Epigallocatechin gallate loaded electrospun silk fibroin scaffold with anti-angiogenic properties for corneal tissue engineering

Journal Pre-proof Epigallocatechin gallate loaded electrospun silk fibroin scaffold with anti-angiogenic properties for corneal tissue engineering Narges Forouzideh, Samad Nadri, Ali Fattahi, Elaheh Dalir Abdolahinia, Mina Habibizadeh, Kobra Rostamizadeh, Alireza Baradaran-Rafii, Haleh Bakhshandeh PII:

S1773-2247(19)31389-9

DOI:

https://doi.org/10.1016/j.jddst.2020.101498

Reference:

JDDST 101498

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 13 September 2019 Revised Date:

30 December 2019

Accepted Date: 2 January 2020

Please cite this article as: N. Forouzideh, S. Nadri, A. Fattahi, E.D. Abdolahinia, M. Habibizadeh, K. Rostamizadeh, A. Baradaran-Rafii, H. Bakhshandeh, Epigallocatechin gallate loaded electrospun silk fibroin scaffold with anti-angiogenic properties for corneal tissue engineering, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/j.jddst.2020.101498. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

AUTHOR STATEMENT

Narges Forouzideh: Conceptualization, Investigation, Writing; Samad Nadri: Supervision; Ali Fattahi: Supervision; Elaheh Dalir Abdolahinia: Investigation; Mina Habibizadeh: Investigation; Kobra Rostamizadeh: Supervision, Project administration; Alireza BaradaranRafii: Methodology; Haleh Bakhshandeh: Supervision, Conceptualization

1

Epigallocatechin Gallate Loaded Electrospun Silk Fibroin

2

Scaffold with Anti-angiogenic Properties for Corneal Tissue

3

Engineering

4

5

Narges Forouzideh1, Samad Nadri2*, Ali Fattahi3,4, Elaheh Dalir Abdolahinia5.6, Mina

6

Habibizadeh7, Kobra Rostamizadeh*7,8,9, Alireza Baradaran-Rafii10, Haleh Bakhshandeh11

7

1

8

Department of Pharmaceutical Nanotechnology, School of Pharmacy, Zanjan University of Medical

Sciences, Zanjan, Iran

9

2

10

Department of Medical, Nanotechnology, School of Medicine, Zanjan University of Medical

Sciences, Zanjan, Iran

11

3

12

Pharmaceutical Sciences Research Center, Health Institute, School of Pharmacy, Kermanshah

University of Medical Sciences, Kermanshah, 6734667149, Iran

13

4

School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, USA

14

5

Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences,

15

Zanjan, Iran

16

6

17

Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University

of Medical Sciences, Tabriz, Iran

18

7

19

Department of Pharmaceutical Biomaterials, School of Pharmacy, Zanjan University of Medical

Sciences, Zanjan, Iran

20

8

21

Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical Sciences, Zanjan,

Iran

22

9

23

Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA

02115, USA

24 1

10

Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

1

11

Nanobiotechnology Department, New Technologies Research Group, Pasteur Institute of Iran,

2

Tehran, Iran

3 4

*Corresponding Authors:

5

Samad Nadri

6

Associate Professor

7

[email protected] (S. Nadri)

8

Department of Medical Nanotechnology, Zanjan University of Medical Sciences, Zanjan, Iran

9

Phone No: +98-24-3314000 ext. 213 Fax No: +98-24-33449553

10

Kobra Rostamizadeh

11

Full Professor

12

[email protected] (K.Rostamizadeh)

13

Department of Pharmaceutical Biomaterials, Zanjan University of Medical Sciences, Zanjan, P.O.

14

Box 45195-1338, Iran

15

Phone: No +98-24- 33473635 ext. 320 Fax No: +98-24-33473639

16 17 18 19

2

Abstract:

1

Avascularity is the first requirement for corneal tissue engineering. The goal of this study was

2

to develop an epigallocatechin gallate (EGCG)-loaded silk fibroin-based scaffold with anti-

3

angiogenesis properties for corneal tissue engineering. Silk nanofibers were prepared by

4

electrospinning, and treated with methanol to enhance water insolubility. Scanning electron

5

microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, tensile testing,

6

differential scanning calorimetry, contact angle testing, and porosimetry were used to

7

characterize the scaffold. The suitability of the scaffold as support for limbal cells was

8

demonstrated by scanning electron microscopy. In situ drug loading of EGCG into the

9

scaffold successfully produced a homogeneous structure of nanofibers and the loaded

10

nanofibers released the drug over 144 hr in a controlled-manner. The appropriate dose of the

11

anti-angiogenic compound to be loaded in the electrospun nanofibers was determined by

12

MTT assay and using human umbilical vein endothelial cells (HUVEC).

The findings

13

showed a dose-dependent inhibition of cell proliferation with the IC50 values of 43.06 ± 2.51

14

µM and 47.1 ± 3.46 µM after 24 and 72 hr treatment, respectively. The MTT results also

15

revealed that the prepared fibrous scaffolds containing EGCG promoted the inhibition of

16

HUVEC during in vitro incubation. It is anticipated that the EGCG-loaded scaffold has the

17

potential for use as a delivery system for corneal tissue engineering.

18

Keywords: Tissue engineering, Silk, Cornea, EGCG, Drug delivery systems, Nanofibers,

19

Electrospinning

20

3

List of Abbreviations:

1

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor

MTT

2

Differential scanning calorimetry

DSC

3

Dimethyl sulfoxide

DMSO

4

Dulbecco's modified Eagle medium

DMEM

5

Epigallocatechin gallate

EGCG

6

Fetal bovine serum

FBS

7

Fourier transform infrared spectroscopy

FTIR

8

Human umbilical vein endothelial cells

HUVEC

9

Phosphate buffer saline

PBS

10

Roswell Park Memorial Institute-1640

RPMI-1640

11

Scanning electron microscopy

SEM

12

Silk fibroin

SF

13

Silk fibroin nanofibers

SFNF

14

Thermogravimetric analysis

TGA

15

Tissue culture polystyrene

TCPS

16

X-ray diffractometry

XRD

17

4

1. Introduction

1

Corneal disease is a leading cause of blindness that affects more than 10 million people

2

worldwide. Allogenic and autogenic cornea transplantation constantly is the best clinical

3

option for patients. Although it has a high rate of success, the shortage of qualified donors in

4

many countries does not equal all demands; thus, there remains a special need for alternative

5

therapies [1].

