Versatile small-molecule motifs for self-assembly in water and the formation of biofunctional supramolecular hydrogels

Article abstract of DOI:10.1021/la1020324

This feature article introduces new structural motifs (referred as "samogen") that serve as the building blocks of hydrogelators for molecular self-assembly in water to result in a series of supramolecular hydrogels. Using a compound that consists of two

Full text of DOI:10.1021/la1020324

pubs.acs.org/Langmuir
© 2010 American Chemical Society
Versatile Small-Molecule Motifs for Self-Assembly in Water and the
Formation of Biofunctional Supramolecular Hydrogels
Ye Zhang, Yi Kuang, Yuan Gao, and Bing Xu*
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02453
Received May 19, 2010. Revised Manuscript Received June 21, 2010
This feature article introduces new structural motifs (referred as “samogen”) that serve as the building blocks of
hydrogelators for molecular self-assembly in water to result in a series of supramolecular hydrogels. Using a compound
that consists of two phenylalanine residues and a naphthyl group (also abbreviated as NapFF (1) in this text) as an
example of the samogens, we demonstrated the ability of the samogens to convert bioactive molecules into molecular
hydrogelators that self-assemble in water to result in nanofibers. By briefly summarizing the properties and applications
(e.g., wound healing, drug delivery, controlling cell fate, typing bacteria, and catalysis) of these molecular hydrogelators
derived from the samogens, we intend to illustrate the basic requirements and promises of the small-molecule
hydrogelators for applications in chemistry, materials science, and biomedicine.
1. Introduction
resulting from the network of cross-linked random polymer
chains, supramolecular hydrogels have three subtle but advanta-
geous features. (i) Despite the random entanglement of the
nanofibers, the molecular arrangement displays significant order
within the nanofibers (as a form of secondary structure). (ii) The
relatively easy structural modification of the small molecules
allows the tailoring of molecular order within the nanofibers.
And more importantly, (iii) small molecules are more accessible to
enzymes and more easily converted into hydrogelators according
to biochemical cues.6 Molecular hydrogels bear similar features to
the extracellular matrix (ECM) and respond to a wide range of
stimuli, thus supramolecular hydrogels are attractive for generat-
ing new biomaterials for tissue engineering, drug delivery, and
other applications.5,7
Self-assembly is ubiquitous in nature, ranging from the gen-
eration of sand dunes to the formation of double helices of
deoxyribonucleic acid (DNA).1 The exploration of self-assembly
on the molecular level has offered scientists a powerful strategy
(i.e., a “bottom-up” method) for developing a range of materials
for many useful applications.2,3 One of these kinds of materials is
supramolecular hydrogels as a consequence of the self-assembly
of certain small molecules (referred to as supramolecular hydro-
gelators or molecular hydrogelators)4,5 in water to form matrices
or networks of nanofibers that immobilize water. Although self-
assembly and hydrogelation are two separate phenomena, the
formation of a supramolecular hydrogel results from the self-
assembly of hydrogelators in water. Unlike polymeric hydrogels6
Although molecular hydrogelators share common features, such
as amphiphilicity and supramolecular interactions (for example,
π-π interactions, hydrogen bonding, and charge interactions
among the molecules), that contribute to the formation of nano-
structures and the 3D networks as the matrices of hydrogels, it
remains a challenge to predict whether a molecule can act as a
hydrogelator. This challenge, unfortunately, limits the research
capability for exploring new molecular hydrogelators for desirable
applications. One short-term solution to this currently intractable
problem is to start the molecular design with a motif known to self-
assemble when generating ordered nanostructures in water and
promoting hydrogelation. We refer to this kind of motif as a
“samogen” in this context. A samogen represents the fundamental
unit of a molecule that promotes self-assembly. Among the samo-
gens used to make hydrogels, a few oligomeric peptides (with and
without lipidlike structures) have received the most extensive
exploration and (probably) are the most successful ones to date,
especially in the development of biomaterials.8 Despite some
success, the oligomeric peptide-based molecular hydrogelators are
expensive and still have to be prepared via multiple-step synthesis,
which limits the exploration of these molecules as samogens for
constructing a wide range of supramolecular materials.
*Corresponding author. Tel: 781-736-5201. Fax: 781-736-2516. E-mail:
bxu@brandeis.edu.
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In this feature article, we introduce a simple samogen, com-
pound 1 (abbreviated as NapFF in this text), that can be easily
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Langmuir 2011, 27(2), 529–537
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DOI: 10.1021/la1020324 529

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Zhang et al.
Scheme 1. Molecular Hydrogelators Derived from NapFF (1)
Figure 1. (A) Optical image, (B) TEM image, and (C) negatively
stained TEM image of the hydrogel of 1 (0.6 wt %).
powerful strategy for promoting the self-assembly of small
molecules in water to form hydrogels.12 To take advantage of
the aromatic-aromatic interaction in water, we chose to make a
dipeptide derivative (i.e., NapFF) that consists of a significant
number of aromatic rings. We connected 2-(naphthalen-2-yl)
acetic acid with Phe-Phe because (i) the X-ray diffraction data
of a β-amyloid analogue13 and the solid-state NMR of a seven-
residue peptide of Aβ14 indicated that the amyloid core likely
contains the hydrophobic Phe-Phe residues for aggregation and
(ii) Phe-Phe self-assembles to form nanotubes.15
As shown in Scheme S1, the synthesis of NapFF (1) is quite
simple and straightforward.
