Single-Dose Electrospun Nanoparticles-in-Nanofibers Wound

May 24, 2016 - Single-Dose Electrospun Nanoparticles-in-Nanofibers Wound Dressings with Enhanced Epithelialization, Collagen Deposition, and Granulati...
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New single-dose electrospun nanoparticles-in-nanofibers wound dressings with enhanced epithelialization, collagen deposition and granulation properties Isra H. Ali, Islam A Khalil, and Ibrahim M. El-Sherbiny ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04369 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 28, 2016

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New single-dose electrospun nanoparticles-in-nanofibers wound dressings with enhanced epithelialization, collagen deposition and granulation properties Isra H. Ali 1§, Islam A. Khalil 1, 2 §, Ibrahim M. El-Sherbiny 1* 1

Nanomaterials Lab., Center of Material Science (CMS), Zewail City of Science and Technology, 6th of October, Giza, Egypt, 2 Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy and Drug Manufacturing, Misr University of Science and Technology (MUST), 6th of October, Giza, Egypt.

§ Both authors contributed equally

*Correspondence to: I. M. El-Sherbiny, PhD Zewail City for Science and Technology. (Email: [email protected]) Tel.:+20238540407, Fax: +20238517181 Nanomaterials Lab, Center for Materials Science, Zewail City of Science and Technology, 6th October City, Giza, Egypt

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ABSTRACT: Phenytoin (Ph), an antiepileptic drug, was reported to exhibit high wound healing activity. However, its limited solubility, bioavailability and inefficient distribution during topical administration limit its use. Therefore, this study aims to develop a new single-dose electrospun nanoparticles-in-nanofibers (NPs-in-NFs) wound dressings that allow a well-controlled release of Ph. These NPs-in-NFs systems are based on enhanced chitosan (CS)/ polyethylene oxide (PEO) electrospun nanofibers (NFs) incorporating optimized Ph-loaded nanocarriers. Firstly, a study was conducted to investigate Ph loading efficiency into polymeric nanocarriers with different natures; pluronic nanomicelles and poly(lactic-co-glycolic) acids nanoparticles (PLGA NPs). The drug release profile from the nanocarriers was further optimized via lecithin coating. Secondly, different electrospinning parameters were manipulated to fabricate beads-free homogeneous NFs with optimized polymer ratios. Plain and Ph-loaded nanocarriers were characterized using FTIR, DSC, TGA, DLS and SEM. Both entrapment efficiency of Ph (EE%) and its release profile in PBS (pH 5.5), simulating wound environment were studied. Biodegradability, swelling, vapor permeability and porosity of the developed Ph-loaded NPs-inNFs wound dressings were investigated. Morphology of the NPs-in-NFs was also studied using SEM and CLSM. Besides, the release profiles of Ph from the optimized NPs-in-NFs were assessed. The newly developed wound dressings were evaluated in-vitro for their cytotoxicity using human fibroblasts and in-vivo using a wound healing mice model. Nanocarriers with particle size ranging from 100 to 180 nm were successfully prepared. All nanocarriers attained a high drug entrapment efficiency exceeding 94%, and showed promising sustained release profiles compared to free Ph. Results also demonstrated that NFs incorporating the optimized lecithin-coated Ph-loaded PLGA NPs could be the most promising candidate for efficient wound healing. These NPs-in-NFs systems conferred a well-controlled and sustained release of Ph over 9 days. Moreover, they showed the best re-epithelization and healing quality during the in vivo study with minimal inflammatory and necrotic cells formation.

Keywords: Single-dose; Electrospun; Nanofibers; Chitosan; Phenytoin; Wound healing.

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1. INTRODUCTION Skin, the soft organ that covers and protects the interior parts of the human body, represents around 8% of the total body weight. Thousands of patients need to graft or repair their skin to compensate skin loss due to injuries, burns, etc. An appropriate recent approach is to develop tissue engineered materials to be used as wound dressings for wound healing and skin regeneration purposes 1. Skin injuries and wounds should heal normally through the natural phenomenon of normal skin cells restoration. However, major clinical challenges act as an obstacle for well time-controlled clean wound healing. These challenges involve microbial infections, limited quality of wound healing and high probability of fluid loss that consequently decreases wound moisture and increases healing time 2. Successful wound dressings should possess some characteristic features such as having a high rate of healing and antimicrobial activity, avoiding scars formation and wound contamination, and providing a convenient wound remedy 3. Additionally, wound dressings should show high porosity and high water vapor permeability to help in rapid fibroblasts spreading, proliferation, and healing through increasing the oxygen and nutrients diffusion

4–6

. Fabrication of wound

dressings in a nanofibrous form rather than solid film can verify the previously mentioned features due to the ability of manipulation of the produced electrospun nanofibers (NFs) to possess higher aspect ratio, higher surface area and consequently higher porosity during the fabrication process 7,8. Several techniques have been reported to produce NFs such as pressurized gyration technique, self-assembly, phase separation and freeze drying. However, all of these techniques are not ideal for wound dressing’s fabrication as the produced nanofibers show large diameters, and consequently low porosity. This would fail in providing adequate diffusion of oxygen and nutrients into cells resulting in failure of cells migration, proliferation and compensation. On a contrary situation, electrospinning technique was reported to overcome all of the previously mentioned drawbacks through producing well-controlled ultrafine nanofibers mimicking the extracellular matrices in human body. In addition, electrospinning is a cost-effective, ecofriendly and facile technique 7–14.

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A broad range of biopolymers either naturally derived such as alginate, chitosan, collagen, dextran and gelatin or synthetic such as polycarpolactone, poly (lactic acid), poly (lactic-coglycolic) acid (PLGA), polyvinyl alcohol (PVA) and polyethylene oxide (PEO) have been electrospun successfully for wound healing applications

2,15–21

. This is attributed to their

biocompatibility, biodegradability, high hydrophilicity, low toxicity, low immunogenicity and low cost 22–25. Chitosan (CS), the most essential derivative of chitin, is the second abundant polysaccharide that is obtained through chitin deacetylation. CS acts as a non-protein matrix that can be fabricated as a 3D scaffold. It is a promising polymer to develop wound dressing matrices due to possessing several unique features including: (a) being a biocompatible, biodegradable and antimicrobial agent, (b) stimulating histoarcehtictureal tissue organization, (c) provoking cell proliferation and (d) conferring hemostating effect that aids in blood clotting as well as nerve ending blockage leading to pain reduction, (e) being of low cytotoxicity and immunogenic without any carcinogenic activity 26–28. However, CS showed poor electrospinnability due to its high positive charges, therefore it has to be blended with other polymers to facilitate its electrospinning in order to generate convenient polymeric drug delivery systems 29. Polymeric drug delivery systems (PDDS) showed superior advantages when compared with other traditional drug carrier systems. These include: a) enhancing drug efficiency, b) minimizing drug side effects and toxicity, and c) the easy use of drug loaded PDDS 30,31. Phenytoin (Ph) is an oral antiepileptic drug that belongs to the barbiturates class. Ph was first reported for its wound healing activity in periodontal injuries in 1958 with almost no postsurgical inflammation

29,32

. It was proven that systemic absorption of topically applied Ph in

wound healing is insignificant. Therefore, it can be applied topically at the targeted wound sites safely without being subjected to the classical metabolic pathways, thus minimizing the dose related side effects that could accompany the oral therapy for wound healing. Moreover, due to metabolic pathways, the amount of orally administered Ph reaching the wound sites could be lower than the required therapeutic level 33. Some reported topical Ph formulations enhanced the wound healing process through: a) accelerating the tissue granulation, b) reducing wound exudates, transudates, inflammation and oedema, c) decreasing the antibiotic requirement due to decreasing the bacterial load within 4 ACS Paragon Plus Environment

