Synthesis and characterisation of doxorubicin-loaded functionalised cobalt ferrite nanoparticles and their in vitro anti-tumour activity under an AC-magnetic field

Purpose: To synthesise and evaluate the anti-tumour properties of doxorubicin-loaded xanthan gumfunctionalised cobalt ferrite nanoparticles (CoFe2O4.NPs@XG-Doxo) under an AC-magnetic field. Methods: Multidimensional magnetic cobalt ferrite (CoFe2O4) nanoparticles (NPs) were synthesised by a co-precipitation method. The synthesised cobalt ferrite nanoparticles (CFNPs) were functionalised with xanthine gum (XG) and subsequently characterised by Fourier transform-infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and contact angle studies. Vibrating sample magnetometry (VSM) was used for magnetic measurements of the native and XG-coated CFNPs. The microstructural morphology of the uncoated and XG-coated CFNPs was established using scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS) studies. Finally, the doxorubicin release profile of the drug-loaded functionalised CFNPs was evaluated using an oscillating magnetic field (OMF) apparatus in the presence of an externally applied magnetic field. Results: XG coating decreased the contact angle of the native CFNPs from 92° to 40°, which indicates that it modified the CFNP surface from hydrophobic to hydrophilic. VSM analysis demonstrated that CoFe2O4.NPs@XG also retained the magnetic characteristics of the bare cobalt ferrite nanocrystals, endorsing its application as a promising magnetic nanovector (MNV). The synthesised CoFe2O4.NPs@XG-Doxo exhibited significantly higher controlled discharge of doxorubicin at acidic pH (5.0) than at neutral pH (7.4). In vitro analysis revealed the remarkable lower systematic toxicity of XGcoated CoFe2O4.NPs compared with uncoated CFNPs against Chinese hamster ovary (CHO) and Huh7 cell lines. Conclusion: These results indicate that XG-coated CFNPs are a biocompatible MNV for doxorubicin.

However, biomedical applications of native CFNPs are restricted because of their hydrophobic surface. Most of the plasma proteins are hydrophobic, which enables the rapid removal of hydrophobic nanostructures from the blood circulatory system through opsonisation [10]. Previously, nanoferrite functionalization was carried out by coating with proteins, polymers, surfactants, starch or chitosan [11]. We report herein the first investigation of encapsulating CFNPs with xanthan gum (XG), which is an anionic polysaccharide that coats the cationic surface of the NPs. The biocompatibility of XG suggests a broad range of applications in the biomedical arena [12].
Herein, we report the synthesis and characterisation of XG-coated CFNPs (CoFe 2 O 4 .NPs@XG) to enhance application as a biocompatible magnetic nanovector (MNV) for targeted delivery of the anti-tumour drug doxorubicin (Doxo). The release of drug from CoFe 2 O 4 .NPs@XG-Doxo was determined in the absence as well as in the presence of an externally applied magnetic field (AMF). The cytocompatibility and toxicity of the naked and XG-coated ferrite NPs toward Chinese hamster ovary (CHO) and Huh7 cell lines are also reported.

Synthesis of the CFNPs
The CFNPs were synthesised using a previously reported method with minor modifications [13]. Briefly, aqueous solutions of ferric chloride (0.4 M, 25 mL) and cobalt chloride (0.2 M, 25 mL) were mixed and stirred in a three-necked flask at pH 1.34. Precipitation occurred immediately as 3 M KOH solution was slowly added drop-wise under an inert atmosphere of nitrogen gas. Then, the reaction temperature was increased to 85 °C and stirring was maintained for 90 min. The resulting dark black precipitate was decanted using a strong permanent magnet, washed multiple times with distilled water and finally oven-dried at 100 °C for 12 h. The synthesised cobalt ferrite nanocrystals were dialysed for 8 h in 100 mL of 0.02 M HNO 3 solution to form a cationic charge over the surface, dried and stored at 4 °C for further functionalization.

Synthesis of MNV (CoFe 2 O 4 .NPs@XG)
XG-coated CFNPs (CoFe 2 O 4 .NPs@XG) were prepared by dispersing the dialysed cobalt ferrite nanocrystals in distilled water, mixing with XG at a 1:2 ratio and stirring for 24 h. The supernatant layer was discarded and the resulting dark brown coated cobalt ferrite nanocrystals were washed with distilled water and finally freeze-dried for further studies.

