Constructing Cross-Linked Nanofibrous Scaffold via Dual-Enzyme-Instructed Hierarchical Assembly

Article abstract of DOI:10.1021/acs.langmuir.0c01023

To explore the potential of step-by-step assembly in the fabrication of biological materials, we designed and synthesized two peptide-based molecules for enzyme-instructed hierarchical assembly. Upon the treatment of alkaline phosphatase, one molecule undergoes enzyme-instructed self-assembly forming uniformed nanofibers. The other one that can self-assemble into vesicles undergoes enzyme-induced transformation of self-assembly converting vesicles into irregular aggregates upon the treatment of carboxylesterase. Coadministration of two enzymes to a mixture of these two molecules in a stage-by-stage fashion leads to a physically knotted nanofibrous scaffold that is applicable as a nanostructured matrix for cell culture.

Full text of DOI:10.1021/acs.langmuir.0c01023

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Article
Constructing Cross-linked Nanofibrous Scaffold
via Dual-Enzyme-Instructed Hierarchical Assembly
Shijin Zhang, William Cortes, and Ye Zhang
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.0c01023 • Publication Date (Web): 17 May 2020
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Constructing Cross-linked Nanofibrous Scaffold via
Dual-Enzyme-Instructed Hierarchical Assembly
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Shijin Zhang, William Cortes, Ye Zhang*
Bioinspired Soft Matter Unit, Okinawa Institute of Science and
Technology Graduate University, 1919-1 Tancha, Onna Son, Okinawa
904-0495, Japan
KEYWORDS Enzyme-instructed self-assembly, Cross-link, Step-by-
step assembly, Alkaline phosphatase, carboxyl esterase.
ABSTRACT
To explore the potential of step-by-step assembly in the
fabrication of biological materials, we designed and synthesized
two peptide-based molecules for enzyme-instructed hierarchical
assembly (EIHA). Upon the treatment of alkaline phosphatase,
one molecule undergoes enzyme-instructed self-assembly (EISA)
forming uniformed nanofibers. The other one that can self-
assemble into vesicles undergoes enzyme-induced transformation
of self-assembly (EITSA) converting vesicles into irregular
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aggregates upon the treatment of carboxylesterase (CES). Co-
administration of two enzymes to a mixture of these two
molecules in a stage-by-stage fashion leads to a physically
knotted nanofibrous scaffold that is applicable as a
nanostructured matrix for cell culture.
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INTRODUCTION
In living systems, advanced biological functions rely on high-
level structural complexity.1 Inspired by biological entities and
processes, hierarchical assembly2-3 is believed to be a promising
bottom-up approach of synthetic chemistry to construct materials
with tunable advanced structures.4 In particular, intensive
studies on special morphology construction via coordination
chemistry5-6 emphasizing the fluorescence properties7-11 have been
reported. Comparatively, hierarchical assembly is rarely applied
in the construction of soft biomaterials. According to
theoretical studies on the correspondence between energetics and
kinetics for optimal design principles,12 we decide to program a
hierarchical assembly pathway to control the structural
energetic stability and kinetic accessibility3 in stage-by-stage
fashion for the fabrication of biomaterials.
Diverse external stimuli have been successfully applied to
instruct molecular self-assembly,13
including chemical
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stimulisolvents, acid/base signals, metal ions, gases,
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biomacromolecules, redox signals, physical stimulitemperature,
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magnetic fields, and light. Among stimuli-responsive self-
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assemblies, enzyme-instructed self-assembly (EISA)14-19
and
enzyme-instructed transformation of self-assembly (EITSA),20 the
processes that integrate enzymatic transformation and molecular
self-assembly in physiological condition, are considered as
practical strategy in biomimetic synthesis of materials.21
Alkaline phosphatase and carboxylesterase (CES) have been
commonly applied in EISA and EITSA because both of them have
broad substrate specificity. To confront the challenge of
bridging hierarchies of multiple length- and time-scales of
self-assembly pathways, these two enzymes with different
reaction dynamics are used as external stimuli to instruct the
step-by-step assembly.
As demonstrated in Figure 1A, one molecule which undergoes ALP-
instructed self-assembly forming nanofibrils, and the other
molecule which undergoes CES-instructed transformation of self-
assembled vesicles into nanoaggregates are mixed together in
aqueous solution. A stage-by-stage administration of two enzymes
in a designated order into the mixture induces co-existence of
nanofibrils and vesicles in close contacts followed by the
conversion of vesicles into clusters of nanoaggregates, and
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leads to a cross-linked scaffold, which is called enzyme-
instructed hierarchical assembly (EIHA).
