Covalent hyaluronic-based coating of magnetite nanoparticles: Preparation, physicochemical and biological characterization

Covalent hyaluronic-based coating of magnetite nanoparticles: Preparation, physicochemical and biological characterization

Materials Science & Engineering C 107 (2020) 110271 Contents lists available at ScienceDirect Materials Science & Engineering C journal homepage: ww...

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Materials Science & Engineering C 107 (2020) 110271

Contents lists available at ScienceDirect

Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec

Covalent hyaluronic-based coating of magnetite nanoparticles: Preparation, physicochemical and biological characterization

T

Andrea Atreia, Claudia Innocentib,c, Stefania Lamponia, Serena Paesanoa, Gemma Leonea, Annalisa Realea, Marco Paolinoa,∗, Andrea Cappellia a

Dipartimento di Biotecnologie, Chimica e Farmacia (Dipartimento di Eccellenza 2018-2022), Università di Siena, via Aldo Moro 2, 53100, Siena, Italy I.C.C.O.M. - C.N.R, I-50019, Sesto Fiorentino, FI, Italy c Dipartimento di Chimica, Università di Firenze and INSTM, I-50019, Sesto F.no, FI, Italy b

ARTICLE INFO

ABSTRACT

Keywords: Magnetic nanoparticles Hyaluronic acid Oligo ethylene glycol Click chemistry Colloidal stability

In this paper we report about the preparation, physicochemical and biological characterization of a magneto responsive nanostructured material based on magnetite nanoparticles (NP) coated with hyaluronic acid (HA). A synthetic approach, based on a Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition “click” reaction between azido-functionalized magnetite NP and a derivative of hyaluronic acid bearing propargylated ferulic acid groups (HA-FA-Pg), was developed to link covalently the polymer layer to the magnetite NP. The functionalization steps of the magnetite NP and their coating with the HA-FA-Pg layer were monitored by Fourier Transform Infrared (FTIR) spectroscopy and Thermal Gravimetric Analysis (TGA) while Dynamic Light Scattering (DLS) and ζpotential measurements were performed to characterize the aqueous dispersions of the HA-coated magnetite NP. Aggregation and sedimentation processes were investigated also by UV-visible spectroscopy and the dispersions of HA-coated magnetite NP were found significantly more stable than those of bare NP. Magnetization and zero field cooled/field cooled curves revealed that both bare and HA-coated magnetite NP are superparamagnetic at room temperature. Moreover, cytotoxicity studies showed that the coating with HA-FA-Pg significantly reduces the cytotoxicity of the magnetite NP providing the rational basis for the application of the HA-coated magnetite NP as healthcare material.

1. Introduction The interest in materials based on iron oxide magnetic nanoparticles (NP) in biomedical applications has significantly grown in the past decade due to their nanometric size, high surface area, and intrinsic magnetic properties. Moreover, magnetic NP have been approved by the US Food and Drug Administration (FDA) [1] and were used as heat mediators in hyperthermia therapy [2], carriers for drug delivery systems [3,4], and contrast agents for magnetic resonance imaging [5–7]. Magnetite (Fe3O4) NP have been prepared through different methods (sol–gel [8], co-precipitation [9], hydrothermal synthesis [10], thermal decomposition [11], microemulsion [12], and colloidal chemistry methods [13]). Among these, the co-precipitation method, based on the co-precipitation from aqueous solutions containing Fe(II) and Fe(III) salts by the addition of a base [14], is considered the most efficient procedure due to its easy and inexpensive process potentially transferable to large-scale production. Interestingly, modification of the Fe(II)/Fe(III) ratio, pH and ionic strength of the solution could be useful



to control the size and shape of the magnetite NP [15–17]. Different hybrid magnetite NP based on magnetite (Fe3O4) core for biomedical applications have been prepared by coating the inorganic core with organic biocompatible layers using covalent or non-covalent synthetic approaches in order to obtain non-toxic and stable colloidal dispersions [18]. Indeed, the organic coating is mainly responsible for the processes of cell recognition and the biocompatibility of the materials. Usually these coatings consist of peptides [19,20], lipids (e. i. lauric acid) [21], saccharides (e. i. dextran or inuline) [22–25] or biocompatible amphiphilic organic molecules (e. i. poly(ethylene glycol)) [26–28], but also of synthetic polymers. In recent studies, magnetite NP stabilized with a non-covalent coating of cationic polymers (i. e. polyallylamine hydrochloride, PAH) were used to fabricate artificial multicellular tissue-mimicking clusters [29]. On the other hand, in an innovative research, magnetite NP were conjugated on nanofibers of suitably modified poly-lactide (PLA) with the aim of magnetizing the polymeric nanofibers through the formation of a homogeneous and continuous

Corresponding author. E-mail address: [email protected] (M. Paolino).

https://doi.org/10.1016/j.msec.2019.110271 Received 17 May 2019; Received in revised form 13 September 2019; Accepted 1 October 2019 Available online 15 October 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Design of the new hybrid material HA-FA-HEG-NP.

magnetic layer [30]. Interesting applications were found also in in vivo bioimaging with magnetite NP coated with chitosan and Gd oxyfluoride luminophore in zebrafish models [31]. Hyaluronic acid (HA) is generally recognized to play an important role in the early processes of cellular adhesion. Moreover, HA coating on cell surface is a key motif of cells in the dispersion state and HAbased coating strategies have been developed in order to confer biocompatibility to materials for biomedical applications [32,33]. Owing to these important functions, in previous studies we functionalized the HA backbone with ferulic acid (FA) residues combining the interesting properties of these two natural structures [34,35]. After that, exploiting this reaction, we developed a coating methodology based on low-molecular weight HA bearing residues of FA with a clickable propargyl group (HA-FA-Pg, Fig. 1) [36]. The conjugation of propargylated FA to the HA backbone confers to the HA-FA-Pg copolymer aggregation-induced emission (AIE) [37] properties and provides an interesting way to combine HA to different substrates by exploiting the Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) click-chemistry reaction [38]. HA-FA-Pg was employed in the covalent HA-based coating (CHABC) of a π-stacked polybenzofulvene derivative [39–44] leading to a biorelevant tri-component polymer brush (TCPB) material [36]. This material was used in the preparation of nanostructured drug delivery systems capable of releasing an anticancer drug (doxorubicin) to cancer cells through an active targeting mechanism based on the interaction of HA with CD-44 receptors overexpressed on the cancer cell surface [45]. Furthermore, HA-FA-Pg copolymer was functionalized with nona(ethylene glycol)-oleamide (NEG-OA) side chains to obtain HA-FA-NEG-OA materials capable of self-assembling to form micelle aggregates in water solution or to interact with the phospholipid bilayer of small unilamellar vesicles (SUV) leading to the formation of a biomimetic perivesicular coating [38]. Interestingly, these studies showed that the push–pull structure of the ferulic linker was sensitive to the restriction of intramolecular motions (RIM) phenomenon conferring fluorescent properties to the material, which can be monitored by fluorescence microscopy [38]. In the present work we explore the CHABC of magnetite NP with

