Rational Design of Short Peptide-Based Hydrogels with MMP-2

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Rational Design of Short Peptide-Based Hydrogels with MMP-2 Responsiveness for Controlled Anticancer Peptide Delivery Cuixia Chen, Yu Zhang, Zhe Hou, Xuejing Cui, Yurong Zhao, and Hai Xu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00911 • Publication Date (Web): 22 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017

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Rational Design of Short Peptide-Based Hydrogels with MMP-2 Responsiveness for Controlled Anticancer Peptide Delivery Cuixia Chen,* Yu Zhang, Zhe Hou, Xuejing Cui, Yurong Zhao, and Hai Xu* State Key Laboratory of Heavy Oil Processing and Center for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China

ACS Paragon Plus Environment

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ABSTRACT

Molecular self-assembly makes it feasible to harness the structures and properties of advanced materials via initial molecular design. To develop short peptide-based hydrogels with stimuli-responsiveness, we here designed short amphiphilic peptides by engineering protease cleavage site motifs into self-assembling peptide sequences. We demonstrated that the designed Ac-I3SLKG-NH2 and Ac-I3SLGK-NH2 self-assembled into fibrillar hydrogels, and the Ac-I3SLKG-NH2 hydrogel showed degradation in response to MMP-2 whereas the Ac-I3SLGK-NH2 hydrogel did not. The cleavage of Ac-I3SLKG-NH2 into Ac-I3S and LKG-NH2 was found to be mechanistically responsible for the enzymatic degradation. Finally, when an anticancer peptide G(IIKK)3I-NH2 (G3) was entrapped into Ac-I3SLKG-NH2 hydrogels, its release was revealed to occur in a “cell-demanded” way in the presence of HeLa cells that over-express MMP-2, therefore leading to a marked inhibitory effect on their growth on the gels.


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INTRODUCTION Molecular self-assembly is of particular significance from the perspective of fundamental and applied sciences. For practical applications, the main advantage of molecular self-assembly is the feasibility of harnessing the structures and properties of sophisticated materials via simple molecular engineering. Due to the biological origin and well-defined nanostructures, synthetic materials formed by peptide and protein self-assembly have received tremendous attention over the past decades. In particular, self-assembling peptide hydrogels have been widely exploited in biomedical applications such as tissue engineering, regenerative medicine, and drug delivery, which is the account of their structural resemblance to the natural extracellular matrix (ECM) (e.g. nanofiber networks and microporous structures), unique properties (e.g. extremely high water content and rapid recovery after thinning), and ease of chemical and biological decoration and functionalization (e.g. by side chain and terminus modification and backbone incorporation).1-7 For example, a nanofiber scaffold from the RADA-16 self-assembly could provide a permissive environment for axon to regenerate and even knit the brain tissue together.8 For another example, self-assembling hydrogels of β-hairpin peptides can be used as injectable agents for local curcumin delivery.9 Towards promoting cell-matrix interactions and guiding cell behaviors, incorporating additional bioactive signals into peptide hydrogels is an ideal strategy. For example, upon incorporation of functional motifs (e.g. RGD-based moieties, laminin derived sequences, and bone marrow homing peptides) into the RADA-16 peptide, the self-assembling nanofiber scaffolds have been successfully used for adult mouse neural stem cell 3-dimensional cultures,10 and more recently for brain tissue engineering.11 In order to endow peptide hydrogels with


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controlled degradation features, matrix metalloproteinase (MMPs) sensitive substrates have been incorporated into self-assembling peptide sequences.12-14 It is well known that MMPs play a crucial role in defining cellular behaviors through degradation and remodeling of the ECM as well as regulated release of growth factors and other signals from the ECM.15-17 As a result, these above peptide hydrogels showed susceptibility to MMPs, thus leading to enhanced cell migration and spreading.12-14 To avoid or alleviate the detrimental effect of additional sequences on peptide self-assembly and gel formation, the parent peptides to be biologically engineered generally contain residues of more than 15 such as ionic complementary peptides,8,10,11 multidomain peptides,13,18 and β-hairpin peptides.9,14 For practical applications, short peptide length always favors large-sale and low-cost production. Furthermore, short peptides are much more stable and robust and their structure-function relationship is readily established, relative to long polypeptides and large proteins.19,20 For these reasons, the development of short peptide sequences containing both strong gelling ability and bioactive motifs remains a challenge. Isoleucine is a hydrophobic amino acid with strong propensity for β-sheet structuring.21 By taking advantage of the intrinsic features of isoleucine, we have designed an ultrashort surfactant-like peptide Ac-I3K-NH2 that showed high self-assembling ability in aqueous solution through the formation of long and stable nanofibers.22,23 Furthermore, the insertion of a functional cysteine (C) or glutamine (Q) residue at the interface of hydrophobic/hydrophilic residues not only produced little impact on the self-assembling ability of the peptides but also endowed them with stimuli-responsiveness.24,25 For example, Ac-I3QGK-NH2 underwent a marked sol-gel transition in the presence of transglutaminase and was thus employed for rapid hemostasis.25 Based on Ac-I3K-NH2, together with the structural feature of MMP cleavage motifs, we here design a short peptide


