Microparticle preparation
The microparticles of a mean of 1 and 10 μm in diameter (Figure 1) were prepared using a double emulsion-solvent evaporation method in accordance with the protocol described by Butoescu and colleagues [7]; a schematic representation of a microparticle is presented in Figure 2. The polymer used as a matrix for the microparticles was poly(D, L-lactide-co-glycolide) (PLGA) with a molecular mass of 19 kDa (Resomer® RG572S; Boehringer Ingelheim GmbH, Ingelheim, Germany). The diameter distribution of the 1-μm microparticle batch ranged from 0.4 to 1.4 μm and that of the 10-μm microparticle ranged from 4 to 14 μm. Blank microparticles were used as a control; the contents of DXM and SPIONs in the batches used as treatment were 2.5% and 1%, respectively. For the in vivo imaging experiment, microparticles were stained with fluorescent (near-infrared) NIR 780 phosphonate (λex/λem = 640/825 nm) purchased from Fluka (Sigma-Aldrich, Buchs, Switzerland). The use of this dye allowed the detection of the microparticles at a wavelength in the NIR domain, where the autofluorescent background of fur and collagen is negligible.
In vivoimaging
Sixteen healthy C57Bl/6 mice (Harlan, Horst, The Netherlands), 8 to 10 weeks old, were put under isofluorane anaesthesia and intra-articularly injected with 10 μL of a 3.6 mg (dry weight)/mL 10-μm microparticle suspension in sterile phosphate-buffered saline (PBS) while four mice were injected with PBS and used as controls. The microparticles were stained prior to injection with fluorescent NIR 780 phosphonate for imaging in the living animals. The left knee was intra-articularly injected with a microparticle suspension, whose quantity was chosen while keeping in mind that the DXM dose that needed to be delivered to the joint would be 1.2 mg/kg, according to El Hakim and colleagues [8]. Four days prior to the experiment, half of the mice were subcutaneously implanted with disc magnets on the external part of the left thigh, near the knee. The other half were used as magnet-free controls. The magnet implantation was verified by micro-computed tomography (CT) (Skyscan-1076; Gloor Instruments AG, Uster, Switzerland) on all animals. The acquired images were analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) to determine the distance and angle between the magnet and the knee, thus permitting calculation of the magnetic flux density exerted on the injected microparticles for each mouse. The right knee was not injected. After injection, all animals were examined via in vivo fluorescence imaging (IVIS-200; Xenogen Corporation, Hopkinton, MA, USA) at days 1, 2, 3, 4, 7, 14 and 21. The image acquisition was done by using an indocyanine green filter, which allows the measurement of an excitation wavelength of 710 to 760 nm and an emission wavelength of 810 to 875 nm. The acquisition time was set at 3 seconds. The fluorescence intensity was expressed as the number of photons per second per square centimetre. At the end of the experiment, mice were sacrificed by CO2 inhalation and the knees were collected for histological analysis.
For the 3-month preliminary study on microparticle retention in the joint, four mice were used: two mice injected with 1-μm (mean diameter) microparticles and two with 10-μm (mean diameter) microparticles. Both knees were intra-articularly injected with a 3.6 mg/mL microparticle suspension. The left knee was implanted with a magnet and the right one was used as a magnet-free control. After 90 days, the mice were sacrificed by CO2 inhalation and the knees were collected for histological analysis.
