Core-sheath gelatin based electrospun nanofibers for dual delivery release of biomolecules and therapeutics

Journal Pre-proof Core-sheath gelatin based electrospun nanofibers for dual delivery release of biomolecules and therapeutics

Nooshin Zandi, Roya Lotfi, Elnaz Tamjid, Mohammd Ali Shokrgozar, Abdolreza Simchi PII:

S0928-4931(19)33031-0

DOI:

https://doi.org/10.1016/j.msec.2019.110432

Reference:

MSC 110432

To appear in:

Materials Science & Engineering C

Received date:

17 August 2019

Revised date:

27 October 2019

Accepted date:

13 November 2019

Please cite this article as: N. Zandi, R. Lotfi, E. Tamjid, et al., Core-sheath gelatin based electrospun nanofibers for dual delivery release of biomolecules and therapeutics, Materials Science & Engineering C (2018), https://doi.org/10.1016/j.msec.2019.110432

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© 2018 Published by Elsevier.

Journal Pre-proof

Core-sheath gelatin based electrospun nanofibers for dual delivery release of biomolecules and therapeutics Nooshin Zandi1, Roya Lotfi1, Elnaz Tamjid2, Mohammd Ali Shokrgozar3, Abdolreza Simchi*1,4 1

Institute for Nanoscience and Nanotechnology, Sharif University of Technology, P.O. Box

Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares

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11365-11155, Tehran, Iran

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University, P.O. Box 14115-175, Tehran, Iran

National Cell Bank Department, Pasteur Institute of Iran, Tehran 13164, Iran

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Department of Materials Science and Engineering, Sharif University of Technology, P.O. Box

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11365-11155, Tehran, Iran

Address correspondence to:

Abdolreza Simchi, Tel: +98 (21) 6616 5226, Fax: 6616 0057, E-mail: [email protected]

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Abstract Coaxial electrospinning with the ability to use simultaneously two separate solvents provides a promising strategy for drug delivery. Nevertheless, controlled release of hydrophilic and sensitive therapeutics from slow biodegradable polymers is still challenging. To address this gap, we fabricated core-sheath fibers for dual delivery of lysozyme, as a model protein, and phenytoin

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sodium as a small therapeutic molecule. The sheath was processed by a gelatin solution while the core fibers was fabricated from an aqueous gelatin/PVA solution. Microstructural studies by

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transmission and scanning electron microscopy reveal the formation of homogeneous core-

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sheath nanofibers with an outer and inner diameter of 180±48 nm and 106±30 nm, respectively.

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Thermal gravimetric analysis determines that the mass loss of the core-sheath fibers fall between

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the mass loss values of individual sheath and core fibers. Swelling studies indicate higher water absorption of the core-sheath mat compared to the separate sheath and core membranes. In vitro

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drug release studies in Phosphate Buffered Saline (PBS) determine sustained release of the

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therapeutics from the core-sheath structure. The release trails three stages including non-Fickian diffusion at the early stage followed by the Fickian diffusion mechanism. The present study

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shows a useful approach to design core-sheath nanofibrous membranes with controlled and programmable drug release profiles.

Keywords: Fibrous membrane; Core-sheath, Dual drug release; Nanocarrier; Biodegradable polymer; Protein

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1. Introduction Drug delivery systems have been developed to enhance the therapeutics effect and reducing the toxicity of their conventional dosage [1, 2]. Nanoscale formulations including polymeric micelles, liposomes, polyelectrolyete complex nanoparticles, and nanofibers have attracted significant attention over the last decade [3-7]. As compared with other methods, electrospinning

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provides great flexibility in selecting biomaterials and therapeutics for drug delivery applications[8, 9]. This is a straightforward and low cost process[10, 11], which allows high drug

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loading and encapsulation efficiency[12] with an ability to tune the release rate[13]. So far,

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different bioactive molecules including growth factors, DNA, and RNA are incorporated into

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electrospun fibers[14, 15]. However, preserving the bioactivity and functionality of these

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macromolecules through electrospinning is still the main challenge of this technique.[3] To achieve a sustained release rate of biomolecules and pharmaceutics, coaxial electrospinning

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techniques have gained interests [16]. Wang et al. [17] utilized a modified coaxial

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electrospinning technique to prepare Hypromellose based composites with better release rate of

