This fact is also confirmed by the decrease in cac with the decrease in temperature.
It could be due to more pronounced ordering effect of the randomly oriented monomers from solvated form into highly organized core of aggregate than disordering effect caused by destruction of water structure around hydrophobic groups of monomer molecule. Moreover, the highly negative value of AH is indicative of greater contribution of electrostatic interactions during aggregation 9. The solutions were allowed to equilibrate for 5 min before every experimental assay. In the present investigations, the interaction of DSP with double stranded-DNA in aqueous solution has been monitored.
The absorbance measurements were performed by keeping the concentration of the drug constant 50 uM while varying the concentration of DNA from 10 uM to uM. The spectrum recorded slight bathochromic and profound hypochromic effects by the addition of DNA. The UV-Vis spectrum Figure. The hypochromism is due to the strong interaction between the electronic states of the DSP chromophore with the bases of DNA 9, Such changes in drug absorption properties as a function of DNA binding were used for the evaluation of the binding constant K b by using the following equation :.
The binding constant K b 8. The value of binding constant obtained here is about twice as the value of K b for the interaction of proflavin 4. The binding constant with a value of 2. The increase in binding constant at high temperature may be attributed to the overall stability of the drug-DNA adduct.
This reveals that the geometry of the drug-DNA complex contributes to anticancer behavior more than the binding affinity. The Gibbs energy change of- However the correct mechanism of drug-DNA binding in eliciting anti-tumor effects remains unresolved due to the reversible nature of drug-DNA complexes, which demands for increased drug level in order to achieve the steady state condition required for detection and thus limits the resolution of such studies apart from additional non specific biologically irrelevant interactions.
Efforts are currently underway to circumvent such problems by the use of different techniques. For the evaluation of thermodynamic parameters of DSP-DNA adduct, the binding constant values were converted to Gibbs energy changes by the expression:. Inspection of table 2 unequivocally reflects an increase in the value of binding constant with the increase in temperature.
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This thermodynamic stabilization of the drug-DNA complex with the increase in temperature can better be explained by the likely overall conformational changes in the whole structure of the conjugate rather than the additional local interaction. Enthalpy of complex formation AH with a value of The high enthalpy value for the complex formation was just the consequence of the interaction between two negatively charged species.
Thermodynamic properties were calculated from critical aggregation concentration cac. The results indicate increase of cac value with rise in temperature due to enhancement in solubility and dehydration of hydrophilic groups as a result of their more repulsion which makes the aggregation difficult thus resulting in increased cac. The increase in binding constant at relatively high temperature suggests thermodynamic stabilization of the drug-DNA complex due to the likely overall conformational changes in the whole structure of the adduct rather than the additional local interaction.
The high enthalpy value for the complex formation was the consequence of the interaction between two negatively charged species. Attwood, Adv. Colloid Interface Sci. Gilpin, Anal. Sambrook, E. Fritsch, T. Jiang, X. Lin, Bioelectrochemistry. Babkina, N. Ulakhovich, Anal. Williams, J. Phillips, K. Mysels, Trans. Evans, J. Khan, S. Shah, J. Kumar, E. Asuncion, J. Janovec, J. Mikes, J. Koval, J. Ungvar-sky, M. Fedorocko, P.
Kristian, J. Imrich, Bioorg. Aslanoglu, Anal. It is necessary to introduce more simple methods for investigating the mechanism of interaction. By understanding the mechanism of interaction, designing of new DNA-targeted drugs and the screening of these in vitro will be possible. A great variety of substances, including several agents of importance in cancer chemotherapy, 3 are known to bind to DNA by intercalation. Chemotherapy is an important part of the program for cancer treatment.
A lot of compounds are developed as potential candidates for anticancer drugs, but only a handful of them have become effective clinical drugs. Many anticancer drugs have been known to interact with DNA to exert their biological activities. Methylene blue MB, Scheme 1 is a phenothiazinyl dye, which is a kind of photosensitizer drug molecules showing promising applications in the photodynamic therapy PDT for anticancer treatment. The interaction of methylene blue with DNA has been studied with various methods.
The interaction of doxorubicin Scheme 2 the main representative of the anthracycline group of antibiotics with DNA is of general interest in connection with questions regarding drug-nucleic acid interactions, since its biological activity is thought to reside in the ability to bind to DNA. In this work, the interaction between doxorubicin and calf thymus DNA has been investigated by absorption and fluorescence spectroscopies as well as by electrochemical methods.
Drug-DNA interactions: structures and spectra.
Because the interaction between MB and DNA is known intercalation mechanism , we used it as a probe for comparing its behavior against DNA in comparison with doxorubicin. The results showed that doxorubicin, similarly to methylene blue can intercalate into the double helix of DNA.
