Biomacromolecules 2004, 5, 2238-2246
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Assessment of in Vitro Bioactivity of Hyaluronic Acid and Sulfated Hyaluronic Acid Functionalized Electroactive Polymer† Lian Cen, K. G. Neoh,* Yali Li, and E. T. Kang Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260 Received May 6, 2004; Revised Manuscript Received June 22, 2004
Electrically conductive polypyrrole (PPY) was surface functionalized with hyaluronic acid (HA) and sulfated hyaluronic acid (SHA) to improve its surface biocompatibility. The immobilization of HA on the PPY film was facilitated by the use of a cross-linker having the appropriate functional groups. The biological activity of the HA functionalized PPY film was assessed by means of an in vitro PC12 cell culture. The cell attachment on different substrates was studied and determined by bicinchoninic acid protein analysis. Cell attachment on the HA functionalized PPY film surface was significantly enhanced in the presence of nerve growth factor. The SHA functionalized PPY film was obtained by the sulfonation of the immobilized HA using pyridinesulfonate. The retention of the biological activity of the immobilized HA after sulfonation was evaluated by the in vitro assessment of the plasma recalcification time (PRT) and platelet adhesion on the substrate. The PRT observed from the SHA functionalized PPY film was significantly prolonged compared with the HA functionalized PPY. Some reduction of platelet adhesion was observed for the SHA functionalized PPY film, compared with that of the HA functionalized PPY film. 1. Introduction In the biomaterials field, there is an ongoing quest to develop materials with excellent biocompatibility. Surface modification of materials with biomolecules to elicit bloodor tissue-compatible properties is of great interest.1 Among various surface modification methods, covalent immobilization has the advantage of nonrelease and long-term stability over other techniques, such as entrapment, ionic bonding, adsorption, or coating.2-7 Heparin (generalized structure shown in Scheme 1a), which acts as an anticoagulant to prevent blood coagulation via catalyzing and enhancing the binding of antithrombin III and thrombin, is an attractive candidate that can be immobilized on the surface of materials. Therefore, heparinized surfaces are often used as bloodcompatible materials. However, it is difficult to achieve a high concentration of heparin on material surfaces via covalent immobilization with good retention of its biological activities.3,4,6 Besides heparin, there is also interest in the heparin-like members in the glycosaminoglycans family.8 Hyaluronic acid (HA), a ubiquitous constituent of the extracellular matrix, has potential applications in wound-healing, tissue regeneration, and angiogenesis.9,10 In addition to its important role in the hydrodynamic properties of the extracellular environment, particularly in embryonic tissues,11 another study has also shown that, in the presence of HA, nerve regeneration and reinnervation show better conduction velocity, higher axon counts, and a trend toward earlier myelination.12 HA, † This paper was presented at the ICMAT 2003 conference, held in Singapore June 29th through July 4th, 2003. * To whom correspondence should be addressed. Tel.: +65 68742176. Fax: +65 67791936. E-mail:
[email protected].
