Engineering Surfaces for Bioconjugation: Developing Strategies and

1146

Bioconjugate Chem. 2004, 15, 1146−1156

TECHNICAL NOTES Engineering Surfaces for Bioconjugation: Developing Strategies and Quantifying the Extent of the Reactions Virginie Gauvreau,† Pascale Chevallier,†,‡ Karine Vallie`res,†,‡ E Ä ric Petitclerc,†,§ †,§ ,†,‡ Rene´ C.-Gaudreault, and Gae´tan Laroche* Unite´ de Biotechnologie et de Bioingeńierie, Centre de Recherche de l’Hoˆpital Saint-Franç ois d’Assise, C.H.U.Q., 10 rue de l’Espinay, Que´bec, Que´bec, Canada, G1L 3L5, De´partement de Geńie des Mines, de la Me´tallurgie et des Mate´riaux, Centre de Recherche en Science et en Ingeńierie des Macromolećules, Faculte´ des sciences et geńie, Universite´ Laval, Que´bec, Que´bec, Canada, G1K 7P4, and De´partement de Me´decine, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Que´bec, Canada, G1K 7P4. Received June 18, 2004

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene) (PTFE) surfaces for subsequent conjugation of biologically relevant molecules. First, PTFE films were treated by a radiofrequency glow discharge (RFGD) ammonia plasma to introduce amino groups on the fluoropolymer surface. This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups, as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy (XPS). In a second step, these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides, carboxylic acids, acetals, aldehydes, and thiols, that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins, DNA, drugs, etc. In the present study, glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface. Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface, providing a diethylacetal function, which is a latent form of aldehyde functionality. Reactions with cross-linkers such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB) were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules. Traut reagent (2-iminothiolane) was also conjugated to introduce a thiol group onto the fluoropolymer surface. PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer. Finally, surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.

INTRODUCTION

Nowadays, biomolecule grafting onto polymer surfaces is often used for biological detection involving viruses, DNA, proteins, etc. (1-5), as well as for improving biomaterial surface properties such as hemocompatibility and biocompatibility (6-16). Most of the time, the conjugation procedures involve the chemical modification of the biomolecules to be grafted with appropriate functionalities and subsequent reactions involving reactive groups on the surface (1-4). However, such biomolecule chemical modifications often lead to denaturation or polymerization phenomena that alter or nullify their biological activity. It seems therefore more suitable to modify the surface onto which the molecule should be conjugated with appropriate chemical functionalities and thereafter proceed to the conjugation of the native and optimally active biomolecule. The first step of the latter * Corresponding author. Phone: (418) 656-2131 ext. 7983. Fax: (418) 656-5343. E-mail: [email protected]. † Centre de Recherche de l’Ho ˆ pital Saint-Franç ois d’Assise. ‡ De ´ partement de Geńie des Mines, Universite´ Laval. § De ´ partement de Me´decine, Universite´ Laval.

strategy involves polymer surface modification with reactive functionalities for further chemical reaction with various reagents that will provide the moieties able to bind biomolecules. In this context, radiofrequency glow discharge (RFGD) plasma treatment was proven in the past to be a technique of choice as it allows the incorporation of reactive moieties onto the polymer surfaces with no modification to the bulk material (17, 18). This approach was successfully used to functionalize the surface of polymers such as polyethylene, polypropylene, polyesters, and poly(tetrafluoroethylene) (PTFE) (19-32). Because of some favorable properties such as high electrical resistivity and good thermal and chemical stability, PTFE has been the subject of several investigations to modify its surface through plasma treatments in order to benefit from its bulk characteristics while modulating its surface behavior (19, 21, 22, 24, 26-35). In the 1960s, Hansen and Schonhorn were the first to employ plasma treatments to modify the hydrophobic PTFE surface and succeeded in rendering it hydrophilic (32). In addition, plasma surface modifications enable the introduction of new chemical functionalities onto the surface depending on the gases employed. For example,

10.1021/bc049858u CCC: $27.50 © 2004 American Chemical Society Published on Web 08/20/2004

Technical Notes

ammonia is used to provide amino groups (20, 24, 28, 31, 36, 37) as well as other nitrogen-containing moieties on the modified surface. Plasma treatments involving CO2, H2O, and CO2/H2O, to introduce carboxylic acid groups onto the surface, were also largely utilized as well as treatments with H2/H2O, O2/H2O and H2/O2 leading to alcohols and other oxygen-containing functionalities (19, 23, 38, 39). However, performing plasma treatments with different gases requires exhaustive optimization of the reaction parameters such as gas pressure and flow, treatment time, power of the radiofrequency field, to maximize the reactive moieties surface concentration while minimizing surface damages. The optimization of these plasma treatments requires extensive characterization of the modified surfaces, control of the plasma composition, and the plasma-polymer interaction through the use of in-situ plasma spectroscopic quantifications and X-ray photoelectron spectroscopy (XPS) surface analyses. In this context, our strategy to introduce various chemical functionalities onto the PTFE surface consists of treating the polymer using a fully optimized radiofrequency glow discharge (RFGD) ammonia plasma reactor during the first step of the procedure (31). Using appropriate experimental conditions, this protocol allows the maximization of the amino group surface concentration while almost completely eliminating surface damages. In addition, this surface treatment was proven to be noncytotoxic (36, 37), therefore enabling its use in several biological applications where living tissues are involved. The nucleophilic surface amino groups are further used for the covalent binding of various chemical reagents which, in turn, allow the grafting of useful functionalities such as maleimides, carboxylic acids, thiols, acetals and aldehydes. The presence of these reactive surface functionalities on the polymer opens the door to a wide variety of biomolecule conjugation strategies. In this study, glutaric anhydride was used to introduce carboxylic groups at the surface of the ammonia plasmatreated PTFE films. Likewise, cis-aconitic anhydride was reacted with the aim to double the surface concentration of carboxylic groups obtained as compared to glutaric anhydride grafting. Following appropriate activation, acidic moieties are able to react with amino groups to form stable amide bonds. Moreover, reaction with bromoacetaldehyde diethylacetal (BADEA) was also evaluated to provide ethylacetal functionalities on the PTFE ammonia plasma-treated surface. Acetals are commonly used as latent protecting groups for aldehydes (40). Once the acetal group is removed, the regenerated aldehyde can react swiftly with aminated entities. Additionally, the reaction of sulfo-succinimidyl-4-(N-maleidomethyl)cyclohexane-1-carboxylate (S-SMCC) and similarly the reaction of sulfo-succinimidyl-4-(p-maleimidophenyl)butyrate (S-SMPB) with the surface amino moieties were assessed to provide a maleimide functionality suitable to carry out further chemical conjugations of thiolated molecules such as fibronectin (41). Finally, 2-iminothiolane was used to introduce a thiol function onto the fluoropolymer surface that can be used afterward for the coupling of either S-SMCC- or S-SMPB-modified molecules. Particular attention was paid to quantify the extent of the chemical reactions performed on the polymer surface. Along with calculations originating from surface derivatization strategies, X-ray photoelectron spectroscopy is particularly well suited to perform such measurements. This spectroscopic technique was also very useful

