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Transformable Nanomaterials as an Artificial Extracellular Matrix for Inhibiting Tumor Invasion and Metastasis Xiao-Xue Hu,†,‡ Ping-Ping He,‡ Guo-Bin Qi,‡ Yu-Juan Gao,‡ Yao-Xin Lin,‡ Chao Yang,‡ Pei-Pei Yang,‡ Hongxun Hao,*,† Lei Wang,*,‡ and Hao Wang*,‡ †

National Engineering Research Center of Industrial Crystallization Technology, State Key Laboratory of Chemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China ‡ CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST) No. 11 Beiyitiao, Zhongguancun, Beijing 100190, China S Supporting Information *

ABSTRACT: Tumor metastasis is one of the big challenges in cancer treatment and is often associated with high patient mortality. Until now, there is an agreement that tumor invasion and metastasis are related to degradation of extracellular matrix (ECM) by enzymes. Inspired by the formation of natural ECM and the in situ self-assembly strategy developed in our group, herein, we in situ constructed an artificial extracellular matrix (AECM) based on transformable Laminin (LN)mimic peptide 1 (BP-KLVFFK-GGDGR-YIGSR) for inhibition of tumor invasion and metastasis. The peptide 1 was composed of three modules including (i) the hydrophobic bis-pyrene (BP) unit for forming and tracing nanoparticles; (ii) the KLVFF peptide motif that was inclined to form and stabilize fibrous structures through intermolecular hydrogen bonds; and (iii) the Y-type RGD-YIGSR motif, derived from LN conserved sequence, served as ligands to bind cancer cell surfaces. The peptide 1 formed nanoparticles (1-NPs) by the rapid precipitation method, owing to strong hydrophobic interactions of BP. Upon intravenous injection, 1-NPs effectively accumulated in the tumor site due to the enhanced permeability and retention (EPR) effect and/or targeting capability of RGD-YIGSR. The accumulated 1-NPs simultaneously transformed into nanofibers (1-NFs) around the solid tumor and further entwined to form AECM upon binding to receptors on the tumor cell surfaces. The AECM stably existed in the primary tumor site over 72 h, which consequently resulted in efficiently inhibiting the lung metastasis in breast and melanoma tumor models. The inhibition rates in two tumor models were 82.3% and 50.0%, respectively. This in vivo self-assembly strategy could be widely utilized to design effective drug-free biomaterials for inhibiting the tumor invasion and metastasis. KEYWORDS: self-assembly, peptide, tumor, metastasis, therapy

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degradation has been confirmed to effectively inhibit tumor invasion and metastasis.14 The other way round, in situ construction of artificial ECM (AECM) in the course of degradation, which conceivably is a promising strategy for inhibition of tumor metastasis, has not been explored yet, probably due to the difficulty in the design of appropriate materials. Naturally, ECM is composed of fibrous proteins and proteoglycans, which are locally secreted from cells and selfassembled into organized mesh.15 Moreover, the self-assembly process can be induced by mechanical forces from in-cell actin

umor metastasis, as one of the major challenges in the clinical management of cancer, is often associated with high patient mortality and is responsible for more than 90% of all cancer-related deaths1−5 because it is difficult to treat surgically or with conventional chemotherapy and radiation therapy.6 Clinical inability to cure tumor metastasis originates from its complexity and multistep process,7 which have been paid extensive effort to study the mechanisms and therapeutic methods.8−11 However, it is commonly believed that tumor invasion and metastasis are related to the degradation of extracellular matrix (ECM) by enzymes, e.g., matrix metalloproteinases (MMPs), which are generally overexpressed in the tumor site.12 The cancer cells start migrating over the barrier of degraded ECM, which allows the cells to pull themselves forward.12,13 Therefore, the inhibition of ECM © 2017 American Chemical Society

Received: February 3, 2017 Accepted: March 23, 2017 Published: March 23, 2017 4086

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Scheme 1. Schematic Illustration of the Biomimic Construction of AECM Based on Transformable 1-NPs for High-Efficient Inhibition of Tumor Invasion and Metastasis

Figure 1. (a) Molecular structure and schematic illustration of peptide 1 and 2. (b) UV−vis and (c) fluorescence spectra characteristics of selfassembly process to form 1-NPs (c = 3.0 × 10−5 M) in mixed H2O/DMSO solutions. A photo of solutions under 365 nm irradiation is shown as an inset in (c).

filament through the binding of protein−ligands to cell-surface receptors, such as RGD with integrins.16 Inspired by the formation of natural ECM, one can mimic the component of ECM to in situ construct AECM as barriers to inhibit migration and metastasis based on the booming in vivo self-assembly strategy (Scheme 1). In our previous studies, we utilized in situ self-assemblied peptides to construct functional biomaterials under specific pathological/physiological conditions.17−21 In this study, Laminin (LN), one of the most important components of ECM,12 which was self-assembled into fibrils with specific peptide sequences (i.e. RGD and YIGSR) for binding to integrins and LN receptors of cancer cells,22−25 acted as a biomimic model for the rational design of AECM building blocks. Herein, we in situ constructed an AECM based on LN-mimic peptide 1 (BP-KLVFFK-GGDGR-YIGSR) for inhibition of tumor invasion and metastasis. The peptide 1 was composed of three modules including (i) the bis-pyrene (BP) unit for fluorescence signals and inducing the formation of nanoparticles, (ii) the KLVFF peptide motif originated from Aβ, as peptide scaffolds for the formation of β-sheet structured fibers,

