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Metformin and Docosahexaenoic Acid Hybrid Micelles for Premetastatic Niche Modulation and Tumor Metastasis Suppression Tianze Jiang, Liang Chen, Yukun Huang, Jiahao Wang, Minjun Xu, Songlei Zhou, Xiao Gu, Yu Chen, Kaifan Liang, Yuanyuan Pei, Qingxiang Song, Shanshan Liu, Fenfen Ma, Huiping Lu, Xiaoling Gao, and Jun Chen Nano Lett., Just Accepted Manuscript • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 26, 2019

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Metformin and Docosahexaenoic Acid Hybrid Micelles for Pre-metastatic Niche Modulation and Tumor Metastasis Suppression Tianze Jiang,† Liang Chen,† Yukun Huang,† Jiahao Wang,† Minjun Xu,† Songlei Zhou,† Xiao Gu,‡ Yu Chen,† Kaifan Liang,† Yuanyuan Pei,† Qingxiang Song,‡ Shanshan Liu,† Fenfen Ma,§ Huiping Lu,§ Xiaoling Gao,‡,* Jun Chen†,§,*

†

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan

University, Lane 826, Zhangheng Road, Shanghai 201203, PR China

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Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of

Medicine, 280 South Chongqing Road, Shanghai 200025, PR China

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Department of Pharmacy, Shanghai Pudong Hospital, Fudan University, 2800 Gongwei Road,

Shanghai, 201399, PR China

ABSTRACT

ACS Paragon Plus Environment

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Metastasis is the major cause of high mortality in cancer patients, thus blocking the metastatic process is of critical importance for cancer treatments. Pre-metastatic niche, a specialized microenvironment with aberrant changes related to inflammation, allows the colonization of circulating tumor cells (CTCs) and serves as a potential target for metastasis prevention. However, little effort has been dedicated to developing nanomedicine to amend the pre-metastatic niche. Here this study reports a pre-metastatic niche targeting micelle for the modulation of pre-metastatic microenvironments and suppression of tumor metastasis. The micelles are self-assembled with the oleate carbon chain derivative of metformin and docosahexaenoic acid, two anti-inflammatory agents with low toxicity, and coated with fucoidan for pre-metastatic niche targeting. The obtained functionalized micelles (FucOMDs) exhibit excellent blood circulation profile and pre-metastatic site-targeting efficiency, inhibit CTC adhesion to activated endothelial cells, alleviate lung vascular permeability and reverse the aberrant expression of key marker proteins in pre-metastatic niches. As a result, FucOMDs prevent metastasis formation, and efficiently suppress both primary tumor growth and metastasis formation when combined with targeted chemotherapy. Collectively, the findings here provide proof of concept that the modulation of pre-metastatic niche with targeted anti-inflammatory agents provides a potent platform and a safe and clinical translational option for the suppression of tumor metastasis.

KEYWORDS: pre-metastatic niche, targeted drug delivery, self-assembly micelles, metformin, docosahexaenoic acid, metastasis suppression

Metastasis is an overwhelming cause of cancer-related high mortality in clinic, thus metastasis suppression is the key and most challenging mission in cancer treatments.1,

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Conventional


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therapeutic options including standard surgery, irradiation and chemotherapy can remove local cancer cells,3 but are far less effective against metastasis. Such situation highlights that the identification of vital cellular and molecular incidents in process of metastasis formation is critical for the development of novel and efficient strategies for metastasis suppression in cancer therapies.1 Metastasis starts from the detachment of cells from primary tumors, followed by the seeding of circulating tumor cells (CTCs) within the microenvironment in distant organs.4 Primary tumorderived factors or extracellular vesicles induce the establishment of microenvironments in distant organs that are favorable for the seeding and survival of CTCs before their arrival at these tissues.4 Such microenvironment is defined as the pre-metastatic niche.5 Characteristic molecular and cellular aberrant changes that arise in the niche support future seeding and growth of CTCs in local niches.4-7 The changes include the raised expression of adhesion molecules (e.g., selectins) on endothelial cells (ECs),8 vascular leakiness,4, 9 remodeling of extracellular matrix (ECM) through the overexpression of metalloproteinase-9 (MMP-9),10,

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recruitment of bone marrow-derived

cells (BMDCs) by the accumulation of fibronectin and inflammation associated with S100 protein family,11, 12 etc. Upon the adhesion of CTCs to activated ECs through adhesion molecules, vascular leakiness and BMDC recruitment promote the extravasation of CTCs across ECs. In addition, the ECM remodeling contributes to future CTC invasion and seeding in pre-metastatic tissues. Therefore, pre-metastatic niche may serve as a target for metastasis prevention. Inflammation is a crucial driver or characteristic for the formation of pre-metastatic niches.5 Tumor-derived cytokines and exosomes, for instance, tumor necrosis factor-α (TNF-α), can prompt the expression of pro-inflammatory mediator S100 protein family in pre-metastatic organs.12,

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Overexpression of S100 proteins (e.g., S100A9) contributes to the formation of


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inflammatory microenvironments via nuclear factor-κB (NF-κB) signaling.12, 14-16 The recruitment of myeloid derived suppressor cells (MDSCs) and activated NF-κB pathway derived from S100A9 promote pre-metastatic niche formation in an inflammatory tissue and enhance metastasis formation.4, 12, 13, 16, 17 Besides, other metastasis related inflammation induced by up-regulation of interleukin-6 (IL-6) and activation of signal transducer and activator of transcription-3 (STAT3) also promotes pre-metastatic niche formation.17,

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Thus, the establishment of inflammatory

microenvironments is a key process for the formation of pre-metastatic niches and thus for the seeding and survival of CTCs.5, 19 Cytotoxic drugs in cancer therapies can facilitate metastasis formation by inducing vascular damage, accelerating MMP-9 expression, up-regulating inflammatory cytokine secretion, enabling tumor cell adhesion to ECs through the overexpression of vascular endothelial growth factor receptor-1 (VEGFR-1) and promoting cancer cell migration or invasion in pre-metastatic organs.20-25 Accordingly, we proposed that the application of the lesstoxic targeted anti-inflammatory agents would provide a new approach to efficiently suppress metastasis by inhibiting the multiple molecular and cellular incidents in the formation of premetastatic niches. Among the various anti-inflammatory agents reported, metformin, a current drug for type II diabetes therapies, displays anti-inflammatory activities by the activation of AMP protein kinase (AMPK) and subsequent inhibition of NF-κB with excellent safety in vivo.26-28 Additionally, docosahexaenoic acid (DHA), a typical ω3-polyunsaturated fatty acid derived from marine organisms without obvious toxicity,29 shows anti-inflammatory effects attributed to various signaling pathways including NF-κB and STAT3 activition, etc.30 As mentioned above, inflammation is closely related to pre-metastatic niche formation, and characteristic aberrant changes in the niche, including adhesion molecules, vascular leakiness, fibronectin and MMP-9,


