Multivalent nanofibers of a controlled length: Regulation of bacterial cell agglutination

Article abstract of DOI:10.1021/ja306802m

Control of the size and shape of molecular assemblies on the nanometer scale in aqueous solutions is very important for the regulation of biological functions. Among the well-defined supramolecular structures of organic amphiphiles, one-dimensional nanofibers have attracted much attention because of their potential applications in biocompatible materials. Although much progress has been made in the field of self-assembled nanofibers, the ability to control the fiber length remains limited. The approach for control of the fiber length presented herein overcomes this limitation through the coassembly of amphiphilic rod-coil molecules in which the crystallinity of the aromatic segment can be regulated by π-π stacking interactions. The introduction of carbohydrate segments into the fiber exterior endows the nanofibers with the ability to adhere to bacterial cells. Notably, the fiber length systematically regulates the agglutination and proliferation of bacterial cells exposed to these fibers.

Full text of DOI:10.1021/ja306802m

Communication
pubs.acs.org/JACS
Multivalent Nanofibers of a Controlled Length: Regulation of
Bacterial Cell Agglutination
Dong-Woo Lee,† Taehoon Kim,† Il-Soo Park, Zhegang Huang, and Myongsoo Lee*
Center for Bio-Responsive Assembly and Department of Chemistry, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul
151-747, Korea
S
* Supporting Information
flexible combination in an amphiphilic system leads to the
ABSTRACT: Control of the size and shape of molecular
assemblies on the nanometer scale in aqueous solutions is
very important for the regulation of biological functions.
Among the well-defined supramolecular structures of
organic amphiphiles, one-dimensional nanofibers have
attracted much attention because of their potential
applications in biocompatible materials. Although much
progress has been made in the field of self-assembled
nanofibers, the ability to control the fiber length remains
limited. The approach for control of the fiber length
presented herein overcomes this limitation through the
coassembly of amphiphilic rod−coil molecules in which
the crystallinity of the aromatic segment can be regulated
by π−π stacking interactions. The introduction of
carbohydrate segments into the fiber exterior endows the
nanofibers with the ability to adhere to bacterial cells.
Notably, the fiber length systematically regulates the
agglutination and proliferation of bacterial cells exposed
to these fibers.
formation of nanofibers with a well-defined width in aqueous
solutions. These nanofibers are based on an amorphous
aromatic core that undergoes a sol−gel interconversion in
response to external stimuli.16−18 We hypothesized that varying
the crystallinity of the aromatic cores in nanofibers would
regulate the fiber length by controlling the π−π stacking
interactions. With this idea in mind, we performed coassembly
experiments to regulate the crystallinity of the aromatic cores of
carbohydrate nanofibers for the purpose of controlling the fiber
length, which might in turn control the biological functions of
bacterial cells by altering the multivalent interactions.
In this communication, we report the ability to control the
length of carbohydrate-coated nanofibers through the coas-
sembly of carbohydrate rod amphiphile 1, which has a highly
crystalline aromatic core, with 2, which has a less crystalline
aromatic core than 1. With increasing content of 2, the fiber
length decreases from a few micrometers to a few tens of
nanometers as the result of the decreasing crystallinity of the
aromatic cores. These nanofibers bind bacterial cells through
multivalent carbohydrate−receptor interactions and systemati-
cally regulate the proliferation of the bacterial cells, with the
effect depending on the fiber length. The amphiphiles that form
these nanofibers consist of aromatic segments that are laterally
grafted by hydrophilic mannose moieties (Figure 1). These
amphiphilic molecules were synthesized in a stepwise fashion
according to the procedure described in the Supporting
Information (SI). The resulting aromatic amphiphiles were
characterized by NMR spectroscopy and MALDI−TOF mass
ne of the fascinating features of carbohydrate-coated
Onanofibers is their multivalent interactions with specific
biomolecules. A recent series of studies revealed that the
carbohydrate-coated one-dimensional nanostructures formed
through the self-assembly of small carbohydrate-containing
molecules, such as discotic compounds1 and peptide
amphiphiles,2−6 specifically bind to multiple mannose-binding
proteins. These fibrillar structures coated by carbohydrates are
excellent multivalent ligands for specific receptors on cell
surfaces7,8 and can modulate carbohydrate−receptor binding
events associated with biological processes.9,10 Although much
progress has been made in the field of carbohydrate nanofibers,
the ability to control the length of carbohydrate-coated
nanofibers remains limited. To date, only a few methods have
been reported to control fiber length in self-assembled systems,
irrespective of the biological functions of these systems. The
research group of Manners and Winnik reported that the length
of monodisperse cylindrical micelles could be well-controlled in
organic solvent by crystallization-driven living self-assembly of
block copolymers.11−13 Besenius et al. reported another
approach, charge repulsion, to control the length of nanofibers
in water.14 Another example is the template approach, in which
dumbbell-shaped rod amphiphiles are used as templates for
peptide assembly on their surfaces to form nanofibers with a
controlled length.15 We previously reported that a rigid−
Figure 1. Chemical structures of amphiphiles 1 and 2.
