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Dual-functional nanoparticles targeting CXCR4 and delivering anti-angiogenic siRNA ameliorate liver fibrosis Chun-Hung Liu, Kun-Ming Chan, Tsaiyu Chiang, Jia-Yu Liu, GuannGen Chern, Fu-Fei Hsu, Yu-Hsuan Wu, Ya-Chi Liu, and Yunching Chen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00913 • Publication Date (Web): 25 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016

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Molecular Pharmaceutics

Dual-functional nanoparticles targeting CXCR4 and delivering anti-angiogenic siRNA ameliorate liver fibrosis Chun-Hung Liu1,#, Kun-Ming Chan2,#, Tsaiyu Chiang1,#, Jia-Yu Liu1, , Guann-Gen Chern1, Fu-Fei Hsu3, Yu-Hsuan Wu2, Ya-Chi Liu1, Yunching Chen1,* #

These authors contributed equally to this work.

1 Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013,

Taiwan, ROC 2 Chang Gung Transplantation Institute, Chang Gung Memorial Hospital at Linkou,

Chang Gung University College of Medicine, Taoyuan, Taiwan 3 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

* Corresponding author: Yunching Chen, PhD, Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.; phone: 886-3-5715131 ext: 35503; email: [email protected] Keywords: siRNA delivery; Liver fibrosis, CXCR4/SDF-1α, VEGF, Nanoparticle, Angiogenesis.

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Abstract The progression of liver fibrosis, an intrinsic response to chronic liver injury, is associated with hepatic hypoxia, angiogenesis, abnormal inflammation, and significant matrix deposition, leading to the development of cirrhosis and hepatocellular carcinoma (HCC). Due to the complex pathogenesis of liver fibrosis, anti-fibrotic drug development has faced the challenge of efficiently and specifically targeting

multiple

pathogenic

mechanisms.

Therefore,

CXCR4-targeted

nanoparticles (NP) were formulated to deliver siRNAs against vascular endothelial growth factor (VEGF) into fibrotic livers to block angiogenesis during the progression of liver fibrosis. AMD3100, a CXCR4 antagonist that was incorporated into the NPs, served dual functions: it acted as a targeting moiety and suppressed the progression of fibrosis by inhibiting the proliferation and activation of hepatic stellate cells (HSCs). We demonstrated that CXCR4-targeted NPs could deliver VEGF siRNAs to fibrotic livers, decrease VEGF expression, suppress angiogenesis and normalize the distorted vessels in the fibrotic livers in the carbon tetrachloride (CCl4)-induced mouse model. Moreover, blocking SDF-1α/CXCR4 by CXCR4-targeted NPs in combination with VEGF siRNA significantly prevented the progression of liver fibrosis in CCl4-treated mice. In conclusion, the multifunctional CXCR4-targeted NPs delivering VEGF siRNAs provide an effective anti-fibrotic therapeutic strategy. Keywords siRNA delivery; Liver fibrosis, CXCR4/SDF-1α, VEGF, Nanoparticle, Angiogenesis.


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Introduction The pathogenesis of liver fibrosis involves abnormal inflammatory wound healing, significant matrix deposition and angiogenesis. With protracted damage, advanced liver fibrosis leads to cirrhosis, loss of liver function, portal hypertension, and ultimately, hepatocellular carcinoma (HCC)1-3. Due to a lack of symptoms in earlyonset liver fibrosis and to limited therapeutic options, liver transplantation is the only clinical treatment for the irreversible liver failure resulting from liver cirrhosis. Thus, there is an urgent clinical need to develop an effective anti-fibrotic treatment to attenuate disease progression and even reverse the fibrotic process 4-8. Therapeutic strategies that can suppress angiogenesis and inhibit hepatic stellate cell (HSC) activation, which play essential roles in liver fibrosis progression, may restore the normal structure and function of the liver and serve as potential approaches for treating liver fibrosis 9, 10. HSC activation and differentiation toward the myofibroblastic lineage during liver injury contribute to the progression of liver fibrosis by increasing extracellular matrix (ECM) deposition and inflammatory cytokine secretion and by stimulating angiogenesis 11-14. Many cytokines and growth factors are actively involved in the process of HSC activation and in the interactions between HSCs and hepatocytes, endothelial cells and other bone marrow-derived cells (BMDCs) in the fibrotic liver 15. According to recent reports, the SDF-1á/CXCR4 axis is upregulated in cirrhotic livers, and activation of the SDF-1á/CXCR4 axis promotes HSC activation and proliferation 16, 17. The blockade of SDF-1á/CXCR4 signaling suppresses HSC activation and proliferation and thus may be a potential treatment option for patients with liver cirrhosis 4.

