Research Article For reprint orders, please contact: [email protected]
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
Aims: This study aimed to examine the efficacy of a nanocarrier (polyethyleneimine [PEI]-superparamagnetic iron oxide nanoparticle [SPIO]), composed of a core of iron oxide and a shell of PEI, in the systemic delivery of therapeutic siRNA to experimental arthritic joints. Materials & methods: PEI-SPIO/siRNA nanoparticles were synthesized and characterized in vitro. Nanoparticles were administered intravenously to arthritic rats to analyze cellular uptake, tissue distribution and the therapeutic effect of a siRNA against the IL‑2/-15 receptor b chain (IL‑2/IL‑15Rb). Results: PEI-SPIOs loaded with siRNA displayed negligible cytotoxicity, improved siRNA stability, efficient uptake by macrophages and the ability to induce specific gene silencing in vitro. PEI-SPIO-delivered siRNA accumulated easily in inflamed joints and was efficiently taken up by joint macrophages and T cells. Although IL‑2/IL‑15Rb siRNA-loaded PEI-SPIOs alone were efficacious in the treatment of experimental arthritis, combination therapy with both PEI-SPIO/IL‑2/IL‑15Rb siRNA and a magnetic field displayed an additive anti-inflammatory effect. Conclusion: PEI-functionalized SPIOs can be employed for systemic siRNA delivery in rheumatoid arthritis and enhanced therapeutic benefit can be achieved by the use of an external magnetic field.
Juanli Duan1, Jinlai Dong2, Tiantian Zhang1, Zhenyi Su3, Jie Ding3, Yu Zhang2 & Xiaohua Mao*,1,3 Key Laboratory of Ministry of Education for Developmental Genes & Human Diseases, School of Life Sciences, Southeast University, Nanjing, China 2 State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials & Devices, School of Biological Science & Medical Engineering, Southeast University, Nanjing, China 3 Department of Biochemistry, School of Medicine, Southeast University, Nanjing, China *Author for correspondence: Tel.: +86 25 83272474 Fax: +86 25 83324887 [email protected] 1
Original submitted 1 November 2013; Revised submitted 12 December 2013 Keywords: IL‑2/IL‑15Rb • macrophage • nanoparticle • rheumatoid arthritis • siRNA
Monoclonal antibodies and soluble receptors targeting major imflammatory cytokines or cells have emerged as a major tool in the treatment of rheumatoid arthritis (RA) and expanded rapidly over the past decade [1–4]. However, each of biologics currently used in RA has associated issues, including only a partial response, and unwanted systemic adverse effects and safety complications after long-term treatment. For this reason, there is an unmet medical need for safer and more efficient treatment regimens for disease remission. Owing to their unique magnetic properties, biocompatibility, comparable size to biologically important objects and easily adaptable surface for bioagent attachment, superparamagnetic iron oxide nanoparticles (SPIOs) have shown great promise in
10.2217/NNM.13.217 © 2014 Future Medicine Ltd
biomedical applications. In particular, their responses to external magnetic fields enable them to be novel vehicles for targeted drug delivery. For gene delivery by SPIOs, their surface is usually modified with a layer of cationic polymers onto which negatively charged nucleic acids can be electrostatically attracted and tethered [5,6]. While magnetic nanoparticle (NP)-based delivery vehicles have been extensively studied in, for example, cancer diagnosis and therapy, to our knowledge, the literature contains few reports of systemically administered SPIOs primed for magnetic targeting in inflammatory diseases. RA has two properties that make it an attractive target for SPIO-mediated therapeutics. First, it is, like solid tumors, an angiogenic disease in which the pathological
Nanomedicine (2014) 9(6), 789–801
part of
ISSN 1743-5889
789
Research Article Duan, Dong, Zhang et al. tissue is lined with abnormal and leaky blood vessels [7]. Second, the tissue is heavily infiltrated with macrophages that, upon activation, are not only the main secretors of pathogenic cytokines and chemokines, but also key players in angiogenesis [8,9] and, therefore, of central importance in the pathogenesis of RA. In this study, polyethylenimine (PEI)-functionalized SPIOs were employed as a carrier to deliver IL‑2/ IL‑15Rb siRNA to inflamed joints in a rat model of RA. We reason that, due to the enhanced permeability and retention effect and their response to an external magnetic field, SPIOs tend to accumulate in and spread inside inflamed joint tissues. These NPs would then be avidly taken up by inflammatory cells, especially macrophages. Given that activated macrophages express IL‑15R and that IL‑15 optimizes interactions between T cells and macrophages, which in turn causes the release of a wide range of inflammatory mediators including the key proinflammatory cytokine TNF‑a and IL‑15 itself [10,11], we believe inhibiting IL‑2/IL‑15Rb expression using siRNA-loaded SPIOs would downregulate a central amplification network of inflammation within the diseased joints. The data, presented below, are consistent with this hypothesis. Materials & methods siRNA duplexes
The siRNA duplex targeting rat IL‑2/IL‑15Rb (sense sequence: 5´GA AGGGAUGUCUACCA AUATT) and a negative control (NC) siRNA (sense sequence: 5´UUCUCCGAACGUGUCACGUTT) were synthesized by GenePharma (Shanghai, China) [12]. NC siRNA labeled with Cy3 or Cy5 (Cy3-/Cy5-siRNA) was from RiboBio (Guangzhou, China). Nanocomposite preparation
PEI-SPIOs were synthesized using the following process. Oleic acid (OA)-capped Fe3O4 was first synthesized by a modified oxidative coprecipitation method in an OA/H2O/dimethylsulfoxide mixing solution, and dimercaptosuccinic acid was then used to replace OA to obtain water-soluble dimercaptosuccinic acid-capped Fe3O4 NPs – SPIOs [13,14]. PEI (10 kDa, Sigma-Aldrich, MO, USA) was further capped onto the surface of SPIOs by electrostatic adsorption. Briefly, 10 ml of 0.2‑mg/ml SPIOs were added into excess PEI solution under stirring. The mixture was sonicated for 20 min and then further stirred for 2 h. The obtained PEI-functionalized SPIOs (PEI-SPIOs) were purified by removing free PEI through an ultrafiltration membrane (molecular weight cut-off: 300 kDa). Fe concentration was determined by the colorimetric method using phenanthroline. The PEI-SPIO/siRNA complexes with different Fe:siRNA (w/w) ratios were prepared by adding an appropriate amount of PEI-SPIO into siRNA, whose
790
Nanomedicine (2014) 9(6)
concentration was kept constant. The resulting mixture was incubated at room temperature (RT) for 30 min to allow composite formation. Physical characterization
z‑potential and the size of PEI-SPIO and PEI-SPIO/ siRNA NPs in solution were measured by dynamic light scattering using Brookhaven ZetaPlus Analyzer (NY, USA). PEI-SPIO/siRNA NPs were prepared by mixing NC siRNA with PEI-SPIOs at a Fe:siRNA weight ratio of 8 for 30 min at RT and then measured by dynamic light scattering with a laser wavelength of 633 nm and a measurement angle of 90°. The morphology of PEISPIO/siRNA and PEI-SPIO NPs was characterized using a transmission electron microscope (TEM). The samples for TEM imaging were prepared by placing a drop of the respective NP suspension onto a copper grid and drying at ambient temperature overnight. The samples were then visualized by a TEM (JEOL JEM 2100, Japan). Gel retardation assay
NC siRNA was mixed with PEI-SPIOs at various Fe:siRNA weight ratios. The resulting mixtures were incubated at RT for 30 min, after which the samples were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide (EtBr) staining. Heparin decomplexation assay
siRNA was complexed with PEI-SPIOs at a Fe:siRNA weight ratio of 8 for 30 min at RT. Various amounts of heparin (heparin:siRNA weight ratio of 0, 20, 80, 160, 200 and 300) were then added, and the mixtures were further incubated for 30 min. After centrifugation (12,000 rpm, 15 min, 4°C), supernatants were analyzed by 3% agarose gel electrophoresis and the released siRNA was visualized by EtBr staining. Serum stability assay
siRNA was complexed with PEI-SPIOs at a Fe:siRNA weight ratio of 8 for 30 min at RT. After adding fetal bovine serum (FBS) at a final 50% concentration, aliquots were incubated for various time periods at 37°C. The resulting samples were treated with 50 µg of heparin (heparin:siRNA weight ratio of 100) at RT for 30 min. The released siRNA fraction was separated and visualized as described above. Peritoneal macrophage isolation
Rats were sacrificed 3 days after intraperitoneal injection of 5 ml of 4% thioglycollate media (SigmaAldrich). Peritoneal lavage was performed with 20 ml of cold phosphate-buffered saline (PBS; 0.1 M, pH 7.4). The resulting peritoneal fluid was centrifuged
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
at 1000 rpm for 10 min at 4°C. Cells were then washed twice with cold PBS and resuspended in high-glucose DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were counted and then seeded. After adhesion to plastic culture dishes for 2 h at 37°C and 5% CO2, the cells were washed three times with PBS to remove nonadherent cells. Adherent macrophages were cultured in fresh medium as described above and used for future experiments. Cell viability assay
Cell viability was determined by MTS assay. Briefly, RAW 264.7 cells were seeded in 96‑well cell culture plates at 1 × 10 4 cells/well in 100 µl DMEM and grown for 24 h to 80–90% confluence. The cells were then exposed to the indicated dose of NPs for 16 h at 37°C. Formazan absorbance at 490 nm was measured 3 h after addition of MTS. Western blotting
Rat peritoneal macrophages were incubated with PEI-SPIO/siRNA NPs (Fe:siRNA weight ratio of 8) at a concentration of 15 µg Fe/ml. Then, 48 h after transfection, cells were lysed in lysis buffer containing 50 mM Tris‑HCl (pH 7.5), 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid, 1% Triton X‑100, 0.1% SDS, 0.5% sodium deoxycholate and protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Cellular debris was removed and proteins were collected by centrifugation. Equal amounts (30 µg) of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 2.5% bovine serum albumin-TBST (1.0 M Tris‑Cl, pH 8.0, 150 mM NaCl, 0.05% Tween‑20 ®, Sigma-Aldrich), incubated with rabbit anti-IL‑2Rb (M‑20) or anti‑b actin (R‑22) antibodies (Santa Cruz Biotechnology, CA, USA), followed by probing with horseradish peroxidase-labeled goat anti-rabbit antibody (Wuhan Boster Biological Technology, China). In vitro assessment of cellular uptake
Rat peritoneal macrophages were seeded in 24‑well plates at 2 × 105 cells/well and grown overnight. Uptake experiments were initiated by adding PEISPIO/Cy3-siRNA complexes (Fe:siRNA weight ratio of 8) at a concentration of 15 µg Fe/ml followed by incubation for 24 h. Cells were then trypsinized into single-cell suspensions with 0.25% trypsin-ethylenediaminetetraacetic acid, harvested by centrifugation, washed with PBS and resuspended in 500 µl of PBS containing 1% FBS. The resulting cell suspensions were analyzed by flow cytometry.
