Poly(vinyl alcohol)-Salicylic Acid Conjugate: Synthesis and Characterization

The polymeric system containing hydrolysable ester bonds linked to salicy lic acid was synthesized for controlled drug release. Po ly(vinyl alcohol) (PVA) functionalized with chlo roacetate groups was obtained by reaction of PVA with chloroacetyl chloride using the N,N-d imethylacetamide/5% lithium chloride system as solvent and pyridine as catalyst. The degree of substitution was calculated from the chloride content and ranged from 37.8 to 98.9 mol%. The coupling of salicylic acid to PVA functionalized with chloroacetate groups was carried out by the reaction with between PVA and the sodium salt of salicylic acid. The structures of chloroacetylated PVA and PVA -salicylic acid conjugates were determined by means FTIR, H-NMR and C-NMR spectra. The hydrolysis in the heterogeneous system of PVA-salicylic acid conjugates were performed in buffer solutions (pH 7.6 and 8.5) at 37°C. Detection of hydrolysis by UV spectroscopy showed that released the drug, can by achieved by the hydrolysis of the ester bond between the drug and the polymeric carrier. The release profiles indicated that the release of the drug (sodium salicy late) from tablets was dependent on hydrophilic character of conjugate and the pH of the buffer solution.


Introduction
In the last decades much attention has been directed to sp eciality p o ly mers fo r b io med ical app lication . One particular approach towards the improved use of drugs is th e d esign o f p o ly mer-d rug co n jug ates o r po ly meric prodrugs [1][2][3][4][5][6][7][8][9]. The chemical attachment of low mo lecular weight d rugs to synthetic or natural po ly mers has been frequ ent ly invest ig ated as a means of imp rov ing t he efficacy of drug control release devices through a constant but prolonged release of drugs with minimu m side effects. The drugs may be lin ked to the poly meric carriers using a n u mb er o f chemical reactio ns with particip ation o f functional groups such as hydroxyl or carbo xyl which are eith er o rig in ally p res en t in th e p o ly mer ch ain o r altern atively fo rmed b y fu n ction alizatio n . A no th er possib ilit y is the use o f funct ionalized mono mers in synthesis of a reactive poly meric precursor. In most cases, drugs bound directly to the polymer chain exh ibit either a reduced or zero bio logical activity. For th is reason, drugs should be separated from the poly meric backbone by means of a spacer. Once the d rug conjugate reaches the target compart ment, the drug can then be split off more read ily in its active form. To facilitate the release of the drug it must be attached to the macro molecu lar chain by covalent bonds of limited stability in a bio logical environ ment.
The advantages of poly(ethylene oxide), poly(vinyl pyrrolidone), poly(2-hydro xypropyl methacrylamide), copolymers of 2-hydro xyethyl methacrylate or polysaccharides as a macro mo lecular carrier for drug immob ilization are well acknowledged, as is apparent from the literature data [10][11][12][13][14][15][16][17][18][19]. In most cases the polymers has been previously transformed into a suitable reactive derivative, in order to achieve the attachment of drug mo lecules and to introduce a spacer between the carrier and the bioactive compounds. Selection of the spacer arm is critical as it opens the possibility of controlling the site and the rate of release of the drug from the conjugates either by hydrolytic or enzy mat ic cleavage of the lin king bond.
Some natural or synthetic polymers possess multiple primary and secondary hydroxyl g roups and there can be easily conjugated with drug molecules with react ive groups either by direct conjugation or by incorporation of a spacer arm. In this paper, PVA with reactive primary hydro xyl groups may be used as a polymeric carrier for coupling pharmaceutical co mpounds.
The aim of the present paper was to synthesize and characterize PVA-salicylic acid conjugates in a two-stage procedure. During the first stage, PVA was chloroacetylated with chloroacetyl ch loride, while in the second stage, chloroacetate groups were reacted with sodium salicylate. A study of the hydrolysis of the resulting conjugates in the heterogeneous system was also conducted in aqueous buffer solutions (pH 7.6 and 8.5) and the quantity of the released drug was detected by UV spectroscopy. The influence of neighbouring groups on the release of the drug from the conjugates was also studied.

Materials
Poly(vinyl alcohol) (PVA) was co mmercial p roduct (Aldrich). The average molecular weight of PVA was M W =31.600-50.000 g/ mol, 98-99% hydrolyzed. N,Ndimethylacetamide (DMAc) (Aldrich), d imethylsulfo xide (DMSO) (Aldrich) was purified by distillation and then stored over 4 Å mo leculare sieves. Lithiu m chlo ride (LiCl) (Aldrich) was dried under reduced pressure in the presence of phosphorus pentoxide. Ch loroacetyl chloride (A ldrich) was purified before use by distillation under reduced pressure. Pyridine (POCh) was reflu xed over CaH 2 under a nitrogen atmosphere and then distilled. Salicylic acid (SA) (Fluka, Buchs, Switzerland) was used without further purification. Sodiu m salicylate (SSA) was obtained by dissolving 8.05 g (0.05 mo l) of the acid in 50 ml of chloroform, then neutralized with 2.0 g (0.05 mol) of NaOH dissolved in 50 ml of ethyl alcohol. The product precipitated by pouring reaction mixtu re into 600 ml of dry acetone. After filtration, the salt was dried under reduced pressure at 50℃ to constant weight.

