Modification of Chitosan–Methylcellulose Composite Films with meso

Article pubs.acs.org/Biomac

Modification of Chitosan−Methylcellulose Composite Films with meso-Tetrakis(4-sulfonatophenyl)porphyrin Alla Synytsya,*,† Michaela Grafová,† Petr Slepicka,‡ Ondrej Gedeon,§ and Andriy Synytsya∥ †

Department of Analytical Chemistry, ‡Department of Solid State Engineering, §Department of Glass and Ceramics, and Department of Carbohydrate Chemistry and Technology, Institute of Chemical Technology in Prague, 166 28 Prague, The Czech Republic ∥

S Supporting Information *

ABSTRACT: Polysaccharide films containing chitosan, methylcellulose, and a mixture of these polysaccharides in various ratios were prepared and modified with meso-tetrakis(4sulfonatophenyl)porphyrin in an aqueous medium at pH 7. The modified films were compared with the initial films using spectroscopic methods and microscopic imaging. Electronic (UV−vis absorption, electronic circular dichroism (ECD)) and vibrational (FTIR and Raman) spectra showed that the porphyrin macrocycles had a strong affinity toward chitosan and did not interact with the methylcellulose. The total porphyrin uptake depended on the chitosan: methylcellulose ratio and pure methylcellulose films did not retain porphyrin macrocycles. ECD measurements detected the presence of optically active porphyrin species bound to the films. SEM and AFM images confirmed that the porphyrin macrocycles caused structural changes on the film surface and within the film layer.

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INTRODUCTION The search for new polymeric matrices that are suitable for drug delivery and can promote wound healing has turned up a variety of new biocompatible materials over the past few decades. The “traditional” dressings used in extensive chronic skin diseases, burns, or postoperative wounds have been replaced by materials that more efficiently absorb exudates and protect the wound from external contamination.1 Modern wound dressings support the healing process by creating a humid medium with adequate fluid and gas transport,2−4 avoiding the formation of a scab, enhancing the restoration of the epithelium, and reducing redness and pain.1,5 Moreover, some materials can protect the skin from bacterial infection.4 Various polysaccharides, such as starch, pectin, or chitosan, can form good quality films both as pure materials and in mixtures.6,7 Polysaccharide films generally have a high permeability to gases, including water vapor and an ability to swell, and they provide good fluid transport with high selectivity for bulky nonpolar compounds.5 These properties, in combination with good mechanical strength, render such films appropriate coverage materials for wound treatment. Among film-forming polysaccharides, chitosan is one of the most widely used in the pharmaceutical industry. It is a natural copolymer of randomly distributed β-(1,4)-linked D-glucosamine and N-acetyl-D-glucosamine, derived from the partial deacetylation of chitin (Chart 1a). The degree of deacetylation determines the solubility of chitosan and its ability to restore supramolecular structures.8 Some reports have described the good tissue compatibility, bioavailability, chemical stability, and © 2011 American Chemical Society

Chart 1. Molecular Formulae of Chitosan (a), Methylcellulose (b), and meso-Tetrakis(4sulfonatophenyl)porphyrin (c)

nontoxicity9 as well the antimicrobial,10 immunomodulating, hemostatic, and even antitumor activities of chitosan.4,11 These properties render this polysaccharide suitable for use in a number of biomedical applications, including artificial skin, tissue regeneration, drug delivery systems, and wet wound dressing covers for the treatment of problematic wounds.8 The Received: October 31, 2011 Revised: December 15, 2011 Published: December 19, 2011 489

