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Biological and Medical Applications of Materials and Interfaces

CCL2 Recruits Macrophage Membrane Camouflaged Hollow Bismuth Selenide Nanoparticles to Facilitate PhotothermalSensitivity and Inhibit Lung Metastasis of Breast Cancer Hongjuan Zhao, Li Li, Junli Zhang, Cuixia Zheng, Kaili Ding, Huifang Xiao, Lei Wang, and Zhenzhong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11645 • Publication Date (Web): 24 Aug 2018 Downloaded from http://pubs.acs.org on August 26, 2018

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ACS Applied Materials & Interfaces

CCL2 Recruits Macrophage Membrane Camouflaged Hollow Bismuth Selenide Nanoparticles to Facilitate Photothermal-Sensitivity and Inhibit Lung Metastasis of Breast Cancer Hongjuan Zhao, †, § Li Li, † Junli Zhang, † Cuixia Zheng, † Kaili Ding, † Huifang Xiao, † Lei Wang, §,* Zhenzhong Zhang §, * †School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, P. R. China §Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, P. R. China *Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, Zhengzhou 450001, P. R. China Keywords: recruitment, macrophage membrane; photothermal-sensitivity, synergistic effect ABSTRACT: Poor tumor accumulation, rapid clearance from blood circulation and high risk of invasive and metastasis are the major barriers which encumber the conventional nanodrug-based tumor therapy. In this work, Macrophage membrane (M) camouflaged quercetin (QE)-loaded hollow bismuth selenide nanoparticles (M@BS-QE NPs) are fabricated for combination therapy of breast cancer. The resulting M@BS-QE NPs are comprehensively characterized, possessing prolonged circulation life, accelerated and enhanced tumoritropic accumulation compared with those of bare BS NPs because of the immune evading capacity, CCL2-mediated recruitment property and active targeting ability. The subsequently QE release under NIR laser irradiation can selectively sensitize cancer cells to photothermal therapy (PTT) by depleting heat shock protein

70

(HSP70,

one

malignancy-specific-overexpressed

thermoresistance-related chaperone) to realize such a cascaded synergistic effect.

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At the same time, M@BS-QE NPs down regulating p-Akt and matrix metalloproteinase-9 (MMP-9, degrade extracellular matrix to promote tumor invasive and metastasis) signal axis to suppress breast cancer lung metastasis. Thus, our results provide a biomimetic strategy via the characteristic of breast cancer and biological properties of macrophages hold great promise to enhance the therapeutic efficacy and improve the accuracy of treatment with minimal side effect on both primary and lung metastasis of breast cancer.

1. Introduction Photothermal therapy (PTT) as a valid cancer-treatment pattern has become considerably attractive on account of its hyperthermia for killing cancer and crucial role for enhancing the treatment efficiency of other therapy patterns, such as chemotherapy and photodynamic therapy. 1-6 In particular, imaging-guided PTT in one nanosystem that allows simultaneous monitoring and killing tumors has attracted extensive research attention of the scientific community due to the conveniences, selectivity, remoteness-control and high efficiency.7-10 In recent years, Bismuth (Bi)-based NPs as the hopeful candidates to achieve multifunctional single nanoagent intrinsically, which is not only as X-ray computed tomography (CT) imaging contrast agents but also as sensitizing agents to enhance the capabilities of PTT cancer treatment.11-14 The excellent performance of Bismuth (Bi)-based NPs is significantly higher than that of the well-known gold-based NPs can be vested in the higher X-ray attenuation coefficient of Bi [Bi > Au > Pt > Ta > I at 100 keV].7.15 Despite the advantages, PTT treated tumor cells can rapidly acquire thermoresistance due to the


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upregulation of heat shock proteins (HSPs), which has been proved to boost tumor cell survival and endurance to stress.16 Heatshock protein 70 (HSP70), as an important apoptotic regulator of signaling pathways, which is can be induced by stress and accumulate in cells to protect tumor cells against apoptosis from hyperthermia.17.18 Moreover, HSP70 enhance cell survival directly by blocking kinase pathways and inhibiting caspase-3 activation.19 Therefore, effectively inhibiting hyperactivated HSP70 may reduce tumor cell resistance to therapy and make tumor cells have a synergistic effect on PTT. Quercetin (3,3′,4′,5,7-pentahydroxyflavone , QE), one of the most common dietary flavonoids, which is not only as a HSP70 inhibitor for heightening PTT efficacy by reduce the expression of HSP70, but a classical protein kinase B (Akt) inhibitor for preventing tumor invasive and metastasis by down-regulation p-Akt/MMP-9 signal pathway, simultaneously inhibiting tumor cell proliferation by inhibit the expression of Akt and active apoptotic protein Caspase-3.20-23 This means QE combine with Bi-based NPs will concurrently sensitizing phototherapy and inhibiting tumor invasive and metastasis. But the rapid clearance of nonself-materials from the blood circulation by the reticuloendothelial systems (RES) or phagocyte system (like macrophages etc.) severely limits the utility of traditional NPs. Therefore, it is advantageous for biomedicine application when NPs with excellent immune escaping ability and blood retention capacity. Cell membrane-mimetic nanoparticles through isolate different cell membranes with particular biological performances as biomimetic nanoscale systems are rapid rapidly developed in the recent years. Those cell membrane-camouflaged systems