with its regular sequenced units, consisting of nonsulfated repeating disaccharide units of glucuronic acid β (1f3) and N-acetylglucosamine β (1f4) linkages (Scheme 1b), can be further sulfonated (Scheme 1c) as an alternative to heparin.13 It has also been documented that the degree of sulfonation is an important functional property that contributes significantly to the anticoagulant effects of glycosaminoglycans.13,14 In our previous work, we have shown that a high concentration of HA can be successfully immobilized on the polypyrrole (PPY) film surface with the retention of its biological activity.15 The current work is focused on the assessment of the bioactivity of the PPY-HA system in an in vitro cell culture, as well as the sulfonation of the immobilized HA on the PPY film and the bioactivity assay of the immobilized sulfated hyaluronic acid (SHA). PPY was chosen as a substrate for our study for its in vitro and in vivo compatibility,16 because it has been shown by previous studies that PPY is not cytotoxic and can be formed into conduits to support the regeneration of damaged peripheral nerves in rats.17-19 Its inherent electrical conductive property facilitates the passage of electrical stimulation, which has been shown to play a positive role in neurite outgrowth and blood compatibility tests.18,20 PC12 was chosen for this preliminary assay of the application of HA immobilized PPY in the neurite outgrowth because it has been used as a model system for neurobiological and neurochemical studies.21 These cells respond reversibly to nerve growth factor (NGF), and in the process, they become electrically excitable and increase catecholamine synthesis and synapses formation with the assumption of many characteristics of sympathetic neurons.21 PC12 cell attachment on pristine and surface modified PPY films was
10.1021/bm040048v CCC: $27.50 © 2004 American Chemical Society Published on Web 08/24/2004
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Scheme 1. Structures of Heparin, HA, and SHA
investigated. The blood compatibility of the SHA modified PPY film surface was evaluated using the plasma recalcification time (PRT) and platelet adhesion assays and compared with those of the PPY film immobilized with HA. 2. Experimental Section 2.1. Materials. Pyrrole (99%) was obtained from the Aldrich Chemical Co. and was distilled before use. 3-Aminopropyl triethoxyl silane [H2N(CH2)3Si(OC2H5)3, denoted as ATS], pyridinesulfonate, and tributylamine were also obtained from Aldrich Chemical Co. Toluene-4-sulfonic acid (TSA), 2-hydroxyethyl acrylate (H2CdCHCO2CH2CH2OH, denoted as HEA), and 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid buffer used for HA immobilization were all purchased from Fluka. HA from human umbilical cord was purchased from Sigma Chemical Co. Water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC) was purchased from Dojindo Chemical Co. and was used as received. Type I collagen (Vitrogen, 3.0 mg/ mL in 0.012 N HCl) was purchased from Cohesion Technologies, Inc., of Palo Alto, CA. The Dulbecco’s phosphate buffer solutions, 10× PBS (containing 1.37 M of sodium chloride, 0.027 M of potassium chloride, 0.081 M of anhydrous disodium phosphate, and 0.0147 M of anhydrous monopotassium phosphate) and its 10× diluted counterpart, or 1× PBS, were obtained from Pierce Chem. Co. of Rockford, IL. Bicinchoninic acid (BCA) protein assay reagent kit was a product from Pierce Biotechnology. Reporter lysis buffer was obtained from Promega Corp., U.S.A. Trypsin-ethylenediaminetetraacetic acid (EDTA) solution, Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, and heat-inactivated horse serum were all from Sigma Chemical Co., U.S.A. Fetal calf serum was obtained from Moregate BioTech, Australia. 2.2. Electrochemical Synthesis of PPY. The electrochemical polymerization of pyrrole was carried out using an Autolab-PGSTAT30 (Metrohm-Schmidt, Ltd.). An electrolyte solution of 0.1 M pyrrole and 0.1 M TSA in acetonitrile containing 1 vol % water was used. The electropolymerization was carried out at a constant voltage of 8 V under a dry nitrogen atmosphere. The PPY film was formed on a stainless steel electrode (usable area of 3.5 cm × 8 cm). The details of the synthesis process have been
