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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 Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b01785 • Publication Date (Web): 07 Jun 2019 Downloaded from http://pubs.acs.org on June 8, 2019
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Tumor-specific silencing of tissue factor suppresses metastasis and prevents
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cancer-associated hypercoagulability
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Shaoli Liu1,2#, Yinlong Zhang 1#, Xiao Zhao1#, Jing Wang1, Chunzhi Di1,2, Ying Zhao1, Tianjiao Ji1, Keman Cheng1, Yongwei Wang1, Long Chen1,2, Yingqiu Qi1,2,3, Suping Li1,2* and Guangjun Nie1,2*
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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 2 Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, China 3 Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
#These
authors contributed equally to this work.
*Address correspondence to: E-mail:
[email protected]; or Email:
[email protected];
39
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Abstract
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Within tumors, the coagulation-inducing protein tissue factor (TF), a major initiator of blood
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coagulation, has been shown to play a critical role in the hematogenous metastasis of tumors, due to
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its effects on tumor hypercoagulability and on the mediation of interactions between platelets and
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tumor cells. Targeting tumor-associated TF has therefore great therapeutic potential for
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anti-metastasis therapy and preventing thrombotic complication in cancer patients. Herein, we
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reported a novel peptide-based nanoparticle that targets delivery and release of small interfering
8
RNA (siRNA) into the tumor site to silence the expression of tumor-associated TF. We showed that
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suppression of TF expression in tumor cells blocks platelet adhesion surrounding tumor cells in vitro.
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The downregulation of TF expression in intravenously administered tumor cells (i.e., simulated
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circulating tumor cells [CTCs]) prevented platelet adhesion around CTCs and decreased CTCs
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survival in the lung. In a breast cancer mouse model, siRNA-containing nanoparticles efficiently
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attenuated TF expression in the tumor microenvironment and remarkably reduced the amount of lung
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metastases in both an experimental lung metastasis model and tumor-bearing mice. What's more, this
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strategy reversed the hypercoagulable state of the tumor bearing mice by decreasing the generation
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of thrombin-antithrombin complexes (TAT) and activated platelets, both of which are downstream
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products of TF. Our study describes a promising approach to combat metastasis and prevent
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cancer-associated thrombosis, which advances TF as a therapeutic target toward the clinic
19
applications.
20 21
Keywords: Peptide self-assembled nanoparticle, Tissue factor, siRNA, Tumor metastasis, Tumor
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hypercoagulability
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Introduction
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Tissue factor (TF), a crucial initiator of the extrinsic blood coagulation pathway, has also long been
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known to play a vital role in tumor progression and metastasis. 1-4 The upregulation of TF was found
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in both tumor cells and vascular endothelial cells in a wide range of solid tumors. 3 In this context,
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TF promotes tumor invasion and metastasis by inducing the production of tumor growth factors such
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as angiogenic factors and chemokines. 5 Emerging evidence from clinical studies also suggests that
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the high levels of TF in tumor tissue are strongly associated with a high incidence of systemic
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hypercoagulability. 6-8 Blood clotting abnormalities are detected in up to 90% of cancer patients, and
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thrombosis are considered to be the second most frequent cause of cancer-associated mortality.9-10
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All these findings have made tumor-associated TF an attractive therapeutic target for anti-cancer
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metastasis and prevention of cancer-associated abnormal hypercoagulability. Approaches that
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suppress TF by antibodies or tissue factor pathway inhibitor (TFPI), have been reported to
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significantly reduce the incidence of metastasis in mice.
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anticoagulation therapy might logically induce tissue bleeding complications.15-16 Novel strategies to
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realize tumor-specific depletion of TF, while avoiding non-specific intervention in normal tissues,
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are urgently needed in order to achieve safe and effective tumor treatment.
11-14
Furthermore, non-specific
17 18
Despite offering a highly efficient mechanism to selectively silence target genes at the
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post-translational level, 17 unmodified siRNA is an ineffective therapeutic agent, suffering from rapid
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clearance from the bloodstream. The circulation half-life of naked siRNA is typically ranges in
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minutes, thus limiting in vivo application of siRNA-based therapy.18-19 Capable of both significantly
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prolonging siRNA circulation time and improving targeting efficiency, the use of targeted
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nanocarriers has potentially revolutionized the delivery of siRNA in vivo.20-26
24 25
Bio-responsive materials for precise design of nanomedicines have been proven to be feasible
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strategies for targeted tumor therapy.
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nanoparticle system to specifically knock-down tumor-associated TF through small interfering RNA
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(siRNA) silencing. An amphipathic peptide which was able to self-assemble into peptide
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nanoparticles (PNPs) was designed as the vehicle and co-assembled with TF siRNA to form
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siRNA-loaded peptide nanoparticles (sPNPs). To further optimize the delivery efficacy, we
27-31
We herein developed a tumor-targeted peptide-based
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integrated into the peptide monomer with a cyclic RGD (cRGD) peptide, which recognizes the αvβ3
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integrins overexpressed on the surface of most tumor cells,32-35 and a polyhistidine sequence
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sensitive to the slightly acidic microenvironment (pH≤6.8) of tumor tissues.