6

Amniotic membrane is one of the best new substitutes for corneal grafts and has attracted

7

much attention. However, this approach has inherent problems, including the difficulty of its

8

handling, heterogeneity related to donor, the preservation process, high cost, and the

9

possibility of disease transmission [2]. Recently, tissue engineering has opened new horizons

10

to overcome these types of challenges. There is a growing interest in tissue engineering for

11

application in corneal disease as well [3]. Corneal tissue engineering has been used to design

12

a scaffold with promising structural, mechanical, and optical properties that is similar to the

13

native cornea. The engineered tissue is composed of a scaffold which is capable of hosting

14

different cells and gradually degrades to let the cells proliferate on it, migrate, and

15

differentiate to produce a new tissue.

16

Nanofibrous scaffolds mimic the native tissue due to the resemblance to extracellular matrix

17

in terms of high surface-area-to-volume ratio, processability, and high-porosity and offer a

18

favorable substrate for cell adhesion, growth and differentiation [4]. Electrospinning is a

19

widely distributed technique for producing nanofibers. Several methods have been developed

20

for electrospinning in the last two decades. Coaxial electrospinning, modified coaxial

21

electrospinning, tri-axial electrospinning, side-by-side electrospinning, and multi-fluid

22

electrospinning are new methods of this technique but single-fluid electrospinning is the

23

easiest and the most extensively used one [5-9]. A variety of natural and organic polymers,

24

5

e.g., collagen, gelatin, polycaprolactone, polyvinyl alcohol, and silk have been used to

1

prepare nanofibrous scaffolds by electrospinning [10].

2

Silk fibroin (SF), a natural product, has superior properties in comparison with other

3

polymers in terms of biodegradability and biocompatibility [11]. Also, silk owing to some

4

unique optical and mechanical properties as well as its versatile processability, is considered

5

to have high potential as a cornea scaffold in engineered tissue devices [12]. Kim et al.

6

showed the suitability of silk as a scaffold for bioengineering of neo-corneas in terms of

7

morphological, structural characteristics and in vitro biological parameters [13]. Silk fibers

8

have shown a suitable niche condition that can compete with the amniotic membrane for

9

limbal cells [12]. However, according to the earlier studies, silk scaffolds have shown the

10

induction of endothelial cell migration and the formation of a capillary network in vivo [14].

11

Moreover, SF induces neovascularization in engineered corneal tissue [15]. It is clear that silk

12

as a scaffold for corneal tissue engineering must prevent vascular proliferation and provide

13

transparency equivalent to native cornea tissue. Thus, there is a need to boost its anti-

14

angiogenic properties.

15

There are a variety of molecules with anti-angiogenic activity that have emerged including

16

epigallocatechin

angiostatin,

17

plasminogen fragment, and endostatin [16]. It has been found that EGCG, as the abundant

18

catechin in green tea, can play a significant role in cancer-preventive and therapeutic

19

activities [17]. Gu et al. have shown that EGCG, prohibits the activation of angiogenesis-

20

related factors like HIF-1α, NFκB, as well as vascular endothelial growth factors expression,

21

which may explain for its anti-angiogenic and anti-proliferative properties in breast tumors

22

[14]. EGCG is a potential bioactive candidate which has been studied in some new clinical

23

trials [18].

24

gallate

(EGCG),

pigment

6

epithelium-derived

factor,

This work aimed to prepare optimized silk fibroin nanofibers (SFNF) to serve as a scaffold

1

with anti-angiogenic properties for engineered corneal tissue. To improve the anti-angiogenic

2

properties of SF, EGCG was also loaded as a bioactive agent into the scaffold.

3

2. Materials and Methods

4

2.1. Materials

5

Bombyx mori cocoons were obtained from the Guilan silkworm breeding development center,

6

Rasht, Iran. Pure formic acid was from Merck (USA). Phosphate buffer saline (PBS) tablets

7

and penicillin/streptomycin were from Invitrogen (USA). Dulbecco's modified Eagle medium

8

(DMEM) was supplied by the Bioidea Company (Iran). Fetal bovine serum (FBS) was

9

purchased from Gibco (USA). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

10

bromidefor) was obtained from Carl Roth (Germany). Roswell Park Memorial Institute

11

(RPMI)-1640 was purchased from Inoclon (Iran). The other solvents and reagents used were

12

of high purity.

13

2.2. Preparation of pure regenerated silk fibroin

14

To prepare pure SF, sericin protein, an impurity in the cocoons, was extracted by the

15

degumming method. Briefly, after manually cleaning of pierced Bombyx mori cocoons, they

16

were degummed with 0.02 M sodium carbonate at 100°C for 30 minutes. The resultant SF

17

was washed with warm water three times to remove any remaining sericin and dried at room

18

temperature. A ternary mixed solvent system of CaCl2/CH3CH2OH/H2O (mole ratio: 1:2:8)

19

was used to dissolve SF at 70 °C for 4 hr. After filtration of SF solution, dialysis (MWCO

20

14,000) against distilled water was carried out at 4 °C until the solution conductivity reached

21

less than 5 µS/cm. The SF solution was centrifuged (9,000 rpm) at 4 °C for 20 min two times,

22

and the supernatant was lyophilized to obtain the regenerated SF sponges [19].

