Stimulation of Wound Healing by Electroactive, Antibacterial, and

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Stimulation of wound healing by electroactive, antibacterial and antioxidant polyurethane/siloxane dressing membranes: in-vitro and in-vivo evaluations Reza Gharibi, Hamid Yeganeh, Alireza Rezapour-Lactoee, and Zuhair Mohammad Hassan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08376 • Publication Date (Web): 16 Oct 2015 Downloaded from http://pubs.acs.org on October 17, 2015

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ACS Applied Materials & Interfaces

Stimulation of wound healing by electroactive, antibacterial and antioxidant polyurethane/siloxane dressing membranes: in-vitro and in-vivo evaluations Reza Gharibiǂ, Hamid Yeganeh*ǂ, Alireza Rezapour-Lactoeeϯ, Zuhair M. Hassanѣ ǂ

Department of Polyurethane, Iran Polymer and Petrochemical Institute, P.O. Box: 14965/115,

Tehran, Iran. ϯ

Department of Tissue Engineering, School of Advanced Medical Technologies, Tehran

University of Medical Sciences, 14177-55469 Tehran, Iran ѣ

Department of Immunology, School of Medical Sciences, Tarbiat Modares University,

P.O. Box: 14115-331, Tehran, Iran.

ABSTRACT

A series of novel polyurethane/siloxane based wound dressing membranes was prepared through sol-gel reaction of methoxysilane end-functionalized urethane-prepolymers composed of castor oil and ricinoleic methyl ester as well as methoxysilane functional aniline tetramer (AT) moieties. The samples were fully characterized and their physico-chemical, mechanical, electrical and biological properties were assayed. The biological activity of these dressings against fibroblast cells and couple of microbes was also studied. It was revealed that samples which displayed electroactivity by introduction of AT moieties showed a broad range of antimicrobial activity towards different microorganisms, promising antioxidant (radical

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scavenging) efficiency and significant activity for stimulation of fibroblast cells growth and proliferation. Meanwhile, these samples showed appropriate tensile strength and ability for maintaining moist environment over wound by controlled equilibrium water absorption and water vapor transmission rate. The selected electroactive dressing was subjected to an in vivo assay using rat animal model and the wound healing process was monitored and compared with analogous dressing without AT moieties. The recorded results showed that the electroactive dressings induced an increase in the rate of wound contraction, promoted collagen deposition and encouraged vascularization in wounded area. Based on the results of in vitro and in vivo assays, the positive influence of designed dressings for accelerated healing of a wound model was confirmed.

KEYWORDS: polyurethane/siloxane, wound healing, antimicrobial, antioxidant, elctroactivity.

INTRODUCTION A wound can be defined as an injury or tear on the skin surface by physical, chemical, mechanical, and thermal damages.1 When skin is damaged, the resulting wound should be protected from further contamination or trauma by covering with a proper dressing. To ensure effective wound healing, the dressing should be nontoxic and biocompatible, promote gaseous exchange and protect wound from external mechanical stress. Dressings should also provide and maintain a moist environment over wounded skin, since it is widely accepted that under such condition acceleration of healing process can occur.2 To construct a dressing with maximum number of ideal factors, several synthetic and natural polymers can be utilized. Among synthetic


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materials, polyurethanes with high biocompatibility, good permeability to oxygen and carbon dioxide, excellent mechanical strength and proper flexibility have been widely used for wound dressing application.3 The attractive characteristics of polyurethanes for wound dressing application are mainly determined by the nature of the starting materials in their synthesis. Preparation of polyurethanes from renewable resources, especially vegetable oil-based polyols has become an area of intensive interest, due to their attractive physico-mechanical and excellent biocompatibility.4,5 Nowadays, these materials have found applications as bone substitute,6 nerve regeneration7 and wound dressing.8 Castor oil (CO) is a very special material among vegetable oils. The major constituent of CO is ricinoleic acid (12-hydroxy-cis-9-octadecenoic acid), a hydroxyl-containing fatty acid, which provides the condition for direct synthesis of polyurethane without any prior functionalization reactions. Interesting biological properties are also reported for CO and its derivatives such as high biocompatibility, remarkable analgesic and anti-inflammatory effects,9,10 as well as positive influence on epithelialization process.11 Therefore, CO has been selected as the main starting material for preparation of polyurethane dressings in the present work. Close inspection of advanced wound dressings recently introduced into the market revealed that these materials have undergone significant changes. In addition to physical protection of wounds the advanced dressing materials should stimulate tissue regeneration and healing process actively. Among several possibilities for active involvement in healing process, we have focused on three characteristic features, i.e. antimicrobial activity, electroactivity and antioxidant properties. The logics behind these prerequisites are described below:



