Electrospun Fibers of Polyester, with Both Nano- and Micron

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Electrospun Fibers of Polyester, with both Nano- and Micron Diameters, Loaded with Antioxidant for Application as Wound Dressing or Tissue Engineered Scaffolds Jorge Fernández, Mario Ruiz-Ruiz, and Jose-Ramon Sarasua ACS Appl. Polym. Mater., Just Accepted Manuscript • DOI: 10.1021/acsapm.9b00108 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

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ACS Applied Polymer Materials

Electrospun Fibers of Polyester, with both Nano- and Micron Diameters, Loaded with Antioxidant for Application as Wound Dressing or Tissue Engineered Scaffolds

Jorge Fernández1*, Mario Ruiz-Ruiz1, Jose-Ramon Sarasua2

1 Polimerbio, 2 Department

S.L, Paseo Mikeletegi 83, 20009 Donostia-San Sebastian, Spain

of Mining-Metallurgy Engineering and Materials Science, POLYMAT,

University of the Basque Country (UPV/EHU), School of Engineering, Alameda de Urquijo s/n. 48013 Bilbao, Spain

* Corresponding author

E-mail: [email protected] (Jorge Fernández Hernández)

1 ACS Paragon Plus Environment

ACS Applied Polymer Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Antioxidant loaded poly(ε-caprolactone) (PCL) scaffolds were fabricated by electrospinning and then tested – mechanically and characterized via microscopy imaging. These mats are intended to be used in tissue repair or for wound healing and their aim is to enhance the biocompatibility by reducing the inflammatory response related to biomaterials via the release of reactive oxygen species, (ROS)-responsive molecules, with antifibrotic properties over a certain length of time. Of the antioxidants studied, only allicin, curcumin, piperine, polydatin and quercetin were found to be miscible with the polyester and gave rise to better processing by electrospinning and a greater homogeneity of the mats. The scaffolds of different fiber morphologies (micro-, nano- or micro-nanostructured, the later more closely mimicking the structure of the extracellular matrix of soft tissue) filled with 5 % of antioxidant, showed a Young´s modulus and tensile strengths lower than those of pure PCL (i.e. elastic modulus of 5-9 MPa vs. 12-27 MPa for PCL) but exhibited very interesting mechanical behavior for soft tissue engineered applications with elongation at breaks higher than 25 % at room temperature. In a preliminary study Human Adipose-derived Mesenchymal Stem Cells were also seeded in several scaffolds and analyzed by fluorescence microscopy and showed that the cells were able to attach, survive and grow in these 3D culture systems.

Keywords: electrospinning, wound dressing, poly(ε-caprolactone), antioxidant, oxidative stress


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1. Introduction Tissue repair after injury is a complex, metabolically demanding process that depends on the tissue´s regenerative capacity and the quality of the inflammatory response. The first stage of the immune system response is inflammation, in three distinct phases [1-2], which helps to restore normal tissue architecture [3]. In an early pro-inflammatory step, necrotic debris, the clotting reaction, and any invading microbes collectively mobilize the recruitment of key inflammatory cells, and so the release of inflammatory substances, including cytokines, free radicals, hormones and other small molecules are triggered. Neutrophils, monocytes, and other innate immune cells are recruited to the wound site to clear cell debris and remove infectious organisms. In the second major phase, the proinflammatory response begins to subside, with key inflammatory cells such as macrophages switching to a reparative phenotype. Finally, tissue homeostasis is restored when the inflammatory cells either exit the site of injury or are eliminated through apoptosis. Polymorphonuclear neutrophils, macrophages and other cells involved in the hostdefense produce Reactive Oxygen Species (ROS) [4-5], partially reduced metabolites of oxygen that possess strong oxidizing capabilities that play an important role as complex signaling functions [6-8] in the progress of inflammatory disorders. However, enhanced ROS generation at the site of inflammation causes oxidative stress, an imbalance between ROS production and the ability to detoxify the reactive oxygen intermediates [9-11]. Chronic or prolonged ROS production is interrelated with the progress of inflammatory diseases [12] and in such cases the wound can become chronic or progressively fibrotic, both outcomes impair tissue function [13].