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Figure 1. A) Schematic illustration of constructing physically
cross-linked scaffold using enzyme-instructed hierarchical
assembly (EIHA). (B) Chemical structures of peptidic molecules 1
and 2, and their enzymatic catalysis reactions, ALP-catalyzed
dephosphorylation of 1 and CES-catalyzed hydrolysis of 2.
EXPERIMENTAL SECTION
Materials
and
Instruments.
Fmoc-Amino acid, 4-
dimethylaminopyridine (DMAP) and 2-chlorotrityl chloride resin
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were purchased from GL Biochem (Shanghai, China);
Dimethylformamide (DMF, 99.5%), Dichloromethane (DCM, 98.0%),
N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA,
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98.0%),
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride (EDC), piperidine (99.0%), Meldrum's acid (98%),
methanol (99.0%), ethanol (99.5%), hexane (95.0%) were purchased
from Nacalai Tesque Inc., Japan; 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (97.0%)
was
purchased
from
Matrix
Scientific;
N,N'-
diisopropylcarbodiimide (DIC, 99.0%) was purchased from FUJIFILM
Wako Pure Chemicals; ethylene glycol (99.8%) was purchased from
Sigma
hydroxysuccinimide (NHS, 98.0%) and biotin (98.0%) were obtained
from Tokyo Chemistry Industry (TCI). 4-
Aldrich.
Piperidinium
acetate
(98.0%),
N-
(diethylamino)salicylaldehyde (98.0%) was purchased from BLD
Pharmatech Ltd; 2-Naphthylacetic acid (NapOH, 98.0%) was
purchased from Combi-Blocks; Organic solvents were dehydrated
before experiment. HPLC purification was performed on Agilent
1260 Infinity Preparative Pump with Agilent 1260 Infinity Diode
Array Detector (Eluent: Acetonitrile and water (0.1% TFA)). Mass
spectra were recorded by Thermo LTQ-ETD mass spectrometer (ESI-
MS). 1H NMR and 13C NMR spectra were recorded on a JEOL 400 (400
MHz and 101MHz, respectively) spectrometer.
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Synthesis. Synthetic procedures and characterizations of the
molecules applied in this study are described in Supporting
Information.
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Transmission electron microscopy imaging. 5 L of sample
solution was dropped on the carbon-coated copper grids with
enhance hydrophilicity via low discharge for about 1 min. After
removing the excess water with filter paper, 5 L of 1% uranyl
acetate was dropped on the grid for about 20s. After removing
the excess solution using a filter paper, the grid was dried in
open air. Observation was carried on JEM-1230R with acceleration
voltage at 100 kV.
Kinetic profiling of ALP-catalyzed dephosphorylation of 1.
Alkaline phosphatase from calf intestine was purchased from
Invitrogen (Cat no. 18009-019). Following the instruction, ALP
stock solution (1u/L) was prepared with the dilution buffer.
Stock solutions of compound 1 or mixture of compound 1 and 2 (40
mM in DMSO) were diluted in borate buffer to the desired
concentration. Proper volume of ALP stock solution was added
into 200 L of compound solution reaching final concentration of
1U. HPLC and LCMS were applied to identify the reaction products
and monitor the progress of hydrolysis at room temperature.
Kinetic
profiling
of
CES-catalyzed
hydrolysis
of
2.
Carboxylesterase (CES) from rabbit liver lyophilized powder was
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purchased from Sigma-Aldrich (E0887-500UN). Following the
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instruction, CES stock solution (1u/5L) was prepared in borate
buffer (pH 8.0). Stock solutions of compound 2 or mixture of
compound 1 and 2 (40 mM in DMSO) were diluted in borate buffer
to the desired concentration. Proper volume of CES stock
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solution was added into 200 L of compound solution reaching
final concentration of 1U. HPLC and LCMS were applied to
identify the reaction products and monitor the progress of
hydrolysis at room temperature.
Circular dichroism (CD) spectroscopy. CD spectra measurements
were carried out on spectrometer JASCO J-820. The bandwidth was
set at 1.0 nm and the measurement range was 190 – 400 nm. All
measurements were carried out in a 1 mm quartz cuvette at room
temperature. Theoretical curve was obtained by a simple sum of
every single components.
