Article pubs.acs.org/Biomac
RVG-Peptide-Linked Trimethylated Chitosan for Delivery of siRNA to the Brain Yikun Gao,† Zhan-You Wang,‡ Jinghai Zhang,§ Youxi Zhang,∥ Hong Huo,† Tianyi Wang,‡ Tongying Jiang,*,† and Siling Wang*,† †
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China College of Life and Health Sciences, Northeastern University, Shenyang 110004, China § School of Medical Devices, Shenyang Pharmaceutical University, Shenyang 110016, China ∥ Department of Pharmacy, The Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China ‡
S Supporting Information *
ABSTRACT: In this work, a peptide derived from the rabies virus glycoprotein (RVG) was linked to siRNA/trimethylated chitosan (TMC) complexes through bifunctional PEG for efficient brain-targeted delivery of siRNA. The physiochemical properties of the complexes, such as siRNA complexing ability, size and ζ potential, morphology, serum stability, and cytotoxicity, were investigated prior to studying the cellular uptake, in vitro gene silencing efficiency, and in vivo biodistribution. The RVG-peptide-linked siRNA/TMC−PEG complexes showed increased serum stability, negligible cytotoxicity, and higher cellular uptake than the unmodified siRNA/TMC−mPEG complexes in acetylcholine receptor positive Neuro2a cells. The potent knockdown of BACE1, a therapeutic target in Alzheimer’s disease, demonstrated the gene silencing efficiency. In vivo imaging analysis showed significant accumulation of Cy5−siRNA in the isolated brain of mice injected with RVG-peptide-linked complexes. Therefore, the RVG-peptide-linked TMC−PEG developed in this study can be used as a potential carrier for delivery of siRNA to the brain.
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INTRODUCTION Since small interfering RNA (siRNA) is able to effectively silence target genes with high specificity, gene therapy using siRNA represents a powerful tool for the treatment of a number of brain conditions such as Alzheimer’s disease and tumors. However, several unfavorable biopharmaceutical properties of siRNA have limited its transvascular delivery to the brain, including rapid degradation by nucleases, low blood stability, and, especially, the restricted permeability of the blood−brain barrier (BBB).1 Due to the tight junctions and lack of fenestration, the BBB acts as a barrier protecting the central nervous system (CNS) and, hence, presents a major problem for delivering therapeutic agents to the brain.2 To overcome this natural barrier, various siRNA delivery systems have been studied, and as a main group of nonviral vectors, cationic polymers have been widely investigated in recent years. Among them, chitosan has been shown to be biodegradable, biocompatible, nontoxic, and noninflammatory.3 However, its poor solubility at physiological pH has limited the interactions between chitosan and siRNA.4 Trimethylated chitosan (TMC) is a derivative of chitosan synthesized by partially quaternizing the amino groups at the C-2 position of chitosan.5 Trimethylation of chitosan improves both the solubility and siRNA complexing ability of chitosan. To overcome the relatively low transfection efficiency of the © 2014 American Chemical Society
cationic polymer vector, a ligand−receptor-mediated targeting strategy can be used. Brain-specific targeting ligand is critical for the effective delivery of siRNA to the brain. A short peptide derived from rabies virus glycoprotein (RVG) can specifically bind to the nicotinic acetylcholine receptor (nAchR) on neuronal cells thus enabling the crossing of the BBB and the entry into neuronal cells.6 Therefore, RVG peptide can be employed as a braintargeting ligand and modified on the surface of siRNA delivery vectors through a poly(ethylene glycol) (PEG) linker that will not only increase the spatial freedom of the RVG peptide molecules and consequently allow efficient binding of the RVG peptide to nAchR but also help increase the biocompatibility, serum stability, and systemic circulation time of the siRNA loaded complexes.7 In this study, RVG peptide was linked by a covalent bond to TMC−PEG either prior to or after loading siRNA to produce RVG-peptide-linked siRNA/TMC−PEG complexes for delivery of siRNA to the brain. This siRNA delivery vector was systematically characterized, and its brain-targeting ability was evaluated both in vitro and in vivo. Received: December 24, 2013 Revised: February 15, 2014 Published: February 18, 2014 1010
dx.doi.org/10.1021/bm401906p | Biomacromolecules 2014, 15, 1010−1018
Biomacromolecules
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Figure 1. Schematic illustration of the synthesis of TMC−PEG. (a) Schematic illustration of the preparation of RVG-peptide-linked siRNA/TMC− PEG complexes. (b) RVG peptide was linked to TMC−PEG either prior to or after loading siRNA.
