Enhancement of magnetofection efficiency using chitosan coated superparamagnetic iron oxide nanoparticles and calf thymus DNA

Colloids and Surfaces B: Biointerfaces 152 (2017) 169–175

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Enhancement of magnetofection efficiency using chitosan coated superparamagnetic iron oxide nanoparticles and calf thymus DNA Z. Sohrabijam, M. Saeidifar ∗ , A. Zamanian Nanotechnology and Advanced Materials, Materials and Energy Research Center, Karaj, Iran

a r t i c l e

i n f o

Article history: Received 2 July 2016 Received in revised form 15 January 2017 Accepted 16 January 2017 Available online 17 January 2017 Keywords: Magnetofection Gene delivery Superparamagnetism Calf thymus DNA Chitosan

a b s t r a c t Superparamagnetic iron oxide nanoparticles (MNPs) were prepared and coated with chitosan (CS). The chitosan-magnetic iron oxide nanoparticles (CS-MNPs) were characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometry (VSM), and the morphology of the particles was studied by transmission electron microscopy (TEM). Our findings show that the magnetic particles were monodisperse (10 nm mean diameter) and exhibited superparamagnetic behavior. The interaction between the particles and calf-thymus DNA (DNA) in physiological buffer was studied with UV–vis, fluorescence and circular dichroism spectroscopy and zeta potential. Spectroscopic studies were indicated DNA conformational changes in the presence of CS-MNPs. Binding and thermodynamic parameters at different temperatures were calculated using the Stern–Volmer, Hill, Scatchard and Van’t Hoff equations. The binding process was spontaneous and interactions were electrostatic with the appropriate binding constant (Kb = 4.52 × 103 M−1 , 3.69 × 103 M−1 and 3.02 × 103 M−1 at 300 K, 310 K and 320 K, respectively). Zeta potential measurements of DNA continually increased with the addition of CS-MNPs, supporting our thermodynamic findings. Moreover, CS-MNPs were able to quench the fluorescence of DNA-intercalated ethidium bromide (DNA-EB) by a static quenching mechanism. Cytotoxicity studies show that the DNA-CS-MNP system is biocompatible with a human foreskin fibroblast cell line, HFFF2. Collectively, these results suggest that surface cationic magnetic chitosan-iron oxide nanoparticles can potentially enhance magnetofection efficiency. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Gene therapy is one of several approaches used to treat incurable diseases and genetic disorders. The success of this approach requires the development of innovative gene delivery vectors [1–3]. Research efforts have focused on the development of safe and effective viral and non-viral vector systems. Although viral vectors have high transduction efficiency, they are plagued with issues such as non-specificity, immunogenicity to target cells, toxicity and enzymatic degradation [2,4]. Non-viral delivery systems are being pursued to facilitate therapeutic gene transfer in the clinic. These carriers, such as cationic lipids and polymers, typically interact with anionic DNA via charged moieties, condensing the long, string- like DNA molecules into compact, nano-sized particles that are suitable for cellular uptake. A range of cationic lipids and polymers have been engineered to accomplish this function effectively and intel-

∗ Corresponding author. E-mail address: [email protected] (M. Saeidifar). http://dx.doi.org/10.1016/j.colsurfb.2017.01.028 0927-7765/© 2017 Elsevier B.V. All rights reserved.

ligent carriers are continually being optimized in order to control the intracellular fate of DNA molecules [5,6]. DNA/polymer complexes with cationic polymers have a number of distinct advantages that make them suitable for gene delivery. These biocompatible complexes are more stable than cationic lipids, have low immunogenicity and minimal cytotoxicity, making these polymers a good alternative to viral- and lipid- mediated delivery methods. Cationic polymers have been used to condense and deliver DNA both in vitro and in vivo. The structural variability and versatility of cationic polymers and the possibility of covalent binding to targeting moieties to mediate gene expression through specific receptors presents interesting possibilities [7–11]. Chitosan (CS) is an inexpensive, biocompatible material with minimal toxicity and high cationic potential that has been widely used for gene delivery. High molecular weight chitosan (100–400 kDa) can form extremely stable polyplexes with DNA [1,12,13]. However, low transfection efficiencies (as compared to viral vectors) and the inability to target specific cells or tissues in vivo remain problematic. In order to overcome these difficulties, magnetofection was developed to enhance the delivery of nucleic acids associated with magnetic nanoparticles [9,14,15]. Magneto-

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fection is a novel and highly efficient method of transfecting cells in culture [16]. In the present study, superparamagnetic iron oxide nanoparticles coated with chitosan were prepared and characterized by FT-IR spectroscopy, X-ray diffraction, VSM and TEM. We studied the interaction between CS-MNPs and DNA by multispectroscopic methods under physiological conditions. These surface cationic magnetic chitosan-iron oxide nanoparticles can potentially enhance magnetofection efficiency to potentially improve gene delivery systems.

