A magnetic nanogel based on O-carboxymethylchitosan for antitumor drug delivery: synthesis, characterization and in vitro drug release.

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Soft Matter Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: C. A. Rodrigues, C. A. Demarchi, A. Debrassi, F. Campos Buzzi, R. Correa, V. Cechinel Filho, N. Nedelko, P. Demchenko, A. lawska-Waniewska and P. Dluzewski, Soft Matter, 2014, DOI: 10.1039/C3SM53157K.

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Soft Matter View Article Online

DOI: 10.1039/C3SM53157K

A magnetic nanogel based on O-carboxymethylchitosan for antitumor

Carla Albetina Demarchia, Aline Debrassia, Fátima de Campos Buzzia, Rogério Corrêaa, Valdir Cechinel Filhoa, Clovis Antonio Rodrigues*a, Nataliya Nedelkob, Pavlo Demchenkob, Anna Ślawska-Waniewskab, Piotr DłuŜewskib, Jean-Marc Grenechec.

a - Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí (UNIVALI), Itajaí, 88302-202, Santa Catarina, Brazil. Fax + 47 341 7601; email: [email protected] b- Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46 PL–02668, Warsaw, Poland. c- Laboratoire de Physique de l’Etat Condensé, UMR CNRS 6087, Université du Maine, 72085, Le Mans Cedex, France.

Abstract This paper studied the synthesis, characterization and use of a nanostructured magnetic chitosan nanogel for carrying meleimidic compounds with antitumor activity. The hydrogel polymer was prepared with O- carboxymethylchitosan, which was crosslinked with epichlorohydrin for subsequent incorporation of iron oxide magnetic nanoparticles. The characterization revealed that the magnetic material comprises about 10% of the hydrogel. This material isomprised of magnetite and maghemite and has ferroferrimagnetic behavior. The average particle size is 4.2 nm. There was high incorporation efficiency of maleimides in the magnetic nanogel. The release was


Published on 11 February 2014. Downloaded by St. Petersburg State University on 12/02/2014 22:48:07.

drug delivery: Synthesis, characterization and in vitro drug release.

Soft Matter

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sustained character and there was a greater release when an external magnetic field applied. The mathematical model that best explained the process of drug release by

an excellent candidate for use in drug-delivery systems.

1. Introduction Hydrogels are formed by three dimensional hydrophilic polymeric structures that are capable of absorbing large quantities of water, biological fluids, and toxic agents1. When the hydrogel has a particle size smaller than 200 nm, it is classified as a nanogel2. Hydrogel nanocomposites involve the incorporation of nanoparticles with hydrophilic matrix, which can improve the properties of conventional hydrogel systems. Many types of nanoparticles have been used in hydrogel nanocomposites systems, including carbon nanotubes, clay, ceramics, metal nanoparticles, and magnetic nanoparticles3, 4. Among the different types of magnetic particles, superparamagnetic nanoparticles of iron oxide, with an average diameter of about 10 nm, have proven to be the best candidates for biomedical applications5. Chitosan has been extensively applied in the biomedical field due to its versatility, biocompatibility, functionality, safety, and biodegradability6, 7. Magnetic hydrogels have been investigated and applied in biomedical procedures, because they can be stimulated by external conditions (variations in pH, ionic strength, temperature, magnetic field, etc.). Their biomedical applications include magnetic separation for protein purification8 and separation of certain cell type9, and are used as contrast agents in magnetic resonance imaging10, in the treatment of tumors by hyperthermia techniques11, and for the vectoring of drugs12, 13. These materials have particularly high potential for vectoring agents for the treatment of cancer. There are many disadvantages in the use of systemic



magnetic hydrogel was that of Peppas-Sahlin. The magnetic nanogel study proved to be

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chemotherapeutic agents for cancer treatment, such as systemic toxicity, low bioavailability of the drug, and difficulties in ensuring the drug targets the tumor site14.

of chemotherapeutic agents in therapeutic doses, increasing treatment efficacy and decreasing adverse effects. Based on the above, this study describes the synthesis, characterization, and in vitro drug release of a nanostructured hydrogel with superparamagnetic characteristics (magnetic nanogel), as a drug delivery system. Three maleimides that present antitumor activity agaist B16-F10 cell line15, HepB3 cell16, N1E115 neuroblastoma cell17 were used as model drugs in this study.

2. Materials and Methods

2.1. Materials Chitosan (Mw 265 g mol-1, deacetylation degree 80%) was obtained from Purifarma. All the reagents used were of analytical reagent grade, purchased from Vetec (Sao Paulo, Brazil) and used as received. Maleimides were prepared and characterized according to a method described in the literature18.

2.2. Synthesis of O-carboxymethylchitosan First, sodium hydroxide (27 g) was dissolved in distilled water (120 mL) and isopropanol (180 mL). Next, chitosan was added (20 g). This mixture was stirred for one hour at 0˚C and stored at the same temperature for 24 hours. A solution of monochloroacetic acid (30 g) in isopropanol (40 mL) was then dripped onto the previously prepared mixture, keeping the temperature at 0˚C. The reaction was



Magnetic hydrogels have the ability to overcome these obstacles through local delivery

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maintained at this temperature for approximately 48 hours and then stopped by adding 70% ethanol (400 mL). The resulting mixture was then filtered, washed with ethanol

2.3. Crosslinking of O-Carboxymethylchitosan with epichorohydrin For the crosslinking reaction, the O-Carboxymethylchitosan in salt form (20 g) was dissolved in approximately 600 mL of distilled water. Epichlorohydrin (13 mL) was then added, and the mixture was heated at 60˚C for 6 hours, with the pH adjusted to precipitate crosslinked O-Carboxymethylchitosan (O-CE). The mixture was then filtered with acetone and dried under vacuum for 24 h.

2.4. In situ synthesis of magnetic nanoparticles (O-CEMg) 13 g O-CE and 13 g of FeSO4.7 H2O were dispersed in distilled water (1.3 L) and the mixture was stirred for 2 h at room temperature to absorb iron ions within the O-CE particles. NaNO2 0.120 g dissolved in 10 ml water was added to the particle suspension and stirred for 15 min to oxidize the Fe2+ for Fe3+. Next 9.17 g of Fe(NH4)2(SO4)2.6 H2O dissolved in 100 mL, were added to the previous dispersion, the pH was adjusted to 9.0 with NH4OH solution and the mixture was stirred and heated for 30 minutes at 60°C. The magnetic nanogel was filtered, washed with distilled water, 95% ethanol, and acetone, and dried under vacuum for 24 h.

2.5. Characterization of magnetic nanogel The magnetic nanogel was characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC),



70% and dried under vacuum for 24 h19.

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magnetization measurements, Mössbauer spectroscopy, and transmission electron microscopy (TEM).

Shimadizu (Japan) in KBr discs. The crystal structure of the samples was studied by the XRD method with Fe Kα radiation (λKα = 1.9373 Å). DSC results were obtained using a Netzsch STA 449 F3 Jupiter thermal analyzer. Sample powders (8–10 mg) were crimped in an aluminum pan and heated at a constant rate of 10 °C min-1over a temperature range of 35 to 700 °C. Indium standard was used to calibrate the DSC temperature. N2 as purging gas at rate of 30 mL min-1. The magnetization measurements were carried using a vibrating sample magnetometer of PPMS (Quantum Design) in the temperature range 2-300 K. Mössbauer spectrometry were performed at 77 and 300 K using a source of

57

Co in Rh

matrix and the spectra were fitted with the Mosfit program assuming Lorentzian shape of the lines. The nanogel was also characterized with a transmission electron microscopy (TEM), using a Titan Cubed 80-300 FEI Cs image corrected instrument.

2.6. Swelling studies Dried nanogel of known mass (0.05g) was placed in contact with a specific volume of water, buffer solution pH 5.0 and 7.4 ( 2.0 mL) for different contact times. At regular time intervals, the swollen samples were removed and weighted after blotting off the excess water from the sample with a filter paper and the degree of swelling (DS), the swelling is calculated using Eq. 1.

(1)



The FTIR spectra were recorded in an Infrared Spectrometer Prestige-21,

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in which Ws and Wd are the masses of swollen at determinate time and dried nanogel,

2.7. Protein adsorption Bovine serum albumin (BSA) was dissolved in PBS (pH 7.4). In each experiment, 20 mg of magnetic nanogel was stirred with 20 mL of BSA solution at different concentrations at room temperature for 1 h. The mixed suspension was then centrifuged and the free BSA concentration in supernatant was measured using a UV spectrophotometer at 595 nm, according to the Bradford method. The amount of adsorbed BSA was calculated by the difference between the initial and final BSA concentrations in the supernatant21.

