Bovine Serum Albumin-Conjugated Ferrimagnetic Iron Oxide Nanoparticles to Enhance the Biocompatibility and Magnetic Hyperthermia Performance
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Magnetic hyperthermia is a fast emerging, non-invasive cancer treatment method which is used synergistically with the existing cancer therapeutics. We have attempted to address the current challenges in clinical magnetic hyperthermia-improved biocompatibility and enhanced heating characteristics, through a single combinatorial approach. Both superparamagnetic iron oxide nanoparticles (SPIONs) of size 10 nm and ferrimagnetic iron oxide nanoparticles (FIONs) of size 30 nm were synthesized by thermal decomposition method for comparison studies. Two different surface modifying agents, viz, Cetyl Trimethyl Ammonium Bromide and 3-Aminopropyltrimethoxysilane, were used to conjugate Bovine Serum Albumin (BSA) over the iron oxide nanoparticles via two different methods—surface charge adsorption and covalent amide bonding, respectively. The preliminary haemolysis and cell viability experiments show that BSA conjugation mitigates the haemolytic effect of the iron oxide nanoparticles on erythrocytes and is non-cytotoxic to the healthy Baby Hamster Kidney cells. It was observed from the results that due to better colloidal stability, the SAR value of the BSA-iron oxide nanoparticles is higher than the iron oxide nanoparticles without BSA, irrespective of the size of the iron oxide nanoparticles and method of conjugation. The BSA-FIONs seem to show improved biocompatibility, as the haemolytic index is less than 2 % and cell viability is up to 120 %, when normalized with the control. The SAR value of BSA-FIONs is 2300 W g−1 when compared to 1700 W g−1 of FIONs without BSA conjugation. Thus, we report here that BSA conjugation over FIONs (with a high saturation magnetization of 87 emu g−1) provide a single combinatorial approach to improve the biocompatibility and enhance the SAR value for magnetic hyperthermia, thus addressing both the current challenges of the same.
KeywordsMagnetic hyperthermia Ferrimagnetic iron oxide nanoparticles Bovine serum albumin Haemolysis Cell viability Specific absorption rate
Cancer is a global killer disease. All the existing cancer treatment strategies like chemotherapy, radiotherapy, and hyperthermia techniques [1, 2] aim to alleviate cancer. Magnetic hyperthermia is a non-invasive technique which is better than the other hyperthermia techniques as it ensures targeted heating of the tumor tissue [3, 4]. Magnetic nanoparticles are a pre-requisite for an efficient magnetic hyperthermia system. Iron oxide nanoparticles with suitable surface modification and functionalization have a plethora of applications in magnetic resonance imaging (MRI), targeted drug delivery, cell separation, cell sorting, especially in magnetic hyperthermia [5, 6, 7], etc. In magnetic hyperthermia technique, iron oxide nanoparticles are injected into the tumor site and are subjected to an external alternating current (AC) magnetic field which raises the temperature of the tumor site up to 42–46 °C. This causes tumor cell death due to necrosis, incase of high dose of temperature–time [8, 9, 10] or apoptosis due to mild exposure and cell sensitization to chemotherapy and radiotherapy incase sublethal dose of temperature–time [11, 12]. Since iron oxide nanoparticles are injected into the biological system to bring about the temperature raise, a system with a very good biocompatibility and heating efficacy is the fundamental requirement for an efficient magnetic hyperthermia system. Thus, the current challenges to be addressed in the field of clinical magnetic hyperthermia are improved biocompatibility and enhanced heating characteristics.
Extensive research has been done to improve the biocompatibility and blood circulation of iron oxide nanoparticles by numerous surface modification and functionalization strategies for various biomedical applications [13, 14, 15, 16, 17, 18]. Similarly, various attempts have been made to enhance the specific absorption rate (SAR) value of the iron oxide nanoparticles by controlling the aggregation, saturation magnetization, anisotropy, etc. [19, 20, 21, 22]. The size and shape of the iron oxide nanoparticles can also be controlled in order to improve the SAR value and thereby enhance the heating characteristics . Achieving both biocompatibility and improved heating efficacy without compromising on either factor poses a huge challenge in the research of clinical magnetic hyperthermia. This paper attempts to address both the pressing challenges by a single combinatorial approach.
