Animals
All animals (n = 5 biologically independent mice per group) were maintained and used in accordance with Guidelines for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, Institutional Animal Care and Use Committee of Seoul National University (Seoul, Republic of Korea; approved animal experimental protocol number, SNU-130129-3-1). Eight-week-old female BALB/c mice and female C57BL/6 mice (Raon Bio Co., Gyeonggi-do, Republic of Korea) were used for in vivo experiments. All mice were housed up to 5 per cage with a 12 h light/dark cycle allowed ad libitum access to food (Rodent Chow; Cat# 38057, Purina Lab, Missouri, USA) and water under the ambient temperature of 23 ± 2 °C and humidity of 50 ± 10%. We have complied with all relevant ethical regulations for animal testing and research.
Preparation of promelittin peptide-modified liposomes
Liposomes surfaces were modified with promelittin peptide by covalently tethering the Cys-promelittin peptide, N-CEPEAEADAEAGPAGIGAVLKVLTTGLPALISWIKRKRQQ-C, via a maleimide reaction. Liposomes were prepared by mixing egg L-α-phosphatidylcholine (PC; Avanti Polar Lipids, Alabaster, AL, USA), egg L-α-phosphatidyl DL-glycerol (PG; Avanti Polar Lipids), cholesterol (Chol; Sigma-Aldrich), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (mal-PEG-DSPE; Avanti Polar Lipids) at a molar ratio of 2:2:2:0.4 in chloroform. For comparison, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-2000] (PEG-DSPE; Avanti Polar Lipids) was used in place of mal-PEG-DSPE in some experiments. After removal of chloroform using a rotary evaporator, the resulting thin lipid films were hydrated with 1 mL of phosphate-buffered saline (PBS; pH 7.4), vortexed, and extruded three times through 0.2 μm polycarbonate membrane filters (Millipore Corp., Billerica, MA, USA). This process resulted ML or PL. The resulting ML were further used to prepare PRL. For preparation of PRL, 0.8 μmol cys-promelittin (Peptron, Daejeon, Republic of Korea) was mixed with 1 mL of ML containing 0.4 μmol of mal-PEG-DSPE. The cysteine in the peptide was reacted with maleimide groups of ML by incubating at room temperature for 6 h.
In some experiments, a scrambled cys-promelittin sequence (N-CEAGAEPAA EPKPATSGDILWVLAARLTVLIGEQKQKRIG-C) or a promelittin-mimicking fluorescence-quenched peptide [CEPEAEADA-E (fluorophore)-AGPAGIGAVLK-quencher], was linked to the surface of ML, resulting in SCL and FQL, respectively. This latter dual-modified promelittin-mimicking peptide, containing 5-[(2-aminoethyl) amino]naphthalene-1-sulfonic acid (EDANS) as a fluorophore and 4-((4-(dimethylamino) phenyl)azo)benzoic acid (DABCYL) as a quencher, was prepared as illustrated in Fig. 2g. The EDANS-DABCYL–linked promelittin-mimicking peptide was tethered to the surface of ML containing 0.4 μmol of mal-PEG-DSPE. The resulting FQL was purified from unreacted peptides by chromatography on a PD-10 column (GE Healthcare Life Science, Cat. No. 17-0851-01, Mickleton, NJ, USA) and stored at 4 °C until use. FAP-sensitive cleavage of promelittin peptide on liposomes was tested by monitoring fluorescence recovery of the quenched fluorophore.
Quantification of peptides on liposomes
The amount of peptides on liposomes was assayed using fluorescamine, a heterocyclic dione that reacts with primary amines of peptides38. Specifically, an aliquot (32 μL) of fluorescamine solution (Sigma, cat. No. F-9015; 3 mg/mL in acetonitrile) was added to 100 μL of peptide-conjugated liposomes, and the mixture was allowed to react at room temperature for 10 min. The fluorescence intensity of fluorescamine-labeled liposomes was assessed at an excitation wavelength of 365 nm and an emission wavelength of 470 nm using a SpectraMAX M5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA). For calculation of peptides on liposomes, a standard curve was generated using serial dilutions of a cys-promelittin standard solution. The amount of phospholipids in liposomes was determined using a phosphate assay as previously described39.