Bioconjugate Chem., Vol. 15, No. 5, 2004 1147

for the detection of specific functionalities present through peak fitting of core level spectra, especially that of the carbon 1s orbitals. This spectral region presents a relatively good sensitivity and a wide literature associating the various binding energies to the corresponding carbon binding states (42). EXPERIMENTAL PROCEDURE

Materials. Poly(tetrafluoroethylene) (Teflon) films were purchased from Goodfellow (Huntingdon, England). Phosphate-buffer-saline (PBS, pH 7.4) was purchased from VWR Canlab (Ville Mont-Royal, QC, Canada) while 2-(N-morpholino)ethanesulfonic acid buffer (MES, pH 4.75) was obtained from Sigma-Aldrich (Milwaukee, WI). Molecules to be grafted such as glutaric anhydride, cisaconitic anhydride, bromoacetaldehyde diethylacetal, sulfo-SMCC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), Lys-Lys dimer, human fibronectin, and reagents such as 5-bromosalicylaldehyde and sodium cyanoborohydride were obtained from SigmaAldrich (Milwaukee, WI). Other molecules to be immobilized onto PTFE such as 2-iminothiolane (Traut reagent) and sulfo-SMPB were acquired from Pierce (Brockville, ON, Canada). Buffer solutions were prepared in filtered, deionized water (Nanopure system) at 0.2 M. Fine adjustments of the pH (7.4 or 8.0, depending on the reaction to be performed) were made by adding either 0.1 M of HCl or NaOH to reach the desired value. All chemicals were of commercial grades of highest purity and were used without further purification. The cell culture experiments were performed with the HT1080 human fibrosarcoma cell line (American Type Cell Culture, Manassas, VA) in Dulbecco’s modified Eagle’s Medium (DMEM) (Sigma-Aldrich, Milwaukee, WI) supplemented with penicillin, streptomycin, and glutamine (Life Technologies, St Paul, MN) using 96-well tissue culture plates (Fisher Scientific, Whitby, ON, Canada). Methods. Preparation of PTFE Surfaces. PTFE surfaces of 3 cm × 3 cm were cleaned ultrasonically for 10 min successively after immersion in high purity methanol, deionized water, and acetone. The films were then air-dried and kept under vacuum until use. The cleaning procedure was validated by XPS showing the presence of only carbon and fluorine in the appropriate stoichiometric ratio. Plasma Treatment. The ammonia plasma treatment and system apparatus were described elsewhere (22, 31). Briefly, the treatments were performed at a power of 20 W and a pressure of 300 mTorr for 250 s using high purity ammonia gas. The PTFE films were held by a Teflon-made circular sample holder and treated in a cylindrical plasma chamber. Only the internal side of the film was treated using such a plasma configuration and geometry. Characterization. Immediately following the ammonia plasma treatment, the aminated-PTFE film was cut into nine one-cm2 samples in a glovebox purged with dry nitrogen in order to minimize the surface oxygen uptake. Three samples, one coming from each row and column, were analyzed by XPS without delay to assess the exact atomic composition of the surface following the plasma treatment. The XPS spectra were recorded using a PHI 5600-ci spectrometer (Physical Electronics, Eden Prairie, MN). A monochromatic aluminum X-ray source (1486.6 eV) at 400 W with neutralizer was used to record the survey spectra while high-resolution spectra were obtained using the monochromatic magnesium X-ray source (1253.6 eV) at 400 W without charge neutralization. The

1148 Bioconjugate Chem., Vol. 15, No. 5, 2004

Technical Notes

detection was performed at 45° with respect to the surface normal. Three other samples were used to quantify the amine surface concentration through vapor-phase chemical derivatization using 5-bromosalicylaldehyde as described previously (31). Briefly, the reaction was performed at 85 °C for 2 h in a sealed glass tube in which a 1 cm-thick bed of soda-lime glass beads was used to separate the reagent from the reactive surface. The surfaces were then vacuum-dried overnight at 40 °C and analyzed by XPS. Taking into account the nine newly bonded atoms upon reaction of 5-bromosalicylaldehyde with the surface amino groups, the amine surface concentration can be determined through eq 1.

[%NH2] ) [%Br]/(100 - 9 × [%Br]) × 100

(1)

Yield Calculations. The atomic percentages following plasma treatment and the amine surface concentration are essential to calculate the theoretical atomic percentages expected for any given chemical reaction aiming at providing a new chemical functionality on the ammonia plasma-treated PTFE. Basically, the efficiency of a surface chemical reaction may be calculated from the knowledge of the surface concentration variation for any atom present on the surface before and after the chemical grafting reaction as determined from XPS as described in eq 2.

[%A] )

[A]0 + y × [NH2]0 100 + w × [NH2]0

× 100

(2)

In this equation, A is the atom for which the surface concentration is measured by XPS, [A]0 is the surface concentration of the atom A on the plasma-treated film surface measured readily after the plasma treatment, [NH2]0 is the amine surface concentration determined from the vapor-phase chemical derivatization, y and w are the number A-type atoms and the total number of atoms in the newly bonded molecule, respectively. The conversion factor, x, is equal to 0 in the case where no reaction occurs and 1 in the case of a complete reaction. The denominator in the right term takes into account for the total number of newly added atoms coming from the reagent as well as the atoms lying underneath and probed by the XPS analyses. The yields of the chemical reactions were obtained from the ratio between the experimental atomic percentage measured by XPS analyses and the calculated theoretical atomic percentage for a particular atom. Indeed, one would expect the atom A experimental relative surface concentration to increase following a chemical reaction with a molecule containing a higher atom A concentration ratio than that measured on the ammonia plasma-treated PTFE surface. Chemical Reactions. The last three samples to be grafted with the various chemical reagents were placed individually in three 5 mL polypropylene tubes in such a manner to avoid mechanical overlay of the samples that would interfere with the chemical reaction. For each of the three PTFE-amino modified samples grafted with a molecule, five punctual areas were analyzed by XPS. Moreover, each grafting was made in triplicate to ensure that the values presented therein are statistically significant. Grafting of Anhydrides. A solution of 55 mg/mL of glutaric anhydride in acetone was prepared in a glovebox under a dry nitrogen atmosphere. The reaction was

