Chem. 1999, 103, 2251-2255.

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Figure 5. Fluorescence emission spectra of 5.16 × 10-5 M AmB as FZ and HTFZ.

uncertainty in designating higher aggregate energy levels is compounded by the possibility of a different packing motif between AmB in the dimer and neighboring dimers within larger aggregates. Figure 4 shows the fluorescence excitation and emission spectra for AmB as FZ. In Figure 4A, the monomer excitation at 408 nm shows weak fluorescence with an emission maximum at 562 nm for FZ. This large energy difference between absorption and emission is typical of linearly conjugated polyenes and has been rationalized by Kohler and co-workers to be the result of emission from the 21Ag energy level below the 11Bu level involved in the absorption.53 The excitation spectrum (Em 560 nm) has three strong bands between 324 and 356 nm. Figure 4B gives corresponding spectra under conditions of dimer excitation and emission. When excited at 350 nm, FZ has a broad fluorescence emission with a maximum at 473 nm. The excitation spectrum (Em 471 nm) again has three strong bands between 324 and 356 nm. The fluorescence excitation and emission spectra of FZ are roughly similar to those observed for purified AmB,27 but with some differences. Our monomer emission spectrum agrees with Gruszecki’s, with the (0-0), (0-1), and (0-2) transitions at 528, 562, and 608 nm, respectively. The fluorescence excitation spectrum measured at 560 nm differs. We record weak (0-0) and (0-1) transitions at 410 and 386 nm, but Gruszecki’s (0-2) transition at 371 nm is absent. This presumably weak fluorescence peak may simply be hidden by more intense neighboring peaks. We additionally record transitions at 324, 339, and 356 nm that Gruszecki observed only in the dimeric AmB excitation spectrum.

Our excitation spectrum (Em 471 nm) agrees with Gruszecki’s results, with transitions at 324, 338, and 356 nm. These bands may originate from excitation of the AmB dimer or higher aggregates, and thus, the vibrational transitions have not been assigned. The corresponding emission spectrum (Ex 350 nm, dimer excitation) also compares favorably, with transitions observed near 452, 473, and 494 nm. Instrumental resolution limits exact determination of these band positions. The low monomer emission is the combined result of the low monomer quantum yield and of AmB in FZ being substantially aggregated. Since the AmB aggregation state affects the selectivity of its sterol binding, characterization of the aggregation state in HTFZ is important to understanding the cause of its decreased toxicity. AmB fluorescence is sensitive to different aggregation states, and it is thus of interest to examine the fluorescence spectra of HTFZ. Figure 4 shows that the fluorescence excitation and emission spectra of HTFZ are very similar to those of FZ but with increased intensity. Increased emission intensity for HTFZ monomeric AmB excitation was unexpected but agrees with Gaboriau’s finding of a slightly increased monomeric AmB content after heating.5 Increased emission intensity for dimeric AmB excitation is likely not due to an increased dimer concentration but may be related to a change in quantum yield for the fluorescence process. Figure 5 shows the fluorescence emission of AmB as FZ and HTFZ when exciting higher aggregates of AmB at 325 nm. The emission scan shows identically those bands that were observed for FZ and HTFZ fluorescence under 350 nm excitation. The fluorescence emission intensity for FZ is increased 26% relative to that of 350 nm excitation, and this increase is nearly constant across the spectrum. HTFZ excited at 325 nm shows an approximately 10% increase relative to that of 350 nm, but this increase is only in the range of 450-540 nm. Petersen has suggested that AmB fluorescence may be due to an impurity, perhaps tetraenes.54 AmB analyzed by thin layer chromatography combined with high-pressure liquid chromatography revealed two fluorescent components (possibly a pentaene and heptaene) in addition to Petersen’s tetraene.55 FZ preparations are permitted under most countries’ pharmacoepia to contain up to 5% of tetraene Amphotericin A (AmA). AmA differs from AmB only in that a single bond replaces the C28d C29 double bond. To gauge a possible fluorescence contribution from AmA, we recorded fluorescence excitation and emission spectra for Nystatin prepared analogously to FZ, denoted Nystatin-deoxycholate (NY-DOC). Nystatin shares a similar structure to AmA except that the C8 and C9 hydroxyls are shifted

(53) Hudson, B. S.; Kohler, B. E.; Schulten, K. In Excited States; Lim, E. C., Ed.; Academic Press: San Diego, 1982; Vol. 6, pp 1-95.

