Recent Progress in Materials is an international peer-reviewed Open Access journal published quarterly online by LIDSEN Publishing Inc. This periodical is devoted to publishing high-quality papers that describe the most significant and cutting-edge research in all areas of Materials. Its aim is to provide timely, authoritative introductions to current thinking, developments and research in carefully selected topics. Also, it aims to enhance the international exchange of scientific activities in materials science and technology.
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Free Publication in 2019
Current Issue: 2019
Open Access Original Research
Formulation-Properties of Novel Ibuprofen-Loaded Soy Protein Wound Dressings

Lia Ofek 1, Daniella Goder 2, Meital Zilberman 1, 2, *

1. Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel

2. Department of Materials Science and Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel

Correspondence: Meital Zilberman

Academic Editor: Hossein Hosseinkhani

Special Issue: Applications and Development of Biomaterials in Medicine

Received: July 31, 2019 | Accepted: September 25, 2019 | Published: October 11, 2019

Recent Progress in Materials 2019, Volume 1, Issue 4, doi:10.21926/rpm.1904004

Recommended citation: Ofek L, Goder D, Zilberman M. Formulation-Properties of Novel Ibuprofen-Loaded Soy Protein Wound Dressings. Recent Progress in Materials 2019;1(4):19; doi:10.21926/rpm.1904004.

© 2019 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.

Abstract

Background: Wound management is an important field in medical care, as annually, millions of wounds require treatment worldwide. Soy protein is highly biocompatible, easily processed, and a relatively cheap natural polymer, which can be used for wound dressing applications.

Methods: In this study, solvent cast soy protein films, plasticized by glycerol and cross-linked by glyoxal, were thermally treated and their mechanical properties were evaluated as a function of various processing parameters. Samples were then loaded with ibuprofen, an analgesi c, and their drug release profiles were assessed.

Results: Cross-linking and plasticizing were found to be essential for high mechanical strength while maintaining sufficient flexibility. Higher treatment temperature and longer duration of treatment resulted in higher tensile strength and Young’s modulus, and lower tensile strain, while higher concentrations of glycerol had the opposite effect. Release profiles of ibuprofen from the films exhibited a medium burst release of 71–83% in the initial 6 h, followed by a decline in the release rate. The glycerol content practically did not affect the drug release profile.

Conclusions: Our research shows that the novel soy protein-based films have a high potential in providing immediate pain relief after injury concomitantly exhibiting adequate tensile properties, and therefore are promising for wound coverage applications.

Keywords

Wound management; soy protein based films; analgesic drug Ibuprofen; wound coverage application

1. Introduction

Wound management is an important field of medical care as each year, millions of wounds are caused worldwide by accidents, surgeries, and burns. Wound healing is a biological process that involves the growth and regeneration of tissues [1,2,3,4]. Pain associated with burns and chronic wounds is a major day-to-day problem for the majority of patients and is described as a sensory emotional experience that is unpleasant and is associated with actual or probable damage to the tissues [5].

Anesthetics (drugs which cause numbness) and analgesics (drugs which cause reduction or relief from pain, keeping normal sensation and muscle ability intact) have proven to be efficient relief medications for acute and chronic pain [6]. The effect of administering systemic pain killers lasts for only several minutes to a few hours, thus making them only partly efficient for the treatment of pain related to chronic wounds [7]. Besides, the addictive nature of such treatment is another major concern. Therefore, local anesthesia is usually required to replace and/or support the systemic treatment. Ibuprofen is an analgesic that inhibits prostaglandin synthesis and may, therefore, inhibit the inflammatory phase of wound healing [8,9]. It is used as an effective adjunct in wound management, for reducing pain during dressings changes. A commercially available foam dressing that releases low doses of ibuprofen, Biatain-Ibu (Coloplast A/S), has demonstrated a reduction in pain intensity when compared to commonly used treatments for chronic exuding wounds, including foams, dressings with active components, hydrogels, hydrofiber, alginates, and hydrocolloids [6].

