hydrophobic nanofibrous network with tunable cell adhesion: Fabrication, characterization and cellular activities

Accepted Manuscript Title: Superhydrophobic/hydrophobic nanofibrous network with tunable cell adhesion: Fabrication, Characterization and Cellular Activities Author: Fangyuan Dong Mi Zhang Wenyu Huang Liping Zhou Man-Sau Wong Yi Wang PII: DOI: Reference:

S0927-7757(15)30108-4 http://dx.doi.org/doi:10.1016/j.colsurfa.2015.07.030 COLSUA 20048

To appear in:

Colloids and Surfaces A: Physicochem. Eng. Aspects

Received date: Revised date: Accepted date:

16-5-2015 7-7-2015 13-7-2015

Please cite this article as: Fangyuan Dong, Mi Zhang, Wenyu Huang, Liping Zhou, Man-Sau Wong, Yi Wang, Superhydrophobic/hydrophobic nanofibrous network with tunable cell adhesion: Fabrication, Characterization and Cellular Activities, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2015.07.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

1

Superhydrophobic/hydrophobic nanofibrous network with tunablecell adhesion:

2

Fabrication, Characterization and Cellular Activities

3 4

Fangyuan Dongb, Mi Zhangb, Wenyu Huangb,Liping Zhou b,Man-Sau Wongb,and Yi Wangab*

5 6

a. Shenzhen Key Laboratory of Food Biological Safety Control, Hong Kong PolyU Shenzhen

7

Research Institute, Shenzhen, PR China.

8

b. Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic

9

University, Hong Hum, Kowloon, Hong Kong SAR.

10 11 12

[email protected], [email protected], [email protected],

13

[email protected], [email protected],[email protected]

14 15

*To whom correspondence should be addressed. Tel: 852-34008673. Fax: 852-23649932. E-

16

mail: [email protected].

17

Graphical abstract

18 19 20 21 22 23 24

Highlights:

zein superhydrophobic/hydrophobic nanofibrous network was formed by electropinning. The highest WCA of the zein electrospun nanofibrous network (ZENN) could reach153.6°. Both the zein concentration and applied voltage had effects on the surface hydrophobicity. ZENN could mimic the extracellular matrix to support the cell growing.

25 26 27 28 1

1 2 3 4 5 6 7

Abstract

8 9

Superhydrophobic/hydrophobicnanofibrous networkshaveattracted much attention because

10

oftheir potential applications in tissue engineering. Cell growth in the scaffold of tissue

11

engineering

12

superhydrophobic/hydrophobic surfaces are usually made from synthesized polymers, which

13

generally are not biocompatible and biodegradable and, thus, not suitable for biomedical

14

applications. Zein is an amphiphilicprotein from corn, and it is potentialfor hydrophobic

15

surface formation. This work aims to make zein superhydrophobic/hydrophobicnanofibrous

16

networkusing electrospinning. The formed zein networks show high hydrophobicity with the

17

water contact angles ranging from 130.5±1.0° to 153.6±2.1°.The cell attachment and

18

growthon the zein networksare studied. It is observedthat the amount of the cells attached and

19

grown in the zein nanofibrous networks are higherthan the ones onthe conventional zein

20

casting films. The results indicate that the electrospun zein nanofibrousnetworkhas great

21

potentialas scaffold in tissue engineeringto support cell growth and tissue regeneration.

can

be

controlled

by

the

hydrophobicity

22 23 24 25 26

Keywords:zein, electrospinning, superhydrophobic, cell adhesion 2

of

the

scaffold.

