Synthesis and aqueous solution behavior of phosphonate-functionalized chitosans

EUROPEAN POLYMER JOURNAL

European Polymer Journal 42 (2006) 2678–2685

www.elsevier.com/locate/europolj

Synthesis and aqueous solution behavior of phosphonate-functionalized chitosans Hongmei Kang, Yuanli Cai *, Junjie Deng, Haijia Zhang, Yufang Tang, Pengsheng Liu Institute of Polymer Science and Engineering, College of Chemistry, Xiangtan University, Xiangtan 411105, PR China Received 5 December 2005; received in revised form 7 May 2006; accepted 8 May 2006 Available online 5 July 2006

Abstract A series of novel phosphonate-functionalized pH-responsive chitosans were directly synthesized via Michael addition of chitosan with mono-(2-acryloyloxyethyl) phosphonate. The conformational and phase transitions of these phosphonatefunctionalized chitosans were investigated by turbidity and UV–vis spectroscopy studies. The results indicated that the inter- or intra-chain electrostatic interactions of the phosphonate-functionalized chitosans could be controlled via adjusting the solution pH, leading to the reversible conformational and phase transitions of these chitosans. These phosphonatefunctionalized chitosans exhibited typical anti-polyelectrolyte effect and polyelectrolyte effect.  2006 Elsevier Ltd. All rights reserved. Keywords: Chitosan; pH-responsive; Polyelectrolyte; Anti-polyelectrolyte; Conformational transition

1. Introduction Chitosan, poly-1,4-b-D-glucosamine, the deacetylated derivatives of chitin, has been extensively applied in the pharmaceutical and cosmetic formulations due to its excellent properties [1], e.g., biocompatibility [2], biodegradability [3], mucoadhesion [4], drug delivery systems [5], protein recognition and separation [6], tissue engineering [7], transplant and cell regeneration [8]. Considerable efforts have been focused on the functionalization of chitosan to improve its solubility and enforce its performances [9]. Michael addition reaction has recently been suc*

Corresponding author. E-mail address: [email protected] (Y. Cai).

cessfully employed for this purpose using acrylic compounds, however, no aqueous solution property was reported in these studies [10,11]. Of particular relevance to the present study is the Schatz et al.’s recent paper on the investigation of the elaboration of biocompatible nanoparticles from the pHinduced self-complexation of the N-sulfated chitosan [12]. These particles were assembled by electrostatic interactions between the protonated amino residues and the sulfate functions and stabilized by an excess of surface sulfate groups. It is of special interest to investigate the aqueous solution behavior of the phosphonate-functionalized chitosans, since their functional groups universally exist in our living body, e.g., phosphorylcholine.

0014-3057/$ - see front matter  2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2006.05.006

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30 C. The yellow liquid crude product was further purified by extraction using 1:1 (v/v) mixture solvent of methanol and petroleum ether. The targeted light yellow liquid MAEP was achieved with a 33% of yield. 1H NMR (D2O, d in ppm): 6.4–5.8 (3H, CH2@CH–), 4.4 (2H, –COOCH2CH2–), 3.8 (2H, –COOCH2CH2O–); 31P NMR (D2O, d in ppm): Only one group of phosphor resonance at d = ca. 0 ppm after purification; potentiometric titration results of the two apparent equilibrium constants confirmed MAEP as a binary acid with pKa,1 = 2.66 and pKa,2 = 7.25, respectively.