2. Experimental section 2.1. Materials and methods Ferrous chloride tetrahydrate (FeCl2 . 4H2 O), ferric chloride hexahydrate (FeCl3 . 6H2 O), ammonium hydroxide (NH4 OH), sodium hydroxide (NaOH), acetic acid and Tris (hydroxymethyl) amino methane hydrocholoride (Tris-HCl) were obtained from Merck Chemical Co. Ethidium bromide (EB), chitosan (CS) and calf thymus DNA (DNA) were purchased from Sigma Chemical Co. Cell lines used in cytotoxicity studies were obtained from the Cell Bank of the Pasteur Institute (Tehran, Iran). All chemicals were of analytical reagent grade and double distilled water was used for all experiments. A powder sample of CS-MNPs was subjected to X-ray diffraction (XRD) on a Buruker X-ray powder diffractometer using Cu-K␣ radiation to check phase purity and average crystallite size. Fourier transform infrared spectra (FT-IR) were recorded from 400 to 4000 cm−1 using a Buruker-Dector FT-IR spectrometer. Magnetization was performed at room temperature using a vibrating sample magnetometer (VSM, MDK6). Transmission electron microscopy (TEM) was carried out on an EM 900 electron microscope (Zeiss). Electronic absorption spectra were recorded on a Lambda 25 UV/Vis spectrophotometer (Perkin Elmer, 1.0 cm quartz cell). DNA concentration was determined by UV absorption at 258 nm using the molar absorption coefficient ␧258 = 6600 M−1 cm−1 [17]. DNA purity was assessed by measuring the ratio of the absorbance at 258 nm and 280 nm (A258 /A280 ), and the ratio was ∼1.8–1.9:1, indicating that the DNA was pure. Fluorescence measurements were carried out on a Hitachi fluorescence spectrophotometer (MPF4) equipped with a thermostat bath and a 1.0 cm quartz cell. The widths of the excitation and emission slits were set at 5.0 nm. The solution was excited at 471 nm and emission wavelengths were recorded between 550 and 700 nm. Circular dichroism spectra were obtained on an Aviv 215 spectropolarimeter using a 1.0 mm cell at 200–320 nm. Observed CD spectra were corrected for buffer signal and results were expressed as mean residue ellipticity in mdeg. pH measurements were obtained using a Metrohm 826 pH meter.

2.2. Solutions for DNA binding studies EB (2 mg/ml) was dissolved in doubly distilled water. DNA (2 mg/ml) was prepared by dissolving appropriate amounts of DNA in Tris-buffer (20 mM Tris–HCl and 20 mM NaCl, pH 7.4).

2.3. Synthesis of Fe3 O4 and chitosan- Fe3 O4 nanoparticles The magnetite (Fe3 O4 ) nanoparticles were synthesized by chemical coprecipitation. FeCl3 and FeCl2 (2:1) were dissolved in 50 ml deionized water and the solution was stirred by strong ultrasonic agitation while heating (343 K). NaOH was added drop-wise to the iron solution under strong ultrasonic agitation for 1 h at 343 K

and bubbling N2 gas. The chemical reaction of Fe3 O4 precipitation is expressed as: FeCl2 ·4H2 O + 2FeCl3 ·6H2 O + 8NaOH → Fe3 O4 + 8NaCl + 20H2 O Black Fe3 O4 particles were separated with a permanent magnet and then washed with deionized water. For subsequent preparation of the chitosan–Fe3 O4 nanoparticles (CS-MNPs), colloidal nanoparticles in water were added to the chitosan solution (0.5 g in 100 ml of 0.2% wt acetic acid solution). The mixture was treated with ultrasound for 10 min, then 5 ml of 25% wt NH4 OH was added and the mixture was again treated with ultrasound for 10 min. The composites were then separated by centrifuge and triple-washed with deionized water. The products were dissolved in 0.5% v/v acetic acid solution. 2.4. Cytotoxicity assay The cytotoxic effects of the DNA-CS-MNPs were measured in the MTT assay. This method is based on the reduction of yellow tetrazolium MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromide) to a purple formazan dye by mitochondrial succinate dehydrogenase as previously reported [11,18]. 2.5. Interaction of magnetic nanoparticles with DNA 2.5.1. UV–vis absorption experiments The absorption spectra of DNA (41 mM) in the presence of different amounts of CS-MNPs (0–0.016 mM) were measured. Each solution was allowed to stand for 5 min to equilibrate. The absorption spectra of corresponding concentrations of solutions were measured over a 230–330 nm wavelength range at room temperature. The equilibrium for the DNA-MNP interaction can be represented by the following equation [17]: K

app DNA + CS − MNP ⇔ DNA − CS − MNP

(1)

