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polyvinylpyrrolidone fibrous materials with complex architecture and antibacterial activity
Materials Science and Engineering C 73 (2017) 206–214
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrospun curcumin-loaded cellulose acetate/polyvinylpyrrolidone fibrous materials with complex architecture and antibacterial activity Petya B. Tsekova a, Mariya G. Spasova a, Nevena E. Manolova a, Nadya D. Markova b, Iliya B. Rashkov a,⁎ a b
Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 103A, BG-1113 Sofia, Bulgaria Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 26, BG-1113 Sofia, Bulgaria
a r t i c l e
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Article history: Received 22 June 2016 Received in revised form 25 October 2016 Accepted 17 December 2016 Available online 20 December 2016 Keywords: Electrospinning Curcumin Cellulose acetate Polyvinylpyrrolidone Antibacterial activity Wound dressings
a b s t r a c t Novel fibrous materials from cellulose acetate (CA) and polyvinylpyrrolidone (PVP) containing curcumin (Curc) with original design were prepared by one-pot electrospinning or dual spinneret electrospinning. The electrospun materials were characterized by scanning electron microscopy (SEM), fluorescence microscopy, Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV–Vis), differential scanning calorimetry (DSC), water contact angle measurements, and microbiological tests. It was found that the incorporation of Curc into the CA and PVP solutions resulted in an increase of the solution viscosity and obtaining fibers with larger diameters (ca. 1.5 μm) compared to the neat CA (ca. 800 nm) and PVP fibers (ca. 500 nm). The incorporation of PVP resulted in increased hydrophilicity of the fibers and in faster Curc release. Curc was found in the amorphous state in the Curc-containing fibers and these mats exhibited antibacterial activity against Staphylococcus aureus (S. aureus). The results suggest that, due to their complex architecture, the obtained new antibacterial materials are suitable for wound dressing applications, which necessitate diverse release behaviors of the bioactive compound. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Wound care dressings play an important role in the wound healing process and form an important part of the global medical wound care products market. Various natural biopolymers such as starch, cellulose, collagen, chitosan, keratin, alginate, and elastin were used for the preparation of wound dressing products [1,2]. Cellulose is the most abundant naturally occurring polysaccharide. Cellulose can be converted upon acetylation to cellulose acetate. The advantage of the resultant cellulose acetate is that it can be easily dissolved or melt and could be shaped into fibers, films, sheets, tubes, pellets or other products. In recent decades, great attention has been paid to fibers from cellulose and cellulose derivatives due to their low cost, lightweight, easy processing, biodegradability, good mechanical and barrier properties and recycling [3]. Bioactive wound dressings have been produced from biomaterials and take active part in the healing process. These types of dressings are sometimes loaded with naturally occurring compounds, antibiotics, vitamins, minerals, enzymes, growth factors and antimicrobials to enhance wound healing process [4]. Curcumin is a biologically active substance found in the roots of Curcuma longa plant. This naturally occurring polyphenolic compound manifests remarkable antibacterial [5], antifungal [6], antioxidant [7], anti-inflammatory [8], anticoagulant [9] and antitumor [10] activity. However, Curc has some ⁎ Corresponding author. E-mail address: [email protected] (I.B. Rashkov).
http://dx.doi.org/10.1016/j.msec.2016.12.086 0928-4931/© 2016 Elsevier B.V. All rights reserved.
drawbacks. It is thermally and chemically instable and easily undergoes photodestruction. The main disadvantage of Curc is its low bioavailability due to its poor aqueous solubility. Incorporation of Curc in polymer matrix is expected to contribute to obviating these drawbacks. Improved dissolution of Curc has been achieved by preparation of CurcPVP solid dispersion by spray drying [11]. In the recent years, electrospinning has emerged as a very suitable technique for preparation of drug-loaded polymeric materials. Nanofibrous nonwoven materials produced by electrospinning have shown great potential as drug-eluting stents and wound dressing materials [12–15]. The large specific surface area of the electrospun materials and the possibility for extended drug release lead to enhancement of the therapeutic effect of the embedded drugs and reducing their side effects. It has been shown that electrospun fibrous materials are suitable carriers for enhancing the bioavailability of Curc. Curcumin has been encapsulated into electrospun material from cellulose acetate [16–18], poly(L-lactide-co-D,L-lactide) [19], poly(ε-caprolactone) [20], poly(εcaprolactone)/polyethylene glycol [21], poly(ethylene glycol)/ poly(butylene succinate) [22], PVP [23], etc. Previously, we have shown that Curc release from electrospun polylactide materials was facilitated by the presence of PVP or polyethylene glycol due to formation of hydrogen bonds with Curc [9]. The aim of the present study was to obtain novel electrospun materials from CA and PVP for Curc delivery. The possibility to modulate the Curc release profile by appropriate selection of the composition of the polymer matrix and the preparation technique (one-pot
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electrospinning or dual spinneret electrospinning) was studied. The effect of the composition and architecture of the mats on the biological behavior upon contact with pathogenic microorganisms (S. aureus) was evaluated.
2. Experimental 2.1. Materials Cellulose acetate (CA, Aldrich) with Mn30,000 g/mol and acetyl content 39.8%, polyvinylpyrrolidone (PVP, Fluka) with Mr = 360.000 g/mol and curcumin (Curc, Merck) were used. Acetone was of analytical grade of purity and was purchased from Sigma-Aldrich.
2.2. Light source Curc was excited with a light emitting-diode (LED) in the blue region of the spectrum [24]. The device (LTM Electronics Ltd., Bulgaria) provided a light emission with predominant central wavelength of 450 nm at intensity (power density) of 11.375 mW cm−2. The irradiances (energy fluency) tested in the study were 82 and 164 J cm−2 and the irradiation was performed in a non-contact mode with a focused beam at 10 cm of working distance (distance between the light source and cell line surface). The system, which was composed of 8 LEDs, could uniformly deliver irradiation without a heating effect. The distance between the LED and the plate was intended to enable an even distribution of light on each well. The power density of the incident radiation was measured using a LabMax TOP PM10 laser power and energy meter (Coherent®, Santa Clara, CA, USA).
2.3. Preparation of fibrous materials by electrospinning In the present study CA, PVP, Curc/CA, Curc/PVP and Curc/CA/PVP fibrous materials were fabricated by one-pot electrospinning. The mats were prepared from the following polymer solutions: CA in acetone/ water 80/20 v/v; PVP in acetone/water 50/50 v/v; Curc/CA in acetone/ water 80/20 v/v; Curc/PVP in acetone/water 50/50 v/v and Curc/CA/ PVP in acetone/water 70/30 v/v. The total polymer concentration was 10 wt%, Curc was 10% in respect to polymer(s) weight. The spinning solutions were loaded in a syringe equipped with a metal needle (gauge: 20GX1½″) connected to the positively charged electrode of the high-voltage power supply (up to 30 kV). Electrospinning was conducted at a constant applied voltage of 25 kV and constant tip-to-collector distance of 15 cm using a grounded rotating aluminum collector (1000 rpm). The spinning suspensions were delivered at a constant rate of 3 ml/h enabled by the use of a pump Syringe Pump NE-300 (New Era Pump Systems, Inc.).
2.4. Preparation of fibrous materials by dual spinneret technique Two hybrid fibrous materials were fabricated by dual spinneret technique. These materials were prepared by using: two pumps for delivering: (i) Curc/CA solution [10 wt% CA and 10 wt% Curc in acetone/ water = 80/20 (v/v)], and (ii) PVP solution - 10 wt% (w/v) in acetone/ water = 50/50 (v/v) or (iii) Curc/CA solution [10 wt% CA and 10 wt% Curc in acetone/water = 80/20 (v/v)], and (iv) Curc/PVP solution [10 wt% PVP and 10 wt% Curc in acetone/water = 50/50 (v/v)]. The pumps for delivering the spinning solutions were located at an angle of 180° in respect to the rotating collector (1000 rpm). The delivery rate of the spinning solutions was 3 ml/h. The tip-to-collector distance was 15 cm. The applied voltage was provided using a common highvoltage power supply at a constant applied voltage of 25 kV.