Biocompatible iron oxide nanoparticles for magnetic hyperthermia using polymers like poly ethylene glycol (PEG), poly vinyl pyrolidone (PVP), poly ethyleneimine (PEI), biomacromolecules like proteins, aptamers, DNA, and surfactants like CTAB are well reported [24, 25, 26]. Combinatorial approach to simultaneously improve the biocompatibility and SAR value using noble metal like Platinum-coated iron oxide core–shell nanoparticles are also reported [27, 28]. Chemical surface modifying agents render good hydrophilicity and stability to the iron oxide nanoparticles. But they are cytotoxic when used beyond the optimum threshold level [29, 30, 31]. Therefore, biomacromolecules like DNA and proteins are preferred surface functionalization agents. For our work, we have chosen bovine serum albumin (BSA) as the biocompatibility agent. Albumin is a versatile protein which forms almost 55 % of blood plasma protein content and helps maintain the pH and osmotic pressure of blood [32, 33]. Thus, BSA conjugation improves the stealth characteristics of iron oxide nanoparticles and hence prolongs the blood circulation time [34, 35]. BSA is also reported to raise the temperature of a nanoparticle system under an applied AC magnetic field by the formation of isotropic clusters . Previous reports of Human Serum Albumin (HSA)-conjugated superparamagnetic iron oxide nanoparticles (SPIONs) by Keshavarz et al. and BSA-conjugated SPIONs by Samanta et al.  also show that albumin conjugation improves the colloidal stability and thereby the SAR value for magnetic hyperthermia. In these reports, SPIONs were synthesized by inorganic co-precipitation method and albumin was conjugated by physical adsorption method. Thus, we have chosen BSA as our single preferred candidate for both improved biocompatibility and enhanced heating efficiency. Moreover ferrimagnetic iron oxide nanoparticles (FIONs) have higher saturation magnetization and hence better SAR value than SPIONs [38, 39]. The unique heat enhancement property of BSA adds further value to heating efficiency of FIONs. Therefore, BSA and FIONs are the candidates of interest for the single combinatorial approach to address the challenges in magnetic hyperthermia.
We have fabricated BSA-conjugated SPIONs of size 10 nm and FIONs of size 30 nm. The iron oxide nanoparticles were synthesized by thermal decomposition method using organic precursors [40, 41]. This is the highly preferred method for synthesizing iron oxide nanoparticles of uniform shape, size, and monodispersity when compared to the inorganic synthesis protocols [42, 43, 44]. Though inorganic synthesis protocols can generate hydrophilic iron oxide nanoparticles, the thermal decomposition method produces better quality hydrophobic iron oxide nanoparticles. The challenges in finding a suitable surface modifying agent to overcome the hydrophobicity of the as-synthesized iron oxide nanoparticles were met out. Two surface modifying agents, viz, Cetyl Trimethyl Ammonium Bromide (CTAB) and 3-aminopropyltrimethoxysilane (APTMS), were used to render preliminary hydrophilicity to the as-synthesized hydrophobic iron oxide nanoparticles and also to facilitate the conjugation of BSA by two different approaches—physical adsorption by surface charge interaction (CTAB-iron oxide nanoparticles) and strong covalent amide bonding by 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) method (APTMS-iron oxide nanoparticles). The haemolytic and cell viability studies show that the biocompatibility of the BSA-iron oxide nanoparticles is improved when compared to the hydrophilic iron oxide nanoparticles without BSA. The SAR value of BSA-iron oxide nanoparticles is also significantly enhanced, irrespective of the size of the iron oxide nanoparticle and method of BSA conjugation.
