Accepted Manuscript

Title: Thermal and magnetic properties of chitosan-iron oxide

nanoparticles

Author: Paula I.P. Soares Diana Machado César Laia Laura

C.J. Pereira Joana T. Coutinho Isabel M.M. Ferreira Carlos
M.M. Novo João Paulo Borges

PII: S0144-8617(16)30500-8
DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2016.04.123
Reference: CARP 11063

To appear in:

Received date: 8-1-2016

Revised date: 1-4-2016
Accepted date: 28-4-2016

Please cite this article as: Soares, Paula IP., Machado, Diana., Laia, César.,
Pereira, Laura CJ., Coutinho, Joana T., Ferreira, Isabel MM., Novo, Carlos MM.,
& Borges, João Paulo., Thermal and magnetic properties of chitosan-iron oxide
nanoparticles.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.04.123

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 1 Thermal and magnetic properties of chitosan-iron oxide

2 nanoparticles

4 Paula I. P. Soares1, Diana Machado1, César Laia2, Laura C. J. Pereira3, Joana T.

3 1 4
5 Coutinho , Isabel M. M. Ferreira *, Carlos M. M. Novo , João Paulo
1
6 Borges *imf@fct.unl.pt, jpb@fct.unl.pt

1
8 i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade

9 NOVA de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal

10 2
REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, FCT, Universidade Nova de

11 Lisboa, 2829-516 Caparica, Portugal.

3
12 IST/C2TN, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10, ao km 139,7, 2695-

13 066 Bobadela LRS, Portugal

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14 Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, IHMT/UNL, 1349-008 Lisboa,

15 Portugal.

16
17 Corresponding Author: *i3N/CENIMAT, Department of Materials Science, Faculty of
18 Science and Technology, Universidade NOVA de Lisboa, Campus de Caparica, 2829-
19 516 Caparica, Portugal.
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32 Highlights

33 • Chitosan is widely used for biomedical applications

34 • Chitosan-iron oxide nanoparticles are suitable for magnetic hyperthermia
35 • Chitosan coating does not affect the properties of iron oxide nanoparticles
36 • Chitosan-iron oxide nanoparticles have a nanometric hydrodynamic diameter
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74 ABSTRACT
75 Chitosan is a biopolymer widely used for biomedical applications such as drug delivery
76 systems, wound healing, and tissue engineering. Chitosan can be used as coating for
77 other types of materials such as iron oxide nanoparticles, improving its biocompatibility
78 while extending its range of applications.
79 In this work iron oxide nanoparticles (Fe3O4 NPs) produced by chemical precipitation
80 and thermal decomposition and coated with chitosan with different molecular weights
81 were studied. Basic characterization on bare and chitosan-Fe3O4 NPs was performed
82 demonstrating that chitosan does not affect the crystallinity, chemical composition, and
83 superparamagnetic properties of the Fe3O4 NPs, and also the incorporation of Fe3O4
84 NPs into chitosan nanoparticles increases the later hydrodynamic diameter without
85 compromising its physical and chemical properties. The nano-composite was tested for
86 magnetic hyperthermia by applying an alternating current magnetic field to the samples
87 demonstrating that the heating ability of the Fe3O4 NPs was not significantly affected by
88 chitosan.
89
90 KEYWORDS
91 Chitosan; Iron oxide nanoparticles; Magnetic hyperthermia; Magnetite.
92
93 1. INTRODUCTION

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 94 Chitosan (CS) is a natural polysaccharide obtained by the partial alkaline deacetylation
95 of chitin, a main component of the exoskeleton of crustacean, arthropod and fungi (Liu
96 et al., 2007; S et al., 2010). Chitosan structure is similar to cellulose: composed of β-
97 1,4-linked 2-acetamino-2-deoxy-D-glucopyranose and 2-amino-2-deoxy glucopyranose
98 subunits, this biopolymer has a positive charge, being soluble in various acids, and can
99 interact with poly-anions to form complexes and gels. Chitosan is also non-toxic,
100 hydrophilic, biocompatible, biodegradable and anti-bacterial, and has been used as a
101 biomaterial and a pharmaceutical excipient in drug formulations (Liu et al., 2007).
102 Biomedical applications of chitosan ranges from drug delivery systems (Janes et al.,
103 2001; Rinaudo, 2006; Yuan et al., 2010), wound healing (Alemdaroglu et al., 2006;
104 Park et al., 2009; Takei et al., 2012), to tissue engineering (Matsuda et al., 2005; Qi et
105 al., 2004; Qin et al., 2009; Yuan et al., 2004; Zhang et al., 2003). It is able to
106 encapsulate not only drug molecules, but also other types of nanoparticles (NPs) adding
107 to them further functionalities. For example, magnetic nanoparticles covered with a
108 chitosan network contributes to the enhancement of their magnetic and thermal
109 properties (Shete et al., 2014; Unsoy et al., 2014; Zamora-Mora et al., 2014).
110 Magnetic nanoparticles are widely used for magnetic hyperthermia (Echeverria et al.,
111 2015; Eneko et al., 2015; Guardia et al., 2012; Kobayashi, 2011; Kolosnjaj-Tabi et al.,
112 2014; Obaidat et al., 2015; Soares et al., 2014b; Soares et al., 2012) and magnetic
113 resonance imaging (Atefeh et al., 2015; Jafari et al., 2014; Ming et al., 2015; Sun et al.,
114 2008). Iron oxide NPs (Fe3O4 NPs) are the most commonly used magnetic NPs due to
115 their biocompatibility. However, their properties are highly dependent on the synthesis
116 method. Among all synthesis methods chemical precipitation is probably the simplest to
117 produce iron oxide NPs, allowing the use of harmless and biocompatible materials and
118 making the resultant nanoparticles suitable for biomedical applications. By controlling
119 the reaction parameters it is possible to obtain iron oxide NPs as small as 5 nm (Indira et
120 al., 2010; McBain et al., 2008; Wu et al., 2008; Wu et al., 2015). Thermal
121 decomposition technique is also often used to synthetize iron oxide NPs which is based
122 on the decomposition of metal oxysalts (e.g., nitrates, carbonates, and acetates) when
123 heated to the solvent ebullition temperature. Since this technique occurs under
124 controlled environment, it is possible to obtain a better size control, narrow size
125 distribution, and crystallinity of grains (Baptista et al., 2013; Bigall et al., 2015; Gubin,
126 2009; Indira et al., 2010; Maity et al., 2007b; Wu et al., 2008).

