Gene Therapy (2015), 1–8 © 2015 Macmillan Publishers Limited All rights reserved 0969-7128/15 www.nature.com/gt
ORIGINAL ARTICLE
TGF-β1-siRNA delivery with nanoparticles inhibits peritoneal fibrosis H Yoshizawa1, Y Morishita1, M Watanabe1, K Ishibashi2, S Muto1, E Kusano1 and D Nagata1 Gene therapies may be promising for the treatment of peritoneal fibrosis (PF) in subjects undergoing peritoneal dialysis (PD). However, a method of delivery of treatment genes to the peritoneum is lacking. We attempted to develop an in vivo small interfering RNA (siRNA) delivery system with liposome-based nanoparticles (NPs) to the peritoneum to inhibit PF. Transforming growth factor (TGF)-β1-siRNAs encapsulated in NPs (TGF-β1-siRNAs-NPs) dissolved in PD fluid were injected into the peritoneum of mice with PF three times a week for 2 weeks. TGF-β1-siRNAs-NPs knocked down TGF-β1 expression significantly in the peritoneum and inhibited peritoneal thickening with fibrous changes. TGF-β1-siRNAs-NPs also inhibited the increase of expression of α-smooth muscle actin-positive myofibroblasts. These results suggest that the TGF-β1-siRNA delivery system with NPs described here could be an effective therapeutic option for PF in subjects undergoing PD. Gene Therapy advance online publication, 8 January 2015; doi:10.1038/gt.2014.116
INTRODUCTION Peritoneal fibrosis (PF) is frequently accompanied by the dysfunction of peritoneal membranes with ultrafiltration failure, resulting in discontinuation of peritoneal dialysis (PD) in patients.1–3 PF is characterized histologically by myofibroblast proliferation and excess accumulation of the extracellular matrix in the form of types of collagen in the peritoneal mesothelium upon undertaking PD.4,5 A therapy to prevent PF has not been established. Hence, the development of treatment options for PF is crucial to improve the prognosis of all PD patients. Gene therapy is a potentially promising strategy for PF treatment because it can target molecules that were previously difficult to set as therapeutic targets using small molecules or antibodies for various reasons (for example, lack of a ligandbinding site; problems relating to protein structure). RNA interference is a sequence-specific gene-silencing mechanism.6,7 It has evolved from being a valuable research tool to a potentially powerful therapeutic approach for various diseases.8–10 RNA interference can be achieved by expressing precursors of small interfering RNAs (siRNAs), such as short hairpin RNAs, with viral vectors, or by incorporating synthetic siRNAs directly into the cell cytoplasm.11 Using siRNAs for target gene silencing has been considered to be a promising therapeutic option because they can be employed without the concerns associated with viral vectors. In addition, siRNAs that enter the late stage of the endogenous RNA interference pathway are less likely to interfere with the gene regulation carried out by the endogenous microRNA machinery.11 Despite the promise of siRNAs as drugs, the biggest hurdle preventing their clinical and in vivo applications is their delivery. siRNAs are subject to rapid renal clearance and can be degraded quickly by serum RNases, thereby shortening their half-life in vivo.8,12 In addition, the efficiency with which siRNAs cross the plasma membrane and enter the cytoplasm is usually very low unless appropriate carriers such as transfection reagents are used.13,14 These lines of evidence suggest that development of a
siRNA delivery system to the peritoneum is required to enable siRNAs to be used for PF treatment, which may be a promising therapeutic option in PD patients. Therefore, we attempted to develop an siRNA delivery system to the peritoneum using non-viral liposome-based nanoparticles (NPs) that can entrap siRNAs in their interior. We entrapped siRNAs targeting transforming growth factor (TGF)-β1 (TGF-β1-siRNAs), which has been considered to have a central role in the development of PF,15,16 using NPs. Then, we investigated the effects of TGF-β1-siRNAs-NPs on PF in a mouse model in vivo.
RESULTS Characterization and in vivo distribution of siRNAs-NPs Polyacrylamide gel electrophoresis showed that NP-entrapped siRNAs were not detected as a band at the expected size of free siRNAs (20 base pairs). However, siRNAs alone was detected as a band at the size of 20 base pairs (Supplementary Figure 1). After treatment of siRNAs-NPs with a surface-acting agent (5% deoxycholate), the band was detected at the size of 20 base pairs (Supplementary Figure 1). These results suggest that siRNAs were encapsulated within NPs. siRNAs-NPs injected into the peritoneum were detected in peritoneal cells. However, siRNAs alone injected into the peritoneum were barely detected in the peritoneum (Figure 1). In terms of other organs, siRNAs-NPs were not delivered to the liver or kidneys. However, siRNAs alone were delivered to the liver and kidneys (Supplementary Figure 2). Then, we investigated interferon (IFN) expression in the peritoneum upon administration of siRNAs-NPs. Expression of IFN-β, STAT1 and 5′-oligoadenylate synthase (OAS1) tended to increase in PF mice compared with Mock mice, but this change did not reach statistical significance. Expression of IFN-β, STAT1 and OAS1 was not increased further by administration of siRNAs-NPs in PF mice (Supplementary Figure 3).
1 Division of Nephrology, Department of Internal Medicine, Jichi Medical University, Tochigi, Japan and 2Department of Medical Physiology, Meiji Pharmaceutical University, Tokyo, Japan. Correspondence: Dr Y Morishita, Division of Nephrology, Department of Medicine, Jichi Medical University, 3311-1, Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail: [email protected] Received 30 April 2014; revised 6 November 2014; accepted 11 November 2014
Nanoparticle-based siRNA delivery for fibrosis H Yoshizawa et al
2 Knockdown of TGF-β1 expression in the peritoneum by TGF-β1siRNAs-NPs Immunofluorescence analyses showed that TGF-β1 expression in the peritoneum was increased in PF mice compared with that in Mock mice (Figure 2a). TGF-β1-siRNAs-NPs significantly knocked down the increase in TGF-β1 expression in the peritoneum in PF mice, whereas Control-siRNAs-NPs did not. TGF-β1-siRNAs alone
did not significantly knock down TGF-β1 expression in the peritoneum in PF mice. The TGF-β1 concentration in peritoneal drainage fluid was increased in PF mice compared with that in Mock mice (Figure 2b). TGF-β1-siRNAs-NPs significantly decreased the increase in the TGF-β1 concentration in peritoneal drainage fluid in PF mice, whereas Control-siRNAs-NPs and TGF-β1-siRNAs alone did not (Figure 2b).