23

7

2.3. Preparation of electrospun SF nanofibers (SFNF) scaffold

1

Electrospinning was carried out using an electrospinning device (Asian Nanostructures

2

Technology Co., Iran) to prepare SFNF scaffolds. The temperature and the relative humidity

3

were 25 ± 5 °C, 35 ± 5 %, respectively. The lyophilized SF was dissolved in formic acid (10

4

to 16%) under magnetic stirring at room temperature. To do electrospinning, the solution

5

was loaded into a polypropylene syringe connected to a high-voltage supply (24 kV) and

6

pumped by a syringe at a rate of 0.1 mL/hr. The fibers were collected on a rotating aluminum

7

foil coated cylindrical drum (2500 rpm). The concentration of the silk fibroin solution, the

8

distance between the needle tip and the collector were optimized, such that there was no

9

spraying of the solution or bead formation.

10

2.4. Post-treatment of electrospun SFNF scaffold

11

To induce crystallinity, the SFNF were immersed in absolute methanol for 10 min. After

12

drying and removing from the backing substrate, the treated SFNF were immersed in PBS for

13

1 hr. A micrometer was used to determine the thickness of the samples.

14

2.5. Characterization of SFNF

15

2.5.1. Scanning electron microscopy (SEM)

16

A scanning electron microscope (SEM: AIS2100, Seron Technologies Inc., South Korea) was

17

used to illustrate the morphology of different nanofibers. The samples were sputter-coated

18

with a gold layer before imaging. Image processing was carried out by the digitization

19

program "Image J" (NIH, USA) of 100 nanofibers from different parts of the sample to

20

determine the diameter of the fibers, the existence of agglomerations (clusters), breaks, and

21

defects of the material.

22

8

2.5.2. Viscosity measurement

1

A rheometer (Brookfield, USA) with a 75 mm cone plate was used to carry out the

2

rheological studies of the optimized SF solution. Viscosity (η) was measured as a function of

3

shear rate (γ̇) in the range of 0.6 to 200 S-1 at room temperature.

4

2.5.3. Fourier transform infrared spectroscopy (FTIR)

5

FTIR spectra (Bruker, Tensor 27, Germany) of the SFNF scaffold before and after the

6

treatment, plain EGCG, and drug-loaded SFNF were investigated to analyze the characteristic

7

bands, assess any change in the functional groups, and the possible existence of chemical

8

changes in the scaffold material due to the electrospinning effect. Spectra were recorded at a

9

resolution of 4 cm-1, in the region of 400–4000 cm-1.

10

2.5.4. Porosity

11

The porosity of the SFNF scaffolds was assessed by the liquid displacement method. Hexane

12

was selected as the liquid because it is a nonsolvent for SFNF and permeates through the

13

interconnected pores without any change in the diameter of fibers. The samples were

14

immersed in hexane, and after the total elimination of trapped air, they were derived out. The

15

porosity was obtained by the following formula:

16

17

W1, W2, and W3 stand for the weight of the dry scaffold in air, the weight of the scaffold

18

impregnated with hexane in air, and the weight of the scaffold while it is suspended in

19

hexane, respectively.

20

2.5.5. Thermogravimetric analysis (TGA)

21

Thermogravimetric analysis was carried out by a thermogravimetric analyzer (STA 409

22

PC/PG, NETZSCH, Germany) at a heating rate of 10 °C/min under a nitrogen atmosphere at

23

9

a 22−500 °C temperature range. This test was done to estimate the thermal stability and

1

degradation temperature of SFNF, EGCG, and EGCG Loaded SFNF.

2

2.5.6. Contact angle

3

The static contact angle of the SFNF was conducted using a contact angle measurement

4

apparatus (OCA 15 plus, Dataphysics Instruments GmbH, Germany) at room temperature.

5

The volume of the distilled water droplet was 4 µL. This analysis was performed before and

6

after methanol treatment, and at least three replicates were averaged. The contact angle was

7

used to evaluate the wettability properties of the nanofibrous scaffold before and after the

8

methanol treatment.

9

2.5.7. X-ray diffractometry (XRD)

10

XRD of the electrospun SFNF scaffolds was carried out by X-ray diffractometer (MPD 3000,

11

Ital-Structure, Italy) with Cu Kα radiation (λ = 0.15406 nm). The existence of crystallinity

12

was analyzed in EGCG, degummed native silk, untreated, and treated SFNF scaffolds and

13

drug-loaded SFNF scaffolds.

14

2.5.8. Differential scanning calorimetry (DSC)

15

Thermal transitions of the EGCG, degummed native silk, SFNF scaffolds, and drug-loaded

16

SFNF scaffolds were studied by a differential scanning calorimeter, DSC (DSC1/STARE

17

SYSTEM, Mettler Toledo, Switzerland). A sealed empty aluminum pan with lid was used as

18

a reference. The samples were heated from 22 °C to 500 °C at a heating rate of 10 °C/min, to

19

determine changes in its physical state such as melting and degradation temperature.

20

2.5.9. Mechanical properties of SFNF scaffold

21

Mechanical properties of the optimized SFNF scaffold (0.15 × 10 × 150 mm) were measured

22

by a universal mechanical test machine (Santam, STM-50, Iran) with a crosshead speed of 10

23

mm/min under a 10 N load cell at room temperature and 65 % RH.

24

10

2.5.10. Swelling behavior and water uptake capability

1

To study the swelling characteristic of SFNF scaffolds, they were cut into 2x2 cm pieces,

2

weighed and immersed in PBS or cell culture medium at 37 °C. The weight of scaffolds at

3

predetermined time intervals was recorded after removing the excess water by a filter paper.

4

The experiment continued until there was no change in the weight of the SFNF scaffolds. The

5

swelling ratio of the scaffold and the percentage of water uptake into the scaffold were

6

calculated as follows:

7

8

9

W0 and Ws stand for the weight of the sample before and after swelling in the buffer or the

10

medium, respectively [20].

