http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–8 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.869635
ORIGINAL ARTICLE
Therapeutic effects of hybrid liposomes without drugs for rheumatoid arthritis Yoko Matsumoto, Hideaki Ichihara, Motoki Hino, Masayo Umebayashi, and Ryuichi Ueoka
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Division of Applied Life Science, Graduate School of Engineering, Sojo University, Kumamoto, Japan
Abstract
Keywords
Hybrid liposomes (HLs) can be prepared by simply sonicating a mixture of vesicular and micellar molecules in buffer solutions. This study aims to demonstrate inhibitory effects of HLs on the growth of fibroblast-like synoviocytes along with apoptosis and therapeutic effects of HLs in a mouse model with rheumatoid arthritis (RA). HLs composed of 95 mol% L--dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene(23)dodecyl ether (C12(EO)23) were prepared by the sonication method. The inhibitory effects of HLs on the growth of human fibroblast-like synoviocytes-RA (HFLS-RA) cells in vitro and their inhibitory mechanism were examined. High inhibitory effects of HLs on the growth of HFLS-RA cells were observed. The induction of apoptosis by HLs was revealed on the basis of flow cytometric analysis. Furthermore, therapeutic effects of HLs in the mouse model with RA were examined in vivo. Our results demonstrate that HLs showed inhibitory effects on the growth of HFLS-RA cells in vitro along with apoptosis and therapeutic effects in mouse models of RA in vivo.
Apoptosis, chemotherapy, fibroblast-like synoviocytes, hybrid liposome, in vivo, rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory, systemic, autoimmune disease of uncertain etiology, which occurs in nearly 1% of the adult population worldwide (Firestein, 2003). RA is characterized by synovial inflammation of multiple joints. The affected synovial tissues contain activated macrophages, fibroblasts, T lymphocytes and B lymphocytes. In response to proinflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a and IL-6, the synovial fibroblasts proliferate and release tissue-degrading enzymes (Feldmann et al., 1996; Scott et al., 2010). The resulting hyperplastic synovial membrane, termed pannus tissue, irreversibly destroys the cartilage and bone of the affected joints. The various biologics such as TNFa-blockers, cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig, anti-CD20 antibody and anti-IL-6 receptor antibody have been proven clinically efficacious and have provided tremendous benefit to RA patients (De Vita & Quartuccio, 2006; Betts et al., 2011). However, because of the relatively high cost, complicated regimen and side effects such as excessive immunosuppression of these drugs, alternative treatments to solve these problems are required. Apoptosis is an evolutionarily conserved cell death pathway that occurs in a variety of physiological situations. An apoptotic stimulus induces an initiation and commitment Address for correspondence: Yoko Matsumoto, Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan. Tel: +81 963263965. Fax: +81 963231331. Email: [email protected]
History Received 23 October 2013 Revised 22 November 2013 Accepted 22 November 2013
phase, followed by a degradation phase (Smith & Walker, 2004). This last stage is regulated by cysteine proteases (caspase-3, -8, -9). There are two central pathways that mediate apoptosis: (1) death receptor pathway generates an apoptotic signal following the aggregation of death ligands and (2) pathway signals through mitochondria (Smith & Walker, 2004). In some cases, death receptor pathway activation may also proceed down the mitochondrial pathway (Vaishnaw et al., 1997; Nakayama et al., 2012). We have prepared hybrid liposomes (HLs) by just the sonication of vesicular and micellar molecules in a buffer solution (Ueoka et al., 1985, 1988). HLs are free from any contamination with organic solvents and remain stable for longer periods. The physical properties of these liposomes such as size, membrane fluidity, phase transition temperature and hydrophobicity can be controlled by changing the constituents and compositional ratios of the HL. In the course of our study for HLs, the following interesting results have been obtained. (a) Inhibitory effects of HLs including antitumor drugs (Kitamura et al., 1996), sugar surfactants (Matsumoto et al., 2000) or polyunsaturated fatty acids (Goto et al., 2008) have been observed on the growth of tumor cells in vitro and in vivo. (b) High inhibitory effects of HLs on the growth of tumor cells in vitro (Iwamoto et al., 2005; Matsumoto et al., 2005) and in vivo (Shimoda et al., 2009; Ichihara et al., 2012) along with the induction of apoptosis have been observed without using any drugs. (c) The mechanistic details of apoptosis of tumor cells induced by HLs (Matsumoto et al., 2005) and the correlation between antitumor effects and membrane fluidity of HLs have been
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clarified (Komizu et al., 2006). (d) After receiving the approval of the Bioethics Committee, successful clinical chemotherapy with drug-free HLs to patients with lymphoma has been reported (Ichihara et al., 2008). In this study, we investigated the induction of apoptosis by HLs composed of 95 mol% L--dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene(23)dodecylether (C12(EO)23) for human fibroblast-like synoviocytes-RA (HFLS-RA) in vitro. Furthermore, the therapeutic effects of HLs in a mouse model with arthritis were examined in vivo.
Materials and methods
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Preparation of HLs
Apoptotic DNA measurements with flow cytometry The HFLS-RA cells treated with HLs for 24 h were centrifuged at 3000 rpm for 5 min, washed with Hanks’ Balanced Salt Solution and then fixed in chilled ethanol. The cells were washed again, treated with RNase (0.25 mg/ml) and then stained with propidium iodide (PI, 0.5 mg/ml) that has 493 nm excitation and 635 nm emission wavelength. The samples were analyzed using flow cytometer (Epics XL System, Beckman Coulter, Inc., Brea, CA) with a single excitation (488 nm) of 15 mW argon laser. The PI signals were detected by FL3 sensor in 605–635 nm. Apoptotic DNA rates were calculated by (apoptotic DNA content/DNA content) 100. Determination of caspase activation
HLs were prepared by the sonication of a mixture containing 95 mol% DMPC (NOF Co. Ltd., Tokyo, Japan) and 5 mol% C12(EO)23 (Sigma Aldrich Co., LLC., St Louis, MO), which is known to be a fusion accelerator (Skelley et al., 2009), using bath-type sonicator (VS-N300, VELVO-CLEAR, Tokyo, Japan) in 5% glucose solution at 45 C with 300 W, and filtered with a 0.20 mm cellulose acetate filter (Advantec, Tokyo, Japan). Dynamic light scattering measurements The diameter of HLs was measured with a light scattering spectrometer (Otsuka Electronics Co., Ltd., Osaka, Japan) using a He–Ne laser (633 nm) at a 90 scattering angle. The diameter (dhy) was calculated using the Stokes–Einstein formula (Equation (1)): dhy ¼ T=3D
ð1Þ
where is the Boltzmann constant, T is the absolute temperature, is the viscosity and D is the diffusion coefficient: Cell culture Human rheumatoid arthritis synoviocytes (HFLS-RA) and normal synoviocytes (HFLS) were obtained from Cell Applications, Inc. (San Diego, CA). Both were cultured in synoviocyte growth medium (Cell Applications, Inc., San Diego, CA). The cells were cultured at 37 C in humidified atmosphere containing 5% CO2. Inhibitory effects of HLs Fifty-percent inhibitory concentration (IC50) on the growth of HFLS-RA and HFLS cells was determined on the basis of WST-1 (2-methoxy-4-nitrophenyl-3-(4-nitrophenyl)-5(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt) assay (Cell Counting Kit-1, Dojindo Laboratories, Kumamoto, Japan). Cells (5.0 104 cells/ml) were seeded in 96-well plates and cultured in a 5% CO2 humidified incubator at 37 C for 24 h. Cells were cultured for 48 h after adding DMPC and HLs. WST-1 solution was added and incubated for 3 h. Absorbance at a wavelength of 450 nm was measured by spectrophotometer (Emax, Molecular Devices, LLC, Sunnyvale, CA). The inhibitory effects of HLs on the growth of HFLS-RA and HFLS cells were evaluated by Amean/Acontrol, where Amean and Acontrol denote the absorbance of water-soluble formazan in the presence and absence of HLs respectively.
