Synthesis, Characterization, and Preliminary Biological Study of

Biomacromolecules 2002, 3, 805-812

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Synthesis, Characterization, and Preliminary Biological Study of Glycoconjugates of Poly(styrene-co-maleic acid) Ivan Donati,* Amelia Gamini, Amedeo Vetere, Cristiana Campa, and Sergio Paoletti Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via L. Giorgieri 1, I-34127, Trieste, Italy Received February 6, 2002; Revised Manuscript Received April 16, 2002

Three derivatives of the biocompatible polymer poly(styrene-co-maleic anhydride) (SMA) were obtained with 1-amino-1-deoxy-β-D-galactose, 1-amino-1-deoxy-β-D-glucose, and 1-amino-1-deoxy-β-D-lactose, respectively. The amino sugars were chemically conjugated via formation of an amide bond between the anomeric amino group of the sugar residue and the anhydride of the copolymer, giving the corresponding glycoconjugate derivatives. Colorimetric assay of the unreacted amino groups and elemental analysis were used to determine the degree of substitution. About 56%, 54%, and 94% of the available anhydride groups reacted to give galactosyl-amide (SMA-Gal), glucosyl-amide (SMA-Gluc), and lactosyl-amide (SMA-Lac) branched polymers, respectively. The synthesized glycopolymers were characterized by Fourier transform infrared spectroscopy, gel permeation chromatography, circular dichroism, and UV and fluorescence spectroscopy. The release of glucosylamine from the glucosyl-amide branched polymer, by basic hydrolysis, was monitored by high-performance anion-exchange chromatography and by capillary electrophoresis, providing for an additional check of the degree of substitution of this specific polymer derivative. Biological activity tests showed that both SMA-Gal and SMA-Lac allow adhesion of HepG2 hepatic cells about five times larger than that of hydrolyzed, underivatized SMA. 1. Introduction There is an increasing interest for the synthesis of the socalled synthetic glycopolymers (i.e., synthetic polymers bearing sugar residues), mainly for their potential use as model compounds covering a wide range of physicochemical properties, modulated by their structural units. In line with the excellent biorecognition properties of naturally occurring glycopolymers (polysaccharides), synthetic glycopolymers have found a variety of biomedical applications such as culture substrates,1,2 tumor diagnosis,3,4 and detection and trapping of viruses.5-7 Poly(styrene-co-maleic anhydride) (SMA) is a synthetic copolymer with interesting features from both the chemical and the biological points of view.8-14 From the chemical point of view, SMA copolymer can be readily subjected to modification. The anhydride groups can easily react with low molecular weight compounds such as water, alcohols, and amines. Cross-linked 3D matrixes can be easily obtained by reaction with polyfunctional reagents, in addition to the classical route of introducing small amounts of, e.g., divinylbenzene during the copolymerization of maleic anhydride and styrene. From the biological point of view, SMA has been used for conjugation with neocarzinostatin (NCS), a potent but very toxic antitumor protein. Conjugation with SMA causes an increase of the NCS plasma half-life and a decrease of its toxicity.13 More recently SMA has been used also for immobilization of Laminin peptide YIGSR resulting * Corresponding author. Tel.: +39-040-5583692. Fax: +39-0405583691. E-mail address: [email protected].

in an increase of its antimetastatic effect.14 On the basis of this work SMA-glycoconjugates have been identified as a group of biocompatible polymer derivatives that might disclose interesting biofunctional properties when compared to pure SMA. Hence, in the present paper we report the synthesis and the characterization of glycosyl derivatives of SMA obtained by reaction with ad hoc prepared glucosylamine (1-amino1-deoxy-β-D-glucose), galactosylamine (1-amino-1-deoxyβ-D-galactose), and lactosylamine (1-amino-1-deoxy-β-Dlactose), respectively. The synthetic glycopolymers have been designed to be used for hepatic cell cultures as a biofunctional matrix, to combine the specific properties of SMA polymers with those of carbohydrate ligands that can be recognized by receptors on cell surfaces. In the normal liver, each hepatocyte performs hundreds of metabolic activities. However, an isolated hepatocyte outside the organism rapidly loses its cellular functions and has only a limited viability. A culture system enabling hepatocytes to survive and proliferate over long periods of time would permit extensive studies of basic cellular phenomena and investigations of the biology of liver diseases. Such a system would also serve as an artificial organ and as a bioreactor to synthesize important proteins. Specific requirements to enhance the viability of hepatocytes have already been addressed. In the first place, hepatocytes need to anchor and adhere to a matrix surface. In addition, hepatocytes display characteristic recognition abilities for carbohydrate sequences. In particular, hepatocytes express on their surface the asialoglycoprotein receptor (ASGPR).