Figure 1. Distribution of siRNAs-NPs in the peritoneum. Immunofluorescence analyses of intraperitoneally injected Cy3-labeled siRNAs-NPs and siRNAs alone in the peritoneum. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 2. Knockdown of TGF-β1 expression in the peritoneum and inhibition of TGF-β1 expression in peritoneal drainage fluid by TGF-β1siRNAs-NPs. (a) Representative immunofluorescence staining and quantitative analyses of TGF-β1-positive cells in peritoneum tissue sections in each group. (b) TGF-β1 concentration was measured by ELISA in the peritoneal drainage fluid in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; NS, not significant; *Po0.05. Gene Therapy (2015) 1 – 8
© 2015 Macmillan Publishers Limited
Nanoparticle-based siRNA delivery for fibrosis H Yoshizawa et al
Effects of TGF-β1-siRNAs-NPs on PF Peritoneal fibrous thickening was observed in PF mice compared with that in Mock mice (Figure 3). TGF-β1-siRNAs-NPs significantly inhibited peritoneal fibrous thickening in PF mice. Control-siRNAsNPs and TGF-β1-siRNAs alone did not significantly inhibit these peritoneal changes in PF mice (Figure 3). qRT-PCR showed that the expression of α-smooth muscle actin (α-SMA) and collagen 1A2 was increased in PF mice compared with that in Mock mice (Figure 4). This increase was inhibited significantly by TGF-β1siRNAs-NPs, but was not inhibited by Control-siRNAs-NPs and TGF-β1-siRNAs alone (Figure 4). Expression of fibronectin 1 and fibroblast-specific protein 1 also showed a similar trend to α-SMA and collagen 1A2, but did not reach statistical significance (Figure 4). In terms of epithelial markers, we investigated changes in the expression of E-cadherin and occludin in each group. These changes did not show statistical significance, most probably because of high variation in each mouse even though they were from the same group (data not shown).
cytokeratin and α-SMA were inhibited by TGF-β1-siRNAs-NPs, but α-SMA single-positive cells were not (Figures 6a–e). ControlsiRNAs-NPs and TGF-β1-siRNAs alone inhibited neither cells double-positive for cytokeratin and α-SMA, for CD45 and α-SMA, nor cells single-positive for α-SMA (Figures 6a–e). The cells doublepositive for CD31 and α-SMA were not detected in PF mice (data not shown).
Effects of TGF-β1-siRNAs-NPs on myofibroblasts derived from multiple origins Immunofluorescence analyses showed that the number of α-SMApositive myofibroblasts increased in the peritoneum of PF mice compared with those in Mock mice. This increased expression of α-SMA-positive myofibroblasts was inhibited significantly by TGFβ1-siRNAs-NPs (Figure 5). Control-siRNAs-NPs and TGF-β1-siRNAs alone did not significantly inhibit these cells in PF mice (Figure 5). Then, we investigated the characteristics of these myofibroblasts by double staining with α-SMA and a marker of peritoneal epithelial cells (cytokeratin), a bone marrow-derived cell marker (CD45) and a marker of endothelial cells (CD31). The number of cells double-positive for cytokeratin and α-SMA (Figures 6a–c) and for CD45 and α-SMA (Figures 6b–d), in addition to small populations of cells single-positive for α-SMA (Figure 6e), was increased in PF mice compared with those in Mock mice. Cells double-positive for CD45 and α-SMA as well as for
DISCUSSION The results of the present study show that NPs delivered siRNAs to the peritoneum, and that NP-encapsulated TGF-β1-siRNAs inhibited PF in a mouse model in vivo. These results suggest that an anti-fibrotic siRNA delivery system with NPs would be a promising therapeutic option for PF in PD. PF is an intractable complication without an established therapy in PD patients.1–3 Gene therapy is a potentially promising option for PF.17 Studies have reported the possible effects of gene therapy for PF. Several studies have reported that viral vectors (for example, adenoviral, lentiviral) can deliver treatment genes to the peritoneum and have shown beneficial effects in terms of PF inhibition.18,19 However, the primary concern with viral vectors in an in vivo therapeutic setting is insertional mutagenesis related to their integrative nature, which may lead to malignancy or immunodeficiency.17 To avoid this disadvantage of viral vectors,
Effects of TGF-β1-siRNAs-NPs on peritoneal functions Peritoneal equilibrium test analyses showed that peritoneal drainage volume was decreased significantly and D/P-Cr increased significantly in PF mice compared with those of Mock mice. TGFβ1-siRNAs-NPs inhibited these peritoneal functional changes in PF mice. Control-siRNAs-NPs inhibited the increase of D/P-Cr, but did not inhibit the decrease of drainage volume in PF mice. TGF-β1siRNAs alone inhibited neither decreased drainage volume nor increased D/P-Cr in PF mice (Figure 7).
Figure 3. Effects of TGF-β1-siRNAs-NPs on peritoneal fibrous thickness. Representative Azan staining of peritoneum tissue and quantitative analyses of peritoneal thickness in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. NS, not significant; *Po 0.05; **Po 0.01. © 2015 Macmillan Publishers Limited
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Figure 4. Effects of TGF-β1-siRNAs-NPs on markers of fibrosis. Changes in expression of α-SMA, collagen 1A2, FN1 and FSP1 in each group was investigated by qRT-PCR. Each group: n = 6, values are the mean ± s.e. (error bars). FN1, fibronectin 1; FSP-1, fibroblast-specific protein 1; NS, not significant; *Po 0.05.
non-viral vectors have also been studied as carriers of treatment genes to the peritoneum to inhibit PF. Nishino et al.20 reported that intraperitoneal injection of heatshock protein (HSP)47 antisense oligonucleotides inhibited PF in a rat model. Guo et al.21 reported that an ultrasound microbubblemediated system can deliver plasmid DNA to peritoneum. We developed a new non-viral vector, liposome-based NPs, to deliver siRNAs to the peritoneum. We also showed significant knockdown of expression of the target gene (TGF-β1) and inhibition of PF in vivo by this system in a mouse model of PF. These results suggest that TGF-β1-siRNA delivery with NPs is a promising therapeutic approach for the inhibition of PF. Liposome-based NPs have been reported to be useful carriers that can deliver target genes by encapsulating them. They have been reported to have high biocompatibility, stability and transfection efficacy as carriers of treatment genes in vivo,22,23 but their effects on PF in vivo have not been evaluated. This is the first report to show the treatment effect on PF by a siRNAs-NPs delivery system. In addition, Gene Therapy (2015) 1 – 8
NP-encapsulated siRNAs were not delivered to the liver or kidneys compared with administration of siRNAs alone. These results suggest that siRNAs-NPs dissolved in PD fluid (PDF) and injected into the peritoneum may enable more specific siRNA delivery to the peritoneum than administration of siRNAs alone. In addition, siRNAs-NPs did not increase IFN expression in the peritoneum of PF mice. Studies have reported that NPs can lead to increases in IFN expression if administrated in vivo.24–26 The administration route, method and dose have been reported to be important factors affecting this immunomodulation of NPs.24–26 The method of administration of siRNAs-NPs in the present study (dissolving siRNAs-NPs in PDF followed by injection into the peritoneum) may have contributed to their lower immunotoxicity. Further studies are needed to elucidate the mechanism of immunomodulation of siRNAs-NPs and its role in PF development. PF is characterized histologically by proliferation of myofibroblasts and excess accumulation of the extracellular matrix as types of collagen in the peritoneal mesothelium.1–3 Recently, Lourerio et al.16 © 2015 Macmillan Publishers Limited
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Figure 5. Effects of TGF-β1-siRNAs-NPs on expression of α-SMA-positive myofibroblasts in the peritoneum. Representative immunofluorescence (a) and quantitative analyses (b) of α-SMA-positive myofibroblasts in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; *Po0.05; **P o0.01.