(54) Petersen, N. O.; Henshaw, P. F. Can. J. Chem. 1981, 59, 3377-3378. (55) Kelly, P. M. J. Chromatogr. 1988, 437, 221-229.

Figure 4. Fluorescence spectra of 5.16 × 10-5 M AmB as FZ and HTFZ. (A) Excitation scan (Em 560 nm) shows peaks at 324, 339, 356, and 386 nm. Emission scan (Ex 408 nm) gives bands at 435, 471, 528, 562, and 608 nm. (B) Excitation scan (Em 471 nm) shows peaks at 324, 338, 356, and 385 nm. Emission scan (Ex 350 nm) gives features at 452, 473, 494, and 525 nm. Peak positions given for FZ; those of HTFZ are similar.

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Figure 6. Fluorescence excitation and emission spectra of 5.74 × 10-5 M Nystatin-DOC under similar conditions to those used in Figure 4. Emission spectrum excited at 408 nm and excitation spectrum measured at 560 nm both magnified 10-fold.

to C7 and C10, respectively. The diene-tetraene moiety responsible for fluorescence is identical in both. Given the similarities in Nystatin and AmA structures, their fluorescence characteristics are expected to be comparable. The fluorescence spectra of 5.74 × 10-5 M NY as NY-DOC (1:1.8 NY/DOC) are presented in Figure 6. Fluorescence excitation and emission wavelengths are identical to those used in Figure 4; reported intensities for excitation at 408 nm (emission scan) and emission at 560 nm (excitation scan) have been magnified 10-fold for clarity. When excited at 408 nm, NY-DOC produces a weak fluorescence that trails off gradually. The excitation scan (Em 560 nm) reveals a broad peak centered at 350 nm. Excitation at 350 nm produces a more intense fluorescence emission with the maximum around 440 nm. Measurement of the fluorescence intensity at 471 nm while scanning the excitation wavelength shows a maximum at 336 nm and lesser peaks at 324 and 306 nm. Comparison of NY-DOC spectra to that of FZ shows that they are clearly different, although there is overlap between the two. Under conditions of monomer AmB excitation and emission, there is no meaningful contribution from AmA. When excited analogously to the AmB dimer, the intensities of NY-DOC are similar to those of FZ. Since FZ contains maximally 5% of AmA, the contribution of AmA to FZ fluorescence is minimal. That our fluorescence spectra are similar to that recorded by Gruszecki for purified AmB suggests that we may neglect the possibility of fluorescent pentaene or heptaene contamination in FZ. The addition of SDS to the system was made with the parallel goals of modulating the AmB monomer/aggregate content and evaluating the relative stability of FZ as compared to HTFZ. The fluorescence excitation and emission spectra of FZ in the presence of varying concentrations of SDS are presented in Figure 7. The equivalent figure for HTFZ fluorescence is provided in the Supporting Information. Figure 7A shows a monomer emission scan (Ex 408 nm) and monomer excitation spectrum (Em 560 nm). Moderate increases in the SDS concentration lead to an increased fluorescence intensity for bands at 339 and 356 nm of the excitation scan. A higher SDS concentration shows a decreased intensity for these bands with a small red-shift. SDS addition dramatically and monotonically increases the fluorescence intensity for the 388 and 410 nm monomer excitation bands, suggesting an increasing AmB monomer concentration. The corresponding emission scan shows large increases in the AmB monomer fluorescence intensity at the 528, 562, and 608 nm peaks and unexpected similar increases at 439 and 460 nm. Figure 7B presents a dimer emission scan (Ex 350 nm) and excitation