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2.5. Characterization of the fibrous materials The rheological measurements of the used solutions and suspensions were performed using a Brookfield, DV – II + Pro, spindle CPE – 52, at 25°С. The morphology of the fibrous materials was evaluated by scanning electron microscopy (SEM). The samples (1 cm2) were vacuum-coated with gold and were observed with Jeol JSM-5510 (Jeol Ltd., Japan). The mean fiber diameter was estimated by Image J software [25], by measuring the diameters of at least 20 random fibers per sample from three different SEM micrographs for a total of 60 measurements and their morphology was assessed applying the criteria for overall evaluation of electrospun materials as described in details in [26]. The mats (2.5 cm2) were observed by a fluorescence microscope (NU-2; Carl Zeiss, Jena, Germany) maximum excitation wavelength (λex) at 420 nm and at maximum emission wavelength (λem) 470 nm. Infrared spectra (Fourier transformed) of the mats (1 cm2) were recorded using an IRAffinity-1 spectrophotometer (Shimadzu Co., Japan), supplied with a MIRacle ATR device (diamond crystal; depth of penetration of the IR beam into the sample - approximately 2 μm) (PIKE Technologies, USA) in the range of 600–4000 cm− 1 with a resolution of 4 cm−1. All spectra were corrected for H2O and CO2 using an IRsolution software program. The thermal behavior of the obtained fibrous materials was evaluated by differential scanning calorimetry (DSC). The samples were heated in the temperature range from 0 to 300 °C at heating rate of 10 °C/min under nitrogen (TA Instruments, DSC Q2000, USA). The crystallinity degree of CA fibers was calculated using the value for the enthalpy of fusion of a perfect crystal of cellulose acetate of 58.8 J/g determined by Cerqueira et al. [27]. X-ray diffraction (XRD) analyses were performed at room temperature using a computer-controlled D8 Bruker Advance ECO powder diffractometer with filtered Cu Kα radiation. Data were collected in the 2θ range from 5° to 60° with a step of 0.02° and counting time of 1 s step−1. The water contact angles of the fibrous materials were measured using an Easy Drop DSA20E KRÜSS GmbH apparatus, Germany. Drops of distilled water with a volume of 10 μl were deposited on the surface of the test specimens (2 cm × 7 cm; cut in the direction of the collector rotation). The mean contact angle value was determined after averaging at least 10 measurements for each specimen. In addition, Curc/CA/PVP mat was placed in a desiccator for 3 days at 20 °C at 96% relative humidity and then the water contact angle was measured. 2.6. Curc release from the fibrous materials The Curc content in the mats was determined after dissolving each mat (1 cm2–6 mg) in 10 ml acetone/water = 80/20 w/w, after which the absorbance was measured by a DU 800 UV spectrophotometer (Beckman Coulter) at a wavelength of 421 nm. The release was studied in vitro at 37 °C, in acetate buffer at pH 5.5 and ionic strength 0.1 (CH3COONa/CH3COOH) containing PVP (acetate buffer/PVP = 98/2 v/v). In a typical process, 6 mg of mats were immersed in 100 ml buffer solution stirred at 150 rpm with an electromagnetic stirrer. Aliquots were withdrawn at determined time intervals and their absorbance was recorded at a wavelength of 440 nm. The amount of released Curc over time was calculated using calibration curves (correlation coefficient R = 0.999) for the mats in acetate buffer/PVP 20 (98/2 v/v), pH = 5.5, ionic strength 0.1. 2.7. Microbiological assays The antibacterial activity of the fibrous materials against Gram-positive S. aureus was evaluated by using the viable cell-counting method as described below. S. aureus (NBIMCC 749) was purchased from the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC,
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Bulgaria). For comparison, the antibacterial activity of CA and PVP against S. aureus was assessed as well. The electrospun mats (0.05 g) were exposed to bacterial suspension (2 ml) with concentration of 105 cells/ml−1 prepared in nutrient broth (Sigma-Aldrich) at 37 °C. In view of the photosensitizing properties of Curc related with its antibacterial activity, the samples were illuminated at λmax = 450 nm with a LED lamp in the blue region of the spectrum during the whole experiment. The distance between the lamp (16 W) and the surface of the broth culture was 10 cm. At specified time intervals (2, 4 and 24 h), aliquots of 50 μl were withdrawn from each broth culture which had been in contact with the mats and after serial ten-fold dilutions with sterilized 0.9% saline solution, then plated on Petri dishes with nutrient agar (Sigma-Aldrich). The plates were incubated at 37 °C for 24 h. The number of the survived bacteria was determined by counting the colony forming units (CFU) in triplicate for each experiment. Evaluation of the adhesion of S. aureus to the surface of the mats was performed by direct SEM observation with Jeol JSM-5510 (Jeol Ltd., Japan). Briefly, the mats were incubated in 2.0 ml broth culture of S. aureus with concentration of 105 cell/ml for 4 h. Then the samples were washed twice with phosphate buffered saline (PBS, pH 7.4) for removal of non-adhered bacteria. The adhered bacteria on the surface of the mats were fixed by immersion of the mats in 2.5 wt% glutaraldehyde solution in PBS at 4 °C for 5 h. Then the samples were washed carefully with PBS and freeze-dried. After 4-h contact of the mats with S. aureus cells and coating of the mats with gold, the bacterial morphology was observed by SEM. 3. Results and discussion In the present work, Curc/CA, Curc/PVP, Curc/CA/PVP, Curc/ CA + PVP and Curc/CA + Curc/PVP electrospun mats were prepared and characterized. For comparison CA, PVP and (CA/PVP) mats were obtained as well. Schematic representation of the cross-section of fibers is shown in Fig. 1. The mats prepared by dual spinneret electrospinning are composed of two types of fibers: CA fiber with Curc and PVP fiber with/without Curc. The distribution was confirmed by the results obtained from SEM and fluorescence microscopy.
Curc/PVP and Curc/CA/PVP solutions was 156 cP, 163 cP and 151 cP, respectively. The increase in solution viscosity was most probably due to the formation of hydrogen bonds between the polymer(s) and Curc [9]. 3.2. Preparation of fibrous materials by electrospinning Fibrous materials of CA, PVP, Curc/CA, Curc/PVP and Curc/CA/PVP were prepared by one-pot electrospinning of solutions of CA, PVP and Curc, respectively. Based on the conducted preliminary experiments the following optimal conditions for electrospinning were found: polymer concentration of 10 wt% for the CA and for the PVP solutions, and tip-to-collector distance - 15 cm in order to obtain defect-free fibers. Representative SEM images of the obtained CA, Curc/CA, PVP, Curc/ PVP and Curc/CA/PVP electrospun mats are shown in Fig. 2. The electrospinning of CA solutions under the selected conditions reproducibly resulted in obtaining fibers with mean fiber diameter of 780 ± 110 nm (Fig. 2A). The mean fiber diameter of the PVP fibers was 495 ± 113 nm (Fig. 2C). However, some defects were detected. The incorporation of Curc into the CA and PVP solutions resulted in obtaining fibers with larger diameters compared to the neat CA and PVP fibers. The increase in fiber diameter and the absence of defect of the hybrid fibers was attributed to the increase in the viscosity of the Curc/CA and Curc/PVP solutions, 156 cP and 163 cP, respectively. It is well known that the increase in solution viscosity results in an increase of the mean fiber diameter [29]. The mean fiber diameter of the hybrid Curc/CA and Curc/PVP fibers was 1150 ± 260 nm and 570 ± 125 nm, respectively (Fig. 2B and D). The mean fiber diameter of the obtained Curc/CA/PVP fibers was 1560 ± 145 nm (Fig. 2E). It is known that Curc possesses fluorescence properties, which enable observation of the prepared mats by fluorescence microscopy [30]. As expected, the CA and PVP fibers did not display any fluorescence (micrographs not shown). In contrast, all the Curc-loaded mats were characterized by continuous intense fluorescence signal all over the fiber length. This provides evidence that the biologically active substance is uniformly distributed into the fibers. Fluorescence micrographs of Curc/CA, Curc/PVP and Curc/CA/PVP mats are shown in Fig. 2 inset.
3.1. Viscosity of the spinning solutions
3.3. Preparation of fibrous materials by dual spinneret electrospinning
It is well known that the viscosity of the spinning solutions has significant influence on the electrospinning process and the resultant fiber morphology [28]. The dynamic viscosity of the solutions used in this study was measured. The viscosity of the CA, PVP and CA/PVP solutions in acetone/water was 122 cP, 153 cP and 142 cP, respectively. The incorporation of Curc into the CA, PVP and CA/PVP solutions resulted in an increase of the viscosity. The measured viscosity for the Curc/CA,
In the present study the dual spinneret electrospinning method was used for the preparation of fibrous materials with different composition and architecture. The following mats were obtained: Curc/CA + PVP and Curc/CA + Curc/PVP. The solution concentration of CA and PVP was 10 wt% and the concentration of Curc was 10 wt% in respect to the polymer weight. SEM micrographs of the obtained Curc/CA + PVP and Curc/CA + Curc/PVP fibers are presented in Fig. 3A and B.
Fig. 1. Schematic representation of the cross-section of fibers: A. Curc/CA, B. Curc/PVP, C. Curc/CA/PVP, D. Curc/CA + PVP, E. Curc/CA + Curc/PVP.
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Fig. 2. SEM micrographs and fluorescence micrographs (insets) of fibrous materials from: A. CA; B. Curc/CA; C. PVP; D. Curc/PVP, E. Curc/CA/PVP; solvent acetone/water (80/20 v/v) for CA and Curc/CA; acetone/water (50/50 v/v) for PVP and Curc/PVP and acetone/water (75/25 v/v) for Curc/CA/PVP;
It can be seen that the dual spinneret electrospinning of Curc/CA and PVP solution resulted in preparation of fibrous materials consisted of Curc/CA ribbon-like fibers with mean ribbon width 1145 ± 320 nm and PVP fibers with defects with mean fiber diameter 215 ± 45 nm. This was confirmed by the fluorescent microscopy as well. In contrast to the PVP fibers, the Curc-containing fibers displayed fluorescence. The dual spinneret electrospinning of Curc/CA and Curc/PVP solutions resulted in obtaining a more uniform mat with mean fiber diameter of 1528 ± 155 nm.
3.4. IR-spectra of the fibrous materials The FTIR-spectra of Curc (powder) and Curc/CA/PVP fibers are presented in Fig. 4. The Curc spectrum showed a band at 3510 cm− 1 which was attributed to the phenolic O\\H stretching vibration (Fig. 4A inset). Strong bands at 1625 cm− 1 (stretching vibrations of the benzene ring), 1506 cm− 1 (C _O and C _C vibration) and 1427 cm−1 (olefinic C\\H bending vibration) were detected as well. Enol C\\O band was detected at 1274 cm−1. The bands at 808 cm− 1
Fig. 3. SEM micrographs and fluorescence micrographs (insets) of: A. Curc/CA + PVP mat, and B. Curc/CA + Curc/PVP mat.