future science group
Research Article
Animal studies
Specific pathogen-free male Wistar rats were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). All animal experiments were performed in accordance with the guideline of the Committee on Animals of Southeast University, China. All surgery and in vivo imaging were performed under anesthesia and all efforts were made to minimize suffering. Induction, treatment & clinical evaluation of adjuvant arthritis
Adjuvant arthritis (AA) was induced by a single intradermal injection (100 µl) of Freund’s complete adjuvant (CFA) containing 10 mg/ml heat-killed Bacillus Calmette–Guérin freeze-dried powder (National Institutes for Food and Drug Control, China) in sterile liquid paraffin into the footpad of the left hind paw on day 0. Clinical evidence of arthritis usually occurred on day 11. For therapeutic treatment studies, PEI-SPIO/IL‑2/ IL‑15Rb siRNA complexes (0.3 mg siRNA/kg) were administered via the tail vein once a week starting on day 10 for 3 weeks. The control rats received either PBS or nonspecific NC siRNA-loaded PEI-SPIOs. To determine whether an external magnetic field is able to enhance the therapeutic effect of IL‑2/IL‑15Rb siRNA, one group treated with a combination of PEISPIO/IL‑2/IL‑15Rb siRNA and a magnetic field was also included. In this group, a round NdFeB magnet (24 mm diameter and 8 mm thick, with a central hole 9 mm in diameter and surface magnetic field of 400 mT) was fixed on the skin of the right hind paw for 2 h after each injection. The animals were anesthetized by isoflurane inhalation during magnetic exposure. Arthritis in each paw was scored as previously described [15]. Three paws except the CFA-injected left hind paw were scored, so the highest possible score per rat was 12. In vivo assessment of cellular uptake
Rats that had developed overt arthritis with a mean clinical score of at least 9 were used. The AA rats were injected intravenously with a single dose of PEI-SPIO/Cy3-siRNA (0.3 mg siRNA/kg) NPs with or without magnetic field exposure and control rats received PBS. To apply a magnetic field, a NdFeB magnet of 400 mT was fixed on the skin of the right hind paw for 2 h after injection. Blood, liver, spleen, kidney and inflamed paws were collected at 2, 8 and 24 h after injection. Mononuclear cells from blood and single-cell suspensions from other tissues were prepared as described [12]. Isolated cells were stained with anti-rat CD11b PerCP-eFluor® 710 or anti-rat CD3 fluorescein
www.futuremedicine.com
791
Research Article Duan, Dong, Zhang et al. isothiocyanate (eBioscience, CA, USA) for 20 min at 4°C and analyzed by flow cytometry. The percentage of CD11b+ macrophages or CD3+ T cells positive for Cy3 fluorescence was given as the percentage of cells in the upper right and lower right quadrants. The data for one time point were obtained from one animal. Histological analysis of iron deposition in tissues
The accumulation of the iron oxide of NPs in the liver, spleen and kidney was histologically monitored by Prussian blue staining [16]. In vivo imaging
AA rats that had developed overt arthritis with a mean clinical score of at least 9 (three paws except the CFA-injected paw) were used for in vivo imaging. Rats were randomly assigned to three groups (n = 3). PEI-SPIO/Cy5-siRNA NPs were injected as a single dose (0.6 mg siRNA/kg) via the tail vein. The right hind paw in one group was exposed to a magnetic field as described above. AA rats that did not receive PEISPIO/Cy5-siRNA served as the controls. At different time points after injection, rats were anesthetized and in vivo images were observed with the Caliper IVIS Spectrum (MA, USA) imaging system (excitation 640 nm, emission 680 nm). After 12 h, rats were killed, and their major organs were excised and imaged. Histologic analysis of ankle joints
Right hind paws were resected and processed for histologic analysis as described previously [15]. Radiography
A week after the third injection (day 31), three AA rats from each treatment group were randomly selected and anesthetized. Radiographs of right hind paws were taken using a lumina XR system (Caliper IVIS Spectrum). Quantitation of mRNA for proinflammatory mediators
Right hind ankle joints were resected on day 31 and stored at -80°C. Total RNA was obtained from homogenized joint tissue using TRIzol extraction (Takara, Japan) according to the manufacturer’s instructions (n = 3 per group). The RNA was reverse transcribed into cDNA with a mix of oligo dT and random primers. The mRNA amounts of inflammatory mediators were quantified by quantitative PCR. The primer pairs for TNF‑a, IL‑1b, IL‑15 and MMP‑3 have been described elsewhere [15]. The primer pair for IL‑2/IL‑15Rb was 5´CTTCTTGTCCTGCGTCTG (forward) and 5´GGATGTGGCACTTGAGA A (reverse). PCR reactions were performed at 95°C for 30 s, followed by
792
Nanomedicine (2014) 9(6)
40 cycles of 95°C for 5 s and 60°C for 34 s. Cytokine expression was normalized to GAPDH and calculated as a percentage of the PBS controls. Statistics
The p‑value was calculated using the Student’s t‑test function in Microsoft Excel®. The results were shown as mean ± standard deviation. Results Properties of PEI-SPIO/siRNA complexes
The hydrodynamic size and z‑potential of SPIOs coated with PEI of 10 kDa (PEI-SPIOs) were 48.0 ± 0.8 nm (polydispersity index = 0.230) and +30.52 ± 1.58 mV, respectively. The PEI-SPIOs were stable for at least 22 days (Figure 1A). The PEI-SPIO formed complexes with NC siRNA at Fe:siRNA weight ratios of 4 and above (Figure 1B). The absence of retarded bands (Fe:siRNA ≥4) may be due to exclusion of EtBr during staining, an indication of the broad and strong complexation capacity of PEI-SPIOs with siRNA. The following in vitro and in vivo experiments were carried out at a Fe:siRNA ratio of 8 at which the resulting PEISPIO/NC siRNA complexes showed an average hydrodynamic diameter of 161.5 ± 3.5 nm (polydispersity index = 0.262) and a z‑potential of +26.32 ± 5.34 mV (Supplementary Figure 1; see online at www.futuremedicine.com/doi/suppl/10.2217/nnm.13.217). It is worth noting that the size of PEI-SPIO/siRNA complexes revealed by TEM was approximately 9–10 nm (Figure 1C), which is consistent with that of the core iron oxide of PEI-SPIOs. The size increase in water and the decrease in the z‑potential of the PEI-SPIO/siRNA complexes suggest successful loading of siRNA onto the PEI-SPIO surface via electrostatic interactions, which was further confirmed by the release of siRNA from PEI-SPIO/siRNA complexes when heparin was added (Figure 2A). The average loading of siRNA per particle was calculated to be 11 duplexes at a Fe:siRNA ratio of 8. To assess whether the nanocarrier PEI-SPIOs could protect the cargo from nuclease degradation during systemic circulation, we subjected PEI-SPIO/siRNA to incubation with 50% FBS at 37°C. PEI-SPIO/siRNA complexes were obviously more stable than naked siRNA in the presence of serum. As shown in Figure 2B, siRNA alone was degraded completely within 12 h, but when complexed with PEI-SPIOs, it could survive beyond 48 h. The transfection efficiency and toxicity of PEI correlate strongly with its molecular weight [17]. As revealed by the MTS assay, while empty PEI-SPIOs exhibited a dose-dependent cytotoxicity on RAW 264.7 cells, siRNA-loaded PEI-SPIOs at concentrations ranging from 10 to 100 µg Fe/ml did not induce cell
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
Research Article
Hydrodynamic size (nm)
60
40
20
0
1 min 1 day 8 days 17 days 22 days Time
50 nm
Figure 1. Physical characterization of polyethyleneimine-superparamagnetic iron oxide/siRNA nanoparticles. (A) Stability of polyethyleneimine (PEI)-functionalized superparamagnetic iron oxide nanoparticles (SPIOs). Hydrodynamic size was measured to verify the stability of PEI-SPIOs dissolved in 75% fetal bovine serum for 22 days. (B) Gel retardation analysis of negative control siRNA binding with PEI-SPIOs at various Fe:siRNA (w/w) ratios. (C) Transmission electron microscopy image of PEI-SPIO/negative control siRNA complexes (Fe:siRNA weight ratio of 8).
