Mussel-inspired encapsulation and functionalization of individual yeast cells

Article abstract of DOI:10.1021/ja1100189

The individual encapsulation of living cells has a great impact on the area of cell-based sensors and devices as well as fundamental studies in cell biology. In this work, living yeast cells were individually encapsulated with functionalizable, artificial

Full text of DOI:10.1021/ja1100189

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Mussel-Inspired Encapsulation and Functionalization of Individual
Yeast Cells
†
†,‡
§
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†,‡
,†,z
Sung Ho Yang, Sung Min Kang, Kyung-Bok Lee, Taek Dong Chung, Haeshin Lee, and Insung S. Choi*
†
Molecular-Level Interface Research Center, Department of Chemistry, KAIST, Daejeon 305-701, Korea
‡
The Graduate School of Nanoscience and Technology, KAIST, Daejeon 305-701, Korea
§
Division of Life Science, Korea Basic Science Institute (KBSI), Daejeon 305-333, Korea
Department of Chemistry, Seoul National University, Seoul 151-747, Korea
z
Department of Bio and Brain Engineering, KAIST, Daejeon 305-701, Korea
S Supporting Information
b
Scheme 1. Schematic Representation of Polydopamine En-
capsulation of Individual Yeast Cells and Functionalization of
the Artificial Shells
ABSTRACT: The individual encapsulation of living cells has
a great impact on the area of cell-based sensors and devices as
well as fundamental studies in cell biology. In this work, living
yeast cells were individually encapsulated with functionaliz-
able, artificial polydopamine shells, inspired by an adhesive
protein in mussels. Yeast cells maintained their viability within
polydopamine, and the cell cycle was controlled by the
thickness of the shells. In addition, the artificial shells aided
the cell in offering much stronger resistance against foreign
aggression, such as lyticase. After formation of the polydopa-
mine shells, the shells were functionalized with streptavidin by
utilizing the chemical reactivity of polydopamine, and the
functionalized cells were biospecifically immobilized onto the
defined surfaces. Our work suggests a biomimetic approach to
the encapsulation and functionalization of individual living
cells with covalently bonded, artificial shells.
7
groups by metabolic or genetic engineering. Noncovalent adsorp-
tion of macromolecules onto the cell surfaces also has been
attempted in order to introduce chemical functionalities into living
cells. However, cell-surface modification has not been achieved
simultaneously with protective encapsulation. In this work, we
report a simple but versatile approach;inspired by nature;for
encapsulating individual living cells with functionalizable, artificial
n nature, certain biological systems, including bacteria, plants,
algae, and fungi, have evolved to preserve their species under
7e,8
I
unfavorable harsh environments by protecting their genetic infor-
mation with a hard shell. Besides the biological uniqueness, the
robustness of such shells was believed to be beneficial in increasing
the stability of cell-based devices, including sensors: for example,
organic shells formed by strong covalent bonds. Polydopamine
9
(
PD), inspired by an adhesive protein in mussels, was chosen as a
1
microbial spores have been used for analyte detection and cell
coating material for introducing organic shells onto living cells
Scheme 1). The polymerization of dopamine occurs under
2
patterns. On the other hand, sporelike structures have recently
(
been generated chemically by encapsulating individual living cells
biologically compatible conditions, and PD itself exhibits negligible
3
4
within artificial shells, such as silica, calcium phosphate, calcium
cytotoxicity, as demonstrated by the fact that it promotes cell
5
6
carbonate, and multilayers of polyelectrolytes. We anticipate that
these artificially formed sporelike structures (“artificial spores”) will
contribute to increasing the long-term stability and performance of
cell-based sensors, bioreactors, microfluidic devices, etc., as well as
to fundamental studies in cell biology. Most of the methods for
forming artificial shells have been limited to electrostatic layer-by-
layer (LbL) self-assembly of polyelectrolytes, where polyelectrolyte
10,11
adhesion on various substrates.
In addition, we envisioned that
the chemical reactivity of the PD shells formed on individual living
9b-9d
cells could be utilized for cell-surface postmodification.
Dopamine was polymerized on the surface of Saccharomyces
9,10
cerevisiae (yeast), leading to the formation of single PD-coated
yeast cells (yeast@PD ). The same procedure was repeated with
1
yeast@PD to form double-PD-coated yeasts (yeast@PD ). The
1
2
6
multilayers acted as a shell material itself or a catalytic template for
yeast cells were encapsulated separately and individually within PD
shells (Figure 1a,b; also see the Supporting Information for
3,4
subsequent formation of inorganic shells.
Cell-surface modification is another important issue for the
application of living cells. It has been achieved mostly by compli-
cated methods, such as the introduction of nonbiogenic functional
Received: November 17, 2010
Published: January 25, 2011
r 2011 American Chemical Society
2795
dx.doi.org/10.1021/ja1100189
|J. Am. Chem. Soc. 2011, 133, 2795–2797

