Many antiviral, anticancer, antibiotic drugs demonstrate their initial biological effects from interaction with Deoxyribonucleic acid (DNA). Hence, these biomacromolecules illustrate the main purpose of drug progress strategies designed to develop next-generation therapeutics for illness such as cancer . Cancer is an important cause of death around the world, each year many people die because they can’t receive appropriate treatment, chemotherapy is expensive and few people can afford it, so finding a new and inexpensive drug with both anticancer effects and saving its own properties can be a good way to treat cancers easily in future . First of all, we need to investigate their interaction with biomacromolecules such as DNA. DNA is the most important part of the cell component that carries on genetical information and mostly is a target for tiny molecules like drug-nanocarriers. Such nanocomposites display DNA-targeted pharmacological functions as they disturb DNA replication, the primary phase of cell division and cell growth. They can also interfere with the synthesis of protein transcription processes . Nowadays the use of magnetic nanoparticles (MNPs) in medical applications has exceeded expectations . Small size and vast surface area of MNPs can cause the aggregation of nanoparticles. So coating the surface of magnetic cores by surface engineering is an important step in synthesis new nanoparticles for drug carrying systems. Fe3O4 nanoparticles as core structure can be coated by various inorganic or organic shell substances, which developed to enhance the effect of NPs on drug loading and keep them versus aggregation and induction the new attributes to the nanoparticles e.g. catalytic properties, biological diagnosis applications, thermo responsiveness. In this study, in order to protect magnetic nanoparticles from aggregation, layered double hydroxides were applied as the shell structure . Layered double hydroxides (LDHs) are biocompatible 2D inorganic compound and receive great attention as drug carriers because of the widespread requirements to improve advanced drug delivery systems (DDS), that is certainly relevant to universal issues like as the well-being and health care of human being, along with extended life, and dominance of illnesses . The most noteworthy feature of LDH as a drug delivery carrier is particle size dependency for cellular uptake behavior. The particle size of LDH nanoparticles smaller than ~250 nm can be transpired into cells via clathrin-mediated endocytosis . Loading drug on nanoparticles can improve the effectiveness and toxicity of drugs on cancer cells and it can also save and protect the drug till achieving the target cells . Lamivudine is called 3TC is an antiretroviral drug and nucleoside analog, nucleoside reverse transcriptase (RT) inhibitors , the main application of Lamivudine refers to prevention and treatment of HIV/AIDS  .In recent studies, results indicated that the binding mode of Lamivudine and CT-DNA was groove binding . The main goal of this study is to determine the binding mode of Fe3O4@LDH@ Lamivudine with CT-DNA by spectroscopic methods.
EXPERIMENTAL AND METHODS MATERIALS
Methylene blue (MB), Hoechst 33258, Deoxyribonucleic acid sodium salt from calf thymus (CT-DNA) purchased from Sigma Aldrich company, used without any purifications. Tris-HCl, phosphate buffer saline (PBS), Iron (III) chloride hexahydrate (FeCl3.6H2O), Iron (II) Chloride tetrahydrate (FeCl2.4H2O), ammonia (25%), Hydrogen chloride 37% (HCl), Sodium hydroxide (NaOH), Calcium Nitrate Tetrahydrate (Ca(NO3)2.4H2O), aluminum nitrate, nonahydrate (Al(NO3).9H2O), Methanol, Ethanol, sodium carbonate (Na2CO3) and Lamivudine, were provided and purchased from Merck Company.
Apparatus and methods
The morphology and particle size of the nanocomposites were characterized by transmission electronic microscopy (TEM, Philips, EM208, 100 kV). Powder X-ray diffraction (XRD, Rigaku, D/Max-RA, Cu Ka) was applied to recognize the crystal structure of the samples. The Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded with KBr pellets using a Bruker ALPHA FT-IR spectrometer. Fluorescence measurements were carried out by JASCO spectrofluorimeter (FP6200) using a 1 cm path length of quartz cuvette. Uv-visible spectra were recorded by Agilent 8453. CD measurements were carried out by a JASCO (J-810) spectropolarimeter at 25 °C. The viscosity was measured (SCHOT AVS 450) at room temperature.
