Article pubs.acs.org/bc
In Vivo Specific Delivery of c-Met siRNA to Glioblastoma Using Cationic Solid Lipid Nanoparticles Juyoun Jin,#,†,‡ Ki Hyun Bae,#,∥ Heekyoung Yang,#,†,‡ Se Jeong Lee,†,‡ Hyein Kim,○ Yonghyun Kim,†,‡ Kyeung Min Joo,‡,§ Soo Won Seo,⊥ Tae Gwan Park,*,∥ and Do-Hyun Nam*,†,‡ †
Department of Neurosurgery, Samsung Medical Center & Sungkyunkwan University School of Medicine, Seoul, South Korea Cancer Stem Cell Research Center, Samsung Biomedical Research Institute, Seoul, South Korea § Department of Anatomy, Seoul National University College of Medicine, Seoul, South Korea ∥ Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea ⊥ Department of Biomedical Engineering, Samsung Medical Center and Biomedical Research Institute, Sungkyunkwan University School of Medicine, Gangnam-gu, Seoul, South Korea ○ Department of Biochemistry and Molecular Biology, Brain Korea 21 Project for Medical Science of Yonsei University, Yonsei University College of Medicine, Seoul, South Korea ‡
ABSTRACT: RNA interference is a powerful strategy that inhibits gene expression through specific mRNA degradation. In vivo, however, the application of small interfering RNAs (siRNAs) is severely limited by their instability and their poor delivery into target cells and tissues. This is especially true with glioblastomas (GBMs), the most frequent and malignant form of brain tumor, that has limited treatment options due to the largely impenetrable bloodbrain barrier. Here, cationic solid lipid nanoparticles (SLN), reconstituted from natural components of protein-free low-density lipoprotein, was conjugated to PEGylated c-Met siRNA. The c-Met siRNA-PEG/SLN complex efficiently down-regulated c-Met expression level, as well as decreased cell proliferation in U-87MG in vitro. In orthotopic U-87MG xenograft tumor model, intravenous administration of the complex significantly inhibited c-Met expression at the tumor tissue and suppressed tumor growth without showing any systemic toxicity in mice. Use of Cy5.5 conjugated SLN revealed enhanced accumulation of the siRNA-PEG/SLN complexes specifically in the brain tumor. Our data demonstrates the feasibility of using siRNA-PEG/SLN complexes as a potential carrier of therapeutic siRNAs for the systemic treatment of GBM in the clinic.
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micelles in vitro.2 This would now allow in vivo systemic administration of siRNA-based therapy possible, and the question remains whether the siRNA-PEG/SLN can also be effective in crossing the BBB. Here, we report an orthotopic human brain tumor mouse model using the siRNA-PEG/SLN delivery system to evaluate its penetration across the BBB to the tumor site. Systemic intravenous administration of the SLN successfully and specifically delivered the siRNA to the tumor and lowered the tumor cell proliferation and tumorigenicity.
INTRODUCTION Glioblastoma multiforme (GBM) is a very aggressive brain tumor, and the prognosis of patients with GBMs remains poor despite multidisciplinary treatment approaches, such as surgical resection, radiotherapy, and chemotherapy. Furthermore, GBM is particularly difficult to treat because the brain is endowed with a uniquely privileged immune environment by the bloodbrain barrier (BBB). To date, the BBB remains largely impenetrable by the existing oncology drugs,1 necessitating the development of novel delivery methods for treating GBM. Toward this end, we previously developed a novel solid lipid nanoparticle (SLN) that can serve as a good carrier of cancer cell targeting moieties.2 In particular, the SLNs were designed to carry small interfering RNAs (siRNAs). Use of siRNAs has been long-desired, because it can specifically silence oncogenes that control proliferation, apoptosis, angiogenesis, or migration of the tumor cells.3 However, its application in the clinic has been limited due to its poor stability in biological fluids and nonspecific cellular uptake.4−6 We overcame these issues by conjugating the siRNAs with poly(ethylene glycol) (PEG) and by mimicking naturally occurring low-density lipoprotein complexes that are common in bodily fluids7 using polyethylenimine (PEI) to form nanoscale polyelectrolyte complex © 2011 American Chemical Society
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MATERIALS AND METHODS Materials. Cholesterol hydrochloride (Chol), L-α-dioleoyl phosphatidylethanolamine (DOPE), and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) were obtained from Avanti Polar Lipids (Alabaster, AL). Cholesteryl oleate and glyceryl trioleate were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical reagent grade.
