Poly (vinyl alcohol)-alginate physically crosslinked hydrogel membranes for wound dressing applications: Characterization and bio-evaluation

2.1 Materials

PVA (typically average M_w = 72,000 g/mol; 98.9% hydrolyzed) was obtained from Biochemica, Germany. SA (medium viscosity, average M_w = 130,000 g/mol as determined by GPC), albumin from bovine serum (BSA, fraction V, minimum 96% electrophoresis, nitrogen content 16.2%), and ampicillin sodium salt were purchased from Sigma–Aldrich Chemie GmbH, Steinheim, Germany. Folin and Ciocalteu’s phenol reagent (FC, 2 N with respect to acid), were purchased from Park Scientific Limited, Northampton, UK. Distilled water was used throughout this research. All other chemicals were used without any further purification.

2.2 Preparation of PVA-SA hydrogels

PVA-SA hydrogel membranes were prepared by freezing–thawing (F–T) cycle according to the reported procedure of (Peppas and Stauffer, 1991). Briefly, aqueous solution containing 10% (w/v) PVA and 1.5% (w/v) SA and 20 mg of sodium ampicillin sodium salt were carefully dissolved in de-ionized water. Different proportions of PVA and SA content (0%, 25%, 33%, 50%, 65%, and 75%) solutions were mixed, sonicating, and vortexing for three hours. Proper amounts of this mixture were poured in Petri dishes, followed by freezing at −20 °C for 18 h and thawing for 6 h at 25 °C for three continuous cycles, to provide mechanically acceptable hydrogels for further experiments. The obtained PVA-SA hydrogel membranes were frozen in liquid nitrogen for 10 min before being lyophilized fractures for SEM investigations. All samples were left in deionized water for 72 h to extract leachable sol fraction or unconnected HES from polymer matrix for further characterizations.

2.3 Determination of gel fraction

The obtained PVA-SA hydrogel membranes were dried in a vacuum oven at 50 °C for 24 h and weighted (W₀), then soaked in distilled water for 24 h up to an equilibrium swelling weight (W_s) for removing the leachable or soluble SA parts from membrane. The gel membrane then dried again at 50 °C in a vacuum oven and weighted again (W_e). The gel fraction (GF%) was calculated by the following Eq. (1) Yang et al., 2008.

2.4 Determination of swelling behavior

In order to measuring the swelling degree of PVA-SA hydrogel membranes, membrane samples were cut into 2 cm × 2 cm pieces and dried at 50 °C in a vacuum oven for 6 h, the weight of dried sample was determined (W_e). The dried samples were soaked in distilled water, maintained and incubated at 37 °C, then weighted (W_s) at specific interval times. The water uptake of PVA-SA hydrogel membranes was determined using the following Eq. (2) Yan et al., 2008.

2.5 Study of protein adsorption onto hydrogel surface

The amount of adsorbed bovine serum albumin (BSA) was detected by UV–visible spectrophotometer (type: Ultrospec 2000, Pharmacia Biotech, Cambridge, England). In order to establish the relationship between the visible absorbance of BSA at 630 nm and the concentration of BSA, a calibration curve was drawn for standard solution of BSA ranging from 3–60 mg/ml. All standard solutions were prepared with distilled water. From the calibration curve a study was made restricting the curve to the linear part that followed Beer’s law in Eq. (3),

Pieces of PVA hydrogel membranes cut into 1 cm × 1 cm were immersed in 10 ml phosphate buffer saline (pH 7.4), and incubated at 37 °C for 24 h until reaching equilibrium swelling weight. The swollen hydrogel pieces were transferred to buffer solution containing BSA (30 mg/ml) and shaken for 4 h at 37 °C. After protein adsorption, the hydrogel pieces were gently removed. The protein adsorption of the each sample was calculated by the difference between protein concentrations before and after immersing hydrogel pieces in protein/phosphate buffer solution using albumin reagent kit (absorbance range at 630 nm), this procedure has been adapted and modified from the procedure of Lin et al. (2006).

2.6 Determination of hydrolytic degradation

Dried membrane samples with size of 15 × 8 mm, were weighted and immersed in 3 ml phosphate buffer saline (0.1 M, pH 7.4, at 37 °C). The samples were removed at timed intervals and then wiped gently with soft paper to remove surface water. The samples were dried under vacuum at room temperature and finally weighted again. All experiments were done in duplicate.