described in an earlier publication.22 PPY films of a thickness of about 40 µm (as determined gravimetrically using an experimentally determined film density of 1.3 g/cm3, and also confirmed from the SEM images of the film’s cross section) were used in the present work after being washed thoroughly with acetonitrile and water and dried under a reduced pressure. 2.3. Surface Functionalization of PPY Film with HA. Briefly, four steps are required for the immobilization of HA: (a) surface graft copolymerization of the argon plasma pretreated (10 s at 35 W and Ar pressure of 80 Pa) PPY with HEA; (b) silanization of HEA graft copolymerized PPY film by ATS; (c) preactivation of HA with WSC; (d) HA immobilization through the amide linkage formation between the primary amine groups introduced on the PPY film via steps a and b and the carboxyl groups of HA preactivated by WSC. The amount of HA immobilized is determined by the toluidine blue method and could be varied by changing the HEA monomer concentration used in step a. The details of the process have been reported in an earlier publication15 and are summarized in Scheme 2. 2.4. Sulfonation of the Immobilized HA. The sulfonation was carried out on the HA immobilized PPY film prepared by graft copolymerization with 5 vol % HEA and subsequently silanized with 1 vol % ATS in dioxane (denoted as PPY-HA in the subsequent discussion). The PPY-HA film (2 cm × 3 cm) was immersed in 30 mL of dimethylformamide (DMF), and 1 mL of tributylamine was then added to form the tributylammonium hyaluronate. This process protects the COOH groups in the subsequent sulfonation reaction. After 1 h, the PPY-HA film was then transferred into another 30 mL of DMF solution. A total of 8 mg of pyridinesulfonate was added, and the reaction proceeded for 1 h under N2 at 0 °C. A total of 1 mL of 1 M NaOH was then added dropwise into the above reaction mixture. After that, the film was taken out, washed with ethanol, and dried. The sulfonation reaction is summarized in Scheme 3. The PPY-HA film after sulfonation is denoted as PPY-SHA. 2.5. Surface Characterization. 2.5.1. X-ray Photoelectron Spectroscopy (XPS) Measurement. XPS measurements were made on a Kratos AXIS HSi spectrometer (Kratos Analytical, Ltd., Manchester, U.K.) with a monochromatized Al KR X-ray source (1486.6 eV photons) at a constant dwell time of 100 ms and a pass energy of 40 eV. The anode current
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Scheme 2. Mechanism of Anchoring HA on the Surface Modified PPY Film
Scheme 3. Sulfonation Reaction of HA30
was 15 mA. The pressure in the analysis chamber was maintained at 7.0 × 10-6 Pa or lower during each measurement. The PPY films were mounted on the standard sample studs by means of double-sided adhesive tapes. The corelevel signals were obtained at a photoelectron takeoff angle of 90° (with respect to the sample surface). To compensate for the surface charging effect, all core-level spectra were referenced to the C(1s) hydrocarbon peak at 284.6 eV. In spectral deconvolution, the line width (full width at halfmaximum) of the Gaussian peaks was maintained constant for all components in a particular spectrum. The peak area ratios for the various elements were corrected using experimentally determined instrumental sensitivity factors. 2.5.2. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM). The surface morphologies of the modified PPY films were characterized on a Nanoscope IIIa atomic force microscope, manufactured by Digital Instruments, Inc. For each sample, an area of 3.5 µm × 3.5 µm was scanned using the tapping mode with the scan rate
of 1.0 Hz. The drive frequency was 330 ( 50 kHz. The applied voltage was between 3.0 and 4.0 V, and the drive amplitude was about 300 mV. An arithmetic mean of the surface roughness (Ra) was calculated from the roughness profile determined by AFM. SEM was carried out on a JEOL JSM 5600LV scanning electron microscope. The samples were sputter-coated with a thin film of platinum for imaging purposes before SEM characterization. 2.6. In Vitro Bioactivity Assay. 2.6.1. Cell Culture and Cell Attachment Assay. Rat PC12 cells from ATCC (American Type Culture Collection) were passaged at least once after thawing and cultured in rat tail collagen-coated tissue culture flasks (Falcon) in DMEM supplemented with 10 vol % heat-inactivated horse serum, 5 vol % fetal calf serum, and 100 U/mL penicillin-streptomycin. The cells were maintained at 37 °C in an incubator with a humidified 5% CO2 atmosphere, and the medium was replaced every 2-3 days. The cells were passaged following detachment with trypsin (0.25 wt % trypsin-EDTA).23,24
