Role of molecular properties of ulvans on their ability to elaborate antiadhesive surfaces Virginie Gadenne,1,2 Laurent Lebrun,1,2 Thierry Jouenne,1,2 Pascal Thebault1,2 1 2
CNRS, UMR 6270, Polyme`res, Biopolyme`res, Surfaces Laboratory, F-76821 Mont-Saint-Aignan, France Normandie Univ, UR, France
Received 18 March 2014; revised 19 May 2014; accepted 30 May 2014 Published online 12 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35245 Abstract: Antiadhesive properties of polysaccharides (such ulvans) once immobilized on a surface are described in the literature but the parameters governing their antifouling properties are not yet well identified. In the present study, the relationship between molecular parameters of ulvans and the inhibition of bacterial adhesion was investigated. To this aim, various ulvans were grafted on silicon wafers under two different experimental immobilization conditions. Results
showed that the experimental immobilization conditions and the polysaccharides molecular weight led to specific layer conformations which exhibited a key role in the surface antiC 2014 Wiley Periodicals, Inc. J Biomed Mater adhesive properties. V Res Part A: 103A: 1021–1028, 2015.
Key Words: biofilms, adhesion, polysaccharides, Staphylococcus aureus
How to cite this article: Gadenne V, Lebrun L, Jouenne T, Thebault P. 2015. Role of molecular properties of ulvans on their ability to elaborate antiadhesive surfaces. J Biomed Mater Res Part A 2015:103A:1021–1028.
INTRODUCTION
Owing to their high resistance to antimicrobials,1 biofilms are a major cause of persistent infections such as those associated with implanted medical devices.2 Adhesion is a prerequisite for the formation of biofilms on surfaces. A lot of studies dealing with the relationship between the physicochemical surface properties of supports and bacterial adhesion have been reported in the literature, pointing out the role of nonspecific interactions such as electrostatic interactions.3,4 Moreover, it has been shown that initial bacterial attachment to materials and early biofilm formation are also subject to the surface roughness of the substratum.5,6 To limit these surface contaminations, different strategies are yet proposed, in particular the elaboration of antimicrobial (biocidal) surfaces and/or that of antiadhesive surfaces,7–10 via for example the covalent immobilization of bioactive molecules.11 It has been shown that the molecular weight and charge of the immobilized molecule, and the hydrophilicity, roughness, and thickness of the layer are key parameters for the bacterial repulsion (or attraction) process.12–18 Though hydrophilic surfaces generally lead to low adhesive surfaces as compared to hydrophobic surfaces,19 some contradictory results are reported due in particular to the hydrophilic feature of some bacterial surfaces.20 It has been also shown that negatively charged surfaces decrease bacterial adhesion by electrostatic repulsion.21,22 Thus, layers of hyaluronic acid (HA) or heparin reduced bacterial attachment thanks the presence of carboxylate and/or sulfo-
nate groups.23–25 However, Hu et al.26 showed that titanium surfaces modified by HA exhibited the same antiadhesive activity before and after addition of growth factor on carboxylic acid functions of HA. Concerning the roughness, the surface topography effect, due to the immobilization of molecules which leads to a nanometer order variation, is difficult to split to the roughness of the native material.18 In the case of modification by polymer brushes, it has been shown that film thickness (and molecular weight of polymers) has an impact on protein adsorption.27,28 While a lot of information is available on the parameters controlling the bacterial adhesion, few concern the immobilization of polysaccharides and the parameters governing their antifouling properties.23–25 Recently, we demonstrated the ability of immobilized ulvan (a natural sulfated polysaccharide) to inhibit the adhesion of Staphylococcus epidermidis and Pseudomonas aeruginosa on titanium surfaces.29 The objective of the present work was to explore the relationship between molecular parameters of the polysaccharide, for example, its molecular weight or sulfate rate, and its ability to inhibit the bacterial adhesion when immobilized on a surface. To this aim, ulvans were covalently grafted on silicon surfaces, previously modified by a selfassembled monolayer of 11-aminoundecyltrimethoxysilane (AUTMS). Two concentrations of activation reagents [Nhydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)N0 -ethyl-carbodiimide hydrochloride (EDC)] which transform carboxylate groups into ester functions were tested.30,31
