Nanoparticles Coated with Neutrophil Membranes ... - ACS Publications

Nanoparticles Coated with Neutrophil Membranes Can Effectively Treat Cancer Metastasis Ting Kang,† Qianqian Zhu,† Dan Wei,‡ Jingxian Feng,† Jianhui Yao,† Tianze Jiang,† Qingxiang Song,§ Xunbin Wei,‡ Hongzhuan Chen,§ Xiaoling Gao,*,§ and Jun Chen*,† †

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Lane 826, Zhangheng Road, Shanghai 201203, P.R. China ‡ Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, P.R. China § Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, P.R. China S Supporting Information *

ABSTRACT: The dissemination, seeding, and colonization of circulating tumor cells (CTCs) serve as the root of distant metastasis. As a key step in the early stage of metastasis formation, colonization of CTCs in the (pre)metastatic niche appears to be a valuable target. Evidence showed that inflammatory neutrophils possess both a CTCand niche-targeting property by the intrinsic cell adhesion molecules on neutrophils. Inspired by this mechanism, we developed a nanosize neutrophil-mimicking drug delivery system (NM-NP) by coating neutrophils membranes on the surface of poly(latic-co-glycolic acid) nanoparticles (NPs). The membrane-associated protein cocktails on neutrophils membrane were mostly translocated to the surface of NM-NP via a nondisruptive approach, and the biobinding activity of neutrophils was highly preserved. Compared with uncoated NP, NM-NP exhibited enhanced cellular association in 4T1 cell models under shear flow in vitro, much higher CTCcapture efficiency in vivo, and improved homing to the premetastatic niche. Following loading with carfilzomib, a second generation of proteasome inhibitor, the NM-NP-based nanoformulation (NM-NP-CFZ) selectively depleted CTCs in the blood, prevented early metastasis and potentially inhibited the progress of already-formed metastasis. Our NP design can neutralize CTCs in the circulation and inhibit the formation of a metastatic niche. KEYWORDS: circulating tumor cells, premetastatic niche, metastasis prevention, drug delivery, neutrophil-mimicking nanoparticles

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Following the dissemination of CTCs from primary sites, a CTC only survives and proliferates in certain organs under favorable microenvironment (soil).9 Such prepared “soil” for the “seeding” of CTC, termed as a “(pre-)metastatic niche”, attracts a CTC to adhere and proliferate with the engagement of inflammatory cells, chemokines, and cytokines.10,11 Therefore, besides CTCs in circulation, we speculated that the premetastatic niche where the seeding and colonization process of CTCs happened would be a more desirable target for the management of cancer metastasis. Very recent progress in metastasis research has shed light on the mysterious

etastasis contributes to over 90% of cancer-related mortality in clinic.1,2 The dissemination, seeding, and colonization of circulating tumor cells (CTCs) serve as the root of distant metastasis. CTCs have emerged as valuable diagnostic tools and therapeutic targets for metastasis prevention. Manipulation of CTCs in the circulation has been technically intractable until the development of CTC-targeted nanomedicine,3 and an antiepithelial cell adhesion molecule (EpCAM) antibody was developed for targeting CTCs or interfering with the interaction between CTCs and the endothelium.4,5 However, the specificity and efficiency of these EpCAM-targeted nanomedicine can be greatly challenged by cancer heterogeneity.6 To address the problem, platelet “camouflaged” approaches have been developed to target CTCs and its microthrombi in circulation.7,8 © 2017 American Chemical Society

Received: September 27, 2016 Accepted: January 11, 2017 Published: January 11, 2017 1397

DOI: 10.1021/acsnano.6b06477 ACS Nano 2017, 11, 1397−1411

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ACS Nano composition of premetastatic niche, in which bulk of neutrophils play fundamental roles in the early stage of the niche formation.12 First, tumor-derived secreted factors and myeloid cells (precursors of neutrophils) initiate the formation of the premetastatic niche.13 Second, activated neutrophils together with CTCs are continuously recruited to the premetastatic niche, in response to a granulocyte colonystimulating factor in the chronic inflammatory microenviron̈ neutrophils can exert CTC ment of the niche. Although naive cytotoxicity after activation, increasing evidence shows that these neutrophils exhibiting a pro-inflammatory phenotype can interact with CTCs in circulation and promote tumor extravasation via the binding of adhesion molecules and the formation of neutrophil extracellular traps (NETs),14,15 suggesting that inflammatory neutrophils possess a CTCtargeting property during the multiple steps in the metastatic cascade.16,17 In coincidence, the mechanism underlying the CTCs and niche-targeting nature of neutrophils depend largely on the distinct adhesion molecules associated with the membrane of neutrophils, which orchestrated synergistically to mediate the inflammatory process that bridge and facilitate CTCs seeding in the premetastatic niche.18 The direct application of neutrophils as a carrier for drug delivery is limited, as the method for in vitro cultivation is currently unavailable because neutrophils are terminal-differentiated cells with only a 7 h half-life span.19 The emerging technique for the development of a biomimetic nanoplatform by endowing synthetic nanoparticles (NPs) with cell-mimicking ̈ targeting abilities represents a promising potential and naive solution to this problem. Biomimetic nanoplatforms have been constructed for targeted chemotherapeutics delivery,8 toxicant neutralization,20 and vaccination development.21−23 Herein, we designed neutrophil-mimicking nanoparticles (NM-NPs) by cloaking the surface of a biodegradable poly(D,L-lactic-coglycolic acid) (PLGA) NP with an inflammatory neutrophilderived membrane (Scheme 1). As a proof of concept, we hypothesize that the cocktail of abundant proteins of neutrophils membrane grafting on NPs would enable NMNPs with “super neutrophils” property to continuously target CTCs in circulation and home to their relevant distant colony. By loading with carfilzomib (CFZ), a second-generation proteasome inhibitor, the carfilzomib-loaded NM-NPs (NMNP-CFZ) hold therapeutic potential for both preventing the de novo metastasis and inhibiting the already-formed metastasis.24 Also from a translational perspective, the top-down controlled approach using the patients’ own neutrophils membrane will provide the “camouflaged” NM-NPs with minimum immunogenicity.

Scheme 1. Schematic Illustration of NM-NPs Loaded with Carfilzomib (NM-NP-CFZ) That Selectively Deplete CTCs and Their Site of Colonizationa

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(A) Protocol for the synthesis of NM-NP-CFZ. (I) Neutrophils extraction from the whole blood using Percoll gradient separation method. (II) Lipopolysaccharide (LPS) stimulation of the isolated neutrophils. (III) Plasma membrane of the LPS-stimulated neutrophils isolated by centrifugation. (IV) NM-NP-CFZ was synthesized by coating the plasma membrane of the LPS-stimulated neutrophils on the poly(latic-co-glycolic acid) (PLGA) NPs. The cocktail of neutrophils membrane-associated proteins enables the resulting NMNP-CFZ to target CTCs in circulation and inflamed endothelial cells in the premetastatic lesion. Three pairs of key interactions including the binding of LFA-1 with ICAM-1, CD44 with L-selectin, and β1 integrin with VCAM-1 were involved in the CTC- and inflamed endothelium-targeting of NM-NPs.

evaporation method as described previously.26 And last, the coating of neutrophils membrane on the surface of NM-NPCFZ was accomplished by mixing them together and subjecting the mixture to sonication with the excess NMVs removed by centrifugation. Average size and zeta potentials of NMVs, NPCFZ, and NM-NP-CFZ were determined by dynamic light scattering (DLS) (Figure 1A and Table S1). Transmission microscopy (TEM) analysis found a single dimmer neutrophil membrane layer (≈10 nm) on the surface of NM-NP-CFZ (Figure 1B, c), which was not seen on that of NP-CFZ (Figure 1B, a). Owing to the membrane cloaking, compared with NPCFZ, NM-NP-CFZ possessed a slight elevation of 10−20 nm in size as detected by DLS. The zeta potential of NM-NP-CFZ was slightly reduced and more close to the zeta potential value of NMVs, confirming the formation of a biomimetic surface charge. Hydrophobic interaction and electrostatic repulsion between the negatively charged PLGA cores and the cellular membrane were considered as the key mechanisms that enable the formation of the structure of polymeric NPs with cell membrane cloaking.27 BCA assay revealed a rather high coating efficiency of the neutrophil membrane on PLGA NPs at the membrane-to-polymer ratio of 1:1 (Figure S1). Similar with surface PEGylation, this membrane coating enhances the NPs stability and prevents their aggregation in both PBS and PBS with 30% serum (Figure S2). Stability of the coating of the neutrophil membrane on the PLGA cores under biological condition was also evaluated by determining their colocalization

RESULTS AND DISCUSSION Synthesis and Characterization of NM-NPs. The synthetic process was divided into three steps. First, neutrophils were highly purified, activated with lipopolysaccharide (LPS), a TLR-4 ligand to induce an inflammatory response in neutrophils. The corresponding neutrophils were then isolated via a Percoll gradient neutrophil separation method according to the density reported (1.09 g/mL).25 Such a method has been reported to be liable for mouse neutrophils isolation with a high purity of neutrophils obtained and negligible damage to their biobinding activity. Neutrophil-derived membrane vesicles (NMVs) were then isolated from the neutrophils homogenate via a series of centrifugations. Second, PLGA NPs loaded with CFZ (NP-CFZ) were prepared via the emulsion/solvent 1398