HA-FA-Pg to prepare an organic/inorganic system able to respond to magnetic fields and potentially useful in nano-theranostic (i.e. therapeutic and diagnostic) applications (Fig. 1). In particular, by using a divergent approach, the surface of magnetite NP was modified by the introduction of a first organic layer consisting in azido terminated hexa(ethylen glycol) chains. Subsequently, a second organic layer of HA was covalently bonded through HA-FA-Pg by exploiting the CuAAC click chemistry reaction [46–50] to obtain HA-FA-HEG-NP material. The various stages of NP functionalization were monitored by Fourier Transform Infrared (FTIR) spectroscopy. Dynamic Light Scattering (DLS) and ζ-potential measurements were used to characterize the aqueous dispersions of the polymer coated NP, while the colloidal stability of HA-FA-HEG-NP dispersions in deionised water and in 0.1 M NaCl was studied by UVvisible spectroscopy. Moreover, the magnetic properties of HA-FAHEG-NP were investigated by measuring magnetization curves and zero-field cooled and field cooled curves. Finally, their potential cytotoxicity was assessed in comparison to the non-treated magnetite NP. 2. Experimental details 2.1. Materials and methods All materials were research grade products (Sigma-Aldrich) and used as received. Yields refer to purified products and are not optimized. Merck TLC plates, silica gel 60 F254 were used for TLC. Compound COOH-HEG-N3 was prepared as reported in ref. [51]. The synthesis and characterization of HA-FA-Pg copolymers was described in ref. [38]. Dulbecco's Modified Eagle's Medium (DMEM), trypsin solution, and all the solvents used for cell culture were purchased from Lonza (Belgium). Mouse immortalized fibroblasts NIH3T3 were from American Type Culture Collection (USA). 2.1.1. Preparation of magnetite NP Fe3O4 NP were prepared by co-precipitation from a solution of Fe(II) and Fe(III) salts by adding a NaOH solution. In particular, 1.17 g of 2

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FeCl3 6H2O and 0.932 g of Fe(NH4)2(SO4)2 6H2O were dissolved in 50 mL of double distilled water (DDW) deoxygenated by N2 bubbling. We used a 10% excess of Fe(NH4)2(SO4)2 6H2O with respect to the stoichiometric Fe(II)/Fe(III) ratio 1:2 to compensate for possible oxidation of Fe(II). The mixture was heated at 60 °C and 22 mL of 1 M NaOH (prepared in deoxygenated water) were rapidly added. Magnetite NP were separated from the solution by means of a magnet and washed several times with DDW. The average size of the primary NP was ca. 10 nm as determined by x-ray diffraction (Figure ESI-1, ESI) NP prepared with this procedures exhibited superparamagnetic behaviour [52,53].

2.1.5. FTIR measurements Fourier Transform Infrared (FTIR) spectra in attenuated total reflection mode were measured by using a Bio-Rad FTS 6000 spectrometer (Bio-Rad, Hercules, CA, USA) equipped with a Ge crystal. All spectra were recorded in the wavenumber range of 750–4000 cm−1 with a resolution of 4 cm−1 2.1.6. DLS, ζ-potential measurements A Zetasizer NanoZS90 instrument (Malvern, Worcestershire, UK) was used to investigate the size of the bare and functionalized NP. The cumulant method was applied to determine the hydrodynamic diameter of the NP. The hydrodynamic diameters are expressed as Z-average. The same apparatus was used for the ζ-potential measurements. The measurements were performed for dispersions of the NP in DDW at a concentration of 0.02 mg/mL at a temperature of 25 °C. DLS measurements as a function of time were performed for dispersions of HA-FA-HEG-NP in DDW and 0.1 M NaCl in order to investigate the aggregation and sedimentation processes. For these measurements, dispersions of 0.028 mg/mL nominal concentration were sonicated and then let to sediment to separate the larger aggregates. DLS measurements were performed as a function of time starting from the sonication of the dispersion.

2.1.2. Preparation of NH2-NP The functionalization of magnetite NP with amino groups was carried out by adding 3-aminopropyltriethoxysilane (APTES, 4.0 mL, 17.3 mmol) to a dispersion of magnetite NP (0.50 g) in an ethanol-water (9:1) mixture (180 mL). The molar ratio of Fe3O4 to APTES was 1:8. The pH was adjusted to 4.5 with 1N HCl and the reaction was carried out under mechanical stirring for one day at room temperature. The functionalized NP were separated from the solution by means of a magnet, washed several times with DDW and ethanol, and dried under a nitrogen stream to obtain the desired material.

2.1.7. UV-visible spectrophotometry The UV-visible spectra were acquired by means of a Perkin Elmer Lambda 650 Spectrophotometer (Perkin Elmer, Waltham, MA, USA). The instrument was not thermostated and the temperature of the sample in the cuvette increased from 25 to 27 °C at the end of the run. The nominal concentration of the NP was 0.4 mg/mL. Dispersions of bare magnetic NP and of HA-FA-HEG-NP were prepared in DDW and in 0.1 M NaCl. The dispersions were sonicated before starting the measurements and the time was counted starting from the end of the sonication. The absorbance (or more correctly the optical density) was measured at 500 nm, a wavelength at which neither the magnetite NP nor the polymer absorb light.

2.1.3. Preparation of azido functionalized HEG-NP The functionalized HEG-NP were prepared either in an aqueous medium (Method A) or in an organic solvent (Method B) as described below. Method A. A mixture of HEG derivative 1 (0.10 g, 0.26 mmol), 1ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.045 g, 0.29 mmol), and N-hydroxysuccinimide (NHS, 0.033 g, 0.29 mmol) in deionised water (5.0 mL) was added to a dispersion of 0.10 g of NH2-NP in 50 mL of MES [2-(N-morpholino)ethanesulfonic] buffer at pH 5. The reaction was carried out at room temperature for 24 h under mechanical stirring. Subsequently, the NP were separated from the reaction mixture by a magnet and washed several times with DDW, methanol, and acetone and dried under a nitrogen stream to obtain 0.11 g of HEGNP. Method B. HEG derivative 1 (0.10 g, 0.26 mmol) was dissolved in 20 mL of anhydrous THF and 1,1’- carbonyldiimidazole (0.043 g, 0.26 mmol) was added. The resulting mixture was heated under reflux in N2 atmosphere for 2 h. Subsequently, the reaction mixture was concentrated under reduced pressure and the residue, consisting in the corresponding imidazolide derivative, was used without further purification. 1H NMR (CDCl3, 400 MHz): 3.13 (t, J = 6.2, 2H), 3.37 (t, J = 5.0, 2H), 3.54–3.69 (m, 22H), 3.92 (t, J = 6.2, 2H), 7.09 (s, 1H), 7.49 (s, 1H), 8.18 (s, 1H). MS(ESI): m/z 451.9 (M+Na+). The imidazolide derivate was dissolved in 30 mL of anhydrous DMF and triethylamine (TEA) (0.20 mL, 1.4 mmol) and 0.10 g of NH2-NP were added to the solution. The reaction mixture was sonicated under N2 atmosphere at room temperature for 24 h. Then, the derivatized magnetite NP were separated from the solution by means of a magnet and washed several times with DDW, methanol, and acetone. The obtained material was dried under vacuum to obtain 0.12 g of HEG-NP as a black powder.