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The apparent binding constant of doxorubicin-DNA complex is found to be 3. The aim of this study is to find the mode of the interaction between doxorubicin and DNA by using spectroscopy and electrochemical methods. This will help us to design new drugs, which have biological and antitumour activities. The advantages of the proposed method involve simplicity and cheap devices.
All the chemicals were of analytical grade and have been purchased from Aldrich. Doubly distilled water was used throughout. Stock solution 3. More dilute solutions were prepared by appropriate dilution with water. Calf thymus DNA was used without further purification. All solutions were adjusted with the phosphate buffer 0. All spectrofluorometric assays were made on a spectrofluoromrtric, JASCO, equipped with a xenon lamp and a 1.
Metal-Based Drug-DNA Interactions
Both excitation and emission slits were set at 5 nm, and the scan rate at nm min A Pentium IV 2. Preparation of the glassy carbon electrode. Before each experiment, glassy carbon GC electrode was first polished to a mirror finish with 0. Then it was transferred into a blank buffer solution phosphate buffer 0. Spectrophotometric studies. Interaction of doxorubicin with ds-DNA. UV-Vis absorption spectra have been obtained by titration of a 1.
There is an absorption band at With the increase of DNA concentration, the absorption band decreases continuously. It is similar to the phenomenon caused by the interaction between DNA and its typical intercalate. The result of fitting the experimental data with equation 1 is shown in Figure 2. It suggests that the complex of doxorubicin with ds-DNA is relative to a complex. In order to clarify the presence of interaction between doxorubicin and ds-DNA, the absorption spectra of methylene blue in the presence of ds-DNA was compared with doxorubicin-DNA spectra.
The changes of the absorption spectra of MB are shown in Figure 3. The absorption spectra of MB upon increasing the concentration of DNA showed gradually decreases in the peak intensities with a red shift. This phenomena is usually associated with molecular intercalation into the base stack of the ds-DNA. This contributes to the hypochromic effect discussed above. This band almost shifts towards the blue end of the spectrum with the appearance of a new peak at nm, which increases progressively in intensity Figure 4.
An isobestic point at nm provides evidence that a new species is forming during the competitive interaction. The observed changes in intensity and position of the bands with increasing amounts of doxorubicin added to the MB-DNA solution, suggest that doxorubicin intercalates into the double helix of the DNA through exchanging with the MB fluorophoric dye probe.
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Typical differential pulse voltammetry behavior of doxorubicin in the absence and presence of DNA has been shown in Figure 5. Addition of DNA causes the peak current of the oxidation wave of doxorubicin to diminish considerably. Additionally, the peak potential E pc shifts to more positive values, from 0. This suggests that the formation constant for the doxorubicin binding to DNA is about 4. A differential pulse voltammogram of the MB dye probe Figure 6 at a glassy carbon electrode exhibits a cathodic peak.
Optical Absorbance and Fluorescence Techniques for Measuring DNA–Drug Interactions
The peak at approximately As the concentration of ds-DNA is increased, no new waves are formed, but a decrease in peak current for reduction peak occurs. Positive shift was observed for reduction peak potential, which again suggests that the MB molecule has intercalated into the DNA.
In these solutions, the ions don't interact with DNA because of coulombic repulsion between their negative charges. Therefore, there are only few effects of the change in viscosity ds-DNA addition on the diffusion, and no significant obstruction on the GC surface from ds-DNA adsorption. The great decrease in current of doxorubicin in the presence of ds-DNA could be attributed to the decrease of diffusion coefficient of the complex and decreasing in the free drug concentration because of the complex formation. Spectrofluorometry studies. Doxorubicin has two maxima fluorescence peaks at ca.
When the ds-DNA solution was added to the doxorubicin solution, the fluorescence intensity decreased quenched fluorescence. Figure 8 shows the fluorescence spectra of doxorubicin in the presence and absence of calf thymus DNA. The inset of Figure 8 shows a Hill plot of fluorescence quenching as a function of increasing ds-DNA concentration. It is clear that the fluorescence of doxorubicin was not completely quenched by ds-DNA, indicating that only a fraction of the binding sites quenches the fluorescence. The fluorescence quenching of doxorubicin in the presence of DNA may be caused by the fact that, by diffusion of drug into the double helix of DNA, the active surface of doxorubicin will be reduced upon excitation radiation.
It can be seen that the fluorescence of MB was efficiently quenched at the maximum emission wavelength nm upon addition of ds-DNA, indicating that the MB binding to DNA changes the excited state electronic structure of MB fluorophore. In equation 2, F 0 and F are the fluorescence intensities in the absence and presence of ds-DNA respectively.
It is clear that the fluorescence of MB was not completely quenched by ds-DNA, indicating that only a fraction of the binding sites quenches the fluorescence. In this research, the interaction of anticancer drug doxorubicin with ds-DNA has been investigated by UV-Vis spectrophotometry, differential pulse voltammetry and spectrofluorometry.
All the acquired spectral data showed that a new adduct between doxorubicin and DNA is formed.