Inflammatory Monocytes Loading Protease-Sensitive Nanoparticles

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Inflammatory Monocytes Loading Protease-Sensitive Nanoparticles Enable Lung Metastasis Targeting and Intelligent Drug Release for Anti-Metastasis Therapy Xinyu He, Haiqiang Cao, Hong Wang, Tao Tan, Haijun Yu, Pengcheng Zhang, Qi Yin, Zhiwen Zhang, and Yaping Li Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b02330 • Publication Date (Web): 31 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017

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Inflammatory Monocytes Loading Protease-Sensitive Nanoparticles Enable Lung Metastasis Targeting and Intelligent Drug Release for Anti-Metastasis Therapy

Xinyu He†,‡,¶, Haiqiang Cao†,‡,¶, Hong Wang†,‡, Tao Tan†, Haijun Yu†, Pengcheng Zhang†, Qi Yin†, Zhiwen Zhang*,†, Yaping Li*,†

†

State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

‡

University of Chinese Academy of Sciences, Beijing 100049, China

¶

These authors contributed equally to this work.

*Corresponding author: Prof. Zhiwen Zhang ([email protected]) and Prof. Yaping Li ([email protected]) Tel/Fax: +86-21-20231979

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Table of Contents


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ABSTRACT Metastasis causes high mortality of breast cancer, and the inability of drug delivery to metastatic sites remains a crucial challenge for anti-metastasis therapy. Herein, we report that inflammatory monocytes loading legumain-activated nanoparticles can actively target lung metastases and initiate metastasis-specific intelligent drug release for anti-metastasis therapy. The cytotoxic mertansine is conjugated to poly(styrene-co-maleic anhydride) with a legumain-sensitive peptide and self-assembled into nanoparticles (SMNs), and then loaded into inflammatory monocytes to prepare the SMNs-loaded monocytes delivery system (MSMNs). M-SMNs would be in living state in circulation to ensure their active targeting to lung metastases, and responsively damaged at the metastatic sites upon the differentiation of monocytes into macrophages. The anti-cancer drugs are intelligently released from M-SMNs as free drug molecules and drug-loaded microvesicles, resulting in considerable inhibition on the proliferation, migration and invasion activities of metastatic 4T1 breast cancer cells. Moreover, M-SMNs significantly improve the delivery to lung metastases and penetrate the metastatic tumors, thus producing a 77.8% inhibition of lung metastases. Taken together, our findings provide an intelligent biomimetic drug delivery strategy via the biological properties of inflammatory monocytes for effective anti-metastasis therapy.

KEYWORDS: Monocyte, Bioinspired, Nanoparticles, Protease, Cancer Metastasis.


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Metastasis is the predominant cause of breast-cancer related deaths, which is extremely difficult to treat in the clinic1-4. Despite recent advances in cancer therapy, results from randomized clinical trials showed little evidence of increased survivals in patients with metastatic breast cancer over the past 30 years2. A crucial challenge is the poor efficiency of drug delivery to the sites of metastases where many current therapies cannot reach, thus painting a dismal picture of anti-metastasis therapy2, 4. The micrometastases smaller than 1-2 mm in diameter are often small clusters of cancer cells with poor vasculature, and present high multiplicity and dispersion in diverse invaded organs, which greatly restricts the specific drug delivery to these metastases2, 5. Moreover, in overt metastases with millions of aggressive cancer cells, they have tortuous vasculature and elevated hydrostatic pressure, causing the poor penetration of anti-cancer drugs2, 5. Accumulating data suggest nanoparticles represent the best efficacy of targeted drug delivery to well-vascularized tumor by capitalizing on the enhanced permeation and retention (EPR) effects, thereby demonstrating great potential for treating cancer metastasis6-12. However, this method would be inadequate when dealing with metastases due to the limited EPR effects in micrometastases and poor penetration nature of overt metastases2, 6. To overcome these issues, novel strategies are greatly needed to improve the metastatic-specific drug delivery for effective anti-metastasis therapy. Recently, bioinspired strategies have attracted more attention in drug delivery applications to improve medical performance on treating tumor progression and metastasis8, 13-28

. During lung metastasis of breast cancer, inflammatory monocytes can be preferentially

recruited to the metastatic niche, and then differentiated into mature macrophage to facilitate the establishment of metastatic tumors4, 29-34. It is worth noting that monocytes are phagocytic cells and can uptake a large variety of nanoparticles35. These unique abilities of monocytes make them attractive drug delivery vehicles for active targeting of lung metastases36. One essential requirement of this delivery is that the intracellular cargo should be quiescent in 4 ACS Paragon Plus Environment

