Bioengineered Macrophages Can Responsively Transform into

Jul 20, 2018 - *E-mail: [email protected] (Z.Z.)., *Phone/Fax: ... as living drug delivery vehicles with controlled drug release to target metast...
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Bioengineered macrophages can responsively transform into nanovesicles to target lung metastasis Haiqiang Cao, Hong Wang, Xinyu He, Tao Tan, Haiyan Hu, Zhiwan Wang, Jing Wang, Jie Li, Zhiwen Zhang, and Yaping Li Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b01236 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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Bioengineered macrophages can responsively transform into nanovesicles to target lung metastasis

Haiqiang Cao†,‡, §, Hong Wang†,‡, §, Xinyu He†,‡, Tao Tan†, Haiyan Hu†, Zhiwan Wang†,‡, Jing Wang†, Jie Li†, 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

School of Pharmacy, Yantai University, Yantai 264005, Shandong, China.

*Corresponding author: Prof. Zhiwen Zhang ([email protected]) and Prof. Yaping Li ([email protected]) Tel/Fax: +86-21-2023-1979

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KEYWORDS: Macrophage; Drug delivery; Cancer metastasis; Nanovesicles

ABSTRACT Specific drug delivery to metastatic tumors remains a great challenge for antimetastasis therapy. We herein report a bioengineered macrophage-based delivery system (LD-MDS) that can be preferentially delivered to lung metastases and intelligently transformed into nanovesicles and secondary nanovesicles for antimetastasis therapy. LD-MDS was prepared by anchoring a legumain-specific propeptide of melittin (legM) and cytotoxic soravtansine (DM4) prodrug onto the membrane of living macrophages. LD-MDS is responsively activated by legumain protease and converted into DM4-loaded exosome-like nanovesicles (DENs), facilitating efficient internalization by metastatic 4T1 cancer cells and considerable cell death. Afterwards, the damaged 4T1 cells can release secondary nanovesicles and free drug molecules to destroy neighboring cancer cells. In vivo, LD-MDS displays superior targeting efficiency for lung metastatic lesions with diameters less than 100 µm and remarkably inhibits lung metastasis. This study provides a new opportunity to explore endogenous macrophages as living drug delivery vehicles with controlled drug release to target metastatic lung tumors.

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Metastasis is the primary cause of the high mortality of breast cancer patients 1-3. Despite recent progress in cancer therapy, the cure rate of patients with metastatic diseases is still low 1

. A crucial challenge is that many drugs cannot reach metastasis sites due to their poor

vasculature, small size and high dispersion in multiple invaded organs 4-6. Nanoparticles have the best efficacy for targeted drug delivery to well-vascularized tumors, but their ability to reach to small unvascularized metastases is limited

7-17

. Recently, living cell-based delivery

systems have attracted increasing attention for tumor-specific delivery owing to their unique tumor-homing ability

18-24

. Compelling evidence has shown that macrophages can be

preferentially recruited by cancer cells to facilitate metastatic tumor establishment

25, 26

.

Moreover, macrophages are one of the most abundant types of circulating cells and comprise 27-29

up to 50% of cells in a tumor mass

. Thus, the use of natural living macrophages as drug

delivery carriers may be a potential strategy for metastasis-targeted drug delivery. The most crucial requirements of delivery are that the macrophage-based delivery system (MDS) should be in a living state in circulation to ensure their metastasis-homing capacity and a massive drug release upon their arrival at tumor sites

30-33

. Because most anticancer

drugs are highly cytotoxic agents, directly loading these drugs into macrophages will quickly kill the cells. Alternatively, loading nanoparticles into macrophages may be a feasible approach, but uncontrolled premature drug release from nanoparticles in macrophages will cause cell death or dysfunction before the cells arrive at the target site. Moreover, after the macrophages reach the targeted tumor, an efficient drug release mechanism is needed and remains an unsolved problem

30, 34

. Therefore, an MDS should be sophisticatedly designed

with precise control of the drug release before and after the macrophages reach the metastatic tumors. Cancer cells have been shown to communicate with neighboring stroma cells via nanometer-sized vesicles of exosomes to promote their metastasis

1, 35-37

. By harnessing the

highly upregulated legumain in metastatic tumors and the reductive milieu in cancer cells 38-40

21,

, we envision that living macrophages can be specifically engineered to undergo an 3 ACS Paragon Plus Environment