The

1 2 3 4 5 6 7

1. Introduction

8

Hydrophobic surface is a surface exhibiting water contact angles (WCA) higher than 90°. It

9

has wide applications in coating, textile, packaging, electronic devices, and biomedical

10

engineering.[1] Superhydrophobic surface is a special kind of hydrophobic surfaces with a

11

WCA higher than 150°. Superhydrophobic surface has attracted much attention over the past

12

decades for its applicationsin many areas includingmicrofluidics[2] andself-cleaning

13

surfaces.[3]Hydrophobic surfaces play an important role in biomedical engineering, especially

14

tissue engineering.Tissue engineering is aiming to create biochemical and physico-chemical

15

substitutes to improve or replace the biological functions of portions or the whole tissue of

16

human body. In tissue engineering, a scaffold is required for the incorporation of living

17

cellsand to support the cellsto adhere, grow, and differentiate.The hydrophobicity and

18

roughness of the surface as well as the microenvironment in the scaffold affect the cell

19

attachment and growth.[4] For example, Valamehr et al. [5] had confirmed that hydrophobic

20

surfaces promoted the proliferation and differentiation of the stem cells.

21

Generally, there are two ways to improve the hydrophobicity of a surface: increasing the

22

surface roughness and reducing the surface energy.[6] Surface roughness at nano- and

23

micrometer scales can be improved through a variety of methods, such as lithography,

24

chemical etching, templating, sol-gel synthesis, controlled crystallization, and phase

25

separation.[7] Hydrophobic surfaces can be formed on various substrates, such as synthetic 3

1

polymers, Si wafers, glass slides, and metals.[8] However, these substrates are usually too rigid

2

for biomedical applications.[9] A loss of hydrophobicity may also occur when the substrates

3

deform.[10] Moreover, for biomedical applications, high biocompatibility and low toxicity are

4

required.Therefore, Foreign synthetic materialsareextremely limitedfor such uses. Natural

5

plant-based biomaterialsare biocompatible, low/non-toxic, inexpensive, sustainable, and

6

biodegradable.[11] However, most of the natural biomaterials are hydrophilic and water

7

absorbing. They may be rapidly solubilized in aqueous environment, which is not good for

8

tissue engineering applications.[12]

9

Zein is a major protein extracted from corn endosperm. It can be dissolved in 40~95%

10

ethanol-water mixture.[13] Zein is capable of self-assembly into various structures, such as

11

microspheres, bicontinuous sponges, films, and fibers.[14] Zein has more than 60%

12

ofhydrophobic amino acids, which makes it amphiphilic and highly potential for hydrophobic

13

surface formation. Zein, as a natural biopolymer, has advantages over manufactured synthetic

14

polymers, such as good biodegradability, low toxicity, and high biocompatibility, and thoseare

15

especially important for applications in biomedical engineering areas. In addition, different

16

from other natural biopolymers, zein is not water-absorbing.

17

In this paper, we report the fabrication of zein superhydrophobic/hydrophobic

18

nanofibrous networkusing electrospinning.We investigate the effects of zein concentration and

19

the electrospinning voltage on the surface hydrophobicity of the zein electrospun nanofibrous

20

network(ZENN). WCA and SEM are used for characterizations. The resultsindicatethat the

21

ZENNcould mimic the extracellular matrix (ECM) to support the cell growingand arehighly

22

potential for tissue engineering applications.

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2. Materials and methods

25

2.1. Materials 4

1

Zein was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Ethanol (96%

2

v/v) was obtained from Guangdong Guanghua Sci-Tech Co., Ltd. (Guangzhou, China).

3 4

2.2. Electrospinning

5

Zein solutions of various concentrations (5, 10, 15, 20, 25, and 30 wt%) were prepared by

6

dissolving zein in 80% (v/v) aqueous ethanol solution followed by 10-minute sonication. A

7

nanofiber electrospinning unit (Kato Tech Co., Ltd, Tokyo, Japan) with a variable high

8

voltage power supply of 0-40kV was used. The positive electrode was attached to a metal

9

needle with an internal diameter of 0.9 mm connected to a 10-ml syringe filled with zein

10

solution. The syringe was placed horizontally on a controlled syringe pump and theflow

11

ratewas kept at0.5 ml/h. And the needle was horizontally directed towards the collector,

12

which is rotating at 1200rpm. Under the applied voltage of 10-24 kV and with the tip-to-

13

collector distance of 15 cm, a positively charged jet of zein solution was formed, travelled

14

through the air gap, and deposited on the collectorcovered with an aluminum foil. After

15

electrospinning for 2 h, the samples (0.7 mm thick) were removedfromthe collector.