10.1021/bm020018x CCC: $22.00 © 2002 American Chemical Society Published on Web 05/23/2002

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ASPGR was the first lectin to be identified in animals; it was shown to bind and internalize glycoproteins with terminal galactosyl residue.2 Indeed, a receptor recognized by an asialoglycoprotein bearing a terminal β-galactose is localized on the hepatocyte cell membrane.2 Polymers having galactosyl pendant groups might then allow fabrication of a cell carrier or matrix which supports hepatic cell adhesion. The new glycopolymers here reported were comparatively tested for hepatic cell adhesion in a preliminary cell culture study. 2. Materials and Methods 2.1. Materials. Poly(styrene-co-maleic anhydride) (SMA 1000P) was a kind gift of ELF ATOCHEM (Paris, France). The polymer sample was purified by reprecipitation with 2-propanol from butanone solutions. The recovered purified polymer (SMAp) was then dried in a vacuum for 2 days. D-Galactose, D-glucose, and D-lactose were from Sigma (St. Louis, MO). The human hepatoma cell line HepG2 was from ATCC (Rockville, MA). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin/ampicillin solution, and fetal calf serum were from GIBCO-BRL (Grand Island, NY). Nunclon Delta MicroWell plates were from NUNC (Rochester, NY). Polystyrene microtest plates were from Sarstedt (Newton, NC). All other chemicals were of analytical grade. For aqueous solutions MilliQ water (Millipore, MA) was used throughout. 2.2. Synthesis of Amino Sugars. 1-Amino-1-deoxy-β-Dgalactose, 1-amino-1-deoxy-β-D-glucose, and 1-amino-1deoxy-β-D-lactose were synthesized following the procedure already described.15 2.3. Preparation of Hydrolyzed SMA Samples (HSMA). A solution of SMAp in DMSO (Cp ≈ 12% w/w) was mixed with a 0.5 M NaHCO3 solution (pH ) 9) to get a final polymer concentration of 0.2% (w/w) and then stirred at room temperature for 3 h. The reaction mixture containing the sodium salt form of the hydrolyzed polymer (HSMA) was exhaustively dialyzed against MilliQ water and then lyophilized to obtain a dialyzed fraction of the hydrolyzed polymer (HSMAd). 2.4. Synthesis of Glycoconjugate-SMA Derivatives. A 12% (w/w) solution of poly(styrene-co-maleic anhydride) (SMAp) in DMSO (0.4 mL.) was mixed with a 0.5 M NaHCO3 buffer solution (24 mL, pH ) 9) to get a final polymer concentration of 0.2% (w/w). Glycosylamine was added to the solution up to the final molar ratio 2:1 of amino sugar-to-polymer repeating unit. The mixture was stirred at room temperature for 3 h. The reaction course was checked by colorimetric assay, monitoring the consumption of amino sugar upon time. The reaction mixture was then dialyzed exhaustively against MilliQ water and then lyophilized. The amidation reaction reported in Scheme 1 produced three different glycoconjugate derivatives, named SMA-Gal, SMAGluc, and SMA-Lac, respectively. 2.5. FT-IR Spectroscopy. Infrared spectra of SMAp, HSMAd, SMA-Gal, SMA-Gluc, and SMA-Lac samples in KBr were recorded with a FT/IR Perkin-Elmer 2000 System spectrometer.

