Cell-surface engineering by a conjugation-and-release approach based on the formation and cleavage of oxime linkages upon mild electrochemical oxidation and reduction

Article abstract of DOI:10.1002/anie.201404099

We report a strategy to rewire cell surfaces for the dynamic control of ligand composition on cell membranes and the modulation of cell-cell interactions to generate three-dimensional (3D) tissue structures applied to stem-cell differentiation, cell-surfa

Full text of DOI:10.1002/anie.201404099

Angewandte
Chemie
DOI: 10.1002/anie.201404099
Electroactive Cell Surfaces
Cell-Surface Engineering by a Conjugation-and-Release Approach
Based on the Formation and Cleavage of Oxime Linkages upon Mild
Electrochemical Oxidation and Reduction**
Abigail Pulsipher, Debjit Dutta, Wei Luo, and Muhammad N. Yousaf*
Abstract: We report a strategy to rewire cell surfaces for the
dynamic control of ligand composition on cell membranes and
the modulation of cell–cell interactions to generate three-
dimensional (3D) tissue structures applied to stem-cell differ-
entiation, cell-surface tailoring, and tissue engineering. We
tailored cell surfaces with bioorthogonal chemical groups on
the basis of a liposome-fusion and -delivery method to create
dynamic, electroactive, and switchable cell-tissue assemblies
through chemistry involving chemoselective conjugation and
release. Each step to modify the cell surface: activation,
conjugation, release, and regeneration, can be monitored and
modulated by noninvasive, label-free analytical techniques. We
demonstrate the utility of this methodology by the conjugation
and release of small molecules to and from cell surfaces and by
the generation of 3D coculture spheroids and multilayered cell
tissues that can be programmed to undergo assembly and
disassembly on demand.
been developed and employed, including dielectrophoresis,[6]
microfabrication,[7] hydrogel,[8] and cell-patterning tech-
niques.[9] The tailoring of cell membranes by cell-surface
engineering methods has also proven to be important for the
development of coculture and multicellular microtissues.[10] In
particular, the integration of bioorthogonal chemical strat-
egies[11] with cell surfaces may enable a range of cell-surface
modifications for the control of ligand presentation and
density, and potentially the spatiotemporal control of cell–cell
interactions. Bioorthogonal chemical reactions (e.g. Diels–
Alder reaction, oxime reaction, Huisgen cycloaddition, Stau-
dinger reaction) have been extensively developed and utilized
owing to their ability to be performed under physiological
conditions without side reactions and in complex protein
mixtures, in cell lysates, and in vivo. Furthermore, these
chemistries have been applied in many fundamental cell
studies,[12] drug-delivery therapies,[13] and diagnostic measur-
ing applications.[14] However, the incorporation of a range of
these chemical groups on the cell surface of a variety of cell
types remains challenging.[15]
Synthetic liposomes and fused liposomes have proven
very useful for numerous studies as cell-membrane model
systems and as microarray platforms for the study of cell-
membrane dynamics and for biotechnology applications.[16]
Methods for liposome-to-liposome and liposome-to-cell
fusion have also been developed for the delivery of ther-
apeutic agents to cells and organelles, and for the study of
cellular interactions.[17] However, until now, there has been no
report on the use of liposome–cell fusion to deliver switchable
and bioorthogonal groups directly to cell surfaces for
subsequent chemoselective conjugation and the release of
ligands or for the temporal programming of controlled cell–
cell assembly. A strategy that combines cell-surface modifi-
cation, without the use of molecular-biology techniques or
biomolecules, with a stable, dynamic, and switchable bioor-
thogonal approach to ligand conjugation and release to direct
tissue formation and subsequent disassembly would greatly
benefit fundamental cell-behavioral studies and tissue-engi-
neering research.
