Programmable Assembly of Peptide Amphiphile via Noncovalent-to-Covalent Bond Conversion

Article abstract of DOI:10.1021/jacs.7b03878

Controlling the number of monomers in a supramolecular polymer has been a great challenge in programmable self-assembly of organic molecules. One approach has been to make use of frustrated growth of the supramolecular assembly by tuning the balance of attractive and repulsive intermolecular forces. We report here on the use of covalent bond formation among monomers, compensating for intermolecular electrostatic repulsion, as a mechanism to control the length of a supramolecular nanofiber formed by self-assembly of peptide amphiphiles. Circular dichroism spectroscopy in combination with dynamic light scattering, size-exclusion chromatography, and transmittance electron microscope analyses revealed that hydrogen bonds between peptides were reinforced by covalent bond formation, enabling the fiber elongation. To examine these materials for their potential biomedical applications, cytotoxicity of nanofibers against C2C12 premyoblast cells was tested. We demonstrated that cell viability increased with an increase in fiber length, presumably because of the suppressed disruption of cell membranes by the fiber end-caps.

Full text of DOI:10.1021/jacs.7b03878

Article
pubs.acs.org/JACS
Programmable Assembly of Peptide Amphiphile via Noncovalent-to-
Covalent Bond Conversion
Kohei Sato,†,‡ Wei Ji,‡,§,∥ Liam C. Palmer,†,‡ Benjamin Weber,⊥ Matthias Barz,⊥
and Samuel I. Stupp*,†,‡,#,⊗,∇
†Department of Chemistry, ‡Simpson Querrey Institute for BioNanotechnology, #Department of Materials Science and Engineering,
⊗Department of Medicine, and ∇Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208, United
States
∥
§Prometheus, Division of Skeletal Tissue Engineering, and Skeletal Biology and Engineering Research Center, Department of
Development and Regeneration, KU Leuven, Leuven 3000, Belgium
⊥Institut fur Organische Chemie, Johannes Gutenberg-Universtitat Mainz, Mainz 55099, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Controlling the number of monomers in a
supramolecular polymer has been a great challenge in
programmable self-assembly of organic molecules. One
approach has been to make use of frustrated growth of the
supramolecular assembly by tuning the balance of attractive
and repulsive intermolecular forces. We report here on the use
of covalent bond formation among monomers, compensating
for intermolecular electrostatic repulsion, as a mechanism to
control the length of a supramolecular nanofiber formed by
self-assembly of peptide amphiphiles. Circular dichroism
spectroscopy in combination with dynamic light scattering,
size-exclusion chromatography, and transmittance electron
microscope analyses revealed that hydrogen bonds between peptides were reinforced by covalent bond formation, enabling the
fiber elongation. To examine these materials for their potential biomedical applications, cytotoxicity of nanofibers against C2C12
premyoblast cells was tested. We demonstrated that cell viability increased with an increase in fiber length, presumably because of
the suppressed disruption of cell membranes by the fiber end-caps.
of noncovalent attraction versus repulsive units within their
chemical structures.19,21 However, it is quite challenging to
modulate the size of a supramolecular polymer formed by a
single type of molecule, since size is thermodynamically
determined by the chemical structure of each monomer.