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nanoparticles arrive at the acidic tumor microenvironment, a pH-triggered surface charge reversion,
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induced by the polyhistidine moiety, facilitates cell uptake.38-41 The subsequent depletion of
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tumor-associated TF decreases the invasive potential and platelets adhesion of cancer cells and
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further prevents its distant metastasis. Also, depletion of tumor-associated TF reversed the
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tumor-induced hypercoagulable state. Our experimental results demonstrate a significantly reduced
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in vitro cellular migration tendency of tumor cells as the result of TF down-regulation by sPNPs.
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Consequently, the TF-suppressed tumor cells showed a remarkable decrease in lung colonization.
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Furthermore, in mice bearing highly metastatic tumors, the administration of sPNPs efficiently
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suppressed the formation of lung metastases and reduced tumor-associated blood coagulation
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parameters.
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When the
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Results and Discussion
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Preparation and characterization of peptide-based, pH sensitive nanoparticles
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We
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(C18)2-Lys-(Gly)2-(Arg-Arg-Ser)10-Lys-(His)8-cRGD (Figure S1), for the construction of the
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co-assembly nanovehicle. The cRGD peptide was engineered at the end of the hydrophilic moiety in
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order to expose it on the surface of the assembled nanoparticle. Next, we added a pH-sensitive
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polyHis sequence whose protonation in response to the acidic tumor microenvironment enables
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sPNPs to convert their surface charge from negative to a cationic form. In the cationic state, they
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were able to electrostatically interact with negatively charged cell membranes and thus promote the
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nanoparticles uptake by tumor cells. To improve the siRNA loading efficiency, a highly cationic
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repeating arginine-arginine-serine (RRS) segment was included to electrostatically absorb the
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negatively RNA molecules during the co-assembly process. Two octadecanoic acid chains at the
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N-terminus serve as the hydrophobic domain and facilitate the assembly formation.
designed
a
novel
amphipathic
peptide
chimera,
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The amphipathic peptide spontaneously self-assembled into nanoparticles (PNPs) in aqueous
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solution (Figure 1a), with a critical micelle concentration (CMC) of 59.8 mg/L (Figure S2). The
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proposed antitumor mechanism of sPNPs nanoparticle were shown in figure 1b.To optimize the
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loading efficiency of siRNA, we tested a series of peptide/siRNA molar ratios to find complete
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encapsulation of siRNA at ≥20:1 as demonstrated by agarose gel electrophoresis (Figure S3). We
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adopted the peptide/siRNA molar ratio of 20:1 in subsequent experiments, at which the sPNPs
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possess a negative charge and an effective siRNA loading efficiency. Results of transmission
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electron microscopy (TEM) examination revealed that both PNPs and sPNPs possessed a typical
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spherical structure (Figure 1c), and the co-assembly of siRNA increased the average diameter of
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PNP from ~43.8 nm to ~ 74.2 nm (Figure 1c and Table S1). Zeta-potential measurements show that
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after encapsulation of siRNA, the surface charge significantly decreased from 13.3 mV of PNPs to
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-9.9 mV of sPNPs (Table S1). Thus, incorporating the siRNA not only equips the nanoparticles with
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effector payload, but also affords them a property that favors an increased circulation half-life since
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nanocarriers with a negative surface charge are less efficiently cleared from the circulation by the
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reticular endothelial system (RES).42-43 We next investigated the protective effects of PNPs on
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siRNA stability in vitro by agarose gel electrophoresis analysis. As shown in Figure S4a, free
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siRNA was almost degraded completely after incubation with RNase for 10 min. However, the
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majority of siRNA loaded in nanoparticles was preserved even after incubation with RNase for 90
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min. The plasma fluorescence intensity of Cy5-labelled siRNA in PNPs was measured after
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intravenous injection at the different time intervals. Compared with free siRNA, the blood circulation
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time of siRNA in sPNPs was significantly prolonged (Figure S4b), indicating siRNA serum stability
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is significantly enhanced via nanoformulation.
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Next, we incubated the sPNPs nanoparticles under different pH values (7.4, 6.8, and 5.5) for 20
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min to verify their pH responsiveness. After incubation at pH 6.8, the average hydrodynamic
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diameter of sPNPs notably increased to 102.7 nm, while they exhibited an apparent zeta potential
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reversal to +4.4 mV, implying a positively charged surface (Table S1). When incubated at pH 5.5,
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the average hydrodynamic diameter and apparent zeta potential of sPNPs further increased to about
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130 nm and +12.4 mV, respectively (Table S1). TEM images of the nanoparticles at the different pH
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values (Figure 1d) further confirm the change in size of the particles. This is likely due to
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protonation of the polyHis sequence under acidic conditions, which subsequently leads to the
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reversion of the nanoparticle’s surface charge and a loosening of the peptide assembly caused by
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electrostatic repulsion. As previously reported, positively charged nanostructures are more readily
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internalized into cells,39, 44-46 hence the reversed charge of sPNPs is expected to enhance their cellular
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uptake and result in more efficient drug delivery into tumor cells that reside in a slightly acidic
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microenvironment. We confirmed this hypothesis by assessing the cellular internalization of sPNPs
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by flow cytometry (Figure S5), which revealed that the uptake of fluorescence-labeled sPNPs by
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MDA-MB-231 tumor cells was significantly higher at pH 6.8 compared to that at pH 7.4.