Antigen-induced arthritis
AIA was induced in male C57Bl/6 mice as previously described [9]. In brief, mice were immunised on day 0 via intradermal injection at the tail root with 100 μL of 2 mg/mL methylated bovine serum albumin (mBSA) (Fluka) emulsified 1:1 with Freund's complete adjuvant (Sigma-Aldrich), containing 1 mg/mL Mycobacterium tuberculosis. A second immunisation was performed on day 7 via intradermal injection of 100 μL of 2 mg/mL mBSA emulsified 1:1 with Freund's incomplete adjuvant (Sigma-Aldrich). On day 16 after the first immunisation, half of the mice were implanted on the external part of the left thigh, near the knee, with 1.2 T permanent disc magnets (4 mm in diameter and 2 mm in height; Maurer Magnetic AG, Grüningen, Switzerland), which produce a 0.14 T magnetic field at the articulation site. Arthritis was induced on day 21 by i-a. injection of 10 μL of 10 mg/mL mBSA in PBS in the right knee. This injection was done along the suprapatellar ligament directly into the joint cavity. Concomitantly with the arthritis induction, the different treatment regimens were started. The right knee was injected with PBS and served as a control. Other controls used in the experiment, in the presence or absence of a magnet, were blank microparticles, SPION-containing microparticles, DXM suspension and SPION suspension. The test drug delivery system consisted of 10-μm microparticles containing DXM and SPIONs ("complete microparticles"). Five animals were used for each group. Joint inflammation was quantified by measuring the accumulation of 99mTc pertechnetate in the knee at days 1 and 4 after arthritis induction (MINI-assay type 6–20 H gamma counter; Uehlinger-Pfiffner AG, Schöftland, Switzerland). Thus, a dose of 10 μCi 99mTc per mouse was subcutaneously injected in the posterior neck region. After 30 minutes, the accumulation of the isotope was measured by external gamma counting by positioning the mice on a custom-made lead platform in which a small opening allows specific counting of the knee region. The acquisition time was set at 10 seconds, and each knee was counted three times, with repositioning of the mouse in between the three measurements. The ratio of 99mTc accumulation in the inflamed arthritic knee to 99mTc uptake in the contralateral control knee was calculated. A ratio higher than 1.1 indicated joint inflammation. Mice were sacrificed 4 days after arthritis induction. Blood was withdrawn by cardiac puncture and was left to coagulate for at least 30 minutes prior to centrifugation at 4,000 revolutions per minute to collect the serum. The knees were dissected, fixed with 4% formaldehyde in PBS and used for histological analysis. All experimental procedures on animals reported in this paper were performed in compliance with Swiss federal law on the protection of animals and in accordance with a protocol approved by the animal ethical committee of the Geneva University School of Medicine and the canton of Geneva authority (Direction Générale de la Santé, authorisation number 1084/3326/2).
Histology
After fixation in 4% formalin, all knee joints were cut in the sagittal direction. After decalcification and embedding in paraffin, 4-μm sections were cut and stained with haematoxylin and eosin, Elastica van Gieson, Masson tri-chrome, toluidine blue and Pearl's Prussian blue to detect the presence of iron using light microscopy. Histological sections were graded by a pathologist (CAS) in a blinded manner. Cartilage erosion and joint destruction as well the intensity of inflammation, including 'pannus' formation, were scored in accordance with the method of Camps and colleagues [10], using a score ranging from 0 to 4 (0 = normal, 1 = minimal, 2 = moderate, 3 = severe and 4 = very severe). In addition, the relative amount of polynuclear neutrophils as part of the inflammation or pannus formation was assessed with a score also ranging from 0 to 4 (0 = no neutrophils present and 4 = maximal neutrophilic infiltration).
Anti-bovine serum albumin antibody measurement in the mouse serum
Ninety-six-well plates (Maxisorp™; Nunc A/S, a brand of Thermo Fischer Scientific, Roskilde, Denmark) were coated overnight at 4°C with 1% BSA in PBS. Serially diluted mouse serum in 1% gelatin in PBS was added to each well and incubated for 2 hours at room temperature. Wells were washed four times with PBS added with 0.05% Tween 20 (PBST). Next, 100 μL of goat anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1:2,000 in PBST was added, and the plate was incubated for 1 hour at room temperature. The wells were washed with PBST and the colour was developed with 100 μL of 1:1 mixture of stabilised hydrogen peroxide and stabilised tetramethylbenzidine (substrate reagent pack; R&D Systems, Abingdon, UK). The reaction was stopped by adding 50 μL/well of 2N H2SO4. Plate reading was performed at 470 nm (Bio-Rad 550 Microplate Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the results were expressed as the percentage of absorbance units of control mice.
Magnetic flux density calculation
The flux density present at different distances from the magnet was calculated by using the electromagnetic modelling software ViziMag (Webskel, Ayrshire, UK).
Statistical analysis
The Mann-Whitney test (Wilcoxon rank sum test) for unpaired variables was used to compare differences between groups with a non-Gaussian distribution. The Student t test was used to compare groups with a Gaussian distribution. A P value of less than 0.05 was considered significant. The data were expressed as the mean ± standard deviation.