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Publication Date (Web): September 21, 2012 ..... Self Assembly Coatings Act as “Fungal Repellents” to Prevent Biofilm Formation on Healthcare Mate...
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Antiadhesive and Antibacterial Multilayer Films via Layer-by-Layer Assembly of TMC/Heparin Complexes Heveline D. M. Follmann,† Alessandro F. Martins,† Adriana P. Gerola,† Thiago A. L. Burgo,‡ Celso V. Nakamura,§ Adley F. Rubira,† and Edvani C. Muniz*,† †

Grupo de Materiais Poliméricos e Compósitos, GMPC, Departamento de Química and §Laboratório de Inovaçaõ Tecnológica no Desenvolvimento de Fármacos e Cosméticos, DBS Bloco B-08, Universidade Estadual de Maringá UEM, Av. Colombo 5790, CEP 87020-900 Maringá, Paraná, Brazil ‡ Instituto de Química, Universidade Estadual de Campinas, CP 6154, CEP 13083-970, Campinas, São Paulo, Brazil S Supporting Information *

ABSTRACT: N-Trimethyl chitosan (TMC), an antibacterial agent, and heparin (HP), an antiadhesive biopolymer, were alternately deposited on modified polystyrene films, as substrates, to built antiadhesive and antibacterial multilayer films. The properties of the multilayer films were investigated by Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy, and Kelvin force microscopy. In vitro studies of controlled release of HP were evaluated in simulated intestinal fluid and simulated gastric fluid. The initial adhesion test of E. coli on multilayer films surface showed effective antiadhesive properties. The in vitro antibacterial test indicated that the multilayer films of TMC/HP based on TMC80 can kill the E. coli bacteria. Therefore, antiadhesive and antibacterial multilayer films may have good potential for coatings and surface modification of biomedical applications.

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INTRODUCTION N-Trimethyl chitosan (TMC) is a quaternary polycation obtained from the N-methylation of chitosan (CHT).1 Similarly to CHT, the TMC also possesses properties of biodegradability, biocompatibility, mucoadhesivity, nontoxicity, and antimicrobial activity. However, recent studies showed that TMC has higher bactericidal capacity than the CHT.2 On the other hand, such antimicrobial activity depends on the quaternary chemical structure, molecular weight, and quaternization degree (DQ) of TMC.3−5 The quaternization degree (DQ) and dimethylation degree (DD) refer, respectively, to the molar percentages of −N(CH3)3+ and −N(CH3)2 groups present in TMC structure.6 Another advantage of TMC related to CHT is its solubility in whole pH range, while the CHT is soluble only at pH < 6.5.7−10 Therefore, in neutral and basic environments, the CHT molecules lose their positive charges and precipitate.11 The limited solubility of CHT in solutions with pH higher than 6.5, hence, restricts its biomedical application, especially in situations where the controlled delivery of drugs in particular condition is required.12 Heparin (HP) is a biocompatible polyanion with anticoagulant activity. This biopolymer has applications in pharmaceutical industry, medical devices, and tissue engineering.13−15 The high negative charge density of HP prevents the adhesion of bacterial cells and, therefore, this biopolymer is an excellent candidate to act as antiadhesive coatings.14 Thus, polyelectrolyte complexes formulations based on TMC (polycation, © XXXX American Chemical Society

antibacterial) and HP (polyanion, antiadhesive) may have potential for application as biomaterials devices.1,16 In this way, Kweon and co-workers17 developed a topical ointment based on CHT and HP which promoted significant increase in the ability of wound healing in rats. Zhu and co-workers18 increased the biocompatibility of the blood with expanded polytetrafluoroethylene (ePTFE), adhering polyelectrolyte complex of CHT and HP on the ePTFE surface. Grafts made of ePTFE are widely used in reconstructive vascular surgery.19 The “layer-by-layer” (LbL) adsorption technique of oppositely charged polyelectrolytes is an efficient method for manufacturing multilayer films of biocompatible macromolecules.20−22 The method involves the sequentially immersion of a substrate loaded in dilute aqueous solutions of opposite electric charged polyelectrolytes, allowing the just-adsorbed polymer to reverse the predominant electric charge existing at the surface before its adsorption. Such a process can be repeated several times. Thus, the thickness of LbL is proportional to the number of immersions. This method presents advantages that include simplicity, universality, low operating costs, and control of the thickness of film.20,23−25 For instance, Fu and co-workers14 developed films by LbL based on CHT and HP on substrates of chemically modified polyReceived: July 27, 2012 Revised: September 19, 2012

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(ethylene terephthalate) (PET). The developed films presented antimicrobial and antiadhesive good properties. This research aimed to obtain multilayer thin films of TMC/ HP by LbL technique, using the chemically modified polystyrene (PS-mod) as substrate. In vitro studies of controlled release of HP were evaluated in simulated intestinal fluid (SIF) and simulated gastric fluid (SGF). The antibacterial and antiadherent capacities of the multilayer thin films, varying the DQ of TMC and the pH for obtaining the layers, were investigated. The strong bactericidal activity of TMC in alkaline medium, relative to CHT has leveraged this study. It is worth to highlight that the application of films of TMC/HP obtained by LbL technique, as aimed in this work, was not previously discussed by the literature.