Therefore, it is of great interest to develop molecules that
enable tunable size supramolecular assemblies based on
frustrated growth. Functionally such molecules would be of
interest in nanomedicine,3−6 since their size and shape can
influence bioactivity such as cellular uptake and targeted drug
delivery.9,23−27 Our group reported recently on one example of
frustrated growth in a positively charged peptide amphiphile
(PA) with an amino acid sequence of V3A3K3 conjugated to
palmitic acid at its N-terminus (Figure 1a) (K3).22 When the
ionic strength of the aqueous PA solution I is above a critical
value (I > Ic) (Ic: critical ionic strength), charge repulsion
among the three lysine residues is suppressed, resulting in the
formation of extremely long cylindrical nanofibers containing β-
sheet secondary structure in the V3A3 peptide domain. In clear
INTRODUCTION
■
Self-assembly of organic molecules into supramolecular
polymers is a powerful method to create easily processable
soft materials with potential for self-healing properties,
recyclability, and bioactivity, among other functionalities.1−6
The preparation of supramolecular polymers with monodis-
perse length, which could be a critical parameter for their
properties, is extremely challenging due to their intrinsically
dynamic nature.7−9 Kinetically controlled seeded-growth and
supramolecular living polymerization have been successfully
used to control growth, but their implementation requires
careful sample preparation and highly sophisticated molecular
designs.10−15 Templated growth achieved by the coassembly of
supramolecular monomers with a rigid template was shown to
form supramolecular nanostructures with lengths determined
by the length of the template.16,17 Another strategy to form
supramolecular nanostructures of finite size is to balance the
interplay between attractive and repulsive intermolecular forces,
the so-called frustrated growth, where repulsive forces are of
steric or electrostatic origin.18−22 It has been relatively
straightforward to molecularly design monomers for frustrated
growth by modulating the fraction of functional groups capable
Received: April 24, 2017
© XXXX American Chemical Society
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the same Ic value reported previously for K3 (Figure S7).22,36
To turn “on” the electrostatic repulsion among lysine residues,
CLK3 was dissolved in deionized H2O below the Ic value
([CLK3] = 440 μM), the same concentration used for our
previous study on K3.22 In order for CLK3 to form nanofibers
with the thermodynamically favored length, the solution was
then annealed at 80 °C for 30 min and subsequently cooled to
room temperature.22 In fact, cryogenic transmission electron
microscopy (cryo-TEM) under these conditions revealed
formation of short fibers with low polydispersity in length
(Figure S12a).36 In contrast, when NaCl ([NaCl] = 10 mM)
was added to the PA solution (([CLK3] = 440 μM but I > Ic) to
suppress charge repulsion among the lysine residues and
annealed through the same procedure, infinitely long fibers
were observed (Figure S12b).36 Similarly, infinitely long fibers
were observed when [CLK3] was above the Ic value ([CLK3] =
4.4 mM) (Figure S12c).36 These results are consistent with
those previously reported for K3, and therefore, we concluded
that the incorporation of double bonds in the hydrophobic
segment of CLK3 does not modify the response of assemblies to
electrostatic repulsion.22
To create covalent bonds within the nanofiber formed by
CLK3, the system was irradiated with ultraviolet (UV) light (λ =
365 nm) in the presence of 2,2-dimethoxy-2-phenylacetophe-
none (DMPA) as a radical initiator.36 Unfortunately, the
reaction could not be monitored by FT-IR due to the overlap
between the characteristic diene stretching band at 1650 cm−1
with the CO vibrational band (Figure S8).33,36 We therefore
turned to electronic absorption spectroscopy, which showed
that the absorption band at 225 nm associated with the 1,3-
diene moiety (Figure S6)36 diminished after photoirradiation
(Figure 2a,b). Likewise, 1H NMR in DMSO-d6 showed that the
1,3-diene peaks disappeared after the irradiation (Figure S9).36
Size-exclusion chromatography (SEC) equipped with a
refractive index (RI) detector was performed to estimate the
molecular weight of PAs following the reaction. Hexafluor-
oisopropanol (HFIP) was chosen as the eluent for its strong
ability to dissociate PA aggregates and dissolve them
individually.38 As shown in Figure 2c, the observed peak
shifted to smaller elution volumes corresponding to higher
molecular weight with increasing irradiation time. These results
indicate that 1,3-diene units successfully formed covalent bonds
with neighboring reactive sites. Considering the monomer to
initiator ratio ([CLK3]/[DMPA] = 8/1) as well as the molecular
weight derived from SEC, the PAs after irradiation contain
monomers and a distribution of oligomers with a conversion
rate of up to 32% (Figure S10).36 We also analyzed the reaction
product of the photoirradiated sample by means of MALDI-
TOF mass spectrometry. As expected, we observed multiple
peaks for CLK3 oligomers (Figure S11).36 In addition, we found
some peaks with m/z values close to those of the CLK3
monomer, which we assume to be the mass peaks of
byproducts generated by termination of radicals. This is
presumably the reason why electronic absorption and NMR
spectroscopy indicated the consumption of 1,3-dienes, while
the SEC trace showed a large content of remaining monomer.