Esterification of PVA with Chloroacetyl Chloride
The typical procedure, of the esterification was as follow: 2.2 g (50.0 mmol, OH groups) of PVA was dissolved in 30 ml DMAc/ 5%LiCl solvent system. The solution was then charged into a three-necked flask equipped with a nit rogen inlet and outlet, d ropping funnel, magnetic stirrer and thermo meter. Pyridine 4.68 ml (60.0 mmol) was added to the flask as an acid acceptor. DMAc solution (10 ml) containing chloroacetyl chloride 5.76 ml (60.0 mmo l) was then added dropwise at about 0℃ with stirring. The reaction mixtu re was heated at 25℃ for 8 h and after the solution was poured into a large amount of co ld 2M HCl to precip itate the product. The precipitated product was filtered and washed several times with co ld distilled water. It was purified by reprecipitation using THF as solvent and cold distilled water as precipitant, then dried under reduced pressure at 50℃ to constant weight. The yield was 79%.
The typical procedure, of the react ion was as follow: The chloroacetylated PVA 2.1 g (19.5 mmo l ClCH 2 CO-groups) was dissolved in 40 ml DM SO at roo m temperature and then 3.68 g (23 mmol) sodium salicy late was added while stirring. The reaction was performed at 30℃ and under stirring for about 5 h. The obtained product was isolated by precipitation using distilled water as precipitant and then ethanol washed to remove unreacted sodium salicylate. All samp les were purified by reprecipitation, using DMSO as solvent and ethanol as precipitant and then dried under reduce pressure at 60℃ to constant weight. The yield was 69%.

Study of The Heterogeneous Hydrolysis of PVA-Salicylic Acid Conjugate
Samples of the PVA-salicylic acid conjugate, about 0.1 g (containing fro m 37.8 to 98.9 mo l-% salicylate groups) in the form of powder were pressed in steel cylindrical cell with a diameter of 12 mm in a hydraulic press under a pressure of about 12 MPa to make d isks. The resulting tablet was placed in conical flasks with 100 ml of an aqueous buffer solution (pH 7.6 and 8.5). The flaskes were put into water bath heated to 37℃. 2 ml samples were taken at appropriate intervals fro m the liquid above of the tablets and equal volu me of same dissolution mediu m was added to maintain a constant volume. The solution contained the released drug, which was quantitatively determined by UV spectroscopy at the absorption wavelength of sodium salicy late (l 295 n m) using calibration curves obtained previously under the same conditions. Tests were performed for different degrees of substitution of the conjugates and various pH values of the reaction mediu m. We noticed that of PVA-salicylic acid conjugates remained insoluble in the reaction environment along the whole hydrolysis process investigated.

Spectroscopic Measurements
Infrared spectra were recorded using Perkin-Elmer 2000 (FTIR) instrument. 1 H-NMR and 13 C-NMR spectra were obtained using Bruker DPX 250 MHz spectrometer with DMSO-d 6 as solvents and TMS as an internal reference. The UV-VIS spectra were obtained using Perkin Elmer UV/ VIS Lambda 2 spectrometer.

Eval uation the Degree of Esterification
The degree of the esterification of the PVA was determined fro m the elemental analysis of chloride. Elemental analysis (Cl) was carried out on a Carlo Erba 1106 EA-instrument.

Results and Discussion
In order to provide a uniform distribution of chloro methyl groups along the polymer chain, the esterification was carried out in homogeneous system, previously dissolving PVA in DMAc/5% LiCl system. PVA modified with chloroacetate group with different degress of substitution was synthesized using the method described for chloroacetylation of starch [19] accord ing to the reaction presented by the Scheme 1.

Scheme 1. Reaction between PVA and chloroacetyl chloride
The effect of react ion conditions on degree of substitution is summarized in Table 1. The degree of the substitution of PVA was calculated fro m the content of chlorine determined by the elemental analysis. As follo ws fro m the date in Tab le 1, the extent of modification increases with an increase in the ratio of chloroacetyl chloride to PVA. For example, the degrees substitution increases from 37.8 to 98.9 mol% chloroacetate groups as chloroacetyl chloride/hydroxyl groups of PVA increase fro m 0.4 to 1.2. The coupling of bioactive carbo xylic acid to PVA functionalized with chloroacetate groups was carried out by using the sodium salicy late according to the following Scheme 2.