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poorly water-soluble drugs. Yang et al. [18] developed a modified tri-axial electrospinning process for the fabrication of pH sensitive polymer/lipid nanocomposites with the ability to enhance dissolution and permeation of poorly water-soluble drugs. Employing a coaxial nozzle provides the possibility of utilizing different solutions via separate syringe pumps. It is also possible to fabricate the sheath from polymeric materials while the core can be made by nonpolymeric Newtonian liquids or even suspensions containing powders [19]. These features provide a novel platform to encapsulate bioactive agents such as proteins, living cells, and DNA into the core structure [19] while the outer shell operates as a protection layer and physical barrier for the drug diffusion[20]. It has been reported that drug release kinetics from nanofibers 3

Journal Pre-proof is invariably controlled by their morphology[21]. Studies have shown that more sustained release profile can be achieved by core–shell configurations fabricated through the coaxial electrospinning technique [22]. Core-sheath fibers also exhibit better biocompatibility and mechanical properties for tissue engineering applications than blended fibers [23]. Moreover, dual drug encapsulation and release profiles from both core and shell can be achieved through a single delivery system [24]. Apart from these advantages, dual drug encapsulation are not a

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straightforward and facile process; variety of instrumental, and solution parameters should be

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optimized to fabricate fibrous mats with desired properties [25].

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So far, wide range of synthetic biopolymers such as poly(ethylene glycol)-poly(DL-lactide)

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(PELA)[26], poly(ε-caprolactone)[27], poly(vinyl alcohol) (PVA[28], poly(D,L-lactide-coglycolide) (PLGA)[29], poly-L-lactic acid (PLLA)[30], poly(vinyl pyrrolidone) PVP[31],

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poly(ethylene oxide) PEO[31], poly(ethylene glycol) (PEG)[32] have been processed by co-axial

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electrospinning for controlled drug release. For this aim, natural polymers are also attractive. It has been shown that natural polymers have better biocompatibility, clinical functionality, and than

synthetic

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non-immunogenicity

polymers

[3].

Although

collagen is

the main

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protein in ECM[33], the strong organic solvents in the electrospinning process denature the collagen into gelatin[34]. Gelatin, a mixture of peptides and proteins, is biodegradable and biocompatible without antigenicity [23, 35].

Nagiah et al. [23] employed coaxial

electrospinning to prepared core-sheath nanofibers of gelatin and different types of synthetic hydrophobic polymer (polyurethane, polycaprolactone, and polylactic acid, as the core). They have shown that the gelatin sheath and the hydrophobic polymer core form scaffolds with tissue like viscoelasticity with high compliance and excellent swellability. Coimbra et al. [36] fabricated core-sheath fibrous mats from PCL and photocurable gelatin through coaxial

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Journal Pre-proof electrospinning for vascular tissue regeneration. They have demonstrated that the gelatin sheath significantly improves the hydrophilicity of the fibers which is essential for the high biologic performance of biomaterials in contact with blood and cells. Apart from many studies that have been performed on the fabrication of core-sheath fibers, mechanisms and kinetics of drug release have lagged behind [17, 37, 38]. The aim of the present study is to fabricate core-sheath fibrous mats with the ability of dual release profile with

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controlled rate. It is hypothesized that this strategy is an effective treatment for tissue injuries

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such as skin wounds that require well-planned incorporation of different therapeutic agents. The

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core is made of a water soluble gelatin/PVA mixture that enables carrying hydrophilic

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biomolecules. Lysozyme as a model protein is encapsulated in the core fibers. Encapsulating lysozyme as bioactive agent in the water soluble core structure prevents denaturation of the

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biomolecules, which commonly occurs in organic solvents[39]. Furthermore, during the release

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process, the encapsulated agent should pass through the matrix of both core and sheath fibers, thereby prolonging the period of release. In fact, the sheath operates as a physical barrier against

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the burst release of the core. It is shown that the sheath effectively contributes in enhancement of

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the physicomechanical properties of the core-sheath architecture. We fabricated the sheath from gelation by dissolving in a mixture of organic (dimethyl formamid and formic acid) and inorganic (water) solvents in order to incorporate phenytoin sodium, a small therapeutic molecule with poor water solubility, for the coaxial electrospinning experiments. The prepared core-sheath fibrous membranes could be used as a promising platform for complex tissue regeneration applications. 2. Material and methods 2.1 Material 5

Journal Pre-proof Gelatin (from bovine skin, type B), poly vinyl alcohol, glutaraldehyde, lysozyme (from chicken egg white), formic acid (FA), and dimethyl formamid (DMF) were purchased from Sigma Aldrich (USA). Phenytoin sodium was obtained from Alhavi Pharma (Tehran, Iran). Deionized (DI) water (18 MΩ.cm) was used to prepare aqueous solutions. 2.2 Preparation of core-sheath fibrous scaffolds

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To prepare the sheath solution, gelatin (20% w/v) was dissolved in a mixture of water/DMF/FA

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(50:25:25 %v/v) and homogenized by magnetic stirring. Phenytoin sodium (20 mg/mL) was then added to the solution. For the preparation of the core solution, aqueous solutions of PVA (12%

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w/v) and gelatin (10% w/v) were prepared separately and then mixed with a volume ratio of 9:1

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(PVA:Gelatin). Afterward, lysozyme (5 mg/mL) was added to the polymeric solution.

