A Rationally Optimized Nanoparticle System for the Delivery of RNA

Article pubs.acs.org/Biomac

A Rationally Optimized Nanoparticle System for the Delivery of RNA Interference Therapeutics into Pancreatic Tumors in Vivo Joann Teo,†,‡,◆ Joshua A. McCarroll,†,‡,◆ Cyrille Boyer,‡,§ Janet Youkhana,∥ Sharon M. Sagnella,†,‡ Hien T. T. Duong,‡,§ Jie Liu,∥ George Sharbeen,∥ David Goldstein,∥,⊥ Thomas P. Davis,∇,○ Maria Kavallaris,*,†,‡,# and Phoebe A. Phillips*,‡,∥

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†

Tumour Biology and Targeting Program, Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, Sydney, New South Wales 2052, Australia ‡ Australian Centre for NanoMedicine, UNSW Australia, Sydney, New South Wales 2052, Australia § Centre for Advanced Macromolecular Design, School of Chemical Engineering, UNSW Australia, Sydney, New South Wales 2052, Australia ∥ Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, Prince of Wales Clinical School, UNSW Australia, Sydney, New South Wales 2052, Australia ⊥ Prince of Wales Hospital, Prince of Wales Clinical School, Sydney, New South Wales 2052, Australia # ARC Centre of Excellence in Convergent Bio-Nano Science and Technology UNSW Australia, Sydney, New South Wales 2052, Australia ∇ ARC Centre of Excellence in Convergent Bio-Nano Science and Technology Monash Institute of Pharmaceutical Sciences, Monash University, Clayton, Victoria 3800, Australia ○ Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom S Supporting Information *

ABSTRACT: Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA (siRNA)-based therapeutics hold promise for the treatment of cancer. However, development of efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using reversible addition−fragmentation chain transfer polymerization (RAFT) for the delivery of siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths of cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-POEGMA polymers were nontoxic to normal cells and delivered siRNA with high efficiency to pancreatic cancer cells to silence a gene (TUBB3/βIII-tubulin) which is currently undruggable using chemical agents, and is involved in regulating tumor growth and metastases. Notably, systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to orthotopic pancreatic tumors in mice and silenced βIII-tubulin expression by 80% at the gene and protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors.

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INTRODUCTION

RNA interference (RNAi) is a powerful gene-silencing mechanism that occurs in mammals.2 In brief, this mechanism involves short-interfering RNA (siRNA) assembling within a multiprotein RNA induced silencing complex (RISC).2

Pancreatic cancer is ranked as the fourth leading cause of cancer-related deaths in Western societies, with a dismal 5-year survival rate of 6%.1 This poor prognosis is due to its resistance to chemotherapy drugs and aggressive growth/metastases. Hence, there is an urgent need to develop novel therapeutic strategies to treat this disease. © 2016 American Chemical Society

Received: February 4, 2016 Revised: June 14, 2016 Published: June 15, 2016 2337

DOI: 10.1021/acs.biomac.6b00185 Biomacromolecules 2016, 17, 2337−2351

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Biomacromolecules

demonstrated that star polymers can be designed to selfassemble DNA or siRNA and deliver it to different cell types in vitro. Cho et al.11,12 showed that star polymers synthesized using atom transfer radical copolymerization (ATRP) can complex siRNA or DNA and deliver them to different nontumor cell types in vitro. A study reported by Pafiti et al.15 showed that star polymers synthesized using “living” polymerization and containing dimethylaminoethyl methacrylate (DMAEMA) were able to deliver siRNA to mouse myoblasts and silence a gene with greater efficiency compared to linear DMAEMA homopolymers. However, while these studies provided proof-of-principle for the use of star-shaped nanoparticles as carriers for siRNA, they did not examine their ability to deliver siRNA and silence target gene expression in vivo. Moreover, there have been no studies to determine whether star polymers could be used as a delivery vehicle for siRNA to solid tumors in mouse models, which closely mimic the human setting. Recently, we reported on the design and synthesis of cationic star polymers using DMAEMA as the monomer via reversible addition−fragmentation chain transfer (RAFT) polymerization, which complexed siRNA resulting in nanoparticles with a size of ∼35 nm.18 These nanoparticle-siRNA complexes were able to deliver siRNA and silence target gene expression in lung and pancreatic cancer cells.18 We also showed that star polymers may be used for local (intratumoral) delivery of siRNA to silence gene expression in tumor cells in vivo.18 However, despite their clinical potential for local delivery of RNAi agents, most cancer therapeutics require systemic administration in an effort to target both primary and metastatic tumor sites.19 A limitation of highly charged cationic nanoparticles for systemic delivery of siRNA is their toxicity and potential to interact with serum proteins present in the bloodstream, which can lead to the formation of large aggregates and poor gene-silencing activity. To overcome this problem researchers have utilized PEGylation [incorporation of poly(ethylene glycol) (PEG) or poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA)] as a strategy to shield the positive charge of cationic nanoparticles, thereby reducing toxicity and masking serum protein aggregation.20,21 However, it is also recognized that PEGylation can impede siRNA gene silencing activity by preventing or retarding the ability of siRNA to complex with nanoparticles, as well as inhibit cellular uptake and endosomal escape within the cell.22 In this study, we describe the synthesis and characterization of a series of core cross-linked star polymers with various compositions. We demonstrate that starPOEGMA-siRNA nanocomplexes are nontoxic and act as highly effective delivery vehicles for siRNA to pancreatic cancer cells. We also show for the first time that star-POEGMA-siRNA nanoparticles accumulate at high levels in pancreatic tumors in mice and potently silence βIII-tubulin expression and decrease tumor growth.