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are closely associated with NF-κB pathway.31-33 These aberrant changes may be repressed by antiinflammatory effects via NF-κB inhibition. Besides, S100A9 is an important stimulus or feature of inflammatory microenvironments and pre-metastatic niches,4, 12, 16 anti-inflammatory agents metformin and DHA may also reduce the expression of S100A9 to modulate inflammatory premetastatic niches. Consequently, we speculated that the combination of metformin and DHA, by inhibiting multiple inflammatory pathways, would efficiently modulate the pre-metastatic microenvironment and suppress tumor metastasis in a synergistic manner. In order to verify the hypothesis that modulation of pre-metastatic niche with targeted antiinflammatory agents would provide an efficient strategy for the suppression of tumor metastasis, here we developed a pre-metastatic niche targeting micelle for the modulation of pre-metastatic microenvironments. Micelles are one of typical applications of nanotechnology for drug delivery. Considering that rapid clearance, poor solubility and insufficient accumulation in targeted tissues may restrict the administration of therapeutic agents in vivo, nanotechnology can facilitate the application of these agents to realize targeted drug delivery, prolong blood circulation and integrate diverse drugs into one system for combination therapy.34-36 In present study, the micelles were loaded with metformin and DHA, two anti-inflammatory agents with low toxicity, and coated with fucoidan for pre-metastatic niche targeting (Figure 1A,B). To realize the combination of the hydrophilic agent metformin and the hydrophobic agent DHA in a simple nanosystem, we synthesized a novel amphiphilic metformin derivative (OA-Met) with the hydrophilic biguanide group of metformin conjugated with a hydrophobic carbon chain of oleic acid. OA-Met formed micelles in water, and DHA was entrapped in hydrophobic core of OA-Met micelles due to the hydrophobic effect and the π-π interaction between DHA and carbon chain of OA-Met. To achieve targeted therapies, the obtained micelles (OMDs) were surface decorated with an algae-derived


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anionic polysaccharide fucoidan, which possesses high affinity for P-selectin,37,

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electrostatic interaction (Figure 1A).39 P-selectin, an adhesion molecule overexpressed on activated ECs in pre-metastatic microenvironments, facilitates the adhesion of CTCs to ECs and accelerates metastasis formation.40-43 The murine triple negative breast cancer (TNBC), a highly metastatic cancer model, was applied to evaluate the metastasis suppression efficacy of the targeted micelles (FucOMDs).7 As far as we know, little effort has been dedicated to developing targeted nanomedicine to modulate the pre-metastatic microenvironment. Collectively, this is the first research to provide proof of concept that the modulation of pre-metastatic niche with targeted antiinflammatory agents (Figure 1B) would serve as a powerful strategy and a safe and clinical translational approach for metastasis suppression in cancer therapies.


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Figure 1. Schematic illustration of fucoidan decorated self-assembly micelles delivering metformin and DHA (FucOMDs) for pre-metastatic niche targeting and modification. (A) Schematic protocol of the FucOMDs preparation. (B) The illustration of the process of FucOMDs targeting P-selectin in the pre-metastatic microenvironment, and the inhibitory effect against typical aberrant alterations in the microenvironment by FucOMDs. ICAM-1: intercellular cell adhesion molecule-1, VCAM-1: vascular cell adhesion molecule-1.


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RESULTS AND DISCUSSION Synthesis and Characterization of OA-Met. OA-Met was synthesized through a one-step reaction between oleylamine and cyanoguanidine.44 Cyanoguanidine was conjugated to the amino group of oleylamine with FeCl3 as the catalyst (Scheme S1). After column chromatography purification, the amphiphilic derivative OA-Met was obtained. The synthesis of OA-Met was confirmed by 1HNMR (600 MHz) and mass spectrometry (MS). As 1H-NMR spectrum shown, the amino peak of oleylamine at the δ value of 1.2 ppm disappeared while the typical biguanide peak at δ 6.5-7.2 ppm was found in the product (Figure S1). Additionally, the mass-to-charge ratio (m/z) of the product presented by ESI-MS was 352.4 [M+H+], confirming the successful synthesis of OA-Met (Figure S2).

Preparation and Characterization of FucOMDs. OMDs were self-assembled by OA-Met with DHA in water. FucOMDs and dextran-decorated OMDs (DexOMDs) were obtained by adsorbing fucoidan and dextran, respectively, onto the surface of OMDs via electrostatic interaction. OMDs without polysaccharide coating were assumed to show positive zeta potential due to biguanide groups, and the dextran on DexOMDs might shield the positive charge of OMDs. Both micelles (OMDs and DexOMDs) were prepared as the control groups without targeted activity for further biology studies. The z-average diameter of OMDs, DexOMDs and FucOMDs was 131.6 ± 3.1 nm, 149.7 ± 2.3 nm and 149.9 ± 1.9 nm with a corresponding polymer dispersity index (PDI) of 0.201 ± 0.013, 0.145 ± 0.016 and 0.138 ± 0.011, respectively. Zeta potential of OMDs, DexOMDs and FucOMDs was 48.6 ± 4.5 mV, -21.2 ± 2.7 mV and -26.7 ± 1.9 mV, respectively (Figure 2A, Table S1). Under a transmission electron microscope (TEM), FucOMDs exhibited a dimmer polysaccharide layer (around 20 nm) that was not found on OMDs (Figure 2B). Such size increase


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(around 20 nm), charge reversal (positive to negative charge) and TEM differences between OMDs and FucOMDs confirmed the successful adsorption of anionic fucoidan on the surface of OMDs. Analysis showed that the encapsulation efficiency (EE) of DHA in OMDs and FucOMDs was 73.4 ± 1.1% and 65.7 ± 2.1%, and the loading capacity (LC) was 21.0 ± 0.3% and 10.2 ± 0.2%, respectively (Table S1). The above data suggested that amphiphilic OA-Met formed micelles in distilled water and the hydrophobic DHA was successfully loaded in the hydrophobic core of OAMet micelles to form cationic OMDs and subsequent FucOMDs. Compared with OA-Met micelles without DHA, we noticed that the addition of well-proportioned DHA (mass ratio of DHA to OAMet = 2:5) formed OMDs with more reasonable z-average diameter and lower PDI, and the selfassembly improvement probably benefited from the conjugative effect among double bounds of OA-Met and DHA (Table S2). Besides, DexOMDs and FucOMDs exhibited good stability with negligible size change when incubated in phosphate buffer saline (PBS, pH 7.4) at 4 °C for more than 14 days, whereas OMDs showed significant augment after 10 days (Figure 2C). In addition, the critical micelle concentration (CMC) of FucOMDs was 4.43 μg/mL (Figure 2D), suggesting that FucOMDs might remain stable when subjected to blood dilution.


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Figure 2. Characterization of FucOMDs. (A) Size distribution, z-average diameter and surface zeta potential of OMDs, DexOMDs and FucOMDs. (B) TEM images of OMDs and FucOMDs. Diagrams on the second line showed the magnification of the first line micelle. Scale bar, 100 nm. (C) Z-average diameter change of OMDs, DexOMDs and FucOMDs with incubation in PBS at 4 °C for 14 days. Data were represented as mean ± SD (n = 3). (D) Plots of the fluorescence intensity ratio of 384 nm and 373 nm to the logarithm concentration of FucOMDs.

