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Epichlorohydrin-Crosslinked Hydroxyethyl Cellulose/ Soy Protein Isolate Composite Films as Biocompatible and Biodegradable Implants for Tissue Engineering Yanteng Zhao, Meng He, Lei Zhao, Shiqun Wang, Yinping Li, Li Gan, Mingming Li, Li Xu, Peter R. Chang, Debbie P Anderson, and Yun Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11152 • Publication Date (Web): 07 Jan 2016 Downloaded from http://pubs.acs.org on January 17, 2016

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ACS Applied Materials & Interfaces

1

Epichlorohydrin-Crosslinked Hydroxyethyl Cellulose/Soy

2

Protein Isolate Composite Films as Biocompatible and

3

Biodegradable Implants for Tissue Engineering

4 5

Yanteng Zhao,1# Meng He,2# Lei Zhao,1 Shiqun Wang,1 Yinping Li,1 Li Gan,1

6

Mingming Li,1 Li Xu,1 Peter R. Chang,3 Debbie P. Anderson3 and Yun Chen1

∗

7 8

1

9

University, Wuhan 430071, China

Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan

10

2

11

Jiangsu 224051, China

12

3

13

Agri-Food Canada, 107 Science Place, Saskatoon, SK, S7N 0X2, Canada

School of Materials Engineering, Yancheng Institute of Technology, Yancheng,

Bioproducts and Bioprocesses National Science Program, Agriculture and

14 15

∗

Corresponding author. Tel.: +86 27 6875 9509; fax: +86 27 6875 9142. E-mail

address: [email protected] (Yun Chen). (# These authors contributed equally to this work.) 1

ACS Paragon Plus Environment


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Abstract

2

A series of epichlorohydrin-crosslinked hydroxyethyl cellulose/soy protein isolate

3

composite films (EHSF) was fabricated from hydroxyethyl cellulose (HEC) and soy

4

protein isolate (SPI) using a process involving blending, crosslinking, solution casting

5

and evaporation. The films were characterized with FTIR, solid-state

6

UV-vis spectroscopy and mechanical testing. The results indicated that crosslinking

7

interactions occurred in the inter- and intra-molecules of HEC and SPI during the

8

fabrication process. The EHSF films exhibited homogenous structure and relative

9

high light transmittance, indicating there was a certain degree of miscibility between

10

HEC and SPI. The EHSF films exhibited a relative high mechanical strength in humid

11

state and an adjustable water uptake ratio and moisture absorption ratio.

12

Cytocompatibility, hemocompatibility and biodegradability were evaluated by a series

13

of in vitro and in vivo experiments. These results showed that the EHSF films had

14

good biocompatibility, hemocompatibility and anticoagulant effect. Furthermore,

15

EHSF films could be degraded in vitro and in vivo, and the degradation rate could be

16

controlled by adjusting the SPI content. Hence, EHSF films might have a great

17

potential for use in the biomedical field.

13

C NMR,

18 19

Key

words:

hydroxyethyl

20

biocompatibility, biodegradability

cellulose,

soy

protein

21

2


isolate,

crosslinking,

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INTRODUCTION

2

As the most abundant biopolymer in nature, cellulose has been used widely in the

3

textiles, papermaking, agriculture, and food industries. In recent decades, cellulose

4

has allowed for its use in specific purpose biomaterials such as hemodialysis devices,

5

hemostatic sponges, wound dressing, bioreactor, drug carrier and tissue engineering

6

scaffolds due to its safety, biocompatibility and biodegradability1-7. Additionally, in

7

order to fully exploit the abundant natural resources and broaden the application of

8

cellulose in the biomedical field, cellulose has been successfully converted into

9

hydrogels, sponges and films using dissolving, crosslinking or coagulation

10

methods8-12. Our previous work used a physical blending process and found that soy

11

protein isolate (SPI) improved biocompatibility and biodegradability of cellulose13,14.

12

Especially, a new variety of nerve guide conduit has be constructed from

13

SPI-modified cellulose and showed potential application for nerve repair in the field

14

of nerve tissue engineering14. Despite improvement in permeability and degradability

15

after SPI modification, the cellulose/SPI composite films maintained their original

16

shape and macrostructures, and had degraded only slightly 3-8 months after

17

implantation in rats, presumably due to the poor in vivo degradability of cellulose

18

itself. Moreover, all the physical modification couldn’t change the cellulose’s original

19

chemical structure. Indeed, the lack of cellulose enzyme in human body makes it is

20

difficult to realize cellulose’s actual degradation in vivo, which has severely hampers

21

its further applications as implanted and biodegradable biomaterials15. Therefore, it is

22

a crucial to improve in vivo biodegradability of cellulose while maintaining proper 3



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mechanical properties, hydrophilicity and biocompatibility of cellulose and its

2

composites.