In Vitro Bioactivity of HA and SHA Polymer
Pristine and modified PPY films were cut into round disks with a diameter ∼15 mm and fixed into a 24-well tissue culture plate by silicone adhesive (Applied Silicone Implant Grade). The plate was then sterilized in 70 vol % ethanol for 3 h and washed three times with PBS. The cell attachment on four types of surfaces was assayed: pristine PPY; PPY film graft copolymerized with 5 vol % HEA and subsequently silanized with 1 vol % ATS in dioxane (P-HEAATS); P-HEA-ATS with subsequent immobilization of HA (PPY-HA); and uncoated tissue culture polystyrene (TCPS). Cells were detached from the tissue culture flasks, collected by centrifugation, and resuspended in DMEM supplemented with 10 vol % heat-inactivated horse serum, 5 vol % fetal calf serum, and 100 U/mL penicillin-streptomycin at a density of 3.3 × 104 cells/mL as determined by a hemocytometer. A total of 300 µL of the above cellcontaining medium was then dispensed on the 24-well tissue culture plate containing different films so as to yield the desired cell density of 1 × 104 cells/well. The plate was then placed in an incubator with a humidified 5% CO2 atmosphere. The above experiment was also performed under the same conditions with the addition of NGF in the above medium at a concentration of 50 ng/mL. This concentration has been shown to induce maximal neurite outgrowth.25 For the cell attachment assay, the plate was taken out from the incubator after 2 h and gently shaken. The medium containing unattached cells in each well was aspirated and transferred to microcentrifuge tubes. The well was washed with 300 µL of PBS, and the washing solution was combined with the medium in the corresponding tube. The cells in the tubes were collected by centrifugation at 4000 rpm for 10 min and washed once with PBS. The PBS was then removed, and 200 µL of 1× reporter lysis buffer was then added into each tube. The tubes were then vortexed and subjected to three freeze-thaw cycles. The protein concentration in the lysate was then determined by BCA protein assay.26-28 The number of attached cells was calculated from the difference in the initial number of cells and the number of unattached cells in the medium (both determined from the BCA protein assay). 2.6.2. PRT. Fresh blood collected from a healthy rabbit was immediately mixed with 3.8 wt % sodium citrate solution in a ratio of 9:1. The mixture was then centrifuged at 3000 rpm at 8 °C for 20 min to obtain the platelet poor plasma (PPP). A total of 0.1 mL of the PPP was introduced onto the PPY film (cut to 2.0 cm × 2.0 cm in size) and incubated at 37 °C under static conditions. A total of 0.1 mL of aqueous 0.025 M CaCl2 solution at 37 °C was then added to the PPP, and the plasma solution was monitored for clotting by manually dipping a stainless steel wire hook coated with silicone into the solution to detect fibrin threads. The clotting time was recorded at the first sign of fibrin formation on the hook.4 The above experiments were all carried out in triplicate for each sample. The differences between the PRT obtained with the pristine PPY film, which acted as the control, and the surface modified PPY film were analyzed statistically, using the two sample t test. The differences observed between samples were considered significant for P < 0.05.
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Figure 1. XPS C(1s) and S(2p) core-level spectra of the (a and b) as-synthesized PPY film; (c and d) PPY film graft copolymerized with 5 vol % HEA and subsequently silanized with 1 vol % ATS in dioxane followed by HA immobilization (PPY-HA); and (e and f) PPY-HA film after sulfonation (PPY-SHA).
