A Novel Strategy To Obtain a Hyaluronan Monolayer on Solid

Biomacromolecules 2007, 8, 3531-3539

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A Novel Strategy To Obtain a Hyaluronan Monolayer on Solid Substrates Daniela Pasqui, Andrea Atrei, and Rolando Barbucci* CRISMA and Department of Chemical and Biosystems Sciences and Technologies, University of Siena, Via A. Moro 2, 53100 Siena, Italy Received July 30, 2007; Revised Manuscript Received August 28, 2007

The aim of this study was to find a novel simple method to obtain polysaccharide ultrathin layers on solid substrates to investigate the interaction between the surface and the biological environment. A Hyaluronan (Hyal) monolayer with a well-defined chemistry was obtained by exploiting the capability of organosilanes to spontaneously adhere onto glass surfaces. A silane alkylic chain was conjugated with Hyal, and the derivatized polysaccharide was allowed to spontaneously adhere onto a glass surface. The elemental analysis of the modified polysaccharide demonstrated that one out of five disaccharide units was conjugated with the alkyl silane chain, corresponding to a substitution degree of the carboxylate groups of ∼20%. The film of the modified polysaccharide was characterized by means of X-ray photoelectron spectroscopy (XPS), water contact angle, and atomic force microscopy (AFM) measurements. XPS analysis demonstrated that we obtained a Hyal layer with a thickness of about 2.0 nm corresponding to a Hyal monolayer. The Hyal-coated surfaces appeared to be rather smooth and highly hydrophilic and showed significant resistance to nonspecific cell adhesion.

Introduction The surface chemistry and topography of a biomaterial are of fundamental importance when its surface encounters a living environment. Generally, the biomolecules of physiological fluids (proteins, oligopeptides, or polysaccharides) have great recognition power through specific receptor bindings, but they are also able to spontaneously adsorb onto a material surface without any specific receptor recognition interaction. Such nonspecific interactions often reduce the material’s functionality and cause undesirable effects such as background interference; therefore interfaces that reduce nonspecific binding are widely required for medical devices.1 Different strategies have been developed for obtaining efficient and stable bio-resistant surfaces. Recently, polysaccharide coatings have attracted much attention in the biomaterials field due to their capability to behave as nonfouling surfaces.2-5 Among polysaccharides, hyaluronan (Hyal) is one of the most studied as shown by the increasing number of articles and communications on the hyaluronan series. Hyal is a linear negatively charged polysaccharide consisting of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronate, linked by β1-4 and β1-3 glycosidic bonds. Most cells synthesize Hyal by specific enzymes and then drive it out through the membrane where it becomes part of the extracellular matrix (ECM) components.6 It is known that, in physiological solution, Hyal binds to some cell membrane proteins named hyaladherins through a specific interaction between “the link module” of the protein (a sequence of about 100 amino acids containing four cysteines) and a precise Hyal sequence. The conformation of the link module domain affects the minimum sequence required for Hyal binding; Hyal repeating units involved in hyaladherin binding range from 6 to 10 disaccharide units.7,8 * Author to whom correspondence should be addressed. Phone: +39 0577 234382. Fax: +39 0577 234383. E-mail: [email protected].

To understand how a Hyal-coated surface behaves toward a biological environment is a more tricky question to answer, because the conformation assumed by the polymer once it is grafted to a surface may affect its interaction with proteins and cells. Different strategies have been attempted to obtain Hyal-coated surfaces. Recent findings reported about the passive adsorption of Hyal onto polar surfaces and the formation of a Hyal film through hydrogen bonds.9 Layer-by-layer self-assembly of Hyal or other polysaccharides can be obtained by exploiting electrostatic interactions between the negatively charged groups of the polysaccharide and a positively charged polymer previously adsorbed to the substrate. In such way, poly(lysine)/Hyal10 or poly(ethylene imine)/Hyal11 multilayers have been prepared, but the possible mixing of the layers of the adsorbed polyelectrolytes and the degradation by hyaluronidase once the surfaces are exposed to physiological fluids make them not suitable for implant devices. Therefore, the best strategy to obtain a stable Hyal coating is the covalent bond between the Hyal chains and specific functional groups of the surface. Cen et al. discussed the covalent binding of Hyal to electrically conductive polypyrrole films,12 while other papers describe a method to covalently graft Hyal to previously functionalized metallic or glass surfaces.13,14 The photografting of a photoreactive derivative of Hyal onto a suitable pretreated substrate is demonstrated to be a good method to prepare homogeneously coated surfaces that are able to prevent the adhesion of several different cell types (including melanocytes, fibroblasts, endothelial cells, etc.).15-17 Basically, by photografting Hyal, we can obtain functionalized surfaces that are resistant to protein adsorption and prevent cell adhesion. Nevertheless, the numerous reactions that occur when the polymer is exposed to UV light prevent the control of surface chemical composition. In fact, UV irradiation induces, besides the polysaccharide surface grafting, a random cross-linking between the polysaccharide chains. This cross-linking leads to the formation of a thick layer of Hyal whose chemical composition, structure, and conformation are

10.1021/bm700834d CCC: $37.00 © 2007 American Chemical Society Published on Web 10/16/2007

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Biomacromolecules, Vol. 8, No. 11, 2007

Pasqui et al.

