Functionalization and Haemolytic analysis of pure superparamagnetic magnetite nanoparticle for hyperthermia application

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONPs) are widely used in clinical research. The single domain nanoparticles are used in magnetic fluid hyperthermia (MFH) to treat cancer. When nanoparticles are exposed to an external magnetic field, it generates heat destroying tumour cells. SPIONPs have a large surface area, so the particles tend to aggregate, which leads to the destabilization of the colloidal system. To enhance the stability and biocompatibility of the nanomaterials, it is necessary to coat the surface with biocompatible material. Magnetite (Fe₃O₄) is a superparamagnetic nanoparticle (SPNPs) that was functionalized with oleic acid (OA) by sol–gel process using ethanol as the solvent. The oleic acid-coated magnetite (OA-Fe₃O₄) was characterized by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), UV–Visible diffuse reflectance spectroscopy (UV-DRS) and vibrating sample magnetometer (VSM). The haemolysis test has been used to investigate the haemocompatibility properties of nanomaterials. Hyperthermia study shows a high SAR value for the concentration of 1 mg/ml at the field of 600 Oe and frequency of 316 kHz. The OA coating enhanced the haemocompatibility of synthesized magnetite nanoparticles which can be used for magnetic fluid hyperthermia applications.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10867-022-09614-y.

Introduction

Tumour cells are more thermosensitive than normal cells. In MFH, the magnetic energy is converted into heat energy when it is exposed to an external magnetic field. The temperature can be varied by altering the range of field and frequency (H*f) [1–4]. The H*f factor should be smaller since higher frequency can generate an eddy current, which is harmful to human beings. The SPIONPs dissipate heat through two processes they are Neel and Brownian relaxation. Neel relaxation occurs due to the reorientation of the magnetic moment of the particle, and the Brownian is due to the movement of the particle itself within the solvent. Heat dissipation of magnetic nanoparticles (MNPs) was characterized by a specific absorption rate (SAR). SAR is the power dissipation of the unit mass of the magnetic nanoparticles in an applied field [5–10].

There are various types of nanoparticles (NPs) used in a wide range of applications. Among them, magnetic nanoparticles (MNPs) are important because their unique properties like size, crystallinity, superparamagnetism and magnetization values enhance MNPs for industrial and biomedical applications. The preparation of nanoparticles should require the size smaller than 50 nm, and it should be superparamagnetic and biocompatible.

However, iron oxide nanoparticles (IONPs) have disadvantages such as agglomeration due to large surface area, easily oxidize and toxicity. The NPs will interact with the blood while delivering by intravenous injection. When NPs injected into the blood, it will cause haemolysis. Haemolysis is the destruction of red blood cells, leading to the release of haemoglobin [11–14]. All the drawbacks mentioned are related to the surface of the material, so by engineering the surface the problems can be overcome. The coating has a dominant effect on the heat efficiency of the MNPs. Surface properties are more important than core properties [15]. It is necessary to coat the MNPs with biocompatible material. Coating with conductors like SiO₂ reduces the outflow of heat and coating thickness and also alters the heating efficiency. Functionalization is the process of adding new functionality or functions to the material by changing the surface chemistry of the material [16–20].

Oleic acid (OA) is a fatty acid with amphiphilic properties. It consists of long-chain hydrocarbon as a hydrophobic group and the carboxylic acid group as hydrophilic. Oleic acid can form a strong chemical bond through the interaction of the carboxyl group with magnetite NPs [21–25]. Due to their biocompatibility and low toxicity, OA can be utilized for biomedical applications. The hydrophobic chain has an important role to carry hydrophobic drugs.

In the present work, pure magnetite was functionalized with oleic acid. By coating with OA, the material shows good biocompatibility. The functionalized superparamagnetic magnetite nanoparticles were characterized by XRD, FTIR, TEM, TGA, UV-DRS and VSM. Haemolysis analysis was used to study haemocompatibility. A hyperthermia test was carried out to find the SAR value of the material for hyperthermia application.

Experimental

The materials used are synthesized pure magnetite (Fe₃O₄), oleic acid and ethanol. 0.5 ml of oleic acid was dissolved in 2 ml of ethanol and stirred at 10 °C. Then, simultaneously add 0.7 gm of pure Fe₃O₄ and keep stirring until it becomes a gel. Then, the gel was dried in a hot air oven at 40 °C for half an hour and sintered at 200 °C for an hour. Finally, oleic acid functionalized Fe₃O₄ was obtained.

