Raman Spectroscopic Study of Aggregation Process of Antibiotic

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Raman Spectroscopic Study of Aggregation Process of Antibiotic Amphotericin B Induced by Hþ, Naþ, and Kþ Ions Mariusz Gagos,*,† Marta Arczewska,† and Wieszaw I. Gruszecki‡ † ‡

Department of Biophysics, University of Life Sciences in Lublin, 20-950 Lublin, Poland Department of Biophysics, Institute of Physics, Maria Curie-Skzodowska University, 20-031 Lublin, Poland ABSTRACT: The normal and the preresonance Raman effects (NR and PRR) of spectroscopy have been used to monitor and explain the aggregation processes of amphotericin B (AmB) in aqueous solution at different pH values and containing the Kþ and Naþ ions. The resonance-enhanced and normal vibrational Raman spectra were recorded with a semiconductor laser (ex 785 nm) and an argon laser (ex 514.5 nm) for investigation of interactions between AmB chromophores. The essential difference between the samples stimulated by resonance-enhanced and by near-infrared was in the CdC stretching mode region of polyene chain. The processes connected with the aggregation of AmB led to changes in the chromophore, which were only visible as a remarkable broadening of the band centered at 1558 cm1. The understanding of possible physical mechanisms responsible for the molecular aggregation of the drug is important from the pharmaceutical applicability standpoint.

’ INTRODUCTION Amphotericin B (AmB) belongs to the group of the polyene macrolide antibiotics and was isolated for the first time in 1953, as the metabolite of the Streptomyces nodosus.1,2 Despite 50 years existence in clinical practice and being considered as the gold standard in the treatment of serious systemic fungal infections, the biological mechanism of its action at the molecular level has not been understood yet.36 The presence of seven conjugated double-bonded species (CdC) determines typical spectroscopy features.7 In the systems where the antibiotic appears in a monomeric form, it shows a well-defined electronic absorption spectrum in the range between 300 and 450 nm, which is connected with the ππ* transition in the chromophore subunit.8,9 The electronic absorption spectrum of the monomeric AmB, which was dissolved in an aqueous solution alkalized to pH 12, has an electron-vibrational structure with the maxima: 408 nm (00), 385 nm (01), and 365 nm (02).10 The position of absorption maxima as well as their intensity strictly depend on the features of the experimental systems.11 In aqueous solution, the drug appears in the form of monomers, dissolved associated oligomers, and insoluble aggregates.1214 The processes of AmB self-association, as well as an aggregation, can be induced by the physical and chemical factors: concentration,15 polarity of the solvent,16 temperature,17 pH value.18 and the changes of ionic strength of solution.19 Amphiphilic structure of AmB promotes formation of cylindrical pores with hydrophilic surface, which could organize into massive molecular aggregates.17 r 2011 American Chemical Society

The molecular aggregation of AmB induced by different physical factors has influence on the spectral shift in both directions: longand short-wavelength.20 The bands, which were created in accordance with the exciton splitting theory, are connected with creating different types of AmB molecular aggregation.11,2123 This organization is related to the dipoledipole interaction (mutual orientation of the electronic dipole moments transition) between chromophores of neighbor molecules involved in the formation of aggregates.21,22 According to the popular hypothesis, the mechanism of AmB action is based on the structural and organizational disturbances of the lipid bilayer, which causes uncontrolled increase in its permeability for the monovalent ions, especially Kþ, as well as small elements from the cell.4,2426 In addition, it was proposed that AmB may have a direct influence on the ATP-proton pump, in the case of the cells of fungi, or it may inhibit the ATP (NaþKþ) activity in the animal cells.3 On the other hand, it was observed that the enrichment of fungal cell cultures with Kþ cations exhibits protective properties against the toxic activity of AmB.27 The latest reports indicate that high sodium intake (>4 mEq/kg) per day may be associated with lower nephrotoxicity in extremely premature infants treated with AmB.28 The preresonance and resonance methods of Raman spectroscopy were used to make analysis of the antibiotic impact in Received: February 22, 2011 Published: April 11, 2011 5032

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Figure 1. Chemical structure of amphotericin B (AmB).