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monocytes and became active upon their arrival in the metastatic sites. In terms of the magic switching of monocytes to macrophages, legumain, a barely expressed protease in monocytes, is abnormally overexpressed in the differentiated macrophages, potentiating it an encouraging candidate for the design of drug delivery carrier37-39. Accordingly, we hypothesize that inflammatory monocytes loading legumain-motivated nanoparticles can actively target lung metastasis and initiate the on-demand drug release in the case of their differentiation into macrophages at metastatic sites, thereby providing an intelligent biomimetic drug delivery platform to treat the deadly metastatic tumors (Scheme 1). To verify this concept, the anti-cancer drug of mertansine is conjugated to poly(styreneco-maleic anhydride) (SMA) with a legumain-sensitive peptide linker to synthesize the SMAAANK-Mertansine conjugate, and self-assembled into legumain-activated nanoparticles (SMNs). Then, we load these SMNs into Ly6c+ inflammatory monocytes to develop SMNsladen monocytes (M-SMNs) to improve targeting ability to lung metastases of breast cancer. In M-SMNs, SMNs would be inactive due to the deficiency of legumain protease in monocytes, which can avoid the undesirable drug release and ensure the living state of monocytes. The monocytes of M-SMNs would be differentiated into macrophages upon their autonomous homing to the metastatic niche. Then, the anti-cancers drugs in M-SMNs can be dissociated from SMNs due to the highly expressed legumain protease, destroy the macrophages and intelligently released at the metastatic sites, thus facilitating their medical performance on treating cancer metastasis. In this manuscript, the physiochemical properties of M-SMNs were characterized. The inhibitory effects of M-SMNs on the proliferation, migration and invasion activities of metastatic 4T1 cells were investigated. Moreover, the specific targeting of M-SMNs to lung metastasis and their anti-metastatic efficacy were evaluated in a lung metastatic breast cancer model to validate the feasibility of our initial hypothesis.


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Initially, we synthesized the polymer-drug conjugate of SMA-AANK-Mertansine for subsequent self-assembly into SMNs (Figure 1A). The cytotoxic mertansine was conjugated to SMA via a legumain-sensitive peptide linker (Sequence: NH2-AANK-Maleimide). It was determined that there were about 2 units of mertansine conjugated to the SMA molecules. The details of the synthesis procedures and characterizations were described in the Supporting Information and Figure S1-2. Then, the SMA-AANK-Mertansine conjugates were selfassembled into legumain-activated nanoparticles of SMNs. The dynamic light scattering (DLS) analysis showed SMNs had a Z-average hydrodynamic diameter of 73.82 nm with a polydispersivity index (PDI) of 0.246 (Figure S3). The morphology was visualized under transmission electronic microscope (TEM), which showed that SMNs were nanometer-sized spherical nanoparticles with the mean diameter of 50.5±7.6 nm (Figure 1B). The difference in diameter could be explained by their states during the measurements. Then, the enzymatic responsiveness of SMNs to legumain protease was investigated. Legumain was a highly conserved lysosomal protease, making it a potential candidate for cancer therapy.40-42 SMNs was incubated with active legumain in phosphate buffer solution (PBS) at pH 5.5 to simulate the lysosomal environments. The TEM images showed the morphology of SMNs was collapsed after their incubation with legumain protease (Figure 1B). Moreover, the drug release from SMNs was obviously increased with the legumain concentration up to 50 ng/mL, and reached a plateau thereafter (Figure 1C). Compared with the drug release of SMNs in PBS (pH 5.5) without legumain, the released drug from SMNs was obviously enhanced over 9-folds at 50 ng/mL and 100 ng/mL of legumain. By contrast, in the absence of legumain, the anti-cancer drug was slightly released from SMNs in PBS at pH 5.5 and pH 7.4, and no difference was detected between them. Thereby, these results effectively verified the formation of SMNs and their sensitivity to legumain protease. Then, the phenotype of inflammatory monocytes, their differentiation into macrophages and the variations of legumain expression were determined for further measurements (Figure 6 ACS Paragon Plus Environment

