# Spectrophotometric Studies on the Thermodynamics of the ds-DNA Interaction with Irinotecan for a Better Understanding of Anticancer Drug-DNA Interactions

Authors: Reza Hajian; Tan Guan Huat
Journal: Journal of Spectroscopy
 (2013)

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## Abstract

The ds-DNA binding properties of irinotecan (CPT-11) including binding constant, thermodynamic parameter, and thermal denaturation (Tm) have been systematically studied by spectrophotometric methods. The binding of CPT-11 to ds-DNA is quite strong as indicated by its remarkable hypochromicity and equilibrium binding constant (Kb). The van't Hoff plot of 1/T versus ln Kb suggests that the CPT-11 binds endothermically to ct-DNA which is characterized by large positive enthalpy and entropy changes. According to the polyelectrolyte theory, the charge release (Z), when ct-DNA interacts with CPT-11, is +0.98, which corresponds very well to the one positive charge carried by CPT-11. The Kb at a low concentration of salt is dominated by electrostatic interaction (98.5%) while that at a high concentration of salt is weakly controlled by nonelectrostatic processes (19.0%). A moderate stabilization of the double helix ds-DNA occurs when CPT-11 binds to ds-DNA as indicated by the increase in Tm of ct-DNA by approximately 15°C in the presence of CPT-11. The CPT-11 is stabilized by intercalation in the DNA (binding constant, K [irinotecan-DNA] = 5.8 × 104 mol−1 L) and displaces the NR dye from the NR-DNA complex (K [NR-DNA] = 2.7 × 104 mol−1 L) in a competitive reaction.


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## Body

## 1. Introduction

Irinotecan (CPT-11), a semisynthetic derivative of camptothecin, has a growing clinical impact due to its effectiveness in treating malignancies of the colon, lung, pancreas, cervix, and ovaries with increased survival benefits for patients. Irinotecan is convertedin vivo to its much higher active metabolite, 7-ethyl-10-hydroxy camptothecin (SN-38) by carboxylesterases. Its disposition by the liver and bile is higher than by any other tissue. Irinotecan is characterized by its relatively poor and highly variable oral bioavailability and therefore this drug is mainly used for intravenous administration. Very few reports exist on irinotecan oral usage and hardly any attempts have been made to improve the oral bioavailability of irinotecan [1–5].DNA plays a major role in the life process because it carries genetic information and instructs the biological synthesis of proteins and enzymes through the process of replication and transcription of genetic information. DNA is quite often the main cellular target for studies with smaller molecules of biological importance like carcinogens, steroids, and several classes of drugs. The investigation of drug-DNA interactions is of current general interest and importance [6–9], especially for the designing of new DNA-targeted drugs and the in vitro screening of these compounds. Early studies indicate that the mechanism of action for irinotecan involves inhibition of the mammalian DNA-topoisomerase-I, thereby causing stabilization of complexes during DNA replication, leading to cell death [4, 10]. However, no reports based on evidence of direct interaction of CPT-11 with ds-DNA have been published. Irinotecan could destroy or have effects on the DNA destruction or transcription.In this respect, our investigation provided a thermodynamic profile, standard free energy(ΔG°), enthalpy (ΔH°), and entropy (ΔS°) changes of the DNA binding of CPT-11 using the van’t Hoff plot by determining the equilibrium binding constant (Kb) at various temperatures. These thermodynamic parameters allow us to further evaluate the enthalpic and entropic contributions to ΔG° of the DNA binding of CPT-11. Moreover, the effect of salt concentration on the binding free energy of small molecules and DNA interactions has been long known. In general, the salt effect arises from the reorganization of the ionic cloud around the DNA and the binding ligand. The binding affinity of small molecules to DNA theoretically decreases when the salt concentration in the solution increases because ion-pair formation between binding ligand and DNA is less favorable in the solution that contains a high concentration of salt due to competition between cationic binding ligand and cationic metal ion released by the salt. However, a systematic study on the effect of salt concentration on the DNA binding of anticancer drugs is limited. The determination of the salt effect on the Kb value of the DNA binding of drugs and then analyzing the results by the polyelectrolyte theory can be used to evaluate systematically the nonelectrostatic contribution to the Kb and separate the total ΔG° into its electrostatic and non-electrostatic contributions. Therefore, in the present study the salt dependence of the DNA binding of CPT-11 has also been characterized by determining its Kb at various concentrations of salt (KCl) using spectrophotometric titration. The data were then evaluated to determine the effects of electrostatic and non-electrostatic forces on the stabilization of the DNA-binding events of CPT-11.In the current work, double stranded calf thymus DNA has been selected because of its low cost, ready availability, and ligand-binding properties. All the results of the study are consistent with the fact that calf thymus and human DNAs are homologous. Interactions with DNA could also be critical for understanding the drug toxicity and its distribution in the organism. The interaction of drug-DNA has been pursued vigorously in the field of life science, and clinical medicine resulting in many interesting studies [11, 12].Neutral red (NR) is a planar phenazine dye and, in general, is structurally similar to other planar dyes, for example, those of the acridine, thiazine, and xanthene varieties. In recent years, the interaction of the fluorescent NR dye with DNA has been demonstrated by spectrophotometric [13–15] and electrochemical [16] techniques. In comparison with a common fluorimetric probe, ethidium bromide (EB) [17, 18], the NR dye offers lower toxicity, higher stability, and ease of use. In addition, its solution remains stable for up to 2 years [19, 20]. In this work, NR was selected as the probe.