The caspase-3 activity was measured as the protease activity of caspase-3 using the cell-permeable substrate of PhiPhiLux G1D2 (OncoImmunin, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Cells (5.0 104 cells) were seeded in 35-mm glass bottom dishes and cultured in 5% CO2 humidified incubator at 37 C for 24 h. Cells were cultured for 6 h after adding HLs ([DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM). Cells were cultured for 1 h after adding 10 mM PhiPhiLux solution including synoviocyte growth supplement. Cells were observed by confocal microscope (Leica TCS-SP, Heidelberg, Germany) using a 488-nm Ar laser (detection, 515–555 nm). Mitochondrial membrane potential Cells (5.0 104 cells) were treated with HLs ([DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM) for 1 h. 3,30 Dihexyloxacarbocyanine iodide (40 nM, DiOC6(3), Molecular Probe, Eugene, OR) was added to evaluate mitochondrial membrane potential (DCm), and the cells were incubated at 37 C for 30 min. The cells were centrifuged at 3000 rpm for 5 min, suspended with 500 ml of phosphate buffered saline (PBS[]) and were used for flow cytometric analysis using flow cytometer (Epics XL System II, Beckman Coulter, Inc., Brea, CA); 15 mW, 488 nm air-cooling Ar laser and FL1 (505–545 nm) (Beckman Coulter, Inc., Brea, CA) were used. Fusion and accumulation of HLs into the cell membrane The fusion and accumulation into the membrane of HFLS-RA and HFLS cells of HLs labeled with a fluorescence probe (1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn- glycero-3-phosphocholine (NBDPC; Avanti Polar Lipids, Inc., Alabama, AL) was performed using confocal laser microscopy (TCS-SP; Leica Microsystems, Berlin, Germany). Cells (3.5 105 cells/ml) were cultured in a 5% CO2-humidified incubator at 37 C for 24 h. The cells were treated with HLs ([DMPC] ¼ 0.45 mM, [C12(EO)23] ¼ 0.025 mM, [NBDPC] ¼ 0.020 mM) coupled with a fluorescence-labeled lipid for 4 h and were observed using confocal laser microscopy with a 488-nm Ar laser line (detection at 505–555 nm). Fluorescence depolarization method Membrane fluidity of intact cells was evaluated on the basis of fluorescence depolarization method with a fluorescent
DOI: 10.3109/10717544.2013.869635
probe 1,6-diphenyl-1,3,5-hexatriene (DPH) (Nacalai Tesque, Inc., Kyoto, Japan). After the preincubation of cells for 24 h, the cells were treated with 0.05% trypsin/EDTA and suspended in PBS(), and then DPH (0.1 mM) was added into the cell suspension (2.0 104 cells/ml). The cell suspension was allowed to stand for 15 min at 37 C, and the fluorescence polarization (P) of DPH was measured using a fluorescence spectrophotometer (F-2000, Hitachi, Japan) as described previously (Komizu et al., 2011a).
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Assessment of anti-RA activity in vivo The mice were handled in accordance with the guidelines for animal experimentation set out in Japanese law. The animal studies were approved by the Committee on Animal Research of Sojo University. SKG/Jcl mice were obtained from CLEA Japan, Inc. (Tokyo, Japan). Mice were reared under the room temperature (25 1 C) and 50 10% of humidity. SKG mice express the human RA-like characteristics such as the invasion of inflammatory cells and synovial cell proliferation with pannus formation and neovascularization (Sakaguchi et al., 2003). Purified beta-glucans such as curdlan and laminarin can trigger severe chronic arthritis in SKG mice. After a quarantine and acclimatization period, the mice were randomly grouped on the basis of body weight using the stratified randomization method. The number of mice was three in each group. Laminarin (Sigma-Aldrich Japan, Tokyo, Japan) was dissolved in sterile saline at 100 mg/ml. Mice were intraperitoneally injected with 0.3 ml (30 mg) laminarin solution. HLs (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) was intravenously administered in mice once a day for 14 days after the injection of laminarin, and then HLs in 5% glucose solution or 5% glucose solution alone (control) was administered once every 2 days for 36 weeks. Joint swelling was monitored by macroscopic inspection. Clinical assessment of arthritis in SKG mice Joint swelling was monitored by inspection and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of the wrist or ankle; and 1.0, severe swelling of the wrist or ankle. Scores for all fingers, toes, wrists, and ankles were totaled for each mouse (Yoshitomi et al., 2005).
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Immunostaining with anti-CD4 and TNF-a antibody Paraffin-embedded sections were cut, dewaxed in xylene and rehydrated through a series of ethanol to water. Tumor sections were heated at 120 C for 10 min for antigen activation and were blocked with a solution of PBS and 1% H2O2 for 5 min. The sections were washed with PBS() and incubated with antihuman/rat/mouse CD4 antibody (R&D Systems, Minneapolis, MN) and TNF-a antibody (R&D Systems) in a humidified box at 4 C overnight. The sections were washed twice with PBS and immunostained with rabbit antigoat immunoglobulin polyclonal antibody (HRP) overnight at 4 C. Finally, the detection of the antigen–antibody link was made through immunoperoxydase followed by 3,30 DAB chromogen. The sections were counterstained with hematoxylin, rinsed in distilled water and mounted. The finger sections were stained with HE and observed by optical microscope. Statistical analysis Results are presented as mean SD. Data were statistically analyzed using Student’s t test. A p value of less than 0.05 was considered to represent a statistically significant difference.
Results Physical properties of HLs We examined the morphology of HLs composed of 95 mol% DMPC and 5 mol% C12(EO)23 on the basis of dynamic light scattering measurements. The results are shown in Figure 1. Hydrodynamic diameter (dhy) of HLs was under 100 nm, which was stable for more than 1 month. On the other hand, DMPC liposomes were unstable and precipitated after 14 days. HLs were kept at the room temperature (25 C) due to the convenience of storage for a long period for clinical application. It is suggested that the diameter of HLs gradually decreased, because membrane fluidity of HLs that kept at room temperature near phase transition temperature (21 C) (Matsumoto et al., 1999) was gradually stabilized after a preparation at 45 C. It is worthy to note that HLs having 50 nm in diameter could avoid the reticular endothelial system
Histological analysis We histologically evaluated the therapeutic effects of HLs using the finger tissue from the mouse models of RA in vivo. HLs (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) was intravenously administered in mice once a day for 14 days after the injection of laminarin, and then HLs in 5% glucose solution or 5% glucose solution alone (control) was administered once every 2 days for 36 weeks. Fingers were removed from anesthetized mice immediately after the treatment with HLs and fixed in 10% formalin solution. The fingers were embedded in paraffin and sectioned at a thickness of 5 mm. The finger sections were stained with hematoxylin and eosin (HE) and observed by optical microscope (Nikon TS-100, Tokyo, Japan).
Figure 1. Time courses of dhy change for HLs. DMPC: [DMPC] ¼ 2.0 102 M, HLs: [DMPC] ¼ 2.0 102 M, [C12(EO)25] ¼ 1.05 103 M, in 5% glucose solution. Stored at 25 C.
