Structural and magnetic characteristics of carboxymethyl dextran coated magnetic nanoparticles: From characterization to immobilization application

Structural and magnetic characteristics of carboxymethyl dextran coated magnetic nanoparticles: From characterization to immobilization application

Reactive and Functional Polymers 148 (2020) 104481 Contents lists available at ScienceDirect Reactive and Functional Polymers journal homepage: www...

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Reactive and Functional Polymers 148 (2020) 104481

Contents lists available at ScienceDirect

Reactive and Functional Polymers journal homepage: www.elsevier.com/locate/react

Structural and magnetic characteristics of carboxymethyl dextran coated magnetic nanoparticles: From characterization to immobilization application

T

Katja Vasića, Željko Kneza,b, Elizaveta A. Konstantinovac,d, Alexander I. Kokorine, ⁎ Sašo Gyergyeka,f, Maja Leitgeba,b, a

University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova 17, SI-2000 Maribor, Slovenia University of Maribor, Faculty of Medicine, Taborska ulica 8, SI-2000 Maribor, Slovenia M. V. Lomonosov Moscow State University, Faculty of Physics, Leninskie Gory 1/2, 119991 Moscow, Russia d National Research Center “Kurchatov Institute”, Kurchatov Square 1, Moscow 123182, Russia e N. N. Semenov Federal Research Center of Chemical Physics, Kosygin st. 4, 119991 Moscow, Russia f Jožef Stefan Institute, Department for Materials Synthesis, Jamova 39, SI-1000 Ljubljana, Slovenia b c

ARTICLE INFO

ABSTRACT

Keywords: Carboxymethyl dextran Magnetic nanoparticles Characterization analysis ADH Immobilization

MNPs were synthesized with co-precipitation of ferrous and ferric ions. Carboxymethyl dextran (CMD) was covalently bound to MNPs and the influence of different concentrations on characteristics of CMD coated magnetic nanoparticles (CMD-MNPs) was studied. Different concentrations of CMD were applied into synthesis process. The surface morphology of CMD-MNPs was monitored by scanning and transmission electron microscopy, where their spherical shape was confirmed. Fourier transform infrared spectroscopy displayed characteristic bonds that confirmed the presence of CMD hydroxyl and carboxyl groups. Thermogravimetric analysis displayed a weight loss, which confirmed the coating weight of CMD. ζ-potential measurements revealed negatively charged hydroxyl groups of CMD, and polydispersity index (PdI) showed the most consistent sizes of CMD3-MNPs. Electron paramagnetic resonance and magnetization measurements confirmed a ferromagnetic system for all CMD-MNPs. Prepared CMD3-MNPs were used as carriers for immobilization of enzyme alcohol dehydrogenase (ADH). Immobilization was carried out at two different temperatures (20 °C and 4 °C) and thermal stability at 20 °C and 40 °C after 24 h was studied. Prepared CMD-MNPs exhibit a layer of CMD coating that provides proper magnetic and structural properties and can therefore be functionalized and used in bioactive compound immobilization, such as ADH.

1. Introduction The ever expanding boom in nanotechnology has widened the horizons for new, unique techniques in nano-drug and nano-carrier technology in the field of biomedicine [1–5]. The main challenge remains in the use of synthetic nanoparticles, which can show side effects in a biological system and can result in some unpredictable manifestations [6,7]. To overcome this obstacle, techniques for binding bioactive, biological, biodegradable and non-toxic substances must be developed. Since such materials belong to a class of materials with numerous potential applications in clinical and sciences, it is necessary that these materials be of appropriate surface chemistry, appropriate sizes in the nano-range, narrow particle size distribution, and most of all, such materials must exhibit biocompatible and non-toxic ⁎

characteristics [8–12]. From this point of view, we decided to examine the use of magnetic nanoparticles (MNPs) as carriers. The properties of MNPs are determined by many factors, such as particle size, chemical composition and interactions with surrounding materials. The main challenge in the synthesis of MNPs is their tendency to aggregate, since they have a large surface-to-volume ratio. It is also important to obtain monodispersed nanoparticles of appropriate size and shape. To avoid these obstacles, special surface coatings with non-toxic and biocompatible characteristics, which also allow targeted delivery for drug-based transport to desired locations in biological systems, must be applied. In the past few years, many polymers have been investigated as suitable coating agents, and many of these have been reported to be efficient materials for MNP preparation, including chitosan, starch,

Corresponding author at: University of Maribor, Faculty of Chemistry and Chemical Engineering, Smetanova 17, SI-2000 Maribor, Slovenia. E-mail address: [email protected] (M. Leitgeb).