2.8. Drug incorporation The studies were done with three maleimides: (Fig. 1): N-phenyl-maleimide (M1), 4-methyl-N-phenyl-maleimide (M2) and 4-methoxy-N-phenyl-maleimide (M3).

Fig 1

The maleimides were incorporated by the solvent evaporation method22. The magnetic nanogel was dispersed in a solution of each maleimide in absolute ethanol, while stirring and heating until total evaporation of the solvent, resulting in OCEMgM1, O-CEMgM2 and O-CEMgM3 respectively. Drug loading content (DLC) and drug loading efficiency (DLE) of the magnetic nanogel were determined using a Spectro Vision DB-18805 spectrophotometer. A precise amount of the nanogel particles containing the maleimides was dispersed in



respectively20.

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ethanol and heated slightly. The particles were magnetically separated, the volume of the solution was completed to 10.0 mL, and the meleimide content was assessed by UV

% %

100 (2)

100 (3)

where dNP is the amount of drug in the nanoparticles, NP is the amount of nanoparticles, and di is the initial amount of drug in the system. The drug-loaded magnetic nanogels were characterized by FTIR spectroscopy, DSC (according to Section 2.5), and scanning electron microscopy (SEM), performed on a scanning electron microscope Philips XL-30.

2.9. In vitro release studies The in vitro release of the maleimides was conducted in phosphate buffer medium at pH 7.4, to simulate body fluid, and in medium with phosphate buffer at pH 5.0, to simulate conditions of mild acidity of tumor tissues24. Each nanogel containing the maleimides (50 mg) was placed in a dialysis membrane (molecular weight cut-off 12,000–14,000 g mol-1) containing 5 mL of medium, and the dialysis membranes were placed in 300 mL of each medium separately, at a temperature of 37°C, with stirring. Aliquots of samples were withdrawn periodically, replaced with an equal amount of medium. This procedure was performed with the maleimides alone for comparison. The concentrations of the maleimides were determined spectrophotometrically at 223 nm for M1, 221 nm for M2, and 274 nm for M3. This procedure was repeated in the presence of an external alternate magnetic field (AMF). The AMF was generated by rotating the sample to cut magnetic induction lines. The rotating speed was 60 rpm 25.



absorption. DLC and DLE were calculated using Eq. 2 and 323:

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3. Results and discussion

O-Carboxymethylchitosan was synthesized from chitosan via a bimolecular nucleophilic substitution reaction. The cross linking reaction with epichlorohydrin occurs by the same reaction mechanism (S1). The synthesis of magnetic nanoparticles was made in situ through the coprecipitation of ferric and ferrous ions (in 2:1 ratio) in a basic medium with the polymer.

3.2. Characterization of magnetic nanogel

3.2.1. Fourier transform infrared (FTIR) spectroscopy The FTIR spectrum of the magnetic nanogel is shown in Fig. 2. A broad band can be seen in the region of 3500 cm-1, characteristic of axial deformation of the O-H bond, a band at 1740 cm-1, characteristic of axial deformation of the C=O bond, and a band at 1600 cm-1, characteristic of axial deformation -COO-. These bands confirm the presence of the carboxylic acid grouping of O-Carboxymethylchitosan. The OH band is probably overlapping the band at 3450 cm-1, characteristic of axial deformation of the N-H bond. There is also the presence of bandwidth between 1150 and 900 cm-1 related to axial deformation of glycosidic linkages C-O and C-O-C. No difference is observed between O-CE and O-CEMg, because due to the amount of magnetic material being small absorption bands for the Fe3O4 were not detected for this technique. Fig 2

3.2.2. Microstructure



3.1. Synthesis of magnetic nanogel

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The X-ray diffraction pattern of O-CEMg is shown in Fig.3 and reveals broad lines that can be indexed with a cubic spinel crystal structure typical of magnetite and

almost overlap, therefore, it is very difficult to distinguish them based solely on X-ray studies. Fig 3

Fig. 4 (a) shows the TEM image of magnetic nanogel deposited on a carbon film with a magnification given by the scale bare of 5 nm. The iron oxide particles are dispersed in the nanogel forming agglomerates. Fourier filtered image of a selected nanoparticle, which is outlined in Fig.4(a) by a white square, is shown in Fig. 4(b). It allows to find the interplanar spacing of 300 pm and 480 pm corresponding to distances between {022} and {111} planes, respectively. Fig. 4(c) presents the Fourier transform from the area selected in (a) together with an electron diffraction pattern calculated and indexed according to iron oxide crystal structure. The observed lattice details demonstrate the crystalline nature of resultant nanoparticles. These well crystallized grains are separated one from each other by a thin disordered layer. The grain size distribution was determined by measuring the mean diameter of more than 1000 particles on the micrographs. Fig. 4d shows the particle size distribution fitted with log-normal distribution. The calculated average particle diameter (well crystallized cores) is ≈ 4.2 nm with a standard deviation σ ≈ 1.2 nm.

Fig 4



maghemite. As the diffractograms of these two phases belonging to a space group Fd3m

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3.2.3. Magnetic properties Magnetic behavior of O-CEMg was characterized with temperature and field

Design). Fig. 5(a) shows the temperature dependences of zero field cooled/field cooled (ZFC/FC) magnetization measured in an external field of 50 Oe. For ZFC/FC measurements, first the sample is cooled in zero field from room temperature to 2 K. Thereafter, 50 Oe magnetic field is applied and the magnetic moment is recorded with increasing temperature (ZFC curve). For the FC curve, the sample is cooled from 300 K to 2 K under the same 50 Oe field and the magnetic moment is recorded again as the temperature increases. Even though the particles are very small and should transit to a superparamagnetic state, no well defined peak is seen in MZFC(T) curve. Moreover, the irreversibility between the ZFC-FC curves are preserved over the whole temperature range and the FC curve is only weakly temperature dependent. The observed behavior shows that, in general, the magnetization process of O-CEMg is dominated by strong interparticle interactions and is analogous to the one observed by Guskos et al 26 for similar set of 4 nm γ-Fe2O3 nanoparticles in a polymer matrix. But a protrusion seen in ZFC curve seen in Fig. 5(a) at lower temperatures may indicate that some nanoparticles remain isolated and display superparamagnetism at temperatures below 100 K. In M(T) dependences any Verwey transition is not observed demonstrating that either the magnetite phase is not stechiometric or the dominant phase is maghemite. Furthermore, in FC curve an additional contribution of a paramagnetic type is seen at the lowest temperature. This contribution can originate from those Fe ions which did not enter to nanocrystals but remain chelated in the mononuclear form in chitosan.

Fig 5



dependences of magnetization performed with a PPMS magnetometer (Quantum

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The magnetization curves of OCEMg recorded at 2 and 298 K are shown in

for the small field range) confirming that the material is not in an entirely superparamagnetic state. Moreover the magnetization does not reach saturation in the field as high as 50 kOe (see the inset in Fig.5(b)). Such behavior has frequently been encountered in small maghemite nanoparticles in the literature 27. At RT the saturation magnetization is Ms = 6.7 emu/g, as estimated with a standard procedure by fitting of the high field data to the function M = Ms (1- a/H – b/H2), where M is the magnetization at the applied field H, a and b are the fitting parameters. The value obtained indicates that the weight fraction of magnetic phase is around 10% (as the magnetization of pure magnetite/ maghemite nanocrystals is in the range 60-80 emu g-1 28, in agreement with TG results, Fig 7. Independent on the temperature the loops are constricted in the middle. Such wasp-waisted loops are characteristic of magnetically coupled either (i) ferro-/ferri- and antiferromagnetic materials or (ii) two ferro-/ferrimagnetic materials with very different anisotropies. In the material studied the case (i) can be excluded because any shift of the loop was observed after cooling the sample in an applied magnetic field. Thus the most probable origin of the loop constriction is the disordered and highly anisotropic surface layer of magnetic particles exchange coupled to magnetically soft crystalline cores 29. Considering that the surface to volume ratio for such small particles is very high, the surface atoms with lower coordination number, lack of the translational symmetry and modified exchange coupling has an important effect on the overall magnetic properties of the granular system leading e.g. to waspwaisted loops, as predicted by the analysis in diluted random-field Ising model 30.



