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DOI: 10.1039/C6CC01693F
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host molecules that are suitable to apply under physiological fluorescence microscopy revealed green emission that was
conditions are the pumpkin-shaped macrocyclic cucurbit[n]uril localized on the cellular membrane (Fig. S11). These results
(CB[n]) host molecules.6, 30-32 For example, we previously used confirm earlier findings that metabolic labeling procedures
CB[8] (n=8) as the host to anchor guest Trp-Gly-Gly-Arg-Gly- yield azido groups at the cell surface that can react with
Asp-Ser (WGGRGDS) peptides via their N-terminal tryptophan fluorescent molecules carrying complementary reactive groups
to surface-bound guest methylviologens as ternary for the SPAAC reaction. To verify the availability of functional
complexes.12 Detachment of the cells that adhered to these groups after metabolic labeling and subsequent SPAAC, the
supramolecular RGD surfaces occurred under electrochemical change in mean fluorescent intensity in the flow cytometry
control. Clearly, this cellular self-assembly strategy relies on data was followed as function of reaction time and storage
the ability to bind RGD ligands to their natural occurring time. After incubation of the cells with azido-functionalized
integrin receptors on the cell membrane. This strategy is mannose (MAN), cells were washed, kept in culture medium
integrin-dependent and therefore lacks in flexibility when for one more day without MAN prior to SPAAC coupling with
dealing with different cell types with different adhesiveness fluoBCN. Similarly, the azido-functionalized mannose was
characteristics. Therefore strategies are required that install administered to cells and directly reacted with fluoBCN, and
non-native supramolecular motifs on cell membranes that then kept in culture medium for an additional day without any
enable programmable assembly protocols orthogonal to reactant. In both cases, most of the fluorescence intensity was
natural cell receptor-ligand interactions. Very recently we have lost, yet remained higher than the intensity of their
incorporated CB[8]-binding motifs at the bacterial surface by corresponding negative controls (see Fig. S12).
genetically modifying
a transmembrane protein to form
multiple intercellular ternary complexes leading to the
assembly of bacteria.15
Here we report the installation of supramolecular CB[8]-
binding ligands on the cellular membrane using metabolic
labeling and the subsequent CB[8]-mediated assembly on a
supported lipid bilayer (SLB). To achieve specific CB[8]-
mediated cell adhesion, this receptor-independent strategy
was combined with the use of a suspension cell line, such as
blood cells. Supramolecular naphthol (Nph) guest moieties
were introduced onto the membrane of white blood cells by
using metabolic oligosaccharide engineering.29 Administration
of peracetylated N-azidoacetyl-D-mannosamine (MAN) to living
Jurkat cells incorporates azido groups into the glycocalyx on
the cell surface (Fig. 1). These azido-functionalized cells were
then reacted with bicyclononyne-functionalized naphthols
(NphBCN, for synthetic details see ESI) in a SPAAC reaction26 to
install the naphthol groups on the cell surface. Finally we show
that specific cellular supramolecular interactions are
established by heteroternary complex formation between
CB[8] and two different guests, i.e. SLB-bound methylviologen
and cell surface-bound naphthol causing cellular assembly at
surfaces. The metabolic introduction of azido groups and the
progress of the subsequent reaction with bicyclononyne
derivatives was validated using flow cytometry. Jurkat cells
were incubated for 3 days with 50 µM MAN in cell culture
medium and subsequently, after washing, a PBS solution of 48
µM bicyclononyne functionalized fluorescein (fluoBCN) was
added for 1 h at 37ºC (see ESI for details) for a SPAAC reaction.
Subsequently, the cells were washed and analyzed by flow
cytometry (Fig. 2a-b). As controls, cells were reacted either
with only MAN (condition +MAN-fluoBCN), or with only
fluoBCN (condition -MAN+fluoBCN), or with neither of these
two (condition -MAN-fluoBCN). All controls consistently
showed low fluorescence intensities with respect to the
condition when both MAN and fluoBCN (condition
+MAN+fluoBCN) were present indicating that the reaction
occurred specifically. Moreover, inspection of the cells that
were treated with both MAN and fluoBCN using confocal
Fig. 2 Analysis of Jurkat cell surface functionalization after 3 day incubation with 50 µM
MAN and subsequent SPAAC with fluoBCN. (a) Distributions of the green fluorescence
intensity of cells labeled with 48 µM fluoBCN and controls as assessed by flow
cytometry. (b) Average fluorescence intensities over three independent experiments is
shown for all conditions: for each individual measurement the mean fluorescence
intensity was normalized against a set of calibration beads (error bars = standard
deviation) (c) FluoBCN concentration dependence of the fluorescence intensity of
labeled Jurkat cells as measured by flow cytometry.
Next, we correlated the concentration of fluoBCN to the
number of fluorophores on the cell. Flow cytometry (Fig. 2c)
shows a linear trend of increasing fluorescence intensity of
cells treated with increasing concentrations of fluoBCN in the
range of 10 to 240 µM. This result indicates that the number of
supramolecular CB[8]-binding ligands on the cell membrane
can be modulated. In fact, the number of molecules at the cell
surface can be determined quantitatively (Fig. S14-S16). When
using for example 50 µM of MAN and 48 µM of fluoBCN, the
number of fluorescein groups per cell was determined to be
1.1*106, in agreement to values found in literature.7 As a result
this approach seems promising to understand the
thermodynamic and kinetic parameters that control cell-cell
2 | J. Name., 2012, 00, 1-3
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