Facile Synthesis of Peptide Cross-Linked Nanogels for Tumor

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Facile Synthesis of Peptide-Crosslinked Nanogels for Tumor Metastasis Inhibition Yi Wang, Sheng-Lin Qiao, and Hao Wang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00203 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 2018

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ACS Applied Nano Materials

Facile Synthesis of Peptide-Crosslinked Nanogels for Tumor Metastasis Inhibition Yi Wanga,b,‡, Sheng-Lin Qiaoa,b,‡and Hao Wanga,b,* a

CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of

Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190, P.R. China b

University of Chinese Academy of Sciences, Beijing 100049, P.R. China

‡

Y. Wang and S.-L. Qiao contributed equally to this work

KEYWORDS: peptide · crosslinker · nanogel ·anti-metastasis · MMP-2

ACS Paragon Plus Environment

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ABSTRACT Cancer metastasis is one of the major factors contributed to high mortality, and here we have designed and synthesized a peptide-crosslinked nanogel for hydrophilic functional molecule encapsulation and tumor metastasis inhibition. In this investigation, MMP2-responsive peptide crosslinker is prepared by coupling acrylate-functional groups at both peptide terminals. The peptide-crosslinked nanogel is then synthesized by free radical precipitation polymerization using N-isopropylacrylamide (NIPAAm) as the scaffold. Meanwhile, the hydrophilic tumor metastasis inhibition peptide P-5m is encapsulated into the nanogel in the one-pot synthesis step without complex cooperation. Importantly, the size-uniformed and narrow-distributed nanogel exhibits high P-5m loading capacity (43.4 %). Interestingly, the nanogel is stable in vitro and in vivo, whereas shows excellent and specific MMP2-triggered payload release property. As expected, the nanogel suppresses cancer cell migration and invasion in high-performance, ultimately achieving remarkable tumor metastasis inhibition in vivo.


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1. INTRODUCTION Cancer remains a high mortality throughout the world despite tremendous efforts has devoted to this research,1 the major cause of death is the high migration and invasion behaviors of cancer.2 Those behaviors enabled cancer cells to spread in the body and finally result in cancer metastasis. Therapies of cancer metastasis face challenges due to the unpredictable property of spreading cancer cells.3-5 Solutions have been exploited to solve this problem, matrix metalloproteinase (MMP) are used as the most prominent proteinases associated with cancer cell migration, invasion and tissue remodeling, and therefore benefit to cancer metastasis.6-7 Targeting MMP or designing medicine based on MMP substrates has become one of the common methods for metastasis cancer therapy.8-9 P-5m peptide (P-5m) is derived from domain 5 of kinin-free molecular weight kininogen (D5H),10 and exhibits anti-migration and anti-invasion properties towards tumor.11-12 However, P-5m, similar to most other peptides, shows poor pharmacological properties as liable to degrade in vivo during blood circulation. Furthermore, originated region of P-5m is rich of His-Gly-Lys motif, which presents positive charge and compromised therapeutic efficacy. So far, a number of drug delivery systems are developed for hydrophobic chemotherapeutic drugs encapsulation and most of them are unsuitable for encapsulation of hydrophilic drugs (e.g. peptide, DNA and RNA).13-17 Nanogels (NGs), as discrete hydrogel nanoparticles, are physically or chemically cross-linked hydrophilic networks.18-19 Compared with other nanocarriers, NGs usually have good biocompatibility, high stability, and well-defined structure.18, 20-21 These merits make them ideal platforms to deliver variable therapeutics, including hydrophilic peptides and proteins.22 Especially, stimuli-responsive NGs enable spatial-temporal delivery of payloads under specific cellular microenvironments, which makes them more promising for biomedical applications.23-25