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A commercially available electrospinning setup (Nano Azma Co., Iran) was used for the

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fabrication of fibrous mats. The processing parameters are presented in Table 1. For coaxial electrospinning, the core and sheath solutions were transferred into separate 5 mL syringes and

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injected through the concentrically coaxial inner and outer needles. Outer/inner diameters (mm)

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of inner capillary are 0.90/0.70 and Outer/inner diameters (mm) of outer capillary are 1.70/1.50. The flow rate of the solutions was 1 mL/h. The distance between the nozzle and the collector was 12 cm. The electrospinning potential was in the range of 17 to 19 kV. Electrospinning of pristine core and sheath fibers was also performed at a flow rate of 1 mL/h. The fibers were collected onto a rotating drum collector having a diameter of 6.5 cm. The electrospinning dwell time was 4 h. All experiments were carried out under ambient temperature with an average humidity of 30%.

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Journal Pre-proof Since gelatin is quickly dissolved in physiological environments, crosslinking is inevitable in order to improve its stability for biomedical applications [40, 41]. For this reason, chemical crosslinking was performed by glutaraldehyde (25% in water) at room temperature. The scaffolds were subjected to the glutaraldehyde vapor in a desiccator for 24 h. The prepared scaffolds were kept in the desiccator before further experiments.

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2.3 Microstructural studies

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The morphology of the fibrous scaffolds was studied by field emission scanning electron microscopy (MIRA3TESCAN-XMU FESEM, Oberkochen, Germany). Fibers were collected on

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an aluminum foil and gold sputtered by a Desk Sputter Coater (Nanostructured Coatings, Iran).

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FESEM images were taken at an accelerating voltage of 15 kV. The diameter distributism of the

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fibers was determined by random selecting of at least 50 individual fibers. The images were analyzed by Image J software (National Institutes of Health, USA). To visualize the core-sheath

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structure, transmission electron microscopy (Zeiss - EM10C TEM, Germany) at an accelerating

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voltage of 80 kV was employed. The fibers were attached to a formvar carbon coated TEM grid

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(Cu Mesh 300) prior to analysis.

2.4 Thermogravimetric analysis Thermogravimetric analysis (TGA) was performed using a Linseis STA PT 1600 instrument (Germany). About 10 mg of the mats were analyzed with a heating rate of 10 ˚C min-1 in a platinum crucible. Differential thermal curves were obtained from TGA plots through calculating the derivative of weight % versus temperature.

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Journal Pre-proof 2.5 Mechanical durability The mechanical properties of electrospun mats under tensile loading were measured before and after crosslinking in dry and wet conditions by a Hiwa 200 universal machine (Iran) in accordance with ASTM D822 standard. Rectangular strips with dimensions of 3× 4 cm2 were used. The thickness of the samples was 85±15 μm. Both ends of the strips were placed between double sided tapes and subjected to tensile testing at ambient temperature. An extension rate of 1

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mm/min was applied. For testing under wet condition, the rectangular samples were immersed in

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PBS before mechanical loading. The first part of the linear region of the stress-strain curves (up

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to 10% strain) was used for determination of the elastic modulus. Experiments were performed

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in triplicate for each sample and the average results were presented.