L-Phenylalanine reacts with the
N-hydroxysuccimimide (NHS)-activated ester of 2-(naphthalene-
2-yloxy) acetic acid to afford (S)-2-(2-(naphthalene-2-yl) acet-
amido)-3-phenylpropanoic acid (NapF). Then, NHS-assisted
prepared on the gram scale. In particular, we demonstrate the
ability of 1 to convert bioactive molecules into molecular hydro-
gelators (Scheme 1) and summarize the properties and applica-
tions of these NapFF-derived molecular hydrogelators. We
arrange the content of this feature article in the following way.
First, we analyze the structural uniqueness of 1 to elucidate the
molecular anatomy, properties of hydrogelation, and possible
self-assembled superstructure of this samogen. Second, we briefly
illustrate the capability of 1 to enable other biofunctional mole-
cules to self-assemble in water as molecular hydrogelators that
exhibit useful properties in applications. Third, we offer several
anecdotal examples of molecular hydrogelators (Scheme 3) de-
rived from the samogens that share the features displayed by 1. In
the last section, we summarize the versatility of the samogens
discussed and the general challenges associated with the explora-
tion of molecular hydrogelators.
coupling between NapF and L-phenylalanine gives the pure com-
pound of (S)-2-((S)-2-(2-(naphthalen-2-yl) acetamido)-3-phenyl-
propanamido)-3-phenylpropanoic acid (NapFF, 1) in 70% yield
after flash column chromatography.
Compound 1 itself also behaves as a molecular hydrogelator
and exhibits a critical gelation concentration (cgc) of as low as 0.4
wt % (0.83 mM) according to the inverting test tube method,
which agrees with the observation that the Phe-Phe motif is prone
to self-assembly in water.15 As shown in Figure 1A, 1 forms a
transparent hydrogel in water, which undergoes a reversible
gel-sol transition at about 323 K. A transmission electron micro-
graph (TEM) reveals that 1 self-assembles to generate nanofibers
with widths of 18-45 nm and lengths longer than a few micro-
meters (Figure 1B). Further negative staining TEM (Figure 1C)
suggests that the fibrils, with a width of 8.5 ( 2.5 nm, twist
together to form the nanofibers.
To understand the molecular arrangement of 1 within the
nanofibers, we grew the crystal of 1 from its ethanol solution.
Although it is unknown whether the obtained crystal structure
exactly reflects the molecular packing in the nanofibers of the
hydrogel, the intermolecular interactions revealed in the single
crystal should offer valuable insight into the possible molecular
superstructures of the nanofibers because the local environment
of the nanofibers should be quite hydrophobic, which may bear a
2. NapFF as a Building Block for Effective Molecular
Self-Assembly in Water
Like the discovery of other molecular gelators,9 the investiga-
tion of NapFF (1) also originates from a serendipitous finding
that a simple dipeptide protected by fluorenylmethyloxycarbonyl
(FMOC), FMOC-Ala-Ala, self-assembles in water to form na-
nofibers and results in a supramolecular hydrogel.10 This result
suggests that aromatic-aromatic interaction not only acts as a
stabilizing force for the structures of proteins11 but also offers a
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530 DOI: 10.1021/la1020324
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Zhang et al.
Invited Feature Article
Figure 2. Molecular packing and hydrogen bonds in the crystal of 1 (recrystallized from ethanol). Views from (A) a, (B) b, and (C) c axes and
(D) view from the c axis to show the hydrogen bonding (green dotted lines) of one molecule with four other molecules and some
aromatic-aromatic interactions (yellow lines).
resemblance to the nucleation of 1 in a nonaqueous polar solvent
(e.g., ethanol in this case). As shown in Figure 2, all of the hydrogen
bonding donors and acceptors participate in the formation of
of 1 via the aromatic-aromatic interaction. These aromatic-
aromatic interactions not only facilitate the long-range order of
the molecules but also provide the hydrophobic environment that
may cooperatively reinforce the intermolecular hydrogen bond-
ing between molecules of 1 even in water, thus greatly increasing
the self-assembly tendency of 1 to form nanofibers and hydrogels.
Overall, it is likely that the extensive multiple interactions,
including hydrogen bonds and aromatic-aromatic interactions,
among the molecules of 1 enhance their self-assembly in water,
which makes 1 an exceptionally efficient samogen.
intermolecular hydrogen bonds (H X = 1.74-2.00 A). The
3 3 3
CdO at the N terminal of 1 forms a hydrogen bond with the
C-terminal NH of another molecule of 1 (Figure 2A). Although the
molecules appear to have “antiparallel” order (being viewed from
the a axis), each molecule of 1 aligns (via crosses) with two other
molecules of 1 and forms hydrogen bonds with them. As viewed
from the baxis (Figure 2B), these crosses extend along the aaxis. As
shown in Figure 2C, the COOH of 1 forms two hydrogen bonds
with the NH and CdO in the middle of another molecule of 1. At
the same time, its own middle section NH and CdO form a pair of
hydrogen bonds with the COOH from a different molecule of 1.
