New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery

Accepted Manuscript Title: New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery Author: Liana Alupei Catalina Anisoara Peptu Andreea-Maria Lungan Jacques Desbrieres Ovidiu Chiscan Sadia Radji Marcel Popa PII: DOI: Reference:

S0141-8130(16)30904-7 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.07.058 BIOMAC 6332

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

4-3-2016 15-7-2016 17-7-2016

Please cite this article as: Liana Alupei, Catalina Anisoara Peptu, AndreeaMaria Lungan, Jacques Desbrieres, Ovidiu Chiscan, Sadia Radji, Marcel Popa, New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.07.058 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

New hybrid magnetic nanoparticles based on chitosan-maltose derivative for antitumor drug delivery

Liana Alupeia, Catalina Anisoara Peptua, Andreea-Maria Lunganb, Jacques Desbrieresc, Ovidiu Chiscand, Sadia Radjic, Marcel Popaa,e,*

a

Technical University “Gheorghe Asachi”, Faculty of Chemical Engineering and Protection of the

Environment, Department of Natural and Synthetic Polymers, 73, Bd. Professor dr. docent Dimitrie Mangeron, 700050 Iasi, Romania. b

Purolite SRL, Research & Development Department, Aleea Uzinei Street, No. 11,Victoria,

Brasov, Romania. c

Pau et Pays de l’Adour University, IPREM (UMR CNRS 5254), Helioparc Pau Pyrénées, 64053

PAU Cedex 09, France. d

Alexandru Ioan Cuza University, Department of Physics, Iasi, 700506, Romania.

e

The Academy of Romanian Scientists, Bd. Independentei, 54, Sector 5, Bucuresti, 50085,

Romania.

* Corresponding author. Tel.: +40232278683; fax : +40232271311. Email address: [email protected], [email protected] (M. Popa).

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Abstract The aim of the present study is to obtain, for the first time, polymer magnetic nanoparticles based on the chitosan-maltose derivative and magnetite. By chemically modifying the chitosan, its solubility in aqueous media was improved, which in turn facilitates the nanoparticles’ preparation. Resulting polymers exhibit enhanced hydrophilia, which is an important factor in increasing the retention time of nanoparticles in the blood flow. The preparation of nanoparticles relied on the double crosslinking technique (ionic and covalent) in reverse emulsion which ensures the mechanical stability of the polymer carrier. The characterization of both the chitosan derivative and nanoparticles was accomplished by Fourier Transform Infrared Spectroscopy, Nuclear Magnetic Resonance Spectroscopy, Scanning Electron Microscopy, Transmission Electron Microscopy, Atomic Force Microscopy, Vibrating Sample Magnetometry, and Thermogravimetric Analysis. The evaluation of morphological, dimensional, structural, and magnetical properties, as well as thermal stability and swelling behavior of nanoparticles was made from the point of view of the polymer/magnetite ratio. The study of 5-Fluorouracil loading and release kinetics as well as evaluating the cytotoxicity and hemocompatibility of nanoparticles justify their adequate behavior in their potential use as devices for targeted transport of antitumor drugs.

Keywords: Chitosan, maltose, nanoparticle, magnetite, 5-Fluorouracil.

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1. Introduction The possibility for investigation and manipulation at the molecular and cellular level allows nanotechnology to have remarkable progress in the biomedical fields. Due to their peculiar properties (biocompatibility, size comparable to that of biological entities, large specific surface, possibility for chemical modifications, etc.), the magnetic nanoparticles with natural polymers matrix demonstrate a significant potential in a large area of medical applications, such as diagnosis, therapy, bioseparation [1-5]. In the majority of cases, the magnetic particles are obtained from hybrids of ferromagnetic elements (Fe, Co, Ni) with other metals, ions, oxygen or carbon dioxide. Among these, the Febased compounds are preponderantly utilized due to the simple obtaining method, chemical stability, biocompatibility, superparamagnetism and lack of toxicity. Forms such as magnetite (Fe3O4), maghemite (γ-Fe2O3), hematite (α-Fe2O3) and other types of ferrites possess a high magnetic moment that allows their physical manipulation by means of an exterior magnetic field [3,6-7]. In order to improve performance, the magnetic particles can be functionalized in surface or embedded in a polymer matrix. The agglomeration of the magnetic nanoparticles as a result of the tendency to decrease the high surface energy can be avoided by the surface functionalization. The amphiphile surfactants’ adsorption allows for a good dispersion of the particles in aqueous mediums, protection against oxidation of the nanoparticles’ surface and a subsequent functionalization. Also, by the inclusion in biocompatible polymer matrices one can improve the drug loading capacity, their solubility in aqueous media, the biocompatibility, stability, as well as the absorption of the therapeutic agents in the biological medium [8-12].