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In vitro functional characterization of sPNPs
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To investigate the capability of sPNPs to deliver TF siRNA into tumor cells and silence TF gene
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expression, we first examined the lysosome escape of sPNPs-delivered TF siRNA. MDA-MB-231
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cells were incubated with sPNPs loaded with FAM-labeled TF siRNA. After a 1 h incubation, the
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siRNA (green) strongly co-localized with the lysosomes (red), indicating the sPNPs are efficiently
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internalized and into lysosomes. However, after a 3 h incubation, this co-localization was markedly
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reduced and much of the intracellular siRNA diffused into the cytoplasm, indicating a lysosomal
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escape of the siRNA (Figure S6). We confirmed these results by examining the cellular gene
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silencing efficiency of sPNPs in vitro. TF protein levels of MDA-MB-231 tumor cells treated with
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sPNPs or control formulations were determined by immunoblotting. As shown in Figure 2a and b,
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sPNPs containing different siRNA concentrations all dramatically down-regulated the levels of TF in
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MDA-MB-231 cells (by 75-80%), while an equivalent concentration of free siRNA (50 nM) had no
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observable effect on TF expression. These results further indicate that sPNPs can transfer siRNA into
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tumor cells and silence the TF gene with satisfactory efficacy.
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One of the known mechanisms underlying the correlation between overexpressed TF and
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metastasis is that TF increases the migration tendency of tumor cells and promotes their malignant
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invasion.47-48 We therefore assessed the effects of sPNPs treatment on the in vitro migration of
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MDA-MB-231 cells using a transwell assay. Cells pre-treated with sPNPs or control formulations
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were allowed to migrate through the transwell filters over a 12 h time period. The sPNPs
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significantly inhibited the migration of tumor cells, while neither free siRNA nor PNPs were able to
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impart any notable effect (Figure 2c and d). Besides its direct impact on the migration and invasion
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of tumor cells, much of the pro-metastatic effect of TF is related to its procoagulant activities. TF has
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been reported to mediate platelet activation and subsequent adhesion to tumor cells, which plays a
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critical role both in supporting the survival of CTCs in the capillary network and in facilitating their
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arrest in distant organs.49-52 Therefore, we investigated the impact of TF knockdown by sPNPs on the
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interaction between platelets and tumor cells in vitro and in vivo. When incubated with platelets, the
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adhesion of fluorescence-labeled platelets to sPNPs-treated green fluorescent protein (GFP) labeled
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MDA-MB-231 cells was reduced by more than 30% compared to cells treated with PNPs (Figure 2e
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and f). We also co-injected (i.v.) tumor cells with platelets into mice and evaluated the formation of
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platelet aggregates around tumor cells in the lung at 1, 4 and 24 h post-injection. The area of platelet
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aggregates around tumor cells pre-treated with sPNPs was markedly reduced compared to the
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PNPs-treated cells at all three time points examined (Figure 2g-i). Moreover, the percentage of
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sPNPs-treated tumor cells that exhibited observable platelet adhesion was significantly lower than in
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PNPs-treated cells, and the size of the platelet aggregates adhering to sPNPs-treated tumor cells was
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also significantly smaller. These data suggest that the silencing of TF expression suppresses the
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interaction between tumor cells and platelets, likely through the inhibition of TF-mediated platelet
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activation.
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Effect of sPNPs treatment on metastatic ability of tumor cells in vivo
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We next investigated the impact of TF deficiency on the metastatic capacity of CTCs by examining
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the pulmonary arrest and colonization after intravenous injection of tumor cells pre-treated with
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sPNPs or control formulations into mice. At 4 and 24 h after the injection of GFP-labeled
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MDA-MB-231 cells, the arrest of sPNPs-treated tumor cells in the lung (estimated by their
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fluorescence) was significantly lower than that of PNPs-treated cells (Figure S 7a-b). A long-term
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experiment was also carried out using two cell lines, MDA-MB-231 and 4T1 cells, to estimate the
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impact of sPNPs treatment on pulmonary colonization. While the lungs of mice injected with
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PNPs-treated cells were heavily infiltrated by metastases a few weeks after tail vein injection with
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these tumoral cells (5 weeks for MDA-MB-231 cells and 2 weeks for 4T1 cells), the numbers of
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observable metastatic foci formed by sPNPs-treated cells were significantly reduced in comparison,
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both of the two used cell lines (Figure S8 a-b). Thus, silencing TF expression in CTCs is able to
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significantly prevent not only their distant organ arrest but also colonization. Since the adhesion of
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platelet/platelet aggregate is believed to protect tumor cells from immune recognition during
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metastasis, and enhanced coagulability was also reported to be an important supporting factor in the
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formation of early metastatic colonies, it is likely that depletion of the procoagulant activities of TF
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blocked these processes to cause the diminished platelets adhesion and colonization of CTCs.