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15% (w/v) were added to the reaction medium, keeping the agitation for about 20 min. The system was maintained under reflux and then 55 mL of methyl iodide were added. After the addition of this reducing agent, the reaction proceeded under reflux for 1 h at 45 °C. The product was collected after precipitation utilizing ethanol (300 mL to each 100 mL of solution). Therefore, the precipitate was separated by centrifugation (3000 rpm for 5 min), washed with diethyl ether (four times), filtered, and dried under vacuum at 40 °C for 48 h. Finally, the product was dissolved in aqueous sodium chloride solution at 10% (w/ v) for iodide ionic exchange. Then, the N-trimethyl chitosan chloride (TMC20) was precipitated in ethanol and finally isolated by centrifugation, following the same methodology as described above for purification of TMC80. Modification of the Polystyrene Surface by Oxidation. Polystyrene (PS) Petri dishes were cut (dimensions of 7.5 × 2.5 cm, thickness = 1.2 mm) and immersed in ethanol for 24 h to remove, as much as possible, the impurities on the virgin PS surface. After this time, the films were washed with an abundance of deionized water and submitted to an oxidation process, as described by Curti and coworkers.27 The label PS-mod was used referring to the oxidized polystyrene films. Preparation of Multilayer Thin Films. The as-obtained PS-mod, with negative surface-charge, was immersed for 60 s in a 2% (w/v) solution of TMC20 (at pH 3.0) or in a TMC80 solution (at pH 3.0 or 7.4). Then, the set PS-mod/TMC was washed by immersing for 30 s in an aqueous solution at the same pH of TMC solution used for the previous immersion. This step aims to remove the material (TMC) weakly adsorbed on the PS-mod surface. After, the material with TMC20 (or TMC80) layer, adsorbed on PS-mod surface, was immersed for 60 s in a 2% (w/v) HP solution (at pH 3.0 or 7.4) and then washed as described above. The immersions were performed with the aid of a semiautomatic “dip-coating” apparatus developed and adapted by our research group. So, multilayer thin films of TMC20/ HP (at pH 3.0) and TMC80/HP (at pH 3.0 and 7.4) with 3, 9, 12, and 18 bilayers were obtained from the conditions described above.

MATERIALS AND METHODS

Materials. Chitosan (CAS 9012−76−4) with a deacetylation degree of 85 mol % and average molecular weight of 87 × 103 g mol−1 was purchased from Golden-Shell Biochemical (China); methyl iodide (CAS 74−88−4) and N-methyl-2-pyrrolidinone (CAS 872−50−4) were both purchased from Sigma (U.S.A.). Heparin sodium (CAS 9041−08−1) was kindly supplied by Kin Master (Brazil). The labels TMC20 and TMC80 were used for correlating the quaternization degree (DQ) of the samples with DQ of 20 and 80 mol %, respectively. The average molecular weights, from intrinsic viscosity measurements, of TMC20 and TMC80 are, respectively, 26 × 103 and 13 × 103 g mol−1. The viscosities were measured in acetic acid/sodium acetate buffer solution using an Ubbelohde-type capillary viscometer (Model Cannon 100/E534), at 25 °C. The constants used on Mark− Houwink−Sakurada equation refer to the TMC with an acetylating degree of 15 mol %, are K = 1.38 × 10−5 and a = 0.85.26 Commercial polystyrene (PS) Petri dishes were purchased from Prolab (Brazil). Other reactants, such as sodium hydroxide, sodium iodide, sodium chloride, sodium persulfate, hydrochloric acid, ethanol, and diethyl ether, were also utilized. All the reactants were used as received from the suppliers. Synthesis of the TMC. TMC20 and TMC80 were synthesized and used with the main objective of evaluating the effect of quaternization degree on the bactericidal activity. The employed methodology for the synthesis of high degree of quaternized TMC was adapted from previously reported procedures.1,7,8 So, the first step to obtain the TMC of high quaternization degree (TMC with DQ equal to 80 mol %) was preceded from the mixture of CHT (10 g) and sodium iodide (24 g). The mixture was dissolved in N-methyl-2pyrrolidinone (400 mL) under magnetic stirring. After the dissolution, 60 mL of an aqueous sodium hydroxide solution 15% (w/v) and methyl iodide (55 mL) were introduced into reaction mixture under constant stirring. The system was maintained at reflux for 1 h at 60 °C. The product N-trimethyl iodide (TMI) obtained at the first step was precipitated with ethanol and isolated by centrifugation (3000 rpm for 5 min). After being washed with diethyl ether (four times), the product was filtered and dried under vacuum at 40 °C for 48 h. In a second step, the TMI obtained at first step was dissolved in 400 mL of N-methyl-2-pyrrolidinone (NMP) under constant stirring. Sodium iodide (24 g), aqueous sodium hydroxide solution (55 mL), and methyl iodide (35 mL) were added to the reactional medium, keeping the system under stirring. The system was maintained at reflux for 30 min at 60 °C. Afterward, methyl iodide (10 mL) and sodium hydroxide pellets (3 g) were added to the mixture and the stirring was continued for 70 min at 60 °C. The TMI was precipitated in ethanol, isolated by centrifugation, washed with diethyl ether, filtered, and dried under vacuum. Finally, TMI was dissolved in 10% (w/v) sodium chloride solution for iodide ionic exchange. Then, the N-trimethyl chitosan chloride (TMC80) was precipitated, isolated, washed, and dried following the same methodology as described above. To obtain the TMC of low quaternization degree (TMC with DQ equal to 20 mol %), 10.0 g of CHT were dissolved in 400 mL of NMP at 45 °C in about 30 min. Then, 24.0 g of NaI and 60 mL of NaOH