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127 The low stability in aqueous solutions of iron oxide NPs is a problem when making
128 colloidal solutions envisaging its manipulation and application. This is however solved
129 using appropriate surfactant molecules (e.g. trisodium citrate or oleic acid) or polymers
130 (e.g. chitosan), but an important aspect to know is how they will affect the thermo-
131 magnetic properties of the iron oxide nanoparticles (Soares et al., 2014a; Soares et al.,
132 2015).
133 The main purpose of this work was to produce iron oxide NPs either by chemical
134 precipitation and thermal decomposition techniques in order to study the effect of
135 chitosan on their properties. A chitosan network was used to incorporate these iron
136 oxide NPs to obtain a nano-composite with a magnetic core and a chitosan coating. To
137 access the influence of chitosan its molecular weight was also varied. Finally, viability
138 of the nano-composite as a magnetic hyperthermia agent was also evaluated.
139
140 2. EXPERIMENTAL SECTION
141 All the chemical reagents used in this research work were of analytical grade and used
142 without further purification.
143
144 2.1. Synthesis of iron oxide nanoparticles
145 Iron oxide nanoparticles were produced by chemical precipitation technique using a
146 method described elsewhere (Soares et al., 2014a; Soares et al., 2015). The
147 nanoparticles without further treatment will be named “Pristine-Fe3O4 NPs”; the ones
148 stabilized with either trisodium citrate dehydrate ( >99%, Sigma-Aldrich) 10 mM or
149 oleic acid (Fisher Chemical) 64 mM will be named “Fe3O4-TC NPs” and Fe3O4-OA
150 NPs”, respectively.
151 To produce Fe3O4 NPs by thermal decomposition technique the following method based
152 on the work of Maity and his coworkers (Maity et al., 2007a; Maity et al., 2009) was
153 used: 2 mmol of Fe(acac)3 (Sigma-Aldrich) were dissolved in 20 mL of Triethylene
154 glycol (TREG, 99%, Sigma-Aldrich) and magnetically stirred under a flow of nitrogen.
155 The mixture was heated at 120ºC for 1 h and then rapidly heated to 280ºC, i.e. TREG
156 boiling temperature, and kept for 30 min using a condenser to prevent the high-boiling
157 temperature solvent from evaporation. The resultant black solution was cooled to room
158 temperature overnight by removing the heat source. The obtained nanoparticles were
159 precipitated by addition of 20 mL of ethyl acetate (Sigma Aldrich) and then isolated by
160 centrifugation (Heraeus Multifuge X1R Centrifuge) for 20 min at 10000 rpm. The

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161 washing process was repeated three times and the obtained nanoparticles were dispersed
162 in ultrapure water. A part of the suspension was freeze-dried (VaCo2, Zirbus) in order
163 to obtain dry nanoparticles for further characterization. These samples will be
164 designated as “Fe3O4-TD NPs”.
165
166 2.2. Chitosan depolymerization
167 Chitosan (DD 75.5%, 469 kDa, Cognis) was depolymerized by chemical reaction with
168 sodium nitrite (NaNO2, Sigma-Aldrich) based on an adapted method of Huang and
169 coworkers (Huang et al., 2004). Briefly, 2.5 g of chitosan was dissolved in 250 mL of
170 acetic acid 1% (V/V) (Panreac), followed by addition of the desired amount of NaNO2
171 dissolved in 10 mL of ultrapure water. The mixture was let to react for 1 h under
172 mechanical agitation. Low molecular weight chitosan was precipitated with sodium
173 hydroxide (NaOH, Eka) 1 M, followed by centrifugation and several washes with
174 ultrapure water. The final obtained product was freeze-dried and stored in a dry place.
175 The molecular weight of depolymerized chitosan was measured by dilute solution
176 viscosity using an Ubbelöhde capillary viscometer (No 0a) at 30ºC. The flow times of
177 CS solutions of different concentrations in 0.2 M acetic acid and 0.1 M sodium acetate
178 (Scharlau) and solvent were recorded in quintuplet and the average value calculated.
179 The intrinsic viscosity [η] was calculated graphically by extrapolating the curve of
180 reduced viscosity versus concentration to zero concentration. The Mark-Houwink-
181 Sakurada (MHS) equation was used to calculate the molecular weight (Mv) of
182 depolymerized chitosan (K=2.26x10-5 dl.g-1, α=0.95) (Kasaai, 2007).
183
184
185 2.3. Preparation of CS-Fe3O4 NPs
186 Chitosan nanoparticles (CS NPs) were produced through ionotropic gelation (Calvo et
187 al., 1997) using tripolyphosphate (TPP, Sigma-Aldrich). TPP solution was rapidly added
188 to the polymer solution with magnetic stirring. The nanoparticles suspension was kept
189 on magnetic stirring for a determined incubation time before any characterization.
190 To produce chitosan coated Fe3O4 NPs (CS-Fe3O4 NPs), an appropriate amount of
191 Fe3O4 NPs suspension (4 mg.mL-1) was added to chitosan solution followed by the
192 addition of TPP. The addition of the polyanion was performed under ultraturrax, and
193 kept for 5 min. For purification, composite NPs where separated using a magnet and