2.7 Hemocompatibility test

Hemolysis experiments were carried out on the surface of the obtained PVA-SA xerogel membranes as described elsewhere (Chhatri et al., 2011; Singh and Ray, 1994). Typically, xerogel membranes were first equilibrated with saline solution (NaCl, 0.9 w/v at 37 °C for 24 h). Before conducting the test, anti-coagulated blood was used for this test, where 1 ml of anti-coagulated acid citrate dextrose solution (ACD) was added to 9 ml of fresh human blood. The equilibrated PVA-SA xerogel membranes (8 cm²) were transferred into polypropylene test tubes and 7 ml of phosphate buffer saline (PBS, pH 7.4) was added and incubated for 72 h. The PBS was removed from the last test tubes and 1 ml of ACD solution was added to each sample and incubated for 37 °C for 3 h. Positive and negative controls were prepared by adding the same amount of ACD to 7 ml of distilled water and PBS, respectively. Each tube was incubated and gently inverted twice each 30 min to guarantee the continuous contacting between membrane and ACD blood. The fluids were transferred to a proper tube then centrifuged at 3000 rpm for 20 min. The hemoglobin released by hemolysis calculation by determination the optical densities (OD) of the supernatant. The supernatant was taken and its absorbance was monitored at 540 nm using a spectrophotometer (model: Ultrospec 2000). The percentage hemolysis was calculated by the following equation: (Chhatri et al., 2011)

2.8 Antibacterial studies

Antibacterial assay of crosslinked PVA-SA xerogel membranes containing sodium ampicillin has been measured according to the methods which are discussed elsewhere (Jeon and Kim, 2000; Jumaa et al., 2002). The antibacterial study was carried out depending on the determination of the zone of inhibition method. Typically, the bacteria were inoculated in a Luria–Bertani medium (LB medium) (1% peptone, 0.5% yeast beef extract, and 1% NaCl). The inoculation was carried out at 37 °C for 24 h with continuous shaking. The obtained bacteria suspension was diluted by the same peptone medium solution for 100 times. 0.1 ml of diluted bacteria suspension was cultured in a 10 ml liquid sterilized peptone medium (sterilization at 120 °C for 20 min.). This peptone medium contains 1 cm × 5 cm PVA-SA membrane pieces using the direct contact method between cultured bacteria and membrane surface. The inoculation medium was maintained at 37 °C for 18 h with continuous shaking. The numbers of inhibited bacteria were counted by using the ultraviolet–visible light spectrophotometry absorbance or optical density value (A) of culture medium at 620 nm. The indicative inhibition ratio (%) results are shown in Table 1, the inhibition ratios (%) of PVA-SA hydrogel membranes were calculated using Eq. (5):

2.9 Release profile study

15 ml of FC reagent was added into a series of 50 ml conical flasks containing sodium ampicillin (0.2–0.8 mg). Sodium ampicillin was chosen as a topical long acting antibiotic which also has good water solubility in an aqueous solution and it has broad-spectrum antibiotic which means that it kills more kinds of bacteria than other penicillin family and gentamicin antibiotics. Sodium ampicillin was utilized as an antibiotic instead of gentamicin sulfate salt which is loaded with PVA-dextran membranes and was discussed elsewhere (Hwang et al., 2010). The contents were completely mixed and kept into a thermostated water bath at 95 °C for 30 min. The flasks were taken out cooled at room temperature at ∼25 °C, then transferred into a 25 ml standard volumetric flask and diluted up to the mark with distilled water. The absorbance of the resulting blue color of dye was measured against a blank reagent at 750 nm using spectrophotometer as previously discussed (Queiroz et al., 2001; Lin et al., 2006; Ahmad et al., 2004).

Two pieces of PVA-SA hydrogel membranes containing sodium ampicillin (each piece size is almost 55 cm²), were immersed in phosphate buffer (pH 8, at 37 °C) and kept in continuous shaking. 200 μL of last solution was withdrawn at timed intervals each 15 min and was added to 3 ml FC reagent. The last mixture was heated at 95 °C for 30 min, and then taken out for cooling at room temperature; 1.8 ml of FC reagent was added to the cooled mixture and carefully vortexed before measuring. The absorbance of sodium ampicillin released from prepared samples was detected by spectrophotometer at 750 nm. All experiments were done in triplicate.