performed for 60 min at room temperature under mechanical agitation. After 20 and 40 min, the concentration of glutaric anhydride was raised to 65 and 75 mg/mL respectively, to ensure completion of the reaction. Similarly, acetonic solution of cis-aconitic anhydride at a concentration of 100 mg/mL was prepared in a glovebox purged with nitrogen. The reaction mixture was mechanically agitated at room temperature for 120 min. After 30 and 60 min, the concentration of cis-aconitic anhydride in the solution was increased to 125 and finally 150 mg/mL. One cm2 films were reacted with 1 mL of the anhydride acetonic solution. Grafting of BADEA. Plasma-treated films were also reacted with bromoacetaldehyde diethylacetal. The aminated-PTFE samples were immersed individually in 1 mL of pure bromoacetaldehyde diethylacetal and agitated for 2 h at room temperature. To ascertain the success of the reaction, the diethylacetal function was further hydrolyzed afterward in a 10% HCl solution stirred at room temperature for 2 h (40). The films were then thoroughly washed with deionized water and the aldehyde functionality was used for the subsequent grafting of the dipeptide Lys-Lys. A solution of 4 mg of Lys-Lys in 1 mL PBS at pH 7.4 was stirred for 5 min at room temperature, and the imine bond formed was immediately reduced to amine by adding 8 mg of NaBH3CN to the reaction mixture that was stirred for 3 h at room temperature. Grafting of Maleimide Derivatives. Sulfo-SMCC and sulfo-SMPB were reacted with the ammonia plasmatreated film samples in 1 mL of 3 mg/mL solutions of reagent in PBS at pH 7.4. The reactions were then allowed to proceed for 2 h under a nitrogen atmosphere and under low light intensity to protect the reactants. Generation of Thiolated Functionalities. Finally, 2-iminothiolane was also reacted with the aminated-PTFE surface immediately after the plasma treatment. The samples were reacted in solutions containing 30 mg/mL of 2-iminothiolane in PBS at pH 8.0. The reaction was allowed to proceed under constant agitation for 2 h under a nitrogen atmosphere. Meanwhile, to ascertain the completion of the reaction, the cross-linking agent SSMPB was conjugated to Lys-Lys in a solution containing 5 mg of S-SMPB (1.09 mmol) and 7.6 mg of Lys-Lys (2.18 mmol) in 1 mL of PBS at pH 7.4 (41). Lys-Lys was added in excess to ensure that all the terminal succinimidyl groups would react exclusively with the dipeptide’s amino groups instead of unreacted amines on the PTFE surface if there were any. This mixture was stirred for 2 h at room temperature. The rinsed thiolated films were then immersed in the solution containing the S-SMPB-derived dipeptide and the reaction allowed to proceed for 2 h under constant mechanical agitation at room temperature. Grafted films were all washed thoroughly with deionized water. BADEA grafted films were first washed with methanol and then with deionized water. Afterward, the films were vacuum-dried overnight at ambient temperature before analysis by XPS. Fibronectin Grafting. Prior to the immobilization of human fibronectin, glutaric anhydride-grafted films were activated with a solution containing 3 mg/mL of EDC in MES buffer (0.1 M, pH 4.75). Two subsequent additions of 3 mg of EDC were made to this solution every 10 min during 20 min at room temperature and under stirring (for a total reaction time of 30 min) to minimize the effect of the water-induced hydrolysis of the activator on the grafting efficiency. These films were then extensively washed with the MES buffer. Both the glutaric anhydride-

Technical Notes


Table 1. Average Atomic Percentages, Amine Surface Concentration, and Atomic Ratios as Probed by XPS for Virgin PTFE and Plasma-Treated PTFE Films films

%C

%F

%N

%O

%NH2a

F/C

O/C

N/C

virgin plasma-treated

32.2 46.6 ( 2.9

66.6 38.8 ( 4.6

11.2 ( 1.8

1.2 2.9 ( 0.9

2.5 ( 0.6

2.07 0.83

0.04 0.06

0.24

a

Determined through vapor-phase chemical derivatization using 5-bromosalicylaldehyde.

grafted films activated with EDC and the S-SMPB grafted films were reacted with a PBS solution containing 10 µg/mL of human fibronectin at pH 7.4 for 3 h under mechanical agitation. They were washed five times with deionized water and cut in 5.9 mm diameter circles with an awl to fit in a 96-well tissue culture plate. Cell Adhesion Experiments. Cell Culture. The HT1080 human fibrosarcoma cell line was used for all the experimentation. The culture medium was Dulbecco’s modified Eagle’s Medium (DMEM) with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.3 mg/mL glutamine, 100 µM citrate, and 0.1% BSA. The cells were cultured at 37 °C and 98% humidity in air containing 5% CO2. Cell Adhesion on Modified PTFE. To check the availability of the cell interaction sites on the PTFE bound fibronectin, adhesion tests were performed with HT1080 cells known for their strong affinity to adhesion sequences of the protein. 5.9 mm diameter circles of clean PTFE, ammonia plasma-treated PTFE, ammonia plasmatreated PTFE conjugated with glutaric anhydride, fibronectin conjugated on ammonia plasma-treated PTFE through glutaric anhydride, ammonia plasma-treated PTFE conjugated with S-SMPB, and fibronectin conjugated on ammonia plasma-treated PTFE through SSMPB were prepared and put in a 96-well tissue culture plate, with the treated face up. Human fibronectin (10 µg/mL in PBS at pH 7.4) was coated 2 h at 37 °C in five empty wells and washed twice with PBS. All wells were blocked with 1% BSA overnight at 4 °C, to prevent any nonspecific cell adhesion, and washed twice with PBS. The HT1080 cells in culture were harvested with trypsin, counted, and diluted in 10% DMEM serum and 0.1% BSA to reach a final concentration of 250 000 cells/mL. An amount of 50 000 cells was seeded in each well, and the plate was incubated for 10 min at 37 °C. The wells were then washed twice with DMEM without serum, and cells were stained with violet crystal for 20 min. After washing, all wells were photographed and the number of cells per square millimeter was counted. Statistical analysis of the difference between each group was performed using the analysis of variance (ANOVA) followed by Dunnett’s test where appropriate. P values lower than 0.01 were considered significant and all data were expressed as mean ( standard error (SE). RESULTS

Surface Composition of the Plasma-Treated Surface. The relative surface concentrations of carbon, fluorine, nitrogen and oxygen atoms as well as the amine concentration were determined for each sample to control the efficiency of each reaction step. The relative atomic percentage of nitrogen detected on the PTFE surface film after plasma treatment was 11.2% on average. Among these nitrogen-containing species, approximately onefifth were shown to be amino moieties as measured by derivatization with 5-bromosalicylaldehyde (Table 1). These results are in agreement with those already available from the literature (22, 27, 31). Selection of the Atom Used as XPS Probe To Follow the Extent of Surface Reaction. For the

Figure 1. Typical XPS spectra of ammonia plasma-treated PTFE surfaces before (a) and after (b) immersion in PBS, showing that the oxygen surface concentration is only slightly affected upon contact with the aqueous solution.

purpose of this study, it seemed that in all cases the oxygen content probed at the modified surface by XPS analyses was the best choice for calculating the yield of the reactions. Indeed, the surface concentration of this atom is only slightly affected upon exposure to solvents such as PBS as demonstrated in Figure 1. In addition, the molecules grafted on the PTFE surface are heavily loaded with oxygen atoms. In fact, the newly bonded molecules contained between one and five oxygen atoms. The ratio O/n (where n is the total number of newly bonded atoms) were of 3/8 or 38% for glutaric anhydride, 5/11 or 45% for cis-aconitic anhydride, 2/8 or 25% for BADEA, 1/6 or 17% for 2-iminothiolane, 3/16 or 19% for SMCC, and finally 3/18 or 17% for SMPB, respectively. The relative oxygen concentration of these molecules is by far higher than that measured on the plasma-treated PTFE surfaces exposed to PBS. In addition, our XPS data (not shown) demonstrated that, once grafted with the various molecules investigated, the surfaces were stable over time. It should be pointed out, on one hand, that both fluorine and carbon surface concentrations, despite leading to less relative variations than those measured for oxygen upon molecule conjugation, provided very similar evaluation of the reaction yields. On the other hand, nitrogen is clearly a less suitable probe, as some of the nitrogen-containing species, other than amino groups, were shown to be readily removed from the surface upon exposure to water, which is the solvent used to perform some of the reactions presented therein. Possible explanations of this phenomenon are related to displacement of polar groups from the surface into the bulk, hydrolysis of surface imine moieties also created during the ammonia plasma treatment, and dissolution in water of low molecular weight fragments known to be formed during the plasma process (22, 31). Therefore, this leads to an overestimation (more than 80% in the present case) of the initial nitrogen surface concentration, required to perform an accurate calculation of the reaction


