Fluorescent Bioactive Corrole Grafted-Chitosan Films - ACS Publications

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Fluorescent bioactive corrole grafted-chitosan films Joana F. B. Barata, Ricardo J. B. Pinto, Vanda Isabel Roldão Canelas Vaz Serra, Armando Jorge Domingues Silvestre, Tito Trindade, Maria Graça P. M. S. Neves, José A. S. Cavaleiro, Sara Daina, Patrizia Sadocco, and Carmen Sofia Rocha Freire Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00006 • Publication Date (Web): 20 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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Multifunctional chitosan films were prepared by chemical modification of chitosan with a corrole macrocycle, viz 5,10,15-tris(pentafluorophenyl)corrole (TPFC), followed by solvent casting. 35x14mm (600 x 600 DPI)

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Fluorescent bioactive corrole grafted-chitosan films Joana F. B. Barata,1,3* Ricardo J. B. Pinto,1 Vanda I. R. C. Vaz Serra,2,3 Armando J. D. Silvestre,1 Tito Trindade,1 Maria Graça P. M. S. Neves,3 José A. S. Cavaleiro,3 Sara Daina,4 Patrizia Sadocco4 and Carmen S. R. Freire1*

1

Department of Chemistry-CICECO, University of Aveiro, 3810-193 Aveiro, Portugal

2

Centro Química Estrutural, Complexo I, Instituto Superior Técnico, 1049-001 Lisboa,

Portugal 3

4

Department of Chemistry-QOPNA, University of Aveiro, 3810-193 Aveiro, Portugal INNOVHUB – Divisione Carta, Piazza Leonardo Da Vinci, 16, 20133 Milan, Italy.

KEYWORDS Chitosan; corroles; multifunctional films; fluorescence; antimicrobial activity; transparency.


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ABSTRACT

Transparent corrole grafted chitosan films were prepared by chemical modification of chitosan with a corrole macrocycle, viz 5,10,15-tris(pentafluorophenyl)corrole (TPFC), followed by solvent casting. The obtained films were characterized in terms of absorption spectra (UV-Vis), FLIM (Fluorescence Lifetime Imaging Microscopy), structure (FTIR, XPS), thermal stability (TGA), thermo-mechanical properties (DMA) and antibacterial activity. The results showed that the chemical grafting of chitosan with corrole units did not affect its film-forming ability and that the grafting yield increased with the reaction time. The obtained transparent films presented fluorescence which increases with the amount of grafted corrole units. Additionally, all films showed bacteriostatic effect against S. aureus, as well as good thermo-mechanical properties and thermal stability. Considering these features, promising applications may be envisaged for these corrole-chitosan films, such as biosensors, bioimaging agents and bioactive optical devices.


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INTRODUCTION In the last decade, rapid and increasing advances in the design and fabrication of functional bio-based materials afforded new solutions for biomedicine and high-tech applications. In particular, extensive research has been carried out on the development of cutting-edge materials through the use of biopolymers, like polysaccharides and proteins, due to their abundance, biodegradability, biocompatibility and specific properties.1-4 Chitosan (CH), the deacetylated derivative of chitin, is one of the most studied biopolymers due to its intrinsic antimicrobial properties, biocompatibility and film-forming ability.5 Chitosan has attracted scientific and industrial interest in several areas as biotechnology, pharmaceutics, food science, biomedicine, among others. The chemical modification of CH or of blends of CH with specific (bio)molecules appears as a strategy to introduce distinct functionalities and properties (luminescence, conductivity, mechanical performance, among many others) and exploit additional areas of application.6,7 For example, tetrapyrrolic macrocycles, like porphyrins and phthalocyanines which have unique recognized properties,8 have been combined with chitosan,to prepare original bio-based functional materials e.g. as photosensitizers delivery systems for photodynamic therapy,9 as wound dressing materials,10 as contrast agents for magnetic resonance imaging,11 as photomicrobiocide films for water disinfection against E.coli,12 as bioinspired enhanced catalytic systems for hydroxylation of cyclohexane13 and as amphiphilic nanocarriers for photochemical internalization.14 Corroles are aromatic tetrapyrrolic macrocycles belonging to the porphyrinoid family, with distinctive structural properties, conferred by their low symmetry attributed to the direct β,β-pyrrole connection. Corroles possess interesting photophysical properties such as high radiative rate constants, large Stokes shift, absorption and emission bands