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Among them, physically-crosslinked NGs are unstable in biological flows,26-27 and most chemically-crosslinked NGs suffer from exhaustive multistep synthesis and poor size distribution.22 Favorably, peptides can be readily designed as responsive crosslinkers through orthogonal chemistry in the solid-phase peptide synthesis processes. Here, we designed and synthesized a MMP-2 responsive peptide crosslinker and constructed NGs for P-5m peptide encapsulation via a one-pot synthesis method (Scheme 1). The peptide crosslinked nanogel was rarely reported to our best knowledge. Peptide crosslinker (AAc-GPLGVRGK-AAc) bearing bis-acryl functional group was orthogonally prepared via standard Fmoc solid-phase peptide synthesis method (Figure S1).28 A series of size-controllable nanogels were synthesized by using peptide crosslinker as a chemical crosslinker and N-isopropylacrylamide (NIPAAm) as the scaffold through precipitation polymerization method (Scheme 1). A hydrophilic peptide P-5m was encapsulated into the nanogel in a high encapsulation efficacy (> 60 %) and loading capacity (> 40 %) accompanied with the synthesis process. And we finally applied these nanogels to inhibit tumor metastasis in mice, the nanogel with MMP-2 responsibility released functional P-5m peptide to metastases, and achieved satisfactory therapeutic effects (Scheme 1). 2. EXPERIMENTAL SECTION 2.1. Peptide synthesis. All peptides were synthesized by solid-phase peptide synthesis methods using a standard Fmoc-Chemistry at a 0.3 mmol scale. The deprotection of Fmoc group on N-terminal was used 1,8-Diazabicyclo(5.4.0)undec-7-ene (20% v/v) in anhydrous DMF. Qualitative Fmoc deprotection was confirmed by a ninhydrin test (ninhydrin, phenol, VC = 1:1:1 v/v). Amino acid activation was achieved by NMM (0.4 M) and HBTU (equally molar ratio to amino acid) in anhydrous DMF. Dde deprotection was accomplished by using hydrazine hydrate


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(2% v/v) in anhydrous DMF. Acrylic acid activation was achieved by NMM (0.4 M) and HBTU (equally molar ratio to amino acid) in anhydrous DMF. Cleavage from the resin and deprotection of the amino acid side chains were performed by reaction with the mixture of TFA: TIS: H2O = 95:2.5:2.5 (v/v) for 2.5 h in ice-bath. After separated from the resin, the above-mixture was vacuum rotary evaporated to remove the TFA. The crude peptides were then precipitated in cold anhydrous diethyl ether, collected by centrifuge and dried under vacuum. 2.2. Mass spectrometry (MS) Measurement. Mass spectra were acquired on a MALDI-TOF-MS using a Microflex LRF System spectrometer (Bruker Daltonics) under positive-ion mode. 2.3. Nanogel fabrication. NGs were synthesized by free radical precipitation polymerization29. In a typical experiment, NIPAAm (113.16 mg) and peptide crosslinkers (18 mg) were dissolved in 250 ml of water purged by nitrogen. Then SDS (2.724 mg, surfactant) was added to the mixture. The solution was degassed by bubbling with nitrogen for one hour at room temperature. After the addition of KPS (2.7 mg, initiator), the polymerization was carried out at 65 oC for 4 h under nitrogen atmosphere. The obtained solution was centrifuged at a speed of 12,000 rpm. The obtained precipitate was purified through dialysis against distilled water at room temperature using a dialysis bag (molecular weight cutoff: 10,000 Da) for 7 days and the water was refreshed twice every day during this period. 2.4. Particle size measurements. The hydrodynamic diameters and particle size distributions of the nanogels were measured on a dynamic light scattering (DLS) analyzer (Zetasizer Nano ZS). The nanogel dispersion (0.5 mg/mL, pH 7.4 PBS) was passed through syringe filters (0.45 µm, Millipore) before measurements.


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2.5. Scanning electron microscope (SEM). High resolution scanning electron microscopy (SEM) images were acquired on Tecnai G2 F20 U-TWIN under an acceleration voltage of 10.0 kV and a working distance of 5.0 mm. 2.6. Transmission electron microscopy (TEM). The morphologies of nanogels were observed by TEM (Tecnai G2 20 S-TWIN) with an acceleration voltage of 200 kV. 10 µL of the nanoparticle dispersions (0.5 mg/mL) was dropped onto a copper mesh, and then most of the liquid was dried by a filter paper after 2 min. The samples were stained with 10 µL of uranyl acetate solution for 1 min followed by drying the spare liquid with the filter. Finally, 10 µL of deionized water was used to wash the copper mesh, which was blotted after 30 s and dried at room temperature. 2.7. Cytotoxicity assay for B16-F10 cells. B16-F10 cell was utilized to evaluate the cytotoxicity of P, NG2-0.6 and P⊂ ⊂NG2-0.6 by the CCK-8 assay. P, NG2-0.6 and P⊂ ⊂NG2-0.6 were dispersed in DMEM medium with a series of different concentrations. A density of 6,000 cells/well were seeded in the 96-well plates in DMEM medium containing 10% FBS and 1% penicillin–streptomycin in a humidified atmosphere with 5% CO2 and then cultured at 37 oC for 16 h. 100 µL of the sample solutions with different concentrations were added to each well, and the cells were incubated for additional 24 h. Then 100 µL of CCK-8 solutions was added to each well and cultured for another 4 h. The UV-Vis absorptions of sample cells (Asample), Ablank and control wells (Acontrol) were measured using a Microplate reader at a test wavelength of 450 nm and a reference wavelength of 690 nm, respectively. Cell viability (%) was equal to (Asample Ablank)/(Acontrol - Ablank) × 100. All the experiments were performed in triplicate.


