Journal of Colloid and Interface Science 650 (2023) 560–572 Available online 4 July 2023 0021-9797/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by- nc/4.0/). Large-scale production of superparamagnetic iron oxide nanoparticles by flame spray pyrolysis: In vitro biological evaluation for biomedical applications Manuel Estévez a,1, Mónica Cicuéndez b,1, Julián Crespo c, Juana Serrano-López d, Montserrat Colilla a,e, Claudio Fernández-Acevedo f, Tamara Oroz-Mateo f, Amaia Rada-Leza f, Blanca González a,e,*, Isabel Izquierdo-Barba a,e,*, María Vallet-Regí a,e,* a Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Instituto de Investigación Sanitaria, Hospital 12 de Octubre i+12, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain b Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Instituto de Investigación Sanitaria del Hospital Clínico San Carlos (IdISSC), 28040 Madrid, Spain c Tecnología Navarra de Nanoproductos S.L. (TECNAN), área industrial PERGUITA, C/A, N◦ 1, 31210 Los Arcos (Navarra), Spain d Experimental Hematology Lab, IIS- Fundación Jiménez Díaz, UAM, Madrid 28040, Spain e Centro de Investigación Biomédica en Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain f Centro Tecnológico ĹUrederra, área industrial PERGUITA, C/A, N◦ 1, 31210 Los Arcos (Navarra), Spain G R A P H I C A L A B S T R A C T A R T I C L E I N F O Keywords: Superparamagnetic iron oxide nanoparticles Flame spray pyrolysis Large-scale production Biocompatibility Hemocompatibility A B S T R A C T Despite the large number of synthesis methodologies described for superparamagnetic iron oxide nanoparticles (SPIONs), the search for their large-scale production for their widespread use in biomedical applications remains a mayor challenge. Flame Spray Pyrolysis (FSP) could be the solution to solve this limitation, since it allows the fabrication of metal oxide nanoparticles with high production yield and low manufacture costs. However, to our knowledge, to date such fabrication method has not been upgraded for biomedical purposes. Herein, SPIONs * Corresponding authors at: Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain. E-mail addresses: manestev@ucm.es (M. Estévez), mcicuendez@ucm.es (M. Cicuéndez), julian.crespo@tecnan-nanomat.es (J. Crespo), juana.serrano@ quironsalud.es (J. Serrano-López), mcolilla@ucm.es (M. Colilla), claudio.fernandez@lurederra.es (C. Fernández-Acevedo), tamara.oroz@lurederra.es (T. Oroz- Mateo), amaia.rada@lurederra.es (A. Rada-Leza), blancaortiz@ucm.es (B. González), ibarba@ucm.es (I. Izquierdo-Barba), vallet@ucm.es (M. Vallet-Regí). 1 Both authors contributed equally to the manuscript. Contents lists available at ScienceDirect Journal of Colloid And Interface Science journal homepage: www.elsevier.com/locate/jcis https://doi.org/10.1016/j.jcis.2023.07.009 Received 29 March 2023; Received in revised form 21 June 2023; Accepted 3 July 2023 mailto:manestev@ucm.es mailto:mcicuendez@ucm.es mailto:julian.crespo@tecnan-nanomat.es mailto:juana.serrano@quironsalud.es mailto:juana.serrano@quironsalud.es mailto:mcolilla@ucm.es mailto:claudio.fernandez@lurederra.es mailto:tamara.oroz@lurederra.es mailto:amaia.rada@lurederra.es mailto:blancaortiz@ucm.es mailto:ibarba@ucm.es mailto:vallet@ucm.es www.sciencedirect.com/science/journal/00219797 https://www.elsevier.com/locate/jcis https://doi.org/10.1016/j.jcis.2023.07.009 https://doi.org/10.1016/j.jcis.2023.07.009 https://doi.org/10.1016/j.jcis.2023.07.009 http://crossmark.crossref.org/dialog/?doi=10.1016/j.jcis.2023.07.009&domain=pdf http://creativecommons.org/licenses/by-nc/4.0/ http://creativecommons.org/licenses/by-nc/4.0/ Journal of Colloid And Interface Science 650 (2023) 560–572 561 Human mesenchymal stem cells Biomedical applications have been fabricated by FSP and their surface has been treated to be subsequently coated with dimercapto- succinic acid (DMSA) to enhance their colloidal stability in aqueous media. The final material presents high quality in terms of nanoparticle size, homogeneous size distribution, long-term colloidal stability and magnetic properties. A thorough in vitro validation has been performed with peripheral blood cells and mesenchymal stem cells (hBM-MSCs). Specifically, hemocompatibility studies show that these functionalized FSP-SPIONs-DMSA nanoparticles do not cause platelet aggregation or impair basal monocyte function. Moreover, in vitro biocom- patibility assays show a dose-dependent cellular uptake while maintaining high cell viability values and cell cycle progression without causing cellular oxidative stress. Taken together, the results suggest that the FSP-SPIONs- DMSA optimized in this work could be a worthy alternative with the benefit of a large-scale production aimed at industrialization for biomedical applications. 1. Introduction Superparamagnetic iron oxide nanoparticles (SPIONs) have been extensively studied and used for medical and biotechnological applica- tions, such as magnetic resonance imaging, tissue repair, cell separation and detection, magnetic hyperthermia, and drug delivery due to their small size, excellent magnetic properties, biocompatibility, and low toxicity [1–9]. Such applications mainly require a precise size and chemical control, good stability in physiological environments, and defined surface properties according to their functionality [7,10,11]. To date, numerous efforts are focusing on the development and optimiza- tion of different lines of research to produce SPIONs employing wet chemical routes such as co-precipitation, thermal decomposition in non- aqueous liquids and microemulsions, sol–gel, among other techniques [12–15], but also particle formation in the gas phase such as thermal decomposition in hot-wall reactors. These technologies allow to better control the particle size, shape, and polydispersity; to obtain populations of small nanoparticles with a narrow size distribution and desired crystalline properties to enhance their magnetic behavior. However, despite the great projection of these technologies in clinical applica- tions, one of the main limitations is the need for large production runs for their industrialization [16,17]. Among the different existing methods to synthesize SPIONs, a bottom-up nanomanufacturing technique with proven scalability and reproducibility [18,19] such as Flame Spray Py- rolysis (FSP) could be the solution to overcome this limitation. FSP technology permits the continuous manufacture of metal oxide nano- particles with high production yields and low production costs at high production rates in a simple and reproducible one-step process on a large scale (up to 10 Kg/h). Furthermore, current studies assess the overall environmental impact of novel optimized FSP technologies to evaluate its potential for pharmaceutical and biotechnological applications. In addition, the versatility of FSP enables the production of metal oxide nanoparticles, such as mixed and doped oxides, complex nanomaterials such as core–shell structures [18–23]. In more detail, FSP technology is based on the preparation of a liquid precursor mixture constituted by organometallic precursors and organic solvents, which is atomized in a nozzle just prior to passing through a flame. In this process all organic matter is burned, and particle nucleation and growth occurs. Because this period is very short, the particles remain in the nanometric range. In addition, since the process occurs at high temperature (2000 ◦C), ob- tained nanoparticles present a great purity, low agglomeration rate, high thermal resistance, and high specific surface area. Furthermore, the variation of key process parameters (composition of precursor mixture, concentrations, oxidative conditions of the flame, feeding flow rate, etc.) allow to exert a high control of particle size, shape and crystalline phase [24] of the nanoparticles, key aspects for their clinical use [25]. Regarding the production of SPIONs by