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Additionally, the secretion of vascular endothelial growth factor (VEGF) by activated HSCs facilitates angiogenesis and progressive fibrogenesis in the fibrotic liver, resulting in abnormal angioarchitecture

18, 19.

Given that the strong interplay

between angiogenesis and fibrosis significantly contributes to the formation and progression of liver fibrosis, we developed a therapeutic strategy to target both angiogenesis and fibrosis via a multifunctional nanoparticle (NP) that co-formulates AMD3100 blocking CXCR4 and small interfering RNA (siRNA) targeting VEGF. siRNAs are double-stranded RNAs containing 19–24 base pairs to efficiently decrease expression of targeted genes with low off-target effects and little unwanted toxicity 20. Accordingly, siRNAs can be developed as safe and effective therapeutic agents. In this study, NPs with the CXCR4 antagonist AMD3100 attached to their surface can deliver therapeutic siRNAs to activated CXCR4-overexpressing HSCs in vitro and to fibrotic livers in vivo at high efficiency. We demonstrated that VEGF siRNAs delivered by AMD3100-modified NPs downregulated the expression of VEGF, reduced the mean vessel density (MVD) and normalized the hepatic vascular structure in the livers of mice with CCl4-induced liver fibrosis. Furthermore, AMD3100 encapsulated in NPs also exhibited anti-fibrotic effects by suppressing the proliferation and activation of HSCs. Thus, blocking SDF-1α/CXCR4 axis activation and VEGF expression via CXCR4-targeted NPs loaded with VEGF siRNA in the fibrotic liver achieved significant anti-fibrotic effects. In conclusion, our multifunctional CXCR4-taretegd NPs that deliver AMD3100 and siRNAs against VEGF may represent a new therapeutic avenue to treat liver fibrosis.

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Experimental Section Materials AMD3100 octahydrochloride hydrate, carbon tetrachloride (CCl4), calf thymus DNA, protamine sulfate salt, siRNAs and olive oil were obtained from Sigma-Aldrich (St Louis, MO) (Table S1). Cholesterol, 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), l,2-dioleoyl-sn-glycero-3- phosphate (DOPA) and 1,2-distearoyl-sn-glycero3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG-2000) were obtained from Avanti Polar Lipids (Alabaster, AL). VEGF siRNA was labeled with FAM at the 5′ end of the sense strand for tracking cellular and tumor uptake. Recombinant SDF-1α was obtained from ProSpec TechnoGene (Rehovot, Israel). Cell culture HSCs were purchased from ScienCell Research Laboratories (San Diego, CA) and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and the antibiotics penicillin and streptomycin (HyClone, Logan, UT). HSCs were further culture-activated on plastic dishes for 7 days. Animals Five-week-old male C3H/HeNCrNarl mice were obtained from the National Laboratory Animal Center (Taipei, Taiwan). To induce liver fibrosis in mice, the mice were administered with 16% CCl4 /olive oil (2 mL/kg, P.O.) by gavage three times per week for 7 weeks. All the animals used in this study received humane care in compliance with institutional animal care guidelines published by the National Academy of Sciences.

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For the in vivo gene silencing studies, we intravenously injected siRNAs formulated in the different NPs into mice with CCl4-induced liver fibrosis (siRNA at 1.5mg/kg, daily). We examined the liver tissue sample followed by three continuous dosing. For evaluation of anti-fibrotic response, we intravenously injected siRNAs formulated in the different NPs into mice with CCl4-induced liver fibrosis (siRNA at 1.5mg/kg, two doses per week) beginning 4 weeks after the start of CCl4 administration, and the changes in hepatic fibrosis and vascularization were evaluated after 3 weeks of treatment (Fig. S1). Patients and liver tissue Human liver tissue was obtained through wedge liver biopsies of patients who had undergone liver resection at the department of general surgery at the Chang Gung Memorial Hospital at Linkou, Taiwan. All study procedures and protocols were approved by the Institutional Review Boards of Chang Gung Memorial Hospital (993855B). Written informed consent was signed by all patients prior to the operation, and 40 patients were enrolled between December 2011 and December 2013. Of those, 24 patients were with liver cirrhosis, and 16 patients had normal liver without cirrhosis. Immunohistochemical stains for expression of VEGF and CXCR4 were performed on paraffin-embedded sections of the liver specimen as the manufacture’s protocol. The expression intensity was analyzed by MetaMorph software (Sunny Vale, CA, USA). Production and characterization of CXCR4-targeted NPs formulated with siRNAs Nanoparticles were prepared as previously described with modifications 21-25. Briefly,

