A versatile large-scale and green process for synthesizing magnetic nanoparticles with tunable magnetic hyperthermia features

Abstract

This work proposes a large-scale synthesis methodology for engineered and functional magnetic nanoparticles (i.e. ferrites, sulfides) designed towards the principles of green and sustainable production combined with biomedical applicability. The experimental setup consists of a two-stage continuous-flow reactor in which single-crystalline nanoparticles are formed by the coprecipitation of metal salts in an aqueous environment. A series of optimized iron-based nanocrystals (Fe₃O₄, Fe₃S₄, CoFe₂O₄ and MnFe₂O₄) with diameters between 18 and 38 nm has been obtained. The samples were validated as potential magnetic hyperthermia agents by their heating efficiency as determined by specific loss power (SLP) in calorimetric experiments. In an effort to enhance colloidal stability and surface functionality, nanoparticles were coated by typical molecules of biomedical interest in a single step process. Finally, two-phase particle systems have been produced by a two-stage procedure to enhance the heating rate by the effective combination of different magnetic features. Results indicate relatively high SLP values for uncoated nanoparticles (420 W g⁻¹ for Fe₃O₄) and a reduction of 20–60% in the heat dissipation rate upon covering by functional groups. Eventually, such effect was more than counterbalanced by the magnetic coupling of different phases in binary systems, since SLP was multiplied up to ∼1700 W g⁻¹ for MnFe₂O₄/Fe₃O₄ suggesting a novel route to tune the efficiency of magnetic hyperthermia agents.

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1. Introduction

Magnetic nanoparticles (MNPs) appear advantageous for a variety of biomedical uses as a consequence of their ability to be manipulated remotely by magnetic fields. Particularly, this class of materials may be utilized as a promising diagnostic (MRI contrast agent), therapeutic (drug delivery, magnetofection, cell separation, local heating) or analytical (biosensing) tool for modern techniques.^1,2 Thus, the fine tuning of the magnetism in such nanoscale objects is one of the requirements for the development of adequate biomedical nanoparticle agents. The incorporation of MNPs in highly regulated and inhomogeneous biological surroundings has a significant impact on their stability and, eventually, defines their overall behavior and efficiency.³ Under this framework, specific combinations of distinct materials and sizes are promoted to overcome biocompatibility and bioaccumulation constraints whereas surface properties of the nanoparticles should be also tuned to optimize their uptake into cells and tissues, as well as diffusion and interaction in biological environments.^4,5

More particularly, for magnetic hyperthermia, one of the most promising and least invasive strategies for cancer treatment, MNPs are used as a mediator for the local heat release under a radio frequency AC magnetic field.⁶ Compared to other biomedical nanoparticle approaches, which are usually based either on their plasmonic response or on their chemical activity, the specific method is the only available with the potential to generate significant heat amounts in regions not directly accessible.⁷ The mechanism of magnetic hyperthermia relies firstly on the selective uptake of MNPs on cancer cells and secondly on the heating of tumors by increasing the local temperature (up to 41–45 °C) leading either to cell degradation or even to apoptotic death.⁸ Up to date, theoretical and experimental studies indicate that nanoparticles consisting of ferrimagnetic iron oxides (Fe₃O₄, γ-Fe₂O₃) remain as the most promising for magnetic hyperthermia since they may combine high heating efficiency with physiological stability, bearable biocompatibility and biodegradability.^9,10 Their performance is governed by their nanoscale magnetic features as dictated by the magnetocrystalline anisotropy, their size and shape and the corresponding tuning with the applied AC field.^11,12 Provided that the hysteresis loss mechanism is by far the most efficient way of generating heat, the optimal nanoparticle diameter should reside in the single-domain region, above the superparamagnetic size limit at temperatures between 290 and 320 K.^13–15 Therefore, a maximum hyperthermia response is expected for iron oxide nanoparticles with a mean diameter in the range of 20–40 nm.^16,17