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mechanical and physical properties of biodegradable chitosan films could potentially be improved by the addition of compatible polysaccharides, such as methylcellulose (Chart 1b). This water-soluble derivative of cellulose has been extensively used as a binder, thickener, or film stabilizer in pharmaceutical, cosmetic, and food applications.12−14 Methylcellulose films are less strong but more flexible than chitosan films, and composite chitosan-methylcellulose films have intermediate tensile properties.15,16 New medicinal materials can be constructed based on porphyrin−polysaccharide composite films. Modification of chitosan with appropriate porphyrin derivatives can enhance the medicinal effects. Porphyrins can act as photosensitizers for photodynamic therapy (PDT), which involves their application at a target position, followed by visible light irradiation. The accessibility of the skin to light exposure has led to the frequent use of PDT in dermatology. Various skin precancerous or malignant tumors (actinic keratoses, carcinomas of basal and squamous cells, etc.) have shown partial or complete clinical response to photodynamic treatment.17,18 PDT also has the potential to cure a variety of nonmalignant skin disorders, such as psoriasis, viral infections, or diseases of the epidermal appendages.17,19,20 Various macrocycles have been incorporated into chitosan membranes by adsorption, by dissolution and casting, or by covalent attachment.21 Water-soluble mesotetrakis(4-sulfonato-phenyl)porphyrin (TPPS4) (Chart 1c) could be interesting in this context because of its apparent antitumor activity and its strong affinity toward chitosan. Application of this porphyrin was effective in the therapy of skin tumors, including basal cell carcinoma lesions,22 and cutaneous metastases of breast cancer.23 Previously, we confirmed that TPPS4 formed complexes and supramolecular structures with chitosan at different pH values in aqueous solutions24 and in the solid state.25 The objective of the present work was to incorporate TPPS4 macrocycles into specially prepared pure chitosan or chitosan− methylcellulose films mixed in various ratios. The structural and physical properties of the modified films were characterized by spectroscopic, microscopic, and other analytical methods.

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Scheme 1. Conversion of Chitosan Acetate into the Free Base Form

an oven at 110 °C until the films had reached a constant weight. The equilibrium moisture content (We, %) was calculated as follows (eq 1) We(%) =

m0 − m × 100 m

(1)

where m0 and m are the initial mass (with moisture) and the ovendried mass, respectively. Samples were analyzed at least in duplicate. Solubility in Water. Film samples of dimensions 0.5 × 0.5 cm2 were cut from dry films, weighed using a Mettler AE 240 digital semimicroanalytical balance with 0.01 mg sensitivity, and soaked in test tubes containing 2 mL of deionized water at 24 °C for 1−144 h. The remaining pieces of film were collected by filtration and dried in an oven at 60 °C until they reached a constant weight. The solubility (S) was calculated as the relative mass of soluble matter (eq 2)

S(%) =

m1 − m2 × 100 m1

(2)

where m1 and m2 are the initial and final masses of the film, respectively. Samples were analyzed at least in duplicate. Incorporation of TPPS4 into the Polysaccharide Films. The water-insoluble films 1−4 were incubated in test tubes containing 2 mL of an aqueous solution of TPPS4 (0.01% for UV−vis and ECD; 0.1% m/m for vibrational spectroscopy and microscopy) at 24 °C for 24 h. The modified films 1−4/P were removed from the solutions, washed with distilled water, and air-dried. The porphyrin content in the films was determined from the UV−vis absorption spectra of the medium based on the maximum intensity of the Soret band corresponding to TPPS4. Samples were analyzed at least in triplicate. Kinetics of the TPPS4 Uptake. The sorption kinetics was measured to determine the contact time required for equilibrium sorption of TPPS4 by films 1−4. The films (m0, 0.0008 to 0.0024 g) were immersed in aqueous solutions (V = 0.002 L) containing porphyrin (ρ0 = 0.002 g L−1). The bulk concentration of porphyrin ρt at various contact times (5−140 min) was determined from the absorbance maximum of the Soret band (λmax = 414 nm, a414 = 396 L g−1 cm−1). Each value reported here corresponds to at least three determinations. The quantity of porphyrin taken up by the films Qt (g g−1) at each contact time interval t (min) was calculated from the eq 3.