2.1.8. TGA measurements Thermal Gravimetric Analysis (TGA) measurements were performed using a SDTQ600 (TA Instruments) apparatus in N2 flux, with a heating rate of 10 °C/minute from RT to 800 °C. 2.1.9. Magnetic measurements Magnetization versus field intensity and ZFC/FC curves were measured by means of Superconducting Quantum Interference Device (SQUID) magnetometer from Quantum Design Ltd. San Diego, CA, USA operating with a maximum magnetic field of 50,000 Oe in the 2–400 K temperature range. Hysteresis loops were measured cyclically varying the applied field from 50,000 Oe. Magnetization curves at 300 K were measured from 50,000 to −300 Oe to check for possible remanence and coercivity. ZFC/FC were measured with an applied field of 50 Oe from 2.5 to 300 K, after having cooled the sample in absence (ZFC) or in the presence of the probe field. 2.1.10. Cell cultures In order to evaluate the in vitro cytotoxicity of the materials, the direct contact tests, proposed by “ISO 10995-5, Biological evaluation of medical devices – Part 5: Tests for cytotoxicity: in vitro methods” was used [54]. This test is suitable for samples with various shapes, sizes or physical status (i.e. liquids or solids). The evaluation of in vitro acute toxicity does not depend on the final use for which the product is intended, and the document ISO 10995-5:2009 recommends many cell lines from American Type Collection. Among them, to test HA-FA-HEGNP and magnetite NP cytotoxicity, NIH3T3 mouse fibroblasts were chosen [55]. NIH3T3 were propagated in DMEM (supplemented with 10% fetal calf serum, 1% L-glutamine-penicillin-streptomycin solution, and 1%

2.1.4. Preparation of HA-FA-HEG-NP A mixture of 0.11 g of HEG-NP (prepared by Method A or Method B) in 50 mL of H2O-t-butanol (1:1) containing 0.11 g of HA-FA-Pg was treated with a solution of the catalyst, consisting in CuSO4 (3.0 mg, 0.018 mmol) and sodium ascorbate (4.0 mg, 0.020 mmol) in H2O-tertbutanol (1:1) (1.0 mL). The resulting mixture was stirred at room temperature in N2 atmosphere for 24 h under sonication. The reaction product was separated from the reaction mixture by a magnet and washed several times with DDW, methanol, and acetone. The obtained material was dried under vacuum to obtain 0.15 g of HA-FA-HEG-NP (H2O) or 0.16 g of HA-FA-HEG-NP(org.) both as a black powder. 3

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MEM non-essential amino acid solution) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. Once at confluence, the cells were washed with PBS 0.1M, separated with trypsin-EDTA solution and centrifuged at 1000 r.p.m. for 5 min. The pellet was re-suspended in complete medium (dilution 1:15).

E = log 2 (a/b)

(1)

Also the second shape descriptor used, circularity, was determined using Image-J software. The circularity parameter describes the characteristics of a certain geometrical shape relative to that of a circle. It assumes the value of 1 for a perfect circle and decreases, progressively, as the shape become more elongated. The dimensionless circularity of cell shapes was computed using the equation:

2.1.11. Evaluation of NIH3T3 viability Cells (1.5 × 104) suspended in 300 μL of complete medium were seeded in each well of a 24 well round multidish and incubated at 37 °C in an atmosphere of 5% CO2. Once reached the 50% of confluence (i.e. after 24 h), the culture medium was discharged and the tested compounds (HA-FA-HEG-NP and magnetite NP), properly diluted in NaCl 0.9%, were added to each well. Concentrations of 5 × 10−3, 1 × 10−2, 2.5 × 10−2, 5 × 10−2 and 1 × 10−1 mg/mL for each sample were tested. All samples were set up in six replicates. Complete medium was used as negative control. The same experiment was performed by applying a permanent magnet (5 mm diameter/ 8 mm height) at the bottom of the wells (the magnet was fixed at the bottom of each wells by a bi-adhesive tape). Cells were treated with and without magnet for 24 and 48 h [56]. After 24 and 48 h of incubation, cell viability was evaluated by Neutral Red (NR) uptake, as follows. First, the following solutions were prepared in order to determine the percentage of viable cells:

C = 4 (area)/(perimeter)2

(2)

For both elongation and circularity, only single cells were analysed excluding cell-cell contacting – and on-edge objects. A minimum of 50 cells from five different regions in each of six biological repeats was analysed. Cells were manually traced for outline. All parameters were measured in μm. 2.1.13. Statistical analysis In vitro results were analysed by multiple comparison performed by one-way ANOVA and individual differences tested by Fisher's test after the demonstration of significant intergroup differences by ANOVA. Differences with p < 0.05 were considered significant. 3. Results and discussion 3.1. Design and synthesis

1. NR stock solution: 0.33 g NR dye powder in 100 mL of sterile H2O; 2. NR medium: 1.0 mL NR stock solution + 99.0 mL routine culture medium pre-warmed to 37 °C; 3. NR desorb solution: 1% glacial acetic acid solution + 50% ethanol + 49% H2O.

