2.1 Materials
Dexamethasone sodium phosphate was supplied by Solarbio Science & Technology (Beijing, China). Methanol and acetonitrile (HPLC grade) were purchased from Kelong Chemical Reagent Factory (Chengdu, China). Rabbit monoclonal antibodies against CD63 or CD9 as well as horseradish peroxidase-conjugated goat anti-rabbit IgG were purchased from Abcam (UK). SDS-PAGE kits and polyvinylidene fluoride membranes were obtained from Sigma (USA). Complete and incomplete Freund’s adjuvant and bovine type II collagen were acquired from Chondrex (USA). ELISA kits were from Thermo Fisher (Austria). Fluorescent dyes PKH26 and PKH67 kits were purchased from Beijing Baiao Laibo Technology; 1, 1'-Dioctadecyl-3, 3, 3', 3'-tetramethylindodicarbocyanine perchlorate (DID), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), FITC-labeled phalloidin DAPI and Lipopolysaccharide (LPS) were supplied by Beijing Solarbio Science & Technology. Other reagents were analytical grade.
2.2 Cells and animals
RAW 264.7 murine macrophages and human umbilical vein endothelial cells (HUVEC) (Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM/HIGH GLUCOSE medium (Hyclone, USA) with 10% fetal bovine serum(FBS) (Gibco, USA) and 1% (v/v) penicillin/streptomycin (Hyclone, USA).
Male DBA/1 mice (8 weeks old) were purchased from Charles River (Beijing, China). All animal experiments were in accordance with The Animal Ethics Committee of Southwest Medical University.
2.3 Isolation and characterization of exosomes
When RAW 264.7 cultures reached the logarithmic phase of growth, cells were switched to FBS-free medium for 24 h, after which the medium was collected and replaced with fresh FBS-free medium. The cultures were incubated for another 24 h, then the medium was collected again. The two volumes of collected medium were pooled, exosomes were isolated using a gradient centrifugation protocol [20] with some modification. Firstly, the medium was centrifuged at 2000 g for 10 min and then at 10,000 g for 30 min to remove cellular debris. Next, the supernatant was concentrated to about 30% of the original volume at 2,000 g for 8 min using ultrafiltration tube (MWCO = 10,000). Finally, the supernatant was centrifuged at 120,000 g for 70 min in an ultracentrifuge (QPTimaMAX-XP Ultra-High, Beckman Coulter, USA). The pellets were washed with large volume cold phosphate-buffered saline (PBS) and centrifuged at 120,000 g for 70 min again to ensure maximal exosome purity. All centrifugation procedures were performed at 4 °C. The pellet was re-suspended in PBS and stored at -80 °C. The amount of exosomes was estimated using a Bradford assay (Bio-Rad Laboratories, Shanghai, China).
Size, polydispersity index (PDI) and zeta potential of purified exosomes were determined using dynamic light scattering (Malvern Zetasizer Nano ZS90, Malvern Instruments, UK). Their morphology was examined using transmission electron microscopy (HT7700, Hitachi, Japan). The presence of CD63 and CD9 on the exosome surface were measured by western blotting. These proteins serve as markers of exosomes derived from mammalian cells [18, 26].
2.4 Preparation of Exo/Dex and FPC-Exo/Dex
To load Dex into exosomes, exosomes (100 µg) were mixed with Dex (300 µg) in PBS with the concentration of trehalose was 80 nM, trehalose was added to avoid the aggregation of exosomes during electroporation. The mixture was subjected to electroporation at room temperature using a double poring pulse (200 V, 5 ms) and transfer pulse of five pulses (20 V, 50 ms) in a 1-cm electroporation cuvette and a NEPA21 Type II electroporater (NEPA genes, Tokyo, Japan). Then un-encapsulated Dex was removed by ultracentrifugation (100,000 g, 60 min, 4 °C). The Dex-loaded exosomes (Exo/Dex) were resuspended in PBS and incubated in 37 °C for 1 h to restore the membrane.
The FA-PEG-Chol conjugate was prepared as described in Supplementary Materials. The Chol end of FA-PEG-Chol was inserted into the lipid bilayer membrane of Exo/Dex by post-insertion [22]. Exo/Dex and FA-PEG-Chol ligands were mixed in a mass ratio of 1:5 and incubated at 37 °C for 2 h. Free ligands were removed by centrifugation at 3000 g for 10 min, giving rise to FPC-Exo/Dex. The amount of FA incorporated was determined by comparison of the UV285 value to a standard curve of folic [27, 28].