Received: July 12, 2012
Published: August 24, 2012
© 2012 American Chemical Society
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Communication
spectrometry, and the results were in full agreement with the
structures presented herein.
The formation of self-assembled nanostructures was initially
investigated by transmission electron microscopy (TEM). In
addition, cryogenic TEM (cryo-TEM) was also undertaken to
investigate the self-assembled structures in bulk solutions
(Figure S2 in the SI). Figure 2 shows micrographs obtained for
on a pentaphenylene rod, self-assembles into short fibers with
an average length of 70 nm. The combination of these two
nanofibers through coassembly might result in the formation of
nanofibers with different lengths. To test this hypothesis, we
performed coassembly experiments with 1 and 2. Interestingly,
the length of the nanofibers systematically decreased from a few
micrometers to less than 70 nm as the amount of 2 increased.
When the length distribution of the nanofibers based on
measurements of >150 fibers in the images was evaluated, the
average lengths of the nanofibers were 1 μm, 300 nm, and 70
nm for 1:2 mole ratios of 85:15, 50:50, and 0:100, respectively.
To gain insight into the packing arrangements of the rod
segments in nanofibers of different lengths, we used UV−vis
(Figure S3) and fluorescence spectroscopy (Figure 3a). When
Figure 3. (a) Fluorescence spectra of amphiphiles (60 μM) upon
excitation at 316 nm. (b) DSC traces for 1, 2, and coassembled
amphiphiles upon heating.
the coassembled samples were excited at the absorption
maximum of 2, the emission peak of 2 was gradually red-
shifted and quenched with increasing 1 content, which
suggested that the energy transfer process between two
amphiphiles occurred more efficiently, indicating that there
are increasing π−π stacking interactions. To assess further the
packing arrangements of the aromatic cores of 1, 2, and the
coassembled amphiphiles, the thermal behavior of the freeze-
dried samples was evaluated by differential scanning calorimetry
(DSC). The melting transition temperatures (Tm) and the
corresponding enthalpy changes of all of the samples obtained
from the DSC heating scans (Figure 3b) are summarized in
Table S1 in the SI. As shown in Figure 3b and Table S1, an
increase in the relative amount of 2 led to a shift in the melting
peak to lower temperatures along with a significant decrease in
the heat of fusion, demonstrating that the crystallinity of the
aromatic core of 1 is disrupted by the addition of 2. These
results suggest that the packing arrangements within the
aromatic core play a critical role in controlling the length of the
self-assembled nanofibers. Indeed, the high crystallinity of the
aromatic core of 1, as reflected by the large heat of fusion, leads
to the formation of long nanofibers with a micrometer-scale
length. In contrast, amorphous aromatic cores with very low
heats of fusion, as in the case of 2, give rise to short fibers with
lengths of only a few tens of nanometers. The systematic
replacement of pyrene units with phenyl units through
coassembly would result in the disruption of the packing of
the aromatic segments, thereby reducing the assembly forces of
the molecules. Consequently, the fiber length of 1 systemati-
cally decreased with the successive addition of 2.