The unceasing concern on the role of MNPs in biomedical studies and clinical practices, is expected to burst out a worldwide demand for well-defined and approved products in the near future.^18,19 Accordingly, the optimization and improvement of existing methods for the preparation of MNPs is a prerequisite for the prescribed physicochemical properties and availability. A very wide variety of synthesis routes resulting in particles with different sizes and surface chemistry has been reported during the last two decades based on traditional or modern techniques.^20,21 Despite the success of most techniques to provide high-quality nanoparticles at a controllable way, only few of them are in position to confront the biocompatibility, technical flexibility and economic viability constraints. For instance, the use of expensive, toxic or aqueous incompatible precursors and solvents, the application of high temperature or pressure conditions and the need for high-cost instrumentation in many wet chemical or physical methodologies are not favorable in terms of green and large-scale production.²² In addition, to our knowledge, none of the reported low-cost aqueous-based chemical approaches^23,24 has been adapted to a continuous-flow operation. Such an upgrade provides absolutely stable reaction conditions, flexibility in parameter modification and a cost reduction of at least 30% in industrial-scale production compared to typical batch synthesis. A series of well-established methods like gas-phase condensation and high-energy ball-milling are also referred as high throughput techniques for the industrial preparation of nanoparticles' powders.²⁵ However, the need for expensive facilities, the elevated energy consumption and the need for secondary consumables limit their competitiveness in the production of nanomaterials with high added value or complicated structures (TiO₂, Si, semiconductors, carbides, nanocomposites).^26,27 In addition, apart from the considerable cost of gas-phase processes (at least one order of magnitude higher than chemical precipitation), the intense heating conditions inhibit the preparation of iron oxide nanoparticles.²⁸

In an effort to bridge the gap between fundamental research and development of commercial nanomaterials for biomedical uses, we report on a novel advanced synthesis technique based on the principles of homogeneous continuous stirred tank reactors targeting to the production of functionalized iron-based nanoparticles.²⁹ The main advantage of this method is the steady state operation which ensures constant concentrations throughout the reaction period as well as complete conversion of reactants into solid products, implying a minimum impact to the environment. Particularly, the reactor is designed to produce various types of spinel-structured nanoparticles including iron oxides and sulfides (Fe₃O₄, CoFe₂O₄, MnFe₂O₄, Fe₃S₄) by coprecipitation of the corresponding metal salts. At the same time, such a reactor configuration provides the option of simultaneous surface functionalization by different organic molecules that assist steric stability and biocompatibility. This technique is further explored for the preparation of two-phase systems that renders high flexibility in tailored magnetic properties. Eventually, the prepared MNPs are evaluated as magnetic hyperthermia agents, in which their size and functionality determine the potential for biomedical applicability.

2. Materials and methods

2.1. Synthesis

Magnetic nanoparticles were synthesized in water using a two-stage continuous stirred-tank reactor (CSTR) by coprecipitation of proper precursor salts in the presence of functionalizing compounds. The setup (Fig. 1) consists of two serially installed CSTR vessels (1 L) with the reaction taking place at the first one while the second one serves as an ageing tank of the produced dispersion. Reagents are continuously pumped in the upper part of the first CSTR while the produced dispersion is transferred to the second one by the overflow, remains there for specific time under mild stirring and gets collected in the outflow for further washing. According to the followed synthetic parameters, samples under study were classified in three groups as follows (see Table 1): group A (primary particles) includes single-phase uncoated MNPs consisting of iron-based ferrites and sulfides, which were examined for the evaluation of structural and magnetic behavior. Group B (functionalized particles) refers to the covering of MNPs by different organic molecules. In this group, the effort is focused on the effect of surface covering in collective features such as magnetic response and heating efficiency. Finally, group C (two-phase particles) combines the results of the previous sample groups and the nanoparticles comprise two magnetically different phases for tuning the magnetic hyperthermia response.^30–32 The synthetic reactions were first developed in batch stirring vessels to optimize preparation parameters and then transferred to the one-for-all continuous flow setup. Due to the simplicity of such configuration, a proportional scale-up at any production rate is possible allowing the significant reduction of nanoparticles cost. Suggestively, for a unit working at a rate of 25 kg h⁻¹, a total production cost of 2–5 euros per kg of MNPs has been estimated, depending on the chemical reagents and the addition of functional molecules. This value includes facilities depreciation, operational (chemicals-70% in total, energy-10% in total), maintenance and labor costs. Another advantage of the process is the total conversion of reagents into the final product ensuring small amount of waste and a minimum toxicity to the outflowing liquid. Thus, this sustainable technique realizes a “green” production of MNPs. Importantly, the system guarantees completely stable conditions throughout the synthesis duration.³³ In particular, when the steady state is achieved, parameters that determine the final product properties such as the concentrations of reagents and solids, the pH, the temperature and the redox potential remain constant. Typically, 1 mol L⁻¹ aqueous solutions of reagents A and B (Table 1) were prepared in separate stirring tanks (5 L) and pumped into the first reactor with a constant rate of 0.5–3.0 L h⁻¹ (streams A and B in Fig. 1). The flowing rate of reagents solutions defined the retention time in the reactor and the reaction period. For example, the preparation of Fe₃O₄ nanoparticles was carried out using a reaction time of 30 min that was achieved by pumping each precipitating reagent (A: FeSO₄, B: Fe₂(SO₄)₃) at a rate 1 L h⁻¹. The temperature was set to 70 °C and the pH was kept constant at a value of 12 by adding dropwise a 10% w/v NaOH solution. The similar dimensions of stirring tanks determined both retention time and ageing stage to 30 min. Finally, the collected dispersion was washed several times by distilled water and dried at 40 °C. To functionalize Fe₃O₄ nanoparticles with citrate ions, the citrate reagent was added at a rate 0.2 L h⁻¹ (stream C) whereas flowrates for streams A and B were proportionally modified (0.9 L h⁻¹) to maintain the reaction time of 30 min.