FucOMDs Targeted Activated ECs in Vitro. In order to evaluate the pre-metastatic niche targeting effect of FucOMDs, the cellular uptake of FucOMDs was firstly measured in activated ECs. Murine endothelial cell lines bEnd.3 cells that was stimulated with inflammatory TNF-α overexpressed P-selectin and thus served as the cell model (Figure S3A,B).45, 46 Due to its surface positive charge, the accumulation of coumarin-6 labeled OMDs (OMDs-Cou) in bEnd.3 cells was the strongest among all formulations. The cellular uptake of coumarin-6 labeled FucOMDs


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(FucOMDs-Cou) was 1.3-fold higher than that of coumarin-6 labeled DexOMDs (DexOMDs-Cou) in the presence of TNF-α stimulation (P < 0.01, Figure S4A,B). In contrast, there was no significant difference of cellular uptake between FucOMDs-Cou and DexOMDs-Cou in the absence of TNF-α (Figure S4B, Figure S5). In addition, P-selectin positive ECs showed higher cellular uptake of FucOMDs-Cou than P-selectin negative ECs (P < 0.0001, Figure S4B), indicating that fucoidan-P-selectin interaction might play an important role in the enhanced uptake of FucOMDs in activated ECs.

Polysaccharide Modification Improved Blood Circulation Profile of OMDs. Encouraged by the promising results in cellular uptake assay, we next studied the targeting effect of FucOMDs in vivo. Primarily, we evaluated the pharmacokinetic behavior of different micelles. The concentrations of metformin, OA-Met and DHA in plasma were detected directly (Figure S6) and main pharmacokinetic parameters including area under curve (AUC0-24h), blood circulation half-life (t1/2), clearance (CL) and mean residence time (MRT0-24h) were analyzed (Table S3, Table S4). For OAMet detection (Table S3), the AUC0-24h of OA-Met in DexOMDs and FucOMDs was found to be 2.48- and 2.42-fold higher than that in OMDs, respectively. In addition, OA-Met in both polysaccharide-modified OMDs (DexOMDs and FucOMDs) possessed longer t1/2 than that in OMDs (P < 0.001). The elimination of DexOMDs and FucOMDs was also slower than OMDs (P < 0.05 for CL), and the MRT0-24h of DexOMDs and FucOMDs was observed to be 1.51- and 1.73fold longer than OMDs, respectively. Moreover, the analysis of DHA in plasma was consistent with the results of OA-Met (Table S4). AUC0-24h and MRT0-24h of DHA in DexOMDs and FucOMDs were significantly higher than that in OMDs. According to t1/2 and CL, the elimination of DHA in DexOMDs and FucOMDs was obviously slower than that in OMDs. In contrast, free


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OA-Met, free metformin and free DHA were all cleared the fastest as expected, and free OA-Met had similar pharmacokinetic behavior to free metformin. OMDs were cleared more quickly than the polysaccharide-modified micelles probably because of its recognition by the mononuclear phagocyte system (MPS) due to the surface positive charge.47, 48 These data collectively justified that polysaccharide (fucoidan or dextran) modification improved the blood circulation profile of OMDs, and the long-term blood circulation of micelles would ensure better targeting effects to pre-metastatic niches.

FucOMDs Targeted Pre-metastatic Niches in Vivo. After demonstrating the excellent blood circulation profile, we then tested the in vivo targeting efficacy of FucOMDs by the biodistribution study. Firstly, in tumor-conditioned medium (TCM) stimulating mice model, TCM from 4T1 cells was injected intravenously into normal Balb/c mice and then the lung targeting efficacy of FucOMDs was investigated. TCM containing abundant tumor cell-derived cytokines and exosomes can induce a pre-metastatic microenvironment with elevated expression of P-selectin (or other adhesion molecules) and lung leakiness, etc,49-54 and the microenvironment would facilitate CTC seeding and metastasis formation.11 As shown in Figure 3A, 1,1’-dioctadecyl3,3,3’,3’-tetramethyl indotricarbocyanine iodide (DiR) labeled FucOMDs (FucOMDs-DiR) showed the highest accumulation in ex vivo lungs among the FucOMDs-DiR, DexOMDs-DiR and OMDs-DiR groups. The results indicated that FucOMDs could target pre-metastatic lungs. To further reveal the mechanism of FucOMDs in lung pre-metastatic niche targeting, we studied the distribution of FucOMDs in lung tissues through immunofluorescent staining of P-selectin and laminin. In addition to P-selectin, laminin abundant lung tissues also represent a pre-metastatic area easily for CTC seeding.55, 56 Accordingly, the fluorescence of FucOMDs-DiR was found to


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restrict to the P-selectin and laminin abundant tissues (Figure 3B), suggesting that the premetastatic niche targeting property of FucOMDs not only benefited from the excellent blood circulation profile but also from the P-selectin targeting ability of surface fucoidan. Next, in the orthotopic TNBC mice model, primary tumor tissues can induce spontaneous development of pre-metastatic niches.4, 5, 51, 52 Our preliminary experimental results in luciferase labeled 4T1 (4T1-Luc) cells derived model showed that lung metastasis began to be observed on day 15 to day 18 after tumor inoculation (day 0), thus the period from day 0 to day 14 could be considered as the pre-metastatic phase.11, 57 Accordingly, we performed the distribution assay on day 14. According to the ex vivo semi-quantitative analysis, the strongest fluorescence of FucOMDs-DiR was observed in lungs compared with DexOMDs-DiR and OMDs-DiR (P < 0.01, Figure 3C,D). Moreover, most FucOMDs-DiR were observed in P-selectin abundant region in lung sections, while OMDs-DiR or DexOMDs-DiR distributed widely in both P-selectin rich area and normal lung tissues with low fluorescence (Figure 3E). FucOMDs-DiR were also limited to laminin abundant area (Figure 3F), whereas DexOMDs-DiR and OMDs-DiR spread broadly in all tissues like P-selectin stained sections. In addition, we also directly determined the concentrations of metformin, OA-Met and DHA in the main organs (heart, liver, spleen, lung and kidney) of this mice model. As shown in Figure S7, lung accumulation of OA-Met and DHA in FucOMDs was significantly stronger than those in DexOMDs and OMDs at both 0.5 h (P < 0.05) and 12 h (P < 0.01). After injection of free metformin, free OA-Met and free DHA, their concentrations in the lungs were the lowest, and free OA-Met had similar distribution to free metformin in lung tissues. Collectively, these results supported that FucOMDs could target premetastatic niches of lungs in different mice model, providing a possibility for inhibition of premetastatic niche and metastasis formation.


