Stability and magnetically induced heating behavior of lipid-coated Fe3O4 nanoparticles
© Allam et al.; licensee Springer. 2013
Received: 23 June 2013
Accepted: 2 October 2013
Published: 17 October 2013
Magnetic nanoparticles that are currently explored for various biomedical applications exhibit a high propensity to minimize total surface energy through aggregation. This study introduces a unique, thermoresponsive nanocomposite design demonstrating substantial colloidal stability of superparamagnetic Fe3O4 nanoparticles (SPIONs) due to a surface-immobilized lipid layer. Lipid coating was accomplished in different buffer systems, pH 7.4, using an equimolar mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and l-α-dipalmitoylphosphatidyl glycerol (DPPG). Particle size and zeta potential were measured by dynamic laser light scattering. Heating behavior within an alternating magnetic field was compared between the commercial MFG-1000 magnetic field generator at 7 mT (1 MHz) and an experimental, laboratory-made magnetic hyperthermia system at 16.6 mT (13.7 MHz). The results revealed that product quality of lipid-coated SPIONs was significantly dependent on the colloidal stability of uncoated SPIONs during the coating process. Greatest stability was achieved at 0.02 mg/mL in citrate buffer (mean diameter = 80.0 ± 1.7 nm; zeta potential = -47.1 ± 2.6 mV). Surface immobilization of an equimolar DPPC/DPPG layer effectively reduced the impact of buffer components on particle aggregation. Most stable suspensions of lipid-coated nanoparticles were obtained at 0.02 mg/mL in citrate buffer (mean diameter = 179.3 ± 13.9 nm; zeta potential = -19.1 ± 2.3 mV). The configuration of the magnetic field generator significantly affected the heating properties of fabricated SPIONs. Heating rates of uncoated nanoparticles were substantially dependent on buffer composition but less influenced by particle concentration. In contrast, thermal behavior of lipid-coated nanoparticles within an alternating magnetic field was less influenced by suspension vehicle but dramatically more sensitive to particle concentration. These results underline the advantages of lipid-coated SPIONs on colloidal stability without compromising magnetically induced hyperthermia properties. Since phospholipids are biocompatible, these unique lipid-coated Fe3O4 nanoparticles offer exciting opportunities as thermoresponsive drug delivery carriers for targeted, stimulus-induced therapeutic interventions.
7550Mw; 7575Cd; 8185Qr
KeywordsSPION Magnetic field generator Hyperthermia Phospholipid Thermoresponsive Colloid
The use of nanosized colloids offers exciting new opportunities for biomedical applications as they have the potential to overcome significant limitations associated with therapeutic drugs (e.g., physical, chemical, or biochemical instability). In addition, encapsulation of pharmacologically active agents into such nanocarriers allows for spatial and temporal control of drug release, which can significantly improve clinical effects (e.g., controlled and targeted delivery) [1, 2].
Superparamagnetic Fe3O4 nanoparticles (SPIONs) are explored as novel drug delivery systems as their orientation within a magnetic field offers new opportunities to manipulate accumulation and/or drug release in desired target tissues by an externally applied magnetic field . Similar to other biomedical applications of SPIONs, including magnetic resonance imaging, biosensing, and cell separation, clinical development critically depends on efficient magnetization and favorable pharmacokinetic properties that minimize clearance by the reticuloendothelial system. It is generally accepted that nanoparticles with hydrophilic surfaces and those less than 200 nm in diameter are compliant with these desired specifications [4, 5].
The large surface-to-volume ratio of small magnetic nanoparticles increases surface energy and, thus, enhancing particle aggregation. As a consequence, chemical reactivity decreases, magnetic properties deteriorate, and clearance within a biological system increases [6–9]. Particle stability in an aqueous vehicle can be augmented by electrostatic repulsion using charged surface coatings and/or surface-associated ions, including OH-, H3O+, or buffer ions . The ability to absorb and convert electromagnetic energy into heat distinguishes SPIONs from other nanoassemblies. As heat or 'hyperthermia’ sensitizes living cells to apoptotic stimuli, this unique feature of SPIONs appears specifically beneficial in cancer therapy where temperatures between 40°C and 45°C have been demonstrated to synergistically enhance or potentiate chemotherapy and radiation efficacy [11, 12]. Hyperthermia generated by SPIONs following exposure to an alternating magnetic field arises from energy loss associated with oscillation and Néel/Brownian relaxation of the nanoparticle magnetic moment . Stimulus-induced heat generation can also be utilized to control dissociation of a therapeutic moiety from a thermoresponsive carrier that undergoes reversible volume or sol-gel phase transition within a desired range of 37°C to 45°C [14–16]. Previously, our laboratory described a novel phospholipid/Fe3O4 nanocomposite designed for stimulus-controlled release of an encapsulated payload via magnetically induced hyperthermia . These results demonstrated the feasibility of immobilizing a 2- to 3-nm-thick layer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) on the surface of SPIONs via high affinity avidin/biotin interactions without negatively affecting magnetically induced heating properties. However, moderate surface charge (zeta potential -5.0 ± 3.0 mV) afforded by the zwitterionic but charge-neutral phospholipid assembly resulted in limited colloidal stability, which rapidly led to particle aggregation into the micrometer range .