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Complex Formation and Linking RVG. The siRNA/TMC− RVG complexes were formed by the electrostatic interaction of the cationic polymer (TMC−PEG or TMC−PEG−RVG) with negatively charged siRNA at a suitable N/P ratio (the ratio of the moles of the amino groups (N) on TMC to the moles of the phosphate groups (P) on siRNA). To evaluate the effects of the sequence of loading siRNA and linking RVG peptide, RVG peptide was linked to TMC−PEG both prior to and after loading siRNA (Figure 1b). In the case of loading siRNA first, TMC−PEG was dissolved in DEPC (diethylpyrocarbonate)-treated water at a concentration of 10 mg/mL, and siRNA was dissolved in DEPC-treated water at a concentration of 0.4 mg/mL. Complexes were obtained by addition of the cationic polymer solution to the siRNA solution and immediate mixing using a vortex mixer for 30 s at room temperature, then allowing 30 min for formation of the complexes.5 Then, the siRNA/TMC−PEG complexes were reacted with RVG-cys peptide in distilled water for 24 h at room temperature to link RVG peptide by means of a specific reaction between the MAL group of TMC−PEG and the thiol group of RVG-cys peptide.10 The unreacted RVG-cys peptide was then removed by centrifugal ultrafiltration (MWCO 30 kDa). In the case of linking RVG peptide first, RVG peptide was initially linked to TMC−PEG to prepare TMC−PEG−RVG and then TMC−PEG−RVG and siRNA formed complexes. The methods of linking RVG peptide and forming complexes were both the same as the methods just described above. TMC−PEG−RVG was analyzed by 1H NMR (300 M, in D2O, ARX300, Bruker, Switzerland). Nontargeted siRNA/TMC-mPEG complexes were prepared in the same way except that MAL−PEG−SCM was replaced by mPEG− SCM. The following terms are used: TPs (siRNA/TMC−PEG−MAL complexes), TsR (siRNA/TMC−RVG complexes loaded with siRNA prior to linking RVG peptide), TRs (siRNA/TMC−RVG complexes linked RVG peptide prior to loading siRNA), and Tms (siRNA/ TMC−mPEG complexes). Gel Retardation Assay. The binding of siRNA to TMC−PEG or TMC−PEG−RVG was examined by gel electrophoresis using a 1.5% agarose gel. Samples of TPs and TRs with different N/P ratios were loaded into the gel. Free siRNA served as a control. Electrophoresis was carried out at 160 V for 30 min. The retardation was visualized by
EXPERIMENTAL SECTION
Materials. Chitosan (MW ∼50 kDa, deacetylation degree 95%) was purchased from HAIDEBEI Marine Bioengineering Co., Ltd. (Jinan, China). Maleimide−poly(ethylene glycol)−succinimidyl carboxy methyl ester (MAL−PEG−SCM, MW 5 kDa) was purchased from Creative PEGWorks (Winston-Salem, NC, USA). Methoxypoly(ethylene glycol)−succinimidyl carboxy methyl ester (mPEG− SCM, MW 5 kDa) was purchased from National Engineering Research Center for Biotechnology (Beijing, China). RVG-cys peptide (sequence YTIWMPENPRPGTPCDIFTNSRGKRASNGC) was purchased from ChinaPeptides Co., Ltd. (Shanghai, China). PPIL2 siRNA (sense 5′-GUGCCUACCUGGACAAGAAdTdT-3′, antisense 5′UUCUUGUCCAGGUAGGCACdTdT-3′), negative control siRNA, and carboxyfluorescein-labeled siRNA (FAM−siRNA) were synthesized by GenePharma Co., Ltd. (Shanghai, China). Cy5-labeled siRNA (Cy5−siRNA) was synthesized by RiboBio Co., Ltd. (Guangzhou, China). Synthesis of TMC−PEG. To improve the solubility and the siRNA complexing ability of chitosan, TMC was synthesized by reductive methylation of chitosan with methyl iodide as previously described.8 In brief, chitosan (1 g) and sodium iodide (2.4 g) were added to a mixture of 15% (w/v) aqueous NaOH solution (5.6 mL) and Nmethyl-2-pyrrolidone (40 mL). The mixture was then heated to 60 °C. Methyl iodide (6 mL) was added, and the mixture was