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2.8. Western blotting. B16-F10 cells were re-suspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton-X 100 and protease inhibitor. The protein content was estimated using a BCA kit (Applygen). Each sample (60 µg of protein) was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. Blots were blocked in a blocking buffer containing 5% (wt/v) non-fat milk, 0.1% (v/v) Tween 20 in 0.01 M TBS, and incubated with primary antibodies overnight at 4 °C and then incubated with an appropriate secondary antibody (ZSGB-BIO) for 1 h at room temperature, subsequently scanned on a Typhoon Trio Variable Mode Imager. Band density was calculated using NIH ImageJ software. 2.9. Fluorescence spectroscopy. Sample (1 mL) was added in a quartz cuvette (1 cm path length)

and

the

fluorescence

spectrum

was

recorded

on

an

F-280

fluorescence

spectrophotometer. The emission spectra (λex = 630 nm) were recorded between 650 and 700 nm. 2.10. Cy5 labeling of peptide. P-5m peptide was dissolved in 1 mL PBS buffer, dissolve Cy5-NHS ester in 500 µL PBS buffer. Add Cy5-NHS ester solution to the solution of P-5m, and vortex well, stirring at room temperature for at least 4 hours. Purify the conjugate by dialysis and obtained the Cy5 labeled P-5m peptide. 2.11. In vivo anti-metastasis activity. All animal experiments were performed complying with the NIH guidelines for the care and use of laboratory animals of National Center for Nanoscience and Technology Animal Study Committee’s requirements and according to the protocol approved by the Institutional Animal Care. The B16-F10 cells were intravenously injected to 6-week female Balb/c mice (100,000 cells). After 2-day-post tumor cell injection, the mice were divided into four groups (n = 4) and treated via the tail vein by saline, P, NG2-0.6, P⊂ ⊂NG2-0.6 (100 µL, ~20 mg/kg for P, ~10 mg/kg for NG2-0.6 and ~30mg/kg for P⊂ ⊂NG2-0.6).


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The nanogel was intravenously administered to mice every two days. After a therapeutic cycle for 2 weeks, mice were sacrificed and dissected for metastasis observation and analysis. 2.12. Statistical analysis. Data are presented as the mean ± standard deviation (SD). Comparison between groups was analysis with the two-tailed 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. 3. RESULITS AND DISCUSSION 3.1 Preparation and characterization of nanogels. We firstly synthesized bis-acryl functional group modified MMP-2 responsive peptide crosslinker (AAc-GPLGVRGK-AAc) and a control peptide crosslinker (AAc-GPMGMRGK-AAc) that unable response to MMP-2 enzyme. Peptide molecular weight and purity were characterized with matrix-assisted laser desorption/ionization

time

of

flight

mass

spectrometry

(MALDI-TOF-MS)

and

high-performance liquid chromatography (HPLC), respectively (Figure S2 and S3). A series of size-controllable

nanogels

were

successfully

synthesized

by

using

peptide

(AAc-GPLGVRGK-AAc) as a chemical crosslinker and N-isopropylacrylamide (NIPAAm) as the scaffold through free radical precipitation polymerization method (Table 1). Size-controlled synthesis of nanogels was confirmed by dynamic light scattering (DLS) (Figure 1a). The data showed that the nanogel size was in a reverse relationship with sodium dodecylsulphate (SDS) concentration. A lower concentration of SDS contributed to a larger size of nanogel. Noticing that small nanoparticles preferred to be phagocytosed and cleared by monocyte and reticuloendothelial system (RES),30 we therefore chose nanogels with a size about 400 nm for further study. Besides size, the nanogel crosslinking density, a factor influence P-5m loading


