Supramolecular Platform Stabilizing Growth Factors

Article abstract of DOI:10.1021/acs.biomac.8b00219

High concentrations of supplemented growth factors can cause oversaturation and adverse effects in in vitro and in vivo studies, though these supraphysiological concentrations are often required due to the low stability of growth factors. Here we demonstrate the stabilization of TGF-β1 and BMP4 using supramolecular polymers. Inspired by heparan sulfate, sulfonated peptides were presented on a supramolecular polymer to allow for noncovalent binding to growth factors in solution. After mixing with excipient molecules, both TGF-β1 and BMP4 were shown to have a prolonged half-life compared to the growth factors free in solution. Moreover, high cellular response was measured by a luciferase assay, indicating that TGF-β1 remained highly active upon binding to the supramolecular assembly. The results demonstrate that significant lower concentrations of growth factors can be used when supramolecular polymers bearing growth factor binding moieties are implemented. This approach can also be exploited in hydrogel systems to control growth factor release.

Full text of DOI:10.1021/acs.biomac.8b00219

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A Supramolecular Platform Stabilizing Growth Factors
Simone I. S. Hendrikse, Sergio Spaans, E. W. Meijer, and Patricia Y. W. Dankers
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00219 • Publication Date (Web): 20 Apr 2018
Downloaded from http://pubs.acs.org on April 22, 2018
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A Supramolecular Platform Stabilizing Growth
Factors
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Simone I. S. Hendrikse, Sergio Spaans, E.W. Meijer* and Patricia Y. W. Dankers*
Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513,
5600 MB Eindhoven, the Netherlands.
KEYWORDS
Supramolecular chemistry, Growth factor stabilization, Transforming growth factorꢀβ1, Bone
morphogenic proteinꢀ4, Heparan sulfate, Sulfonated peptide.
ABSTRACT: High concentrations of supplemented growth factors can cause oversaturation and
adverse effects in in vitro and in vivo studies. Though, these supraphysiological concentrations
are often required due to the low stability of growth factors. Here we demonstrate the
stabilization of TGFꢀβ1 and BMP4 using supramolecular polymers. Inspired by heparan sulfate,
sulfonated peptides were presented on a supramolecular polymer to allow for nonꢀcovalent
binding to growth factors in solution. After mixing with excipient molecules, both TGFꢀβ1 and
BMP4 were shown to have a prolonged halfꢀlife compared to the growth factors free in solution.
Moreover, high cellular response was measured by a luciferase assay, indicating that TGFꢀβ1
remained highly active upon binding to the supramolecular assembly. The results demonstrate
that significant lower concentrations of growth factors can be used when supramolecular
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polymers bearing growth factor binding moieties are implemented. This approach can also be
exploited in hydrogel systems to control growth factor release.
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INTRODUCTION
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Growth factors are involved in many cellular processes such as cell survival, proliferation,
differentiation and migration.1,2 In the extracellular matrix (ECM), sulfated glycosaminoglycans
(GAGs) present on proteoglycans are known to bind various heparan sulfate/heparinꢀbinding
growth factors, that mediate cellular delivery and subsequent intracellular signaling.3 The
negatively charged sulfates and carboxylic acids present on e.g. heparan sulfate interact via
electrostatic interactions with positively charged regions, that are rich in lysine and arginine,
within the growth factor.4 Depending on the growth factor, hydrogen bonding and hydrophobic
effects can have a significant contribution as well.5 Immobilization of growth factors to heparan
sulfate results in protection from degradation and inactivation. Moreover, the local high
concentration as a result of this binding allows for multivalent interactions, contributing to
spatiotemporal presentation. In contrast, for in vitro and in vivo studies, growth factors are
typically administrated in supraphysiological concentrations due to a lack of controlled delivery,
slow diffusion and low stability over time, leading to subꢀoptimal conditions and possibly
adverse effects.6 In addition, growth factors are usually diluted in high concentrations of bovine
serum albumin (BSA) to enhance the halfꢀlife. However, BSA binds to several other components
as well and is known to influence both metabolic and biosynthetic processes in cells.7 Therefore,
there is an increasing need to develop scaffolds capable of binding growth factors, maintaining
their activity, and presenting the growth factors to the cell in a spatiotemporal manner.
In order to improve the efficiency of growth factor presentation compared to soluble
supplementation, growth factors can be immobilized by either physical entrapment in a hydrogel
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system8 or by nonꢀcovalent9 or covalent10 conjugation.11 To elucidate the role of participating
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functional groups in protein binding and to investigate the sustained release of growth factors,
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heparin based systems,12 polymers,13 monosaccharides,14 synthetic peptides,15,
multiꢀ
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component assemblies,17 and molecules18 have been developed, that have different degrees of
sulfates and sulfonates. Moreover synthetic peptides which specifically target certain growth
factors are also established.19 Although successful conjugation strategies have been developed to
covalently attach growth factors to polymers,10 care has to be taken considering the fact that
conformational changes and inactivation are likely to occur. Another consideration to take into
account is that growth factor receptor presentation might be hampered when the binding is too
strong or encapsulation too efficient. Therefore, tuning the number of nonꢀcovalent interactions
might be beneficial to enhance both growth factor stabilization and cell receptor binding.