Rheological measurement. Rheology tests were conducted on
Anton-Paar MCR302; parallel-plate geometry with an upper plate
diameter of 25 mm was used during the experiment, and the gap
was 0.1 mm. Throughout the experiment, the stage temperature was
maintained at 25 ºC. The specimen was transferred to the stage
by spatula, dynamic strain (0.1 – 100%) was performed at 6.28
rad/s, maximum storage moduli in the linear range of the strain
sweep test was selected for frequency sweep test (0.1 – 200
rad/s).
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Cell viability assay. HeLa cells in exponential growth phase
were seeded in a 96 well plate at a concentration of 8000
cells/well for all cell lines. The cells were allowed to attach
to the wells for 12 h at 37 ºC, 5% CO2. The culture medium was
removed followed by addition of 100 μL culture medium containing
different concentrations (50, 100, 200, 500 and 1000 μM) of
compound 1 and 2. After the desired time of incubation, 10 μL
MTT solution (5 mg/mL) was added to each well and incubated at
37 °C for another 4 h, and then 100 μL of SDS solution (10% in
Milli-Q water) was added to stop the reduction reaction and
dissolve the purple formazan. The absorbance at 570 nm was
measured using Nivo3 microplate reader (PerkinElmer). All
experiments were performed in triplicate and repeated three
times.
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Multicellular spheroid culture assay. To introduce various
nanostructures into the culture medium, we first prepared the
culture medium containing both molecule 1 (200 M) and molecule 2
(200 M). And then the enzymes were introduced to the solutions
in different orders. For spheroid formation, a fixed number of
HeLa cells (2000) was seeded in 100 L of complete culture medium
(with and without the nanostructures) in round bottomed, ultra-
low attachment 96-well plates (ThermoScientific, 174929 96U
Bottom Plate) and incubated for 72 h. For each culture
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condition, a minimum of three independent repeat experiments was
performed (n3). The growth of spheroids was recorded using
IncuCyte S3.
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RESULTS AND DISCUSSION
Molecular design and synthesis. Regarding the broad impact of
peptide-based scaffolds in biomedical applications,22-25 two
peptidic molecules 1 and 2 are designed as precursors for EIHA.
As shown in Figure 1B, 7-(diethyamino)-coumarin-3-carboxylic
acid26-27 that is predominantly used as a biomedical inhibitor is
applied as the aromatic building block facilitating
intermolecular - stacking for both molecules. Based on the
instructions of previous design on EISA induced nanofibril
formation, by coupling the aromatic building block to the N-
terminal of Tyr(PO3H2)-Phe-Lys(biotin), we obtained hydrophilic
molecule 1. The phosphor-tyrosine unit of molecule 1 response to
enzyme leading to ALP-catalyzed dephosphorylation. Coupling
biotin to the side chain of lysine is to enhance the molecular
interaction with cell surface via biotin-avidin binding.28-29 Upon
the treatment of ALP, molecule 1 will transform into relatively
hydrophobic derivative 1a triggering molecular self-assembly. To
have self-assembled vesicles,20 molecule 2 was synthesized by
linking naphthalene-Phe-Phe, the classic peptide building block
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for - interaction and hydrogen bonding oriented self-assembly,
to 7-(diethyamino)-coumarin-3-carboxylic acid via ethylene
glycol. Upon the treatment of CES, molecule 2 is hydrolyzed into
the self-assembly building block 2a and the aromatic building
block 2b.
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EISA of molecule 1 and EITSA of molecule 2. Molecule 1 is
highly soluble in water. Upon the treatment of ALP,
dephosphorylation of molecule 1 to 1a triggers molecular self-
assembly forming uniform nanofibrils (Figure 2A). Different from
that, molecule 2 self-assembles in borate buffer forming
vesicles with a broad range of diameters. Upon the treatment of
CES, hydrolysis of 2 to 2a and 2b triggers disassembly of
vesicles into clusters of nanoaggregates in irregular shapes
(Figure 2B).
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Figure 2. (A) TEM images of ALP-catalyzed dephosphorylation of
molecule 1 (1 mM) induced self-assembly. Scale bar represents
100 nm. (B) TEM images of CES-catalyzed hydrolysis of molecule 2
(1 mM) induced transformation of self-assembly. Scale bars
represent 100 nm. (C) Kinetic profiles of ALP-catalyzed
dephosphorylation of molecule 1, and CES-catalyzed hydrolysis of
molecule 2 separately in borate buffer at 25 C. (D) UV-vis
absorption spectra and emission spectra (excited at 405 nm) of
molecule 1 (0.2 mM) in borate buffer at 25 C before and after
treatment with ALP (1 U/mL) for 12 hours. (E) UV-vis absorption
spectra and emission spectra (excited at 405 nm) of molecule 2
(0.2 mM) in borate buffer at 25 C before and after treatment
with CES (0.2 U/mL) for 12 hours.