Since oxidative stress affects cellular structures (including lipids, membranes, proteins and DNA) leading to inflammation, cellular apoptosis and senescence, cells make use of a wide-range of molecules and enzymes to prevent the accumulation of ROS [14-17]. Nevertheless, the oxidative stress can also be effectively neutralized by enhancing cellular defenses with the delivery of natural and synthetic antioxidants. These compounds can be loaded into biomaterials for prolonged and targeted delivery; thereby increasing their bioavailability. The design of tissue engineered scaffolds with antioxidants incorporated in them aims to ensure a continuous release at the implant site so as to aid the healing process [18-20] and at the same time attenuate the inflammation reactions related to bioabsorbable polymers (implantation and local accumulation of degradation products that also may generate ROS) [21-24]. On the other hand, attention has recently turned towards the potential use of natural antioxidants as stabilizers for polymers [25] because the risk of formation of harmful by-products is much lower with their use than with other compounds. Apart from tissue engineering, delivery of bioactive molecules is one of the most promising applications of electrospun fibers due to their large specific surface area, which gives them a high loading capacity [26-27]. During wound healing, the wound dressings [28-30] are designed to be in contact with the wound and may be interacting with the extracellular matrix. Highly biocompatible polymeric fibers are particularly desirable since they would not cause a negative reaction from the body upon application. In the current paper poly(ε-caprolactone) (PCL) was used as a model bio-polyester for electrospinning [31] while a 5% weight content of several antioxidant compounds were added to the polymer solution for the fabrication of polymer-drug fibers. The scaffolds, with differing morphologies, were then thoroughly characterized by microscopy imaging, encapsulation efficiency measurements and mechanical testing. 4 ACS Paragon Plus Environment

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Based on their activity, antioxidants can be categorized as enzymatic and non-enzymatic [32]. The former act by breaking down and removing free radicals after converting oxidative products to hydrogen peroxide (H2O2) and then to water in a multi-step process. Examples of enzymatic antioxidants are catalase (CAT), glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and peroxiredoxin I-IV. On the contrary, nonenzymatic antioxidants work by interrupting free radical chain reactions and are the subject of this article. The main antioxidants in this category are vitamins (vitamin E [33], vitamin C and vitamin A), bioflavonoids (a group of natural benzo-γ-pyran derivatives widely distributed in fruits and vegetables including flavonols, flavones, flavonones, anthocyanidin and isoflavones), carotenoids (lycopene and β-carotene are the most prominent among the other 600 different compounds), hydroxycinnamates (ferulic acid, caffeic acid, sinapic acid and ρ-coumaric acid), other natural antioxidants (theaflavin, theaflavin-3-gallate, allicin, piperine and curcumin), other polyphenols (tannic acid, polydatin, resveratrol), physiological antioxidants (uric acid in plasma and glutathione (GSH)) or synthetic antioxidants such as cinnamic acid derivatives, melatonin or selegiline. In this study, N-acetyl-L-cysteine, allicin, crocin, curcumin, piperine, polydatin, quercetin and rutin were selected from the myriad antioxidants

mentioned

above and their thermal properties and miscibility with poly(ε-caprolactone) were studied by means of differential scanning calorimetry and thermogravimetric analysis prior to being incorporated into the electrospun mats. Furthermore, as a proof of concept, human adipose-derived mesenchymal stem cells (ADMSCs) were cultured in the antioxidant loaded PCL scaffolds and observed by means of fluorescence microscope. MSCs are multipotent cells, meaning that they are capable of differentiation into a limited number of distinct lineages depending on the nature of the environmental signals that they receive, and bring several advantages for clinical use [34]. 5 ACS Paragon Plus Environment


2. Experimental 2.1. Materials Poly(ε-caprolactone) (PCL) Purasorb PC12, with an weight average molecular weight (Mw) of 128.7 Kg mol-1 and a dispersity (D) of 1.76, was provided by Corbion. Allicin (mixture of diallyl disulfide and diallyl trisulfide) and Rutin were supplied by Cymit Quimica, while the rest of antioxidants were purchased from Sigma Aldrich: Curcumin (#C1386), Crocin (#17304), N-Acetyl-L-cysteine (#A7250), Piperine (#P49007) and Polydatin (#15721). Regarding the solvents used, formic acid (> 98% assay) was supplied by Sigma Aldrich (#33015), tetrahydrofuran and methanol were purchased from Labbox and 2,2,2-trifluoroethanol (> 99% assay), used in the electrospinning tests, was obtained from Alfa Aesar. For the in vitro assays, Human Adipose-derived Mesenchymal Stem Cells (AD-MSCs), the Mesenchymal Stem Cell Basal Medium, a Mesenchymal Stem Cell Growth Kit, Dulbecco’s Phosphate Buffered Saline (D-PBS), Trypsin-EDTA for Primary Cells, Trypsin Neutralizing Solution, Penicillin-Streptomycin-Amphotericin B solution and Phenol Red were obtained from the American Type Culture Collection (ATCC). Paraformaldehyde, Rhodamin-phalloidin and DAPI used in cell staining were purchased from Thermo Fisher Scientific. Triton X-100 was used as received from Sigma-Aldrich and both Tween® 20 and Fluoromount™ Aqueous Mounting Medium were obtained from Sigma Life Science.