death (Figure 3A). Since PEI toxicity is mainly associated with the high positive charge of the polymer, little toxicity of PEI-SPIO/siRNA can be partially attributed to the decreased positive charge upon siRNA binding. On the other hand, cytotoxicity of NPs can be size dependent. RAW 264.7 cells are probably not sensitive to the particles approximately 161 nm in size. To examine cellular uptake of siRNA when complexed with PEI-SPIOs, flow cytometric analyses were performed. As shown in Figure 3B, approximately 96.6% of rat peritoneal macrophages engulfed the siRNA. To evaluate the potential application of PEI-SPIOs for siRNA delivery, in vitro transfection experiments were conducted with rat peritoneal macrophages. Gene silencing efficiency of the PEI-SPIO/IL‑2/IL‑15Rb siRNA complexes was determined by measuring the amount of IL‑2/IL‑15Rb protein and mRNA transcripts. Clearly, PEI-SPIO/IL‑2/IL‑15Rb siRNA complexes showed a significant silencing effect, while PEI-SPIO/NC siRNA did not (Figure 3C). Thus, we indirectly demonstrated that IL‑2/IL‑15Rb siRNA was released from the endocytosis vesicles. Tissue distribution of systemically administered PEI-SPIO/siRNA complexes
To determine tissue uptake of the iron core of PEISPIO/siRNA complexes, AA rats were intravenously injected with NC siRNA-loaded PEI-SPIOs. Organs of elimination, including the liver, spleen and kidney, were analyzed for iron content via Prussian blue staining of histology slides at 8 and 24 h after injection. As shown in Figure 4, Prussian blue deposits can be detected in the liver and spleen, but not in the kidney. In the liver and spleen, biodistribution profiles of iron at a given time point are similar between AA rats with and without a magnet adhered to the right hind paw (Figure 4)
future science group
(some data not shown). Since bone decalcification may remove iron, we did not histologically analyze iron accumulation in arthritic joints. To investigate tissue uptake of siRNA in the context of the inflammatory disorder, PEI-SPIOs loaded with Cy5-siRNA were injected into AA rats via the tail vein, and in vivo fluorescence was visualized at 6 and 12 h. Clearly, Cy5-siRNA accumulated in the paws within 6 h and, compared with the paw to which a magnet was
Figure 2. Agarose gel electrophoresis. (A) siRNA release assayed by heparin decomplexation. siRNA (0.5 µg) was incubated with polyethyleneiminesuperparamagnetic iron oxide nanoparticles at a weight ratio of 1:8 for 30 min, then different amounts of heparin were added and the mixtures were further incubated for 30 min. (B) siRNA serum stability. siRNA (0.5 µg per sample) either in naked form or complexed with polyethyleneimine-superparamagnetic iron oxide nanoparticles was mixed with fresh fetal bovine serum (1:1, v/v) to give a 50% serum concentration and incubated at 37°C for various periods of time. C: Complexed with polyethyleneiminesuperparamagnetic iron oxide nanoparticles; N: Naked form.
www.futuremedicine.com
793
Research Article Duan, Dong, Zhang et al.
120
Cell viability (% of control)
100
80
60
40 PEI-SPIO
20
PEI-SPIO/siRNA 0 0
20
40
60 Dose (µg/ml)
Negative control
Cy3
80
100
120
PEI-SPIO/Cy3-siRNA
104
104
103
103
102
102
101
101
100
96.6%
100 10
0
10
10
1
2
10
3
10
4
100
101
102
103
104
FL1-H
Relative IL-2/15Rβ expression
120 100 NC siRNA
IL-15Rβ siRNA
80 IL-15Rβ 60
β-actin
40 20 0
NC siRNA
IL-2/15Rβ siRNA
Figure 3. Biological characterization of polyethyleneimine-superparamagnetic iron oxide/siRNA complexes. The PEI-SPIOs were complexed with siRNA at a Fe:siRNA (w/w) ratio of 8. (A) Cytotoxic effect. RAW 264.7 cells were treated with either PEI-SPIOs or PEI-SPIO/NC siRNA at doses ranging from 10 to 100 µg Fe/ml for 16 h and the MTS assay was performed. Absorbance (y‑axis) values were normalized by dividing over the absorbance of the control (no particle exposure). (B) Uptake of PEI-SPIO/Cy3-siRNA analyzed by flow cytometry. Rat peritoneal macrophages were incubated with 15 µg Fe/ml particles for 24 h. (C) Gene silencing effect of PEI-SPIO/IL‑2/IL‑15Rb siRNA complexes in rat peritoneal macrophages assessed by quantitative PCR (left) and western blotting (right). Cells were incubated with the complexes at 15 µg Fe/ml.
794
Nanomedicine (2014) 9(6)
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
Research Article
Figure 3. Biological characterization of polyethyleneimine-superparamagnetic iron oxide/siRNA complexes (cont.). Total RNA and proteins were prepared after 24 and 48 h of incubation, respectively. NC siRNA was used as a nontargeting negative control. Results of quantitative PCR were normalized to GAPDH and are presented as the percentage of NC siRNA. NC: Negative control; PEI: Polyethyleneimine; SPIO: Superparamagnetic iron oxide.
not applied, application of a magnet overlaying the right hind paw apparently increased siRNA accumulation in this tissue (Figure 5A & B). To determine fluorescence signals in other tissues, major organs were isolated at 12 h and examined using the same equipment as for the whole-animal assay. Strong fluorescence was observed in the kidney and liver, whereas fluorescence was almost absent from the heart, lung and spleen (Figure 5C). In these organs, distribution profiles of Cy5-siRNA are similar between AA rats with and without a magnet adhered to the right hind paw (data not shown). In vivo cellular uptake of systemically administered siRNA
Two predominant cell types in the synovial infiltrate of RA, the phagocytic macrophages and the Liver
non-phagocytic T lymphocytes were chosen to assess the preferential immune cell type targeted by PEISPIO/siRNA NPs. To this end, we collected blood, spleen, liver, kidney and inflamed joints at 2, 8 and 24 h after intravenous injection of PEI-SPIO/Cy3-siRNA complexes to AA rats, and performed flow cytometry to examine in vivo cellular uptake of the nanocomplexes. The Cy3-siRNA was detected in all of the tissues investigated. Flow cytometry staining for CD11b+ cells and CD3+ cells in the right hind paw at 24 h is shown in Figure 6A & B. As expected, CD11b+ cells took up the siRNA more efficiently than CD3+ cells at any time point in all of the organs examined (Figure 6C & D), indicating that the siRNA showed specific targeting to macrophages in the PEI-SPIO formulation. At a given time point, the percentage of Cy3-positive CD11b+ or Spleen
Kidney
0h
8h
24 h
Figure 4. Tissue distribution of the iron core of polyethyleneimine‑superparamagnetic iron oxide/negative control siRNA nanoparticles in the absence of a magnetic field. The nanoparticles were administered to adjuvant arthritis rats via tail veins at 0.3 mg siRNA/kg. At 8 and 24 h after single-dose administration, rats were killed and organs of elimination were collected. 0 h represents noninjected controls. Representative tissue sections were stained by Prussian blue to visualize iron deposits. As the spleen is a primary site for destruction of old red blood cells and subsequent recycling of hemoglobin‑bound iron, small amounts of iron can be found in the blank spleen.
future science group
www.futuremedicine.com
795
Research Article Duan, Dong, Zhang et al.