The coating of magnetic NP with suitable polymer layers is a commonly used strategy to modulate their chemical and biological properties. In fact, the polymer coating allows the preparation of stable colloidal dispersions of NP in water and, in view of their biomedical applications, may reduce their potential toxicity [59]. Thus, this strategy is largely used to obtain the approval by regulatory authorities for their use in humans [60]. Moreover, the covalent binding, instead of the simpler and most commonly used non-covalent adsorption of the polymer layer, guarantees the adhesion of the coating in media with relatively high ionic strength [18]. In order to achieve this goal by using a divergent approach [61], we decided to build, step by step, a biocompatible organic coating on the surface of the magnetite NP by exploiting the formation of stable covalent bonds. In the first synthetic step (Scheme 1), the surface of magnetite NP was functionalized with amino terminated propyl side chains by reaction with 3-aminopropyltriethoxysilane (APTES) [62]. Subsequently, the amino groups were linked via amide bond to an hetero-bifunctional azido and carboxylic terminated hexa(ethylene glycol) (HEG) spacer to obtain the material indicated as HEG-NP. The amide bonds between the carboxylic group of HEG (compound 1, in Scheme 1) and the amino groups of the functionalized NP were formed in both aqueous and organic environments (see materials and method section for details). Reagents: (i) APTES, ethanol-water (95:5 v/v), pH 4.5; (ii) method A: EDC, NHS, water (pH 5.0); method B: a) CDI, THF; b) TEA, DMF, ultrasound; (iii) CuSO4, ascorbic acid, water-t-butanol (1:1 v/v). The PEGylation strategy to functionalize organic and inorganic NP is largely reported in literature [33,43,63–65]. Usually, PEG coatings improve the hydrophilicity and wettability of NP, decrease immunogenicity, prevent their aggregation, opsonisation, and phagocytosis and improve their systemic circulation time [66]. Magnetite NP functionalized with azido groups are flexible substrates to which a variety of molecules can be conjugated by exploiting the CuAAC “click” chemistry reaction [67–69]. Therefore, CuAAC coupling reaction involved the azide groups on the surface of the magnetite NP coated with HEG and propargyl residues of previously reported HA-FA-Pg graft copolymer was exploited for the CHABC procedure to obtain the hybrid material HA-FA-HEG-NP [38]. Among the available HA-FA-Pg graft copolymer we select a samples bearing around 2-3 ferulate residues each macromolecule in order to lessen the possible cross-linking among

At the end of incubation, the routine culture medium was removed from each plate and the cells were carefully rinsed with 1 mL prewarmed D-PBS 0.1M. Plates were then gently blotted with paper towels. 1.0 mL NR medium was added to each dish and further incubated at 37 °C, 95% humidity, 5.0% CO2 for 3 h. The cells were checked during incubation for NR crystal formation. After incubation, the NR medium was removed and the cells were carefully rinsed with 1 mL pre-warmed D-PBS 0.1M. PBS was decanted and blotted from the dishes and exactly 1 mL NR desorb solution was added to each sample. Plates were placed on a shaker for 20–45 min to extract NR from the cells and form a homogeneous solution. During this step the samples were covered to protect them from light. Five minutes after removal from the shaker, absorbance was read at 540 nm with a UV/visible spectrophotometer (Lambda 25, Perkin Elmer). 2.1.12. Morphological analysis In order to evaluate changes in cell morphology as a function of different concentration of both HA-FA-HEG-NP and magnetite NP, NIH3T3 were analysed by an inverted optical microscope (Olympus IX 70) equipped with a Sony CCD-IRIS videocamera. Changes of fibroblasts shape were evaluated by Image-J software (National Institute of Health, NIH). Following image acquisition, the 2-dimensional conformation of single cells was digitally characterised using two parameters describing shape that are invariant to both, size and orientation [57]. Firstly, cell elongation was established by calculating the ratio between the major and minor axis of the best fitting ellipse of the cell profile. According to Dunn and Heath [58], the elongation represents the numerical value required to minimise the extension of a particular profile and may, therefore, provide a quantitative parameter in assessing cell morphological responses to the test materials. Optical images were processed using Image-J software whereby the best fitting ellipse was obtained representing the uniform distribution of the exact number of pixels enclosed by the cell perimeter. Following the establishment of the ellipses’ axis (a and b), the elongation (dimensionless) was calculated by: 4

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Scheme 1. Synthesis of HA-FA-HEG-NP.

the magnetite NP [38].

sample was shown in Fig. 2 and the assignment of the peaks was reported in Table 1. The spectrum of NH2-NP is consistent with those reported in previous works [70,71]. The band at ca. 1000 cm−1 is due to the Si-O-Si stretching vibrational modes (Fig. 2A, bottom curve) whereas the peak at 1516 cm−1 can be ascribed to the NH2 bending mode. On the other hand, the spectrum of HEG-NP obtained after reaction with HEG

3.2. FTIR, DLS and TGA measurements All the synthetic steps, starting from the silanization up to the conjugation with hyaluronic derivative HA-FA-Pg by “click” reaction, were characterized by FTIR spectroscopy. The spectrum of the NH2-NP 5

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Table 1 Positions and assignment of the most relevant peaks in the FTIR spectra measured for the magnetite NP functionalized with APTES (NH2-NP), with azido terminated HEG (HEG-NP), and subsequently with HA-FA-Pg copolymer (HAFA-HEG-NP) together with that of the intermediate HEG derivative 1 and the hyaluronic acid derivative (HA-FA-Pg). Sample NH2-NP

HEG-NP

HA-FA-HEG-NP

HEG derivative 1

HA-FA-Pg

Fig. 2. A) FTIR spectra measured for Fe3O4 NP functionalized with APTES (NH2-NP, bottom curve), for NH2-NP after reaction with HEG derivative 1 in organic solvent (HEG-NP, middle curve) and for HEG derivative 1 (top curve). B) Results of the curve-fitting analysis of the region around 1500 cm−1 of the spectrum measured for HEG-NP.

derivative 1 in organic solvent shows the peaks corresponding to the amide I (1650 cm−1) and to the amide II (1550 cm−1) vibrational modes (Fig. 2A, middle curve) together with the band due to the azide group, which result visible at ca. 2115 cm−1. The region around 1650 cm−1 was analysed by a curve fitting procedure in order to determine the components contributing to the observed band. The results of the curve fitting are shown in Fig. 2B. This analysis indicates that the band centred at 1550 cm−1 has a contribution from the peak due to the amine groups (ca. 1510 cm−1), which did not react with HEG derivative 1. The presence of free amino groups can be attributed to the presence of a partial derivatization of the amino groups, which can remain trapped in the siloxane multilayer. Therefore, only the amino groups at the outermost layer are accessible to HEG derivative 1 to form the amide bonds. Similar FTIR spectrum was measured for HEG-NP prepared carrying out the reaction in aqueous solution. The main difference is that the bands of the amide and of the azide groups were less intense suggesting a less amount of the azido terminated side chains when the reaction is carried out in aqueous media. In Fig. 3, the spectrum of HA-FA-HEG-NP (middle curve) was compared with those of their precursors HEG-NP material obtained via organic solvent (bottom curve) and of the hyaluronic acid derivative HA-FA-Pg (top curve). The spectrum of HA-FA-Pg is very similar to that of pure HA (not show), besides the small peak at 1500 cm−1 due to the

Peak position (cm−1)

Assignment

992 1100 1510 1615 2930 3380

Si-O-Si Si-O-Si Bending N-H Bending H2O Stretch. –CH2 Stretch. N-H and O-H

1452 1550 1650 2117 2932 3387

-CH2 bending Amide II + bending N-H Amide I Stretch. -N3 Stretch. C-H Stretch. N-H and O-H

1044, 1080, 1158 1510 1560 1640 1740 2936 3380

-C-O-C ring HA Aromatic ring FA II Amide Amide I C=O in COOH Stretch. C-H Stretch. O-H

1129, 1196 1685 2110 2898

Stretch. Stretch. Stretch. Stretch.

