Photoactive Modified Chitosan - American Chemical Society

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Biomacromolecules 2008, 9, 1631–1636

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Photoactive Modified Chitosan Maria Nowakowska,* Łukasz Moczek, and Krzysztof Szczubiałka Faculty of Chemistry, Jagiellonian University, 30-060 Krako´w, Ingardena 3, Poland Received February 10, 2008; Revised Manuscript Received March 26, 2008

A water-soluble polymeric photosensitizer that contains naphthyl chromophores and absorbs light in the near UV region was obtained by modification of chitosan. The excitation energy can be used to induce photochemical reactions via energy and electron transfer.

Introduction The interest in photoactive polymers results from their wide potential applications. They are considered important components of photocurable paints and resins, formulations for holographic recording, xerography, photoresists, data storage and light-emitting devices, or solar energy conversion systems.1–7 A relatively new area of studies is related to the development of polymeric photosensitizers and polymeric photocatalysts. Among their possible applications are those in biomedical (as drug carriers, sensitizers, and sensors)7 or environmental field (for water purification).8–14 Majority of the systems synthesized and studied so far are based on synthetic polymers. Considering the environmental issues, we have been interested in development of photosensitizers based on natural polymers.15–21 In the current paper, we present the studies on the synthesis and properties of the photosensitizers based on chitosan (CH). CH is a natural polysaccharide that can be easily obtained from chitin, the most abundant natural polymer in biosphere, by its Ndeacetylation. CH is a low cost material that possesses many useful properties and has found a number of practical applications. Due to its biodegradability, biocompatibility, nontoxicity, antibacterial, and antiviral properties, it is getting a great deal of interest for medical, pharmaceutical, and agricultural applications. Both the properties and the function of CH can be adjusted according to the needs by its modification. The aim of our current studies was to obtain photosensitizers that could be used to photosensitize reactions of organic compounds in water using light from the near UV-visible spectral region. For that purpose we have modified CH by covalent attachment of naphthyl chromophores.

Experimental Section 1

Apparatus. The H NMR spectra of the polymers were measured in D2O/CF3COOD solution using a Bruker AMX 500 spectrometer. The IR spectra were recorded in KBr pellets using a Bruker IFS 48 spectrometer (Bruker, Rheinstetten, Germany). The dynamic light scattering (DLS) measurements were performed using Malvern Nano ZS light-scattering apparatus (Malvern Instruments Ltd. Worcestershire, U.K.). Atomic force microscopy (AFM) images were obtained with a Multi Mode scanning probe microscope with a NanoScope IVA controller working in tapping mode equipped with a silicone cantilever (Digital Instruments, Santa Barbara, CA). The * To whom correspondence should be addressed. Tel.: +48 12 6632250. Fax: +48 12 6340515. E-mail: [email protected].