Figure 2. (a−d) TEM images of (a) 1 in water (60 μM) and (b−d)
coassembled samples with decreasing nanofiber length and increasing
proportion of 2: (b) 1:2 = 85:15; (c) 1:2 = 50:50; (d) 2. (e) Cryo-
TEM image of 1. (f−h) Contour length distributions of samples (b−
d), respectively.
60 μM aqueous solutions of 1, 2, and coassembled amphiphiles
cast onto TEM grids. The TEM image of 1, which is based on
pyrene units and was negatively stained with uranyl acetate,
clearly shows long, rigid nanofibers with a uniform width of ∼6
nm and lengths of >2 μm. We could not accurately measure the
lengths of the nanofibers because the fibers extended beyond
the boundary of the TEM image. In contrast, 2, which is based
To gain further insight into the variation in the length of the
fibers, we measured the solution viscosity and performed static
light scattering (SLS) experiments. The solution viscosities of 1,
2, and the coassembled amphiphiles were measured at 27 °C
using a capillary viscometer. The results showed that the
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viscosity dramatically increases with an increase in the relative
amount of 1 (Figure 4a). This result could be attributed to an
Figure 4. (a) Reduced viscosity and (b) absolute molecular weight
(from SLS) as functions of the content of 1 (in mol %).
increase in the fiber length. This hypothesis was confirmed by
molecular weight measurements with SLS. Analysis of the
molecular weight using a Zimm plot showed a dramatic
decrease from 1.2 × 108 g/mol for 1 to 9.6 × 106 g/mol for 2,
and this trend is consistent with the viscosity results (Figure
4b). These results, together with the TEM data, demonstrate
that the nanofiber length can be controlled by varying the 1:2
mole ratio.
The simultaneous presentation of mannose epitopes on a
nanofiber scaffold creates a multivalent ligand that has a high
affinity for carbohydrate receptors.19 For this reason, multi-
valent carbohydrate-coated molecules and nanofibers have been
utilized as competitive inhibitors targeting several biological
interactions.20 Recent reports have shown that multivalent
carbohydrate-coated nanofibers can induce agglutination and
inhibit the motility of pathogenic cells.21−23 To investigate the
multivalent interactions between nanofibers and Escherichia coli
cells, we choose an E. coli strain expressing the mannose-
binding adhesion protein FimH in its type-1 pili (ORN 178-
GFP). As shown in Figure 5, when the E. coli were incubated
with pure 1 and with coassembled samples, clusters of
fluorescent bacteria of different sizes were observed. The
cluster size decreased with decreasing fiber length, indicating
that the length of the mannose nanofibers plays a critical role in
the agglutination of bacterial cells. In contrast, we did not
observe bacterial agglutination with 2, most likely because of
the low aggregation stability of the fibers. To examine the
ability of the nanofibers to agglutinate bacterial cells,
agglutination index (AI) assays were performed (Figure 6a).
Significant differences among the samples were observed in
terms of the ability to agglutinate E. coli. The evaluated AI
systematically increased with increasing 1 content. These
results demonstrate that the length of the nanofibers has a
significant influence on the formation of bacterial clusters and is
a critical factor controlling agglutination. This result suggests
that long nanofibers are able to aggregate E. coli cells dispersed
in media, forming large bacterial clusters. However, the ability
to aggregate E. coli decreases with decreasing fiber length
(Scheme 1).
Figure 5. Microscopy images from fluorescence colocalization studies
of E. coli (yellow) with nanofibers (blue): (a) bright-field, (b)
fluorescence (excitation filter at λex = 450−490 nm), and (c)
fluorescence (excitation filter at λex = 340−380 nm) images for
incubation with (1) 1, (2) 1:2 = 85:15, (3) 1:2 = 50:50, and (4) 2.