Technical Notes

Table 2. Glutaric Anhydride Grafting. The [NH2]0 Average for These Plasma Treatments Was 2.4% plasma treatment glutaric anhydride theoretical values ∆ experimental ∆ expected % yield

%C

%F

%N

%O

47.1 ( 1.3 52.5 ( 0.6 49.6 +5.4 +2.5 106 ( 3

39.7 ( 3.0 35.1 ( 1.8 33.3 -4.6 -6.4 95 ( 9

11.3 ( 1.1 5.1 ( 0.9 9.5 -6.2 -1.8 54 ( 9

1.9 ( 0.2 7.3 ( 0.7 7.6 +5.4 +5.7 96 ( 6

yields. As XPS probes relative surface concentrations, the loss of nitrogen (8.2% from Figure 1) is mainly reported in the relative concentrations of both carbon and fluorine (Figure 1) and therefore leads to less important relative overestimations, as the surface concentration of these two atoms is inherently higher than that of nitrogen. Grafted Molecules. These latter observations are further supported through the XPS data recorded in the case of glutaric anhydride conjugation on ammonia plasma-treated PTFE (Table 2). As depicted in Table 2, the oxygen surface concentration increased by 5.4% upon glutaric anhydride grafting. Therefore, based on this atom surface concentration, it can be confirmed that the glutaric anhydride grafting is very successful, the yield of the reaction being near 100%. Both carbon and fluorine XPS signals, with surface variations of +5.4% and -4.6%, respectively, lead to similar conclusions with calculated reaction yields reasonably close to 100% (106 ( 3% and 95% ( 9 using the carbon and fluorine XPS signal, respectively). Performing the calculations with the nitrogen signal leads to a very different trend with an extent of reaction of 54 ( 9%, in agreement with the fact that the initial nitrogen surface concentration is overestimated due to the removal of some of the nitrogencontaining species upon exposure to water. The successfulness of this reaction is confirmed more visually by the comparison of the XPS spectrum for each steps of the glutaric anhydride grafting process (Figure 2). Nitrogen is obviously not observed on the virgin PTFE surface and presents a surface concentration of 11.3% immediately after the ammonia plasma treatment. The amino group surface concentration on this particular aminated-surface was assessed to be 2.4% through surface derivatization and allowed the grafting of glutaric anhydride thereafter raising the carbon and oxygen peaks from 47.1% and 1.9% to 52.5% and 7.3%, respectively, while diminishing the fluorine and nitrogen peaks from 39.7% and 11.3% to 35.1% and 5.1%, respectively. The conjugation of glutaric anhydride to aminatedPTFE being successful, further studies were made using cis-aconitic anhydride. Reaction of cis-aconitic anhydride with the amino groups provides two free carboxylic groups for every grafted molecule (Figure 3a), hence theoretically doubling the density of carboxylic acid group surface concentration as compared to the glutaric anhydride conjugation. In addition, cis-aconitic anhydride is recognized to form labile amide bonds with drugs such as daunorubicin that slowly hydrolyze in aqueous solutions to release the drug in its native form (43). On the basis of the oxygen surface concentration, the cis-aconitic anhydride yielded to a 76% reaction with the amines. The expected 2-fold increase of the carboxylic acid functionalities was therefore not reached. Instead, conjugation with cis-aconitic anhydride allows adding approximately 1.5 times more carboxylic acid moieties onto the polymer surface as compared to the aforementioned grafting of glutaric anhydride. As predicted from the molecular structure of cis-aconitic anhydride, XPS spectra showed an increase in carbon and oxygen atomic content, from 47.2% and 3.0% to 54.4% and 9.7%, respectively, whereas

Figure 2. (a) Synthetic scheme of glutaric anhydride grafting onto an ammonia plasma-treated PTFE film. (b) Spectra for plasma-treated PTFE film and glutaric anhydride-grafted PTFE film. [NH2]0 was 2.4% and the yield of the reaction 96 ( 6% based on the oxygen surface concentration.

the fluorine and nitrogen content decreased along from 35.6% and 13.8% to 30.5% and 5.3%, respectively (Figure 3b). Grafting of both anhydrides was successful, and thereafter the free carboxylic groups generated could further be activated by several carboxyl-activating agents (5, 10, 41, 44) such as N-hydroxysuccinimide (NHS), 1-ethyl-3(3-(dimethylamino)propyl)carbodiimide (EDC), and carbonyldiimidazole (CDI) to conjugate numerous organic or biologic molecules containing amine functionalities, therefore leading to the formation of an amide link between the biomolecule and the polymer surface. Bromoacetaldehyde diethylacetal (BADEA) also allows the conjugation of amino group-containing biomolecules. However, in this case, the reaction of the imine group generated in the reaction between the PTFE-aldehyde and the amine-bearing biological molecule with the borohydride leads to the formation of a substituted amine, which is stable to hydrolysis. Grafting of BADEA was also successful, with a reaction yield of 57% according to the oxygen surface concentration (Figure 4). Although the atomic percentages obtained from XPS seem to point toward at least a partially grafted surface,

Technical Notes

Figure 3. (a) Synthetic scheme of cis-aconitic anhydride grafting onto an ammonia plasma-treated PTFE film. (b) Spectra for plasma-treated PTFE film and cis-aconitic anhydridegrafted PTFE film. [NH2]0 was 2.7% and the yield of the reaction 76 ( 15%.

Figure 4. (a) XPS spectra for each step of bromoacetaldehyde diethylacetal grafting and subsequent reaction with dipeptide Lys-Lys. (b) Synthetic scheme of bromoacetaldehyde diethylacetal grafting onto an ammonia plasma-treated PTFE film, subsequent reduction of the diethylacetal into aldehyde functionality, and further conjugation with Lys-Lys. [NH2]0 was 2.5% and the yield of the reaction of BADEA with the amino groups 57 ( 8%

the reaction was not complete enough to ascertain the grafting. Therefore, to establish whether the BADEA grafting was successful and provided ethylacetal functionality, this functionality was hydrolyzed into the more nucleophilic aldehydic group. The aldehyde moiety provides only three newly bonded atoms to the PTFE surface compared to the plasma-treated analyzed surface. In


