Supramolecular Design of Unsymmetric Reverse Bolaamphiphiles for Cell-Sensitive Hydrogel Degradation and Drug Release

Article abstract of DOI:10.1002/anie.201913087

Self-assembly of peptide-based building units into supramolecular nanostructures creates an important class of biomaterials with robust mechanical properties and improved resistance to premature degradation. Yet, upon aggregation, substrate–enzyme interactions are often compromised because of the limited access of macromolecular proteins to the peptide substrate, leading to either a reduction or loss of responsiveness to biomolecular cues. Reported here is the supramolecular design of unsymmetric reverse bolaamphiphiles (RBA) capable of exposing a matrix metalloproteinase (MMP) substrate on the surface of their filamentous assemblies. Upon addition of MMP-2, these filaments rapidly break into fragments prior to reassembling into spherical micelles. Using 3D cell culture, it is shown that drug release is commensurate with cell density, revealing more effective cell killing when more cancer cells are present. This design platform could serve as a cell-responsive therapeutic depot for local chemotherapy.

Full text of DOI:10.1002/anie.201913087

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Supramolecular Design of Unsymmetric Reverse
Bolaamphiphiles for Cell-Sensitive Hydrogel Degradation and
Drug Release
Authors: Rami W Chakroun, Alexandra Sneider, Caleb F Anderson,
Feihu Wang, Pei-Hsun Wu, Denis Wirtz, and Honggang Cui
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201913087
Angew. Chem. 10.1002/ange.201913087
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10.1002/anie.201913087
Angewandte Chemie International Edition
RESEARCH ARTICLE
Supramolecular Design of Unsymmetric Reverse
Bolaamphiphiles for Cell-Sensitive Hydrogel Degradation and
Drug Release
Rami W Chakroun,[a] Alexandra Sneider,[a] Caleb F Anderson,[a] Feihu Wang, [a] Pei-Hsun Wu,[a] [b]
Denis Wirtz,[a] [b] and Honggang Cui*[a] [b] [c]
Abstract: Self-assembly of peptide-based building units into
supramolecular nanostructures creates an important class of
biomaterials with robust mechanical properties and improved
resistance to premature degradation. Yet, upon aggregation,
substrate-enzyme interactions are often compromised due to the
limited access of macromolecular proteins to the peptide substrate,
leading to a reduction or loss of responsiveness to biomolecular cues.
We report here the supramolecular design of unsymmetric reverse
been developed, consisting of multiple hydrophobic and
hydrophilic segments. Linear bolaamphiphiles with symmetric[7]
and asymmetric[8] designs have also been investigated, forming a
variety of morphologies. Dendritic bolaamphiphiles are another
important class of branched molecules possessing a central
hydrophobic region with hydrophilic dendrons at each terminal.[9]
These innovative amphiphile designs have expanded the
morphology library and functional space of classical surfactant
assembly, leading to the discovery of a plethora of interesting
nanostructures with unique functionalities.[9a, 9b, 10] In addition to
architecture variations, numerous functional units have also been
introduced to the amphiphile design.[11] One notable example is
the peptide amphiphiles (PAs) that exploit both the structural and
bolaamphiphiles (RBA) capable of exposing
a
matrix
metalloproteinase (MMP) substrate on the surface of their filamentous
assemblies. Upon addition of MMP-2, these filaments rapidly break
into fragments prior to re-assembling into spherical micelles. Using 3D
cell culture, we demonstrate that drug release is commensurate with
cell density, revealing more effective cell killing when more cancer
cells are present. We elucidate the important role of RBA
supramolecular assemblies in specific response to enzyme-
expressing cells, and envision this design platform as a cell-
responsive therapeutic depot for local chemotherapy.
biological roles of small molecule peptides to construct protein
11d, 12]
analogous micelles[11c,
and biologically active peptide
amphiphile nanofibers.[13] Along the lines, the incorporation of
therapeutic agents creates an emerging class of self-assembling
prodrugs, termed drug amphiphiles (DA) that can assume a
variety of supramolecular morphologies in aqueous
environment.[14] It is in this context that we report a reverse
bolaamphiphile (RBA) design with asymmetric hydrophobic units
as terminal groups to enable specific enzyme- and cell-
responsive hydrogel degradation and drug release.