Figure 7. Fluorescence excitation and emission spectra of 5.16 × 10-5 M AmB as FZ with varying concentrations of added SDS as labeled. (A) Emission scan (Ex 408 nm) and excitation scan (Em 560 nm). (B) Emission scan (Ex 350 nm) and excitation scan (Em 471 nm). Corresponding data for HTFZ are provided as Supporting Information.

scan (Em 471 nm) showing that increasing the SDS/AmB ratio leads to an initial increase in fluorescence emission intensity, while higher SDS concentrations reverse this trend. The largest increase occurs at the main 473 nm band. At high SDS concentrations, the monomer emission bands at 562 and 608 nm become apparent. The fluorescence excitation scan shows that the 325, 338, and 356 nm bands increase with moderate SDS concentration but then begin to decrease at high SDS concentrations. Monomer excitation bands at 386 and 410 nm grow monotonically with increasing SDS concentration. The emission at 437 and 460 nm that is observed in Figure 7A after monomer excitation at 408 nm can be rationalized in terms of S2 f S0 fluorescence emission from the 11Bu level to the 11Ag ground state. In Figure 7B, the monomer excitation bands that appear for high SDS concentrations at 386 and 410 nm are a further expression of S2 f S0 fluorescence; the bandwidth of our 471 nm measurement allows detection of the edge of the 460 nm S2 f S0 transition. As shown in Figure 3, there are two important excited states for UV-vis absorption and fluorescence spectroscopy of linear all-trans polyenes. Excitation to the 11Bu (S2) level is strongly allowed, while excitation to 21Ag (S1) is symmetry forbidden. The energy gap between the two excited states increases with a longer polyene conjugation length, resulting in shorter polyenes fluorescing from the 21Ag level and longer polyenes fluorescing from 11Bu due to decreased internal conversion.53,56 This S2 f S0 fluorescence is called anti-Kasha since it violates Kasha’s rule that fluorescence is observed from the lowest singlet energy state. Some polyenes exhibit both S1 f S0 and S2 f S0 emission, termed dual fluorescence. Whether (56) Cosgrove, S. A.; Guite, M. A.; Burnell, T. B.; Christensen, R. L. J. Phys. Chem. 1990, 94, 8118-8124.

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Figure 8. Ratio of fluorescence emission intensity from S2 (437 nm band) and S1 (562 nm band) energy levels for 5.16 × 10-5 M AmB as FZ and HTFZ with increasing surfactant concentration. (9) FZ with added SDS and (b) HTFZ with added SDS. Fluorescence excitation at 408 nm.

a polyene emits from S2 or S1 depends on the rate of internal conversion between S2 and S1, which is very sensitive to the energy difference between the states and thus sensitive to the chemical structure of the molecule. For example, 1,3,5,7,9,11,13-tetradecaheptaene has dual fluorescence with S2 f S0 bands at 430, 440, and 460 nm, while 2,4,6,8,10,12-hexadecaheptaene shows only S1 f S0 fluorescence.56 Examination of the ratio of S2 f S0 to S1 f S0 fluorescence can give details about the rate of internal conversion. Figure 8 shows the ratio of S2 f S0 to S1 f S0 emission intensity for FZ and HTFZ as a function of SDS concentration. S2 fluorescence was taken as the peak intensity of the 437 nm band, while S1 fluorescence was taken as the peak intensity of the 562 nm band. At zero added SDS, the S2/S1 emission ratio for FZ is close to unity. A slight decrease in the ratio is observed at low SDS concentrations, but the overall trend is a gradual increase with increasing SDS. For this increase to occur as AmB is forced to the monomeric state, the quantum yield of the S2 f S0 transition must be increasing, likely due to a decrease in the rate of S2 to S1 internal conversion. The corresponding ratio for HTFZ shows significantly more S2 than S1 emission at zero SDS concentration, followed by a rapid decrease at moderate SDS concentrations. At a 109 SDS/AmB mol ratio, the emission ratio for HTFZ matches that of FZ and remains so at higher SDS concentrations. The environment of AmB in the super-aggregates of HTFZ must be such that the S2 f S0 transition is more favored. It is known that solvent stabilization preferentially effects the 11Bu (S2) state more than the 21Ag (S1) state due to the larger transition dipole,57 and any change in the S2-S1 energy gap will be reflected in the emission ratio. The controlling factor for S2 f S0 versus S1 f S0 fluorescence from AmB is not the aggregation state of the drug but the solvating influence of the neighboring AmB or SDS. Accordingly, the ratio of S2 f S0 to S1 f S0 fluorescence may serve as a useful tool for measuring the super-aggregated nature of AmB solutions; we note that the strong similarity in the FZ and HTFZ electronic absorption spectra precludes the use of the UV-vis absorption technique for this purpose. It is interesting to note that there are instances of monomer emission features appearing after dimer excitation (dimer to monomer crossover). Figure 4 shows that emission from the AmB monomer (560 nm) can occur after stimulation of the excitation bands of 324, 339, and 356 nm. Additionally, dimer

