Vinyl Polymers Based on l-Histidine Residues. Part 1. The

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Biomacromolecules 2004, 5, 1325-1332

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Vinyl Polymers Based on L-Histidine Residues. Part 1. The Thermodynamics of Poly(ampholyte)s in the Free and in the Cross-Linked Gel Form Mario Casolaro,*,† Severino Bottari,‡ Andrea Cappelli,§ Raniero Mendichi,| and Yoshihiro Ito⊥ Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi, Via Aldo Moro, Dipartimento di Fisica, Via Roma, and Dipartimento Farmaco-Chimico Tecnologico, Via Aldo Moro, Universita` di Siena, I-53100 Siena, Italy, Istituto per lo Studio delle Macromolecole (CNR), Via E.Bassini 15, I-20133 Milano, Italy, and Kanagawa Academy of Science and Technology, KSP East 309, Sakado 3-2-1, Takatsu-ku, Kawasaki, 213-0012, Japan Received February 5, 2004; Revised Manuscript Received April 7, 2004

Amphiphilic vinyl polymers (in the free and cross-linked forms), carrying carboxyl and imidazole groups, were prepared by a radical polymerization of the purposely synthesized N-acryloyl-L-histidine. The protonation thermodynamic studies (at 25 °C in 0.15 M NaCl) showed high polyelectrolyte character of the soluble polymer. Unlike the linear decreasing trend of the basicity constant, over the whole range of R (degree of protonation), the enthalpy changes for the protonation of the imidazole nitrogen in the polymer showed a decreasing pattern only at R > 0.5. This was ascribed to the formation of hydrogen bonds between protonated and free neighboring monomer units. Viscometric data revealed a minimum hydrodynamic volume of the polymer at its isoelectric point (pH 5), whereas at higher or lower pHs, the macromolecule expanded greatly as a consequence of the charged sites formation. This produced a preferential solvation of the protonated imidazole and carboxylate ions, the latter being surrounded by more water molecules in the hydration shell. The peculiar hydration behavior was confirmed in the cross-linked polymer. The hydrogel showed an equilibrium degree of swelling (EDS), strongly dependent on pH, in a similar manner as viscometric data of the soluble polymer. A linear relationship between the reduced viscosity and the EDS was found. The polymer was non toxic against the RAW264 cell line. Introduction In the past few years, several stimuli-responsive polymers were synthesized in view of their application in the biotechnological field.1-6 The polymers, sensitive to pH and temperature, were obtained by the corresponding vinyl monomers carrying purposely introduced amino acid residues.7-9 The compounds contain, besides the carboxyl group, amido and isopropyl groups in a similar way to the temperature-sensitive poly(N-isopropylacrylamide) (PNIPAAm).10 The latter is a well-known polymer showing a lower critical solution temperature (LCST) of 32 °C. An interesting feature of PNIPAAm lies in the possibility of tuning the LCST by incorporating comonomers with variable degree of hydrophilicity.11,12 In previous papers, we reported the peculiar acid-base behavior of poly(carboxyl acid) homopolymers and copolymers with NIPAAm.7-9,13 The effect of pH and temperature showed that high pH led to * To whom correspondence should be addressed. Phone: +39 0577 234388. Fax +39 0577 234177. E-mail: [email protected]. † Dipartimento di Scienze e Tecnologie Chimiche e dei Biosistemi, Universita` di Siena. ‡ Dipartimento di Fisica, Universita ` di Siena. § Dipartimento Farmaco-Chimico Tecnologico, Universita ` di Siena. | Istituto per lo Studio delle Macromolecole. ⊥ Kanagawa Academy of Science and Technology.

Chart 1. Structure of the Monomeric Unit of Poly(Hist)

increasing LCST values, in line with hydrophilic copolymers of NIPAAm.9,14 In this paper, we report the synthesis of a new polymer, namely poly(N-acryloyl-L-histidine), poly(Hist), and copolymers with NIPAAm, having an amphotheric character (Chart 1). Ampholyte polymers are a special class of polyelectrolytes which contains both positive and negative charges along the macromolecular chain.15 Some of them, of poly(amidoamine) structure, are recently being studied as endosomolytic agents for their low toxicity.16,17 A number of studies of synthetic polyampholytes, which are very similar to proteins in structure and behavior, has been reviewed in ref 18. The poly(Hist) was synthesized to provide a model compound for the investigation of the histidine residue properties in

10.1021/bm049929s CCC: $27.50 © 2004 American Chemical Society Published on Web 05/14/2004