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Fig. 4. IR-spectra of: (A) Curc and (B) Curc/CA/PVP mat in the range from 1800 to 800 cm−1 and from 4000 to 3200 cm−1 (inset).
and 962 cm−1 were attributed to the bending vibrations of the C\\H bond of alkene groups (RCH = CH2). The FTIR spectrum of Curc/CA/PVP electrospun mat is shown in Fig. 4B. In the presented spectrum, peaks for the C _O functional groups at 1740 cm− 1, the O\\H functional groups at ~ 3500 cm− 1, the CH3 groups at 1369 and 1226 cm−1, and well as the ether C–O–C functional groups at 1037 cm−1 characteristic for the CA were observed [31]. In addition, bands of PVP at 1651 cm−1, 1286 cm−1 and 1422 cm−1 characteristic for C _O, С\\N and СН stretching vibrations were detected. The broad peak at about 3050–3620 cm−1 with at a maximum at 3414 cm− 1 attributed to stretching vibrations of the N\\H group of PVP is observed [32,33]. In the spectra of Curc/CA/PVP mat characteristics bands for the Curc are also detected. As expected they are with low intensity due to the relatively low Curc quantity in the polymer mat (10 wt% in respect to the
polymer weight). It should be noted that in Curc/CA/PVP mat, the O\\H stretching vibration at 3510 cm− 1 characteristic for the Curc was disappeared. Therefore, it can be concluded that hydrogen bonding between Curc and PVP takes place. 3.5. Thermal characteristics of the fibrous materials Representative DSC thermograms of the prepared fibrous materials are shown in Fig. 5. Thermograms of CA, PVP and Curc/СА mat as well as of Curc powder are presented in Fig. 5A. CA fibers showed a broad endothermic peak between ambient temperature (25 °C) and 100 °C with a peak maximum at 70° С. This event is attributed to desorption of water from cellulose acetate [34,35]. Endothermic transition corresponding to Tg at 195° С and endothermic peak at 225° С corresponding to Tm of the cellulose acetate were observed. The obtained results are in
Fig. 5. DSC thermograms (first heating run) of: A. Curc powder and CA, PVP and Curc/CA mat and B. Curc powder and CA/PVP, Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP mats.
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very good agreement with literature data presenting thermal characteristics of cellulose acetate fibers prepared by electrospinning [36]. The calculated crystallinity of the CA fibers was 16%. The incorporation of Curc into CA fibers resulted in a decrease to 162 °C and 206 °C for the Tg and Tm, respectively. However, Curc incorporation did not lead to a significant change in degree of crystallinity of CA - 14%. It can be assumed that there is an interaction between CA and Curc resulting in decrease of Tg and Tm, Interaction based on hydrogen bonds between CA and Curc has been reported by other authors [31]. It is noteworthy that the incorporated Curc in the CA fibers is in the amorphous state as evidenced by the lack of an endothermic peak corresponding to the melting of Curc at about 175 °C. The amorphous state of Curc exhibits a disordered structure in comparison to its crystalline state and possesses higher free energy. Thus it provides enhanced apparent solubility, dissolution rate and bioavailability [37]. DSC thermogram of PVP mat is shown in Fig. 5A. A broad peak was observed between ambient temperature and 100° С due to the water absorbed. Moreover, the PVP used in the present study was a high-molecular-weight polymer with Tg of 173° С (Fig. 5A). The incorporation of Curc into PVP fibers resulted in decrease of the glass transition temperature to 149°С (DSC curves not shown). We assumed that the shift of the Tg to lower temperatures can be attributed to interaction between PVP and Curc. Curc presented into the PVP fibers was in amorphous state (no peak corresponding to the melting point of Curc was observed). This result is consistent with previously reported data [9]. DSC thermograms of CA/PVP, Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP mats are presented in Fig. 5B. All CA and PVP mats showed broad endothermic peak between ambient temperature (25 °C) and 100 °C, corresponding to the loss of moisture. It could be seen that the incorporated Curc into the fibrous mats was in the amorphous state. The crystallinity degree of the Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP mats was ca. 9% - a value which is lower than the degree of crystallinity of the neat CA fibers (16%). 3.6. X-ray diffraction XRD pattern of Curc powder, CA and Curc/CA/PVP mats recorded in 2θ range from 5 to 60° are presented in Fig. 6. The presence of the main diffraction peaks of pure Curc powder located at 2θ = 8.9°, 14.5°, 17.2°, 18.2°, and 23.3° were detected. Characteristic peaks for Curc were absent in the XRD pattern of Curc/CA/PVP mat, thus implying that Curc was in amorphous state. This result was in accordance with the DSC results where no peak corresponding to Curc melting was observed.
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depend on the mat composition. CA fibrous materials were hydrophobic (123.10 ± 2.0°, Fig. 7A) and the water droplet preserved its spherical shape on the CA mats within more than 2 h. The measured contact angle value of the Curc/CA mats was even higher with equilibrium contact angle 129.4 ± 3.8° (Fig. 7C). The addition of a water-soluble polymer – PVP led to hydrophilization of the fibers and to more rapid absorption of the droplet by the mat (Fig. 7B, D). The starting contact angles of CA/PVP and Curc/CA + PVP were 36° ± 3.5° and 14.8 ± 1.7°, respectively. However, within 5 min. after droplet deposition the contact angle decreased to 0°. The equilibrium contact angle values of the mats containing PVP were rapidly reached thus implying that these values were directly related to surface structure reorganization. It is known that when a droplet is deposited onto a polymer material, the hydrophilic component of polymer is reorganized toward the surface in order to reduce the surface tension which results in decreased contact angle value [38]. The effect of depositing time on the contact angles of Curc/CA/PVP and Curc/CA + Curc/PVP mats is shown in Fig. 8. The incorporation of Curc into the fibers resulted in hydrophobization of the Curc/CA/PVP (117.04 ± 1.8°) (Fig. 8A) and Curc/CA + Curc/PVP (121.8 ± 3.4°) fibrous materials (Fig. 8C). However, as seen from Fig. 8, within 120 min. the contact angle value reached 0°. This behavior is in accordance with findings for other systems [38] where the starting contact angle is contributed by the hydrophobic component on polymer surface and the equilibrium contact angle mainly by the hydrophilic component of polymer. Additional experiments were performed in order to prove this statement. Curc/CA/PVP mat was placed for 3 days at 20 °C at 96% relative humidity. Then, the contact angle was measured. The starting contact angle was lower (84.3 ± 5.4°) compared to the measured starting contact angle of the same mat not placed in water vapour medium (117.04 ± 1.8°). This evidenced that surface reorganization occurs and fragments of the hydrophilic PVP are oriented toward the mat surface which resulted in decrease of the measured contact angle. Moreover, Curc/CA/PVP mat which was placed in medium rich in water vapour reached the value of the contact angle 0° more rapidly. It could be concluded that time needed to reach the equilibrium contact angle value is very short for the hydrophobic CA and Curc/CA mats and for the water-soluble PVP and Curc/PVP mats (1 min), fast for the CA/PVP and Curc/CA + PVP (5 min) and much longer for Curc/CA/PVP and Curc/CA + Curc/PVP mats (2 h). Measuring the equilibrium contact angle is important for finding a relationship between composition of the mat and potential application of the mats as wound healing dressings. For example, it is well known
3.7. Water contact angle and sorption kinetics of the fibrous materials The time dependence of contact angles was investigated by the dynamic contact angle measurements over time using static sessile drop method. It was found that the starting and equilibrium contact angles
Fig. 6. XRD patterns of Curc powder, CA mat and Curc/CA/PVP mat.
Fig. 7. Digital photographs of water droplets (10 μl) on the surface of mats: A. CA; B. CA/ PVP, C. Curc/CA and D. Curc/CA + PVP (The measurements were performed immediately after water droplet deposition).
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Fig. 8. Dependence of the contact angle as a function of time: A. Curc/CA/PVP, B. Curc/CA/PVP, placed in a desiccator over water for 3 days and C. Curc/CA + Curc/PVP mat.
that in wound different amounts of exudate rich in dead cells, tissue fragments, dirt and bacteria, is initially produced. If large amounts of exudate remain in the wound, the recovery is hampered and the risk of infection increases. For this reason, an excessive amount of exudate should be absorbed by the dressing [39]. Thus, bacteria, harmful metabolic products, devitalized tissue, dirt and foreign bodies are removed from the wound and do not need to be eliminated from the body phagocytosis. It is anticipated that the mats containing PVP, which have low contact angle values and quickly reach equilibrium value will successfully find potential application as wound dressings for open wounds, where more exudate leakage is expected. Producing a set of mats with diverse complex architecture based on cellulose acetate, water-soluble polymer - PVP and biologically active substance - Curc will enable their application depending on the specific medical case (type of wound) to achieve proper treatment and successful treatment outcome. 3.8. In vitro release studies of Curc Based on the fact that PVP is used as solubilizing agent of poorly water-soluble drugs to improve their dissolution rate and oral absorption [40], and on our previous findings [9] that Curc release from polylactide materials was facilitated by the presence of PVP, in this study PVP was used as solubilizing agent of Curc. To check and visualize the improved solubility of Curc in the presence of PVP, two solutions with the same concentration of Curc (0.2% wt/v) were prepared: in water and in aqueous solution of PVP (2% wt/v). As seen from Fig. 9, Curc was practically insoluble in water and stayed on the top of the liquid phase. In contrast, when PVP was present in the aqueous phase, Curc
was well wetted and the solution had the characteristic yellowish colouration of Curc which proved that PVP enhanced Curc solubility. Curc release from Curc/CA, Curc/CA/PVP, Curc/CA + PVP and Curc/ CA + Curc/PVP mats was studied in acetate buffer/PVP (98/2 v/v). As seen (Fig. 10), Curc was released most slowly and in the smallest amount (ca. 10% or 0.5 μg/ml for 120 min) from the hydrophobic Curc/CA mat even in the presence of water-soluble polymer - PVP in the dissolution medium. The Curc was more rapidly released when PVP was included in the fibers. The amount of the released bioactive substance from the Curc/CA/PVP fibers was about ca. 22% (1.2 μg/ml) for 120 min. Furthermore, Curc was released to the greatest extent from the mats obtained by dual spinneret electrospinning – c.a. 56% (3 μg/ml) and 68% (3.7 μg/ml), from the Curc/CA + PVP and Curc/ CA + Curc/PVP mats, respectively. In the present study it was found that the greater hydrophilicity of the PVP-containing mats as compared with Curc/CA fibers assists the penetration of the dissolution medium in the mats. This result is with accordance with our previous results that show that Curc was released more rapidly when the water soluble polymer (PVP or PEG) was included in the fibers of PLA/PVP or PLA/PEG [9]. The obtained results showed that the electrospun PVP-containing fibrous materials could be used to create advanced release systems with enhanced solubility of the poorly water-soluble Curc. Moreover, the use of electrospinning or dualelectrospinning enables the preparation of fibrous materials with diverse architecture and hence – with different release profile of Curc. This allows for obtaining materials suitable for different types of wounds, e.g. mats containing PVP for strongly exuding wounds or Curc-containing mats for infected wounds.