FSP, such technology has been also employed to produce superparamagnetic nanomaterials [26–31]. It is worth mentioning that the oxidative conditions of the flame result in obtaining metals in their highest oxidation state and maghemite mate- rials are obtained. Nevertheless, Pratsinis et al. [32] have demonstrated the versatility of the technology by being able to modify key process parameters to control iron oxidation and to produce different batches of maghemite, magnetite and even wustite nanoparticles. In this sense, although FSP technology presents great advantages for the production on an industrial scale of this type of superparamagnetic nanomaterials, to date only small production processes have been carried out at labo- ratory research scale and only Teleki et al. in 2022 [33] have been involved in the production of undoped and doped SPIONs using FSP technology [34,35] to induce amorphization of a poorly aqueous soluble crystalline drug (celecoxib) in tablets for oral administration. In this work, we demonstrate the suitability of large-scale production by FSP technique of SPIONs appropriate for biomedical applications in terms of narrow size distribution, superparamagnetic properties, colloidal stability, hemocompatibility and cytotoxicity. Thus, in a first stage, the key parameters of FSP to produce small nanoparticles of about 15 nm with high homogeneous size distribution were optimized since this is one of the main challenges for the implementation of the FSP nanoparticle production method in the pharmaceutical industry. Then, the obtained FSP-SPIONs were further processed via an acid treatment to clean and activate their surface, followed by a coating with dimer- captosuccinic acid (DMSA) to obtain stable aqueous colloidal solutions. The surface optimization demonstrated in this work specifically with SPIONs becomes the starting point for other more complex compositions of nanoparticles prepared also by FSP technology. Hence, properties such as morphology, size, composition, magnetism, colloidal stability and in vitro validation with peripheral blood cells and human bone marrow-mesenchymal stem cells (hBM-MSCs) were evaluated, endorsing the future use of these nanoparticles for biomedical applications. 2. Materials and methods 2.1. Reagents Iron (III) acetylacetonate 97%, iron (III) naphtenate < 99.9%, eth- ylhexanoic acid 99%, xylene 78%, dimercaptosuccinic acid (DMSA) 98% and 12 kDa cellulose dialysis bags were purchased from Sigma- Aldrich (Madrid, Spain). Tert-butyl acetate 99% was purchased from Cymit Química S.L. (Barcelona, Spain). Fe(NO3)3⋅9H2O 98% and abso- lute ethanol were purchased from PanReac (Barcelona, Spain). All other chemicals (KOH, NaOH, HNO3 65%, HCl 37%, etc.) were of the highest quality commercially available and were used as received. Milli-Q water (resistivity 18.2 MΩ⋅cm at 25 ◦C) was used in all experiments. Phosphate buffered saline (PBS, pH 7.4) and trypsin/EDTA 0.25% were purchased from Lonza and Gibco, Thermo Fisher Scientific, Wil- mington, DE, USA. NH4Cl to make erythrocytes lysis buffer was obtained from Sigma-Aldrich. Mesenchymal stem cell basal medium (MSCBM), mesenchymal cell growth supplement (MCGS), L-glutamine 200 mM and gentamicin sulphate and amphotericin-B (GA-1000) were purchased from Lonza, Bioscience (Switzerland). Propidium iodide 1.0 mg/mL solution in water was purchased from life technologies molecular probes (Thermo Fisher Scientific). 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) was purchased from Invitrogen, Thermo Fisher Scientific (Waltham, MA, USA). RNAse was purchased by molecular biology (Thermo Scientific GeneJET). M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 562 2.2. Characterization The analytical methods used to characterize the synthesized mate- rials were as follows: nitrogen sorption, inductively coupled plasma atomic emission spectroscopy (ICP-AES), powder X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transformed infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA) and chemical microanalysis, vibrating sample magnetometry, electropho- retic mobility measurements to calculate the values of zeta-potential (ζ-potential), dynamic light scattering (DLS). The equipment and con- ditions used are described in the Supporting Information. 2.3. Large-scale production of superparamagnetic iron oxide nanoparticles Superparamagnetic iron oxide nanoparticles have been synthesized by TECNAN and Lurederra Technological Centre employing the one-step Liquid Flame Spray Pyrolysis (FSP) technology. The liquid precursor mixture consists of a two-precursor system, based on two solutions that were prepared by dissolving, on the one hand, iron (III) acetylacetonate in xylene and, on the other hand, iron (III) naphthenate in 1:2 mixture of xylene and 2-ethylhexanoic acid, to obtain a final iron concentration of 0.6 and 0.5 M, respectively. Once prepared, both solutions were mixed under vigorous stirring. If needed, thinners such as tert-butyl-acetate were used as additives for an optimum droplet atomization in the FSP nozzle. The precursor mixture was employed to feed the equipment with a feeding flow rate 15–25 mL/min. Oxygen was selected as dispersion gas and set at 30–40 mL/min. The ratio of methane to oxygen gas flow rate was 3–6 L/h to form a self-maintaining main core flame. Finally, the produced nanoparticles were collected in a specific sleeve filter system. SPIONs with a narrow size distribution were produced using the same FSP technique under the following conditions: the solutions for the precursor mixture were 0.35 M iron (III) acetylacetonate in xylene and 0.23 M iron (III) naphthenate in xylene/2-ethylhexanoic acid (1:2); precursor feeding flow rate was established in the range of 10–30 mL/ min; O2 flow was set at 20–30 mL/min; the ratio of methane to oxygen gas flow rate was 4–8 L/h. The resulting sample was denoted as FSP- SPIONs. Table S1 summarizes the FSP process parameters used in both synthesis (see Supporting Information). 2.4. Surface activation and DMSA coating of as-synthetized SPIONs 2.4.1. Surface activation 75 mL of 2 M HNO3 were added to 1 g of as-synthetized SPIONs in a 250 mL one-neck round bottom flask equipped with mechanical stirring (stirrer shaft) and the mixture was vigorously stirred for 15 min. The nitric acid solution was then removed by magnetic decantation. Subse- quently, 20 mL of 1 M Fe(NO3)3 and 33 mL of Milli-Q water were added to the nanoparticles and the mixture was refluxed under stirring for 30 min. The suspension was then cooled down to room temperature. Then, the supernatant was replaced with 75 mL of 2 M HNO3 by magnetic decantation and stirred for 15 min. The resulting product was washed three times with water and dried in the oven at 70 ◦C. The resulting nanoparticles were denoted as FSP-SPIONs*. 2.4.2. DMSA coating A solution of 14.7 mg of DMSA 98% in 10 mL of distilled water was added to a suspension of the surface activated SPIONs in 20 mL of distilled water at pH 3, to achieve a final iron concentration of 3 mg/mL. The mixture was gently shaken and the pH was adjusted to 11 with KOH. Then, the sample was sonicated for 20 min and dialyzed using a 12 kDa dialysis membrane against 5 L of distilled water for 48 h changing the water twice a day. Finally, the pH was adjusted to 7 and filtered through 0.22 µm, affording FSP-SPIONs-DMSA. 2.5. In vitro hemocompatibility tests Peripheral blood (PB) samples were collected from 3 healthy vol- unteers (sodium citrate 3.8% tubes, 0.129 M, BD, Biosciences, Franklin Lakes, NJ, USA) and processed within 3 h post extraction. SPIONs were sonicated for 20 min and diluted to the final concentration in PBS pH 7.4. 