Our results show that BSA-conjugated FIONs show better biocompatibility and heating characteristics when compared to the BSA-conjugated SPIONs and hydrophilic SPIONs/FIONs without BSA conjugation. It is also evident from our results that BSA conjugation over iron oxide nanoparticles through covalent amide bond formation is better than physical adsorption in improving the biocompatibility and SAR value. To the best of our best knowledge, there are no previous attempts to fabricate a single combinatorial system of BSA-FIONs to address the two main challenges in clinical magnetic hyperthermia-biocompatibility and high heating efficiency.
2 Materials and Methods
2.1 Synthesis of Iron Oxide Nanoparticles
The iron oxide nanoparticles were chemically synthesized by the thermal decomposition method as described elsewhere [45, 46]. The chemical composition and physical parameters were modified as per the requirements. Briefly, iron(III) acetylacetonate (Sigma Aldrich) was added to oleic acid (Sigma Aldrich) and benzyl ether (Sigma Aldrich) in required composition and heated under nitrogen purging to 110 °C in order to remove moisture. The temperature was later increased to 160 °C, to initiate nucleation. The reaction was maintained at 280 °C with reflux to promote growth of the iron oxide nanoparticles. The size of the iron oxide nanoparticles depends on the duration of the reaction which is maintained at 280 °C.
We have synthesized SPIONs of size 10 nm and FIONs of size 30 nm. The as-synthesized iron oxide nanoparticles were characterized for their shape and size using Transmission Electron Microscope (TEM, JEOL 100CX). The magnetization properties of the two different sizes—10 nm SPIONs and 30 nm FIONs—were investigated using Vibrating Sample Magnetometer (VSM, Lake Shore Model 7407) and Superconducting Quantum Interference Device (SQUID, QuantumDesign). The size, crystallinity, and purity of the sample were studied using X-ray diffractometer (XRD, Bruker D8 Advanced Diffractometer System with Cu Kα (1.5418 Å) source).
2.2 Surface Modification of Iron Oxide Nanoparticles
2.2.1 Conversion to Hydrophilic Phase
The as-synthesized iron oxide nanoparticles were hydrophobic in nature due to the oleic acid capping. The iron oxide nanoparticles have to be converted to hydrophilic phase so as to use it for in vitro and in vivo applications.
184.108.40.206 Cationic Capping Using CTAB
The strategy of phase transfer using additional hydrophilic molecular layer over the original ligand (oleic acid) of iron oxide nanoparticles was used in this approach. CTAB is a quarternary salt whose hydrocarbon chains adsorb onto the oleic moiety of the iron oxide nanoparticles, allowing the cationic ammonium moiety to face out into the solution, making the iron oxide nanoparticles hydrophilic .
Briefly, 0.1 M of CTAB was added to 10 mg iron oxide nanoparticles. The mixture was vortexed and heated to 80 °C. The reaction was stopped after 3 h, and the mixture was washed thrice with ethanol and distilled water. The product was well dispersed and stored in de-ionised water.
220.127.116.11 Ligand Exchange by APTMS
In the ligand exchange strategy, the hydrophilic ligand with more affinity towards the inorganic iron core replaces the original hydrophobic ligand capping the iron oxide nanoparticles, thus rendering hydrophilicity .
In our experiments, the silane group of APTMS was exchanged for oleic moiety on the iron oxide nanoparticles. Briefly, 1 mg of iron oxide nanoparticles was dispersed in 10 mL toluene. To the dispersion, 90 µL of APTMS was added and vortexed thoroughly. The mixture was kept in the shaker at room temperature for 72 h for the ligand exchange reaction to take place. The reaction mixture was washed with ethanol and later with distilled water. The product was found to be well dispersed in water.