endow nanoparticles with biological properties that have been created and perfected in the original cells, so as to render powerful advantages to nanoparticles that can hardly be achieved with any physics or chemistry surface modification.24-28 For instance, Red blood cell (RBC) membrane coated NPs were designed to prolonging prolong blood retention and alleviating clearance by the macrophages.29-31 Platelet membrane-camouflaged NPs were fabricated for tumor targeting treatment and bacterial infection therapy.32-34 Macrophage, a type of white blood cell in the tissues, which have aroused the design of many functional nanocarriers owing to their role as circulating sentinels for phagocytose foreign materials and for immunologic function. The interactions between the α4 integrin of the macrophages and the vascular cell adhesion molecule-1 (VCAM-1) of cancer cells endow macrophages native actively tumor-targeting to lung metastasis of breast cancer is reported by Li and co-workers.35 Furthermore, He and co-workers constructed macrophage cell membrane coated NPs for recognizing tumor endothelium and thus improved tumoritropic accumulation.36 More importantly, secretion of C-C chemokine ligand 2 (CCL2) from breast tumors and stromata was proved to be able to facilitate recruitment of CCR2-expressing monocytes and macrophages derived from monocytes and it is reported that macrophages are preferentially recruited while in pulmonary metastases.37-39 Tumor associated macrophages (TAMs) become the largest immunosuppression cell subpopuiation in TME just because of the valid recruits and chemotaxis of some chemokine axis, especially CCL2/CCR2 and colony stimulating factor 1 (CSF-1)/ CSF-1 receptor (CSF-1R). Some researcher improved anticancer efficiency by


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targeting TAMs with CCR2 or CSF-1 inhibition.40.41 Therefore, we assume that the combination of macrophage membrane with Bi-based NPs will not provide Bi-based NPs excellent immune escaping property, blood retention capacity, and active targeting ability by simultaneously recognizing tumor endothelium cells and cancer cells through α4/VCAM-1 interaction, but breast cancer utilize the CCL2/CCR2 chemokine

axis

to

proactive

recruit

macrophage

membrane-camouflaged

nanoparticles, which will exhibits strong accumulate in both primary and lung metastasis accompanied by TAMs resetting, which is because macrophage membrane-camouflaged nanoparticles are not real macrophages and can’t be TAMs. Herein, we design a macrophage membrane camouflaged quercetin-loaded hollow Bi2Se3 nanoparticles with hyperthermia-triggered drug release and CCL2-mediated recruit to inhibit growth and lung metastasis of breast cancer. At first, macrophage membranes as outer shells taking advantages of excellent biocompatibility and versatile functionality of macrophage membranes. Then, BS-QE NPs as inner cores could significantly sensitize cancer cells to PTT by inhibiting HSP70 expression, suppress lung metastasis of breast cancer by down-regulating p-Akt/MMP-9 signal and promote cancer cell apoptosis by repressing Akt and activating Cas-3. And BS NPs with strong absorbance of both X-ray and NIR light, useful for CT imaging (high resolution and facile three-dimensional visualization on tissues of interest) and infrared imaging (IRT imaging), achieve tumor monitoring. The excellent immune evading property, CCL2/CCR2 recruitment ability and α4/VCAM-1 identification ability combined with excellent HSP70 inhibition and p-Akt/MMP-9 down-regulation



led to outstanding therapeutic efficiency of M@BS-QE NPs. It is expected that M@BS-QE NPs will be helpful for future clinical breast cancer therapy (Scheme 1).

Scheme 1. Illustration of the preparation of biomimetic synergist M@BS-QE with tumor proactive recruitment/targeting ability (CCL2/CCR2 chemokine axis and α4/VCAM-1 interaction) and an additive anti-tumor effect for simultaneously sensitizing PTT and blocking invasion and metastasis of breast cancer.

