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Novel copper (II) alginate hydrogels and their potential for use as anti-bacterial wound dressings
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045008 (http://iopscience.iop.org/1748-605X/9/4/045008) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 155.97.178.73 This content was downloaded on 13/10/2014 at 18:18
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 045008 (11pp)
doi:10.1088/1748-6041/9/4/045008
Novel copper (II) alginate hydrogels and their potential for use as anti-bacterial wound dressings Wimonwan Klinkajon1,2 and Pitt Supaphol1,2 1
The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand The Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Bangkok 10330, Thailand 2
E-mail: [email protected] Received 19 January 2014, revised 31 May 2014 Accepted for publication 4 June 2014 Published 16 July 2014 Abstract
The incorporation of a metal ion, with antimicrobial activity, into an alginate dressing is an attractive approach to minimize infection in a wound. In this work, copper (II) cross-linked alginate hydrogels were successfully prepared using a two-step cross-linking procedure. In the first step, solid alginate films were prepared using a solvent-casting method from soft gels of alginate solutions that had been lightly cross-linked using a copper (II) (Cu2+) sulfate solution. In the second step, the films were further cross-linked in a corresponding Cu2+ sulfate solution using a dipping method to further improve their dimensional stability. Alginate solution (at 2%w/v) and Cu2+ sulfate solution (at 2%w/v) in acetate buffer at a low pH provided soft films with excellent swelling behavior. An increase in either Cu2+ ion concentration or cross-linking time led to hydrogels with more densely-cross-linked networks that limited water absorption. The hydrogels clearly showed antibacterial activity against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis and Streptococcus pyogenes, which was proportional to the Cu2+ ion concentration. Blood coagulation studies showed that the tested copper (II) cross-linked alginate hydrogels had a tendency to coagulate fibrin, and possibly had an effect on pro-thrombotic coagulation and platelet activation. Conclusively, the prepared films are likely candidates as antibacterial wound dressings. Keywords: alginate hydrogel, copper (II) ions, antibacterial activity, blood coagulation, wound dressing (Some figures may appear in colour only in the online journal)
1. Introduction
acid (M) and L-guluronic acid (G), which is derived from seaweed, can produce strong hydrophilic gels that offer high water absorption which limits wound secretions and minimizes bacterial contamination [6]. The gelation of an alginate simply occurs in the presence of multivalent cations, and has been widely used in pharmaceutical and medical applications, including wound dressing materials [7–9]. The incorporation of antimicrobial metals into alginate dressings is an attractive approach to control the inflammatory reaction and prevent and/or eliminate infections in wounds. Because silver ions are effective against a wide range of
Providing a moist environment to the wound bed is the currently accepted way of promoting wound healing [1, 2]. In moist wounds, an environment that is rich in white blood cells, enzymes, cytokines and growth factors can be easily generated and maintained throughout the healing process [3–5]. Much research has focused on the development of materials that provide a moist environment for a wound. One of the most important classes of materials is hydrogels. Alginate, a biocompatible natural polymer consisting of D-mannuronic 1748-6041/14/045008+11$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
W Klinkajon and P Supaphol
Biomed. Mater. 9 (2014) 045008
(CuSO4.5H2O) were purchased from Sigma-Aldrich Corp. (St. Louis, USA). Sodium acetate (anhydrous) was purchased from Fluka (Buchs, Switzerland). Acetic acid (glacial) was purchased from Mallinckrodt Chemicals, USA. All other chemicals were of analytical reagent grade and used without further purification.