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capacity was also investigated. The influence was measured by an established method using HPLC. We established a calibration curve with a certain concentrations of P-5m (Figure S4). As shown in Table 2, nanogel with a moderate amount of peptide-crosslinker contributed to the largest encapsulation efficacy (65.1 ± 1.9 %) and loading capacity (43.4 ± 1.3 %), nanogels with lower concentration of peptide crosslinker have the second largest encapsulation efficacy (56.0 ± 1.7 %) and loading capacity (37.4 ± 1.1 %), and the highest concentration of peptide crosslinker constructed nanogels exhibited the lowest encapsulation efficacy (41.8 ± 1.6 %) and loading capacity(27.8 ± 1.0 %). One of the possible reasons was that higher crosslinker density restricted the encapsulation of P-5m during the nanogel fabrication process and the lower ratio of crosslinker resulted in releasing of the encapsulated payload easily. After comprehensively evaluating the nanogel size and crosslinking density, we chose P⊂ ⊂NG2-0.6 as the optimal nanogel for the in-depth investigation. The morphology of nanogels were observed by scanning electron microscopy (SEM), nanogels NG2-0.6 and P⊂ ⊂NG2-0.6 both exhibited a uniform morphologies (Figure 1b and 1c). To meet our requirement for inhibiting metastasis in vivo, we identified their stability in plasma. Blood were collected and serum were isolated from normal mice at 2h, 4h, 12h and 24h post administration of P⊂ ⊂NG2-0.6 nanogels, P-5m were labeled with Cy5 and encapsulated in nanogels for fluorescent measurement.31 The fluorescence of P-5m-Cy5 were analyzed, the percentages of released P-5m were negligible (less than 3%) (Figure 1d). 3.2 Microenvironment responsibility of nanogels. Except for the responsive nanogel P⊂ ⊂NG2-0.6 (P-5m encapsulated nanogels), we also fabricated a non-responsive nanogel P⊂ ⊂NG2-0.6 (C) as a control based on the MMP-2 non-responsive peptide. The nanogels P⊂ ⊂NG2-0.6 and P⊂ ⊂NG2-0.6 (C) with a concentration of 15 mg mL-1 in buffer solution were


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exposed to the active MMP-2 enzyme with a concentration of 20 U, and the reaction was carried out at 37 oC for 24 h (Figure S5). The morphology change of nanogels with responsive property was observed by transmission electron microscopy (TEM) (Figure 2). This was in sharp contrast with MMP-2 untreated nanogel or MMP-2 insensitive nanogel P⊂ ⊂NG2-0.6 (C) that remained spherical morphology. Besides, we observed the hydrodynamic size of nanogels after incubated with MMP-2. The nanogels were disintegrated and the diameters were decreased (Figure S6). The above-mentioned results implied P⊂ ⊂NG2-0.6 nanogel could accurately deliver P-5m in metastasis areas where MMP-2 was overexpressed. 3.3 Nanogels effectively inhibit B16-F10 cell migration and invasion. To evaluate the metastasis inhibition effects of P⊂ ⊂NG2-0.6 nanogels, we first investigated their cytotoxicity. No detectable cytotoxicity was observed when the concentration of nanogels below 2 mg mL-1 (Figure S7). We first performed wound healing tests in the cellular level.15 The results of wound healing tests indicated the cell motility under the different nanogels treating (Figure 3a). By defining the scratch area as 1, we calculated that the non-treated highly metastatic B16-F10 cell (blank control) wound healing up to 61.3%. B16-F10 cells treated with P-5m and P-5m non-encapsulated nanogel (NG2-0.6) healed 41.8 % and 55.6 %, respectively. Significantly, P-5m encapsulated nanogel (P⊂ ⊂NG2-0.6) slowed down the wound healing of B16-F10 cell (27.8 %). The wound healing areas were analysis and the significant differences were represented in Figure 3b. Moreover, the migration and invasion inhibition abilities of P⊂ ⊂NG2-0.6 nanogels were verified by transwell assay.32 B16-F10 cells were seed in transwell chamber and with poly(ethylene terephthalate) (PET) membrane, P⊂ ⊂NG2-0.6 nanogels displayed consistent migration inhibition towards B16-F10 cells after 24 h incubation. The migration inhibition ability of P⊂ ⊂NG2-0.6 nanogel was evaluated in comparison with B16-F10 cells treated with


