Introduction

Due to their unique physical properties, magnetic nanoparticles (MNPs), have been widely used in various biomedical applications, such as, targeted drug delivery (Zhao et al. 2013; Zheng et al. 2005), hyperthermia (Simioni et al. 2007; Kumar and Mohamad 2011; Feuser et al. 2015a), and magnetic resonance imaging (MRI) (Gupta and Gupta 2005; Abulateefeh et al. 2011). For these applications, MNPs must have combined properties of high magnetic saturation, superparamagnetic properties and biocompatibility (Zheng et al. 2005; Kumar and Mohamad 2011). MNPs represented by magnetite (Fe3O4) are often made in sizes smaller than 15 nm in diameter and their superparamagnetic properties are sufficient to cause tumor cell death by hyperthermia (Feuser et al. 2015a, b). However, the use of pure MNPs for these applications is challenging due to the high surface to volume ratio and strong dipole–dipole interaction between the particles, which makes MNPs agglomeration possible (Kumar et al. 2014; Tang et al. 2013). The agglomeration of MNPs can be attenuated with the encapsulation of MNPs in polymeric nanoparticles (NPs). The encapsulation of MNPs in polymeric NPs improves their chemical and physical stability, solubility, biological stability, target delivery and reduces the side effects of the encapsulated material (Fan et al. 2009; Zhang et al. 2011; Akbarzadeh et al. 2012). A large number of strategies have been proposed in the literature to encapsulate MNPs in polymeric colloidal particles including, inverse miniemulsion polymerization (Romio et al. 2013) and direct miniemulsion polymerization (He et al. 2009; Yan et al. 2011; Feuser et al. 2015a).

Polymeric NPs can be prepared from natural or synthetic polymers (Landfester and Mailander 2013). They have been studied extensively as drugs carriers, where the main advantages are the protection of the drug, controlled release and the possibility of drug delivery within the targeted cell or tissue (Feuser et al. 2016; Landfester and Musyanovych 2010; Landfester and Mailander 2013). Miniemulsion polymerization allows the production of polymeric NPs with unique characteristics of great interest for biomedical application, producing stable aqueous dispersions of droplets (50–500 nm) containing monomer, surfactant, ultrahydrophobe, initiator, and water-insoluble compounds (e.g., hydrophobic MNPs) by applying high shear stress (Higuchi and Mira 1962; Landfester 2009; Crespy and Landfester 2010; Asua 2014). The main advantage of the miniemulsion polymerization process is the ability to produce complex nanostructures, including inorganic nanoparticle encapsulation, in a single-reaction step with fast polymerization rates (Qiu et al. 2007; Mahdavian et al. 2008).

The NPs surface modification with folic acid (FA) can increase drug uptake in tumor cells that express high levels of type α or β folate receptors (FR). The FR-α is significantly overexpressed on the surface of tumor cells, while the FR-β is overexpressed in leukemic cells and macrophages. FRs exhibit limited expression on non-tumor cells and tissues, where one of the main advantages is the targeted drug delivery by folate receptors (Bhattacharya et al. 2007; Jia-Jyun et al. 2009; Wibowo et al. 2013; Yang et al. 2014; Lee et al. 2015). FR-mediated drug delivery is facilitated by endocytosis, which may enable drug delivery (Sudimack and Lee 2000; Pengcheng et al. 2013; Chen et al. 2013). The NPs can be internalized across a plasmatic membrane (passive diffusion) and through endocytosis (NPs are engulfed by endocytic vesicles) (Wang et al. 2012). Several in vitro studies have demonstrated that the modified surface of nanoparticles with FA increases the cellular uptake in tumor cells that overexpressed FR (Zhang et al. 2009; Duan et al. 2012; Sahoo et al. 2013; Yang et al. 2014). In vivo studies were also performed, as for instance by Arosio et al. (2015) who showed that NPs functionalized with FA increased the therapeutic efficacy of drugs due to the targeting ability and prolonged retention in breast tumor tissues. However, the functionalization or adsorption of the ligands cannot ensure drug delivery when it is intravenously administered (in vivo). When the functionalized nanoparticles enter the bloodstream they need to cross the blood vessel endothelial cells, which do not express FR, in order to reach the interstitial space of tumor sites (Chin and Ferreira 1999; Andhariya et al. 2013). To overcome this, a drug delivery system with superparamagnetic proprieties was proposed that could be attracted by external magnetic field (Mody et al. 2014) and uptake in vivo by FR mediated by endocytosis (Zheng et al. 2005; Xu and Su 2013). Chen et al. (2012) developed a nanoplatform for guided drug delivery by conjugation quantum dots with carbon nanotubes filled with Fe3O4. The authors demonstrated in vitro that this nanoplatform was capable to transport doxorubicin into HeLa cells by means of an external magnetic field. Chertok et al. (2008) showed in vivo that magnetic targeting induced a 5-fold increase accumulation of MNPs over non-targeted tumors. Furthermore, MNPs can also induce cell death by hyperthermia, when external alternating magnetic fields are applied (Xu et al. 2014, Mody et al. 2014; Feuser et al. 2015a, b). In a previous study, Feuser et al. (2015a) showed that superparamagnetic poly(methyl methacrylate) nanoparticles obtained by miniemulsion polymerization induced glioblastoma (U87MG) cell death when alternating magnetic fields were applied for 6 min.