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Tumor targeting capability of sPNPs
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To investigate the tumor targeting capacity of the sPNPs system, we first examined its
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cRGD-mediated tumor cell association in vitro. Flow cytometry assays indicate that when
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FAM-labeled sPNPs were incubated with MDA-MB-231 cells, the cellular fluorescence significantly
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increased over time (1, 2 or 3 h incubation), indicating that sPNPs readily associate with the tumor
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cells (Figure 3a). However, when the cells were pre-treated with free cRGD peptide for 1 h, their
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association with sPNPs significantly decreased (by approximately 50% after a 3 h co-incubation)
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compared with the untreated cells (Figure 3a&b). This is probably due to competitive binding
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between free cRGD and the cRGD-modified sPNPs with cRGD receptors on the cell surface,
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implying that the specific interaction between the cRGD and their receptors was likely responsible
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for the association of the sPNPs with tumor cells.
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In vivo tumor targeting of sPNPs was tested by intravenously administering free Cy5-siRNA or
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Cy5-siRNA loaded nanocomplexes (Cy5-sPNPs) into tumor-bearing mice. In vivo fluorescence
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imaging indicated that from 4 to 24 h post-injection, the Cy5-sPNPs accumulated in the tumor
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region, while in the free Cy5-siRNA-treated mice, the apparent fluorescence signal almost entirely
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disappeared after 4 h (Figure 3c). Ex vivo imaging of excised tumors and major organs at 24 h
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post-injection also demonstrated a much stronger fluorescence in Cy5-sPNPs treated tumors
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compared to those treated with free Cy5-siRNA (Figure 3d&e), confirming that sPNPs were able to
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effectively deliver their cargo to the tumor tissue.
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In vivo anti-metastatic activity of sPNPs
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The anti-metastatic activity of sPNPs was investigated in a 4T1 breast tumor bearing mouse model.
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Female BALB/c mice bearing tumors (~100 mm3) were treated with sPNPs or control formulations
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every three days for 6 total treatments. Histological examination showed that sPNPs treatment
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notably inhibited the formation of metastatic nodules in the lung (Figure 4a and b; almost 80%
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inhibition compared to the saline group), while free siRNA and PNPs did not elicit any significant
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anti-metastatic effect. Further analysis of TF levels in the tumor tissues using immunoblotting and
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immunohistochemistry staining confirmed that sPNPs suppressed the expression of tumor-associated
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TF by approximately 75% compared to the other groups (Figure 4c-e). These results indicate that
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sPNPs treatment is able to efficiently knockdown tumor-associated TF and has the potential to
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significantly reduce the pulmonary metastases of breast cancer.
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Depletion of TF decreases tumor-associated blood hypercoagulable state
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Aberrant expression of TF in aggressive tumors plays a key role in the formation of a
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hypercoagulable microenvironment, which facilitates tumor cell survival, immune escape and
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metastasis.53-54 Although a strong correlation between TF overproduction in the tumor and the
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occurrence of metastasis has been reported,6,
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inhibition on tumor microenvironment coagulability. We next examined whether TF silencing by
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sPNPs could reverse tumor hypercoagulability in 4T1 breast cancer mouse model. After treating the
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animals with sPNPs or control formulations, we administered Cy5-labeled fibrinogen to visualize the
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coagulant activity in the tumor tissue. Compared with the saline, PNPs and free siRNA treated
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groups, the fibrinogen accumulation in the tumor of sPNPs-treated mice was significantly reduced,
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as evidenced by the lowest fluorescence intensity (Figure 4f), indicating a less active coagulation
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state. Quantification of tumor fluorescent signals revealed a 60% decrease of Cy5-fibrinogen in the
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sPNPs-treated mice compared to the other groups (Figure 4g). We also characterized thrombin
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generation in the plasma and tumor tissue using a thrombin-antithrombin complex (TAT) assay. The
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plasma TAT concentration of tumor bearing mice is significantly higher than normal mice, which is
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congruent with tumor-associated hypercoagulability. However, after treatment with sPNPs, the
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plasma TAT concentration of the tumor bearing mice decreased nearly to normal levels (Figure 4h).
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Moreover, the local TAT levels within the tumor tissue of sPNPs treated mice was significantly
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lower (by approximately 50%) than those of the control groups (Figure 4i). Immunostaining for the
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platelet activation marker P-selectin also indicates that sPNPs treatment effectively reduces the
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number of activated platelets within tumor tissue (Figure 4j). Altogether, these results suggest that
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TF silencing by sPNPs can effectively reverse the procoagulant state of tumor microenvironment by
30
decreasing fibrinogen accumulation, thrombin generation and platelet activation.
54-55
few studies have examined the impact of TF
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Safety evaluation of sPNPs
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We comprehensively assessed the biocompatibility of sPNPs both in vitro and in vivo. Neither PNPs
4
nor sPNPs exhibited any detectable cytotoxicity in MDA-MB-231, 4T1 or human umbilical vein
5
endothelial cells (HUVECs) even when the equivalent siRNA concentration in sPNPs reached up to
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500 nM (Figure 5a). In addition, we also investigated the immunostimulating activity of sPNPs by
7
evaluating the levels of immunostimulatory cytokines (IL-2, IFN-γ and IL-6) secreted by splenic
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cells isolated from treated mice. No significant changes were detected (Figure 5b). All these results
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are suggestive of favorable biocompatibility of sPNPs. To examine the in vivo safety of the
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nanoparticle formulation, healthy BALB/c mice were administered (i.v.) with saline, siRNA, PNPs
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or sPNPs once every other day for 5 total injections. H&E staining of the major organs and blood
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biochemical analysis revealed no overt histological change and organ function damages were
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observed (Figure 5c and Figure S9). TF is generally considered as a crucial initiator of the extrinsic
14
blood coagulation pathway.