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P-5m or NG2-0.6 nanogels (Figure 3c). The migration was significantly inhibited by P⊂ ⊂NG2-0.6 nanogels up to 15% (Figure 3d). Besides, the invasion inhibition property of nanogels was evaluated by transwell assay while the PET membranes were covered with a matrix gel layer. Cell traverse the membrane required the secretion of MMP that in assistance to broke up the obstruction of matrix gel. B16-F10 cells with P⊂ ⊂NG2-0.6 nanogels treatment displayed fewer cells traversing, and P-5m peptide also made contribution to the invasion inhibition. In contrast with P⊂ ⊂NG2-0.6 nanogels and P-5m, more invasion cells were displayed with NG2-0.6 treating (Figure 3e). The number of migration and invasion cells were counted and analyzed, the P⊂ ⊂NG2-0.6 nanogel displayed significant inhibition of B16-F10 cells mobility in migration (15 %) and invasion (9 %) (Figure 3d and 3f), and contribute to the metastasis inhibition in vivo. 3.4 Mechanisms of nanogels inhibiting metastasis. Except for the evaluation of mobility, we further investigated the expression of MMP-2 and vascular endothelial growth factor (VEGF), which implied the anti-adhesion and angiogenesis properties and correlated to the metastasis ability of cancer cells.33 The expression of MMP-2 and VEGF were detected by western blot (Figure 4a), the band intensities were analyzed based on actin, the expression of MMP-2 and VEGF with P⊂ ⊂NG2-0.6 nanogels incubation were decreased one half of those cells without any treatment; moreover, MMP-2 were significantly decreased with P⊂ ⊂NG2-0.6 nanogels treating compared to P-5m, while VEGF expression had no significant difference (Figure 4b). Those results demonstrated that the P⊂ ⊂NG2-0.6 nanogels hampered the motor ability through inhibiting the migration and invasion ability of cells; moreover, P⊂ ⊂NG2-0.6 nanogels inhibited the cells metastasis by decreasing the expression of MMP-2 and VEGF. The motility inhibition of cells by P⊂ ⊂NG2-0.6 nanogels directed the potential for anti-metastasis therapy in vivo.


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3.5 Bio-distribution and metastasis inhibition of nanogels in vivo. The bio-distribution of P⊂ ⊂NG2-0.6 nanogels was monitored prior to in vivo tests. The bio-distribution was measured at 24 h post injection, mice were sacrificed and the major organs were dissected for tissues homogenate, and emission fluorescent spectra of homogenate analysis displayed accumulation of P⊂ ⊂NG2-0.6 nanogels in liver and kidney (Figure S8). According to the biodistribution of nanogels, we further evaluated the potential systemic toxicity through blood biochemical index of AST, ALT, ALP and BUN, the biochemical index was in the normal range (Figure S9), and the pathological analysis of major organs by hematoxylineosin (H&E) staining further indicated that P⊂ ⊂NG2-0.6 was hypotoxic and safe for bio-applications (Figure S10). Based on the biosafety of nanogels, metastasis inhibition experiment was employed in vivo for confirming the functions of P⊂ ⊂NG2-0.6 nanogel. B16-F10 tumor bearing mice were started therapy after 3 d of tumor cells injected and intravenously injected with P-5m, NG2-0.6, P⊂ ⊂NG2-0.6, respectively. P⊂ ⊂NG2-0.6 nanogel was intravenously injected every other day and lasted for two weeks. Mice were sacrificed and major tumor metastatic organ (i.e., lungs) were dissected (Figure 5a) and fixed for H&E staining and immunohistochemical analysis (Figure 5b). The metastasis and the fold of MMP-2 change were analyzed (Figures 5c and d). In accordance to the results of inhibiting migration and invasion, P⊂ ⊂NG2-0.6 nanogel treated mice had fewer metastases (~6 metastases) and MMP-2 experssion in lung tissues were significantly decreased compared to control group, which revealed that P⊂ ⊂NG2-0.6 nanogel was able to significantly inhibit B16-F10 metastasis. 4. CONCLUSION