Synthesis of Fe3O4
The Fe3O4 magnetic nanoparticles were synthesis trough a co-precipitation method. In order to achieve this purpose, ferric chloride hexahydrate FeCl3.6H2O (3.25 g) and ferrous chloride tetrahydrate FeCl2.4H2O (1.6 g) with the molar ratio of 2Fe(III):1Fe(II) were dissolved in 250 mL of distilled water under N2 atmosphere by vigorous stirring (60 °C). Then pH value of the solution was adjusted (pH=10), by subjoining 15 mL of 25% NaOH solution quickly and reaction was performed under continuous stirring (500 rpm) for 1 h at 60 °C. The black precipitation was cooled down to the temperature of room and washed 3 times with double distilled water and ethanol to remove unreacted chemicals. The magnetite precipitates were dried at room temperature for 24 h under vacuum .
Synthesis of Fe3O4@LDH
First of all, the slick suspension got ready by ultrasonication of Fe3O4 (0.3 g) into 75 mL solvent (Vmethanol/Vwater=1/1) for 20 min. In the following step, 50 mL alkaline solution of Na2CO3 and sodium hydroxide (NaOH) (0.16 g and 0.24 g respectively) was added dropwise into the ready suspension until pH ca. 10.0 and hold for 10 min. Then 50 mL of the salt solution of 0.45 g of Aluminum (III) nitrate nonahydrate (Al(NO3)3⋅9H2O), and 0.85 g of calcium (II) nitrate tetrahydrate (Ca(NO3)2⋅4H2O) was added to the former suspension and applying the alkaline solution of Na2CO3 and NaOH, the pH of above mixture was adjusted to 10.0. The obtained slurry dispersion was stirred vigorously for 5 min then collected by a magnet and washed three times with deionized water and ethanol. Finally, Fe3O4@CaAl LDH was dried 24 h at 60 °C and the desired product was collected.
Preparing Fe3O4@LDH@ Lamivudine
Loading the Lamivudine into the structure and on the surface of Fe3O4@CaAl LDH was accomplished by appending 0.01 g of Fe3O4@CaAl LDH to 30 mL of 2.5 mM Lamivudine solution in distilled water and stirred for 24 h at 25 ᴼC. In this level, the pH value of the solution adjusted to 7.4. The resulting solid was separated by applying a magnet, then washed three times with distilled water and dried under vacuum condition at 25 ᴼC and the final product was achieved (Fe3O4@CaAl@Lamivudine) .
DNA binding studies
In the first step, the concentration of CT-DNA was measured by UV-vis absorbance at 260 nm, then calculating the concentration of CT-DNA by using (ℇP) of 6600 M-1cm-1. ℇP is the extinction coefficient at 260 nm. The entire experiments of CT-DNA were done with Tris-HCl buffer solution (pH 7.4) at room temperature. In order to investigate the binding mode of Fe3O4@CaAl@Lamivudine with CT-DNA, fluorescence and Uv-vis spectroscopy, viscosity and CD spectropolarimetry were applied .
RESULTS AND DISCUSSIONS
Characterization of the Fe3O4@LDH@Lamivudine
The XRD patterns of Fe3O4, Fe3O4@CaAl LDH, and Fe3O4@CaAl@Lamivudine were demonstrated in Fig. 1. In the XRD pattern of Fe3O4 (curve a), the diffraction peaks were identified with cubic-phase Fe3O4 crystalline structure [JCPDS-019-0629]. The (220), (311), (400), (422), (511) and (440) reflections of typical Fe3O4 in 2θ = 30.2°, 35.7°, 43.1°, 53.3°, 56.8° and 62.8° affirm the great crystallinity of the Fe3O4 nanoparticles. The XRD pattern of Fe3O4@CaAl LDH obviously illustrated the formation of CaAl LDH shell around Fe3O4 nanoparticles (curve b). moreover, the Fe3O4 reflections, the diffraction pattern of resulting Fe3O4@CaAl LDH core-shell structure, represents specified absorption peaks at 10.1°, 20.2°, 30.6°, 37.3°, 48.2°, 62.6°, 63.1° which are ascribed to the (003), (006), (009), (015), (018), (110) and (113) facets of LDH structures .