Bioresorbable polymers can be used for wound dressing applications to eliminate the need for a dressing change and need not be removed from the wound as they can degrade. Furthermore, their byproducts are exerted or absorbed by the body, and any remaining dressing can be easily washed with saline. Degradation of the dressing or implant may also facilitate localized controlled release of incorporated bioactive agents such as drugs or proteins, for promoting tissue regeneration [10]. Bioresorbable natural polymers have been extensively studied in wound dressing applications and were found to be favorable compared to synthetic bioresorbable dressings, due to significantly better biocompatibility. These polymers show high similarity to the extracellular matrix (ECM) and intrinsic cellular interactions make them highly suitable for biomedical applications, especially in the fields of tissue engineering and drug delivery systems [11]. These advantages concur with the growing trend in recent years to replace synthetic polymers with natural, abundant, non-animal origin, and low-cost biodegradable products such as soy protein for biomedical uses.

Soy protein has been studied mainly for its utility in the polymer, food and agriculture sectors and was selected for the current study due to its unique combination of hydrophilicity, biocompatibility, and tissue-like properties, besides being safe, low cost and versatile in processing [12]. Soy protein is not only non-cytotoxic but also contains a high concentration of polar amino acids, which stimulate cell proliferation and collagen deposition, promote tissue regeneration, therefore, has a significant potential for wound healing applications [13,14,15,16,17,18].

Soy protein has a large number of polar groups, such as hydroxyl, amide, and carboxyl which enable it to associate with different types of compounds, such as cross-linking agents and drugs. Crosslinking of soy protein is essential to achieve the necessary mechanical properties, assuring integrity and water stability during an implantation period [19]. For natural polymers, the most effective and commonly used plasticizers are sorbitol and glycerol [20]. In this study, glycerol was chosen as the preferred plasticizer due to the results obtained in the initial stages of this study. Analgesic drugs, such as ibuprofen, can be incorporated into the soy-based wound dressing and thereby add an additional attribute.

The current study focused on the development and characterization of a novel ibuprofen-loaded soy protein film for wound dressing applications. The resulting biodegradable wound dressing combines with a local pain management solution and forms a support system for wound healing. The benefit of local release, as opposed to systemic release, is the reduction in the drug amount needed for achieving a therapeutic effect, thus preventing unwanted side effects. Moreover, it also relieves the attending medical staff from the responsibility of administering active medication to the patient.

The suggested study is novel and noteworthy in several aspects: (a) soy protein is newly acknowledged in the medical field and soy wound dressings are not yet available commercially, (b) although there are various wound dressings which release antibacterial agents, there are not any formats for controlled release of analgesics to the wound site, and (c) understanding the relationships between processing parameters and formulation with the mechanical properties, drug release profile and bio-compatibility of the soy protein films, with and without the incorporation of ibuprofen, is of relevance in wound healing.

2. Materials and Methods

2.1 Materials

Soy Protein Source: Non-genetically modified soy protein (Solpro 910™, minimum 90% w/w dry protein) produced by Solbar™ (Ashdod, Israel) was obtained as a donation.

Plasticizer: Glycerol (G–7893) was purchased from Sigma-Aldrich (Rehovot, Israel).

Cross-linking agent: Glyoxal, 40 wt.% in H2O (50650), was purchased from Sigma-Aldrich (Rehovot, Israel).

Drug: Ibuprofen sodium salt (I1892) was purchased from Sigma-Aldrich (Rehovot, Israel).

Others: Phosphate buffered saline (PBS), pH 7.0 was purchased from J.T. Baker (Beith Dekel, Ra’anana, Israel). Sodium azide was purchased from Sigma-Aldrich (Rehovot, Israel). Methanol (99.8%) was purchased from Sigma-Aldrich (Rehovot, Israel). Acetonitrile (CH3CN, HPLC grade) was purchased from J.T. Baker.

2.2 Preparation of Ibuprofen-Loaded Soy Protein Films

Soy protein films were prepared using the solvent casting method. Double Distilled Water (DDW) was heated until the desired temperature (55 °C or 70 °C) was achieved. The soy protein was then slowly dissolved with constant stirring in hot water, followed by the addition of glycerol and glyoxal to the mixture.

