GIANT RAMAN SCATTERING SPECTRA OF THEMOLECULAR COMPLEX DOXORUBICIN–DNA

A. E. German,* N. D. Strekal’, S. A. Maskevich,and G. A. Gachko

UDC 543.424+535.370+547.963

We have investigated the giant Raman scattering spectra of the doxorubicin–DNA complex adsorbed on thesurface of annealed thin silver films modified by sodium chloride. It is found that doxorubicin interacts withDNA via carbon groups with involvement of their π-electron system into interaction. Heating of a substrate inthe process of adsorption from 20 to 37oC leads to a sharp increase in the number of chemisorbed moleculesas a result of the thermally activated formation of specific adsorption sites. It has been found that in adsorp-tion of the doxorubicin–DNA complex on the substrate surface, the chromophore molecules are oriented pre-dominantly in parallel with the surface of the metal.

Keywords: giant Raman scattering, doxorubicin, deoxyribonucleic acid, thin silver film.

Introduction. Doxorubicin (see Fig. 1) is an efficient antitumoral antibiotic of the anthracycline series and astrong intercalator of DNA [1–5]. In chemical structure the preparation is close to the widely known rubomycine, dif-fering from the latter only in the presence of a hydroxyacetyl group in position 14 instead of an acetyl one, i.e., it canbe considered as oxyrubomycine.

It is assumed that the mechanism of the action of anthracyclines is associated with the inhibition of the syn-thesis of nucleic acids due to their ability to be incorporated into the double spiral of DNA and to split DNA due tothe formation of free radicals. The indicated processes lead to the disturbance of the readout of information from thecomplementary filament of the DNA by cell enzymes. Moreover, it is assumed that the interaction with the cell mem-branes is of great importance for the antitumoral effect. This mechanism is also responsible for the cytotoxic action ofpreparations on healthy cells. In view of this, the study of the distribution of a drug in a cell and of the mechanismsof its transport through the cell membranes is of great importance.

Among the physicochemical methods of analysis only fluorescence spectroscopy and the spectroscopy of giantRaman scattering (GRS) of light have a rather high sensitivity for studying the enumerated mechanisms of the interac-tion of doxorubicin with a biological system. The high selectivity of the GRS spectroscopy has been demonstrated ear-lier in relation to the GRS spectra of doxorubicin adsorbed on silver hydrosol [2, 4]. Nevertheless, a number ofproblems are still without final explanation predominantly due to the short-range mechanisms of the enhancement ofthe GRS light by the molecules adsorbed on the surface of colloidal particles. In view of this it is necessary to de-velop new techniques of investigation of the chemical and biophysical properties of anthracyclines both in the freestate and in the state bound with a biological system.

It is known [2–4] that the method dealing with the analysis of the second radiation spectra of the chromo-phore incorporated in the DNA, when metal colloids are used as a substrate, is not characterized by a high sensitivity.In [2], where the GRS of the doxorubicin–DNA complex adsorbed on silver hydrosol was observed, it was noted thateven with a ratio of the number of molecules of the preparation to the number of DNA bases of 1:20 there was agreat (by an order of magnitude or more) decrease in the intensity of the recorded signal. Here, the resulting spectrum

Ya. Kupala Grodno State University, 22 Ozheshko Str., Grodno, 230023, Belarus; e-mail: ger-man@grsu.grodno.by. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 69, No. 1, pp. 28–31, January–February,2002. Original article submitted August 1, 2001.

Journal of Applied Spectroscopy, Vol. 69, No. 1, 2002

0021-9037/01/6901-0028$27.00 2002 Plenum Publishing Corporation28

*To whom correspondence should be addressed.

virtually did not differ from the GRS spectrum of doxorubicin on DNA-free hydrosol. At concentrations of the orderof one molecule of doxorubicin per 100 pairs of bases, the GRS signal disappeared entirely. It was stated in [2] thatthe GRS observed at smaller concentrations is due to the presence of a certain portions of the molecules which hadno time as yet to bind with the DNA and precisely which gave the GRS spectrum that was recorded. In [3, 4], similareffects are explained by short-range mechanisms of enhancement during adsorption of anthracyclines on the surface ofthe hydrosol particles.