refluxed for 45 min. Subsequently, 15% NaOH solution (5.6 mL) and methyl iodide (2.5 mL) were added, and the mixture was stirred for another 45 min. The reaction was stopped by adding the mixture to ethanol (200 mL). The obtained precipitate was subsequently washed thoroughly with diethyl ether. Finally the product was dissolved in 10% (w/v) aqueous NaCl solution (40 mL) and stirred for 3 h to allow ion-exchange. The obtained solution was dialyzed (MWCO 10 kDa) against deionized water and then lyophilized to obtain TMC. TMC (10 mg) was reacted with MAL−PEG−SCM (15 mg) in distilled water for 6 h at room temperature to allow the primary amino groups on TMC to specifically react with the SCM group of the heterobifunctional PEG (Figure 1a). After removal of the unreacted MAL−PEG−SCM by centrifugal ultrafiltration (MWCO 30 kDa), TMC−PEG−MAL was obtained by lyophilization.9 1011
dx.doi.org/10.1021/bm401906p | Biomacromolecules 2014, 15, 1010−1018
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Figure 2. 1H NMR spectra of TMC (a), TMC−PEG (b), and TMC−PEG−RVG (c) in D2O at 300 MHz. the cells in each well were trypsinized and resuspended in 300 μL of PBS. The mean fluorescence intensity (MFI) was analyzed by flow cytometry (FCM) using a FACS flow cytometer (FACSCalibur, Becton Dickinson, USA). The cellular uptake of TsR-24 was further investigated by confocal laser scanning microscopy (CLSM). Neuro2a cells were seeded on glass coverslips in 6-well plates and incubated overnight. Then, cells were treated with TsR-24 prepared with FAM−siRNA at an siRNA concentration of 150 nM and then incubated for 4 h. Subsequently, the cells were washed three times with PBS, fixed using 4% paraformaldehyde in PBS and stained with DAPI (Beyotime, China).14 Fluorescence images were acquired using an Olympus FV1000S-SIM/IX81 laser confocal scanning microscope (Olympus, Japan). For DAPI imaging, the employed excitation wavelength was 405 nm and the emission wavelength was 461 nm. For FAM−siRNA imaging, the employed excitation wavelength was 488 nm and the emission wavelength was 519 nm. In Vitro Gene Silencing. The siRNA-mediated knockdown of target gene was detected at a protein level by Western blot analysis.15 Neuro2a cells were treated with unmodified complexes (Tms-24) or RVG-peptide-linked complexes (TsR-24) prepared with PPIL2 siRNA at an siRNA concentration of 100 nM for 48 h. Lysates of whole cells (50 μg of total protein) were separated on a 10% SDS-PAGE gel. The primary antibodies used for Western blot analysis were rabbit antiBACE1 (1:1000, Sigma) and mouse anti-GAPDH (1:10000, KC-5G5, Kang Chen). Images were obtained using BIORAD Universal hood II (BIORAD Laboratories Inc., CA). The percentage BACE1 protein expression was calculated from the band intensities quantified using ImageJ software taking the BACE1 expression of an untreated control as 100%. In Vivo Evaluation of Brain Targeting. For the in vivo animal experiments, KM mice (male, 20−25 g) were used and treated according to the guidelines of the Animal Care and Use Committee of Shenyang Pharmaceutical University. Tms-24 or TsR-24 complexed with Cy5−siRNA was injected into the tail vein of KM mice at a dose of 30 μg of siRNA per mouse. The mice were killed 12 h after injection, and all the major organs, including the brains, were isolated. Images were obtained using an in-vivo imaging system, FX Pro (Carestream Health, USA). To further confirm the brain targeted delivery of siRNA, the brains were collected from the mice sacrificed 12 h post-iv injection and fixed in a PBS solution of 4% (w/v) paraformaldehyde overnight. After being dehydrated in 30% sucrose solution for 48 h, the brains were frozen in Tissue Tek O.C.T. compound (Mc Cormick, USA) and then cut into slices of 15 μm thickness. After washing with deionized water, the sections were stained with DAPI (Beyotime, China) and mounted using mounting medium.16 The images of the brain sections were
