pH-Controlled Self-Assembling of meso-Tetrakis(4-sulfonatophenyl

Biomacromolecules 2009, 10, 1067–1076

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pH-Controlled Self-Assembling of meso-Tetrakis(4-sulfonatophenyl)porphyrin-Chitosan Complexes Alla Synytsya,*,† Andriy Synytsya,‡ Petra Blafkova´,‡ Jana Ederova´,§ Jirˇi Speˇvacˇek,⊥ Petr Slepicˇka,# Vladimı´r Kra´l,† and Karel Volka† Department of Analytical Chemistry, Department of Carbohydrate Chemistry and Technology, Central Laboratories, Department of Solid State Engineering, Institute of Chemical Technology, Prague 16628, Czech Republic, and Institute of Macromolecular Chemistry, Academy of Science of the Czech Republic, Prague 16206, Czech Republic Received October 15, 2008; Revised Manuscript Received February 16, 2009

Solid meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4)-chitosan supramolecular complexes were prepared by addition of porphyrin to an aqueous solution of chitosan at pH values. The precipitates obtained were assigned as 1 (pH 6.8) and 2 (pH 2.5) and characterized by spectroscopic, thermal, and microscopic methods. Spectroscopic investigation confirmed the presence of TPPS4 and chitosan in both products and that the porphyrin is highly self-associated. H-type (stacked) of TPPS4 aggregation was proposed for 1 and J-type (tilted) for 2. Thermal analysis revealed different pyrolysis routes of the complexes depending on their structural diversity. Light microscopic analysis indicated fibrous and lamellar microstructures, respectively, for 1 and 2. SEM and AFM analysis showed that both complexes consist of compact nanostructures; their size and interconnection is different for 1 and 2. Based on structural inferences, self-assembling hierarchy models were proposed for both of the TPPS4-chitosan supramolecular complexes.

Introduction Supramolecular structures based on organized assemblies of macrocyclic chromophores have attracted widespread interest as molecular devices with potential applications in nanotechnology and related research areas like molecular electronics, light harvesting and pharmacology.1-4 These structures may involve covalent links between the components or be stabilized by weak interactions (electrostatic, polar, nonpolar, etc.). Selfassembly provides a primary mechanism through which molecular aggregates are created in both natural and artificial systems. The manipulation with self-assembled structures could be an alternative method for nanofabrication technologies. The attachment of such structures on solid surfaces is one of the fundamental processes for the development of molecule-based nanodevices. Porphyrins and other closely related macrocycles have been used as suitable chromophores for preparation of supramolecular systems. Self-aggregation in aqueous systems is a common feature of water-soluble porphyrins carrying polar or charged substituents. Dissolved in water, these macrocycles are able to self-assemble into dimers or larger structures: face-to-face stacked (H-type) and edge-to-edge tilted (J-type) aggregates.2,5-8 Under appropriate conditions, porphyrins arrange into larger assemblies of nano and micro scale dependent on ionic strength, pH and concentration. Increasing the ionic strength supports the formation of fractal structures at relatively low porphyrin concentration, while rod-shaped mesoscopic ag* To whom correspondence should be addressed. Phone: +420 220 444 106. Fax: +420 220 440 352. E-mail: [email protected]. † Department of Analytical Chemistry, ICT Prague. ‡ Department of Carbohydrate Chemistry and Technology, ICT Prague. § Central Laboratories, ICT Prague. ⊥ Institute of Macromolecular Chemistry, ASCR. # Department of Solid State Engineering, ICT Prague.

gregates arise at low ionic strength and higher porphyrin concentration.9,10 Fractal supramolecular clusters have been observed both for stacked11,12 and for tilted9 aggregates of substituted porphyrins. It has been described in many investigations that water-soluble porphyrins and related macrocycles are able to form supramolecular complexes and ordered structures on the surface of inorganic substrates2,13 as well as on polar or oppositely charged molecular scaffolds, polypeptides,14,15 proteins,4,16-18 nucleic acids,19-21 polysaccharides,22,23 cyclodextrins,24 dendrimers,25 synthetic polymers,26,27 surfactants,28,29 or mitochondrial membranes.30,31 Being achiral molecules, these macrocycles can demonstrate induced chirality in their complexes and aggregates obtained by interaction with chiral environment.23 Therefore, scaffold biological macromolecules promote self-forming of chiral ordered porphyrin arrays. Chitosan, a natural polysaccharide, has been found to be a useful biomaterial. Due to the presence of amino groups in the molecule, chitosan is soluble in aqueous acidic media. It forms viscous solutions that can be applied to produce gels, membranes, beads, coatings, fibers, and sponges. Chitosan is a biocompatible, biodegradable, biologically inert, and stable material. These properties make it suitable for use in a number of biomedical applications, including artificial skin, tissue regeneration, and drug delivery systems.32 Chitosan is an interesting candidate for the electrostatic self-assembly of macrocycles due to its positive charge (pKa ∼ 6.2).33 The polysaccharide itself and its derivatives have been used for self-assembling nanoparticles,34-36 nanofibers,37 and membranes.38 Various macrocyclic compounds have been incorporated into chitosan membranes by adsorption, by dissolution and casting, and by covalent attachment.39 Self-assembled sulfonated C60-porphyrin complexes have been obtained on the surface of