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2.2. Methods Electrospinning To begin, the antioxidant was dissolved in tetrahydrofuran (THF), trifluoroethanol (TFE) or in solvent mixes of TFE with formic acid (FA) or methanol (MET) (added to increase the conductivity) to prepare the PCL-antioxidant scaffolds. Then, the appropriate amount of PCL (4.0 g in 15 mL of solvent) was added to obtain a 5 % w/w drug/polymer. After the preparation of the polymer solution, the mixtures were subsequently vortexed to ensure proper mixing. The 5 % w/w concentration was chosen for this study because at higher antioxidant concentrations the mechanical performance of the mats greatly worsened and they were difficult to handle. Electrospinning was performed at room temperature (21±2ºC) with controlled humidity (~ 40%) in a Nanospinner Ne-200 (Inovenso) system. The tunable high-voltage power supply was connected to the tip of the needle (0.5 mm in diameter, positive lead) and attached to the collector (negative lead) with an alligator clip. The needle-to-collector distance was 20 cm and the polymer solutions were sprayed using a syringe pump at an adjusted flow rate. Polymer mats were spun directly onto a plate-shaped collector (aluminium) for 45 min to achieve rectangular samples (6 x 5 cm) with a thickness of 100-150 μm. The electrospun mats were examined using Field Emission Scanning Electron Microscopy (FE-SEM). The PCL-antioxidant scaffolds were sputter-coated with a thin layer of gold (~ 15 nm) in a Emitech K550X and observed in a Hitachi SEM (Hitachi S4800N). The voltage used was 10-15 kV and the working distance was 7.0-9.0 mm with a magnification of 100×, 1000x, 5000x and 20000x. To assess the average diameter, over



50 individual fibers were measured with Image J software [35] using SEM images from at least 5 different sections of each sample. Characterization The mechanical properties were determined by tensile tests with an Instron 5565 testing machine at a crosshead displacement rate of 10 mm min-1. These tests were performed at room temperature (21 ± 2ºC) following ISO 527-3/1995. The specimens had the following dimensions: distance between marks = 50 mm and width =10 mm; and were cut from 100-150 µm thick polymer mats. The mechanical properties reported correspond to average values of at least 5 determinations. The loading capacity of the scaffolds was calculated by Ultraviolet-Visible spectroscopy. The spectra of the antioxidants were recorded using Lambda 265 from PerkinElmer. The bands in the 250-450 nm (see Supporting Information S.1.) were employed to build a standard curve with known concentrations of the antioxidant. The measurements were carried out at concentrations under 25 ppm, using calibration curves in THF (for allicin, piperine, polydatin and quercetin), chloroform/methanol (10:1) (for curcumin and rutin) and chloroform/formic acid (10:1) (for crocin). It was not possible to obtain a standard curve for the acetyl cysteine because the different solvents (that dissolve both the polymer and the drug) give signals in the 200-250 nm range, which are within the absorption band of the reagent. The thermal properties of the antioxidants were studied on a DSC Q200 (TA Instruments). Samples of 5-9 mg were heated at 20 ºC min-1 from -80ºC to the end of the melting peak. After this first scan, the samples were quenched in the DSC and a second scan was made from -80ºC at the same rate. For the miscibility studies, DSC analysis was conducted on