In this study, we describe a facile synthesis of the phosphonate-functionalized zwitterionic chitosans via the Michael addition of mono-(2-acryloyloxyethyl) phosphonate (MAEP) to chitosan (Scheme 1). The pH-responsive behavior and anti-polyelectrolyte effect/polyelectrolyte effect of these chitosan derivatives were investigated by turbidity and UV– vis spectroscopy studies. 2. Experimental section 2.1. Materials Chitosan, product of Yuhuan Chitin Company, was purified according to that described in the literature [13]. The degree of deacetylation of the purified chitosan was 0.80 (1H NMR result); and the viscosity-average molecular weight Mg was 8.9 · 105 g/mol (determined using a Ubbelohde viscometer according to the literature [14]). 2Hydroxyethyl acrylate (HEA, used without further purification) and dialysis membrane (molecular weight cutoff 12,000 g/mol) were purchased from ACROS. Other reagents were from Shanghai Reagent Company except where otherwise stated, and purified according to common methods. Mono-(2-acryloyloxyethyl) phosphonate (MAEP) was synthesized in our laboratory. The detailed procedure was as follows: phosphorous pentoxide (P2O5, 21.3 g, 0.15 mol) and anhydrous benzene (60 mL) were charged into a 250 mL three-necked round bottom flask equipped with a mechanical stirrer, followed by charging HEA (5.8 g, 0.05 mol) and hydroquinone inhibitor (5.5 mg, 0.05 mmol). The reaction was carried out under stirring in dried nitrogen atmosphere at 20 C for 72 h. The reaction mixture was filtrated to remove the residual P2O5; benzene was removed by rotary evaporation at

2.2. Synthesis of phosphonate-functionalized chitosan The synthesis protocol was as follows: in a 10 mL vial equipped with a magnetic stirrer, 3.698 g aqueous solution of MAEP (0.37 g, 1.89 mmol) was adjusted from pH 0.96 to pH 3.06 using a 1 wt.% NaOH aqueous solution. Chitosan (0.10 g, 0.47 mmol amino groups) was added into the vial under stirring at 25 C to generate a colorless, transparent and viscous solution. The Michael addition reaction was performed at 36 C for 3 h. The crude product was purified by dialysis against distilled water for 3 days. Precipitation of the chitosan derivatives from acetone and drying under vacuum at 40 C overnight afforded the white powdery solid. 2.3. Characterization 1

H NMR and 31P NMR studies were carried out using a Bruker 400 MHz NMR spectrometer at room temperature. D2O, 1 wt.% NaOD/D2O and 0.5 M DCl/D2O were used for the 1H NMR and 31 P NMR studies for MAEP and chitosan derivatives respectively. O

O

CH2OH O O

O

O

HO

O MAEP

P

OH OH O

Acidic aqueous solution

NH2

CH2OH O

Chitosan

HO

O NHR

Phosphonate-functioanalized chitosan O O

P O

R:

OH OH

O Scheme 1. Synthesis of phosphonate-functionalized chitosan.

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2.4. Turbidity measurements The pH-responsive behavior of the phosphonatefunctionalized chitosans was determined by turbidity measurements. The solution transmittances at k = 500 nm were recorded using a PerkinElmer Lambda 25 UV–vis spectrometer with the slit width of 1.00 nm at room temperature. Phosphonate-functionalized chitosan aqueous solutions were prepared using doubly distilled water in nitrogen atmosphere at room temperature. The solution pH was adjusted using either dilute HCl or NaOH aqueous solution. A PHS-3C pH Meter was employed to monitor the solution pH. Anti-polyelectrolyte effect of the phosphonatefunctionalized chitosans at their isoelectric points was investigated by turbidity measurements. The ionic strength of the solutions was controlled by adjusting the sodium chloride concentration. The solution transmittances at k = 500 nm were recorded using the above-mentioned UV–vis spectrometer. 2.5. UV–vis spectroscopy UV–vis absorption of phosphonate-functionalized chitosan in basic medium was investigated at room temperature. The procedure was as follows: the diluted vitamin B1 (VB1) aqueous solutions with various sodium chloride concentrations were prepared and used as solvents. The phosphonate-functionalized chitosans were dissolved in these solvents, followed by adjusting the solution to pH 9.65 using a diluted NaOH aqueous solution. The UV–vis spectra were recorded using a PerkinElmer Lambda 25 UV–vis spectrometer at room temperature. As control experiments, the UV–vis spectra of the diluted VB1 aqueous solutions with various sodium chloride concentrations were also recorded. 3. Results and discussion 3.1. Michael addition of chitosan with MAEP The predominant structural units of 1,4-b-D-glucosamine in chitosan backbone provide us a highly reactive primary amine and therefore can be applied as an ideal precursor to facilitate further functionalization to covalently bind phosphonates. Among the reactions relative to amine, the Michael addition reaction is one of the most suitable reactions for this purpose. Thanks to its high reactivity of primary