Introduction
Low-molecular-weight surfactants[1] and macromolecular
surfactants[2] such as block copolymers are known to form a great
diversity of discrete supramolecular morphologies in solution and
nanostructured materials in bulk.[3] Conventionally, amphiphiles
are linear molecules formed by covalent linkage of a hydrophobic
moiety to a hydrophilic headgroup or segment. To create more
sophisticated functionalities, the molecular design of linear
amphiphile has evolved over the past three decades to those
containing various functional units and with complex chain
architectures.[4] For example, branched amphiphiles such as
mikto-arm star block copolymers[5] or dendritic amphiphiles[6] have
Abnormal enzymatic activities are implicated in many
human diseases such as cancer and represent a popular target
for disease treatment.[15] In particular, the expression and release
of matrix metalloproteinases (MMPs) is linked to tumor
aggressiveness and metastasis,[15a, 16] and has been a subject of
heavy interest in the development of enzyme-responsive
biomaterials. For instance, the Xu lab has pioneered the use of
enzymes as a trigger for self-assembly into biologically relevant
nanomaterials.[17] For MMP-responsiveness, it has been
demonstrated by a number of labs that the enzymatic cleavage at
a monomeric level transforms the molecule into an effective
hydrogelator[18] or triggers
a
change in nanostructure
morphology.[19] From the perspective of molecular design,
success of this strategy relies heavily on the accessibility of the
enzyme to the target molecular substrate, which occurs easily in
its monomeric state but is greatly hindered in the assembled state.
One way to overcome this issue is through post-assembly
crosslinking, where the cleavage sites are chemically conjugated
onto the peptide nanostructures, ensuring surface degradation.[20]
However, this method suffers from the complicated procedure of
crosslinking chemistry and subsequent removal of the potentially
toxic initiators. Another method is to incorporate MMP degradable
sequences into the main peptide design such as RADA
peptides,[21] multi-domain peptides[22] and β-hairpin peptides[23] to
form enzyme-responsive hydrogel systems. However, in these
systems, it is not clear whether disassembly is a result of MMPs
[a]
R. Chakroun, A. Sneider, C. Anderson, Prof. D. Wirtz, Prof. H. Cui*
Department of Chemical and Biomolecular Engineering, and
Institute for NanoBiotechnology
The Johns Hopkins University
3400 North Charles Street, Baltimore, MD, 21218, United States
E-mail: hcui6@jhu.edu
[b]
[c]
Department of Oncology and Sidney Kimmel Comprehensive
Cancer Center
Johns Hopkins University School of Medicine
400 North Broadway, Baltimore, Maryland 21231, United States
Center for Nanomedicine, The Wilmer Eye Institute
Johns Hopkins University School of Medicine,
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201913087
Angewandte Chemie International Edition
RESEARCH ARTICLE
acting on the fibers themselves, or a compensation for the shift in
the equilibrium balance between monomers and their respective
assemblies. In this manuscript, we demonstrate the importance of
RBA design in exposing the MMP-2 cleavable sequence on the
filament surface. Through a series of 2D and 3D in vitro
experiments, we illustrate the functionality and efficiency of the
self-assembling RBA hydrogel system as a supramolecular local
depot for their specific response to enzymes and enzyme-
expressing cancer cells.
hydrophilic segment consists of an Arg-Gly-Asp-Arg which helps
promote solubility through multiple charged amino acids. The
second part serves as the MMP cleavable sequence, Pro-Leu-
Gly-Val-Arg (PLGVR).[26] This substrate has a Km value of 290 µM
and Kcat value of 4.1 s-1 for MMP-2, with the cleavage site between
G and V.[26] Upon MMP-2 cleavage, it is expected that removal of
the peptide, Val-Arg-Val-Val-Val sequence, from the RBA-1 would
significantly weaken the intermolecular interactions so as to alter
the assembly landscape. Details of synthesis, purification and
characterization of all the studied molecules can be found in
online supporting materials (Figures S1-S9).
