D-amino acids modulate the cellular response of enzymatic-instructed supramolecular nanofibers of small peptides

Article abstract of DOI:10.1021/bm5010355

Peptides made of d-amino acids, as the enantiomer of corresponding l-peptides, are able to resist proteolysis. It is, however, unclear or much less explored whether or how d-amino acids affect the cellular response of supramolecular nanofibers formed by e

Full text of DOI:10.1021/bm5010355

Article
pubs.acs.org/Biomac
D‑Amino Acids Modulate the Cellular Response of Enzymatic-
Instructed Supramolecular Nanofibers of Small Peptides
Junfeng Shi, Xuewen Du, Dan Yuan, Jie Zhou, Ning Zhou, Yibing Huang,† and Bing Xu*
Department of Chemistry, Brandeis University, 415 South Street, MS 015, Waltham, Massachusetts 02453, United States
S
* Supporting Information
ABSTRACT: Peptides made of D-amino acids, as the
enantiomer of corresponding L-peptides, are able to resist
proteolysis. It is, however, unclear or much less explored
whether or how D-amino acids affect the cellular response of
supramolecular nanofibers formed by enzyme-triggered self-
assembly of D-peptides. In this work, we choose a cell
compatible molecule, Nap-L-Phe-L-Phe-L-pTyr (LLL-1P), and
systematically replace the L-amino acids in this tripeptidic
precursor or its hydrogelator by the corresponding D-amino
acid(s). The replacement of even one D-amino acid in this
tripeptidic precursor increases its proteolytic resistance. The
results of static light scattering and TEM images show the
formation of nanostructures upon the addition of alkaline
phosphatase, even at concentrations below the minimum gelation concentration (mgc). All these isomers are able to form
ordered nanostructures and exhibit different morphologies. According to the cell viability assay on these stereochemical isomers,
cells exhibit drastically different responses to the enantiomeric precursors, but almost same responses to the enantiomeric
hydrogelators. Furthermore, the different cellular responses of LLL-1P and DDD-1P largely originate from the ecto-phosphatases
catalyzed self-assembly of DDD-1 on the surface of cells. Therefore, this report not only illustrates a new way for tailoring the
properties of supramolecular assemblies, but also provides new insights to answering the fundamental question of how
mammalian cells respond to enzymatic formation of nanoscale supramolecular assemblies (e.g., nanofibers) of D-peptides.
conformation control of cyclic-RGD25 for binding integrin,
INTRODUCTION
■
destabilization of peptide helices,26 sustaining drug or dye
This article reports that the incorporation of D-amino acids into
small peptides not only changes the stereochemistry of the
molecules, but also modulates the biological activities (e.g.,
delivery via participation to the supramolecular structure,27,28
triggering three-stranded β-sheet to form β-sheet-rich fibrils,29
cytotoxicity) of the supramolecular assemblies1−3 (e.g., nano-
and constraining hydrogen bonding of linear peptides in
water.30 These advantages have also stimulated the recent
fibers) of the small peptides formed by enzyme-instructed
molecular self-assembly. Being the enantiomers of L-amino
acids, D-amino acids rarely serve as the building blocks of
naturally occurring proteins. This feature allows D-peptides to
successful development of in vitro translation of D-amino acids
into proteins via charging tRNA with D-peptides.31,32
Besides expanding the stereochemical space of molecules, the
use of D-amino acids has led to novel supramolecular structures.
resist proteolysis catalyzed by endogenous proteases in vivo.
For example, the elegant design of D,L-peptides allows the
Such a relatively long-term biostability has stimulated the
formation of nanotubes,33 as well as nanoribbons, nanotapes,
exploration of a variety of biological or biomedical applications
twisted fibers, and bundles,24,28,34,35 the incorporation of a D-
of D-amino acids. For example, D-amino acids have served as
building blocks of D-peptides for tracing the lineage of cells4
proline establishes β-hairpin for a novel class of peptide
hydrogels,36 and the coassembly of D-peptides with L-peptides
has generated rippled β-sheet.37 Encouraged by these results,
we have been using D-amino acids for developing supra-
molecular nanofibers/hydrogels.15,35,38−43 Our previous works
show that the integration of D-amino acid with D-glucosamine
afford a supramolecular hydrogel for wound healing,42 the
hydrogels made from D-amino acids are suitable for sustained
and the growth of axons,5 disrupting protein interactions,6−8
preventing HIV-1 entry,9−11 blocking mechanosensitive
channels,12 decreasing the freezing point,13 targeting DNA,14
reducing adverse drug reactions (ADR) of anti-inflammatory
drugs,15 and inhibiting the aggregation of β-amyloid (Aβ).16
The integration of D-amino acids with other molecular motifs
has resulted in bacterial peptidoglycan,17 antimicrobial
agents,18−20 and natural products.21 More importantly, the
combination of D-amino acids with L-amino acids to form
peptides or proteins offers new and rich structures or
functions22−24 that are otherwise difficult to access, such as
Received: June 2, 2014
Revised: September 2, 2014
Published: September 5, 2014
© 2014 American Chemical Society
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drug release in vivo,40 and the incorporation of D-amino acid
residues in small peptide hydrogelators enhances their
resistance to proteolysis.39,43 Moreover, our recent studies
show that, not only do the D-peptides boost the selectivity of a
nonsteroidal anti-inflammatory drug (NSAIDs),15 but also
certain D-peptides (e.g., peptides containing D-tyrosine
phosphate) can serve as the substrates of appropriate
endogenous enzymes (e.g., phosphatases), without affecting
the rate of dephosphorylation.38 While these advances by our
group as well as others27,44,45 highlight the promises of the use
of D-amino acid-based materials for potential biomedical
applications, they also underscore the importance of evaluating
the cellular responses to the self-assembly of small peptides
containing D-amino acids, which has just begun to be explored
systematically.
