Antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory, and anticancer activities of propolis and its ability to stimulate the immune system and promote wound healing make it a proper component for wound dressing materials. Silver nanoparticles are recognized to demonstrate strong antiseptic and antimicrobial activity; thus, it also could be considered in the development of products for wound healing. Combining propolis and silver nanoparticles can result in improved characteristics of products designed for wound healing and care. The aim of this study was to formulate electrospun fast dissolving mats for wound dressing containing propolis ethanolic extract and silver nanoparticles. Produced electrospun nano/microfiber mats were evaluated studying their structure, dissolution rate, release of propolis phenolic compounds and silver nanoparticles, and antimicrobial activity. Biopharmaceutical characterization of electrospun mats demonstrated fast release of propolis phenolic compounds and silver nanoparticles. Evaluation of antimicrobial activity on
Current advances in formulation of novel drug delivery systems offer a great opportunity to develop new therapies or to enhance the effectiveness of available medical treatments. These advances are particularly relevant to the field of regenerative medicine, including challenging healthcare issues in wound healing and skin repair [
Fast disintegration and release of nonsteroidal anti-inflammatory drug meloxicam have been achieved by incorporating it into PVP-based electrospun mat produced by electrospinning [
Antibacterial protection is a necessary prerequisite for efficient wound healing. Antibacterial agents should be incorporated into wound dressing materials to prevent bacterial colonization and subsequent wound infections [
The aim of this study was to produce and evaluate biopharmaceutical characteristics and antimicrobial activity of electrospun fast releasing antibacterial mats for wound dressings. The structure of electrospun mats, release kinetics of propolis components and Ag NPs, and antimicrobial activity of electrospun PVP mats were evaluated to determine their potential for application in wound healing. Water soluble polymer was chosen to achieve fast disintegration, dissolution, and release of biologically active substances, promoting epithelization and ensuring antibacterial protection. These arguments supported choice and testing of PVP, which is hygroscopic in nature, induces water absorption up to 40% of its weight under atmospheric conditions, and demonstrates appropriate biocompatibility characteristics adequate for biomedical applications [
PVP (Sigma Aldrich; Mw, 1300 000 g/mol) was used in electrospinning of mats.
Propolis ethanolic extract (PEE) was produced by using 70% ethanol aqueous solution for extraction of raw propolis under mixing with a magnetic stirrer IKAMAG® C-MAG HS7 (IKA-Werke GmbH & Co.KG, Staufen, Germany) for 1 h at room temperature. The stirrer rotation speed was 250 rpm. The ratio of raw propolis to extractive agent was 1 : 10. PEE was filtered through a paper filter with 20 to 25
PVP polymer was dissolved in ethanol and PEE for 7 h under stirring. The concentration of PVP in PEE was 6% (mean viscosity of PVP solution was 100 mPa·s) or 8% (mean viscosity of PVP solution was 160 mPa·s).
Colloidal solution of Ag nano/microparticles was prepared by dissolving 10 g of PVP (Sigma Aldrich; Mw, 40 000 g/mol) in 80 mL of ethanol, and after full dissolution, 2 g of AgNO3, 10 mL of water, and 100 mL of ethanol were added to the prepared PVP solution.
Electrospun mats were formed with roller rotating electrospinning equipment “Nanospider
Scheme of electrospinning apparatus “Nanospider” [
Electrospun mats from PVP nano/microfibers were formed by applying 30 kV voltage; the distance between electrodes (4 and 5) was 13 cm; environmental temperature, 21°C ± 2°C; and humidity, 60% ± 5%.
The compositions of PVP solutions used in the production of nano/microfiber mats by electrospinning are shown in Table
Composition of electrospun PVP solutions.
Code of sample | Composition of electrospun solutions |
---|---|
A | PVP (8%) in ethanol |
B | PVP (8%) in PEE |
C | PVP (6%) in ethanol |
D | PVP (6%) in PEE |
B1 | PVP (8%) in PEE with 10% Ag colloidal solution |
B2 | PVP (8%) in PEE with 20% Ag colloidal solution |
D1 | PVP (6%) in PEE with 10% Ag colloidal solution |
The structure of electrospun mats was confirmed by a scanning electron microscope SEM S-3400N (Hitachi, Japan) by applying 10 000-fold (5
A scanning electron microscope (Hitachi S-3400N) with an energy dispersive X-ray spectrometer (Bruker Quad 5040) was used to investigate the composition of electrospun mats.
An infrared absorption spectrum was obtained by using a Fourier transform infrared spectrometer FT-IR Spectrum GX (Perkin Elmer, USA).
Phenolic acids (coumaric, ferulic, caffeic, and vanillic acids) and vanillin were quantified by high-performance liquid chromatography (HPLC) using Agilent 1260 Infinity capillary LC (Agilent Technologies, Inc., Santa Clara, CA, USA) with an Agilent diode array detector and performing separation on ACE C18 column (150 × 0.5 mm, 5
The mobile phase was composed of acetonitrile (solvent A) and 0.5% (v/v) acetic acid in water (solvent B). The linear elution gradient was applied from 1% to 21% of solvent A in B for 25 min. The column temperature was 25°C, the injection volume was 0.2
All the samples were filtered through 0.20
The precise amounts of nano/microfibers (70–300 mg) were placed into 25 mL of aqueous acceptor medium, which was stirred periodically.
