Wool Keratin 3D Scaffolds with Light-Triggered Antimicrobial Activity

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Wool keratin 3D scaffolds with light-triggered antimicrobial activity Claudia Ferroni, Giovanna Sotgiu, Anna Sagnella, Greta Varchi, Andrea Guerrini, Demetra Giuri, Eleonora Polo, Viviana Teresa Orlandi, Emanuela Marras, Marzia Bruna Gariboldi, Elena Monti, and Annalisa Aluigi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00697 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Wool keratin 3D scaffolds with light-triggered antimicrobial activity Claudia Ferroni,¶,* Giovanna Sotgiu,¶ Anna Sagnella,§ Greta Varchi,¶ Andrea Guerrini,¶ Demetra Giuri, ¶ Eleonora Polo,♯ Viviana Teresa Orlandi,† Emanuela Marras,† Marzia Gariboldi,† Elena Monti,† and Annalisa Aluigi¶,* ¶

Institute of Organic Synthesis and Photoreactivity – Italian National Research Council, Via P. Gobetti 101, 40129 Bologna, Italy §

♯

MIST E-R Laboratory, via P. Gobetti 101, 40129 Bologna, Italy

Institute of Organic Synthesis and Photoreactivity – Italian National Research Council, UOS Ferrara, Via L. Borsari 46, 44121 Ferrara, Italy †

Dipartimento di Biotecnologie e Scienze della Vita (DBSV), Università degli Studi dell'Insubria, Via Dunant 3, Varese, Italy

KEYWORDS Keratin, Sponges, Antimicrobial Photodynamic Therapy, Reactive Oxygen Species, Tissue Engineering.

ACS Paragon Plus Environment

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ABSTRACT

Photo-activatable keratin sponges were prepared from protein aqueous solutions by freeze-drying method, followed by photo-functionalization with two different photosensitizers (PS): Azure A (AzA)

and

5,10,15,20-tetrakis

[4-(2-N,N,N-trimethylethylthio)-2,3,5,6-

tetrafluorophenyl]porphyrin tetraiodide salt (TTFAP). The prepared sponges have a porosity of between 49% and 80% and a mean pore size in the 37 - 80 µm range. As compared to AzA, TTFAP interacts more strongly with the sponges as demonstrated by a lower PS release (6% vs 20%), a decreased swelling ratio (1.6 vs 7.4) and a slower biodegradation rate. Nevertheless, AzA loaded sponges showed the highest photo-activity, as also demonstrated by their higher anti-bactericidal activity towards both Gram-positive and Gram-negative bacteria. The obtained results suggest that the antimicrobial photodynamic effect can be finely triggered through a proper selection of the amount and type of photosensitizer, as well as through the irradiation time. Finally, all the prepared sponges support human fibroblast cells growth, while no significant cells viability impairment is observed upon light irradiation.

INTRODUCTION The use of three-dimensional (3D) scaffolds as templates for cells growth is underpinning the tissue-engineering field. Regardless of the tissue type, scaffolds intended for tissue engineering should (a) be biocompatible, since following implantation, the scaffold must elicit a negligible immune reaction; (b) have a biodegradation rate corresponding to the rate of new tissue formation; and (c) show low toxicity of the degradation by-products and (d) suitable mechanical properties. Moreover, it should mimic the extracellular matrix architecture (ECM-like structure) of interconnected pore structure and high porosity to ensure cell penetration and diffusion of