reported that myofibroblasts in the peritoneum may be derived from different origins, such as mesothelium cells via mesothelialto-mesenchymal transition, bone-marrow-derived cells and endothelial cells via endothelial-to-mesenchymal transition, in addition to proliferation of resident fibroblasts. TGF-β1 has been considered to play central parts in mesothelial-to-mesenchymal transition, endothelial-to-mesenchymal transition and fibrocyte differentiation.27–29 Lourerio et al.16 also reported that blockade of the TGF-β1 signaling pathway using TGF-β1-blocking peptide inhibited PF by decreasing the number of myofibroblasts suggested to be derived from mesothelium cells, bone-marrowderived cells and endothelial cells; however, TGF-β1-blocking peptide did not decrease the increase in expression of resident fibroblasts. We also observed that TGF-β1-siRNAs-NPs decreased the number of myofibroblasts that were double-positive for CD45 or cytokeratin and α-SMA; however, TGF-β1-siRNAs-NPs did not decrease the number of α-SMA single-positive myofibroblasts. These results are consistent with previous results, and suggest that TGF-β1-siRNAs-NPs knockdown of TGF-β1 expression in different types of cell, such as mesothelium cells and bone-marrow-derived cells, inhibits the change in their cellular phenotype. Increased expression of α-SMA single-positive myofibroblasts was not suppressed by TGF-β1-siRNAs-NPs. Hence, whether NPs could not deliver TGF-β1-siRNAs to those cells, TGF-β1 knockdown with this system did not sufficiently inhibit proliferation of those cells, or those cells were refractory to TGF-β1 knockdown for inhibition of proliferation, merits further investigation. It was also reported that the inhibition of TGF-β1 signaling may enhance acute peritoneal inflammation induced by a bacterial infection in a rat model in vivo.30 Hence, the effects of TGF-β1 knockdown of peritoneum by TGF-β1-siRNAs-NPs for acute peritoneal inflammation also should be further investigated because it may lead to adverse effects. CONCLUSION The results of the present study suggest that the TGF-β1-siRNAs delivery system using NPs inhibited PF in vivo by knockdown of © 2015 Macmillan Publishers Limited
expression of the target gene, TGF-β1. This system is a potentially promising therapeutic option for PF in PD. MATERIALS AND METHODS The experimental protocol was approved by the Animal Ethics Committee of Jichi Medical University (Tochigi, Japan).
Creation of a model of PF in mice C57BL/6 male mice (10 weeks; 20–25 g) were purchased from CLEA Japan, Inc. (Tokyo, Japan). They were housed in a room with controlled temperature, humidity and a 12-h light–dark cycle. To induce PF, the mice were injected with PDF (100 ml kg − 1, i.p.) containing 40 mM methylglyoxal solution (Sigma–Aldrich Corp., Saint Louis, MO, USA) every day for 2 weeks.31 PDF contained 2.5% glucose, 100 mM NaCl, 35 mM sodium lactate, 2 mM CaCl2 and 0.7 mM MgCl2 (Midperic; Terumo, Tokyo, Japan).
siRNAs siRNAs targeting mouse TGF-β1 (TGF-β1-siRNAs) and universal nontargeted siRNAs (Control-siRNAs) were purchased from Sigma–Aldrich Japan (Ishikari, Hokkaido, Japan). The sequences of TGF-β1-siRNAs were: 5′-GCAACAACGCCAUCUAUGATT-3′ (sense strand) and 5′-UCAUAGA UGGCGUUGUUGCTT-3′ (antisense strand). The following siRNAs were used as control siRNAs: Mission_SIC_001s (sense strand) and Mission_SIC_001as (antisense) (the actual sequence was not disclosed).
Preparation of siRNAs entrapped in NPs Lyophilized NPs composed of phosphatidylcholine, dipalmitoylphosphatidylethanolamine and cholesterol were purchased from Hokkaido System Science Co., Ltd. (Sapporo, Hokkaido, Japan). siRNAs were dissolved in DNase/RNase-free distilled water at 50 μM (Life Technologies, Carlsbad, CA, USA). Then, 500 nmol lyophilized NPs were rehydrated by adding 100 μl of distilled water containing siRNAs. The diameter of NPs entrapping siRNAs as measured by Hokkaido System Science Co., Ltd., was 100–200 nm. A total of 100 μl of siRNAs entrapped in NPs were dissolved in 100 ml kg − 1 PDF. Finally, they were injected, via the intraperitoneal route, into mice three times per week for 2 weeks. Gene Therapy (2015) 1 – 8
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Figure 6. Effects of TGF-β1-siRNAs-NPs on myofibroblasts derived from multiple origins. Representative immunohistochemistry of cells doublepositive (yellow) for cytokeratin and α-SMA (a) and for CD45 and α-SMA (b), and quantitative analyses of cells double-positive for cytokeratin and α-SMA (c) and for CD45 and α-SMA (d), and cells single-positive for α-SMA (e) in the peritoneum in each group. Each group: n = 4, values are the mean ± s.e. (error bars) of at least two independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; *Po 0.05; **P o0.01.
Delivery of siRNAs entrapped in NPs to the peritoneum for PF treatment in vivo To investigate the distribution of siRNAs with NPs in the peritoneum, 100 μl of Cy3-labeled siRNAs (5 nmol) (Mirus Bio, Madison, WI, USA) were entrapped in 500 nmol NPs, dissolved in PDF, and injected, via the intraperitoneal route, into mice. One hour after injection, tissues from the peritoneum, liver and kidneys were purified, cut and fixed with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, containing 25 mM CaCl2 at room temperature. Fixed tissues were immersed and embedded in Tissue-Tek optimal cutting temperature compound which is a formulation of water soluble glycols and resins, and frozen in liquid nitrogen. Cryostat sections of thickness 7–8 μm were mounted on silanecoated glass slides (Matsunami, Osaka, Japan) and air-dried. Then, fluorescence was detected by observation under a Fluorescence Microscope (BH2-RFL-T3 and BX50; Olympus, Shinjuku, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA). To investigate the treatment effects of siRNAs entrapped in NPs in PF, 100 μl of TGF-β1-siRNAs (5 nmol) entrapped in 500 nmol NPs (TGF-β1siRNAs-NPs) were dissolved in 100 ml kg − 1 PDF, and injected, via the intraperitoneal route, into mice (PF+TGF-β1-siRNAs-NPs) from the one day before methylglyoxal injection at a frequency of three times per week for 2 weeks separately from methylglyoxal injection. The following groups served as controls: mice without any treatment (Mock); mice injected with PDF without NPs or siRNAs (PF); mice injected with control-siRNAs entrapped in NPs by the same methods as for the TGF-β1-siRNAs-NPs (PF +control-siRNAs-NPs); and mice injected with 100 μl of TGF-β1-siRNAs (5 nmol) alone dissolved in PDF (PF+TGF-β1-siRNAs). Mice in all groups were killed 14 days after carrying out peritoneal equilibrium tests (as described Gene Therapy (2015) 1 – 8
below). Then, peritoneal tissues were removed and purified after washing with phosphate-buffered saline (PBS).
Peritoneal equilibrium test The details of the peritoneal equilibrium test have been described previously.31 Briefly, 5 ml of standard PDF containing 2.5% glucose (Midperic; Terumo) was injected via the intraperitoneal route. The dialysate was collected 1 h later to analyze the peritoneal drainage volume and concentration of creatinine (D-Cr). At that time, blood samples were also collected from the inferior vena cava to measure plasma creatinine concentration (P-Cr). Then, the ratio of creatinine concentration in the dialysate to that in plasma (D/P-Cr) was calculated.
