water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efficiency...

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International Journal of Pharmaceutics 454 (2013) 233– 240

Contents lists available at SciVerse ScienceDirect

International Journal of Pharmaceutics

j o ur nal ho me page: www.elsev ier .com/ locate / i jpharm

Pharmaceutical nanotechnology

Water dispersible cross-linked magnetic chitosan beads for increasingthe antimicrobial efficiency of aminoglycoside antibiotics

Alexandru Mihai Grumezescua, Ecaterina Andronescua, Alina Maria Holbanb,Anton Ficaia, Denisa Ficai c, Georgeta Voicua, Valentina Grumezescua,d,Paul Catalin Balauree,∗, Carmen Mariana Chifiriucb

a Department of Science and Engineering of Oxidic Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, University Politehnicaof Bucharest, Polizu Street no. 1-7, 011061 Bucharest, Romaniab University of Bucharest, Faculty of Biology, Microbiology Immunology Department, Aleea Portocalelor no. 1-3, 060101 Bucharest, Romaniac Department of Inorganic Chemistry, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Polizu Street no. 1-7, 011061Bucharest, Romaniad Laser-Surface-Plasma Interactions Laboratory, Lasers Department, National Institute for Lasers, Plasma and Radiation Physics, Institute of Atomic Physics,MG-36 RO-77125 Magurele, Bucharest, Romaniae Department of Organic Chemistry, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Polizu Street no. 1-7, 011061Bucharest, Romania

a r t i c l e i n f o

Article history:Received 5 March 2013Received in revised form 17 June 2013Accepted 19 June 2013Available online 3 July 2013

Keywords:Magnetic chitosan beadsEntrapped drugsAntimicrobial therapyCross-linked

a b s t r a c t

The aim of this study was to obtain a nano-active system to improve antibiotic activity of certain drugs bycontrolling their release. Magnetic composite nanomaterials based on magnetite core and cross-linkedchitosan shell were synthesized via the co-precipitation method and characterized by Fourier transforminfrared spectroscopy (FT-IR), infrared microscopy (IRM), scanning electron microscopy (SEM), dynamiclight scattering (DLS), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). The prepared mag-netic composite nanomaterials exhibit a significant potentiating effect on the activity of two cationic(kanamycin and neomycin) drugs, reducing the amount of antibiotics necessary for the antimicrobialeffect. The increase in the antimicrobial activity was explained by the fact that the obtained nanosystemsprovide higher surface area to volume ratio, resulting into higher surface charge density thus increas-ing affinity to microbial cell and also by controlling their release. In addition to the nano-effect, thepositive zeta potential of the synthesized magnetite/cross-linked chitosan core/shell magnetic nanopar-ticles allows for a more favorable interaction with the usually negatively charged cell wall of bacteria.The novelty of the present contribution is just the revealing of this synergistic effect exhibited by thesynthesized water dispersible magnetic nanocomposites on the activity of different antibiotics againstGram-positive and Gram-negative bacterial strains. The results obtained in this study recommend thesemagnetic water dispersible nanocomposite materials for applications in the prevention and treatmentof infectious diseases.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Magnetic nanoparticles (NPs) have a quite broad area of appli-cations covering various scientific research fields, such as magneticfluids (Jin et al., 2012; Saviuc et al., 2011), catalysis (Ranjbakhshet al., 2012), biotechnology/biomedicine including targeted drugdelivery (Oestreicher et al., 2012; Mihaiescu et al., 2011), magneticresonance imaging (Takahashi et al., 2012) and environmentalengineering (Tamilarasan and Ramaprabhu, 2012; Chung et al.,2012). In the last decade a tremendous effort has been focused on

∗ Corresponding author. Tel.: +40 726259170.E-mail addresses: [email protected], [email protected] (P.C. Balaure).

the possible applications of iron oxide NPs, especially magnetite,Fe3O4, which is ferrimagnetic and exhibits superparamagnetismwhen the particles are less than 15 nm in size (Wu et al., 2008).However, the practical use of magnetite NPs raises a series of prob-lems, the major one being their intrinsic instability due to highsurface energy associated with the high surface area to volumeratio (Lu et al., 2007). Consequently, they tend to aggregate in orderto reduce the surface energy (Wu et al., 2008). Moreover, nakedmagnetite NPs are chemically reactive and prone to oxidation inair with loss of magnetic properties and dispersibility (Wu et al.,2008). Fortunately, they can be chemically stabilized by graftingor coating with organic molecules including surfactants, polymersand biologically active molecules, or by inorganic coatings of silica,metals, or metal oxides or sulfides (Lu et al., 2007). These protective