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Ac-I3SLKG-NH2. We demonstrate that the peptide can self-assemble into fibrillar hydrogels in aqueous solution, which subsequently show a quick recovery property after thinning. Furthermore, the self-assembled nanofibers and the entangled network can be significantly disrupted upon treatment with MMP-2, leading to a gradual reduction in the mechanical properties of Ac-I3SLKG-NH2 hydrogels, thus suggesting a susceptibility to enzymatic cleavage. Finally, when an anticancer peptide is physically entrapped into the hydrogel, its release occurs in a “cell-demanded” fashion in the presence of HeLa cells and the growth of the cancer cells can be inhibited significantly. 2. MATERIALS AND METHODS 2.1. Materials The materials used in peptide synthesis, including Rink amide-MBHA resin, protected amino acids, deprotection, coupling, and cleavage reagents, acetic anhydride, and solvents, were purchased from GL Biochem Ltd. (Shanghai, China) and Bo Maijie Technology (Beijing, China).

Calcein

acetoxymethyl

ester

(calcein-AM),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and MMP-2 were obtained

from

Sigma

(St.

Louis,

MO).

Piperidine,

dichloromethane

(DCM),

and

N,N′-dimethylformamide (DMF) were redistilled prior to use and other materials were used as received. Water used in all experiments was processed through a Millipore Milli-Q system, with a resistivity of 18.2 MΩ·cm. Mouse embryonic fibroblast NIH 3T3 cell and human cervical cancer HeLa cells were obtained from the Cell Bank in Shanghai Institute of Cell Biology. Cells were cultured in Iscove’s modified Dubelcco’s medium (IMDM) with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2.


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2.2. Peptides Synthesis and Hydrogel Preparation Peptides used in the present study were synthesized on a CEM Liberty microwave peptide synthesizer. The detailed procedures for the peptide synthesis and purification have been described in our previous work.22 The final products were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and reversed phase high-performance liquid chromatography (RP-HPLC) analyses, indicating their correct sequences and high purity (>98%). The synthesized peptides were dissolved in 25 mM Hepes (pH 7.4) at a concentration of 8 mM. Peptide solutions were first sonicated for ~30 min and then transferred into a water bath. After incubation for 2 h at 80 oC, they were taken out and further incubated for 24 h at room temperature. Self-supporting hydrogels were observed to rapidly form upon attaining room temperature. 2.3. Peptide Hydrogel Degradation The peptide hydrogels prepared as above showed shear-thinning and quick recovery properties (see below). For degradation assays, 1 mL of peptide gel was mixed by pipette with 10 µL of MMP-2 (human, recombinant, CHO cells), which was activated in a testing buffer (25 mM Hepes, pH 7.5, 40 µM ZnSO4, and 20 mM CaCl2), giving rise to a final MMP-2 concentration of 100 ng/mL in the gel. After incubation at 37 oC for the indicated times, the peptide hydrogel was subjected to instrumental characterizations to determine its degradation behaviors. 2.4. Atomic Force Microscopy (AFM) and Transmission Electron Microscopy (TEM) AFM measurements were performed on a commercial Nanoscope IVa MultiMode AFM (Digital Instruments, Santa Barbara, CA) in tapping mode, with silica wafers as the substrate. The silica surface was cleaned with 5% neutral Decon90 solution and then rinsed copiously with


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water prior to use. 10 µL of peptide hydrogel was pipetted onto the silica surface, immediately followed by addition of 140 µL water. After adsorption for ~30 s, excess solution was removed and the substrate surface was dried by purging with N2. AFM height images (512 × 512 pixel) were acquired at a scan rate of 1 Hz and a scan angle of 0°. TEM micrographs were obtained on a JEOL JEM-1400 electron microscope operated at an accelerating voltage of 120 kV. For negative staining TEM measurements, a drop of peptide hydrogel was pipetted on a small piece of Parafilm, and a 300-mesh copper grid was then placed on top of the gel. After contact for ~ 5 s, the copper grid was negatively stained with 2% uranyl acetate aqueous solution for 8 min, followed by removal of excess solution with filter paper. For cryogenic TEM (cryo-TEM) measurements, samples were prepared in a controlled environment vitrification system (CEVS).26,27 Peptide gel (~20 µL) was pipetted onto the TEM copper grid coated with a laced support film and after adsorption for ~8 s, the gel was wicked away with filter paper, leaving a thin film suspending on the mesh holes. The film was quickly plunged into a reservoir of liquid ethane at -165 oC. The vitrified sample was stored in liquid nitrogen, followed by transferring it to a sample holder (Gatan 626) for TEM imaging at approximately -174 oC. 2.5. Circular Dichroism (CD) Spectroscopy CD spectra were recorded on a Biologic Mos-450/AF-CD spectrophotometer by using a thin quartz cell (optical path length: 0.05 mm). The wavelength scans were performed from 250 to 190 nm with a 0.05 nm step. The presented CD signals were the average of six individual measurements and expressed as [θ] (deg·cm2·dmol-1). 2.6. ThT Binding Assay