Correspondence to: P. Thebault; e-mail: [email protected] Contract grant sponsor: Interreg IVA France (Channel)—England; contract grant number: 4119 GIMs
C 2014 WILEY PERIODICALS, INC. V
1021
The parameters controlling the bacterial repulsion, that is, surface wettability, roughness and chemical composition, were characterized by contact angle, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS), respectively. The antiadhesive properties of the modified surfaces were then evaluated against Staphylococcus aureus, one of the major pathogens involved in biofilm contaminations.32 EXPERIMENTAL
Chemicals materials All the solvents used (methanol, H2O2, and sulfuric acid), NHS and EDC were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Polysaccharides were obtained from the Centre d’Etude et de Valorization des Algues (CEVA, Pleubian, France). Three ulvans were used: the ulvan P1, extracted from ulva compressa, and ulvans P2 and P3 coming from ulva armoricana. P3 was recovered by chemical degradation to reduce its molecular weight whereas P2 was desulfated by acid hydrolysis in MeOH/HCl solution. AUTMS was purchased from Sikemia (Clapiers, France). All products were used as received without further purification. Surface preparation Silicon preparation method was adapted from the protocol which we used previously for titanium.29 The smooth silicon surface (rms < 4 Å) was chosen to limit substrate roughness effect on bacterial adhesion. Briefly, silicon wafers (Siltronix, France) were cleaned in a piranha solution (3:7 H2O2:H2SO4) for 30 min at 120 C, then extensively rinsed with Milli-Q water, and immediately immersed in a methanol solution with 1 mM of AUTMS for 24 h at room temperature to incorporate amino functions on silicon surface. Samples were rinsed and sonicated in methanol for removing all ungrafted molecules, and dried under a nitrogen flow. Simultaneously, polysaccharide solutions at 1 mg mL21 in phosphatebuffered saline (PBS) were prepared with a mixture of EDC/NHS at 0.03/0.015 and 0.1/0.05 M for normal and excess activation conditions, respectively. After 15 min of activation, the AUTMS-modified silicon samples were immersed in these solutions overnight, rinsed with deionized water in an ultrasonic bath and dried under a nitrogen flow. The coupling agents (EDC/NHS) allowing activation of carboxylic functions were used for anchoring polysaccharide on the surface by amide bond formation. The functionalized surfaces were called SiP1, SiP2, and SiP3 when a normal concentration of activation reagents was used, and SiP1ex, SiP2ex, and SiP3ex in the case of activation reagents excess. The use of coupling agents in excess concentrations allowed the conversion of the major amount of the carboxylic acid groups of the polysaccharides into succiinimide ester and to observe only sulfate negative charges effect on surfaces antiadhesive properties. Chemical and surface characterization Chemical characterizations of ulvans (i.e., molecular weight and chemical composition), except for infrared analyses, were performed by the CEVA.
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The molecular weights were determined by highperformance liquid chromatography analyses with a refractometer detector. The chromatographic peaks were identified by comparison with pullulan reference. The chemical composition of polysaccharides was evaluated by an internal CEVA method. Fourier transform infrared (FTIR) spectra were acquired using a Nicolet Avatar 360 spectrophotometer equipped with a DTGS detector. Spectra were recorded in ATR-mode at 4 cm21 resolution and 58 scans. Each spectrum was performed on polysaccharide powder. Surface hydrophobicity was measured by static water contact angle measurements (Multiskop from Optrel). Angle values were the average of four measurements (1 mL drops) on three different samples each. XPS measurements were carried out with a Thermo Electron K-Alpha Spectrometer using a monochromatic Al-Ka XRay source (1486.6 eV) with a spot size of 400 mm. The survey spectra were collected over a range of 210 to 1350 eV with pass energy of 200 eV. The high resolution spectra over the C 1s, O 1s, N 1s, Si 2p, regions were acquired with a pass energy of 50 eV. The binding energy was referenced by setting the maximum C 1s peak at 285.0 eV. The peaks deconvolution and the proportion of component composition from peak areas were performed using a