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the core complex of NPs was formulated by mixing protamine (16 μl, 2 mg/ml), AMD3100 (18 μl, 2 mg/ml), calf thymus DNA (10 μl, 2 mg/ml) and siRNA (12 μl, 3 mg/ml) in 120 μl of double-distilled water and was incubated for 5 minutes. Next, liposomes composed of cholesterol, DOPA and DOPC (1:2:1 molar ratio) were mixed with the core complex and incubated for 10 minutes. To enhance NP internalization in activated CXCR4-expressing HSCs, 30 µl of AMD3100 (2 mg/ml) in phosphatebuffered saline (PBS) was added into the solution and incubated at RT for 5 min. To prolong the blood circulation time, the DSPE-PEG-2000 (20 μl, 10 mg/ml) was postinserted into the formulation, resulting in AMD-NPs. We observed the geometry of the NPs by the transmission electron microscope (TEM) in Microscopy Center at Chang Gung University (Taipei, Taiwan). The colloid solution (10 μL) was dropped onto the polished silicon wafer and dried at RT. We obtained the images using a Hitachi H-7500 TEM operated at 75 kV. To measure the size and zeta potential of NPs, NPs were resuspended in doubledistilled water with a pH of 7, and the physicochemical properties were analyzed using an electrophoretic light scattering spectrometer (Zetasizer 3000HS, Malvern Instruments Ltd., Worcestershire, UK). Cellular viability assays MTT assay was used for detection of cellular viability. HSCs (2,000 cells/well) were seeded in 96-well plates. Twelve hours after cell seeding, HSCs were incubated in serum-free medium and were treated with different formulations. Forty-eight hours after treatments, MTT (0.5 mg/ml) was added to HSCs, followed by incubation for 3 hours at 37°C. The medium was aspirated, and 50 µl of dimethylsulfoxide (DMSO)

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was added to each well. The cell viability was evaluated by measuring the absorbance at 570 nm using a Multiskan reader (Thermo, USA). Immunofluorescence Ten µm liver tissue sections were fixed in ice-cold acetone, rinsed with PBS, and blocked with 5% BSA. The liver tissue sections were then incubated with antiCXCR4, anti-VEGF-A, anti-collagen I (Abcam, Cambridge, MA) and anti-von Willebrand Factor (vWF; Dako, Denmark) antibodies (1:100 dilution). After overnight incubation, the sections were then rinsed with PBS and incubated for 1 h with Alexa Fluor® 488 conjugated anti-rabbit IgG- antibodies (Life Technologies, Grand Island, NY) (1:200 dilution). The sections were counterstained with 4'6diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA), and immunofluorescence images were captured using a laser scanning confocal microscope (LSM 780, Zeiss, Germany). Protein expression was quantified by measuring the area occupied by the staining of interest normalized by the area of DAPI-stained nuclei as the ratio of green/blue (Alexa Fluor 488/DAPI) relative fluorescence units. The fluorescence intensity was analyzed using Image J software. Identical analysis settings and thresholds were applied for all liver sections. Vessel diameters were measured with confocal line scans across the blood vessels (n=150~200). Cellular uptake study HSCs (1 × 105 cells per well) were seeded into 12-well plates and exposed to FAMsiRNAs formulated in various NPs. The cells were rinsed with PBS and fixed in 4% paraformaldehyde. The nuclei were stained with DAPI. The siRNA uptake in HSCs

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was examined with a confocal microscope (Zeiss LSM 780, Carl Zeiss, Oberkochen, Germany). To examine the competitive siRNA uptake, HSCs were pretreated with free AMD3100 at different concentrations for 10 mins. Next, the HSCs were further treated with FAM-siRNAs encapsulated in AMD-NPs. The reduction in siRNA uptake in HSCs was observed using a confocal microscope. Liver uptake study Healthy C3H mice or CCl4-treated C3H mice with fibrotic livers were administered with NPs loaded with FAM-siRNAs (1.5 mg/kg). Four hours after injection, the liver tissues were obtained. To evaluate the siRNA uptake in the fibrotic livers, ten μm frozen liver sections were fixed in ice-cold acetone, rinsed with PBS, and counterstained with DAPI. The images were captured using a laser scanning confocal microscope (LSM 780, Zeiss, Germany).