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~ 150 μm films that were prepared by solvent casting with TFE (THF in the case of allicin) as solvent. The thermal degradation of the drugs was studied under nitrogen by means of thermogravimetric analysis (TGA) into a TGA model Q50-0545 (TA Instruments). Samples of 10-15 mg were heated from room temperature to 500 °C at a heating rate (β) of 20 °C min-1, with the heat flow, sample temperature, residual sample weight and its time derivative being continuously recorded. Cell culture and cell-staining Adipose-derived mesenchymal stem cells were kept on polystyrene culture T75 flasks in complete cell growth medium (Mesenchymal Stem Cell Basal Medium with 5 ng/mL rh FGF basic, 5 ng/mL rh FGF acidic, 5 ng/mL rh EGF, 2% FBS and 2.4 nM L-Alanyl-LGlutamine) and grown at 37°C in a 5% CO2 humidified incubator. Circle scaffolds, previously sterilized with ultraviolet light, were placed in 48-well plates and attached to the bottom of the plate with silicone rings to prevent them from floating in the medium. Once cells reached 80 % confluence, they were subcultured and seeded into the different scaffolds at a density of 10 000 cells per scaffold using 75 μL drops. Two hours after plating, the complete medium was added to reach a total volume of 400 μL. The medium was changed one day after seeding. Cells were fixed with 4 % paraformaldehyde for 5 minutes at room temperature three days after seeding. The scaffolds were then washed twice with D-PBS and permeabilized with 0.5 % Triton X-100 in PBS for 10 minutes. After removing Triton X-100, the samples were washed twice with PBS for 5 minutes and incubated in 1% BSA in PBS for 30 minutes to block unspecific binding. Later, the scaffolds were incubated with 1 % 9 ACS Paragon Plus Environment


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Rhodamine-Phalloidin in BSA in a dark room for 15 minutes. Following this incubation, the samples were washed three times with 0.1 % PBS-Tween for 10 minutes. After washing, the scaffolds were again incubated with 300μM DAPI in a dark room for 2-3 minutes and washed three times with PBS. Finally, the samples were mounted with Fluoromount ™ Aqueous Mounting Medium and the following day analyzed in a fluorescence microscope Nikon Eclipse Ts2-FL. The images were captured using a Nikon DS-Qi2 camera.

3. Results and Discussion 3.1. Antioxidant characterization and miscibility with poly(ε-caprolactone) (PCL) Table 1. Properties of the antioxidants used in this paper

Water

DSC b

TGA

Solubility a Antioxidant

Miscibility with poly(εcaprolactone)

Predicted by ALOGPS

Tm

∆Hm

Tg ºC

Tonset

Tpeak

ºC

J g-1

∆CP

ºC

ºC

T50% of weight loss

ºC

(J g-1) ºC-1)

(mg/mL)

Acetyl Cysteine

Inmiscible

5.09

116.4

189.7

Allicin

Miscible

6.13

< 25

Crocin

Partially miscible

0.567

Curcumin

Miscible

0.006

110

222

316

-

25

162

173

191.5

50.5

182

330

335

179.5

134.4

193

398

400


2.6 (0.79)

66.7 (0.60)

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Piperine

Miscible

0.149

133.6

106.7

15.1 (0.45)

193

347

346

Polydatin

Miscible

0.766

226.6

164.1

106.6 (0.71)

246

318

399

Quercetin

Miscible

0.261

327.7

140.4

200

357

500

Rutin

Inmiscible

3.54

182.0

270.0

45 / 200

277

465

a Aqueous

solubility data are predicted properties obtained from ALOGPS (Virtual Computational

Chemistry Laboratory) [36]. Acetyl cysteine, piperine, quercetin and rutin present water solubility experimental results of 277 mg/mL and 40, 60 and 125 ppm, respectively. b The Tgs were obtained from the second scan but in the cases of allicin, crocin, quercetin and rutin, they were already degraded in the first.

Figure 1. Chemical structure (from top to bottom, see the right curves for the names) (a) and first scans of Differential Scanning Calorimetry (DSC) (b) and Derivative Thermogravimetric Analysis (DTG) (c) for the different antioxidants in this study