In our experiments to study its assembly and MMP
responsiveness, the RBA-1 was first dissolved in water at a
concentration of 2 mM, and the solution was then heated to 80oC
for one hour and left overnight. Transmission electron microscopy
(TEM) imaging reveals dominant filamentous structures (Figure
2A). Upon the addition of PBS, the RBA-1 solution was triggered
to form a self-supporting hydrogel (Figure 2B). In the presence of
0.2 µg of MMP-2 enzyme (0.01 mg/ml in PBS), the hydrogel was
observed to break down completely within 24 h (Figure 2B). We
also synthesized the molecule that would be formed after MMP
cleavage of the PLG*VR (C12-GGRGDRPLG) where * denotes
the target cleavage site. This cleaved RBA-1 control molecule
Results and Discussion
Molecular Design and Assembly. The reverse peptide
bolaamphiphile reported here consists of a hydrophilic peptide
and an MMP cleavable peptide in the middle with two hydrophobic
terminals that are structurally different: C-terminus contains a
triple valine sequence, while the N-terminus is a twelve-carbon
alkyl chain (Figure 1). Valine was chosen because of its known
high propensity to form β–sheet conformations,[24] and also
because our previous studies also suggested valine represents a
strong contributor to filament formation.[25] The first part of the
Figure 1. Molecular design and assembly of unsymmetric reverse bolaamphiphile (RBA-1). (A) Chemical structure of a representative RBA-1 design with two
different hydrophobic ends (black) consisting of an alkyl chain on the N-terminus (region 1), and a triple valine sequence on the C-terminus (region 2) and, a
hydrophilic region (blue) consisting of a double glycine spacer and an RGDR sequence, and an MMP-2 cleavable sequence PLGVR (orange). (B) The RBA-1
monomers assemble into supramolecular filaments in water with the MMP-2 substrates displayed on the surface. At elevated concentrations or in the presence of
counterions, these filaments entangle into a hydrogel. Upon exposure to enzymes or enzyme-expressing cells, the supramolecular filament hydrogel is expected to
break down, with the possibility of reassembly by the cleaved molecule.
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10.1002/anie.201913087
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RESEARCH ARTICLE
exhibited no gelation capabilities when subjected to PBS, forming
spherical micelles of ~ 11.7 nm in diameter upon aging in water
at a 2 mM concentration (Figure 2C-E). Circular dichroism (CD)
spectra of this cleaved RBA-1 control revealed a random coil
secondary structure, as evident by the negative peak at ~200 nm
(Figure S10). These results demonstrate the importance of
including a valine rich peptide in the C-terminus of RBA-1 design
to facilitate the filamentous growth. The formation of spherical
assemblies after enzymatic removal may account for the hydrogel
breakdown illustrated in Figure 2F.
Enzyme Degradation. To develop a deeper understanding of the
hydrogel breakdown process, TEM and CD were carried out on
RBA-1 hydrogels at different time points, following the addition of
MMP-2. Initially, RBA-1 showed great gelation capabilities
(Figure 3A) with dominant filamentous nanostructures observed
throughout the TEM grid (Figure 3B). The CD spectrum of the
molecule (Figure S11) displayed a negative peak around ~220
nm, attributed to intermolecular hydrogen bonding among
peptides. After 24 h by MMP-2 treatment, the hydrogel became a
cloudy suspension of gel particles. TEM displayed longer
filaments accompanied by numerous shorter fragments (Figure
S12). Concurrently, CD displayed a decrease in the β-sheet peak
and a sign of random coil absorption, as indicated by a minor
negative peak shown at ~205 nm. Upon further aging for
additional 48 h, spherical assemblies were observed, similar to
those formed by the control molecule (Figure 2B). As expected,
CD study revealed random coils as the dominant secondary
structure. After a total of 72 h of MMP treatment, a sample solution
of the final product was removed and changed, and replaced with
multiple other peaks, including the expected cleaved molecule
(G*V). The production of other species is likely a result of the
cleaved molecule undergoing further degradation by the MMP
enzyme.