(iii) the Y-type RGD-YIGSR motif (Figure 1a), derived from LN conserved sequence, served as ligands to bind cancer cell surfaces and subsequently induced structural transformation.22−25 The as-prepared nanoparticles of 1 (1-NPs) at the initial state were intravenously (i.v.) injected into tumorbearing mice and effectively accumulated in the tumor site based on the EPR effect20 and/or targeting capability of RGDYIGSR.26,27 The 1-NPs simultaneously transformed into nanofibers of 1 (1-NFs) around the tumor upon binding to receptors on tumor cell surfaces and further entwined to form AECM. The idea and the biological mechanism of this design was inspired by the self-assembly process of natural ECM induced by ligand−receptor interactions.28 For the purpose of comparison, the nontransformable control peptide 2 (BPKAAGGK-GGDGR-YIGSR) was synthesized by the mutation of a self-assembly motif.29 The structural transformation from 1-NPs to 1-NFs was unambiguously confirmed in solution by transmission electron microscope (TEM) and circular dichroism (CD) studies. Moreover, similar structural changes were also observed by coculture of 1-NPs with cancer cells and multicellular tumor 4087

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Figure 2. (a) DLS and (b) CD of 1 nanoaggregates structural evolution in the absence/presence of Ca2+ in aqueous solutions (c = 3.0 × 10−5 M) in 6 days. (c) TEM images for the corresponding morphologies of 1 nanoaggregates.

self-assembly process was monitored by UV−vis and fluorescence spectral analysis (Figure 1b). The absorption bands centered at 232, 265, and 350 nm, which were ascribed to the peptide backbone, aromatic residues, and BP modules, respectively, decreased and broadened, indicating the formation of peptide 1 aggregates.18 Especially, the decreased absorption at 265 nm indicated the aggregation of aromatic residues of peptide via π−π interactions. Meanwhile, as shown in Figure 1c, a nonfluorescent solution of peptide 1 (3.0 × 10−5 M) in DMSO showed a dramatic fluorescence increase with a maximum at 535 nm upon the addition of water due to the formation of J-type BP aggregation.32,33 The peptide 2 showed the similar aggregation behavior with peptide 1 based on UV− vis absorption and fluorescence studies (Figures S3−S4). Overall, due to the large π-conjugation and strong hydrophobicity of BP motif, peptides 1 and 2 were supposed to form nanoparticles with a hydrophilic peptide motif as an outer layer. To further verify the formation of nanoparticles and determine their sizes, dynamic light scattering (DLS) and TEM experiments were carried out. The spherical structure with a diameter of 26.5 ± 5.5 nm was observed with TEM (Figure 2c), which was in accordance with the radius of 28.2 ± 10.2 nm from DLS (Figure 2a). The peptide 2 also formed nanoparticles in solution with similar diameter (24.4 ± 8.7 nm, Figure S5). The Structural Transformation from 1-NPs into 1-NFs in the Presence of Ca2+ in Solution. The structural transformation of 1-NPs into 1-NFs by RGD ligand−intergrin receptor interactions was stimulated by Ca2+ in the 1-NPs solution because the binding site of RGD to integrins mainly relied on the coordination interactions between RGD and the metal ions (Ca2+, Mg2+) at the “metal ion-dependent adhesion

spheroids (MCTs) by using variable techniques such as confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Importantly, 1-NPs efficiently restrained migration and invasion of highly metastatic cancer cells (breast cancer cells MDA-MB-231 and melanoma cells B16-F10) in vitro, and the percentages of migrated and invasive cells were lowered to 21.4% and 8.6% for MDA-MB-231 and 58.5% and 59.5% for B16-F10, respectively. Moreover, in vivo studies further showed that AECM stably existed in the tumor site for over 72 h, which consequently showed inhibition rates of 82.3% and 50.0% for metastatic breast and melanoma cancers, respectively, in lung metastatic mice models. Besides, the formed 1-NFs also somewhat induced the apoptosis of cancer cells and therefore delayed the progression of primary tumors. Altogether, the in situ constructed biomimic materials showed great potential as an effective drug-free strategy for inhibiting the tumor invasion and metastasis.

RESULTS AND DISCUSSION Preparation of Peptides 1 and 2 and Their SelfAssembled Nanoparticles. The peptide 1 and its control analogue 2 were prepared according to the standard solid-phase peptide synthesis techniques using Fmoc-coupling chemistry (Scheme S1),29 which were finally cleaved from Wang resin beads and purified by reprecipitation for 3 times. The molecular structures of peptides 1 and 2 were confirmed by matrixassisted laser desorption/ionization time-of-flight (MALDITOF) mass spectra (Figures S1−S2).30 The peptide 1 selfassembled into spherical structures by the rapid precipitation method.31 In our case, peptide 1 in DMSO (1.5 mM, 20 μL) was quickly injected into water (980 μL) under stirring. The 4088

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Figure 3. CLSM images of MDA-MB-231 cells incubated with (a) 1-NPs (30 μM) and (c) 2-NPs (30 μM) for 2 h, respectively, and quantitative analysis (b, d). (e) SEM images of cell surfaces of MDA-MB-231 treated with 1-NPs and 2-NPs and untreated MDA-MB-231 as a control. (f) The schematic illustration of ligand−receptor interaction induced structural transformation of 1-NPs to 1-NFs on cell surfaces.

