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Bioconjugate Chem. 1999, 10, 678−686
TECHNICAL NOTES Bioactive Surfaces via Immobilization of Self-Assembling Polymers onto Hydrophobic Materials Lev Bromberg* and Lawrence Salvati, Jr. Abbott Laboratories, MediSense Products. Received August 5, 1998; Revised Manuscript Received December 29, 1998
Conjugation of proteins to copolymers from poly(acrylic acid) grafted onto PEO-PPO-PEO backbone (Pluronic-PAA) following adsorption of the conjugates onto hydrophobic surfaces is reported. InsulinPluronic-PAA conjugates show negligible internalization of insulin into human uterine smooth muscle cells as well as enhancement of mitogenic activity. Glucose-induced release of glycated albumin complexed with a Pluronic-PAA-concanavalin conjugate and adsorbed onto polystyrene nanospheres may provide a model for a glucose-responsive protein delivery system or a heterogeneous diagnostic device.
INTRODUCTION
Immobilization of biomolecules such as enzymes, antibodies, and nucleic acids onto various particulates and films is a widespread method of producing bioactive surfaces for use in immunoassays, chromatography, cell proliferation, drug delivery, etc. (1). Hydrophobic polymers, and particularly polystyrene, are often quite useful as neutral carriers readily available in a variety of shapes and sizes. Since polystyrene causes nonspecific adsorption and also lacks functional groups, it is customary to modify it with hydrophilic polymers (2). This modification is usually accomplished by surface treatment, e.g., by glow discharge (3) or copolymerization (4). In this study, a novel strategy toward bioactive surfaces by adsorption of self-assembling copolymers modified by proteins and enzymes onto polystyrene was explored. The self-assembling copolymers possess sufficiently hydrophobic blocks that strongly interact with polystyrene and yet carry polyelectrolyte segments, which are accessible for modification. Recently, a novel class of hydrophobically modified polyelectrolytes was introduced whereby poly(propylene oxide) (PPO) group acts as a temperaturesensitive hydrophobe (5-20). Attachment of the PPO group onto a polyelectrolyte is a means of creating a dually responsive, self-assembling material by adding temperature sensitivity to an already pH- and ionsensitive polymer. In the present work, ongoing study is continued that is concerned with a distinct species of such dually responsive polymers whereby multiple poly(acrylic acid) (PAA) segments are grafted onto Pluronic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers via C-C bonding (10). High molecular weights and extreme sensitivity to temperature distinguish these PEO-PPOPEO-PAA copolymers. Above certain temperatures, their semidilute aqueous solutions can form reversible * To whom correspondence should be addressed: 15 Sherwood Road, Swampscott, MA 01907. E-mail
[email protected].
gels with significant elastic moduli (9). Gelation is due to appearance of micelle-like aggregates that serve as physical cross-links between Pluronic-PAA chains, as shown by rheological methods (8), probe solubilization (17), light scattering (7, 15), NMR (20), SANS (12), SEC (11), and DSC (11) techniques. Herein, we have taken interest in the use of the self-assembling polymers as matrixes for conjugation of biomolecules. The conjugates appear to adsorb strongly onto hydrophobic surfaces, as described below. EXPERIMENTAL SECTION
Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich Co. and were used as received. Pluronic F127 NF (EO100PO65EO100) was purchased from BASF Corp. (Parsippany, NJ). Poly(ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide)-g-poly(acrylic acid) (PEO-PPO-PEO-PAA) (CAS no. 18681081-1) was synthesized by dispersion/emulsion polymerization of acrylic acid along with simultaneous grafting of poly(acrylic acid) onto the Pluronic backbone using initiators lauroyl peroxide and 2,2′-azobis(2,4-dimethylpentanenitrile) (DuPont Specialty Chemicals) as described in detail elsewhere (14). Copolymer fraction used in this study had a Mw of 3.2 × 106 and PAA and Pluronic contents of 55 and 45%, respectively (11, 14). Polystyrene (PS) nanospheres were obtained from Seradyn, Inc. (Indianapolis, IN) and had a nominal diameter of 69 nm. Albumin-2-amido-deoxy-D-glucose (bovine, ∼15-20 mol of glucose/mol of albumin) and R-D-glucosylated-FITCalbumin (bovine, ∼15-20 mol of glucopyranose and 2-3 mol of FITC/mol of protein) were obtained from Sigma and dissolved at 1:1 molar ratio in 10 mM phosphate buffer following lyophilization prior to use. Conjugation of Proteins with Pluronic-PAA Copolymers. Conjugation of fluorescein isothiocyanate (FITC)-labeled concanavalin A (Con A, 1.5 mol of FITC/ mol of protein, ∼95% protein) onto Pluronic-PAA was carried out by simultaneous activation and coupling with a water-soluble condensing agent, 1-ethyl-3-(3-dimethy-
10.1021/bc9800973 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/15/1999
Technical Notes
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Scheme 1. Conjugation of a Protein and PluronicPAA Copolymer
laminopropyl)carbodiimide (EDA) (Scheme 1). The Pluronic-PAA, FITC-Con A, and EDA components were dissolved in 10 mM phosphate buffer (pH 7.4) at 4 °C to result in final concentrations of 10 mg/mL, 2 mg/mL, and 2 mg/mL, respectively. The mixture was then gently shaken for 24 h at 4 °C. The resulting conjugate was precipitated by addition of 1 M HCl at pH 4.8, quickly filtered off, and redissolved in chilled 10 mM phosphate buffer. The conjugated FITC-Con A was assayed fluorometrically (typical λex 490 nm, λem 520 nm). To verify that the FITC-Con A was covalently attached to PluronicPAA, a control experiment was conducted as follows. A mixture of FITC-Con A and the polymer was run without addition of the EDC activation reagent. After the same treatment as that with EDC, a negligible amount of protein and FITC-Con A was detected in the precipitate. Gel-permeation chromatography (GPC) of Pluronic-PAA and Con A-Pluronic-PAA conjugates was run at 15 °C on a Shimadzu LC-10A Series HPLC set up which included an RF-551 fluorescence HPLC detector, LC-10 AD solvent delivery unit, an SCL-10A system controller, and a Viscotek differential laser refractometer. An aliquot of 50 µL of 1.0 mg/g sample of conjugate solution in 0.05 M NaNO3 was loaded onto a PL aquagel-OH analytical temperature-controlled 3-column system (particle size 8 or 15 µm; dimensions 300 × 7.5 mm, Polymer Laboratories, Inc.) and then eluted with 0.05 M NaNO3 using a pump speed of 0.8 mL/min. Representative chromatograms of FITC-Con A-Pluronic-PAA conjugate are shown in Figure 1. GPC confirmed covalent binding of Con A and Pluronic-PAA. The conjugates were lyophilized and kept in sealed vials at 4 °C. Zn2+-insulin (from porcine pancreas, crystalline) and [125I]insulin (porcine, 80-120 µCi/µg, NEN Life Science Products, Boston, MA) were conjugated to Pluronic-PAA copolymers as follows. Stock solution of Zn2+-insulin and [125I]insulin (50:1 mol ratio, total insulin concentration 500 µg/mL) was prepared in 10 mM phosphate buffer (pH 7.4). Pluronic-PAA and EDA were added to the stock solution at 4 °C to result in final concentrations of 10 and 2 mg/mL, respectively, and the mixture was gently shaken for 24 h at 4 °C. Then an equal volume of 10 mM glycine in phosphate buffer was added, and the solution was stirred at 4 °C for 6 h and filtered off using 0.45 µm membrane filter. The conjugate was then dialyzed against phosphate buffer at 4 °C for 24 h using Spectra/Por cellulose ester membrane (molecular weight cutoff 50 000, Spectrum, Laguna Hills, CA), lyophilized, and stored at
Figure 1. Gel-permeation chromatograms illustrating the conjugation of FITC-Con A with Pluronic-PAA copolymers. (1, 2) FITC-Con A-Pluronic-PAA; (3) FITC-Con A-Pluronic-PAA as in chromatogram 1 blended with FITC-Con A. Fluorescence at 515 nm (1 and 3) and refractive index (2) detection was used. Unbound FITC-Con A appears as a small peak in chromatogram 3.
Figure 2. Gel-permeation chromatograms illustrating the conjugation of [125I]insulin with Pluronic-PAA copolymers. (1) Insulin-Pluronic-PAA synthesized in the presence of EDA following dialysis; (2) Pluronic-PAA blended with [125I]insulin without EDA following dialysis; (3) insulin.