Figure 2. Self-assembly and degradation characteristics of our RBA-1 molecule.
(A) Transmission electron microscopy micrograph of the supramolecular
filaments formed, (B) gel images before and after MMP-2 treatment of 0.2 µg
enzyme (0.01 mg/ml in PBS), aged at 37oC. Upon addition of charge-screening
phosphate buffer saline (PBS) directly after aging in water, the molecule
spontaneously formed
a hydrogel. (C) Cleaved RBA-1 control molecule
resembling the segment that would be left after cleavage of our original design
displayed spherical micelle morphology using TEM, (D) no hydrogel formation
was observed after addition of PBS and (E) DLS measured 11.7 nm particles.
The molecules were dissolved in water and heated at 80oC for 1 hour, after
which they were aged for 1 day, at 2 mM concentration, before carrying out TEM,
DLS or gel formation and degradation studies. (F) Descriptive illustrations for
our main reverse bolaamphiphile design breaking down and reassembling into
spherical micelles.
Figure 3. MMP-2 triggered RBA-1 supramolecular filament breakdown and re-assembly of the fragmented RBA-1 at different time points. (A) Gel images of the
RBA-1 molecule at different time points (0 h, 24 h and 72 h) after being treated with 0.2 µg (0.01 mg/ml in PBS) of MMP-2 enzyme. (B) The respective TEM images
display the breakdown of the originally formed filaments into smaller ones, followed by re-assembly of the fragmented RBA-1 into spherical micelles.
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The RBA-1 gelation behavior and rheological properties
before and after MMP-2 treatment was also assessed using an
oscillatory time sweep test (Figure S13). Before PBS addition,
the RBA-1 filaments demonstrated solution-like rheological
properties while the addition of 10 X PBS at the 150 s time point
resulted in an increase in the storage modulus (G’) and
stabilization of the loss modulus (G”), with the crossover point
(G’>G”) occurring at ~170 s (i.e. 20 s after PBS treatment),
suggestive of forming a gel. The stiffnes of the gel was observed
to increase slightly with time. After 24 hour treatment with a large
amount of MMP-2, rheology test clearly suggested a transition
back to solution phase, as evidenced by a destabilization of the
storage modulus (G’) and loss modulus (G”).
The key in developing enzyme-responsive supramolecular
filaments is the presentation of the cleavage site to the target
enzyme. Using a traditional amphiphilic design, the cleavage site
would largely remain embedded within the core or become overly
crowded in the corona, not easily accessible to macromolecular
proteins. The formation of shortened filaments in Figure 3 after
24 h treatment with the MMP-2 enzyme, provides direct evidence
for concurrent cleavage on the filament surface, not only on the
monomeric units that shift the assembly equilibrium. If the latter
case dominated the cleavage mechanism, one would expect to
observe a large population of spherical assemblies, instead of
shortened filaments, formed by the cleaved molecule.