EXPERIMENTAL SECTION

Materials. The sodium salt of meso-tetrakis(4-sulfonato-phenyl)porphyrin (TPPS4) was purchased from Sigma-Aldrich (St. Louis, MO). Chitosan from crab shell α-chitin (Mw 400 kD, degree of acetylation 21.2 mol %) was purchased from Fluka (Germany). Methylcellulose was purchased from Carl Roth (Germany). Preparation of the Polysaccharide Films. Pure and composite polysaccharide films containing chitosan (1), methylcellulose (5), or a mixture of these polysaccharides (2−4) were prepared according to Garcia et al. (2004)15 and Pinotti et al. (2007).16Solutions containing pure chitosan (2% m/m in 1% aqueous acetic acid) or methylcellulose (1% m/m in water) were prepared, and the solutions were mixed in volume proportions of 3:1 (2), 1:1 (3), and 1:3 (4). The pure and mixed solutions (4 g) were then poured into 5 × 5 cm polyethylene forms and air-dried. The films 1−4 were removed from the forms and immersed in an aqueous solution of sodium hydroxide (0.2 mol L−1) for 30 min. Therefore, these films we converted into a water-insoluble free base form of chitosan (Scheme 1). After alkali treatment, the films 1−4 were washed thoroughly with distilled water until the solution reached a neutral pH and then dried in an oven at 60 °C for 24 h. The films obtained were stored at 24 °C without any apparent changes and were used in subsequent experiments. Humidity. The water content in the treated polysaccharide films was determined by measuring the weight loss of films upon drying in

Qt =

(ρ0 − ρt )· V m0

(3)

A logistic function (eq 4) was chosen as a suitable model for fitting to the kinetic dependence of Qt on t

Qt =

Q e·(t /t0.5) p

1 + (t /t0.5) p

(4)

where Qe is the equilibrium uptake, t0.5 is the time for achieving half the total uptake, and p is a parameter that characterizes the nature of the process: p exceeds 1 for a sigmoidal dependence and equals 1 for a hyperbolic dependence. Sorption Isotherms of the TPPS4 Uptake. Films 1−4 (m0, 0.0005 g) were immersed in aqueous solutions containing porphyrin (ρ 0, 0.01 to 1.00 g L−1) for 24 h. The films were removed, and the medium was analyzed. The bulk concentration of porphyrin ρe at 490

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equilibrium was determined from the UV−vis absorption spectra based on the maximum intensity of the TPPS4 Soret band. Equilibrium porphyrin uptake by the films Qe (g g−1) was calculated according to the eq 5. ρ − ρe Qe = 0 m0

(2−4) at various ratios. The solutions and obtained films were homogeneous and transparent. The physical properties of the films, such as the moisture retention and the solubility in water (Supporting Information, Table S1), depended on the chitosan: methylcellulose ratio that is in agreement with the results reported in literature.15,16 Dried chitosan films 1 were brittle and rigid, whereas composite and pure methylcellulose films 2− 5 were much more flexible. Film 5 exhibited the lowest equilibrium moisture retention (1.21%), whereas the highest moisture retention was observed for film 1 (7.48%). Washing of films 1−4 with 0.2 mol L−1 NaOH decreased the moisture content by 0.8 to 1.2%. Dissolution curves for these films are shown in Figure 1. Film 1 in a free base chitosan form had a

(5)

Triplicate determinations were made for each case, and their mean values were computed for quality assurance. The obtained data were fit to a Marczewski−Jaroniec sorption isotherm26,27 to describe the process of monolayer single-solute adsorption from a dilute solution onto an energetically heterogeneous solid. This equation comprises all isotherm equations and is an extension of a simple Langmuir isotherm, that is, a generalized Freundlich, Langmuir−Freundlich, and Tóth isotherms. Therefore, these isotherms are special cases of the general equation (eq 6)

⎡ (K ·ρ )n ⎤m / n e Q e = Q m·⎢ n⎥ ⎣⎢ 1 + (K ·ρe) ⎥⎦

(6)

