Preparation, physical-chemical and biological characterization of chitosan nanoparticles loaded with lysozyme.

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Preparation, physical–chemical and biological characterization of chitosan nanoparticles loaded with lysozyme

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Anna Maria Piras a,1 , Giuseppantonio Maisetta b,1 , Stefania Sandreschi a , Semih Esin b , Matteo Gazzarri a , Giovanna Batoni b , Federica Chiellini a,∗ a Laboratory of Bioactive Polymeric Materials for Biomedical and Environmental Applications (BIOlab), UdR INSTM, Department of Chemistry and Industrial Chemistry, University of Pisa, via Vecchia Livornese 1291, 56010 San Piero a Grado (Pi), Pisa, Italy b Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy

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Article history: Received 16 October 2013 Received in revised form 13 March 2014 Accepted 15 March 2014 Available online xxx

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Keywords: Chitosan Lysozyme Nanoparticles Deacetylation degree Sustained antibacterial activity

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1. Introduction

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A commercially available chitosan (CS) was employed in the formulation of nanoparticles loaded with lysozyme (LZ) as antimicrobial protein drug model. Due to the variability of commercially available batches of chitosans and to the strict dependence of their physical and biological properties to the molecular weight (Mw ) and deacetylation degree (DD) of the material, the CS was fully characterized resulting in weight-average molecular weight of 108,120 g/mol and DD of 92%. LZ-loaded nanoparticles (LZ-NPs) of 150 nm diameter were prepared by inotropic gelation. The nanoparticles were effectively preserving the antibacterial activity of the loaded enzyme, which was slowly released over 3 weeks in vitro and remained active toward Staphylococcus epidermidis up to 5 days of incubation. Beyond the intrinsic antibacterial activity of CS and LZ, the LZ-NPs evidenced a sustained antibacterial activity that resulted in about 2 log reduction of the number of viable S. epidermidis compared to plain CS nanoparticles. Furthermore, the LZ-NPs showed a full in vitro cytocompatibility toward murine fibroblasts and, in addition to the potential antimicrobial applications of the developed system, the proposed study could serve as an optimal model for development of CS nanoparticles carrying antimicrobial peptides for biomedical applications. © 2014 Published by Elsevier B.V.

Chitosan (CS) is a linear copolymer of ˇ-(1–4) linked 2-acetamido-2-deoxy-ˇ-d-glucopyranose and 2-amino-2-deoxyˇ-d-glucopyranose. It is obtained by deacetylation of its parent polymer Chitin, the second most abundant natural polymer in nature after cellulose, found in the structure of a wide number of invertebrates (crustaceans’ exoskeleton, insects’ cuticles) and in the cell walls of fungi. Due to its natural origin, CS can not be defined as a unique chemical structure but as a family of polymers, namely chitosans, which present a high variability in their chemical and physical properties. This variability is related not only to the origin of the pristine Chitin, but also to the method adopted for their preparation. Chitosans, with the main structural differences being represented by the relative proportions of N-acetyl-d-glucosamine/dglucosamine residues and the molecular weight (Mw ), provide specific chemical and physical features affecting their biological

∗ Corresponding author. Tel.: +39 50 2210305; fax: +39 50 2210332. E-mail address: [email protected] (F. Chiellini). 1 These authors contributed equally to the work.

properties. Chitosans are used in various fields of applications comprising food, biomedicine and agriculture, among others [1]. The bactericidal action of chitosans is well known and it has been widely investigated especially in the recent years. The mechanism of the bactericidal effect of chitosans has been attributed to an electrostatic interaction between NH3 + groups of CS and phosphoryl groups of phospholipid components and lipopolysaccharides (LPS) of bacterial cell membranes, which increases the permeability, forms pores, and ultimately disrupts the bacterial cell membranes, with the release of cellular contents [2,3]. The bactericidal effect has also been correlated to the structural properties of chitosans [4–8], leading also to the determination of alternative bactericidal mechanisms as the blocking of the RNA transcription by direct interaction of DNA with low Mw chitosans penetrated into the bacterial cell [9]. Bactericidal effects have been described also for chitosan constructs such as, membranes [10], and composites [11] (e.g. with silver [12,13], and copper [14]). Furthermore, several studies report on the use of CS for the formulation of micro/nanoparticles as drug delivery systems, either alone [15] or combined into polyelectrolyte complexes (PEC) with alginate [16] or poly()-glutamic acid (PGA) [17]. The latter PEC form has also been applied for the loading of lysozyme (LZ) for bactericidal purpose [18]. LZ is a 14.3 kDa

http://dx.doi.org/10.1016/j.ijbiomac.2014.03.016 0141-8130/© 2014 Published by Elsevier B.V.