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194 washed several times with ultrapure water. The final product was freeze-dried and
195 stored in a dry place for further characterization.
196
197 2.4. Characterization
198 Transmission electron microscopy (TEM) images were obtained using a Hitachi H-8100
199 II with thermo-ionic emission LaB6. TEM analysis was performed in a little quantity of
200 nanoparticles suspended in pure water that were placed in a Kevlar 25 mesh grid.
201 The crystalline phases of the samples were verified using powder X-ray diffraction.
202 X’Pert PRO PANAlytical X-ray diffractometer was used to obtain X-ray diffraction
203 patterns of the iron oxide nanoparticles previously freeze-dried. The 2θ values were
204 taken from 15º to 80º using a Cu-Kα radiation (k=1.54060 Å) with a step size of 0.033.
205 The Scherrer’s equation was used to measure the average crystallite size.
206 The iron content of the samples was determined using the 1,10-phenantroline
207 colorimetric method (Talelli et al., 2009). To obtain the nanoparticles concentration the
208 formula [Fe]=0.7x[NPs] was used, obtained from control experiments.
209 FTIR spectra of the samples were obtained using a Nicolet 6700 – Thermo Electron
210 Corporation Attenuated Total Reflectance-Fourier Transform Infrared spectrometer
211 (ATR-FTIR). Measurements were performed in freeze-dried samples in the range of
212 480 to 4500 cm-1.
213 Dynamic Light Scattering (DLS) measurements were performed using a SZ-100
214 nanopartica series (Horiba, Lda) with a laser of 532 nm and controlling temperature
215 with a Peltier system (25ºC). DLS measurements were carried out for diluted NPs
216 suspensions in triplicates using a disposable cell with a scattering angle equal to 90º.
217 Data analysis was performed using cumulants statistics to measure hydrodynamic
218 diameter (Dh) and polydispersity unless stated otherwise.
219 Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) studies
220 were carried out using a Thermal Analyzer NETZSCH STA 449 F3 Jupiter® at a rate of
221 10ºC.min-1 in a N2 atmosphere
222 The DC magnetic properties were performed using a 7 T SQUID magnetometer
223 (S700X; Cryogenic Ltd.). The zero-field cooled (ZFC) and field-cooled (FC)
224 measurements were performed by cooling the sample to 5 K at zero fields or in the
225 presence of an external field of 8 kA.m-1, respectively. All the magnetic measurements
226 were carried out in increasing temperature range 5–320 K. Isothermal magnetization
227 curves were obtained for fields up to 5 T for temperatures of 10 and 320 K.

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228
229 2.5. Magnetic hyperthermia measurements
230 Magnetic hyperthermia measurements were obtained using a DM100 series from Nb
231 Nanoscale Biomagnetics apparatus. This apparatus allows measurements at different
232 magnetic field intensities up to 24 kA.m-1 with a fixed frequency of 418.5 kHz. The
233 heating ability of 1 mL sample of freshly prepared NPs solution was measured using an
234 alternating current (AC) magnetic field of 24 kA.m-1, with a frequency of 418.5 kHz for
235 10 minutes.
236
237 3. Results and discussion
238 The magnetic and thermal properties of chitosan-Fe3O4 NPs are closely dependent on
239 the properties of the Fe3O4 NPs. As such, two methods were used to synthesize Fe3O4
240 NPs: chemical precipitation and thermal decomposition. Although chemical
241 precipitation technique is a facile and rapid method to produce Fe3O4 NPs, the obtained
242 NPs are highly dependent upon the synthesis parameters. Consequently, the low
243 stability of these NPs in aqueous medium generally leads to the need of using adjuvants,
244 such as surfactants, to maximize the stabilization.
245
246 3.1. Iron oxide nanoparticles
247 In a previous work, we have successfully used trisodium citrate and oleic acid to obtain
248 Fe3O4 NPs stable in aqueous medium. Pristine-Fe3O4 NPs, i.e. Fe3O4 NPs obtained by
249 chemical precipitation technique without any added stabilizer, have an average diameter
250 of 8.5±2 nm with a significant aggregation between them (Soares et al., 2014a; Soares
251 et al., 2015). TEM image of Fe3O4 nanoparticles obtained from thermal decomposition
252 (Fe3O4-TD NPs) (Figure 1) shows monodisperse nanoparticles with an average diameter
253 of 8±1.8 nm without significant aggregation. Based on the data obtained from TEM, it
254 is clear that the chosen technique to synthesize the nanoparticles has not significant
255 influence in their size, although it contributes to the aggregation state of the
256 nanoparticles.
257
258
259 To compare the structure of both samples, XRD patterns were obtained and are shown
260 in Figure 1C. The comparison of the obtained XRD patterns with the standard
261 diffraction peaks for magnetite (JCPDS 00-019-0629) and maghemite (JCPDS 00-039-

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262 1346) powders clearly identifies the diffraction peaks of crystalline cubic magnetite
263 structure on both samples. The average crystallite size (τ) was determined by the
264 Scherrer’s equation τ = K λ / (β cosθ ) , where K is the grain shape factor (K=0.94), λ is
265 the incident X-ray wavelength, β is the full width at half-maximum (in radians) of the
266 highest intensity, and θ is the corresponding diffraction angle. Fe3O4 NPs obtained by
267 chemical precipitation and thermal decomposition have similar average crystallite size
268 of 9.7 nm. This confirms the TEM results and effectively equivalent structure and
269 particle size are obtained by both methods.
270 Concerning composition, FTIR measurements were performed to compare the main
271 chemical bonds of Fe3O4 NPs produced by each method. The spectra of Figure 1D
272 shows that pristine-Fe3O4 NPs are composed of three characteristic bands: a broad band
273 between 3000 cm-1 and 3500 cm-1 related to the –OH stretching vibration modes of
274 water vapor, a band at 1630 cm-1 attributed to the O-H stretching vibration modes, and a
275 band at 560 cm-1 which is characteristic of the Fe-O stretching vibration mode. The
276 spectrum of the Fe3O4-TD NPs have the same absorbance characteristic bands. An extra
277 band is present at 1421 cm-1, attributed to the C-H bending vibration mode of the
278 triethylene glycol used during synthesis.
279
280 3.2. Chitosan-Fe3O4 NPs
281 The Fe3O4 NPs were incorporated into chitosan’s structure aiming to protect the iron
282 oxide core, maximize its stability and also to add functionalities to the composite, such
283 as the possibility to incorporate drugs or to functionalize the surface.
284 Chitosan-Fe3O4 NPs were produced by ionotropic gelation, a simple and rapid method.
285 In this method, sodium tripolyphosphate (TPP) complexes with chitosan to form
286 ionically cross-linked nanoparticles (Calvo et al., 1997; Fernandez-Urrusuno et al.,
287 1999). Fe3O4 NPs were added to chitosan’ solution prior to crosslinking; when TPP is
288 added to the solution, Fe3O4 NPs are entrapped into CS network, creating a core-shell
289 structure (Figure 2). The production of composite NPs was performed by using chitosan
290 with different Mv from 469 kDa (initial CS) to 38 kDa (lowest Mv obtained after
291 depolymerisation).
292
293 The bonding between chitosan of higher Mv (469 kDa) and Fe3O4 NPs was confirmed
294 by FTIR measurements. FTIR spectrum of CS-Pristine-Fe3O4 NPs (Figure 3A) is very

