**Silanization with APTES for Controlling the Interactions Between Stainless Steel and Biocomponents: Reality vs Expectation**

Jessem Landoulsi1, Michel J. Genet2, Karim El Kirat3, Caroline Richard4, Sylviane Pulvin5 and Paul G. Rouxhet2 *1Laboratoire de Réactivité de Surface, Université Pierre & Marie Curie -Paris VI, 2Institute of Condensed Matter and Nanosciences – Bio & Soft Matter, Université Catholique de Louvain, 3Laboratoire de Biomécanique et Bioingénierie, 4Laboratoire Roberval, 5Génie Enzymatique et Cellulaire, Université de Technologie de Compiègne, 1,3,4,5France 2Belgium* 

### **1. Introduction**

98 Biomaterials – Physics and Chemistry

Zhang, H.J., Mao, W.J., Fang, F., Li, H.Y., Sun, H.H., Gehen, Y., Qi, X.H., Chemical

(February 2008), pp. 428-434, ISSN 0144-8617

characteristics and anticoagulant activities of a sulphated polysaccharide and its fragments from Monostroma latissimum, *Carbohydrate Polymers,* Vol.71, No.3,

> The surface of biomaterials is frequently chemically modified with the aim to modify the physicochemical properties (hydrophobicity, electrical charge, solvation) which control the interactions with biomolecules and consequently with cell surfaces, or to retain biochemical entities which are specifically recognized by the cells (Williams, 2010). Regarding inorganic materials, widespread procedures involve self-assembly of alcane thiols on gold, silver, copper or platinum (Wink et al., 1997). However, these substrates have limited interest in biomedical applications. Other procedures consist in grafting organosilanes on silica and other metal oxides (Weetall, 1993). The use of silane coupling agents has been reported in various biomaterials researches, such as surface modification of titanium (Nanci et al., 1998), natural fiber/polymer composites (Xie et al., 2010) or dental ceramics (Matinlinna et al., 2004; Matinlinna & Vallittu, 2007).

> The silanization reaction at interfaces is complex and there is still considerable debate on the retention mechanisms and on the organization of the interface (Gooding & Ciampi, 2011; Haensch et al., 2010, Suzuki & Ishida, 1996). Depending on the nature of reactive moieties bound to Si in the silane (typically Cl or alkoxy group) and their number, and on the reaction conditions (particularly the presence of water), the relative importance of covalent binding to the surface, oligomerization, polymerization along the surface plane, threedimensional polymerization may possibly vary. The efficiency of the surface modification is often demonstrated by its influence on biochemical or biological activity. However the nature of the interface produced is difficult to characterize, which limits the guidelines

Silanization with APTES for Controlling the Interactions

of the presence of contaminants and the perspectives of avoiding it.

a. Sonication in acetone, 5 min; heating 2 h at 500°C in air.

**a. Substrate preparation b. APTES treatment** 

curing 10 min at 105°C.

+ enzyme.

24 h at 30 °C.

consistent evolution.

**d. Interface characterization** 

Between Stainless Steel and Biocomponents: Reality vs Expectation 101

Stainless steels (SS) are extensively used in biomaterials researches and other applications involving contact with biologic compounds, owing to their adaptable mechanical properties, their manufacturability and their outstanding corrosion resistance. For instance, SS may be used in the manufacture of vascular stents, guide wires, or other orthopedic implants (Hanawa, 2002, Ratner, 2004). In these conditions, SS are subjected to the adsorption of biomolecules (proteins, polysaccharides, lipids) and biological materials (cellular debris). The surface modification of SS may thus be important to orient the host response as desired. The present work is dedicated to the surface composition of 316L SS surfaces at different stages of the procedure used to graft a protein via the use of APTES and of a bifunctional agent expected to link the NH2-terminated silane with NH2 groups of the protein. Glucose oxidase was chosen as a model protein for reasons of convenience owing to previous works related to microbiologically influenced corrosion (Dupont et al., 1998, Landoulsi et al., 2009, Landoulsi et al., 2008b, Landoulsi et al., 2008c). A particular attention is given to (i) the real state of the interface (composition, depth distribution of constituents) at different stages and (ii) the mode of protein retention. Therefore, X-ray photoelectron spectroscopy is used in a way (angle resolved measurements, reasoned peak decomposition, validation by quantitative relationships between spectral data) to provide a speciation in terms of classes of compounds (silane, protein, contaminants), using guidelines established in previous works (Genet et al., 2008; Rouxhet & Genet, 2011). Water contact angle measurements are used to address the issue