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Results

CXCR4 expression is increased in the liver after fibrotic induction We previously demonstrated that SDF-1α expression is elevated both in HCC and in cirrhotic liver tissues of HCC patients 17. In this study, we further asked whether the expression of CXCR4, a receptor specific for SDF-1α, is also increased in the liver tissues of patients with cirrhosis. The expression of CXCR4 and of VEGF, an angiogenic marker, was nearly undetectable in normal livers but markedly increased in the parenchyma of cirrhotic livers. Specifically, the expression of both CXCR4 and VEGF was elevated in the early cirrhotic liver as well as in the severe cirrhotic liver (Fig. 1), indicating that CXCR4 and VEGF expression increases with the progression of liver fibrosis. To establish a murine model of liver fibrosis, we treated mice with CCl4 for 7 weeks. CXCR4 expression was elevated 4- to 15-fold, accompanied by increased collagen I, α-SMA and VEGF expression, in response to CCl4 treatment during progressive fibrosis (Fig. 2A-E). The CXCR4 staining pattern in fibrotic livers was consistent with collagen I and α-SMA expression around the fibrous septa, indicating the correlation between increased SDF-1α/CXCR4 axis activation and fibrosis progression. Inhibition of the SDF-1α/CXCR4 axis by free-form AMD3100 or AMD3100loaded NPs suppresses the proliferation and activation of HSCs To determine the role of the SDF-1α/CXCR4 axis in regulating the proliferation and activation of HSCs, we first exposed human HSCs to recombinant SDF-1α. Consistent with previous finding, we found that SDF-1α enhanced the activation of ERK and

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AKT, increased the viability of HSCs and increased the expression of α-SMA and collagen I, which are markers of HSC activation (Fig. 3A-C). We further demonstrated that SDF-1α activates NF-kB and the pro-fibrotic effect of SDF-1α was mediated through NF-kB activation (Fig. S2). Moreover, CXCR4 inhibition by freeform AMD3100, a CXCR4 antagonist, prevented the effects of SDF-1α on both the proliferation and activation of HSCs (Fig. 3A-B). Next, we evaluated the effect of AMD3100 encapsulated in the NPs (AMD-NPs) on proliferation and differentiation of HSCs. Similar to free-form AMD3100, AMD-NPs reversed the pro-proliferative response mediated by SDF-1α in HSCs (Fig. 3A). In addition, CXCR4 inhibition by AMD3100 in NPs prevented the effects of SDF-1α on HSC activation, indicated by reduced α-SMA and collagen I expression accompanied by decreased activation of Akt and MAPK in HSCs (Fig. 3B-C). AMD-NPs deliver siRNAs and achieve profound downregulation of the targeted gene in HSCs in vitro and in the fibrotic liver in vivo In order to suppress both fibrogenesis and angiogenesis in the fibrotic liver, we load siRNAs against VEGF in AMD-NPs with the structure shown in Fig 4A as previously described with modifications21-25. A condensed core complex was composed when mixing the calf thymus DNA, protamine and AMD3100. In this formulation, the calf thymus DNA was used as a carrier DNA to encapsulate siRNAs and AMD3100 in the condensed core with presence of protamine. The core was further encapsulated in the negatively charged liposome containing DOPA. NPs wrapped by anionic liposomes showed an average zeta-potential value of -52.9 ± 1.5 at pH 7.4. To deliver siRNAs into activated CXCR4-expressing HSCs, the negatively charged surfaces of the