Overall, each molecule of 1 can form six hydrogen bonds with four
neighboring molecules of 1 (Figure 2D). The capability to form
multiple hydrogen bonds with multiple partners possibly facilitates
the self-assembly of 1.
3. Molecular Hydrogelators Based on NapFF
The capability of 1 to form multiple noncovalent interactions
with several partners suggests that 1 should be able to promote
molecular self-assembly in water after it conjugates with bioactive
or other molecules, though such conjugation could cost a few
noncovalent interactions because of the loss of carboxylic acid at
the C terminal and/or the increase in steric congestion on the side of
1. Figure 3 shows two modes for conjugating 1 to a bioactive
molecule: end-on or side-on. In this section, we begin the discussion
with the end-on mode because it was explored prior to the side-on
mode (although the side-on samogen might be more versatile).
NapFF-aminobisphosphonate (2). To evaluate the functions
and applications of bisphosphonate-based hydrogels, we conju-
gated 1 to a bisphosphonate in end-on mode. Bisphosphonates
are small molecules with a P-C-P structure that has structural
similarities to the P-O-P structure of pyrophosphate. The P-
C-P and P-O-P groups both show a high affinity for calcium
phosphate crystal surfaces, and their binding to these surfaces
inhibits further calcium phosphate accretion or dissolution. Thus,
Besides displaying the possible modes of the hydrogen bonding
originating from the amide bonds and the carboxylic acid group
of 1, the crystal structure of 1 reveals extensive aromatic-aro-
matic interactions between the aromatic rings along both the
a and b axes (with the average ring distance of 5.31 A). As shown
in Figure 2D, the naphthyl group of 1 interacts with the middle
phenyl group of its neighboring molecules of 1 in the same plane.
The middle phenyl group simultaneously interacts with the
terminal phenyl group of the molecules of 1 below and above.
Reciprocally, the terminal phenyl group of 1 interacts with the
middle phenyl group of a pair of molecules of 1 below and above.
Thus, each molecule of 1 interacts with six neighboring molecules
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Zhang et al.
Figure 3. Illustration of the modes of linking bioactive molecules
to NapFF (1), the samogen.
Figure 4. Optical (A) and TEM (B) imagesof the hydrogel formed
by 2 (1.0 wt % and pH 6.5).
bisphosphonates are potent inhibitors of bone resorbing cells
(osteoclasts) and are of clinical benefit in a variety of metabolic
bone disorders.16 Besides the applications for bone diseases, the
phosphate group on the bisphosphonate exhibits a high affinity
for uranyl cations. Therefore, hydrogels consisting of a bisphos-
phonate group for binding to uranium and other transuranium
metal ions may provide unique advantages for the treatment of
wounds contaminated with radionuclides because (i) nanofibers
have a high surface/volume ratio with which to provide excellent
binding capacity and (ii) hydrogels should confine the radioactive
uranium to the wound, in contrast to liquid-based treatments,
because they barely flow. These potential applications of bispho-
sphonate-based hydrogels lead us to conjugate 1 to a bispho-
sphonate in end-on mode. Scheme S2 shows the synthesis
consisting of an HBTU-assisted coupling reaction linking tetra-
ethyl 3-amino-propane-1,1-bisphosphonate to 1 in DMF to
afford the ester-protected compound in 55% yield. The subse-
quent deprotection by the treatment of TMSBr produces 2 in
good yield (51%).
As expected, 2 self-assembles in water to result in a molecular
hydrogel at a concentration of 1.0 wt %. Typically, the hydrogel
(Figure 4A) forms upon carefully adjusting the pH of the solution
from 10 to 6.5. The hydrogels are stable at room temperature for
months. According to the TEM images of the hydrogel of 2, the
hydrogel contains irregular fibers with widths ranging from 30 nm
to about 250 nm, and the fibers show a strong tendency to
aggregate into bundles and leave large pores in the matrices of
the gel.
produce multifunctional hydrogels for the topical treatment of
various wounds.
NapFF-tyrosine Phosphate (3a) and NapFF-tyrosine
(3b). The connection of 1 to tyrosine not only provides an
example of the hydrogelator via end-on modification but also
greatly expands the scope of the applications of the hydrogelators
because it allows the enzymatic formation of the hydrogelators
and molecular hydrogels. As shown in Figure S1A, it is quite
simple and easy to make the precursor (3a) of the hydrogelator
(3b). After the activation of NapFF by NHS, the coupling
between NapFF-NHS and O-phosphotyrosine under a weakly
basic condition affords the precursor (3a). According to the
rheological measurement, the addition of acid phosphatase (5.88
U/mL) to the aqueous solution of 3a (0.5 wt %) triggers imme-
diate hydrogelation to afford a transparent hydrogel. Two hours
after the enzyme is added, 84.4% of 3a transform into 3b. The
TEM image (FigureS1C) shows quite uniform nanofibers (20-40
nm) in the enzymatically formed hydrogels.