10.1021/bm8011715 CCC: $40.75  2009 American Chemical Society Published on Web 03/20/2009

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Scheme 1. Molecular Formulae of meso-Tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) (A) and Chitosan (B) Used in the Preparation of Complexes

chitosan thin films.40 Precipitation of solid ionic chlorophyllinchitosan complexes has been described by Arimoto-Kobayashi et al.41 In our previous study, we showed that chitosan induces and enhances self-aggregation of meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS4) in aqueous solution.23 The type of aggregation depends strongly on pH: the H-type predominates at neutral and weakly acidic conditions (pH 6.8 and 5.6), while the J-type is favored under more acidic conditions (pH 2.5). Both types of aggregated TPPS4-chitosan species exist in dynamic equilibrium and can transform into each other and to monomers dependent on pH value and porphyrin to chitosan ratio. An excess of chitosan leads to destruction of the aggregates and monomeric porphyrin-polysaccharide complexes appear in the solution. According to the models described for the TPPS4 aggregation onto the chitosan scaffold in solution,23 it has been assumed that the polysaccharide chains involved in porphyrin aggregate binding have free amino groups at the nonbinding side. These groups can interact with free macrocycles forming larger aggregated structures. Such self-assembling takes place at higher porphyrin concentrations and may lead to precipitation of mixed TPPS4-chitosan aggregates. The present work is devoted to the investigation of this phenomenon. Solid aggregates of TPPS4 on chitosan scaffold were isolated and analyzed by spectroscopic, thermal and image analysis.

Experimental Section Materials. Studies were performed with meso-tetrakis(4-sulfonatophenyl)porphyrin commercially available as the sodium salt (Scheme 1A) and chitosan of medium molecular weight (Mr ) 400 kD) originated from crab shell R-chitin (Scheme 1B). Both compounds were purchased from Fluka. The degree of polymerization (n) of the chitosan can be obtained from the equation:

n)

Mr(chitosan) 400000 ) ) 2351.5 Mr(unit) 170

The average mass of chitosan monomeric unit is

Mr(unit) )

Mr(GlcN)·(100 - DA) + Mr(GlcNAc)·DA - 18 ) 170 100

Figure 1. UV-vis absorption spectra of TPPS4-chitosan complexes 1, 2 (solid), and free TPPS4 (dash).

The molecular masses Mr of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) units are 179.2 and 221.2, respectively. The degree of acetylation (DA, mol %) of the chitosan used was determined by a first derivative UV spectroscopic method.42 The DA value obtained was 21.2%. Redistilled water was used as the solvent for all sample preparation. The pH values of the chitosan solutions were adjusted by dropwise addition of either 0.1 mol L-1 HCl or 0.1 mol L-1 NaOH and measured by a Marrison model 90 pH/temperature meter. Preparation of Solid Porphyrin-Chitosan Complexes. Small amounts (50 mg) of solid TPPS4 were successively added to flasks containing aqueous solutions of chitosan (10 mL, 2% m/m), pH 6.8 and 2.5, with continuous mixing. The colored precipitates formed were isolated, washed twice with aqueous medium, and then washed with ethanol. Finally, the precipitates were dried on a glass surface. The solid products obtained were assigned as chitosan-TPPS4 conjugates 1 (pH 6.8) and 2 (pH 2.5). Organic Elemental Analysis. The solid chitosan-TPPS4 complexes obtained were analyzed for their carbon, hydrogen, nitrogen, and sulfur contents using an EL III Universal CHNOS Elemental Analyzer (Elementar Analysensysteme GmbH, Germany). The contents of porphyrin and polysaccharide components in the complexes were calculated from the sulfur and nitrogen contents

wporph )

wS·Mporph 4·32

(

)

wchit )

wS wN ·Mr(unit) 14 32

The chitosan to porphyrin molar ratio R of the complexes was calculated according to the equation

Self-Assembling of TPPS4-Chitosan Complexes R)


wchit·Mporf wporf·Mr(unit)