To evaluate how the D-amino acids modulate the biological
activities of supramolecular nanofibers46−49 formed by enzyme-
instructed self-assembly,50−58 we systematically replaced the L-
amino acids in a tripeptidic hydrogelator59 (LLL-1) or its
precursor60 (LLL-1P) by D-amino acids and examined the
viability of mammalian cells incubated with the stereochemical
isomers of the precursors and the hydrogelators. Our results
show that all of these isomers (Scheme 1) are able to form
upon the addition of alkaline phosphatase, even at concen-
trations (e.g., 200 μM) below the minimum gelation
concentrations (mgc; e.g., DDD-1: 1.0 mM). Unexpectedly,
while most of the enantiomer pairs of the hydrogelators lead to
almost the same cellular responses, the enantiomer pairs of the
precursors result in different cellular responses. For example,
while the precursor LLL-1P exhibits little inhibition on cell
proliferation at a concentration as high as 500 μM, the IC50 of
its enantiomer (precursor DDD-1P) is about 279 μM against
HeLa cells. In contrast, both enantiomers of the hydrogelators
(e.g., LLL-1 and DDD-1) appear to be cell compatible even at
500 μM. These results indicate that these stereochemical
isomers exhibit quite different biological properties due to the
introduction of D-amino acids and the enzyme-triggered self-
assembly. These results contribute new insights to answering
the fundamental question on how mammalian cells respond to
D-amino acids or D-peptides (e.g., containing aromatic group(s)
as the self-assembly promoter40,42,60,61) when the self-assembly
of the D-peptides integrates with endogenous enzymatic
reactions. In addition, these results indicate that judicious
incorporation of D-amino acid(s) into peptidic hydrogelators
(and their precursors) is a feasible and useful method to
modulate the morphological and biological properties of
supramolecular assemblies of small peptides.48,50,62−66 The
fundamental conceptual advance of this work is that ecto-
phosphatases (e.g., placental alkaline phosphatases) have
spatiotemporal control over the formation of the nanofibers
of the small peptides, thus inhibiting cancer cells.86
Scheme 1. Molecular Structures of the Precursors and the
Corresponding Hydrogelators that are Enantiomeric
Isomers
MATERIALS AND METHODS
■
A. Materials. Alkaline phosphatase (ALP) was purchased from
Biomatik USA, 2-naphthylacetic acid from Alfa Aesar, N,N-
diisopropylethylamine (DIPEA), and O-benzotriazole-N,N,N′,N′-
tetramethyl-uronium-hexafluoro-phosphate (HBTU) from Acros
Organics USA, all amino acid derivatives from GL Biochem
(Shanghai) Ltd.
B. Instrument. LC-MS on Waters Acquity ultra Performance LC
with Waters MICROMASS detector, rheological measurement on
ARES-G2 rheometer; electron microscopy was performed on a FEI
Morgagni 268 TEM with a 1k CCD camera (GATAN, Inc.,
Pleasanton, CA); MTT assay for cell toxicity test on DTX880
Multimode Detector.
C. Peptide Synthesis and Purification. According to the
structures shown in Scheme 1, we synthesized the precursors and
the hydrogelators using conventional SPPS.67 The procedure reported
by Alewood68 gives tyrosine phosphate in 90% yield. Following an
established procedure,69 it is easy to obtain Fmoc-protected tyrosine
phosphate for further reaction, which starts with loading Fmoc-PTyr-
OH (or Fmoc-Tyr-OH for synthesis of hydrogelators) at the C-
terminal onto 2-chlorotrityl chloride resin for SPPS.67 The removal of
the protecting group by 20% piperidine allows the coupling of Fmoc-
Phe-OH to the free amine group by using N,N-diisopropylethylamine/
O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phos-
phate (DIPEA/HBTU) as the coupling agent. At the final step, 2-
naphthelene acetic acid reacts to the N-terminal tripeptide. The resin-
bound peptide was cleaved using a cocktail of TFA/triisopropylsilane/
water (95:2.5:2.5) for 2 h under nitrogen, then collecting the filtrate,
and washing the resin twice using TFA. Crude product was obtained
after the addition of cold diethyl ether into concentrated filtrate. The
crude product was purified by reverse phase high performance liquid
chromatography (HPLC) using a semiprepare C18 column. HPLC
solvents consisted of solvent A (0.1% TFA in water) and solvent B
(0.1% TFA in acetonitrile). The precursors were purified by linear
gradient of 20−60% B in 22 min, the desired compound eluted at 17
min. The resulting peptide solution was frozen by liquid nitrogen and
lyophilized to afford purified precursors in fair yields (40−60%). A
supramolecular hydrogels when alkaline phosphatase catalyzes
the dephosphorylation of the precursors to result in the
hydrogelators. As expected, the molecules of different isomers
self-assemble in water to form nanofibers that exhibit different
morphologies. The use of proteinase K, a powerful
endopeptidase, to treat the precursors reveals that the
incorporation of even one D-amino acid in this tripeptidic
precursor decreases the proteolysis catalyzed by proteinase K
and the incorporation of D-amino acid residues in the middle
position of the tripeptidic backbone also enhances the
proteolytic resistance of the hydrogelators. Static light
scattering and TEM images reveal the formation of nanofibers
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similar SPPS procedure affords the corresponding hydrogelators in
around 80% yields after purification.
incubated the cells at 37 °C and 5% CO2 for 24 h. The culture
medium was collected and diluted with methanol for LC-MS analysis.