Fig.5(b). Even at the room temperature the loop displays irreversibility (see the image

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3.2.4. Mössbauer spectrometry

are composed of a magnetically split component superimposed on a paramagnetic doublet which intensity increases from ~8 % (at 77 K) to ~17% (at 300 K). Similar behavior has often been found in the literature for polymer coated maghemite nanoparticles 27,31. The average hyperfine field of the magnetically split sextet is ≈ 50.3 and 40.2 T at 77 and 300 K, respectively, and these values are typical of both magnetite and maghemite. Considering, however, the isomer shift, i.e. a parameter highly sensitive to the electron density (valency state), which in the sample studied is ≈ 47 and 0.36 mm/s (at 77 and 300 K, respectively), it can be concluded that the nanoparticles consist mainly of maghemite 28. A quadrupolar contribution seen at 77 K may originate from paramagnetic Fe ions spread in chitosan (seen also in zfc-fc curves) and/or small fraction of isolated particles that are in superparamagnetic state due to very small sizes. The increase of the relative fraction of this contribution at 300 K along with broadening of the magnetically split resonance lines, reflects the increasing fraction of superparamagnetic particles.

Fig 6

3.2.6. Differential scanning calorimetry (DSC) and thermogravimetry (TG) In the TG curve of O-CE and O-CEMg there is a mass loss of approximately 15% and 30%, respectively, caused by the loss of residual water in the samples, and the second at 300˚C, caused by polymer degradation (Fig. 7). O-CEMg presented 10% lower mass loss when compared with the loss of O-CE, which represents the amount of inorganic material (magnetic particles) contained in the material. DSC curves show



Mössbauer spectra of OCMg recorded at 77 and 300 K are shown in Fig.6. They

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endothermic peak at approximately 100 ˚C and exothermic peak at 300˚C for the two materials, corresponding to loss of residual water and polymer degradation,

Fig 7

3.3. Swelling studies The degree of swelling is an important property for the characterization of a nanogel because it evaluates the behavior of the polymer matrix in a given medium. The degree of swelling of a crosslinked polymer depends on the polymer, the degree of crosslinking, and the solvent in which the polymer is immersed 32. Fig. 8 and S2 show the degree of swelling of the magnetic nanogel when placed in contact with water, phosphate buffer pH 5.0, and 7.4.

Fig 8

Rapid swelling of the particles is observed, which tends to stabilize at around 120 minutes. This rapid swelling is due to the presence of hydrophilic groups (carboxyl and amino) on the structure of O-Carboxymethylchitosan, which in the absence of crosslinking points, dissolve in water, absorbing large quantities of water. When the degree of swelling is compared in three different media, water, phosphate buffer pH 5.0 (slightly acid as the tumor environment), and phosphate buffer pH 7.4, a high degree of swelling is observed in phosphate buffer pH 7.4. Due to the basicity of this media, the ionization of the carboxyl groups of the Ocarboxymethylchitosan increases water absorption. The lowest degree of swelling was observed in water, because the nanogel is not ionized. At pH 5.0 some protonation of



respectively.

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amino groups present in the O-Carboxymethylchitosan may occur, which explains the higher swelling compared to water. Another study of the nanogel based O-

due to the presence of carboxyl and amino groups that change the ionization state, depending on the pH33.

3.4. Protein adsorption Fig. 9 shows the adsorption isotherm of albumin (the major plasma protein) by magnetic nanogel after one hour of contact with a solution of bovine serum albumin at concentrations of 0.5 to 5.0 mg mL-1 in phosphate buffer pH 7.4, with the application of the Langmuir-Freundlich mathematical model.

Fig 9

Nanogel has a low adsorption capacity of albumin (80 mg g-1) and the maximum adsorption capacity is 143.6 mg g-1 with the application of the Langmuir-Freundlich isotherm (correlation coefficient = 0.99996). This low capacity is required for use in biological systems because the binding of plasma proteins on the surface of the nanogel (opsonization) is the bridge between the carrier and phagocytosis, so that the nanogel particles are easily captured by the reticuloendothelial system. Low adsorption capacities of plasma proteins reduce opsonization and prolong the circulation time of the carrier in vivo34, 35. The magnetic nanogel based on O-Carboxymethylchitosan presented lower adsorption capacity of plasma proteins than other carriers described in the literature21, 36.



Carboxymethylchitosan also showed that this swelling behavior influenced by the pH

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3.5. Drug loading content (DLC) and drug loading efficiency (DLE)

presented in Table 1. Table 1 High DLE and good DLC were observed, since the suggested value for DLC is between 5-25% 37. The hydroxyl and carboxyl groups present in Ocarboxymethylchitosan chains can form hydrogen bonds with the maleimide group in M1, M2 and M3 molecules to form intermolecular complexes, resulting in a high drug loading efficiency. Characterization of the maleimides loaded into the magnetic nanogel was performed by FTIR spectroscopy. In the FTIR spectra of M1, M2 and M3 loaded in the magnetic nanogel (Fig S3, Fig S4 and Fig S5, respectively), it is possible to observe the incorporation of the maleimides in the magnetic nanogel. There is a clear overlapping of the characteristic absorption bands in the spectra of maleimide derivatives alone, and those incorporated in the magnetic nanogel. In addition to the bands related to OCarboxymethylchitosan previously mentioned in item 3.2.1, the spectra show the band at 3100-3000 cm-1, characteristic of axial deformation of the C-H bond of the aromatic ring. The characteristic bands of the maleimide group at 1710 cm-1, characteristic of axial deformation of the C=O, two weak bands at about 1300 and 1200 cm-1, characteristic of axial deformation of the C-N aromatic bond, at approximately 1380 and 1145 cm-1, characteristic of axial symmetric and asymmetric deformation of the CN-C bonds, respectively, and at 830 cm-1, characteristic of symmetrical angular deformation out of the plane of the C-H maleimide bonds. In the spectrum of the



The DLC and DLE of maleimidics derivates loaded in the magnetic nanogel are

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derivative M3 (Fig S3), a band is observed at 1250 cm-1, characteristic to axial deformation of C-O bond of the methoxy group.

loaded in the magnetic nanogel. In TG analyses (Fig S6), compounds M1 and M2 have a great loss of mass (60 and 80%, respectively) at approximately 200˚C and compound M3 has a loss of 60% at 250˚C, corresponding to the thermal degradation of the compounds. TG curves of the magnetic nanogel containing the maleimides have a small initial loss of mass of between 50 and 100˚C, related to water loss. The mass loss of between 200 and 250˚C corresponds to the degradation of the maleimides, and after 300˚C, the mass loss corresponds to the degradation of the polymer. Fig S7 shows the DSC curves of the maleimides and these incorporated in the magnetic nanogel. DSC curves of derivatives M2 and M3 have an endothermic peak at approximately 150˚C and the derivative M1 presents this peak at 100˚C, which relates to the melting point of the crystalline form of the derivatives. DSC curves of M2 and M3 incorporated in the magnetic nanogel have an endothermic peak in the same region of 150˚C, indicating that the compounds remain in crystalline form and are therefore only physically deposited on the surface of the hydrogen. This peak does not appear in curve M1, probably because its melting point is in the same temperature range of the loss of residual water present in the sample. In SEM analyses (Fig 10), it is possible to observe an agglomerate of particles with irregular surface and the presence of a few crystals of the maleimides, indicating that the compounds are only deposited on the surface of the magnetic nanogel. These results confirm those obtained in the DSC experiments shown above.