(b)

(a)

10

5

0 -5 Chemical Shift (ppm)

-10

-15

Fig. 1. 31P NMR spectra of (a) MAEP in D2O and (b) phosphonate-functionalized chitosan in 1 wt.% NaOD/D2O.

amino groups with acrylic compounds, this reaction exhibits high reactivity under mild conditions even in vivo [15], which has recently been successfully employed for the functionalization of chitosans for the synthesis of chitosan N-carboxybetainates without the resource of protecting group chemistry [11]. It is reasonable to expect that the phosphonatefunctionalized chitosan can be synthesized via Michael addition of chitosan with an acrylic compound containing phosphonate, e.g., mono-(2-acryloyloxyethyl) phosphonate (MAEP). As shown in Fig. 1, 31P NMR spectrum of the phosphonate-functionalized chitosan (b) was similar to that of MAEP (a) except a slight shift of d, suggesting that covalent-connection of phosphonate moieties to chitosan led to slight variation of the phosphorous chemical environment. Fig. 2 showed the 1H NMR spectra of (a) the chitosan precursor and (b) its corresponding phosphonate-functionalized derivatives in DCl/D2O. Comparing the 1H NMR of chitosan precursor, clear evidence of the Michael addition reaction was the appearance of the typical signal at d = 2.69–2.91 ppm (peak 1) as shown in Fig. 2b, which was assigned to the methylene proton in Nalkylated groups. Furthermore, the resonances of H-2 (peak 2) attributed to the GlcN and N-alkylated GlcN residues were shifted from d = ca. 2.98 to 3.07–3.21 ppm and cracked into two parts. The changes were due to the substitution of the N-alkylated group to the amino groups of the GlcN residues. Resonance of –NHCOCH3 proton in

H. Kang et al. / European Polymer Journal 42 (2006) 2678–2685

D2O

1

2

3

b 2

3

a

6

5

4

3

2

1

Chemical shift (ppm) Fig. 2. 1H NMR spectra of (a) chitosan precursor and (b) phosphonate-functionalized chitosan in 0.5 M DCl/D2O.

GlcNAc residues (peak 3) appeared at d = 2.10 ppm. Based on the integral of resonance peaks at d = 2.69–2.91 ppm (S1) and 3.07–3.21 ppm (S2), the degree of substitution (DS) of phosphonatefunctionalized chitosan could be calculated according to the equation of DS = S1/4S2, here, DS was defined as the molar ratio of the reacted hydrogen in the amino groups to the initial primary amino groups of chitosan, i.e., the theoretical maximum degree of substitution was 2. The experimental results in Table 1 showed that this system was quite reactive in aqueous solution even at low reaction temperature. Among the four parameters affecting the DS of this Michael addition

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reaction, the solution pH was a crucial factor. Under the conditions of [MAEP]0: [–NH2]0 = 4:1 at 36 C for 3 h (see Fig. 3), DS increased with the increase of the solution pH in low pH range, while decreased as increasing the solution pH above 3.12. The maximum DS was 1.3 at pH 3.12. This was not surprising, strongly acidic solution led to protonation of the primary amino groups of chitosan precursors, i.e., decreased the amount of the neutral primary amino groups, accordingly retarding the rate of Michael addition reaction, thus unavoidably leading to relatively low DS. As increasing the solution pH, DS increased due to partial deprotonation of the protonated amino groups of chitosan, thus increasing the concentration of reactive neutral primary amines of chitosans; whereas, further increasing the solution pH above 3.12 led to the decrease of DS due to the decreased solubility of chitosan. Please note that relatively high DS (see Table 1) was achieved in a short reaction time, e.g., 3 h, while for the conventional acrylic compounds, several days were needed to achieve the similar DS as reported in other literatures [16]. The viscosity-average molecular weight (Mg) of the phosphonate-functionalized chitosans was assessed under the same conditions as described in the experimental section for assessment of Mg of chitosan precursor except at the solution pH 2.46. The Mg of phosphonate-functionalized chitosan with DS = 1.2 (sample 2 in Table 1) was 8.5 · 105 g/mol, while the Mg of chitosan precursor was 8.9 · 105 g/mol. Considering the experimental errors and calculation inaccuracy due to the