**Substrate Linker Biomolecule Reference** 

Stainless steel EDC Alginate Yoshioka et al., 2003

b. In toluene, 1h; rinsing in toluene and ethanol; sonication in ethanol, 5 min; drying in air;

d. XPS: elemental concentration, consistent evolution according to reaction steps; C 1s peak, demonstration of alginate retention, majority of carbon of C-(C,H) type at all stages. Stainless steel GA Lysozyme Minier et al., 2005

c. Increase of the enzymatic activity in bacterial lysis. Blanks = native + enzyme, silanized

a. Cleaning in ethanol/water (1/1, v/v); rinsing in water; drying under reduced pressure,

LbL film

Meng et al., 2009

c. Preventing adsorption of blood-clotting proteins. Blanks = native, silanized.

b. In ethanol/water, 3 min; curing 1 h at 100-150 °C in air; rinsing with water.

d. IRRAS: Characteristic bands of APTES. XPS: elemental concentration, consistent evolution according to reaction steps; N/Si ratio vs photoelectron collection angle,

a. Acid etching at 60 °C; rinsing in water; drying under N2 gas flow.

Stainless steel stent none Chitosan/heparin

b. In ethanol, 4 h at 37 °C; rinsing in water, drying in air at 50 °C.

c. Promoting re-endothelialization after stent implantation, improvement of

**c. Evaluation of efficiency regarding biomolecule activity. Substrate taken as blank** 

available to improve the procedures. Moreover organic contaminants are always present on high energy solids. They are mainly of hydrocarbon nature and are readily adsorbed from surrounding air or in surface analysis spectrometers (Caillou et al., 2008; Landoulsi et al., 2008a). The possible influence of contaminants on the silanization process and product is usually not considered. In the case of silicon wafer silanized with 3-[methoxy(polyethyleneoxy)]propyl trimethoxysilane and trichlorosilane in organic solvents under a controlled atmosphere, the surface obtained was described as a 1 to 2 nm thick grafted silane layer covered by a thin layer of adventitious contaminants, suggesting that contamination was posterior to the silanization reaction. On the other hand, the silane layer was not stable in phosphate buffered saline at 37°C (Dekeyser et al., 2008).

Aminopropylalkoxysilanes are attractive for surface modification (Plueddemann, 1991), as their bifunctional nature is expected to offer the possibility of covalently attaching a biomolecule, either directly or through a linker. 3-Aminopropyl(triethoxysilane) (APTES) is one of the most frequently used organosilane agents for the preparation of amineterminated films (Asenath Smith & Chen, 2008; Howarter & Youngblood, 2006; Kim et al., 2009a; Lapin & Chabal, 2009; Pasternack et al., 2008).