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Bacterial infections delay healing process, increase exudate formation and facilitate improper collagen deposition.12 Wound dressing itself can also cause infection if it is improperly sterilized.12 There are interesting reports in literature which reveal that antimicrobial agents like silver nanoparticles have the ability of reducing the number of pathogens and the inflammatory response the of the wound site can promote wound healing process.13 Therefore, utilization of antimicrobial wound dressings is a good method to avoid the above mentioned problems. Some recent studies have confirmed the broad spectrum antimicrobial activity of polyaniline and its oligomeric analogous.14,15 To take advantage of this characteristics oligoaniline was examined in the present work. There are interesting research findings regarding significant role of electroactive materials, such as conductive polymers, played in cellular activities such as cell adhesion, proliferation, migration, and differentiation of electrically excitable cells such as nerve, bone, muscle, keratinocytes, fibroblasts, cardiac, and mesenchymal stem cells.16 Since the regulation of cellular behavior is critical for the regeneration of new tissue, therefore, utilization of this advantage in dressing membrane is considered in this study. Reactive oxygen species (ROS) are important inflammatory mediators under pathological condition and overproduction of these species can disrupt the cellular oxidant/antioxidant balance leading to tissue damage, infection, and slow wound healing.17 The conducting polymers are widely studied to scavenge free radicals,18,19 therefore, wound dressings containing conducting polymers can reduce the excessive levels of free radicals and consequently, protect tissue from oxidative damage during healing process. Utilizing radical scavenging activity of oiligoaniline for modification of dressing membranes was considered in the present work.


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In our previous work, we have shown that simultaneous utilization of these features (antimicrobial activity, electroactivity and antioxidant property) in a wound dressing material could have positive influence on wound healing process under in vitro condition.20 To expand this strategy and widen the wound types that can be healed by this active category of dressings, new polyurethane/siloxane membranes were prepared using a renewable resource raw material, CO, and its derivative ricinoleic methyl ester (RM). An oligoaniline material, aniline tetramer (AT), was also properly functionalized and incorporated into the dressing formulations as an active ingredient. These novel materials with tailor-made physical and mechanical properties were fully characterized by conventional methods. Their biological activities including their influence on proliferation of fibroblast cells and inhibition of microbial growth were examined under in vitro condition. Also, the designed active dressing was applied on full thickness skin wound created on rat animal model to acquire a better insight regarding its potential influence on wound healing process. For comparison, a similar dressing without active AT moieties was prepared and its performance on similar wound model was explored.

EXPERIMENTAL SECTION Materials CO with hydroxyl number 154.5 mg KOH/g was purchased from Sigma. It was dried at 80 ᵒC under vacuum for 24 h just before use. Isophorone diisocyanate (IPDI) from Merck was purified via

vacuum

distillation.

phenylenediamine,

(3-Aminopropyl)

methanol,

trimethoxysilane

dibutyltindilaurate

(DBTDL),

(APS),

N-phenyl-1,

ammonium

4-

persulfate,

glutaraldehyde (GA) (50 wt% in H2O), sodium metal, diethyl ether and camphorsulfonic acid



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(CSA) were purchased from Aldrich. N,N-Dimethylformamide (DMF), chloroform and methyl ethyl ketone (MEK) were distilled over CaH2. All other chemicals were of analytical grade and used as received. S. aureus (ATCC 6538), P. aeruginosa (ATCC 15449) bacteria and C. albicans (ATCC 10231) were purchased from Iranian Research Organization for Science and Technology (IROST). Mouse L929 fibroblast cells were also received from Pasteur Institute of Iran and used as obtained Synthesis of methoxysilane-terminated CO-based polyurethane prepolymer (Si-CPU) A three-necked polymerization reactor equipped with a mechanical stirrer, condenser, dropping funnel and a nitrogen inlet was charged with IPDI (37.36 g) and CHCl3 (100 ml). A solution of CO (40.00 g) in CHCl3 (100 ml) was slowly dropped into the reactor during 30 min under ambient temperature. After that, a drop of DBTDL catalyst was introduced into the system and the temperature increased. The reaction continued under reflux condition until the free NCO content of the product reached the calculated theoretical value as determined by back titration method (ASTMD-2572) using standard solution of dibutylamine. The heating mantel was removed, and the reactor content was cooled to 5 ᵒC. Then APS (60.23 g) was dropped into the reactor. The mixture was stirred at the same temperature for 30 min, and then the temperature increased to 50 ᵒC for 3 h. The reaction product with pre-determined solid content (30% w/w) was transferred to a glass bottle, sealed and kept in refrigerator. Synthesis of methoxysilane-terminated ricinoleic methyl ester urethane prepolymer (SiRM) Firstly, ricinoleic methyl ester (RM) was synthesized and characterized according to the procedure reported by Romero21 with some modifications. A 250 ml two-necked round-bottomed