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gases, metabolites, nutrients and molecular signals, both within the scaffold and between the scaffold and the surrounding environment.1,2 A common approach in the design of a scaffold is the use of natural-occurring materials that besides being biodegradable are also biologically active promoting excellent cell adhesion and growth. So far, various biopolymers such as collagen, proteoglycans, chitosan, fibroin, etc., have been successfully used for the production of scaffolds for tissue engineering.3-5 Among natural biopolymers of interest for biomedical applications, the non-food protein keratin is one of the most promising, due to its unique molecular structure and biological properties.6 Keratin is the major component of hair, wool, feathers, horns and nails and it can be easily recovered from a variety of waste sources, such as textile, dairy and poultry industries. In particular, wool keratin is available in large extent from those sources, if considering the amount of non-spinnable and short wool fibres deriving from textile industry and dairy processing and meat slaughtering. These wool wastes pose a growing environmental problem, because burning for fuel is inefficient (Limiting Oxygen Index, LOI, of keratin-rich materials is close to 25) and co-firing is polluting (wool contains 3-4 % wt of sulphur); therefore, possible alternative uses are attracting the interest of researchers.7,8 Wool keratin has been demonstrated to have great potential in regenerative medicine, not only for the presence of cell adhesion sequences such as RGD (arginine-glycine-aspartic acid) and LDV (leucine-aspartic acid-valine),9 but also because it improves the production of antiinflammatory cytokines and reduce the amount of pro-inflammatory cytokines, thereby promoting a positive remodeling response.10 Porous sponges and hydrogels made from keratin have been widely studied and increasing evidence from the literature supports their potential for clinically relevant applications.11,12


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In order to prevent the attachment of bacteria to an implant surface, which can result in colonization (biofilm) with consequent infections and material deterioration,13 it is crucial to confer antiseptic/antibacterial properties to the scaffold. Moreover, the growing incidence of antimicrobial resistance (AMR) has spurred a quest for novel antimicrobial treatments against which microorganisms might find it difficult to develop resistance. As an instance, antimicrobial materials have been produced using cationic polymers and/or specific functional groups (such as quaternary ammonium, quaternary pyridinium, phosphonium, etc.),14 or by loading the scaffold with silver metal cations or nanoparticles, peroxides, antibacterial peptides and other chemicals.15 However, the cytotoxicity of most of these agents (such as silver nanoparticles) hinders their clinical use. In recent years, excited state singlet oxygen (1O2) and reactive oxygen species (ROS) have been identified as potential alternatives for bacteria inactivation. 1O2 and ROS are highly cytotoxic species that are generated in situ when a photosensitizer (PS) is excited by exposure to lowpower visible light in the presence of oxygen.16 In particular, the excited PS can react with bioorganic molecules producing ROS such as superoxide, hydroxyl radicals and hydrogen peroxide (type I mechanism); whereas, an energy transfer from the excited PS to molecular oxygen results in the generation of 1O2 (type II mechanism). Recently, many studies have employed porous scaffolds functionalized with PSs in order to achieve antimicrobial photodynamic activity (APDT). The PSs were simply encapsulated into the scaffold,17,18 either electrostatically or covalently bonded to their surface.19,20 Wool keratin has been demonstrated to be a good support for PSs and for the design of photoactive and biodegradable scaffolds, since the ROS and 1O2 do not induce any photodegradation of the protein.21


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In the present study, photo-activable porous sponges made of wool keratin were prepared by freeze-drying method followed by functionalization with different amounts of two different PSs, e.g.

Azure

A

(AzA)

and

5,10,15,20-tetrakis

[4-(2-N,N,N-trimethylethylthio)-2,3,5,6-

tetrafluorophenyl]porphyrin tetraiodide (TTFAP). The photo-functionalized keratin sponges were characterized in terms of physico-chemical properties, antibacterial activity and ability to function as scaffolds for fibroblasts growth in vitro. MATERIALS AND METHODS Materials. Australian Merino wool (21 µm) was kindly supplied by Cariaggi Fine Yarns S.p.A., whereas, Azure A Acetone, Dimethyl Sulphoxide (DMSO) and glutaraldehyde were purchased from Sigma-Aldrich. 5,10,15,20-tetrakis [4-(2-N,N,N-trimethylethylthio)-2,3,5,6tetrafluorophenyl]porphyrin tetraiodide salt indicated as TTFAP, was synthetized and characterized as described in the supporting information section. All standard chemicals and cell culture reagents, unless otherwise indicated, were purchased from Sigma-Aldrich (Milan, Italy). Preparation of keratin sponges, PS loading and in vitro release. Keratin was extracted from wool fibres by a sulphitolysis reaction, as previously described.21 The obtained keratin powder was dissolved in water at a concentration of 20% w/v. Keratin solutions (200 µL) were placed in round bottom vials (1 cm diameter), frozen at -18°C for 3 days and lyophilized overnight. For PS loading, the sponges were immersed in acetone and the desired amounts of AzA (10 mg/mL in water) and TTFAP (10 mg/mL in DMSO) were added (Table 1). The final liquor ratio was adjusted to 40:1 (mg of sponge per mL of PS solution). Following two-hour incubation at room temperature, glutaraldehyde 25% (0.5 µL per mg of keratin) was added and allowed to react overnight. Thereafter, the supernatant was analyzed for residual PS concentration using UV-Vis spectrometry. For PS quantification, the amount of PS loaded onto the sponges was determined