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2.5 Swelling and degradation

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In vitro swelling tests were carried out on rectangular electrospun samples with dimensions of 1.5×4.5 cm2. The specimens were placed in 3 mL PBS at 37°C (pH=7.4) for different times up to

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24 h. After selected time intervals, the samples were taken out and placed on a piece of tissue

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paper to remove excess PBS, according to the method reported elsewhere [6]. The degree of swelling (%) was determined by: Water retention(%) = (𝑊𝑠𝑡 − 𝑊𝑑𝑡 ) × 100%

(1)

Wst and Wdt are the weight of wet and dry samples, respectively. In vitro degradation of the electrospun mats was measured by placing rectangular samples with dimensions of 6.5 × 9 cm2 (n≥3) in 3 mL PBS at 37°C for 28 days. The samples were weighted before the experiments. Following incubation for 1, 3, 5, 7, 14 and 28 days, the weight of the

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Journal Pre-proof samples was measured after freeze drying for 24 h. The weight loss was measured using the following equation[25]: Weight loss (%) = (𝑊𝑖 − 𝑊𝑡 )/𝑊𝑖 × 100%

(2)

where (Wi) is the initial weight, and (Wt) the weight after the designated time points. 2.6 In vitro cytotoxicity assay

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2.6.1 Cell Culture

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L929 cells (NCBI C161, cell bank of Pasteur Institute of Iran, Tehran) were used to evaluate cell

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toxicity of electrospun mats. The thawed cells were transferred into a tissue culture flask containing Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal

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bovine serum (FBS). The flask was then incubated in 5% oxygen humidified atmosphere (90%)

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at 37°C.

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2.6.2 MTT Assay

The dimethyl-thiazole diphenyl tetrazolium bromide (MTT, Sigma, USA) assay was used to

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determine the cell viability. The electrospun mats with 4 cm2 surface area were sterilized by

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ultraviolet irradiation for 45 min. Then, the mats were transferred to 24 well plates and 2 × 104 cells in 100 μL culture medium were added. After incubation at 37°C for 4 h, 200 μL of the culture medium were added to each well and incubated for 1, 3 and 5 days. Next, 300 μL of MTT solution in PBS (0.5 mg/mL) was added to each well and incubated for 4 h. The reagent was then removed and isopropanol was added to dissolve the resulting purple crystals for 15 min. Finally, the absorbance was measured using an Elisa Reader (STAT FAX 2100, St. Louis, MO, USA) at a wavelength of 545 nm. The cell viability was determined in comparison with the control (RPMI).

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Journal Pre-proof 2.7 Drug release studies In vitro drug release profiles were evaluated in PBS (pH 7.4) at 37°C. Rectangular samples (10 mg) were immersed into 3 mL of PBS and incubated at 37°C with shaking (150 rpm) in a DKZ-2 orbital rotator (YIHENG, China). At each time points, 200 µL of the medium was withdrawn for analysis of drug content and replaced by an equal volume of fresh PBS buffer. To quantify the phenytoin release, a Lambda 35 UV-spectrophotometer (Perkinelmer, USA) at 245 nm was used.

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The cumulative drug release was measured from a standard calibration curve. At least three

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replicates were done and the results were reported as mean±S.D. QuantiProTM BCA assays

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were employed for the quantification of lysozyme release based the manufacturer’s protocols. Briefly, 150 μL of the reagent was added to 100 μL of the collected medium, and the mixtures

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were incubated at 37°C for 2 h. We used a release media suitable to dissolve around 5 times the

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initial dose to maintain sink condition during release. The absorbance at 562 nm was determined

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by a plate reader (LEDETECT 96, EU). The data were normalized based on the degradation of electrospun mats.

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2 Results and discussion 3.1 Morphological studies

Core-sheath fibers containing two different biomolecules were prepared using the coaxial electrospinning method. A schematic illustration of the procedure is shown Figure 1. Representative SEM images of the prepared fibers before and after incubation in PBS are also demonstrated. SEM studies have shown that uniform and bead-free fibers were obtained (Figure 2A to 2C). The size histograms determines that separate electrospining of the core and sheath solutions forms fibers with an average diameter of 190±25 and 205±45 nm, respectively. The average diameter of the core-sheath fibers was 213±80 nm. TEM studies (Figure 2D) revealed 10

Journal Pre-proof successful formation of the core-sheath structure with a mean outer and inner diameter of 180±48 nm and106±30 nm, respectively. The strong contrasts in TEM image could be attributed to the higher density of polymers in the core, which fewer electrons could transmit through this

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region.

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Figure 1. Schematic illustration of the procedure used for the preparation of electrospun coresheath fibers containing phenytoin sodium in the sheath and lysozyme in the core. FESEM images of electrospun nanofibers before and after immersing in PBS are shown in the right side of the figure.

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Figure 2. SEM and TEM images and size distribution histograms of electrospun fibers: (A) Gelatin fibers; (B) PVA/gelation fibers; (C) Coaxial electrospinning. (D) TEM image of coresheath fibers.