The crosslinking reaction of glyoxal with the amino acids of the soy protein occurs as follows:

To obtain drug-loaded films, the ibuprofen sodium salt was then added. The solution was kept at a constant temperature of 55 °C or 70 °C with continuous stirring for 30 min on a digital hot plate (M.R.C labs, GHS) and then cooled at room temperature while still being stirred for additional 30 min to eliminate bubble formation. The solution, now at room temperature, was cast into low-density polyethylene plates and dried at ambient conditions for 72 h (approximately 25 °C, 40–60% relative humidity). The specimens were stored in desiccators at room temperature and 30% relative humidity until use. For thermal treatment, the specimens were placed in glass Petri dishes in a sealed oven at 60, 80 or 100 °C, and heated for the specified duration. All processing parameters are presented in Table 1.

The films’ thickness was controlled by casting the same volume of solution (50 mL) per plate, which was determined to be approximately 0.5 mm (using a micrometer). Dried films were removed from the plates and specimens were cut to size for each test.

Table 1 Processing parameters of the studied films.

2.3 Measurement of Tensile Properties

The tensile tests were performed on soy protein film samples having the shape of a dog bone (neck length of 20.6 mm and width of 4.6 mm), according to standard test method ASTM 188 D638–03. Measurements were carried out using a 5500 Instron universal testing machine (Instron Engineering Corp) with a 2 kN load cell. The tested samples were subjected to unidirectional tension, at a rate of 50 mm/min at room temperature.

The tensile strength was defined as the maximum strength at failure in the stress-strain curve. The maximal strain was defined as the breaking strain. The slope of the stress-strain curve in the elastic (linear) region was used to define Young’s modulus. At least three samples were tested for each type of specimen.

2.4 Evaluation of Drug Release Profile

In vitro release studies were performed to determine the release kinetics of ibuprofen from soy protein films with varying plasticizer concentrations. The soy protein films used for the release study were cast from 5% w/v solutions (55 °C, pH=7.2), cross-linked using 1% w/w glyoxal at 80 °C for 24 h. Films plasticized with four glycerol concentrations (25%, 30%, 40% or 50% w/w glycerol) were studied. All films were loaded with ibuprofen at 3% w/w (1.5 mg/mL in solution).

Soy protein film discs (1.5 cm diameter) were weighed and immersed in 1.5 mL PBS (pH=7.0) in scintillation bottles. Sodium azide (0.02% w/v) was added to the medium to prevent bacterial and fungal growth. The immersed samples were placed in a static incubator (Heraeus, B12) at 37 °C and 100% humidity for 42 days during which the medium was replaced at predetermined time points. The medium was completely removed at each sampling time (1 h, 6 h, 12 h, 1, 2, 3, 5, 7, 14, 21, 28, 35 and 42 days) and fresh medium was added. The removed medium was filtered using a disposable filter unit (Whatman, 0.2 μm) and kept in HPLC glass vials at –20 °C until analysis by High-Performance Liquid Chromatography (HPLC).

The concentration of ibuprofen in the media samples was determined by Jasco HPLC using a UV 2075 Plus detector and a reverse-phase column (ACE 5 C18, inner diameter d=4.6 mm, length=250 mm), with a guard cartridge (ACE 5 C18, inner diameter d=3.0 mm, length=10 mm), at a constant temperature of 40 °C. The mobile phase consisted of a mixture of PBS (pH 3.3) and acetonitrile (40/60, v/v) at a flow rate of 2 mL/min, with a quaternary gradient pump (PU 2089 plus) without gradient. Samples (20–40 μL) were injected with an autosampler (AS 2057 Plus). The area of each eluted peak was integrated using EZstart software version 3.1.7 according to a pre-determined calibration curve and the amount of drug in the vial was determined. The result for each sampling time was multiplied by a dilution factor to determine the amount of drug released in the scintillation bottles. The dilution factor was calculated using the following formula:

$$Dilution\ Factor= \frac{Volume\ of\ medium\ in\ scintillation\ bottle}{Volume\ of\ injected\ HPLC\ sample}$$
(1)

Residual drug recovery from the SPI films was measured by cleaving the SPI film completely in trypsin A solution at 40 °C for 24 h and drug concentration was measured using the HPLC method described above. For all the sampling points, any residual drug was added to the cumulative amounts of ibuprofen and the total amount obtained was used to normalize the results.