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high molecular weight compounds. The imidazole group of the histidine residue is responsible for most of the buffering capacity of proteins in the physiological pH range. It is also capable of combining with various metal ions and appears to constitute the principal site for metal binding in proteins.19 Metal ion complexes with linear oligopeptides were extensively examined, to clarify the binding modes of the histidine residues located at different positions of the peptide chains.20-23 Indeed, copper (II) complexes of histidine-containing peptides are strikingly interesting as enzyme activating systems24 and for mimicking superoxide dismutase (SOD) activity.25 Cells are protected against the superoxide radical26 by SOD, a metalloenzyme which very efficiently catalyses the dismutation of superoxide radicals into hydrogen peroxide and molecular oxygen. The same coordination compounds are considered as models for studying the biological activity of proteins involved in fatal disorders such as Alzheimer’s disease27 or prion infection.28 This preliminary study is devoted to the synthesis and to the solution properties of the poly(Hist) and the corresponding copolymers with NIPAAm, either in the free and in the cross-linked form. In this case the LCST of copolymers may be changed by tuning pH at low or high values, i.e., below and above its isoelectric point. To this purpose, we evaluated the thermodynamic functions for the protonation of basic groups in the soluble and cross-linked (hydrogel) polymer and in a corresponding copolymer with NIPAAm, by using mainly potentiometric and calorimetric techniques. We believe that these polymers may be of interest as polymer therapeutics in the more sophisticated bionanotechnologies that are needed to realize the full potential of the post-genomic age.29 Experimental Section Materials. L-Histidine (99.5%), acryloyl chloride (97%), ammonium peroxo-disulfate (APS, 98%), triethylamine (TEA, 99%), 2,6-di-tert-butyl-p-cresol, R,R′-azoisobutironitrile (AIBN), and N,N′-ethylenebis-acrylamide (EBA, 98%) were purchased from Fluka Co. N-Isopropylacrylamide (NIPAAm, 97%) was purchased from Aldrich Co. Syntheses. Monomer. N-Acryloyl-L-histidine (Hist) was prepared by the general synthetic route to introduce the vinyl group in monomers.30 To a well-stirred aqueous solution of L-histidine (31.0 g, 0.20 mol), sodium hydroxide (16.0 g, 0.40 mol), and 2,6-di-tert-butyl-p-cresol (0.02 g) in twice distilled water (65 mL) was added dropwise acryloyl chloride (17.9 g, 0.20 mol) over a 30 min period. The reaction mixture was kept at -5 °C by external ice-bath cooling, and then the temperature was raised to room temperature for 30 min more. The mixture was acidified to pH 2 with concentrated hydrochloric acid (34 mL). The clear solution was concentrated in vacuo (temperature ca. 30 °C) to obtain crude N-acryloyl-L-histidine. The crude monomer was purified by repeated reprecipitation in acetone. The product was of analytical grade, as shown by acid-base titrations (potentiometry and solution calorimetry) and spectroscopic analysis (proton NMR in DMSO-d6 and FT-IR). Polymers. Poly(N-acryloyl-L-histidine) [poly(Hist)] and poly(N-acryloyl-L-histidine-co-N-isopropylacrylamide) [poly-

Casolaro et al.