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in the visible region and high luminescent quantum yields.1516 The chemistry of corroles has gained great relevance,17 after the development in 1999, of simple and efficient procedures for the synthesis of meso-substituted corroles.18,19 This breakthrough combined with extensive studies on their chemical properties, endorsed corroles to be explored in several applications such as sensors,202122 contrast agents,23 dye sensitized solar cells,24 as catalysts25,26 or photosensitizers in photodynamic therapy.27,28 However, the combination of corroles with (bio)polymeric materials is an almost unexplored area. To the best of our knowledge, only one study was recently described by our group20 where corroles were embedded in a polymethylmethacrylate (PMMA)/ polyacrylamide gel aiming to prepare sensitive solid supported sensors to cyanide and fluoride anions. In the present report, we describe for the first time, the preparation and characterization of corrole grafted-chitosan films with emissive features and bacteriostatic effect, as well as good mechanical performance and chemical stability.

MATERIALS AND METHODS

2.1 Chemicals Ethanol (Panreac, 99.8%), dichloromethane (CH2Cl2, 99.5%, Panreac), acetic acid (Aldrich, 99.8%), trifluoroacetic acid (TFA, 99%, reagent plus), sodium hydroxide (granulates, 97%), pentafluorobenzaldehyde (C6F5CHO, 95%, Sigma-Aldrich), 2.3dichloro-5,6-dicyano-p-benzoquinone (DDQ, 98%, Sigma-Aldrich), deuterated water (D2O, Acros Organics) and deuterated acetic acid (CD3COOD, Aldrich) were used as received. Pyrrole (C4H4NH, 95%, Sigma-Aldrich) was distilled before use and dimethyl sulfoxide (DMSO, Fluka, 99.98%) was dried using standard procedures.


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2.2. Materials The chitosan (CH) used in this work was kindly provided by Mahtani Chitosan Pvt. Ltd. (India). This commercial sample was purified by dissolution in a 1% (v/v) aqueous acetic acid solution, filtered and precipitated by neutralizing with NaOH 0.5 M up to a pH of 8.5. The ensuing precipitate was washed with distilled water until neutral pH and air dried. Its viscosity-average molar mass, obtained at 25 ºC from a 0.3 M CH3CO2H/ 0.2 M CH3CO2Na solution, using the published Mark–Houwink constants,29 was 350000 g mol-1. Its degree of deacetylation (DDA), determined by 1H NMR, (in D2O containing 1% of CD3COOD) using a DRX-300 Brüker spectrometer, was found to be 97%.30

2.3. Preparation of 5,10,15-tris(pentafluorophenyl)corrole (TPFC) 5,10,15-tris(pentafluorophenyl)corrole (TPFC) was synthesized according to the literature.31

Briefly,

this

macrocycle

was

synthesized

by

condensation

of

pentafluorobenzaldehyde and pyrrole under acid conditions followed by oxidation with 2,3-dicloro-4,5-dicyanobenzoquinone (DDQ). TPFC was obtained after purification by column chromatography with 20% yield. The analytical data is in accordance with the literature.31

2.4. Preparation of corrole grafted-chitosan derivatives Typically, for each synthesis, 100 mg of chitosan powder was suspended on a Schlenk tube and 15 mL of dried DMSO were added under N2 atmosphere and then the mixture was stirred for 24 h. Afterwards, 3.5 mg of TPFC were added to the tube and the


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mixture was heated at 80 ºC over distinct times (6, 24 and 48h) in order to promote the grafting of corrole units into chitosan backbone. The resulting conjugates were washed several times with dichloromethane and the resulting suspensions centrifuged, at 6000 rpm for 10 min, in order to remove the unbound corrole units. The centrifugation cycles were repeated until the washing solutions did not show the typical bands of corrole in the UV-Vis spectra. The ultimate washing cycle was made with ethanol. Finally, the grafted chitosan samples were dried in an oven at 40 ºC overnight. The identification of the obtained grafted chitosan samples (CH/TPFC6h, CH/TPFC24h and CH/TPFC48h) is displayed in Table 1.