The possibility of studying the anthracycline antibiotics by the GRS spectroscopy methods with thin silverfilms (TSF) used as substrates was demonstrated earlier in [6, 7]. In the present work, we analyzed substrates basedon annealed thin silver films (ATSF) that were much more sensitive than silver colloids and were applied for studyingthe GRS spectra of the doxorubicin–DNA complex.

Experimental Technique. The doxorubicin, silver wire (99.99%), sodium chloride, DNA, and PBS-buffer (pH7.4) were supplied by Sigma, USA, and were used without further purification. The DNA was obtained from theerythrocytes of the blood of a chicken. The original solution of DNA (10−2 M pairs of nucleotides) was prepared inthe PBS-buffer and a 0.01-N solution of NaCl directly before the experiment. To attain the needed ratio of doxoru-bicin molecules on conversion to the quantity of pairs of the DNA bases, the working solution was dissolved by thePBS-buffer or NaCl solution on a basis of the final concentration of the drug of 10−6 M. The concentration of theDNA (as the number of pairs of bases) was evaluated photometrically on the basis of the well-known extinction factorεDNA = 12,500 M−1⋅cm−1 at λ = 260 nm.

Incubation of doxorubicin with the DNA was carried out after the addition of the preparation to the DNA so-lution. The incubation lasted for 30 min. This time was sufficient to complete the process of the incorporation in DNAof the majority of intercalating molecules. In the process of incubation a constant temperature of the solution of 37oCwas sustained, which is typical of actual biological systems in which the drug studied is usually applied.

The silver films were obtained by the method of vacuum deposition of metal on quartz substrates accordingto [8, 9]. The chemical modification of annealed thin silver films was done by placing a film-coated substrate into a0.001-N solution of NaCl for 2 min with subsequent washing with distilled water and drying in air in an inclined po-sition in a thermostated desiccator at 60oC.

Adsorption of the doxorubicin–DNA complex on the surface of substrates was carried out by a method ofsoaking film-coated substrates in a solution of the complex for 30 min at 37oC. The substrates were washed withbidistilled water at room temperature with subsequent drying for 1 h at the indicated temperature.

Fig. 1. Spectra of the OD (a) and fluorescence (b) of doxorubicin in the PBSbuffer pH 7.4 (1) of doxorubicin incubated with DNA (2–4). The concentra-tion of doxorubicin in a 10−6 M DNA solution: 15 (2), 50 (3), and 100 (4)pairs of bases per doxorubicin molecule; λexc = 488 nm. The structural for-mula of doxorubicin is given in the inset.

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The optical density (OD) spectra were obtained with the aid of a Specord 200 spectrophotometer (Carl Zeiss,Jena). The GRS spectra were recorded on a LOMO (Leningrad Opticomechanical Association) DFS-52 spectrometer(the discretization step is 1 cm−1) on excitation by radiation from an ILA-120 argon laser (Carl Zeiss, Jena) operatingat λ = 488 nm. The radiation power measured directly on a specimen was 5 mW. The luminescence spectra in solu-tions were recorded on a LOMO SDL-2 spectrometer. All the measurements were made no fewer than three times.

Discussion of Results. To evaluate the degree of the intercalation of doxorubicin in DNA we employed thewell-known fact of the substantial decrease in the intensity of fluorescence of doxorubicin bound with DNA and theaccompanying typical changes in the optical density spectra of the solution [5]. Figure 1a shows the optical densityspectra of the doxorubicin–DNA complex for different concentrations of the preparation relative to the number of pairsof DNA bases. On formation of the complex one observes substantial weakening of the absorption band at about 480nm and a general (by 10–15 nm) bathochromic shift of the spectrum. The fluorescence spectra of the preparation in asolution (Fig. 1b) are also very informative relative to intercalation processes. The intensity of the fluorescence of dox-orubicin bound with the DNA decreases by an order of magnitude in comparison with the fluorescence of the prepa-ration in the free state, and the spectrum itself undergoes a red shift (by up to 3–4 nm). From the data given it is seenthat the observed processes of intercalation are saturated at concentrations of about 100 pairs of DNA bases per mole-cule of the drug. Thus, the indicated concentration is optimal for radiation, since we found that virtually all the chro-mophore molecules are involved in the interaction with the DNA.

We have obtained very informative GRS spectra of the doxorubicin–DNA complex at a concentration of1:100 and less on the surface of annealed thin silver films modified with NaCl. These substrates were applied becauseof their affinity for biological systems (since the majority of solutions for intravenous injections of doxorubicin areprepared with the use of aqueous solutions of NaCl). And, moreover, the highest gains of GRS are attained on sub-strates modified with chlorides [10].

