Supporting Information

 Wiley-VCH 2014
69451 Weinheim, Germany

A Cytoprotective and Degradable Metal–Polyphenol Nanoshell for

Single-Cell Encapsulation**
Ji Hun Park, Kyunghwan Kim, Juno Lee, Ji Yu Choi, Daewha Hong, Sung Ho Yang,
Frank Caruso,* Younghoon Lee,* and Insung S. Choi*

anie_201405905_sm_miscellaneous_information.pdf
 SUPPORTING INFORMATION

CONTENTS

 Experimental Section.
 Table S1. Mean values of logCFU/mL for native yeast and yeast@TA-Fe(III) before and after
HCl treatment.
 Figure S1. Resazurin assay for native yeast and yeast@[TA-Fe(III)]4.
 Figure S2. Raman spectra of native yeast and yeast@[TA-Fe(III)]4.
 Figure S3. CLSM images of yeast@[TA-Fe(III)]4 after treatment of BSA-Alexa Fluor® 488.
 Figure S4. TEM micrographs of microtomed yeast@[TA-Fe(III)]2.
 Figure S5. Color changes of yeast@[TA-Fe(III)]4 after treatment of different amount of HCl
 Figure S6. Linear-fitted graphs of lnOD600 vs. time for yeast@[TA-Fe(III)]2 and yeast@[TA-
Fe(III)]4 before and after 30-min HCl treatment (20 mM) for 30 min.
 Figure S7. Repetition of shell formation and degradation in the control of cell division in a
solution-phase culture.
 Figure S8. Characterizations of HeLa@TA-Fe(III).
 Figure S9. Pattern generation of avidin-cojugated yeast@[TA-Fe(III)]4 on a biotin-patterned
gold surface.
 Figure S10. Magnetic functionalization of yeast cells.

1
Experimental Section

Materials. Tannic acid (TA, Sigma), iron(II) chloride hydrate (Sigma), iron(III) chloride
hexahydrate (FeCl3∙6H2O, Sigma), 3-(N-morpholino)propanesulfonic acid (MOPS, Sigma),
fluorescein diacetate (FDA, Sigma), propidium iodide (PI, Sigma), adenine hemisulfate salt
(C5H5N5∙1/2H2SO4, Sigma), Tris-EDTA buffer solution (pH 7.4, Sigma), glycerol (Junsei),
surfuric acid (H2SO4, Junsei), hydrochloric acid (HCl, 37 wt%, Junsei), Difco™ YPD broth
(YPD broth, BD), Bacto™ agar (BD), Luria-Bertani broth (LB broth, Difco), lyticase from
Arthrobacter luteus (≥ 2,000 units/mg, Sigma), Alexa Fluor® 647 and 488 conjugated albumin
from bovine serum (BSA-Alexa Fluor® 647 and 488, Life Technologies), CellTiter-Blue®
(Promega), Live/Dead® viability/cytotoxicity kit (Life Technologies), silver nanoparticles
(diameter: 20 nm, 60 nm, and 100 nm; 0.02 mg∙mL-1, Sigma), fatal bovine serum (FBS,
Welgene), penicillin-streptomycin (5,000 U∙mL-1 of penicillin and 5,000 g∙mL-1 of streptomycin,
Welgene), phosphate-buffered saline (PBS, pH 7.4, Welgene), Dulbecco’s modified eagle
medium (DMEM, Welgene), dopamine hydrochloride ((HO)2C6H3CH2CH2NH2∙HCl, Sigma),
diethylene glycol ((HOCH2CH2)2O, Sigma), absolute ethanol (CH3CH2OH, Merck), anhydrous
N,N’-dimethylformamide (DMF, Sigma), methylene chloride (CH2Cl2, Sigma), copper(I)
bromide (Cu(I)Br), 2,2’-dipyridyl (bpy, Sigma), N,N’-disuccinimidyl carbonate (DSC, Sigma), 4-
(dimethylamino)pyridine (DMAP, Sigma), 2-(2-aminoethoxy)ethanol (EG2-NH2, Aldrich), (+)-
biotinyl-3,6,9-trioxaundecanediamine (biotin-NH2, Pierce) were used as received. YPAD broth
liquid media were prepared with 50 g of YPD broth and 100 mg of adenine hemisulfate in 950
mL of deionized (DI) water and used after autoclaved (15 min, 121 oC). YPAD agar plates were
prepared on Petri dishes (dimensions: 87 × 15 mm) with 20 mL of YPAD agar solution (50 g of
YPD broth, 15 g of Bacto™ agar, and 100 mg of adenine hemisulfate in 935 mL of DI water).
Poly(ethylene glycol) methacrylate (OEGMA, Mn: ~360, Sigma) was passed through a column
of activated, basic aluminum oxide to remove inhibitors. [BrC(CH3)2COO(CH2)11S]2, which was
the initiator for surface-initiated polymerization, was prepared according to the literature (Shah,
R. R., Merreceyes, D., Husemann, M., Rees, I., Abbott, N. L., Hawker, C. J. & Hedrick, J. L.
Using atom transfer radical polymerization to amplify monolayers of initiators patterned by
microcontact printing into polymer brushes for pattern transfer. Macromolecules 2000, 33, 597-
605). Ultrapure water (18.3 M·cm) from the Human Ultrapure System (Human Corp., Korea)
was used.