1043, 1077, 1150 1510 1550 1613 1650 1740 2936 3350

-C-O-C ring HA acid Aromatic ring FA II Amide Bending H2O Amide I C=O in COOH Stretch. C-H Stretch. O-H and N-H

-C-O-C C]O in COOH -N3 C-H

Fig. 3. Comparison of the FTIR spectrum measured for HEG-NP (bottom curve) with those measured for HA-FA-HEG-NP (middle curve), and for the hyaluronic acid derivative HA-FA-Pg (top curve).

aromatic ring of the FA chromophore. The comparison of the two starting materials highlights that the band of the O-C-O of the HA rings (around 1000 cm−1) is overlapped with the main band present in the spectrum of HEG-NP due to the Si-O-Si bonds. However, in the 6

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spectrum of HA-FA-HEG-NP, the presence of an intense and well detectable peak attributed to the non-dissociated carboxylic groups of HA residue, together with the absence of bands at about 2100 cm−1 related to the azido group, confirm the successful occurrence of the CuAAC reaction. The most representative size and ζ-potential distribution curves obtained by DLS measurements for water dispersions of HEG-NP and HA-FA-HEG-NP are shown in Fig. 4. Particle sizes and ζ-potential values for the various synthetic intermediates are summarized in Table 2. The hydrodynamic diameters of Fe3O4 NP and of NH2-NP determined by DLS are much larger than the size (ca. 10 nm) determined by X-ray diffraction in previous studies [52]. Hence, DLS results suggest that the dispersions contain aggregates of individual (primary) NP. This behaviour has been highlighted during the preparation of magnetite NP by co-precipitation that results in multicore aggregates with a broad size distribution [72]. However, it should be taken into account that larger particles contribute more than smaller ones to the scattered intensity. Hence, for polydispersed systems, the sizes derived from intensity distribution tend to be overestimated. This conclusion is confirmed by the AFM images of the HA-FA-HEG-NP (Figure S-2, see Supplementary Material). As expected, the ζ-potential of NH2-NP is significantly higher than that of bare NP due to the protonation of the NH2 groups at neutral pH. The size of the NP increases after coating with HEG. This size increase is probably due to the aggregation of particles rather than to the thickness of the polymer layer, which is negligible compared to the diameter of the aggregates. The hydrodynamic diameter of the NP after the “click” reaction to bind HA-FA-Pg is larger when starting from HEG-NP prepared in organic solvent (492 nm) than those prepared in water (259 nm). This would be consistent with a larger polymer surface coverage for HEG-NP prepared in the organic solvent. However, we cannot rule out that the formation of aggregates is, at least in part, responsible for the observed size increase after binding of HA-FA-Pg. The ζ-potential of HA-FA-HEGNP is negative both in the case of the hybrid material obtained from HEG-NP(H2O) (−18 mV) and in the hybrid material obtained from

Table 2 Z-average, Poly Dispersity Index (PDI) and ζ-potential of bare magnetite NP, NH2-NP, HEG-NP prepared in water (H2O) and in organic solvent (org.), and HA-FA-HEG-NP derived from HEG-NP prepared in water suspension (H2O) or in organic solvent (org.). Measurements were performed in double distilled water, at neutral pH and 25 °C. The concentration of the NP was 0.02 mg/mL. Sample

Z-average (nm)

PDI

ζ-potential (mV)

Fe3O4 NP

217 ± 4

0.36 ± 0.01

18 ± 1

NH2-NP

185 ± 5

0.46 ± 0.09

45 ± 1

HEG-NP(H2O) HEG-NP(org.)

266 ± 25 345 ± 8

0.33 ± 0.01 0.23 ± 0.04

13 ± 1 9±2

HA-FA-HEG-NP(H2O) HA-FA-HEG-NP(org.)

259 ± 3 492 ± 15

0.38 ± 0.01 0.39 ± 0.01

−18 ± 1 −30 ± 1

HEG-NP(org.) (−30 mV). The negative ζ-potential of HA-FA-HEG-NP is due to the negatively charged carboxylates groups of HA. Although the ζ-potential depends on many factors, the more negative ζ-potential measured for HA-FA-HEG-NP prepared from HEG-NP(org.) suggests a larger amount of HA in these particles than in those prepared from HEG-NP(H2O) in line with the higher concentration of azido terminated HEG side chains observed by FTIR in HEG-NP(org.). In order to further investigate this aspect, TGA measurements were performed on the two HEG-NP and on their corresponding derivative materials HA-FA-HEG-NP to estimate the amount of organic material attached to the NP. The TGA curves measured for these materials are shown in Fig. 5, while the weight loss as a percentage are reported in Table 3. The weight decreased below 120 °C can be attributed to the loss of water while the losses at higher temperatures are due to the decomposition of the organic layers [73]. Indeed, the TGA results confirmed that the coating of magnetic NP with HEG derivative 1 is more efficient in terms of surface coverage when it is carried out in the organic solvent than in water and the larger amount of azido terminated HEG side chains results in an increase of HA bonded to the

Fig. 4. Scattered intensity versus hydrodynamic diameter (left) and ζ-potential (right) curves measured for the HEG-NP prepared in organic solvent and for the its derived HA-FA-HEG-NP. 7

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Fig. 5. TGA curve measured for HEG-NP prepared in aqueous solution (black line, H2O) or organic solvent (dashed black line, org.) and for HA-FA-HEG-NP obtained starting from HEG-NP(H2O) (red line) or HEG-NP(org.) (dashed red line). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article).

Table 3 Results of the TGA measurements performed for HEG-NP prepared in water (HEG-NP(H2O)) and in the organic solvent (HEG-NP(org.)) and for HA-FAHEG-NP prepared starting from HEG-NP(H2O) or HEG-NP(org.). The weight losses between 100 and 800 °C correspond to the pyrolysis of the organic coating. Sample

Weight loss (100–800 °C) %

HEG-NP(H2O) HEG-NP(org.) HA-FA-HEG-NP(H2O) HA-FA-HEG-NP(org.)