Table 1 presents a list of references in which APTES was used to hopefully graft biomolecules on different substrates. The survey is exhaustive for stainless steeel substrates relevant for the field of biomaterials and illustrative for other substrates. Additional references are: El-Ghannam et al., 2004; Kim et al., 2010; Sasou et al., 2003; Sarath Babu et al., 2004; Quan et al., 2004 ; Subramanian et al., 1999 ; Jin et al., 2003 ; Cho & Ivanisevic, 2004 ; Katsikogianni & Missirlis, 2010 ; Sordel et al., 2007 ; Toworfe et al., 2006 ; Balasundaram et al., 2006 ; Doh & Irvine, 2006 ; Palestino et al., 2008 ; Son et al., 2011 ; Koh et al., 2006 ; Mosse et al., 2009 ; Weng et al., 2008 ; Charbonneau et al., 2011 ; Iucci et al., 2007 ; Chuang et al., 2006 ; Schuessele et al., 2009 ; Ma et al., 2007 ; Toworfe et al., 2009 ; Zile et al., 2011 ; Sargeant et al., 2008 ; Lapin & Chabal, 2009. Table 1 indicates the substrate and linker used, the main conditions of the APTES treatment and the evaluation of the surface treatment regarding biomolecule activity with the blank used for comparison. The table also presents the main data obtained by surface characterization. In some systems, no covalent grafting was aimed. In other systems, although it was aimed, there is no direct evidence for the formation of covalent bonds between the biomolecules and the substrate surface. On the other hand, the evaluation of the bio-efficacy was never based on comparisons involving a complete set of blanks: treatment with the biomolecule without silanization, without linker, without silanization and linker. In a study of surface modification with the aim to enhance mineralization, it has been demonstrated that APTES-coated glass retains a homopolymer with monoester phosphate groups, poly[(2-methacryloyloxy)ethyl phosphate], by proton transfer and electrostatic interaction, while the retention of a neutral homopolymer, poly[2- (acatoacetoxy)ethyl methacrylate], was attributed to covalent linkage by reductive amination between the keto groups of the polymer and the surface amine functions (Jasienak et al., 2009). The retention of the diblock copolymer seemed to occur via segments allowing covalent bonds to be formed. In Table 1, several systems show an improved behavior which may only be attributed to non covalent bonding between the biomolecule and the silanized substrate. In contradiction with frequent implicit considerations, the occurrence or improvement of bioactivity as a result of surface treatments does not demonstrate that the chemical schemes which motivated the treatments worked in reality. This question is crucial as many organic reactions that work well in solution are difficult to apply at solid surfaces (Kohli et al., 1998).

available to improve the procedures. Moreover organic contaminants are always present on high energy solids. They are mainly of hydrocarbon nature and are readily adsorbed from surrounding air or in surface analysis spectrometers (Caillou et al., 2008; Landoulsi et al., 2008a). The possible influence of contaminants on the silanization process and product is usually not considered. In the case of silicon wafer silanized with 3-[methoxy(polyethyleneoxy)]propyl trimethoxysilane and trichlorosilane in organic solvents under a controlled atmosphere, the surface obtained was described as a 1 to 2 nm thick grafted silane layer covered by a thin layer of adventitious contaminants, suggesting that contamination was posterior to the silanization reaction. On the other hand, the silane

Aminopropylalkoxysilanes are attractive for surface modification (Plueddemann, 1991), as their bifunctional nature is expected to offer the possibility of covalently attaching a biomolecule, either directly or through a linker. 3-Aminopropyl(triethoxysilane) (APTES) is one of the most frequently used organosilane agents for the preparation of amineterminated films (Asenath Smith & Chen, 2008; Howarter & Youngblood, 2006; Kim et al.,

Table 1 presents a list of references in which APTES was used to hopefully graft biomolecules on different substrates. The survey is exhaustive for stainless steeel substrates relevant for the field of biomaterials and illustrative for other substrates. Additional references are: El-Ghannam et al., 2004; Kim et al., 2010; Sasou et al., 2003; Sarath Babu et al., 2004; Quan et al., 2004 ; Subramanian et al., 1999 ; Jin et al., 2003 ; Cho & Ivanisevic, 2004 ; Katsikogianni & Missirlis, 2010 ; Sordel et al., 2007 ; Toworfe et al., 2006 ; Balasundaram et al., 2006 ; Doh & Irvine, 2006 ; Palestino et al., 2008 ; Son et al., 2011 ; Koh et al., 2006 ; Mosse et al., 2009 ; Weng et al., 2008 ; Charbonneau et al., 2011 ; Iucci et al., 2007 ; Chuang et al., 2006 ; Schuessele et al., 2009 ; Ma et al., 2007 ; Toworfe et al., 2009 ; Zile et al., 2011 ; Sargeant et al., 2008 ; Lapin & Chabal, 2009. Table 1 indicates the substrate and linker used, the main conditions of the APTES treatment and the evaluation of the surface treatment regarding biomolecule activity with the blank used for comparison. The table also presents the main data obtained by surface characterization. In some systems, no covalent grafting was aimed. In other systems, although it was aimed, there is no direct evidence for the formation of covalent bonds between the biomolecules and the substrate surface. On the other hand, the evaluation of the bio-efficacy was never based on comparisons involving a complete set of blanks: treatment with the biomolecule without silanization, without linker, without silanization and linker. In a study of surface modification with the aim to enhance mineralization, it has been demonstrated that APTES-coated glass retains a homopolymer with monoester phosphate groups, poly[(2-methacryloyloxy)ethyl phosphate], by proton transfer and electrostatic interaction, while the retention of a neutral homopolymer, poly[2- (acatoacetoxy)ethyl methacrylate], was attributed to covalent linkage by reductive amination between the keto groups of the polymer and the surface amine functions (Jasienak et al., 2009). The retention of the diblock copolymer seemed to occur via segments allowing covalent bonds to be formed. In Table 1, several systems show an improved behavior which may only be attributed to non covalent bonding between the biomolecule and the silanized substrate. In contradiction with frequent implicit considerations, the occurrence or improvement of bioactivity as a result of surface treatments does not demonstrate that the chemical schemes which motivated the treatments worked in reality. This question is crucial as many organic reactions that work well in solution are difficult to