2.5.1. Monocyte activation assay PB samples were incubated with SPIONs at 50 and 100 µg/mL at 37 ◦C for 2 h. NH4Cl was then added into each tube and incubated for 20 min at 37 ◦C, followed by 5 min centrifugation at 1500 rpm and 2x PBS wash. Cells in the pellet were resuspended in 100 µL of PBS and stained with 5 µL of fluorescein isothiocyanate (FITC)-conjugated anti-CD16 (clon 3G8), phycoerythrin dye-(PE) conjugated anti-CD14 (clon M5E2) and allophycocyanin (APC)-conjugated anti-CD11B (clon ICRF44) (BD, Biosciences) for 15 min. Cells were analyzed by Flow Cytometry in FACSCanto II cytometer equipped with FACSDIVA™ software (BD, Biosciences) for further multiparameter analysis of the data. 2.5.2. Platelet aggregation assay Platelet-rich plasma (PRP) was obtained by centrifugation at 200 g for 10 min at room temperature. Platelet-poor plasma (PPP) was ob- tained by centrifugation for 15 min at 2500 g at room temperature. Platelets aggregation was measured using a TA-4 V/8V Aggregometer (Stago, Saint-Ouen-l’Aumône, France) linked to Thrombosoft 1.6. soft- ware. First, a volume of 270 µL of PPP was used to calibrate the aggregometer channels. Second, 270 µL of PRP were incubated for 3 min in aggregometer channels and then measured for calibration, following industry recommendations with minor modifications. After this process, 30 µL of SPIONs at 50 and 100 µg/mL were added in the different PRP- containing cuvettes and incubated for 20 min at 37 ◦C. Positive controls were performed by adding of 30 µL of 5 µM epinephrine (EPI), (25 µM, Stago, Asnières sur Seine, France) and 50 µM thrombin-like peptides (TRAP), (50 µM, Stago) as soluble agonists for platelet activation. The effect of the different conditions was recorded in real-time to study the ability of NPs to stimulate platelet aggregation. 2.6. In vitro cytocompatibility tests Human bone marrow derived mesenchymal stem cells (hBM-MSCs) (Lonza, donor 38157, passage 5) were cultured in MSCBM supplemented with MCGS, GA-1000 and L-glutamine. The cells were washed with PBS and then trypsinized with 3 mL of trypsin/EDTA 0.25%. Cells were then centrifuged at 1000 rpm for 5 min. The resulting pellet was suspended in 1 mL of culture medium, measured for cell number, and seeded into 24- well plates (1 × 105 cells/well) with a final volume of 1 mL of medium per well. The cells were then incubated at 37 ◦C in 5% CO2 for 24 h. Afterwards, SPIONs (25 and 50 µg/mL FSP-SPIONs-DMSA nano- particles) were added and kept in contact with the cells for 24 h. After this, the cells were washed with 1 × PBS, harvested with trypsin/EDTA 0.25% and processed for flow cytometry analysis. As a control, cells were cultured at the same cell density but in the absence of SPIONs. Moreover, the authors note that the doses studied (25 and 50 µg/mL) are within the range usually tested for nanoparticles with diverse biological applications [36]. 2.6.1. Evaluation of FSP-SPIONs-DMSA nanoparticles uptake by hBM- MSCs through flow cytometry and confocal microscopy Flow cytometry. The flow cytometer is an excellent instrument for investigations of cellular and molecular biochemistry. Most beneficial is the high-speed analysis of each cell, e.g., tens of thousands of cells can be analyzed in a second. In flow cytometry, light scattering at 90◦ disper- sion angle is called side-scatter light (SSC) and reflects the intracellular complexity. Specifically, the SSC intensity is proportional to the complexity of intracellular structures such as the cellular cytoplasm, M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 563 mitochondria, and pinocytic vesicles, among other [37]. For example, when apoptotic cell death is induced, which is one pattern of cell death, the intensity of SSC increases significantly because the cells shrink and intracellular density rises [38]. Thus, in our study, the incorporation of FSP-SPIONs-DMSA nanoparticles by hBM-MSCs after 24 h of treatment was evaluated by flow cytometry analyzing the SSC intensity because it is a simple method to evaluate the uptake of nanomaterials by mammalian cells when nanomaterials are not labelled with any fluo- rochrome [39,40]. Flow cytometric quantification of SSC also under- represents the particle amount because aggregates of detached particle were gated out and apoptotic and necrotic cells, which may contain large amounts of particles, were excluded from SSC determination to avoid including the increased SSC of dying cells. Data acquisition and analysis conditions were established with negative and positive controls using the CellQuest Program of Becton Dickinson, and these conditions were maintained in all the experiments. Each experiment was performed in triplicate, and single representative experiments were displayed. For statistical significance, at least 10,000 cells were analyzed by flow cytometry in each sample. A FACScalibur Becton Dickinson flow cy- tometer was used (Becton Dickinson, San Jose, CA, USA). Confocal microscopy. Cells were fixed with 3.7% para- formaldehyde (Sigma-Aldrich Corporation, St. Louis, MO, USA) in PBS for 10 min, washed with PBS and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 5 min. The samples were then washed with PBS and preincubated with PBS containing 1% BSA (Sigma-Aldrich Corporation, St. Louis, MO, USA) for 30 min to prevent non-specific binding. Samples were incubated for 20 min with Phalloidin-Atto 550 (Sigma-Aldrich Corporation, St. Louis, MO, USA) 1:40 (100 μL) to stain the F-actin filaments. Samples were then washed with PBS and the cell nuclei were stained with 4′,6-diamidine-2′-phe- nylindole dihydrochloride (DAPI) (Sigma-Aldrich Corporation, St. Louis, MO, USA) 1:1000 for 5 min. The samples were examined in an OLYMPUS FV1200 Confocal Laser Scanning Microscope (OLYMPUS, Tokyo, Japan). The fluorescence Phalloidin-Atto was excited at 550 nm and the emitted fluorescence was measured at 565–675 nm. DAPI fluorescence was excited at 405 nm and measured at 420–480 nm. 2.6.2. Cell viability Cell viability was measured by adding 0.005% (wt/vol) propidium iodide (PI) in PBS (Sigma-Aldrich, St. Louis, MO, USA) into the samples to stain the dead cells. The PI exclusion indicates the plasma membrane integrity. PI fluorescence was detected in a FACScalibur Becton Dick- inson flow cytometer with a 530/30 filter, exciting the sample at 488 nm. 2.6.3. Intracellular reactive oxygen species (ROS) content After exposure to the FSP-SPIONs-DMSA nanoparticles for 24 h, hBM-MSCs were incubated with 10 μM of 2′,7′-dichlorodihydro fluo- rescein diacetate (DCF-H2-DA, Serva, Heidelberg, Germany) during 45 min at 37 ◦C. The non-fluorescent DCF-H2-DA is converted into 2′,7′- dichlorofluorescein (DCF) after hydrolysis by cellular esterases and oxidation by ROS. When DCF is excited at 488 nm emission wave- lengths, green fluorescence is emitted that can be detected at 525 nm. DCF fluorescence was measured in a FACScalibur Becton Dickinson flow cytometer with a 530/30 filter, exciting the sample at 488 nm. 2.6.4. Cell-cycle analysis Cells in 0.5 mL of PBS were mixed with 4.5 mL of ethanol 70% and maintained at 4 ◦C overnight. The cell suspensions were then centri- fuged for 10 min at 1500 rpm and resuspended in 0.5 mL of RNAse solution containing 0.1% Triton X-100, 20 μg/mL of IP and 0.2 mg/mL of RNAse (Sigma-Aldrich, St. Louis, MO, USA). After 30 min of incuba- tion at 37 ◦C, PI fluorescence was detected in a FACScan Becton Dick- inson flow cytometer with a 585/42 filter, exciting the sample at 488 nm. The CellQuest Program from Becton Dickinson was used to calculate the percentage of cells in each cycle phase: G0/G1 (growth), S (DNA synthesis) and G2/M (growth and mitosis). 2.6.5. Gene expression PCR hBM-MSCs were seeded in 6-well plates and incubated for 4 and 7 days (n = 3) in osteogenic differentiation medium. Total RNA was iso- lated from the cells using a standard procedure (Trizol, Invitrogen, Groningen, The Netherlands), and cDNA synthesis was performed using a high-capacity RNA-to-cDNA kit (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). Gene expression was analyzed by real- time PCR using a QuantStudio 5 Real-Time PCR System. Unlabelled human-specific primers for Runx2 and ALP, and TaqManTM MGB Probe (Applied Biosystems) were used to perform qRT-PCR assay. The mRNA copy numbers were calculated for each sample by using the cycle threshold (Ct) value. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rRNA (a housekeeping gene) was amplified in parallel with tested genes. The relative gene expression was represented as 2− ΔΔCt, where ΔΔCt = ΔCttarget gene − ΔCtGAPDH. 2.7. Statistical analysis Data were expressed as means ± standard deviations of a represen- tative of three experiments carried out in triplicate. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software, version 22. Statistical comparisons were made by analysis of variance (ANOVA). Scheffé test was used for post-hoc eval- uations of differences among groups. In all the statistical evaluations, p < 0.05 was considered as statistically significant. 3. Results and discussion 3.1. Large-scale production of superparamagnetic iron oxide nanoparticles Superparamagnetic iron oxide nanoparticles (SPIONs) were synthe- sized by TECNAN and Lurederra Technological Centre employing the one-step Liquid Flame Spray Pyrolysis (FSP) technology (Fig. 1A). To do so, an FSP pilot plant, developed by themselves, with a production ca- pacity of 100 g/h was used and optimized to produce FSP-SPIONs (Fig. 1B). More in detail, a liquid precursor mixture constituted by different iron coordination complexes, featuring organic ligands, and organic solvents are introduced into a high temperature flame through an atomizer. This precursor mixture must be homogeneous and stable, being necessary to pay special attention to the miscibility of the solvents and solubility of the iron precursors, avoiding possible precipitations or secondary reactions that may affect the SPION by varying the iron concentration, viscosity or heat capacity, that vary the characteristics of the final product. During the production process, in addition to the pa- rameters related to the precursor mixture, the precursor feed rate, the flow rate of the dispersion gas and the spraying conditions, including pressure, etc. also have a direct consequence on the final properties of the produced nanoparticles. So the main difficulty is to find the balance between an adequate production speed and obtaining a population with a narrow size distribution and a high specific surface area [18–20,24]. These characteristics must be optimized when the nanoparticles are aimed at biomedical applications [6]. The first attempt to obtain FSP-SPIONs was made by using a liquid precursor mixture composed of iron (III) acetylacetonate in xylene and iron (III) naphthenate in a mixture 1:2 of xylene and 2-ethylhexanoic acid, with iron concentration of 0.6 and 0.5 M, respectively. The feeding flow rate of the precursor mixture into the equipment was 15–25 mL/min using O2 as dispersion gas with a flow set at 30–40 L/min and a methane to oxygen gas flow rate of 3–6 L/h to form a self- maintaining main core flame. These operating conditions led to a sam- ple of SPIONs exhibiting heterogeneous particle sizes ranging from ca. 5 to 35 nm, as shown in the TEM images (see Fig. S1 in Supporting Information). M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 564 In order to ensure the production of a single population of small nanoparticles while avoiding the growth of larger nanoparticles certain parameters were adjusted (see section 2.3). An exhaustive control was exerted in the production process with the aim to ensure suitable flame properties as temperature, morphology, size and/or oxidative condi- tions. More specifically, with the aim to reduce the particle contact in the flame that leads to agglomeration and nanoparticle size growth, the iron content in both initial precursor mixtures was reduced to 0.35 and 0.23 M, respectively. Also, with the aim to reduce the residence time of the mixture inside the flame, a wider feeding flow rate (10–30 mL/min) was studied, revealing that the use of smaller fluxes results in smaller flames, therefore implying smaller areas for particle growth and/or agglomeration, and favoring the production of smaller nanoparticles. Additionally, the oxygen dispersion gas flow was reduced to 20–30 L/ min in order to exert an extra control in the reaction through a more stable temperature of the flame, avoiding potential cooling of this parameter and potential temperature ramps. Complementary to this adjustment, support flame gases flow was increased using a higher methane and oxygen flow (4–8 L/h) to maintain more stable flame morphology of the flame more stable, as well as the conditions of the specific zone where the nanoparticles are generated. TEM images of the obtained sample show FSP-SPIONs exhibiting pseudo-spherical nano- particle morphologies with sizes between ca. 10 and 20 nm. The sta- tistical treatment of size diameters reveals a single population being 12.5 nm the most abundant size (Fig. S2). The N2 sorption analysis of samples was used to estimate the particle size of FSP-SPIONs by using Equation (1) [41], where d is the particle diameter in nm; SSA the specific surface area in m2/g and ρ the density (4.9 g/mL). From the N2 adsorption–desorption analysis, a specific surface area of 75 m2/g and 92 m2/g is calculated for both samples, respectively, revealing an average particle size of approximately 10–18 nm for the sample obtained with optimized parameters, which completely correlates with particle size observed in the TEM images. d = 6000 SSA × ρ (1) Therefore, the optimized operating conditions produced SPIONs that were suitable in size and homogeneity and hence selected for the further bioanalytical characterization. These nanoparticles will be denoted as FSP-SPIONs onwards in the manuscript. 3.2. Surface modification of as-synthetized FSP-SPIONs Maintaining the stability of SPIONs over time in aqueous media without agglomeration or precipitation is a crucial requirement with regard to biomedical applications. Aiming at translation into the clinic there is a great advantage in the large-scale production of SPIONs by FSP, although the aqueous stability of these superparamagnetic nano- particles is an important issue to be resolved. In this work, the produced FSP-SPIONs were further processed by exploiting a treatment based on nitric acid and iron (III) nitrate to activate their surface, followed by a DMSA coating to obtain stable aqueous colloidal solutions (Fig. 1C). Magnetite nanoparticles obtained by coprecipitation methods are usu- ally subjected to a surface treatment and stabilization procedure to improve their colloidal stability in water. In a first stage, dispersion in nitric acid creates positive surface charges that provide sufficient elec- trostatic repulsion between the particles since the surface hydroxyl groups are protonated in the acidic medium. Subsequently, to prevent the gradual dissolution of the nanoparticles, oxidation to maghemite by iron (III) nitrate is performed to obtain the aqueous ferrofluids [42–44]. Fig. 1. A) Scheme of synthesis of SPIONs employing flame spray pyrolysis technology. B) Photography of the pilot FSP plant at the TECNAN and Lurederra Technological Centre facilities for the synthesis of FSP-SPIONs. C) Surface activation of as-synthetized FSP-SPIONs and subsequent DMSA coating. M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 565 As well, iron oxide nanoparticles prepared by laser pyrolysis were ob- tained with a significant enhancement of colloidal properties through an optimized acid treatment. This chemical protocol is consistent with a reduction of nanoparticle surface disorder induced by a dis- solution–recrystallization mechanism [45]. On the other hand, the sur- face modification of iron oxide nanoparticles through a ligand substitution process with meso-2,3-dimercaptosuccinic acid (DMSA) is usually performed with very good results to transfer hydrophobic SPIONs obtained through thermal