L. Light Scattering Measurement. The static light scattering
experiments were performed using an ALV (Langen, Germany)
goniometer and correlator system with a 22 mW HeNe (λ = 633 nm)
laser and an avalanche photodiode detector. All samples were filtered
by using 0.22 μm filters. The addition of ALP (0.5 U/mL) to the
solution of precursors for 24 h, we obtained corresponding samples of
hydrogelators. The SLS tests were carried out at room temperature,
and the angles of light scattering we chose were 30, 60, 90, and 120°,
respectively. The resulting intensity ratios are proportional to the
amount of aggregates in the samples.
D. General Procedure for Hydrogel Preparation. All
precursors (2.4 mg) were dissolved in 400 μL of PBS buffer (pH
7.4), the hydrogels formed after the addition of ALP (12.5 U/mL).
The samples were aged for 2 days before measurement;70 we choose
to age the gels to allow adequate time for completely converting the
precursors into hydrogelators.
E. Circular Dichroism Measurement (CD). CD spectra were
recorded (180−350 nm) using a JASCO 810 spectrometer under a
nitrogen atmosphere. The hydrogel (0.6%, 200 μL) was placed evenly
on the 1 mm thick quartz cuvette and scanned with 0.5 nm interval
three times. The CD spectra in Figure S9 confirm the chirality of
enantiomeric pairs of the peptides.
F. Rheological Measurement. Rheological tests were conducted
on TA ARES-G2 rheometer, parallel-plate geometry with an upper
plate diameter of 25 mm was used during the experiment, and the gap
was 0.4 mm. During the measurement, the stage temperature was
maintained at 25 °C by Peltier heating cooling system. The hydrogel
was loaded into stage by spatula, and then we performed dynamic
strain (0.1−100%) at 6.28 rad/s, the strain for maximum G′ in the
linear range of strain sweep test was picked for frequency sweep test
(0.1−200 rad/s).
G. TEM Measurement. Aliquots (3−5 μL) of sample solutions
were added into glow discharge thin carbon-coated copper grids (400
meshes, Pacific Grid-Tech) and incubated for 30 s at room
temperature. Excess sample solution was removed by blotting with
filter paper touched to the edge of the grid. After removing excess
fluid, the grid was washed with three successive drops of deionized
water and then exposed to three successive drop 2.0% (w/v) uranyl
acetate. Data were collected at high vacuum on Morgagni 268
transmission electron microscope.
RESULTS AND DISCUSSION
■
Molecular Design. In our recent work, we found that a
dipeptide derivative, Nap-L-Phe-L-Phe (NapFF),40,72 not only
exhibits remarkable ability to self-assemble in water to form
supramolecular nanofibers and a hydrogel, but also, after being
uptaken into cells, disrupts the dynamics of microtubules and
induces the apoptosis of glioblastoma cells. More importantly,
the nanofibers of NapFF are innocuous to model neuron cells
(e.g., PC-12).73,77 This result suggests that it is possible to use
the nanofibers of small molecules to target cancer cells
selectively. Since some more metastatic cancer cells overexpress
phosphatase,74 we chose to attach a phosphatase substrate,
tyrosine phosphate (L-pTyr), to a self-assembly motif (e.g.,
NapFF72) for generating nanofibers upon the action of
phosphatases from cancer cells as a way to inhibit cancer
cells. Thus, we decided to examine the precursor Nap-L-Phe-L-
Phe-L-p-Tyr (LLL-1P), which is an easily accessible tripeptide
derivative known to form nanofibers upon the treatment of
phosphatase (e.g., ALP).60 Because our previous studies prove
that peptides containing D-tyrosine phosphate (D-pTyr) are able
to act as the substrates of phosphatase without reducing the
rate of dephosphorylation,15,38,41 we also chose D-pTyr to
connect with NapFF. This choice leads to the question about
the role of D-amino acid in the nanofibers of the tripeptidic
derivatives. To investigate how D-amino acids affect the
biological activity of supramolecular nanofibers formed by
enzyme-triggered self-assembly of the tripeptides, we used D-
Phe,75,76 and D-Tyr to replace the corresponding L-amino
acid(s) in the precursor LLL-1P (or hydrogelator LLL-1). Such
a systematic design requires the synthesis of eight precursors
and eight hydrogelators (Scheme 1). Among them, there are
four enantiomer pairs in either the precursors or their
corresponding hydrogelators.
H. Dephosphorylation Assay. Typically, 4 mL of precursor
solution in PBS buffer (500 μM, pH = 7.4) was treated with ALP (0.1
U/mL) at 37 °C. A total of 100 μL of sample was taken out at the
desired time and mixed with 100 μL of methanol. The obtained
samples were analyzed by analytic HPLC to determine the amount of
precursor and hydrogelator.