Please cite this article in press as: A.M. Piras, et al., Int. J. Biol. Macromol. (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.03.016

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enzyme, which catalyzes the hydrolysis of ˇ-1,4-linkages between N-acetylmuramic acid and N-acetyl-d-glucosamine residues in a peptidoglycan [19]. It is a relatively small protein (129 aa) with an optimal pH in the range 6–9, an isoelectric point near 9.2 and antimicrobial, antiviral, antitumor and immune-modulatory activities. Besides being a good model protein for its structural and enzymatic properties [20,21], LZ received attention also for its use as food preservative and in the cosmetic field. In the present work, a commercially available CS has been employed in the formulation of nanoparticles loaded with LZ as antimicrobial protein drug model. Due to the variability of commercially available batches of CS and to the strict dependence of their physical and biological properties to the structural features (Mw and deacetylation degree—DD) of the material, this study was conducted starting from the characterization of the employed material, followed by the determination of the physical and biological properties of the developed CS–LZ nanosystem (LZ-NPs), with particular attention to its enhanced bactericidal effect compared to the unloaded CS nanoparticles (CS-NPs). It is our convincement that this approach should be followed especially when CS is applied for the development of bactericidal constructs, for reproducibility aims and scientific significance of the data collected. To our knowledge, this is the first time that a CS–LZ nanoparticulate system is investigated starting from the determination of the most important material properties to correctly identify the CS used. 2. Materials and methods 2.1. Materials CS (weight-average molecular weight Mw 50–190 kDa and DD 75–85%), sodium tripolyphosphate (TPP) LZ from chicken egg white (∼100,000 U/mg), d-(+)-glucosamine hydrochloride (GlcN) purity 99%, N-acetyl-d-glucosamine (GlcNAc) purity 99%, and potassium bromide (KBr) FTIR grade, were all purchased from Sigma-Aldrich, Milan, Italy. Pullulan (Molecular weight 5.8–853k) was obtained from Showa Denko K.K., Tokyo, Japan. Acetic acid analytical grade was obtained from Carlo Erba, Milan, Italy. Deionized water (Milli-Q, ddH2 O) was used throughout the experiments. Cell line 3T3/BALB-C Clone A31 mouse embryo fibroblast (CCL163) was purchased from American Type Culture Collection (ATCC, LGC standards, Milan, Italy) and propagated as indicated by the supplier; Dulbecco’s Modified Eagles Medium (DMEM), 0.01 M pH 7.4 Dulbecco’s Phosphate Buffer Saline without Ca2+ and Mg2+ (DPBS), bovine calf serum (BCS), glutamine and antibiotics (penicillin/streptomycin) were purchased from GIBCO/Brl, Monza, Italy; Cell proliferation reagent WST-1 was provided by Roche diagnostic, Milan, Italy. The S. epidermidis strain used in the study was purchased from the American Type Culture Collection (S. epidermidis ATCC 35984). For preparation of stock cultures, the bacterial strain was grown in Tryptone soya broth (TSB) (Oxoid Basingstoke, United Kingdom) at 37 ◦ C until mid-log phase, subdivided in aliquots, and kept frozen at −80 ◦ C until future use. For colony forming units (CFU) count serially diluted bacterial suspensions were plated on Tryptone soya agar (TSA) (Oxoid, Basingstoke, United Kingdom) and incubated for 48 h at 37 ◦ C.

6–13 ␮m columns (7.8 × 300 mm) (Waters, Milford, USA) were employed. The eluent was 0.5 M sodium acetate buffer and the flow rate was maintained at 1 ml/min. Column temperature was kept at 40 ◦ C in Jones oven 7971 (Jones, USA). Pullulan standards (Polymer Laboratories, UK) were used to obtain a calibration curve. CS samples were dissolved in the eluent at a concentration of 2 mg/ml. Fourier transform infrared spectroscopy (FTIR) measurements were carried out by a Perkin-Elmer Spectrum One spectrophotometer (Perkin-Elmer, Monza, Italy). Absorbance spectra of 1.5 wt% CS pellets were taken as an average of 32 scans with 2 cm−1 resolution in the frequency range 4000–400 cm−1 . Prior to analysis, CS and KBr were dried at 60 ◦ C for 2 h. Ultraviolet spectra were recorded in the range 200–250 nm using a Jasco V-530 UV/V spectrophotometer. Calibration curves for GlcN and GlcNAc were drawn through a linear regression between the concentration and first derivative UV signal arising from each one at 205 nm (0.6–2 mM, R2 = 0.9921 and 0.02–0.3 mM, R2 = 0.9904, respectively). A solution of acetic acid 0.01 M was used as blank. Accurately weighed (10 mg) CS samples were dissolved in 1 ml of acetic acid 0.1 M and diluted 10-fold with distilled water to obtain a final acetic acid concentration of 0.01 M. CS was not dissolved directly in acetic acid 0.01 M since it would be difficult to get a complete dissolution of the sample in a reasonable short time. Thermogravimetric analysis (TGA) evaluations were performed using a Mettler TA 4000 System instrument (Mettler Toledo, Milan, Italy) consisting of TGA-50 furnace with a M3 microbalance, and STARe software 9.01 (Mettler Toledo). Samples of ca. 5 mg were scanned at 10 ◦ C/min from 25 to 700 ◦ C, under 300 ml/min flow rate of nitrogen. Differential scanning calorimetry (DSC) measurements were performed using a Mettler DSC-882 instrument. The DSC curves were performed using 8 mg samples under nitrogen atmosphere on aluminium pans. The scanning rate was 5 ◦ C/min in the range 25–500 ◦ C. 2.3. Preparation of blank or lysozyme-loaded nanoparticles Several nanoparticles (NPs) formulations were prepared using a simple ionic gelation process [22] (Table 1). Briefly, CS was dissolved in 1% (v/v) acetic acid (1 mg/ml) and TPP was dissolved in water (0.5–1 mg/ml); for LZ-NPs, 2.5 mg of the enzyme were added to the CS solution. NPs formed spontaneously upon addition of 2 ml of TPP aqueous solution to 5 ml of CS solution under magnetic stirring; the mixture was stirred at room temperature for 2 h. NP suspensions were purified by centrifugation in ALC® (Milan, Italy) PK121R centrifuge at 8500 rpm for 60 min, at 4 ◦ C. 2.4. Characterization of NPs