(Hist-co-NIPAAm)] were obtained by a radical polymerization of the corresponding monomers. The homopolymer was obtained as follows. To a well-degassed and nitrogenpurged solution of 3.00 g of Hist in 20 mL of water was added 26 mg of recrystallized (from methanol) R,R′azobisisobutyronitrile (AIBN). The mixture was purged with nitrogen and allowed to stand in a thermostated oil bath at 60 °C for 24 h. The polymer was precipitated in a large excess of acetone (200 mL). It was washed three times with fresh acetone and dried in vacuo to give a light-yellow powder. The polymer was dissolved in a small amount of water and purified by dialysis by use of cellophane tubingseamless as a membrane (nominal cutoff of 10 000 g/mol) and water as a solvent for 2 days. The pure polymer was obtained by reprecipitation in acetone (yield 60 wt %). The poly(Hist-co-NIPAAm) was obtained with a similar procedure by using 2.00 g (8.14 mmol) of Hist dissolved in 10 mL of twice distilled water and 0.82 g (7.25 mmol) of NIPAAm dissolved in 10 mL of twice distilled water. To this mixture was added 31 mg of AIBN. FT-IR and 1H NMR spectra (D2O) confirmed the proposed structure. Cross-Linked Polymers (Hydrogels). Two samples of poly(Hist), H5 and H10, cross-linked with different amounts of EBA (5 and 10 mol %) were synthesized. Their preparation was carried out in a glass tube, under nitrogen atmosphere, by the following procedure. Sample H5 (15 wt % monomer concentration): The monomer Hist (3.00 g, 11.93 mmol) was dissolved in twice distilled water (20 mL) containing 127 mg (1.26 mmol) of TEA and 102 mg (0.60 mmol) of EBA. Sample H10 (13 wt % monomer concentration): The monomer Hist (2.02 g, 5.80 mmol) was dissolved in twice distilled water (15 mL) containing 70 mg (0.69 mmol) of TEA and 117 mg (0.68 mmol) of EBA. Both of the mixtures were repeatedly flushed with nitrogen, and then APS (25 mg, 0.11 mmol) was added. The reaction mixture was kept at room temperature for 24 h even if the gelation was observed within 1 h. Afterward, the gels were removed, thoroughly washed with twice distilled water for 1 week, and then slowly dried at r.t. up to constant weight in a desiccating cabinet. Unlike the sample H5, that was cut in small disks, sample H10 was treated with acetone to give a fine powder for potentiometric analysis. Instruments. Proton NMR spectra were recorded in DMSO-d6 or D2O on a Bruker AC 200 spectrometer. FT-IR spectra were recorded on a FTS 6000 Biorad spectrophotometer. Molecular Characterization. Fractionation and characterization of polymers was performed by a multi-detector size exclusion chromatography (SEC) system. The analytical system consisted of an Alliance 2690 separation module, a differential refractometer from Waters (Milford, MA), and an additional multi-angle laser light scattering (MALS) Dawn DSP-F photometer from Wyatt (Santa Barbara, CA). This multi-detector SEC system was described in details elsewhere.31 Two aqueous Ultrahydrogel SEC columns (100 and 250 Å of pore size) from Waters were used. In the case of poly(Hist), the running SEC conditions were 0.2 M NaCl + 0.1 M Tris buffer pH 8.0 as mobile phase, temperature 35 °C, flow rate 0.8 mL/min, injection volume 200 µL, whereas

Vinyl Polymers Based on L-Histidine Residues

in the case of poly(Hist-co-NIPAAm), the Zimm Plot was obtained using N,N-dimethylformamide + 0.05 M LiBr as solvent at 25 °C. The wavelength of the MALS laser was 632.8 nm. The light scattering signal was simultaneously detected at 15 scattering angles ranging from 14.5° to 151.3°. The calibration constant was calculated using toluene as standard assuming a Rayleigh Factor of 1.406 × 10-5 cm-1. The angular normalization was performed by measuring the scattering intensity of a concentrated solution of BSA globular protein in the mobile phase assumed to act as an isotropic scatterer. The MALS photometer was described in detail elsewhere.31,32 The refractive index increment, dn/dc, of poly(Hist) and poly(Hist-co-NIPAAm), with respect to the solvent, was measured by a KMX-16 differential refractometer from LDC Milton Roy (Riviera Beach, FL). The dn/dc values for poly(Hist) and for poly(Hist-co-NIPAAm) were 0.176 and 0.072 mL/g, respectively. Viscometric Measurements. Viscometric titrations were carried out in aqueous solution at 25 °C by using an AVS 310 automatic Schott-Gerate viscometer. The polymer solution was freshly prepared by weighing and dissolving ca. 50 mg of compound in 25 mL of 0.15 M NaCl containing a known amount of standard 0.1 M hydrochloric acid solution. A standard sodium hydroxide solution (0.1 M) was stepwise delivered by a Metrohm Multidosimat piston buret. The program Fith33 was used to calculate the pH values, at each neutralization step, from the log K’s. Potentiometric Measurements. The potentiometric titrations were carried out in aqueous solution at 25 °C by using a TitraLab 90 titration system from Radiometer Analytical. Titralab 90 consists of three components: the TIM900, a powerful Titration Manager; the ABU901, a high-precision autoburet; and the SAM7, a convenient sample stand. A Windows based software (TimTalk 9) was used in connection with the TIM900 Titration Manager for remote control. The titrations of the compounds were performed in a thermostated glass cell filled with 100 mL of 0.15 M NaCl in which a weighed amount of solid material (monomer, 0.12 mmol; polymer, 0.20 mmol; copolymer and hydrogel, 0.18 mmol) and a measured amount of standard hydrochloric acid solution were dispersed by magnetic stirring, under a presaturated nitrogen stream. Forward and backward titrations, with standard 0.1 M NaOH and 0.1 M HCl, respectively, showed rielable results. The equilibration time was 300 s for each titration step in the case of polymers and the gel H10, whereas it reached 1500 s in the case of gel H5. The basicity constants were evaluated with the Superquad34 and ApparK33 programs, running on PC. Calorimetric Measurements. Calorimetric titrations were carried out in aqueous solution at 25 °C with a Tronac calorimeter (mod. 1250) operating in the isothermal mode. The aqueous solution (25 mL of 0.15 M NaCl), containing a weighed amount of compound (monomer, 0.2 mmol; polymer and copolymer, 0.10-0.16 mmol) and a measured volume of standard sodium hydroxide, was titrated with standard 0.1 M HCl solution with a constant BDR (Buret Delivery Rate) of 0.1000 mL/min through a Gilmont buret. All of the experiments were automatically controlled by the