11

2.6. Preparation of EGCG-loaded SFNF scaffolds

12

The preparation procedure of drug-loaded SFNF scaffolds was as follows: first, EGCG (0.5

13

mg/mL) was fully dissolved in formic acid containing SF (10%), then the electrospinning was

14

carried out at a flow rate of 0.1 mL/hr by applying a voltage of 24 kV with the distance

15

between the tip of the needle and the collector set at 125 mm.

16

2.7. In vitro drug release

17

All SFNF scaffolds were put into a dialysis bag (MWCO 14000) and immersed in 5 mL of

18

PBS (pH 7.4) under continues stirring at 100 rpm at 37 °C. The samples (0.5 mL) were taken

19

out at certain time points and replaced by fresh warm buffer. The amount of released EGCG

20

from scaffolds was followed by the determination of the concentration of EGCG until

21

equilibrium was reached. The extent of drug release was normalized to the drugs finally

22

11

released at the equilibrium point. Drug loading was calculated by immersing the defined

1

weight of loaded SFNF in PBS until the complete dissolution of the drug. The percentage of

2

drug loading was calculated by [21]:

3

4

The experiment was carried out in triplicate and reported as mean ± SD. The amount of drug

5

was analyzed using a spectrophotometer at 278 nm. The method was linear in the range of 1–

6

50 µg/mL (R2˃0.996). The precision of all the data was less than 15%, and the accuracy

7

ranged from 97.78% to 108.37%. The limit of detection and limit of quantification for EGCG

8

were determined to be 1 and 0.3 µg/mL, respectively. All the samples showed an absorbance

9

above the limit of quantification.

10

2.8. Cell viability and adhesion assay of the limbal cells on SFNF scaffolds

11

The limbal cells (isolated from corneal limbus as a kind gift from Labbafinejad Medical

12

Center, Iran) were grown in DMEM supplemented with 2 mM glutamine and 10% FBS, 100

13

unit/mL penicillin and 100 µg/mL streptomycin at 37 °C with 5% CO2. To evaluate the

14

influence of the treated SFNF scaffolds on the limbal cells, an MTT assay was used. Briefly,

15

7×103 limbal cells were seeded on the silk scaffold placed into a 24-well plate (the cultured

16

cells in the plate served as the control group) for 2 and 4 days. Then, the cells were incubated

17

with MTT solution (0.5 mg/mL in DMEM) for 4 hr. Following the removal of supernatants,

18

dimethyl sulfoxide (DMSO) was used to dissolve the formazan crystals. After transferring the

19

suspension into a 96-well plate, the absorbance was read at λ = 570 nm by a microplate

20

reader (ELX808, Biotek, US). To visualize the adhesion of the limbal cells on the treated

21

SFNF, SEM was applied. After 24 hr, the seeded cells on the methanol-treated SF were fixed

22

in 4% glutaraldehyde for 10 min at room temperature and dehydrated in a series of ethanol

23

12

solutions (70-100%, for 10 min). Finally, the samples were coated with a gold layer prior to

1

imaging.

2

2.9. Cell viability assay of HUVEC against EGCG

3

To investigate the influence of the EGCG on the growth of HUVEC, we used the MTT assay.

4

HUVEC (National Cell Bank, Pasteur Institute, Iran) were cultivated in RPMI-1640 with

5

10% FBS. Then, the cells were seeded into a 96-well culture plate at a density of 1.2 × 103

6

cells/mL. After 24 hr, the cells were exposed to EGCG (10-540 µM) for 24 and 72 hr. The

7

viability of the cells was determined by the MTT assay, as described in the previous section.

8

2.10. Anti-angiogenic activity of EGCG loaded scaffold on HUVEC

9

The MTT assay was performed to evaluate the effects of the loaded EGCG on the growth of

10

HUVEC. Briefly, 3 × 104 cells/well were seeded into a 24-well plates in RPMI-1640 medium

11

containing 10% FBS. After 24 hr, the cells were classified into 6 groups as follows: I: control

12

or untreated cells, II: cells received SFNF scaffolds, III: cells given free EGCG (43 µg/mL),

13

IV: cells treated with EGCG loaded in the SFNF scaffolds, V: SFNF scaffolds placed on a

14

transwell insert, and VI: EGCG loaded in the SFNF scaffolds located on a transwell insert

15

membrane. The influence of released EGCG from the loaded SFNF scaffolds was determined

16

on the 2nd, 4th, and 6th day using the MTT assay as described above.

17

2.11.

Statistical Analysis

18

The statistical tests were performed to compare different sample groups in each assay. The

19

porosity results were statistically analyzed by the t-test. The MTT results were statistically

20

analyzed by the t-test and one-way ANOVA tests. Sigma Plot 12.0 software was used for

21

analysis. A P-value less than 0.05 was considered statistically significant, and all data are

22

presented as mean ± SD unless otherwise noted.

23

13

3. Results and Discussion

1

3.1. The effect of parameters on electrospinning of silk fibers

2

To obtain homogenous nanofibers with the proper size, morphology, and diameter

3

distribution, the most determinant parameters, including the concentration of SF solution, and

4

the distance between collector and nozzle were optimized. The other parameters, like relative

5

humidity and flow rate, were set constant. The flow rate of the SF solution was set to the

6

lowest available value of the device (0.1 ml/hr) which was matched to the applied voltage to

7

inhibit droplet forming [23]. The relative humidity was kept constant close to the room

8

atmosphere (35 ± 5%). It is expected that increasing of both of these variables would lead to

9

the thicker fibers [24, 25]. Figure 1. A illustrates the morphological appearance of the

10

electrospun nanofibers generated from different concentrations of SF solution (10%, 12%,

11

and 16%). As the SEM images show the average fiber diameter increases from 245 ± 42 nm

12

to 687 ± 210 nm when the concentration of SF solution is increased (Figure 1.B). This

13

finding can be related to the high viscosity of the SF solution at high concentrations [26].