Electrophoresis To investigate the encapsulation of siRNAs in NPs, 1 μl of siRNAs (50 μM) encapsulated in NPs (500 nmol) diluted with 4 μl of DNase/RNase-free distilled water, 1 μl of siRNAs (50 μM) encapsulated in NPs (500 nmol) treated with 4 μl of 5% deoxycholate (Sigma–Aldrich) and 1 μl of siRNAs (50 μM) diluted with 4 μl of DNase/RNase-free distilled water were subjected to electrophoresis on polyacrylamide gels.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Sections of peritoneum tissue were homogenized using a glass homogenizer and a filter column shredder (QIA Shredder; Qiagen, Valencia, CA, USA). © 2015 Macmillan Publishers Limited
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Figure 7. Effects of TGF-β1-siRNA-NPs on peritoneal functions. Results of PET analyses in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. PET, peritoneal equilibrium test; D/P-Cr, the ratio of creatinine concentration (Cr) in the dialysate (D) to that in plasma (P); NS, not significant; *P o0.05; **Po 0.01. Then, total RNA from the peritoneum tissue was isolated using an RNeasy Total RNA Isolation kit (Qiagen). A total of 100 ng of isolated RNA was reverse-transcribed using a Superscript III First-strand Synthesis system (Life Technologies) according to the manufacturer's instructions. Real-time RT-PCR was undertaken using SYBR Green ER qPCR Supermix (Life Technologies). All reactions were carried out in a reaction volume of 20 μl in duplicate. After initial denaturation at 95 °C for 10 s, real-time qRTPCR was carried out for 40 cycles by denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 60 s. Primers for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-SMA, collagen 1A2, fibronectin 1, fibroblast-specific protein 1, IFN-β, STAT1 and OAS1 were purchased from Takara Bio Inc (Otsu, Shiga, Japan). Quantification of expression of the target gene was achieved using Step One software (Life Technologies). mRNA expression was normalized with GAPDH (endogenous control). Data are expressed as relative quantities compared with the Mock group.
Enzyme-linked immunosorbent assay (ELISA) for TGF-β1 An ELISA kit specific for mouse TGF-β1 (TGF-β1 ELISA; IBL, Hamburg, Germany) was used according to the manufacturer instructions. Briefly, 100 μl of dialysate sample was added to a plate pre-coated with monoclonal antibody against mouse TGF-β1. Then, anti-mouse TGF-β1 antibody conjugated to horseradish peroxidase was added and incubated for 4 h at room temperature. After washing, color was developed with 100 μl of substrate (tetramethyl benzidine). The reaction was terminated by addition of 1 M of phosphoric acid and absorbance measured at 450 nm.
Histologic analyses After perfusion with PBS, peritoneum tissues were removed and fixed in 4% paraformaldehyde overnight at 4 °C. They were embedded in paraffin, sectioned and subjected to Azan staining to evaluate the intensity of fibrotic changes. Collagen fibers were stained blue upon Azan staining. © 2015 Macmillan Publishers Limited
For evaluation of the degree of peritoneal thickening, the thickness of the submesothelial compact zone (membrane area extending from the lower limit of the mesothelial layer to the upper limit of the muscle layer) was measured in 10 fields of each Azan-stained sample chosen randomly at × 400 magnification using computerized image-analysis software (Image Pro 5.1, Media Cybernetics Inc., Rockville, MD, USA). The mean thickness of the submesothelial compact zone in each mouse was calculated and defined as the peritoneal thickness.
Immunofluorescent microscopy To evaluate cells that were positive for TGF-β1 in each group, paraffinembedded sections of peritoneum tissue were deparaffinized, rehydrated and autoclaved for 15 min at 121 °C in 10 mM citrate buffer (pH 6.0) to retrieve antigens, followed by incubation with 10% normal goat serum in PBS to block non-specific binding of antibodies. Then, sections of pretreated peritoneum tissue were incubated overnight at 4 °C with a rabbit polyclonal antibody against mouse TGF-β1 (Abcam, Cambridge, UK) at 1:100 dilution. After washing with PBS, sections were incubated for 2 h at room temperature with a monoclonal antibody against anti-rabbit IgG conjugated to Alexa 488 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 1:100 dilution. To evaluate cells that were positive for α-SMA, cytokeratin, CD45 and CD31, sections of pretreated peritoneum tissue were incubated overnight at 4 °C with a monoclonal antibody against mouse α-SMA conjugated to Cy3 (Sigma–Aldrich) at 1:400 dilution, cytokeratin (Acris Antibodies, San Diego, CA, USA) at 1:50 dilution, CD45 (Abcam) at 1:400 dilution or CD31 (Abcam) at 1:50 dilution. After washing with PBS, sections were incubated for 2 h at room temperature with a monoclonal antibody against anti-rabbit IgG conjugated to Alexa 488 (Jackson ImmunoResearch Laboratories) at 1:100 dilution. Then, all sections were mounted with VECTASHIELD mounting medium (Vector Laboratories Inc., Burlingame, CA, USA) using 4′,6-diamidino-2-phenylindole to stain the nucleus. Gene Therapy (2015) 1 – 8
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8 Laboratory methods Blood analyses were carried out by a clinical chemistry laboratory (SRL, Tokyo, Japan).
Statistical analyses Data are the mean ± s.e. Analysis of variance was employed to investigate differences among groups. If statistical significance was detected by analysis of variance, Tukey’s test was carried out as post hoc analyses to compare the mean values of two groups. SPSS v21 (IBM, Armonk, NY, USA) was used for statistical analyses. Po0.05 was considered significant.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank Motomu Shimaoka (Professor of Molecular Pathobiology, Mie University School of Medicine) for discussion and comments regarding the manuscript. This work was supported by JSPS KAKENHI (grant number 25461252) and MEXTSupported Program for Strategic Research Foundations at Private Universities, 2013– 2017 (S1211029).
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13 Palliser D, Chowdhury D, Wang QY, Lee SJ, Bronson RT, Knipe DM et al. An siRNAbased microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 2006; 439: 89–94. 14 Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS et al. Lentivirusdelivered stable gene silencing by RNAi in primary cells. RNA 2003; 9: 493–501. 15 Yao Q, Pawlaczyk K, Ayala ER, Styszynski A, Breborowicz A, Heimburger O et al. The role of the TGF/Smad signaling pathway in peritoneal fibrosis induced by peritoneal dialysis solutions. Nephron Exp Nephrol 2008; 109: e71–e78. 16 Loureiro J, Aguilera A, Selgas R, Sandoval P, Albar-Vizcaino P, Perez-Lozano ML et al. Blocking TGF-beta1 protects the peritoneal membrane from dialysateinduced damage. J Am Soc Nephrol 2011; 22: 1682–1695. 17 Li XJ, Sun L, Xiao L, Liu FY. Gene delivery in peritoneal dialysis related peritoneal fibrosis research. Chin Med J (Engl) 2012; 125: 2219–2224. 18 Motomura Y, Kanbayashi H, Khan WI, Deng Y, Blennerhassett PA, Margetts PJ et al. The gene transfer of soluble VEGF type I receptor (Flt-1) attenuates peritoneal fibrosis formation in mice but not soluble TGF-beta type II receptor gene transfer. Am J Physiol Gastrointest Liver Physiol 2005; 288: G143–G150. 19 Margetts PJ, Gyorffy S, Kolb M, Yu L, Hoff CM, Holmes CJ et al. Antiangiogenic and antifibrotic gene therapy in a chronic infusion model of peritoneal dialysis in rats. J Am Soc Nephrol 2002; 13: 721–728. 20 Nishino T, Miyazaki M, Abe K, Furusu A, Mishima Y, Harada T et al. Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats. Kidney Int 2003; 64: 887–896. 21 Guo H, Leung JC, Chan LY, Tsang AW, Lam MF, Lan HY et al. Ultrasound-contrast agent mediated naked gene delivery in the peritoneal cavity of adult rat. Gene Therapy 2007; 14: 1712–1720. 22 Kikuchi A, Aoki Y, Sugaya S, Serikawa T, Takakuwa K, Tanaka K et al. Development of novel cationic liposomes for efficient gene transfer into peritoneal disseminated tumor. Hum Gene Ther 1999; 10: 947–955. 23 Sato Y, Murase K, Kato J, Kobune M, Sato T, Kawano Y et al. Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagenspecific chaperone. Nat Biotechnol 2008; 26: 431–442. 24 Wang X, Podila R, Shannahan JH, Rao AM, Brown JM. Intravenously delivered graphene nanosheets and multiwalled carbon nanotubes induce site-specific Th2 inflammatory responses via the IL-33/ST2 axis. Int J Nanomedicine 2013; 8: 1733–1748. 25 Ban M, Langonne I, Huguet N, Guichard Y, Goutet M. Iron oxide particles modulate the ovalbumin-induced Th2 immune response in mice. Toxicol Lett 2013; 216: 31–39. 26 Morishige T, Yoshioka Y, Inakura H, Tanabe A, Narimatsu S, Yao X et al. Suppression of nanosilica particle-induced inflammation by surface modification of the particles. Arch Toxicol 2012; 86: 1297–1307. 27 Li J, Qu X, Bertram JF. Endothelial-myofibroblast transition contributes to the early development of diabetic renal interstitial fibrosis in streptozotocin-induced diabetic mice. Am J Pathol 2009; 175: 1380–1388. 28 Bellini A, Mattoli S. The role of the fibrocyte, a bone marrow-derived mesenchymal progenitor, in reactive and reparative fibroses. Lab Invest 2007; 87: 858–870. 29 Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G. Transforming growth factorbeta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993; 122: 103–111. 30 Wang X, Nie J, Jia Z, Feng M, Zheng Z, Chen W et al. Impaired TGF-beta signalling enhances peritoneal inflammation induced by E. coli in rats. Nephrol Dial Transplant 2010; 25: 399–412. 31 Hirahara I, Kusano E, Yanagiba S, Miyata Y, Ando Y, Muto S et al. Peritoneal injury by methylglyoxal in peritoneal dialysis. Perit Dial Int 2006; 26: 380–392.