Fig 10



Thermal analyses were also performed for the characterization of meleimides

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3.6. In vitro drug release In phosphate buffer pH 5.0 there was a release of 59% for M1, 85% for M2 and

there was a release of 57% for M1, 73% for M2, and 67% for M3, Fig. 12 (a). There is a large difference in the final amount released, but when they are incorporated into the nanogel, the maleimides are released more slowly, indicating sustained release. The pH 5.0 medium stimulates the release at the site of action, since the tumor environment is mildly acidic. For the proper action of the maleimides, it is important that they are released in a sustained way. However, this release should not be too slow, as this would involve maintenance of the external magnetic field at the affected site for a very long period, creating discomfort for the patient. Fig. 11(b) shows the release profiles of the maleimides alone and incorporated in the magnetic nanogel in phosphate buffer pH 7.4. The final amount of maleimides released was lower when these are incorporated into the magnetic nanogel (67% for M1, 57% for M2, and 66% for M3). In comparison, for the derivatives alone, the amount released was 80% for M1, 74% for M2, and 85% for M3. For magnetically vectorized release, it is important that most of the drug is released at the site of action. The release profiles for a physiological pH of 7.4 corroborates with this, because there is slower release (sustained). Thus, the drug, when incorporated into magnetic nanogel, reaches the site of action in greater quantity, and therefore can be more slowly released. This behavior shows that there is an interaction between the magnetic nanogel and the maleimides, delaying their release. Note that the derivatives begin to be released after about 10 minutes. Considering that the normal blood circulation time is one



60% for M3 when incorporated into the magnetic nanogel. For the maleimides alone,

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minute38, the material presents an appropriate release time, which is enough for the nanogel particles to reach the site of action and be trapped by the magnetic field.

The data obtained from the release curve were fitted to six kinetic equations (zero order, first order, Hixson–Crowell, Higuchi, Korsmeyer–Peppas, and PeppasSahlin) to determine the mechanism of drug release and the release rate. The following plots were made, which are presented in S7 and S8: Qt vs. t (zero order), log Qt vs. t (first order), Qt1/3 vs. t (Hixson–Crowell), Qt/Qinf vs. t1/2 (Higuchi), Qt/Qinf vs. t (Korsmeyer–Peppas non-linear model), and Qt/Qinf vs. t (Peppas-Sahlin non-linear model), where Qt is the amount of drug released at time t and Qinf is the amount of drug released at infinite time. The correlation coefficient (r) and constant release are show in Table S10. Through the values of correlation coefficients, the mathematical model that best explained the maleimide release process by the magnetic nanogel was the Peppas-Sahlin model. This kinetic model indicates a release mechanism that has the effect of Fickian diffusion and relaxation of the polymer chain (anomalous transport mechanism). To evaluate whether the presence of a magnetic field affects the release profile of maleimides, the release study was performed in phosphate buffer pH 5.0 with the application of an external magnetic field (EMF). Fig 11 (c) shows that there is a greater final amount released, which is favorable for the delivery system under study, because it leads to an increase in the accumulated amount of the drug at the site of action. The amount of maleimide, released in the presence of magnetic field is higher than those observed in the absence of magnetic field, specially for the M1 and M2 compounds, Table S11.



Fig 11

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For comparison the rate of release of compounds, with and without magnetic field, was chosen kinetic model of Korsmeyer-Peppas, since this model showed good

As can be observed, the values of K for the three nanogels are higher than those found for the release of the compounds without the magnetic field, Table S10. Increasing amount of the drug release has been observed in the presence of a high frequency alternating (AC) external magnetic field by Oliveira et al 39 and Samson et al 40 and generally ascribed to hyperthermia effect that causes a collapse of the nanogel polymer network promoting the release of trapped compounds 41. Considering however that in the studies of the maleimides release, the rotation rate was of an order of 1 Hz, the magnetic relaxation was too slow to allow the magnetic moments of particles to follow the oscillating magnetic field and heating effects can be excluded. The magnetic nanogels are also sensitive to static (DC) magnetic fields as shown by Liu et al. 42 being dependent on the concentration of magnetite particles, their dimensions, and the amplitude of magnetic field and properties of the polymer. For big maghemite particles (~200 nm) introduced to the gel by a freezing–thawing technique, the DC field caused a decrease of the drug release and this effect was ascribed to Fe3O4 aggregate formation of and related reduction of the gel porosity and drug diffusion 43. This mechanism is not very probable for very small iron oxide particles (~ 4 nm) in the nanogel studied as the magnetostatic attraction forces are in this case very small. For two adjacent identical particles, each one with a moment µ located at the center, the dipolar energy can be estimated from the formula Ed / kB = (µ0 / 4π kB) µ2 / d3 where d is the center-to-center distance and kB is the Boltzmann constant. For the magnetic nanogel studied µ ≈ 1200 µB and d ≈ 4.2 nm that gives Ed /kB (A) ≈ 14 K which is more than 4 orders of magnitude smaller than in the ferrogel discussed in Liu et al 42 .



correlation with the experimental and the calculated parameters are shown in Table S12.

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4. Conclusions

developed. The nanogel was characterized for various techniques showing ferroferrimagnetic behavior and average particle size of 4.2 nm. The magnetic material is about 10% of the nanogel. The nanogel showed rapid swelling due to the presence of hydrophilic groups (carboxyl and amino groups) in the structure of O-carboxymethyl and a low adsorption of plasma protein. There was a high incorporation efficiency of maleimides the magnetic nanogel. The release was sustained character and there was a greater release when an external magnetic field applied. The mathematical model that best explained the process of releasing derivatives maleimídicos by magnetic nanogel was to Peppas-Sahlin, pointing to a release mechanism that includes the effects of Fickian diffusion and relaxation of the polymer chain.

Acknowledgements C.A.R acknowledges the financial support given by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). C.A.D. thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brasil), for master fellowship. This work was partially performed in the laboratories founded by the European Union within the Innovative Economy Operational Programme POIG.02.02.00-00-025/09 and No POIG.02.01-00-14-032/08.



In our study, a magnetic nanogel for targeting and delivery of a therapeutic agent was

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228-236.

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Figure caption Fig.1 Maleimidics derivates.

Fig. 3. X-ray diffraction pattern of O-CEMg. Fig. 4.(a) TEM image of iron oxide nanoparticles in OCEchMagn; (b) Fourier filtered image of a selected nanoparticle outlined by a white square in (a); (c) Fourier transform from the selected area together with an electron diffraction pattern calculated and indexed according to Fe3O4 magnetite crystal structure; Histogram of the particle size distribution; the solid line is the best fit using a log-normal distribution function (d). Fig. 5. (a) Thermal evolution of ZFC/FC magnetization in the field of 50 Oe; (b) Enlarged parts of the hysteresis loops of OCEchMagn at 298 K and 2 K in a small field range selected from the full magnetization curves shown in the inset. Fig. 6. Mössbauer spectra of O-CEMg at 77 and 300 K. Fig 7. Thermal analysis of O-CE and O-CEMg. TG O-CE (1), TG O-CEMg (2), DSC O-CE (3) and DSC O-CEMg (4). Fig. 8. Degree of swelling of the O-CEMg in different pH conditions. Fig. 9. Adsorption isotherm of albumin by O-CEMg in phosphate buffer pH 7.4, with the application of Langmuir-Freundlich mathematical model. Fig. 10. SEM analyses of maleimides loaded in the magnetic nanogel, M1 (a), M2 (b) e M3 (c). Fig. 11. In vitro release of the meleimides at pH 5.0 (A), pH 7.4 (B) and pH 5.0 with application of external magnetic field (C).



Fig. 2. FTIR spectra of O-CE and O-CEMg

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Maleimide derivate

DLC

DLE

M1

5.96

74.50

M2

6.30

78.75

M3

5.80

72.50



Table 1. DLC and DLE of maleimides loaded in the magnetic nanogel.



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Fig 1

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105 100

Transmittance

90 85 80 75 70

4000

3500

3000

900

55

O-CEMg O-CE 2500

2000

1500 -1

Wave number (cm )

1150

60

1600

65

1000

500


95

1740


Fig 2

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(111)

2000

(311)

1600

(220) (...) magnetite & maghemite * maghemite

1400 1200

(400)

1000

*

(511) (422) (440) *

800 600 400 20

30

40

50

60

70

80

2theta (deg)

90

100

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120


1800

Intensity (a.u.)


Fig 3

Number of particles 60

50

d)

40

0




30

20

10

0

2

Partilce size (nm)

4

6

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Fig 4

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Fig 5

1,02

FC

0,90 0,84

0,72 0,66 0,60

ZFC

0,54

Hext = 50 Oe

0,48 0

100

200

300

Temperature (K)

5

298K

4 3 8 2

2K

Moment (emu/g)

6

1 0 -1

b

4 2 0 -2 -4 -6 -1

0

1

-200

2

3

4

5

6

Field (kOe)

-2 0

200

400

Field (Oe)

600

800


a

0,78

Moment (emu/g)

Moment (emu/g)


0,96

Relative transmission

Relative transmission


1,00

0,99

77 K

-12

1,00

-12 -9

-9 -6

-6 -3

V (mm/s)

-3 0

1,00

1,00

1,00

S23 300K

V (mm/s) 0 3 6 9 12

3 6 9 12



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Fig 6

1,00



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Fig 7

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6

5

4

3 Water Phosphate buffer pH 7,4 Phosphate buffer 5,0

2

1

0 0

20

40

60

80

100

120

140

160

180

200

Time (min)

Fig. 8. Degree of swelling of the magnetic hydrogel in different pH conditions.


