Abstract
The chemical, geometric and thermal properties of nanofluid obtained dispersing the same type of nanoparticles in water, octane and buffer solution after inclusion in polylactic and glycolic acid (PLGA) were investigated by means of an experimental set-up in adiabatic conditions. The Mn-ferrite nanoparticle were obtained by thermal decomposition. The aim is to obtain some very thermally performant nanoparticles suitable to treat cancer by Magnetic Fluid Hyperthermia (MFH). The heating properties of 1 ml suspension samples of the NPs were studied applying a time varying magnetic field of 27.1 kA/m at 245 kHz, a typical frequency for MFH, using a power generator and a cylindrical inductor.
Introduction
MFH uses magnetic nanofluid to increase locally the temperature in the target area using a time varying magnetic field, in order to induce cell apoptosis [1–4]. Nanoparticles can be dispersed in water or other fluid. Nevertheless, in some conditions not all type of nanoparticles could be well dispersed in the fluid without surface treatments or inclusion in a polymer since they can aggregate and form macroscopic particles [5–7]. The solubility and stability of the NPs in polar or not-polar solvents depends on their surface properties. In MFH water soluble NPs are preferred for the need to operate in biological media. Nevertheless, well-performant not-polar NPs can be used if their surface is suitably modified [8–10]. A typical cover layer able to allow water dispersion is made of dextrose, which is also compliant with the biological environment, or of silica; otherwise, for compatibilization, NPs can be included in a polymeric matrix such as Poly Lactic and Glycolic Acid (PLGA) [11–18].
PLGA is a biodegradable FDA (U.S. Food and Drug Administration) approved polymer markedly used for drug delivery [11,13,14]. This polymer form spherical particles with a hydrophobic core that can host iron oxide particles coated with oleate or other hydrophobic chains, thus allowing for their dispersion in polar solvents. In this study, the thermal properties of manganese doped iron oxide nanoparticles (Fe/Mn-NPs) were considered. The SAR for the NPs and the NPs embedded in PLGA is computed considering the mass of NPs or the mass of magnetic material. In particular, the thermal properties of Fe/Mn-NPs embedded in the PLGA are compared with those observed in two different solvents (water and octane) at different concentrations.
Materials and methods
Synthesis
The considered magnetic NPs are an iron oxide doped with manganese. They were prepared by a two steps thermal decomposition method starting from organometallic precursors devised by Sun et al. [19]. Typically, 2.80 g of iron(III) acetylacetonate, [Fe(acac)3], and 1.00 g of manganese(II) acetylacetonate, [Mn(acac)2] were mixed in inert atmosphere (N2) with oleylamine (6.40 g), oleic acid (6.80 g) and 1,2-tetradecanediol (9.20 g) in 100 ml of benzyl ether as solvent. The dark red homogeneous mixture was vigorously magnetically stirred and heated at 200 °C for and 120 min and then at 300 °C for 90 min. After cooling the system to room temperature, the black suspension was poured in a 3-fold volume of ethanol and mechanically stirred. The NPs were magnetically separated, washed three times with ethanol (50 ml), and dried under vacuum. 150 mg of the just prepared NPs were dispersed in 50 ml of benzyl ether and treated in similar conditions with 0.70 g of [Fe(acac)3], 0.25 g [Mn(acac)2], 1.60 g of oleylamine, 1.7 g of oleic acid, and 2.30 g 1,2-tetradecanediol. The purification process was performed as in the first step to obtain the desired product, stored as a solid powder. For heating tests and PLGA inclusion, the solid NPs were dispersed either in n-octane or n-hexane respectively. For dispersion in water, an equal mass of cetyltrimethylammonium bromide (CTAB) have been grinded with the NPs until a homogeneous mixture was obtained and then dispersed in the solvent (deionized water).
PLGA with NPs were prepared as described in [11]. Briefly, Poly(D,L-lactide-co-glicolide) (PLGA) RG 502H 50:50, average molecular weight (MW) 30 000–60 000 Da, and poly (vinyl alcohol) (PVA), MW 31000–50000 Da (98%–99% hydrolyzed) was provided by Sigma-Aldrich. PLGA-NPs were obtained using an oil-in-water (o/w) emulsion solvent extraction method. The emulsion was prepared by dissolving 25 mg of PLGA and Fe/Mn nanoparticles (2.5, 5, 10 mg of NP in PLGA1, PLGA2 and PLGA3, respectively) in 0.5 ml of chloroform; this solution was called phase 1. Phase 2 consisted of 3% w/v PVA aqueous solution (3 ml). Phase 1 was added into phase 2 drop by drop and sonicated for 300 s at 100% power. The final emulsion was transferred to a 100 ml round-bottomed flask and put into a rotary evaporator at 740 mmHg and 30 rpm for 150 min to remove the organic solvent. The excess of PVA and non entrapped NPs were removed by washing the emulsion with vivaspin filters (Sartorius) (cutoff 1 × 106 Da) by centrifugation at 5000 rpm three times with 20 ml of buffer HBS.