https://doi.org/10.1016/j.reactfunctpolym.2020.104481 Received 14 November 2019; Received in revised form 1 January 2020; Accepted 3 January 2020 Available online 07 January 2020 1381-5148/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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albumin, alginate, polyethylene glycol (PEG), polyvinyl alcohol (PVA) and different polyoxamines [8,13–22]. Also, the use of amphiphilic polymers has been studied. For example, hydrophobically capped nanocrystals are transferred from an organic to an aqueous solution by wrapping an amphiphilic polymer around the particles [23]; encapsulation inside amphiphilic polymers was also studied and proved to be a versatile and effective method of providing water-solubility and colloidal stability [24–26]. Among these agents, dextran is often used as a choice of polymer coating because of its degradability by dextranase, biocompatibility and non-toxicity [27,28]. It has also been shown that coating MNPs with hydrophilic organic polymers such as dextran can improve their characteristics for drug delivery and colloidal stability, which is considered a valuable strategy in synthesizing MNPs [1,12,29,30]. Dextran is a water-soluble hydrophilic polymer made of many glucose molecules consisting of mostly α-1, 6-glycosidic linkages, which in alkaline solutions are physically adsorbed with non-covalent interactions onto MNPs. Hydroxylic groups can easily be cross-linked to dextran and functionalized with primary amines, and therefore be able to bind to targeted ligands. Dextran is also used in medicine as an antithrombotic agent to reduce blood viscosity and as a volume expander in hypovolaemia, because of its simple and non-immunogenic biopolymeric nature. It can be found in certain lactic acid bacteria, such as Leuconostoc mesenteroides and Streptococcus mutans [31–33]. The established platform of dextran MNPs has been proven to be an important part of personalized medicine, since they play a crucial role in the development of novel therapeutics and diagnostics [16,29,34]. MNPs with coatings such as dextran are widely utilized in many experiments for different reasons: they are biocompatible with favorable properties, the MNP surfaces can easily be modified and can be further developed with functionalization of different antibodies, peptides or enzymes [35,36]. Dextran, as a coating polymer which is used in precipitation from alkaline solutions containing a mixture of Fe2+ and Fe3+ ions, can result in biocompatible materials for in vivo use [37,38]. A recent study reports on chemical synthesis of a class of dextran superparamagnetic NPs that bear hydrophilic porphyrin units covalently grafted by a click chemistry reaction in aqueous solutions. Such MNPs are used in MRI and hyperthermia [39]. The current research on superparamagnetic iron oxide nanoparticles (SPIONs) is widening their use as diagnostic agents in MRI, as well as for drug delivery vehicles. Arora et al. reviewed the understanding of SPIONs with regard to their method of preparation and their utility as drug delivery systems [40] and Harivardhan et al. conducted a comprehensive study on the design, characterization, toxicity, biocompatibility, pharmaceutical and biomedical applications of MNPs [41]. Some research articles investigate different polymers used as coating materials for MNPs, varying from chitosan, collagen or chitosan-collagen are used as supports for enzyme immobilization, in particular for immobilization of lipase on such coated materials crosslinked with squaric acid [42]. Other studies use heparin as coating material for MNPs. Such MNPs (Hep-MION) can effectively promote transcellular transport [43]. Moreover, toxicity of MNPs coated with tetraethyl orthosilicate (TEOS), 3-aminopropyltrimethoxysilane (APTMS) or TEOS/APTMS have been investigated against human normal fibroblasts and fibrosarcoma cells [44]. A study that uses diethylaminoethyl (DEAE) dextran as coating material for MNPs synthesis investigated several procedures for sterilization and preservation of such MNPs. In this manner, DEAE dextran coated MNPs were treated by freezing, autoclaving, lyophilization and UV radiation [45]. There is also a NanoMag project study [46] that presents classification of different magnetic single- and multi-core particle systems using their measured dynamic magnetic properties. Coating materials for MNPs used in this study are silica, polystyrene and also dextran. ADH, being an important biological catalyst and an enzyme with many potential applications in various chemical industries, has low stability, which limits its use in these industrial applications. Therefore

it is important to improve ADH's stability in form of providing the appropriate carrier for immobilization to enhance the enzyme's activity and most importantly, to improve the enzyme's stability [47–49]. As there are many research studies on synthesis of MNPs using different dextrans, some dated even in the early 80's and 90's from Molday et al., Jung et al. to Jordan et al. [50–52], there are no research reports that investigate the use of its derivative carboxymethyl dextran (CMD) in the synthesis process of MNPs for enzyme immobilization. CMD provides not only hydroxyl functional groups but also additional carboxyl functional groups. These multiple functional groups allow facile chemical modification. CMD has also excellent water solubility, high biocompatibility and biodegradability [53]. However, an investigation of superparamagnetic NPs functionalized with CMD is reported, but synthesized in a two-step method. Firstly, MNPs were synthesized with co-precipitation of Fe2+ and Fe3+ ions and coated with dextran. Later on, dextran on the surface was reacted with monochloroacetic acid (MCA) in an alkaline solution in order to prepare CMD coated MNPs. The prepared CMD coated MNPs were investigated for MRI applications [54]. There are also studies describing CMD-based polymeric conjugates for cancer immunotherapy [53], magnetofluorescent nanoprobes chelated on CMD coated MNPs for MRI applications [55], CMD-based hypoxia-responsive MNPs for doxorubicin delivery [56] and also some studies describing complexes of CMD and carboxymethyl chitosan [57–60]. Our study describes synthesis of MNPs using CMD directly in the synthesis process, where the final product in a form of prepared nano-carrier (CMD3-MNPs) is applied in immobilization process of ADH. Finally, dextran coated MNPs are approved for human in vivo use by the Food and Drug Administration [61], dextran as coating material has been shown, together with polyethylene glycol, to reduce cytotoxicity in aortic endothelial cells [62]. Such promising properties of polymer dextran provide a wide range of possible applications for the use of our MNPs coated with CMD in fields of targeted drug delivery systems. In this manner, in order to investigate MNPs coated with CMD, the aim of our study was to find a suitable concentration of CMD coating on MNPs to form a biocompatible and non-toxic compound that can prevent the degradation of iron oxide nanoparticles in an aggressive environment and provide functional groups for possible bioactive compound attachment, as is a successful ADH immobilization. 2. Materials and methods 2.1. Materials Iron (III) chloride hexahydrate (FeCl3∙6H2O) and iron (II) chloride tetrahydrate (FeCl2∙4H2O) were obtained from Merck, and CMD sodium salt (MW = 10,000–20,000 Da), EClH (1-Chloro-2,3-epoxypropane), sodium pyrophosphate, sodium phosphate, ethanol, β-Nicotinamide adenine dinucleotide (NAD) and ADH were obtained from Sigma Aldrich. Ammonium hydroxide was purchased from Chem-Lab, Belgium. All reagents were of analytical grade and used without further purification. In all experiments, deionized water was used. 2.2. Experimental 2.2.1. Preparation of CMD solutions Three CMD solutions with different concentrations were prepared using CMD sodium salt in water solution. 0.25 g/mL, 0.4 g/mL and 0.5 g/mL concentrations of CMD were prepared in deionized water heated to 40 °C for 30 min when clear solution of the polymer was obtained. 2.2.2. Synthesis of CMD coated MNPs Ferric (III) ions (FeCl3∙6H2O) and ferrous (II) ions (FeCl2∙4H2O) were combined in a 2:1 M ratio and dissolved completely in 40 mL of deionized water. 20 mL of CMD solution (each of a different concentration) 2

