Yeast cells as microcapsules. Analytical tools and process variables in the encapsulation of hydrophobes in S. cerevisiae

Supplementary Information

Federica Ciamponia, Craig Duckhamb, Nicola Tirellic,d*

a School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

b Cara Technology, Leatherhead Enterprise Centre, Randalls Road, Leatherhead, KT22 7RY, United Kingdom

c School of Materials, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

d School of Biomedicine, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

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Nile Red characteristics and use

In a previous study (Bishop et al. 1998), the water-based process of encapsulation of

terpenes was monitored using a phenoxazine lipid-staining dye, Nile Blue (NB), as a

fluorescent probe.

NB, however, contains oxidized products (phenoxazones) in various proportions, in

particular Nile Red (NR), 9-diethylamino-5H-benzo[α]phenoxazine-5-one (Fig 1SI).

Both compounds are solvatochromic, with green or blue fluorescence in a polar

environment, which red shifts once incorporated into lipidic compartments. NB is

positively charged at neutral pH and typically co-localizes with negatively charged

lipids such as phosphatidyl-inositol and cardiolipin. NR is non-ionic and its emission

does not depend on the local pH, or on the presence of specific chemical compounds.

Additionally, because of its poor water solubility (c.a. 1 μg/mL, i.e. = 3 μM), NR

exhibits bright red-yellow fluorescence in a hydrophobic environment, but negligible

fluorescence in water (Greenspan et al. 1985). Owing to its greater purity and stability,

to the reproducibility of its spectral properties and to the better signal-to-noise ratio, NR

has been extensively used for microscopic imaging purposes; in particular, intracellular

lipidic droplets, which are ubiquitous storage organelles formed by a surface monolayer

of phospholipids and a core of neutral lipids such as triglycerides and esters of

ergosterol (in yeast), or of cholesterol (in mammalian cells) (Greenspan et al. 1985),

have been successfully imaged with NR both in mammalian (Greenspan et al. 1985) and

yeast cells (Kurat et al. 2006).

Fig. 1SI. Molecular structures and spectral properties (Jose and Burgess 2006) of Nile Blue (NB, left) and Nile Red (NR, right).

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Fig. 2SI. Solvatochromic behaviour of NR. NR was dissolved in (from left to right) 50:50 CH3OH:H2O; CH3OH; CH3CN; THF; toluene; cyclohexane; hexane.

Fig. 3SI. Emission spectra of untreated yeast cells, of NR-stained control yeast cells and of the same cells after encapsulation of limonene (20% limonene, 2h). The emission of NR in ethanol is also shown for reference. The concentration of NR and the excitation wavelength (480 nm) was the same in all samples.

Fig. 4SI. Chromatogram of a methanol solution containing 0.05 mg/mL NR, 0.5 mg limonene and 0.55 mg/mL ISD (linalool). The peak at 2.9 min corresponds to linalool, that at 3.7 min to Nile Red and that at 4.4 min to limonene. HPLC analysis was performed using a Reprosil 100 C18 (5µm), 250mm X 4.6 mm column, T = 40°C, eluent: MeOH: Hex 90:10; the UV-detector was set at 210 nm.

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[min.]Time

2 4 6 8

[mV]

Volta

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0

100

200

300

400

nile_red_MeOH-hex_90_1015_02_2010 16_16_09

1.01

7

2

1.89

3

3

2.99

3

4

3.75

0

5

4.42

7

6

0.00 0.05 0.10 0.15 0.20 0.250.0

0.2

0.4

0.6

0.8

1.0

heig

th N

R/ h

eigt

h IS

D

weigth NR/ weigth ISD (mg/ml)

R2 = 0.998

Fig. 5SI. Calibration curve of NR by HPLC. The analysis was performed using a Reprosil 100 C18 (5µm), 250mm X 4.6 mm column, temperature of the column was 40°C, MeOH: Hex 90:10 was the mobile phase and the UV-detector was set at 210 nm.

Fig. 6SI. Control (A) and limonene-loaded (B) log phase (48 hours culture) S. cerevisiae cells stained with NR (0.01 mg/mL, red fluorescence) and Calcofluor white (25 μM, blue). In the control (A) NR stained naturally occurring intracellular lipidic domains, while Calcofluor white allowed the identification of the cell wall (crystalline chitin domains). After treatment with a 20% wt. limonene water dispersion

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(B), the number and size of NR-stained bodies. Deconvolved fluorescence pictures were obtained using DAPI (blue) and TXR-TRITC-PE (red) filters.

Fig. 7SI. Using limonene loading curves as an example, a time log scale (right) allows to better appreciate the more accurate fit of the early time points offered by the biexponential model.