Next, the photoirradiated samples were diluted to the
concentration below the Ic value ([CLK3] = 440 μM), then
annealed at 80 °C for 30 min and subsequently cooled to room
temperature to turn “on” electrostatic repulsion.22 Without
photoirradiation, the hydrophobic effect and intermolecular
hydrogen bonding are not strong enough to compensate for
electrostatic repulsion so that the observed CD spectrum
Figure 1. (a) Schematic molecular structures of covalently linkable
peptide amphiphile CLK3 and its nonlinkable analogue K3. (b)
Schematic illustration of covalent bond formation via ultraviolet
(UV) light irradiation. The covalent bond formation compensates for
electrostatic repulsion, thus resulting in longer fiber length.
contrast, when the ionic strength is below the critical ionic
strength (I < Ic), charge repulsion among lysine residues
dominates, leading to the formation of monodisperse short
nanofibers. The peptide segments in these nanofibers have
random coil conformations and are therefore weakly hydrogen
bonded.22 In order to develop the potential for nanostructures
with variable lengths, we investigate here a new molecule that
could undergo frustrated growth. The strategy involves the use
of covalent bond formation among the amphiphilic molecules
as a mechanism to compensate electrostatic repulsion (Figure
1b).
RESULTS AND DISCUSSION
■
In order to maintain the characteristic assembly behavior of K3
where electrostatic repulsion controls nanofiber length,22 we
synthesized PA CLK3 which is able to form covalent bonds by
radical cross-linking with neighboring 1,3-dienes in the
assembly (Figure 1a). Among varieties of molecular mo-
tifs,28−32 1,3-diene was selected as a covalent bond forming unit
for minimizing the structural difference from saturated palmitic
acid.33−35 The peptide backbone of CLK3 was synthesized using
solid-phase peptide synthesis (SPPS), and 1,3-diene palmitic
acid was conjugated to the peptide sequence by amide bond
condensation (synthetic details for CLK3 are described in the
Supporting Information).36 The resulting CLK3 molecule was
1
unambiguously characterized by H NMR spectroscopy, ESI-
mass spectrometry, and analytical HPLC (Figures S3−S5).36
To understand how the dienes affected the assembly, we first
investigated how the electrostatic repulsion controls the
assembly behavior of CLK3 prior to covalent bond formation
among PA molecules. As we reported in our previous work on
K3, electrostatic repulsion among lysine residues of CLK3 is
expected to be highly responsive to I.22 Therefore, we used a
fluorescence assay with the Nile Red dye to determine the Ic
value of CLK3 in H2O.37 As expected, CLK3 showed effectively
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Figure 2. (a) Electronic absorption spectra of CLK3 ([CLK3] = 440
μM) in H2O at 25 °C and (b) plots of ΔAbs at 225 nm as a function
of UV light irradiation time (red: 0 h irradiation, blue: 144 h
irradiation). (c) SEC-RI traces of CLK3 with HFIP as an eluent (red: 0
h irradiation, blue: 144 h irradiation). (d) Circular dichroism (CD)
spectra of CLK3 ([CLK3] = 440 μM) in H2O at 25 °C and (e) plots of
Δε at 203 nm as a function of irradiation time (red: 0 h irradiation,
blue: 144 h irradiation). (f) Molar scattering intensity of CLK3 ([CLK3]
= 440 μM) in H2O at 25 °C as a function of irradiation time (red: 0 h
irradiation, blue: 144 h irradiation).
reveals peptide segments largely in random coil conformations
(Figure 2d,e, red). Dynamic light scattering (DLS) was used to
evaluate the assembly size,22,39 and a relatively low light
scattering intensity was observed (Figure 2f, red). As expected,
short fibers with a low polydispersity (Figure 3a and S14, Ln =
94 nm, Lw = 114 nm, Lw/Ln = 1.22 and σ/Ln = 0.47 where Ln is
the number-average and Lw is the weight-average contour
length, and σ is the standard deviation) were observed by cryo-
TEM (incorporation of photoinitiator did not significantly
affect the self-assembly behavior of CLK3).36 Nonirradiated
CLK3 showed effectively the same length dispersity observed for
K3 under the same conditions (Lw/Ln = 1.19) (Figure S18).36
We point out that in supramolecular polymerization a
polydispersity below 1.4 has been regarded as a well-controlled
polymerization.11 Interestingly, procedures in photoirradiated
samples, the CD signal eventually shifted from random coil to a
β-sheet rich signature with increasing irradiation time (Figure
2d). Concomitant with the CD intensity change at 203 nm as a
marker for β-sheet content (Figure 2e), a gradual increase in
light scattering intensity was observed (Figure 2f). These
results indicate that covalent linking between neighboring CLK3
molecules induces stronger hydrogen bonding and at the same
time leads to the formation of longer nanofibers. In fact, cryo-
TEM enabled visualization of longer nanofiber formation with
Figure 3. Cryogenic TEM micrographs of CLK3 ([CLK3] = 440 μM) in
H2O at different cross-linking time and their corresponding histograms
of the contour length of randomly selected 100 fibers for (a) 0 h, (b) 4
h, (c) 8 h, and (d) 24 h of UV light irradiation, respectively.