In vitro drug loading and release studies
The prepared MNV (CoFe 2 O 4 .NPs@XG) was loaded with Doxo by dissolving 10 mg of dried CoFe 2 O 4 .NPs@XG in 25 mL of a drug solution (0.2 mg/mL) at room temperature and gently stirring for 2 days in the dark. The drug solution was prepared in phosphate-buffered solution (PBS) at pH 7.4. Thereafter, the drug-loaded magnetic nanoassembly, CoFe 2 O 4 .NPs@XG-Doxo, was separated using a magnet, washed several times with PBS and finally freeze-dried. The supernatant and washed solutions were collected and analysed to determine the unloaded drug concentration by UV-visible (UV-Vis) spectroscopy at a wavelength of 481 nm. The drug loading efficiency (L) was calculated using Eq 1.
where Co is the total amount of the added drug and C is the unencapsulated amount of drug.
To determine the drug release profile, 6  In a similar fashion, the drug release experiment was repeated when the drug release system was placed in a 40-mL glass tube in the presence of an external oscillating magnetic field (OMF) (ca. 1000 Oe) generated by a custommade instrument called a Teslamate. This device ( Figure 1) consisted of 96 turns of a 76.5-mm length of 0.8-mm-diameter copper wire that operated over a frequency of 1-500 kHz at a current of 1-3 amperes and a magnetic flux density of 0-100 mG.

Cell cultures
The CHO and Huh7 cell lines were provided by the Centre of Excellence and Molecular Biology (CEMB), Lahore, Pakistan. To obtain culture growth, the cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal bovine serum (FBS) and incubated at 37 °C under a humidified atmosphere containing 5 % CO 2. The cells lines were routinely harvested by adding 0.25 % trypsin-ethylenediaminetetraacetic acid (EDTA) solution.

Cytotoxicity of the MNV (CoFe 2 O 4 .NP@XG)
To analyse the in vitro cytotoxicity of the bare and coated cobalt ferrite NPs against CHO and Huh7, cells were harvested in 96-well plates at a density of 10,000 cells per well. Different concentrations of native CoFe 2 O 4 .NPs and CoFe 2 O 4 .NPs@XG (0-2.0 mg/mL) were added to each well, in duplicate. Thereafter, the culture plate was incubated at 37 °C in humidified air containing 5 % CO 2 for 48 h. After culturing sufficient cells, the medium in all wells was exchanged with fresh medium. Finally, the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay was used to measure the cell viability relative to that of the control untreated cells.

In vitro cytotoxicity of the magnetic nanoassembly (CoFe 2 O 4 .NPs@XG-Doxo)
The in vitro cytotoxicity of the drug-loaded magnetic nanoassembly, CoFe 2 O 4 .NPs@XG-Doxo, was tested against the Huh7 cell line by MTT assay. Briefly, 1 × 10 4 cells/well were harvested in a 96-well plate and incubated at 37 °C with humidified air containing 5 % CO 2 for 24 h. Then, different concentrations (0-0.20 mg/mL) of the drug-loaded magnetic nanoassembly were added to each well medium, in triplicate, and incubated in 5 % CO 2 humidified air at 37 °C for 24 h to determine cell viability.

UV-visible spectroscopy
The UV-vis spectra of the pure cobalt ferrite NPs were recorded in the range of 100-600 nm using a spectrometer (model UV-2600; Shimadzu, Japan). The drug loading and release profile was also obtained with the same instrument at 481 nm.

Fourier transform-infrared (FT-IR) spectroscopy
The FT-IR spectra of the cobalt ferrite NPs and its nanocomposites were recorded on a spectrophotometer (model 983; Perkin-Elmer, USA) using the KBr technique in the range of 400 -4,000 cm -1 . The thermal properties of the native cobalt ferrite NPs and the weight percentage of XG coated on the NPs were determined by TGA (model TGA-50; Shimadzu, Japan) over the range of 20 -800 °C.