2.2. Characterization of chitosan

2.4.1. Physical–chemical characterizations The size distribution of the developed NPs was measured by mean of dynamic light scattering (DLS) (Coulter LS230 Laser Diffraction Particle Size Analyzer, Beckman Coulter, Nyon, Switzerland). The Zeta-potential of the developed formulations was evaluated using a Beckman-Coulter DelsaTM Nano C, at 25 ◦ C and aqueous solutions pH 6.6. The morphology of the nanoparticles was assessed through Scanning Transmission Electron Microscopy (STEM) by using a GEMINI® Multi-Mode STEM (Carl Zeiss Microscopy GmbH). Samples were diluted in ethanol (1/200) and directly air dried on the grid, before the analysis.

The Gel Permeation Chromatography (GPC) apparatus consisted of a WatersTM 600 Controller equipped with a Waters 410 Differential Refractometer and a Waters TM 600 Pump, managed by ChromNAV software (Jasco Europe, Lecco, Italy). An UltrahydrogelTM guard column and two UltrahydrogelTM linear

2.4.2. Evaluation of LZ loading capacity of NPs The loading content and encapsulation efficiency of the NPs were determined as previously described [23]. Briefly, LZ-NPs were collected by centrifugation at 8500 rpm 4 ◦ C for 60 min; the amount of free LZ in the supernatant was measured recording


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Table 1 Particle size, Z potential values, and LZ loading capacity of NPs. Run

[CS] (mg/ml)

[TPP] (mg/ml)

[LZ] (mg/ml)

Diameter (nm ± SD)

Zeta pot. (mV ± SD)

Loading (% ± SD)

EE (% ± SD)

CS-NPs1 CS-NPs2 CS-NPs LZ-NPs

1 1 1 1

0.5 0.75 1 1

– – – 0.5

N.A.* Aggregates 140 ± 20 159 ± 24

– – 37.2 ± 1.1 41.6 ± 1.1

– – – 8.3 ± 0.1

– – – 10.0 ± 0.2

*

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Not assessed, the sample was too limpid to induce light scattering.

the characteristic absorbance of LZ at 281 nm and compared with a standard curve generated from LZ acetic acid solutions (2.5–200 ␮g/ml, R2 = 0.9999). The Loading content was defined as amount of LZ per LZ-NPs dry weight; the Encapsulation efficiency (EE) was define as the amount of LZ recovered in the LZ-NPs compared to the total amount of enzyme used in the formulation protocol.

2.4.3. Evaluation of LZ release from the LZ-NPs in vitro Purified LZ-NPs were re-dispersed in 1 ml of SPB (Sodium Phosphate Buffer, 10 mM, pH 6.6) or PBS (Phosphate Buffer Saline, pH 7.4) and placed into test tubes at 37 ◦ C under magnetic stirring. At appropriate time intervals, samples were centrifuged at 13,000 rpm 4 ◦ C for 30 min and a fixed volume of the supernatant was collected and replaced by fresh medium. The amount of LZ released from the LZ-NPs was evaluated by UV–Vis analysis of the supernatants, based on calibration curves of the enzyme in SPB (2.5–200 ␮g/ml; R2 0.9992) or PBS (2.5–200 ␮g/ml; R2 0.9995), accordingly.

2.4.4. Stability evaluation In order to evaluate the stability of the NPs during bactericidal assays, both blank (CS-NPs) and LZ-NPs were resuspended in SPB and incubated at 37 ◦ C. After 1, 3 and 5 days, the samples were centrifuged at 8500 rpm 4 ◦ C for 60 min and the pellets were first lyophilized and then re-dispersed (2 mg/ml) in 0.5 M sodium acetate buffer for the evaluation of CS molecular weight. Moreover, a dimensional characterization was performed by mean of DLS on the re-dispersed NPs at the beginning of the experiment and after 1, 3 and 5 days of incubation.

2.5. Biological evaluation of NPs 2.5.1. Cytotoxicity tests Cytotoxicity evaluation of both blank (CS-NPs) and LZ-NPs was carried out using the 3T3/BALB-C Clone A31 cell line. Cells were grown in DMEM containing 10% (v/v) calf serum, 4 mM glutamine, 100 U/ml of penicillin and 100 ␮g/ml of streptomycin. For the determination of the cytocompatibility of the NPs, a subconfluent monolayer of 3T3 fibroblasts was trypsinized using a 0.25% trypsin, 1 mM EDTA solution, centrifuged at 200 × g for 5 min, re-suspended in growth medium and counted. Appropriate dilution was made in order to obtain 3 × 103 cells per 100 ␮l of medium, the final volume present in each well of a 96 well plate. Cells were incubated at 37 ◦ C, 5% CO2 for 24 h until 60–70% confluence was reached. The medium was then removed from each well and replaced with complete DMEM containing purified CS-NPs or LZ-NPs at different concentrations (75, 100 or 150 ␮g/ml). After 24 h of incubation, cells were analyzed for viability with Cell Proliferation Reagent WST-1. Briefly, cells were incubated with WST-1 reagent diluted 1:10 (as indicated by the manufacturer) for 4 h at 37 ◦ C, 5% CO2 . Plates were then analyzed with a Bio-Rad Benchmark Microplate Reader (Bio-Rad, Hercules, CA, USA); measurements of formazan dye absorbance were carried out at 450 nm, with the reference wavelength at 655 nm.