Where Kapp is the apparent association constant. Kapp was obtained from the following equation: 1/(Aobs − A0 ) = 1/(Ac − A0 ) + 1/K app (Ac − A0 ) [CS-MNPs]

(2)

Aobs represents the observed absorbance of the solution containing different concentrations of MNP at 258 nm and A0 and Ac are the absorbance of DNA and CS-MNPs at 258 nm, respectively. The value of the apparent association constant, Kapp , wasdetermined from the linear relationship between 1/(Aobs − A0 ) versus 1/[CSMNPs], where the slope is equal to 1/Kapp (Ac − A0 ) and the intercept is equal to 1/(Ac − A0 ). 2.5.2. Fluorescence spectroscopy studies Fluorescence titration was performed by successively adding CS-MNPs (0–298 ␮M) into the quartz cuvette containing a fixed amount of DNA (60 ␮M) and EB (2 ␮M) at 293 K, 303 K and 313 K in Tris-HCl buffer. Quenching mechanisms are usually classified as dynamic or static [19]. Dynamic quenching refers to a process where the fluorophore and the quencher come into contact during the lifetime of the excited state, whereas static quenching refers to fluorophore–quencher complex formation [20]. In order to ascertain the quenching mechanism between CS-MNPs and DNA, we used the Stern-Volmer equation [21]: F 0 /F = 1 + kq  0 [CS-MNP] = 1+ K SV [CS-MNP]

(3)

F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively. KSV is the Stern-Volmer dynamic quench-

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171

ing constant and [CS-MNP] is the concentration of MNP. kq is the quenching rate constant of the biomolecule that can be written as:

based on log Kb versus 1/T. The value of free energy change (G) can be determined from the following equation [27,28]:

kq = K sv / 0

G = −R T ln K b

(4)

␶0 is the average quencher and its value is around 10−8 s for most biomolecules [22,23]. Therefore, KSV at different temperatures can be determined from the linear regression plot of F0 /Fversus [CSMNP]. 2.5.3. Calculation of binding constant and number of binding sites The binding constant (Kb ) and the number of binding sites in base pairs (n) were determined using the following equation [11,24]: log F 0 − F/F = log K + n log [CS-MNP]

(5)

Where F, F0 and [CS-MNP] are the same as in Eq. (4). The binding constants and the number of binding sites were also calculated using the Scatchard equation to confirm how many molecules of the MNP bound to DNA-intercalated EB [17]: r/Df = nK b − rK b

(6)

r is the number of moles of CS-MNPs bound per mole of DNAintercalated EB, Df is the molar concentration of free CS-MNPs; n and Kb are the number of binding sites and the binding constant, respectively. The binding constant and number of binding sites were the slopes and the slopes divided by intercepts, respectively [17]. 2.5.4. Thermodynamic analysis A thermodynamic process was considered to be responsible for the interaction between DNA and CS-MNPs. Generally speaking, the main binding forces between small molecules and biomacromolecules can be divided into Hydrogen-bonding, Van der Waals forces, electrostatic and hydrophobic interactions [25]. The thermodynamic parameters can be estimated from the Van’t Hoff equation as follows [26]: ln K b = H/RT + S/R

(7)

H is the value of enthalpy change, S is the value of entropy change, R is the gas constant (8.314 J/mol k−1 ) and temperatures were 293 K, 303 K and 313 K. The values of H and S were calculated from the slope and intercept of the linear Van’t Hoff equation

(8)

2.5.5. Zeta potential measurements Zeta potential is proportional to the surface charge density and can be used to monitor the binding of negatively charged DNA to positively charged chitosan-MNPs. Zeta potential measurements were taken for naked DNA and DNA in the presence of increasing concentrations of CS-MNPs [29]. 2.5.6. Circular dichroism studies Circular dichroism (CD) spectra of DNA in the absence and presence of various amounts of CS-MNPs were measured at pH 7.4. The concentration of DNA was fixed at 103 mM and the molar ratio values of CS-MNPs to DNA were 0.025, 0.03 and 0.035 mM, respectively. 3. Results and discussion 3.1. Characterization of magnetic nanoparticles and chitosan-MNPs Magnetic nanoparticles and chitosan-magnetic nanoparticles were prepared as described (Section 2.3.). Fig. S1 shows the XRD pattern of CS-MNPs synthesized by co-precipitation. The Rietveld refinement of the XRD pattern was used to calculate the average grain size from the broadening of the XRD peaks of CS-MNPs. The average grain size of the chitosan coated magnetic nanoparticles is 15 nm, which is in line with previous studies [30,31]. To confirm the structure of the samples, the FT-IR spectrum of MNPs (a) and CS-MNPs (b) were recorded at room temperature. Fig. S2 indicates a strong absorption band at 584 cm−1 that can be assigned to the vibration of the Fe O functional groups. The peak observed around 3418 cm−1 indicates an OH group and the peak ∼1630 cm−1 for the a spectrum and 1567 cm−1 for the b spectrum can be assigned to N H binding vibrations. The peak at 1410 cm−1 for the b spectrum that did not appear in the a spectrum corresponds to C O stretching of the primary alcohol group of chitosan [6]. Magnetic materials about 12 nm in size are used to show superparamagnetic behavior in which coercivity was below 150 Oe [7]. The hysteresis loop of CS-MNPs at room temperature is shown in Fig. 1a. This phenomenon indicates that the magnetic nanoparticles produced in this study are superparamagnetic because of the coercivity value

Fig. 1. (a) Measured magnetization moments of CS-MNPs. (b) The TEM images of the CS-MNPs.