Together, siRNA-RISC binds perfectly complementary to target mRNA, which results in its degradation and reduced translation.2 RNAi can be manipulated by the introduction of chemically synthesized siRNA to suppress genes that are involved in regulating human disease.3 An attractive feature for exploiting the therapeutic potential of RNAi is its ability to silence any gene, including those difficult to inhibit using chemical inhibitors.3−5 This has led to an intense research effort to design RNAi therapeutics for the treatment of many types of human disease, including cancer. Microtubules are cytoskeletal proteins that comprise α- and β-tubulin heterodimers which contribute to essential cellular processes such as maintenance of cell shape, intracellular transport and mitosis.6 β-tubulins’ have been extensively studied in cancer given they are the target site for tubulinbinding chemotherapy drugs such as taxanes, vinca alkaloids and epothilones.6 However, development of drug resistance and potential off-target toxicity has limited their efficacy.6 βtubulin has 7 different isotypes (βI, βII, βIII, βIVa, βIVb, βV and βVI), which share high sequence homology, encoded by different genes and are distinguished by their unique carboxy terminal tail.6 The different isotypes have tissue-specific expression.6 High βIII-tubulin (encoded by the TUBB3 gene) expression has been reported in different tumor types and is associated with poor patient survival and increased chemoresistance.6 Recently, we demonstrated that human pancreatic tumors and pancreatic cancer cells expressed high levels of βIIItubulin.7 Importantly, stable suppression of βIII-tubulin using shRNA in pancreatic cancer cells reduced tumor growth and metastases in a clinically relevant orthotopic pancreatic cancer xenograft mouse model.7 Moreover, inhibition of βIII-tubulin expression in pancreatic cancer cells increased their sensitivity to chemotherapy drugs in vitro.7 Despite the promise of βIIItubulin as a novel therapeutic target for pancreatic cancer, there are no pharmacological agents which can specifically inhibit βIII-tubulin and design of such inhibitors is problematic due to its high sequence homology with the other β-tubulin proteins. Therefore, a unique opportunity exists to develop siRNA-based therapies to selectively silence βIII-tubulin expression in pancreatic cancer cells. siRNA requires a delivery vehicle to protect it from degradation within the circulatory system and allow it to enter cells.3−5 To overcome this problem nanotechnology has been used to encapsulate or complex siRNA and deliver it to a host of different cell types.5 Nanoparticle-siRNA therapies are in clinical trial to treat a number of diseases and have been shown to be safe.8−10 However, despite their potential there is still a need to develop nanomaterials that are easy to produce, can be synthesized in large-scale amounts with high reproducibility for clinical translation, are nontoxic, can selfassemble with siRNA, and have high tumor bioavailability and retention time. Star polymers have received increased attention over the last several years as delivery vehicles for different therapeutic agents.11−17 They are cost-effective to produce, can be synthesized in large-scale quantities, and, importantly, their structure is well-defined and can be easily tailored for a desired application.16 For example, their internal core and/or peripheral side-arms can be modified to control their size or to incorporate biodegradable components that allow them to be processed within a cell.16 Their side-arms can also be manipulated to increase their stability or contain moieties to actively target specific cell types.16 Notably, studies have