Fig. 9. Photographs of: A. Curc (0.02 g) in water (10 ml) and B. Curc (0.02 g) in PVP solution (PVP concentration - 2 wt% in water).
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Fig. 10. Curc release from the Curc/CA (▲), Curc/CA/PVP (◊), Curc/CA + PVP (□) and Curc/CA + Curc/PVP (■) mats. The results are presented as average values from three separate measurements with the respective standard deviation; acetate buffer/PVP (98/ 2 v/v), pH 5.5, 37 °C, ionic strength 0.1.
3.9. Microbiological tests Curc has potential as a photosensitizer. By irradiation of Curc with light in the visible range of the spectrum, oxygen species, such as singlet oxygen and free radicals, are formed, which then produce an effect that is toxic to the bacterial cell [41]. Therefore, the antibacterial activity of the fibrous CA, CA/PVP, Curc/CA, Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP materials against S. aureus was tested in nutrient broth (Sigma-Aldrich) by irradiation with blue light during the whole experiment. The number of survived bacteria was subsequently assessed by plating and counting of CFU in solid medium. The log of the survived bacteria versus the exposure time for the electrospun mats is presented in Fig. 11. For comparison the growth of a control of the S. aureus was assessed as well. It was found that the control developed normally during the experiment. As seen from Fig. 11, significant decrease in the number of the viable cells was detected for the exposure time of 2 h for the mats containing Curc. The Curc/CA, Curc/CA/PVP and Curc/CA + PVP mats manifest antibacterial activity and for the contact of 4 h, a decrease of S. aureus titer by 2.3, 2.8 and 3 log units was attained. This is due to Curc release
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from the mats. Curc release from Curc/CA, Curc/CA/PVP, Curc/CA + PVP mats was 10, 22 and 56% for 120 min, respectively. This result is in accordance with our previous finding on the antibacterial activity of PLA/PVP/curcumin fibrous mats [9]. Moreover, the Curc/CA + Curc/ PVP mat kills all the bacteria at the 4th h. This is due to fact that the Curc was released to the greatest extent from the Curc/CA + Curc/PVP mat – 68%. The studies of Fallah [42] have also revealed that nanofibers of PCL/gelatin/Curc were 99.9% antibacterial against methicillin-resistant S. aureus. In contrast, neat CA and CA/PVP mats not containing the biologically active substance did not alter the bacterial growth and did not exhibit antibacterial activity. A slight increase in the number of viable cells was detected at the 24th h for the Curc/CA, Curc/CA/PVP and Curc/CA + PVP mats. This is most probably due to fact that Curc was released to the greatest extent in the first 2–3 h. A significant increase in the number of the viable bacteria was determined for the CA and CA/ PVP mats which was close to the growth of the S. aureus control. It may be noticed that while the release profile of Curc for Curc/CA/PVP and Curc/CA + PVP mat was different, the antibacterial activity was the same. This behavior may be attributed to the fact that both types of mats release Curc (up to 24 h) in the range which is above the MIC against S. aureus, thus resulting in reduction of survived cells with 3 log. In order to evaluate the attachment of the bacterial cells to the surface of the fibrous materials, the CA, CA/PVP, Curc/CA, Curc/CA/ PVP, Curc/CA + PVP and Curc/CA + Curc/PVP materials were incubated for 4 h in a broth culture of S. aureus. Then, the bacterial cells were fixed with glutaraldehyde solution, washed with distilled water, freeze-dried and characterized by SEM. It was found that S. aureus cells adhered to the CA mat not containing the biologically active substance (Fig. 12A). However, the cell morphology was not altered (cells remained round and with smooth surfaces). This indicated that the CA mats did not alter the growth of pathogenic microorganisms. This result was in accordance with the tests for the antimicrobial activity which demonstrated that the number of the bacterial cells increased up to ca. 5.5 log units after 4 h contact of the CA and CA/PVP mats with S. aureus. In contrast, non-adhered bacterial cells were detected onto the surface of the electrospun mats containing Curc (Curc/CA + Curc/PVP) (Fig. 12B). This was due to the antibacterial properties of the incorporated biologically active substance. This result was in agreement with the results obtained from the microbiological test where it was shown that the incorporation of Curc into the CA and CA/PVP mats resulted in obtaining fibrous materials with antibacterial activity.
Fig. 11. Logarithmic plot of the number of viable bacteria versus the exposure time (in h). Test has been carried out against S. аureus. Curc content was 10 wt% in respect to the polymer weight.
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Fig. 12. SEM micrographs of A. CA mat and B. Curc/CA + Curc/PVP mat that were incubated in broth culture of S. aureus with concentration of 105 cells/ml for 4 h at 37 °C. Nutrient Broth (Sigma-Aldrich) was used for cultivation of S. aureus.
4. Conclusion Novel CA and CA/PVP fibrous materials containing biologically active substance – Curc were prepared by one-pot electrospinning or dual spinneret electrospinning. The composition of the polymer matrix and the preparation method influenced the design of the mat, the hydrophilic/hydrophobic properties and the Curc release profile. The incorporation of a water-soluble polymer resulted in hydrophilization of the mat and more rapid release of Curc. The incorporation of Curc into the CA and CA/PVP fibrous materials imparted them antibacterial properties against S. aureus. Moreover, the use of dual-spinneret electrospinning enabled the preparation of fibers with more complex architecture allowing more easily to modulate the Curc release profile and hence the antibacterial properties of the obtained materials. The Curc/ CA + Curc/PVP mat prepared by dual-spinneret electrospinning killed all the bacteria at the 4th h. The results suggest that the obtained novel antibacterial fibrous materials containing Curc are potential candidates for wound dressing applications. Acknowledgments The authors thank the National Science Fund of Bulgaria for the financial support (Grant number DFNI–T02/1-2014). References [1] X. Liu, T. Lin, Y. Gao, Z. Xu, C. Huang, G. Yao, L. Jiang, Y. Tang, X. Wang, Antimicrobial electrospun nanofibers of cellulose acetate and polyester urethane composite for wound dressing, J. Biomed. Mater. Res., Part B 100B (2012) 1556–1565. [2] Z. Cao, X. Luo, H. Zhang, Z. Fu, Z. Shen, N. Cai, Y. Xue, F. Yu, A facile and green strategy for the preparation of porous chitosan-coated cellulose composite membranes for potential applications as wound dressing, Cellulose (2016) http://dx.doi.org/10. 1007/s10570-016-0860-y (available online). [3] V. Thakur, M. Thakur, Processing and characterization of natural cellulose fibers/ thermoset polymer composites, Carbohydr. Polym. 109 (2014) 102–117. [4] J. Boateng, K. Matthews, H. Stevens, G. Eccleston, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci. 97 (2008) 2892–2923. [5] S. Moghadamtousi, H. Kadir, P. Hassandarvish, H. Tajik, S. Abubakar, K. Zandi, A review on antibacterial, antiviral, and antifungal activity of Curc, Biomed. Res. Int. 1 (2014) 1–12, Article ID 186864. [6] O. Khalil, O. Oliveira, J. Vellosa, A. Urba de Quadros, L. Dalposso, T. Karam, R. Mainardes, N. Khalil, Curc antifungal and antioxidant activities are increased in the presence of ascorbic acid, Food Chem. 133 (2012) 1001–1005. [7] T. Ak, I. Gulcin, Antioxidant and radical scavenging properties of Curc, Chem. Biol. Interact. 174 (2008) 27–37. [8] P. Basnet, N. Skalko-Basnet, Curc: an anti-inflammatory molecule from a curry spice on the path to cancer treatment, Molecules 16 (2011) 4567–4598. [9] G. Yakub, A. Toncheva, N. Manolova, I. Rashkov, D. Danchev, V. Kussovski, Electrospun polylactide-based materials for Curc release: photostability, antimicrobial activity, and anticoagulant effect, J. Appl. Polym. Sci. 133 (2015) 42940. [10] P.R. Sarika, N.R. James, P.R. Anil Kumar, D.K. Raj, Preparation, characterization and biological evaluation of Curc loaded alginate aldehyde–gelatin nanogels, Mater. Sci. Eng. C 68 (2016) 251–257. [11] A. Paradkar, A. Ambike, B. Jadhav, K.R. Mahadik, Characterization of Curc–PVP solid dispersion obtained by spray drying, Int. J. Pharm. 271 (2004) 281–286. [12] J. Quirós, K. Boltes, R. Rosal, Bioactive applications for electrospun fibers, Polym. Rev. (2016) http://dx.doi.org/10.1080/15583724.2015.1136641 (available online).