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CHARACTERIZATION Characterization of TMC through 1H NMR and Determination of DQ. The synthesis of the TMC from CHT was characterized through 1H NMR (spectra are shown in Supporting Information, Figure S1). The 1H NMR spectra were performed on a Varian, Mercury Plus 300 BB NMR spectrometer (U.S.A.), operating at 300.06 MHz for 1H frequency. For acquisition of 1H NMR spectra, 10 mg of sample (CHT, TMC20, and TMC80) were dissolved in 1.0 mL of D2O/HCl (100/1 v/v) and D2O, respectively. 1H NMR spectra were acquired at room temperature and the main acquisition parameters were as follows: a pulse of 45°, a recycle delay of 10 s, and acquisition of 128 transients. Time-domain data were apodized with a 0.2 Hz exponential function (lb) to improve the signal-to-noise ratio before Fourier transformation. The 1H NMR technique was also used for determination of quaternization degree (DQ) of the TMC20 and TMC80 through the ratio between the areas of the signals due to the hydrogen of the methyl groups of the acetamide moieties [(NHCOCH3)], relative to the hydrogen of methyl groups of the dimethylated site [(CH3)2], and the areas of the methyl hydrogen of the quaternized [(CH3)3] sites, according to methodology described by Martins and co-workers.1,11 Surface Analysis. Drop Water Contact Angle Measurement. The contact angle of drop water (Milli-Q) deposited in the surface of virgin PS, PS-mod, and TMC/HP thin films (18 bilayers) was measured at room temperature, using a Tantec Contact Angle Meter (model Cam Micro, U.S.A.). The value of contact angle was taken as an average of six different measures, B

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Tryptic Soy Agar (TSA). After incubation for 24 h, the colonyforming units were counted. The experiments were performed in triplicate. In Vitro Heparin Release. The in vitro heparin (HP) release studies were performed in two different environments, both without the presence of enzymes: simulated intestinal fluid (SIF, 6.8 g of KH2PO4 and 77 mL of aqueous NaOH 0.20 mol L−1 in 1000 mL of water, pH = 6.8) and simulated gastric fluid (SGF, 2.0 g NaCl and 7.0 mL of concentrated aqueous solution of HCl 37% (v/v) in 1000 mL of water, pH = 1.2). Five samples (1.0 cm2) of each TMC/HP multilayer thin film of 18 bilayers was deposited in a sealed flask with 25 mL of SGF or SIF at 37 °C. At a desired time interval, an aliquot (100 μL) was removed from the flask and directly added to 4.0 mL of aqueous methylene blue (MB) solution (5.0 mg L−1 at pH ∼ 5.5) for quantifying through UV measures the amount of HP released. The measurements of absorbance of the methylene blue/heparin complex (MB/HP) were performed immediately after homogenization of the samples that occurred quickly after mixing, according to methodology described by Martins and co-workers.11 To quantify the solute released from TMC/HP multilayer films, two analytical curves correlating the absorption of MB/HP complex to the concentration of HP, from 0.20 to 7.0 mg L−1 using SGF (R2 = 0.987) and SIF (R2 = 0.989), as solvents, were built. The analysis was performed with the use of an ultraviolet−visible (UV−vis) spectrophotometer (Femto, Brazil) model 800Xi and the readings were done at 567 nm.

under the same experimental conditions, performed in different parts of a given sample. Infrared Spectroscopy. The chemical structures of the multilayer thin films of TMC/HP, as well the growing number of bilayers, were characterized by infrared spectroscopy (FTIR) using the equipment Shimadzu Scientific Instruments (model 8300, Japan). The analysis was performed using the technique of attenuated total reflection (FTIR-ATR), in the range 4000 to 625 cm−1. Resolution of 4 cm−1 was obtained after cumulating 64 scans. Atomic Force Microscopy (AFM). AFM images of virgin PS, PS-mod, and multilayer thin films of TMC/HP surfaces were obtained in Atomic Force Microscope equipment, from Shimadzu (model SPM-9500 J3, Japan), at room temperature. Scanning Electron Microscopy (SEM). The surface morphologies of multilayer films prepared in this work were investigated by SEM from Shimadzu (model SS 550, Japan). The films were sputter-coated with a thin layer of gold for allowing the SEM visualization. The images were taken by applying an electron accelerating voltage of 10 and 12 kV. All the SEM analyses were performed at 20 °C. Virgin PS and PSmod films were used as controls. Kelvin Force Microscopy (KFM). Samples for KFM measurements were cut into squared small pieces (∼5 mm edge) and imaged in noncontact mode. A scanning probe microscope Shimadzu SPM-9600 was used, with a silicon tip Nanoworld EFM-20 (resonance frequency = 79 kHz and force constant = 2.6 N m−1). KFM scanning system is enclosed within an environmental chamber that allows controlling temperature (25 ± 1 °C) and relative humidity during electric potential scanning. Initial Adhesion of Escherichia coli on PS Substrates with TMC/HP Multilayer Thin Films. One sample (1.0 cm2) of each TMC/HP multilayer thin film was placed in a culture plate (24wells). Thus, 1.0 mL of bacterial suspension 5 × 107 CFU mL−1 in PBS was added to each well. The culture plate was incubated at 37 °C for 4 h. Then, the bacterial suspension was removed and the sample washed three times with sterile PBS. Sterilized filter paper was used to suck-up the moisture on the film surface. For the fixation of the E. coli, the samples were immersed into 4 vol % formaldehyde in PBS at 4 °C for 4 h. After this period, the formaldehyde solution was removed and the samples were washed with PBS and dehydrated with 25, 50, 70, 95 and 100% ethanol (v/v) for 10 min. In sequence, the substrates were dried and sputter-coated with a thin film of gold for imaging purposes for scanning electron microscope Shimadzu (model SS 550, Japan). The samples were also analyzed by Normaski interference contrast microscopy (Olympus BX51, Japan) and pictures were captured with a UC30° camera (Olympus). Virgin PS and PS-mod films were used as controls.14 In Vitro Antibacterial Test. Tests for antibacterial activity of the thin films against the Escherichia coli (E. coli; ATCC 26922) were carried out according to viable-cell-counting method.14 One sample (1.0 cm2) of each TMC/HP multilayer thin film was placed in a culture plate (24-wells), and 1.0 mL of inoculum of E. coli 5 × 104 CFU mL−1 in PBS was added on the surface of the substrates. The suspension of E. coli completely covered the surface of the film. In parallel, E. coli 5 × 104 CFU mL−1 in PBS was used as positive control. The plates were incubated at 37 °C for 6 h. Then, aliquots of bacterial suspensions were diluted with sterile PBS to a concentration of 5 × 103 CFU mL−1. From this solution, 20 μL were spread on

