Accepted Manuscript Title: Preparation of Transparent Zein Films for Cell Culture Applications Author: Yi-Long Han Qin Xu Zhi-Qian Lu Jin-Ye Wang PII: DOI: Reference:
S0927-7765(14)00217-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.019 COLSUB 6396
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-11-2013 1-4-2014 16-4-2014
Please cite this article as: Y.-L. Han, Q. Xu, Z.-Q. Lu, J.-Y. Wang, Preparation of Transparent Zein Films for Cell Culture Applications, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.019 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.
Preparation of Transparent Zein Films for Cell
2
Culture Applications
3
Yi-Long Han1, Qin Xu1, Zhi-Qian Lu2*, Jin-Ye Wang1*
cr
ip t
1
1
School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China;
5
2
Department of Cardio-thoracic Surgery, Shanghai No.6 People Hospital, Affiliated Shanghai
6
Jiao Tong University, Shanghai, 200233, China.
9
*Corresponding author: Prof. Jin-Ye Wang, School of Biomedical Engineering, Shanghai Jiao
10
Tong
11
[email protected].
12
Cardio-thoracic Surgery, Shanghai No.6 People Hospital, Affiliated Shanghai Jiao Tong
13
University, Shanghai, 200233, China.
15
800
Dongchuan
Fax/Tel:
Ac ce p
14
University,
te
d
8
M
7
an
us
4
Road,
021-34205824;
Shanghai, Prof.
200240,
Zhi-Qian
Lu,
China.
E-mail:
Department
of
16 17 18 1
Page 1 of 28
18
Abstract In our previous studies, zein has shown good cell compatibility in both films and porous
20
scaffolds. To use the zein film in microfluidic devices or as a cell culture substrate, swelling and
21
excessive degradation should be avoided. Moreover, the film should be transparent. In this study,
22
we found that the zein film could maintain good transparency even after swelling when it was
23
treated for 20 min at 121°C, 100% relative humidity and 103.4 kPa, especially for the zein film
24
without the addition of a plasticizer. FT-IR and XRD analysis showed that the ratio of α-helix of
25
zein structure decreased and the ratio of its β-sheet increased. Proliferation of NIH 3T3 cells on
26
the transparent zein film was as cytocompatible as the non-treated one. The improved
27
transparency of zein films after swelling will expand the application field of zein.
M
an
us
cr
ip t
19
31 32 33 34
te
30
Keywords: zein; transparent; degradation; swelling; cell culture
Ac ce p
29
d
28
35
2
Page 2 of 28
35
1.
Introduction Optically transparent polymers have been used in many applications, such as for
37
microfluidic devices and cell culture substrates. In microfluidic applications, properties of the
38
material that may be of fundamental importance include optical properties, biocompatibility, and
39
many others.[1] The use of transparent biomaterials is mandatory for live-imaging of cellular
40
dynamics.[2] These new microfluidic devices are made primarily of poly(dimethylsiloxane)
41
(PDMS) due to attractive physical and mechanical properties, such as biocompatibility, low cost,
42
optical transparency, and ease of fabrication.[3] However, petroleum-based polymers and plastic
43
materials pose potential environmental concerns, especially when they are used as disposable
44
devices. Biodegradable materials that have been used as substrates in microfluidic devices
45
include silk fibroin, gelatin, calcium alginate and poly (DL-lactic-co-glycolide) (PLGA).[4-7]
46
Among these, the natural materials have unique environmental advantages.
d
M
an
us
cr
ip t
36
Due to its solubility in alcohol–water mixtures, zein belongs to a family of proteins known
48
as prolamins.[8] It has a large amount of hydrophobic residues, such as leucine, proline, alanine,
49
and phenylalanine, which contribute to its significant hydrophobic properties. A structural model
50
of zein has been developed under the assumption that each of the tandem repeat units form a
51
single α-helix, and these units are joined by glutamine-rich “turns” or loops.[9] Wang et al.