bacteria, a number of silver-containing wound dressings have been developed. Le and coworkers introduced the method to incorporate silver sulfadiazine into alginate fibers by extruding a spinning solution of sodium alginate containing sodium sulfadiazine into a calcium/silver solution [10]. Although silver ions are highly effective to disinfect bacteria growth, it also can cause discoloration and irritation to the skin. Antibacterial zinc alginate fibers have also been developed by extruding a sodium alginate solution into a zinc (II) chloride bath [11]. However, Zn (II) ions can be bound to alginate chains with less binding affinity and less gel strength leading to an unstable matrix [12]. Among the various types of multivalent cations, copper (II) ions (Cu2+) have been well known for their antibacterial activity since ancient times [13, 14]. Interestingly, alginates with incorporated Cu2+ were previously fabricated in various forms in order to improve the antibacterial activity of materials [15–17]. As a nutrient, copper complexes are essential in the catalysis of a wide range of enzymatic activities including the remodeling of the extracellular matrix (lysyl oxidase) and blood clotting processes (Factors V and VIII), etc. In wound management, Cu2+ been used to enhance wound healing, most likely by stabilizing (dimerizing) the angiogenic factor (FGF [18]). However, Cu2+ have been reported to have high binding affinity, but non-selectivity with the carboxylate moieties in the alginate molecules, resulting in an alginate-copper complex that is less flexible, producing a stiff matrix [19, 20]. In order to overcome this problem, one solution is to provide an optimal environment that allows the G blocks to be readily dissociable. One means is by controlling the pH during the formation of the alginate gels. The presence of protons would decrease the number of free carboxylate moieties, hence there would be less tendency of ionic bridging. As a result, the obtained alginate gels would be more flexible and have better swelling properties. In this work, Cu2+ cross-linked alginate hydrogels were prepared in an acidic condition using a two-step cross-linking procedure. In the first step, solid alginate films were prepared using a solvent-casting method from soft gels of alginate solutions that had been lightly cross-linked using a Cu2+ sulfate solution. In the second step, the films were further cross-linked in a corresponding Cu2+ sulfate solution using a dipping method that aimed to improve their dimensional stability. This system was designed to simultaneously meet three performance requirements: moisture control, which can be evaluated by water content, water absorption and water vapor transmission rate (WVTR); flexibility without losing physical integrity, which can be related to absorption ability/swelling with minimal weight loss; and antimicrobial control with minimal toxicity, which is evaluated using a disc diffusion method that confirms the release ability of copper (II) from the alginate hydrogels to inhibit bacteria growth. In addition, the hydrogels were tested for their ability to accelerate blood coagulation.
2.2. Preparation of copper (II) alginate film
In the first step, the acetate buffer, at pH 3.0, 4.0, 5.0, and distilled water were used as the alginate solvent at different concentrations (0.5%w/v, 1.0%w/v and 2.0%w/v). Copper (II) sulfate solutions (source of Cu2+) in various concentrations (0.5– 2.5%w/v) at various cross-linking times (5–15 min) were added dropwise into the sodium alginate solution to initiate gelation. The volumetric ratio between each of the alginate solutions and each of the Cu2+ sulfate solutions was 10:1 v/v. The soft gels of the alginate solutions, which had been lightly cross-linked using a Cu2+ sulfate solution, were dried under vacuum for three to five days in culture dishes to finally obtain solid films (hereafter, Cu2+ crosslinked alginate films/hydrogels). In the second step, the alginate films were re-immersed in the corresponding Cu2+ sulfate solutions for 5, 10 and 15 min to improve the dimensional stability of the hydrogel films. All of the immersed films were washed in distilled water for 10 s to eliminate excess copper. After preparation, the hydrogels were cut into discs (14 and 1 mm thickness) and were further dried at 60 °C to a constant weight. The hydrogels were kept in sealed bags prior to use. 2.3. Physical and chemical characterization
After the initial addition of Cu2+ sulfate solution to the sodium alginate solution, the free Cu2+ in the clear solution, which was left from the crosslinking of the alginate chains, was quantified using a Shimadzu UV-1800 spectrophotometer. The chemical interaction between the Cu2+ and certain chemical functional groups of alginate was examined on a copper (II) crosslinked alginate film using a Nicolet Fourier transform infrared (FT-IR) 360 spectrophotometer. FT-IR spectra of the sodium alginate were also recorded for comparison, using the KBr pellet method. Morphologies of the Cu2+ alginate films, both before and after subsequent immersion in the corresponding Cu2+ sulfate solutions, were observed with a JEOL JSM-5200 scanning electron microscope (SEM). 2.4. Evaluation of the dressing properties 2.4.1. Determination of equilibrium water content and water absorption. The films were immersed in pH 7.4 simulated
2.1. Materials
body fluid (SBF) at 37 °C, which imitated the physiological conditions of the human body. They were allowed to swell for 24 h, until reaching constant weight. The obtained hydrogels were removed from the droplets of liquid (the excess of SBF on the hydrogel surface) using filter paper prior to being weighed. The equilibrium water content and water absorption were calculated as follows:
Alginic acid (sodium salt, Brookfield viscosity 20 000– 40 000 cps; hereafter, sodium alginate) and copper (II) sulfate
Ws − Wd Equilibrium water content (%) = × 100, (1) Ws
2. Experiment details
2
W Klinkajon and P Supaphol
Biomed. Mater. 9 (2014) 045008
where Ws is the weight of the hydrogel at the equilibrium swollen state. It should be noted that Wd was obtained after the swollen hydrogels were dried in an oven at 60 °C for 24 h.