3

It is known that a series of cellulose derivatives, such as cellulose acetate,

4

hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), carboxymethyl

5

cellulose (CMC), methylcellulose, and cyanoethyl cellulose, are derived from

6

cellulose through chemical reaction16-20. Among these cellulose derivatives, HEC and

7

CMC had been produced commercially and were widely used as water soluble

8

additives. One of the most interesting points is that both HEC and CMC are easy to be

9

dissolved in water. That means they can be degraded in vivo or at least could be

10

dissolved in body liquid, which show very promising applications as biodegradable or

11

bio-absorbable biomaterials. We postulated that using water-dissolved cellulose

12

derivatives (e.g. HEC) instead of cellulose for the preparation of cellulose/SPI

13

composites would improve the in vivo biodegradability due to the water-solubility of

14

HEC and the biodegradability of SPI. At the same time, HEC itself exhibits other

15

features such as good thickening, suspension, dispersion, emulsification and

16

film-forming similar to those of cellulose21. Conceivably, composite films fabricated

17

from HEC/SPI would retain the good processibility, relatively high mechanical

18

strength, and hydrophilicity of cellulose/SPI composite films, but have a much higher

19

in vivo biodegradability. However, another problem will exist for the HEC/SPI

20

composite films when they are used as implantations in body. In this case, HEC might

21

be quickly dissolved in body liquid and SPI is fast degraded in vivo, the HEC/SPI

22

composite

material

may

disintegrate

(or

degrade)

4


before

their

intended

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biofucntionality are still needed for living body. As such, the required biodegradation

2

rate, mechanical properties, and biofunctionalities need to be carefully considered

3

before a practical strategy is devised. To solve this problem, chemical crosslinking

4

reactions could be applied. Thus, in this work, HEC was blended with SPI and

5

epichlorohydrin (ECH) was used to crosslink the HEC and SPI. It was expected that

6

the solubility and biodegradability of the composite materials could be controlled by

7

adjusting the degree of crosslinking, and the mechanical properties, hydropilibility

8

and biocompatibility of HEC/SPI composite films maintained at levels previously

9

reported for cellulose/SPI composites13,14. The structure and properties of the prepared

10

ECH-crosslinked HEC/SPI composite films (EHSF) were characterized by Fourier

11

transform infrared spectroscopy (FTIR), scanning electron microscope (SEM),

12

ultraviolet (UV) spectrometer and mechanical properties testing. Moreover,

13

cytocompatibility was evaluated through cell culture experiment and determined by

14

MTT assay. Hemocompatibility was tested by the plasma recalcification time (PRT)

15

testing, platelet adhesion, and hemolysis rate measurement. Finally, biodegradability

16

and histocompatibility were also evaluated by in vitro degradation and in vivo

17

implantation experiments. Results were expected to show that novel biomaterials with

18

excellent physical and chemical properties, good biocompatibility, and controllable in

19

vivo biodegradability based on cellulose derivative and SPI could be obtained.

20 21

EXPERIMENTAL SECTION

22

Materials Hydroxyethyl cellulose (viscosity, 30000 mPa), was purchased from 5



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1

Shandong Head Reagent Co. Ltd. (Shandong, China). Commercial soy protein isolate

2

(SPI), with weight-average molecular weight (Mw) of 2.05×105, was obtained from

3

DuPont-Yunmeng Protein Technology (Yunmeng, China). HEC and SPI were

4

vacuum-dried for 24 h at 60 ºC before use. Epichlorohydrin (ECH, analytical grade,

5

liquid, 1.18 g/mL), NaOH and acetic acid were purchased from Sinopharm Chemical

6

Reagent Co. Ltd. (Shanghai, China). Modified Eagle’s Medium (MEM), fetal bovine

7

serum (FBS) and 3-[4,5-dimethyl-2- thiazoly1]-2,5-diphenyl-2H-tetrazolium bromide

8

(MTT) were obtained from Invitrogen Corporation (Gibco BRL, Grand Island, NY,

9

USA). Other chemicals were of analytical grade agents and used without further

10

treatment.

11

Preparation of Epichlorohydrin-crosslinked Hydroxyethyl cellulose/ Soy protein

12

isolate films. Epichlorohydrin-crosslinked HEC/SPI films were prepared as follows:

13

Briefly, a 2 wt% HEC aqueous solution was prepared by dissolving HEC in deionized

14

water. A 10 wt% SPI aqueous solution was prepared by dispersing 10 g SPI powders

15

into 60 g water and then 30 g NaOH aqueous solution (5 wt%) were added to dissolve

16

SPI. And then HEC and SPI solutions were mixed mechanically to get a series of

17

HEC/SPI blend solutions with SPI content (WSPI, calculated as the weight percent of

18

the amount weight of original SPI and HEC powders) of 0, 10, 30, 50, 70, 90 and 100

19

wt%, respectively. ECH (20 % of the weight based on the total weight of original SPI

20

and HEC powders) was added dropwisely in the blend solutions at room temperature

21

with stirring for 30 min, which were further degassed at 10 ºC by centrifugation for 10

22

min at 6000 rpm. Then, the degassed solutions were poured into molds (square 6


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1

polypropylene plastic plate with side width of 10 cm and height of 0.7 cm) and

2

air-dried at room temperature to form films. The films were immersed in 5 % acetic

3

acid solutions for 20 min to neutralize the NaOH, and then soaked into deionized

4

water to remove residue acetic acid. The resultant films were dried at 25 ºC and coded

5

as EHSF-n, where EHSF means ECH-crosslinked HEC/SPI films, “n” corresponds to

6

the initial SPI content (n=10, 30, 50, 70 and 90, respectively). For example, EHSF-10

7

means the ECH-crosslinked HEC/SPI film with 10 wt% SPI content (WSPI). The codes

8

and compositions of the films are listed in Table 1. The air-dried ECH-crosslinked

9

HEC film without SPI was washed with 5 wt% acetic acid/ethanol and followed by

10

ethanol to remove residual ECH, and was coded as EHSF-0. The ECH-crosslinked

11

SPI film without HEC was coded as EHSF-100. The he final weight of the resultant

12

films was around 75-79% of the theoretical weight based on the initial weight of HEC,

13

SPI and ECH. In some cases, the raw materials of SPI and HEC powders were used as

14

controls for comparison, which were coded as “SPI powder” and “HEC powder”,

15

respectively.

