Proc. of Microelectronics & Nanotechnology (2014) Received 2 July 2013; accepted 7 October 2013

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Surface Properties of Cholesteryl Ester Liquid Crystal

Wan Ibtisam Wan Omar1,*, Hatijah Basri2, Chin Fhong Soon1 1Biosensor and Bioengineering Laboratory, Microelectronic and Nanotechnology Shamsuddin Research Center

(MinT-SRC), Universiti Tun Hussein Onn Malaysia, 86400, Parit Raja, Batu Pahat, Johor.

2Faculty of Science, Technology and Human Development, Universiti Tun Hussein Onn Malaysia, 86400, Parit Raja, Batu Pahat, Johor.

1. Introduction

Bio-compatible materials are thought to be very

important in many biomedical applications [1]. Recently,

a research found that cholesteryl ester liquid crystal (CELC) as a new biomaterial could be used for culturing

cells without pre-treatment with extracellular matrix

protein [2]. The amphiphilic molecules in the mesogenic

nature have the ability to self-order into liquid crystalline

structures. Shear sensitive CELC had been shown to be

non-toxic and provide cell affinity for cell attachment

with thermal stability between 20 °C and 50 °C [2].

Rheological characterizations of this material showed that

CELC after incubated in the cell culture media at 37 °C

for 48 hours exhibits a linear viscoelastic behaviour under

low rates of shear (<1 s-1) and within 10% of shear strain [3].

Cholesteric liquid crystals containing cholesteryl

moieties were found supporting cell adhesion and had

similar chemical structures to cholesteryl esters in a lipid

core hence serve as a bio-compatible biomaterial [4].

Cholesteryl moieties homeostasis is critical to cell

survival [5]. Therefore, this type of biomaterial is

attractive substrates because it provides universally

fundamental molecules that support adhesion to

mammalian cells. As part of this study, ester based and

cholesterol are both considered to be easily biodegradable

thus making the system suitable for the preparation of tissue engineering that sustain cell proliferation and

migration.

The advantage of using liquid crystal as the

biomaterials is because it provides a means of label-free

observations of biological phenomenon and this give

great interest in the area of biomedical engineering [6].

Thus, this study reported on the synthesis and

characterization of surface properties of CELC design to

interact with human keratinocytes (HaCaT) cells. In this

paper, the surface properties of cholesteryl ester liquid crystal materials that enable cell affinity were

investigated. The surface characterization was carried out

primarily through polarizing optical microscopy then

followed by fourier transform infrared spectroscopy

(FTIR). In addition, studies on culturing of human

keratinocytes (HaCaT) cells on this biomaterial were

described.

2. Materials and Methods

HaCaT cells were cultured in Dulbecco’s Modified Eagles’s Medium (DMEM, Sigma Aldrich, UK) media

supplemented with fetal calf serum (PromoCell, UK), L-

glutamine (2mm, Sigma Aldrich), and fungizone (2.5

mg/l, Sigma Aldrich). These cells were harvested using

standard cell culture procedures and seeded onto the LC

substrates in the Petri Dish (Fig.1) with density of

2.4x104 cells/ml for 24, 48 and 72 hours. Preparation of

ester based cholesteryl liquid crystal gel and coating of

the LC substrates were as reported in [3]. A LC substrate

at a thickness of ~100µm were prepared and was placed

in a petri dish containing DMEM culture media and

incubated at 37 °C for 24 hours. After incubation, the LC substrates was ready for imaging under polarizing

microscopy to study the changes of LC phase in cell

culture media.

Fig. 1: Cell suspension plated on LC substrates in Petri

dish.

Abstract: Nematic and cholesteric liquid crystals were recently explored their potentials in biosensing. Cholesteryl ester liquid crystal was shown to be a biocompatible material which enabled cell adhesion and it may be used to

functionalize a surface for cell affinity. In this paper, we characterized the surface properties of the liquid crystal

using polarizing optical microscopy and Fourier Transform Infrared Spectroscopy. This compound extracted from

the surface of the bulk cholesteric liquid crystal after interaction with the cell culture media was found rich in

cholesteryl moieties and displayed with lyotropic smectic phases that are very similar to the natural cholesteryl

ester found in the human physiological system. The surface layer was shown to promote adhesion and proliferation

of cells. The hydrophilic layer of lyotropic phase could be a useful means to be applied in cell or tissue

engineering.

Keywords: Liquid crystal, cell adhesion, lyotropic phase

Proc. of Microelectronics & Nanotechnology (2014)

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(a) (b)

Fig. 2: Image comparison using (a) phase contrast and (b) polarizing optical microscopy. (Scale bar: 20µm)

The interaction between CELC and DMEM (Sigma

Aldrich, UK) was investigated using FTIR spectrometer

(Perkin Elmer Spectrum 2000). 10µl of CELC was

incubated with DMEM in a glass vial for 24 hours at

37 °C before being analyzed. After incubation, the CELC

samples were sandwiched between a pair of circular cell

windows, CAF2 disk and scanned in the FTIR

spectrometer. The 32 scans were taken, and the resolution

in wave number was set at 4 cm-1. The CELC were

examined in the FTIR to investigate the functional groups of CELC incubated with and without DMEM culture

media. Omnic software was used to control spectral

acquisition and to process the acquired data.