Figure 1. Reaction scheme of the synthesis of the organo-silane Hyal derivative (Hyal-Sil).

difficult to foresee. The development of methods alternative to the photoimmobilization process to prepare polysaccharide layers with a well-defined chemistry is very important for a better understanding of the biological properties of Hyal bound to a substrate. In this paper we exploit a novel method to graft Hyal as well as other polysaccharides to silicon oxide or glass surfaces through silane chemistry. It is known that silane-containing molecules spontaneously adhere onto glass or silicon surfaces so that to form a uniform layer with a well-defined chemistry. Our idea was to conjugate an alkylsilane to the polysaccharide and then let the derivatized polysaccharide adhere to the bare substrate. The surface chemistry was characterized by Xphotoelectron spectroscopy (XPS), the surface wettability was determined by water contact angle measurements, and information about surface roughness and Hyal layer thickness were obtained by atomic force microscopy (AFM). This strategy allowed us to obtain a Hyal-coated surface with a well-defined chemistry.

Experimental Section Materials. Hyaluronan sodium salt (Hyal-Na; Mw ) 240.000) was provided by Biophil S.p.A (Germany). The (3-amino-propyl) trimethoxysilane, N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC), 2-morpholinoethanesulfonic acid monohydrate (MES) and all of the solvents were purchased from Fluka (Switzerland). Glass coverslips (diameter of 12 mm, thickness of 0.13-0.17 mm) were provided by VWR International. Synthesis of Hyal-Organosilane Conjugate. Hyaluronan sodium salt (Hyal-Na) was dissolved in MES buffer (0.1 M, pH 4.5) at a concentration of 0.5% w/v. EDC was added to this solution in a molar ratio of 1:1, and afterward (3-amino-propyl) trimethoxysilane was added in a large excess (500 µL for 25 mL of reaction mixture). The pH was adjusted to 4.75 and the reaction was allowed to proceed for 4 h at room temperature. The reaction mixture was dialyzed (Medicell

International Ltd. molecular weight cutoff of 12-14.000 Da) against double-distilled water for 15 days, changing the water twice a day, and then lyophilized. The derivatized polysaccharide was referred to as Hyal-Sil. The yield of the product was about 70%. The reaction scheme is reported in Figure 1. The grafting ratio of organosilane to Hyal chains was estimated by elemental analysis. The analysis was carried out by the Mikroanalytishes Labor Pasher (Germany) once the product of the reaction had been dialyzed for 15 days. Infrared Spectroscopy. The reaction product was characterized by infrared spectroscopy. Infrared analysis was performed in a Fourier transform infrared attenuated total reflectance (FT-IR-ATR) spectrometer Bio-Rad FTS 6000 purged with nitrogen. ATR spectra of dry samples were acquired with a horizontal (PIKE) ATR accessory equipped with a 45° Ge ATR crystal and a mercury cadmium telluride (MCT) detector. Sixty-four scans at a resolution of 4 cm-1 were averaged for each spectrum. Spectra were elaborated by WIN-IR PRO, version 2.6, recorded by baseline correction and smoothing (boxcar function; 9 N of P). Spectra were deconvolved according to the following parameters: K factor of 1.4 and a full maximum at half-width of 36.000. Surface Grafting of Hyal-Sil. To clean the bare substrates, glass coverslips were sonicated in Caro’s acid solution (H2SO4/H2O2 1:1 v/v) for 20 min at 40 °C and 75% of power, rinsed with a large amount of double-distilled water, sonicated again in NaOH 0.5 M (same conditions), rinsed with double-distilled water, dried with 0.22 µm filtered nitrogen stream, and finally baked at 90 °C for 30 min. Hyal-Sil polymer was dissolved in water/ethanol 1:1 v/v solution at a concentration of 10 mg/mL. Freshly cleaned glass coverslips were dipped in the solution for 4 h at room temperature, rinsed with absolute ethanol and double-distilled water, and finally dried with 0.22 µm filtered nitrogen stream. A Hyal-Sil thick layer was prepared to have a reference surface for XPS measurements. Approximately 100 µL of the same Hyal-Sil solution was spin-coated on bare clean glass coverslips at 1500 rpm for 30 s. The coated surfaces were left at room temperature and analyzed by XPS without any rinsing.

