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Procedia Chemistry Procedia Chemistry 2 (2010) 26–33

www.elsevier.com/locate/procedia

5th Conference by Nordic Separation Science Society (NoSSS2009) Study of cell membrane based coatings in capillary electrochromatography

Kert Martma, Sandra Zetterström Fernaeus#, Tiit Land, Ruth Shimmo¤*

Department of Natural Sciences, Institute of Mathematics and Natural Sciences, Tallinn University, Narva mnt. 25, 10120 Tallinn, Estonia

Abstract

Neuronal cell line based membrane models were studied as capillary coatings by capillary electrochromatography. The membrane models were based either on membrane suspensions or on membrane lipid extractions. The stability of the coatings at different pH values and buffer compositions was studied. The results showed that the cell membrane suspension based coatings were stable over pH range of 6.5–10.8. The use of Hepes instead of TE buffer did not improve the coating performance.

Keywords: Membrane models, open tubular capillary electrochromatography,human glioma cells

1. Introduction

The study of different aspects and properties of biological membranes has fascinated many researchers from different fields over a century and the interest is not ceasing in time. The dominating model for biological membranes is still the one of Singer and Nicolson describing the membrane as a two-dimensional bilayer of fluid lipids with adsorbed or embedded proteins [1]. The role of membrane proteins has been extensively studied and their importance seems to be relatively clear. The information on membrane lipids composition and the reason for the vast diversity of membrane lipids is still much less known. However, the lipid-related research has gained more and more attention during last decade and the crucial role of membrane lipids has been established [2,3]. It is becoming more evident that the spatial and temporal regulation of lipids in a biological membrane is closely related to the health of cells, tissues and organs in human body and that deregulation of the lipid membrane would cause a variety of human diseases, including neurodegenerative and infectious diseases, cancer, diabetes and many others [4,5]. For

# Formerly Sandra Fernaeus ¤ Formerly Ruth Kuldvee * Corresponding author: R. Shimmo. Tel.: +372-640-9405; fax.: +372 6409 418

E-mail address: [email protected].

1876-6196/09/$– See front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.proche.2009.12.007 K. Martma et al. / Procedia Chemistry 2 (2010) 26–33 27 example, Alzheimer’s disease is associated with abnormal metabolism of cholesterol [6]. Gangliosides are known to promote the aggregation of β-amyloid protein on membrane surface [7]. Mutations in α-synuclein are associated with familial cases of early-onset Parkinson’s disease, etc [8]. There is also strong evidence that one of the ways of how lipid deregulation and the neurodegenerative diseases are related is the oxidative damage of membrane lipids by the overproduction of reactive oxygen species (ROS). Yet, very few studies have been performed on oxidation studies of intact membrane phospholipids in biological membranes [9]. Currently many powerful analytical methods are applied to lipid research [4]. Even though the most often used approach has been the hyphenation of liquid chromatography and mass spectrometry, capillary electromigration methods, including capillary electrochromatography (CEC), have gained their importance on the field. The unique feature of CEC is that at favorable conditions most of lipids relevant in mammalians form a supported lipid bilayer on the capillary wall which enables to study not just single lipid species but to a certain extent the membrane as a whole. It is advantageous as it is known that the biological membrane can have properties which are not explained on the level of its single constituents. This way it is possible to study the influence of different environmental factors on the membrane model as well as to study special lipid-protein and lipid-lipid interactions. CEC is especially attractive method for the membrane studies as it enables to attach the subject of interest to the capillary wall and change the environment around it effortlessly, therefore, saving the time and material. Several research groups have carried out extensive studies to explore the nature of different membrane models by CEC [10–14]. However, nearly all of the studies have concentrated on well-controlled mixtures of two or three different lipids, often synthetical phospholipids. One of the few exceptions has been a study on human red blood cell (RBC) ghost lipids as a capillary coating [15]. Biological membrane derived coatings contain great number of components and may therefore differ dramatically in stability, interactions and response to different environmental factors from the coatings which are based on single lipid species. The aim of the present work was to estimate the stability and reproducibility of natural membrane derived coating materials under different environmental conditions (pH, buffer composition). In a recent study (submitted for publication in Electrophoresis) we demonstrated that neuronal cell lines can be immobilized to fused silica capillary wall and that different cell lines enable to achieve similar coatings. The current study is an extension of the before mentioned work. The coating solutions were prepared according to two different protocols – one of them enabling to have both membrane proteins and membrane lipids in the coating solution and the other where the proteins were excluded by chloroform extraction. The performances of the different coatings are compared.