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wounds, and d) facilitating the nerve regeneration

34–37

. In a previous in vivo study, it was

reported that Ph base powder demonstrated more enhanced wound healing activity when compared to an equivalent amount of its sodium salt form

33,38

. However, an optimal

administered dose of Ph base powder and its topical distribution by the patient may not be adequate or sufficient for a successful wound treatment

33

. Therefore, fabrication of a well-

designed single-dose wound dressing loaded with optimum concentration of Ph base is considered to be an ideal solution. In the present study, we report the development of an optimized single-dose controlled delivery system of the most potent form of Ph (Ph base) for efficient wound healing. The delivery system is based on CS/ PEO electrospun NFs mats incorporating optimized Ph-loaded nanocarriers. A preliminary study was firstly carried out to investigate the loading efficiency of Ph into two polymeric nanocarriers of different natures; pluronic nanomicelles and PLGA nanoparticles (NPs). This nano-incorporation of the hydrophobic drug, Ph, was carried out to allow its sustained release as well as to enhance its distribution into the hydrophilic electrospinning CS/PEO aqueous solution. Optimization of the drug release profile form the developed nanocarriers was also achieved via a lecithin coating process. In addition, all the different electrospinning parameters were investigated to produce optimized homogeneous NFs mats. The newly developed NPs-in-NFs wound dressing mats were characterized and evaluated in-vitro for their cytotoxicity using human fibroblasts and in-vivo using a wound healing mice animal model.

2. Experimental 2.1 Materials Chitosan (low molecular weight), pluronic F–127 “poly(ethylene glycol)–block–poly(propylene glycol)–block–poly(ethylene glycol)”, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), disodium hydrogen phosphate and dipotassium hydrogen phosphate were purchased from Sigma-Aldrich in China and Germany. Poly (lactic–co–glycolic acid) copolymer (PLGA) was obtained from Purac, Holland. Polyethylene oxide (MW 900 kDa) was purchased from Acros, USA. Phenytoin base (Ph) was obtained as a gift from El-Nasr Pharmaceutical Co. Lecithin was purchased from Thermofischer, Germany. Acetone (99.5%) and absolute ethanol were obtained from Piochem, Egypt. All other reagents were of analytical grade and used as received. 5 ACS Paragon Plus Environment

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2.2 Polymeric Nanocarriers 2.2.1 Preparation of Polymeric Nanocarriers Phenytoin (Ph) was loaded into two polymeric nanocarrier systems; pluronic nanomicelles and PLGA nanoparticles (NPs) through nanoprecipitation technique (Table 1) as reported previously 39

. The loaded optimized formulas consisted of a polymer: drug ratio of 2:1. The incorporation

into nanocarrier systems was done to facilitate the homogenous distribution of the extremely hydrophobic drug, Ph within the aqueous electrospinning solution and to sustain its release from the electrospun nanofibers mats. The organic phase, in which the polymer and the drug were dissolved, was either pure acetone or 95% acetone in distilled water, while the aqueous phase was either 0.05% PVA aqueous solution or pure distilled water during the preparation of PLGA NPs and pluronic micelles, respectively. The volume ratio of the organic phase to aqueous phase was kept at 1:2. PLGA NPs were further optimized to enhance the releasing profile of Ph from them. This was achieved via coating of the Ph-loaded PLGA NPs with lecithin using the previously reported thin film hydration method with a PLGA: lecithin ratio of 1:1

40

. Plain

PLGA, lecithin-coated PLGA and pluronic nanocarriers were prepared using the same abovementioned procedure and used as controls. 2.2.2 Characterization of Nanocarriers Particle size, polydispersity index and zeta potential measurements of unloaded and Ph-loaded nanocarriers were done using zetasizer (Nano-ZS, Malvern, UK). Diluted samples were run at room temperature in triplicates. Morphological investigations were performed using scanning electron microscope (SEM) under low vacuum (Nona Nano SEM, FEI, USA). Few drops of each sample were added on a foil sheet then left to be completely dried before investigation. Chemical characterization was performed for Ph, unloaded and Ph-loaded nanocarriers using Fourier Transform Infrared (FTIR) spectroscopy (Thermoscientific, USA) in the range of 600 – 4000 cm1

. Thermal properties of Ph and the different unloaded and Ph-loaded nanocarriers were studied

using differential scanning calorimetry, DSC (Q20, TA instrument, USA). Five mg of each sample was heated up to 350 о C at the rate of 10 о C/min in the presence of nitrogen as an inert carrier gas flowing at the rate of 25 ml/min. The thermal behavior of samples was confirmed using thermogravimetric analysis TGA (Q500, Thermoscientific, USA). The change in weight of 5 mg sample was determined as a function of temperature. The samples were heated up to 500 о C in air at ramp rate of 10 о C/min. 6 ACS Paragon Plus Environment

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2.2.3 Entrapment Efficiency A known amount of Ph-loaded nanocarriers was re-suspended in 3 ml of absolute ethanol and left in a shaking incubator for 3 days to extract Ph from nanocarriers. The samples were then filtered using a syringe filter, and Ph concentration in ethanol was estimated by measuring the absorbance at 258 nm using UV-Visible spectrophotometer (Evolution UV 600, Thermo Scientific, USA). The Ph entrapment efficiency was then calculated using equation (1) 41. % Entrapment Effeciency = 

 

  

! × 100

(1)

2.2.4 In Vitro Drug Release Study from Nanocarriers 2.2.4.1 Estimation of Cumulative Release Percent In vitro drug release tests were performed using the dialysis bag method with phosphate buffer saline (PBS) pH 5.5 /absolute alcohol (1:1) as the receptor medium. PBS pH 5.5/absolute ethanol mixture was used to mimic the wound real environment and maintain sink conditions during the experiments. Briefly, 1 ml of each Ph-loaded nanocarrier suspensions (P2, P4, and P5 containing 2 mg/ml Ph base) was added into the dialysis bag (Spectra Por7,10 Kd) which was then placed into 45 ml falcon tubes containing 10 ml of the receptor medium. Ph release from the nanocarriers was tested during 48 h as a preliminary study before being incorporated into the nanofibrous matrices. Typically, a 1 ml aliquot of the medium was withdrawn at specific intervals (0.25, 0.5, 1, 2, 4, 6, 24 and 48 h) and replaced by fresh medium. The Ph concentration in the withdrawn samples was determined using UV spectrophotometry at 258 nm. In order to compare the drug release behavior from the nanocarriers with the non-encapsulated drug, a 1 ml of Ph solution with a concentration of 2 mg/ml was prepared. The concentration of Ph at different time intervals was monitored then calculated using equation (2). Each Ph-loaded nanocarrier was tested in triplicates, and the cumulative release percentage was plotted against time. %& = %& '()&* + A/V ∑012 342 %* '()&*

(2)

where Cn is the expected nth sample concentration, Cn means is the measured concentration, A is the volume of withdrawn aliquot, V is the volume of the dissolution medium, n-1 is the total volume of all the previously withdrawn samples before the currently measured sample, and Cs is the total concentration of all previously measured samples before the currently measured sample.