2.6.3. Platelet Adhesion. Fresh blood diluted with a 3.8 wt % sodium citrate solution as mentioned above was centrifuged at 700 rpm at 8 °C for 10 min to obtain the platelet rich plasma (PRP). The PRP was then diluted with PBS in a 1:1 (v/v) ratio. A total of 0.1 mL of the diluted PRP was introduced onto the PPY film and spread to cover approximately 0.8 cm × 0.8 cm. After incubation at 37 °C for 30 min under static conditions, the film was gently washed three times with PBS. An aqueous 2.0 vol % glutaraldehyde solution was then added on the above PPY film for 2 h at 4 °C to fix the adhered platelets. After fixation, the film was gently washed with PBS, water, and ethanol for three times each and dried under a vacuum before being subjected to SEM observation.29 3. Results and Discussion 3.1. Surface Functionalization of the PPY Film with HA and SHA. The XPS C(1s) core-level spectrum of the pristine PPY indicates a small amount of oxidized C, primarily CsO (286.2 eV) and CdO (287.6 eV; Figure 1a). The presence of these species may have resulted from surface oxidation or charge-transfer complexing with oxygen. These species are desirable for the subsequent surface grafting process. The S(2p) core-level spectrum of the pristine PPY (Figure 1b) shows a spin-orbit split doublet [S(2p3/2) and S(2p1/2)] with peaks at 168.1 and 169.3 eV attributable to
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-SO3- groups of the TSA counterions of the PPY film. The molar ratio of the -SO3- groups to total nitrogen, estimated from the corrected area ratio of the S(2p) peak and N(1s) peak (expressed as the [S]/[N] ratio), is 0.27. The ratio of the negatively charged TSA counterions to the positively charged nitrogen of PPY is 0.93, which indicates that charge neutrality is maintained. 3.1.1. HA Immobilization. The appearance of the Si(2p) core-level signal after silanization indicates that the ATS must have been successfully chemisorbed onto the HEAgrafted PPY film surfaces as shown in our previous work.15 For the PPY-HA film, the enhancement of the peak attributable to the CsO species and the appearance of a new peak attributable to the CdO species in Figure 1c, as compared with those of the PPY film after HEA grafting and subsequent silanization but before HA immobilization (results not shown), indicates the success of the HA immobilization. The CH/CsO/CdO/COO ratio of the PPY film after HEA grafting and subsequent silanization as determined by XPS analysis is 1:0.6:0:0.2, whereas the corresponding ratio of the PPY film after HA immobilization is 1:1.6:0.5:0.2. The increase in the proportions of CsO and CdO species is consistent with the abundance of these groups in HA (Scheme 1b). The signal due to the TSA counterions is hardly discernible in the S(2p) spectrum of the PPY-HA film (Figure 1d) because of the surface coverage of grafted HEA, ATS, and immobilized HA. The dependence of the amount of HA immobilized (as determined by the toluidine method) on the monomer concentration of HEA used for grafting was shown in previous work.15 Under the experimental conditions used, the highest amount of HA immobilized on the PPY film surface is ∼70 µg/cm2. The PPY-HA film used in the present work has 50 µg/cm2 of immobilized HA, and this film was used for the subsequent sulfonation. 3.1.2. Sulfonation of the Immobilized HA. The sulfonation reaction occurs through a nucleophilic attack of the alcoholic oxygen of HA on the sulfur atom of the pyridinesulfonate. It has been shown that this reaction is easily controllable, and different degrees of sulfonation ranging from one to four sulfate groups per disaccharide unit of HA can be obtained by varying the HA/pyridinesulfonate molar ratio. A molar ratio of OH in HA/pyridinesulfonate of 1:8 was reported to be sufficient to result in approximately four sulfate groups on each repeating unit of free HA.30 Because it has also been shown that heparin-like activities increase with the sulfonation degree, a ratio of OH in HA/pyridinesulfonate of 1:15 was used in the present work. XPS characterization of the PPY-HA film before and after sulfonation shows an increase in the intensity of the peak component at 284.6 eV attributable to the CH species after sulfonation (Figure 1e) compared with Figure 1c. This may be due to the presence of some residual tributylamine and pyridine groups. The appearance of the S(2p) core-level signal after sulfonation as shown in Figure 1f confirms the success of the reaction, because almost no S(2p) signal can be detected from the PPY film after HA immobilization because of the surface coverage of this functional layer (Figure 1d). The extent of sulfonation can be estimated from the [S]/[N] ratio [computed from the area ratio of the S(2p)
Cen et al.