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295 similar to the one of CS-Fe3O4-TD NPs (Figure 3B). In both cases, typical bands of
296 chitosan are present: the band at 3357 cm-1 corresponds to the O-H stretching
297 overlapping the N-H stretching, while the band at 2874 cm-1 is attributed to the C-H
298 stretching. The amide II band can be found at 1649 cm-1 corresponding to the C-O
299 stretching of the acetyl group. The sharp bands at 1415 cm-1, 1374 cm-1 and 1316 cm-1
300 are attributed to the –CH2 bending, the asymmetrical C-H bending of the CH2 group,
301 and the amide III band, respectively. The bands related to the C-O-C bridge are located
302 at 1150 cm-1 (anti-symmetric stretching of C-O-C bridge), 1023 cm-1 (skeleton
303 vibrations involving the C-O-C stretching bands), and 892 cm-1 (C-O-C stretching of the
304 glycosidic linkage). The band at 1064 cm-1 is attributed to the C-N stretching vibration
305 mode (Kumirska et al., 2010). Additionally, the second band of the amide II (usually
306 found around 1587 cm-1) disappeared and a new band at 1531 cm-1 appeared which
307 corresponds to the N-O asymmetric stretching vibration mode (Wu et al., 2005),
308 confirming the ionic bond between chitosan and TPP. The band at 560 cm-1 is attributed
309 to Fe-O stretching vibration mode related to the presence of iron oxide. When pristine-
310 Fe3O4 NPs stabilized with either TC or OA are incorporated into chitosan, the
311 characteristic absorbance bands of Fe3O4 are also present in FTIR spectra (Figure 3C
312 and D) (Soares et al., 2014a; Soares et al., 2015). The spectra of composite NPs with CS
313 of 38 kDa show similar absorbance bands, which indicates that chitosan Mv does not
314 affect the bonding between CS, Fe3O4 NPs, and TPP.
315
316 DLS studies were performed to compare the differences in the hydrodynamic diameter
317 between CS NPs and CS-Fe3O4 NPs. So far, composite NPs were produced using
318 chitosan of 469 kDa. Since the density of the polymeric structure can affect the
319 properties of the composite NPs, different Mv of CS were tested in a range of 38 kDa to
320 469 kDa. Figure 4A compares the Dh of CS NPs vs. CS-Fe3O4 NPs for the tested range
321 of chitosan Mv. The Dh of CS-Fe3O4 NPs has the tendency to increase with chitosan Mv.
322 However, for the highest molecular weight the Dh is significantly smaller, except for
323 CS-pristine-Fe3O4 NPs.
324
325 The observed differences between the obtained Dh were analyzed by autocorrelation
326 functions (ACF) of the two types of CS-Fe3O4 NPs (Figure 4). ACF of CS-pristine-
327 Fe3O4 NPs (Figure 4B) show two different behaviors dependent on the Mv. For the three
328 lowest Mv, the ACF curves show only one relaxation mode as a typical single-

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329 exponential relaxation. Higher Mv show a slow relaxation mode attributed to very large
330 aggregates of NPs, and contributes largely to the overall measurement. Moreover, the
331 contribution of the slow relaxation mode increases with the increase of the Mv.
332 ACF of CS-Fe3O4-TD NPs (Figure 4C) show only one relaxation mode for all cases
333 except for the highest Mv that show a slow relaxation mode attributed to large
334 aggregates. The presence of larger aggregates in some samples may be attributed to
335 chitosan chains that are not cross-linked into nanoparticles. Consequently, the presence
336 of such polymeric chains in the solution causes scattering in DLS measurements
337 represented as the slow relaxation modes in ACF curves.
338 For better understanding the physical and chemical changes that may have occurred
339 during coating, thermogravimetric analysis was performed. Thermogravimetric curves
340 (sample weight % as a function of temperature) and its derivative (DTA) of Fe3O4 NPs
341 with and without CS are displayed in Figure 5. As expected, Fe3O4 NP are not degraded
342 within the tested range of temperature (Pristine-Fe3O4 NPs and Fe3O4-TD NPs). The
343 small reduction in wt.% is related to the conversion of Fe3O4 to γ-Fe2O3 and FeO, which
344 are the stable phases of diagram of Fe-O system above 570ºC (Darken et al., 1946).
345 However, when Fe3O4 NPs are stabilized with a surfactant, typical degradation stages of
346 those molecules appear in the TGA curve. For example, Fe3O4-OA NPs show three
347 degradation peaks in the DTA graph: a peak at 214ºC, close to the boiling temperature
348 of OA (b.p. 94-195ºC/1.2 mmHg) and can be correlated to the removal of free OA on
349 the Fe3O4 NPs; the second peak is at about 338ºC and corresponds to the removal of OA
350 molecules that are bounded to Fe3O4 NPs surface (Sahoo et al., 2001); the third peak at
351 712ºC is related to the carbon from the OA that is oxidized to CO and CO2 during the
352 annealing and the reduction mechanism may be as follows (Ayyappan et al., 2008):
353
354
355 Fe3O4 + CO → 3FeO + CO2 (1)
356
357 3FeO ↔ Fe2O3 + α Fe (2)
358
1
359 CO + O2 → CO2 (3)
2
360