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2.2.4.2 Release Data Analysis (Model Fitting Kinetics) Various release kinetics models such as zero order, first order, Higuchi, Korsmeyer–Peppas, Hixson-Crowell, Hopfenberg and Baker-Lonsdale were fitted using equations (3-9), respectively. The coefficient of determination (r2) was considered for assessing the appropriateness of a model that was able to describe Ph release behavior from the different nanocarriers. GraphPad Prism Software Version 6 was used to analyze the obtained results. Zero order: Qt = Q0 + K0 t

(3)

First order: log Qt = log Q0 – K1 t / 2.303

(4)

Higuchi: Qt = KH t 0.5

(5)

Korsmeyer–Peppas: log Qt / Q∞ = n log t + log k

(6)

Hixson-Crowell: Q0 1/3 - Qt 1/3 = log t + log κ

(7)

Hopfenberg: Qt =100 [1- (1- kHB t) n] ; kHB=k0 / (C0 A0)

(8)

Baker-Lonsdale: (3/2) [1 - (1 - (Qt /100)) 2/3] - [Qt /100] = kBL t

; kBL= [3D Cs / (2r0×C0)]

(9)

where, Qt is the fraction of drug released at time t, Q0 is initial fraction of drug in solution, K0 is zero order release constant, K1 is first order release constant, KH is Higuchi dissolution constant. In Korsmeyer–Peppas, Qt/Q∞ is the fraction of drug released at time t, Q∞ is the total drug released, k is a kinetic constant, and n is the exponent explaining the drug release mechanism. For instance, (n ≤ 0.5) corresponds Fickian diffusion, (n= 1) represents case-II transport, (0.5 < n 1) is considered super case-II transport. In Hixson-Crowell, κ (kappa) is a constant describing the surface-volume relation. In Hopfenberg, kHB is the combined constant and n is 1, 2, and 3 for a slab, cylinder, and sphere, respectively. In BakerLonsdale, kBL is the combined constant, D is the diffusion coefficient, Cs is the saturation solubility, r0 is the initial radius for a sphere or cylinder or the half-thickness for a slab, and C0 is the initial drug loading in the matrix 42. To assess the difference between dissolution profiles, the model independent approach was applied. The nanocarriers were compared to free Ph. The difference (f1) and similarity factors (f2) were determined using Equations (10) and (11), respectively. Generally, the difference factor less than 15 and similarity factor greater than 50 refer to dissimilarity of the two dissolution profiles, 52 = 6∑0942|89 − ;9 | ÷ ∑0942 89 = × 100

(10)

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2

5> = 50 @AB CD1 + 0 ∑0942E89 − ;9 F> G

1H.J

× 100K

(11)

where, n is the number of time points in dissolution profile, Rt and Tt are the cumulative Ph release at time t for plain Ph and tested Ph-loaded NPs, respectively. 2.3 Polymeric Nanofibrous Mats 2.3.1 Preparation and Fabrication of Unloaded and Ph-loaded Wound Dressings 2.3.1.1 Preparation of Electrospinning CS/PEO Blend Solution Chitosan (CS) solution (3% w/v) and polyethylene oxide (PEO) solution (7.5% w/v) were prepared separately by dissolving the required amount of polymer in 1% aqueous acetic acid, and distilled water, respectively at room temperature for 6 h to ensure complete dissolution. Then, CS/PEO polymer blend solution was prepared through mixing the two polymer solutions at the ratio of 1:1 for 1 h to ensure full homogeneity. The final solution was left without stirring overnight to get rid of any existing air bubbles. The CS/PEO polymer mixture was used to fabricate the drug-free CS/PEO electrospun NFs (F1) (negative control). To develop the Phloaded CS/PEO NFs, a predetermined amount of each type of Ph-loaded nanocarriers was added to the previously prepared CS/PEO polymer solution. Upon optimization of electrospinning parameters, it was found that a Ph weight of 10% relative to the total weight of polymers in the solutions corresponds to the maximum value of Ph-loaded nanocarriers that could be incorporated within the electrospinning solution and capable of generating uniform electrospun NFs. The calculated amounts of each of the three types of the Ph-loaded nanocarriers (P2, P4 and P5) were added each separately to CS/PEO blend solution to obtain the corresponding CS/PEO electrospinning blend solution for F2, F3 and F4 respectively. After addition of the Ph-loaded nanocarriers, the solutions were stirred for 20 min to obtain homogenous well dispersed nanoparticles in CS/PEO blend solution. 2.3.1.2 Preparation of Electrospun CS/PEO NFs: Optimization of electrospinning parameters The fabricated wound dressing nanofibrous mats were obtained using a NANON–O1A electrospinner (MECC, Japan). Each polymer solution was fed separate in a 5 ml plastic syringe (13.1 mm diameter) connected with 18 gauge stainless steel needle through a 1.5 mm wide plastic nozzle. Optimization of electrospinning parameters was achieved through adjustment of the flow rate of the electrospinning solution, the amount of the applied voltage between the spinneret tip and the receiving collector, the distance between the spinneret tip and the receiving 9 ACS Paragon Plus Environment

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collector and the speed of the moving spinneret. Electrospinning parameters were adjusted to obtain the most reliable uniform non-beaded NFs. The optimum electrospinning parameters were found to be: 0.8-1 ml/h flow rate, 13-17 kv applied voltage, and 13 cm distance between the spinneret tip and the ground stationary collector. Upon adjusting the different aforementioned parameters, the applied voltage was able to overcome the surface tension of the electrospinning solution leading to the formation of a Taylor cone at the tip of the spinneret. This ended up with ejection of ultrafine electrospun nanofibers from the moving spinneret to be received on the aluminum foil sheets covering the stationary collector. The moving spinneret was adjusted to move with a speed of 100 mm/sec forming a wound dressing film of 100 mm width and 0.5 mm thickness. The recorded humidity and temperature inside the electrospinner during the fabrication process were 30-35% and 32 оC, respectively. The NFs were dried in a Labconco freeze dryer for 48 h to ensure getting rid of any residual solvents. 2.3.2 Physicochemical Characterization of Electrospun Nanofibrous Mats Biodegradability was studied by placing samples of NFs in PBS pH 5.5 in a shaking incubator for 7 days. The weight remaining percentage was assessed daily using Equation (12), where Wi is the initial weight and Wf is the final weight measured each day 43. PQ

Reminaing Weight % = P! × 100

(12)

Swelling profiles of the developed nanofibrous mats were also determined. NFs were weighed (Wd), placed in 5.5 pH PBS, and left in a shaking incubator at room temperature. At specific time intervals, the samples were removed outside the buffer, plotted with a dry filter paper to remove any excess PBS and weighed again (Ws) till no change in weight was observed. The swelling percentage was calculated according to equation (13) 43. Swelling % = 

PU1P P

! × 100

(13)

Water vapor permeability of the different NFs was evaluated using ASTM E96 desiccant method as reported previously 44,45. A setup of an Erlenmeyer flask containing a known amount of silica gel powder as a desiccant and sealed with a membrane of the nanofiber was placed in a 75% humidity environment for 7 days. Water vapor permeation (WVP) across the NFs membranes was then calculated using Equation (14): VWX = 

∆ Z [

! \ ∆]

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(14)