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and 5 mL of 25% ammonium solution were added into a three-neck flask equipped with a mechanical stirrer. Later on, solutions of FeCl3∙6H2O and FeCl2∙4H2O were added to the mixture. The solution was vigorously stirred at 500 rpm in a nitrogen atmosphere and heated to 85 °C for 1 h. After synthesis, the CMD coated MNPs obtained using coating concentrations of 0.25 g/mL of CMD (CMD1-MNPs), 0.4 g/mL of CMD (CMD2-MNPs) and 0.5 g/mL of CMD (CMD3-MNPs), which were obtained in a solution, were washed three times with deionized water and two times with ethanol, using a magnet to remove excess ammonia and CMD, and later on dried at 40 °C.

2.3.7. Magnetization measurements The room-temperature magnetization curves of all CMD-MNPs and uncoated MNPs were measured with a Lake Shore 7307 vibratingsample magnetometer (VSM). 2.3.8. Immobilization of ADH onto CMD3-MNPs 20 mg of CMD3-MNPs were added to 1 mL of 0.1 M sodium acetate buffer with pH 7.5. ADH (7.4 U) was added to the solution and stirred for 2 h at 300 rpm/min. First immobilization was performed at 20 °C and second immobilization was performed at 4 °C. After 2 h immobilization the obtained ADH immobilized CMD3-MNPs were washed 2 times with distilled water and magnetically separated using a permanent magnet. Activity assay for ADH was performed.

2.3. MNP characterization 2.3.1. Fourier transform infrared spectroscopy (FT-IR) To study chemical bonds formed between the MNPs and different CMD coatings, FT-IR analysis of the samples was performed by pressing the samples to form a tablet using KBr as the matrix. The spectra were detected over a range of 4000–500 cm−1 and recorded by a FT-IR spectrophotometer (Perkin Elmer 1600 Fourier transform infrared spectroscopy).

2.3.9. Thermal stability of immobilized ADH After immobilization of ADH onto CMD3-MNPs when applying 4 °C as immobilization temperature with conditions stated in chapter 2.3.8, thermal stability of such immobilized ADH was investigated at 20 °C and 40 °C. Residual activity was determined after 3 h, 5 h and 24 h of incubation time at certain temperature. Residual activity was calculated using Eq. (1):

2.3.2. Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) was performed on a TGA/DSC1 (Shimadzu, IRAffinity-1, Mettler Toledo, Switzerland), with a heating rate of 10 °C/min from room temperature to 600 °C in an N2 atmosphere. Temperature precision was ± 0.3 °C and temperature accuracy was ± 0.5 °C.

Residual activity (%) =

activity of immobilized ADHafter incubation activity of immobilized ADHbefore incubation

100 (1)

2.3.10. Assay for ADH activity The activity of soluble and immobilized ADH was determined spectrophotometrically using ethanol as substrate. The standard reaction mixture in a total volume of 3 mL contained 22 mM sodium pyrophosphate, 3.2% (v/v) ethanol, 7.5 mM β-NAD, 0.3 mM sodium phosphate and 0.003% (w/v) BSA. The reaction was initiated by the addition of ethanol and β-NAD to soluble or immobilized ADH, and subsequently the increase in absorbance at 340 nm due to formation of β-NADH was measured. The activity of soluble and immobilized ADH was calculated using Eq. (2):

2.3.3. Scanning electron microscopy (SEM) SEM analysis was performed using a scanning electronic microscope (FE, SEM SIRION, 400 NC, FEI) to investigate morphology and size of prepared CMD-MNPs. The samples were measured on a gold (Au) substrate. Also, energy-dispersive x-ray spectroscopy (EDX) microanalysis was performed for chemical composition analysis. 2.3.4. Transmission electron microscopy (TEM) TEM images of uncoated MNPs and CMD3-MNPs were acquired using Jeol 2010F transmission electron microscope. A field emission source was operated at 200 kV. Nanoparticles were deposited on a copper-grid-supported holey carbon foil.

