Well Defined Poly(Methyl Methacrylate)-Fe3o4/Poly

Article Well Deﬁned Poly(Methyl Methacrylate)- Fe3O4/Poly(Vinyl Pivalate) Core–Shell Superparamagnetic Nanoparticles: Design and Evaluation of In Vitro Cytotoxicity Activity against Cancer Cells

Graciane Resende 1, Gabriel V. S. Dutra 1, Maria S. B. Neta 2, Olacir A. Araújo 3 , 2 1, , Sacha B. Chaves and Fabricio Machado * † 1 Laboratório de Desenvolvimento de Processos Químicos, Instituto de Química, Universidade de Brasília, Campus Universitário Darcy Ribeiro, CEP 70910-900 Brasília, DF, Brazil; [email protected] (G.R.); [email protected] (G.V.S.D.) 2 Departamento de Genética e Morfologia, Instituto de Biologia, Universidade de Brasília, Campus Universitário Darcy Ribeiro, CEP 70910-900 Brasília, DF, Brazil; [email protected] (M.S.B.N.); [email protected] (S.B.C.) 3 Universidade Estadual de Goiás, Campus Central—Ciências Exatas e Tecnológicas, CP 459, CEP 75132-903 Anápolis, GO, Brazil; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +55-61-3107-3868 Current address: School of Chemistry, University of Nottingham, University Park, † Nottingham NG7 2RD, UK.

Received: 24 October 2020; Accepted: 23 November 2020; Published: 30 November 2020

Abstract: The objective of this work is to develop and characterize polymeric nanoparticles with core–shell morphology through miniemulsion polymerization combined with seeded emulsion polymerization, aiming at the application in the treatment of vascular tumors via intravascular embolization. The synthesis of the core–shell nanocomposites was divided into two main steps: (i) Formation of the core structure, consisting of poly(methyl methacrylate)/magnetic oxide coated with oleic acid (OM-OA) via miniemulsion and (ii) shell structure produced through seeded emulsion polymerization of vinyl pivalate. Nanocomposites containing about 8 wt.% of OM-OA showed high colloidal stability, mean diameter of 216.8 nm, spherical morphology, saturation magnetization (Ms) of 4.65 emu g 1 (57.41 emu g 1 of Fe O ), preserved superparamagnetic behavior and glass transition · − · − 3 4 temperature (Tg) of 111.8 ◦C. TEM micrographs conﬁrmed the obtaining of uniformly dispersed magnetic nanoparticles in the PMMA and that the core–shell structure was obtained by seeded 1 1 emulsion with Ms of 1.35 emu g (56.25 emu g of Fe O ) and Tg of 114.7 C. In vitro cytotoxicity · − · − 3 4 ◦ assays against murine tumor of melanoma (B16F10) and human Keratinocytes (HaCaT) cell lines were carried out showing that the core–shell magnetic polymeric materials (a core, consisting of poly(methyl methacrylate)/Fe3O4 and, a shell, formed by poly(vinyl pivalate)) presented high cell viabilities for both murine melanoma tumor cell lines, B16F10, and human keratinocyte cells, HaCaT.

Keywords: methyl methacrylate; vinyl pivalate; magnetic nanoparticles; embolization; miniemulsion; seeded emulsion; core–shell polymer particles

1. Introduction The development of polymeric materials is undoubtedly attractive in the manufacture of many biomedical devices, given their structural versatility, ease of manufacture, biocompatibility,

Polymers 2020, 12, 2868; doi:10.3390/polym12122868 www.mdpi.com/journal/polymers Polymers 2020, 12, 2868 2 of 18 low cytotoxicity, flexibility, biodegrability, porosity, and controlled morphology, among others [1,2]. In this field, highlight the nanocomposites that combine the thermoplastic properties of polymers and the superparamagnetic behavior of nanoparticles, presenting great potential in the treatment of vascular tumors [3,4]. Iron oxide nanoparticles possessing various characteristics such as biocompatibility, superparamagnetic properties, and recyclability [5] have been widely used in controlled drug delivery systems, facilitating drug channeling to specific target cells by externally controlling their path using a field [6], and as a contrast agent for magnetic resonance, hyperthermia treatment of tumors and detoxification of biological fluids [7–9], etc. In biomedical applications, the encapsulation of nanoparticles with polymeric matrices ensures their stability in biological media, provides functionality, protects and prevents agglomeration [10] and results in increased local drug concentrations, enabling their use for targeted delivery of therapeutic agents, reducing toxic effects in cells or tissues healthy [11,12]. The polymeric matrices can be prepared from synthetic or natural polymers, obtaining colloidal particles by several synthetic routes, including emulsion [13–15], suspension [16–18], and miniemulsion polymerization [19–21]. Embolization therapy involves deliberate blocking of blood vessels due to the introduction of embolic agents, with the aim of decreasing or preventing blood transport through the blood vessels towards the injured area [22]. The choice of the adequate embolizing agent is important for the safety and effectiveness of the treatment [23]. Embolic agents can be classified into two main categories: Temporary and permanent. Although temporary agents block blood flow, they will be dissolved or eliminated by the immune system within a prescribed period of time. For the temporary embolic materials, the most used materials are autologous agents and resorbable gelatin sponge. Permanent agents are designed to provide irreversible blood flow obstruction to target lesions or final organs. In particular for the permanent embolic agent, the most used materials are coils, plugs, detachable balloons (solid agents), polyvinyl alcohol, compressible microspheres, thrombin, dehydrated alcohol, sodium tetradecyl sulfate, glues, and onyx (liquid agents) [23,24]. The miniemulsion polymerization process has excelled in recent years due to the intrinsic characteristics of the materials obtained, such as narrow particle size distributions, high colloidal stability, high molar mass, obtaining a wide range of polymers and mainly, the ability to encapsulate materials inorganics in a single reaction step with fast polymerization rates [25–28]. A versatile class of nanocomposites can be developed by combining the peculiarities of the miniemulsion with other polymerization techniques, obtaining polymeric nanoparticles of the core–shell type, the core being produced from the polymer initially polymerized, and the polymers formed later are incorporated into the layer external [29]. These materials have final properties dependent not only on the composition of each polymeric phase, but also on the morphology of these particles [30]. Core–shell polymer nanoparticles have attracted great interest due to their unique structure in which two different components are combined into a single material [31]. In biological applications, core–shell nanoparticles have great advantages over conventional nanoparticles, thus improving performance, such as low cytotoxicity, good dispersibility, high biocompatibility and cell compatibility, better connection with other biologically active molecules, as well as improved thermal and chemical stability [6,32,33]. With this perspective, Rahman et al. [34] developed a thermo-responsive magnetic polymer, synthesizing the magnetic seeds of cross-linked polystyrene by emulsion polymerization. The thermosensitive poly(N-isopropylacrylamide) shell (PNIPAM) was obtained via precipitation polymerization and then the polymer was functionalized with aminoethylmethacrylate hydrochloride (AEMH). The TEM micrographs confirmed the core–shell morphology of the particles. Chen et al. [35] synthesized hybrid epoxy-acrylic latex with core–shell morphology, using an epoxy dispersion as seed and, subsequently, carried out the seeded emulsion polymerization in acrylic monomer. Recently, Li et al. [36] synthesized cross-linked polystyrene nanospheres via dispersion polymerization and, subsequently, these seeds were swollen in tert-butyl acrylate and styrene monomers and Polymers 2020, 12, 2868 3 of 18 polymerized by seeded emulsion. The authors investigated the effect of methanol concentration, used in the synthesis of seeds, and the degree of crosslinking on the morphology of the nanoparticles. For the synthesis of core–shell nanoparticles to be successful, the polymerization technique and the operating conditions used are highly fundamental [37]. In the current work, the technique used for the formation of the shell is the seeded emulsion, which consists of the formation of a new polymer layer grafted on the seed surface (N.B. herein, the core nanoparticles consisting of poly(methyl methacrylate)/Fe3O4 is synthesized through miniemulsion polymerization process), through addition of fresh monomer feed following a semibatch mode operation, leading to the formation of a uniform shell and promoting easy control of final core–shell morphology [38,39]. The focus of this work is to obtain core–shell particles through combination of a batch mode (used to form the core structure via miniemulsion polymerization) and a semicontinuous mode operation (to form the particle shell structure through seeded emulsion polymerization), in which the feed composition is adequately controlled, allowing for the formation of a new polymer phase on the previously formed polymer particle. These particular core–shell polymer particles, intended for to be used as an embolic agent, consist basically of (i) a core, consisting of oleic acid functionalized magnetic nanoparticles (OM-OA), properly dispersed into spherical poly(methyl methacrylate) nanoparticles (PMMA), and (ii) a shell, consisting of poly(vinyl pivalate) (PVPi). By the combination of vinyl pivalate and methyl methacrylate, this new magnetic embolic agent based on PVPi/PMMA exhibits low susceptibility to undesirable hydrolysis reactions, which is strongly responsible for the recanalization of blood vessels, since they are minimized by the steric effect caused by the insertion of PVPi into the particles due to the tert-butyl group present in its chemical structure [17,40]. In addition, these nanocomposites have good chemical and mechanical resistance and high thermal stability, conferred to the methyl methacrylate based core [41]. As an additional study, the cytotoxicity of PVPi/PMMA core–shell particles was evaluated in vitro for tumoral cell lines B16F10 (murine melanoma) and HaCat (human keratinocyte). In vitro studies are of fundamental importance to give insight into the cytotoxicity ability of the magnetic nanocomposites on tumoral and non-tumoral cell lines. In the current work, B16F10 and HaCat were selected because murine B16F10 melanoma model is widely used as metastatic melanoma model for preclinical studies, as well as, human keratinocyte HaCaT cell line model is extensively used in epidermal homeostasis and pathophysiology studies.