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234 A.M. Grumezescu et al. / International Journal of Pharmaceutics 454 (2013) 233– 240

shells can be further functionalized, thus enlarging the spectrum ofpossible applications of magnetite NPs. Specific therapeutic agentssuch as antibiotics or anticancer drugs can be physically loaded onor covalently grafted, via specific linkers, to the organic protectiveshell surrounding the magnetite nanoparticle core (Rangel-Yaguiet al., 2005). In principle, the use of core/shell magnetic NPs in drugdelivery allows for the movement within the body of the wholenanostructured assembly to be controlled with the aid of an exter-nally applied magnetic field (Akbarzadeh et al., 2012). If magnetitecore/organic shell NPs are to be used in biomedical applications,the main requests for the shell are to be biocompatible andbiodegradable (Grumezescu et al., 2012a,b). Therefore polymerssuch as poly(ethylene glycol) (Mondini et al., 2008; Paul et al., 2004)which protects the NPs against opsonization by reticuloendothelialsystem (Papisov, 1998), poly(vinyl alcohol; Chastellain et al., 2004;D’Souza et al., 2004), poly(acrylic acid) (Shan et al., 2003; Arbabet al., 2003), poly(lactic acid; Gomez-Lopera et al., 2006), poly(lacticco glycolic acid), and polysaccharides (Berry and Curtis, 2003; Berryet al., 2003; Grumezescu et al., 2011a) are widely used as organicshells for biomedical purposes of core/shell magnetite NPs. Amongother natural polysaccharides such as dextran or starch, the uniquefeatures of chitosan (CS) make it particularly suited for biomedicalapplications. Chitosan, poly[�-(1-4)-linked-2-amino-2-deoxy-d-glucose] is non-toxic, hydrophilic, biocompatible, biodegradableand exhibits anti-bacterial properties (Dhanasingh et al., 2001;Shen et al., 2012). CS coated magnetic nano- or microparticles havea large scale of actual and potential applications. On its polymericbackbone, CS bears free amino and hydroxyl groups through whichCS coated NPs are able to bind to a variety of other chemical groupsleading to a scale of applications such as targeted drug and genedelivery (Osuna et al., 2012; Li et al., 2007), magnetic resonanceimaging (Hong et al., 2010), tissue engineering (Sasaki et al.,2008), antibiofilm strategies (Grumezescu et al., 2011b, 2012c)and enzyme immobilization (Kuo et al., 2012; Xie and Wang,2012). Our research group has recently initiated and developed aseries of studies on CS coated magnetite micro- and nanoparticles,including their applications in controlled antibiotics release aswell as the improvement they produce on the efficacy of antibiotictherapy (Grumezescu et al., 2011c, 2012d; Chifiriuc et al., 2012).In the present contribution we report the preparation of newwater dispersible magnetic nanosystems, based on magnetite andcross-linked CS and we demonstrate the improvement effect theyhave on the efficiency of several antibiotics against Gram-positiveand Gram-negative bacterial strains.

2. Materials and methods

2.1. Materials

All chemicals were used as received. Chitosan (M60,000–120,000), FeCl3, FeSO4·7H2O, NH4OH (25%), Kanamycin(K), Neomycin (N) and CH3OH were purchased from Sigma–AldrichChemieGmbh (Munich, Germany).

2.2. Synthesis of chitosan cross-linked magnetic beads

In the present paper, magnetic materials were prepared bya modified co-precipitation method (Grumezescu et al., 2012e;Mihaiescu et al., 2012). To 300 mL of ultrapure water, 10 mL of28% NH3 aqueous solution were added under stirring at roomtemperature. After that, 500 mL solution consisting of 1 g of CS,5 mL CH3COOH (1 N), 1 g of FeCl3 and 1.6 g of FeSO4·7H2O weredropped under permanent stirring up to pH = 8, leading to the for-mation of a black precipitate. The product was repeatedly washedwith ultrapure water and divided into three parts. One part was

cross-linked with glutaraldehyde (1%), thus the nanostructuredmagnetic material denoted as Fe3O4@CS being obtained.

2.3. Immobilization of antibiotics into cross-linked chitosanmagnetic beads

The other two parts (500 mg each) were dispersed into 100 mLultrapure water and 50 mg of kanamycin sulfate (K) and 50 mgneomycin sulfate (N), respectively, were added. Then, these solu-tions were cross-linked with glutaraldehyde (1%) in order toobtain two novel magnetic materials denoted as Fe3O4@CS-K andFe3O4@CS-N, respectively.