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361 The incorporation of Fe3O4 into CS network does not seem to influence chitosan
362 degradation stages, as can be seen in Figure 5C and D. The degradation of CS took
363 place in three stages. The first stage occurred between 30ºC to 160ºC, corresponds to a
364 mass loss of 21% that is associated with the removal of water. The second stage resulted
365 in a mass loss of 24% correspondent to chitosan degradation between 210ºC and 380ºC.
366 The total degradation occurred in the final stage, with a remaining amount of product of
367 27% at 900ºC. The remaining product of CS-Fe3O4 NPs is inferior in percentage
368 compared to CS NPs. This seams contradictory to what was expected since Fe3O4 is not
369 degraded in the tested range of temperature. However, the total percentage of
370 nanoparticles is not only composed of chitosan, but also of Fe3O4 NPs. As such, if the
371 remaining amount is normalized taking into consideration the partial amount of
372 chitosan, the remaining amount is composed of 3% of Fe3O4 NPs. This means that in
373 this case chitosan was able to encapsulate 3% of Fe3O4 NPs.
374 SQUID magnetic measurements were performed to access the influence of chitosan in
375 the Fe3O4 NPs magnetic properties. Magnetic saturation values are presented in emu per
376 gram of the whole particle (magnetic and non-magnetic material). Figure 6A shows the
377 temperature dependence of magnetization with the ZFC and FC curves of pristine-Fe3O4
378 NPs and Fe3O4-TD NPs measured under an applied DC field of 8 kA.m-1. The blocking
379 temperature (TB) was determined as the maximum value of ZFC curve being around 155
380 K for pristine-Fe3O4 NPs and 182 K for Fe3O4-TD. In both cases, the TB is well below
381 room temperature. Figure 6B shows the hysteresis loops of pristine-Fe3O4 NPs and
382 Fe3O4-TD NPs at 320 K, with inset corresponding to the magnification at lower fields.
383 At this temperature both samples show the same behavior with only a slight difference
384 in their saturation magnetization (MS): 58 emu.g-1 (Soares et al., 2014a; Soares et al.,
385 2015), and 61 emu.g-1 for pristine-Fe3O4 NPs and Fe3O4-TD NPs, respectively.
386 Nevertheless, above the TB both samples of Fe3O4 NPs are superparamagnetic,
387 confirmed by the absence of coercivity and remanence at 320 K.
388 When Fe3O4 NPs are coated with low (38 kDa) or high (469 kDa) Mv chitosan (Figure
389 6C and D, respectively), the MS of the nanoparticles is greatly reduced to 29 and 18
390 emu.g-1, respectively, in the case of pristine-Fe3O4 NPs, and 30 and 25 emu.g-1,
391 respectively, in the case of Fe3O4-TD NPs. The significant decrease on the MS may be
392 attributed to surface effects caused by coating (Sanjai et al., 2014). The reduction
393 caused by chitosan seems to be also dependent upon its Mv. Again the absence of

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394 coercivity and remanence at 320 K is visible, which confirms that CS does not change
395 the superparamagnetic behavior of the Fe3O4 NPs above TB.
396
397 3.3. Magnetic hyperthermia
398 A possible biomedical application for this type of composite NPs is magnetic
399 hyperthermia (Soares et al., 2012; Unsoy et al., 2012; Zamora-Mora et al., 2014;
400 Zamora-Mora et al., 2015), but the influence of the coating in the heating ability of the
401 Fe3O4 NPs is often omitted. In a previous work (Soares et al., 2015), magnetic
402 hyperthermia measurements were performed on Fe3O4 NPs obtained from chemical
403 precipitation technique. In the present work, the heating ability of the nanoparticles
404 obtained is analyzed for different iron concentrations.
405 The heating ability of pristine-Fe3O4 NPs and Fe3O4-TD NPs, in the tested range of iron
406 concentration, is depicted in Figure 7A where a huge difference is observed between
407 both samples. So far, the performed characterizations led to the conclusion that both
408 NPs were similar, excepting the higher tendency to aggregate in the case of pristine-
409 Fe3O4 NPs. Consequently, although Fe3O4-TD NPs are expected to be highly stable and
410 monodisperse in size, its application in magnetic hyperthermia is compromised by its
411 low heating ability. Consequently, magnetic hyperthermia measurements of CS-Fe3O4
412 NPs were performed only using Fe3O4 NPs obtained by chemical precipitation. CS of
413 38 kDa was used as a polymeric coating for three types of Fe3O4 NPs: pristine-Fe3O4,
414 Fe3O4-TC, and Fe3O4-OA (Figure 7B, C and D, respectively). The amount of CS and
415 tripolyphosphate was kept constant, while the amount of Fe3O4 NPs was varied. As a
416 reference, bare Fe3O4 NPs results in the same tested conditions were added to each case.
417
418 The presence of CS coating on the pristine-Fe3O4 NPs (Figure 7B) does not influence
419 the generated temperature by the Fe3O4 NPs. On the other hand, CS-Fe3O4-TC NPs
420 (Figure 7C) shows a reduction in the generated temperature compared to the reference.
421 As such, chitosan has a negative influence for the application of Fe3O4-TC NPs in
422 magnetic hyperthermia. The same was observed for CS-Fe3O4-OA NPs (Figure 7D).
423 To evaluate the influence of chitosan Mv, the Fe3O4 NPs were coated with CS 38 kDa
424 and CS 469 kDa. Figure 8 shows the SAR (specific absorbance rate) values for each
425 case. SAR is defined as the quantity of power absorbed by the sample as per mass unit
426 (W.g-1) and was calculated using the following equation:
427

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 CNP mFe + Cl ml
428 ( )
SAR W .g −1 =
mFe
dT
dt
(4)
max

429 where (dT/dt)max is the maximum gradient of the temperature curve, CNP is the specific
430 heat of the nanoparticles, Cl is the specific heat of the liquid, ml is the fluid mass, and
431 mFe is the iron mass in the colloid. SAR values were taken under adiabatic conditions,
432 i.e. by using the maximum of the derivative of (dT/dt), which is present in the first few
433 seconds of the experiment.
434
435 The results show that SAR values decrease with the increase of chitosan Mv and this
436 behavior is even more evident for Fe3O4-TD NPs, were the difference is around 7-fold.
437 However, this difference is less evident in the Fe3O4 NPs stabilized with trisodium
438 citrate and oleic acid. One possible explanation is that the surfactant already reduced the
439 heating ability of the Fe3O4 NPs, therefore cloaking the effect of chitosan coating.
440 Nevertheless, the produced Fe3O4 NPs are still able to rise significantly the surrounding
441 temperature.
442
443 4. CONCLUSIONS
444 With the present research work we were able to produce and characterize composite
445 nanoparticles composed by a magnetic core and a polymeric coating suitable for
446 magnetic hyperthermia applications. The effect of chitosan, a biopolymer widely used
447 for biomedical applications, on the magnetic and thermal properties of iron oxide
448 nanoparticles produced by two different synthesis methods was evaluated. The
449 polymeric coating does not affect the magnetic properties of the produced iron oxide
450 NPs since iron oxide NPs do not change their basic composition (magnetite), also
451 keeping superparamagnetism. From the magnetic hyperthermia measurements it was
452 possible to conclude that these iron oxide NPs were able to generate heat once an
453 alternating magnetic field is applied. Moreover, although the incorporation of Fe3O4
454 NPs increases the hydrodynamic diameter of CS NPs, it does not change the physical
455 and chemical properties of the CS NPs.
456 Furthermore, chitosan coating may function as a platform for drug incorporation,
457 creating a drug delivery system based on the developed nano-composite system.
458
459 ACKNOWLEDGMENT