Spectroscopic Analysis. UV-vis absorption spectra (300 - 800 nm) of thin solid samples deposited onto the glass surface were recorded using a UV 4 UNICAM UV-vis spectrometer (Unicam, Great Britain). Absorption FT-IR (400-4000 cm-1, KBr discs) and diffuse reflectance FT-NIR (4000-10000 cm-1) spectra were obtained using a Nicolet 6700 spectrometer (Nicolet Analytical Instruments, U.S.A.); 64 scans were accumulated with a spectral resolution of 4.0 cm-1. FT-Raman spectra were recorded using a Bruker FT-Raman (FRA 106/S, Equinox 55/S) spectrometer equipped with a quartz beamsplitter, a liquid nitrogen cooled germanium detector, and excitation at 1064 nm from a Nd: YAG laser. The laser power was set at 250 mW, and 1026 scans were accumulated with a spectral resolution of 2.0 cm-1. Laser Raman spectra were recorded on a Dilor-Jobin Yvon-Spex Raman spectrometer equipped with an Olympus BX 40 system microscope with 100× objective, which is capable of providing a laser spot diameter of approximately 10-12 µm. An argon ion laser system with an excitation line at 488 nm and excitation power 2.5 mW was used in the measurements. The exposure time for one accumulation was 600 s. Average spectra were smoothed by a 5 cm-1 filter and were corrected by polynomial baseline using LabSpec (Dilor-Jobin Yvon-Spex) software. High-resolution 13C and 15N CP/MAS (cross polarization/ magic angle spinning) NMR spectra were measured using a Bruker Avance 500 spectrometer in 4 mm ZrO2 rotors at the frequency 125.8 and 50.7 MHz, respectively, with contact time 2 ms, repetition delay 4 and 1 s, respectively, and spinning frequency 11 and 10 kHz, respectively. The number of scans in 13C CP/MAS NMR spectra was 1800-5400; the number of scans in 15N CP/MAS NMR spectra was ∼54000. Chemical shifts in the 13C NMR spectra were referred to the carbonyl line of glycine (with a signal at 176.0 ppm from TMS), chemical shifts in 15N NMR spectra were referred to the NH4Cl (signal at 40.7 ppm), in both cases by sample replacement. The chitosan to porphyrin molar ratio R of the complexes was calculated on the basis of carbon peak areas Achit and Aporph of the corresponding components, where the intensities of the spinning sidebands of porphyrin signals were also taken into account

R ) (Achit·44)/(Aporph·6) Thermal Analysis. Thermogravimetric analysis of TPPS4-chitosan complexes was carried out using Setsys evolution TG-DTA/DSC 18 (SETARAM, France). DSC analysis of these samples was performed using calorimetry (DSC 131; SETARAM, France). The samples (2-10 mg) were heated in closed platinum cuvettes under nitrogen from 20 to 900 °C (TG) or to 700 °C (DSC) at a heating rate of 10 °C/min. Indium standard was used for calibration. Microstructural Analysis. Solid TPPS4-chitosan complexes were deposited onto the glass surface and analyzed by light microscopy (LM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The microscopic samples were placed on the sample glass under the objective 4 of a Nicon SM2-2T optical microscope (Nicon, Japan) equipped with an Intralux 4000-1 lamp, color camera, and digitizer Micro-Movies. Images were observed under transmitted light, focused, digitized and decomposed into pixels using 256 dot scale. The image processing was performed by the LUCIA software. The microscopic

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samples were also analyzed by scanning electronic microscopy (Hitachi S-4700, Hitachi, Japan) at the accelerating voltage of 10 kV, with a working distance of 12 mm and a resolution of 2.1 nm. For surface morphology studies, the samples were dried in vacuum at room temperature, mounted on a metal stub, and sputtered with gold to make the sample conducting, and the SEM images recorded at 5000× magnification. The surface morphology of samples was examined using AFM (tapping mode), performed under ambient conditions on a CP II Veeco microscope (Veeco Metrology, U.S.A.). Probe RTESPA-CP was used. Mean roughness (Ra) represents the arithmetic average of the deviations from the central plane of the sample.

Results and Discussion Preparation of TPPS4-Chitosan Complexes. TPPS4, a synthetic porphyrin carrying four anionic sulfonato groups, and chitosan, a cationic polyelectrolyte, were used for the preparation of solid supramolecular complexes at different pH. It was observed that a purple colored fibrous (pH 6.8) or green colored lamellar (pH 2.5) precipitate appeared in the solutions depending on initial pH. Porphyrin was added to the chitosan solution until precipitation completed and the medium slightly stained by a small excess of dissolved TPPS4. This suggests that the solid complexes probably formed between TPPS4 and chitosan, and the mode of binding depended on the pH value. The precipitates obtained were washed with an aqueous medium of appropriate pH and distilled water to remove uncomplexed TPPS4 and chitosan, and then air-dried on a glass surface. The results of organic elemental analysis were used for calculation of the molar ratio R between the polysaccharide and porphyrin components in the complexes (Table 1). Comparison of the sulfur and nitrogen contents confirmed that both complexes consisted of TPPS4 and chitosan. The experimental molar ratios R confirmed that complex 2 prepared at more acidic conditions contained less chitosan (R ) 5.8 mol/mol) than complex 1 (R ) 8.3 mol/ mol). Thus, the pH conditions of the reaction medium seem to be a principal factor controlling the mechanism of selfassembling and the stoichiometry of precipitated TPPS4chitosan complexes. Spectroscopic Analysis of TPPS4-Chitosan Complexes. Spectroscopic methods are effective tools to confirm the presence of both polysaccharide and porphyrin components in the complexes and, furthermore, are able to provide information about the mode of binding between these components (Figures 1-3). UV-vis absorption spectra of the TPPS4-chitosan complexes and of the solid porphyrin deposited onto the glass surface at appropriate pH are shown in Figure 1. Complex 1 demonstrated a Soret band at 409 nm with a shoulder near 428 nm and four Q bands at 522, 558, 594, and 650 nm. These bands are typical for H-aggregates and have similar maxima as those of free TPPS4 (pH 6.8).23,43 The shoulder could be assigned to the monomeric porphyrin bound to chitosan; a similar band at 420 nm has been found for TPPS4-chitosan solution (pH 6.8) in the presence of a high excess of the polysaccharide.23 The Soret band of complex 1 underwent pronounced hypochromicity in