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ThT stock solution (5 mM ThT in 25 mM Hepes) was mixed with hydrogel at a volume ratio of 1:100, followed by gently vortexing for 5 min. Then, the ThT fluorescence emission spectra from 460 to 550 nm were collected in a 2 mm quartz cuvette at room temperature on a Horiba Jobin Yvon FluoroMax-4 spectrofluorometer. The excitation wavelength was set at 442 nm, with the excitation and emission slits being 2 nm. 2.7. Rheology Rheological properties of peptide hydrogels were analyzed at 25 °C on a Thermo Scientific Haake MARS III modular rheometer operating in a cone-plate mode (cone angle: 2.0°, diameter: 34.995 mm, truncation: 0.105 mm). Dynamic strain sweeps from 0.01% to 100% strain at 1 Hz were first conducted to determine the linear viscoelastic regime.25,28 Then, dynamic frequency sweeps from 0.01 to 10 Hz were performed at 1% strain. For gel shear-thinning and recovery measurements, after loading gels, a dynamic time sweeping (1 HZ and 1% strain) was first taken for 30 min, and 1000% strain was then applied for 2 min, followed by an instant strain reduction to 1% and another dynamic time sweeping at 1 Hz for 30 min. Such a cycle was repeated three times. 2.8. MALDI-TOF MS Mass measurements were conducted on a Bruker Mircoflex MALDI-TOF mass spectrometer. After incubation for 15 days in the presence or absence of MMP-2, peptide gels were subjected to

lyophilization,

followed

by

completely

dissolving

lyophilized

powders

in

hexafluoroisopropanol (HFIP). After removal of HFIP by evaporation at room temperature, a certain amount of 30% acetonitrile/water was introduced, giving rise to the peptide solutions with an 8-fold dilution. For example, the peptide concentration of the peptide hydrogel without MMP-2 was decreased from 8 to 1 mM after such a treatment. The treatment was also performed


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in the sample preparation for HPLC analyses, as indicated below. The objective was to eliminate the possible adverse effect on MS and HPLC results of aggregated oligomers or degradation products with low solubility. Then, the newly prepared peptide solution was mixed with a saturated matrix (4-hydroxy-α-cyanocinnamic acid) solution in 30% acetonitrile/water at an equal volume ratio. Finally, 1 µL of the mixture was dropped on a polished steel sample plate and air-dried, immediately followed by mass measurements. The instrumental conditions for the mass measurement have been previously given.28 Each of the MS spectra presented was the integration of 512 individual measurements. 2.9. RP-HPLC To assess the degradation extent of peptide hydrogels induced by MMP-2, they were subject to RP-HPLC analyses on a Waters 2695 Alliance HPLC system equipped with an XBridge BEH C18 column (4.6 mm × 150 mm, 5 µm). Samples for HPLC measurements were prepared using the same procedure as for the above mass characterizations. The instrumental parameters were the same as described in our previous study.25,28 According to the area reduction of the substrate peak, the degradation ratio of peptide hydrogels was calculated with time and expressed as ( − )/ , where and are the substrate peak area in the absence and

presence of MMP-2, respectively. 2.10. Encapsulation of G(IIKK)3I-NH2 in Peptide Hydrogels and Release An anticancer peptide G(IIKK)3I-NH2 (denoted by G3) was used as the drug for delivery experiments. In order to monitor the release of G3, it was first labelled with FITC, thus giving rise to the conjugate FITC-G(IIKK)3I-NH2 (FITC-G3). Then, FITC-G3 was accurately weighed and dissolved in 1 mL of 25 mM Hepes (pH 7.4) with Ac-I3SLKG-NH2 or Ac-I3SLGK-NH2, and the resulting peptide solutions had a FITC-G3 and Ac-I3SLKG-NH2 or Ac-I3SLGK-NH2


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concentration of 50 µM and 8 mM, respectively. After sonication for ~30 min and incubation for 2 h in a water bath (80 oC) in the dark, the peptide solutions were immediately transferred into the wells of a 24-well tissue culture polystyrene (TCPS) plate, followed by further incubation for 24 h at room temperature. For the delivery experiments in response to MMP-2, the enzyme was mixed with the peptide gels by pipette after the above 24-h incubation at room temperature. Finally, 1 mL of 25 mM Hepes (pH 7.4) was added on the gel surface. After incubation at 37oC for the indicated time, the buffer was removed to determine the FITC-G3 concentration by fluorescence measurements. Once the buffer was removed out, 1 mL of fresh Hepes buffer was immediately added into the wells. The standard curve of FITC-G3 in 25 mM Hepes (pH 7.4) and its concentration in the released samples were determined by using a Horiba Jobin Yvon FluoroMax-4 Spectrofluorometer. The emission fluorescence intensity at 514 nm (emission slit=2 nm) was recorded under an excitation at 490 nm (excitation slit=2 nm). The release ratio of FITC-G3 from the peptide hydrogels was calculated based on the followed equation: Release ratio =