Units enzyme mL ( A340 nm/ min) of SAMPLE =

2.3.5. Dynamic light scattering (DLS) Dynamic light scattering (DLS) was performed to analyze hydrodynamic size, size distribution and ζ-potential of prepared CMD-MNPs. The measurements were performed on a Zetasizer Nano ZS. Each diameter value was the average of three consecutive measurements. Higher values indicate a very broad size distribution, whereas lower values correspond to more or less monodisperse particle size distributions. Measurement was carried out under equilibrium conditions. Measured samples were dispersed in water with neutral pH at room temperature. Concentration of all CMD-MNPs and uncoated MNPs were 2 mg/mL.

( A340 nm / min ) of BLANK 6.22 0.1

3

df (2)

where:3 - total volume (mL) of assaydf - dilution factor6.22 millimolar extinction coefficient of β-NADH at 340 nm0.1 - volume (mL) of enzyme used ADH activity was measured by Sigma-Aldrich enzymatic assay [63], and the residual activity was calculated using Eq. (3):

Residual activity (%) =

activity of immobilized ADH activity of soluble ADH

100

(3)

3. Results and discussion

2.3.6. Electron paramagnetic resonance (EPR) EPR spectra were detected using the standard Bruker ELEXSYS-500 X-band EPR spectrometer with a modulation frequency of 100 kHz at room temperature (25 °C) in thin quartz tubes 1.0 mm in diameter in air. EPR spectra at 77 K were recorded using an X-band Bruker EMX-8 spectrometer. For precise calibration of the magnetic field, the external reference standard of DPPH (g0 = 2.0036) and of Mn2+ ions in a matrix of MgO were used. For calculating the effective g-value from experimental EPR spectra, a magnetic field value in the middle of the spectrum line amplitude was used. The amount of all samples was in the range of 0.3–0.7 mg.

3.1. Synthesis of CMD-MNPs CMD-MNPs were prepared with three different concentrations of CMD applied to the synthesis process (0.25 mg/mL, 0.4 g/mL and 0.5 g/ mL). Concentrations of 0.25 g/mL, 0.4 g/mL and 0.5 g/mL of CMD were applied to the synthesis process of CMD-MNPs in order to obtain particles of nano-scale, since higher concentrations (above 0.5 g/mL) of CMD can cause increased thickening of the CMD-MNP coating layer, which could result in particles of micro-scale. As lower concentrations of CMD were used, all CMD-MNPs obtained exhibited nano-sized properties. Characterization of such CMD-MNPs was investigated. 3

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Fig. 1. FT-IR spectra of CMD1-MNPs, CMD2-MNPs, CMD3-MNPs, uncoated MNPs and plain CMD.

groups in CMD chains, as well as carboxylic groups around 1600 cm−1. The stretching vibration of the CeH bond is found at 2900 cm−1. Peaks at 1410, 1330 and 1150 cm−1 for CMD2-MNPs and CMD3-MNPs can be assigned to the deformation vibration of CeH of CMD chains. Both spectra also indicate the characteristic νC–O vibrations at 1010 cm−1. CMD was similarly confirmed also by Liu et al. in [54]. Quantification of different amounts of CMD coatings were analyzed using thermogravimetric analysis (TGA). The results are shown in Fig. 2. In the thermal decomposition curve, the initial weight loss for all samples occurs in the temperature range of 70–150 °C because of physically adsorbed water. The second remarkable weight loss occurs in the temperature range of 150–350 °C, which is a typically high enough temperature to induce thermal degradation of ordinary polymers. This corresponds to the decomposition of CMD, since the thermal decomposition of pure CMD (Fig. 2d) shows that CMD degrades between 250 and 350 °C. The results confirm that CMD has been coated to the nanoparticle surface, as more energy is required to break down the

3.2. Characterization of CMD-MNPs After preparation and synthesis of the CMD-MNPs, the presence of CMD was confirmed with various analytical methods. Firstly, FT-IR and TGA analysis were performed, which revealed and confirmed successful binding of CMD onto the surface of MNPs. Secondly, characteristics of the obtained CMD-MNPs, observing size and morphology of MNPs, were investigated using SEM, TEM and DLS techniques. EPR spectroscopy of the CMD-MNPs was performed to study the paramagnetic centers of the metal complexes in the CMD-MNPs. Lastly, VSM measurements were performed to investigate magnetic properties of all CMD-MNPs. Fig. 1 illustrates the FT-IR spectra of CMD1-MNPs, CMD2-MNPs, CMD3-MNPs, uncoated MNPs and plain carboxymethyl dextran. The FT-IR spectra of uncoated MNPs show that the characteristic absorption peaks at 569 cm−1 and 682 cm−1 belong to the stretching vibration modes of the FeeO bond. In addition, the peak centered around 1624 cm−1 can be ascribed to the H-O-H bending mode of water adsorbed at the Fe3O4 surface [64,65]. The intensity of absorption peaks at 569 cm−1 and at 682 cm−1 were slightly reduced after functionalization of uncoated MNPs with CMD polymer. The FT-IR spectrum of CMD1-MNPs shows a strong adsorption peak at 2900 cm−1, which is assigned to CMD νC–H and δC–H vibrational modes. Also, absorption peaks at 1375–1460 cm−1 can be observed, which also correspond to the afore-mentioned vibrational modes. Absorption peaks at 1600 cm−1 are attributed to carboxylic groups of CMD. FT-IR spectrum of plain CMD shows absorption peaks of carboxyl (1600 cm−1) and hydroxyl (3417 cm−1) groups, which are clearly visible. The absorption peak at 1010–1012 cm−1 corresponds to CMD νC–O vibrations, which can be seen in all of the CMD-MNP, whereas the broad absorption peak at 3408 cm−1 corresponds to the characteristic νO–H stretching and δO–H deformation modes of CMD hydroxyl groups, also seen in all CMDMNPs. Both CMD2-MNPs and CMD3-MNPs show similar spectra, differing in the intensity of absorption peaks caused by different coating amounts of CMD. In both CMD2-MNPs and CMD3-MNPs, spectra presence of CMD is observed and confirmed via FT-IR spectroscopy. The different amounts of CMD resulted in similarities of the spectra, containing broad absorption peaks at 3400 cm−1, indicating the structural OeH hydroxyl