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrospun curcumin-loaded cellulose acetate/polyvinylpyrrolidone fibrous materials with complex architecture and antibacterial activity Petya B. Tsekova a, Mariya G. Spasova a, Nevena E. Manolova a, Nadya D. Markova b, Iliya B. Rashkov a,⁎ a b
Laboratory of Bioactive Polymers, Institute of Polymers, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 103A, BG-1113 Sofia, Bulgaria Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev St, bl. 26, BG-1113 Sofia, Bulgaria
a r t i c l e
i n f o
Article history: Received 22 June 2016 Received in revised form 25 October 2016 Accepted 17 December 2016 Available online 20 December 2016 Keywords: Electrospinning Curcumin Cellulose acetate Polyvinylpyrrolidone Antibacterial activity Wound dressings
a b s t r a c t Novel fibrous materials from cellulose acetate (CA) and polyvinylpyrrolidone (PVP) containing curcumin (Curc) with original design were prepared by one-pot electrospinning or dual spinneret electrospinning. The electrospun materials were characterized by scanning electron microscopy (SEM), fluorescence microscopy, Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV–Vis), differential scanning calorimetry (DSC), water contact angle measurements, and microbiological tests. It was found that the incorporation of Curc into the CA and PVP solutions resulted in an increase of the solution viscosity and obtaining fibers with larger diameters (ca. 1.5 μm) compared to the neat CA (ca. 800 nm) and PVP fibers (ca. 500 nm). The incorporation of PVP resulted in increased hydrophilicity of the fibers and in faster Curc release. Curc was found in the amorphous state in the Curc-containing fibers and these mats exhibited antibacterial activity against Staphylococcus aureus (S. aureus). The results suggest that, due to their complex architecture, the obtained new antibacterial materials are suitable for wound dressing applications, which necessitate diverse release behaviors of the bioactive compound. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Wound care dressings play an important role in the wound healing process and form an important part of the global medical wound care products market. Various natural biopolymers such as starch, cellulose, collagen, chitosan, keratin, alginate, and elastin were used for the preparation of wound dressing products [1,2]. Cellulose is the most abundant naturally occurring polysaccharide. Cellulose can be converted upon acetylation to cellulose acetate. The advantage of the resultant cellulose acetate is that it can be easily dissolved or melt and could be shaped into fibers, films, sheets, tubes, pellets or other products. In recent decades, great attention has been paid to fibers from cellulose and cellulose derivatives due to their low cost, lightweight, easy processing, biodegradability, good mechanical and barrier properties and recycling [3]. Bioactive wound dressings have been produced from biomaterials and take active part in the healing process. These types of dressings are sometimes loaded with naturally occurring compounds, antibiotics, vitamins, minerals, enzymes, growth factors and antimicrobials to enhance wound healing process [4]. Curcumin is a biologically active substance found in the roots of Curcuma longa plant. This naturally occurring polyphenolic compound manifests remarkable antibacterial [5], antifungal [6], antioxidant [7], anti-inflammatory [8], anticoagulant [9] and antitumor [10] activity. However, Curc has some ⁎ Corresponding author. E-mail address: [email protected] (I.B. Rashkov).
http://dx.doi.org/10.1016/j.msec.2016.12.086 0928-4931/© 2016 Elsevier B.V. All rights reserved.
drawbacks. It is thermally and chemically instable and easily undergoes photodestruction. The main disadvantage of Curc is its low bioavailability due to its poor aqueous solubility. Incorporation of Curc in polymer matrix is expected to contribute to obviating these drawbacks. Improved dissolution of Curc has been achieved by preparation of CurcPVP solid dispersion by spray drying [11]. In the recent years, electrospinning has emerged as a very suitable technique for preparation of drug-loaded polymeric materials. Nanofibrous nonwoven materials produced by electrospinning have shown great potential as drug-eluting stents and wound dressing materials [12–15]. The large specific surface area of the electrospun materials and the possibility for extended drug release lead to enhancement of the therapeutic effect of the embedded drugs and reducing their side effects. It has been shown that electrospun fibrous materials are suitable carriers for enhancing the bioavailability of Curc. Curcumin has been encapsulated into electrospun material from cellulose acetate [16–18], poly(L-lactide-co-D,L-lactide) [19], poly(ε-caprolactone) [20], poly(εcaprolactone)/polyethylene glycol [21], poly(ethylene glycol)/ poly(butylene succinate) [22], PVP [23], etc. Previously, we have shown that Curc release from electrospun polylactide materials was facilitated by the presence of PVP or polyethylene glycol due to formation of hydrogen bonds with Curc [9]. The aim of the present study was to obtain novel electrospun materials from CA and PVP for Curc delivery. The possibility to modulate the Curc release profile by appropriate selection of the composition of the polymer matrix and the preparation technique (one-pot
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electrospinning or dual spinneret electrospinning) was studied. The effect of the composition and architecture of the mats on the biological behavior upon contact with pathogenic microorganisms (S. aureus) was evaluated.
2. Experimental 2.1. Materials Cellulose acetate (CA, Aldrich) with Mn30,000 g/mol and acetyl content 39.8%, polyvinylpyrrolidone (PVP, Fluka) with Mr = 360.000 g/mol and curcumin (Curc, Merck) were used. Acetone was of analytical grade of purity and was purchased from Sigma-Aldrich.
2.2. Light source Curc was excited with a light emitting-diode (LED) in the blue region of the spectrum [24]. The device (LTM Electronics Ltd., Bulgaria) provided a light emission with predominant central wavelength of 450 nm at intensity (power density) of 11.375 mW cm−2. The irradiances (energy fluency) tested in the study were 82 and 164 J cm−2 and the irradiation was performed in a non-contact mode with a focused beam at 10 cm of working distance (distance between the light source and cell line surface). The system, which was composed of 8 LEDs, could uniformly deliver irradiation without a heating effect. The distance between the LED and the plate was intended to enable an even distribution of light on each well. The power density of the incident radiation was measured using a LabMax TOP PM10 laser power and energy meter (Coherent®, Santa Clara, CA, USA).
2.3. Preparation of fibrous materials by electrospinning In the present study CA, PVP, Curc/CA, Curc/PVP and Curc/CA/PVP fibrous materials were fabricated by one-pot electrospinning. The mats were prepared from the following polymer solutions: CA in acetone/ water 80/20 v/v; PVP in acetone/water 50/50 v/v; Curc/CA in acetone/ water 80/20 v/v; Curc/PVP in acetone/water 50/50 v/v and Curc/CA/ PVP in acetone/water 70/30 v/v. The total polymer concentration was 10 wt%, Curc was 10% in respect to polymer(s) weight. The spinning solutions were loaded in a syringe equipped with a metal needle (gauge: 20GX1½″) connected to the positively charged electrode of the high-voltage power supply (up to 30 kV). Electrospinning was conducted at a constant applied voltage of 25 kV and constant tip-to-collector distance of 15 cm using a grounded rotating aluminum collector (1000 rpm). The spinning suspensions were delivered at a constant rate of 3 ml/h enabled by the use of a pump Syringe Pump NE-300 (New Era Pump Systems, Inc.).
2.4. Preparation of fibrous materials by dual spinneret technique Two hybrid fibrous materials were fabricated by dual spinneret technique. These materials were prepared by using: two pumps for delivering: (i) Curc/CA solution [10 wt% CA and 10 wt% Curc in acetone/ water = 80/20 (v/v)], and (ii) PVP solution - 10 wt% (w/v) in acetone/ water = 50/50 (v/v) or (iii) Curc/CA solution [10 wt% CA and 10 wt% Curc in acetone/water = 80/20 (v/v)], and (iv) Curc/PVP solution [10 wt% PVP and 10 wt% Curc in acetone/water = 50/50 (v/v)]. The pumps for delivering the spinning solutions were located at an angle of 180° in respect to the rotating collector (1000 rpm). The delivery rate of the spinning solutions was 3 ml/h. The tip-to-collector distance was 15 cm. The applied voltage was provided using a common highvoltage power supply at a constant applied voltage of 25 kV.