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partial thromboplastin time (APTT) of the mice treated with sPNPs. As indicated in Figure S10,
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sPNPs treatment did not affect the blood clotting function of mice.
2-3
Therefore, we next evaluated the tail bleeding time and activated
17 18
Conclusions
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The hypercoagulable state in solid tumors has long been thought to be a crucial supporting factor for
20
the metastasis and colonization of tumor cells. The blockage of local hypercoagulability therefore
21
may represent a particularly attractive approach for anti-metastatic therapy. In our current work, we
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report a peptide-based assembly vehicle to specifically deliver TF siRNA to tumor sites and realize
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an inhibition of tumor-associated TF, which plays a central role in the formation of tumor
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hypercoagulability. With the ability to successfully target tumor tissue and knockdown tumoral TF
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expression, this nanoparticle system significantly inhibits the interactions of CTCs with platelets and
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reverses the tumor coagulant state. As an anti-metastatic agent, our system significantly inhibits
27
metastasis to the lung in a mouse breast cancer model. This nanosystem represents a possibility to
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safely and specifically knockdown tumor-associated TF for preventing metastasis and may provide
29
useful insight for the development of new oncotherapies.
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Materials and methods
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Materials
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All chemicals were purchased from Sigma-Aldrich (Shanghai, China) unless otherwise stated.
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Peptides used in this study were synthesized and purified by Top-peptide (Shanghai, China). Small
5
interfering RNAs, including FAM- and Cy5-labeled siRNAs were purchased from GenePharma
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(Shanghai, China). The anti-tissue factor monoclonal antibodies (cat. no. ab151748) and
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thrombin-antithrombin complex (TAT) ELISA kits (no. ab137994) were purchased from Abcam
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(Cambridge, MA, USA). Anti-CD62p rabbit polyclonal antibodies (cat. no. or b11315) was
9
purchased from Biorbyt (Cambridge, UK). Anti-β-actin mouse monoclonal antibody, goat
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anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP were purchased from YEASEN (Shanghai,
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China) and Santa Cruz Biotechnologies (Santa Cruz, CA). Interferon-γ (IFN-γ), interleukin-2 (IL-2)
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and interleukin-6 (IL-6) ELISA kits were purchased from eBioscience (Santa Cruz, CA).
13 14
Cell culture
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The human breast cancer cell lines MDA-MB-231 (American Type Culture Collection, ATCC,
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Manassas, VA, USA) and GFP-labeled MDA-MB-231 (National Infrastructure of Cell Line
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Resources, Beijing, China) were cultured in DMEM medium. Mouse breast cancer cell lines 4T1
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(ATCC) and human umbilical vein endothelial cells (HUVECs, ATCC) were cultured in RPMI-1640
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and F12K medium respectively. All the culture media were supplemented with 10% fetal bovine
20
serum (FBS), 100 U/mL penicillin and 100 μg/ mL streptomycin. Cells were maintained in 5% CO2
21
at 37°C.
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Preparation and characterization of nanoparticles
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For the preparation of sPNPs, 1.75 mg peptide[(C18)2-Lys-(Gly)2-(Arg-Arg-Ser)10-Lys-(His)8-cRGD]
25
dissolved in 10 μl DMSO was diluted into 1 mL DEPC water solution containing 181.5 ug siRNA
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(peptide: siRNA molar ratio = 20:1) and the mixture was ultrasonicated at 100 W for 5 min. After a 1
27
h incubation at room temperature, the solution was dialyzed against DEPC water at room
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temperature for 6 h. For the preparation of PNPs, DEPC water was used in place of the siRNA
29
solution. The zeta potential of the resultant nanoparticles was determined using a ZetaSizer Nano
30
series Nano-ZS (Malvern Instruments Ltd., Malvern, U.K.). The size and morphology of the
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nanoparticles were characterized by transmission electron microscopy (TEM) using an EM-200CX
2
microscope (JEOL Ltd., Tokyo, Japan) after negative staining with 4% uranyl acetate for 10 min.
3
The siRNA sequence used is shown below:
4
5’-GCGCUUCAGGCACUACAAATT
5
TTCGCGAAGUCCGUGAUGUUU-5’
6 7
Determination of critical micelle concentration (CMC)
8
The stability of the peptide assembly-based nanoparticles was evaluated by determining the CMC of
9
the amphipathic peptide. The peptide, at different concentrations (0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 25,
10
50, 100 or 200 μM), was incubated with pyrene (24 μg/L) for 1 h in phosphate buffered saline (PBS,
11
pH 7.4). The solutions were then analyzed with an F-4600 fluorescence spectrophotometer (Hitachi,
12
Japan) using an excitation wavelength of 334 nm. The ratio of the fluorescence intensity between
13
373 nm and 384 nm to the peptide concentration was plotted. The inflection point of the resulting
14
curve was taken as the CMC. 56
15 16
Stability evaluation of sPNPs.