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Besides, we found that the fluorescence of FucOMDs-DiR at orthotopic tumor sites increased gradually within 24 h and was stronger than that of OMDs-DiR and DexOMDs-DiR at each time points (Figure S8). The fluorescence images and semi-quantitative data of ex vivo organs also showed more accumulation of FucOMDs at tumor sites (P < 0.01, Figure 3C,D). Similarly, tumor accumulation of OA-Met and DHA in FucOMDs was the strongest at both 0.5 h (P < 0.05) and 12 h (P < 0.05 for OA-Met, P < 0.01 for DHA, Figure S7). The orthotopic targeting effect could be attributed to the expression of P-selectin on the endothelial membrane of breast tumor activated by endogenous cytokines.58


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Figure 3. FucOMDs targeted pre-metastatic microenvironments in vivo. (A) Ex vivo fluorescence


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imaging and semi-quantitative analysis of fluorescence intensity of lungs in TCM stimulating mice model. Data were represented as mean ± SD (n = 3), ****P < 0.0001. (B) Fluorescence microscopy images of FucOMDs targeting P-selectin abundant site (left column) and laminin abundant area (right column) in lung tissues of TCM stimulating mice. The white dotted line separates premetastatic tissues from normal tissues. Blue, Hoechst 33258; green, laminin-positive or P-selectin positive pre-metastatic tissues, the PE orange color of P-selectin was transferred to green color; red, DiR. Scale bar, 100 µm. (C) Ex vivo fluorescence imaging of main organs and tumors obtained from orthotopic breast tumor mice after in vivo fluorescence imaging. (D) Semi-quantitative analysis of the fluorescence intensity of tumors and organs. Data were represented as mean ± SD (n = 3), **P < 0.01. Fluorescence microscopy analysis of micelles targeting (E) P-selectin abundant site and (F) laminin abundant area in lung tissues of orthotopic breast tumor mice. The white dotted line separates pre-metastatic tissues from normal tissues. Blue, Hoechst 33258; green, laminin-positive or P-selectin positive pre-metastatic tissues, the PE orange color of P-selectin was transferred to green color; red, DiR. Scale bar, 100 µm.

OA-Met and DHA Suppressed Inflammation in a Synergistic Manner. To investigate the inhibitory effects of pre-metastatic niches of FucOMDs, the combination anti-inflammatory efficacy of OA-Met and DHA was evaluated in vitro. Lipopolysaccharide (LPS) induces the activation of NF-κB in macrophages, up-regulating expression of various inflammatory cytokines, such as TNF-α and IL-1β.59, 60 Therefore, we investigated the synergistic anti-inflammatory effects of OA-Met and DHA by the analysis of cytokine secretion (TNF-α and IL-1β).60 For this detection, RAW264.7 cells were treated with OA-Met, DHA and the combination of OA-Met and DHA (with OA-Met and DHA at 5:2 mass ratio), and the cells were stimulated with LPS. The mass ratio of


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OA-Met and DHA combination was consistent with the ratio in micelles. Culture medium from the cells was then collected and detected by ELISA kits. The combination index (CI) was finally calculated by CompuSyn software.61 As shown in Table S5, we found that CI of OA-Met and DHA combinations at different dosing concentrations were all less than 1 (CI < 1). These data suggested that the combination of OA-Met and DHA was potently synergistic, providing a basis for the subsequent combination therapies of these two agents in FucOMDs.

FucOMDs Inhibited CTC Adhesion to Activated ECs and Suppressed Elevated Expression of endothelial adhesion molecules. The adhesion of CTCs to activated ECs in pre-metastatic microenvironments is an initial and vital step for CTC colonization.41, 62 For this reason, we next analyzed whether FucOMDs could inhibit tumor cell adhesion to activated ECs in vitro. For the analysis, bEnd.3 cells were firstly stimulated with TNF-α, and then exposed to metformin, OAMet, DHA, OMDs, DexOMDs or FucOMDs, respectively. After incubation with drug formulations,49 green fluorescent protein-labeled 4T1 (4T1-GFP) cells were added to conduct the adhesion assay with ECs. It was found that the adhesion of 4T1-GFP cells to the TNF-α activated ECs was much higher than that to the TNF-α free control ECs (P < 0.0001, Figure S9A,B). Metformin, OA-Met and DHA individually mitigated such adhesion, while micelles with the combination of OA-Met and DHA (OMDs, DexOMDs or FucOMDs) achieved more effective inhibition. FucOMDs showed the strongest effect among the micelle groups on the basis of the role of fucoidan in cellular internalization (Figure S9A,B). Overall, the adhesion study suggested that FucOMDs could greatly inhibit the adhesion of tumor cells to ECs, reducing the possibility of metastasis establishment. We then continued to uncover the molecular mechanism for the reduction of adhesive tumor


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cells to ECs through flow cytometry assay. In pre-metastatic niches, the up-regulation of endothelial adhesion molecules such as intercellular cell adhesion molecule-1 (ICAM-1), Eselectin and vascular cell adhesion molecule-1 (VCAM-1) plays significant roles in the adhesion of CTCs.41, 49, 62-64 To simulate such microenvironment, the bEnd.3 cells were pre-stimulated with TNF-α and then incubated with various drug formulations.65 Those ECs activated with TNF-α showed higher expression of adhesion molecules (ICAM-1, E-selectin and VCAM-1) than the TNF-α negative group (P < 0.0001, Figure S10A,B). Such overexpression of adhesion molecules was inhibited by all therapeutics. Metformin showed the lowest effect probably because of its hydrophilic property and less cellular uptake. OA-Met achieved higher inhibitory effects than metformin due to its amphiphilicity and promoted cellular uptake (P < 0.05 for E-selectin, P < 0.0001 for ICAM-1 and VCAM-1). All micelle groups (OMDs, DexOMDs or FucOMDs) showed much higher inhibition than metformin, OA-Met and DHA, suggesting the synergistic effect of OA-Met and DHA. Particularly, FucOMDs achieved the highest inhibition efficiency among the micelle groups (Figure S10A,B). The expression profile of adhesion molecules in activated ECs was well correlated with the adhesion of CTCs to activated ECs, confirming that FucOMDs could efficiently block the expression of adhesion molecules in activated ECs and inhibit the interaction between CTCs and ECs.