## 2. Experimental

### 2.1. Chemicals

Deoxyribonucleic acid sodium salt from calf thymus (Sigma Chem. Co., USA) was used without further purification, and its stock solution was prepared by dissolving an appropriate amount of DNA in doubly distilled water and stored at 4°C. The concentration of DNA in stock solution was determined by UV absorption at 260 nm using a molar absorption coefficientε260=6600 L mol−1cm−1. Purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of >1.8 at A260/A280, which indicates that the DNA was sufficiently protein-free [13]. Irinotecan hydrochloride (CPT-11) stock solution (1.0 × 10−3mol L−1) was prepared by dissolving its powder (Sigma Chem. Co., USA) in deionized water and stored in a cool and dark place. Phosphate buffer (20 mmol L−1, pH 7.5) and potassium chloride (KCl) for adjusting the ionic strength were purchased from Fluka (USA). Neutral Red dye stock solution (1.0 × 10−3mol L−1) was prepared by dissolving its crystals (Sigma-Aldrich) in water and diluted to the required volume.


### 2.2. Spectrophotometric Measurements

The equilibrium binding constant (Kb) for the ct-DNA binding of CPT-11 was determined by spectrophotometric titration at various temperatures and salt concentrations. A fixed amount of CPT-11 in 2.0 mmol L−1 phosphate buffer at pH 7.5 was titrated with increasing amounts of ds-DNA stock solutions at various temperatures, that is, 30, 35, 40, and 45°C or at various salt concentrations, for example, 10, 20, 50, and 100 mmol L−1. The spectra in the wavelength range of 200–700 nm were recorded by using a Shimadzu model 1650 UV-Vis spectrophotometer equipped with a FISONS model HAAKE D1 cell-temperature controller. For the determination of Kb at various salt concentrations, the cell compartments were thermostated at 25±0.1°C and for the determination of Kb at various temperatures the salt concentration was kept constant at 10 mmol L−1.


## 2.1. Chemicals

Deoxyribonucleic acid sodium salt from calf thymus (Sigma Chem. Co., USA) was used without further purification, and its stock solution was prepared by dissolving an appropriate amount of DNA in doubly distilled water and stored at 4°C. The concentration of DNA in stock solution was determined by UV absorption at 260 nm using a molar absorption coefficientε260=6600 L mol−1cm−1. Purity of the DNA was checked by monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of >1.8 at A260/A280, which indicates that the DNA was sufficiently protein-free [13]. Irinotecan hydrochloride (CPT-11) stock solution (1.0 × 10−3mol L−1) was prepared by dissolving its powder (Sigma Chem. Co., USA) in deionized water and stored in a cool and dark place. Phosphate buffer (20 mmol L−1, pH 7.5) and potassium chloride (KCl) for adjusting the ionic strength were purchased from Fluka (USA). Neutral Red dye stock solution (1.0 × 10−3mol L−1) was prepared by dissolving its crystals (Sigma-Aldrich) in water and diluted to the required volume.


## 2.2. Spectrophotometric Measurements

The equilibrium binding constant (Kb) for the ct-DNA binding of CPT-11 was determined by spectrophotometric titration at various temperatures and salt concentrations. A fixed amount of CPT-11 in 2.0 mmol L−1 phosphate buffer at pH 7.5 was titrated with increasing amounts of ds-DNA stock solutions at various temperatures, that is, 30, 35, 40, and 45°C or at various salt concentrations, for example, 10, 20, 50, and 100 mmol L−1. The spectra in the wavelength range of 200–700 nm were recorded by using a Shimadzu model 1650 UV-Vis spectrophotometer equipped with a FISONS model HAAKE D1 cell-temperature controller. For the determination of Kb at various salt concentrations, the cell compartments were thermostated at 25±0.1°C and for the determination of Kb at various temperatures the salt concentration was kept constant at 10 mmol L−1.