∑

where ci denotes the FITC-G3 concentration in the ith supernatant removed from the wells and Vi is its volume, being 1 mL in our experiments; c0 denotes the initial concentration (50 µM) of FITC-G3 in the gels and V0 is the gel volume (1 mL) in the wells. 2.11. MTT Assay and Fluorescence Microscopy According to the above procedure, the Ac-I3SLGK-NH2 and Ac-I3SLKG-NH2 hydrogels (100 µL) were prepared in the wells of a 48-well TCPS plate. Then, NIH 3T3 cells (5 × 105 cells/mL, 150 µL) were introduced into the wells with and without the gels, respectively, and incubated in IMDM with 10% fetal bovine serum (FBS) at 37 °C under 5% CO2. The cell culture medium


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was refreshed every 24 h. After incubation for 4 and 7 days, 30 µL of MTT (5 mg/mL) was added to each well, followed by further incubation for 4 h at 37 °C. Finally, the supernatant was removed and the precipitated formazan was dissolved with 200 µL of dimethyl sulfoxide (DMSO), followed by measuring the absorbance at 490 nm (A490) on a microplate autoreader (Molecular Devices, M2e). The wells in the absence of cells acted as the blank for those with cells during the A490 measurement. The cell cytotoxicity was expressed as the proliferation ratio, which was calculated based on the following equation: Proliferation ratio =

(!"#, %&')

where A490 (TCPS, 4 days) denotes the absorbance at 490 nm of formazan for the cells seeded in the TCPS wells after incubation for 4 days and acted as the control. After the G3‒loaded hydrogels (100 µL) were prepared in the wells of a 48-well TCPS plate, HeLa or NIH 3T3 cells (5 × 105 cells/mL, 150 µL) were seeded on the gel surface. After incubation for 24 h, the cell viability was determined by the MTT assay as described above and expressed as the survival ratio, which was calculated based on the following equation: Survival ratio =

(+,- .!.,/ 0.)

where A490 (NIH 3T3, no G3) denotes the absorbance at 490 nm of formazan for the NIH 3T3cells on the gel without G3. The morphology of NIH 3T3 and HeLa cells on the gel surface were observed using fluorescence microscopy. After incubation for 24 h, the seeded cells were washed with IMDM for 3 times and stained with 100 µL of calcein-AM (1 µM) for 30 min in the dark at 37 oC. The unbound dye was discarded by washing with PBS for at least 3 times, followed by observation of the cells using a fluorescence microscope (DMI3000, Leica).


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3. RESULTS AND DISCUSSION 3.1. Peptide Design As indicated above, the ultrashort I3K may be an ideal parent peptide for incorporation of additional sequences, due to its strong self-assembling ability. For a protease, the cleavage site motif includes residues both N- and C-terminal to the scissile bond, denoted as …P3-P2-P1-P1'-P2'-P3'…, with cleavage occurring between the P1 and P1' residues.15 MMP-2, also known as gelatinase A or type IV collagenase, plays an important role in angiogenesis and tumor cell invasion, in addition to degrading and remodeling the ECM.17,29-31 Thus, the enzyme is frequently upregulated in human cancer cells such as HeLa cells.32 For MMP-2, there is a strong preference for serine (S) at P1 and leucine (L) at P1', respectively, and hydrophobic amino acids such as isoleucine (I) and valine (V) on the N-terminal side of the scissile bond (S-L) are thought to enhance the cleavage site specificity.13,15,16 Furthermore, MMP-2 generally requires basic amino acids (e.g. arginine (R) and lysine (K)) at P2' and small residues (e.g. glycine (G) and alanine (A)) at P3', respectively.13,15 Taken together, we design a short amphiphilic peptide Ac-IIISLKG-NH2 (Ac-I3SLKG-NH2), with the expected cleavage site (SL) being at the interface of the hydrophobic III and hydrophilic KG segments (Figure 1b). The three consecutive isoleucine residues not only provide hydrophobic interactions and β-sheet hydrogen bonding for self-assembly but also increase the substrate specificity. Besides acting as the hydrophilic head, the specific role of KG will be demonstrated by the design of an isomeric Ac-I3SLGK-NH2, in which lysine and glycine at the C-terminal are swapped (Figure 1a). Additionally, because the expected cleavage site is at the hydrophobic-hydrophilic interface, it is most unlikely to be deeply embedded inside the formed nanostructures upon self-assembly in aqueous solutions, thus being readily assessable to enzymes.33


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3.2. Self-Assembled Nanostructure and Hydrogels To promote the formation of well-defined nanostructures and gelation, the peptide solutions (8 mM in 25 mM Hepes) were first incubated in 80oC water bath for 2 h and then cooled to room temperature. During the cooling process, self-supporting hydrogels were observed to form (inset of Figure 1). After further aging at room temperature for 24 h, the morphology of the peptide assemblies was observed using AFM and TEM.