site” (MIDAS).34 The fresh solutions of 1-NPs and 2-NPs (3.0 × 10−5 M, H2O/DMSO = 98:2) were incubated with a CaCl2 aqueous solution (3.0 × 10−5 M), independently. TEM was utilized to observe morphological changes in different solutions over a period of 6 days. Particulate structures for 1-NPs with Ca2+ were shown with a size of 25.8 ± 7.2 nm for the first day. The DLS data (32.4 ± 11.7 nm for 1-NPs with Ca2+) further validated the TEM results. The 1-NPs without Ca2+ were larger than that of 1-NPs with Ca2+, possibly because the addition of metal ions compressed the electrostatic double layer and decreased the diameter of 1-NPs.35 The short fibers (Φ = 10.3 ± 3.2 nm) were observed for 2 days, which were further transformed into long fibrous bundles (1-NFs, Φ = 245.6 ± 180.2 nm) upon incubation with Ca2+ for 6 days, implying that the Ca2+ induced the structural transformation (Figure 2c).36 DLS results (Φ = 11.4 ± 5.6 nm for short fibers, and Φ = 295.0 ± 105.6 nm for fibrous bundles) also verified the sizes of the aggregation obtained from TEM observations. In contrast, the control peptide 2 did not show transformable capability when incubated with Ca2+ ions under the same condition (Figure S6), indicating the hydrogen bonds played an important role in the structural transformation of peptides. CD spectra were further utilized to study the secondary structure of peptides during the structural transformation.21 As shown in Figures 2b and S7, the fresh 1-NPs and 2-NPs with/without Ca2+ showed no obvious signals, indicating that there were no remarkable hydrogen bonds formed in 1-NPs and 2-NPs. It was ascribed that the aggregation of peptides 1 and 2 induced by the BP motif with strong hydrophobic interactions was too fast to form intermolecular hydrogen bonds. The characteristic CD signals of peptides 1 and 2 after 2 days exhibited a positive CD signal at 198 nm and a negative one at 219 nm, which were typical CD features of β-sheet structures.30,31 The results indicated that

the peptides 1 and 2 could gradually form intermolecular hydrogen bonds with an ordered β-sheet structure regardless of the morphology. The 1-NFs formed with Ca2+ showed stronger negative peaks (−6.2 mdeg) than that (−4.2 mdeg) of 1-NPs without Ca2+ in 6 days, probably proving the more highly ordered β-sheet structure formation in fibrous morphologies.18 The above experimental results validated the morphology transformation from 1-NPs to 1-NFs via interactions between the ligand (RGD) and the receptor via stimulation of Ca2+. The other way round, the stability of 1-NPs in blood plasma was investigated by time-dependent TEM observation of 1-NPs (Figure S8). The results displayed that the 1-NPs remained in the particulate structures up to 24 h and transformed into 1NFs at 48 h. The 1-NPs were stable enough for in vitro and in vivo applications. The Structural Transformation from 1-NPs into 1-NFs on Cell Surfaces and MCTs. In order to study the receptorbinding induced transformation of 1-NPs, 1-NPs and 2-NPs (30 μM) were incubated with MDA-MB-231 cells for 2 h and observed by CLSM and SEM. As shown in Figure 3c and d, 2NPs treated MDA-MB-231 cells showed strong green fluorescence intracellularly, implying 2-NPs were internalized into cells, which was usually observed for other nanoparticles when incubated with living cells. In contrast, as shown in Figure 3a and b, 1-NPs treated MDA-MB-231 cells showed green fluorescence on cell surfaces, probably due to the in situ transformation from 1-NPs to 1-NFs and the inhibition of internalization of 1-NPs into cells. The SEM measurements were further carried out to characterize the morphology of 1NPs and 2-NPs treated MDA-MB-231 cells. As shown in Figure 3e, the membrane of an untreated MDA-MB-231 cell exhibited some irregular protrusions. For 1-NPs treated cells, the fibrous network surrounded cells were observed, potentially 4089

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Figure 4. Microscopy images of wound healing (a), migration (c), and invasion (e) and quantitative analysis (b, d, f) of MDA-MB-231 cells. The first group was the control group without any treatment. The second and third groups were incubated with 2-NPs and 1-NPs for 24 h, respectively. Data are given as mean ± SD (n = 3). **p < 0.01, ***p < 0.001.