Supplementary Information accompanies this paper on Gene Therapy website (http://www.nature.com/gt)
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ORIGINAL ARTICLE
TGF-β1-siRNA delivery with nanoparticles inhibits peritoneal fibrosis H Yoshizawa1, Y Morishita1, M Watanabe1, K Ishibashi2, S Muto1, E Kusano1 and D Nagata1 Gene therapies may be promising for the treatment of peritoneal fibrosis (PF) in subjects undergoing peritoneal dialysis (PD). However, a method of delivery of treatment genes to the peritoneum is lacking. We attempted to develop an in vivo small interfering RNA (siRNA) delivery system with liposome-based nanoparticles (NPs) to the peritoneum to inhibit PF. Transforming growth factor (TGF)-β1-siRNAs encapsulated in NPs (TGF-β1-siRNAs-NPs) dissolved in PD fluid were injected into the peritoneum of mice with PF three times a week for 2 weeks. TGF-β1-siRNAs-NPs knocked down TGF-β1 expression significantly in the peritoneum and inhibited peritoneal thickening with fibrous changes. TGF-β1-siRNAs-NPs also inhibited the increase of expression of α-smooth muscle actin-positive myofibroblasts. These results suggest that the TGF-β1-siRNA delivery system with NPs described here could be an effective therapeutic option for PF in subjects undergoing PD. Gene Therapy advance online publication, 8 January 2015; doi:10.1038/gt.2014.116
INTRODUCTION Peritoneal fibrosis (PF) is frequently accompanied by the dysfunction of peritoneal membranes with ultrafiltration failure, resulting in discontinuation of peritoneal dialysis (PD) in patients.1–3 PF is characterized histologically by myofibroblast proliferation and excess accumulation of the extracellular matrix in the form of types of collagen in the peritoneal mesothelium upon undertaking PD.4,5 A therapy to prevent PF has not been established. Hence, the development of treatment options for PF is crucial to improve the prognosis of all PD patients. Gene therapy is a potentially promising strategy for PF treatment because it can target molecules that were previously difficult to set as therapeutic targets using small molecules or antibodies for various reasons (for example, lack of a ligandbinding site; problems relating to protein structure). RNA interference is a sequence-specific gene-silencing mechanism.6,7 It has evolved from being a valuable research tool to a potentially powerful therapeutic approach for various diseases.8–10 RNA interference can be achieved by expressing precursors of small interfering RNAs (siRNAs), such as short hairpin RNAs, with viral vectors, or by incorporating synthetic siRNAs directly into the cell cytoplasm.11 Using siRNAs for target gene silencing has been considered to be a promising therapeutic option because they can be employed without the concerns associated with viral vectors. In addition, siRNAs that enter the late stage of the endogenous RNA interference pathway are less likely to interfere with the gene regulation carried out by the endogenous microRNA machinery.11 Despite the promise of siRNAs as drugs, the biggest hurdle preventing their clinical and in vivo applications is their delivery. siRNAs are subject to rapid renal clearance and can be degraded quickly by serum RNases, thereby shortening their half-life in vivo.8,12 In addition, the efficiency with which siRNAs cross the plasma membrane and enter the cytoplasm is usually very low unless appropriate carriers such as transfection reagents are used.13,14 These lines of evidence suggest that development of a
siRNA delivery system to the peritoneum is required to enable siRNAs to be used for PF treatment, which may be a promising therapeutic option in PD patients. Therefore, we attempted to develop an siRNA delivery system to the peritoneum using non-viral liposome-based nanoparticles (NPs) that can entrap siRNAs in their interior. We entrapped siRNAs targeting transforming growth factor (TGF)-β1 (TGF-β1-siRNAs), which has been considered to have a central role in the development of PF,15,16 using NPs. Then, we investigated the effects of TGF-β1-siRNAs-NPs on PF in a mouse model in vivo.
RESULTS Characterization and in vivo distribution of siRNAs-NPs Polyacrylamide gel electrophoresis showed that NP-entrapped siRNAs were not detected as a band at the expected size of free siRNAs (20 base pairs). However, siRNAs alone was detected as a band at the size of 20 base pairs (Supplementary Figure 1). After treatment of siRNAs-NPs with a surface-acting agent (5% deoxycholate), the band was detected at the size of 20 base pairs (Supplementary Figure 1). These results suggest that siRNAs were encapsulated within NPs. siRNAs-NPs injected into the peritoneum were detected in peritoneal cells. However, siRNAs alone injected into the peritoneum were barely detected in the peritoneum (Figure 1). In terms of other organs, siRNAs-NPs were not delivered to the liver or kidneys. However, siRNAs alone were delivered to the liver and kidneys (Supplementary Figure 2). Then, we investigated interferon (IFN) expression in the peritoneum upon administration of siRNAs-NPs. Expression of IFN-β, STAT1 and 5′-oligoadenylate synthase (OAS1) tended to increase in PF mice compared with Mock mice, but this change did not reach statistical significance. Expression of IFN-β, STAT1 and OAS1 was not increased further by administration of siRNAs-NPs in PF mice (Supplementary Figure 3).
