Comparative FT SERS, resonance Raman and SERRS studies of doxorubicin and its complex with DNA

SPECTROCHIMICA ACTA PART A Spectrochimica Acta Part A 51 (1995) 2083-2090

ELSEVIER

Comparative FT SERS, resonance Raman and SERRS studies of doxorubicin and its complex with D N A A. Beljebbar, G.D. Sockalingum, J.F. Angiboust, M. Manfait * Laboratoire tie Spectroscopie BiomolOculaire, UFR de Pharmacie, Universit~ de Reims Champagne - Ardenne, 51 rue Cognacq Jay, F-51096 Reims Cedex, France

Received 27 June 1995; accepted 28 June 1995

Abstract Fourier transform surface enhanced Raman scattering (FT SERS) coupled with a microscope has been used as a probe to obtain information on the interaction of a drug and of its complex with DNA. Micro-FT SERS spectra of the antitumour agent doxorubicin (DOX) at 10 -5 M and of this complex with D N A have been recorded in aqueous silver hydrosol and compared with the corresponding resonance Raman (RR) and SERS spectra at concentrations of 5 x 10 -4 M and 5 × 10-8 M, respectively. The interactions between the drug and calf thymus D N A induced identical effects in the RR and visible SERS spectra. The data show that the adsorption of the d r u g - D N A complex on the silver hydrosol does not induce detectable perturbations of the molecular interactions within the complex. Micro-FT SERS spectra were found to be partially different from those obtained in visible SERS spectra. These differences concern the relative enhancement of some vibrational modes which could hardly be observed when resonance excitation was used. The FT SERS approach enables further information to be obtained and additional details on the geometry of the d r u g - D N A interaction to be revealed. An analysis of the FT SERS spectra of the drug and of its complex with D N A not only confirms the model of interaction proposed using RR and SERS data in the visible, but brings about new information, especially on the vibrations of ring A of the molecule, which are usually masked by the vibrations of rings B and C dominant in the visible SERS spectra.

Abbreviations DNA DOX FT NIR PBS RR SERRS SERS

D e o x y r i b o n u c l e i c acid Doxurubicin Fourier transform N e a r infrared P o t a s s i u m buffered saline Resonance Raman Surface e n h a n c e d r e s o n a n c e R a m a n scattering Surface e n h a n c e d R a m a n scattering

* Corresponding author. 0584-8539/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved S S D I 0584-8539(95)01515-9

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1. Introduction

Many DNA intercalators have been shown to have antitumour activity [1]. This biological property has been attributed to the formation of the intercalation complexes between the chromophore and the base pairs of DNA [2,3]. The changes in the overall structure of the d r u g - D N A complexes provide a possible explanation for the differences in the clinical activity of the drugs [4]. Much research has been carried out in order to elucidate the structural basis of the intercalation mechanism [4-6]. There are few techniques available for the selective study of molecular interactions within complicated high-molecular-weight supramolecular complexes. Compared with these, Raman spectroscopy has excellent fingerprinting capabilities. In particular, resonance Raman (RR) spectroscopy has been widely used because of its selectivity which permits the observation of bands corresponding only to the vibration of the cbromophore and its sensitivity in monitoring the structure of d r u g - D N A complexes [7]. Unfortunately, it is usually quite difficult to record good quality resonance Raman spectra because of strong fluorescence of these molecules. Surface enhanced Raman scattering (SERS) has been used as a powerful method to obtain information from fluorescent chromophores [8,9]. It allows total quenching of the chromophore fluorescence and enhances Raman scattering by several orders of magnitude. The Raman scattering can be further enhanced by combining SERS with RR. Some research teams [10-12] have published data undoubtedly demonstrating that the interaction with the metal surface in hydrosols is quite smooth and does not disturb the native structure of the molecular of their interactions within the complexes. It is known that in RR spectroscopy, the bands due to the vibrational modes of chromophore are selectively enhanced at the expense of a loss in information. Because FT Raman spectroscopy is a nonresonant technique, the spectra of this drug at very far-off resonance conditions of excitation at 1064 nm may reveal relative enhancement of some deformation vibrations which could hardly be observed when resonance excitation is used. In order to improve the sensitivity when in aqueous conditions, this technique has been combined with SERS [13-20]. The use of this technique as a probe for the study of the molecular interaction of doxorubicin (DOX) (Fig. l) and its complexes with DNA may allow us to have additional information on the changes in the spectra of DOX and on its interaction with DNA. In order to reduce the analysed volume (because a small amount of drug is available), all FT Raman measurements were carried out under a microscope. Details of this set-up were given in an earlier report [21]. Thus, in these conditions only 5 - l 0 pl of the drug were enough to record the micro-FT SERS spectra. In this paper, we report a detailed comparative study of RR, SERS and micro-FT SERS of doxorubicin and its complex with DNA and evaluate the influence of the silver surface on the molecular interactions within the d r u g - D N A complexes. Doxorubicin is an anticancer agent used in the treatment of acute leukaemias and solid tumours in man [22-25]. The biological activity of this drug seems to be due to its complexation with DNA. In previous studies, normal RR and SERS have been used as probes for the characterisation of DOX and its complex with DNA [7,8,10]. Normal RR spectra of

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OH Fig. 1. Chemical structure of the anti-tumour drug, doxorubicin.