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intelligent transformation into exosome-like nanovesicles upon their arrival at metastatic sites for antimetastasis therapy. To confirm this assumption, we engineered macrophages with the legumain-specific propeptide melittin (legM) and the redox-sensitive prodrug of cytotoxic soravtansine (DM4) on the macrophage membrane to prepare legM- and DM4-loaded MDS (LD-MDS). Legumain is an asparaginyl endopeptidase that is highly upregulated in tumor microenvironments, especially tumor-associated macrophages, making it an ideal candidate for use in drug delivery carriers38. Legumain protease can specifically recognize a substrate peptide (NH2-alanine–alanine–asparagine-COOH, AAN) and efficiently cleave the bond at the COOH terminus of asparagine. Upon cleavage of the AAN substrate, the legM peptide in LD-MDS can transform into active melittin, facilitating the transformation of LD-MDS into nanovesicles for antimetastasis therapy. LD-MDS will facilitate metastasis-specific drug delivery in a unique manner (Scheme 1): (1) Living LD-MDS can be actively recruited to lung metastasis sites and then responsively transform into DM4-loaded exosome-like nanovesicles (DENs); (2) DENs can be efficiently internalized by metastatic 4T1 cancer cells and cause considerable cell death; (3) The 4T1 cells damaged by DENs can release secondary DM4 nanovesicles and free drug molecules to kill neighboring cancer cells, thereby transporting anticancer drugs to other lung metastasis sites. In this manuscript, the responsive transformation of living LD-MDS into nanovesicles was comprehensively characterized. The in vivo specific targeting of lung metastasis and therapeutic effects were evaluated to validate the feasibility of treating cancer metastasis. Initially, the LD-MDS was fabricated by anchoring the DM4 prodrug and legM peptide onto the surface of a macrophage membrane. In the LD-MDS, DM4 was used as a highly cytotoxic agent that can exert antitumor effects by disrupting microtubule assemblies, and legM was used as a response-regulation agent to induce the formation of exosome-like 4 ACS Paragon Plus Environment

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nanovesicles from the LD-MDS at metastatic sites. To ensure their loading into the macrophages, DM4 was linked to 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-Nmethoxy (polyethylene glycol) (DMPE-PEG) via disulfide bonds to form the redox-sensitive prodrug DMPE-PEG-S-S-DM4 (Supporting information, Figure S1-3), while the legM peptide was conjugated with DMPE-PEG to form the DMPE-PEG-legM conjugate, as previously described 41. The macrophages were derived from myeloid progenitor cells in bone marrow from healthy nude mice, incubated with macrophage colony-stimulating factor (MCSF) and conditioned in culture media of metastatic 4T1 breast cancer cells, and the macrophages were determined to be the M2 phenotype by measuring the expression of typical markers including F4/80, CD11b and CD206 (Figure 1A). M2 macrophages are one of the major components of stromal cells in tumor and can prime tumor microenvironments to facilitate tumor progression and metastasis.28,

42

Moreover, macrophages can interact with

breast cancer cells to promote their metastasis in the lungs,25 making them a good candidate delivery vehicle to target lung metastasis. These conjugates were directly inserted into the lipid bilayers of the macrophage membrane via the DMPE segments to prepare the LD-MDS. For comparison, the insertion of DMPE-PEG-legM or DMPE-PEG-S-S-DM4 into the macrophage membrane was also performed to prepare the legM-loaded macrophage system (legM-MDS) or DM4-loaded macrophage system (DM4-MDS). The insertion of these conjugates into the macrophage membrane was evaluated using laser confocal scanning microscopy (LCSM). The fluorescence signals of the Cyanine5-linked DMPE-PEG conjugate (DMPE-PEG-Cy5) could be readily visualized on the surface of the macrophages (Figure 1B), suggesting effective anchoring onto the macrophage membrane was accomplished. The loading capacity of DM4 in LD-MDS depended on the incubation concentration (Table S1). When the macrophages were incubated with 1.0 mg/mL DMPE-PEG-S-S-DM4 for 2 h, the DM4 loaded in the LD-MDS was approximately 2.0 µg per million cells, which was determined by high-performance liquid chromatography (HPLC). 5 ACS Paragon Plus Environment