In contrast to [2–4], we did not observe a sharp decrease in the intensity of the GRS signal from bound dox-orubicin. Moreover, in some cases a signal from the complex exceeds in intensity the signal for an unbound prepara-tion of the same concentration. Figure 2 presents the GRS spectra of doxorubicin adsorbed on the surface of anonmodified annealed thin silver film at room temperature and at 37oC. The GRS spectrum typical of a higher tem-perature is much more intense and very close to the GRS spectra of doxorubicin on the surface of annealed thin silverfilms modified with chlorides [7]. It is likely that the degree of the binding of a molecule with a surface increasesbecause of the thermally activated process of the formation of GRS-active sites with specific properties. Here, the con-tribution of the molecular mechanisms of amplification increases. Moreover, at a higher temperature the processes ofsurface modification are intensified; they may lead to extra roughening of the surface and thus stimulate electromag-netic amplification. This effect is also probable when the chromophore–DNA complex is adsorbed on the surface of anannealed thin silver film. In addition to the influence of temperature, the absence of the weakening of the GRS spectraof the doxorubicin–DNA complex can be attributed to the substantially more far-ranging character of the amplificationof substrates based on annealed thin silver films in comparison with hydrosols.

The formation of a specific complex between doxorubicin and DNA is clearly manifested in the GRS spectraof the preparation that are shown in Fig. 3. Both spectra demonstrate a very strong drop in the fluorescence (in com-parison with the spectra of secondary radiation of unbound doxorubicin [7]), pointing to the presence of intercalationalso in the adsorbed state. The analysis of the obtained GRS spectra of the doxorubicin–DNA complex allows someconclusions on the process of incorporation of the drug into the double spiral of DNA. We found that the most sen-sitive to the processes of interaction with DNA are the vibrations assigned to δ(C=O), δ(O—H), and (C=O). Notewor-thy is the presence and behavior of two bands in the range 1500–1700 cm−1. The deconvolution carried out shows thatthe indicated group involves the ν(C=O) band with a maximum near 1628–1630 cm−1 shifting in binding from 1642cm−1 by 12–14 cm−1. The relative intensity of the 450-cm−1 band (δ(C=O)) also decreases, pointing to the formationof a hydrogen bond by one of the carbon groups of the chelating system of the chromophore with one of the func-tional groups of DNA.

The strong decrease in the relative intensity of the bands D1210 (δ(O—H)), 1309 (ν(C=O)), and 1433 cm−1

(deformation of rings) allows the statement that there is active incorporation of the chromophore in the DNA with theinvolvement into the interaction of the π-electron system of the chromophore. This process is much more manifestedon inhibition of the DNA in a solution of NaCl in comparison with the PBS buffer.

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Based on the analysis of the GRS spectra of dauno- and pyrorubicin, the orientation of the preparation mole-cule relative to a metal surface was examined in [11, 12] and it was shown that the interaction with various polargroups of phospholipids can lead to the reorientation of the studied molecules adsorbed on the metal (see also [13]).According to those works, the basic criterion for the position of a doxorubicin molecule relative to the metal surfaceis the relative intensities of the bands at about 1260 cm−1 manifesting themselves in the case of the "plane" geometryof adsorption and at about 1430 cm−1 active in the case of "perpendicular" geometry.

It has been found that on binding of doxorubicin with the DNA and adsorption of the resulting complex onan annealed thin silver film a considerable part of the chromophore molecules lies in a "plane" configuration relativeto the metal surface in contrast to the "perpendicular" orientation in a free state [7]. The conclusion was drawn on thebasis of the comparative analysis of the relative intensities of the 1412/1428 cm−1 doublet bands in the free (Fig. 2,spectrum 1) and intercalated (Fig. 3) states and also on the fact of the appearance of a sufficiently amplified band ofdeformation vibrations at about 1260 cm−1 (Fig. 3). Here, the number of molecules occurring in the "plane" configu-ration increases on adsorption of the doxorubicin–DNA complex incubated in NaCl in comparison with incubation inthe PBS buffer.

This work was carried out with support from the Ministry of Education of the Republic of Belarus and Inter-national grant INTAS No. 97-O-522.

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