Besides being converted to 3b by the acid phosphatase, 3a also
serves as the substrate of alkaline phosphatase. As shown in
Figure 5, an alkaline phosphatase converts 3a to 3b, which leads
to the formation of nanofibers and results in hydrogelation. The
addition of alkaline phosphatase to a phosphate saline buffer
(PBS) solution of 3a (0.5 wt %, 6.91 mM) affords a hydrogel.
Rheological tests reveal that the hydrogel starts to form almost
instantly after the phosphatase is added at room temperature,
as indicated by the storage modulus (G0) dominating the loss
modulus (G00) (Figure 5A). According to HPLC analysis, about
48% of 3a transformed to 3b at the gelling point. The transpar-
ency of hydrogel 3b (Figure 5B) suggests that no microcrystalline
aggregates formed in the hydrogel to scatter visible light. The
TEM image of the hydrogel (Figure 5C) shows that the diameter
of the nanofibers formed by the self-assembly of 3b is around
26 nm. The addition of phosphatase to solutions containing
different concentrations of 3a (Figure 5D) allows the estima-
tion of the minimum gelation concentration (mgc) of 3a between
0.025 and 0.05 wt % (0.35 and 0.69 mM) in the PBS buffer.
Because the molecular weight of 1 is rather low (480 g/mol), it
brings down the molecular weight of the molecular precursor (3a,
mw = 723 g/mol). The low molecular weight of3a permits passive
We administered the hydrogel of 2 to wound sites on the backs
of mice after contamination of the wounds by uranyl nitrate and
found that the survival rates of the mice improved significantly
and their body weight returned to normal after treatment with the
hydrogel.17 Although the applications of the hydrogel of 2 still
remain to befully established, the further development ofthis type
of hydrogel could provide a general strategy for removing toxic
substances (e.g., radionuclides or chemical poisons) from external
wound sites on human bodies. Moreover, the supramolecular
hydrogels should allow the incorporation of other therapeutics to
(16) Yuasa, T.; Nogawa, M.; Kimura, S.; Yokota, A.; Sato, K.; Segawa, H.;
Kuroda, J.; Maekawa, T. Clin. Cancer Res. 2005, 11, 853–859.
(17) Yang, Z. M.; Xu, K. M.; Wang, L.; Gu, H. W.; Wei, H.; Zhang, M. J.; Xu,
B. Chem. Commun. 2005, 4414–4416.
532 DOI: 10.1021/la1020324
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Invited Feature Article
Figure 5. (a) Oscillatory rheology of a PBS buffer solution contain-
ing 6.91 mM (0.5 wt %) 3a and 10 μL of enzyme solution (14U/μL)
at pH 7.4 and 25.8 °C. (b) Optical and (c) TEM images of the
hydrogel formed by 3b through enzymatic gelation in PBS buffer
solution (0.5 wt %). (d) Enzymatic conversion of 3a to 3b with the
addition of alkaline phosphatase (700 U/mL) to PBS buffer solu-
tions containing different concentrations of 3a.
diffusion and facilitates the exploration of the hydrogelation of
small molecules inside a cell (Figure 6).18 The enzyme then
converts the precursor into the hydrogelator that self-assembles
to form nanofibers, which could induce hydrogelation inside the
cells. The hydrogelation may change the viscosity of the cyto-
plasm and cause stress on the cell. Using E. coli that overexpress
soluble phosphatase as the model system, we treated the E. coli
with 3a. When the bacteria overexpress the enzyme (IPTGþ), the
formation of the nanofibers of 3b in the bacteria result in the high
intracellular concentration of 3b (Figure 6B), and the subsequent
intracellular hydrogelation inhibited the growth of E. coli.18
Besides, as the first example of the intrabacterial formation of a
molecular hydrogel, this result suggests that the up-regulated
expression or high activity of an enzyme may confer the specificity
needed to control the cell’s fate. In addition, these results clearly
indicate the compatibility of the samogen (1) to enzymatic
reaction, that is, 1 exhibits little inhibitory effect toward the
enzyme and it can enable molecular self-assembly in a compli-
cated environment, such as cytosol.
After hydrogelation, the enzyme remains in the hydrogel and
should be functional if there is no inhibition caused by the
hydrogelators derived from 1. Our recent study19 of the catalytic
activity of the hydrogel-immobilized acid phosphatase has indeed
validated this notion. As shown in Figure 7, we chose an acid
phosphatase (AP) to catalyze molecular hydrogelation and have
examined the catalytic activity of the hydrogel-immobilized AP in
both organic and aqueous media. Upon the addition of AP to the
solution of the precursor (3a) at room temperature, AP catalyzed
the hydrolysis of 3a to produce 3b and the self-assembly of 3b
afforded the hydrogel that immobilized the AP. The test of the
stability and activity of AP in different solvents reveals that the
self-immobilized AP exhibited activity in chloroform that was
about 100 times greater than the activity of the corresponding free
AP in water (Figure 7C). Moreover, the stability of the immobi-
lized AP increases significantly (Figure 7D). This study further
demonstrated the versatility of 1.
