Tumor-Specific Silencing of Tissue Factor Suppresses Metastasis and

Jun 7, 2019 - Effect of sPNPs Treatment on Metastatic Ability of Tumor Cells in Vivo .... The antitissue factor monoclonal antibodies (cat. no. ab1517...
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Letter Cite This: Nano Lett. 2019, 19, 4721−4730

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Tumor-Specific Silencing of Tissue Factor Suppresses Metastasis and Prevents Cancer-Associated Hypercoagulability Shaoli Liu,†,‡,# Yinlong Zhang,†,# Xiao Zhao,†,# Jing Wang,† Chunzhi Di,†,‡ Ying Zhao,† Tianjiao Ji,† Keman Cheng,† Yongwei Wang,† Long Chen,†,‡ Yingqiu Qi,†,‡,§ Suping Li,*,†,‡ and Guangjun Nie*,†,‡

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CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China ‡ Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, China § Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China S Supporting Information *

ABSTRACT: Within tumors, the coagulation-inducing protein tissue factor (TF), a major initiator of blood coagulation, has been shown to play a critical role in the hematogenous metastasis of tumors, due to its effects on tumor hypercoagulability and on the mediation of interactions between platelets and tumor cells. Targeting tumor-associated TF has therefore great therapeutic potential for antimetastasis therapy and preventing thrombotic complication in cancer patients. Herein, we reported a novel peptide-based nanoparticle that targets delivery and release of small interfering RNA (siRNA) into the tumor site to silence the expression of tumorassociated TF. We showed that suppression of TF expression in tumor cells blocks platelet adhesion surrounding tumor cells in vitro. The downregulation of TF expression in intravenously administered tumor cells (i.e., simulated circulating tumor cells [CTCs]) prevented platelet adhesion around CTCs and decreased CTCs survival in the lung. In a breast cancer mouse model, siRNA-containing nanoparticles efficiently attenuated TF expression in the tumor microenvironment and remarkably reduced the amount of lung metastases in both an experimental lung metastasis model and tumor-bearing mice. What’s more, this strategy reversed the hypercoagulable state of the tumor bearing mice by decreasing the generation of thrombinantithrombin complexes (TAT) and activated platelets, both of which are downstream products of TF. Our study describes a promising approach to combat metastasis and prevent cancer-associated thrombosis, which advances TF as a therapeutic target toward clinic applications. KEYWORDS: peptide self-assembled nanoparticle, tissue factor, siRNA, tumor metastasis, tumor hypercoagulability

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or tissue factor pathway inhibitor (TFPI) have been reported to significantly reduce the incidence of metastasis in mice.11−14 Furthermore, nonspecific anticoagulation therapy might logically induce tissue bleeding complications.15,16 Novel strategies to realize tumor-specific depletion of TF, while avoiding nonspecific intervention in normal tissues, are urgently needed in order to achieve safe and effective tumor treatment. Despite offering a highly efficient mechanism to selectively silence target genes at the post-translational level,17 unmodified siRNA is an ineffective therapeutic agent, suffering from rapid clearance from the bloodstream. The circulation half-life of naked siRNA typically ranges in minutes, thus limiting in vivo

issue factor (TF), a crucial initiator of the extrinsic blood coagulation pathway, has also long been known to play a vital role in tumor progression and metastasis.1−4 The upregulation of TF was found in both tumor cells and vascular endothelial cells in a wide range of solid tumors.3 In this context, TF promotes tumor invasion and metastasis by inducing the production of tumor growth factors such as angiogenic factors and chemokines.5 Emerging evidence from clinical studies also suggests that the high levels of TF in tumor tissue are strongly associated with a high incidence of systemic hypercoagulability.6−8 Blood clotting abnormalities are detected in up to 90% of cancer patients, and thrombosis is considered to be the second most frequent cause of cancerassociated mortality.9,10 All these findings have made tumorassociated TF an attractive therapeutic target for anticancer metastasis and prevention of cancer-associated abnormal hypercoagulability. Approaches that suppress TF by antibodies © 2019 American Chemical Society

Received: May 1, 2019 Revised: June 5, 2019 Published: June 7, 2019 4721

DOI: 10.1021/acs.nanolett.9b01785 Nano Lett. 2019, 19, 4721−4730

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Figure 1. Design and characterization of pH-sensitive sPNPs. (a) Schematic self-assembly of the nanoparticles. (b) Proposed antitumor mechanism of sPNPs nanoparticle. Upon penetration into the tumor microenvironment, the sPNPs nanoparticles in response to the acidic tumor environment allow sPNPs to possess a positive surface charge; at the same time, the cRGD motif on the surface of sPNPs will recognize the integrin αvβ3. Both of these conditions promote cell uptake of sPNPs. Under the acidic conditions in lysosomes, polyhistidine sequence undergoes a protonation effect, depolymerizing the nanoparticles and releasing siRNA. (c) TEM images of PNPs and sPNPs. Scale bar: 200 nm. (d) TEM images of sPNPs at pH 7.4, 6.8, and 5.5; scale bars, 200 nm (left) and 50 nm (right).