Supramolecular polymers are proposed to serve as excellent ECM mimics, since their selfꢀ
assembly of monomers, which is driven by nonꢀcovalent interactions, gives rise to fibrous
structures with an inherent dynamic nature closely resembling ECM properties.20 Due to their
tunability, functional monomers can be simply coꢀassembled with scaffolding monomers to
arrive at multiꢀcomponent functional biomaterials. Supramolecular polymers based on peptide
amphiphiles have previously been shown to bind the growth factor TGFꢀβ1 by a short peptide
sequence (HSNGLPL)21 and several other growth factors by a sulfated monosaccharide.14
Inspired by heparan sulfate, which is able to bind different growth factors by distinct binding
affinities,5 the short synthetic tetrapeptide discovered by Maynard and Hubbell22 also serves as
an attractive candidate. This peptide displays the three functional groups of heparan sulfate and
heparin, i.e. sulfates, carboxylates and hydroxyl groups, and can be easily synthesized and
incorporated into artificial systems. Moreover, they showed the importance of a carboxylate
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between the two sulfated tyrosines compared to a solely negatively charged peptide on the
binding to vascular endothelial growth factor (VEGF). In addition, Kim and Kiick investigated
the influence of more sulfated tyrosines included in this peptide, and confirmed that this shorter
peptide has the highest binding affinity to VEGF and heparin binding peptides due to a reduced
steric hindrance and/or repulsion upon binding.23
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Rather than investigating the sustained release of growth factors from hydrogels, we here
sought to elucidate the stabilization effects of excipient molecules in the diluted state using selfꢀ
assembled ureidopyrimidinone (UPy) based supramolecular polymers. The selfꢀcomplementary
UPy selfꢀassembles into fibrillar 1D fibers by first dimerization due to quadruple hydrogen
bonding and subsequent lateral stacking guided by both hydrophobic effects and hydrogen
bonding between the urea groups.24 In order to equip the supramolecular polymers with growth
factor binding sites, UPy polymers were coꢀassembled with a sulfonated peptide to prolong the
stability of transforming growth factorꢀβ1 (TGFꢀβ1) and bone morphogenic proteinꢀ4 (BMP4).
The stability of both growth factors was assessed at 37 °C with different scaffolds (i.e.
excipients) in solution. Upon incorporation of sulfonated peptides in a supramolecular fiber, nonꢀ
covalent protein interactions enhance growth factor stability over physical adsorption. As a
proofꢀofꢀprinciple, cell experiments were carried out with TGFꢀβ1 sensitive cells to assess
whether the stabilized TGFꢀβ1 was still able to induce a cellular response, i.e. the conversion of
active TGFꢀβ1 into luciferase.
EXPERIMENTAL SECTION
Materials. Chemicals and reagents were purchased from Sigma and Novabiochem, and used
as received unless otherwise indicated. (S)ꢀFmocꢀphenylalanineꢀ4ꢀsulfonic acid was purchased
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from PepTech Corporation. Peptides and conjugates were purified on a C18 automated column
with a gradient of 5 to 100% acetonitrile in water using a Buchi Reveleris system. Recombinant
human TGFꢀβ1 (HEK293 derived) was purchased from Peprotech, and Recombinant human
BMPꢀ4 (carrier free) from R&D systems. Enzymeꢀlinked immunosorbent assays (ELISA) were
performed using human TGFꢀβ1 Quantikine ELISA kits or human BMPꢀ4 Quantikine ELISA
kits from R&D systems. Luciferase assay kit was purchased from Promega. Phosphate buffered
saline (PBS) tablets were purchased from SigmaꢀAldrich (pH 7.20ꢀ7.60). TrypsinꢀEDTA
solution was purchased from Sigma (0.5 g/L porcine trypsin and 0.2 g/L EDTA in Hank’s
Balanced Salt Solution with phenol red).
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Methods. Analytical Techniques. Reversedꢀphase high performance liquid chromatography–
mass spectrometry (RPꢀHPLCꢀMS) was performed using a Shimadzu instrument. The amount of
active TGFꢀβ1 bound to the ELISA kit was assessed by measuring the absorbance at 450 nm,
with a wavelength correction set to 570 nm, using a microplate reader from Safire II. Ultravioletꢀ
visible (UVꢀvis) spectrophotometry was performed on a Jasco Vꢀ650 instrument. The relative
amount of luciferase expressed by cells was evaluated by measuring the luminescence intensity
using a microplate reader from Synergy HT.