16

Characterization The EHSF-n films were vacuum-dried at 60 ºC for 24 h, and cut

17

into 2 × 2 cm2 pieces for the measurements by attenuated total reflectance Fourier

18

transform infrared spectroscopy (ATR-FTIR). Spectra were recorded on an FTIR

19

spectrometer (TNZ1-5700, Nicolet, USA) over the wavenumber range from 4000 to

20

600 cm−1. The infrared spectra of the raw materials (SPI and HEC powders) were

21

measured with a FTIR spectrometer (1600, Perkin−Elmer Co., UAS) over the

22

wavelength range from 4000 to 400 cm−1. The dry SPI and HEC powders were 7



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1

respectively mixed with KBr to laminate for the measurements of FTIR. The FTIR of

2

a physical blend containing 50% HEC and 50% SPI (coded as HEC/SPI-50) was

3

obtained by the same method. Solid-state 13C NMR spectra of the film samples were

4

recorded on a BRUKER AVANCE III spectrometer operated at a 13C frequency of 75

5

MHz using the combined technique of cross-polarization and magic angle spinning

6

(CP/MAS). Spinning speed was set at 5 KHz for all samples. The contact time was 3

7

ms, the acquisition time 50 ms and the recycle delay 3 s. A typical number of 1024

8

scans were acquired for each spectrum. The samples were cut into powder and dried

9

in a vacuum oven for 48 h before NMR testing. X-ray diffraction measurements were

10

measured with a WAXD diffractometer (D8-Advance, Bruker, USA). The patterns

11

with Cu Kα radiation (λ = 0.15405 nm) at 40 kV and 30 mA were recorded over the

12

region of 2θ from 5 to 40°, scanning rate was 2 o/min. The dried EHSF films were cut

13

into powders, and then the powders of EHSF, HEC, SPI and HEC/SPI-50 powders

14

were used for XRD analysis, respectively. Transmittance of the EHSF-n (n =0, 10, 30,

15

50, 70, 90 and 100) films was measured with a Shimadzu UV 2401 PC

16

spectrophotometer. The films, with a thickness of 0.22 mm, were cut into pieces (5

17

mm × 20 mm) and analyzed with UV spectrophotometer over a wavelength range

18

200-800 nm. The transmittance spectra were acquired using air as the background.

19

The resolution of the spectrophotometer was 1.5 nm and the photometric accuracy

20

was ± 0.01 Abs. The morphology of the films was observed on a scanning electron

21

microscope (SEM, VEGA3, TESCAN, Czech Republic) with 20 kV as the

22

accelerating voltage. The films were frozen in liquid nitrogen, and then fractured 8


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1

immediately. The surfaces and cross-sections of the films were coated with gold for

2

SEM observation. The mechanical properties of the humid films (films were placed

3

for 1 week in sealed container with a saturated K2CO3 water vapor atmosphere) were

4

tested on a universal testing machine (CMT6503, Shenzhen SANS Test Machine,

5

China) according to ISO6239-1986 (E) at a tensile rate of 5 mm/min. The mean

6

values of the tensile strength, the elongation at break, and the standard deviation of

7

the films were calculated from four specimens, respectively. Because the mechanical

8

properties are affected by environmental temperature and humidity, all measurements

9

were made under the same conditions. To measure the water uptake ratio, the EHSF-n

10

films were cut into disc (20 mm radius), dried in a vacuum oven at 70 oC for 24 h, and

11

then weighed to determine the initial dry mass (W0). The film samples were soaked in

12

deionized water at 37 oC for a specific time, and then carefully wiped with filter paper

13

to remove the excess water from the surface and weighed again (Wt). The water

14

uptake ratio was calculated according to equation (1). To measure the moisture

15

absorption ratio, the films were kept at 25 ºC in a sealed container with an atmosphere

16

of saturated CuSO4 solution for a set time (t). The moisture absorption ratio for each

17

specimen was calculated from its initial weight (W0) and its weight at the time t (Wt)

18

using equation (1)22:

19

Water uptake ratio or moisture absorption ratio (%) ＝[(Wt –W0)/W0] × 100 (1)

20

Cytocompatibility evaluation

21

Preparation of extracts from the films

22

Extracts from the EHSF-n films were prepared according to ISO 10993-12: 2007. The 9



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films were immersed in cell culture medium (Dulbecco’s Modified Eagle’s Medium

2

(MEM) 1640, 0.2 g film per 1 mL MEM 1640) at 37 ºC for 72 h. The extracts were

3

stored at 4 ºC and steam-sterilized before use.

4

Cell viability evaluation by MTT assay

5

According to ISO 10993-5: 2007, L929, a cell line of mouse lung fibroblasts

6

(provided by China Centre for Type Culture Collection, Wuhan University, Wuhan,

7

China), was re-suspended in culture medium and plated (100 µL/well) into 96-well

8

microtitre plates at a density of 1×103 cells/well. The medium in the plates were

9

incubated at 37 ºC in a humidified atmosphere of 5% CO2 for 24 h. The medium was

10

then replaced with sterilized extracts from the films. Culture medium with similar

11

cells was used as a control. After incubating for 24, 48 and 72 h, the cells were treated

12

with 10 µL/well of MTT and incubated for another 4 h at 37 ºC in a humidified 5%

13

CO2 atmosphere. Absorbance values were read in triplicate against a reagent blank at

14

a test wavelength of 570 nm using a multiwell microplate reader (Tecan GENios,

15

Tecan Austria GmbH, Salzburg, Austria). Cell viability was calculated using equation

16

(2): Cell viability (%) = (Atest/Acontrol) ×100

17

(2)

18

where Atest and Acontrol are the absorption values of the test and control groups,

19

respectively.