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RESULTS AND DISCUSSION Chemical Modification of the Polystyrene (PS) Surface To Obtain the PS-mod. Contact Angle Measurements. Contact angle measurements of the virgin PS and modified PS (PS-mod) were evaluated with the aim of verifying changes on surface wettability due to the oxidation process. The virgin PS has hydrophobic characteristics, leading to high value of contact angle, about 81° (Table 1). However, the oxidation process Table 1. Values of Contact Angle of the Virgin PS and PSmod Surfaces PS film before modification 82° 81° 84° 81° 78° 81°

± ± ± ± ± ±

1.63 2.75 1.63 1.51 3.28 1.00

PS film after modification 66° 58° 64° 68° 65° 66°

± ± ± ± ± ±

3.47 4.19 4.68 3.41 4.69 3.03

significantly changes the surface wettability related to the virgin PS, as verified by the decrease of contact angle to about 65° (Table 1). This decrease confirms the chemical modification of the PS surface. The results presented are consistent with the literature.27 AFM Analysis. AFM images of the virgin PS, PS-mod, and PS-mod with a monolayer of TMC80 adsorbed at pH 7.4 are presented in Figure 1. The virgin PS, as expected, presented a smooth, compact, and more homogeneous surface. The topography of PS-mod presented a rough surface with projections and high heterogeneity. So, the roughness is higher in PS-mod than in virgin PS. This also confirms the chemical modification of the PS surface. Even so, EDS analysis was performed to characterize the presence of oxygen in the surface of PS-mod and, thus, strengthen the occurrence of chemical C

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its structure, thus, revealing the effective oxidation after treatment of virgin PS using sodium persulfate.27 The adsorption of TMC80 provided topographical changes on the PS-mod surface (see the Figure 1b,c). The topography of PSmod surface containing absorbed TMC80 tends to be less flat and some places show roughness spots in contrast to the PSmod. This fact enables the formation of a layer of TMC on the surface of PS-mod. Characterization of Multilayers Absorbed on PS-mod Surfaces. FTIR-ATR Spectroscopy. The FTIR-ATR spectra of the multilayer films of TMC20/HP and TMC80/HP obtained at pHs 3.0 and 7.4 are presented in Figure 2 (a,b), respectively.

Figure 2. (a) FTIR-ATR spectra of the TMC20/HP multilayer films prepared at pH 3.0. (b) FTIR-ATR spectra of the TMC80/HP multilayer films prepared at pH 7.4.

The stretching attributed to angular deformation of CC bonds (1598 and 1492 cm−1); H−C−H (1450 and 1459 cm−1) and C−H (754 and 659 cm−1) are characteristic of the repetitive unit of PS on PS-mod.28 The increase the number of bilayers (TMC/HP) results in a decrease on intensity of all above-mentioned bands (Figure 2). However, the adsorption of polysaccharides (HP and TMC) at PS-mod surface films was ratified by the increase in intensity of the bands attributed to the CO stretching of carboxylic acids (1735 cm−1), secondary amide groups and carboxylate anions (1655 to 1601 cm−1); SO (1214 and 1225 cm−1) and C−O (1030 cm−1) of primary alcohols, as shown in Figure 2 (a,b),

Figure 1. AFM images of PS: (a) virgin, (b) oxidized, and (c) oxidized with a monolayer of TMC80 adsorbed at pH 7.4.

modification at polymer surface. The EDS spectrum of the PSmod (spectrum are shown in Supporting Information, Figure S2) confirmed the presence of oxygen atoms on the sample surface. The virgin PS does not contain such kinds of atoms in D

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Figure 3. continued

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Figure 3. (A) AFM images of the TMC20/HP multilayer films assembled at pH 3.0 with: 3 (a), 9 (b), 12 (c) and 18 (d) bilayers. (B) AFM images of the TMC80/HP multilayer films assembled at pH 3.0 with: 3 (a), 9 (b), 12 (c) and 18 (d) bilayers. (C) AFM images of the TMC80/HP multilayer films assembled at pH 7.4 with: 3 (a), 9 (b), 12 (c) and 18 (d) bilayers.

respectively. It is worth mentioning the increase of the band intensities at 1214 and 1225 cm−1 attributed to the axial stretching of SO bonds present in the structure of HP (Figure 4). The band at 1475 cm−1 (Figure 2a) is related to angular deformation of C−H bonds of methyl groups existing on the structure of TMC20 in higher proportion. This band is observed only in spectra of Figure 2a. In Figure 2b there is an overlap of bands at 1459 and 1492 cm−1 related to PS-mod structure that hides the band at 1475 cm−1. The main distinction observed between the FTIR-ATR spectra (Figure 2) was due to the different pH conditions that the TMC/HP multilayers were assembled. For instance, the band at 1735 cm−1 (Figure 2a) was assigned to carboxylic acids groups that are partially ionized in structure of HP when in acidic medium (see Figure 4). However, this band was not observed in FTIR-ATR spectrum of the Figure 2b due to full ionization of carboxylic groups at pH 7.4. AFM Analysis. The Figure 3 shows the AFM images of the TMC20/HP multilayer films obtained at pH 3.0 (Figure 3A) and multilayers of TMC80/HP formed at pHs 3.0 (Figure 3B) and 7.4 (Figure 3C). The decrease of the undulations and projections and the minimization of the depressions on the surface of the multilayer films are favored by increasing the number of bilayers. The topography of the films in Figure 3A−C presented significant differences that are dependent on pH and structural heterogeneity of TMC. After analysis of Figure 3 it can be inferred that the TMC20/HP system exhibits higher compatibility with the surface of PS-mod related to the