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As expected, the LD-MDS should be in a living state in blood circulation and responsively release the anticancer drugs upon exposure to legumain at metastatic sites to result in therapeutic effects. The death or lysis of macrophages is an efficient approach to ensure drug release. Once the legM in the LD-MDS is transformed by legumain into active melittin and reaches a sufficient concentration, it causes extensive damage to the carrier macrophages, resulting in the release of the anticancer drugs. The transformation greatly depends on the legM amount in the LD-MDS and the legumain incubation concentration. When the LD-MDS was incubated with 400 ng/mL of legumain for 24 h, the number of dead macrophages significantly increased with the concentration of DMPE-PEG-legM during the preparation of the LD-MDS (Figure S4). Moreover, the number of dead macrophages in the LD-MDS was evidently augmented with legumain concentrations ranging from 50 ng/mL to 400 ng/mL (Figure S5). In addition, the serum legumain concentration in healthy nude mice and lung metastatic mice model was respectively around 4.1±0.3 ng/mL and 6.0±0.4 ng/mL. When LD-MDS were incubated with leugmain at 7 ng/mL for 6 h, the damages of carrier macrophages were barely detected (Figure S6), ensuring their living state in blood circulation. Then, the live/dead states of the macrophages in the LD-MDS, legM-MDS and DM4-MDS groups were measured after 6 h and 24 h of incubation with the culture media without legumain, culture media with legumain (400 ng/mL) and conditioned media from 4T1 cells (Figure 1C and Figure S7) to confirm their responsiveness to legumain. After 6 h of incubation, the macrophages in the LD-MDS and legM-MDS showed high viability in the culture media without legumain and the conditioned media from 4T1 cells, but the macrophages were largely damaged in the culture media with legumain (400 ng/mL), suggesting their specific response to the legumain protease. The remarkable reduction of the cell numbers and cell death can be attributed to the lysis or death of the carrier macrophages by the activated melittin. After 24 h of incubation, a remarkable reduction in the macrophage numbers and cell death were observed in the LD-MDS and legM-MDS for all three media 6 ACS Paragon Plus Environment

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types (Figure S7), which ensures responsive drug release at metastatic sites. The cell death in the LD-MDS and legM-MDS in media without legumain and the conditioned media from 4T1 cells can be ascribed to the legumain secreted by the carrier macrophages. In contrast, cell damage was barely detectable in the DM4-MDS group. Accordingly, the damage of macrophages in the LD-MDS can mainly be ascribed to the legM peptide rather than the DM4 prodrug. The legM peptide in the LD-MDS can be specifically activated by the legumain protease into active melittin, which can disrupt macrophages due to its pore-forming activity and lead to drug release at metastatic sites. Notably, active melittin can induce large phospholipid vesicles to bud into nanometersized vesicles due to its pore-forming ability 43, which can presumably cause the LD-MDS to transform into DENs after specific activation by legumain protease. The possibility of the LDMDS transforming into DENs was investigated after their activation (Figure 1D). The total culture media from an activated LD-MDS was harvested and centrifuged in a programmed manner to collect the supernatant and sediments in the bottom of the centrifuge tube. The sediments were resuspended in a phosphate-buffered solution (PBS, pH 7.4) and analyzed by dynamic light scattering (DLS) and field emission transmission electronic microscopy (FETEM) (Figure 1E and F). The DLS analysis showed a hydrodynamic diameter of 113.8±34.8 nm, and the morphology measurements by FE-TEM also verified the existence of spherical nanometer particles, which confirmed the activated LD-MDS formed nanometer-size vesicles. Afterwards, we determined whether cytotoxic DM4 was released as free drug molecules in the supernatant or into the DENs. The drug concentration in the supernatant, DENs and total cell lysates was determined by HPLC. The DM4 peak signals were detected in the total cell lysates and DENs, but they were not detected in the supernatant (Figure 1G and Figure S8). Moreover, the drug concentration in the DENs was responsible for approximately 96.72% of that of the total cell lysates, indicating that the DM4 in the LD-MDS was predominantly released as nanovesicles in DENs rather than free drug molecules in the supernatant (Figure 7 ACS Paragon Plus Environment