2.5. Evaluation of the antibacterial hydrogel wound dressing
Good antimicrobial activity with minimal toxicity is most desirable for optimal performance of the hydrogels. The antibacterial activity of the material was evaluated using a disc diffusion method, which confirmed the release ability of Cu2+.
Ws – Wi Water absorption (%) = × 100 (2) Wi Wi – Wd Weight loss (%) = × 100 (3) Wi
2.5.1. Cumulative copper (II) ions release. The Cu2+ cross-
linked alginate hydrogels were cut into discs (9 mm diameter, 1 mm thickness, 102.02 ± 14.0 mg) and each individual disc was immersed in a bottle containing 10 ml of SBF solution at 37 °C under agitation at 60 rpm. At various time intervals, the immersion solution was collected for investigation of the cumulative release of copper species. Replacement of 10 ml of SBF solution into each bottle was performed after each collection interval. The concentration of copper species was measured using a Varian SpectrAA300. The initial content of Cu2+ in Cu2+ cross-linked alginate hydrogel was calculated corresponding to amounts of added Cu2+.
where Wi is the initial dry weight of each film (before being immersed in SBF). 2.4.2. Water vapor transmission rate (WVTR). The WVTR was measured following the monograph of the European Pharmacopeia. The experiment measured the rate of water transport from a glass bottle containing 25 ml of water. The glass bottle, with a 14 mm hydrogel disc cap, was kept in an oven at 35 °C for 24 h. WVTR was calculated using the following equation
( W − Wt ) WVTR = i × 106 g (m2 .h)−1 (4) A × 24
2.5.2. Antibacterial evaluation. Specimens of hydrogel were
where WVTR is expressed in g (m2 · h)−1, A is the area of the diameter of the bottle (mm2), Wi is the weight of bottle before and Wt is the weight after being placed in an oven.
cut into discs (9 mm diameter), which were further studied via the disc diffusion method (The US Clinical and Laboratory Standards Institute (CLSI) disc diffusion method). Both gram-negative bacteria, E. coli, ATCC 25922 and grampositive bacteria, S. aureus, ATCC 25923, were used to test antibacterial activity of the Cu2+ alginate hydrogels. MRSA, DMST 20654, S. epidermidis, ATCC 12228 and S. pyogenes, DMST 17020, all of which can cause dermal infection, were also selected for evaluation of antibacterial activity. All of the bacteria were diluted until the colonies equaled 108 CFU (colony forming unit) ml−1, and then a 200 μl bacteria solution was transferred onto DifcoTM Mueller Hinton agar dishes. The hydrogel specimens were then placed on the agar culture dishes and incubated at 37 °C for 24 h. An inhibition zone was clearly seen around each specimen, whenever there was antibacterial activity.