14

Moreover, electrospinning at a low concentration of SF (10%) produced uniform nanofibers

15

with low diameter distribution (Figure 1 B). The effect of spinning distance, on the size,

16

diameter distribution, and morphology of the nanofibers was also studied. According to the

17

SEM micrographs, an increase in the spinning distance from 100 to 125 mm decreased the

18

average fiber diameter from 289 ± 97 nm to 245 ± 42 nm presumably due to more stretching

19

of the jet and consequently reducing the jet diameter (Figure 1.B) [27]. Electrospinning of the

20

SF solution at the optimum conditions resulted in bead-free and continuous nanofibers with a

21

relatively narrow fiber diameter distribution and rounded cross-section. The average fiber

22

diameter was 245 ± 42 nm with a rough surface suitable for use as biomaterials for tissue

23

engineering.

24

14

Figure 1. (A) SEM images of electrospun SFNF scaffolds with different concentrations at 24 kV and with a flow rate of 0.1

1

ml/hr (B) Fiber diameter distribution of relevant SF scaffolds.

2

The ability of the polymer solution to produce desirable nanofibers with few protein

3

aggregate structures depends on the rheological behavior of the SF solution [28]. Figure S.1

4

(see Supplementary Material) shows the rheological behavior of the SF solution in formic

5

acid. The SF solution was evidently non-Newtonian, and the fluid shear thinning behavior is

6

most probably due to the orientation of macromolecular chains [29]. For the shear thinning

7

behavior, the apparent viscosity changes according to a power-law model, η = m(γ̇)n-1, which

8

"m" is called the consistency index and the dimensionless parameter "n" is referred as the

9

degree of non-Newtonian behavior [29, 30]. The degree of non-Newtonian behavior of this

10

solution was less than 1 that confirmed shear-thinning behavior and a shear-sensitive network

11

of silk in formic acid [31]. The shear-thinning behavior of SF solution ensured that we

12

selected the proper concentration for obtaining bead-free fibers [32].

13

3.2. Fiber treatment

14

Generally, SFNF scaffolds as biomaterial for tissue engineering suffer from curling,

15

shrinking, and water solubility [33]. To overcome these shortcomings, there is a need to

16

improve the structural stability and crystallinity of SFNF [34]. There are mainly three

17

different conformations for SF, including random coil, silk I, and silk II (β-sheet). Among

18

them, β-sheet conformation is known as the most crystallized and insoluble conformation. To

19

convert the random coil or silk I conformation into β-sheet (silk II), the most widely used

20

method is to treat the SFNF scaffolds with an organic solvent, particularly methanol [35]. In

21

this study, the electrospun SFNF scaffolds were treated with methanol to obtain the solvent‐

22

induced crystallization and reduce the water solubility of the scaffolds.

23

To prove β-sheet conformation, FTIR analysis of the SFNF and the treated SFNF were used

24

to show the molecular conformation of SFNF considering the amide I and II bonds.

25

15

Furthermore, FTIR was used to confirm the degumming process for degummed native silk.

1

The removal of sericin (degumming) is evidenced by the absence of the characteristic bands

2

at 3279, 2930, 1442, 1400, and 1235 cm-1, as shown in Figure 2 [36]. Generally, the

3

electrospinning of SF into nanofibers results in the change of fibroin conformation into a

4

semicrystalline structure containing almost 50% β-sheet crystallites [37]. So, both spectra of

5

as-spun SFNF and treated SFNF show the characteristic bonds of β-sheet conformation,

6

including amide I, amide II, and amide III. However, the bonds of treated SFNF shifted from

7

1652 cm-1 and 1542 cm-1 to 1629 cm-1 and 1521 cm-1 for amide I (C=O stretch) and amide II

8

bands (N-H deformation), respectively [38, 39].

9

Figure 2. FTIR spectrum of samples: (a) Untreated SFNF (b) Treated SFNF(c) Degummed native silk.

10

Figure S.2 (Supplementary Material) shows the SEM image of the electrospun SFNF after

11

methanol treatment. As shown, the treatment has no remarkable influence on the morphology

12

of the nanofibers. The insolubility of the treated SFNF scaffold in water can also demonstrate

13

the silk conformational change. Thus, these findings confirm that the change in the

14

conformation of the random coil to β-sheet on SFNF has been successfully achieved.

15

Pore properties of both untreated and treated scaffold were determined by the hexane

16

replacement method. A high percentage porosity of the SFNF provides three-dimensional

17

structure, high surface-area-to-volume ratio, and consequently more opportunity to interact

18

with the biological molecule and to proliferate cells in in vivo condition [40]. The porosity of

19

the untreated and treated SFNF scaffold was 95.52 ± 0.92%, and 94.18 ± 0.60%, respectively

20

(p-value>0.05), indicating its suitability for tissue engineering applications.

21

3.3. Thermal analysis

22

Thermogravimetric analysis of the treated SFNF nanofibers determines the thermal stability

23

and degradation temperature of the samples. Figure 3 presents the TGA curves of the SFNF

24

16

scaffold. The thermograms of the samples show that the initial weight loss of the scaffold

1

occurred around 100 °C due to the loss of water adsorbed by physical interaction. As the

2

temperature reaches around 256 °C, the side chain groups cleavage of amino acid residues

3

and peptide bonds in the amorphous structure of the SFNF takes place [41]. The weight loss

4

related to the high molecular orientation and crystallinity of SFNF was about 26%, which

5

started at 300 °C.

6

Figure 3. TGA thermogram of SFNF scaffolds.