Biomacromolecules, Vol. 8, No. 11, 2007 3533

Hyaluronan Monolayers on Solid Substrates X-ray Photoelectron Spectroscopy. The surface chemical composition of bare and Hyal-Sil-modified glass substrates was determined by XPS. The same analysis was performed also on a Hyal-Sil thick layer deposited by spin coating (see Materials and Methods, Surface Grafting of Hyal-Sil). XPS spectra were recorded in an ultrahigh vacuum chamber equipped with a hemispherical electron energy analyzer (VSW HA150) and a non-monochromatized Al KR (1486.6 eV) X-ray source. The X-ray gun was operated with a power of 180 W. The electron takeoff angle was 90°. Low-resolution survey spectra were collected from 400 to 1490 eV of electron kinetic energy with a constant pass energy was of 90 eV. Higher-resolution spectra were acquired for the most relevant peaks with a constant pass energy of 44 eV. The analyzed area was a circle of 5 mm in diameter. For the angle-resolved measurements the sample was transferred on a manipulator that allowed the rotation of the sample to vary the takeoff angle of the photoelectrons. For the spectra of the bare glass and Hyal-Sil-modified glass surfaces, the binding energy (BE) scale was calibrated setting the Si 2p BE at 103.3 eV according to the value reported in the literature for the Si 2p peak of glass surfaces.18 For the Hyal-Sil thick layer, the BE scale was calibrated setting the C-C component of the C 1s peak (as determined by the curve fitting analysis) to 285.0 eV.18,19 For the quantification of the XPS intensities, we used the atomic sensitivity factors reported in ref 18. The data analysis was performed by Casa-XPS software. A modification of the XPS spectra was observed after prolonged exposure of the Hyal-Sil layer to X-rays. To avoid a possible radiation damage of the organic film, the acquisition time was reduced as much as possible. Static Water Contact Angle. Measurements (three different positions for each sample) were performed on six glass coverslips before and after surface coating with an automated contact angle goniometer (Rame´-Hart, Mountain Lakes, NJ) on both sides of 3 µL drops of Milli-Q water (pH 5.6, resistance 18.2 MΩ/cm) positioned onto each sample. The system was entirely controlled by software (RHI 2001 Imaging), and it consisted of a light source, a sample holder, a charge coupled device camera, and an automated pipetting system to drop the liquid onto the surface. The system allowed us to obtain the measurements in the same positions for each sample thanks to a grid on the sample holder. Atomic Force Microscopy. Freshly cleaned glass coverslips and Hyal-Sil-functionalized surfaces (Hyal-Sil thick layer, Hyal-Sil-modified surfaces) were viewed by atomic force microscopy (Solver Pro, NT MDT Instruments, Russia). AFM images were acquired in air in noncontact mode on five different areas for the samples (three samples for each type) with a sharpened gold-coated silicon tip with a spring constant of 2.5-10 N/m and using a nominal resonance frequency between 120 and 180 kHz. Surface roughness parameters Ra and Rq of five characteristic scan areas (scan size of 5 × 5 µm2) of the different substrates were determined using SPLM-Lab, version 5.01, software. The arithmetic average (Ra) and the root-mean-squares (Rq) roughness are defined as follows: Ra is the arithmetic average of the absolute height values of the m points of the profile and determined by eq 120

Ra )

m

1

∑|z(xi)|

m i)1

(1)

Rq, also called root-mean-square (rms), is the root-mean-square of all of the values of the profile and defined by eq 220

Rq )

x

1

m

∑ z2(xi) m i)1

(2)

Cell Adhesion Experiments. Human fibroblasts with diploid nature from skin biopsies of normal individuals were cultured on Hyal-Sil-

functionalized and bare cleaned glass surfaces. Samples were sterilized in 70% ethanol (30% double-distilled water) for 20 min and air-dried under a laminar flow hood. Three samples per surface type were placed in 24-well plates; a cell suspension of 30.000 cells/mL was plated on each surface. The same amount of fibroblasts was plated on uncoated tissue culture polystyrene (TCPS) wells (as an internal control). Cultures were maintained for 72 h in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal calf serum (FCS) and 1% penicillin/ streptomycin, replacing the medium every 48 h. Cells were monitored by the time and fixed after 72 h of culture with 2.5% of glutaraldehyde in 100 mM sodium cacodylate. Each sample was viewed by phase contrast microscopy.

Results and Discussion Synthesis of Hyal-Sil Conjugate. The coating of Hyal on glass was obtained by dipping clean glass substrates in a solution of an alkyl silane derivative of the polysaccharide. The conjugation of Hyal with (3-amino-propyl) trimethoxysilane involved the formation of an amidic bond between the carboxylate groups of the polysaccharide and the primary amine of the organosilane. EDC was used as an activating agent for the carboxylate groups. The carbodiimide reacts with carboxylic groups to form readily an unstable intermediate O-acylurea, which, in the presence of a nucleophile such as a primary amine, allows the formation of a stable amidic bond.21 Also, the unstable O-acylurea partially rearranges to a N-acylurea, which cannot be converted anymore and remains as a secondary product of the reaction. An acid environment is needed to catalyze the reaction through the protonation of carbodiimide nitrogen.22 In Figures 2a and 2b, original and deconvolved spectra of native Hyal and organo-silane Hyal conjugate are reported in the region between 1700 and 1500 cm-1. Deconvolution was necessary to point out the differences between the two spectra. Table 1 summarizes the main wavenumbers observed in both spectra (Hyal and Hyal-Sil) and their assignments. In the spectrum of Hyal-Sil, a wide band at 1644 cm-1 assigned to amidic CdO stretching was observed. Under these conditions, it was not possible to distinguish the CdO stretching of the native acetamidic group of the Hyal and those of the newly formed amidic bond. The peak at 1605 cm-1, ascribed to carboxylate CdO stretching, suggested the presence of free carboxylate groups that are not bound to the aminopropylsilane. This means that, despite the excess of alkylsilane and EDC added to the reaction mixture, the grafting ratio was lower than 100%. Spectra of the same compound were collected at pH ) 3 to visualize the possible presence of the two amidic bonds. Figure 2c shows the original spectrum of organo-silane Hyal compound (pH ) 3) in the region between 1750 and 1500 cm-1 together with the spectrum of native Hyal collected under the same conditions. The assignment of the detected peaks is reported in Table 1. Under these conditions, the peak at 1605 cm-1 (carboxylate CdO stretching) completely disappeared, while a new peak at 1736 cm-1, which belonged to carboxylic group CdO stretching, was revealed. The disappearance of the band belonging to CdO carboxylate stretching highlighted two peaks in the amidic CdO stretching region that were previously covered by the COO- band: one at 1660 cm-1 and another at 1639 cm-1. Two peaks were also noticed in the amidic N-H bending region: one at 1562 cm-1 and another at 1554 cm-1. The appearance of these peaks in the CdO stretching and N-H bending regions showed the presence of two amidic bonds in the derivatized Hyal: one belonging to the acetamidic group of the native polysaccharide and the other formed during the