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Fig. 2. (a) UV–vis absorption spectra of DNA (41 mM) with various concentrations of CS-MNPs (0–0.016 mM) in Tris-HCl buffer at pH 7.4 and room temperature. (b) The plot of 1/(Aobs − A0 ) versus 1/[CS-MNPs] (Mean ± 1 SD).

of 8.5 Oe. In addition, the saturated magnetization MS of magnetic nanoparticles is 67.44 emu/g, which is lower than that of the bulk and naked MNPs (Fig. 1a). The size and shape of the CS-MNPs were investigated using TEM. The TEM image of CS-MNPs is shown in Fig. 1b. We observed that the nanoparticles were monodisperse and had a mean diameter of 10 nm which corresponds with XRD results.

3.2. In vitro cytotoxicity studies In vitro cytoxicity studies are performed to measure the effects of chemical substances on mammalian cells [18]. The toxicity of DNACS-MNPs was tested in the HFFF2 human foreskin fibroblast normal cell line. The number of growing cancer cells was significantly attenuated following 24 and 48 h treatment with DNA-CS-MNPs. DNA-CS-MNPs were not toxic at the same dosage range in a normal human cell line (Fig. S3).

Fig. 3. Fluorescence emission spectra of DNA-intercalated EB in the presence of CS-MNPs at 303 K in Tris-HCl buffer, pH 7.0.

3.4. Fluorescence quenching mechanism of the interaction between DNA and CS-MNPs Fluorescence titration is used to study the binding properties of small molecules to DNA [35]. In the present study, CS-MNPs did not show fluorescence at room temperature in the buffer solution or in the presence of DNA and DNA-binding cannot be directly predicted from the emission spectra. To further investigate the interaction of the CS-MNPs with DNA, a competitive binding experiment using EB as a probe was performed. EB emits intense fluorescence at ∼615 nm in the presence of DNA, however the addition of a second molecule could quench this enhanced fluorescence [11]. The fluorescence emission spectra of DNA-intercalated EB upon the successive addition of CS-MNPs are shown in Fig. 3. There is a significant reduction in fluorescence emission intensity with the increasing addition of CS-MNPs, indicating that CS-MNPs can compete with EB and bind to DNA. The curves of F0 /F versus [CS-MNPs] at three temperatures (293 K, 303 K and 313 K) are shown in Fig. S4 and the corresponding KSV and kq are listed in Table 1. The values of kq were greater than the values of the maximum scattering collision quenching constant (2.0 × 1010 M−1 s−1 ) and the corresponding KSV decreased

3.3. UV–vis studies of the interaction between DNA and CS-MNPs UV–vis absorption spectroscopy is used to detect conformational changes in DNA and is also used to distinguish between static and dynamic quenching. Dynamic quenching affects the excited state of a fluorophore and does not change the absorption spectrum. However, the formation of a non-fluorescent ground-state complex results in a change in the absorption spectrum of the fluorophore [32,33]. Fig. 2a shows a red shift from 258 to 259 nm which is suggestive of static quenching; a significant decrease in the intensity of the maximum absorption wavelength of DNA (hypochromicity) with increasing concentrations of CS-MNPs is due to the interaction between nanoparticles and DNA. In addition, the binding constant Kapp for the DNA–CS-MNP interaction was 2.83 (±0.4) × 105 M−1 (R2 = 0.99) according to Eq. (2) and Fig. 2b. The binding constant of the system was lower in comparison to those observed for other intercalators (EB-DNA, 2.6 × 106 M−1 ), indicating that CSMNPs bind to DNA in a different manner [34].

Table 1 Stern–Volmer quenching constants, binding constants and number of binding sites for DNA–CS-MNPs system at different temperatures. T (K)

Stern-volmer method 3

−1

KSV (×10 M 293 303 313 a b

)

4.20 ± 0.6 3.40 ± 0.5 2.92 ± 0.3

b

R2 is the correlation coefficient. S.D. is standard deviation.