## 3. Results and Discussion

### 3.1. Spectrophotometric Confirmation on the Interaction of CPT-11 with ds-DNA

UV visible spectrophotometry is the most common and convenient way to study the interaction between small molecules or rare earth complexes and nucleic acids. Molecules containing aromatic or phosphate chromophore groups can interact with the double helix structure of DNA; therefore, the interaction between them can be investigated according to changes in the absorption spectra before and after the reaction.The red shift (or blue shift), hyperchromic (or hypochromic) effects, and the isochromatic point are spectral properties of DNA-drug interaction, which are closely related with the double helix structure [21]. The hypochromicity at the maximum absorption of DNA (260 nm) is indicating the compaction of DNA due to the electrostatic interaction. Intercalation induces the hyperchromicity at this wavelength [22].In order to validate the interaction between CPT-11 and ds-DNA, the UV-Vis absorption spectra of CPT-11 in the presence of different concentrations of ct-DNA were measured (Figure1). A remarkable decrease in the absorption intensity of CPT-11 at 368 nm followed by hyperchromic effect at 280 nm was especially for the solution with a lower ionic strength, that is, 4.0 mM. The degree of hypochromicity (H in %) increases with temperature. For example, the hypochromic effect of CPT-11 absorption spectra goes up from 10.06% to 12.84% by increasing the temperature from 30°C to 45°C in the solution containing 2 mM phosphate buffer at pH 7.5 (Table 1). In contrast, the hypochromic effect of CPT-11 absorption spectra in the presence of DNA is found to decrease from 9.3% to 5.7% when the KCl concentration in the solution containing 2 mmol L−1 phosphate buffer (pH 7.5) at 25°C is increased from 4 mmol L−1 to 100 mmol L−1 (Table 2).Table 1
Some thermodynamic parameters for the CPT-11 binding to ds-DNA at various temperatures.
T(K)
H
* (%)
K
b (L mol−1)
Δ
H
o (KJ mol−1)
Δ
S
o (KJ mol−1 K−1)
303
10.07
5.77 × 103
51.15
241.08
308
10.85
8.77 × 103
313
11.82
1.11 × 104
318
12.84
1.54 × 104
*Hypochromicity.Table 2
Hypochromicity (H), total equilibrium binding constant (Kb), nonelectrostatic equilibrium binding constant (Kt0), and ratio of Kt0 to Kb at various salt concentrations.
[K+] (mol L−1)
H (%)
K
b (L mol−1)
K
t
0 (L mol−1)
K
t
0
/
K
b
(
%
)
0.01
8.21
6.37 × 104
8.81 × 102
1.38
0.02
7.24
3.06 × 104
8.90 × 102
2.91
0.05
6.27
2.56 × 104
9.06 × 102
3.53
0.10
5.74
4.90 × 103
9.22 × 102
18.81Figure 1
Absorption spectra of CPT-11 in the presence of ds-DNA at different concentrations.CDNA = 0.0, 6.1, 12.2, 17.9, 23.8, 29.5, 35.2, 40.8, 46.3, 51.8, 57.2, 62.5, 67.8, 73.0, and 78.1 μmol·L−1 for curves 1–15, and CCPT-11 = 30.0 μmol L−1 in phosphate buffer (0.002 mol L−1, pH 7.5).Spectrophotometric titration curves measured in the wavelength region between 200 and 450 nm revealed that an isosbestic point is present at 296 nm, indicating that there is an equilibrium state between CPT-11 and ds-DNA. These remarkable hypochromic effects without a red shift indicate the existence of a strong interaction between CPT-11 and ds-DNA, most probably via the electrostatic mode of CPT-11 within the ds-DNA double helix as well as intercalation especially at high ionic strength.


### 3.2. Thermodynamic Constants

In order to further investigate the interaction mode of CPT-11 with ds-DNA, the binding constant between drug-DNA at different temperatures and ionic strengths was calculated according to the double-reciprocal equation [23–25]:
(1)A0A-A0=εGεH-G-εG+εGεH-G-εG×1Kb[DNA],
where “A0” and “A” are the absorbances of CPT-11 in the absence and presence of DNA, respectively, and εG and εH-G are their extinction coefficients. The double reciprocal plots of A0/(A-A0) versus 1/[DNA] were linear and the binding constants (Kb) were calculated from the ratio of the intercept to the slope equations.Although there have been many studies on the DNA binding of drugs to DNA, the measurements of thermodynamic parameters such as standard enthalpy(ΔH°), entropy (ΔS°), and free energy (ΔG°) changes upon binding of drugs to ds-DNA have been exceptionally rare. In fact, the thermodynamic parameters of DNA-drug complex are essential for a thorough understanding of the driving forces governing the binding of drugs to DNA. To study the thermodynamic parameters of the ct-DNA binding of CPT-11, the binding constant of CPT-11 to ct-DNA has been determined at various temperatures, that is, 30, 35, 40, and 45°C by spectrophotometric titration and the data were analyzed by (1). Typical reciprocal plots to determine the binding constant (Kb) at different temperatures are shown in Figure 2 and the results of the Kb determination are tabulated in Table 1. The values of the Kb for the binding of CPT-11 with ds-DNA are nearly in accordance with the electrochemical method reported with Hajian and Huat [13]. The determination of the binding constants at various temperatures provides a good means to indirectly calculate the thermodynamic parameter of the DNA binding through the van’t Hoff plot of 1/T versus lnKb in the corresponding temperature range (see Figure 3) [26–28]. Assuming that the enthalpy change (ΔH°) is independent of temperature over the range of employed temperatures, ΔH° of DNA-binding reaction of CPT-11 is immediately obtained from the van’t Hoff plot (Table 1). The striking observation of this table is that CPT-11 binds endothermically to ds-DNA because the Kb value increases with temperature.Figure 2
Typical plots ofA0/(A-A0) versus 1/[ds-DNA] for the determination of the equilibrium binding constant (Kb) based on (1) of the ds-DNA binding of CPT-11 at various temperatures.Figure 3
The van’t Hoff plot oflnKb versus 1/T for the binding of CPT-11 to ds-DNA. All of conditions are similar to Figure 1.