Degree of swelling (g/g)


Fig 8

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Fig 9

80

70

60

50

40

0

1

2

3

4

5

Concentration (mg/mL)

Fig. 9. Adsorption isotherm of albumin by magnetic hydrogel in phosphate buffer pH 7.4, with the application of Langmuir-Freundlich mathematical model.


Adsorption capacity (mg/g)


90

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Fig. 10. SEM analyses of maleimides loaded in the magnetic hydrogel, M1 (a), M2 (b) e M3 (c).



Fig 10

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Fig 11

100 90

60 50

(a)

40 30 20

M1 M1 with EMF M2 M2 with EMF M3 M3 with EMF

0 -10 0

50

100

150

200

250

300

350

400

Time (min)

90 80

Amount released (%)

70 60 50

(b)

40 30 20

M1 O-CEMgM1 M2 O-CEMgM2 M3 O-CEMgM3

10 0 -10 0

50

100

150

200

250

300

350

400

Time (min)

100 90 80 70 60

(c)

50 40 30 O-CEMgM1 O-CEMgM2 O-CEMgM3 O-CEMgM1 /EMF O-CEMgM2 /EMF O-CEMgM3 /EMF

20 10 0 -10 0

50

100

150

200

Time (min)

250

300

350

400


Amount released (%)

70

10

Amount released (%)


80

Number of particles


60

50

40

30

20

10



0

2

4

Partilce size (nm)

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0

6

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A magnetic nanogel based on O-carboxymethylchitosan for antitumor

Carla Albetina Demarchia, Aline Debrassia, Fátima de Campos Buzzia, Rogério Corrêaa, Valdir Cechinel Filhoa, Clovis Antonio Rodrigues*a, Nataliya Nedelkob, Pavlo Demchenkob, Anna Ślawska-Waniewskab, Piotr DłuŜewskib, Jean-Marc Grenechec.

a - Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí (UNIVALI), Itajaí, 88302-202, Santa Catarina, Brazil. Fax + 47 341 7601; email: [email protected] b- Institute of Physics, Polish Academy of Sciences, Al. Lotników 32/46 PL–02668, Warsaw, Poland. c- Laboratoire de Physique de l’Etat Condensé, UMR CNRS 6087, Université du Maine, 72085, Le Mans Cedex, France.

Abstract This paper studied the synthesis, characterization and use of a nanostructured magnetic chitosan nanogel for carrying meleimidic compounds with antitumor activity. The hydrogel polymer was prepared with O- carboxymethylchitosan, which was crosslinked with epichlorohydrin for subsequent incorporation of iron oxide magnetic nanoparticles. The characterization revealed that the magnetic material comprises about 10% of the hydrogel. This material isomprised of magnetite and maghemite and has ferroferrimagnetic behavior. The average particle size is 4.2 nm. There was high incorporation efficiency of maleimides in the magnetic nanogel. The release was



drug delivery: Synthesis, characterization and in vitro drug release.

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sustained character and there was a greater release when an external magnetic field applied. The mathematical model that best explained the process of drug release by

an excellent candidate for use in drug-delivery systems.

1. Introduction Hydrogels are formed by three dimensional hydrophilic polymeric structures that are capable of absorbing large quantities of water, biological fluids, and toxic agents1. When the hydrogel has a particle size smaller than 200 nm, it is classified as a nanogel2. Hydrogel nanocomposites involve the incorporation of nanoparticles with hydrophilic matrix, which can improve the properties of conventional hydrogel systems. Many types of nanoparticles have been used in hydrogel nanocomposites systems, including carbon nanotubes, clay, ceramics, metal nanoparticles, and magnetic nanoparticles3, 4. Among the different types of magnetic particles, superparamagnetic nanoparticles of iron oxide, with an average diameter of about 10 nm, have proven to be the best candidates for biomedical applications5. Chitosan has been extensively applied in the biomedical field due to its versatility, biocompatibility, functionality, safety, and biodegradability6, 7. Magnetic hydrogels have been investigated and applied in biomedical procedures, because they can be stimulated by external conditions (variations in pH, ionic strength, temperature, magnetic field, etc.). Their biomedical applications include magnetic separation for protein purification8 and separation of certain cell type9, and are used as contrast agents in magnetic resonance imaging10, in the treatment of tumors by hyperthermia techniques11, and for the vectoring of drugs12, 13. These materials have particularly high potential for vectoring agents for the treatment of cancer. There are many disadvantages in the use of systemic



magnetic hydrogel was that of Peppas-Sahlin. The magnetic nanogel study proved to be

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chemotherapeutic agents for cancer treatment, such as systemic toxicity, low bioavailability of the drug, and difficulties in ensuring the drug targets the tumor site14.

of chemotherapeutic agents in therapeutic doses, increasing treatment efficacy and decreasing adverse effects. Based on the above, this study describes the synthesis, characterization, and in vitro drug release of a nanostructured hydrogel with superparamagnetic characteristics (magnetic nanogel), as a drug delivery system. Three maleimides that present antitumor activity agaist B16-F10 cell line15, HepB3 cell16, N1E115 neuroblastoma cell17 were used as model drugs in this study.

2. Materials and Methods

2.1. Materials Chitosan (Mw 265 g mol-1, deacetylation degree 80%) was obtained from Purifarma. All the reagents used were of analytical reagent grade, purchased from Vetec (Sao Paulo, Brazil) and used as received. Maleimides were prepared and characterized according to a method described in the literature18.

2.2. Synthesis of O-carboxymethylchitosan First, sodium hydroxide (27 g) was dissolved in distilled water (120 mL) and isopropanol (180 mL). Next, chitosan was added (20 g). This mixture was stirred for one hour at 0˚C and stored at the same temperature for 24 hours. A solution of monochloroacetic acid (30 g) in isopropanol (40 mL) was then dripped onto the previously prepared mixture, keeping the temperature at 0˚C. The reaction was



Magnetic hydrogels have the ability to overcome these obstacles through local delivery

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maintained at this temperature for approximately 48 hours and then stopped by adding 70% ethanol (400 mL). The resulting mixture was then filtered, washed with ethanol

2.3. Crosslinking of O-Carboxymethylchitosan with epichorohydrin For the crosslinking reaction, the O-Carboxymethylchitosan in salt form (20 g) was dissolved in approximately 600 mL of distilled water. Epichlorohydrin (13 mL) was then added, and the mixture was heated at 60˚C for 6 hours, with the pH adjusted to precipitate crosslinked O-Carboxymethylchitosan (O-CE). The mixture was then filtered with acetone and dried under vacuum for 24 h.

2.4. In situ synthesis of magnetic nanoparticles (O-CEMg) 13 g O-CE and 13 g of FeSO4.7 H2O were dispersed in distilled water (1.3 L) and the mixture was stirred for 2 h at room temperature to absorb iron ions within the O-CE particles. NaNO2 0.120 g dissolved in 10 ml water was added to the particle suspension and stirred for 15 min to oxidize the Fe2+ for Fe3+. Next 9.17 g of Fe(NH4)2(SO4)2.6 H2O dissolved in 100 mL, were added to the previous dispersion, the pH was adjusted to 9.0 with NH4OH solution and the mixture was stirred and heated for 30 minutes at 60°C. The magnetic nanogel was filtered, washed with distilled water, 95% ethanol, and acetone, and dried under vacuum for 24 h.

2.5. Characterization of magnetic nanogel The magnetic nanogel was characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), differential scanning calorimetry (DSC),



70% and dried under vacuum for 24 h19.

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magnetization measurements, Mössbauer spectroscopy, and transmission electron microscopy (TEM).