Characterizations. Raman spectra were obtained with a Jobin Yvon/HORIBA LabRAM

spectrometer equipped with an integral microscope (Olympus BX 41, Japan). The 632.8 nm
line of an air-cooled He/Ne laser was used as excitation source. Zeta potentials were measured
with Zetasizer Nano ZS (Malvern, UK) and calculated based on Smoluchowski model. Field-
emission scanning electron microscopy (FE-SEM) imaging was performed with a FEI Inspect
F50 microscope (FEI, the Netherlands) with an accelerating voltage of 10 kV, after sputter-
coating with platinum. The cells were observed with a LSM 700 confocal microscope (Carl Zeiss,
Germany). For resazurin assay, fluorescence intensity was measured with a Synerge™ MX
multi-mode microplate reader (BioTek Instruments, USA). Optical density was measured with an
Ultrospec 7000 spectrophotometer (GE Healthcare Life Science, UK). Transmission electron
microscopy (TEM) imaging was performed by using a JEM-2100 (JEOL, Japan). Specimens
were fixed with glutaraldehyde and OsO4, and then dehydrated in ethanol. The fixed samples
2
were embedded in Epon 812/Araldite M resin. Thin sections were cut by using ULTRACUT
UCT ultramicrotome (Leica, Austria) and stained with uranyl acetate and lead citrate.

Encapsulation of Individual S. cerevisiae with TA-Fe(III) Shells. A single colony of S.

cerevisiae (Baker’s yeast) was picked from a YPAD agar plate and cultured in the YPAD broth
liquid media with continuous shaking at 30 oC for 60 h. The yeast cells were washed with
deionized (DI) water three times and dispersed in DI water. The 5 μL aqueous solution of TA (40
mg∙mL-1) and the 5 μL aqueous solution of FeCl3∙6H2O (10 mg∙mL-1) were added sequentially to
the aqueous suspension of yeast cells (490 L) with 10-sec vigorous mixing between the
additions. After addition of the FeCl3∙6H2O solution, the resulting suspension was mixed
vigorously for 10 sec, and 0.5 mL of 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (20
mM, pH 7.4) was added to the yeast@TA-Fe(III) suspension for the stabilization of the pH,
resulting in the formation of stable TA-Fe(III) shell. Yeast@TA-Fe(III) was washed with DI
water three times to remove any residual TA and FeCl3. The coating process from addition of TA
and FeCl3 to washing with DI water was repeated two or four times.

Viability Test. The cell viability was investigated by the fluorescein diacetate (FDA) assay,
where FDA is hydrolyzed to the green-fluorescent fluorescein by esterases in the metabolically
active cells. The FDA stock solution (5 mg∙mL-1) was prepared in acetone due to the insolubility
of FDA in water, and 4 μL of the stock solution was mixed with 0.5 mL of the yeast suspension.
After 20 min, the cells were washed with DI water three times and then visualized by confocal
laser-scanning microscopy.