application of siRNA-based therapy.18,19 Capable of both significantly prolonging siRNA circulation time and improving targeting efficiency, the use of targeted nanocarriers has potentially revolutionized the delivery of siRNA in vivo.20−26 Bioresponsive materials for precise design of nanomedicines have been proven to be feasible strategies for targeted tumor therapy.27−31 We herein developed a tumor-targeted peptidebased nanoparticle system to specifically knock-down tumorassociated TF through small interfering RNA (siRNA) silencing. An amphipathic peptide, which was able to selfassemble into peptide nanoparticles (PNPs), was designed as the vehicle and coassembled with TF siRNA to form siRNAloaded peptide nanoparticles (sPNPs). To further optimize the delivery efficacy, we integrated into the peptide monomer with a cyclic RGD (cRGD) peptide, which recognizes the αvβ3 integrins overexpressed on the surface of most tumor cells,32−35 and a polyhistidine sequence sensitive to the slightly acidic microenvironment (pH ≤ 6.8) of tumor tissues.36,37 When the nanoparticles arrive at the acidic tumor microenvironment, a pH-triggered surface charge reversion, induced by the polyhistidine moiety, facilitates cell uptake.38−41 The subsequent depletion of tumor-associated TF decreases the invasive potential and platelets adhesion of cancer cells and further prevents its distant metastasis. Also, depletion of tumor-associated TF reversed the tumor-induced hypercoagulable state. Our experimental results demonstrate a significantly reduced in vitro cellular migration tendency of

tumor cells as the result of TF down-regulation by sPNPs. Consequently, the TF-suppressed tumor cells showed a remarkable decrease in lung colonization. Furthermore, in mice bearing highly metastatic tumors, the administration of sPNPs efficiently suppressed the formation of lung metastases and reduced tumor-associated blood coagulation parameters. Results and Discussion. Preparation and Characterization of Peptide-based, pH Sensitive Nanoparticles. We designed a novel amphipathic peptide chimera, (C18)2-Lys(Gly)2-(Arg-Arg-Ser)10-Lys-(His)8-cRGD (Figure S1), for the construction of the coassembly nanovehicle. The cRGD peptide was engineered at the end of the hydrophilic moiety in order to expose it on the surface of the assembled nanoparticle. Next, we added a pH-sensitive polyHis sequence whose protonation in response to the acidic tumor microenvironment enables sPNPs to convert their surface charge from negative to cationic form. In the cationic state, they were able to electrostatically interact with negatively charged cell membranes and thus promote the nanoparticles uptake by tumor cells. To improve the siRNA loading efficiency, a highly cationic repeating arginine-arginine-serine (RRS) segment was included to electrostatically absorb the negatively RNA molecules during the coassembly process. Two octadecanoic acid chains at the N-terminus serve as the hydrophobic domain and facilitate the assembly formation. The amphipathic peptide spontaneously self-assembled into nanoparticles (PNPs) in aqueous solution (Figure 1a), with a 4722

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Figure 2. In vitro functional characterization of sPNPs. (a,b) TF expression of MBA-MB-231 cells after two successive treatments (24 h each) with sPNPs and controls (NC indicates a nonfunctional control siRNA). The cells were harvested 24 h after the second treatment, at which time the TF expression was analyzed by immunoblotting (a) and quantified using ImageJ (b). β-Actin was used as the loading control. (c,d) Migration ability of MDA-MB-231 cells after treatment with free siRNA, PNPs, or sPNPs, as analyzed by transwell assay. The number of cells migrating across the transwell filter over 12 h was estimated using crystal violet staining photographed by an EVOS inverted microscope. Scale bars: 100 μm, ***p < 0.001. (e) Percentage of platelets adhered to tumor cells after 1 h coincubation of sPNPs-treated tumoral cells with DIL-labeled platelets; quantification of DIL fluorescence. (f) Adhesion of DIL-labeled platelets (red) to GFP-labeled MDA-MB-231 cells (green) treated with PNPs or sPNPs; images acquired by confocal microscopy. The tumor cell nuclei were stained with Hoechst nuclear dye. Scale bars: 50 μm. (g−i) Platelet adhesion to tumor cells in vivo. GFP-labeled MDA-MB-231 cells (5 × 105 cells per mouse) were intravenously coinjected with DIL-labeled platelets into BALB/c nude mice. After 1 h (g), 4 h (h), and 24 h (i), the lungs were excised and sectioned for confocal microscopy analysis. Scale bars: 20 μm. To quantify the proportion of cells with platelet adhesion and the area of adhered platelet aggregates, 15 tumor cells were selected randomly from each lung.

plasma fluorescence intensity of Cy5-labeled siRNA in PNPs was measured after intravenous injection at the different time intervals. Compared with free siRNA, the blood circulation time of siRNA in sPNPs was significantly prolonged (Figure S4b), indicating siRNA serum stability is significantly enhanced via nanoformulation. Next, we incubated the sPNPs nanoparticles under different pH values (7.4, 6.8, and 5.5) for 20 min to verify their pH responsiveness. After incubation at pH 6.8, the average hydrodynamic diameter of sPNPs notably increased to 102.7 nm, while they exhibited an apparent zeta potential reversal to +4.4 mV, implying a positively charged surface (Table S1). When incubated at pH 5.5, the average hydrodynamic diameter and apparent zeta potential of sPNPs further increased to about 130 nm and +12.4 mV, respectively (Table S1). TEM images of the nanoparticles at the different pH values (Figure 1d) further confirm the change in size of the particles. This is likely due to protonation of the polyHis sequence under acidic conditions, which subsequently leads to the reversion of the nanoparticle’s surface charge and a loosening of the peptide assembly caused by electrostatic repulsion. As previously reported, positively charged nanostructures are more readily internalized into cells;39,44−46 hence, the reversed charge of sPNPs is expected to enhance their cellular uptake and result in more efficient drug delivery into tumor cells that reside in a slightly acidic microenvironment. We confirmed this hypothesis by assessing the cellular