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Journal Pre-proof 3.2 Effect of chemical crosslinking Thermogravimetric analysis (TGA) was employed to study the effect of crosslinking on the thermal stability of the core-sheath fibers. The thermal behavior of the individual core and sheath fibers were also examined. Figure 3 shows TGA and derivative thermogravimetric (DTG) graphs of the fibrous mats. All the examined materials exhibited similar thermal degradation behavior between 30 to 700°C. The initial weight loss occurred at temperatures <150°C and ascribed to

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the decomposition of small compounds like water (bonded and adsorbed). The second step of

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weight loss appeared between 200 and 500°C. Thermal-oxidative decomposition of polymer

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chains caused significant weight loss in this temperature range (Table 1). At higher temperatures

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(>500°C), thermally stable structures were degraded. It was also noticed that the sheath fibers

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underwent a single-step weight loss while the core and core-sheath fibers exhibited a two-step weight loss (Figure 3B).

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In the case of un-crosslinked fibers, the gelatin chains were relatively stable up to 275°C

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(Tmax1=310 ˚C). The sheath fibers lost 65.9% of their weight between 200-500˚C, which was

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attributed to the hydrolysis and oxidation of the polymer chains [42]. Gradual degradation of gelatin occurred beyond 500°C, which could be related to the breaking of imide and amide bonds. The un-crosslinked core fibers exhibited 88.4% degradation between 200-500°C. The first step degradation occurred at 312˚C due to the scission or cleavage of the PVA/gelatin sidechains. Breakage of C-C backbone of PVA caused thhe second step degradation at 402˚C [42]. The degradation peaks of the crosslinked fibers were less intensive and they were shifted toward higher temperatures (Figure 3B). These results indicated that the stability of the fibers was significantly improved due to chemical crosslinking between OH groups of PVA and/or NH2

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groups of gelatin with CHO groups of GA.

Figure 3. (A) TGA and (B) DTA curves showing thermal degradation of electrospun fibers. The

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notations are: c-c (crosslinked core fibers); u-c (uncrosslinked core fibers); c-s (crosslinked sheath fibers); u-s (uncrosslinked sheath fibers); c-cs (crosslinked core-sheath fibers); un-cs

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(uncrosslinked core-sheath fibers)

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It was also interesting to note that the weight loss of the core-sheath fibers was felt between the weight loss values of the sheath and core fibers. The initial weight loss occured between 248370°C with a Tmax1 value of 323°C for the un-crosslinked core-sheath fibers. The second step weight loss occurred in the range of 375-451°C with a Tmax2 value of 432°C. Tmax2 of the coresheath fibers showed a broader peak that could be due to the formation of a weak hydrogen bond between PVA and gelatin. As a result, the crosslinked fibers exhibited higher stability (Figure 3A-B and Table 1). We have found that the crosslinked sheath fibers have the highest thermal stability followed by the core-sheath ones. In the temperature range of 200 to 500°C, the weight 14

Journal Pre-proof loss could be ranked as sheath (59.08%) < core-sheath (63.1%) < core (78.8%). It appeared that inter- and intra-molecular bonds induced by chemical interactions between the polymer chains and the crosslinker improved the thermal stability of fibers[43]. Table1. Thermal properties of electrospun fibers

After crosslinking

Core

Core-sheath

Tmax1 (˚C)

310.0

312.0

323.0

Tmax2 (˚C)

-

402.0

432.0

T-5% (˚C)

74.3

69.2

Weight loss (%) at

9.8

6.1

200–500°C

Residue(%) at

Core-sheath

339.0

324.0

331.0

-

441.0

445.0

81.2

132.0

102.3

101.4

12.7

5.6

10.0

11.0

88.5

79.1

59.1

78.9

63.1

2.5

2.1

34.2

9.8

21.6

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66.0

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Weight loss (%) at

Core

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30–150°C

sheath

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sheath

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Without crosslinking

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Characteristics

22.4

500°C

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Journal Pre-proof 3.3 Mechanical properties Figure 4A shows the tensile stress-strain curves of the electrospun fibrous mats. The mechanical properties of the examined materials including the elastic modulus (E), elongation (EL), and ultimate tensile strength (UTS) are reported in Table 2. The data are the average values of triplicate tensile tests. For the un-crosslinked mats, both core and core-sheath samples exhibited a type of plastic failure while the mat prepared from the sheath polymer showed a stiffer and

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more brittle behavior. The core-sheath fibrous mats had higher elongation before fracture (32%)

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as compared with the other (12%). It appeared that the presence of PVA preceded interactions

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between hydroxyl, amino, and carbonyl groups of the polymers. After crosslinking, the elastic