16 17

2.3. Water contact angle (WCA)

18

WCA was used to measure the surface hydrophobicity. A hydrophobic surface has a WCA

19

larger than 90° and a superhydrophobic surface has a WCA larger than 150°. WCA was

20

measured using a standard goniometer (Kruss GmbH DSA 100, Hamburg, Germany). The

21

water droplets were introduced by a micro-syringe, and images were captured usinga camera

22

system. The WCAs were calculated according to the Young-Laplace equation by the software.

23 24

2.4. Scanning electron microscopy (SEM)

25

The surface morphology of the ZENN was examined using SEM. The ZENNwere gold coated 5

1

usingan Edwards S150B sputter coater to improve the electrical conductivity. SEM images

2

were obtained using a JEOL JSM-6490 SEM (Tokyo, Japan).

3 4

2.5.Oxygenplasma treatment

5

Oxygen plasma was used to introduce the oxidation of the hydroxyl groups on

6

ZENNsurfacestoincrease the surface wettability.A PDC-32Gplasma cleaner(Harrick Plasma,

7

USA) with low-pressure mercury vapor lamp was used to treat the ZENN. The

8

ZENNwasplaced in a vacuum chamber, the pressure in which is 110–115 mTorr, and exposed

9

to oxygen plasma for 5-10 min.

10 11

2.6.Cell culture

12

Human liver hepatocellular carcinoma cells (HepG2) and rat osteoblastic UMR106 cells were

13

incubatedin Dulbecco’s Modified

14

mg/mlpenicillin and 100 mg/mlstreptomycin. The cells were supplemented with 10% fetal

15

bovine serum (FBS), and maintained in a humidified incubator at 37℃ in anatmosphere of 5%

16

CO2. Then, thecells were trypsinized using a 0.25% trypsin solution in PBS buffer for 5 min

17

and resuspended in the completeculture medium.

Eagle

Medium (DMEM),

which

contains 100

18 19

2.7. Cell adhesion assay

20

The cell attachment assays of HepG2 and UMR106 were conducted on three samples, the

21

ZENNs, the oxygen plasma treated ZENNs (OZENNs), and the zein casting films (ZCFs, 0.7

22

mm thick) were collected on a rotated collector covered with an aluminum foil.), respectively.

23

The ZENN wasmade from the zein solution (30 wt%). It was cut into pieces in the size of

24

10×10 mm2. Each ZENN sample was then attached on a microscope cover glass and hold in a

25

6-cell plate. The ZCF sample was prepared by cast dryingofthe 30 wt% zein solution. All the 6

1

samples were sterilized using UV light for 30 min before the cell experiments. SEM was used

2

for the cell adhesion study. For sample preparation of SEM, cells were seeded on the 6-well

3

plate at a density of 2×105 cells/ml and allowed for cellattachment for 24 h. After incubation,

4

the samples were washed three times by PBS and fixed by the 4% paraformaldehyde solution.

5

The samples were freeze-dried overnight before the SEM observation. The fluorescence-

6

based assay was also used to study the cell adhesion. The cells were first stained by 4',6-

7

diamidino-2-phenylindole (DAPI) before cell seeding onto the samples. For staining, the

8

attached cells in the culture dish were immersed in 5 ml of 10 μg/ml DAPI solution, and it

9

was performed at 37℃ for 20 min with light protection. The stained cells were then washed

10

using PBS for 5 times to remove any residue of DAPI on the cell surface. After that, the cells

11

were detached using trypsin and collected bycentrifugation.The DAPI stained cells were then

12

dispersed in DMEM and seeded on the ZENN, OZENN, and ZCF samples, respectively, at a

13

density of 2×10 5 cells/ml. After 24 h of incubation, the cells were fixed with the 4%

14

paraformaldehyde solution.