T
he control of cell–cell interactions and cellular architecture
in three-dimensional (3D) space and time is critical for the
proper development[1] and survival of higher-order organ-
isms.[2] These dynamic interactions are complex but essential
for correct cell behavior and tissue function based on a myriad
of physical, mechanical, and hydroynamic forces, as well as
autocrine and paracrine signaling.[3] However, the reproduc-
tion of these processes in vitro while maintaining these
dynamic and discrete cell–cell contacts is difficult and
requires a multidisciplinary coordinated effort.[4] Thus, the
ability to modulate cell–cell interactions in space and time
would allow unprecedented control of cell behavior and
enable the design of new useful dynamic tissue-engineering
scaffolds, in vivo imaging systems, high-throughput tissue-
based screening assays, and drug-delivery therapies.[5]
Several approaches to generate coculture tissue structures
in two and three dimensions (2D and 3D, respectively) have
[*] A. Pulsipher, D. Dutta, W. Luo, Prof. M. N. Yousaf
Department of Chemistry
University of North Carolina at Chapel Hill (USA)
and
Department of Chemistry and Biology, York University (Canada)
E-mail: chrchem@yorku.ca
Herein, we report a liposome-based methodology to tailor
cell surfaces with dynamic and switchable bioorthogonal
chemical functionalities and to direct the assembly and
disassembly of 3D tissues for applications in stem-cell differ-
entiation and tissue engineering. We show that this strategy is
redox-responsive and allows for multiple rounds of the
controlled conjugation and release of molecules to and from
cell surfaces in situ. This chemical methodology can be used
simultaneously as an analytical probe for monitoring cellular
[**] This research was supported by the Carolina Center for Cancer
Nanotechnology Excellence (NCI), the Burroughs Wellcome Foun-
dation (Interface Career Award), the National Science Foundation
(Career Award), the National Science and Engineering Research
Council of Canada (NSERC), and the Canadian Foundation for
Innovation (CFI).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201404099.
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Figure 1. Scheme and transmission electron micrographs showing dynamic liposome–liposome fusion and liposome–cell fusion for tailoring cell
surfaces. a) Hydroquinone (HQ)-containing liposomes 1 are in the “off state”. They are activated to the “on-state” quinone (Q) 2 by mild
chemical oxidation and mixed with aminooxy (AO)-containing liposomes 3. The mixing of these two populations results in the formation (driven
by oxime-bond formation) of b) multiadherent (left), partially fused (middle), and completely fused liposomal structures (right). c) General
scheme for the dynamic control of cell surfaces on the basis of liposome–cell fusion for the delivery and tailoring of bioorthogonal groups. POPC,
DOTAP, and a HQ-functionalized alkane are mixed to form liposomes. The addition of the resulting HQ-tethered liposomes 4 to cells in culture
leads to fusion and the subsequent presentation of HQ groups on the cell surface (in 5). HQ is then activated to Q (in 6), which reacts
chemoselectively with a range of AO-tethered (or RONH2-tethered) ligands or cells to form a covalent and stable oxime linkage (in 7). Upon
a mild change in the redox environment, the oxime bond is cleaved to release the ligand or cell, and the HQ-presenting cell 5 is regenerated for
subsequent rounds of dynamic and controlled conjugation and release.
interactions, as well as a trigger to alter cell-surface ligands
and cell–cell contacts.
(Figure 1b). We found that over time, an oxime-driven
process directed liposomes to first aggregate and then fuse
to form larger assemblies (Figure 1b). We used fluorescence
resonance energy transfer (FRET) to characterize liposome
adhesion and observed a clear FRET signal only when the
complementary oxime pair was present in the liposomes (see
Figure S6). Furthermore, dynamic light scattering (DLS)
showed spontaneous liposomal growth due to this oxime-
driven liposomal fusion (see Figure S7). Aggregated or fused
liposomal structures were not observed in control experi-
ments in which HQ-containing liposomes were not activated
or when either member of the oxime pair was missing.