2. Results and Discussion 2.1 Characterization of M@BS-QE. M@BS-QE was prepared with the following four steps: preparing hollow mesoporous BS NPs, encapsulating anticancer drug QE into BS NPs, isolating the macrophage membrane from freshly harvested macrophages, camouflaging BS-QE


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NPs with macrophage membrane to obtain M@BS-QE (Scheme 1). To prepare hollow mesoporous BS NPs, Bi2O3 NPs were first prepared from Bi(NO3)3·5H2O and the transmission electron microscopy (TEM) (Figure S1) show that Bi2O3 NPs were homogeneous spherical with an average diameter of ~ 110 nm. Then, hollow mesoporous BS NPs were obtained through utilized the Bi2O3 NPs as bismuth template and precursor via the hydrothermal process method.7 TEM (Figure 1A) results reveal its well hollow mesoporous structure with an average diameter ~120 nm and the porosity was measured via N2 adsorption-desorption isotherms (Figure 1C), showing that hollow mesoporous BS NPs have a surface area of 71.03 m2 g-1 and the pore size ~ 8.98 nm. Such a large surface areas and highly hollow mesoporous nanostructures allow it is an ideal carrier to encapsulate anticancer drug. Moreover, Energy-dispersive spectroscopy (EDS) analysis reveals the presence of Bi and Se (Figure S2) and the X-ray diffraction (XRD) pattern (Figure S3) with all the diffraction peaks of BS NPs can be highly indexed its purity.7 Herein, QE was chosen to load into BS NPs with good drug entrapment efficiency and the amount of QE within the BS NPs was not significantly altered in the process used for macrophage membrane coating (Figure S4). After QE loading, the characteristic absorption peak at 370 nm of QE in the Ultraviolet-visible-near-infrared (UV-Vis-NIR) absorption spectrum was red shifted to 420 nm because of the broad and strong absorption between 200 and 800 nm of BS NPs (Figure 1D). Upon macrophage membrane coating, M@BS-QE were characterized by TEM, obvious core-shell structure that verify the camouflaging of BS-QE NPs by macrophage membrane thin shells. The



dynamic light scattering (DLS) data of NPs before and after macrophage membrane coating were investigated (Figure 1E) to further confirm the macrophage membrane coating. The surface zeta potential changes from -27.8 to -19.1 mV following macrophage membrane coating, which is close to the zeta potential of macrophage membrane (-17.5 mV). And the particles size slightly increased from 145.6 to 155.3 nm after macrophage membrane coating with a polydispersity index (PDI) of 0.312 (Figure S5).

The stability of the BS-QE NPs and M@BS-QE NPs were monitored

by DLS and the highly improved stability of M@BS-QE NPs is demonstrated with negligible increase particle size over a span of 48 h, whereas the particle size of BS-QE NPs is increased from 145.6 to 552.1 nm (Figure S6). In addition, the UV-Vis-NIR spectrum of M@BS-QE shows hardly any change, which is indicates macrophage membrane will not affect the property of QE. The above results validate the successful camouflaging of macrophage membrane on BS-QE NPs. The photothermal performance of BS NPs was investigated by irradiated with a NIR laser (808 nm, 2 Wcm-2) for 5 min at different concentrations (0.1, 0.25, 0.5 mg mL-1 in PBS). The high thermal contrast produced by BS NPs (Figure 1B) proved the high IR thermal imaging properties. PBS was used to as negative control. Next, the photothermal property and photothermal stability of NPs was investigated after anticancer drug QE encapsulating and macrophage membrane camouflaging. The temperature up-down of the NPs (BS NPs concentration 100 µg mL-1) for four NIR laser on/off cycles was recorded ~ 100 min. As exhibited in Figure 1 F and G, BS-QE NPs and M@BS-QE show remarkable photostability and reproducibility as that BS


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NPs, indicating that macrophage membrane camouflaging has no impact on photothermal conversion efficiency of BS NPs. Meanwhile, the QE release from M@BS-QE exhibits typical photo-responsive release profiles. As shown in Figure 1H, QE can rapid release from BS-QE NPs and M@BS-QE upon a 5 min NIR irradiation, which is probably due to the accelerated thermal vibration from rapid increase of local temperature generated by BS NPs. Such release profile may benefit in reducing the side effects of anti-cancer drugs and strengthen the antitumor effect.

Figure 1. Characterization of BS and M@BS-QE NPs. (A) TEM images of a) BS NPs, b) macrophage membrane and c) M@BS-QE. (B) Infrared thermal images of BS NPs during NIR laser irradiation. (C) N2 adsorption-desorption isotherm (inset: corresponding pore size distribution) of BS NPs. (D) UV-vis-NIR absorption spectra. (E) Hydrodynamic size and surface charge of (a) BS-QE NPs, (b) macrophage membrane and (c) M@BS-QE NPs. (F) Temperature increases and (G) photostability of BS NPs, BS-QE NPs and M@BS-QE during NIR laser irradiation. (H) QE release profile. The data presented as Mean ± SD (n = 3), *P < 0.05, **P < 0.01 and ***P < 0.005.