2.5. Preparation of corrole grafted-chitosan films Solutions (1% w/v) of the grafted chitosan derivatives, CH/TPFC6h, CH/TPFC24h and CH/TPFC48h (and also of unmodified chitosan for comparison), were prepared by dissolving 20 mg of grafted chitosan in 2mL of aqueous acetic acid (1% v/v). The mixtures were homogenized by mechanical stirring for 30 minutes and then degassed to remove the entrapped air. The cast films of corrole grafted-chitosan derivatives were prepared by placing the solutions in acrylic plate molds (5 x 5 cm). The plate molds were maintained in a ventilated oven set, at 40 ºC overnight. The obtained thin films were then removed from the molds and kept in a desiccator containing P2O5. In addition, for comparative proposes a set of films containing non-grafted corrole were prepared by adding different amounts of corrole (10 to 500 µg) to the 1% unmodified chitosan solution before the casting step in order to determine the grafting yields.

2.8. Characterization of the corrole grafted-chitosan films


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All the obtained films were characterized in terms of transparency, chemical structure, thermal stability, thermomechanical performance, fluorescence and antimicrobial activity. The thickness of the films was determined using a digimatic micrometer (model MDE-25TJ, Mitutoyo Corp., Tokyo, Japan). Mean thicknesses were calculated from 10 measurements taken at different locations in each film. The optical spectra of the films were recorded using a Jasco V-560 UV–Vis spectrophotometer; for solid samples the spectra were recorded in the diffuse reflectance mode using MgO as reference. FTIR-ATR spectra were collected using a Perkin Elmer FT-IR System Spectrum BX spectrometer equipped with a single horizontal Golden Gate ATR cell. Each spectrum was an average of 32 scans taken with 4 cm−1 resolution in the 500–4000 cm−1 range. The XPS analysis was performed using a Kratos AXIS Ultra HSA, with VISION software for data acquisition and CASAXPS software for data analysis. The analysis was carried out with a monochromatic Al Kα X-ray source (1486.7 eV), operating at 15kV (90 W), in FAT mode (Fixed Analyser Transmission), with a pass energy of 40 eV for regions ROI and 80 eV for survey. Data acquisition was performed with a pressure lower than 1 x 10E-6 Pa, and a charge neutralisation system was used. The deconvolution of spectra was performed using the CasaXPS program, in which an adjustment of the peaks was performed using peak fitting with Gaussian-Lorentzian peak shape and Shirley type background subtraction. Thermal decomposition temperatures were determined by thermogravimetric analyses (TGA) on a Shimadzu TGA-50 analyzer equipped with a platinum cell. The thermograms were run under nitrogen atmosphere at constant heating rate of 10 ºC.min−1 from room temperature to 800 ºC.