Received: July 26, 2011 Revised: November 7, 2011 Published: November 9, 2011 2568
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Synthesis of Cationic Solid Lipid Nanoparticles (SLN). Cationic solid lipid nanoparticles (SLNs) were produced by a modified emulsification-solvent evaporation method, as reported previously.2 Briefly, cholesteryl oleate (22.5 mg), glyceryl trioleate (1.5 mg), DOPE (7 mg), Chol (5 mg), and DC-Chol (14 mg) were codissolved in 2 mL of chloroform/methanol mixture (2:1, v/v). After 10 mL of deionized water was added, the mixture was vortexed for 2 min and subsequently sonicated for 5 min using a Bronson Sonifier 450 equipped with a microtip (20 kHz, duty cycle = 35, output control = 3.5). The resulting emulsion was transferred to a round-bottom flask and then the solvent was rapidly evaporated at 60 °C at 20 mmHg using a rotary evaporator (EYELA, Japan). The produced SLNs were purified by extensive dialysis against deionized water (Mw cutoff of 100 kDa). Formation of siRNA-PEG/SLN Complexes. An siRNA targeting human c-Met (sense; 5′-GUGCAGUAUCCUCUGACAGUU-(CH2)6 NH2−3′, antisense; 5′-CUG UCA GAG GAU ACU GCA CUU-3′) and a nontargeting (NT) siRNA (sense; 5′-GUUCAGCGUGUCCGGCGAGTT-(CH2)6NH2−3′, antisense; 5′-CUCGCCGGACACGCUGCUGAACTT-3′), both modified with 3′-hexylamine on the sense strand, were obtained from Samchunlly Inc. siRNA was conjugated with PEG through a disulfide bond as described previously.8 SLN was incubated with siRNA-PEG conjugate at room temperature for 15 min in deionized water. To minimize interference from autofluorescence in in vivo imaging studies, siRNA-PEG/SLN complex was labeled with near-infrared Cy5.5. The diameter and surface zeta-potential were measured using a dynamic light scattering (DLS) instrument (Zeta-Plus, Brookhaven Instrument Co., NY) as described previously. 2 The SLN was observed by atomic force microscopy (AFM; PSIA XE-100, Park Systems, Santa Clara, CA) and transmission electron microscopy (TEM; Zeiss Omega 912, Carl Zeiss, Oberkochen, Germany). Cell Culture and Transfection. U-87MG human GBM cells (ATCC) were grown in EMEM supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL). For siRNA-PEG/SLN transfection, c-Met expressing U-87MG ells were plated on a 6-well plate at a density of 2.5 × 105 cells/well. After 24 h of incubation, the medium was replaced by fresh medium containing 0.5% FBS and 20 nM NT or 40 nM c-Met siRNA-PEG/SLN complexes were added to the cells. After 6 h incubation at 37 °C, the transfection medium was discarded and supplemented with medium containing 0.5% FBS. After an additional 24 h of incubation, the cells were harvested for Western blotting. Western Blotting. Cell lysates were prepared from transiently transfected U-87MG cells by scraping 90% confluent 10 cm dishes into NP40 Cell Lysis Buffer (50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% NP40, 0.02% NaN3) (Invitrogen Corporation, Camarillo, CA) containing protease inhibitor cocktail tablets (Roche, Laval, QC). The protein concentrations were determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) according to the manufacturer’s directions. Proteins were separated by SDS-PAGE, transferred onto PVDF membrane (GE Healthcare, Piscataway, NJ), and immunoblotted with primary antibodies overnight at 4 °C. A rabbit polyclonal antibody raised against a C-terminal cytoplasmic domain of human c-Met (1:200; Abcam, San Francisco, CA) was used. A mouse monoclonal antibody against β-actin (1:5000; Sigma, Saint Louis, MO) was used for the internal control. Bound
antibodies were detected using horse radish peroxidaseconjugated goat antirabbit IgG (1:5000, Invitrogen) or goat antimouse IgG (1:5000, Zymed, San Francisco, CA) and were visualized via enhanced chemiluminescence (GE Healthcare). In Vitro Proliferation Analysis. Cells were seeded at a density of 0.5 × 104 cells/well in 96-well plates and were transfected with NT or c-Met siRNA-PEG/SLN as described above. After transfection, cells were treated with 200 ng/mL of HGF for 24 h, 48 h, 72 h, or 96 h. Cell proliferation was quantified using Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technology, Gaithersburg, MD, USA), which assays cellular mitochondrial dehydrogenase activity as a measure of cell viability. GBM Orthotopic Model and siRNA Treatment. Animal experiments were approved by the appropriate Institutional Review Boards of the Samsung Medical Center (Seoul, Korea) and conducted in accord with the “National Institute of Health Guide for the Care and Use of Laboratory Animals” (NIH publication no. 80−23, revised in 1996). For orthotopic GBM animal