I. MTT Assays. We seeded 2 × 104 (cells/well)71 of HeLa cells into
a 96-well plate (Obtained from Falcon) with 100 μL of MEM medium
supplemented with 10% fetal bovine serum (FBS), 100 U/mL
penicillin, and 100 μg/mL streptomycin. Incubation at 37 °C and 5%
CO2 for 12 h allowed HeLa cells to attach to the bottom of the 96-well
plate. Then we replaced the medium with another 100 μL of growth
medium that contained serial diluents of our compounds and then
incubated the cells at 37 °C and 5% CO2 for an additional 72 h.
During the viability measurement of HeLa cells, which were assayed
for 3 days, we added 10 μL of (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT, 0.5 mg/mL) into the assigned
wells in their corresponding day every 24 h, which was followed by
adding 100 μL of 0.1% sodium dodecyl sulfate (SDS) 4 h later. We
then collected the assay results after 24 h incubation. Since the
mitochondrial reductase in living cells reduced MTT to purple
formazan, the absorbance at 595 nm of the whole solution was finally
measured by DTX 880 Multimode Detector. With MEM medium as
blank and untreated HeLa cells as control, we measured each
concentration of these compounds in triplicate. The IC50 values of our
hydrogelators were read from their activity curves on day 2.
J. General Procedure for Digestion Experiment. A total of 3
mL of solution of different compounds in HEPES buffer (10 mM, pH
= 7.5) were treated with proteinase K (3.0 U/mL) at 37 °C. A 100 μL
aliquot of sample was taken out at the desired time and mixed with
100 μL of methanol. The obtained samples were analyzed by HPLC to
determine the amount of compound remaining in solution.
K. Identification and Quantification of Residue Compounds
in Culture Medium. A total of 4.0 × 106 of cells in exponential
growth phase were seeded into 10 mL Petri dish with 10 mL culture
medium. After 4 h attachment, we replaced the medium with another
10 mL culture medium containing precursors at 300 μM and then
Enzyme-Triggered Self-Assembly and Hydrogelation.
To evaluate the effects of D-amino acids on the enzymatic
hydrogelation process, we treated the solutions of the
precursors with alkaline phosphatase (ALP) and examined
the resulting hydrogels. As shown in the insets of Figure 1, the
addition of ALP (12.5 U/mL) to the solutions of the
stereochemical isomers of 1P (0.6 wt % in PBS buffer)
converts the precursors to the corresponding hydrogelators of
1. All the hydrogelators resulted in hydrogels, except the
hydrogel of DLD-1, which is relatively weak. For example, LLL-
1 and DDD-1 are able to form transparent hydrogels that
support their own weights and are stable for more than six
months. Like the hydrogels of LLL-1 and DDD-1, the
hydrogels of LLD-1 and DDL-1 are transparent, but they
exhibit slight syneresis and shrink a little after several weeks.
Both the solutions of DLL-1P and LDD-1P turn into stable
hydrogels after the treatment of ALP. While the hydrogels of
DLL-1 and LDD-1 recover quickly after shearing, the hydrogel
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Figure 1. TEM images of the hydrogels (inset: optical images) of (a)
LLL-1, (b) DDD-1, (c) LLD-1, (d) DDL-1, (e) DLL-1, (f) LDD-1
(g), LDL-1, and (h) DLD-1, formed by the addition of alkaline
phosphatase (12.5 U/mL) to the solution of their corresponding
precursors (LLL-1P, DDD-1P, LLD-1P, DDL-1P, DLL-1P, LDD-1P,
LDL-1P, and DLD-1P) at the concentration of 0.6 wt % in PBS buffer
(scale bar = 100 nm).
of LDL-1 fails to restore quickly to gel state after being
disrupted mechanically (by spatula). Interestingly, at the same
concentration, DLD-1 forms a slightly weaker hydrogel than
the hydrogel formed by LDL-1, which is consistent with their
small storage moduli and critical strain for LDL-1 (0.0067 Pa,
2.5%) and DLD-1 (0.022 Pa, 0.9%). These results, though
indicating the subtle effects of the position of the D-amino acids
in these tripeptidic derivatives, confirm that the peptide
containing D-tyrosine phosphate can serve as the substrate of
alkaline phosphatase (ALP).38
Rheometry. We used rheometry to compare the hydrogels
made by the stereochemical isomers shown in Scheme 1. As
shown in Table 1, the values of storage moduli are always larger
than those of loss moduli (Figure S2), indicating that all
samples (including the hydrogel of DLD-1) behave as solid-like
materials. Additionally, the modulus-strain profile provides the
maximum modulus G0 in the linear range and the value of
critical strain (Y0) at which the value G′ starts to decrease
sharply due to the loss of cross-linking within the gel network.78
The hydrogel of LLL-1 has G0 of 0.28 KPa and Y0 of 3.0%,
agreeing with our previous report that LLL-1 is able to form a
stable hydrogel.76 The hydrogel of DDD-1 has the largest value
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of critical strain (4.0%) among all the hydrogels, while its G0 is
0.097 KPa. Due to the heterogeneity of the hydrogels, the G′ of
the hydrogel of LLL-1 is larger than that of the hydrogel of
DDD-1, which agrees with observation of their TEM (Figure
1a,b). Furthermore, since the presence of the enzyme in the
hydrogels, the enzymatically formed hydrogels of DDD-1 and
LLL-1 are diastereomeric systems; thus, it is reasonable to
observe a rheological difference between them. The hydrogels
of LLD-1 and DDL-1 have close values of G0, 1.1 KPa (LLD-1)
and 1.8 KPa (DDL-1), as well as close values of Y0 (3.0% for
LLD-1 and 2.3% for DDL-1). The storage moduli of the
hydrogels of LLD-1 and DDL-1 increase around 4−5-fold
compared to that of the hydrogel of LLL-1, indicating that the
replacement of the L-amino acid (or D-amino acid) by
corresponding D-amino acid (or L-amino acid) could change
the rheological properties of the hydrogels. The values of G0 of
the hydrogels of DLL-1 and LDD-1 are 0.93 and 1.1 KPa,
respectively, and the corresponding values of critical strain (Y0)
are 2.7 and 2.1%. In contrast, after changing the position of D-
phenylalanine in the tripeptide of DLL-1P and LDD-1P, the
resulting hydrogel of LDL-1 has the lowest G0 (6.8 Pa), while
gel DLD-1 has the lowest values of critical strain (0.9%). This
implies that the position of D-amino acid also could affect the
rheological properties of the hydrogels. These results also
indicate that the hydrogels of the enantiomer pairs exhibit
similar viscoelastic properties and the incorporation of D-amino
acid likely modulates the elasticity of the hydrogels via forming
different supramolecular structures, as confirmed by TEM (vide
infra) and observed in previous self-assembled tripeptide
hydrogels.24,75,76,79
hydrogelators, likely originating from the alternation of D-amino
acid and L-amino acid residues in the tripetides because such
alternation results in the side-chain of the amino acids on the
same side of the peptides. These results suggest that
incorporation of D-amino acid is able to modulate the
morphologies of the matrices of supramolecular hydrogels.