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Dynamic mechanical analyses (DMA) were performed on a Tritec 2000 DMA (Triton Technologies) equipment operating in the tension mode. For the temperature sweeps, a ramp rate of 2 °C/min was used and samples were heated from -60 to 175 ºC, at a frequency of 1 Hz, with a displacement of 0.04 mm. Films average dimensions were approximately 5 x 5 x 0.02 mm. Tg values were determined using the maximum of the tan δ curve. Fluorescence-lifetime imaging microscopy (FLIM) was performed using a Microtime 200 from Picoquant GmbH and a more detailed description may be found elsewhere.32 Briefly, the 638 nm excitation light from a pulsed diode laser was focused by an oil immersion objective 100 with 1.3 NA, into the sample. Fluorescence was collected by the same microscope objective, passed through the dichroic mirror and an appropriate band-pass filter (695AF55 Omega optical) focused through a pinhole (30 µm), to reject out-of-focus light, onto a single-photon counting avalanche photodiode SPAD (Perkin-Elmer) whose signal was processed by the TimeHarp 200 TC-SPC PCboard (PicoQuant) working in the special Time-Tagged Time-Resolved Mode, which stores all relevant information for every detected photon. Fluorescence lifetimes were obtained in the same equipment with a time-correlated single-photon counting (TCSPC) technique. The goodness of the fit was evaluated by the usual statistical criteria, by visual inspection of the weighted residual distribution and the autocorrelation function. Several images were obtained from different regions of the quartz slide in order to achieve properly distributions of the corrole molecules within the films. For each one of the collected images several point-by-point emission decays were measured and further analysed.


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The microbial strain used in this work to assess the antibacterial activity of the films was provided by DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures). Staphylococcus aureus ATCC 6538 (DSM799) were maintained frozen (−80 ºC) and transferred monthly on TSA (Tryptone Soya Agar) made of 15 g/L tryptone, 5 g/L soya peptone, 5 g/L NaCl, and 15 g/L neutralized bacteriological agar. All bacterial pre-inoculum cultures were grown overnight at 37 ºC in 20 mL of nutrient broth (made of 1 g/L beef extract; 5 g/L neutralized peptone; 2 g/L yeast extract; 5 g/L NaCl) subjected to horizontal shaking at 100 rpm. The film samples were placed in contact with a microbial liquid suspension, subjected to vigorous shaking in order to assure the best contact between bacteria and sample. At 0 h and 24 h contact times, the bacterial concentration (CFU/mL) of the microbial suspension was determined by plating serial dilution on Plate Count Agar to obtain the overall number of bacteria (CFU—Colony Forming Units). For the antibacterial tests the specific conditions were as follows: Microbial liquid suspension: 5 mL of 5% nutrient broth in phosphate buffer (0.3 mM, pH 7.2) inoculated with 10−4–10−5 CFU/mL bacteria; total flask volume: 25 mL; sample incubation: 24 hours at 37 ºC under vigorous shaking. It was used a solar lamp GE ARC70/UVC/730 - 6000 lux and the assay was carried in a period of 4 hours. The quantity of tested material was 10 mg of sample. The film samples were cut in small piece and tested in duplicate; Control samples: chitosan pristine film and corrole (TPFC) powder were tested as blank references, while as internal reference of the method, the bacteria growth was tested on flasks only containing inoculated broth media. All the samples were subjected to sterilization by autoclave.


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The bacteria log reduction of the samples was calculated as follows: log reduction = log (CFU T24 control sample) − log (CFU T24 film). As mentioned in the standard dynamic shake flask method, at least a 1 log reduction of bacteria load is required to claim antibacterial property.

RESULTS AND DISCUSSION The grafting of corrole units into chitosan was performed in DMSO, at 80 ºC, for different reaction times (6, 24 and 48h), yielding chitosan-corrole conjugates, namely CH/TPFC6h, CH/TPFC24h and CH/TPFC48h, with distinct degrees of substitution. The formation of the covalent linkage between chitosan and corrole units, involves the nucleophilic

substitution

of

a

para-fluorine

atom

from

the

5,10,15-

tris(pentafluorophenyl)corrole (TPFC)27,28,33 by amino groups of chitosan (Scheme 1).


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Scheme 1 - Schematic representation of the chemical grafting of CH with TPFC.