staining the gel with ethidium bromide. Images were acquired using an ImageQuant 300 gel documentation system (GE healthcare, USA). Size and ζ Potential Measurements. The sizes and ζ potentials of TRs (with different N/P ratios), TPs, and TsR (with an N/P ratio of 24/1) were examined by dynamic light scattering (DLS) using a Zetasizer Nano ZS 90 (Malvern Instruments, Ltd., UK) at a concentration of approximate 1 mg/mL in PBS buffer (pH 7.4, 137 mM NaCl). To evaluate the influence of pH on the ζ potential, the ζ potential of TRs was also measured in PBS buffer (pH 5.0, 137 mM NaCl). The size (by intensity) and ζ potentials are presented as the mean values of three measurements ± SD (standard deviation). Transmission Electron Microscopy (TEM). The morphology of TsR-24 (TsR prepared at an N/P ratio of 24/1) was examined by TEM (JEM 1200EX, JEOL, Japan). Briefly, the TEM sample was prepared by placing one drop of TsR-24 dispersion on a carbon membrane coated copper grid. Then the sample was stained with 2% phosphotungstic acid (PTA) and dried.11 siRNA Serum Stability. The serum stability of siRNA in TsR-24 was characterized by gel electrophoresis.11 Naked siRNA and TsR-24 were incubated in PBS (pH 7.4, 137 mM NaCl) containing 50% fetal bovine serum (FBS) at 37 °C. At each predetermined time point (0, 3, 6, 9, 12, 24, 48, and 72 h), 30 μL of each sample was collected and stored at −20 °C until gel electrophoresis was performed. To displace siRNA from TsR-24, 5 μL of heparin (500 U/mL) was added to each sample and then the integrity of the siRNA was examined by gel electrophoresis.12 Cell Culture. Mouse neuroblastoma Neuro2a cells were cultured in MEM (minimum essential medium) supplemented with 10% FBS and antibiotics. HeLa cells were maintained in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% FBS and antibiotics. All cells were incubated at 37 °C in 5% CO2. Cytotoxicity. The cytotoxicity of TMC−mPEG polymer, TsR-24, and Tms-24 (siRNA/TMC−mPEG complexes with an N/P ratio of 24/1) was determined in triplicate by MTT assay according to the method of Mossman.13 Neuro2a cells were treated with samples at concentrations ranging from 50 to 500 μg/mL in 96-well plates. After a 24 h incubation, the cell viability was analyzed by MTT assay, and the IC50 values, which represent the concentration of the complexes resulting in 50% inhibition of cell growth, were calculated. Cellular Uptake of siRNA Loaded Complexes. To determine the cellular uptake and neuronal specificity of siRNA delivery, HeLa and Neuro2a cells were seeded in 12-well plates at a density of 3 × 105 cells per well and incubated overnight. Tms-24 and TsR-24 were prepared with FAM−siRNA and mixed in the cell culture medium at final siRNA concentrations of 300 and 600 nM. Cells were incubated with the samples for 4 h before being washed twice with PBS and subsequently incubated in complete culture medium for 16 h. Then, 1012
dx.doi.org/10.1021/bm401906p | Biomacromolecules 2014, 15, 1010−1018
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visualized using an Olympus FV1000S-SIM/IX81 laser confocal scanning microscope (Olympus, Japan). For Cy5−siRNA imaging, the employed excitation wavelength was 635 nm and the emission wavelength was 664 nm. Statistical Analysis. Comparison between two groups was analyzed by Student’s t test for unpaired samples using IBM SPSS statistics software (version 21). Statistical significance was set at P values