where Qm is the sorption capacity, K is the Langmuir-type constant representing the sorption affinity, and m and n are heterogeneity parameters. Because this equation has four fitting constants (Qe, K, m, and n), it provides a better description of the adsorption process than the more simple isotherms mentioned above. Spectroscopic Analyses. UV−vis absorption and electronic circular dichroism (ECD) measurements were conducted by placing the films between the quartz plates of cuvettes with a light path of 0.1 mm (Hellma, type 106-QS, USA). Absorption spectra (350−800 nm) were recorded using a single-beam Cary 50 UV−vis spectrophotometer (Varian, USA). ECD spectra were recorded using a J-850 ECD spectrometer (Jasco, Japan) over the spectral ranges 190−300 and 350−550 nm, corresponding to the electronic transitions of the polysaccharides and the porphyrin (Soret band), respectively. FTIR spectra (4000−650 cm−1) were recorded using a Nicolet 6700 spectrometer (Nicolet Analytical Instruments, USA) using HATR equipment. Sixty-four scans were accumulated with a spectral resolution of 2.0 cm−1. Dispersion Raman spectra were recorded on a Dilor−Jobin Yvon−Spex Raman spectrometer (Dilor−Jobin Yvon− Spex, France) equipped with an Olympus BX 40 system microscope with a 100× objective. This objective was capable of focusing the laser to a spot of diameter 10−12 μm. An argon ion laser system with an excitation line at 488 nm and an excitation power of 2.5 mW was used in the measurements. The exposure time for one accumulated spectrum was 600 s at 24 °C. The average spectra were smoothed using a 5 cm−1 filter, and the baselines were corrected using a polynomial function in the LabSpec software (Dilor−Jobin Yvon− Spex). Smoothing and baseline correction of the spectra were performed using the Origin 6.0 (Microcal Origin, USA) software. Microscopic Analyses. The polysaccharide films were analyzed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The samples used for imaging were analyzed by SEM using a Hitachi S-4700 (Hitachi, Japan) at an acceleration voltage of 10 kV with a working distance of 12 mm and a resolution of 2.1 nm. Surface morphology studies and thickness estimations were performed using samples that had been dried under vacuum at room temperature, mounted on a metal stub, and sputtered with gold to make the sample conductive. SEM images were collected at 5000× magnification. The surface morphologies of the samples were examined using AFM (tapping mode), performed under ambient conditions using a CP II Veeco microscope (Veeco Metrology, USA). A RTESPA-CP probe was used. The mean roughness (Ra) indicated the arithmetic average of the deviations from the center plane of the sample.

Figure 1. Dissolution curves for the films 1−4.

very low solubility (2.96%), even after 6 days. These films became rubbery after dipping into water, but they maintained their integrity. The composite films 2−4 displayed intermediate water solubility, which decreased with increasing chitosan/ methylcellulose ratio. Among all films, film 4 gave the highest solubility (37.6%) and yielded a gel-like structure at the end of the solubility test. By contrast, the methylcellulose film 5 was completely soluble in water. These observations demonstrated that chitosan is able to retain moisture, whereas methylcellulose enhanced the swelling properties of all composite films. FTIR and Raman spectra of the representative films 1−4 (initial films and after alkali treatment) and 5 are shown in Figure 2a,b. Band assignment was made according to the literature.28−34 The spectra of pure polysaccharide films 1 and 5 revealed characteristic IR and Raman bands of chitosan and methylcellulose, respectively. The spectra of the composite films 2−4 were more complex and revealed overlapping bands of both polysaccharides. The positions and intensities of these characteristic bands were sensitive to the chitosan/methylcellulose ratio. Two IR bands of the initial films 1−4 at 1560 and 1408 cm−1 were assigned to carboxylate stretching vibrations of acetate anions. These bands disappeared after treatment with NaOH, and a new band of NH2 in-plane scissoring vibration arose at 1586 cm−1, confirming the conversion of chitosan acetate to a free base form. The IR bands at 1650 cm−1 (amide I) and a shoulder near 1540 cm−1 (amide II) are characteristic of the Nacetyl groups in chitosan and did not depend on the state of the free amino groups.30,31 The bands from the methylcellulose film 5 at 1454 and 1372 cm−1 were assigned to the bending vibrations of CH3.32 Corresponding bands of chitosan film 1

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RESULTS AND DISCUSSION Preparation of the Polysaccharide Films. All polysaccharide films were prepared from pure chitosan (1), methylcellulose (5), or a chitosan: methylcellulose mixture 491

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Figure 2. FTIR (a) and Raman (b) spectra of the films 1−5: the initial films (dashed lines) and films after alkaline treatment (solid).