All modified chitosan samples (CH/TPFC) displayed a green color, typical of TPFC34 (Figure 1) suggesting that the corrole moieties were successfully grafted to the chitosan chains, as the samples were intensely washed with different solvents to assure the total removal of the unbound corrole molecules. The green tone of the chitosan derivatives increased with the reaction time which suggests an increment of the grafting yield with time. In order to verify the effect of the grafting reaction on the filmogenic properties of chitosan, the obtained CH/TPFC derivatives were dissolved in a 1% acetic acid


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solution and casted in a ventilated oven. All grafted chitosan samples were filmogenic and the resulting films also display a greenish colour, as well as the intrinsic transparency of pristine chitosan films. CH/TPFC films were also prepared by direct mixing of unmodified chitosan and established amounts of TPFC (10, 25, 50, 75, 100 and 500 µg) before casting, in order to determine the grafting yield of the corrole in grafted films (as will be discussed below) (Table S1). The corresponding direct mixing casted films presented a final green colour, identical to the covalently linked corrole-chitosan films where the ones with higher TPFC contents presented a stronger colour intensity (Figure S1).

Figure 1. Digital photographs of a) chitosan and CH/TPFC derivatives for different reaction times: b) 6, c) 24, and d) 48 h, and corresponding films obtained by casting.

The amount of TPFC incorporated in the grafted chitosan films was, then, determined using a calibration curve made by plotting the absorbance maximum of the Soret band in function of the amount of TPFC (see absorption spectroscopy discussion), added in


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the films prepared by simple mixing of chitosan and TPFC (Figure S2b). Based on that curve, it was determined that the corrole contents were 0.461, 0.711 and 1.30 µg of corrole/mg of chitosan for CH/TPFC6h, CH/TPFC24h and CH/TPFC48h, respectively.

Table 1 summarizes the basic parameters of TPFC grafted chitosan films prepared in this study. The thicknesses of the films weights are about the same order of magnitude for all samples.

Table 1. Identification and characterization of corrole grafted chitosan films prepared in this work. Thickness errors correspond to standard deviations (SD, n = 10).

Sample

CH

Reaction

Thickness

Film

TPFC

TPFC content on

(mg)

time (h)

(µ µm)

weight

content (µ µg)

the film (µ µg/mg)

(mg) CH

20

-

15.6 ± 2.0

22.4

-

CH/TPFC6h

20

6

12.9 ± 1.4

22.6

10.4*

0.461

CH/TPFC24h

20

24

13.1 ± 1.2

22.1

15.7*

0.711

CH/TPFC48h

20

48

13.3 ± 0.9

18.7

24.3*

1.30

* The TPFC content grafted was calculated using the equation obtained on the Figure S2b.

Optical properties The optical properties of the grafted CH/TPFC films were assessed by UV-Vis absorption spectroscopy. The absorption spectra of the films showed two absorption bands in the visible region, around 426 and 609 nm (Figure 2, and Figure S2a), following the characteristic trend observed for corroles in polar solvents.28,35


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However, in these spectra those bands appeared red-shifted and this is probably due to molecular environment effects or due to the chemical attachment of the corrole moieties to the chitosan polymeric backbone.

Figure 2. UV-Vis spectra of chitosan and chemical grafted CH/TPFC films.

In Figure 2 an enhancement of the absorption bands intensity for higher reaction times is also perceived. This absorption increase confirmed that longer reaction times promote a higher degree of grafting, as previously suggested by the visual aspect (color intensity) of the films. For the samples prepared by direct mixture an increase of the corresponding absorption bands was also observed (Figure S2a), in this last case related with the increasing amount of corrole added before casting.

Fluorescence lifetime microscopy FLIM was used to study in detail the emissive properties of the corrole grafted chitosan films and to recognize the corrole moieties spatial distribution in the bulk of the films. The FLIM images shown in Figure 3 were obtained in different regions of each one of the grafted chitosan films (24 and 48 hours


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of reaction) in order to properly achieve corrole distributions. FLIM images of nongrafted chitosan films were also obtained. As expected, non-modified chitosan films are non-emissive under the experimental conditions used to confirm corrole incorporation (Figures 3 A-D). The emission shown by the colored points of the map code of modified chitosan films (Figures 3E-L) can only be due to the fluorescence of TPFC. In DMSO solution, the fluorescence decay of TPFC is monoexponential with a fluorescence lifetime of 3.7 ns.

