The double-stranded siRNAs targeting human caspase-3 (GenBank accession numbers NM_004346, NM_032991) and scrambled siRNA were designed and synthesized by Dharmacon (Lafatette, CO). The siRNA was modified to incorporate a thiol group on the 5′ end of the sense strand. There was no modification of the antisense strand.
Probe synthesis and characterization.
The probe consisted of magnetic nanoparticles (for MRI, synthesized as described in Ref. 20
) conjugated to the near-infrared (NIRF) Cy5.5 dye (for correlative microscopy) and to siRNA (for gene silencing) (). The synthesis of the probe involved four distinct steps: synthesis of dextran-coated magnetic nanoparticles (MN), conjugation of the fluorescent dye Cy5.5-N
-hydroxy succinimide ester to MN, conjugation of the N
-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to MN-Cy5.5, and conjugation of siRNA to MN through SPDP linker. In brief, a solution of monoactivated Cy5.5 succinimide ester (Amersham Biosciences, Piscataway, NJ) in 20 mmol/L sodium citrate and 0.15 mol/L NaCl was allowed to react with the previously dialyzed immunopure, amino-derivatized dextran-coated iron oxide (aminated iron oxide, MN-NH2
) at pH 8.5, with constant agitation over a period of 12 h at room temperature. The Cy5.5-labeled aminated iron oxide (MN-NIRF) was purified from unreacted dye using a Sephadex G-25, PD-10 column (Amersham Biosciences). A ratio of 2 Cy5.5 molecules per nanoparticle was obtained. MN-NIRF was then conjugated to the heterobifunctional cross-linker SPDP (Pierce Biotechnology, Rockford, IL) by means of the N
-hydroxy succinimide ester, followed by purification using a Sephadex G-25, PD-10 column in phosphate-buffered saline (PBS)/EDTA, pH 7.5. The labeling of SPDP per crystal was determined based on the release of pyridine-2-thione at 343 nm (e
= 8.08 × 103
) after the addition of the reducing agent, tris-(2-carboxyethyl) phosphine hydrochloride (TCEP; 35 mmol/L in DMSO). A ratio of 31 SPDP molecules per nanoparticle was obtained.
FIG. 1. A: Schematic representation of the MN-NIRF-siCaspase-3 probes. B: Agarose gel electrophoresis of MN-near-infrared fluorophore-siCaspase-3 probe. C: Quantitative analysis showed that 105 pmol siRNA was conjugated to MN-NIRF-siCaspase-3 probe. (A high-quality (more ...)
The caspase-3-targeting and scrambled siRNA duplexes were then conjugated to MN-Cy5.5-SPDP through its 5′-sense thiol group. Before conjugation, the disulfide protecting group on 5′-disulfide bond was deprotected using TCEP according to the manufacturer’s instructions. The double-stranded RNA (dsRNA) was then reacted overnight (4°C) with the previously activated MN-Cy5.5-SPDP product via the SPDP cross-linker in PBS/EDTA, pH 8, followed by purification using a Quick Spin Column G-50 Sephadex Column (Roche Applied Science, Indianapolis, IN). The probes designated MN-siCaspase-3/MN-siSCR () were next purified using magnetic separation columns as described by the manufacturer (Miltenyi Biotec, Auburn CA). The amount of conjugated siRNA was assayed using agarose gel electrophoresis. To assess siRNA dissociation from the nanoparticles under reducing conditions, the probe was pretreated with 15 mmol/L TCEP for 30 min. The siRNA standard probes, untreated probes, and probes treated with reducing agents were applied on a 2% agarose gel in Tris borate-EDTA buffer (Invitrogen, Carlsbad, CA) at 145 V for 1 h. After electrophoresis, the gel was stained with 0.5 μg/ml ethidium bromide for 30 min and visualized using a Molecular Imager FX scanner (Bio-Rad Laboratories, Hercules, CA). The image was quantitated using the software ImageJ, version 1.43u.
Human islet culture and labeling.
Human islets were obtained from the Islet Cell Resource Centers (National Institutes of Health and Juvenile Diabetes Research Foundation). The viability and purity of the islets were both higher than 90%. On arrival at our facility, islets were cultured in CMRL-1066 medium (GIBCO, Grand Island, NY) supplemented with 10% FBS and 100 μg/mL penicillin-streptomycin.
For labeling experiments, 1,000 islet equivalents were counted and incubated for 48 h with either MN-siCaspase-3 or MN-siSCR probe in the same medium (0.025 mg Fe, 105 pmol siRNA, in 1 mL medium). To mimic the apoptotic condition, we cultured a group of islets in CMRL-1066 medium supplemented with 100 μg/mL penicillin-streptomycin without serum (serum starvation conditions). After incubation, islets were washed three times in culture medium and used for in vitro and in vivo experiments.
In vitro characterization of labeled islets.
Glucose-stimulated insulin secretion was evaluated using static incubation of experimental MN-siCaspase- and control MN-siSCR–labeled islets at low (1.7 mmol/L) and high (20 mmol/L) glucose concentrations. Insulin was measured in supernatants and islet extracts using a human insulin ELISA kit (Mercodia, Uppsala, Sweden). A stimulation index was calculated as the ratio of stimulated to basal insulin secretion normalized by the insulin content.