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1D). The Ly6c+F4/80low inflammatory monocytes were derived from myeloid progenitor cells by incubation with macrophage colony-stimulating factor (M-CSF) for 48 h, and isolated by flow cytometry. Then, the inflammatory monocytes were further incubated with M-CSF for 45 days to promote their differentiation into mature macrophages, which were characterized by Ly6CLowF4/80+ phenotype cells. In Figure 1D, the percentage of Ly6CLowF4/80+ cells (R1 region) were dramatically increased to 97.77% in differentiated macrophages, which confirmed the transformation of inflammatory monocytes to macrophages. Thereafter, the expression of legumain protease in monocytes and macrophages were evaluated by western blotting analysis (Figure 1E). The protein signals were barely detected in monocytes, but obviously observed in the differentiated macrophages. These results suggested the high variations of legumain expression in inflammatory monocytes and mature macrophages, providing a primary foundation for the design of metastasis-targeting drug delivery vehicles. M-SMNs was fabricated by loading SMNs into inflammatory monocytes, which was visualized under laser confocal scanning microscopy (LCSM, LSM 700, Zeiss, Germany). SMNs was fluorescently labeled with hydrophobic dye of Nile red by physical entrapment for the observations. As shown in Figure 1G, the red fluorescence signals of SMNs could be largely observed in monocytes, indicating the efficient loading of SMNs into monocytes and the formation of M-SMNs. In particular, the essential requirement of this delivery system is that the M-SMNs should be alive when the monocytes are not differentiated, and would be responsively damaged upon their shift into macrophages to release the anti-cancer drugs. The cell viability of M-SMNs was evaluated by Live/Dead assays, in which the living cells presented as green fluorescence signals and the dead cells were denoted as red colors. In Figure 1F, the green fluorescence signals were extensively detected in monocytes and MSMNs, suggesting the living state of the monocytes in M-SMNs. Then, we used the conditioned culture media from highly metastatic 4T1 breast cancer cells to imitate the metastatic microenvironment. After 24 h of incubation with the conditioned culture media, the 7 ACS Paragon Plus Environment

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red fluorescence signals were obviously observed in M-SMNs, indicating the living cellsbased delivery system were largely damaged (Figure 1F). The cell damages of M-SMNs could be resulted from the protease-responsive drug release from SMNs since the monocytes would be activated into macrophages under the given conditions (Figure S4), hereafter denoted as activated M-SMNs. Afterwards, the culture media from activated M-SMNs was analyzed by liquid chromatograph-mass spectrometer method (LC-MS, Agilent G6520, QTOF, USA) (Figure S5), which verified that the derivative of mertansine (K-mertansine) would be released from the M-SMNs system for anti-metastasis therapy. It was reported that cancer cells incubated with anti-cancer drugs can package these drugs into microvesicles.43 Then, we attempted to clarify whether the anti-cancer drugs would be released from M-SMNs as free drug molecules or drug-loaded microvesicles. The media were centrifuged to separate the possible microvesicles and free anti-cancer drugs, and then measured by high performance liquid chromatography (HPLC) analysis. The drug amount in the centrifuged pellets and supernatant were both about 50% of the total drug amount in the culture media (Figure 1H). The centrifuged pellets were re-suspended in PBS and the morphology was visualized under field emission transmission electronic microscope (FE-TEM), which presented as nanometersized particles with the mean diameter of 132 ±45.2 nm (Figure 1I). Statistical images analysis of over 100 individuals revealed that the particle size of the microvesicles was ranging from 70 nm to 300 nm, and mainly within 70-150 nm (Figure S6), which would facilitate their penetration into the metastatic tumors. These results indicated that both free drug molecules and drug loaded microvesicles would be released from M-SMNs. Summarizing, in M-SMNs system, upon the differentiation of monocytes into macrophages, the anti-cancer drugs would be responsively degraded from SMNs in respond to the highly expressed legumain protease. Afterwards, the dissociated mertansine derivatives would destroy the macrophages of M-SMNs, and then released into the surrounding environments as


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free drug molecules and microvesicles, thereby holding great premise for anti-metastasis therapy. Next, the inhibitory effects of M-SMNs on cell proliferation were evaluated in metastatic 4T1 breast cancer cells. As shown in Figure 2A, the 4T1 cells were seeded to each well of plate while various samples of monocytes, free mertansine, SMNs and M-SMNs were respectively added to the upper chambers of inserts (pore size, 0.4 µm). The cell viability of 4T1 cells was detected by Live/Dead assays and the cell proliferation profiles were monitored using the xCELLigence RTCA (label-free real-time cell-based assays) DP system (ACEA Biosciences, USA). In the captured images (Figure 2A), the green fluorescence signals were readily detected in each group, but the cell numbers in mertansine, SMNs and M-SMNs groups were much less than that of control and monocyte groups. Due to the tubulininhibitory effects of mertansine, SMNs and M-SMNs did not induce obvious damages of cancer cells, but produced considerable inhibition on cell proliferation. Likewise, in the realtime cell proliferation profiles in Figure 2B, free mertansine, SMNs and M-SMNs displayed significant anti-proliferation activities in comparison with the negative control, but monocytes alone had no inhibitory effects. Typically, the anti-proliferation effects of M-SMNs significantly depended on the cell numbers in the upper chambers of inserts, and was slightly lower than that of free mertansine and SMNs (Figure 2B and Figure S7). This could be mainly ascribed to the unique drug release behaviors from M-SMNs, which would be exclusively motivated upon the differentiation of monocytes into macrophages. Of note, the differentiation could be accomplished when M-SMNs was co-cultured with 4T1 cells (Figure S4), but this transition was a gradual transforming process, which would partially restrict the velocity of drug release from M-SMNs, thereby comprising their inhibitory effects on the proliferation of 4T1 cells. Furthermore, to clarify the possible mechanism, we respectively evaluated the anti-proliferation activities of total culture media from activated M-SMNs, the separated microvesicles and free drug supernatant. The cell viability index of each group was 9 ACS Paragon Plus Environment