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1H). As denoted by the DM4 content, the efficiency of converting the LD-MDS into DENs was 85.9±1.2%. Therefore, DENs were released from the LD-MDS upon activation by legumain, providing a platform for further antimetastasis therapy. The inhibition of cell viability and migration activity by the LD-MDS was measured in metastatic 4T1 breast cancer cells using transwell-mediated assays. For the cytotoxicity assay, various groups of macrophages, legM-MDS, DM4-MDS and LD-MDS were added to the upper chambers of inserts (pore size, 0.8 µm) (equivalent to 666.7 ng/mL of DM4), while 4T1 cells were seed to each well of the plate (Figure 2A and Figure S9). The small pore size of the transwell membrane (0.8 µm) only permits the cancer cells to interact with the DENs rather than the whole MDS. Figure 2A shows that the viability of the 4T1 cells obviously decreased by 61.5% in the LD-MDS treated group but was not affected in the other groups. Similarly, to evaluate the cell migration inhibition, 4T1 cells were seeded in the upper chambers of the transwell inserts (pore size, 8 µm), while various groups of MDS were added to each well of the plate (Figure S10). To avoid the possible interference of LD-MDS mediated cytotoxicity on the antimigration assay and clarify their suppression on the migratory capacity of cancer cells, the 4T1 cancer cells were treated with lower concentration of LD-MDS (equivalent to 13.3 ng/mL of DM4). At this concentration, the number of 4T1 cells did not obviously decrease in the LD-MDS group compared to that in the negative control group (Figure S11), excluding the possible interference of LD-MDS mediated cell death on their antimigration activities. The large pore size of the transwell membrane (8.0 µm) allows free migration of the cancer cells for the antimigration assay. The cells that migrated across the transwell membrane were scarcely visible for the LD-MDS groups but extensively observed for the other groups (Figure S11). The LD-MDS treatment resulted in a 98.8% inhibition of cell migration, which was enormously higher than that of the other groups (Figure 2B). As a result, the LD-MDS treatment could cause considerable cell death and remarkably suppress the migratory capacity of cancer cells, demonstrating its great potential for antimetastasis therapy. 8 ACS Paragon Plus Environment

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The LD-MDS can be intelligently transformed into DENs upon exposure to legumain protease. To clarify the possible mechanism of LD-MDS in antimetastasis therapy, the cellular uptake of the DENs was evaluated in 4T1 cells by LCSM (Figure 2C). Fluorescent DENs were derived from the DiR (1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide)-loaded LD-MDS and incubated with 4T1 cells before the measurements. After 2 h of incubation, the red fluorescence signals of the DENs could be detected with a strong intensity, indicating efficient internalization by the 4T1 cells (Figure 2C). DENs have been identified as nanometer-sized vesicles from macrophage membranes, and their exosome-like properties would facilitate interaction with 4T1 cancer cells. Additionally, melittin can be effectively taken up by cancer cells via receptor-mediated endocytosis 44. Thereby, the active melittin in DENs can promote their internalization by 4T1 cells to exert anticancer activity. Moreover, the antiproliferation activities of the DENs, supernatant and total media from the activated LD-MDS were measured in 4T1 cells (Figure 2D and E). The total media refers to the cultured media collected from the activated LD-MDS, which can be centrifuged to collect the DENs and supernatant for measurements. The DENs were resupsended in the culture media with a volume equal to that of the supernatant. Both the DENs and total media displayed concentration-dependent, significant inhibition of cell viability, but the supernatant showed negligible inhibition of cell viability (Figure 2D). Typically, 20 µL of DENs (equal to 0.84 µg/mL of DM4) resulted in a 93.1% inhibition of cell proliferation, which was comparable to that observed for the total media group. The distinguished inhibitory effect of the DENs may be due to the predominant loading of DM4 into the DENs after activation of the LD-MDS. Meanwhile, the live/dead assay indicated that the cell numbers in the DEN and total media treatment groups were markedly reduced compared to those of the supernatant groups (Figure 2E), which can be ascribed to the efficient antiproliferative activity of DM4. Moreover, the inhibitory effects of the DENs were much more effective than those of free

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legM and DM4 at equivalent doses (Figure 2F). These results confirmed the prominent therapeutic effects of the DENs that were released from the LD-MDS upon activation. Of note, cancer cells incubated with chemotherapeutic drugs can package the drugs into microvesicles upon their death