UV-vis spectra of the samples were obtained using a HP 8452A diode-array spectrophotometer (Hewlett-Packard, Palo Alto, CA). The steady-state fluorescence spectra were obtained using an SLM Aminco 8100 spectrofluorimeter (SLM, Rochester, NY) set up in the L-type geometry. The gel permeation chromatography (GPC) analyses of the polymers were carried out using a Waters GPC system (Waters, Millford, MA) equipped with an Ultrahydrogel Linear 8 µm column. Detection was done using a Waters 2410 refractive index (RI) detector, a Waters 474 scanning fluorescence detector, and a Waters 2996 photodiode array (PDA) detector. A 0.25 M acetic acid (AA)/0.25 M sodium acetate solution was used as the mobile phase at a flow rate of 0.5 mL/min. The viscosity of the polymer solutions was measured using an Ubbelohde capillary viscometer equipped with an electronic time-measuring unit ViscoClock (Schott, Mainz, Germany). Irradiations of the samples were performed with a mercury lamp (150 W) equipped with 280 nm interference filter. Materials. A chitosan (CH) sample (a shrimp-based product) was obtained from Novachem Limited, Dartmouth, Nova Scotia, Canada. The viscosity and weight average molecular weights, MV and MW, of the polymer were determined to be 1230 and 1300 kDa, respectively, based on the Mark-Houwink-Sakurada equation using the literature values of K ) 1.57 × 10 mL/g, R ) 0.79, and qMHS ) 0.95.22 The degree of acetylation (DA) of chitosan was determined to be 12% based on NMR spectrum. 1-Naphtylacetic acid (NA, Sigma, 97%) was purified by crystallization from a water-ethanol mixture (80:20 v/v). 4,4Bipyridinium-1,1-bis(trimethylenesulfonate) (SPV) was prepared according to the procedure described in the literature.23 The aqueous polymer solutions were prepared using deionized water. Synthesis of the Polymers (CHNA). The CHNA polymers characterized by various degrees of substitution with naphthyl chromophores were obtained. The polymers were synthesized in reaction between amine groups of CH and carboxylic groups of NA in aqueous solution. In the first step, the chitosan amino NA salt is formed, which is then dehydrated (Scheme 1). The general procedure was as follows: CH (0.5 g) was dissolved in 1% AA aqueous solution (25 mL) and was stirred at room temperature for 12 h. Then a solution of NA in water (0.25, 0.127, or 0.025 g in 10 mL, respectively) was added dropwise over a period of about 30 min. The mixture was intensively stirred using a magnetic stirring bar for 24 h. After the reaction was completed, the mixture was kept for 12 h under vacuum at 80 °C to dehydrate the CH salt formed and to obtain an amide bond between CH and NA. The product was rinsed several times with methanol, dissolved in water, and exhaustively dialyzed (Sigma, cellulose tubing, cutoff molecular weight 12000-14000 g/mol) against water for a week to remove nonbound NA. The dialysis was finished when no NA residues were detected in the UV-vis spectrum of the dialysate. The polymers were subsequently freeze-dried. The content of naphthyl units in the polymers was determined using NMR and UV-vis spectroscopy.

10.1021/bm800141v CCC: $40.75  2008 American Chemical Society Published on Web 05/06/2008

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Scheme 1. Reaction between CH and NA

Results and Discussion Properties of CHNA Polymers. The CHNA polymers were obtained as white pellets. They were very well soluble in water at pH values lower than 6. For all further studies, the polymers were dissolved in 1% AA aqueous solution. The polymers were characterized by elemental analysis, DLS, AFM, viscometry, spectroscopic methods (1H NMR, Fourier transform infrared (FTIR), and absorption and emission in the UV-vis spectral region), and by GPC. Using these techniques, we have confirmed that NA was covalently attached to the CH polymer chains. The 1H NMR spectra show the characteristic signals in the range of 7.4-8.1 ppm, indicating the presence of naphthalene aromatic rings, and at 4.1 ppm, confirming the presence of methylene bridge (Figure 1). Based on these spectra, the content of naphthyl groups in polymers was calculated (Table 1). The values obtained from the NMR were verified by calculations done from the electronic absorption spectra of CHNA solutions, using the experimentally determined value of molar extinction coefficient for naphthyl groups, 282 ) 6000 M-1 cm-1 . FTIR spectra of the substituted polymers showed bands that could be assigned to the amide bonds between CH and NA (Figure 2). There are two characteristic peaks at 1650 cm-1 (amide I, carbonyl stretching vibration) and 1600 cm-1 that correspond to N-acetylated units and free amino groups, respectively. The ratio of the intensity of these bands (1650/ 1600) is considerably higher for CHNA than for CH polymer, which indicates that the content of the amide groups is higher in the substituted polymers. Moreover, the amide band is shifted toward lower wave numbers, suggesting that the aggregation induced by hydrogen bonding is diminished. In the CHNA spectrum, a weak band at 1250 cm-1 assigned to the deformation N-H vibration is observed (so-called II amide band). The spectrum indicates that there is no ester bonds in the CHNA

Nowakowska et al.