CasaXPS software system. Surface morphology was visualized by AFM measurements in tapping mode (resonance frequency of 350 kHz) with a Nanoscope III controller from Veeco Instruments. Antibacterial and adhesion tests. S. aureus strain ATCC 29213 was used. The strain was maintained as glycerol stocks and stored at 280 C. Precultures were performed for 18 h in brain heart infusion (BHI) broth. Biocidal tests. The antimicrobial activity of ulvans was evaluated by MIC measurements through the microdilution method based on ISO standard 20776-1.33 Briefly, a solution of each ulvan at various concentrations, between 100 and 1 mg mL21 in a mixture of PBS/BHI (50/50; v/v), was inoculated by a bacterial suspension (106 colony forming units CFU mL21) of S. aureus. Positive (without polysaccharide) and negative (without bacteria) controls were included in each assay. After 24 h of incubation at 37 C, the bacterial growth was visualized by the broth turbidity for each solution. MIC is defined as the lowest concentration of ulvan that inhibits the visible bacterial growth after 24 h. Adhesion tests. After a preculture for 18h in BHI broth, bacteria were harvested by centrifugation (2683g for 15 min) and resuspended in PBS solution. Wafers were previously decontaminated in ethanol, rinsed with sterile distilled water, and dried under sterile atmosphere. They were then covered with 100 mL of a bacterial suspension (106 CFU mL21). After incubation for 4 h at room temperature, wafers were rinsed with sterile distilled water, immersed in 2mL of a sterile PBS solution and sonicated for 3 min to recover adherent bacteria as previously described.34–36 Decimal dilutions of the resulting bacterial suspension were spread on BHI agar plates. After incubation for 24 h at 37 C, the
MOLECULAR PROPERTIES OF ULVANS
ORIGINAL ARTICLE
TABLE I. Principal Physicochemical Features of the Three Studied Ulvans
Polysaccharides
Molecular Weight (g mol21)
Iduronic and Glucuronic Acida (%)
Sulfate Groups (%)
Nitrogen (%)
876,250 38,800 10,159
22.8 32.6 28.4
12.9 2.7 15.3
1.87 0.95 0.97
P1 P2 P3 a
Others sugars are mainly rhamnose.
number of colonies was enumerated and expressed as CFU cm22. Three independent enumerations were carried out for each assay. The inhibition values (%) are given compared with SiO2 surface. Colonization tests. After preculture for 18h in BHI broth, bacteria were harvested by centrifugation (2683 x g for 15 min) and resuspended in BHI medium. Bacterial suspension of 100 mL (106 CFU mL21) was deposited on each wafer (prepared as described previously). After incubation at 37 C for 24 h under agitation (150 rpm), samples were fixed in a 2% glutaraldehyde, 0.1 M cacodylate buffer (pH 7.4) for 30 min and rinsed (3 3 10 min) in 0.2 M cacodylate buffer (pH 7.4). Samples were then dehydrated by passing them through the following ethanol series: 30, 50, 80%, each for 10 min; 100% ethanol, 2 3 10 min. Support samples were then dried at 37 C for 24 h. The bacterial colonization of the surface was evaluated by scanning electronic microscopy (SEM, evo 40 eps, Zeiss). The rate of bacterial coverage was determined by the MatLab program and represented the average of 20 analyzed images. RESULTS
Chemical characterization The molecular weight and the principal chemical composition of each polysaccharide are given in Table I. Data have been supplied by the CEVA.
FIGURE 1. ATR-FTIR spectrum of each polysaccharide powder. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MAR 2015 VOL 103A, ISSUE 3
The content of iduronic and glucuronic acid was similar for polysaccharides P2 and P3 with, respectively, 32.6 and 28.4%. P1 was characterized by the lowest acid content (22.8%) and the highest molecular weight (876,250 g mol21), whereas P2 exhibited the lowest percentage of sulfated groups (2.7%). A low nitrogen content was observed for the three polysaccharides. A complementary characterization of polysaccharides by ATR-IR spectroscopy was performed to confirm the desulfation of polysaccharide P2. Results are shown in Figure 1. Whatever the ulvan, a strong band at about 1050 cm21, attributed to C–O stretching from the sugar ring, and two bands at 1630 and 1420 cm21 were observed due to asymmetrical and symmetric stretching band of carboxylate groups, respectively.37 IR spectra of polysaccharides P1 and P3 exhibited an intense band at 1230 cm21 and two weak bands at 790 and 850 cm21, corresponding to sulfate groups.38 On P2 spectrum, two bands disappeared at lowest frequency and the intensity of the band at 1230 cm21 decreased.