2.5 Statistical Analysis

Data were processed using Excel software. Statistical comparison between more than two groups was made using the ANOVA (Tukey-Kramer) method via the IBM SPSS Statistics software (ver. 19). A value of p<0.05 was considered statistically significant for all types of statistical comparisons. All errors and error bars indicate standard deviation (SD) from the mean.

3. Results

Soy protein films were prepared using solution casting technique under varying processing parameters and were homogenous and transparent with a light yellowish color. The cast soy protein films were assessed for their potential to serve as drug-eluting wound dressings by studying the two important characteristics of drug-eluting wound dressings, effects of the process parameters on the tensile properties and drug release profile.

3.1 Tensile Properties

Wound dressings ideally combine strength with ductility and flexibility. The parameters that were studied to determine their effect on the films’ tensile properties were cross-linking agent concentration, duration, and temperature of the thermal treatment, soy protein concentration, plasticizer concentration, and drug concentration. Understanding the effect of the processing parameters on the mechanical properties is crucial in choosing the most suitable samples for their use in the following studies.

3.1.1 The Effect of Concentration of Cross-Linking Agent

Since soy protein is a natural polymer, we initially hypothesized that to create durable films, cross-linking by chemical and/or physical means is required, and therefore selected glyoxal as the preferred crosslinking agent.

To determine the ideal glyoxal concentration to prepare soy protein films, film samples were cast from soy protein solutions with 50% w/w glycerol as a plasticizer and glyoxal at different concentrations as the cross-linking agent followed by a thermal treatment as per the protocol mentioned earlier in this article.

The results presented in Figure 1 show that the higher concentration of glyoxal enhanced the soy protein film’s strength with a concurrent decrease in its maximal strain. The soy protein films cast using the highest concentration of glyoxal (2% w/w relative to soy protein) were significantly stronger than the films cast using 0%, 0.5%, and 1% (w/w relative to soy protein) of glyoxal. There was no significant difference in films with 0%, 0.5%, and 1% glyoxal, probably due to the high variation within the samples prepared with 0.5% glyoxal. Likewise, the changes in Young’s modulus and maximal strain due to changes in the glyoxal concentration were also not significant.

3.1.2 The Effect of Thermal Treatment

Two parameters of the thermal treatment process, i.e., temperature and duration of treatment, were assessed for their effect on the soy protein films’ tensile properties.

Soy protein films thermally treated at 100 °C showed higher tensile strength and Young’s modulus and lower values of maximal strain compared to the films treated at 60 °C and 80 °C. However, no significant difference in mechanical characteristics was observed in the films treated at 60 °C and 80 °C (Figure 2).

With the increase in the duration of thermal treatment, a gradual increase in tensile strength and Young's modulus of the soy protein films as well as a decrease in the maximal strain were obtained (Figure 3), and these changes were statistically significant. Figure 4 presents stress-strain curves for selected samples that were subjected to thermal treatment at varying temperatures and durations. The differences in strength, stiffness and elongation during thermal treatment are clearly presented as a function of the temperature and duration.

Therefore, an increase in the temperature and duration of the thermal treatment resulted in steeper elastic region (indicating higher stiffness, higher yield point, higher tensile strength, and lower maximal strain.

Figure 1 The effect of the crosslinking agent (glyoxal) concentration on the properties of the soy protein films: (a) Tensile strength, (b) Young's Modulus and (c) Maximal strain. Films were cast from 5% w/v soy protein solutions (70 °C, pH 7.2) with 50% w/w glycerol and 0, 0.5, 1 or 2% w/w glyoxal, relative to soy content. The films were heat-treated for 24 h at 80 °C. Statistical significance is marked with an asterisk (*).

Figure 2 The effect of thermal treatment temperature on (a) Tensile strength, (b) Young's Modulus and (c) Maximal strain of soy protein films. Soy protein films were cast from 5% w/v SPI solutions (70 °C, pH=7.2) with 50% w/w glycerol and 1% w/w glyoxal. The dried films were then subjected to a 24 h long thermal treatment at temperatures of 60, 80 and 100 °C. Statistical significance is marked with an asterisk (*).