2.5.2. Antibacterial activity of the prepared NPs Antibacterial activity of blank CS-NPs was evaluated according to standard liquid microdilution susceptibility assays and procedures previously described [24–27]. To this aim, S. epidermidis ATCC 35984 was grown in TSB until the exponential growth phase, at 37◦ with shaking. Bacteria were resuspended in SPB to obtain a density of approximately 1 × 107 CFU/ml. Ten microliters of the bacterial suspension were exposed for 4 h at 37 ◦ C to different concentrations of CS-NPs, in 100 ␮l of SPB enriched with 1.25% TSB. The adopted incubation time (4 h) is in the range of those reported in the literature for antimicrobial agents likely to act by an electrostatic interaction with bacterial cells, as is the case of chitosan [24,25,28]. Samples containing bacterial cells incubated in the absence of CSNPs were set up as positive control of cell viability. Following incubation, samples were diluted 10-fold in TSB, and 0.2 ml of each dilution was plated onto TSA [26,27]. After 48 h of incubation at 37 ◦ C the CFU number was assessed. Antibacterial activity was evaluated in terms of minimal bactericidal concentration (MBC) defined as the CS-NPs concentration able to cause a reduction of the CFU/ml numbers of ≥3 log, after 4 h incubation. The antibacterial properties of selected concentrations of LZ-NPs were evaluated at 5 days, following the same protocol described above. The reduction in the CFU number was compared to that caused by the same concentrations of blank CS-NPs and by free LZ. Control bacteria included untreated S. epidermidis cells incubated in the same conditions as test samples. 2.6. Statistical analysis All the characterizations were performed at least on three CS or NPs replicates, unless otherwise specified. The data were statistically analyzed using the Student’s t-test or one-way ANOVA. Statistical significance was set at the level of p < 0.05 or p < 0.01. 3. Results and discussion 3.1. Physicochemical characterization of commercially available CS The success of chitosans in different fields of application is directly related to their physicochemical properties such as the Mw and the DD. Typically, for research purposes, commercial CS is available in two grades: medium and low Mw . As sold by Sigma-Aldrich, the low Mw CS used in the present work is described as animal derived CS with weight-average Mw in the range of 50k–190k g/mol and DD of 75–85%. To better identify the starting raw CS, the Mw , the decomposition temperature and the DD were assessed. The GPC data collected and evaluated over the pullulan standard curve confirmed an average Mw of 108,120 g/mol with a polydispersity index (Mw /Mn ) of 2.4. The high polydispersity index measured for the investigated batch of commercial CS could be related to the presence of three main populations of CS polymer chains with Mw of 164,642 g/mol (PI 1.6), 102,179 g/mol (PI 1.7) and 56,144 g/mol (PI 2.6), respectively. The thermogravimetric analysis (Table 2) evidenced a first step in the temperature range 30–120 ◦ C corresponding to the


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4 Table 2 TGA data of CS in nitrogen atmosphere.

CS

Volatile (%)a

Td (◦ C)

Tp (◦ C)

R700 (%)

5.8 ± 1.1

264.1 ± 1.5

302.3 ± 0.1

33.9 ± 1.2

Volatile means the weight loss up to 120 ◦ C; Td is the pyrolysis decomposition temperature defined at 2% of weight loss from 150 ◦ C; Tp is the first derivative peak; R700 is the residual weight at 700 ◦ C. a

Fig. 1. FT-IR absorbance spectrum of CS.

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evaporation of the residual sample moisture. The thermo-oxidation temperature defined at 2 wt% of weight loss after that of volatile (Td ) for CS was 264 ◦ C. The white residue at 700 ◦ C (R700 ) of 34% is generally associated to the inorganic components present in the crustaceous shells. The DD of chitosan is the most important parameter that influences its biological, physicochemical and mechanical properties. The effectiveness and behavior of chitosan have been found to be dependent on its DD, as well as the expansion and stiffness of the macromolecular chain conformation, the tendency of the macromolecule chains to aggregate and the ability of the polymer to form NPs [29]. The methods to assess the DD can be essentially confined in three groups: spectroscopic, conventional and destructive. In this study, the DD of CS was assessed by mean of two spectroscopic (FT-IR and UV–Vis) and one destructive (DSC) methods. FT-IR technique needs a calibration versus an absolute technique like nuclear magnetic resonance (NMR), which allows a direct determination of CS deacetylation. A big effort was devoted to identify the right combination of bands and respective baselines, which led to a large amount of proposed methods present in the literature. In this study, the calibration curve obtained by Brugnerotto et al. was employed [30]. In the FT-IR spectrum in absorbance of CS (Fig. 1), the band at 1320 cm−1 was assigned to the C-N stretching of the N-acetylglucosamine (probe, green arrow) while the band centered at 1420 cm−1 corresponded to –CH2 bending (internal reference, red arrow). Using Brugnerotto’s equation the obtained value of DD% (100-DA%) for CS was 91.5 ± 1.2% (Table 3). The method of the first derivative UV spectrophotometry is one of the most used for the determination of the DD, being it the simplest and most convenient among all the available techniques. In this work the DD degree of chitosan was calculated as previously Table 3 Deacetylation degree of chitosan. Technique

Equation

Ref.