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2.6. Elemental Analysis. Determination of C, H, and N content was performed on the Na+-forms of SMA-Gal, SMALac, and SMA-Gluc derivatives. The obtained elemental percentage compositions were as follows: Calcd for SMAGal: C, 48.9; H, 4.53; N, 2.01. Calcd for SMA-Gluc: C, 53.9; H, 5.15; N, 2.16. Calcd for SMA-Lac: C, 52.2; H, 5.05; N, 2.47. From the above-reported percentages the molar degrees of substitution (DS) with respect to the anhydride comonomer were calculated to be 0.56 for SMA-Gal, 0.54 for SMA-Gluc, and 0.94 for SMA-Lac, respectively. 2.7. UV and Fluorescence Spectroscopy. Absorption measurements were carried out using a double-beam Cary4E spectrophotometer. Quartz cells of 1 cm path length were used. The interaction existing in aqueous basic conditions between free amines and 2,4,6-trinitrobenzenesulfonic acid (TNBS)16 was exploited to follow upon time the consumption of glycosylamine during the synthesis of the glycoconjugate derivatives. Polymer-free glycosylamine solutions in 0.5 M NaHCO3 (pH ) 9) were used as reference. Absorbance readings were made at 335 nm. Following the procedure reported elsewhere,16 the amount of unreacted glycosylamine was determined using a calibration curve obtained with solutions of the galactosylamine-TNBS derivative in the amino sugar concentration range from 1.1 × 10-5 to 5.6 × 10-5 mol/L, in aqueous 0.5 M NaHCO3. Absorbance measurements of the sodium salt form of the polymer derivatives (HSMA, SMA-Gal, SMA-Gluc, and SMA-Lac) were performed in aqueous solution, at neutral pH. Polymer concentrations of 2 × 10-3 monomol/L were used for spectra recorded in the 320-240 nm range. For spectra recorded in the 240-200 nm range, polymer concentrations of an order of magnitude lower were used (i.e., 1 × 10-4 monomol/L). A Perkin-Elmer single-beam LS50B spectrofluorimeter was used to perform fluorescence measurements on HSMA and SMA-Lac aqueous solutions. Polymer concentrations were 2 × 10-4 monomol/L in water. 2.8. Potentiometry. Potentiometric titrations were performed to determine the equivalent weight of the HSMAd copolymer. A Radiometer pHM240 pH-meter equipped with a glass electrode was used. The H+ form of the hydrolyzed HSMAd sample was prepared by dialyzing relatively concentrated polymer solutions (Cp ≈ 3 g/L) against 0.1 M HCl. The excess of HCl was removed by exhaustive dialysis against MilliQ water. The polymer was recovered by freezedrying. Aqueous solutions of known polymer specific concentration were titrated with 0.1 M NaOH standard solution (Titrisol, Merck). A repeating unit molar mass of 219 ( 6 g/mol was calculated for HSMAd (H+-form), which compared rather well with the theoretically expected value of 220 g/mol. 2.9. High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAECPAD). The system (Dionex, Sunnyvale, CA) consisted of an isocratic pump (DQP-1), with a pulsed amperometric detector (100 µL loop). A CarboPac PA1 anion-exchange column (250 × 4 mm i.d.) plus guard column (50 × 4 mm

Glycoconjugates of Poly(styrene-co-maleic acid)

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i.d.) were used. The flow-through detection cell contained a gold working electrode and an Ag/AgCl reference electrode. The waveform parameters were Edet ) +0.06 V, tdet ) 440 ms (tint ) 240 ms), Eox ) +0.8 V, tox ) 180 ms, Ered ) -0.3V, tred ) 360 ms. The eluent was 0.1 M NaOH flowing at 1.0 mL/min. The analysis was performed on solutions of HSMA (8.8 mg/mL) and SMA-Gluc under hydrolytic conditions (8.8 mg/mL in 3 M NaOH at 70 °C for 1.5 h). The latter solution was neutralized with 1.5 M H2SO4 and injected after 24 h in order to attain full conversion of the released glucosylamine into glucose.15 2.10. Capillary Electrophoresis (CE). The system was an Applied Biosystems HPCE model 270A-HT with Turbochrom Navigator (4.0) software. The fused silica column (72 cm (50 cm to detector) × 50 µm i.d. × 375 µm o.d.) was from Supelco (St. Louis, MO). All runs were done at 27 °C. Samples were loaded under vacuum at a pressure of 16.9 kPa (1.5 s). Before sample injection, a 4-min conditioning of the capillary with the buffer followed a 2-min washing with 0.1 N NaOH (vacuum pressure 67.6 kPa). CE/indirect UV on HSMA (4 mg/mL) and SMA-Gluc hydrolysis solutions (4 mg/mL in 3 M NaOH at 70 °C) was performed using 6 mM sorbate at pH 12.5 (10 kV, 256 nm). The hydrolysis solutions were neutralized with 6 M HCl and analyzed after 24 h in order to attain full conversion of the released glucosylamine into glucose.15 2.11. Viscometry. Reduced capillary viscosities were measured by using a Schott-Gera¨te AVS/G automatic apparatus mounting a Ubbelohde type viscometer. Measurements were performed at 25 °C on solutions of the purified starting material (SMAp) as well as of the hydrolyzed HSMAd sample. For SMA copolymer in THF with 5% acetic acid the following [η]-Mv relationship holds:8 [η] ) (8.724 × 10-2)Mv1/2 + (10.01 × 10-5)Mv

(1)

where Mv is the viscosity-average molar mass. According to eq 1 and using the value [η] ) 6.59 mL/g obtained in THF doped with acetic acid (5%), an average molar mass (Mv) of 5700 g/mol was estimated for the purified SMA sample, corresponding to a viscosity-average degree of polymerization DPv = 28. Viscosity measurements on the hydrolyzed sample were performed in 0.15 M NaCl in the polymer concentration range from 9 to 30 g/L; the intrinsic viscosity was 9.0 mL/ g. 2.12. Gel Permeation Chromatography (GPC). GPC measurements were performed on salt solutions (0.15 M NaCl) of HSMAd as well as of the SMA-Gal derivative using a Jasco PU-880 HPLC, with a Rheodyne 9125 injector and coupled with a RI detector (Waters model 410). A set of TSK Pwxl columns (G6000, G5000, and G3000) was used. Standard pullulans were used for the (molecular weight vs elution time) calibration curve. 2.13. CD Spectroscopy. Circular dichroism spectra on HSMAd, SMA-Gal, SMA-Gluc, SMA-Lac, and aqueous solutions were recorded in the 200-300 nm range of wavelengths with a Jasco J-700 spectropolarimeter. A quartz cell of 1-cm optical path length was used. The raw data of