FucOMDs Reduced Lung Vascular Permeability and Reversed Aberrant Expression of Key Marker Proteins in Pre-metastatic Niches. Extravasation is an indispensable process that CTCs pass through the endothelial barriers to form metastasis following the adhesion of CTC to ECs.5 Such process is significantly related to high vascular permeability that is promoted by primary tumor-derived factors such as vascular endothelial growth factor (VEGF) and TNF-α.49,

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Therefore, we next investigated whether high vascular permeability in lungs could be remitted by FucOMDs. One day after the final drug injection (on day 14 after tumor inoculation), mice were infused i.v. with Evans Blue (EB).49 Two hours later (Figure 4A), EB leakage in the lungs of those breast tumor bearing mice was 2.51-fold higher than that in the lungs of normal mice (Figure 4B, Figure S11), confirming the elevated lung vasculature permeability in pre-metastatic niches. DexOMDs and FucOMDs treatments inhibited EB leakage observably while other individual agents (metformin, OA-Met and DHA) exhibited slight inhibitory effects, highlighting the importance of the combination of the anti-inflammatory agents and the long circulation of the nanosystem. Furthermore, FucOMDs showed lower EB leakage than DexOMDs on account of its pre-metastatic niche targeting capability (P < 0.05, Figure 4B, Figure S11). These data confirmed that FucOMDs could mitigate the high vascular permeability microenvironment efficiently, reducing the possibility in the extravasation and seeding of CTCs. `Furthermore, except for up-regulated expression of adhesion molecules and high vascular permeability that occur in pre-metastatic niches, abnormal accumulation of other key marker proteins like fibronectin, MMP-9 or S100 proteins (e.g., S100A9) also contributes to the premetastatic niche formation.4, 6, 67 As an ECM protein, fibronectin facilitates the homing of VEGFR+ BMDCs that promote the formation of pre-metastatic niches and the extravasation of tumor cells.6, 11, 68

The increased expression of MMP-9 in lung ECs and macrophages accelerates the migration

of CTCs into the lung tissue by degrading ECM, resulting in tissue remodeling during the development of metastasis.6, 10, 11 Additionally, S100A9 promotes the formation of inflammatory microenvironments via NF-κB signaling. The protein is related to the recruitment of MDSCs, leading to the suppression of anti-tumor immune response. Moreover, MDSCs and activated NFκB pathway derived from S100A9 induce the formation of pre-metastatic niches.12, 16, 69 Therefore,


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we examined whether FucOMDs would display a potential role in regulation of these proteins by immunofluorescence staining (Figure 4A). In the case of individual agents, metformin, OA-Met and DHA exhibited inhibitory effects compared with the saline group in the inhibition of fibronectin and MMP-9. In contrast, only DHA and OA-Met showed obvious effect in S100A9 inhibition (Figure 4C-E). Furthermore, DexOMDs and FucOMDs showed better effects than individual agents, and FucOMDs showed the strongest inhibition among all the formulations (Figure 4C-E). Thus, the aberrant expression of fibronectin, MMP-9 and S100A9 in lungs was reversed efficiently, suggesting the pre-metastatic niche inhibition and potential metastasis prevention activity of FucOMDs.


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Figure 4. FucOMDs alleviated lung vascular permeability and reversed aberrant expression of


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fibronectin, MMP-9 and S100A9 in pre-metastatic niches in vivo. (A) Timeline and treatment schedule for lung leakage assay and evaluation of fibronectin, MMP-9 and S100A9 in lung tissues. (B) Quantitative analysis of EB leakage in lungs. The unit of EB level (μg/g) means the mass of EB per gram of lung. Confocal microscopy imaging and semi-quantitative analysis of the expression of (C) fibronectin, (D) MMP-9 and (E) S100A9 in lung tissue frozen sections. Blue, Hoechst 33258; green, fibronectin-positive, MMP-9-positive or S100A9-positive lung tissues. Scale bar, 100 µm. Data were represented as mean ± SD (n = 4), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FucOMDs Prevented Metastasis Formation. To evaluate the inhibitory efficacy of FucOMDs against lung metastasis formation in TCM stimulating mice model, TCM was injected intravenously into normal Balb/c mice, and then different drug formulations were injected via tail vein for the niche treatment on the same day. Following the treatment, 4T1-Luc cells were injected i.v. and the lung metastasis was monitored by an IVIS spectrum imaging system every three or four days (Figure 5A). Mice injected with TCM began to show metastatic foci before day 4 while mice without TCM injection started to appear on day 7 with lower luciferin radiance (Figure S12A,B). The results confirmed that TCM could stimulate the establishment of pre-metastatic niche to accelerate the formation of metastasis. Additionally, no metastatic foci were found on day 4 in all treatment groups. From day 4 to day 18, mice with metastatic foci could be gradually detected in metformin, DHA, OA-Met and DexOMDs groups, and the number of mice with metastasis at 4, 11 and 18 days was, respectively, 0, 3 and 3 for metformin, 0, 2 and 3 for OA-Met, 0, 2 and 3 for DHA, and 0, 1 and 2 for DexOMDs. Mice with metastasis began to be found in the FucOMDs group on day 18 (Figure 5B,C). Moreover, the count of lung metastatic nodules stained


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by Bouin's fluid on day 21 showed that the inhibitory effects of DexOMDs and FucOMDs against metastatic foci formation were stronger than that of metformin, DHA and OA-Met groups (Figure 5D,E). Further, FucOMDs displayed less metastatic nodules than DexOMDs (P < 0.05, Figure 5D,E). In conclusion, we demonstrated that, by modulating pre-metastatic niches, FucOMDs exhibited the best inhibitory activity against CTC seeding and metastasis formation among all drug formulations.


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Figure 5. FucOMDs prevented metastasis formation in TCM stimulating mice model with 4T1-


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Luc cell intravenous injection. (A) Treatment schedule and timeline for TCM stimulation, treatments with various drug formulations and lung metastasis evaluation. Days were recounted from day 0 after all TCM stimulations and treatments. (B) In vivo bioluminescent images of lung metastasis on day 4, day 11 and day 18. (C) Mice with bioluminescent signal and bioluminescent intensity in each group detected by bioluminescent imaging. Dots and transverse lines mean mice with bioluminescent signal and average bioluminescence radiance in each group, respectively. The number of mice with metastasis at 4, 11 and 18 days was, respectively, 2, 4 and 4 for saline, 0, 3 and 3 for metformin, 0, 2 and 3 for OA-Met, 0, 2 and 3 for DHA, 0, 1 and 2 for DexOMDs and 0, 0 and 1 for FucOMDs. (D) The number of metastatic nodules of ex vivo lungs stained by Bouin's fluid. Data were represented as mean ± SD (n = 4), *P < 0.05, **P < 0.01. (E) Representative images of lungs with metastatic nodules stained by Bouin's fluid. Red cycles mean representative metastatic nodules on the surface of lungs.

Combination of FucOMDs and CK-PTX NPs Suppressed Primary Tumor Growth and Metastasis Formation. After demonstrating the inhibitory effect of FucOMDs against metastasis formation, we finally determined whether FucOMDs could prevent the metastasis formation in 4T1-Luc cell derived orthotopic breast tumor mice model. The development of metastasis is a complicated process affected by interactions among primary tumor-derived cytokines or exosomes, BMDCs, CTCs and local microenvironments.5 The orthotopic breast tumor can simulate almost all phases during metastasis formation, and the therapeutic effect of FucOMDs should be evaluated in this model. Besides, although many targeted nanoparticles loading chemotherapeutic drugs are effective for primary tumor therapies, metastatic cancers are still difficult to be cured due to noneffective treatments for preventing metastasis. Our group has demonstrated that CK peptide