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Pasqui et al. Table 1. Main Wavenumbers and Peak Assignment Observed in the IR Spectra of Native Hyal and Hyal-Sil Compounds wavenumber (cm-1)

compounds Hyal polymer

1650 1605

Hyal-Sil polymer

Hyal-Sil polymer (pH ) 3)

assignment

1558 1736

amidic CdO stretching carboxylate CdO symmetrical stretching acetamidic N-H bending amidic CdO stretching carboxylate CdO symmetrical stretching amidic N-H bending carboxylic acid CdO stretching

1660 1639 1562 1554

amidic CdO stretching amidic CdO stretching amidic N-H bending amidic N-H bending

1560 1645 1605

Table 2. Elemental Weight Percent Composition of Hyal-Sil Compound after 15 Days of Dialysis against Double-Distilled Water and Its Theoretical Composition with a Substitution Degree Corresponding to 20%a

element

determined composition (15 days of dialysis)

theoretical composition (molecular formula 5 disaccharide units plus 1 alkylsilane chain) (C76H116O62SiN6‚5H2O)

C H N O Si

39.8% 6.2% 3.6% 46.5% 1.2%

40.9% 5.5% 3.7% 46.8% 1.2%

a

Figure 2. FT-IR spectra of (a) native Hyal, (b) Hyal-Sil conjugate, and (c) Hyal-Sil conjugate at pH ) 3. Spectra have been acquired on dry polymers (lyophilized state, content of water ∼10-15% w/w). Spectral region: 1700-1500 cm-1 for parts a and b and 1750-1500 cm-1 for part c.

reaction. In the spectrum of native Hyal, the presence of a single amidic bond belonging to the acetamidic group of the polysaccharide was observed. Elemental analysis of Hyal-Sil demonstrated that the grafting ratio of the alkylsilane chain to Hyal was 20%. In Table 2, the elemental percentage composition determined by the analysis as well as the theoretical composition for a grafting ratio of 20% are summarized. Previous tests demonstrated that 1 week of dialysis was not sufficient to remove all of the free EDC

The confidence interval for the reported percentages is (0.1%.

and silane from the mixture. On the contrary, by extending the dialysis time over 15 days, the percentage of detected Si remained unchanged. Starting from the given elemental percentage, the nitrogen-to-silicon ratio turned out to be 5.9. Taking into consideration that there would be one silicon atom and two nitrogen atoms for each disaccharide unit after silane binding (one belonging to the native polysaccharide and one introduced after the amidic bond formation), a nitrogen-to-silane ratio of 5.9 means that, on average, one out of five disaccharide units was conjugated with the alkyl silane, which corresponded to a substitution degree of 20%. Moreover, by comparing the percentages of O and H in the theoretical structure of the disaccharide unit with the experimental values, it was clear that there was one molecule of water for each disaccharide unit of Hyal-Sil. The presence of water is due to the high hydrophilicity of Hyal. Surface Chemistry: XPS Analysis. The chemical compositions of the Hyal-Sil-coated glass surfaces, bare glass, and HyalSil thick layer prepared by spin coating were investigated. The detection of nitrogen in the spectrum of the Hyal-Sil-modified glass surface demonstrated the presence of a thin layer of polysaccharide grafted to the surface that remained even after an extensive washing (Figure 3). In Table 3, the normalized intensities (i.e., the experimental peak areas divided by the corresponding sensitivity factors) are summarized and expressed as a percentage of the summed normalized peak areas of each surface element of the Hyal-Sil thick layer, Hyal-Sil-modified glass, and bare glass. The presence of nitrogen was detected also in the spectrum of the Hyal-Sil thick layer obtained by


Hyaluronan Monolayers on Solid Substrates

Table 4. XPS Binding Energy ((0.2 eV), the Chemical Assignments of the Different Peak Components, and the Areas of the Different Peak Components Expressed as Relative Percentages of the Total Area

Figure 3. XPS survey spectra of bare cleaned glass, Hyal-Sil thick layer (obtained by spin coating), and Hyal-Sil monolayer.