### 3.3. Salt-Dependent Binding of CPT-11 to ds-DNA

By using the 3D-Chem draw software on the total molecule charges, the results show that CPT-11 is a cationic molecule. Therefore it can be expected that its binding to DNA is thermodynamically linked to the amount of K+ bound to DNA. As a result, the DNA binding constant (Kb) of CPT-11 will depend on the total of K+ concentration in the solution. In order to investigate such effect, we have determined the Kb of the DNA binding of CPT-11 at various concentrations of KCl by means of spectrophotometric titration and the results are tabulated in Table 2. The binding constants reported here were obtained over the concentration range 0.010–0.100 mol L−1 KCl in order to apply the polyelectrolyte theory to calculate the nonelectrostatic binding constants and separate the total change in binding free energy into its electrostatic and non-electrostatic contributions. The salt concentrations of 0.010–0.100 mol L−1 were selected in this study because the polyelectrolyte theories used for subsequent analysis are based on limiting laws that are strictly applicable to salt concentrations lower than 0.100 mol L−1. It has been reported that the dependence of Kb on salt concentration becomes nonlinear at higher concentrations of salt [29]. The plot of ln [K+] against lnKb for the binding of CPT-11 to ds-DNA is shown in Figure 4. It is clear that the binding constant decreases by increasing the salt concentration. This is due to the stoichiometric amount of counter ion release that follows the binding of the cationic molecule [30], suggesting that electrostatic interactions are also involved in the DNA binding event. Using the slope from the linear fitting of Figure 4, the non-electrostatic binding constant (Kt0) at various concentrations of KCl ([M+]) can be calculated according to the following polyelectrolyte theory [30, 31]:
(2)lnKb=lnKt0+Zξ-1{ln(γ±δ)}+Zψ(ln[M+]),
where Z is estimated from the slope of the regression line in Figure 4. Z is the partial charge on the binding ligand involved in the DNA interaction as predicted by polyelectrolyte theory, ψ is the fraction of counterions associated with each DNA phosphate (ψ=0.88 for double-stranded B form DNA), γ± is the mean activity coefficient for M+, and the remaining terms are constants for the double-stranded DNA in the B form, that is, ζ=4.2 and δ=0.56. The results of the calculations are summarized in Table 2 along with the percentage of non-electrostatic binding constant (Kt0) contribution to the total binding constants (Kb) at various concentrations of K+. These Kt0 can be taken as a measure of how large the non-electrostatic forces can stabilize the ligand-DNA interaction. In contrast to the Kb values which are salt dependent, the magnitude of Kt0 is constant throughout the concentration of KCl employed with the average value of 7.37±0.99  (×103) mol−1 L. This is consistent with the expectation for the salt independency of this parameter. Although the values of Kt0 are constant throughout the concentrations of salt, the percentage of Kt0 contributions to the Kb increases significantly and reaches a maximum of 97.6% at [K+] = 0.1 mol L−1.Figure 4
The effect of [K+] on the equilibrium binding constant (Kb) for the binding of CPT-11 to ds-DNA.Further analysis is also possible to dissect the standard binding free energy change(ΔG°) for the binding of CPT-11 to ds-DNA into its electrostatic (ΔGpe0) and non-electrostatic (ΔGt0) contributions at a given concentration of KCl [31]. Table 3 summarizes the results of the calculation of energetics for the binding of CPT-11 to ds-DNA at various concentrations of salt. The total binding free energy changes listed in Table 3 were calculated based on the standard Gibbs relation:
(3)ΔG0=-RTlnKb,
where R is the gas constant and T is the temperature in Kelvin. The salt dependence of the binding constant (Figure 4) is defined as the slope, SK:
(4)SK=δlnKbδln[K+]=-ZΨ.
The quantity of SK is equivalent to the number of counterions released upon binding of the molecule with net charge Z. Based on Figure 4 it is found that approximately 0.98 counterion is liberated from DNA polymer upon binding of CPT-11. Using this value, which is equal to Zψ  (ψ=0.88), the charge on CPT-11 molecule (Z) can then be calculated and the value of +0.47 is obtained. The SK value can also be used to calculate the polyelectrolyte standard free energy change (ΔGpe0) contribution to the overall free energy change (ΔG°) at a given KCl concentration by the relation [13, 21–34]:
(5)ΔGpe0=(SK)RTln[K+].
The difference between the Gibbs free energy changes (ΔG°) and ΔGpe0 is defined as the non-electrostatic free energy change (ΔGt0):
(6)ΔGt0=ΔG0-ΔGpe0.
It is clearly observed from Table 3 that the standard electrostatic DNA binding free energy change (ΔGpe0) of CPT-11 as predicted by the electrolyte theory decreases with the increase in salt concentration. In contrast, the non-electrostatic binding free energy change (ΔGt0) is not affected by salt concentration in the solution and remains constant at −16.35±1.63 kJ mol−1 in the KCl concentrations 0.01 to 0.1 mol L−1. This value of non-electrostatic binding free energy contributes approximately 58.9% to total binding free energy change (ΔG°) for the solution containing 0.004 mol L−1 K+ and increases to 73.23% for solution with 0.1 mol L−1 K+, suggesting that the stabilization of the DNA binding event both at low and high concentrations of salt is clearly dominated by non-electrostatic processes.Table 3
The effect of salt concentration (KCl) on free energy changes (ΔG0, ΔGpe0 and ΔGt0).
[K+] (mol L−1)
Δ
G
0 (KJ mol−1)
Δ
G
pe
0 (kJ mol−1)
Δ
G
t
0 (kJ mol−1)
Δ
G
t
0
/
Δ
G
0
(
%
)
0.01
−27.41
−11.27
−16.14
58.88
0.02
−25.59
-
9.57
−16.02
62.59
0.05
−25.15
-
7.33
−17.82
70.85
0.10
−21.05
-
5.64
−15.42
73.23