The XRD pattern of Fe3O4@CaAl@Lamivudine nanostructure undoubtedly shows the characteristic peaks of CaAl LDH and Fe3O4 nanoparticles. The broadening and shift of (003) basal reflection of Fe3O4@CaAl LDH indicates that the Lamivudine molecules are perfectly loaded on the surface and intercalated into the layers of the Fe3O4@CaAl LDH structure (curve c).
Transmission electron microscopy (TEM) images obviously demonstrated MNPs. It shows the core-shell structure of Fe3O4@LDH and Lamivudine drug which was loaded on the surface and into the structure of nanoparticles (Fig. 2). It can be clearly perceived that nanoparticles were mostly semi-spherical with similar particle size. The particle size was averagely about 125 nm in the TEM image .
The FT-IR spectra of the Lamivudine, Fe3O4, Fe3O4@CaAl-LDH, Fe3O4@CaAl@Lamivudine were shown in Fig. 3. Lamivudine exhibits main characteristic bands of carbonyl (C=O-NR2) stretching at 1632 cm-1. This band overlaps the band due to N-H bending at 1607 cm-1. The band due to stretching vibration of the imine group (R2-C=NR) is observed at 1648 cm-1. Broad bands due to the stretching vibration of -NH2 and -OH group are observed at 3300-3500 cm-1 [17, 18]. The absorption peaks at 568 cm-1 belonged to the stretching vibration mode of Fe-O bonds in Fe3O4. The FT-IR spectrum of Fe3O4@CaAl LDH, in addition to Fe3O4 absorption bands which the Fe-O vibrations of magnetite phase absorption bands at about 575 cm-1 were observed and shows the bands at 496 and 521 cm-1 that are related to the M-O vibration modes of CaAl-LDH structure (curve c). It is notable that the characteristic absorption band around 1388 cm-1 is related to the presence of CO32- in the LDH structure . NPs exhibit a strong band due to stretching mode of the -OH group at 3,400 cm−1. Further the peak at 540 cm−1 corresponds to the inherent characteristic of the MNPs . The decrease in the intensity of this band in Fe3O4@CaAl@Lamivudine spectrum clearly indicates the replacement of CO32- anions by Lamivudine molecules. Finally, the FT-IR spectrum of Fe3O4@CaAl@Lamivudine shows the characteristic peaks of Fe3O4 nanoparticles, CaAl-LDH and Lamivudine which is in agreement with the formation of Fe3O4@CaAl@Lamivudine structure (curve d) .
Interaction with calf-thymus DNA
In the first step, binding constant measurements were carried out by keeping CT-DNA concentration constant (5×10-5 M) during the experiments, Fe3O4@LDH@Lamivudine concentration varies from 0 to 13 mg mL-1. The binding constant (Kb) of Fe3O4@LDH @Lamivudine with CT-DNA was calculated by UV-Vis absorption spectral analysis and using this equation :
Kb is the binding constant, A and A0 are the absorbance of nanocomposite-DNA and DNA respectively, ℇD and ℇD-DNA are the molar extinction coefficient of nanocomposite and nanocomposite-DNA complex also respectively . The binding constant, (Kb 4.9×103 M-1) of Fe3O4@LDH@Lamivudine with DNA was calculated from ratio of intercept to slop (1/A-A0 vs. 1/[nanocomposite]). The absorbance was enhanced by increasing the concentration of nanocomposite (Fig. 4). Generally, the hyperchromism occurred due to non-covalent interactions outside the helix of DNA , the absorption band of DNA was observed at 261 nm while the λ max value for Fe3O4@LDH@Lamivudine was 268 nm, so we observed 7nm shift to higher wavelengths. The moderate red shift (bathochromic effect) was observed due to the interaction of Fe3O4@LDH@Lamivudine nanoparticles with CT-DNA. The noticeable shift in UV-visible wavelength (red shift ≥15 nm) shows the intercalative binding mode of compound to DNA helix because of the interaction of a DNA π stack with compound, on the other hand, outside binders (groove binders) indicate a minor red shift (Δλ ≤ 8 nm) .