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flask, equipped with condenser and magnetic stirrer, was charged with CO (20.0 g) and methanol (64.0 g). Then, a solution of sodium methoxide in methanol (0.6 ml, 16% w/v) was added to the above solution and the reaction mixture was heated to 65-70°C and stirring was continued for 2 h. After this stage the excess amount of methanol was removed by using a rotary evaporator. The crude product was dissolved in diethyl ether (40 ml) and transferred into a separating funnel. The ether solution was repeatedly washed with distilled water, until pH value of the water phase was neutral. The solvent was evaporated and the final product was subjected to high vacuum at 85 °C for 12 h to ensure complete removal of any residual solvent and water molecule. The OH number of synthesized RM was measured as 168.5 mg KOH/g. In the second step, appropriate amount of RM (11.1 g), IPDI (7.33 g), dried MEK (40 ml) and a drop of DBTDL catalyst were added to a 100 ml two-necked round-bottomed flask, equipped with a condenser, magnetic stirrer and nitrogen inlet. While stirring, the temperature was increased to 80 °C. The reaction continued until the free NCO content of the product reached its calculated theoretical value. In this stage, the reactor content was cooled to room temperature and APS (6.55 g) was dropped into the reactor. Then, the temperature was increased to 50 °C and reaction was continued for 3 h. Finally, the reaction product with a pre-determined solid content (30% w/w) was transferred to a glass bottle, sealed and kept in refrigerator.

Synthesis of methoxysilane-terminated aniline tetramer (Si-AT) Si-AT was synthesized according to the procedure described in our previous article.20



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Preparation of polyurethane/siloxane cross-linked networks composed of Si-CPU and SiRM (NESiPU1-4) NESiPU1-4 films were prepared according to the formulations given in Table 1. The Si-CPU was mixed with different amounts of Si-RM, and the total volume of mixtures was adjusted to 20 ml by addition of MEK. After removal of trapped air bubbles by application of partial vacuum, the solution was cast slowly into a clean Teflon mold and kept at room temperature overnight. It was then heated in an oven at 80 ᵒC for 12 h and at 100 ᵒC for 2 h. The resulting films were finally subjected to high vacuum at 70 °C to ensure removal of any residual solvent. Preparation of electroactive polyurethane/siloxane wound dressing membranes (EASiPU13) The dressing membranes were prepared according to the formulations given in Table 2. The appropriate amounts of Si-CPU and Si-RM were mixed with different portions of Si-AT and the total volumes of mixtures were adjusted to 20 ml by addition of DMF. After removal of trapped air bubbles by application of partial vacuum, the solution was cast slowly into a clean Teflon mold and kept at room temperature overnight. It was then heated in an oven at 80 ᵒC for 12 h and at 100 ᵒC for 2 h. The resulting films were finally subjected to high vacuum at 70 ᵒC to ensure removal of any residual solvent. Some of the prepared membranes were doped by immersion in a CSA solution (2 mol/l) for 48 h, and then cut to the desired shape for further experiments. It is worth mentioning that the final dressing membranes were subjected to solvent extraction/purification procedure by immersion into ethanol (70 % w/w) for 24 h and subsequently into distilled water for another 24 h before further characterization and assay tests.


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Spectroscopic, mechanical and dynamic-mechanical analysis The details are presented in Supplementary Info, S1. Conductivity and electroactivity analysis The electrical conductivity (σ, S.cm-1) of the samples, at both doped and undoped states, were measured according to the procedure described at Supplementary Info, S2. Equilibrium water absorption (EWA) and water vapor transmission rate (WVTR) of membranes The details are presented in Supplementary Info, S3. Gel content and surface hydrophilicity of membranes These characteristics of membranes were evaluated according to the procedure described in Supplementary Info, S4. In vitro cytocompatibility assays The details are presented in Supplementary Info, S5. Evaluation of cell adhesion and proliferation The numbers of attached cells onto the surface of dressing membranes, with and without electroconductive aniline tetramer moieties, was evaluated in order to find an estimation of the possible participation of dressing material on cell growth and proliferation. The details of procedures are described in Supplementary Info, S6. Antibacterial activity of membranes



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Antibacterial activity of the prepared membranes was studied using "colony forming count" method according to the procedure described in Supplementary Info, S7. Antioxidant efficiency of membranes The details are presented in Supplementary Info, S8 In vivo studies The wound healing performance of the wound dressings was evaluated under in vivo condition using rat animal model with full thickness wound. Details of followed procedures are described in Supplementary Info, S9. Statistical analysis Statistical analyses were performed by a PASW statistics program package, version 18 (SPSS Inc., Chicago, IL, USA). Comparison of the obtained data for different samples was performed with One-Way ANOVA with Tukey posthoc test. The significance level was set at p ≤ 0.05.