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MATERIALS AND METHODS

Materials. Dimethylaminoethyl methacrylate (DMAEMA, 99%, Sigma-Aldrich), oligoethylene glycol methyl ether methacrylate (OEGMA, Sigma-Aldrich) and dimethylaminoethyl acrylate (DMAEA, Sigma-Aldrich) were deinhibited by passing through a basic alumina column. N,N′-methylenebis(acrylamide) (Sigma-Aldrich, 98%) and N,N′-bis(acryloyl)cystamine (Sigma-Aldrich, 99%) were used without additional purification. 4-cyanopentanoic acid dithiobenzoate was prepared as previously described23 (a brief description of its preparation is provided in the Supporting Information). The initiator, 2,2′-azobis(isobutyronitrile) (AIBN), 2338


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Biomacromolecules

acetonitrile (30 mL) were added to a round-bottom flask (100 mL) and stirred. The reaction was cooled and degassed using nitrogen at 0 °C for 1 h. This solution was then stirred for 14 h at 65 °C. Following this the reaction was quenched and an aliquot assessed by GPC and 1 H NMR. The monomer conversion was ∼70%. Rotary evaporation was used to concentrate the mixture and the polymer precipitated twice in petroleum ether to remove any traces of monomer. The purified PDMAEMA was analyzed by NMR, UV−vis and GPC. The molecular weight was calculated using NMR spectroscopy (Mn,NMR = 10 000 g/mol) and was close to the theoretical value (Mn,theor. = 10 500 g/mol) and in good agreement with the GPC results (Mn,GPC = 10 700 g/mol, PDI = 1.09). Synthesis of PDMAEMA/POEGMA Core Cross-Linked Star via the Arm First Methodology (Star 1). PDMAEMA (Mn,GPC = 5100 g/mol, 5.1 g, 1 × 10−3 mol) and POEGMA (Mn,GPC = 11 500 g/mol, 5.1 g, 4.43 × 10−4 mol) were added to a vial containing a magnetic stirrer, together with AIBN (78 mg, 4.76 × 10−4 mol) and toluene (100 mL). Cross-linker [(N,N′-bisacryloyl(cystamine), 3.0 g, 1.15 × 10−2 mol] and dimethylaminoethyl acrylate (DMAEA, 0.80 g, 5.59 × 10−3 mol) were then added and the vials sealed and purged with nitrogen for 1 h at 0 °C. The vials were then incubated in an oil bath for 24 h at 70 °C. At the end of the polymerization process, aliquots were collected and analyzed by 1H NMR and GPC. 1H NMR analysis confirmed the high conversion of cross-linker and DMAEA. Next, the reaction mixture was concentrated under vacuum. Core cross-linked star polymers were precipitated twice in cold petroleum ether/diethyl ether mixture (05/ 95 v/v) to remove the residual arms to yield a yellow product (yield ∼60%). The purified core cross-linked star polymer was analyzed by GPC (Mn,GPC = 125 000 g/mol and PDI = 1.19), FTIR and NMR spectroscopy to determine its composition (feed ratio: f OEGMA/ f DMAEMA 34/66 mol % (or 50/50 w-%), final composition: f OEGMA/ f DMAEMA 48/52 mol % (or 64/36 wt %). The purified core cross-linked star polymer was solubilized in methanol and then stored in a refrigerator to avoid irreversible cross-linking. Core cross-linked star polymers were dissolved in methanol and dialyzed with water/HCl (pH = 3.0) for 24 h. A further dialysis against water was then carried out for 48 h. Finally, the solution was freeze-dried to yield POEGMA/ PDMAEMA star polymers and characterized by NMR, FTIR spectroscopy, DLS, and TEM. Synthesis of PDMAEMA/POEGMA Star Using an Arm First Approach (Star 2). The synthesis of star 2 was carried out using similar techniques as described above for star 1. In brief, PDMAEMA (Mn = 10 700 g/mol, 5.0 g, 4.67 × 10−4 mol) and POEGMA (Mn = 11 500 g/mol, 5.0 g, 4.34 × 10−4 mol) were added to a vial containing a magnetic stir bar, together with AIBN (50 mg, 3.06 × 10−4 mol) and toluene (100 mL). Cross-linker (N,N′-bisacryloyl(cystamine) (1.910 g, 7.34 × 10−3 mol) and DMAEA (0.500 g, 3.49 × 10−3 mol) were added. The mixture was polymerized according to the previous condition. The core cross-linked star polymer was precipitated twice in cold mixture petroleum ether/diethyl ether (5/95 v/v) to remove the residual arms to yield a yellow product (yield ∼65%). The purified core cross-linked star polymer was analyzed by GPC (Mn,GPC = 145 000 g/mol and PDI = 1.29), and by NMR and FTIR after purification to determine its composition (feed ratio: f OEGMA/f DMAEMA 34/66 mol % (or 50/50 w-%), final composition: f OEGMA/f DMAEMA 51/49 mol % (or 66/34 w-%). The purified core cross-linked star polymer was dissolved in methanol and stored in the fridge to avoid irreversible cross-linking. Core cross-linked star polymers were solubilized in methanol and dialyzed with water/HCl (pH = 3.0) for 24 h. A further dialysis against water was then carried out for 48 h. The resulting solution was freeze-dried to yield PDMAEMA/POEGMA core crosslinked star polymers and characterized by NMR, FTIR spectroscopy, DLS, and TEM. Synthesis of PDMAEMA/POEGMA Star via the Arm-First Methodology (Star 3). The synthesis of star 3 was carried out using similar techniques as above. In brief, PDMAEMA (Mn = 10 700 g/mol, 5.25 g, 4.91 × 10−4 mol) and POEGMA (Mn = 11 500 g/mol, 0.83 g, 7.21 × 10−5 mol) were added into a vial containing a magnetic stir bar along with AIBN (33 mg, 2× 10−4 mol) and toluene (100 mL). Cross-linker (N,N′-bisacryloyl(cystamine) (1.139 g, 4.38 × 10−3 mol) and DMAEA