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NPs were further coated with AMD3100 with a positive charge via charge-charge interaction to assemble CXCR4-targeted NPs. Accordingly, AMD3100 could serve in both blocking CXCR4 and targeting siRNA delivery. To improve blood circulation and biodistribution, polyethylene glycol (PEG) was post -inserted into the lipid bilayer to modify the surface of AMD3100-coated NPs. The PEG-modified AMD-NPs were spherical in shape as indicated by the TEM image (Fig. 4A). The size and the surface charge of siRNA loaded AMD-NPs were determined by Zetasizer, which showed an average diameter of 81.62 ± 20.26, with a PDI of 0.312±0.058 and an average zetapotential value of -14.7 ± 0.9 at pH 7.4 (Fig. S3). The encapsulation efficiencies of AMD3100 and siRNA in NPs were around 80% and 90%, respectively. As shown in Figure 4B and C, the FAM-siRNA delivered by AMD-NPs achieved higher uptake in HSCs than that delivered by NPs without modification of AMD3100. Free AMD3100 competitively inhibited the siRNA uptake mediated by AMD-NPs in a dose-dependent manner, indicating that the cellular uptake of siRNAs delivered by AMD-NPs involved ligand (AMD3100)-dependent internalization (Fig. 4D). Furthermore, delivery of VEGF siRNAs mediated by AMD-NPs knocked down the expression of VEGF in HSCs (Fig. 4E). However, VEGF expression remained unchanged while siRNAs were delivered by NPs without modification of AMD3100 (Fig. 4E). The data indicated that the CXCR4-targeted NPs could deliver siRNAs and suppressed the targeted gene expression in CXCR4-expressing HSCs in vitro. For the in vivo evaluation, we first examined the tissue distribution of siRNA-loaded NPs in mice with CCl4-induced liver fibrosis after systemic administration of FAMsiRNAs in different formulations. In Figure 5A-C, AMD-NPs delivered siRNAs into

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the fibrotic livers more efficiently compared with non-targeted NPs or free siRNAs. The distribution of siRNA delivered by AMD-NPs in the fibrotic liver was heterogeneous. Robust delivery of siRNAs by AMD-NPs occurred close to the vasculature and fibrotic region. In addition, strong colocalization of FAM-labeled siRNAs with α-SMA+ myofibroblast was observed (Fig. 5D). Furthermore, VEGF siRNAs delivered into the fibrotic liver via AMD-NPs significantly decreased VEGF expression in the fibrotic livers of CCl4-treated mice (Fig. 5E-G). VEGF siRNAs loaded in non-targeted NPs induced partial downregulation of VEGF expression in fibrotic livers (Fig. 5E-G). However, VEGF expression remained unchanged in the fibrotic liver tissues of mice treated with AMD-NPs containing control siRNAs or free VEGF siRNAs (Fig. 5E-G). Thus, AMD-NPs delivered VEGF siRNAs and silenced VEGF expression efficiently in the fibrotic liver in vivo. Moreover, both in vitro and in vivo studies indicated that the delivery and gene silencing effect of the siRNA-loaded AMD-NPs were ligand (AMD3100) dependent. VEGF siRNAs delivered by AMD-NPs suppressed angiogenesis in livers of mice with CCl4-induced liver fibrosis. Angiogenesis and distortion of the vascular architecture and function have occurred in association with the progression of liver fibrosis 2, 26. Given that VEGF plays an important role in regulating fibrosis-associated angiogenesis in the liver, we further examined the anti-angiogenic effect of VEGF siRNA-loaded AMD-NPs in the CCl4induced mouse model of liver fibrosis. VEGF siRNAs loaded in different NPs were intravenously administered to mice with CCl4-induced liver fibrosis (1.5 mg/kg, 2 doses per week) beginning 4 weeks after the start of CCl4 administration, and the

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changes in hepatic vascularization were evaluated after 3 weeks of treatment (Fig. S1). The immunostaining of blood vessels revealed that VEGF siRNAs delivered by AMD-NPs significantly reduced the mean vessel density (MVD) in fibrotic livers compared with control siRNA and free VEGF siRNA treatments (Fig. 6A and C). The systemic delivery of VEGF siRNAs via non-targeted NPs induced a mild reduction in the MVD in fibrotic livers (Fig. 6A and C). Moreover, as indicated by the reduced mean vessel diameters and carbonic anhydrase 9 (CAIX) expression – an indicator of hypoxia, VEGF siRNA encapsulated in AMD-NPs normalized vascular structure of livers and alleviated hepatic hypoxia efficiently in mice with CCl4-induced liver fibrosis (Fig. 6B-E). Thus, VEGF siRNAs in AMD-NPs suppressed angiogenesis and normalized the distorted vessels in the fibrotic livers of CCl4-treated mice. VEGF siRNAs delivered by AMD-NPs ameliorated liver fibrosis in the CCl4induced murine model of liver fibrosis Finally, we examined whether VEGF siRNA-loaded AMD-NPs reduced liver fibrosis in the CCl4-treated mouse model. H&E, Masson’s trichrome and collagen I staining indicated that VEGF siRNA-loaded AMD-NPs significantly suppressed liver fibrosis (Fig. 7A-B). In addition, we observed decreased α-SMA+ myofibroblast infiltration in the livers of CCl4-treated mice after administration with VEGF siRNA loaded in AMD-NPs compared with that in the livers of control-treated mice (Fig. 7A and CD). AMD-NPs containing control siRNAs showed a moderate therapeutic effect on liver fibrosis, indicating that AMD3100 attached to the NPs could block SDF1α/CXCR4, leading to reduced HSC activation and enhanced anti-fibrotic effects in the CCl4-induced mouse model of liver fibrosis. α-SMA and collagen I expression