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It is well known that chemotherapeutical substances cause undesired effects by their general systemic distribution, nonselectivity, and the weak control of release in the tumor areas. After intravenous administration, the selective targeting of nanoparticles by means of the external electric field represents a promising alternative in cancer therapy by concentrating the antineoplastic drug doses in the proximity of the target tissues/cells [8,13-14]. A significant interest in the obtaining of the coating materials for the magnetic particles is given by the hydrophilic natural and synthetic polymers through their capacity for preventing agglomeration, improving solubility and the stability of particles. Classical examples of natural polymers used to this end are dextrane [15], alginate and chitosan [16]. The chitosan (CS) is a cationic polysaccharide whose chemical structure is made up from alternating units of β-(1→4)-2-acetamido-D-glucose and β-(1→4)-2-acetamino-D-glucose, obtained through the partial deacetilation of chitin that originate from the shell of crustaceans. Due to the unique biopharmaceutical properties CS is one of the most versatile biopolymers used for obtaining nanoparticles with applications in cancer therapy. Chitosan’s solubility is strictly dependent on the distribution of N-acetyl and free amino groups, such that in acid solutions (pH<6.5) the amino groups become protonated, determining the solubilization of the macromolecules. By the presence of amino reactive functional groups (C2) CS can be chemically modified in a variety of derivatives with new, improved properties [17-21]. Among these, the ones obtained by modification with disaccharides are very attractive for potential biomedical applications, having as immediate effect the improvement of the resulted derivatives’ solubility in aqueous media through the increase of their hydrophilicity. Moreover, this last effect represents an important factor in increasing the retention time of particles based on this type of derivatives, in the blood flow, preventing their elimination by the macrophage cells [18]. Yang et al. proved, 4

as well, the increase of antimicrobial activity of CS derivative with maltose (on E. Coli and S. aureus cultures) comparing to that of native CS, at the same time with the increase of the substitution degree [22]. Lin reported the improvement of the antioxidant activity on 2,2diphenyl-1-picrylhydrazyl radicals, hydrogen peroxide and superoxide anions of the CS derivative with disaccharides (lactose, maltose or cellobiose). The authors proved that this is dependent both on the substitution degree of CS and on concentration [23]. The goal of the present work consists in improving the solubility of CS in weakly acid aqueous media by introducing the disaccharide segments of maltose on the polymer chains, which can facilitate the obtaining of polymer nanoparticles through the double crosslinking technique (ionic and covalent), in reverse emulsion, previously elaborated by our team [24]. Also, the presence of maltose as a substituent of chitosan has the effect of improving the derivative’s hydrophilicity. It is known that by using neutral and hydrophilic compounds as coating for nanoparticles, the circulatory half-life in the body can be improved from minutes to hours, even days. [25] Hydrophilicity is an important factor also by preventing nanoparticles from being cleared out by macrophages and plasma protein adsorption. [26,27]. These polymer matrix characteristics are among the most important parameters that must be considered in light of the intended nanoparticles’ application, namely the injectable administration into the blood stream for the active targeting of tumors through an exernal magnetic field. Our work also proposes, for the first time, the embedding of magnetite particles in the crosslinked matrix of the CS derivative, with the development of hybrid nanoparticles with magnetic properties.

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2. Materials and methods 2.1.

Materials

Low molecular weight chitosan (CS, MW = 70.000 g/mol, DD = 80.3 %), D-(+)-Maltose monohydrate, glutaraldehyde 25% aqueous solution (GA), 5-fluorouracil (5-FU), Dulbecco’s modified eagle medium (DMEM), sodium cyanoborohydride (NaBH3CN), Pluronic F127 and fetal bovine serum were obtained from Sigma Aldrich. Tween 80, Span 80, ethylic ether and hexane were provided by Merck. Acetic acid, toluene, acetone, Na2HPO4·12H2O, NaH2PO4·2H2O and methanol came from Chemical Company. Sodium hydroxide (NaOH), sodium sulfate (Na2SO4) and iron (III) chloride anhydrous originated from Lachner. Iron (II) chloride tetrahydrate (FeCl2·4H2O) was purchased from Fluka. Double distilled water was freshly produced in our laboratory. TritonX-100 was purchased from Scharlau Chemicals. GA was previously extracted from toluene and then used in the preparation process. The chemicals used in this study were of analytical grade purity and were used without further purification. The human blood samples used were freshly obtained from one healthy nonsmoker volunteer. Fibroblast cells were extracted from rabbit dermis in the Bioengineering Department of “Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania.

2.2.

Methods

2.2.1. Magnetite preparation The synthesis procedure used in the magnetite preparation was based on the co-precipitation method described by Hriţcu and collaborators [28], with slight modifications. Briefly, 0.055 moles FeCl3 anhydrous were dissolved in 90 mL H2O in which were added 36 mL Pluronic F127 solution of concentration 2% as a non-ionic surfactant. Separately, 0.0275 moles FeCl2·4H2O 6

were dissolved in 84 mL H2O with the adding of the same volume of surfactant solution. Afterwards, the two solutions were introduced in a 500 mL flask, equipped with mechanical stirrer, placed in a heating bath. Under energetic continuous stirring at 65 °C, in nitrogen inert atmosphere, an aqueous sodium hydroxide solution (12.8 g/120 mL H2O) was added. Reaction continued for 30 minutes, eventually the Fe3O4 particles being washed with double distilled water until pH=7. The concentration of the final aqueous suspension was 5.7 % (w/v). The average diameter of the magnetic nanoparticles was 5-15 nm. 2.2.2. Preparation of the chitosan derivative with maltose In order to obtain the CS derivative with maltose we considered a modified protocol reported by Yang and collaborators. [29] 1 g CS was completely dissolved in 100 mL acetic acid solution (1%, w/w) then the solution’s pH was raised to 5.4 using NaOH 1M. Subsequently, a maltose monohydrate solution was added at a final molar ratio NH2/maltose=1/0.7, followed by the addition of the appropriate quantity of NaCNBH3 (NH2/NaCNBH3=1/2), the reaction taking place at room temperature, under stirring, for 30 hours. Finally, the reaction mass was precipitated with NaOH 1M up to pH=9, the solid product being washed with methanol and ethylic ether, before being dried out under vacuum. 2.2.3. Preparation of nanoparticles A certain volume of magnetite aqueous suspension was added in 50 mL CS-Malt solution (of desired concentration) in acetic acid solution 1%, in which the adequate quantity of surfactant (2% from the polymer quantity) has been homogenized (Tween 80). The mix was ultrasonated then added dropwise in 200 mL toluene, containing the appropriate quantity of surfactant, Span 80. Following the stabilization time the ionic crosslinker solution (Na2SO4 5%) was added, after which the reaction mix was slopped in a mechanical stirrer-equipped reactor where the ionic 7