Table 3. Surface Elemental Composition (Atomic Percentages) for Bare Glass, Hyal-Sil-Modified Glass, and Hyal Thick Layer sample bare glass Hyal-Sil-modified glass Hyal-Sil thick layer

% O 1s % N 1s %K 2p % C 1s %Si 2p 71.0 39.0 36.7

1.1 7.0 7.3

7.0 49.0 54.3

20.9 5.0 1.7

spin coating. As expected, no nitrogen was found in the spectrum of the bare clean glass surface. This spectrum is consistent with the typical XPS spectra of glass surfaces. The C concentration found was found to be 7%, which was within the range for “clean” glass, and it was due to hydrocarbon contaminants from the atmosphere. The Si 2p signal is present both in the spectrum of the Hyal-Sil-grafted layer and in that of the Hyal-Sil thick layer. In the latter case, the thickness of the film (∼100 nm) is much larger than the depth probed by Si 2p photoelectrons (∼10 nm, taking into consideration the electron mean free path).22 Hence, the Si 2p signal comes from Si atoms belonging the modified polysaccharide molecule. For the Hyal-Sil-modified glass surface, the Si 2p peak is due to the Si atoms of glass and to those at the interface between the polymer and the substrate. In Tables 4a and 4b, the experimental binding energies of the C 1s, O 1s, N 1s, and Si 2p XPS peaks for the different tested surfaces are summarized. The nitrogen peak shows two components: one at ∼400 eV and the other located at a higher binding energy (∼401.5 eV). The first component can be assigned to N in amines or amides18,19 (Figure 4b). The second component, according to previous findings,9 can be attributed to the protonation or hydrogen bonds of nitrogen. The hydrogen bonds might be formed with -Si(OH) groups of the substrate surface or with other protons of the polysaccharide chains. The relative percentages of these two N 1s components in the HyalSil-grafted and the spin-coated layers are significantly different. A decrease from 40.0% down to 13% for the component at higher binding energies was noticed on the Hyal-Sil-grafted layer. This may suggest a different conformation (and hence a

element

peak

oxygen carbon

O 1s C 1s(1) C 1s(2) C 1s(3)

nitrogen silicon

N 1s(1) N 1s(2) Si 2p

oxygen

O 1s(1)

carbon

C 1s(1) C 1s(2) C 1s(3)

nitrogen silicon

N 1s(1) N 1s(2) Si 2p

oxygen silicon

O 1s Si 2p

binding energy (eV)

assignment

(a) Hyal-Sil Thick Layer 532.9 O bound to carbon 285.0 CsC 286.7 -C-O288.5 -N-CdO, O-CdO, O-C-O 399.8 -NH2 401.7 -NH2sH 102.7 -Si-(OCH3)3 (b) Hyal-Sil-Modified Glass 532.8 O bound to carbon, O in SiO2 284.8 CsC 286.4 -C-O288.3 -N-CdO, O-CdO, O-C-O 400.2 -NH2 401.5 -NH2sH 103.3 SiO2 C-Si

area

a 11.9% 68.4% 19.7% 60.8% 39.2% 100%

a 20.7% 60.4% 18.9% 87.8% 12.2% 100%

(c) Bare Glass 532.5 O bound to carbon 103.3 SiO2, C-Si

a The O 1s signal appeared as a broad symmetrical peak; no components were detected.

different possibility to form hydrogen bonds) of the polysaccharide in the spin-coated and grafted layers. The C 1s signal of the Hyal-Sil-modified surfaces as well as of the spin-coated layers appears as a broad peak. The curve fitting analysis shows that there are at least three different components contributing to the C 1s spectra of the two films (Figure 4a). The present findings are consistent with those reported in the literature for films of Hyal and similar polymers.9,23 The results of the curve fitting analysis are summarized in Table 4. The component at a BE of ∼285.0 eV is due to aliphatic carbon in the polymer. However, there is also a contribution from the carbon contamination (mainly consisting of aliphatic carbon, as shown by the C 1s spectrum measured for the bare glass). The contribution of adventitious carbon is generally on the order of 10% of the total amount of carbon. The main component (located at a BE of ∼286.5 eV) can be assigned to carbon singly bonded to oxygen (-C-OC- and -C-OH groups). In a previous XPS study of Hyal films, an additional component at 286.0 eV corresponding to carbon in C-N groups was found in the curve fitting analysis.9 The energy resolution used in the present work did not allow us to distinguish this component from the peaks due to aliphatic carbon and carbon singly bound to oxygen. The last component at 288 eV has contributions from carbon atoms in N-CdO, -O-C-O-, and O-CdO groups. The BEs of the various components do not vary (within (0.2 eV) in the Hyal-Sil-grafted and the spin-coated layers. The relative intensities of the various components do not change significantly in the spectra of the grafted and the spin-coated layers with the exception of the C-C component, which increases from 11% to 21% in the Hyal-Silgrafted layer. This can be due to the fact that the presence of

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Pasqui et al.