7

3.4. Contact angle

8

The contact angle is a valuable index for the hydrophilic and hydrophobic properties of a

9

substrate. Surprisingly, the contact angle for the untreated and treated fibers was 102.9 ± 9°

10

and 64 ± 28°, respectively (Figure 4). The lower contact angle of the treated SFNF scaffold

11

compared to the non-treated one can be related to the presence of water in the treated SFNF

12

as confirmed by the TGA results, which can be responsible for the plasticization of the

13

treated scaffolds and the β-sheet conformation of SF during the electrospinning process and

14

treatment [37].

15

Figure 4. The contact angle of SFNF scaffold (A) before and (B) after methanol treatment.

16

3.5. Crystallinity

17

The crystalline structure of the degummed native silk, untreated, and treated SFNF were

18

studied by X-Ray diffractometry (Supplementary Material, Figure S.3). X-ray diffractogram

19

of degummed silk shows peaks at 13.9, 18.5, 25.5, and 29.25°, which is related to 55, 44, 31,

20

3 Å in silk I and II structure [33]. For the untreated and methanol-treated samples, there is a

21

strong diffraction peak at 16.8° and three relatively weak peaks at 14.8°, 22.6°, and 25.5

22

correspondings to silk II, I, I and I [42]. The results demonstrate that degummed native silk

23

was composed of two types of silk and electrospun silk before and after treatment had a

24

17

similar composition. These results are consistent with the previous FTIR results. Thus, as

1

mentioned before, there was a relatively low amount of the silk II structure in untreated SFNF

2

due to the structural transition of the SFNF from amorphous to a silk II structure by solvent

3

evaporation in the course of electrospinning process as previously discussed.

4

3.6. Differential scanning calorimetry (DSC)

5

DSC is commonly used to explain the molecular structure and physicochemical interaction

6

between different substances. Thermodynamic data from DSC for degummed SF and treated

7

SFNF are shown in Figure 5. The DSC curve of degummed silk fibers is different from that

8

of SFNF. The thermogram of degummed silk shows a broad endothermic peak at 281-306 ˚C

9

attributed to the thermal decomposition of the degummed native silk with unoriented β-sheet

10

conformation of fibroin [43]. In treated scaffold, a sharp endothermic decomposition peak at

11

284 ˚C, as well as the lack of a distinct peak around 230-250 ˚C indicates the absence of α-

12

helix structure [44]. The sharp endothermic peak of the treated SFNF at about 284 ˚C shows

13

the non-oriented β-sheet structure of the nanofibers, providing further support for a

14

crystalline structure of the SFNF scaffolds [45].

15

Figure 5. DSC curves of different samples, a) EGCG, b) EGCG loaded SFNF scaffold, c) Treated SFNF scaffold, d)

16

Degummed native silk.

17

3.7. Mechanical Properties

18

The mechanical properties of the electrospun SFNF scaffold play a determinant role in

19

various tissue engineering applications. Figure S.4 (Supplementary Material) presents the

20

results of the mechanical properties as stress-strain curves studied on the samples with 20 µm

21

thickness. As shown, the mean strain at breaking point and Young’s modulus of the SFNF

22

scaffold were 3.5 N/m2 and 704 MPa, respectively. This result indicates that the SFNF

23

scaffold possesses desirable mechanical properties. Since the mechanical properties of

24

nanofibers depend on multiple factors, including the molecular weight, the physicochemical

25

18

properties of the respective fibers, the distributions of fiber diameter, the orientations of

1

fibers, and the fiber density, it is difficult to compare the data with the other studies,

2

quantitatively. However, it may be concluded that SFNF is suitable for use in corneal tissue

3

engineering because of its greater Young’s modulus compared to the normal cornea (2.45 ×

4

10-2 MPa) [46].

5

3.8. Swelling properties

6

The degree of water uptake of the treated SFNF scaffolds was measured at predetermined

7

time intervals after immersion in different media, including PBS, and cell culture media. As

8

shown in Figure 6, it took less than 2 hr for the SFNF scaffolds to reach equilibrium fluid

9

uptake. The water content in the final swollen samples was determined to be 85.65 % and

10

81.67 %, for PBS and cell culture media, respectively. The maximum attainable swelling

11

ratio for SFNF scaffolds was determined to be 6.03 and 4.81 for PBS and cell culture media,

12

respectively. Additionally, there was no remarkable change in the size of swollen SFNF

13

scaffolds compared to the dried ones. The porosity and hydrophilic/ hydrophobic balance are

14

determinant factors in the swelling ratio. The very porous structure of the SFNF scaffold and

15

a high proportion of hydrophilic parts in SF protein have made this nanofibrous structure able

16

to absorb liquid in large quantities. The excellent water-binding ability of the SFNF enhances

17

cell adhesion properties [47, 48].

18

Figure 6. (A) Water uptake and (B) swelling ratio curves of treated SFNF scaffold in different media (n=3 and data are

19

shown as mean ± SE).

20 21

3.9. Drug loading

22

FTIR spectroscopy analysis was used to characterize the intermolecular interactions of ECGC

23

and the SFNF. The spectra of the drug, SFNF, and EGCG loaded SFNF are shown in Figure

24

19

7. The FTIR spectra of the EGCG loaded SFNF revealed the characteristic bands of EGCG

1

and SFNF. EGCG showed characteristic peaks within 3600–3400 cm-1 for the phenyl-OH

2

stretching mode, 1543 cm-1 for the C-C stretch in the aromatic ring, 1447 cm-1 for the C-H

3

group present in the chroman ring, 1692 cm-1 for the C=O group that links the

4

trihydroxybenzoate group and chroman. Peaks below 1000 cm-1 for the aromatic C-H group

5

in the chroman ring and the other aromatic ring (Figure 7) [49, 50]. The shift of amide peaks

6

(I, II, and III) in the FTIR spectrum of EGCG loaded SFNF indicates the interaction of the

7

hydroxyl group of EGCG with the scaffold and successful loading of EGCG. Also, the band

8

of the stretching vibration of OH bands of ECGC at 3433 cm-1 shifted to 3267 cm-1,

9

indicating the presence of intermolecular hydrogen bonding between drugs and the SFNF

10

scaffold (Figure 7) [51].