2. Materials and Methods All reagents were analytical grade (PA) and used without prior puriﬁcation. Methyl methacrylate (MMA) 99%, vinyl pivalate (VPi) 99%, hexadecane 99%, benzoyl peroxide 75% and ethylene glycol dimethylacrylate (EGDMA) 98% were purchased from Sigma-Aldrich Brasil Ltda. (Rio de Janeiro, Brazil). Hydrochloric acid (HCl) 36.5–38% and oleic acid 99% were purchased from Synth (São Paulo, Brazil). Ferric chloride hexahydrate (FeCl 6H O) 97%, ferrous sulfate heptahydrate (FeSO 7H O) 3· 2 4· 2 99%, sodium hydroxide (NaOH) 99% and potassium persulfate (KPS) 99% were purchased from Vetec Química Fina Ltda. (Rio de Janeiro, Brazil) and 90% pure sodium dodecyl sulfate (SDS) was supplied by Reagen Produtos para Laboratórios Ltda. (Parana, Brazil). Nitrogen gas was supplied by White Martins Gases Industriais Ltda. (Rio de Janeiro, Brazil) with 99.5% purity. Scheme1 illustrates the experimental protocol for the synthesis of poly(methyl methacrylate)/Fe3O4/poly(vinyl pivalate) magnetic core–shell nanoparticles. Polymers 2020, 12, 2868 4 of 18 Polymers 2020, 12, x FOR PEER REVIEW 4 of 17

SchemeScheme 1. 1. FlowchartFlowchart summarizing summarizing the experimental the experimental protocol for protocolformation forof poly(methyl formation of methacrylate)/Fe3O4/poly(vinyl pivalate) magnetic core–shell nanoparticles. poly(methyl methacrylate)/Fe3O4/poly(vinyl pivalate) magnetic core–shell nanoparticles. 2.1. Synthesis2.1. Synthesis of Magnetic of Magnetic Nanoparticles Nanoparticles (OM) (OM) MagneticMagnetic nanoparticles nanoparticles (OM) (OM) were were obtained obtained by by thethe alkalinealkaline hydrolysis coprecipitation coprecipitation method method of 2+ 3+ Fe2+ ofand Fe Fe and3+ ions Fe inions watery in watery environment, environment, asdescribed as described by by Neto Neto et et al. al. [42 [42]] in in 500 500 mL mL of of distilled distilled water water were added 2 mL of a 10% HCl solution and 0.03 mol of Fe2+ and Fe3+ metal ions in a were added 2 mL of a 10% HCl solution and 0.03 mol of Fe2+ and Fe3+ metal ions in a stoichiometric stoichiometric molar ratio of 1:2 with 0.01 mol of FeSO4·7H2O and 0.02 mol of FeCl3·6H2O. The molar ratio of 1:2 with 0.01 mol of FeSO 7H O and 0.02 mol of FeCl 6H O. The mixture was heated mixture was heated to 70 °C under constant4· 2 stirring at 500 rpm and under3· 2nitrogen atmosphere for to 7030◦C min. under Then constant the precipitating stirring agent, at 500 NaOH, rpm and with under a concentration nitrogen of atmosphere 5 mol L−1 was for added 30 min. until Then the the 1 precipitatingsolution reached agent, NaOH,a pH ofwith approximately a concentration 12. Afte ofr 5precipitation, mol L− was the added suspension until theremained solution under reached a pHstirring of approximately at 70 °C and 12.bubbling After nitrogen precipitation, for 30 min. the suspensionThe precipitate remained was washed under with stirring distilled at water 70 ◦C and bubblingand decanted nitrogen with for 30the min. aid of The a magnet precipitate until it was reached washed neutral with pH. distilled water and decanted with the aid of a magnet until it reached neutral pH. 2.2. Coating of Magnetic Nanoparticles with Oleic Acid (OM-OA) 2.2. CoatingThe of magnetic Magnetic nanoparticles Nanoparticles were with suspended Oleic Acid in (OM-OA)170 mL of distilled water and then subjected to mechanicalThe magnetic stirring nanoparticles under nitrogen were bubbling suspended and 85 in 170°C heating. mL of distilledThen the watercoating and material, then subjected2.5 mL to oleic acid, was added dropwise at a maximum rate of 0.5 mL min−1. After 30 min of the addition of mechanical stirring under nitrogen bubbling and 85 C heating. Then the coating material, 2.5 mL oleic acid, the nanoparticles were washed with distilled◦ water to neutral pH and treated with acetone oleic acid, was added dropwise at a maximum rate of 0.5 mL min 1. After 30 min of the addition of to remove free coating material [16,42]. − oleic acid, the nanoparticles were washed with distilled water to neutral pH and treated with acetone to remove2.3. Synthesis free coating of PMMA/OM-OA material [16 Magnetic,42]. Nanocomposite

2.3. SynthesisFor the of PMMAproduction/OM-OA of nanocomposites, Magnetic Nanocomposite different parameters such as temperature, reaction time, amount of initiator and OM-OA content were studied. The concentration of surfactant, SDS, used in theFor synthesis the production was below of nanocomposites,the critical micellar di concentrationﬀerent parameters (CMC) at such a temperature as temperature, of 85 °C, reaction which time, amountcorresponds of initiator to 4.35 and g OM-OA L−1 [43] and content 3.68 g were L−1 at studied. 70 °C (N.B. The different concentration from emulsion of surfactant, polymerization SDS, used in process, the amount of surfactant is kept below CMC in order to avoid the formation of micelles in the synthesis was below the critical micellar concentration (CMC) at a temperature of 85 ◦C, the continuous aqueous phase,1 which guarantees1 that the polymerization reactions take place which corresponds to 4.35 g L− [43] and 3.68 g L− at 70 ◦C (N.B. diﬀerent from emulsion polymerization preferably inside the monomer nanodroplets). process, the amount of surfactant is kept below CMC in order to avoid the formation of micelles in the The nanocomposite was synthesized by miniemulsion polymerization as follows: In one case continuous aqueous phase, which guarantees that the polymerization reactions take place preferably the aqueous phase was homogenized with 153 g of distilled water and 0.6 g of SDS under constant insidestirring the monomer at 300 rpm nanodroplets). for 10 min. Separately, the organic phase consisting of 27 g MMA, 0.81 g hexadecane,The nanocomposite 0.72 g BPO was and synthesized 4.05 g OM-OA by miniemulsion was prepared polymerizationin a beaker and homogenized as follows: In using one casean the aqueousultrasonic phase disperser was homogenized (Cole-Parmer with® 750 153 W, g Vernon of distilled Hills, water IL, USA) and for 0.6 5g min of SDSat 40% under amplitude constant in an stirring at 300ice rpm bath. for Subsequently, 10 min. Separately, the organic the phase organic was phase mixed consisting into the aqueous of 27 gphase MMA, and 0.81 kept g under hexadecane, constant 0.72 g