2.4.3. Swelling behavior. Studies were carried out based on the kinetics of water sorption dynamics method utilized by Balakrishnan and Lee [21, 22]. Briefly, the films were weighed before being immersed in distilled water at 37 °C. After a specific immersion time, the films were taken out and the water droplets on the surface of the films were removed with filter paper sheets prior to being weighed again. The swelling ratio (Qm) was calculated using the following expression:
Ws − Wi Qm = , (5) Wi
where Ws is the weight of the film hydrogels in their swollen state and Wi is the initial dry state. The crosslink density (ve, mol cm−3) of the copper (II) cross-linked hydrogels was calculated based on the FloryRehner equation:
2.5.3. Indirect cytotoxicity evaluation. The ISO 10993–5
standard test method was used to evaluate toxicity of the Cu2+ alginate hydrogel, which was used in both L929 cells (mouse fibroblast) and NHDF cells (normal human dermal fibroblast). The cells were cultured in Dulbecco’s modified Eagle’s medium, DMEM (composition of 10% fetal bovine serum (FBS; Invitrogen Corp., USA), 1% l-glutamine (Invitrogen Corp., USA), and 1% antibiotic and antimycotic formulation which contained penicillin G sodium, streptomycin sulfate, and amphotericin B (Invitrogen Corp., USA)). The disc specimens (14 mm diameter) were sterilized with 70%v/v ethanol for 30 min. The extraction medium was prepared using immersing each disc specimen (weighed in mg of hydrogel) in serum free media (SFM; DMEM containing 1% l-glutamine, 1% lactalbumin and 1% antibiotic and antimycotic formulation) for 1, 2 and 3 days with extraction ratios of 15, 25 and 75 mg ml−1, respectively. The cells were then separately cultured using SFM in 24-well tissue-culture polystyrene plates (TCPS; NunclonTM, Denmark) which
−1 ⎡ ⎛ 2ν2 ⎞ ⎤ 1/3 2 νe = − ln(1 − ν2 ) + ν2 + χ1 ν2 ⎢ V1⎜ ν2 − (6) ⎟⎥
[
]⎣
⎝
f ⎠⎦
where χ1 is the interaction parameter, f is the cross-linking functionality, V1 is the molar volume of water (18.062 cm3 mol−1), and v2 is the volume fraction of polymer in the hydrogels at the equilibrium swollen state. According to Balakrishnan et al [21] and Lee et al [22], χ1 and f were assumed to be 0.35 and 2, respectively, for the alginate-water systems. The value of the polymer volume fraction (v2) was calculated using the following equation: ⎛ ⎞⎡ ⎛ ⎞ ⎤−1 ν2 = ⎜ 1 ρ ⎟ ⎢ Qm ρ + ⎜ 1 ρ ⎟ ⎥ (7) s ⎝ p⎠⎣ ⎝ p⎠ ⎦
(
)
where ρp is the polymer density (0.8755 g cm−3), ρs is the water density (0.9933 g cm−3 at 37 °C) and Qm is the swelling ratio. 3
W Klinkajon and P Supaphol
Biomed. Mater. 9 (2014) 045008
were incubated at 37 °C for 24 h. Since L 929 had a higher growth rate compared to NHDF, it was seeded with a lower cell number than NHDF and the cell density was optimized in each well (5000 cells per well for L929 and 10 000 cells per well for NHDF). The obtained extraction media was used as the growth media. The relative cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
Absorbance
(a)
(b) Absorbance
2.6. Blood clot formation assay
Blood clot formation initiated by the hydrogels was demonstrated via fibrin formation on the surface of the hydrogels. For the first step, human blood from Chulalongkorn Hospital (Thailand) was dropped on the surface of each sample including 6 mm in diameter of the obtained hydrogels (i.e. the Cu2+ alginate films after cross-linking the surface by 1.0% CuSO4 solution and 2.5% CuSO4 solution, respectively) and the glass slides as controls. The samples were immediately covered with glass cover slides and then incubated for 5 min at room temperature. Afterwards, the samples were gently washed to remove excess blood using PBS buffer (pH 7.2). In order to observe fibrin formation, all of the samples were fixed with 3%v/v glutaraldehyde in PBS buffer for 30 min at room temperature, and rinsed with PBS buffer. Finally, the samples were dehydrated with a series of ethanol solutions (30, 50, 70, 90 and 100%) for 2 min each and air dried. The morphology of the fibrin formation was observed by SEM.
Absorbance
(c)
600
900
0.33 0.22 0.11 0.00 1.00 0.75 0.50 0.25 1.00 0.00 0.75 0.50 0.25 0.00
600
900 Wavelength (nm)
Figure 1. The absorbance spectrum of (a) 2.5%w/v CuSO4 solution, (b) copper (II) alginate film with 0.1% CuSO4 solution, and (c) copper (II) alginate film with 0.5% CuSO4 solution.
2.7. Statistical analysis
Standard deviations (SD) from triplicate end point data were plotted as error bars on all graphs. Differences between the control and samples were determined using one-way analysis of variance (ANOVA) and the Scheffe’s post hoc test with SPSS 11.5 for Windows software (SPSS). Statistically significant differences were set at p
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My IOPscience
Novel copper (II) alginate hydrogels and their potential for use as anti-bacterial wound dressings
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Biomed. Mater. 9 045008 (http://iopscience.iop.org/1748-605X/9/4/045008) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 155.97.178.73 This content was downloaded on 13/10/2014 at 18:18
Please note that terms and conditions apply.