DD (%)

UV–Vis FT-IR DSC

DD = [GlcN]/[GlcN + GlcNAc] (A1320 /A1420 ) = 0.3822 + 0.03133 × DA Peak area (J/g) = 257.98 − 3.25 × DA

[25] [24] [28]

97.6 ± 0.4 91.5 ± 1.2 92.7 ± 0.8

described by Dash et al. [31], based on the assumption that the molar absorptions of both GlcN and GlcNAc chromophoric groups change when they are covalently bound through ␤-(1,4) glycosidic linkages. The DD of commercially available CS obtained by mean of UV spectroscopy was 97.6 ± 0.4% (Table 3). Thermal methods, such as differential scanning calorimetry (DSC), have emerged as powerful thermoanalytical techniques to monitor characteristic physicochemical features in biopolymers, including DD determination. It is known that DSC peak area ascribed to the amine (GlcN) groups decrease as the DD of CS increase [32]. The DSC thermogram for CS (Fig. 2) displayed an exothermic peak at 295 ◦ C (53 min) attributed to the decomposition of the high content of amine (GlcN) groups; since the integration of the peak was not calculable by mean of linear extrapolation procedure, an integral tangential baseline (STARe software 9.01) was employed [33]. The DD calculated as previously described by Guinesi and Cavalheiro [34], resulted to be 92.7 ± 0.8% (Table 3). The difference between the DD values obtained from FT-IR and DSC techniques was not statistically significant (p > 0.05, Student’s t test), even if the data were obtained from calibration curves reported by the literature in two separate papers [30,34]; these methods appear reliable and useful to characterize the DD of CS. The difference between the DD values obtained from FT-IR/DSC and UV–Vis techniques was statistically significant (p < 0.05, Student’s t test). UV–Vis analysis was correctly performed, with a low arising standard deviation: the major limitation of this method was believed to be an instrumental one, mainly due to very low absorbance values recorded for GlcN calibration curve. Moreover, it was very important to carefully set the analytical wavelength for GlcN/GlcNAc calibration and CS samples at a value at which the determination of the monosaccharides concentration was insensitive to the fluctuations of the adopted acetic acid concentration [35]. 3.2. Preparation and evaluation of CS based NPs The prepared NPs batches were compared in terms of NPs size, lactescence and reproducibility (Table 1). With optimal mean diameters of 140 ± 20 nm and 159 ± 24 nm, monomodal distribution and good reproducibility, formulations CS-NPs (Blank) and LZ-NPs were selected for further studies. As confirmed by the STEM micrographs of the CS-NPs and LZ-NPs samples (Fig. 3), the nanoparticles appeared round in shape and with a regular distribution of the LZ payload within the CS matrix. The Zeta potential values obtained for CS-NPs and LZ-NPs are shown in Table 1: the positive values arising are due to the presence of cationic CS on the NPs surface. Moreover the two values are statistically different (p < 0.01, Student’s t test) suggesting that part of the positively charged LZ (pH of analysis 6.6) is adsorbed onto the NPs surface. The LZ loading capacity and encapsulation efficacy of NPs, evaluated by mean of UV–Vis spectroscopy, are shown in Table 1. The obtained results revealed a LZ loading of 8.3% and an encapsulation efficacy of 10%. These relatively low values of EE% and loading% were mostly due to the high solubility of the protein in the aqueous medium in which the NPs are formed and to the scarce electrostatic interaction of LZ with CS, as they both are positively charged at the applied conditions. The release kinetic of LZ from LZ-NPs was studied in SPB pH 6.6, the medium used for bactericidal assays, and in PBS pH 7.4, to mimic physiological conditions (Fig. 4). The system displayed an initial burst corresponding to the 5% of the total LZ loaded in the carrier due to the desorption of the LZ molecules dispersed onto to the NPs surface [36]. The cumulative release profile of LZ proceeded with a second burst (between 1.5 and 2 h) related to the swelling of the NPs once in equilibrium with the release medium and continued with a slow release sustained over


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Fig. 2. DSC curve for CS.

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22 days. After the burst release period, the release rate decreased because the dominant release mechanism changed to drug diffusion through the CS matrix outer layer of NPs. The release kinetic of LZ was faster in SPB than in PBS, indicating that the two buffers affected the amount of LZ released from the NPs. Indeed, the two different pHs had the main influence since the CS hydration was higher at acidic pH when the amines were more protonated and the swelling of the nano-hydrogel system was greater. Furthermore, LZ is a basic protein and also its water solubility is higher at acidic pH, leading to a faster release from the CS matrix. The influence of the pH on the interaction between LZ and CS (in form of microparticles) was also reported by Torres et al. [37]. The features of the prepared nanocarriers were in agreement with the findings of Deng et al. [38], in which a full formulation study was performed and appeared useful for the preparation of CS LZ loaded nanoparticles with small mean diameter and sustained LZ release. Although the modest expected EE%, the amount of LZ loaded in the NPs resulted adequate to proceed to the antibacterial investigations.