The goal of this study was to synthesize and characterize superparamagnetic poly(methyl methacrylate) (PMMA) NPs obtained by miniemulsion polymerization surface modified with folic acid. In vitro cytotoxicity and cellular uptake assays were performed with the surface modified superparamagnetic poly(methyl methacrylate) (PMMA) with FA (MNPsPMMA-FA) and without FA (MNPsPMMA) on non-tumor cells, murine fibroblast cells (L929), cells that overexpressed FR-β, and chronic myeloid leukemia (K562) cells at two different temperatures (4 and 37 °C). Lastly, hemolysis assays were performed on human red blood cells.

Experimental procedure

Materials

For synthesis of magnetic nanoparticles coated with oleic acid (MNPsOA), the following reagents were used (high purity grade): ferrous sulfate (FeSO4·4H2O), iron (III) chloride hexahydrate (FeCl3·6H2O), ammonium hydroxide (99 %), and oleic acid (OA), all purchased from chemistry Vetec. For the preparation of surface modified superparamagnetic PMMA NPs with FA (MNPsPMMA-FA), the following reagents were used: methyl methacrylate (MMA) obtained from chemistry Arinos, azobisisobutyronitrile (AIBN) and folic acid (FA) purchased from chemistry Vetec, lecithin obtained from Alpha Aesar, and Crodamol GTCC (as co-stabilizer) purchased from Croda. Distilled water was used throughout the experiments.

Methods

Synthesis of MNPs(OA)

MNPs(OA) were prepared by co-precipitation in aqueous phase methods as described by Feuser et al. (2015a). Momentarily, FeCl3·6H2O and FeSO4·7H2O (mole ratio of 1:1.2) were dissolved in a beaker containing distilled water under mechanical stirring in the range of 800 rpm. Afterwards an ammonium hydroxide solution (11 mL) was rapidly added to the solution. After 1 h, 30 mL was added to the OA and the stirring process was continued (800 rpm) for 1 h. The MNPs(OA) produced was centrifuged and washed three times with ethanol to remove the unreacted OA.

Preparation of MNPsPMMA-FA by miniemulsion polymerization

For encapsulation of MNPs(OA) by miniemulsion polymerization, an organic phase containing 2 g of MMA with 0.4 g of MNPs(OA), 0.1 g of lecithin, 0.1 g of crodamol, and 0.04 g of AIBN were added to a beaker containing 20 mL of distilled water and 0.02 g of FA (aqueous phase). The organic phase was added dropwise under higher shear with an amplitude of 70 % (Fisher Scientific, Sonic Dismembrator, 500 W). The high shear was maintained for 5 min (10 s on and 1 s off) in an ice bath immersed beaker to avoid an increased temperature during sonication. The miniemulsion product was transferred to glass tubes (10 mL) at 70 °C for polymerization during 3 h. Afterwards the material was cooled, centrifuged and washed three times with phosphate buffered saline (PBS) at pH 7.4. Subsequently, NPs were resuspended in 20 mL of PBS buffer. The magnetic fluid concentration used to produce the FA-MNPsPMMA was about 7 × 1016 particles  mL−1. For a comparative study, PMMA NPs coated MNPs were prepared without folic acid (MNPsPMMA). The method of preparation was the same already described by Feuser et al. (2015a).