In this work the antioxidant molecules, whose chemical formulas are given in Figure 1a, were studied; from top to bottom: N-acetyl-L-cysteine [37], allicin [38-40], crocin [4144], curcumin [45-48], piperine [49-52], polydatin [53-54], quercetin and rutin [55-59]. These compounds are a representative sample of non-enzymatic antioxidants with antiinflammatory properties (see Supporting Information S.1.). Table 1 summarizes the thermal properties obtained from the DSC and TGA analysis, while Figures 1b and 1c show their curves. Miscibility with PCL Depending on the interactions between the functional groups of these small molecules and the poly(ε-caprolactone) chains, the antioxidants could mix homogeneously or not with the polymer. In the cases of curcumin, piperine and polydatin (Figure 2), they form miscible composites with the polyester. Thus, in the second scans a single Tg were observed in all three cases, demonstrating the presence of a single amorphous phase. As an example, the Tg of PCL (at -64ºC for the pure compound) rose to -53, -33 and -3 ºC when 5%, 25% or 50% of curcumin (Tg = 67ºC) was added. Moreover, in the first scans the melting temperature of the antioxidants shifted to lower temperatures at contents of 50 % or 25 %, with lower melting enthalpies associated to both PCL and the drug (see the dotted curves). This also occurs for quercetin although a second scan is not shown in Figure 3 since the flavonoid partially degraded in the first heat treatment. However, both quercetin and allicin also appear to be miscible with PCL. The bio-composite films were homogeneous and took a color typical of biological molecules. In the case of allicin, this sulfur compound did not melt in any of the mixtures but the PCL peak underwent changes to its melting peak and enthalpy (Tm = 63ºC and ∆Hm = 89 J/g for the 5% allicin composite vs. Tm = 55ºC and ∆Hm = 63 J/g for the 50% allicin composite).


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On the contrary, acetyl cysteine, crocin and rutin were not miscible with PCL although crocin can be classified as partially miscible because the melting peak of the saffron carotenoid fell and moved towards lower temperatures when PCL was added. However, the films were not homogeneous and even though they had a reddish-orange color, some pigment aggregates were observed. Figure S.3 shows the DSC scans of 50-50 blends of acetyl cysteine and rutin with PCL and as can be seen, the antioxidant melting peaks appear at the same temperature as those of the pure molecules. Moreover, for the acetyl cysteine containing sample a double Tg behavior (at ~ -60 and 0ºC) was seen, a clear indication of the presence of two phases. These miscibility aspects could play a critical role not only in the fabrication of suitable PCL-antioxidant fibers by electrospinning, but also in producing bio-composites with suitable mechanical properties and controlled drug release profiles.

Figure 2. Miscibility study: DSC scans of different bio-composites of Curcumin, Piperine and Polydatin with PCL. First scans are in dashed line and second scans in solid line.



Figure 3. Miscibility study: First DSC scans of different bio-composites of Allicin and Quercetin. 3.2. Electrospinning and mechanical characterization The electrospinning process was first optimized for PCL Purasorb PC12 before incorporating antioxidants into the polymer matrix. Figure 4 and Table S.3 in the supporting information, summarize the electrospinning conditions and mechanical properties obtained for scaffolds of ~ 200 nm nanofibers (formic acid), ~ 200 nm / ~ 1 μm fiber mix (TFE-methanol 14:1 v:v) and ~ 2 μm microfibers (TFE). Different morphologies affected the mechanical performance of PCL so that the nanofiber mats presented a linear elastic behavior until break (at 27 %) with Young´s modulus of 17.2 MPa, while those of 2 μm fibers showed a change in the slope of the stress-strain curve at around 5 % and then broke progressively by tearing. The elongation at break of the latter (1.3 MPa at 62%) was approximately half of the nanofiber mats, in which fiber orientation is probably boosted, but the elastic modulus was a little higher (E = 27.2 MPa). Mixing nano-fibers with microfibers led to a more complex structure that had a lower 14 ACS Paragon Plus Environment

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mechanical response at the beginning of the curve (E = 11.8 MPa) but showed an intermediate behavior.

Figure 4. Typical stress-strain curves of PCL electrospun mats with different fiber morphologies. The encapsulation of antioxidants using electrospun polymer fibers is not an entirely new phenomenon, as curcumin has already been incorporated into polylactide [60], PCLtragacanth gum [61-62] and PVA fibers [63], as has piperine in gelatin fibers [64-65] and quercetin in poly(lactide-co-glycolide)-PCL nanofibers [66]. However, such cases are not particularly common and are viewed only as drug delivery systems and the mechanical properties are ignored. In this work PCL fibers of different fiber diameter loaded with several antioxidants were successfully prepared. Figure 5 shows a macroscopic view of them in which the red or yellow tinge of crocin (A3), curcumin (B1 and B2), quercetin (C2) and rutin (C3) can be appreciated.