validating the transformation of 1-NPs to 1-NFs on cell surfaces, which was in accordance with the observation in solution and was induced by ligand−receptor interactions (Figure 3f). The control 2-NPs treated cells showed a similar morphology with untreated cells besides some particulate structures. In order to simulate the tumor tissue, MCTs were prepared in 96-well plates according to standard protocol.37 Agarose (50 μL) was added at the bottom of each well, followed by inoculation of 1600 cells per well, leading to the formation of MCTs after 4 days. 1-NPs and 2-NPs (30 μM, 200 μL) were added and incubated for 24 h and washed away with PBS for 3 times. 1-NP treated MCTs exhibited a green fluorescence covering the surfaces of MCTs as a circle in CLSM images (Figure S9). However, only green spots were scattered on the surfaces of MCTs treated with 2-NPs. The in vitro experiment results on living cells and MCTs indicated that 1-NPs targeted the receptors on cell surfaces and induced structural transformation, which was driven and stabilized by strong hydrogen bonds from KLVFF. In parallel, 1-NPs were incubated with a panel of cell lines including B16-F10 (melanoma cells), HUVECs (endothelial cells), and L929 (normal fibroblasts) to study the specificity of transformation by CLSM and SEM measurements (Figures S10−15). CLSM images showed the green fluorescence on cell surfaces of B16-F10, HUVECs, and L929. However, the 2-NP incubated cells (control groups) showed green fluorescence signals in the cytoplasm of the cell. These results indicated that 1-NPs transformed into 1-NFs induced by ligand−receptor interactions and attached to all cell surfaces, and 2-NPs were internalized into cells without structural transformation. The intensity of green fluorescence showed similar trends for both 1-NP and 2-NP treated cells, i.e. MDA-MB-231 > B16-F10 > HUVECs > L929. The difference of fluorescence intensity

probably revealed that the highly metastatic cancer cells MDAMB-231 and B16-F10 expressed higher integrins and/or LN receptors than HUVECs and L929. The morphology of the MDA-MB-231 and B16-F10 cells became round-like, which were significantly different from their spindle morphology after treatment with 2-NPs. The HUVECs and L929 remained similar in morphology upon incubation with 1-NPs or 2-NPs. The morphology changes of MDA-MB-231 and B16-F10 cells incubated with 1-NPs were probably ascribed to the formation of 1-NFs network, i.e., AECM. The cytotoxicity of four cell lines with different concentrations of 1-NPs and 2-NPs were further carried out, respectively (Figure S16). Basically, the cell viability of 1-NPs treated groups was lower than that of 2-NPs treated groups. Given the fact that peptides 1 and 2 were designed with quite similar molecular structures, it was excluded that the cytotoxicity difference arose from interactions between 1NPs/2-NPs and cells based on the above experiment results. The 1-NPs could form a 1-NF network on cell surfaces as an AECM, affecting the physiological activity of cells. However, the nontransformable 2-NPs were mainly internalized into cells. Furthermore, 1-NPs showed a higher toxicity to MDAMB-231 and B16-F10 than that of HUVECs and L929. The IC50 of 1-NPs to MDA-MB-231 and B16-F10 was lower than 100 μM. The IC50 of 1-NPs to HUVECs and L929 was higher than 100 μM. The results validated that the in situ constructed 1-NF network on cell surfaces could affect the physiological activity of cells. The more 1-NFs on cell surfaces (MDA-MB231 > B16-F10 > HUVECs > L929), which were observed from CLSM and SEM, the lower the cell viability. Aiming at the inhibition effect of tumor invasion and metastasis, the 30 μM of 1-NPs was utilized for the in vitro experiment to eliminate the influence of cytotoxicity. 4090

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Figure 5. Biodistribution of 1-NPs in MDA-MB-231 tumor-bearing mice. (a) In vivo fluorescence images for construction of an AECM by 1NPs and 2-NPs in a tumor and (b) corresponding quantitative analysis in tumor. (c) The concentration of peptide 1 and 2 in blood determined by fluorescence of BP. (d) The fluorescence images and (e) the corresponding quantitative results of ex vivo biodistribution of peptides 1 and 2 in major organs heart, liver, spleen, lung, kidney and tumor 24 h post-administration. *p < 0.05, ***p < 0.001.

Antimigration and Invasion on MDA-MB-231 and B16-F10 Cells Based on 1-NPs in Vitro. To evaluate the inhibition effect of 1-NPs for tumor invasion and metastasis, a series of experiments including wound healing, transwell migration, and invasion assays were performed. The results of the wound healing assay for the inhibitory capability study of 1NPs and 2-NPs on cell motility are shown in Figures 4a and S17. Highly metastatic MDA-MB-231 cells of the control group showed a strong migration healing ability 24 h after scratching, which was defined as the 100% wound healing rate. The 1-NPs treatment decreased the wound healing rate to 32.7%, much lower than that (51.0%) of the 2-NPs treated group (Figure 4b), indicating the inhibitory effect of 1-NPs was stronger than that of 2-NPs. Transwell migration assays were performed to further investigate the inhibition of longitudinal motility ability of MDA-MB-231 cells by 1-NPs. As shown in Figure 4c, inconsistent with the wound healing assay, cells of the control group showed a high migration ability with cells passing through the poly(ethylene terephthalate) (PET) membrane. 1NPs showed a high inhibition on longitudinal motility ability of MDA-MB-231 cells with a coverage rate of 21.4%. The inhibition effect of 2-NPs was evaluated with a coverage rate of 67.8% as the control (Figure 4d). The inhibition capability of 1NPs and 2-NPs originated from the blockage of integrin/LN receptors of cancer cells by RGD/YIGSR ligands. A higher population of 2-NPs was proved to be internalized into the