52
indicated that the basic α-helix structure in zein was stable, as it was not easily changed by
53
heating, dissolution, or mechanical treatment.[10] Zein has also been shown to be a natural,
54
non-toxic, and biocompatible material that is compatible with human umbilical vein endothelial
55
cells, human liver cells and mice fibroblast cells.[11, 12] However, zein can lose and gain water
56
due to changes in relative humidity, and once in contact with water, it becomes opaque due to
57
protein precipitation.[13] This phenomenon makes zein films unacceptable for some
Ac ce p
te
47
3
Page 3 of 28
applications. For use as a food coating or for other uses, zein must be made less susceptible to
59
water.[14] If we can develop methods to improve transparency, its application field will be
60
extended.
ip t
58
Zein films can be plasticised with glycerol (GLY), triethylene glycol (TEG), dibutyl
62
tartrate (DBT), levulinic acid (LA), polyethylene glycol 300 (PEG), and oleic acid (OA).[15]
63
Water, which is the most important solvent of proteins in nature, interacts with protein through
64
hydrogen bonds and promotes solvent-induced crystallisation.[16] Research on water plasticising
65
process has mostly focused on improving the mechanical properties of the material, with little
66
focus on the transparency of the resulting film. However, for microfluidic devices and cell
67
culture substrates, transparency is an important factor. Macoshi et al. found that an amorphous
68
zein film was converted to β-form crystals by steam treating at 120°C for 15 min.[17] In gluten,
69
the α-sheet content has been reported to change as hydration increases.[18] In silk, to increase
70
the amount of crystalline β–sheets (to ～60%), the silk is steam-autoclaved, causing induction of
71
β-sheets by both high temperature and pressure.[19, 20]
Ac ce p
te
d
M
an
us
cr
61
72
The natural properties of zein allow for easy swelling and degradation. Zein films can be
73
easily prepared by the evaporation of an opaque ethanol-water solution containing zein. One of
74
the aim of this study is to improve transparency of the zein film. The changes in the structure of
75
the film were characterised by fourier transform infrared (FT-IR) spectroscopy and X-ray
76
diffraction (XRD) analysis. The in vitro degradation, cell adhesion and optical transparency of
77
these films were also evaluated.
78 79
2. Materials and Methods
4
Page 4 of 28
80
2.1 Preparation of zein films Firstly, 0.2 g of zein (Wako Pure Chemical Industries LTD., Osaka, Japan) was dissolved
82
in 1 ml of a 90% (v/v) anhydrous ethanol/water mixture by magnetic stirring at ambient
83
temperature. Zein films of 50 to 100 μm in thickness were prepared by casting the protein
84
solution on coverslips.[21] The solvent was evaporated at room temperature under ambient
85
conditions. Film thickness was controlled by the amount of protein solution used in the sample
86
preparation and measured by a screw micrometer. The treated films were obtained by
87
steam-autoclaving (121°C, 100% relative humidity and 103.4 kPa for 20 min). The mass ratio of
88
glycerol (GLY) or oleic acid (OA) to zein solution was 3% (w/v).
89
2.2 Optical characterization
M
an
us
cr
ip t
81
A dry or hydrated sample film was taped onto the sample holder facing to the incident
91
beam, and transmittance was measured using a spectrometer (U-3010, Hitachi, Japan). Hydrated
92
samples were prepared by wetting films in a secondary solution of water, equilibrating these
93
films overnight, and then blotting away any excess water. Bare glass cover slip without film was
94
used as a blank.
95
2.3 Swelling experiment
Ac ce p
te
d
90
96
Zein solution was cast onto the glass wafer with a diameter of 1 cm and the thickness of
97
zein film was about 50 μm. The swelling characteristic of the films was then determined by
98
immersing dried samples to swell in Milli Q water at 25°C for 6 h. Samples were taken out and
99
their weights were measured at intervals. The swelling ratio was calculated from the following
100
equation:
5
Page 5 of 28
101 102
Water uptake = (m1- m0)/ m0×100% m1 is the weight of the swollen sample at certain times and m0 is the weight of the dried sample. All experiments were carried out in triplicate.
104
2.4 In vitro degradation
ip t
103
Treated and untreated zein films (n=4) were subjected to a degradation condition in which
106
samples were digested with 2.5 mg/ml trypsin (from porcine pancreas, Amerson, USA) in PBS
107
(pH 7.4, 100 units/ml penicillin and 100 μg/ml streptomycin) at 37°C. The degradation ratio was
108
calculated by weighing the samples.
109
2.5 Contact angle measurements
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an
us
cr
105
Sessile drop air–water contact angles of glass (1 cm in diameter) coated with treated and
111
untreated zein films (n=5) were measured using an Optical Contact Angle Measuring Device
112
OCA20 (Dataphysics, Inc., Stuttgart, Germany).
113
2.6 FT-IR spectroscopy and XRD
Ac ce p
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114
FT-IR spectra of treated and untreated zein films were measured with a Bruker FT-IR
115
Equinqx55 spectrometer. This analysis was performed in triplicate. XRD patterns of the zein
116
films were obtained by a D8 ADVANCE X-ray diffractometer (Bruker, Germany). A Theta
117
goniometer was used to scan from 2-90°2θ with a step size of 0.06°2θ at a time of 2700 s.
118
2.7 SDS-PAGE
119
SDS-PAGE was carried out on polyacrylamide gels 12% (w/v) (Bio-Rad Laboratories,
120
USA). The untreated zein powder (up), the treated zein film (tf) and the untreated zein film (uf)
6
Page 6 of 28
were as the samples. 10 μg samples dissolved in 10 μl electrophoretic sample buffer (0.125 M
122
Tris–HCl, 4% SDS, 20% glycerol and bromophenol blue 0.01%, 10% 2-mercaptoethanol, pH
123
6.8). These samples were heated at 95°C for 5 min before submitted to SDS-PAGE The gel was
124
stained with Coomassie blue R250 for band visualization. The molecular weight standards were
125
Phosphorylase b (97.4 kDa), Bovine serum albumin (66.2 kDa), Ovalbumin (45 kDa), Carbonic
126
anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa) and Lysozyme (14.4 kDa).
127
2.8 Cell culture
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Mice fibroblast cells (NIH 3T3) were grown in DMEM (Dulbecco's Modified Eagle
129
Medium) with a high glucose content (Invitrogen, UK), containing 10% foetal bovine serum, 100
130
units/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a humidified
131
incubator at 37°C in an atmosphere of 95% air/5% CO2. The cells were trypsinised using a
132
0.25% trypsin solution in PBS for 2 min, resuspended in complete culture medium, and seeded
133
onto the glass wafers with the untreated and treated zein films at a concentration of 105 cells/ml.
134
The growth curve was drawn after the experiment had been carried out in 35 mm plates.
135
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to the culture
136
medium to obtain quantitative values of NIH 3T3 cell proliferation on the untreated and treated
137
zein films. The morphologies and proliferation of the cells were observed with a fluorescence
138
microscope (IX71, Olympus, Japan).
139
2.9 Statistical analysis
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140
Origin 7.5 (Origin Lab Co. Ltd., USA) was used to compare data to determine variability.
141
A paired t-test post hoc analysis was conducted to determine p values. All data are reported as
142
the mean ± standard deviation (SD).
7
Page 7 of 28
143 3. Results and Discussion
145
3.1 Optical characterization
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144
Tapioca starch films have high transparency of 85% transmittance in visible light
147
district.[22] Perfluoropolyethers (PFPE) films showed a high transmittance of 98% at 550
148
nm.[23] The transmittance of zein films was over 92% between 450-800 nm in dry state, which
149
could be considered as good transparency.[24] OA or GLY was added as a plasticiser (3% w/v).
150
The transparency of the zein film with OA was similar to that of the pure zein film. However,
151
GLY reduced the film’s transparency. This loss of transparency was presumably due to the
152
wettability of the two plasticisers. OA is a hydrophobic substance and is almost non-miscible
153
with water. By contrast, GLY is hydrophilic and can therefore be infinitely miscible with water.
154
OA and zein could act as similar barriers to water, while GLY could easily absorb water from air
155
to form small droplets on the film surface and in internal voids. The light scattering of these
156
water droplets may reduce film transparency.
Ac ce p
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157
As microfluidic devices and cell culture substrates are always used in a hydrated state, it is
158
necessary for zein films to maintain good transparency when hydrated. However, zein films
159
swelled when immersing in ultra-pure Milli-Q water, which induced a sharp decrease of
160
transparency of the films. Although OA in zein films can retard the process of swelling due to its
161
hydrophobic property, an opaque white film eventually formed with time (shown in
162
supplemental data). Besides, GLY accelerated the rate of swelling.
163
To solve the problem, various processing conditions such as heating and pressuring were
164
tested. It was found that even the heating temperature was elevated to 160°C for 20 minutes, the 8
Page 8 of 28
swelling characteristic of the zein film could not be improved. Additionally, heating at lower
166
temperature such as 100°C under a humid condition couldn’t change the swelling characteristic
167
of the zein film either. However, conditions of heating temperature 121°C and pressure 103.4
168
kPa improved the transparency of the zein film significantly. Addition of 3% GLY also showed a
169
similar result, while the transparency of the zein film containing 3% OA significantly decreased
170
in the same condition. As oleic acid is embedded in zein film. It could be considered that
171
expansion volume of zein and OA is different when heating, a small gap would appear inside the
172
film, causing light scattering and reducing transparency.
an
us
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165
The untreated zein films displayed a better optical transmittance than the treated ones in
174
the dry state (Fig. 1 a1, a2, a3 and a4). While it became a reverse tendency that the treated zein
175
films possessed a much better transmittance than the untreated ones when fully hydrated (Fig. 1
176
b1, b2, b3 and b4). Before swelling, the untreated films displayed transmittances of 97% at 800
177
nm, 92% at 450 nm, and 57% at 350 nm, whereas the treated film displayed transmittances of
178
98% at 800 nm, 91% at 450 nm, and 56% at 350 nm, as shown in Fig. 1’s a1 and a3. After
179
swelling, the untreated films displayed transmittances of 4% at 800 nm, 4% at 450 nm, and 2% at
180
350 nm, whereas the treated films displayed transmittances of 98% at 800 nm, 94% at 450 nm,
181
and 58% at 350 nm, as shown in Fig. 1’s b1 and b3. It shows the same result for zein films with
182
thickness of 100 μm. i.e., the untreated films displayed transmittances of 99% at 800 nm, 91% at
183
450 nm, and 41% at 350 nm, and the treated films displayed transmittances of 99% at 800 nm,
184
88% at 450 nm, and 36% at 350 nm before swelling, as shown in Fig.1’s a2 and a4. After
185
swelling, the untreated films displayed transmittances of 0.8% at 800 nm, 0.4% at 450 nm, and
186
0.2% at 350 nm, and the treated films displayed transmittances of 98% at 800 nm, 87% at 450
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9
Page 9 of 28
187
nm, and 37% at 350 nm, as shown in Fig. 1’s b2 and b4. The significant improvement in
188
transparency when hydrated after treatment could be clearly seen from Figure 2. Soaking in water caused the untreated films swelling and opaque. One reason might be the
ip t
189
embedded small water droplets in the films
Soaking may also increase the zein-zein interaction
191
and cause the aggregation of zein molecules. Both lead to increased scattering. Heating and
192
humidity prevents water entering into the films in the form of water droplets when swelling, so
193
the films remained transparent. This improved transparent property may be useful in microfluidic
194
devices and cell culture substrates. As a consideration for a more environmental friendly and
195
agricultural based alternative to PDMS, zein microfluidic device has been studied to show
196
comparable bond strength and similar processes of fabrication and distinct permeability to small
197
molecules. But the deficiency in transparency limits the application.[13]
198
3.2 Swelling of zein films
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d
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190
Fig. 3 shows the swelling kinetic curves of the treated and untreated zein films in water.
200
The untreated zein films show a quick water uptake rate, and the rate was obviously lowered
201
after the heat-moist treatment. Moreover, the time to reach a plateau of water uptake (equilibrium
202
water uptake) was shorter for the treated zein films than that of the untreated ones. This might be
203
due to the heat-moist process, but how this process restrains the swelling behaviour of the zein
204
films is still unclear. The lower volume of water uptake plus the hydrophobic property of zein
205
would be beneficial to a material used in microfluidic devices and cell culture substrates.
206
Additionally, the lower rate of water uptake may be the reason of the high transparency observed
207
after swelling.
208
3.3 In vitro degradation
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199
10
Page 10 of 28
Biodegradable zein films used as cell culture substrates should be resistant to degradation
210
during cell culture. Therefore, the enzymatic hydrolysis of the zein films was studied using
211
trypsin. The degradation rate of the zein films with different incubation times is shown in Fig. 4.
212
During the entire period, the treated zein films showed significant low degradation rate. After 2
213
hours, the untreated zein film was degraded approximately 10%, while the treated zein film was
214
approximately 3.8% (p
S0927-7765(14)00217-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.04.019 COLSUB 6396
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
22-11-2013 1-4-2014 16-4-2014
Please cite this article as: Y.-L. Han, Q. Xu, Z.-Q. Lu, J.-Y. Wang, Preparation of Transparent Zein Films for Cell Culture Applications, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.04.019 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.
Preparation of Transparent Zein Films for Cell
2
Culture Applications
3
Yi-Long Han1, Qin Xu1, Zhi-Qian Lu2*, Jin-Ye Wang1*
cr
ip t
1
1
School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China;
5
2
Department of Cardio-thoracic Surgery, Shanghai No.6 People Hospital, Affiliated Shanghai
6
Jiao Tong University, Shanghai, 200233, China.
9
*Corresponding author: Prof. Jin-Ye Wang, School of Biomedical Engineering, Shanghai Jiao
10
Tong
11
[email protected].
12
Cardio-thoracic Surgery, Shanghai No.6 People Hospital, Affiliated Shanghai Jiao Tong
13
University, Shanghai, 200233, China.
15
800
Dongchuan
Fax/Tel:
Ac ce p
14
University,
te
d
8
M
7
an
us
4
Road,
021-34205824;
Shanghai, Prof.
200240,
Zhi-Qian
Lu,
China.
E-mail:
Department
of
16 17 18 1
Page 1 of 28
18
Abstract In our previous studies, zein has shown good cell compatibility in both films and porous
20
scaffolds. To use the zein film in microfluidic devices or as a cell culture substrate, swelling and
21
excessive degradation should be avoided. Moreover, the film should be transparent. In this study,
22
we found that the zein film could maintain good transparency even after swelling when it was
23
treated for 20 min at 121°C, 100% relative humidity and 103.4 kPa, especially for the zein film
24
without the addition of a plasticizer. FT-IR and XRD analysis showed that the ratio of α-helix of
25
zein structure decreased and the ratio of its β-sheet increased. Proliferation of NIH 3T3 cells on
26
the transparent zein film was as cytocompatible as the non-treated one. The improved
27
transparency of zein films after swelling will expand the application field of zein.
M
an
us
cr
ip t
19
31 32 33 34
te
30
Keywords: zein; transparent; degradation; swelling; cell culture
Ac ce p
29
d
28
35
2
Page 2 of 28
35
1.
Introduction Optically transparent polymers have been used in many applications, such as for
37
microfluidic devices and cell culture substrates. In microfluidic applications, properties of the
38
material that may be of fundamental importance include optical properties, biocompatibility, and
39
many others.[1] The use of transparent biomaterials is mandatory for live-imaging of cellular
40
dynamics.[2] These new microfluidic devices are made primarily of poly(dimethylsiloxane)
41
(PDMS) due to attractive physical and mechanical properties, such as biocompatibility, low cost,
42
optical transparency, and ease of fabrication.[3] However, petroleum-based polymers and plastic
43
materials pose potential environmental concerns, especially when they are used as disposable
44
devices. Biodegradable materials that have been used as substrates in microfluidic devices
45
include silk fibroin, gelatin, calcium alginate and poly (DL-lactic-co-glycolide) (PLGA).[4-7]
46
Among these, the natural materials have unique environmental advantages.
d
M
an
us
cr
ip t
36
Due to its solubility in alcohol–water mixtures, zein belongs to a family of proteins known
48
as prolamins.[8] It has a large amount of hydrophobic residues, such as leucine, proline, alanine,
49
and phenylalanine, which contribute to its significant hydrophobic properties. A structural model
50
of zein has been developed under the assumption that each of the tandem repeat units form a
51
single α-helix, and these units are joined by glutamine-rich “turns” or loops.[9] Wang et al.
52
indicated that the basic α-helix structure in zein was stable, as it was not easily changed by
53
heating, dissolution, or mechanical treatment.[10] Zein has also been shown to be a natural,
54
non-toxic, and biocompatible material that is compatible with human umbilical vein endothelial
55
cells, human liver cells and mice fibroblast cells.[11, 12] However, zein can lose and gain water
56
due to changes in relative humidity, and once in contact with water, it becomes opaque due to
57
protein precipitation.[13] This phenomenon makes zein films unacceptable for some
Ac ce p
te
47
3
Page 3 of 28
applications. For use as a food coating or for other uses, zein must be made less susceptible to
59
water.[14] If we can develop methods to improve transparency, its application field will be
60
extended.
ip t
58
Zein films can be plasticised with glycerol (GLY), triethylene glycol (TEG), dibutyl
62
tartrate (DBT), levulinic acid (LA), polyethylene glycol 300 (PEG), and oleic acid (OA).[15]
63
Water, which is the most important solvent of proteins in nature, interacts with protein through
64
hydrogen bonds and promotes solvent-induced crystallisation.[16] Research on water plasticising
65
process has mostly focused on improving the mechanical properties of the material, with little
66
focus on the transparency of the resulting film. However, for microfluidic devices and cell
67
culture substrates, transparency is an important factor. Macoshi et al. found that an amorphous
68
zein film was converted to β-form crystals by steam treating at 120°C for 15 min.[17] In gluten,
69
the α-sheet content has been reported to change as hydration increases.[18] In silk, to increase
70
the amount of crystalline β–sheets (to ～60%), the silk is steam-autoclaved, causing induction of
71
β-sheets by both high temperature and pressure.[19, 20]
Ac ce p
te
d
M
an
us
cr
61
72
The natural properties of zein allow for easy swelling and degradation. Zein films can be
73
easily prepared by the evaporation of an opaque ethanol-water solution containing zein. One of
74
the aim of this study is to improve transparency of the zein film. The changes in the structure of
75
the film were characterised by fourier transform infrared (FT-IR) spectroscopy and X-ray
76
diffraction (XRD) analysis. The in vitro degradation, cell adhesion and optical transparency of
77
these films were also evaluated.
78 79
2. Materials and Methods
4
Page 4 of 28
80
2.1 Preparation of zein films Firstly, 0.2 g of zein (Wako Pure Chemical Industries LTD., Osaka, Japan) was dissolved
82
in 1 ml of a 90% (v/v) anhydrous ethanol/water mixture by magnetic stirring at ambient
83
temperature. Zein films of 50 to 100 μm in thickness were prepared by casting the protein
84
solution on coverslips.[21] The solvent was evaporated at room temperature under ambient
85
conditions. Film thickness was controlled by the amount of protein solution used in the sample
86
preparation and measured by a screw micrometer. The treated films were obtained by
87
steam-autoclaving (121°C, 100% relative humidity and 103.4 kPa for 20 min). The mass ratio of
88
glycerol (GLY) or oleic acid (OA) to zein solution was 3% (w/v).
89
2.2 Optical characterization
M
an
us
cr
ip t
81
A dry or hydrated sample film was taped onto the sample holder facing to the incident
91
beam, and transmittance was measured using a spectrometer (U-3010, Hitachi, Japan). Hydrated
92
samples were prepared by wetting films in a secondary solution of water, equilibrating these
93
films overnight, and then blotting away any excess water. Bare glass cover slip without film was
94
used as a blank.
95
2.3 Swelling experiment
Ac ce p
te
d
90
96
Zein solution was cast onto the glass wafer with a diameter of 1 cm and the thickness of
97
zein film was about 50 μm. The swelling characteristic of the films was then determined by
98
immersing dried samples to swell in Milli Q water at 25°C for 6 h. Samples were taken out and
99
their weights were measured at intervals. The swelling ratio was calculated from the following
100
equation:
5
Page 5 of 28
101 102
Water uptake = (m1- m0)/ m0×100% m1 is the weight of the swollen sample at certain times and m0 is the weight of the dried sample. All experiments were carried out in triplicate.
104
2.4 In vitro degradation
ip t
103
Treated and untreated zein films (n=4) were subjected to a degradation condition in which
106
samples were digested with 2.5 mg/ml trypsin (from porcine pancreas, Amerson, USA) in PBS
107
(pH 7.4, 100 units/ml penicillin and 100 μg/ml streptomycin) at 37°C. The degradation ratio was
108
calculated by weighing the samples.
109
2.5 Contact angle measurements
M
an
us
cr
105
Sessile drop air–water contact angles of glass (1 cm in diameter) coated with treated and
111
untreated zein films (n=5) were measured using an Optical Contact Angle Measuring Device
112
OCA20 (Dataphysics, Inc., Stuttgart, Germany).
113
2.6 FT-IR spectroscopy and XRD
Ac ce p
te
d
110
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FT-IR spectra of treated and untreated zein films were measured with a Bruker FT-IR
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Equinqx55 spectrometer. This analysis was performed in triplicate. XRD patterns of the zein
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films were obtained by a D8 ADVANCE X-ray diffractometer (Bruker, Germany). A Theta
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goniometer was used to scan from 2-90°2θ with a step size of 0.06°2θ at a time of 2700 s.
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2.7 SDS-PAGE
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SDS-PAGE was carried out on polyacrylamide gels 12% (w/v) (Bio-Rad Laboratories,
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USA). The untreated zein powder (up), the treated zein film (tf) and the untreated zein film (uf)
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were as the samples. 10 μg samples dissolved in 10 μl electrophoretic sample buffer (0.125 M
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Tris–HCl, 4% SDS, 20% glycerol and bromophenol blue 0.01%, 10% 2-mercaptoethanol, pH
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6.8). These samples were heated at 95°C for 5 min before submitted to SDS-PAGE The gel was
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stained with Coomassie blue R250 for band visualization. The molecular weight standards were
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Phosphorylase b (97.4 kDa), Bovine serum albumin (66.2 kDa), Ovalbumin (45 kDa), Carbonic
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anhydrase (31 kDa), Soybean trypsin inhibitor (21.5 kDa) and Lysozyme (14.4 kDa).
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2.8 Cell culture
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Mice fibroblast cells (NIH 3T3) were grown in DMEM (Dulbecco's Modified Eagle
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Medium) with a high glucose content (Invitrogen, UK), containing 10% foetal bovine serum, 100
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units/ml penicillin and 100 μg/ml streptomycin. The cells were maintained in a humidified
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incubator at 37°C in an atmosphere of 95% air/5% CO2. The cells were trypsinised using a
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0.25% trypsin solution in PBS for 2 min, resuspended in complete culture medium, and seeded
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onto the glass wafers with the untreated and treated zein films at a concentration of 105 cells/ml.
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The growth curve was drawn after the experiment had been carried out in 35 mm plates.
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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added to the culture
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medium to obtain quantitative values of NIH 3T3 cell proliferation on the untreated and treated
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zein films. The morphologies and proliferation of the cells were observed with a fluorescence
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microscope (IX71, Olympus, Japan).
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2.9 Statistical analysis
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Origin 7.5 (Origin Lab Co. Ltd., USA) was used to compare data to determine variability.
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A paired t-test post hoc analysis was conducted to determine p values. All data are reported as
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the mean ± standard deviation (SD).
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143 3. Results and Discussion
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3.1 Optical characterization
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Tapioca starch films have high transparency of 85% transmittance in visible light
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district.[22] Perfluoropolyethers (PFPE) films showed a high transmittance of 98% at 550
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nm.[23] The transmittance of zein films was over 92% between 450-800 nm in dry state, which
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could be considered as good transparency.[24] OA or GLY was added as a plasticiser (3% w/v).
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The transparency of the zein film with OA was similar to that of the pure zein film. However,
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GLY reduced the film’s transparency. This loss of transparency was presumably due to the
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wettability of the two plasticisers. OA is a hydrophobic substance and is almost non-miscible
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with water. By contrast, GLY is hydrophilic and can therefore be infinitely miscible with water.
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OA and zein could act as similar barriers to water, while GLY could easily absorb water from air
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to form small droplets on the film surface and in internal voids. The light scattering of these
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water droplets may reduce film transparency.
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As microfluidic devices and cell culture substrates are always used in a hydrated state, it is
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necessary for zein films to maintain good transparency when hydrated. However, zein films
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swelled when immersing in ultra-pure Milli-Q water, which induced a sharp decrease of
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transparency of the films. Although OA in zein films can retard the process of swelling due to its
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hydrophobic property, an opaque white film eventually formed with time (shown in
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supplemental data). Besides, GLY accelerated the rate of swelling.
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To solve the problem, various processing conditions such as heating and pressuring were
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tested. It was found that even the heating temperature was elevated to 160°C for 20 minutes, the 8
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swelling characteristic of the zein film could not be improved. Additionally, heating at lower
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temperature such as 100°C under a humid condition couldn’t change the swelling characteristic
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of the zein film either. However, conditions of heating temperature 121°C and pressure 103.4
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kPa improved the transparency of the zein film significantly. Addition of 3% GLY also showed a
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similar result, while the transparency of the zein film containing 3% OA significantly decreased
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in the same condition. As oleic acid is embedded in zein film. It could be considered that
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expansion volume of zein and OA is different when heating, a small gap would appear inside the
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film, causing light scattering and reducing transparency.
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The untreated zein films displayed a better optical transmittance than the treated ones in
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the dry state (Fig. 1 a1, a2, a3 and a4). While it became a reverse tendency that the treated zein
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films possessed a much better transmittance than the untreated ones when fully hydrated (Fig. 1
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b1, b2, b3 and b4). Before swelling, the untreated films displayed transmittances of 97% at 800
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nm, 92% at 450 nm, and 57% at 350 nm, whereas the treated film displayed transmittances of
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98% at 800 nm, 91% at 450 nm, and 56% at 350 nm, as shown in Fig. 1’s a1 and a3. After
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swelling, the untreated films displayed transmittances of 4% at 800 nm, 4% at 450 nm, and 2% at
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350 nm, whereas the treated films displayed transmittances of 98% at 800 nm, 94% at 450 nm,
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and 58% at 350 nm, as shown in Fig. 1’s b1 and b3. It shows the same result for zein films with
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thickness of 100 μm. i.e., the untreated films displayed transmittances of 99% at 800 nm, 91% at
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450 nm, and 41% at 350 nm, and the treated films displayed transmittances of 99% at 800 nm,
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88% at 450 nm, and 36% at 350 nm before swelling, as shown in Fig.1’s a2 and a4. After
185
swelling, the untreated films displayed transmittances of 0.8% at 800 nm, 0.4% at 450 nm, and
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0.2% at 350 nm, and the treated films displayed transmittances of 98% at 800 nm, 87% at 450
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nm, and 37% at 350 nm, as shown in Fig. 1’s b2 and b4. The significant improvement in
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transparency when hydrated after treatment could be clearly seen from Figure 2. Soaking in water caused the untreated films swelling and opaque. One reason might be the
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embedded small water droplets in the films
Soaking may also increase the zein-zein interaction
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and cause the aggregation of zein molecules. Both lead to increased scattering. Heating and
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humidity prevents water entering into the films in the form of water droplets when swelling, so
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the films remained transparent. This improved transparent property may be useful in microfluidic
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devices and cell culture substrates. As a consideration for a more environmental friendly and
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agricultural based alternative to PDMS, zein microfluidic device has been studied to show
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comparable bond strength and similar processes of fabrication and distinct permeability to small
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molecules. But the deficiency in transparency limits the application.[13]
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3.2 Swelling of zein films
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Fig. 3 shows the swelling kinetic curves of the treated and untreated zein films in water.
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The untreated zein films show a quick water uptake rate, and the rate was obviously lowered
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after the heat-moist treatment. Moreover, the time to reach a plateau of water uptake (equilibrium
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water uptake) was shorter for the treated zein films than that of the untreated ones. This might be
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due to the heat-moist process, but how this process restrains the swelling behaviour of the zein
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films is still unclear. The lower volume of water uptake plus the hydrophobic property of zein
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would be beneficial to a material used in microfluidic devices and cell culture substrates.
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Additionally, the lower rate of water uptake may be the reason of the high transparency observed
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after swelling.
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3.3 In vitro degradation
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Biodegradable zein films used as cell culture substrates should be resistant to degradation
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during cell culture. Therefore, the enzymatic hydrolysis of the zein films was studied using
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trypsin. The degradation rate of the zein films with different incubation times is shown in Fig. 4.
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During the entire period, the treated zein films showed significant low degradation rate. After 2
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hours, the untreated zein film was degraded approximately 10%, while the treated zein film was
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approximately 3.8% (p