3. Characterization

3.1. Fourier transform-infrared spectrometry (FT-IR)

A Nicolet 6700 FT-IR spectrometer (Thermo Nicolet, Madison,WI) running the OMNIC operating system software (Version 8.2Thermo Nicolet) was used to obtain FT-IR spectra of the preparedmagnetic materials. The samples were placed on a multibounceplate of ZnSe crystal with attenuated total reflectance (ATR) at con-trolled ambient temperature (25 ◦C). FT-IR spectra were collectedin the frequency range of 4.000–650 cm−1 by co-adding 32 scansat a resolution of 4 cm−1 with strong apodization. All spectra wereregistered against a background of an air spectrum.

3.2. Infrared microscopy (IRM)

IR mapping were recorded on a Nicolet iN10 MX FT-IRMicroscope with MCT liquid nitrogen cooled detector in the mea-surement range 4000–600 cm−1. Spectral collection was made inreflection mode at 4 cm−1 resolution, 32 scans were co-added andconverted to absorbance using Ominc Picta software (Thermo Sci-entific).

3.3. Scanning electron microscopy (SEM)

SEM analysis was performed on a HITACHI S2600N electronmicroscope, using primary electron beams with energies of 15 and25 keV, respectively, on samples covered with a thin silver layer.

3.4. X-ray diffraction (XRD)

X-ray diffraction analysis was performed on a Shimadzu XRD6000 diffractometer at room temperature. In all the cases, Cu K�radiation (� = 15,406 A at 15 mA and 30 kV) was used. The sampleswere scanned in the Bragg angle 2� range of 10–80 degrees.

3.5. Dynamic light scattering (DLS)

Particles size analysis was performed using intensity distribu-tion by dynamic light scattering technique (Zetasizer Nano ZS,Malvern Instruments Ltd., UK), at scattering angles of 90◦ and 25 ◦C.The average diameters (based on Stokes–Einstein equation) werecalculated from three individual measurements. The zeta potentialwas measured using the Zetasizer Nano ZS.

3.6. Thermogravimetric analysis (TGA)

The thermogravimetric (TG) analysis of the prepared magneticchitosan beads was assessed with a Shimadzu DTG-TA-50H instru-ment. Samples were screened to 200 mesh prior to analysis, wereplaced in alumina crucible, and heated with 10 K min−1 from room


A.M. Grumezescu et al. / International Journal of Pharmaceutics 454 (2013) 233– 240 235

temperature to 800 ◦C, under the flow of 20 mL min−1 dried syn-thetic air (80% N2 and 20% O2).

3.7. Antimicrobial susceptibility test

Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosaATCC 27853 reference bacterial strains were used in this study.Quantitative testing of the antimicrobial activity of compositenanosystems was performed by microdilution method in liquidmedium (Mueller Hinton broth), using 96 multiwell plates in orderto establish the minimum inhibitory concentration (MIC) values.Two-fold serial microdilutions were achieved in 200 �L medium,the dilution range being 500–0.24 �M. Subsequently, the wellswere seeded with 50 �L of each bacterial suspension, adjusted to0.5 MacFarland densities. Positive and negative controls were used.After incubating the plates at 37 ◦C for 24 h, the results were macro-scopically assessed for bacterial growth, MIC corresponding to thewell with clear content, thus without no visible microbial growth.For determining the dynamic of controlled release of the antibioticsembedded in the nanosystems the optical density (OD 600 nm) ofthe samples treated with plain or nanosystem embedded antibi-otics was achieved using a spectrophotometer.

4. Results and discussion

X-ray diffraction was used to identify the crystallinity of themineral phase dispersed in CS matrix. XRD patterns of Fe3O4@CS,Fe3O4@CS-N andFe3O4@CS-K are plotted in Fig. 1. The magneticmaterials show sharp diffraction peaks at 2� = 30.31, 35.71, 43.31,53.90, 57.61 and 62.81 that can be assigned to (2 2 0), (3 1 1), (4 0 0),(4 2 2), (5 1 1) and (440) plane of magnetite lattice, respectively. Noother diffraction peaks corresponding to other iron oxide, such as�-Fe2O3 and �-Fe2O3, could be observed. These results are in goodagreement with reported literature (Li et al., 2006; Xu et al., 2007;Zhai et al., 2009).