The hydrodynamic radius and thereby the stability of the hydrophilic iron oxide nanoparticles over a period of time were studied using Dynamic Light Scattering technique (DLS, Malvern Zetasizer Nano-ZS). The coating of surface modified agents over the oleic acid-capped iron oxide nanoparticles was confirmed by Fourier Transform Infra Red spectroscopy (FTIR, Varian 3100).
2.2.2 Conjugation of BSA
Conjugation of biomacromolecules like proteins on to the hydrophilic iron oxide nanoparticles, improves the colloidal stability and specificity of the system [49, 50, 51]. Generally, the inner side of a protein is hydrophobic while it exposes its hydrophilic amino acid side into the solution. This attribute makes it favourable for researchers to make biocompatible protein-nanoparticle systems . In our experiment, BSA (Sigma Aldrich) was conjugated over the hydrophilic iron oxide nanoparticles in order to render improved biocompatibility and blood circulation.
18.104.22.168 Physical Adsorption using CTAB
In this method of physical adsorption, the cationic CTAB-iron oxide nanoparticles were incubated with anionic BSA. BSA is anionic in nature due to the carboxylate moiety in the amino acids. It can be physically adsorbed on to the cationic CTAB-iron oxide nanoparticles by surface charge interaction. 2 mg mL−1 of BSA in 1X Phosphate Buffer Saline (PBS) buffer was added to the hydrophilic CTAB-iron oxide nanoparticles and was left in shaker overnight and washed thoroughly with distilled water.
22.214.171.124 Covalent Amide Bond Formation using APTMS
The APTMS-capped iron oxide nanoparticles have exposed amino groups. The carboxylic groups on the BSA can be activated by the EDC method . A strong amide bond was formed between the carboxylic group of BSA and amino group of APTMS-capped iron oxide nanoparticles. Briefly, 26 mM EDC and 10 mM NHS were prepared in MES (2-(N-morpholino) ethane sulfonic acid) buffer. 200 µL of EDC/NHS/MES mixture was added to 1 mL BSA (2 mg mL−1) to activate the carboxylic group. 2 mL of APTMS-iron oxide nanoparticles was dispersed in 1X PBS (0.1 mg mL−1). The pH was maintained around 7.2–7.4. Both the solutions were then mixed and left in the shaker overnight at room temperature. The reaction was terminated and the product was washed.
2.3 Improvement of Biocompatibility
2.3.1 Blood Aggregation and Haemolytic Studies
The haemolytic index was also calculated according to ASTM F756-00 standards, according to which, 0–2 % is non-haemolytic; 2–5 % is mildly haemolytic and >5 % is haemolytic .
2.3.2 Cell Viability Studies
The cell viability assay was performed on healthy Baby Hamster Kidney (BHK) cells (ATCC) in order to compare the biocompatibility of the BSA-conjugated FIONs and hydrophilic FIONs without BSA. Cell Counting Kit-8 (CCK-8) was used to perform the cell viability studies.
Briefly, 100 µL of cell suspension was dispensed into a 96-well plate and pre-incubated at optimum conditions. To the well, 10 µL of BSA-APTMS-FIONs and 10 µL of APTMS-FIONs of final ferric ion concentration of 12.5, 25, 50, and 100 µg mL−1 were added. The plate was co-incubated for 24 h. CCK-8 solution of 10 µL was added and further incubated for 4 h. The absorbance at 450 nm was measured using FluoStar Optima microplate reader.
2.4 Enhancement of Heating Efficiency
3 Results and Discussion
3.1 Characterization of the As-Synthesized Iron Oxide Nanoparticles
3.2 Surface Modification of Iron Oxide Nanoparticles
3.2.1 Conversion to Hydrophilic Phase
For the APTMS-coated iron oxide nanoparticles, the peaks at 1700 and 1648 cm−1 are due to the stretching vibration of C = O and stretching vibration of C = C bonds, respectively. The peaks at 1172 and 1017 cm−1 are due to the stretching vibration of C–O. The asymmetric stretching vibration and scissoring bending vibration of CH2 group are represented by peaks at 2926 and 1460 cm−1, respectively . This is evident from Fig. 6b.
