Stereochemical effects of chiral monolayers on enhancing the resistance to mammalian cell adhesion

Article abstract of DOI:10.1039/c1cc10855g

This work describes the different durations of surface confinement of adhered mammalian cells by monolayers comprised of enantiomers of bio-inert polyol-terminated alkanethiols. Enhanced resistance to protein adsorption and cell adhesion is obtained on monolayers formed by a racemic mixture of the enantiomeric alkanethiols.

Full text of DOI:10.1039/c1cc10855g

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Stereochemical effects of chiral monolayers on enhancing the resistance
to mammalian cell adhesionw
Debjyoti Bandyopadhyay, Deepali Prashar and Yan-Yeung Luk*
Received 14th February 2011, Accepted 4th April 2011
DOI: 10.1039/c1cc10855g
This work describes the different durations of surface confine-
ment of adhered mammalian cells by monolayers comprised of
enantiomers of bio-inert polyol-terminated alkanethiols.
Enhanced resistance to protein adsorption and cell adhesion is
obtained on monolayers formed by a racemic mixture of the
enantiomeric alkanethiols.
We created patterned monolayers by microcontact printing,
in which circular regions of pentadecanethiol (135 mm in
diameter) are surrounded by SAMs formed by 1, 10 or SAMs
formed by racemic mixture of the enantiomers (Fig. 1). The
patterned SAMs were cultured with Swiss albino 3T3
fibroblasts, and monitored using optical microscopy. Cell
adhesion confined to circular patterns was observed for SAMs
presenting the enantiomeric alkanethiols, 1 and 10, and the
racemic mixture of the enantiomers. Surprisingly, the
D-gulitol-terminated monolayers were more resistant to cell
adhesion than the L-gulitol-terminated monolayers. While
D-gulitol-terminated monolayers confined cell adhesion in
circular patterns up to 19 days, L-gulitol-terminated mono-
layers failed at 13 days. This observation demonstrated that
mammalian cells have a higher propensity to overcome the
bioinertness of a surface formed by one enantiomer over the
other. Interestingly, SAMs formed by the racemic mixture
of the enantiomers, 1 and 10, confined cell adhesion up
to 23 days, longer than SAMs formed by either of the
enantiomers (Fig. 2). This result suggests an enhanced anti-
fouling surface chemistry by using a racemic mixture of chiral
molecules that can form bioinert monolayers.
Biofoulings, due to protein adsorption,1 mammalian cell
adhesion2 and biofilm formation,3 occurs ubiquitously on a
wide variety of surfaces. Developing effective anti-fouling
surface chemistries requires the understanding of fundamental
science that governs the interaction of surfaces with proteins,
mammalian cells and microbes. Self-assembled monolayers
(SAMs) on surfaces,4–6,7 particularly on gold films, have
provided a chemically and structurally well-defined platform
for studying both protein adsorption and mammalian cell
adhesion. In this work, we show that the resistance of
mammalian cell (3T3 fibroblasts) adhesion is different on
monolayers comprised of chiral polyol-terminated alkane-
thiols on gold films, and that the anti-biofouling chemistry
can be enhanced by using monolayers formed by a racemic
mixture of the enantiomeric alkanethiols.
Mammalian cell adhesion on surface is mediated by
selective molecular binding including cell surface receptor
integrin binding to a small tripeptide Arg-Gly-Asp (RGD)
on extra cellular matrix (ECM) proteins fibronectin or
Antifouling chemistry has been long sought after beca
use of the intriguing fundamental science and applications
that impacts multiple industries. Among different bioinert
surfaces, sugar and sugar derivatized SAMs5,8–10 have been
shown to be highly effective at resisting all aspects of bio-
fouling (protein adsorption, mammalian cell adhesion and
biofilm formation). However, the rich stereochemistry of sugar
derivatives and the mechanism of their antifouling chemistry
remains unexplored. Here, we synthesized two pairs of
enantiomers11 of alkanethiols, D-gulitol (1) and L-gulitol (10),
and D-mannonamide (2) and L-mannonamide (20), and two
diastereomeric alkanethiols, D-gulonamide (5), D-gluconamide
(6) and examined their effectiveness at resisting mammalian
cell adhesion, and at confining cells adhered in micrometre-
scale patterns.