Scheme 1. Reaction Scheme for the Production of SMA Glycopolymers

optical ellipticity were corrected for the water baseline. For all samples the concentration of the solutions were in the range from 2 × 10-4 to 3 × 10-4 monomol/L. 2.14. Biological test. 2.14.1. Preparation of Microwell Plates. Polystyrene-made microwell plates were used. In preliminary control experiments, that kind of material was demonstrated not to give rise to any hepatocyte adhesion. Coating of the 96-well polystyrene microtest plates was done by exposure with 12 × 300 µL of sterile solutions of 3.8 × 10-4 monomol/L of HSMAd, SMA-Gal, SMA-Glc, and SMA-Lac in 0.1 M bicarbonate buffer (pH ) 9.6) at 4 °C for 12 h and washing them with 2 × 300 µL of PBS just prior to use. 2.14.2. Cell Cultures. HepG2 cell culture was performed in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL of penicillin, and 100 µL/mL of streptomicyn at 37 °C in a 5% CO2 atmosphere. 2.14.3. Cell Adhesion Assay. Cell adhesion assay was performed according to a slight modification of a method reported by Riikonen.17 Confluent cultures of HepG2 cells were detached by using 0.01% trypsin and 0.02% EDTA and then suspended in DMEM. Fifty thousand cells were transferred into each well of the polymer-treated polystyrene microtest plate and incubated at 37 °C for 2 h. Nonadherent cells were removed by twice rinsing the wells with 100 µL of DMEM. Adherent cells were fixed for 20 min in the dark with 100 µL of a 2% solution of paraformaldehyde in PBS. Cells were then stained for 10 min with 100 µL of a 0.5% solution of crystal violet in 20% of ethanol and then washed twice with 300 µL of water. The plates were allowed to airdry, the stained cells were dissolved with 10% acetic acid, and absorbance was measured spectrophotometrically at 600 nm with a microplate reader. 3. Results and Discussion SMA-Gal, SMA-Lac, and SMA-Gluc derivatives were obtained via formation of an amide bond between the polymer anhydride functionality and the anomeric amino group of the glycosylamine, as shown in Scheme 1. Despite identical conditions applied to all synthetic procedures, the obtained derivatives are characterized by a different degree of substitution, as shown below. Contrary to what could be

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Figure 1. IR spectra of SMA (a), HSMA (b), SMA-Gal (c), and SMA-Lac (d) polymer samples.

expected on the basis of the identical stability of the used glycosylamines, lactosylamine showed the highest reactivity being the only one for which nearly 100% of amidation was achieved. 3.1. FT-IR. IR spectroscopy confirmed that the synthesized compounds were the glycoconjugate derivatives, according to reaction Scheme 1. As an example, in Figure 1 the FT-IR spectrum of the purified SMA copolymer (a) is compared with the spectra recorded for the HSMAd (b), SMA-Gal (c), and SMA-Lac (d) polymer samples, respectively. The symmetric CdO stretching absorption band (1760 cm-1) and the asymmetric one (1860 cm-1) of the anhydride monomers (a) disappeared in spectra b, c, and d where the signal corresponding to the carboxylate CdO stretching vibrations (≈1560 cm-1) developed instead. The vibrational band corresponding to the CdO stretching of the amide group located at ≈1670 cm-1 appeared in both SMA-Gal (Figure 1c) and SMA-Lac spectra (Figure 1d). In the latter case the amide band is better developed than in the former, as expected on the basis of the higher degree of substitu tion. 3.2. Elemental Analysis. The extent of derivatization for the Na+ form of SMA-Gluc, SMA-Gal, and SMA-Lac was estimated by elemental analysis. For the SMA-Gal derivative, one galactosyl-amide residue was found for 1.8 SMA repeating units (degree of substitution, DS, ) 0.56). Similarly, 0.54 and 0.94 were the values of the degree of substitution calculated for SMA-Gluc and SMA-Lac, respectively. 3.3. Potentiometry and NMR. The determination of the value of the degree of substitution was attempted also by potentiometry. Surprisingly, potentiometric measurements, performed on 0.1 M NaCl aqueous solution of the polymers in the H+-form, gave values of degree of substitution which differed roughly by 40% from the above-reported elemental