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Figure 1. Characterization of NM-NP. (A) Size distribution, Z-average diameter, and zeta potentials of CFZ-loaded particles (NP-CFZ, NMNP-CFZ) and NMV in buffer measured by DLS. (B) Morphology of (a) bare PLGA NPs, (b) NMVs, and (c) NM-NPs. Scale bar, 100 nm. (C) Stability of NM-NPs following its internalization in 4T1 cells at 37 °C for 2 h: (a) fluorescein-labeled neutrophil membranes (green); (b) DiD-labeled PLGA core (red); (c) Hoechst stained nuclei; and (d) colocalization (yellow) of the green and red fluorescent signals, indicating that NM-NPs kept stable in the biological environment of 4T1 cells. Scale bars, 10 μm.

endothelium.30 In order to confirm if these functional adhesion proteins were highly preserved, Western blot assay was conducted. Several representative adhesion proteins (L-selectin, LFA-1, β1 integrin, CXCR4), which were derived from a neutrophils membrane, were all detected on NM-NPs (Figure 2B). To endow the neutrophils with maximum adhesion molecules expression, LPS, a stimulus reported to play essential roles in neutrophils activation, was used to treat neutrophils before the membrane isolation. Western analysis showed that the expression levels of L-selectin and LFA-1 (αLβ2 integrin) on LPS-activated neutrophils were much higher than those of untreated neutrophils, except for CXCR4 (Figure 2B,C). As the relative levels of membrane proteins could be changed during the formulation process, these discrepancies indicated that variation in source cells and stimulation method could change the quality of NM-NP. The amount of most of the adhesion protein detected on NM-NP derived from the LPS-activated neutrophils was higher than that from the untreated neutrophils. Notably, before LPS stimulation, two bands for β1 integrin were detected, suggesting that degradation of the protein occurs during the homogenization process, owing to the activation of proteases and phospholipases. After LPS stimulation, only one band for β1 integrin was detected on neutrophils and NMVs, probably because the LPS mediated integrin activation with higher β1 integrin stabilization and clustering and thereby minimized protease degradation. Taken together, most adhesion proteins were enriched on NM-NPs derived from LPS-activated neutrophils, providing a prerequisite for the neutrophil-mimicking properties of NM-NPs. Specific Targeting of NM-NPs to CTCs in Vitro. For malignant transformation, glycoproteins and glycans like CD44 and carbohydrate sialyl Lewis X are found aberrantly expressed in tumor cells and serve as ligands of selectins to promote metastasis.31 Recent studies have also shown that breast cancer

following cellular internalization (Figure 1C). Notably, colocalization (yellow) of fluorescein-labeled membrane with DiD-labeled core in 4T1 cells revealed that the binding of membrane and hydrophobic PLGA core kept stable upon cellular internalization (Figure 1C, d) Similar to that of NP-CFZ and PLGA-PEG-NP, the drugloading capacity and encapsulation efficacy of NM-NP-CFZ were found to be 3.74 ± 0.28% and 39.38 ± 5.32%, respectively, as determined via the HPLC method previously described (Table S1).28 As the cytoplasm and nuclei were deprived during the membrane isolation process, the biological functions of neutrophils such as phagocytosis to digest bacteria were lost during the formulation process. The aim of this study was to mimic the biobinding properties of neutrophils, which were mediated by the abundant molecules on the neutrophils membrane.29 To see if the proteins on the neutrophils membrane can be fully translocated to the surface of PLGA NPs via our method, protein content was measured by Coomassie blue assay (Figure 2A). Compared with the whole neutrophils sample, neutrophils membrane materials resulted in a modulated protein profile with low-weight molecules removed after membrane isolation. Crucially, the profile of proteins tracked on NM-NP was similar to that of the neutrophils membrane, suggesting that our method can translocate most of the protein of a neutrophils membrane to the surface of PLGA NPs. Meanwhile, neutrophils serve as crucial mediators that regulate the interactions between CTCs and the premetastatic endothelium. To achieve this, coordinated corporation needs to be accomplished among the various adhesion molecules and chemotaxis signaling molecules that expressed on the membrane of neutrophils such as selectins, α2, β1 integrins, and chemokine receptors, which conjointly act as linkers for the interaction between CTCs and the inflamed 1399


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Figure 3. Specific targeting of NM-NP to CTC in vitro. (A) Fluorescent images of 4T1 cells following the incubation with 400 μg/mL coumarin-6-labeled NPs and NM-NPs under a static condition or sheared condition (in a cone-and-plate viscometer at the rate of 188 s−1) at 37 °C for 2 h. Scale bar, 50 μm. (B) Quantitative analysis of cellular association of coumarin-6-labeled NPs and NM-NPs in 4T1 cells at various concentrations from 50 to 800 μg/mL for 2 h at 37 and 4 °C, respectively. Data represented mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 significantly higher than the association of NPs in static 4T1 cells for 2 h at 37 °C. (C) Quantitative analysis of cellular association of coumarin-6-labeled NPs and NM-NPs in sheared 4T1 cells (in a cone-and-plate viscometer at various concentrations from 50 to 800 μg/mL for 2 h at 37 and 4 °C, respectively. Data represented mean ± SD (n = 3). #p < 0.05, ##p < 0.01, ###p < 0.001 significantly higher than the association of sheared NPs in flowing 4T1 cells for 2 h at 37 °C. (D) Cellular association of coumarin-6-labeled NPs and NM-NPs in 4T1 cells following pre-incubation with various endocytosis inhibitors. Fluorescence intensity in the noninhibited cells, representing the maximum internalized amount of coumarin6-labeled NPs, was taken as control. Data represented mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 significantly lower than that of NPs control. Ip < 0.05, ##p < 0.01, ###p < 0.001 significantly lower than that of NM-NPs control.

Figure 2. Characterization of membrane proteins on NM-NPs. Samples of the associated membranes and membrane-coated NMNPs were prepared from neutrophils with and without LPS stimulation for 4 h, respectively. (A) SDS-PAGE protein tracking of the neutrophils membrane, NM-NPs, and neutrophils, respectively. An equal concentration of protein was added to each well, and total proteins were stained by Coomassie blue. (B) Western-blot identification and comparison of membrane-associated adhesive proteins including LFA-1, L-selectin, β1 integrin, CXCR4, with and without LPS stimulation. (C) Quantitative analysis of expression levels of four proteins on a neutrophils membrane, NM-NPs, and neutrophils, respectively. Data represented mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 significantly higher than that of NM-NPs without LPS stimulation.

cells express a high level of intercellular cell adhesion molecule1 (ICAM-1), a well-known molecule that mediates the attachment between neutrophils and the endothelium during the recruitment of neutrophils to the sites of inflammation.32 To evaluate the targeting efficiency of NM-NPs, 4T1 cells were used as a CTC model since the metastasis-promoting glycoproteins and glycans, such as CD44 and intercellular cell adhesion molecule-1 (ICAM-1), were found aberrantly expressed on 4T1 cells (Figure S3). Under a static condition at 37 °C, after incubation with the nanoformulations for 2 h, the fluorescent signal of NM-NP in 4T1 cells monolayer was 1.26and 2.59-fold compared with that of NP and PLGA-PEG-NP in 4T1 cells, respectively (Figures 3A,B and S5). However, the microenvironment for CTCs and primary tumor cells are remarkably different, as shear flow changes once CTCs enter the circulation. Therefore, the efficiency of NM-NP to target CTCs was also studied on 4T1 cells under a shear flow

condition. When sheared in a cone-and-plate viscometer at 37 °C at the uniform rate of 188 s−1 for 2 h, the fluorescent intensity of NM-NPs associated with 4T1 cells was 1.89- and 3.58-fold compared with that of NP and PLGA-PEG-NP, respectively (Figures 3B,C and S4). An Antibody blocking assay (Figure S5) showed that the association of NM-NPs in 4T1 cells was reduced by 47.2% and 33.9% following the preincubation of anti-ICAM-1 and anti-CD44, respectively. This observation confirmed that the presence of adhesion molecules on NM-NPs could specifically enhance the binding and sequentially induce a stronger cellular uptake in flowing 4T1 cells. Notably, the cellular association of NM-NPs (400 μg/ mL) in both static or sheared 4T1 cells was significantly reduced, compared with that of NM-NPs incubated at 37 °C for 2 h. Such temperature-dependent cellular association of 1400


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Figure 4. The precise CTC-targeting effect of NM-NPs in vivo. The interaction between GFP+ 4T1 cells and NM-NPs/NPs measured by in vivo flow cytometry. A laser slit was covered across the mouse ear artery to monitor the GFP+ 4T1 cells passed through it. Representative 10 min data are shown after intravenous injection (n = 3) with GFP+ 4T1 cells and the following DiD-labeled NPs. Each green peak represents one CTC or CTC cluster, while the red peak represents cells internalized with DiD-labeled NM-NPs/NPs. Once the nanoformulations were internalized by GFP+ 4T1 cells, an overlay of GFP and DiD signals was recorded, generating double positive peaks. Arrows and blocks are pointed to the double positive signal of GFP and DiD. Images shown on the right are augmented double positive signals.