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Summarily, we developed a facile method for synthesizing peptide-crosslinked nanogels which exhibited uniform size and narrow distribution. The nanogels possess high loading capacity (up to 43.4 %) for the hydrophilic peptide P-5m. By introducing MMP-2 cleavable peptide for stabilizing and crosslinking nanogels, the resultant nanogels showed satisfactory responsive and release properties in the presence of MMP-2 enzyme. Finally, the nanogels presented remarkable suppression capability towards migration and invasion of cancer cells, and ultimately showed therapeutic efficacy for tumor metastasis in vivo.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xx. Additional experimental details and Figures S1-S9 (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Hao Wang: 0000-0002-1961-0787 Yi Wang: 0000-0003-3077-2899 Sheng-Lin Qiao: 0000-0001-5006-5257 Author Contributions ‡

Y. Wang and S.-L. Qiao contributed equally to this work.


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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 (51573032) and Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), Key Project of Chinese Academy of Sciences in Cooperation with Foreign Enterprises

(GJHZ1541),

CAS

Key

Research

Program

for

Frontier

Sciences

(QYZDJ-SSW-SLH022) and CAS Interdisciplinary Innovation Team. REFERENCES (1) Ma, Y.; Qiao, S.-L.; Wang, Y.; Lin, Y.-X.; An, H.-W.; Wu, X.-C.; Wang, L.; Wang, H., Nanoantagonists with nanophase-segregated surfaces for improved cancer immunotherapy. Biomaterials 2018, 156, 248-257. (2) Hanahan, D.; Weinberg, R. A., Hallmarks of Cancer: The Next Generation. Cell 2011, 144, 646-674. (3) Desgrosellier, J. S.; Cheresh, D. A., Integrins in cancer: biological implications and therapeutic opportunities. Nat. Rev. Cancer 2010, 10, 9-22. (4) Chaffer, C. L.; Weinberg, R. A., A Perspective on Cancer Cell Metastasis. Science 2011, 331, 1559-1564. (5) Mantovani, A.; Allavena, P.; Sica, A.; Balkwill, F., Cancer-related inflammation. Nature 2008, 454, 436-444. (6) Curran, S.; Murray, G. I., Matrix metalloproteinases in tumour invasion and metastasis. J. Pathol. 1999, 189, 300-308. (7) Deryugina, E. I.; Quigley, J. P., Matrix metalloproteinases and tumor metastasis. Cancer Metast. Rev. 2006, 25, 9-34. (8) Egeblad, M.; Werb, Z., New functions for the matrix metalloproteinases in cancer progression. Nat. Rev. Cancer 2002, 2, 161-174.


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(20) Jiang, Y.; Chen, J.; Deng, C.; Suuronen, E. J.; Zhong, Z., Click hydrogels, microgels and nanogels: Emerging platforms for drug delivery and tissue engineering. Biomaterials 2014, 35 (18), 4969-4985. (21) Li, L.-L.; Qiao, S.-L.; Liu, W.-J.; Ma, Y.; Wan, D.; Pan, J.; Wang, H., Intracellular construction of topology-controlled polypeptide nanostructures with diverse biological functions. Nat. Commun. 2017, 8, 1276. (22) Smith, M. H.; Lyon, L. A., Multifunctional Nanogels for siRNA Delivery. Accounts Chem. Res. 2012, 45, 985-993. (23) Shen, X.; Zhang, L.; Jiang, X.; Hu, Y.; Guo, J., Reversible surface switching of nanogel triggered by external stimuli. Angew. Chem. Int. Ed. 2007, 46, 7104-7107. (24) Park, S. Y.; Baik, H. J.; Oh, Y. T.; Oh, K. T.; Youn, Y. S.; Lee, E. S., A Smart Polysaccharide/Drug Conjugate for Photodynamic Therapy. Angew. Chem. Int. Ed. 2011, 50, 1644-1647. (25) Du, J.-Z.; Sun, T.-M.; Song, W.-J.; Wu, J.; Wang, J., A Tumor-Acidity-Activated Charge-Conversional Nanogel as an Intelligent Vehicle for Promoted Tumoral-Cell Uptake and Drug Delivery. Angew. Chem. Int. Ed. 2010, 49, 3621-3626. (26) Shimoda, A.; Sawada, S.-i.; Kano, A.; Maruyama, A.; Moquin, A.; Winnik, F. M.; Akiyoshi, K., Dual crosslinked hydrogel nanoparticles by nanogel bottom-up method for sustained-release delivery. Colloids and Surf B-Biointerfaces 2012, 99, 38-44. (27) Kabanov, A. V.; Vinogradov, S. V., Nanogels as Pharmaceutical Carriers: Finite Networks of Infinite Capabilities. Angew. Chem. Int. Ed. 2009, 48, 5418-5429. (28) Qiao, S.-L.; Ma, Y.; Wang, Y.; Lin, Y.-X.; An, H.-W.; Li, L.-L.; Wang, H., General Approach of Stimuli-Induced Aggregation for Monitoring Tumor Therapy. ACS Nano 2017, 11, 7301−7311. (29) Su, S.; Wang, H.; Liu, X.; Wu, Y.; Nie, G., iRGD-coupled responsive fluorescent nanogel for targeted drug delivery. Biomaterials 2013, 34, 3523-3533. (30) Guo, Z.; Chen, J.; Lin, L.; Guan, X.; Sun, P.; Chen, M.; Tian, H.; Chen, X., pH Triggered Size Increasing Gene Carrier for Efficient Tumor Accumulation and Excellent Antitumor Effect. ACS Appl. Mater. Interfaces 2017, 9, 15297-15306.