other words, hydrolysis of the ethylacetal functionality decreases by five the number of atoms as compared to the surface grafted with BADEA before the hydrolysis that is too small to ascertain its presence on the surface. Thus, it was further reacted with the dipeptide Lys-Lys, and immediately the imine group was reduced into a stable amine using sodium cyanoborohydride (Figure 4b). This reaction provides 18 supplementary atoms on the analyzed surface, well enough to confirm its presence and therefore provided indirect solid evidences for the bromoacetaldehyde diethylacetal grafting successfulness. This is confirmed by the atomic percentages obtained at each step of the process and more visually by the XPS spectra of each of these steps (Figure 4a). The oxygen content increases from 3.3% to 4.4% upon hydrolysis of the diethylacetal moieties and finally to 6.4% after conjugation of Lys-Lys to the BADEA-grafted surface. The small increase of the relative atomic oxygen percentage upon hydrolysis of the acetal groups, despite being surprising is normal because the relative oxygen content increases from 25% (two oxygen atoms out of eight atoms total) to 33% (one oxygen atom out of three atoms total). Grafting of sulfo-succinimidyl-4-(N-maleidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) on the aminated-PTFE (Figure 5a) was also studied in order to bind the biomolecules through their thiol groups (41). It appeared that the reaction between sulfo-SMCC and the amine moieties present at the surface of the PTFE plasma-treated film was very successful, with a quantitative reaction yield, considering the experimental error. The efficacy of the reaction is depicted in Figure 5c: the carbon content increased from 46.1% to 54.5% while the oxygen content increased from 2.2% to 7.9%. Accordingly, the fluorine and nitrogen content decreased from 42.1% and 9.7% to 30.9% and 6.0%, respectively. The ability of sulfo-succinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB) to react with the amine moieties (Figure 5b) was also tested since, as a cross-linker, it provides the same double bond functionality as SSMCC with a longer spacer arm, allowing to conjugate a second molecule further apart from the PTFE surface. XPS analyses showed the presence of 0.6% of sulfur (Figure 5d). These sulfur atoms must be originating from the sulfo group of the cross-linker. As aforementioned, sulfur exhibits a low XPS sensibility index and its presence added to an oxygen content superior than expected indicates that a competitive and unexpected reaction takes place. This reaction would allow the SO3 part of S-SMPB to be grafted or adsorbed on the polymer surface, hence increasing the oxygen and sulfur content. The oxygen atomic percentage reported is therefore widely increased by this unknown reaction that explains the 163% yield calculated according to the oxygen content. Nevertheless the S-SMPB reaction is successful and the double bond function is available for subsequent reactions with for example sulfhydryl containing proteins such as fibronectin, which is known for enhancing cellular adhesion (45). The C1s high-resolution spectra for SMCC and SMPB (Figure 5e) were curve-fitted into four components at 291.1, 288.4, 285.9, and 284.8 eV which were assigned to CF2 components, CdO groups, CN groups, and CHx, CC, and CdC moieties, respectively (27, 46). These spectra clearly indicate, either qualitatively and quantitatively, that both SMCC and SMPB are bonded to the PTFE surface. From a qualitative point of view, the relative peak area of the CF2 components is largely reduced compared to that measured for the plasmatreated surface as the contribution of the other carbon


Technical Notes

Figure 5. Synthetic schemes for S-SMCC (a) and S-SMPB (b) grafting on an ammonia plasma-treated PTFE film. Spectra for plasma-treated PTFE films and for SMCC- (c) and SMPB- (d) grafted PTFE films. [NH2]0 was 2.8% for the S-SMCC reaction and 1.9% in the case of S-SMPB grafting reaction. The former reaction presents a yield of 114 ( 25% and the latter reaction 163 ( 20%. C1s high-resolution spectrum (e) for S-SMCC and S-SMPB grafting. Panel e (top to bottom) shows the curve fits of the C1s XPS spectra of the ammonia plasma-treated PTFE surface and S-SMCC and S-SMPB PTFE-grafted surfaces, respectively.

spectral components increases when the cross-linkers are bonded to the surface. Quantitatively, the relative peak area of the curve fittings matches closely with the stoichiometric ratios of the carbon components in the grafted molecules. Moreover, the area of the CdO band located at 288.4 eV is useful to calculate the yield of the reactions. In the case of SMCC grafting, the CdO component corresponds to 17.0% of the total carbon contribution on the surface. Combining this value with the carbon content of 54.5% measured from the XPS survey spectrum lead to conclude that 9.3% of the atoms on the modified surface are CdO groups. As one SMCCgrafted molecule to an amino group provides three carboxylic groups, it means that the initial amino group surface concentration should be one-third of 9.3% that is 3.1%. This simplistic calculation closely matches with the value of 2.8% of amino groups determined through vapor-phase chemical derivatization and leads to a SMCC grafting reaction yield of 111% (3.1/2.8), which is in good agreement with the value of 114 ( 25% presented in

Figure 4c. A similar calculation may be performed for the SMPB grafting reaction, as the CdO component at 288.4 eV correspond to 18.0% of the 56.9% carbon atom contribution after SMPB grafting. Again, if three CdO groups are covalently attached to the surface for every SMPB molecule, there would be 3.4% [NH2]0 on the plasma-treated PTFE film. For this plasma-treated film, derivatization with 5-bromosalicylaldehyde allowed measurement of an amino group surface concentration of 1.9%, therefore leading to a reaction yield of 179%, in fairly good agreement with the value reported in Figure 5d and therefore confirming the hypothesis of the occurrence of a competitive and unexpected reaction. Nevertheless, as aforementioned, this competitive reaction does not impede further conjugation of other molecules. Grafting of the 2-iminothiolane was also performed since a successful grafting would provide a thiol functionality on the PTFE-ammonia plasma-treated surface ready to further react with a maleimidyl-containing molecule (47) (Figure 6a). Our XPS analysis first revealed

Technical Notes


Figure 7. XPS spectra of fibronectin conjugated on ammonia plasma-treated PTFE previously activated with S-SMPB (a) or glutaric anhydride (b).

Figure 6. (a) Synthetic scheme of 2-iminothiolane grafting on an ammonia plasma-treated PTFE film and hydrolysis of the CdNH2+Cl- into an amide bond. (b) Synthetic scheme of the conjugation of sulfo-SMPB to the dipeptide Lys-Lys and reaction of the succinimidyl moiety of the cross-linker to the thiol function resulting from the reaction of 2-iminothiolane to the aminated-PTFE. (c) Spectra for plasma treated-PTFE film, 2-iminothiolane grafted-PTFE film and 2-iminothiolane-SMPBLys-Lys grafted-PTFE film. [NH2]0 was 2.7%. The yield of the reaction of 2-iminothiolane with the amino groups is 94 ( 12%, and the yield of the conjugation of SMPB-Lys-Lys to the thiol group of 2-iminothiolane is 82 ( 6%

that only 0.6% of sulfur was detected, and since this atom is hardly perceived by XPS spectroscopy, having a very low atomic sensitivity factor of 0.570 (48), we proceeded the same way as we did for bromoacetaldehyde diethylacetal to verify the presence of 2-iminothiolane on the plasma-treated PTFE surface. The thiol function was