We aimed to employ a similar strategy to generate
electroactive, dynamic, and switchable cell surfaces. There-
fore, we applied our liposome method to deliver the
bioorthogonal HQ (in 4) and AO groups (in 12) for
subsequent fusion and presentation on cell membranes. For
these studies, an HQ alkane was mixed with 1-palmitoyl-2-
oleoylphosphatidylcholine (POPC) and DOTAP (a cationic
lipid: N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammo-
nium) in a 10:88:2 ratio, and an AO alkane was mixed with
POPC and DOTAP (5:93:2; see Figure S5). After their
addition to cells (Swiss 3T3 fibroblasts (Fbs)) in culture, the
liposomes first fuse, thus delivering the chemical groups to
cell surfaces (Figure 1c). HQ can then be activated to
generate Q, which will conjugate chemoselectively with
AO-tethered ligands or cells to form a stable, interfacial
oxime linkage. The oxime bond at the cell membrane can then
be selectively cleaved with the simultaneous release of the
ligand or cell to regenerate the HQ-presenting cell. This
To deliver and tailor cell surfaces with dynamic and
bioorthogonal chemical groups for cell-membrane manipu-
lation and the control of cell–cell interactions, we first
developed a liposome-fusion-based model system. Hydro-
quinone (HQ) and aminooxy (AO) alkanes were synthesized
and incorporated into liposomes (see Figures S1–S5 in the
Supporting Information). We have previously shown in
solution and on conducting substrates how HQ is in the “off
state” and can be activated to the “on-state” quinone (Q) by
mild chemical or electrochemical oxidation.[18] Q can then
chemoselectively react with AO-tethered ligands to form
stable oxime linkages under physiological conditions (see
Figure S1). The oxime linkage can then be selectively cleaved
under reductive conditions to regenerate HQ with simulta-
neous release of the AO-tethered groups. This redox-active,
oxime-based conjugation-and-release chemistry is bioorthog-
onal and can be carried out in complex protein mixtures, cell
lysates, and cell culture.[18] Our rationale was to combine this
dynamic, switchable, and bioorthogonal conjugation-and-
release strategy with liposome fusion to tailor cell surfaces
in vitro for studies in cell-surface manipulation and tissue
engineering.
To evaluate whether liposome-to-liposome adhesion can
occur through oxime chemistry, we activated HQ- to Q-
containing liposomes (1!2) and then mixed them with AO-
containing liposomes 3 (see Figure S5). We observed rapid
liposome aggregation and fusion (Figure 1a), as shown by
transimission electron microscopy (TEM) image analysis
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dynamic cycle is noncytotoxic (see Fig-
ure S8), redox-triggered, and switchable
(see Figure S1), can be performed in situ
and under physiological conditions, and
provides unprecedented control of cell–
cell and cell–membrane interactions
through the conjugation and release of
ligands and cells.
Figure 2 demonstrates the dynamic
and switchable conjugation and release
of ligands to and from the cell mem-
brane. After HQ-containing liposomes 4
were fused with Fbs to give 5 on a con-
ductive substrate, cyclic voltammetry
(CV) was performed, and distinct HQ-
to-Q redox peaks were observed (Fig-
ure 2b). Upon activation to Q (in 6;
400 mV, 10 s) and the conjugation of
AO-tethered rhodamine to cells (conju-
gates 7 and 8), a shift in the CV peaks,
characterisitic of oxime formation,[19]
was observed. The rhodamine-labeled
Fbs were also imaged, and red fluores-
cence was observed (Figure 2c). Upon
the application of a noncytotoxic, reduc-
tive potential (ꢀ100 mV, 10 s; see Fig-
ure S10), rhodamine was released from
the cells, and HQ was regenerated on the
cell surface, as indicated by CV and
fluorescence microscopy (Figure 2d).