Reconstitution of TGFꢀβ1 and BMP4. TGFꢀβ1 and BMP4 were reconstituted according to
manufacturer’s protocol. Briefly, TGFꢀ β1 was dissolved in 10 mM citric acid, whereas BMP4
was dissolved in 4 mM HCl. After 30 minutes, the growth factors were aliquoted in 0.1% BSA in
PBS, or PBS (BSA free) at a 5 µg/ml concentration and stored at ꢀ20 °C before usage.
Synthesis of sulfated tyrosine for SP1. FmocꢀTyr(tBu)ꢀOH (4.35 mmole) was deprotected in a
cleavage cocktail containing TFA, TIS and water (95:2.5:2.5) for 2 hours at room temperature.
Subsequently, the TFA was partly evaporated, precipitated in cold hexane/ether (1:1), incubated
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for 15 min at ꢀ20 °C and centrifuged for 10 minutes at 20k RPM. The supernatant was removed
and the pellet was redissolved in water/acetonitrile, and after lyophilization a white solid was
obtained in 91.1% yield (1.60 g). LCꢀMS: Mwcalc = 403.43 g/mol, m/zobs = 404.08 [M+H]+.
Subsequently, FmocꢀTyr(OH)ꢀOH (6.63 mmole) was dissolved in DMF and stirred under argon
atmosphere for 10 minutes before sulfur trioxide pyridine complex (19.88 mmole, 3 eq) was
added. The reaction was stirred for 2 hours under argon atmosphere, followed by cooling to 0 °C
and the slow addition of cold saturated sodium bicarbonate (100 ml, 0 °C) under vigorously
stirring the solution. Subsequently, tetrabutylammonium hydrogensulfate (9.94 mmole, 1.5 eq)
was added and the pH was lowered to pH 5/6 by the slow addition of cold 0.1 M citric acid (150
ml, 0 °C). The solution was extracted 3 times with chloroform, and the organic layer was
evaporated. The product was redissolved in water/acetonitrile pH 8 (by addition of 1M NaOH)
and lyophilized twice to obtain a white solid in 82.9 % yield (4.0 g). LCꢀMS: Mwcalc = 482.48
g/mol (FmocꢀY(SO3)ꢀOH), Mwcalc = 242.47 g/mol (N+Bu4), m/zobs = 242.42 [NBu4]+, 482.58
[FmocꢀY(SO3)ꢀOH]ꢀ.
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Synthesis of sulfated serine for SP2. FmocꢀSer(tBu)ꢀOH (13.08 mmole) was deprotected in a
cleavage cocktail containing TFA, TIS and water (95:2.5:2.5) for 4 hours at room temperature.
Subsequently, the TFA was evaporated and the residue was redissolved in water/acetonitrile and
lyophilized. A yellow oil was obtained in 116% yield (4.96 g) with traces of solvents. LCꢀMS:
Mwcalc = 327.33 g/mol, m/zobs = 328.00 [M+H]+. Subsequently, FmocꢀSer(OH)ꢀOH (9.17
mmole) was dissolved in DMF and stirred under argon atmosphere for 15 minutes before sulfur
trioxide pyridine complex (27.50 mmole, 3 eq) was added. The reaction was stirred for 3 hours
under argon atmosphere, followed by cooling to 0 °C and the slow addition of cold saturated
sodium bicarbonate (60 ml, 0 °C) under vigorously stirring the solution. Subsequently,
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tetrabutylammonium hydrogensulfate (13.75 mmole, 1.5 eq) was added and the pH was lowered
to pH 5/6 by the slow addition of cold 0.1 M citric acid (10 ml, 0 °C). The solution was extracted
3 times with chloroform, and the organic layer was evaporated. The product was redissolved in
water/acetonitrile pH 8 (by addition of 1 M NaOH) and lyophilized twice to obtain an oil in 109
% yield (6.5 g) with traces of solvent. LCꢀMS: Mwcalc = 406.39 g/mol (FmocꢀS(SO3)ꢀOH),
Mwcalc = 242.47 g/mol (N+Bu4), m/zobs = 242.33 [NBu4]+, 406.33 [FmocꢀY(SO3)ꢀOH]ꢀ.
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ꢀ
Synthesis of peptide derivatives UF, SP1, SP2 and SP3. GSYDYG (UF), GSY(OSO3
ꢀ
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ꢀ
)DY(OSO3 )G (SP1), GSS(OSO3 )DS(OSO3 )G (SP2), and GSF(SO3H)DF(SO3H)G (SP3)
peptides were manually prepared using Solid Phase Peptide Synthesis (SPPS), Fmoc chemistry
and Rink amide MBHA resin. 250 ꢁmol resin was allowed to swell in NMP for 1 hour.