critical micelle concentration (CMC) of 59.8 mg/L (Figure S2). The proposed antitumor mechanism of sPNPs nanoparticle were shown in Figure 1b. To optimize the loading efficiency of siRNA, we tested a series of peptide/siRNA molar ratios to find complete encapsulation of siRNA at ≥20:1 as demonstrated by agarose gel electrophoresis (Figure S3). We adopted the peptide/siRNA molar ratio of 20:1 in subsequent experiments, at which the sPNPs possess a negative charge and an effective siRNA loading efficiency. Results of transmission electron microscopy (TEM) examination revealed that both PNPs and sPNPs possessed a typical spherical structure (Figure 1c), and the coassembly of siRNA increased the average diameter of PNP from ∼43.8 to ∼ 74.2 nm (Figure 1c and Table S1). Zeta-potential measurements show that, after encapsulation of siRNA, the surface charge significantly decreased from 13.3 mV of PNPs to −9.9 mV of sPNPs (Table S1). Thus, incorporating the siRNA not only equips the nanoparticles with effector payload but also affords them a property that favors an increased circulation half-life since nanocarriers with a negative surface charge are less efficiently cleared from the circulation by the reticular endothelial system (RES).42,43 We next investigated the protective effects of PNPs on siRNA stability in vitro by agarose gel electrophoresis analysis. As shown in Figure S4a, free siRNA was almost degraded completely after incubation with RNase for 10 min. However, the majority of siRNA loaded in nanoparticles was preserved even after incubation with RNase for 90 min. The 4723

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Figure 3. Cell binding and in vivo tumor targeting of sPNPs. (a) MDA-MB-231 cells were pretreated with cRGD peptide for 1 h. Then the cells were treated with sPNPs (containing FAM-siRNA) in serum-free medium for 1, 2, and 3 h, respectively, and analyzed using flow cytometry. Flow cytometry showed that pretreated with cRGD peptide significantly inhibited the cellular uptake of sPNPs. (b) Fluorescence intensity was quantified. **p < 0.01; ***p < 0.001. (c) Mice bearing MDA-MB-231 tumors were administered a single intravenous injection of free Cy5-siRNA (middle) or Cy5-sPNPs (right). In vivo fluorescence was imaged at 0.5, 2, 4, 6, 12, and 24 h postinjection. In mice injected with sPNPs, the Cy5 fluorescence specifically accumulated in the tumor region (white circle), peaking at 4 h postinjection, while no fluorescence associated with the tumors in the Cy5-siRNA-treated mice at any time point. Mice not injected with any fluorescent agent were used as a reference (left). Representative images are shown. (d) Ex vivo imaging of the tumor and major organs from tumor bearing mice 24 h after intravenous injection with Cy5-siRNA or Cy5-sPNPs. The sPNPs group showed significant tumor accumulation, while the signal from the free Cy5-siRNA group was almost completely cleared from the tumor by this time point. (e) Fluorescence intensity of the tumor region in d was quantified ***p < 0.001.

internalization of sPNPs by flow cytometry (Figure S5), which revealed that the uptake of fluorescence-labeled sPNPs by MDA-MB-231 tumor cells was significantly higher at pH 6.8 compared to that at pH 7.4. In Vitro Functional Characterization of sPNPs. To investigate the capability of sPNPs to deliver TF siRNA into tumor cells and silence TF gene expression, we first examined the lysosome escape of sPNPs-delivered TF siRNA. MDA-MB231 cells were incubated with sPNPs loaded with FAM-labeled TF siRNA. After a 1 h incubation, the siRNA (green) strongly colocalized with the lysosomes (red), indicating the sPNPs are efficiently internalized and into lysosomes. However, after a 3 h incubation, this colocalization was markedly reduced, and much of the intracellular siRNA diffused into the cytoplasm, indicating a lysosomal escape of the siRNA (Figure S6). We confirmed these results by examining the cellular gene silencing efficiency of sPNPs in vitro. TF protein levels of MDA-MB-231 tumor cells treated with sPNPs or control formulations were determined by immunoblotting. As shown in Figure 2a,b, sPNPs containing different siRNA concentrations all dramatically down-regulated the levels of TF in MDA-MB-231 cells (by 75−80%), while an equivalent concentration of free siRNA (50 nM) had no observable effect on TF expression. These results further indicate that sPNPs can transfer siRNA into tumor cells and silence the TF gene with satisfactory efficacy. One of the known mechanisms underlying the correlation between overexpressed TF and metastasis is that TF increases the migration tendency of tumor cells and promotes their malignant invasion.47,48 We therefore assessed the effects of