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modulus of the mats increased significantly (Figure 4B). The highest improvement was attained

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for the core-sheath fibers (1100%) followed by the sheath polymer (300%). The modulus enhancement for the core polymer was only 40%. It should be noted that the sheath material

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contained more gelatin than the core. Therefore crosslinking of gelation was more effective. As it

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will be discussed in following sections, the swelling and degradation of the core-sheath fibers are also influenced by the chemical crosslinking. The higher mechanical durability of the core-

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sheath nanofibers could be attributed to interactions between different functional groups such as hydroxyl, amino, and carbonyl of the polymers in the core and the sheath [44, 45]. Diffusion and interactions of the polymers during coaxial electrospinning might also have an influence[23].

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Figure 4. Mechanical characterizations of electrospun mats including (c) core fibers, (s)

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sheath fibers, (cs) core-sheath fibers: (A) strain/stress curves; (B) elastic modulus; (C) ultimate strength; (D) elongation. Data are represented as mean ±SD. Ns denotes nonsignificant (*p <

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0.05, ****p < 0.0001 and n> 3).

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We also examined the mechanical strength of the fibrous mats under wet condition and measured lower elastic modulus (Figure 4C). The ultimate tensile strength (UTS) of the sheath mat and the core-sheath mat was 4.4±1.0 MPa and 2.5±2.0 MPa under dry condition, respectively. These values are statistically higher than the ultimate strength of the wet mats (2.6±0.4 MPa for the sheath mat and 1.8±0.1 MPa for the core-sheath mat). Although tensile testing of the core material under the wet condition did not yield measurable values, shielding of the core with the sheath increased the mechanical durability. Meanwhile, the elongation was enhanced under wet condition. For instance, the elongation of the core-sheath mat increased from 6.0±3% to

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Journal Pre-proof 23.8±0.2%. This enhancement was ascribed to a change in the brittle nature of the mats to hydrogel deformation after immersion in PBS (Figure 4D). Table 2. Mechanical properties of electrospun mats

EL (%)

UTS (MPa)

Dry

-

50.7±3

53.0±10

3±0.6

Dry

+

67.6±4

Wet

+

N/A

Dry

-

Dry

+

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12.0±0.6

4.3±0.2

106.9±4

6.0±3

4.4±1

6.0±0.3

23.8±0.2

2.6±0.4

-

14.7±0.2

31.8±5

2.2±1

Dry

+

173±3

16.8±0.9

4.98±2

Wet

+

5.95±0.7

24.3±3

1.8±0.1

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3.42±0.3

N/A

25.9±2

+

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Dry

sheath

50.6±9

N/A

Wet

Core-

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E (MPa)

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sheath

Crosslinked

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core

Condition

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Fibers

3.4 Swelling and degradation Water absorption capability of the crosslinked fibers was evaluated by examining their swelling behavior in PBS (pH=7.4) at 37°C for a period of 24 h (Figure 5A). The swelling profiles were found to vary with the fiber configuration and their composition. PVA and gelatin are hydrophilic biopolymers which should enhance the swelling ratio. Therefore, the highest 18

Journal Pre-proof swelling ratio was attained after 4 h for the core fiber (445%) but degraded due to the dissolution of PVA in PBS (Figure 5B). The lowest swelling ratio was attained for the sheath fibers (260%) without measurable weight loss over 24 h immersions in PBS. The decreased swelling ratio determined the effect of crosslinking on the water uptake[46]. Interestingly, the core-sheath electrospun mat exhibited higher swelling ratio than the others (e.g. 360±40% after 12 h). Meanwhile, the degradation rate was not significant even after 24 h. It seemed that the fast

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degradable core fibers were shielded by the sheath which prevented rapid hydrolysis of PVA and

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gelation in contact with PBS. The high reactivity of aldehyde groups of glutaraldehyde forms

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imine bonds with amino groups (Schiff’s base)[47]. On the other hand, the formation of acetyl

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bonds with hydroxyl groups[48] provides effective crosslinking with gelatin and PVA. It should be noted that crosslinking of hydroxyl groups with glutaraldehyde must be carried out in acidic

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conditions while crosslinking requires neutral or basic conditions[49]. Since PVA is the

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predominant matrix of the core fiber, the sheath should most likely be crosslinked under the neutral condition. Anyway, the high swelling behavior of the core-sheath fibers provides a great

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opportunity for the fibrous scaffolds to be used as hydrogel biomaterials in physiological

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environments. Herein, the controlled degradation rate is of crucial importance because the mechanical stability of a tissue engineering scaffold should be compromised with the neotissue growth[50]. Regarding degradation results (Figure 5B), the weight loss of the core, sheath, and core-sheath fibers after 28 days were 77.7±0.1, 54.7±1.5, and 52.9±5.6 %, respectively. Therefore, it could be concluded that the degradability of the core polymer was reduced by the sheath.