Shimadizu (Japan) in KBr discs. The crystal structure of the samples was studied by the XRD method with Fe Kα radiation (λKα = 1.9373 Å). DSC results were obtained using a Netzsch STA 449 F3 Jupiter thermal analyzer. Sample powders (8–10 mg) were crimped in an aluminum pan and heated at a constant rate of 10 °C min-1over a temperature range of 35 to 700 °C. Indium standard was used to calibrate the DSC temperature. N2 as purging gas at rate of 30 mL min-1. The magnetization measurements were carried using a vibrating sample magnetometer of PPMS (Quantum Design) in the temperature range 2-300 K. Mössbauer spectrometry were performed at 77 and 300 K using a source of

57

Co in Rh

matrix and the spectra were fitted with the Mosfit program assuming Lorentzian shape of the lines. The nanogel was also characterized with a transmission electron microscopy (TEM), using a Titan Cubed 80-300 FEI Cs image corrected instrument.

2.6. Swelling studies Dried nanogel of known mass (0.05g) was placed in contact with a specific volume of water, buffer solution pH 5.0 and 7.4 ( 2.0 mL) for different contact times. At regular time intervals, the swollen samples were removed and weighted after blotting off the excess water from the sample with a filter paper and the degree of swelling (DS), the swelling is calculated using Eq. 1.

(1)



The FTIR spectra were recorded in an Infrared Spectrometer Prestige-21,

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in which Ws and Wd are the masses of swollen at determinate time and dried nanogel,

2.7. Protein adsorption Bovine serum albumin (BSA) was dissolved in PBS (pH 7.4). In each experiment, 20 mg of magnetic nanogel was stirred with 20 mL of BSA solution at different concentrations at room temperature for 1 h. The mixed suspension was then centrifuged and the free BSA concentration in supernatant was measured using a UV spectrophotometer at 595 nm, according to the Bradford method. The amount of adsorbed BSA was calculated by the difference between the initial and final BSA concentrations in the supernatant21.

2.8. Drug incorporation The studies were done with three maleimides: (Fig. 1): N-phenyl-maleimide (M1), 4-methyl-N-phenyl-maleimide (M2) and 4-methoxy-N-phenyl-maleimide (M3).

Fig 1

The maleimides were incorporated by the solvent evaporation method22. The magnetic nanogel was dispersed in a solution of each maleimide in absolute ethanol, while stirring and heating until total evaporation of the solvent, resulting in OCEMgM1, O-CEMgM2 and O-CEMgM3 respectively. Drug loading content (DLC) and drug loading efficiency (DLE) of the magnetic nanogel were determined using a Spectro Vision DB-18805 spectrophotometer. A precise amount of the nanogel particles containing the maleimides was dispersed in



respectively20.

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ethanol and heated slightly. The particles were magnetically separated, the volume of the solution was completed to 10.0 mL, and the meleimide content was assessed by UV

% %

100 (2)

100 (3)

where dNP is the amount of drug in the nanoparticles, NP is the amount of nanoparticles, and di is the initial amount of drug in the system. The drug-loaded magnetic nanogels were characterized by FTIR spectroscopy, DSC (according to Section 2.5), and scanning electron microscopy (SEM), performed on a scanning electron microscope Philips XL-30.

2.9. In vitro release studies The in vitro release of the maleimides was conducted in phosphate buffer medium at pH 7.4, to simulate body fluid, and in medium with phosphate buffer at pH 5.0, to simulate conditions of mild acidity of tumor tissues24. Each nanogel containing the maleimides (50 mg) was placed in a dialysis membrane (molecular weight cut-off 12,000–14,000 g mol-1) containing 5 mL of medium, and the dialysis membranes were placed in 300 mL of each medium separately, at a temperature of 37°C, with stirring. Aliquots of samples were withdrawn periodically, replaced with an equal amount of medium. This procedure was performed with the maleimides alone for comparison. The concentrations of the maleimides were determined spectrophotometrically at 223 nm for M1, 221 nm for M2, and 274 nm for M3. This procedure was repeated in the presence of an external alternate magnetic field (AMF). The AMF was generated by rotating the sample to cut magnetic induction lines. The rotating speed was 60 rpm 25.



absorption. DLC and DLE were calculated using Eq. 2 and 323:

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DOI: 10.1039/C3SM53157K

3. Results and discussion

O-Carboxymethylchitosan was synthesized from chitosan via a bimolecular nucleophilic substitution reaction. The cross linking reaction with epichlorohydrin occurs by the same reaction mechanism (S1). The synthesis of magnetic nanoparticles was made in situ through the coprecipitation of ferric and ferrous ions (in 2:1 ratio) in a basic medium with the polymer.

3.2. Characterization of magnetic nanogel

3.2.1. Fourier transform infrared (FTIR) spectroscopy The FTIR spectrum of the magnetic nanogel is shown in Fig. 2. A broad band can be seen in the region of 3500 cm-1, characteristic of axial deformation of the O-H bond, a band at 1740 cm-1, characteristic of axial deformation of the C=O bond, and a band at 1600 cm-1, characteristic of axial deformation -COO-. These bands confirm the presence of the carboxylic acid grouping of O-Carboxymethylchitosan. The OH band is probably overlapping the band at 3450 cm-1, characteristic of axial deformation of the N-H bond. There is also the presence of bandwidth between 1150 and 900 cm-1 related to axial deformation of glycosidic linkages C-O and C-O-C. No difference is observed between O-CE and O-CEMg, because due to the amount of magnetic material being small absorption bands for the Fe3O4 were not detected for this technique. Fig 2

3.2.2. Microstructure



3.1. Synthesis of magnetic nanogel

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The X-ray diffraction pattern of O-CEMg is shown in Fig.3 and reveals broad lines that can be indexed with a cubic spinel crystal structure typical of magnetite and

almost overlap, therefore, it is very difficult to distinguish them based solely on X-ray studies. Fig 3

Fig. 4 (a) shows the TEM image of magnetic nanogel deposited on a carbon film with a magnification given by the scale bare of 5 nm. The iron oxide particles are dispersed in the nanogel forming agglomerates. Fourier filtered image of a selected nanoparticle, which is outlined in Fig.4(a) by a white square, is shown in Fig. 4(b). It allows to find the interplanar spacing of 300 pm and 480 pm corresponding to distances between {022} and {111} planes, respectively. Fig. 4(c) presents the Fourier transform from the area selected in (a) together with an electron diffraction pattern calculated and indexed according to iron oxide crystal structure. The observed lattice details demonstrate the crystalline nature of resultant nanoparticles. These well crystallized grains are separated one from each other by a thin disordered layer. The grain size distribution was determined by measuring the mean diameter of more than 1000 particles on the micrographs. Fig. 4d shows the particle size distribution fitted with log-normal distribution. The calculated average particle diameter (well crystallized cores) is ≈ 4.2 nm with a standard deviation σ ≈ 1.2 nm.

Fig 4



maghemite. As the diffractograms of these two phases belonging to a space group Fd3m

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3.2.3. Magnetic properties Magnetic behavior of O-CEMg was characterized with temperature and field

Design). Fig. 5(a) shows the temperature dependences of zero field cooled/field cooled (ZFC/FC) magnetization measured in an external field of 50 Oe. For ZFC/FC measurements, first the sample is cooled in zero field from room temperature to 2 K. Thereafter, 50 Oe magnetic field is applied and the magnetic moment is recorded with increasing temperature (ZFC curve). For the FC curve, the sample is cooled from 300 K to 2 K under the same 50 Oe field and the magnetic moment is recorded again as the temperature increases. Even though the particles are very small and should transit to a superparamagnetic state, no well defined peak is seen in MZFC(T) curve. Moreover, the irreversibility between the ZFC-FC curves are preserved over the whole temperature range and the FC curve is only weakly temperature dependent. The observed behavior shows that, in general, the magnetization process of O-CEMg is dominated by strong interparticle interactions and is analogous to the one observed by Guskos et al 26 for similar set of 4 nm γ-Fe2O3 nanoparticles in a polymer matrix. But a protrusion seen in ZFC curve seen in Fig. 5(a) at lower temperatures may indicate that some nanoparticles remain isolated and display superparamagnetism at temperatures below 100 K. In M(T) dependences any Verwey transition is not observed demonstrating that either the magnetite phase is not stechiometric or the dominant phase is maghemite. Furthermore, in FC curve an additional contribution of a paramagnetic type is seen at the lowest temperature. This contribution can originate from those Fe ions which did not enter to nanocrystals but remain chelated in the mononuclear form in chitosan.