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on the basis of the difference between the initial ([PS]i µg/mL) and the final ([PS]f µg/mL) PS concentration, and the loading efficiency was evaluated using the following equation:

% =

× 100

[1]

Control (PS-free) sponges were also prepared, by omitting the PS loading step. The in vitro release of the PSs was evaluated only for the sponges with the highest PS loading. The sponges (40 mg) were placed in a test tube and immersed in a phosphate buffer (PBS), pH 7.4, at 37°C for 72 h under shaking. Aliquots of 100 µL were taken from the supernatant at given times, while an equal amount of fresh buffer solution was added back to the incubation media (sink conditions). The PS concentration in the withdrawn solution was determined by UV-Vis spectroscopy, based on calibration curves prepared by adding to PBS solutions desired amounts of each PS dissolved in a proper solvent (water for AzA and DMSO for TTFAP). Drug release experiments were repeated in triplicate. Microstructural morphology. The scaffold surface was investigated with a Zeiss EVO LS 10 LaB6 scanning electron microscopy (SEM) instrument with an acceleration voltage of 5 kV and a working distance of 5 mm. Samples were mounted on a specimen stab with doublesided adhesive tape and sputtered with a gold layer for 1 min before analysis. Pore size was estimated using Gimp 2.8 Image analysis software; average pore size and its standard deviation were determined by measuring 50 pore diameters randomly collected from several sponges. Swelling ratio. The swelling ratio (SR) of PS-free and PS-loaded sponges was determined by immersing the dried samples in PBS at pH 7.4, at 37°C for 24 h. Excess buffer was removed and the wet weight of the samples was determined. The SR was then calculated as follows: =

[2]


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where Ww (mg) and Wd (mg) indicate the mass of the wet and dried material, respectively. Porosity. Porosity (ε) was measured by liquid displacement method. The scaffolds were immersed in a known volume (V1) of hexane in a graduated cylinder for five minutes. The total volume of hexane and the hexane-impregnated sponge was recorded as V2. The hexaneimpregnated sponge was then removed from the cylinder and the residual hexane volume was recorded as V3. The porosity of the scaffold was obtained on the basis of the following equation: !% =

"# "$ "% "$

× 100

[3]

Tests were carried out in triplicate and values are expressed as mean ± standard deviation. Thermal analysis and infrared spectroscopy. The thermogravimetric analysis was carried out by a SDT Q600 V20 instrument, calibrated by an indium standard, under nitrogen atmosphere in the 25-600°C. The infrared spectra were acquired using the attenuated total reflectance technique (ATR), with a Bruker Vertex 70 interferometer equipped with a diamond crystal single reflection Platinum ATR accessory, in the 4000-400 cm-1, with 100 scans and a resolution of 4 cm-1. In Vitro Biodegradability. The biodegradation profiles of the PS-free and PS-loaded sponges were obtained according to the gravimetric method.22 Briefly, the sponges (about 25 mg) were incubated at 37°C in 300 µL of a solution containing 1 mg/mL of protease XIV (protease from Streptomyces griseus type XIV, ≥ 3.5 unites per mg solid) in PBS and in 300 µL of protease-free PBS as control, at pH 7.4. Solutions were changed and collected daily. At designed time points, samples were washed thoroughly with milli-Q water, dried in a oven at 50°C and weighted in order to determine the percentage of weight loss of the sponges. Each experiment was performed in triplicate and values are expressed as mean ± standard deviation.