polymers (no bands at ∼1730 cm-1). These observations confirm that naphthyl chromophores are attached to the CH chain exclusively by the amide bonds. The formation of the amide bonds was confirmed by 13C NMR spectra, which correlate with those obtained by simulation done with ChemOffice software. In experimental spectrum we have observed a peak at 39.8 ppm, which could be ascribed, based on simulation, to the carbon adjacent to the amide carbonyl group. Moreover, in 13C NMR experimental spectrum there was no signal at 44.6 ppm, which would be expected for carbon adjacent to the ester group. Also, the sample used for the measuring of 13C NMR spectra was dissolved in DCl + D2O solution. Under these acidic conditions, the ester bond, if present, is expected to hydrolyze with the formation of carboxylic acid (1-naphthylacetic acid). We have not observed a signal that could be ascribed to carboxyl carbon in that sample. That seems to rule out formation of the ester bonds in naphthyl substituted chitosan and supports the conclusion that the chromophore is attached to polymer chain via amide linkage, indeed. GPC chromatograms for the substituted CHs recorded using simultaneously two detectors, that is, refractive index (RI) and PDA detectors, demonstrated that both signals obtained overlap (Figure 3). That observation confirms the conclusion that naphthyl chromophores are indeed covalently attached to the CH chain. Dynamic light scattering measurements (DLS) for CH and substituted CHs (data not shown) indicated that the substitution of CH results in decrease of hydrodynamic diameters of polymer chains; from 22 nm for CH to around 15 ( 2 nm for the substituted ones. That is confirmed by the viscometric measurements. Figure 4 shows dependence of reduced viscosities on concentration of aqueous CH and CHNAs solutions. One can observe that substitution of CH chain with hydrophobic naphthalene molecules induces the conformational changes; the polymer chain becomes more compact. Interestingly, even relatively low content of naphthyl groups induces dramatic conformational changes, as reflected in viscosity. The AFM measurements supported the above-mentioned observation made by other techniques. The AFM images obtained for CH and the CHNAs are depicted in Figure 5. The AFM picture of CH shows the presence of the isolated domains of a mean diameter of approximately 30 nm . The hydrogen bonds play an essential role in organizing the macromolecules of CH. The structure of CHNA films is quite different. The films are considerably smoother and the domains are much smaller; the size of the grain is about 15 nm. That can be explained considering two effects: first, the partial elimination of the hydrogen bonds in substituted CH, and second, the hydrophobic interactions between naphthyl substituents, resulting in a decrease of the hydrodynamic volume of the polymer chain (as seen by DLS and viscosimetry). Electronic absorption and steady-state emission spectra of CHNAs in aqueous solutions are characteristic of naphthyl chromophores. Comparison of the absorption spectrum of CHNA3 with that for NA, the model compound, and the shape of the emission spectrum for CHNA3 indicates that there is no aggregation of the chromophores in that system (see Figure 6). That is confirmed by the measurements of the excitation and synchronous emission spectra (not shown). Photosensitizing Activity of CHNAs. To demonstrate the usefulness of CHNA polymers as photosensitizers for reactions initiated via photoinduced electron transfer, the experiments in

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Figure 1. 1H NMR spectrum of CHNA3 (1% CD3COOD in D2 O).

Figure 2. FTIR spectra of CH (upper) and CHNA3 (lower). Figure 3. Normalized GPC chromatograms for CHNA3 recorded using RI (solid line) and PDA (dotted line, λabs ) 280 nm) detectors; eluent, 0.25 M acetic acid/0.25 M sodium acetate, pH ) 4.7.

Table 1. Content of Naphthyl Groups in CHNA Polymers Determined by 1H NMR and UV-Vis Spectroscopy polymer CHNA1 CHNA2 CHNA3

1

H NMR (% mol)

UV-vis (% mol)

2.0 ( 0.2 3.1 ( 0.3 17.4 ( 1

1.8 ( 0.1 3.6 ( 0.3 17.0 ( 1

which the aqueous polymer solutions containing SPV, (viologen water-soluble compound) were irradiated with light at 280 nm, absorbed exclusively by naphthyl chromophores. The reduction of the SPV occurred, as evidenced by the appearance of absorption characteristic of radical anion SPV•- (absorption maxima at 399 and 602 nm, see Figure 7) and blue color of irradiated solution. The concentration of the SPV was determined from the value of absorption at 602 nm using the extinction coefficient  602 ) 12800 M cm.24 The kinetics of the process could be described by the pseudofirst-order kinetic equation (data not shown). The free energy of that process can be determined using a Rehm-Weller equation25

∆G0 ) EDox - EAred - E/ - C

(1)

ox where ED is the oxidation potential of the electron donor, ox red (ED ) 2.29 eV for naphthalene),26 EA is the reduction

Figure 4. Reduced viscosity versus concentration of aqueous solutions of CH (9), CHNA1 (2), CHNA2 (1), and CHNA3 (f).

red potential of the electron acceptor (EA ) >0.37 eV for 27 / SPV), E is the energy of the electronically excited-state of donor (E/ ) 3.99 eV for naphthalene)28 and C is an electrostatic correction term, which is typically equal to 0.1 eV for polar solvent.