model, anesthetized 6-week-old male Balb/c-nu mice were secured in a rodent stereotactic frame, a hollow guide screw was implanted into a small drill hole made at 2 mm left and 1 mm anterior to the bregma, and 2 × 105 U-87MG cells in 5 μL HBSS were injected through this guide screw into the white matter at a depth of 2 mm [anterior/posterior (AP) +0.5 mm, medial/lateral (ML) +1.7 mm, dorsal/ventral (DV) −3.2 mm]. Two weeks after tumor cell implantation, mice were randomized into four groups (n = 7 for each group) and were given intravenous injection of SLN alone (control), 0.125 mg/ kg, 0.5 mg/kg, or 2 mg/kg of c-Met siRNA-PEG/SLN complex intravenously three times once a week (Figure 3A). Brains were harvested and processed for paraffin embedding at six hours after the final injection. For tumor mass volume quantification, standard H&E staining was performed in the paraffin sections and observed under optical microscope. The tumor volume was calculated by measuring the section with the largest tumor portion and applying the formula: (width)2 × length × 0.5. The c-Met expression in tumor was determined by immunohistological staining using mouse antihuman c-Met monoclonal antibody (Zymed). Peroxidase-conjugated secondary antibodies were used and visualized by incubating the slides with stable 3,3′diaminobenzidine (DAB) for 10−20 min. The sections were rinsed with distilled water, counterstained with Gill’s hematoxylin for 1 min, and mounted with Universal Mount (Research Genetics, Huntsville, AL). In Vivo Quantification of c-Met siRNA-PEG/SLN Tumor-Tropism into the Brain. To evaluate tumor targeting of c-Met siRNA-PEG/SLN complex in U-87MG tumor-bearing mouse, siRNA-PEG/SLN complex conjugated with Cy5.5 was utilized. Three weeks after tumor cell implantation, mice were randomized into two groups (n = 3 for each group) and were given single intravenous injection of SLN alone (control) or 0.5 mg/kg c-Met siRNA-PEG/SLNCy5.5 complex (Figure 4A). Two days after injection, the mice were anesthetized with 2−3% isoflurane. The signal from Cy5.5 was detected in the region of the brain using a prototype Xenogen IVIS Spectrum in vivo imaging system (Caliper Life Science). Fluorescence intensity was analyzed as photons per second (p/s) by Living Image 3.1 software (Caliper Life Science). 2569
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Figure 1. Characterization of solid lipid nanoparticle (SLN). (A) Size distribution of SLN and (B) zeta-potential value were measured by dynamic light scattering (DLS) and (C) TEM (scale bar = 500 nm) and (D) AFM images.
Figure 2. Effects of c-Met blockade in U87MG cell proliferation. (A) Western Blot analysis of U87MG transfected either with non-targeting (NT) siRNA-PEG/SLN complex or c-Met (Met) siRNA-PEG/SLN complex. c-Met siRNA-PEG/SLN complex efficiently reduced c-Met protein level. Endogenous human ß-actin was used as a control. (B) Inhibition of cell proliferation by transfection of c-Met siRNA-PEG/SLN complex. Values are mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001 vs Control.
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can readily cross the BBB for a targeted therapy of GBM. We chose to target c-Met, since its overexpression is associated with poor prognosis and invasiveness of tumors in GBM patients, 9 and we demonstrate that c-Met siRNA treatment can successfully attenuate GBM tumor growth in vivo. Synthesis and Characterization of siRNA-PEG/SLN Complex. Our RNAi delivery system is based on the formation of SLN complex with PEGylated siRNA. PEGylated
RESULTS AND DISCUSSION
Although siRNA is a promising nucleic acid drug in gene therapy, various intra- and extracellular barriers hamper therapeutic applications. Negatively charged siRNAs have extremely low cellular uptake and transfection efficiency, and undergo rapid chemical degradation when administered intravenously. To overcome the in vivo stability problem of siRNA, we report here a new delivery system for siRNAs that 2570
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Figure 3. In vivo c-Met targeting in xenograft tumor. (A) The timeline for the assessment of in vivo antitumor activities of c-Met siRNA-PEG/SLN complex in an orthotopic xenograft model. (B) c-Met siRNA-PEG/SLN complex reduced U-87MG tumor volumes in a dose-dependent manner. Data are expressed as the mean ± SE. (C) Representative microphotographs of immunohistochemistry determining c-Met expression in the brains of control siRNA or c-Met siRNA treated mice. Immunopositive cells were visualized by brown DAB staining. Values are mean ± SEM *p < 0.05, **p < 0.01, vs Control.