TMC80/HP system, owing to the high homogeneity of the TMC20/HP multilayer films obtained at pH 3.0 (compare Figure 3A,B). This fact is explained based on the greater hydrophilicity of TMC20 compared TMC80. These statements are clarified after analysis of the scheme presented in Figure 4. The reduction reaction of TMC is not fully controlled; therefore, the methylation process does not occur just on −NH2 groups, but also on −OH sites of CHT.1,6,11 The greater number of steps employed in the reaction synthesis of TMC favors the increase of DQ.1,6,11 The TMC20 was obtained using

Figure 4. Predominant structures of TMC80, TMC20, and HP. F

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one-step methodology and presented MV of 26 × 103 g mol−1, while the TMC80 was prepared using two-step methodology and presented MV equal to 13 × 103 g mol−1. Therefore, the methodology utilized for TMC synthesis directly influences the average molecular weight of the final product.1,6,11 The excess of sodium hydroxide employed in the TMC80 synthesis considerably decreases the average molecular weight of CHTderivative (TMC80) to 13 × 103 g mol−1. Moreover, the lowest concentration of hydroxide ions employed in a one-step synthesis (TMC20) related to the two-step synthesis (TMC80) decreased the average molecular weight of the CHT derivative (TMC20) to 26 × 10−3 g mol−1. The elevated concentration of hydroxide ions allows the cleavage of the glycosidic bonds and decreases the average molecular weight of TMC as compared to CHT.6 According to Verheul et al.,29 the alkylation reaction conditions of primary amines of CHT by reaction of this polymer in strong alkaline conditions with excess of iodomethane and using NMP as solvent provides the polymer chain scission and, importantly, the partial and uncontrolled methylation of the C3 and C6 hydroxyl groups of the CHT chains (Figure 4). Thus, due to high DQ, the TMC80 has mostly Ntrimethylated and O-methylated sites in its saccharide structure (Figure 4a,b). The high density of O-methyl groups contributes to the increased hydrophobicity of the TMC80 as compared to TMC20.1,6,11 This characteristic significantly reduces the association among the TMC80 and HP (biopolymer of high hydrophilicity).1,11 Therefore, it is still visible on images of Figure 3 of the existence of depressions and projections in the TMC80/HP films with 12 and 18 bilayers (Figure 3B (c,d) and 3C (c,d)), a fact not observed in TMC20/HP with a respective number of bilayers (Figure 3A (c,d)). The TMC20 has greater hydrophilicity when compared to the TMC80 mainly due to the low proportion of O-methylated sites in its structure (Figure 4c). Therefore, the high density of methyl groups at C3−OCH3 and C6−OCH3 was responsible for the higher hydrophobicity of TMC80 compared to TMC20. Contact angle measurements confirmed the higher hydrophilicity of the TMC20/HP surface related to TMC80/HP system. The surfaces of TMC20/HP (pH 3.0), TMC80/HP (pH 3.0), and TMC80/HP (pH 7.4) multilayer films presented contact angle of 25° ± 2.2, 45° ± 3.2, and 51° ± 2.3, respectively. Another factor that favors the association between the HP and TMC20 is the existence of elevated proportion of amino groups, N-monomethylated and N-dimethylated sites on the TMC20 structure, when associated with the TMC80 (Figure 4a−c). The lower hydrophobic capability and steric effects of the amino groups, N-methyl and N-dimethyl sites as compared to the N-trimethyl groups provided greater association of the TMC20 with the HP at pH 3.0. The higher density of −NH3+, −NHCH3+, and −N(CH3)2+ groups on TMC20 at pH 3.0 increased its capability for complexing with the HP, as compared to higher amount of N-trimethylated sites on TMC80.1,11 Therefore, the strongest association among the TMC20 and HP molecules contributed to multilayer films obtained with more homogeneous surfaces (Figure 3A), while the multilayer films of TMC80/HP showed a surface with bigger heterogeneity (Figure 3B,C). Three important observations can be made after analysis of AFM images from the surface of TMC/HP films with 18 bilayers (Figures 3A (d), 3B (d), and 3C (d)). The multilayers of TMC20/HP presented smooth surfaces (Figure 3A (d)), while the multilayers of TMC80/HP, prepared in acidic

condition, presented a surface with depressions and projections (Figure 3B (d)). However, what drew most of our attention was the existence of small pores on the surface of the multilayers of TMC80/HP, prepared in alkaline conditions (Figure 3C (d)). The topographical differences of the multilayer films of TMC80/HP with 18 bilayers were related to the ionization process of the −COO− and -OSO3− groups existing in the structure of HP (Figure 4). This fact is strengthened because the charge density of TMC80 is almost independent of pH due to its high DQ (Figure 4a). It is worth mentioning that at alkaline medium (pH 7.4) the HP is fully ionized (Figure 4d).1,11 Nevertheless, the presence of small pores can be related to the low molecular weight of TMC80 as compared to TMC20. Polymers with high molecular weight show a stronger adsorption than low molecular weight polymers. The adsorption of these polymeric materials on the solid surface is originated from the entropic gain due to displacement of solvent molecules from the surface into the solution: each molecule of solvent gains mobility as it leaves the surface, while the polymer chain loses only a little mobility, compared to solvent, as it is absorbed at the surface. The difference in gainlose mobility is higher as the molecular weight of polymer is increased. It is worth emphasizing that the number of molecules of solvent leaving the surface is higher than the number of polymer segments arriving on the surface during the adsorption process. In the adsorption of polymers with higher molecular weight, the configuration at equilibrium will produce a layer with an approximately 3−30 nm thickness,30 and the adsorption is almost monomolecular and the process is irreversible because there will always be a polymer segment adsorbed at the surface. For fractions with lower molecular weight, the process would be more reversible because the existence of a few points of contact between the single polymer chain and the surface.30 SEM Analysis. The multilayer films of TMC/HP were also characterized by SEM (Figure 5A−C). So, it can be checked whether the AFM and SEM techniques are in agreement with each other regarding the topographical features of the TMC/ HP multilayer films. Analyzing Figure 5, it was observed that the TMC20 is more compatible with the HP compared to TMC80. The complete overlapping of the oxidized regions on PS-mod film occurs after nine bilayers of TMC20/HP have been alternately absorbed (Figure 5A (a)). The same behavior was observed in TMC80/ HP system only after 12 bilayers have been alternately absorbed, in acid medium (Figure 5B (b)). However, for the TMC80/HP multilayers prepared at pH 7.4, the complete overlapping of erosion points on PS-mod film occurs only after 18 bilayers (Figure 5C (c)) have been adsorbed. This indicates that in alkaline conditions the TMC80 possesses lower hydrophilicity and lesser capability for associating with the HP. The occurrence of nanopores on the surface of TMC80/ HP multilayers was observed by SEM analyses and the micrographs are shown in Figure 5C (c). This matches the data provided by AFM analysis (Figure 3C (d)). The size of the nanopores was about 170 nm. All the results related to morphology discussed above were observed through both techniques, AFM and SEM, and this confirms the dependence of topographical TMC/HP multilayers to the pH of immersing solutions and to the DQ of TMC. KFM Analysis. Kelvin force microscopy (KFM) is frequently used to investigate electric properties of surfaces and to map G