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The kinetic profiles of both catalytic reactions under single
component condition were recorded (Figure 2C). At room
temperature (25 ºC), ALP-catalyzed dephosphorylation of molecule
1 into 1a is completed within half an hour (Figure S1), which is
more than 20 times faster than the CES-catalyzed hydrolysis of 2
into 2a and 2b that takes about 12 hours (Figure S2).
Consequently, the formation of nanofibrils via EISA is much
faster than the disassembly of vesicles into nanoaggregates via
EITSA. Regarding the blue fluorescence of aromatic building
block 2b shared by both molecules, the absorption and emission
spectra of molecules 1 and 2 before and after the catalytic
reactions were characterized. For molecule 1 in borate buffer,
the absorption and emission peaks remain at 434 nm and 484 nm,
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respectively,
before
and
after
the
ALP-catalyzed
dephosphorylation (Figure 2D). While, both intensities decrease
after EISA due to self-assembly shielding effect.30 While,
molecule 2 in borate buffer has an absorption peak at 421 nm,
and a fluorescent peak at 546 nm. After the treatment with CES,
the absorption peak shifts to 412 nm, and the fluorescent peak
shifts to 468 nm (Figure 2E). Compare to the absorption and
emission spectra of 2b that have peaks at 424 nm and 472 nm
(Figure S3), respectively, CES-induced EITSA causes blue shifts
via both morphological change and component change.
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Stage-by-stage administration of enzymes to the mixture of 1
and 2 leads to EIHA. Similar as the self-assembly of molecule 2
in borate buffer, the 1:1 mixture of molecules 1 and 2 at the
same total concentration forms vesicles of wide range of
diameters (Figure 3A). The CD spectrum of the mixture is similar
to the simple sum of both single-component spectra. Considering
the solubility of molecule 1, the addition of molecule 1 may
induce alteration of the solution environment of molecule 2
slightly affecting its self-assembly instead of inducing co-
assembly with molecule 2 (Figure 3B). The administration of two
enzymes to the mixture in different orders leads to distinct
nanostructures. For example, the co-administration of ALP and
CES simultaneously to the mixture of two molecules leads to
scattered beaded nanofibrils (Figure 3A right panel) via
molecular co-assembly confirmed by the CD spectrum which is
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distinct from the theoretical calculationthe sum of CD spectra
of 1a, 2a and 2b (Figure 3B). While, the administration of two
enzymes in an order of ALP first and CES second triggers the
formation of uniform nanofibrils around vesicles in close
contacts (Figure 3C) first, and then the transition of vesicles
into clusters crosslinking the nanofibrous into scaffold (Figure
3D indicated by blue arrow head) via co-assembly confirmed by
the comparisons between the experimental and theoretical CD
spectra (Figure 3E). Distinct from that, the administration of
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two enzymes in an order of CES first and ALP second induces the
disassembly of vesicles into scattered irregular aggregates
first (Figure 3F), and then the formation of nanofibrils
tangling with irregular nanoaggregates (Figure 3G) via co-
assembly, which is also confirmed the CD spectra comparisons
(Figure 3H). Consistent with the morphology difference induced
by three enzyme administration orders, the CD spectra of final
nanostructures are distinct from each other. Scanning electron
microscopy revealed the difference of final structures in
macroscopic scale. As shown in Figure 3I, co-administration of
the two enzymes simultaneously into the mixture of two molecules
lead to piles of aggregates. Administration of enzymes in the
order of ALP then CES leads to fibrous scaffolds (Figure 3J).
And the administration of CES first then ALP leads to plies of
short sticks (Figure 3K).