The Kb of Fe3O4@LDH@Lamivudine and DNA complex (4.9×103 M-1) was compared with complex of Lamivudine and DNA (5±0.3 × 104 M-1), binding constant of nanocomposite is one order lower than it’s in complex of Lamivudine and DNA. Recent studies reported that, the interaction of Lamivudine with DNA was groove binding , we can conclude that, Fe3O4@LDH@Lamivudine was created a complex with DNA by groove binding.
Fluorescence emission spectroscopy
The fluorescence Spectroscopy is the most common and reliable techniques that use to investigate the interaction between DNA-compounds and specifies the binding modes . Some compounds show fluorescence properties, due to the special organic functional groups in their structures containing alicyclic carbonyl or aliphatic structures or double-bond conjugated structures. These aromatic groups show low levels of energy in π-π* transition level . Other compounds don’t show any fluorescence properties and we have to use some special kind of dyes as the probe . Ethidium bromide (EB) is a fluorophore that bind to DNA as an intercalative agent. Increasing The fluorescence of EB in the presence of DNA, because of its well-appointed intercalation between the DNA and base pairs . The other intercalative agent is Methylene Blue (MB), this probe has an important application in fluorescence spectroscopy. The intercalation of MB between two near base pairs need unstacking of the base pairs making the binding pocket, and unwrapping the helix of DNA . The most commonly used groove binding agents is Hoechst. This molecule chooses to bind to A-T rich regions of double strand DNA and interact with DNA as a minor groove binding agent. After binding to DNA, Hoechst will withstand an approximately 30-fold enhancement in fluorescence . In this study, we focused on two important probes in fluorescence (MB and Hoechst 33258), to determine the interaction of Fe3O4@LDH@Lamivudine with DNA, is associated with which kind of binding modes. Competitive fluorescence studies were carried out by using MB (5×10-6 M) as an intercalating agent, shown in Fig. 5. The DNA concentration was kept constant, while increasing the concentration of Fe3O4@LDH@Lamivudine, if compound was an intercalative one, it has to replace MB and decreasing fluorescence spectroscopy , while there weren’t observed any changes or decrement in fluorescence intensity. In the next experiment, Hoechst 33258 (5×10-6 M) was used to assure that, Fe3O4@LDH@Lamivudine interact with CT-DNA by groove binding mood (Fig. 5b). By increasing the amount of Fe3O4@LDH@Lamivudine (0 to 400 μg mL-1), excitation was observed at 412 nm and the intensity of fluorescence was decreased and fill the gap between the spectra of Hoechst and DNA Hoechst. This quenching was occurred due to the replacement of Hoechst by compound and forming minor groove binding with DNA .
Quenching can occur by various mechanisms, which are generally classified as static and dynamic quenching . Commonly, dynamic and static quenching can be diagnosed by their diver’s affiliation on temperature and excited-state lifetime [32, 33]. The Ksv value (the quenching constant of stern-volmer) was calculated by stern-volmer (Eq. 2):
In here F0 and F respectively indicate the fluorescence intensities in the absence and presence of DNA . Kq is the fluorophore quenching rate constant , the lifetime of the fluorophore in the absence of a quencher (τ0= 10-8), Ksv is quenching constant and [Q] is the concentration of compound [36, 37], which here is Fe3O4@LDH@Lamivudine. This experiment was carried out in three different temperatures; 288, 298 and 310 K. The Ksv decrease from 2.69×103 M-1 to 1.25×103 M-1 by increasing the temperature, this evidence indicates that the quenching mechanism of nanocomposite with CT-DNA involves static quenching, because Ksv decrease with increasing of temperature.
The binding constant (Kb) and the stoichiometry of binding (n) , for the formation of new complex between Fe3O4@LDH@Lamivudine and DNA were calculated using Eq. 3 :
In here, F0 and F are the intensities of fluorescence’s fluorophore in the absence and presence of various amount of CT-DNA [39, 40], n is the number of equivalent binding sites, that can be determined by the slope based on the Eq.3. The corresponding results at various temperatures (288, 298 and 310 K) are summarized in Table 1. The Kb value at 298 K was 6.43×103 L mol-1, which corresponding considerably with Kb (4.9×103 L mol-1) that was obtained from the UV-visible technique.