cells so that the blocking effect and the resultant inhibition of migration were significantly decreased. In a sharp contrast, the supramolecular transformation of 1-NPs into the 1-NFs network (AECM) on cell surfaces as barriers showed more highly efficient blocking of the integrin/LN receptors, leading to a significant inhibition of migration. To simulate the in vivo environment, the invasion assay based on an ECM (Matrigel) coated transwell chamber was carried out. The highly metastatic cells were supposed to degrade the ECM barrier, migrate, and settle in a new location.12 Similarly, both 1-NPs and 2-NPs showed an inhibitory effect of the cell invasion with invasion rates of 8.6% and 45.8%, respectively (Figure 4e and f). Besides MDA-MB-231, 1-NPs also inhibited the migration and invasion of B16-F10 cells dramatically. As shown in Figure S18, the wound healing rate of B16-F10 by 1-NPs was 44.6%. The high inhibition of longitudinal motility and the invasion of B16-F10 cells treated with 1-NPs were observed with a coverage rate of 58.5% and an invasion rate of 59.5% by transwell migration and invasion assays. In contrast, the 2-NPs showed a lower inhibitory effect for migration and invasion of B16-F10 cells. All in vitro experimental results above revealed that transformable 1-NPs showed a much stronger inhibitory effect in tumor invasion and metastasis than that of control 2NPs, indicating that the in situ transformation into 1-NFs to 4091

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Figure 6. In vivo therapeutic effect of transformable 1-NPs in a MDA-MB-231 orthotropic mammary tumor metastasis model and 2-NPs, RGD +YIGSR, PBS treated groups as controls. (a) The schematic tumor implantation and treatment protocol of mice. (b) Photos of original tumors (upper panel) and representative photomicrographs of immuno-histochemical staining analyses for CD31 of the tumor (lower panel) on day 19 after the first administration. (c) The quantitative analysis of MVD from (b). (d) Photos of lungs (upper panel) and H&E stained lung slices (lower panel) on day 19 after the first administration. (e) The quantitative analysis of lungs from (d). The tumor volume (f) and body weight (g) of 1-NPs, 2-NPs, RGD+YIGSR and PBS treated mice. (h) Inhibition rate of lung metastases treated with different concentrations of 1-NPs. Data are given as mean ± SD (n = 4). *p < 0.05 and **p < 0.01.

maximum at 10 h post-injection for 1-NPs treated group. As shown in Figure 5a, the long retention time indicated that the fibrous network (AECM) based on 1-NFs in situ transformed from 1-NPs. In contrast, the nontransformable 2-NPs almost totally cleared from the tumor at 24 h, which was usually observed for organic or polymeric nanoparticles accumulation and excretion in tumor-bearing mice.39 Quantitative analysis (Figure 5b) based on in vivo fluorescence images further validated the remarkably enhanced retention time of in situ converted 1-NFs and resulting fibrous network (AECM). At the same time, the concentration of peptides 1 and 2 in blood was studied by measuring the time-dependent BP fluorescence in mice after i.v. injection of 1-NPs and 2-NPs, respectively. The blood (20 μL) was collected from the tail veil of a mouse,

construct AECM was critical to the inhibition capability of highly metastatic cells. The Evaluation of AECM Construction Based on 1-NPs in Vivo. The construction of AECM based on in situ transformed 1-NFs was investigated in a MDA-MB-231 orthotropic mammary tumor metastasis model, which was monitored by a fluorescence imaging in vivo imaging system to identify the frequency of administration of 1-NPs for the inhibition of tumor invasion and metastasis (Figure 5a).38 The tumor metastasis mice were randomly divided into three groups and were treated with 200 μL of 1-NPs (100 μM), 2-NPs (100 μM), and PBS through i.v. injection when the tumor volume reached 100 mm3. The in vivo fluorescence imaging results showed green fluorescence in the tumor site over 72 h with a 4092

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ACS Nano and the blood plasma (40 μL) was further separated by centrifugation upon the addition of anticoagulation (40 μL). The resultant blood plasma was diluted with 120 μL PBS for fluorescence measurements. As shown in Figure 5c, the peak concentration of 1-NPs in blood appeared at 26 h postadministration and then slowly decreased to the background at 72 h. In parallel, the 2-NPs showed the highest concentration in blood at 10 h and dramatically decreased to zero at 28 h. The longer circulation time of 1-NPs than that of 2-NPs in blood may imply a higher accumulation and long-term retention in the tumor of 1-NPs, which was in accordance with results obtained by the in vivo fluorescence imaging. In order to further confirm the targeting accumulation and long-term retention of 1-NPs, the tumor bearing mice were sacrificed 24 h post-i.v. injection, and the major organs such as heart, liver, spleen, lung, and kidney and tumor were harvested for ex vivo fluorescence imaging. As shown in Figure 5d and e, the heart, liver, spleen, lung, and kidney showed weak fluorescence except for the tumor, indicating this strategy achieved the tumor preferred organ distribution. Moreover, the 1-NPs showed a 4-fold higher fluorescence in tumors than the 2-NP treated group, probably indicating that transformed 1-NFs had a longer retention time than the nontransformable 2-NPs, since both nanoparticles had similar sizes and surfaces for tumor targeting. Finally, the 1-NPs for in vivo antimetastasis experiment were i.v. injected into mice every 72 h in the following experiment. The Evaluation of Antimetastasis Effect Based on 1NPs in Vivo. To evaluate the antimetastasis effect, the 1-NPs were injected into a MDA-MB-231 orthotropic mammary tumor metastasis model via i.v. administration at 72 h, and 2NPs, RGD+YIGSR, and PBS were utilized as controls. The schematic of tumor implantation and treatment protocol of mice was shown in Figure 6a. 1-NPs treatment could prevent lung metastasis with high efficiency. As shown in Figure 6d, the excised lung tissue of mice treated with PBS had an average of 26.0 metastatic nodules in lungs and a high density variation in hematoxylin and eosin (H&E) staining images of lung tissue, which corresponded to the high degree of lung metastasis of MDA-MB-231 cells. For 1-NPs treated group, the lung metastasis nodules were significantly reduced with an average of 4.6 nodules in lungs (inhibition rate of 82.3%), and smaller area of the tumor burden in H&E staining images.40 Mice treated with 2-NPs and RGD+YIGSR displayed 12.3 and 16.1 nodules (Figure 6e), respectively, indicating the inhibitory effect on tumor metastasis, which was obviously not as high as that of the 1-NPs treated group. The results were in agreement with other reports that the YIGSR and RGD peptide inhibited the invasion and metastasis via competing binding to receptors on cancer cells with natural ECM and inhibiting following signal pathways. Therefore, RGD-YIGSR-based nanostructures showed higher inhibition efficacy due to the multivalent effect of nanomaterials. Especially, the in situ constructed AECM as long-term barriers around the tumor showed highest inhibition effect for tumor invasion and metastasis by the repair of degraded ECM with effective binding. The antimetastasis effect of 1-NPs was further investigated by CD-31IHC staining assay of tumor (Figure 6b and c).38 The microvessel density (MVD) in sections of primary tumors treated with 1-NPs significantly decreased compared to the PBS, RGD+YIGSR, and 2-NPs treated groups.41 The results also indicated the highly efficient metastasis inhibition ability of 1-NPs. Besides the antimetastasis of tumor, the growth of primary tumors was also inhibited in the 1-NPs treated group. As