methods with low CTCs isolation efficacy from the rare blood sample, this method could quantitatively monitor every individual GFP+ 4T1 cell that passed through the laser slit across the artery. The illumination image of the mouse ear microcirculation is shown in Figure 4, and green peaks represent GFP+ 4T1 cells flowing across the artery of the mouse ear, while red signals represent the signal of DiD-labeled NPs. Typical data of mice showed 29 ± 10 double positive signal peaks in 30 min after the administration of NM-NPs, accounting for approximately 25% of total GFP+ 4T1 cells passing by, whereas only 1 ± 1 double positive signal peaks were detected in mice following the treatment with NPs. Confocal intravital microscopy (IVM) imaging was also performed to visualize the interaction between NM-NPs and CTCs in vivo in real time (Supporting Movie 1 and Figure S6). Consistent with IVFC findings, most of the flowing GFP-4T1 cells detected simultaneously exhibit a DiD and GFP signal. This observation confirmed that DiD-labeled NM-NPs could capture CTCs more efficiently. Homing of NM-NP-CFZ to Premetastasis in Vitro and in Vivo. Having determined the CTC-targeting efficiency of NM-NPs, we next investigated its homing capacity to the premetastatic site. In such a microenvironment, metastatic seeding of CTC usually happens at the cytokine-activated endothelium associated with up-regulation of adhesion molecules, including ICAM-1and VCAM-1.34 To mimic the physical environment, the efficiency of NM-NP homing to premetastatic endothelial “soil” was studied using an in vitro model by shearing nanoformulations with human umbilical vein

NM-NP indicated that cellular association of NM-NP was an energy-dependent and active trafficking process. To study the endocytosis pathway of NM-NP, the cellular uptake analysis was also conducted in the presence of various inhibitors. As shown in Figure 3D, the cellular internalization of NP in sheared 4T1 cells was significantly inhibited by chlorpromazine (p < 0.001), BFA (p < 0.05), NaN3 with deoxglucose (p < 0.01), cyto-D (p < 0.01), and nocodazole (p < 0.001), indicating that it was a clathrin-mediated and energydependent endocytic process, with the involvement of the Golgi apparatus. In contrast, in the case of NM-NPs, decreased internalization was observed in the presence of NaN3 with cytoD (p < 0.001), filipin (p < 0.001), genistein (p < 0.001), and monensin (p < 0.001), but not chlorpromazine (p > 0.1), indicating a caveolae-mediated intracellular trafficking of NMNPs and the involvement of lysosome therein. This was probably because the membrane attachment induced a differential endocytic pattern for NM-NPs. Precise CTC-Targeting Effect of NM-NP in Vivo. Since promising results had been achieved by in vitro experiments, the CTC-targeting ability of NM-NP in mice circulation was investigated using an in vivo flow cytometry assay.33 Briefly, mice were injected with 1 × 106 4T1 cells that stably express green fluorescent protein (GFP+ 4T1 cells) and DiD-labeled NPs sequentially. After that, the mice were fixed with ear microcirculation immobilized and visualized under illumination with a 535 ± 15 nm light emitting diode (LED). After selecting the artery of interest, images were captured, and fluorescent signals were recorded. Different from traditional in vitro 1401


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significantly increased by 2.9-fold compared with that of NPs, suggesting that NM-NPs can potentially bind to the inflamed endothelium under systemic circulation. The blockage of ICAM-1 with specific antibodies induced a 32.9% decrease in NM-NP binding efficiency in the inflamed HUVEC, indicating that these adhesion molecules on NM-NPs play key roles in its homing to the activated endothelia. To study the premetastatic niche-homing efficiency of NMNPs in vivo, we analyzed the distribution of NM-NPs in a laminin-abundant region of the premetastatic mouse model. As an extracellular matrix (ECM) component in lung, laminin, the main ligand for β1 integrin, provides an important target for cancer cells to attach.37 To prepare the premetastasis model in mice, 4T1 cells were intravenously injected, and mice were perfused at different time points (8, 16, 24, 32, 40 h) postinoculation for immunofluorescent analysis of laminin in the lung. Enhanced expression of laminin was observed in the time range from 16 to 24 h post 4T1 cells injection. Therefore, within this period after 4T1 cells inoculation, the mice could serve as animal models of premetastasis. After intravenous injection, substantial NM-NPs were found in the lamininabundant area, while a much smaller cluster of NPs was found in this area (Figure 5C). This result supported our inspection that membrane protein cocktails would drive this biomimetic NP to specifically bind to the laminin-rich premetastatic region, providing a prerequisite for early intervention of metastasis. Further, analysis of the NM-NPs homing to different premetastatic niche components was performed, following an intravenous injection of red fluorescent protein (RFP)-labeled tumor cells and immunostaining of CD31 (marker of neovasculature), F4/80 (marker of macrophages), laminin (marker of extracellular matrix), and αSMA (marker of fibroblast), respectively. As shown in Figure S7, a high extent of colocalization of NM-NPs with CD31, laminin, and tumor cells was found, consistent with our hypothesis that the neutrophil-mimicking NM-NPs may target the premetastatic niche via tethering to the neovasculature, laminin, and tumor cells in a similar manner as the recruitment of neutrophils to the inflammatory sites. Targeting of NM-NP to Already Formed Lung Metastasis. Pharmacokinetic analysis showed that the blood circulation half-life (t1/2) of CFZ loaded by NPs, NM-NPs, and PLGA-PEG-NPs was 0.77, 6.59, and 4.73 h, respectively. In addition, the AUCs for CFZ-encapsulated PLGA-PEG-NPs and NM-NPs were 134.4- and 207.2-fold higher than that of NPs (n = 3) (Figure S8, Table S2). Collectively, these data indicated that NM-NPs exhibited a superior blood circulation profile compared with both bare NPs and PEGylated NPs. We next investigated the targeting capability of NM-NPs in the established lung metastatic mouse model.38 Nodules in lung were already found at 10 days after an intravenous injection of highly aggressive luciferase-labeled 4T1 cells (luc+ 4T1 cells), confirmed by bioluminescent (BLI) imaging. In order to see if the NM-NPs can enter these metastasis lesions efficiently, a dual-mode in vivo imaging assay was conducted to analyze the colocalization of a near-infrared (NIR) fluorescent signal and BIL signal in the same mouse with the timeline and the schedule depicted in Figure 6A. Improved accumulation, along with a much better correlation of NM-NPs, was found in the metastatic site, compared to that of NPs (Figure 6B). To further justify the targeting ability of NM-NPs, mice were immediately sacrificed with the organs harvested for ex vivo imaging. NM-NPs were found consistently captured in nearly

endothelial cells (HUVEC cells) pre-incubated with TNF-α overnight.35,36 Consistent with previous work, HUVEC was strongly activated by TNF-α with upregulation of ICAM-1, Eselectin, and CD44 (major adhesion ligands for integrins and Lselectin) observed by flow cytometry (Figure S3).36 As shown in Figure 5A,B, following TNF-α stimulation, the cellular association of NPs remained unchanged, while that of NM-NPs

Figure 5. Premetastasis homing of NM-NP-CFZ in vitro and in vivo. (A) Adhesion of flowing NM-NP and NP to HUVEC monolayer under a mimetic physiological condition. 400 μg/mL of coumarin6-labeled NPs and NM-NPs (green) were sheared with a HUVEC monolayer pretreated with or without TNF-α (3 ng/mL, for 12 h), respectively, for 2 h at the shear rare of 188 s−1 at 37 °C. For a mechanism study, an anti-ICAM-1 antibody was incubated with HUVEC for 2 h before shearing with nanoformulations. After that, HUVEC on coverslips were transferred back for nuclei staining (blue) before imaging. Bar, 20 μm. (B) Quantification of HUVEC binding efficiency of NP and NM-NP under various condition. Results were expressed as the average fluorescent intensity, and data represent the mean ± SD (n = 3), ***p < 0.001 significantly lower than that in the TNF-α stimulated HUVEC. (C) Confocal microscopy analysis of NPs targeting premetastatic region in a mouse model (n = 3). Mice were intravenously injected with coumarin-6-labeled NPs and NM-NPs (coumarin-6 dosage of 1 mg/kg), respectively, and expression of laminin in mice lungs was examined by immunofluorescent staining. Blue: Hoechst stained nuclei; red: laminin-positive premetastatic niche; green: coumarin6-labeled NPs. Scale bar, 50 μm. 1402