Contact angle measurements
The contact angles of uncoated and XG-coated cobalt ferrite NPs were measured using a contact angle meter with the sessile drop method (model HO-ED-M-01, Holmarc Opto-Mechatronics, India).

X-ray diffraction (XRD) studies
XRD patterns were measured with a diffractometer (model 6000; Shimadzu, Japan) in the 2 range of 5-70° using Ni-filtered Cu-K radiation. The crystallite size was determined from the broad diffraction plane (311) using the Debye-Scherer equation (Eq 2) [14]: where k is a constant (0.9), β is the full-width at half-maximum (FWHM) of the high-intensity diffraction peak corresponding to the (311) plane, λ is the wavelength of the Cu Kα radiation (1.54 Å) and θ is the Bragg angle.

Vibrating sample magnetometry (VSM)
The magnetic potential of coated and uncoated CoFe 2 O 4 .NPs was evaluated using a vibrating sample magnetometer (model 7404; Lake Shore Cryotronics, USA) at 300 K at an AMF ranging from −15 to 15 KOe.

Microstructural analysis
The microstructures of prepared samples were studied by scanning electron microscopy (SEM; model TM-1000; Hitachi, Japan). Atomic force microscopy (AFM; Park NX10, Korea) was used to measure the size of native and coated ferrite particles dispersed in hexane. The hydrodynamic diameter and polydispersity index (PDI) of a sample was measured by dynamic light scattering (DLS; Zetasizer Nano S; Malvern, UK).

UV-vis spectra
The successful synthesis of the cobalt ferrite NPs by co-precipitation was initially confirmed by UV-Vis spectroscopy. The nanoferrite spectra displayed a characteristic shoulder band in the range of 330-500 nm that was attributed to their synthesis [15] (Figure 2).

Thermal characteristics
Thermal analysis of the uncoated and coated CFNPs was performed from 25 to 800 °C to obtain quantitative evidence of the nanoferrite coating. The TGA curves of the pure and XGcoated ferrite NPs displayed different thermal behaviours (Figure 4). The percentage weight loss of the uncoated ferrite nanocrystals was just 3 % over the range of 20 -200 °C. However, the XG-coated CFNPs exhibited a ca. 8% mass loss below 150°C, which increased to ca. 16 % from 150 to 380 °C.

Contact angle
The wettability and surface interactions of the coated and uncoated CFNPs with water were determined by contact angle measurements. CFNPs should be hydrophilic for biomedical applications. Figure 4 shows the sessile water droplet (2 μL) contact angles over thick solid sample surfaces. The contact angles of the native and XG-coated ferrite NPs were 92.40° and 39.90°, respectively ( Figure 5 (a, b)).

Morphology
SEM, AFM and DLS were used to investigate the microstructure of the uncoated and XG-coated CFNPs. SEM images (Figure 7 (a, b)) reveal the elongated, oval-shaped morphology of the native ferrite NPs. The average size of the native and coated CFNPs ranged from 20 to 60 nm. Topographical images of the uncoated and coated CoFe 2 O 4 .NPs are shown in Figure 8 (a,  b). Furthermore, the 3-D AFM image (Figure 8c) confirmed the oval-shaped texture of the uncoated CFNPs, as previously noted [18]. DLS measurements ( Figure 9) established a PDI of less than 0.16 and a nanosized range for all of the prepared samples [9].

Magnetic properties
The magnetic properties of the uncoated and XG-coated CFNPs were measured by VSM at room temperature. Figure 10 shows the magnetisation as a function of the applied field (M-H curves) of the coated and uncoated CFNPs for magnetisation varying from −15 to 15 kOe. VSM study produced Ms values of 62.55 and 50.27 emu/g for the uncoated and coated CFNPs, respectively. A suspension of pure CFNPs showed a strong attraction toward a magnetic field (Figure 11).