sPNPs treatment on the in vitro migration of MDA-MB-231 cells using a transwell assay. Cells pretreated with sPNPs or control formulations were allowed to migrate through the transwell filters over a 12 h time period. The sPNPs significantly inhibited the migration of tumor cells, while neither free siRNA nor PNPs were able to impart any notable effect (Figure 2c,d). Besides its direct impact on the migration and invasion of tumor cells, much of the pro-metastatic effect of TF is related to its procoagulant activities. TF has been reported to mediate platelet activation and subsequent adhesion to tumor cells, which plays a critical role both in supporting the survival of CTCs in the capillary network and in facilitating their arrest in distant organs.49−52 Therefore, we investigated the impact of TF knockdown by sPNPs on the interaction between platelets and tumor cells in vitro and in vivo. When incubated with platelets, the adhesion of fluorescence-labeled platelets to sPNPs-treated green fluorescent protein (GFP) labeled MDA-MB-231 cells was reduced by more than 30% compared to cells treated with PNPs (Figure 2e,f). We also coinjected (i.v.) tumor cells with platelets into mice and evaluated the formation of platelet aggregates around tumor cells in the lung at 1, 4, and 24 h postinjection. The area of platelet aggregates around tumor cells pretreated with sPNPs was markedly reduced compared to the PNPs-treated cells at all three time points examined (Figure 2g−i). Moreover, the percentage of sPNPs-treated tumor cells that exhibited observable platelet adhesion was significantly lower than in PNPs-treated cells, and the size of the platelet aggregates adhering to sPNPs-treated tumor cells was also significantly smaller. These data suggest that the 4724

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Figure 4. Antimetastatic efficacy of sPNPs in vivo. (a) Effects of PNP, free TF siRNA, and sPNPs on metastasis in 4T1 tumor bearing mice. The mice were treated once every 3 days for 6 total treatments, after which the lungs were isolated for histological examination. Scale bars: 1 mm (n = 3). (b) Quantification of the area of metastases. Error bars indicate SD (n = 3). ***p < 0.001. (c) Immunoblot analysis of TF levels in the tumor tissue after treatment. (d) Quantification of TF levels in tumor tissue. Error bars indicate SD (n = 3). ***p < 0.001. (e) Immunohistochemistry staining for TF in the tumor tissue. Scale bars: 50 μm. (f) Ex vivo fluorescence imaging and (g) tissue fluorescence intensity quantification of 4T1 tumors (pretreated with saline, PNPs, free TF siRNA, or sPNPs) 1 h after administration of Cy5-labeled fibrinogen. The lower fibrinogen accumulation in the sPNPs-treated tumors indicates a reduced coagulant activity. A representative image from three animals is shown. Error bars indicate SD; ***p < 0.001. (h) Plasma concentration of TAT as measured using an ELISA kit. Error bars indicate SD (n = 3). *p < 0.05. (i) Measurement of TAT concentration in tumor tissue using an ELISA kit. Error bars indicate SD (n = 3). ***p < 0.001. (j) Immunohistochemistry assay for P-selectin (marker for activated platelets) in tumor tissue after treatment with sPNPs or controls. Scale bars: 50 μm.

not only their distant organ arrest but also colonization. Since the adhesion of platelet/platelet aggregate is believed to protect tumor cells from immune recognition during metastasis and enhanced coagulability was also reported to be an important supporting factor in the formation of early metastatic colonies, it is likely that depletion of the procoagulant activities of TF blocked these processes to cause the diminished platelet adhesion and colonization of CTCs. Tumor Targeting Capability of sPNPs. To investigate the tumor targeting capacity of the sPNPs system, we first examined its cRGD-mediated tumor cell association in vitro. Flow cytometry assays indicate that when FAM-labeled sPNPs were incubated with MDA-MB-231 cells, the cellular fluorescence significantly increased over time (1, 2, or 3 h incubation), indicating that sPNPs readily associate with the tumor cells (Figure 3a). However, when the cells were pretreated with free cRGD peptide for 1 h, their association with sPNPs significantly decreased (by approximately 50% after a 3 h coincubation) compared with the untreated cells (Figure 3a,b). This is probably due to competitive binding between free cRGD and cRGD-modified sPNPs with cRGD

silencing of TF expression suppresses the interaction between tumor cells and platelets, likely through the inhibition of TFmediated platelet activation. Effect of sPNPs Treatment on Metastatic Ability of Tumor Cells in Vivo. We next investigated the impact of TF deficiency on the metastatic capacity of CTCs by examining the pulmonary arrest and colonization after intravenous injection of tumor cells pretreated with sPNPs or control formulations into mice. At 4 and 24 h after the injection of GFP-labeled MDA-MB-231 cells, the arrest of sPNPs-treated tumor cells in the lung (estimated by their fluorescence) was significantly lower than that of PNPs-treated cells (Figure S7a,b). A longterm experiment was also carried out using two cell lines, MDA-MB-231 and 4T1 cells, to estimate the impact of sPNPs treatment on pulmonary colonization. While the lungs of mice injected with PNPs-treated cells were heavily infiltrated by metastases a few weeks after tail vein injection with these tumoral cells (5 weeks for MDA-MB-231 cells and 2 weeks for 4T1 cells), the numbers of observable metastatic foci formed by sPNPs-treated cells were significantly reduced in comparison to both of the two used cell lines (Figure S8 a,b). Thus, silencing TF expression in CTCs is able to significantly prevent 4725

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Figure 5. Cytotoxicity and immunogenicity of sPNPs. (a) Cell viability of MDA-MB-231, 4T1, and HUVEC cells after incubation with PNPs, free TF siRNA (50 nM), or sPNPs for 24 h. Error bars indicate SD. (b) Secretion of immune related cytokines by splenic cells isolated from PNP, free siRNA, or sPNPs-treated BALB/c mice. Error bars indicate SD. (c) BALB/c female mice were intravenously injected with saline, siRNA (1.5 mg/ kg), PNPs (peptide, 25 mg/kg), and sPNPs (peptide, 25 mg/kg; siRNA, 1.5 mg/kg) every other day for 5 total treatments (n = 3). Major organs (heart, liver, spleen, lung, and kidney) harvested after the last treatment were subjected to histological examination. No visible bleeding spots or other pathological changes were observed in the sPNPs treated group. Scale bars: 1 mm.

