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Study on surface morphology and phase separation of polymer/cellulose liquid crystal composite membranes after post-treatment

Mei Tua,b* Wanqing Hana,b Rong Zenga,b Serena M. Bestc Ruth E. Cameronac aDepartment of Materials Science and Engineering, Jinan University, Guangzhou, 510632, P.

R. China bEngineering Research Center of Artificial Organs and Materials, Ministry of Education,

Guangzhou, 510632, P. R. China cDepartment of Material Science and Metallurgy, University of Cambridge, CB2 3QZ, UK

*Corresponding author: Mei Tu

Address: Department of Material Science and Engineering, College of Science and

Engineering, Jinan University, Huangpu Road 601, Guangzhou, 510632, P. R. China

Tel.: +86-20-85223271; Fax: +86-20-85223271

E-mail address: [email protected]

Abstract: This paper explores the effects of the incorporation of liquid crystalline (LC) phases

1 into polyurethane with the aim of creating composites with biomimetic surfaces. The surface morphology and phase separation structure of polyurethane(PU)/butyl hydroxypropyl cellulose ester (BPC) composite membranes with different BPC contents and different post- treatments were investigated by using polarized optical microscopy (POM), scattering electron microscopy (SEM) and small angle X-ray scattering (SAXS).

For the PU/BPC composite membranes observed in their as prepared state, the surface morphology showed a well-dispersed liquid crystal ( strip birefringent textureLC) phases.

With increasing BPC content, the liquid crystal (LC) domains tended to form quasispherical aggregates of LC-rich phase and the size of the aggregates became larger. The LC molecules inside the domains poorly oriented and there was little regular phase separation between LC domains and substrate material in this case. However, the surface morphology of membranes showed obvious changes after post-treatment. A sharper peak emerged in the SAXS pattern for those samples with different LC contents after heat treating and cooling under three different conditions. This indicated that the arrangement of LC molecular chains in the domain and the phase separation structure between substrate and LC domain had changed.

The SAXS peaks were sharper and more intense for the membranes cooled by natural cooling in oven, indicating that the LC domains had more regular arrangement and more obvious phase separation than those in membranes cooled at to 20 ºC and at to -20 ºC respectively. It is suggested that the surface morphology of polymer/LC membranes could be controlled through adjusting LC contents and performing post treatment under different conditions.

Keywords: polymer/liquid crystal composite membrane; surface morphology; post-treatment; phase separation structure

2 1. Introduction

It has long been recognized that the response of host organisms to biomaterals at macroscopic, at cellular and protein levels is, in most cases, closely associated with the materials’ surface properties, and that the surface of the material has a critical influence on the biological response [1-7]. This means that a crucial consideration is the control the surface properties to obtain an optimum interaction between cells and biomaterials [8-10]. Considerable efforts have thus been focused on surface engineering to endow polymer surfaces with the ability to maintain cell normal phenotype and function [11-15] and to promote cell adhesion,

deposition, activation, growth, differentiation and secretion [16-18].

Many physiological and biochemical responses in the body rely on ordered structure [19,20].

Cell membranes have liquid crystal like characteristics and one strategy is to mimic this structure with the design of polymer/LC biomimetic engineering surfaces.

Previous studies have shown that addition of a LC compound with low molecular weight into a polymer substrate could improve the blood compatibility of biomaterials due to formation of the LC domains on the material surfaces [21,22]. Furthermore, a cholesteric LC phase was shown to be more effective than a nematic one. The distribution, size and morphology of LC domains on the material surfaces could be adjusted through change of LC content and the application of electric fields during the process of film preparation [23,24]. However, it was also found that low molecular LC escapes at a rate of 0.2wt% per day in simulated body fluid at 37˚C, which would lead to a decrease in the antithrombogenicity of the material. The escaped molecules may also produce a change in osmotic pressure in the serum and cause

3 hemolysis. To solve this problem, cellulose LCs with high molecular weight were chosen as the dispersed phase in this work. Preliminary studies have shown that these large molecule

LCs could also form LC domains in the matrix. Furthermore, the surface morphologies of the polymer/LC composites were affected by LC content and the conditions of film forming, which would have effects on the protein adsorption characteristics of the biomaterial surfaces

[25-29].