### 3.4. Interaction of NR with ds-DNA by Spectrophotometric Titration

Figure5 shows the absorption spectra of the NR with the addition of DNA. It can be seen that the absorption peak of the NR at approximately 455 nm exhibits a gradual decrease and a slight red shift with the increasing concentration of DNA, and a new band at about 545 nm appears. An isosbestic point at 490 nm provides evidence of the new DNA-NR complex formation. According to the double-reciprocal equation, the binding constant for NR binding to ds-DNA is calculated as 2.74 × 104 mol−1 L. NR can intercalate into the base pairs of the double helix DNA uniquely [34], so it was employed as the molecule probe in the study.Figure 5
Absorption spectra for NR dye in the presence of ds-DNA at different concentrations.CDNA = 0.0, 6.7, 13.4, 20.1, 26.8, 33.5, 40.2, 46.9, 53.6, 60.3, and 67.0 μmol L−1 for curves 1–11, CNR = 10.0 μmol L−1 in phosphate buffer (0.002 mol L−1, pH 7.5).


### 3.5. Competitive Interaction of CPT-11 with the NR-DNA Complex

Further support for the mode of binding between CPT-11 and DNA is given through the competitive interaction of CPT-11 and NR with DNA. The observed band of DNA-NR complex at 531 nm gradually decreases in intensity with the increasing concentration of the added CPT-11. This band shifted slightly towards the blue end of the spectrum with the appearance of a new peak at 353 nm, which increased progressively in intensity (Figure6). This new band is the sum of the changing absorption of the NR dye (531 nm) and CPT-11 (353 nm). An isosbestic point at 414 nm provides evidence that a new species is formed during the competitive interaction and that the reaction is homogeneous. When compared with the absorption spectra of the NR dye in the absence or presence of DNA at pH 7.5 (Figure 5), the results represented the reverse process. The observed changes in intensity and position of the bands with increasing concentration of CPT-11 added to the DNA-NR solution suggested that CPT-11 diffuses into the double helix of the DNA structure by substitution of NR in the DNA-NR system. These changes in the competitive interactions have been observed in several other studies [35].Absorption spectra of the competitive interaction between CPT-11 and NR bonded to ds-DNA in the wavelength ranges of (a) 220–600 nm and (b) 400–600 nm. Conditions:CCPT-11 = 0.0, 6.7, 13.4, 20.1, 26.8, 33.5, 40.2, 46.9, 53.6, 60.3, and 67.0 μmol L−1 for curves 1–11, CNR = 10.0 μmol L−1, and CNA = 30.5 μmol L−1 in phosphate buffer (0.002 mol L−1, pH 7.5).
(a)
(b)


### 3.6. Melting Studies

Heat and alkali can destroy the double helix structure of DNA and change it into a single helix at the melting temperature(Tm). Interaction of small molecules with DNA can affect the Tm value. Intercalation binding can stabilize the double helix structure and Tm value increases above 5–8°C, but the nonintercalation binding causes no obvious increase in Tm [36, 37]. The values of Tm for ds-DNA in the absence and presence of CPT-11 were determined, respectively, by monitoring the maximum absorbance values of the systems as a function of temperature ranging from 25°C to 80°C. The melting curves are shown in Figure 7. It can be seen that the Tm value of ds DNA in the absence of CPT-11 is approximately 64°C. The observed melting temperature of DNA in the presence of CPT-11 is 67.0°C. The interaction of CPT-11 with ds-DNA does not increase the Tm value obviously, suggesting that intercalation binding is one of the minor mechanisms involved in the interaction of CPT-11 and ds-DNA.Figure 7
Thermal denaturation profile of ds-DNA in the absence and presence of CPT-11. Conditions:CDNA = 27.5 μmol L−1, CIR = 30.0 μmol L−1, phosphate buffer (0.002 mol L−1, pH 7.5).