20

Cell culture and morphology of the cells cultured on the films

21

The selected EHSF-n films were cut into 10 mm × 10 mm pieces and sterilized by

22

autoclave without soaking into water to avoid the dissolution of the compositions. The 10


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1

cells were digested with 0.25% trypsin and cell density was adjusted to 1 × 106

2

cells/mL. The sterilized films were transferred into 24-well plastic culture plates. The

3

cell-culture medium was MEM supplemented with 10% (v/v) fetal calf serum (FCS,

4

Gibco, USA). After pre-wetting with culture medium overnight, 100 µL cell

5

suspensions were seeded onto each film. Another 900 µL of culture medium was

6

added after 3 h, followed by incubation at 37 ºC for 72 h in a 5% CO2 humidified

7

atmosphere. To observe the morphology of L929 cells, cells cultured on the EHSF-n

8

films were stained by using a 3, 3′-dioctadecyloxacarbocyanineperchlorate (DiO, 5

9

µM) dye solution for 20 min at 37 °C, and then washed with PBS for 4 times. The

10

films with DiO-stained cells were observed and photographed using a fluorescence

11

microscope (Nikon ECLIPSETE 2000-U) at the corresponding excitation wavelength:

12

(Ex/Em = 484/501 nm)23. The micromorphology of the cells cultured on the films was

13

observed by SEM with 20 kV as the accelerating voltage. Briefly, the films with

14

cultured cells were fixed in 2.5 wt% glutaraldehyde in pH 7.4 phosphate buffer. Then,

15

the films were washed with deionized water, immersed into 50, 70, 80, 90 and 100%

16

(v/v) ethanol solution in sequence, frozen in liquid nitrogen, vaccum-dried, and coated

17

with gold for SEM observation.

18

Hemcompatibility evaluations

19

Platelet adhesion

20

Whole blood from a normal healthy rabbit was collected in a syringe preloaded with

21

anticoagulant (3.8% (w/v) citrate sodium). Platelet-rich plasma (PRP) was obtained

22

by centrifugation of the whole blood at 1000 rpm for 10 min at 4 oC24. EHSF-n 11



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1

composite films were immersed in normal saline for 24 h, and then incubated with

2

PRP for 30 min at 37 oC, under static conditions. After washing with PBS, the

3

samples were fixed for 4 h in a 2.5 % (v/v) glutaraldehyde solution in pH 7.4

4

phosphate buffer.. The films were progressively dehydrated in ethanol (as mentioned

5

previously), frozen in liquid nitrogen, and vaccum-dried. The samples were coated

6

with gold and observed by SEM.

7

Plasma recalcification time.

8

Whole blood from a healthy rabbit was collected in a syringe preloaded with

9

anticoagulant (3.8% (w/v) citrate sodium). Platelet-poor plasma (PPP) was obtained

10

by centrifugation of the whole blood at 3000 rpm for 20 min at 4 oC25. To measure the

11

plasma recalcification time (PRT), EHSF-n films were pre-equilibrated with normal

12

saline, and then incubated with 0.1 mL PPP for 10 min. The plasma was then

13

recalcified by adding 0.1 mL 0.025 M CaCl2 solution. The PRT record was the time

14

required for fibrin clot formation. Glass was used in place of the film for the control.

15

The experiments were performed in triplicate.

16

Hemolysis testing

17

Fresh rabbit blood was collected in anti-coagulant heparin-containing tubes for

18

standard hemolysis testing26. Film samples were immersed in 10 mL extraction

19

medium of sterilized physiological saline and incubated at 37 ºC for 30 min under

20

static conditions. The negative control in this experiment was sterilized physiological

21

saline and the positive control was distilled water. The anti-coagulated rabbit blood

22

was diluted by sterilized physiological saline Then a 1 cm × 1 cm piece of each 12


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1

sample was added and incubated for 60 min in water bath at 37 ºC. Thereafter, each

2

tube was centrifuged and the absorbance of the supernatant was measured at 545 nm.

3

The hemolysis ratio (HR) was calculated using equation (3):

4

HR (%) = [(AS−AN) / (AP −AN)] ×100

(3)

5

where AS, AP and AN are the average absorbance values of the samples, negative

6

control and positive control, respectively. HR values of >5, 5-2, and 2-0, correspond

7

to hemolytic grade of hemolytic, slightly hemolytic and non-hemolytic27,28,

8

respectively. The spectral absorption was recorded on Shimadzu UV-1601 at 545 nm.

9

In vitro degradation study

10

In order to investigate the degradation behavior of the films, film samples (1 cm × 4

11

cm strips) were sterilized by autoclave and then incubated at 37 ºC in PBS for 56 days.

12

Buffer solution was exchanged every third day. After 3, 7, 14, 28, 42 and 56 days,

13

three samples at each point in time were dried and weighed, and the in vitro

14

degradation ratio was calculated using equation (4):

15

In vitro degradation ratio (%) = [(m0 – mt) / m0] × 100

(4)

16

where mt and m0 are the mass at time t and the mass before degradation, respectively.