the lipid multilayers.29 It was noticed that the intensity of the Raman band corresponding to the CdC symmetrical stretching vibrations showed the sensitivity on the structural changes, which occur during the phase transition in the DMPC-cholesterol layers containing AmB.29 The Raman spectrum of AmB has, similarly to other linear-conjugated polyenes, the all-trans configuration, due to the phenomenon of resonance enhancement.30 Thus, the frequency between 1500 and 1600 cm1 is dominated by the strong band corresponding to the CdC symmetric vibrations of the polyene chain (see Figure 1). The unique features of this band were used to monitor therapeutic and subtherapeutic drug concentration in a model system mimicking the anterior chamber by La Via et al.31 Measurements of AmB levels in the aqueous humor of the eye by Resonant Raman spectroscopy were used to develop a correlation curve for prediction of drug concentration.31 The weak lines in the 16001650 cm1 region correspond to the carbonyl stretching band. However, it should be noticed that all of those modes are admixed with the CdC asymmetric vibrations. This is in distinct contrast to the behavior of the nearly isostructural nystatin including two carbonyl groups in the molecule, as the CdO modes are strongly resonance enhanced.32 In the range of 11501200 cm1, there are the bands corresponding to the CC skeletal vibrations coupled with the in-plane CCH bending deformations. The relatively weak band at 1010 cm1 can be assigned to the CC vibrations of the CCH group coupled with the CdCC bending distortion in the chromophore of AmB.29,30,3335 The normal and preresonance Raman spectroscopy has been applied in the present work to monitor aggregation processes of AmB induced by pH changes and by presence of the Kþ and Naþ ions. On the basis of the preresonance effect, it was possible to preferentially enhance the long-wavelength spectral forms related to the AmB aggregated structures.

’ MATERIALS AND METHODS Materials. Amphotericin B (AmB) in a crystalline form was purchased from Sigma Chemical Co. AmB was further purified by means of HPLC on a YMC C-30 coated phase reversed column (length 250 mm, internal diameter 4.6 mm) with 40% 2-propanol in H2O as a mobile phase. The potassium and sodium salts (KCl and NaCl) were of analytical grade. Preparation of AmB Experimental Formulations. AmB was dissolved in water alkalized to pH 12 (with KOH and NaOH) and then centrifuged for 15 min at 15 000g to remove microcrystals of the drug, which still remained in the sample. To record Raman spectra at different pH values and in the presence of KCl and NaCl (140 mM), all solutions (15 μM) were dissolved in

Figure 2. Electronic absorption spectra of the aqueous solution of AmB at pH 7 (dotted line) and alkalized to pH 12 (solid line). Relatively low intensity spectral features (absorbance multiply by 10) in the absorption spectra of the same sample can be observed.

pure water. The crystalline form of AmB was deposited on a platinum plate. Spectroscopic Measurements. Electronic absorption spectra were recorded with a Cary 300 Bio UVvis spectrophotometer from Varian (Australia). All Raman spectra were recorded with a Raman microscope inVia Reflex from Renishaw (UK), which used a charge-coupled device (CCD) detector with a resolution of 1 cm1. The Raman spectra were collected using the excitation wavelengths from both a visible range (514.5 nm, Arþ laser) and a near-infrared range (785 nm). The laser power has been tuned in the range 0.420 mW (an Arþ laser) and 1.5 mW (a semiconductor laser). The laser beam diameter was defocused to 20 μm. The spectra have been accumulated within 10 s integration time. All types of the spectra were recorded at least four times, and the spectral effects reported were found to be reproducible. The preresonance Raman scattering spectra were corrected by subtracting a background signal originating from the fluorescence.

’ RESULTS AND DISCUSSION The chromophore of AmB is composed of the conjugated CdC bond system. Because conjugated double bonds are highly polarizable, Raman spectroscopy is particularly useful in characterizing such a system. Because of the fact that AmB chromophore is involved in formation of aggregated structures, Raman spectroscopy seems to be a technique particularly suited to examining molecular organization of the drug. 5033

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Table 1. Frequencies of Raman Transitions of Amphotericin B in the Solid Phase and an Aqueous Solutiona Raman shift [cm1] solid

solution

ex 514.5 and 785 nm

ex 514.5 nm

1010b

1015

δ(CCH)

1139 1156

1158

δ(CCH) þ γ(CdCC) ν(CC) þ δ(CCH)

1196

1198b

assignment [310]

1297

1298

1558b

1559

νs(CdC)

1603

1609

νas(CdC) mixed with ν(CdO)

1638

1637b

ν, stretching vibrations; δ, bending vibrations; γ, distortion vibrations; s, symmetrical; as, asymmetrical. b Infrared band frequencies recorded in KBr and in D2O solutions. a

Figure 3. Raman spectra of crystalline form of AmB deposited at the platinum glass. Spectra were obtained using the 514.5 and 785 nm excitation wavelengths. The spectral changes between preresonance and NR Raman are represented by the difference spectrum for the same sample. All spectra were normalized with respect to integrated area under the bands in the region of 14001800 cm1.