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(Allen et al., 1991) and should be appropriate for in vivo and clinical applications after the intravenous administration.
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Inhibitory effects of HLs on the growth of human rheumatoid arthritis synoviocytes We examined the 50% inhibitory concentration of HLs on the growth of HFLS-RA in vitro. The results are shown in Figure 2. The IC50 value of HLs for HFLS-RA cells was 0.44 0.06 mM, whereas that for HFLS cells was 0.62 0.04 mM. The IC50 values of HLs on the growth of HFLS-RA cells were less than those of HFLS cells. How does HLs distinguish between HFLS-RA and HFLS cells? The fusion and accumulation of HLs toward HFLS-RA cells was examined using confocal laser microscope. The results are shown in Figure 3. An increase in the accumulation of NBDPC-labeled HLs into HFLS-RA cells was observed, though no accumulation of HLs into normal HFLS cells was obtained. Next, we examined membrane fluidity of HFLS-RA and HFLS cells using the fluorescent lipid probe DPH. As shown in Figure 4, the mean P values of DPH-labeled HFLS-RA cells were significantly decreased as compared to normal HFLS cells, indicating that HFLS-RA cell membrane was more fluid than normal HFLS ones.
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Induction of apoptosis We examined apoptotic DNA rates in HFLS-RA cells treated with HLs using flow cytometry (Figure 5). The apoptotic DNA rate of HLs for HFLS-RA cells was 24.8%, although that of DMPC liposomes was 1.5%. A high apoptotic DNA rate was obtained after the treatment with HLs, although fairly low apoptotic DNA rates were obtained in the case of DMPC liposomes. To investigate the apoptotic pathways of HLs on HFLS-RA cells, caspase-3 activity in HFLS-RA cells treated with HLs was measured using the cell-permeable fluorescence substrate by confocal microscope. Fluorescence micrographs of HFLSRA cells stained with the cell-permeable fluorescence caspase3 substrate after the treatment with HLs are shown in Figure 6. Interestingly, HFLS-RA cells were dyed in green after adding HLs, indicating that HLs activated caspase-3 of HFLS-RA cells, whereas the cells were not dyed when using the DMPC liposomes. Next, we examined the mitochondrial pathway for apoptotic signal transduction by HLs using flow cytometry. The results are shown in Figure 7. Interestingly, mitochondrial transmembrane potential was decreased after the treatment with HLs. Therapeutic effects of HLs mouse models of RA We examined the therapeutic effects of HLs using SKG mice as models of RA in vivo. The results are shown in Figure 8.
Figure 2. About 50% inhibitory concentration (IC50) for HLs on the growth of HFLS and HFLS-RA cells. Data presented are mean SD.
Figure 4. Fluorescence polarization (p values) change for DPH-labeled HFLS and HFLS-RA cells.
Figure 3. Fluorescence micrographs of HFLS and HFLS-RA cells treated with HLs coupled with NBDPC as fluorescence probe using confocal laser microscopy. Magnification: 400. [DMPC] ¼ 0.45 mM, [C12(EO)23] ¼ 0.025 mM, [NBDPC] ¼ 0.020 mM.
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DOI: 10.3109/10717544.2013.869635
No detection of rheumatic clinical score indicating joint swelling and deformity of fingers was obtained in the mice treated with HLs, although increased rheumatic score was observed in the control group. Furthermore, we histologically evaluated the therapeutic effects of HLs using the finger tissue from the mouse models of RA. HE staining images of the finger section of mouse models of RA treated with HLs are shown in Figure 9. It was confirmed that pannus, thickening synovial tissue, that covers articular cartilage was observed in the fingers of the untreated control group, indicating synoviocytic overgrowth after the supersecretion of inflammatory cytokine. On the other hand, no abnormal findings were observed in the fingers of the group treated with HLs. To elucidate the reduction of inflammatory infiltrate lymphocytes in pannus by HLs, we examined therapeutic effects of HLs by immunostaining using CD4 antibody. The results are shown in Figure 10. Many CD4-positive cells (brown color and arrows) group of control were observed apart from the cases of HLs. With regard to inflammatory cytokine, we examined finger sections by immunostaining using TNF-a antibody as shown in Figure 11. Interestingly,
Figure 5. Apoptotic DNA rates of HFLS-RA cells treated with HLs for 24 h. [DMPC] ¼ 0.45 mM, [C12(EO)23] ¼ 0.025 mM.
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Figure 7. Mitochondrial transmembrane potential (D m) disruption of mitochondria in HFLS-RA cells treated with HLs for 1 h. [DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM.
Figure 8. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis using score method. Data presented are mean SD. Dose for DMPC: 136 mg/kg.
Figure 6. Activation of caspase-3 of HFLS-RA cells treated with HLs for 6 h. Magnification: 800. [DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM.
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TNF-a positive cells were observed in fingers of mice models with RA in control, although the cells were not dyed when using HLs.
Discussion
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RA is a chronic immune disease that affects the joints and promotes joint destruction. The characteristics of RA are synovial fibroblast proliferation and macrophage infiltration, which were induced by chemokines and cytokines. Cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-a, are important mediators of inflammation and
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tissue destruction in RA. Chemotherapy with anti-RA drug such as disease modifying anti-rheumatic drugs (DMARDS) is effective for the treatment of patients with RA. Although anticancer drugs kill RA synoviocytes, they also damage normal synoviocytes, causing side effects. Therefore, chemotherapy along with the induction of apoptosis without any side effects should be desirable. It is well known that apoptosis is essential in many aspects of normal development and is required for maintaining tissue homeostasis. Consequently, control of apoptosis is an important potential target for therapeutic intervention.
Figure 9. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis on the basis of HE staining of tissue sections in fingers. Arrow: inflammatory infiltrate lymphocytes in pannus. Dose for DMPC: 136 mg/kg.
Figure 10. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis in tissue sections in fingers using immunostaining of CD4. Dose for DMPC: 136 mg/kg. Magnification: 400.
Figure 11. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis in tissue sections in fingers using immunostaining of TNF-a. Arrows: TNF-a positive cells. Dose for DMPC: 136 mg/kg. Magnification: 400.