Figure 4. High-resolution XPS spectra for (a) carbon C 1s and (b) nitrogen N 1s peaks of a Hyal-Sil-modified glass surface. Peak fitting was performed with Casa-XPS software, using a least-squares fit routine following Shirley background subtraction.

contaminants has a larger effect on a thin layer with respect to a thicker one. The atomic composition of the spin-coated layer determined by XPS was consistent with the results of the elemental analysis as far as the C and O atomic percents were concerned. However, there were significant differences in the N and Si atomic percents. For instance, the nitrogen-to-silicon atomic ratio turned out to be 4.2 ( 0.2, which was lower than the value (5.9) determined by elemental analysis of the polymer. XPS measurements were performed on several Hyal spin-coated thick layers and gave the same results. The differences in the results of the elemental and XPS analyses may be attributed to the fact that the former gives the bulk composition whereas the latter is sensitive to the surface composition. A thick layer of the polymer deposited on a solid substrate may expose preferentially some functional groups at the outermost surface and thus leads to a composition different from that of the bulk as determined by XPS.24,25 The thickness of the grafted Hyal-Sil layer was assessed by two methods: (i) the attenuation of the Si 2p signal of the substrate produced by the polymer film; (ii) the C 1s and Si 2p intensity ratio. We assumed that the polymer film was a layer of uniform thickness thatcompletely covered the substrate surface. The roughness of the film (as determined by AFM, see below) suggested that this was a reasonable assumption. The mean free path for the Si 2p photoelectrons in the organic layer was calculated to be 3.0 nm according to the method reported in ref 26.

The area of the Si 2p peak ISi 2p measured for the glass substrate covered by a polymer layer of thickness d is given by eq 3

( )

ISi 2p ) ISi 2p0 exp -

d λ1

(3)

where ISi 2p0 is the area of the Si 2p peak measured for the bare glass surface and λ1 is the inelastic mean free path of the Si 2p photoelectrons in the polymer. The film thickness was determined on the basis of the C 1s and Si 2p intensity ratio using eq 4

IC 1s IC 1s0 ) ISi 2p I 0 Si 2p

[ ] ( ) ( )

-d λ2 -d exp λ1

1 - exp

(4)

where IC 1s is the area of the C 1s peak of the Hyal-Sil-grafted surface, IC 1s0 is the area of the C 1s peak of the Hyal-Sil thick layer, ISi 2p is the area of the Si 2p peak of the Hyal-Sil-grafted surface, ISi 2p0 is the area of the Si 2p peak of bare glass, d is the layer thickness, and λ2 and λ1 are the inelastic mean free paths for the C 1s and Si 2p photoelectrons in the polymer layer, respectively. Because the kinetic energies of the C 1s and Si 2p photoelectrons do not differ much, we used the same inelastic mean free path (λ1) for both kinds of photoelectrons to simplify the calculations.


Figure 5. Fitting of the experimental data (solid line) for angleresolved XPS measurements.

The values of d calculated using the two methods (2.3 and 2.2 nm, respectively) are the same within the experimental error of (0.5 nm The calculated thickness is consistent with that measured by ellipsometry for similar surfaces13 and that determined for a monolayer of a polysaccharide film immobilized on PEI.11 To check whether the polymer film completely covers the substrate surface, we performed XPS measurements collecting the C 1s and Si 2p intensities at various takeoff angles of the photoelectrons. The results of the angle-resolved measurements are shown in Figure 5. There is some scattering of the data due to the short acquisition time to avoid possible damage of the film induced by the X-rays. These data were fitted using eq 2 but including a variable takeoff angle and a fraction coverage of the film (which was taken equal to one in eq 2). The fitting parameters optimized by the least-squares method were the thickness of the film and its fractional coverage. The best fit of the experimental data (solid line in Figure 5) was obtained for a thickness of 3.0 ( 0.5 nm and a coverage of substrate of 0.7 ( 0.1. This thickness is slightly larger than the value found by analyzing the XPS intensities with the previous methods. Those methods assume that the polymer film covers the whole substrate surface and therefore tend to underestimate the actual thickness of the layer. On the basis of these results, we can conclude that the polymer film covers a large fraction of the substrate and with an average thickness corresponding to a monolayer of Hyal. Surface Wettability. The WCA of bare cleaned glass surfaces was 18° ( 2°, whereas the WCA of Hyal-Sil-modified surfaces was 25° ( 3°, demonstrating that the polysaccharide functionalization slightly decreased the surface wettability even if both surfaces had a high degree of hydrophilicity. The mean WCA is reported because the values between left and right θ were very similar: ∆θ ) (2°. Results were analyzed in detail, by taking into consideration the difference of WCA values before and after Hyal grafting of the same surfaces on the same position to minimize the small differences due to single sample characteristics. The average difference in WCA values before and after Hyal-Sil grafting for the six samples for each measured point was 7°, meaning that surface modification slightly decreased the surface wettability. The WCA of the Hyal coating obtained by photografting was higher (40° ( 3°). This effect may be related to how the