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Thermal program, from Tronac Inc., that was renewed to operate through a NI-DAQ driver software in Windows, from National Instruments. The graphical programming language LabVIEW was used to create the application. The calibration of the apparatus and the corrections for the titrant heats of dilution were made before each titration run. The enthalpy change values were computed with the Fith program.33 Swelling Measurements. The swelling behavior of the hydrogel was investigated at 25 °C as a function of pH and ionic strength of the bathing medium. Specifically, the dried sample of the gel contained in a Stainer cell (40 µm pore size) was first swollen in twice distilled water or 0.15 M NaCl until equilibrium was reached within 24 h. The degree of swelling (DS) was monitored at intervals, till a plateau of DS in relation to time was reached. Afterward, for swelling studies in hydrochloric acid (0.01 M), sodium acetate (0.01 M), phosphate (0.01 M), or Tris (0.01 M) buffer solutions contained in a thermostated glass cell (100 mL) and having pH values ranging from 2 to 9, the gel sample was placed in the proper medium and allowed to equilibrate for a further 24 h, under stirring. The sample was removed from the bath at intervals, quickly blotted with tissue paper to remove any surface droplets, and weighed (recorded as wet weight, Wwet). This procedure was repeated to a constant weight. The equilibrium degree of swelling (EDS) was calculate by: EDS ) (Wwet - Wdry)/Wdry, where Wdry is the weight of the dried sample. Evaluation of Cytotoxicity. RAW264 cells were cultured in a minimum essential medium (Sigma) with 10% fetal bovine serum (FBS) and 1% nonessential amino acids (Invitrogen Life Technologies). The cells were harvested with a 0.25% trypsin solution containing 0.5 mM EDTA. The recovered cells were washed with the culture medium and suspended in the medium. The cell suspension was added to the well of the 24-well plate in the presence of polymer and was allowed to stand for 3 days at 37 °C. After the incubation, the cell number was counted by microscopy. The results were expressed as viability (%) relative to a control containing no polymer. The means ((SD) of four experiments, each containing three replicates, are reported. Results and Discussion Synthesis and Characterization. Monomer. The monomer N-acryloyl-L-histidine (Hist) was obtained following the usual acylation reaction of acryloyl chloride with the corresponding R-amino acid, in alkaline solution (Scheme 1).7,8,30 The reaction temperature was controlled below -5 °C, while the solution mixture remained always clear. Unlike the previously synthesized acrylates bearing only carboxyl groups, that gave voluminous solid phase separation on adding concentrated hydrochloric acid, the solution of this ampholyte monomer continued to be clear till pH 2. The presence of a net charge enabled greater hydrophilicity. The stronger hydrophilic character of the monomer limited its crystallization from water. In fact, the crystallization from concentrated monomer solution led to a phase separation containing only ca. 20 wt % of the pure product that

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Scheme 1. Synthetic Route of Hist

Table 1. Proton NMR Chemical Shifts (δ, ppm; J, Hz) and Main IR Frequencies (cm-1) of N-Acryloyl-L-histidine

assignments NMR: 8.91 (s, 1H imidazole 7); 8.74 (d, J ) 8.3, 1H amide 3); 7.33 (s, 1H imidazole 6); 6.22-6.36 (m, 1H, vinyl 1); 5.98-6.09 (m, 1H vinyl 2); 5.52-5.60 (m, vinyl 2); 4.49-4.61 (m, 1H 4); 3.00-3.25 (m, 2H 5) IR: 1729 (vs) CdO strech of COOH group; 1658 (vs) Amide I band; 1621 (vs) CdC; 1543 (vs) Amide II band