Fig. 1 Sketch of the large-scale production setup for the synthesis of MNPs. The nanoparticles are prepared by coprecipitation of corresponding metal salts in aqueous solution in a continuous-flow reactor. Streams A and B correspond to the feed of reactor by metal and sulfide reagents whereas stream C indicates the addition of functional molecules or seed nanoparticles. A photo of the laboratory setup is presented in Fig. S1.†

Table 1 Synthetic parameters and properties of MNPs under study^a

Product	Reagent A	Reagent B	Reaction pH	Functional group	tr/ta (min)	Tr/Td (°C)	Diameter (nm)	Ms (A m^2 kg^−1)	IEP
a tr/ta reaction/ageing time, Tr/Td reaction/drying temperature, Ms saturation magnetization normalized to the magnetic material content, IEP isoelectric point, * condition for each reaction step.
Group A. Primary nanoparticles
Fe3O4	FeSO4	Fe2(SO4)3	12	—	30	70/40	21	68	6.2
Fe3S4	FeSO4	Na2S	3.5	—	10	20/100	35	14	7.0
CoFe2O4	CoSO4	Fe2(SO4)3	10.5	—	30	70/40	18	47	6.6
MnFe2O4	MnSO4	Fe2(SO4)3	10.5	—	30	70/40	34	49	6.4
Group B. Functionalized nanoparticles
Fe3O4	FeSO4	Fe2(SO4)3	12	Citrate	30	70/40	8	58	5.4
Fe3O4	FeSO4	Fe2(SO4)3	12	CTAB	30	70/40	20	48	8.0
Fe3O4	FeSO4	Fe2(SO4)3	12	Dextran	30	70/40	22	51	6.6
Fe3S4	FeSO4	Na2S	3.5	Citrate	10	20/100	32	25	6.0
Fe3S4	FeSO4	Na2S	3.5	CTAB	10	20/100	38	1.5	7.2
Fe3S4	FeSO4	Na2S	3.5	Dextran	10	20/100	31	26	6.7
CoFe2O4	CoSO4	Fe2(SO4)3	10.5	Citrate	30	70/40	9	6	5.7
MnFe2O4	MnSO4	Fe2(SO4)3	10.5	Citrate	30	70/40	33	42	5.4
Group C. Binary nanoparticles
CoFe2O4/Fe3O4	CoSO4/FeSO4	Fe2(SO4)3	10.5/12*	Citrate	30/30*	70/40	9/8	43	5.7
MnFe2O4/Fe3O4	MnSO4/FeSO4	Fe2(SO4)3	10.5/12	Citrate	30/30	70/40	34/8	65	5.5

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2.1.1. Primary nanoparticles. Single-phase surfactant-free magnetic ferrite (Fe₃O₄, CoFe₂O₄, MnFe₂O₄) or sulfide (greigite, Fe₃S₄) nanoparticles are prepared by coprecipitation of proper precursor salts working as metal ions or sulfide sources (FeSO₄, Fe₂(SO₄)₃, MnSO₄, CoSO₄, Na₂S). Ferrite nanoparticles were synthesized by the simultaneous precipitation of bivalent metal ions (M²⁺, M = Fe, Mn, Co) and Fe³⁺ under alkaline conditions according to the following reaction:

The retention time for all samples was 30 min and the reaction temperature was set to 70 °C. Note that for the sake of comparison and validation of critical reaction parameters in our experiments, similar reagent ratios and conditions were applied for all preparations. Nevertheless, further individual tuning of each product regarding morphological and magnetic properties is possible considering obvious limitations of aqueous synthesis, such as temperature and polarity.

For the preparation of iron sulfide nanoparticles, the precipitation of Fe²⁺ initially proceeds by the reaction with Na₂S in solution at acidic conditions and the solid state transformation of formed FeS to Fe₃S₄:

The second reactor was designed to provide an equal period of ageing for the MNPs under slow stirring. To avoid decomposition effects,³⁴ a rapid reaction time of 10 min was allowed for Fe₃S₄ nanoparticles preparation, followed by immediate drying of the solid product at 100 °C. The pH in the reacting mixture was stabilized by the addition of NaOH or HCl.