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2.5. Characterization of the fibrous materials The rheological measurements of the used solutions and suspensions were performed using a Brookfield, DV – II + Pro, spindle CPE – 52, at 25°С. The morphology of the fibrous materials was evaluated by scanning electron microscopy (SEM). The samples (1 cm2) were vacuum-coated with gold and were observed with Jeol JSM-5510 (Jeol Ltd., Japan). The mean fiber diameter was estimated by Image J software [25], by measuring the diameters of at least 20 random fibers per sample from three different SEM micrographs for a total of 60 measurements and their morphology was assessed applying the criteria for overall evaluation of electrospun materials as described in details in [26]. The mats (2.5 cm2) were observed by a fluorescence microscope (NU-2; Carl Zeiss, Jena, Germany) maximum excitation wavelength (λex) at 420 nm and at maximum emission wavelength (λem) 470 nm. Infrared spectra (Fourier transformed) of the mats (1 cm2) were recorded using an IRAffinity-1 spectrophotometer (Shimadzu Co., Japan), supplied with a MIRacle ATR device (diamond crystal; depth of penetration of the IR beam into the sample - approximately 2 μm) (PIKE Technologies, USA) in the range of 600–4000 cm− 1 with a resolution of 4 cm−1. All spectra were corrected for H2O and CO2 using an IRsolution software program. The thermal behavior of the obtained fibrous materials was evaluated by differential scanning calorimetry (DSC). The samples were heated in the temperature range from 0 to 300 °C at heating rate of 10 °C/min under nitrogen (TA Instruments, DSC Q2000, USA). The crystallinity degree of CA fibers was calculated using the value for the enthalpy of fusion of a perfect crystal of cellulose acetate of 58.8 J/g determined by Cerqueira et al. [27]. X-ray diffraction (XRD) analyses were performed at room temperature using a computer-controlled D8 Bruker Advance ECO powder diffractometer with filtered Cu Kα radiation. Data were collected in the 2θ range from 5° to 60° with a step of 0.02° and counting time of 1 s step−1. The water contact angles of the fibrous materials were measured using an Easy Drop DSA20E KRÜSS GmbH apparatus, Germany. Drops of distilled water with a volume of 10 μl were deposited on the surface of the test specimens (2 cm × 7 cm; cut in the direction of the collector rotation). The mean contact angle value was determined after averaging at least 10 measurements for each specimen. In addition, Curc/CA/PVP mat was placed in a desiccator for 3 days at 20 °C at 96% relative humidity and then the water contact angle was measured. 2.6. Curc release from the fibrous materials The Curc content in the mats was determined after dissolving each mat (1 cm2–6 mg) in 10 ml acetone/water = 80/20 w/w, after which the absorbance was measured by a DU 800 UV spectrophotometer (Beckman Coulter) at a wavelength of 421 nm. The release was studied in vitro at 37 °C, in acetate buffer at pH 5.5 and ionic strength 0.1 (CH3COONa/CH3COOH) containing PVP (acetate buffer/PVP = 98/2 v/v). In a typical process, 6 mg of mats were immersed in 100 ml buffer solution stirred at 150 rpm with an electromagnetic stirrer. Aliquots were withdrawn at determined time intervals and their absorbance was recorded at a wavelength of 440 nm. The amount of released Curc over time was calculated using calibration curves (correlation coefficient R = 0.999) for the mats in acetate buffer/PVP 20 (98/2 v/v), pH = 5.5, ionic strength 0.1. 2.7. Microbiological assays The antibacterial activity of the fibrous materials against Gram-positive S. aureus was evaluated by using the viable cell-counting method as described below. S. aureus (NBIMCC 749) was purchased from the National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC,
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Bulgaria). For comparison, the antibacterial activity of CA and PVP against S. aureus was assessed as well. The electrospun mats (0.05 g) were exposed to bacterial suspension (2 ml) with concentration of 105 cells/ml−1 prepared in nutrient broth (Sigma-Aldrich) at 37 °C. In view of the photosensitizing properties of Curc related with its antibacterial activity, the samples were illuminated at λmax = 450 nm with a LED lamp in the blue region of the spectrum during the whole experiment. The distance between the lamp (16 W) and the surface of the broth culture was 10 cm. At specified time intervals (2, 4 and 24 h), aliquots of 50 μl were withdrawn from each broth culture which had been in contact with the mats and after serial ten-fold dilutions with sterilized 0.9% saline solution, then plated on Petri dishes with nutrient agar (Sigma-Aldrich). The plates were incubated at 37 °C for 24 h. The number of the survived bacteria was determined by counting the colony forming units (CFU) in triplicate for each experiment. Evaluation of the adhesion of S. aureus to the surface of the mats was performed by direct SEM observation with Jeol JSM-5510 (Jeol Ltd., Japan). Briefly, the mats were incubated in 2.0 ml broth culture of S. aureus with concentration of 105 cell/ml for 4 h. Then the samples were washed twice with phosphate buffered saline (PBS, pH 7.4) for removal of non-adhered bacteria. The adhered bacteria on the surface of the mats were fixed by immersion of the mats in 2.5 wt% glutaraldehyde solution in PBS at 4 °C for 5 h. Then the samples were washed carefully with PBS and freeze-dried. After 4-h contact of the mats with S. aureus cells and coating of the mats with gold, the bacterial morphology was observed by SEM. 3. Results and discussion In the present work, Curc/CA, Curc/PVP, Curc/CA/PVP, Curc/ CA + PVP and Curc/CA + Curc/PVP electrospun mats were prepared and characterized. For comparison CA, PVP and (CA/PVP) mats were obtained as well. Schematic representation of the cross-section of fibers is shown in Fig. 1. The mats prepared by dual spinneret electrospinning are composed of two types of fibers: CA fiber with Curc and PVP fiber with/without Curc. The distribution was confirmed by the results obtained from SEM and fluorescence microscopy.
Curc/PVP and Curc/CA/PVP solutions was 156 cP, 163 cP and 151 cP, respectively. The increase in solution viscosity was most probably due to the formation of hydrogen bonds between the polymer(s) and Curc [9]. 3.2. Preparation of fibrous materials by electrospinning Fibrous materials of CA, PVP, Curc/CA, Curc/PVP and Curc/CA/PVP were prepared by one-pot electrospinning of solutions of CA, PVP and Curc, respectively. Based on the conducted preliminary experiments the following optimal conditions for electrospinning were found: polymer concentration of 10 wt% for the CA and for the PVP solutions, and tip-to-collector distance - 15 cm in order to obtain defect-free fibers. Representative SEM images of the obtained CA, Curc/CA, PVP, Curc/ PVP and Curc/CA/PVP electrospun mats are shown in Fig. 2. The electrospinning of CA solutions under the selected conditions reproducibly resulted in obtaining fibers with mean fiber diameter of 780 ± 110 nm (Fig. 2A). The mean fiber diameter of the PVP fibers was 495 ± 113 nm (Fig. 2C). However, some defects were detected. The incorporation of Curc into the CA and PVP solutions resulted in obtaining fibers with larger diameters compared to the neat CA and PVP fibers. The increase in fiber diameter and the absence of defect of the hybrid fibers was attributed to the increase in the viscosity of the Curc/CA and Curc/PVP solutions, 156 cP and 163 cP, respectively. It is well known that the increase in solution viscosity results in an increase of the mean fiber diameter [29]. The mean fiber diameter of the hybrid Curc/CA and Curc/PVP fibers was 1150 ± 260 nm and 570 ± 125 nm, respectively (Fig. 2B and D). The mean fiber diameter of the obtained Curc/CA/PVP fibers was 1560 ± 145 nm (Fig. 2E). It is known that Curc possesses fluorescence properties, which enable observation of the prepared mats by fluorescence microscopy [30]. As expected, the CA and PVP fibers did not display any fluorescence (micrographs not shown). In contrast, all the Curc-loaded mats were characterized by continuous intense fluorescence signal all over the fiber length. This provides evidence that the biologically active substance is uniformly distributed into the fibers. Fluorescence micrographs of Curc/CA, Curc/PVP and Curc/CA/PVP mats are shown in Fig. 2 inset.
3.1. Viscosity of the spinning solutions
3.3. Preparation of fibrous materials by dual spinneret electrospinning
It is well known that the viscosity of the spinning solutions has significant influence on the electrospinning process and the resultant fiber morphology [28]. The dynamic viscosity of the solutions used in this study was measured. The viscosity of the CA, PVP and CA/PVP solutions in acetone/water was 122 cP, 153 cP and 142 cP, respectively. The incorporation of Curc into the CA, PVP and CA/PVP solutions resulted in an increase of the viscosity. The measured viscosity for the Curc/CA,
In the present study the dual spinneret electrospinning method was used for the preparation of fibrous materials with different composition and architecture. The following mats were obtained: Curc/CA + PVP and Curc/CA + Curc/PVP. The solution concentration of CA and PVP was 10 wt% and the concentration of Curc was 10 wt% in respect to the polymer weight. SEM micrographs of the obtained Curc/CA + PVP and Curc/CA + Curc/PVP fibers are presented in Fig. 3A and B.
Fig. 1. Schematic representation of the cross-section of fibers: A. Curc/CA, B. Curc/PVP, C. Curc/CA/PVP, D. Curc/CA + PVP, E. Curc/CA + Curc/PVP.
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Fig. 2. SEM micrographs and fluorescence micrographs (insets) of fibrous materials from: A. CA; B. Curc/CA; C. PVP; D. Curc/PVP, E. Curc/CA/PVP; solvent acetone/water (80/20 v/v) for CA and Curc/CA; acetone/water (50/50 v/v) for PVP and Curc/PVP and acetone/water (75/25 v/v) for Curc/CA/PVP;
It can be seen that the dual spinneret electrospinning of Curc/CA and PVP solution resulted in preparation of fibrous materials consisted of Curc/CA ribbon-like fibers with mean ribbon width 1145 ± 320 nm and PVP fibers with defects with mean fiber diameter 215 ± 45 nm. This was confirmed by the fluorescent microscopy as well. In contrast to the PVP fibers, the Curc-containing fibers displayed fluorescence. The dual spinneret electrospinning of Curc/CA and Curc/PVP solutions resulted in obtaining a more uniform mat with mean fiber diameter of 1528 ± 155 nm.
3.4. IR-spectra of the fibrous materials The FTIR-spectra of Curc (powder) and Curc/CA/PVP fibers are presented in Fig. 4. The Curc spectrum showed a band at 3510 cm− 1 which was attributed to the phenolic O\\H stretching vibration (Fig. 4A inset). Strong bands at 1625 cm− 1 (stretching vibrations of the benzene ring), 1506 cm− 1 (C _O and C _C vibration) and 1427 cm−1 (olefinic C\\H bending vibration) were detected as well. Enol C\\O band was detected at 1274 cm−1. The bands at 808 cm− 1
Fig. 3. SEM micrographs and fluorescence micrographs (insets) of: A. Curc/CA + PVP mat, and B. Curc/CA + Curc/PVP mat.