1H

cocrystallized with sodium chloride. The purity of this compound was determined by spectroscopy (1H NMR, FT-IR) and potentiometric analysis. The chemical shifts and the main infrared frequencies are reported in Table 1. Due to a limited yield during crystallization, a larger amount of monomer was obtained by repeated reprecipitation in acetone. The so obtained product showed a very close similarity to that obtained by crystallization. The analysis of potentiometric and calorimetric titration curves showed basicity and enthalpy changes compatible with the protonation of the imidazole nitrogen (see protonation section). Polymers. Poly(N-acryloyl-L-histidine) and related copolymers with NIPAAm and hydrogels were synthesized by radical polymerization of the corresponding monomers. In all cases, the polymerization was readily accomplished with radical initiators in water solution. In this solvent, either the linear polymer and the copolymer remained in solution, whereas the cross-linked polymer formed a transparent gel. Poly(Hist), being soluble only in water, was precipitated in acetone with good yield. Its 1H NMR spectrum in D2O showed the complete disappearance of the vinyl double bond (in the 5.5-6.5 ppm region) and the presence of broad lines for backbone and side-chain resonances, consistent with the

Casolaro et al. Table 2. Molecular Characterization Data Obtained from SEC Experiments sample

Mw‚10-3 (g/mol)

Mn‚10-3 (g/mol)

D ) Mw/Mn

poly(Hist) dialyzed poly(Hist)

507.5 871.1

181.3 436.0

2.8 2.0

presence of a slowly tumbling macromolecular species in solution. In fact, high-molecular-weight polymers were obtained as shown by SEC light scattering and viscometric measurements. The molecular characterization of poly(Hist) is summarized in Table 2. The table reports the weight- (Mw) and the number-average (Mn) molecular weight and the polydispersity index D ) Mw/Mn. The poly(Hist) revealed a relatively high polydispersity index (D ) 2.8) and an average molecular weight Mw closer to that showed by the previously reported vinyl polymers containing different amino acid residues.9 However, after dialysis through the membrane with a nominal cutoff 10 000 g/mol, the poly(Hist) polymer showed a more narrow molecular weight distribution (MWD) and a meaningful lower polydispersity index (D ) 2.0). The disappearance of the 1621 cm-1 very strong band of the IR spectrum, in conjunction with the NMR results, indicated a total conversion of the monomer into the corresponding polymer. A sample of copolymer with NIPAAm, poly(Hist-coNIPAAm), was obtained with a molar ratio Hist/NIPAAm of 1; its average molecular weight Mw was 30.080 g/mol, as obtained using the Zimm-plot method. The composition was determined by the potentiometric titration of the protonated imidazole group. The amount of the latter was found to be very close to the feed composition (1.12). The proton NMR spectrum was consistent with the expected structure. The methyl protons of the NIPAAm unit drop at 0.9 ppm; from the integrated signals of the two broad lines of the CH2 side chain of the Hist unit and the CH side chain of the NIPAAm unit respectively falling at 3.1-3.3 and 3.7 ppm, a NIPAAm/ Hist molar ratio very close to 1 was determined. The result, taken in conjunction with the potentiometric analysis, suggested that the comonomer feed ratio reflected the relative comonomer incorporation level, with a presumably total conversion of the monomers into the copolymer being the reaction of radical type. This was already observed for acrylic polymers of similar structure,1,3,7,8 probably obeying Bernouillian statistics and forming copolymers with a random distribution of histidine residues along the chain.35 Moreover, two hydrogels H5 and H10 were obtained with a different amount of cross-linking agents14 (EBA, 5 and 10 mol %). The water solution containing the monomers (15 wt % total monomer concentration) gelified at r.t. within 1 h, giving a soft product at lower EBA content. Any attempt to decrease the amount of EBA failed the gelification. The two cross-linked polymers were treated in a different way. Unlike H5, that was cut in small disks, the hydrogel H10 was precipitated in acetone to give it a fine powder for potentiometric measurements. In both cases, the samples were slowly dried at r.t. for 1 week and then under vacuum to reach a constant weight. SEM analysis revealed a homogeneous H5, instead of the microporous H10 material.36

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Vinyl Polymers Based on L-Histidine Residues Table 3. Basicity Constants of the N-Acryloyl-L-histidine (Hist) and Related Polymers in 0.15 M NaCl at 25 °C compd Hist

reaction step L- +

H+ )

LH(

LH( + H+ ) LH2+ poly(Hist) L- + H+ ) LH( LH( + H + ) LH2+ b H10 L- + H+ ) LH( LH( + H + ) LH2+ H5c L- + H+ ) LH( LH( + H+ ) LH2+ poly(Hist-co-NIPAAm) L- + H+ ) LH( LH( + H + ) LH2+

log K° a

na

6.48 ( 0.02 7.64 ( 0.07 2.3 7.74 ( 0.10 2.9 7.60 ( 0.12 2.4 7.11 ( 0.03 2.9

2.22 ( 0.09 1.66 ( 0.06 1.96 ( 0.08 1.76 ( 0.02

a log K ) log K0 + (n - 1)log[(1 - R)/R]. b Cross-linked with 10 mol % of EBA. c Cross-linked with 5 mol % of EBA.