Figs. 3–5 present second derivative infrared micrographs ofFe3O4@CS, Fe3O4@CS-N and Fe3O4@CS-K surfaces. Second deriva-tive infrared mapping can be used as a quick, easy, reproducible,inexpensive, and non-destructive tool to evaluate the purity andstructural integrity of samples (Byler et al., 1995). Absorbanceintensities of IR spectra maps are proportional to color changesstarting with blue (the lowest intensity) and gradually increas-ing through green, yellow to finally red (the highest intensity).

Fig. 1. XRD patterns of fabricated nanomaterials.

Approximately 600 spectra were analyzed for each magnetic mate-rial sample.

Four absorptions known as being characteristics for the chitosanmacromolecule were selected as spectral markers of CS presencein the prepared nanostructured magnetic materials (Fig. 2). Theseselected FT-IR absorptions were the following ones: the absorp-tion at 1028 cm−1 due to stretching vibrations of CO single bondsof COH, COC and CH2OH groups in the pyranose ring, the absorp-tion at 1645 cm−1 due to the presence of amide carbonyl groups,C( O)NHCH3, and indicating an incomplete deacetylation of chitin,the absorption at 2922 cm−1 which is related to the stretchingvibrations of CH single bonds in CH2OH groups, and the absorptionat 3300 cm−1 corresponding to the stretching vibrations of associ-ated OH and NH2 groups. Fig. 4 shows the distribution and relativedensity of the above characteristic functional groups on the surfaceof the prepared magnetite core/cross-linked CS shell NPs.

Fig. 6 shows the size distribution histograms of the Fe3O4@CS,Fe3O4@CS-N and Fe3O4@CS-K. The size distribution of the sam-ples at 25 ◦C showed a hydrodynamic particle size below 100 nm,the mean particle size being situated in the range between 30 and50 nm.

Fig. 2. FT-IR spectra of CS, N, K, Fe3O4@CS-N and Fe3O4@CS-K.



Fig. 3. Second derivate IR mappings of Fe3O4@CS surface: intensity distribution of (a) 1028 cm−1, (b) 1645 cm−1, (c) 2922 cm−1, (d) 3300 cm−1. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

The zeta potential measurements (Fig. 7) offered an insighttoward the synergistic effect between the obtained nanosystemsand both cationic and anionic antibiotics. Both newly synthesizedmagnetic nanocomposite materials, Fe3O4@CS-N and Fe3O4@CS-K,respectively, exhibited a positive zeta potential of about 30 mV. Thisvalue was considered to be favorable for the electrostatic interac-tion with the negatively charged bacterial wall allowing the betterrelease of therapeutic agents inside the bacterial cell. Also, elec-trostatic repulsion between particles is higher at high positive ornegative zeta potential values, so that it results in higher colloidalstability (Vidojkovic et al., 2011).

The morphology of magnetic materials was evaluated by SEM, asseen in Fig. 8. Fe3O4@CS-N and Fe3O4@CS-K showed spherical mor-phology with porosity at micro scale dimensions (Fig. 8: b2 and c2),as opposed to the smooth and regular surface of Fe3O4@CS (Fig. 8:a2).

The TGA thermograms reveals continuous weight loss for CS, Nand K (Fig. 9). The weight losses are ∼44% for Fe3O4@CS, ∼45.13%for Fe3O4@CS-K and 45.48% for Fe3O4@CS-N, respectively. The

results confirmed the entrapped of K and N on Fe3O4@CS. The Kand N contents were estimated as the difference between weightloss at approximately 600 ◦C for Fe3O4@CS-K or Fe3O4@CS-N andFe3O4@CS, and they are approximately 1.13% (for K) and 1.48% (forN).

The potential of magnetic CS NPs to act as drug carriers andtargeted drug delivery systems has been demonstrated in the litera-ture for different therapeutic substances (Grumezescu et al., 2012e;Mihaiescu et al., 2012). Magnetite chitosan microspheres conju-gated with methotrexate (MTX) for the controlled release of MTXhave been prepared by suspension cross-linking technique usingglutaraldehyde as the cross-linker (Zhang et al., 2009). Biocompat-ible magnetically driven nanocomposites with high antibacterialand antifungal activities against ten tested bacterial strains andfour Candida species were also prepared (Mihaiescu et al., 2012).In the synthesis of these nanocomposites, biogenic magnetite NPsisolated from magnetotactic bacteria have been stabilized by cover-ing with a CS shell that also works as reducing agent for a silver saltresulting in the formation of silver NPs. The Fe3O4NPs/CS-AgNPs

Fig. 4. Second derivative IR mappings of Fe3O4@CS-N surface: intensity distribution of (a) 1028 cm−1, (b) 1645 cm−1, (c) 2922 cm−1, (d) 3300 cm−1. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)



Fig. 5. Second derivative IR mappings of Fe3O4@CS-K surface: intensity distribution of (a) 1028 cm−1, (b) 1645 cm−1, (c) 2922 cm−1, (d) 3300 cm−1. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6. Size distribution histogram of (a) Fe3O4@CS, (b) Fe3O4@CS-N and (c) Fe3O4@CS-K.

composites prepared by this way revealed an increased bactericidaland antifungal activity (Marková et al., 2012).