Characterization studies
Liposomes were characterized by size, zeta potential, and morphology. The size and zeta potential of various liposome preparations were measured using dynamic light scattering and laser-Doppler micro-electrophoresis, respectively. The hydrodynamic diameters of liposomes were measured at an angle of 90° at 24.1 °C using a HeNe laser (10 mW) and an ELSZ-1000 dynamic light-scattering instrument (Photal Otsuka Electronics Co., Osaka, Japan). Zeta potential was measured at an angle of 22°. Data were analyzed using the ELSZ-1000 software (ver. 5.10) package supplied by the manufacturer. TEM images were acquired using a Talos L120C TEM system (FEI, Brno, Czech) operating at 120 kV. Liposomes were loaded onto a 300-mesh copper grid coated with formvar-carbon (01753-F; Ted Pella, Redding, CA, USA). The grid was washed twice with distilled water and then negative-stained with 1% (w/v) uranyl acetate, after which images of negatively stained liposomes were obtained.
Stability test
The stability of liposomes was monitored up to 18 days in the presence and absence of FAP (5 nM). For 18 days, liposomes were stored with or without FAP at 4 °C or room temperature. The hydrodynamic diameters of liposomes were measured 3 day intervals using an ELSZ-1000 (ver.5.10) instrument (Photal Otsuka Electronics Co.).
FAP-mediated fluorescence recovery test
The cleavage activity of FAP towards promelittin sequences was tested using the quenching and dequenching feature of the promelittin-mimicking fluorescence-quenched peptide on FQL. FQL (0.9 mg phospholipid/mL) were incubated with 1 μM FAP or matrix metalloproteinase 9 (MMP9; R&D Systems Inc.) at 37 °C for 1 h. Recovery of the fluorescence of EDANS liberated from the nearby quencher DABCYL was assessed by fluorometry at an excitation wavelength of 335 nm and an emission wavelength of 490 nm using a SpectraMAX M5 system (Molecular Devices).
Hemolysis assay
The hemolysis activity of various liposomes was tested using mouse RBCs. RBCs (2 × 104 cells/mL) were isolated from BALB/c mice and incubated with various liposome preparations in the absence or presence of 1 μM FAP. RBCs treated with 1% Triton X-100 served as a 100% hemolysis positive control, and a suspension of RBCs in PBS was used as a negative control. After centrifuging samples at 1000 × g for 10 min, supernatants containing hemoglobin released from lysed RBCs were transferred to a clear, flat-bottomed, 96-well polystyrene plate (SPL Life Sciences, Pocheon, Republic of Korea). The percentage of hemolysis was calculated by measuring the absorbance of each sample at 540 nm using a SpectraMax Plus plate reader (Molecular Devices).
Release kinetics of PRL
To evaluate the release kinetics of melittin by FAP, PRL (0.9 mg phospholipid/mL) was incubated without or with 5 nM of FAP. At various time points, the released melittin was separated from PRL by filtration with a 50-kDa membrane filter (Millipore, Billerica, MA, USA). The amounts of released melittin were measured using a fluorescamine assay38.
FAP-mediated release kinetics of DABCYL-modified peptides from FQL
To evaluate the FAP-mediated release kinetics of fluorescent melittin from FQL, we incubated FQL (0.9 mg phospholipid/mL) without or with 5 nM of FAP or matrix metalloproteinase 9 (MMP9; R&D Systems, Inc.) at 37 °C for various periods. The recovery of fluorescence representing EDANS-labeled melittin peptide liberated from FQL was assessed by fluorometry at an excitation wavelength of 335 nm and an emission wavelength of 490 nm using a SpectraMAX M5 system (Molecular Devices).
Cell culture
LX-2 human hepatic stellate cells (#SCC064, Sigma-Aldrich, kindly provided by Professor Sang Geon Kim, College of Pharmacy, Dongguk University, Republic of Korea) and FAP-negative Chang cells (ATCC CCL-13; kindly provided by Professor Mi-Ock Lee, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Republic of Korea) were used for testing FAP-mediated cleavage of peptides on various liposome preparations. Chang cells are listed in the ICLAC register as a misidentified cell line. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a humidified 5% CO2 atmosphere. For activation, LX-2 cells were stimulated with TGF-β1 (transforming growth factor-β1) (10 ng/ml; GenScript Biotechnology, Nanjing, China) for 24 h. In some experiments, LX-2 cells treated with or without small interfering RNA (siRNA) targeting FAP (siFAP) were seeded onto six-well plates (SPL Life Sciences, Pocheon, Republic of Korea) at a density of 4 × 105 cells/well and then incubated for 24 h to ~80% confluence.