Preparation of MNPsPMMA-FA by miniemulsion polymerization labeled with 6-coumarin

A highly fluorescent molecule, 6-coumarin, was dissolved in Miglyol to form a uniform solution (0.5 mg mL−1). This solution (6-coumarin/crodamol) was used in the preparation of NPs to assess cellular uptake by fluorescence microscopy. In a beaker MNPs(OA) containing 20 mL of distilled water and 0.02 g of FA (aqueous phase) the organic phase was added (2 g of MMA with 0.4 g of MNPs(OA), 0.1 g of lecithin, 0.1 g of solution crodamol/6-coumarin and 0.04 g of AIBN) dropwise under higher shear (amplitude of 70 %). The following steps are the same as previously described.

Characterization

Monomer consumption was monitored by gravimetric analysis of samples withdrawn from the polymerization medium at different times and the reaction was stopped with addition of a 1 % hydroquinone (w/w) solution. The determination of the concentration of residual monomer after the polymerization reaction in miniemulsion was performed by GC (Shimadzu GC2010AF). Particle size and surface charge were measured by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS analyzer. The NPs morphology was observed using a JEM 2100F transmission electron microscope (TEM) operating at 80 kV. Average particle size and polydispersity index (PdI) were also measured by dynamic light scattering and the surface charge of the NPs was investigated through zeta potential measurements (in both cases using the same Zetasizer). All samples were analyzed three times, from which the average and standard deviation (SD) were calculated. Chemical characterization was performed by Fourier transform infrared spectroscopy (FT-IR) using KBr pellets. X-ray diffraction (XRD) experiments were performed to identify the crystallographic structure of MNPs(OA) and MNPsPMMA-FA NPs. The crystalline phase of NPs was identified by XRD measurements using an XPert-Pro diffractometer, Cu-Kα (alpha) radiation (45 kV/40 mA) in the 2θ (theta) range of 20–80o. Thermogravimetric analysis (TGA) was performed. The MNPs(OA) and MNPsPMMA-FA NPs were measured through TGA runs under nitrogen atmosphere at a heating rate of 10 °C/min. A MicroSense model EV9 vibrating sample magnetometer (VSM) was used to measure the hysteresis loops of MNPs(OA) and MNPsPMMA-FA NPs. All analyses were performed at room temperature.

In vitro studies

Cell culture and maintenance

K562 cells were cultured in Roswell Park Memorial Institute Medium (RPMI) (GIBCO, São Paulo, SP, Brazil) supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg mL−1 streptomycin and 10 mM HEPES under 5 % CO2 humidified atmosphere in 75 cm2 flasks at 37 °C. Cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). L929 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO, São Paulo, SP, Brazil) supplemented with 10 % heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg mL−1streptomycin and 10 mM HEPES under 5 % CO2 humidified atmosphere in 75 cm2 flasks at 37 °C. Cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA).

Viability assay (MTT assay)

The MTT cell proliferation assay was employed to assess cell viability after the cytotoxic assay. Briefly, 150 μL of medium was removed and 50 μL mL−1 of MTT solution (5 mg mL−1) was added to each well. The cells were then incubated for 4 h, at 37 °C and 5 % CO2 to allow the formazan-formation reaction. Following incubation, the medium containing the MTT solution was removed, and the formazan crystals were dissolved in 2-propanol. The optical density was measured at 570 and 690 nm using a Safire2 microplate reader (Tecan Group Ltd.). The results are presented as survival percentage, where the control (untreated cells) was 100 %.