45

. After the 4T1 cells were damaged by the DENs, we

attempted to clarify whether cytotoxic DM4 would be released as drug-loaded secondary microvesicles or free drug molecules (Figure 2G and H). The 4T1 cells were incubated with the DENs for 8 h, and then, the medium was removed and replaced with fresh culture medium for an additional 24 h of incubation. Afterwards, the media were collected to separate the supernatant and sediments as described above for further analysis. The DLS analysis confirmed the existence of nanometer-sized vesicles with a mean diameter of 138.7±77.9 nm (Figure S12). Meanwhile, in the FE-TEM images, spherical nanometric particles can be distinctly observed (Figure 2G). These results verified the formation of secondary DM4loaded nanovesicles (DNs) upon 4T1 cell damage. The HPLC analysis showed that the proportion of DM4 was 65.05% in the supernatant and 36.41% in the secondary DNs relative to the total drug amount in the media (Figure 2H). Thus, cytotoxic DM4 is released from the DEN-damaged 4T1 cells as free drug molecules and secondary DNs at a ratio of 1.8:1. The secondary DNs can be further internalized by other 4T1 cancer cells to allow additional therapeutic effects (Figure S13). The cytotoxicity of the supernatant and secondary DNs from the 4T1 cells was retested in 4T1 cells. In Figure 2I, both the supernatant and secondary DNs demonstrate considerable inhibition of the viability of 4T1 cells in a concentration-dependent manner. In summary, the LD-MDS can be specifically transformed into DENs in respond to legumain protease, which causes extensive damage to 4T1 cells after internalization. Then, cytotoxic DM4 can be released from the DEN-damaged 4T1 cells as secondary DNs and free drug molecules to kill neighboring cancer cells (Figure 2J). Importantly, the specific targeting of the LD-MDS for metastatic tumors was measured in a lung metastatic breast cancer model, which was induced by tail vein injection of 4T1 cells 10 ACS Paragon Plus Environment

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with stable expression of green fluorescence protein (GFP). For imaging, the living macrophages in legM-MDS, DM4-MDS and LD-MDS were labeled with the near-infrared membrane probe of DiR. At 6.0 h after tail vein injection of these formulations, the mice were sacrificed, and their major organs were collected for imaging under an in vivo imaging system (Spectrum, Perkin-Elmer). The fluorescence signals were easily detected in the liver, lung and spleen but barely detected in other organs (Figure S14). In lungs with metastatic tumors, the four groups showed no significant differences. The specific targeting of the LD-MDS for metastatic lesions in lungs was determined by LCSM (Figure 3). In the captured images, the metastatic lesions are denoted as cell clusters with green signals, and the MDS is represented by red signals. In each group, the sites of the red signals from the MDS coincide with the green fluorescence of the metastatic lesions, even when the diameter of the metastatic lesion is less than 100 µm. Throughout the lung tissues (Figure 3A), when green cell clusters were present, the red signals of MDS were also present. Moreover, the MDS signals were extensively detected in all metastatic foci regions, including interior sides and exterior surfaces, suggesting efficient permeation throughout the whole metastatic lesion. In the enlarged images (Figure 3B), bright yellow signals, which merge with the red signals of the MDS and green color of the cancer cells, can be observed in the legM-MDS and LD-MDS groups, but these signals are only slightly detected in the macrophage and DM4-MDS groups. The formation of DENs has been evidenced by TEM and DLS measurements (Figure 1E-H), moreover, the internalization of DiR-loaded DENs from DiR-labeled LD-MDS has been evaluated in 4T1 cancer cells (Figure 2C). In view of the highly upregulated legumain in metastatic niches, the legM peptide in the LD-MDS and legM-MDS groups can be transformed into active melittin, resulting in the formation of DiR-loaded exosome-like nanovesicles. Rationally, the yellow signals could be considered as the efficient internalization of DiR-labeled nanovesicles from LD-MDS or legM-MDS by GFP-expressing 4T1 cancer cells in metastatic foci. In contrast, in macrophages and DM4-MDS, the legM 11 ACS Paragon Plus Environment

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deficiency limits the formation of exosome-like nanovesicles, resulting in fewer yellow signals. The results show that the LD-MDS can be efficiently delivered to metastatic lesion sites with a deep permeation capacity and then be transformed into DENs to facilitate their internalization by cancer cells, which will undoubtedly provide an essential platform for future antimetastasis therapy. Finally, the in vivo antimetastasis efficacy of the LD-MDS was measured in a lung metastatic breast cancer model (Figure 4). The tumor-bearing mice were nude mice with an immune-deficiency to avoid immunogenicity interferences, and they were treated with saline, macrophages, legM-MDS, DM4-MDS and LD-MDS with 0.5 mg/kg of DM4 (approximately 10 µg per mouse) or an equivalent number of macrophages (approximately five million macrophages per mouse) via tail vein injection. During these treatments, variations in the body weight in each group were not detectable (Figure S15). On day 14 after inoculation, the mice were autopsied, and their lung tissues were collected to evaluate inhibitory effects on lung metastasis (Figure 4A, C and Figure S16). Metastatic nodules were extensively detected in the macrophage and legM-MDS groups, indicating their inability to suppress lung metastasis. In contrast, the incidence of lung metastasis slightly decreased by 29.6% in the DM4-MDS treated group, suggesting its potential for suppressing cancer metastasis (Figure 4A-C). In the LD-MDS group, the average number of lung metastatic nodules was markedly reduced to 6.2±3.1, which was only 10.5%, 11.4% and 19.0% of the nodules observed in the free macrophage, legM-MDS and DM4-MDS groups, respectively. Compared to the saline control, the LD-MDS treatment resulted in an 86.7% inhibition of lung metastasis, which was remarkably higher than that of the DM4-MDS (Figure 4B). Moreover, histological examinations of the lung tissues were performed using hematoxylin and eosin (H&E) staining assays. In the whole lung tissue sections, metastatic lesions were rarely visualized in the LDMDS group but were detected in the other four groups (Figure 4D). In addition, histological examinations of other organs, including the heart, liver, spleen and kidneys, were performed 12 ACS Paragon Plus Environment