Table 1. Organic Element (C, H, N, S), Moisture Content (% m/m), and Chitosan to Porphyrin Molar Ratios R (mol/mol) of TPPS4-Chitosan Complexes 1 and 2 content (% m/m)

a

sample

C

H

N

S

H2Oa

porphyrin

chitosan

R (mol/mol)b

1 2

43.15 39.47

5.84 5.47

6.48 5.74

4.81 5.38

10.43 11.75

35.02 39.22

52.61 40.93

8.3 5.8

Calculated from TG analysis.

b

Calculated on the average of monomeric chitosan units, that is, anhydro GlcN(Ac).

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Figure 3. 13C (A) and 15N (B) CP/MAS NMR spectra of TPPS4chitosan complexes 1, 2, and their parent compounds.

Figure 2. Absorption FT-IR (A), FT NIR and visible Raman (B, C), and diffuse reflectance FT-NIR (D) spectra of TPPS4-chitosan complexes 1, 2, and their parent compounds.

comparison with that of solid TPPS4. This effect is caused by H-aggregation; therefore, complex 1 probably consists of larger stacked aggregates of macrocycles than those in the free base porphyrin (pH 6.8). In contrast, complex 2 showed three peaks at 426, 493, and 704 nm assigned to J-aggregated macrocycles.8,23,44 Corresponding peaks of diacid TPPS4 (pH 2.5) were somewhat narrower and found at very similar positions. An additional broadening of the Soret and Q bands of complex 2 may be caused by small alteration of molecular environment as a result of interaction with chitosan. FT-IR spectra of TPPS4-chitosan complexes 1 and 2 are shown in Figure 2A. The spectra of complexes contain bands originating from both porphyrin and polysaccharide components. Broad chitosan bands are overlapped by sharp bands of TPPS4. IR bands of complex 1 assigned to the porphyrin core and phenyl vibrations showed similarities to those of the free base TPPS4 (pH 6.8). In contrast, the corresponding bands of complex 2 were similar to those of diacid porphyrin (pH 2.5). A strong band at ∼1488 cm-1 and a medial band at ∼984 cm-1 found in complex 2 are characteristic for the dicationic form of macrocycles.45,46 The band at 1004 cm-1 is significantly shifted

Self-Assembling of TPPS4-Chitosan Complexes

to lower frequencies in comparison to the corresponding bands of the free base and diacid TPPS4 (1014 and 1012 cm-1). This band position is characteristic of unsubstituted TPP macrocycles45 and, in the case of complex 2, might be explained by the conformational change in the porphyrin core due to weakening of sulfonato-phenyl bonds as a result of complexation. The shift of several phenyl bands at 1034, 734, 704, and 626 cm-1 to lower frequencies (6-14 cm-1) as well as the appearance of new phenyl bands at 1226, 824, and 662 cm-1 indicates perturbation of the macrocyclic core and the different microenvironment of phenyls in complex 2. N-Protonation of the macrocycle under acidic conditions led to its deformation and orientation of NH bonds out of the ring plane. This configuration supports intermolecular interaction between the porphyrin core and sulfonato groups of a neighboring macrocycle resulting in J-type aggregation.47 The porphyrin macrocycle in J-aggregates, however, changes slightly in comparison to monomeric diacid.45 The IR spectrum of free base TPPS4 has two strong bands at 1192 cm-1 (with a shoulder at 1219 cm-1) and 1128 cm-1 assigned to νas(SO3-) and νs(SO3-) vibrations of ionized sulfonato groups.45,46 These bands changed significantly after complexation: the high frequency shoulder increased and, in the case of complex 2, overlapped the phenyl band at 1226 cm-1, while the latter band shifted by 4-6 cm-1 to lower frequencies in both cases. These spectral changes support the assumption that sulfonato groups are involved in the complexation. IR bands of chitosan at 1380, 1320-1322, 1070-1080, 1034-1036, 945 (shoulder), and 902 cm-1 were found in the spectra of the complexes.48 In both complexes, the band at 1170-1176 cm-1, assigned to the COC stretching vibration of glycosidic bonds, was broadened and shifted by 16-20 cm-1 to higher frequencies in comparison with that of untreated chitosan. This change might be explained by conformational alterations in chitosan upon complexation with TPPS4. A broadband at 1526 cm-1, assigned to νs(NH3+), indicated ionized amino groups in complex 2, while a shoulder at 1600 cm-1, found in the spectrum of complex 1, is characteristic for the scissoring vibration of NH2 groups. Therefore, it appears that complex 1 contains chitosan macromolecules mainly in free base (NH2) form and complex 2 in cationic (NH3+) form. FT Raman and visible-excited Raman (vis-Raman) spectra of TPPS4-chitosan complexes are shown in Figure 2B,C. Porphyrin bands predominate in both types of Raman spectra, while chitosan bands were insignificant and completely overlapped by much stronger porphyrin peaks.45,49-52 In the spectra of TPPS4-chitosan complexes, the position and intensity of these porphyrin bands were similar to those in the spectra of free porphyrin at appropriate pH. The ν(Cβ-Cβ) band of free base TPPS4 (pH 6.8) at 1547-1549 cm-1 showed a significant shift to low frequencies (15-20 cm-1) in the case of diacid porphyrin (pH 2.5) and complex 2, while the position of this band in complex 1 was unchanged. Similar but less pronounced shift to low frequencies (3-7 cm-1) was observed for another ν(Cβ-Cβ) band at 1499 cm-1. The ν(CR-Cm) band of free base TPPS4 at 1450-1453 cm-1 shifted to 1457 cm-1 in complex 1 and to 1474 cm-1 in complex 2. Similarly, the ν(CR-N) band of free porphyrin at 1327 cm-1 shifts to 1330 cm-1 for 1 and to 1358 cm-1 for 2 on complexation; the corresponding band of diacid porphyrin (pH 2.5) was found at 1361 cm-1. A large shift to high frequencies (14-22 cm-1) was also observed for the CR-Cβ/CR-N stretching bands of protonated TPPS4 (pH 2.5) and complex 2 found at 1005-1015 and 979-986 cm-1. Marked resonance enhancement of the low frequency vis-Raman bands at ∼237 and 311 cm-1 assigned to out-of-plane vibrations