Protonation Study. The acid-base behavior of all of the compounds was studied in aqueous media (0.15 M NaCl) at 25 °C by potentiometry, solution calorimetry, and viscometry. Basicity Constants. The analysis of forward and backward potentiometric titration curves enabled us to evaluate only a single basicity constant (log K) for the monomer. On the other hand, polymeric compounds showed two log K values in different buffering regions. In Table 3 is reported the result of each protonation step. Regarding the monomer, the magnitude of basicity was compatible with the protonation of the imidazole nitrogen.37 In fact, the log K of 6.48 lay in the range between the simple histidine (6.02) and the imidazole (7.03). The presence of the amido group in the monomer weakened the electronic withdrawing properties, shown by the protonated primary amino group in the simple histidine. Closer data were usually reported for short polypeptides molecules bearing histidine residues.38,39 As the double bond of the monomer opened to form the polymeric species, the basicity constant values increased more than 1 order of magnitude (Table 3) and the log K became “apparent”, i.e., strongly dependent on the degree of protonation R. This behavior, similar to that shown in vinyl poly(carboxyl acid)s previously reported, was observed in all of the compounds considered.2,3,8 The well-known modified Henderson-Hasselbalch equation40 log K ) log K° + (n - 1) log[(1 - R)/R] well described the linear decreasing pattern of log K in relation to R (Figure 1), where the n value was a measure of the electrostatic interactions magnitude, as well as the hydrophilicity.41 The main reason of the greater log K was due to electrostatic effects exerted by the ionized carboxylate ions spread along the macromolecular chain. The poly(Hist) showed in fact a strong polyelectrolyte character, as evidenced by the high n value and the sharp decreasing pattern in Figure 1a. The presence of the randomly distributed NIPAAm units in the copolymer drastically reduced both log K° and n (Table 3). The uncharged monomer acted as a shield of the imidazole nitrogen, because it was it protonated.42 On the other hand, the hydrogel showed closer log K° as the corresponding homopolymer but a lower n value. This may be attributed to the reduced

Figure 1. Basicity constants in relation to R for the protonation of the imidazole nitrogen in poly(Hist) (a) and hydrogels H10 (b) and H5 (c) in 0.15 M NaCl at 25 °C.

Figure 2. Reduced viscosity (η/C, dL/g) in relation to pH of poly(Nacryloyl-L-histidine) in 0.15 M NaCl at 25 °C.

conformational freedom, with the macromolecule now being a cross-linked network.1 Unlike the protonation of the imidazole nitrogen, either in the free or the cross-linked polymer, the trend of the basicity constant for the protonation of COO- groups cannot be well depicted, because of the rather low degree of protonation reached in these conditions. The amphotheric nature of the polymer is well described by viscometric data (Figure 2). The plot of the reduced viscosity η/C in relation to pH showed the major role played by charges in the conformational arrangements of the macromolecule.16,43 Low and high pHs were respectively compatible with the predominance of positive and negative charges, the isoelectric point lay at pH 5. It is evident that the macromolecule should extend more when being free of protons, i.e., in the negatively charged state due to COO- groups. At low pH, when the imidazole nitrogen was positively charged, the coil seemed closer, probably because the carboxylate group was not completely protonated. The lower logK value of the latter is responsible for the limited charge neutralization. Enthalpy and Entropy Changes. Calorimetric titration data for the protonation of the basic nitrogen and the carboxylate group in the monomer and polymers showed only one inflection point. The exothermicity of the linear curve was attributed to the protonation of the imidazole nitrogen. This was consistent with the evaluated -∆H° values reported in Table 4 along with the -∆G° and the calculated ∆S°. In all cases, the enthalpy changes are similar and closer to that reported for the simple histidine and the imidazole,44 suggesting that the protonation really involved the imidazole nitrogen. Unlike the monomer and the copolymer, the poly(Hist) showed a peculiar behavior during the protonation process (Figure 3).

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Table 4. Enthalpy and Entropy Changes of N-Acryloyl-L-histidine (Hist) and Related Polymers in 0.15 M NaCl at 25 °C compd Hist poly(Hist) poly(Hist-co-NIPAAm) a

reaction step L-

H+

LH(

+ ) L- + H+ ) LH( L- + H+ ) LH(

-∆G°, a kJ/mol

-∆H°,a kJ/mol

∆S°,a J/mol K

37.0 ( 0.1 43.6 ( 0.4 40.6 ( 0.2

30.5 ( 0.2 30.6 29.5

21.8 ( 0.8 43.5 37.2

Obtained at R ) 0.5.