BPO and 4.05 g OM-OA was prepared in a beaker and homogenized using an ultrasonic disperser (Cole-Parmer® 750 W, Vernon Hills, IL, USA) for 5 min at 40% amplitude in an ice bath. Subsequently, the organic phase was mixed into the aqueous phase and kept under constant stirring at 2700 rpm Polymers 2020, 12, 2868 5 of 18 for 20 min in a mechanical disperser (IKA T25 digital ULTRA TURRAX®, Wilmington, NC, USA). Then the mixture was homogenized again through the ultrasonic disperser for 4 min at 70% amplitude in an ice bath. Finally, the mixture was added to a jacketed reactor at 85 ◦C and maintained by constant stirring at 400 rpm for 2 h.

2.4. Production of Poly(Vinyl Pivalate) Shell via Seeded Emulsion Polymerization Core–shell morphology nanoparticles (PMMA/OM-OA/PVPi) were obtained using the seeded emulsion method [44], where PMMA/OM-OA nanocomposite seeds were fed with vinyl pivalate (VPi), initiator (KPS), and cross-linking agent (EGDMA) for shell formation. In a 500 mL tritubulated ﬂask was added 200 g of PMMA/OM-OA colloidal dispersion (N.B. mass fraction of PMMA/OM-OA nanoparticles equal to 13.6%). The main components of the seeded emulsion polymerization step were initially separated into two phases, added separately into the PMMA/OM-AO colloidal dispersion: (i) The organic phase consisting of approximately 5.155 g VPi and 0.103 g EGDMA and (ii) the aqueous phase 20.5 g distilled water, 0.12 g SDS and 0.103 g KPS. The PMMA/OM-OA colloidal dispersion was kept under constant agitation at 500 rpm at 70 ◦C. Organic phase feeding was performed using a Solenoid Metering pump (ProMinent®, Gala series gamma/L 1000, São Paulo, Brazil) with a rate of 1 2.56 g h− . After 1h of feeding the organic phase, the aqueous phase was slowly added for about 1 h. Then the remainder of the organic phase was added at the same rate. Subsequently, the polymerization was maintained for a further 2 h at 70 ◦C in inert nitrogen atmosphere.

2.5. Sample Characterization X-ray diffraction measurements were used to identify the crystallographic phases present in the dimensions and the average size of the crystals. The diffractograms were acquired using a Rigaku-D/MAX X-Ray diffractometer (Austin, TX, USA), with CuKα (λ = 1.542510 Å) as a radiation source and a Ni-filtrate with CBO monochromator operating at 35 kV and 15 mA. The measurements performed in the variation range of 25◦ 2θ 80◦ with an increment of 0.05◦ and angular velocity of 1 ≤ ≤ 1.0◦ min− . The magnetization measurements were obtained using a vibrating sample magnetometer (VSM, MicroSense, Lowell, MA, USA), ADE Magnetics model EV-9, with a magnetic field from 18 to +18 (kOe), at room temperature. − Energy dispersive X-ray spectroscopy (EDX) analyses measurements were carried out on an EDX-720 fluorescence spectrometer (Shimadzu Europa GmbH, Duisburg, Germany). EDX spectra were acquired under vacuum with a 10 mm collimator at accelerating voltage of 50 keV with steps of 0.02 and a live time of 100 s, using the Ti-U channel. Fourier transform infrared spectra (FTIR) were displayed by dispersing bottles in KBr and presented as pellets. The spectra were acquired on a Varian Model 640 spectrometer (Varian Instruments, 1 Santa Clara, CA, USA) with a resolution of 4 cm− , updating 16 scans per sample. The average diameter of monomer droplets (Dg) and polymeric particles (Dp) were determined using the dynamic light scattering technique (DLS) using a Malvern model Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK). For the analyzes, the samples were diluted with distilled water. Transmission electron micrographs (TEM) were obtained using a JEOL JEM-2100 (Jeol Ltd., Tokyo, Japan) microscope operated at 200 kV. The samples were dispersed in water and sonicated for 5 min, where a drop of the suspension was applied on a copper screen containing a 200 mesh supported carbon film and dried at room temperature. The differential scanning calorimetry (DSC) curves were obtained using a Shimadzu equipment (model DSC-60, Shimadzu Scientific Instruments, Columbia, MD, USA). The initial masses used were approximately 5.0 mg and aluminum crucibles were used. For analysis, two heating ramps were performed, using a temperature range 1 -1 of 30 to 180 C, with a temperature ramp of 10 C min , He flow of 30 mL min and cooling was − ◦ ◦ · − · performed using liquid N2. The second heating ramp was used to determine the glass transition temperature (Tg) of the nanocomposites. Polymers 2020, 12, 2868 6 of 18

The conversions were determined by gravimetry, and the latexes were added in previously weighed aluminum foil capsules, treated with 2 g L 1 hydroquinone solution and dried in an oven at · − 60 ◦C for approximately 48 h. The conversion was obtained by the ratio of the dry polymer mass to the monomer mass used.

2.6. In Vitro Cytotoxicity Assays The polymer materials were evaluated through in vitro cytotoxicity tests with murine melanoma (B16F10) and human keratinocyte (HaCat) tumor lines provided by Cell Bank of Rio de Janeiro, Brazil and maintained in culture at Nanobiotechnology Laboratory of the University of Brasília, UnB. All cell lines were cultured in DMEM (Dulbecco’s Modified Eagle Medium, provided by Sigma-Aldrich Brasil, São Paulo, Brazil) medium buffered with sodium bicarbonate and supplemented with 1% antibiotic (100 IU mL 1 penicillin and 100 µg mL 1 streptomycin) and 10% fetal bovine serum (FBS) at pH 7.2. · − · − Cells were maintained in cell culture flasks into an incubator at 37 ◦C, under 95% humidified air and 5% CO2 atmosphere, and the assays were performed in the logarithmic growth phase of the cells. Viable cells were quantified by using 40 µL of solution of Trypan Blue dye added in 10 microliters of cell suspension. The samples were evaluated for 24 h, with concentrations equal to 0.01 mg mL 1, · − 0.05 mg mL 1, 0.1 mg mL 1, 0.25 mg mL 1, and 0.5 mg mL 1. The tumor cell lines (B16F10 and HaCat) · − · − · − · − were seeded in 96-well plates at a concentration of 2 103 and 5 103 cells per well with DMEM medium. · · Cell viability was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-reducing colorimetric method (MTT) through spectrophotometry measurements using a 595 nm wavelength, whose results correspond to an average of three independent experiments in triplicates.

3. Results and Discussion Figure1 shows the powder X-ray di ffractograms and their diffraction patterns of OM and OM-OA samples. All peaks were analyzed and compared using data from the Joint Committee on Powder Diffraction Standards (JCPDS). The characteristic peaks of magnetic particles were observed at 2θ = 30.2◦, 35.6◦, 43.3◦, 53.6◦, 57.3◦, 62.8◦, and 74.5◦, respectively, corresponding to the reflections of the crystalline planes (220), (311), (400), (422), (511), (440), and (533) of typical magnetite and/or maghemite spinel type cubic crystal structure. The diffraction patterns of the samples were similar, showing that the presence of oleic acid did not influence the structural and crystalline properties of nanoparticles [45]. The identification of the magnetite (Fe3O4) and maghemite (γ-Fe2O3) phases by XRD is quite complex, as both have the same spinel-like cubic crystal structure and therefore exhibit similar diffraction patterns. One way to determine the predominant crystalline phase is to use the peak position corresponding to the crystalline plane (440), with a value of 62.5◦ for the magnetite phase and 62.9◦ for maghemite [46]. The samples synthesized here have this peak located at 62.8◦, indicating the presence of both phases. The mean crystallite size (DDRX) was calculated from the width at half height of the highest intensity peak (FWHM) from the Scherrer equation (Equation (1)).