Liver Lung Heart Kidney Spleen Fluorescence (arbitrary units)
300 Untreated
Treated, M-
Treated, M+
250
MM+
200
*
Untreated * Treated 1
150 100
Treated 2
50 0
6h
12 h
Treated 3
Figure 5. In vivo imaging of siRNA biodistribution. The polyethyleneimine-superparamagnetic iron oxide/Cy5-siRNA nanoparticles were administered as a single dose (0.6 mg siRNA/kg) to six adjuvant arthritis rats via the tail vein. Right hind paws of three rats were exposed to a magnetic field for 2 h after injection. Images were taken with the Caliper IVIS Spectrum imaging system. Untreated represents the noninjected control. (A) Representative fluorescence images of right hind paws at 6 and 12 h after injection. (B) Measurement of fluorescence in the right hind paws at 6 and 12 h after injection. Areas of identical size were chosen and background fluorescence obtained from the noninjected control was subtracted. (C) Fluorescence images of isolated organs. Three rats exposed to a magnetic field were killed 12 h after injection. The heart, liver, spleen, lung and right kidney were isolated and imaged. *p < 0.05. M+: Presence of a magnet fixed to the right hind paw; M-: Absence of a magnet.
CD3+ cells in the arthritic joint was similar to that in the liver, but higher than in the spleen, blood and kidney. This is in contrast to an animal model injected intravenously with siRNA lipoplexes, where the proportion of CD11b+ cells that entrapped the siRNA was much lower in joints than in the blood, liver and spleen [18]. Notably, a 2‑h treatment with a magnetic field significantly increased Cy3-siRNA uptake by CD11b+ and CD3+ cells isolated from the arthritic joint throughout the experimental period (Figure 6). Systemic delivery of PEI-SPIO/IL‑2/IL‑15Rb siRNA NPs provides protection against AA
To investigate the therapeutic effect of systemically administered PEI-SPIO/IL‑2/IL‑15Rb siRNA, AA rats were injected with the NPs every 7 days (day 10, 17 and 24) after arthritis induction on day 0. On day 31, rats were subjected to radiography before being killed, and paws were then isolated for histological evaluation. Compared with PBS- and NC siRNAtreated groups, rats treated with IL‑2/IL‑15Rb siRNA showed a decreased severity of AA, as assessed by clinical scores (Figure 7A). Histological and radiographic examination of IL‑2/IL‑15Rb siRNA-treated animals confirmed these findings and revealed a strong reduction in articular inflammation and destruction (Figure 7B & C). Notably, when a magnetic field was applied to the inflamed joint in an attempt to increase the local concentration of the particles, the therapeutic effect of IL‑2/IL‑15Rb siRNA was further improved.
796
Nanomedicine (2014) 9(6)
Specifically, clinical scores in the group receiving combination treatment with IL‑2/IL‑15Rb siRNA and a magnetic field decreased rapidly and were always lower than those in the IL‑2/IL‑15Rb siRNA-treated group. While IL‑2/IL‑15Rb siRNA monotherapy greatly inhibited bone damage of the arthritic joints, it did not completely protect against it, as shown by the presence of mild bone erosions in two of the three right hind paws examined (arrows in Figure 7C). On the other hand, right hind paws (three out of three) from the combination treatment group appeared completely normal. Therefore, treatment with PEI-SPIOdelivered IL‑2/IL‑15Rb siRNA clearly inhibits disease progression in AA rats and addition of a magnet on the diseased joint even for a short time can lead to a more profound enhancement of RNAi-mediated therapeutic effect. Quantitative PCR was used to assess the effect of PEI-SPIO-delivered IL‑2/IL‑15Rb siRNA treatment on the expression of IL‑2/IL‑15Rb, as well as several wellknown proinflammatory mediators in the joints of AA rats. As shown in Figure 7D, expression of IL‑2/IL‑15Rb, TNF‑a, IL‑1b and MMP‑3 in the group receiving IL‑2/IL‑15Rb siRNA decreased sharply compared with PBS-treated arthritic controls. Interestingly, reduction in IL‑15 mRNA production upon IL‑2/IL‑15Rb knockdown was not as drastic, consistent with the role of IL‑15 signaling in the amplification of the inflammatory network in RA. Combination treatment with PEI-SPIO/IL‑2/IL‑15Rb siRNA and a magnet field
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
produced a stronger inhibitory effect on the expression of both IL‑2/IL‑15Rb and the cytokines examined.
PLGA-coated superparamagnetic microparticles as carriers for local treatment of arthritis [21,22]. Although an intra-articular injection of RA therapeutics can be used to avoid systemic side effects, it is not patient friendly due to the associated pain and repeated injections into multiple joints. Therefore, we investigated the PEIfunctionalized SPIOs for systemic siRNA delivery in experimental arthritis. NPs are usually taken up by the liver, spleen and other organs of the reticuloendothelial system. Consistent with this, tissue distribution analysis showed
Discussion Various types of drug carriers including liposomes and biodegradable polymers have been used for the delivery of RA therapeutics [19,20]. Considering that SPIO is the prevailing nanomaterial in biomedical research, there are very few reports in the literature describing superparamagnetic iron oxide-based micro- or nanoparticles for treating RA. In 2009, Butoescu et al. used PBS
Cy3-siRNA
Cy3-siRNA + magnet
104
104
104
103
103
103
2 Cy3 10
102
102
101
101
101
100 100
101
102
103
100 104 100
Research Article
101
102
103
100 104 100
101
102
103
104
CD3 PBS
Cy3-siRNA
Cy3-siRNA + magnet
104
104
104
103
103
103
2 Cy3 10
102
102
101
101
101
101
102 103 CD11b
100 104 100
101
30 2h 8h 24 h
25 20 15 10 5 0
M- M+ M- M+ M- M+ M- M+ M- M+ Kidney Liver Spleen Paw Blood
102
Cy3-siRNA-positive CD11b cells
Cy3-siRNA-positive CD3 cells
100 100
103
100 104 100
101
102
103
104
60 50
2h 8h 24 h
40 30 20 10 0
M- M+ M- M+ M- M+ M- M+ M- M+ Kidney Liver Spleen Paw Blood
Figure 6. In vivo cellular uptake of siRNA nanocomplexes after single systemic administration. Six arthritic rats were injected intravenously with 0.3 mg/kg Cy3-siRNA formulated with polyethyleneimine-coated superparamagnetic iron oxide. Following injection, the right hind paws of three rats were exposed to a magnetic field for 2 h. The rat receiving PBS was used as a control. Cellular uptake of Cy3-siRNA was evaluated by flow cytometry 2, 8 and 24 h after injection using anti-CD3 and anti-CD11b monoclonal antibodies. (A & B) Flow cytometry dot plots of cells in the right hind paw at 24 h. (C & D) Histograms show percentages of Cy3-siRNA uptake within the gated CD3 + or CD11b + cells in the M+ or M-. M+: Presence of a magnet fixed to the right hind paw; M-: Absence of a magnet; PBS: Phosphate-buffered saline.
future science group
www.futuremedicine.com
797
Research Article Duan, Dong, Zhang et al. 14 PBS NC siRNA IL-2/IL-15Rβ siRNA IL-2/IL-15Rβ siRNA + magnet
Arthritis score
12 10 8 6
***
4 2 0
**
**
** *** ** ** ***
*
** **
*** ***
***
*** *** *** *** *** ** *** *** *** *** *** *** *** *** 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 21 22 23 26 28 30 Time after immunization (days) IL-2/IL-15Rβ IL-2/IL-15Rβ siRNA siRNA + magnet PBS NC siRNA
Normal
PBS
NC siRNA
IL-2/IL-15Rβ siRNA
IL-2/IL-15Rβ siRNA + magnet
mRNA expression (%)
140 120 100 80
p < 0.05
60 40
p < 0.001
20 0
**
**
TNF-α
p < 0.001 **
**
MMP3
p < 0.01 ** IL-1β
*
p < 0.05 **
** IL-15
NC siRNA IL-2/IL-15Rβ siRNA IL-2/IL-15Rβ siRNA + magnet
** ** IL-2/IL-15Rβ
Figure 7. Systemic delivery of polyethyleneimine-superparamagnetic iron oxide/IL‑2/IL‑15Rb siRNA complexes inhibits arthritis progression in adjuvant arthritis rats. Male Wistar rats (n = 6 per group) were given Freund’s complete adjuvant in the left hind footpad on day 0. PBS and IL‑2/IL‑15Rb or NC siRNA nanocomplexes were separately administered on days 10, 17 and 24 at 0.3 mg siRNA/kg via the tail vein. For combination therapy, the right hind paw was exposed to a static magnetic field for 2 h after each injection. (A) Arthritis score. Values are the mean and standard deviation. (B) Representative histopathologies of the right hind ankle joints stained with hematoxylin and eosin. On day 31, rats were sacrificed and subjected to histopathological examination. (C) Representative radiographs of right hind paws. On day 31, three rats from each group were subjected to radiography. Arrow heads indicate sites with mild bone erosion in the IL‑2/IL‑15Rb siRNA treatment group. (D) Effects of IL‑2/IL‑15Rb siRNA nanocomplexes on mRNA expression of certain inflammatory factors. On day 31, RNA was prepared from the right hind ankle joints. mRNA levels of each factor were determined by quantitative PCR, normalized against GAPDH and expressed as a percentage of the PBS controls. The results are presented as values (the mean and standard deviation) obtained from three different samples randomly selected in each group. (A) *p
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
Aims: This study aimed to examine the efficacy of a nanocarrier (polyethyleneimine [PEI]-superparamagnetic iron oxide nanoparticle [SPIO]), composed of a core of iron oxide and a shell of PEI, in the systemic delivery of therapeutic siRNA to experimental arthritic joints. Materials & methods: PEI-SPIO/siRNA nanoparticles were synthesized and characterized in vitro. Nanoparticles were administered intravenously to arthritic rats to analyze cellular uptake, tissue distribution and the therapeutic effect of a siRNA against the IL‑2/-15 receptor b chain (IL‑2/IL‑15Rb). Results: PEI-SPIOs loaded with siRNA displayed negligible cytotoxicity, improved siRNA stability, efficient uptake by macrophages and the ability to induce specific gene silencing in vitro. PEI-SPIO-delivered siRNA accumulated easily in inflamed joints and was efficiently taken up by joint macrophages and T cells. Although IL‑2/IL‑15Rb siRNA-loaded PEI-SPIOs alone were efficacious in the treatment of experimental arthritis, combination therapy with both PEI-SPIO/IL‑2/IL‑15Rb siRNA and a magnetic field displayed an additive anti-inflammatory effect. Conclusion: PEI-functionalized SPIOs can be employed for systemic siRNA delivery in rheumatoid arthritis and enhanced therapeutic benefit can be achieved by the use of an external magnetic field.