1 Division of Nephrology, Department of Internal Medicine, Jichi Medical University, Tochigi, Japan and 2Department of Medical Physiology, Meiji Pharmaceutical University, Tokyo, Japan. Correspondence: Dr Y Morishita, Division of Nephrology, Department of Medicine, Jichi Medical University, 3311-1, Yakushiji, Shimotsuke, Tochigi 329-0498, Japan. E-mail: [email protected] Received 30 April 2014; revised 6 November 2014; accepted 11 November 2014
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2 Knockdown of TGF-β1 expression in the peritoneum by TGF-β1siRNAs-NPs Immunofluorescence analyses showed that TGF-β1 expression in the peritoneum was increased in PF mice compared with that in Mock mice (Figure 2a). TGF-β1-siRNAs-NPs significantly knocked down the increase in TGF-β1 expression in the peritoneum in PF mice, whereas Control-siRNAs-NPs did not. TGF-β1-siRNAs alone
did not significantly knock down TGF-β1 expression in the peritoneum in PF mice. The TGF-β1 concentration in peritoneal drainage fluid was increased in PF mice compared with that in Mock mice (Figure 2b). TGF-β1-siRNAs-NPs significantly decreased the increase in the TGF-β1 concentration in peritoneal drainage fluid in PF mice, whereas Control-siRNAs-NPs and TGF-β1-siRNAs alone did not (Figure 2b).
Figure 1. Distribution of siRNAs-NPs in the peritoneum. Immunofluorescence analyses of intraperitoneally injected Cy3-labeled siRNAs-NPs and siRNAs alone in the peritoneum. DAPI, 4′,6-diamidino-2-phenylindole.
Figure 2. Knockdown of TGF-β1 expression in the peritoneum and inhibition of TGF-β1 expression in peritoneal drainage fluid by TGF-β1siRNAs-NPs. (a) Representative immunofluorescence staining and quantitative analyses of TGF-β1-positive cells in peritoneum tissue sections in each group. (b) TGF-β1 concentration was measured by ELISA in the peritoneal drainage fluid in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; NS, not significant; *Po0.05. Gene Therapy (2015) 1 – 8
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Effects of TGF-β1-siRNAs-NPs on PF Peritoneal fibrous thickening was observed in PF mice compared with that in Mock mice (Figure 3). TGF-β1-siRNAs-NPs significantly inhibited peritoneal fibrous thickening in PF mice. Control-siRNAsNPs and TGF-β1-siRNAs alone did not significantly inhibit these peritoneal changes in PF mice (Figure 3). qRT-PCR showed that the expression of α-smooth muscle actin (α-SMA) and collagen 1A2 was increased in PF mice compared with that in Mock mice (Figure 4). This increase was inhibited significantly by TGF-β1siRNAs-NPs, but was not inhibited by Control-siRNAs-NPs and TGF-β1-siRNAs alone (Figure 4). Expression of fibronectin 1 and fibroblast-specific protein 1 also showed a similar trend to α-SMA and collagen 1A2, but did not reach statistical significance (Figure 4). In terms of epithelial markers, we investigated changes in the expression of E-cadherin and occludin in each group. These changes did not show statistical significance, most probably because of high variation in each mouse even though they were from the same group (data not shown).
cytokeratin and α-SMA were inhibited by TGF-β1-siRNAs-NPs, but α-SMA single-positive cells were not (Figures 6a–e). ControlsiRNAs-NPs and TGF-β1-siRNAs alone inhibited neither cells double-positive for cytokeratin and α-SMA, for CD45 and α-SMA, nor cells single-positive for α-SMA (Figures 6a–e). The cells doublepositive for CD31 and α-SMA were not detected in PF mice (data not shown).
Effects of TGF-β1-siRNAs-NPs on myofibroblasts derived from multiple origins Immunofluorescence analyses showed that the number of α-SMApositive myofibroblasts increased in the peritoneum of PF mice compared with those in Mock mice. This increased expression of α-SMA-positive myofibroblasts was inhibited significantly by TGFβ1-siRNAs-NPs (Figure 5). Control-siRNAs-NPs and TGF-β1-siRNAs alone did not significantly inhibit these cells in PF mice (Figure 5). Then, we investigated the characteristics of these myofibroblasts by double staining with α-SMA and a marker of peritoneal epithelial cells (cytokeratin), a bone marrow-derived cell marker (CD45) and a marker of endothelial cells (CD31). The number of cells double-positive for cytokeratin and α-SMA (Figures 6a–c) and for CD45 and α-SMA (Figures 6b–d), in addition to small populations of cells single-positive for α-SMA (Figure 6e), was increased in PF mice compared with those in Mock mice. Cells double-positive for CD45 and α-SMA as well as for
DISCUSSION The results of the present study show that NPs delivered siRNAs to the peritoneum, and that NP-encapsulated TGF-β1-siRNAs inhibited PF in a mouse model in vivo. These results suggest that an anti-fibrotic siRNA delivery system with NPs would be a promising therapeutic option for PF in PD. PF is an intractable complication without an established therapy in PD patients.1–3 Gene therapy is a potentially promising option for PF.17 Studies have reported the possible effects of gene therapy for PF. Several studies have reported that viral vectors (for example, adenoviral, lentiviral) can deliver treatment genes to the peritoneum and have shown beneficial effects in terms of PF inhibition.18,19 However, the primary concern with viral vectors in an in vivo therapeutic setting is insertional mutagenesis related to their integrative nature, which may lead to malignancy or immunodeficiency.17 To avoid this disadvantage of viral vectors,
Effects of TGF-β1-siRNAs-NPs on peritoneal functions Peritoneal equilibrium test analyses showed that peritoneal drainage volume was decreased significantly and D/P-Cr increased significantly in PF mice compared with those of Mock mice. TGFβ1-siRNAs-NPs inhibited these peritoneal functional changes in PF mice. Control-siRNAs-NPs inhibited the increase of D/P-Cr, but did not inhibit the decrease of drainage volume in PF mice. TGF-β1siRNAs alone inhibited neither decreased drainage volume nor increased D/P-Cr in PF mice (Figure 7).
Figure 3. Effects of TGF-β1-siRNAs-NPs on peritoneal fibrous thickness. Representative Azan staining of peritoneum tissue and quantitative analyses of peritoneal thickness in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. NS, not significant; *Po 0.05; **Po 0.01. © 2015 Macmillan Publishers Limited
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Figure 4. Effects of TGF-β1-siRNAs-NPs on markers of fibrosis. Changes in expression of α-SMA, collagen 1A2, FN1 and FSP1 in each group was investigated by qRT-PCR. Each group: n = 6, values are the mean ± s.e. (error bars). FN1, fibronectin 1; FSP-1, fibroblast-specific protein 1; NS, not significant; *Po 0.05.