Fig 5



dependences of magnetization performed with a PPMS magnetometer (Quantum

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The magnetization curves of OCEMg recorded at 2 and 298 K are shown in

for the small field range) confirming that the material is not in an entirely superparamagnetic state. Moreover the magnetization does not reach saturation in the field as high as 50 kOe (see the inset in Fig.5(b)). Such behavior has frequently been encountered in small maghemite nanoparticles in the literature 27. At RT the saturation magnetization is Ms = 6.7 emu/g, as estimated with a standard procedure by fitting of the high field data to the function M = Ms (1- a/H – b/H2), where M is the magnetization at the applied field H, a and b are the fitting parameters. The value obtained indicates that the weight fraction of magnetic phase is around 10% (as the magnetization of pure magnetite/ maghemite nanocrystals is in the range 60-80 emu g-1 28, in agreement with TG results, Fig 7. Independent on the temperature the loops are constricted in the middle. Such wasp-waisted loops are characteristic of magnetically coupled either (i) ferro-/ferri- and antiferromagnetic materials or (ii) two ferro-/ferrimagnetic materials with very different anisotropies. In the material studied the case (i) can be excluded because any shift of the loop was observed after cooling the sample in an applied magnetic field. Thus the most probable origin of the loop constriction is the disordered and highly anisotropic surface layer of magnetic particles exchange coupled to magnetically soft crystalline cores 29. Considering that the surface to volume ratio for such small particles is very high, the surface atoms with lower coordination number, lack of the translational symmetry and modified exchange coupling has an important effect on the overall magnetic properties of the granular system leading e.g. to waspwaisted loops, as predicted by the analysis in diluted random-field Ising model 30.



Fig.5(b). Even at the room temperature the loop displays irreversibility (see the image

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3.2.4. Mössbauer spectrometry

are composed of a magnetically split component superimposed on a paramagnetic doublet which intensity increases from ~8 % (at 77 K) to ~17% (at 300 K). Similar behavior has often been found in the literature for polymer coated maghemite nanoparticles 27,31. The average hyperfine field of the magnetically split sextet is ≈ 50.3 and 40.2 T at 77 and 300 K, respectively, and these values are typical of both magnetite and maghemite. Considering, however, the isomer shift, i.e. a parameter highly sensitive to the electron density (valency state), which in the sample studied is ≈ 47 and 0.36 mm/s (at 77 and 300 K, respectively), it can be concluded that the nanoparticles consist mainly of maghemite 28. A quadrupolar contribution seen at 77 K may originate from paramagnetic Fe ions spread in chitosan (seen also in zfc-fc curves) and/or small fraction of isolated particles that are in superparamagnetic state due to very small sizes. The increase of the relative fraction of this contribution at 300 K along with broadening of the magnetically split resonance lines, reflects the increasing fraction of superparamagnetic particles.

Fig 6

3.2.6. Differential scanning calorimetry (DSC) and thermogravimetry (TG) In the TG curve of O-CE and O-CEMg there is a mass loss of approximately 15% and 30%, respectively, caused by the loss of residual water in the samples, and the second at 300˚C, caused by polymer degradation (Fig. 7). O-CEMg presented 10% lower mass loss when compared with the loss of O-CE, which represents the amount of inorganic material (magnetic particles) contained in the material. DSC curves show



Mössbauer spectra of OCMg recorded at 77 and 300 K are shown in Fig.6. They

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endothermic peak at approximately 100 ˚C and exothermic peak at 300˚C for the two materials, corresponding to loss of residual water and polymer degradation,

Fig 7

3.3. Swelling studies The degree of swelling is an important property for the characterization of a nanogel because it evaluates the behavior of the polymer matrix in a given medium. The degree of swelling of a crosslinked polymer depends on the polymer, the degree of crosslinking, and the solvent in which the polymer is immersed 32. Fig. 8 and S2 show the degree of swelling of the magnetic nanogel when placed in contact with water, phosphate buffer pH 5.0, and 7.4.

Fig 8

Rapid swelling of the particles is observed, which tends to stabilize at around 120 minutes. This rapid swelling is due to the presence of hydrophilic groups (carboxyl and amino) on the structure of O-Carboxymethylchitosan, which in the absence of crosslinking points, dissolve in water, absorbing large quantities of water. When the degree of swelling is compared in three different media, water, phosphate buffer pH 5.0 (slightly acid as the tumor environment), and phosphate buffer pH 7.4, a high degree of swelling is observed in phosphate buffer pH 7.4. Due to the basicity of this media, the ionization of the carboxyl groups of the Ocarboxymethylchitosan increases water absorption. The lowest degree of swelling was observed in water, because the nanogel is not ionized. At pH 5.0 some protonation of



respectively.

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amino groups present in the O-Carboxymethylchitosan may occur, which explains the higher swelling compared to water. Another study of the nanogel based O-

due to the presence of carboxyl and amino groups that change the ionization state, depending on the pH33.

3.4. Protein adsorption Fig. 9 shows the adsorption isotherm of albumin (the major plasma protein) by magnetic nanogel after one hour of contact with a solution of bovine serum albumin at concentrations of 0.5 to 5.0 mg mL-1 in phosphate buffer pH 7.4, with the application of the Langmuir-Freundlich mathematical model.

Fig 9

Nanogel has a low adsorption capacity of albumin (80 mg g-1) and the maximum adsorption capacity is 143.6 mg g-1 with the application of the Langmuir-Freundlich isotherm (correlation coefficient = 0.99996). This low capacity is required for use in biological systems because the binding of plasma proteins on the surface of the nanogel (opsonization) is the bridge between the carrier and phagocytosis, so that the nanogel particles are easily captured by the reticuloendothelial system. Low adsorption capacities of plasma proteins reduce opsonization and prolong the circulation time of the carrier in vivo34, 35. The magnetic nanogel based on O-Carboxymethylchitosan presented lower adsorption capacity of plasma proteins than other carriers described in the literature21, 36.



Carboxymethylchitosan also showed that this swelling behavior influenced by the pH

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3.5. Drug loading content (DLC) and drug loading efficiency (DLE)

presented in Table 1. Table 1 High DLE and good DLC were observed, since the suggested value for DLC is between 5-25% 37. The hydroxyl and carboxyl groups present in Ocarboxymethylchitosan chains can form hydrogen bonds with the maleimide group in M1, M2 and M3 molecules to form intermolecular complexes, resulting in a high drug loading efficiency. Characterization of the maleimides loaded into the magnetic nanogel was performed by FTIR spectroscopy. In the FTIR spectra of M1, M2 and M3 loaded in the magnetic nanogel (Fig S3, Fig S4 and Fig S5, respectively), it is possible to observe the incorporation of the maleimides in the magnetic nanogel. There is a clear overlapping of the characteristic absorption bands in the spectra of maleimide derivatives alone, and those incorporated in the magnetic nanogel. In addition to the bands related to OCarboxymethylchitosan previously mentioned in item 3.2.1, the spectra show the band at 3100-3000 cm-1, characteristic of axial deformation of the C-H bond of the aromatic ring. The characteristic bands of the maleimide group at 1710 cm-1, characteristic of axial deformation of the C=O, two weak bands at about 1300 and 1200 cm-1, characteristic of axial deformation of the C-N aromatic bond, at approximately 1380 and 1145 cm-1, characteristic of axial symmetric and asymmetric deformation of the CN-C bonds, respectively, and at 830 cm-1, characteristic of symmetrical angular deformation out of the plane of the C-H maleimide bonds. In the spectrum of the



The DLC and DLE of maleimidics derivates loaded in the magnetic nanogel are

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derivative M3 (Fig S3), a band is observed at 1250 cm-1, characteristic to axial deformation of C-O bond of the methoxy group.

loaded in the magnetic nanogel. In TG analyses (Fig S6), compounds M1 and M2 have a great loss of mass (60 and 80%, respectively) at approximately 200˚C and compound M3 has a loss of 60% at 250˚C, corresponding to the thermal degradation of the compounds. TG curves of the magnetic nanogel containing the maleimides have a small initial loss of mass of between 50 and 100˚C, related to water loss. The mass loss of between 200 and 250˚C corresponds to the degradation of the maleimides, and after 300˚C, the mass loss corresponds to the degradation of the polymer. Fig S7 shows the DSC curves of the maleimides and these incorporated in the magnetic nanogel. DSC curves of derivatives M2 and M3 have an endothermic peak at approximately 150˚C and the derivative M1 presents this peak at 100˚C, which relates to the melting point of the crystalline form of the derivatives. DSC curves of M2 and M3 incorporated in the magnetic nanogel have an endothermic peak in the same region of 150˚C, indicating that the compounds remain in crystalline form and are therefore only physically deposited on the surface of the hydrogen. This peak does not appear in curve M1, probably because its melting point is in the same temperature range of the loss of residual water present in the sample. In SEM analyses (Fig 10), it is possible to observe an agglomerate of particles with irregular surface and the presence of a few crystals of the maleimides, indicating that the compounds are only deposited on the surface of the magnetic nanogel. These results confirm those obtained in the DSC experiments shown above.