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DOI: 10.3109/10717544.2013.869635
Morphological change in human promyelocytic leukemia (HL-60) cells after the treatment with HLs composed of phospholipids having the same hydrophilic head group (phosphatidylcholine group) and different hydrophobic alkyl chains (L--dilauroylphosphatidylcholine, C12: DLPC; C14: DMPC; L--dipalmitoylphosphatidylcholine, C16: DPPC) and C12(EO)23 using a time-laps video has been investigated (Nagami et al., 2006b). The formation of bleb and corpuscle indicating the characteristic feature of apoptosis was observed for HLs of 90 mol% DMPC/10 mol% C12(EO)23. On the other hand, swelling of cells and dissolving of cell membrane, that is, necrosis, were observed for HLs of 90 mol% DLPC/ 10 mol% C12(EO)23. Neither apoptosis nor necrosis was observed for HLs of 90 mol% DPPC/10 mol% C12(EO)23. Thus, two methylene groups of acyl chains in phosphatidylcholines could distinguish between apoptosis, and necrosis has already been reported (Nagami et al., 2006b). HFLS-RA treated with HLs had been positive on the basis of TUNEL method, indicating that HLs induced apoptosis for HFLS-RA cells, although apoptotic cells were not obtained using the DMPC liposomes (Ichihara et al., 2011). However, the apoptotic cells were not observed in normal HFLS cells after the treatment with HLs. Thus, HLs could distinguish between normal and HFLS-RA cells and induce apoptosis for HFLS-RA cells without affecting normal cells. The pathways of apoptosis induced by HLs of DMPC/ 10 mol% C12(EO)10 in human promyelocytic leukemia (HL60) cells have already been reported (Matsumoto et al., 2005). HLs fused and accumulated in HL-60 cell membranes, and the apoptotic signal first passed through the mitochondria, then caspase-9 and caspase-3, second through FAS, caspase-8 and caspase-3 and then reached the nucleus. We examined the induction of apoptosis for HFLS-RA cells by HLs. We investigated the apoptotic DNA rates in HFLS-RA cells treated with HLs using flow cytometer. A high apoptotic DNA rate was obtained after the treatment with HLs, although fairly low apoptotic DNA rates were obtained in the case of DMPC liposomes. To investigate the apoptotic pathways of HLs on HFLS-RA cells, caspase-3 activity in HFLS-RA cells treated with HLs was measured using a cell-permeable fluorescence substrate. Interestingly, HFLS-RA cells were dyed in green after adding HLs, indicating that HLs activated caspase-3 of HFLS-RA cells, although the cells were not dyed when using the DMPC liposomes. With regard to the mitochondrial pathway for apoptotic signal transduction by HLs, we examined using flow cytometry. Interestingly, mitochondrial transmembrane potential was decreased after the treatment with HLs, suggesting that the mitochondrial pathway should be also implicated in apoptosis induced by HLs. It is well known that the fluidity of cell membranes of disease such as tumor is generally larger than that of normal cells (Deliconstantinos, 1987; Sok et al., 1999). Steady-state fluorescence polarization studies with the fluorescent lipid probe (DPH) are utilized to determine the degree of fluidity of cellular membrane lipids in cells (Liebes et al., 1981; Komizu et al., 2006). Recently, we have found that the degree of membrane fluidity of human adult T-cell leukemia cells was greater than that of normal lymphocyte cells on the basis of fluorescence depolarization method (Komizu et al., 2011b).
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On the other hand, it is well known that a membrane fusion of lipid vesicles with mammalian cells could influence lipid fluidity in these membranes (Papahadjopoulos et al., 1973). Therefore, we examined the membrane fluidity of HFLS-RA and normal HFLS cells. The mean P values of DPH-labeled HFLS-RA cells were significantly decreased as compared to normal HFLS cells, indicating that HFLS-RA cell membranes are more fluid than those of normal synoviocytes. These results suggest that the inhibitory effects of the HLs on the growth of HFLS-RA cells should be related to membrane fluidity. We examined the therapeutic effects of HLs composed of 95 mol% DMPC and 5 mol% C12(EO)23 using SKG mice as models of RA in vivo. No detection of rheumatic score indicating joint swelling and deformity of fingers was obtained in the mice treated with HLs, although increased rheumatic score was observed in the control group. It is noteworthy that remarkable therapeutic effects were obtained in the mouse models of RA intravenously treated with HLs without drugs. Next, to elucidate reduction of inflammatory infiltrate lymphocytes in pannus by HLs, we examined therapeutic effects of HLs by immunostaining using CD4 antibody. Many CD4-positive cells in the group of control were observed apart from the cases of HLs. In inflammatory cytokine, we examined therapeutic effects of HLs by immunostaining using TNF-a antibody. Interestingly, TNF-a positive cells were observed in fingers of mice models of RA in control, although the cells were not dyed when using HLs. Elucidation of the molecular mechanisms of inflammatory cytokine downregulation by immunosuppressive functions of HLs is very important. Therefore, immunoregulation mechanism of HLs is under investigation in detail at present. These results suggest that the HL could inhibit the production of pannus by downregulation of inflammatory cytokine. No side effects of HLs on healthy rats have been obtained in vivo (Nagami et al., 2006a; Ichihara et al., 2008). HLs have been metabolized in the liver for 3 h after intravenous administration to healthy mice as described previously (Ichihara et al., 2008, 2012). In this study, no abnormal findings such as severe arthritis were observed in SKG mice for 36 weeks after the treatment with HLs. These results indicate that HLs circulated in blood for 3 h after intravenous administration to mice and affected in a short period of time RA-background synoviocyte and inflammatory cells along with apoptosis and then immediately metabolized in the liver. Thus, the successful therapy for model mice of RA using HLs without side effect and any anti-RA drug in this study should be important for clinical applications of patients with RA in the near future.
Conclusion We clearly demonstrated that HLs showed inhibitory effects on the growth of HFLS-RA cells in vitro along with apoptosis and therapeutic effects for mouse models of RA in vivo. The noteworthy aspects are as follows. (a) The IC50 values of HLs on the growth of HFLS-RA cells were remarkably less than those of the DMPC liposomes. (b) The induction of apoptosis by HLs was verified for HFLS-RA cells on the basis of flow cytometry and TUNEL method. (c) Therapeutic effects of
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HLs were obtained in mouse models of RA. It is noteworthy that remarkable inhibitory effects of drug-free HLs leading to apoptosis on the growth of synoviocyte causing rheumatoid arthritis were obtained for the first time. The results in this study should be advantageous in the chemotherapy for patients with RA in the near-future clinical applications.
Declaration of interest This work was supported in part by a Grant-in Aid for Science Research from the Ministry of Education, Science, and Culture of Japan (No. 23300173, 25289299, 25420843).
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Komizu Y, Yukihara M, Kariya R, et al. (2011b). Selective accumulation of hybrid liposomes into adult T-cell leukemia cells along with induction of apoptosis. Bioorg Med Chem Lett 21:3962–5. Liebes LF, Pelle E, Zucker-Franklin D, Silber R. (1981). Comparison of lipid composition and 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization measurements of hairy cells with monocytes and lymphocytes from normal subjects and patients with chronic lymphocytic leukemia. Cancer Res 41:4050–6. Matsumoto Y, Kato T, Iseki S, et al. (1999). Remarkably enhanced inhibitory effects of hybrid liposomes on the growth of specific tumor cells. Bioorg Med Chem Lett 9:1937–40. Matsumoto Y, Kato T, Suzuki H, et al. (2000). Highly specific inhibitory effect of three-component hybrid liposomes including sugar surfactants on the growth of glioma cells. Bioorg Med Chem Lett 10: 2617–19. Matsumoto Y, Iwamoto Y, Matsushita T, Ueoka R. (2005). Novel mechanism of hybrid liposomes-induced apoptosis in human tumor cells. Int J Cancer 115:377–82. Nagami H, Matsumoto Y, Ueoka R. (2006a). Chemotherapy with hybrid liposomes for lymphoma without drugs in vivo. Int J Pharm 315: 167–72. Nagami H, Nakano K, Ichihara H, et al. (2006b). Two methylene groups in phospholipids distinguish between apoptosis and necrosis for tumor cells. Bioorg Med Chem Lett 16:782–5. Nakayama H, Yaguchi T, Yoshiya S, Nishizaki T. (2012). Resveratrol induces apoptosis MH7A human rheumatoid arthritis synovial cells in a sirtuin 1-dependent manner. Rheumatol Int 32:151–7. Papahadjopoulos D, Poste G, Schaeffer BE. (1973). Fusion of mammalian cells by unilamellar lipid vesicles: influence of lipid surface charge, fluidity and cholesterol. Biochim Biophys Acta 323: 23–42. Sakaguchi N, Takahashi T, Hata H, et al. (2003). Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature 426:454–60. Scott DL, Wolfe F, Huizinga TW. (2010). Rheumatoid arthritis. Lancet 376:1094–108. Shimoda S, Ichihara H, Matsumoto Y, Ueoka R. (2009). Chemotherapy with hybrid liposomes for human breast tumors along with apoptosis in vivo. Int J Pharm 372:162–8. Skelley AM, Kirak O, Suh H, et al. (2009). Microfluidic control of cell pairing and fusion. Nat Methods 6:147–52. Smith MD, Walker JG. (2004). Apoptosis: a relevant therapeutic target in rheumatoid arthritis? Rheumatology 43:405–7. Sok M, Sentjurc M, Schara M. (1999). Membrane fluidity characteristics of human lung cancer. Cancer Lett 139:215–20. Ueoka R, Moss RA, Swarup S, et al. (1985). Extraordinary micellar enantioselectivity coupled to altered aggregate structure. J Am Chem Soc 107:2185–6. Ueoka R, Matsumoto Y, Moss RA, et al. (1988). Membrane matrix for the hydrolysis of amino acid esters with marked enantioselectivity. J Am Chem Soc 110:1588–95. Vaishnaw AK, McNally JD, Elkon KB. (1997). Apoptosis in the rheumatic diseases. Arthritis Rheum 40:1917–27. Yoshitomi H, Sakaguchi N, Kobayashi K, et al. (2005). A role for fungal b-glucans and their receptor Dectin-1 in the induction of autoimmune arthritis in genetically susceptible mice. J Exp Med 201: 949–60.