2. Materials and methods

2.1. Materials

Chlroform and Tris were purchased from AppliChem (Darmstadt, Germany), EDTA from Sharlau Chemie S.A. (Barcelona, Spain), DMSO was from Fluka (Buchs, Switzerland), acetic acid (99.7 %, d 1.049 kg/L), leupeptin was from Sigma (St Louis, Mo, USA). PBS from Naxo OÜ (Tartu, Estonia), methanol from Rathburn (Scotland), 1 M sodium hydroxide from Agilent Technologies (Germany) and hydrochloric acid from Lach-Ner (Czech Republic). Fetal bovine serum “GOLD”, d-glucose and streptomycin were from PAA (Austria). The murine microglial BV-2 cells were from Dr. Katarina Bedecs and human glioma U87-MG cells were generously provided by Dr. Kerstin Iverfelt, Dept. Neurochemistry, Stockholm University.

2.2. Solutions

PBS buffer contained of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH was 7.4. TE buffer contained 10 mM Tris and 0.1 mM EDTA. The lysis buffer contained leupeptine 0.5 µg/ml, PMSF 1 mM in TE buffer. Western Blotting Detection Reagents (GE Healthcare, Germany) for Western Blotting. DMEM with Glutamax II and 4.5 g/l d-glucose supplemented with 5 % fetal bovine serum “GOLD” (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin for cell cultivation. The ionic strength of the BGE solutions (phosphate, acetate and 28 K. Martma et al. / Procedia Chemistry 2 (2010) 26–33

CAPS buffers) was 10 mM with pH adjusted to 4.5–10.8 with 1.0 M sodium hydroxide. The BGE solution did not contain any membrane solution. EOF measurements were carried out with 0.1 % DMSO aqueous solution.

2.3. Equipment

Fused silica capillaries of 75 µm i.d., total length of 40 cm, length to detector 31.5 cm were from Composite Metal Services (Worchestershire, UK). A Hewlett-Packard 3DCE system (Agilent, Waldbronn, Germany) was used for the CEC experiments. A Mettler Toledo MP225 pH Meter was used for pH adjustments in BGEs. A Motic Inverted Microscope AE 21 was used to get cell images. Ultrasonic bath J.P.Selecta Ultrasons Medi-II was used to produce small unilamellar vesicles and also to hydrate liposomes into the buffer solution after chloroform extraction. Karl Hecht GmbH & Co. KG vortexer was used to accelerate the hydration. Eppendorf Centrifuge 5804R was used after cell homogenization for fractionation. Nitrogen gas (99.996 %) from Elme Messer Gaas (Tallinn, Estonia) was used to evaporate chloroform from lipid extractions. The gel electrophoresis systems used for Western blotting were Bio-Rad Mini-Protean Tetra Cell and Bio-Rad Trans-Blot SD, Semi-Dry Transfer Cell.

2.4. Methods

2.4.1. Cell cultivation

Bv-2 and U-87 MG were generated as previously described by Pontén and Macentyre[16] and Biedler and o collegues [17], respectively. Cells were maintained at 37 C under an atmosphere of 5 % CO2 in DMEM with Glutamax II and 4.5 g/l d-glucose supplemented with 5 % fetal bovine serum “GOLD” (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were sub-cultivated 1:10 once per week, and medium was changed 3–4 days after sub-cultivation. For cell-membrane extracts, two 75 cm2 cell-culture flasks were seeded 1:10, seven days before the preparation.