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where ∆ W is the change in weight due to water vapor permeation, A is the surface area and ∆t is the change in time. Porosity per volume of the developed NFs was estimated using pycnometer (Ultrapyc 1200 e, Quantachrome instruments, USA). A piece of nanofibrous sample with known dimensions was weighed then placed in the pycnometer container 46. Then, the porosity per volume and porosity percentage of each nanofiber were calculated using Equations (15) and (16), respectively: XA^A*_]` a(^ WA@b'( = 1 −

cd

ef9gh cfhijk Elk0m9n o Zpq9n o enprs0k33

XA^A*_]` % = XA^A*_]` a(^ WA@b'( \ 100

(15) (16)

where, VF is the volume of the nanofiber estimated by the pycnometer using helium gas. 2.3.3 Morphological Characterization of Electrospun Nanofibrous Mats Morphological investigation of the different produced NFs was performed using a scanning electron microscope (Nova Nano SEM, FEI, USA). A nanofiber sample was examined on a SEM grid under low vacuum. SEM images of each sample were analyzed using Image J software to estimate the NFs diameter and polydispersity index. In addition, confocal laser microscopy, CLSM (Nikon, USA) was used to investigate the distribution of loaded NPs within the NFs. Fluorescein dye-loaded NPs were prepared and incorporated into NFs with the same previously mentioned amounts and procedures. 2.3.4 In vitro Ph Release Profile The release behavior of Ph from different nanofibrous mats was studied using the same abovementioned procedure in section 2.2.4. Cumulative release percentages of Ph were measured and different kinetic models were determined. The release from each of the NFs was performed in triplicates. 2.4 Cell Viability Study Cell viability experiments of the different electrospun nanofibrous mats were performed according to the protocol of ISO 10993-5 using the MTT (3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazolium bromide) assay as previously reported 47. Using a human dermal fibroblasts cell line (ATCC, CRL-2522) in a 96-well plate, an amount of 200 µl of a cell suspension of 1 × 105 count were plated along with each type of the NFs individually per well. After 24 h incubation, the medium was withdrawn carefully out of the well-plate, then the wells were 11 ACS Paragon Plus Environment

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washed with MEM (w/o) FCS for 2-3 times. Afterwards, an amount of 200 µl of the MTT (5 mg/ml) agent was added in the wells and incubated for 6-7 h inside a 5% CO2 incubator. After incubation, 1 ml of DMSO was added to each well and mixed well using a micropipette. The plate was left for 45 seconds before visualizing the viable cells remarked by the purple color development of formazan crystals. Optical density of the viable cells was assessed using a spectrophotometer by detecting the absorbance of the cell suspension at λ of 595 nm using DMSO as a blank 32. The cell viability percentage was expressed by Equation (17): %(@@ W_)t_@_]` % =  2.5

ukg0 vw9prgh xk03p9y

zf09{fh vw9prgh xk03p9y

! × 100

(17)

In vivo Assessment

2.5.1 Mice Wound Healing Model Animal experiments were performed according to institutional ethical guidelines. Approval of wound healing animal model study was obtained from the Animal Research Committee of Misr University for Science and Technology. The in-vivo wound healing experiments were performed using bulb-c male mice weighing 20-25 grams to evaluate the wound healing ability of the different produced nanofibrous mats on regeneration of the injured skin. Mice were anaesthetized using ketamine and xylazine, and then their back hair was shaved using a depilatory cream. A 9 mm diameter biopsy puncher was used to perform a full thickness wound along the dorsal side of each mice skin. Two wounds were created on each mice; one to be treated by one of the formulations while the other one as a control wound (untreated). All of the induced injuries were of the same depth, where all of the skin tissues were removed to reach the muscles. Six groups of mice (n=15) were tested for the change in the induced wound areas, where three mice were assigned for one of the 5 tested time points. Four groups were assigned for applying the four produced NFs at the wound site. The fifth group represented a negative control (no treatment), while the sixth group represented the positive control (Ph ointment). It was adjusted in such a way that the concentration of drug (Ph) in the applied ointment and NFs is the same. Change in wound healing area was monitored over 2 weeks, where the wound area was measured using a caliper at 3, 5, 7 10 and 14 days. Finally, the wound healing contraction was noticed through calculating the wound healing area percentage using Equation (18): VAb&| }^() % = 

Z9 Zp

! × 100

(18)

where, Wt is the wound area at certain time point and Wi is the initial wound healing area 29. 12 ACS Paragon Plus Environment

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2.5.2 Histopathology Assessment At each time point, three mice were euthanized in each group, and then the surrounding skin and muscle including wound areas were carefully dissected, fixed using 10% neutral buffered formalin, and finally embedded in paraffin to be preserved. Hematoxylin and eosin (H&E) and Masson’s Trichrome staining were performed to histologically investigate the skin tissue sections. Histopathological lesions scores were used to evaluate presence of necrosis, inflammatory cells, hemorrhage, granulation tissue (GT) extent, re-epithelization and thick epidermis formation in the H&E images. Moreover, angioblasts, fibroblasts and collagen fiber deposition were evaluated from Masson’s Trichrome staining images 24. 2.6 Statistical analysis Results are expressed in mean ± standard deviation. Significant differences were examined applying Student's t-test and one-way analysis of variance (ANOVA) to all data obtained for all types of NPs and NFs mats. All tests were calculated using the software GraphPad Prism Software Version 6.

3. Results and Discussion The fundamental aim of the present study is to prepare a single-dose nanofibrous patch loaded with sufficient amount of Ph as a wound healing agent along with controlling its release from the NFs. This was achieved through two stages as illustrated in Scheme 1. The first stage involves the optimization of the release profile of Ph via its incorporation into polymeric nanocarriers of different natures (pluronic nanomicelles and PLGA NPs). Then, lecithin coating of the developed Ph-loaded PLGA NPs was performed to further enhance the Ph release. The target of the first stage was also to enhance Ph distribution within the hydrophilic polymeric electrospinning solution. The second stage involved the fabrication and optimization of CS-PEO NFs matrices reinforced with each of the previously optimized Ph-loaded nanocarriers. 3.1 Preparation and Characterization of Polymeric Nanocarriers All the Ph-loaded polymeric nanocarriers of different natures; pluronic nanomicelles (P2), PLGA NPs (P4) and the optimized lecithin-coated PLGA NPs (P5) were prepared successfully using the nanoprecipitation technique

39

. Ph-free pluronic nanomicelles (P1) and PLGA NPs (P3) were

prepared using the same technique and used as controls (Table 1).

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Table 1. Composition, particle size, polydispersity index (PDI), zeta potential, entrapment efficiency (EE) and Ph release kinetics from the different prepared polymeric nanocarriers. f1

f2

Model

-

Drug Dissolved % 6h 48 h 95.8 ± 4.8 -

-

-

-17.3 ± 1.4

95.2 ± 1.5

26.1 ± 4.2*

53.9 ± 3.7*

73.7

18.1

0.22

-16.9 ± 1.0

-

-

-

-

-

105 ± 30

0.08

-16.4 ± 1.9

98.4 ± 1.3

26.0 ± 4.9*

33.4 ± 3.9*

72.9

18.3

KorsmeyerPeppas KorsmeyerPeppas

133 ± 06

0.18

-16.8 ± 1.6

94.4 ± 1.8

11.8 ± 1.2*

33.5 ± 3.5*

94.9

12.9

Free Ph Control pluronic nanomicelles

Size (nm) 161 ± 31

0.92

Zeta Potential -15.7 ± 1.0

P2

Ph-loaded pluronic nanomicelles

139 ± 22

0.85

P3

Control PLGA NPs

114 ± 32

P4

Ph-loaded PLGA NPs

P5

Ph-loaded lecithin coated PLGA NPs

Code

Formula#

Ph P1

#

PDI

EE%

Polymer: phenytoin (Ph) ratio is 2:1 in P2, P4 and P5; Polymer: lecithin ratio is 1:1 in P5, * Significant difference to plain phenytoin release profile

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Scheme 1. A schematic representation of the preparation and optimization stages of the Phloaded polymeric nanocarriers, and the fabrication of the newly developed medicated NFs wound dressing mats.