The different morphologies are not only responsible for the
difference in the rheological properties of the supramolecular
hydrogels, but also imply that the position of D-amino acids in
the tripeptides likely affects the self-assembly of the hydro-
gelators.
Cell Viability. To investigate how D-amino acids affect the
cellular response to the tripeptidic precursors and hydro-
gelators, we used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) assay to examine cell viability
of the HeLa cells incubated with the stereochemical isomers
shown in Scheme 1. One unique feature of the results of the
cell viability test is that the cytotoxicities of the precursors or
hydrogelators usually deviate from the sigmoidal dose response
law.80 Thus, we define the calculated concentration of 50%
inhibition from these experiments as “apparent IC50”, though it
is denoted as IC50. As shown in Figure 2, the IC50 values of the
TEM of the Hydrogels. We used TEM to examine the
nanoscale morphologies of the matrices of the hydrogels. As
shown in Figure 1a, the TEM images in the hydrogel of LLL-1
shows long, flexible nanofibers with diameters of 8
2 nm.
Similarly, the hydrogel of DDD-1 (Figure 1b) also consists of
nanofibers with width around 8 nm. In both case, the
nanofibers tend to form a considerable amount of bundles,
reflecting the significant interfiber interactions. TEM image in
the hydrogel of LLD-1 (Figure 1c) reveals two kinds of
morphologies: large nanofibers and slim nanofibers. The slim
nanofibers have lengths around several micrometers and
diameter of 8
2 nm; the width of large nanofibers ranges
from 13 to 20 nm. Like LLD-1, hydrogel of DDL-1 (Figure 1d)
also contains large nanofibers and slim nanofibers. The slim
nanofibers have similar width (around 8 nm) with that in the
hydrogel of LLD-1. The width of large nanofibers, ranging from
25 to 50 nm, is almost twice of that of the hydrogel of LLD-1.
These large nanofibers, which consist of the slim nanofibers,
likely contribute to the relatively large storage moduli of the
hydrogels of LLD-1 and DDL-1. In Figure 1e, TEM image of
the hydrogel of DLL-1 shows individual nanofibers and helical
nanofibers. The individual nanofibers have width 6
2 nm.
Figure 2. IC50 values of (a) the precursors and (b) the hydrogelators
against HeLa cells at 48 h.
Apparently, two single nanofibers twist each other to form
helical nanofibers with a diameter of 12 nm and a helical pitch
of around 50 nm (Figure S3A). Similarly, the hydrogel of LDD-
1 (Figure 1f) contains twisted nanofibers with width of 10 nm
and helical pitch of 45 nm (Figure S3B). The enantiomer pair
of LDL-1 and DLD-1 self-assemble to form similar nanotubes
with widths ranging from 46 to 70 nm (Figure 1g and 1h).
These nanotubes form poorly cross-linked networks, which
explain the weak stability of gel LDL-1 and DLD-1. The
morphology of the nanofibers of LDL-1 (or DLD-1) differs
significantly from those of the other three pairs of enantiomeric
precursors LLL-1P and DDD-1P are >500 and 279 μM,
respectively. Although this result apparently suggests that the
incorporation of D-amino acids increases the cytotoxicity, the
corresponding hydrogelators of LLL-1 and DDD-1, which are
the products of the enzyme-catalyzed dephosphorylation of the
precursors LLL-1P and DDD-1P, hardly inhibit cell prolifer-
ation even at 500 μM. Similar to the case of LLL-1P and DDD-
1P, while the values of IC50 of LLD-1P and DDL-1P are
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different, 428 and 335 μM, respectively, their corresponding
hydrogelators exhibit similar values of IC50, 412 μM for LLD-1
and 400 μM for DDL-1. Like LLL-1P and DDD-1P, the
enantiomer pair, DLL-1P and LDD-1P, inhibits cell prolifer-
ation differently. That is, at 500 μM, DLL-1P is cell compatible,
but LDD-1P inhibits around 50% of cells. Their corresponding
hydrogelators DLL-1 and LDD-1, again, show similar
cytotoxicity: the values of IC50 are 401 μM (DLL-1) and 400
μM (LDD-1), respectively. While the enantiomer pair, LDL-1P
and DLD-1P, are cell compatible (IC50 values >500 μM), their
corresponding hydrogelators, LDL-1 and DLD-1, also show
close cytotoxicity, the values of IC50 (287 μM for LDL-1 and
311 μM for DLD-1). Obviously, these results indicate that
enantiomeric precursors exhibit dramatically different cellular
responses, while enantiomeric hydrogelators show similar
cytotoxicities. Particularly, the precursors with more D-amino
acid substitution are more toxic than their corresponding
enantiomers (except enantiomeric pair LDL-1P and DLD-1P).