decompositions methods to water. In these cases, DMSA coating yields hydrophilic nanoparticles very stable in water dispersions that preserve their superparamagnetic properties [46]. Furthermore, DMSA-coated SPIONs were assessed in terms of biocompatibility with human mesenchymal stem cells and studied in numerous biomedical applications confirming promising clinical use [47–52]. To the best of our knowledge, this combination of treatments has not been further applied to SPIONs obtained at a large scale by the FSP technique to increase their colloidal stability in aqueous media and therefore enable their use for biomedical applications. Before under- taking the bioanalytical study of our FSP-SPIONS-DMSA nanoparticles, a comprehensive physicochemical characterization of the as-synthetized and the DMSA-coated FSP-SPIONs was performed and it is detailed in the following paragraphs. The XRD patterns of both FSP-SPIONs and FSP-SPIONs-DMSA nanoparticles (Fig. 2A, left graph) show six maxima that are indexed as (220), (311), (400), (422), (511) and (440) of a cubic spinel structure. The lattice parameters calculated from the (311) reflection were 8.39 Å for the as-synthetized nanoparticles and 8.34 Å for the acid nitric treated and subsequently functionalized with DMSA nano- particles. These values lie between that of magnetite (Fe3O4, a = 8.40 Å) and maghemite (γ-Fe2O3, a = 8.35 Å), according to JCPDS cards numbers 19–629 and 39–1346, respectively. These results point to our iron oxide nanoparticles consisting in a mixture of both phases. There- fore, to estimate the relative amount of each phase, we applied the peak deconvolution routine for (511) reflection (Fig. 2A, right), as described by Kim et al. [53]. The results revealed that as-synthetized FSP-SPIONs contained 84 wt% of Fe3O4 and 16 wt% of γ-Fe2O3, therefore being magnetite the most abundant phase. On the other hand, FSP-SPIONs- DMSA sample corresponds to a mixture consisting in 17 wt% of Fe3O4 and 83 wt% of γ-Fe2O3, which necessarily implies that partial oxidation of magnetite to maghemite has taken place during the nitric acid treatment previous to the DMSA coating. The magnetic properties of iron oxide nanoparticles were investi- gated by vibrating sample magnetometry. The magnetization curves normalized to the mass of iron are displayed in Fig. 2B, showing similar shape and coercivity for both FSP-SPIONs and FSP-SPIONs-DMSA. The as-synthetized FSP-SPIONs exhibit superparamagnetic properties, which are preserved after surface activation and functionalization with DMSA. This characteristic superparamagnetic behavior is indicated by coer- civity values lower than 3 mT registered in both cases, which can be considered as absence of a hysteresis loop on the magnetization curve, i. e., coercivity and remanence close to zero. Moreover, the initial slopes show a rapid approach to saturation, suggesting a high magnetic susceptibility. The saturation magnetization values (M) at 5 T resulted to be consistent, being 97 and 90 emu/gFe for the as-synthetized and the DMSA-coated nanoparticles, respectively. The value for as-synthetized FSP-SPIONs is close to the value for bulk magnetite (~100 emu/gFe) [54], while the value for FSP-SPIONs-DMSA is somehow lower. This decrease in the saturation magnetization to 90 emu/gFe can be attrib- uted to the partial oxidation of magnetite to maghemite that takes place during the surface activation treatment, as also evidenced in the crys- tallographic analysis by XRD (vide supra). Therefore, this value is still higher than that of bulk maghemite (74–80 emu/gFe) [55,56], which is consistent with the presence of the maghemite/magnetite mixture in the FSP-SPIONs-DMSA composition identified by XRD. The efficient DMSA coating of the SPIONs was confirmed by FTIR spectroscopy, TGA, elemental chemical analysis and DLS measurements. FTIR spectra of FSP-SPIONs and FSP-SPIONs-DMSA (Fig. 2C) display a broad band in the 3000–3500 cm− 1 region, which can be attributed to the overlapping of the O–H stretching bands of hydrogen-bonded water molecules (H–O–H) and surface FeO-H groups. The bands at ca. 533 and 400 cm− 1 correspond to the two fundamental modes for the spinel structure of magnetite [57]. The many extra signals at 694, 623, 582 and Fig. 2. A) Powder XRD patterns of FSP-SPIONs and FSP-SPIONs-DMSA (left). Smoothed step scan patterns of (511) reflection for both FSP-SPIONs and FSP- SPIONs-DMSA samples (right). The maximum corresponding to (511) reflec- tion for each sample is deconvolutioned into magnetite (Fe3O4) and maghemite (γ-Fe2O3) and the lattice parameters, a, are also displayed. B) Magnetization curves of FSP-SPIONs and FSP-SPIONs-DMSA samples measured by vibrating sample magnetometry. The magnification of the magnetization curves near the origin are shown in the right side. C) FTIR spectra of as-synthesized (FSP- SPIONs) and surface-activated plus DMSA-coated (FSP-SPIONs-DMSA) nanoparticles. M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 566 440 cm− 1, were explained by assuming a tetragonal symmetry derived from the ordering of the vacancies produced in the partial oxidation of magnetite to maghemite. This is in good agreement with XRD studies, which revealed the coexistence of both crystalline phases in pristine and DMSA-coated FSP-SPIONs. FTIR spectrum of as-prepared FSP-SPIONs exhibits vibration bands attributed to asymmetric and symmetric C–H stretching vibrations (2960, 2924, 2881 and 2855 cm− 1). In the 1550 to 1000 cm− 1 range, the measured absorption bands can be attributed to the asymmetric and symmetric O–C––O stretches (1530 and 1421 cm− 1) and asymmetric and symmetric deformation of C–H groups (1464, 1358 and 1311 cm− 1). The bands at 1105 and 1068 cm− 1 were assigned to C–O stretching vibrations. All these bands exhibit rather similar patterns to those of FTIR spectra reported by Alkan et al., [58] suggesting that they probably arise from remaining acetate and carboxylate groups from the precursor solutions used in the flame spray synthesis. The FTIR spectrum of FSP-SPIONs-DMSA presents additional broad bands at 1573 and 1360 cm− 1 attributable to asymmetric and symmetric stretching vibrations of coordinated carboxylate groups of DMSA, respectively. The presence of such bands suggests that DMSA molecules were coordinating to iron ions located in the surface of SPIONs via –COO− groups. Ac- cording to reported studies [52], the large splitting between the –COO− bands (Δν = 213 cm− 1) points to the carboxylate groups bounded to SPIONs through a monodentate interaction, where one iron ion is bound to one carboxylic oxygen atom. Nonetheless, the possible presence of bridging bidentate structures should not be overruled (see Fig. 2C). Besides, broad absorption bands appeared at 1100 and 1046 cm− 1 (S–CH bond rocking vibrations) due to DMSA. The absence of sharp peaks in the 2500–2600 cm− 1 region in the spectrum of the DMSA coated SPIONs, which could be originated from the vibrations of the thiol groups (S–H) from DMSA (see Fig. S3 in Supporting Information), could be explained due to partial oxidation of the thiol groups into di- sulfide (S–S) during the ligand exchange process, as previously reported by Fauconnier et al. [59] However, the disulfide vibration bands at 500–540 cm− 1 could not be appreciated in the FTIR analysis due to the overlapping with Fe-O bands. The weight loss and elemental composition of as-synthetized, surface activated and DMSA-functionalized FSP-SPIONs were analyzed by TGA and elemental chemical analysis (Table 1). A slight decrease in weight loss as well as in the percentage of C in sample FSP-SPIONs* was observed comparing FSP-SPIONs with surface activated FSP-SPIONs*. It may be attributed to the presence of carbon residues and organic com- pounds on the surface of the nanoparticles, as evidenced by FTIR. These residues, from the synthetic FSP procedure, would be partially removed during the