0.9λ D = (1) DRX β cos θ where: 0.9 is the proportionality constant for the spherical shape of the particles; λ is the wavelength of radiation CuKα (1.54056 Å); β is the half-height peak width corrected by the standard sample; θ is the Bragg diﬀraction angle of the most intense peak relative to the reﬂection of the crystalline plane (311). Polymers 2020, 12, 2868 7 of 18

Polymers 2020, 12, x FOR PEER REVIEW 7 of 17

Figure 1. FigureX-ray 1. di X-rayﬀractograms diffractograms of samplesof samples ofof magneticmagnetic oxide oxide (OM) (OM) and oleic and acid oleic functionalized acid functionalized magnetic oxide (OM-OA). magnetic oxide (OM-OA). Figure 2 shows the magnetization curve of OM and OM-OA samples as a function of the applied The averagefield at room crystallite temperature. sizes The obtained saturation weremagnet 10.5ization nm (Ms) (OM) of the and OM12.2 and OM-OA nm (OM-OA), samples were this increase was also observed63.7 and 57.4 in emu·g other−1, studiesrespectively. [47 The–49 ].OM-OA These sample values shows are a importantlower Ms value from compared the point to the of view of applicationOM in sample, intravascular a reduction embolization, of approximately since 10.4%. the This superparamagnetic decrease can be explained character by the fact is essentialthat the for this value of this measurement corresponds to the sum of all magnetic moments present in the sample, in medical procedure and is characteristic of particles with an average size smaller than the critical size, this case, the magnetic material (iron oxides) and the non-magnetic coating material (oleic acid). Thus, generally agreed to be less than 20 nm [50]. this result shows that the non-magnetic coating layer present in the OM-OA sample has a ratio of Figureapproximately2 shows the 10%. magnetization In addition, the curve nanoparticles of OM andshowed OM-OA superparamagnetic samples as behavior, a function since of the the applied Figure 1. X-ray diffractograms of samples of magnetic oxide (OM) and oleic acid functionalized coercivity and magnetization values of remnant are close to zero [51], corroborating the XRD results. ﬁeld at room temperature.magnetic oxide (OM-OA). The saturation magnetization (Ms) of the OM and OM-OA samples were Thus, besides the fact that the Fe3O4 nanoparticles become hydrophobic, the chemical 63.7 and 57.4 emu g 1, respectively. The OM-OA sample shows a lower Ms value compared to the functionalization· − of the magnetic oxide surfaces with oleic acid preserved the magnetic properties. OM sample, aFigure reduction 2 shows of the approximately magnetization curve 10.4%. of OM This and decreaseOM-OA samples can be as a explained function of the by applied the fact that the field at room temperature. The saturation magnetization (Ms) of the OM and OM-OA samples were value of this63.7 measurementand 57.4 emu·g−1, corresponds respectively. The to OM-OA the sum sample of all shows magnetic a lower moments Ms value compared present to in the the sample, in this case,OM the sample, magnetic a reduction material of approximately (iron oxides) 10.4%. and This the decrease non-magnetic can be explained coating by the material fact that the (oleic acid). Thus, thisvalue result of showsthis measurement that the non-magneticcorresponds to the coating sum of all layer magnetic present moments in the present OM-OA in the sample, sample in has a ratio of approximatelythis case, the 10%. magnetic In addition, material (iron the nanoparticlesoxides) and the non-magnetic showed superparamagnetic coating material (oleic acid). behavior, Thus, since the this result shows that the non-magnetic coating layer present in the OM-OA sample has a ratio of coercivityapproximately and magnetization 10%. In addition, values ofthe remnant nanoparticles are showed close to superparamagnetic zero [51], corroborating behavior, since the the XRD results. Thus, besidescoercivity the fact and that magnetization the Fe3O values4 nanoparticles of remnant are become close to hydrophobic, zero [51], corroborating the chemical the XRD functionalization results. of the magneticThus, oxidebesides surfacesthe fact withthat oleicthe Fe acid3O4 preservednanoparticles the become magnetic hydrophobic, properties. the chemical functionalization of the magnetic oxide surfaces with oleic acid preserved the magnetic properties.

Figure 2. Magnetization curves as a function of the magnetic field applied at room temperature of samples OM and OM-OA.

Figure 2. MagnetizationFigure 2. Magnetization curves curves as aas function a function ofof the ma magneticgnetic field ﬁeld applied applied at room at temperature room temperature of of samples OM and OM-OA. samples OM and OM-OA.

Polymers 2020, 12, 2868 8 of 18

Polymers 2020, 12, x FOR PEER REVIEW 8 of 17 PMMA/OM-OA nanocomposites were obtained by in situ incorporation of OM-OA nanoparticles into poly(methylPMMA/OM-OA methacrylate) nanocomposites matrices bywere miniemulsion obtained by technique. in situ incorporation This polymerization of OM-OA process favorsnanoparticles the formation into of highpoly(methyl molar massmethacrylate) lattices, narrowmatrices particle by miniemulsion size distributions, technique. high colloidalThis stabilitypolymerization and proper process encapsulation favors the of inorganicformation nanoparticlesof high molar [ 27mass]. All lattices, miniemulsions narrow particle were preparedsize with 15%distributions, monomer high phase colloidal (MMA) stability and 15% and by proper weight encapsulation OM-OA relative of inorganic to monomer nanoparticles content. [27]. In addition, All 3% byminiemulsions weight hexadecane were prepared and 2% with initiator 15% monomer (BPO), bothphase relative (MMA) toand monomer 15% by weight mass. OM-OA relative to monomer content. In addition, 3% by weight hexadecane and 2% initiator (BPO), both relative to Figure3 shows the micrographs from TEM measurements and histogram of sample size distribution monomer mass. (A) PMMA and (B) PMMA/PVPi. The particles showed spherical morphology and uniform size, where Figure 3 shows the micrographs from TEM measurements and histogram of sample size 326 and 307 particles were analyzed and found average sizes of 70 10 nm and 85 9 nm for PMMA distribution (A) PMMA and (B) PMMA/PVPi. The particles showed± spherical morphology± and and PMMAuniform/PVPi size, respectively.where 326 and Figure 307 particles3 clearly were shows analyzed that aand core-structure found average type sizes structure of 70 ± 10 was nm properlyand obtained85 ± through9 nm for PMMA seeded and emulsion PMMA/PVPi polymerization respectively. by Figures using 3 precursor clearly shows PMMA that nanoparticlesa core-structure from miniemulsiontype structure prolymerization was properly obtained process. through According seeded to emulsion average polymerization particle size, theby using fraction precursor of PVPi in the outerPMMA shell nanoparticles of polymer from particles miniemulsion is expected prolymerization to be in the intervalprocess. fromAccording approximately to average 14.3particle wt.% to 19.0 wt.%,size, the which fraction agrees of PVPi very in wellthe outer with shell the of theoretical polymer particles PVPi mass is expected fraction to equalbe in the to interval 17.7% forfrom a total consumptionapproximately of the 14.3 vinyl wt.% pivalate. to 19.0 wt.%, which agrees very well with the theoretical PVPi mass fraction equal to 17.7% for a total consumption of the vinyl pivalate.