Subsequently, the resin was washed with NMP (6x) and deprotected twice with a 20% piperidine
solution in NMP for 5 minutes. The impurities were washed away with NMP (6x) and a cocktail
of the desired amino acid was prepared of 200 mM amino acid, 1600 mM DIPEA, and 0.4 M
HBTU in NMP (2:1:1, 5:2.5:2.5 ml). The cocktail was added to the resin and coupled for 30 min
at room temperature. This cycle of washingꢀdeprotectionꢀwashingꢀcoupling was repeated for
every amino acid. After the last amino acid was coupled, the remaining Fmoc protecting group
was removed and the resin was washed extensively with NMP and DCM, followed by drying.
Cleavage of the resin and amino acid protecting groups was performed in TFA/H2O/TIS
(94:2.5:2.5) for 2 hours on ice (except for SP3 and UF, which was 4 hours at room temperature).
Subsequently, the peptides were precipitated in 20% cold hexane/diethyl ether, incubated for 15
minutes at ꢀ20 °C, and centrifuged for 10 minutes at 20k RPM. The supernatant was removed
and the pellet was redissolved in water and lyophilized. The peptides (except UF) were purified
using RP column chromatography using a gradient of 5 – 100 % acetonitrile in water yielding the
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peptides in about 10% yield (98% yield for UF). SP1 and SP2 were defined as having about 1.5
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sulfates present based on LCꢀMS interpretation. LCꢀMS: MWcalc UF = 659.65 g/mol, m/zobs UF
=
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660.42 [M+H]+, MWcalc SP1 = 817.65 g/mol, m/zobs SP1 = 818.25 [MꢀH]ꢀ, 408.75 [Mꢀ2H]ꢀ, and
738.33 [MꢀSO3]ꢀ, MWcalc SP2 = 665.56 g/mol, m/zobs SP2 = 666.33 [MꢀH]ꢀ, and 586.50 [MꢀSO3]ꢀ,
MWcalc SP3 = 787.77 g/mol, m/zobs SP3 = 786.67 [MꢀH]ꢀ, and 393.00 [Mꢀ2H]2ꢀ.
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Synthesis of the supramolecular peptide conjugate UPyꢀSP3. The UPyꢀCOOH precursor
molecule was synthesized as previously reported.25 The peptide resin (31.0 ꢁmole) was allowed
to swell for 1 hour in DMF. Meanwhile, the reaction mixture containing UPyꢀCOOH (60 mg,
52.7 ꢁmol, 1.7 eq), HATU (43.4 ꢁmol, 1.4 eq), DIPEA (93.0 ꢁmol, 3 eq) and 4 ml of DMF was
preꢀactivated for 30 minutes. Subsequently, the preꢀactivated reaction mixture was added to the
peptide resin and agitated overnight at room temperature. Afterwards, the resin was washed with
DMF (6x) and DCM (6x) and cleaved from the resin using a cocktail mixture containing
TFA/TIS/H2O (94:2.5:2.5) for 4 hours at room temperature. The UPyꢀpeptide was precipitated
in cold 50% hexane/ether, incubated for 15 minutes at ꢀ20 °C, and centrifuged for 10 minutes at
20k RPM. The supernatant was removed, the pellet was redissolved in 10% acetonitrile/water
and the solvent was lyophilized. Purification with RP column chromatography using a gradient
of 5ꢀ100% acetonitrile in water yielded in pure UPyꢀSP3 (10.1 mg, 17.1% yield). LCꢀMS:
MWcalc = 1908.14 g/mol, m/zobs = 954.4 [Mꢀ2H]2ꢀ.
ELISA experiments. Samples were dissolved in PBS in the right concentration. Because of the
ꢀ
undefined state of both SP1 and SP2, a molecular weight of 738.70 (1*OSO3 ) and 626.03 g/mol
ꢀ
(1.5*OSO3 ) was used, respectively. The UPy samples were prepared by heating the solid
dissolved in PBS to 72 °C for 1 hour, and UPyꢀSP3 was incorporated by an additional incubation
step of 15 minutes at 45 °C. Annealing overnight at room temperature resulted in selfꢀassembled
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polymers with UV patterns similar to reference spectra.26 A stock of every condition was
prepared by the addition of TGFꢀβ1 (0.8 µl for 1 ml to obtain a 4 ng/ml stock, or 0.5 µl for 1 ml
to obtain a 2.5 ng/ml stock). For each condition, 100 µl was pipetted into a 96 wells plate (in
duplicate or triplicate for each time point), incubated at 37 °C with 5% CO2, removed at
indicated time points and stored at ꢀ20 °C until the ELISA experiment was conducted. The
ELISA experiments were performed following the manufacturer’s protocol. Briefly, standard or
sample was incubated for 2 hours, washed 4 times with wash buffer, incubated with growth
factor specific conjugate for 2 hours, washed 4 times with wash buffer, and incubated for 30
minutes with substrate solution. Then the reaction was quenched with stop solution. The optical
density was measured at 450 nm with a wavelength correction set to 570 nm.