2.10 Characterizations

• FT-IR

Vacuum dried samples of freeze–thawed PVA-SA xerogels were analyzed by FT-IR on an EQUINOX 55 instrument (BRUKER, Germany). Translucent KBr-disks were prepared by grinding the dried sample materials together with infrared grade KBr and then pressing. The FTIR spectrums were obtained by recording 64 scans between 4000 and 400 cm⁻¹ with a resolution of 2 cm⁻¹. All samples were freeze-dried using liquid nitrogen, crushed to a fine powder (KBr: sample = 140 mg: 2 mg) respectively, and pressed by applying a force 105 N into transparent disk (maximum disk weight = 150 mg) with a diameter 13 mm. All samples were measured in absorbance mode.

• Mechanical property measurements

The maximum tensile strength and the elongation degree to break of PVA-SA blend hydrogel membranes have been conducted using a tensile test machine (model: AG-I/ 50 N-10KN, Japan). PVA-SA membranes were cut into a specific dog-bone shape (6 cm long, 2 cm wide at the ends, and 1 cm at the middle). The analysis was performed at stretching rate 20 mm/min with pre-load of 0.5 N to determine load for each sample (Alencar et al., 2003). The thickness of membrane samples was measured with a digimatic caliper before examination.

• Scanning electron microscope

The surface and internal structure of the xerogel membrane samples were investigated by Analytical-SEM (type: JEOL, JSM-6360LA, Japan) with 15 kV voltage for secondary electron imaging. The xerogel membranes were dehydrated by freeze-dryer and coated with Au using an ion sputter coater (model: 11430, USA, combined with vacuum base unit or SPi module control, model: 11425, USA).

• Surface roughness

The surface roughness of the substrate of PVA-SA hydrogel membranes which are designed for wound dressing is a very important parameter. The surface upper-side of PVA-SA hydrogel membranes was measured by surface roughness tester (model: Mattito SJ-201P, Japan). The samples were mounted onto a glass slide with double-sides. The minimum sample dimensions were 25 mm × 25 mm. All results are measured in triplicate.

3.1 FTIR spectroscopy

Poly (vinyl alcohol)-alginate (PVA-SA) blend hydrogel membranes were synthesized using the freeze–thawing technique, while the crosslinking was accomplished physically by crystallization step. In Fig. 1, the IR spectra of freeze–thawed PVA-SA blend xerogel membranes are shown, while the IR spectra of crosslinked PVA and virgin SA were discussed elsewhere, respectively (Kim et al., 2008; Chhatri et al., 2011). The IR spectrum of SA exhibited characteristic absorption bands for hydroxyl groups (–OH) at 3445 cm⁻¹ and carboxylic groups (–COOH) at 1419 cm⁻¹. It clearly reveals the main peaks associated with freeze–thawed PVA. For example, it can be easily observed C−H broad alkyl stretching band (ν = 2925 cm⁻¹) and the typical strong –OH group bands for free unreacted alcohol (non-bonded –OH stretching band at ν = 3650–3590 cm⁻¹) and hydrogen bonded bands (bonded –OH stretching bands at ν = 3590–3200 cm⁻¹). The hydrogen bonding between –OH groups at (ν = 3445 cm⁻¹) can occur among PVA chains due to high hydrophilic forces Mansur et al., 2004. Also, the presence of sharp absorption peak was noted at ν = 1150–1050 cm⁻¹. This stretching band has been used as an indicator for PVA structure, because it is a semi-crystalline synthetic polymer able to form some domains depending on several process parameters such as the F–T cycle number, the molecular weight and concentration of used PVA (Mansur et al., 2004). Additionally, it was found that a notable stretching band at ν = 1549–1453 cm⁻¹ of –CH₂ groups which are regarded as feature groups for chemical structure of PVA and PVA-SA blend polymer. All last mentioned stretching peaks, have been detected in structure of both PVA and PVA-SA blend polymer.