2.5 Preparation of Dex-loaded anion liposomes (Lip/Dex) as control group
Dex-loaded anionic liposomes were prepared in order to compare with exosome-based drug delivery systems. Anionic liposomes were prepared using ethanol injection as described [29]. Firstly, 40 mg of yolk lecithin, 10 mg of cholesterol and 10 mg of Dex were dissolved in 3 mL ethanol, and the solution was slowly injected into 5 mL PBS (pH 7.4) while stirring. The solution was slowly stirred under bath conditions at 40 °C for 2 h, and the solution was filtered through 0.45-µm and 0.22-µm membrane successively, yielding Lip/Dex.
2.6 Characterization of different Dex preparations
Size, PDI and zeta potential of all preparations were determined using dynamic light scattering, and morphology of FPC-Exo/Dex were examined by transmission electron microscopy. Encapsulation efficiency (EE) and drug loading efficiency (DLE) were measured using high-performance liquid chromatography (HPLC). Briefly, each formulation was divided into two equal portions, one of which was demulsified with 10% methanol, and the amount of total drug (Wt) was measured by HPLC. The free drug in the second portion (We) was pelleted by ultracentrifugation and weighed. EE was calculated using the equation EE = We / Wt × 100%. EE of Lip/Dex was determined in the same way.
The amount of exosomes (Ws) was estimated by BCA assay to calculate DLE according to the equation DLE = We / (Wt + Ws) × 100%. An equal amount of Lip/Dex was dried and weighed (Wp) to calculate DLE according to the equation DLE = We / Wp × 100%.
2.7 In vitro cumulative drug release study
FPC-Exo/Dex, Exo/Dex, Lip/Dex, or free Dex (40 µg of Dex contained in all preparations) were added to 1 mL PBS (pH 7.4) in a dialysis bag with a molecular weight cut-off of 3000 Da. The bag was placed in 30 mL PBS and shaken at 37 °C at 1000 rpm. At predetermined time points, 200 µL of release medium was collected and immediately replaced with an equal volume of fresh medium. Dex concentration was determined by HPLC, and the cumulative amount released was calculated.
2.8. Toxicity assay of nanoparticles by MTT
RAW264.7 cells and HUVEC in logarithmic growth phase were digested into single-cell suspensions and seeded in 96-well plates at 1 × 104 cells per well, then incubated at 37 °C overnight. FPC-Exo/Dex, Exo/Dex and Dex were prepared in culture medium without serum or antibiotics, and 200 µL of each preparation was added to wells at Dex concentrations of 5–25 µg/mL. After 24 h, the medium was discarded, and 20 µL of MTT (5 mg/mL) solution and 180 µL of complete medium were added. Cells were cultured for another 4 h, then medium was replaced with 150 µL DMSO and cultures were shaken for 15 min at 37 °C. Absorbance at 490 nm was measured using a Varioskan Flash microplate reader (Thermo Fisher, USA). Relative cell viability was calculated using the equation: Cell viability = (sample − blank) / (negative control − blank) × 100%.
2.9 Cellular uptake study by flow cytometry and confocal laser scanning microscopy
Lip/Dex, Exo/Dex and FPC-Exo/Dex were labeled with PKH67 or PKH26 according to the dye manufacturer’s protocol. PKH67-labeled Dex formulations were incubated for 2 h with resting or lipopolysaccharide (LPS)-activated (stimulated for 24 h with LPS at a final concentration of 100 ng/mL) RAW264.7 cells. Uptake by cells was measured using a Verse cytometer (BD, USA).
In order to visually observe the situation of the formulations entering the cell, the three Dex formulations labeled by PKH26 were incubated with resting or LPS-activated RAW264.7 cells for 2 h, then analyzed using confocal laser scanning microscopy (Leica SP8, Germany) after staining the cytoskeleton with FITC-phalloidin and the nucleus with DAPI[18].
2.10 Anti-inflammatory effects of nanoparticles to LPS-activated RAW264.7 cells
RAW264.7 cells were seeded into a 24-well plate at a density of 5 × 105 cells per well and allowed to adhere, then stimulated for 24 h with LPS at a final concentration of 100 ng/mL. The medium was replaced with fresh medium containing FPC-Exo/Dex, Exo/Dex, Lip/Dex or free Dex at a final Dex concentration of 20 µg/mL. Negative control wells were incubated in culture contain PBS. After 24 h, the culture medium was collected, centrifuged at 2000 g for 5 min, and the supernatant was assayed for IL-β, TNF-α and IL-10 using ELISA kits according to the manufacturer’s instructions [21].