0.25 PMMA (A)

0.20

0.15

0.10

0.05 Relative Frequency(a.u.)

0.00 40 50 60 70 80 90 100 110 Diameter (nm)

0.25 PMMA/PPVi (B)

0.20

0.15

0.10

0.05 Relative Frequency(a.u.)

0.00 50 60 70 80 90 100 110 120 Diameter (nm) Figure 3. Micrographs from TEM measurements and histogram of particle size distribution (A) Figure 3. Micrographs from TEM measurements and histogram of particle size distribution (A) PMMA PMMA homopolymer and (B) PMMA/PVPi core–shell nanoparticles. homopolymer and (B) PMMA/PVPi core–shell nanoparticles. In the particular case of magnetic core–shell particles (PMMA/OM-OA and PMMA/OM- InOA/PVPi), the particular high conversion case of magnetic was achieved, core–shell around particles 89%, after (PMMA 2 h of/OM-OA polymerization. and PMMA Comparatively,/OM-OA/PVPi), high conversionusing DLS analysis, was achieved, the polymer around particle 89%, after diameter 2 h of polymerization.of the core structure Comparatively, obtained through using DLS analysis,miniemulsion the polymer polymeriza particletion diameterremained ofvirtually the core unchanged, structure exhibiting obtained 190 through nm for miniemulsionmonomer polymerizationdroplets (Dg, remainedwith PdI = 0.229) virtually and 217 unchanged, nm polymer particles exhibiting (Dp, 190with nmPdI = for0.254), monomer indicating dropletsthat (Dg, withthe nucleation PdI = 0.229) mechanism and217 preferably nm polymer occurs within particles the monomer (Dp, with droplets PdI = and0.254), that indicatingthe coalescence that the nucleationand Ostwald mechanism ripening preferably effects were occurs minimized within during the monomer polymerization droplets (N.B. and PdI that values the around coalescence 0.248 and Ostwaldindicate ripening that enanoparticlesﬀects were minimized with relatively during narrow polymerization distribution (N.B. size PdIare valuesobtained). around The 0.248 average indicate that nanoparticles with relatively narrow distribution size are obtained). The average particles size Polymers 2020, 12, 2868 9 of 18

Polymers 2020, 12, x FOR PEER REVIEW 9 of 17 from DLS measurements reveals an increase in the particles diameter as a result of the incorporation of particles size from DLS measurements reveals an increase in the particles diameter as a result of the Fe3O4 magnetic nanoparticles. incorporation of Fe3O4 magnetic nanoparticles. Figure4 shows the TEM of the PMMA /OM-OA and PMMA/OM-OA/PVPi samples, in which it is Figure 4 shows the TEM of the PMMA/OM-OA and PMMA/OM-OA/PVPi samples, in which it possible to observe the presence of magnetic nanoparticles uniformly dispersed in spherical polymer is possible to observe the presence of magnetic nanoparticles uniformly dispersed in spherical nanoparticles, containing an outer layer acquiring a structure of the type core–shell. Figure4D shows polymer nanoparticles, containing an outer layer acquiring a structure of the type core–shell. Figure a darker part (core) and a lighter part (shell), showing the formation of a particle with core–shell 4D shows a darker part (core) and a lighter part (shell), showing the formation of a particle with core– morphology. Since these are polymers with a close refractive index, the TEM cannot clearly distinguish shell morphology. Since these are polymers with a close refractive index, the TEM cannot clearly the shell and core layers. Considering the efficiency of formation of the shell structure and an average distinguish the shell and core layers. Considering the efficiency of formation of the shell structure number of PMMA/OM-OA equal to 4.37 1015 particles in the final latex, it is expected to form the final and an average number of PMMA/OM-OA· equal to 4.37·1015 particles in the final latex, it is expected magnetic core–shell particles with an average diameter of approximately 232 nm. to form the final magnetic core–shell particles with an average diameter of approximately 232 nm.

Figure 4.4. (A(A,B) )and Micrographs (B) Micrographs captured bycaptured TEM of by the TEM PMMA of/ OM-OAthe PMMA/OM-OA and (C,D) micrographs and (C) recordedand (D) micrographsby TEM of the core–shellrecorded poly(methylby TEM methacrylate)of the core–she nanoparticlesll poly(methyl (PMMA) methacrylate)/OM-OA/PVPi nanoparticles.nanoparticles (PMMA)/OM-OA/PVPi nanoparticles. In order to demonstrate that the nanoparticles observed are mainly composed of iron, EDX analysis was performed,In order to being demonstrate possible tothat observe the nanoparticle the dominants observed Kα and K areβ peaks mainly related composed to iron inof theiron, interval EDX analysisfrom 6.40 was Kev performed, to 7.06 Kev being (Figure poss5).ible The to fraction observe of the Fe dominant observed K inα the and samples Kβ peaks was related approximately to iron in the97% interval along with from small 6.40 amountsKev to 7.06 of impurities Kev (Figure associated 5). The withfraction Si, Ca,of Fe S, Zn,observed and Mn in fromthe samples the reagents was approximatelyused in the syntheses. 97% along The resultswith small observed amounts in Figure of impurities5 are in agreement associated with with the Si, experimental Ca, S, Zn, and results Mn fromdescribed the reagents elsewhere used [17 in,18 the]. syntheses. The results observed in Figure 5 are in agreement with the experimental results described elsewhere [17,18].

100 56 (A) (B) FeK α FeK 48 α 80 40

60 32

24 40

16 Intensity (cps/uA) Intensity Intensity (cps/uA) Intensity 20 8 FeKβ FeKβ

0 0 03691215 03691215 Energy (keV) Energy (keV)

Figure 5. Energy dispersive X-ray spectra of the nanoparticles. (A) PMMA/OM-OA; (B) PMMA/OM- OA/PVPi.

Polymers 2020, 12, x FOR PEER REVIEW 9 of 17 particles size from DLS measurements reveals an increase in the particles diameter as a result of the incorporation of Fe3O4 magnetic nanoparticles. Figure 4 shows the TEM of the PMMA/OM-OA and PMMA/OM-OA/PVPi samples, in which it is possible to observe the presence of magnetic nanoparticles uniformly dispersed in spherical polymer nanoparticles, containing an outer layer acquiring a structure of the type core–shell. Figure 4D shows a darker part (core) and a lighter part (shell), showing the formation of a particle with core– shell morphology. Since these are polymers with a close refractive index, the TEM cannot clearly distinguish the shell and core layers. Considering the efficiency of formation of the shell structure and an average number of PMMA/OM-OA equal to 4.37·1015 particles in the final latex, it is expected to form the final magnetic core–shell particles with an average diameter of approximately 232 nm.

Figure 4. (A) and (B) Micrographs captured by TEM of the PMMA/OM-OA and (C) and (D) micrographs recorded by TEM of the core–shell poly(methyl methacrylate) nanoparticles (PMMA)/OM-OA/PVPi nanoparticles.

In order to demonstrate that the nanoparticles observed are mainly composed of iron, EDX analysis was performed, being possible to observe the dominant Kα and Kβ peaks related to iron in the interval from 6.40 Kev to 7.06 Kev (Figure 5). The fraction of Fe observed in the samples was approximately 97% along with small amounts of impurities associated with Si, Ca, S, Zn, and Mn Polymersfrom the2020 reagents, 12, 2868 used in the syntheses. The results observed in Figure 5 are in agreement with10 ofthe 18 experimental results described elsewhere [17,18].

100 56 (A) (B) FeK α FeK 48 α 80 40

60 32

24 40

16 Intensity (cps/uA) Intensity Intensity (cps/uA) Intensity 20 8 FeKβ FeKβ

0 0 03691215 03691215 Energy (keV) Energy (keV)