Figure 6. (A)Schematic representationofintrabacterialnanofiber
formation leading to hydrogelation and the inhibition of bacterial
growth. (B) Concentrations of3a and 3b inthe culture medium and
within E. coli (BL21, plasmidþ, IPTGþ, or IPTG-). Adapted
with permission from ref 18. Copyright Wiley-VCH 2007.
supramolecular hydrogels.20 As shown in Scheme 2, we synthe-
sized a pentapeptidic hydrogelator, NapFFGEY (4b), which
formed hydrogels at 0.6 wt % via the self-assembly of 4b
(Figure 8A). The addition of a tyrosine kinase to the hydrogel in
the presence of adenosine triphosphates (ATP) phosphorylates 4b
to give the corresponding peptide tyrosine phosphate (4a), thus
disrupting the self-assembly to induce a gel-sol phase transition
(Figure 8B); treating the resulting solution with a phosphatase
dephosphorylates 4a to form 4b, thus restoring the self-assembly
to form the hydrogel (Figure 8C). The TEM image (Figure 8D)
also confirmed the formation of the network of the nanotubes.
After using an MTT assay to verify the biocompatibility of 4a or
4b,20 we injected 4a into the mice (subcutaneously) and found the
formation of the supramolecular hydrogel in vivo (Figure 8E). As
the first demonstration of an enzyme-switch-regulated supramo-
lecular hydrogel and the first formation of supramolecular
NapFF to Conjugate with the Substrate of Kinase/Phos-
phatase. The compatibility of 1 with phosphatase allows us
to explore the use of a kinase/phosphatase switch to regulate
(18) Yang, Z.; Liang, G.; Guo, Z.; Xu, B. Angew. Chem., Int. Ed. 2007, 46,
8216–8219.
(19) Wang, Q. G.; Yang, Z. M.; Gao, Y.; Ge, W. W.; Wang, L.; Xu, B. Soft
(20) Yang, Z. M.; Liang, G. L.; Wang, L.; Xu, B. J. Am. Chem. Soc. 2006, 128,
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Matter 2008, 4, 550–553.
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DOI: 10.1021/la1020324 533

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Figure 8. Optical imagesof(A) the hydrogelof4b, (B) the solution
obtained after adding a kinase to the gel of 4b, and (C) the hydrogel
of 4b formed by using phosphatase to treat 4a. (D)TEM images
of the hydrogel of 4b formed by using phosphatase to treat 4a.
(E) Optical image of the hydrogel formed subcutaneously 1 h after
injecting 4a into the mice. Adapted with permission from ref 20.
Copyright American Chemical Society 2006.
Figure 7. (A) Illustration to show the location of the enzyme after
the formation of the nanofibers in the hydrogel. (B) Acid phospha-
tase in the hydrogel that catalyzes the conversion of the substrate
(O-phospho-nitrophenol, represented by the blue sphere plus red
dot) to the product (nitrophenol, represented by the blue sphere) in
an organic medium. (C) Hydrolysis of the substrate (10 mM)
catalyzed by AP (within the gel, 20 μg/L) in chloroform (b), toluene
(2), n-octane (1), and AP (free) in water (9). (D) Remainingactivity
of AP (within the gel, b) and AP (free) (9) in water after incubation
at 60 °C at various times. Adapted with permission from ref 19.
Copyright Royal Society of Chemistry 2008.
Figure 9. (A) Illustration of the design of a substrate of β-lacta-
mase (Bla) as the precursor of a hydrogelator and the opening of
the β-lactam ring catalyzed by Bla. (B) One possible mode of self-
assembly of the hydrogelator and the formation of the hydrogel.
Scheme 2. Structure of the Substrates of Kinase and Phosphatase
versa, which leads us to determine if the use of a relatively large
soluble group can achieve a similar phase transition. To evaluate
the possible application of the visually observable change (i.e., gel-
to-sol or sol-to-gel phase transition), we chose to conjugate 1 with a
soluble β-lactam ring because a major class of antimicrobial agents
relies on the strained β-lactam ring to react with penicillin-binding
proteins (PBPs) to inhibit the cell wall synthesis and growth of
bacteria. However, β-lactamases hydrolyze the four-membered
β-lactam ring, render the antibiotics ineffective, and cause the most
widespread antimicrobial drug resistance. To detect β-lactamases
and screen their inhibitors, we chose to use the event of hydro-
gelation to report the presence of β-lactamases.21 Figure 9 outlines
the general principle and molecular design for a β-lactamase-
catalyzed hydrogelation. Using the cephem nucleus as the linker,
a hydrophilic group (e.g., glycine) connects a hydrogelator (5b)
derived from 1 to afford the precursor (5a), which is too soluble to
form a hydrogel. Upon the action of a β-lactamase, the β-lactam
ring opens to release the hydrogelator (5b), which self-assembles in
water into nanofibers to generate a hydrogel.
As shown in Figure S2A,C, the precursor (5a) is too soluble to
form a hydrogel (i.e., the precursor supplies too few hydrophobic
interactions to self-assemble and gel water, Figure S2A,C). Upon
the action of a β-lactamase, the β-lactam ring opens to release the
hydrogelator (5b), which self-assembles in water and forms
nanofibers that result in a hydrogel (Figure S2B,D,). The key
feature of the design is to use a β-lactamase to generate a
hydrogels in vivo by an enzymatic reaction, this enzyme-catalyzed
reversible self-assembly and gelation of the hydrogelators could
lead to a new type of medium for drug delivery because it allows
the hydrogels to respond to the expressions of specific enzymes
associated with certain tissues, organs, or diseases.