3.1.1 Nanocarriers Yield, Particle Size and Zeta Potential The yield (percentage recovery) of the prepared plain and Ph-loaded nanocarrier systems exceeded 95%. The yield was calculated through comparing the amount of obtained NPs with the initially used polymer weight. The loss was attributed to the repeated centrifugation and retrieval from lyophilized tubes. Analysis of the obtained particle size results, illustrated in Table 1 and Figure 1a, demonstrated a non-significant difference between the plain nanocarriers and their Ph-loaded counterparts. The size of all of the prepared nanocarriers ranged from 105 – 160 nm. As shown in Figure 1b, it can be inferred that zeta potential did not show any significant

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difference related to neither the type of used polymeric nanocarrier nor the presence of Ph within the nanocarrier matrices. The recorded range of zeta potential of the different plain and Phloaded polymeric nanocarriers was -15.7 to -17.3 mV. Zeta potential of the prepared nanocarriers depends mainly on the end groups of either PLGA or pluronic. Being highly charged nanocarriers have the advantage of forming highly stable colloidal nanosuspensions since the surrounding charge could induce a Columbic force between the nanocarriers that could overcome the Van Der Waals attraction among them 48.

Figure 1. Zetasizer results for the different prepared polymeric nanocarrier systems: a) Size and PDI, and b) Zeta surface charge.

3.1.2 Morphology of Ph-Loaded Polymeric Nanocarriers Scanning electron micrographs revealed the successful formation of spherical Ph-loaded polymeric nanocarriers as shown in Figure 2. The obtained particle sizes for P2, P4 and P5 were found to be in agreement with the results obtained using DLS measurements (Figure 1a).

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Figure 2. SEM images of a) Ph-loaded pluronic nanomicelles, b) Ph-loaded PLGA NPs and c) lecithin coated Ph-loaded PLGA NPs. 3.1.3 Fourier Transform Infrared Spectroscopy (FTIR) The IR spectra of free drug (Ph), plain (P1) and Ph-loaded pluronic nanomicelles (P2) scanned in the range of 600 – 4000 cm-1 are shown in Figure 3a. As apparent from the figure, the spectrum of Ph shows two characteristic peaks around 3195 cm-1 and 1770 cm-1 that could be attributed to the N-H

stretching and C=O stretching, respectively. In addition, the spectra shows a

characteristic absorption peak at around 1400 cm-1 which is assigned to the C-N stretching 49. On the other hand, the spectrum of plain pluronic nanomicelles (P1) demonstrated two characteristic peaks at 2858 cm-1 and 1100 cm-1 attributed to CH2 stretching and the ether linkage, respectively 50,51

. The successful assembly of Ph-loaded pluronic nanomicelles (P3) was confirmed through

detecting the characteristic peaks of both free drug, Ph and the plain pluronic in its spectra. FTIR spectra of plain PLGA NPs (P3), Ph-loaded PLGA NPs (P4), lecithin and lecithin-coated Ph-loaded PLGA NPs (P5) were also examined (Figure 3b). PLGA NPs (P3) spectrum showed a broad characteristic absorption peak at around 3300 cm-1 corresponding to the stretching vibration of OH of the terminal carboxylic group of PLGA. The spectra also showed several weak absorption peaks at around 2940 cm-1 corresponding to the stretching vibrations of CH, and CH2 abundant within PLGA structure. Another characteristic peak at around 1755 cm-1 was detected due to the C=O stretching. Two distinct absorption peaks were also noted at about 1420 cm-1 owing to CH, CH2 and CH3 bending. The stretching of C–O ester linkage was detected through the appearance of an absorption band at 1180 cm-1

52

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. Successful Ph loading within

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PLGA NPs was confirmed by detecting the characteristic peaks of both Ph and P3 in the spectrum of P4. Lecithin spectrum showed a broad characteristic absorption peak at around 3200 cm-1 corresponding to the OH stretching of the lipid structure. Absorption peaks that appeared in the range 2800 - 3000 cm-1 could be assigned to CH, CH2 and CH3 stretching. The spectrum also revealed an absorption peak around 1745 cm-1 due to the stretching vibration of C=O bond of the glicophosphatide ester linkage. An absorption peak appeared around 1465 cm-1 that could be assigned for CH, CH2 and CH3 bending vibrations. Moreover, several characteristic absorption peaks were recorded at around 1190 cm-1, 1160 cm-1, and 1100 cm-1 owing to P=O stretching, C– O–C stretching and the P–O stretching, respectively. These results are in an agreement with what has been previously reported

53,54

. By comparing the spectra of lecithin, P4 and P5, it can be

inferred that successful coating of Ph-loaded PLGA NPs with lecithin has been achieved.

Figure 3. FTIR spectra of: a) Ph and Ph-loaded pluronic nanomicellar system and b) Ph as compared to Ph-loaded PLGA NPs system. 18 ACS Paragon Plus Environment

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3.1.4 Thermal Characteristics DSC analysis, displayed in Figure 4a, was performed to confirm the successful loading of Ph into the nanocarriers (P2, P4 and P5) as well as studying any crystallinity change. DSC thermograms demonstrate how the heat flow varies as a function of temperature for Ph, plain pluronic nanomicelles (P1), plain PLGA NPs (P3), and the corresponding Ph-loaded nanocarriers (P2, P4 and P5). DSC curve of plain Ph showed a sharp endothermic event at 297.1 оC owing to its melting point. It can be observed that in P2, the corresponding melting event of Ph totally disappeared proving the complete and homogeneous incorporation of Ph within the pluronic nanomicellar matrix. On the other hand, Ph melting event has been slightly shifted to 293.6 оC and 296.2 оC in the DSC thermograms of Ph-loaded PLGA NPs (P4) and lecithin-coated Phloaded PLGA NPs (P5), respectively. The change in the temperature of Ph melting event among free Ph, P4 and P5 is insignificant indicating a minor change in the crystallinity 50,52,55.

Figure 4b shows the TGA of Ph, plain (P1 and P3), and the Ph-loaded (P2, P4 and P5) nanocarriers. The curves describe again the thermal characteristics of the prepared formulas but through studying the variation of weight loss (%) as a function of temperature. It can be inferred from the TGA curves that Ph, P1, P4 and P5 undergo a single stage decomposition while P2 and P3 showed multistage decomposition with stable intermediates. It can be also noted that the calculated ∆Tc of P2, P4 and P5 is higher than their Ph-free counterparts due to the decomposition of both the drug and the polymer. This indicates the successful loading of Ph within the polymeric nanocarriers 51,52.

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Figure 4. Thermal behavior of the different plain and Ph-loaded polymeric nanocarriers: a) DSC and b) TGA thermograms.

3.1.5 Entrapment Efficiency (EE %) The entrapment efficiency (EE %) of Ph was calculated for P2, P4 and P5 using equation (1), and it was found to exceed 95 %, 98 % and 94 %, respectively (Table 1). The slight variation in Ph loading between P2 and P4 demonstrates the effect of the polymer type. For instance, PLGA was able to relatively accommodate more drug than the pluronic F-127. The slight decrease in Ph loading observed in P5 as compared to P4 could be attributed to a loss of a small amount of the drug during the lecithin coating step of the PLGA NPs using the thin film hydration technique.