We then reactivated the HQ-presenting
Fbs to Q-presenting 6, to which we
conjugated biotin hydrazide, followed
by fluorescein isothiocyanate (FITC)–
streptavidin. We found that the cells
could once again conjugate and release
molecules from the cell surface, as shown
by CVand fluorescence microscopy (Fig-
ure 2e). The oxime linkage is stable, and
only upon the application of a mild
reductive potential does the oxime
cleave and release ligands.[19] In general,
this dynamic strategy can be used for
controlling the chemical structure of cell
surfaces in 3D space and time with
micro- or nanoelectrode arrays, whereby
the cell-surface ligands can be replaced
Figure 2. Electrochemical characterization of cyclical cell-surface tailoring and the release of
ligands on the basis of redox-responsive chemoselective chemistry. a) Fibroblasts (Fbs) not
fused with liposomes presenting HQ groups show no redox signal or fluorescence. b) HQ-
containing liposomes 4 are added to Fbs, thus resulting in membrane fusion (to give 5) and
the presentation of HQ on the surface. Interconversion of stable HQ (in 5) into Q (in 6) can be
monitored by cyclic voltammetry (CV) owing to its diagnostic redox peaks (black trace, HQ:
130 mV, Q: 258 mV). c) Activated Q-presenting Fbs 6 undergo chemoselective reaction with
rhodamine–AO for cell-surface tailoring (7 and 8). This reaction results in stable, fluorescently
labeled cells (red) and a diagnostic shift in the redox signal (red trace, HQ: 252 mv, Q:
284 mV). d) In a reductive environment, the oxime bond is cleaved with the release of
rhodamine and the regeneration of HQ-presenting Fbs 5, as indicated by a loss of fluorescence
and the redox peaks of the HQ-to-Q cycle (black trace). e) Cell surfaces be conjugated a second
time with hydrazide-tethered biotin (9 and 10) and fluorescein-presenting streptavidin, thus
resulting in fluorescently labeled cells (green) and a shift in the redox peaks (green trace).
with any biomolecule of interest, thus creating a new tool for
the modulation of cell interactions.
surface was observed. Furthermore, when chemical or
electrochemical activation did not occur, the conjugation
and release of ligands was not observed. We determined the
amount of HQ molecules at the cell membrane upon initial
liposome fusion by fluoresence-activated cell sorting (FACS).
FACS analysis also demonstrated that HQ remains incorpo-
rated in the membrane after several rounds of cell growth and
division, and that HQ can still be actived for the conjugation
and release of ligands (see Figure S9). These results may lead
to new ways to tailor and monitor in vitro and in vivo events
that occur at cell membranes and may enable the develop-
Several assays were performed to evaluate cell viability as
a function of applied redox potential (see Figure S10). No
change in cell viability was observed after the application of
different electrochemical potentials (ꢀ100 to 650 mV, 10 s) to
cells cultured with HQ for up to 7 days. From other control
experiments, we could conclude that the removal of either
HQ or AO from the fusion liposomes resulted in no ligand
conjugation at the cell surface. When an AO alkane or HQ
alkane was added directly (not in liposome form) to cells in
culture, no incorporation of AO or HQ groups on the cell
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ment of new types of pulse-
and-chase-type experiments
for cell imaging and for
tracking cell movement.[20]
We further investigated
the incorporation and utility
of HQ on the cell surface by
attaching cells to an AO-
patterned substrate and
releasing them again (Fig-
ure 3a). HQ-presenting Fbs
5 were activated to Q (in 6)
and then seeded onto an
inert substrate presenting
AO groups.[21] The Q groups
on the cell surface (in 6)
reacted biospecifically with
the patterned AO ligands to
form an interfacial oxime
linkage. The cells attached
and then proliferated to fill
Figure 3. Fluorescence characterization of cyclical cell-surface tailoring and the release of ligands on the
basis of redox-responsive chemoselective chemistry. a) Fbs were cultured with HQ-containing liposomes 4,
thus resulting in the membrane display of HQ on cell surfaces (cells 5). Mild chemical oxidation converts
HQ into Q groups on the cell surface (in 6). Q-presenting Fbs 6 (104 cellsmLꢀ1, 2 h) were then added to
a substrate patterned with AO-terminated ligands. Cells adhered to the substrate owing to a biospecific
interfacial oxime ligation and then proliferated within the patterned region, as shown by fluorescence
microscopy at b) lower and c) higher magnification. Upon electrochemical reduction, the interfacial oxime is
cleaved, and the cells are released from the substrate (d). Cells were stained for actin (red, phalloidin), the
out the patterned regions. An nucleus (blue, DAPI), and anti-vinculin (green, Cy-2).
electrochemical trigger was
then applied to cleave the
oxime linkage, and cells were released from the substrate. A
novel method for MALDI mass spectrometry analysis of cell-
membrane incorporation showed oxime conjugation between
Q-presenting cells and AO-terminated surfaces (see Fig-
ure S11).[22] This strategy enables spatial and temporal control
of cell interactions in 2D and may be extended to other
materials and nanoparticles for the design of new cell-based
assays and renewable microarray platforms.[23]
autocrine and paracrine signaling events, and can be com-
bined with microfabricated scaffolds as a tissue-engineering
platform.