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Reactive oxygen species (ROS) and singlet oxygen (1O2) detection. For the detection of both ROS and 1O2 production, each sponge (about 40 mg) was immersed in a glass well containing 13µM of 2’,7’-dichlorodihydrofluoresceindiacetate (DCFH), dissolved in phosphate buffer (pH 7.4) and irradiated with a tungsten lamp (Phillips, 300W) at a distance of 20 cm (energy density 29 mW/cm2) for 5, 10, and 15 min corresponding to fluence of 9, 17, and 26 J/cm2, respectively. In this method, the non-fluorescent DHCF is oxidized into fluorescent 2’,7’-dichlorofluorescein (DCF) in the presence of ROS and 1O2, generated by light irradiation of PS-loaded sponges. The DCF shows its characteristic adsorption band at 500 nm whose intensity is correlated to the amount of generated ROS and 1O2. The sponges were subjected to three cycles of different irradiation times (5, 10, and 15 min). For each irradiation cycle the DHCF solution was replaced with a freshly prepared one. Antimicrobial photoinactivation assay. Staphylococcus aureus (MSSA) ATCC 25293 and Pseudomonas aeruginosa PAO1 were grown 24 h in Luria Bertani (LB) broth under aerobic conditions at 37°C. Cell suspensions were obtained by diluting overnight cultures in phosphate buffer (KH2PO4 /K2HPO4 10 mM pH 7.4) to reach a cell density of ~108 CFU mL−1. Keratin sponges and keratin sponges loaded with the two PSs were transferred into a 12 wells microplate containing bacterial suspensions. Slices of ~1 mg of KS-AzA and slices of ~10 mg of KSTTFAP were incubated with 1 mL of bacterial suspension. Keratin sponges (PS-free and PS-loaded) or free PS, were incubated in the dark or irradiated with a 500 W halogen-tungsten lamp (energy density 48 mW/cm2, considering the 400 nm of the whole width of the lamp emission spectrum) for 3, 6, 9, 12, 15, 30, 60, 90, and 120 min corresponding to fluence of 9, 17, 26, 35, 43, 86, 173, 259; and 346 J/cm2, respectively. The lamp was placed at a distance of 20 cm above the sample, and a 1.5 cm thick circulating


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water/glass filter was interposed to avoid overheating. A panel of controls was set for each experiment: keratin sponges and keratin sponges loaded with PSs incubated in the dark, keratin sponges irradiated. The APDT efficacy was evaluated as viable counts of bacterial suspensions collected after dark incubation or irradiation. Viable counts, expressed as colony forming units (CFU mL−1) were estimated by a plate count technique: a volume (0.1 or 0.01 mL) of undiluted or serially diluted samples was plated on LB agar plates, and incubated for 24 h at 37°C. The experiment was performed in triplicate. Cyto-compatibility and cellular behavior. Cell culture experiments were conducted on WH1 human fibroblasts, kindly provided by Dr. Guven.23 Cells were maintained in ISCOVE medium supplemented with 10% FBS, 1% sodium pyruvate, 1% glutamine and 1% antibiotics mixture, in standard culture conditions at 37°C, in a humidified 5% CO2 atmosphere. Before evaluating sponges cyto-compatibility and the effect of irradiation on cell growth, dry control (PS-free) and PS-loaded sponges were weighed, placed onto 96 wells plates, sterilized in 70% ethanol, washed 3 times with PBS, and conditioned with complete culture medium for 3h at 37°C. 15000 cells in 5 µl culture medium were then seeded onto the sponges and incubated at 37°C to allow an initial cell attachment. After 1 h, 200 µl of fresh medium were added to each well and cells were allowed to growth. After 72 h, cells were irradiated under visible light (halogen lamp 500 W) for 1 h, and then incubated for 24 h at 37°C. The growth of WH1 cells onto control and PS-loaded sponges was evaluated using an Olympus IX81 fluorescence microscope. The sponges were then incubated with the bis-benzimidine compound Hoechst33342 (1 mg/mL), a cell permeable nucleic acid stain that emits blue fluorescence when bound to dsDNA; following 45 min incubation at 37°C, the samples were washed with PBS, transferred onto microscope slides, and observed after excitation at 350 nm.