crosslinking continued. The GA solution extracted in toluene (c=1.12 mg/mL) was added to fulfill the covalent crosslinking process. After crosslinking ended the emulsion was centrifuged, particles being repeatedly washed with double distilled water, acetone and hexane, before being dried out in vacuum at 40 °C. The experimental protocol with the observed parameters in the obtaining of hybrid nanoparticles is presented in Table 1.

2.3.

Techniques of characterization

2.3.1. FT-IR spectroscopy The structural characterization of the chitosan derivative and nanoparticles was accomplished spectrally by FT-IR. The spectra were obtained on a Bruker Vertex FT-IR spectrometer, of resolution 2 cm-1, in the range of 4000-400 cm-1. All samples were analyzed by the KBr pellet technique. The relevant bands in the absorption spectrum have been attributed to corresponding functional groups. 2.3.2. 1

1

H-NMR spectroscopy

H NMR spectra of CS, maltose and CS-Malt were recorded at 85 °C on a Bruker Avance 400

NMR spectrometer in D2O in which a few drops of 1 M HCl were added (pH =4). The number of scans was 64 and the latency time was 5 seconds. Chemical shifts were referred to the residual solvent peak. Polymer concentration was 10 mg/mL. 2.3.3. Thermogravimetric analysis The thermogravimetric analysis of the CS derivative and nanoparticles was accomplished with a Mettler Toledo TGA/SDTA 851 system for monitoring the mass losses in the destructive process as the temperature rose. Determinations were made in the interval 25-700 °C with a heating speed 8

of 10 °C/min, in inert atmosphere (N2) or air. 2.3.4. Evaluating the magnetic properties In terms of magnetization, three properties were studied for Fe3O4 and composite nanoparticles. i) The saturation magnetization (Ms) appears when all the magnetic dipoles are aligned with an external magnetic field. ii) The remanent magnetization (Mr) is the remanent induced magnetization after the removal of the magnetic field. ii) Coercivity (Hc) is required for reducing the magnetization to zero [30]. Properties were evaluated by vibrating sample magnetometry (VSM; Micromag TM 3900, Princeton Measurement Systems, USA) on dried powders. 2.3.5. Electronic microscopy The morphological analysis of the magnetite and composite nanoparticles was investigated by TEM and SEM technique, respectively. SEM images were recorded with a HITACHI SU 1510 scanning electronic microscope after deposition of a 6 nm thick gold layer onto the nanoparticles using a Cressington 108 device. TEM images have been obtained using a HITACHI HT 7700 microscope which operates in high contrast at a 100 kV voltage acceleration. The probes were prepared by placing small drops of diluted nanoparticle dispersion (~1 g/L) on copper grills covered with carbon, of 300 mesh, then dried out in vacuum at 50 °C. 2.3.6. Atomic force microscopy The hybrid nanoparticles were diluted in Milli-Q water and dispersed on a silicon surface previously cleaned and treated by UV-ozone to remove organic contaminants. The obtained substrate surface was imaged with an atomic force microscope (AFM), the MultimodeNanoscope VIII (BRUKER). It operated in the intermittent contact mode under ambient conditions. A standard rectangular cantilever was used for imaging, with a free resonance frequency of 81.87 kHz and a typical spring constant of about 3 N/m. To analyze the inner 9

structure of the nanoparticles, they were embedded in a resin and dried at ambient area. The block was clamped in a vise and cryo-microformed with the Leica EM-FC7 ultramicrotome. The cuts were performed by using an ultra 35° knife, with speed of 1mm/sec and cut thickness of 50 nm. The obtained surface was analyzed by AFM under the same conditions described above. 2.3.7. Evaluating the swelling degree Determinations were accomplished by the gravimetric method in double distilled water for anticipating the behavior of hybrid nanoparticles in the loading-release process. 57 mg of composite nanoparticles were immersed in 1 mL H2O and maintained at room temperature under mechanical stirring. After preset times, nanoparticle suspension was ultracentrifuged and the swollen samples were weighted after the supernatant removal. The swelling degree was determined with equation (1): Q (%)=(W1-W0)/W0 x 100