Surface characterization The surface topographies were investigated by AFM. The roughness values, expressed by a root mean square (rms), are recapitulated in Table II. In normal condition, rms value increased with the decrease in the polysaccharides molecular weight, that is, from 0.816 to 1.19 nm for SiP1 and SiP3, respectively. For surfaces elaborated in excess of coupling agents, rms values were very close, no molecular weight dependence being observed. Furthermore, the increase in the activation reagents concentration induced a slight decrease in rms values whatever the surface. The surface hydrophobicity was evaluated after polysaccharides immobilization by water contact angle measurements. Results are listed in Table II. After surface modification, a decrease in contact angle was observed (from 80 for AUTMS layer to
CNRS, UMR 6270, Polyme`res, Biopolyme`res, Surfaces Laboratory, F-76821 Mont-Saint-Aignan, France Normandie Univ, UR, France
Received 18 March 2014; revised 19 May 2014; accepted 30 May 2014 Published online 12 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35245 Abstract: Antiadhesive properties of polysaccharides (such ulvans) once immobilized on a surface are described in the literature but the parameters governing their antifouling properties are not yet well identified. In the present study, the relationship between molecular parameters of ulvans and the inhibition of bacterial adhesion was investigated. To this aim, various ulvans were grafted on silicon wafers under two different experimental immobilization conditions. Results
showed that the experimental immobilization conditions and the polysaccharides molecular weight led to specific layer conformations which exhibited a key role in the surface antiC 2014 Wiley Periodicals, Inc. J Biomed Mater adhesive properties. V Res Part A: 103A: 1021–1028, 2015.
Key Words: biofilms, adhesion, polysaccharides, Staphylococcus aureus
How to cite this article: Gadenne V, Lebrun L, Jouenne T, Thebault P. 2015. Role of molecular properties of ulvans on their ability to elaborate antiadhesive surfaces. J Biomed Mater Res Part A 2015:103A:1021–1028.
INTRODUCTION
Owing to their high resistance to antimicrobials,1 biofilms are a major cause of persistent infections such as those associated with implanted medical devices.2 Adhesion is a prerequisite for the formation of biofilms on surfaces. A lot of studies dealing with the relationship between the physicochemical surface properties of supports and bacterial adhesion have been reported in the literature, pointing out the role of nonspecific interactions such as electrostatic interactions.3,4 Moreover, it has been shown that initial bacterial attachment to materials and early biofilm formation are also subject to the surface roughness of the substratum.5,6 To limit these surface contaminations, different strategies are yet proposed, in particular the elaboration of antimicrobial (biocidal) surfaces and/or that of antiadhesive surfaces,7–10 via for example the covalent immobilization of bioactive molecules.11 It has been shown that the molecular weight and charge of the immobilized molecule, and the hydrophilicity, roughness, and thickness of the layer are key parameters for the bacterial repulsion (or attraction) process.12–18 Though hydrophilic surfaces generally lead to low adhesive surfaces as compared to hydrophobic surfaces,19 some contradictory results are reported due in particular to the hydrophilic feature of some bacterial surfaces.20 It has been also shown that negatively charged surfaces decrease bacterial adhesion by electrostatic repulsion.21,22 Thus, layers of hyaluronic acid (HA) or heparin reduced bacterial attachment thanks the presence of carboxylate and/or sulfo-
nate groups.23–25 However, Hu et al.26 showed that titanium surfaces modified by HA exhibited the same antiadhesive activity before and after addition of growth factor on carboxylic acid functions of HA. Concerning the roughness, the surface topography effect, due to the immobilization of molecules which leads to a nanometer order variation, is difficult to split to the roughness of the native material.18 In the case of modification by polymer brushes, it has been shown that film thickness (and molecular weight of polymers) has an impact on protein adsorption.27,28 While a lot of information is available on the parameters controlling the bacterial adhesion, few concern the immobilization of polysaccharides and the parameters governing their antifouling properties.23–25 Recently, we demonstrated the ability of immobilized ulvan (a natural sulfated polysaccharide) to inhibit the adhesion of Staphylococcus epidermidis and Pseudomonas aeruginosa on titanium surfaces.29 The objective of the present work was to explore the relationship between molecular parameters of the polysaccharide, for example, its molecular weight or sulfate rate, and its ability to inhibit the bacterial adhesion when immobilized on a surface. To this aim, ulvans were covalently grafted on silicon surfaces, previously modified by a selfassembled monolayer of 11-aminoundecyltrimethoxysilane (AUTMS). Two concentrations of activation reagents [Nhydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)N0 -ethyl-carbodiimide hydrochloride (EDC)] which transform carboxylate groups into ester functions were tested.30,31