## 3.1. Spectrophotometric Confirmation on the Interaction of CPT-11 with ds-DNA

UV visible spectrophotometry is the most common and convenient way to study the interaction between small molecules or rare earth complexes and nucleic acids. Molecules containing aromatic or phosphate chromophore groups can interact with the double helix structure of DNA; therefore, the interaction between them can be investigated according to changes in the absorption spectra before and after the reaction.The red shift (or blue shift), hyperchromic (or hypochromic) effects, and the isochromatic point are spectral properties of DNA-drug interaction, which are closely related with the double helix structure [21]. The hypochromicity at the maximum absorption of DNA (260 nm) is indicating the compaction of DNA due to the electrostatic interaction. Intercalation induces the hyperchromicity at this wavelength [22].In order to validate the interaction between CPT-11 and ds-DNA, the UV-Vis absorption spectra of CPT-11 in the presence of different concentrations of ct-DNA were measured (Figure1). A remarkable decrease in the absorption intensity of CPT-11 at 368 nm followed by hyperchromic effect at 280 nm was especially for the solution with a lower ionic strength, that is, 4.0 mM. The degree of hypochromicity (H in %) increases with temperature. For example, the hypochromic effect of CPT-11 absorption spectra goes up from 10.06% to 12.84% by increasing the temperature from 30°C to 45°C in the solution containing 2 mM phosphate buffer at pH 7.5 (Table 1). In contrast, the hypochromic effect of CPT-11 absorption spectra in the presence of DNA is found to decrease from 9.3% to 5.7% when the KCl concentration in the solution containing 2 mmol L−1 phosphate buffer (pH 7.5) at 25°C is increased from 4 mmol L−1 to 100 mmol L−1 (Table 2).Table 1
Some thermodynamic parameters for the CPT-11 binding to ds-DNA at various temperatures.
T(K)
H
* (%)
K
b (L mol−1)
Δ
H
o (KJ mol−1)
Δ
S
o (KJ mol−1 K−1)
303
10.07
5.77 × 103
51.15
241.08
308
10.85
8.77 × 103
313
11.82
1.11 × 104
318
12.84
1.54 × 104
*Hypochromicity.Table 2
Hypochromicity (H), total equilibrium binding constant (Kb), nonelectrostatic equilibrium binding constant (Kt0), and ratio of Kt0 to Kb at various salt concentrations.
[K+] (mol L−1)
H (%)
K
b (L mol−1)
K
t
0 (L mol−1)
K
t
0
/
K
b
(
%
)
0.01
8.21
6.37 × 104
8.81 × 102
1.38
0.02
7.24
3.06 × 104
8.90 × 102
2.91
0.05
6.27
2.56 × 104
9.06 × 102
3.53
0.10
5.74
4.90 × 103
9.22 × 102
18.81Figure 1
Absorption spectra of CPT-11 in the presence of ds-DNA at different concentrations.CDNA = 0.0, 6.1, 12.2, 17.9, 23.8, 29.5, 35.2, 40.8, 46.3, 51.8, 57.2, 62.5, 67.8, 73.0, and 78.1 μmol·L−1 for curves 1–15, and CCPT-11 = 30.0 μmol L−1 in phosphate buffer (0.002 mol L−1, pH 7.5).Spectrophotometric titration curves measured in the wavelength region between 200 and 450 nm revealed that an isosbestic point is present at 296 nm, indicating that there is an equilibrium state between CPT-11 and ds-DNA. These remarkable hypochromic effects without a red shift indicate the existence of a strong interaction between CPT-11 and ds-DNA, most probably via the electrostatic mode of CPT-11 within the ds-DNA double helix as well as intercalation especially at high ionic strength.


## 3.2. Thermodynamic Constants

In order to further investigate the interaction mode of CPT-11 with ds-DNA, the binding constant between drug-DNA at different temperatures and ionic strengths was calculated according to the double-reciprocal equation [23–25]:
(1)A0A-A0=εGεH-G-εG+εGεH-G-εG×1Kb[DNA],
where “A0” and “A” are the absorbances of CPT-11 in the absence and presence of DNA, respectively, and εG and εH-G are their extinction coefficients. The double reciprocal plots of A0/(A-A0) versus 1/[DNA] were linear and the binding constants (Kb) were calculated from the ratio of the intercept to the slope equations.Although there have been many studies on the DNA binding of drugs to DNA, the measurements of thermodynamic parameters such as standard enthalpy(ΔH°), entropy (ΔS°), and free energy (ΔG°) changes upon binding of drugs to ds-DNA have been exceptionally rare. In fact, the thermodynamic parameters of DNA-drug complex are essential for a thorough understanding of the driving forces governing the binding of drugs to DNA. To study the thermodynamic parameters of the ct-DNA binding of CPT-11, the binding constant of CPT-11 to ct-DNA has been determined at various temperatures, that is, 30, 35, 40, and 45°C by spectrophotometric titration and the data were analyzed by (1). Typical reciprocal plots to determine the binding constant (Kb) at different temperatures are shown in Figure 2 and the results of the Kb determination are tabulated in Table 1. The values of the Kb for the binding of CPT-11 with ds-DNA are nearly in accordance with the electrochemical method reported with Hajian and Huat [13]. The determination of the binding constants at various temperatures provides a good means to indirectly calculate the thermodynamic parameter of the DNA binding through the van’t Hoff plot of 1/T versus lnKb in the corresponding temperature range (see Figure 3) [26–28]. Assuming that the enthalpy change (ΔH°) is independent of temperature over the range of employed temperatures, ΔH° of DNA-binding reaction of CPT-11 is immediately obtained from the van’t Hoff plot (Table 1). The striking observation of this table is that CPT-11 binds endothermically to ds-DNA because the Kb value increases with temperature.Figure 2
Typical plots ofA0/(A-A0) versus 1/[ds-DNA] for the determination of the equilibrium binding constant (Kb) based on (1) of the ds-DNA binding of CPT-11 at various temperatures.Figure 3
The van’t Hoff plot oflnKb versus 1/T for the binding of CPT-11 to ds-DNA. All of conditions are similar to Figure 1.