To further understand the cleavage mechanism and specificity,
we synthesized two more control molecules seen in Figure 4. In
the first molecular design, the triple valine sequence originally on
the C-terminus of the peptide was shifted towards the N-terminus
while the hydrophilic RGDR was moved to the C-terminus. The
resultant molecule can assemble into filamentous structures, as
shown by TEM imaging (Figure 4A). However, even after aging
with MMP-2 for 2 weeks, no noticeable sign of hydrogel
degradation was observed by naked eyes (Figure 4B). This
further confirms the significance of the reverse bolaamphiphile
design that enables the display of the MMP-2 substrate on the
filament surface. It is reasonable to assume that after self-
assembly, the MMP-2 substrate incorporated in the PA-1 would
be embedded within the resultant filaments, inaccessible to
macroscale enzymes. Given that both molecules exhibit low
critical micelle concentrations (Figure S14), only a very small
percentage of monomer exists in solution and thus cleavage by
MMP on the monomeric units is not expected to contribute
significantly to the hydrogel degradation process.To provide more
insight into the MMP cleavage process, Nile Red was used to
compare filament cleavage difference in RBA-1 and PA-1
assemblies (Figure S15). When encapsulated in a hydrophobic
environment, Nile Red emits at 635 nm as opposed to 660 nm in
aqueous solution. Since spherical micelles are able to
accommodate more Nile Red (Figure S15B), the rate of filament
breakdown by MMP-2 would be reflected by an increase in
fluorescence signal at 635 nm as a result of forming spherical
micelles that accommodate more Nile Red. As shown in Figure
S15C, adding MMP into RBA-1 filaments led to a rapid increase
over time in Nile Red encapsulated in micelles, which reached a
plateau after 24 hours. In comparison, PA-1 did not reveal any
significant change in encapsulation over the period of 72 hours.
In the second molecular design, we replaced the PLGVR
with a scrambled PVGLR sequence (presumably not cleavable by
MMP). This molecule also forms a supramolecular filament
hydrogel, and again, no disassembly or hydrogel breakdown was
observed upon aging with MMP-2 enzyme (Figure 4A, B). MALDI
further confirmed the resistance of both PA-1 and Scrambled
RBA-1 hydrogels for MMP responsiveness and chemical
breakdown (Figure S16). Collectively, these experiments suggest
Figure 4. Resistance to enzyme degradation of two control molecules to
highlight the significance of RBA-1 design. (A) Traditional linear amphiphile
design by placing the RGDR hydrophilic peptide at the C-terminus (PA-1
control), and (B) RBA-1 design with a modified non-cleavable MMP substrate
(Scrambled RBA-1 control). (A, D) TEM images of the filaments formed
assembly of the respective molecules, (B, E) gel images before and after MMP-
2 treatment of 0.2 µg enzyme, aged at 37oC and (C, F) descriptive illustrations.
Gelation was triggered by the addition of phosphate buffer saline (PBS). Both
molecules were treated the same as our original design: dissolved at 2 mM
concentration in water and heated at 80oC for 1 hour, then aged for 1 day.
Figure 5. Surface erosion of our RBA-1 hydrogel treated with cell-expressed
MMP. (A) Metastatic breast cancer cell lines, MDA-MB-231, were cultured by
imbedding them within a collagen gel matrix, to mimic a 3D environment. The
cell media used to replenish the cells was removed from the top of the collagen
matrix with high cell density (100,000 cells) and placed on the top of our reverse
bola amphiphile gel (B). At 2 and 4 week time points, gel images were taken to
demonstrate the difference in degradation speed of our hydrogel under high cell
density cell environments and a control consisting of fresh cell media.
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10.1002/anie.201913087
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RESEARCH ARTICLE
that the PLGVR sequence of the RBA-1 in the assembled states
can be accessed and effectively cleaved by target MMP.
showed no noticeable changes even up to four weeks, neither
PA-1 nor Scrambled RBA-1 hydrogels showed visual signs of
degradation (Figure S17).
Since enzymatic reaction is
Hydrogel Degradation by Enzyme-Expressing Cells. We next
investigated the degradability of RBA-1 supramolecular hydrogel
by MMP-expressing cells. It has been previously shown that
certain cancer cell types at higher densities in 3D collagen type I
matrices tend to secrete more MMPs than those at lower
densities.[12a] We therefore cultured the highly metastatic breast
concentration-dependent and MMPs are typically expressed by
cells at much lower concentrations[27],[28] slower degradation rates
were expected and observed in these experiments relative to the
results reported in Figures 2-4. The change in color observed
after 4 weeks could be a result of proteins denaturing and/or
degradation within the cell media.