Hill method 11

kq (×10

4.20 ± 0.6 3.40 ± 0.5 2.92 ± 0.3

−1

M

−1

s

)

2a

3

−1

R

Kb (×10 M

0.99 0.99 0.99

4.52 ± 0.4 3.69 ± 0.3 3.02 ± 0.5

Scatchard method )

n

R

Kb (×103 M−1 )

n

R2

1.10 ± 0.3 1.20 ± 0.5 1.18 ± 0.2

0.99 0.99 0.99

4.14 ± 0.3 3.37 ± 0.4 2.81 ± 0.7

1.05 ± 0.5 1.05 ± 0.3 1.07 ± 0.6

0.99 0.99 0.98

2

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Table 2 Thermodynamic parameters for DNA-CS-MNPs system at different temperatures. T(K)

G (kJ/mol)

H (kJ/mol)

S (kJ/mol K)

R2 a

293 303 313

−3.67 −4.27 −4.53

−2.37 ± 0.4b

4.55 ± 0.6.

0.98

a b

R2 is the correlation coefficient. S.D. is standard deviation.

with increasing temperatures. The evidence demonstrates that the probable quenching mechanism of the DNA-CS-MNP interaction is static quenching [36]. This result was consistent with absorbance spectroscopy studies.

Fig. 4. Zeta potential Variation of (1) CS-MNPs (2) DNA (3) DNA+ 5 ␮M CS-MNPs (4) DNA+ 50 ␮M CS-MNPs and (5) DNA+ 100 ␮M CS-MNPs (Mean ± 1 SD).

3.5. Binding constant and binding sites The binding parameters can be obtained from the slope of log (F0 -F/F) versus log [CS-MNPs] at 293 K, 303 K and 313 K and Eq. (5) (Fig. S5a and Table 1). The values of n for DNA were approximately equal to 1, indicating that there was a single interaction site between each molecule of DNA and CS-MNP. The Kb values were in the order of 103 M−1 , suggesting that CS-MNPs can bind to DNA with low affinity. This finding is in line with other reports, for example, DNA conjugated with chitosan (15 kD) has an observed binding constant of 1 × 103 M−1 [28]. Similarly, Chai et al. developed ractopamine as a carrier of DNA with Kb = 4.13 × 103 M−1 at 298 K [37]. Therefore, CS-MNPs can be suitable for gene delivery because they are released rapidly in the unbound form and can reach intended sites. Increasing temperature demonstrated that the affinity between CS-MNPs and DNA decreased indicating that the binding of CS-MNPs to DNA decreased the stability of the DNA–CS-MNP system. Binding constants and the number of binding sites were calculated using the Scatchard equation. There was linear dependence between r/Df and r (Fig. S5b) and the slopes decreased with increasing temperatures, indicating a static quenching mechanism. These results were consistent with the conclusions obtained from the Hill equation (Table 1). 3.6. Thermodynamic analysis and nature of the binding forces The signs and magnitudes of thermodynamic parameters, the enthalpy change (H), entropy change (S) and Gibbs free energy (G) can elucidate the primary binding mode between small molecules and macromolecules. That is, if H > 0 and S > 0, hydrophobic interactions are implicated. If H < 0 and S < 0, van der Waals and hydrogen-bonding interactions play major roles in the reaction. Electrostatic forces are more important when H ≈ 0 and S > 0 [27]. The thermodynamic parameters for the interaction between CS-MNPs with DNA (Eq. (7) and Fig. S6) are listed in Table 2. In the present case, the values of H (≈0) and S (>0), indicate that electrostatic interactions are predominant during the binding process, i.e. the interaction between the polymer cationic charged NH2 of chitosan and the negatively charged phosphate groups on the DNA backbone [28]. A negative G value indicates that the interaction between CS-MNPs and DNA is spontaneous [27,28]. 3.7. Zeta potential measurements Zeta potential changes of DNA molecules with increasing concentrations of CS-MNPs are shown in Fig. 4. As the concentration of the complex increased, the negative charges on DNA decreased, therefore, the zeta potential increased as chitosan-MNP concen-

Fig. 5. CD spectra of DNA (103 mM) in the presence of increasing amounts of CSMNPs at room temperature and pH 7.4 ([CS-MNPs]/[DNA] = (a) 0; (b) 0.025; (c) 0.03 and (d) 0.035 mM).

trations increased from −8.90 to 4.97 mV. Negative surface charge distribution stabilizes DNA particles through electrostatic repulsion and therefore prevents DNA from condensing. Increasing positive charges increases electrostatic interactions with negatively charged DNA, resulting in DNA condensation. This finding is consistent with our thermodynamic data and data from other studies [38–40]. 3.8. CD studies of the interaction between DNA and CS-MNPs CD spectra are sensitive to DNA interactions and are used to determine conformational changes in DNA during ligand-DNA interactions [17,41]. The CD spectra of DNA after the addition of CS-MNPs showed obvious changes in the positive band due to basestacking and in the negative band due to right-handed helicity, indicating an overall change in DNA structure (Fig. 5). 4. Conclusions We describe the formulation of superparamagnetic chitosan iron oxide nanoparticles for magnetofection and characterized these nanoparticles using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometry (VSM) and transmission electron microscopy (TEM). Particle size was calculated by XRD and TEM with similar results. The superparamagnetic properties of CS-MNPs significantly increased the transfection efficiency. Studying DNA-CS-MNPs in human fibroblast HFFF2 cells suggests that these particles are not cytotoxic.