ORIGINAL ARTICLE
Therapeutic effects of hybrid liposomes without drugs for rheumatoid arthritis Yoko Matsumoto, Hideaki Ichihara, Motoki Hino, Masayo Umebayashi, and Ryuichi Ueoka
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Division of Applied Life Science, Graduate School of Engineering, Sojo University, Kumamoto, Japan
Abstract
Keywords
Hybrid liposomes (HLs) can be prepared by simply sonicating a mixture of vesicular and micellar molecules in buffer solutions. This study aims to demonstrate inhibitory effects of HLs on the growth of fibroblast-like synoviocytes along with apoptosis and therapeutic effects of HLs in a mouse model with rheumatoid arthritis (RA). HLs composed of 95 mol% L--dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene(23)dodecyl ether (C12(EO)23) were prepared by the sonication method. The inhibitory effects of HLs on the growth of human fibroblast-like synoviocytes-RA (HFLS-RA) cells in vitro and their inhibitory mechanism were examined. High inhibitory effects of HLs on the growth of HFLS-RA cells were observed. The induction of apoptosis by HLs was revealed on the basis of flow cytometric analysis. Furthermore, therapeutic effects of HLs in the mouse model with RA were examined in vivo. Our results demonstrate that HLs showed inhibitory effects on the growth of HFLS-RA cells in vitro along with apoptosis and therapeutic effects in mouse models of RA in vivo.
Apoptosis, chemotherapy, fibroblast-like synoviocytes, hybrid liposome, in vivo, rheumatoid arthritis
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory, systemic, autoimmune disease of uncertain etiology, which occurs in nearly 1% of the adult population worldwide (Firestein, 2003). RA is characterized by synovial inflammation of multiple joints. The affected synovial tissues contain activated macrophages, fibroblasts, T lymphocytes and B lymphocytes. In response to proinflammatory cytokines such as interleukin (IL)-1b, tumor necrosis factor (TNF)-a and IL-6, the synovial fibroblasts proliferate and release tissue-degrading enzymes (Feldmann et al., 1996; Scott et al., 2010). The resulting hyperplastic synovial membrane, termed pannus tissue, irreversibly destroys the cartilage and bone of the affected joints. The various biologics such as TNFa-blockers, cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig, anti-CD20 antibody and anti-IL-6 receptor antibody have been proven clinically efficacious and have provided tremendous benefit to RA patients (De Vita & Quartuccio, 2006; Betts et al., 2011). However, because of the relatively high cost, complicated regimen and side effects such as excessive immunosuppression of these drugs, alternative treatments to solve these problems are required. Apoptosis is an evolutionarily conserved cell death pathway that occurs in a variety of physiological situations. An apoptotic stimulus induces an initiation and commitment Address for correspondence: Yoko Matsumoto, Division of Applied Life Science, Graduate School of Engineering, Sojo University, 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082, Japan. Tel: +81 963263965. Fax: +81 963231331. Email: [email protected]
History Received 23 October 2013 Revised 22 November 2013 Accepted 22 November 2013
phase, followed by a degradation phase (Smith & Walker, 2004). This last stage is regulated by cysteine proteases (caspase-3, -8, -9). There are two central pathways that mediate apoptosis: (1) death receptor pathway generates an apoptotic signal following the aggregation of death ligands and (2) pathway signals through mitochondria (Smith & Walker, 2004). In some cases, death receptor pathway activation may also proceed down the mitochondrial pathway (Vaishnaw et al., 1997; Nakayama et al., 2012). We have prepared hybrid liposomes (HLs) by just the sonication of vesicular and micellar molecules in a buffer solution (Ueoka et al., 1985, 1988). HLs are free from any contamination with organic solvents and remain stable for longer periods. The physical properties of these liposomes such as size, membrane fluidity, phase transition temperature and hydrophobicity can be controlled by changing the constituents and compositional ratios of the HL. In the course of our study for HLs, the following interesting results have been obtained. (a) Inhibitory effects of HLs including antitumor drugs (Kitamura et al., 1996), sugar surfactants (Matsumoto et al., 2000) or polyunsaturated fatty acids (Goto et al., 2008) have been observed on the growth of tumor cells in vitro and in vivo. (b) High inhibitory effects of HLs on the growth of tumor cells in vitro (Iwamoto et al., 2005; Matsumoto et al., 2005) and in vivo (Shimoda et al., 2009; Ichihara et al., 2012) along with the induction of apoptosis have been observed without using any drugs. (c) The mechanistic details of apoptosis of tumor cells induced by HLs (Matsumoto et al., 2005) and the correlation between antitumor effects and membrane fluidity of HLs have been
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clarified (Komizu et al., 2006). (d) After receiving the approval of the Bioethics Committee, successful clinical chemotherapy with drug-free HLs to patients with lymphoma has been reported (Ichihara et al., 2008). In this study, we investigated the induction of apoptosis by HLs composed of 95 mol% L--dimyristoylphosphatidylcholine (DMPC) and 5 mol% polyoxyethylene(23)dodecylether (C12(EO)23) for human fibroblast-like synoviocytes-RA (HFLS-RA) in vitro. Furthermore, the therapeutic effects of HLs in a mouse model with arthritis were examined in vivo.