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Fig. 4. IR-spectra of: (A) Curc and (B) Curc/CA/PVP mat in the range from 1800 to 800 cm−1 and from 4000 to 3200 cm−1 (inset).
and 962 cm−1 were attributed to the bending vibrations of the C\\H bond of alkene groups (RCH = CH2). The FTIR spectrum of Curc/CA/PVP electrospun mat is shown in Fig. 4B. In the presented spectrum, peaks for the C _O functional groups at 1740 cm− 1, the O\\H functional groups at ~ 3500 cm− 1, the CH3 groups at 1369 and 1226 cm−1, and well as the ether C–O–C functional groups at 1037 cm−1 characteristic for the CA were observed [31]. In addition, bands of PVP at 1651 cm−1, 1286 cm−1 and 1422 cm−1 characteristic for C _O, С\\N and СН stretching vibrations were detected. The broad peak at about 3050–3620 cm−1 with at a maximum at 3414 cm− 1 attributed to stretching vibrations of the N\\H group of PVP is observed [32,33]. In the spectra of Curc/CA/PVP mat characteristics bands for the Curc are also detected. As expected they are with low intensity due to the relatively low Curc quantity in the polymer mat (10 wt% in respect to the
polymer weight). It should be noted that in Curc/CA/PVP mat, the O\\H stretching vibration at 3510 cm− 1 characteristic for the Curc was disappeared. Therefore, it can be concluded that hydrogen bonding between Curc and PVP takes place. 3.5. Thermal characteristics of the fibrous materials Representative DSC thermograms of the prepared fibrous materials are shown in Fig. 5. Thermograms of CA, PVP and Curc/СА mat as well as of Curc powder are presented in Fig. 5A. CA fibers showed a broad endothermic peak between ambient temperature (25 °C) and 100 °C with a peak maximum at 70° С. This event is attributed to desorption of water from cellulose acetate [34,35]. Endothermic transition corresponding to Tg at 195° С and endothermic peak at 225° С corresponding to Tm of the cellulose acetate were observed. The obtained results are in
Fig. 5. DSC thermograms (first heating run) of: A. Curc powder and CA, PVP and Curc/CA mat and B. Curc powder and CA/PVP, Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP mats.
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very good agreement with literature data presenting thermal characteristics of cellulose acetate fibers prepared by electrospinning [36]. The calculated crystallinity of the CA fibers was 16%. The incorporation of Curc into CA fibers resulted in a decrease to 162 °C and 206 °C for the Tg and Tm, respectively. However, Curc incorporation did not lead to a significant change in degree of crystallinity of CA - 14%. It can be assumed that there is an interaction between CA and Curc resulting in decrease of Tg and Tm, Interaction based on hydrogen bonds between CA and Curc has been reported by other authors [31]. It is noteworthy that the incorporated Curc in the CA fibers is in the amorphous state as evidenced by the lack of an endothermic peak corresponding to the melting of Curc at about 175 °C. The amorphous state of Curc exhibits a disordered structure in comparison to its crystalline state and possesses higher free energy. Thus it provides enhanced apparent solubility, dissolution rate and bioavailability [37]. DSC thermogram of PVP mat is shown in Fig. 5A. A broad peak was observed between ambient temperature and 100° С due to the water absorbed. Moreover, the PVP used in the present study was a high-molecular-weight polymer with Tg of 173° С (Fig. 5A). The incorporation of Curc into PVP fibers resulted in decrease of the glass transition temperature to 149°С (DSC curves not shown). We assumed that the shift of the Tg to lower temperatures can be attributed to interaction between PVP and Curc. Curc presented into the PVP fibers was in amorphous state (no peak corresponding to the melting point of Curc was observed). This result is consistent with previously reported data [9]. DSC thermograms of CA/PVP, Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP mats are presented in Fig. 5B. All CA and PVP mats showed broad endothermic peak between ambient temperature (25 °C) and 100 °C, corresponding to the loss of moisture. It could be seen that the incorporated Curc into the fibrous mats was in the amorphous state. The crystallinity degree of the Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP mats was ca. 9% - a value which is lower than the degree of crystallinity of the neat CA fibers (16%). 3.6. X-ray diffraction XRD pattern of Curc powder, CA and Curc/CA/PVP mats recorded in 2θ range from 5 to 60° are presented in Fig. 6. The presence of the main diffraction peaks of pure Curc powder located at 2θ = 8.9°, 14.5°, 17.2°, 18.2°, and 23.3° were detected. Characteristic peaks for Curc were absent in the XRD pattern of Curc/CA/PVP mat, thus implying that Curc was in amorphous state. This result was in accordance with the DSC results where no peak corresponding to Curc melting was observed.
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depend on the mat composition. CA fibrous materials were hydrophobic (123.10 ± 2.0°, Fig. 7A) and the water droplet preserved its spherical shape on the CA mats within more than 2 h. The measured contact angle value of the Curc/CA mats was even higher with equilibrium contact angle 129.4 ± 3.8° (Fig. 7C). The addition of a water-soluble polymer – PVP led to hydrophilization of the fibers and to more rapid absorption of the droplet by the mat (Fig. 7B, D). The starting contact angles of CA/PVP and Curc/CA + PVP were 36° ± 3.5° and 14.8 ± 1.7°, respectively. However, within 5 min. after droplet deposition the contact angle decreased to 0°. The equilibrium contact angle values of the mats containing PVP were rapidly reached thus implying that these values were directly related to surface structure reorganization. It is known that when a droplet is deposited onto a polymer material, the hydrophilic component of polymer is reorganized toward the surface in order to reduce the surface tension which results in decreased contact angle value [38]. The effect of depositing time on the contact angles of Curc/CA/PVP and Curc/CA + Curc/PVP mats is shown in Fig. 8. The incorporation of Curc into the fibers resulted in hydrophobization of the Curc/CA/PVP (117.04 ± 1.8°) (Fig. 8A) and Curc/CA + Curc/PVP (121.8 ± 3.4°) fibrous materials (Fig. 8C). However, as seen from Fig. 8, within 120 min. the contact angle value reached 0°. This behavior is in accordance with findings for other systems [38] where the starting contact angle is contributed by the hydrophobic component on polymer surface and the equilibrium contact angle mainly by the hydrophilic component of polymer. Additional experiments were performed in order to prove this statement. Curc/CA/PVP mat was placed for 3 days at 20 °C at 96% relative humidity. Then, the contact angle was measured. The starting contact angle was lower (84.3 ± 5.4°) compared to the measured starting contact angle of the same mat not placed in water vapour medium (117.04 ± 1.8°). This evidenced that surface reorganization occurs and fragments of the hydrophilic PVP are oriented toward the mat surface which resulted in decrease of the measured contact angle. Moreover, Curc/CA/PVP mat which was placed in medium rich in water vapour reached the value of the contact angle 0° more rapidly. It could be concluded that time needed to reach the equilibrium contact angle value is very short for the hydrophobic CA and Curc/CA mats and for the water-soluble PVP and Curc/PVP mats (1 min), fast for the CA/PVP and Curc/CA + PVP (5 min) and much longer for Curc/CA/PVP and Curc/CA + Curc/PVP mats (2 h). Measuring the equilibrium contact angle is important for finding a relationship between composition of the mat and potential application of the mats as wound healing dressings. For example, it is well known
3.7. Water contact angle and sorption kinetics of the fibrous materials The time dependence of contact angles was investigated by the dynamic contact angle measurements over time using static sessile drop method. It was found that the starting and equilibrium contact angles
Fig. 6. XRD patterns of Curc powder, CA mat and Curc/CA/PVP mat.
Fig. 7. Digital photographs of water droplets (10 μl) on the surface of mats: A. CA; B. CA/ PVP, C. Curc/CA and D. Curc/CA + PVP (The measurements were performed immediately after water droplet deposition).
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Fig. 8. Dependence of the contact angle as a function of time: A. Curc/CA/PVP, B. Curc/CA/PVP, placed in a desiccator over water for 3 days and C. Curc/CA + Curc/PVP mat.
that in wound different amounts of exudate rich in dead cells, tissue fragments, dirt and bacteria, is initially produced. If large amounts of exudate remain in the wound, the recovery is hampered and the risk of infection increases. For this reason, an excessive amount of exudate should be absorbed by the dressing [39]. Thus, bacteria, harmful metabolic products, devitalized tissue, dirt and foreign bodies are removed from the wound and do not need to be eliminated from the body phagocytosis. It is anticipated that the mats containing PVP, which have low contact angle values and quickly reach equilibrium value will successfully find potential application as wound dressings for open wounds, where more exudate leakage is expected. Producing a set of mats with diverse complex architecture based on cellulose acetate, water-soluble polymer - PVP and biologically active substance - Curc will enable their application depending on the specific medical case (type of wound) to achieve proper treatment and successful treatment outcome. 3.8. In vitro release studies of Curc Based on the fact that PVP is used as solubilizing agent of poorly water-soluble drugs to improve their dissolution rate and oral absorption [40], and on our previous findings [9] that Curc release from polylactide materials was facilitated by the presence of PVP, in this study PVP was used as solubilizing agent of Curc. To check and visualize the improved solubility of Curc in the presence of PVP, two solutions with the same concentration of Curc (0.2% wt/v) were prepared: in water and in aqueous solution of PVP (2% wt/v). As seen from Fig. 9, Curc was practically insoluble in water and stayed on the top of the liquid phase. In contrast, when PVP was present in the aqueous phase, Curc
was well wetted and the solution had the characteristic yellowish colouration of Curc which proved that PVP enhanced Curc solubility. Curc release from Curc/CA, Curc/CA/PVP, Curc/CA + PVP and Curc/ CA + Curc/PVP mats was studied in acetate buffer/PVP (98/2 v/v). As seen (Fig. 10), Curc was released most slowly and in the smallest amount (ca. 10% or 0.5 μg/ml for 120 min) from the hydrophobic Curc/CA mat even in the presence of water-soluble polymer - PVP in the dissolution medium. The Curc was more rapidly released when PVP was included in the fibers. The amount of the released bioactive substance from the Curc/CA/PVP fibers was about ca. 22% (1.2 μg/ml) for 120 min. Furthermore, Curc was released to the greatest extent from the mats obtained by dual spinneret electrospinning – c.a. 56% (3 μg/ml) and 68% (3.7 μg/ml), from the Curc/CA + PVP and Curc/ CA + Curc/PVP mats, respectively. In the present study it was found that the greater hydrophilicity of the PVP-containing mats as compared with Curc/CA fibers assists the penetration of the dissolution medium in the mats. This result is with accordance with our previous results that show that Curc was released more rapidly when the water soluble polymer (PVP or PEG) was included in the fibers of PLA/PVP or PLA/PEG [9]. The obtained results showed that the electrospun PVP-containing fibrous materials could be used to create advanced release systems with enhanced solubility of the poorly water-soluble Curc. Moreover, the use of electrospinning or dualelectrospinning enables the preparation of fibrous materials with diverse architecture and hence – with different release profile of Curc. This allows for obtaining materials suitable for different types of wounds, e.g. mats containing PVP for strongly exuding wounds or Curc-containing mats for infected wounds.