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Cell Culture. Human fibrosarcoma cells (HT1080, kindly provided by MarieꢀJosé Goumans),
transfected with (CAGA)9 MLPꢀluc using FuGENE6,27 were cultured in DMEM (Gibco)
supplemented with 1% penicillin/streptomycin (Lonza), 10% fetal bovine serum (Bovogen) and
1% nonꢀessential amino acids. Cells were routinely cultured at 37 °C and 5% CO2. Medium was
changed every 2ꢀ3 days and passaged at 80ꢀ90% confluency. This cell line expresses luciferase
upon exposure to extracellular TGFꢀβ1 which is regulated via Smad3/Smad4 signaling and
binding to the CAGA box located in the plasminogen activator inhibitorꢀ1 (PAIꢀ1) gene.
Luciferase Assay. HT1080 cells were seeded at a density of 50,000 cell/cm2 in a 12 wells plate
and allowed to adhere overnight at 37 °C and 5% CO2. The excipient molecules were dissolved
in DMEM (containing 1% pen/strep) in the correct concentration. The UPy samples were
prepared by heating the solution to 72 °C for 1 hour, and UPyꢀSP3 was incorporated by an
additional incubation step of 15 minutes at 45 °C. Annealing overnight at room temperature
resulted in selfꢀassembled polymers with similar UV patterns. The next day medium was
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replaced by serumꢀfree DMEM supplemented with 1% pen/strep and incubated for 7 hours. A
stock of every condition was prepared by the addition of TGFꢀβ1 (0.8 µl for 1 ml to obtain a 4
ng/ml stock, or 0.5 µl for 1 ml to obtain a 2.5 ng/ml stock). Four hours before cell seeding, 1.2
ml of the stock solutions was pipetted (in triplicate) in a plain 12 wells plate and incubated at 37
°C with 5% CO2 to yield into the 4 hours preꢀincubated samples. Subsequently, either 1 ml fresh
stock was added onto the cells or 1 ml of the 4 hours preꢀincubated samples (in triplicate). Cells
were then incubated an additional 19 hours, lysed and the luciferase expression was measured (in
duplicate of the triplicate) according to the manufacturer’s protocol (Promega). Relative
luciferase intensities were normalized for the DNA content using the CyQUANT cell
proliferation assay (Invitrogen).
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RESULTS AND DISCUSSION
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Selection of the peptide derivative. The stability of the growth factor TGFꢀβ1 was studied
over time and heparin peptide mimics were selected to assess their binding. TGFꢀβ1 is known to
modulate behavior of many cell types, including immune cells. Furthermore, altered signaling is
associated with several disorders, such as cancer and fibrosis.28, 29 TGFꢀβ1 is synthesized as an
inactive precursor, which is activated upon cleavage from the ECM by proteases followed by
acidic conditions to remove the latent TGFꢀβ binding protein.30 We assessed the stability of
activated TGFꢀβ1 over time at 37 °C with an enzymeꢀlinked immunosorbent assay (ELISA)
revealing full degradation within 5 hours (figure 1A, PBS condition, halfꢀlife of about 0.4 hours).
Then four peptide derivatives, based on the tetrapeptide of Maynard and Hubbell (here named
SP1)20, were synthesized and assessed for their ability to stabilize TGFꢀβ1 (figure 1). In this
small library, unfunctionalized peptide (UF) was included to investigate the absence of the
sulfate groups. Sulfated peptide 2 (SP2) lacks aromatic rings hence hydrophobic effects as
compared to sulfated peptide 1 (SP1), and finally, since sulfate groups are labile, a more stable
sulfonated peptide (SP3) was included.
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The four peptides were synthesized with manual solid phase peptide synthesis using sulfated or
sulfonated amino acids. Due to the acid sensitive sulfate groups,31 both SP1 and SP2 were
difficult to obtain in a fully defined state (about 1.5 sulfate groups instead of 2 were obtained).
The purified excipient peptides were mixed with TGFꢀβ1 and incubated for different time points
(figure 1A and ESI, fig. S1). In the ELISA, UF and SP2 showed a low relative absorbance
whereas SP1 and SP3 showed a more enhanced optical density over time. The lower stabilization
effect of SP1 might be due to the labile sulfate group and/or the lessꢀdefined state of only 1ꢀ1.5
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sulfate groups per molecule instead of the maximum of 2, and the presence of more salt as
counter ions yielding a slightly lower concentration as intended. Peptide SP2 and the UF peptide
showed similar behavior highlighting the importance of both a sulfate or sulfonate and an
aromatic ring for TGFꢀβ1 binding. All peptides showed a significant enhancement of the halfꢀlife
of TGFꢀβ1, of which SP3 was shown to have the most pronounced stabilization potential of the
peptides studied. Moreover, increasing or decreasing the concentration of the excipient peptides
has an enhanced or reduced effect, respectively (ESI, fig. S2). In addition, it was shown that SP1
at 10 µM has a higher stabilizing effect as compared to heparin. This introductory study
indicated that a combination of hydrophobic effects, hydrogen bonding and electrostatic effects
contribute to growth factor binding and is concentration dependent.