Materials and methods
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Preparation of HLs
Apoptotic DNA measurements with flow cytometry The HFLS-RA cells treated with HLs for 24 h were centrifuged at 3000 rpm for 5 min, washed with Hanks’ Balanced Salt Solution and then fixed in chilled ethanol. The cells were washed again, treated with RNase (0.25 mg/ml) and then stained with propidium iodide (PI, 0.5 mg/ml) that has 493 nm excitation and 635 nm emission wavelength. The samples were analyzed using flow cytometer (Epics XL System, Beckman Coulter, Inc., Brea, CA) with a single excitation (488 nm) of 15 mW argon laser. The PI signals were detected by FL3 sensor in 605–635 nm. Apoptotic DNA rates were calculated by (apoptotic DNA content/DNA content) 100. Determination of caspase activation
HLs were prepared by the sonication of a mixture containing 95 mol% DMPC (NOF Co. Ltd., Tokyo, Japan) and 5 mol% C12(EO)23 (Sigma Aldrich Co., LLC., St Louis, MO), which is known to be a fusion accelerator (Skelley et al., 2009), using bath-type sonicator (VS-N300, VELVO-CLEAR, Tokyo, Japan) in 5% glucose solution at 45 C with 300 W, and filtered with a 0.20 mm cellulose acetate filter (Advantec, Tokyo, Japan). Dynamic light scattering measurements The diameter of HLs was measured with a light scattering spectrometer (Otsuka Electronics Co., Ltd., Osaka, Japan) using a He–Ne laser (633 nm) at a 90 scattering angle. The diameter (dhy) was calculated using the Stokes–Einstein formula (Equation (1)): dhy ¼ T=3D
ð1Þ
where is the Boltzmann constant, T is the absolute temperature, is the viscosity and D is the diffusion coefficient: Cell culture Human rheumatoid arthritis synoviocytes (HFLS-RA) and normal synoviocytes (HFLS) were obtained from Cell Applications, Inc. (San Diego, CA). Both were cultured in synoviocyte growth medium (Cell Applications, Inc., San Diego, CA). The cells were cultured at 37 C in humidified atmosphere containing 5% CO2. Inhibitory effects of HLs Fifty-percent inhibitory concentration (IC50) on the growth of HFLS-RA and HFLS cells was determined on the basis of WST-1 (2-methoxy-4-nitrophenyl-3-(4-nitrophenyl)-5(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt) assay (Cell Counting Kit-1, Dojindo Laboratories, Kumamoto, Japan). Cells (5.0 104 cells/ml) were seeded in 96-well plates and cultured in a 5% CO2 humidified incubator at 37 C for 24 h. Cells were cultured for 48 h after adding DMPC and HLs. WST-1 solution was added and incubated for 3 h. Absorbance at a wavelength of 450 nm was measured by spectrophotometer (Emax, Molecular Devices, LLC, Sunnyvale, CA). The inhibitory effects of HLs on the growth of HFLS-RA and HFLS cells were evaluated by Amean/Acontrol, where Amean and Acontrol denote the absorbance of water-soluble formazan in the presence and absence of HLs respectively.
The caspase-3 activity was measured as the protease activity of caspase-3 using the cell-permeable substrate of PhiPhiLux G1D2 (OncoImmunin, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Cells (5.0 104 cells) were seeded in 35-mm glass bottom dishes and cultured in 5% CO2 humidified incubator at 37 C for 24 h. Cells were cultured for 6 h after adding HLs ([DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM). Cells were cultured for 1 h after adding 10 mM PhiPhiLux solution including synoviocyte growth supplement. Cells were observed by confocal microscope (Leica TCS-SP, Heidelberg, Germany) using a 488-nm Ar laser (detection, 515–555 nm). Mitochondrial membrane potential Cells (5.0 104 cells) were treated with HLs ([DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM) for 1 h. 3,30 Dihexyloxacarbocyanine iodide (40 nM, DiOC6(3), Molecular Probe, Eugene, OR) was added to evaluate mitochondrial membrane potential (DCm), and the cells were incubated at 37 C for 30 min. The cells were centrifuged at 3000 rpm for 5 min, suspended with 500 ml of phosphate buffered saline (PBS[]) and were used for flow cytometric analysis using flow cytometer (Epics XL System II, Beckman Coulter, Inc., Brea, CA); 15 mW, 488 nm air-cooling Ar laser and FL1 (505–545 nm) (Beckman Coulter, Inc., Brea, CA) were used. Fusion and accumulation of HLs into the cell membrane The fusion and accumulation into the membrane of HFLS-RA and HFLS cells of HLs labeled with a fluorescence probe (1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn- glycero-3-phosphocholine (NBDPC; Avanti Polar Lipids, Inc., Alabama, AL) was performed using confocal laser microscopy (TCS-SP; Leica Microsystems, Berlin, Germany). Cells (3.5 105 cells/ml) were cultured in a 5% CO2-humidified incubator at 37 C for 24 h. The cells were treated with HLs ([DMPC] ¼ 0.45 mM, [C12(EO)23] ¼ 0.025 mM, [NBDPC] ¼ 0.020 mM) coupled with a fluorescence-labeled lipid for 4 h and were observed using confocal laser microscopy with a 488-nm Ar laser line (detection at 505–555 nm). Fluorescence depolarization method Membrane fluidity of intact cells was evaluated on the basis of fluorescence depolarization method with a fluorescent
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probe 1,6-diphenyl-1,3,5-hexatriene (DPH) (Nacalai Tesque, Inc., Kyoto, Japan). After the preincubation of cells for 24 h, the cells were treated with 0.05% trypsin/EDTA and suspended in PBS(), and then DPH (0.1 mM) was added into the cell suspension (2.0 104 cells/ml). The cell suspension was allowed to stand for 15 min at 37 C, and the fluorescence polarization (P) of DPH was measured using a fluorescence spectrophotometer (F-2000, Hitachi, Japan) as described previously (Komizu et al., 2011a).
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Assessment of anti-RA activity in vivo The mice were handled in accordance with the guidelines for animal experimentation set out in Japanese law. The animal studies were approved by the Committee on Animal Research of Sojo University. SKG/Jcl mice were obtained from CLEA Japan, Inc. (Tokyo, Japan). Mice were reared under the room temperature (25 1 C) and 50 10% of humidity. SKG mice express the human RA-like characteristics such as the invasion of inflammatory cells and synovial cell proliferation with pannus formation and neovascularization (Sakaguchi et al., 2003). Purified beta-glucans such as curdlan and laminarin can trigger severe chronic arthritis in SKG mice. After a quarantine and acclimatization period, the mice were randomly grouped on the basis of body weight using the stratified randomization method. The number of mice was three in each group. Laminarin (Sigma-Aldrich Japan, Tokyo, Japan) was dissolved in sterile saline at 100 mg/ml. Mice were intraperitoneally injected with 0.3 ml (30 mg) laminarin solution. HLs (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) was intravenously administered in mice once a day for 14 days after the injection of laminarin, and then HLs in 5% glucose solution or 5% glucose solution alone (control) was administered once every 2 days for 36 weeks. Joint swelling was monitored by macroscopic inspection. Clinical assessment of arthritis in SKG mice Joint swelling was monitored by inspection and scored as follows: 0, no joint swelling; 0.1, swelling of one finger joint; 0.5, mild swelling of the wrist or ankle; and 1.0, severe swelling of the wrist or ankle. Scores for all fingers, toes, wrists, and ankles were totaled for each mouse (Yoshitomi et al., 2005).
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Immunostaining with anti-CD4 and TNF-a antibody Paraffin-embedded sections were cut, dewaxed in xylene and rehydrated through a series of ethanol to water. Tumor sections were heated at 120 C for 10 min for antigen activation and were blocked with a solution of PBS and 1% H2O2 for 5 min. The sections were washed with PBS() and incubated with antihuman/rat/mouse CD4 antibody (R&D Systems, Minneapolis, MN) and TNF-a antibody (R&D Systems) in a humidified box at 4 C overnight. The sections were washed twice with PBS and immunostained with rabbit antigoat immunoglobulin polyclonal antibody (HRP) overnight at 4 C. Finally, the detection of the antigen–antibody link was made through immunoperoxydase followed by 3,30 DAB chromogen. The sections were counterstained with hematoxylin, rinsed in distilled water and mounted. The finger sections were stained with HE and observed by optical microscope. Statistical analysis Results are presented as mean SD. Data were statistically analyzed using Student’s t test. A p value of less than 0.05 was considered to represent a statistically significant difference.