17

Implantation study: in vivo biodegradability and tissue compatibility evaluations

18

Sixty-five Sprague-Dawley (SD) female rats, weighing 200–250 g, were obtained

19

from the Wuhan University Laboratory Animal Center (WHULAC, China) and

20

acclimatized in the animal care facility for two weeks prior to surgery. These rats

21

were randomly divided into four experimental groups (15 rats per group) and one

22

control group (5 rats). In the preliminary experiments, EHSF-0 and EHSF-10 films 13



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Page 14 of 57

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quickly disappeared in the rats due to their high water-solubility in body liquid, so

2

these two samples were not used for in vivo degradation study. Animals in

3

experimental groups were designated for the implantation of the EHSF-30, EHSF-50,

4

EHSF-70 and EHSF-100 films, respectively. All experimental procedures were

5

carried out in accordance with the “Guidelines and Regulations for the use and care of

6

Animals of the Review Board of Hubei Medical Laboratory Animal Center”, based on

7

the Experimental Animal Management Ordinance (National Science and Technology

8

Committee of the People's Republic of China, 1998). The rats were anesthetized by

9

intraperitoneal administration of Chloral hydrate at a dose of 1 mL per 200 g body

10

weight. After the anesthesia had taken effect, the hair on the back of the rats was

11

shaven and the skin was treated with 70% alcohol solution. Two small incision

12

wounds were made in the skin on the back of each animal to prepare bilateral

13

subcutaneous pockets along the backbone. Two steam-sterilized films, moistened with

14

0.9% NaCl solution, were inserted bilaterally through both incisions into

15

subcutaneous pockets without fixation. The skin was closed with interrupted

16

absorbable sutures. Rats without implantations were regarded as blank controls. The

17

animals were killed sequentially at 15, 30, 60，90 and 120 days after implantation, and

18

the implantation sites excised completely then processed to harvest the specimens..

19

Half of the implants were fixed in 4 wt% formaldehyde buffered solution for 6 h,

20

dehydrated,

21

hematoxylineosin (HE) for viewing with an optical microscope. The other half of the

22

specimens were fixed in 2.5 wt% glutaraldehyde29 in pH 7.4 phosphate buffer, and

embedded

in

wax,

vertically

sectioned,

14


and

stained

with

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1

then progressively dehydrated in ethanol, as mentioned previously. The dehydrated

2

samples were then frozen in liquid nitrogen, vaccum-dried, and coated with gold for

3

SEM observation at an accelerated voltage of 20 kV.

4 5

RESULTS AND DISCUSSION

6

Crosslinking interactions in EHSF-n films

7

Figure 1a (HEC) shows the FTIR spectrum of the original HEC powder. The band at

8

3443 cm-1 was attributed to stretching vibrations of –OH of HEC (Figure 1a, HEC

9

powder), which shifted to 3429 cm-1 in EHSF-0 (Figure 1b, EHSF-0), confirming the

10

formation of crosslinking network among the –OH of HEC30,31. Furthermore, by

11

comparison the FTIR of HEC powder and EHSF-0 film, it was observed that the

12

absorption band of O–H band around 3443 cm-1 decreased and that of C–O–C

13

stretching vibration at 1060-1150 cm-1 in EHSF-0 increased, indicating the

14

crosslinking effect. Figure 1a (SPI powder) shows the FTIR spectrum of original SPI

15

powder. The absorption band at 3298 cm-1 was attributed to hydrogen bonding

16

between protein chains and bound water in the protein. There were obvious –NH

17

bands at 1630–1678 and 1505–1561 cm-1, which agreed with the reported soy protein

18

spectrum that had amide I and amide II bands at 1632 and 1536 cm-1, respectively32.

19

The peak around 1630 cm-1 was attributed to C=O stretching vibration. The absorption

20

band at 1241–1472 cm-1 was due to C–N stretching and N–H bending (amide I)

21

vibrations, while the band at 1060 cm-1 was attributed to vibrations such as

22

out-of-plane C–H bending (from an aromatic structure)33. In Figure 1b (EHSF-100), 15



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1

the –NH bands of SPI obviously weakened, indicating that most of the –NH groups of

2

SPI has reacted with epoxy groups from ECH. A schematic depiction of the putative

3

crosslinking reactions of SPI and HEC with ECH is shown in Scheme 1. In this case,

4

the –NH groups from SPI molecules could be crosslinked with the –OH groups in

5

HEC molecules to form crosslinked structure (Scheme 1a)34, the –NH and –OH

6

groups from SPI molecules could form inter-molecular crosslink structure with the

7

other SPI molecules (Scheme 1b), and the –OH groups from HEC molecules could

8

form inter-molecule crosslinked structure with other HEC molecules (Scheme 1c). As

9

shown in Scheme 1, the crosslinking reaction for the EHSF-n composite films is

10

complex because there is competition among the three potential reactions (Scheme 1a,

11

b and c); thereofore, it is hard to confirm the crosslinking degree and the ratio of the

12

final products in the films. To further prove the successful crosslinking reaction with

13

ECH, the FTIR spectra of HEC/SPI-50 powder (the physical blend of 50% HEC and

14

50% SPI powders) and EHSF-50 film are shown in Figure S1. The peaks at 3357,

15

2924, 2869 and 1394 cm-1 for the stretching vibrations of hydroxyl groups, the

16

stretching vibrations of C–H from the –CH2– and N–H bending (amide I) vibrations

17

in HEC/SPI-50 powder shifted to 3424, 2928, 2874 and 1405 in EHSF-50 film,

18

respectively, especially the intensity of the peaks for the –CH2– increased obviously