Cellular uptake by fluorescence microscopy

The intracellular fate of carrier systems marked with 6-coumarin (MNPsPMMA and MNPsPMMA-FA) were evaluated with fluorescence microscopy. L929 and K562 cells were cultured at a density of 5 × 105 cells/mL, 200 mL/well. MNPsPMMA and MNPsPMMA-FA NPs were incubated at a low temperature (4 °C), blocking receptor-mediated endocytosis mechanisms (Leamon and Low 1991; García-Días et al. 2011), and at 37 °C with a concentration of 100 µg mL−1. After 2 h of incubation, L929 and K562 cells were washed with PBS (three times) and the coverslips covering the bottom of the plate were removed and placed on a glass slide. Cell morphology and cellular uptake were evaluated by optical microscopy and the fluorescence of NPs was monitored by fluorescence microscopy. Cell images were acquired using emission mode with 20× objective after exciting the sample from 450 to 490 nm using barrier filters. Cells were observed under a fluorescence microscope (Olympus BX41) and representative fields were photographed with a digital camera. Three different fields with cell homogeneity of the glass slide were measured and quantified by ImageJ software (Collins 2007). The experiments were performed in triplicate. Fluorescence intensity was calculated as fluorescence intensity of the NPs divided by the fluorescence intensity of control group (Dong et al. 2014). The image background was subtracts by ImageJ software. Statistical analysis was performed using one-way ANOVA followed by post-test Bonferroni’s.

Hemolysis assay

In order to analyze the effect of MNPsPMMA-AF in normal red blood cells (RBC), five human blood samples were collected according to the ethics committee requirements (CEPSH no 913/2010). Briefly, 4 mL of whole blood was added to 8 mL of a sterile solution of sodium chloride in water (saline) and the RBCs were isolated from serum by centrifugation at 10,000×g for 5 min. The RBCs were further washed five times with saline solution; after that, the RBCs were diluted in 2 mL of saline. Then 120 µL of the diluted RBC suspension was added to 880 µL of water or saline. Samples were treated with MNPsPMMA-AF at a concentration of 50 and 100 µg mL−1. All samples were prepared in triplicate and the suspension was briefly vortexed before undergoing a gentle stirring at 37 °C for 60 min. Afterwards, the mixture was briefly vortexed again and centrifuged at 10,000×g for 5 min. 100 μL of supernatant was transferred to a 96-well plate. The absorbance value of hemoglobin at 570 nm was measured with the reference wavelength at 540 nm. 120 µL of the diluted RBC suspension incubated with 880 µL of water and saline was used as the positive and negative control, respectively (Yu et al. 2011; Wang et al. 2009). Hemolysis percentage was calculated according to Eq. 1:

$${\text{Hemolysis}} \left( \% \right) = \frac{{{\text{Sample absorbance}} - {\text{negative control}}}}{{{\text{Positive control}} - {\text{negative control}}}} \times 100$$
(1)

Results and discussion

The polymerization kinetics of NPs with and without FA obtained by miniemulsion polymerization was verified by gravimetric conversion. Analysis of residual monomer is an important parameter to be evaluated, since the total conversion of the monomer to polymer avoids possible toxic effects of the residual monomer, allowing application of the PMMA NPs in the biomedical field (Bettencourt and Almeida 2010; Gosavi et al. 2010). The gravimetric conversion presented in Fig. 1 showed that around 200 min of reaction the conversion of MNPsPMMA and MNPsPMMA-FA reached approximately 98 %. As shown in Fig. 1, the final conversion did not reach 100 %, probably due to the loss of monomer (MMA) by evaporation during the emulsification and polymerization stages. Monomer evaporation can occur due to high vapor pressure of the MMA (Feuser et al. 2016). It is important to mention that FA did not interfere in the polymerization rate.