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Cells viability was evaluated by the MTT assay24. Following 1h irradiation and 24 h incubation, the sponges were transferred to fresh wells and 250 µl of a 0.4 mg/mL MTT solution in PBS were added to each well for 3 h at 37°C and the formazan crystals formed through MTT metabolism by viable cells were dissolved in DMSO. Optical densities were measured using a universal microplate reader (Biotek Instruments) at 570 nm. Optical density (OD) values obtained for each sponge were normalized on the basis of the weight of the sponge present in each sample; normalized OD values obtained for cell-free sponges, that were included in the experiment to correct for background colour due to PS release, were then subtracted. Possible intrinsic (i.e. non photodynamic) cytotoxic effects of the PS were assessed by omitting the irradiation step. RESULTS AND DISCUSSION Photo-active keratin sponges. The sulphitolysis reaction, used to extract keratin from wool, is based on the cleavage of disulphide covalent bonds of cystine (S-S) into a cysteine-Ssulphonated (S-SO3-) residue and a cysteine thiol (S-H). The high solubility of sulphonated keratin allows the preparation of concentrated keratin aqueous solution (20%wt), which can be transformed into porous sponges by freeze-drying technique.


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Figure 1. Chemical structure of AzA (a) in water and TTFAP (b) in DMSO. Normalized absorption spectra of AzA (blue line) and TTFAP (red line) acquired at 25°C at a photosensitizer concentration of 1µg/mL. For the present study, keratin sponges were functionalized with two different photosensitizers, AzA (Figure 1a) and TTFAP (Figure 1b). The former shows a maximum adsorption peak at 632 nm, while TTFAP has the maximum adsorption peak (Soret band) at 401 nm and other adsorption peaks (Q-bands) at 510 and 586 nm (Figure 1c). AzA was linked to keratin sponges through both electrostatic and covalent interactions. In particular, the positive charges of the PS were exploited for electrostatic interaction with the negative groups (COO- and SSO3-) present on the protein backbone; while the covalent conjugation was achieved by cross-linking the amino groups of the PS and the protein with


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glutaraldehyde (Figure 2a). Moreover, TTFAP was loaded onto keratin sponges through strong electrostatic interactions between the sulphonated groups of the protein and the quaternary ammonium cations of the PS (Figure 2b). Two different loading ratios were considered for the two PSs, as summarized in Table 1. The PS loading efficiency was always over the 50%, with TTFAP slightly higher than that of AzA (Table 1).

Table 1. List of prepared samples. Sample

PS

PS/KS x 10-2 µmol/mg

Loading Efficiency (%)

KS

none

#

#

KS-TTFAP-1

TTFAP

1.41±0.06

86±4

KS-TTFAP-2

TTFAP

6.90±0.04

84±1

KS-AzA-1

AzA

1.6±0.1

66±4

KS-AzA-2

AzA

7.5±0.2

68±2

The prepared sponges are cylindrical in shape (Figure 3a) and their cross-sectional morphology is characterized by an interconnected 3D porous structure made of circular shaped pores (Figure 3b). As shown in Table 2, the unloaded sponge shows a porosity of about 49% with an average pore size of ca. 37 µm. However, while the AzA loading does not significantly change the porosity and the pore size of keratin sponges, the TTFAP induces a significant increase of both mean pore size and porosity. This behavior could be ascribed to the effect of the DMSO used for dissolving the porphyrin. However, the obtained sponge’s architecture is adequate for the regeneration of adult mammalian skin cells.25