Department of Chemistry, Department of Biomedical Engineering,
Syracuse University, Syracuse, NY 13244, USA.
E-mail: yluk@syr.edu
w Electronic supplementary information (ESI) available: Experimental
details and spectral data for all new compounds. See DOI: 10.1039/
c1cc10855g
Fig. 1 Schematic representation of patterned SAMs of HS(CH2)14CH3,
surrounded by the chiral polyol- or amine- or EG3OH-terminated SAMs
on gold films.
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These results enforce the observed racemic enhancement effect
on bioinertness.
Because protein adsorption is intimately accompanied with,
and sometimes a prerequisite for,4,13 cell adhesion, we used
surface plasmon resonance (SPR)4 to examine protein
adsorption, and the resistance of which, by SAMs presenting
D-mannonamide, L-mannonamide and racemic mixture of the
enantiomers, and compared them to SAMs presenting methyl
groups. Fig. 4 shows the data for adsorption of bovine serum
albumin (BSA) on the four SAMs formed on a SPR array chip
(16 spot per chip). Buffer (1 ꢀ PBS, pH 7.42) was first
introduced to the SAMs, followed by PBS containing BSA
(0.5 mg mLꢁ1). Pure PBS was introduced again to observe the
irreversibly adsorbed protein on SAMs. The percentage of the
monolayer covered by the adsorbed proteins (%ML)
was compared to the amount of protein adsorbed on the
methyl-terminated SAMs, and was estimated by %ML =
(DPIUmannonamide/DPIUmethyl) ꢀ 100, for which DPIU is the
difference in the pixel intensity units before and after the
surfaces were exposed to the protein solution.8,15
Fig. 2 Optical micrographs for the adhesion of Swiss albino 3T3
fibroblast on circular patterns (135 mm in diameter) of HS(CH2)14CH3,
surrounded by alditol-terminated SAMs on gold films. The number
of days the substrates were in the culture is indicated to the left. Scale
bar = 152 mm.
vitronectin,12 which is the key event for initiating vital cell
activities.13 Interestingly, Addadi and co-workers have shown
that adhesion of epithelial cells (A6 Xenopus laevis) on
(S,S)-tartrate tetrahydrate crystal was slow and mediated by
RGD containing adhesive proteins, but attachment and
spreading on the (R,R) crystal was fast, RGD-independent
and led to cell death.14 Recently, macrophages were reported
to have reduced adhesion with round morphology on a
monolayer of N-isobutyryl-D-cysteine on gold surface, but
with a larger number of cells and well-spread morphology
on a monolayer of N-isobutyryl-L-cysteine.7 In contrast to
these findings, the different ability of enantiomeric monolayers
to resist cell adhesion suggests that cells can distinguish
chirality of the surface, even when the surface chemistry is
resistant to cell adhesion.
We observed that SAMs presenting D-mannonamide and
L-mannonamide were both protein-resistant, supporting about
21.18 ꢂ 7.13% and 6.95 ꢂ 0.71% of monolayer (%ML),
respectively. Interestingly, SAMs presenting the racemic mixture
of the two enantiomers exhibited little protein adsorption
(%ML = 1.11 ꢂ 0.42) that was within the noise of the SPR
signals. This result suggests that SAMs presenting racemic mixture
of enantiomeric alkanethiols is likely more resistant to protein
adsorption than SAMs presenting either of the enantiomers.