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DOX can be recorded at concentrations of not less than 10 - 4 M because an increase in the drug concentration leads to an increase in the fluorescence background [26]. The detection limits for the SERS spectra of this drug have been obtained at concentrations as low as 10-m M. The interaction of this molecule with DNA has also been studied by other techniques such as Fourier transform infrared (FT IR) [27,28], circular dichroism (CD) [29], and fluorescence spectroscopy [30-32]. We show here that by combining information obtained in the visible region (pre-RR and SERS) with that in the near infrared (NIR), it is possible to have a better and more complete explanation of the interaction modes of the drug with DNA. Models of the interaction of doxorubicin with the DNA double helix proposed on the basis of FT results are compared with those already obtained using the RR and SERS techniques.

2. Materials and methods

2. I. Apparatus Micro-FT SERS spectra of doxorubicin were recorded with a Fourier transform infrared spectrometer (Bomem, DA 3.02) modified to work in the NIR [21]. The detecting device was a liquid nitrogen-cooled InGaAs detector. A Nd/YAG laser (CVI, Model C-95), operating at typically 800 mW output, was used at an excitation wavelength of 1064 nm. This spectrometer was interfaced with a microscope (Olympus, BH-2) equipped with a 60 × objective (BBT, Krauss). This objective focused the laser beam onto the sample, and collected and redirected the backscattered light onto the entrance of the spectrometer. With this set-up, the size of the laser spot at the sample was about 8 ~tm and only a few microlitres of the sample were needed to acquire a spectrum. Spectra were recorded at 4 cm-~ resolution, with a laser power at the samples of about 100 mW. Pre-RR and SERS spectra were obtained with a DILOR model Omars-89 Raman spectrometer, supplied with a multichannel (1024 photodiodes) detector. A SpectraPhysics, model 2020-03 argon laser was used for excitation (514.5 nm). All band intensities were corrected for the monochromator-detector response. The high-frequency bands at about 3400 cm- ~ of water or at about 2500 cm- l of deuterium oxide were used as references for the intensity measurements. The bands due to the ring breathing vibrations of the chromophores were used for normalisation of the resonance and SERS spectra of free drug and drug-target complexes in order to obtain the difference spectra (these bands are not changed upon interaction of the drugs with DNA). The procedure of normalisation has been previously described [8,33]. All spectra were reproduced at least three times for different preparations of the drug and drug-DNA complexes.

2.2. Hydrosol preparation Aqueous silver hydrosol was prepared by reduction of silver nitrate (Prolabo, France) with trisodium citrate (Sigma, St. Louis, USA). The preparation and also the spectral and morphological properties of these colloids have been dealt with elsewhere [34,35]. To work in the NIR, the hydrosol was pre-aggregated by the addition of sodium perchlorate solution (Sigma, St. Louis, USA) up to a final concentration of 0.06 M. Hydrosol preaggregation increased both absorption of the colloids in the NIR region and the level of the SERS signals from compounds introduced into the preaggregated sols. D20 colloids were made in exactly the same way, substituting D20 (99.75%, Sigma, St. Louis, USA) for water.

2.3. Chemicals Stock solutions of doxorubicin (Laboratoire Bellon, Paris, France) were prepared in phosphate-buffered saline (PBS) at concentration of 10 -3 M and diluted to the desired ~(A) 5t:i2-s

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~';fi

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Fig. 2. Visible SERS (b, c) and micro-FT SERS (a) spectra of doxorubicin in aqueous solution

(a, b) and in deuterium oxide (c). Experimentalconditions: spectral resolution4 cm - ~(b) and (c): excitation 514.5nm; concentrations 5 x 10-6M and 5 x 10-aM, respectively; laser power, 10 mW; 100 accumulations (1 s each). (a): excitation 1064nm, concentration 10-5 M; laser power 100 mW, 400 accumulations. concentration before each experiment. Drug concentrations in PBS solution were determined by their absorbances at 480 nm. Calf thymus D N A was purchased from Sigma and was also dissolved in PBS. The concentration of D N A was estimated on the basis of a molar extinction coefficient of 6600 M - t c m - ~ at 260 nm.