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decorated nanoparticles are able to target primary tumors with VEGFR-2 and sonic hedgehog (Shh) expression.70, 71 Therefore, in order to treat metastatic cancers (e.g., TNBC) more efficiently, we adopted CK peptide modified PEG-PLA nanoparticles loading paclitaxel (CK-PTX NPs) for primary tumor targeting therapy and FucOMDs for metastasis inhibition simultaneously (Figure 6A).71 Two days after the last injection (on day 30), the FucOMDs group displayed wider apoptosis area by hematoxylin and eosin (H&E) staining (Figure S13A), less tumor burden (P < 0.05) and less tumor volume (P < 0.01) than DexOMDs (Figure 6B,C, Figure S13B). The inhibition of FucOMDs against primary tumor was consistent with the primary tumor targeting effect and the anti-tumor effects of metformin and DHA.28,

72

The treatment with CK-PTX NPs exhibited

stronger anti-tumor effect than FucOMDs and DexOMDs, confirming the crucial role of chemotherapy drugs in the treatment of primary tumors. FucOMDs plus CK-PTX NPs exhibited the most obvious anti-tumor effect, which suggested that FucOMDs could improve the therapeutic effect of chemotherapies (Figure 6B,C, Figure S13A,B). To analyze the inhibitory effect of the treatments against metastasis formation, the lungs excised from all mice were subjected to IVIS bioluminescence analysis. Only three mice developed slight lung metastasis from the FucOMDs group and the FucOMDs plus CK-PTX NPs group, while all mice from the saline, DexOMDs, CK-PTX NPs and CK-PTX NPs plus DexOMDs group developed serious lung metastasis based on the lung bioluminescent signals (Figure 6D). Further semi-quantitive data suggested that the FucOMDs group and the FucOMDs plus CK-PTX NPs group significantly reduced the lung metastasis establishment (Figure 6E). The suppressive effects against pre-metastatic niches confirmed that FucOMDs showed efficient inhibitory activities against spontaneous metastasis formation in this model. Besides, the survival analysis stated that the CK-PTX NPs plus FucOMDs group displayed the longest medium survival time (64 days),


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while the medium survival time of saline, DexOMDs, FucOMDs, CK-PTX NPs and CK-PTX NPs plus DexOMDs group was 36, 41, 50, 52, and 54 days, respectively (Figure S13C). Collectively, the anti-metastatic cancer therapy could be improved by the combination of FucOMDs and chemotherapy.

Safety of FucOMDs in vivo. The safety of our micelles was also assessed in vivo. No weight loss was found in all groups during the same treatment process in the orthotopic model mentioned above (Figure 6F). Furthermore, the in vivo toxicity of DexOMDs and FucOMDs was analyzed by H&E staining of major organ (heart, liver, spleen, lung and kidney) sections, and no obvious damage was found in these organs after treatments with DexOMDs and FucOMDs (Figure S14). The results indicated that the biocompatible FucOMD was an appropriate choice for the prevention of metastasis.


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Figure 6. Combination of FucOMDs and CK-PTX NPs suppressed primary tumor growth and metastasis formation in orthotopic breast tumor mice model. (A) Treatment schedule and timeline for treatments of DexOMDs or FucOMDs with CK-PTX NPs. (B) Primary tumor growth curves of mice in 30 days. (C) The weight of ex vivo primary tumor tissues after all treatments. Data were


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represented as mean ± SD (n = 5), *P < 0.05, **P < 0.01. (D) The ex vivo images of lungs with bioluminescent signals excised from mice at the end of treatment. (E) Ex vivo semi-quantitative analysis of lungs with bioluminescent radiance. (F) Mice body weight curves during the treatment period. Data were represented as mean ± SD (n = 5), *P < 0.05.

CONCLUSION In this study, we developed pre-metastatic niche targeting micelles (FucOMDs) to restrain the premetastatic microenvironment and suppress the metastasis formation. OA-Met, as a novel derivative of metformin, was synthesized and self-assembled with DHA to form micelles for antimetastasis studies. Fucoidan decoration on the surface of micelles enabled their significant targeting effect to activated ECs, long circulation and outstanding targeting efficacy to the premetastatic niches. In addition, FucOMDs reduced CTC adhesion to activated ECs by downregulating adhesion molecules, mitigated high vascular permeability and reversed aberrant expression of fibronectin, MMP-9 and S100A9 in the pre-metastatic niches. As a result, FucOMDs repressed metastasis formation, and efficiently suppressed both primary tumor growth and metastasis formation when combined with targeted chemotherapy. Besides, OA-Met exhibited similar or even better pre-metastatic lung distribution and therapeutic activities compared with metformin, suggesting the successful application of OA-Met instead of metformin. In summary, here we provide proof of concept that early intervention against the pre-metastatic niche with targeted anti-inflammatory agents would afford a potent platform and a safe and clinical translational selection for metastasis prevention in cancer therapies.


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EXPERIMENTAL METHODS Synthesis and Characterization of OA-Met. Anhydrous FeCl3 (1.12 mmol) was added to a solution of Oleylamine (OA, 1.12 mmol) and cyanoguanide (1.68 mmol) in 1,4-dioxane (15 mL), then the reaction mixture was stirred at 90 °C overnight. Since the reaction completed, the solvent was removed in vacuo and the crude product was purified by column chromatography to obtain OA-Met with a yield of 58.9%. The chemical structure of OA-Met was confirmed by 1H-NMR (600 MHz, BRUKER) in DMSO- d6 and ESI-MS. Preparation and Characterization of FucOMDs. OMDs were prepared through self-assembly method firstly.73, 74 Briefly, a solution of OA-Met in ethanol (375 μL at 10 mg/mL) mixed with DHA (1.5 mg) at an OA-Met to DHA mass ratio of 5:2 was added dropwise into distilled water (750 μL) under stirring (500 rpm). The mixture was stirred for another 3 h at room temperature and then OMDs occurred spontaneously. Ethanol was completely removed under vacuo at 25 °C to obtain an aqueous suspension of OMDs (7.35 mg/mL). Next, the FucOMDs were prepared by electrostatic adsorption. Briefly, aqueous OMDs suspension (152 μL) was slowly added into fucoidan solution (1 mL, 0.8 mg/mL) under stirring (500 rpm). The dispersion was stirred for another 1 h at room temperature and an aqueous suspension of FucOMDs was obtained. Coumarin6 and DiR labeled OMDs, DexOMDs or FucOMDs were prepared by the same protocol except that coumrin-6 or DiR was mixed with DHA and OA-Met in ethanol before the addition into distilled water. Z-average diameter, PDI and zeta potential of various micelles were determined by a dynamic light scattering detector (DLS) (Zetasizer, Nano-ZS, Malvern, UK). The stability of various micelles was tested in PBS at 4 °C for 14 days and the z-average diameter of micelles was detected at pre-designed time points. To investigate EE and LC of DHA, OMDs, DexOMDs or FucOMDs


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were dissolved in methanol and then subjected to the high performance liquid chromatography (HPLC) system at the detector wavelength of 220 nm. The EE and LC were calculated by Equation (1) and (2) (n = 3): 𝐸𝐸 (%) = 𝐿𝐶 (%) =

amount of DHA in the micelles total amount of DHA added amount of DHA in the micelles micelles weight

× 100%

(1)