reacted with the maleimide functionality of sulfo-succinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB) previously conjugated to Lys-Lys (Figure 6b). The increase of the molecular weight of the molecule grafted on the PTFE surface allows ascertaining the successfulness of the Traut reagent conjugation through XPS, as it provides 43 new atoms upon conjugation to the surface. The spectra and the atomic percentages obtained from XPS analyses clearly indicate that the reaction occurred (Figure 6c). 2-Iminothiolane was grafted on the fluoropolymer surface with a yield of reaction estimated to 94% according to the oxygen content, taking into account that the oxygen atom considered in the percentage yield equation comes from hydrolysis of CdNH2+Cl- groups into amide moieties. This estimate is also supported by the atomic percentages obtained following the grafting of the 43-atom molecule where again, if this latter reaction is complete, we get a higher than 80% yield for 2-iminothiolane grafting and the subsequent reaction with the dipeptide. The conjugation reaction of 2-iminothiolane to SMPB-lysine-lysine is a two-step reaction, where sulfo-SMPB first reacts with the dipeptide and then reacts with 2-iminothiolane, and both reactions may not be complete. Therefore, it is once more reasonable to assert a 94% yield for the grafting reaction of 2-iminothiolane to the plasma-treated PTFE film. Grafting Fibronectin on Plasma-Treated PTFE Surfaces: Influence of the Conjugation Strategy on Cell Adhesion. A convincing example of the usefulness of the conjugation strategies and quantification methods presented above is seen through the immobilization of fibronectin on PTFE surfaces. The attachment of this protein on various material surfaces was shown to promote cell attachment and proliferation (9). In this context, fibronectin was covalently attached on plasmatreated PTFE samples previously reacted either with glutaric anhydride or S-SMPB. The XPS-spectra (Figure 7) of both surface samples clearly put in evidence that the same amount of protein was grafted with either surface activator, therefore eliminating the possibility of a fibronectin surface concentration effect on the cell adhesion experiments. However, using these surfaces in cell adhesion experiments leads to very different behaviors. As illustrated in Figure 8, a clean PTFE surface does not support cell adhesion. On the other hand, fibronectin


Figure 8. Adhesion of human endothelial vascular cells on a clean PTFE surface (0), in a well coated with fibronectin (right diagonal stripe), on ammonia plasma-treated PTFE conjugated with glutaric anhydride (left diagonal stripe), on fibronectin conjugated on ammonia plasma-treated PTFE through glutaric anhydride (dotted), on ammonia plasma-treated PTFE conjugated with S-SMPB (horizontal stripe), and on fibronectin conjugated on ammonia plasma-treated PTFE through S-SMPB (vertical stripe). (*) indicates a significant difference with respect to the clean PTFE surface with p < 0.01 according to Dunnett’s test.

conjugated on ammonia plasma-treated PTFE through glutaric anhydride clearly supports cell adhesion, with about one-third of the cells observed for a well coated with fibronectin. In the opposite way, immobilization of fibronectin on ammonia plasma-treated PTFE using SSMPB as activator gives rise only to slightly more cell adhesion than the clean PTFE surface. This surprising result tends to demonstrate that an inappropriate surface conjugation strategy of fibronectin may lead to conformational changes and/or hiding of biologically relevant protein adhesion sites. DISCUSSION

Many research groups have focused their efforts for the past decades on modifying polymer surfaces using different types of plasma treatments (19, 20, 28, 30, 38, 49). However, these treatments require sustained work of characterization over prolonged periods of time in order to determine the most effective plasma treatment conditions for a particular plasma apparatus and a specific polymer. The plasma procedure used in the present study has been the subject of a complete set of characterization while treating PTFE surfaces (22, 31). These previously published data demonstrated that the surface plasma treatments were homogeneous and that it was possible to exert a control over the amine surface concentration through an appropriate selection of experimental parameters such as ammonia gas pressure and flow, treatment time, and radiofrequency power. From these works and considering various parameters intrinsic to the XPS analytical technique (such as the surface elements electron mean free path), it can be concluded that the 2-3.5% amino group relative surface concentration corresponds to about 0.5 to 2 amine every nm2. This amino surface concentration is by far sufficient to conjugate biological molecules such as proteins, enzymes, or growth factors, considering that the surface plasma modification occurs

Technical Notes

on the first atomic layers of the polymer surface (50). For example, albumin, with a molecular weight of 66 kDa, has an elliptical shape and can cover 12 nm2 in the best conditions (51). It means that one HSA molecule could cover between 6 and 24 amino groups on the PTFE surface, depending on the protein orientation on the surface, therefore showing that the amine surface concentration reached through radiofrequency glow discharge is by far sufficient to allow the grafting of molecules of biological interest. This study reports a solid proof-of-concept for biotechnological applications of modified polymer-surfaces since only one type of plasma treatment is now required to optimally enable further reaction with chemical reagents and generation of chemical functionalities for biomolecule conjugation. Moreover, it appears that most of the chemical reactions that we have studied are very simple, straightforward, and easily performed using readily available reagents. Conjugation of molecules onto polymer surfaces is commonly used in various spheres of biotechnology such as biochips and biomaterials fabrication. Most of the time, the developments in these fields are based on the awareness of the biological function and activity of biomolecules and on the assumption that they should preserve their characteristics once conjugated onto surfaces. However, such reasoning does not take into account some important aspects of biomolecule interactions with the surrounding environment. Indeed, the surface concentration of the conjugated biomolecule may play a key role to ensure the proper function of the surfaceengineered material. Surface grafting of the vascular endothelial growth factor (VEGF) for improving vascular prostheses self-endothelialization is a good example of this assertion, as one of the characteristics of this molecule regulatory pathway is a cellular down-regulation in response to an excess of the growth factor (52, 53). This issue points toward the fact that the construction of biotechnologically relevant surfaces requires the knowledge and control over the concentration of the linking arms aimed at binding the biomolecules. To our knowledge, the present study is the first to provide quantitative data on the reaction yields and chemical functionality surface concentrations obtained after chemical synthesis on surfaces. The importance of accurately measuring the surface concentration of chemical moieties on surfaces raises the question of the choice of the surface analytical technique to perform a quantitative characterization. During the course of this study, the potential of other spectroscopic techniques, namely FTIR-ATR and Raman confocal spectroscopies, was also investigated in order to reach our objectives. On one hand, it turned out that FTIR-ATR allowed the measurement of spectral characteristics of the moieties grafted onto the plasma treated PTFE; however, the signal-to-noise ratio was too poor to allow any quantitative analyses. On the other hand, Raman confocal spectroscopy completely failed to provide any data because of the difficulty of precisely focusing the laser beam on the modified layer surface for which the thickness does probably not exceed a few nanometers. In this context, surface derivatization strategies coupled with the surface sensitivity of X-ray photoelectron spectroscopy, that probes about 10 nm in depth, despite being cumbersome, remains one of the best techniques to perform quantitative analyses on surfaces. The second constraint to overcome upon conjugation of biomolecules onto surfaces is the ability of proteins, enzymes, or nucleic acids to retain their active confor-

Technical Notes

mational structure during the surface grafting process. In this context, it is strategically more relevant to perform the first steps of the conjugation protocol on the polymer surface, as these procedures often require the utilization of harsh reaction conditions such as the use of organic solvents, hydrolysis in acidic aqueous solution, or direct contact with chemical reagents. Performing the surface conjugation through the reaction of the biomolecule with the cross-linker as the first step of the reaction may lead (and in fact often lead) to a complete loss of the biomolecule-targeted activity. In connection with this idea, the various surface conjugation schemes presented in this publication open the way to the possibility of selecting appropriate synthetic pathways for preserving molecules bioactivity. Indeed, the endothelial cell adhesion data presented therein on fibronectin-grafted PTFE surfaces are good examples of the importance of selecting appropriate strategies for grafting biologically active molecules in an attempt to preserve their function. Most of the time, biological molecules are grafted on surfaces without any consideration for the conjugation scheme with the assumption that they will continue to play their biological roles. The Sheardown’s group was the first to demonstrate that the surface orientation of cell signaling peptides such as RGD are of prime importance for promoting cell adhesion (54). The present publication goes a step further by unambiguously demonstrating that even larger molecules require to be conjugated with appropriate orientation/conformation on surfaces to ensure their biological function. CONCLUSION