Biomedical Materials Biomed. Mater. 9 (2014) 045008 (11pp)
doi:10.1088/1748-6041/9/4/045008
Novel copper (II) alginate hydrogels and their potential for use as anti-bacterial wound dressings Wimonwan Klinkajon1,2 and Pitt Supaphol1,2 1
The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand The Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Bangkok 10330, Thailand 2
E-mail: [email protected] Received 19 January 2014, revised 31 May 2014 Accepted for publication 4 June 2014 Published 16 July 2014 Abstract
The incorporation of a metal ion, with antimicrobial activity, into an alginate dressing is an attractive approach to minimize infection in a wound. In this work, copper (II) cross-linked alginate hydrogels were successfully prepared using a two-step cross-linking procedure. In the first step, solid alginate films were prepared using a solvent-casting method from soft gels of alginate solutions that had been lightly cross-linked using a copper (II) (Cu2+) sulfate solution. In the second step, the films were further cross-linked in a corresponding Cu2+ sulfate solution using a dipping method to further improve their dimensional stability. Alginate solution (at 2%w/v) and Cu2+ sulfate solution (at 2%w/v) in acetate buffer at a low pH provided soft films with excellent swelling behavior. An increase in either Cu2+ ion concentration or cross-linking time led to hydrogels with more densely-cross-linked networks that limited water absorption. The hydrogels clearly showed antibacterial activity against Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis and Streptococcus pyogenes, which was proportional to the Cu2+ ion concentration. Blood coagulation studies showed that the tested copper (II) cross-linked alginate hydrogels had a tendency to coagulate fibrin, and possibly had an effect on pro-thrombotic coagulation and platelet activation. Conclusively, the prepared films are likely candidates as antibacterial wound dressings. Keywords: alginate hydrogel, copper (II) ions, antibacterial activity, blood coagulation, wound dressing (Some figures may appear in colour only in the online journal)
1. Introduction
acid (M) and L-guluronic acid (G), which is derived from seaweed, can produce strong hydrophilic gels that offer high water absorption which limits wound secretions and minimizes bacterial contamination [6]. The gelation of an alginate simply occurs in the presence of multivalent cations, and has been widely used in pharmaceutical and medical applications, including wound dressing materials [7–9]. The incorporation of antimicrobial metals into alginate dressings is an attractive approach to control the inflammatory reaction and prevent and/or eliminate infections in wounds. Because silver ions are effective against a wide range of
Providing a moist environment to the wound bed is the currently accepted way of promoting wound healing [1, 2]. In moist wounds, an environment that is rich in white blood cells, enzymes, cytokines and growth factors can be easily generated and maintained throughout the healing process [3–5]. Much research has focused on the development of materials that provide a moist environment for a wound. One of the most important classes of materials is hydrogels. Alginate, a biocompatible natural polymer consisting of D-mannuronic 1748-6041/14/045008+11$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
W Klinkajon and P Supaphol
Biomed. Mater. 9 (2014) 045008
(CuSO4.5H2O) were purchased from Sigma-Aldrich Corp. (St. Louis, USA). Sodium acetate (anhydrous) was purchased from Fluka (Buchs, Switzerland). Acetic acid (glacial) was purchased from Mallinckrodt Chemicals, USA. All other chemicals were of analytical reagent grade and used without further purification.