3. Results and discussion

3.1. Comparative studies of RR and SERS spectra of D O X SERS spectra of DOX have been recorded at concentrations of 5 x 10 -8 M. Comparison of this spectrum with the RR spectrum of DOX [7] shows a very high contribution of the fluorescence background for RR spectrum and a strong quenching of fluorescence in the SERS spectrum. These spectra were found to be nearly identical both in frequencies and relative intensities of the bands. The adsoprtion of the drug on the surface of silver hydrosol does not induce detectable perturbation on the structure of this molecule. Thus, the changes observed in the SERS spectra of D O X - D N A complexes correspond well to the interactions of the drug with double helical DNA.

3.2. Vibrational assigment of visible SERS and micro-FT SERS spectra of D O X and its complexes with DNA Because the vibrations of the skeletal ring and of OH and C - O H groups bring considerable contributions to the SERS spectra of DOX, analysis of the effect of deuteration can help to evaluate the extent of coupling between ring stretching vibrations of the chromophore and C---O, C-O, and O H motions. Fig. 2 displays the visible SERS (b, c) and micro-FT SERS (a) spectra of free doxorubicin in aqueous (a, b) and deuterium oxide (c) solutions. After close inspection of these data, the band at 1642 c m can be assigned to the C---O group (ring B) hydrogen-bonded to the hydroxyl group. In the visible, this band is very weak and does not exist in deuterium oxide but is relatively intense in the NIR. The position of the band of the hydrogen chelated C--O coupled to the ring C---C stretching vibrations is at 1590 era-I and 1579 c m - I for visible and NIR excitation respectively [11]. In D20, this band is downshifted to 1542 cm-~. The Raman

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bands in the region 1462-1436 cm-~ are assigned to the C--C and C - C stretches of the aromatic hydrocarbons. These bands shift only slightly upon deuteration of the phenolic hydroxyl groups indicating some coupling with the C--O" • • H motion also. In NIR this region is blue shifted to 1445-1411 cm -1. The group of bands in the 1200-1300cm -~ region (with two different excitations) are assigned to the vibrations involving in-plane C-O, C - O - H and C - H bending modes. These bands disappear completely upon deuteration [7,11,17,36,37]. At low frequencies, the visible SERS band at 447cm -I is not perturbed after deuteration. The assignment of the NIR bands below 600 c m - ~ was not reliable because the cut-off behaviour to the filter prevents observation of bands below 300 cm-1 and significantly perturbs the intensities of those between 300 and 600 cm -~. The comparison between the spectra of the free DOX in the visible and N I R excitation shows intensity differences in the 1610-1400 cm - t region (vibrations corresponding to the phenolic rings B and C of the chromophore). The band at 1462 cm -1 is very strong with pre-resonance conditions. The biological activity of the DOX molecule has been attributed to the formation of a complex between the chromophore and base pairs of DNA. The comparison between the SERS spectrum of free DOX and that of the D O X - D N A complex in the visible shows a loss in intensity together with the disappearance of some bands upon complexation (see Fig. 3a, b). In the latter spectrum, the intensities of the bands at 1226cm -j and 1255 cm - l (region involving v ( C - O ) + ~ ( C - O - H ) + ~(C-H) motions), and at 1461 cm -~ (skeletal ring stretching coupled with C 4 ) - H vibrations) are strongly decreased. Because vibrations are characteristic of rings B and C of the chromophore, this is indicative that these entities are intercalated inside the D N A double helix and thus no longer accessible to the hydrosol surface. Another important observation is that the band at 1642 cm -~ (C--O--. H hydrogen-bonded) is comparatively more intense. Following the above argument, this would mean that this moiety remains outside the helix. The other bands in the spectra do not change significantly. The SERS data correspond well with X-ray results [6] and demonstrate an intercalation of the C - O H side groups and rings B and C of the chromophore (see Fig. 1) inside the DNA double helix, leaving the A-ring accessible to the silver hydrosol surface. This geometry should lead to a decrease in intensities of the bands of C - O - H together with the bands due to ring vibrations strongly coupled with the vibrations of these exogenous groups.

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Fig. 3. SERS spectra of doxorubicin (curve a) and of its complex with calf-thymus DNA (curve b). Drug-DNA complex: ratio of 1 moleculeof drug for 750 base pairs of DNA. Concentration of DOX in the free form, 5 x 10-8 M; concentration of DOX in complex with DNA, 8 x 10-8 M. All other conditions as for Fig. 2, curve b.