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remained unchanged in mice that were treated with VEGF siRNAs in NPs without modification of AMD3100. Our results demonstrated that VEGF siRNAs loaded in CXCR4-targeted NPs efficiently blocked angiogenesis, inhibited HSC activation and decreased collagen deposition in fibrotic livers, leading to the amelioration of liver fibrosis. The inhibition of the SDF-1α/CXCR4 axis and VEGF expression in the fibrotic livers achieved by a single formulation synergistically suppressed the progression of liver fibrosis in the CCl4-induced mouse model of liver fibrosis (Fig. 8). To this end, AMD3100 and VEGF siRNAs co-delivered by the multi-functional NPs (VEGF siRNAloaded AMD-NPs) may serve as potent anti-fibrotic agents, which may supplant liver transplantation as a clinical treatment option. Discussion Given that hepatic fibrogenesis is a complicated process associated with the release of various cytokines and with inflammation, ECM deposition and angiogenesis, a combination therapy targeting multiple properties will be an effective treatment for liver fibrosis. However, the major obstacles to a combination therapy are the lack of potent inhibitors to specifically suppress the molecules regulating the progression of fibrosis and the increase in unwanted toxicity that accompanies multiple drug effects. Thus, we developed a dual-functional nanoparticle that co-delivers a CXCR4 antagonist and siRNA against VEGF into fibrotic livers to suppress angiogenesis and yield enhanced anti-fibrotic efficacy without adding severe side effects. Here, we demonstrated that chronic liver injury increased the activity of the SDF1α/CXCR4 axis, which mediates the activation of downstream molecules such as

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MAPK and Akt and thus promotes fibrogenesis. A previous study reported that FGFR1 signaling activation increased CXCR4 expression in liver sinusoidal endothelial cells and accelerated the pro-fibrotic transition of the angiocrine response during chronic liver injury 27. Moreover, SDF-1α directly stimulated the proliferation and myofibroblast differentiation of CXCR4-expressing HSCs, resulting in pro-fibrogenic activation

16.

In our study, we detected constitutive CXCR4

overexpression in the fibrotic livers of patients with cirrhosis and of mice treated with CCl4; accordingly, CXCR4 could serve as a therapeutic target. Nanoparticles coated with AMD3100, a CXCR4 antagonist, efficiently suppressed HSC activation in vitro and attenuated liver fibrosis in CCl4-treated mice. Angiogenesis, which is induced by the hypoxic microenvironment, is a key feature involved in the development of liver fibrosis. Hepatic angiogenesis and fibrosis are highly correlated in both clinical and experimental settings 28. Among the angiogenic factors, VEGF, which is elevated in response to chronic liver injury, plays a key role in regulating the angiogenesis and sinusoidal remodeling, resulting in immature and non-functional vessel formation and acceleration of fibrosis progression. Therefore, VEGF has become an attractive target for the treatment of liver fibrosis. Antiangiogenic agents such as bevacizumab block VEGF and attenuate the development of hepatic fibrosis, leading to the restoration of liver function. In this study, we showed that VEGF siRNAs delivered by CXCR4-targeted NPs decreased ECM deposition and normalized the hepatic vascular structure indicated by reduced vascular diameter and tortuosity in the fibrotic livers of CCl4-treated mice, resulting in the amelioration of hepatic hypoxia. This effect contributed to a negative feedback

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loop leading to the resolution of hypoxia-induced angiogenesis and fibrogenesis. However, although blocking angiogenesis could be a promising therapeutic strategy for the treatment of patients with advanced fibrosis, other evidence indicates that sinusoidal angiogenesis may contribute to the amelioration of liver fibrosis 29. For example, myeloid cell-derived VEGF has been reported to serve as a critical regulator of ECM degradation by liver endothelial cells, thereby leading to the resolution of fibrosis 30. In addition, blocking integrin αvβ3 has been reported to decrease angiogenesis but worsen hepatic fibrosis in a bile duct ligation (BDL) mouse model

31.