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of Cm-Cφ bonds and pyrrole rings was also observed in these cases.8 These low-frequency vibrations are characteristic of the diprotonated core and are forbidden in the Raman spectra of free base TPPS4 (pH 6.8). The intensity increase of these bands is evidence of tilted J-type aggregation of macrocycles.45,53 FT-NIR spectra of the TPPS4-chitosan complexes 1 and 2 are shown in comparison with the corresponding spectra of the initial compounds (Figure 2D). The complexes showed several characteristic bands of the porphyrin and polysaccharide components.48,54 Porphyrin bands at 4058-4065, 4655-4659, 5986-6001, and 6133-6136 cm-1 were pronounced in both complexes; the position of the last band seems to be sensitive to the protonation of the macrocycle core (6125-6133 cm-1 for the free base form, 6136-6144 cm-1 for the diacid form). Two bands of complex 2 at 8821 and 9006 cm-1 (second overtone of CH stretching vibrations) are indicative of the protonated macrocycles. The bands of free base chitosan near 4933 and 6541 cm-1 assigned to combination and overtone vibrations of NH2 groups were found in the spectrum of complex 1. NIR bands of chitosan salt are broad or weak, so they contribute slightly to the spectrum of complex 2. 13 C CP/MAS NMR spectra of TPPS4-chitosan complexes are shown in Figure 3A. Assignments of carbon peaks were made according to the literature.55,56 The NMR spectra showed carbon resonance signals of chitosan (polysaccharide carbons at 50-110 ppm, acetyl carbons at ∼174 ppm and ∼23.5 ppm) and porphyrin (aromatic carbons at 110-160 ppm). The values of chitosan to porphyrin molar ratios R obtained from the areas of corresponding carbon signals (including spinning sidebands of porphyrin signals) were 8.1 for 1 and 5.3 for 2, in good agreement with results of organic elemental analysis. Comparison of the polysaccharide carbon signals (50-110 ppm) of the complexes with those of chitosan in free base and salt forms confirmed that complex 1 contains a mix of both chitosan forms (broadening of C-1 and C-4, upfield shift of C-1 and a shoulder near 71 ppm), while complex 2 contains the salt form only (splitting of C-5 and C-3 signals, C-1 at 97 ppm and less pronounced C-6 shoulder). Therefore, nonamidated units of chitosan macromolecules are partially ionized in complex 1 and completely ionized in complex 2. It is also evident from the intensities of the CdO and OCH3 signals that complexation did not affect N-acetylation of chitosan. The porphyrin region (110-160 ppm) is less resolved for both the complexes in comparison to free TPPS4 (pH 6.8). In complex 1, all the resonance signals of porphyrin core carbons are markedly broadened and downfield shifted by ∼2-5 ppm, whereas the signals of phenyl carbons demonstrated weaker broadening and slight shifts. In complex 2, the resonances of pyrrolic carbons are slightly changed in their positions, while the signal of mesocarbon is markedly (8 ppm) downfield shifted. Small downfield shifts (1-3 ppm) of phenyl carbon signals were observed for both complexes. Observed changes in signal positions could be explained by the decrease in the angle between the macrocycle and the phenyl rings as a result of aggregation and protonation. 15 N CP/MAS NMR spectra of solid TPPS4-chitosan complexes 1 and 2 are shown in Figure 3B. The spectrum of complex 2 demonstrates two sharp peaks of comparable intensity at 35.2 and 132.7 ppm. The former signal was assigned to free amino groups in chitosan,56,57 while the latter one corresponds to the nitrogens of protonated TPPS4 macrocycles.58,59 The presence of the sharp singlet peak of porphyrin nitrogens confirms that all macrocycles are in the dicationic form. In contrast, the spectrum of complex 1 exhibits only one peak at

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Scheme 2. Two-Step Thermal Degradation of TPPS4-Chitosan Complex 2