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monitored after 24 h incubation (Figure 2C). When compared to the initial time points of incubation, the cell viability index was obviously increased 3.6 folds in negative control, but significantly reduced by 54.5% and 39.2% in total media and microvesicles group, and slightly decreased in free drug supernatant group. Compared with the negative control at 24 h of incubation, the cell proliferation of 4T1 cells in total culture media, microvesicles and free drug supernatant was respectively reduced to 12.5%, 16.6% and 24.0%, suggesting that both microvesicles and free drug supernatant from activated M-SMNs were responsible for the anti-proliferation activities (Figure 2C). As a result, M-SMNs presented significant inhibition on the proliferation of 4T1 cells, which could be owing to the intelligent drug release from the M-SMNs system. Moreover, the inhibition of M-SMNs on cell migration and invasion of metastatic 4T1 cells were measured by transwell-mediated assays. The cell migration and invasion are two fundamental steps during metastasis of cancer cells13,

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. The proliferation of 4T1 cells

treated with monocytes and M-SMNs (0.01 mg/mL) was monitored as described above, and no difference were detected between them, which would not interfere with the migration and invasion assays (Figure S8). The 4T1 cells were pretreated with monocytes, free mertansine, SMNs and M-SMNs according to the method in cell proliferation assays. After 12 h incubation, 4T1 cells were harvested and added to the upper chambers of transwell inserts (pore size, 8.0 µm). The migrated or invaded cells were denoted as the cluster of violet regions in the captured images. In the control groups, the migrated or invaded cells were extensively detected, indicating the high metastatic activity of 4T1 cells. The cell migration and invasion activities of 4T1 cells were obviously inhibited in free mertansine, SMNs and M-SMNs treated groups, but not impacted by monocytes alone (Figure 3A). Compared with the negative control, the migrated cells in mertansine, SMNs and M-SMNs treated groups were respectively reduced by 85.3%, 74.6% and 70.3% (Figure 3B), while the invaded cells across the matrigel coated transwell membrane was obviously reduced to only 9.8%, 18.8% 10 ACS Paragon Plus Environment

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and 8.6% (Figure 3C). In addition, the anti-migration and anti-invasion effects of M-SMNs were comparable with that of free mertansine, which could be owing to the unique drug release profiles from M-SMNs. During the gradual transition of monocytes into macrophages the anti-cancer drugs would be successively released from M-SMNs with lower concentration than free mertansine group, making their inhibitory effects no better than the free mertansine. Therefore, the cell migration and invasion activities of 4T1 cells were efficiently suppressed by M-SMNs, indicating great potential for anti-metastasis efficacy. It was worth noting that the inflammatory monocytes would be preferentially recruited to the sites of metastasis to facilitate the establishing of lung metastases

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. The in vivo

distribution of monocytes and M-SMNs were measured in a lung metastatic breast cancer model. For the imaging detections, monocytes were fluorescently labeled with DiR (1,1dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide) by incubation (MonocyteDiR), and SMNs were loaded into MonocyteDiR to prepare fluorescent M-SMNs (MDiR-SMNs). At 4 h after injection, the fluorescence signals of MonocyteDiR and MDiR-SMNs were obviously detected in liver, lung and spleen, but no significant difference was detected between monocytes and M-SMNs treated group (Figure 4A). Moreover, to compare the in vivo distribution of SMNs and M-SMNs, SMNs was labeled with DiR by physical entrapment (SMNsDiR), and then incubated with monocytes to prepare the DiR labeled M-SMNs (MSMNsDiR). The ex vivo imaging results indicated that both SMNs and M-SMNs were mainly distributed in liver, spleen and lung tissues, and the fluorescence signals of M-SMNs in lungs was significantly higher than that of SMNs (Figure S9). In particular, the specific targeting of M-SMNs to lung metastases were further determined by LCSM detections (Figure 4B), in which monocyte and M-SMNs were fluorescently labeled with DiIC18(3) (DiI) for the measurements. At 4.0 h after injection, the lung tissues were carefully removed, sectioned, and respectively stained with DAPI (blue, Beyotime) and phalloidin-FITC (blue, Beyotime) for visualization. In the captured images, MonocyteDiI and MDiI-SMNs were denoted as red 11 ACS Paragon Plus Environment