shown in Figure 6f, the tumor growth process of 1-NPs treated group was much slower than that of the PBS treated group. In addition, at the end of treatment, the tumor volume of PBS treated group was about 3-fold larger than that of the 1-NPs treated group, which was confirmed by the ex vivo tumor tissue collected from sacrificed mice. Similarly, the 2-NPs and RGD +YIGSR treated groups inhibited the growth of tumor, which showed less efficacy than the transformable 1-NPs treated group. The previous cell viabilities of 1-NPs and 2-NPs showed that a high concentration (higher than 30 μM) of nanoparticles had obvious toxicity. Therefore, in vivo inhibition of tumor growth was ascribed to a high concentration i.v. administration (100 μM). The flow cytometry measurements had a similar trend with cell viability and showed that the 1-NPs treatment induced cellular apoptosis (Figure S19). In the whole treatment experiment, 1-NPs, 2-NPs, RGD+YIGSR, and PBS did not induce symptoms of dehydration and obvious body loss of mice (Figure 6g). The systemic toxicity of 1-NPs, 2-NPs, RGD +YIGSR, and PBS was further investigated by hematology and histopathology assays (Figure S20). No significant differences in pathological signs (heart, liver, spleen, and kidney) were detected between the mice treated with 1-NPs, 2-NPs, RGD +YIGSR, and the PBS treated control groups. All of the above results meant that 1-NPs did not cause the obvious side effects and could be used as safe biomaterials for the inhibition of tumor invasion and metastasis. The dose-dependent antimetastatic activity of 1-NPs was further investigated in vivo. The suspension of 1-NPs (300 μM) was not stable enough and easily precipitated, resulting in high systemic toxicity. Considering the safety issues, we chose 1-NPs with concentrations from 50 to 200 μM (based on peptide 1 in 200 μL sterilized PBS) for evaluating dose-dependent metastatic inhibition performance. All three dosages were able to inhibit the tumor invasion and metastasis as well as growth to some extent (Figures 6h, S21, and S22). The best metastatic inhibition rate (82.3%) of 1-NPs toward the MDA-MB-231 orthotropic mammary tumor metastasis model was achieved upon administrative concentration of 100 μM. Moreover, lower (50 μM) and higher (200 μM) dosages gave 54.4% and 42.4% inhibition rates, respectively. The poor inhibition rate at higher concentration was probably ascribed to the instability of 1-NPs in blood after i.v. injection, leading to the formation of larger aggregates. The larger aggregates were quickly cleared by the reticuloendothelial system. To validate the reliability of this strategy, melanoma (B16F10) tumor bearing mice as another lung metastasis model were studied to demonstrate the metastatic inhibition capability of 1-NPs (Figure S23). The experimental results indicated that the 1-NPs treated group showed remarkably decreased metastatic nodules in the lung with a inhibition rate of 50.0% compared to the control group treated with PBS. Additionally, the smaller area of tumor burden was clearly observed in the H&E staining images. Finally, the progression of the primary tumor was also slower after being treated with 1-NPs and the tumor size shrunk down to half compared to the control group treated with PBS after 20 days.