Figure 5. Samples of PCL-antioxidant cut from mats fabricated using TFE-MET as solvent mix (THF in the case of PCL-allicin): (A1) Acetyl cysteine, (A2) Allicin, (A3) Crocin, (B1) Curcumin, (B2) Curcumin-piperine, (B3) Piperine, (C1) Polydatin, (C2) Quercetin, (C3) Rutin.

Figure 6. SEM images at 1000x of PCL fibers with allicin, piperine, polydatin, acetyl cysteine, crocin and rutin. 16 ACS Paragon Plus Environment

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The processing by electrospinning of the PCL-miscible molecules worked very well in steady state and was much easier than when the solutions of PCL and the PCL-immiscible antioxidants (acetyl cysteine, crocin and rutin) were employed. Consequently, in the latter cases the scaffolds were more heterogeneous with regions of pure PCL fibers and others rich in antioxidant, leading to a larger variance in the measurements of the mechanical properties (see Table S.4 in supporting information). So, PCL-Crocin scaffolds exhibited red aggregates of crocin (not incorporated into the polymer fibers) and balloon-like fibers were observed in the SEM images of PCL-rutin (see Figure 6). As has been mentioned above, the loading capacity of the PCL-acetyl cysteine mats could not be obtained because the different solvents that dissolve both the polymer and the drug give signals within the range of the absorption band of acetyl cysteine.

Table 2. Electrospinning data and mechanical properties of the miscible PCL-antioxidant systems.



Electrospinning conditions

Mechanical properties

Sample*

Solvent mix

Allicin 5%

% Antioxid.

Piperine 1%

Tensile Strength at 15%

Ultimate Tensile Strength

Elongation

(MPa)

(MPa)

(MPa)

(%)

9.9 ± 0.6

0.6 ± 0.1

0.7 ± 0.1

27.3 ± 7

5.2 ± 0.9

0.6 ± 0.1

0.9 ± 0.1

30.9 ± 6

5.5 ± 1.1

1.1 ± 0.2

1.7 ± 0.4

27.7 ± 6

6.1 ± 1.0

0.8 ± 0.1

1.0 ± 0.2

25.7 ± 6

Fiber morphology

2.9

~ 5 μm

14 KV

(14:1)

1 mL h-1

~ 200nm 5.5

17 KV

TFE-FA

1 mL/h

(11:4)

19 KV

TFEMET

1 mL h-1

~ 1 μm

5.7

Curcumin 4%

Young´s Modulus (E)

2 mL h-1 THF*

TFEMET Curcumin 5%

Flow rate and Voltage

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(14:1)

~ 200nm

~ 200nm 3.9 / 1.4

16 KV

TFE-FA

1 mL h-1

(11:4)

18 KV

~ 1 μm

3.5 / 1.3

~ 200 nm

9.3 ± 1.4

1.5 ± 0.1

3.1 ± 0.4

37.9 ± 3

5.1

~ 2 μm

14.2 ± 2.7

0.9 ± 0.1

1.3 ± 0.1

91.8 ± 32

14.4 ± 1.1

1.3 ± 0.1

2.1 ± 0.4

49.5 ± 14

14.0 ± 1.8

1.6 ± 0.2

2.9 ± 0.7

32.2 ± 9

1 mL h-1 TFE 17 KV

Piperine 5%

TFEMET (14:1)

1 mL h-1

~ 200nm 4.7

14 KV

TFE-FA

1 mL h-1

(11:4)

19 KV

~ 1 μm

3.7

~ 200nm


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TFEMET Polydatin 5%

(14:1)

1 mL h-1

~ 200nm 4.8

17 KV

TFE-FA

1 mL h-1

(11:4)

19 KV

6.0 ± 1.1

0.7 ± 0.1

1.0 ± 0.1

33.1 ± 6

~ 1 μm

3.9

~ 200 nm

5.2 ± 1.0

1.1 ± 0.1

2.0 ± 0.2

37.8 ± 4

4.9

~ 2 μm

5.8 ± 0.4

0.6 ± 0.1

0.9 ± 0.1

55.6 ± 5

3.3 ± 0.6

0.4 ± 0.1

0.6 ± 0.1

33.5 ± 4

6.5 ± 0.7

0.5 ± 0.1

0.8 ± 0.1

37.3 ± 8

5.3 ± 0.7

0.8 ± 0.1

1.9 ± 0.3

49.4 ± 7

2 mL h-1 THF

*

15 KV

~ 200nm 1 mL h-1 TFE

5.2

~ 1 μm

15 KV ~ 10 μm Quercetin 5% TFEMET (14:1)

1 mL h-1

~ 200nm 5.0

18 KV

TFE-FA

1 mL h-1

(11:4)

20 KV

~ 1 μm

5.3

~ 200nm

* With allicin and quercetin in TFH, 3.5 g and 4.5 g of PCL were added instead of 4.0 g.