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The interaction between the particles and DNA was explored using UV–vis spectroscopy, fluorescence spectroscopy, zeta potential measurements and circular dichroism. The absorption spectra of CS-MNPs with DNA showed a slight red shift and hypochromic effect. The fluorescence quenching of the emission peak was seen in the DNA-intercalated EB upon the addition of CS-MNPs. The binding and thermodynamic parameters of the interaction at different temperatures were calculated using the Stern–Volmer, Hill, Scatchard and Van’t Hoff equations. These results indicate that the CS-MNPs could interact with DNA via static quenching and electrostatic interactions. This study describes a novel system that can be used to enhance magnetofection efficiency because of the advantages posed by its magnetic properties and DNA-binding ability. However, further in vitro and in vivo studies are required to characterize the usefulness of these particles as non-viral gene delivery systems. Acknowledgements We are grateful for financial support from the Materials and Energy Research Center. We thank Dr. Roya Pedram Fatemi for careful reading of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.01. 028. References [1] H. Peng, J. Ye, S. Loong, Y. Wang, W. Min, X. Sheng, J. Min, S. Wang, Biomaterials Gene transfer using self-assembled ternary complexes of cationic magnetic nanoparticles, plasmid DNA and cell-penetrating Tat peptide, Biomaterials 31 (2010) 769, http://dx.doi.org/10.1016/j.biomaterials. 2009.09.085. [2] M.S. Al-Dosari, X. Gao, Nonviral gene delivery: principle, limitations, and recent progress, AAPS J. 11 (2009) 671, http://dx.doi.org/10.1208/s12248009-9143-y. [3] H. Xu, Z. Li, J. Si, Nanocarriers in gene therapy: a review, J. Biomed. Nanotechnol. 10 (2014) 3483, http://dx.doi.org/10.1166/jbn.2014.2044. [4] S. Xenariou, U. Griesenbach, S. Ferrari, P. Dean, R.K. Scheule, S.H. Cheng, D.M. Geddes, C. Plank, E.W.F.W. Alton, Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo, Gene Ther. 13 (2006) 1545, http:// dx.doi.org/10.1038/sj.gt.3302803. [5] C.Y.M. Hsu, H. Uluda˘g, Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers, BMC Biotechnol. 8 (2008) 23, http://dx.doi.org/10.1186/1472-6750-8-23. [6] M. Julia, A. Rechtenbach, Synthesis and physical characterization of magnetite nanoparticles for biomedical applications, Mater. Chem. Phys. 110 (2008) 426, http://dx.doi.org/10.1016/j.matchemphys.2008.02.037. [7] G. Borchard, Chitosans for gene delivery, Adv. Drug Deliv. Rev. 52 (2001) 145, S0169-409X(01)00198-3. [8] H. Zhang, M.Y. Lee, M.G. Hogg, J.S. Dordick, S.T. Sharfstein, Gene delivery in three-dimensional nanoparticles, ACS Nano 4 (2010) 4733, http://dx.doi.org/ 10.1021/nn9018812. [9] D. Kami, S. Takeda, Y. Itakura, S. Gojo, M. Watanabe, M. Toyoda, Application of magnetic nanoparticles to gene delivery, Int. J. Mol. Sci. 21 (2011) 3705, http://dx.doi.org/10.3390/ijms12063705. [10] S.C. McBain, H.H.P. Yiu, A. El Haj, J. Dobson, Polyethyleneimine functionalized iron oxide nanoparticles as agents for DNA delivery and transfection, J. Mater. Chem. 17 (2007) 2561, http://dx.doi.org/10.1039/b617402g. [11] M. Saeidifar, H. Mansouri-Torshizi, Y. Palizdar, M. Eslami-Moghaddam, A.A. Saboury, Synthesis, characterization, cytotoxicity and DNA binding studies of a novel anionic organopalladium(II) complex, Acta Chim. Slov. 61 (2014) 126, http://dx.doi.org/10.1080/15257770.2013.790552. [12] S. Tang, Z. Huang, H. Zhang, Y. Wang, Q. Hu, H. Jiang, Design and formulation of trimethylated chitosan-graft-poly(␧-caprolactone) nanoparticles used for gene delivery, Carbohydr. Polym. 101 (2014) 104, http://dx.doi.org/10.1016/j. carbpol.2013.09.053. [13] N. Duceppe, M. Tabrizian, Advances in using chitosan-based nanoparticles for in vitro and in vivo drug and gene delivery, Exp. Opin. Drug Deliv. 7 (2010) 1191, http://dx.doi.org/10.1517/17425247.2010.514604. [14] Y. Shi, L. Zhou, R. Wang, Y. Pang, In situ preparation of magnetic nonviral gene vectors and magnetofection in vitro, Nanotechnology 21 (2010) 115103, http://dx.doi.org/10.1088/0957-4484/21/11/115103.