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specifically internalized by neutrophils.40 In addition, the accumulation of NM-NPs in the spleen might result from a ̈ neutrophils. certain splenic homing property of naive NM-NP-CFZ Exhibited Selective Elimination of CTCs in Blood. Since a promising targeting effect of NM-NPs was achieved, we tested the therapeutic potential of NM-NPs after loading with CFZ, a second-generation proteasome inhibitor in a Phase II clinical trial. Proteasome inhibition resulted in the disruption of the homeostatic mechanism within the cells that lead to cell death. Therefore, the in vitro antiproliferative property of NM-NP-CFZ was analyzed via MTT assay, compared to that of free carfilzomib (free CFZ) and NPCFZ. The IC50 of free CFZ, NP-CFZ, and NM-NP-CFZ in 4T1 cells was found to be 58.2 ng/mL, 178 ng/mL, and 69.4 ng/mL, following a 48 h incubation, respectively (Figure S11). This indicated that the encapsulation of CFZ in NPs could significantly reduce the cytotoxicity of free CFZ to 4T1 cells, while the enhanced internalization of NM-NP-CFZ in 4T1 cells could result in a much higher cytotoxicity to 4T1 cells. However, the activity of NM-NP-CFZ in selective elimination of CTCs remained to be addressed under biological environment. We then further determined the apoptosis rate of GFP+ 4T1 cells and leukocytes following incubation with NM-NPCFZ in the presence of blood (Figure 7). Interestingly, populations of early apoptotic GFP+ 4T1 cells after treatment with NM-NP-CFZ (23.0 ± 3.2%) were significantly higher than that of cells treated with NP-CFZ and free CFZ (1.88 ± 0.30% and 1.58 ± 0.27%, respectively) (Figure 7A). In addition, following the treatment of NM-NP-CFZ, 94.6% of leukocytes was kept viable, which suggested that the toxicity of NM-NPCFZ to the leukocytes is greatly minimized. In contrast, about 16.8% of necrosis was induced by free CFZ in 4T1 cells (Figure 7B). All of these data indicated that the cytotoxicity of NM-NPCFZ to CTCs was largely maintained in blood, which was probably attributed to the selective binding and internalization of NM-NP-CFZ in CTCs, while the lost cytotoxicity of free CFZ to CTCs could be attributed to its nonspecific interaction with the abundant blood cells. NM-NP-CFZ Prevented the Formation of Early Metastasis. After confirming the ability of NM-NP-CFZ in the selective CTCs depletion in blood, we set out to determine the early metastasis prevention effect of NM-NP-CFZ in the mouse model injected with 4T1 cells. The treatment was initiated right after 4T1 cells were inoculated four times with a CFZ dosage of 0.5 mg/kg (on days 0, 7, 14, and 21). The formation of metastatic nodules was found to be significantly reduced (p < 0.001), compared to that of the mice treated with saline or free CFZ (Figure 8A,D). Interestingly, tumor cell proliferation was also significantly reduced in the lymph nodes of mice following the NM-NP-CFZ treatment, compared with that of mice treated with NP-CFZ and free CFZ (Figure 8B). This complete prevention of growth arrest of 4T1 cells in the formation of lymph node metastasis implied that besides eliminating disseminated CTCs in blood, NM-NP-CFZ could also alleviate the metastatic burden by reducing their incidence of successful colonization of CTCs in both lymph nodes and tissues. Furthermore, we next examined the recruitment of neutrophils to metastatic lungs by immunohistochemistry. As a cytoplasm protein secreted by neutrophils at the site of inflammation, S100A9 has recently been identified as a niche promoting molecule, recruiting different subsets of myeloid cells to the niche, and its level was closely associated with the development of distant metastasis.41 Consistent with previous

Figure 6. Neutrophil-mimicking NM-NPs efficiently target metastasis in the 4T1 mouse model (n = 3). (A) Timeline and imaging schedule for evaluating the targeting property of NM-NPs and NPs in vivo and ex vivo via (bioluminescent) BLI imaging and NIR imaging. (B) Dual-mode imaging of mice with obvious metastasis 14 days after luc+ 4T1 cells injection in vivo and ex vivo. The mice were intravenously administered with DiR-labeled NPs and NMNPs before subjection to NIR imaging. Immediately after that, BIL imaging was conducted on the same mice. To obtain the detailed information on the targeting efficiency, mice were sacrificed, and the organs harvested and imaged. The major lung metastasis lesions are shown with red arrows and circles.

all the metastasis lesions in the lung, while NPs showed a much poorer colocolization with the metastatic sites. Despite the long blood circulation time, PLGA-PEG-NPs displayed a much inferior targeting property to metastatic foci, compared to that of NM-NPs (Figure S9). Quantitative analysis (Figure S10) showed that the accumulation of NM-NPs in metastatic foci was, respectively, 2.12- and 3.02-fold compared to that of NPs and PLGA-PEG-NPs ex vivo 24 h after intravenous administration. This evidence collectively confirmed the targeting capacity of the biomimetic NM-NPs to the already formed lung metastasis. Notably, NM-NPs showed a higher affinity with certain organs, like liver, in the mice model bearing an already formed metastasis, compared to that of NPs. A possible reason was that neutrophils possess Fc receptors binding to immunoglobulin G (IgG) in the blood, which directed NM-NPs to the mononuclear phagocyte system (MPS) in the liver.39 Such a phenomenon has also been observed for bovine serum albumin-coated NPs that are 1403


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Figure 7. NM-NP-CFZ exhibited selective elimination of CTCs in blood. (A) DAPI/Annexin-V flow cytometry gates of GFP+ 4T1 cells and leukocytes sheared with 800 ng/mL free CFZ, NP-CFZ, and NM-NP-CFZ in the presence of blood, at the rate of 188 s−1, 37 °C for 2 h. Representative flow cytometry plots of four GFP+ 4T1 cells (and leukocytes) subpopulations: viable cells (double negative), early apoptotic cells (only positive for Annexin-V PE), late apoptotic (positive for DAPI and Annexin-V PE), and necrotic cells (only positive for DAPI). (B) Percentage of viable, early apoptotic, late apoptotic, and necrotic cells among GFP+ 4T1 cells and leukocytes after the same treatment. Data represented mean ± SD (n = 3). ***p < 0.001, *p < 0.05, significantly different with the apoptosis rate of 4T1 treated with NP-CFZ and ##p < 0.01 for apoptosis rate of leukocytes under the same treatment.

findings, after 4T1 cells inoculation, 37 ± 4% of total cells was S100A9-positive in the metastatic lungs of those saline-treated mice (Figure 8C,E). Strikingly, the aberrant expression of S100A9 in the lung was reversed more obviously following the intravenous administration of NM-NP-CFZ (reduced by 71%), compared to that in the mice following the treatment of NPCFZ (reduced by 44%) and free CFZ (reduced by 41%), confirming its potential role in regulation of the microenvironment in the metastatic lung. Apart from eliminating immunesuppressed neutrophils in the tumor context, expression levels of neutrophils and stromal cell-derived cytokines including MMP2, CXCL12 and TNF-α were also significantly inhibited after the treatment with NM-NP-CFZ, but it was not obvious in the mice administered with NP-CFZ and free CFZ (Figure 8F). As we know, proteasome inhibition results in the blocking of NF-κB activation during the process of IκB protein degradation. As a cell survival signal, NF-κB activation not only promotes cell adhesion, angiogenesis, and metastasis but also provides mechanism for cancer cells’ immune surveillance.42 Compounds with a proteasome-inhibitory property are of great importance in relieving inflammation and preventing cancer metastasis.43 Therefore, we suspect, by down-regulating levels of these cytokines in a niche microenvironment, NM-NPCFZ displayed the most obvious metastasis prevention activity among all formulations.

NM-NP-CFZ Inhibited the Progression of Already Formed 4T1 Lung Metastasis. To determine whether NM-NP-CFZ affect the spread of the already-formed metastasis, we began the treatment after the formation of metastasis on day 7 following the inoculation of luc+ 4T1 cells. As in Figure 9A, BLI imaging was performed after the treatment with free CFZ, NP-CFZ, and NM-NP-CFZ at a CFZ dosage of 1 mg/kg, to monitor the progress of lung metastasis in mice. As shown in Figure 9B, mice treated with free CFZ had expeditious tumor growth from days 8 to 16, despite the most obvious cytotoxicity observed in vitro (Figure S11). An obvious inhibitory effect was observed in mice following the treatment with NP-CFZ, which potentially reduced approximately 42.8% metastasis foci on day 16, compared with that of the same mice measured on day 8 (Figure 9C). Most obviously, NM-NP-CFZ induced an 87.2% reduction of metastasis foci at the end of the treatment. After that, mice were sacrificed, and the lungs were harvested for the analysis of the metastatic deposits. Ex vivo imaging confirmed that minimal metastasis deposits were found in the lungs of mice receiving NM-NPCFZ treatment, compared to those of mice treated with NPCFZ and free CFZ. Free CFZ failed to inhibit the progress of metastasis probably because of its lower selectivity, compared with the nanoformulations. In particular, the fact that NM-NPCFZ diminished the spread of metastasis growth at a greater level than NP-CFZ could be resulted from its powerful niche1404


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Figure 8. NM-NP-CFZ prevented the formation of early metastasis. (A) Histological examination (H&E staining) of lung tissues after mice were treated with saline and low-CFZ dosage (0.5 mg/kg) of free CFZ, NP-CFZ, and NM-NP-CFZ, respectively, on days 0, 7, 14, and 21 after inoculation (n = 3). Arrows show metastatic nodules. Scale bar, 5 mm. (B) Representative histological analysis of cancer cells infiltration in lymph node after the same treatment, n = 3. Scale bar, 100 μm. (C) Inhibition of NM-NP-CFZ, NP-CFZ, and free CFZ on neutrophils recruitment in mice lungs by immunological staining with S100A9 (brown), n = 3. Arrows were pointed to S100A9-positive cells. Scale bar, 100 μm. (D) Quantification of metastasis nodules in lung tissue slides. (E) Quantification of S100A9-positive neutrophils in lung sections, n = 3. (F) Concentration of CXCL12, MMP2, and TNF-α in culture supernatant of each lungs measured by ELISA assay, n = 3. ***p < 0.001, *p < 0.05, compared with that of mice treated with saline (control).

targeting effect. Collectively, the strongest inhibitory effect of NM-NP-CFZ confirmed our hypothesis that these biomimetic NM-NPs possess a whole-stage therapeutic potential for management of metastasis. Several studies have demonstrated that metastatic tumor cells were highly surrounded by various subsets of stromal cells and leukocytes, including neutrophils, macrophages, and lympho-

cytes. To further determine the CTC-targeting specificity of treatment, we used a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to measure the apoptosis of the successfully colonized GFP+ 4T1 cells in the lung (Figure 9D). As shown in Figure 9E, NM-NP-CFZ induced the most significant rate of apoptotic cells (accounting for 84.3 ± 7.4% of total numbers of GFP+ 4T1 cells), compared 1405