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Figure 1

The IR spectra of PVA-SA xerogel membranes without ampicillin.

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3.2 Study of physicochemical properties of PVA-SA hydrogel membranes

The consecutive F–T cycles produced entangled PVA-SA polymer hydrogel membranes. The influence of blending of sodium alginate (SA) contents (0%, 25%, 33%, 50%, 65%, and 75%) and sodium ampicillin introduction as a drug model on the gel fraction percentage (GF%), is calculated by Eq. (1) and shown in Fig. 2. Generally, the lower gel fraction was observed and accompanied to the less flexibility of obtained gel. In the absence of SA and drug (0% SA content and without drug), the gel fraction increased apparently to the maximum value, which was about 80%, suggesting that the PVA was almost crystallized in the highest degree and consequently entangled. This result is consistent with the obtained results by Yokoyama et al. (1986) While, GF% monotonically decreased with increasing SA content or addition of ampicillin in PVA hydrogel and decreased drastically to less than 39% at 75% of SA content. This behavior can be attributed to SA content and additions of ampicillin in PVA hydrogel might reduce the entanglement reaction and consequently the gelation process is reduced clearly. The obtained results have provided the principle of wound dressing materials in terms of saving a moist local environment with blending SA in varied ratios with PVA hydrogel membranes.

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Figure 2

Effect of SA content in PVA hydrogel membranes on gel fraction (GF%).

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Fig. 3 presents the water uptake percent or swelling behavior of PVA-SA hydrogel membranes versus SA contents using Eq. (2). In the light of our swelling study, when PVA-SA hydrogel membrane was immersed in distilled water for 20 min, a small amount of blended SA was dissolved in swelling medium. The dissolved amount of SA is sharply dependent on the initial blended SA in PVA hydrogel. Moreover, the dissolved amount of SA significantly affected the swelling test. As shown in Fig. 3, the maximum swelling ability increases with an increase in the SA content in PVA hydrogel up to a certain limit of huge swelling, the hydrogel structure was then destructed. This is due to SA as a blended material does not crosslink and has high ability to be soluble in water of swelling medium. While, in the absence of SA (0%, SA content); high crosslinked structure for PVA hydrogel has been obtained and this structure could not retain water amount within which result in low swelling ability, is about 1500% with a drug. After increasing SA content to 75%, the water uptake % progressively increases to 4200%. This is due to the high content of SA in PVA film increases the wettability and hydrophilicity characters of hydrogel which sometimes result in partial or complete destruction of hydrogel with much higher SA contents. These results are compatible with the reported results by Balakrishnan et al. (2005) and Choi et al. (1999).

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Figure 3

Effect of SA content in PVA hydrogel membranes on swelling degree or water uptake (%).

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3.3 Mechanical properties of PVA-SA hydrogel membranes

To investigate the blending influence of SA on the mechanical properties of PVA hydrogel membranes, the maximum tensile strength and elongation at break have been carried out and presented in Fig. 4. As shown, the maximum tensile strength and elongation at break of PVA-SA hydrogel membranes progressively decreased with increasing SA contents. Proportionally, the maximum tensile strength at break possessed the same pattern of behavior to elongation at break of hydrogel membranes. These results can be ascribed to the addition of SA into PVA hydrogels that might accelerate and destabilize the break elongation of hydrogel, which result in decreasing and deconstructing the maximum tensile strength. Furthermore, the obtained humble mechanical properties of PVA-SA due to the addition of high SA content might increase the hydrophilic susceptibility and decrease the entanglement degree which results in very weak mechanical resistance. These results are consistent with the obtained results by Rosiak et al. (2001). They have revealed that the maximum tensile strength of PVA hydrogel decreased with increasing the blended materials due to decreased crosslinking density. Similarly, our results are completely consistent with the reported results by Hwang et al. (2010). They have demonstrated that the maximum tensile strength of PVA hydrogel has sharply decreased with increasing dextran portions in the hydrogel.

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Figure 4

Effect of SA content in PVA hydrogel membranes on maximum strength and elongation to break.