CONCLUSIONS In summary, we developed a biomimic strategy to construct AECM for inhibition of tumor invasion and metastasis. The designed LN-mimic peptide building block 1 self-assembled into 1-NPs, which were delivered to tumor sites through i.v. administration via passive and active accumulation mechanisms. 4093

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solidified with glutaraldehyde (4%) for 10 min and washed with PBS for 3 times. Finally, the cells were cultured with PBS for measurements. The specimens were examined by confocal laser scanning microscopy with a 40× immersion objective lens using a 405 nm laser for 1-NPs and 2-NPs. MCTs Experiment. MCTs were prepared in 96-well plates. At the bottom of each well was added 50 μL agarose, and after sterilization, 1600 cells per well were inoculated. It was observed that cells turned into balls after 4 days. 1-NPs or 2-NPs (30 μM) were added to incubate with cell balls for 24 h. Finally, the cell balls were washed with PBS for 3 times and further cultured with DMEM for CLSM measurements. Inhibition Effect of 1-NPs and 2-NPs for Tumor Cells Invasion and Metastasis in Vitro. The Cell Viability of 1-NPs and 2-NPs for Different Cells. Cells were seeded in a 96-well plate (1 × 104 cells/well) and incubated for 24 h. Then cells were treated with 1-NPs or 2-NPs at serial concentrations at 37 °C in a humidified environment containing 5% CO2 for another 24 h. After washing with PBS twice, 10 μL of CCK-8 solution was added to each well and cultured for 2 h. The UV−vis absorptions of each well were measured using a Microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm, respectively. Cell viability (%) was calculated, and data were presented as mean ± standard deviation (SD) in triplicate. Flow cytometry analysis of MDA-MB-231 cells upon treatment 1-NPs and 2-NPs was carried out on BD FACSAriaIII. Wound Healing Assay. For the wound healing assay, MDA-MB231 cells were seeded in 12-well plates and incubated 24 h, while cells cover 70−80% of the dish surface. Then the confluent cell monolayers were wounded with a P200 pipet tip, and cells were washed with PBS twice and incubated with 1-NPs or 2-NPs at a concentration of 30 μM for another 24 h. The wound healing area was photographed under a microscope. We utilized the wound width to calculate the degree of wound healing ability. Migration and Invasion Assay. In the migration experiment, the number of MDA-MB-231 cells was 1 × 106 plated to the top chambers of transwell without Matrigel. MDA-MB-231 cells were removed from the serum medium and suspended in a serum-free medium for 12 h. Then the lower chambers were added with 500 μL of medium as a chemoattractant. After incubation with 1-NPs, 2-NPs or culture medium for 24 h, the cells that did not migrate or invade in the upper wells were removed with a cotton swab. Cells that passed through the membrane on the lower surface were fixed with anhydrous methanol and stained with crystal violet. In the invasion experiment, MDA-MB231 cells were removed from the serum medium and suspended in a serum-free medium for 12 h. After trypsinized, 1 × 105 cells suspended in serum-free medium were plated to the top chambers of transwell coated with Matrigel (BD Biosciences), and 500 μL of 10% FBS in medium was added to lower chamber and incubated with 1-NPs, 2NPs, or culture medium for 24 h. The area of migration and invasion was photographed under a microscope. In parallel, the inhibition of wound healing, migration, and invasion was carried out by using the B16-F10 cell line as another model with a similar experimental protocol. Biological Effect of 1-NPs and 2-NPs in Vivo. Distribution of 1NPs and 2-NPs in Tumor in Vivo. The mice were treated with 200 μL of 1-NPs (100 μM per mouse), 2-NPs (100 μM per mouse), and PBS through i.v. injection until the tumor volume reached 100 mm3. The fluorescence imaging of mice at 4, 10, 24, 48, 72 h was monitored by a CRI maestro 2 multispectral fluorescence small animal in vivo imaging system. The biodistribution was further confirmed by ex vivo fluorescence images and their quantification results at 24 h postinjection. The major organs heart, liver, spleen, lung, and kidney and tumor were harvested at 24 h post-administration. The in vivo stability of nanoparticles was studied by measuring the time-dependent BP fluorescence in the blood of mice after (i.v.) injection of 1-NPs and 2NPs, respectively. The blood (20 μL) was collected from the tail vein of mice, and blood plasma (40 μL) was further separated by centrifugation upon the addition of anticoagulation (40 μL, 15 g/L

The gathered 1-NPs transformed into the 1-NFs network as an AECM by a biomimic assembly strategy through the ligand− receptor binding. The AECM acted as a long-term barrier with a competing binding capability with natural ECM, resulting in highly efficient inhibition of tumor metastasis and tumor growth. Therefore, this in situ bioinspired construction of AECM showed great potential for inhibition of tumor invasion and metastasis.