bacteria, a number of silver-containing wound dressings have been developed. Le and coworkers introduced the method to incorporate silver sulfadiazine into alginate fibers by extruding a spinning solution of sodium alginate containing sodium sulfadiazine into a calcium/silver solution [10]. Although silver ions are highly effective to disinfect bacteria growth, it also can cause discoloration and irritation to the skin. Antibacterial zinc alginate fibers have also been developed by extruding a sodium alginate solution into a zinc (II) chloride bath [11]. However, Zn (II) ions can be bound to alginate chains with less binding affinity and less gel strength leading to an unstable matrix [12]. Among the various types of multivalent cations, copper (II) ions (Cu2+) have been well known for their antibacterial activity since ancient times [13, 14]. Interestingly, alginates with incorporated Cu2+ were previously fabricated in various forms in order to improve the antibacterial activity of materials [15–17]. As a nutrient, copper complexes are essential in the catalysis of a wide range of enzymatic activities including the remodeling of the extracellular matrix (lysyl oxidase) and blood clotting processes (Factors V and VIII), etc. In wound management, Cu2+ been used to enhance wound healing, most likely by stabilizing (dimerizing) the angiogenic factor (FGF [18]). However, Cu2+ have been reported to have high binding affinity, but non-selectivity with the carboxylate moieties in the alginate molecules, resulting in an alginate-copper complex that is less flexible, producing a stiff matrix [19, 20]. In order to overcome this problem, one solution is to provide an optimal environment that allows the G blocks to be readily dissociable. One means is by controlling the pH during the formation of the alginate gels. The presence of protons would decrease the number of free carboxylate moieties, hence there would be less tendency of ionic bridging. As a result, the obtained alginate gels would be more flexible and have better swelling properties. In this work, Cu2+ cross-linked alginate hydrogels were prepared in an acidic condition using a two-step cross-linking procedure. In the first step, solid alginate films were prepared using a solvent-casting method from soft gels of alginate solutions that had been lightly cross-linked using a Cu2+ sulfate solution. In the second step, the films were further cross-linked in a corresponding Cu2+ sulfate solution using a dipping method that aimed to improve their dimensional stability. This system was designed to simultaneously meet three performance requirements: moisture control, which can be evaluated by water content, water absorption and water vapor transmission rate (WVTR); flexibility without losing physical integrity, which can be related to absorption ability/swelling with minimal weight loss; and antimicrobial control with minimal toxicity, which is evaluated using a disc diffusion method that confirms the release ability of copper (II) from the alginate hydrogels to inhibit bacteria growth. In addition, the hydrogels were tested for their ability to accelerate blood coagulation.
2.2. Preparation of copper (II) alginate film
In the first step, the acetate buffer, at pH 3.0, 4.0, 5.0, and distilled water were used as the alginate solvent at different concentrations (0.5%w/v, 1.0%w/v and 2.0%w/v). Copper (II) sulfate solutions (source of Cu2+) in various concentrations (0.5– 2.5%w/v) at various cross-linking times (5–15 min) were added dropwise into the sodium alginate solution to initiate gelation. The volumetric ratio between each of the alginate solutions and each of the Cu2+ sulfate solutions was 10:1 v/v. The soft gels of the alginate solutions, which had been lightly cross-linked using a Cu2+ sulfate solution, were dried under vacuum for three to five days in culture dishes to finally obtain solid films (hereafter, Cu2+ crosslinked alginate films/hydrogels). In the second step, the alginate films were re-immersed in the corresponding Cu2+ sulfate solutions for 5, 10 and 15 min to improve the dimensional stability of the hydrogel films. All of the immersed films were washed in distilled water for 10 s to eliminate excess copper. After preparation, the hydrogels were cut into discs (14 and 1 mm thickness) and were further dried at 60 °C to a constant weight. The hydrogels were kept in sealed bags prior to use. 2.3. Physical and chemical characterization
After the initial addition of Cu2+ sulfate solution to the sodium alginate solution, the free Cu2+ in the clear solution, which was left from the crosslinking of the alginate chains, was quantified using a Shimadzu UV-1800 spectrophotometer. The chemical interaction between the Cu2+ and certain chemical functional groups of alginate was examined on a copper (II) crosslinked alginate film using a Nicolet Fourier transform infrared (FT-IR) 360 spectrophotometer. FT-IR spectra of the sodium alginate were also recorded for comparison, using the KBr pellet method. Morphologies of the Cu2+ alginate films, both before and after subsequent immersion in the corresponding Cu2+ sulfate solutions, were observed with a JEOL JSM-5200 scanning electron microscope (SEM). 2.4. Evaluation of the dressing properties 2.4.1. Determination of equilibrium water content and water absorption. The films were immersed in pH 7.4 simulated
2.1. Materials
body fluid (SBF) at 37 °C, which imitated the physiological conditions of the human body. They were allowed to swell for 24 h, until reaching constant weight. The obtained hydrogels were removed from the droplets of liquid (the excess of SBF on the hydrogel surface) using filter paper prior to being weighed. The equilibrium water content and water absorption were calculated as follows:
Alginic acid (sodium salt, Brookfield viscosity 20 000– 40 000 cps; hereafter, sodium alginate) and copper (II) sulfate
Ws − Wd Equilibrium water content (%) = × 100, (1) Ws
2. Experiment details
2
W Klinkajon and P Supaphol
Biomed. Mater. 9 (2014) 045008
where Ws is the weight of the hydrogel at the equilibrium swollen state. It should be noted that Wd was obtained after the swollen hydrogels were dried in an oven at 60 °C for 24 h.