Figure 3 The effect of thermal treatment duration on (a) tensile strength, (b) Young's Modulus and (c) maximal strain of soy protein films. Films were cast from 5% w/v SPI solutions (70 °C, pH=7.2) with 50% w/w glycerol and 1% w/w glyoxal. The dried films were then subjected to a thermal treatment at 80 ºC, for varying durations of 1, 3, 6 and 24 h. Statistical significance is marked with an asterisk (*).

Figure 4 Examples of stress-strain curves for soy protein films following thermal treatments. The temperature and duration used in the thermal treatment are presented adjacent to the plot.

3.1.3 The Incorporation of Plasticizer and Ibuprofen

The effect of the plasticizer (glycerol) concentration was studied on films loaded with 3% ibuprofen and unloaded films. In all types of wounds, dressings with controlled release of analgesic drugs are beneficial to patients. Therefore, we loaded our soy-based films with 1.5 mg/mL ibuprofen (3% w/w relative to soy protein) and evaluated the effect of the drug incorporation on the properties of films plasticized with glycerol at varying concentrations. The results are presented in Figure 5.

An increase in plasticizer concentration resulted in reduced tensile strength and Young's modulus and enhanced maximal strain of the film. Significant differences in mechanical properties were found in the ibuprofen loaded group for Young’s modulus as well as in the ultimate tensile stress values for 25% glycerol and all other plasticizer concentrations. Likewise, significant differences in maximal strain were observed between the treatments with 25%, 40% and 50% glycerol and between 30% and 50% glycerol. In unloaded films, significant differences in Young’s modulus for all plasticizer concentrations except between 40% and 50% glycerol were observed, while ultimate tensile stress was significantly different between all groups except for 30% and 40% glycerol and between 40% and 50% glycerol. Besides, unloaded films also showed a significant difference in maximal strain between 25% to 40% and 50% plasticizer contents. Thus, incorporation of ibuprofen did not significantly change these properties, except for the films loaded with 50% plasticizer, which showed significantly lower Young’s modulus and higher maximal strain in the ibuprofen loaded samples, compared to the unloaded ones.

Figure 5 The effect of plasticizer content on (a) ultimate tensile strength, (b) Young’s modulus, (c) maximal strain of ibuprofen loaded films, compared to the unloaded films. Films were cast from 5% w/v SPI solutions (70 °C, pH=7.2), with 1% w/w glyoxal and 25, 30, 40 or 50% w/w glycerol. Statistical significance is described in the text.

3.2 Ibuprofen Release Kinetics from Soy Protein Films

The cumulative release profiles of ibuprofen from films plasticized with various glycerol concentrations are presented in Figure 6.

The first stage in the drug release process is a medium burst release, followed by a decline in release rate during the first two days (Figure 6a). The majority of the ibuprofen was released on the first day. The extraction procedure of all samples retrieved no residual drug, which is attributed to the release of the entire amount of encapsulated drug during the study. The burst release values for all studied samples are presented in Table 2. When the cumulative release profiles are presented as absolute quantity (micrograms), differences between the samples of different plasticizer concentrations were observed mainly due to variations in the initial weights of the film samples (Figure 6b).

Figure 6 The cumulative release profiles of ibuprofen from soy protein films at varying plasticizer concentration: 25% (), 30% (), 40% (), and 50% () w/w. Ibuprofen concentration: 3% w/w ibuprofen (1.5 mg/mL). The results are given in (a) percentage and (b) absolute quantity.

Table 2 Burst effect for Ibuprofen release from soy protein films with varying plasticizer concentrations.

4. Discussion

The current research focuses on the development and study of novel biodegradable wound dressings, based on soy protein films. Soy protein is an excellent choice due to its high biocompatibility and biodegradability. It also has commercial advantages, such as being economically competitive and presenting good water-resistance and storage-stability.

We prepared soy protein films, using the solution casting technique and varying processing parameters. Following the mechanical characterization of these films, reference specimens were selected for the controlled release study of the analgesic drug, ibuprofen. Ibuprofen is well-proven for its efficiency in both acute and chronic pain therapy [6]. It is widely used in the pain management of wounds as an effective adjunct for reducing pain during the replacement of dressings.

