A Novel Bilayer Propionyl-L-carnitine Loaded Polyvinyl Alcohol/Calcium Alginate/Carboxymethyl Cellulose wound dressing for the treatment of diabetic wounds: an in vitro and in vivo study

In the current study a drug delivering bilayer porous/nanofibrous wound dressing was developed using a combination 29 of electrospinning and freeze-drying methods. The wound dressings were prepared by lyophilization of 1:1 weight 30 ratios of calcium alginate and carboxymethyl cellulose (CMC) solutions. Drug delivering nanofibrous sheets were 31 fabricated by electrospinning of polyvinyl alcohol (PVA) solution incorporated with 1%,3%,5%, and 10% of 32 Propionyl-L-carnitine. The dressings were studied regarding their microstructure, swelling capacity, mechanical 33 strength, surface wettability, water vapor permeability, drug release profile, in vitro degradation, cell viability assay, 34 hemocompatibility, porosity measurement, microbial penetration assay, and protein adsorption assay. Based on in 35 vitro studies, PVA sheets loaded with 5% Propionyl-L-carnitine was chosen for the preparation of wound dressings. 36 The healing potential of the produced constructs was studied in rat model of diabetic wound. Our results showed that 37 the drug delivering dressings demonstrated significantly higher wound closure and better histological regeneration 38 compared to drug free constructs and sterile gauze. Our results suggest potential applicability of Propionyl-L-carnitine 39 delivering Calcium Alginate/CMC/PVA dressing for the treatment of diabetic wounds in clinic. 40


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Wounds are defined by integrity loss of skin tissue which can be caused by thermal, physical, or chemical injuries 85 (Tarassoli et al. 2018). Given the fact that skin tissue has an inherent healing potential following injuries; in most 86 cases, this tissue can repair its damages (Yu et al. 2019). However, in case of critical-sized defects or in disordered 87 health conditions such as diabetes mellitus, wounds may turn into chronic non-healing wounds (  biopolymer-based wound dressings has gained significant attention during the past decades (Shah et al. 2019). In this Firstly, sodium alginate of medium-viscosity (61% of mannuronic and 39% of guluronic acid) and CMC of medium 118 viscosity (degree of substitution 0.7) were dissolved separately in glycerol containing distilled water (glycerol: 119 polymer weight ratio was 60%) at final concentration of 1.5% (wt%) and stirred for 24 hours at room temperature.

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Then, Calcium chloride at final concentration 2% (wt%) in distilled water was added to the alginate solution at volume 121 ration of 1:4 to begin the cross linking process and stirred for further 24 h at room temperature. The equal amounts of 122 each solution were blended to obtain a 1;1 alginate/CMC solution and stirred for 24 h. The prepared mixture was 123 transferred into -20 º C and incubated for 24 h. The solidified sample was then kept at -80 ºC for further 24 h. Then, 124 the samples were freeze-dried (Telstar, Terrassa, Spain) for 48 h.  electrospinning began by applying a positive high voltage of 16 to 18 kv. The needle to mandrel distance set at 15 cm 131 and the polymer feeding rate was 1 ml/h. The electrospinning continued until the mandrel was fully covered by the 132 nanofibers. The prepared sheets were cross-linked according to a method described previously (Stone et al. 2013).

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To analyze the microstructure of the constructs, they were imaged under and SEM device (AIS2100, Seron

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Technology, South Korea) at an accelerating voltage of 20 kV, after the sputter coating with gold for 250 s using a 136 sputter coater (SC7620, Quorum Technologies, England).

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Swelling studies on Calcium alginate/CMC films

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The swelling properties of the Calcium alginate/CMC films was studied using a method as described previously

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Swelling ratio: Where m1 is the swollen weight of samples and m0 is the dry weight of the films.

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The mechanical properties of the scaffolds were studied using a uniaxial tensile testing device (Softon Technologies,

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USA) at an extension rate of 1 mm/min,

Contact angle measurement
Hamburg, Germany). A water droplet was placed on different spots of each scaffold and the angle between its surface 150 and the scaffolds was calculated and averaged.

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Water vapor permeability study

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To assess the dressing's capability for gas transfer, this experiment was performed on scaffolds. 10 ml of distilled 153 water was poured into an empty bottle and then capped by the fabricated dressings and then incubated at 33 °C for 12 154 hours, the evaporated water through the scaffolds was calculated using the following equation.

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Where w is mass of evaporated water, A is surface area, and T is the time of incubation.

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In vitro degradation profile of the samples

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The degradation rate of the scaffolds was studied by immersing predetermined amounts of each scaffold in 10 ml of 159 PBS solution at 37 ºc under gentle shaking over a period of 10 days. After each time point, the samples were taken 160 out, dried, and weighed. Weight loss was measured according to the following equation.    To evaluate the release profile of Propionyl-L-carnitine from PVA films, high-performance liquid chromatography 173 method was exploited as described previously (Marzo et al. 1988). Briefly, the drug loaded PVA fibers were immersed In each group, 10 ml bottles filled with 5 ml of BHI broth culture medium was covered by the prepared constructs and 181 incubated at room temperature. The invasion of the bacteria into the growth medium was studied at 3 and 7 days' time 182 points. Vials capped with the sterile gauze and open vials were used as negative and positive controls, respectively.