3.1.6 In vitro Release of Ph from the Nanocarriers A preliminary study of drug release from the developed polymeric nanocarriers (P2, P4 and P5) was performed for 48 h as an optimization step before being incorporated into the CS-PEO NFs matrices, and also to understand the model and kinetics of the Ph release. The fundamental goal

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is to develop a single-dose topical dosage form for wound healing, thus the release study was carried out in PBS with pH 5.5 to mimic the natural environment of wound. As apparent from Figure 5a and Table 2, almost 100% of the free drug, Ph was released during the first 6 h. However, Ph-loaded nanocarriers (P2, P4 and P5) showed sustained release patterns. From the figure, it can also be noted that P2 and P4 exhibit initial burst release of almost 20% of Ph in the first 2 h, followed by sustained release profiles. The kinetics of drug release from the nanocarriers were analyzed using various in vitro kinetic models. The obtained fitting parameters are demonstrated in Table 2. Based on the observed r2 values, P2 and P4 followed the Korsmeyer-Peppas model. To determine the Ph release mechanism, the Korsmeyer-Peppas model was further applied. It was observed that the release of Ph from P2 (n= 0.381) and P4 (n= 0.238) followed Fickian diffusion (n ˂ 0.5). The f1 (~73) and f2 (~18) values for P2 and P4 in the model-independent approach were calculated using Eq. 10 and 11, respectively. The results showed a significant difference in the release profiles of P2 and P4 as compared to free Ph as f1 value was greater than 15, whereas, f2 value was less than 50. By the end of two days, Ph-loaded pluronic nanocarrier (P2) showed a faster release rate as more than 50% of the initial Ph amount was released. This could be attributed to the relative hydrophilicity of pluronic nanocarrier as compared to the high hydrophobicity of PLGA one (P4). In order to reduce the burst release from P2 and P4, and at the same time confer a further delay in the Ph release, an optimization of the PLGA nanocarriers was carried out through their coating with lecithin using the thin film hydration method. The optimized formula (P5) demonstrated a delayed release profile where Ph release started after 2 h, and only 12% and 33% of the initial Ph amount were released after 6 and 48 h, respectively. Based on the observed r2 values, P5 followed the Higuchi model. Higuchi model expresses the drug release from insoluble matrix depending on Fickian diffusion. The f1 (94) and f2 (13) values in the model-independent approach prove that there was a highly significant difference between the release profiles of P5 and the free Ph. This could be attributed to the high hydrophobicity of both the drug (Ph) and the polymer (PLGA) that would delay the release of the drug from the polymer matrix. Moreover, it can be noted from Figure 5a that P5 showed an enhanced release pattern than P4 owing to the presence of lecithin coating which facilitates the Ph release out of the polymeric matrix.

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Table 2. Drug release models and correlation coefficient of plain Ph and the Ph-loaded polymeric NPs and NFs mats. The best fitting of each model was performed based on correlation coefficient (r2) values. Kinetic Models Zero

First

Higuchi

Korsmeyer- HixsonPeppas Crowell

Hopfenberg

BakerLonsdale

Fitted kinetic model

0.933 0.804 0.969 0.762 0.968 0.901

0.968 0.829 0.977 0.956 0.961 0.957

0.987 0.907 0.989 0.855 0.891 0.967

0.993 0.962 0.986 0.943 0.996 0.977

0.968 0.829 0.977 0.956 0.990 0.957

0.992 0.917 0.988 0.898 0.887 0.976

Korsmeyer-Peppas Korsmeyer-Peppas Higuchi Hopfenberg Korsmeyer-Peppas Korsmeyer-Peppas

NPs

P2 P4 P5 F2 F3 F4

NFs

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0.957 0.820 0.975 0.854 0.963 0.954

Table 3. Composition, fiber diameter, Ph release kinetics, porosity, biodegradability, swelling and water vapor permeability of the different electrospun NFs mats. Code Ph F1 F2 F3 F4

Wound Dressing Matrix Free Ph CS-PEO NFs CS-PEO/ P2 NFs CS-PEO/ P4 NFs CS-PEO/ P5 NFs

-

Nanofibers Diameter (nm) -

95.8 ± 4.8

-

-

107 ± 23

-

P2

129 ± 22

P4 P5

P

Drug Dissolved % f1

f2

Model

Porosity %

-

-

-

-

-

7 Days Biodegrada bility % -

-

-

-

-

-

99.9 ± 0.01

83.1 ± 4.2*

89.5 ± 3.2*

100.0*

12.8

50.8

Hopfenberg

106 ± 17

0.0 ± 0.0*

0.0 ± 0.0*

28.1±7*

100.0

11.5

115 ± 20

44.5 ± 2.3*

57.1 ± 2.9*

100.0*

75.3

18.7

6h

48 h

288 h

KorsmeyerPeppas KorsmeyerPeppas

3h S%

24 h WVP

-

-

14.1 ± 5.8

217.5 ± 20.2

640 ± 27

99.6 ± 0.06

6.7 ± 1.7*

204.7 ± 6.0

526 ± 55*

99.8 ± 0.02

16.4 ±1.2

179.7 ± 18.1*

0*

99.7 ± 0.05

19.7 ± 3.7

153.9 ± 19.6*

381 ± 65*

CS: PEO ratio is 4:6 in F1-F4; Phenytoin is 10% (w/w) of CS-PEO matrix in F2-F4; * Significant difference to plain phenytoin release profile

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Figure 5. Cumulative release profiles of Ph from: a) the different polymeric nanocarriers systems and b) the different medicated NFs matrices.

3.2 Preparation and Characterization of Electrospun Nanofibrous Wound Dressing Mats All the electrospun NFs mats were prepared through tailoring the various electrospinning parameters as discussed earlier. The previously developed Ph-loaded nanocarriers (P2, P4 and P5) were successfully incorporated into CS-PEO NFs to produce the corresponding nanocomposites (F2, F3 and F4, respectively) as described in Table 3. Here, we report for the first time, to the best of our knowledge, the development and characterization of well-designed CS-PEO based NFs mats loaded with the more potent form of Ph (Ph base). Moreover, the NFs obtained in the present study were able, after a series of optimizations, to incorporate 10% equivalent of the drug, Ph base. This is 5 – 10 folds of the amount of its less potent salt counterpart (Ph sodium) loaded in previous studies into other polymeric matrices

24,29,32

. Neat

CS-PEO NFs matrix (F1) was prepared and used as a control during the characterization, in vitro and the in-vivo study. 3.2.1 Morphological Investigation Morphological investigation of the developed electrospun NFs was performed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Figure 6 (a – d) shows the SEM micrographs of the NFs; F1, F2, F3 and F4, respectively and their corresponding fiber diameters histograms. As apparent from the figure and Table 3, incorporation of the 23 ACS Paragon Plus Environment

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different Ph-loaded nanocarriers (P2, P4 and P5) into the CS-PEO NFs does not show a significant effect on the NFs diameter. SEM micrographs of F1 and F2 showed uniform NFs, however those of F3 and F4 showed some beaded regions (pointed by yellow arrows). This could be attributed to the higher hydrophobicity of PLGA NPs incorporated into F3 and F4 NFs as compared to that of the pluronic nanomicelles incorporated into F2 resulting in its non-uniform distribution within the hydrophilic matrix of the electrospun CS-PEO NFs. However, F4 showed less beaded regions than F3 which could be attributed to the presence of lecithin that decreases the hydrophobicity of PLGA NPs, and consequently enhances its distribution within the hydrophilic NFs matrices.