Scheme 2. Synthesis of PVA-salicylic acid conjugate
The elementary analysis of the products obtained from chloroacetylated PVA with various degrees of substitutions and sodium salicylate showed the absence of chlorine, which allo wed one to assume its total rep lacement with the substitution degree of the conjugate being the same as that for corresponding chloroacetylated derivatives of PVA. Figure 1a-c shows exemplary the FTIR spectra of unmodified PVA, chloroacetylated PVA (98.9 mo l% of chloroacetate groups) and PVA-salicy lic acid adduct (98.9mo l% of salicylate groups). As is seen, the spectrum of chloroacetylated PVA (Fig. 1b), unlike the spectrum of PVA (Fig. 1a), has a new absorption band at 1749 cm -1 of carbonyl group (in -COO-CH 2 -Cl). On the other hand, there disappears the band within the range 3934 -3099 cm -1 derived fro m hydro xyl groups. Moreover in spectrum o f the conjugate PVA-salicylic acid (Fig. 1c) absorption band appears at 1683, 1615, 1585 and 761 cm -1 , which results fro m scissoring vibrations bands of >C=C< and C-H in the benzene ring. The 1 H-NM R spectrum of the same chloroacetylated PVA (Fig. 2a) shows a characteristic peak of protons of chloroacetate groups at 4.05 pp m. There is also visible peak at 1.72 pp m, which belong to protons of -CH 2 -in the main chain. The spectrum of PVA-salicylic acid conjugate shows characteristic signals at 6.72 -7.83 pp m and at 10.2 ppm, which can be assigned to the protons of the benzene ring and -OH groups, respectively. The 13 C-NM R spectrum of chloroacetylated PVA is characterized by chemical shifts at 39.8 and 166.1 pp m, which correspond to chloromethyl and carbonyl carbon atoms of chloroacetate groups, respectively. The spectrum of the PVA-salicylic acid conjugate shows additional peaks between 111.4 and 134.9 pp m, which due to the resonance of carbon atoms in the benzene ring and the signals at 158.4 ppm can be assigned to the C 6 H 4 -CO-groups. All these spectroscopic results confirm the p resence of chloroacetate and salicylate groups in the modified PVA .
Fro m the literature reports it follows that the side chain hydrolysis of drug pendent polymers depends on the strength and chemical nature of the polymer-drug bonds, the structure of polymer and mediu m conditions. The hydrolysis of the bond is also dependent on its distance fro m the main poly mer chain. The length and hydrophilicity of the spacer between the drug and polymer backbone can also influence the drug release rate [33]. The in vitro hydrolysis behavior of polymer-drug conjugate was studied in buffer solutions (pH 7.6 and 8.5) at 37℃. Two hydrolyzable ester groups were present in PVA-salicylic acid conjugates. The investigation of the hydrolyzing solution by UV spectroscopy showed that the polymer-drug conjugates released the sodium salicylate gradually under mild conditions, by the hydrolysis of the ester bond between the drug and side chain of the polymer during the reaction. The direct ester lin kage to the main chain of the polymer was less susceptible towards hydrolysis. This may be connected with the steric hindrance of the poly meric chain that reduces the bond mobility [34].  Figure 4 shows the release behaviour of sodium salicy late at 37 ℃ and pH 8.5 fro m three PVA-salicylic acid conjugates with various compositions, containing fro m 37.8 to 98.9 mo l% of salicylate groups. Fro m the kinetic curves it follows that the release of the act ive co mpound is the quickest in the case of the conjugate with the lowest content of salicylate groups. This seems to be connected with the interaction between the polymer and water. The decreased content of salicylate groups makes the polymer mo re hydrophilic and consequently facilitates the penetration of hydroxyl ions to ester groups in the conjugate, effectively increasing the relative hydrolysis rates. This is consistent with the results obtained by Babazadeh [13] for conjugates of copolymers 2-hydro xyethyl methacrylate.  Figure 5 shows a typical course of the heterogeneous hydrolysis of PVA-salicylic acid conjugate (containing 98.9 mo l% of salicylate groups) in slightly alkaline mediu m fro m pH 7.6 to 8.5 at 37℃. The presented results clearly indicate the increase in the release of sodium salicylate with the increase in the alkalinity of react ion med iu m. The hydrolysis rate of conjugate is the lowest at pH 7.6. This is consistent with the results obtained by Sanchez-Chaves et al. [14] for the 2-aceto xybenzoic-dext ran conjugates.

Conclusions
The studied performed have shown that as a result of the reaction between PVA functionalized with chloroacetate groups and sodium salts of salicylic acid a new conjugate PVA-salicylic acid is obtained. The chemical structures of all the modificat ion products of PVA were assessed by means of FT-IR , 1 H-NM R and 13 C-NM R spectroscopy. The hydrolysis of the conjugates was examined under physiological conditions. The results obtained have shown that the procentage of the drug resleased increases with increasing conjugate hydrophilicity and pH of buffer solutions. The results suggest that the PVA-salicy lic acid conjugate can be a useful carrier for controlled release of the drug.