layer was not stable in phosphate buffered saline at 37°C (Dekeyser et al., 2008).

2009a; Lapin & Chabal, 2009; Pasternack et al., 2008).

apply at solid surfaces (Kohli et al., 1998).

Stainless steels (SS) are extensively used in biomaterials researches and other applications involving contact with biologic compounds, owing to their adaptable mechanical properties, their manufacturability and their outstanding corrosion resistance. For instance, SS may be used in the manufacture of vascular stents, guide wires, or other orthopedic implants (Hanawa, 2002, Ratner, 2004). In these conditions, SS are subjected to the adsorption of biomolecules (proteins, polysaccharides, lipids) and biological materials (cellular debris). The surface modification of SS may thus be important to orient the host response as desired. The present work is dedicated to the surface composition of 316L SS surfaces at different stages of the procedure used to graft a protein via the use of APTES and of a bifunctional agent expected to link the NH2-terminated silane with NH2 groups of the protein. Glucose oxidase was chosen as a model protein for reasons of convenience owing to previous works related to microbiologically influenced corrosion (Dupont et al., 1998, Landoulsi et al., 2009, Landoulsi et al., 2008b, Landoulsi et al., 2008c). A particular attention is given to (i) the real state of the interface (composition, depth distribution of constituents) at different stages and (ii) the mode of protein retention. Therefore, X-ray photoelectron spectroscopy is used in a way (angle resolved measurements, reasoned peak decomposition, validation by quantitative relationships between spectral data) to provide a speciation in terms of classes of compounds (silane, protein, contaminants), using guidelines established in previous works (Genet et al., 2008; Rouxhet & Genet, 2011). Water contact angle measurements are used to address the issue of the presence of contaminants and the perspectives of avoiding it.


Silanization with APTES for Controlling the Interactions

a. Surface polishing; rinsing in water and ethanol.

Tantalum coating DS3; DSS; DSC;

implantation). Blanks = native, silanized.

typical structure of fibrillar collagen.

a. Plasma treatment.

c. -

for Ti-6Al-4V. Blank = native + enzyme, silanized + enzyme.

SIMS: demonstration of silanization and protein retention.

d. Wet chemical analysis of amino groups on silanized substrate only.

CDI

a. Acid etching; rinsing with different solvents; drying in vacuum.

c. Antigen/antibody test. Influence of plasma exposure time.

a. Oxidation and hydroxylation; rinsing with different solvents.

a. Oxidation in a solution of ammonia and hydrogen peroxide. b. Vapor deposition; curing 40 min at 80 °C under vacuum.

c. Increase of fibroblast proliferation. Blanks = no RGD.

silanized + LC-SPDP with blocked termination.

a. Cleaning in different solvent.

curing 15 min at 120°C.

b. In toluene, 24 h at 70 °C; rinsing in different solvents; sonication in ethanol.

d. XPS: elemental concentration, consistent evolution according to reaction steps. Tof-

b. In boiling toluene under argon; rinsing in chloroform, methanol, and in water. c. Increase of cell adhesion and proliferation (stem cell culture and subcutaneous

b. In ethanol/water (95/5, v/v), rinsing with ethanol ; curing 15 min at 120 °C.

b. In dry toluene, 120 °C, 3 h; sonication in various organic solvents and water.

small clumps after APTES step ; larger clumps after APTES+SMP step.

elemental concentration, consistent evolution according to reaction steps.