NapFF-Based Hydrogelators for β-Lactamase Screen-
ing. As shown in the two previous cases, the attachment and
detachment of a relatively small soluble group (e.g., phosphate)
of 1 can convert the precursor solution to the hydrogel and vice
(21) Yang, Z. M.; Ho, P. L.; Liang, G. L.; Chow, K. H.; Wang, Q. G.; Cao, Y.;
Guo, Z. H.; Xu, B. J. Am. Chem. Soc. 2007, 129, 266–267.
534 DOI: 10.1021/la1020324
Langmuir 2011, 27(2), 529–537

Zhang et al.
Invited Feature Article
expression of the esterases in the HeLa cell promotes the quick
buildup of 6b, which self-assembles to form nanofibers. In
addition, this result also indicates that self-assembly may amplify
the relatively small difference in the expression of enzymes. In a
broad sense, the target of 6b in this case is water in the cytosol;
therefore, it is possible to design a substrate to be susceptible to
multiple enzymes to achieve more sophisticated controls (e.g.,
both spatial and temporal) for regulating cell fate.
NapFF Conjugates with Taxol for Self-Drug Delivery.
The exceptional capability of 1 to induce other molecules to self-
assemble in water leads us to conjugate it with taxol, a sophisticated
molecular anticancer agent.23 Considering the bulky size of taxol,
we decided to connect taxol to NapFF via the side-on mode. To
help create the site for the connection, a lysine residue links 1 to
afford NapFFK. A tyrosine phosphate also extends from the
C-terminal of NapFFK to afford 7, which provides the enzymatic
trigger and improves the solubility of the final conjugate (8a).
Scheme S3 shows the synthesis route and the structure of the
conjugate (8a), which consists of the samogen (blue), an enzyme-
cleavable group (red), a linker (pink), and a taxol molecule (orange).
As shown in Figure 12, upon the action of the enzyme, the precursor
(8a) transforms into a hydrogelator (8b), which self-assembles to
form nanofibers and affords a supramolecular hydrogel of taxol.
According to the TEM in Figure 12E, 5 min after the addition of the
enzyme, the mixture already contains nanofibers with a diameter of
20 nm, in addition to particle aggregates. The hydrogel of 8b exhibits
well-dispersed nanofiber networks with a uniform fiber width of
29 nm (Figure 12F).
Figure 10. Illustration of enzyme-catalyzed intracellular hydroge-
lation and chemical structures and graphic representations of the
precursor (6a) and the hydrogelator (6b).
hydrogelator. By treating the solution of 5a with different types of
bacteria cell lysates, we accurately identified the lysates containing
β-lactamase on the basis of this visual assay.21 This success not
only promises an alternative approach to assaying β-lactamase
(e.g., ESBL) in a more specific way via tailoring the structure of
the precursors but also confirms the versatility of 1 in offering a
general motif to enable the self-assembly of other biofunctional
molecules or groups.
As shown in Figure S3A, the activity of taxol after conjugating
with NapFFK is successfully conserved in the precursor and the
hydrogelator. Figure S3B shows the release profiles of 8b from its
own gel. The slow release and cytotoxicity of 8b suggest a method of
drug self-delivery. By taking advantage of the samogen (NapFFK
in this case), this result represents the first example of enzyme-
instructed self-assembly and hydrogelation of complex, bioactive
small molecules, which demonstrates a new, facile way to formulate
highly hydrophobic drugs, such as taxol, into an aqueous form (e.g.,
hydrogel) without comprising their activity and promises a general
methodology for creating therapeutic molecules that play a dual
role as the delivery vehicle and the drug itself. This result also
suggests that drug molecules are excellent candidates for conjugat-
ing with the samogens in the development of functional hydrogels
or soft materials that promise various biomedical applications,
including sustained or controlled drug release.
NapFF-NHCH2CH2OH for Selective Cell Inhibition.
Another hydrogelator (6b) derived from 1, generated by an enzyme
from its precursor (6a), can form the hydrogel inside cancer cells
and inhibit cell growth.22 After verification that 6b is an effective
hydrogelator exhibiting an mgc of as low as 0.08 wt %, we designed
and synthesized the precursor (6a) containing the hydrogelator and
a cleavable butyric diacid, which forms an ester bond with the
hydroxyl group on the aminoethanol end of 6b. The butyric acid
group acts as the enzymatic trigger for changing the overall balance
of the hydrophobic and hydrophilic interactions and prevents the
formation of the nanofibers of 6b without enzymatic hydrolysis.
The addition of the esterase to the solution of 6a (0.08 wt %) at
physiological temperature (37 °C) results in the formation of the
hydrogel. Because the resulting hydrogelator (6b) lacks the car-
boxylic acid group, the hydrogel is stable over wide range of pH
1
(from 0 to 14). According to the rheological experiments and H
NMR analysis, the hydrogelation starts 6 min after the addition of
the esterase when 68% of 6a transforms to 6b. The formed hydrogel
of 6b is transparent, which agrees with the TEM image of the
hydrogel that shows the width of the nanofibers to be about 25 nm.