## 3.3. Salt-Dependent Binding of CPT-11 to ds-DNA

By using the 3D-Chem draw software on the total molecule charges, the results show that CPT-11 is a cationic molecule. Therefore it can be expected that its binding to DNA is thermodynamically linked to the amount of K+ bound to DNA. As a result, the DNA binding constant (Kb) of CPT-11 will depend on the total of K+ concentration in the solution. In order to investigate such effect, we have determined the Kb of the DNA binding of CPT-11 at various concentrations of KCl by means of spectrophotometric titration and the results are tabulated in Table 2. The binding constants reported here were obtained over the concentration range 0.010–0.100 mol L−1 KCl in order to apply the polyelectrolyte theory to calculate the nonelectrostatic binding constants and separate the total change in binding free energy into its electrostatic and non-electrostatic contributions. The salt concentrations of 0.010–0.100 mol L−1 were selected in this study because the polyelectrolyte theories used for subsequent analysis are based on limiting laws that are strictly applicable to salt concentrations lower than 0.100 mol L−1. It has been reported that the dependence of Kb on salt concentration becomes nonlinear at higher concentrations of salt [29]. The plot of ln [K+] against lnKb for the binding of CPT-11 to ds-DNA is shown in Figure 4. It is clear that the binding constant decreases by increasing the salt concentration. This is due to the stoichiometric amount of counter ion release that follows the binding of the cationic molecule [30], suggesting that electrostatic interactions are also involved in the DNA binding event. Using the slope from the linear fitting of Figure 4, the non-electrostatic binding constant (Kt0) at various concentrations of KCl ([M+]) can be calculated according to the following polyelectrolyte theory [30, 31]:
(2)lnKb=lnKt0+Zξ-1{ln(γ±δ)}+Zψ(ln[M+]),
where Z is estimated from the slope of the regression line in Figure 4. Z is the partial charge on the binding ligand involved in the DNA interaction as predicted by polyelectrolyte theory, ψ is the fraction of counterions associated with each DNA phosphate (ψ=0.88 for double-stranded B form DNA), γ± is the mean activity coefficient for M+, and the remaining terms are constants for the double-stranded DNA in the B form, that is, ζ=4.2 and δ=0.56. The results of the calculations are summarized in Table 2 along with the percentage of non-electrostatic binding constant (Kt0) contribution to the total binding constants (Kb) at various concentrations of K+. These Kt0 can be taken as a measure of how large the non-electrostatic forces can stabilize the ligand-DNA interaction. In contrast to the Kb values which are salt dependent, the magnitude of Kt0 is constant throughout the concentration of KCl employed with the average value of 7.37±0.99  (×103) mol−1 L. This is consistent with the expectation for the salt independency of this parameter. Although the values of Kt0 are constant throughout the concentrations of salt, the percentage of Kt0 contributions to the Kb increases significantly and reaches a maximum of 97.6% at [K+] = 0.1 mol L−1.Figure 4
The effect of [K+] on the equilibrium binding constant (Kb) for the binding of CPT-11 to ds-DNA.Further analysis is also possible to dissect the standard binding free energy change(ΔG°) for the binding of CPT-11 to ds-DNA into its electrostatic (ΔGpe0) and non-electrostatic (ΔGt0) contributions at a given concentration of KCl [31]. Table 3 summarizes the results of the calculation of energetics for the binding of CPT-11 to ds-DNA at various concentrations of salt. The total binding free energy changes listed in Table 3 were calculated based on the standard Gibbs relation:
(3)ΔG0=-RTlnKb,
where R is the gas constant and T is the temperature in Kelvin. The salt dependence of the binding constant (Figure 4) is defined as the slope, SK:
(4)SK=δlnKbδln[K+]=-ZΨ.
The quantity of SK is equivalent to the number of counterions released upon binding of the molecule with net charge Z. Based on Figure 4 it is found that approximately 0.98 counterion is liberated from DNA polymer upon binding of CPT-11. Using this value, which is equal to Zψ  (ψ=0.88), the charge on CPT-11 molecule (Z) can then be calculated and the value of +0.47 is obtained. The SK value can also be used to calculate the polyelectrolyte standard free energy change (ΔGpe0) contribution to the overall free energy change (ΔG°) at a given KCl concentration by the relation [13, 21–34]:
(5)ΔGpe0=(SK)RTln[K+].
The difference between the Gibbs free energy changes (ΔG°) and ΔGpe0 is defined as the non-electrostatic free energy change (ΔGt0):
(6)ΔGt0=ΔG0-ΔGpe0.
It is clearly observed from Table 3 that the standard electrostatic DNA binding free energy change (ΔGpe0) of CPT-11 as predicted by the electrolyte theory decreases with the increase in salt concentration. In contrast, the non-electrostatic binding free energy change (ΔGt0) is not affected by salt concentration in the solution and remains constant at −16.35±1.63 kJ mol−1 in the KCl concentrations 0.01 to 0.1 mol L−1. This value of non-electrostatic binding free energy contributes approximately 58.9% to total binding free energy change (ΔG°) for the solution containing 0.004 mol L−1 K+ and increases to 73.23% for solution with 0.1 mol L−1 K+, suggesting that the stabilization of the DNA binding event both at low and high concentrations of salt is clearly dominated by non-electrostatic processes.Table 3
The effect of salt concentration (KCl) on free energy changes (ΔG0, ΔGpe0 and ΔGt0).
[K+] (mol L−1)
Δ
G
0 (KJ mol−1)
Δ
G
pe
0 (kJ mol−1)
Δ
G
t
0 (kJ mol−1)
Δ
G
t
0
/
Δ
G
0
(
%
)
0.01
−27.41
−11.27
−16.14
58.88
0.02
−25.59
-
9.57
−16.02
62.59
0.05
−25.15
-
7.33
−17.82
70.85
0.10
−21.05
-
5.64
−15.42
73.23