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The blurred growth mediums as the indication of bacterial growth was analyzed using a spectroscopy method at 600 184 nm using a microplate spectrophotometer.

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To evaluate the ability of the constructs to adsorb protein they were studied via batch contact method as described   Where c1 is the initial protein concentration of BSA, C0 is the protein concentration after scaffolds soaking and 194 removal, w is the weight of swollen scaffold, and V is the volume of BSA.

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8ml of whole blood was obtained from a healthy volunteer and mixed with 1 ml of 3.8% Sodium citrate anticoagulant.

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This sample was diluted with 2.5 ml normal saline solution. 200 μl of this blood was poured onto the samples and   dressing was fixed in place by using an elastic adhesive bandage. The wound dressings were changed on daily basis.

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To evaluate the wound size reduction, the macroscopic appearance of the wounds was imaged at day 7 and 14 using with a digital camera (Olympus, Tokyo, Japan). Epithelial tissue thickness, recruitment of macrophages, fibroplasia, and new blood vessel formation were studied. Furthermore, collagen deposition was also measured according to 242 histopathological images.

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The microstructure of the scaffolds was observed using scanning electron microscopy imaging. The results (Fig. 2 a) 247 showed the Calcium alginate/CMC films had porous structure with interconnectivity of the pores. The pore size  The ability of the bioactive dressing to sustain release the loaded drug in the wound bed is of paramount importance.

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As shown in Fig. 4, the cumulative release of Propionyl-L-carnitine from PVA films could reach to 80.06 ± 4.38 %

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after 24 h of incubation. Therefore, the designed delivery system will be able to deliver almost all the loaded drug to

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Positive control had significantly higher absorbance values compared to other groups.

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Cell viability assay

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To study the metabolic activity and proliferation rate of L929 cells on different scaffolds, MTT assay was conducted 289 at days 1, 3, and 7. As shown in Fig. 6 a, there was no significant deference between Calcium alginate/CMC films and 290 control group at different time intervals, implying that the prepared construct was not toxic against L929 cells. In 291 drug-loaded PVA groups (Fig. 6 b), at day 1 PVA fibers incorporated with 5% Propionyl-L-carnitine had significantly

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higher absorbance values compared to other groups. This trend was almost unchanged towards the day 7. According

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to this assay, we assumed that 5% of Propionyl-L-carnitine was optimal concentration which had a beneficial effect 294 on fibroblasts' metabolic activity. Therefore, this concentration was chosen for treating skin wounds.

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An ideal wound care product should prevent microbial invasion into the wound bed (Samadian et al. 2020). Our results

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In vivo wound healing

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The healing potential of the bilayer wound dressing was investigated in a rat model od diabetic wound. Fig. 8   that the epithelial thickness (Fig. 8 b) in drug delivering wound dressing was significantly higher compared to other 342 groups. The epithelial thickness for Calcium alginate/CMC/PVA 5% Propionyl-L-carnitine group was 48.13 ± 5.49 343 µm. While, Calcium alginate/CMC/PVA and negative control groups exhibited the epithelial thickness of 31.52 ± 344 6.87 µm and 6.19 ± 1.61 µm respectively. Wound tissue in the negative control group had immature granulation tissue 345 which was evidenced by ineffective wound closure. The wound contraction heavily depends on deposition of collagen 346 molecules. Study revealed that the percentage of collagen deposition (Fig. 8 c) in Calcium alginate/CMC/PVA 5%

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The main goal of skin tissue engineering is to develop products which results in prompt aesthetic and functional

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Alginate is a natural biomaterial that is harvested from brown seaweed. Its low cost, biocompatibility and non-toxicity

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Lyophilization results in a porous structure which facilitates gas exchange and exudate absorption (Gonzaga et al.

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2020). While, the electrospinning method produce scaffolds with structural resemblance to native extracellular matrix.

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In addition, the fabricated fibers have a high surface to volume ratio which make them ideal carriers for a variety of

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Therefore, we assume that part of the observed healing effect could be due to inhibiting endothelial dysfunction and    Scanning electron microscopy images of (a) Calcium alginate/CMC lms, (b) PVA bers lloaded with 5% Propionyl-L-carnitine The swelling percentages of Calcium alginate/CMC lms over a period of 72 h Figure 4 Cumulative release pro le of Propionyl-L-carnitine from PVA nano bers Microbial barrier property of the bilayer wound dressing after 3 and 7 days of incubation measured by Spectrophotometer at 600 nm