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Figure 5. Effect of immersion time on swelling and degradation of fibrous mats in PBS. S,

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C and CS denote sheath, core and core-sheath fibers, respectively.

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3.5 In vitro cytotoxicity

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After physico-mechanical characterizations, the cytocompatibility of the core-sheath electrospun

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mats were examined by MTT assay. The results of L929 cell viability up to 5 days are shown in Figure 6. Each time point is presented relative to the cell culture medium on tissue culture plate

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cytotoxic.

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as the control. The cell viability was about 90±2%, which indicated that the mats are not

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Journal Pre-proof Figure 6. Cell viability of core-sheath mats as assessed by MTT after 1, 3, and 5 days incubation. 3.6 Drug release The release kinetics of two different fiber configurations was examined: (A) the core mat consisting of lysozyme; (B) the core-sheath fibers, which lysozyme is encapsulated in the core

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and phenytoin in the sheath. For both configurations, the release occurs through molecules

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diffusion in the polymer matrices, dissolution of PVA, and swelling of gelatin. Noted that, the rate of molecular diffusion within polymeric materials depends on the size and shape of the

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diffusing molecules, internal nanostructures, pore and mesh size of the matrix material[51, 52].

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The cumulative release of lysozyme from two types of electrospun mats in PBS at 37°C was

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assessed by bicinchoninic acid assay (BCA) kit. The amount of phenytoin sodium release was determined by UV spectroscopy at the absorbance peak of 245 nm using a predetermined linear

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calibration curve (absorbance= 10.097 × drug concentration - 0.0896; R = 0.993). The release

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profiles are shown in Figure 7. As seen, the release profiles can be considered in three stages

Jo

including (I) an initial burst release, (II) decreased release rate, and (III) constant release regime. The first 8 h can be considered as stage I. The second stage lasted 33 h. Finally, a nearly steadystate release profile was noticed.

21

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ro

of

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re

Figure 7. Cumulative drug release (37°C, pH=7.4) from lysozyme loaded electrospun core fibers

na

lP

(configuration A) and dual purpose core-sheath fibers (configuration B) over a period of 167 h.

The drug release profile of phenytoin sodium in the configuration B and lysozyme in the both A

Jo

Mt/M0 = Ktn (3)

ur

and B configurations follows the Peppas equation[53]:

where M denotes the drug concentration at the initial stage (M0) and at time t (Mt). K is a constant reflecting the structural and geometric characteristics of the fibers and n the release exponent that indicates the drug release mechanism. From the slope and intercept of ln (Mt/M0)-ln (t) plots, these characteristic values were determined (Table 3).

22

Journal Pre-proof Table 3. Effect of fiber configuration on the mechanism and release kinetics of lysozyme and phenytoin sodium from electrospun mats

Model parameter

R2

I; 0.93

0.99

II; 0.52

0.98

non-Fickian

0.96

Fickian

0.96

non-Fickian

0.97

Fickian

III; 0.12

0.98

Fickian

Phenytoin sodium

I; 0.98

0.98

non-Fickian

II; 0.37

0.99

Fickian

III; 0.27

0.93

Fickian

ro

Lysozyme

of

Stage; n

re

-p

III; 0.1

Core-sheath

Lysozyme

I; 0.66

ur

na

II; 0.22

Core-sheath

Model mechanism

lP

Core

Bioactive agent

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Fiber

non-Fickian

Analysis of the data indicated that the core fibers (configuration A) exhibited a significant burst release (50%) within the first few hours after immersing in PBS. After 8 h, almost 90% of lysozyme was released. This fast release profile is ascribed to the hydrophilic properties of PVA 23

Journal Pre-proof and stronger polymer–solvent interactions compare to the polymer–polymer attraction forces[54]. As a result, the polymer chains rapidly absorb solvent molecules which are accompanied by fast swelling and degradation. Additionally, the release of accumulated drug molecules at or near the surface of the fibers during the electrospinning process could contribute to the high rate at the initial stage[55]. After the burst release and at stage II, the value of n for the lysozyme release in configuration A is 0.52. Therefore, the release path from the fibers

of

should be controlled via a typical non-Fickian diffusion mechanism (n>0.45).