3.3. Stability study The stability of the prepared blank and LZ-NPs was studied in SPB at 37 ◦ C to mimic the bactericidal assay conditions and to check the behavior of the nanosystems during the activity tests. The stability was followed in terms of NPs size distribution and eventually occurring polymer degradation (GPC analysis, Table 4). The dimensional analysis carried out on blank CS-NPs and LZ-NPs after 5 days in SPB at 37 ◦ C highlighted a small increase in the nanoparticles dimensions, probably due to the swelling and water adsorption of the system. Concerning the degradation of the CS entangled in the NPs, it was first observed that the GPC analysis carried out on the purified NPs pellets revealed that only the low Mw chains of the commercial polymer batch were actually involved in the formation of the nanocarrier. As matter of fact, by comparing the average Mw of the pristine CS (108,120 g/mol PI 2.4) with that of the CS of the NPs pellets (CS-NPs: 53,137 g/mol PI 2.7; LZ-NPs: 50,206 g/mol PI 2.47), the obtained values were about halved. Although LZ is generally applied for the digestion of CS into low Mw chains, both blank

Fig. 3. STEM micrographs of plain CS-NPs (A) and LZ-NPs (B).


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Fig. 4. LZ release profile of the NPs in SPB and PBS.

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and loaded formulations showed no difference in their CS molecular weight after 1, 3 or 5 days of incubation. This was assumed as an evidence that no hydrolysis or degradation of the CS constituting the NPs occurred under the applied conditions even in the presence of LZ, confirming that highly deacetylated CS is scarcely susceptible to LZ enzymatic degradation [39].

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3.4.1. NPs cytocompatibility evaluation The cytocompatibility of the NPs was evaluated using 3T3/BALBC Clone A31 murine fibroblast as a cell line, as indicated by international ISO 10993 guidelines. Quantitative evaluation of cell viability performed by WST-1 assay indicated no toxicity for the developed blank CS-NPs and LZ-NPs batches; cell viability in the presence of CS-NPs was comparable to the control cell profile up to a NPs concentration of 150 ␮g/ml, as shown in Fig. 5. A full in vitro cytocompatibility was indicated for both NPs kinds at the tested concentrations. 3.4.2. Antibacterial properties of CS-NPs While it is well established that polymeric CS exerts antibacterial activity against a wide variety of bacterial species [40], far less investigated are the antibacterial properties of CS-NPs. Table 4 Stability study of CS-NPs and LZ-NPs.

CS-NPs

LZ-NPs

Time (days)

Mn

Mw

Mw /Mn

Diameter (nm ± S.D.)

0 1 3 5 0 1 3 5

19,440 19,804 19,807 21,104 21,043 22,407 22,060 21,061

53,137 47,890 45,082 50,183 50,206 50,852 49,968 47,102

2.7 2.4 2.3 2.4 2.4 2.3 2.3 2.2

101 ± 17 – – 156 ± 29 120 ± 16 – – 151 ± 30

In the present study, the antibacterial activity of the well characterized CS-NPs was assessed against S. epidermidis, a Gram-positive bacterial species that has been recently become one of the most important cause of nosocomial infections and one of the major human pathogen associated with the use of indwelling medical device [41]. As compared to untreated control, no reduction in the number of viable cells was observed following incubation of S. epidermidis for 4 h with CS-NPs, in the concentration range of 2.3–9.37 ␮g/ml (Fig. 6A). In contrast, a striking dose-dependent killing capacity of CSNPs was recorded in the concentration range of 18.75–150 ␮g/ml. In particular, at the concentration of 75 g/ml a reduction of 3 log (99.9%) in the number of viable S. epidermidis cells was observed, corresponding to the MBC value. Since the stability study of CS-NPs proved that there is no hydrolysis or degradation of CS in the bactericidal assay conditions (Table 4), the shown antimicrobial activity is likely to be attributed to CS-NPs and not to free CS. These results are in agreement with recent studies reporting the ability of CSNPs to exert antibacterial activity against both Gram-positive and Gram-negative bacteria [14]. Bactericidal concentrations identified in the present study are much lower than those previously reported in some [42] but not in other studies [14] highlighting that differences related to the CS physical–chemical features (Mw and DD), NPs preparation procedures, and/or bactericidal assay conditions (e.g. pH, assay medium, time of exposure, bacterial species tested) may greatly influence the antibacterial properties of CS-NPs. 3.4.3. Antibacterial properties of LZ-loaded CS-NPs LZ-NPs were tested in bactericidal assays. In order to test the ability of LZ-NPsto exert a prolonged antibacterial activity, killing assays were carried out at 5 days of incubation with S. epidermidis cells and the results compared with those obtained using unloaded CS-NPs or free LZ. Concentrations of free LZ were chosen according to the theoretical amount of protein loaded in the corresponding concentrations of LZ-NPs. The number of CFU obtained in the presence of blank CS-NPs as well as free LZ was only slightly reduced as compared to untreated control, after 5 days of incubation. In


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Fig. 5. Cytocompatibility of (a) CS-NPs and (b) LZ-NPs evaluated by WST-1 assay.