(1)

where: W1- weight of swollen probe, W0- weight of dry probe. 2.3.8. Evaluating the nanoparticles’ capacity for drug loading and release The drug loading-release process was done through diffusional mechanism. To this end, 5fluorouracil was used as model drug. In the study of loading kinetics, 57 mg of composite nanoparticles (MG1) were suspended in 1 mL 5-FU (10 mg/mL) aqueous solution and dispersed by sonication. The suspension was then maintained under mechanical stirring for 96 hours. At preset times the probe was centrifuged and the quantity of 5-FU retained by the nanoparticles was calculated by determining the drug content from supernatant based on a calibration curve previously obtained, whose equation is represented by relation (2). We used for this a spectrophotometer UV-Vis HITACHI U-5100, at the wavelength of 265 nm of 5-FU. y= 0.535x, R2=0.999

(2) 10

In the study of the release kinetics, nanoparticles loaded with 5-FU were immersed in 1.5 mL phosphate buffer solution (pH=6.7 similar to that of the tumor area) at 37 °C, under continuous stirring, after which the 5-FU concentration in supernatant was spectrophotometrically determined. 2.3.9. Evaluating cytotoxicity The cell viability has been estimated by the 3-(4, 5-Dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay. Fibroblast cells were placed in the number of 10000 in 3 24-well plates (one plate each day). The medium in which they were cultivated was DMEM supplemented with fetal bovine serum 10% and antibiotics 1%. Subsequently, volumes of nanoparticle suspensions to be tested (50 and 100 µg/mL) were added onto the cells, such that the final volume of the mix was 500 µL/well. In each plate, three control wells were left (over which no suspension was added) and three well for each suspension to be analyzed. It was used one plate for each evaluation (24 h, 48 h, and 72 h). After each of the three time intervals, the culture medium and the UV-sterilized nanoparticles from the control and the analyzed cells were removed and a solution of MTT 5% was added; then the plate was left in the incubator for 2 hours (37 °C, CO2 5% and 95% humidity) in the dark. After 2 hours, the MTT solution was removed from the wells and the isopropanol was added, the formed crystals being left to be solubilized. 100 L from each sample were collected and the absorbance for both control and analyzed samples were read out at 570 nm using a TECAN plate reader.

2.3.10. Evaluating hemocompatibility Hemolysis experiments were performed using a method adapted from Vuddanda et al [31]. First, 5 mL blood was centrifuged at 2000 rpm for 5 min. Supernatant plasma surface layer was 11

removed and the red blood cells (RBC) were separated and washed with normal saline solution. The purified RBC were resuspended in saline solution to obtain 25 mL of suspension. 2 mL of nanoparticles suspension in saline solution at different concentrations were added to 2mL of RBC suspension (final concentrations 100, 200 and 400 µg nanoparticles/mL). Positive (100% lysis) and negative (0% lysis) control samples were prepared by adding equal volumes of Triton X-100 2% and normal saline, respectively, to RBC suspension. Samples were incubated at 37 °C for 2, 4 and 6 hrs and slightly shaken once for every 30 min for resuspension. After the incubation time, samples were centrifuged and 1.5 mL of supernatants was incubated for 30 min at room temperature to allow hemoglobin oxidation. Oxyhemoglobin absorbance in supernatants was measured spectrophotometrically (PG Instruments T60 UV-Vis Spectrophotometer) at 540 nm. Hemolysis percentages of the RBC were calculated using the following formula (4): %Hemolysis=(Abssample-Absnegative control)/(Abspositive control-Absnegative control)·100 (3) The experiments were performed in triplicate. 3. Results and discussions The present work proposes obtaining, for the first time, composite magnetic nanoparticles based on magnetite embedded in the polymer matrix constituted of maltose modified CS, to be used as targeting delivery systems for the antitumor drugs. The study of the influence of reaction parameters - such as the polymer solution concentration, the ratio between polymer and crosslinkers, or the reticulation time - on the morphological properties, on the swelling degree or on the capacity for drug including/release of nanoparticles has been studied in detail and previously reported by our research group [24,32]. As such, the

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present study focuses on the influence the magnetite content has on the morphology and physical properties of the hybrid nanoparticles. Replacing the hydrogen atoms from the CS’s primary amino groups with maltose hydrophilic moieties improves the solubility of CS in aqueous mediums. The chemical modification of CS with maltose targets both improving the hydrophilicity in weakly acid aqueous media (which can determine the increase of retention time of particles in the blood flow) and enhancing some biomedical properties of CS. The method for obtaining the composite nanoparticles through double crosslinking reverse simple emulsion is based on the protocol reported by our research group [24]. The mechanical stability of nanoparticles is ensured by the introduction of a minimal quantity of covalent crosslinker (GA), after these were formed during the ionic crosslinking phase (Na2SO4). a) Obtaining the magnetite suspension The magnetite suspension was conventionally obtained by the method of co-precipitation of a Fe3+/Fe2+ (2:1) aqueous mix in an alkaline non-oxidant medium in accordance with the reaction (I) [33]. Reaction I Because the colloidal aqueous suspension is not stable, we found it necessary to in situ use a nonionic surfactant, Pluronic F127. Its hydrophobic part attaches to the surface of the magnetic particles, while the hydrophilic part ensures their stabilization [28]. A suspension of 5.7% concentration was thus obtained.