We have tested the potential synergistic effect between theprepared Fe3O4@CS NPs and different antibiotics (K and N) rec-ommended by CSLI to be tested against S. aureus and P. aeruginosareference strains. The analyzed bacterial strains have been chosenas experimental models for the study of the interaction betweenthe CS-magnetite beads and the Gram-positive, respectively Gram-negative bacteria cells.

During microbial susceptibility test we observed that in thepresence of CS-magnetite beads, both antibiotics exhibited animproved anti-microbial activity on both S. aureus and P. aeruginosatested strains. The results revealed that MIC values of the

Table 1MIC values of controls (K and N) and prepared nanostructures (Fe3O4@CS-K andFe3O4@CS-N).

MIC (�g/mL) N Fe3O4@CS-N K Fe3O4@CS-K

S. aureus 15.62 7.81 15.62 3.9P. aeruginosa 31.25 7.81 15.62 7.81

antibiotics are significantly reduced (at least half) when using thecontrolled released nanosystem comparing with plain antibiotic(Table 1).

This result is explained by the property of newly synthe-sized nanosystem to control the release of the active drug.

Fig. 7. Zeta potential distribution of (a) Fe3O4@CS, (b) Fe3O4@CS-N and (c) Fe3O4@CS-K.



Fig. 8. SEM micrographs of (a) Fe3O4@CS, (b) Fe3O4@CS-K and (c) Fe3O4@CS-N.

Fig. 9. TGA analysis of Fe3O4@CS, Fe3O4@CS-N and Fe3O4@CS-K.

Figs. 10 and 11 reveal S. aureus (Fig. 10) and P. aeruginosa (Fig. 11)growth curves depending on the amount of plain/nanosystemembedded antibiotics after 24 h at 37 ◦C. Even though bacteriagrowth is impaired in a dose dependent manner when using bothplain and nano-attached antibiotics, bacteria cells grow muchslower in the presence of nanosystem embedded antibiotics atalmost all concentrations used. Since chitosan nanoparticles withno antibiotic has proved no significant effect against both S. aureus(Fig. 10) and P. aeruginosa (Fig. 11) growth, the improved growthinhibition is due to the ability of the nanosystem to enhance antimi-crobial properties of the drugs, most likely by controlling theirrelease.

Our results showed that the nano-scaled Fe3O4@CS beads exhib-ited a significant potentiating effect on the activity of cationicantibiotics K and N, which could be explained also by the fact thatthe nanosystem provides higher surface to volume ratio, translat-ing into higher positive surface charge density, so that the affinityto microbial cell is increased.



Fig. 10. Graphic representation of S. aureus growth in the presence of different concentrations of plain neomycin (N) (1) and kanamycin (K) (2), or nanosystem embeddedneomycin (Fe3O4@CS-N) (1) and kanamycin (Fe3O4@CS-K) (2), and control nanosystem Fe3O4@CS.

Fig. 11. Graphic representation of P. aeruginosa growth in the presence of different concentrations of plain neomycin (N) (1) and kanamycin (K) (2), or nanosystem embeddedneomycin (Fe3O4@CS-N) (1) and kanamycin (Fe3O4@CS-K) (2), and control nanosystem Fe3O4@CS.



5. Conclusions

The magnetic and nano-effects of magnetite/cross-linked CScore/shell NPs, the control of antibiotic loading, the biocompati-ble character of CS matrix, and the increased antimicrobial effectare strong arguments for the use of the developed nano- andmicrocomposites for targeted applications in the management ofinfectious diseases. Our results demonstrate that the preparednanosystems significantly potentate the activity of antibioticsagainst both Gram-positive and Gram-negative bacteria strainsoffering new insights for developing efficient antimicrobial ther-apeutic strategies by lowering the amount of antibiotics, butmaintaining the same activity.

Acknowledgments

This paper is supported by the Sectoral Operational ProgrammeHuman Resources Development, financed from the European SocialFund and by the Romanian Government under the contract numberPOSDRU/86/1.2/S/58146 (MASTERMAT).

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water dispersible cross-linked magnetic chitosan beads for increasing the antimicrobial efficiency...

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