4.1 Mechanical Properties

The mechanical properties of a wound dressing are of high importance for its function and performance. To withstand the stresses generated during its handling and application and to avoid tearing, the dressing must combine strength with ductility and flexibility. For wound dressings made of natural polymers, such as collagen or gelatin, mechanical properties are expected to deteriorate rapidly due to hydration and enzymatic activities, characteristic of the dressing’s clinical environment. Therefore, cross-linking is important to relatively maintain the strength of the dressing.

Several research groups have produced biopolymer films made of soy protein and gelatin, using different processing conditions and cross-linking agents [18,21,22,23]. Compared to these films, our solution cast soy protein films demonstrated excellent tensile strength and elongation at break. The tensile strength of our soy protein films is 1.1-2 times higher than other soy protein films [21,22] and seven times higher than gelatin mats, based on electrospun fibers [23]. Similarly, the maximal strain of our soy protein films is 1.4­­-2.3 times higher than other soy protein films [18,22] and eight times higher than gelatin mats [23]. Hence, we succeeded in increasing the overall flexibility of the film compared to other soy protein films, with a slight increase in the tensile strength.

We studied various processing parameters to determine their effects on the tensile mechanical properties of the film, the results of which are discussed below.

4.1.1 The Effect of Crosslinking

Cross-linking of hydrophilic biopolymers is known to improve their mechanical strength as well as their susceptibility to hydrolytic and enzymatic degradation [24,25]. Hence, we aimed to induce this synergistic effect on our soy protein films by cross-linking. In this study, we employed two cross-linking methods-(i) chemical agent (glyoxal) and (ii) thermal treatment. In our opinion, the synergistic effect is achieved due to the different mechanisms in which the cross-linking is created in each method. Chemical cross-linking using glyoxal mostly involves free e-amino groups; whereas, the heat treatment encourages the formation of disulfide bonds (S-S bonds), new hydrogen bonds and hydrophobic interactions [26]. In addition, heat treatment may enhance the chemical reaction of glyoxal with the protein chains, resulting in a greater number of cross-links via amine groups.

Previous studies have reported that cross-linking via glyoxal increases the tensile strength and Young's modulus while decreasing elongation in soy protein films [24,27]. It induces intramolecular and intermolecular cross-links by reacting with the free e-amino groups (-NH2), sulfhydryl groups (-SH) and the sidechains of histidine and tyrosine [28]. Therefore, soy protein is susceptible to glyoxal as it is rich in the basic amino acids, lysine, and arginine.

We have previously investigated the effects of cross-linking agents like glyoxal and L-cysteine on soy protein films, cast from 5% w/v solution. Our results demonstrated that glyoxal imparted better and less variant mechanical properties on the soy protein films (not statistically significant) compared to L–cysteine [29]. Since glyoxal is considered the least toxic among the dialdehyde cross-linking agents, we decided to use it for the current study.

This study exhibited that higher concentrations of glyoxal increased the film strength and decreased its maximal strain, with no significant difference between the groups (Figure 1). The small differences in glyoxal amounts used (due to its low concentration in the solution) and the variant results probably contributed to the statistical insignificance of the results. The samples with 0.5% w/w glyoxal varied vastly; whereas, the samples with 2% w/w glyoxal were expected to have a toxic effect and decreased flexibility. Hence, we continued our study using a concentration of 1% w/w glyoxal relative to soy protein.

To ensure that the dressing components and the processing methods did not cause any cytotoxic effects, a cytotoxicity study was performed on fibroblast cells [30]. Fibroblasts are a major cell type in the dermis and are actively involved in the wound healing process. Fibroblasts produce remodeling enzymes such as proteases and collagenases, which play an important role in the healing of a wound. We assessed cell viability in the presence of soy protein dressing extracts by observing cell morphology and by assaying with Alamar-Blue. The latter is similar to the MTT assay, measuring changes in cellular metabolic activity. The morphology and viability of cells cultured in the presence of soy protein film extracts were similar to the control group. There was also a constant increase in cell viability over a period of 72 h. This was also the case in extracts from 0–24 h and 24–48 h. Hence, our soy protein films are biocompatible and do not induce cytotoxic effects.