Figure 6. (a) Effect of the length of mannose-coated nanofibers on
bacterial agglutination and proliferation. The degree of agglutination is
represented by the agglutination index (AI). Each value represents the
mean standard deviation (SD) of two independent experiments. (b)
Growth curves based on the optical density (OD) at 600 nm for E. coli
grown in the presence of 1, 2, and coassembled amphiphiles for 12 h.
Each value represents the mean
SD of three independent
experiments. (c) E. coli growth curves after addition of α-methyl-D-
mannopyranoside.
Scheme 1. Schematic Representation of the Regulation of
Agglutination and Proliferation of Bacterial Cells by
Variation of the Nanofiber Length
Given adequate nutrition conditions and a pleasant environ-
ment, single-cell organisms such as E. coli can survive by
proliferating, but uncontrolled bacterial proliferation is closely
related to abnormal responses such as inflammation and
autoimmune diseases. Therefore, the controlled proliferation of
bacterial cells has emerged as an important issue. As previously
reported,2,3,7,8 there is a close relationship between the form of
bacterial clusters and the repression of the proliferation activity
of bacteria cells. On the basis of these results, we anticipated
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that the length of the nanofibers would affect the size of the
bacterial clusters and, subsequently, control bacteria prolifer-
ation. To confirm this hypothesis, we examined E. coli
proliferation in the presence of mannose-coated nanofibers of
different lengths. Spectrophotometric analysis based on
turbidity or optical density (OD) is widely used to estimate
the number of bacteria in liquid cultures.24,25 As the population
of bacterial cells grows, the intensity of transmitted light
decreases. As the first step in proliferation experiments, an
overnight culture of E. coli strain ORN 178 in Luria−Bertani
(LB) medium was diluted in phosphate-buffered saline (PBS)
until the OD at 600 nm (OD600) was 1.1−1.2. The E. coli
suspension was mixed with aliquots of 1, 2, and coassembled
samples in PBS. We measured the variation in the size of the E.
coli population by measuring OD600 every 30 min. As shown in
Figure 6b, a normal bacterial growth curve was observed only in
the presence of 2. In contrast, we did not observe an increase in
the cell population for 1 during our experimental time range.
For the coassembled amphiphiles, the slope of the cell growth
curve decreased with increasing 1 content. This result indicates
that the proliferation of bacterial cells is regulated by the length
of the carbohydrate-coated nanofibers, and this effect is
attributed to the different agglutination forces for 1, 2, and
the coassembled samples. This result indicates that the length
of the nanofibers plays a critical role in regulating the
proliferation of bacterial cells.
To determine whether this nanofiber is bacteriocidal or
bacteriostatic, we added α-methyl-D-mannopyranoside (Man)
as a specific competitor in high excess (1000-fold) to an
agglutinated solution containing amphiphile 1 that had been
incubated for 4 h. After the addition of excess Man, the
bacterial cells started to proliferate, demonstrating that the
agglutination was reversible (Figure 6c).
In summary, we were able to control the fiber length of
carbohydrate-coated nanofibers by the coassembly of carbohy-
drate-conjugated rod amphiphiles with different aromatic
segments. The major driving force controlling the length of
the fibers is the level of crystallinity of the fiber cores. The
resulting carbohydrate-coated nanofibers with controlled
lengths can systematically regulate biological functions, such
as the agglutination and proliferation of specific bacterial cells.
(MEST) (2012-0001240). We acknowledge a fellow of the
BK21 Program of the Ministry of Education and Human
Resource Development.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Materials, methods, detailed experimental procedures (syn-
thesis and cellular assays), compound characterization, and
supporting figures. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
myongslee@snu.ac.kr
■
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2010, 16, 12168.
Author Contributions
†D.-W.L. and T.K. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Y.-b. Lim (Yonsei Univ.) for supplying E. coli strains
ORN 178-GFP and ORN 208-RFP and for advice. We
gratefully acknowledge the National Research Foundation of
Korea (NRF) grant funded by the Korean Government
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