RESULT AND DISCUTION Synthesis and spectroscopic characterization It is well known that the dressing membrane should protect wounds from external stress and preserve their dimensional stability during the healing period.22 It has been proved that introduction of an inorganic siloxane domain is a versatile approach for enhancing the mechanical properties of polyurethane matrices.23 To impart suitable mechanical strength in the


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dressing membrane, preparation of siloxane cross-linked CO-based polyurethane via sol-gel process was considered. The chemical route followed for the preparation of starting materials is depicted in Scheme 1. Firstly, CO was reacted with an excess amount of IPDI to prepare NCOterminated urethane intermediate compound. The reaction of this compound with three equivalents of APS led to formation of Si-CPU as a reactive moisture sensitive methoxysilaneterminated urethane prepolymer. For tuning the crosslink density and consequently optimizing the tensile strength of the final network, a monofunctional methoxysilane material was prepared from methyl ricinolate via the same procedure followed for functionalization of CO. FTIR spectra of these materials are shown in Figure 1. The broad band centered at 3328 cm-1, was related to the stretching vibration of urethane and urea N-H groups present in the structure of SiCPU. The urethane (NH-CO-O-), urea (-NH-CO-NH-) and ester carbonyl (-CO-O) groups were detected as a distinct peak at 1709, 1634 and 1741 cm-1, respectively. The peak observed at 1566 cm-1 was attributed to C-N stretching, combined with N-H out-of-the-plane bending of urethane and urea groups. The stretching vibration of (O-Si-O) groups appeared at 771 cm-1. As well, the absence of peak at 2230 cm-1 related to NCO group and the presence of urea carbonyl peak at 1634 cm-1 confirmed the complete reaction of isocyanate and amine groups. The same spectral pattern with similar details was also recorded for Si-RM compound (Figure 1b).



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Figure 1: FTIR spectra of (a) Si-CPU and (b) Si-RM.


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Scheme 1: Synthesis route for the preparation of Si-CPU and Si-RM. Having proper tensile property is an essential feature of dressing membranes. To control this factor, different weight ratios of monofunctional Si-RM was added to Si-CPU and the mixtures



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were subjected to sol-gel reaction. Different formulations of the prepared networks are collected in Table 1. The resulting membranes were visually inspected and subjected to DMA study to evaluate their viscoelastic behavior and quantify their crosslink density. The network obtained from neat Si-CPU was hard and brittle with highest crosslink density; however, with increasing the concentration of Si-RM, films with lower crosslink density and better flexibility were obtained. Close inspection of DMA curves offered valuable information (Figure 2). All membranes showed two thermal transitions. The first transition at lower temperature in the range of 15 to 30 ᵒC (from tan δ maximum) was related to glass transition temperature (Tg) of fatty ester structure of CO and RM moieties. The second transition at about 70-98 ᵒC was attributed to the glass transition of hard segment composed of siloxane and urethane/urea linkages. With incorporation of Si-RM into the networks, both Tg peaks were shifted to lower temperature and also the height of tan δ peaks were intensified. The shape and position of tan δ peaks were known to be determined by the factors like cross-linking density and the presence of plasticizer and fillers.24,25 The increasing in height of the tan δ peaks, decreasing in rubbery plateau modulus and shift of Tg peaks to lower temperature with raising Si-RM concentration were indications of lowered cross-linking density for the corresponding networks. Lower methoxysilane functionality of Si-RM and interplasticization effect of bulky and long alkyl chain moiety (dangling chains) of Si-RM are main factors affecting viscoelastic properties of these materials. The contribution of the latter factor on reducing the rigidity, increasing the segmental motion and improving flexibility of polyurethane materials containing dangling moieties is also reported by other scientists.24 Based on viscoelastic properties, the NESiPU4 containing equal weight ratio of Si-CPU and Si-RM with lowest Tg and crosslink density as well as proper handling and


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flexibility was selected as the base formulation for preparation of electroactive analogous of wound dressing membranes.

Figure 2: DMA curves of the NESiPU1-4 membranes. Table 1: Formulation of sample with different amounts of Si-RM x)

NESiPU1

Si-CPU solution (g) 3.33

Si-RM solution (g) -

NESiPU2

3.33

NESiPU3 NESiPU4

Samples

H2O (g)

Gel content (%)

υc (mol/cm3)z

Appearance

0.10

99.5±0.2a

1.3×10-1

Brittle

1.66

0.10

99.6±0.1a

6.7×10-2

Brittle

3.33

2.50

0.10

99.3±0.4a

1.9×10-2

Semi flexible

3.33

3.33

0.10

98.7±0.2a

6.5×10-3

Flexible

x)

According to analysis of variances P-values of

Stimulation of Wound Healing by Electroactive, Antibacterial, and

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