2.4.2. Preparation of membrane solution

Cell membrane suspensions were prepared as by a modified procedure of Karelson et al [18]. Shortly, the complete culture medium is removed from 250 ml CELLSTAR Tissue Culture Flask and the cells are washed with ice cold PBS two times (10 ml + 10 ml). Then 1.5 ml of lysis buffer is added. After that the cells are scraped out of the flask with plastic cell scraper, collected into a tube and centrifuged at 1000 rpm for 5 min at 4 oC. The pellet is resuspended in approx 1 ml of lysis buffer and vortexed. Cell are then homogenized with 15 strokes in Dounce cell homogenizer on ice. After that the solution is centrifuged again at 1600 rpm for 10 min at 4 oC to remove cell nucleus. The supernatant is saved and centrifuged again at 16000 rpm for 20 min at 4 oC to remove mitochondria. The supernatant is sonicated for 3x15 min. After that the solution is ready to use for coating.

2.4.3. Estimation of phospholipid concentration of the sample

The concentration of phospholipids was estimated by measuring the total concentration of phosphorus of the samples according to the procedure of Murphy and Riley [19] modified by Koroleff [20].

2.4.4. Preparation of lipid solutions

Extraction of lipids was done by the method of Bligh and Dyer [21]. The cells were first treated the same way as in the membrane suspension procedure. After breaking the cells and removing larger pieces of membrane by centrifugation the membrane suspension was mixed with methanol/chloroform solution. The lipid containing bottom K. Martma et al. / Procedia Chemistry 2 (2010) 26–33 29 layer was removed and the solvent was evaporated to dryness under a stream of nitrogen. The solid residue was either redissolved in chloroform and stored at –18 oC or directly dissolved in the BGE.

2.4.5. Capillary coating

Unused capillary was flushed for 45 min with 1 M NaOH, 10 min with H2O and 10 min with BGE. Then analysis with uncoated capillary was carried out. Then the capillary was flushed 10 min with 0.5 M HCl, 20 min with H2O and 5 min with BGE. Membrane coating was applied by rinsing the capillary for 40 min with membrane solution at 50 mbar. Then the membrane solution was kept in capillary for 15 min. Finally the capillary was flushed for 2 min with BGE solution to remove unbound coating material.

2.4.6. EOF measurements

After coating, the capillary was flushed with BGE solution for 15 min. For stability measurements of the coatings, twenty successive runs with DMSO as EOF marker were performed in freshly coated capillaries. TE buffer was used throughout the experiments as coating and running solution except when the influence of HEPES was investigated. Between runs the capillary was flushed for 2 min. If not stated differently a fresh BGE solution was used for every run. CE conditions were as follows: the voltage was 20 kV, temperature of the capillary cassette 25 oC; injection was done for 8 sec at 20 mbar. Detection was carried out at 210 nm.

2.4.7. Western blot analysis

In order to confirm the presence of membrane proteins in the membrane suspension 7–10 µg of total cell extracts prepared in TE buffer were analyzed by Western blotting [22]. Briefly, the cell extract was separated on a 10 % SDS-PAGE before transfer onto a nitrocellulose membrane. Proteins were detected with primary antibodies against c-terminal fragment of amyloid precursor protein (APP), followed by secondary horse radish peroxidase (HRP) labeled goat anti-mouse antibodies. Membranes were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare, Germany).

3. Results and discussion

The aim of the work was to evaluate the stability and reproducibility of the biological cell membrane based OT- CEC coatings under different experimental conditions (the impact of the pH of the analysis media and buffer composition). Another purpose of the study was to compare membrane suspension based coatings (contain both proteins and lipids) to the coatings which were based on the lipids of the membrane.

3.1. Comparison of membrane based coatings of different origin

Under current study the cell membranes of two different types of eukaryotic cells were studied: murine microglial Bv-2 and human glioma U87-MG cells. The cells are different by outlook (Figure 1), origin and functions. U-87 MG is a cell line originally derived from brain malignant glioma. Murine microglial BV-2 cells were immortalized by infection with v-raf/c-myc recombinant retrovirus and have retained microglial phenotype and morphology. It was demonstrated already by our first study that neuronal cell line derived coatings have similar properties. The repeated experiments confirmed that both the stability and the charge of the coating were similar for the cell lines under study (Figure 2).

30 K. Martma et al. / Procedia Chemistry 2 (2010) 26–33

A B

Fig. 1. Images of different cells with an inverted light microscope. Magnification 100 times. Cell solution consists DMEM (Dulbecco’s Modified Eagle Medium) and FBS (Fetal Bovine Serum) (A – Bv-2; B – U87 cells).