19

in EHSF-50 compared with HEC/SPI-50 powder because the crosslinker ECH could

20

provide abundant –CH2– groups, indicating the successful crosslinking reaction. The

21

ATR-FTIR spectra for EHSF-n films in Figure 1b show the characteristic peaks of

22

HEC and SPI both appeared in the composite films from EHSF-10 to EHSF-70, 16


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1

indicating the presence of both components in the composite films. Moreover, the

2

peak shape and intensity for EHSF-n films varied with SPI content, showing that

3

different network structures also formed with different SPI content, which could

4

affect their physical and biological properties.

5

Figure S2 shows the XRD patterns of EHSF-n films (0, 10, 30, 50, 70, 90 and 100)

6

and SPI, HEC and HEC/SPI-50 powders. The SPI exhibited two peaks at 8.6 and

7

19.5°, suggesting that there was some ordered structure. The HEC powder exhibited a

8

dominant peak at 20.4°, indicating the crystal structure of cellulose II and the

9

cellulose structure changed during the etherification process for the preparation of

10

HEC35. Obviously, the intensity of the peaks for EHSF-n films was very weak,

11

confirming further that the crosslinking reaction truly occurred and the original

12

ordered structures of SPI and HEC were broken during the crosslinking process.

13

The cross-polarization/magic-angle spinning (CP/MAS) 13C NMR is an effective way

14

to provide information on the micro-chemical environment surrounding individual C

15

and could also be used to prove the presence of C from ECH linkage. The 13C NMR

16

spectra of the original SPI，HEC and HEC/SPI-50 powders as well as EHSF-0,

17

EHSF-30, EHSF-70 and EHSF-100 films are shown in Figure 2. The

18

spectrum of SPI powder displayed 5 major signals corresponding to carbonyl

19

(168-183 ppm), aromatic (129-141 ppm), α-C (48-68 ppm), β-C (24-39 ppm),

20

methylene and methyl groups (16-24 ppm), which are far away from the backbone36-38.

21

These signals shifted to (166-179 ppm), (126-133 ppm), (46-63 ppm), (19-35 ppm)

22

and (12-17 ppm) in EHSF-100, respectively. The signal at 70-81 ppm for SPI powder 17


13

C NMR


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1

lost its resolution greatly in EHSF-100, indicating that the SPI was crosslinked with

2

ECH. The resonance of anomeric carbon C1 of anhydrous D-glucose units is observed

3

at 106.2 ppm in the spectra of HEC powder. The resonance at 85.2 ppm was mostly

4

due to C4 and the band centered at 77.3 ppm was assigned to C2, C3 and C5, and the

5

resonance at 63.8 ppm corresponded to C6, although there was also resonances due to

6

the oxyethylene chains of substituents resonate in this frequency range for HEC

7

powder. The resonances for C1, C4, C2,3,5 and C6 for HEC in EHSF-0 changed

8

compared with HEC powder, indicating the crosslinking process changed the original

9

HEC structure. Moreover, the resonance at 64.4 ppm could be attributed to C6 of

10

EHSF-30 film. The C4 and C6 signals showed a large loss of resolution, while the peak

11

C2,3,5 became broadened for EHSF-30 and EHSF-70. This could be explained by that

12

the mobility of C lower mobility of carbons from anhydrous D-glucose units was

13

lowered induced by the chemical crosslinking of HEC with other HEC molecules or

14

SPI to form linkage (Scheme 1a, c)39, which further confirmed the FTIR result. The

15

α-C and β-C peaks became weaken and almost disappeared in EHSF-30, confirming

16

the existence of the ECH crosslinking. The HEC/SPI-50 powder exhibited both the

17

signals of HEC and SPI. Its resonances were obviously different from those of

18

EHSF-30 and EHSF-70, further confirming the existence of chemical crosslinking in

19

the EHSF films.

20 21 22

Structure and morphologies of the EHSF-n films SEM images of the surface and cross-section of EHSF-n films (n = 0, 10, 30, 50, 18


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1

70, 90 and 100) are shown in Figure 3. Both of the EHSF-0 and EHSF-100 films

2

exhibited relative smooth surface. In composite films with HEC as the main

3

component (n ≤ 50), the incorporation of SPI caused the appearance of

4

microprotrusions on the surface to increase the surface roughness of EHSF-n film.

5

Surface roughness increased as SPI content increased and was highest on the

6

EHSF-50 film (Figure 3a). As SPI became the main ingredient, the surface roughness

7

decreased and was relative smooth. This phenomenon may be explained by the fact

8

that the HEC is more hydrophilic than SPI, and dehydration of SPI was faster than

9

that of HEC during the drying process, which led to microphase separation in the

10

composite films and the formation of microprotrusions. However, the surfaces of all

11

samples were relative homogenous, which indicated certain miscibility between HEC

12

and SPI. The SEM images of all samples show a homogeneous structure on the

13

cross-sections (Figure 3b), confirming the good compatibility between SPI and HEC.