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Figure 1. Molecular structures and self-assembled nanostructures of the designed peptides: (a-c) Ac-I3SLGK-NH2 and (d-e) Ac-I3SLKG-NH2. (a,d) Molecular structures, (b,e) cryo-TEM images, and (c,f) height AFM images. Insets of (b) and (c) indicate the formation of self-supporting peptide hydrogels (Rhodamine B was introduced to aid visualization under UV light). The peptide concentration was fixed at 8 mM in 25 mM Hepes (pH 7.4). As shown in Figures 1c and 1f, AFM imaging showed that both Ac-I3SLGK-NH2 and Ac-I3SLKG-NH2 self-assembled into nanofibers with diameters of ~7 nm and lengths up to several micrometers at a concentration of 8 mM, indicating little influence of the swapping of two hydrophilic residues K and G at the C-terminal on their self-assembly. Cryo-TEM imaging, in which the nanostructures of interest in liquid solution can be well preserved through cryogenic vitrification, also verified the formation of long and thin nanofibers from the two peptides (Figures 1b and 1e). Furthermore, these nanofibers were heavily entangled, thus causing the hydrogelation of the two peptide solutions. The physical properties of the peptide hydrogels were further assessed by oscillatory rheology. Their storage moduli (G') remained around 1000 Pa in a dynamic frequency sweep from 0.01 to 10 Hz at 1% strain and always an order of magnitude larger than their loss moduli (G'') (Figures S1a and S1b), characteristic of rigid and solid-like gels with a significantly cross-linked network. Moreover, the two hydrogels displayed excellent shear-thinning and quick recovery properties (Figures S1c and S1d). When application of 1000% strain, they underwent a gel-sol transition and became fluid as a liquid, with its G' being around 10 Pa and even less than G''. Upon cessation of shear, i.e. lowering oscillatory strain from 1000% to 1%, the two gels rapidly went back to their initial mechanical states. Additionally, the two hydrogels were very stable at room temperature over 1 month with little change in their nanostructures and rheological properties.


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2.3. Enzymatic Responsiveness As described by Galler et al., mixing MMP-2 with peptide hydrogels can increase its contact with the substrate, therefore resulting in a more rapid degradation, relative to what was observed when the enzyme was only placed on top of gels.13 Therefore, we mixed MMP-2 with peptide hydrogels by pipette and monitored their bulk degradation behaviors. After incubation with MMP-2 for 15 days, G' remained almost unchanged for the Ac-I3SLGK-NH2 hydrogel whereas G' of the Ac-I3SLKG-NH2 hydrogel changed remarkably from ~1000 Pa to ~100 Pa (Figure 2a), indicating its susceptibility to MMP-2. Note that the rheological properties of the Ac-I3SLKG-NH2 hydrogel were little changed after incubation for 15 days in the absence of MMP-2.


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Figure 2. (a) Storage moduli (G') and (b) CD spectra of peptide hydrogels in the presence and absence of MMP-2. (c) Fluorescence emission spectra (excitation at 442 nm) of ~0.05 mM ThT in Hepes and in the presence of peptide hydrogels. Peptides were dissolved in 25 mM Hepes (pH 7.4) at a concentration of 8 mM to form hydrogels. After incubation at 37 oC for 15 days in the absence and presence of MMP-2, the hydrogels were subjected to rheology, CD, and ThT binding measurements at 25 oC or room temperature. Then, secondary structures were followed using CD in the presence and absence of MMP-2. As shown in Figure 2b, the two peptide hydrogels in the absence of MMP-2 exhibited well defined negative peaks around ~218 nm, characteristic of β-sheet conformations. Upon mixing with MMP-2, there was little variation in the CD signals of Ac-I3SLGK-NH2 hydrogels, even after incubation for 15 days. For the Ac-I3SLKG-NH2 gel in the presence of MMP-2, however, the CD spectra changed significantly, with the characteristic peak of ~218 nm decreasing markedly in intensity after enzymatic incubation for 15 days. Because β-sheet hydrogen bonding has been widely demonstrated to drive the axial growth of peptide assemblies,23,26,34 the degeneration of β-sheet secondary structures may cause the disruption of self-assembled one-dimensional nanofibers and their network, eventually resulting in a major decrease in mechanical strength. This can be exemplified by the Ac-I3SLKG-NH2 hydrogel in the presence of MMP-2 (Figures 2a and 2b). Upon mixing with the two peptide hydrogels, ThT showed marked increases in fluorescence emission intensity at ~482 nm (Figure 2c), well consistent with the formation of amyloid-like nanofibers with β-sheet confomations.35,36 In the case of Ac-I3SLGK-NH2, the ThT fluorescence enhancement remained unchanged in the presence of MMP-2 (Figure 2c). For Ac-I3SLKG-NH2 hydrogels, however, such an enhanced effect of fluorescence emission decreased considerably in