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b) Chemical modification of chitosan with maltose The reductive amination reaction represents a simple and versatile procedure for modifying the chitosan by means of covalent bound of a substrate to the primary amino groups of the polysaccharide. In the case of our present study, the chitosan is modified by introducing the hydrophilic maltose segments, using NaBH3CN as reducing agent, according to the reaction (II) shown below. The advantage consists in its behavior in acid media. The hydrolysis rate of NaBH3CN at pH = 3 is smaller than other usual reducing agents and at a pH of 7 it is only 0.5 mol% after 24 h. In addition, the reduction of iminium ion by BH3CN- anion is rapid at pH values of 6-7 and the reduction of aldehydes or ketones is negligible in this pH range [34]. Reaction II 3.1.

FT-IR analysis

A first structural characterization of the maltose-modified chitosan (CS-Malt) is given by the FTIR spectroscopy. Fig. 1 presents the spectra of maltose, native CS and modified CS. The secondary amino group that forms with the chitosan derivative cannot be emphasized because its signal in the interval 3300-3400 cm-1 overlaps with the wide bands of the stretching vibrations of the –OH groups, present both in CS and in maltose. Although the spectral profiles of CS and modified CS are similar, one notices in the derivative spectrum a widening of the absorption band in the interval 3300-3600 cm-1 explained by the increase of the contribution of the –OH groups brought by maltose involved in a large number of H bounds [35]. Moreover, unlike the simple CS spectrum, the derivative’s spectrum presents slight drifts in the absorption bands. A clear example is given by the signal from 1659.65 cm-1 in the CS spectrum characteristic to the C=O bound in the amide group that appears slightly shifted in the derivative’s spectrum, at 1662.51 cm-1. Shifts occur also in the case of signals specific to the deformation vibrations of methylene 14

groups which, in the case of the simple CS, appear in the interval 1261.31-1422.42 cm-1, as well as stretching vibration signals of the etheric bounds at 1078.14-1155.29 cm-1 and of vibrations specific to saccharide cycles ranging in 896.85-1033.18 cm-1.

Fig. 1 3.2.

NMR spectroscopy

The recorded spectra offer useful information in determining the substitution degree (DS) of the derivative. From the chitosan’s 1H-NMR spectrum (Fig. 2) the signal at 3.13 ppm is characteristic to the C-2 position of the glucopyranose ring. Between 3.58 – 3.83 ppm, the peaks of the protons belonging to C-3, C-4, C-5, C-6 carbons from the glucopyranose ring are present. The signal at 4.53 ppm is characteristic to N-acetylglucosamine unit, while the peaks from 4.81 to 4.83 ppm belong to the protons of the glucosamine unit [36]. From the substituted chitosan spectra (CSMalt) with an initial molar ration of NH2/maltose=1/0.7, it is possible to identify the signal of the following protons: 3.12 ppm characteristic to the 1H proton of the C2 from the glucopyranose ring; 5.01 ppm belonging to the protons of the substituted unit; the doublet at 4.95 and 4.97 ppm assigned to β-anomer of maltose (present in 61% ratio compared to α-anomer, according to Roslund et al.) [37]. We can determine thus the substitution degree as the ratio between the integral value and the abundance of the β anomer: 0,08/0.61=0.13 that means 13% substituted units by maltose. The value is very close to 11% (0.11) of the integral at 5.01 ppm, so we can consider a substitution degree of about 12%. The acetylation degree of CS was determined by comparing the integral value of the signal generated by the –CH3 group at 1.956 ppm with the integrals of the protons considered as internal reference [36].

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Fig. 2 c) Obtaining the magnetic polymer nanoparticles The present work reports the obtaining, for the first time, of magnetic polymer nanoparticles based on the maltose modified chitosan matrix. By the presence of the amino groups left available following the maltose modification, CS may react with both the sulphate groups from Na2SO4 forming ionic bonds and also with the carbonil groups from GA forming iminic bonds. With these aspects in mind, the method for obtaining nanoparticles relies on the double crosslinking in reverse emulsion (w/o) developed by our research group. The possible structure of the polymer network that includes the magnetic material is represented in Fig. 3. Fig. 3 In order to accomplish the dimensional and mechanical stabilization of the nanoparticles formed in the main phase of ionic crosslinking with sodium sulphate, the process continued by crosslinking with a minimal quantity of covalent crosslinker (GA). 3.3.

Spectral characterization of magnetic polymer nanoparticles

The FT-IR spectra recorded for all magnetic polymer particles confirm the formation of the structure proposed in Fig. 3. To exemplify, we chose the MG1 sample (Fig. 1). In the spectrum we first find all of the absorption bands specific to CS-Malt. The band from  = 1632.68 cm-1 corresponds to stretching vibrations of the –C=N- inimic bound as a result of the covalent crosslinking, while the absorption band from 617.2 cm-1 is characteristic to the ionic crosslinking through the sulphate groups (-SO42-). The presence of the magnetic material in the structure of the composite nanoparticles is substantiated by the occurrence of the signal from 585.37 cm-1 corresponding to the Fe-O/Fe-O-Fe bounds from magnetite (Fe3O4) [38]. 16

3.4.