Fig. 1
figure 1

Gravimetric conversion analyses of the MNPsPMMA and MNPsPMMA-FA NPs obtained by miniemulsion polymerization

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The results of the DLS and TEM analyses are presented in Fig. 2. DLS analyses (Fig. 2a, c) showed that the MNPsPMMA and MNPsPMMA-FA NPs have intensity average particle diameters of 104 ± 3.9 nm and 134 ± 3.5 nm, and a polydispersity index of 0.11 ± 0.02 and 0.14 ± 0.02, respectively. The FA adsorption on NPs surface increased the average size by approximately 30 nm, when compared to NPs without FA. TEM images of MNPsPMMA-FA showed dark regions (black arrow) (Fig. 2b, d), which are attributed to the presence of MNPs (6-12 nm in diameter) encapsulated in PMMA NPs. The MNPsPMMA-FA obtained by miniemulsion polymerization presented a sub-micrometric size with a spherical morphology and unimodal size distribution. Other important factor in the preparation of NPs for biomedical application is the zeta potential, since particle size can influence their ability to interact with cells. At pH 7.4 the zeta potential of MNPsPMMA (−37 mV ± 4.8) and MNPsPMMA-FA NPs (−38 mV ± 4.5) is not significantly different. The negative zeta potential can be attributed to the carboxylic groups of oleic acid and folic acid on the NPs surface. The high negative values of zeta potential are related to the good colloidal stability of NPs and their possible interactions with biological medium (He et al. 2010).

Fig. 2
figure 2

a and c dynamic light scattering; b and d transmission electron microscopy images of MNPsPMMA and MNPsPMMA-FA NPs, respectively. Data refer mean ± standard deviation (n = 3) different experiments

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The FTIR analyses (Fig. 3) were performed, in order to evaluate possible chemical interactions of MNPs and FA on PMMA NPs. FTIR spectrum (Fig. 3a) showed a band at 1736 cm−1 corresponding to C=O groups of PMMA. In addition, the bands in the 3000–2800 cm−1 region are attributed to the stretching of C-H bonds of the saturated alkane in PMMA (Feuser et al. 2016). The peak at approximately 580 cm−1 corresponds to asymmetric stretching vibrations of Fe–O ligands, which is attributed to the MNPs (Andhariya et al. 2013; Feuser et al. 2015a). The absorption bands at 1694 (amide I), 1603 (amide II) and 1482 cm−1 (ring phenyl) are characteristic peaks of FA (Pan et al. 2013; Sahoo et al. 2013; Yoo et al. 2013; Duan et al. 2012; Zhang et al. 2007). The FTIR spectrum of MNPsPMMA-FA NPs suggested an adsorption of FA onto NPs due to the appearance of an additional band at 1694 cm−1, as shown in Fig. 3b.

Fig. 3
figure 3

a FTIR spectrum: A MNPs(AO), PMMA, FA, MNPsPMMA and MNPsPMMA-FA and b FTIR spectrum at low scale: FA and MNPsPMMA-FA NPs

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X-ray diffraction (XRD) patterns for the naked MNPs(OA) and MNPsPMMA-FA NPs are shown in Fig. 4. Figure 4 shows six characteristic peaks from the cubic inverse spinel structure of bulk magnetite/maghemite, observed at 2θ = (220), (311), (400), (422), (511) and (440) (Dorniani et al. 2012; Yang et al. 2014). These results demonstrate that the MNPsPMMA-FA has the expected crystalline structure of magnetite/maghemite. An additional broad peak for the MNPsPMMA-FA NPs was observed at an angle between 10 and 20°, which are characteristic of amorphous polymers. (Feuser et al. 2015a; Feuser et al. 2016; Sahoo et al. 2013).

Fig. 4
figure 4

XRD powder diffraction patterns of MNPs(OA) and MNPsPMMA-FA NPs

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TGA analysis was performed estimating the total concentration of MNPs in formulation. Figure 5 shows mass loss in the temperature range from 20 to 220 °C, which can be attributed to the evaporation of water (Nan et al. 2013). The weight loss between 230 and 420 °C corresponds to the polymer PMMA, which are completely degraded when reaching approximately 410 °C, co-stabilizer (Crodamol); OA and FA contribute to the major portion of the total weight. (Landfester and Ramires 2003; Mohapatra et al. 2007; Feuser et al. 2015a). The residual weight of 16 % corresponds to the encapsulated MNPs.