Juanli Duan1, Jinlai Dong2, Tiantian Zhang1, Zhenyi Su3, Jie Ding3, Yu Zhang2 & Xiaohua Mao*,1,3 Key Laboratory of Ministry of Education for Developmental Genes & Human Diseases, School of Life Sciences, Southeast University, Nanjing, China 2 State Key Laboratory of Bioelectronics, Jiangsu Key Laboratory for Biomaterials & Devices, School of Biological Science & Medical Engineering, Southeast University, Nanjing, China 3 Department of Biochemistry, School of Medicine, Southeast University, Nanjing, China *Author for correspondence: Tel.: +86 25 83272474 Fax: +86 25 83324887 [email protected] 1
Original submitted 1 November 2013; Revised submitted 12 December 2013 Keywords: IL‑2/IL‑15Rb • macrophage • nanoparticle • rheumatoid arthritis • siRNA
Monoclonal antibodies and soluble receptors targeting major imflammatory cytokines or cells have emerged as a major tool in the treatment of rheumatoid arthritis (RA) and expanded rapidly over the past decade [1–4]. However, each of biologics currently used in RA has associated issues, including only a partial response, and unwanted systemic adverse effects and safety complications after long-term treatment. For this reason, there is an unmet medical need for safer and more efficient treatment regimens for disease remission. Owing to their unique magnetic properties, biocompatibility, comparable size to biologically important objects and easily adaptable surface for bioagent attachment, superparamagnetic iron oxide nanoparticles (SPIOs) have shown great promise in
10.2217/NNM.13.217 © 2014 Future Medicine Ltd
biomedical applications. In particular, their responses to external magnetic fields enable them to be novel vehicles for targeted drug delivery. For gene delivery by SPIOs, their surface is usually modified with a layer of cationic polymers onto which negatively charged nucleic acids can be electrostatically attracted and tethered [5,6]. While magnetic nanoparticle (NP)-based delivery vehicles have been extensively studied in, for example, cancer diagnosis and therapy, to our knowledge, the literature contains few reports of systemically administered SPIOs primed for magnetic targeting in inflammatory diseases. RA has two properties that make it an attractive target for SPIO-mediated therapeutics. First, it is, like solid tumors, an angiogenic disease in which the pathological
Nanomedicine (2014) 9(6), 789–801
part of
ISSN 1743-5889
789
Research Article Duan, Dong, Zhang et al. tissue is lined with abnormal and leaky blood vessels [7]. Second, the tissue is heavily infiltrated with macrophages that, upon activation, are not only the main secretors of pathogenic cytokines and chemokines, but also key players in angiogenesis [8,9] and, therefore, of central importance in the pathogenesis of RA. In this study, polyethylenimine (PEI)-functionalized SPIOs were employed as a carrier to deliver IL‑2/ IL‑15Rb siRNA to inflamed joints in a rat model of RA. We reason that, due to the enhanced permeability and retention effect and their response to an external magnetic field, SPIOs tend to accumulate in and spread inside inflamed joint tissues. These NPs would then be avidly taken up by inflammatory cells, especially macrophages. Given that activated macrophages express IL‑15R and that IL‑15 optimizes interactions between T cells and macrophages, which in turn causes the release of a wide range of inflammatory mediators including the key proinflammatory cytokine TNF‑a and IL‑15 itself [10,11], we believe inhibiting IL‑2/IL‑15Rb expression using siRNA-loaded SPIOs would downregulate a central amplification network of inflammation within the diseased joints. The data, presented below, are consistent with this hypothesis. Materials & methods siRNA duplexes
The siRNA duplex targeting rat IL‑2/IL‑15Rb (sense sequence: 5´GA AGGGAUGUCUACCA AUATT) and a negative control (NC) siRNA (sense sequence: 5´UUCUCCGAACGUGUCACGUTT) were synthesized by GenePharma (Shanghai, China) [12]. NC siRNA labeled with Cy3 or Cy5 (Cy3-/Cy5-siRNA) was from RiboBio (Guangzhou, China). Nanocomposite preparation
PEI-SPIOs were synthesized using the following process. Oleic acid (OA)-capped Fe3O4 was first synthesized by a modified oxidative coprecipitation method in an OA/H2O/dimethylsulfoxide mixing solution, and dimercaptosuccinic acid was then used to replace OA to obtain water-soluble dimercaptosuccinic acid-capped Fe3O4 NPs – SPIOs [13,14]. PEI (10 kDa, Sigma-Aldrich, MO, USA) was further capped onto the surface of SPIOs by electrostatic adsorption. Briefly, 10 ml of 0.2‑mg/ml SPIOs were added into excess PEI solution under stirring. The mixture was sonicated for 20 min and then further stirred for 2 h. The obtained PEI-functionalized SPIOs (PEI-SPIOs) were purified by removing free PEI through an ultrafiltration membrane (molecular weight cut-off: 300 kDa). Fe concentration was determined by the colorimetric method using phenanthroline. The PEI-SPIO/siRNA complexes with different Fe:siRNA (w/w) ratios were prepared by adding an appropriate amount of PEI-SPIO into siRNA, whose
790
Nanomedicine (2014) 9(6)
concentration was kept constant. The resulting mixture was incubated at room temperature (RT) for 30 min to allow composite formation. Physical characterization
z‑potential and the size of PEI-SPIO and PEI-SPIO/ siRNA NPs in solution were measured by dynamic light scattering using Brookhaven ZetaPlus Analyzer (NY, USA). PEI-SPIO/siRNA NPs were prepared by mixing NC siRNA with PEI-SPIOs at a Fe:siRNA weight ratio of 8 for 30 min at RT and then measured by dynamic light scattering with a laser wavelength of 633 nm and a measurement angle of 90°. The morphology of PEISPIO/siRNA and PEI-SPIO NPs was characterized using a transmission electron microscope (TEM). The samples for TEM imaging were prepared by placing a drop of the respective NP suspension onto a copper grid and drying at ambient temperature overnight. The samples were then visualized by a TEM (JEOL JEM 2100, Japan). Gel retardation assay
NC siRNA was mixed with PEI-SPIOs at various Fe:siRNA weight ratios. The resulting mixtures were incubated at RT for 30 min, after which the samples were analyzed by 3% agarose gel electrophoresis and visualized by ethidium bromide (EtBr) staining. Heparin decomplexation assay
siRNA was complexed with PEI-SPIOs at a Fe:siRNA weight ratio of 8 for 30 min at RT. Various amounts of heparin (heparin:siRNA weight ratio of 0, 20, 80, 160, 200 and 300) were then added, and the mixtures were further incubated for 30 min. After centrifugation (12,000 rpm, 15 min, 4°C), supernatants were analyzed by 3% agarose gel electrophoresis and the released siRNA was visualized by EtBr staining. Serum stability assay
siRNA was complexed with PEI-SPIOs at a Fe:siRNA weight ratio of 8 for 30 min at RT. After adding fetal bovine serum (FBS) at a final 50% concentration, aliquots were incubated for various time periods at 37°C. The resulting samples were treated with 50 µg of heparin (heparin:siRNA weight ratio of 100) at RT for 30 min. The released siRNA fraction was separated and visualized as described above. Peritoneal macrophage isolation
Rats were sacrificed 3 days after intraperitoneal injection of 5 ml of 4% thioglycollate media (SigmaAldrich). Peritoneal lavage was performed with 20 ml of cold phosphate-buffered saline (PBS; 0.1 M, pH 7.4). The resulting peritoneal fluid was centrifuged
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
at 1000 rpm for 10 min at 4°C. Cells were then washed twice with cold PBS and resuspended in high-glucose DMEM medium containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were counted and then seeded. After adhesion to plastic culture dishes for 2 h at 37°C and 5% CO2, the cells were washed three times with PBS to remove nonadherent cells. Adherent macrophages were cultured in fresh medium as described above and used for future experiments. Cell viability assay
Cell viability was determined by MTS assay. Briefly, RAW 264.7 cells were seeded in 96‑well cell culture plates at 1 × 10 4 cells/well in 100 µl DMEM and grown for 24 h to 80–90% confluence. The cells were then exposed to the indicated dose of NPs for 16 h at 37°C. Formazan absorbance at 490 nm was measured 3 h after addition of MTS. Western blotting
Rat peritoneal macrophages were incubated with PEI-SPIO/siRNA NPs (Fe:siRNA weight ratio of 8) at a concentration of 15 µg Fe/ml. Then, 48 h after transfection, cells were lysed in lysis buffer containing 50 mM Tris‑HCl (pH 7.5), 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid, 1% Triton X‑100, 0.1% SDS, 0.5% sodium deoxycholate and protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Cellular debris was removed and proteins were collected by centrifugation. Equal amounts (30 µg) of protein were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 2.5% bovine serum albumin-TBST (1.0 M Tris‑Cl, pH 8.0, 150 mM NaCl, 0.05% Tween‑20 ®, Sigma-Aldrich), incubated with rabbit anti-IL‑2Rb (M‑20) or anti‑b actin (R‑22) antibodies (Santa Cruz Biotechnology, CA, USA), followed by probing with horseradish peroxidase-labeled goat anti-rabbit antibody (Wuhan Boster Biological Technology, China). In vitro assessment of cellular uptake
Rat peritoneal macrophages were seeded in 24‑well plates at 2 × 105 cells/well and grown overnight. Uptake experiments were initiated by adding PEISPIO/Cy3-siRNA complexes (Fe:siRNA weight ratio of 8) at a concentration of 15 µg Fe/ml followed by incubation for 24 h. Cells were then trypsinized into single-cell suspensions with 0.25% trypsin-ethylenediaminetetraacetic acid, harvested by centrifugation, washed with PBS and resuspended in 500 µl of PBS containing 1% FBS. The resulting cell suspensions were analyzed by flow cytometry.