Scheme 2. Protonation Mechanism of the Imidazole Nitrogen of Poly(Hist)

At low R values, the entropy changes were higher, probably because of the extended and disordered macromolecular coil in a fully hydrated state. While the protonation of the imidazole nitrogen proceeded, the ∆S° value regularly

Figure 3. Free energy (-∆G°, kJ/mol), enthalpy (-∆H°, kJ/mol), and entropy (∆S°, J/mol K) changes in relation to R for the protonation of poly(N-acryloyl-L-histidine) in 0.15M NaCl and 25 °C.

decreased even if the -∆H°/R trend remained flatter. This process went on till R ) 0.5, i.e., when half of the monomer units were protonated. The decrease of the entropy may be due to a gradual ordering state assumed by the macromolecule. The process probably involved the formation of hydrogen bonds between adjacent monomer units being protonated. When R > 0.5, the -∆H° decreased more sharply than the ∆S°. The slight endothermicity that was of the order of 5 kJ/mol may be attributed to the breaking of the H bonds to allow further protonation of the imidazole nitrogen. The mechanism can be depicted as in Scheme 2, where the proton was shared between two adjacent monomer units. Besides the well-known poly(vinylamine) system,45 this was a further example of such spatial approaching systems that behaved in a manner similar to that already reported for a vinyl polymer containing a piperazine residue in the side chain.46 Swelling BehaVior of the Hydrogel. The degree of the hydrogel hydration was studied at 25 °C in 0.15 M NaCl. In the current study, the effect of pH was investigated over a wide range, against a backdrop of substantially different

Vinyl Polymers Based on L-Histidine Residues

Figure 4. Equilibrium degree of swelling (EDS) in relation to pH of the gels H5 and H10, cross-linked with 5 mol % and 10 mol % of EBA, in 0.15 M NaCl at 25 °C.

buffers. Figure 4 shows the results obtained in the equilibrium degree of swelling (EDS) conditions for the two hydrogels H5 and H10, containing a different amount of cross-linking agent. The plot of the cross-linked polymer was very similar to that observed for the viscometric measurements of the corresponding linear polymer. It is evident that charges played a significant role in the hydration process. At high pHs, the swelling was the highest, meaning that carboxylate anions formed greater solvation shells compared to that showed by the “onium” ion at low pH values.47 At the isoelectric point, the gel shrank to its minimum hydration. From this result, we can reasonably give a better explanation of the conformational change observed in the linear macromolecule. In fact, the extension of the coil at high/low pHs was not only due to charge repulsion but also to the hydration shell magnitude of the ionized groups.43,47 It is evident, from Figure 4, that the amount of cross-links played a role in the swelling process of the poly(Hist). Any increase of the crosslinking amount led to a decrease of the EDS at pH beyond the isoelectric point of poly(Hist). This result may be quite trivial, even considering the closer log K01 values (Table 3). On the other hand, at low pHs, the EDS/pH plots almost overlap each other. The reasons for this characteristic feature may be found on the net charge present at a given pH, by taking into account the basicity constant of the COO- group. In fact, log K02 of H10 is higher than that of H5 (Table 3); thus, at the same low pH value, a greater protonation of H10 is expected with respect to H5. This improved a more positively charged macromolecule that led to an increase of EDS. However, the expected EDS decrease, due to the higher cross-linking density in H10, is compensated by the positive net charge that enabled a greater swelling. It is worthy to mention that the relative magnitude of the log Ks may further on clarify the small shift of the minimum EDS value of H10 at higher pHs, as the isoelectric point slightly increased. On the basis of the above results, we found a linear relation when a plot of the free polymer reduced viscosity (η/C) in connection with the EDS of the corresponding cross-linked gel was evaluated. Figure 5 shows that the linearity obeyed for both gels H5 and H10, even at the two different molecular weights of poly(Hist). Any increase of Mw led to a decrease in the slope. It is known that the reduced viscosity and EDS are proportional to intrinsic volumes, respectively, per linear macromolecule and per much shorter chain fragments between cross-links

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Figure 5. Plot of the swelling equilibrium degree (EDS) of the crosslinked gels H5 (4, [) and H10 (×, 9) in relation to the reduced viscosity (η/C, dL/g) of the poly(Hist) at two different molecular weights (Mw).