complex in U-87MG orthotopic models. Two weeks after tumor cell implantation, mice were randomized into four groups (n = 7 for each group) and were given intravenous injection of SLN alone (control), 0.125 mg/kg, 0.5 mg/kg, or 2 mg/kg of c-Met siRNA-PEG/SLN complex intravenously three times once a week (Figure 3A). The tumor volumes were determined at the day of last c-Met siRNA-PEG/SLN complex injection. Treatment with c-Met siRNA-PEG/SLN complex significantly inhibited U-87MG tumor growth in a dosedependent manner; 0.125 mg/kg, 0.5 mg/kg, and 2 mg/kg groups showed 50%, 62%, and 91% tumor volume reduction, respectively, compared with the control group (*P < 0.05 and **P < 0.01 vs control, Figure 3B). Immunohistochemical staining of the tumor sections demonstrated that there was a down-regulation of c-Met with c-Met siRNA-PEG/SLN complex administration (Figure 3C). These results indicate that a SLN-based siRNA delivery system can overcome the current problems facing siRNA therapy. Here, we showed that GBM tumor volume reduction can be effectively achieved via RNAi-mediated down-regulation of c-Met. More importantly, we show that the SLN complex employed here allows for a systemic in vivo application of c-Met siRNAs in glioblastoma orthotopic models that can effectively control tumor growth. It should be noted that no significant difference in body weight changes was observed between mice with and without treatment of siRNA-PEG/SLN complex during the indicated experimental period (data not shown). This suggests that there was no apparent critical systemic toxicity caused by the formulations. c-Met siRNA-PEG/SLN Complex Can Cross the BloodTumor Barrier and Specifically Target the Tumor Site. To investigate the in vivo BBB-permeability of the c-Met siRNA-PEG/SLN complex, we intravenously administered Cy5.5-labeled c-Met siRNA-PEG/SLN complex to mice bearing the U-87MG tumor (Figure 4A). At 48 h postinjection,
c-Met siRNA or NT siRNA was conjugated with SLN at room temperature in deionized water. The mean diameter of SLN as measured by laser light scattering was 117.4 ± 11.7 nm (Figure 1A), and the zeta potential value of SLN was 37.3 ± 2.3 mV, indicating good stability of the complex (Figure 1B). The TEM (Figure 1C) and AFM (Figure 1D) observations confirmed the size and the spherical shape of the SLN. We demonstrated previously the ability to target the delivery of siRNA-PEG complex specifically to cancer cells in vitro.2 Here, we show for the first time that these SLN could be utilized as nontoxic, serum-stable, and highly effective carriers for delivery of siRNA specifically to tumor site in vivo. c-Met siRNA-PEG/SLN Complex Down-Regulates cMet Protein and Tumor Cell Proliferation. To establish the efficacy and specificity of this targeting method, we used U87MG glioblastoma cell lines positive for expression of c-Met. The c-Met siRNA-PEG/SLN complex exhibited profound inhibition extent of c-Met expression (32.5 ± 1.9% reduction, as compared to the no treatment control) than the NT siRNAPEG/SLN complex (2.9 ± 3.0% reduction, as compared to the no-treatment control) (P < 0.01; Figure 2A). In the proliferation assay, treatment with the c-Met siRNA-PEG/ SLN complex significantly reduced tumor cell proliferation in a dose dependent manner; 20 nM and 40 nM groups showed 13.1% and 23.4% tumor cell proliferation reduction, respectively, compared with NT siRNA-PEG/SLN complex group. No such reduction was observed with the NT siRNAPEG/SLN complex compared to the control (Figure 2B). These results indicate that our c-Met siRNA-PEG/SLN complex specifically reduces proliferation of glioblastoma cells by controlling the expression of c-Met in vitro. Antitumor Effects of c-Met siRNA-PEG/SLN Complex in a Glioma Xenograft Orthotopic Model. The antitumor effects of systemic administration of c-Met siRNA-PEG/SLN complex were evaluated by giving intravenous injection of the 2571
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Article
AUTHOR INFORMATION
Corresponding Author *Department of Neurosurgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwondong Gangnam-gu, Seoul, South Korea 135-710. Tel: +82-23410-3497 Fax: +82-2-3410-0048 E-mail:
[email protected]. Author Contributions # Equal contribution.
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ACKNOWLEDGMENTS This work was supported by the National Research Foundation of KOREA (NRF) grant funded by the Korea government (MEST) (No.20090093731).