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Figure 9. NM-NP-CFZ inhibited the development of already formed 4T1 lung metastasis, n = 3. (A) Timeline for 4T1 mouse models establishment and treatment schedule. (B) BLI imaging of mice treated with free CFZ, NP-CFZ, and NM-NP-CFZ for three times on days 7, 11 and 15, respectively, at the CFZ dosage of 1 mg/kg, and mice were imaged on days 8, 12, and 16. (C) Quantitative analysis of total BLI signal detected in vivo and ex vivo in isolated major organs including heart, liver, spleen, lung, and kidney at the end of treatment. (D) In vivo apoptosis of metastatic GFP+ tumor cells (green) indicated by TUNEL assay (red). GFP-labeled 4T1-tumor metastasis models were established by intravenous injection with GFP+ 4T1 cells. On 16th day, lungs were resected for TUNEL analysis. Nuclei were stained with Hoechst 33258 (blue). Scale bar, 100 μm. (E) Quantitative analysis of GFP positive nodules per slice and percentage of TUNEL positive cells of six randomly chosen microscopic fields. ***p < 0.001, compared with that of mice treated with NP-CFZ.

to that of mice treated with NP-CFZ (41.2 ± 7.5%) and free CFZ (19.5 ± 5.2%). Only approximately 2.3 ± 0.6% of GFP+ 4T1 cells were apoptotic in the control mice treated with saline. Consistently, GFP+ clusters in six randomly selected fields were counted under microscope (Figure 9C), and only 2.4 nodules on average were found after the treatment with NM-NP-CFZ, significantly lower than that following the administration of NP-CFZ (average nodules, 7.1) and free CFZ (average nodules, 8.4). This observation indicated that the biomimetic

NM-NP-CFZ could also selectively facilitate the depletion of tumor cells in already formed metastasis. Body weight data of mice were also recorded under the same treatment modality (Figure S12). Mice injected with free CFZ kept losing weight in the following days. When intravenously administered with NP-CFZ and NM-NP-CFZ, fluctuation of body weight was significantly reduced. In particular, mice receiving the NM-NP-CFZ treatment showed a steady rise in body weight. This also supported our hypothesis that biomimetic NM-NPs formulation was the most suitable choice 1406


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layer. After the lysis of residual erythrocytes by lysis buffer at 4 °C, neutrophils with high purity were obtained. To obtain activated neutrophils, lipopolysaccharide (LPS, 100 ng/mL) was added to the culture of neutrophils for 4 h, followed by a final wash in PBS. Isolation of Neutrophil Plasma Membrane. The plasma membrane isolation process was originally described by Pilchler et al.45 To isolate the plasma membrane from the LPS-stimulated neutrophils, the cells were suspended in the ice-cold Isolation Buffer-1 (IB-1) containing 225 mannitol, 75 mM sucrose, 0.5% (w/v) BSA, 0.5 mM EDTA, and 30 mM Tris-HCl and supplemented with protease inhibitor cocktail. Thereafter, they were homogenized for 50−100 strokes using a dounce homogenizer with a tight pestle. The homogenate was then centrifuged at 800×g, 4 °C, for 10 min, to remove the unbroken cells and nuclei. The supernatant was then centrifuged at 10,000×g, 4 °C to remove the mitochondria by discarding the pellet. The supernatant was centrifuged again at 100,000×g, 4 °C for 1 h. Finally, the plasma membrane containing pellet was washed with 10 mM Tris-HCl and 0.5 mM EDTA with protease inhibitor cocktail and then freeze-dried, weighted, and stored at −80 °C for further use. Synthesis and Characterization of NM-NP. The polymeric cores were prepared with carboxyl-terminated 50:50 poly(latic-coglycolic) acid (PLGA) via the emulsion/solvent evaporation method, as described previously.26 Briefly, 10 mg of PLGA was dissolved in 1 mL dichloromethane and then added to 2 mL of 1% sodium cholate aqueous solution. The mixture was then sonicated at 240 w for 80 s on ice (Ningbo Scientz Biotechnology Co. Ltd., China) and immediately added into 10 mL of stirring 0.5% sodium cholate solution for 5 min. After evaporation under a vacuum, the resulting PLGA NPs were collected by centrifugation (14500 rpm, 50 min) at 4 °C. As another control, PEGylated nanoparticles (PLGA-PEG-NPs) were prepared with PLGA-PEG3500-COOH using the same emulsion/solvent evaporation method.26 For in vitro experiments, DiD, coumarin-6, or CFZ were loaded into NPs at a weight ratio of 0.1% and then purified with 0.01 M HEPES buffer (pH 7.0) through a 1.5 × 20 cm sepharose CL-4B column to remove the free DiD, coumarin-6, or carfilzomib. For in vivo experiments, coumarin-6, DiR, and CFZ were encapsulated in NPs at a weight ratio of 1% with the same purify procedure. At room temperature, 1 mg of the above NMV was suspended in 1 mL water and sonicated in a capped centrifuge tube at 100 w for 30 s (sonication for 1 s, intervals for 2 s) on ice. For the synthesis of NMNPs, NMV (1 mg/mL) was mixed with NPs (1 mg/mL) at membrane-to-core weight ratios of 1:1, and the mixture was then sonicated at 100 w for 30 s (sonication for 1 s, intervals for 2 s) on ice. In order to remove any excess NMV, the product was centrifuged at 14500 rpm at 4 °C for 50 min. The size and zeta potentials of fabricated NMV, NPs, and NM-NPs were determined by DLS using a Zeta Potential/Particle Sizer NICOMP 380 ZLS (Santa Barbara, California, USA), and the morphology was examined under a field emission TEM (Tecnai G2 F20 S-Twin). First, NPs at a concentration of 1 mg/mL were dropped on the carbon-coated grid. Five min after that, the excess NPs were removed, and the grid was rinsed with 10 drops of distilled water. Afterward, a drop of 1% (w/v) uranyl acetate staining solution was added for negative staining, and the grid was dried and imaged under a TEM. Identification of Membrane Associated Protein. The presence of membrane-associated proteins on the particles was verified by Coomassie blue staining and Western blot. NM-NPs were collected by centrifugation at 14500 rpm, 50 min, 4 °C to remove the uncoated NMV. For the analysis, the prehomogenated neutrophils, NMV, and NM-NPs were first lysed in a RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing Protease Inhibitor Cocktail (cOmplete, Roche, Germany) on ice for 5 min. Thereafter, the lysates were centrifuged at 13,000×g for 5 min at 4 °C, and the supernatant was then subjected to enhanced BCA protein assay (Beyotime Biotechnology, Haimen, China) for the quantification of the total protein. After that, the supernatant protein was mixed with SDS loading buffer and heated at 100 °C for 5 min. An equivalent 20 μg of protein per sample was loaded into each well of a

for management of metastasis with advanced therapeutic potential.

CONCLUSIONS A neutrophil-mimicking nanoparticle (NM-NP-CFZ) was developed to alleviate the metastatic burden by targeting both CTCs in circulation and premetastatic niche. It displayed a diameter of around 100 nm and a surface coating by the cocktails of neutrophil membrane-associated adhesive proteins. The resulting NM-NP was capable of binding with CTCs under shear stress and specifically homing to the premetastatic endothelium model in vitro. More crucially, single CTCs cellular targeting effect and improved accumulation in premetastatic niche of NM-NP were observed using in vivo flow cytometry technology and confocal imaging. The selective targeting efficiency of NM-NP in the already formed metastatic model was also confirmed by dual-mode imaging. Loading with a proteasome inhibitor, carfilzomib, the nanoformulation facilitated selective CTC apoptosis in blood, prevented the formation of nodules at the early stage, and induced apoptosis and inhibition effects in both GFP-labeled and luciferaselabeled metastatic 4T1 models. This biomimetic drug delivery system will provide an advanced strategy for cancer metastasis prevention and therapy. MATERIALS AND METHODS Materials. Acid-terminated PLGA polymer (50/50, Lakeshore Biomaterials) was kindly gifted by Birmingham Laboratories (Evonik Degussa Corp, Birmingham AL, USA). 1,19-dioctadecyl-3,3,39,39tetramethylindodicarbocyanine perchlorate (DiD) was purchased from Invitrogen Co., USA. NHS-fluorescein was the Annexin V-FITC Apoptosis Detection Kit I and was provided by BD Bioscience (San Diego, CA, USA). Coumarin-6, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide (DiR), Hoechst 33258, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and protease inhibitor cocktail were all obtained from - (St. Louis, MO, USA). Penicillin, streptomycin, and 0.25% trypsin-EDTA were purchased from Invitrogen Co., USA. Copper grids were bought from Beijing Xinxing Braim Technology Co., Ltd. (Beijing, China). All other chemicals were provided by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless mentioned otherwise. Cell Lines and Animals. Primary human umbilical vein endothelial cells (HUVEC cells) were provided by Cascade Biologics (Portland, OR, USA), and (GFP+ or luc+) 4T1 cell line were provided by Shanghai University of Traditional Chinese Medicine (Shanghai, China). HUVEC cells and 4T1 cells were maintained in DMEM and RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2, respectively. Female Balb/c nude mice (20 ± 2 g) and male ICR mice were provided by BK Lab Animal Ltd. (Shanghai, China) and maintained under standard housing conditions of Department of Experimental Animals, Fudan University (Shanghai, China). All the animal experiments were performed in accordance with guidelines evaluated and approved by Institutional Animal Care and Use Committee (IACUC), School of Pharmacy, Fudan University (Shanghai, China). Isolation and Activation of Peripheral Neutrophils. Percoll gradient method described by Boxio et al. was used for the isolation of neutrophils from the whole blood of ICR mice (male).44 Blood samples were collected in heparin tubes, purified by centrifugation (400 g, 10 min, 4 °C), and then diluted in PBS containing ethylene diamine tetraacetic acid (EDTA). The cell pellets were then carefully added into the top of a three-layer Percoll gradient of 78%, 69%, and 52% diluted in PBS and then centrifuged at 1500×g for 30 min at room temperature according to the manufacturer’s instructions (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The neutrophils were withdrawn from the 69/78% interface and the upper part of the 78% 1407