Figure 3. Fluorescence lifetime images obtained for different regions of: (A-D) Non modified chitosan film; (E-H) CH/TPFC24h film and (I-L) CH/TPFC48h film.

The FLIM images obtained after 24 hours reaction, foresee a highly homogeneous corrole grafted chitosan film as revealed by the regularity of the intensity of the images obtained (Figure 3 E-H). Nevertheless, the histograms for the


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fluorescence lifetimes related with the images show a broader distribution (Figure 3M) which is probably due to different orientations and environments sensed by the fluorophore. After 48 hours of reaction, a slight increase in the fluorescence intensity with the adsorption time occurs as a result of a higher concentration of grafted corrole units, in accordance with the previously described absorption measurements. Also, the appearance of several brighter green spots, with only a few micrometers and spread to, is observed with the increase of the reaction time. The fluorescence lifetime histograms of these are narrowed and shifted to the shorter fluorescence lifetimes (1.0 ns) than the previous (Figure S3). It is possible that a higher functionalization of adjacent amino groups of chitosan with corrole will be able to promote π-π stacking between nearby fluorophores, leading to a decrease in fluorescence lifetimes in accordance with the exciton theory.36 Nevertheless, some other mechanism such as electron or charge transfer processes could be involved in fluorescence lifetime decrease. It is noticed that the fluorescence intensity of the films can be easily increased by directly mixing unmodified chitosan and higher amounts of corrole (500 µg) before casting (Figure S4). In this case, a fairly homogeneous spatial corrole distribution is obtained with narrow fluorescence lifetimes histograms (centered at 1.0 ns) quite comparable with the ones obtained for the green spots of CH/TPFC48h in Figure 3.

Structural analysis The FTIR spectra of the grafted CH/TPFC films (Figure S5) were very similar to that of the unmodified chitosan matrix37 corroborating the low contents of grafted corrole as determined by UV-Vis. In fact, the typical corrole bands, at 980 and 922 cm-1,38 were only noticeable in the spectra of the CH/TPFC films prepared by simple mixing, where higher amounts of corrole were incorporated (Figure S6).


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In order to ascertain the covalent linkage of the new materials, XPS analysis, including high resolution analysis of C1s, N1s and F1s, was performed for CH/TPFC48h (grafted film) and CH/TPFC100 (non-grafted film) with 100 µg of TPFC. The XPS high resolution spectra of C1s region of the pristine chitosan (Figure 4a), as expected, showed essentially three components: at 285.0 eV, typical of C-C and C-H carbon atoms, at 286.7 eV associated with C-N and C-O bonds and at 288.3 eV typical of carbon atoms involved in acetal and amide linkages [O–C-O, N–C=O].39 This chitosan signature was also observed in both CH/TPFC100 and CH/TPFC48h (mixed and grafted CH/corrole films, respectively, Figure 4b and c) along with a four component C1s peak at 289.1 eV (see Table S2) typical of the C–F linkage of the pentafluorophenyl rings of the corrole macrocycle,34 reinforcing the presence of corrole in these films. However the C-N covalent bonding of corrole macrocycle to chitosan cannot be unambiguously confirmed since it is expected to appear at the same energy (286.7 eV)34 as that observed for the C-N of corrole macrocycle and the C-N chitosan moiety. Additionally, the low grafting yields obtained turn their detection even more difficult .The higher intensity of the peak at 285.0 eV, typical of C-C and C-H carbon atoms, in the spectra of the unmodified chitosan films and corrole-chitosan films prepared by simple mixing is normally attributed to intrinsic impurities (fatty acids, sterols, etc) present in the chitosan samples that migrate to the films surface during drying; and that were in part removed during the grafting/washing process.40 The N1s peak of pristine chitosan (Figure 4d) was deconvoluted into two peaks. The first peak at 399.6 eV was attributed to non-protonated amine or amide and the second peak at 401.7 eV was assigned to the protonated amine nitrogen atoms.40 Notice that two N1s components are also expected for corrole macrocycles: one related to the pyrrolic aminic type (400.5) and the other to the pyrrolic iminic type (398.3).34 Having


