COMMUNICATION
Intracellular protein delivery by glucose-coated polymeric beadsw
Suhyun Jung,a Seong Huh,b Yong-Pil Cheon*c and Seongsoon Park*a
Received (in Cambridge, UK) 30th March 2009, Accepted 23rd June 2009
First published as an Advance Article on the web 13th July 2009
DOI: 10.1039/b906268h
Glucose-coated polymeric beads have been prepared and applied
to delivery of a model protein (enhanced green fluorescent
protein) into mouse embryonic stem cells as well as Hela cells.
The intracellular delivery of proteins is garnering more interest
because proteins can be used as therapeutic agents1 and research
tools for studying intracellular protein–protein, protein–lipid,
and protein–DNA interactions.2 Moreover, the development of
a protein delivery system may benefit researchers in a variety of
applications including drug delivery,3 and the control of cell
differentiation using signaling peptides or functional proteins.4
However, proteins are generally unable to cross the membrane
barrier of eukaryotic cells without using protein-specific trans-
port systems.5 For intracellular protein delivery, researchers
have used recognition molecules that can interact with cell
membrane molecules. One example is cationic lipids.6 The
cationic head group of cationic lipids is able to interact with
negatively charged cell membrane molecules through electro-
static attractions. Another approach for protein delivery is the
usage of cell-permeable peptides or proteins that can interact
with cell membranes and penetrate into cells.7
Scheme 1 Synthesis of 6-O-glucosyl methacrylate and monodispersed
polymeric beads.
methacrylate, styrene, and acrylic acid. Acrylic acid provided
the functional group with which to conjugate proteins
(see below). Glucose was ligated to methacrylate by CAL-B
(Candida antarctica lipase B, Novozyme 435) to form 6-O-
glucosyl methacrylate ((1) in Scheme 1). The 6-O-glucosyl
methacrylate was then polymerized with styrene and acrylic
acid in aqueous methanol to form submicro-sized beads ((2) in
Scheme 1).11
The amount of glucose units on the beads was limited to
5–10% molar ratio compared to the amount of styrene used.
Preparation of glucose-free acrylic acid beads for the control
experiment was attempted by polymerization of styrene and
acrylic acid without the addition of 6-O-glucosyl methacrylate.
Scanning electron microscopy (SEM) (Fig. 1) was used to show
that the glucose-coated beads were uniformly of an average
diameter of about 150 nm (Fig. 1a). However, the acrylic acid
beads were larger than the glucose-coated beads, having an
average diameter of about 300 nm (Fig. 1b). Because we were
unable to prepare acrylic acid beads with the same diameter as
the glucose-coated beads, and transduction efficiency may vary
with size, an alternative approach was employed. Similar-sized
non-glucose-coated beads (0%-glucose-coated beads) for the
control experiment were prepared by removing glucose units
from the glucose-coated beads via acid hydrolysis (Fig. 1c).
EGFP (enhanced green fluorescent protein) was chosen as a
model protein because it can easily be tracked in cells and its
folding status may easily be recognized by fluorescence. The
fluorescent protein EGFP was covalently attached to the
beads by a simple chemical treatment. The carboxylic acid
groups on the beads were utilized to bind proteins after
activation with EDC (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide) (eqn (1)). The free amino groups of EGFP react
The development of protein carriers requires the usage of a
recognition molecule to establish interactions with the cell
membrane. Glucose is a good candidate for such a molecule
because most cells take up glucose to use as a fuel molecule as
well as a metabolic intermediate. Many mechanisms for the uptake
of glucose into cells have recently been described,8 although the
detailed mechanisms are still a matter of debate.9 Thus, glucose
may be used to establish interactions between the cell membrane
and the carrier. In this communication, we tested this idea with a
view to developing a novel protein delivery system using polymeric
beads. We show that glucose-coated polymeric beads can
efficiently deliver native, properly folded proteins into cells.
The monodispersed polymeric beads were prepared by
dispersion polymerization. Dispersion polymerization is an
appropriate method for attaining small-sized monodispersed
beads in a single step. In this procedure, various functional groups
can be introduced to the beads by using proper monomers.10
Glucose-coated beads were prepared using 6-O-glucosyl
a Department of Chemistry, Center for NanoBio Applied Technology,
and Institute of Basic Sciences, Sungshin Women’s University, Seoul
136-742, Korea. E-mail: spark@sungshin.ac.kr; Fax: 82 2 9202047;
Tel: 82 2 920 7646
b Department of Chemistry and Protein Research Center for
Bio-Industry, Hankuk University of Foreign Studies,
Yongin 449-791, Korea
c Department of Biology, Sungshin Women’s University, Seoul 136-742,
Korea. E-mail: ypcheon@sungshin.ac.kr; Tel: 82 2 920 7639
w Electronic supplementary information (ESI) available: Experimental
details, analysis by transmission electron microscopy, and the bead
transduction into Hela cells at 37 1C and into mES cells at 4 1C. See
DOI: 10.1039/b906268h
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This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 5003–5005 | 5003