4 9 8 16

Fig. 6. A) Hydrodynamic diameter versus time measured by DLS for dispersions of HA-FA-HEG-NP(org.) in DDW (full circles) and 0.1 M NaCl (empty circles). The nominal concentration of the HA-FA-HEG-NP was 0.028 mg/mL. B) Size distribution curves measured after overnight sedimentation of HA-FA-HEG-NP water dispersion (black solid curve) and after sonication (red dashed curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article).

magnetite NP. On the basis of the TGA analysis a rough estimation of the amount of HA on the NP can made. 3.3. Colloidal stability of HA-FA-HEG-NP dispersions It is well known that magnetite NP aggregate during the preparation and the aggregates grow in size and sediment in aqueous dispersions at neutral pH [74]. The aggregation and sedimentation processes occur even faster upon increasing the ionic strength of the solution. One of the purposes of the polymer coating is to increase the colloidal stability of the NP dispersions by preventing the aggregation of the particles. Therefore, in order to investigate the effect of the HA coating on the magnetite NP, DLS spectra were acquired at increasing times after sonication of HA-FA-HEG-NP(org.) water dispersions. DLS measurements do not shown any significant increase of the size of the particles in the dispersions. On the contrary, a slight decrease of the size was observed at longer times (Fig. 6A) and, after overnight sedimentation, the hydrodynamic diameter reduced from 473 ± 5 nm to 312 ± 12 (Fig. 6B). Since, after sonication, we do not observe any increase of the particle size with time, two hypothesis are possible: 1) HA-FA-HEG-NP aggregate faster than the time needed to acquire the first DLS curve or 2) aggregates of HA-FA-HEG-NP, with a size of a few hundred nm, were already formed during the synthesis. The sedimentation of the larger aggregates could be responsible for the decrease in size of the particles detected by DLS. Essentially, a similar behaviour was observed in 0.1 M NaCl, with an increase of the sedimentation rate due to the higher ionic strength of the solution. The larger hydrodynamic size of the aggregates of HA-FA-HEG-NP in 0.1 M NaCl solution than in DDW is probably due to the reduction of the electrostatic repulsion between the particles,

which makes the aggregation process faster and more effective. DLS measurements performed after 24 h of sedimentation show size values of the HA-FA-HEG-NP aggregates remaining in the surnatant around 320 nm. Further sonication of this dispersions produces a significant decrease of the size of the HA-FA-HEG-NP aggregates, which reaches 213 nm with a PDI of 0.24 (Fig. 6B). Hence, the aggregation of HA-FA-HEG-NP results reversible and the aggregates can be easily redispersed by sonication. Since DLS cannot be applied to study relatively concentrated dispersions because, in addition to multiple scattering effects, a large sedimentation rate negatively affects the results of the measurements, UV-visible spectrophotometry was used to investigate aggregation and sedimentation processes of HA-FA-HEG-NP at higher concentrations. Even if the intensity of light crossing a particles dispersion is measured at a wavelength at which the materials of the samples do not absorb, an absorbance or, more correctly, an optical density is measured since the particles in the dispersion scatter light in all directions [75,76]. The optical density depends on the number of particles per unit volume. Hence, the sedimentation of the aggregates should result in a decrease of the optical density with time. On the other hand, if aggregation of particles takes place, the optical density of the dispersions should increase with time due to the formation of larger aggregates, which 8

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scatter light more strongly. As a result, the overall behaviour of the optical density of the dispersions vs. the time depends on the rates of particle aggregation and sedimentation as well as on the particle size. The modelling of the optical density versus time curves to extract quantitative information is rather complicated due to the various contributions to the measured quantity. Nonetheless, some semi-quantitative information can be gathered from optical density vs. time curves. Optical density versus time after sonication curves measured for dispersions of bare Fe3O4 NP and HA-FA-HEG-NP in DDW (top panel) and in NaCl 0.1 M (bottom panel) are shown in Fig. 7A. For all curves the optical density decreases as a function of time after sonication of the dispersions. These results, consistently with those obtained by DLS, indicate the presence of aggregates of HA-FA-HEG-NP, which sediment. The comparison of the sedimentation curves shows that the coating with the polymer layer increases the colloidal stability of the NP and the effect is more prominent in 0.1 M NaCl solutions than in DDW. The results are consistent with the reduction of the electrostatic repulsion between the HA-FA-HEG-NP nanoparticles (which are negatively charged) due to the presence of electrolytes in solution. Hence, the adhesion of particle, leading to larger aggregates which sediment faster, is more probable in presence of ions than in pure water. We tried to model the optical density vs. time curves measured for HA-FA-HEG-NP in DDW and 0.1 M NaCl. We assumed that the optical density is proportional to the number of aggregates per unit volume, N, and that the aggregates can be represented as spheres of radius R. The decrease of N due to sedimentation is:

dN (t ) =

1 N (t ) vdt h

(3)

where h is the vertical size of the light beam crossing the cuvette, N(t) is the number of aggregates per cm3 at the time t, v is the steady rate of sedimentation given by:

v=

2g (

2 l) R

p

9

(4)

where g is the gravity acceleration, ρp and ρl are the densities of the particles and of the liquid, respectively, η is the viscosity of the dispersion and R the radius of the particles. N (and the optical density) of the dispersion decreases exponentially with time:

N(t) = N0 exp( k t)

(5)

where N0 is the concentration of aggregates at t=0 and k=v/h. On the basis of this model, a straight line should be obtained by plotting ln(A/A0) vs. time when v is constant, i.e. when R does not change in time. As a first approximation, the plots ln(A/A0) vs. time measured for HA-FA-HEG-NP in DDW and 0.1 M NaCl can be divided in two regions of linear behaviour that can be fitted by straight lines with different slopes (Fig. 7B). The slope is steep in the initial stages o because larger particles (with a faster sedimentation rate) sediment first while it becomes smooth in the later stages when smaller aggregates (with a slower sedimentation rate) sediment. 3.4. Magnetic properties Magnetization versus magnetic field curves measured for the bare magnetite NP and for HA-FA-HEG-NP at 300 and 2.5 K are shown in Fig. 8, top and middle panel. At 300 K no remanent magnetization is detectable, within the uncertainty of the measurement, either for bare Fe3O4 NP or HA-FA-HEGNP whereas a remanence and coercivity were observed at 2.5 K (Fig. 8, middle panel). Hence, both bare Fe3O4 NP and HA-FA-HEG-NP exhibit a superparamagnetic behaviour. The values of the most relevant magnetic parameters for the two samples are reported in Table 4. For the bare Fe3O4 NP the saturation magnetization (Ms) is slightly lower than that of the bulk magnetite but comparable with the best results reported for magnetite NP of similar size [77]. A decrease of Ms is expected for nanosized systems because of the relevance of surface layer where the spins are not well ordered as in the bulk region. The observed decrease of Ms of HA-FA-HEG-NP with respect to the bare NP is due to the weight fraction of the polymer layer which is diamagnetic and thus its contribution to the magnetization is negligible. The Ms values are in good agreement when the 15% in weight of polymer determined by TGA for HA-FA-HEG-NP is considered. For both temperature measurements, the magnetization curve of HA-FA-HEG-NP is almost coincident with that of bare NP when the curves are normalized to the respective Ms values. This suggests that the covalent bonding of APTES, directly on the magnetite surface, and of the polymeric organic layers does not affect significantly the magnetic properties of the bare magnetite NP. In order to obtain additional information about the magnetic properties, ZFC/FC curves, collected by measuring the magnetization as a function of temperature with (FC) and without (ZFC) an applied weak magnetic field, for the bare Fe3O4 NP and for HA-FA-HEG-NP are shown in the bottom panel of Fig. 8. ZFC curves do not show any clear maximum that could be associated with the blocking temperature (Tb) of the NP. Tb of non-interacting magnetite NP with a size of 10–15 nm should be well below 200–250 K. In the present case, however, the strong magnetic interactions of NP within the clusters could be responsible for the shift of Tb, which can be roughly identified with the deflection of the curves around 200 K and 300 K, for the magnetite NP and for HA-FA-HEG-NP, respectively. Usually, single magnetite particles with a size in the range of