Assessment of siFAP efficiency
The efficiency of siFAP was assessed by measuring the expression levels of FAP using flow cytometry. LX-2 cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. LX-2 cells (4 × 105 cells/well) were seeded to a six-well plate (SPL Life Sciences), incubated for 24 h, and transfected for 20 min with 50 nM siFAP (Bioneer Corporation, Daejeon, Republic of Korea) complexed with 5 μL of Lipofectamine 2000. The sequences of siFAP were 5′-CUC UAU GCA GUG UAU CGA AdTdT-3′ (sense) and 5′-UUC GAU ACA CUG CAU AGA gdTdT-3′ (antisense). In some experiments, scrambled-sequence siRNA (siSCR) was used as a control. After transfection, cells were incubated for an additional 48 h. Next, cells were stained with a rabbit anti-FAP primary IgG antibody (1:50, Abcam) for 1 h, followed by an allophycocyanin (APC)-conjugated goat anti-rabbit IgG antibody (1:100, Abcam). After staining, cells were analyzed via BD LSR Fortessa (BD Bioscience) and Leica TCS8 confocal microscope (Leica, Bensheim, Germany). Flow cytometry data were acquired with BD FACSDIVA™ (v8.0.1., BD Bioscience). LEICA Application Suite X (v3.6.0., Leica) was used to collect confocal images. Control cells were stained with rabbit IgG isotype antibody (1:50, Abcam) followed by an APC-conjugated goat anti-rabbit IgG antibody (1:100, Abcam).
MTT assay
Cell viability after treatment with various liposome preparations was assessed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, LX-2 cells were seeded onto 24-well plates at a density of 6 × 104 cells/well. After cells reached 70% confluence, they were treated with various liposome preparations for 24 h. MTT solution (500 μM) was then added to each well and plates were incubated for 2 h. The medium was then aspirated and 200 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to dissolve formazan crystals generated by metabolically active (live) cells. The viability of cells was quantified by measuring optical density at 570 nm using a microplate reader (Tecan Group Ltd., Seestrasse, Mannedorf, Switzerland).
Establishment of the bile duct-ligation model
Liver fibrosis was induced in mice by performing BDL surgery under anesthesia (isoflurane 1.5%)40 with slight modifications. The surgical area was shaved and cleaned preoperatively using a povidone-iodine solution, then an incision was made in the upper midline and the bile duct was exposed using a wet swab. The bile duct was ligated with silk thread knots at two sites and then cut between the knots, after which the abdominal wall was closed using nonabsorbable silk thread. On days 1 and 3 after BDL surgery, mice were intravenously injected with various liposome preparations.
In vivo efficacy test in carbon tetrachloride induced liver fibrosis model
To test the in vivo antifibrotic effect of PRL, the CCl4-induced chronic liver fibrosis model was established. Eight-week-old female C57BL/6 mice were intraperitoneally injected with CCl4 1 ml/kg (25% CCl4 in olive oil) twice a week for 6 weeks16. The liver fibrosis-bearing mice were then intravenously treated with the various liposomes every other day for 2 weeks and sacrificed at 2 weeks after the first treatment. Blood and liver tissues were collected for further analysis. To test the in vivo antifibrotic effect of PRL in an earlier phase of the CCl4-induced liver fibrosis model, 8-week-old female C57BL/6 mice were intraperitoneally injected with CCl4 at 1 ml/kg (25% CCl4 in olive oil) twice a week for 4 weeks. The liver fibrosis-bearing mice were then intravenously treated with PRL every other day for 2 weeks and sacrificed at 4 weeks after the first PRL treatment. Liver tissues were collected for analysis.
In vivo efficacy test in high fat diet-induced liver fibrosis model
The high fat diet-induced liver fibrosis model was established41. Mice were fed with CDAHFD (Cat#A06071302; Research Diets, New Brunswick, NJ) for 10 weeks. For normal control group, mice were fed with chow diet (Rodent Chow; Cat# 38057, Purina Lab, Missouri, USA). The mice were then treated with the various liposomes (dose = 0.27 mg phospholipid/mouse) every other day for 2 weeks and sacrificed 1 day after the last treatment. Blood and liver tissues were collected for further analysis.