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Figure 2. (a) Growth curve of native yeast (2), yeast@PD
yeast@PD (b). (b) Survival of native yeast (2), yeast@PD
yeast@PD
1
(9), and
(9), and
2
1
2
(b) in the presence of lyticase. Optical density was measured
using absorbance measurements at 600 nm (OD600).
Figure 1. Confocal micrographs of (a) native yeasts and (b) yeast@PD2.
The insets show magnified images. (c) TEM micrograph of microtome-
sliced yeast@PD
e) yeast@PD , and (f) yeast@PD
indicate the polydopamine shells.
1
. (d-f) Magnified TEM images of (d) native yeast,
(
1
2
(scale bar = 100 nm). The arrows
confocal images of yeast@PD ). Yeast@PD was opaque to light
1
because of its PD shell. The opacity results from the molecular
structure of PD, which is thought to be composed of highly
9d
Figure 3. Optical micrographs of (a) yeast@PD and (b) avidin-linked
1
conjugated aromatic rings. While the native yeast was noticeably
shrunk, yeast@PD maintained its original round shape even after
drying for 12 h at room temperature (see the Supporting Informa-
tion for scanning electron microscopy images). The maintenance
of the shape indicated that the PD shells could act as a physically
yeast@PD
micrograph of avidin-linked yeast@PD
PEGMA) surface that was composed of a poly(PEGMA) region (left)
1
on biotin-presenting poly(PEGMA) surfaces. (c) Optical
1
on a biotin-patterned poly-
(
and a biotin-presenting region (right). The scale bars in the main panels
represent 100 μm, and that in inset of (b) represents 10 μm.
3-5
protective shell for living cells.
Transmission electron micros-
copy (TEM) micrographs showed that the PD shell consisted of
two parts, a uniform thin film firmly coating the cell wall and big
particulates (Figure 1c-f; also see the Supporting Information for a
TEM image of the native yeast). The robust coating was achieved
presumably by covalent bonding between PD and amine or thiol
moieties of (glyco)proteins in the cell wall during the course
native yeast was lysed (Figure 2b; also see the Supporting
Information for full UV spectra). The results clearly show that
the PD shells retarded the penetration of lyticase, probably by
stabilizing cellular membranes through the formation of covalent
bonds between PD and cell-wall constituents. Furthermore, the
resistance against lyticase was controlled simply by the thickness of
9
b-9d,12
of polymerization.
In addition, the results showed that
the PD shell. The optical density of yeast@PD remained higher
2
the thickness of the PD shell was controlled facilely by multi-
coating: the average thicknesses were estimated to be ∼30 nm for
yeast@PD and ∼80 nm for yeast@PD These values corre-
than that of yeast@PD until 15 h. The control of cell-division
1
cycles or chemical/biological protection was not reported for
6
organic multilayers of polyelectrolytes, implying that the multi-
1
2
sponded well with those for previously reported PD films that
layers were too weak to protect the cells and suppress cell division
because they were formed by electrostatic interactions. Therefore,
the PD encapsulation, which utilizes covalent bonds, could serve as
a new strategyfor controlling cell division and protection of artificial
sporelike structures in a designed way.
9d,10
had been formed on flat surfaces and microspheres.
The PD encapsulation of yeast cells was found to control cell
division, which would be one of the essential characteristics to be
drawn from the artificially formed sporelike structures. The cell-
culture experiments showed that yeast cells kept the capability of
dividing themselves under culture conditions even after PD
encapsulation (Figure 2a; also see the Supporting Information
for confocal micrographs of duplicating cells). Native yeast cells
immediately proliferated without a lag phase, but the growth curves
of yeast@PD and yeast@PD remained in the lag phase for more
We utilized the chemical reactivity of PD toward amine and thiol
9b-d
13
functionalities
for the surface modification of yeast@PD1.
Yeast@PD was functionalized with avidin and then immobilized
1
on a biotin-presenting surface (see the Supporting Information for
