Effective Delivery of siRNA into Cancer Cells and Tumors Using Well

Apr 23, 2013 - Tumour Biology and Targeting Program, Children's Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales...
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Article pubs.acs.org/molecularpharmaceutics

Effective Delivery of siRNA into Cancer Cells and Tumors Using WellDefined Biodegradable Cationic Star Polymers Cyrille Boyer,∥,‡ Joann Teo,∥,†,‡ Phoebe Phillips,∥,§ Rafael B. Erlich,†,‡ Sharon Sagnella,†,‡ George Sharbeen,§ Tanya Dwarte,† Hien T. T. Duong,‡ David Goldstein,§ Thomas P. Davis,*,†,‡ Maria Kavallaris,*,†,‡ and Joshua McCarroll*,†,‡ †

Tumour Biology and Targeting Program, Children’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, NSW, Australia ‡ Australian Centre for Nanomedicine, School of Chemical Engineering, University of New South Wales, NSW, Australia § Pancreatic Cancer Translational Research Group, School of Medical Sciences, University of New South Wales, NSW, Australia

ABSTRACT: Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition−fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer−siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases. KEYWORDS: controlled radical polymerization, self-assembling biodegradable nanoparticles, siRNA, cancer cells



INTRODUCTION Despite improvements in our understanding of cancer and the rapidly emerging concept of personalized medicine, it is still a major cause of death worldwide. Resistance to conventional chemotherapy and/or radiotherapy and the presence of local and distant metastases are often associated with a poor survival outcome. This scenario is most evident in both lung and pancreatic cancer. Lung cancer is currently the most common © 2013 American Chemical Society

cause of cancer death worldwide, while pancreatic cancer is the fourth most common cause of cancer death in the western world.1,2 Hence, there is an urgent need to develop novel Received: Revised: Accepted: Published: 2435

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Molecular Pharmaceutics



therapies which specifically target and silence key genes involved in regulating both chemotherapy resistance and metastases. RNA interference (RNAi) is a naturally occurring gene silencing mechanism which holds great potential for the development of a new class of therapeutics for the treatment of cancer.3 This process utilizes small double-stranded RNA (siRNA) to silence target genes with a high level of potency and specificity.4 Moreover, genes which are not traditionally amenable to inhibition using chemical inhibitors can be targeted by siRNA.4 However, before siRNA can become a reality in the clinic, there are a number of biological challenges that need to be addressed. First, siRNA cannot enter a cell by itself and requires a vehicle and, second, the vehicle needs to be nontoxic and to be able to deliver siRNA with a high level of efficiency to allow for its release so it can interact with the RNAi machinery.5 To overcome these challenges, there has been intense research into the development of nonviral nanoparticles as delivery vehicles for siRNA. To date, lipidbased nanoparticles have been the most widely used agents to deliver siRNA. However, despite progress in the development of siRNA delivery vehicles, new chemistry which allows for easy and highly efficient complexation of siRNA is still urgently required. In addition, nanoparticles designed for siRNA delivery to tumor cells in an in vivo setting should ideally be uniformly small in size (approximately 100 nm) to enable them to take advantage of the enhanced permeability and retention effect and be passively taken up by the tumor cells as well as have the capacity to allow for targeting moieties to be conjugated to their surface.6 Different polymeric structures have been developed for the delivery of siRNA.7−11 One group of polymers which have shown great promise are cationic star polymers.7 Indeed, star polymers possess singular properties, originating from their multiarmed structures, leading to unique solution behavior and enhanced cell uptake.12 Star polymers have been used in nanomedicine for the delivery of chemotherapy agents, nitric oxide,13 catalysis,14,15 and in emulsion.16 More recently, starshaped nanoparticles have also been used to deliver siRNA to a number of different cells types.7,8 Well-defined star polymers can be prepared using several different methodologies. For example, the core first approach is a process whereby the arm is grown out from a functional core, while the arm first approach involves polymers being linked together using a cross-linker. The arm first approach presents several advantages. Indeed, it is easier to achieve higher molecular weight star polymers as well as limit the formation of coupling star structures. However, this method can also result in the formation of broad polydisperse star polymers when reversible addition−fragmentation transfer polymerization (RAFT) is employed.17 Recent studies have demonstrated the synthesis of well-defined poly(ethylene glycol) methyl acrylate star polymers using RAFT.18,19 Our laboratory has recently demonstrated that star polymers can deliver chemotherapy agents with high levels of efficacy to cancer cells.20 Therefore, in this study, we set out to design and synthesize cationic star polymers using dimethylaminoethyl methacrylate (DMAE-MA) as a monomer and RAFT polymerization to yield well-defined nanoparticles which uniformly selfassemble with siRNA and silence target genes in cancer cells both in vitro and in vivo.

Article

EXPERIMENTAL SECTION

Materials. Dimethylaminoethyl methacrylate (DMAE-MA, 99%, Sigma-Aldrich) was deinhibited by passing through a basic alumina column. The initiator, 2,2′-azobisisobutyronitrile (AIBN), was crystallized twice from methanol. High purity N2 (Linde gases) was used for reaction solution purging. All other chemical reactants as well as both firefly luciferase (Luc2) and green fluorescent protein (GFP) forward and reverse realtime PCR primers were purchased from Sigma-Aldrich. All reagents were supplied at the highest purity available. All nonmodified, double-stranded 21-nucleotide siRNAs were purchased from Thermo Fisher Scientific (Lafayette, CO, USA). The siRNAs were solubilized in X1 siRNA buffer (ThermoFisher Scientific, Lafayette, CO, USA) to a 20 μM (in vitro) or 250 μM (in vivo) concentration following the manufacturer’s resuspension protocol. Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) or Roswell Park Memorial Institute medium (RPMI, Invitrogen), heat inactivated fetal calf serum (FCS), and 2 mM L-glutamine were purchased from SAFC Biosciences (Lenexa, KS, USA). Cell culture flasks, plates, and Trypsin-EDTA were purchased from Thermo Fisher Scientific. Lipofectamine 2000 was purchased from Invitrogen. Colorimetric cell viability assay kit was purchased from Dojindo. Power SYBR green PCR master mix and high capacity cDNA reverse transcription kits used in real-time PCR experiments were purchased from Applied Biosystems. β2-Microglobulin Quantitect primer assay was purchased from Qiagen (Valencia, CA, USA). The PGL4-Luc2 plasmid construct and luciferase assay system used to measure luc2 expression was purchased from Promega (Madison, WI, USA). BCA protein assay kit and Rabbit polyclonal antibody against GFP were purchased from Pierce and Cell Signaling Technology (Danvers, MA, USA), respectively. Methods. Synthesis of 4-Cyanopentanoic Acid Dithiobenzoate (RAFT Agent). Synthesis of Dithiobenzoic Acid (DTBA). Sodium methoxide (30% solution in methanol) was added to a thoroughly dried 1 L, three-necked round-bottomed flask equipped with a magnetic stir bar, addition funnel (250 mL), thermometer, and rubber septum for liquid transfers. Anhydrous methanol (250 mL) was added to the flask via a cannula, followed by rapid addition of elemental sulfur (32 g, 1.0 mol). Benzyl chloride (63 g, 0.5 mol) was then added dropwise via the addition funnel over a period of 1 h at room temperature under a dry nitrogen atmosphere. The reaction mixture was heated in an oil bath at 67 °C for 10 h. After this time, the reaction mixture was cooled to 0 °C using an ice bath. The precipitated salt was removed by filtration, and the solvent was removed in vacuum. Deionized water (500 mL) was then added to the residue. The solution was filtered a second time and transferred to a 2 L extraction funnel. The crude sodium dithiobenzoate solution was washed with diethyl ether (3 × 200 mL). Diethyl ether (200 mL) and 1.0 N HCl (500 mL) were added, and dithiobenzoic acid was extracted into the ethereal layer. Deionized water (300 mL) and 1.0 N NaOH (600 mL) were added, and sodium dithiobenzoate was extracted to the aqueous layer. This washing process was repeated two more times to finally yield a solution of sodium dithiobenzoate. Synthesis of Di(thiobenzoyl) Disulfide. Potassium ferricyanide(III) (32.93 g, 0.1 mol) was dissolved in deionized water (500 mL). Sodium dithiobenzoate solution (350 mL) was transferred to a 1 L conical flask equipped with a magnetic stir bar. Potassium ferricyanide solution was added dropwise to 2436

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finally freeze-dried to yield cationic star polymers. The purified star polymer was analyzed by GPC (Mn,GPC = 155000 g/mol and PDI = 1.09). Gel Permeation Chromatography (GPC) Measurements. DMAc GPC analyses of the polymers were performed in N,N-dimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% 2,6dibutyl-4-methylphenol (BHT)] at 50 °C (flow rate = 1 mL·min−1) using a Shimadzu modular system comprised of an SIL-10AD autoinjector, a PL 5.0-mm bead-size guard column (50 mm × 7.8 mm), followed by four linear PL (Styragel) columns (105, 104, 103, and 500 Å) . A RID-10A differential refractive-index detector was used. Calibration was achieved with commercial polystyrene standards ranging from 500 to 106 g/mol. Nuclear Magnetic Resonance (NMR). Structures of the synthesized compounds were analyzed by NMR spectroscopy using a Bruker DPX 300 spectrometer at 300 MHz for hydrogen nuclei. UV−Visible Spectroscopy. UV−visible spectra were recorded using a CARY 300 spectrophotometer (Bruker) equipped with a temperature controller. Dynamic Light Scattering (DLS). DLS measurements were performed using a Malvern Zetasizer Nano series running DTS software and using a 4 mW He−Ne laser operating at a wavelength of 633 nm and an avalanche photodiode (APD) detector. The scattered light was detected at an angle of 173°. The temperature was stabilized to ±0.1 °C of the set temperature. To reduce the influence of larger aggregates, the number-average hydrodynamic particle size is reported. The polydispersity index (PDI) is used to describe the width of the particle size distribution, as calculated from the DTS software using a Cumulants analysis of the measured intensity autocorrelation function; it is related to the standard deviation of the hypothetical Gaussian distribution (i.e., PDI = s2/ZD2, where s is the standard deviation and ZD is the Z average mean size). Zeta Potential Measurement. The particle ζ potential was measured by means of electrophoretic mobility using a Malvern Zetasizer Nano series. A particle concentration of 15 μM of siRNA was used. The ζ potential values have been measured in triplicate for two different samples (star polymer alone and star polymer complexed to siRNA). Transmission Electron Microscopy (TEM). The sizes and morphologies of the star polymers were observed using a transmission electron microscopy JEOL1400 TEM at an accelerating voltage of 100 kV. The particles were dispersed in water (0.0125 mg/mL) and deposited onto 200 mesh, holey film, copper grid (ProSciTech). The sample was treated using positive staining agent by application of osmium tetroxide vapor. siRNA Binding and Gel Retardation Assay. The ability of the star polymer to complex siRNA was assessed by using a gel retardation assay. Briefly, increasing amounts of the star polymer were complexed to a fixed amount of siRNA (60 ng) in OptiMEM I reduced serum culture medium for 20 min at room temperature. The complexes were then electrophoresed on a 2% (w/v) agarose gel containing the nucleic acid gel stain GelRed for 30 min. The gel was then visualized using the BioRad Gel Doc System. Cell Culture. The human pancreatic cancer cell line (MiaPaCa-2) was generated to stably express the firefly luciferase 2 (Luc2) gene using the PGL4-Luc2 reporter construct (Promega, Inc.) according to the manufacturer’s

the sodium dithiobenzoate via an addition funnel over a period of 1 h under vigorous stirring. The red precipitate was filtered and washed with deionized water until the washings became colorless. The solid was dried in vacuum at room temperature overnight. Synthesis of 4-Cyanopentanoic Acid Dithiobenzoate. Distilled ethyl acetate (80 mL) was added to a 250 mL round-bottomed flask. Dry 4,4-azobis(4-cyanopentanoic acid) (5.84 g, 21.0 mmol) and di(thiobenzoyl) disulfide (4.25 g, 14.0 mmol) were added to the flask. The reaction solution was heated at 75 °C for 14 h. The ethyl acetate was removed in vacuum. The crude product was isolated by column chromatography (silicagel 60 Å, 70−230 mesh) using ethyl acetate:hexane (2:3) as eluent. Fractions that were red in color were combined and dried over anhydrous sodium sulfate overnight. The solvent mixture was removed in vacuum, and the red oily residue placed in a freezer at −20 °C, whereupon it crystallized. The target compound was recrystallized from benzene. 1H NMR confirmed the structure expected. 1H NMR (300 MHz, CDCl3), δ (ppm from TMS): 2.1 (3H, s, CH3), 2.5 (2H, dt, −CH2-CH2CO2H), 2.7 (2H, t, −CH2-COOH), 7.2 (2H, aromatic group), 7.6 (1H, aromatic group), 7.8 ppm (2H, aromatic group). 13C NMR (75 MHz, CDCl3) δ (ppm from TMS): 224, 175, 145, 135, 130, 128, 126, 120, 45, 35, 32, 25. Synthesis of the Poly(DMAE-MA) Arm Homopolymer. Briefly, dimethylaminoethyl methacrylate (DMAE-MA, 15.7 g, 0.1 mol), 2,2′-azobisisobutyronitrile (AIBN), 4-cyanopentanoic acid dithiobenzoate (CPAD, 0.310 g, 1.12 × 10−3 mol), and dry toluene (50 mL) were placed into a round-bottom flask (100 mL), equipped with a magnetic stir bar. The cooled reaction mixture was degassed using nitrogen at 0 °C for 1 h. The degassed solution was stirred at 65 °C for 14 h, after which the reaction was quenched and an aliquot was sampled for GPC and 1H NMR analyses. The monomer conversion was determined to be around 70%. The reaction medium was concentrated using rotary evaporation and the polymer purified via dialysis against methanol to remove any traces of monomer (molecular weight membrane cut off 3500 Da) and then precipitated in petroleum ether. The purified poly(DMAEMA) was analyzed by UV−visible spectroscopy, NMR, and GPC. The molecular weight calculated by UV−visible (Mn,UV−visible = 10500 g/mol) and NMR spectroscopy (Mn,NMR = 10200 g/ mol) are close to the theoretical value (Mn,theor = 9700 g/mol). However, the molecular weight assessed by GPC is slightly higher (Mn,GPC = 12500 g/mol, PDI = 1.15). The difference between the theoretical and GPC values is attributed to the poly(styrene) calibration. Synthesis of Poly(DMAE-MA) Star via the Arm First Methodology. Poly(DMAE-MA) (10.2 g, 1 mmol) was introduced into a vial equipped with a magnetic stirrer, together with AIBN (33 mg, 0.2 mmol) and toluene (50 mL). Crosslinker (N,N′-bis(acryloyl)cystamine) (2.1 g, 8 mM) and DMAE-MA (1.3 g, 8.3 mmol) were added, and the vials were sealed and purged with nitrogen for 1 h at 0 °C. The reaction vials were then placed in an oil bath at 70 °C for 24 h. Following polymerization, the polymers were sampled for 1H NMR and GPC analyses. 1H NMR analysis confirms the full conversion of cross-linker and DMAE-MA. The arm incorporation was determined to be around 80%. Star polymers were precipitated in petroleum ether/diethyl ether mixture (80/20 v/v) to remove residual arms. Star polymers were then dissolved in methanol and dialyzed against water/HCl (pH = 3.0) for 24 h and then against water for 24 h. The solution was 2437

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post-transfection, the cells were fixed using fresh 4% paraformaldehyde and the slides were mounted with VECTASHIELD antifade mounting media with DAPI. siRNA uptake was then visualized using an Olympus FluoView FV1000 confocal microscope. Real-Time Quantitative PCR (RT-qPCR) Analysis. The expression of Luc2 and GFP mRNA in MiaPaCa-2 and H460 cells was examined using RT-qPCR. Total RNA was isolated as previously described.22,23 RT-qPCR was performed using the Applied Biosystems high capacity cDNA reverse transcription and SYBR green kits. The primers for Luc2 and GFP mRNA were as follows: Luc2, forward 5′-GCTCAGCAAGGAGGTAGGTG-3′, reverse 5′-TCTTACCGGTGTCCAAGTCC-3′, and GFP, forward 5′-ATGGTGAGCAAGGGCGAGGA-3′, reverse 5′-ACTTGTGCCCGTTTACGTCGC-3′. All data were normalized to the housekeeping genes β2-microglobulin and 18S (β2-Microglobulin and 18S QuantiTect Primer Assays, Qiagen). Western Blotting. Cells were harvested and solubilized in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors on ice. Equal amounts of whole cell lysates were loaded onto 10% SDS PAGE gels, and GFP protein expression was determined as described previously.22 Luminescence Measurement. MiaPaCa-2-Luc 2 expressing cells were seeded into 6-well plates and transfected with star polymer−siRNA complexes targeting the Luc2 gene. Then 24 h after transfection, cells were harvested and lysed and equal amounts of protein plated directly into 96-well tissue culture plates. Luciferase expression was measured using the luciferase assay system (Promega, Inc.) according to the manufacturer’s instructions. All data were normalized to total protein content. In Vivo Mouse Tumor Model. Female Balb/c nude mice (6−8 weeks of age) were obtained from the Animal Resource Center at the University of New South Wales (Sydney, New South Wales, Australia) and maintained under specific pathogen-free conditions for the studies. All animal experiments were approved by the Animal Ethics Committee, University of New South Wales (ACEC no. 12/9B). Mice were inoculated subcutaneously into the flank with H460-GFP expressing NSCLC cells (1 × 106) as described previously.23 To assess siRNA uptake in vivo, mice were injected intratumorally (once tumors reached 200 mm3) with Alexa Fluor-647 labeled siRNA (20 μg) alone or when complexed to the star polymer. Then 24 h postinjection, mice were sacrificed and tumor tissue harvested and snap-frozen in OCT embedding media. Frozen sections (5 μm) of the tumor tissue were then placed onto saline-coated histological slides and mounted with VECTASHIELD antifade mounting media with DAPI. siRNA uptake was visualized using an Olympus FluoView FV1000 confocal microscope. To determine gene silencing efficiency, mice were randomized into two treatment groups (three mice per group). One group was treated with the star polymer complexed to GFP siRNA, while the control group was treated with the star polymer complexed to a commercially available nonsilencing (control) siRNA. Mice were injected three times (every second day) intratumorally with 20 μg (approximately 1 mg/kg) siRNA. Then 48 h after the final injection, mice were sacrificed and tumor tissue was harvested for RNA analysis and GFP mRNA levels were assessed by RT-qPCR. Statistics. Statistical analyses were performed using the GraphPad Prism program. Results are expressed as means of at least three independent experiments ± standard error of the

instructions. Cells were grown in DMEM culture medium containing 10% fetal bovine serum (FCS), 2% horse serum, 2 mM of L-glutamine, and the selection antibiotic hygromyocin (200 μg/mL) to enrich a population of cells that had retained the PGL4-Luc2 plasmid. Then 12−15 individual colonies were selected and examined for luciferase expression using a Victor2 plate reader (Perkin-Elmer, Waltham MA). The clone which contained the highest expression of Luc2 was used for this study. The H460 nonsmall cell lung cancer (NSCLC) cell line which stably expresses enhanced green fluorescent protein (GFP) as described previously21 was maintained in RPMI containing 10% FCS and 2 mM of L-glutamine. Both cell lines were grown at 37 °C in a humidified atmosphere with 5% CO2 and were routinely screened and found to be free of mycoplasma. Toxicity Analysis. To assess the toxicity of the star polymer, MiaPaCa-2 and H460 cells were plated into 6-well tissue culture plates for 16 h before transfection. The following day, cells were treated with the star polymer alone or when complexed to nonsilencing (control) siRNA [8:1−10:1 (w/ w)]. Then 24 h post-treatment, cell viability was measured by direct counting of viable and nonviable cells using the trypan blue stain or by using a colorimetric cell viability assay (Dojindo Cell Counting Kit) according to the manufacturer’s instructions. Small-Interfering RNA (siRNA) Transfection. To transfect cells with siRNA complexed to the star polymer, cells were plated into 6-well tissue culture plates (1.5 × 105 for MiaPaCa2; 6 × 104 for H460) for 16 h before transfection. The following day cells were transfected with the star polymer− siRNA complexes [8:1−10:1 (w/w)] with siRNA directed against Luc2, sense strand 5′-GCUAUGGGCUGAAUACAAAUUUU-3′, antisense 5′-AAUUUGUAUUCAGCCCAUAGCUU-3′; or GFP, sense strand 5′-GCAAGCUGACCCUGAAGUUCAU-3′, antisense 5′-GAACUUCAGGGUCAGCUUGCCG-3′ (the final concentration of all siRNA for these experiments was 100 nM). Cells treated with a commercially available nonsilencing siRNA (negative control) or with siRNA against GFP or Luc2 complexed to the commercial transfection reagent lipofectamine 2000 (L2K) (positive control) served as controls. Total RNA and whole cell lysates were collected 24− 72 h post-treatment, and Luc2 and GFP mRNA and protein levels were assessed using real-time PCR, Western blotting, and luminescence assays. Intracellular Cell Uptake. To measure the cell uptake of the star polymer−siRNA complexes in both pancreatic and lung cancer cells, MiaPaCa-2 and H460 cells were plated into 6well tissue culture plates as described above, and the following day, cells were transfected with the star polymer complexed to siRNA labeled with Alexa Fluor-488 for 4 h at 37 °C. Then 24 h post-transfection, the cells were washed three times in warm phosphate buffered saline (PBS) and harvested. The fluorescent intensity within the cells was measured using the Becton and Dickenson Canto flow cytometer. Cells treated with Alexa Fluor-488 siRNA alone or complexed to lipofectamine 2000 served as controls. To observe the cellular distribution of the star polymer−siRNA complexes, MiaPaCa-2 and H460 cells were plated into 4-well Nunc Lab-Tek tissue culture chamber slides. The following day, cells were transfected with the star polymer complexed to Alexa Fluor-488-labeled siRNA (MiaPaCa-2 cells) or Alexa Fluor-647-labeled siRNA (H460 cells). Cells transfected with fluorescent siRNA alone or siRNA complexed to lipofectamine 2000 served as controls. Then 24 h 2438

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Figure 1. Synthesis of poly(DMAE-MA) biodegradable cationic star polymers. (A) A schematic diagram showing the synthesis steps required to produce the biodegradable cationic star polymer. (B) H NMR spectrum of the star polymer (recorded in D2O). (C) GPC trace confirming the formation of the star polymer.

mean (SEM) (error bars). Unpaired two-tailed Student’s t tests were used to determine the statistical differences between various experimental and control groups, with p < 0.05 considered statistically significant.

groups at 7.2−7.6 ppm. The molecular weight calculated by NMR using the following equation: [Mn, NMR = MWDMAE‑MA × (I4.2 ppm/I7.2−7.4 ppm)) + MWRAFT, with I4.2 and I7.2−7.4 ppm corresponding to the intensity of CH2O (from DMAE-MA) and CH of the benzyl ring (RAFT end group)] is in good agreement with the theoretical values. However, a slight difference between GPC and NMR values was observed. This difference is most likely explained by the use of the polystyrene calibration. Subsequently, the linear polymers were chain extended in the presence of a cross-linking molecule (N,N′bis(acryloyl)cystamine), AIBN, and DMAE-MA, with a [RAFT end group]:[AIBN]:[DMAE-MA]:[cross-linker] ratio of 1.0:0.3:X:8.0, in toluene for 24 h. X was varied from 4 to 16. For X equal to 4−12, we obtained soluble star polymers in toluene. However, for X = 16, we observed the formation of a gel (data not shown). After the star formation, the crude star polymers were analyzed by GPC and 1H NMR spectroscopy to determine the arm incorporation efficiency and monomer conversions. Full DMAE-MA and cross-linker conversions were observed by 1H NMR spectroscopy using the signal between 5.0 and 6.5 ppm originating from acrylamide cross-linker and methacrylate groups (Figure 1B). GPC confirmed the formation of star polymers with a very low PDI (1.15, Mn = 155 000 g/mol) for X = 10 (Figure 1C). For X < 10, the arm incorporation is lower than X = 10, while X > 12 results by the formation of broad star polymers (PDI > 1.5). For the remainder of the study, we decided to use star polymers



RESULTS AND DISCUSSION Herein, we describe for the first time the characterization and synthesis of a biodegradable cationic star polymer using RAFT polymerization which is able to self-assemble with siRNA via a simple and quick process to form uniform and well-defined nanoparticles which can efficiently deliver siRNA to both pancreatic and lung cancer cells. Synthesis and Characterization of the Star Polymer. The synthesis of star polymer is depicted in Figure 1A. First, the poly(DMAE-MA) arm was synthesized via RAFT polymerization controlled by 4-cyanopentanoic acid dithiobenzoate as the RAFT agent and AIBN as initiator at 60 °C in toluene overnight. The polymerization was stopped at 70% monomer conversion to limit the formation of dead polymers. After purification by precipitation, the RAFT end group was determined to be greater than 95% by UV−visible spectroscopy, using the following equation: RAFT end-group f unctionality = (Abs305·l/ε305)/[Polymer]0), where Abs305, l, ε305, and [Polymer]0 correspond to absorbance, cuvette path-length, extinction coefficient at 305 nm, and polymer concentration, respectively (data not shown). 1H NMR analyses confirmed the presence of RAFT end-groups by the signals of the benzyl 2439

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Figure 2. Physiochemical properties of the star polymer. (A) A representative agarose gel showing a fixed concentration of free unmodified siRNA (60 ng) alone (lane 1) or when complexed to increasing amounts of the star polymer (w/w ratio with siRNA) (lanes 2−10) (n = 3 individual experiments). (B) Transmission electron microscopy (TEM) of the star polymer alone (a) and after complexing siRNA (b). Dynamic light scattering (DLS) graph of the star polymers alone (blue line) and after complexing siRNA (red line). (C) A representative graph demonstrating cell viability of MiaPaCa-2 pancreatic cancer cells (black columns) and H460 nonsmall cell lung cancer (NSCLC) cells (white columns) when incubated in culture medium alone (control), star polymer alone [8:1−10:1 (w/w) ratio with siRNA (100 nM)] or star polymer complexed to siRNA [8:1− 10:1 (w/w) ratio with siRNA (100 nM)]. Cell viability was measured 24 h post-transfection and was expressed as a percent of control (no transfection). Each value represents the mean ± standard deviation of triplicate wells (n = 3−5 individual experiments).

obtained with X = 10. The number of arms per star polymer was calculated using the molecular weight assessed by DLS (MW = 260 000 g/mol). For X = 10, the star is constituted by 20 arms. Physiochemical Characteristics of the Star Polymer. To examine the physiochemical properties of the star polymers, they were freeze-dried and then dispersed in water. The star polymers were readily soluble and stable in water. Next, we determined its ability to self-assemble with siRNA (electrostatic interaction) via a simple and quick mixing process (10−20 min) using agarose gel electrophoresis. As expected, naked unmodified siRNA migrated to the bottom of the gel due to its small size and net negative charge (Figure 2A). In contrast, increasing amounts of the star polymer complexed to a fixed amount of siRNA completely prevented its migration thereby, indicating that the star polymer was able to fully complex with the siRNA (Figure 2A). It was apparent from these results that an 8:1−10:1 (w/w) ratio was optimal for fully complexing siRNA. To confirm that the star polymer was cationic, we measured the ζ potential of the star polymer alone and when complexed to siRNA. As expected, the star polymer was positively charged with a charge of 50 mV (±2 mV). However, after complexation with siRNA, the ζ potential was reduced to 29 (±2 mV) and is in agreement with a mechanism of

association of the siRNA with the star polymer via an electrostatic interaction. An important feature for efficient delivery of siRNA (both in vitro and in vivo) when complexed to nanoparticles via electrostatic interactions is its overall size. To observe the morphology and size of the star polymer alone and when complexed to siRNA, we performed TEM and DLS. The star polymer alone was readily dispersed and formed welldefined spherical nanoparticles (Figure 2B, panel a). DLS also confirmed the presence of well-defined nanoparticles with an overall size of 40 nm (Figure 2B). Finally, we examined the shape, dispersity, and the size of the nanoparticle when fully complexed with siRNA (8:1 w/w ratio). Importantly, the shape and size of the nanoparticle−siRNA complex did not significantly change (DLS, 35 nm) and small uniform nanoparticle−siRNA complexes were observed (Figure 2B, panel b). It is to be noted that the size of our siRNA−star polymer complexes are smaller than other cationic polymer− siRNA systems which complex siRNA by an electrostatic interaction.24,25 This is an important feature of our system as it is an ideal size (60%) 24 h post-transfection when compared to cells treated with nonsilencing (control) siRNA (100 nM) (Figure 4B). To assess whether the star polymer could also deliver active siRNA to other cancer cell types, H460 NSCLC cells stably expressing high levels of GFP were also examined. Importantly, the star polymer complexed to GFP siRNA (100 nM) potently suppressed GFP mRNA expression by greater than 80% 48 h post-transfection when compared to cells transfected with star polymer alone (results not shown) or when complexed to nonsilencing control siRNA (100 nM) (Figure 5A). Moreover, a significant decrease at the protein level (72 h posttransfection) was also observed (Figure 5B). Notably, the ability of the star polymer to deliver siRNA to its target in both cancer cell lines were not too dissimilar to the commercially available transfection reagent lipofectamine 2000 (L2K) (Figures 4 and 5). Finally, to determine whether this first-generation star polymer could deliver siRNA to tumor cells and induce specific gene silencing activity in vivo, mice were injected subcutaneously into the flank with H460 NSCLC cells stably expressing high levels of GFP. For this series of experiments, we chose to deliver the star polymer−siRNA complex intratumorally given that the unmodified star polymers could be potentially taken up by the reticuloendothelial system if administered systemically. To assess cell uptake, tumors were allowed to grow until they reached 200 mm3 (approximately 9 days postinjection). Mice were then injected intratumorally with siRNA (20 μg) labeled with a near-infrared dye (Alexa Fluor-647) alone or when complexed to the star polymer [10:1 w/w)]. Then 24 h postinjection, tumor tissue was collected and siRNA uptake assessed by confocal microscopy. As expected, tumors injected with siRNA alone showed no cell uptake (Figure 6A). In contrast, tumors injected with fluorescent siRNA complexed to the star polymer demonstrated a high

level of cell uptake into the tumor cells. To confirm that the star polymer could also deliver active siRNA and silence a target gene in vivo, we injected tumors with siRNA against GFP complexed to the star polymer. GFP is stably expressed at high levels in our lung cancer cell line and serves as a good gene marker to measure the gene-silencing activity of the star polymer−siRNA complexes. Mice were administered intratumorally three times (every second day) with siRNA (20 μg). Then 48 h after the final injection, tumor tissue was collected and GFP mRNA levels measured by RT-qPCR. Star polymer− siRNA complexes were able to significantly silence GFP mRNA expression by 50% when compared to mice injected with the star polymer complexed to nonsilencing control siRNA (Figure 6B). This result confirms that the decrease in GFP mRNA expression is due to RNAi activity within the tumor cells and not a nonspecific effect of the star polymer. Furthermore, the three treatments with the star polymer−siRNA complexes did not affect total mouse body weight and/or gross anatomical appearance (results not shown). Collectively, these results highlight the potential of star polymers to deliver siRNA and induce RNAi activity in tumor cells in vivo and maybe ideal for local administration of siRNA to cancer cells. Further investigations in our laboratory are examining their potential when administered systemically. For these studies, we are currently assessing the biodistribution of the star polymers with poly(ethylene glycol) (PEG) and cancer-cell targeting moieties attached to their surface in clinically relevant orthotopic pancreatic and lung cancer mouse models.



CONCLUSIONS In this study, we have designed and synthesized cationic biodegradable star polymers using DMAEMA as the monomer and RAFT polymerization to produce well-defined star polymers which are soluble and stable in water. The polymers contained tertiary amino groups along the polymer arms, which allowed for rapid and efficient self-assembly with siRNA. The star polymer−siRNA complexes were small and uniform in size and were nontoxic in both pancreatic and lung cancer cells. Moreover, the star polymer efficiently delivered siRNA to silence target genes in both cancer cell types. Finally, the star polymer was able to deliver active siRNA and silence its target gene in an in vivo mouse tumor model. Future studies are ongoing in our laboratory using star polymers to deliver therapeutic siRNA as a novel treatment strategy to silence key genes involved in regulating chemotherapy drug sensitivity and tumor growth in preclinical cancer mouse models.



AUTHOR INFORMATION

Corresponding Author

*Phone: 612-9885-1556 (J.M.). E-mail: [email protected]. edu.au (J.M.); [email protected] (M.K.); t.davis@ unsw.edu.au (T.P.D.). For J.M. and M.K.: Tumour Biology and Targeting Program, Children’s Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, NSW, Australia; Australian Centre for Nanomedicine, University of New South Wales, NSW Australia. For T.P.D.: Australian Centre for Nanomedicine, University of New South Wales, NSW Australia. Author Contributions ∥

Authors contributed equally to this work.

Notes

The authors declare no competing financial interest. 2443

dx.doi.org/10.1021/mp400049e | Mol. Pharmaceutics 2013, 10, 2435−2444

Molecular Pharmaceutics



Article

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ACKNOWLEDGMENTS P.P., M.K., D.G., and J.M. are supported by grants from the National Health and Medical Research Council (NHMRC), Cancer Council New South Wales (MK), Cure Cancer Australia Foundation Grant (P.P., J.M.), Balnaves Young Researcher Award (J.M., S.G.), Cancer Institute New South Wales Career Development Fellowship (J.M.), NHMRC Career Development Fellowship (P.P.), and NHMRC Senior Research Fellowship (M.K.). J.T. is supported by a UNSW International Postgraduate Research Scholarship. C.B. and T.D. are thankful for funding from Australian Research Council (ARC, DP110104251), and C.B. holds an ARC-Future Fellowship (FT 120100096) and ARC-APD Fellowship. We also acknowledge the DVCR, Professor Les Field, at UNSW, for significant strategic funding to set up the Australian Centre for Nanomedicine.



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dx.doi.org/10.1021/mp400049e | Mol. Pharmaceutics 2013, 10, 2435−2444