15 16

3. Results and discussion

17

3.1. Effects of the zein concentration on the surface hydrophobicity of ZENN

18

Zein solutions of various concentrations (5, 10, 15, 20, 25, and 30 wt%) were electrospun to

19

prepareZENNs. All the other parameters of the electrospinning were kept constant in this

20

study.Table 1 showed the WCA values of the ZENNs prepared from different zein

21

concentrations. All the ZENNs were highly hydrophobic as they all had a WCA larger than

22

130°. The WCA of ZCF was also measured, and itwas76.5±1.1°(Figure not shown) and the

23

ZCF was hydrophilic. The electrospinning made the zein structure changed from hydrophilic

24

to hydrophobic. Among the ZENN samples, the ones made from 10 and 15 wt% zein

25

solutions were superhydrophobic with the WCAs of 153.6±2.1° and 150.1±1.3°, 7

1

respectively. The surfacemorphology of the ZENNs was studied using SEM, and the images

2

were shown inFigure 1.Itshowed that there were two kinds of zein structures on the surface of

3

ZENNs: beads andfibers.It was considered that the beads were formed when the concentration

4

(5 and 10wt%) as well as the viscosity ofthe zein solution was low.[15]When the zein

5

concentration was 30 wt%, the morphology of the ZENNwere fibers.And 15 wt% of zein

6

concentrationwas a transition state between the beads and fibers.It was also observed that

7

there were two kinds of beads: solid beads and collapsed beads. When the zein concentration

8

was increased from 5 to 15 wt%, the size of thezein beads increased and the amount of

9

collapsed beads also increased. Because the collapsed beads had rough surface while the solid

10

beads had smooth surface, the roughness of the collapsed beads was higher. So the roughness

11

of the ZENN surfaces increased when the zein concentration was increased from 5 to 15 wt%,

12

and the WCAalso increased. It was also observed that there were two kinds of fibers on

13

ZENN: solid fibers and collapsed fibers. When the zein concentration was increased from 15

14

to 30 wt%,the diameter of the fibersincreased and the amount of collapsed fibers decreased.

15

Because the roughness of the collapsedfibers was higher than that of the solidfibers, sothe

16

roughness decreased when the zein concentration was increased from 15 to 30 wt%. So the

17

WCAs of the ZENNsamples decreased.

18 19 20 21 22

3.2.Effects of the electrospinning voltage on the surface hydrophobicity of ZENN

23

The effects of the applied voltage on the surface hydrophobicity of the ZENN samples were

24

also investigated for the electrospinning process. Samples were prepared undervarious

25

voltages: 10, 18, 21, and 24 kV, and the WCA values of the ZENN samples were shown in

26

Table 2. The WCA of the ZENN samples increased from 130.5±1.0° to 153.6±2.1° as the 8

1

voltageincreased from 10 kV to 18 kV, but decreased from 153.6±2.1° to 135.5±1.5° as the

2

voltage further increased from 18 kV to 24 kV. The morphology of the ZENN samples was

3

examined using SEM, and the images were shown in Figure 2.It was reported that the

4

increasing voltage increased the electrostatic stresses, which had strong influence on the

5

morphology of formed structures.[16] Whenthe voltageswere10 and 18 kV,the surface is

6

covered with collapsed zein beads with wrinkled surfacesas well as small amount of solid

7

beads. When the voltage was increased from 10kV to 18kV, the amount of the collapsed beads

8

increased while the amount of the solid beads decreased, which lead to increased surface

9

roughness. When the voltage was increased from 18 to 21 kV, more solid beads with smooth

10

surfaces were formed instead of collapsed beads with wrinkled surfaces, so the surface

11

roughness decreased.When the voltage reached24 kV, fibers started to appear and more solid

12

spheres with smooth surfaces formed. The increase of the amount of fibers and solid beads

13

further decreased the surface roughnessanddecreased WCAs.