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DiD-labeled NPs were captured by a 4T1+ cell, signals of DiD and GFP would be detected simultaneously and overlapped at the same time point. Premetastatic Endothelial Binding Assay. HUVEC cells were seeded on coverslips which were placed in the bottom of 24-well plates at the density of 5000 cells for 24 h. After that, the cells were then stimulated with 3 ng/mL TNF-α for 8 h. HUVEC culture without TNF-α stimulation was used as the negative control. The coverslips were then washed three times with PBS and adhered to the bottom of a cone-and-plate viscometer using vacuum grease. Sequentially, the HUVEC monolayer was sheared with 1 mL of NPs and NM-NPs at a concentration of 400 μg/mL in DMEM medium, respectively, for 2 h at the shear rate of 188 s−1 at 37 °C. Thereafter, the HUVEC coverslips were transferred back to a 24-well plate, washed three times with PBS and fixed in 4% paraformaldehyde for 15 min, washed for additional three times with PBS before nuclear staining with Hoechst33258, and finally subjected to confocal microscopy (LSM710, Leica, Germany) for visualization. Metastasis Models Establishment. To prepare the premetastatic mouse model, we perfused mice at different time points (8, 16, 24, 32, 40 h) after intravenous injection of 4T1 cells, and laminin was immunofluorescently stained using an antibody (Abcam, ab11575). The mouse with the highest expression level of laminin in a particular time was chosen as the premetastatic mouse model by comparing its expression between different time points after 4T1 cells injection (n = 3). For already formed metastatic model establishment, we inoculated 1 × 106 luciferase-expressed 4T1 cells (luc+ 4T1 cells) intravenously to the 6−8 week-old female Balb/c nude mice, respectively. Premetastatic Region Homing Assay. To further reveal the distribution of nanoformulations in the premetastasis lesion, 200 μL of coumarin-6-labeled NPs and NM-NPs (1 mg/kg of coumarin-6) was intravenously given to premetastatic mouse models via the tail vein (n = 3). Two hours later, lungs were resected, fixed, embedded, frozen, and sliced. The sections were permeabilized with 0.1% Triton (v/v), immunostained with a rabbit antibody against laminin (Abcam, ab11575), F4/80 (Abcam, ab6640), αSMA (Abcam, ab5694), and CD31 (Biolegend, 910005), washed with PBS, and stained with an Alexa 555-labeled secondary antibody and Hoechst 33258 before the subjection to confocal microscopy. Premetastatic mouse models established by red-fluorescent labeled 4T1 cells were used to investigate the NM-NPs distribution with tumor cells in the niche. Dual-Mode Imaging. NIR imaging and BLI were simultaneously performed, with the colocalization of the targeted NM-NPs and metastasis nodules determined. Fourteen days after models establishment, mice were divided in two groups and injected with equal volumes of DiR-labeled NM-NPs and NPs (1 mg/kg of DiR) (n = 3). After 24 h, the mice were intraperitoneally administered with 200 μL of 5 mg/mL D-luciferin 10 min before their imaging under an in vivo IVIS spectrum imaging system (PerkinElmer, USA). Major organs were then collected and imaged immediately under the same system. Cell Viability Assay. The in vitro cytotoxicity of carfilzomib formulations on 4T1 cells was measured by MTT assay. Briefly, cells were seeded in 96-well plates at a density of 3000 cells per well and cultured overnight for attachment. The cells medium was changed with series of CFZ formulations including free CFZ, NP-CFZ, and NM-NP-CFZ solution diluted with RPMI 1640 without serum, respectively (at the CFZ concentrations from 1 ng/mL to 10000 ng/ mL). For comparison, drug-free medium was used as a control. After 48 h, the cells were incubated with MTT reagent for 4 h at 37 °C, with formazan crystals production in metabolically active cells determined at an absorbance of 570 nm with the microplate reader (Thermo Multiskan MK3, USA). IC50 values were determined by nonlinear regression analysis using a GraphPad Prism software. Apoptosis Assay. 4T1 cells were seeded in a 6-well plate and allowed to grow to 70−80% confluence. After that, the cells were trypsinized and sheared with free CFZ, NP-CFZ, and NM-NP-CFZ (at the carfilzomib concentration of 400 ng/mL) in a cone-and-plate viscometer, respectively, at 188 s−1, 37 °C for 2 h. Cells without shearing were used as a negative control. For immediate apoptotic cells detection, PI/Annexin V-FITC assay was used to measure the

6% Tris/glycine SDS-polyacrylamide gelatin in an electrophoresis chamber system (Bio-Rad Laboratories, PA, USA). For total imaging, the protein blots were stained with Coomassie blue fast staining solution (Beyotime Biotechnology, Haimen, China). For Western blot analysis, the protein was transferred to polyvinylidene fluoride membranes (Cell Signaling technology, MA, USA), which was then blocked with 5% nonfat milk in TBS-T (Tris-HCl 50 mM, NaCl 150 mM, Tween-80 0.1%) for 1 h at room temperature. The blots were probed by antibodies against L-selection (R&D systems, MAB1534), CXCR4 (Abcam, ab124824), LFA-1 (Abcam, ab13219), β1 integrin (Abcam, EPR16895), and β-actin overnight at 4 °C and then incubated with the corresponding horseradish peroxidase (HRP)conjugated antimouse (Cell Signaling technology, 7076) or antirabbit IgG (Cell Signaling technology, 7074) before the final visualization on film. Binding of NM-NPs with Static 4T1 Cells. The 4T1 cells were seeded in 96-well plates at a density of 8 × 103 per well, cultured for 24 h, and then incubated with coumarin-6-labeled NPs and NM-NPs that were diluted with serum-free RPMI 1640 at concentrations ranging from 50 μg/mL to 800 μg/mL for 2 h at 37 and 4 °C, respectively. After that, the cells were washed three times with PBS and fixed with 4% formaldehyde for 15 min. For quantitative study, nuclei were then stained with 2 μg/mL Hoechst 33258 before scanning under a Kinetic Scan HCS Reader (version 3.1, Cellomics Inc., Pittsburgh, PA, USA). All the fluorescence images were taken under identical conditions with the same exposure time. Binding of NM-NP with Circulating 4T1 Cells under Shear Flow. As CTCs were exposed to a considerable physical stress under flow in vivo, a cone-and-plate viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA) was chosen to imitate this condition. Briefly, 4T1 cells were digested with trypsin-EDTA, washed twice with PBS, and suspended in RPMI 1640 at the density of 1 × 106 cells per milliliter. NPs and NM-NPs at concentrations ranging from 100 μg/mL to 1600 μg/mL were gently mixed with an equal volume of 4T1 cells suspension before the subjection to the cone-and-plate viscometer at the shear rate of 188 s−1 for 2 h at 37 and 4 °C, respectively. To ensure cell viability and avoid media evaporation, the temperature was carefully controlled and monitored. Although constant CO2 levels could not be maintained during the shearing process, little apoptosis in 4T1 cells was found after shearing for 2 h, suggesting the method could be applied for in vitro study. Cells pellets were washed twice with PBS by centrifugation at 1000 rpm for 4 min and immediately transferred to 96-well plates for culture for 24 h in complete media. After incubation, cells were washed and fixed and stained via the method described in Binding of NM-NP with Static 4T1 Cells section. Endocytosis Mechanism Study. For endocytosis mechanism study, we utilized various inhibitors to reveal whether membrane grafting regulates the endocytosis pathway of the NPs. Briefly, cells were seeded in a 96-well plate at a density of 8 × 103 per well 24 h before experiment and were then incubated with endocytic inhibitors for 2 h, including 10 μg/mL chlorpromazine, 4 μg/mL colchicines, 10 μg/mL cyto-D, 5 μg/mL BFA, 5 μg/mL filipin, 10 μM NaN3 with 50 μM deoxyglucose, 2.5 μM methyl-β-cyclodextrin (M-β-CD), 200 μM monensin, 20 μM nocodazole, and genistein. After incubation, cells were treated as the quantitative study above. In Vivo Flow Cytometry Assay. To monitor the CTC-targeting ability of NM-NPs in vivo, an in vivo flow cytometry technique with well-established sensitivity and specificity was applied.33 Briefly, 1 × 106 of GFP+ 4T1 cells were injected via the tail vein of 6−8 week-old female Balb/c mice, sequentially followed by intravenous injection with 200 μL of DiD-labeled NP and NM-NP, respectively (n = 3). The mice were then anesthetized and fixed on the sample stage. Major veins and arteries of the mouse ear were then visualized under illumination with a 535 ± 15 nm LED using a CCD (charge-coupled device) camera. Lasers of 488 and 635 nm were focused onto this artery with a slit. When a GFP+ 4T1 cell flowed through the slit, a peak signal would appear in the GFP detection channel. Similarly, when a cell internalized DiD-labeled NPs flowed through the laser slit, a peak signal would also appear in the DiD detection channel. Thus, when 1408