was crystallized twice using methanol. High purity N2 (Linde gases) was used for reaction solution purging. All other reactants were used as received without further purification. Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, USA), heat inactivated fetal calf serum (FCS), horse serum and 2 nM L-glutamine were purchased from SAFC Biosciences (Lexena, KS, USA). Cell culture consumables were obtained from Thermo Fisher Scientific (Lafayette, CO, USA). Lipofectamine 2000 was purchased from Life Technologies (Carlsbad, CA, USA). All reagents used to measure gene-silencing activity using real-time PCR (qPCR) were purchased from Life Technologies (Carlsbad, CA, USA). 18S Quantitect housekeeping gene primer assay was purchased from Qiagen (Valencia, CA, USA). BCA protein assay kit was purchased from Pierce (Danvers, MA. USA). siRNAs were purchased from GE Lifesciences (Lafayette, CO, USA). AlexaFlour488 conjugated and AlexaFlour-647 conjugated siRNAs for nanoparticle cell uptake studies were purchased from Qiagen (Valencia, CA, USA). Primary antibodies: anti-βIII-tubulin (clone TUJ1) was purchased from Covance (Richmond, CA, USA), anti-GAPDH (clone 6C5) was purchased from Sapphire Bioscience (Waterloo, NSW, Australia) and anti-CD31 was purchased from AbCam, Ltd., Australia. Horseradish peroxidase (HRP)-conjugated IgG goat antimouse secondary antibody was purchased from GE Healthcare (Buckinghamshire, UK). Eight well chamber slides were purchased from Ibidi (Martinsried, Germany). ProLong Gold Antifade Reagent with DAPI, LysoTracker Red DND-99, Hoechst 33342 were purchased from Life Technologies (Carlsbad, CA, USA), D-Luciferin potassium salt was purchased from Gold Biotechnology (St Louis, MO, USA) and growth-factor reduced matrigel from BD Biosciences (San Jose, CA, USA). Synthesis of Star-POEGMA Polymers. Synthesis of the POEGMA Arm Homopolymer. Oligo(ethylene glycol) methyl ether methacrylate [OEGMA (MW 300 g/mol), 15.0 g, 0.05 mol], 2,2′azobis(isobutyronitrile) (AIBN, 33.0 mg, 2 × 10−4 mol), 4cyanopentanoic acid dithiobenzoate (CPADB, 0.310 g, 1.11 × 10−3 mol) and dry toluene (50 mL) were added to a round-bottom flask, (100 mL), and stirred. The mixture was then degassed with nitrogen at 0 °C for 1 h. The solution was stirred at 65 °C for 14 h and quenched. An aliquot was then collected and analyzed using GPC and 1H NMR. The monomer conversion was ∼70%. The mixture was concentrated by rotary evaporation and the polymer precipitated in petroleum ether (low boiling point) to remove trace amounts of monomer. The purified POEGMA was analyzed by NMR, GPC, and UV−visible spectroscopy. The molecular weight was calculated by NMR spectroscopy (Mn,NMR = 10 600 g/mol) to be close to the theoretical value (Mn,theor. = 10 100 g/mol) and corresponded with GPC results (Mn,GPC = 11 500 g/mol, PDI = 1.14). Synthesis of PDMAEMA Homopolymers. Two homopolymers of PDMAEMA were prepared by RAFT polymerization with a molecular weight of ∼5000 and 10 000 g/mol. Dimethylaminoethyl methacrylate (DMAEMA, Mw = 157 g/mol, 7.78 g, 0.05 mol), 2,2′-azobis(isobutyronitrile) (AIBN, 31.8 mg, 1.94 × 10−4 mol), 4-cyanopentanoic acid dithiobenzoate (CPADB, 0.271 g, 9.7 × 10−4 mol) and dry acetonitrile (30 mL) were added to a round-bottom flask (100 mL), and stirred. The reaction mixture was cooled and degassed using nitrogen at 0 °C for 1 h. The solution was stirred at 65 °C for 14 h and then quenched. An aliquot was collected for GPC and 1H NMR analyses (monomer conversion ∼70%). The solvent was removed using rotary evaporation and the red polymer mixture was dissolved in acetone. The polymer was precipitated twice in petroleum ether to remove any traces of monomer. The polymer was collected and dried under vacuum. The purified PDMAEMA was analyzed by NMR, UV− vis and GPC. The molecular weight was determined using NMR spectroscopy (Mn,NMR = 5000 g/mol) was close to the theoretical value (Mn,theor. = 5600 g/mol) and correlated with GPC results (Mn,GPC = 5100 g/mol, PDI = 1.09). A similar procedure was employed for the synthesis of PDMAEMA (Mn = 10 700 g/mol), dimethylaminoethyl methacrylate [DMAEMA (Mw = 157 g/mol), 7.78 g, 0.05 mol], 2,2′-azobis(isobutyronitrile) (AIBN, 17.0 mg, 1.03 × 10−4 mol), 4-cyanopentanoic acid dithiobenzoate (CPADB, 0.145 g, 5.19 × 10−4 mol) and dry 2339