cancer MDA-MB-231 cell line in
a
3D in vitro collagen
To further demonstrate the specificity of our hydrogel to
MMPs, we carried out experiments of using an MMP inhibitor,
Batismastat to block the MMP activities (Figure S17). Our results
suggest that upon inhibition of MMP function no RBA-1 hydrogel
degradation was observed. On basis of these obervations, we can
confirm that degradation of the RBA-1 hydrogel in Figure 5 is a
result of MMP cleavage as opposed to other non-specific
proteolysis reactions. Given that MMPs expressed by cancer cells
can accelerate the breakdown of our hydrogel, we speculate this
cell-responsive characteristic can be usesful in an in vivo setting
to treat tumors of various sizes and aggressiveness.
environment at a high cell (100,000 cells/ml) density. Over a 72
h period, cells were allowed to migrate, communicate, express
MMPs, and proliferate. After 72 h, the cell media of each plate
was removed and added to the top of our MMP-2 responsive
hydrogel. Fresh cell medium was used as a control. After two
weeks, the hydrogel treated with the media showed significant
breakdown. After four weeks, numerous fragmented hydrogel
pieces can be observed to suspend in the solution, indicative of a
bulk erosion mechanism enabled by cell-expressed MMPs. For
degradation mechanism dominated by monomer cleavage, we
would expect the cleavage reaction to be heavily dependent upon
the monomer dissociation kinetics from supramolecular filaments.
This dissociation-dependent release often led to an apparent
surface erosion mechanism.[25] In comparison, the control media
Drug-Based RBA Design and Assembly. Inspired by the cell-
regulated hydrogel breakdown, we sought to validate the use of
Figure 6. The design, assembly, and enzyme responsiveness of a drug-bearing RBA (DRBA-1). (A) Chemical structure of the drug version of our reverse
bolaamphiphile design with the alkyl chain of our peptide amphiphile (PA) replaced with paclitaxel (red), and RDRD serving as the hydrophilic region (blue). All other
design features remain similar to RBA-1. (B) Cytotoxicity comparison of our DRBA-1 with free PTX at different concentration, in MDA-MB-231 breast cancer cells.
RBA-1 and cleaved RBA-1 control was also tested. Cells were incubated with the PTX or conjugates for 72 h and cell viability was determined by MTT assay.
Values of 56 nM and 29 nM represent the IC50 value. Data are given as mean ± s.d (n=3). (C) Gel images of our drug amphiphile at different time points (0 h, 24 h
and 72 h) after being treated with 0.2 µg of MMP-2 enzyme (0.01 mg/ml in PBS). The respective TEM images (D) for each time point display the breakdown of the
original filaments into smaller fragments, followed by reassembly into spherical micelles. To form the filaments, the DRBA-1 was dissolved at 2 mM and heated for
1 h at 80oC followed by overnight aging. After the addition of PBS, the filament solution solidified into a gel, from which the TEM, CD and degradation studies were
conducted.
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Figure 7. Controlled release of paclitaxel by enzyme-expressing cancer cells. (A) Illustration detailing the setup procedure for our 3D in vitro cell studies. 20 µL
droplets of 20% drug loading were placed on a metal spatula and PBS was added to trigger gelation, producing solid gel pieces of similar gel volume. The gel pieces
were then deposited within a 3D collagen matrix of different cell densities. The release kinetics and cytotoxic effect of our hydrogel under high and low cell densities
was assessed. Our gel was implanted in the collagen matrix containing an equal distribution of MDAMB 231 cells at different densities. The hydrogel was placed on
one side of the well and both quantitative (B, D) and qualitative (C, E) data was obtained revealing cell death across the matrix, away from the gel, at different time
points. Separate plates were prepared for analysis of each time point, at which cells were stained with Hoechst and propidium iodide, and then imaged. Data are
given as mean ± s.d (n=3). (F) Schematic Illustration depicting the fragmentation of filaments within our hydrogel upon contact with MMP-2 enzyme, into monomers
followed by their consequent diffusion throughout the tumour microenvironment.