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3.4 Morphological investigation of PVA-SA hydrogel membranes

The surface morphology of PVA-SA hydrogel membranes was investigated by SEM. The SEM micrographs of the surface of PVA hydrogel membranes versus SA portions are shown in Fig. 5. According to SEM micrographs, the absence of SA presents a very smooth, uniform, and non pores shape surface structure. However, addition of SA into PVA hydrogel in different potions 25% and 65% provides very tiny pores at surface; these pores notably increase with increasing SA contents. Furthermore, the pore diameters, the number of pores, and pore size distribution are significantly noticed at the surface of PVA hydrogel, when high SA contents were incorporated. This morphological change can be attributed to the extraction of SA particles in different agglomeration numbers and this explanation has been previously verified in swelling study (Fig. 3). Although, excess amount of SA in PVA hydrogel can perturb the formation of PVA crystallites because of partial miscibility with PVA, these parts of excess SA can be dissolved again and transfer from the hydrogel network into swelling solution. Also, it can be speculated that morphological changes are due to the presence of a big difference in the homogeneity, hydrophilicity, or miscibility degrees between two components of membrane (i.e. PVA and SA), which resulted in ordered-crystalline phase and uniform shape structure in case of 0% of SA, due to high entanglement of PVA and disordered-crystalline phase for PVA hydrogel blended with high portions of SA. The morphological results and our speculation are compatible with obtained results by Fathi et al. (2011) and Cascone et al. (1999).

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Figure 5

SEM micrographs showing the surface morphology distinctions of different SA contents in PVA hydrogel membranes, (original magnification was ×2500).

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The distinctions of the surface roughness of PVA hydrogel membranes versus SA portions are drawn in Fig. 6. It was easily noticed that the surface roughness drastically increased with increasing the blended SA contents regardless of the presence or absence of the loaded ampicillin as a drug model. This behavior is explained as the addition of high SA contents, might result in the formation of high amorphous like structure and formation of more structural pores due to the big difference in the miscibility degree between PVA and SA which produced significant surface roughness differences.

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Figure 6

Effect of SA contents in PVA hydrogel membranes on roughness (μm) of membrane’s surface.

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3.5 Drug release profile study

The cumulative ampicillin percentage release profile from PVA-SA hydrogel membranes was conducted in phosphate buffer solution (pH 8.0 at 37 °C) and the obtained data are drawn in Fig. 7. The initial release profile of ampicillin exhibited rapid release behavior in the first 15 min as known the fact of burst effect of drug release (Huang and Brazel, 2001). However, the release behavior increased slightly after several hours. The burst release of ampicillin varies in the range 38–45%, can be attributed to the rapid diffusion of ampicillin which was loaded close to the membranes’ surface. After the first 15 min, the increase of ampicillin release profile from PVA-SA hydrogel membranes can be ignored and no significant distinctions in the total release percentage were observed after 6 h of release. Furthermore, addition of SA in different portions to PVA hydrogel membranes has adverse influence on the release behavior of ampicillin. It is clearly noticed that an increase in SA content hindered the diffusion of ampicillin into phosphate buffer solution. It is likely that a bonding might occur between negatively charged COO⁻ groups of sodium alginate and positively charged ${\text{NH}}_3^{+}$ , Na⁺, or H⁺ groups of sodium ampicillin. This potential bridging agent can decrease and impede the release behavior of ampicillin. The current explanation is consistent with the obtained results of Takamura et al. (1992). They found that incorporation of SA in different portions decreased the released drug percent.

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Figure 7

Effect of SA contents on accumulative release percentage of ampicillin from PVA-SA hydrogel membranes in phosphate buffer solution (at 37 °C, pH 8.0).

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3.6 Wound dressing evaluation (in-vitro studies)

Essential characters for wound dressing films, such as protein adsorption, hydrolytic degradation, hemocompatibility, and antibacterial activity behavior were investigated under in vitro conditions to evaluate using our membranes as wound dressing materials. The obtained data are discussed in the following.