Such differences are clearly associated with enzyme-instructed
self-assembly, which exclude that the cytotoxicity is due to the
amphiphilic precursors behaving as surfactants.
innocuous to cells even at 500 μM, the proteolysis of LLL-1P
likely contributes to its cell compatibility. In contrast, DDD-1P
hardly undergoes proteolysis in the presence of proteinase K.
As shown in Figure 3a, more than 94% of LLD-1P, DDL-1P,
DLL-1P, LDD-1P, LDL-1P, and DLD-1P remain upon
treatment with proteinase K for 24 h. These results indicate
that the incorporation of one D-amino acid to the tripeptidic
precursors, regardless of position, renders the precursors to
have proteolytic resistance.
Interestingly, unlike their corresponding precursors, the
hydrogelators exhibit quite different proteolytic stability in
the presence of proteinase K. As shown in Figure 3b, the
hydrogelators having D-amino acid residues in the middle
position of the tripeptides (e.g., DDD-1, DDL-1, LDD-1, and
LDL-1) exhibit excellent proteolytic resistance. That is, almost
100% of those tripeptide derivatives remain after incubation
with proteinase K for 24 h. However, the hydrogelators having
L-amino acid residues in the middle position of the tripeptides
(e.g., LLL-1, LLD-1, DLL-1, and DLD-1) undergo, albeit at
different rates, proteolysis in the presence of proteinase K. The
rate of proteolysis decreases in the order of LLL-1, LLD-1,
DLL-1, and DLD-1 which appears to agree with the trend of
the IC50 values of these four hydrogelators. The proteolytic
resistant hydrogelators, however, exhibit the same trend of the
decrease of IC50 values as that of proteolytic susceptible
hydrogelators. These results indicate that the IC50 values of the
tripeptidic hydrogelators unlikely correlate with their proteo-
lytic susceptibilities only. Particularly, although these results
suggest that the difference in proteolytic resistance likely
contributes to the different cellular responses to LLL-1P and
DDD-1P, it is unable to explain the same cell compatibility of
LLL-1 and DDD-1.
Biostability. To understand the results from the cell
viability assay (Figure 2) and to investigate the influence of
the D-amino acid(s) on the proteolytic stability of the
precursors, we used a powerful endopeptidase, proteinase K,
to treat these precursors at a concentration of 500 μM. As
revealed in Figure 3a, all precursors, except LLL-1P, exhibit
Due to the existence of many endopeptidases in cells,81 it is
necessary to know the stability of the precursors in a cellular
environment to establish the spatiotemporal profiles of the
tripeptidic derivatives and their assemblies. Thus, we used
HeLa cell to incubate with precursors (300 μM) at 37 °C for 24
h and then collected culture medium for LC-MS analysis. As
shown in Figure 4, except for LLL-1P (which became L-2), all
the residue compounds in the culture medium are their
corresponding hydrogelators. This result is not only consistent
with the digestion curve in Figure 3a, but also confirms that the
Figure 3. Digestion curve of precursors (a) and hydrogelators (b)
upon treatment with proteinase K (3 U/mL) for 24 h. All compounds
are at the concentration of 500 μM; L-2 and DL-2 indicates Nap-L-Phe
and Nap-D-Phe-L-Phe, respectively.
excellent resistance toward proteolytic digestion. This result
indicates that the incorporation of even one D-amino acid in
this tripeptide precursor is able to reduce proteolytic hydrolysis
of the precursors. LC-MS indicates that LLL-1P proteolytically
hydrolyzes to L-2 upon treatment with proteinase K for 12 h,
suggesting that the phosphate group on the tripeptide, alone, is
unable to prevent the digestion by proteinase K. Since L-2 is
Figure 4. Concentrations of residue compounds in the culture
medium after incubation of HeLa cells with precursors (300 μM) at 37
°C for 24 h. LC-MS was used to identify and quantify the residue
compounds in culture medium.