Fig. 2. TGA curves for (a) CMD1-MNPs, (b) CMD2-MNPs, (c) CMD3-MNPs and (d) CMD. 4

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Fig. 3. SEM of CMD-MNPs coated with CMD concentrations of (a) 2.5 mg/mL (CMD1-MNPs), (b) 4 mg/mL (CMD2-MNPs), (c) 5 mg/mL (CMD3-MNPs) and (d) uncoated MNPs.

amount of CMD, which prevents agglomeration of nanoparticles to some extent. CMD1-MNPs gave the smallest nanoparticles, most likely because the high viscosity of the solution inhibited iron ion transport to the surface of the nanoparticles. Size distribution analysis (Fig. 4) of each prepared CMD-MNP revealed that the nanoparticle size of CMD1-MNPs varies from 10 to 60 nm with an average of 27 nm, for CMD2-MNPs from 15 to 50 nm with an average of 30 nm, and for CMD3-MNPs from 10 to 50 nm with an average of 28 nm, according to the most frequent size of each nanoparticle. CMD3-MNPs presented the highest consistency in particle size, which was confirmed by the lowest polydispersity index (PdI) measurements for CMD3-MNPs (see paragraph 3.1.4). Additional valuable information was obtained with the use of EDX spectral analysis of CMD3-MNPs (Fig. 5), and the results are presented in Table 1. EDX analysis revealed peaks of C, O and Fe with (w/w) % of approx. 15, 29 and 56, respectively. This clearly suggests that C, O and Fe are the main constituents in the MNPs. In both samples (uncoated MNPs and CMD3-MNPs) nanoparticles deposited as a rather thick agglomerates (Fig. 6 a and b). Only on the edges of the agglomerates the number density of the nanoparticles was low enough that individual nanoparticles can be resolved, thus making estimation of their size distribution rather unreliable. However, by inspecting a large number of images we are able to conclude that in both samples the majority of the nanoparticles are in the size range between 8 nm to 38 nm. High-resolution imaging showed that individual nanoparticles are monocrystals (insets in Fig. 6 a and b). The hydrodynamic diameter of all prepared CMD coated and uncoated MNPs was measured by DLS. The average hydrodynamic sizes were reported as the z-average, while the variability of particle size within different amounts of CMD was quantified by the PdI. The results are presented in Table 2. The size measurements obtained by DLS are of water contained

covalent bonds prior to degradation of the coating [66–69]. When CMD is decomposed completely, the residue that remains is composed mostly of iron MNPs. From the results obtained by TGA measurements, we can also estimate the polymer content (CMD) for each of the CMD-MNPs. The coating efficiency is therefore calculated to be 34.84% (CMD1MNPs), 25.56% (CMD2-MNPs) and 37.80% (CMD3-MNPs). The highest polymer content occurs in CMD3-MNPs since the highest concentration of CMD has more chance to come in contact with MNPs. With increasing CMD concentration, CMD chains become more tangled on the surface of MNPs, which allows more CMD to covalently bind to the nanoparticle surface. For CMD2-MNPs, lower polymer content appears. Nevertheless, all profiles of weight losses display a single drop in mass with increasing temperature, which indicates a single layer of CMD on the nanoparticle surface. CMD coated MNPs, reported by Liu et al., displayed different polymer content, since the synthesis protocol differed from the one we investigated. Lower polymer contents are a result of different CMD concentrations, as well as different synthesis protocols [54]. The surface morphology and particle size of CMD coated and uncoated MNPs were observed by SEM, as shown in Fig. 3. From Fig. 3 we can clearly see that most of the particles are spherical in shape with estimated mean diameters of 27 nm (CMD1-MNPs), 30 nm (CMD2MNPs), 28 nm (CMD3-MNPs) and 37 nm (uncoated MNPs). Compared to research published by Liu et al., the sizes of our CMD-MNPs are more uniform [54]. Since MNPs in general have large specific surface area and high surface energy (large surface-to-volume ratio), they tend to aggregate. Therefore, there can still be some agglomerates observed in SEM images. SEM images also show the reduction in MNP diameter when they are coated with CMD (CMD1-MNPs, CMD2-MNPs and CMD3-MNPs). The diameter of uncoated MNPs was reduced from 37 nm to 27 nm in the case of CMD1-MNPs, to 30 nm in the case of CMD2-MNPs and to 28 nm in the case of CMD3-MNPs. This is likely a result of the larger 5

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Fig. 4. Size distribution graphs of (a) CMD1-MNPs, (b) CMD2-MNPs, (c) CMD3-MNPs and (d) uncoated MNPs. (Size distribution graphs were created with software supported statistical analysis (using ImageJ and Origin) of SEM images.).