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Figure 5. AFM height (left) and 3D (right) images of CH (A), CHNA2 (B), and CHNA3 (C).

Using these values one can find that

∆G ) -2.17 eV This indicates that the process is thermodynamically favorable and can be described as follows 1

NA* + SPV f NA•+ + SPV•-

(2)

The occurrence of the photoinduced electron transfer process was confirmed by the observation that SPV efficiently quenches the CHNA fluorescence in aqueous solution (Figure 8). The quenching of excited naphthyl chromophores in CHNA by SPV resulting from the singlet-singlet energy transfer can be excluded considering the values of the respective energy levels; ESPV ) 4.97 eV, ENA ) 3.99 eV28 (∆E ) ENA - ESPV ) 0.98 eV). Considering the possibility of practical application of CHNAs as photosensitizers the oxidation of perylene (Pe), a model compound for polynuclear aromatics, was studied. It was found that in aqueous solution CHNAs can solubilize scarcely watersoluble perylene molecules. That was concluded from the measurements of Pe fluorescence in the system. It is known that Pe emission in water is negligible, while its quantum efficiency in polar organic solvent is very high, Φ ) 0.87.28

Figure 6. Normalized electronic absorption (dotted line) and steadystate emission (solid line) spectra of aqueous solution of CHNA3 (c ) 0.1 g/L, pH ) 3.5) and absorption spectrum of NA (dashed line) in aqueous solution (c ) 5 × 10-5 M).

Very intensive emission of Pe solubilized in aqueous solutions of CHNAs indicates that Pe experiences highly hydrophobic microenvironment created by the clustering of naphthyl chromophores. That was confirmed by the efficient quenching of CHNA emission by solubilized Pe. The process is thermody-

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Figure 7. Changes in electronic absorption spectrum of CHNA1/SPV/ EDTA on irradiation with light at 280 nm (Cpol ) 0.1 g/L), cSPV ) 8.57 × 10-4 M, cEDTA ) 1.14 × 10-3 M).

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Figure 9. Perylene fluorescence decay on irradiation of aqueous solution of CHNA3 containing Pe with light at λirr ) 280 nm (Cp ) 0.1 g/L, cPe ) 3 × 10-5 M). Table 2. Rate Constants for Photosensitized Oxidation of Pe Solubilized in Aqueous Solutions of CHNAs polymer (Cp ) 0.1 g/L)

kq (min-1) × 102

CHNA1 CHNA2 CHNA3

0.26 ( 0.02 0.95 ( 0.05 1.3 ( 0.1

excitation energy is transferred from NA chromophores to Pe, while in the second the electron transfer from Pe molecules to the suitable acceptor. Naphthyl chromophores or naphthoquinone chromophores, formed as a result of oxidation of the former, can serve as electron acceptors. The Pe radical cations formed are very reactive toward oxygen and reaction leads to the formation of perylenequinones as final molecular products. Figure 8. Steady-state fluorescence spectra of CHNA1 in aqueous solution in the absence and in the presence of SPV (Cp ) 0.1 g/L, λexc ) 280 nm).

Conclusions

Scheme 2. Photosensitized Oxidation of Perylene

Series of photoactive naphthyl-substituted chitosans (CHNA) was prepared. The polymers are water-soluble and absorb light from the near UV spectral region. The electronically excited Np chromophores can serve as energy and electron donors to the molecules of suitable acceptors. CHNAs are quite promising as photosensitizers for reactions of organic compounds in aqueous solutions. Considering these and biodegradability of polysaccharide-based polymers studied, they can be useful in water purification.

namically feasible, considering the energy levels of the respective singlet states; ENA ) 3.99 eV and EPe ) 2.90 eV28 and it occurs according to the Fo¨rster resonance mechanism.1 The radius of interaction R0 between naphthyl chromophores in CHNA and Pe was calculated from the experimental data and found to be ∼31 Å. Irradiation of oxygen saturated aqueous solution of CHNAs containing solubilized Pe with light absorbed exclusively by the naphthyl chromophores, λ ) 280 nm, leads to rapid oxidation of Pe with a formation of perylene quinones (Scheme 2). The reaction was followed by the measurements of Pe fluorescence decay, as the oxidation products are nonfluorescent (Figure 9). The value of pseudofirst-order rate constant for Pe oxidation was found to be dependent on the degree of CH substitution. That confirms that the process is indeed sensitized by NA chromophores (see Table 2). Based on literature29 and results of our experiments it was suggested that the process occurs in two stages; in first the