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The interactions between the α4 integrin of the macrophages and the VCAM-1 of breast cancer cells can endow macrophage native actively tumor-targeting and tumor-binding to breast cancer. More strikingly, CCL2 secretion in tumor tissue efficiently

recruits

macrophages

based

on

the

expression

of

CCR2

on

macrophages.35.39.42 So the expression of α4 integrin and CCR2 on RAW 264.7 cells and VCAM-1 of breast cancer cells were first measured by immunofluorescence detection and flow cytometry (FCM) analysis (Figure 2A). The laser confocal scanning microscope (LCSM) images shows strong fluorescence on the cell membrane of RAW 264.7 cells and 4T1 cells, which are confirmed the expression of

α4/CCR2 and VCAM-1 integrins on the cell membrane of RAW 264.7 cells and 4T1 cells, respectively (Figure S7). And the FCM analysis data were verified the high expression of α4/CCR2 and VCAM-1 integrins. Then we tested the CCL2 secretion in tumor tissue and lung metastatic sites by immunofluorescence, the large-scale strong red fluorescence area reflected the abundant CCL2 secretion, which is definitely important to recruit M@BS-QE NPs (Figure 2B). To determine whether M@BS-QE NPs could be recruited in a CCL2-dependent mechanism, we further investigated their chemotactic properties by using a dual-chamber transwell system with 5-µm and 0.4-µm-sized microporous membranes (macrophages are able to migrate through 5-µm pores and macrophage membranes are able to migrate through 0.4-µm pores). We evaluated the migration of DiI-labelled macrophages and macrophage membranes towards free medium, CCL2 cytokine, cancer cells and both (Figure S8). Representative images were taken by a fluorescence microscope show increased


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migration of red fluorescent macrophages and macrophage membrane to bottom chambers that contained CCL2 cytokine compared to cancer cells alone or free medium (Figure 2C). Quantified analysis confirmed that the DiI fluorescence area of macrophage membrane towards CCL2 and CCL2 + cancer cells were four- and fivefold greater than free medium (Figure S9). The results of chemotaxis assay have shown that macrophage membrane can be recruited by CCL-2 dependent mechanism, and the merits of macrophage membrane will be transferred to M@BS-QE NPs which almost have the same protein profiles as the macrophage membrane (Figure S10). Thus, it is expected that M@BS-QE NPs could not only significantly improve the immune evading capability and blood circulation time, but receive recruitment signal and tumor-targeting abilities.

2.2 In Vitro Cellular Uptake and Cytotoxicity To assess the efficacy of the M@BS-QE to target and bind cancer cells, we cocultured equivalent numbers of FITC-labeled NPs and free FITC with 4T1 cells, which is replace QE because of its fluorescence could be tracked. And the nucleus of 4T1 cells was stained with DAPI for easy observation and localization in CLSM. CLSM results (Figure 2D) demonstrate that although free FITC can rapid uptake by 4T1cells at 3 h, FITC-labeled NPs can significantly increase the uptake at 6 h and enhance the retention of NPs in 4T1 cells at 12 h. This illustrates that macrophage membrane camouflaging significantly enhance the internalization and retention of BS NPs in 4T1 breast cells owing to the most retained membrane protein on the M@BS-QE, provided active internalization capacity by recognizing 4T1 breast cells.



Meanwhile, we examine the ability of macrophage membrane camouflaging NPs to escape from the capture with macrophage phagocytosis. By contrast, we cocultured equivalent numbers of FITC-labeled NPs with RAW264.7 cells. The captured images (Figure S10) show that BS-FITC NPs were extensively uptake in macrophage cells with strong green fluorescence. However, green fluorescence in macrophage cells was evidently reduced in M@BS-FITC group, which is verified the immune evade ability of M@BS-QE.

Figure 2. (A) The α4/CCR2 integrin in RAW 264.7 cells and VCAM-1 integrin in 4T1 cells were detected by immunofluorescence staining under a LCSM and FCM analysis. (B) The CCL2 secretion in 4T1 breast tumor and lung metastases of breast cancer (Scale bar = 50 µm). (C) Diagram shows the design of chemotaxis assay and fluorescence images of macrophages membrane. Macrophage membranes were seeded in the upper chamber and different formations (with/without CCL2 cytokine) were in the lower chamber (Scale bar = 100 µm). (D) Cellular uptake in 4T1 cancer cells of different groups in preset points detected under LCSM (Scale bar = 5 µm).