longer irradiation time (Figure 3 and Figures S14−S17).36
Remarkably, the polydispersity of fiber length was maintained
around Lw/Ln = 1.22 upon elongation, regardless of the
photoirradiation time as well as peptide secondary structure.
We assume that electrostatic repulsion among lysine residues,
which frustrates assembly, is likely to be responsible for the low
polydispersity. Covalent-bond formation and peptide secondary
structural changes seem to be responsible for fiber elongation.
As demonstrated by the SEC measurements (Figure 2c and
Figure S10),36 molecules within the photoirradiated PA fibers
are not completely cross-linked and the fibers should contain
instead a mixture of monomers and oligomers. The observed
low polydispersity in fiber length may suggest that monomers
and oligomers are coassembled, since self-sorting of these
components is likely to result in bimodal distributions. When
monomers and oligomers coassemble, we hypothesize that
oligomers with the least mobility and highest density of
hydrogen bonding may work as templates for monomers to
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Figure 4. (a−h) Calcein AM (green, live cells) and ethidium homodimer (red, dead cells) staining of C2C12 premyoblasts 24 h after treated with
media containing CLK3 ([CLK3] = 27.5 μM) at different irradiation times: (a) 0 h, (b) 8 h, (c) 24 h, (d) 72 h, (e) 144 h. (f) Nonirradiated infinitely
long fibers (long, 0 h irradiation), (g) irradiated infinitely long fibers (long, 144 h irradiation) and (h) tissue culture plate as control experiments. (i)
Cell viability was quantified as percentage of live cells (green) in total number of cells (green + red). ****p < 0.0001, *p < 0.05 (0 h irradiation:
nonirradiated CLK3. 8 h irradiation: CLK3 with 8 h of irradiation. 24 h irradiation: CLK3 with 24 h of irradiation. 72 h irradiation: CLK3 with 72 h of
irradiation. 144 h irradiation: CLK3 with 144 h of irradiation. Infinitely long, 0 h irradiation: nonirradiated infinitely long fibers of CLK3. Infinitely long,
144 h irradiation: infinitely long fibers of CLK3 with 144 h of irradiation. Control: cells in a tissue culture plate. (j, k) Phase micrographs of C2C12
premyoblasts 24 h after treated with media containing CLK3 ([CLK3] = 27.5 μM), irradiated for (j) 0 h or (k) 144 h. Arrows indicate dead cells.
support the formation of stable β-sheets within the fibers that
can compensate for electrostatic repulsion (Figure 1b). As
photoirradiation time increases, the content of oligomers as
well as the repeating length of oligomers increases, resulting in
longer supramolecular fibers.
Given the enormous potential of PAs as bioactive
nanostructures for regenerative medicine,3−6 we used CLK3 to
examine how cells interact with fibers of different lengths.