Sample analysis
ICP-MS (Inductively Coupled Plasma Mass Spectrometry): NP Fe and Mn concentrations were measured by ICP-MS. Digestion was performed by heating under microwave (Milestone Ethos Up Microwave Digestion System) NP suspensions (10 μl) for 10 ′ at 160 °C, after the addition of 1 ml of concentrated HNO3 (70%). After mineralization, 3 ml of ultrapure water were added to the remaining sample volumes for ICP-MS (Element-2; Thermo-Finnigan, Rodano (MI), Italy).
Dynamic Light Scattering (DLS): The hydrated mean diameter of magnetic particles alone and embedded into PLGA-NPs were determined using a dynamic light scattering (DLS) Malvern Zetasizer 3000HS (Malvern, U.K.) All samples were analyzed at 25 °C in filtered (cutoff, 200 nm) NaCl 10 mM buffer (pH 7.4).
All the NPs samples, pristine and PLGA-embedded, were observed using a Transmission electron microscopy (TEM, TECNAI FEI G2 microscope, Hillsboro, Oregon, USA). The size of nanoparticles was measured using the tools provided by ImageJ software (National Institutes of Health (NIH), USA, https://imagej.nih.gov/ij/index.html).
Thermal characterization
The PLGA NPs were dispersed in a buffer (an isotonic NaCl/Hepes solution (HBS)); whereas pristine nanoparticles were dispersed, considering the different powder concentrations, in n-octane or deionized water (after treatment with CTAB) obtaining homogeneous suspensions. The specific heat of deionized water is 4.180 Jg−1 K−1 while that of n-octane is 2.135 Jg−1 K−1. The heat capacity of the buffer is close to that of deionized water and the density is 1.0046 g/ml.
The nanoparticles, included or not included in PLGA, and dispersed in a fluid were treated with a time-varying magnetic field at 245 kHz with amplitude 27.1 kA/m. The magnetic filed was generated by means of a voltage generator EASYHEAT 8310 LI (Ambrell, Rochester, NY, USA) connected to a cylindrical inductor (7 copper turns with internal diameter of 8 cm and a length of 15 cm) designed by authors. Temperature was recorded by means of a fibre optic thermometer (Optocon Fotemp-1H thermometer with a TS3/2 fiber optic, Dresden, Germany, accuracy ±0.1 °C). The measured sample was formed by a 1 mL of solution were three different NPs concentrations were dispersed in case of nude NPs, or PLGA loaded with three different NPs content are considered. For each sample 50 μL of the fluid were preserved to be observed at TEM. The glass 5 mL test tube was fixed to a holed plastic cylinder (external diameter 7 cm and internal diameter 4 cm) in order to maintain adiabatic condition in the first minutes of the experiment. The magnetic field was applied for 15 min and the temperature was recorded. After magnetic field application 50 μL of solution were preserved for TEM observation.
The thermal properties were studied analysing initial slope of the temperature curve in the 1 min time interval for which adiabatic condition are considered. The SAR in [Wg−1] was evaluated as [20]:
Figure 1 shows the TEM images of the Mn–Fe NPs dispersed in octane and the corresponding NPs included in PLGA. Table 1 reports the Mn and Fe content of the pristine nanoparticle evaluated by ICP and the magnetic core diameter and DLS in the water or octane dispersion. The average diameter of the magnetic core of the NPs is 11.8 nm; whereas the average diameter of PLGA is 60 nm. The hydrodynamic diameter, DLS, of pristine NPs dispersed in n-octane or water are reported in Table 1, as well as the DLS of PLGA.
TEM images of (a) NP dispersed in octane and (b) PLGA made with the same NPs.
NPs characteristics in size and Fe and Mn content
Table 2 reports the temperature increment in 1 min due to application of a time varying magnetic field (in case of PLGA temperature increment of the buffer only was subtracted; any thermal effect was revealed applying the magnetic field to deionized water or octane for 1 min). In the same time interval of magnetic field the PLGA buffer (not containing iron oxide NPs) treated in the same magnetic field condition did not show any temperature increment.
Temperature increment in different conditions and SAR
Figures 2 and 3 show TEM images acquired before and after the application of the magnetic field. Both images show the presence of magnetic particle aggregates that before the magnetic field application appear more regular and round shaped, thus demonstrating their loading into PLGA particles. Whereas after the magnetic field application the aggregates have less regular shape and size. This could be due to a partial degradation of the organic component of PLGA due to the solution heating. The computed SAR for NPs in water and PLGA in buffer, which have similar thermal properties of water, are both lower than those for NPs in octane.
TEM images of PLGA before the magnetic field application considering the three composition described in Table 1. (a) PLGA_2 and (b) PLGA_3.
TEM images of PLGA after the magnetic field application considering the three composition described in Table 1. (a) PLGA_2 and (b) PLGA_3.
We can conclude that PLGA embedding of magnetic nanoparticles improves significantly their efficiency in thermal properties, stability and water dispersion. PLGA nanoparticles have the advantage to be highly biocompatible and dissolve oleate coated magnetic particles. Moreover, the presence of a small % of Mn in the magnetic particle can improve the efficiency in reducing NMR relaxation time of water protons, making them good candidates to be considered as Magnetic Resonance Imaging contrast agents.
Footnotes
Acknowledgements
This research was funded by the grant ‘Progetto di Ateneo’ of Padova University (CPDA114144). The authors are grateful to Dr. Federico Caicci and Dr. Francesco Boldrin of the Electronic Microscopy Service of the University of Padova for TEM images.