Thermogravimetric analysis of nanoparticles

The thermogravimetric analysis of the magnetic polymer nanoparticles was done for two reasons. First, we wanted to simulate the process of thermal sterilization in the case of materials with biomedical applications and, secondly, we aimed at evaluating the magnetite content included in the polymer matrix as a function of the polymer/magnetite ratio used in the obtaining process. The probes’ thermal behavior in isothermal conditions was analyzed in air, by keeping them at the constant temperature of 120 ºC for 40 minutes, in order to simulate the way in which the sterilization of the materials takes place. The heating continued with the speed of 10 ºC/min until 700 ºC. In accordance with the experimental protocol presented in section 2 (Materials and methods), the materials were preheated from 25 ºC at the rate of 10 ºC/min up to the temperature at which isothermal conditions were maintained, a stage when water has been removed (Fig. 4). We notice the fact that in the stage of maintaining the constant temperature, there are no mass losses. Fig. 4 The percentage losses of mass from temperatures greater than 120 ºC were compared in Table 2 with those obtained from the curves recorded in dynamical conditions in the temperature interval 25-700 ºC, with the rate 10 ºC/min. We noticed variations of the lost mass percentage in the limit of 2%. The results show that the analyzed materials have a very good thermal stability so that they may be safely subjected to the sterilization process at a temperature of 120 ºC for 40 minutes. To evaluate the magnetite content in the polymer matrix, the study of the thermal degradation was done in the interval 25 °C – 700 °C at a heating rate of 10 °C/min, in inert atmosphere (N2).

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The thermal analysis (Fig. 5) revealed that all samples present three degradation stages, the residue quantity ranging from 45.75-53.91%. The first degradation stage takes place in the interval 46.09-114.71 °C when there is a mass loss of 8.55-12.9% associated to the removal of water from particles. The second stage occurs in the interval 212.4-451.35 °C accompanied by a mass loss of 13.21-16.26% that characterizes the beginning of the particles’ degradation, fact explained by the degradation of the saccharidic rings from the base chain of the CS derivative. The maximum mass loss (22.62 – 28.11%) is in the range 450-700 °C and corresponds to the degradation of the polymer matrix. One can notice that the residue mass varies as expected: the smaller the polymer/Fe3O4 ratio used in synthesis becomes (quantity of Fe3O4 rises), the more the degradation residue increases (MG1 and MG2). Fig. 5 We can state that the mass loss in the degradation process of the magnetic polymer particles occurs as a result of the polymer’s degradation, the total residue being composed of the magnetic material and the polymer residue [39]. 3.5.

Analysis of the magnetic properties

In Fig. 6 we represent the magnetization curves for the pure magnetite and also for the samples MG, MG1 and MG3 determined through VSM at room temperature. As one can notice, in all four cases, the magnetization curves do not present hysteresis when an exterior magnetic field is applied. Both the coercivity (Hc) and the remanence (Mr) are zero, fact that proves the nanoparticles’ superparamagnetism [40]. The concept is explained by the fact that when an exterior magnetic field is applied, the nanoparticles become magnetic, while returning to the nonmagnetic state once the magnetic field is stopped. This feature makes possible the 18

applications of the nanoparticles in the targeted drug delivery. By making possible the prevention of their “active” behavior once the magnetic field is off, the nanoparticles can be excreted from the body [41]. Determining the saturation magnetizations values (Ms) for each sample, we record Ms= 65.6 emu/g for Fe3O4, Ms=29.32 emu/g for sample MG, and the values 22 emu/g and 20 emu/g for MG1 and MG3, respectively. The comparative analysis of the values for the saturation magnetization reinforces the idea that the polymer matrix has successfully embedded the magnetic material. The highest magnetization value is recorded for the simple magnetite, while the value decreases as the polymer/ Fe3O4 ratio used in the obtaining protocol increases (from 1/1 in the case of probe MG, to 1/0.5 for probe MG3). In other words, as the quantity of magnetic material embedded in the polymer matrix lowers, the magnetization will decrease, this being determined by the diminishing of the polymer’s magnetic properties. Fig. 6 3.6.

Morphological and dimensional analysis of nanoparticles

The morphology of the particles (Fe3O4 and composite) was studied by TEM and SEM analyses whose images are represented in Fig. 7. As can be seen from the TEM micrograph image of the simple magnetite, the nanoparticles’ sizes range in the interval 5-15 nm, their well emphasized shape being spherical. Analyzing the SEM images of the magnetic polymer nanoparticles led to the following conclusions: in the case of the MG sample containing the highest amount of magnetite, we notice the occurrence of aggregates in the nanoparticles’ structure, explained by the presence of magnetite, not included in the polymer matrix, which destabilizes the particles. The decrease of the Fe3O4 quantity from the composite particles led to the obtaining of well individualized nanoparticles of spherical shape (samples MG1 and MG2). Maintaining constant 19

the polymer/Fe3O4 ratio and increasing the concentration of the polymer solution from 0.5% to 0.8% led to an increase of the average diameter of nanoparticles from 300-500 nm for the sample MG1, to 700-1500 nm in the case of sample MG2. When changing the polymer/Fe3O4 ratio from 1/0.7 to 1/0.5 (for the samples MG1 and MG4), one cannot notice a significant difference in the shape of the particles but only a slight decrease of the average diameter from 300-500 nm to 200400 nm. In the case of the samples MG3 and MG4, by maintaining constant the polymer/Fe3O4 ratio to 1/0.5 and diluting the polymer solution to 0.3% for the MG3 sample led to the alteration of the particles’ shape with formation of aggregate. This can be explained by the fact that at too high dilutions of the polymer solution, the crosslinking bridges between the primary amino groups of the CS derivative and the functional groups of the crosslinkers become difficult to achieve. Fig. 7 Since the final goal of the obtained hybrid polymer nanoparticles is the administration as an injectable suspension in the blood flow near the tumors, their size is an important factor. Following the morphological, SEM dimensional as well as zeta potential (ζ = - 52.43 mV for MG1) analyses, sample MG1 was chosen as representative, with an average diameter ranging between 300-500 nm. As such, for reasons of space considerations, the following studies present the data obtained for this sample. 3.7. Analysis of the morphology and inner structure of the hybrid nanoparticles In order to emphasize the entrapment of magnetite nanoparticles in the polymer matrix, the hybrid nanoparticles were morphologically analyzed by atomic force microscopy. The recording of topographic and phase images offered information on the internal structure and surface