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fluorescence signals. As shown in Figure 4B, MonocyteDiI could be infiltrated into the sites of metastasis niche, but were mainly distributed in the outer region of the metastases. However, for MDiI-SMNs, the red fluorescence signals were largely detected in the sites of metastases with strong intensity, and could also be observed in the inner sides of metastatic lesions. As a result, M-SMNs could be efficiently delivered to the sites of lung metastases and penetrate the metastatic foci. The active targeting of M-SMNs to lung metastases could be owing to the autonomous metastasis-homing ability of inflammatory monocytes, while the deeppenetration in metastatic lesions could be ascribed to the nanometer-sized microvesicles derived from the M-SMNs upon their arrival at the metastatic sites. The deep penetration of nanoparticles in tumors is an important factor that would affect the therapeutic efficacy13, 14, 44, 47-50

. The specific targeting of M-SMNs in lung metastases and their deep penetration in the

metastatic tumors could provide an essential prerequisite for the anti-metastasis therapy. Finally, to validate the merit of M-SMNs on the anti-metastasis efficacy, the in vivo assays were performed in lung metastatic breast cancer models. The lung metastatic models were treated with saline, mertansine, SMNs, monocytes and M-SMNs (n=5) (0.5 mg/kg of mertansine or equivalent dose), respectively. The body weight of mice from each group was not significantly changed during the treatment (Figure S10). At day 14 after the inoculation, mice were autopsied and the lung tissues from each group were stained with India ink to detect the metastatic nodules. As shown in Figure 5A and Figure S11, the metastatic nodules which were denoted as white spots, were extensively detected in lungs from saline, mertansine, SMNs and monocytes groups, but barely observed in M-SMNs treated group. Compared with saline control, free mertansine and SMNs treatments showed a slight suppression of lung metastasis, while the monocyte treatment had no inhibition on lung metastasis (Figure 5C&D). In particular, in M-SMNs treated group, the average number of metastatic nodules per lung was 17.4±6.9, which was only 22.2%, 23.3%, 28.5% and 29.7% of the saline control, monocyte, mertansine and SMNs treated group, respectively (Figure 5C). 12 ACS Paragon Plus Environment

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Thus, the M-SMNs treatment produced a 77.8% inhibition of lung metastases, which was 3.5and 3.1-fold higher than that of free mertansine and SMNs (Figure 5D). Moreover, the histological examinations showed that the metastatic lesions were rarely observed in M-SMNs group, but obviously detected in other groups (Figure 5B). Therefore ， the M-SMNs treatment could result in a considerable inhibition on the incidence of lung metastasis. In the in vitro evaluations, M-SMNs showed significant inhibition on the proliferation, migration and invasion activities of metastatic 4T1 breast cancer cells. Moreover, in vivo, M-SMNs could be specifically delivered to the sites of lung metastasis via the biomimetic metastasishoming effects, intelligently activated at the metastatic niches to release the anti-cancer drugs, and penetrate into the deep of metastatic tumors, thereby leading to the significant inhibition of lung metastasis. To a larger content, the specific targeting of M-SMNs to lung metastases and their intelligent drug release at the metastatic sites would be crucial for the effective antimetastatic therapy. In summary, we developed an inflammatory monocytes-based biomimetic drug delivery system (M-SMNs) that can actively target lung metastases of breast cancer with metastasistriggered drug release for effective anti-metastasis therapy. M-SMNs were prepared by loading legumain-responsive SMNs into inflammatory monocytes, which would be in living state to facilitate their active targeting to lung metastasis. Once the monocytes of M-SMNs were differentiated into macrophages, the anti-cancer drugs would be dissociated from SMNs due to the highly expressed legumain protease, destroy the macrophages, and released from M-SMNs as free drug molecules and nanometer-sized vesicles. M-SMNs showed considerable inhibition on the proliferation, migration and invasion activities of metastatic 4T1 cancer cells. Moreover, in lung metastatic breast model, M-SMNs could be efficiently delivered to the sites of lung metastases and penetrate into the inner sides of metastatic tumors, resulting in a considerable inhibition of lung metastases. Therefore, this proof-of-concept 13 ACS Paragon Plus Environment

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study provides an intelligent biomimetic drug delivery strategy by exploring the biological properties of inflammatory monocytes to treat lung metastasis of breast cancer.