Correspondence to: P. Thebault; e-mail: [email protected] Contract grant sponsor: Interreg IVA France (Channel)—England; contract grant number: 4119 GIMs
C 2014 WILEY PERIODICALS, INC. V
1021
The parameters controlling the bacterial repulsion, that is, surface wettability, roughness and chemical composition, were characterized by contact angle, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS), respectively. The antiadhesive properties of the modified surfaces were then evaluated against Staphylococcus aureus, one of the major pathogens involved in biofilm contaminations.32 EXPERIMENTAL
Chemicals materials All the solvents used (methanol, H2O2, and sulfuric acid), NHS and EDC were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Polysaccharides were obtained from the Centre d’Etude et de Valorization des Algues (CEVA, Pleubian, France). Three ulvans were used: the ulvan P1, extracted from ulva compressa, and ulvans P2 and P3 coming from ulva armoricana. P3 was recovered by chemical degradation to reduce its molecular weight whereas P2 was desulfated by acid hydrolysis in MeOH/HCl solution. AUTMS was purchased from Sikemia (Clapiers, France). All products were used as received without further purification. Surface preparation Silicon preparation method was adapted from the protocol which we used previously for titanium.29 The smooth silicon surface (rms < 4 Å) was chosen to limit substrate roughness effect on bacterial adhesion. Briefly, silicon wafers (Siltronix, France) were cleaned in a piranha solution (3:7 H2O2:H2SO4) for 30 min at 120 C, then extensively rinsed with Milli-Q water, and immediately immersed in a methanol solution with 1 mM of AUTMS for 24 h at room temperature to incorporate amino functions on silicon surface. Samples were rinsed and sonicated in methanol for removing all ungrafted molecules, and dried under a nitrogen flow. Simultaneously, polysaccharide solutions at 1 mg mL21 in phosphatebuffered saline (PBS) were prepared with a mixture of EDC/NHS at 0.03/0.015 and 0.1/0.05 M for normal and excess activation conditions, respectively. After 15 min of activation, the AUTMS-modified silicon samples were immersed in these solutions overnight, rinsed with deionized water in an ultrasonic bath and dried under a nitrogen flow. The coupling agents (EDC/NHS) allowing activation of carboxylic functions were used for anchoring polysaccharide on the surface by amide bond formation. The functionalized surfaces were called SiP1, SiP2, and SiP3 when a normal concentration of activation reagents was used, and SiP1ex, SiP2ex, and SiP3ex in the case of activation reagents excess. The use of coupling agents in excess concentrations allowed the conversion of the major amount of the carboxylic acid groups of the polysaccharides into succiinimide ester and to observe only sulfate negative charges effect on surfaces antiadhesive properties. Chemical and surface characterization Chemical characterizations of ulvans (i.e., molecular weight and chemical composition), except for infrared analyses, were performed by the CEVA.
1022
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The molecular weights were determined by highperformance liquid chromatography analyses with a refractometer detector. The chromatographic peaks were identified by comparison with pullulan reference. The chemical composition of polysaccharides was evaluated by an internal CEVA method. Fourier transform infrared (FTIR) spectra were acquired using a Nicolet Avatar 360 spectrophotometer equipped with a DTGS detector. Spectra were recorded in ATR-mode at 4 cm21 resolution and 58 scans. Each spectrum was performed on polysaccharide powder. Surface hydrophobicity was measured by static water contact angle measurements (Multiskop from Optrel). Angle values were the average of four measurements (1 mL drops) on three different samples each. XPS measurements were carried out with a Thermo Electron K-Alpha Spectrometer using a monochromatic Al-Ka XRay source (1486.6 eV) with a spot size of 400 mm. The survey spectra were collected over a range of 210 to 1350 eV with pass energy of 200 eV. The high resolution spectra over the C 1s, O 1s, N 1s, Si 2p, regions were acquired with a pass energy of 50 eV. The binding energy was referenced by setting the maximum C 1s peak at 285.0 eV. The peaks deconvolution and the proportion of component composition from peak areas were performed using a CasaXPS software system. Surface morphology was visualized by AFM measurements in tapping mode (resonance frequency of 350 kHz) with a Nanoscope III controller from Veeco Instruments. Antibacterial and adhesion tests. S. aureus strain ATCC 29213 was used. The strain was maintained as glycerol stocks and stored at 280 C. Precultures