Glass none Proteins from ECM,

c. Improvement of osteoblast adhesion and growth. Blank = silanized.

d. Wet chemical analysis: amino groups, collagen, consistent with expectation. AFM:

SiO2, TiO2, Si3N4 GA Antigen Kim et al., 2009b

d. Water contact angle and XPS on cleaned substrate, influence of plasma exposure time. Silicon dioxide SMP RGD peptide Davis et al., 2002

d. XPS: elemental concentration, consistent evolution according to reaction steps. AFM:

c. Slight decrease of the enzyme activity when the surface is not oxidized (a). No blank. d. AFM: consistent evolution of surface roughness according to reaction steps. XPS:

d. Ellipsometry and water contact angle: consistent evolution according to reaction steps.

b. In anhydrous ethanol + acetic acid, followed by addition of ultrapure water, 5 min;

RGD peptide

Siperko et al., 2006

Silicon dioxide GA Glucose oxidase Libertino et al., 2008

Silicon dioxide LC-SPDP Tagged Kcoil peptides Boucher et al., 2009 a. Piranha treatment, 10 min at 100°C; rinsing in water; drying in air; storing in vacuum. b. In anhydrous toluene, 3 h; curing 1.5 h at 120°C; sonication in freshly distilled toluene. c. Improvement of binding efficiency (amount, affinity*)* of Ecoil-tagged EGF. Blank =

Between Stainless Steel and Biocomponents: Reality vs Expectation 103

Magnesium Ascorbic acid BSA Killian et al., 2010

Collagen Müller et al., 2005


bisphosphonate

North et al., 2004

2004

haemocompatibility (in vivo and in vitro tests). Blank = native stainless steel stent,

b. Vapor deposition, 10 min at 60 °C; curing 1 h at 150°C; sonication in xylene.

Stainless steel screw GA Fibrinogen +

a. Acid etching; rinsing with different solvents; outgassing.

b. In toluene, 24 h; sonication in toluene, ethanol and water.

d. QCM on model substrate slide: in situ monitoring of the LbL film growth on APTES-

a. Sonication in acetone; acid etching; treatment in H2O2/NH4OH solution, 5 min at 80 °C.

c. Improvement of fixation of screws in rat tibia. Blank = test of bisphosphonate action. d. Ellipsometry on model substrate slide: consistent increase of the film thickness

b. In dry toluene, 120 °C, 3 h plus variants; sonication in various organic solvents and

a. Polishing; cleaning in different solvents followed by nitric acid passivation or piranha

d. Ellipsometry and radiolabeling: growth of silane surface layer upon repeating treatments. XPS peak shapes: semi-quantitatively consistent evolution according to

d. XPS: elemental concentration, consistent evolution according to reaction steps.

c. Improvement of blood compatibility and promotion of endothelialization. Blanks =

d. XPS : survey spectra consistent with treatments. AFM: small clumps after APTES step, and after APTES + protein step. Wet chemical analysis of proteins: consistent evolution

Ti-6Al-4V SMP Cyclic peptides Porté-Durrieu et al.,

b. In anhydrous ethanol, 10 h, sonication in ethanol; curing 6 h at 120 °C.

b. In dry hexane under argon; rinsing with dry hexane; outgassing.

c. Increase of osteoprogenitor cells attachment. Blanks = native, native + peptide. d. XPS: elemental concentration, consistent evolution according to reaction steps; N 1s peak, demonstration of peptide retention; C 1s peak, majority of carbon of C-(C,H) type at

Co-Cr-Mo; Ti-6Al-4V GA Trypsin Puleo, 1997

c. Decrease of the loss of enzymatic activity as a function of time for Co-Cr-Mo, no effect

Titanium SMP RGDC peptide Xiao et al., 1997

Titanium GA Chitosan Martin et al., 2007

Titanium none Heparin/fibronectin Li et al., 2011 a. Polishing; sonication in different solvents, drying 2 h at 60 °C ; NaOH treatment, 2h at

native+chitosan.

water. c. -

reaction steps.

treatment.

c. -

80°C.

native, silanized.

all stages.

according to reaction steps.

a. Substrate oxidation; outgassing at 150 °C.

a. Cleaning in different solvent, acid passivation.

b. In water 3 h or acetone 10 min; curing at 45 °C overnight.

coated silicon substrate.

according to reaction steps.