ASSOCIATED CONTENT Supporting Information Materials,

synthesis

of

SMA-AANK-Mertansine

conjugate,

preparation

and

characterization of SMNs and M-SMNs, in vitro evaluation on cell proliferation, migration and invasion activities, in vivo targeting to lung metastasis of breast cancer, and in vivo inhibitory effects on lung metastasis. Additional data of Figures 1-11. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]

*

E-mail: [email protected]

Author Contributions X. He and H. Cao contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial supports by the National Basic Research Program of China (2015CB932103), the National Natural Science Foundation of China (81690265, 81521005, 81630052) and the Youth Innovation Promotion Association of CAS are greatly acknowledged.

REFERENCES 14 ACS Paragon Plus Environment

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Ghajar, C. M. Nat Rev Cancer 2015, 15, 238-247. Steeg, P. S. Nat Rev Cancer 2016, 16, 201-218. Eckhardt, B. L.; Francis, P. A.; Parker, B. S.; Anderson, R. L. Nat Rev Drug Discov 2012, 11, 479-497. Wan, L. L.; Pantel, K.; Kang, Y. B. Nat Med 2013, 19, 1450-1464. Quail, D. F.; Joyce, J. A. Nat Med 2013, 19, 1423-1437. Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.; Jacks, T.; Anderson, D. G. Nat Rev Cancer 2011, 12, 39-50. Huang, B.; Abraham, W. D.; Zheng, Y.; Bustamante Lopez, S. C.; Luo, S. S.; Irvine, D. J. Sci Transl Med 2015, 7, 291ra94. Chen, Q.; Liang, C.; Wang, C.; Liu, Z. Adv Mater 2015, 27, 903-910. Chauhan, V. P.; Jain, R. K. Nat Mater 2013, 12, 958-962. Liang, C.; Xu, L. G.; Song, G. S.; Liu, Z. Chem Soc Rev 2016, 45, 6250-6269. Pelaz, B.; Alexiou, C.; Alvarez-Puebla, R. A.; Alves, F.; Andrews, A. M.; Ashraf, S.; Balogh, L. P.; Ballerini, L.; Bestetti, A.; Brendel, C.; Bosi, S.; Carril, M.; Chan, W. C. W.; Chen, C.; Chen, X.; Chen, X.; Cheng, Z.; Cui, D.; Du, J.; Dullin, C.; Escudero, A.; Feliu, N.; Gao, M.; George, M.; Gogotsi, Y.; Grünweller, A.; Gu, Z.; Halas, N. J.; Hampp, N.; Hartmann, R. K.; Hersam, M. C.; Hunziker, P.; Jian, J.; Jiang, X.; Jungebluth, P.; Kadhiresan, P.; Kataoka, K.; Khademhosseini, A.; Kopeček, J.; Kotov, N. A.; Krug, H. F.; Lee, D. S.; Lehr, C.-M.; Leong, K. W.; Liang, X.-J.; Ling Lim, M.; Liz-Marzán, L. M.; Ma, X.; Macchiarini, P.; Meng, H.; Möhwald, H.; Mulvaney, P.; Nel, A. E.; Nie, S.; Nordlander, P.; Okano, T.; Oliveira, J.; Park, T. H.; Penner, R. M.; Prato, M.; Puntes, V.; Rotello, V. M.; Samarakoon, A.; Schaak, R. E.; Shen, Y.; Sjöqvist, S.; Skirtach, A. G.; Soliman, M. G.; Stevens, M. M.; Sung, H.-W.; Tang, B. Z.; Tietze, R.; Udugama, B. N.; VanEpps, J. S.; Weil, T.; Weiss, P. S.; Willner, I.; Wu, Y.; Yang, L.; Yue, Z.; Zhang, Q.; Zhang, Q.; Zhang, X.-E.; Zhao, Y.; Zhou, X.; Parak, W. J. ACS Nano 2017, 11, 2313-2381. Armstrong, J. P. K.; Holme, M. N.; Stevens, M. M. ACS Nano 2017, 11, 69-83. Cao, H. Q.; Zou, L. L.; He, B.; Zeng, L. J.; Huang, Y. Z.; Yu, H. J.; Zhang, P. C.; Yin, Q.; Zhang, Z. W.; Li, Y. P. Adv Funct Mater 2017, 27, DOI: 10.1002/adfm.201605679. Cao, H. Q.; Dan, Z. L.; He, X. Y.; Zhang, Z. W.; Yu, H. J.; Yin, Q.; Li, Y. P. ACS Nano 2016, 10, 7738-7748. Kang, T.; Zhu, Q. Q.; Wei, D.; Feng, J. X.; Yao, J. H.; Jiang, T. Z.; Song, Q. X.; Wei, X. B.; Chen, H. Z.; Gao, X. L.; Chen, J. ACS Nano 2017, 11, 1397-1411. Cao, B. R.; Yang, M. Y.; Zhu, Y.; Qu, X. W.; Mao, C. B. Adv Mater 2014, 26, 46274631. Rodriguez, P. L.; Harada, T.; Christian, D. A.; Pantano, D. A.; Tsai, R. K.; Discher, D. E. Science 2013, 339, 971-975. Hu, Q. Y.; Sun, W. J.; Qian, C. G.; Wang, C.; Bomba, H. N.; Gu, Z. Adv Mater 2015, 27, 7043-7050. Choi, M. R.; Stanton-Maxey, K. J.; Stanley, J. K.; Levin, C. S.; Bardhan, R.; Akin, D.; Badve, S.; Sturgis, J.; Robinson, J. P.; Bashir, R.; Halas, N. J.; Clare, S. E. Nano Lett 2007, 7, 3759-3765. Sun, H.; Su, J.; Meng, Q.; Yin, Q.; Chen, L.; Gu, W.; Zhang, P.; Zhang, Z.; Yu, H.; Wang, S.; Li, Y. Adv Mater 2016, 28, 9581-9588. Guo, Y. Y.; Wang, D.; Song, Q. L.; Wu, T. T.; Zhuang, X. T.; Bao, Y. L.; Kong, M.; Qj, Y.; Tan, S. W.; Zhang, Z. P. ACS Nano 2015, 9, 6918-6933. Fang, R. H.; Hu, C. M. J.; Luk, B. T.; Gao, W. W.; Copp, J. A.; Tai, Y. Y.; O'Connor, D. E.; Zhang, L. F. Nano Lett 2014, 14, 2181-2188.