Fig. 9. Photographs of: A. Curc (0.02 g) in water (10 ml) and B. Curc (0.02 g) in PVP solution (PVP concentration - 2 wt% in water).
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Fig. 10. Curc release from the Curc/CA (▲), Curc/CA/PVP (◊), Curc/CA + PVP (□) and Curc/CA + Curc/PVP (■) mats. The results are presented as average values from three separate measurements with the respective standard deviation; acetate buffer/PVP (98/ 2 v/v), pH 5.5, 37 °C, ionic strength 0.1.
3.9. Microbiological tests Curc has potential as a photosensitizer. By irradiation of Curc with light in the visible range of the spectrum, oxygen species, such as singlet oxygen and free radicals, are formed, which then produce an effect that is toxic to the bacterial cell [41]. Therefore, the antibacterial activity of the fibrous CA, CA/PVP, Curc/CA, Curc/CA/PVP, Curc/CA + PVP and Curc/CA + Curc/PVP materials against S. aureus was tested in nutrient broth (Sigma-Aldrich) by irradiation with blue light during the whole experiment. The number of survived bacteria was subsequently assessed by plating and counting of CFU in solid medium. The log of the survived bacteria versus the exposure time for the electrospun mats is presented in Fig. 11. For comparison the growth of a control of the S. aureus was assessed as well. It was found that the control developed normally during the experiment. As seen from Fig. 11, significant decrease in the number of the viable cells was detected for the exposure time of 2 h for the mats containing Curc. The Curc/CA, Curc/CA/PVP and Curc/CA + PVP mats manifest antibacterial activity and for the contact of 4 h, a decrease of S. aureus titer by 2.3, 2.8 and 3 log units was attained. This is due to Curc release
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from the mats. Curc release from Curc/CA, Curc/CA/PVP, Curc/CA + PVP mats was 10, 22 and 56% for 120 min, respectively. This result is in accordance with our previous finding on the antibacterial activity of PLA/PVP/curcumin fibrous mats [9]. Moreover, the Curc/CA + Curc/ PVP mat kills all the bacteria at the 4th h. This is due to fact that the Curc was released to the greatest extent from the Curc/CA + Curc/PVP mat – 68%. The studies of Fallah [42] have also revealed that nanofibers of PCL/gelatin/Curc were 99.9% antibacterial against methicillin-resistant S. aureus. In contrast, neat CA and CA/PVP mats not containing the biologically active substance did not alter the bacterial growth and did not exhibit antibacterial activity. A slight increase in the number of viable cells was detected at the 24th h for the Curc/CA, Curc/CA/PVP and Curc/CA + PVP mats. This is most probably due to fact that Curc was released to the greatest extent in the first 2–3 h. A significant increase in the number of the viable bacteria was determined for the CA and CA/ PVP mats which was close to the growth of the S. aureus control. It may be noticed that while the release profile of Curc for Curc/CA/PVP and Curc/CA + PVP mat was different, the antibacterial activity was the same. This behavior may be attributed to the fact that both types of mats release Curc (up to 24 h) in the range which is above the MIC against S. aureus, thus resulting in reduction of survived cells with 3 log. In order to evaluate the attachment of the bacterial cells to the surface of the fibrous materials, the CA, CA/PVP, Curc/CA, Curc/CA/ PVP, Curc/CA + PVP and Curc/CA + Curc/PVP materials were incubated for 4 h in a broth culture of S. aureus. Then, the bacterial cells were fixed with glutaraldehyde solution, washed with distilled water, freeze-dried and characterized by SEM. It was found that S. aureus cells adhered to the CA mat not containing the biologically active substance (Fig. 12A). However, the cell morphology was not altered (cells remained round and with smooth surfaces). This indicated that the CA mats did not alter the growth of pathogenic microorganisms. This result was in accordance with the tests for the antimicrobial activity which demonstrated that the number of the bacterial cells increased up to ca. 5.5 log units after 4 h contact of the CA and CA/PVP mats with S. aureus. In contrast, non-adhered bacterial cells were detected onto the surface of the electrospun mats containing Curc (Curc/CA + Curc/PVP) (Fig. 12B). This was due to the antibacterial properties of the incorporated biologically active substance. This result was in agreement with the results obtained from the microbiological test where it was shown that the incorporation of Curc into the CA and CA/PVP mats resulted in obtaining fibrous materials with antibacterial activity.
Fig. 11. Logarithmic plot of the number of viable bacteria versus the exposure time (in h). Test has been carried out against S. аureus. Curc content was 10 wt% in respect to the polymer weight.
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Fig. 12. SEM micrographs of A. CA mat and B. Curc/CA + Curc/PVP mat that were incubated in broth culture of S. aureus with concentration of 105 cells/ml for 4 h at 37 °C. Nutrient Broth (Sigma-Aldrich) was used for cultivation of S. aureus.
4. Conclusion Novel CA and CA/PVP fibrous materials containing biologically active substance – Curc were prepared by one-pot electrospinning or dual spinneret electrospinning. The composition of the polymer matrix and the preparation method influenced the design of the mat, the hydrophilic/hydrophobic properties and the Curc release profile. The incorporation of a water-soluble polymer resulted in hydrophilization of the mat and more rapid release of Curc. The incorporation of Curc into the CA and CA/PVP fibrous materials imparted them antibacterial properties against S. aureus. Moreover, the use of dual-spinneret electrospinning enabled the preparation of fibers with more complex architecture allowing more easily to modulate the Curc release profile and hence the antibacterial properties of the obtained materials. The Curc/ CA + Curc/PVP mat prepared by dual-spinneret electrospinning killed all the bacteria at the 4th h. The results suggest that the obtained novel antibacterial fibrous materials containing Curc are potential candidates for wound dressing applications. Acknowledgments The authors thank the National Science Fund of Bulgaria for the financial support (Grant number DFNI–T02/1-2014). References [1] X. Liu, T. Lin, Y. Gao, Z. Xu, C. Huang, G. Yao, L. Jiang, Y. Tang, X. Wang, Antimicrobial electrospun nanofibers of cellulose acetate and polyester urethane composite for wound dressing, J. Biomed. Mater. Res., Part B 100B (2012) 1556–1565. [2] Z. Cao, X. Luo, H. Zhang, Z. Fu, Z. Shen, N. Cai, Y. Xue, F. Yu, A facile and green strategy for the preparation of porous chitosan-coated cellulose composite membranes for potential applications as wound dressing, Cellulose (2016) http://dx.doi.org/10. 1007/s10570-016-0860-y (available online). [3] V. Thakur, M. Thakur, Processing and characterization of natural cellulose fibers/ thermoset polymer composites, Carbohydr. Polym. 109 (2014) 102–117. [4] J. Boateng, K. Matthews, H. Stevens, G. Eccleston, Wound healing dressings and drug delivery systems: a review, J. Pharm. Sci. 97 (2008) 2892–2923. [5] S. Moghadamtousi, H. Kadir, P. Hassandarvish, H. Tajik, S. Abubakar, K. Zandi, A review on antibacterial, antiviral, and antifungal activity of Curc, Biomed. Res. Int. 1 (2014) 1–12, Article ID 186864. [6] O. Khalil, O. Oliveira, J. Vellosa, A. Urba de Quadros, L. Dalposso, T. Karam, R. Mainardes, N. Khalil, Curc antifungal and antioxidant activities are increased in the presence of ascorbic acid, Food Chem. 133 (2012) 1001–1005. [7] T. Ak, I. Gulcin, Antioxidant and radical scavenging properties of Curc, Chem. Biol. Interact. 174 (2008) 27–37. [8] P. Basnet, N. Skalko-Basnet, Curc: an anti-inflammatory molecule from a curry spice on the path to cancer treatment, Molecules 16 (2011) 4567–4598. [9] G. Yakub, A. Toncheva, N. Manolova, I. Rashkov, D. Danchev, V. Kussovski, Electrospun polylactide-based materials for Curc release: photostability, antimicrobial activity, and anticoagulant effect, J. Appl. Polym. Sci. 133 (2015) 42940. [10] P.R. Sarika, N.R. James, P.R. Anil Kumar, D.K. Raj, Preparation, characterization and biological evaluation of Curc loaded alginate aldehyde–gelatin nanogels, Mater. Sci. Eng. C 68 (2016) 251–257. [11] A. Paradkar, A. Ambike, B. Jadhav, K.R. Mahadik, Characterization of Curc–PVP solid dispersion obtained by spray drying, Int. J. Pharm. 271 (2004) 281–286. [12] J. Quirós, K. Boltes, R. Rosal, Bioactive applications for electrospun fibers, Polym. Rev. (2016) http://dx.doi.org/10.1080/15583724.2015.1136641 (available online).
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