As can be seen from the absorption spectra presented in Figure 2, application of the 514.5 nm line of Arþ laser creates a resonance condition for the long-wavelength spectral forms of aggregated AmB (in the solution at pH 7) but not for the sample of monomeric AmB (at pH 12). Figure 3 presents both the Raman scattering spectra recorded with the excitation laser beam at 514.5 and 785 nm, beyond the resonance or even the preresonance spectral region. The assignment of the bands is presented in Table 1.2935 The essential differences between the spectra of the samples recorded with the excitation lines of 514.5 and 785 nm can be seen in the region of the CdC vibrations in the polyene chain. Interestingly, the processes of AmB aggregation influence considerably on the principal band, which centers at 1558 cm1. A pronounced broadening of the band can be observed, which is associated with the slight shift of the center of gravity of the band toward lower frequencies. This effect can be seen in the difference spectrum presented in Figure 3B. Such an effect can be explained in terms of a disturbance of the excitonic state due to the aggregation, induced by interactions between chromophores.10 It should be noted here that the band centered at 1558 cm1 was also observed in the infrared absorption spectra of AmB in the KBr disk.36,37 The main maximum centered at 1558 cm1 is highly sensitive to structural changes of AmB chromophores. However, its intensity depends on the concentration, the presence of ions in solution, and selection of the excitation laser line.35 As can be seen from the analysis of the difference spectrum (Figure 3B), the aggregation of AmB results in a decrease of the bands intensity representing the CC skeletal vibrations.

Figure 4 shows the effect of changes in pH (12, 7, and 2) on preresonance Raman spectra of AmB in aqueous solution (15 μM). As can be seen, acidification of the AmB solution results in the intensity decrease and in the small shift of the main band toward lower frequencies (by 1 cm1 at pH 2 and by 2 cm1 at pH 7). The bandwidth of the maximum centered at 1603 cm1, which was also observed in the spectrum of the crystalline AmB (Figure 3), was relatively low intensive in the spectrum of the compound in the aqueous solution and was shifted to the higher frequencies up to 1609 cm1; see Figure 5. On the other hand, it was easy to notice the remarkable broadening together with the intensity increase in the typical region of the CdC mixed with the CdO modes for pH 7 and 2.32 This effect is further analyzed in Figure 5A for the CdC stretching region. As can be seen, the most pronounced differences with respect to the Raman spectra of AmB in the monomeric form (pH 12) are observed at pH 7. Such process is related to the aggregation of AmB, which was also manifested by marked differences in electronic absorption spectra.10 The differences were mostly limited to the intensity increase of the bands from the region of 16001800 cm1 and the appearance of a shoulder centered at 1540 cm1 on the 1558 cm1. Interestingly, the presence of the low-frequency shoulder was also observed in Raman spectra of AmB in acidic and basic methanol by Bunow et al.29 Comparing the difference spectra presented in Figure 5B, it was easy to notice the remarkable increase of the intensity in the region of 15501800 cm1 at pH 7 with respect to the spectrum of the sample, which was recorded at pH 2. In the difference spectra appeared the weak band in the region between 1450 and 1550 cm1 and the additional maxima in the range of 16001700 cm1 representing the CdO stretching vibrations in the COOH group (see the inset of Figure 5B). This effect may be connected with the involvement of the COO group in the interaction with other molecules of AmB. The band centered at 1637 cm1 coincides quite closely with that observed in the infrared and assignable to the CdO in the carboxylic group (see Table 1).37 The participation of this group as well as the conjugated double-bond systems seems to be of crucial importance in the aggregation process of AmB in aqueous environment at physiological pH.10,37 Because of the above fact, the analysis of the spectral effects connected with the Naþ and Kþ influence on the AmB 5034

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Figure 4. Preresonance Raman spectra of AmB in water solution (15 μM) obtained at pH 12, 7, and 2. Spectra were obtained using the 514.5 excitation wavelength.