Fig. 6. Antibacterial activity of CS-NPs against S. epidermidis ATCC 35984. (A) Bacterial cells (1 × 106 CFU/ml) were incubated in Sodium phosphate buffer (SPB, 10 mM, pH 6.6) enriched with 1.25% Triptone Soy Broth (TSB) for 4 h at 37 ◦ C in the presence of different concentrations of Cs-NPs. Control cells (CTRL) represents un-treated S. epidermidis incubated in the same conditions as test samples; a representative experiment is depicted. (B) Antibacterial activity of LZ-NPs as compared to LZ and CS-NPs alone at 5 days of incubation with S. epidermidis. Bacterial cells (106 CFU/ml) were incubated in sodium phosphate buffer (SPB) enriched with 1.25% Triptone Soy Broth (TSB) for 5 days at 37 ◦ C in the presence of different concentrations of CS-NPs, LZ or LZ-NPs. CTRL: un-treated S. epidermidis, cells incubated in the same conditions of test samples. Values represent the mean ± SEM of 4–5 experiments. * p < 0.05, ANOVA.

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contrast, at the concentration of 75 and 37.5 ␮g/ml, LZ-NPs caused, a statistically significant reduction in the number of CFU as compared to blank nanoparticles or LZ used alone (p < 0.05, ANOVA) (Fig. 6B). These results suggest that besides being bactericidal themselves, LZ-NPs may provide a controlled and long lasting release of LZ molecules able to provide a continuous antibacterial activity. The increase in the nanoparticles dimensions after 5 days of incubation at 37 ◦ C in SPB was probably due to the swelling capacity of the NPs that may facilitate the release of LZ in these conditions, as seen with the release kinetics in vitro, and contributing to the antimicrobial properties of the developed nanosystem. 4. Conclusions This work presents a systematic study on the physical–chemical and biological characterization of NPs loaded with LZ and based on a CS matrix. Commercial CS was fully characterized for its molecular weight and DD, key parameters for the development of CS-NPs. LZNPs with good dimensional features were successfully prepared by means of a mild ionic gelation technique. LZ loading in the NPs was up to 8% and the release kinetic studies showed that up to 20% of the loaded enzyme was slowly released over 3 weeks, in a controlled

and sustained manner. The developed formulation showed a full in vitro cytocompatibility and LZ-NPs exhibited a good antimicrobial activity on S. epidermidis. The controlled delivery system for LZ developed in this study has potential antimicrobial applications and could serve as an optimal model for the encapsulation of antimicrobial peptides in Cs based NPs. The choice of using LZ as model drug was made since its shares key features (e.g. isoelectric point, antimicrobial activity) with antimicrobial peptides, a class of bioactive macromolecules that are gaining increasing interest as new and highly effective antimicrobial compound [43,44]. However the employment of antimicrobial peptides as therapeutic agents is hampered by primarily by their poor stability in physiological conditions [45], thus their loading into polymeric nanovectors may represent a promising strategy for their successful employment in clinical applications.

Acknowledgments The authors would like to acknowledge the contribution given by Dr. Giovanni Baldi, Dr. Filippo Mazzantini and Dr. Costanza


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Ravagli in recording STEM images of nanoparticles and Ms. Giulia Pirastu for her support during the experiments. References

[1] N.M. Alves, J.F. Mano, Int. J. Biol. Macromol. 43 (2008) 401–414. 517 [2] L. Hui, D. Yumin, W. Xiaohui, S. Liping, Int. J. Food Microbiol. 95 (2004) 147–155. 518 [3] A.B. Vishu Kumar, M.C. Varadaraj, L.R. Gowda, R.N. Tharanathan, Biochim. Bio519 phys. Acta 1770 (2007) 495–505. 520 [4] H.K. No, N.Y. Park, S.H. Lee, S.P. Meyers, Int. J. Food Microbiol. 74 (2002) 65–72. 521 [5] E.M. Costa, S. Silva, C. Pina, F.K. Tavaria, M.M. Pintado, Anaerobe 18 (2012) 522 305–309. 523 [6] F.S. Kittur, A.B. Vishu Kumar, M.C. Varadaraj, R.N. Tharanathan, Carbohydr. Res. 524 340 (2005) 1239–1245. 525 [7] M. Benhabiles, R. Salah, H. Lounici, N. Drouiche, M. Goosen, N. Mameri, Food 526 Hydrocolloids 29 (2012) 48–56. 527 [8] João C. Fernandes, P. Eaton, Isabel Franco, Óscar S. Ramos, Sérgio Sousa, Hen528 rique Nascimento, Ana Gomes, Alice Santos-Silva, F. Xavier Malcata, Manuela 529 Q2 E. Pintado, Int. J. Biol. Macromol. 50 (2012) 148–152 530 [9] X. Liu, L. Yun, Z. Dong, L. Zhi, D. Kang, J. Appl. Polym. Sci. 79 (2001) 1324–1325. 531 [10] T. Takahashi, M. Imai, I. Suzuki, J. Sawai, Biochem. Eng. J. 40 (2008) 485–491. 532 [11] W.-L. Du, S.-S. Niu, Y.-L. Xu, Z.-R. Xu, C.-L. Fan, Carbohydr. Polym. 75 (2009) 533 385–389. 534 [12] P. Sanpui, A. Murugadoss, P.V.D. Prasad, S.S. Ghosh, A. Chattopadhyay, Int. J. 535 Food Microbiol. 124 (2008) 142–146. 536 [13] H.V. Tran, L.D. Tran, C.T. Ba, H.D. Vu, T.N. Nguyen, D.G. Pham, P.X. Nguyen, 537 Colloids Surf., A 360 (2010) 32–40. 538 [14] L. Qi, Z. Xu, X. Jiang, C. Hu, X. Zou, Carbohydr. Res. 339 (2004) 2693–2700. 539 [15] M. Amidi, E. Mastrobattista, W. Jiskoot, W.E. Hennink, Adv. Drug Del. Rev. 62 540 (2010) 59–82. 541 [16] S.-J. Yang, F.-H. Lin, H.-M. Tsai, C.-F. Lin, H.-C. Chin, J.-M. Wong, M.-J. Shieh, 542 Biomaterials 32 (2011) 2174–2182. 543 [17] F.-Y. Su, K.-J. Lin, K. Sonaje, S.-P. Wey, T.-C. Yen, Y.-C. Ho, N. Panda, E.-Y. Chuang, 544 B. Maiti, H.-W. Sung, Biomaterials 33 (2012) 2801. 545 [18] Y. Liu, Y. Sun, Y. Xu, H. Feng, S. Fu, J. Tang, W. Liu, D. Sun, H. Jiang, S. Xu, Int. J. 546 Biol. Macromol. 59 (2013) 201–207. 547 [19] Y. Ito, O.H. Kwon, M. Ueda, A. Tanaka, Y. Imanishi, J. Bioact. Compat. Polym. 15 (2000) 376–395. 516