Silanization with APTES for Controlling the Interactions

purchased from Pierce (Rockford, IL, USA).

**2.2 Stainless steel surface preparation** 

**2.3 Surface treatment procedure** 

the enzyme (Gox) as detailed below:

under nitrogen gas flow.

nat and sil samples, respectively,

excess of non attached silanes. These are called "sil".

contact angle measurements as a function of time.

**2.4 X-ray photoelectron spectroscopy** 

pH~6.8) for 2 h, on nat and sil samples, respectively,

Between Stainless Steel and Biocomponents: Reality vs Expectation 105

were purchased from Sigma-Aldrich (France). Bis(sulfosuccinimidyl) suberate (BS) was

The samples (both faces and perimeter) were polished with 1 µm diamond suspension (Struers, Denmark), rinsed in binary mixture of milliQ water/ethanol (1/1, v/v) in a sonication bath (70W, 40 kHz, Branson, USA) and dried under nitrogen gas flow.The samples were then immediately immersed for 48 h in synthetic aqueous medium (NaCl 0.46 mmol.L-1, Na2SO4 0.26 mmol.L-1, NaNO3 0.2 mmol.L-1, NaHCO3 3.15 mmol.L-1, pH about 8), abundantly rinsed with milliQ water (Millipore, Molsheim, France) and dried under nitrogen gas flow. These samples are considered as native SS and designated as "nat".

The silanization of nat samples was performed in the gas phase with APTES. To this end, nat samples were placed, together with a small vial containing 100 µL APTES, in a 7 dm3 closed recipient under vacuum for 30 min at room temperature. The samples were cured for 1 h at 100°C under vacuum, rinsed and incubated for 6 h in milliQ water to eliminate the

Both nat and sil samples were subjected to treatments with the coupling agent (BS) and/or

i. "+BS" and "sil+BS" obtained after BS treatment (10 mM in milliQ water) for 30 min on

ii. "+Gox" and "sil+Gox" obtained after Gox treatment (0.1 mg.mL-1 in phosphate buffer

iii. "+BS+Gox" and "sil+BS+Gox" obtained after both BS and Gox treatment (according to

After BS or Gox treatment, the samples were rinsed three times with milliQ water and dried

In order to clarify the issue of surface contamination, nat samples were further cleaned by UV-ozone treatment (UVO Cleaner, Jelight Co, Irvine, Ca, USA) during 20 minutes. They were then placed in a Petri dish, left in the laboratory environment, and submitted to water

XPS analyses were performed using a Kratos Axis Ultra spectrometer (Kratos Analytical, UK), equipped with a monochromatized aluminum X-ray source (powered at 10 mA and 15 kV) and an eight channeltrons detector. No charge stabilization device was used on these conductive samples. Analyses were performed in the hybrid lens mode with the slot aperture; the resulting analyzed area was 700 µm ¯ 300 µm. A pass energy of 40 eV was used for narrow scans. In these conditions, the full width at half maximum (FWHM) of the Ag 3d5/2 peak of clean silver reference sample was about 0.9 eV. The samples were fixed on the support using a double-sided adhesive conducting tape. The pressure in the analysis chamber was around 10-6 Pa. The photoelectron collection angle *θ* between the normal to the sample surface and the electrostatic lens axis was 0° or 60°. The following sequence of spectra was recorded: survey spectrum, C 1s, O 1s, N 1s, P 2p, Cr 2p, Fe 2p, Ni 2p, Mo 3d, Na 1s, S 2p, Si 2p and C 1s again to check for charge stability as a function of time, and absence of sample degradation. The binding energy scale was set by fixing the C 1s

the procedure described above) on nat and sil samples, respectively.