× 100%

(2)

The CMC of FucOMDs was determined by pyrene as the fluorescence probe. Pyrene solution in acetone (100 μL, 0.125 mg/mL) was added into several tubes and the solvent was dried. Several concentrations of FucOMDs (0.0001 to 0.2 mg/mL) were added to the tubes containing dried pyrene, respectively. The tubes were then kept in a shaker at 37 °C for 24 h at 120 rpm. The excitation spectra of pyrene were scanned at 335 nm and the emission wavelength was fixed at 373 nm and 384 nm via a fluorescence spectrophotometer. The CMC of FucOMDs was the cross point in the plots of the fluorescence intensity ratio of 384 nm and 373 nm to the logarithm concentration of FucOMDs. The morphology of OMDs and FucOMDs was analyzed by a field emission TEM (TEM-1400 Plus Electron Microscope, Leica). OMDs or FucOMDs (1 mg/mL) were dropped on the carboncoated grid. When water was dried, a drop of 2% (w/v) phosphotungstic acid staining solution was dropped on the grid for negative staining. Five minutes later, the solution was removed and the grid was subjected to a TEM. Cellular uptake of FucOMDs in vitro. bEnd.3 cells were seeded into 96-well plates at a density of 5,000 cells per well and allowed to be incubated for 24 h (n = 3). Then the cells were stimulated with TNF-α (50 ng/mL) for 4 h. Afterwards, the media was replaced with free coumarin-6, OMDs-Cou, DexOMDs-Cou and FucOMDs-Cou (200 ng/mL for coumarin-6), respectively. After 2 h incubation in the dark, the cells were then washed three times with PBS


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buffer, fixed with 4% paraformaldehyde (PFA) for 10 min and stained with the solution of Hoechst 33258 (2.5 μg/mL) at room temperature for 10 min away from light. Finally, the cells were washed another three times with PBS buffer, subjected to a Kinetic Scan HCS Reader (version 3.1, Cellomics Inc., Pittsburgh, PA, USA) for quantitative analysis, and imaged under a fluorescence microscope (Leica DMI 4000B, Germany) for qualitative observation. Pharmacokinetic study of FucOMDs. To investigate whether fucoidan or dextran could prolong the blood circulation time of OMDs in vivo, eighteen SD rats were randomly divided into six groups (n = 3) and i.v. injected with metformin, DHA, OA-Met, OMDs, DexOMDs and FucOMDs (at an equivalent metformin dose of 8 mg/kg), respectively. Blood samples (100 μL) were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after injection, and then centrifuged at 4000 rpm for 10 min immediately. The supernatant plasma samples were stored at -20 °C until analysis. To detect the concentration of metformin, OA-Met and DHA in plasma, 150 μL methanol containing 100 ng/mL warfarin (internal standard) was added to 30 μL plasma samples in order to precipitate proteins. Then the samples were vortexed for 1 min and centrifuged at 12000 rpm for 5 min. The supernatant was subsequently subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS, API 4000, Applied Biosystems, Ontario, Canada) for analysis. Quantification of the ions was achieved by the multiple reaction monitoring (MRM) mode, in positive mode for metformin (monitoring the transition of the m/z 130.1 precursor ion to the m/z 71), OA-Met (monitoring the transition of the m/z 352.3 precursor ion to the m/z 60.0) and in negative mode for DHA (monitoring the transition of the m/z 327.2 precursor ion to the m/z 283.1). The pharmacokinetic parameters were calculated by Drug and Statistics (DAS) software (Version 2.0, Mathematical Pharmacology Professional Committee of China).


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Preparation of TCM. 4T1 cells were cultured in RPMI 1640 medium with 10% FBS (v/v), 100 mg/mL streptomycin and 100 U/mL penicillin at 37 °C in a humidified atmosphere containing 5% CO2. When the cells formed a consecutive monolayer, the media was replaced with serumfree RPMI 1640 media and the cells were incubated overnight. Then the medium was collected, centrifuged, and the supernatant was filtered through 0.22 μm syringe filters. The obtained TCM was stored at -20 °C. In vivo biodistribution study of FucOMDs. The in vivo distribution study was designed to demonstrate the pre-metastatic niche targeting effect of FucOMDs. In TCM stimulating mice model, TCM (200 μL) was intravenously injected into normal Balb/c mice. Thirty minutes later, the mice were randomly divided into three groups (n = 3) and intravenously injected with OMDsDiR, DexOMDs-DiR and FucOMDs-DiR (1 mg/kg for DiR). After 3 h, the mice were sacrificed and lungs were harvested for ex vivo imaging by the IVIS spectrum imaging system (Cailper PerkinElemer, USA). To further investigate the distribution of FucOMDs in the lung premetastatic niche, lung tissues were fixed, embedded in optimal cutting temperature (OCT) compound and sliced into 18-μm frozen sections. Sections were then stained with PE labeled antiP-selectin antibodies, or primary rabbit anti-mouse laminin antibodies followed by secondary Alexa Flour 488-labeled goat anti-rabbit IgG antibodies according to latter immunofluorescence methods. Finally, the distribution of DiR signals was detected after the subjection to a fluorescence microscope (Leica DMI 4000B, Germany). In 4T1 cell-derived orthotopic breast tumor mice model, 4T1 cells were inoculated into one mammary fat pad of Balb/c mice. On day 14 after tumor inoculation, the mice were randomly divided into three groups (n = 3) and intravenously injected with OMDs-DiR, DexOMDs-DiR and FucOMDs-DiR (1 mg/kg for DiR). The in vivo biodistribution of micelles was monitored by an in


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vivo IVIS spectrum imaging system (Cailper PerkinElemer, USA) at different pre-designed time points. After 24 h, the mice were sacrificed immediately, and major organs (heart, liver, spleen, lung and kidney) and orthotopic tumors were harvested for ex vivo imaging. The distribution of FucOMDs-DiR in the lung pre-metastatic microenvironment was detected in keeping with TCM stimulating mice model. Tumor cell adhesion to endothelial cells in vitro. To conduct the study of tumor cell adhesion to ECs,49 bEnd.3 cells were seeded into 6-well plates at a density of 500,000 cells per well (n = 3). After 24 h incubation, the cells were stimulated with TNF-α for 4 h, washed twice with PBS buffer and incubated with metformin, OA-Met, DHA, OMDs, DexOMDs, FucOMDs (1.8 μg/mL for OAMet) or control serum-free DMEM for 24 h. Cells were washed with PBS, and 4T1-GFP cells were then added at a tumor/endothelial cell ratio of 1:1. After incubation for 40 min, wells were washed with PBS to remove unattached tumor cells, then the residue cells were fixed with 4% PFA and counterstained by Hoechst 33258, and attached tumor cells were counted by using a fluorescence microscope (Leica DMI 4000B, Germany). Flow cytometry assay for adhesion molecules in vitro. The expression of ICAM-1 and VCAM-1 on bEnd.3 surface was examined by flow cytometry. Briefly, bEnd.3 cells were seeded into 6-well plates at a density of 500,000 cells per well (n = 3). After 24 h incubation, the cells were stimulated with TNF-α for 4 h and incubated with metformin, OA-Met, DHA, OMDs, DexOMDs, FucOMDs (1.8 μg/mL for OA-Met) or control serum-free DMEM for 24 h. Cells were then washed twice with PBS buffer and treated with trypsin-EDTA for cell detachment. After washing with PBS buffer, cells were stained with PE labeled anti-VCAM-1 antibodies or FITC labeled anti-ICAM-1 antibodies in darkness at 4 °C for 30 min. Finally, the stained cells were suspended in PBS buffer (200 μL) and subjected to a BD FACScan (BD FACSAriaⅡ) for flow