In addition to the formation of spheroid assemblies, we
demonstrated that 3D multilayered coculture tissue struc-
tures can be generated on a solid support. We cultured
activated, Q-presenting hMSCs 11 on a substrate to form a 2D
monolayer (Figure 4e,f) and then added AO-displaying Fbs
15. Chemoselective ligation occurred between the two cell
populations, followed by 3D multilayer tissue growth after
3 days (Figure 4h). When the proper oxime pair was not
present, only a single monolayer of hMSCs was observed,
with no Fbs adhering. We found that cells were viable for
many days (> 7 days; see Figure S12) and that the HQ and
AO groups could be carried forward on the cell surface
(FACS analysis over time; see Figure S8), even through cell
growth and proliferation. The multilayers could be disassem-
bled by applying a mild reductive potential to the substrate
(ꢀ100 mV, 10 s, pH 7.4) to cleave the oxime linkage and
release the interactions between cells. Overall, this dynamic
method to generate 3D multilayer tissue structures may be
used to control cell–cell interactions for many coculture-
based cell-behavioral and cell-tissue applications.[24]
We further employed this dynamic strategy to study stem-
cell differentiation by applying our liposome-fusion-based
delivery of activatable bioorthogonal groups to cell surfaces
to generate 3D multilayered cell tissues and induce adipocyte
differentiation (Figure 5). HQ-presenting hMSCs were acti-
vated and cocultured with Fbs as described previously. This
tissue was grown in media that induced adipocyte differ-
entiation after 10 days, thus resulting in 3D multilayered
coculture tissues of adipocytes and Fbs (Figure 5d). The
application of a mild reductive potential disassembled the 3D
tissue after mild agitation to leave a relatively pure, 2D
adipocyte monolayer (Figure 5e,f). The dynamic and con-
We extended this methodology to demonstrate dynamic
control over cell–cell interactions by coculturing HQ-pre-
senting human mesenchymal stem cells (hMSCs) 13 with AO-
displaying Fbs 15 to form 3D tissue structures. Upon chemical
activation (see the Supporting Information) and mixing in
solution, 3D spheroid assemblies could be rapidly generated
(Figure 4a). By increasing the mixing duration, control of the
spheroid size was possible (see Figure S12). The intercon-
nected cells that make up the spheroids could then be
disassembled into individual cells by mild electrochemical
reduction (ꢀ100 mV, 10 s; Figure 4d). Additionally, spheroids
were formed when Swiss 3T3 albino mouse Fbs presenting
HQ groups were activated to 6 and cocultured with nuclear-
mCherry-labeled Rat2 Fbs 14 displaying AO groups (Fig-
ure 4c). Figure 4e exhibits a cryo scanning electron micro-
graph (SEM) of an oxime-ligated, spheroid assembly of
hMSCs (11) and Fbs (14) attached to a substrate. The viability
of the cells in the spheroids was analyzed over time (1–5 h,
trypan blue assay, blue false-colored) and found to be above
99% (see Figure S13). Control experiments without activa-
tion or when one of the oxime complements is not present in
a cell type showed no spheroid formation. These results
indicate that 3D coculture assemblies in solution can be
generated in a straightforward manner with the ability to
control both the size and composition of the assembly, as well
as the duration of cell–cell interactions. This strategy is
general and may be used for numerous studies, including
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trolled 3D multilayer cell disassembly
indicates that cell–cell interactions,
even for complex stem-cell-differen-
tiation processes over long time peri-
ods, can be precisely manipulated. By
the assembly and disassembly of the
cocultures on demand, a time course
of cell behavior, owing to the length of
cell–cell interactions, can be deter-
mined for a range of cell lines and
coculture-based applications.[25]
In summary, we have developed
a new general and straightforward
liposome-based methodology for the
delivery of dynamic and switchable
bioorthogonal chemical functionali-
ties to tailor cell membranes and
direct the formation of 3D coculture
tissue structures. We demonstrated
and extensively characterized the con-
jugation and release of molecules to
and from cell surfaces in situ, as well
as the triggered assembly and disas-
sembly of 3D spheroid and multilay-
ered tissues. Additionally, dynamic
cocultures of hMSCs and Fbs were
able to be generated and differenti-
ated by this redox oxime strategy.