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the distribution of electric potential at the surface of a given material. The topography and surface potential images of PSmod and TMC/HP self-assemblies with 18 bilayers are presented in Figure 6. The deposition of the polymers (18 bilayers of TMC and HP) on the PS-mod surface is responsible for changing the negative surface potential from −4.02 (PSmod) to −0.015 (TMC20/HP), −1.075 (TMC80/HP, pH 3.0) and −0.030 V (TMC80/HP, pH 7.4), as observed in Figure 6. It should be emphasized that the KFM technique takes into account a small region of the surface and, due to the heterogeneous morphology of the TMC/HP self-assemblies, the value of electric potential may present discrepancies in relation to another region of the same film. The potential differences between the samples are related to the chemical structure of polymers (TMC80 and TMC20) and the possible interactions that exist in the TMC/HP multilayers. The potential surface of TMC80/HP assembled at pH 3.0 was more negative, as compared to the potential surfaces of the others samples. However, it is worth highlighting that deposition of TMC and HP on the PS-mod considerably increases the surface electrical potential related to PS-mod. On the other hand, this property did not change significantly when compared to the different TMC/HP multilayer films obtained at varied conditions (Figure 6). Additionally, the surface topography of the samples also influenced the surface electric potential, because the different values of surface electrical

Figure 5. (A) SEM images of the TMC20/HP multilayer films prepared at pH 3.0 with: 9 (a), 12 (b) and 18 (c) bilayers. (B) SEM images of the TMC80/HP multilayer films assembled at pH 3.0 with: 9 (a), 12 (b) and 18 (c) bilayers. (C) SEM images of the TMC80/HP multilayer films prepared at pH 7.4 with: 9 (a), 12 (b) and 18 (c) bilayers.

Figure 6. KFM images of PS-mod (a) and thin films of 18 bilayers: (b) TMC20/HP, (c) TMC80/HP (pH 3.0), and (d) TMC80/HP (pH 7.4). H

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potential were related to the intrinsic characteristic of each system. The TMC/HP multilayer (with 18 bilayers) presented smooth topography related to the other samples. Thus, increasing the number of bilayers also increased the electrical potential at surface of the films, because an increase of homogeneity on the TMC/HP surfaces was observed. In general, the surface electrical potential presented for samples of 3, 9, and 12 bilayers was more negative as compared to the electrical potential found at the surface of TMC/HP multilayer films with 18 bilayers. Initial Adhesion of E. coli on PS-mod and TMC-HP Multilayer Surfaces. The microbial adhesion to different substrates is influenced by chemical and physicochemical properties of the substrate, including the degree of hydrophobicity, one of the most important factors. E. coli (Gramnegative) possesses a thin layer of glycoprotein covered by a thick layer formed mainly by lipoproteins and lipids.14,31 Thus, the hydrophobic character at the surface should favor the interaction of E. coli with the lipid bilayers. Figure 7 shows the SEM images of PS-mod (used as control) and TMC/HP multilayer films (18 bilayers), after being

Figure 8. Normaski interference contrast microscopy (400 x) of: (a) PS-mod, (b) (TMC20/HP)18 multilayer film assembled at pH = 3.0, (c) (TMC80/HP)18 multilayer film assembled at pH = 3.0, (d) (TMC80/HP)18 multilayer film assembled at pH = 7.4 after exposure to 5 × 107 cells mL−1 E. coli for 4 h.

In Vitro Bactericidal Test. The comparison of antibacterial activity of different TMC/HP multilayer systems prepared in this work can be made by analysis of Figure 9. It appears that

Figure 7. Scanning electron micrographs of (a) PS-mod, (b) (TMC20/HP)18 multilayer film assembled at pH = 3.0, (c) (TMC80/HP)18 multilayer film assembled at pH = 3.0, and (d) (TMC80/HP)18 multilayer film assembled at pH = 7.4 after exposure to 5 × 107 cells mL−1 E. coli for 4 h. Figure 9. Changing of the viability of E. coli cells with exposure time (6 h) for the TMC20/HP (at pH 3.0), TMC80/HP (at pH 7.4), and TMC80/HP (at pH 3.0) films.