Characterization

The crystalline nature of OA-coated magnetite (OA-Fe₃O₄) nanoparticles was characterized using powder X-ray diffraction (XRD). The crystallite size of OA-Fe₃O₄ was calculated using the Scherrer formula. The functional group was examined by Fourier transform infrared spectrometer (FTIR) with the sample dispersed in a KBr pellet. The surface morphology and SAED pattern were studied using a transmission electron microscope. A thermogravimetric analyzer (Simultaneous DTA–TGA system, DTG 60 M Shimadzu, 2020) with a heating capacity of up to 600 °C was used to find the weight loss and the stability of the sample. The vibrating sample magnetometer (VSM) is used to study the magnetic property of the sample. A hyperthermia study was carried out to find the SAR value of the sample. In linear response theory (LRT), the SAR is given by Watts per gram and its determined by the equation,

$$\mathrm{SAR}\left(\mathrm{f},,,\mathrm{H}\right)=\frac{\pi {\mu }_0{\chi }^{//}{H}^{2}f}{\rho }$$ 1

Here, ρ is the mass density of the nanomaterial, ${\mu }_0$ is the permeability of free space, ${\chi }^{//}$ is the imaginary part of susceptibility, and H and f are the field and frequency of the AMF. The imaginary part of susceptibility is given by,

$${\chi }^{//}=\frac{2\pi f\tau }{1+{(2\pi f)}^2}{\chi }_0$$ 2

$${\chi }_0=\frac{{\mu }_0{M}_s^{2}V}{k_{B}T}$$ 3

$$\frac{1}{\tau }=\frac{1}{{\tau }_N}+\frac{1}{{\tau }_B}$$ 4

Hence in addition to field and frequency, SAR depends on other factors like magnetic saturation, relaxation time ($\tau$), the magnetic anisotropy of the MNPs and the viscosity of the liquid in which MNPs were dispersed [26].

Haemolytic analysis

Haemolysis analysis was used to study the haemolytic effect of the NPs. Phosphate buffer saline (PBS) and sodium dodecyl sulphate (SDS) were taken as negative control and positive control, respectively. A total of 100 μl of erythrocytes suspension was mixed with different concentration of OA-Fe₃O₄ (100, 90, 80, 70, 60, 50, 40, 30, 20 and 10 μg/ml). The suspension was incubated in a 37 °C water bath for 60 min. The volume of the suspension was made up to 300 μl by adding PBS and it was centrifuged at 1500 rpm for 5 min and the resulting haemoglobin in the supernatant was measured at 540 nm by spectrophotometer to determine the concentration of haemoglobin. The average value was calculated from triplicate assays. Haemolysis percentage for each sample was calculated from the formula:

$$\mathrm{Haemolysis}(\%)=\frac{\mathrm{OD}\mathrm{of}\mathrm{the}\mathrm{sample}\mathrm{to}\mathrm{be}\mathrm{tested}-\mathrm{OD}\mathrm{of}\mathrm{negative}\mathrm{control}}{\mathrm{OD}\mathrm{of}\mathrm{positive}\mathrm{control}-\mathrm{OD}\mathrm{of}\mathrm{negative}\mathrm{control}}$$

Results and discussion

XRD analysis

XRD is used to utilize the crystal structure of the material. XRD patterns of pure Fe₃O₄ and OA-Fe₃O₄ are shown in Fig. 1a, b. XRD pattern shows pure phase magnetite nanoparticles. XRD which have miller indices of (311), (400), (511) and (440) are reliable with reference peaks of magnetite (PDF #19–0629) and there is no other presence of a second face. The decrease in intensity and broadening of the peaks was due to the coating of OA. The average crystallite size was calculated using Scherrer’s formula. The lattice parameter was determined by Bragg’s law.

Fig. 1.

a, b XRD pattern of pure Fe₃O₄ and OA-Fe₃O₄

The average crystallite size of OA-Fe₃O₄ was found to be 7.6 nm and the lattice parameter a = 8.455 Å. The crystallite size of pure Fe₃O₄ was 16 nm and a = 8.348 Å [27–29]. Here, the size decreases for OA-Fe₃O₄. As the size decreases, the lattice parameter increases. The intensity of the peak decreases due to the presence of OA on the surface which forms a protective organic layer. Even after coating, the shape remains unchanged (spherical). The oleic acid reduces the attraction between the particles, leading to decreases in crystallite size.