2.1.2. Functionalized nanoparticles. For surface functionalization, the proposed synthetic procedure was modified for the preparation of MNPs coated by citrate groups, cetyltrimethyl-ammonium bromide (CTAB), and dextran in order to examine representative systems already incorporated in biomedical applications.^35,36 Functionalizing agents were dissolved in water (10%) and continuously added (0.2 L h⁻¹) in the first reactor (stream C in Fig. 1). It turned out that the presence of the coating agents during particle growth, instead of their addition in the second ageing stage, is more efficient to form isolated functionalized particles.³⁷ In particular, citrate groups show high chemical affinity to iron-based nanoparticles and they are often used as an excellent substrate for the fluorescence labeling of MNPs or drug delivery.^38–41 Here, sodium citrate (C₆H₇NaO₇) was chosen as a citrate source, because it avoids the regulatory influence of citric acid in the pH of the reaction and overcomes issues related to its corrosive and oxidizing behavior on produced solids during ageing. CTAB is a cationic quaternary surfactant commonly used for the hydrophilization of nanoparticles and its antiseptic characteristics.⁴² Finally, dextran was tested as a biocompatible long-chain molecule that enables the isolation of MNPs inside the formed organic matrix facilitating antithrombotic activity.^43,44

2.1.3. Binary nanoparticles. The possibility to prepare binary systems of ferrite nanoparticles combining different magnetic properties is also demonstrated using the same experimental setup. This procedure includes a two-step process in which citrate-coated CoFe₂O₄ or MnFe₂O₄ nanoparticles were separately synthesized by the co-precipitation of the corresponding salts, then collected and added in dispersion (15 g L⁻¹) during the synthesis of Fe₃O₄ (stream C). The obtained systems were tested for their potential to enhance the heating response during magnetic hyperthermia by tuning nanoscale magnetic interactions as recently exhibited by the corresponding core–shell nanostructures.^30,31

2.2. Characterization

The structure of the nanoparticles was determined by X-ray diffraction (XRD) with a Rigaku Ultima + powder diffractometer using Cu-K_α radiation. Further, the particles' morphology and size distribution were studied by transmission electron microscopy (TEM). A Philips Tecnai F20 Supertwin microscope operating at 200 kV with a field emission gun was employed for high-resolution TEM (HRTEM). Fourier-transformed infrared (FT-IR) spectra of adsorbents were recorded in KBr media using a Perkin-Elmer Spectrum 100 spectrophotometer. The fine powders were dried and pelletized with KBr powder for the FT-IR study.

The Fe, Mn, Co content of ferrite nanoparticles was determined by graphite-furnace atomic absorption spectrophotometry using a Perkin-Elmer AAnalyst 800 instrument. The samples were dissolved in HCl prior to analysis. The net oxide content of dried samples, excluding naturally adsorbed humidity and attached coating agents, was estimated by the weight loss during thermogravimetric analysis (TGA) obtained by a water-cooled Perkin-Elmer STA 6000 instrument in the temperature range 50–900 °C, at a heating rate of 20 °C min⁻¹ and nitrogen gas flow. Isoelectric points of particles dispersions were determined by the zeta-potential curve recorded by a Rank Brothers Micro-electrophoresis Mk II device.

Magnetic measurements were performed on powder samples using a superconducting quantum interference device (SQUID) magnetometer (MPMS XL, Quantum Design) operating at a maximum field of 5 T and variable temperature. The heating efficiency of nanoparticle dispersions (4 mg mL⁻¹) was measured in a 4.5 kW commercial inductive apparatus at a frequency of 765 kHz and AC magnetic field of 30 mT. The specific loss power (SLP) was derived from the slope of the temperature versus time curve as previously described.^45,46 The temperature was monitored using a GaAs-based fiber optic probe immersed in a test tube containing 1 mL of dispersion.