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Fig. 4. Micro-FT SERS spectra of doxorubicin (a) and its complex with calf-thymus DNA (b) and their difference spectrum (c = a - b). Durg-DNA complex: ratio of 1 molecule of drug for 1 base pair of DNA. This ratio is very strong in the spectrum of DOX DNA complex, so the contribution of the free DOX is important; therefore subtraction of the spectrum of DOX DNA from that of free DOX has been carried out to obtain the spectrum of this complex alone. The absolute concentration of the drug in the complex in the hydrosol is 10 5 M. All conditions are as for Fig. 2, curve a.

3.3. M i c r o - F T S E R S spectra o f complexes o f D O X with D N A

In the NIR, FT SERS spectra of the drug at the very far-off resonance conditions of excitation at 1064 nm, showed a relative enhancement of some vibrations (originating from ring A) which could hardly be observed when resonance excitation was used. Micro-FT SERS spectra of free D O X at 10 s M and of its complex with calf-thymus D N A (ratio of 1 molecule of drug for 1 base pair of DNA) have been recorded and are displayed in Fig. 4a, b. Because this ratio is very high, the contribution of the free DOX is important in the spectrum of the coomplex, and we therefore subtracted the spectrum of D O X - D N A complex from that of free DOX in order to have the spectrum of the complex only. The comparison of the spectrum of free DOX with the difference spectrum (corresponding only to D O X - D N A complex) reveals modifications in vibrational frequencies and intensities of several bands (see Fig. 4c). These disparities are listed below. (i) The interaction of DOX with D N A shows the appearance of a new band at 1273 cm 1. This corresponds to the ring stretching mode coupled with the v(C-O) of ring A which is not intercalated within DNA. This band is hardly observable in the spectrum of D O X - D N A complex in the visible region because the other chromophore bands are more enhanced in the pre-resonance SERS conditions. Upon deuteration, it appears weakly because the chromophore bands are less enhanced. (ii) An additional band appears at 1507 cm i corresponding to the vibration of the C=C of ring A. (iii) An additional band appears at 985 c m - I corresponding to the vibration of the C - C of ring A. (iv) The band at 1642 c m - I (assigned to hydrogen-bonding to the C--O) is very weak upon complexation. (v) Loss in intensity of the bands at 1214 and 1246 cm -1, assigned to the vibrations involving the in-plane C-O, C - O - H and C - H bending modes, is observed. This confirms the intercalation of rings B and C within the double helix, thus preventing any contact with the silver colloids.

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M i c r o - F T S E R S with N I R excitation has p r o v e d to be a powerful p r o b e to o b t a i n m o l e c u l a r i n f o r m a t i o n f r o m the free d r u g a n d f r o m its c o m p l e x with D N A . A n analysis o f the F T S E R S spectra o f D O X a n d the D O X - D N A c o m p l e x confirms the m o d e l o f i n t e r a c t i o n p r o p o s e d by R R a n d S E R S in the visible a n d b r i n g a d d i t i o n a l i n f o r m a t i o n , especially on the v i b r a t i o n s o f ring A o f the d r u g molecule which are m a s k e d by the strong v i b r a t i o n s o f rings B a n d C in the S E R S spectra at 514.5 nm. T h e S E R S spectra o f D O X r e c o r d e d on the silver h y d r o s o l are identical to the c o r r e s p o n d i n g R R spectra. F T S E R S d a t a c o m p l e m e n t the visible R R a n d S E R S techniques very well with a view to o b t a i n i n g a b e t t e r a n d m o r e c o m p l e t e e x p l a n a t i o n o f the interaction m o d e s o f the d r u g with D N A . F T S E R S spectra o f the d r u g at very far-off resonance c o n d i t i o n s o f excitation at 1064 n m show relative e n h a n c e m e n t o f some v i b r a t i o n s which c o u l d h a r d l y be o b s e r v e d when resonance excitation was used. A l t h o u g h this technique suffers f r o m low sensitivity, it allows us to o b t a i n m o r e detailed i n f o r m a t i o n c o n c e r n i n g the interaction o f the d r u g with c a l f t h y m u s D N A . M o r e o v e r , using N I R excitation, the p r o b l e m s o f fluorescence a n d p h o t o d e c o m p o s i t i o n associated with biological samples are easily circumvented. Finally, the absence o f d e t e c t a b l e p e r t u r b a t i o n s o f m o l e c u l a r interactions o f l o w - m o l e c u l a r - w e i g h t c o m p o u n d s a n d their targets when a d s o r b e d at the silver surface, m a k e s this technique a very p r o m i s i n g a p p r o a c h by which to follow drugs within living cells.

Acknowledgement W e t h a n k Professor I g o r N a b i e v a n d Dr. H a m i d M o r j a n i for their c o n t r i b u t i o n to this work.

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