Figure 3. Kinetic curves (a,b) and sorption isotherms (c) describing TPPS4 uptake by the films 1−4.

were found at 1460 and 1376 cm−1. The films display several intense overlapping IR bands in the region of CO and CC stretching vibrations (950−1200 cm−1). Among these bands, those at 1152, 1076, and 1034 cm−1 arose from chitosan and those at 1196, 1110, 1063, and 1027 cm−1 arose from methylcellulose. The OH stretching bands of chitosan and methylcellulose were found at 3362 and 3450 cm −1 ,

respectively; the band at 3300 cm−1 was assigned to the NH stretching vibrations of chitosan (Figure 2a).30,33,34 The Raman spectra of the initial films 1−4 display several bands of acetate anion vibrations at 1408, 1340, 922, and 470 cm−1 (Figure 2b).28,29 The treatment of these films with NaOH led to disappearance of these bands, and a new band arose at 1593 cm−1 (NH2 scissoring). Raman bands at 1460, 1376, 492

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Figure 4. UV−vis absorption spectra (350−800 nm) of the films 1−4/P (a) and the 2nd derivatives of the spectra (b) in the Soret region (350−500 nm). ECD spectra of the films 1−5 and 1−4/P in the UV region (190−300 nm) (c,d) and in the Soret region (350−500 nm) (e).

1112, 1096, and 1034 cm−1 were characteristic of chitosan, whereas those at 1454, 1369, 1151, 1121, 1027, 947, and 851 cm−1 were typical of methylcellulose. Incorporation of TPPS4 into the Polysaccharide Films. The kinetic curves corresponding to the TPPS4 uptake by films 1−4 from aqueous porphyrin (2 mg L−1) solutions are shown in Figure 3a. A comparison of the curves revealed significant differences between the films, depending on the composition. The TPPS4 uptake curves intercepted at 33 min. This point divided the curves into initial and final steps. Most curves displayed a sigmoidal character that was less pronounced for films with higher methylcellulose contents. The initial uptake rate (0−33 min) was determined by the process of film swelling in aqueous media. Water penetrated the film, allowing the macrocycles to reach the chitosan binding sites. Swelling of the pure chitosan film 1 was relatively slow, whereas the presence of methylcellulose enhanced the rate of swelling of the composite films 2−4, depending on the ratio between the chitosan and the methylcellulose. A logistic function was chosen as a suitable model for fitting the observed kinetic dependence (eq 4). The main model parameters are listed in Table S2 of the Supporting Information. The equilibrium uptake Qe values calculated

from the models were in the range 0.79 to 1.80 g g−1 and increased with the amount of chitosan in the film. The time required to take up half the total porphyrin uptake, t0.5, decreased from 47.1 to 27.6 min as the amount of methylcellulose in the film increased. The parameter p was in the range 1.14−3.15 for the films 1−4, and p decreased as the methylcellulose content increased. The rate constant for the final step (after 33 min) depended on the number and availability of binding sites. All curves reached a plateau at 90 min contact time. The first derivative of this model function (Figure 3b) described the dependence of the rate of porphyrin uptake on the contact time. The maximal rate of uptake (Vm) was 0.016 to 0.033 mg g−1 min−1. The contact time (tm) decreased from 38 to 0.02 min as the methylcellulose content in the films increased. The values p ≈ 1 and tm ≈ 0 for film 4 indicated that porphyrin uptake followed a hyperbolic function. Sorption isotherms plot the amount of porphyrin bound to a film (Qe) as a function of the residual porphyrin concentration in solution (ρe) at equilibrium. The equilibrium distributions of TPPS4 between solid and liquid phases were determined by varying the initial porphyrin concentration (ρ0). Figure 3c shows the equilibrium sorption data for films 1−4 and the corresponding model curves. The Marczewski−Jaroniec 493

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Figure 5. FTIR (a) and Raman (b) spectra of the films 1−4/P.