The aim of the present study was to explore the impact of a modified phospholipid composition and different fabrication parameters during the lipid coating process on colloidal stability of these thermoresponsive nanocomposites. In addition, the concentration-dependent heating behavior of these nanoassemblies was compared using two magnetic field generators of different designs. Surface immobilization of an equimolar mixture of DPPC and l-α-dipalmitoylphosphatidyl glycerol (DPPG) on SPIONs significantly increased colloidal stability of these nanocomposites in physiological buffer systems. Exposure to an alternating magnetic field rapidly increased the temperature of the surrounding vehicle as a consequence of magnetically induced hyperthermia. Heating rates were dependent on particle concentration, suspension vehicle, and magnetic field generator design. These results underline the importance of standardized in vitro assessment of SPIONs for magnetically induced hyperthermia applications in order to effectively facilitate clinical development of these promising nanocarriers.
SPIONs were synthesized following a previously published coprecipitation method . Briefly, 4.44 g of FeCl3·6H2O and 1.73 g of FeCl2·4H2O (Thermo-Fischer Scientific, Pittsburgh, PA, USA) were dissolved in deionized water at a molar ratio of 1:2. Temperature was increased to 70°C while stirring under N2 protection before 20 mL of an aqueous 0.5 M NaOH solution was added under continuous stirring. Precipitation was completed after 30 min at 90°C, and SPIONs were collected by magnetic separation following three washes with deionized water.
Fabrication of lipid-coated Fe3O4 nanoparticles
A DPPC/DPPG (50:50, mol/mol) lipid coat was immobilized on the surface of SPIONs via high-affinity avidin/biotin interactions as described previously by this laboratory . For a standard fabrication batch, 1 mL of Fe3O4 nanoparticles suspended at 0.024 mg/mL in citrate buffer, pH 7.4, was incubated with 0.05 mg/mL of avidin at 4°C for 24 h. Excess avidin was removed by three consecutive wash cycles using the same citrate buffer. In a separate 1.5 mL microcentrifuge tube, 95 μL of an equimolar DPPC/DPPG mixture (NOF America, White Plains, NY, USA) prepared in CHCl3 was combined with 5 μL of 0.6 mM DSPE-PEG2000-biotin (Avanti Polar Lipids, Alabaster, AL, USA) solution prepared in the same organic solvent. CHCl3 was removed under vacuum forming a dry phospholipid film along the centrifuge tube wall. Affinity-stabilized immobilization of a phospholipid layer on avidin-coated SPIONs was induced at room temperature by a 15-min continuous exposure to ultrasonic waves (60 Hz) followed by an additional stabilization period of 30 min at 4°C. Phospholipid-modified Fe3O4 nanoparticles were washed three times with the buffer solution of interest before used for experiments.
Physicochemical particle properties
Particle size distribution and electrokinetic potential of uncoated and lipid-coated SPIONs were determined by dynamic laser light scattering (DLS) using the Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK) equipped with a 4-mW helium/neon laser (λ = 633 nm) and a thermoelectric temperature controller. Particle suspensions prepared in different buffer solutions were preincubated at 25°C for 5 min before each measurement. Particle size values reported in this study correspond to hydrodynamic diameters.
Magnetically induced hyperthermia
Experiments were performed in triplicate unless otherwise noted. Statistical assessment of differences between experimental groups was performed by one-way ANOVA or two-sided Student's t test for pairwise comparison. A probability value of p < 0.05 was considered statistically significant (GraphPad Prism 6.0, GraphPad, San Diego, CA, USA).