In this work, a series of polyurethane/butyl hydroxypropyl cellulose ester composite membranes with different LC content were prepared, and post-treated under three different conditions. The surface morphology and phase separation structure of these membranes was characterized using polarized light optical microscopy (POM), scanning electron microscopy

(SEM) and small angle X-ray scattering (SAXS) to observe the texture, shape, size and distribution of the LC domains on the materials surfaces. The relationship between the phase separation, LC content and post-treatment conditions is discussed.

2. Experimental section

2.1 Chemical and materials

Medical applied grade polyurethane, (PU) [Mw=270,000, containing 35% (wt) hard segment

4,4@-methylene diphenylene diisocyanate and 65%(wt) soft segment polyether], was purchased from Yin Tesheng Co. of Guangzhou in China. Butyl hydroxypropyl cellulose

ester, (BPC) with Mw=100,000, Tg=18.4°Cand TNI= 156.3°C was prepared as described elsewhere [30].

2.2 Preparation of the Polymer/LC composite membranes

4 A series of composite membranes composed of PU and BPC were prepared by a solution- casting method. PU and BPC were dissolved in tetrahydrofuran at room temperature (20C), with weight ratios of the PU:BPC of 9:1, 8:2, 7:3, 6:4 and 5:5. The mixing solution was cast onto clean glass plates and the solvent was allowed to evaporate for 72h. The membranes were further dried under vacuum at 20C for 10 h to eliminate the residual solvent. After the membranes were dried completely, they had a thickness of 200m.

The composite membranes were then put into an oven at 140C for 10 h, before being cooled down under three different conditions for 12h. The cooling conditions were air cooling to 20

C, quenching to -20C and oven-cooling to room temperature.

2.3 Characterization

The phase separation structure and surface texture of the PU/BPC composite membranes were observed using a polarized light optical microscope (XPL3230, Guangdong Silique

International Croup Maufar Co., Ltd) equipped with a digital camera system. Small angle X- ray scattering measurements were carried out using a Nanostar camera (Bruker AXS Inc.

Madison, WI, USA), fitted with a sealed microbeam source, at 40 kV, 35 mA and filtered to

give CuKα radiation (wavelength, λX=0.154 nm). The sample-to-detector distance was approximately 1.05 m and the entire optical path, including sample chamber, was evacuated.

The exposure time was 3600 s for all samples. The scattering vector was defined as q=(4/)sin(/2), where  and  are the scattering angle and the wavelength of the X-ray, respectively. The centre of the 2D-SAXS pattern was found and the scattering angle (2θ) was calibrated using a silver behenate reference standard. The morphology of composite membranes were investigated using a scanning electron microscope (PHILIPSXL-30ESEM).

5 A gold layer was coated on the specimen surface in a sputter coater (BAL-TEC, SCD005) prior to observation. The static contact angles of the air side of the polymer/liquid crystal films against water were measured by using a Drop Shape Analysis System DSA100 (Kruss,

Germany) at room temperature, and the average contact angle value was calculated based on measurements at five different positions in each sample.

3. Results and discussion

3.1 Surface texture of PU/BPC composite membranes

The surface textures of the PU/BPC composite membranes were observed using POM. Figure

1 shows that all pure PU films showed no orientation when viewed under cross polarization, while composite membranes showed a well-dispersed birefringent texture. The samples which had not been heat treated had LC domains dispersed throughout the PU matrix forming a strip texture (Fig.1 e,i). When the LC content was smaller than 20%, the LC domains tended to form quasispherical aggregates of LC-rich phase. When LC content was larger than 20%, the size of quasispherical aggregates were observed, which became larger with increasing of LC content (Fig.1 m,q,u) and the visual field under the POM became brighter. However, the surface morphology of the membranes showed clear changes after post-treatment. In general, the phase separation and texture of the LC domains became more homogeneous, with the LCs apparently becoming more completely dispersed and incorporated into PU matrix. The quasispherical aggregates of LC domains which occurred on the surfaces of pre-treatmentas prepared composite membranes disappeared when LC content was larger than 20% for all post-treated samplesafter heat treatment.