## 3.4. Interaction of NR with ds-DNA by Spectrophotometric Titration

Figure5 shows the absorption spectra of the NR with the addition of DNA. It can be seen that the absorption peak of the NR at approximately 455 nm exhibits a gradual decrease and a slight red shift with the increasing concentration of DNA, and a new band at about 545 nm appears. An isosbestic point at 490 nm provides evidence of the new DNA-NR complex formation. According to the double-reciprocal equation, the binding constant for NR binding to ds-DNA is calculated as 2.74 × 104 mol−1 L. NR can intercalate into the base pairs of the double helix DNA uniquely [34], so it was employed as the molecule probe in the study.Figure 5
Absorption spectra for NR dye in the presence of ds-DNA at different concentrations.CDNA = 0.0, 6.7, 13.4, 20.1, 26.8, 33.5, 40.2, 46.9, 53.6, 60.3, and 67.0 μmol L−1 for curves 1–11, CNR = 10.0 μmol L−1 in phosphate buffer (0.002 mol L−1, pH 7.5).


## 3.5. Competitive Interaction of CPT-11 with the NR-DNA Complex

Further support for the mode of binding between CPT-11 and DNA is given through the competitive interaction of CPT-11 and NR with DNA. The observed band of DNA-NR complex at 531 nm gradually decreases in intensity with the increasing concentration of the added CPT-11. This band shifted slightly towards the blue end of the spectrum with the appearance of a new peak at 353 nm, which increased progressively in intensity (Figure6). This new band is the sum of the changing absorption of the NR dye (531 nm) and CPT-11 (353 nm). An isosbestic point at 414 nm provides evidence that a new species is formed during the competitive interaction and that the reaction is homogeneous. When compared with the absorption spectra of the NR dye in the absence or presence of DNA at pH 7.5 (Figure 5), the results represented the reverse process. The observed changes in intensity and position of the bands with increasing concentration of CPT-11 added to the DNA-NR solution suggested that CPT-11 diffuses into the double helix of the DNA structure by substitution of NR in the DNA-NR system. These changes in the competitive interactions have been observed in several other studies [35].Absorption spectra of the competitive interaction between CPT-11 and NR bonded to ds-DNA in the wavelength ranges of (a) 220–600 nm and (b) 400–600 nm. Conditions:CCPT-11 = 0.0, 6.7, 13.4, 20.1, 26.8, 33.5, 40.2, 46.9, 53.6, 60.3, and 67.0 μmol L−1 for curves 1–11, CNR = 10.0 μmol L−1, and CNA = 30.5 μmol L−1 in phosphate buffer (0.002 mol L−1, pH 7.5).
(a)
(b)


## 3.6. Melting Studies

Heat and alkali can destroy the double helix structure of DNA and change it into a single helix at the melting temperature(Tm). Interaction of small molecules with DNA can affect the Tm value. Intercalation binding can stabilize the double helix structure and Tm value increases above 5–8°C, but the nonintercalation binding causes no obvious increase in Tm [36, 37]. The values of Tm for ds-DNA in the absence and presence of CPT-11 were determined, respectively, by monitoring the maximum absorbance values of the systems as a function of temperature ranging from 25°C to 80°C. The melting curves are shown in Figure 7. It can be seen that the Tm value of ds DNA in the absence of CPT-11 is approximately 64°C. The observed melting temperature of DNA in the presence of CPT-11 is 67.0°C. The interaction of CPT-11 with ds-DNA does not increase the Tm value obviously, suggesting that intercalation binding is one of the minor mechanisms involved in the interaction of CPT-11 and ds-DNA.Figure 7
Thermal denaturation profile of ds-DNA in the absence and presence of CPT-11. Conditions:CDNA = 27.5 μmol L−1, CIR = 30.0 μmol L−1, phosphate buffer (0.002 mol L−1, pH 7.5).


## 4. Conclusion

It has been demonstrated that the binding of CPT-11 to ds-DNA is quite strong as indicated by the remarkable hypochromicity and equilibrium binding constant depending on the temperature and salt concentration in the medium. From the van’t Hoff plot, it has been revealed that the CPT-11 binds endothermically to ds-DNA, and therefore, its DNA binding is enthalpically driven as indicated by the large positive enthalpy and entropy changes. The salt dependence of the DNA binding of CPT-11 indicates that the charge release (Z) when ds-DNA interacts with CPT-11 in accordance with the polyelectrolyte theory is +0.47. The DNA binding constant of CPT-11 at a low concentration of salt (0.01 mol L−1) is dominated by electrostatic interaction (87.38%) while that at a high concentration of salt is partially controlled by non-electrostatic processes (97.57%). Nevertheless, the stabilization of the DNA binding in both cases is governed by electrostatic and non-electrostatic processes as reflected by the ratio of ΔGt0/ΔG0 which are in the range of 58.88% (low salt) to 73.23% (high salt). A thermal denaturation study has suggested that the Tm value of ds-DNA increased approximately by 3.0°C in the presence of CPT-11. The small value of ΔTm suggests that irinotecan binds to ds-DNA by non-intercalation mode.



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*Source: 380352-2012-09-29.xml*