treatment with nitric acid and ferric nitrate. Comparing FSP-SPIONs-DMSA with both the preceding sample (FSP- SPIONs*) and the as-synthetized FSP-SPIONs, a marked increase in the percentage of weight loss of ca. 7%, as well as in the percentage of C, ca. 3.5%, was observed. This increase, together with the presence of S, ca. 3%, in FSP-SPIONs-DMSA, is attributed to the organic matter gained after DMSA coating. After confirming the successful DMSA coating of the nanoparticles, FSP-SPIONs and FSP-SPIONs-DMSA samples were analyzed by TEM, finding no significant differences in either the morphology or size of the nanoparticles (Fig. 3A and 3B, respectively). The DLS measurements of the resulting aqueous suspensions dis- played monomodal hydrodynamic size distributions centered at 61.4 nm for FSP-SPIONs and at 30.9 nm for FSP-SPIONs-DMSA (Fig. 3C and 3D, respectively). Since the size of the inorganic nanoparticle is not affected after functionalization, according to TEM images, this shift in the maximum of the hydrodynamic size distribution indicates that coating of SPIONs with DMSA causes a decrease in the size of the nanoparticle aggregates, reflecting a marked improvement of dispersion in water. Indeed, a change in the appearance of the aqueous suspensions before and after coating was observed (Fig. 3E and 3F respectively) reflecting the enhanced colloidal stability after DMSA coating, since FSP-SPIONs- DMSA in aqueous media showed brighter appearance and greater sta- bility over time than FSP-SPIONs. Moreover, ζ-potential values measured in water shift to more negative after DMSA coating, from − 15.8 mV for FSP-SPIONs to − 30.9 mV for FSP-SPIONs-DMSA (Fig. S4). The more negative ζ-potential value in the DMSA-coated nanoparticles compared to pristine ones indicates the presence of the carboxylate groups at the surface of the nanoparticles which are undergoing their acid-base equilibrium in the aqueous medium (–COOH + H2O ⇌ –COO¡ + H3O+). All these facts suggest that the colloidal stability in water of the FSP-SPIONs-DMSA can be ascribed to the electrostatic repulsion among –COO− groups, in addition to the steric stabilization provided by the DMSA molecules themselves on the surface of the nanoparticles. Moreover, the visual appearance of water suspensions of unmodified FSP-SPIONs and DMSA-functionalized FSP-SPIONs and their evolution with time, at short times (t = 0, 1 and 24 h) has been compared. The as- synthetized unmodified FSP-SPIONs start to sediment within an hour after dispersion in water, and have completely settled by 24 h. However, the DMSA-functionalized FSP-SPIONs remain in colloidal suspension at the same times (Fig. S5 and Video V1 in supporting Information). The hydrodynamic size distribution obtained by dynamic light scattering of a sample of FSP-SPIONs-DMSA which has been kept in aqueous sus- pension eleven months after synthesis shows that the SPIONs do not undergo aggregation since a narrow single-modal distribution, in the range from about 20 to 80 nm, is preserved (Fig. S6). Moreover, the maximum of the hydrodynamic size distribution barely shifts from 30.9 nm for the newly synthesized sample to 32.8 nm for the long-term sample in aqueous suspension. Both facts indicate long-term stability of FSP-SPIONs-DMSA, which is a critical requirement for biomedical applications. Regarding biomedical applications, the effect of a high ionic strength on the colloidal stability of the nanoparticles has been evaluated in PBS buffer, a medium which represents the conditions where the bio- analytical characterization is performed. The hydrodynamic size dis- tribution of the nanoparticles was measured by DLS in PBS 1x (Fig. S7 in Supporting Information). The hydrodynamic size distribution for FSP- SPIONs shows a bimodal pattern with the maxima centered at ca. 223 and 1461 nm, clearly indicating that the increase of ionic strength produces large aggregates. However, the monomodal hydrodynamic size distributions centered at 23.3 nm of FSP-SPIONs-DMSA clearly rules out particle aggregation in PBS. 3.3. Nanoparticles impact on formed components of blood To determine the clinical translational value of SPIONs produced by FSP technique, it is mandatory to evaluate the effects of close contact between the formed elements of blood and the nanomaterial [60]. The blood has two main components, the blood cells and the plasma, i.e., the Table 1 Organic content and elemental composition from thermogravimetric and chemical analyses (atomic percentages) of as-synthesized (FSP-SPIONs), surface-activated (FSP-SPIONs*) and DMSA-coated (FSP-SPIONs-DMSA) nanoparticles. Sample Weight loss (%) a %C %N %S FSP-SPIONs 2.70 ± 0.13 1.24 ± 0.06 0.11 ± 0.01 – FSP-SPIONs* 2.50 ± 0.12 0.82 ± 0.04 0.24 ± 0.01 – FSP-SPIONs-DMSA 9.75 ± 0.49 4.57 ± 0.22 0.95 ± 0.05 2.95 ± 0.15 a Weight loss (wt%) is determined from the TGA excluding the weight loss due to the desorption of water (up to 125 ◦C). M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 567 fluid phase of the blood [61]. Among the blood cells, myeloid compartment instead lymphoid compartment is supporting primary immune responses (PIR) and also plays a critical role in the regulation of hemostasis [62]. One of the main participants in the PIR are monocytes, with a normal count in blood of 2.5–8.5%. They are highly phagocytic and the main source of tissue-resident macrophages [62]. Platelets or also called thrombocytes are the smallest anucleate formed elements of blood, full of granules and highly sensitive to clotting factors [62,63]. Herein, we focus on testing the hemocompatibility of monocytes and platelets (Fig. 4). To determine the activating effect on classical circulating monocyte population (CD14+CD16+), we analyzed the expression of CD11B by flow cytometry [64] (Fig. 4A). CD11B is an integrin member family which is expressed on the surface of many leukocytes and regulates adhesion and migration of these types of cells in an inflammatory response [65,66]. The presence of both FSP-SPIONs and FSP-SPIONs- DMSA at 100 µg/mL resulted in overexpression of CD11B when compared to basal CD11B expression. These data suggest an in vitro activation of classical monocytes in presence of both iron oxide nano- materials. Interestingly, FSP-SPIONs-DMSA (in red) attenuates CD11B up-regulation compared to the bare FSP-SPIONs (in blue). The func- tionalized FSP-SPIONs-DMSA nanoparticles have shown to down- regulate CD11B at doses of 50 and 100 µg/mL, reporting a reduction up to 40% in CD11B expression compared to FSP-SPIONs without sta- tistical differences though (Fig. 4B). In other hand, the incubation of unwashed PRP with both FSP-SPIONs and FSP-SPIONs-DMSA NPs at 50 and 100 µg/mL resulted in negligible platelet aggregation (less than ± 5% sedimentation rate). As expected, positive controls with platelets agonists led to platelet aggregation up to 85%, demonstrating that donor platelets responded in a normal manner and that FSP-SPIONs and FSP- SPIONs-DMSA do not affect the platelet aggregation process (Fig. 4C). Furthermore, there is no difference neither between the two materials nor between the two doses used as<5% of sedimentation is produced in all conditions [67]. Fig. 3. TEM images of as-synthetized FSP-SPIONs (A) and functionalized FSP-SPIONs-DMSA (B). Hydrodynamic size distributions obtained by dynamic light scattering of FSP-SPIONs (C) and FSP-SPIONs-DMSA (D). Photographs of the aqueous suspensions of the FSP-SPIONs before (E) and after (F) DMSA coating. Fig. 4. A) Representative unidimensional histogram of CD11B expression on monocyte in presence of 100 µg/mL of bare FSP-SPIONs (blue) or functional- ized FSP-SPIONs-DMSA (red). B) N-fold increase of CD11B expression (Mean Fluorescent Intensity) on monocyte surface in presence of FSP-SPIONs (in blue) and FSP-SPIONs-DMSA nanoparticles (in red) compared to basal monocyte expression. N = 3. C) Representative platelet aggregation chart in platelet-rich plasma in presence of agonists EPI (black) and TRAP (red), FSP-SPIONs (green and blue), and FSP-SPIONs-DMSA nanoparticles (orange and yellow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 568 3.4. In vitro cytocompatibility assays Generally, in vitro screening assays with nanomaterials/nano- particles can be classified into three general categories, all of them being covered in this research: (i) uptake analysis; (ii) viability assays and (iii) functional assays to evaluate the effects of nanomaterials on various cellular processes [68]. Specifically, we have evaluated the impact of FSP-SPIONs-DMSA, selected for their colloidal stability in water media and to their hemocompatible response, on the cellular incorporation process, cell viability, reactive oxygen species (ROS) intracellular con- tent, cell-cycle progression, and gene expression of human bone marrow derived mesenchymal stem cells (hBM-MSCs). 