excitation at 350 nm leads to a broad monomer emission near 528 nm, presumably the (0-0) fluorescence transition. Clearly, dimer excitation may lead to monomer emission. Energy transfer from dimer to monomer may occur by either transfer from the dimer’s upper exciton level into the 11Bu monomer level or from the lower exciton level into the 21Ag monomer level. Identical AmB emission bands when excited at 325 and 350 nm suggest that there is a further energy transfer from the higher aggregate upper exciton level to the dimer lower exciton level. Excitation of either higher aggregates or dimers results in dimer emission. The most likely explanation of the energy transfer processes is that of resonance energy transfer via Coulombic coupling. The separation distance, which provides 50% efficiency of energy transfer between donor and acceptor, is termed the Fo¨rster distance and may be in the tens of angstroms. A rule-of-thumb for the Fo¨rster-type energy transfer is that significant spectral overlap between donor emission and acceptor absorption is required. We note that although there is no significant overlap between monomer absorption spectra and dimer emission spectra, it has been shown that the usual requirements for Fo¨rster energy transfer, including spectral overlap, need not be met for energy transferal in cases of confined molecular assemblies.58 Briefly, if the donoracceptor separation is smaller or on the order of the donor or acceptor aggregate size, the electric dipole-dipole coupling model used in Fo¨rster’s theory breaks down because localized Coulombic interactions within the aggregate become important.58 Figure 9 summarizes fluorescence emission intensity and UVvis absorbance data as a function of the added surfactant/AmB ratio. The effect of added SDS to FZ and HTFZ is shown along with the effect of DOC added to FZ for comparison. Figure 9A,B show the fluorescence emission intensity after excitation at 408 nm (monomer, left panel) and 325 nm (aggregate, right panel). Figure 9C,D shows the corresponding absorbance data. Addition of SDS has little effect on the FZ and HTFZ monomer absorbance

(57) Snyder, R.; Arvidson, E.; Foote, C.; Harrigan, L.; Christensen, R. L. J. Am. Chem. Soc. 1985, 107, 4117-4122.

(58) Scholes, G. D.; Jordanides, X. J.; Fleming, G. R. J. Phys. Chem. B 2001, 105, 1640-1651.

Figure 9. Summarized fluorescence emission and absorbance of 5.16 × 10-5 M AmB as FZ and HTFZ with increasing surfactant concentration. (9) FZ with added SDS; (b) HTFZ with added SDS; and (sideways triangle) FZ with added DOC. Peak fluorescence emission after excitation at 408 nm (A) and 325 nm (B). Absorbance at 408 nm (C) and 325 nm (D).