FAP expression analysis by flow cytometry
FAP expression on aHSC of liver tissues was analyzed via flow cytometry. Single-cell suspensions of liver tissue were obtained as previously described with slight modification treatment42. Briefly, liver tissues were perfused with HEPES buffer (Sigma-Aldrich; cat. No. H4034) containing collagenase D (Sigma-Aldrich; cat. No. C5138-1G) and pronase (Sigma-Aldrich), and digested with stirring at 37 °C for 30 min. Cells were collected by centrifugation at 580 × g for 10 min, and dead cells were excluded using a Zombie Red Fixable Viability Kit (BioLegend). The cells were then stained with PE/Cyanine7-conjugated rat anti-mouse CD26 antibody (1:100, BioLegend; cat. No. 137810), PE-conjugated rat anti-mouse CD31 antibody (1:100, BioLegend; cat. No. 102407, Lot. No. B261070), PerCP/Cy5.5-conjugated rat anti-mouse CD45 antibody (1:50, BioLegend; cat. No. 103132, Lot. No. B282872), and rabbit anti-mouse FAP antibody (1:50, Abcam; cat. No. ab28244, Lot No. GR217381-54) for 1 h at 4 °C. Secondary antibody staining was performed with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (1:200, Abcam; cat. No. ab150083, Lot No. GR3370563-1) for 30 min at 4 °C. Cells were analyzed via BD LSR Fortessa (BD Bioscience). Flow cytometry data were acquired with BD FACSDIVA™ (v8.0.1., BD Bioscience). The HSC population was gated as CD26-CD31-CD45-VitA+ cells using FACS; quadrants for FAP + HSC were selected relative to the fluorescence minus one (FMO) control.
Molecular imaging
The organ distribution of FQL was determined by molecular imaging. Five days after BDL surgery, BALB/c mice (5 mice per group) were intravenously injected with FQL at a dose of 0.27 mg phospholipid/mouse. One hour later, vital organs (i.e., liver, heart, lung, spleen, and kidney) were isolated and their fluorescence intensities were assessed using an In Vivo Imaging System (IVIS; PerkinElmer, Hopkinton, MA, USA) (n = 5 independent samples per group). Living image (v4.5.2., PerkinElmer) was used to analyze fluorescent images of organs.
Assay of collagen in liver tissue
The amount of collagen in liver tissues was determined by measuring the content of hydroxyproline using a Hydroxyproline assay kit (Abcam, Cambridge, UK) as described by the manufacturer. Briefly, 4 days after BDL surgery, mice treated with various formulations were sacrificed and their liver tissues were extracted and homogenized. The homogenate was then treated with 10 N NaOH, incubated at 120 °C for 1 h, and neutralized with 10 N HCl. After centrifugation at 10,000 × g for 5 min, the supernatant was added to wells of a 96-well plate and dried at room temperature. The resulting crystalline residue was dissolved with 0.1 mL of oxidation-buffered chloramine T solution and incubated at room temperature for 20 min. After addition of 50 μL of acidic developer solution, provided in the kit, the plate was incubated at 65 °C for 45 min and treated with 50 μL of the provided DMAB solution. Absorbance was measured at 540 nm using a SpectraMax Plus plate reader (Molecular Devices).
In vivo assessment of liver function
The antifibrotic effects of liposomes were tested in vivo using a hematological parameter assay and histological staining. BDL surgery was performed on 8-week-old female BALB/C mice (Raon Bio). On days 1 and 3 after BDL surgery, mice were intravenously administered liposomes at a phospholipid dose of 2.7 mg/mL. Four days after BDL surgery, blood samples were collected and assayed for ALT, AST, bile acid, and total bilirubin levels by the Neodin VET Diagnostics Institute (Seoul, Republic of Korea). For histological assessment of liver tissues, the liver was extracted 4 days after BDL surgery, fixed in 10% formalin for 48 h, and paraffin-embedded. The tissues sections were analyzed with H&E staining, and Masson’s trichrome staining. For H&E staining, tissue sections were immersed in filtered Harris hematoxylin for 10 s and then in eosin for 30 s. The percentage of connective tissue areas in fibrotic regions on stained slides was calculated using a Vectra 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer, Hopkinton, MA, USA). Images were analyzed using the InForm v2.4.11. software (Perkin-Elmer).
Immunohistochemistry of liver tissues
FAP expression on aHSC in the liver was evaluated with immunofluorescent staining of formalin-fixed paraffin-embedded tissues. Liver tissues were sectioned at 4 µm thickness, de-paraffinized with xylene, and rehydrated with an ethanol series. Tissue slides were incubated with a Target Retrieval Solution (pH 6.0) (Agilent Dako, Santa Clara, CA, USA). After being washed, the slides were incubated with 0.1% Triton-X 100, blocked with 10% goat serum in PBS, and stained overnight at 4 °C with rabbit anti-mouse FAP antibody (1:100, Abcam; cat. No. ab28244, Lot No. GR217381-54) and mouse anti-mouse alpha-smooth muscle actin (αSMA) antibody (1:100, Abcam; cat. No. ab7817, Lot No. GR3356520-4). Alexa Fluor 594-conjugated goat anti-mouse IgG antibody (1:100, BioLegend; cat. No. 405326, Lot. No. B324994) and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (1:200, Abcam; cat. No. ab150083, Lot No. GR3370563-1) were applied for 1 h at room temperature, and the slides were imaged using a Thunder imager 3D assay (Leica Microsystems GmbH, Wetzlar, Germany).