3.4.1. Cell uptake and viability of hBM-MSCs exposed to FSP-SPIONs- DMSA nanoparticles A key factor when considering the potential biological applications of a specific nanoparticle is its ability to internalize into cells [69–72]. In many cases, the interaction of nanoparticles, including SPIONs, with cell membranes lipids is considered to be one of the major causes of cyto- toxicity, as this interaction can lead to cell death [73]. Generally, the SPIONs cell uptake can occur after just 1 to 4 h of incubation. However, higher uptake by mammalian cells is normally observed in prolonged incubation times of around 24 h. In fact, Lee et al found that more than 90% of the cells internalized SPIONs after incubating at 1 μg/mL for 1 h, but the maximum incorporation was observed at 24 h of exposure [74]. Herein, we studied the viability of hBM-MSCs to assess whether the nano-biointerface interactions with the SPIONs during their incorpora- tion provokes the loss of cellular plasma membrane integrity. Moreover, to guarantee the incorporation of these SPIONs by hBM-MSCs and determine their effects on cell, 24 h were chosen as the exposure time. Fig. 5A displays the cell uptake of FSP-SPIONs-DMSA by hBM-MSCs when exposed to 25 and 50 μg/mL and Fig. 5B shows their cell viability after 24 h of exposure. The results of cellular incorporation, through changes in the side-scatter intensity, indicate that the internal cellular complexity (SSC) of hBM-MSCs significantly increases ≈ twofold and ≈ threefold compared to that of control cells when exposed to 25 and 50 μg/mL of SPIONs for 24 h, respectively. Furthermore, there is a significant increase in the SSC parameter of these cells at the highest concentration (50 μg/mL). Specifically, the SSC significantly increased ≈ 43%, compared to the lowest concentration of nanoparticles. Regarding the cell viability, hBM-MSCs exposed to both concentrations of nanoparticles, 25 and 50 μg/mL, showed high cell viability (≥95%), without significant differences with respect to control cells cultured in the absence of nanoparticles (98%). These results highlight that the FSP- SPIONs-DMSA are incorporated by hBM-MSCs in a dose-dependent manner and that such cellular uptake process does not cause the loss of cellular plasma membrane integrity, in the tested conditions. These results are in agreement with literature regarding MSs cells exposed to DMSA-coated SPIONs synthetized by other methodologies [47]. Cell morphology is one of the powerful indicators of cell health, which is very important for understanding cell behavior, including the tracking of intracellular molecules/organelles and the cell deformation after nanomaterial incorporation. It is known that the presence of nanoparticles, including SPIONs, inside cell could cause damage to the cytoskeletal structure (F-actin filaments). Thus, in this study, the cellular morphology of hBM-MSCs after FSP-SPIONs-DMSA nanoparticles up- take as well as their intracellular localization was evaluated after 24 h by confocal microscopy (Fig. 5C), since these filaments are essential ele- ments in maintaining cellular and structural morphology. Confocal im- ages allowed us to observe the characteristic morphology of hBM-MSCs in all conditions of the culture tested [75]. In addition, the staining of the F-actin filaments revealed the correct cytoskeletal assembly on spreading hBM-MSCs exposed to both concentrations (25 and 50 µg/mL) similar to control cells. Finally, the confocal images also show that these nanoparticles have been preferentially incorporated and localized in the perinuclear area, clearly outside of the nucleus and without obvious signs of nuclear shrinkage (pyknosis), as indicated by the white arrows. 3.4.2. Intracellular ROS content in hBM-MSCs exposed to FSP-SPIONs- DMSA nanoparticles According to the literature, several studies have proposed oxidative stress as a key mechanism involved in the nanoparticle’s toxicity [76,77]. Nanoparticles enter to the intracellular environment and interact with the proteins, organelles, and even DNA, inducing over- production of ROS. This overproduction limits the cellular capacity to maintain the normal physiological redox-regulated functions, leading to adverse biological effects such as membrane lipid peroxidation, protein denaturation, mitochondrial dysfunction, formation of apoptotic bodies, leakage of lactate dehydrogenase and DNA and RNA damage [78]. The reactive molecules contain oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2 – (superoxide anion), peroxynitrite (ONOO− ), hypochlorous acid (HOCl) and the radical hydroxyl (HO•− ). In this study, the ROS intracellular content of hBM-MSCs after exposure Fig. 5. Cell uptake of FSP-SPIONs-DMSA by hBM-MSCs (A) and cell viability of hBM-MSCs exposed to FSP-SPIONs-DMSA (B), evaluated by flow cytometry after 24 h of exposure. Cell uptake is shown through intracellular complexity, side angle scatter (SSC-A), where the bars represent the geometric mean of SSC data as mean ± SD. Cell viability was evaluated through propidium iodide exclusion assay. Statistical significance: ***p < 0.005 (vs Control). ##p < 0.01 (vs 25 µg/mL). C) Morphology evaluation by confocal microscopy of hBM-MSCs after 24 h of culture with FSP-SPIONs-DMSA nanoparticles. Cells were stained with DAPI for the visualization of the cell nuclei in blue and rhodamine- phalloidin for the visualization of cytoplasmic F-actin filaments in red. FSP- SPIONs-DMSA nanoparticles appear as black deposits (transmission) inside the cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 569 to 25 and 50 μg/mL of FSP-SPIONs-DMSA nanoparticles for 24 h was investigated (Fig. 6A and 6B). The obtained results regarding the percentage of the ROS intracel- lular content show no significant differences when the cells were cultured with both concentrations compared to control cells. Further- more, the results of fluorescence intensity showed a significant decrease of ROS intracellular content of hBM-MSCs exposed to both concentra- tions with respect to control, being this decrease being dose-dependent. This indicates the low or no cytotoxicity of SPIONs, which means that the ROS produced by the SPIONs labelled cells are maintained in a controllable range [79–81]. Recent studies have suggested an effective reduction of ROS through an up-regulation of the cell’s antioxidant defense after the initial ROS production induced by SPIONs treatment in cells. Such studies evaluated glutathione and superoxide dismutase (SOD) as oxidative stress markers to evaluate the damage of SPIONs to the intracellular antioxidant system. Moreover, Gao et al. attributed it to intact iron oxide particles, but not to iron leaching, an intrinsic peroxidase-like activity capable of catalyzing the breakdown of H2O2. Therefore, one of the potential mechanisms involved in the stimulation of hBM-MSCs growth could be the diminishing of intracellular H2O2 [82]. These preliminary assays give information on the biosafety of FSP- SPIONs-DMSA, which indeed do not impede the inherent protective antioxidative mechanisms of hBM-MSCs while maintaining low levels of cytotoxicity. 