Figure 5. 5. EnergyEnergy dispersive dispersive X-ray X-rayspectra spectra of the nanoparticles. of the nanoparticles. (A) PMMA/OM-OA; (A) PMMA (B)/ OM-OA;PMMA/OM- (B) OA/PVPi.PMMA/OM-OA /PVPi. Polymers 2020, 12, x FOR PEER REVIEW 10 of 17 Figure6 shows the FTIR spectrum of PMMA /OM-OA and PMMA/OM-OA/PVPi samples. The mainFigure absorption 6 shows the bands FTIR and spectrum their respective of PMMA vibrational/OM-OA and assignments PMMA/OM-OA/PVPi are presented samples. in Table The1. 1 Themain absorption absorption bands bands between and their 2995 respective and 2852 cmvibrational− are characteristic assignments of asymmetricalare presented and in Table symmetrical 1. The 3 1 stretchingabsorption C-H bands sp between. The peak 2995 at 1731and 2852 cm− cmis− attributed1 are characteristic to the carbonyl of asymmetrical group stretch and andsymmetrical the 1272 1 andstretching 1149 cm C-H− bandssp3. The to peak the C-O at 1731 stretch cm−1 corresponding is attributed to to the the carbonyl ester group group present stretch in and the the structure 1272 and of 1 MMA, VPi,−1 and EGDMA. Absorption at 1381 and 1365 cm are attributed to the CH symmetrical 1149 cm bands to the C-O stretch corresponding to the ester− group present in the −structure3 of MMA, angularVPi, and deformation EGDMA. Absorption of PVi tert-butyl at 1381 groups and 1365 [52]. cm The−1 are obtaining attributed of the to polymeric the −CH3 materialsymmetrical is conﬁrmed angular 1 bydeformation the 1446 cmof PVi− band tert-butyl referring groups to the[52]. angular The obtaining deformation of the ofpolymeric the methylene material group is confirmed ( CH2) by of 1 − thethe polymeric1446 cm−1 chainband andreferring the absence to the ofangular absorption deformation bands between of the methylene 3100 and 3000group cm (−−CHand2) bandsof the 1 2 betweenpolymeric 990 chain and and 910 the cm− absenceattributed, of absorption respectively, bands to between the stretching 3100 and 3000 angular cm-1 deformation and bands between CH sp of990 vinyl and groups910 cm-1 [ 53attributed,]. In the spectra,respectively, the presence to the stretching of magnetic and oxidesangular was deformation not conﬁrmed CH sp due2 of to vinyl the 1 absencegroups [53]. of absorption In the spectra, in the the 578 presence cm− region of magnet attributedic oxides to was Fe-O not stretching confirmed of tetrahedraldue to the absence sites [54 of]. Nevertheless,absorption in the approaching 578 cm−1 region a magnet attributed to the to latex Fe-O shows stretching that theof tetrahedral nanoparticles sites are [54]. attracted Nevertheless, to the containerapproaching wall. a magnet These resultsto the latex indicate shows that that OM-OA the nanoparticles are dispersed are in attracted the polymer to the matrix container in a smallwall. amountThese results [55]. indicate that OM-OA are dispersed in the polymer matrix in a small amount [55].

PMMA - OM-OA

PMMA/OM-OA - PVPi

2852 985 2995 1489 Transmmitance(%) 1446 2949 1365 1272

1149 1381 1731

3500 3000 2000 1500 1000 500 Wavenumbers (cm-1)

Figure 6.6. FTIRFTIR spectra spectra of of PMMA PMMA/OM-OA/OM-OA and and PMMA PMMA/OM-OA/PVPi/OM-OA/PVPi samples samples obtained obtained from KBr from pellets. KBr pellets.

Table 1. Main absorption bands obtained in FTIR spectra for PMMA/OM-OA and PMMA/OM- OA/PVPi samples and their vibrational assignments.

Wavenumbers (cm−1) Vibrational Assignments 2995–2852 Asymmetrical and symmetrical stretch C-H sp3 1731 Stretch C=O of ester 1489 and 1446 Angular deformation of methylene groups (–CH2) 1381 and 1365 Symmetrical angular deformation –CH3 of tert-butyl groups 1272 and 1149 Stretch C-O of ester 985 Out of plane angular deformation C-H

The glass transition temperatures (Tg) of PMMA/OM-OA and PMMA/OM-OA/PVPi samples were determined by the DSC curves shown in Figure 7. The Tg reached values greater than 110 °C, which are higher than those reported in the literature, around 104 °C [56]. This increase is attributed to the presence of OM-OA nanoparticles, restricting the mobility of the polymer chains [57] or due to a change in the tacticity of the PMMA chains [58], with major interactions between polymer chains and, consequently, increasing the Tg values. The Tg values of the PMMA/OM-OA nanocomposites

Polymers 2020, 12, 2868 11 of 18

Table 1. Main absorption bands obtained in FTIR spectra for PMMA/OM-OA and PMMA/OM-OA/PVPi samples and their vibrational assignments.

1 Wavenumbers (cm− ) Vibrational Assignments Polymers 2020, 12, x FOR PEER2995–2852 REVIEW Asymmetrical and symmetrical stretch C-H sp3 11 of 17 1731 Stretch C=O of ester were 108 °C, while 1489those and of 1446the PMMA/OM-OA/PVPi Angular deformation were of 113 methylene °C. It is groups also worth (–CH2 mentioning) that Symmetrical angular deformation –CH of the increase in Tg observed1381 and here 1365 for the polymer resulted from seed emulsion process3 is closely related tert-butyl groups to the EGDMA used as cross-linking agent. 1272 and 1149 Stretch C-O of ester ℑ The mass fraction of985 vinyl pivalate ( VPi Out) estimated of plane angular based deformationon the Fox C-Hmodel (Equation (2)) is equal to 0.16, indicating that vinyl pivalate was successfully incorporated onto the surface of the precursors PMMA particles, which explains the dominant effect of the composition on the Tg of the The glass transition temperatures (Tg) of PMMA/OM-OA and PMMA/OM-OA/PVPi samples copolymers. It is important to emphasize that the DSC measurements corroborate the vinyl pivalate were determined by the DSC curves shown in Figure7. The Tg reached values greater than 110 ◦C, mass fraction estimated from TEM measurements, which was determined to be in the interval from which are higher than those reported in the literature, around 104 ◦C[56]. This increase is attributed to approximately 0.14 to 0.19. the presence of OM-OA nanoparticles, restricting the mobility of the polymer chains [57] or due to a change in the tacticity of the PMMA chains1 [581−], withℑ majorℑ interactions between polymer chains = + (2) and, consequently, increasing the Tg values. The Tgvalues of the PMMA/OM-OA nanocomposites were 108 ◦C, while those of the PMMA/OM-OA/PVPi were 113 ◦C. It is also worth mentioning that the where, , and are the glass transition temperatures of the PMMA/PVPi, and in the literature increase in Tg observed here for the polymer resulted from seed emulsion process is closely related to of poly(methyl methacrylate) with Tg = 110 °C and poly(vinyl pivalate) with Tg = 80 °C, respectively the EGDMA used as cross-linking agent. [59,60].

PMMA/PVPi

Tg = 105 °C

PMMA/OM-OA

Tg = 108 °C

PMMA/OM-OA - PVPi

Tg = 113 °C ENDOTHERMIC Heat Flow (a.u.) 80 90 100 110 120 130 140

Temperature (°C) FigureFigure 7. DSC 7. DSC curves curves of the of PMMAthe PMMA/OM-OA/OM-OA and and PMMA PMMA/OM-OA/poly(vinyl/OM-OA/poly(vinyl pivalate) pivalate) (PVPi) (PVPi) samples. samples. The mass fraction of vinyl pivalate ( ) estimated based on the Fox model (Equation (2)) is equal =VPi to 0.16,The indicating magnetic that behavior vinyl pivalate of the wasPMMA/OM-OA successfully incorporatedand PMMA/OM-OA/PVPi onto the surface samples of the precursors at room temperaturePMMA particles, is shown which in explainsFigure 8. theThe dominant samples show eﬀected of saturation the composition magnetization on the T g(Ms)of the of 4.65 copolymers. emu·g−1 (MsIt is important= 57.4 emu·g to− emphasize1 of nanoparticles) that the DSCand 1.35 measurements emu·g−1 (56.25 corroborate emu·g−1 theof nanoparticles), vinyl pivalate massrespectively. fraction Theestimated magnetization from TEM curves measurements, have no hy whichsteresis was cycle determined and are reversible to be in theat room interval temperature, from approximately indicating that0.14 the to 0.19. superparamagnetic behavior of the nanoparticles was preserved during polymerization and 1 (1 VPi) their magnetic properties were successfully= incorporated− = + into=VPi the nanocomposites. (2) T T T The Ms values of the nanocompositesg12 were significantlyg1 g2 lower than that of OM-OA (Ms = 57.4 emu·g−1), due to the existence of non-magnetic polymeric material present in the samples. Considering these Ms values, it is possible to have an estimate of the OM and OM-OA content incorporated in each nanocomposite, being, respectively, 7.3% and 8.1% for PMMA/OM-OA and 2.1% and 2.4% for PMMA/OM-OA/PVPi. In fact, these values are slightly higher, since during polymerization part of the magnetic oxides, such as Fe3O4, are converted into other iron oxides that have less magnetization, such as γ-Fe2O3, due to the presence of strong oxidants, such as BPO [17,47].