Nano Letters

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23.

24.

25. 26. 27. 28. 29. 30. 31. 32.

33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

44. 45. 46. 47.

Xu, C.; Miranda-Nieves, D.; Ankrum, J. A.; Matthiesen, M. E.; Phillips, J. A.; Roes, I.; Wojtkiewicz, G. R.; Juneja, V.; Kultima, J. R.; Zhao, W.; Vemula, P. K.; Lin, C. P.; Nahrendorf, M.; Karp, J. M. Nano Lett 2012, 12, 4131-4139. Kaneti, L.; Bronshtein, T.; Malkah Dayan, N.; Kovregina, I.; Letko Khait, N.; LupuHaber, Y.; Fliman, M.; Schoen, B. W.; Kaneti, G.; Machluf, M. Nano Lett 2016, 16, 1574-1582. Toledano Furman, N. E.; Lupu-Haber, Y.; Bronshtein, T.; Kaneti, L.; Letko, N.; Weinstein, E.; Baruch, L.; Machluf, M. Nano Lett 2013, 13, 3248-3255. Li, L.; Guan, Y.; Liu, H.; Hao, N.; Liu, T.; Meng, X.; Fu, C.; Li, Y.; Qu, Q.; Zhang, Y.; Ji, S.; Chen, L.; Chen, D.; Tang, F. ACS Nano 2011, 5, 7462-7470. Kang, S.; Bhang, S. H.; Hwang, S.; Yoon, J.-K.; Song, J.; Jang, H.K.; Kim, S.; Kim, B.S. ACS Nano 2015, 9, 9678-9690. Huang, X.; Zhang, F.; Wang, Y.; Sun, X.; Choi, K. Y.; Liu, D.; Choi, J.-s.; Shin, T.H.; Cheon, J.; Niu, G.; Chen, X. ACS Nano 2014, 8, 4403-4414. Chen, Q.; Zhang, X. H.; Massague, J. Cancer Cell 2011, 20, 538-549. Qian, B. Z.; Li, J.; Zhang, H.; Kitamura, T.; Zhang, J.; Campion, L. R.; Kaiser, E. A.; Snyder, L. A.; Pollard, J. W. Nature 2011, 475, 222-225. Bonapace, L.; Coissieux, M. M.; Wyckoff, J.; Mertz, K. D.; Varga, Z.; Junt, T.; Bentires-Alj, M. Nature 2014, 515, 130-133. Zhang, Y.; Yang, P.; Sun, T.; Li, D.; Xu, X.; Rui, Y.; Li, C.; Chong, M.; Ibrahim, T.; Mercatali, L.; Amadori, D.; Lu, X.; Xie, D.; Li, Q. J.; Wang, X. F. Nat Cell Biol 2013, 15, 284-294. Franklin, R. A.; Liao, W.; Sarkar, A.; Kim, M. V.; Bivona, M. R.; Liu, K.; Pamer, E. G.; Li, M. O. Science 2014, 344, 921-925. Kitamura, T.; Qian, B. Z.; Pollard, J. W. Nat Rev Immunol 2015, 15, 73-86. Geissmann, F.; Manz, M. G.; Jung, S.; Sieweke, M. H.; Merad, M.; Ley, K. Science 2010, 327, 656-661. Fliervoet, L. A. L.; Mastrobattista, E. Adv Drug Deliver Rev 2016, 106, 63-72. Liu, Z.; Xiong, M.; Gong, J. B.; Zhang, Y.; Bai, N.; Luo, Y. P.; Li, L. Y.; Wei, Y. Q.; Liu, Y. H.; Tan, X. Y.; Xiang, R. Nat Commun 2014, 5, 4280. Vandooren, J.; Opdenakker, G.; Loadman, P. M.; Edwards, D. R. Adv Drug Deliver Rev 2016, 97, 144-155. Jiang, Y. F.; Lu, J. Y.; Wang, Y. P.; Zeng, F.; Wang, H. Y.; Peng, H. G.; Huang, M.; Jiang, H. L.; Luo, C.; Huang, Y. Z. Adv Mater 2014, 26, 