future science group
Research Article
Animal studies
Specific pathogen-free male Wistar rats were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). All animal experiments were performed in accordance with the guideline of the Committee on Animals of Southeast University, China. All surgery and in vivo imaging were performed under anesthesia and all efforts were made to minimize suffering. Induction, treatment & clinical evaluation of adjuvant arthritis
Adjuvant arthritis (AA) was induced by a single intradermal injection (100 µl) of Freund’s complete adjuvant (CFA) containing 10 mg/ml heat-killed Bacillus Calmette–Guérin freeze-dried powder (National Institutes for Food and Drug Control, China) in sterile liquid paraffin into the footpad of the left hind paw on day 0. Clinical evidence of arthritis usually occurred on day 11. For therapeutic treatment studies, PEI-SPIO/IL‑2/ IL‑15Rb siRNA complexes (0.3 mg siRNA/kg) were administered via the tail vein once a week starting on day 10 for 3 weeks. The control rats received either PBS or nonspecific NC siRNA-loaded PEI-SPIOs. To determine whether an external magnetic field is able to enhance the therapeutic effect of IL‑2/IL‑15Rb siRNA, one group treated with a combination of PEISPIO/IL‑2/IL‑15Rb siRNA and a magnetic field was also included. In this group, a round NdFeB magnet (24 mm diameter and 8 mm thick, with a central hole 9 mm in diameter and surface magnetic field of 400 mT) was fixed on the skin of the right hind paw for 2 h after each injection. The animals were anesthetized by isoflurane inhalation during magnetic exposure. Arthritis in each paw was scored as previously described [15]. Three paws except the CFA-injected left hind paw were scored, so the highest possible score per rat was 12. In vivo assessment of cellular uptake
Rats that had developed overt arthritis with a mean clinical score of at least 9 were used. The AA rats were injected intravenously with a single dose of PEI-SPIO/Cy3-siRNA (0.3 mg siRNA/kg) NPs with or without magnetic field exposure and control rats received PBS. To apply a magnetic field, a NdFeB magnet of 400 mT was fixed on the skin of the right hind paw for 2 h after injection. Blood, liver, spleen, kidney and inflamed paws were collected at 2, 8 and 24 h after injection. Mononuclear cells from blood and single-cell suspensions from other tissues were prepared as described [12]. Isolated cells were stained with anti-rat CD11b PerCP-eFluor® 710 or anti-rat CD3 fluorescein
www.futuremedicine.com
791
Research Article Duan, Dong, Zhang et al. isothiocyanate (eBioscience, CA, USA) for 20 min at 4°C and analyzed by flow cytometry. The percentage of CD11b+ macrophages or CD3+ T cells positive for Cy3 fluorescence was given as the percentage of cells in the upper right and lower right quadrants. The data for one time point were obtained from one animal. Histological analysis of iron deposition in tissues
The accumulation of the iron oxide of NPs in the liver, spleen and kidney was histologically monitored by Prussian blue staining [16]. In vivo imaging
AA rats that had developed overt arthritis with a mean clinical score of at least 9 (three paws except the CFA-injected paw) were used for in vivo imaging. Rats were randomly assigned to three groups (n = 3). PEI-SPIO/Cy5-siRNA NPs were injected as a single dose (0.6 mg siRNA/kg) via the tail vein. The right hind paw in one group was exposed to a magnetic field as described above. AA rats that did not receive PEISPIO/Cy5-siRNA served as the controls. At different time points after injection, rats were anesthetized and in vivo images were observed with the Caliper IVIS Spectrum (MA, USA) imaging system (excitation 640 nm, emission 680 nm). After 12 h, rats were killed, and their major organs were excised and imaged. Histologic analysis of ankle joints
Right hind paws were resected and processed for histologic analysis as described previously [15]. Radiography
A week after the third injection (day 31), three AA rats from each treatment group were randomly selected and anesthetized. Radiographs of right hind paws were taken using a lumina XR system (Caliper IVIS Spectrum). Quantitation of mRNA for proinflammatory mediators
Right hind ankle joints were resected on day 31 and stored at -80°C. Total RNA was obtained from homogenized joint tissue using TRIzol extraction (Takara, Japan) according to the manufacturer’s instructions (n = 3 per group). The RNA was reverse transcribed into cDNA with a mix of oligo dT and random primers. The mRNA amounts of inflammatory mediators were quantified by quantitative PCR. The primer pairs for TNF‑a, IL‑1b, IL‑15 and MMP‑3 have been described elsewhere [15]. The primer pair for IL‑2/IL‑15Rb was 5´CTTCTTGTCCTGCGTCTG (forward) and 5´GGATGTGGCACTTGAGA A (reverse). PCR reactions were performed at 95°C for 30 s, followed by
792
Nanomedicine (2014) 9(6)
40 cycles of 95°C for 5 s and 60°C for 34 s. Cytokine expression was normalized to GAPDH and calculated as a percentage of the PBS controls. Statistics
The p‑value was calculated using the Student’s t‑test function in Microsoft Excel®. The results were shown as mean ± standard deviation. Results Properties of PEI-SPIO/siRNA complexes
The hydrodynamic size and z‑potential of SPIOs coated with PEI of 10 kDa (PEI-SPIOs) were 48.0 ± 0.8 nm (polydispersity index = 0.230) and +30.52 ± 1.58 mV, respectively. The PEI-SPIOs were stable for at least 22 days (Figure 1A). The PEI-SPIO formed complexes with NC siRNA at Fe:siRNA weight ratios of 4 and above (Figure 1B). The absence of retarded bands (Fe:siRNA ≥4) may be due to exclusion of EtBr during staining, an indication of the broad and strong complexation capacity of PEI-SPIOs with siRNA. The following in vitro and in vivo experiments were carried out at a Fe:siRNA ratio of 8 at which the resulting PEISPIO/NC siRNA complexes showed an average hydrodynamic diameter of 161.5 ± 3.5 nm (polydispersity index = 0.262) and a z‑potential of +26.32 ± 5.34 mV (Supplementary Figure 1; see online at www.futuremedicine.com/doi/suppl/10.2217/nnm.13.217). It is worth noting that the size of PEI-SPIO/siRNA complexes revealed by TEM was approximately 9–10 nm (Figure 1C), which is consistent with that of the core iron oxide of PEI-SPIOs. The size increase in water and the decrease in the z‑potential of the PEI-SPIO/siRNA complexes suggest successful loading of siRNA onto the PEI-SPIO surface via electrostatic interactions, which was further confirmed by the release of siRNA from PEI-SPIO/siRNA complexes when heparin was added (Figure 2A). The average loading of siRNA per particle was calculated to be 11 duplexes at a Fe:siRNA ratio of 8. To assess whether the nanocarrier PEI-SPIOs could protect the cargo from nuclease degradation during systemic circulation, we subjected PEI-SPIO/siRNA to incubation with 50% FBS at 37°C. PEI-SPIO/siRNA complexes were obviously more stable than naked siRNA in the presence of serum. As shown in Figure 2B, siRNA alone was degraded completely within 12 h, but when complexed with PEI-SPIOs, it could survive beyond 48 h. The transfection efficiency and toxicity of PEI correlate strongly with its molecular weight [17]. As revealed by the MTS assay, while empty PEI-SPIOs exhibited a dose-dependent cytotoxicity on RAW 264.7 cells, siRNA-loaded PEI-SPIOs at concentrations ranging from 10 to 100 µg Fe/ml did not induce cell
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
Research Article
Hydrodynamic size (nm)
60
40
20
0
1 min 1 day 8 days 17 days 22 days Time
50 nm
Figure 1. Physical characterization of polyethyleneimine-superparamagnetic iron oxide/siRNA nanoparticles. (A) Stability of polyethyleneimine (PEI)-functionalized superparamagnetic iron oxide nanoparticles (SPIOs). Hydrodynamic size was measured to verify the stability of PEI-SPIOs dissolved in 75% fetal bovine serum for 22 days. (B) Gel retardation analysis of negative control siRNA binding with PEI-SPIOs at various Fe:siRNA (w/w) ratios. (C) Transmission electron microscopy image of PEI-SPIO/negative control siRNA complexes (Fe:siRNA weight ratio of 8).