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endogenous phosphatases from HeLa cells,74 indeed, catalyze
the dephosphorylation of the precursors within 24 h. The
results in Figure 4 also agree with the enhanced biostability of
tripeptidic precursors after incorporation of D-amino acid(s),
even in vivo.40
Dephosphorylation. One possibility is that DDD-1P and
LLL-1P have different rates of dephosphorylation, which results
in different cytotoxicities. To evaluate the effect of D-amino
acid(s) on the rate of dephosphorylation of the precursors, we
used ALP (0.1 U/mL) to treat the precursor at 500 μM. As
shown in Figure 5, the rate of dephosphorylation of DDD-1P is
Figure 6. (a) Viability of HeLa cell incubated with L-Phe and (L-Phe +
DDD-1P) for 48 h; (b) Cytotoxicity of DDD-1P after addition of
different amounts of ALP after 48 h.
phenylalanine is cell compatible below the concentration of 1.0
mM, the reduced cytotoxicity of DDD-1P is likely due to the
inhibition of ALPP by L-phenylalanine.85 This result suggests
that ecto-phosphatases likely catalyze the formation of DDD-1
and its self-assembly on cell surface for inhibiting the growth of
HeLa cells, indicating that the spatiotemporal control of the
self-assembly of DDD-1 is a critical factor for determining the
cytotoxicity of DDD-1P and DDD-1.
Figure 5. Increase of hydrogelators with time shows the dephosphor-
ylation process of the precursors after incubation with ALP (0.1 U/
mL) at 37 °C. The precursors dissolve in pH 7.4 PBS buffer at a
concentration of 500 μM.
To further confirm the critical role of endogenous ecto-
phosphatases, we added ALP (at 10, 1.0, 0.1, 0.01, or 0.001 U/
mL), as an exogenous enzyme, to DDD-1P instantly before
treating the HeLa cells. As shown in Figure 6b, the addition of
exogenous ALP (at 0.1 U/mL or above) completely abrogates
the cytotoxicity of DDD-1P. This result therefore proves that
endogenous enzymatic dephosphorylation is critical for the
cellular response to DDD-1P. Our recent study confirms that
the dephosphorylation of DDD-1P by the endogenous
phosphatases on the cell surface, indeed, causes the self-
assembly of DDD-1 on the cell surface to form a hydrogel in
the pericellular space. Meanwhile, the incubation of HeLa cell
with DDD-1 at 560 μM fail to form pericellular hydrogel since
it distributed evenly in the cell culture medium.86 This result
also explains why DDD-1P and DDD-1 exhibit quite different
cellular responses. The concentrations of other hydrogelators in
culture medium are at least 260 μM, suggesting that the
positions and the numbers of the D-amino acids also affect
distribution of the hydrogelators in cellular environment.
Static Light Scattering and TEM below mgc. To
understand the different cell viabilities exhibited by non-
enantiomeric precursors and hydrogelators, we examined their
self-assembly below mgc. To evaluate the self-assembly of the
hydrogelators (or the precursors) below the mgc, we used static
light scattering (SLS) to investigate the extent of self-assembly
in the solution of precursors before and after the addition of
ALP. As a statistical method to characterize the aggregates, SLS
provides the qualitative comparison of aggregates or self-
assemblies of precursors in the solution before and after
dephosphorylation. All the precursors (1P) in the solution
exhibit negligible scattering signal (Figure S7), suggesting that
there are no detectable assemblies formed by the precursors,
even at 500 μM. After the addition of ALP to the solution of
1P, all of samples exhibit sharp increases of light scattering
signals starting at the concentration as low as 200 μM. This
result confirms that the dephosphorylation catalyzed by ALP
results in the formation of supramolecular assemblies of 1.
Because some hydrogelators (e.g., DDL-1) form large
assemblies that precipitate to the bottom of test tube (Figure
comparable (appearing slightly faster at the initial stage) to that
of LLL-1P under the same conditions. This result is consistent
with our previous study that shows ALP will dephosphorylate
D-tyrosine phosphate and L-tyrosine phosphate at almost the
same rate.38 Like LLL-1P and DDD-1P, enantiomer pair LDD-
1P and DLL-1P also shows similar dephosphorylation rates.
About 90% LDD-1P and DLL-1P convert to their correspond-
ing hydrogelators after being incubated with ALP for 12 h. In
the case of DDL-1P/LLD-1P and LDL-1P/DLD-1P, the rates
of the dephosphorylation of DDL-1P and LDL-1P are higher
than those of their corresponding enantiomers (i.e., LLD-1P
and DLD-1P). Except the enantiomeric pair of LDL-1P/DLD-
1P, the precursors with higher rates of dephosphorylation
exhibit higher cytotoxicity than their enantiomers. This result
suggests that the difference in enzymatic dephosphorylation
rate contributes to the difference in the rate of nanofiber
formation, thus, resulting in different cellular responses of
enantiomeric precursors.
Interestingly, the concentration of DDD-1 (208 μM) in the
culture medium is the lowest among all the hydrogelators
formed by the dephosphorylation of the precursors (Figure 4).
Despite its proteolytic susceptibility, as the enantiomer of
DDD-1, the concentration of LLL-1 in the culture medium is
287 μM. Such a discrepancy suggests that a significant amount
of DDD-1 molecules are present either inside cells or on the
cell surface (i.e., in the pericellular space of HeLa cells). Indeed,
the DDD-1P at 300 μM could form hydrogel on HeLa cells
after 24 h incubation at 37 °C (as shown in Figure S11). Since
it is known that the overexpression of placental alkaline
phosphatase (ALPP, as an ecto-phosphatase) on the surface of
HeLa cell,82−84 we used L-phenylalanine to inhibit ALPP during
cell culture.85 As shown in Figure 6a, the cytotoxicity of DDD-
1P against HeLa cell decreases after the addition of certain
amount L-phenylalanine (e.g., 0.3 mM and 1.0 mM). Since L-
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S8), it is impossible to correlate the intensity of SLS with the
amount of assemblies of the hydrogelators in a quantitative
manner.