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Biomacromolecules Table 1. Summary of the Physicochemical Properties of Polymers and Star-POEGMA Polymers composition (mol %)a polymers

OEGMA

DMAEMA

MnGPC (g/mol)b

PDIc

size (nm)d

NArme

PDIDLSf

ξ (mV)g

POEGMA PDMAEMA1 PDMAEMA2 Star 1 Star 2 Star 3

100 48 51 12.5

100 100 52 49 87.5

11 500 5 100 10 700 125 000 145 000 120 000

1.14 1.09 1.09 1.19 1.29 1.35

6 4h 7h 25 (±10) 25 (±5) 35 (±10)

16 14 12

0.12 0.11 0.10 0.18 0.18 0.22

+18 (±8) +25 (±10) +25 (±10)

a

Molar composition was determined by NMR. bMolecular weight was determined by GPC [calibration poly(methyl methacrylate), DMAc as eluent]. cPolydispersity index (Mw/Mn) was determined by GPC. dSize (number distribution) was determined by DLS in water after filtration using 0.45 μm filter (average of three measurements of three different samples). eNumber of arms was calculated by GPC. fDispersity was measured by DLS after filtration using 0.45 μm filter. gZeta-potential (mV). hDLS was carried out in water (pH = 4.0) after filtration using 0.45 μm filter. (0.308 g, 2.15 × 10−3 mol) were then added. The mixture was polymerized according to the previous conditions. The core crosslinked star polymer was precipitated twice in cold diethyl ether/ petroleum spirit (90/10 v/v) to remove the unreacted arm to yield a yellow product (yield ∼80%). The purified core cross-linked star polymer was analyzed by GPC (Mn,GPC = 145 000 g/mol and PDI = 1.29), and by NMR and FTIR after purification to determine its composition (feed ratio: f OEGMA/f DMAEMA 7.7/92.3 mol % (or 14/86 wt %), final composition: f OEGMA/f DMAEMA 12.5/87.5 mol % (or 21.5/ 78.5 wt %). The purified core cross-linked star polymer was then analyzed by GPC (Mn,GPC = 120 000 g/mol and PDI = 1.35) and by NMR. The polymer was solubilized in methanol and dialyzed with acidic water (pH = 3.0) for 24 h, and then further dialyzed using water (pH = 6.5) for 48 h. Finally, the freeze-dried polymer was analyzed by NMR and FTIR. Physicochemical Characterization of Star-POEGMA Polymers. Gel Permeation Chromatography (GPC) Measurements. DMAc GPC analyses of the polymers were performed in N,Ndimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% 2, 6-dibutyl-4methylphenol (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 × 7.8 mm) followed by four linear PL (Styragel) columns (105, 104, and 103). A RID-10A differential refractive-index detector was used. Calibration was achieved using commercial poly(methyl methacrylate) and polystyrene standards ranging from 500 to 106 g/mol. The star polymer molecular weight was determined using poly(methyl methacrylate) calibration. The star polymer solutions were filtered before analysis using 0.45 μm filter. Nuclear Magnetic Resonance (NMR). The structures of the polymers which were synthesized were analyzed by NMR spectroscopy as described previously by Boyer et al.18 UV−Visible Spectroscopy. UV−visible spectra were recorded using methods described previously by Boyer et al.18 Dynamic Light Scattering (DLS). DLS measurements were performed as described previously by Boyer et al.18 The numberaverage hydrodynamic particle size was reported to reduce the influence of larger aggregates (Table 1). Intensity and volume distributions are also presented in Figure S16. The polydispersity index (PDI) was used to describe the width and size distribution of star polymers with and without siRNA using the DTS software along with 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). Transmission Electron Microscopy (TEM). The size and shape of the star-POEGMA polymers were examined using a transmission electron microscope (TEM, JEOL1400) at an accelerating voltage of 100 kV. The star-POEGMA polymers were dispersed in water (1 mg/ mL). The solution was deposited onto 200 mesh, holey film, copper grid (ProSciTech) and dried at room temperature for 4 h before analysis.