2.5. Evaluation of the antibacterial hydrogel wound dressing
Good antimicrobial activity with minimal toxicity is most desirable for optimal performance of the hydrogels. The antibacterial activity of the material was evaluated using a disc diffusion method, which confirmed the release ability of Cu2+.
Ws – Wi Water absorption (%) = × 100 (2) Wi Wi – Wd Weight loss (%) = × 100 (3) Wi
2.5.1. Cumulative copper (II) ions release. The Cu2+ cross-
linked alginate hydrogels were cut into discs (9 mm diameter, 1 mm thickness, 102.02 ± 14.0 mg) and each individual disc was immersed in a bottle containing 10 ml of SBF solution at 37 °C under agitation at 60 rpm. At various time intervals, the immersion solution was collected for investigation of the cumulative release of copper species. Replacement of 10 ml of SBF solution into each bottle was performed after each collection interval. The concentration of copper species was measured using a Varian SpectrAA300. The initial content of Cu2+ in Cu2+ cross-linked alginate hydrogel was calculated corresponding to amounts of added Cu2+.
where Wi is the initial dry weight of each film (before being immersed in SBF). 2.4.2. Water vapor transmission rate (WVTR). The WVTR was measured following the monograph of the European Pharmacopeia. The experiment measured the rate of water transport from a glass bottle containing 25 ml of water. The glass bottle, with a 14 mm hydrogel disc cap, was kept in an oven at 35 °C for 24 h. WVTR was calculated using the following equation
( W − Wt ) WVTR = i × 106 g (m2 .h)−1 (4) A × 24
2.5.2. Antibacterial evaluation. Specimens of hydrogel were
where WVTR is expressed in g (m2 · h)−1, A is the area of the diameter of the bottle (mm2), Wi is the weight of bottle before and Wt is the weight after being placed in an oven.
cut into discs (9 mm diameter), which were further studied via the disc diffusion method (The US Clinical and Laboratory Standards Institute (CLSI) disc diffusion method). Both gram-negative bacteria, E. coli, ATCC 25922 and grampositive bacteria, S. aureus, ATCC 25923, were used to test antibacterial activity of the Cu2+ alginate hydrogels. MRSA, DMST 20654, S. epidermidis, ATCC 12228 and S. pyogenes, DMST 17020, all of which can cause dermal infection, were also selected for evaluation of antibacterial activity. All of the bacteria were diluted until the colonies equaled 108 CFU (colony forming unit) ml−1, and then a 200 μl bacteria solution was transferred onto DifcoTM Mueller Hinton agar dishes. The hydrogel specimens were then placed on the agar culture dishes and incubated at 37 °C for 24 h. An inhibition zone was clearly seen around each specimen, whenever there was antibacterial activity.