The dissolution of electrospun mats produced from 8% PVP in PEE (B) and 8% PVP in PEE with 10% Ag colloidal solution (B1) was visualized by means of an inverted microscope Olympus IX71 equipped with LCAchN40xPH lens (total visual magnification, 40 × 10).
A microscopic slide with a small section of the mat was placed on a microscopic table. In order to identify changes during dissolution, mats were irrigated with distilled water drops until complete dissolution. Changes in the structure were documented after every irrigation step.
The precise amounts of nano/microfibers (70–300 mg) were placed into 25 mL of aqueous acceptor medium, which was stirred periodically.
The solutions and produced electrospun mats were tested for their antibacterial and antifungal activity: 8% PVP in ethanol (A), 8% PVP in PEE (B), 8% PVP in PEE containing 10% Ag colloidal solution (B1), and 8% PVP in PEE containing 20% Ag colloidal solution (B2).
The antibacterial and antifungal activity was determined
Standard strains of nonsporing bacteria
Standard cultures of spore-forming bacteria
The standard fungal
A, B, B1, and B2 sample solutions (1 mL) were placed into sterile Petri dishes under aseptic conditions. Liquid Mueller-Hinton agar (10 mL) preheated to 45°C was added, and both liquids were mixed. The area in the Petri dish was divided into 9 sectors, and each of them was inoculated with standardized suspensions of microbial strains and cultivated for 20–24 h at 35°C. Growth of tested strains indicated no antimicrobial activity.
Positive control was performed by addition of 7% ethanol aqueous solution (1 mL) to standard microorganism cultures in the Mueller-Hinton II agar medium. Negative control was performed by addition of 15% ethanol aqueous solution (1 mL) to standard microorganism cultures in the Mueller-Hinton II agar medium.
Samples of electrospun mats were prepared by cutting the electrospun mats to 30 × 30 mm sheets. All the samples were sterilized with ultraviolet radiation. Microbial cell suspensions at the density of 1 × 108 CFU/mL were used for inoculation of Mueller-Hinton agar media. The samples of sterilized electrospun mats were placed on the surface of Mueller-Hinton agar inoculated with microbial cultures under aseptic conditions. The samples were incubated at 36°C for 24 h for bacteria and at 30°C for 24 h for fungi. The antimicrobial activity of samples was determined by formation the sterile zone around the inserted sample.
The structure of electrospun mats is affected mostly by technological parameters such as applied voltage and distance between electrodes, and especially by properties of the polymer solution, including concentration, surface tension, and nature of solvent [
Figure
SEM images of electrospun nano/microfibers. Magnification ×1000, scale 50
Distribution of electrospun PVP nano/microfibers, produced from 8% PVP solution (a), 6% PVP solution (b), and 8% PVP solution with Ag NPs (c), and 6% PVP solution with Ag NPs (d) by diameter.
Inclusion of Ag NPs into the electrospun PVP solution was associated with the formation of thinner fibers (Figures
Thinner fibers were electrospun using 6% PVP solution, and this could be explained by lower viscosity of solution. When the concentration of PVP in ethanol solution was 6% (C), 53% of the fibers had a diameter ranging from 200 to 400 nm. When the concentration of PVP in the solution was 8% (A), 57% of the produced fibers had a diameter of 400–600 nm.
Inclusion of colloidal Ag NPs into the electrospun solution of PVP in PEE resulted in the formation of thinner and more uniform nano/microfibers. The uniformity of nano/microfiber mats increased when the concentration of PVP in the solution was 6%.
Elemental analysis of PVP mats containing PEE and Ag NPs was performed by SEM-EDS. Spectra of 8% PVP in PEE containing colloidal Ag solution are displayed in Figure
EDS spectra of electrospun PVP mats containing PEE and 10% (a) or 20% (b) colloidal Ag.
FT-IR spectroscopy is considered as an efficient tool to analyze the solid phase structure as FT-IR spectra can provide additional information about possible interactions between PVP, PEE, and Ag NPs in the produced electrospun mats (Figure
FT-IR spectra of PVP and PVP with Ag NPs.
The FT-IR characteristic absorption peaks of PVP nano/microfibers were observed at 1665, 1440, 1370, and 1290 cm−1, which are attributed to the vibration of the carbonyl group (C-O), O-H bending, lactone structure, and -C-N- stretching, respectively [
The quality of produced nano/microfibers was defined by release profile of propolis phenolic compounds and Ag NPs. Biopharmaceutical characterization of nano/microfiber mats was performed by determining the dissolved amounts of vanillic, caffeic, p-coumaric, and ferulic acids and vanillin thus evaluating possible complex interactions of phenolic compounds with a fiber-forming polymer and incorporated Ag NPs. The released fraction of phenolic acids (vanillic, caffeic, p-coumaric, and ferulic) and vanillin differed depending both on the concentration of PVP and presence of Ag NPs in the fibers (Table
Maximum quantities of phenolic acids released from nano/microfibers in 6 h.