Results and discussion
Fabrications of lipid-coated Fe3O4 nanoparticles
Physicochemical properties of uncoated and lipid-coated SPIONs in different buffer solutions at pH 7.4
Particle concentration (mg/mL)
Particle size (nm)
Zeta potential (mV)
520.0 ± 45.4
651.6 ± 25.3
-32.4 ± 1.0
-11.9 ± 1.4
Citrate, pH 7.4
286.6 ± 25.4
460.3 ± 15.4
-40.7 ± 1.4
-15.6 ± 1.4
80.0 ± 1.7*
179.3 ± 35.0**
-47.1 ± 2.6*
-19.1 ± 1.3**
1860.0 ± 180.9a
2422.0 ± 223.5a
-11.2 ± 1.0
-4.5 ± 0.9
HBSS, pH 7.4
1255.0 ± 35.2a
1560.0 ± 135.2a
-12.3 ± 1.1
-5.5 ± 1.0
580.0 ± 8.5
193.5 ± 32.6**
-23.3 ± 0.8
-7.4 ± 1.4
2800.0 ± 320.4a
2990.0 ± 412.5a
-10.3 ± 0.5
-2.2 ± 0.6
PBS, pH 7.4
2214.0 ± 45.3a
2500.0 ± 245.3a
-10.8 ± 1.0
-3.4 ± 1.1
931.0 ± 4.5
229.9 ± 12.42**
-22.5 ± 0.8
-5.2 ± 1.6
Earlier experiments performed in our laboratory with DPPC-coated SPIONs revealed limited colloidal stability in physiological buffer systems due to low surface charge (zeta potential -5.0 mV) . DPPG is a negatively charged phosphatidyl glycerol with the same transition temperature as DPPC (i.e., 41°C). Stability of liposomes prepared with mixtures of these two phospholipids has been studied previously, and an equimolar lipid ratio was demonstrated to enhance colloidal stability . When comparing physicochemical properties of lipid-coated SPIONs suspended in different buffer systems (Table 1), it was notable that the mean particle size significantly increased to 179 nm after the coating procedure. It is conceivable that the modified avidin coating protocol using citrate buffer altered the charge distribution at the steric layer, thus augmenting the negative surface charge of avidin-coated SPIONs. With the introduction of the negatively charged DPPG into the lipid mixture, charge repulsion may have resulted in less tight association of the lipid layer with the avidin-coated Fe3O4 surface. Further assessment of the nanoassembly using high-resolution transmission electron microscopy (HRTEM) and atomic force microscopy could provide additional experimental support for this hypothesis. Nevertheless, it is relevant to emphasize that DLS measurements are performed in the presence of a liquid suspension vehicle (e.g., citrate buffer) and determine hydrodynamic particle size distributions. HRTEM requires dry samples and may result in different quantitative size information due to the absence of a surface-associated hydration layer. The incorporation of a 50% molar ratio of DPPG into the lipid layer effectively augmented the negative surface charge of the lipid coat from -5.0  to -19.1 mV. The enhanced negative charge associated with the nanoparticle surface is expected to increase colloidal stability of the suspension. Furthermore, it is predicted that this favorable zeta potential reduces surface adsorption of serum components such as proteins and lipoproteins . Ultimately, these improved physicochemical properties of lipid-coated SPIONs may significantly increase biological circulation time after systemic administration allowing more effective delivery of therapeutic payload to desired target cells.
Magnetically induced hyperthermia
Initial heating rates of uncoated and lipid-coated SPIONs following exposure to an alternating magnetic field
Particle concentration/suspension vehicle
Initial heating rate (°C/min)
MFG-1000 at 7.0 mT (1 MHz)
MHS at 16.6 mT (13.6 MHz)
1 mg/mL (Citrate buffer)
0.88 ± 0.02
1.26 ± 0.03**
0.35 ± 0.01
0.61 ± 0.02**
0.24 mg/mL (Citrate buffer)
0.90 ± 0.02
1.05 ± 0.04
0.36 ± 0.02
0.56 ± 0.01
0.02 mg/mL (Citrate buffer)
0.95 ± 0.03*
0.94 ± 0.02
0.47 ± 0.01*
0.46 ± 0.01
0.02 mg/mL (HBSS)
0.66 ± 0.02
0.94 ± 0.01
0.33 ± 0.01
0.44 ± 0.02
0.02 mg/mL (PBS)
0.55 ± 0.02
0.92 ± 0.02
0.20 ± 0.01
0.43 ± 0.01
The results from this study demonstrate that surface immobilization of an equimolar DPPC/DPPG mixture on SPIONs via high-affinity avidin-biotin interactions increases colloidal stability in the presence of different buffer ions. Citrate buffer, pH 7.4, provides a significant advantage during avidin coating due to efficient colloid dispersion as a consequence of negative surface charge. Magnetically induced heating properties of uncoated and lipid-coated SPIONs were significantly dependent on the design of the magnetic field generator used. However, therapeutically relevant hyperthermia (>40°C was achieved within 10 min following exposure to an alternative magnetic field between 7 and 17 mT. These results underline that biocompatible, phospholipid-coated SPIONs offer exciting opportunities as thermoresponsive drug delivery carriers for targeted, stimulus-induced therapeutic interventions.
Dynamic laser light scattering
High-resolution transmission electron microscopy
Magnetic hyperthermia system
Superparamagnetic iron oxide nanoparticles.
The authors would like to thank Richard (Jason) Sookoor (University of Cincinnati, Department of Physics) for his assistance with the SPION synthesis. This research was supported in part by a predoctoral fellowship from the Egyptian Ministry of Higher Education awarded to Ayat A. Allam.
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