analysis values (i.e., 0.32, 0.31, and 0.51 for galactosyl-, glucosyl-, and lactosyl-amide derivatives, respectively). Such a discrepancy was accounted for by a partial hydrolysis of the amide bond induced by the low pH at which the dialysis procedure was performed (see Experimental Section). Indeed, elemental analysis of the glycosyl-amide derivatives in the H+ form18 provided values of DS almost coinciding with those from the potentiometric data (i.e., 0.32, 0.30, and 0.51 for SMA-Gal, SMA-Gluc, and SMA-Lac, respectively). Also 1H NMR spectra were recorded, trying to confirm the chemical structure of the polymer derivatives. However, the very low resolution of the spectra prevented any signal attribution, due to the pronounced broadness of the resonances (data not shown). A properly addressed NMR study is certainly required and will be the subject of a future work. 3.4. UV and Fluorescence Spectroscopy. The derivatives formed between free amines and TNBS in aqueous basic conditions16 are UV absorbing, thus allowing the monitoring with time of the consumption of glycosylamine and hence the reaction of amide formation. The relatively high value of pH (pH ) 9) prevented the hydrolysis of the glycosylamines.15 Figure 2 reports the case of the galactosylamineTNBS complex. The consumption of galactosylamine reacting with the anhydride groups is clearly indicated by the decrease of the optical density (triangles in Figure 2). For comparison, the time-independent absorbance of a (polymer free) galactosylamine reference solution is also reported (circles). In this specific case, it was estimated that ≈54% of the SMA repeating units bear a galactosylamine moiety, in very good agreement with the above-reported elemental analysis data. Beside monitoring the amidation reaction course, UV measurements, as well as fluorescence spectroscopy, were used also to characterize the purified polymer derivatives.


Figure 2. Dependence of the optical density on the time of reaction for galactosylamine-TNBS in the presence of SMA (triangles) and for the galactosylamine-TNBS (polymer-free) solution (circles).


Figure 4. Absorption (left) and fluorescence intensity spectra (right) of HSMA (s) and SMA-Lac (- -) samples in aqueous solution. Polymer concentrations were 2 × 10-3 monomol/L for the absorption spectra and 2 × 10-4 monomol/L for the fluorescence spectra (λex ) 270 nm), respectively.

Figure 3. (left) UV spectra of 1 × 10-4 monomol/L aqueous solutions of HSMA (s), SMA-Gluc (‚ ‚ ‚), SMA-Gal (- -), SMA-Lac (‚ -), and EMA (‚ ‚ -) samples. (right) UV spectra of 2 × 10-3 monomol/L aqueous solutions of HSMA (s), SMA-Gluc (‚ ‚ ‚), SMA-Gal (- -), and SMA-Lac (‚ -).

Figure 3 reports the UV spectra obtained for HSMA, SMAGluc, SMA-Gal, and SMA-Lac aqueous solutions, respectively. In the spectrum of HSMA, the excitation of π electrons in the aromatic ring19 gives rise to the absorption band centered around 260 nm as well as that of much higher intensity occurring below 200 nm and the one centered around 210 nm (as a shoulder). Below 220 nm a much smaller contribution due to carboxylated groups is additionally overimposed. The small absorbance contribution due to the carboxylated chromophors is easily verified by comparing the UV data of HSMA with the spectrum recorded for poly(ethylene-co-maleic acid) (EMA) at a comparable polymer concentration (left side of Figure 3). As shown in Figure 3, the absorption intensity increased upon derivatization, roughly proportional to the degree of substitution. Beside the expected contribution below 220 nm of the newly formed amide linkages, the observed absorbance increase that characterizes the entire spectra should mainly arise from the different environment of the chromophors created by the insertion of sugar residues along the polymer chains. It is known that, as a result of loss of symmetry, an extinction coefficient higher for phenol than for benzene is observed for the symmetry-forbidden π f π* transitions, similarly to what found for tyrosine in comparison with phenylalanine.20 In line with these findings, the data of Figure 3 can be safely interpreted as due to a strong effect of the sugar moiety on

Figure 5. HPAEC-PAD chromatogram obtained after neutralization (HCl) of a SMA-Gluc solution treated with 3 M NaOH for 1.5 h at 70 °C. Peaks 1 and 2 are those of glucose and fructose, respectively.