submitted to contact with E. coli. The presence of the E. coli was only detected in the control group. This fact can be explained by the low hydrophilicity of PS-mod surface as compared to TMC/HP multilayer surfaces. The contact angle decreases significantly after the deposition of TMC/HP bilayers on the PS-mod surface. So, the TMC/HP multilayers presented higher hydrophilicity as compared to the PS-mod surface. Therefore, the excellent antiadhesive properties presented by the TMC/ HP multilayer films were attributed to the increase of the wettability surfaces of these films. It is worthy to mention that the last layer of films on TMC/HP is formed by HP (hydrophilic and antiadhesive biopolymer). These results suggest that the TMC/HP surfaces using different DQ of TMC and pH of immersion are potential candidates to surface coatings for anti-infection purposes. The observation by Normaski interference contrast microscopy (Figure 8) shows the same results observed in the SEM analyses (Figure 7).

the TMC80 film in acid medium showed no significant influence on antibacterial activity as compared to control (PSmod). On the other hand, the TMC20/HP (prepared at pH 3.0) and TMC80/HP (prepared at pH 7.4) presented a bactericidal effect of about 32.7 and 64.6% inhibition, respectively. By comparison of TMC20/HP to the TMC80/ HP multilayers, being both prepared at pH 3.0, more expressive bactericidal effect was observed on TMC20/HP and it was due to structural differences on the TMC, as discussed earlier and illustrated in Figure 4. The antimicrobial activity of TMC/alginate complexes is strongly affected by pH-forming of the new biomaterials.32 So, the pH of preparation of the thin films explains the differences of bactericidal activity for the systems showed in Figure 9. Compared to TMC20 ones, the chains of TMC80 are more I

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flexible and easier to interact with the E. coli cell envelope. This fact is related to the high DQ of TMC 80. Therefore, according to data from Figure 9, the TMC80/HP system obtained at pH 7.4 was more efficient as bactericide than the TMC20/HP system, because DQ of TMC20 is lower than that of TMC80. However, the bactericidal activity depends on the pH-forming of the systems TMC/HP self-assemblies (pH-condition of TMC/HP complexes formation). The TMC80 has elevated DQ and behaves as a strong acidic salt, because the −N(CH3)3+Cl− groups of TMC80 could ionize as the following equations33 −N(CH3)3+ Cl− ↔ −N(CH3)3+ + Cl−

(1)

−N(CH3)3+ + H 2O ↔ −N(CH3)3 OH + H+

(2)

the electric potential of PS-mod surface were more bactericidal. According to Sadeghi et al.,34 CHT molecules with the least positive surface charge showed the least bacterial inhibition; conversely, the C2−C6 trimethylated 6-amino-6-deoxy chitosan, with the highest zeta potential, showed the highest antibacterial activity. Materials with high zeta potential, probably have the highest ability to bind to the negative peptidoglycans on the bacterial cell wall and may induce severe morphological alterations in the bacteria envelope.34 The bactericidal tests also were performed for the TMC/HP system in which the outer layer is TMC20 or TMC80 (results not shown). It was found that there is a small increase in the bactericidal action of these films (ca. 4.6%) as compared to films having HP as the outer layer. Fu et al.,14 obtained multilayer films of chitosan/heparin (CHT/HP) at different pH conditions (2.9, 3.8, and 6.0) and studied the antibacterial activity of these films against E. coli at pH 7.4. The pH-assembly of the CHT/HP thin films has a remarkable effect on the antibacterial property of the multilayers. The in vitro antibacterial assay indicated that the CHT/HP multilayer could effectively kill the bacteria. However, the samples assembled at pH 2.9, 3.8, and 6.0 presented bactericidal inhibitions of 58, 68, and 46%, respectively. Therefore, the CHT/HP thin films were more bactericidal when these were assembled in acid medium (pH < 4). On the other hand, the TMC80/HP multilayer prepared in this work at pH 7.4 showed 64.6% of bactericidal inhibition at physiological pH (7.4). This result confirms that the TMC80/ HP system, assembled at pH 7.4, has good potential for surface modification of cardiovascular devices due to excellent bactericidal activity of this system at pH-assembling of the respective multilayer film. In Vitro Heparin Release. The released curve of HP from TMC/HP multilayers (18 bilayers) in SIF is presented in Figure 10a. Considering the TMC20/HP multilayer and the

On the other hand, due to the low DQ, the ionization process of TMC20 depends mainly on the −NH2, −NHCH3, and −N(CH3)2 groups and could be performed as the following equations32 −NH 2 + H+ ↔ −NH3+

(3)

−NHCH3 + H+ ↔ −NH 2CH3+

(4)

−N(CH3)2 + H+ ↔ −NH(CH3)2+

(5)

The lower pH-forming of the thin films could benefit the protonation of −NH2, −NHCH3, and −N(CH3)2 groups,32,33 but repress the ionization of −N(CH3)3+Cl− sites.33 According to Xu et al.,33 the −N(CH3)3+Cl− groups could not interact with the negatively charged sites on cell envelope and TMC with DQ = 95% present strong bactericidal activity at pH 7.2. So, a thin film of TMC80/HP prepared at pH 3.0 showed low antibacterial activities, because the −N(CH3)3+Cl− sites are not initially ionized. Therefore, at pH 3.0, the chains of TMC80 are not flexible enough to interact with the cell envelope. On the other hand, the TMC80/HP system obtained at pH 7.4 showed high bactericidal action, because at this pH-condition the −N(CH3)3+Cl− sites are initially ionized, providing enough mobility the chains of TMC80. This fact allows interaction with the cellular envelope of bacteria and increases the bactericidal inhibition to 64.6% (Figure 9). Thus, the inhibition of 32.7% observed to the TMC20/HP system is mainly due to the ionization process of the −NH2, NHCH3, and N(CH3)2 sites, as represented in eqs 3−5. In general, the interaction between positively charged −NH3+, NH2CH3+, NH(CH3)2+, and −N(CH3)3+ groups and negatively charged microbial cell membranes (E. coli) leads to the leakage of proteinaceous and other intracellular constituents.32 The bactericidal tests were conducted at physiologic pH (7.4); therefore, the pH-obtaining of the films was the responsible factor by results shown in Figure 9. Besides the factors already mentioned, others may have also influenced the obtained results, such as that related to the parent CHT (the source, the molecular weight, and the degree of deacetylation) used in the synthesis of TMC, the methodology used for TMC synthesis (proportion of Nmonomethylated, N-dimethylated, and N-trimethylated sites and ion exchange of TMC), and the assay conditions such as culture temperature and ion strength.32,33 Additionally, the different bactericidal effects observed in Figure 9 are associated with the electric potential at surface of the films, reported in section of KFM analysis. The films that presented a higher increase in the surface electric potential (TMC80/HP, pH 7.4, and TMC20/HP, pH 3.0) in relation to

Figure 10. Released curves of HP from TMC/HP films in SIF (a) and in SGF (b).