4.1.2 The Effect of Thermal Treatment

The soy protein films, thermally treated at 100 °C, showed higher tensile strength and Young’s modulus and lower values of maximal strain, compared to the films treated at 60 °C and 80 °C (Figure 2). There was no significant difference between the mechanical characteristics of the films treated at 60 °C and 80 °C. The increased strength and toughness and decreased flexibility, resulting from heating, can be partially attributed to the heat-induced intramolecular and intermolecular cross-links formed within the film structure, via disulfide bonding, hydrogen bonding and hydrophobic interactions between lysine and cysteine sidechains [26,31].

Apart from the heat-induced cross-linking, differences in moisture content contribute to the effect of heating on the tensile mechanical properties of the films. Earlier studies reported that water plasticizes hydrophilic protein-based films, such as wheat gluten and whey protein [21]. The heat-induced cross-links detailed above increase the hydrophobicity of the films, thereby decreasing their moisture content. Therefore, heat treatment reduces the plasticizing effect of water, resulting in an increase in the films’ strength and a decrease in its flexibility.

Thermal curing for 24 h at 80 °C resulted in excellent tensile mechanical properties. Thus to reduce the preparation time of films, we investigated whether shorter periods of thermal treatments would be sufficient. We treated the films with heat for 1, 3, and 6 h as opposed to the usual 24 h period. The tensile strength and Young's modulus of the films gradually increased and the maximal strain decreased with an increase in the duration of thermal treatment (Figure 3). Longer treatment is therefore expected to result in higher cross-linking density. While a 24-hour treatment resulted in a stronger film, the slightly decreased strain is still sufficient for the films’ purpose. Therefore, we demonstrate that the ideal thermal treatment should last 24 h.

4.1.3 The Effect of Plasticizer and Ibuprofen Incorporation

A plasticizer is an essential component in soy protein films. Without plasticizing, the films are brittle and impossible to handle [32]. Incorporation of a plasticizer, such as glycerol, is also necessary for longer storage to avoid cracking of films even before their use. Soy protein films, prepared without plasticizer or with glycerol concentrations lower than 25% w/w relative to soy protein, were highly brittle and difficult to handle. On the contrary, films cast from solutions containing 70% w/w glycerol were very sticky. It was difficult to prepare the dog bones for determining tensile mechanical properties and the samples remained sticky after the thermal treatment. This posed a problem in conducting the tensile tests. From these preliminary experiments, we concluded that glycerol concentrations of 25–50% needed to be investigated to determine its optimal concentration in the solution for casting films. An increase in plasticizer content resulted in higher maximal stress and Young's modulus and a lower maximal strain of the films (Figure 5). As is widely known, polyol-based plasticizers reduce stiffness and induce flexibility by penetrating between protein chains, forming hydrogen bonds and lowering the glass transition temperature (Tg) [32]. Thus, the addition of a plasticizer enables easy handling and good flexibility that prevents cracking during processing, storage, and application.

The incorporation of the drug in the film barely changed the properties of films plasticized with 25%, 30% or 40% glycerol (Figure 5). Some difference was observed only for films plasticized with 50% glycerol. Therefore, we deduce that ibuprofen is chemically inert in the soy protein film structure and hardly affects its mechanical properties. The glycerol content of 50% is relatively high. When ibuprofen was incorporated in such a formulation, it probably enhanced its plasticizing effect, thus resulting in lower modulus and higher maximal strain. Therefore, we investigated its effect on the drug release profile.

4.2 Ibuprofen Release Kinetics

We prepared films from solutions of 5% w/v soy protein at a temperature of 55 °C, crosslinked with 1% w/w glyoxal, plasticized with 50% w/w glycerol and then thermally treated them at 80 °C for 24 h. These films displayed an ideal combination of strength and flexibility and hence were used for studying the drug release kinetics. As presented above, plasticizing had a significant effect on the mechanical properties of the film.

All studied formulations exhibited a medium burst release of 30–40%, followed by a decrease in the release-rate during the first day. There was no significant effect of the glycerol content on the ibuprofen-release profile in our study.