sorption isotherm model parameters26 (eq 6) are listed in Table S2 of the Supporting Information. The sorption data were well-described by this model over the range of concentrations considered here. In general, high-performance sorbents should have both high uptake capacity Qm and high affinity to sorbate indicated by low values of K.27 The comparison of sorption performance should be made on whole sorption isotherm plots. The plots revealed that the sorption capacity Qm of the films fell from 1.23 to 0.40 g g−1 as the chitosan content decreased. Higher chitosan concentrations in a film yielded higher uptake capacities. The parameter K was found to vary from 1.25 (1) to 2.92 (2), so pure chitosan films demonstrated the highest affinity to TPPS4. In the case of films 2, small amounts of methylcellulose is not able to create strong own network system, and inclusions of this inert polysaccharide may impede porphyrin molecules to reach binding sites inside the film. Such steric hindrance by methylcellulose was less pronounced for films 3 and 4 containing higher amounts of this polysaccharide. This could be probably due to the formation of strong methylcellulose network supporting the penetration of water and TPPS4. The heterogeneous parameters m and n subsequently increased in the films as the methylcellulose content increased. Heterogeneity among the binding sites in the pure chitosan film 1 could be explained in terms of the partial ionization, distribution, and availability of free NH2 groups in the polysaccharides. In the composite films 2−4,

methylcellulose formed its own network within the film alongside the chitosan network.24,25,27 These two networks superimposed on each other and thereby increased the heterogeneity of the film structure. Structure of the TPPS4-Modified Polysaccharide Films. The porphyrin-modified polysaccharide films 1−4/P were prepared using a protocol similar to that used for the sorption experiments described above at the two initial porphyrin concentrations. The TPPS4 contents in the films are summarized in Table S3 of the Supporting Information. The 1−4/P films prepared at a higher porphyrin concentration (0.1% m/m) contained about six times the TPPS4 content in the films prepared at a lower porphyrin concentration (0.01% m/m). The 1/P films showed the highest porphyrin content (852.9 mg g−1), and the content subsequently fell to 363.8 mg g−1 in the raw and mixed films 2−4/P with increasing methylcellulose content. The water-soluble 5/P films contained negligible amounts of TPPS4 (2.3 mg g−1) when water was replaced with ethanol as the reaction medium. These results indicated that only chitosan bound to the porphyrin effectively, whereas methylcellulose remained inert. The absorption spectra (350−800 nm) of TPPS4 and the porphyrin-containing polysaccharide films 1−4/P are shown in Figure 4a. The Soret and Q-bands of TPPS4 were markedly shifted toward the red in comparison with the free base porphyrin, providing evidence of a complexation. In addition, 494

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Figure 6. Raman spectra of the film 3/P measured at a position perpendicular to the film surface (a) and at a cut of the film (b) in comparison with the spectrum of the solid TPPS4 (c).

the ECD spectra of films 1−4/P (Figure 4e) demonstrated a complex system of highly overlapping positive and negative couplets, which gradually shifted toward the red as the methylcellulose content in the films increased. The complex CD features in the Soret region could be explained by the presence of several optically active forms of the porphyrin macrocycles bound to chitosan. The FTIR and Raman spectra of the films 1−4/P are shown in Figure 5. The FTIR spectra consisted of highly overlapping bands that originated from both the porphyrin and the polysaccharides. The polysaccharide contribution was much more pronounced than the porphyrin contribution (Figure 5a). By contrast, the porphyrin bands dominated the Raman spectra due to preresonance enhancing effects, whereas the polysaccharide bands were insignificant (Figure 5b). Three IR bands at 718, 739, and 857 cm−1 and a shoulder at 962 cm−1 were assigned to the core and phenyl vibrations of TPPS4, whereas IR features at 1220, 1195, and 1122 cm−1 were assigned to the sulfonato groups.25,37−39 The bands characteristic of chitosan were observed at 1420, 1378, 1322, 1152, 1034, 948, and 896 cm−1. The maximum position of the broad peak at 1065−1078 cm−1 depended on the relative contributions of chitosan and methylcellulose. The region 1500−1700 cm−1 underwent some changes upon TPPS4 binding. Two chitosan bands at 1586 (NH2 scissoring) and 1651 cm−1 (amide I) shifted, respectively, to 1592 and 1647 cm−1. The latter band broadened and decreased in intensity relative to the former band, and a shoulder became more prominent near 1543 cm−1 (amide II). These effects were more pronounced for the films containing higher quantities of methylcellulose. The observed spectroscopic changes could be explained in terms of the contributions of porphyrin bands at 1633, 1597, and 1566 cm−1, partial ionization of NH2 groups by the formation of TPPS4 salts containing sulfonato groups, and changes in the