3.2.2 Conjugation of BSA
3.3 Improvement of Biocompatibility
3.3.1 Blood Aggregation and Haemolytic Studies
Showing the absorbance of haemoglobin at 540 nm
Absorbance @ 540 nm
Positive control (distilled water)
Negative control (0.1 M NaCl)
10 nm SPIONs
30 nm FIONs
CTAB-10 nm SPIONs
BSA-CTAB-10 nm SPIONs
CTAB-30 nm FIONs
BSA-CTAB-30 nm FIONs
APTMS-10 nm SPIONs
BSA-APTMS-10 nm SPIONs
APTMS-30 nm FIONs
3.3.2 Cell Viability Studies
From the haemolytic studies and the cell viability studies, it is evident that BSA conjugation improves the biocompatibility of the iron oxide nanoparticles system. BSA-FIONs show better results when compared BSA-SPIONs or as-synthesized particles or the hydrophilic particles. Thus, the first requisite for an efficient magnetic hyperthermia system is met out.
3.4 Enhancement of Heating Efficiency
The SAR value plots show that, irrespective of the method of conjugation, BSA improves the heating efficiency of the system. While here we report the phenomenon, we are to further investigate the possible reasons behind this interesting phenomenon. It is stated elsewhere that isotropic clusters of BSA are formed under AC magnetic field . Isotropic clusters might have possibly prevented the fibrous aggregation of iron oxide nanoparticles under the AC magnetic field. In general, fibrous aggregation increases the critical size of the nanoparticles in a solution and hence decreases the specific heat of the system. It is well reported that aggregation of nanoparticles decreases the SAR value significantly [40, 64]. Prevention of aggregation by BSA conjugation might have thus enhanced the SAR value of the system. As reported by Samanta et al. in similar studies with SPIONs, this phenomenon could also be attributed to the increased colloidal stability of the BSA-conjugated iron oxide nanoparticles . The BSA-conjugated iron oxide nanoparticles were well separated and well suspended, even under applied magnetic field, whereas the hydrophilic iron oxide nanoparticles without BSA conjugation aggregate in the presence of magnetic field. This interesting phenomenon of improved heating efficiency relating to the colloidal stability imparted by the BSA to the iron oxide nanoparticles will be studied further.
BSA conjugation over FIONs show better SAR value than the BSA-SPIONS, due to the high saturation magnetization. Thus, the second requisite for an efficient magnetic hyperthermia system is also well established.
We have studied the biocompatibility and heating characteristics of both BSA-SPIONS and BSA-FIONs. The iron oxide nanoparticles were synthesized by the preferred thermal decomposition method using organic solvents. BSA conjugation was done by both physical adsorption and strong covalent amide bond formation.
The haemolytic studies and cell viability studies discussed in the paper confirm that the biocompatibility of the iron oxide nanoparticles increased after BSA conjugation. Particularly BSA-FIONs show better biocompatibility than that of BSA-SPIONs. The improved SAR value of the BSA-conjugated iron oxide nanoparticles system is due to the enhanced colloidal stability and prevention of aggregation. Though the study of efficiency of the surface modifying agents used is beyond the scope of this paper, we still report that BSA conjugation by covalent bonding using APTMS has better colloidal stability and hence better biocompatibility and heating efficiency, as BSA-APTMS-FIONs show better results than that of BSA-CTAB-FIONs. Also the higher magnetic saturation of FIONs leads to higher SAR value and hence better heating efficiency than SPIONs.
We thus conclude that the two key challenges of a very good magnetic hyperthermia system-improved biocompatibility and heating enhancement were addressed through the fabrication of BSA-FIONs. To the best of our knowledge, there are no previous systematic studies on the same. A multivariate therapeutic strategy combining magnetic hyperthermia and chemotherapy using targeted, ligand-conjugated FIONs, could be an extension of our work.
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