In this study, surface modification strategies for PTFE surfaces were developed to provide various chemical functionalities for further molecule immobilization. In a first step, a radiofrequency glow discharge (RFGD) ammonia plasma treatment was applied to PTFE films to covalently bind amino groups onto their surface. In a second step, various chemical reactions were performed, generating carboxylic acid, thiol, maleimide, ethylacetal, and aldehyde functionalities on the PTFE surfaces. The grafting reactions were carefully controlled at each step by XPS analyses, allowing quantifying the yield of each reaction. Some examples were also provided to confirm the accessibility and the reactivity of these new functional groups used also as a linking arm for relevant proteins, aiming at increasing the compatibility/function of the grafted material. ACKNOWLEDGMENT

This study was supported by the National Science and Engineering Research Council (NSERC) of Canada and the Fonds pour la Recherche en Sante´ du Que´bec (FRSQ) (G.L.). V. Gauvreau acknowledges a postgraduate scholarship from NSERC. LITERATURE CITED (1) Wang, C. C., Seo, T. S., Li, Z., Ruparel, H., and Ju, J. (2003). Site-specific fluorescent labeling of DNA using staudinger ligation. Bioconjugate Chem. 14, 697-701. (2) Zhu, H. W., Xu, C. L., Wu, D. H., Wei, B. Q., Vajtai, R., and Ajayan, P. M. (2002). Direct synthesis of long single-walled carbon nanotube strands. Science (Washington, D. C.) 296, 884-886. (3) Kumar, P., and Gupta, K. C. (2003). A rapid method for the construction of oligonucleotide arrays. Bioconjugate Chem. 14, 507-512.

Bioconjugate Chem., Vol. 15, No. 5, 2004 1155 (4) Skrzypczynski, Z., and Wayland, S. (2003). New reagents for the introduction of reactive functional groups into chemically synthesized DNA probes. Bioconjugate Chem. 14, 642652. (5) Wagner, P., Kernen, P., Hegner, M., Ungewickell, E., and Semenza, G. (1994). Covalent anchoring of proteins onto golddirected NHS-terminated self-assembled monolayers in aqueous buffers: SFM images of clathrin cages and triskelia. FEBS Lett. 356, 267-271. (6) Allain, L. R., Askari, M., Stokes, D. L., and Vo-Dinh, T. (2001). Microarray sampling-platform fabrication using bubblejet technology for a biochip system. Fresenius’ J. Anal. Chem. 371, 146-150. (7) Blawas, A. S., and Reichert, W. M. (1998). Protein patterning. Biomaterials 19, 595-609. (8) Chandy, T., Das, G. S., Wilson, R. F., and Rao, G. H. R. (2000). Use of plasma glow for surface-engineering biomolecules to enhance blood compatibility of Dacron and PTFE vascular prosthesis. Biomaterials 21, 699-712. (9) Chen, C. S., Mrksich, M., Huang, S., Whitesides, G. M., and Ingber, D. E. (1998). Micropatterned surfaces for control of cell shape, position, and function. Biotechnol. Prog. 14, 356363. (10) Favia, P., Palumbo, F., D’Agostino, R., Lamponi, S., Magnani, A., and Barbucci, R. (1998). Immobilization of heparin and highly sulfated hyaluronic acid onto plasma-treated polyethylene. Plasmas Polym. 3, 77-96. (11) Ito, Y. (1999). Surface micropatterning to regulate cell functions. Biomaterials 20, 2333-2342. (12) Keusgen, M., Glodek, J., Milka, P., and Krest, I. (2001). Immobilization of enzymes on PTFE surfaces. Biotechnol., Bioeng. 72, 530-540. (13) Levesque, S., Thibault, J., Castonguay, M., C.-Gaudreault, R., and Laroche, G. (2002). Modification of lipid transport through a microporous PTFE membrane wall grafted with poly(ethylene glycol). Colloids Surf., B: Biointerf. 25, 205217. (14) Massia, S. P., and Stark, J. (2001). Immobilized RGD peptides on surface-grafted dextran promote biospecific cell attachment. J. Biomed. Mater. Res. 56, 390-399. (15) Reichmuth, P., Sigrist, H., Badertscher, M., Morf, W. E., de Rooij, N. F., and Pretsch, E. (2002). Immobilization of biomolecules on polyurethane membrane surfaces. Bioconjugate Chem. 13, 90-96. (16) Zhang, Q., Wang, C., Babukutty, Y., Ohyama, T., Kogoma, M., and Kodama, M. (2002). Biocompatibility evaluation of ePTFE membrane modified with PEG in atmospheric pressure glow discharge. J. Biomed. Mater. Res. 60, 502-509. (17) Ratner, B. D., Chilkoti, A., and Lopez, G. P. (1990). Plasma deposition and treatment for biomedical applications, in Plasma Deposition, Treatment, and Etching of Polymers (D’Agotino, R., Ed.) p 463, Academic Press, New York. (18) Ratner, B. D. (1993). New ideas in biomaterials science. A path to engineered biomaterials. J. Biomed. Mater. Res. 27, 837-850. (19) Da, Y., Griesser, H. J., Mau, A. W. H., Schmidt, R., and Liesegang, J. (1991). Surface modification of poly(tetrafluoroethylene) by gas plasma treatment. Polymer 32, 1126-1130. (20) Gengenbach, T. R., Xie, X., Chatelier, R. C., and Griesser, H. J. (1994). Evolution of the surface composition and topography of perfluorinated polymers following ammoniaplasma treatment. J. Adhes. Sci., Technol. 8, 305-328. (21) Inagaki, N., Tasaka, S., Narushima, K., and Teranishi, K. (2002). Surface modification of poly(tetrafluoroethylene) with pulsed hydrogen plasma. J. Appl. Polym. Sci. 83, 340-348. (22) Mantovani, D., Castonguay, M., Pageau, J. F., Fiset, M., and Laroche, G. (1999). Ammonia RF-plasma treatment of tubular ePTFE vascular prostheses. Plasmas Polym. 4, 207228. (23) Me´dard, N., Soutif, J.-C., and Poncin-Epaillard, F. (2002). CO2, H2O, and CO2/H2O plasma chemistry for polyethylene surface modification. Langmuir 18, 2246-2253. (24) Pringle, S. D., Joss, V. S., and Jones, C. (1996). Ammonia plasma treatment of PTFE under known plasma conditions. Surf. Interface Anal. 24, 821-829.