17
Free siRNA or sPNPs were mixed with RNase A and incubated at 37℃ for 0, 10, 20,30,60 and 90
18
min. Then the mixed solution was incubated with gel loading buffer containing 1% SDS for 10 min
19
and detected by 1% EtBr agarose gel. For in vivo stability, BALB/c mice were injected intravenously
20
with free Cy5-siRNA or sPNPs loaded with Cy5-siRNA, respectively. Blood was collected from tail
21
veins at different time intervals and used for fluorescent imaging.
22 23
In vitro cellular uptake
24
MDA-MB-231 cells were seeded into 12-well plates at a density of 1 × 105 cells per well and
25
maintained in 5% CO2, 37°C for 24 h. The culture medium was then replaced with serum-free
26
medium (pH 7.4 or 6.8) containing sPNPs loaded with FAM-labeled siRNA. After incubating the
27
plates for varying time periods, the cells were collected, washed with PBS and resuspended in 200
28
μL PBS. The total fluorescence intensity of the cells was detected using a BD C6 flow cytometer.
29 30
In vitro imaging of tumor cells
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MDA-MB-231 cells were seeded onto 35 mm chambered, borosilicate cover glasses (Nunc, USA).
2
The cells were treated with sPNPs loaded with FAM-siRNA for different time periods. We then
3
stained the cells with LysoTracker Red to visualize lysosomes, according to the manufacturer's
4
instructions, for 2 h, washed the samples three times with PBS and observed the cells with an LSM
5
710 confocal microscope (Carl Zeiss, USA).
6 7
Immunoblotting
8
Total protein-containing lysates from cell and tissue samples were extracted using a RIPA Lysis and
9
Extraction Buffer (Solarbio) and quantified using a BCA Protein Assay Kit (Thermo Scientific)
10
according to the manufacturers’ protocols. The extracted protein was then incubated at 100°C in
11
SDS-PAGE loading buffer for 5 min and resolved by SDS-PAGE electrophoresis. After transferring
12
the proteins to a PVDF transfer membrane (Merck Millipore, Germany) using a Trans-Blot Transfer
13
System (BIO-RAD, US), the membrane was blocked with 5% non-fat milk in Tris buffered saline
14
tween (TBST) for 1 h. Then the membrane was incubated with the appropriate antibodies for 2 h
15
under room temperature and washed the membrane three times (10 min per time) using TBST buffer.
16
Next, the membrane was incubated with corresponding secondary antibodies for an hour under room
17
temperature and washed again for three times as mentioned above. Signals were visualized using a
18
ChemiDoc Touch Imaging System (BIO-RAD, US).
19 20
Cell migration assay
21
MDA-MB-231 tumor cells were cultured for 24 h in serum-free medium to achieve a steady-state.
22
The cells were then plated onto 8 μm-pore transwell filters (2 × 104 cells per well maintained in
23
serum-free medium) that were placed in a 12-well plate filled with medium containing 10% FBS.
24
After a 12 h incubation, the cells were washed three times with serum-free medium and fixed with 4%
25
formaldehyde for 30 min. The transwell filters were washed three times with PBS and cells on the
26
upper side of the polycarbonate membrane were scraped off before the filter was stained in 0.1%
27
crystal violet. The number of cells that migrated to the bottom chamber over 12 h was counted under
28
an EVOS inverted microscope.
29 30
Isolation of platelets from volunteer blood
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Whole blood from healthy volunteers was collected in vacuum blood collection tubes and
2
centrifuged for 10 min at 180 × g at room temperature to obtain platelet-rich plasma (PRP). The PRP
3
was then centrifuged at 1200 × g for another 10 min to collect a platelet pellet. The pellet was
4
resuspended and washed three times in CGS buffer (13 mM trisodium citrate, 30 mM dextrose and
5
120 mM NaCl, pH 7.0) in the present of 1 μg/mL PGE1. Washed platelets were resuspended in
6
Tyrode’s buffer (containing 0.3% BSA) at a concentration of 1 × 109 platelets per mL and stained
7
with the membrane dye DIL.
8 9
In vitro platelet adhesion assay
10
MDA-MB-231 cells were seeded in 96-well plates (black plate with clear bottom; Corning, NY) at a
11
concentration of 4 × 104 cells per 100 μL per well and grown until confluent. The cells were washed
12
three times with serum-free medium. Washed platelets (3 × 108 per mL) preloaded with DIL as
13
described above were allowed to adhere to the cells for 60 min at 37°C in the presence of 10%
14
human plasma. The wells were imaged by a fluorescence imaging system (CRi, Woburn, MA) using
15
excitation and emission wavelengths of 553 nm and 570 nm, respectively. To remove the
16
non-adhered platelets, the cells were then washed three times with PBS and supplied with 100 μL
17
serum-free medium and imaged again. The fluorescence intensity of well containing 100 μL medium
18
with 10% human plasma or serum-free medium were used as the respective blank samples. The
19
percentage of adhered platelets was calculated as (fluorescence after PBS wash -
20
blank)/(fluorescence before PBS wash-blank) × 100%.