MATERIALS AND METHODS Materials. All reagents and solvents for organic synthesis were purchased from commercially available sources and used without further purification unless otherwise stated. O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), piperidine, 4-methylmorpholine (NMM), 2, 5-dihydroxybenzoic acid (DHB), and trifluoroacetic acid (TFA) were purchased from SigmaAldrich Chemical Co. All Fmoc amino acids and Wang resins were obtained from GL Biochem (Shanghai) Ltd. Cell counting kit-8 assay (CCK-8) was obtained from Beyotime Institute of Biotechnology, China. The cell lines MDA-MB-231, B16-F10, HUVECs, and L929 were purchased from the cell culture center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Cell culture medium and fetal bovine serum (FBS) were purchased from Wisent Inc. (Multicell, Wisent Inc., St. Bruno, Quebec, Canada). 0.25% Trypsin-EDTA and antibiotic solution (penicillin and streptomycin) were purchased from Invitrogen (Invitrogen, Carlsbad, CA). Culture dishes and plates were purchased from Corning (Corning, New York, USA). Dichloromethane (DCM) and N,Ndimethylformamide (DMF) were distilled over CaH2 and stored under Ar; silica gel (200−300 mesh). Distilled and deionized water was used throughout the work. Self-Assembly Characterization and Preparation of 1-NPs and 2-NPs. The peptide 1 was dissolved in DMSO to form a solution (1.5 × 10−3 M). The 20 μL of peptide 1 solution was further diluted with DMSO (880, 680, 480, 280, 80, 0 μL) and mixed with deionized water (100, 300, 500, 700, 900, 980 μL), respectively. The UV−vis and fluorescence of 0%, 10%, 30%, 50%, 70%, 90%, 98% aqueous solution were measured to validate the formation of 1-NPs and 2-NPs. Morphological Transformation in Solution and in Vitro. TEM Charaterization in Solution. The peptides 1 or 2 were dissolved in DMSO with a concentration of 1.5 × 10−3 M. The 1 or 2 solution (20 μL) was added into deionized water (980 μL), followed by addition of CaCl2 aqueous solutions with a concentration of 3 × 10−5 M. The resulting samples (at 0, 2, and 6 d) were drop-coated onto carboncoated copper grids. After contacting the droplets with copper grids for another 5 min, excess droplets were removed using filter papers. The uranyl acetate was drop-coated onto carbon-coated copper grids for another 40 s, and excess droplets were removed using filter papers. All samples were dried under vacuum before the TEM studies. DLS Charaterization in Solution. The sizes of samples were calculated by average values of at least triple measurements. The measurement was performed at 25 °C with a detection angle of 90°, and the raw data were subsequently correlated to a Z average mean size using a cumulative analysis by the Zeta Sizer software package. SEM for Morphological Transformation on Cell Surfaces. The morphologies of 1-NPs or 2-NPs treated cells were directly examined using SEM. The SEM samples were prepared by performing 1-NPs or 2-NPs (3.0 × 10−5 M) incubation with cells in DMEM (1 mL) on a silicon wafer. After contacting the solution with a silicon wafer for 1 h, an excess amount of solution was removed. Subsequently, the cells were solidified with glutaraldehyde (4%) overnight and then coated with gold for 2 min. CLSM Validation of 1-NPs and 2-NPs Structural Transformation on Cell Surfaces. The 1-NPs or 2-NPs transformation on cells surfaces was investigated on a Zeiss LSM710 confocal laser scanning microscope (Jena, Germany). The cells were cultured for 12 h in glass bottom dishes. The same concentration of 1-NPs or 2-NPs (3 × 10−5 M) was incubated with cells (105 cells/mL) in DMEM at 37 °C for 2 h, followed by washing with PBS for 3 times. Then, the cells were 4094

DOI: 10.1021/acsnano.7b00781 ACS Nano 2017, 11, 4086−4096

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ACS Nano EDTA). The resultant blood plasma was diluted with 120 μL of PBS for fluorescence measurements. In Vivo Therapeutic Effect. All animal experiments were performed complying with the NIH guidelines for the care and use of laboratory animals of Peking University Animal Study Committee’s requirements and according to the protocol approved by the Institutional Animal Care. Balb/c nude mice with MDA-MB-231 cell (5 × 106 cells per mouse) tumors inoculated on their right side of the mammary gland were used in our experiments. The mice were divided into four groups at 7 days post-tumor inoculation. Each of them treated with 1-NPs, 2NPs, RGD+YIGSR, and PBS every 72 h via i.v. administration. During the process of the treatment, the tumor volumes and body weight were measured once every 2 days. In parallel, the inhibition of tumor invasion and metastasis on mice bearing B16-F10 tumors as another model was carried out with similar experimental method mentioned above. Statistical analysis. Data are presented as the mean ± standard deviation (SD). The comparison between groups was analyzed with the Student’s t test. Differences were considered statistically significant when the p values were less than 0.05 (p < 0.05). The level of significance was defined at *p < 0.05, **p < 0.01, and ***p < 0.001.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00781. MALDI-TOF spectra of peptides 1 and 2, UV−vis, FL, CD and DLS spectra and TEM images of peptide 2; CLSM and SEM images of peptides 1 and 2 on cell surfaces and MCTs; cell viability and flow cytometry analysis of peptides 1 and 2 on cells (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Hongxun Hao: 0000-0001-6445-7737 Hao Wang: 0000-0002-1961-0787 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was also supported by National Basic Research Program of China (973 Program, 2013CB932701), National Natural Science Foundation of China (51573031, 21374026, 21304023, 51303036, and 21376165), Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), Key Project of Tianjin Science and Technology Supporting Program (no. 13ZCZDNC08900). REFERENCES (1) Gupta, G. P.; Massague, J. Cancer Metastasis: Building a Framework. Cell 2006, 127, 679−695. (2) Gilkes, D. M.; Semenza, G. L.; Wirtz, D. Hypoxia and the Extracellular Matrix: Drivers of Tumour Metastasis. Nat. Rev. Cancer 2014, 14, 430−439. (3) Mehlen, P.; Puisieux, A. Metastasis: a Question of Life or Death. Nat. Rev. Cancer 2006, 6, 449−458. (4) Li, M.; Tang, Z.; Lv, S.; Song, W.; Hong, H.; Jing, X.; Zhang, Y.; Chen, X. Cisplatin Crosslinked pH-Sensitive Nanoparticles for Efficient Delivery of Doxorubicin. Biomaterials 2014, 35, 3851−3864. 4095

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