14 15 16 17 18 19

3.3. Cell attachment behaviors

20

To further evaluate whether the ZENN was suitable for tissue engineering applications, a

21

series of in vitro cell attachment experiments were conducted. The ZENNsample made from

22

30 wt% zein solution was chosen for the cell attachment study.[17]The cell growth behavior of

23

the ZENN samples was investigated using Human liver hepatocellular carcinoma cells

24

(HepG2). The cells were seeded on the sample surfaces and incubated for 24 hours. SEM was

25

used to study the cell adhesion (Figure 3a).Figure 3a showed that the cells adhered and grew 9

1

between the electrospun zein fibers. The cell attachment behaviors on ZENN can also be

2

visualized through florescence microscopy images (Figure 3b). The DAPI stained cell

3

nucleuses showed blue color under laser excitation. Figure3b showed thatthe cells grew not

4

only on the surface but also into the fibrous network, which indicated that the porous ZENN

5

successfully mimicked the in vivoextracellularenvironment.

6

The cell attachments of HepG-2 on 3D ZENN and 2D ZCF were compared and the result

7

showed that 3D ZENN had structural advantages over 2D ZCF oncell adhesion.Figure 4

8

showed that, compared to ZCF, much more cells were attached to the porous ZENN. The

9

better cell adhesion behaviors on ZENN could be attributed to both the surface morphology

10

and the 3D structure. Compared to the flat and solid surface of ZCF, ZENN had a fibrous and

11

porous 3D structure. The 3D structure of ZENN resulted in a large superficial area with a high

12

surface to volume ratio, a large porosity and a large interconnectivity, which favored cell

13

adhesion.

14 15

3.4. Effects of plasma treatment induced tunable wettability on cell attachment

16

The oxygen plasma treatment is a widely used technique to change the surface hydrophobicity

17

of various materials. The plasma treatment introduces the desired functional groups to the

18

material surface by oxidation.It can lead to the formation of oxidized chemical groups, such

19

as carbonyl, carboxyl, and ester groups, which increase the polarity and wettability of the

20

polymer surface.[18]To better understand the relation betweensurface wettability andcell

21

adhesion, the oxygen plasma treatment was applied to change the surface hydrophobicity and

22

the attachments of HepG2 and UMR106 cells on the treated samples were studied.ZENN

23

samples (made from 30 wt% zein) were treated with oxygen plasma for varioustime

24

lengths.The WCA values of ZENN for samples treated with oxygen plasma for 5 min and 10

25

min were 106.1±2.7° and 8.2±1.4°, respectively. The result showed that the longer the

26

treatment time, the higher the surface hydrophilicity. What’s more, the cell attachment could 10

1

be controlled by changing the surface wettability.From the SEM images, it was observed that

2

a considerable amount of cells were attached to the highly hydrophobic ZENN (Figure5a and

3

5d). The amount of attached cell decreased after ZENN treated by oxygen plasma for 5 min

4

(Figure 5b and 5e).The amount of the attached cells wasfurther decreased when the ZENN

5

was treated with oxygenplasma for 10 min (Figure 5c and 5f). The decreased cell attachment

6

on ZENN after oxygen plasma treatment can be attributed to the decrease of the amount of the

7

cell adhesion-mediating proteins absorbed to the treated ZENN surface. It has been reported

8

that the amount of the absorption of cell adhesion-mediating protein, fibronectin, decreased in

9

the following order of the chemical groups: NH2>CH3>COOH>OH.[19]The carboxyl groupson

10

the ZENN created by the oxygen plasma, compared to the original CH3 and NH2groups on the

11

ZENN surface, leaded to less adsorbed proteins, thus less cell adhesion.