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Figure 1. Peptide derivatives were assessed on their stabilization behavior on TGFꢀβ1. A) The
optical density of TGFꢀβ1 after incubation with different peptide derivatives as measured with an
ELISA assay. Sulfonated peptide SP3 showed the highest stabilizing effect (cTGFꢀβ1 ≈ 4 ng/ml,
cpeptide = 100 µM, cBSA ≈ 0.8 µg/ml, N=2, error bars indicate standard deviation). B) Molecular
structures of the peptide derivatives assuming a pH of 7.4.
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Implementation into the supramolecular platform. Subsequently, the sulfonated peptide
SP3 was integrated in a UPyꢀbased supramolecular system to investigate increased local
concentration on the stabilization of TGFꢀβ1. It was previously shown that a subtle change in the
design and coꢀassembly of the supramolecular polymer system has a profound effect on the
internal dynamics of the system.26 Moreover, the size of the hydration shell of the ethylene
glycol might have an effect on growth factor stabilization as well. Therefore, three different UPy
scaffolding molecules (UPyꢀOMe, UPyꢀ10kꢀUPy and UPyꢀ20kꢀUPy, figure 3B) were used. SP3
was coupled onto a UPy precursor molecule to allow incorporation in the scaffolding molecules
by coꢀassembly (figure 2). Different percentages of UPyꢀSP3 conjugate were coꢀassembled into
monovalent UPy stacks and the stacking behavior was investigated using UVꢀvis spectroscopy
(ESI, fig. S3). Since the absorbance of the peptide overlapped with the absorption spectrum of
the UPy, bare SP3 at different concentrations was measured and subtracted from the
corresponding coꢀassembled spectra. The spectra obtained after subtraction overlapped with each
other, thus indicating the successful incorporation, though increased intensity at 270 nm for
100% UPyꢀSP3 was observed. This is probably due to steric and repulsive effects of the peptides
generating smaller and less defined aggregates at a 100% functionalization. As a result, a coꢀ
assembly percentage of 25 mol% was chosen to include in a TGFꢀβ1 stabilization assay. The
three bare UPy scaffolding molecules (i.e. without UPyꢀSP3) were also included to investigate
the effect of physical adsorption on TGFꢀβ1 stabilization.
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Figure 2. Synthesis of the UPyꢀSP3 conjugate. The UPy precursor was coupled onto the peptide
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resin, followed by the cleavage of the protecting groups and the resin (green).
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Investigation of TGFꢀβ1 stabilization. TGFꢀβ1 was mixed with preꢀassembled UPy samples
with different compositions and incubated for different time points (figure 3, and ESI, fig. S4).
The concentration of the functional epitope was kept constant (cSP3=cUPyꢀSP3=100 µM) resulting
in a total UPy concentration of 400 µM. All studies were performed in solution, however, the
UPyꢀ10kꢀUPy excipient formed a thin viscous layer on the bottom of the well plate indicative of
minor gel formation at a 400 µM concentration. For the bare UPy scaffolds (UPyꢀOMe, UPyꢀ
10kꢀUPy and UPyꢀ20kꢀUPy) a slightly higher relative absorbance over time was observed as
compared to the reference SP3, probably due to the 4x higher concentration. Upon incorporating
25 mol% of UPyꢀSP3 in the different scaffolding monomers, a synergistic effect was observed,
with the highest stabilizing effect of UPyꢀ10kꢀUPy scaffold with 25 mol% UPyꢀSP3
incorporated. The same trend was observed at lower concentration TGFꢀβ1 (2.5 ng/ml) mixed
with 1.6 fold lower UPy concentration (ESI, fig. S5).
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The binding of TGFꢀβ1 to the excipient molecules was proposed to occur via electrostatic
interactions to binding sites at the interface between the TGFꢀβ1 dimers rather than wrapping
around the growth factor based on previous studies32,33 (figure 3C). In contrast, binding to nonꢀ
functionalized supramolecular polymers might be due to nonꢀspecific PEG binding and/or
hydrophobic interactions. Importantly, growth factor binding has a minor influence on the
stacking behavior as shown with UVꢀvis spectroscopy (ESI, fig. S6). Moreover, it was proposed
that due to the temporal nonꢀcovalent interactions between the excipient and growth factor,
dissociation of the growth factor might result in presentation to the cell receptor (important for in
vitro assays) or prone to conformational changes due to its free occurrence in solution and/or
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adherence to the plastic environment leading to denaturation. Nevertheless, the results obtained
here indicate that the halfꢀlife of TGFꢀβ1 was significantly enhanced by binding to the UPy
fibers without changing the fiber organization.