4c−e). These results indicate that sPNPs treatment is able to efficiently knockdown tumor-associated TF and has the potential to significantly reduce the pulmonary metastases of breast cancer. Depletion of TF Decreases Tumor-associated Blood Hypercoagulable State. Aberrant expression of TF in aggressive tumors plays a key role in the formation of a hypercoagulable microenvironment, which facilitates tumor cell survival, immune escape, and metastasis.53,54 Although a strong correlation between TF overproduction in the tumor and the occurrence of metastasis has been reported,6,54,55 few studies have examined the impact of TF inhibition on tumor microenvironment coagulability. We next examined whether TF silencing by sPNPs could reverse tumor hypercoagulability in the 4T1 breast cancer mouse model. After treating the animals with sPNPs or control formulations, we administered Cy5-labeled fibrinogen to visualize the coagulant activity in the tumor tissue. Compared with the saline, PNPs, and free siRNA treated groups, the fibrinogen accumulation in the tumor of sPNPs-treated mice was significantly reduced, as evidenced by the lowest fluorescence intensity (Figure 4f), indicating a less active coagulation state. Quantification of tumor fluorescent signals revealed a 60% decrease of Cy5-fibrinogen in the sPNPs-treated mice compared to the other groups (Figure 4g). We also characterized thrombin generation in the plasma and tumor tissue using a thrombin-antithrombin complex (TAT) assay. The plasma TAT concentration of tumor bearing mice is significantly higher than normal mice, which is congruent with tumor-associated hypercoagulability. However, after treatment

receptors on the cell surface, implying that the specific interaction between the cRGD and their receptors was likely responsible for the association of the sPNPs with tumor cells. In vivo tumor targeting of sPNPs was tested by intravenously administering free Cy5-siRNA or Cy5-siRNA loaded nanocomplexes (Cy5-sPNPs) into tumor-bearing mice. In vivo fluorescence imaging indicated that from 4 to 24 h postinjection, the Cy5-sPNPs accumulated in the tumor region, while in the free Cy5-siRNA-treated mice, the apparent fluorescence signal almost entirely disappeared after 4 h (Figure 3c). Ex vivo imaging of excised tumors and major organs at 24 h postinjection also demonstrated a much stronger fluorescence in Cy5-sPNPs treated tumors compared to those treated with free Cy5-siRNA (Figure 3d,e), confirming that sPNPs were able to effectively deliver their cargo to the tumor tissue. In Vivo Antimetastatic Activity of sPNPs. The antimetastatic activity of sPNPs was investigated in a 4T1 breast tumor bearing mouse model. Female BALB/c mice bearing tumors (∼100 mm3) were treated with sPNPs or control formulations every 3 days for 6 total treatments. Histological examination showed that sPNPs treatment notably inhibited the formation of metastatic nodules in the lung (Figure 4a,b; almost 80% inhibition compared to the saline group), while free siRNA and PNPs did not elicit any significant antimetastatic effect. Further analysis of TF levels in the tumor tissues using immunoblotting and immunohistochemistry staining confirmed that sPNPs suppressed the expression of tumor-associated TF by approximately 75% compared to the other groups (Figure 4726