2.11 Establishing Mouse model of collagen-induced arthritis (CIA)
Bovine type II collagen was thoroughly emulsified with an equal volume of complete Freund’s adjuvant by vortex, and 100 µL of emulsion was administered intradermally at the base of the mouse tail. After 21 days, mice received an intradermal booster injection of type II collagen with an equal volume of incomplete Freund’s adjuvant.
2.12 Biodistribution of nanoparticles in CIA mice
A total of 12 CIA mice were randomly divided into 4 groups (3 animals per group), which were intravenously administered DID-labeled Lip/Dex, Exo/Dex, FPC-Exo/Dex, or free DID (1 µg DID per mouse). At 1, 4, 8, and 24 h later, mice were anesthetized with 10% chloral hydrate (0.04 mL per 10 g) and analyzed using the IVIS® Spectrum system (Caliper, Hopkinton, MA, USA).
At 24 h, mice were euthanized and the blood, heart, liver, spleen, lung, and kidney were removed. Blood was sampled and centrifuged at 3000 g for 7 min to obtain plasma. Fluorescence of plasma and organs was measured using the IVIS® Spectrum system (PerkinElmer, USA).
2.13 Measurement of weight, paw thickness, foot volume and articular index (AI) score of CIA mice
On day 21 after the booster immunization, CIA mice were randomly assigned to five groups (3 animals per group) and injected intravenously with free Dex, Lip/Dex, Exo/Dex or FPC-Exo/Dex at a Dex dose of 1.2 mg/kg in all cases. Negative control mice were injected with the same volume of Saline. AI scores were determined for each limb as described [8]. Body weight, hind paw thickness and foot volume were measured every 3 days during treatment. Foot volume is measured with drainage method.
2.14 Micro-computed tomography (Micro-CT) analyses of articular bone
After mice were sacrificed, the left hind limbs were removed and immediately fixed in 4% paraformaldehyde for 48 h. Then the microstructure of each limb was analyzed using a SIEMENS Inveon PET/CT computed tomography system (SIEMENS, Germany) with the following parameters: voltage, 80 kV; current, 500 µA; exposure time, 1800 ms; total rotation, 220°; and projections, 120 sheets. A region of interest (ROI) of the trabecular bone within the calcaneus was defined by aligning the calcaneus bone along the sagittal plane using the Data Viewer, starting 0.2 mm away from the epiphyseal plate and continuing for 40–50 slides (1 mm)[30, 31]. The bone mineral density (BMD), percent bone volume (BV/TV), bone surface density (BS/BV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp) of the ROI were calculated using SIEMENS Inveon Research Workplace software 4.2.
2.15 Histological evaluation of joint tissues
Ankle joints were dissected from each group, fixed in 4% paraformaldehyde for 48 h, and decalcified in 10% neutral EDTA solution for 15 days at room temperature. Then decalcified tissue was embedded in paraffin. Thin sections (5 µm) were cut and stained with hematoxylin-eosin (H&E) or safranin O (SO) combines with chondroitin sulfate to stain articular cartilage red [32]. An H&E score from 0 to 3 was determined for each of the following aspects: inflammatory cell infiltration, synovial tissue proliferation, fibrous tissue hyperplasia, and macrophage infiltration. Then all these scores were summed for a given ankle joint, and the overall scores for all ankle joints were summed to obtain H&E scores for a given animal.
2.16 Evaluation of inflammatory cytokines in serum
Blood samples and joint tissues were collected from mice on day 45 after induction of CIA. Serum levels of inflammatory cytokines TNF-α, IL-1β and IL-10 were measured using ELISA kits according to the manufacturer’s instructions.
2.17 In vivo safety evaluation of nanoparticles in CIA mice
Aspartate transaminase (AST) and alanine transaminase (ALT) levels of serum collecting from different treatment groups were assayed using a commercial kit (Nanjing Institute of Biological Engineering, Nanjing, China) according to the manufacturer’s protocol.
2.18 Statistical analysis
Statistical analysis was performed using Graphpad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Statistical comparisons were performed by one-way ANOVA (Dunnett's multiple comparisons test) for multiple groups, except for the analyses in Fig. 4B and Fig. 6B, which were performed using two-way ANOVA. Differences associated with p < 0.05 were considered significant. All results were expressed as mean ± SD.