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Figure 3. The effect of supramolecular polymers on TGFꢀβ1 stabilization. A) The optical density
of TGFꢀβ1 after incubation with different supramolecular excipients as measured with an ELISA
assay. Physical entanglement of TGFꢀβ1 with bare UPy fibers showed a similar stabilizing effect
as compared to SP3, whereas the incorporation of UPyꢀSP3 in supramolecular fibers enhanced
the stabilization significantly (cTGFꢀβ1 ≈ 4 ng/ml, cpeptide = 100 µM, cUPy = 400 µM, c25%UPyꢀSP3 in
= 100 µM, cBSA ≈ 0.8 µg/ml, N=2, error bars indicate standard deviation). B) Molecular
UPy
structures of the excipient molecules. C) Cartoon representing the proposed binding of TGFꢀβ1
(orange) to supramolecular polymers (blue) coꢀassembled with sulfonated peptides (red).
Investigation of BMP4 stabilization. In order to investigate whether the excipient molecules
are also applicable to other growth factors, the stabilization of bone morphogenic proteinꢀ4
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(BMP4) was evaluated (figure 4, and ESI, fig. S7 and S8). BMP4 plays an important role in
cardiac development during the embryonic stage and it is known to be upregulated during
impaired remodeling of the adult heart.34,35 Despite being a member of the transforming growth
factor beta superfamily with similarities in secondary structure, binding of BMP4 to heparan
sulfate might occur on different positions compared to TGFꢀβ1.36 Therefore excipient binding
and stabilization of BMP4 might be altered. In addition to investigation of the stabilization of
BMP4, also the influence of BSA was evaluated, since supraphysiological amounts of BSA are
typically added to enhance growth factor stability. All previous experiments were carried out in
the presence of BSA. Here, we investigate the stability of a growth factor both in the presence
and in the absence of BSA. In both cases, with or without BSA added, growth factor binding by
monovalent UPyꢀOMe functionalized with 25 mol% UPyꢀSP3 showed a synergistic effect as
compared to the nonꢀfunctionalized supramolecular polymer (UPyꢀOMe) and reference SP3,
which remained relatively constant over a few hours (figure 4A and 4B, respectively).
Interestingly, when BSA was added, the relative absorbance of BMP4 after 1 hour was slightly
higher than the 0 hour sample, probably due to experimental error and/or conformational changes
of the UPy at 37 °C leading to more optimal binding hence stabilization. Without BSA, BMP4
has a low stability, with about 6 fold lower initial relative absorbance as compared to BMP4
containing BSA (cBSA ≈ 0.5 µg/ml, figure 4B). This lower concentration could be explained by
the acid treatment necessary to activate BMP4, which most likely degraded BMP4 much more in
the absence of BSA. As a result, BSA has a strong and short initial stabilization effect which
quickly diminishes after an hour. In contrast, upon addition of the supramolecular platform after
activation, the percentage active BMP4 was better retained. Therefore, the stability of BMP4
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might be further enhanced when the supramolecular platform would be added during growth
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factor activation, i.e. acid treatment.
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Figure 4. Supramolecular polymers also stabilize BMP4. A) with BSA (+BSA) and B) without
BSA (ꢀBSA). Without BSA, BMP4 has a low stability, however, when mixed with
supramolecular polymers and BSA, the activity of BMP4 was enhanced for several hours. (cBMP4
≈ 2.5 ng/ml, cpeptide = 62.5 µM, cUPy = 250 µM, cBSA ≈ 0.5 µg/ml, error bars indicate standard
deviation, N=2).
Cellular readꢀout of active TGFꢀβ1. To investigate, as a proofꢀofꢀprinciple, whether the
stabilized TGFꢀβ1 with excipient molecules is still able to induce cellular responses, HT1080
fibrosarcoma cells were exposed to different conditions. These cells have a luciferase reporter,
which can convert active TGFꢀβ1 into luciferase.27 The converted amount of luciferase is directly
proportional to the amount of active TGFꢀβ1. Moreover, the luciferase activity corresponds to
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the initial concentration of TGFꢀβ1 rather than the concentration during incubation with the cells
(ESI, fig. S9 and S10). For this reason, excipient molecules mixed with TGFꢀβ1 were preꢀ
incubated for 4 hours prior to cell exposure (2.5 ng/ml TGFꢀβ1, 62.5 µM SP3, and 250 µM UPy,
figure 5, and ESI, fig. S11 and S12). As expected, a high luciferase activity was observed when
the cells were exposed to fresh TGFꢀβ1 (i.e. TGFꢀβ1 addition directly after thawing) and the
activity decreased when TGFꢀβ1 was preꢀincubated before addition to cells (figure 5A). Both
SP3 and UPyꢀ10kꢀUPy showed the highest luciferase activity. However, ELISA clearly
confirmed the presence of more active TGFꢀβ1 when mixed with UPy excipients (UPyꢀ10kꢀUPy
+ 25% UPyꢀSP3 and 100% UPyꢀSP3, figure 5B). This might be due to the poly(ethylene glycol)
surrounding the fibers, which can partly shield the TGFꢀβ1 presentation towards cells owing to
their hydration shell.37 Although the 100% UPyꢀSP3 has the same concentration as the reference
experiment with SP3, which should increase the local concentration, a lower cellular response
was observed in contradiction to the ELISA results. Due to multivalent effects, the binding
affinity to the excipient might be increased, lowering receptor binding availability. Moreover, a
thin layer of gel was observed on the bottom of the well plate (during preꢀincubation) for the
UPy samples, which might contain local higher growth factor concentration. Though, this layer
was not transferred onto the cells, therefore probably some TGFꢀβ1 was lost during this step.