The biocompatibility of M@BS-QE was examined by hemolysis assay and there is


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no significant hemolysis (less than 8%) in the presence of M@BS-QE for 3 h (Figure S11). Then the photothermal effect of BS NPs on 4T1 cells was first detected by Calcein-AM/PI assay. PI can only stain apoptotic/dead cells with red fluorescence and Calcein-AM stain the living cells with green fluorescence. Results present in Figure 3A show that 4T1 cells cultured with only BS NPs upon NIR laser irradiation present strong red fluorescence, which indicates most 4T1 cells were dead. Since HSP70 as an important apoptotic regulator of signaling pathways, which is can be induced by stress and accumulate in cells to protect tumor cells against apoptosis from hyperthermia. In this way, we test the expression of HSP70 of 4T1 breast cells incubated with blank BS NPs upon NIR irradiation with different time. As presented in Figure 3B-b, the expression of HSP70 is really elevated in hyperthermia and the expression of HSP70 is highest when cells treated with BS NPs (40 µg mL-1) and NIR irradiated 2 min. Next, CCK-8 method was utilized to assess the cytotoxicity of all the QE formulations and BS NPs. As shown in Figure 3B-a, blank BS NPs (concentrations range 0.1-200 µg mL-1) have negligible cytotoxicity without NIR irradiation, indicating their prominent biocompatibility. And BS-QE NPs and M@BS-QE (QE concentrations range 10-200µg mL-1) show similar activity as free QE solution (P > 0.05). The cell inhibition gradually elevated in all the QE formulations and BS NPs with NIR irradiated 2 min, and M@BS-QE +NIR group shows the greatest cell inhibition (P < 0.05). The IC50 values in BS+NIR and free QE groups are 152.1µg mL-1 and 0.512 mg mL-1, respectively. With the presence of QE, the IC50 values of BS in BS-QE+NIR and M@BS-QE+NIR groups are decreased to



92.26 and 51.34 µg mL-1, respectively. These are attributable to the released QE not only an Akt inhibitor but selectively inhibiting on HSP70, leading to cells more sensitive to hyperthermia and distinctly make a synergistic PTT effect of M@BS-QE. Western blotting results of HSP70 in M@BS-QE +NIR group shown in Figure 3C-b verified that the raised HSP70 after NIR irradiation is actually reduced by anticancer drug QE. The combination index (CI) values of BS-QE and M@BS-QE NPs under NIR irradiation condition were calculated using IC50 are 0.8671 and 0.4583, respectively. These CI values were less than 1 which indicated a synergistic effect between BS and QE under NIR irradiation condition (CI < 1, synergism; CI = 1, additive effect; CI > 1, antagonism). Furthermore, the Annexin-v/PI assay was utilized to evaluate the percentage of apoptosis cells in all the QE formulations by FCM analysis. The percentage of apoptosis cells was largest in M@BS-QE +NIR group because of the Akt inhibition and PTT synergy (Figure 3D).


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Figure 3. (A) Photothermal destruction of 4T1 cells treated with BS NPs (Scale bar = 100 µm). (B, C) Cell viability and Western blotting assays showing the corresponding HSP70 expression levels after treatments various formations. The data presented as Mean ± SD (n = 3). (D) FCM assays the cell apoptosis under different treatments.

2.3 In Vitro Anti-metastasis Activity Cell invasive and metastasis are highly complex process and involve sequential steps. There is one step that via metalloproteinase-9 (MMP-9) degrades the extracellular matrix (ECM). However, ECM can prevent tumor metastasis by forming a physical barrier and a self-protective, apoptosis-resistant microenvironment. Hence, down-regulation MMP-9 could effectively prevent breast cancer metastasis.

Notably,

Akt is a typical signal regulator essential for cell proliferation, apoptosis, and metastasis. Anticancer drug QE as Akt inhibitor can block cell proliferation via inhibit