In order to illustrate the interaction of Fe3O4@LDH@Lamivudine with DNA, the thermodynamic parameters were estimated . The binding force of Fe3O4@LDH@Lamivudine with CT-DNA was calculated by Kb. The plot of log Kb versus 1/T (Fig. 6) helped us to compute the enthalpy (ΔH), entropy (ΔS) (Eq. 4) and free energy (ΔG) changes by Eq. 5 [31, 42].
Which Kb is the binding constant at three different temperatures (288, 298 and 310 K) and R is the gas constant (8.314 J mol-1 K-1). Table 1, also display the amount of ΔH, ΔS and ΔG. According to obtained results ΔH<0 and ΔS<0, this results indicated that the interaction between DNA and nanocomposite is hydrogen bond and Vander-Waals force , and the negative amount of ΔG indicated the spontaneous interaction between nanocomposite and DNA .
DNA viscosity experiment
Binding of Compounds to DNA can cause various changes in the length of DNA helix, intercalating binding mode typically increase the length of DNA so a significant change in viscosity measurements will occur , increasing the viscosity of DNA is due to compound’s intercalation binding with DNA that causes the separation of base pairs in DNA molecules . Groove binding and electrostatic agents don’t make significant changes in the viscosity and length of DNA . The changes in the length of DNA were evaluated, by the values of relative specific viscosity (η/η0)1/3 that were plotted against r (r = [compound]/[DNA]) . The viscosity of the samples (η), the relative specific viscosity (η/η 0)1/3, which η0 and η are the specific viscosity contributions of CT-DNA in the absence and in the presence of the compound [47, 48]. In this study the concentration of DNA keeps constant (5×10-5 M) while varying the concentration of compound (0 to 1 M), by increasing the amount of compound, no change in viscosity was observed (Fig. 7), and the binding mode was described as groove binding .
Circular dichroism spectroscopy
CD spectroscopy is a beneficial instrument to recognize alterations in CT-DNA . Morphology changes during the drug–DNA intercalations, since the positive band along with base stacking (273 nm) and the negative one owing to right-handed helicity (246 nm) are entirely sensitive to the interaction mode of DNA with small molecules [46, 50]. intercalation interaction between drug and DNA cause an enhancement in the intensities of both bands owing to potent base stacking interactions and permanent DNA conformations (right-handed B conformation of CT-DNA), whilst simple groove binding and electrostatic interactions with compounds represent less of a disturbance or no perturbation whatever on the base stacking and helicity bands [51, 52]. By increasing the concentration of the Fe3O4@LDH@Lamivudine (Fig. 8), both positive and negative bands show little changes, which imply a non-intercalative mode between DNA and Fe3O4@LDH@Lamivudine. So in refer to the evidence we can conclude that, the new complex was formed by interaction of nanocomposite and CT-DNA is mainly by groove binding [32, 52].
In this study, Fe3O4@LDH@Lamivudine was synthesized and then characterized by TEM, XRD and FT-IR spectroscopy, afterward the interaction of these nanoparticles with CT-DNA has been investigated by fluorescence spectroscopy, Uv-visible spectroscopy, viscosity, circular dichroism (CD). The absorbance of UV-visible spectroscopy was increased by adding the Fe3O4@LDH@Lamivudine, which indicated hyperchromism with the binding constant of (4.9×103 M-1). According to obtained results, fluorescence spectroscopy illustrated that the nanocomposite was quenched the substantial fluorescence intensity of CT-DNA through a static quenching procedure. Furthermore, the values of ΔH, ΔS and ΔG were negative, which indicated hydrogen bonding and vander-waals force. Moreover, the viscosity measurements were applied and the results showed that, by enhancing the amount of the nanocomposite, no changes were observed. The changes which were observed in the CD spectra, display confirmation of the right-handed B form of CT-DNA and suggested the interaction between the nanocomposite and CT-DNA. These experimental results illustrated groove binding interaction of Fe3O4@LDH@Lamivudine to CT-DNA.
We gratefully acknowledge the support of this research by the Razi University of Kermanshah, Iran.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.
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