References and Notes (1) Guillet, J. E. Polymer Photophysics and Photochemistry; Cambridge University Press: New York, 1985. (2) Mizoguchi, K.; Hsegawa, E. Polym. AdV. Technol. 1996, 7, 471. (3) Morin, J.-F.; Boudreault, P.-L.; Leclerc, M. Macromol. Rapid Commun. 2002, 23, 1032. (4) Shibaev, B.; Boiko, N. Prog. Polym. Sci. 2003, 28, 729. (5) Zhao, Y.; Bai, Z.; Asatryan, K.; Galstin, T. AdV. Funct. Mater. 2003, 13, 781. (6) Yang, X.; Loos, J.; Veenstra, S. C.; Verhees, W. J. H.; Wienk, M. M.; Kroon, J. M.; Michels, M. A. J.; Janssen, R. A. J. Nano Lett. 2005, 5, 579. (7) Tyagi, R.; Pandey, M. K.; Malhotra, S.; Kumar, R.; Kumar, J.; Parmar, V. S.; Watterson, A. C. J. Macromol. Sci., Chem. 2007, 44, 1283. (8) Nowakowska, M.; Guillet, J. E. Chem. Br. 1991, 27, 327. (9) Nowakowska, M.; Sustar, E.; Guillet, J. E. J. Am. Chem. Soc. 1991, 113, 253. (10) Guillet, J. E.; Burke, N. A. D.; Nowakowska, M.; Reese, H.; Gravett, D. Macromol. Symp. 1995, 98, 53. (11) Nowakowska, M.; Burke, N. A. D.; Guillet, J. E. Chemosphere 1999, 39, 2249.

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(12) Nowakowska, M.; Keˆpczyn˜ski, M. J. Photochem. Photobiol., A 1998, 116, 251. (13) Nowakowska, M.; Keˆpczyn˜ski, M.; Szczubiałlka, K. Pure Appl. Chem. 2001, 73, 491. (14) Torres, J. D.; Faria, E. A.; SouzaDe, J. R.; Prado, A. G. S. J. Photochem. Photobiol., A 2006, 182, 202. (15) Nowakowska, M.; Sterzel, M.; Szczubiałlka, K.; Guillet, J. E. Macromol. Rapid Commun. 2002, 23, 972. (16) Nowakowska, M.; Zapotoczny, S.; Sterzel, M.; Kot, E. Biomacromolecules 2004, 5, 1009. (17) Nowakowska, M.; Sterzel, M.; Zapotoczny, S.; Kot, E. Appl. Catal., B 2005, 57, 1. (18) Nowakowska, M.; Sterzel, M.; Zapotoczny, S. Photochem. Photobiol. 2005, 81, 1227. (19) Nowakowska, M.; Sterzel, M.; Szczubiałlka, K. J. Polym. EnViron. 2006, 14, 59. (20) Moczek, Ł.; Nowakowska, M. Biomacromolecules 2007, 8, 433.

Nowakowska et al. (21) Nowakowska, M.; Zapotoczny, S.; Sterzel, M. Polym. Int. 2007, 56, 635. (22) Chirkov, S. N. Appl. Biochem. Microbiol. 2002, 38, 5. (23) Brugger, P. A.; Gratzel, M.; Guarr, T.; McLendow, G. J. Phys. Chem. 1982, 86, 944. (24) Krasna, A. I. Photochem. Photobiol. 1979, 29, 267. (25) Weller, A. Z. Phys. Chem. 1982, 133, 93. (26) Kavarnos, G. J.; Turro, N. J. Chem. ReV. 1986, 86, 401. (27) Brugger, P. A.; Gra¨tzel, M.; Guarr, T.; McLendon, G. J. Phys. Chem. 1982, 86, 944. (28) Murrov, S. L.; Carmichael, I.; Hug, G. L. Handbook of Photochemistry, 2nd ed.; Marcel Dekker, Inc.: New York, 1993. (29) Burke, N. A. D.; Templin, M.; Guillet, J. E. J. Photochem. Photobiol., A 1996, 100, 93.

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