siRNA Binding to Star-POEGMA Polymers. Agarose gel electrophoresis was used to assess the ability of star polymers (star 1, star 2, star 3) to complex with siRNA as described.18 Biological Characterization of Star-POEGMA Polymers. Cell Culture. The human pancreatic cancer cell line MiaPaCa-2 was maintained in DMEM culture medium supplemented with 10% fetal calf serum, 2.5% horse serum, and 2 mM of L-glutamine as described.24−26 Human HPAF-II pancreatic cancer cells were grown in DMEM with 10% fetal calf serum and 2 mM of L-glutamine as described.24−26 Normal nontumorigenic human pancreatic ductal epithelial (HPDE) cells (a kind gift from Ming Tsao, Ontario Cancer Institute) were grown in keratinocyte-serum-free (KSF) medium supplemented with 50 mg/mL bovine pituitary extract (BPE) and 5 ng/mL epidermal growth factor (EGF) as previously described.24,27 All cells were regularly tested for mycoplasma and were negative. Toxicity Analysis. The toxicity profile of star POEGMA-siRNA complexes were examined in MiaPaCa-2 (1.1 × 105) cells. In brief, cells were plated into 6-well plates the day (16 h) before transfection. The next day, cells were transfected with star, star 1, star 2 or star 3 at increasing w/w ratios with siRNA (100 nM). Cells incubated in cell culture media alone served as controls. Cells were trypsinized, collected, and trypan blue exclusion studies were performed to determine the number of viable cells 24 h post-transfection. siRNA Transfection. To examine star-POEGMA-siRNA gene silencing activity, MiaPaCa-2 and HPAF-II cells were transfected with siRNA targeted against the microtubule protein, βIII-tubulin (encoded by the TUBB3 gene) which is expressed at high levels in pancreatic cancer cells.24,28 Briefly, cells were seeded into 6-well plates (1.1 × 105) the day prior to transfection. Cells were then transfected with increasing amounts of star-POEGMA-siRNA complexes [8:1− 30:1 (w/w) ratio with siRNA]. TUBB3 siRNA (100 nM) sense strand: 5′ -G UACG UG CC UC GAG CC AU UU U- 3 ′, antis e nse : 5′ UUCAUGCACGGAGAGCUCGGUAA-3′. Cells treated with nonsilencing (Control) siRNA sense strand: 5′-GCUAUGGCUGAAUACAAAUUUU-3′, antisense: 5′-UUCGAUACCGACUUAUGUUUAA-3′ or cells transfected with TUBB3 siRNA (also referred to as βIII-tubulin siRNA) complexed to Lipofectamine 2000 (L2K) served as positive and negative controls, respectively. Total RNA and whole cell lysates were collected 72 h post-transfection, and βIIItubulin mRNA and protein levels measured using qPCR and Western blotting as described.24 Intracellular Uptake. To observe the cellular uptake and pattern of distribution of star-POEGMA-siRNA complexes, MiaPaCa-2 cells were seeded into 8-well chambers slides (1.2 × 103 cells) the day prior to transfection. The next day, cells were transfected with fluorescently labeled (AlexaFlour-488, green) siRNA (100 nM) complexed to starPOEGMA polymers (star 1, star 2, star 3) for 4 h at 37 °C. Cells transfected with fluorescent siRNA complexed to L2K or fluorescent siRNA by itself served as positive and negative controls, respectively. Twenty-four hours post-transfection, cells were incubated with 1 μM LysoTracker (diluted with culture medium) for 1 h. The cells were washed three times in warm PBS for 5 min, and then fixed with 4% paraformaldehyde for 3 min. Slides were mounted using ProLong 2340