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ACS Nano apoptosis rate via flow cytometry according to the manufacturer’s instruction (BD Biosciences, CA, USA). Briefly, cells collected following trypsinization were washed for three times with ice-cold PBS and then incubated in 300 μL of binding buffer containing 5 μL FITC-Annexin V and 5 μL PI for 30 min at room temperature away from light. The extent of apoptosis was detected via a flow cytometer (FACS Calibur, BD, USA). Data analysis was performed using CellQuest software (Becton Dickinson, USA). Apoptosis Detection of CTC Spiked in Blood under Flow. Harvested by trypsinization, 1 × 106 GFP+ 4T1 cells were spiked in the 500 μL of prepared fresh blood and sheared with an equal volume of free CFZ, NP-CFZ, and NM-NP-CFZ at the carfilzomib concentration of 800 ng/mL for 2 h at 37 °C. GFP+ 4T1 cells without shearing or mixing with blood were used as a control. Before fluorescent labeling, erythrocytes were lysed with NH4Cl-NaHCO3 buffer, and the remaining leukocytes were then stained with DAPI and PE-Annexin-V to determine the variability of cells, according to the manufacturer’s instruction (BD Biosciences, CA, USA). Briefly, cells were incubated in 300 μL of binding buffer containing 5 μL PEAnnexin V and 5 μL DAPI for 30 min at room temperature away from light. The extent of apoptosis was detected via a flow cytometer (FACS Calibur, BD, USA). Data analysis was performed using CellQuest software (Becton Dickinson, USA). Metastasis Prevention Assay. Mice were injected with 2 × 106 GFP+4T1 cells through the tail vein (n = 3). Sequentially, a low dosage of free CFZ and CFZ-loaded nanoformulations (at the CFZ concentration of 0.5 mg/kg) was intravenously injected on days 0, 7, 14, 21 after GFP+4T1 cells injection. Mice treated with saline were taken as a control. Thirty days after treatment, lungs of animals were imbedded in paraffin and sectioned at 10 μm, after which the H&E staining and immunohistochemistry analysis were performed, respectively. The micrometastatic nodules and lymph node were then counted and morphologically examined. Association of neutrophils with cancer cells in the nodules and neutrophils in premetastatic niche were evaluated by S100A9 staining (Abcam, ab63818) to indicate the accumulation of lung neutrophils. ELISA Assay. We measured the levels of neutrophil-produced cytokines and other tumorigenic factors in vivo after the treatment with saline and a low-dosage of free CFZ and CFZ-loaded nanoformulations (at the CFZ concentration of 0.5 mg/kg) on days 0, 7, 14, 21 post-GFP+ 4T1 cells injection (n = 3). On day 30, an enzymelinked immunosorbent assay (ELISA) was conducted. Concentrations of TGF-β, MMP 2, and CXCL12 in each lung’s culture supernatant were determined according to the instruction provided by the manufacturer (Cusabio, Wuhan, China). After the reaction, a value at wavelength of 450 nm was measured with microplate reader (Thermo Multiskan MK3, USA), and the concentration was calculated from the standard curve. Already Formed Metastasis Inhibition Assay. We next evaluated the therapeutic potential of NM-NP-CFZ on already-formed metastatic models by BIL imaging. After luc+ 4T1 metastatic models were established, the mice were randomly divided into four groups (n = 3). On days 7, 11 and 15, 200 μL of saline, free CFZ, NP-CFZ, and NM-NP-CFZ (at the CFZ dosage of 1 mg/kg) were intravenously administered. On days 8, 12, and 16 after inoculation, bioluminescence imaging was performed 10 min after intraperitoneal administration of 200 μL of 5 mg/mL D-luciferin using an in vivo IVIS spectrum imaging system (PerkinElmer, USA). At the end of BLI in vivo imaging on day 16, major organs were collected for ex vivo imaging using the same system. TUNEL Assay. The apoptosis of tumor cells on already formed metastatic models following the carfilzomib treatment was evaluated via TdT-mediated dUTP nick end labeling (TUNEL) assay. Briefly, 200 μL of saline, free CFZ, NP-CFZ, and NM-NP-CFZ were intravenously administered on days 7, 11, and 15 after mice were injected with GFP+ 4T1 cells (n = 3). On the day 16, lungs were resected, fixed, embedded, frozen, and sliced. For TUNEL assay, the sections were fixed in 4% paraformaldehyde for 20 min, washed for 30 min with PBS, and then permeabilized for 2 min on ice, according to the manufacturer’s instructions (In Situ Cell Death Detection Kit,

Roche, Germany). After washing with PBS, each section was incubated with 50 μL of TUNEL reaction mixture prepared, before finally stained with Hoechst 33258 and subjected to confocal microscopy imaging. Statistical Analysis. All the data were presented as mean ± standard deviation, and a comparison among multiple groups was performed by one-way ANOVA analysis followed by Bonferroni test. Statistical significance was defined as p < 0.05.

ASSOCIATED CONTENT S Supporting Information *

Supporting Information is available free of charge from the ACS Nano home page (http://pubs.acs.org/journal/ancac3). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b06477. Additional experimental details and data (PDF) Movie: IVM imaging was also performed to visualize the interaction between NM-NPs and CTCs in vivo in real time (AVI)

AUTHOR INFORMATION Corresponding Authors

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

Jun Chen: 0000-0003-1330-9616 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (nos. 81673019, 81573382, 61227017), grant from Shanghai Science and Technology Committee (15540723700), “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (15SG14), National Major Scientific Research Program of China (2011CB910404, 2012CB966801), and the National Science Fund for Distinguished Young Scholars (no. 61425006). REFERENCES (1) Mantovani, A. Cancer: Inflaming Metastasis. Nature 2009, 457, 36−37. (2) Gupta, G. P.; Massague, J. Cancer Metastasis: Building a Framework. Cell 2006, 127, 679−695. (3) Mitchell, M. J.; Wayne, E.; Rana, K.; Schaffer, C. B.; King, M. R. TRAIL-Coated Leukocytes That Kill Cancer Cells in the Circulation. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 930−935. (4) Myung, J. H.; Gajjar, K. A.; Saric, J.; Eddington, D. T.; Hong, S. Dendrimer-Mediated Multivalent Binding for the Enhanced Capture of Tumor Cells. Angew. Chem., Int. Ed. 2011, 50, 11769−11772. (5) Gao, Y.; Gu, S.; Zhang, Y.; Xie, X.; Yu, T.; Lu, Y.; Zhu, Y.; Chen, W.; Zhang, H.; Dong, H.; Sinko, P. J.; Jia, L. The Architecture and Function of Monoclonal Antibody-Functionalized Mesoporous Silica Nanoparticles Loaded with Mifepristone: Repurposing Abortifacient for Cancer Metastatic Chemoprevention. Small 2016, 12, 2595−2608. (6) Alix-Panabieres, C.; Pantel, K. Challenges in Circulating Tumour Cell Research. Nat. Rev. Cancer 2014, 14, 623−631. (7) Hu, Q.; Sun, W.; Qian, C.; Wang, C.; Bomba, H. N.; Gu, Z. Anticancer Platelet-Mimicking Nanovehicles. Adv. Mater. 2015, 27, 7043−7050. (8) Li, J.; Ai, Y.; Wang, L.; Bu, P.; Sharkey, C. C.; Wu, Q.; Wun, B.; Roy, S.; Shen, X.; King, M. R. Targeted Drug Delivery to Circulating Tumor Cells Via Platelet Membrane-Functionalized Particles. Biomaterials 2016, 76, 52−65. 1409