Figure 5. Preresonance Raman spectra showing the CdC stretching region of AmB in an aqueous solution (15 μM) obtained at pH 12, 7, and 2 (A) and their difference spectra. Spectra were obtained using the 514.5 excitation wavelength. All spectra were normalized with respect to integrated area under the bands in the region of 14001800 cm1. The inset presents an enlarged frequency region of 14501800 cm1 for the difference spectra presented in Figure 5B.

aggregation may have an important role.38 Raman spectra of a thin film of AmB containing NaCl and KCl salts were applied to monitor the aggregation of drug. The most remarkable differences in the spectra of AmB were noticed in the typical region of the CdC stretching vibrations of a polyene chain (see

Figure 6. Preresonance Raman spectra showing the CdC stretching region of AmB in water solution (15 μM) contain potassium and sodium salts (140 mM) (A) and their difference spectra (A). Samples was deposited at the platinum plate and evaporated by N2. The spectra presented in panel A were obtained using the 514.5 nm excitation wavelength. All spectra were normalized with respect to integrated area under the bands in the region of 14001800 cm1. The inset presents an enlarged frequency region of 15251555 cm1 for the difference spectra presented in Figure 6B.

Figure 6A). The differences were mainly related to the broadening of the main band and a slight spectral shift (by 1 cm1) in the direction of lower frequencies, which was connected with a slight delocalization of the polyene π electron system.29 Because of the presence of the above ions in aqueous solution of AmB, the structural alternation has led to the changes in the chromophore. 5035

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The Journal of Physical Chemistry B Similarly as in the case illustrated in Figure 5, the difference spectra presented in Figure 6B gave additional information about the band centered at 1545 cm1. Using the enlarged frequency region of 15101555 cm1 for the difference spectra presented in Figure 6B, it can be seen that the appearance of low-frequency shoulders on the CdC modes has a higher intensity in the presence of Kþ ions than in the presence of Naþ ions (see the inset of Figure 6). The results of these studies together with the previously discussed changes in the PRR spectra of AmB in aqueous solution of varying acidity indicate a significant effect of the Kþ ions in the process of formation of aggregated structures of AmB stabilized by interactions between the chromophores.

’ CONCLUSIONS The preresonance and normal vibrational Raman spectra of amphotericin B presented in this work show that the position and the shape of the principal spectral band corresponding to the CdC stretching mode are highly sensitive to the aggregation state. Formation of the aggregates of AmB was induced by different pH values and by the presence of Naþ and Kþ in aqueous solution. Each procedure of AmB aggregation applied gave rise to spectral changes, which can be applied as diagnostic in examination of molecular organization of the drug. ’ AUTHOR INFORMATION Corresponding Author

*Phone: þ(48 81) 4456899. Fax: þ(48 81) 4456684. E-mail: [email protected].

’ ACKNOWLEDGMENT This research was financed by the Ministry of Education and Science of Poland from the budget funds for science in the years 20082011 within the research project N N401 015035. ’ REFERENCES (1) Dutcher, J. D.; Gold, W.; Pagano, J. F.; Vandepatte, J. Amphotericin B, its production and its salts. U.S. patent 2,908,611, 1959. (2) Gold, W.; Stout, H. A.; Pagano, J. F.; Donovick, R. Antibiot. Annu. 1956, 579–585. (3) Brajtburg, J.; Bolard, J. Clin. Microbiol. Rev. 1996, 9, 512–&. (4) Brajtburg, J.; Powderly, W. G.; Kobayashi, G. S.; Medoff, G. Antimicrob. Agents Chemother. 1990, 34, 183–188. (5) Cereghetti, D. M.; Carreira, E. M. Synthesis 2006, 6, 914–942. (6) Hartsel, S.; Bolard, J. Trends Pharmacol. Sci. 1996, 17, 445–449. (7) Baginski, M.; Gariboldi, P.; Bruni, P.; Borowski, E. Biophys. Chem. 1997, 65, 91–100. (8) Millie, P.; Langlet, J.; Berges, J.; Caillet, J.; Demaret, J.-Ph. J. Phys. Chem. B 1999, 103, 10883–10891. (9) Caillet, J.; Berges, J.; Langlet, J. Biochim. Biophys. Acta 1995, 1240, 179–195. (10) Gagos, M.; Herec, M.; Arczewska, M.; Czernel, G.; Dalla Serra, M.; Gruszecki, W. I. Biophys. Chem. 2008, 136, 44–49. (11) Gagos, M.; Koper, R.; Gruszecki, W. I. Biochim. Biophys. Acta 2001, 1511, 90–98. (12) Mazerski, J.; Bolard, J.; Borowski, E. Biochim. Biophys. Acta 1982, 719, 11–17. (13) Mazerski, J.; Grzybowska, J.; Borowski, E. Eur. Biophys. J. 1990, 18, 159–164. (14) Strauss, G.; Kral, F. Biopolymers 1982, 21, 459–470.

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