6 Fig.1. Surface texture of pure PU membrane and PU/BPC composite membranes, there are six groups of a-d, e-h, i-l, m-p, p-t and u-x, the LC content of each group is 0%,10%,20%,30%,40%, 50% respectively; the treatment condition for four membranes in each group is followed by as-prepared, cooling at to 20°C, cooling at to -20°C and natural oven-cooled However, the surface morphology of the oven-cooled samples cooled was different from those cooled at to 20°C and at to -20°C respectively. It could be seen from Fig.1h that there were a series of isolated uniformly distributed dots on the membrane surface when the LC content

7 was 10% This effect was an obvious sign of the phase separation of the LC domains and the

PU matrix. With increasing LC content, the amount number of dots decreased and the membrane surface exhibited a characteristic “stripe” texture decreased. The surface morphologies of membranes air-cooled to room temperature were basically similar over the different range of LC contents, although there were was a low volume fraction of dots distributed on the membrane surfaces. For those samples prepared by cooling to -20°C, when the LC content was lower than 40%, there were was a very small number of dots dispersed on the membrane surface, however, when LC content was larger than 40%, the small dots aggregated together to present bright spots of a stripy texture on the membrane surface..

The POM observations described above indicated that the LC contents and post-treatment conditions significantly affect the configuration of LC domains embedded into the polymer/LC composites. Before heat-treatment, thermotropic liquid-crystal molecules self- assembled into phases in their simplest form with orientational order, but often without positional order[15]. The process of texture formation with the temporal evolution of a given

LC in a certain polymeric matrix was produced by competition between the liquid crystal– solvent and liquid crystal–polymer phases (INCLUDE REFERENCE?). When heat-treatment was performed,, the fluidity of the LC macromolecular chains increased markedly, and the LC segments rearranged and tended to become more regular. Because of the high relaxation times of polymer LCs, their regular alignment inside the matrix led to ordered structures and an anisotropic character [31] during the cooling process. Hence the membranes showed multifarious textures under the POM.

3.2 Surface morphology and phase separation behavior of composite membrane

8 To gain further information and understanding of the microstructure of composite membrane surfaces, SEM observation was carried out and the results are shown in Fig.2. The SEM image revealed that LC domains with a radiating, flower-like shape appeared on the

membrane surfaces. The shape of LC domains was similar to the hexagonal HⅡ phase(a type of texture of LC domain) reported in the literature[32]. The size of ‘flower’ became larger with increasing LC content, and this implied that the volume of the LC domain increased. After post-treatment, the LC domains tended to form an interwoven net-like texture. The above results revealed that LC content and heat-treatment impacted on the morphological features, including both the LC domain size and shape.

Fig.2. SEM image of PU/BPC composite membranes，there are three groups of a-d, e-h, i-l, the LC content of each group is 10%, 30%, 50% respectively; the treatment condition for four membranes in each group is followed by as- prepared, cooling at to 20°C, cooling at to -20°C and natural oven-cooled

To elucidate the effects of LC content and post-treatment on phase separation behavior, SAXS

9 measurement was performed. In Fig.3, SAXS profiles are shown for all samples. The SAXS curves showed that there were no clear scattering peaks for as-prepared polymer/LC composite membranes with different LC contents (Fig.3a). However, when the membranes were post treated using heat-treatment and cooling under the three different cooling conditions, a new peak emerged for every sample shown in Fig.3b, c and d. The peak appeared to be sharpest for the samples prepared by natural oven-cooling and the peak position shifted slightly for each cooling condition at the same LC content (Fig.3e,f). The appearance of the sharpened peak in the SAXS pattern after post-treatment suggested the development of a more highly ordered structure within the membranes. This implied that the

LC domains rearranged during the cooling process, but their regularity varied under the three different cooling conditions. The SAXS curves for those membranes cooled to -20 °C presented a broader and less intense peak than those cooled under the other conditions. It was also found that the LC content had no systematic effect on the scattering intensity (I(q)) and had no effect on the peak position for the samples prepared under the same treatment regime.