11

The drug-loaded SFNF was also studied by the XRD technique (Figure S.3, Supplementary

12

Material). Lack of any significant difference in pattern and absence of any distinct diffraction

13

peaks indicates that the EGCG was encapsulated in amorphous forms in the SFNF scaffold.

14

An amorphous or disordered crystalline form of EGCG in the loaded SFNF was also

15

confirmed by the DSC thermogram (Figure 5). The DSC curve shows the absence of EGCG

16

peaks at 220 and 250 ˚C, which correspond to the glass transition temperature and the melting

17

point of EGCG, respectively [50, 52]. Also, the shift of the melting point of the loaded SFNF

18

to a higher temperature revealed that EGCG might induce more β-sheet content after drug

19

loading [53]. SEM images of EGCG loaded nanofibers did not show any remarkable change

20

in the morphology of the SFNF (Figure 7), although the average diameter was reduced to 142

21

± 41 nm probably as a result of increasing the surface charge density [49, 54]. The extent of

22

drug loading in the loaded SFNF was determined to be 8.0 %.

23

Figure 7. FTIR spectra of (a) EGCG (b) EGCG Loaded SFNF (c) Treated SFNF.

24

Figure 8. SEM image of EGCG loaded SFNF scaffolds.

25 20

3.10. Drug release

1

Figure 9 shows the cumulative percentage of drug release from EGCG-loaded SFNF

2

scaffolds. As shown, a burst release was observed during the first hours of release that

3

reflects the high dispersion of EGCG near the fiber surfaces as well as the swelling of SFNF

4

[54]. The sustained release of EGCG and controlled diffusion supports the potential of SFNF

5

as a suitable carrier. The maximum attainable drug release (98%) was reached in 144 hr.

6

Clearly, delivering the drug at a slow and controlled rate offers the possibility of keeping

7

EGCG concentration at an effective dose for a long time.

8

Figure 9. The cumulative percentage drug release profile from the scaffold during 144 h (data are shown as mean ± SD and

9

n=3).

10

3.11. Cell adhesion and viability assay of limbal cells on the SFNF scaffolds

11

SEM images revealed the limbal cells adhered very well to the treated SFNF scaffolds

12

(Figure 10.A). This result proves that the SFNF provides supportive conditions for hosting

13

cells and forming a tissue [12]. The MTT assay was used to study the viability, proliferation

14

of attached, and seeded cells on the SFNF scaffolds in the cell culture experiment period. The

15

results revealed that the SFNF scaffolds supported limbal proliferation and the proliferation

16

rate of the cells was maintained during 4 days of cell culture (Figure 10.B). On the other

17

hand, the increase in the cell viability indicates that the cells adhered and proliferated

18

vigorously on the surface of the SFNF scaffold during the time of culture. This finding also

19

indicates the high biocompatibility of the SFNF for limbal cells.

20

Figure 10. (A) SEM image of attached limbal cells to the SFNF scaffold. (B)The MTT assay of SFNF scaffold on limbal

21

cells. The *** symbol indicates the statistical significance of the SFNF group to the control group each day and P-

22

value<0.001. The data are triplicate and reported as mean ± SD.

23

21

3.12. Inhibition effect of EGCG on the HUVEC proliferation

1

The MTT assay was used to evaluate the influence of EGCG at different concentrations on

2

the inhibition of HUVEC proliferation over time. HUVEC were treated with 10-540 µM

3

EGCG for 24 and 72 hr. Figure 11 shows the inhibition effect of EGCG on HUVEC. The

4

findings revealed that the treatment with EGCG resulted in a dose-dependent inhibition of the

5

cell proliferation of HUVEC. The IC50 value of EGCG after 24 and 72 hr treatment was

6

43.06 and 47.1 µM, respectively. These results indicate the dependency of HUVEC viability

7

on the concentration of EGCG and incubation time. Moreover, our findings showed that IC50

8

of EGCG at 72hr is more than the IC50 in 24h; this may be due to the degradation and

9

oxidation of EGCG. This phenomenon is affected by high temperature, the length of the

10

incubation period, and higher values of pH [56-58]. To handle this challenge, EGCG was

11

loaded into the silk scaffold as a carrier to control drug release and to protect from physical

12

changes in media during the experiment. To obtain the best anti-angiogenic effects of loaded

13

EGCG on HUVEC, the loading and treatment have to be carried out at the specific

14

concentrations and exposure time.

15

Figure 11. Dose-response of HUVEC to EGCG measured using MTT assay to determine the IC50 values for 24 and 72 hr.

16

Data are mean ± SD from triplicate experiments.

17

3.13. Inhibition effect of the EGCG-loaded SFNF scaffold on the proliferation of HUVEC

18 19

Angiogenesis is defined as the generation of new blood vessels from pre-existing vessels. It

20

can cause blindness if it occurs in the cornea. There are some methods to manage

21

vascularization of corneal tissue, such as using anti-angiogenic compounds. In this study,

22

loading of EGCG in a SFNF scaffold was selected, because it can control vascularization

23

through modulating ROS, inhibiting nuclear factor-κB signaling (NF-κB), and regulating

24

MAPKs signaling [14]. Also, HUVEC was used as an angiogenesis model to evaluate the

25

22

effect of the bioactive compound. For this purpose, the MTT assay was used to evaluate the

1

HUVEC viability on the treated SFNF scaffold and the cell metabolic activity as an indicator

2

of survival and growth characteristics of HUVEC against released EGCG. Figure 12

3

summarizes the cell viability at different conditions after 2, 4, and 6 days of incubation. The

4

cells seeded on tissue culture polystyrene (TCPS) were used as the control group. As

5

expected, the growth of angiogenic cells was inhibited through EGCG’s action. In addition to

6

this result, the released EGCG from the scaffold had the same effect, but the HUVEC were

7

inhibited less due to the slow-release conditions. On the 6th day, the difference between free

8

EGCG and the loaded scaffold was disappeared. It can be explained by the chemical change

9

of EGCG, such as oxidation or degradation in aqueous media, which can be decreased by

10

using SF as a carrier [59].