14

When the SPI content was lower than 50%, there were fish scales-like plates appeared

15

on the fractured cross-sections that became larger and looser with as SPI content

16

increased (Figure 3b). These plates disappeared in the EHSF-50 film, which had a

17

layered structure that was even more obvious in EHSF-70 and EHSF-90 films. This

18

layered structure had almost disappeared in the EHSF-100, whose cross-section was

19

relatively smooth due to the single component SPI. The structure of the film

20

determines its properties, so different properties (e.g. light transmittance and

21

mechanical properties) were anticipated for these films.

22

Light transmittance, mechanical properties and swelling behaviors of the 19



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1

EHSF-n films

2

Figure 4a shows the UV transmittance curves of EHSF-n films. Light transparency is

3

associated with the surface of the material. In our case, all films exhibited some

4

transparency that increased with the wavelength. The EHSF-0 and EHSF-100 films

5

exhibited good transparency due to the formation of homogenous and smooth

6

structure of the single chemical crosslinking reaction. The transparency of EHSF-0

7

was higher than that of EHSF-100 due to its smoother surface (as shown in the SEM

8

images in Figure 3). Figure 4b shows the transparency at 700 nm of EHSF-n films

9

changed with SPI content. As mentioned previously, crosslinking in the composite

10

films was complex with at least three potential crosslinking reactions (Scheme 1), so

11

the surfaces of the composite films were not homogenous like the EHSF-0 and

12

EHSF-100 films. Moreover, during the drying process, the different hydrophilicities

13

of HEC and SPI may cause certain microphase separation and microprotrusions in the

14

composite films (Figure 3). Such protrusions may lead light refraction and diffraction,

15

resulting in a loss of light and a decrease in transparency. Interestingly, when SPI

16

content was lower than 50%, the light transmittance of the composite films decreased

17

as SPI content increased (Figure 4b), because the homogenous structure of HEC was

18

partially destroyed by the incorporation of SPI. Similarly, when SPI content was

19

higher than 50% and became the main component of the composite films, its

20

homogenous structure was affected by the introduction of HEC (Figure 4b). As

21

expected, the film with the lowest light transmittance was the EHSF-50 film, which

22

had the roughest surface (Figure 3). Photographs in Figure 4c show the EHSF-n films 20


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1

covering papers with the letters “ABC” printed on them. Clearly, the final products

2

were light yellow films with a certain transparency and their color and transparency

3

varied with the SPI content, demonstrating the change of transparency indicated in

4

Figure 4b. The results from transparency measurement agreed with those from SEM

5

observation.

6

Generally, the films will be used wet or in a humid state for the cell culturing or

7

implantation in animals, so the mechanical properties of the wet or humid films are

8

particularly important. The tensile strength (σb) and elongation at break (εb) of humid

9

films are shown in Figure 5. The EHSF-100 film was very fragile and it was difficult

10

to test its mechanical property, so the corresponding data is not shown here. The

11

tensile strength of EHSF-0 was about 3.8 MPa and increased with the incorporation of

12

SPI as long as WSPI ≤ 50 wt%. The tensile strength at break for EHSF-50 reached 9.82

13

MPa, indicating the reinforcement role of SPI at a suitable content. However, further

14

increase in the SPI content caused the tensile strength to decrease and it was 3.74 MPa

15

for EHSF-90 film. In this case, SPI became the main component and acted as the

16

“matrix” rather than “reinforcement”. The elongation at break decreased as SPI

17

content increased because water acted as a plasticizer in humid conditions, and the

18

plasticizer effect was greater on HEC than on SPI because HEC is more hydrophilic.

19

This micro-structure of the films also affected its properties, especially the mechanical

20

property, in that the fish scales-like structure affects flexibility, while the layered

21

structure affects strength due to load dispersion40. Therefore, the mechanical

22

properties for most of the composite films were enough to satisfy requirements for 21



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1

practical application in biomedical field, especially EHSF-50 could be used as a

2

band-aid or wound dress because it exhibited the highest strength and elongation at

3

break even in humid state.

4

Figure 6a shows the water uptake ratio curves of the EHSF-n films. The EHSF-0

5

and EHSF-10 films were completely dissolved in water after 4 h. So the water uptake

6

ratios for the EHSF-0 and EHSF-10 after 4 h were not measured as shown in the inset

7

of Figure 6a. The water uptake ratio for the other films increased with time and

8

reached equilibrium at 4 h. It is known that HEC is a water-dissoluble polymer which

9

could be dissolved in water in 20 minutes. However, the EHSF-0 film was not totally

10

dissolved in water during 4 h, indicating that the chemical reactions had happened in

11

the HEC/SPI-0 film during film formation process. Although both HEC and SPI are

12

hydrophilic materials, the water uptake ratio decreased as the SPI content increased

13

because HEC is more hydrophilic than SPI. The EHSF-30, EHSF-50, EHSF-70,

14

EHSF-90 and EHSF-100 were insoluble in water though they were fabricated from

15

water soluble raw materials (HEC and SPI), which indicated that the crosslinking

16

reaction had effectively enhanced the water resistance of the composite films. As a

17

result, the crosslinked EHSF-n films exhibited good mechanical properties, water

18

resistance and suitable biodegradability.

19

Figure 6b shows the moisture absorption ratio curves of the EHSF-n films. The

20

EHSF-n films could absorb moisture quickly within in 48 h, and then the absorption

21

rate decreased obviously with time for the films. The moisture absorption ratio of the

22

EHSF-n films except for EHSF-100 have reached equilibrium after about 96 h, while 22


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1

that of EHSF-100 film had reached equilibrium after 168 h, indicating very high

2

hydrophilicity. Similar to the water uptake ratio, the moisture absorption ratio

3

decreased as SPI content increased. Therefore, the water up-take ratio and moisture

4

absorption ratio of the EHSF-n films can be controlled by adjusting the ratio of HEC

5

and SPI and through crosslinking reaction to help broaden their practical application

6

as biomaterials in wet or moist state.