the Soret band of film 1/P was significantly broadened, and the broadening was less pronounced for films 2−4/P, which had higher methylcellulose contents. A second derivative analysis of the Soret band showed the presence of three components at 416−419, 423, and 428−430 nm (Figure 4b). Each of these components indicated specific forms of the porphyrin macrocycles, which had different conformations and orientations in the films as a result of binding to the polysaccharides. The former and latter peaks were shifted toward the red and blue, respectively, as the methylcellulose content increased. As these components drew together, the full Soret band across the visible absorption spectrum narrowed. The peak near 423 nm, which was close to the main maximum of the Soret band, was weak and overlapped with the main band. Its position was difficult to determine for films 2−4/P. The ECD spectrum (UV region) of the pure chitosan film 1 (Figure 4c) displayed a broad negative band near 220 nm. This band was assigned to the n→π electronic transition of the C O bond in the N-acetyl groups and has been used previously to determine the degree of chitosan acetylation.24,35,36 The intensity of this band in the spectra of the composite films 2−4 decreased and was blue-shifted to 210−212 nm. The spectrum of film 1/P demonstrated a similar blue shift to 213 nm (Figure 4d). In the spectra of films 2−4/P, this blue shift was more pronounced (up to 209 nm) and was accompanied by an intensity decrease that depended on the methylcellulose content. The intensity of this negative CD peak was a good marker of the presence of chitosan because the degree of Nacetylation of this polysaccharide is expected to be stable under mild film preparation conditions. The blue shift of this peak, observed for the films 1−4/P, could be explained in terms of the hydrophobic interactions of the N-acetyl groups in chitosan, with methyl groups in the methylcellulose or porphyrin macrocycle. The region of the Soret band (410−430 nm) in 495

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amide I and II bands, caused by interactions between the porphyrin macrocycles and the N-acetyl groups of chitosan. The Raman bands of the porphyrin core displayed vibrations at 1567, 1549, 1367, 1340, 1327, 1135, 1056, and 807 cm−1, which were shifted to lower wavenumbers relative to those of the free TPPS4 (Figure 5b). By contrast, the porphyrin bands at 1449, 999, and 838 cm−1 were shifted to higher wavenumbers by 9−19 cm−1. Several low-frequency TPPS4 bands in the range 200−700 cm−1 were found to be shifted as well, and two new porphyrin core vibrational bands arose at 1648 and 780 cm−1. Phenyl vibration bands at 1589, 1283, 1230, 716, 669, and 631 cm−1 underwent significant changes upon porphyrin incorporation into the films. These spectroscopic changes could be explained in terms of porphyrin core deformations and changes in the phenyl orientations caused by interactions with chitosan. Significant differences in the position and intensity of the TPPS4 bands were evident from the Raman spectra of the 3/P film, measured perpendicularly with respect to the film surface at the cut of the film (Figure 6). The peaks corresponding to CϕCϕ and CβCβ vibrations at 1589 and 1542 cm−1 were markedly decreased in intensity or even undetectable in the Raman measurements at the cut, but those corresponding to the CαCm vibrations at 1646−1648, 1558−1562, and 1461 cm−1 were shifted slightly relative to the corresponding peaks observed in the perpendicular measurements. The CmCϕ vibrational peak was shifted to 1240 cm−1. Several new peaks, observed at 1415, 1374, and 932 cm−1, were assigned to the inplane vibrations of pyrrolic rings. The peaks at 1050−1160 cm−1 (in-plane symmetric CβH vibrations), 800−900 cm−1 (pyrrolic ring in-plane deformations) and below 700 cm−1 (pyrrolic ring out-of-plane deformations) also differed significantly between the two measurements. Therefore, the orientation of the TPPS4 macrocycles within the films was not random; rather, the TPPS4 macrocycles were ordered relative to the polysaccharide chains. SEM and AFM images of the representative films are shown in Figures 7 and 8. SEM analysis confirmed that the films 1−4 had smooth homogeneous surfaces (Figure 7a−d), whereas porphyrin uptake led to the appearance of compact lamellar structures on the surfaces of the films 1−4/P (Figure 7e−h). These lamellar structures were more evident for the films 2−4/ P, which included higher quantities of methylcellulose. SEM cut images of the films 2−4 revealed multilayer structures (Figure 7i−l) that were even more apparent after porphyrin incorporation, as in films 2−4/P (Figure 8m−p). The AFM images revealed some surface irregularities for the composite films 2−4, whereas the surface of the pure chitosan film 1 was smooth (Figure 8a−d). The roughness of the films Ra calculated from the AFM data (Supporting Information Table 4) were markedly higher for the composite films 2−4 (71.2− 77.7 nm) in comparison with film 1 (3.5 nm). Porphyrin incorporation led to an increase in this value for film 1/P of up to 9.4 nm, whereas Ra of the composite films 2−4/P dropped to 24.8−48.5 nm. The AFM images of the films containing TPPS4 illustrate these changes (Figure 8e−h). If the lamellar structures observed by SEM consisted of ensembles of macrocycles bound to the chitosan strains, then the differences in the surface properties of the films after porphyrin incorporation could be explained. Macrocycles can bind to the smooth surfaces of films that form such structures, thereby increasing Ra for the film 1/P. In the case of films 2−4/P, porphyrin molecules probably fill the surface cavities and smooth the film surface.