14
460 This work is funded by FEDER funds through the COMPETE 2020 Program and
461 National Funds through FCT - Portuguese Foundation for Science and Technology
462 under the project number POCI-01-0145-FEDER-007688, Reference UID/CTM/50025.
463 P.I.P. Soares and J.T. Coutinho acknowledge FCT for PhD grants,
464 SFRH/BD/81711/2011 and SFRH/BD/84628/2012, respectively.
465
466 REFERENCES
467 Alemdaroglu, C., Degim, Z., Celebi, N., Zor, F., Ozturk, S., & Erdogan, D. (2006). An investigation
468 on burn wound healing in rats with chitosan gel formulation containing epidermal growth
469 factor. Burns, 32(3), 319-327.
470 Atefeh, J., Mojtaba, S., Saber Farjami, S., Zahra, H., Ahmad Bitarafan, R., Komail, B., & Ali, N.
471 (2015). Synthesis and characterization of Bombesin-superparamagnetic iron oxide
472 nanoparticles as a targeted contrast agent for imaging of breast cancer using MRI.
473 Nanotechnology, 26(7), 075101.
474 Ayyappan, S., Gnanaprakash, G., Panneerselvam, G., Antony, M. P., & Philip, J. (2008). Effect of
475 Surfactant Monolayer on Reduction of Fe3O4 Nanoparticles under Vacuum. The Journal of
476 Physical Chemistry C, 112(47), 18376-18383.
477 Baptista, A., Soares, P., Ferreira, I., & Borges, J. P. (2013). Nanofibers and Nanoparticles in
478 Biomedical Applications. In A. Tiwari & A. Tiwari (Eds.). Bioengineered Nanomaterials (pp. 98-
479 100). New York: CRC Press.
480 Bigall, N. C., Dilena, E., Dorfs, D., Beoutis, M.-L., Pugliese, G., Wilhelm, C., Gazeau, F., Khan, A.
481 A., Bittner, A. M., Garcia, M. A., Garcia-Hernandez, M., Manna, L., & Pellegrino, T. (2015).
482 Hollow Iron Oxide Nanoparticles in Polymer Nanobeads as MRI Contrast Agents. The Journal of
483 Physical Chemistry C, 119(11), 6246-6253.
484 Calvo, P., Remuñan-López, C., Vila-Jato, J. L., & Alonso, M. J. (1997). Chitosan and
485 chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for
486 proteins and vaccines. Pharmaceutical research (Vol. 14, pp. 1431-1436).
487 Darken, L. S., & Gurry, R. W. (1946). The System Iron—Oxygen. II. Equilibrium and
488 Thermodynamics of Liquid Oxide and Other Phases. Journal of the American Chemical Society,
489 68(5), 798-816.
490 Echeverria, C., Soares, P., Robalo, A., Pereira, L., Novo, C. M. M., Ferreira, I., & Borges, J. P.
491 (2015). One-pot synthesis of dual-stimuli responsive hybrid PNIPAAm-chitosan microgels.
492 Materials & Design, 86, 745-751.
493 Eneko, G., Olivier, S., Juan-Mari, C., Jose Angel, G., Stéphane, M., & Fernando, P. (2015).
494 Specific absorption rate dependence on temperature in magnetic field hyperthermia measured
495 by dynamic hysteresis losses (ac magnetometry). Nanotechnology, 26(1), 015704.
496 Fernandez-Urrusuno, R., Calvo, P., Remunan-Lopez, C., Vila-Jato, J. L., & Alonso, M. J. (1999).
497 Enhancement of nasal absorption of insulin using chitosan nanoparticles. Pharm Res, 16(10),
498 1576-1581.
499 Guardia, P., Di Corato, R., Lartigue, L., Wilhelm, C., Espinosa, A., Garcia-Hernandez, M., Gazeau,
500 F., Manna, L., & Pellegrino, T. (2012). Water-soluble iron oxide nanocubes with high values of
501 specific absorption rate for cancer cell hyperthermia treatment. ACS Nano, 6(4), 3080-3091.
502 Gubin, S. P. (2009). Magnetic Nanoparticles. Federal Republic of Germany: WILEY-VCH Verlag
503 GmbH & Co.
504 Huang, M., Khor, E., & Lim, L.-Y. (2004). Uptake and cytotoxicity of chitosan molecules and
505 nanoparticles: effects of molecular weight and degree of deacetylation. Pharmaceutical
506 research, 21, 344-353.
507 Indira, T. K., & Lakshmi, P. K. (2010). Magnetic Nanoparticles – A Review. 3.