the symmetry-forbidden n f π* and π f π* transitions of both the amide and the aromatic chromophors. Fluorescence data collected for polymer derivatives in aqueous solutions are in line with UV data. As an example, the emission spectra of HSMA and SMA-Lac samples are reported in Figure 4 and compared with the corresponding absorption spectra in the same wavelength range. Changes in the molecular environment of the aromatic ring are clearly indicated by the notably higher intensity of the light emitted by SMA-Lac as compared to the reference polymer HSMA, as well as by the appearance of a better developed band at lower energies (λ ≈ 337 nm). Newly established solvent interactionssalso driving conformational rearrangementss would then likely be responsible for the parallel increased probability of both absorption and emission. 3.5. HPAEC-PAD and CE. HPAEC-PAD and CE were used to confirm the chemical structure of the derivatives. Figure 5 reports the HPAEC-PAD chromatogram of the products of amide hydrolysis, obtained by treating SMAGluc with 3 M NaOH at 70 °C for 1.5 h, followed by neutralization with HCl. The addition of HCl ensures that the dissociation equilibrium of glucosylamine, released by the hydrolysis of the glycopolymer, is completely shifted

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Figure 6. Glucose concentration (mM) as a function of the time of alkaline hydrolysis of SMA-Gluc as determined by CE. The solid line represents the best-fit curve calculated by using a (pseudo) first-order kinetics for the hydrolysis reaction.

Figure 7. MWD distribution curves for aqueous 0.15 M NaCl solutions of HSMAd (full line) and SMA-Gal (dashed line).

toward glucose (peak 1 of Figure 5), according to the following series of reactions: OH-

H+

-CONH-S 9 8 -COO- + H2N-S 9 8 (slow) (fast) -COOH + OH-S + NH4+ S represents the sugar ring. Fructose (peak 2 of Figure 5) is known to originate from glucose isomerization.15 A quantitative check of the amount of glucose released was performed at different times, granting support to the elemental analysis values (data not reported). Also CE was used to quantify the extent of substitution. In Figure 6 the amount of glucose (mM) released from SMAGluc is reported as a function of hydrolysis time. The solid line represents the best-fit curve calculated by using (pseudo) first-order kinetics for the hydrolysis reaction. The calculated asymptotic value of glucose concentration (i.e., 8.1 mM) corresponds to a degree of substitution of 0.5, in good agreement with the elemental analysis results. 3.6. GPC Measurements. Figure 7 reports the molar mass distribution (MWD) curves for HSMAd and SMA-Gal obtained from GPC performed in 0.15 M NaCl. Apparent weight-average molar mass values (Mw) of 12700 and 10400 g/mol were obtained for HSMAd and SMA-Gal, respectively.

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Figure 8. CD spectra recorded at neutral pH for aqueous solutions of HSMA (‚ ‚ ‚) and EMA (O O O) (right) and of SMA-Gal (- -), SMAGluc (- ‚ ‚-), SMA-Lac (s) (left). The polymer concentration was 2 × 10-4 monomol/L for SMA-Lac and 3 × 10-4 monomol/L for the other derivatives.

The polydispersity index was 1.84 for HSMAd and 1.68 for SMA-Gal. Due to the identical conditions used for the whole set of the performed synthesis as well as for sample purification, a molar mass of the order of 104 g/mol also for the SMA-Gluc and SMA-Lac samples should be considered as a reasonable estimate. The Mw values of HSMAd and SMA-Gal are larger than that of the not-dialyzed starting copolymer (SMAp); this fact can be reasonably accounted for by an enrichment in higher molar mass fractions of the two former polymers, through the dialysis performed to purify them. As far as data of Figure 7 are concerned, an apparent shift of the MWD curve of the SMA-Gal toward low molar masses is clearly evident. The mildness of the hydrolysis conditions (i.e., 0.5 M NaHCO3), identically applied to obtain both HSMAd and SMA-Gal derivatives would hardly justify the occurrence of chain degradation. Instead, such an apparent shift might be justified considering for SMA-Gal a reduced hydrodynamic volume with respect to that of the hydrolyzed copolymer (HSMAd), as a consequence of the smaller number of net charges due to amidation. The decreased electrostatic repulsion, due to the decrease of the linear charge density upon chain substitution, likely resulted in a decrease of the coil expansion sufficiently large to be detected by GPC. 3.7. CD Spectroscopy. Interestingly enough, the aqueous solutions of the Na+ salt form of the underivatized HSMA copolymer showed nonzero optical activity in the 230-210 nm wavelength range. The consistent recording of a complex pattern of ellipticity for HSMA, well above the instrumental detection limit, is at total variance with the results from aqueous solutions of the simple analogue poly(ethylene-comaleic acid) (EMA), for which a signal of approximately zero is observed at neutral pH (see Figure 8, left). The latter result is expected for an alternating copolymer in which the anhydride ring opening is essentially random, thereby canceling any possible preferential stereochemistry with ensuing chirooptical properties. The surprising result shown by HSMA might simultaneously stem from (i) a regular insertion (head-to-head or head-to-tail) and opening of the


Figure 9. Relative hepatocyte adhesion efficiency of SMA derivatives to that of hepatocytes on EMA.