Fourier transform infrared spectroscopy

Figure 2 shows the FTIR of OA-Fe₃O₄. The peak at 2925 cm⁻¹ denotes the presence of CH₃ stretching which confirms the presence of OA. The peak at 1710 cm⁻¹ shows C = O stretching. The peak at 1529 cm⁻¹ was attributed to Vs (–COO −) stretch. The peak at 451 corresponds to Fe–O. From this, it was confirmed that there were no additional components present.

Fig. 2.

FTIR of OA-Fe₃O₄

Transmission electron microscopy (TEM)

Figure 3a shows the TEM image of OA-Fe₃O₄. The particle size was narrow. The pure Fe₃O₄ NPs tend to agglomerate but OA-coated NPs were separated from the adjacent particle. Figure 3b shows the SAED pattern from which we calculated interplanar distance d. The diffraction rings correspond to (400), (511) and (440) planes with d values 2.1 Å, 1.6 Å and 1.5 Å, respectively. From Fig. 4c, using inverse FFT, we have calculated the interplanar distance d. For OA-Fe₃O₄, the value of d is 1.31 Å which corresponds to the (440) plane of cubic magnetite. Figure 4c shows the inverse FFT of OA-Fe₃O₄ at the core that indicates continuous fringes of magnetite at the core. Figure 5b shows the cropped image at the shell and Fig. 5c shows its FFT pattern. Figure 5d shows inverse FFT of OA-Fe₃O₄ at the shell which indicates the fringes are discontinuous at the shell. This elucidates that the surface was coated with OA acid.

Fig. 3.

a, b TEM image and SAED pattern of OA-Fe₃O₄

Fig. 4.

a HRTEM image of OA-Fe₃O₄, b FFT of OA-Fe₃O₄ at the core, c inverse FFT of OA-Fe₃O₄ at the core

Fig. 5.

a, b Crop image at shell, c FFT of OA-Fe₃O₄, d inverse FFT of OA-Fe₃O₄

UV-DRS spectroscopy

The bandgap is the energy gap (E_g eV) between the conduction band and the valence band. In this, DRS UV is used to measure the optical bandgap energy. Equation (5) gives the value of bandgap energy.

$$E_g(\mathrm{eV})\mathrm{hv}=1240/\lambda$$ 5

where λ is the wavelength, and E_g is optical bandgap energy.

The absorption coefficient (α) was calculated using the following equation,

$$\alpha =2.{303}^{\ast }d/A$$ 6

where d is the sample thickness and A is the absorption data. The figure represents the.

Figure 6 shows the UV-DRS of OA-Fe₃O₄ and its bandgap energy was found to be 2.3 eV. The bandgap value for pure magnetite was found to be 2 eV. As the particle size reduces, the electron got confined. The width of the band gets narrower. This cause an increase in the energy gap between the VB and CB.

Fig. 6.

Tauc’s plot calculated using UV-DRS data of OA-Fe₃O₄

Thermogravimetric analysis

Figure 7 shows the percentage of weight loss as a function of the temperature of OA-Fe₃O₄. The TGA plot of OA-Fe₃O₄ shows the sample was stable till 196 °C without any weight loss. The weight loss of 3.6% from 196 to 433 °C was due to the decomposition of oleic acid on the surface of Fe₃O₄ and also shows the stability of the sample.

Fig. 7.

Thermo gravimetric analysis of OA-Fe₃O₄

Magnetic property

At room temperature, M vs H was calculated using a vibrating sample magnetometer. Figure 8 shows the magnetic property of OA-Fe₃O₄ nanoparticles. The figure shows that the hysteresis loop was absent and it shows superparamagnetic behaviour. The value of magnetic saturation of pure magnetite was 41.31 (emu/g) and for oleic acid-coated magnetite was found to be 14.85 (emu/g). After coating with OA, the Ms value decreases. OA on the surface of the magnetite reduce the interaction between the particles and does not affect the magnetic property which shows superparamagnetism.

Fig. 8.

Magnetic moment against applied magnetic field at room temperature of OA-Fe₃O₄

Haemolytic analysis

The haemolytic activity of Fe₃O₄ and OA-Fe₃O₄ was shown in Fig. 9a, b. According to ASTM E2524-08 (ASTM E2524–08, 2013) standard protocol, the haemolysis % should be less than 5% [27]. Blood erythrocytes were mixed with different concentrations of OA-Fe₃O₄ (10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µg/ml). Haemolytic analysis of pure Fe₃O₄ shows 4.14% of haemolysis activity at 100 µg/ml and on further increases in concentration the, haemolysis % was high to 47.43%. From this study, it was clear that up to 100 µg/ml, there was less haemolysis activity with a rate of 1.8% which was less than 5%. The sample showed zero haemolysis activity up to μg/ml. Haemolysis % for different concentrations of pure Fe₃O₄ OA-Fe₃O₄ is tabulated in Table 1 (a, b)_. Hence, from the result, it was clear that, compared to pure Fe₃O₄, OA-Fe₃O₄ shows good haemocompatibility that can be used for hyperthermia applications.