Table 2 summarizes the solvent mix used, the flow rate, voltage, percentage of antioxidant encapsulated, fiber morphology and mechanical properties of the PCL- antioxidant electrospun scaffolds that are miscible and as we have demonstrated are of more interest for practical purposes. In the case of allicin, this sulfur compound only dissolved in THF and ~ 5 μm fibers were obtained encapsulating 2.9 % (when initially 5 % was added). These mats displayed a mechanical curve similar to PCL microfiber scaffolds with lower values in the stress related properties (E = 9.9 MPa and tensile strength at break of 0.7 MPa). The mechanical fracture of these samples, along with the other microfiber mats (~ 2 μm fiber diameter) containing piperine and quercetin, was by tearing after a stage of constant stress with respect to strain. Conversely, TFE-MET (14:1 v:v) was successfully



employed to produce nano-microstructured PCL scaffolds (with fibers of ~ 200 nm and ~ 1 μm) loaded with curcumin, curcumin-piperine, piperine, polydatin and quercetin, whereas nanofibers ~ 200 nm diameter were fabricated when TFE-FA (11:4 v:v) was used as solvent mix. The TFE-MET samples exhibited Young´s Modulus in the range of 5-7 MPa, tensile strength at 15 % of strain of 0.6-0-8 MPa, ultimate tensile strength of 0.8-1.0 MPa and deformations at break of 25-40 %. On the other hand, the nanofiber scaffolds of the antioxidants mentioned above presented a similar elastic modulus (5-9 MPa) but higher values of tensile strength at 15 % and at break (0.8-1.5 MPa and 1.7-3.1 MPa). In all cases, ~ 5 % weight content of antioxidant (the desired concentration) was encapsulated, with the exception of piperine and polydatin using formic acid (~ 4 %). A good example of the different fiber morphologies is illustrated in Figure 7, this shows SEM images and the corresponding mechanical curve of several fiber structures of PCL loaded with ~ 5 % quercetin. Those mats with a fiber distribution of the same diameter (microfibers produced using THF or nanofibers fabricated with TFE-FA) exhibited higher strains at break than the heterogeneous mix of micro- and nanofibers. However, the latter also displayed interesting mechanical behavior for soft tissue engineering applications, for example, the presence of fibers with different diameters may be a suitable solution to the drawbacks of the scaffolds that are formed only by micro- or nanofibers. Nowadays, there are two main types of electrospun scaffolds for tissue engineering: microfibrous and nanofibrous. Microfibrous scaffolds provide topography and orientation; and promote 3D-space for cell growth and cell penetration. On the other hand, nanofibrous structures improve cell adhesion through their high surface area, promoting proliferation and differentiation. However, they only have 2D-space for cell growth and low porosity [67], one of the main characteristics of the synthetic scaffolds, providing the diffusion and cellular infiltration of nutrients and metabolites [68]. However, native 20 ACS Paragon Plus Environment

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extracellular matrix (ECM) of soft tissues consists of different shapes and sizes fibers such as collagen (1-20 μm, depending on the type of tissue) [69-70], elastin (0.1-0.2 μm), fibronectin (10 nm-1 μm), and laminin (5-10 nm), which have both structural and adhesive functions [71]. It was therefore concluded that the electrospinning conditions of our study [72] have an advantage over previously known methods. As they are prepared using a simple process (one that does not require separate process steps for each diameter) a mixture of electrospun fibers of submicron and micron diameters is prepared in one step, and these diameters more closely mimick the structure of the ECM in soft tissue.