Populations of cells in the liver tissues were analyzed via immunohistochemistry using various cell markers. Fibrosis-induced mice were treated with various liposomes. One day after liposome treatment, mice were anesthetized using isoflurane and the liver was perfused via the vena cava with 30 mL of PBS, and then collected. Excised liver tissues were fixed in 4% paraformaldehyde for 48 h at 4 °C. After dehydration in 30% sucrose solution overnight, liver tissues were embedded in OCT compound (Sakura Finetek, Japan) and frozen at −80 °C. Frozen liver sections (8 μm thickness) were blocked with 10% goat serum (Abcam, Cambridge, UK) and stained with primary antibodies overnight at 4 °C. Alexa Fluor 594 rabbit anti-mouse alpha-smooth muscle actin (αSMA) antibody (1:100, Cell Signaling Technology, Danvers, MA, USA; cat. No. 36110S), phycoerythrin (PE)-conjugated rat anti-mouse F4/80 antibody (1:50, BioLegend, San Diego, CA, USA; cat. No. 123110), APC-conjugated rat anti-mouse CD26 antibody (1:100, BioLegend; cat. No. 137807), PE-conjugated rat anti-mouse CD31 antibody (1:50, Invitrogen, Waltham, MA, USA; cat. No. 12-0311-82), and rabbit anti-mouse cytokeratin 7 antibody (1:100, Abcam; cat. No. ab181598) with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (1:200, Abcam; cat. No. ab150083, Lot No. GR3370563-1) were used as markers of HSCs, macrophages, hepatocytes, endothelial cells, and cholangiocytes, respectively.
After secondary antibody staining, tissues were counterstained with DAPI and examined by a VECTRA tissue analyzer (v3.0.5., Perkin-Elmer). Image analysis was applied using the InForm 2.2.1 image analysis software (Perkin-Elmer).
qRT-PCR for analysis of cell types
Populations of cells in the liver tissues were also evaluated by running qRT-PCR. Total RNA was extracted from liver tissue using the TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA by incubating at 42 °C for 60 min and 70 °C for 5 min using RT PreMix (Intron Biotechnology Inc., Seoul, Republic of Korea). The sequences of primers for qRT-PCR are listed in Supplementary Table 1. qRT-PCR was performed at Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using TOPreal qPCR 2x premix (Enzynomics, Daejeon, Republic of Korea, cat. No. RT501M). The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, was used for normalization of each mRNA expression level. Applied Biosystems® 7300 Real-Time PCR System (v1.4.0.) was used to collect relative gene expression values.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
To evaluate apoptosis of cells in liver tissues, the TUNEL assay was performed. In brief, frozen liver tissue sections (8 μm thickness) were stained with a DeadEnd™ Fluorometric TUNEL System (Promega) according to the manufacturer’s protocols. To identify the types of TUNEL-positive apoptotic cells, immunofluorescence staining was sequentially done using markers for various cells. Alexa 594-conjugated anti-αSMA antibody (1:100, Cell Signaling Technology, cat. No. 36110S), PE-conjugated anti-F4/80 antibody (1:50, BioLegend, cat. No. 123110), APC-conjugated anti-CD26 antibody (1:100, BioLegend; cat. No. 137807) and rabbit anti-mouse cytokeratin 7 antibody (1:100, Abcam; cat. No. ab181598) with Alexa Fluor 647-conjugated goat anti-rabbit IgG antibody (1:200, Abcam; cat. No. ab150083, Lot No. GR3370563-1) were used to mark aHSC, macrophages, and hepatocytes, respectively. After overnight incubation at 4 °C, DAPI staining and mounting were performed on the slides, and fluorescence images of slide were obtained by VECTRA (Perkin-Elmer). Then, images were analyzed by InForm 2.2.1 image analysis software (Perkin-Elmer) followed by quantification of co-localization of TUNEL signals with each cell marker.
Safety study
To evaluate the toxicity of PRL, a survival study was conducted in normal mice. Eight-week-old BALB/c mice received repeated intravenous administrations of PL or PRL at a dose of 0.27 mg phospholipid/mouse. Mice were administered with PL or PRL four times over 2 weeks. The survival and body weight of mice were monitored over 60 days.
Statistics
Experimental data were statistically analyzed by one-way analysis of variance (ANOVA) with Tukey test. All statistical analyses were performed using GraphPad Prism software (v8.0, GraphPad Software, San Diego, CA, USA). A P-value less than 0.05 was considered statistically significant.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