Polymers 2020, 12, 2868 12 of 18

where, Tg12 , Tg1 and Tg2 are the glass transition temperatures of the PMMA/PVPi, and in the literature of poly(methyl methacrylate) with Tg = 110 ◦C and poly(vinyl pivalate) with Tg = 80 ◦C, respectively [59,60]. The magnetic behavior of the PMMA/OM-OA and PMMA/OM-OA/PVPi samples at room temperature is shown in Figure8. The samples showed saturation magnetization (Ms) of 4.65 emu g 1 (Ms = 57.4 emu g 1 of nanoparticles) and 1.35 emu g 1 (56.25 emu g 1 of nanoparticles), · − · − · − · − Polymersrespectively. 2020, 12, xThe FOR PEER magnetization REVIEW curves have no hysteresis cycle and are reversible at12 room of 17 temperature, indicating that the superparamagnetic behavior of the nanoparticles was preserved during These Ms values corroborate the results of FTIR and DSC in which they show the presence of the poly polymerization and their magnetic properties were successfully incorporated into the nanocomposites. (vinyl pivalate) matrix present in the nanocomposites obtained by seeded emulsion.

Figure 8. 8. MagnetizationMagnetization curves curves as as a a function function of of the the magn magneticetic field ﬁeld applied applied to to the the ambient ambient temperature temperature of the PMMA/OM-OA PMMA/OM-OA and PMMA/OM-OA/PVPi PMMA/OM-OA/PVPi samples.

FiguresThe Ms 9 and values 10 depict of the cytotoxicity nanocomposites results of were in vitro signiﬁcantly tests performed lower with than polymeric that of particles. OM-OA 1 This(Ms kind= 57.4 of emubiologicalg− ), test due is toof fundamental the existence importance, of non-magnetic as it is polymericclosely related material to product present viability, in the · accordingsamples. Consideringto its level of these toxicity. Ms values,Considering it is possible that both to havehuman an and estimate animal of thecells OM were and used, OM-OA and knowingcontent incorporated that lower percentage in each nanocomposite, of cell viability, being, the greater respectively, its cytotoxic 7.3% and potential, 8.1% for it PMMA is necessary/OM-OA to evaluateand 2.1% influence and 2.4% of for material PMMA /concentrationOM-OA/PVPi. on In ce fact,ll viability these values in order are slightlyto determine higher, its since borderline during concentration.polymerization part of the magnetic oxides, such as Fe3O4, are converted into other iron oxides that haveFigure less magnetization, 9 illustrates performance such as γ-Fe 2ofO tested3, due topoly themeric presence materials of strong against oxidants, murine such melanoma as BPO [tumor17,47]. lineThese (B16F10) Ms values in cell corroborate viability at the an results exposure of FTIR of 24 and h when DSC incompared which they to the show control the presence (p < 0.05). of For the each poly polymeric(vinyl pivalate) material, matrix assays present were in carried the nanocomposites out with polymer obtained concentrations by seeded emulsion.lying in interval from 0.01 mg·mLFigures−1 to 90.50 and mg·mL 10 depict−1. cytotoxicityAccording to results Figure of 9,in vitroconcentrationtests performed of polymer with polymericsamples plays particles. an importantThis kind ofeffect biological on cell viability. test is of fundamentalIt is observed importance,that cell viability as it is is slightly closely affected related to when product low polymer viability, concentrationsaccording to its levelare employed of toxicity. (i.e., Considering from 0.01 that mg·mL both human−1 to 0.10 and mg·mL animal− cells1), leading were used, to aand decrease knowing in averagethat lower cell percentage viability of of at cell most viability, 18%. In the general greater for its other cytotoxic polymer potential, materials, it is it necessary seems reasonable to evaluate to assumeinﬂuence that of materialborderline concentration concentration on corresponds cell viability to in concentration order to determine of 0.25 its mg·mL borderline−1, which concentration. leads to a decrease in cell viability of approximately 45%. It is also important to bear in mind that best results were observed for core–shell magnetic polymer materials (PMMA/OM-OA and PMMA/OM- OA/PVPi), presenting a cell viability comparable to control, at concentrations of 0.01 to 0.25 mg·mL−1 and from 0.01 and 0.05 mg·mL−1, respectively. This result is very promising, considering that as an embolic agent this versatile material present a twofold action, as they can be used to both obstruct blood vessels interrupting supply of oxygen and nutrients to tumor region and hyperthermia treatment of injured area.

Polymers 2020, 12, x FOR PEER REVIEW 13 of 17

Figure 9. Evaluation of cytotoxic activity of poly(methyl methacrylate)/magnetic oxide coated with oleic acid/poly(vinyl pivalate)—(PMMA/OM-OA/PVPi), poly(methyl methacrylate)/poly(vinyl pivalate)—(PMMA/PVPi) and poly(methyl methacrylate)/magnetic oxide coated with oleic acid— Polymers 2020, 12, 2868 13 of 18 Polymers(PMMA/OM-OA) 2020, 12, x FOR PEER in REVIEWmurine melanoma tumor cell lines, B16F10, after 24h exposure in the13 of 17 concentrations of 0.01 mg·mL−1, 0.05 mg·mL−1, 0.10 mg·mL−1, 0.25 mg·mL−1, and 0.50 mg·mL−1. The asterisks are related to the statistical significance associated with different confidence levels: for 95% with p ≤ 0.05, * 99% with p ≤ 0.01, ** 99.9% with p ≤ 0.001 and *** 99.99% with p ≤ 0.0001.