The coatings showed randomly a significant variation in the EOF mobility (variation was up to 11 %) during 10 successive runs. The fluctuation disappeared when a fresh BGE was used for every run (Figure 2). According to the literature, that frequent change has not been needed when a lipid coating, used as a membrane model, has consisted of few chosen lipid species [14]. The results indicate that the stabilization time of the membrane based coating might be longer that it is for pure lipid based coatings and that loose parts of a membrane leak in and out of the capillary during first runs. When the BGE solution was changed after every run the EOF mobility did not fluctuate but showed very slight change either downwards or upwards which decreased in time. Both the decrease and increase in EOF mobility during first runs could be explained by reorganizations in the coating.

3.50

3.00 -1 V

-1 2.50 s 2

m Bv-2 I -8 2.00 Bv-2 II U-87 1.50 Bv-2 III

1.00 EOF mobilityEOF [10 0.50

0.00 0 4 8 12 16 20 Number of experiments

Fig. 2. Repeatability of the EOF mobility in columns coated with different membrane based solutions. Bv-2 I stands for the coating where the BGE was changed after 6 runs; for Bv-2 II, Bv-2 III and U-87 coatings the BGE was changed after every run. BGE: TE buffer, pH 7.4. 0.1 % DMSO was used as neutral marker. Running conditions: voltage 20 kV, injection 8 s at 20 mbar, detection 210 nm, length of the capillary 31.5/40 cm.

K. Martma et al. / Procedia Chemistry 2 (2010) 26–33 31

The overall reproducibility of the procedure (measured by EOF mobility) including six different cell cultivation actions (different batches of U-87 cells, different membrane suspension preparations and coatings) was around 9 %. If the same solution was used for parallel coatings during three weeks then the RSD of EOF mobility in different capillaries was 6 %.

3.2. The influence of pH on the stability of the coating

Further, the influence of the pH on a membrane coating was studied. The capillary was coated at pH 7.4 and the pH of the BGE was varied in range of 4.5–10.8. Two different approaches were used: the pH of the BGE solution was either changed in the same capillary (Figure 3A) or the capillary was coated freshly for every pH series (Figure 3B). The pH values (4.5, 6.5, 7.4, 8.5 and 10.8) were chosen according to Hautala, et al where an extensive study was carried out on the influence of pH in the formation and stability of PC/PS coatings [23]. The capillary was always coated at pH 7.4, the EOF mobility was measured and then the BGE was changed to have the desired pH value. For evaluating the stability of a single coating, the pH values were changed in the following order: 6.5, 8.5, 4.5 and 10.8. Figure 3A demonstrate that the membrane coating is stable over the chosen pH range. The RSDs for parallel experiments were less than 3 %. These results indicate that the membrane is not deteriorated by short time use of relatively extreme pH values, yet it is not clear if they introduce any irreversible changes into the membrane or not. To study further the influence of pH and to avoid memory effects the influence of pH was consequently studied during longer time periods and using fresh membrane coating for every pH. As seen from Figure 3B the EOF mobility change from pH 4.5 to pH 7.4 was very similar to the situation where the pH change was carried out on the same coating. The average EOF mobilities were remarkably close. At higher pH values the EOF mobility increased with pH in freshly coated capillaries while it stayed at approximately same level if the same coating was submitted to different pH conditions. These results indicate that application of extreme pH values (4.5 and 10.8) on a lipid membrane might introduce irreversible changes. The stability of coating was the poorest at most acidic pH in freshly coated capillary (RSD 25 % during 10 runs). At the most basic pH value, however, the stability was quite good (RSD 6.5 % during 10 runs).