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ACS Nano (9) Massague, J.; Obenauf, A. C. Metastatic Colonization by Circulating Tumour Cells. Nature 2016, 529, 298−306. (10) Nguyen, D. X.; Bos, P. D.; Massague, J. Metastasis: From Dissemination to Organ-Specific Colonization. Nat. Rev. Cancer 2009, 9, 274−284. (11) Mantovani, A.; Allavena, P.; Sica, A.; Balkwill, F. Cancer-Related Inflammation. Nature 2008, 454, 436−444. (12) Wculek, S. K.; Malanchi, I. Neutrophils Support Lung Colonization of Metastasis-Initiating Breast Cancer Cells. Nature 2015, 528, 413−417. (13) Coffelt, S. B.; Kersten, K.; Doornebal, C. W.; Weiden, J.; Vrijland, K.; Hau, C. S.; Verstegen, N. J.; Ciampricotti, M.; Hawinkels, L. J.; Jonkers, J.; de Visser, K. E. Il-17-Producing Gammadelta T Cells and Neutrophils Conspire to Promote Breast Cancer Metastasis. Nature 2015, 522, 345−348. (14) Wang, H. S.; Hung, Y.; Su, C. H.; Peng, S. T.; Guo, Y. J.; Lai, M. C.; Liu, C. Y.; Hsu, J. W. CD44 Cross-Linking Induces IntegrinMediated Adhesion and Transendothelial Migration in Breast Cancer Cell Line by up-Regulation of LFA-1 (Alpha L Beta 2) and VLA-4 (Alpha 4 beta 1). Exp. Cell Res. 2005, 304, 116−126. (15) Park, J.; Wysocki, R. W.; Amoozgar, Z.; Maiorino, L.; Fein, M. R.; Jorns, J.; Schott, A. F.; Kinugasa-Katayama, Y.; Lee, Y.; Won, N. H.; Nakasone, E. S.; Hearn, S. A.; Kuttner, V.; Qiu, J.; Almeida, A. S.; Perurena, N.; Kessenbrock, K.; Goldberg, M. S.; Egeblad, M. Cancer Cells Induce Metastasis-Supporting Neutrophil Extracellular DNA Traps. Sci. Transl. Med. 2016, 8, 361ra138. (16) Spicer, J. D.; McDonald, B.; Cools-Lartigue, J. J.; Chow, S. C.; Giannias, B.; Kubes, P.; Ferri, L. E. Neutrophils Promote Liver Metastasis Via Mac-1-Mediated Interactions with Circulating Tumor Cells. Cancer Res. 2012, 72, 3919−3927. (17) Strell, C.; Lang, K.; Niggemann, B.; Zaenker, K. S.; Entschladen, F. Surface Molecules Regulating Rolling and Adhesion to Endothelium of Neutrophil Granulocytes and MDA-MB-468 Breast Carcinoma Cells and Their Interaction. Cell. Mol. Life Sci. 2007, 64, 3306−3316. (18) Muller, W. A. Getting Leukocytes to the Site of Inflammation. Vet. Pathol. 2013, 50, 7−22. (19) Coffelt, S. B.; Wellenstein, M. D.; de Visser, K. E. Neutrophils in Cancer: Neutral No More. Nat. Rev. Cancer 2016, 16, 431−446. (20) Wang, F.; Gao, W.; Thamphiwatana, S.; Luk, B. T.; Angsantikul, P.; Zhang, Q.; Hu, C. M.; Fang, R. H.; Copp, J. A.; Pornpattananangkul, D.; Lu, W.; Zhang, L. Hydrogel Retaining Toxin-Absorbing Nanosponges for Local Treatment of MethicillinResistant Staphylococcus Aureus Infection. Adv. Mater. 2015, 27, 3437−3443. (21) Hu, C. M.; Fang, R. H.; Wang, K. C.; Luk, B. T.; Thamphiwatana, S.; Dehaini, D.; Nguyen, P.; Angsantikul, P.; Wen, C. H.; Kroll, A. V.; Carpenter, C.; Ramesh, M.; Qu, V.; Patel, S. H.; Zhu, J.; Shi, W.; Hofman, F. M.; Chen, T. C.; Gao, W.; Zhang, K.; et al. Nanoparticle Biointerfacing by Platelet Membrane Cloaking. Nature 2015, 526, 118−121. (22) Gao, W.; Fang, R. H.; Thamphiwatana, S.; Luk, B. T.; Li, J.; Angsantikul, P.; Zhang, Q.; Hu, C. M.; Zhang, L. Modulating Antibacterial Immunity Via Bacterial Membrane-Coated Nanoparticles. Nano Lett. 2015, 15, 1403−1409. (23) Guo, Y.; Wang, D.; Song, Q.; Wu, T.; Zhuang, X.; Bao, Y.; Kong, M.; Qi, Y.; Tan, S.; Zhang, Z. Erythrocyte MembraneEnveloped Polymeric Nanoparticles as Nanovaccine for Induction of Antitumor Immunity against Melanoma. ACS Nano 2015, 9, 6918− 6933. (24) Adams, J. The Development of Proteasome Inhibitors as Anticancer Drugs. Cancer Cell 2004, 5, 417−421. (25) Marchi, L. F.; Sesti-Costa, R.; Chedraoui-Silva, S.; Mantovani, B. Comparison of Four Methods for the Isolation of Murine Blood Neutrophils with Respect to the Release of Reactive Oxygen and Nitrogen Species and the Expression of Immunological Receptors. Comp. Clin. Pathol. 2014, 23, 1469−1476. (26) Kang, T.; Gao, X.; Hu, Q.; Jiang, D.; Feng, X.; Zhang, X.; Song, Q.; Yao, L.; Huang, M.; Jiang, X.; Pang, Z.; Chen, H.; Chen, J. iNGR-

Modified PEG-PLGA Nanoparticles That Recognize Tumor Vasculature and Penetrate Gliomas. Biomaterials 2014, 35, 4319−4332. (27) Luk, B. T.; Hu, C. M.; Fang, R. H.; Dehaini, D.; Carpenter, C.; Gao, W.; Zhang, L. Interfacial Interactions between Natural Rbc Membranes and Synthetic Polymeric Nanoparticles. Nanoscale 2014, 6, 2730−2737. (28) Kang, T.; Zhu, Q.; Jiang, D.; Feng, X.; Feng, J.; Jiang, T.; Yao, J.; Jing, Y.; Song, Q.; Jiang, X.; Gao, X.; Chen, J. Synergistic Targeting Tenascin C and Neuropilin-1 for Specific Penetration of Nanoparticles for Anti-Glioblastoma Treatment. Biomaterials 2016, 101, 60−75. (29) Molinaro, R.; Corbo, C.; Martinez, J. O.; Taraballi, F.; Evangelopoulos, M.; Minardi, S.; Yazdi, I. K.; et al. Biomimetic Proteolipid Vesicles for Targeting Inflamed Tissues. Nat. Mater. 2016, 15, 1037−1046. (30) Futosi, K.; Fodor, S.; Mócsai, A. Neutrophil Cell Surface Receptors and Their Intracellular Signal Transduction Pathways(). Int. Immunopharmacol. 2013, 17, 638−650. (31) Hauselmann, I.; Borsig, L. Altered Tumor-Cell Glycosylation Promotes Metastasis. Front. Oncol. 2014, 4, 28. (32) Di, D.; Chen, L.; Wang, L.; Sun, P.; Liu, Y.; Xu, Z.; Ju, J. Downregulation of Human Intercellular Adhesion Molecule-1 Attenuates the Metastatic Ability in Human Breast Cancer Cell Lines. Oncol. Rep. 2016, 35, 1541−1548. (33) Fan, Z. C.; Yan, J.; Liu, G. D.; Tan, X. Y.; Weng, X. F.; Wu, W. Z.; Zhou, J.; Wei, X. B. Real-Time Monitoring of Rare Circulating Hepatocellular Carcinoma Cells in an Orthotopic Model by in Vivo Flow Cytometry Assesses Resection on Metastasis. Cancer Res. 2012, 72, 2683−2691. (34) Roblek, M.; Calin, M.; Schlesinger, M.; Stan, D.; Zeisig, R.; Simionescu, M.; Bendas, G.; Borsig, L. Targeted Delivery of Ccr2 Antagonist to Activated Pulmonary Endothelium Prevents Metastasis. J. Controlled Release 2015, 220, 341−347. (35) Sakhalkar, H. S.; Dalal, M. K.; Salem, A. K.; Ansari, R.; Fu, J.; Kiani, M. F.; Kurjiaka, D. T.; Hanes, J.; Shakesheff, K. M.; Goetz, D. J. Leukocyte-Inspired Biodegradable Particles That Selectively and Avidly Adhere to Inflamed Endothelium in Vitro and in Vivo. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 15895−15900. (36) Zen, K.; Liu, D. Q.; Guo, Y. L.; Wang, C.; Shan, J.; Fang, M.; Zhang, C. Y.; Liu, Y. Cd44v4 Is a Major E-Selectin Ligand That Mediates Breast Cancer Cell Transendothelial Migration. PLoS One 2008, 3, e1826. (37) Ross, J. B.; Huh, D.; Noble, L. B.; Tavazoie, S. F. Identification of Molecular Determinants of Primary and Metastatic Tumour ReInitiation in Breast Cancer. Nat. Cell Biol. 2015, 17, 651−664. (38) Tang, S.; Yin, Q.; Su, J.; Sun, H.; Meng, Q.; Chen, Y.; Chen, L.; Huang, Y.; Gu, W.; Xu, M.; Yu, H.; Zhang, Z.; Li, Y. Inhibition of Metastasis and Growth of Breast Cancer by Ph-Sensitive Poly (BetaAmino Ester) Nanoparticles Co-Delivering Two Sirna and Paclitaxel. Biomaterials 2015, 48, 1−15. (39) Wang, Z.; Li, J.; Cho, J.; Malik, A. B. Prevention of Vascular Inflammation by Nanoparticle Targeting of Adherent Neutrophils. Nat. Nanotechnol. 2014, 9, 204−210. (40) Chu, D.; Gao, J.; Wang, Z. Neutrophil-Mediated Delivery of Therapeutic Nanoparticles across Blood Vessel Barrier for Treatment of Inflammation and Infection. ACS Nano 2015, 9, 11800−11811. (41) Hiratsuka, S.; Watanabe, A.; Sakurai, Y.; Akashi-Takamura, S.; Ishibashi, S.; Miyake, K.; Shibuya, M.; Akira, S.; Aburatani, H.; Maru, Y. The S100A8-Serum Amyloid A3-Tlr4 Paracrine Cascade Establishes a Pre-Metastatic Phase. Nat. Cell Biol. 2008, 10, 1349−1355. (42) Karin, M.; Greten, F. R. Nf-Kappab: Linking Inflammation and Immunity to Cancer Development and Progression. Nat. Rev. Immunol. 2005, 5, 749−759. (43) Hallett, W. H.; Ames, E.; Motarjemi, M.; Barao, I.; Shanker, A.; Tamang, D. L.; Sayers, T. J.; Hudig, D.; Murphy, W. J. Sensitization of Tumor Cells to Nk Cell-Mediated Killing by Proteasome Inhibition. J. Immunol. 2008, 180, 163−170. (44) Boxio, R.; Bossenmeyer-Pourie, C.; Steinckwich, N.; Dournon, C.; Nusse, O. Mouse Bone Marrow Contains Large Numbers of 1410


Article

ACS Nano Functionally Competent Neutrophils. J. Leukocyte Biol. 2004, 75, 604− 611. (45) Suski, J. M.; Lebiedzinska, M.; Wojtala, A.; Duszynski, J.; Giorgi, C.; Pinton, P.; Wieckowski, M. R. Isolation of Plasma MembraneAssociated Membranes from Rat Liver. Nat. Protoc. 2014, 9, 312−322.

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