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this in mind, the binding energies of CH/TPFC100 (Figure 4e) and CH/TPFC48h (Figure 4f) were assigned to the expected chitosan peaks and to the pyrrolic nitrogen atoms typical of the corrole moieties. In fact, the N1s peaks for CH/TPFC100 (Figure 4e) and CH/TPFC48h are different from those of pure chitosan, indicating that the contribution of the pyrrolic iminic nitrogens is probably overlapped with the peak of the non-protonated amine nitrogen atoms of chitosan, at 399.5eV. Regarding the F1s peak, for the corrole chitosan films only one contribution was obtained concerning the C-F bonds of the pentafluorophenyl groups of the corrole macrocycle.


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Figure 4. Fitted high-resolution photoemission spectra of C1s (left column) and N1s (right column) of: a,d) chitosan film, b,e) CH/TPFC100, and c,f) CH/TPFC48h films.

Thermal stability The effect of the chitosan grafting with corrole moieties on the thermal degradation profile and stability of chitosan based films was evaluated by termogravimetry. The typical TGA curves of CH and CH/TPFC are presented in Figure 5. The thermogram of the pure CH film displayed two mass losses, the first between 100200 ºC related with the volatilization of water and acetic acid and the other one with a maximum degradation at 285 ºC typically assigned to the degradation of the chitosan chains.41 The grafted CH/TPFC films showed a degradation profile very similar to that of unmodified chitosan, however a small displacement of the maximum degradation temperature was observed for the films obtained with increased reaction times. This is certainly due to the presence of the corrole moieties that are more thermally stable than chitosan. In fact, in the thermogram of the CH/TPFC100 (prepared by direct mixing and with a higher content of corrole – Figure S7) this displacement is obviously perceptible (up to 12 ºC).


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Figure 5. Thermogravimetric curves of CH, TPFC and CH/corrole derivatives. The inset showed the respective -dTGA curves.

Dynamic mechanical properties The thermo-mechanical properties of the corrole grafted chitosan films were studied by DMA. Figure 6 shows the temperature dependence of the storages modulus at 1Hz of CH and CH/TPFC48h films. The curves of the storage modulus vs temperature of both CH and CH/TPFC48h exhibited two main relaxations, at 0-40 ºC and 125-155 ºC, characteristic of chitosan films.42 The first relaxation (β relaxation) is associated with the hydration of side groups on chitosan, though the transition occurring at higher temperatures (α transition) reveals the glass transition of chitosan. The storage modulus of the chitosan films slightly decreases after the chemical grafting with corrole moieties. However, this drop on the storage modulus was not very significant because of the low grafting yield. In fact, this tendency is clearly visible in the CH/corrole films prepared by direct mixing that contain higher corrole contents (Figure S8). This behavior is probably associated with the incorporation of bulky


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corrole molecules promoting a decrease on the mobility of the chitosan chains and consequently on the flexibility of the films.

Figure 6. Temperature dependency of the modulus of CH and CH/TPFC48h films.

Antibacterial activity In order to evaluate the antibacterial activity of the corrole grafted-chitosan films, selected samples have been tested towards the bacterial strain S. aureus. Figure 7 shows the results obtained for all grafted CH/TPFC samples, as well as for chitosan and corrole for comparison. In Figure S9 are also presented the results obtained for the films prepared by direct mixing.


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Figure 7. Antibacterial activity of CH, TPFC, and corrole grafted chitosan films against S. aureus. The log CFU values were determined in the testing broth after 24 h contact time and were compared to those of pure CH and TPFC matrices. Horizontal dark line refers to the initial inoculum (log CFU at time 0).