21 22
To further visualize the interaction between tumor cells and platelets, GFP-labeled MDA-MB-231
23
cells were seeded in 35-mm confocal dishes and grown until confluent. DIL-stained platelets (3 × 108
24
per mL) were added into the dish and the samples were incubated for 60 min at 37°C in the presence
25
of 10% human plasma. The cells were then washed three times with PBS, fixed with 4%
26
formaldehyde and imaged using an LSM 710 confocal microscope (Carl Zeiss, USA).
27 28
In vivo platelet adhesion assay
29
Female BALB/c nude mice were injected with 5 × 105 GFP-labeled MDA-MB-231 cells (untreated
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or treated with sPNPs for 48 h) and 5 × 109 DIL-stained platelets isolated from human blood
2
(separately into opposite lateral tail veins). After 1, 4 and 24 h, the lungs were excised and
3
freeze-sectioned and the sections were imaged using a confocal microscope.
4 5
In vivo pulmonary colonization of tumor cells
6
Female BALB/c nude mice were intravenously injected with 5 × 105 MDA-MB-231 cells either
7
untreated or pre-treated with sPNPs for 48 h. Female BALB/c mice were intravenously injected with
8
2 × 105 4T1 cells either untreated or pre-treated with sPNPs for 48 h. For the MDA-MB-231 group,
9
the lungs were excised five weeks after injection and sectioned for histological examination for
10
pulmonary metastasis foci. For the 4T1 group, the lungs were excised after two weeks and sectioned
11
for histological examination.
12 13
In vivo imaging and biodistribution
14
About 1 × 106 4T1 mouse breast cancer cells (suspended in 50 μL PBS) mixed with 50 μL matrigel
15
were transplanted into the second mammary fat pads of female BALB/c nude mice; tumors were
16
allowed to grow to about 100 mm3. The mice were then intravenously injected with saline, free
17
Cy5-labeled negative control siRNA (33 g per mouse) or sPNPs (loaded with Cy5-labeled negative
18
control siRNA, 33 g per mouse). At different time points post administration, the mice were imaged
19
using an ex/in vivo optical imaging system (Maestro, CRi, Woburn, MA) to visualize the Cy5
20
fluorescence. To examine tissue biodistribution, the mice were sacrificed 24 h post-injection, the
21
tumor and major organs were excised and imaged.
22 23
In vivo anti-metastasis experiments
24
Mice bearing 4T1 tumors (about 100 mm3) were intravenously injected with saline, siRNA (1.5
25
mg/kg), PNPs (15 mg/kg peptide) or sPNPs (containing 1.5 mg/kg siRNA and 15 mg/kg peptide)
26
every three days for a total of 6 injections. After the final treatment the mice were sacrificed and the
27
tumors were excised for immunoblotting and immunohistochemistry analysis of TF expression. The
28
lungs were excised and sectioned for histological examination using H&E staining.
29 30
Tumor coagulation state assay
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Mice bearing 4T1 tumors (about 100 mm3) were intravenously injected with saline, siRNA (1.5
2
mg/kg), PNPs (15 mg/kg peptide) or sPNPs (containing 1.5 mg/kg siRNA and 15 mg/kg peptide)
3
every three days for a total of 6 injections. After the final treatment the mice were intravenously
4
injected with Cy5-labeled fibrinogen (0.5 mg per mouse). An hour later, the tumors were excised for
5
fluorescence imaging.
6 7
TAT concentration assay
8
After treatment, whole blood was collected using EDTA coated tubes and centrifuged for 10 min at
9
850 xg under room temperature to obtain the plasma. The total protein from tumor tissue was
10
collected as described above. The TAT concentration in the plasma and tumor tissue was measured
11
using a TAT ELISA kit according to the manufacturer's protocols.
12 13
Cytotoxicity assay
14
MDA-MB-231 cells, 4T1 cells or HUVECs were maintained in 96-well plates (1 × 104 per well) at
15
37°C for 24 h. The cells were then treated with PNPs, siRNA (500 nM), or sPNPs formulations
16
containing different concentrations of siRNA for another 24 h. Cell viability was determined using a
17
CCK-8 kit.
18 19
Immunogenicity test
20
Splenic cells isolated from BALB/c mice were cultured in 12-well plates and stimulated with PNPs,
21
siRNA (200 nM) or sPNPs (containing 200 nM siRNA) for 24 h. The medium was then replaced
22
with fresh medium containing 1% FBS, and 12 h later, the conditioned medium was collected and
23
the concentrations of interferon-gamma, interleukin 2 and interleukin 6 were determining using
24
ELISA kits.
25 26
Blood clotting hemostatic function evaluation.
27
BALB/c mice were randomly divided into 4 groups and intravenously injected with saline, siRNA
28
(1.5 mg/kg), PNPs (15 mg/kg peptide) or sPNPs (15 mg/kg peptide and 1.5 mg/kg siRNA) every the
29
other day for 5 total treatments. Two more days after the last treatment, the mice were anesthetized
30
with sodium pentobarbital and the tail were cut about one millimeter from the tip side. Then the tail
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was quickly inserted into the 37 ℃ PBS solution and the bleeding time was recorded. Then the
2
plasmas of the mice were collected and the activated partial thromboplastin time (APTT) was
3
detected using a semi-automatic coagulation analyzer (LG-PABER-I, Taizhou, China).