Figure 7. SEM images of the surface (a−h) and cut (i−p) of the films 1−4 (a−d, i−l) and 1−4/P (e−h, m−p).

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CONCLUSIONS

In the present Article, pure chitosan and chitosan−methylcellulose films were obtained and successfully modified using water-soluble porphyrin TPPS4. Each polysaccharide component of the composite films played a specific role in the interactions with water/chitosan retained the moisture necessary for wet wound dressings, and methylcellulose enhanced the swelling properties and supported water penetration into the film. The results of our investigations demonstrate that porphyrin molecules have a strong affinity for chitosan and do not interact with methylcellulose. In the composite chitosan−methylcellulose films, the porphyrin uptake depended on the amount of chitosan in the films, and pure methylcellulose films retained no macrocycles. The spectroscopic analyses indicate that TPPS4 macrocycles are bound to chitosan. Raman and ECD measurements detected the optically active TPPS4 species, which may assume a specific orientation in a film relative to the chitosan chains. SEM and AFM images demonstrated that porphyrin macrocycles arranged onto film surfaces as well as penetrated the film. Incorporation of TPPS4 caused the formation of specific lamellar structures, which were more pronounced for films containing methylcellulose. The porphyrin package, however, should be quite different from that previously proposed for the chitosan−TPPS4 precipitates.25 No highly H- and/or J-type aggregated porphyrin species was detected in the modified 496

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Figure 8. AFM images of the films 1−4 (a−d) and 1−4/P (e−h).

films, although the interaction between macrocycles cannot be entirely excluded. The polysaccharide−porphyrin films presented in this work could be interesting for the construction of novel wound dressings. The photodynamic effects of porphyrin macrocycles can potentially enhance the overall therapeutic impact of the composite wound-dressing material.

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ASSOCIATED CONTENT

S Supporting Information *

Water retention, solubility, sorption kinetic and isotherm parameters of TPPS4 uptake, porphyrin contents, and the Ra values for the polysaccharide and/or polysaccharide − porphyrin films are summarized. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +420 220 443 762. Fax: +420 220 440 352. E-mail: [email protected].

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ACKNOWLEDGMENTS This work was supported by the Ministry of Education of the Czech Republic (project no. CEZ: MSM6046137307) and the Czech Science Foundation (project no. 525/09/1133).

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REFERENCES

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