Protein Conjugation. To attest the protein conjugation ability of the TA-Fe(III) nanoshell, the
aqueous suspension of native yeast or yeast@[TA-Fe(III)]4 was mixed with the aqueous solution
of BSA-Alexa 647 (0.4 mg∙mL-1), and the resulting solution was incubated for 15 min. After that,
the viability of the resulting yeast@[TA-Fe(III)]4 was investigated by the FDA assay, showing
the core-shell structures.

Control of Cell Divsion. Before cell-division test, the optical density of native yeast or
yeast@TA-Fe(III) was adjusted to 2.0 at 600 nm by dilution with DI water. For the degradation
of the TA-Fe(III) nanoshell, the yeast cells (OD600 = 2.0) were incubated in 5 or 20 mM of HCl
for 90 min. (a) On Agar plates: The resulting yeast cells were washed with DI water three times
and dispersed in DI water. The colony-forming unit (CFU) values were obtained by the cell
culture on YPAD agar plates. The 15 L of the yeast suspension was diluted to 300 L with DI
water, and 10 L of the diluted suspension was diluted further to 1 mL with DI water (total
dilution factor: 2000). The 150 L of the resulting yeast suspension was spread on a YPAD agar
plate. After 2-d incubation in a thermostat incubator at 30 oC, the colonies were enumerated, and
the CFU values were converted into log ones. (b) In the Liquid Media: The value was
calculated based on the linear-fitted plot of lnOD600 vs. time. Native yeast and yeast@TA-Fe(III)
(OD600 = 0.01) were suspended, respectively, in the YPAD broth liquid media and cultured in a
shaking incubator at 30 oC. The 400 L of the mixture was picked at the predetermined time, and
the optical density was measured at 600 nm by UV-visible spectroscopy. The growth curve of the
HCl-treated yeast@TA-Fe(III) was also obtained by the same protocol.

3
Protection Tests: E. coli-induced Agglutination, UV Irradiation, Lyticase, and Silver
Nanoparticles. For fimbriae expression of E. coli, a single colony of MG1655, wild type strain
of E. coli, was inoculated in LB liquid media and grown statically at 37 oC for 16 h. The E. coli
cells were centrifuged (5000 g, 5 min, 4 oC), washed with DI water twice, and dispersed in PBS
(pH 7.4). The optical densities of yeast and E. coli were adjusted to ca. 5 and 3 at 600 nm,
respectively. The yeast and E. coli suspensions (20 L each) were mixed together on a slide glass
with gentle shaking. After 1 min, the agglutination was visualized with a confocal microscope.
For UV-C resistance test, the optical density of native yeast or yeast@[TA-Fe(III)]4 was adjusted
to ~1.0, and 3 mL of the yeast suspension was prepared in a quartz cuvette (Hellma Co.,
Germany). The cuvette was placed in a home-made dark chamber equipped with a 4-W filtered
UV lamp, VL-4.LC (Vilber Lourmat Co., France). The distance between the cuvette and the UV
lamp was adjusted to be 5 cm. UV-C light (: 254 nm) was irradiated for the predetermined time
(irradiation energy: 8 J or 12 J). The irradiation energy was calculated based on the reported
intensity of the UV lamp (intensity at 15 cm = 265 W∙cm-2). After UV-C irradiation, the
viability of the resulting yeasts was measured by the FDA assay (with at least 300 cells). For
cell-lysis test, the stock solution of lyticase was prepared by dissolving lyticase (~3.8 mg, ≥
2,000 units/mg protein, from Arthrobacter luteus) in a mixture of glycerol (500 μL) and Tris-
EDTA (TE) buffer solution (500 μL, pH 7.4). The 10 μL of the stock solution was added to the
yeast suspension (TE buffer solution, pH 7.4), and the suspension was incubated while shaking
at 37 oC. The 400 L of the mixture was picked after 3 h, and the optical density was measured
at 600 nm by UV-visible spectroscopy. For protection test against silver nanoparticles, the optical
density of native yeast or yeast@[TA-Fe(III)]4 was adjusted to ~2.0 at 600 nm by dilution with
DI water. The 500 L of yeast suspension was mixed with 500 L of the three different silver
nanoparticle suspensions each (20 nm, 60 nm, or 100 nm; 0.02 mg∙mL-1). The mixture was
concentrated to 50 L by centrifugation (10,000 rpm, 1 min) and incubated at room temperature
for 20 h with gentle mixing. The cells were stained by FDA (4 L∙mL-1 of the stock solution (5
mg∙mL-1 in acetone) for live cells) and propidium iodide (PI) (2 L∙mL-1 of the stock solution (1
mg∙mL-1 in DI water) for dead or dying cells) and visualized with a confocal laser-scanning
microscope.