4 °C in a sealed vial. To quantify conjugation of insulin onto Pluronic-PAA, identical synthetic procedure (but without EDA) was conducted followed by dialysis. A 0.01 mg/mL solution of the insulin-Pluronic-PAA conjugate or the initial mixture of [125I]insulin and unlabeled insulin was passed through a TSK-gel G3000PW GPC column (600 × 7.5 mm, Toyo Soda) at 15 °C. The flow rate of 0.2 mL/min with 10 mM phosphate buffer as an eluent was used and a Gilson FC205 automatic fraction collector was applied. Collected fractions were kept in sealed vials at 4 °C and subjected to γ-radioactivity measurement. Figure 2 shows chromatograms pertaining to the insulinPluronic-PAA conjugate and Pluronic-PAA made with and without EDA, respectively, as well as insulin mixture. As can be seen, the polymer that participated in the control experiment without EDA did not contain detectable level of insulin, whereas the conjugate made
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Table 1. Effect of Conjugate Adsorption on Surface Characteristics of Polystyrene Films
polystyrene
composition by XPS/ESCA (%) C O N
as received, bare surface 97.4 2.6 nd Pluronic-PAA 73.3 26.7 nd insulin-Pluronic-PAA 71.7 18.4 9.9
air-in-water contact angle (deg) 86 ( 4 (6) 33 ( 4 (10) 33 ( 6 (10)
with EDA exhibited well-defined peak clearly distinguishable from the original insulin. Fractions around the peak were collected and total radioactivity was measured. The yield of conjugation reaction of insulin onto PluronicPAA in the presence of EDA was estimated to be about 60%, based on relative radioactivity levels. Conjugate Adsorption onto Polystyrene Surfaces. Polystyrene nanospheres were coated with FITC-Con A-Pluronic-PAA conjugates by first dispersing 2 mg/mL nanospheres in 10 mM chilled phosphate buffer following addition of equal volume of 2 mg/mL FITC-Con APluronic-PAA conjugate. The resulting suspension was kept at 37 °C for 16 h, then snap-frozen and lyophilized. The resulting material was resuspended in 10 mM phosphate buffer (pH 7.4). The conjugate-coated particles were concentrated by centrifugation at 37 °C using Micropure Filtration Device (pore size 0.22 µm) (Millipore Co.), and the supernatant was removed. After resuspension in phosphate-buffered saline the particles were again concentrated and the supernatant collected for fluorescence measurement. Washing cycles were repeated until no FITC was detected in the washouts. Surface concentration of the conjugate was estimated spectrofluorometrically to be about 0.6 µg of FITC-Con A/cm2. Adsorption of unconjugated FITC-Con A onto polystyrene particles under identical conditions was below 0.1 µg/cm2. Flat-well, polystyrene plates (Corning) with untreated surfaces were modified with insulin-Pluronic-PAA conjugates by placing 2 µL of 2 mg/mL conjugate solution in 10 mM phosphate buffer on the bottom of the well following evaporation of aqueous solution on air at 37 °C. A mixture of [125I]insulin and unlabeled insulin was used for the conjugate preparation. Adsorption procedure was repeated up to 20 times for each well. The wells were gently rinsed by deionized water at 37 °C until γ-radioactivity in the washouts did not exceed that of a blank solution. In the control experiments, polystyrene surfaces were treated by identical Pluronic-PAA conjugate solutions without insulin or by insulin without conjugate. Insulin contents were calculated by comparing the radioactivity in the wells with that of [125I]insulin per unit weight, as described elsewhere (3, 21, 22). The effect of conjugate adsorption on the properties of the polystyrene surfaces is presented in Table 1. Figure 3 shows adsorption isotherms (37 °C) of the insulin conjugated with Pluronic-PAA and that without conjugation. It can be seen that the amount of the adsorbed conjugated insulin at saturation by an order of magnitude exceeded that of unconjugated insulin. Quantification of the Polymer Adsorption onto PS Particles. The mass of Pluronic-PAA conjugates and Pluronic F127 adsorbed on PS nanoparticles was determined by radioisotope method as follows (23, 24). Aminoterminated Pluronic F127 was synthesized via the sequence of quantitative modifications (25):
Pluronic-OH f Pluronic-SO2CH3 f Pluronic-N3 f Pluronic-NH2 with the amino group attached to one end of the Pluronic
Figure 3. Adsorption isotherms (37 °C) of insulin and insulinPluronic-PAA conjugates onto polystyrene plate wells. Each point represents an average of six independent measurements.
molecule. Activation of only one terminal hydroxyl out of two present in a polyether is ensured by the proper ratio of equivalents of OH groups and an activation agent and is a commonly used technique (25-27). Marginal fractions with both end-groups modified are separated by HPLC (27). The 4-hydroxyphenyl moiety was then introduced into Pluronic-NH2 by reaction with N-succinimidyl-3-[4-hydroxyphenyl]propionate (Bolton-Hunter reagent) (Pierce Chemical, Rockford, IL) in tetrahydrofuran following iodination reaction with Na125I as described elsewhere (28). For 125I labeling, an aqueous solution of 3 w/v% Pluronic-PAA and 5 mg/mL N-hydroxysulfosuccinimidyl4-azidosalicylic acid (sulfo-NHS-ASA) (Pierce) was deaerated under vacuum and illuminated by a LightWelder 3010 EC UV spot/wand lamp (spectral output