2.4.3. Swelling behavior. Studies were carried out based on the kinetics of water sorption dynamics method utilized by Balakrishnan and Lee [21, 22]. Briefly, the films were weighed before being immersed in distilled water at 37 °C. After a specific immersion time, the films were taken out and the water droplets on the surface of the films were removed with filter paper sheets prior to being weighed again. The swelling ratio (Qm) was calculated using the following expression:
Ws − Wi Qm = , (5) Wi
where Ws is the weight of the film hydrogels in their swollen state and Wi is the initial dry state. The crosslink density (ve, mol cm−3) of the copper (II) cross-linked hydrogels was calculated based on the FloryRehner equation:
2.5.3. Indirect cytotoxicity evaluation. The ISO 10993–5
standard test method was used to evaluate toxicity of the Cu2+ alginate hydrogel, which was used in both L929 cells (mouse fibroblast) and NHDF cells (normal human dermal fibroblast). The cells were cultured in Dulbecco’s modified Eagle’s medium, DMEM (composition of 10% fetal bovine serum (FBS; Invitrogen Corp., USA), 1% l-glutamine (Invitrogen Corp., USA), and 1% antibiotic and antimycotic formulation which contained penicillin G sodium, streptomycin sulfate, and amphotericin B (Invitrogen Corp., USA)). The disc specimens (14 mm diameter) were sterilized with 70%v/v ethanol for 30 min. The extraction medium was prepared using immersing each disc specimen (weighed in mg of hydrogel) in serum free media (SFM; DMEM containing 1% l-glutamine, 1% lactalbumin and 1% antibiotic and antimycotic formulation) for 1, 2 and 3 days with extraction ratios of 15, 25 and 75 mg ml−1, respectively. The cells were then separately cultured using SFM in 24-well tissue-culture polystyrene plates (TCPS; NunclonTM, Denmark) which
−1 ⎡ ⎛ 2ν2 ⎞ ⎤ 1/3 2 νe = − ln(1 − ν2 ) + ν2 + χ1 ν2 ⎢ V1⎜ ν2 − (6) ⎟⎥
[
]⎣
⎝
f ⎠⎦
where χ1 is the interaction parameter, f is the cross-linking functionality, V1 is the molar volume of water (18.062 cm3 mol−1), and v2 is the volume fraction of polymer in the hydrogels at the equilibrium swollen state. According to Balakrishnan et al [21] and Lee et al [22], χ1 and f were assumed to be 0.35 and 2, respectively, for the alginate-water systems. The value of the polymer volume fraction (v2) was calculated using the following equation: ⎛ ⎞⎡ ⎛ ⎞ ⎤−1 ν2 = ⎜ 1 ρ ⎟ ⎢ Qm ρ + ⎜ 1 ρ ⎟ ⎥ (7) s ⎝ p⎠⎣ ⎝ p⎠ ⎦
(
)
where ρp is the polymer density (0.8755 g cm−3), ρs is the water density (0.9933 g cm−3 at 37 °C) and Qm is the swelling ratio. 3
W Klinkajon and P Supaphol
Biomed. Mater. 9 (2014) 045008
were incubated at 37 °C for 24 h. Since L 929 had a higher growth rate compared to NHDF, it was seeded with a lower cell number than NHDF and the cell density was optimized in each well (5000 cells per well for L929 and 10 000 cells per well for NHDF). The obtained extraction media was used as the growth media. The relative cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay.
Absorbance
(a)
(b) Absorbance
2.6. Blood clot formation assay
Blood clot formation initiated by the hydrogels was demonstrated via fibrin formation on the surface of the hydrogels. For the first step, human blood from Chulalongkorn Hospital (Thailand) was dropped on the surface of each sample including 6 mm in diameter of the obtained hydrogels (i.e. the Cu2+ alginate films after cross-linking the surface by 1.0% CuSO4 solution and 2.5% CuSO4 solution, respectively) and the glass slides as controls. The samples were immediately covered with glass cover slides and then incubated for 5 min at room temperature. Afterwards, the samples were gently washed to remove excess blood using PBS buffer (pH 7.2). In order to observe fibrin formation, all of the samples were fixed with 3%v/v glutaraldehyde in PBS buffer for 30 min at room temperature, and rinsed with PBS buffer. Finally, the samples were dehydrated with a series of ethanol solutions (30, 50, 70, 90 and 100%) for 2 min each and air dried. The morphology of the fibrin formation was observed by SEM.
Absorbance
(c)
600
900
0.33 0.22 0.11 0.00 1.00 0.75 0.50 0.25 1.00 0.00 0.75 0.50 0.25 0.00
600
900 Wavelength (nm)
Figure 1. The absorbance spectrum of (a) 2.5%w/v CuSO4 solution, (b) copper (II) alginate film with 0.1% CuSO4 solution, and (c) copper (II) alginate film with 0.5% CuSO4 solution.
2.7. Statistical analysis
Standard deviations (SD) from triplicate end point data were plotted as error bars on all graphs. Differences between the control and samples were determined using one-way analysis of variance (ANOVA) and the Scheffe’s post hoc test with SPSS 11.5 for Windows software (SPSS). Statistically significant differences were set at p