the expression of Akt and prevent cell metastasis via down regulate MMP-9 protein level through the inhibition of Akt phosphorylation (p-Akt).21,43 Moreover, QE induce cell apoptosis in a mitochondrial-dependent manner, such as active caspase-3.44 M@BS-QE also could damage the tumor vasculature through the accumulation via the active tumor recruitment and targeting ability. We first performed wound healing assays and transwell invasive assays (Figure 4A and Figure 4B), as in vitro model to evaluate the inhibition capability of all the QE formations on 4T1 cell motility. The wound healing results have demonstrated that untreated cells healed greatest after 24 h and the wounds of all QE groups shown poor healing (P < 0.05). And the invasive rate in the untreated group almost 100%, but the invasive rates in all QE groups are 60.5%, 29.67%, 19.3%, 15.5%, and 13.5%, respectively (Figure 4C) (P < 0.05). The results are basically similar as wound healing assays. Those mean that 4T1 breast cells exhibited strong invasive and migration capacity, but the ability could be suppressed by M@BS-QE. And tube formation assay results exhibited in Figure 4D showing the extensively broken vasculature in M@BS-QE+NIR group, which further impede the invasive and migration of breast cancer. To investigate the cell invasive and migration inhibition mechanism underlying the all QE formations, we first examined Akt and p-Akt expression in 4T1 cells by immunofluorescence staining (Figure 4E). CLSM images showed strong red and green fluorescence on 4T1 cells with untreated group, which effectively confirmed high expression of Akt and p-Akt. However, Akt protein decreased in all QE groups and p-Akt reduced more (Figure 4F). Then western blotting was investigated to detect the expression of Akt, p-Akt, and


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MMP-9, β-actin signals in these formations were detected as a control. As presented in Figure 4G, the results of western blotting are consistent with immunofluorescence. Moreover, we also detected the influence of caspase-3 on QE-induced mitochondrial pathway. The western blotting results show the activated caspase-3 after the treatment of M@BS-QE.

Figure 4. (A) The wound healing images (Scale bar = 200 µm), (B) microscopy images of invasive (Scale bar = 80 µm) and (C) the corresponding quantitative analysis after different treatments. (D) Tube formation assay (Scale bar = 80 µm). (E) Representative immunofluorescence images (Scale bar = 25 µm) and (F) the corresponding quantitative analysis of Akt and p-Akt after different treatments. (G) Western blotting assays showing the expression level of Akt, p-Akt and Cas-3 after different treatments. The data presented as Mean ± SD (n = 3), *P < 0.05, **P < 0.01 and ***P < 0.005.

2.4 In Vivo Distribution and Phototherapy of M@BS-QE It is well known that macrophages have active tumor targeting ability via the



α4/VCAM-1 interaction and CCL2 secretion in tumor microenvironment could recruit more macrophages. To evaluate whether macrophage camouflaging NPs can improve the tumor accumulation and long circulating capacity of NPs by means of the ability of receiving CCL2 signal and α4/VCAM-1 targeting capacity, we carried out in vivo fluorescence imaging by utilizing 4T1-bearing BALB/c mouse model. 10 days after subcutaneous injection into forelimbs of 4T1 tumor, IR780 labeled NPs were then intravenous injection (i.v.) into 4T1-bearing BALB/c mouse. As shown in Figure 5A, M@BS-IR780 realize tumor accumulation after i.v. 4 h and maintain strong tumor retention up to 24h, whereas BS-IR780 NPs achieve tumor site after i.v. 6 h and free IR780 is 10 h, which suggested the successfully macrophage membrane camouflaging with strong recruited capacity and active tumor targeting ability of M@BS-QE. The ex vivo fluorescence images in Figure 5B exhibited the major organs (heart, liver, spleen, lung, kidney, brain) and tumors of three different groups after i.v. 36 h. It is clearly observed the apparent fluorescence in the tumor of M@BS-IR780 group, which could be attributed the synergistic effect of CCL2/CCR2 recruitment and α4/VCAM-1 targeting abilities as demonstrated above. The in vivo antitumor effects on the primary tumor were evaluated in the 4T1-bearing BALB/c mouse model because of the outstanding in vitro antitumor effect of M@BS-QE. First, the in vivo photothermal effect of M@BS-QE was measured. The results in Figure 5C revealed the tumor site became brighter in all BS formations upon NIR irradiation compared with the PBS group. The local temperatures of tumors in all BS formations under NIR irradiation were up to 70 ff,


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whereas 40 ff for mice injected with PBS (Figure 5D). These results reflect that M@BS-QE can play efficiently PTT role via absorb NIR light energy. Then the in vivo tumor growth behaviors in all formations were demonstrated as Figure 5E. The mice treated with PBS as control showed rapid tumor growth during the test. The mice treated with blank BS NPs exhibited a similar growth behavior as the PBS group without antitumor effect and little cell apoptosis, which could result from negligible cytotoxicity of BS NPs. The antitumor effect of BS NPs with NIR irradiation is mainly attributes to the PTT effect of the BS NPs in the tumor site. After loading QE in BS NPs, the tumor volume was obviously decreased in BS-QE NPs with NIR irradiation group, indicating the sensitive PTT effect and Akt inhibition for antiproliferation. When irradiated with NIR laser after i.v. of M@BS-QE, the tumor volume was sharply reduced compared with other groups and the strongest cell apoptosis with the largest brown area distributed around the whole tumor tissues (Figure 5E), which should result from the macrophage membrane-induced high tumor accumulation of M@BS-QE