The mechanism of an antifouling chemistry is not trivial;
hydrophilic surfaces are not necessarily beneficial for
achieving bioinertness. Proteins are known to adsorb on
surfaces presenting hydrophilic amine groups.16 An interesting
theoretical study by Sharp and co-workers suggests that
similar water organization is induced by methyl and amino
moieties.17 To examine the effect of potentially protein-
denaturing, yet hydrophilic surface on mammalian cell
adhesion, we prepared patterned squares (600 mm-wide) of
HS(CH2)14CH3 surrounded by SAMs presenting amine
group,3,18 and tri(ethylene glycol) (EG3OH).4,19 At the early
stage, cell adhesion was onlycell adhesion was only seen on
We also examined diastereomeric polyol-terminated mono-
layers, for which the polyols are linked by amide bonds instead
of ether bonds. We observed that mannonamide-terminated
monolayers, 2 and 20 failed to resist cell adhesion, but
D-gulonamide, 5, and D-gluconamide, 6, confined cell adhesion
up to 2 days and 5 days, respectively (Fig. 3). Interestingly,
while monolayers presenting either D-mannonamide (2) or
L-mannonamide (20) were not adhesion-resistant, mono-
layers formed by a racemic mixture of the enantiomers
(50/50 : 2/20) were adhesion-resistant and confined adhered
cell up to 10 days (Fig. 3 and supporting informationw).
Fig. 3 Optical micrographs for the adhesion of Swiss albino 3T3
fibroblast on circular patterns (135 mm in diameter) of HS(CH2)14CH3,
surrounded by aldonamide-terminated SAMs on gold films. The
number of days the substrates were in the culture is indicated to the
left. Scale bar = 152 mm.
Fig. 4 Intensity of surface plasmon resonance (SPR) for adsorption
of bovine serum albumin (0.5 mg mLꢁ1 in 1 ꢀ PBS, pH 7.42) over time on
four SAMs presenting methyl groups, D-mannonamide; L-mannonamide;
or a racemic mixture of D & L-mannonamide alkanethiols.
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Fig. 5 Optical micrographs for the adhesion of Swiss albino 3T3
fibroblast on 600 mm-wide squares of pentadecanethiols, surrounded
by (A) amine-terminated monolayer, 3, for 1 day, (B) for 3 days, and
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methyl-terminated squares, giving
a ‘‘reversed’’ pattern
(Fig. 5A). Over a short period of 3 days, a confluent mono-
layer of cells was observed, covering the entire monolayer
(Fig. 5B). In contrast, EG3OH-terminated monolayer resisted
cell adhesion from the beginning of cell culture (Fig. 5C).
These results support the notion that amine groups are likely
chaotropic in nature, and supported rapid cell adhesion.
Several theories exist for bioinert chemistries that resist
protein adsorption.8,9,15,20,21,28 To understand the bioinert
surface chemistry, we note that polyols are similar in structure
to glycerol and sucrose that are known to stabilize protein
structure.22 Other small molecules such as urea and
guanidinium are known to denature proteins.23–25 While the
stabilizing or destabilizing effects of added ions or molecules
on protein folding may be due to direct25,26 or indirect24,25
interactions between the additives and the proteins, the
stabilizing effect by nonionic organic osmolytes on protein
structure is likely indirect.25 Together, these results are
consistent with the notion that organic molecules that stabilize
protein structure can also resist biofouling when immobilized
on a surface.9,27 One plausible mechanism for such anti-
fouling chemistry is an extensively hydrogen bonded water
network on the monolayer, which prevents biological entities
from contacting or attaching to the surface.9,27,28
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In summary, SAMs presenting chiral polyols that have the
same chemical composition have different abilities to resist
mammalian cell adhesion. While cells can distinguish chiral
surfaces that support adhesion,7,14 our results show that cells
can also distinguish chirality of the surface when the surface
chemistry is to resist cell adhesion. We observed enhanced
bioinertness on monolayer formed by racemic mixtures of both
gulitol- and mannonamide-terminated alkanethiols, suggesting
that this racemic effect on bioinertness is likely general for
other bioinert chiral monolayers. These results suggest an
approach for potentially enhancing antifouling chemistry on
materials beyond gold films. We believe that this bioinert
chemistry is influenced strongly by the water solvation and
organization at the interface.20,29
Notes and references
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215101/1–215101/7; D. J. Vanderah, R. J. Vierling and
M. L. Walker, Langmuir, 2009, 25, 5026–5030.
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Chem. Commun., 2011, 47, 6165–6167 6167