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RBA design in controlled drug release. In this context, paclitaxel
was chosen and conjugated to the N-terminus of our RBA peptide
to replace the alkyl tail. A reducible disulphide linker (buSS) was
used to bridge the drug to the peptide and also to facilitate the
release of free drug after cell internalization. Figure 6A displays
the chemical structure of the drug-based reverse bolaamphiphile
(DRBA-1). The hydrophilic Arg-Gly-Asp-Arg was substituted with
a more charged Arg-Asp-Arg-Asp sequence to help counter
solubility issues. In an in vitro assay, MDA-MB-231 cells were
respectively treated with the monomeric form of our DRBA-1 and
free PTX at different concentrations (Figure 6B and Figure S18).
We observed comparable cytotoxicity between both drug
molecules; DRBA-1 and free PTX exhibited an IC50 of 56 nM and
29 nM, respectively. These results imply the efficient reduction of
the buSS linker by cells, maintaining the cytotoxic potency of the
DRBA-1. In addition, RBA-1 and its major cleaved product, RBA-
1 control, did not exhibit any noticeable effect on cell viability
under the studied conditions indicating a non-toxic nature (Table
S1). Our in vitro studies also suggest the potential use of RBA-1
hydrogels as coatings for 2D cell culture (Figure S19).
environment, cell death was localized to areas around the
hydrogel, whereas in a higher cell (100,000 cells/ml) density, an
expanding impact to cells farther away was observed as time
progressed. At a higher cell density, cells at the far end of the well
exhibited an increase in cell death over time, showing 78%
viability over 72 h. In a low cell density environment no cell death
was observed beyond areas prominent to the gel even after 72 h
of treatment. We attribute the difference in cell killing to the MMP-
responsive nature of our hydrogel and its ability to degrade more
rapidly in the presence of an elevated level of MMP. In an
environment with a larger number of cancer cells, more MMP will
result in a faster breakdown of the RBA/DRBA hydrogel, leading
to more rapid drug release. This elevated drug level may also
promote a deeper distribution into the tumor intersitium, attributed
to the spherical morphology of the cleaved DRBA-1 product after
cleavage. In an environment with fewer cancer cells, the RBA
hydrogel will not breakdown as fast due to the limited MMP
supplies, and the drug release will be restricted to adjacent areas
around the hydrogel.
A proposed action mechanism of DRBA-1 is illustrated in
Figure 7F showing the multipe stages of cell-controlled release
of the therapeutic agents. Upon MMP cleavage, DRBA-1
filaments could fragment into short pieces that eventaully
dissociate into monomeric drug amphiphiles. Delivering paclitaxel
in the form of shortened filaments and/or potentially the
reassembled spherical assemblies, as opposed to the monomeric
individual drug conjugates, could lead to an enhanced distribution
throughout the tumor intersitium with a better controlled drug
release, as revealed by other laboratories.[29] Furthermore, drugs
in the nanoparticle form could exhibit an enhanced cellular uptake
and are also protected from rapid intracellular breakdown.[30]
To confirm its cell-responsive breakdown, the DRBA-1
hydrogel was treated with 0.2 µg of MMP-2 (0.01 mg/ml in PBS)
and analyzed at different time points using TEM (Figure 6D) and
CD (Figure S7B). In these experiments, the DRBA-1 monomers
were dissolved in water at a 2 mM concentration. After aging
overnight, TEM imaging displayed long and flexible filamentous
structures capable of forming a hydrogel upon addition of PBS.
These filaments displayed a strong dominant β-sheet peak at
~220 nm in CD with a strong positive peak at ~237 nm
corresponding to the paclitaxel absorbtion. Similar to the previous
studies on RBA-1, these filaments were also observed to break
down into shorter fragments after 24 h in an enzyme environment, Nanoparticles could also help overcome multi-drug resistance.[31]
eventually converting to a solution form (Figure 6D). CD spectra
displayed mixed absorption of random coils and β-sheet,
indicating the cleavage of the triple-valine segment and a change
in assembly hehavior. After a total of 72 h incubation, only
spherical micelles could be observed according to TEM imaging,
and the random coil secondary structure measured dominated the
collected CD spectra. MALDI-TOF studies (Figure S16) revealed
the dissapearance of the original DRBA-1 molecular mass, again
confirming the action of the enzyme. The consistency of these
Although we have yet to demonstrate all these features in our
current studies, it is reasonable to assume that the potentially re-
assembled nanoparticles may exhibit some of these properties.