Figure 7. Typical stress-strain curves and SEM images at 1000x of PCL-quercetin mats with different fiber distributions. (a) THF; (b); TFE (c) TFE-MET (14:1 v:v) and (d) TFEFA (11:4 v:v), used as solvent mix. It has been demonstrated that the combination of piperine with curcumin possesses a synergistic effect, enhancing the antioxidant activity of curcumin by doubling its absorption [73-74]. In this study we were able to successfully incorporate 4 % of curcumin and 1% of piperine to PCL fibers. Figure 8 shows typical stress strain curves of 21 ACS Paragon Plus Environment


the PCL-curcumin and the PCL-curcumin-piperine scaffolds and SEM images at 5000x and 20000x magnification of those containing both biomolecules. As can be observed, the curcumin-piperine samples formed by a micro-nanofiber mix (c´) or only by nanofibers (d´) possessed a higher elastic modulus, ultimate tensile strengths and elongations at break than the PCL-curcumin mats. This may be due to the presence of piperine, since the PCL-piperine fibers displayed the highest Young´s modulus (~ 14 MPa) and had greater values for strength than the rest of bio-composite scaffolds.

Figure 8. SEM images and mechanical performance of PCL-Curcumin-piperine electrospun scaffolds. 3.3. Cell studies In a preliminary study Human Adipose-derived Mesenchymal Stem Cells (AD-MSCs) were cultured in different electrospun scaffolds (those obtained with TFE-MET as solvent mix) to assess their cytocompatibility. MSCs were selected because they are considered 22 ACS Paragon Plus Environment

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one of the most promising tools in regenerative medicine due to their easy extraction and because they avoid the ethical issues surrounding embryonic stem cell research [75-77].

Figure 9. Fluorescence microscope images at 20-40x on Day 3 after AD-MSCs seeding in A) 2D plastic flask and B) PCL scaffold; C) PCL-polydatin scaffold and D) PCL-crocin scaffold. The scaffolds are those fabricated using TFE-MET as solvent mix. Blue dye is DAPI (specific dye to nuclear compartment due to its ability to bind to AT-rich areas of DNA) while red dye is Rhodamine-Phalloidin (dye that selectively binds to F-actin and therefore shows the actin filaments present within the cell cytoplasm). The electrospun scaffolds showed cytocompatible features; the cells were able to attach to the fibers of PCL in the presence or absence of antioxidants, as well as able to survive and grow in the different types of mats. Three days after the seeding, AD-MSCs were present in the pure PCL scaffold (Figure 9B) and in those loaded with antioxidants, such as polydatin or crocin (Figure 9C and D). As can be seen in those figures, the morphology 23 ACS Paragon Plus Environment


of the cells was similar to those found when grown in a 2D plastic flask (Figure 9A). However, due to the 3D-structure of these scaffolds, cells could be observed at different levels of detail according to their spatial distribution, filling the gaps between the nanoand microfibers that form the scaffold. In comparison to the 2D monolayer culture, the presence of pores of diverse sizes of this culture system allows the diffusion of nutrients and molecules needed for the correct growth and development of cells. Moreover, it would facilitate the possible future vascularization of the scaffold. Cells would also be able to replace the bio-absorbable scaffold with a new extracellular matrix, leading to the creation of a new tissue.


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4. Conclusions The fabrication of electrospun scaffolds formed by polyester fibers filled with antioxidants has the potential to become a practical therapeutic approach for wound healing or tissue repair after injury. However, the miscibility of the polymer and the different biological molecules must be studied in greater depth and work only with those that are miscible, because high quality fiber mats with suitable mechanical properties are only achieved in these cases. On the other hand, the antioxidant dose must be better adjusted. In this work only 5 % weight content of antioxidant was added but the drug release profiles have not yet been studied (this should be carried out in a non-oxidizing medium). From our own experience 10% seems to be the maximum amount of antioxidant that can be incorporated without damaging the structure of the fiber. In this paper, the electrospun scaffolds of different fiber morphologies (micro-, nano- or micro-nanostructured) exhibited very interesting mechanical behavior and good cytocompatibility with Human Adipose-derived Mesenchymal Stem Cells. Nevertheless, cell survival in H2O2 rich media will be studied in the future (with different % weight content of antioxidant), along with more complex research into other cellular models (i.e. inflammatory response of monocytes/macrophages, stimulating them to produce ROS) before considering any further in vivo evaluation with a polyester that degrades faster than poly(ε-caprolactone) (only used in the current paper as a model polyester for electrospinning).



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TOC – Graphical Abstract



TOC 254x190mm (300 x 300 DPI)

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Electrospun Fibers of Polyester, with Both Nano- and Micron

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