8174-8178. Luo, Y. P.; Zhou, H.; Krueger, J.; Kaplan, C.; Lee, S. H.; Dolman, C.; Markowitz, D.; Wu, W. Y.; Liu, C.; Reisfeld, R. A.; Xiang, R. J Clin Invest 2006, 116, 2132-2141. Solberg, R.; Smith, R.; Almlof, M.; Tewolde, E.; Nilsen, H.; Johansen, H. T. Biol Chem 2015, 396, 71-80. Liu, C.; Sun, C. Z.; Huang, H. N.; Janda, K.; Edgington, T. Cancer Res 2003, 63, 2957-2964. Tang, K.; Zhang, Y.; Zhang, H. F.; Xu, P. W.; Liu, J.; Ma, J. W.; Lv, M.; Li, D. P.; Katirai, F.; Shen, G. X.; Zhang, G. M.; Feng, Z. H.; Ye, D. Y.; Huang, B. Nat Commun 2012, 3, 1282. He, X. Y.; Bao, X. Y.; Cao, H. Q.; Zhang, Z. W.; Yin, Q.; Gu, W. W.; Chen, L. L.; Yu, H. J.; Li, Y. P. Adv Funct Mater 2015, 25, 2831-2839. He, X. Y.; Yu, H. J.; Bao, X. Y.; Cao, H. Q.; Yin, Q.; Zhang, Z. W.; Li, Y. P. Adv Healthc Mater 2016, 5, 439-448. Zhang, Z. W.; Cao, H. Q.; Jiang, S. J.; Liu, Z. Y.; He, X. Y.; Yu, H. J.; Li, Y. P. Small 2014, 10, 4735-4745. Jain, R. K.; Stylianopoulos, T. Nat Rev Clin Oncol 2010, 7, 653-664. 16 ACS Paragon Plus Environment

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48. 49. 50.

Sharma, S.; Kotamraju, V. R.; Molder, T.; Tobi, A.; Teesalu, T.; Ruoslahti, E. Nano Lett 2017, 17, 1356-1364. Zhou, H.; Fan, Z.; Deng, J.; Lemons, P. K.; Arhontoulis, D. C.; Bowne, W. B.; Cheng, H. Nano Lett 2016, 16, 3268-3277. Wei, T.; Liu, J.; Ma, H.; Cheng, Q.; Huang, Y.; Zhao, J.; Huo, S.; Xue, X.; Liang, Z.; Liang, X. J. Nano Lett 2013, 13, 2528-2534.


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Scheme 1 Schematic illustration of inflammatory monocytes loading legumain-sensitive nanoparticles (M-SMNs) to target lung metastasis of breast cancer and initiate metastaticspecific drug release to achieve the anti-metastasis efficacy. M-SMNs could be preferentially delivered to the sites of metastases due to the autonomous metastasis-homing effects of monocytes, and then activate the on-demand drug release as free drug molecules and drug loaded microvesicles upon the differentiation into macrophages, thereby enhancing the medical performance on breast cancer metastasis.


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Figure 1 Preparation and characterization of M-SMNs. (A) Schematic illustration of the preparation of M-SMNs and metastasis-triggered drug release from M-SMNs. (B) typical TEM images of SMNs in the absence and presence of legumain, scale bar=200 nm; (C) the enzymatic responsive drug release from SMNs at various legumain concentration, *p

Inflammatory Monocytes Loading Protease-Sensitive Nanoparticles

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