Fig. 5. EDX elemental analysis of CMD3-MNPs. 6

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Table 1 EDX spectral analysis of CMD3-MNPs.

Table 2 Hydrodynamic size and PdI of uncoated and different CMD-MNPs.

Element

Weight %

Atomic %

Sample

Mean diameter (nm)

PdI

C O Fe

15 29 56

31 45 24

CMD1-MNPs CMD2-MNPs CMD3-MNPs Uncoated MNPs

369 307 290 225

0.49 0.47 0.31 0.72

particles (hydrodynamic radius), whereas the size measurements observed by SEM correspond to particles in a dried state. Therefore, particle sizes observed by SEM cannot be compared to the particle sizes measured with DLS, since measurements with DLS give much higher values. This observation can be attributed to the effect of large particles on hydrodynamic radius, while DLS provides z-average of the radius. Another reason can also be attributed to the fact that iron magnetic nanoparticles have a large surface-to-volume ratio, which results in nanoparticle tendency to aggregate in order to reduce their surface energy. The difference in mean sizes obtained by DLS and SEM (or TEM) has been observed before and is reported in many research papers [8,12,30,70]. The size distribution of samples is expressed in the PdI, which corresponds to the variance of the size distribution of the nanoparticles. Samples with wider range of particle sizes have higher PdI values, therefore consisting of more unevenly sized particles [71], as it can be observed in uncoated MNPs, giving the highest PdI value of 0.72, as presented in Table 2. It can be observed that CMD3-MNPs gave the lowest PdI values, which reflects more consistent and evenly sized particles, than the other CMD1-MNPs and CMD2-MNPs. Therefore, CMD3-MNPs indicate more monodisperse MNPs and grater particle stability, than CMD1-MNPs and CMD2-MNPs. CMD1-MNPs and CMD2MNPs gave a slightly higher PdI values of 0.49 and 0.47, respectively. The results correspond to a more or less monodispersed size distribution of CMD3-MNPs, which indicates the most consistent sizes of all CMDMNPs that can occur because of high CMD concentration. The higher PdI value of uncoated MNPs (0.72) also corresponds with the theory of MNPs having a tendency to aggregate, since they have a large surfaceto-volume ratio, therefore, giving the highest PdI values. While increasing CMD concentration in CMD-MNPs synthesis, the particles in solution may result in more interparticle-chain interactions, as well as are more aggregated. This observation is in accordance with size distribution analysis observed with SEM, TEM, as well as with TGA analysis. Hydrodynamic diameter of all CMD-MNPs and uncoated MNPs is expressed in size per number as well as in size per intensity and is presented in Fig. 7. The surface charge on each of the prepared MNPs stabilized by the electrostatic method was analyzed with ζ-potential measurements. Fig. 8 presents ζ-potential values for uncoated MNPs and all CMD coated MNPs (CMD1-MNPs, CMD2-MNPs, CMD3-MNPs). The uncoated MNPs exhibit positive values of 25.5, which can be explained by water molecules being absorbed in the maghemite. This can cause partial protonation of surface groups on uncoated MNPs, which generates a small positive charge. On the other hand, CMD coated MNPs exhibit

± ± ± ±

33 95 26 61

negative ζ-potential values of −44.37 (CMD1-MNPs), −31.9 (CMD2MNPs) and −33.9 (CMD-MNPs), which indicates negatively charged hydroxyl and carboxyl groups of CMD present on the surface of the nanoparticles. During synthesis of CMD-MNPs, the negative charges of hydroxyl and carboxyl groups form a larger electron cloud around nanoparticles obtained from CMD, which contributes to negatively charged surface, which is expressed in negative values of ζ-potential measurements. This confirms the presence of CMD on all of our CMDMNPs. Typical EPR spectra of all iron-containing samples at 25 °C and −196 °C are shown in Fig. 9. The spectra recorded are very broad asymmetric single lines, which allows us to assume the anisotropy of these spectra, i.e., that they characterize superimposition of EPR spectra which belong to spins of both paramagnetic centers (PCs) located in one nanoparticle as well as in different ones. The total amount of these PCs in the samples was estimated by double integration of the spectra and their comparison with a standard - the EPR spectrum of a CuCl2·2H2O monocrystal with a known quantity of spins. We have to stress that this estimation is not precise because (a) EPR lines are very broad and do not really achieve the zero line at low fields, and (b) the spin transitions in Fe3+ ions are very complex regarding its electron (S = 5/2) and nuclear (I = 5/2) spins. Anyway, from the results of double integration we can conclude that practically all iron ions contribute to the EPR spectra. One can see from Fig. 9 that EPR spectra of the samples: 1) differ strongly with temperature changes, and 2) are noticeably different in the case of various PCs at any certain temperature. These differences are evidently seen not only from Fig. 9 but also from the effective parameters listed in Table 3. Such EPR spectra and their temperature changes are characteristic of many ferromagnetic magnetite-type systems, both inorganic or containing different organic molecules, or macromolecules including biopolymers [72–78]. Indeed, changes in the EPR spectra of uncoated MNPs and CMD1MNPs, CMD2-MNPs and CMD3-MNPs are analogous to those observed in iron-oxide containing compounds [72–76]. In the case of CMD1MNPs, CMD2-MNPs and CMD3-MNPs, line widths ΔH increase with the increase in CMD content at both temperatures, and are always markedly broader at low temperature (Table 3). The position of these lines in the magnetic field also changes, and this reflects a noticeable decrease in g-values in the CMD1-MNPs to CMD3-MNPs sample at 25 °C. To understand the nature of these changes and to provide a correct description, we need to carry out precise EPR measurements in helium temperatures of −263 °C up to 77–87 °C, also measuring the magnetic