ro

For configuration B, the burst release of lysozyme (within the first 8 h) through diffusion from

-p

the polymer matrix and degradation of the fibers was comparatively lower. During the second

re

stage, a lower amount of drug was also released (n=0.37). Thereby, the sheath provided a more

lP

sustainable release of lysozyme in configuration B through a Fickian diffusion process (n<0.45). After 7 days (168 h), the amount of lysozyme released from the core-sheath fibers reached

na

83.5%. In the case of phenytoin sodium, three phasic release profiles were noticed for the core-

ur

sheath mat. After the initial burst release, a sustained drug release was achieved during stage II (n=0.22). Finally, a gradual decrease in the release rate was observed in stage III. The primary

Jo

reason for the faster release of lysozyme in both configurations compare to the phenytoin sodium is likely due to the hydrophilic nature of PVA and its dissolution in the physiological fluid. This difference is also related to the size of molecules, i.e. lysozyme is a macromolecule (14.4 kDa) while phenytoin sodium is small molecule (Mw 252 g mol-1). It is also expected that during the electrospinning process, a certain portion of positively charged lysozyme are agglomerated close to or upon the surface of fibers under the electrical driving force. Once immersed in PBS, most of the lysozyme located on or near the surface is quickly released[56, 57]. The burst release of lysozyme from the core structure could also be happened through the imperfections of core–shell 24

Journal Pre-proof structure[22]. Thereafter, the encapsulated lysozyme in the core is released via diffusion mechanism with a more sustainable rate[22, 57]. When configuration B is utilized, more time is required for water molecules to penetrate into the polymer matrix, as the sheath operates as a physical barrier. The PVA-rich core is also under radial stress and Laplace pressure which affect the release rate[58]. Figure 8 show changes in the morphology of the core-sheath electrospun fibers after incubation

of

in PBS at 37˚C at different times. After 1 day of incubation, the fibers were swelled and lost their

ro

initial surface smoothness (Figure 8A). After 7 days, the dissolution of PVA in the buffer led to

-p

structural changes in the fibrous mat (Figure 8B). At the prolonged time (14 days), most of the

Jo

ur

na

lP

re

fibers were collapsed and lost their initial shape (Figure 8C).

Figure 8. Effect of incubation in PBS (37˚C) for (A) 1, (B) 7, and (C) 14 days on morphology of core-sheath fibers

3 Conclusions Core-sheath electrospun fibrous mats were successfully fabricated by the coaxial electrospinning technique for dual delivery purpose. The core consisted of gelatin/PVA containing lysozyme 25

Journal Pre-proof biomolecules. The gelatin sheath contained phenytoin sodium and operated as a physical barrier against burst release from the core. The sheath also enhanced physicomechanical characteristics of the core-sheath fibers. TEM studies determined that the average diameter of the core and sheath was 106±30 nm and 180±48 nm, respectively. TGA showed that the thermal stability of the core-sheath fibers was improved by inter- and intra- molecular bonds formed between the core and the sheath. The swelling ratio and the amount of degradation of the fibers in PBS was

of

also changed by coaxial electrospinning. Evaluations of the mechanical characterization of the

ro

fibrous mats in dry and wet conditions revealed that the mechanical strength of the core-sheath

-p

membranes was superior to the individual electrospun fibers in both conditions. Higher

re

elongation was measured in wet samples. On the other hand, the release profile of lysozyme and phenytoin sodium from the fibrous mats exhibited a three-stage behavior. The release rate from

lP

the core-sheath fibers was substantially lower and more sustainable than the others. The first

na

stage of the release for the core-sheath fibers was ascribed to the non-Fickian diffusion mechanism. Following the swelling of the sheath and degradation of the water soluble

ur

components, Fickian diffusion was governed. The developed core-sheath electrospun fibers with

applications.

Jo

dual delivery capacity can be used for specific healthcare needs such as wound healing

Conflicts of interest: Authors have no conflicts to declare. Acknowledgements: We wish to thank funding support from Sharif University of Technology (Grant No. QA970816) and Iran National Science Foundation (INSF No. 95-S-48740).

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Journal Pre-proof Highlights

Dual delivery of therapeutics from core-shell fibers was studied.



Core-shell fibers were fabricated by coaxial electrospinning of gelation (core) and gelatin/PVA (sheath) solutions.



Three-stage release kinetics from the core-sheath fibers was shown.



Improved mechanical properties and swelling behavior were reported.

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30