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Figure 2. Schematic representation of the glutaraldehyde crosslinking and chemical reactions keratin-AzA (a) and keratin-TTFAP (b). Table 2. Mean pore size and porosity of unloaded and PS loaded sponges Sample

Pore size (µm) (n = 50)

Porosity (%)

KS

37 ± 6

49 ± 2

KS-TTFAP-2

78 ± 42

75 ± 2

KS-AzA-2

39 ± 8

45 ± 5


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Figure 3. Visual appearance of keratin sponges loaded with AzA and TTFAP (a) and scanning electron micrographs of keratin sponges (b). The thermal properties of the samples were determined by thermogravimetric analysis (TG) carried out in the 100-600°C range. In Figure 4a, the TG profiles of unloaded and PS loaded sponges are reported. As shown, for the KS sponge, the onset temperature related to the first weight loss is found at 203°C and it shifts towards 139°C and 100°C for KS-AzA2 and KSTTFAP-2 sponges, respectively. This behavior suggests a lower thermal stability of the PSloaded sponges as compared to the unloaded one. The residual material at the end of the test, e.g. 26% for KS, 25% for the KS-AzA-2 and 21% KS-TTFAP-2, also confirms this result.


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Figure 4. Thermogravimetric curves (a) and FTIR spectra (b) of KS, KS-AzA-2 and KSTTFAP-2 sponges. The chemical interactions between keratin and selected PSs were studied by FT-IR spectroscopy. The infrared spectra of keratin sponges (Figure 4b), acquired in the 1800-860 cm-1 range, show the characteristics keratin adsorption bands, such as the amide I in the 1700-1600 cm-1 range (C=O stretching vibrations), the amide II in the 1510-1580 cm-1 range (in plane N-H blending and C-N stretching vibrations) and the amide III in the 1220-1300 cm-1 range (more complex vibrational band).26 The two intense bands at 1190 and 1021 cm-1 are due to the asymmetric and symmetric vibration stretching of sulphonated groups.27 As can be seen in Figure 4b (box on the left), the adsorption peak at 1021 cm-1 of KS sponge is shifted to 1017 cm-1 in the KS-TTFAP-2 sample, demonstrating that the sulphonated groups are strongly involved in the electrostatic interactions with quaternary ammonium cations of porphyrin. Moreover, the KS-AzA-2 sponges show an intense adsorption peak at 1515 cm-1 due to the – NH3+ symmetric deformation of lysine and arginine, which is reduced in intensity in the KS and KS-TTFAP-2 samples (Figure 4b, box on the right). A possible explanation for the higher signal observed for KS-AzA-2 sponges, could be attributed to a lower amount of crosslinking between


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the amino groups of the protein side chain, due to the presence of the amino groups of the dye, which act as competitors in the reaction with glutaraldehyde. In vitro PS release and kinetic study. PS release was only assessed for sponges at the highest PS loading (KS-TTFAP-2 and KS-AzA-2), in phosphate buffer at pH 7.4 and at a temperature of 37°C for 72 h. As shown in Figure 5, a burst release occurred within the first 1.5 h, due to the migration of the PS on the scaffold surface (upper box of Figure 5), followed by a continuous slow release for the remaining observation time (72 h). Moreover, during the considered timeframe, the total TTFAP released was 6%, while AzA reached the 20%.

Figure 5. Cumulative release of AzA and TTFAP from KS-AzA-2 and KS-TTFAP-2 sponges. This suggests that bonding to keratin is stronger for TTFAP than for AzA. In order to describe the mechanisms of PS release, the experimental data were fitted with known kinetic models such as zero-order, Higuchi and Korsemeyer-Peppas models.28 The results are listed in Table 3. By comparing the correlation coefficients obtained for the different kinetic models, the best fit of the kinetic data was obtained for the Korsmeyer-Peppas. In this model, the n value characterizes the release mechanism and in the case of a cylindrical tablet, n

Wool Keratin 3D Scaffolds with Light-Triggered Antimicrobial Activity

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