and

sensitization

to PTT followed

by the

hyperthermia-triggered release of QE in the tumor site. The body weight in all formations was measured during the test as an indicator of systemic toxicity (Figure 5D). The mice treated with free QE showed severe weight loss compared with other groups, which are demonstrated the absence of obvious systemic toxicity. The hematoxylin and eosin (H&E) staining of major organs after test in all formations were exhibited in Figure S12, further demonstrating no obvious systemic toxicity. And the histological analysis of tumors exhibited in Figure 5E confirmed the highest



antitumor efficiency of M@BS-QE with NIR irradiation, which is indicated by more condensed nuclei and vacuoles. In addition, the results of CD31 and MMP9 immunofluorescence images exhibited the lowest vascular proliferation and MMP9 expression in M@BS-QE with NIR irradiation group, as indicated by less red and green fluorescence. The anti-angiogenesis effect may be attributed to the tumor endothelium recognize and thus improved tumoritropic accumulation for vascular damage of M@BS-QE. And the inhibition of MMP-9 may owe to the released QE, which can down regulate the expression of MMP-9 to prevent breast cancer metastasis.

Figure 5. (A) Representative images for in vivo fluorescent imaging. (B) Typical ex vivo imaging of major organs from each group at 36 h after intravenous injection with free IR 780, BS-IR780 NPs and M@BS-IR780. (C) In vivo IR thermal images and (D) the corresponding temperature variation of tumor-bearing mice i.v. injected by PBS, BS NPs, BS-QE NPs and M@BS-QE upon NIR laser radiation. (E) Body weights and (F) Tumor volume growth curves of 4T1 tumor-bearing mice in different groups. (G) TUNEL stained images of tumors showing apoptotic cells in brown.


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H&E stained images of tumors (Scale bar = 200 µm) showing cytoplasm in red. Representative immunofluorescence images of tumor slices (The blood vessels and MMP-9 distribution were visualized by anti-CD31 antibody (red) and anti-MMP-9 antibody (green), cell nucleus in blue, respectively). (Scale bar = 80 µm). The data presented as Mean ± SD (n = 6), *P < 0.05, **P < 0.01 and ***P < 0.005.

2.5 CT Imaging CT imaging, which is a frequently-used biomedical imaging featured with the advantages of high resolution, no depth limitation and the capability for 3D reconstruction.11,45 Given the higher X-ray attenuation coefficient of Bi than other reported and broadly applied elements [Bi > Au > Pt > Ta > I at 100 keV], it is expected that M@BS could also provide strong contrast under CT imaging. The enhanced CT signal intensities along with the increased NPs concentration is detected in vitro (Figure 6A), and the calculated CT value (Hounsfield Unit, HU) reveals a good linear increase with the NPs concentrations (Figure 6C). The X-ray absorption coefficient of M@BS was calculated to be 25.66 HU·mL·mg-1, which is obviously higher than that of commercial contrast agent (iohexol, 12.96 HU·mL·mg-1). The increased CT contrast efficacy of M@BS is benefit to reduc the potential side-effects and complications caused by the high dose of contrast agents in clinical applications. We then investigated the performance of M@BS as the CT imaging contrast agent in vivo. 4T1 tumor-bearing Balb/c mice were i.t. injected with M@BS and iohexol (10 mg kg-1), respectively. As shown in Figure 6B, the imaging contrast can be strongly enhanced after the i.t. injection, and a much higher CT value in the tumor site is observed in the mice treated with M@BS (~295 HU) than the mice treated with iohexol (~183 HU), suggesting the excellent CT contrasting efficiency of M@BS (Figure 6D) (P < 0.05). Moreover, compared with the disappeared CT signal in mice



after i.v. injection iohexol 6 h, there is strong CT signal in mice treated with M@BS (Figure 6B).

Figure 6. (A) In vitro CT contrast images and (C) Linear fitting of CT values of the M@BS and lohexol contrast agent at the different concentrations. (B) In vivo three-dimensional CT images of a 4T1 tumor-bearing mouse before (Pre) and after (Post) i.t. injection and i.v. injection at predesigned point of M@BS (10.0 mg mL-1, 200 µL) and lohexol contrast agent. The CT contrast at the tumor site was largely enhanced after injection. (D) The corresponding CT values at the tumor site. The data presented as Mean ± SD (n = 6), *P < 0.05, **P < 0.01 and ***P < 0.005.