Like compound 3a, the 6a molecule is small enough to enter
cells via passive diffusion (Figure 10). To evaluate the effect of 6a
on mammalian cell lines, we added 6a to the culture of fibroblast
cells (NIH3T3) or HeLa cells and measured the proliferation of
the cells. As the MTT assay shows in Figure 11A, the number of
NIH3T3 cells barely decrease from the first day to the third day.
On the contrary, the HeLa cells treated by 6a display decreased
cell proliferation with the increase in the concentration of 6a or
time (Figure 11B), which is consistent with the observation of the
change in cell morphology. A simple qualitative assay of the
esterase expression in HeLa and fibroblast cells indicates that the
level of expression of esterase in HeLa cells is higher than that in
NIH3T3 cells.22 This result suggests that the relatively high-level
4. Several Samogens Share Common Features with
NapFF
As discussed in previous sections, the key feature of 1 is the
cooperative enhancement of noncovalent interactions due to
multiple aromatic-aromatic interaction and hydrogen bonds.
Therefore, if multiple aromatic-aromatic interactions and hy-
drogen bonds originate from their molecular structure, other
small molecules should be able to self-assemble in water. In this
section, we introduce a few examples (Scheme 3) bearing struc-
tural characteristics of 1.
Enantiomer of NapFF. The structural isomer that would
have (almost) identical behavior to that of 1 is its enantiomer, Napff
(9, f=D D-phenyl-
-phenylalanine).24 Thus, we synthesized 9 using
alanine and examined its properties. Compound 9 forms a clear
(23) Gao, Y.; Kuang, Y.; Guo, Z. F.; Guo, Z. H.; Krauss, I. J.; Xu, B. J. Am.
Chem. Soc. 2009, 131, 13576-13577.
(24) Liang, G. L.; Yang, Z. M.; Zhang, R. J.; Li, L. H.; Fan, Y. J.; Kuang, Y.;
Gao, Y.; Wang, T.; Lu, W. W.; Xu, B. Langmuir 2009, 25, 8419–8422.
(22) Yang, Z. M.; Xu, K. M.; Guo, Z. F.; Guo, Z. H.; Xu, B. Adv. Mater. 2007,
19, 3152-3156.
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DOI: 10.1021/la1020324 535

Invited Feature Article
Zhang et al.
Figure 11. MTT assays of (A) NIH3T3 cells and (B) HeLa cells treated with 6a at concentrations of 0.08, 0.04, and 0.02 wt %. Adapted with
permission from ref 22. Copyright Wiley-VCH 2007.
Figure 12. Optical (A-C) and corresponding transmission elec-
tron microscopy (TEM) images (D-F) of the solution of 8a with
[8a] = 1.0 wt % (A, D); the solution of 8a 5 min after the addition
of alkaline phosphatase (ALP) (B, E); and the hydrogel of 8b
overnight after the addition of ALP (C, F). Adapted with permis-
sion from ref 23. Copyright American Chemical Society 2009.
Figure 13. TEM images of the nanofiber matrices in the hydrogel
of Napff (9). The scale bar is 500 nm.
Scheme 3. Hydrogelators Similar to NapFF
Figure 14. (A) Optical image of the hydrogel formed by 10. (B)
TEM image of the hydrogel of 10.
gel-encapsulated 131I-NaI remained. Using the clinically used
hydrogel at a concentration of 1% and exhibits similar rheological
behavior to that of 1 at the same concentration. The circular
dichroism spectra of the hydrogels of 1 and 9 display a mirror
image that agrees with their chirality.24 TEM images also reveal
that 9 form nanofibers in water with a diameter of 20-46 nm
(Figure 13), which is close to that of the nanofibers formed by 1.
However, there is a significant difference between the hydrogels
of 1 and 9. The hydrogel of 9 resists protease-based digestive
enzymes (e.g., proteinase K),24 a very useful property that allows
the hydrogel of 9 to act as a potential medium for drug delivery.
Using a mice model and radioactive tracers, we found that the
hydrogel formed by 9 exhibited good controlled-release character-
istics in vivo. For example, after subcutaneously injecting the
hydrogel of 9 into the abdomens of rats, gel-encapsulated 125I-NaI
was released and thereafter the blood concentration of 125I-NaI was
maintained within a narrow range during the first 12 h after
administration. SPECT images also showed that 4 h after sub-
cutaneous injection only about one-third of the 131I-NaI solution
remained in the injection site whereas nearly two-thirds of the
drug epidepride to replace NaI, we also found that gel-encapsulated
125I-epidepride and 131I-epidepride showed controlled release beha-
vior in vivo. These results suggest that supramolecular hydrogels
based on D-amino acids or D-peptides may lead to new types of
biomaterials for sustained drug release.
β-Peptide as a Structural Isomer of NapFF. One structural
isomer of NapFF investigated by us belongs to β-peptides. Despite
considerable progress in the design and synthesis of β-peptides, the
application of β-amino acids to control the bioavailability of
supramolecular hydrogels remains little explored because the func-
tion of the β-amino acid derivative as a hydrogelator was unknown.