Figure 6. Cytotoxycity of poly(Hist) against RAW264 cells.

in the gel. The reported linearity indicated a similarity of the linear macromolecule and the gel. In Vitro Cytotoxicity. The cytotoxicity was usually evaluated by culture of mammalian cells, L929 mouse fibroblast.48,49 In the present investigation, RAW264, which is a leukemic monocyte from a mouse, was employed: the cells exposed to the polymer proliferated at the same rate as cells grown in polymer-free solution up to 48 h. Cytotoxicity was not observed (Figure 6) at any concentration up to 2 × 10-4 M. Schmalenberg et al.48 indicated no cytotoxicity of poly(ethylene glycol) (PEG) from the result that the cell growth was not inhibited by PEG up to 10-4 M. Therefore, the present investigation demonstrated that the polymer had no cytotoxicity. Conclusions This first preliminary paper of polymers carrying Lhistidine residues deals with the synthesis of a novel acrylic monomer of ampholyte character. It may be easily polymerized to obtain free and/or cross-linked polymers that are useful in the field of biomacromolecules, since the imidazole of the histidine residues is a well-known agent responsible for most of the buffering capacity and metal ion complex ability of proteins in the physiological pH range. The thermodynamic data for the protonation of the imidazole nitrogen and the carboxylate group in polymers revealed a greater polyelectrolytes character for either free or cross-linked hydrogels. The free polymer showed the formation of H-bonding interaction between adjacent monomer units, which were absent in the copolymer with NIPAAm. The latter, acting as a shield, reduced the polyelectrolyte character of the poly(ampholyte) that, how-

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Figure 7. Evaluated species distribution curves of poly(Hist) in 0.15 M NaCl at 25 °C (L- is the complete ionized form of the polymer; LH( is the zwitterionic neutral form of the polymer; LH2+ is the complete protonated form of the polymer).

ever, was worthy in the gels. The basicity constants enabled us to evaluate species distribution curves (Figure 7), which allowed a determination of the prevailing charged or neutral species at given pHs. The figure is a useful guide to better understand the thermodynamic behavior of poly(Hist) and its corresponding hydrogels, even at different degrees of cross-linking. The poly(Hist) revealed zero cytotoxicity against the RAW264 cell line, in the range of polymer concentrations that were usually evaluated for polymeric biomaterials. This result was confirmed also in copolymers with NIPAAm when in the form of hydrogels.36 The absence of toxicity allows us to propose these materials as useful candidates as polymer therapeutics.29 Acknowledgment. This work was supported by a research program (PAR 2000) of Siena University. Miss Ilaria Casolaro’s careful reading of the manuscript is also acknowledged. References and Notes (1) Casolaro, M. In Frontiers in Biomedical Polymer Applications; Ottenbrite, R. M., Ed.; Technomic Publishing Co., Inc.: Lancaster, 1998; Vol. 1, 109-122. (2) Casolaro, M. In Polymeric Materials Encyclopedia; Salamone, J. C., Ed.; CRC Press: Boca Raton, FL, 1996; Vol. 10, 7979-7992. (3) Casolaro, M.; Barbucci, R. Polym. AdV. Technol. 1996, 7, 831. (4) Casolaro, M. In Properties and Chemistry of Biomolecular Systems; Russo, N. et al., Eds.; Kluwer Academic Publishers: 1994; 127141. (5) Kuckling, D.; Vo, C. D.; Wohlrad, S. E. Langmuir 2002, 18, 4263. (6) Yoshida, M.; Asano, M.; Suwa, T.; Katakai, R. Radiat. Phys. Chem. 1999, 55, 677. (7) Casolaro, M. React. Polym. 1994, 23, 71. (8) Casolaro, M. Macromolecules 1995, 28, 2351. (9) Bignotti, F.; Penco, M.; Sartore, L.; Peroni, I.; Mendichi, R.; Casolaro, M.; D’Amore, A. Polymer 2000, 41, 8247. (10) Heskins, M.; Guillet, J. E. J. Macromol. Sci., Chem. 1968, A2, 1441. (11) Feil, H.; Bae, Y. H.; Feijen, J.; Kim, S. W. Macromolecules 1993, 26, 2496. (12) Iwata, H.; Oodate, M.; Uyama, Y.; Amemiya, H.; Ikada, Y. J. Membr. Sci. 1991, 55, 119. (13) Casolaro, M. Polymer 1997, 38, 4215. (14) Penco, M.; Bignotti, F.; Sartore, L.; Peroni, I.; Casolaro, M.; D’Amore, A. Macromol. Chem. Phys. 2001, 202, 1150. (15) McCormick, C. L.; Brent Johnson, C. Macromolecules 1988, 21, 686.

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