Fig. 6. TEM images of the samples MNPs (a) and CMD3-MNPs (b). Insets are high-resolution TEM images of individual nanoparticles. 7

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Fig. 7. Hydrodynamic diameter of a) CMD1-MNPs, b) CMD2-MNPs, c) CMD3-MNPs and d) uncoated MNPs expressed in size per number (a, b, c, d) and per intensity (a1, b1, c1, d1). (Comparison of Size distribution by Intensity of all MNPs combined in one graph is available in Supplementary Data S1.)

8

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Fig. 8. ζ-potential of uncoated MNPs, CMD1-MNPs, CMD2-MNPs and CMD3-MNPs (a). Values are presented in a graph (b) and are the average of three independent measurements. Table 3 EPR parameters of the samples. Sample

Uncoated MNPs CMD1-MNPs CMD2-MNPs CMD3-MNPs a

25 °C

−196 °C

ΔH ± , G

g ± 0.01

960 ± 40 790 ± 30 1040 ± 40 1020 ± 40

2.01 2.02 1.98 1.92

a

g ± 0.01a

ΔH, G 1600 1240 1270 1400

± ± ± ±

60 50 50 55

2.91 2.65 2.70 2.63

g-value is measured at ½ of the EPR line height.

distribution [81–83]. The room-temperature magnetization curves of all CMD-MNPs and uncoated MNPs were measured and are presented in Fig. 10. Uncoated MNPs display saturation magnetization of ca. 64 emu/g, typical for iron oxide nanoparticles in this size range [81,84,85]. The measured coercivity value of 13 Oe is low, however above the error of measurement (approx. 2 Oe) indicating the presence of small amount of larger, ferrimagnetic nanoparticles, consistent with our results of the EPR spectroscopy. The TEM analysis suggests as well presence of nanoparticles larger than 15 nm in diameter, an approximate size limit above which the magnetic iron oxide nanoparticles become blocked and ferrimagnetic at room temperature [86]. Samples of CMD1-MNPs, CMD2-MNPs and CMD3-MNPs show low but variable coercivity (62 Oe, 4 Oe and 11 Oe, respectively) indicating slight variations in the size distributions between the samples, which were most likely modified during processing. The saturation magnetizations of CMD coated magnetic nanoparticles CMD1-MNPs, CMD2-MNPs and CMD3-MNPs are of 19.7 emu/g, 12.2 emu/g and 47.0 emu/g, respectively. They are lower than those of the MNPs due to dilution of the

Fig. 9. EPR spectra of CMD1-MNPs (1), CMD2-MNPs (2), CMD3-MNPs (3), and uncoated MNPs (4) at 25 °C (short dashed lines) and −196 °C (solid lines). Spectra are normalized by the amplitude.

susceptibility over the same temperature interval. Theoretical approaches of such quantitative calculations were published in [79,80]. We intend to conduct appropriate studies in the future. The magnetic properties of nanoparticles reflect important features of any Fe3O4-containing material, e.g., saturation magnetization reflects their structural peculiarities, an average size and broadness of the size 9

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24 h at 20 °C. On the other hand, immobilized ADH onto CMD3-MNPs managed to maintain 76.3% of its initial residual activity after 3 h. After 5 h of incubation at 20 °C, the residual activity slightly decreased to 53.1% and was kept constant even after 24 h. When increasing the temperature for investigation of thermal stability to 40 °C, immobilized ADH onto CMD3-MNPs managed to obtain 53.6% after 3 h and after 5 h further decrease to 32.2% was observed. After 24 h a significant increase to 75.3% was observed, while soluble ADH lost all of its initial activity after just 3 h at 40 °C. The results suggest that our immobilization protocol, using CMD3-MNPs as support for ADH managed to improve enzymes' stability. Immobilized ADH was stable even after 24 h of incubation at 20 °C and 40 °C, while soluble ADH at 20 °C kept 28.3% of initial activity and was completely inactivated at 40 °C after 3 h. The decrease of residual activity of immobilized ADH-CMD-MNPs after 3 h of incubation and increase after 5 h might be attributed to a combination of certain temperature and incubation time with synthesized CMD3-MNPs, which can cause a change of catalysis capability of the enzyme [87]. Results are presented in Fig. 12. Since immobilized enzymes are more stable at wider physical conditions and are also more easily recovered and recycled, the increase in residual activity as well as thermal stability at 20 °C and 40 °C of immobilized ADH onto CMD3-MNPs provides an essential feature that allows immobilized ADH to be applied into different applications.

Fig. 10. Room-temperature magnetization curves for CMD1-MNPs, CMD2MNPs, CMD3-MNPs and uncoated MNPs.