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to confirm the good compatibility of these treatments (Figure S17). The incidence of lung metastasis was considerably suppressed by the LD-MDS. In view of the ineffectiveness of the legM-MDS and the mild inhibitory effect of DM4-MDS alone, the notable inhibition of LDMDS on lung metastasis can be mainly ascribed to the synergistic combination of legM and DM4 in macrophages, which can intelligently transform into DENs upon arriving at metastatic niches to exert anticancer activity. In summary, we developed a living macrophage-based delivery system (LD-MDS) that can specifically target metastatic lesions in lungs with controlled drug release for antimetastasis therapy. Harnessing the highly expressed legumain in metastatic niches, the LD-MDS can be specifically transformed into exosome-like nanovesicles of DENs, which are efficiently internalized by cancer cells and cause extensive cell damage. Afterwards, cytotoxic DM4 is released from the damaged 4T1 cancer cells as secondary DNs and free drug molecules to kill neighboring cancer cells, delivering anticancer drugs to additional cells. In a lung metastatic model, the LD-MDS treatment notably inhibited the incidence of lung metastases. Therefore, this study provides a new opportunity to explore endogenous macrophages as living drug delivery vehicles with controlled drug release to target metastatic tumors and can avoid the drawbacks of traditional nanomedicine. Moreover, this approach may hold great potential to facilitate clinical translation by utilizing macrophages from cancer patients for future antimetastasis therapy.

ASSOCIATED CONTENT Supporting Information Additional data, including supplementary methods, in vitro characterizations, in vitro inhibition of cell viability and migration activity of metastatic 4T1 cells, ex vivo distribution and in vivo therapeutic results, is available free of charge via the internet at http://pubs.acs.org. 13 ACS Paragon Plus Environment

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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions § H.

Cao and H. Wang contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2015CB932103), the National Natural Science Foundation of China (31771092, 81521005, 81690265), the Youth Innovation Promotion Association of CAS and the Shanghai Sailing Program (18YF1428400).

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Scheme 1 Schematic illustration of the bioengineered macrophage delivery system (LDMDS) for specific targeting of lung metastasis. The LD-MDS is preferentially recruited to lung metastases and responsively transformed into nanovesicles and secondary nanovesicles for an effective antimetastasis therapy. (I) Fabrication of the LD-MDS and its responsive transformation into nanovesicles in response to legumain protease; (II) The specific targeting of LD-MDS for lung metastases and drug delivery to cancer cells.

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Figure 1 Fabrication and characterization of the LD-MDS. (A) The expression of typical markers in macrophages determined by flow cytometry; (B) Macrophages stained with CFSE and inserted with DMPE-PEG-Cy5 were imaged with LCSM, scale bar=20 µm; (C) The responsiveness of the LD-MDS to legumain, which was determined by live/dead assays, scale bar=200 µm; (D) Schematic illustration of the transformation of the LD-MDS into DENs; (E) The size distribution of the DENs derived from the activated LD-MDS; (F) The typical TEM images of DENs from activated LD-MDS, scale bar=100 nm. (G) The typical HPLC spectra of DM4 in the supernatant and DENs from the activated LD-MDS; red arrow denotes the peak of DM4 in the HPLC profiles; (H) The percentage of DM4 in the supernatant and DENs from the activated LD-MDS.

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Figure 2 In vitro therapeutic effects and possible mechanism of the LD-MDS. (A) The inhibitory effects of the LD-MDS on the viability of metastatic 4T1 cancer cells,**p