7

Cytocompatibility evaluations

8

The principle of MTT assay is the measurement of mitochondrial activity of

9

living cells. Since for most cell populations mitochondrial activity is proportional to

10

the number of viable cells, this assay is one of the most sensitive and broadly used for

11

detecting in vitro cytotoxic events. In this work, L929 cells were incubated with

12

extracts from EHSF-n films for 24, 48 and 72 h. Cell viability was expressed as a ratio

13

against a control (cells incubated without extract). In Figure 7, the cell viability of

14

L929 cells in the extracts from both EHSF-0 and EHSF-10 films was much higher

15

than that of the control over the cell culture periods of 24, 48 and 72 h at 37 ºC in a

16

humidified atmosphere of 5% CO2. The test results of EHSF-30 and EHSF-50 were

17

close to that of the control under the same conditions. Conversely, the cell viability of

18

EHSF-70, EHSF-90, and EHSF-100 decreased compared with the control and

19

maintained around 80%, suggesting very slight toxicity of the corresponding extracts.

20

These MTT results are consistent with previous reports that an appropriate amount of

21

SPI hydrolysate in extracts promotes cell growth and proliferation, whereas high

22

concentration of SPI hydrolysate in extracts has side-effect on cell viability41. 23



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1

Nevertheless, the overall viability of the cells for all films in this experiment was in

2

the normal range, showing low or no cytotoxicity.

3

As shown in Figure 8, most of the cells on the surfaces of EHSF-n films stained

4

by DiO exhibited a normal shuttle-shape like that of the control, confirming that the

5

EHSF-n films were compatible with L929 cells. The surface of EHSF-30, -50 and -70

6

was homogenous and relative rough (Figure 3), which was beneficial for cell

7

adherence and the cells could grow uniformly on the surface of the films. The

8

non-cytotoxicity of SPI and HEC and the resulting cytocompatibility of the EHSF-n

9

films to L929 cells indicated the chemical reactions and fabrication for EHSF-n films

10

were “green” process and the produced films were without residual toxic material.

11

To further evaluate the potential applications of the EHSF-n films (n = 30, 50, 70,

12

90 and 100) as cell reactors or biomaterials, L929 cells were used as seed cells to

13

observe their adhesion, proliferation and distribution. SEM images of L929 cells

14

cultured for 72 h on the surfaces of EHSF-n films are shown in Figure 9. The L929

15

cells of the control (Figure 9, Control) exhibited a morphology that was shuttle-shape.

16

L929 cells seeded on the EHSF-n films were found to adhere, spread, and proliferate

17

uniformly, as shown in Figure 9. It was noted that the surfaces of EHSF-n films (n=30,

18

50, 70 and 90) were relative rough compared with the EHSF-100 film, so there was

19

increased area for cells to adhere and adsorb nutrition and more protein-binding sites

20

on the surface42. With increased SPI content, more and more cells were in

21

shuttle-shape like those in control group. This indicated that the cell shape partially

22

depended on the original HEC or SPI content. SPI perhaps contributed more to 24


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1

maintain normal shuttle-shape for the cells. These results indicated that the EHSF-n

2

films were good candidates for cell culture and proliferation, and therefore have wide

3

application as biomaterials43,44.

4

Hemocompatibility evaluation

5

Platelet adhesion is often the first point of contact for a biomaterial with blood

6

because platelets play an important role in the clotting process. When a blood vessel is

7

injured, circulating platelets quickly approach the wounded site and aggregate to form

8

a fibrin network45, gradually forming a thrombus. A similar phenomenon occurs when

9

blood contacts the surface of a biomaterial and platelet adhesion on the surface can

10

trigger platelet activation and thrombosis. Much effort have been devoted to

11

developing surfaces that will minimize platelet adhesion and meet the requirement for

12

biomedical applications46,47. Figure 10 shows the SEM images of the EHSF-n films (n

13

= 0, 10, 30, 50, 70, 90 and 100) after contact with PRP for 30 min. The surface of the

14

control was fully covered with platelets that were not highly activated due to the lack

15

of aggregation and pseudopodia. In contrast, the numbers of platelets distributed on

16

the surfaces of the EHSF-n films were much lower than that on the control, and these

17

platelets remained inactivated as well. These results show that EHSF-n films

18

themselves had an anticoagulation effect on platelets and did not activate platelet

19

deformation and aggregation.

20

Plasma recalcification time (PRT) mimics the intrinsic coagulation pathway in

21

vitro and is another important indicator of the anticoagulant properties of biomaterials.

22

Figure 11 shows the PRT values of EHSF-n films. All of the EHSF-n films were 25



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1

found to prolong PRT compared to the control. The PRT value of the control group

2

was 144 s, and the PRT values of EHSF-0 and EHSF-100 films were close to that of

3

the control. For the other EHSF-n films, when the SPI content was below 50%, PRT

4

increased as SPI content increased. The maximum PRT value of 210 s was for

5

EHSF-50 film. For the films where n > 50, PRT decreased SPI content increased.

6

However, the PRTs of all EHSF-n films (n was from 10 to 90) were significantly

7

higher than that of the control (P

Soy Protein

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