34.6 ppm. As in complex 2, this chemical shift corresponds to free amino groups in chitosan. No peak of porphyrin nitrogens was observed in this case that could be explained by the lower amount of porphyrin and by broadening of the TPPS4 signal following chemical exchange of the central hydrogens in the macrocyclic core.58,59 Thus, the porphyrin molecules in complex 1 are mainly in free base (nonprotonated) form. Thermal Properties of TPPS4-Chitosan Complexes. TG, DTG, and DSC thermograms of TPPS4-chitosan complexes 1 and 2 are shown in Figure 3A-C. The temperatures of significant weight loss (DTG minima) were similar to the positions of corresponding DSC peaks. The first decline of the TG curves (Figure 3A) and endothermic DSC peak (Figure 3C) below 100 °C are associated with water evaporation, and the second thermal event at ∼200-260 °C with the polysaccharide degradation.60 According to DTG curves (Figure 3B), the latter process occurs at lower temperatures in the complexes (260 °C for 1 and 205 °C for 2) than in initial chitosan (310 °C). The lower thermal stability could be due to the porphyrin-chitosan arrangement: the macrocyclic aggregates are inserted between chitosan macromolecules that weaken the interconnection of the polysaccharide chains. As a result, the chitosan part of the complexes is more susceptible to thermal degradation. Complex 2 contains a high amount of TPPS4, thus, causing more pronounced disruption in the chitosan intermolecular bonding, so this complex is thermally less stable. DSC curves of the complexes are different in the region of chitosan degradation. Complex 1 has a broad exothermic peak at 262 °C corresponding to that of pure chitosan at 310 °C. Similarly to free chitosan, this peak indicates cross-linking reactions between the products of -NH2 group pyrolysis.60,61 In contrast, complex 2 has no peak at 250-350 °C, but an intense and sharp exo/endo couplet at 199 and 212 °C. A similar couplet of complex 1 is very weak and shifted to 238-240 °C. To aid in the assignment of structural changes during the thermal decomposition, FTIR spectra of the TPPS4-chitosan complexes (Figure 3D) were measured before and after heating to 200 and 250 °C. A significant decrease of broad chitosan bands was observed in the spectra of heated samples, while the intense and narrower porphyrin bands showed no evident changes. An exception was that two sharp bands of complex 2 in the region of 400-420 cm-1 vanished after heating. These bands are characteristic for J-aggregated TPPS4 macrocycles,8,50 and their decrease confirms dissociation of J-aggregates. Furthermore, heating of complex 2 to 200 °C was accompanied by the rise of a new band at 1694 cm-1, which significantly declined

to a weak shoulder after heating to 250 °C. This band, assigned to CdO stretching vibrations, indicated the presence of carbonyl intermediates. Comparing the results of FTIR and thermal analysis, we assume that the exo/endo DSC couplet indicates ionic crosslinkage between chitosan and porphyrin parts and arises from two-step pyrolysis of the complexes (Scheme 2): (a) formation of sulfonamide linkages SO2NH instead of salt bridges SO3-NH3+ (exothermic peak) and (b) pericyclic cis-elimination of the porphyrin-sulfonamide adduct (endothermic peak). These reactions are similar to those observed for thermal modification of chitosan films62 and thermal degradation of chitin61,63,64 and chitosan.65 DSC thermograms with an exo/endo couplet have been reported for chitosan cross-linked with various amounts of sodium polytriphosphate.66 In this case, heating led to elimination of a water molecule and formation of a covalent phosphamide bond. Thus, the ionic cross-linking is substituted by a covalent one that is similar to cross-linking reactions observed in chitosan pyrolysis.65 In all three cases (TPPS4chitosan, chitosan-polytriphosphate, and chitosan itself), the exothermic process corresponds to the formation of new covalent bonds between macromolecules. Further elimination of porphyrin-sulfonamide at higher temperatures is, in turn, similar to the endothermic elimination of acetamide during thermal degradation of chitin.63 The exo/endo couplet is weak for complex 1 and strong for complex 2 confirming the prevalence of ionic binding in the latter case. The detection of carbonyl intermediates by FTIR confirms the subsequent degradation of sugar units via water elimination (Figure 2D, Scheme 2). Micro- and Nanostructure of the TPPS4-Chitosan Complexes. Figure 5 shows LM and SEM images of the TPPS4-chitosan complexes. Complex 1 displays wreathed thread-like microstructures (Figure 5A). According to the image scale, the average diameter of a single thread is ∼5 µm. In contrast, complex 2 forms crumpled layer microstructures (Figure 5B), with layers of thickness ∼3 µm. The SEM images of the complexes (Figure 5C,D) revealed a smoother surface structure for complex 2 than for complex 1. Both complexes consist of variable sized roundish nanostructures, which are more pronounced for complex 1. It is difficult to estimate the thickness and width of these nanostructures owing to their high overlapping. Their diameter was ∼50-70 nm (1) and ∼10-30 nm (2; Table 2). These nanostructures are combined into larger clusters, which are more distinct for complex 2. Tapping mode AFM images (Figure 6) reveal information about the surface topography of the TPPS4-chitosan complexes. Like SEM analysis,



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Scheme 3. Self-Assembling and Structural Hierarchy of Solid TPPS4-Chitosan Complexes 1 (Left) and 2 (Right)a

a Primary aggregates (A), expanded aggregates (B), nanostructures (C), and microstructures (D).