20

morphology of composite material. For illustrative purposes we chose the sample MG1 for which cryo-sections were provided. From the study of the recorded images we concluded the following: 

topographic image 8a indicates the presence of well shaped spherical particles having an

average diameter of approx. 450 nm, value which is in agreement with the SEM data; 

phase image 8b shows the presence of a phase contrast at the surface of individual hybrid

compounds which proves the existence of magnetic nanoparticles on the surface of polymer particles; 

cryo-sections 8c and 8d of the sample certifies the occurrence of a phase contrast in

the hybrid particles. This fact justifies the presence of the magnetic material in agglomerate form inside the polymer particles. The dark color regions represent the magnetic material, more rigid than the polymer. Fig. 8 3.7.

The study of the swelling degree of nanoparticles

Because the drug loading and release diffusional mechanism in/from the polymer matrix depends on the polymer network’s swelling, we proceeded with the evaluation of the swelling degree of the composite particles in order to observe the effect the magnetite content has on it. Additionally, this can help in predicting the behavior at drug loading. As such, for the magnetic polymer particles MG1, MG2, MG3, MG4, the swelling degree in aqueous medium (similar to the drug solution) recorded at 24 hours was 303%, 335%, 539%, 310%, respectively. We can therefore state that the swelling degree slightly increases with the decrease of the magnetite quantity from the polymer matrix. This is perfectly explainable because the polymer quantity from the composite particles is determinant for their swelling degree: the larger the polymer quantity (against the magnetite), the greater the swelling degree. The large value of swelling 21

degree for the MG3 sample is explained by the retaining of a large water quantity in the polymer agglomerates that did not succeed to uniformly incorporate the magnetic material due to the excessive dilution of the polymer solution. Fig. 9 shows a representative example of the kinetic swelling curve for sample MG1 with the standard deviation of 23.83. Fig. 9 For comparison, a nonmagnetic sample obtained from the CS-Malt derivative, by the same crosslinking technique, was subjected to the same swelling process in aqueous medium, registering a swelling degree of 654.9% after 24 hours. The much higher swelling degree in this case is justified by the lack of the magnetic material from the nanoparticles’ structure. This fact supports our previous statements about the influence of the polymer/magnetite ratio on the swelling degree. 3.8.

Nanoparticles’ capacity of loading and releasing 5-FU

The diffusional process for loading the particles with 5-FU in aqueous medium follows the behavior of the samples at swelling, the drug quantity retained by the particles depending on the polymer/magnetite ratio used. We determined that the analyzed samples (MG1, MG2, MG3, and MG4) retained, after 24 hours, between 46.5-72.6 mg 5-FU/g nanoparticles. Fig. 10a presents the loading kinetics for the sample MG1 up to 72 hours when the maximum amount of loaded drug is reached, namely 49.12 mg 5-FU/g nanoparticles. Fig. 10 Concerning the drug release process from the hybrid particles (Fig. 10b) a first quick stage is witnessed in the first 300 minutes explained by the release of the drug adsorbed at the surface and

22

the superficial layers of the particles, followed by a slow stage (characterized by a constant release) until 1440 minutes, when it reaches the maximum of 81.14% from the retained quantity. The analysis of the release kinetics was accomplished based on the Korsmeyer-Peppas mathematical model (4): Mt/M ∞=k*tn

(4)

where k is a kinetic parameter that characterizes the interactions between drug and polymer, n is the parameter that specifies the release mechanism and was determined from experimental data. By analyzing the release kinetics data the following equation was obtained: y=0.408x-0.985

(5)

where: k=0.103, n=0.408 and R2=0.966 The value of the exponent n, quite close to 0.5, is indicative of a transport/release mechanism dominated by diffusion [42,43]. 3.9.

Cytotoxicity

Cytotoxicity tests were accomplished for all types of particles, but we exemplify it with the results for the MG1 sample. The toxicity test was accomplished at two different concentrations of the suspension: 50 µg/mL and 100 µg/mL. The absorbances were read out at 24, 48 and 72 h, the tests being performed in triplicate. The values for cell viability registered for the two concentrations are listed in Fig. 11, and confirm the including of the analyzed particles in the category of those with low toxicity [44]. Fig. 11 3.10.