non-viral vectors have also been studied as carriers of treatment genes to the peritoneum to inhibit PF. Nishino et al.20 reported that intraperitoneal injection of heatshock protein (HSP)47 antisense oligonucleotides inhibited PF in a rat model. Guo et al.21 reported that an ultrasound microbubblemediated system can deliver plasmid DNA to peritoneum. We developed a new non-viral vector, liposome-based NPs, to deliver siRNAs to the peritoneum. We also showed significant knockdown of expression of the target gene (TGF-β1) and inhibition of PF in vivo by this system in a mouse model of PF. These results suggest that TGF-β1-siRNA delivery with NPs is a promising therapeutic approach for the inhibition of PF. Liposome-based NPs have been reported to be useful carriers that can deliver target genes by encapsulating them. They have been reported to have high biocompatibility, stability and transfection efficacy as carriers of treatment genes in vivo,22,23 but their effects on PF in vivo have not been evaluated. This is the first report to show the treatment effect on PF by a siRNAs-NPs delivery system. In addition, Gene Therapy (2015) 1 – 8
NP-encapsulated siRNAs were not delivered to the liver or kidneys compared with administration of siRNAs alone. These results suggest that siRNAs-NPs dissolved in PD fluid (PDF) and injected into the peritoneum may enable more specific siRNA delivery to the peritoneum than administration of siRNAs alone. In addition, siRNAs-NPs did not increase IFN expression in the peritoneum of PF mice. Studies have reported that NPs can lead to increases in IFN expression if administrated in vivo.24–26 The administration route, method and dose have been reported to be important factors affecting this immunomodulation of NPs.24–26 The method of administration of siRNAs-NPs in the present study (dissolving siRNAs-NPs in PDF followed by injection into the peritoneum) may have contributed to their lower immunotoxicity. Further studies are needed to elucidate the mechanism of immunomodulation of siRNAs-NPs and its role in PF development. PF is characterized histologically by proliferation of myofibroblasts and excess accumulation of the extracellular matrix as types of collagen in the peritoneal mesothelium.1–3 Recently, Lourerio et al.16 © 2015 Macmillan Publishers Limited
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Figure 5. Effects of TGF-β1-siRNAs-NPs on expression of α-SMA-positive myofibroblasts in the peritoneum. Representative immunofluorescence (a) and quantitative analyses (b) of α-SMA-positive myofibroblasts in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; *Po0.05; **P o0.01.
reported that myofibroblasts in the peritoneum may be derived from different origins, such as mesothelium cells via mesothelialto-mesenchymal transition, bone-marrow-derived cells and endothelial cells via endothelial-to-mesenchymal transition, in addition to proliferation of resident fibroblasts. TGF-β1 has been considered to play central parts in mesothelial-to-mesenchymal transition, endothelial-to-mesenchymal transition and fibrocyte differentiation.27–29 Lourerio et al.16 also reported that blockade of the TGF-β1 signaling pathway using TGF-β1-blocking peptide inhibited PF by decreasing the number of myofibroblasts suggested to be derived from mesothelium cells, bone-marrowderived cells and endothelial cells; however, TGF-β1-blocking peptide did not decrease the increase in expression of resident fibroblasts. We also observed that TGF-β1-siRNAs-NPs decreased the number of myofibroblasts that were double-positive for CD45 or cytokeratin and α-SMA; however, TGF-β1-siRNAs-NPs did not decrease the number of α-SMA single-positive myofibroblasts. These results are consistent with previous results, and suggest that TGF-β1-siRNAs-NPs knockdown of TGF-β1 expression in different types of cell, such as mesothelium cells and bone-marrow-derived cells, inhibits the change in their cellular phenotype. Increased expression of α-SMA single-positive myofibroblasts was not suppressed by TGF-β1-siRNAs-NPs. Hence, whether NPs could not deliver TGF-β1-siRNAs to those cells, TGF-β1 knockdown with this system did not sufficiently inhibit proliferation of those cells, or those cells were refractory to TGF-β1 knockdown for inhibition of proliferation, merits further investigation. It was also reported that the inhibition of TGF-β1 signaling may enhance acute peritoneal inflammation induced by a bacterial infection in a rat model in vivo.30 Hence, the effects of TGF-β1 knockdown of peritoneum by TGF-β1-siRNAs-NPs for acute peritoneal inflammation also should be further investigated because it may lead to adverse effects. CONCLUSION The results of the present study suggest that the TGF-β1-siRNAs delivery system using NPs inhibited PF in vivo by knockdown of © 2015 Macmillan Publishers Limited
expression of the target gene, TGF-β1. This system is a potentially promising therapeutic option for PF in PD. MATERIALS AND METHODS The experimental protocol was approved by the Animal Ethics Committee of Jichi Medical University (Tochigi, Japan).
Creation of a model of PF in mice C57BL/6 male mice (10 weeks; 20–25 g) were purchased from CLEA Japan, Inc. (Tokyo, Japan). They were housed in a room with controlled temperature, humidity and a 12-h light–dark cycle. To induce PF, the mice were injected with PDF (100 ml kg − 1, i.p.) containing 40 mM methylglyoxal solution (Sigma–Aldrich Corp., Saint Louis, MO, USA) every day for 2 weeks.31 PDF contained 2.5% glucose, 100 mM NaCl, 35 mM sodium lactate, 2 mM CaCl2 and 0.7 mM MgCl2 (Midperic; Terumo, Tokyo, Japan).
siRNAs siRNAs targeting mouse TGF-β1 (TGF-β1-siRNAs) and universal nontargeted siRNAs (Control-siRNAs) were purchased from Sigma–Aldrich Japan (Ishikari, Hokkaido, Japan). The sequences of TGF-β1-siRNAs were: 5′-GCAACAACGCCAUCUAUGATT-3′ (sense strand) and 5′-UCAUAGA UGGCGUUGUUGCTT-3′ (antisense strand). The following siRNAs were used as control siRNAs: Mission_SIC_001s (sense strand) and Mission_SIC_001as (antisense) (the actual sequence was not disclosed).
Preparation of siRNAs entrapped in NPs Lyophilized NPs composed of phosphatidylcholine, dipalmitoylphosphatidylethanolamine and cholesterol were purchased from Hokkaido System Science Co., Ltd. (Sapporo, Hokkaido, Japan). siRNAs were dissolved in DNase/RNase-free distilled water at 50 μM (Life Technologies, Carlsbad, CA, USA). Then, 500 nmol lyophilized NPs were rehydrated by adding 100 μl of distilled water containing siRNAs. The diameter of NPs entrapping siRNAs as measured by Hokkaido System Science Co., Ltd., was 100–200 nm. A total of 100 μl of siRNAs entrapped in NPs were dissolved in 100 ml kg − 1 PDF. Finally, they were injected, via the intraperitoneal route, into mice three times per week for 2 weeks. Gene Therapy (2015) 1 – 8
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Figure 6. Effects of TGF-β1-siRNAs-NPs on myofibroblasts derived from multiple origins. Representative immunohistochemistry of cells doublepositive (yellow) for cytokeratin and α-SMA (a) and for CD45 and α-SMA (b), and quantitative analyses of cells double-positive for cytokeratin and α-SMA (c) and for CD45 and α-SMA (d), and cells single-positive for α-SMA (e) in the peritoneum in each group. Each group: n = 4, values are the mean ± s.e. (error bars) of at least two independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; *Po 0.05; **P o0.01.
Delivery of siRNAs entrapped in NPs to the peritoneum for PF treatment in vivo To investigate the distribution of siRNAs with NPs in the peritoneum, 100 μl of Cy3-labeled siRNAs (5 nmol) (Mirus Bio, Madison, WI, USA) were entrapped in 500 nmol NPs, dissolved in PDF, and injected, via the intraperitoneal route, into mice. One hour after injection, tissues from the peritoneum, liver and kidneys were purified, cut and fixed with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, containing 25 mM CaCl2 at room temperature. Fixed tissues were immersed and embedded in Tissue-Tek optimal cutting temperature compound which is a formulation of water soluble glycols and resins, and frozen in liquid nitrogen. Cryostat sections of thickness 7–8 μm were mounted on silanecoated glass slides (Matsunami, Osaka, Japan) and air-dried. Then, fluorescence was detected by observation under a Fluorescence Microscope (BH2-RFL-T3 and BX50; Olympus, Shinjuku, Tokyo, Japan), and processed with Adobe Photoshop software (Adobe Systems, San Jose, CA, USA). To investigate the treatment effects of siRNAs entrapped in NPs in PF, 100 μl of TGF-β1-siRNAs (5 nmol) entrapped in 500 nmol NPs (TGF-β1siRNAs-NPs) were dissolved in 100 ml kg − 1 PDF, and injected, via the intraperitoneal route, into mice (PF+TGF-β1-siRNAs-NPs) from the one day before methylglyoxal injection at a frequency of three times per week for 2 weeks separately from methylglyoxal injection. The following groups served as controls: mice without any treatment (Mock); mice injected with PDF without NPs or siRNAs (PF); mice injected with control-siRNAs entrapped in NPs by the same methods as for the TGF-β1-siRNAs-NPs (PF +control-siRNAs-NPs); and mice injected with 100 μl of TGF-β1-siRNAs (5 nmol) alone dissolved in PDF (PF+TGF-β1-siRNAs). Mice in all groups were killed 14 days after carrying out peritoneal equilibrium tests (as described Gene Therapy (2015) 1 – 8
below). Then, peritoneal tissues were removed and purified after washing with phosphate-buffered saline (PBS).