3. Results and discussion

3.1. Primary nanoparticles

The operation of the reactor for 3 h under steady state conditions (product is collected after a period equal to the retention time starting from inflow initialization) resulted in the production of around 1 kg of MNPs on dry basis for iron oxides and ferrites while the same quantity was received in less than 1 h for the case of iron sulfide. In spite of the high production rate of such kilogram-scale process and the absence of any surfactant agent, the synthesized MNPs consist of distinct units retaining good size monodispersity, high crystallinity and the designated composition. In Fig. 2a and c, the TEM images of the uncoated Fe₃O₄ and Fe₃S₄ nanoparticles are indicative of the assembly of MNPs into agglomerates during drying whereas representative high-resolution observations verify the absence of coalescence during the growth, ageing or drying period (Fig. 2b and d). Magnetite nanoparticles possess a mean diameter of 20 nm while the growth stage in Fe₃S₄ ones reaches a final size of 35 nm. The difference may be attributed to the two-step process for the formation of greigite which involves the solid state reaction of FeS. Under identical parameters of synthesis, the coprecipitation of CoFe₂O₄ results in nanoparticles of 18 nm in diameter whereas MnFe₂O₄ are stabilized at around 34 nm (Table 1). Following the same preparation method, other studies also report smaller diameters for the CoFe₂O₄ particles growth.^47,48 In this case, the variation in the precipitation rate of Co²⁺, Mn²⁺, Fe²⁺ and Fe³⁺ and the higher activation energy required to form the mixed phase are considered the main reasons for the observed diameter diversity.⁴⁹

Fig. 2 Low-magnification and high-resolution TEM images of uncoated Fe₃O₄ (a and b) and Fe₃S₄ (c and d) nanoparticles synthesized by continuous flow aqueous precipitation.

Fig. 3 presents an overview of the structural and the magnetic characterization of uncoated MNPs. The XRD analysis in panel (a) verifies that coprecipitation provides well-established results for each synthetic procedure without significant secondary crystalline products. The sole appearance of Fe₃O₄, Fe₃S₄, CoFe₂O₄ and MnFe₂O₄ is identified by the corresponding diagrams whereas the degree of crystallinity, an indirect indication of particle size, is signified by the peak broadening. Average grain sizes calculated by Scherrer's equation were found to be 16.4, 22.3, 15.7 and 19.6 nm for Fe₃O₄, Fe₃S₄, CoFe₂O₄ and MnFe₂O₄ nanoparticles, respectively. Thus, the designed setup delivers crystalline ferrimagnetic Fe₃O₄, Fe₃S₄, CoFe₂O₄ and MnFe₂O₄ nanoparticles with around 10–15% wt of magnetically inert constituents (humidity, adsorbed ions) as determined by TG analysis (Fig. S2 in ESI†). Among the prepared MNPs, Fe₃O₄ particles present the highest saturation magnetization (68 A m² kg⁻¹) and coercive field (28 mT) implying to a higher potential for hysteresis losses during hyperthermia. Both MnFe₂O₄ and CoFe₂O₄ nanoparticles indicate a magnetization of around 50 A m² kg⁻¹, which is lower than the bulk values (80–90 A m² kg⁻¹)⁵⁰ due to size effects, a similar coercive field of around 150 mT but much different saturation fields. Finally, Fe₃S₄ nanoparticles possess a saturation magnetization of 14 A m² kg⁻¹ (bulk value calculated for ideal crystal 59 A m² kg⁻¹)⁵¹ but a coercive field very close to that of magnetite indicative of adequate hysteresis loss production for magnetic hyperthermia.

Fig. 3 XRD diagrams (a) and corresponding room temperature hysteresis loops (b) of uncoated Fe₃O₄, Fe₃S₄, CoFe₂O₄ and MnFe₂O₄ nanoparticles synthesized by continuous flow aqueous precipitation. The reference peak positions from the ICDD-JCPDS powder diffraction database are included for comparison.

3.2. Functionalized nanoparticles

The application of nanoparticles in biomedicine usually requires their integration with functional molecules which, depending on their chemistry and their specific role, can be attached on the particle's surface or form a matrix of encapsulated nanoparticles. In this study, the functional molecules compounds were added in the particles' growth stage, therefore, it is crucial to understand their incorporation mechanism, the potential interfering activity in the precipitation reaction and the impact in obtained MNPs properties. Magnetite nanoparticles are discussed in more detail as a representative case. Fig. 4 shows the TEM images of Fe₃O₄ nanoparticles synthesized by the coprecipitation of Fe²⁺ and Fe³⁺ salts in the presence of citrate, CTAB or dextran. Compared to the primary particles (Fig. 4a), the functionalized MNPs present some differences in the size and the degree of agglomeration. Citrate adsorption results in a significant decrease of particles' size to around 8 nm (Fig. 4b) suggesting an interference in the rate of nucleation and growth as described by LaMer's model,⁵² through the intermediate formation of an iron–citrate complex. Such mechanism determines the precipitation rate and contributes to a more homogeneous growth at lower dimensions.^53,54 Citrate-coated nanoparticles obtain a near-spherical morphology while aggregation effects are not fully confronted due to the small chain length of citrate molecule which is not sufficient to significantly increase particle spacing. On the other hand, in CTAB- and dextran-coated samples, the diameter is preserved (∼20 nm as shown in Fig. 4c and d) but the particles show some deviation from spherical shape, a higher packing density and aggregation degree. From such observations one may surmise the participation of these groups only in the shape formation and isolation of nanoparticles but not directly in the precipitation reaction. For CTAB, this can be interpreted by an assembly of CTAB molecules in elongated micelles under alkaline conditions, which promotes the deviation of growth from the spherical shape.⁵⁵ To this end, the high temperature, the high conductivity and presence of SO₄²⁻ ions also contribute. In addition, the network provided by dextran macromolecules and the subsequent kinetic limitations in the particle formation should be responsible for the close-pack assembling of MNPs hindering their effective manipulation within a biological environment.