4. Conclusions Magnetic nanoparticles were successfully synthesized by co-precipitation of ferric and ferrous ions. Solutions of CMD were prepared with different concentrations of CMD (0.25 g/mL, 0.4 g/mL and 0.5 g/ mL) dissolved in water and used as coating material on the MNPs. Obtained CMD-MNPs (CMD1-MNPs, CMD2-MNPs and CMD3-MNPs) were characterized using various techniques, including FT-IR, TGA, SEM, TEM, DLS, EPR and VSM. FT-IR analysis of all CMD-MNPs confirmed the presence of CMD with characteristic vibrational modes, which correspond to CMD hydroxyl and carboxyl groups. TGA analysis revealed that all CMD-MNPs display a single drop in mass with increasing temperature, which indicates a single layer of CMD on the nanoparticle surface. Moreover, the highest CMD content occurs in CMD3-MNPs (37.80%). SEM analysis revealed that CMd1-MNPs, CMD2-MNPs and CMD3-MNPs gave the smallest sizes of 27 nm, 30 nm and 28 nm, respectively. However, TEM analysis of uncoated MNPs and CMD3-MNPs was able to conclude that the majority of MNPs in both samples have a size range between 8 nm to 38 nm. DLS analysis confirmed the most consistent sizes of CMD3-MNPs with the lowest PdI (0.31); also, ζ-potential was measured, confirming the negatively charged hydroxyl and carboxyl groups of CMD on the surface of MNPs. EPR measurements show a typical ferromagnetic system, and line widths ΔH increasing with the increase of CMD content at both temperatures. In the future, additional EPR measurements of magnetic susceptibility need to be performed to learn more about the nature of EPR changes in CMD-MNPs samples. Consistent with EPR measurements, VSM analysis indicated the presence of a small amount of larger, ferromagnetic nanoparticles. Based on the results obtained, it can be concluded that all CMD-MNPs exhibit a layer of CMD coating; CMD3MNPs possess the most preferable characteristics and were therefore applied as nano-carrier in immobilization of ADH. Investigation of thermal stability revealed preferable characteristics of immobilized ADH onto CMD3-MNPs when applying low temperature (4 °C) into immobilization protocol. Compared to soluble ADH that lost around 70% of its initial activity at 20 °C and all of its activity at 40 °C after 24 h, immobilized ADH obtained 53% of its residual activity at 20 °C and 75% at 40 °C after 24 h of incubation time. All obtained results are very promising and give us an optimistic outlook on synthesized and characterized CMD-MNPs, which will be focused on improving the residual activity of immobilized ADH, using these synthesized and characterized CMD-MNPs. Our further work will

Fig. 11. Residual activity of ADH immobilized onto CMD3-MNPs at 20 °C and at 4 °C. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ± 1%.)

magnetic phase by the diamagnetic CMD. 3.3. Immobilization of ADH onto CMD3-MNPs After detailed characterization analysis, CMD3-MNPs were selected to be tested as nano-carriers for immobilization of ADH. Two immobilization processes were carried out at two different temperatures (the first at 20 °C and the second at 4 °C). When immobilizing ADH onto CMD3-MNPs at 20 °C only 14.9% of initial ADH activity was achieved, but when decreasing the immobilization temperature to 4 °C there was an increase of enzyme activity, resulting in 26.4% of initial ADH activity. Results are given in Fig. 11. 3.3.1. Thermal stability of immobilized ADH onto CMD3-MNPs As implementing immobilization temperature of 4 °C into immobilization protocol showed higher residual activity, immobilized ADH onto CMD3-MNPs was further investigated for thermal stability at two different temperatures: at 20 °C and 40 °C. For the comparison, thermal stability of soluble ADH has been also studied. Initial activity of soluble ADH decreased in time being only 28.3% of it maintained after 10

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Fig. 12. Thermal stability of ADH immobilized onto CMD3-MNPs at 20 °C and at 40 °C. (Each experiment was performed in triplicate. Data were expressed as the means ± standard deviations of three replicates and vary for less than ± 1%.)

Acknowledgments

be focused on finding the appropriate immobilization conditions as well as finding the appropriate cross-linker that will significally improve activity and stability of immobilized ADH. In that manner, developed CMD-MNPs in this work provide very useful platform for further functionalization of CMD-MNPs for enzyme immobilization.

The authors acknowledge the Slovenian Research Agency, Slovenia (Program “Separation Processes and Product Design”, Contract No. P20046 and Program “Advanced magnetic and multifunctional materials”, Contract No. P2-0089). A. I. K. and E. A. K. thank the Russian Foundation, Russia for Basic Research (grant No. 18-53-00020-Bel-a) for financial support. EPR measurements were partially done at the equipment of the User Facilities Center of M. V. Lomonosov Moscow State University.

Author contributions KV performed the majority of the experiments, together with ML performed data analysis, performed statistical analysis, wrote the manuscript and designed the study together with ML. AIK and EAK designed and performed the EPR measurements and data analysis. SG performed TEM and VSM measurements and data analysis. ML, AIK and ŽK reviewed and revised the manuscript. All authors read and approved the manuscript.

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.reactfunctpolym.2020.104481. References

Data availability

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The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study. Author statement KV performed the majority of the experiments, together with ML performed data analysis, performed statistical analysis, wrote the manuscript and designed the study together with ML. AIK and EAK designed and performed the EPR measurements and data analysis. SG performed TEM and VSM measurements and data analysis. ML, AIK and ŽK reviewed and revised the manuscript. All authors read and approved the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 11

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