Peritoneal equilibrium test The details of the peritoneal equilibrium test have been described previously.31 Briefly, 5 ml of standard PDF containing 2.5% glucose (Midperic; Terumo) was injected via the intraperitoneal route. The dialysate was collected 1 h later to analyze the peritoneal drainage volume and concentration of creatinine (D-Cr). At that time, blood samples were also collected from the inferior vena cava to measure plasma creatinine concentration (P-Cr). Then, the ratio of creatinine concentration in the dialysate to that in plasma (D/P-Cr) was calculated.
Electrophoresis To investigate the encapsulation of siRNAs in NPs, 1 μl of siRNAs (50 μM) encapsulated in NPs (500 nmol) diluted with 4 μl of DNase/RNase-free distilled water, 1 μl of siRNAs (50 μM) encapsulated in NPs (500 nmol) treated with 4 μl of 5% deoxycholate (Sigma–Aldrich) and 1 μl of siRNAs (50 μM) diluted with 4 μl of DNase/RNase-free distilled water were subjected to electrophoresis on polyacrylamide gels.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) Sections of peritoneum tissue were homogenized using a glass homogenizer and a filter column shredder (QIA Shredder; Qiagen, Valencia, CA, USA). © 2015 Macmillan Publishers Limited
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Figure 7. Effects of TGF-β1-siRNA-NPs on peritoneal functions. Results of PET analyses in each group. Each group: n = 6, values are the mean ± s.e. (error bars) of at least three independent experiments. PET, peritoneal equilibrium test; D/P-Cr, the ratio of creatinine concentration (Cr) in the dialysate (D) to that in plasma (P); NS, not significant; *P o0.05; **Po 0.01. Then, total RNA from the peritoneum tissue was isolated using an RNeasy Total RNA Isolation kit (Qiagen). A total of 100 ng of isolated RNA was reverse-transcribed using a Superscript III First-strand Synthesis system (Life Technologies) according to the manufacturer's instructions. Real-time RT-PCR was undertaken using SYBR Green ER qPCR Supermix (Life Technologies). All reactions were carried out in a reaction volume of 20 μl in duplicate. After initial denaturation at 95 °C for 10 s, real-time qRTPCR was carried out for 40 cycles by denaturation at 95 °C for 15 s and annealing and extension at 60 °C for 60 s. Primers for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-SMA, collagen 1A2, fibronectin 1, fibroblast-specific protein 1, IFN-β, STAT1 and OAS1 were purchased from Takara Bio Inc (Otsu, Shiga, Japan). Quantification of expression of the target gene was achieved using Step One software (Life Technologies). mRNA expression was normalized with GAPDH (endogenous control). Data are expressed as relative quantities compared with the Mock group.
Enzyme-linked immunosorbent assay (ELISA) for TGF-β1 An ELISA kit specific for mouse TGF-β1 (TGF-β1 ELISA; IBL, Hamburg, Germany) was used according to the manufacturer instructions. Briefly, 100 μl of dialysate sample was added to a plate pre-coated with monoclonal antibody against mouse TGF-β1. Then, anti-mouse TGF-β1 antibody conjugated to horseradish peroxidase was added and incubated for 4 h at room temperature. After washing, color was developed with 100 μl of substrate (tetramethyl benzidine). The reaction was terminated by addition of 1 M of phosphoric acid and absorbance measured at 450 nm.
Histologic analyses After perfusion with PBS, peritoneum tissues were removed and fixed in 4% paraformaldehyde overnight at 4 °C. They were embedded in paraffin, sectioned and subjected to Azan staining to evaluate the intensity of fibrotic changes. Collagen fibers were stained blue upon Azan staining. © 2015 Macmillan Publishers Limited
For evaluation of the degree of peritoneal thickening, the thickness of the submesothelial compact zone (membrane area extending from the lower limit of the mesothelial layer to the upper limit of the muscle layer) was measured in 10 fields of each Azan-stained sample chosen randomly at × 400 magnification using computerized image-analysis software (Image Pro 5.1, Media Cybernetics Inc., Rockville, MD, USA). The mean thickness of the submesothelial compact zone in each mouse was calculated and defined as the peritoneal thickness.
Immunofluorescent microscopy To evaluate cells that were positive for TGF-β1 in each group, paraffinembedded sections of peritoneum tissue were deparaffinized, rehydrated and autoclaved for 15 min at 121 °C in 10 mM citrate buffer (pH 6.0) to retrieve antigens, followed by incubation with 10% normal goat serum in PBS to block non-specific binding of antibodies. Then, sections of pretreated peritoneum tissue were incubated overnight at 4 °C with a rabbit polyclonal antibody against mouse TGF-β1 (Abcam, Cambridge, UK) at 1:100 dilution. After washing with PBS, sections were incubated for 2 h at room temperature with a monoclonal antibody against anti-rabbit IgG conjugated to Alexa 488 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) at 1:100 dilution. To evaluate cells that were positive for α-SMA, cytokeratin, CD45 and CD31, sections of pretreated peritoneum tissue were incubated overnight at 4 °C with a monoclonal antibody against mouse α-SMA conjugated to Cy3 (Sigma–Aldrich) at 1:400 dilution, cytokeratin (Acris Antibodies, San Diego, CA, USA) at 1:50 dilution, CD45 (Abcam) at 1:400 dilution or CD31 (Abcam) at 1:50 dilution. After washing with PBS, sections were incubated for 2 h at room temperature with a monoclonal antibody against anti-rabbit IgG conjugated to Alexa 488 (Jackson ImmunoResearch Laboratories) at 1:100 dilution. Then, all sections were mounted with VECTASHIELD mounting medium (Vector Laboratories Inc., Burlingame, CA, USA) using 4′,6-diamidino-2-phenylindole to stain the nucleus. Gene Therapy (2015) 1 – 8
Nanoparticle-based siRNA delivery for fibrosis H Yoshizawa et al
8 Laboratory methods Blood analyses were carried out by a clinical chemistry laboratory (SRL, Tokyo, Japan).
Statistical analyses Data are the mean ± s.e. Analysis of variance was employed to investigate differences among groups. If statistical significance was detected by analysis of variance, Tukey’s test was carried out as post hoc analyses to compare the mean values of two groups. SPSS v21 (IBM, Armonk, NY, USA) was used for statistical analyses. Po0.05 was considered significant.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We thank Motomu Shimaoka (Professor of Molecular Pathobiology, Mie University School of Medicine) for discussion and comments regarding the manuscript. This work was supported by JSPS KAKENHI (grant number 25461252) and MEXTSupported Program for Strategic Research Foundations at Private Universities, 2013– 2017 (S1211029).
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