15
508 Jafari, A., Farjami Shayesteh, S., Salouti, M., & Boustani, K. (2014). Dependence of structural
509 phase transition and lattice strain of Fe3O4 nanoparticles on calcination temperature. Indian
510 Journal of Physics, 89(6), 551-560.
511 Janes, K. A., Fresneau, M. P., Marazuela, A., Fabra, A., & Alonso, M. J. (2001). Chitosan
512 nanoparticles as delivery systems for doxorubicin. J Control Release, 73(2-3), 255-267.
513 Kasaai, M. R. (2007). Calculation of Mark–Houwink–Sakurada (MHS) equation viscometric
514 constants for chitosan in any solvent–temperature system using experimental reported
515 viscometric constants data. Carbohydrate Polymers, 68(3), 477-488.
516 Kobayashi, T. (2011). Cancer hyperthermia using magnetic nanoparticles. Biotechnol J, 6(11),
517 1342-1347.
518 Kolosnjaj-Tabi, J., Di Corato, R., Lartigue, L., Marangon, I., Guardia, P., Silva, A. K., Luciani, N.,
519 Clement, O., Flaud, P., Singh, J. V., Decuzzi, P., Pellegrino, T., Wilhelm, C., & Gazeau, F. (2014).
520 Heat-generating iron oxide nanocubes: subtle "destructurators" of the tumoral
521 microenvironment. ACS Nano, 8(5), 4268-4283.
522 Kumirska, J., Czerwicka, M., Kaczynski, Z., Bychowska, A., Brzozowski, K., Thoming, J., &
523 Stepnowski, P. (2010). Application of spectroscopic methods for structural analysis of chitin
524 and chitosan. Mar Drugs, 8(5), 1567-1636.
525 Liu, C., Tan, Y., Liu, C., Chen, X., & Yu, L. (2007). Preparations, characterizations and
526 applications of chitosan-based nanoparticles. Journal of Ocean University of China, 6(3), 237-
527 243.
528 Maity, D., & Agrawal, D. (2007a). Synthesis of iron oxide nanoparticles under oxidizing
529 environment and their stabilization in aqueous and non-aqueous media. Journal of Magnetism
530 and Magnetic Materials, 308, 46-55.
531 Maity, D., & Agrawal, D. C. (2007b). Synthesis of iron oxide nanoparticles under oxidizing
532 environment and their stabilization in aqueous and non-aqueous media. Journal of Magnetism
533 and Magnetic Materials, 308(1), 46-55.
534 Maity, D., Kale, S. N., Kaul-Ghanekar, R., Xue, J.-M., & Ding, J. (2009). Studies of magnetite
535 nanoparticles synthesized by thermal decomposition of iron (III) acetylacetonate in
536 tri(ethylene glycol). Journal of Magnetism and Magnetic Materials, 321(19), 3093-3098.
537 Matsuda, A., Kobayashi, H., Itoh, S., Kataoka, K., & Tanaka, J. (2005). Immobilization of laminin
538 peptide in molecularly aligned chitosan by covalent bonding. Biomaterials, 26(15), 2273-2279.
539 McBain, S. C., Yiu, H. H., & Dobson, J. (2008). Magnetic nanoparticles for gene and drug
540 delivery. Int J Nanomedicine, 3(2), 169-180.
541 Ming, W., Da, Z., Yongyi, Z., Lingjie, W., Xiaolong, L., & Jingfeng, L. (2015). Nanocluster of
542 superparamagnetic iron oxide nanoparticles coated with poly (dopamine) for magnetic field-
543 targeting, highly sensitive MRI and photothermal cancer therapy. Nanotechnology, 26(11),
544 115102.
545 Obaidat, I., Issa, B., & Haik, Y. (2015). Magnetic Properties of Magnetic Nanoparticles for
546 Efficient Hyperthermia. Nanomaterials, 5(1), 63-89.
547 Park, C. J., Clark, S. G., Lichtensteiger, C. A., Jamison, R. D., & Johnson, A. J. (2009). Accelerated
548 wound closure of pressure ulcers in aged mice by chitosan scaffolds with and without bFGF.
549 Acta Biomater, 5(6), 1926-1936.
550 Qi, L., Xu, Z., Jiang, X., Hu, C., & Zou, X. (2004). Preparation and antibacterial activity of
551 chitosan nanoparticles. Carbohydr Res, 339(16), 2693-2700.
552 Qin, L., Dichen, L., Zhongmin, J., Jue, W., Aimin, L., & Zhen, W. (2009). Fabrication and In Vitro
553 Evaluation of Calcium Phosphate Combined with Chitosan Fibers for Scaffold Structures.
554 Journal of Bioactive and Compatible Polymers, 24(1 Suppl), 113-124.
555 Rinaudo, M. (2006). Chitin and chitosan: Properties and applications. Progress in Polymer
556 Science, 31(7), 603-632.
557 S, K. S. K., K, R. A., & Satyawan, S. (2010). Chitosan : A Platform for Targeted Drug Delivery.
558 International Journal, 2, 2271-2282.