7e,f). Differing from other isomers, LDL-1 or DLD-1 self-
assembles to form disordered and nonfibrillar hydrogelators
(formed by ALP after dephosphorylation of precursors (500
μM)), indicating that the supramolecular assemblies of the
enantiomeric pairs of the hydrogelators exhibit similar nano-
scale morphology (e.g., similar widths of nanofibers), which
agrees with the similar cytotoxicities of the enantiomeric pairs
of the hydrogelators. It is interesting to note that the
morphology at 500 μM is totally different with that at the gel
state (0.6 wt %). Since self-assembly is a result of the balance
between hydration forces and intermolecular hydrophobic
interactions, LDL-1 and DLD-1 interact with more water
molecules at low concentration than at high concentration,
which likely results in LDL-1 and DLD-1 forming distinct
nanostructures at different concentrations. Apparently, the
disordered and nonfibrillar nanoscale aggregates of hydro-
gelators (e.g., the case of LDL-1 and DLD-1) exhibit lower IC50
values than those of ordered nanofibers of hydrogelators. This
result suggests that the morphology of nanoscale assemblies,79
rather than the stereochemistry of individual molecules,
determines the cytotoxicity of the supramolecular assemblies
of these tripeptidic hydrogelators. This result also agrees with
the earlier observation that the morphology of nanoscale
assemblies dictates the interaction between proteins and
supramolecular assemblies of small molecules.87
Despite the existence of the multiscattering issue of
hydrogelator assemblies for light scattering, the result from
SLS measurements still confirms the formation of supra-
molecular assemblies of the hydrogelators upon treatment of
precursors below mgc (at 500 μM) with ALP. Thus, we used
TEM to evaluate the morphology of nanoscale supramolecular
assemblies of the hydrogelators (at 500 μM) formed by
dephosphorylation. While being consistent with SLS measure-
ment, the TEM images of the precursors at 500 μM (Figure
S4) hardly show large amounts of ordered nanostructures, the
TEM of the hydrogelators reveals a considerable amount of
nanoscale assemblies in different morphologies. For example,
upon dephosphorylation catalyzed by ALP, the resulting LLL-1
or DDD-1 self-assembles to form uniform nanofibers with
widths of 8 2 nm (Figure 7a,b). The self-assemblies of LLD-1
CONCLUSION
■
This work examines the cellular response to the enzymatic
formation of molecular nanofibers that contain D-amino acid
residues. The most noteworthy result is that even though
phosphatases (e.g., ALP) quickly convert precursors to the
hydrogelators, the precursors exhibit completely different
cytotoxicity from those of the hydrogelators due to the location
of the endogenous enzymes that convert the precursors to the
hydrogelators. Comparing to the use of ligand−receptor
interaction, the spatiotemporal control of the formation of
molecular nanofibers represents an unprecedented approach to
control the fate of cells.86 Since the nanofibers formed by the
self-assembly of DDD-1 would eventually dissociate to
monomeric DDD-1, the cytotoxicity (or other properties) of
the nanofibers likely will be transient, which should be a useful
feature for designing nanomedicines that function via molecular
self-assembly. Therefore, this work may ultimately lead to a
new paradigm of supramolecular chemistry. In addition, this
work also suggests that judicious incorporation of D-amino
acid(s) into peptides is a feasible and useful approach to
modulate the morphological and biological properties of
supramolecular assemblies of small peptides.24,35,45,79,88
Although the detailed mechanism of cytotoxicity of the
nanofibers on the cell surface remains to be elucidated, one
possible reason for the nanofibers of different conjugates to
exhibit different cytotoxicities likely would be that the abilities
of the nanofibers to block cell mass exchange with surrounding
of cells are varied. Furthermore, introducing D-amino acid(s) to
supramolecular self-assemblies of peptides45,79,89−95 may result
in other unexpected biological properties, which is less
investigated and warrants further exploration.
Figure 7. TEM images of the hydrogelators, formed by treating the
solution of the precursors (500 μM) with ALP (0.5 U/mL). Scale bar
= 100 nm.
and DDL-1 result in uniform nanofibers with widths around 8
2 nm (Figure 7c,d). Some of the nanofibers of DDL-1 exist
as bundles, suggesting strong interfiber interactions. The
formation of bundles likely causes aggregates to precipitate to
the bottom of the test tube (Figure S8). Upon dephosphor-
ylation by ALP, the resulting DLL-1 (or LDD-1) self-assembles
to form uniform nanofibers with widths of 9 2 nm (Figure
ASSOCIATED CONTENT
■
S
* Supporting Information
Details of the synthesis, NMR spectra, and LC-MS data for all
compounds, rheological data, cytotoxicity data, and optical
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at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: bxu@brandeis.edu.
(27) Marchesan, S.; Qu, Y.; Waddington, L. J.; Easton, C. D.;
Glattauer, V.; Lithgow, T. J.; McLean, K. M.; Forsythe, J. S.; Hartley, P.
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Present Address
†Key laboratory for Molecular Enzymology and Engineering of
the Ministry of Education, Jilin University, 2599 Qianjin St.,
Changchun 130012, China (Y.H.).
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Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partially supported by NIH (R01CA142746)
and Fellowship from Chinese scholar council (2008638092 for
J.F.S. and 2010638002 for D.Y.). J.F.S. thanks Mr. R. Haburcak
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