A similar study has been carried out for HaCaT lineage in order to evaluate the polymer materials toxicity to non-pathological cells. According to Figure 10, target magnetic polymer with core–shell structure (PMMA/OM-OA/PVPi) exhibits highest cell viability in comparison to the other tested polymer materials, and whose values of cell viability can be considered comparable and very close to control, except for concentration of 0.01 mg·mL−1. It is also interesting to notice that PMMA/OM-OA/PVPi performance seems not to be dependent on concentration in experimental range evaluated here. In particular case of poly(methyl methacrylate) magnetic particles −1 (PMMA/OM-OA), high cell viability were observed for concentration of 0.25 mg·mL and 0.50 mg·mLFigure-1 equivalent 10. Evaluation to 108% of and cytotoxic 93%, respectively. activity of poly(methyl methacrylate)/magnetic oxide coated Figure 10. Evaluation of cytotoxic activity of poly(methyl methacrylate)/magnetic oxide coated with withNon-magnetic oleic acid /polycore–shell (vinyl nanoparticles pivalate)—(PMMA (PMMA/PVPi)/OM-OA/PVPi), presented poly(methyl an average methacrylate) cell viability/poly of oleic acid/poly (vinyl pivalate)—(PMMA/OM-OA/PVPi), poly(methyl methacrylate)/poly (vinyl 84%;(vinyl however, pivalate)—(PMMA the highest cell/PVPi) viability and poly(methyl very close methacrylate) control was/magnetic approximately oxide coated 99% withobserved oleic for pivalate)—(PMMA/PVPi) and poly(methyl methacrylate)/magnetic oxide coated with oleic acid— concentrationacid—(PMMA of 0.10/OM-OA) mg·mL in−1. human keratinocyte cells, HaCaT, after 24 h in the concentrations of (PMMA/OM-OA) in human keratinocyte cells, HaCaT, after 24 h in the concentrations of 0.01 0.01 mg mL 1, 0.05 mg mL 1, 0.10 mg mL 1, 0.25 mg mL 1, and 0.50 mg mL 1. The asterisks are mg·mL−1·, 0.05− mg·mL−1, 0.10· mg·mL− −1, 0.25· mg·mL− −1, and· 0.50− mg·mL−1. The asterisks· − are related to the related to the statistical significance associated with different confidence levels: * for 95% with p 0.05, statistical significance associated with different confidence levels: for 95% with p ≤ 0.05, * 99% with≤ p ** 99% with p 0.01, *** 99.9% with p 0.001 and **** 99.99% with p 0.0001. ≤ 0.01, ** 99.9%≤ with p ≤ 0.001 and *** ≤99.99% with p ≤ 0.0001. ≤ Figure9 illustrates performance of tested polymeric materials against murine melanoma tumor Feuser et al. [61] evaluated in vitro cytotoxicity in murine fibroblast (L929) and normal human line (B16F10) in cell viability at an exposure of 24 h when compared to the control (p < 0.05). For each fibroblast (NIH-3T3) by MTT assay for 24 h of PMMA nanoparticles and nanocapsules obtained by polymeric material, assays were carried out with polymer concentrations lying in interval from miniemulsion1 polymerization.1 Both cells presented high cell viability, between 81 to 84% even at 0.01 mg mL− to 0.50 mg mL− . According to Figure9, concentration of polymer samples plays an highest tested· concentration· of 750 μg·mL−1. Guindani et al. [62] also investigated in vitro cytotoxicity important effect on cell viability. It is observed that cell viability is slightly affected when low polymer in mouse embryonic fibroblasts (NIH3T3) breast cancer (MDAMB231) and human cervical cancer concentrations are employed (i.e., from 0.01 mg mL 1 to 0.10 mg mL 1), leading to a decrease in · − · − average cell viability of at most 18%. In general for other polymer materials, it seems reasonable to 1 assumeFigure that 10. borderline Evaluation concentration of cytotoxic activity corresponds of poly(met to concentrationhyl methacrylate)/magnetic of 0.25 mg mL oxide− , whichcoated leadswith to a · decreaseoleic inacid/poly cell viability (vinyl of pivalate)—(PMMA/OM-OA/P approximately 45%. It is alsoVPi), important poly(methyl to bearmethacrylate)/poly in mind that best(vinyl results werepivalate)—(PMMA/PVPi) observed for core–shell magneticand poly(methyl polymer methacrylate)/magnetic materials (PMMA/OM-OA oxide coated and PMMA with oleic/OM-OA acid—/PVPi), presenting(PMMA/OM-OA) a cell viability in human comparable keratinocy to control,te cells, atHaCaT, concentrations after 24 h of in 0.01 the toconcentrations 0.25 mg mL of1 and0.01 from · − 0.01 andmg·mL 0.05−1, 0.05 mg mg·mLmL 1,− respectively.1, 0.10 mg·mL−1, This 0.25 mg·mL result− is1, and very 0.50 promising, mg·mL−1. The considering asterisks are that related as an to embolicthe · − agentstatistical this versatile significance material associated present with a different twofold confidence action, as levels: they canfor 95% be usedwith p to ≤ 0.05, both * obstruct99% with p blood vessels≤ 0.01, interrupting ** 99.9% with supply p ≤ 0.001 of oxygen and *** 99.99% and nutrients with p ≤ to0.0001. tumor region and hyperthermia treatment of injured area. AFeuser similar et al. study [61] evaluated has been in carried vitro cytotoxicity out for HaCaT in murine lineage fibroblast in order (L929) to evaluate and normal the polymer human materialsfibroblast toxicity(NIH-3T3) to non-pathologicalby MTT assay forcells. 24 h of According PMMA nanoparticles to Figure 10, and target nanocapsules magnetic polymer obtained with by core–shellminiemulsion structure polymerization. (PMMA/OM-OA Both cells/PVPi) presented exhibits highesthigh cell cell viability, viability between in comparison 81 to 84% to the even other at highest tested concentration of 750 μg·mL−1. Guindani et al. [62] also investigated in vitro cytotoxicity in mouse embryonic fibroblasts (NIH3T3) breast cancer (MDAMB231) and human cervical cancer

Polymers 2020, 12, 2868 14 of 18 tested polymer materials, and whose values of cell viability can be considered comparable and very close to control, except for concentration of 0.01 mg mL 1. It is also interesting to notice that · − PMMA/OM-OA/PVPi performance seems not to be dependent on concentration in experimental range evaluated here. In particular case of poly(methyl methacrylate) magnetic particles (PMMA/OM-OA), high cell viability were observed for concentration of 0.25 mg mL 1 and 0.50 mg mL 1 equivalent to · − · − 108% and 93%, respectively. Non-magnetic core–shell nanoparticles (PMMA/PVPi) presented an average cell viability of 84%; however, the highest cell viability very close control was approximately 99% observed for concentration of 0.10 mg mL 1. · − Feuser et al. [61] evaluated in vitro cytotoxicity in murine fibroblast (L929) and normal human fibroblast (NIH-3T3) by MTT assay for 24 h of PMMA nanoparticles and nanocapsules obtained by miniemulsion polymerization. Both cells presented high cell viability, between 81 to 84% even at highest tested concentration of 750 µg mL 1. Guindani et al. [62] also investigated in vitro cytotoxicity · − in mouse embryonic fibroblasts (NIH3T3) breast cancer (MDAMB231) and human cervical cancer (HeLa) by MTT assay for 24 h of PMMA nanoparticles and nanocapsules of PMMA conjugated with bovine serum albumin (BSA) obtained by miniemulsion polymerization at four concentrations: 100, 200, 300, and 400 µg mL 1. According to authors, polymeric materials can be considered biocompatible · − in all concentrations tested. Mendes et al. [63] evaluated cytotoxic activity of PMMA nanoparticles obtained by miniemulsion polymerization in human leukemic cell line K562 by MTT assay for 24 h. Cytotoxic activity of polymeric nanoparticles did not induce cell death even at highest tested concentration of 1500 µg mL 1. · − According the Chinese standard (GB/T 16886.5-2003) materials with cell viability greater than 75% represent no cytotoxicity [64,65]. Therefore, based on the results obtained in this study, it is possible to consider that nanoparticles achieved high cell viability for both murine melanoma tumor cell lines, B16F10, and human keratinocyte cells, HaCaT., and can be considered promising for other biological studies such as in vivo tests.

4. Conclusions The results showed that functionalized magnetic nanoparticles presented good dispersibility with a uniform average size of 10.5 nm, saturation magnetization of 57.4 emu g 1 and superparamagnetic · − behavior. Miniemulsion polymerization process ensured that magnetic nanoparticles were encapsulated in polymer matrix uniformly, maintaining their superparamagnetic properties with 56.25 emu g 1 of · − Fe3O4 nanoparticles. Results obtained are promising, since from point of view of medical application of intravascular embolization. Superparamagnetic character, essential for this medical procedure, remains present in nanocomposite and in result of seeded emulsion, in addition to presence of core–shell morphology. This new polymeric material designed to contain, as a core–shell structure, poly(methyl methacrylate)/magnetic Fe3O4/poly(vinyl pivalate), exhibited high colloidal stability, which can be linked to the desired properties of methyl methacrylate/vinyl pivalate-based polymers such as good chemical, mechanical and thermal stability. Based on cell viability assays, the core–shell magnetic polymeric materials [a core, consisting of poly(methyl methacrylate)/Fe3O4 and a shell, formed by poly(vinyl pivalate)] can be regarded as a very promising material, as they achieved high cell viabilities for both murine melanoma tumor cell lines, B16F10, and human keratinocyte cells, HaCaT.

Author Contributions: F.M. and S.B.C. conceived and designed the experiments; G.R., G.V.S.D., and M.S.B.N. performed the experiments; G.R., G.V.S.D, M.S.B.N., O.A.A., S.B.C., and F.M. analyzed the data and wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This work has been supported by Fundação de Apoio à Pesquisa do Distrito Federal (FAPDF), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento Polymers 2020, 12, 2868 15 of 18 de Pessoal de Nível Superior (CAPES)—Finance Code 001. The authors thank the Laboratório Multiusuário de Microscopia de Alta Resolução (LabMic) for the TEM micrographs. Conflicts of Interest: The authors declare no conflict of interest.

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