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Figure 3. TEM images of molecule 1 (1 mM) and molecule 2 (1 mM)
mixture in borate buffer at 25 C before and after treatment with
ALP and CES simultaneously for 16 hours (A); treated with ALP
for 30 minutes (C) first, and then with CES for 14 hours (D);
treated first with CES for 12 hours (F) and then with ALP for 40
mins (G). Scale bars represent 100 nm. (B) CD spectra of
molecule 1 and molecule 2 mixture in borate buffer at 25 C
before and after treated by ALP and CES simultaneously. (E) CD
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spectra of molecule 1 and molecule 2 mixture in borate buffer at
25 C treated by ALP for 30 minutes, and after the second stage
treatment with CES. (H) CD spectra of molecule 1 and molecule 2
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mixture in borate buffer at 25 C treated by CES for 12 hours,
and after the second stage treatment with ALP. Solid and dash
lines in panels B, E, and H represent experimental (Exp.) and
theoretical (Theo.) CD spectra, respectively. SEM image of end
stage of molecule 1 (1 mM) and molecule 2 (1 mM) mixture in
borate buffer at 25 C treated with ALP and CES simultaneously
(I), treated first with ALP for 30mins and then with CES for 14
hours (J), and treated first with CES for 12 hours and then with
ALP for 40 mins (K). The scale bars of panel I-K represent 2 m.
Dynamics and transitional morphology study of Stage-by-stage
assembly. By comparing the kinetics of ALP-catalyzed
dephosphorylation of molecule 1 and CES-catalyzed hydrolysis of
molecule 2 in three different enzyme-administration processes,
and the correlated TEM characterizations of molecular assembly
at transition stages, we try to explore the general implications
underlying the stage-by-stage hierarchical assembly. During the
co-administration of both enzymes simultaneously to the mixture
of molecules 1 and 2, ALP-catalyzed dephosphorylation of
molecule 1 remains the same reaction speed as during the
administration of ALP solely in molecule 1. While the CES-
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catalyzed hydrolysis of molecule 2 was slowed down by requiring
4 more hours to complete the reaction that takes 12 hours in
single component condition (Figure 4A). In the middle of the
reaction when ALP-catalyzed dephosphorylation is completed but
CES-catalyzed hydrolysis is still ongoing, which introduces the
surface interaction among 1a-assembled-nanofibers, 2-assembled-
vesicles, and disassembled vesicles. TEM imaging reveals
consistent results that nano-aggregates and deformed vesicles
attach to nanofibrils (Figure 4B). Besides the ‘solid effect’
induced by the nanofibrils formation, the physical interactions
between the different nanofilaments may also slow down the CES-
catalyzed hydrolysis. And scattered beaded nanofibrils are
formed in the end of the process.
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During stage-by-stage administration of two enzymes in the
order of ALP and CES, ALP-catalyzed dephosphorylation of
molecule 1 remains the same reaction speed as during the
administration of ALP solely in molecule 1. While the CES-
catalyzed hydrolysis of molecule 2 required about 14 hours,
which is 2-hour slower than the single component condition, but
2-hour faster than the co-administration process (Figure 4C).
During the first stage enzyme administration, the TEM image
reveals short nanofibrils forming around scattered vesicles. One
hour after the second stage enzyme administration, TEM imaging
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reveals dense nanofibrils tangling around vesicles with close
surface contacts. The two TEM images confirm that the formation
of nanofibers induced by ALP-catalyzed dephosphorylation does
not affect the morphology of 2-assembled-vesicles. The second
stage EITSA under the condition of close surface interactions
between nanofibers and vesicles leads to the formation of dense
nanofibrous scaffold tied up by the nanoaggregates (Figure 4D).
During stage-by-stage administration of two enzymes in the order
of CES and ALP, the CES-catalyzed hydrolysis of 2 sped up to
complete the reaction in 10 hours suggesting that the
hydrophilic molecule 1 may moderate the ‘solid effect’ induced
by assembled vesicles. While the following ALP-catalyzed
hydrolysis of 1 remained the same speed as in single component
condition (Figure 4E). Shortly after the first stage enzyme
administration, TEM images showed vesicles with rough edges and
disrupt shapes indicating the initiation of disassembly. Shortly
after the second stage of enzyme administration, the TEM image
reveals the formation of short nanofibrils surrounded by
irregular nanoaggregates, which eventually leads to elongated
nanofibrils with attached nanoaggregates (Figure 4F). Comparing
these three enzyme-administration processes, we found that the
coexistence of nanofibrils and nanovesicles in close contacts
prior to the second stage of assembly is critical to scaffold
formation. Disassembling these well inserted vesicles can
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physically knot the nanofibrils together by transformed
nanoaggregates. And the stage-by-stage administration
successfully control the interface interactions among various
assembled nanostructures leading to distinct hierarchical
morphologies that are confirmed by TEM imaging and CD
characterization.