polysaccharide is bound to the substrate. In the case of the organo-silane derivative, the polymer is bound to the substrate but rather free to interact with water molecules of the environment. However, when Hyal is photografted to glass, the additional cross-linking between the polymer chains leads to a more close-packed structure, which reduces water-surface interactions and the exposure of hydrophilic groups at the interface. Other possible reasons (such as the different chemistry of the modified Hyal or the different substitution degree) could be responsible for the diffferent wettability of the Hyal-Sil coating and the photografted Hyal film. Surface Topography. Surface topography and surface roughness of bare glass and Hyal-Sil-modified glass surfaces were evaluated by AFM. AFM scans are shown in Figure 6a. The reported images are representative of the whole surface. The bare glass surface was very smooth as expected. (Ra was 0.40 ( 0.06 nm, and Rq was 0.63 ( 0.08 nm.) The Hyal-Sil monolayer appeared smooth as well (Ra was 0.90 ( 0.09, nm and Rq was 1.21 ( 0.20 nm), while the average roughness of the Hyal-Sil thick layer was much higher (Ra was 5.5 ( 0.7 nm and Rq 8.9 ( 0.9 nm). The measured roughness of the HyalSil-modified surfaces, which was between that of bare glass and that of the Hyal-Sil thick layer, is further evidence of the presence of a Hyal-Sil monolayer grafted to the surface. AFM analysis of the Hyal-Sil-modified surface topography showed that the surface topography was not completely homogeneous. Even if most of the surface exhibited a flat uniform topography, there were some regions that showed some holes or irregularities. To verify whether the holes corresponded to uncovered glass regions, AFM analysis in lateral force mode was performed. The differences of friction force (Figure 6b) demonstrated that the irregularities corresponded to glass uncovered regions and that their depth was about 2 nm, which corresponded to the polysaccharide layer thickness as calculated by XPS measurements. Cell Adhesion Experiments: Preliminary Results. Figure 7 shows the different fibroblast behaviors on Hyal-modified surfaces and bare clean glass. Fibroblasts do not adhere on HyalSil-modified surfaces (Figure 7a); the few adhered cells showed a round suffering morphology even after 72 h of culture, which is unusual for fibroblasts. On the contrary, good cell adhesion was observed, as expected, on bare cleaned glass coverslips (Figure 7b). These data are perfectly consistent with those reported in previous works concerning the biological performance of Hyal covalently bound to a surface. Despite the methods used for surface binding, all of these Hyal-functionalized surfaces act as good protein and cell-resistant coatings. Our previous results demonstrated that a continuous uniform layer of Hyal obtained by a photografting procedure is able to prevent the adhesion of many different types of cells.15,16 However, Hyal simply adsorbed by electrostatic interaction, although at first shows a certain degree of cell resistance, with time increasing, loses its properties and becomes covered by cells.12 From all of these data and data reported in the literature, it seems that Hyal works differently according to whether it is covalently bound or simply adsorbed to a surface. Generally, polysaccharide coatings are viewed as highly hydratated antiadhesive coatings that somehow prevent protein adsorption, but this cannot be considered as a general rule. Indeed, it has been demonstrated that photografted chitosan coatings behave as celladhesive surfaces due to the fact that the surface promotes the adsorption of the medium proteins, probabily thanks to its positive surface charge.27 Hyaluronan-modified surfaces (both the monolayer and the photografted coatings) are negatively

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Pasqui et al.

Figure 6. (A) AFM images (scan size of 5 × 5 µm2) of (a) Hyal-Sil thick layer obtained by spin coating, (b) Hyal-Sil monolayer, and (c) bare cleaned glass. (B) AFM images (scan size of 5 × 5 µm2) of a Hyal-Sil monolayer: (a) surface topography; (b) lateral force mode of the same area.

Figure 7. Phase contrast microscopy images (original magnification 20×) of human fibroblasts plated on (a) Hyal-Sil monolayer and (b) bare cleaned glass surface after 72 h of culture.

charged due to the presence of free carboxylic groups; hence this is could be one of the possible explanations for the protein resistance of such surfaces. It is also worth noting that sulfated hyaluronic-acid-coated surfaces, which are negatively charged as well, are good platforms for cell adhesion and protein adsorption;17 hence the surface charge itself is not sufficient to explain why Hyal-modified surfaces are cell-resistant. It should be considered also the amount and the type of adsorbed serum protein and also the mechanical properties of a material (softness, stifness etc). In the case of Hyal, we have to take into consideration that the existence of a specific direct interaction between some cell membrane receptors (such as CD44 and lyve1) and a precise sequence of the polysaccharide, which consists at least of eight repeating units, has been demonstrated.28,29 The same type of interaction has not been demonstrated for other polysaccharides so far. We can imagine

that the interaction between Hyal and the receptor site may be prevented when the polymer is bound to the surface and the minimum size required for the receptor interaction may be not available anymore. In the case of a Hyal monolayer, the presence of the alkyl silane chain may reduce the degree of freedom of the polymer and change its conformation when the polysaccharide is bound to the surface. Hence, the binding of Hyal to the surface may reduce the interaction capability with CD44 receptor even if eight free disaccharide units may be present due to the random distribution of the alkyl chains on Hyal.

Summary and Conclusions We developed a novel and versatile method for the grafting of polysaccharides to a surface with controlled chemistry. A stable monolayer of Hyal was obtained by the spontaneous


assembly of the polysaccharide conjugated with (3-aminopropyl) trimethoxysilane to glass. The thickness of this layer, as determined by XPS, is consistent with the presence of a monolayer of the polymer. The Hyal-modified surfaces are rather smooth and highly hydrophilic and show a significant resistance to cell adhesion. The proposed method could be applied to graft hyaluronan as well as other polysaccharides having carboxylic groups to glass, silicon, or other surfaces. Acknowledgment. The authors gratefully acknowledge Dott. Gabriella Caminati (Department of Chemistry, University of Florence) for WCA measurements, the MIUR FIRB 2001 Project “Technologies for Nanometric Scale Manufacturing of Materials and Their Biomedical Application” and the PRISMA project (PC 23-2005) for financial support.