16
559 Sahoo, Y., Pizem, H., Fried, T., Golodnitsky, D., Burstein, L., Sukenik, C. N., & Markovich, G.
560 (2001). Alkyl Phosphonate/Phosphate Coating on Magnetite Nanoparticles: A Comparison
561 with Fatty Acids. Langmuir, 17(25), 7907-7911.
562 Sanjai, C., Kothan, S., Gonil, P., Saesoo, S., & Sajomsang, W. (2014). Chitosan-triphosphate
563 nanoparticles for encapsulation of super-paramagnetic iron oxide as an MRI contrast agent.
564 Carbohydrate Polymers, 104, 231-237.
565 Shete, P. B., Patil, R. M., Thorat, N. D., Prasad, A., Ningthoujam, R. S., Ghosh, S. J., & Pawar, S.
566 H. (2014). Magnetic chitosan nanocomposite for hyperthermia therapy application:
567 Preparation, characterization and in vitro experiments. Applied Surface Science, 288, 149157.
568 Soares, P. I., Alves, A. M., Pereira, L. C., Coutinho, J. T., Ferreira, I. M., Novo, C. M., & Borges, J.
569 P. (2014a). Effects of surfactants on the magnetic properties of iron oxide colloids. J Colloid
570 Interface Sci, 419, 46-51.
571 Soares, P. I. P., Ferreira, I. M. M., & Borges, J. P. M. R. (2014b). Application of hyperthermia for
572 cancer treatment: recent patents review. In Atta-ur-Rahman & K. Zaman (Eds.). Topics in Anti-
573 Cancer Research (Vol. 3, pp. 342-383 (342)). USA: Bentham Science Publishers.
574 Soares, P. I. P., Ferreira, I. M. M., Igreja, R. A. G. B. N., Novo, C. M. M., & Borges, J. P. M. R.
575 (2012). Application of Hyperthermia for Cancer Treatment: Recent Patents Review. Recent
576 patents on anti-cancer drug discovery, 7(1), 64-73.
577 Soares, P. I. P., Lochte, F., Echeverria, C., Pereira, L. C. J., Coutinho, J. T., Ferreira, I. M. M.,
578 Novo, C. M. M., & Borges, J. P. M. R. (2015). Thermal and magnetic properties of iron oxide
579 colloids: influence of surfactants. Nanotechnology, 26(42), 425704.
580 Sun, C., Fang, C., Stephen, Z., Veiseh, O., Hansen, S., Lee, D., Ellenbogen, R. G., Olson, J., &
581 Zhang, M. (2008). Tumor-targeted drug delivery and MRI contrast enhancement by
582 chlorotoxin-conjugated iron oxide nanoparticles. Nanomedicine (Lond), 3(4), 495-505.
583 Takei, T., Nakahara, H., Ijima, H., & Kawakami, K. (2012). Synthesis of a chitosan derivative
584 soluble at neutral pH and gellable by freeze-thawing, and its application in wound care. Acta
585 Biomater, 8(2), 686-693.
586 Talelli, M., Rijcken, C. J., Lammers, T., Seevinck, P. R., Storm, G., van Nostrum, C. F., & Hennink,
587 W. E. (2009). Superparamagnetic iron oxide nanoparticles encapsulated in biodegradable
588 thermosensitive polymeric micelles: toward a targeted nanomedicine suitable for image-
589 guided drug delivery. Langmuir, 25(4), 2060-2067.
590 Unsoy, G., Khodadust, R., Yalcin, S., Mutlu, P., & Gunduz, U. (2014). Synthesis of Doxorubicin
591 loaded magnetic chitosan nanoparticles for pH responsive targeted drug delivery. Eur J Pharm
592 Sci, 62, 243-250.
593 Unsoy, G., Yalcin, S., Khodadust, R., Gunduz, G., & Gunduz, U. (2012). Synthesis optimization
594 and characterization of chitosan-coated iron oxide nanoparticles produced for biomedical
595 applications. Journal of Nanoparticle Research, 14(11).
596 Wu, W., He, Q., & Jiang, C. (2008). Magnetic iron oxide nanoparticles: synthesis and surface
597 functionalization strategies. Nanoscale Res Lett, 3(11), 397-415.
598 Wu, W., Wu, Z., Yu, T., Jiang, C., & Kim, W.-S. (2015). Recent progress on magnetic iron oxide
599 nanoparticles: synthesis, surface functional strategies and biomedical applications. Science and
600 Technology of Advanced Materials, 16(2), 023501.
601 Wu, Y., Yang, W., Wang, C., Hu, J., & Fu, S. (2005). Chitosan nanoparticles as a novel delivery
602 system for ammonium glycyrrhizinate. Int J Pharm, 295(1-2), 235-245.
603 Yuan, Q., Shah, J., Hein, S., & Misra, R. D. (2010). Controlled and extended drug release
604 behavior of chitosan-based nanoparticle carrier. Acta Biomater, 6(3), 1140-1148.
605 Yuan, Y., Zhang, P., Yang, Y., Wang, X., & Gu, X. (2004). The interaction of Schwann cells with
606 chitosan membranes and fibers in vitro. Biomaterials, 25(18), 4273-4278.
607 Zamora-Mora, V., Fernández-Gutiérrez, M., Román, J., Goya, G., Hernández, R., & Mijangos, C.
608 (2014). Magnetic core–shell chitosan nanoparticles: Rheological characterization and
609 hyperthermia application. Carbohydrate Polymers, 102, 691698.

17
610 Zamora-Mora, V., Soares, P., Echeverria, C., Hernández, R., & Mijangos, C. (2015). Composite
611 Chitosan/Agarose Ferrogels for Potential Applications in Magnetic Hyperthermia. Gels, 1(1),
612 69.
613 Zhang, Y., Ni, M., Zhang, M., & Ratner, B. (2003). Calcium phosphate-chitosan composite
614 scaffolds for bone tissue engineering. Tissue Eng, 9(2), 337-345.
615
616
617
618 Figure captions
619

620

621
622 Figure 1 – A) TEM image of Fe3O4 NPs obtained by thermal decomposition technique (Fe3O4-TD) and
623 (B) the respective size distribution graph; C) XRD patterns of pristine-Fe3O4 NPs and Fe3O4-TD NPs; D)
624 FTIR spectra of pristine-Fe3O4 NPs and Fe3O4-TD NPs.
625
626
627

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628
629 Figure 2 – Schematic representation of the ionic interaction between chitosan and tripolyphosphate, and
630 between chitosan and iron oxide nanoparticles (represented as the black dots).
631
632

633

634
635 Figure 3 – FTIR spectra of chitosan-Fe3O4 NPs: A) CS-pristine-Fe3O4 NPs; B) CS-Fe3O4-TD NPs; C)
636 CS-Fe3O4-TC NPs; D) CS-Fe3O4-OA NPs.
637
638

19
639

640
641 Figure 4 – (A) Hydrodynamic diameter of CS NPs and chitosan-pristine-Fe3O4 and Fe3O4-TD NPs as a
642 function of chitosan Mv. Comparison of measured (symbols) and adjusted (lines) autocorrelation curves
643 of DLS measurements between (B) CS-pristine-Fe3O4 NPs and (C) CS-Fe3O4-TD NPs.
644

20
645

646
647 Figure 5 - TGA and DTA of Fe3O4 NPs (A and B, respectively), and chitosan nanoparticles (NP CS), CS-
648 pristine-Fe3O4 NPs (CS-Fe3O4) and pristine-Fe3O4 NPs (C and D, respectively).
649
650

21
651
652 Figure 6 - Magnetic characterization of Fe3O4 NPs obtained by chemical precipitation or thermal
653 decomposition technique: (A) ZFC and FC results for Fe3O4 NPs; Magnetic field dependence of the
654 magnetization at 320 K for (B) Fe3O4 NPs, (C) Fe3O4 NPs coated with chitosan of 38 kDa, and (D) Fe3O4
655 NPs coated with chitosan of 469 kDa.
656
657

658

22
659
660 Figure 7 - Generated temperature as a function of iron concentration during 10 minutes of an AC
661 magnetic field application with intensity of 24 kA.m-1 and 418.5 kHz of frequency by: A) pristine-Fe3O4
662 NPs and Fe3O4-TD NPs, B) pristine-Fe3O4 NPs with and without CS coating, C) Fe3O4-TC NPs with and
663 without CS coating, and D) Fe3O4-OA NPs with and without CS coating.
664
665

666
667 Figure 8 – Comparison of SAR values of CS-Fe3O4 NPs. Fe3O4 NPs were coated with CS 38 kDa or CS
668 469 kDa. The results are expressed as the average ± standard deviation for 3 independent experiments.
669
670

23