Fig. 8SI. Arrhenius plot of the kinetic data for the internalisation of limonene, linalool and carvone (all 20% wt.) in S. cerevisiae log phase cells. It is apparent that the two terpenes with lower log P (carvone and limonene) do not follow a simple Arrhenius behaviour

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Experimental conditions for viability assaysFUN-1

The dye was dissolved in the staining buffer, consisting of 10 mM Na-HEPES (pH 7.2)

containing 2% glucose, usually in stock solutions of 100 μM and stored at -25°C. 200 μl

samples of control untreated cells, control dead cells (autoclaving at 120°C for 20 min)

and encapsulated cells in log or stationary phase (108 cells/ mL) were centrifuged at

10.000 rpm for 3 minutes in a Eppendorf bench centrifuge and resuspended in the

staining buffer to the same starting volume. The stock solution of FUN-1 was added to

the yeast suspension to reach the final concentration of 10 μM and then incubated in the

dark for 30 min at 37°C in an Eppendorf Thermomixer set at 600 rpm. In some

experiments, the cell wall was stained with Calcofluor White (Molecular Probes) at the

final concentration of 25 μM in staining buffer, following the protocol provided in

http://probes.invitrogen.com/media/pis/mp07009.pdf.

A) Viability assessment through optical microscopy: on a standard microscope slide, 5

μl of Na-alginate 2% (w/v) were deposited and then mixed with 5 μl of the stained cell

suspension. Subsequently, 5 μl of CaCl2 0.1 M were added to induce gelification of the

alginate mixture, which was covered with a 18mm by 18 mm by 1.5 mm cover slip and

then allowed to dry for 1 h at room temperature in darkness. Deconvolved pictures were

analysed by three different observers and scored for the fraction of viable cells (defined

as those presenting a sharp red intracellular element without green cytoplasmic

staining). The number of cells was automatically determined for each field using the

appropriate routine in Image J program and a minimal of 200 cells was evaluated for

each sample.

B) Viability assessment through spectrofluorimetry: The fluorescence emission was

recorded 1) in plate readers (Biotech Synergy 2; 485/20 nm excitation filter and either

528/20 nm (green) or 620/40 (red) emission filters; or TECAN Saffire, excitation at

480nm. And emission recorded in 500 - 700 nm) after transferring aliquots of the

stained suspensions in 96-well plates (bottom-clear, black Greiner Bio One) and

incubating them in the dark for 30 min at 37°C. 2) in a Perkin Elmer LS55

spectrofluorimeter, diluting the yeast suspenssions in 2 mL of staining buffer (to a final

concentration of approximately 107 cells/mL; O.D ca. 1) in disposable plastic cuvettes

(Kartell, 4 clear sides of 4.5 mL capacity). The excitation wavelength was set at 480 nm

and the emission spectra were recorded at T= 25 ºC between 500 and 750 nm with a 10

nm slit. The viability of loaded yeast was calculated on the basis of the red/green

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emission ratio (620 nm / 528 nm), as [Ratio R/G (terpene) - Ratio R/G (autoclaved cells

incubated with terpene) / Ratio R/G (control) - Ratio R/G (autoclaved cells)]*100.

LIVE/DEAD® BacLight

The measurements were carried by flow cytometry (Zhang and Fang 2004) using a

Dako Cyan ADP 488/633 nm dual laser flow cytometer (core facility of the Faculty of

Life Sciences, University of Manchester, technical assistance of Mr. Mike Jackson).

Samples of control untreated cells, control dead cells (autoclaving at 120°C for 20 min)

and encapsulate cells in log or stationary phase were diluted in distilled water to reach a

final concentration of 106 cells/mL in 10 mL. 2 mL aliquots were then transferred to

different 10 mL glass vials and stained with a mixture containing 100 µM SYTO 9 and

600 µM PI. Samples were incubated at room temperature in the dark for 15 min and

then processed for flow cytometry collecting 20,000 cells per sample. The percentage of

viable cells was calculated as the ratio of prevalently green-emitting cells, which was

selected by an appropriate gate in the dot plot obtained by the flow cytometer, over the

total number of emitting cells, which was selected by a different gate in the same dot

plot. A calibration curve was previously obtained using mixtures of live cells with

different proportions of autoclaved cells.

References

Bishop JRP, Nelson G, Lamb J (1998) Microencapsulation in yeast cells. J Microencapsul 15:761-773

Greenspan P, Mayer EP, Fowler SD (1985) Nile Red - A selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100:965-973

Jose J, Burgess K (2006) Benzophenoxazine-based fluorescent dyes for labeling biomolecules. Tetrahedron 62:11021-11037

Kurat CF, Natter K, Petschnigg J, Wolinski H, Scheuringer K, Scholz H, Zimmermann R, Leber R, Zechner R, Kohlwein SD (2006) Obese yeast: Triglyceride lipolysis is functionally conserved from mammals to yeast. J Biol Chem 281:491-500

Zhang T, Fang HHP (2004) Quantification of Saccharomyces cerevisiae viability using BacLight. Biotechnol Lett 26:989-992

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