3. Results and Discussion

3.1 Polarizing Optical Microscopy

In order to analyze the adhesion of cells after incubated

for 24 hours, 48 hours and 72 hours with temperature of

37 °C on the CELC, this study was conducted by using

crossed-polarizing optical microscopy (POM), to view

the phase change of the CELC and compare the LC phase

change in cell culture media. Under the POM, CELC

after immersion with cell culture media was observed

with multi-color reflectivity. Fig.2 shows the image

comparison using phase contrast and polarizing optical

microscopy. Fig.2 (a) shows a translucent film formed on

the surface of the cholesteryl ester liquid crystal in phase

contrast microscopy which is the adhesion layer to the keratinocytes. In cross polarizing microscopy, a similar

translucent film formed on top of colorful cholesteric

liquid crystals substrate (Fig. 2b).

These experiments showed that the cell culture media

changed the physical properties and structure of the

surface cholesteryl ester liquid crystal in order to enable

cell adhesion. The cells secreted their own adhesive

proteins on the LC to facilitate integrins mediated cell

adhesion. This probably due to cholesteryl moieties

present in this compound. Fig.3 (a-c) shows the

immersion of liquid crystals in the cell culture media for 24 hours, 48 hours and 72 hours. Amphiphilic liquid

crystals molecules were found self-assembled into a

lyotropic or lamellar system. This system indicates that

the lipid molecules were interlaced by water molecules

[7].

After immersion in cell culture media, the

hydrophilic head and hydrophilic tails of the liquid crystal

molecules reoriented to interface with the water at the

surface of the bulk liquid crystal as reported in previous

literature [8]. However, the chemical compounds of the

cholesteryl ester liquid crystals in attracting the adhesion

of cells have not been clearly identified and understood.

Therefore, an investigation on the effect of immersion in cell culture media to the cholesteric liquid crystals by

using FTIR was essential.

3.2 Fourier Transform Infrared

Spectroscopy Analysis

Formation of lyotropic layer at the surface of the

cholesteryl liquid crystal substrate as discussed in [9] is

an adhesion layer to the keratinocytes. The study of how

lyotropic layer was formed has already been discussed,

but further study on surface properties of CELC that

enabled cell adhesion required further information. Fig. 4

shows the FTIR spectrum of CELC. The peaks at 2950

cm-1 and 2852 cm-1 are corresponding to C-H groups of

Aldehyde. The presence of ester group was denoted by low intensity peak wavenumber 1739 cm-1 which was

assigned to C=O. While peak at 1466 cm-1 was assigned

to C=C of aromatic ring. The appearance of S=O

stretching peaks at 1377, 1265, 1253 and 1169 cm-1 were

evidences of graft finger prints of CELC. From the FTIR spectrum, the ester and C-H groups were

found in the LC which was related to the cholesterol

group of the CELC. In the human skin barrier system,

cholesterol and its ester group is the major sterol in

stratum corneum. These sterol affects the membrane

trafficking which was found at large amount in myelin filament and skin [10]. They are also important

constituent of the cellular membranes and as an enhancer

for transdermal delivery [10]. From previous report, Unna

& Goldsdetz, suggested the association of keratinization

came from the esterification of cholesterol [11]. The

cholesterol ester is derived from phospholipids tissue in

the stratum corneum by esterification of cholesterol with

fatty acids [12]. In addition, cholesterol is an important

Proc. of Microelectronics & Nanotechnology (2014)

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component of the lipid membrane of cells which enhance

the mechanical durability of cells, critical cell survival

from its homeostasis and improve the permeability of the

membrane [5]. Therefore, the similarity of CELC to the

cholesteryl ester in-vivo that may provide universally

fundamental molecules to interface with the cells making

the liquid crystal substrate an attractive substrates for cell

attachment and proliferation.

Fig.3 : The surface of CELC after immersion in cell culture media for (a) 24 hours, (b) 48 hours and (c) 72 hours.

(Scale bar: 20 µm)

Fig.4 : FTIR spectrum of Cholesteryl Ester Liquid Crystal.

Conclusion In summary, POM observed the phase change of the

CELC and compared the LC phase change in cell culture media. Different incubated time could change the

physical properties of the CELC surfaces. In order to find

the properties of CELC after incubated with DMEM that

could be the causes of the physical properties changes,

FTIR analysis was applied. Thus, the discovery of

cholesteryl ester groups which consist of hydrophilic

head would re-orient themselves in the cell culture media

and hence enabled a biocompatible surface for cell

adhesion.

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