Fig 10



Thermal analyses were also performed for the characterization of meleimides

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3.6. In vitro drug release In phosphate buffer pH 5.0 there was a release of 59% for M1, 85% for M2 and

there was a release of 57% for M1, 73% for M2, and 67% for M3, Fig. 12 (a). There is a large difference in the final amount released, but when they are incorporated into the nanogel, the maleimides are released more slowly, indicating sustained release. The pH 5.0 medium stimulates the release at the site of action, since the tumor environment is mildly acidic. For the proper action of the maleimides, it is important that they are released in a sustained way. However, this release should not be too slow, as this would involve maintenance of the external magnetic field at the affected site for a very long period, creating discomfort for the patient. Fig. 11(b) shows the release profiles of the maleimides alone and incorporated in the magnetic nanogel in phosphate buffer pH 7.4. The final amount of maleimides released was lower when these are incorporated into the magnetic nanogel (67% for M1, 57% for M2, and 66% for M3). In comparison, for the derivatives alone, the amount released was 80% for M1, 74% for M2, and 85% for M3. For magnetically vectorized release, it is important that most of the drug is released at the site of action. The release profiles for a physiological pH of 7.4 corroborates with this, because there is slower release (sustained). Thus, the drug, when incorporated into magnetic nanogel, reaches the site of action in greater quantity, and therefore can be more slowly released. This behavior shows that there is an interaction between the magnetic nanogel and the maleimides, delaying their release. Note that the derivatives begin to be released after about 10 minutes. Considering that the normal blood circulation time is one



60% for M3 when incorporated into the magnetic nanogel. For the maleimides alone,

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minute38, the material presents an appropriate release time, which is enough for the nanogel particles to reach the site of action and be trapped by the magnetic field.

The data obtained from the release curve were fitted to six kinetic equations (zero order, first order, Hixson–Crowell, Higuchi, Korsmeyer–Peppas, and PeppasSahlin) to determine the mechanism of drug release and the release rate. The following plots were made, which are presented in S7 and S8: Qt vs. t (zero order), log Qt vs. t (first order), Qt1/3 vs. t (Hixson–Crowell), Qt/Qinf vs. t1/2 (Higuchi), Qt/Qinf vs. t (Korsmeyer–Peppas non-linear model), and Qt/Qinf vs. t (Peppas-Sahlin non-linear model), where Qt is the amount of drug released at time t and Qinf is the amount of drug released at infinite time. The correlation coefficient (r) and constant release are show in Table S10. Through the values of correlation coefficients, the mathematical model that best explained the maleimide release process by the magnetic nanogel was the Peppas-Sahlin model. This kinetic model indicates a release mechanism that has the effect of Fickian diffusion and relaxation of the polymer chain (anomalous transport mechanism). To evaluate whether the presence of a magnetic field affects the release profile of maleimides, the release study was performed in phosphate buffer pH 5.0 with the application of an external magnetic field (EMF). Fig 11 (c) shows that there is a greater final amount released, which is favorable for the delivery system under study, because it leads to an increase in the accumulated amount of the drug at the site of action. The amount of maleimide, released in the presence of magnetic field is higher than those observed in the absence of magnetic field, specially for the M1 and M2 compounds, Table S11.



Fig 11

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For comparison the rate of release of compounds, with and without magnetic field, was chosen kinetic model of Korsmeyer-Peppas, since this model showed good

As can be observed, the values of K for the three nanogels are higher than those found for the release of the compounds without the magnetic field, Table S10. Increasing amount of the drug release has been observed in the presence of a high frequency alternating (AC) external magnetic field by Oliveira et al 39 and Samson et al 40 and generally ascribed to hyperthermia effect that causes a collapse of the nanogel polymer network promoting the release of trapped compounds 41. Considering however that in the studies of the maleimides release, the rotation rate was of an order of 1 Hz, the magnetic relaxation was too slow to allow the magnetic moments of particles to follow the oscillating magnetic field and heating effects can be excluded. The magnetic nanogels are also sensitive to static (DC) magnetic fields as shown by Liu et al. 42 being dependent on the concentration of magnetite particles, their dimensions, and the amplitude of magnetic field and properties of the polymer. For big maghemite particles (~200 nm) introduced to the gel by a freezing–thawing technique, the DC field caused a decrease of the drug release and this effect was ascribed to Fe3O4 aggregate formation of and related reduction of the gel porosity and drug diffusion 43. This mechanism is not very probable for very small iron oxide particles (~ 4 nm) in the nanogel studied as the magnetostatic attraction forces are in this case very small. For two adjacent identical particles, each one with a moment µ located at the center, the dipolar energy can be estimated from the formula Ed / kB = (µ0 / 4π kB) µ2 / d3 where d is the center-to-center distance and kB is the Boltzmann constant. For the magnetic nanogel studied µ ≈ 1200 µB and d ≈ 4.2 nm that gives Ed /kB (A) ≈ 14 K which is more than 4 orders of magnitude smaller than in the ferrogel discussed in Liu et al 42 .



correlation with the experimental and the calculated parameters are shown in Table S12.

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4. Conclusions

developed. The nanogel was characterized for various techniques showing ferroferrimagnetic behavior and average particle size of 4.2 nm. The magnetic material is about 10% of the nanogel. The nanogel showed rapid swelling due to the presence of hydrophilic groups (carboxyl and amino groups) in the structure of O-carboxymethyl and a low adsorption of plasma protein. There was a high incorporation efficiency of maleimides the magnetic nanogel. The release was sustained character and there was a greater release when an external magnetic field applied. The mathematical model that best explained the process of releasing derivatives maleimídicos by magnetic nanogel was to Peppas-Sahlin, pointing to a release mechanism that includes the effects of Fickian diffusion and relaxation of the polymer chain.

Acknowledgements C.A.R acknowledges the financial support given by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). C.A.D. thanks the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brasil), for master fellowship. This work was partially performed in the laboratories founded by the European Union within the Innovative Economy Operational Programme POIG.02.02.00-00-025/09 and No POIG.02.01-00-14-032/08.



In our study, a magnetic nanogel for targeting and delivery of a therapeutic agent was

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228-236.

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Figure caption Fig.1 Maleimidics derivates.

Fig. 3. X-ray diffraction pattern of O-CEMg. Fig. 4.(a) TEM image of iron oxide nanoparticles in OCEchMagn; (b) Fourier filtered image of a selected nanoparticle outlined by a white square in (a); (c) Fourier transform from the selected area together with an electron diffraction pattern calculated and indexed according to Fe3O4 magnetite crystal structure; Histogram of the particle size distribution; the solid line is the best fit using a log-normal distribution function (d). Fig. 5. (a) Thermal evolution of ZFC/FC magnetization in the field of 50 Oe; (b) Enlarged parts of the hysteresis loops of OCEchMagn at 298 K and 2 K in a small field range selected from the full magnetization curves shown in the inset. Fig. 6. Mössbauer spectra of O-CEMg at 77 and 300 K. Fig 7. Thermal analysis of O-CE and O-CEMg. TG O-CE (1), TG O-CEMg (2), DSC O-CE (3) and DSC O-CEMg (4). Fig. 8. Degree of swelling of the O-CEMg in different pH conditions. Fig. 9. Adsorption isotherm of albumin by O-CEMg in phosphate buffer pH 7.4, with the application of Langmuir-Freundlich mathematical model. Fig. 10. SEM analyses of maleimides loaded in the magnetic nanogel, M1 (a), M2 (b) e M3 (c). Fig. 11. In vitro release of the meleimides at pH 5.0 (A), pH 7.4 (B) and pH 5.0 with application of external magnetic field (C).



Fig. 2. FTIR spectra of O-CE and O-CEMg

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Maleimide derivate

DLC

DLE

M1

5.96

74.50

M2

6.30

78.75

M3

5.80

72.50



Table 1. DLC and DLE of maleimides loaded in the magnetic nanogel.

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