11

The inhibiting effect of SFNF on HUVEC was investigated by the direct contact method,

12

which is a highly sensitive procedure in comparison to other methods. After 4 days, a

13

reduction of cell proliferation was observed. It is of great importance to find out whether the

14

real cause of this decrease arose from an inhibiting SFNF effect or other factors (e.g.,

15

physical trauma or crushing of the cells) [60]. The extraction method could be a suitable

16

method, but limitations such as the influence of applied time and temperature, and instability

17

of EGCG in aqueous media make it an inappropriate method for our purposes. We used

18

transwell for in situ investigation of the loaded and unloaded SFNF inhibition effect on the

19

cell growth. Transwell represents a gradient concentration system and successive extraction

20

from fibers during the total culture period that ensures simultaneous exposure to the testing

21

drug [60-62]. The results showed that there was no remarkable difference between the direct

22

contact method and transwell. According to Hakimi’s study, the growth arrest could be

23

caused by enzymatic degradation of SF, whether in the direct contact or transwell method

24

23

[63]. The large difference of HUVEC viability between the SFNF and the EGCG loaded

1

scaffold confirmed that EGCG acts as the main factor in promoting of anti-angiogenesis.

2

Figure 2.The anti-angiogenic activity of SF and Loaded scaffolds on HUVEC. Each symbol indicates statistical significance

3

at certain time points (one symbol: 0.01
4

symbols:P<0.0001 and *: free EGCG as reference group, #: SF scaffold as reference group, ¥: Loaded SF scaffold as

5

reference group and $: SF scaffold (Transwell) as reference group). Data are mean ± SD from triplicate experiments.

6

4. Conclusion The present study aimed to develop an EGCG-loaded SF-based scaffold with anti-angiogenic

7 8

properties for corneal tissue engineering. An optimized electrospun scaffold was obtained

9

with a homogenous structure by manipulation of different parameters. The SEM images

10

revealed the formation of bead-free and continuous nanofibers. Crystallinity and water

11

insolubility of the scaffold was successfully induced by methanol treatment. The

12

characterization of the scaffold in terms of contact angle, mechanical strength, crystallinity,

13

swelling, porosity, and thermal analysis, confirmed SFNF desirable properties as a scaffold

14

for tissue engineering. The extent of the drug loading of EGCG into the SFNF scaffold was

15

determined to be 8.0%. The drug release study showed the controlled release of the drug for

16

144 hr. The homogeneity and high growth of limbal cells on the scaffold was demonstrated

17

by SEM images indicating the suitability of the electrospun SFNF scaffold as a support for

18

limbal cells. The inhibition of EGCG on HUVEC proliferation studied by the MTT assay

19

showed a dose-dependent inhibition of cell proliferation. The MTT results also revealed that

20

the prepared nanofibrous scaffolds inhibited the survival of HUVEC during in vitro

21

incubation. It is anticipated that with further development, this scaffold has the potential for

22

being used as a delivery system and engineered tissue for cornea.

23

24

Acknowledgment

1

This project was supported by Zanjan University of Medical Sciences, Deputy of Research

2

and Technology (Grant No A-12-227-14). This article has been derived from the Ph.D. thesis

3

of Narges Forouzideh. The authors would like to appreciate Dr. William C. Hartner for

4

providing invaluable help in language editing of the manuscript.

5

Conflicts of Interest

6

All the authors declare that they have no financial conflict of interest.

7

Ethical Statement

8

The applied protocols in this research were approved by the committee of ethics at Zanjan

9

University of Medical Sciences (Ethical Code: ZUMS.REC.1394.323). No in vivo testing has

10

been done in this research.

11

Appendix A. Supplementary data

12 13

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Figure legends Figure 1. (A) SEM images of electrospun SFNF scaffolds with different concentration at 24 kV and with flow rate 0.1 ml/hr (B) Fiber diameter distribution of relevant SF scaffolds. Figure 2. FTIR spectrum of the samples: (a) Untreated SFNF (b) treated SFNF (c) Degummed native silk. Figure 3. TGA thermogram of SFNF scaffolds. Figure 4. The contact angle of SFNF scaffold (A) before and (B) after methanol treatment. Figure 5. DSC curves of different samples, a) EGCG, b) EGCG loaded SFNF scaffold, c) Treated SFNF scaffold and d) Degummed native silk. Figure 6. (A) Water uptake and (B) swelling ratio curves of treated SFNF scaffold in different media (n=3 and data are shown as mean ± SD). Figure 7. FTIR spectra of (a) EGCG (b) EGCG Loaded SFNF (c) treated SFNF Figure 8. SEM image of EGCG loaded SFNF scaffolds. Figure 9. The cumulative percentage drug release profile from the scaffold during 144 h (data are shown as mean ± SD and n=3). Figure 10. (A) SEM image of attached limbal cells on the SFNF scaffold. (B) The MTT assay of SFNF scaffold on limbal cells. The *** symbol indicates the statistical significance of the SFNF group to the control group each day and P-value<0.001. The data are triplicate and reported as mean ± SD. Figure 11. Dose-response of HUVEC to EGCG measured using MTT assay to determine the IC50 values for 24 and 72 hr. Data are mean ± SD from triplicate experiments. Figure 12. The anti-angiogenic activity of SFNF and the loaded scaffolds on HUVEC. Each symbol indicates statistical significance at certain time points (one symbol: 0.01
symbols: 0.001
experiments.

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Declaration of interests *The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: