Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy

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Journal of Controlled Release j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j c o n r e l

Systemic delivery of siRNA with cationic lipid assisted PEG-PLA nanoparticles for cancer therapy Xian-Zhu Yang a, 1, Shuang Dou b, c, 1, Tian-Meng Sun a, Cheng-Qiong Mao b, c, Hong-Xia Wang b, c, Jun Wang b, c,⁎ a b c

Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230027, PR China CAS Key Laboratory of Brain Function and Disease, University of Science and Technology of China, Hefei, Anhui 230027, PR China School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China

a r t i c l e

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Article history: Received 2 May 2011 Accepted 26 July 2011 Available online 1 August 2011 Keywords: siRNA delivery Nanoparticle RNA interference Cancer therapy Polo-like kinase 1

a b s t r a c t Delivery of small interfering RNA (siRNA) has been one of the major hurdles for the application of RNA interference in therapeutics. Here, we describe a cationic lipid assisted polymeric nanoparticle system with stealthy property for efficient siRNA encapsulation and delivery, which was fabricated with poly(ethylene glycol)-b-poly(d,l-lactide), siRNA and a cationic lipid, using a double emulsion-solvent evaporation technique. By incorporation of the cationic lipid, the encapsulation efficiency of siRNA into the nanoparticles could be above 90% and the siRNA loading weight ratio was up to 4.47%, while the diameter of the nanoparticles was around 170 to 200 nm. The siRNA retained its integrity within the nanoparticles, which were effectively internalized by cancer cells and escaped from the endosome, resulting in significant gene knockdown. This effect was demonstrated by significant down-regulation of luciferase expression in HepG2-luciferase cells which stably express luciferase, and suppression of polo-like kinase 1 (Plk1) expression in HepG2 cells, following delivery of specific siRNAs by the nanoparticles. Furthermore, the nanoparticles carrying siRNA targeting the Plk1 gene were found to induce remarkable apoptosis in both HepG2 and MDA-MB-435s cancer cells. Systemic delivery of specific siRNA by nanoparticles significantly inhibited luciferase expression in an orthotopic murine liver cancer model and suppressed tumor growth in a MDA-MB-435s murine xenograft model, suggesting its therapeutic promise in disease treatment. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Since the discovery of RNA interference (RNAi) in 1998 by Fire et al., small interfering RNA (siRNA) has rapidly emerged as a promising candidate for the treatment of numerous diseases (e.g. neurodegenerative disorders, cancer and infectious diseases), due to its posttranscriptional gene-silencing ability [1–9]. For example, delivery of siRNA specifically targeting an oncogene or the gene for vascular endothelial growth factor (VEGF) in tumor cells has shown therapeutic potential in cancer therapy [10–12]. However, siRNAs are relatively large molecules with a polyanionic nature, rendering it difficult for these molecules to enter cells by a passive diffusion mechanism [13]. Moreover, the in vivo delivery of siRNA to targeted tissue and cells remains the biggest challenge for clinical applications, owing to the rapid enzymatic digestion of siRNA in plasma, its renal elimination, limited penetration across the capillary endothelium and

⁎ Corresponding author at: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, PR China. Tel.: + 86 551 3600335; fax: + 86 551 3600402. E-mail address: [email protected] (J. Wang). 1 Contributed equally to this work. 0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2011.07.035

inefficient cellular uptake [14]. To overcome these obstacles, a number of delivery systems have been developed, including lipid-based and polymer-based systems, peptide conjugates and single-chain fragment variable antibody fusion protein systems [15–20]. They have shown promising efficacy in specific gene silencing and the treatment of various diseases [21–25]. Biodegradable poly(d,l-lactide) (PLA) and its copolymers have been widely used in biomaterial research and biomedical applications [26–30]. For examples, by utilizing PLA polymers, Lupron Depot has become the first parenteral sustained-release formulation, which was approved in 1989 by the US Food and Drug Administration [31]. Block copolymer of poly(ethylene glycol) and poly(d,l-lactide-co-glycolide) has been formulated with paclitaxel into nanoparticles, known as Genexol-PM, which is being used for the treatment of breast cancer in South Korea and is undergoing phase II human clinical investigation in the USA in a selected cancer [32]. Poly(d,l-lactide) and poly(d,llactide-co-glycolide) (PLGA) have also demonstrated the potential for sustained nucleic acid delivery [33–35]. Recently, they have been formulated into microspheres or nanoparticles for siRNA delivery [36–43]. As a typical example, Saltzman and co-workers have reported that PLGA nanoparticles can be densely loaded with siRNA in the presence of spermidine and, when applied topically to the

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vaginal mucosa, lead to efficient and sustained gene silencing [36]. In another example, Panyam and co-workers have demonstrated that the introduction of polyethylenimine (PEI), a cationic polymer commonly used in gene delivery applications, in the PLGA matrix can improve the retention of anionic siRNA molecules [39]. The encapsulation efficiency of siRNA in PLGA nanoparticles has been reported to reach 79%. Such nanoparticles can mediate targeted gene silencing and drug delivery to overcome tumor drug resistance [42]. In this study, we reported the fabrication of a nanoparticular delivery system with siRNA encapsulation, using the amphiphilic block copolymer of poly(ethylene glycol)-b-poly(d,l-lactide) (mPEGPLA) as the matrix under the assistance of a cationic lipid. The siRNAencapsulated nanoparticles were prepared by a non-condensation process and a double-emulsion solvent evaporation technique. This process and formulation achieved high siRNA encapsulation efficiency, and meanwhile, with the presence of a hydrophilic PEG component, the nanoparticles provided steric stabilization and protection from the physiological surroundings. We investigated the effect of siRNA encapsulation by the composition of the formulation, cellular uptake of nanoparticles by cancer cells and gene silencing efficacy in vitro and in vivo in cancer models. 2. Materials and methods 2.1. Materials The diblock copolymer of mPEG with poly(d,l-lactide) (mPEG5K– PLA25K) was synthesized by ring-opening polymerization of d,l,-lactide using monomethyl ether of poly(ethylene glycol) (mPEG5k-OH, Mw = 5000) as the macroinitiator and Sn(Oct)2 as the catalyst according to a previously reported method [44], and the detailed information was given in the supplementary material. Cationic lipid N, N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl aminoethyl) ammonium bromide (BHEM-Chol) was synthesized as described in the supplementary material. 3-{4,5-Dimethylthiazol-2yl}-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Poly(vinyl alcohol) (PVA, 88% hydrolyzed, Mw = 22,000) was purchased from Acros Organics Co. (Morris Plains, NJ). Ultra-purified water was prepared using a Milli-Q Synthesis System (Millipore, Bedford, MA). Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island) and L-glutamine were purchased from Gibco BRL (Eggenstein, Germany). The Lipofectamine 2000 transfection kit from Invitrogen (Carlsbad, USA) was used as suggested by the supplier. Fluorescently labeled FAM-siRNA and cy5-siRNA, negative control siRNA with a scrambled sequence (siN.C., antisense strand, 5′-ACGUGACACGUUCGGAGAAdTdT-3′), siRNA targeting luciferase mRNA (siLuci, antisense strand, 5′-CUUACGCUGAGUACUUCGAdTdT-3′) and siRNA targeting Plk1 mRNA (siPlk1, antisense strand, 5′-UAAGGAGGGUGAUCUUCUUCAdTdT-3′) were synthesized by Suzhou Ribo Life Science Co. (Kunshan, China).

by sonication for 30 s over an ice bath in 0.5 ml of chloroform containing 1.0 mg of BHEM-Chol and 25 mg of mPEG5K–PLA25K. This primary emulsion was further emulsified in 1.5 mL of PVA solution (1.0%, w/v) by sonication (80 W for 2 min) over an ice bath to form a water-in-oil-in-water emulsion. The mixture was then added to 25 mL of PVA solution (0.3%, w/v), which was further stirred for 3 h to allow for the evaporation of chloroform. The particles were collected by centrifugation (30,000 ×g, 1 h) and washed twice with sterile water to remove PVA and possible un-encapsulated siRNA. The supernatant was also collected for encapsulation efficiency analyses. This siRNA-loaded nanoparticle formulation was further denoted as NP0.2/1.0/25.0, where NP represents nanoparticles and the subscript represents the weight ratio of siRNA, BHEM-Chol and mPEG5K–PLA25K in this preparation, which was 0.2 mg: 1.0 mg: 25 mg.

2.4. Evaluation of encapsulation efficiency of siRNA to the nanoparticles The encapsulation efficiency (E.E.) of siRNA in the formulation was evaluated by measuring the concentration of un-encapsulated siRNA in the above-mentioned supernatant following centrifugation, by high-performance liquid chromatography (HPLC) analyses, using a Waters HPLC system consisting of a Waters 1525 binary pump, a Waters 2475 fluorescence detector, a 1500 column heater and a Symmetry C18 column. FAM-siRNA was used in the experiment. HPLC grade triethylammonium acetate buffer (0.1 M, pH 7.4) with acetonitrile at a ratio of 72:28 (v/v) was used as the mobile phase at 30 °C with a flow rate of 0.5 mL min − 1. The fluorescence detector was set at 485 nm for excitation and 535 nm for emission and linked to Breeze software for data analysis. Linear calibration curves for concentrations in the range of 0.078–40 μg/mL were constructed using the peak areas by linear regression analysis. The content of siRNA encapsulated into nanoparticles was calculated by subtracting the amount of siRNA in the supernatant from the total amount used for loading, and the E.E. was calculated accordingly by dividing it by the total amount of siRNA used for loading. The loading content of siRNA in the nanoparticles was calculated by dividing the amount of siRNA in the nanoparticles with the weight of polymer.

2.5. Characterization of the stability of nanoparticles As a typical example, NP0.2/1.0/25.0 with FAM-siRNA encapsulation was incubated in DMEM containing 10% serum (v/v) at 37 °C for 3 days. The mean diameters of nanoparticles were monitored by a Malvern Zetasizer Nano ZS90 apparatus, which were analyzed in triplicate at a concentration of 1.0 mg/mL in a total volume of 1 mL. The average values from a total of 20 runs of 30 s each were used.

2.6. In vitro release of siRNA from nanoparticles 2.2. Characterizations Zeta potentials and particle size measurements were conducted using a zeta potential analyzer with dynamic light-scattering (DLS) capability as previously described [45], with a Malvern Zetasizer Nano ZS90, a He-Ne laser (633 nm) and 90° collecting optics. All samples were prepared in aqueous solution at a concentration of 1.0 mg mL − 1 and the measurements were carried out at 25 °C. Data was analyzed using Malvern Dispersion Technology Software 4.20. 2.3. Preparation of nanoparticles with siRNA encapsulation Nanoparticles loaded with siRNA were prepared by a double emulsion-solvent evaporation technique. As an example, an aqueous solution of siRNA (0.2 mg) in 25 μL of RNase free water was emulsified

NP0.2/1.0/25.0 and NP0.2/0.0/25.0 encapsulated with FAM-siRNA were suspended in 5.0 mL of phosphate buffer (0.01 M, pH 7.4) in triplicate, and incubated at 37 °C with gentle shaking (70 rpm). The release of siRNA was monitored at different time intervals. Samples were taken at predetermined intervals and centrifuged (30,000 ×g, 1 h). The concentration of FAM-siRNA in the supernatant was determined by HPLC as described above and the released amount of FAM-siRNA was calculated accordingly. The released FAM-siRNA from the nanoparticles was also identified by electrophoresis. The supernatant (20 μL) with a loading dye was electrophoresed in 1.5% agarose gel at a constant voltage of 120 V for 5 min in Tris/Borate/EDTA (TBE) buffer. The siRNA bands were visualized with ethidium bromide staining under a UV transilluminator at a wavelength of 365 nm.

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2.7. Cell culture

2.10. In vitro gene silencing with siRNA-encapsulated nanoparticles

The human hepatocellular liver carcinoma cell line HepG2 and the human breast cancer cell line MDA-MB-435s from the American Type Culture Collection (ATCC) were maintained in DMEM medium supplemented with 10% FBS (Hyclone, Waltham, USA), 4 mM Lglutamine, 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, USA). Cells were incubated at 37 °C in a 5% CO2 atmosphere. The HepG2luciferase cell line, which stably expresses luciferase, was obtained by transfection with a retrovirus carrying the luciferase gene according to a standard protocol and clones derived from discrete colonies were isolated and amplified in medium.

HepG2-luciferase cells (5×104) were seeded into 24-well plates and allowed to grow until 50% confluence. The cells were treated with siLuciencapsulated NP0.2/1.0/25.0 at different siLuci doses from 50 nM to 300 nM, corresponding to polymer concentrations from 0.092 mg mL− 1 to 0.549 mg mL− 1. The control was treated with NP0.2/1.0/25.0 encapsulated with siN.C. at a polymer concentration of 0.549 mg mL− 1 containing 300 nM siN.C., empty nanoparticles at a polymer concentration of 0.549 mg mL− 1, free siLuci at 300 nM or PBS. Lipofectamine 2000 carrying 50 nM of siLuci was used as the positive control. After 48 h incubation, cells were washed and lysed with the reported lysis buffer and the luciferase activity was measured for 10 s on a Veritas microplate luminometer (Model 9100–102, Turner BioSystems, Sunnyvale, CA), using a Promega luciferase assay kit according to the standard protocol provided by the supplier. The relative light units (RLU) were normalized against protein concentrations in the cell extracts, which were measured using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). To assess the silencing capability of the nanoparticles encapsulating siPlk1, HepG2 cells were seeded on 6-well plates (1.0× 105 per well) and allowed to grow until 50% confluence. The cells were treated for 24 h with NP0.2/1.0/25.0 encapsulating siPlk1. The polymer concentrations were varied from 0.183 mg mL− 1 to 0.549 mg mL− 1, and the corresponding concentration of siPlk1 were from 100 nM to 300 nM. The control formulations for cell treatments were NP0.2/1.0/25.0 encapsulating 300 nM siN.C. at a polymer concentration of 0.549 mg mL− 1, the empty nanoparticles at a concentration of 0.549 mg mL− 1, free siPlk1 at 300 nM and PBS. Lipofectamine 2000 carrying 50 nM of siPlk1 was used as the positive control. Total RNA from transfected cells was isolated using an RNeasy mini-kit (Qiagen, Valencia, CA) according to the protocol of the manufacturer. Two micrograms of total RNA were transcribed into cDNA using the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Thereafter, 2 μL of cDNA was subjected to quantitative real-time PCR analysis targeting Plk1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the SYBR® Premix Ex Taq™ (Perfect Real Time) (Takara). Analysis was performed using the Applied Biosystems StepOne™ Real-Time PCR Systems. Relative gene expression values were determined by the ΔΔCT method using StepOne™ Software v2.1 (Applied Biosystems). Data are presented as the fold difference in Plk1 expression normalized to the housekeeping gene GAPDH as the endogenous reference, and relative to the untreated control cells. The primers used in the quantitative realtime PCR for Plk1 and GAPDH were: Plk1-forward 5′-AGCCTGAGGCCCGATACTACCTAC-3′, Plk1-reverse 5′-ATTAGGAGTCCCACACAGGGTCTTC-3′, and GAPDH-forward 5′-TTCACCACCATGGAGAAGGC3′, GAPDH-reverse 5′-GGCATGGACTGTGGTCATGA-3′. PCR parameters consisted of 30 s of Taq activation at 95 °C, followed by 40 cycles of PCR at 95 °C × 5 s, 60 °C× 30 s, and 1 cycle of 95 °C× 15 s, 60 °C× 60 s and 95 °C × 15 s.

2.8. Cytotoxicity measurement The cytotoxicity of nanoparticles with or without BHEM-Chol (NP0.0/1.0/25.0, NP0.0/0.5/25.0, NP0.0/0.1/25.0 or NP0.0/0.0/25.0) was assessed with a MTT viability assay against HepG2 cells. Cells were seeded in 96-well plates at 10,000 cells per well in 100 μL of complete DMEM medium supplemented with 10% FBS, and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. To determine the cytotoxicity of nanoparticles, 100 μL of NP0.0/1.0/25.0, NP0.0/0.5/25.0, NP0.0/0.1/25.0 or NP0.0/0.0/25.0 in complete DMEM at different concentrations were used to replace the culture medium and cells were further cultured for 24 h. MTT stock solution was then added to each well to achieve a final concentration of 1 g L − 1, with the exception of the wells used as a blank, to which the same volume of phosphate buffered saline (PBS, 0.01 M, pH 7.4) was added. After incubation for an additional 2 h, 100 μL of the extraction buffer (20% SDS in 50% DMF, pH 4.7, prepared at 37 °C) was added to the wells and incubated overnight at 37 °C. The absorbance was measured at 570 nm using a Bio-Rad 680 microplate reader. Cell viability was normalized to that of HepG2 cells cultured in the culture medium with PBS treatment. 2.9. Cellular uptake of nanoparticles and intracellular trafficking HepG2 cells (5× 10 4) were seeded into 24-well plates and cultured in complete DMEM to reach 50% confluency. The cells were then incubated at 37 °C with FAM-siRNA-encapsulated NP0.2/1.0/25.0 suspended in complete DMEM at a polymer concentration of 0.366 mg mL− 1 and a siRNA dose of 200 nM. At different time points, the medium was aspirated and cells were rinsed twice with cold PBS. The cells were then trypsinized, washed with cold PBS, filtered through 35 μm nylon mesh, and subjected to flow cytometric analysis using a BD FACSCalibur flow cytometer (BD Bioscience, Bedford, MA). For microscopic observation, after 2 h incubation with FAM-siRNAencapsulated NP0.2/1.0/25.0 at identical formulation as described above, HepG2 cells were washed twice with PBS and fixed with 4% formaldehyde for 15 min at room temperature. The cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, Haimen, China) for cell nuclei and Alexa Fluor® 568 phalloidin (Invitrogen, Carlsbad, USA) for the cytoskeleton according to the standard protocol provided by the suppliers. Cover slips were mounted on glass microscope slides with a drop of anti-fade mounting media (SigmaAldrich Co.) to reduce fluorescence photobleaching. The cellular uptake of nanoparticles was visualized by a laser scanning confocal microscope (LSCM; LSM 700 Meta, Carl Zeiss Inc., Thornwood, NY). To follow the internalization and endosomal release of nanoparticles, after 4 h or 24 h of incubation with FAM-siRNA-encapsulated NP0.2/1.0/25.0 as described above, HepG2 cells were washed with PBS twice and stained with LysoTrackerTM Red (Molecular Probe, Eugene, OR) following the manufacturer's instructions. The cells were then washed twice with PBS, fixed with 4% formaldehyde for 15 min and counterstained with DAPI. The cells were imaged by LSCM to determine the localization of siRNA inside the cells.

2.11. In vitro apoptosis induction post siPlk1 transfection within nanoparticles HepG2 or MDA-MB-435s cells were seeded into 24-well plates and allowed to grow until 50% confluence. The cells were treated with the above-mentioned formulations. After 48 h of treatment, apoptotic cells were detected by flow cytometry using the Annexin V-FITC apoptosis detection kit I (BD Biosciences) and the results were analyzed using WinMDI 2.9 software. 2.12. Animal tumor models and treatments BALB/c nude mice (4–6 weeks old) were purchased from the Shanghai Experimental Animal Centre of the Chinese Academy of Sciences (Shanghai, China) and all animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of

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Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. To establish an orthotopic liver tumor model, male nude mice were anesthetized and opened via a midline incision to expose the liver, then HepG2-luciferase cells (5 × 10 5 cells) in 25 μL of Matrigel basement membrane matrix (BD Biosciences) were implanted into a lobe of the liver using a 28 gauge needle. Ten days after tumor cell implantation (day 0), the mice were treated with PBS, free siLuci, NP0.2/1.0/25.0 encapsulating siN.C. or siLuci, respectively, by intravenous injection once every day at a dose of 20 μg siRNA per mouse. The dose of polymer per injection was 2.745 mg when applied. The animals were imaged on day 0 and day 2 on a Xenogen IVIS® Lumina system (Caliper Life Sciences, Hopkinton, MA) after receiving 200 μL of Dluciferin (15 mg/mL, Xenogen) via intraperitoneal injection. Measurement of total flux (photons/second) of the emitted light reflects the relative luciferase expression of cells in the tumor. Data were analyzed using Xenogen Living Image software. To establish the human breast cancer xenograft tumor model, a singlecell suspension of MDA-MB-435s cells in PBS (5×106 per mouse) was injected subcutaneously into the mammary fat pads of female mice. The animals were used 14 days post tumor inoculation. The tumor volume was around 50 mm3 at 14 days after cell implantation, calculated according to the formula: tumor volume (mm3)=0.5×length×width2. Tumor-bearing mice were randomly divided into five groups (six mice per group), and treated using PBS, free siPlk1, blank NP0.0/1.0/25.0, NP0.2/1.0/25.0 encapsulating siN.C. or NP0.2/1.0/25.0 encapsulating siPlk1 by intravenous injection once every other day. The dose of siRNA was 20 μg per mouse per injection, while the dose of polymer was 2.745 mg per mouse per injection. Tumor growth was monitored by measuring the perpendicular diameter of the tumor using calipers. The estimated volume was calculated based on the equation described above. 3. Results and discussion 3.1. Preparation and characterization of nanoparticles The success of RNAi-based therapy is highly dependent on the delivery system, while the therapeutic efficiency of siRNA is significantly affected by the physicochemical properties of nanoparticles. Although a few studies have demonstrated that siRNA can be encapsulated into PLA nanoparticles by a double-emulsion method [36,38,39,41], the encapsulation efficiency of siRNA and the loading content of siRNA have not been satisfactory, which may limit its application in therapeutics. As reported, the encapsulation efficiency of siRNA was about only 20% without incorporating PEI, and the siRNA content was 0.26% (w/w) at most [39,41]. Several groups have reported that incorporation of PEI by

Scheme 1. Schematic illustration of the fabrication of cationic lipid assisted nanoparticles with siRNA encapsulation. mPEG-PLA: poly(ethylene glycol)-b-poly(d,llactide); BHEM-Chol: N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl aminoethyl) ammonium bromide; W and O represent water phase and oil phase, respectively.

either a condensation process with siRNA or a non-condensation process can improve siRNA loading efficiency through a doubleemulsion method [37–39,41]. However, PEI can not be degraded within the body and could potentially lead to severe toxicity [46]. Moreover, PEI, which is a hydrophilic polyelectrolyte, may diffuse into the external water phase during nanoparticle preparation, resulting in lower siRNA encapsulation efficiency. Meanwhile, for systemic application, it is expected that the nanoparticle formulation of siRNA could contain a hydrophilic coating (e.g. PEG) to provide steric stabilization and to evade immune system recognition. In this study, with the double emulsion method shown in Scheme 1, by incorporating an amphiphilic cationic lipid BHEM-Chol in a solution of the mPEG5K-PLA25K matrix, we demonstrated that siRNA could be immobilized in the formed nanoparticles at high encapsulation efficiency, which was dependent on the content of BHEM-Chol in the formulation. As summarized in Table 1, without the addition of BHEMChol, the encapsulation efficiency of siRNA was only 35.4% in a formulation of NP0.04/0.0/25.0, where the weights of siRNA to mPEG5KPLA25K were 0.04 mg and 25 mg in the formulation, respectively. The encapsulation efficiency became even lower when the loading ratio of siRNA to mPEG5K-PLA25K increased to 0.2: 25.0, with an E.E. of 26.2% in the formulation of NP0.2/0.0/25.0. However, addition of the cationic lipid to the solution of mPEG5K-PLA25K dramatically improved the E.E. of siRNA encapsulated in the nanoparticles, and such an improvement was correlative to the increase of cationic lipid content in the formulation. As typical examples, the addition of 1.0 mg of BHEM-Chol in the formulations NP0.04/1.0/25.0 and NP0.2/1.0/25.0 resulted in 96.4% and

Table 1 Cationic lipid-polymer formulations and properties. Formulation

NP0.0/0.0/25.0 NP0.0/0.1/25.0 NP0.0/0.5/25.0 NP0.0/1.0/25.0 NP0.04/0.0/25.0 NP0.04/0.1/25.0 NP0.04/0.5/25.0 NP0.04/1.0/25.0 NP0.2/0.0/25.0 NP0.2/0.1/25.0 NP0.2/0.5/25.0 NP0.2/1.0/25.0 NP0.6/1.0/25.0 NP1.8/1.0/25.0 * N.A.: not available.

Feeding weight siRNA (mg)

BHEM-Chol (mg)

mPEG5K-PLA25K (mg)

0.0 0.0 0.0 0.0 0.04 0.04 0.04 0.04 0.20 0.20 0.20 0.20 0.60 1.80

0.0 0.1 0.5 1.0 0.0 0.1 0.5 1.0 0.0 0.1 0.5 1.0 1.0 1.0

25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0 25.0

E.E. (%)

siRNA Loading Content (%)

Diameter (nm)

PDI of nanoparticles

Zeta potential (mV)

N.A. N.A. N.A. N.A. 35.4 67.2 90.2 96.4 26.2 56.6 90.7 95.7 85.3 64.5

N.A. N.A. N.A. N.A. 0.057 0.107 0.141 0.148 0.210 0.451 0.711 0.736 1.97 4.47

199 185 171 175 192 188 189 168 199 196 202 170 186 203

0.162 0.179 0.153 0.210 0.218 0.237 0.216 0.183 0.153 0.197 0.221 0.216 0.231 0.257

− 16.5 − 9.8 38.7 39.1 − 17.4 − 13.8 6.7 17.9 − 16.6 − 16.7 − 11.7 13.8 1.7 − 8.3

95.7% encapsulation efficiencies. On the other hand, although it was observed that increasing siRNA feeding may reduce the encapsulation efficiency (e.g. 67.2% in NP0.04/0.1/25.0 ∝ 56.6% in NP0.2/0.1/25.0), the loading content of siRNA to polymer was significantly enhanced at a fixed weight ratio of BHEM-Chol in the formulation (0.103% in NP0.04/0.1/25.0 ∝ 0.435% in NP0.2/0.1/25.0). By increasing the feeding ratio of siRNA, we have successfully encapsulated 4.47% of siRNA (w/w) into the nanoparticles though the encapsulation efficiency was decreased. The measurements of nanoparticles by dynamic light scattering indicated that the diameters and the polydispersity indices (PDIs) of nanoparticles were not strongly associated with the components of the initial feeding ratios. Regardless of the feeding level of siRNA, the nanoparticles exhibited an average size around 170 to 200 nm, independent on the addition of BHEM-Chol. However, the zeta potential of nanoparticles was found to be dependent on the feeding of BHEM-Chol as well as the feeding of siRNA. Increasing the feeding ratio of BHEM-Chol increased the zeta potential of nanoparticles at a fixed siRNA feeding ratio, due to the cationic nature of BHEM-Chol, while increasing the siRNA feeding ratio reduced the zeta potential of nanoparticles when the weight of incorporated BHEM-Chol was unchanged. Recent data show that nanoparticles less than 200 nm in size that carry a very slight positive charge can penetrate throughout large tumors following systemic administration [32]. Additionally, on the basis of the encapsulation efficiency and siRNA loading, NP0.2/1.0/25.0 was used for subsequent experiments. 3.2. Colloidal stability of siRNA-encapsulated nanoparticles and the in vitro release of siRNA The stability of nanoparticles is closely correlated to the surface property of nanoparticles [47]. To study the stability of siRNAencapsulated nanoparticles, we incubated NP0.2/1.0/25.0 in DMEM culture medium, containing 10% FBS at 37 °C. At different time intervals, the particle size of nanoparticles was measured. As shown in Fig. 1, although a very small increase of the hydrodynamic diameter of nanoparticles was observed from 170 nm to about 180 nm in the initial incubation, the size of the nanoparticles was consistently maintained at ca. 180 nm for about two days. It is also worth noting that the PDI of the nanoparticles only slightly increased from 0.216 to 0.323 after 72 h incubation in DMEM culture medium. It can be speculated that PEG blocks are presented at the surface of nanoparticles, which are known to prevent non-specific protein adsorption and aggregation of nanoparticles. The in vitro release profile of siRNA from nanoparticles was investigated at pH 7.4 and 37 °C, by comparing two formulations NP0.2/0.0/25.0 and NP0.2/1.0/25.0 at 1 mg/mL of nanoparticles, where the former did not contain BHEM-Chol. As shown in Fig. 2A, the release of siRNA from nanoparticles was partially retarded by the incorporated cationic lipid. However, NP0.2/1.0/25.0 exhibited sustained siRNA release, without showing a significant burst release, in contrast to

Fig. 1. Effect of incubation in serum (10%)-containing DMEM media on the particle size of NP0.2/1.0/25.0.

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Fig. 2. In vitro release of FAM-siRNA from nanoparticles. (A) Cumulative released amount of siRNA from 1 mg of siRNA-encapsulated nanoparticles; (B) Percentage of siRNA released from siRNA-encapsulated nanoparticles; (C) and (D) Integrity of released siRNA analyzed by HPLC (C) and gel retardation assay (D).

NP0.2/0.0/25.0, which exhibited a significant burst release of ~18% in 12 h. Due to the higher loading content of siRNA in NP0.2/1.0/25.0, the cumulatively released amount of siRNA from NP0.2/1.0/25.0 was much higher than that from NP0.2/0.0/25.0 (Fig. 2B), which was about four times of that released from NP0.2/0.0/25.0 over 13 days. The integrity of released siRNA from nanoparticles was examined by HPLC and gel electrophoresis. As shown in Fig. 2C, siRNA released from both NP0.2/0.0/25.0 and NP0.2/1.0/25.0 showed the same elution time as the control siRNA. In addition, Fig. 2D demonstrates that the electrophoretic mobility of the released siRNA was as same as the control siRNA. It is noteworthy that the lower density of the siRNA band corresponding to siRNA released from NP0.2/0.0/25.0 was due to the lower released amount of siRNA from the nanoparticles. These data implied that the integrity of the siRNA was maintained during the preparation of nanoparticles and the release process. 3.3. In vitro cytotoxicity of the nanoparticles The cytotoxicity of nanoparticles (NP0.0/0.0/25.0) and BHEM-Chol incorporated nanoparticles (NP0.0/0.1/25.0, NP0.0/0.5/25.0 and NP0.0/1.0/25.0) to HepG2 cells was evaluated using the MTT method. As shown in Fig. 3, after 24 h incubation, cell viability with NP0.0/0.0/25.0 up to 1 g L− 1 remained nearly 100% when compared with untreated cells, suggesting its good biocompatibility with HepG2 cells. Similarly, nanoparticles with the incorporation of various weights of BHEM-Chol (NP0.0/0.1/25.0,

Fig. 3. Cytotoxicity of nanoparticles to HepG2 cells without (NP0.0/0.0/25.0) or with BHEM-Chol incorporation (NP0.0/0.1/25.0, NP0.0/0.5/25.0 and NP0.0/1.0/25.0).

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phalloidin and DAPI, respectively. As shown in Fig. 4A, it was clearly demonstrated that FAM-siRNA-encapsulated NP0.2/1.0/25.0 had been internalized and observed in the cytoplasma of HepG2 cells after 2 h of incubation. To more precisely understand how the incubation time affected cell internalization of NP0.2/1.0/25.0, we incubated FAM-siRNAencapsulated NP0.2/1.0/25.0 with HepG2 cells for different periods of time, and subsequently analyzed the cells by FACS. Incubation of FAMsiRNA alone exhibited very minimal cell fluorescence, while stronger fluorescence was observed in cells when they were cultured with FAM-siRNA-encapsulated NP0.2/1.0/25.0, and the fluorescence intensity of cells increased with the extension of culturing time from 0.5 to 4 h, as indicated in Fig. 4B. Nanoparticles enter cells by an endocytosis pathway, while the lysosomal escape of nanoparticles is important for the subsequent post-transcriptional gene silencing in the cytoplasm [39]. To investigate whether siRNA had escaped from the endosome/lysosome with the delivery of nanoparticles, we incubated FAM-siRNA-encapsulated NP0.2/1.0/25.0 with HepG2 cells, and observed the localization of nanoparticles in cells by staining the acidic organelles (including endosomes and early lysosomes) with LysoTracker TM Red after 4 h and 24 h of culture. The cell nuclei were counterstained with DAPI. As

Fig. 4. Uptake of siRNA-encapsulated nanoparticles (0.366 mg/mL nanoparticles, FAMsiRNA dose of 200 nM) by HepG2 cells. (A) LSCM imaged cell internalization after 2 h of incubation. Cell nuclei and F-actin were counterstained with DAPI (blue) and Alexa Fluor® 568 phalloidin (Alex-568, red), respectively; (B) Flow cytometric analyses of cells treated with FAM-siRNA-encapsulated nanoparticles (NP0.2/1.0/25.0) for different periods of time.

NP0.0/0.5/25.0 and NP0.0/1.0/25.0) did not exhibit notable cytotoxicity to HepG2 cells either.

3.4. Cellular uptake of siRNA-encapsulated nanoparticles Internalization of siRNA-encapsulated nanoparticles was evaluated by confocal microscopic observation after culturing nanoparticles with HepG2 cells. We used FAM-labeled siRNA, and the cytoskeleton F-actin and the cell nuclei were counterstained with Alexa Fluor® 568

Fig. 5. Assessment of endosomal escape of FAM-siRNA-encapsulated NP0.0/1.0/25.0 (0.366 mg/mL nanoparticles, FAM-siRNA dose of 200 nM) after 4 and 24 h of incubation by LSCM. Cell nuclei and endosomes were counterstained with DAPI (blue) and LysoTrackerTM Red (red), respectively.

Fig. 6. Gene silencing by siRNA-encapsulated nanoparticles. (A) Relative luciferase expression in HepG2-luciferase cells after treatment with different formulations; (B) Plk1 mRNA expression in HepG2 cells determined by quantitative RT-PCR analysis following treatment with different formulations. Lipo/siLuci and Lipo/siPlk1 represent the complexes of Lipofectamine 2000 with siLuci (50 nM) and siPlk1 (50 nM), respectively. Free siLuci and free siPlk1 represent that cells were incubated with siLuci and siPlk1 at a dose of 300 nM, respectively. NP0.2/1.0/25.0 and NP0.2/1.0/25.0/siN.C. represent that cells were administered empty nanoparticles (NP0.2/1.0/25.0, 0.549 mg mL− 1 of polymer) and nanoparticles encapsulating siN.C. (NP0.2/1.0/25.0/siN.C., 300 nM siRNA and 0.549 mg mL− 1 of polymer), respectively.

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shown in Fig. 5, confocal laser microscopic observation indicated that after 4 h of incubation, the FAM-siRNA-encapsulated NP0.2/1.0/25.0 were mainly colocalized with the LysoTracker TM Red stained organelles, suggesting that those nanoparticles still resided in the endosomes or early lysosomes. However, it was clearly shown that incubation of cells with FAM-siRNA-encapsulated NP0.2/1.0/25.0 for 24 h resulted in separate localization of green and red fluorescence inside the cells, with minimal co-localization, which demonstrated that FAM siRNA appeared to have escaped from the lysosomal vesicles. 3.5. Gene expression knockdown and induction of cell apoptosis in vitro To evaluate the gene silencing function of siRNA-encapsulated nanoparticles, we incubated HepG2-luciferase cells, which stably expresses luciferase, with NP0.2/1.0/25.0 carrying siLuci at different doses from 50 nM to 300 nM, and analyzed the luciferase expression knockdown efficiency after 48 h. As shown in Fig. 6A, the negative controls (free siLuci, blank nanoparticles NP0.2/1.0/25.0 and NP0.2/1.0/25.0 carrying siN.C.) did not exhibit notable efficiency in terms of silencing luciferase expression. The treatment of HepG2-luciferase cells with Lipofectamine 2000 complex carrying 50 nM of siLuci exhibited ~50% knockdown of luciferase expression. Although NP0.2/1.0/25.0 carrying 50 nM siLuci was less effective than Lipofectamine 2000 carrying the same dose of siLuci for gene silencing, increasing the dose of siLuci significantly improved the luciferase expression knockdown activity, reaching a similar level to Lipofectamine 2000 transfected cells when the dose of siLuci was 200 nM or above. We further evaluated the gene silencing function by examining the suppression of an endogenous Plk1 gene in HepG2 cells following the delivery of siPlk1. As reported, Plk1 is a key regulator for the mitotic progression of mammalian cells, and the activity of Plk1 is elevated in cancer cells, which contributes to the oncogenic transformation [48–50]. The level of Plk1 mRNA was analyzed by real time quantitative RT-PCR 24 h after transfection. As shown in Fig. 6B. Free siPlk1, empty nanoparticles and NP0.2/1.0/25.0 encapsulated with siN.C. failed to inhibit Plk1 expression, but NP0.2/1.0/25.0 carrying siPlk1 significantly downregulated the mRNA expression of Plk1 in HepG2 cells, which was also dose-dependent. A comparable Plk1 gene silencing level to that seen with the Lipofectamine 2000 complex with siPlk1 (50 nM) was achieved with the transfection of NP0.2/1.0/25.0 carrying 200 nM of siPlk1 or above, similar to that observed in the luciferase expression knockdown experiments. Plk1 inhibition has been shown to be associated with apoptosis induction [51]. Apoptosis of HepG2 cells was then evaluated after

Fig. 7. The influence of siPlk1 delivery by nanoparticles on apoptosis in HepG2 cells. Apoptosis was evaluated after treating HepG2 cells for 48 h. Early apoptotic cells are presented in the lower right quadrant and late apoptotic cells are presented in the upper right quadrant. Lipo/siPlk1 represents the complexes of Lipofectamine 2000 with siPlk1 (50 nM). Free siPlk1 represent that cells were incubated with siPlk1 at a dose of 300 nM. NP0.2/1.0/25.0 and NP0.2/1.0/25.0/siN.C. represent that cells were administered empty nanoparticles (NP0.2/1.0/25.0, 0.549 mg mL− 1 of polymer) and nanoparticles encapsulating siN.C. (NP0.2/1.0/25.0/siN.C., 300 nM siRNA and 0.549 mg mL− 1 of polymer), respectively.

Fig. 8. Downregulation of luciferase expression in HepG2-luciferase cells inoculated in the orthotopic liver tumor model by systemically delivering siLuci with nanoparticles: (A) Images of luciferase expression obtained immediately before the first i.v. injection (day 0) and after receiving two injections (day 2) of siLuci-encapsulated nanoparticles; (B) Quantification of luciferase expression as performed in (A). Free siLuci represents that mouse was injected with siLuci at a dose of 20 μg per mouse per injection. NP0.2/1.0/25.0/siN.C. and NP0.2/1.0/25.0/siLuci represent that mice were administered 2.745 mg NP0.2/1.0/25.0 encapsulating siN.C. and siLuci at a siRNA dose of 20 μg per mouse per injection, respectively. The mice received one injection per day. A pseudo-color image representing the intensity of emitted light (red, most intense; blue, least intense) superimposed on a grayscale reference image (for orientation) shows luciferase expression in mice.

transfection with different formulations, and the cells were analyzed for cell apoptosis after staining with Annexin-V-FITC and propidium iodide (PI). Fig. 7 demonstrated that delivery of siPlk1 into HepG2 cells with NP0.2/1.0/25.0 induced siPlk1 dose-dependent cell apoptosis. Complexes of Lipofectamine 2000 with siPlk1 at 50 nM led to 21.04% cell apoptosis (including early apoptotic cells and the late apoptotic cells); this value was achieved when using NP0.2/1.0/25.0 at a siPlk1 dose of 200 nM. It should be mentioned that the empty nanoparticles NP0.0/1.0/25.0, NP0.2/1.0/25.0 carrying siN.C., and free siPlk1 did not induce remarkable cell apoptosis with the same dose of siRNA. A similar phenomenon was observed in MDA-MB-435s cells when siPlk1 was delivered with NP0.2/1.0/25.0 (Figure S1). 3.6. In vivo gene silencing and tumor growth suppression The ability to effectively silence gene expression in vivo using nanoparticles was demonstrated in an orthotopic liver tumor model and a breast cancer tumor model. The orthotopic liver tumor model

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was established in situ with the injection of HepG2-luciferase cells into the liver of nude mice. Following i.v. injection of 2.745 mg NP0.2/1.0/25.0 carrying 20 μg siLuci, the animals were imaged with a Xenogen IVIS®Lumina System to examine luciferase expression, which was compared with different negative controls, including i.v. injection of PBS, free siLuci at the same dose and NP0.2/1.0/25.0 carrying 20 μg siN.C. As shown in Fig. 8, the treatment of liver tumor-bearing mice with NP0.2/1.0/25.0 carrying siLuci significantly reduced luciferase expression, while treatment with PBS, free siLuci or NP0.2/1.0/25.0 carrying siN.C. did not downregulate luciferase expression, while in fact the expression of luciferase was slightly elevated in these groups, likely due to the differentiation of HepG2-luciferase cells inoculated into the liver. To further reveal the potential of such a delivery system in cancer therapy, we examined the anti-tumor growth effect of treatment with NP0.2/1.0/25.0 carrying siPlk1 in a murine tumor xenograft model. The MDA-MB-435s tumor-bearing mice received an i.v. injection of NP0.2/1.0/25.0 carrying siPlk1 once every other day at a siPlk1 dose of 20 μg per mouse per injection. As indicated in Fig. 9, treatment of tumor-bearing mice with free siPlk1 (20 μg siRNA), blank nanoparticles NP0.0/1.0/25.0, or NP0.2/1.0/25.0 carrying siN.C. (20 μg siRNA) could not inhibit tumor growth in comparison with PBS-treated mice. However, tumor growth was significantly hindered by the i.v. injection of NP0.2/1.0/25.0 carrying siPlk1, indicating that the antitumor effect is sequence-specific and that such a delivery system can be used in cancer therapy by delivering a siRNA targeting an oncogene.

4. Conclusions We have developed a novel nanoparticular system for the systemic delivery of siRNA, using a biocompatible and biodegradable mPEGPLA copolymer, with the assistance of an amphiphilic and cationic lipid. Such nanoparticles exhibit high efficiency of siRNA encapsulation. It has been demonstrated that the siRNA-encapsulated nanoparticles can successfully enter cells and escape from endosomes, which in turn leads to remarkable and specific gene knockdown efficiency in cancer cells. Moreover, such a delivery system has demonstrated the ability to down-regulate gene expression in an orthotopic liver tumor model and to inhibit the tumor growth in a MDA-MB-435s xenograft tumor model following systemic administration, suggesting its potential for siRNA delivery in cancer therapy.

Fig. 9. Inhibition of tumor growth in a MDA-MB-435s xenograft murine model following i.v. injection of siPlk1-encapsulated nanoparticles. Free siPlk1 represent that mouse was injected with siPlk1 at a dose of 20 μg per mouse per injection. NP0.2/1.0/25.0, NP0.2/1.0/25.0/siN.C. and NP0.2/1.0/25.0/siPlk1 represent that mice were administered empty nanoparticles, NP0.2/1.0/25.0 encapsulating siN.C. (NP0.2/1.0/25.0/siN.C.) and siPlk1 (NP0.2/1.0/25.0/siPlk1) at a siRNA dose of 20 μg per mouse per injection, respectively. The dose of polymer was 2.745 mg per mouse per injection when applied. The mice received one injection every two days.

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