[20] G. Mocanu, D. Mihai, M. Legros, L. Picton, D. Lecerf, J. Bioact. Compat. Polym. 23 (2008) 82–94. [21] V.K. Garripelli, S. Jo, J. Bioact. Compat. Polym. 27 (2012) 198–209. [22] M. Dash, F. Chiellini, R.M. Ottenbrite, E. Chiellini, Prog. Polym. Sci. 36 (2011) 981–1014. [23] L. Wei, C. Cai, J. Lin, L. Wang, X. Zhang, Polymer 52 (2011) 5139–5148. [24] X. Geng, R. Yang, J. Huang, X. Zhang, X. Wang, J. Food Sci. 78 (2013) M90–M97. [25] J. Huang, H. Jiang, M. Qiu, X. Geng, R. Yang, J. Li, C. Zhang, Int. J. Biol. Macromol. 52 (2013) 85–91. [26] C.a.L.S. Institute, in: P.C. Wayne (Ed.), CLSI Document M23-A3: Development of In Vitro Susceptibility Testing Criteria and Quality Control Parameters: Approved Guideline, CLSI, Wayne, PA, 2008. [27] G. Maisetta, G. Batoni, S. Esin, W. Florio, D. Bottai, F. Favilli, M. Campa, Antimicrob. Agents Chemother. 50 (2006) 806–809. [28] H. Tan, R. Ma, C. Lin, Z.T.T. Liu, Int. J. Mol. Sci. 14 (2013) 1854–1869. [29] M. Kasaai, J. Agricultural and food chemistry 57 (2009) 1667–1676. [30] J. Brugnerotto, J. Lizardi, F.M. Goycoolea, W. Argüelles-Monal, J. Desbrières, M. Rinaudo, Polymer 42 (2001) 3569–3580. [31] M. Dash, F. Chiellini, E. Fernandez, A.M. Piras, E. Chiellini, Carbohydr. Polym. 86 (2011) 65–71. [32] F.S. Kittur, K.V. Harish Prashanth, K. Udaya Sankar, R.N. Tharanathan, Carbohydr. Polym. 49 (2002) 185–193. [33] J. dos Santos, E. Dockal, E. Cavalheiro, Carbohydr. Polym. 60 (2005) 277–282. [34] L. Guinesi, E. Cavalheiro, Thermochim. Acta 444 (2006) 128–133. [35] R.A.A. Muzzarelli, R. Rocchetti, Carbohydr. Polym. 5 (1985) 461–472. [36] S. Zhou, X. Deng, X. Li, J. Controlled Release 75 (2001) 27–36. [37] M.A. Torres, M.M. Beppu, C.C. Santana, Braz. J. Chem. Eng. 24 (2007) 325–336. [38] Q. Deng, C. Zhou, B. Luo, Pharm. Biol. 44 (2006) 336–342. [39] R.J. Verheul, M. Amidi, M.J. van Steenbergen, E. van Riet, W. Jiskoot, W.E. Hennink, Biomaterials 30 (2009) 3129–3135. [40] M. Kong, X. Chen, K. Xing, H. Park, Int. J. Food Microbiol. 144 (2010) 51–63. [41] M. Otto, Nat. Rev. Microbiol. 7 (2009) 555–567. [42] A. Shrestha, Z. Shi, K. Neoh, A. Kishen, J. Endod. 36 (2010) 1030–1035. [43] G. Batoni, G. Maisetta, F.L. Brancatisano, S. Esin, M. Campa, Curr. Med. Chem. 18 (2011) 256–279. [44] G. Maisetta, A. Vitali, M.A. Scorciapino, A.C. Rinaldi, R. Petruzzelli, F.L. Brancatisano, S. Esin, A. Stringaro, M. Colone, C. Luzi, A. Bozzi, M. Campa, G. Batoni, FEBS J. 280 (2013) 2842–2854. [45] N.K. Brogden, K.A. Brogden, Int. J. Antimicrob. Agents 38 (2011) 217–225.


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