References and Notes (1) Integrated Biomaterials Science; Barbucci, R., Ed.; Kluwer Academic/ Plenum: New York, 2002. (2) Krishna, O. D.; Kwangmeyung, K.; Youngro, B. Biomaterials 2005, 26, 7115. (3) Iwanaga, S.; Akiyama, Y.; Kikuchi, A.; Yamato, M.; Sakai, K.; Okano, T. Biomaterials 2005, 26, 5395. (4) Luk, Y.; Kato, M.; Mrksich, M Langmuir 2000, 16, 9604. (5) Osterberg, E.; Bergstro¨m, K.; Holmberg, K.; Riggs, J. A.; Van, Alstine, J. M.; Schuman, T. P; Norman, L. B; Harris, J. M. Colloids Surf., A 1993, 77, 159. (6) Weigel, P. H.; Hascall, V. C.; Tammi, M. J. Biol. Chem. 1997, 272, 13997. (7) Teriete, P.; Banerji, S.; Noble, M.; Blundell, C. D.; Wright A. J.; Pickford, A.; Lowe, E.; Mahoney, D. J.; Tammi, M. I.; Kahmann, J. D.; Campbell, I. D.; Day, A. J.; Jackson, D. G. Mol. Cell 2004, 13, 483. (8) Lesley, J.; English, N.; Hascall, V. C.; Tammi, M.; Hyamn, R. Hyaluronan binding by cell surface CD44. In Hyaluronan; Kennedy J. F., Philips G. O., Williams P. A., Hascall V., Eds.; Woodhead Publishing: Cambridge, U. K., 2002; Vol. 1, p 341. (9) Suh, K. Y.; Yang, J. M.; Khademhosseini, A.; Berry, D.; Tran, T. T.; Park, H.; Langer R. J. Biomed. Mater. Res., Part B 2005, 72, 292.

Biomacromolecules, Vol. 8, No. 11, 2007 3539 (10) Picart C., Butterer, J.; Richert, L., Luo, Y.; Prestwich, G. D.; Schaaf, P.; Voegel, J. C.; Lavalle, P. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12531. (11) Morra, M.; Cassinelli, C.; Pavesio, A.; Renier, D. J. Colloid Interface Sci. 2003, 259, 236. (12) Cen, L.; Neoh, K. G.; Li, Y.; Kang, E. T. Langmuir 2002, 18, 8633. (13) Pitt, W. G.; Morris, R. N.; Mason, M. L.; Hall, M.W.; Luo, Y.; Prestwich, G. D. J. Biomed. Mater. Res., Part A 2004, 68, 95. (14) Stile R. A; Barber T. A.; Castner D. G.; Healy K. E. J. Biomed. Mater. Res. 2002, 61, 391. (15) Barbucci, R.; Magnani, A.; Chiumiento, A.; Pasqui, D.; Cangioli, J.; Lamponi, S. Biomacromolecules 2005, 6, 638. (16) Barbucci, R.; Lamponi, S.; Magnani, A,. Pasqui D., Bryan S. Biomaterials 2003, 24, 915. (17) Pasqui, D.; Rossi, A.; Barbucci, R.; Lamponi, S.; Gerli, R.; Weber E. Lymphology 2005, 38, 50. (18) Briggs D. Surface Analysis of Polymers by XPS and Static SIMS Cambridge University Press: Cambridge, U. K., 1998. (19) Practical Surface Analysis: By Auger and X-ray Photo-Electron Spectroscopy; Briggs, D., Seah, M. P., Eds.; Wiley: New York, 1983. (20) Thomas, R. T. Rough Surfaces; Imperial College Press: London, 1999. (21) Carraway, K. L.; Koshland, D. E. Methods Enzymol. 1972, 25, 616. (22) Kurzer, F.; Douraghi-Zadeh, K. Chem. ReV. 1967, 67, 107. (23) Shard, A. G.; Davies, M. C.; Tendler, S. J. B; Benedetti, L.; Purbrick, M. D.; Paul, A. J.; Beamson, G. Langmuir 1997, 13, 2808. (24) Shang, J.; Geva, E. J. Phys. Chem. B 2005, 109, 16340. (25) Beamson, G. J. Electron Spectrosc. Relat. Phenom. 2001, 121, 163. (26) Tanuma, S.; Powell, C. J.; Penn, D.R. Surf. Interface Anal. 1991, 17, 927. (27) Fukuda, J.; Khademhosseini, A.; Yeo, Y.; Yang, X.; Yeh, J.; Eng, G., Blumling, J.; Wang, C.; Kohane, D. S.; Langer, R. Biomaterials 2006, 27, 5259. (28) Bajorath, P. F.; Grenfield, B.; Munro, S. B.; Day, A. J.; Aruffo, A. J. Biol. Chem. 1998, 273, 338. (29) Peach, R. J.; Hollenbauugh, D.; Stamenkovick, I.; Aruffo, A. J. Cell. Biol. 1993, 122, 257.

BM700834D

A Novel Strategy To Obtain a Hyaluronan Monolayer on Solid

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