Figure 6. SEM micrographs of the developed NFs: a) Neat CS-PEO NFs, b), c) and d) CS-PEO NFs incorporating Ph-loaded pluronic nanomicelles, Ph-loaded PLGA NPs, and lecithin coated Ph-loaded PLGA NPs, respectively. (Arrows are pointing at the beaded regions)

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The morphology of the developed NPs-in-NFs composites was confirmed through CLSM scans of F2, F3 and F4, shown in Figure 7(a–c), respectively. Fluorescein dye was used instead of Ph to confirm the successful incorporation and homogeneous distribution of P2, P4 and P5 into F2, F3 and F4 NFs, respectively. The distribution of the polymeric nanocarriers within F2, F3 and F4 was in agreement with the SEM micrographs, where P2 showed the best distribution within the NFs (no beaded regions), followed by P5 that showed good distribution with very limited beaded regions (pointed by the yellow arrows, Figure 7c) due to the presence of lecithin coating, followed by P4 that showed the worst distribution and relatively large beaded areas (pointed by the yellow arrows, Figure 7b) due to the high hydrophobicity of the incorporated PLGA NPs. Figure 7d illustrates a summary for the average diameters of the different developed NPs-in-NFs matrices.

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Figure 7. CLSM images of the prepared NFs: a), b) and c) CS-PEO NFs incorporated fluorescein-loaded pluronic nanomicelles, fluorescein-loaded PLGA NPs, and lecithin coated fluorescein-loaded PLGA NPs, respectively. d) Average diameter of the plain and Ph-loaded NFs. (Arrows are pointing at the beaded regions).

3.2.2 Physicochemical Characterization Physicochemical characterization of the developed NFs mats involves the assessment of their in vitro biodegradability, swelling profiles, water vapor permeability, and porosity extent. Biodegradability study was performed to ensure the easy and safe decomposition of the wound dressing materials. Figure 8a illustrates the degradation profiles of the developed plain and drugloaded NFs mats in PBS (pH 5.5) over 7 days. As apparent from the figure, more than 80% of 26 ACS Paragon Plus Environment

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the NFs mats were degraded during the first 7 days. It was also noted that F3 and F4 have attained a relatively lower biodegradability than F1 and F2 which could be attributed to the presence of the less biodegradable PLGA NPs. Swelling study was performed to estimate the hydrophilicity extent of the developed wound dressing NFs mats along with understanding the drug release mechanism. As can be noted from Figure 8b and Table 3, the swelling percentage (S%) of all the different types of NFs exceeds 150% at equilibrium. It is also apparent that there is no significant difference in the S% values between F1 and F2. However, F3 and F4 showed a slight decrease in their S% attained at equilibrium. This can be attributed again to the presence of PLGA NPs that possess limited swellability due to their high hydrophobicity. These results are in agreement with the drug release profiles shown in Figure 5b, where F3 and F4 showed more sustained release than F2. Materials possessing high water vapor permeability (WVP) are considered to be ideal wound dressing candidates due to their ability to permit oxygen perfusion, maintain moist exudate under the dressing, prevent absorption and evaporation of wound fluids, prohibit wound bed dissection, and retain the wound comfortable for convenient wound healing

44,45

. WVP test was performed

for the developed NFs mats according to ASTM standard for 7 days as illustrated in Figure 8c. As can be noted from the figure, there is no significance difference in WVP values between F1 and F2. This could be attributed to the similar hydrophilicity of the two matrices since the pluronic nanomicelles have considerable hydrophilicity. On the other hand, F3 showed a dramatic decrease of WVP, and there is no permeation at all was observed during the first 48 h. This could be returned back to the high hydrophobicity of PLGA NPs that hinders the moisture passage across the NFs membrane. Interestingly, it can be observed that the optimized formula, F4 showed an increase again in the WVP when compared to F3. This could be attributed to the lecithin coating of the PLGA NPs. Accurate porosity measurement of wound dressing mats is essential to demonstrate their convenient performance in simulating cell proliferation and inducing oxygen and nutrient diffusion into cells 43. The porosity extents of the prepared plain and Ph-loaded NPs-in-NFs mats were obtained using a pycnometer and the results are displayed as in Figure 8d. It was found that all of the investigated NFs mats are highly porous with a porosity per volume % exceeds 99%. Therefore, the developed NFs could be ideal candidates for wound dressing fabrication due to 27 ACS Paragon Plus Environment

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their high porosity that exceeded the suggested porosity for ideal scaffolds for cell proliferation 56,57

.

Figure 8. Physicochemical characterization of the newly developed NFs mats: a) Biodegradability, b) Swelling Profiles, c) Water Vapor Permeability (WVP), and d) Porosity extents (per volume %).

3.2.3 In vitro Release Profiles of Ph from the Electrospun NFs Mats Since, the prepared NFs are considered the final dosage form as wound dressing mats, a prolonged release study of Ph from the NFs was performed for 12 days. The in vitro cumulative release profiles of Ph from the developed F2, F3 and F4 NFs matrices are illustrated in Figure 5b and Table 3. As apparent from the figure, the three NFs mats, F2, F3 and F4 showed sustained release of Ph as compared to the free drug. Similar to the release profile of Ph from P2, the F2

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showed the most rapid drug release due to the presence of the hydrophilic pluronic nanomicelles as discussed previously. The drug release kinetics of F2 was fitting to Hopfenberg model (Table 2). The Hopfenberg model is used to describe the mechanism of release from slab, cylinder and spheres, which mainly demonstrated biphasic release kinetics. This biphasic release profile could be attributed to the diffusion of Ph from the spherical nanocarriers as the first matrix followed by its release from the cylindrical NFs. The f1 (~12) and f2 (~50) values in the model-independent approach prove the similarity of the release profiles of F2, P2 and free Ph. The bi-nature of pluronic as a surfactant could represent weakness points in the NFs matrix that could consequently release the drug very rapidly. As apparent from Figure 5b, 100% of Ph was released from F2 during the first 5 days. In the case of F3, Ph was highly retained in the NFs and almost no release of Ph was noted in the first 48 h, then the overall amount released after 12 days was minimal (only 28% of the initially loaded drug). Although, F3 showed a controlled release profile of Ph, it is too slow release to be convenient for a patient to be used as a single patch for such long period. On the other hand, the optimization achieved in F4 via lecithin coating of PLGA NPs before their incorporated into the NFs has enhanced the overall Ph release pattern, where Ph was released steadily in a controlled manner from F4 matrix until its complete release attained after 9 days. The drug release kinetics of F3 and F4 followed the Korsmeyer-Peppas model (see Table 2). To determine the Ph release mechanism, the Korsmeyer-Peppas model was further applied. It was observed that the Ph release from F3 (n= 1.9) and F4 (n= 0.38) followed super case-II transport (n >1) and Fickian diffusion (n ˂ 0.5), respectively. For F3, the f1 (100) and f2 (11) values prove the significant difference to plain Ph with the delayed release profile. While, the f1 (75) and f2 (18) values for F4 demonstrated the significant sustain release with 45% released after 6 h. It was obvious at this experiment that lecithin coating has enhanced the Ph release from PLGA NPs. Therefore, it can be concluded that F4 could be an ideal candidate for fabrication of a controlled release wound dressing patch. 3.3 Cell Viability Study MTT assay was carried out to prove the nontoxicity and biocompatibility of the fabricated NFs matrices as wound dressings. As can be noted from Figure 9, all the tested NFs samples are nontoxic when compared to the negative control. No significant difference in cell viability % was also noted between the different tested NFs. Interestingly, it was observed that the tested 29 ACS Paragon Plus Environment

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NFs mats showed an increased cell optical density when compared to the negative control (no material). This reveals that the NFs fabricated in the current study could have a potential to stimulate cells proliferation and seeding, and consequently regenerate damaged skin cells due to injuries.