Thus, treating liver fibrosis with anti-angiogenic therapy has

remained controversial. Dose and time dependence of anti-fibrotic effects and vascular normalization may occur in the fibrotic livers treated with anti-VEGF agents. Thus, the clinically effective dose of anti-VEGF treatment is an important consideration for future clinical treatment of liver fibrosis. Our siRNA delivery system may provide a platform to deliver siRNAs into the fibrotic livers and unmask the pro- and anti-fibrotic mechanisms of angiogenesis-related factors by introducing siRNAs corresponding to different molecules. These results will make possible the rational design of a targeted anti-fibrotic therapy. Because siRNAs can specifically suppress disease-associated gene expression, they are potential therapeutic agents for the treatment of various diseases 32-35. However, the efficient delivery of siRNAs into diseased organs or targeted cells has remained challenging. The CXCR4-targeted NPs that we developed in this study could efficiently encapsulate and deliver siRNAs into activated HSCs in vitro and fibrotic liver tissues in vivo to silence target genes. Although CXCR4 is expressed in various

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cells such as activated HSCs, various cancer cells and other normal cells such as bone marrow-derived cells (BMDC), its antagonist induces significant inhibition of cell proliferation only in cancer cells and activated HSCs 16, 17. Furthermore, NPs can achieve the predominant accumulation in the liver tissues, leading to the enhanced retention of CXCR4-targeted NPs within the fibrotic livers and the reduction in offtarget effects. In conclusion, the CXCR4 antagonist AMD3100 incorporated into the NPs displays dual functions, working as a targeting ligand for the intracellular delivery of siRNA into fibrotic livers and as an inhibitor to block the SDF-1α/CXCR4 axis, synergistically inhibiting liver fibrosis progression when combined with therapeutic siRNAs against pro-angiogenic or fibrogenic molecules. Anti-angiogenic siRNAs delivered by CXCR4-targeted NPs can modulate the angiogenic and fibrotic microenvironment toward a normalized phenotype, providing a new strategy to treat early stage liver fibrosis and prevent the progression of liver cirrhosis. Acknowledgments This work was supported by Ministry of Science and Technology (MOST 103-2221E-007-032-MY2, MOST 104-2628-B-007-001-MY3), Chang Gung Memorial HospitalNational Tsing Hua University Joint Research Grant (104N2744E1) to Yunching Chen and the Chang Gung Medical Research Program (CMRPG3A1393) to Chan K.M.. This work was partially supported by Dr. Shiou-Han Wang, Department of Dermatology, National Taiwan University Hospital. We thank the members of the Microscopy Center of Chang Gung University Center for their assistance to obtain TEM images.

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Figure legends Figure 1: CXCR4 and VEGF expression is increased in liver tissue samples from patients with cirrhosis. Paraffin-embedded tissue sections were stained for CXCR4 and VEGF by immunohistochemistry. A, CXCR4 and VEGF expression in normal liver tissues was nearly undetectable but was consistently elevated in cirrhotic liver tissues. The expression of both CXCR4 and VEGF was co-localized with ECM components/fibrous septa, indicating correlations among SDF-1α/CXCR4 axis activation, angiogenesis and fibrogenesis (scale bar=50 μm). B, Quantification of the expression intensity revealed a tremendous increase in CXCR4 and VEGF expression in cirrhotic livers. Error bars represent the score ± the standard error of the mean (S.E.M.; n=10 regions of interest per sample).

Figure 2. CXCR4 expression increases in the liver after fibrotic induction. A-E, Liver sections from mice treated with CCl4 at different time points were analyzed for collagen I (B), α-SMA (C), VEGF (D) and CXCR4 (E) expression by immunofluorescence (IF). CXCR4 expression increased in response to CCl4 treatment during progressive fibrosis (n=7-10). Scale bar=100 μm. The data are the mean values ± S.E.M. *p

Dual-Functional Nanoparticles Targeting CXCR4 ... - ACS Publications

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