Specifically, it is known that cationic peptides can be
cytotoxic,22,40,41 and therefore, we investigated the viability of
myoblast C2C12 cells after CLK3 solutions ([CLK3] = 27.5 μM)
exposed to different photoirradiation times were added to their
media. Note that the concentration used for the assay is above
the critical association concentration so that fibers can still be
formed (Figure S7).36 After 24 h, the solutions containing
nonirradiated samples, which possess the shortest fiber lengths,
led to the viability of the cells as low as 40 2% (Figure 4a,i),
(Figure S13).22,36 Cells cultured with such infinitely long fibers
showed comparable viability to those cultured on a tissue
culture plate (Figure 4f−i). Therefore, the observed increase in
cell viability is most likely due to the increase in fiber length
rather than the covalent bond formation itself. Our group
recently demonstrated that short fibers formed by K3 that are
hundreds of nanometers long are highly cytotoxic, while
infinitely long fibers formed by the same molecules exhibit less
cytotoxicity. In these experiments, cell death was triggered by
cell membrane disruption caused by PA fibers as opposed to
disassembled PA monomers.22 To assess the mechanism of cell
death in the current system, we monitored the phase transition
behavior of dipalmitoylphosphatidylcholine liposomes, as a
model system for the lipid bilayer, in the presence of CLK3 using
differential scanning calorimetry. As shown in Figure S19,36 the
phase-transition temperature did not show any changes in the
presence of nonirradiated or irradiated CLK3, indicating that no
significant amount of CLK3 monomers had incorporated into
the membrane. Therefore, in the current system, the difference
in cell viability is most likely caused by the different lengths of
PA fibers, although the biological mechanism is not perfectly
clear. Previously, shorter amyloid fibrils were reported to
display higher cytotoxicity compared with longer fibrils due to
their enhanced ability of fiber termini to damage lipid
bilayers.42,43 We assume that the same mechanism took place
as compared with the viability of 90
2% from the cells
cultured in a tissue culture plate as controls (Figure 4h,i). Quite
strikingly, the CLK3 solution showed significantly increased cell
viability with increasing irradiation time compared to the
nonirradiated sample. Furthermore, we observed a clear
correlation between cell viability and irradiation time (Figure
4a−e,i,k). We also tested irradiated and nonirradiated infinitely
long fibers prepared using a previously reported procedure
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when cells were exposed to short CLK3 fibers, resulting in the
low cell viability (Figure 4j). Taking advantage of the covalent
linking strategy investigated here, we could possibly control the
cytotoxicity of cationic assemblies.
assistance with cryogenic TEM. This work made use of the
IMSERC at Northwestern University, which has received
support from the SHyNE Resource; the State of Illinois; and
the IIN. Biological and light-scattering experiments were
performed in the Analytical BioNanoTechnology Core Facility
of the Simpson Querrey Institute at Northwestern University,
and peptide synthesis was performed in the Peptide Synthesis
Core Facility of the Simpson Querrey Institute at Northwestern
University. The U.S. Army Research Office, the U.S. Army
Medical Research and Materiel Command, and Northwestern
University provided funding to develop both of these facilities,
and ongoing support is received from the SHyNE Resource. Dr.
Wei Ji is a postdoctoral fellow of the Research Foundation
Flanders (12G2715N), and received a travel grant of long stay
abroad (V468915N) from the Research Foundation Flanders
(FWO-Vlaanderen), and Junior Mobility Programme (JuMo)
of KU Leuven (JUMO-15-0514). Benjamin Weber and Dr.
Matthias Barz acknowledge support by the German Research
Council (CRC 1066-1). We thank Dr. Job Boekhoven and Dr.
Faifan Tantakitti for helpful discussions.
CONCLUSIONS
■
We have developed a strategy to control the length of charged
peptide amphiphile supramolecular assemblies. In this strategy,
covalent bond formation among PA molecules in these
assemblies alter the balance between hydrogen bond formation
and compensation of repulsive electrostatic interactions,
enabling an elongation of fibers that is not otherwise
energetically possible. We were also able to learn from these
supramolecular systems that cell viability has a clear depend-
ency on fiber length. The strategy should be useful in
controlling the size of supramolecular assemblies and the
optimization of their functions.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications Web site. Details of synthesis, character-
ization, spectroscopic studies, additional cryo-TEM images, and
experimental procedures (PDF). The Supporting Information
is available free of charge on the ACS Publications website at
DOI: 10.1021/jacs.7b03878.
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AUTHOR INFORMATION
Corresponding Author
*s-stupp@northwestern.edu
■
ORCID
Kohei Sato: 0000-0002-8948-8537
Liam C. Palmer: 0000-0003-0804-1168
Samuel I. Stupp: 0000-0002-5491-7442
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This work was primarily supported by the Center for Bio-
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DOI: 10.1021/jacs.7b03878
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