Results Physical properties of HLs We examined the morphology of HLs composed of 95 mol% DMPC and 5 mol% C12(EO)23 on the basis of dynamic light scattering measurements. The results are shown in Figure 1. Hydrodynamic diameter (dhy) of HLs was under 100 nm, which was stable for more than 1 month. On the other hand, DMPC liposomes were unstable and precipitated after 14 days. HLs were kept at the room temperature (25 C) due to the convenience of storage for a long period for clinical application. It is suggested that the diameter of HLs gradually decreased, because membrane fluidity of HLs that kept at room temperature near phase transition temperature (21 C) (Matsumoto et al., 1999) was gradually stabilized after a preparation at 45 C. It is worthy to note that HLs having 50 nm in diameter could avoid the reticular endothelial system
Histological analysis We histologically evaluated the therapeutic effects of HLs using the finger tissue from the mouse models of RA in vivo. HLs (dose for DMPC, 136 mg/kg) in 5% glucose solution or 5% glucose solution alone (control) was intravenously administered in mice once a day for 14 days after the injection of laminarin, and then HLs in 5% glucose solution or 5% glucose solution alone (control) was administered once every 2 days for 36 weeks. Fingers were removed from anesthetized mice immediately after the treatment with HLs and fixed in 10% formalin solution. The fingers were embedded in paraffin and sectioned at a thickness of 5 mm. The finger sections were stained with hematoxylin and eosin (HE) and observed by optical microscope (Nikon TS-100, Tokyo, Japan).
Figure 1. Time courses of dhy change for HLs. DMPC: [DMPC] ¼ 2.0 102 M, HLs: [DMPC] ¼ 2.0 102 M, [C12(EO)25] ¼ 1.05 103 M, in 5% glucose solution. Stored at 25 C.
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(Allen et al., 1991) and should be appropriate for in vivo and clinical applications after the intravenous administration.
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Inhibitory effects of HLs on the growth of human rheumatoid arthritis synoviocytes We examined the 50% inhibitory concentration of HLs on the growth of HFLS-RA in vitro. The results are shown in Figure 2. The IC50 value of HLs for HFLS-RA cells was 0.44 0.06 mM, whereas that for HFLS cells was 0.62 0.04 mM. The IC50 values of HLs on the growth of HFLS-RA cells were less than those of HFLS cells. How does HLs distinguish between HFLS-RA and HFLS cells? The fusion and accumulation of HLs toward HFLS-RA cells was examined using confocal laser microscope. The results are shown in Figure 3. An increase in the accumulation of NBDPC-labeled HLs into HFLS-RA cells was observed, though no accumulation of HLs into normal HFLS cells was obtained. Next, we examined membrane fluidity of HFLS-RA and HFLS cells using the fluorescent lipid probe DPH. As shown in Figure 4, the mean P values of DPH-labeled HFLS-RA cells were significantly decreased as compared to normal HFLS cells, indicating that HFLS-RA cell membrane was more fluid than normal HFLS ones.
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Induction of apoptosis We examined apoptotic DNA rates in HFLS-RA cells treated with HLs using flow cytometry (Figure 5). The apoptotic DNA rate of HLs for HFLS-RA cells was 24.8%, although that of DMPC liposomes was 1.5%. A high apoptotic DNA rate was obtained after the treatment with HLs, although fairly low apoptotic DNA rates were obtained in the case of DMPC liposomes. To investigate the apoptotic pathways of HLs on HFLS-RA cells, caspase-3 activity in HFLS-RA cells treated with HLs was measured using the cell-permeable fluorescence substrate by confocal microscope. Fluorescence micrographs of HFLSRA cells stained with the cell-permeable fluorescence caspase3 substrate after the treatment with HLs are shown in Figure 6. Interestingly, HFLS-RA cells were dyed in green after adding HLs, indicating that HLs activated caspase-3 of HFLS-RA cells, whereas the cells were not dyed when using the DMPC liposomes. Next, we examined the mitochondrial pathway for apoptotic signal transduction by HLs using flow cytometry. The results are shown in Figure 7. Interestingly, mitochondrial transmembrane potential was decreased after the treatment with HLs. Therapeutic effects of HLs mouse models of RA We examined the therapeutic effects of HLs using SKG mice as models of RA in vivo. The results are shown in Figure 8.
Figure 2. About 50% inhibitory concentration (IC50) for HLs on the growth of HFLS and HFLS-RA cells. Data presented are mean SD.
Figure 4. Fluorescence polarization (p values) change for DPH-labeled HFLS and HFLS-RA cells.
Figure 3. Fluorescence micrographs of HFLS and HFLS-RA cells treated with HLs coupled with NBDPC as fluorescence probe using confocal laser microscopy. Magnification: 400. [DMPC] ¼ 0.45 mM, [C12(EO)23] ¼ 0.025 mM, [NBDPC] ¼ 0.020 mM.
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DOI: 10.3109/10717544.2013.869635
No detection of rheumatic clinical score indicating joint swelling and deformity of fingers was obtained in the mice treated with HLs, although increased rheumatic score was observed in the control group. Furthermore, we histologically evaluated the therapeutic effects of HLs using the finger tissue from the mouse models of RA. HE staining images of the finger section of mouse models of RA treated with HLs are shown in Figure 9. It was confirmed that pannus, thickening synovial tissue, that covers articular cartilage was observed in the fingers of the untreated control group, indicating synoviocytic overgrowth after the supersecretion of inflammatory cytokine. On the other hand, no abnormal findings were observed in the fingers of the group treated with HLs. To elucidate the reduction of inflammatory infiltrate lymphocytes in pannus by HLs, we examined therapeutic effects of HLs by immunostaining using CD4 antibody. The results are shown in Figure 10. Many CD4-positive cells (brown color and arrows) group of control were observed apart from the cases of HLs. With regard to inflammatory cytokine, we examined finger sections by immunostaining using TNF-a antibody as shown in Figure 11. Interestingly,
Figure 5. Apoptotic DNA rates of HFLS-RA cells treated with HLs for 24 h. [DMPC] ¼ 0.45 mM, [C12(EO)23] ¼ 0.025 mM.
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Figure 7. Mitochondrial transmembrane potential (D m) disruption of mitochondria in HFLS-RA cells treated with HLs for 1 h. [DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM.
Figure 8. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis using score method. Data presented are mean SD. Dose for DMPC: 136 mg/kg.
Figure 6. Activation of caspase-3 of HFLS-RA cells treated with HLs for 6 h. Magnification: 800. [DMPC] ¼ 11.5 mM, [C12(EO)23] ¼ 0.61 mM.
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TNF-a positive cells were observed in fingers of mice models with RA in control, although the cells were not dyed when using HLs.
Discussion
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RA is a chronic immune disease that affects the joints and promotes joint destruction. The characteristics of RA are synovial fibroblast proliferation and macrophage infiltration, which were induced by chemokines and cytokines. Cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF)-a, are important mediators of inflammation and
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tissue destruction in RA. Chemotherapy with anti-RA drug such as disease modifying anti-rheumatic drugs (DMARDS) is effective for the treatment of patients with RA. Although anticancer drugs kill RA synoviocytes, they also damage normal synoviocytes, causing side effects. Therefore, chemotherapy along with the induction of apoptosis without any side effects should be desirable. It is well known that apoptosis is essential in many aspects of normal development and is required for maintaining tissue homeostasis. Consequently, control of apoptosis is an important potential target for therapeutic intervention.