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orb11315) was purchased from Biorbyt (Cambridge, UK). Anti-β-actin mouse monoclonal antibody, goat antimouse IgGHRP, and goat antirabbit IgG-HRP were purchased from YEASEN (Shanghai, China) and Santa Cruz Biotechnologies (Santa Cruz, CA). Interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-6 (IL-6) ELISA kits were purchased from eBioscience (Santa Cruz, CA). Cell Culture. The human breast cancer cell lines MDA-MB231 (American Type Culture Collection, ATCC, Manassas, VA, USA) and GFP-labeled MDA-MB-231 (National Infrastructure of Cell Line Resources, Beijing, China) were cultured in DMEM medium. Mouse breast cancer cell lines 4T1 (ATCC) and human umbilical vein endothelial cells (HUVECs, ATCC) were cultured in RPMI-1640 and F12K medium, respectively. All the culture media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in 5% CO2 at 37 °C. Preparation and Characterization of Nanoparticles. For the preparation of sPNPs, 1.75 mg of peptide[(C18)2-Lys(Gly)2-(Arg-Arg-Ser)10-Lys-(His)8-cRGD] dissolved in 10 μL of DMSO was diluted into 1 mL of DEPC water solution containing 181.5 μg of siRNA (peptide/siRNA molar ratio = 20:1), and the mixture was ultrasonicated at 100 W for 5 min. After a 1 h incubation at room temperature, the solution was dialyzed against DEPC water at room temperature for 6 h. For the preparation of PNPs, DEPC water was used in place of the siRNA solution. The zeta potential of the resultant nanoparticles was determined using a ZetaSizer Nano series NanoZS (Malvern Instruments Ltd., Malvern, U.K.). The size and morphology of the nanoparticles were characterized by transmission electron microscopy (TEM) using an EM200CX microscope (JEOL Ltd., Tokyo, Japan) after negative staining with 4% uranyl acetate for 10 min. The siRNA sequence used is 5′-GCGCUUCAGGCACUACAAATTTTCGCGAAGUCCGUGAUGUUU-5′ Determination of Critical Micelle Concentration (CMC). The stability of the peptide-assembly-based nanoparticles was evaluated by determining the CMC of the amphipathic peptide. The peptide, at different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100, or 200 μM), was incubated with pyrene (24 μg/L) for 1 h in phosphate buffered saline (PBS, pH 7.4). The solutions were then analyzed with an F4600 fluorescence spectrophotometer (Hitachi, Japan) using an excitation wavelength of 334 nm. The ratio of the fluorescence intensity between 373 and 384 nm to the peptide concentration was plotted. The inflection point of the resulting curve was taken as the CMC.56 Stability Evaluation of sPNPs. Free siRNA or sPNPs were mixed with RNase A and incubated at 37 °C for 0, 10, 20, 30, 60, and 90 min. Then the mixed solution was incubated with gel loading buffer containing 1% SDS for 10 min and detected by 1% EtBr agarose gel. For in vivo stability, BALB/c mice were injected intravenously with free Cy5-siRNA or sPNPs loaded with Cy5-siRNA, respectively. Blood was collected from tail veins at different time intervals and used for fluorescent imaging. In Vitro Cellular Uptake. MDA-MB-231 cells were seeded into 12-well plates at a density of 1 × 105 cells per well and maintained in 5% CO2, 37 °C for 24 h. The culture medium was then replaced with serum-free medium (pH 7.4 or 6.8) containing sPNPs loaded with FAM-labeled siRNA. After incubating the plates for varying time periods, the cells were

with sPNPs, the plasma TAT concentration of the tumor bearing mice decreased nearly to normal levels (Figure 4h). Moreover, the local TAT levels within the tumor tissue of sPNPs treated mice were significantly lower (by approximately 50%) than those of the control groups (Figure 4i). Immunostaining for the platelet activation marker P-selectin also indicates that sPNPs treatment effectively reduces the number of activated platelets within tumor tissue (Figure 4j). Altogether, these results suggest that TF silencing by sPNPs can effectively reverse the procoagulant state of tumor microenvironment by decreasing fibrinogen accumulation, thrombin generation, and platelet activation. Safety Evaluation of sPNPs. We comprehensively assessed the biocompatibility of sPNPs both in vitro and in vivo. Neither PNPs nor sPNPs exhibited any detectable cytotoxicity in MDA-MB-231, 4T1, or human umbilical vein endothelial cells (HUVECs) even when the equivalent siRNA concentration in sPNPs reached up to 500 nM (Figure 5a). In addition, we also investigated the immunostimulating activity of sPNPs by evaluating the levels of immunostimulatory cytokines (IL-2, IFN-γ, and IL-6) secreted by splenic cells isolated from treated mice. No significant changes were detected (Figure 5b). All these results are suggestive of favorable biocompatibility of sPNPs. To examine the in vivo safety of the nanoparticle formulation, healthy BALB/c mice were administered (i.v.) with saline, siRNA, PNPs, or sPNPs once every other day for 5 total injections. Hematoxylin and eosin (H&E) staining of the major organs and blood biochemical analysis revealed no overt histological change, and organ function damages were observed (Figures 5c and S9). TF is generally considered as a crucial initiator of the extrinsic blood coagulation pathway.2,3 Therefore, we next evaluated the tail bleeding time and activated partial thromboplastin time (APTT) of the mice treated with sPNPs. As indicated in Figure S10, sPNPs treatment did not affect the blood clotting function of mice. Conclusions. The hypercoagulable state in solid tumors has long been thought to be a crucial supporting factor for the metastasis and colonization of tumor cells. The blockage of local hypercoagulability therefore may represent a particularly attractive approach for antimetastatic therapy. In our current work, we report a peptide-based assembly vehicle to specifically deliver TF siRNA to tumor sites and realize an inhibition of tumor-associated TF, which plays a central role in the formation of tumor hypercoagulability. With the ability to successfully target tumor tissue and knockdown tumoral TF expression, this nanoparticle system significantly inhibits the interactions of CTCs with platelets and reverses the tumor coagulant state. As an antimetastatic agent, our system significantly inhibits metastasis to the lung in a mouse breast cancer model. This nanosystem represents a possibility to safely and specifically knock down tumor-associated TF for preventing metastasis and may provide useful insight for the development of new oncotherapies. Materials and Methods. Materials. All chemicals were purchased from Sigma-Aldrich (Shanghai, China) unless otherwise stated. Peptides used in this study were synthesized and purified by Top-peptide (Shanghai, China). Small interfering RNAs, including FAM- and Cy5-labeled siRNAs were purchased from GenePharma (Shanghai, China). The antitissue factor monoclonal antibodies (cat. no. ab151748) and thrombin-antithrombin complex (TAT) ELISA kits (no. ab137994) were purchased from Abcam (Cambridge, MA, USA). Anti-CD62p rabbit polyclonal antibodies (cat. no. 4727