Figure 9. Cell viability percentage of the different fabricated NFs mats using human dermal fibroblasts cell line. 3.4 In Vivo Study In this part of the study, the appropriateness of the developed NFs mats as wound dressing candidates was investigated using a bulb/C mice animal model. Two wounds were induced on the hairless dorsal back of each mice. The first wound was treated while the second one was left untreated as a control as demonstrated in Figure 10a. The four fabricated NFs mats were applied to cure these wounds. Ointment that contains the same amount of Ph as in the fabricated NFs was used as a positive control, while non-treated wounds were used as a negative control group. The results were investigated both macroscopically and microscopically as will be discussed in the following sections.

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(a)

(b)

Figure 10. a) Demonstration for the method of applying the developed NFs as a wound dressing in an in vivo animal model and b) Macroscopic investigation of the healing progress during 14 days for the different mice groups. 3.4.1 Macroscopic Investigation of Wound Closure

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Photographic images of the wound healing progress in the investigated 6 groups of mice are summarized in Figure 10b. The images were captured at five different time points (3, 5, 7, 10 and 14 days). It could be noted from the figure that, in general, the developed F2, F3 and F4 NFs are more effective in wound closure when compared to the neat F1 NFs and the Ph ointment throughout the study period. This could be attributed to controlled release of the same dose of Ph over the study time. In addition, the presence of lecithin in F4 around the incorporated PLGA NPs has enhanced the wound healing performance. This could be because of the reported wound healing activity of lecithin in addition to its role in improving the Ph release from PLGA NPs as discussed previously 58. The healing results were confirmed through measuring the wound area at same time intervals then calculating the percentage of wound area (Figure 11a). It was found that on the 3rd day, the wound areas covered with F2, F3 and F4 NFs mats decreased to 78%, 83% and 78%, respectively, and further decreased to 67%, 44% and 39% on the 5th day, respectively. On the 7th day, the wound areas reduced to be 50%, 39% and 28%, then further decreased to reach 39%, 28% and 17% on the 10th day for F2, F3 and F4, respectively. After 14 days, the wound areas attained 28%, 11% and 11% for F2, F3 and F4, respectively. Although the positive control (ointment) and the neat NFs (F1) have some positive results, the healing progress was a way more similar to the negative control. Moreover, the healing progress was not in a controlled steady manner as what was observed in healing processes of F2, F3 and F4. Therefore, it can be concluded again that the optimized NFs formulation, F4 could be the most promising candidate material for the wound dressing fabrication.

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Figure 11. a) Wound area reduction percentage for the different mice groups, and b) Cumulative score for the histological assessment indicating the positive signs, negative signs and their difference. 3.4.2 Histological Investigation of Wound Closure In order to investigate the healing effect of the different developed NFs mats on the wound tissues microscopically, Hematoxylin and Eosin (H & E) staining as well as Masson Trichrome staining were performed on the dissected tissues from the wounds as shown in Figures 12 and 14, respectively. Microscopic histological assessment was carried out to investigate the quality of the healing process through the quantitative investigation of several parameters such as epithelization, granulation tissue, haemorrhage, inflammatory cells, necrosis and thick epidermis formation 24. Granulation tissue (GT) is the newly constructed skin layer consisting of connective tissues and tiny blood vessels that are built up on the surfaces of a wound during the healing process. GT typically grows from the base of a wound and is able to fill wounds of almost any size. The length of the newly formed epithelium, collagen deposition and the granulation thickness are considered the positive signs of the wound healing process. Figure 12 illustrates the H & E staining of dissected tissues from each of the six animal groups. Although the macroscopic investigation of the wound area of the negative control group showed that it has been healed apparently, microscopic investigation proved that the quality of the healing process was very poor. For instance, moderate epithelization did not start until the 10th day, while inflammatory necrotic cells continued until the 10th day. This was due to the continuity of necrosis and subepidermal haemorrhage within the wound tissues till the 7th day and 14th day, respectively. 33 ACS Paragon Plus Environment

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Microscopic investigation of the wound area did not show epithelization and granulation formation till the 10th day. Moreover, mild to severe abundance of necrotic and inflammatory cells were detected along the whole period of the experiment. The group treated by the neat F1 NFs showed mild to intense granulation and epithelization after the 5th and 7th days, respectively, mild to intense haemorrhage after the 3rd day, mild to severe necrotic cells till the 5th day and mild to severe inflammatory cells along the whole period. Groups treated by F2 and F3 showed similar results to some extent as they showed moderate to intense granulation and epithelization after the 3rd and the 5th days, respectively. Moreover, necrotic cells started to disappear after the 5th day. However, the two groups continued showing some moderate to mild inflammatory cells along the whole period of the experiment in addition to mild to severe haemorrhage till the 3rd day. In the case of the last group treated by F4 NFs, it showed intense epithelization and granulation along the whole period of experiment. Moreover, it was the only NFs group that did not show any haemorrhage, while intense thick epidermis formation was recorded. In addition, it showed very minor inflammatory and necrotic cells compared to the other groups.

Figure 12. Microscopic histological investigation of desiccated H & E stained tissues of a sample from each mice group throughout the 14 days of investigation.

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The cumulative scores were calculated for each group as shown in Figure 11b. It was concluded that F4 had the largest difference between the positive and the negative signs, hence confirming that F4 could be the ideal candidate for wound dressing fabrication. In addition, each of the previously mentioned parameters was demonstrated separately in Figure 13 where scores 0,1,2 and 3 represented no, mild, moderate and severe change, respectively in the different tested histological assessments such as epithelization, granulation, haemorrhage, inflammatory and necrotic cells formation, etc. Table S1 in the supplementary information file also provide more illustration and clarification of scoring of the change in histological assessments.

Figure 13. Detailed score results for: a) epithelization, b) granulation, c) haemorrhage, d) inflammatory cells, e) necrosis, and f) thick epidermis formation for each mice group during the 14 days.

Finally, Figure 14 illustrates the microscopic investigation of the collagen stained dissected tissues from each of the investigated animal groups. It can be inferred from the figure that the negative control is the only group that showed haphazardly granulation of tissues. In a contrary situation, all the other groups showed well-oriented granulation tissues formation. Moreover,

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collagen fibers deposition was noticed in all the NFs groups. This confirms the capability of the newly developed NFs matrices to play the scaffold role in preparation of skin regenerating wound dressing.

Figure 14. Microscopic histological investigation of desiccated collagen stained tissues of a sample from each mice group treated by: A) negative control, B) positive control, C) F1, D) F2, E) F3 and F) F4 after the 14 days.

4. Conclusion and Future Aspects This study reports the incorporation of the insoluble Ph base within different polymeric nanocarriers systems (pluronic nanomicelles, PLGA NPs and optimized lecithin coated PLGA NPs) in order to control the Ph release and to enhance its distribution within the aqueous CSPEO electrospinning solution. The different Ph-loaded nanocarriers were then homogeneously incorporated into CS-PEO NFs through electrospinning. The newly developed Ph-loaded NPs36 ACS Paragon Plus Environment

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in-NFs were then investigated as single-dose wound dressing patches. The fabricated NFs-based wound dressings exhibited several advantages including controlled release of Ph, high biodegradability, high biocompatibility, optimum porosity, very promising water vapor permeability, and high cell viability. These features made the NFs developed in the present study promising materials for wound dressings. Through in vivo experiments, it was found that the developed CS-PEO NFs reinforced with Ph-loaded lecithin coated PLGA NPs not only exhibited the best wound healing activity but a good healing quality as well. This was clearly observed through the histological assessment of the cured wounds that showed highest epidermis and granulation formation with minimal necrotic and inflammatory tissues and no haemorrhage.

Conflict of Interest The authors report no conflict of interest.

Funding Sources All the experiments expenses were supported and covered by the fund provided by the Center for Material Science at Zewail City for Science and Technology, Giza, Egypt.

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