12 13

4. Conclusions

14

This research work provides a facile method to fabricate superhydrophobic/hydrophobic

15

surfaces.Zein

16

fabricated using electropinning, andthe highest WCA of the prepared ZENNs could reach

17

153.6±2.1°.

18

hydrophobicity of the ZENNs were studied. The increase of the zein concentration from 5 to

19

10 wt% resulted in an increase in the surface hydrophobicity, while its furtherincrease from 10

20

to 30 wt% resulted in a decrease in the surface hydrophobicity.The increase of

21

electrospinningvoltage from 10 to 18 kV resulted in an increase in the surface hydrophobicity,

22

while its furtherincrease from 18 to 24kV resulted in a decrease in the surface hydrophobicity.

23

The difference on the surfacehydrophobicity was mainly attributed to difference in surface

24

morphology. The cell adhesion and cell growth on the ZENNs were also studied. It was

25

observed that the cell successfully attached onto the nanofibrous network. There were more

superhydrophobic/hydrophobic

The

effects

nanofibrous

of zeinconcentration

11

and

networkswere

successfully

electrospinningvoltage

on

the

1

cells attached ontoZENN compared to ZCF. When the surface hydrophobicity of ZENN

2

decreased, the amount of the cells attached on the ZENN dramatically reduced. It

3

isdemonstratedthat the zein electrospun nanofibrous structure has greatpotentialin various

4

applications of tissue engineering.

5 6 7

Acknowledgements

8

This work is financially supported by the National Natural Science Foundation of China

9

(project number: 51303153) and the Hong Kong Polytechnic University (project number: 1-

10

ZVA9, 5-ZDAJ, G-UC07, and G-YK99). We appreciate the help from the Material Research

11

Center of the Hong Kong Polytechnic University.

12 13 14 15 16 17

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1

2 3

Figure 1. SEM and WCA images of zein films obtained by electrospinning from a solution

4

containing (a) 5, (b) 10, (c) 15, (d) 20, (e) 25, and (f) 30 wt% zein in 80% ethanol,

5

respectively.

6 7 14

1 2

Figure 2. SEM and WCA images of the morphology of the zein films obtained by

3

electrospinning at (a) 10, (b) 18, (c) 21kV, and (d) 24kV from a solution containing 10 wt%

4

zein in 80% ethanol, respectively.

5

6 7

Figure 3.(a) SEM image and (b) Fluorescence image of HepG2 cell growth on the ZENN.

8 15

1

2 3

Figure 4. Fluorescence images of HepG2 cell attachment on (a) ZENN and (b) ZCF.

4 5

6 7

Figure 5.Fluorescence images of HepG2 cell attachment on the ZENNs treated with oxygen

8

plasma for (a) 0 min, (b) 5 min, and (c) 10 min, respectively. Fluorescence images of

9

UMR106 cell attachment on the ZENNs treated with oxygen plasma for (d) 0 min, (e) 5 min,

10

and (f) 10 min, respectively.

11 12

16

1 2 3 4 5 6 7 8 9 10

Table 1. The WCAs of ZENNs obtained from a solution containing 5, 10, 15, 20, 25, and 30

11

wt% zein in 80% ethanol, respectively. Zein (wt%) Morphology

5 bead

10 bead

15 bead-fiber

20 bead-fiber

25 fiber

30 fiber

WCA (°)

138.2±3.0

153.6±2.1

150.1±1.3

143.8±2.4

141.0±1.6

136.4±2.1

12 13 14 15

Table 2. The WCAs of zein surfaces obtained by electrospinning at applied voltages of 10,

16

18, 21, and 24kV from a solution containing 10 wt% zein in 80% ethanol, respectively. Voltage (kV) Shape WCA (°)

10 bead

18 bead

21 bead

24 bead-fiber

130.5±1.0

153.6±2.1

139.4±2.3

135.5±1.5

17

17