[15] S. Aliramaji, A. Zamanian, M. Mozafari, Super-paramagnetic responsive silk fibroin/chitosan/magnetite scaffolds with tunable pore structures for bone tissue engineering applications, Mater. Sci. Eng. C 70 (2017) 736, http://dx. doi.org/10.1016/j.msec.2016.09.039. [16] C. Plank, O. Zelphati, O. Mykhaylyk, Magnetically enhanced nucleic acid delivery. Ten years of magnetofection—progress and prospects, Adv. Drug Deliv. Rev. 63 (2011) 1300, http://dx.doi.org/10.1016/j.addr.2011.08.002. [17] Q. Wang, S.R. Zhang, X. Ji, Investigation of interaction of antibacterial drug sulfamethoxazole with human serum albumin by molecular modeling and multi-spectroscopic method, Spectrochim. Acta Part A 124 (2014) 84, http:// dx.doi.org/10.1016/j.saa.2013.12.100. [18] H. Xu, L. Gong, Y. Xia, L. Qu, Q. Li, L. Pang, J. Si, Z. Li, Frizzled-7 promoter is highly active in tumors and promoter-driven Shiga-like toxin I inhibits hepatocellular carcinoma growth, Oncotarget 6 (39908) (2015), http://dx.doi. org/10.18632/oncotarget.5516. [19] G.Z. Pan Zhu, Y. Ma, Y. Zhang, H. Miao, Y. Wub, Study of DNA interactions with bifenthrin by spectroscopic techniques and molecular modeling, Spectrochim. Acta Part A 112 (2013) 7, http://dx.doi.org/10.1016/j.saa.2013.04.022. [20] T. Xu, X. Guo, L. Zhang, F. Pan, J. Lv, Y. Zhang, H. Jin, Multiple spectroscopic studies on the interaction between olaquindox, a feed additive, and bovine serum albumin, Food Chem. Toxicol. 50 (2012) 2540, http://dx.doi.org/10. 1016/j.fct.2012.04.007. [21] Y.M. Guowen Zhang, L. Wang, Y. Zhang, J. Zhou, Multispectroscopic studies on the interaction of maltol, a food additive, with bovine serum albumin, Food Chem. 133 (2012) 264, http://dx.doi.org/10.1016/j.foodchem.2012.01.014. [22] X.H. Guowen Zhang, N. Zhao, W. Li, L. He, Studies on the interaction of aminocarb with calf thymus DNA by spectroscopic methods, Pestic. Biochem. Phys. 98 (2010) 206, http://dx.doi.org/10.1016/j.pestbp.2010.06.008. [23] L. Bouffier, H.H. Yiu, M.J. Rosseinsky, Chemical grafting of a DNA intercalator probe onto functional iron oxide nanoparticles: a physicochemical study, Langmuir 27 (2011) 6185, http://dx.doi.org/10.1021/la104745x. [24] G.Z. Yepeng Zhang, Peng Fu, Yadi Ma, Jia Zhou, Study on the interaction of triadimenol with calf thymus DNA by multispectroscopic methods and molecular modeling, Spectrochim. Acta Part A 96 (2012) 1012, http://dx.doi. org/10.1016/j.saa.2012.08.002. [25] D. Lu, X. Zhao, Y. Zhao, B. Zhang, B. Zhang, M. Geng, R. Liu, Binding of Sudan II and Sudan IV to bovine serum albumin: comparison studies, Food Chem. Toxicol. 49 (2011) 3158, http://dx.doi.org/10.1016/j.fct.2011.09.011. [26] L.L.a. aQiong Guo, J. Dong, H. Liu, T. Xu, J. Li, Synthesis, crystal structure and interaction of L-valine Schiff base divanadium(V) complex containing a V2O3 core with DNA and BSA, Spectrochim. Acta Part A 106 (2013) 155, http://dx. doi.org/10.1016/j.saa.2012.12.089. [27] X. Pan, P. Qin, R. Liu, J. Wang, Characterizing the Interaction between tartrazine and two serum albumins by a hybrid spectroscopic approach, J. Agric. Food Chem. 59 (2011) 6650, http://dx.doi.org/10.1021/jf200907x. [28] D. Agudelo, L. Kreplak, H.A. Tajmir-Riahi, Microscopic and spectroscopic analysis of chitosan-DNA conjugates, Carbohydr. Polym. 137 (2016) 207, http://dx.doi.org/10.1016/j.carbpol.2015.09.080. [29] M. Mamusa, C. Resta, F. Barbero, D. Carta, D. Codoni, K. Hatzixanthis, M. McArthur, D. Berti, Interaction between a cationic bolaamphiphile and DNA: The route towards nanovectors for oligonucleotide antimicrobials, Colloid Surf. B 143 (2016) 139, http://dx.doi.org/10.1016/j.colsurfb.2016.03.031. [30] H. Yang, W. Liang, N. He, Y. Deng, Z. Li, Chemiluminescent labels released from long spacer arm functionalized magnetic particles: a novel strategy for ultrasensitive and highly selective detection of pathogen infections, ACS Appl. Mater. Interfaces 7 (2015) 774, http://dx.doi.org/10.1021/am507203s. [31] I. Sharifi, A. Zamanian, A. Behnamghader, Synthesis and characterization of Fe0.6Zn0.4Fe2O4 ferrite magnetic nanoclusters using simple thermal decomposition method, J. Magn. Magn. Mater. 412 (2016) 107, http://dx.doi. org/10.1016/j.jmmm.2016.03.091. [32] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Study on the interaction between antibacterial drug and bovine serum albumin: a spectroscopic approach, Spectrochim. Acta A 73 (2009) 841, http://dx.doi.org/10.1016/j.saa. 2009.04.018. [33] R. Prabhakaran, P. Kalaivani, P. Poornima, F. Dallemer, R. Huang, V. Vijaya Padma, K. Natarajan, Synthesis, DNA/protein binding and in vitro cytotoxic studies of new palladium metallothiosemicarbazones, Bioorg. Med. Chem. 21 (2013) 6742, http://dx.doi.org/10.1016/j.bmc.2013.08.005. [34] G. Zhang, Y. Zhang, Y. Zhang, Y. Li, Spectroscopic studies of cyanazine binding to calf thymus DNA with the use of ethidium bromide as a probe, Sens. Actuators B 182 (2013) 453, http://dx.doi.org/10.1016/j.snb.2013.03.038. [35] M. Razmi, A. Divsalar, A.A. Saboury, Z. Izadi, T. Haertle, H. Mansuri-Torshizi, Beta-casein and its complexes with chitosan as nanovehicles for delivery of a platinum anticancer drug, Colloid Surf. B 112 (2013) 362, http://dx.doi.org/10. 1016/j.colsurfb.2013.08.022. [36] X. Li, G. Liu, W. Yan, P.K. Chu, K.W.K. Yeung, S. Wu, C. Yi, Z. Xu, Preparation of Fe3O4/poly (styrene –butylacrylate – [2-(methacryloxy) ethyl] trimethylammoniumchloride) by emulsifier – free emulsion polymerization and its interaction with DNA, J. Magn. Magn. Mater. 324 (2012) 1410–1418, http://dx.doi.org/10.1016/j.jmmm.2011.11.056. [37] J. Chai, J. Wang, Q. Xu, F. Hao, R. Liu, Multi-spectroscopic methods combined with molecular modeling dissect the interaction mechanisms of ractopamine and calf thymus DNA, Mol. Biosyst. 8 (2012) 1902, http://dx.doi.org/10.1039/ c2mb25095k.