death (Figure 3A). Since PEI toxicity is mainly associated with the high positive charge of the polymer, little toxicity of PEI-SPIO/siRNA can be partially attributed to the decreased positive charge upon siRNA binding. On the other hand, cytotoxicity of NPs can be size dependent. RAW 264.7 cells are probably not sensitive to the particles approximately 161 nm in size. To examine cellular uptake of siRNA when complexed with PEI-SPIOs, flow cytometric analyses were performed. As shown in Figure 3B, approximately 96.6% of rat peritoneal macrophages engulfed the siRNA. To evaluate the potential application of PEI-SPIOs for siRNA delivery, in vitro transfection experiments were conducted with rat peritoneal macrophages. Gene silencing efficiency of the PEI-SPIO/IL‑2/IL‑15Rb siRNA complexes was determined by measuring the amount of IL‑2/IL‑15Rb protein and mRNA transcripts. Clearly, PEI-SPIO/IL‑2/IL‑15Rb siRNA complexes showed a significant silencing effect, while PEI-SPIO/NC siRNA did not (Figure 3C). Thus, we indirectly demonstrated that IL‑2/IL‑15Rb siRNA was released from the endocytosis vesicles. Tissue distribution of systemically administered PEI-SPIO/siRNA complexes
To determine tissue uptake of the iron core of PEISPIO/siRNA complexes, AA rats were intravenously injected with NC siRNA-loaded PEI-SPIOs. Organs of elimination, including the liver, spleen and kidney, were analyzed for iron content via Prussian blue staining of histology slides at 8 and 24 h after injection. As shown in Figure 4, Prussian blue deposits can be detected in the liver and spleen, but not in the kidney. In the liver and spleen, biodistribution profiles of iron at a given time point are similar between AA rats with and without a magnet adhered to the right hind paw (Figure 4)
future science group
(some data not shown). Since bone decalcification may remove iron, we did not histologically analyze iron accumulation in arthritic joints. To investigate tissue uptake of siRNA in the context of the inflammatory disorder, PEI-SPIOs loaded with Cy5-siRNA were injected into AA rats via the tail vein, and in vivo fluorescence was visualized at 6 and 12 h. Clearly, Cy5-siRNA accumulated in the paws within 6 h and, compared with the paw to which a magnet was
Figure 2. Agarose gel electrophoresis. (A) siRNA release assayed by heparin decomplexation. siRNA (0.5 µg) was incubated with polyethyleneiminesuperparamagnetic iron oxide nanoparticles at a weight ratio of 1:8 for 30 min, then different amounts of heparin were added and the mixtures were further incubated for 30 min. (B) siRNA serum stability. siRNA (0.5 µg per sample) either in naked form or complexed with polyethyleneimine-superparamagnetic iron oxide nanoparticles was mixed with fresh fetal bovine serum (1:1, v/v) to give a 50% serum concentration and incubated at 37°C for various periods of time. C: Complexed with polyethyleneiminesuperparamagnetic iron oxide nanoparticles; N: Naked form.
www.futuremedicine.com
793
Research Article Duan, Dong, Zhang et al.
120
Cell viability (% of control)
100
80
60
40 PEI-SPIO
20
PEI-SPIO/siRNA 0 0
20
40
60 Dose (µg/ml)
Negative control
Cy3
80
100
120
PEI-SPIO/Cy3-siRNA
104
104
103
103
102
102
101
101
100
96.6%
100 10
0
10
10
1
2
10
3
10
4
100
101
102
103
104
FL1-H
Relative IL-2/15Rβ expression
120 100 NC siRNA
IL-15Rβ siRNA
80 IL-15Rβ 60
β-actin
40 20 0
NC siRNA
IL-2/15Rβ siRNA
Figure 3. Biological characterization of polyethyleneimine-superparamagnetic iron oxide/siRNA complexes. The PEI-SPIOs were complexed with siRNA at a Fe:siRNA (w/w) ratio of 8. (A) Cytotoxic effect. RAW 264.7 cells were treated with either PEI-SPIOs or PEI-SPIO/NC siRNA at doses ranging from 10 to 100 µg Fe/ml for 16 h and the MTS assay was performed. Absorbance (y‑axis) values were normalized by dividing over the absorbance of the control (no particle exposure). (B) Uptake of PEI-SPIO/Cy3-siRNA analyzed by flow cytometry. Rat peritoneal macrophages were incubated with 15 µg Fe/ml particles for 24 h. (C) Gene silencing effect of PEI-SPIO/IL‑2/IL‑15Rb siRNA complexes in rat peritoneal macrophages assessed by quantitative PCR (left) and western blotting (right). Cells were incubated with the complexes at 15 µg Fe/ml.
794
Nanomedicine (2014) 9(6)
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
Research Article
Figure 3. Biological characterization of polyethyleneimine-superparamagnetic iron oxide/siRNA complexes (cont.). Total RNA and proteins were prepared after 24 and 48 h of incubation, respectively. NC siRNA was used as a nontargeting negative control. Results of quantitative PCR were normalized to GAPDH and are presented as the percentage of NC siRNA. NC: Negative control; PEI: Polyethyleneimine; SPIO: Superparamagnetic iron oxide.
not applied, application of a magnet overlaying the right hind paw apparently increased siRNA accumulation in this tissue (Figure 5A & B). To determine fluorescence signals in other tissues, major organs were isolated at 12 h and examined using the same equipment as for the whole-animal assay. Strong fluorescence was observed in the kidney and liver, whereas fluorescence was almost absent from the heart, lung and spleen (Figure 5C). In these organs, distribution profiles of Cy5-siRNA are similar between AA rats with and without a magnet adhered to the right hind paw (data not shown). In vivo cellular uptake of systemically administered siRNA
Two predominant cell types in the synovial infiltrate of RA, the phagocytic macrophages and the Liver
non-phagocytic T lymphocytes were chosen to assess the preferential immune cell type targeted by PEISPIO/siRNA NPs. To this end, we collected blood, spleen, liver, kidney and inflamed joints at 2, 8 and 24 h after intravenous injection of PEI-SPIO/Cy3-siRNA complexes to AA rats, and performed flow cytometry to examine in vivo cellular uptake of the nanocomplexes. The Cy3-siRNA was detected in all of the tissues investigated. Flow cytometry staining for CD11b+ cells and CD3+ cells in the right hind paw at 24 h is shown in Figure 6A & B. As expected, CD11b+ cells took up the siRNA more efficiently than CD3+ cells at any time point in all of the organs examined (Figure 6C & D), indicating that the siRNA showed specific targeting to macrophages in the PEI-SPIO formulation. At a given time point, the percentage of Cy3-positive CD11b+ or Spleen
Kidney
0h
8h
24 h
Figure 4. Tissue distribution of the iron core of polyethyleneimine‑superparamagnetic iron oxide/negative control siRNA nanoparticles in the absence of a magnetic field. The nanoparticles were administered to adjuvant arthritis rats via tail veins at 0.3 mg siRNA/kg. At 8 and 24 h after single-dose administration, rats were killed and organs of elimination were collected. 0 h represents noninjected controls. Representative tissue sections were stained by Prussian blue to visualize iron deposits. As the spleen is a primary site for destruction of old red blood cells and subsequent recycling of hemoglobin‑bound iron, small amounts of iron can be found in the blank spleen.
future science group
www.futuremedicine.com
795
Research Article Duan, Dong, Zhang et al.