3.4.3. Effect of FSP-SPIONs-DMSA nanoparticles on cell cycle of hBM- MSCs Cell cycle is defined as the ordered set of events that leads to cell growth. The cell cycle in eukaryote cells comprises four consecutive stages, the most important of which is the DNA replication phase known as the synthesis (S) phase. The cell cycle phases are defined as G0/G1 phase (Quiescence/Gap 1), S phase (Synthesis) and finally to the G2/M phase (Gap 2 and Mitosis). A cell can be in the state of non-division or interphase, where it performs its specific functions or in state of division, called M phase. During G1 phase, cell doubles its size and mass due to the continuous synthesis of protein and RNA. The second cycle stage, S phase, is characterized by the DNA replication. During G2 phase con- tinues the synthesis of proteins and RNA. Finally, M phase encompasses mitosis (distribution of nuclear genetic material) and cytokinesis (divi- sion of the cytoplasm). The cell cycle is mediated by different factors, the most important of which are perhaps cycle-dependent kinases (CDKs) [83]. Fig. 7 shows the cell cycle phases of hBM-MSCs exposed at 25 and 50 μg/mL FSP-SPIONs-DMSA for 24 h. Results highlight that there is a significant increase of the S and G2/M phases in the cells exposed to 25 μg/mL FSP-SPIONs-DMSA compared to the phases showed by hBM- MSCs cultured in absence of nanoparticles. In addition, when hBM- MSCs are cultured with 50 μg/mL FSP-SPIONs-DMSA nanoparticles for 24 h, the S phase experienced a slight decrease, while the G0/G1 and G2/ M phases are kept practically constant with respect to the control. These results evidence the no cytotoxicity of FSP-SPIONs-DMSA nanoparticles, and indeed the slight increase in cell growth for 25 μg/mL can be explained due to the FSP-SPIONs-DMSA ability to diminish intracellular ROS content through intrinsic peroxidase-like activity, also found by other authors [84]. In this regard, it is worth noting that this increase in cell proliferation is a required phase for mesenchymal stem cells prior to differentiation into another cell lineage [85]. Considering this increase in proliferation of hBM-MSCs, and to verify their ability to differentiate as inherent property of these cells, a pre- liminary differentiation assay by Runx2 and ALP gene expression have been carried out [86,87]. Fig. 8 shows the Runx2 and ALP gene expression, as bone osteogenic markers, of hBM-MSCs exposed to 25 μg/ mL of FSP-SPIONs-DMSA nanoparticles quantitatively evaluated by PCR. No significant differences in Runx2 gene expression were observed compared to that shown by cells in absence of SPIONs after 4 and 7 days of exposure. On the other hand, osteogenic marker ALP expression was significantly increased both 4 and 7 days compared to cells cultured in absence of nanoparticles (control). Therefore, results highlight that the FSP-SPIONs-DMSA nanoparticles do not block the expression of early markers of osteogenic differentiation in human bone marrow derived mesenchymal stem cells, keeping their cell functionality. Fig. 6. ROS intracellular content of hBM-MSCs evaluated by flow cytometry after 24 h of exposure to FSP-SPIONs-DMSA nanoparticles. Data expressed in percentage (A) and fluorescence intensity (B). Statistical significance: **p < 0.01, ***p < 0.005 (vs Control). #p < 0.01 (vs 25 µg/mL). Fig. 7. Cell cycle phases of hBM-MSCs after 24 h of exposure to FSP-SPIONs- DMSA evaluated by flow cytometry. Up to down: control, 25 and 50 μg/mL. Statistical significance: **p < 0.01 (vs Control); ##p < 0.01 (vs 25 µg/mL). M. Estévez et al. Journal of Colloid And Interface Science 650 (2023) 560–572 570 4. Conclusions In this work, large-scale production of superparamagnetic iron oxide nanoparticles (SPIONs) by flame spray pyrolysis (FSP) technique was accomplished for biomedical applications. This bottom-up nano- manufacturing technique has recognized scalability and reproducibility, enabling the production of metal oxide nanoparticles with high yields and low production costs [18–23]. The novelty of this manuscript lies fundamentally in the validation of SPIONs synthesized by FSP meth- odology for medical applications. For this purpose, a thorough charac- terization was carried out in terms of superparamagnetic properties, colloidal stability, hemocompatibility and cytotoxicity. First, very ho- mogeneous SPIONs with a particle size of ca. 12 nm and a yield of 100 g/ h have been obtained by FSP. Then, as-synthetized FSP-SPIONs were further processed via an acid treatment to clean and activate their sur- face, followed by a coating with dimercaptosuccinic acid (DMSA) to obtain stable aqueous colloidal solutions [46,49]. The final product shows high quality in terms of nanoparticle size, homogeneous size distribution, long-term colloidal stability and magnetic properties. Hemocompatibility studies with monocytes and platelets demonstrate that functionalized FSP-SPIONs-DMSA nanoparticles allow normal basal monocyte function and do not cause platelet aggregation. Moreover, in vitro biocompatibility assays with mesenchymal stem cells (hBM-MSCs) show a dose-dependent cellular uptake while maintaining high values of cell viability and cell cycle progression, without producing cellular oxidative stress, which could be an added value in SPIONs synthetized by FSP. The successful in vitro biological validation of the FSP-SPIONs- DMSA nanoparticles produced in this work, which benefit from large- scale production, constitutes a significant advance in the challenging race towards the industrial development of SPIONs for biomedical ap- plications. In this sense, we are currently conducting further studies on the anti-oxidative mechanisms [84] as well as the cellular internaliza- tion pathways of these nanoparticles to stablish their most suitable clinical applications. Funding This project has received funding from the European Union’s Hori- zon 2020 Research and Innovation Program under grant agreement No 814410 (GIOTTO) and ERC-2015-AdG (VERDI) Proposal No. 694160. This study was also supported by the Ministerio de Ciencia e Innovación (MICINN) PID2020-117091RB-I00 grant. CRediT authorship contribution statement Manuel Estévez: Methodology, Validation, Formal analysis, Inves- tigation, Data curation, Writing – original draft, Visualization. Mónica Cicuéndez: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Julián Crespo: Conceptualization, Methodology, Validation, Formal analysis, Investi- gation, Data curation, Writing – original draft, Visualization. Juana Serrano-López: Methodology, Validation, Formal analysis, Investiga- tion, Resources, Data curation, Writing – original draft, Visualization. Montserrat Colilla: Methodology, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, Visualization. Claudio Fernández-Acevedo: Conceptualization, Investigation, Writing – review & editing, Visualization, Supervision, Project admin- istration, Funding acquisition. Tamara Oroz-Mateo: Conceptualization, Methodology, Investigation, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. Amaia Rada- Leza: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft. Blanca González: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration. Isabel Izquierdo-Barba: Conceptualization, Methodology, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization, Supervision, Project administration, Funding acquisition. María Vallet-Regí: Writing – review & editing, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgements We thank the Materials for Medicine and Biotechnology Group (María del Puerto Morales, Alvaro Gallo-Cordova) for advice and assis- tance in the characterization in the analysis services of the ICMM-CSIC for the characterization of the magnetic properties. 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