Encapsulation of HeLa Cells. HeLa cells were seeded in a cell culture flask with 10 mL of the
serum-free DMEM solution containing 10% FBS and 1% penicillin-streptomycin, and the cells
were incubated at 37 oC under humidified atmosphere of 5% CO2. When the cells were grown to
80% confluency, they were washed with PBS twice. The 2 mL of trypsin was added to the
culture flask, and the cells were placed at 37 °C for 5 min. After the cells were detached from the
flask, 3 mL of DMEM was added and the cells were collected by centrifugation and cells were
washed with PBS twice. The detached cells were encased with a TA-Fe(III) nanoshell by
incubating them in the DMEM solution of TA (0.4 mg∙mL-1) and FeCl3 (0.1 mg∙mL-1) for 10 sec.
The viability of HeLa@TA-Fe(III) was measured with Live/Dead® viability/cytotoxicity kit.

Generation of Cell Patterns. Yeast@[TA-Fe(III)]4 was immersed in PBS (10 mM, pH 7.4) of
0.5 mg/mL avidin (10-15 units/mg protein, from Egg White) for 1 h with gentle stirring. The
washing with the PBS solution gave the avidin-conjugated yeast@TA-Fe(III). The line pattern of
biotin on gold was generated by following the reported procedure (Lee, B. S., Chi, Y. S., Lee, K.-
B., Kim, Y.-G. & Choi, I. S. Functionalization of poly(oligo(ethylene glycol) methacrylate) films

4
on gold and Si/SiO2 for immobilization of proteins and cells: SPR and QCM Studies.
Biomacromolecules 2007, 8, 3922-3929). Briefly, poly(oligo(ethylene glycol) methacrylate)
(pOEGMA) was formed on a gold substrate by surface-initiated, atom transfer radical
polymerization. To activate the terminal hydroxyl groups of side chains in the pOEGMA films,
the films were immersed in dry DMF solution containing 0.1 M DSC and 0.1 M DMAP for 14 h
at room temperature under an argon atmosphere. The resulting substrates were rinsed with DMF
and CH2Cl2, and then dried in a stream of argon. Line-patterned PDMS stamps (width: 50 m,
spacing: 100 m) were prepared according to the literature method using Sylgard 184 silicone
elastomer (Dow Corning, USA). After curing for 6 h at 60 oC, the PDMS stamp was peeled off
from the master. Before use, the PDMS stamp was cleaned and oxidized by an oxygen plasma
cleaner (Harrick PDC-002 at medium setting) for 1 min. For the pattern generation, the PDMS
stamp was inked by spin-casting with an ethanolic solution of biotin-NH2 (1 mg∙mL-1). The
inked stamp was brought into contact with the DSC-activated pOEGMA film for 60 s. The stamp
was carefully peeled off and then immersed immediately in a solution of EG2-NH2 (0.1 mg∙mL-1,
0.1 M sodium bicarbonate) for 1 h and rinsed with DI water. The biotin-patterned gold substrate
was immersed in PBS containing the avidin-conjugated yeast@[TA-Fe(III)]4. The cell pattern
was readily formed and observed with an optical microscope (Eclipse ME 600, Nikon, Japan).