Table 1 Synthesis parameters for Michael addition reaction and the resulting degree of substitutions (DS) Sample

Solution pH

Temperature (C)

Reaction time (h)

½MAEP0 : ½–NH2 0 a

DSb

1 2 3 4 5 6 7 8 9 10 11 12

3.06 3.06 3.06 2.21 3.12 4.25 3.06 3.06 3.06 3.06 3.06 3.06

36 36 36 36 36 36 25 50 36 36 36 45

3 3 3 3 3 3 3 3 1 2 4 3

2:1 4:1 5:1 4:1 4:1 4:1 4:1 4:1 4:1 4:1 4:1 8:1

–c 1.2d 1.2 0.9 1.3 0.9 –c 0.6 –c 1.1 1.0 0.7

a b c d

The feed molar ratio of MAEP to amino groups. Degree of substitution for Michael addition reaction. Insoluble in 1 wt.% NaOD/D2O. Mg of phosphonate-functionalized chitosan is 8.5 · 105 g/mol, determined by viscometry measurements at 25 ± 0.1 C at pH 2.46.

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1.0

1.2

0.9 Transmittance

1.4

DS

1.0

0.8

IEP range

0.8

0.7 0.6 0.6 0.4 0.6

1.2

1.8

2.4

3.0 pH

3.6

4.2

4.8

5.4

Fig. 3. Effect of the solution pH on degree of substitution of Michael addition reaction ([MAEP]0:[–NH2]0 = 4:1 at 36 C for 3 h).

difficulty in determining the equation parameters, negligible hydrolysis of chitosan backbones occurred after Michael addition reaction in the solutions at pH 3.06 and 36 C for 3 h. 3.2. Turbidity studies of phosphonate-functionalized chitosan in various solution pH: determination of isoelectric point (IEP) Turbidity of these aqueous solutions of zwitterionic phosphonate-functionalized chitosan was determined by monitoring of the transmittance of the solutions at various solution pH at k = 500 nm. Based on our experimental results, these phosphonate-functionalized chitosans were molecularly dissolved in either basic or acidic medium, whereas flocculated or precipitated from the aqueous solutions in the intermediate pH range, which was a typical feature of polyampholytes, where these chitosan derivatives were essentially electrically neutral. The pH in the middle of the flocculation range was defined as isoelectric point (IEP) [17]. As shown in Fig. 4, the transmittance of the solutions was quite high at either high or low solution pH, while it dramatically decreased at or around the flocculation range or IEP range. Table 2 illustrated the dependence of IEP on the different DS of chitosan derivatives. The experimental results were quite reasonable within the experimental errors [18]. This phase transition of chitosan derivatives from molecularly soluble to macroscopic phase separation with varying the solution pH was strongly

3

4

5

6

7

8

9

pH Fig. 4. Turbidity measurements for 0.628 mg/mL phosphonatefunctionalized chitosan (DS = 0.6) aqueous solutions with different solution pH at ambient temperature judged by variation of solution transmittance at k = 500 nm.

dependent on the macromolecular architectures and compositions, although the complex structural nature of these phosphonate-functionalized chitosans, e.g., the statistically distributed units along the macromolecular backbones including N-acetylated glucosamines (20 mol.%), glucosamines and that containing second/tertiary amines and phosphonates, made it difficult to clarify the specific mechanisms of conformational and phase transitions, these transitions were undoubtedly caused by the formation of attractive intra- and inter-chain electrostatic interactions, resulting in the formation of ionically cross-linking networks at or around the IEP, which was confirmed by the pH-responsive properties and anti-polyelectrolyte effect of these phosphonate-functionalized chitosans on addition of sodium chloride as discussed below. Our experimental results also showed that the particular phase transitions of these phosphonatefunctionalized chitosans were fully reversible on adjusting the solution pH, implying the potential application of these chitosan derivatives in separation and purification of anionic protein [6]. 3.3. Anti-polyelectrolyte effect of the phosphonatefunctionalized chitosans at IEP The effect of the solution ionic strength on the phase behavior of these phosphonate-functionalized chitosan chains in aqueous solution was investigated by turbidity studies. As above-mentioned,