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Figure 4. (A) Kinetic profiles of ALP-catalyzed hydrolysis of 1
and CES-catalyzed hydrolysis of 2, when ALP and CES are added
into the mixture of 1 and 2 simultaneously. (B) TEM image of 1
and 2 mixture reacts with ALP and CES simultaneously for 6
hours. Scale bare represents 100 nm. (C) Kinetic profiles of
ALP-catalyzed hydrolysis of 1 and CES-catalyzed hydrolysis of 2
when 1and 2 mixture reacts with ALP first, then CES. (D) TEM
image of 1 and 2 mixture reacts with ALP for 15 minutes, and TEM
image of 1 and 2 mixture first reacts with ALP for 30 minutes,
then with CES for 1 hour. Scale bare represents 100 nm. (E)
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Kinetic profiles of ALP-catalyzed hydrolysis of 1 and CES-
catalyzed hydrolysis of 2 when 1and 2 mixture reacts with CES
first, then ALP. (F) TEM image of 1 and 2 mixture reacts with
CES for 15 minutes, and TEM image of 1 and 2 mixture first
reacts with CES for 10 hours, then with ALP for 15 minutes.
Scale bare represents 100 nm.
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Multicellular spheroid culture in stage-by-stage assembled
nanostructures. The mechanical properties of the final
nanostructures obtained via three pathways were evaluated via
rheology measurements. The oscillatory frequency sweep (Figure
5A) confirms that the nanostructures obtained via stage-by-stage
administration of ALP then CES to the mixture of 1 and 2 is much
stronger than the nanostructures obtained via other pathways,
which is consistent with the TEM image of physically cross-
linked nanofibrous scaffold.
Various three-dimensional (3D) matrices have been applied31-33 in
cancer spheroid culture to adjust 3D cell-cell contracts to
resemble the physiological microenvironments of different solid
tumors for cancer research.34 Regarding the biocompatibility of
both molecules 1 and 2 (Figure S4), the nanostructures obtained
via various pathways are applied in multicellular spheroid
culture. By examining the diameters of the spheroids cultured
from the same number of HeLa cells, we found that tightly packed
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spheroids were obtained upon the treatment of nanostructures
compare to the control condition (Figure 5B) due to the cell-
nanostructure interaction via biotin-avidin binding. By
summarizing the length of long diameter and short diameter of
spheroids cultured in various nanostructures, we also found that
the crosslinked scaffold obtained via stage-by-stage treatment
of ALP and CES facilitated the growth of the most tightly packed
spheroids with less variations. Besides the accurate size
control of cancer spheroids, adjusting spheroid packing density
via synthetic matrix will also facilitate the tumor cell
microenvironment mimicking that both are potentially helpful to
the drug screening.
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Figure 5. (A) The frequency sweep tests of co-assembled
nanostructures obtained by administration of two enzymes in
three different orders. (B) Optical images of HeLa cell
spheroids cultured under various conditions exhibiting the
influence of nanostructures obtained by stage-by-stage assembly.
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CONCLUSIONS
We introduced here the construction of a variable assembly
system guided by two enzymes, ALP and CES. The alteration of
enzyme administration orders leads to distinct nanostructures.
And a stage-by-stage administration of ALP and CES leads to
hierarchical assembly forming physically cross-linked
nanofibrous scaffold which is potentially applicable in the
control of 3D cell-cell contacts in multicellular spheroid
culture. Besides that, the underlining mechanism studies also
reveal that at defined temperature and concentration, the ALP-
catalyzed dephosphorylation is barely affected by the
surrounding nanostructures. While, the CES-catalyzed hydrolysis
of
2 is affected by the surrounding molecules and
nanostructures. By regulating the enzyme administration order,
the hydrolysis reaction speed is altered which influences the
formation and distribution of various nanofilaments leading to
prospective hierarchical assembly. By demonstrating such
practical design of step-by-step assembly for constructing
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nanostructured soft material, it’s proved that hierarchical
assembly offers promising potentials to the engineering of
synthetic extracellular matrix (ECM). Advanced extension of this
design is programed assembly of multiple functional peptides for
fine adjustment of cell-cell and cell-ECM interactions.
ASSOCIATED CONTENT
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Supporting Information. The Supporting Information is available
free of charge.
Synthesis and characterization of molecules, UV-vis absorption
and emission measurements, cell viabilities (PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: ye.zhang@oist.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the Proof-of-Concept program of OIST,
Okinawa Institute of Science and Technology Graduate University
(OIST), and Takeda Science Foundation.
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