4 5
Biosafety assessment
6
BALB/c mice were randomly divided into 4 groups and intravenously injected with saline, siRNA
7
(1.5 mg/kg), PNPs (15 mg/kg peptide) or sPNPs (15 mg/kg peptide and 1.5 mg/kg siRNA) every
8
other day for 5 total treatments. After the last treatment, blood and the major organs were collected
9
for serum biochemical examination and tissue histological examination (H&E staining).
10 11
Statistical analysis. Data were examined statistically using the SPSS17.0 statistical analysis
12
software. Student t-test or one-way ANOVA followed by Tukey’s HSD test was applied for
13
comparison between two groups or among multiple groups, respectively.
14 15
ASSOCIATED CONTENT AVAILABLE: Supporting Information available. [Hydrodynamic
16
diameter and surface charge of PNPs and sPNPs, chemical structure of peptide complex, critical
17
micelle concentration of the peptide complex, loading efficiency of siRNA, the protective effects of
18
PNPs on siRNA, cellular uptake of sPNPs at different pH conditions, intracellular distribution and
19
lysosomal escape of sPNPs-delivered siRNA, cell arrest in the lung, histological analysis of lungs,
20
biocompatibility of sPNPs, blood clotting hemostatic function evaluation.]
21 22
Acknowledgements: This work was supported by the National Key R&D Program of China (Grant
23
No. 2018YFA0208900) , the National Natural Science Foundation of China (Grant Nos. 31730032,
24
31661130152, 31471035, 51673051, 81630068, 21877023, and 51861145302), a Key Research
25
Project of Frontier Science of the Chinese Academy of Sciences (Grant No. QYZDJ‐SSW‐SLH022),
26
Beijing Nova Program (Grant No. Z171100001117010), Beijing Natural Science Foundation (Grant
27
No. 7172164), National Postdoctoral Program for Innovative Talents (BX20180083) and Youth
28
Innovation Promotion Association CAS (Grant No. 2017056) and K. C. Wong Education Foundation
29
(GJTD-2018-03).
30
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Figure 1. Design and characterization of pH-sensitive sPNPs. (a) Schematic self-assembly of the
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nanoparticles. (b) The proposed antitumor mechanism of sPNPs nanoparticle. Upon penetration into
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the tumor microenvironment, the sPNPs nanoparticle in response to the acidic tumor environment
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and allow sPNPs to possess a positive surface charge; at the same time, the cRGD motif on the
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surface of sPNPs will recognize the integrin αvβ3. Both of these conditions promote cell uptake of
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sPNPs. Under the acidic conditions in lysosomes, polyhistidine sequence undergoes a protonation
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effect, depolymerizing the nanoparticles and releasing siRNA. (c) TEM images of PNPs and sPNPs.
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Scale bar: 200 nm. (d) TEM images of sPNPs at pH 7.4, 6.8 and 5.5. Scale bars, 200 nm (above) and
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50 nm (below).
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Figure 2. In vitro functional characterization of sPNPs. (a&b) TF expression of MBA-MB-231
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cells after two successive treatments (24 h each) with sPNPs and controls (NC indicates a
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non-functional control siRNA). The cells were harvested 24 h after the second treatment, at which
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time the TF expression was analyzed by immunoblotting (a) and quantified using Image J (b).
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β-actin was used as the loading control. (c&d) Migration ability of MDA-MB-231 cells after
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treatment with free siRNA, PNPs or sPNPs, as analyzed by transwell assay. The number of cells
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migrating across the transwell filter over 12 h was estimated using crystal violet staining
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photographed by an EVOS inverted microscope. Scale bars: 100 μm, *** p < 0.001. (e) Percentage
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of platelets adhered to tumor cells after 1 h co-incubation of sPNPs-treated tumoral cells with
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DIL-labeled platelets; quantification of DIL fluorescence. (f) Adhesion of DIL-labeled platelets (red)
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to GFP-labeled MDA-MB-231 cells (green) treated with PNPs or sPNPs; images acquired by
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confocal microscopy. The tumor cell nuclei were stained with Hoechst nuclear dye. Scale bars: 50
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μm. (g-i) Platelet adhesion to tumor cells in vivo. GFP-labeled MDA-MB-231 cells (5 × 105 cells per
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mouse) were intravenously co-injected with DIL-labeled platelets into BALB/c nude mice. After 1 h
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(g), 4 h (h) and 24 h (i), the lungs were excised and sectioned for confocal microscopy analysis.
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Scale bars: 20 μm. To quantify the proportion of cells with platelet adhesion and the area of adhered
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platelet aggregates, 15 tumor cells were selected randomly from each lung.
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Figure 3. Cell binding and In vivo tumor targeting of sPNPs. (a) MDA-MB-231 cells were
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pre-treated with cRGD peptide for 1 h. Then the cells were treated with sPNPs (containing
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FAM-siRNA) in serum-free medium for 1, 2 and 3 h respectively and analyzed using flow
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cytometry. Flow cytometry showed that pre-treated with cRGD peptide significantly inhibited the
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cellular uptake of sPNPs. (b) The fluorescence intensity in a was quantified. ** p < 0.01; *** p