Fig. 7. A) Optical density (absorbance) at 500 nm (normalized to the initial value A0) versus the time after sonication for dispersions of bare magnetite NP and HA-FA-HEG-NP in DDW (top panel) and in 0.1 M NaCl (bottom panel). B) Fitting of the curves ln(A/A0) versus time after sonication with two straight lines of different slopes. Top panel: HA-FA-HEG-NP in DDW. Bottom panel: HAFA-HEG-NP in 0.1 M NaCl. 9

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100–200 nm show ferrimagnetic properties. In our case, on the contrary, the aggregates of similar size are superparamagnetic, with no residual magnetization at 300 K because each nanoparticle behaves as an individual building block with its crystallographic orientation. A similar behaviour was observed by Ge et al. for colloidal nanocrystal clusters of magnetite NP [78]. These authors found that clusters with sizes ranging from 30 to 180 nm, composed of 10 nm size magnetite NP,

Fig. 8. Top panel: Magnetization (emu per g of sample) versus field intensity (Oe) for the bare magnetite NP (black curve) and for HA-FA-HEG-NP (red curve) measured at 300 K. In the inset the region around the zero intensity of the field is enlarged for magnetization curves normalized to the respective saturation magnetization (Ms). Middle panel: Magnetization curves (normalized to the respective Ms) versus the field intensity (Oe) for the bare magnetite NP (black curve) and for HA-FA-HEG-NP (red curve) measured at 2.5 K. The plot reports the low-field region to evidence the hysteretic behaviour. Bottom panel: ZFC (empty circles) and FC (full circles) curves measured with a 50 Oe probe field for the bare magnetite NP (black curves) and for HA-FA-HEG-NP (red curve). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article). Table 4 Results of the magnetic measurements performed for the bare Fe3O4 NP and for HA-FA-HEG-NP. Ms is the saturation magnetization measured at the specified temperature. Mr is the residual magnetization measured at 2.5 K. Hc is the coercitive field. Magnetization values are reported per grams of measured sample. Sample

Fe3O4-NP HA-FA-HEG-NP

Ms at 300 K

Ms a 2.5 K

Mr at 2.5 K

(emu/g)

(emu/g)

(emu/g)

71 61

79 69

26 23

Mr/Ms

Hc at 2.5 K (Oe)

0.33 0.33

310 370

showed superparamagnetic properties. 3.5. In vitro cytotoxicity: cell viability and morphology 3.5.1. Cell viability Cytotoxicity of HA-FA-HEG-NP and bare magnetite NP was evaluated by Neutral Red Uptake (NRU) on immortalized mouse fibroblasts NIH3T3, a cell line used as a model to screen cytotoxicity of novel compounds or carriers [79]. These cells were incubated with HA-FAHEG-NP and bare magnetite NP at concentrations of 5 × 10−3, 1 × 10−2, 2.5 × 10−2 , 5 × 10−2 and 1 × 10−1 mg/mL (mg of NP refers always to dry weight nanomaterial). Results, in terms of cell viability (%) as a function of samples concentration, are reported in Fig. 9A,B. The same experiment was performed by applying a permanent magnet at the bottom of the wells, in order to evaluate the effect of magnetic field in cell viability (Fig. 9C,D). The reported data after 24 h (Figs. 9A) and 48 h (Fig. 9B), showed that NIH3T3 viability in presence of HA-FA-HEG-NP was not statistically different from the control for concentration values ranging from 5 × 10−3 to 2.5 × 10−2 mg/mL while a cell viability reduction of about 20% (24 h) and 40% (48 h) was observed, with the highest concentration tested, i.e. 2.5 × 10−2 and 1 × 10−1 mg/mL. On the contrary, magnetite NP demonstrated to have a dose-dependent toxic effect towards mouse fibroblasts reducing drastically cell viability in comparison to control (Fig. 9A). The cytotoxicity of magnetite NP resulted increased after 48 h of incubation when the percentage of viable NIH3T3 in contact with bare NP decreased to about 15–20% (Fig. 9B). In vitro cytotoxicity data are consistent with the cytotoxicity studies reported in literature in which the toxic effects of bare magnetite nanoparticles are related to concentration and exposure time [80]. In fact, it is well documented that magnetite nanoparticles are capable of interacting with cells by different uptake mechanisms. The most important are passive diffusion, receptor mediated endocytosis, clatharin- or caveoline-mediated endocytosis. Inside the cells, magnetite NP are degraded by lysosomial enzymes generating ions capable to form reactive oxygen species (ROS), which alter mitochondrial and other organelle functions inducing the inflammatory process [81]. The same experiment, repeated applying a permanent magnet to cells in contact with both HA-FA-HEG-NP and magnetite NP, showed 10

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Fig. 9. Percentage of viable NIH3T3 cells after incubation for 24 h (A) and 48 h (B) and for 24 h (C) and 48 h (D) in presence of external magnet with HAFA-HEG-NP and bare magnetite NP as determined by the NRU assay. Data are mean ± SD of three experiments run in six replicates. *Values are statistically different versus negative control (complete medium), p < 0.05.

that cell viability was not affected by the applied magnetic field (Fig. 9C,D). Indeed, the percentage of viable NIH3T3 was not statistically different from that determined without the magnet for both the HA-FA-HEG-NP and magnetite NP at any tested concentration.