8724 Langmuir, Vol. 23, No. 17, 2007

and fluorescence emission until about a 100 surfactant/AmB ratio, where a sigmoidal increase is observed. The rate of increase in the FZ and HTFZ monomeric absorption slows above 127 surfactant/AmB but still continues to rise at the highest SDS concentration, suggesting that AmB is largely but not completely monomeric above 127 surfactant/AmB. Mixtures of 1:16 AmB/ SDS previously have been reported to hold AmB in a monomeric form for SDS concentrations above its CMC of 8 mM.59 Forcement of monomeric AmB is determined by SDS concentration more than the surfactant/AmB ratio. The higher aggregate absorbance of FZ and HTFZ shows a sigmoidal trend with a high initial absorbance at low SDS concentrations, then a rapid decrease between 100 and 127 surfactant/AmB ratio. Further increases in the SDS concentration do not lead to a lower absorbance, suggesting that the higher aggregates are entirely broken up. Addition of DOC also gives an increased monomer fluorescence emission and absorbance, but the changes are smaller and more gradual as compared to added SDS. Even at 209 DOC/AmB ratio, FZ is still partially in the aggregated form. Absorbance of FZ with added DOC also leads to a decreased absorbance but is more gradual. The absorbance at the highest surfactant/AmB ratio is higher for DOC than SDS, suggesting incomplete breakup of higher aggregates by DOC. The fluorescence emission intensity of FZ and HTFZ higher aggregates with added SDS shows a different trend than that predicted from absorbance measurements. At low surfactant concentrations, the intensity is low and decreases on initial surfactant addition. At an 85 SDS/ AmB ratio, the fluorescence intensity undergoes a rapid increase, peaking near a 107 SDS/AmB ratio. Further increases of SDS result in a decreased fluorescence intensity to a level similar to the zero added surfactant case. FZ with added DOC does not show a peak in fluorescence intensity. Rather, an initial decrease and then a moderate stepwise increase is observed between 85 and 100 DOC/AmB ratio, followed by an unchanging fluorescence with continued DOC addition. These results may be rationalized in part by considering the behavior commonly observed in dye-aggregation studies using surfactants to modulate aggregation. Premicellar surfactantdye complexes are formed, and with continued surfactant addition, the aggregate is broken up to give a micelle incorporated dye monomer.60,61 Tancrede et al.45 found that surfactants added to solutions above AmB’s CMC of about 10-7 M first yielded an increase in scattered light intensity as the surfactant penetrated the AmB micelles, changing their size or shape. Further addition of surfactant gave a decrease in scattering, indicative of micelle break-up and dispersal of AmB in solution. Above the CMC of the added surfactant, scattering increased again due to the formation of surfactant-only micelles. Accordingly, we may expect strong absorbance and fluorescence from AmB higher aggregates at zero added surfactant concentration, followed by a large increase in monomer absorbance and fluorescence and corresponding decreases for aggregates near the CMC. The CMC of our SDS/FZ system was found using surface tension measurements to be 6.4 mM SDS (total surfactant concentration 7.4 mM), corresponding to a SDS/AmB ratio of 124. The CMC of the DOC/FZ system is expected to be near 4.5 mM,45 or a 78 DOC/AmB ratio. We observe dramatic increases in monomer fluorescence and absorbance slightly below the CMC when SDS is added, indicative of SDS intercalation into AmB micelle leading to eventual break-up of the micelle, confirmed by the corre(59) Matsumori, N.; Houdai, T.; Murata, M. J. Org. Chem. 2007, 72, 700706. (60) Mandal, A. K.; Pal, M. K. Spectrochim. Acta, Part A 1999, 55, 13471358. (61) Goswami, A.; Pal, M. K. Colloids Surf., B 1998, 10, 149-159.

Stoodley et al.