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Table 2 Effect of degree of substitution (DS) on apparent isoelectric point (IEP) and critical salt concentration (CSC) of the phosphonatefunctionalized chitosan aqueous solution Sample

DS

[MAEP-CTS]a (mg/mL)

IEP

CSC (mol/L)

8 10 5

0.6 1.1 1.3

0.628 0.528 0.604

6.5 6.0 5.4

0.023 0.067 0.117

a

Concentration of phosphonate-functionalized chitosans.

these phosphonate-functionalized chitosans were flocculated and finally precipitated from water around their IEPs. Whereas, on addition of small amount of sodium chloride, the precipitates were gradually swelled, ‘‘homogenously’’ dispersed in aqueous solutions and further became emulsions. Addition of more sodium chloride ultimately led to a molecularly dissolved transparent solution. These transitions were precisely monitored by the observation of the transmittance variation as increasing the NaCl concentration (see Fig. 5). These particular transitions for polyampholytes were well-documented as anti-polyelectrolyte effect [19,20]. This dissolution process was understood in terms of the penetration of small molecular weight electrolyte into the ionic network, screening the net attractive electrostatic interactions between polymer chains, thus promoting the solubility. The critical salt concentration (CSC) for these phosphonate-functionalized chitosans was strongly dependent on the degree of substitution (see Table 2):

1.0

Transmitance

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.00

0.02

0.04

0.06

0.08

0.10

0.12

[NaCl] (mol/L) Fig. 5. Turbidity measurements for 0.628 mg/mL phosphonatefunctionalized chitosan (DS = 0.6) aqueous solutions at isoelectric point (pH 6.5) with different concentration of NaCl at ambient temperature judged by variation of solution transmittance at k = 500 nm.

larger DS led to more hydrophilic nature of these chitosans, thus relatively higher CSC. 3.4. Polyelectrolyte effect of phosphonatefunctionalized chitosans in basic medium Increase of the ionic strength of the polyelectrolyte aqueous solution led to the conformational transition of macromolecular chains from extended states to collapsed coils due to electrostatic screening, i.e., polyelectrolyte effect, which unavoidably caused the variation of the solution viscosity and other properties [20]. Here, the effect of the solution ionic strength on the chain conformational behavior of the phosphonate-functionalized chitosans in basic solution (pH = 9.65) was investigated by using vitamin B1 (VB1), a water-soluble cationic aryl ammonium, as the UV–vis probe. The polyelectrolyte effect of the chitosan derivative (DS = 1.3, sample 5 in Table 1) in basic medium was summarized in Fig. 6a. It was clearly observed that the absorption of solution at the characteristic wavelength of 235–232 nm decreased upon increasing the NaCl concentration. Please note that the phosphonatefunctionalized chitosan aqueous solutions had no absorption in the wavelength range of 200–700 nm (see the dotted plot in Fig. 6a). Thus, the UV absorption was attributed to the existence of VB1, and the decrease of absorbance was caused by the decrease in the concentration of fully hydrated VB1 in aqueous solutions according to Beer–Lambert law. It was not surprising if we considered the effect of conformational transition of the phosphonate-functionalized chitosans: the phosphonate groups of these chitosans were fully ionized while the amino groups were completely deprotonated in basic solution at pH 9.65, i.e., chitosan chains were predominantly anionic under these conditions. On addition of sodium chloride, the additional electrostatic screening unavoidably led to the conformational transition from extended state to collapsed coils, i.e., polyelectrolyte effect, thus causing the local-enrichment of anionic phosphonates. This led