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Nano Letters collected, washed with PBS, and resuspended in 200 μL of PBS. The total fluorescence intensity of the cells was detected using a BD C6 flow cytometer. In Vitro Imaging of Tumor Cells. MDA-MB-231 cells were seeded onto 35 mm chambered, borosilicate cover glasses (Nunc, USA). The cells were treated with sPNPs loaded with FAM-siRNA for different time periods. We then stained the cells with LysoTracker Red to visualize lysosomes, according to the manufacturer’s instructions, for 2 h, washed the samples three times with PBS, and observed the cells with an LSM 710 confocal microscope (Carl Zeiss, USA). Immunoblotting. Total protein-containing lysates from cell and tissue samples were extracted using a RIPA Lysis and Extraction Buffer (Solarbio) and quantified using a BCA Protein Assay Kit (Thermo Scientific) according to the manufacturers’ protocols. The extracted protein was then incubated at 100 °C in SDS-PAGE loading buffer for 5 min and resolved by SDS-PAGE electrophoresis. After transferring the proteins to a PVDF transfer membrane (Merck Millipore, Germany) using a Trans-Blot Transfer System (BIO-RAD, US), the membrane was blocked with 5% nonfat milk in trisbuffered saline Tween (TBST) for 1 h. Then the membrane was incubated with the appropriate antibodies for 2 h under room temperature and washed three times (10 min per time) using TBST buffer. Next, the membrane was incubated with corresponding secondary antibodies for an hour under room temperature and washed again for three times as mentioned above. Signals were visualized using a ChemiDoc Touch Imaging System (BIO-RAD, US). Cell Migration Assay. MDA-MB-231 tumor cells were cultured for 24 h in serum-free medium to achieve a steadystate. The cells were then plated onto 8 μm-pore transwell filters (2 × 104 cells per well maintained in serum-free medium) that were placed in a 12-well plate filled with medium containing 10% FBS. After a 12 h incubation, the cells were washed three times with serum-free medium and fixed with 4% formaldehyde for 30 min. The transwell filters were washed three times with PBS, and cells on the upper side of the polycarbonate membrane were scraped off before the filter was stained in 0.1% crystal violet. The number of cells that migrated to the bottom chamber over 12 h was counted under an EVOS inverted microscope. Isolation of Platelets from Volunteer Blood. Whole blood from healthy volunteers was collected in vacuum blood collection tubes and centrifuged for 10 min at 180 × g at room temperature to obtain platelet-rich plasma (PRP). The PRP was then centrifuged at 1200 × g for another 10 min to collect a platelet pellet. The pellet was resuspended and washed three times in CGS buffer (13 mM trisodium citrate, 30 mM dextrose and 120 mM NaCl, pH 7.0) in the presence of 1 μg/mL PGE1. Washed platelets were resuspended in Tyrode’s buffer (containing 0.3% BSA) at a concentration of 1 × 109 platelets per mL and stained with the membrane dye DIL. In Vitro Platelet Adhesion Assay. MDA-MB-231 cells were seeded in 96-well plates (black plate with clear bottom; Corning, NY) at a concentration of 4 × 104 cells per 100 μL per well and grown until confluent. The cells were washed three times with serum-free medium. Washed platelets (3 × 108 per mL) preloaded with DIL as described above were allowed to adhere to the cells for 60 min at 37 °C in the presence of 10% human plasma. The wells were imaged by a fluorescence imaging system (CRi, Woburn, MA) using

excitation and emission wavelengths of 553 and 570 nm, respectively. To remove the nonadhered platelets, the cells were then washed three times with PBS and supplied with 100 μL of serum-free medium and imaged again. The fluorescence intensity of a well containing 100 μL of medium with 10% human plasma or serum-free medium was used as the blank sample. The percentage of adhered platelets was calculated as (fluorescence after PBS wash − blank)/(fluorescence before PBS wash − blank) × 100%. To further visualize the interaction between tumor cells and platelets, GFP-labeled MDA-MB-231 cells were seeded in 35 mm confocal dishes and grown until confluent. DIL-stained platelets (3 × 108 per mL) were added into the dish, and the samples were incubated for 60 min at 37 °C in the presence of 10% human plasma. The cells were then washed three times with PBS, fixed with 4% formaldehyde, and imaged using an LSM 710 confocal microscope (Carl Zeiss, USA). In Vivo Platelet Adhesion Assay. Female BALB/c nude mice were injected with 5 × 105 GFP-labeled MDA-MB-231 cells (untreated or treated with sPNPs for 48 h) and 5 × 109 DIL-stained platelets isolated from human blood (separately into opposite lateral tail veins). After 1, 4, and 24 h, the lungs were excised and freeze-sectioned, and the sections were imaged using a confocal microscope. In Vivo Pulmonary Colonization of Tumor Cells. Female BALB/c nude mice were intravenously injected with 5 × 105 MDA-MB-231 cells either untreated or pretreated with sPNPs for 48 h. Female BALB/c mice were intravenously injected with 2 × 105 4T1 cells either untreated or pretreated with sPNPs for 48 h. For the MDA-MB-231 group, the lungs were excised 5 weeks after injection and sectioned for histological examination for pulmonary metastasis foci. For the 4T1 group, the lungs were excised after 2 weeks and sectioned for histological examination. In Vivo Imaging and Biodistribution. About 1 × 106 4T1 mouse breast cancer cells (suspended in 50 μL of PBS) mixed with 50 μL of matrigel were transplanted into the second mammary fat pads of female BALB/c nude mice; tumors were allowed to grow to about 100 mm3. The mice were then intravenously injected with saline, free Cy5-labeled negative control siRNA (33 μg per mouse) or sPNPs (loaded with Cy5labeled negative control siRNA, 33 μg per mouse). At different time points post administration, the mice were imaged using an ex/in vivo optical imaging system (Maestro, CRi, Woburn, MA) to visualize the Cy5 fluorescence. To examine tissue biodistribution, the mice were sacrificed 24 h postinjection, and the tumor and major organs were excised and imaged. In Vivo Antimetastasis Experiments. Mice bearing 4T1 tumors (about 100 mm3) were intravenously injected with saline, siRNA (1.5 mg/kg), PNPs (15 mg/kg peptide), or sPNPs (containing 1.5 mg/kg siRNA and 15 mg/kg peptide) every 3 days for a total of 6 injections. After the final treatment the mice were sacrificed and the tumors were excised for immunoblotting and immunohistochemistry analysis of TF expression. The lungs were excised and sectioned for histological examination using H&E staining. Tumor Coagulation State Assay. Mice bearing 4T1 tumors (about 100 mm3) were intravenously injected with saline, siRNA (1.5 mg/kg), PNPs (15 mg/kg peptide), or sPNPs (containing 1.5 mg/kg siRNA and 15 mg/kg peptide) every 3 days for a total of 6 injections. After the final treatment, the mice were intravenously injected with Cy5-labeled fibrinogen 4728