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Biomacromolecules

Figure 1. A schematic diagram showing the structure and synthetic steps required to produce different star-POEGMA polymers. treatment groups and administered intratumorally with star 3-βIIItubulin siRNA (2 mg/kg) twice weekly for 2 weeks. Mice injected with star 3-control siRNA served as controls. Tumor size was measured twice weekly as previously described.18 At the end of the experiment, tumors were harvested and RNA and protein lysates prepared as previously described.24 Total RNA integrity was confirmed using an Agilent Technologies 2100 Bioanalyzer with an RNA integrity number (RIN) a value >7. βIII-tubulin expression was measured by qPCR, Western blotting and immunohistochemistry as described.24 (ii) Orthotopic Model: Eight-week old BALB/c nude mice were used. In brief, human pancreatic cancer cells (MiaPaCa-2 and HPAF-II) were xenografted orthotopically into the tail of the pancreas as we have described previously.24−26 Star 3-siRNA Biodistribution in Vivo. To examine the biodistribution of siRNA complexed to star 3 in vivo, mice with orthotopic pancreatic MiaPaCa-2 (6 weeks post-tumor cell implantation) or HPAF-II (3 weeks post-tumor implantation) tumors were administered (via the tail vein) with star 3 complexed to fluorescently labeled siRNA (AlexaFluor-647, Red). Mice injected with PBS or siRNA alone served as controls. One, four and 24 s postinjection mice were humanely euthanized and hearts, lungs, livers, spleens, kidneys, and pancreatic tumors were collected and infrared fluorescence measured using the IVIS imaging system as described.24 After imaging, pancreatic tumor tissue was snap-frozen in OCT embedding media and siRNA uptake and extravasation from tumor vessels in tissue sections visualized by immunofluorescence staining and confocal microscopy. Gene-Silencing Activity of Star 3 in Vivo. To examine the genesilencing activity of star 3-βIII-tubulin in vivo, MiaPaCa-2 pancreatic tumors were established as described above. Six-weeks post-tumor cell

Gold Antifade Reagent with DAPI. Uptake of siRNA was visualized as previously described by Boyer et al.18 Real-time PCR (qPCR). βIII-tubulin (TUBB3) mRNA expression in MiaPaCa-2 and HPAF-II cells was measured using qPCR as described.24 All data were normalized to the housekeeping gene 18S (18S Quantitect primer assays, Qiagen). Western Blotting. Seventy-two hours post-transfection with starPOEGMA-βIII-tubulin siRNA, MiaPaCa-2 and HPAF-II cells were harvested, and βIII-tubulin and GAPDH expression was measured in whole cell lysates as previously described.24 Inhibition of Endocytosis. To examine which endocytic pathways may be involved in the internalization of star-POEGMA-siRNA, MiaPaCa-2 cells were plated into 8 well chambers slides (1.2 × 103 cells) the day before transfection. The next day, MiaPaCa-2 cells were incubated with a clathrin-dependent inhibitor chlorpromazine (5 μg/ mL) or two different clathrin-independent inhibitors, methyl-βcyclodextrin (MβCD, 5 mM) and genestein (200 μM) for 2 h. The specificity and effectiveness of each inhibitor was confirmed as previously described by our laboratory.29 Subsequently, the cells were transfected with AlexaFlour-647 labeled siRNA (red) complexed to star-POEGMA polymers (star 1, star 2, star 3) for 1 h at 37 °C. The nucleus was then labeled with Hoechst 33342 (blue). Live cell images were taken to visualize siRNA uptake using a Zeiss LSM 780 confocal microscope. Pancreatic Cancer Mouse Models. (i) Subcutaneous Model: Eight-week old BALB/c nude mice were used. All animal experiments were approved by the Animal ethics committee, UNSW Australia (ACEC 13/130B). In brief, 4 × 106 MiaPaCa-2 cells were injected subcutaneously into the flank of mice as previously described.18 Once tumors reached the size of ≥50 mm3, mice were randomized into 2341