This is evident in our 3D in vitro studies, which not only shows a
tumor-responsive drug release, but for a limited amount of drug,
a higher percentage of cell death was observed in areas with a
greater cell density.
results with the previous RBA-1 studies furhter confirms the ability Conclusion
of our sytem to retain its self-arranged structure and enzyme
sensitivity, independent of the hydrophobic segment used.
In summary, we have designed and synthesized peptide-
based reverse bolaamphiphiles with unsymmetric hydrophobic
segments, and demonstrated the importance of the reverse
bolaamphiphile design in constructing enzyme-responsive
supramolecular filament hydrogels, with effective display of MMP-
2 substrate on the surface. By replacing the hydrocarbon with an
anticancer drug, we developed a drug-based RBA hydrogel with
potentially improved cancer treatment capabilities. Our in vitro 3D
cell studies revealed a greater drug release rate and more
effective cancer cell killing when a higher density of cancer cells
were present as a result of an increased level of MMP expression.
We believe this RBA design plaform is a new addition to the
amphiphile family, and envision that after further optimization a
selective release of chemotherapeutic agent can be achieved in
response to the tumor size and aggresiveness, thus enabling
improved local treatment of cancer.
Cell-Controlled Drug Release. To further demonstrate the
enhanced cytotoxicity in response to cell density, we developed
an in vitro 3D cell assay (Figure 7). The hydrogels used in these
experiments were created by mixing DRBA-1 with RBA-1 at a
molecular ratio of 1 to 4. A drop of 20 µL mixed solution was
placed on a metal spatula, followed by gelation using PBS (Figure
7A). Next, these hydrogel pellets were embedded within a
collagen matrix containing different densities of the breast cancer
cells (MDA-MB-231). At specific time points (0, 24, 48 and 72 h),
cells from different locations of the petri dish were removed and
treated with Hoechst stain and propidium iodide to examine their
viability. This assay enables us to measure drug distribution,
represented by cell death, as a function of position and time. As
seen in Figure 7B-D, in a lower cell (10,000 cells/ml) density
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10.1002/anie.201913087
Angewandte Chemie International Edition
RESEARCH ARTICLE
NMR Center for acquisition of NMR spectra and consultation at
The Johns Hopkins University. In addition, we are very thankful to
Dr Siva P. Kambhampati and Dr Rangaramanujam M. Kannan for
their help in obtaining and analyzing the rhelogical properties of
our hydrogel.
Acknowledgements
The work reported here is supported by the National Science
Foundation (DMR 1255281) and 1746891 (Wirtz). R.W.C. and
A.S. acknowledge the support of NSF Graduate Research
Fellowships Program (DGE 1746891). We also extend our
sincere gratitude to the Integrated Imaging Center (IIC) for TEM
imaging, the Mass Spectrometry Facility (MSF) for help aquiring
high resolution mass spectrometry data and the Biomolecular
Keywords: bolaamphiphile • drug delivery • hydrogel • prodrug •
self-assembly
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RESEARCH ARTICLE
R. Chakroun, A. Sneider, C. Anderson,
D. Wirtz, and H. Cui*
Page No. – Page No.
Supramolecular Design of
Unsymmetric Reverse
Bolaamphiphiles for Cell-Sensitive
Hydrogel Degradation and Drug
Release
Reverse bolaamphiphile with a chemotherapeutic agent and an MMP-2 substrate
was shown to self-assemble into a cell-responsive supramolecular filament hydrogel.
Cancer-expressed MMP-2 triggered hydrogel degradation through filament
fragmentation, eventually releasing therapeutics against cancer cells.
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