Fig. 9.

a, b Haemolytic activity of Fe₃O₄ and OA-Fe₃O₄

Table 1.

(a, b) Haemolysis % for different concentrations of Fe₃O₄ and OA-Fe₃O₄

S. No	Tested sample concentration (μg/ml)	Haemolysis (%) (in triplicates)			Mean value (%)
a
1	Control	100	100	100		100
2	100 μg/ml	2.56	1.61	1.24		1.8
3	90 μg/ml	0	0	0		0
4	80 μg/ml	0	0	0		0
5	70 μg/ml	0	0	0		0
6	60 μg/ml	0	0	0		0
7	50 μg/ml	0	0	0		0
8	40 μg/ml	0	0	0		0
9	30 μg/ml	0	0	0		0
10	20 μg/ml	0	0	0		0
11	10 μg/ml	0	0	0		0

b
1	Control	100	100	100		100
2	500 μg/ml	47.67	43.24	51.37		47.43
3	250 μg/ml	11.70	8.016	14.34		11.35
4	100 μg/ml	3.79	3.90	4.74		4.14
5	50 μg/ml	3.58	3.16	3.16		3.30
6	10 μg/ml	0.84	0	2.53		1.12

Magnetic hyperthermia

Hyperthermia works on the principle of the heat dissipation mechanism of MNPs. There were two processes by which MNPs dissipate heat to the tumour cells, namely Neel and Brownian relaxation time. Neel relaxation occurs when the magnetic moment is reoriented parallel to the magnetic field, whereas Brownian relaxation occurs due to the rotation of the particle as a whole concerning the applied field [29, 30]. Heat dissipation was calculated by SAR value. SAR value was calculated from the initial slope method of the temperature–time graph:

$$\mathrm{SAR}=\frac{C}{m}\times \frac{\mathit{dT}}{\mathit{dt}}$$ 7

where C is the specific heat capacity of water, m is the mass of nanoparticles in the suspension and $\frac{\mathit{dT}}{\mathit{dt}}$ is the initial slope of the temperature–time curve.

Magnetic hyperthermia for OA-Fe₃O₄ was performed at 316 kHz under an AMF of 200, 400 and 600 Oe for different concentrations of 0.25, 0.5 and 1 mg/ml. Figure 10a and b show the temperature–time-dependent graph and SAR value for different fields of OA-Fe₃O₄. The temperature increases to 46 °C with an increase in field and concentration. The SAR value depends on the concentration, field and temperature. So we got the highest SAR value of 4200 W/g for 1 mg/ml concentration at 600 Oe. It has been reported that for higher saturation the SAR value increases. But in this study, OA-Fe₃O₄ has a high SAR value despite its low M_s value [31–34]. SAR value depends on various parameters like field, frequency, concentration, size, shape and magnetic property of the material.

Fig. 10.

a, b Temperature–time-dependent graph and c SAR value at different fields (Fe₃O₄)

Conclusion

Magnetite nanoparticles have been functionalized with oleic acid by the sol–gel technique. Coating with oleic acid showed a small crystallite size and reduced the agglomeration of nanoparticles. From surface morphology, the magnetite was successfully coated with OA as the fringes at the shell were discontinuous. Coating with OA reduces magnetic saturation but does not show any impact on the magnetic property (superparamagnetism). From hyperthermia analysis, the OA-Fe₃O₄ showed a high SAR value and it showed good haemocompatibility compared to pure Fe₃O₄. Thus oleic acid-coated magnetite improves the quality of the nanomaterial for hyperthermia applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

Dr. Muralidharan Rajaram, Dr. Pathipan Govindasamy and Hemalatha Kothandaraman: conceptualization. Hemalatha Kothandaraman and Alamelumangai Kaliyamoorthy: methodology. Hemalatha Kothandaraman: software, data curation, writing original draft preparation. Dr.Arulmozhi Rajaram, Chandunika R Kalaiselvan and Niroj Kumar Sahu: formal analysis. Dr. Muralidharan Rajaram: writing review and editing. Dr. Muralidharan Rajaram and Dr. Pathipan Govindasamy: supervision.

Declarations

Ethical approval

This article does not contain any studies involving human participants performed by any of the authors.

Competing interests

The authors declare no competing interests.