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the presence of MMP-2, again indicating the degeneration of β-sheet secondary structures as well as the most likely dissociation of Ac-I3SLKG-NH2 nanofiber network. Consistent with these variations in physical properties and secondary structures, TEM and AFM imaging showed that the Ac-I3SLKG-NH2 nanofibers and entangled network were destroyed seriously in the presence of MMP-2. As shown in Figure 3, short nanofibers rather than long and entangled ones were found to dictate after the Ac-I3SLKG-NH2 gel was incubated with MMP-2 for 15 days. In contrast, the Ac-I3SLKG-NH2 nanofibers and network remained intact after incubation of the Ac-I3SLKG-NH2 hydrogel with MMP-2 for 15 days.

Figure 3. Negative-staining TEM and AFM images of the Ac-I3SLKG-NH2 hydrogel after incubation with MMP-2 for 15 days.


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For the two peptide hydrogels, the above alterations induced by MMP-2 in their physical properties, secondary structures, and supramolecular nanostructures demonstrated a strict specificity of MMP-2 to substrate sequence. To explore the molecular mechanism underpinning the specific susceptibility, we then performed MALDI-TOF MS and RP-HPLC measurements. As shown in Figure 4a, there was a HPLC peak at ~17.4 min for the Ac-I3SLKG-NH2 hydrogel, and its mass spectrum displayed three peaks at m/z of 785.2, 807.2, and 821.1, which are assigned to the ion adducts of Ac-I3SLKG-NH2 ([M+H+], [M+Na+], and [M+K+], respectively). Upon addition of MMP-2, two new HPLC peaks appeared at ~16.1 and ~19.0 min in addition to the one at ~17.4 min (Figure 4b), suggesting the possible formation of two new compounds. The compound of ~16.1 min must be more hydrophobic than the one of ~19.0 min. Furthermore, the corresponding mass spectrum indicated the occurrence of two new peaks at m/z of 487.6 and 316.4, in addition to the three peaks of Ac-I3SLKG-NH2 (Figure 4b). Because the theoretical molecular weights of Ac-I3S and LKG-NH2 are 486.6 and 315.4, the two new mass peaks are most likely to belong to Ac-I3S and LKG-NH2, respectively. The degradation of Ac-I3SLKG-NH2 at the expected cleavage site S-L is anticipated to lead to the formation of Ac-I3S and LKG-NH2. Additionally, we synthesized Ac-I3S and LKG-NH2 and then recorded their HPLC profiles under the exact same RP-HPLC conditions. As shown in Figure 4c and 4d, Ac-I3S and LKG-NH2 showed a peak at ~16.1 and 19.0 min, respectively, and the former was more hydrophobic than the latter. Taken together, we think that cleavage of Ac-I3SLKG-NH2 by MMP-2 at the desired cleavage site (S-L) is mechanistically responsible for the enzymatic degradation of Ac-I3SLKG-NH2 hydrogels. However, HPLC measurements on Ac-I3SLGK-NH2 hydrogels indicated that in the presence of MMP-2, no peak was observed except for the one of Ac-I3SLGK-NH2 at ~17.4 min and the peak area of Ac-I3SLGK-NH2 remained little changed, in


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comparison with that in the absence of MMP-2 (Figure S2). It is evident from the HPLC and MS results that the combination of basic amino acid lysine at P2' and small amino acid glycine at P3' is crucial to the specificity of MMP-2 to cleavage motif, and upon swapping the two C-terminal residues, it appeared that the cleavage motif was completely deactivated.

Figure 4. RP-HPLC profiles and MALDI-TOF mass spectra of Ac-I3SLKG-NH2 in the (a) absence and (b) presence of MMP-2. The peptide was dissolved in 25 mM Hepes (pH 7.4) at a concentration of 8 mM to attain hydrogels. After incubation at 37 oC for 15 days in the absence and presence of MMP-2, HPLC and MS measurements were performed. (c and d) RP-HPLC profiles of Ac-I3S and LKG-NH2.


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As shown in Figure 5, the degradation ratio of Ac-I3SLKG-NH2 hydrogels increased with incubation time, broadly in a linear mode. After incubation for 15 days, approximately 60% of Ac-I3SLKG-NH2 was degraded whereas no degradation occurred for Ac-I3SLGK-NH2. The enzymatic responsiveness and degradation mode of Ac-I3SLKG-NH2 hydrogels endow them with great potential as a scaffold for the controllable release of drugs in therapeutics and regenerative medicine.