Fig. 4 TEM micrographs of Fe₃O₄ nanoparticles prepared without functional groups (a) and in the presence of citrate ions (b), CTAB (c) and dextran (d). Insets show the corresponding diffraction patterns that can be recognized as spinel-type structures.

The structural characterization of the coated samples by XRD (Fig. 5a) verifies that the presence of functional molecules during synthesis does not cause any significant variation on the particles' structure. However, the saturation magnetization follows a small decreasing trend in the functionalized samples (Fig. 5c) which can be partially attributed to the different mass percentage of added groups as determined by TG analysis (18, 34, 27% losses for citrate, CTAB, dextran, respectively. See Fig. S3† for details). However, the reduction of the coercive field in citrate- and CTAB-coated Fe₃O₄ nanoparticles indicates that surface modification has some influence in the magnetic order of the surface layers probably related to particle size or the formation of γ-Fe₂O₃.⁵⁶ The effect is even more intense in citrate-coated CoFe₂O₄ nanoparticles were the decreased crystallinity and the superparamagnetic properties (see Fig. S4†) suggest that citrate complexation strongly influences the growth progress. The observed magnetic properties are in good agreement with an experimental report on CoFe₂O₄ nanoparticles prepared by the similar route and comparable dimensions.⁵⁷ For CTAB coating, it was found that this molecule inhibits the preparation of Fe₃S₄ nanoparticles because of competitive reactions with Na₂S under acidic conditions resulting in the formation of non-magnetic iron sulfides and other byproducts (see Fig. S5†).

Fig. 5 XRD (a) and FTIR (b) diagrams of Fe₃O₄ nanoparticles prepared without functional groups and in the presence of citrate ions, CTAB and dextran. Corresponding magnetic hysteresis loops at room temperature (c). Inset indicates magnified range around axes center.

The FTIR measurements of the functionalized particles are consistent with the presence of adsorbed functional groups on the particles' surface (Fig. 5b). MNPs prepared without additives exhibit a number of peaks related to the strong adsorption of charged ions from the solution to the Stern layer. Particularly, the bands at around 1170, 1450 and 1670 cm⁻¹ are assigned to coordinated SO₄²⁻, CO₃²⁻ and OH⁻ ions, respectively.³³ Sulfate ions originate from the used iron salts while carbonate and hydroxyl presence is favored by alkaline conditions of precipitation. Another vibration located at around 600 cm⁻¹ corresponds to the Fe–O bonds of iron oxide. An indication of the surface modification induced by functional groups is the attenuation of these peaks for the other samples. In addition, the existence of characteristic bands for each functional compound are also identified. The complexation of citrates with Fe atoms is verified by the asymmetric stretching of C–O from carboxylate group observed at 1550 cm⁻¹.⁵⁸ Another peak at 1310 cm⁻¹ could be identified as the symmetric stretching of C–O bond or the CH₂ wagging mode. In the same range, CTAB-coated particles provide scissoring vibrations at around 1500 cm⁻¹ related to the δ–(CH₂) or the characteristic asymmetric δ(N–CH₃) mode.⁵⁹ Finally the presence of dextran is signified by the characteristic C–O stretching band at around 1050 cm⁻¹.⁶⁰