Liver Lung Heart Kidney Spleen Fluorescence (arbitrary units)
300 Untreated
Treated, M-
Treated, M+
250
MM+
200
*
Untreated * Treated 1
150 100
Treated 2
50 0
6h
12 h
Treated 3
Figure 5. In vivo imaging of siRNA biodistribution. The polyethyleneimine-superparamagnetic iron oxide/Cy5-siRNA nanoparticles were administered as a single dose (0.6 mg siRNA/kg) to six adjuvant arthritis rats via the tail vein. Right hind paws of three rats were exposed to a magnetic field for 2 h after injection. Images were taken with the Caliper IVIS Spectrum imaging system. Untreated represents the noninjected control. (A) Representative fluorescence images of right hind paws at 6 and 12 h after injection. (B) Measurement of fluorescence in the right hind paws at 6 and 12 h after injection. Areas of identical size were chosen and background fluorescence obtained from the noninjected control was subtracted. (C) Fluorescence images of isolated organs. Three rats exposed to a magnetic field were killed 12 h after injection. The heart, liver, spleen, lung and right kidney were isolated and imaged. *p < 0.05. M+: Presence of a magnet fixed to the right hind paw; M-: Absence of a magnet.
CD3+ cells in the arthritic joint was similar to that in the liver, but higher than in the spleen, blood and kidney. This is in contrast to an animal model injected intravenously with siRNA lipoplexes, where the proportion of CD11b+ cells that entrapped the siRNA was much lower in joints than in the blood, liver and spleen [18]. Notably, a 2‑h treatment with a magnetic field significantly increased Cy3-siRNA uptake by CD11b+ and CD3+ cells isolated from the arthritic joint throughout the experimental period (Figure 6). Systemic delivery of PEI-SPIO/IL‑2/IL‑15Rb siRNA NPs provides protection against AA
To investigate the therapeutic effect of systemically administered PEI-SPIO/IL‑2/IL‑15Rb siRNA, AA rats were injected with the NPs every 7 days (day 10, 17 and 24) after arthritis induction on day 0. On day 31, rats were subjected to radiography before being killed, and paws were then isolated for histological evaluation. Compared with PBS- and NC siRNAtreated groups, rats treated with IL‑2/IL‑15Rb siRNA showed a decreased severity of AA, as assessed by clinical scores (Figure 7A). Histological and radiographic examination of IL‑2/IL‑15Rb siRNA-treated animals confirmed these findings and revealed a strong reduction in articular inflammation and destruction (Figure 7B & C). Notably, when a magnetic field was applied to the inflamed joint in an attempt to increase the local concentration of the particles, the therapeutic effect of IL‑2/IL‑15Rb siRNA was further improved.
796
Nanomedicine (2014) 9(6)
Specifically, clinical scores in the group receiving combination treatment with IL‑2/IL‑15Rb siRNA and a magnetic field decreased rapidly and were always lower than those in the IL‑2/IL‑15Rb siRNA-treated group. While IL‑2/IL‑15Rb siRNA monotherapy greatly inhibited bone damage of the arthritic joints, it did not completely protect against it, as shown by the presence of mild bone erosions in two of the three right hind paws examined (arrows in Figure 7C). On the other hand, right hind paws (three out of three) from the combination treatment group appeared completely normal. Therefore, treatment with PEI-SPIOdelivered IL‑2/IL‑15Rb siRNA clearly inhibits disease progression in AA rats and addition of a magnet on the diseased joint even for a short time can lead to a more profound enhancement of RNAi-mediated therapeutic effect. Quantitative PCR was used to assess the effect of PEI-SPIO-delivered IL‑2/IL‑15Rb siRNA treatment on the expression of IL‑2/IL‑15Rb, as well as several wellknown proinflammatory mediators in the joints of AA rats. As shown in Figure 7D, expression of IL‑2/IL‑15Rb, TNF‑a, IL‑1b and MMP‑3 in the group receiving IL‑2/IL‑15Rb siRNA decreased sharply compared with PBS-treated arthritic controls. Interestingly, reduction in IL‑15 mRNA production upon IL‑2/IL‑15Rb knockdown was not as drastic, consistent with the role of IL‑15 signaling in the amplification of the inflammatory network in RA. Combination treatment with PEI-SPIO/IL‑2/IL‑15Rb siRNA and a magnet field
future science group
Polyethyleneimine-functionalized iron oxide nanoparticles for systemic siRNA delivery in experimental arthritis
produced a stronger inhibitory effect on the expression of both IL‑2/IL‑15Rb and the cytokines examined.
PLGA-coated superparamagnetic microparticles as carriers for local treatment of arthritis [21,22]. Although an intra-articular injection of RA therapeutics can be used to avoid systemic side effects, it is not patient friendly due to the associated pain and repeated injections into multiple joints. Therefore, we investigated the PEIfunctionalized SPIOs for systemic siRNA delivery in experimental arthritis. NPs are usually taken up by the liver, spleen and other organs of the reticuloendothelial system. Consistent with this, tissue distribution analysis showed
Discussion Various types of drug carriers including liposomes and biodegradable polymers have been used for the delivery of RA therapeutics [19,20]. Considering that SPIO is the prevailing nanomaterial in biomedical research, there are very few reports in the literature describing superparamagnetic iron oxide-based micro- or nanoparticles for treating RA. In 2009, Butoescu et al. used PBS
Cy3-siRNA
Cy3-siRNA + magnet
104
104
104
103
103
103
2 Cy3 10
102
102
101
101
101
100 100
101
102
103
100 104 100
Research Article
101
102
103
100 104 100
101
102
103
104
CD3 PBS
Cy3-siRNA
Cy3-siRNA + magnet
104
104
104
103
103
103
2 Cy3 10
102
102
101
101
101
101
102 103 CD11b
100 104 100
101
30 2h 8h 24 h
25 20 15 10 5 0
M- M+ M- M+ M- M+ M- M+ M- M+ Kidney Liver Spleen Paw Blood
102
Cy3-siRNA-positive CD11b cells
Cy3-siRNA-positive CD3 cells
100 100
103
100 104 100
101
102
103
104
60 50
2h 8h 24 h
40 30 20 10 0
M- M+ M- M+ M- M+ M- M+ M- M+ Kidney Liver Spleen Paw Blood
Figure 6. In vivo cellular uptake of siRNA nanocomplexes after single systemic administration. Six arthritic rats were injected intravenously with 0.3 mg/kg Cy3-siRNA formulated with polyethyleneimine-coated superparamagnetic iron oxide. Following injection, the right hind paws of three rats were exposed to a magnetic field for 2 h. The rat receiving PBS was used as a control. Cellular uptake of Cy3-siRNA was evaluated by flow cytometry 2, 8 and 24 h after injection using anti-CD3 and anti-CD11b monoclonal antibodies. (A & B) Flow cytometry dot plots of cells in the right hind paw at 24 h. (C & D) Histograms show percentages of Cy3-siRNA uptake within the gated CD3 + or CD11b + cells in the M+ or M-. M+: Presence of a magnet fixed to the right hind paw; M-: Absence of a magnet; PBS: Phosphate-buffered saline.
future science group
www.futuremedicine.com
797
Research Article Duan, Dong, Zhang et al. 14 PBS NC siRNA IL-2/IL-15Rβ siRNA IL-2/IL-15Rβ siRNA + magnet
Arthritis score
12 10 8 6
***
4 2 0
**
**
** *** ** ** ***
*
** **
*** ***
***
*** *** *** *** *** ** *** *** *** *** *** *** *** *** 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 21 22 23 26 28 30 Time after immunization (days) IL-2/IL-15Rβ IL-2/IL-15Rβ siRNA siRNA + magnet PBS NC siRNA
Normal
PBS
NC siRNA
IL-2/IL-15Rβ siRNA
IL-2/IL-15Rβ siRNA + magnet
mRNA expression (%)
140 120 100 80
p < 0.05
60 40
p < 0.001
20 0
**
**
TNF-α
p < 0.001 **
**
MMP3
p < 0.01 ** IL-1β
*
p < 0.05 **
** IL-15
NC siRNA IL-2/IL-15Rβ siRNA IL-2/IL-15Rβ siRNA + magnet
** ** IL-2/IL-15Rβ
Figure 7. Systemic delivery of polyethyleneimine-superparamagnetic iron oxide/IL‑2/IL‑15Rb siRNA complexes inhibits arthritis progression in adjuvant arthritis rats. Male Wistar rats (n = 6 per group) were given Freund’s complete adjuvant in the left hind footpad on day 0. PBS and IL‑2/IL‑15Rb or NC siRNA nanocomplexes were separately administered on days 10, 17 and 24 at 0.3 mg siRNA/kg via the tail vein. For combination therapy, the right hind paw was exposed to a static magnetic field for 2 h after each injection. (A) Arthritis score. Values are the mean and standard deviation. (B) Representative histopathologies of the right hind ankle joints stained with hematoxylin and eosin. On day 31, rats were sacrificed and subjected to histopathological examination. (C) Representative radiographs of right hind paws. On day 31, three rats from each group were subjected to radiography. Arrow heads indicate sites with mild bone erosion in the IL‑2/IL‑15Rb siRNA treatment group. (D) Effects of IL‑2/IL‑15Rb siRNA nanocomplexes on mRNA expression of certain inflammatory factors. On day 31, RNA was prepared from the right hind ankle joints. mRNA levels of each factor were determined by quantitative PCR, normalized against GAPDH and expressed as a percentage of the PBS controls. The results are presented as values (the mean and standard deviation) obtained from three different samples randomly selected in each group. (A) *p