We synthesized and characterized compound 10, which consists of
two β-amino acids (β3-homophenylglycine and β3-Hphg). 10 acts as
a hydrogelator under the proper conditions (e.g., concentration,
temperature, and pH). For example, 5 mg of 10 in 1.0 mL of water
forms a slightly opaque hydrogel (Figure 14A) by careful adjust-
ment of the pH or temperature. (The pH range and temperature of
the gel-sol phase transition are 6.2-6.5 and ∼48 °C, respectively.)
This hydrogel is stable at room temperature for several months. As
536 DOI: 10.1021/la1020324
Langmuir 2011, 27(2), 529–537

Zhang et al.
Invited Feature Article
shown in Figure 14B, a wide range of fibril sizes (ranging from 20 to
80 nm) constitute the matrices of the hydrogel. The small fibrils
exhibit a tendency to tangle with each other and form large bundles,
which further confirms the stronger π-π stacking ability of
β3-HPhg and agrees with its relative high storage modulus.25
On the basis ofthe knowledge that Nap-β3-HPhg-β3-HPhg(10)
is a hydrogelator, we designed and synthesized a chimera of a
tripeptide derivative that consists of two β-amino acids (i.e., β3-
homophenylglycine) and one R-amino acid residue (i.e., tyrosine
phosphate). The tripeptide derivative undergoes enzymatic hy-
drogelation in vitro and in vivo.26 Moreover, in vivo experiments
revealed that the hydrogels formed by β-amino acid derivatives
had a longer lifetime than hydrogels formed by R-amino acid
derivatives. These results suggest that supramolecular hydrogels
derived from the β-amino acid-derived samogen, Nap-β3-HPhg-
β3-HPhg, could evolve into promising candidates for biomedical
applications when long-term stability is required and the long-
term biostability of the β-peptide is established.
in wound healing and prevents the formation of scars on a mouse
model. This result also supports the notion that the self-assembly
of bioactive molecules, to form networks of nanofibers in the
hydrogel, could offer a useful and effective way to generate
biomaterials.
5. Conclusion and Perspective
We have demonstrated the versatility of several simple samo-
gens, which serve as the building blocks of hydrogelators that
promise many useful applications, including drug release, bacter-
ial typing, wound healing, cell inhibition, and catalysis. An
obvious opportunity is to use the known or potential samogens
to conjugate with the vast pool of bioactive molecules and to
enable molecular self-assembly in water for the development of
molecular hydrogels. In fact, the recent development of molecular
gelators has provided an array of possible samogens that are
based on either the aromatic-aromatic interaction3,28 or hydro-
phobic forces among alkyl chains.29 Because of their small sizes,
the samogens discussed here can be modified easily. Therefore, it
is feasible to make a large variety of molecular hydrogelators.
Because the molecular arrangement in the nanofibers remains
a challenge, the accumulation of knowledge that is necessary
for establishing, validating, and refining theoretical models of
molecular self-assembly becomes necessary. The use of samogens
for self-assembly not only provides rich opportunities to develop
supramolecular materials but also contributes to the interplay
of the modeling30 and experimental design, which ultimately
could lead to a more predictive theory or models for molecular
self-assembly. We expect that the collaboration of experimental
scientists and theoreticians on the study of samogen-based hydro-
gelators will lead to the rational design and development of
molecular hydrogels for a variety of applications.
NapF-glucosamine. One interesting case is that NapF can
also act as a samogen to induce the self-assembly of a glycoside.27
As shown in Scheme 3, either Nap-L-phenylalaninae (NapF) or
Nap- -phenylalaninae (Napf) can connect to glucosamine via the
D
formation of an amide bond to afford 11 or 12. Both 11 and 12 are
effective hydrogelators that form transparent hydrogels at 0.2 wt
% (Figure S4A,B) and a pH of around 7. These two hydrogels are
stable at room temperature for several months. Rheological
measurement (Figure S4C,D) confirms that the value of the
storage modulus (G0) of the hydrogel of 11 is larger than that of
the hydrogel of 12 and the range of the plateau of the hydrogel of
12 is wider than that of the hydrogel of 11, suggesting that the
hydrogel of 11 is slightly more viscoelastic and the hydrogel of 12
is more tolerant to external force. The TEM images of the
hydrogels, as shown in Figure S4E,F, reveal that the small,
irregular ribbons form large bundles and tangle with each other
in the hydrogel of 11, in addition to a small number of helical
fibers with diameters ranging from 27 to 55 nm. In the hydrogel of
12, small rigid ribbons with diameters of 35-50 nm form well-
distributed matrices. The different morphologies in both gels
likely result from their different structures. According to TEM
Acknowledgment. The work described was partially sup-
ported by the Research Grant Council (Hong Kong), the HFSP
(RGP0056/2008), start-up funds from Brandeis University, the
NSF (MRSEC 0820492), and the NIH (R01CA142746-01). Some
of the images were taken at the Brandeis EM facility.
images, compound 11 with an
L
-phenylalanine tends to aggregate
Supporting Information Available: CIF file of the crystal
structure of 1 and additional schemes and figures. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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the resulting supramolecular hydrogelators exhibit good biocom-
patibility. More importantly, the resulting hydrogel of 11 assists
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