TMC80/HP multilayer assembled at pH 3.0, it was verified that the maximum concentration of HP released in SIF was about 30 and 20 mg mL−1, respectively, and it was achieved after 1 h in both cases (Figure 10a). This result shows that TMC20/HP and TMC80/HP (formed at pH 3.0) films present great potential to be used as devices for sustained release of HP at neutral pH, the almost same condition found in the intestinal region. On the other hand, the TMC80/HP sample prepared at pH 7.4 does not release HP in SIF (Figure 10a). The profile of HP released in SGF is presented in Figure 10b. The thin films presented stability in SGF, and the extension of HP releasing was not as high as observed in SIF (Figure 10b). J

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The release of HP is strongly pronounced in SIF medium due to ionization of the −NH2, −NHCH3, and −N(CH3)2 groups. At the conditions that such TMC20/HP and TMC80/ HP multilayers were obtained, these sites were initially positively charged and effectively interacted with the HP molecules. After being placed in contact with the SIF (pH 6.8), the sites (−NH2, −NHCH 3, and −N(CH 3)2 ) became deprotonated and part of HP complexed with TMC molecules was released into the middle. The TMC20 has a higher content of −NH2, NHCH3, and −N(CH3)2 groups in its structure compared to TMC80, so that the concentration of HP released from the TMC20/HP multilayers was superior to TMC80/HP (Figure 10a). On the other hand, the TMC80/HP multilayers obtained at pH 7.4 are extremely stable. At this pH condition (7.4) the complexation of TMC/HP occurs exclusively through −N(CH3)3+ ionized sites. The complexation with these groups was permanent and represses the release of HP (Figure 10). When the HP release test was evaluated in SGF (pH 1.2), the −NH 2 , −NHCH 3 , and −N(CH 3 ) 2 sites of TMC are protonated and strongly interact with HP through electrostatic interactions. This fact substantially reduced the amount of HP released in SGF (Figure 10b). The greater hydrophobicity of TMC80 compared to TMC20 can minimize the spread of HP in the whole TMC80 surface. But HP strongly interacts with TMC80, even in few points at surface. Thus, due to that, a low amount of HP is released compared to the TMC20/HP system. The amount of HP released in SIF reaches the equilibrium after about 1 h, and the antiadhesive and bactericidal assays were investigated at pH 7.4 during 4 h. This fact, associated with the less effective complexation among HP and TMC80 chains, at pH 7.4, compared to the TMC20/HP system should also be related to the strong antiadhesive and bactericidal effect observed in this work.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank CNPq/CAPES, Brazil, for the doctorate fellowship (H.D.M.F.) and for the financial support (Proc. 481424/2010-5 and 309005/2009-4).

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REFERENCES

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CONCLUSIONS TMC were successfully synthesized and their DQ (20 and 80 mol %) were determined by 1H NMR spectroscopy. It was possible to obtain multilayers of TMC/HP through the LbL technique, at different pH (3.0 and 7.4), based on positive charges along the TMC chains and negative charges in HP chains. The alternated adsorption of TMC and HP forming multilayers on the PS-mod surface was confirmed through FTIR-ATR, AFM, and SEM techniques, and the electrical potential surface was measured by KFM. Decreasing in roughness and depressions minimizing on the multilayer surface as the number of TMC/HP bilayers increase were observed by AFM and SEM images. The TMC/HP multilayers reduced the bacterial adhesion and the TMC80/HP system assembled at pH 7.4 showed effective bactericidal activity, which makes the TMC/HP system suitable for use as coatings for anti-infection purposes.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting figures included (1) 1H NMR spectra of the chitosan and its derivative with different degrees of quaternization (TMC20 and TMC80) and (2) EDS analysis confirming the chemical oxidation of the polystyrene. This material is available free of charge via the Internet at http:// pubs.acs.org. K

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(28) Botan, R.; Nogueira, T. R.; Lona, L. M. F.; Wypych, F. Polymer 2011, 21, 34−38. (29) Verheul, J. R.; Amidi, M.; Van der Wal, S.; Van Riet, E.; Jiskoot, W.; Hennink, W. E. Biomaterials 2008, 29, 3642−3649. (30) Adamsom, A. W.; Gast, A. P. Physical Chemistry of Surfaces, 6th ed.; Wiley: New York, 1997, 390−419. (31) Kenawy, El-R.; Worley, S. D.; Broughton, R. Biomacromolecules 2007, 8, 1359−1384. (32) Martins, A. F.; Follmann, H. D. M.; Rodrigues, F. H. A.; Gerola, A. P.; Kimura, E.; Rubira, A. F.; Muniz, E. C. Carbohydr. Polym. 2012, submitted for publication. (33) Xu, T.; Xin, M.; Li, M.; Huang, H.; Zhou, S. Carbohydr. Polym. 2010, 81, 931−936. (34) Sadeghi, A. M. M.; Amini, M.; Avadi, M. R.; Siedi, F.; RafieeTehrani, M.; Junginger, H. E. J. Bioact. Compat. Polym. 2008, 23, 262− 275.

L

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