Fig. 5
figure 5

TGA analyses of MNPs(OA) and MNPsPMMA-FA NPs

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The magnetic properties of MNPs(OA) and MNPsPMMA-FA NPs were ascertained by VSM (Fig. 6). The encapsulation of MNPs in PMMA NPs with FA-modified surface showed typical superparamagnetic behavior at room temperature (Fig. 6a, b), with the absence of hysteresis loop and a low remanent magnetization/saturation magnetization (Mr/Ms) ratio value, and a coercive field (Hc) of 5.0 × 10−2 and 0.4, respectively (Chandrasekharana et al. 2011; Nan et al. 2013; Mody et al. 2014; Yang et al. 2014). The superparamagnetic proprieties are very important for biomedical field. Thus, when an external magnetic field is not applied, their overall magnetization value is randomized to zero, avoiding the interactive behavior of the nanoparticles (Mody et al. 2014). The value of magnetization saturation (Ms) in emu/g of MNPs was obtained considering the iron oxide mass determined by TGA (Fig. 5). The Ms value was 37 and 66 emu/g for MNPsPMMA-FA NPs and MNPs(OA). The decrease in Ms value can be attributed to the dense coating of PMMA, as well as oxidation processes during the sonication, leading to the formation of some nonmagnetic or low Ms iron oxide, since maghemite (-Fe2O3) has a lower Ms of 60–80 emu/g compared to 92–100 emu/g for magnetite (Landfester and Ramires 2003; Chandrasekharana et al. 2011; Feuser et al. 2015b). However, this Ms value was enough to move encapsulated MNPs quickly and can be easily separated from the aqueous phase by the action of an external magnetic field gradient, which is shown in Fig. 6c, d. Hence, MNPsPMMA-FA NPs can be directed towards a specific target in the human body, with application of an external magnetic field.

Fig. 6
figure 6

VSM analyses of magnetic properties of MNPs(OA) and MNPsPMMA-FA NPs with magnetic field of a 20 KOe and b 4 KOe. MNPsPMMA-FA NPs in PBS (7.4) c without application of an external magnetic field and d with application of an external magnetic field

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To verify the cytotoxic effect of MNPsPMMA-FA NPs on L929 and K562 cells, the MTT assay was performed. As shown in Fig. 7, the MNPsPMMA-FA NPs did not significantly reduce cell viability (p > 0.05) on L929 cells at different concentrations (20, 40, 60, 80, and 100 ug mL−1). These results are very important because they will facilitate innovative research of new nanomaterials for hyperthermia-based treatment and drug release systems. The non-cytotoxicity on L929 cells is important, since ISO 10993-5 recommends these cells for in vitro biocompatibility assays of new materials for biomedical application. The same results were observed when the cytotoxicity of MNPsPMMA-FA NPs was evaluated on K562 cells at different concentrations (40, 60, 80, 100 and 200 ug mL−1of NPs/mL), as shown in Fig. 7.

Fig. 7
figure 7

Cytotoxicity assay. Cytotoxicity effects of different concentrations of MNPsPMMA-FA on L929 and K562 cells exposed 24 h. The cell viability was monitored through MTT assay. (p > 0.05) using one-way ANOVA followed by post-test Bonferroni’s