The different liquid crystallization behavior in the as-prepared membranes and the post- treated ones with the three different cooling conditions may be attributed to the changes in the arrangement and phase separation structure of the LC domains [33,34].

For the as-prepared membranes, when the LC content was lower, the LC phase was dispersed as particles in the PU matrix. With increasing LC content, the LC particles gradually aggregated to form LC domains and began to phase separate from the PU. However, the LC molecules were poorly oriented inside these domains, and the phase separated structure was not obvious – as indicated by the low intensity and broad peaks in the SAXS patterns. When

10 those membranes were heated at 140°C (close to clearing temperature of BPC) for 10 hr, and subsequently cooled, the chains re-arranged and re-aggregated, and the LC particles in each

1400 1400 AS-prepared Cooling at 20℃ 1200 a LC:10% 1200 b LC:10% LC:20% LC:20% ) ) 1000 s 1000 s t LC:30% t LC:30% i i n n u u

LC:40% LC:40%

y 800 y 800 r r a a LC:50%

LC:50% r r t t i i b b r r a a 600 600 ( ( ) ) q q ( ( I I 400 400

200 200

0 0 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 q/nm-1 q/nm-1

1400 1400 Cooling at -20℃ Oven cooled 1200 c LC:10% 1200 d LC:10% LC:20% LC:20% ) ) s 1000 1000 LC:30% s t t i LC:30% i n n

u LC:40% u

LC:40% y y

r 800 800 r LC:50% a a

r LC:50% r t t i i b b r r a 600 a 600 ( ( ) ) q q ( ( I I

400 400

200 200

0 0 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 q/nm-1 q/nm-1

1000 1000 LC:30% LC:20% 900 900 e f As prepared 800 As-prepared 800 ℃

) Cooling at 20 )

700 s 700 s t t i i Cooling at 20℃ n n

u u Cooling at -20℃ 600 600 y y r r a Cooling at -20℃ a r r

t Oven cooled t i 500 i 500 b b r r a a (

Oven cooled ( ) 400 ) 400 q q ( ( I I

300 300 200 200 100 100 0 0 0 0.5 1 1.5 2 2.5 0 0.5 1 1.5 2 2.5 q/nm-1 q/nm-1 Figure 3 SAXS profiles for PU/BPC composite membranes with different LC contents, a is as-prepared one, b-d are post-treated ones followed by cooling at to 20°C, cooling at to -20°C and natural oven-cooling, e,f are those with the same LC content through post-treating in different conditions domain tended to become regular and oriented, while the surface texture of membranes changed (showed in POM photos) and sharper peaks occurred in the SAXS patterns from the microphase separation structure [35]. The membranes possessed contrasting surface texture

11 and extent of microphase separation due to the disparity of the speed nature of macromolecular re-arrangement under the different cooling conditions.

3.3 Change of surface energy of composite membranes

Investigating the interaction between water and any material to be used as a biological matrix is very important. Therefore, the static water contact angles of the composite membrane surfaces were evaluated to explore the effect of the liquid crystal phases on the surface energy.

It was confirmed that all of the membranes used in this study had flat surfaces and showed no evidence of a porous structure in the bulk phase. The surface contact angles of the as prepared and post-treated membranes with different LC contents are listed in Table 1. It was found that the maximum values of water contact angles for all membranes were obtained when the LC content reached 50%. In addition, the values of water contact angle for all membranes increased after post-treatment. Indeed, the membranes prepared by natural cooling in the oven after heating exhibited significantly higher surface contact angles as compared with the other two membranes cooling at to 20°C and at to -20°C respectively. For the as-prepared membranes, the contact angles of the composite membranes were higher than those of pure

PU, the contact angles increased with increasing of LC content. However, for the post-treated composite membranes, only when LC content was larger than 30% were the contact angles higher than those of pure PU prepared under the same condition.