Z. Sohrabijam et al. / Colloids and Surfaces B: Biointerfaces 152 (2017) 169–175 [38] S. Liu, A. Shibata, S. Ueno, F. Xu, Y. Baba, D. Jiang, Y. Li, Investigation of interaction of Leu-enkephalin with lipid membranes, Colloid Surf. B 48 (2006) 148, http://dx.doi.org/10.1016/j.colsurfb.2006.02.005. [39] Y. Zhang, M. Yang, N.G. Portney, D. Cui, G. Budak, E. Ozbay, M. Ozkan, C.S. Ozkan, Zeta potential: a surface electrical characteristic to probe the interaction of nanoparticles with normal and cancer human breast epithelial cells, Biomed. Microdevice 10 (2008) 321, http://dx.doi.org/10.1007/s10544007-9139-2.

175

[40] M. Alatorre-Meda, P. Taboada, J. Sabin, B. Krajewska, L.M. Varela, J.R. Rodriguez, DNA-chitosan complexation: a dynamic light scattering study, Colloid Surf. A 339 (2009) 145, http://dx.doi.org/10.1016/j.colsurfa.2009.02. 014. [41] W. Liu, S. Sun, Z. Cao, X. Zhang, K. Yao, W.W. Lu, K.D.K. Luk, An investigation on the physicochemical properties of chitosan/DNA polyelectrolyte complexes, Biomaterials 26 (2005) 2705, http://dx.doi.org/10.1016/j. biomaterials.2004.07.038.