Magnetic Functionalization. Dopamine-terminated superparamagnetic iron oxide nanoparticles

(Fe3O4∙dopamine) were synthesized according to the previous literature (Qu, H., Caruntu, D., Liu,
H. & O’Connor, C. J. Water-dispersible iron oxide magnetic nanoparticles with versatile surface
functionalities. Langmuir 2011, 27, 2271-2278). Yeast cells were coated with TA-Fe(III) by
repeating the deposition process twice, and the [TA-Fe(III)]2-coated yeast cells were dispersed in
the 500 L of DI water, followed by mixing with 500 L of magnetic nanoparticle suspension (1
mg∙mL-1) for 5 min with vigorous shaking. The resulting yeast@[TA-Fe(III)]2∙Fe3O4∙dopamine
was washed with DI water three times, and the deposition of TA-Fe(III) was performed twice,
generating yeast@[TA-Fe(III)]4∙Fe3O4∙dopamine.

5
Table S1. Mean values of logCFU/mLa for native yeast and yeast@TA-Fe(III) before and after
HCl treatment.

Native Yeast@ Yeast@

Conc. of HCl
Yeast [TA-Fe(III)]2 [TA-Fe(III)]4
0 7.12±0.02 5.71±0.02 4.47±0.00

5 mM 7.07±0.03 6.79±0.00 6.39±0.02

20 mM 7.01±0.00 6.81±0.02 6.91±0.02

a
LogCFU/mL: log-transformed colony forming unit (CFU) value per milliliter.

6
Figure S1. Resazurin assay: fluorescence intensity plot of resorufin (reduced form of resazurin)
generated by the biochemical reduction of resazurin inside viable cells (excitation wavelength:
560 nm). RFU: relative fluorescence unit.

7
Figure S2. Raman spectra of (blue) native yeast and (red) yeast@[TA-Fe(III)]4 (data acquisition
time: 60 sec). Black arrows indicate the strong bands attributed to the ring structures of TA.

8
Figure S3. CLSM images of yeast@[TA-Fe(III)]4 after treatment of BSA-Alexa Fluor® 488 (left:
differential interference contrast microscopy image merged with fluorescent image; right:
fluorescent image).

9
Figure S4. TEM micrographs of microtomed yeast@[TA-Fe(III)]2 at different magnifications.

10
Figure S5. Color changes of the yeast@[TA-Fe(III)]4 solution after treatment of different
amounts of HCl (final concentration of HCl: 0 mM, 5 mM, 10 mM, 20 mM, or 100 mM).

11
Figure S6. Linear-fitted graphs of lnOD600 vs. time for yeast@[TA-Fe(III)]2 and yeast@[TA-
Fe(III)]4 before and after 30-min HCl treatment (20 mM). The growth rate () is defined as the
slope of the linear-fitted line. Blue and red linear-fitted lines are drawn based on the average
values of intercept and slope for yeast@[TA-Fe(III)]2 and yeast@[TA-Fe(III)]4.

12
Figure S7. Repetition of shell formation and degradation in the control of cell division in a
solution-phase culture for yeast[TA-Fe(III)]4.

13
Figure S8. FE-SEM micrographs of (a) native HeLa cell and (b) HeLa@TA-Fe(III). (c)
Live/dead assay of HeLa@TA-Fe(III). The HeLa cells, detached from a culture flask and
collected by centrifugation, were incubated in the DMEM solution of tannic acid (0.4 mg/mL)
and FeCl3 (0.1 mg/mL) for 10 sec. The live/dead assay of HeLa@TA-Fe(III) was performed with
the LIVE/DEAD® cell viability kit (Life Technologies). The green cells were considered to be
alive, and the red ones to be dead.

14
Figure S9. Pattern generation of avidin-cojugated yeast@[TA-Fe(III)]4 on a biotin-patterned
gold surface. pOEGMA: poly(oligo(ethylene glycol) methacrylate). The line pattern of biotin on
gold was generated by following the reported procedure (Lee, B. S., Chi, Y. S., Lee, K.-B., Kim,
Y.-G. & Choi, I. S. Functionalization of poly(oligo(ethylene glycol) methacrylate) films on gold
and Si/SiO2 for immobilization of proteins and cells: SPR and QCM Studies. Biomacromolecules
2007, 8, 3922-3929).

15
Figure S10. Magnetic functionalization of yeast cells. Yeast@[TA-Fe(III)]4∙Fe3O4 was viable
after magnetic functionalization (evidenced by the FDA assay). Magnetic separation was
demonstrated by applying magnetic field with a neodymium magnet (Nd2Fe14B).

16