Drug release profile
The anti-tumour drug release profile of the drugloaded MNV was determined in the absence and presence of an external AMF. The Doxo encapsulation efficiency of the  [20]. The drug release behaviour of the MNV was studied for 120 h in phosphatebuffered saline (0.1 M) at two different pHs (7.4 and 5.0) at 37 °C (Figure 12 (a, b)). The pH of 7.4 corresponds to the normal environment of body tissue and blood, while pH 5.0 is more appropriate for tumour-affected cells [21]. The drug-loaded MNV (CoFe 2 O 4 .NPs@XG-Doxo) did not show a significant release of Doxo at neutral pH 7.4 in the absence (13.5 %) as well as presence (32 %) of AMF after 120 h. In contrast, at pH 5.0, the discharge of drug was slow (ca. 15 %) in the first 6 h, which increased to 39 % after 120 h. However, an increased, controlled and regular release of drug up to 69% was observed in the presence of the external AMF after 120 h.

In vitro toxicity of CoFe 2 O 4 .NPs@XG
The MTT assay against the CHO and Huh7 cell lines was used to establish the in vitro toxicity of the XG-coated ferrite NPs. Figure 13 exhibits the cell viability profiles of the CHO and Huh7 cells incubated at various concentrations (0-2.0 mg/mL) of MNV. After 48 h of dose exposure, cell viability exceeding 80 %, even at a high concentration (1.6 mg/mL), confirmed the biocompatibility of the CoFe 2 O 4 .NPs@XG. This is likely because XG is a food-grade polysaccharide whose degradation products are not toxic to the exposed cells.

Pharmacological activity of CoFe 2 O 4 .NPs@XG-Doxo
A 0-0.18 mg/mL dose of the drug-loaded MNA (CoFe 2 O 4 .NPs@XG-Doxo) was applied to Huh7 cells over 48 h of incubation to investigate its pharmacological activity. Figure 14 reveals the significant decrease in the percentage cell viability with increasing concentration of the magnetic nanoassembly. More than 50 % of the cells were dead even at the low concentration of 0.16 mg/mL. This marked decrease in cell viability was attributed to the anti-proliferative effect of the Doxo discharged from the MNA.

DISCUSSION
The UV-vis absorption band in the range of 330-500 nm was attributed to the homogenised electronic motion of the CFNPs. Generally, metal nanoparticles display a characteristic plasmon resonance band in their UV-vis absorbance spectra due to the collective resonance of their conduction electrons [19]. Peaks in the range of 400-600 cm −1 for metal ferrite NPs indicate the presence of a metal-oxygen bond. The splitting of the low-frequency band for the octahedral site (B) reveals the presence of different metal ions including Fe 3+ , Fe 2+ and Co 2+ .   (Figure 3c) revealed drug adsorption in two ways: first, by the partial interaction between the XG carboxylate (COO − ) and amine (-NH 2 ) groups of the drug via amide formation and, second, by the -OH vibrational bands [21].
The percentage weight loss determined by TGA of the pure CFNPs was attributed to the evaporation of absorbed water and ethanol from the nanoferrite powder surface. Such a small change in mass was consistent with the pure spinel phase of the CFNPs. The mass loss of the XG-coated CFNPs was mostly observed in two regions. The first mass loss was due to the loss of loosely bound water molecules and the coated layer of polymer. However, the second step of a sharp mass loss corresponded to the combustion of the polymer coating from the NP surface [22].
Coating with XG is an effective way to reduce the hydrophobicity of ferrites and increase their water dispersibility. Indeed, the presence of abundant hydroxyl (-OH) and carboxyl (-COOH) groups in XG increases the porosity and surface energy of XG-coated magnetic NPs, and increases their hydrophilicity [23]. SEM images of the uncoated CFNPs revealed some agglomeration that was attributed to van der Waals forces and dipole-dipole magnetic attractions [25]. However, the XG-coated CFNPs were less agglomerated and had lower polydespersity.
The absence of a hysteresis loop, and negligible remanence (Mr) and coercivity (Hc) reflected the superparamagnetic behaviour of the CFNPs. The lower Ms value of the coated CFNPs (CoFe 2 O 4 .NPs@XG) may be correlated with various factors such as the low mass and small size of the magnetic core and the presence of the magnetically inactive XG layer, which reduced the exchange coupling energy of the magnetic field [26]. Additionally, the high magnetisation value of the native CFNPs reflected their uniform distribution and small degree of agglomeration.