Figure 9. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis on the basis of HE staining of tissue sections in fingers. Arrow: inflammatory infiltrate lymphocytes in pannus. Dose for DMPC: 136 mg/kg.
Figure 10. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis in tissue sections in fingers using immunostaining of CD4. Dose for DMPC: 136 mg/kg. Magnification: 400.
Figure 11. Therapeutic effects of HLs on the SKG mice with rheumatoid arthritis in tissue sections in fingers using immunostaining of TNF-a. Arrows: TNF-a positive cells. Dose for DMPC: 136 mg/kg. Magnification: 400.
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DOI: 10.3109/10717544.2013.869635
Morphological change in human promyelocytic leukemia (HL-60) cells after the treatment with HLs composed of phospholipids having the same hydrophilic head group (phosphatidylcholine group) and different hydrophobic alkyl chains (L--dilauroylphosphatidylcholine, C12: DLPC; C14: DMPC; L--dipalmitoylphosphatidylcholine, C16: DPPC) and C12(EO)23 using a time-laps video has been investigated (Nagami et al., 2006b). The formation of bleb and corpuscle indicating the characteristic feature of apoptosis was observed for HLs of 90 mol% DMPC/10 mol% C12(EO)23. On the other hand, swelling of cells and dissolving of cell membrane, that is, necrosis, were observed for HLs of 90 mol% DLPC/ 10 mol% C12(EO)23. Neither apoptosis nor necrosis was observed for HLs of 90 mol% DPPC/10 mol% C12(EO)23. Thus, two methylene groups of acyl chains in phosphatidylcholines could distinguish between apoptosis, and necrosis has already been reported (Nagami et al., 2006b). HFLS-RA treated with HLs had been positive on the basis of TUNEL method, indicating that HLs induced apoptosis for HFLS-RA cells, although apoptotic cells were not obtained using the DMPC liposomes (Ichihara et al., 2011). However, the apoptotic cells were not observed in normal HFLS cells after the treatment with HLs. Thus, HLs could distinguish between normal and HFLS-RA cells and induce apoptosis for HFLS-RA cells without affecting normal cells. The pathways of apoptosis induced by HLs of DMPC/ 10 mol% C12(EO)10 in human promyelocytic leukemia (HL60) cells have already been reported (Matsumoto et al., 2005). HLs fused and accumulated in HL-60 cell membranes, and the apoptotic signal first passed through the mitochondria, then caspase-9 and caspase-3, second through FAS, caspase-8 and caspase-3 and then reached the nucleus. We examined the induction of apoptosis for HFLS-RA cells by HLs. We investigated the apoptotic DNA rates in HFLS-RA cells treated with HLs using flow cytometer. A high apoptotic DNA rate was obtained after the treatment with HLs, although fairly low apoptotic DNA rates were obtained in the case of DMPC liposomes. To investigate the apoptotic pathways of HLs on HFLS-RA cells, caspase-3 activity in HFLS-RA cells treated with HLs was measured using a cell-permeable fluorescence substrate. Interestingly, HFLS-RA cells were dyed in green after adding HLs, indicating that HLs activated caspase-3 of HFLS-RA cells, although the cells were not dyed when using the DMPC liposomes. With regard to the mitochondrial pathway for apoptotic signal transduction by HLs, we examined using flow cytometry. Interestingly, mitochondrial transmembrane potential was decreased after the treatment with HLs, suggesting that the mitochondrial pathway should be also implicated in apoptosis induced by HLs. It is well known that the fluidity of cell membranes of disease such as tumor is generally larger than that of normal cells (Deliconstantinos, 1987; Sok et al., 1999). Steady-state fluorescence polarization studies with the fluorescent lipid probe (DPH) are utilized to determine the degree of fluidity of cellular membrane lipids in cells (Liebes et al., 1981; Komizu et al., 2006). Recently, we have found that the degree of membrane fluidity of human adult T-cell leukemia cells was greater than that of normal lymphocyte cells on the basis of fluorescence depolarization method (Komizu et al., 2011b).
Therapeutic effects of hybrid liposomes for rheumatoid arthritis
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On the other hand, it is well known that a membrane fusion of lipid vesicles with mammalian cells could influence lipid fluidity in these membranes (Papahadjopoulos et al., 1973). Therefore, we examined the membrane fluidity of HFLS-RA and normal HFLS cells. The mean P values of DPH-labeled HFLS-RA cells were significantly decreased as compared to normal HFLS cells, indicating that HFLS-RA cell membranes are more fluid than those of normal synoviocytes. These results suggest that the inhibitory effects of the HLs on the growth of HFLS-RA cells should be related to membrane fluidity. We examined the therapeutic effects of HLs composed of 95 mol% DMPC and 5 mol% C12(EO)23 using SKG mice as models of RA in vivo. No detection of rheumatic score indicating joint swelling and deformity of fingers was obtained in the mice treated with HLs, although increased rheumatic score was observed in the control group. It is noteworthy that remarkable therapeutic effects were obtained in the mouse models of RA intravenously treated with HLs without drugs. Next, to elucidate reduction of inflammatory infiltrate lymphocytes in pannus by HLs, we examined therapeutic effects of HLs by immunostaining using CD4 antibody. Many CD4-positive cells in the group of control were observed apart from the cases of HLs. In inflammatory cytokine, we examined therapeutic effects of HLs by immunostaining using TNF-a antibody. Interestingly, TNF-a positive cells were observed in fingers of mice models of RA in control, although the cells were not dyed when using HLs. Elucidation of the molecular mechanisms of inflammatory cytokine downregulation by immunosuppressive functions of HLs is very important. Therefore, immunoregulation mechanism of HLs is under investigation in detail at present. These results suggest that the HL could inhibit the production of pannus by downregulation of inflammatory cytokine. No side effects of HLs on healthy rats have been obtained in vivo (Nagami et al., 2006a; Ichihara et al., 2008). HLs have been metabolized in the liver for 3 h after intravenous administration to healthy mice as described previously (Ichihara et al., 2008, 2012). In this study, no abnormal findings such as severe arthritis were observed in SKG mice for 36 weeks after the treatment with HLs. These results indicate that HLs circulated in blood for 3 h after intravenous administration to mice and affected in a short period of time RA-background synoviocyte and inflammatory cells along with apoptosis and then immediately metabolized in the liver. Thus, the successful therapy for model mice of RA using HLs without side effect and any anti-RA drug in this study should be important for clinical applications of patients with RA in the near future.
Conclusion We clearly demonstrated that HLs showed inhibitory effects on the growth of HFLS-RA cells in vitro along with apoptosis and therapeutic effects for mouse models of RA in vivo. The noteworthy aspects are as follows. (a) The IC50 values of HLs on the growth of HFLS-RA cells were remarkably less than those of the DMPC liposomes. (b) The induction of apoptosis by HLs was verified for HFLS-RA cells on the basis of flow cytometry and TUNEL method. (c) Therapeutic effects of
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HLs were obtained in mouse models of RA. It is noteworthy that remarkable inhibitory effects of drug-free HLs leading to apoptosis on the growth of synoviocyte causing rheumatoid arthritis were obtained for the first time. The results in this study should be advantageous in the chemotherapy for patients with RA in the near-future clinical applications.
Declaration of interest This work was supported in part by a Grant-in Aid for Science Research from the Ministry of Education, Science, and Culture of Japan (No. 23300173, 25289299, 25420843).
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