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2.0

0.10

a

b

Absorbance

0.08

increase NaCl concentration 1.5

0.06

increase NaCl concentration

0.04 0.02 0.00 -0.02

Absorbance

210

225

240

255

270

285 300

1.0

0.5

0.0 225

250

275

300

325

350

Wavelength (nm) Fig. 6. Effect of ionic strength of NaCl on the UV–vis absorption of (a) the mixture solution of VB1 and phosphonate-functionalized chitosan and (b) VB1 aqueous solution (a: DS = 1.3, concentration of phosphonate-functionalized chitosan: 0.47 mg/mL; [VB1] = 0.0034 mmol/L; spectra from top to bottom: [NaCl] = 0.031, 0.059, 0.093, 0.148, 0.216, 0.306, 0.393, 0.486, 0.597, 0.733, 0.858, 0.985, 1.13 and 1.308 mol/L respectively at pH = 9.65. The dotted plot was the UV–vis spectroscopy of aqueous solution of phosphonatefunctionalized chitosan without VB1 and NaCl; b: [VB1] = 0.0034 mmol/L, from top to bottom: [NaCl] = 1.383, 1.161, 0.928, 0.694, 0.461, 0.226 and 0 mol/L at pH = 9.65).

to the entrapment of permanently cationic VB1 into the collapsed coils via attractive electrostatic interactions. Accordingly, the concentration of fully hydrated or ‘‘free’’ VB1 in aqueous solution decreased. As the control experiments, UV–vis spectroscopy studies for VB1 aqueous solutions with variation of NaCl concentration were also carried out. As shown in Fig. 6b, the absorption at characteristic wavelength of 235–232 nm slightly increased as increasing the NaCl concentration, rather than decreased as observed for the phosphonate-functionalized chitosan solutions. This confirmed that the decrease of UV absorbance was attributed to the conformational transition of these chitosan backbones to collapsed coils, which led to the entrapment of VB1 into the collapsed chitosan coils. 4. Conclusions In this paper, we described a facile synthetic route for novel phosphonate-functionalized pHresponsive chitosans. The attractive inter- or intra-chain electrostatic interactions of the phos-

phonate-functionalized chitosans could be controlled via adjusting the solution pH, leading to the reversible transitions of these chitosan backbones from conformational extended state to collapsed coils and followed by the flocculation or precipitation around the isoelectric point. The typical anti-polyelectrolyte effect of these phosphonatefunctionalized chitosans exhibited at IEP was confirmed by the existence of the critical salt concentration (CSC). The results showed that both IEP and CSC were influenced by the degree of substitution (DS). The polyelectrolyte effect of these phosphonate-functionalized chitosans was also illustrated by using vitamin B1 as the UV–vis probe in a basic medium with the solution pH 9.65, far from the IEP. These phosphonate-functionalized chitosans have potential applications in separation and purification of anionic proteins and pharmaceutical formulations. We are well aware that the biocompatibility and stability of these phosphonate-functionalized pH-responsive chitosans are essential for these applications. Further studies are in progress but beyond the scope of this paper.

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Acknowledgements This research was financially supported by the state education ministry scientific research foundation for the returned overseas Chinese scholars, Hunan provincial natural science foundation of China (05JJ40023) and the start-up research foundation of Xiangtan University (04QDZ31). References [1] Felt O, Buri P, Gurny R. Drug Dev Ind Pharm 1998;24:979. [2] Vande-Vord PJ, Matthew HWT, Desilva SP, Mayton L, Wu B, Wooley PH. J Biomed Mater Res 2002;59:585. [3] Onishi H, Machida Y. Biomaterials 1999;20:175. [4] Lehr CM, Bouwstra JA, Schacht EH, Junginger HE. Int J Pharm 1992;78:43. [5] Langer RA. Chem Res 2000;33:94. [6] Black FE, Hartshorne M, Davies MC, Roberts CJ, Tendler SJB, Williams PM, et al. Langmuir 1999;15:3157. [7] Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, et al. Science 1999;284:489. [8] Ishaug-Riley S, Okun LLE, Prado G, Applegate MA, Ratcliffe A. Biomaterials 1999;20:2245.

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