Figure 5. Degradation ratio of peptide hydrogels in the presence of MMP-2 over 15-day incubation. The degradation ratio was derived from HPLC measurements under the identical measuring conditions, expressed as ( − are the )/ , where and

substrate peak area in the absence and presence of MMP-2, respectively. 2.4. Cytotoxicity For biological applications such as drug delivery and regenerative medicine, ideal peptide hydrogels should have excellent biocompatibility. We first used the MTT assay to assess the cytotoxicity of Ac-I3SLGK-NH2 and Ac-I3SLKG-NH2 hydrogels against NIH 3T3 cells, with the


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results shown in Figure 6a. After incubation for 4 days, the proliferation of NIH 3T3 fibroblasts seeded on the gels was comparable to that of NIH 3T3 fibroblasts seeded on bare TCPS plates. With further increasing the incubation time to 7 days, a higher level of cell growth and proliferation was observed for the cells on the peptide gels and TCPS plates. Then, the morphology of the cells on the two peptide gels was observed using inverted fluorescence microscopy. As shown in Figure 6b, nearly all of the cells were well stained with calcein-AM after incubation for 24 h. Note that the dye is widely used to stain living cells and excites green fluorescence. Furthermore, most of the cells spread well on the peptide gels. These results suggest that the two peptide hydrogels have low cytotoxicity. 2.5. Release of Anticancer Peptide G3 Mediated by MMP-2 Anticancer peptides (ACPs) have been regarded as a potential source of new anticancer agents because of their broad anticancer activity, low cytotoxicity, and low probability of inducing resistance.37,38 When ACPs are administered systemically, however, their poor proteolytic stability significantly decrease their efficacy and thus hinder their clinical applications.39-41 To circumvent the critical issue, encapsulation of liable drugs by smart materials and their controlled delivery represent a powerful strategy.42-46 Self-assembling peptide hydrogels have been widely exploited for the encapsulation and delivery of drugs, proteins, and even cells, not only due to their inherent biocompatibility but also the ease of engineering responsiveness into such delivery systems.3-7,9,18 As indicated above, the Ac-I3SLKG-NH2 hydrogels not only showed low cytotoxicity but also were engineered with MMP-2 responsiveness, implying their potential as drug delivery vehicles.


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Figure 6. a) Cytotoxicity of the Ac-I3SLKG-NH2 and Ac-I3SLGK-NH2 hydrogels against NIH 3T3 cells, which was determined by the MTT assay and expressed as the proliferation ratio. The cells seeded in the TCPS wells after incubation for 4 days acted as the control. b) Fluorescence images of NIH 3T3 cells on the two peptide hydrogels. After incubation for 24 h on the gels, the cells were stained with calcein-AM for imaging. c) G3 (G(IIKK)3-NH2) release from the G3‒ loaded Ac-I3SLKG-NH2 and Ac-I3SLGK-NH2 gels in the presence and absence of MMP-2. d) Viability of NIH 3T3 and HeLa cells on the two peptide gels with and without G3 loading. After incubation for 24 h, the cell viability was determined by the MTT assay.


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The designed helical peptide G3 has been demonstrated to kill HeLa cells efficiently in vitro and inhibit HeLa tumor growth in human cervical carcinoma xenografts by intraperitoneal injection in vivo whilst exhibiting low cytotoxicity against normal cells.47 Furthermore, the anticancer peptide adopted random coil conformation in aqueous solutions, thus unlikely producing major impact on the self-assembly and gelation of Ac-I3SLKG-NH2 and Ac-I3SLGK-NH2. As a result, the FITC-labelled G3 (FITC-G3) was encapsulated into peptide hydrogels for our controlled release study. As shown in Figure 6c, after incubation for 15 days at 37 oC, less than 10% of entrapped FITC-G3 was released from the two peptide hydrogels in the absence of MMP-2, indicating that the diffusion-controlled FITC-G3 release was negligible for the two gels. The result further suggests that the two peptide gels have a high-density nanofiber network, thus making their pore sizes either be comparable to or smaller than the molecular dimensions of the drug.48 In the presence of MMP-2, the release ratio of FITC-G3 from the Ac-I3SLGK-NH2 gel was still less than 10% whereas that from the Ac-I3SLKG-NH2 gel displayed a completely different profile (Figure 6c). For example, ~25% release was attained after incubation for 7 days and with further incubation for 15 days, ~70% release was achieved. Such a releasing trend of ACPs from the Ac-I3SLKG-NH2 gel is reminiscent of the gel’s degradation profile in the presence of MMP-2 (Figure 5), suggesting a degradation-controlled release of ACPs from the gel by MMP-2. Note that such a bulk-degradation behavior should be closely coupled to molecule diffusion.48. Additionally, the negligible release from the control peptide gel again suggests the sequence specificity of MMP-2. In fact, MMP-2 has been found to be over-expressed by human cervical cancer cell lines such as HeLa cells.32 Thus, we cultured HeLa cells and NIH 3T3 fibroblasts on the surface of G3‒ loaded hydrogels and the cell viability was followed using the MTT assay. After incubation for