The dynamic and bioorthogonal
oxime chemistry reported has several
key advantages as a cell-surface-engi-
neering and cell-tissue-generating
system. First, the oxime complemen-
tary pair is synthetically straightfor-
ward, and ketone-, hydrazide-, and
AO-tethered ligands are commeri-
cially available. Second, oxime reac-
tivity can be switched “on” and “off”
through a change in the redox envi-
ronment and can therefore be used to
monitor the cell-surface incorporation
of molecules and cell-surface interac-
tions. Third, the oxime bond forms
rapidly and is stable under physiolog-
ical conditions until subjected to
a chemical or electrochemical reduc-
ing potential. Fourth, the redox
manipulation is noncytotoxic. Fifth,
this liposome-fusion-based method is
general and can be used to deliver the
oxime pair to a range of cell lines for
a variety of applications. This method-
ology can be used to deliver a variety
of other bioorthogonal “click” func-
tionalities to cell surfaces. Finally, by
tailoring and controlling cell surfaces
and cell–cell interactions, new types of
autocrine and paracrine signaling
studies for fundamental cell-behavio-
Figure 4. Scheme and corresponding images showing 3D dynamic spheroid and multilayered-
tissue assembly and disassembly through liposome fusion and chemoselective cell-surface
tailoring. a) Human mesenchymal stem cells (hMSCs) are functionalized with HQ groups to give
13 upon liposome fusion and are then activated to Q-functionalized hMSCs 11. Fbs 14 presenting
AO groups are then cocultured with Q-displaying hMSCs 11 to produce 3D spheroid assemblies
(b, c, and e) through chemoselective oxime formation. Mild electrochemical reduction causes
oxime cleavage and the dynamic disassembly of cells (d). f) Activated, Q-tethered hMSCs 11 are
cultured on a substrate (105 cellsmLꢀ1) to give a 2D cell monolayer (g). When AO-presenting Fbs
15 are added (105 cellsmLꢀ1) to hMSCs 11, a 3D interconnected multilayered structure forms (h).
A reductive potential applied to the substrate cleaves the oxime bond and induces the dynamic
release of Fbs from the multilayer, thus regenerating the 2D monolayer of hMSCs (i). The nuclei of
AO-tethered Fbs 14 shown in (c) were stained with mCherry for enhanced visualization. The
HMSCs 11 and Fbs 15 displayed in (g–i) were stained for actin (red, phalloidin) and the nucleus
(blue, DAPI).
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Figure 5. Scheme and corresponding phase-contrast images showing the formation, differentiation, and release of 3D dynamic tissues by the use
of a Fb/hMSC coculture. a) Activated, Q-tethered hMSCs 11 are cultured on a substrate (105 cells mLꢀ1) and form a 2D monolayer, as shown by
the image in (b). AO-presenting Fbs 15 are then added (105 cellsmLꢀ1) to produce a 3D multilayered, interconnected coculture (c). When the
appropriate induction medium is delivered to the coculture, hMSCs differentiate into adipocytes, thus resulting in a 3D multilayered coculture of
Fbs and adipocytes (d). The dynamic release of Fbs leaves only the adhered adipocytes on the surface as a 2D monolayer, as shown by images at
lower (e) and higher magnification (f). Adipocytes were stained for lipid vacuoles (red, Oil Red O) and the nucleus (purple, Harris hemotoxylin).
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Keywords: cell-surface engineering · electrochemical oxidation ·
electrochemical reduction · liposomes · smart surfaces
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