styrene comonomer in the growing chain and (ii) a correlated preferential stereochemistry of quaternarization of the maleic anhydride comonomer. The presence of many peaks and troughs in the CD spectrum seems to suggest the occurrence of different, not-too-long stereoregular sequences, but with a net prevalence of some as indicated by the nonzero ellipticity. Certainly, further and more detailed investigations are required to better clarify the conformational origins of the peculiar optical activity features shown by HSMA. The introduction of sugar moieties as side chains of HSMA produces a significant enhancement of the above-discussed CD properties. As shown in Figure 8 (right), a rather complex circular dichroism spectrum was observed for SMA-Gal, SMA-Gluc, and SMA-Lac aqueous solutions at neutral pH. It is worth remembering that, besides the already addressed transitions of the aromatic ring, the amide n f π* transition (symmetry-forbidden), generally appearing as a shoulder of the more intense π f π* UV absorption band centered at ≈190 nm, occurs in the 220-210 nm range.20 The amide group is directly linked to the chiral sugar moiety. Therefore, the amide transitions are most obviously expected to bring a significant CD contribution, such as in polypeptides and proteins. Moreover, the higher degree of substitution and the dimeric nature of the carbohydrate substituent might easily account for the greater ellipticity observed for SMA-Lac with respect to SMA-Gal and SMA-Gluc. However, the nonzero CD value of HSMA likely also suggests the possibility that, to some extent, a specific mutual orientation of the comonomer chromophors (i.e., carboxylic and aromatic groups) be present along the polymer chain. In line with UV and fluorescence data, this specific mutual orientation (together with a stiffening by the hindering chiral sugar side chains) should contribute to the enhancement of the circular dichroism bands of the polymer derivatives, over and above the addressed group contribution to CD. 3.8. Cell Adhesion. To measure the cell adhesion due to the glycoconjugate coating, polystyrene-made microwell plates were used. In fact, in preliminary control experiments that kind of material was demonstrated not to give rise to any hepatocyte adhesion. As shown in Figure 9, the performed test indicated that all polyanions favor hepatic cell adhesion. EMA showed the least efficiency, followed by HSMA. The latter showed an increase of about 30% over the former polyanion, addressing


to a modest contribution of the hydrophobic phenyl moiety to adhesion. On the contrary, the relative adhesion of both SMA-Gal and SMA-Lac was approximately five times higher than that of HSMA. At variance with that, only a 2-fold increase of the efficiency was observed for SMA-Gluc still with respect to HSMA. Obviously, further biological studies are needed to properly quantify all possible contributions governing the mechanism of cell adhesion. Still, some qualitative conclusions can be drawn: (i) Ionic interactions between the ionizable groups of the polymer backbone with the positiVely charged groups of membrane proteins play an important role in the mechanism of adhesion. Over and above, the hydrophobic nature of the styrene comonomer in HSMA seems to add a slight favorable effect onto hepatocyte adhesion. (ii) Given the large difference of the pKa of first and second ionization of HSMA, one can safely conclude that its net charge at around neutral pH is mostly due to the first ionization. This implies that both HSMA and the glycopolymers bear a very similar amount of charge. Therefore, the obserVed increase in the adhesion efficiency of the latter ones with respect to unsubstituted HSMA can be safely attributed to the pendant saccharidic moiety. (iii) The highest (and same) adhesion efficiency was shown by glycopolymers bearing galactose as the most exposed sugar in the side chain. We suggest that in both cases the attachment mechanism likely involves the galactose receptor located on the surface of the cell membrane.2 Also glucose as a pendant residue favors hepatocytes adhesion, albeit to a significantly lower extent. This finding is not surprising: polyacrylamide containing glucose as a side chain has been reported to show approximately the same attachment efficiency for rat hepatocytes as galactose-bearing polyacrylamide.21 Acknowledgment. C.C. is grateful to the University of Trieste for her Post-Doc Fellowship. The financial contributions of the University of Trieste (“ex-60%-Funds” to A.G. and to S.P.), of the Italian National Research Council, C.N.R. (“Young Scientists” Funds to C.C.), and of F.B.C. S.r.l., Area Science Park, Trieste, are gratefully acknowledged. References and Notes (1) Kobayashi, A.; Akaike, H. Enhanced adhesion and survival efficiency of liver cells in culture dishes coated with a lactose carrying styrene homopolymer. Makromol. Chem. Rapid Commun. 1986, 7, 64550. (2) Kobayashi, K.; Kobayashi, A.; Akaike, T. Culturing hepatocytes on lactose-carrying polystyrene layer via asialoglycoprotein receptormediated interactions. In Methods in Enzymology; Lee, Y. C., Lee R. T., Eds.; Academic Press: San Diego, CA, 1994; Vol. 247, p 409-18. (3) Bovin, N. V.; Gabius, H. J. Polymer-immobilized carbohydrate ligands: versatile chemical tools for biochemistry and medical sciences. Chem. Soc. ReV. 1995, 413. (4) Rye, P. D.; Bovin, N. V. Selection of carbohydrate-binding cell phenotypes using oligosaccharide-coated magnetic particles. Glycobiology 1997, 7, 179-82. (5) Choi, S. K.; Mammen, M.; Whitesides, G. M. Generation and in situ evaluation of libraries of poly(acrylic acid) presenting sialosides as side chains as polyvalent inhibitors of influenza-mediated hemagglutination. J. Am. Chem. Soc. 1997, 119, 4103-11.