and N(1s) peaks after correction with the appropriate sensitivity factors], which was found to be 1.05, because the [S]/[N] ratio of the film before sulfonation is ∼0. As shown in Scheme 1c, complete sulfonation should yield a [S]/[N] ratio of 4. Because complete sulfonation is possible with free HA at the HA/pyridinesulfonate reactant molar ratio used in this work,30 the relatively low value obtained in the PPYSHA film may be due to the steric hindrance resulting from the immobilization of the HA. It is possible that HA is more sulfated on the outermost part and less so in the inner portion of the layer. Hence, the [S]/[N] ratio of 1.05 gives an average value across the sampling depth of the XPS technique. 3.2. Surface Morphologies of the Functionalized PPY Films. The change in the surface morphology of the PPY film after functionalization can be observed by means of AFM. Figure 2a-c shows the AFM images of the pristine PPY film, PPY-HA, and PPY-SHA, respectively. The surface morphology of PPY (Figure 2a) has changed after HA immobilization (Figure 2b) with Ra increasing from 7.8 to 12.8 nm. This change occurs mainly after HA immobilization (step d in Scheme 2), because the AFM image of the PPY sample after HEA grafting and ATS silanization before HA immobilization shows a similar morphology as that of the pristine PPY film. The surface of the PPY-HA film shows clusters of nodules (Figure 2b). After sulfonation (Figure 2c), Ra decreased from 12.8 to 10.6 nm and the film surface becomes striated. While the cause for this morphological change is not clear, the dense negative charge of SHA (Scheme 1c) may play a role in affecting the surface morphology. A strong electrostatic repulsion can be expected in the presence of a large number of sulfate groups, resulting in a more rigid and stretched structure of the immobilized chains of SHA compared with a coiling conformation of HA.14,30 Because the roughness of a surface that contacts the blood may produce turbulence in the bloodstream which in turn may induce hemolysis and platelet activation and aggregation, it is claimed that the ideal surface used for blood contacting should be perfectly smooth.31 Biomaterials, for example vascular prosthesis, must have a smooth inner surface that corresponds to the endothelial covering of natural blood vessels.31 As the Ra values of PPY-HA and PPY-SHA surfaces did not change significantly from that of pristine PPY, the effect of surface morphology on the following blood compatibility assays is expected to be minor. A comparison of the surface characteristics of the various modified PPY films with the pristine PPY is given in Table 1. An increase in hydrophilicity is observed upon immobilization with HA, and the sulfonation process does not change the contact angle significantly. The significant decrease in electrical conductivity after sulfonation of the PPY-HA film is due to the addition of NaOH to the reaction mixture during sulfonation. Nevertheless, the PPY-SHA film still retains significant electrical conductivity. 3.3. Cell Attachment. Our previous work15 has shown that HA immobilized on the PPY surface was biologically active as evaluated by a protein binding assay.11 In the present work, the cellular response from the in vitro PC12 cell culture was assayed. Figure 3 shows the percentage of attachment of PC12 cells on the pristine PPY, P-HEA-ATS, PPY-HA,
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Table 1. Surface Properties of the PPY Film before and after Surface Functionalization
film typea
surface [S]/[N] ratiob
surface roughness Rac (nm)
water contact angle (θ°)
conductivity σd (S cm-1)
PPY PPY-P PPY-HA PPY-SHA
0.27 0.27 0 1.05
7.8 8.7 12.8 10.6
68 ( 1 50 ( 1 29 ( 2 32 ( 2
71 ( 1 63 ( 3 55 ( 1 7(2
a PPY denotes the as-synthesized PPY film; PPY-P denotes the argon plasma pretreated (10 s at 35 W and Ar pressure of 80 Pa) PPY; PPYHA denotes the PPY film with 50.2 µg/cm2 immobilized HA; and PPYSHA denotes the PPY-HA film after the sulfonation of the immobilized HA. b As determined by XPS. c As determined by AFM. d As calculated from the four-point probe technique.41
Figure 3. PC12 cell attachment on pristine PPY, HEA graft copolymerized and silanized PPY film, P-HEA-ATS (carried out with 5 vol % HEA monomer in dioxane solution and subsequent silanization with 1 vol % ATS in dioxane), PPY-HA film, and TCPS. The attachment was assessed 2 h after cell seeding both with and without 50 ng/mL NGF.
Figure 2. AFM images of (a) pristine PPY, (b) PPY-HA, and (c) PPYSHA films.
and TCPS after 2 h of incubation (after 2 h, the cell attachment reached a plateau for all four substrates) at 37 °C in the absence of NGF. More than 70% of the cells are attached on TCPS, while the PC12 cells show the least affinity for the PPY-HA surface (18%), followed by P-HEAATS (22%) and PPY (40%). The highly hydrophilic nature of HA may contribute significantly to the poor adhesion of the PC12 cells on the HA functionalized PPY surface. In the presence of NGF, the cell attachment increases substantially for all four surfaces with the PPY-HA surface
showing the largest increase, from