The cumulative amounts of ibuprofen observed were approximately 0.074 mg/cm2 in the first hour and 1.59 mg/cm2 during the first 6 h (Table 2).

The hydrophilic nature of both, soy protein and ibuprofen, induces drug diffusion from the soy protein matrix before any significant degradation of the drug occurs. As a result, the burst effect and the release profile are typical of such diffusion-controlled systems. The hydrophilicity of soy protein accelerates water uptake, leading to full swelling of the matrix during the first few hours of immersion [29]. Additionally, the hydrophilicity of ibuprofen accelerates the diffusion of the drug from the film. The release rate decreases following the initial burst effect because the drug has a longer path to pass through, while simultaneously, the driving force for diffusion decreases.

It should be noted that the method used in this in vitro drug release study was designed to resemble the conditions in the clinical application. The immersed samples were stored at 37 °C and 100% humidity. These conditions can be considered "worst-case" in terms of water uptake and weight loss since in clinical application the wound dressing is not fully immersed in liquid. The medium replacement method (a complete extraction at each sampling point) was selected to simulate the removal of the drug from the wound area in a clinical environment, affecting the driving force for diffusion. Nonetheless, this study did not simulate the enzymatic degradation of the SPI film, expected during clinical application.

Pain management is a crucial factor for the entire rehabilitation period. Administration of pain relief medications is required for varying periods of time, depending on the type and severity of the wound. The only commercially available dressing with a pain relief effect is a constant, low-dose, ibuprofen-eluting foam dressing-Biatain Ibu (Coloplast A/S, Humlebaek, Denmark). The 10×10 cm dressing contains 50 mg ibuprofen, with 0.5 mg/cm ibuprofen homogeneously dispersed throughout the foam. This is equivalent to a quarter of an ibuprofen tablet and can exert adequate pain-reducing effects for up to seven days.

The burst effect demonstrated in our drug release profile can be beneficial for immediate pain relief within the first hours of wound treatment. For example, it will be applicable in pains associated with cutaneous wounds, specifically in burns reported to occur immediately after the injury. The rate of drug release, following the burst effect, shall be sufficient for local pain management, which is often complementary to systemically administrated pain-killers. In treating various chronic wounds, the dressing is usually replaced every 1–2 days. Hence, our soy-based wound dressing with ibuprofen-release is optimal. In order to achieve various release profiles suitable for different wound types and severities, varying amounts of ibuprofen could be loaded into the film.

5. Conclusions

Our study explores the concept of soy protein wound dressings with ibuprofen-release and displays its feasibility. The dressings we have developed combine the desired tensile mechanical properties and drug release capabilities. Cross-linking and plasticizing are essential for achieving strong yet flexible films, which are suitable for wound dressing purposes. The combination of chemical cross-linking using glyoxal and thermal cross-linking has a synergic effect that strengthens the film. In addition, glycerol contributes to the flexibility and ease of handling of the films. Longer and higher temperature thermal treatments increase the tensile strength and Young's modulus of the films and decrease their maximal strain, probably due to the higher cross-linking density. A temperature of 80 °C is favorable for a combination of strength and flexibility.

The ibuprofen-release profiles of soy protein films, with varying plasticizer concentrations, display a characteristic medium burst release, followed by a decrease in release-rate, during the first three days. These profiles are typical for diffusion-controlled systems, due to the hydrophilic nature of both, soy protein and ibuprofen. Most of ibuprofen is released during the first six hours after application. This is an important feature for instant pain relief, immediately after a severe injury. Glycerol concentrations do not affect the ibuprofen-release kinetics.

We successfully demonstrate that selecting suitable components and processing conditions enables the production of cost-effective soy protein films with desired tensile properties. These films can be loaded with analgesic drugs like ibuprofen and optimized for drug release, making them potential wound dressings for use in clinical applications.

Acknowledgments

The authors are grateful to the Israel Science Foundation, ISF, grant no. 1055/11/for supporting this research, and thank Solbar™ (Ashdod, Israel) for kindly providing the soy protein isolate used in this study.

Author Contributions

Lia Ofek and Daniella Goder designed and conducted the study and framed the manuscript, under the supervision of Prof. Meital Zilberman.

Competing Interests

The authors have declared that no competing interests exist.

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