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(6) Lees, W. J.; Spaltenstein, A.; Kingery-Wood, J. E.; Whitesides, G. M. Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems J. Med. Chem. 1994, 37, 3419-33. (7) Mammen, M.; Dahmann, G.; Whitesides, G. M. Effective inhibitors of hemagglutination by influenza virus synthesized from polymers having active ester groups. Insight into mechanism of inhibition. J. Med. Chem. 1995, 38, 4179-90. (8) Klumperman, L. Free radical copolymerization of styrene and maleic anhydride. Ph.D. Thesis, Technical University Eindhoven, 1994. (9) De Brouwer, H.; Schellekens, M. A. J.; Klumperman, B.; Monteiro, M. J.; German, A. L. Controlled radical copolymerization of styrene and maleic anhydride and the synthesis of novel polyolefin-based block copolymers by reversible addition-fragmentation chain-transfer (RAFT) polymerization. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 3596-3603. (10) Bruch, M.; Ma¨der, D.; Bauers, F.; Loontjes, T.; Mu¨lhaupt, R. Melt modification of poly(styrene-co-maleic anhydride) with alcohols in the presence of 1,3-oxazolines. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 1222-31. (11) Basu, D.; Banerjee, A. Determination of optimum compatibilizer (SMA) concentration for PBT/ABS (70/30) blend using tensile strength data. J. Appl. Polym. Sci. 1997, 64, 1485-87. (12) Kilwon, C.; Kyung, H. S.; Tae, O. A.; Jungahn, K.; Kwang, U. K. Enhancement of interfacial adhesion between polystyrene and styrene maleic anhydride random copolymer via reactive reinforcement. Polymer 1997, 38, 4825-4830. (13) Maeda, H.; Ueda, M.; Morinaga, T.; Matsumoto, T. Conjugation of poly(styrene-co-maleic acid) derivatives to the antitumor protein neocarzinostatin: pronounced improvements in pharmacological properties. J. Med. Chem. 1985, 28, 455.

Donati et al. (14) Mu, Y.; Kamada, H.; Kaneda, Y.; Yamamoto, Y.; Kodaira, H.; Tsunoda, S.; Tsutsumi, Y.; Maeda, M.; Kawasaki, K.; Nomizu, M.; Yamada, Y.; Mayumi, T. Bioconjugation of laminin peptide YIGSR with poly(styrene co-maleic acid) increases its antimetastatic effect on lung metastasis of B16-BL6 melanoma cells. Biochem. Biophys. Res. Commun. 1999, 255, 75-79. (15) Campa, C.; Donati, I.; Vetere, A.; Gamini, A.; Paoletti, S. Synthesis of glycosylamines: identification and quantification of side products. J. Carbohydr. Chem. 2001, 20 (3&4), 263-271. (16) Habeeb, A. F. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal. Biochem. 1966, 14, 328-336. (17) Riikonen, T.; Vihinen, P.; Potila, M.; Rettig, W.; Heino, J. Antibody against human R1β1 integrin inhibits hela cell adhesion to laminin and to type I, IV and V collagens. Biochem. Biophys. Res. Commun. 1995, 209 (1), 205-212. (18) N/C ratios were 0.023, 0.022, and 0.028 for the H+-form of SMAGal, SMA-Gluc and SMA-Lac, respectively. (19) Silverstein, Bassler and Morrill, Spectrometric identification of Organic Compunds, IVth ed.; John Wiley & Sons: New York, Chichester, Brisbane, Toronto, and Singapore, 1981. (20) Cantor, C. R.; Shimmel, P. R. In Biophysical Chemistry, Part II; W. H. Freeman and Company Publisher: New York, 1998. (21) Bahulekar, R.; Tokiwa, T.; Kano, J.; Matsumura, T.; Kopjima, I.; Kodama, M. Polyacrylamide containing sugar residues: synthesis, characterization and cell compatibility studies. Carbohydr. Polym. 1998, 37, 71-78.

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Synthesis, Characterization, and Preliminary Biological Study of

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