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24 h, the survival ratio of HeLa cells was comparable to that of NIH 3T3 cells on the Ac-I3SLKG-NH2 hydrogel without G3 (Figure 6d) and both were close to 1.0, which was consistent with the low cytotoxicity of the peptide gel. On the G3‒loaded Ac-I3SLKG-NH2 hydrogel, the survival ratio of HeLa cells was greatly reduced to ~0.2 after incubation for 24 h whereas that of NIH 3T3 was still close to 1.0 (Figure 6d). Because HeLa cells can secrete more MMP-2, the inhibitory effect on HeLa cells was ascribed to the degradation of the G3‒loaded Ac-I3SLKG-NH2 hydrogel by MMP-2 and the consequent release of active G3 molecules. On the G3‒loaded Ac-I3SLGK-NH2 hydrogel, however, the survival ratio of HeLa cells declined slightly relative to that of NIH 3T3 after incubation for 24 h. It is evident that the Ac-I3SLKG-NH2 gel could sense the signal caused by the disease and the entrapped agent G3 could be released from the gel in a “cell-demanded” way. 4. CONCLUSIONS Through engineering protease cleavage site motifs into self-assembling peptide sequences, we designed short amphiphilic peptides Ac-I3SLKG-NH2 and Ac-I3SLGK-NH2. The two peptides were demonstrated to undergo self-assembly in aqueous solutions to form hydrogels with rapid shear-thinning and recovery properties. Furthermore, the Ac-I3SLKG-NH2 hydrogel showed high enzymatic susceptibility, with secondary structures, nanofiber network, and rheological properties being compromised significantly by MMP-2. MS and HPLC measurements revealed that the cleavage of Ac-I3SLKG-NH2 into the Ac-I3S and LKG-NH2 fragments was mechanistically responsible for the enzymatic degradation behaviors. As a control, the Ac-I3SLGK-NH2 hydrogel did not show any responsiveness to MMP-2. Finally, when an anticancer peptide G3 was encapsulated into the Ac-I3SLKG-NH2 hydrogel, its release could be well controlled by MMP-2. Importantly, the release of G3 from the Ac-I3SLKG-NH2 gel


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followed a “cell-demanded” way in the presence of HeLa cells, which typically over-express MMP-2, therefore leading to a marked inhibitory effect on their growth on the gel surface. Collectively, these specific features of Ac-I3SLKG-NH2 hydrogels make them suitable as smart materials for a variety of biological applications such as controlled drug delivery and regenerative medicine, although a thorough investigation on the gel is highly needed in the near future.

ASSOCIATED CONTENT Supporting Information Figures S1-S2 including the rheological measurements of the peptide gels before addition of MMP-2 and RP-HPLC profile of Ac-I3SLGK-NH2 hydrogels in the presence and absence of MMP-2. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (C.C); [email protected] (H.X) Notes The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China under grant numbers 21373270 and 31271497, and the Fundamental Research Funds for the Central Universities (14CX02189A). REFERENCES (1) Nowak, A. P.; Breedveld, V.; Pakstis, L.; Ozbas, B.; Pine, D. J.; Pochan, D.; Deming, T. J. Rapidly Recovering Hydrogel Scaffolds from Self-Assembling Diblock Copolypeptide Amphiphiles. Nature 2002, 417, 424-428. (2) Collier, J. H.; Rudra, J. S.; Gasiorowski, J. Z.; Jung, J. P. Multi-Component Extracellular Matrices Based on Peptide Self-Assembly. Chem. Soc. Rev. 2010, 39, 3413-3424. (3) Matson, J. B.; Stupp, S. I. Self-Assembling Peptide Scaffolds for Regenerative Medicine. Chem. Commun. 2012, 48, 26-33. (4) Wu, E. C.; Zhang, S.; Hauser, C. A. Self-Assembling Peptides as Cell-Interactive Scaffolds. Adv. Funct. Mater. 2012, 22, 456-468. (5) Jung, J. P.; Gasiorowski, J. Z.; Collier, J. H. Fibrillar Peptide Gels in Biotechnology and Biomedicine. Biopolymers 2010, 94, 49-59.


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(46) Kang, M. K.; Colombo, J. S.; D’Souza, R. N.; Hartgerink, J. D. Sequence Effects of Self-Assembling

Multidomain

Peptide

Hydrogels

on

Encapsulated

SHED

Cells.

Biomacromolecules 2014, 15, 2004-2011. (47) Chen, C.; Hu, J.; Zeng, P.; Pan, F.; Yaseen, M.; Xu, H.; Lu, J. R. Molecular Mechanisms of Anticancer Action and Cell selectivity of Short α-Helical Peptides. Biomaterials 2014, 35, 1552-1561. (48) Lin, C. -C.; Metters, A. T. Hydrogel in Controlled Release Formulations: Network Design and Mathematical Modeling. Adv. Drug Delivery Rev. 2006, 58, 1379-1408.


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Rational Design of Short Peptide-Based Hydrogels with MMP-2

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