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(1) Rajadhyaksha, M., Boyden, T., Liras, J., El-Kattan, A., and Brodfuehrer, J. (2011) Current advances in delivery of biotherapeutics across the blood-brain barrier. Curr. Drug Discovery Technol. 8, 87− 101. (2) Kim, H. R., Kim, I. K., Bae, K. H., Lee, S. H., Lee, Y., and Park, T. G. (2008) Cationic solid lipid nanoparticles reconstituted from low density lipoprotein components for delivery of siRNA. Mol. Pharm. 5, 622−31. (3) Hannon, G. J., and Rossi, J. J. (2004) Unlocking the potential of the human genome with RNA interference. Nature 431, 371−8. (4) Oishi, M., Nagasaki, Y., Itaka, K., Nishiyama, N., and Kataoka, K. (2005) Lactosylated poly(ethylene glycol)-siRNA conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive polyion complex micelles achieving enhanced gene silencing in hepatoma cells. J. Am. Chem. Soc. 127, 1624−5. (5) Lu, P. Y., Xie, F., and Woodle, M. C. (2005) In vivo application of RNA interference: from functional genomics to therapeutics. Adv. Genet. 54, 117−42. (6) Kim, S. H., Mok, H., Jeong, J. H., Kim, S. W., and Park, T. G. (2006) Comparative evaluation of target-specific GFP gene silencing efficiencies for antisense ODN, synthetic siRNA, and siRNA plasmid complexed with PEI-PEG-FOL conjugate. Bioconjugate Chem. 17, 241−4. (7) Chung, N. S., and Wasan, K. M. (2004) Potential role of the lowdensity lipoprotein receptor family as mediators of cellular drug uptake. Adv. Drug Delivery Rev. 56, 1315−34. (8) Kim, S. H., Jeong, J. H., Lee, S. H., Kim, S. W., and Park, T. G. (2006) PEG conjugated VEGF siRNA for anti-angiogenic gene therapy. J. Controlled Release 116, 123−9. (9) Kong, D. S., Song, S. Y., Kim, D. H., Joo, K. M., Yoo, J. S., Koh, J. S., Dong, S. M., Suh, Y. L., Lee, J. I., Park, K., Kim, J. H., and Nam, D. H. (2009) Prognostic significance of c-Met expression in glioblastomas. Cancer 115, 140−8. (10) Grzelinski, M., Urban-Klein, B., Martens, T., Lamszus, K., Bakowsky, U., Hobel, S., Czubayko, F., and Aigner, A. (2006) RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts. Hum. Gene Ther. 17, 751−66.
Figure 4. In vivo optical imaging of c-Met siRNA-PEG/SLN complex in tumor-bearing mice. (A) Detailed experimental schedules using the glioblastoma animal model are illustrated. (B) Distribution of Cy5.5labed c-Met siRNA-PEG/SLN complex in brain bearing glioblastoma cells was visualized by in vivo fluorescent optical imaging. (C) Fluorescence intensity (FI) was analyzed. Values are mean ± SEM **p < 0.01 vs Control.
Cy5.5-labeled c-Met siRNA-PEG/SLN complex showed higher fluorescence intensity in the brain compared to the notreatment control (Figure 4 B,C). Grzelinski et al.10 previously reported a similar delivery system of gene silencing but with local intratumor injection of siRNA/polyethlenimine complex in subcutaneous U-87MG model. Our work illustrates an improved delivery system that is effective even with systemic intravenous administration of the RNAi/nanoparticle complex in an orthotopic model. We speculate that siRNA-based therapy in orthotopic models may have been difficult to evaluate in the past due to the difficulty of delivery across the BBB. The system employed here showed that the siRNA-PEG/ SLN can specifically cross the BBB to the tumor site (Figure 4) with no apparent systemic toxicity. This is highly significant since it provides proof-of-concept that in vivo diagnostics can also be a potential future application of the technology employed here. Caution should be taken by noting that the mice model used here lacks an intact immune system, so the innate immune response to RNAi will need to be addressed in future studies. Nevertheless, the tumor-tropic delivery of siRNAs across the BBB and the outstanding tumor volume reduction achieved by our system provide great promise for a clinical application in the near future.
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REFERENCES
CONCLUSIONS
Here, we present the SLN complexed with siRNAs as a viable platform for RNAi-based gene targeting, providing clinical rationale for the use of siRNAs against optimal targets of a given tumor. Specifically for GBM, we introduce SLNcomplexed, c-Met-specific siRNAs as an efficient tool for attenuating tumor growth in vitro and in vivo. There was a successful tumor control via intravenous administration of the siRNA complex in the orthotopic GBM xenograft tumor model, along with specific tumor-tropic localization of the SLN per in vivo imaging. The model described here indicates that this approach is a highly feasible novel therapeutic option for GBM in the clinic. 2572
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