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Fluorescence microscopy assay was performed in order to evaluate the morphology and cellular uptake on L929 (non-tumor) and K562 (tumor) cells overexpressing FR-β. The cells were treated with 13 ug mL−1 of MNPsPMMA-FA and MNPsPMMA at two different temperatures (4 °C and 37 °C). As shown in Fig. 8, L929 and K562 cells incubated for 24 h at 37 and 4 °C, and did not present significant changes in their morphology. The cellular uptake assays showed a higher internalization of MNPsPMMA-FA than MNPsPMMA at 37 °C in K562 cells, which can be shown by their bright fluorescence (Fig. 8). These results were corroborated with the quantification of fluorescence, which showed a significant increase in fluorescence intensity of MNPsPMMA-FA when compared with MNPsPMMA. These increases in cellular uptake suggested that MNPsPMMA-FA entered into cancer cells by folate receptor-mediated endocytosis (Zheng et al. 2014; Kam et al. 2005; García-Días et al. 2011). On the other hand, when folate receptor-mediated endocytosis was blocked by low temperature at 4 °C, there was no significant difference in cellular uptake, as shown in Figs. 8 and 9. However, when L929 cells were used, similar results were observed for L929 cells at 37 and 4 °C (Figs. 8 and 9). These results were expected for L929 cells, since they do not express FR, suggesting that the NPs entered into cells by energy-independent mechanisms (Kam et al. 2005; Zheng et al. 2014). All endocytosis pathways are energy- and temperature-dependent mechanisms, which involve the uptake of NPs in small invaginations of membrane vesicles (Rastogi et al. 2014). Finally, our results showed that the superparamagnetic PMMA NPs with an FA modified surface are possibly an attractive target for cancer selective delivery by FR (Moriyama et al. 1986; Moulin et al. 2007; Wibowo et al. 2013; Van der Heijden et al. 2009; Qi and Ratnam 2006).

Fig. 8
figure 8figure 8

Optical (MO) and fluorescence (MF) microscopy images of L929 and K562 cells. The cells were treated with MNPsPMMA or MNPsPMMA-FA at concentration of 100 µg mL−1 and incubated at 37 °C or at 4 °C for 2 h, after this period the cell morphology was evaluated by optical microscopy and the fluorescence of NPs was monitored by fluorescence microscopy (Olympus BX41)

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Fig. 9
figure 9

Quantification of mean fluorescence intensity by ImageJ software. Cells (K562 and L929) were incubated with MNPsPMMA and MNPsPMMA-FA NPs at 37 °C and 4 °C for 2 h, labeled with 6-coumarin at concentration of 100 µg mL−1. **p < 0.001, ***p < 0.001 compared to control groups, using one-way ANOVA followed by Bonferroni’s post-test. Scale bar 100 µm

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In order, to evaluate the biocompatibility on human red blood cells, hemolysis assays were carried out, which is shown in Fig. 9. The hemolysis assay is an important test to assess cytotoxicity in red blood cells (Chen et al. 2012; Zhou et al. 2011). According to the criterion of Standard Test Method for Analysis of Hemolytic Properties of Nanoparticles (ASTM E2524–08), a percentage hemolysis >5 % indicates that the test material causes damage. As shown in Fig. 10, high concentrations of MNPsPMMA-FA incubated for 24 h with human red blood cells did not exceed 5 %. The non-hemolytic character confirms the hemocompatibility of NPs obtained by miniemulsion polymerization, which can be an alternative for drug delivery systems administered systemically for leukemic treatment.

Fig. 10
figure 10

Hemolysis Assay of MNPsPMMA-FA. Relative rate of hemolysis in human RBCs upon incubation with MNPsPMMA-FA at concentration of 50 and 100 µg mL−1. The presence of hemoglobin in the supernatant (turbid) was observed at 540 nm. Data are mean ± SD (n = 3)

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Conclusion

The synthesis of surface modified superparamagnetic PMMA with FA obtained by miniemulsion polymerization resulted in a stable polymeric system in aqueous dispersion, with good polydispersity index and biocompatibility. MNPsPMMA-FA presented a significant increase in the K562 cell uptake at 37 °C, when compared with the MNPsPMMA, suggesting that it enters into cancer cells by folate receptor-mediated endocytosis. When the MNPsPMMA-FA was incubated at 4 °C, there was a reduction in cellular uptake, demonstrating that the mechanism of entering into K562 cells is energy dependent (endocytosis). The hemolysis assay demonstrated the hemocompatibility of MNPsPMMA-FA, which can be administered at 100 µg mL−1 without causing any damage to human red blood cells. Moreover, other studies are being performed, such as, drug encapsulation for cancer treatment and cellular uptake assays to improve and understand the entering mechanism of these NPs in cells overexpressing FR-α and FR-β.