Sample code | Composition of electrospun solutions | Released quantity (%) of phenolic compounds | ||||
---|---|---|---|---|---|---|
Vanillic acid | Caffeic acid | p-Coumaric acid | Ferulic acid | Vanillin | ||
B | PVP in PEE ( |
99.9 | 52.1 | 42.1 | 58.6 | 74.3 |
B1 | PVP in PEE ( |
71.1 | 48.6 | 38.0 | 49.3 | 53.6 |
B2 | PVP in PEE ( |
66.9 | 32.2 | 37.9 | 43.0 | 45.4 |
D | PVP in PEE ( |
65.0 | 50.7 | 35.6 | 47.1 | 48.9 |
D1 | PVP in PEE ( |
100.0 | 100.0 | 82.9 | 100.0 | 79.1 |
Unexpectedly high release of propolis phenolic compounds was identified for nano/microfibers produced by electrospinning from 6% PVP solution containing 10% colloidal Ag. All amount of vanillic, caffeic, and ferulic acids and around 80% of p-coumaric acid and vanillin present in nano/microfibers were released in 6 h. The release of the same phenolic compounds from nano/microfibers without colloidal Ag was significantly lower during the same time interval of dissolution testing.
The mean size of Ag NPs in the initial solution was
The disintegration and dissolution process of electrospun mats was analyzed in order to obtain general information on the behavior of PEE and colloidal Ag containing nano/microfibers in the presence of aqueous fluids. This should contribute to the understanding of mechanisms involved in the release of propolis phenolic acids and Ag NPs from electrospun PVP nano/microfibers. The changes in the mat structure during the dissolution process are depicted in Figure
Disintegration and dissolution of PVP-based nano/microfiber mats containing PEE and Ag NPs.
The release and presence of Ag NPs in the acceptor liquid during mat dissolution testing were confirmed by determining the size of dominating Ag NPs. Table
Size of Ag NPs released from nano/microfibers.
Sample | Particle size, mean ± SD, nm | |||||
---|---|---|---|---|---|---|
0.25 h | 0.5 h | 1 h | 2 h | 4 h | 6 h | |
B1 | 37.53 ± 0.75 | 42.08 ± 0.50 | 41.34 ± 0.07 | 61.87 ± 1.02 | NA | 65.15 ± 0.87 |
B2 | 38.62 ± 2.56 | 38.71 ± 0.28 | 41.37 ± 1.08 | 52.64 ± 1.09 | 50.32 ± 0.19 | 55.62 ± 0.67 |
D1 | 30.85 ± 0.58 | 33.77 ± 0.15 | 37.45 ± 0.35 | 49.98 ± 0.68 | 71.95 ± 1.06 | 76.34 ± 1.09 |
NA, not available.
The application of the light scattering technique for characterization of Ag NPs provided the data of fractional distribution of NPs by the mean particle size in the aqueous medium following PVP nano/microfiber dissolution. The total number of Ag NPs in the aqueous medium increased when dissolution and release characteristics of nano/microfibers containing 10% Ag colloidal solution were evaluated. For these nano/microfibers, it was determined that the fraction of particles with a smaller diameter increased during 6 h period of observation. Dissolution testing of PVP nano/microfibers containing 20% colloidal Ag showed a decreased number of particles and an increased mean particle size in the aqueous medium. The mean particle size varied in the individual fractions during nano/microfiber dissolution testing, but generally it resulted in an increase in the mean particle size from 44% to 147%.
The antibacterial and antifungal activity of electrospun solutions was tested on standard microbial strains
Antimicrobial activity of electrospun solutions and nano/microfiber mats.
Number | Standard strains of microorganisms | Electrospun solutions | Produced mats | ||||||
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A | B | B1 | B2 | A | B | B1 | B2 | ||
1 |
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− |
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2 |
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3 |
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4 |
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− | − | + | + | − | − | + | + |
5 |
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6 |
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7 |
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8 |
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− | + | + | + | − | + | + | + |
9 |
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− | + | + | + | − | + | + | + |
“−” no antibacterial activity determined, “+” antibacterial activity determined.
Antimicrobial activity testing also confirmed the efficient release of propolis phenolic compounds and Ag NPs from formulated nano/microfibers, as demonstrated similar pattern of inactivation of microorganism strains to that of PVP solutions in PEE and containing Ag NPs.
The results of present study confirmed the possibility to incorporate biological active compounds of PEE into PVP mats by the electrospinning technique. Produced nano/microfiber mats exhibited fast release of propolis phenolic compounds and it may be used in the further development of products stimulating wound healing. Inclusion of PEE into the electrospun PVP solution demonstrated no significant influence on the structure of electrospun mats. Additional inclusion of Ag NP colloidal solution into a mixture of PVP and PEE led to the formation of homogeneous and relatively thin nano/microfibers. Solutions of PVP with PEE and electrospun mats demonstrated low antimicrobial activity and affected the growth of
The authors declare that there is no conflict of interests.
This research was funded by Grant (no. MIP-020/2014) from the Research Council of Lithuania.