Table1 The surface contact angles of the as-prepared and post-treated membranes with different LC content Content of BPC(%) Cooling conditions As-prepared Cooling at to Cooling at to Natural oven-cooled 20°C -20°C 0(pure PU) 80.6±5.5 84.5±2.2 87.2±2.2 95.1±3.7 10 82.2±4.5 83.8±2.2 85.0±3.8 85.3±2.6 30 86.0±2.8 87.7±5.1 88.7±1.3 95.8±4.7 50 97.5±3.5 101.4±2.0 102.1±2.5 104.3±1.5

12 These results revealed that introduction of LC into substrate affected the surface contact angles, and LC contents and that post-treatment conditions also had a significant influence. In other words, the LC phase formed on the polymer surface could influence surface energy. It is well known, from previous observations of POM and SEM, that the LC domains on the surfaces of composite membranes exhibited different sizes, distributions, phase separation and surface morphology with varying LC content and post-treatment conditions, and these consequently changed the surface energy of membrane [ADD REFERENCE]. When the LC content was higher than 30%, the surface contact angles of all composite membranes increased markedly in comparison with the PU film produced under similar treatment conditions. This suggests that the surface energy decreased. It is suggested that PU/BPC composite membranes which have a surface liquid crystalline phase might be expected to show effective cellular attachment[36].

4. Summary

The effects of LC content and post-treatment conditions on surface morphology and microphase separation structure were investigated by means of POM, SEM and SAXS.

The surface morphology of the as prepared PU/BPC composite membranes, was a well- dispersed “strip-like” birefringent texture under cross polarization when the LC content was less than 20%. The LC domains tended to form quasispherical aggregates of the LC-rich phase, and the size of the aggregates became larger with increasing LC content. No obvious scattering peaks in the SAXS pattern were observed with any LC content, which indicated that the arrangement of LC molecules inside the domains was in random orientation and no regular phase separation was observed between the LC domains and the substrate material.

13 However, after post-treatment the surface morphology of membranes was obviously different from the morphology before heat treatment. The LC domains dispersed more homogeneously into the matrix and the quasispherical aggregates of LC domains that occurred on the surfaces of pre-treated composite membranes disappeared when LC content was larger than 20% for all post-treatment samples. An obvious peak emerged in the SAXS pattern for those samples with different LC contents through cooling in three different conditions after heat-treatment.

The SAXS peaks were sharper and more intense for the membranes prepared by natural cooling in the oven, and revealed that the LC domains embedded into the matrix had a more regular arrangement and obvious phase separation characteristics than those cooled to 20°C and -20°C respectively.

The results of water contact angle tests showed that the contact angles of the composite membranes were higher than those of pure PU, the contact angles increased with increasing

LC content. Moreover, the values of water contact angle for all membranes increased with post-treatment. It revealed that the introduction of LCs into the substrate material affected the surface contact angles, and the LC domains formed on the surfaces of the composite membranes were altered, indicating a profound influence of LC content and post-treatment.

The results above indicated that the surface morphology of the polymer/LC membranes could be adjusted by changing the LC content and post treatment performed, and, consequently, that the surface properties could be controlled with considerable potential importance for specific biointeractions. The effects of surface morphology, phase separation and surface energy on the protein adsorption and cell adhesion on the surfaces of composite membranes will be investigated further.

14 Acknowledgement

This work was supported by National Nature Science Foundation of China(30870613 and

31040027), Scientific Research Cultivation and Innovation Funds of Jinan University

(21610607), Special Funds for Base Construction of Industry, Education and Research of

Guangdong Province(20090918). The SAXS experiments were performed in the Department of Materials Science and Metallurgy, Cambridge, UK, and thanks are given to Dr Peter R.

Laity for his kind help for this work.

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