As already discussed, coating by citrate ions is a very simple way to produce Fe₃O₄ nanoparticles with appreciable monodispersity and minimum aggregation effects following the coprecipitation in a continuous-flow reactor. Moreover, there are other important advantages related to the strong binding of citrates on particles surface. Nanoparticles are becoming really functional and they can be applied as flexible systems for ligand attachment, opening a wide range of possibilities based on the chemistry of citrate molecule. In addition, the non-reversible adsorption modifies the surface charge of MNPs as represented by their isoelectric point. For instance, citrate-coated Fe₃O₄ nanoparticles present an IEP of 5.4 which is lower than the corresponding value of the uncoated sample 6.2 (Table 1). Thus, it broadens the window for electrostatically stable nanoparticles under physiological conditions due to their negative charging. A similar trend is found for citrate-coated Fe₃S₄, MnFe₂O₄ and CoFe₂O₄ nanoparticles. For these systems, the presence of citrate ions is expected to modify the complexation of cations and affect the formation of mixed phases.⁴⁹ As described, such mechanism appears to have a dramatic impact to the size of CoFe₂O₄ nanoparticles (9 nm) but no influence is observed for Fe₃S₄ and MnFe₂O₄. For the latter cases, the diameters remain close to the values of non-coated particles (Fig. 6a–c). On the other hand, CTAB and dextran coating define a higher IEP, at 8.0 and 6.6, respectively, potentially exploitable for diverse applications. The important outcome of citrate and CTAB adsorption on the surface charge as evidenced by the change of IEP, should be considered as an additional aspect of the observed coercive field variations.

Fig. 6 TEM micrographs of citrate-coated Fe₃S₄ (a), MnFe₂O₄ (b), CoFe₂O₄ (c), binary MnFe₂O₄/Fe₃O₄ (d) and CoFe₂O₄/Fe₃O₄ (e) nanoparticles.

3.3. Binary systems

The sequential preparation of multi-phase systems in the continuous flow reactor was also attempted to examine the versatility of the proposed up-scalable, low-cost approach. Applications like magnetic hyperthermia benefit from such two-phase systems as discussed below. Here, the citrate-coated MnFe₂O₄ and CoFe₂O₄ nanoparticles were used as seeds in the Fe₃O₄ precipitation. The growth of Fe₃O₄ in the presence of preformed nanoparticle seeds with similar structures implies the possible development of epitaxial interfaces in the nanoscale able to lead in strongly modified magnetic properties.³¹ The preparation of Fe₃O₄ nanoparticles on either MnFe₂O₄ or CoFe₂O₄ seeds resulted in two-phase binary systems with noticeable homogeneity in their intermixing (Fig. 6d and e). In particular, the MnFe₂O₄/Fe₃O₄ binary system consists of larger MnFe₂O₄ nanoparticles surrounded by smaller Fe₃O₄ particles (Fig. 6d). This uniform intermixing between the two types of nanoparticles is related to the particle surface modification by citrate coating that inhibits agglomeration of nanoparticles caused by short-range electrostatic interactions.

Since the mass ratio of MnFe₂O₄ or CoFe₂O₄ with respect to Fe₃O₄ was estimated almost equal according to the results obtained from the chemical analysis of each binary sample, a comparable contribution of coexisting phases is also expected in the collective physical properties of the two-phase system. Particularly, the Mn and Co content of the corresponding ferrite samples was determined to be 9.7 and 9.2% wt while Fe was 50.4 and 51.2% wt, respectively. Such values and ratios are very close to the stoichiometric ones for a 1 : 1 mixture of the two materials.

3.4. Magnetic hyperthermia response

The success of the proposed methodology for producing MNPs for biomedical applications was evaluated by measuring their heating efficiency in calorimetric hyperthermia experiments. In particular, the role of phase combination and functional group selection was investigated. Fig. 7 summarizes the SLP values of primary single-phase MNPs. In order to make a reliable and accurate comparison of the heating rate between samples, the SLP was normalized to the metal content of each system (Fe, Mn, Co) as determined from chemical analysis. The SLP is proportional to the magnitude of the applied field reaching relatively high values of 300 W g⁻¹ for Fe₃O₄, Fe₃S₄ and MnFe₂O₄ nanoparticles at 30 mT and 765 kHz. On the contrary, CoFe₂O₄ nanoparticles hardly generate a heating power of 40 W g⁻¹. This finding may be correlated to the higher hysteresis losses per cycle at the working field of 30 mT in Fe₃O₄, Fe₃S₄ and MnFe₂O₄ nanoparticles compared to the CoFe₂O₄ ones as indicated by the hysteresis losses in corresponding minor loops (see Fig. S6†).

Fig. 7 Specific loss power of Fe₃O₄, Fe₃S₄, CoFe₂O₄ and MnFe₂O₄ uncoated nanoparticles at 765 kHz and field intensities (20, 25 and 30 mT).