2.6 Antitumor Effect on Lung metastasis of Breast Cancer Finally, to validate the in vivo therapeutic efficacy on lung metastasis of breast cancer of M@BS-QE, the 4T1 tumor bearing Balb/c lung metastasis modal were investigated with PBS, free QE, BS-QE NPs and M@BS-QE. During the treatment, we recorded the body weight of every group every two days and it was slightly rose (Figure 7B). At the end point, the major organs of every group were collected and showing no significantly changed (Figure S13) except lung tissues. As shown in Figure 7C, the mice treated with PBS had the maximum number of macroscopic metastatic nodules, moderately decreased of metastatic nodules in the mice treated with free QE and BS-QE (P < 0.05). In particular, the average number of metastatic


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nodules of M@BS-QE was only 17%, which were 100, 75, and 66% of the PBS, free QE, and BS-QE NPs group, respectively (Figure) (P < 0.05). H&E staining results (Figure 7D) showed that the metastatic lesions were stained with dark nuclei. Compared with PBS, free QE, and BS-QE NPs group with large dark areas, there were barely visualized metastatic lesions in the M@BS-QE group, indicating the promising antimetastatic performance of M@BS-QE. As a result, the lung metastasis of breast cancer was obviously inhibited by M@BS-QE could be attributed to the combined action from active targeting ability of M@BS-QE via α4/VCAM-1 interaction and proactive recruitment capacity from CCL2/CCR2, thus significantly improve the accumulation of M@BS-QE in the metastatic foci.

Figure 7. In vivo therapeutic efficacy of M@BS-QE to lung metastasis of breast cancer. (A) Pulmonary metastasis model were established by tail vein injection in BABL/c mice. (B) Body weights of 4T1 tumor-bearing mice in different groups. Data were presented as Mean ± SD (n = 6). (C) Typical photographs of lung tissues from mice treated with control, free QE, BS-QE NPs and M@BS-QE (the black arrow denotes the visually detected metastatic nodules in each lung tissue). (D) The corresponding quantitative analysis of macroscopic lung metastatic nodules for each



group. (E) Histological examination of metastatic lesions in lung tissues after H&E staining. (Scale bar = 500 µm). *P < 0.05, **P < 0.01 and ***P < 0.005.

3. Conclusion In summary, a comprehensive therapy platform based on macrophage membrane camouflaged QE-loaded hollow BS NPs was developed. The merit of macrophage membrane was successfully grafted to BS NPs through the simple cloaking approach. The superior tumoritropic accumulation demonstrated that M@BS-QE NPs were successfully synthesized with CCL2/CCR2 proactive recruitment capacity and α4/VCAM-1 active targeting property. The rapid release of QE under NIR irradiation and obvious decrease of HSP70 subsequently further strength breast cancer sensitivity to PTT and enhance anticancer efficiency. Great antitumor growth and metastasis effects indicated that the combination of HSP70-inhibition and p-Akt/MMP-9-down regulation rather than any of them alone showed great potential in the breast cancer therapy. Moreover, M@BS-QE showed excellent CT and IRT imaging performances due to the high X-ray attenuation coefficient of BS NPs. Taking together, M@BS-QE NPs could serve as a promising nanodrug platform with tumor-recruited, photothermal-sensitized and metastasis-inhibited ability for the future treatment of breast cancer.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental Section, representative TEM image of Bi2O3 NPs, EDS analysis of Bi2Se3 NPs, XRD analysis of Bi2Se3 NPs, particle size distribution and stability of M@BS-QE NPs, QE entrapment efficiency (EE) at different feeding amounts of QE, the α4/CCR2 integrin in RAW 264.7 cells and VCAM-1 integrin in 4T1 cells were


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detected by immunofluorescence staining under a LCSM, chemotaxis assay and fluorescence images of macrophage cells and macrophages membrane, SDS-PAGE protein analysis of M@BS-QE and macrophage membrane, cellular uptake in RAW264.7 cells of different groups, hemolysis quantifcation of red blood cells at various concentrations of M@BS-QE, histology analysis of the mice major organs after various treatments, typical photographs of major organs from mice treated with control, free QE, BS-QE NPs and M@BS-QE. (PDF)

Author Information Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions The experimental work was designed and carried out by H.Z. under the support of Prof. Z. Z. The manuscript was written by H. Z. and reviewed by all authors. All authors have given approval to the final version of the manuscript.

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81673021, 81573364), the scientific and technological project of Henan Province (182102310117) and Modern Testing Technology and Methods centre of Zhengzhou University.

Notes The authors declare no competing financial interest

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