were performed for 18 h in brain heart infusion (BHI) broth. Biocidal tests. The antimicrobial activity of ulvans was evaluated by MIC measurements through the microdilution method based on ISO standard 20776-1.33 Briefly, a solution of each ulvan at various concentrations, between 100 and 1 mg mL21 in a mixture of PBS/BHI (50/50; v/v), was inoculated by a bacterial suspension (106 colony forming units CFU mL21) of S. aureus. Positive (without polysaccharide) and negative (without bacteria) controls were included in each assay. After 24 h of incubation at 37 C, the bacterial growth was visualized by the broth turbidity for each solution. MIC is defined as the lowest concentration of ulvan that inhibits the visible bacterial growth after 24 h. Adhesion tests. After a preculture for 18h in BHI broth, bacteria were harvested by centrifugation (2683g for 15 min) and resuspended in PBS solution. Wafers were previously decontaminated in ethanol, rinsed with sterile distilled water, and dried under sterile atmosphere. They were then covered with 100 mL of a bacterial suspension (106 CFU mL21). After incubation for 4 h at room temperature, wafers were rinsed with sterile distilled water, immersed in 2mL of a sterile PBS solution and sonicated for 3 min to recover adherent bacteria as previously described.34–36 Decimal dilutions of the resulting bacterial suspension were spread on BHI agar plates. After incubation for 24 h at 37 C, the
MOLECULAR PROPERTIES OF ULVANS
ORIGINAL ARTICLE
TABLE I. Principal Physicochemical Features of the Three Studied Ulvans
Polysaccharides
Molecular Weight (g mol21)
Iduronic and Glucuronic Acida (%)
Sulfate Groups (%)
Nitrogen (%)
876,250 38,800 10,159
22.8 32.6 28.4
12.9 2.7 15.3
1.87 0.95 0.97
P1 P2 P3 a
Others sugars are mainly rhamnose.
number of colonies was enumerated and expressed as CFU cm22. Three independent enumerations were carried out for each assay. The inhibition values (%) are given compared with SiO2 surface. Colonization tests. After preculture for 18h in BHI broth, bacteria were harvested by centrifugation (2683 x g for 15 min) and resuspended in BHI medium. Bacterial suspension of 100 mL (106 CFU mL21) was deposited on each wafer (prepared as described previously). After incubation at 37 C for 24 h under agitation (150 rpm), samples were fixed in a 2% glutaraldehyde, 0.1 M cacodylate buffer (pH 7.4) for 30 min and rinsed (3 3 10 min) in 0.2 M cacodylate buffer (pH 7.4). Samples were then dehydrated by passing them through the following ethanol series: 30, 50, 80%, each for 10 min; 100% ethanol, 2 3 10 min. Support samples were then dried at 37 C for 24 h. The bacterial colonization of the surface was evaluated by scanning electronic microscopy (SEM, evo 40 eps, Zeiss). The rate of bacterial coverage was determined by the MatLab program and represented the average of 20 analyzed images. RESULTS
Chemical characterization The molecular weight and the principal chemical composition of each polysaccharide are given in Table I. Data have been supplied by the CEVA.
FIGURE 1. ATR-FTIR spectrum of each polysaccharide powder. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | MAR 2015 VOL 103A, ISSUE 3
The content of iduronic and glucuronic acid was similar for polysaccharides P2 and P3 with, respectively, 32.6 and 28.4%. P1 was characterized by the lowest acid content (22.8%) and the highest molecular weight (876,250 g mol21), whereas P2 exhibited the lowest percentage of sulfated groups (2.7%). A low nitrogen content was observed for the three polysaccharides. A complementary characterization of polysaccharides by ATR-IR spectroscopy was performed to confirm the desulfation of polysaccharide P2. Results are shown in Figure 1. Whatever the ulvan, a strong band at about 1050 cm21, attributed to C–O stretching from the sugar ring, and two bands at 1630 and 1420 cm21 were observed due to asymmetrical and symmetric stretching band of carboxylate groups, respectively.37 IR spectra of polysaccharides P1 and P3 exhibited an intense band at 1230 cm21 and two weak bands at 790 and 850 cm21, corresponding to sulfate groups.38 On P2 spectrum, two bands disappeared at lowest frequency and the intensity of the band at 1230 cm21 decreased.
Surface characterization The surface topographies were investigated by AFM. The roughness values, expressed by a root mean square (rms), are recapitulated in Table II. In normal condition, rms value increased with the decrease in the polysaccharides molecular weight, that is, from 0.816 to 1.19 nm for SiP1 and SiP3, respectively. For surfaces elaborated in excess of coupling agents, rms values were very close, no molecular weight dependence being observed. Furthermore, the increase in the activation reagents concentration induced a slight decrease in rms values whatever the surface. The surface hydrophobicity was evaluated after polysaccharides immobilization by water contact angle measurements. Results are listed in Table II. After surface modification, a decrease in contact angle was observed (from 80 for AUTMS layer to