Under the tested experimental conditions, both CH and TPFC control samples presented a bacteriostatic effect (inhibition of growth) against S. aureus, since the log CFU was lower than the initial bacteria broth used in the antimicrobial experiments (Figure 7). As expected, due to its antimicrobial properties,43 CH showed a lower log CFU value (more than 2 log bacterial growth over 24 h incubation time). A more modest bacteriostatic effect was obtained with TPFC. In fact, other studies with positively- and negatively-charged antimony and phosphorus corrole derivatives already revealed a photodynamic inhibition of the growth of mold fungi44 and green algae.45

The results obtained for the corrole grafted-films (CH/TPFC6h, CH/TPFC24h, and CH/TPFC48h) also revealed a bacteriostatic effect, which increased with the increasing of corrole grafted to chitosan in the films. In this way, it is observed that the chemical grafting of chitosan with corrole moieties improved its bioactivity. These results are


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quite interesting because the TPFC control is less effective than chitosan. In fact, CH/TPFC6h presents a higher bacteriostatic effect (less 1 log on bacterial growth) when compared with the material obtained by direct mixing (CH/TPFC10) that present a similar corrole load (Figure S9). These results together with the fact that the corresponding films prepared by direct mixing showed a lower activity, are a clear evidence that the chemical grafting of chitosan with corrole enhances the tetrapyrrolic macrocycle activity. However, this behaviour must be studied in more detail in the near future.

CONCLUSIONS Transparent corrole grafted chitosan films were prepared by chemical modification of chitosan with 5,10,15-tris(pentafluorophenyl)corrole followed by solvent casting. The films obtained were macroscopically homogeneous, fluorescent, flexible and thermally stable. Corrole grafted chitosan films also demonstrated a bacteriostatic effect against S. aureus with an enhance response when compared with chitosan and corrole controls. These properties could be exploited for several applications, including as bioactive fluorescent films for bioimaging or biosensing.

ASSOCIATED CONTENT Digital photographs of chitosan/TPFC films prepared by direct mixture of the components with distinct TPFC amounts, Optical spectra of CH/TPFC composites films prepared from direct mixture and plot of maximum of absorbance of Soret band vs TPFC amount added, Lifetime histogram of representative images E and K, Fluorescence lifetime images obtained for corrol-chitosan films obtained by direct mixing, FTIR spectra of pristine chitosan and correspondent corrole grafted chitosan


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films, Thermogravimetric and respective -dTGA curves of the sample CH/TPFC100, Temperature dependency of the modulus of CH and CH/TPFC48h films in comparison with the ones prepared by direct mixture, Antibacterial activity of CH, TPFC, and chitosan/corrole derivatives against S. aureus. Identification of all CH/TPFC films prepared by direct mixing and Binding energies of C1s, O1s, N1s and F1s and bond assignments determined by XPS for CH, CH/TPFC100, CH/TPFC48h (pdf). The supporting information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] (00351 234 370064), [email protected] (00351 234 370360)

ACKNOWLEDGMENTS The authors wish to thank to FCT (Fundação para a Ciência e Tecnologia) and POPH/FSE for the postdoctoral grants to J.F.B. Barata (SFRH/BPD/63237/2009), R.J.B.

Pinto

(SFRH/BPD/89982/2012)

and

V.I.R.C.

Vaz

Serra

(SFRH/BPD/74270/2010). Carmen S.R. Freire also acknowledges FCT for the research grants under the program Investigador FCT 2012 contract number IF/01407/2012. This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (FCT Ref. UID /CTM /50011/2013), and QOPNA research Unit (FCT UID/QUI/00062/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020


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Partnership

Agreement

and

Projects

REEQ/115/QUI/2005

OE/QUI/UI0100/2013/2014.


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and

Pest-

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REFERENCES

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Fluorescent Bioactive Corrole Grafted-Chitosan Films - ACS Publications

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