1156 Bioconjugate Chem., Vol. 15, No. 5, 2004 (25) Ramires, P. A., Mirenghi, L., Romano, A. R., Palumbo, F., and Nicolardi, G. (2000). Plasma-treated PET surfaces improve the biocompatibility of human endothelial cells. J. Biomed. Mater. Res. 51, 535-539. (26) Wilson, D. J., Williams, R. L., and Pond, R. C. (2001). Plasma modification of PTFE surfaces Part II: plasmatreated surfaces following storage in air or PBS. Surf. Interface Anal. 31, 397-408. (27) Wilson, D. J., Williams, R. L., and Pond, R. C. (2001). Plasma modification of PTFE surfaces Part I: surfaces immediately following plasma treatment. Surf. Interface Anal. 31, 385-396. (28) Xie, X., Gengenbach, T. R., and Griesser, H. J. (1992). Changes in wettability with time of plasma-modified perfluorinated polymers. J. Adhes. Sci., Technol. 6, 1411-1431. (29) Yamada, Y., Yamada, T., Tasaka, S., and Inagaki, N. (1996). Surface Modification of Poly(tetrafluoroethylene) by Remote Hydrogen Plasma. Macromolecules 29, 4331-4339. (30) Yu, Q. S., Reddy, C. M., Meives, M. F., and Yasuda, H. K. (1999). Surface modification of poly(tetrafluoroethylene) by a low-temperature cascade arc torch and radiofrequency plasmas. J. Polym. Sci., Part A: Polym. Chem. 37, 44324441. (31) Chevallier, P., Castonguay, M., Turgeon, S., Dubrulle, N., Mantovani, D., McBreen, P. H., Wittmann, J. C., and Laroche, G. (2001). Ammonia RF-plasma on PTFE surfaces: chemical characterization of the species created on the surface by vapor-phase chemical derivatization. J. Phys. Chem. B 105, 12490-12497. (32) Hansen, R. H., and Schonhorn, H. (1966). A new technique for preparing low surface energy polymers for adhesive bonding. J. Polym. Sci. 4, 203-209. (33) Inagaki, N., Tasaka, S., and Umehara, T. (1999). Effects of surface modification by remote hydrogen plasma on adhesion in poly(tetrafluoroethylene)/copper composites. J. Appl. Polym. Sci. 71, 2191-2200. (34) Inagaki, N., Tasaka, S., and Goto, Y. (1997). Surface modification of poly(tetrafluoroethylene) film by plasma graft polymerization of sodium vinylsulfonate. J. Appl. Polym. Sci. 66, 77-84. (35) Kim, S. R. (2000). Surface modification of poly(tetrafluoroethylene) film by chemical etching, plasma, and ion beam treatments. J. Appl. Polym. Sci. 77, 1913-1920. (36) Sipehia, R. (1990). The enhanced attachment and growth of endothelial cells on anhydrous ammonia gaseous plasma modified surfaces of polystyrene and poly(tetrafluoroethylene). Biomat. Art. Cells, Art. Org. 18, 437-446. (37) Sipehia, R. (1993). Enhanced attachment and growth of human endothelial cells derived from umbilical veins on ammonia plasma modified surfaces on PTFE and ePTFE synthetic vascular graft biomaterials. Biomat., Art. Cells Immob. Biotech. 21, 455-468. (38) Vargo, T. G., Gardella, J. A., Jr., Meyer, A. E., and Baier, R. E. (1991). Hydrogen/liquid vapor radio frequency glow discharge plasma oxidation/hydrolysis of expanded poly(tetrafluoroethylene) (ePTFE) and poly(vinylidene fluoride) (PVDF) surfaces. J. Polym. Sci., Part A: Polym. Chem. 29, 555-570. (39) Vargo, T. G., Bekos, E. J., Kim, Y. S., Ranieri, J. P., Bellamkonda, R., Aebischer, P., Margevich, D. E., Thompson,

Technical Notes P. M., Bright, F. V., and Gardella, J. A., Jr. (1995). Synthesis and characterization of fluoropolymeric substrata with immobilized minimal peptide sequences for cell adhesion studies. I. J. Biomed. Mater. Res. 29, 767-778. (40) Baldwin, S. W., Aube, J., and McPhail, A. T. (1991). Studies directed at the synthesis of optically active pretazettine via intramolecular nitrone/alkene cycloaddition reactions. J. Org. Chem. 56, 6546-6550. (41) Hermanson, G. T. (1996). Bioconjugate Techniques, Academic Press, San Diego. (42) Beamson, G., and Briggs, D. (1992). High-Resolution XPS of Organic Polymers: The Scienta ESCA300 Database, John Wiley & Sons, New York. (43) Shen, W. C., and Ryser, H. J. (1981). cis-Aconityl spacer between daunomycin and macromolecular carriers: a model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate. Biochem. Biophys. Res. Commun. 102, 1048-1054. (44) Vermette, P., and Meagher, L. (2002). Immobilization and characterization of poly(acrilyc acid) graft layers. Langmuir 18, 10137-10145. (45) Dekker, A., Reitsma, K., Beugeling, T., Bantjes, A., Feijen, J., and van Aken, W. G. (1991). Adhesion of endothelial cells and adsorption of serum proteins on gas plasma-treated poly(tetrafluoroethylene). Biomaterials 12, 130-138. (46) Stile, R. A., Barber, T. A., Castner, D. G., and Healy, K. E. (2002). Sequential robust design methodology and X-ray photoelectron spectroscopy to analyze the grafting of hyaluronic acid to glass substrates. J. Biomed. Mater. Res. 61, 391-398. (47) Leggett, G. J., Roberts, C. J., Williams, P. M., Davies, M. C., Jackson, D. E., and Tendler, S. J. B. (1993). Approaches to the immobilization of proteins at surfaces for analysis by scanning tunneling microscopy. Langmuir 9, 2356-2362. (48) Moulder, J. F., Stickle, W. F., Sobol, P. E., and Bomben, K. D. (1992). Handbook of X-ray Photoelectron Spectroscopy, Perkin-Elmer Corporation, Physical Electronics Division, Eden Prairie. (49) Hook, D. J., Vargo, T. G., Gardella, J. A., Jr., Litwiler, K. S., and Bright, F. V. (1991). Silanization of radio frequency glow discharge modified expanded poly(tetrafluoroethylene) using (aminopropyl)triethoxysilane. Langmuir 7, 142-151. (50) Gerenser, L. J. (1987). J. Adhesion Sci. Technol. 1, 303318. (51) Sukhishvili, S. A., and Granick, G. (1999). J. Chem. Phys. 110, 10153-10161. (52) Tassiopoulos, A. K., and Greisler, H. P. (2000). Angiogenic mechanisms of endothelialization of cardiovascular implants: a review of recent investigative strategies. J. Biomater. Sci. Polym. Ed. 11, 1275-1284. (53) Crombez, M., and Mantovani, D. (2003). Biomaterials in tissue engineering and repair - Progresses in synthetic vascular prostheses: toward the endothelialization, in Tissue engineering, stem cells and gene therapies (Elç in, M. Y., Ed.) Kluwer Academic Publishers, New York. (54) McMillan, R., Meeks, B., Bensebaa, F., Deslandes, Y., and Sheardown, H. (2001). J. Biomed. Mater. Res. 54, 272-283.

BC049858U

Engineering Surfaces for Bioconjugation: Developing Strategies and

Recommend Documents