Figure 4. TG (A), DTG (B), and DSC (C) thermograms of TPPS4-chitosan complexes 1 (solid) and 2 (dash). (D) Changes in FT-IR spectra of TPPS4-chitosan complexes 1 and 2 (solid) after heating to 200 (dashes) and 250 °C (dots). Table 2. Nanostructural Parameters of TPPS4-Chitosan Complexes 1 and 2 According to SEM and AFM SEM

1 2

AFM

diameter (nm)

diameter (nm)

height (nm)

roughness (nm)

∼50-70 ∼10-30

∼60-100 ∼20-50

∼20-50 ∼3-8

∼10.5-21.5 ∼7.5-9.0

this method confirmed that complex 2 has a smoother structure than complex 1. The roughness Ra measured at several 2 × 2 µm sites of the samples varied in the range of 10.5-21.5 and 7.5-9.0 nm, respectively, for 1 and 2 (Table 2). According to AFM 3D and topographic images (Figure 6A,B), the nanostructures of complex 1 are larger than those of complex 2. The

Figure 5. LM (A, B) and SEM (C, D) micrographs of TPPS4-chitosan complexes 1 (left panels) and 2 (right panels).

typical profiles of the AFM topographic images illustrate the difference in their arrangements (Figure 6C). The profile of complex 1 consists of a number of ∼50-100 nm wide peaks, which are single in general or joined into very small groups of

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Figure 6. Nanostructure of TPPS4-chitosan complexes 1 (left panel) and 2 (right panel) according to AFM: 3D (A) and topographic (B) images, their profiles (C), and the arrangement of nanoparticles (D).

2-3 peaks; the height of these peaks (20-50 nm) is comparable with their width. The profile of complex 2 includes several large clusters of smaller high peaks of ∼3-8 nm height. Thus, the nanostructures of complex 1 are rather globular, while those of complex 2 seem to be something flattened. According to the AFM 3D images (Figure 6D), the globules are grouped into a chain network in the former complex, while in the latter complex the globules are joined into bunch-like clusters. The values of the linear size of nanostructures obtained by AFM are comparable with, but somewhat higher than, those obtained by SEM. This probably results from site variability. Unfortunately, the nanostructures of both complexes overlay each other rendering the estimation of their size complicated. Self-Assembling Architecture of TPPS4-Chitosan Complexes. It is evident from the results mentioned above that precipitates 1 and 2 are TPPS4-chitosan complexes in which porphyrin macrocycles are arrayed onto the polysaccharide scaffold. As was noted earlier for TPPS4-chitosan interaction in solution23 and TPPS4-protein coprecipitation,4 in this case the biopolymer possesses net positive charge for electrostatic binding to anionic sulfonato groups of the porphyrin. The mode of macrocycle arrangement, however, depends on conditions in the medium (pH and ionic strength) owing to the equilibria

between dicationic and free base cores as well as between ionized and protonated functions, namely, sulfonato groups in TPPS4 and free amino groups in chitosan.2,23 Possible structures of the TPPS4-chitosan complexes forming in aqueous solutions of pH 2.5-6.8 have been proposed earlier.23 At first approximation these models are apt to the basic structure of solid coprecipitates (Scheme 3). Under neutral conditions, the macrocyclic core of TPPS4 (free base) is nearly planar and four phenyl rings are near orthogonal to the core.67 This conformation supports face-to-face stacking of porphyrin macrocycles in complex 1. To minimize the repulsion between anionic sulfonato groups, each macrocycle is rotated by 45° in relation to the neighbors. According to the proposed model (Scheme 3A, left), up to eight sulfonato groups are situated at the periphery of the stacked cores and the same number of chitosan chains is attached to them. Under neutral conditions, the amino groups of chitosan are in free base form, so macrocyclic arrays are stabilized mainly by polar interaction of the sulfonato groups of TPPS4 with amine and hydroxyl groups of the polysaccharide. Water molecules and sodium counter cations may be involved in such interactions. At pH 2.5, the core of TPPS4 is saddle-distorted (D2d symmetry) due to protonation: pyrrole rings are tilted up and down alternatively with respect to the porphyrin mean plane, pairs of