maintained bipolar and multipolar shape with regular borders, and a major change of cell morphology was observed only for the highest concentrations tested (5 × 10−2 and 1 × 10−1 mg/mL). The same trend was observed after 48 h of contact. In this case, however, as the concentrations of HA-FA-HEG-NP increased, there was a more marked increase in circularity and an equally marked decrease in elongation (Fig. 10C and Table 5B). In fact, many cells maintained fibroblastic morphology at concentration values of 5 × 10−3, 1.0 and 2.5 × 10−2 mg/mL, but at 5 × 10−2 and 1 × 10−1 mg/mL NIH3T3 showed mainly round morphology with irregular borders (Fig. 10). The decrease of cell elongation and the increase of cell circularity was observed also in cells in contact with bare magnetite NP, but in this case cell morphology resulted significantly modified already after 24 h (Table 5A, Fig. 11). Only a few cells maintained the typical fibroblastic morphology and most of them assumed a round or irregular shape as demonstrated by optical microscope images reported in Fig. 10B. By increasing the incubation time till 48 h, elongation and circularity resulted dramatically modified (Table 5B) at all the bare magnetite NP concentrations tested. All the few viable fibroblasts resulted to have rounded and, mainly, irregular morphology with very irregular

3.5.2. Cell morphology NIH3T3 fibroblasts are bipolar or multipolar cells with an elongated shape, which grow attached to a substrate. Changes in NIH3T3 morphology (i.e. elongation and circularity) after exposure to materials is correlated to the toxic effect of the test sample towards cells. Therefore, morphological analysis results important to investigate the physiological state of the cells, and it can be commonly used as a qualitative and quantitative measure of biological assays. In particular, a relative tendency for decreased elongation appears to be associated with increased circularity and with the ability of the material to interfere with cell viability [57]. After 24 h of incubation, the increase of HA-FA-HEG-NP concentration induced the decrease of cell elongation and the increase of cell circularity (Table 5A). Anyway, as demonstrated also by optical microscope images reported in Fig. 10A and Fig. 11, most of cells

Table 5 Cell elongation and circularity after 24 h (A) and 48 h (B) of contact with HA-FA-HEG-NP and magnetite NP. *Values are statistically different versus negative control (complete medium), p < 0.05. A)

HA-FA-HEG-NP

Magnetite NP

Concentration (mg/mL)

Elongation (mean ± SD)

Circolarity (mean ± SD)

Elongation (mean ± SD)

Circolarity (mean ± SD)

Control 5.0 × 10−3 1.0 × 10−2 2.5 × 10−2 5.0 × 10−2 1.0 × 10−1

1.43 ± 0.07 1.38 ± 0.06 *1.18 ± 0.06 *9.41 × 10−1 ± 0.08 *8.22 × 10−1 ± 0.09 *7.73 × 10−1 ± 0.05

0.369 ± 0.115 0.399 ± 0.106 *0.457 ± 0.105 *0.497 ± 0.129 *0.562 ± 0.116 *0.579 ± 0.119

1.43 ± 0.07 *6.13 × 10−1 ± 0.08 *5.89 × 10−1 ± 0.06 *5.96 × 10−1 ± 0.09 *5.34 × 10−1 ± 0.08 *4.45 × 10−1 ± 0.05

0.369 ± 0.115 *0.703 ± 0.141 *0.700 ± 0.141 *0.734 ± 0.119 *0.647 ± 0.110 *0.676 ± 0.135

B) Concentration (mg/mL)

HA-FA-HEG-NP Elongation (mean ± SD)

Circolarity (mean ± SD)

Magnetite NP Elongation (mean ± SD)

Circolarity (mean ± SD)

Control 5.0 × 10−3 1.0 × 10−2 2.5 × 10−2 5.0 × 10−2 1.0 × 10−1

1.45 ± 0.04 1.31 ± 0.08 *9.94 × 10−1 ± 0.05 *7.63 × 10−1 ± 0.07 *5.38 × 10−1 ± 0.09 *5.59 × 10−1 ± 0.08

0.378 ± 0.139 0.357 ± 0.150 *0.679 ± 0.171 *0.658 ± 0.183 *0.794 ± 0.098 *0.734 ± 0.125

1,45 ± 0.04 *5.31 × 10−1 ± 0.08 *2.94 × 10−1 ± 0.07 *2.85 × 10−1 ± 0.07 *2.96 × 10−1 ± 0.13 *2.88 × 10−1 ± 0.11

0,378 ± 0.139 *0.826 ± 0.065 *0.786 ± 0.068 *0.783 ± 0.072 *0.799 ± 0.085 *0.780 ± 0.104

11

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Fig. 10. Optical microscope images (20x) of NIH3T3 after 24 h of contact with different concentrations (mg/mL) of HA-FA-HEG-NP (line A), magnetite NP (line B), and after 48 h of contact with different concentrations (mg/mL) of HA-FA-HEG-NP (line C), magnetite NP (line D). The scale bars correspond to 5 μm.

bearing azido and carboxylic groups at the ends. We found that when the amidation reaction is carried out in organic solvent instead of in water, a larger amount of organic molecules was attached to NH2-NP. DLS results showed that the size of the particles in the aqueous dispersions of bare and coated magnetite NP ranges from 200 to 500 nm. The size is much larger than that of single core magnetite NP as determined previously by XRD [52]. Hence, DLS results suggest that the particles consist of multicore aggregates of individual magnetite NP. The formation of these clusters occurs during the co-precipitation stage, prior to the surface functionalization and coating, and the clusters are not disaggregated during the functionalization steps. Aqueous dispersions of HA-FA-HEG-NP are significantly more stable than those of bare NP, in particular in 0.1 M NaCl solutions, proving that the coating with the HA layer provides an electrostatic and steric stabilization. Magnetization and FC/ZFC curves showed that HA-FA-HEG-NP has a superparamagnetic behaviour. Since magnetite particles with a size of hundreds of nanometers should be ferrimagnetic, these results suggest that the magnetic properties of the clusters, for both bare and coated NP, are determined by the size of the primary particles rather than by that of the multicore aggregates. In vitro cytotoxicity studies revealed a significant increase in cell vitality of the cells exposed to the magnetic NP derivatized with HA respect to the bare magnetite NP, confirming the quality of our covalent HA masking strategy capable of providing, even to an inorganic material such as magnetite, useful properties for the development of healthcare materials.

Fig. 11. Details of the optical microscope images (20x) of NIH3T3 after 24 h and 48 h of contact with 5.0EXP-3 mg/mL of HA-FA-HEG-NP and the same amount of magnetite NP.

borders (Fig. 10D). The obtained morphometric results suggest that HAFA-HEG-NP induce a moderate decrease of cell elongation in a concentration-dependent manner, while bare magnetite NP are capable of disrupting the cytoskeleton proteins and the dynamic meshwork of Factin producing cell death.

Declaration of competing interest There is no conflict of interest to be declared.

4. Conclusions

Acknowledgments

We have shown that the hyaluronic acid derivative HA-FA-Pg can be covalently linked to magnetite NP via CuAAC “click” reaction. For this purpose magnetite NP were firstly functionalized with amino groups, which were reacted with an hexa(ethylene glycol) derivative

Thanks are due to Italian MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca) for financial support, Grant “Dipartimento di Eccellenza 2018–2022”. 12

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Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110271.

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