sponding drop in higher aggregate absorbance. The low fluorescence emission intensity from higher aggregates at zero added surfactant concentration does not fit our expectations. The 325 nm excitation here is into a strongly absorbing band, and we expected to see strong dimer emission at 471 nm. Instead, the emission is relatively weak, and this is presumably due to fluorescence quenching of the excited higher aggregate or excited dimer. Any process that decreases the quantum yield by favoring alternate relaxation pathway(s) can be considered a quench. The source of this quenching is unclear, although self-quenching seems a likely possibility. Self-quenching occurs by energy transfer to a nonfluorescent state on a like molecule either by collisional orbital interaction (Dexter transfer) or by Coulombic coupling between molecules (Fo¨rster transfer). Higher aggregates of AmB are likely to have AmB in a variety of microstates, raising the possibility of successful Coulombic coupling and resultant Fo¨rster energy transfer. We hypothesize that further surfactant intercalation into the higher aggregate relieves the quench, resulting in an increased fluorescence near a 100 surfactant/AmB ratio (Figure 8B). Decreased quenching may be related to the rigid conformation AmB was found to adopt in SDS micelles,59 which may restrict the modes available for Fo¨rster energy transfer. The peak shape of the fluorescence as a function of added surfactant is the result of a competition between decreased quenching and aggregate break-up as the SDS concentration is increased. The steady fluorescence emission for DOC/FZ above a 100 DOC/AmB ratio reflects DOC being less effective than SDS at breaking up the higher aggregates. PreCMC complex formation between surfactant and AmB, which changes the shape or structure of the AmB micelle such that AmB is more closely packed, is consistent with initial increases in absorbance and initial decreases in higher aggregate fluorescence due to quenching. The SDS/AmB ratio required to instigate the break-up of AmB aggregates and form monomers can be compared for FZ and HTFZ to determine their relative stabilities to disaggregation by surfactant. A decrease in absorbance at 325 nm measures the break-up of aggregates, while increases in monomer absorption at 408 nm and increases in fluorescence emission at 560 nm (Ex 408 nm) measure the AmB monomer concentration. The trend of increasing monomer content with increasing SDS/AmB is virtually identical for FZ and HTFZ, with the highest rate of change at 115 SDS/AmB. The corresponding decrease in higher aggregate content shows a similar trend for FZ and HTFZ absorbance, while the fluorescence intensity peaks at a slightly lower SDS/AmB ratio for HTFZ. The slight difference in peak fluorescence intensity is not significant, so we conclude that FZ and HTFZ have a similar stability toward disaggregation by SDS. This result is relevant to understanding how AmB interacts with cell membranes since the AmB interaction with lipid may require an analogous disaggregation of FZ and HTFZ.

Conclusion We have measured the fluorescence spectra of FZ and HTFZ and found that the fluorescence is indeed that of AmB and not of a contaminant. The fluorescence spectra of monomeric and dimeric AmB are distinct, but Fo¨rster energy transfer is observed to occur from excited dimer to monomer. Additionally, excitation of the AmB monomer can give both S2 f S0 as well as the previously observed S1 f S0 fluorescence emission. Application of AmB fluorescence measurements to the study of monomeric or dimeric AmB interactions with membranes will thus require careful selection of the excitation and emission wavelengths to ensure a species-specific response. FZ and HTFZ have similar

Fluorescence of Amphotericin B-Deoxycholate

S1 f S0 fluorescence spectra. The ratio of S2 f S0 to S1 f S0 fluorescence differs for FZ and HTFZ at zero and low surfactant concentrations because the solvating environment around the AmB molecules is different in FZ and HTFZ. Use of this ratio may therefore prove to be an efficient way to characterize the super-aggregation of AmB solutions. SDS forms complexes with micelles of FZ or HTFZ at concentrations below the CMC of SDS. Complexes at low surfactant concentration featured decreased higher aggregate fluorescence due to increased fluorescence quenching, while the addition of further surfactant decreased the quenching and increased the fluorescence. At surfactant concentrations close to the CMC, higher aggregates are broken up to form the AmB monomer. FZ and HTFZ higher aggregates are equally stable toward break-up, with similar surfactant concentrations required for both.

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Acknowledgment. The Cary 4000 spectrophotometer and the ISS K2 fluorometer were provided through a grant from the Canada Foundation for Innovation to the Centre for Blood Research and the Laboratory for Molecular Biophysics of UBC. R.S. thanks the National Science and Engineering Research Council for a Canada Graduate Scholarship and the Michael Smith Foundation for a Trainee Award. We also thank one of the reviewers for their constructive criticism that has undoubtedly helped us to improve this manuscript. Supporting Information Available: Plot of the fluorescence excitation and emission spectra of 5.16 × 10-5 M AmB as HTFZ with varying concentrations of added SDS and UV-vis absorbance spectra of 5.16 × 10-5 M AmB as HTFZ with varying concentrations of added SDS. This material is available free of charge via the Internet at http://pubs.acs.org. LA7008573