nitrogen atoms are extended outward, and phenyl rings are near parallel.68,69 Stacking of macrocycles is complicated in this case, but positively charged core nitrogens are able to interact with anionic sulfonato groups of neighbor macrocycles. This electrostatic interaction stabilizes a tilted J-aggregation of TPPS4 macrocycles. Chitosan macromolecules support this process by formation of salt bridges between free sulfonato and amino groups. The “spread desk of cards” arrangement, in which two chitosan molecules are situated along the row of macrocycles, is the most probable for J-aggregation of protonated TPPS4,70,71 and it is acceptable for complex 2 (Scheme 3A, right). In the models proposed for complexes 1 and 2, chitosan molecules bound to porphyrin have charged (NH3+) and polar (NH2, OH) functional groups at the side opposite to an array of aggregated macrocycles. Under the conditions of our experiments, the local concentration of TPPS4 is extremely high and free macrocycles are able to attach to these groups of chitosan. In addition, macrocyclic arrays can directly bind to each other. In both cases, secondary aggregates are forming (Scheme 3B). The growth of primary and secondary aggregates, however, is restricted by the presence of N-acetyl groups in the chitosan scaffold. If several aggregation sites are situated along the same polysaccharide chains, the spaces between them will subsequently fill because of the involvement of free polysaccharide chains in the complexation. As a result, the TPPS4-chitosan aggregates expand until they reach each other forming larger structures visible in SEM and AFM images (Figures 5 and 6 and Scheme 3C). The size of these structures varied significantly for both complexes depending on local conditions (pH, ionic strength, porphyrin concentration, length and N-acetylation of chitosan macromolecules, hindrance by neighboring aggregates, etc.); their arrangement depends on the topology of the selfassembling process, the mechanism of the aggregate expansion, and the nature of the interconnection between aggregates. According to our model of complex 1, the expansion of H-aggregates leads to interlacement of cylinder-like structures in which chitosan macromolecules are situated along the axes of macrocycle arrays. Polysaccharide chains may connect neighboring agglomerates of such structures into a branched network (Scheme 3C, left). In contrast, complex 2 consists of expanded J-aggregates, in which chitosan macromolecules are located on the sides of the macrocycle arrays. In this case, neighboring aggregates can stratify with each other forming bunch-like clusters (Scheme 3C, right). Observed mesoscopic structures of both types, in turn, determine precipitation of larger micro- and macrostructures, fibers (complex 1) or layers (complex 2), which are visible by the unaided eye or light microscope (Scheme 3D). It is interesting to compare the structural inferences of TPPS4-chitosan complexes of this study with earlier reported solid cis-bis(4-sulfonatophenyl)diphenylporphyrin (TPPS2op) clustering on a glass surface.72 Disk or short-stripe shape nanostructures have been observed in these clusters, while single nanostructures have not been found. In contrast, on solid surfaces TPPS4 forms separated rod-like nanostructures, which are flattened or submerged depending on the nature of the surface.71,72 These nanostructures have been defined as hollow tubes consisting of stacked rings of J-aggregated macrocycles. Stacking of such rings is possible at low pH due to polar interaction between free protonated sulfonato groups, which are absent in similar J-aggregates of TPPS2op. In our experiments, the formation of stacked rings was probably inhibited by the association of these groups with chitosan. Polysaccharide chains are flexible and, according to our model, are able to connect


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neighboring TPPS4 aggregates, but they are too long (mean length of ∼2.42 µm) to act as a scaffold for assembling closed rings. Another factor supporting the formation of rod-like porphyrin aggregates in solution is low ionic strength.9,10 As a cationic polyelectrolyte, chitosan contributes to the ionic strength of its concentrated aqueous solutions. Thus, we suggest that the growth of clusters similar to those supported by ionic strength9,11,12 is more probable in TPPS4-chitosan solutions and could cause precipitation of the solid complexes studied in our investigation.

Conclusions Two solid TPPS4-chitosan complexes were prepared by precipitation under different pH conditions. Analysis of the products obtained confirms the following: (a) both precipitates contain TPPS4 and chitosan; (b) complex 1 prepared at pH 2.5 contains less porphyrin and more polysaccharide than complex 2 prepared at pH 6.8; (c) the complexes contain porphyrin macrocycles in self-aggregation states, that is, H-type (face-toface stacking) aggregates for 1 and J-type (edge-to-edge tilted) aggregates for 2, and both these forms are structurally different from the free TPPS4 at appropriate pH. In these complexes chitosan macromolecules play a role as scaffold for the formation of TPPS4 aggregates, self-assembling of primary aggregates into nanostructures, and integration of the nanostructures into mesoscopic clusters and larger macrostructures. Evident structural difference between complexes 1 and 2 is a result of diverse modes of porphyrin binding onto chitosan macromolecules under neutral and acidic conditions. The pH conditions define the preferred TPPS4 aggregation (H- or J-types), of aggregate expansion and of interconnection between nanostructures as well as the topology of mesoscopic clusters and macrostructures (fibers or layers). Solid TPPS4-chitosan complexes are interesting for the preparation of new materials for electronics and photodynamic therapy. Acknowledgment. We are grateful to the Academy of Science, the Czech Republic (KAN 400480701, KAN200100801, KAN200200651, and AVOZ 40500505), the Ministry of Education of the Czech Republic (research program No. LC 06041; projectsNo.CEZ:MSM6046137305andCEZ:MSM6046137307), and the Grant Agency of the Czech Republic (203/02/0420 and 525/05/0273) for financial support. We also thank Dr. Martin Marysˇka (Department of Glass and Ceramics, ICT Prague) for measuring of the SEM images. Supporting Information Available. Assignment of UV-vis, NIR, IR, and Raman bands and 13C NMR chemical shifts for the TPPS4-chitosan complexes and the pure compounds (Tables 3-7). This material is available free of charge via the Internet at http://pubs.acs.org.

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pH-Controlled Self-Assembling of meso-Tetrakis(4-sulfonatophenyl

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