3.7 Protein adsorption

The protein adsorption onto PVA-SA blend hydrogel membranes has been conducted via in vitro experiments and calculated using Eq. (3), the protein adsorption results have been shown in Fig. 8. As shown in Fig. 8, the adsorption of BSA increased from 0.7–1.8 mg/cm² as the amounts of SA increased in the PVA hydrogels from 0–75 (%, w/w). Interestingly, the PVA hydrogels with loaded ampicillin showed a feasible adsorption of BSA as compared to free-ampicillin hydrogels, indicating that SA content affected the protein adsorption behavior onto the surface of PVA hydrogel. These results are completely consistent with the reported results by Kim et al. (2008) they have revealed that adsorption of protein increased with increasing blended alginate in PVA hydrogels. Although the hydrophilicity of membranes increased with SA content which exhibited a decrease of non-specific adsorption of proteins, the obtained results may be revealed to the incorporation of BSA molecules into the resulted pores in the membrane structure.

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Figure 8

Effect of SA content in PVA hydrogel membranes on protein adsorption values.

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3.8 Hydrolytic degradation

Gravimetric determination was used to study the in vitro hydrolytic degradation of PVA-SA hydrogel membranes (Gan et al., 1999). Due to the hydrolytic cleavage of hydrogen bonding among –OH groups of PVA chains, apparently weight loss values can be observed within the depicted results in Fig. 9. Also, this weight loss was previously noticed and expected during experiments in Fig. 3, due to the high water solubility of SA as a blend material and high hydrophilic forces among PVA chains. As shown in Fig. 9, it was found that the weight loss of pure freeze–thawed PVA membrane (0% SA) is relatively limited; almost 18%. While for PVA-SA membrane 75% SA content reaches 60%. These results refer to the weight loss of PVA-SA hydrogels that is dramatically increased with increasing SA contents. This phenomenon can be ascribed to the degradation of PVA-SA hydrogel membranes are predominantly the cleavage of entanglement segments of PVA and is consistent with the fact that the degradation of PVA is quite limited, whereas the degradation of PVA-SA is quite high. In addition, as PVA and SA are nontoxic materials, (Kamoun and Menzel, 2012; Xiao and Zhou, 2003) the PVA-SA hydrogel membranes and their degraded by-products might be expected to be nontoxic too.

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Figure 9

Effect of SA contents on weight loss of the PVA-SA hydrogel membranes versus different degrading times in phosphate buffer saline (PBS) (0.1 M, pH 7.4, at 37 °C).

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3.9 Hemocompatibility

The in vitro blood compatibility of the prepared PVA-SA xerogel membranes was determined here by the method of hemocompatibility test using compensation in Eq. (4). Table 1 represents the obtained hemolysis (%) values from PVA-SA xerogel membranes. According to the classification of hemolytic tendency of polymeric materials, (American Society for Testing and Materials, 2000) it is clear from the depicted data of hemolysis (%) in Table 1 that the hemolysis values vary in the range 0.06–1.22% with SA contents increasing, which indicate for a good compatibility of the xerogel membranes and non-hemolysis materials were found. These results can be explained by the fact that both PVA and SA are highly hydrophilic and biocompatible polymeric materials. Furthermore, no chemical crosslinking was used; the xerogel membranes thus showed excellent hemocompatibility property too. The hemocompatibility results are consistent with the obtained results by Chhatri et al. (2011).

3.10 Antibacterial activity

The in vitro antibacterial activity of PVA-SA hydrogel membranes containing sodium ampicillin was conducted and the data of the zone inhibition of bacteria are summarized in Table 1. The Gram positive bacteria (e.g. Staphylococcus pyogenes and Staphylococcus aureus) and the Gram negative bacteria (e.g. Pseudomonas aeruginosa and Proteus vulgaris) were utilized in this assay. Using antimicrobial drugs or antibiotic like ampicillin is necessary that play a vital role to stop microbes from growing or killing them outright (Chhatri et al., 2011). Moreover, these disinfectants can control of infectious disease spreading and inhibit the microbe activity till dead. Therefore, the wound dressing applications adapted from polymeric materials should be loaded with antimicrobial drugs, even the used polymeric material possesses antimicrobial activity itself e.g. chitosan (Kanatt et al., 2012; Yang et al., 2008). In the current investigation, the depicted data in Table 1 indicate that the loaded disinfectants (i.e. ampicillin) have broad antibacterial activity against pathogenic organisms such as Gram positive and negative bacteria, however addition of SA in different portions has no significant impact on antibacterial activity. These results clearly demonstrate that the prepared PVA-SA hydrogel membranes possess sufficient antibacterial activity.