the detailed experimental procedures with native yeast asa control).
Avidin-linked yeast@PD was densely immobilized onto a biotin-
1
2
1
than 24 h. Since the viability was much above 50% (see the
Supporting Information for the viability test), we thought that
the PD shell prevented yeast cells from dividing and let them
remain in a quiescent state, because even 50% loss of the living cells
would just extend the lag phase slightly. More interestingly, the
period of the quiescent state was controlled by the thickness of the
presenting poly(PEGMA) surface, while yeast@PD was rarely
adsorbed onto the same surface (Figure 3a,b). When avidin-linked
1
yeast@PD was exposed to the biotin-patterned poly(PEGMA)
1
surface, it was conjugated spatioselectively only onto the biotin-
presenting area via the biospecific interaction between avidin and
biotin (Figure 3c).
PD shell: the lag phase of yeast@PD lasted for 36 h, while that of
Insummary, wehavedemonstrated thereactiveencapsulation of
individual yeast cells with polydopamine, which is a biocompatible
coating material inspired by the adhesive protein in mussels.
The individual encapsulation with polydopamine is of importance
in the realization of artificialspores:(1) Itisthefirst approach to the
encapsulation of living cells within covalently bonded organic
materials. (2) The polydopamine coating was found to be physi-
cally stable in comparison with polyelectrolyte multilayers and to be
1
yeast@PD lasted for 84 h.
2
It is one of the important roles of hard shells in natural spores to
preserve their species against foreign aggression. Although both
native and PD-coated yeasts were lysed as a result of the digestion
activity of lyticase, the PD-coated yeasts were much more resistant
against lysis than the native yeast: ∼70% of yeast@PD and ∼90%
1
of yeast@PD still survived after 1 h, while more than 90% of the
2
2
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Journal of the American Chemical Society
COMMUNICATION
effective in protecting living cells and controlling cell division.
(d) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science
2007, 318, 426.
(
3) The polydopamine shell could be further functionalized for
3-7
(
10) Postma, A.; Yan, Y.; Wang, Y.; Zelikin, A. N.; Tjipto, E.; Caruso,
F. Chem. Mater. 2009, 21, 30424.
11) (a) Ku, S. H.; Ryu, J.; Hong, S. K.; Lee, H.; Park, C. B.
applications of interest. In comparison with previous reports,
polydopamine encapsulation is a simple and versatile method for
introducing various functionalities onto the cell surface under
physiologically compatible conditions. We believe that polydopa-
mine encapsulation would be a good starting point for both
fundamental research and applications based on artificial spores,
as it endows living cells with durability against harsh environ-
ments, controllability in cell cycles, and reactivity for cell-surface
modification.
(
Biomaterials 2010, 31, 2535. (b) Hwang, D. S.; Waite, J. H.; Tirrell,
M. V. Biomaterials 2010, 31, 1080.
(12) (a) Anderson, T. H.; Yu, J.; Estrada, E.; Hammer, M.; Waite,
J. H.; Israelachvili, J. N. Adv. Funct. Mater. 2010, 20, 4196. (b) Lee, H.;
Scherer, N. F.; Messersmith, P. B. Proc. Natl. Acad. Sci. U.S.A. 2006, 103,
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(13) (a) Kang, S. M.; Kong, B.; Oh, E.; Choi, J. S.; Choi, I. S. Colloids
Surf., B 2010, 75, 385. (b) Kang, S. M.; Lee, K.-B.; Kim, Y.; Choi, I. S.
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’
ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
additional figures. This material is available free of charge via the
Internet at http://pubs.acs.org.
’
AUTHOR INFORMATION
Corresponding Author
ischoi@kaist.ac.kr
’
ACKNOWLEDGMENT
This work was supported by a Korea Research Foundation
grant funded by the Korean Government (MOEHRD, KRF-
008-313-C00496), WCU (R31-2008-000-10071-0), and the
2
Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Educa-
tion, Science and Technology (2010-0001953; 2010-0001954).
We thank Dr. H. S. Kweon and H. J. Cho at KBSI for the TEM
analyses and K. B. Kim and H. Choi at Seoul National University
for the cell-viability analyses.
’
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