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cytometric analysis. To evaluate the expression of E-selectin, bEnd.3 cells were incubated in 6-well plates as mentioned above. After detachment from plates, cells were washed with PBS buffer, and incubated with primary rat anti-mouse E-selectin antibodies at 4 °C for 1 h. Following primary incubation, cells were washed with PBS buffer three times, corresponding secondary Alexa Fluor 647 labeled goat anti-rat IgG antibodies were then applied to the cell suspension, and the cells were incubated for 30 min at 4 °C. After three subsequent washes with PBS buffer, the stained cells were analyzed by flow cytometry. Lung leakage assay with EB in vivo. The lung vascular leakage was investigated by the degrees of EB leakage.49, 57 Breifly, 4T1 cells were carefully inoculated into one mammary fat pad of Balb/c mice. On day 5 after tumor inoculation, the mice were divided randomly into six groups (n = 4) and intravenously injected with saline, metformin, OA-Met, DHA, DexOMDs and FucOMDs for five times in 10 days (8 mg/kg for metformin), then mice were treated with EB solution (20 mg/kg) via the tail vein. Two hours later, lungs were excised, rinsed with PBS and incubated in formamide for 24 h at 37 °C to extract EB. Finally, the supernatant absorbance was measured by a microplate reader (Thermo Multiskan MK3, USA) at 620 nm and compared with a standard curve of EB in formamide to calculate tissue EB concentrations (μg/g, the mass of EB per gram of lung). Immunofluorescence for lung frozen sections. The establishment of orthotopic breast cancer mice model and treatment options referred to the lung leakage assay. One day after the end of administration, lungs were harvested, fixed and then embedded in OCT compound. Immunofluorescence staining was then performed on 10-μm frozen lung tissue sections. Briefly, frozen sections were incubated with a permeable solution containing 0.25% Triton X-100 (v/v) for


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10 min and then blocked with 10% goat serum (v/v) for 2 h at room temperature. Tissue sections were then incubated with primary rabbit anti-mouse fibronectin, MMP-9 or S100A9 antibodies at 4 °C overnight. After washing with PBS buffer for three times, the sections were reacted with corresponding secondary Alexa Fluor 488-labeled goat anti-rabbit IgG antibodies at room temperature for 1 h. After stained with Hoechst 33258, the sections were subjected to the confocal microscope (LSM710, Leica, Germany) analysis and immunofluorescence quantitation was analyzed by using ImageJ 1.46 version program. Metastasis prevention assay in TCM stimulating mice model. TCM (200 μL) was intravenously injected into mice one time a day for three days. The mice were treated with saline, metformin, OA-Met, DHA, DexOMDs and FucOMDs (8 mg/kg for metformin) after each TCM injection on the same day (n = 4). Then 400,000 4T1-Luc cells in serum-free RPMI 1640 media (200 μL) were injected via the tail vein. The lung metastasis was monitored every three or four days by using an in vivo IVIS spectrum imaging system (Cailper Perkin Elmer, USA) after intraperitoneal injection of D-luciferin (50 mg/kg).75 Twenty-one days later, the lungs were harvested and stained by Bouin's fluid, then those lungs were rinsed with 75% ethanol and the number of metastatic nodules was counted by photographing. Combination therapy of FucOMDs with CK-PTX NPs in orthotopic breast tumor mice. We next studied whether the combination of FucOMDs with CK-PTX NPs could simultaneously inhibit the growth of orthotopic tumor and spontaneous metastasis in 4T1-Luc cell-derived orthotopic breast tumor mice model. Briefly, 4T1-Luc cells were inoculated into mammary fat pad of Balb/c mice. When the orthotopic tumors grew to around 100 mm3, the mice were divided randomly into six groups (n = 10), and injected with saline, DexOMDs, FucOMDs (8 mg/kg for metformin), CK-PTX NPs (5 mg/kg for PTX), DexOMDs + CK-PTX NPs and FucOMDs + CK-


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PTX NPs one time every three days via tail veins. The anti-tumor effect was evaluated by measuring the orthotopic tumor volume using Vernier calipers. Thirty days later, five mice from each group were anesthetized and perfused with saline, and major organs (heart, liver, spleen, lung and kidney) and tumors were harvested. In addition, tumor tissues were fixed, embedded in paraffin followed by section and stained with H&E. To evaluate the metastasis prevention effect, lung tissues were then incubated immediately in D-luciferin solution (0.5 mg/mL) for 5 min. Luminescence images and quantitative data of lung tissues were collected by using an in vivo IVIS spectrum imaging system.76 The survival time of the rest five mice in each group was monitored and recorded. Toxicity evaluation in vivo. To evaluate the toxicity of our micelles, the weight of mice was monitored every three days during the treatment. At the end of systematic administration, major organs (heart, liver, spleen, lung and kidney) were harvested as mentioned above and served for H&E staining. Statistical analysis. All results were expressed as the mean ± standard deviation (SD). Student’s t-test was used for a comparison between two groups, and the difference among multiple groups was performed by one-way ANOVA analysis in Graphpad Prism 6.02. Statistical significance was analyzed with P value lower than 0.05.

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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications Web site. Supplementary experimental methods, synthetic route of OA-Met, 1H-NMR spectrum and ESI-MS result of OA-Met, flow cytometry results of endothelial P-selectin, cellular uptake


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of FucOMDs, plasma concentration-time curves, drug concentrations in different tissues, in vivo fluorescence imaging of orthotopic breast tumor mice, results of tumor cell adhesion to endothelial cells, flow cytometry analysis of endothelial adhesion molecules, lung images of lung leakage assay, bioluminescence images of TCM stimulating mice metastasis, H&E staining of tumor sections, primary tumor image, Kaplan–Meier survival analysis, H&E staining of major organ sections, characterization of OMDs, DexOMDs and FucOMDs, optimization of the parameters for the preparation of OMDs, tables of pharmacokinetic parameters, and table of combination index (PDF)



AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] *E-mail: [email protected] ORCID Jun Chen: 0000-0003-1330-9616 Notes The authors declare no competing financial interest.



ACKNOWLEDGMENT

This work was supported by National Natural Science Foundation of China (No. 81673019, 81690265, 81872498, 81573382, 81722043), “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (15SG14), and the Leaders Training Program of Pudong Health Bureau of Shanghai (No. PWRd2017-11).


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