Hemolysis

Nanoparticles can react with red blood cells and induce some undesirable reactions such as hemolysis [45]. Since the obtained nanoparticles are intended for in vivo use, it was necessary to 23

evaluate their hemocompatibility. The hemolytic potential of MG1 nanoparticles was evaluated for concentrations between 100 and 400 µg/mL, using a spectrophotometric method. The results of hemolysis assay are shown in Fig. 12 and are expressed as means ± SD (n = 3). It can be seen in the graphs (Fig. 12) that the hemolytic percentage increases with the increasing of nanoparticle concentration. The hemolytic percentage was lower than 5% for all tested concentrations (100, 200 and 400 µg/mL) at all three tested times. The magnetic nanoparticles present a hemolytic percentage close to the 5% limit, without exceeding it. The results suggest that MG1 nanoparticles are suitable for intravenous administration if their concentration in the bloodstream does not exceed 400µg/mL. Fig. 12 4. Conclusions A new delivery system for antitumor drugs has been proposed and developed, based on the active targeting by means of an external magnetic field. The magnetic material (magnetite) was embedded in the polymer matrix composed of the chitosan modified with maltose. The double crosslinking in reverse emulsion of the derivative allowed us to obtain stable magnetic polymer particles with submicronic size. The analysis of the magnetic properties led to the conclusion that the particles exhibit superparamagnetism with sufficiently high saturation magnetizations and zero remanence. The morphological characteristics of the particles proved to be dependent on the polymer/magnetite ratio and the polymer solution concentration. Atomic force microscopy demonstrated the entrapment of magnetite in the polymer matrix. The resulted nanoparticles are biocompatible, a fact confirmed by the cytotoxicity and hemocompatibility tests. The capacity for

24

loading and sustained release of 5-FU makes them potentially useful in the cancer therapy through active targeting, mediated by an external magnetic field.

25

Disclosure In the present work all the authors have materially participated in the research and article preparation. Liana Alupei proceeded in the synthesis of the chitosan-maltose derivative as well as the hybrid nanoparticles preparation. She accomplished the swelling and loading/release studies contributing to the design, analysis and interpretation of data and also drafting and critically reading the manuscript. Catalina Anisoara Peptu recorded and thoroughly analyzed the SEM and TEM data completing the interpretation of the morphological and dimensional characteristics of nanoparticles. She also contributed to the design and critically read the manuscript. Maria Andreea Lungan registered the FT-IR spectra and contributed to the evaluation of the structural characteristics of the derivative and nanoparticles giving her help in designing the manuscript as well as seriously and severely reading it. Jacques Desbrieres proceeded in recording NMR data together with the determination of the substitution degree of the derivative. He also supervised the process of the chitosan-maltose derivative synthesis giving precious suggestions and revising the manuscript. Ovidiu Chiscan was responsible for providing the magnetic spectra of the hybrid nanoparticles and their detailed analysis and interpretation. He, as well, critically read the article. Sadia Radji performed, processed and interpreted the AFM data of nanoparticles. She also meticulously read the article. Marcel Popa supervised the experimental part of the study and supported data analysis and interpretation. He intensively contributed intellectually to the conception, design and preparation of the article. All authors have approved the final form of the article.

26

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33

Reaction I

Reaction I

Reaction II

Reaction II

34

Figure 1. FT-IR spectra of chitosan, maltose, CS-Malt derivative and sample MG1 Figure 2. 1H-NMR spectra of chitosan (CS), maltose (Malt) and chitosan’s derivative with

maltose (CS-Malt)

Figure 3. Schematic representation of the structure resulted through double crosslinking in

reverse emulsion; red: covalent crosslinking through GA; green: ionic crosslinking through

Na2SO4.

Figure 4. Mass loss in the simulation of particles’ thermal sterilization

Figure 5. TG and DTG curves of the MG1, MG2, MG3, and MG4 samples

Figure 6. Magnetization curves for magnetite and MG, MG1 and MG3 samples

Figure 7. TEM and SEM images for magnetite and composite particles MG, MG1, MG2, MG3,

and MG4.

Figure 8. Atomic force microscopy images for sample MG1; a) topographical image and b)

phase image taken in parallel; c) topographical cryo-section and d) phase cryo-section image

taken in parallel

35

Figure 9. The swelling degree kinetics for the MG1 sample in water

Figure 10. (a) loading kinetics of 5-FU in MG1 particles in water; (b) release kinetics of 5-FU in

MG1 particles in phosphate buffer, pH=6.7

Figure 11. Cellular viability for sample MG1 registered at 24, 48, and 72 h

Figure 12. Hemolysis percentage after 2, 4, and 6 h exposures to MG1 nanoparticles.

36

37

38

39

40

41

42

43

44

45

46

47

48

Table 1. Coding the obtained hybrid particles and varied parameters Sample code

Polymer

solution Polymer/Fe3O4 (g/g) ratio

concentration (%) MG

0.5

1/1

MG1

0.5

1/0.7

MG2

0.8

1/0.7

MG3

0.3

1/0.5

MG4

0.5

1/0.5

*

for all samples the following parameters have been used: 2 h ionic crosslinking time; 2.5 h

covalent crosslinking; 1/7.2 -NH3+/Na2SO4 molar ratio; 1/0.26 -NH2/GA molar ratio.

49

Table 2. Test results for thermal resilience in isothermal conditions MG1 Sample/Conditions

MG2

MG3

MG4

Mass loss (%) at temperatures higher than 120 ºC

10C/min (25-700C)

48.98

51.85

52.30

54.37

50.86

51.34

54.29

56.00

49.14

48.66

45.71

44

10C/min (25-120C) 40 min (120C) 10C/min (120-700C) Residue* (%)

*Residue corresponds to the simulation of sterilization process.

50