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Nano Letters (0.5 mg per mouse). An hour later, the tumors were excised for fluorescence imaging. TAT Concentration Assay. After treatment, whole blood was collected using EDTA coated tubes and centrifuged for 10 min at 850 × g under room temperature to obtain the plasma. The total protein from tumor tissue was collected as described above. The TAT concentration in the plasma and tumor tissue was measured using a TAT ELISA kit according to the manufacturer’s protocols. Cytotoxicity Assay. MDA-MB-231 cells, 4T1 cells, or HUVECs were maintained in 96-well plates (1 × 104 per well) at 37 °C for 24 h. The cells were then treated with PNPs, siRNA (500 nM), or sPNPs formulations containing different concentrations of siRNA for another 24 h. Cell viability was determined using a CCK-8 kit. Immunogenicity Test. Splenic cells isolated from BALB/c mice were cultured in 12-well plates and stimulated with PNPs, siRNA (200 nM), or sPNPs (containing 200 nM siRNA) for 24 h. The medium was then replaced with fresh medium containing 1% FBS, and 12 h later, the conditioned medium was collected and the concentrations of interferon-gamma, interleukin 2, and interleukin 6 were determining using ELISA kits. Blood Clotting Hemostatic Function Evaluation. BALB/c mice were randomly divided into 4 groups and intravenously injected with saline, siRNA (1.5 mg/kg), PNPs (15 mg/kg peptide), or sPNPs (15 mg/kg peptide and 1.5 mg/kg siRNA) every the other day for 5 total treatments. Two more days after the last treatment, the mice were anesthetized with sodium pentobarbital, and the tails were cut about one millimeter from the tip. Then the tail was quickly inserted into the 37 °C PBS solution, and the bleeding time was recorded. Then the plasmas of the mice were collected, and the activated partial thromboplastin time (APTT) was detected using a semiautomatic coagulation analyzer (LG-PABER-I, Taizhou, China). Biosafety Assessment. BALB/c mice were randomly divided into 4 groups and intravenously injected with saline, siRNA (1.5 mg/kg), PNPs (15 mg/kg peptide), or sPNPs (15 mg/kg peptide and 1.5 mg/kg siRNA) every other day for 5 total treatments. After the last treatment, blood and the major organs were collected for serum biochemical examination and tissue histological examination (H&E staining). Statistical Analysis. Data were examined statistically using the SPSS17.0 statistical analysis software. Student t test or oneway ANOVA followed by Tukey’s HSD test was applied for comparison between two groups or among multiple groups, respectively.

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sPNPs, and blood clotting hemostatic function evaluation (PDF)

AUTHOR INFORMATION

Corresponding Authors

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

Suping Li: 0000-0002-0294-8861 Guangjun Nie: 0000-0001-5040-9793 Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (Grant No. 2018YFA0208900), the National Natural Science Foundation of China (Grant Nos. 31730032, 31661130152, 31471035, 51673051, 81630068, 21877023, and 51861145302), a Key Research Project of Frontier Science of the Chinese Academy of Sciences (Grant No. QYZDJ-SSWSLH022), Beijing Nova Program (Grant No. Z171100001117010), Beijing Natural Science Foundation (Grant No. 7172164), National Postdoctoral Program for Innovative Talents (BX20180083), Youth Innovation Promotion Association CAS (Grant No. 2017056), and K. C. Wong Education Foundation (GJTD-2018-03).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b01785. Hydrodynamic diameter and surface charge of PNPs and sPNPs, chemical structure of peptide complex, critical micelle concentration of the peptide complex, loading efficiency of siRNA, protective effects of PNPs on siRNA, cellular uptake of sPNPs at different pH conditions, intracellular distribution and lysosomal escape of sPNPs-delivered siRNA, cell arrest in the lung, histological analysis of lungs, biocompatibility of 4729

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DOI: 10.1021/acs.nanolett.9b01785 Nano Lett. 2019, 19, 4721−4730