Interestingly, heparin even showed a lower cellular response than PBS, in corroboration with the
ELISA results. The reason that heparin has no stabilizing effect might be due to a subꢀoptimal
sulfateꢀpattern present, failing to support TGFꢀβ1 binding. This observation is supported by a
study of Gallagher et al. where heparan sulfate originating from porcine mucosa was proved to
have low binding affinity.33
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Taking the results all together, a strong cellular response was observed in all cases. However,
coꢀassembling the sulfonated peptide in the supramolecular platform (UPyꢀ10kꢀUPy + 25%
UPyꢀSP3) did not increase the luciferase activity as compared to the bare supramolecular
polymer (UPyꢀ10kꢀUPy) when TGFꢀβ1 was preꢀincubated for 4 hours, though, more active
TGFꢀβ1 was detected with ELISA. Probably the concentration plays an important role, which is
in this case close to receptor saturation levels (ESI, fig. S9), and also the preꢀincubation time
could be extended in order to induce more differences between the conditions. Further extensive
studies are necessary to find the optimal concentration and incubation time for these cell
experiments.
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Figure 5. A) TGFꢀβ1 response after cell incubation in the form of luciferase and B) the
corresponding optical density of TGFꢀβ1 as measured with ELISA. In contrast to the cell
experiments, the UPy samples have a slightly higher TGFꢀβ1 optical density as compared to SP3
indicating that the PEG shell decreases TGFꢀβ1 receptor presentation. (cTGFꢀβ1 ≈ 2.5 ng/ml, cpeptide
= 62.5 µM, cUPy = 250 µM, cBSA ≈ 0.5 µg/ml, error bars indicate standard deviation, N=3 (A),
N=2 (B), A) data was corrected for the DNA content).
CONCLUSION
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Materials based on supramolecular polymers are proposed to be ideal platforms for mimicking
the natural extracellular matrix due to their dynamic, responsive and adaptable properties.
Supramolecular polymers based on peptide amphiphiles functionalized with bioactive cues were
already proven to be successful in binding growth factors. Here, we introduced a sulfonated
peptide coꢀassembled in ureidopyrimidinoneꢀbased supramolecular polymer platforms for the
stabilization of the growth factor TGFꢀβ1. Different excipient molecules were shown to prolong
the halfꢀlife of the growth factor, and proofꢀofꢀprinciple cell experiments confirmed high cellular
response in the form of luciferase, indicating the maintenance of TGFꢀβ1 activity upon excipient
binding. Although there is a slight difference in cellular readꢀout and the corresponding ELISA
experiments – maybe due to steric effects of the PEG preventing optimal cell presentation – all
results show a significant stabilization in all experimental systems. Moreover, it was shown that
the excipient molecules can also be used for the stabilization of other heparan sulfate binding
proteins, as was revealed with BMP4. This approach can be used to extend the activity of
important growth factors, but can also be implemented in hydrogel systems. In hydrogel systems
the sustained release can be controlled by the binding strength to the protein and by tuning the
physical properties of the hydrogel. Gaining more control over growth factor concentrations is
beneficial in biomedical applications, e.g. in directing specific processes during different stages
of tissue regeneration.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website at
DOI: xxx. LCMS spectra of the molecules, additional ELISA results, UVꢀvis experiments, and
statistical analysis (PDF).
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: E.W.meijer@tue.nl, P.Y.W.Dankers@tue.nl
Author Contributions
The study was designed and the results were obtained by S.I.S.H and S.S. The experiments were
proposed and results were discussed by all authors. The manuscript was written through
contributions of all authors. All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was funded by the NWO/DPI program NEWPOL (project #731.015.503), partially
financed with TKI (Topconsortia for Knowledge Innovation) allowance provided by the Dutch
Ministry of Economic Affairs. EWM acknowledges the Ministry of Education, Culture and
Science (Gravity program FMS, 024.001.035). PYWD acknowledges the European Research
Council (FP7/2007ꢀ2013) ERC Grant Agreement 308045. We also acknowledge the support
from the Netherlands Cardiovascular Research Initiative (CVON 2012ꢀ01), the Dutch Heart
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Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for
Health Research and Development, and the Royal Netherlands Academy of Sciences. The
authors thank Prof. MarieꢀJosé Goumans (Leiden University Medical Center, the Netherlands)
for providing the HT1080 cells.
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