) 8,00 ) 8,00 -1 -1 V V -1 -1 6,00 Uncoated 6,00 Uncoated s s

2 Coated 2 Coated 4,00 4,00 *(m *(m -8 -8 2,00 2,00

EOF 10 0,00 EOF 10 0,00 4,5 6,5 7,4 8,5 10,8 4,5 6,5 7,4 8,5 10,8

pH pH

Fig. 3. Average EOF mobility in uncoated and coated capillaries at different pH values. The coating pH was 7.4. A – the pH change was carried out on the same coating; B – the capillary was freshly coated for every pH study. Running conditions as in Fig. 2

3.3. The impact of the use of HEPES

Hautala et al. has demonstrated that the use of HEPES, a piperazine ring containing buffer, has a significant effect on the success of the phosphatidyl choline/phosphatidyl serine (PC/PS) 80:20 mol% coating [11]. In the 32 K. Martma et al. / Procedia Chemistry 2 (2010) 26–33 present work, the influence of HEPES on the coating performance was studied as well. The results showed that HEPES in the coating solution did not improve the performance of membrane suspension based coatings. The stability of the coating as well as the EOF mobility was in the same range as with TE buffer or inferior (data not shown). It is not too unexpected taking into consideration that the dramatic effect of HEPES was noted for anionic phospholipids but the membrane suspension includes a vast variety of different lipids and proteins which are very likely having an impact on the coating mechanism. In present work TE buffer was used in further study.

3.4. Membrane suspension vs. membrane lipid extraction

Membrane studies by OT-CEC could be roughly divided into three approaches: studies of specific protein interactions, study of the intact (or close-to-intact) biological membrane and studies of lipid bilayer. For the first two approaches the membrane suspension based coating described in the current study would be a good candidate. However, for lipid peroxidation the use of only the lipid part of the membrane would be more appropriate for capillary coating. The membrane suspension was submitted to chloroform/methanol extraction [21] which has been demonstrated to extract both the complex and simple lipids. The lipid extract derived from the suspension of U87 cells yielded a stable coating with an EOF mobility of 2.3·10-8 m2s-1V-1. The RSD of EOF mobility was around 2 % over 20 successive runs. As seen from Figure 4 the coating based on lipid extraction is well comparable with the coating based on membrane suspensions in terms of surface charge and stability.

Membrane suspension 6.00 Lipid coating uncoated 5.00 ] -1

V 4.00 -1 s 2

[m 3.00 -8

2.00 EOF*10

1.00

0.00 0 2 4 6 8 10 12 Number of experiments

Fig. 4. Repeatability of the EOF mobility in uncoated capillary and in columns coated with membrane suspension or lipid extraction of U-87 cell based solutions. Running conditions as in Fig. 2

The results are also in good agreement with the red blood cells (RBC) [15] studies by Lindén et al. which according to our knowledge are the only OT-CEC studies where a lipid extract of a cell membrane has been used for capillary coating. Repetition of the entire procedure with a new cell cultivation gave a coating with faster EOF mobility (2.8·10-8 m2s-1V-1) which indicates that the lipid concentration in the coating solution might have been too low. The analysis of phospholipids content confirmed that the lipid concentration was lower in the second coating K. Martma et al. / Procedia Chemistry 2 (2010) 26–33 33 solution (7 µM vs. 9 µM). The stability of the second coating was also inferior to the coating from the first batch (RSD of EOF mobility over 20 runs was 6.5 %). These results suggest that lipid concentrations should be around 8 µM or higher to ensure successful coating of the capillary. The limiting concentration was found to be lower in our experiments compared to the work of Lindén et al. [ 24 ]. They demonstrated that for single lipid (phosphatidylcholine, POPC) coating the lowest concentration enabling the formation of a stable coating was above 100 µM. A possible reason for the difference is the fact that the lipid mixture used in our experiments contained numerous spieces of different lipids which changed the nature and mechanism of the coating formation.

4. Concluding remarks

Murine microglial and human glioma cell membrane suspension based coatings could be used for studies of membrane interactions and lipid peroxidation under pH range of 6.5 to 10.8. The coatings were instable at more acidic pH value (4.5) and showed constant decrease in the EOF mobility (25 % over 10 successive runs). Differently from coatings based on artificial mixtures of anionic phospholipids the coatings’ stability was not improved by the use of HEPES. The coating based on lipid extractions gave similar results to the coatings based on membrane suspensions in terms of surface charge and stability. The limiting lipid concentration in the coating solution was shown to be around 10 µM.

Acknowledgments

Financial support was provided by the Estonian Ministry of Education, targeted financing no. 0132723s06 (RS), no. SF0130171s08 (all authors), ESF grants no. ETF7338 (SZF and RS) and no.ETF7333 (TL).

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