Although the influence of particles' functionalization on the magnetic and structural behavior was found to be moderate, its impact in the heat generation and dissipation under an AC magnetic field is apparent.⁶¹ As mentioned above, the presence of citrate ions, CTAB or dextran molecules during synthesis may affect the growth mechanism and the physical properties of the MNPs in various ways (size, shape, degree of agglomeration, complexation, surface reconstruction and charge inversion). Here, it appears that surface modification by functional groups is responsible for a decrease in magnetocrystalline anisotropy of nanoparticles which is reflected to the lower hysteresis losses signified by the corresponding minor loops (see Fig. S7†) as well as to the decrease in calorimetrically observed heating rate. Suggestively, for Fe₃O₄ nanoparticles, the SLP value drops more than 50% for citrates (190 W g⁻¹) and CTAB (95 W g⁻¹) while dextran was found to have the minimum effect in hyperthermia efficiency keeping an SLP of 360 W g⁻¹ (Fig. 8a) probably because the negative influence is balanced by increased agglomeration effects.

Fig. 8 Specific loss power values of uncoated and functionalized Fe₃O₄ nanoparticles (a) and comparison of binary systems to their single phase counterparts (b) at 765 kHz and 30 mT.

The incorporation of binary two-phase systems instead of single phase nanoparticles has been proposed as a novel approach to counterbalance the attenuation of power losses due to functionalization or size-effects.^32,62 Here, we comparatively evaluate the performance in hyperthermia for the binary systems CoFe₂O₄/Fe₃O₄ and MnFe₂O₄/Fe₃O₄ with respect to their citrate-coated single-phase counterparts (Fig. 8b). It is remarkable that the heating efficiency of citrate-coated CoFe₂O₄/Fe₃O₄ is almost two orders of magnitude higher than the corresponding citrate-coated CoFe₂O₄ (280 and 25 W g⁻¹, respectively) while in corresponding MnFe₂O₄/Fe₃O₄ SLP is elevated up to 1700 W g⁻¹ which is one of the highest values reported for aqueous-dispersed MNPs.

These findings can be interpreted by analyzing the minor DC hysteresis loops of the specimen at identical fields as compared to AC heating experiments (30 mT). Fig. 9 summarizes the minor loops of the MNPs functionalized by citrate ions. Among the single-phase nanoparticles prepared in the continuous-flow reactor, Fe₃O₄ nanoparticles present the highest demarcate area which is consistent with their AC heating response in calorimetric experiments. On the opposite, the hysteresis losses in superparamagnetic CoFe₂O₄ nanoparticles are practically diminishing (Fig. S8†). However, the two-phase combination of Co or Mn ferrite nanoparticles with Fe₃O₄ results in the multiplication of hysteresis losses as directly illustrated by the proportional SLP values. Such enhanced response, is the beneficiary outcome, steaming from the different contributions of the two coexisting phases, which interface can cause exchange-spring effects at the nanoscale.³²

Fig. 9 Minor hysteresis loops obtained at 30 mT for citrate-coated Fe₃O₄, MnFe₂O₄, Fe₃S₄, binary Fe₃O₄/MnFe₂O₄ and Fe₃O₄/CoFe₂O₄ nanoparticles.

4. Conclusions

Chemical precipitation of metal salts is reported as a facile way to produce MNPs of appreciable quality allowing up-scaling to kg h⁻¹ production rates at competitive costs. Nevertheless, commercial compounds based on such nanomaterials are still considered as high-value products especially when addressing biomedical aspects. The main achievement of this study was to demonstrate a methodology for the large-scale synthesis of various iron-based nanoparticles following a green and low-cost method derived from a continuous stirring tank setup typically operating in industrial facilities. The process includes numerous advantages such as total conversion of reactants, zero environmental impact, constant reaction conditions and water compatibility, but most importantly, it provides an incredibly low-cost per kg of nanomaterials that drops down to 2 euros for the case of Fe₃O₄ nanoparticles. The proposed synthetic methodology was successfully developed for the preparation of single-phase nanoparticles covered with functional molecules as a way to improve their biocompatibility and two-phase particles to maximize heating performance. Produced samples were validated as magnetic hyperthermia agents indicating adequate heating efficiency depending on ferrite selection while their functionalization seems to maintain SLP values at reasonable levels. Eventually, the preparation of two-phase systems of magnetically interacting phases with elevated heating performance (1.7 kW g⁻¹) suggests that a very wide field for tuning and improvement is still open.

Acknowledgements

Financial support by IKYDA2013 bilateral Greek-German collaboration scheme is acknowledged. S.L.V. acknowledges funding from the European Community's Seventh Framework Programme (FP7-NMP) under grant agreement no. 280670 (REFREEPERMAG).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra09362k

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