Islet cell viability was determined after labeling by colorimetric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay according to the manufacturer’s protocol (Promega, Madison, WI). To determine silencing effect after islet treatment with the probes, we performed real-time RT-PCR (TaqMan protocol).
Total RNA was isolated from the experimental and control islets with the RNeasy Mini kit, according to the manufacturer’s protocol (QIAGEN, Valencia, CA). The PCR primers and TaqMan probe specific for caspase-3 were designed with Primer Express software 1.5. Primer and probe sequences were as follows: Forward primer, 5′-TGTTCCATGAAGGCAGAGCC-3′; Reverse primer, 5′-TGCGTATGGAGAAATGGGC-3′; and TaqMan Probe 5′-TGGACCACGCAGGAAGGGCCT-3′.
Eukaryotic 18S rRNA TaqMan PDAR Endogenous Control reagent mixture (PE Applied Biosystems, Foster City, CA) was used to amplify 18S rRNA as an internal control, according to the manufacturer’s protocol. All samples were run in duplicate.
Caspase-3 protein levels in pancreatic islets were determined by Western blot with rabbit monoclonal antibody to caspase-3 (Abcam, Cambridge, MA) followed by horseradish peroxidase-conjugated goat anti-rabbit antibodies (Cell Signaling Technology, Danvers, MA). Staining of β-actin was used as an internal control. In situ apoptosis detection was performed using transferase-mediated dUTP nick-end labeling (TUNEL) assay (Chemicon, Temecula, CA) according to the manufacturer’s protocol. The percentage of cells undergoing apoptosis was calculated from the number of nuclei positive for DNA fragmentation (fluorescein isothiocyanate [FITC] channel) versus the total number of cells present (diaminido phenylindol [DAPI] channel).
Fluorescence microscopy of labeled islets was performed with anticleaved caspase-3 rabbit monoclonal IgG antibody (clone 269518; R&D, Minneapolis, MN) followed by FITC-labeled anti-rabbit IgG (H+L) secondary antibody (Vector Laboratories, Burlingame, CA). Slides were mounted with DAPI-containing mounting medium (Vector Laboratories) and examined under Nikon Eclipse 50i microscope.
For iron staining, we used Prussian Blue stain as described previously (14
). All images were acquired using a CCD camera (SPOT 7.4 Slider RTKE; Diagnostic Instruments, Sterling Heights, MI) and analyzed using iVision 4.015 version software.
All animal experiments were performed in compliance with institutional guidelines and approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. MN-siCaspase-3–labeled human pancreatic islets were implanted under the left kidney capsule (1,000 islet equivalents/kidney) of 6-week-old NOD.scid mice (n = 6). The same number of islets labeled with parental nanoparticles (MN) was transplanted under the right kidney capsule of the same animal.
Islet phantoms were prepared by fixing islet pellets in 2% paraformaldehyde and sedimenting them in 1% agarose gel. Imaging was performed using a 9.4T AVANCE scanner (Bruker BioSpin, Billerica, MA) equipped with ParaVision 3.0.1 software. The imaging protocol consisted of coronal T2 weighted spin echo (SE) pulse sequences with the following parameters: repetition time (TR)/echo time (TE) = 3,000/8, 16, 24, 32, 40, 48, 56, 64 ms, field of view (FOV) = 3.2 cm2, matrix size 128, resolution 250 μm2, and slice thickness = 0.5 mm. Image reconstruction and analysis were performed using Marevisi 3.5 software (Institute for Biodiagnostics, National Research Council, Canada). T2 relaxation times were determined by T2 map analysis of regions of interest drawn around the islet pellets.
In vivo MRI was performed on a 9.4T scanner 1, 3, 5, 7, and 14 days after islet transplantation. The imaging protocols consisted of Multi-Slice Multi Echo T2-weighted map (for volume and T2 relaxivity measurement). Parameters: TR = 2,000 ms and multiecho TE = 8, 16, 24, 32, 40, 48, 56, and 64; number of averages (NA) = 4; rapid acquisition with relaxation enhancement factor = 8; FOV = 4.4 cm2, matrix size 128, spatial resolution 312 μm2, and slice thickness = 0.5 mm. We calculated graft volumes by counting the area in each slice of the region of interest (ROI) outlining the graft and multiplying by the number of slices.
Ex vivo histology.
After the last imaging time point, kidneys were removed, fixed in 4% formaldehyde, and embedded in paraffin. Sections were stained for insulin with anti-human insulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and for caspase-3 with anticleaved caspase-3 rabbit monoclonal IgG antibody (269518; R&D, Minneapolis, MN). After incubation with Alexa Fluor 594-conjugated secondary goat anti-mouse IgG (H+L; Invitrogen) and FITC-labeled secondary goat anti-rabbit IgG (Vector Laboratories) and counterstaining with DAPI, the sections were used for microscopy as described above. In situ apoptosis detection of kidney sections was performed using TUNEL assay similar to the procedure described above.
Data are presented as means ± SD. Statistical differences were analyzed by Student t test (SigmaStat 3.0; Systat Software, Richmond, CA). A value of P < 0.05 was considered statistically significant.