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Scheme 1. Schemetic illustrating the fabrication of hydrophilic payloads encapsulated nanogel for inhibiting melanoma metastasis in vivo.


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Table 1 Characterization of the diameters nanogels that prepared under different SDS concentrations in the PBS buffer (pH 7.4).

Entry

Peptide:NIPAAm

SDS (mg)

Size (nm)

NG1-0

6:94

0

-

NG1-0.6

6:94

0.6

390

NG1-5

6:94

2

317

NG1-10

6:94

10

266

NG1-50

6:94

50

71

NG2-0.6

4:96

0.6

407

NG3-0.6

1:99

0.6

414


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Figure 1. Characterizations of size controlled nanogels a) The hydrodynamic size distribution of nangels NG1-0.6, NG1-5, NG1-10 and NG1-50 with a series SDS concentration; b) The morphology of NG2-0.6. Inset, the hydrodynamic size of PNG2-0.6. c) The morphology of peptide P-5m encapsulated NG2-0.6. Inset, the hydrodynamic size of P⊂ ⊂PNG2-0.6. d) The released percentage of peptide P-5m in plasma at different time point.


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Table 2 Encapsulation efficacy (EE %) and loading capacity (LC %) of nanogels with different crosslinking intensities in the PBS buffer (pH 7.4)

Entry

Peptide:NIPAAm

EE %

LC %

P⊂NG1-0.6

6:94

41.8 ± 1.6

27.9 ± 1.0

P⊂NG2-0.6

4:96

65.1 ± 1.9

43.4 ± 1.3

P⊂NG3-0.6

1:99

56.1 ± 1.7

37.4 ± 1.1


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Figure 2. MMP-2 responsibility of P⊂ ⊂NG2-0.6 nanogels a) The morphology of PNG2-0.6 and PNG2-0.6(C) before MMP-2 enzyme addition. b) The morphology of the nanogels after incubation with MMP-2 enzyme. Scale bars were indicated in representative images.


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Figure 3. Nanogels inhibit tumor migration and invasion a) Microscopy images of wound healing (a), migration (c), and invasion (e) and quantitative analysis (b, d, f) with different treating towards B16-F10 cells for 24h incubation, respectively; Data were given as mean ± SD (n = 5), **p < 0.01, ***p < 0.001.


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Figure 4 Nanogels down-regulation of metastasis related molecules. a) Expression of MMP-2, VEGF and actin and b) their quantitative analysis in B16-F10 cells with different treatment for 24h. Data were given as mean ± SD (n = 3). **p < 0.01, ***p < 0.001.


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Figure 5. Nanogels inhibit metastasis in vivo a) Representative images of lungs with indicated treatment for two weeks after B16-F10 cancer cells were xenografted for 2 d, tissues were fixed with Bouin’s solution; b) Pathological images of lungs (upper) and the immunohistochemical staining of MMP-2 (lower). Black circles, metastases; black arrows, MMP-2 positive dots; whited arrows, the melanoma B16-F10 cells. c) The quantitative analysis of the number of metastases, d) The fold of change of MMP-2 enzyme in metastasis tumor tissue. Data were given as mean ± SD (n = 5). **p < 0.01, ***p < 0.001.


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TOC


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Facile Synthesis of Peptide Cross-Linked Nanogels for Tumor

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