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Figure 2. Physicochemical properties of star-POEGMA polymers. (A) Gel permeation chromatography (GPC) traces of the three different starPOEGMA polymers (star 1, star 2 and star 3) after purification. (B) 1H NMR spectra of purified core cross-linked star polymers (star 1, star 2, and star 3) after quaternization with HCl [recorded in D2O, using a recovery time of 5 s, number of scans = 128] (Note 1: the core cross-linked is represented by the black sphere (the structure of the core cross-linked is shown in Supporting Figure 5). The signals of the cross-linkers are overlapped with the signal of polymers. The successful incorporation of cross-linker was confirmed by FTIR spectroscopy by the presence of amide band at 1650 cm−1; Note 2: R corresponds to 4-cyanopentanoic acid group). (C) FTIR spectra of purified star polymers after quaternization with HCl (star 1, star 2, and star 3). (D) Dynamic light scattering (DLS) of the different star-POEGMA polymers in water (star 1, star 2, and star 3); volume and intensity distribution are given in Supporting Figure 16. (E) TEM pictures of the different Star-POEGMA polymers (star 1, star 2, and star 3). injection mice were randomized into treatment groups and administered systemically (via the tail vein) with star 3-βIII-tubulin siRNA (4 mg/kg) once daily for 3 days. Mice treated with control siRNA served as controls. Twenty-four h after the final injection, pancreatic tumors were harvested and βIII-tubulin expression was measured by qPCR, Western blotting, and immunohistochemistry as described.24 Statistics. Data are presented as the means of at least three independent experiments ± standard error of the mean (SEM) (error bars). Graphpad Prism-6 was used to perform ANOVA or paired twotailed Student’s t tests followed by Tukey’s or Dunnet post tests were used to measure statistical differences between experimental and control groups, with p < 0.05 considered statistically significant.

EGMA) and poly(dimethylaminoethyl methacrylate), (PDMAEMA) was carried out using 4-cyanopentanoic acid dithiobenzoate as the chain transfer agent and 2,2′-azobis(isobutyronitrile) (AIBN) as the radical initiator. The chosen POEGMA polymer is closely related to poly(ethylene glycol) (PEG) and has similar low-fouling properties. The polymerizations were performed for 14 h at 65 °C to yield a monomer conversion of around ∼70%. The ratio between the monomer and RAFT agent was varied to control the molecular weight of polymers. PDMAEMA with two different molecular weights, 5100 and 10 700 g/mol and POEGMA with a molecular weight of 11 500 g/mol were prepared with a narrow molecular weight distribution (PDI < 1.20) (Figure S1−S3). After several rounds of purification by precipitation, homopolymer chains were extended using a diacrylamide monomer [(N,N′-bis(acryloyl)cystamine)] as a cross-linker, AIBN as the initiator and dimethylaminoethyl acrylate (DMAEA) as a comonomer at 70 °C in toluene for 24 h (Figure S4). To obtain the greatest incorporation of arm into the star polymers with the lowest molecular weight distribution, we used a [Arm]:[DMAEA]:

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RESULTS AND DISCUSSION Synthesis and Characterization of Star Polymers. The core cross-linked star-POEGMA polymers were prepared using an arm-first approach and living radical polymerization (reversible addition−fragmentation chain transfer polymerization, RAFT). 30−32 In a first step, the synthesis of poly[oligo(ethylene glycol) methyl ether methacrylate] (PO2342


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Figure 3. siRNA binding efficiency and cell uptake of star-POEGMA-siRNA complexes in pancreatic cancer cells. (A−C) Agarose gels showing a fixed concentration of free siRNA (siRNA, 60 ng) (lane 1) or when complexed to increasing amounts of star 1, star 2, star 3 or star polymer without POEGMA (star) (8:1−30:1 w/w ratio with siRNA) (n = 3 individual experiments). (D) Confocal microscope images demonstrating cell uptake of fluorescently labeled-siRNA (green) complexed to star 1 (panel I), star 2 (panel II) or star 3 (panel III), (Blue, nuclear DNA, DAPI stained). Cells were stained with lysotracker red to stain for lysosomes. Panels IV, V, and VI are high power (zoomed) confocal microscope images showing that star 1- and star 2-siRNA readily colocalize with endosomes (yellow-orange indicated by white arrows) compared to star 3-siRNA (white arrows indicate free siRNA, green) in MiaPaCa-2 cells.

[cross-linker] ratio of ∼1:4:8. High monomer and cross-linker conversion (≥85% for both monomers) were observed by NMR analysis after 24 h (Figure S5 exhibits a typical NMR of unpurified star polymer). GPC analysis showed the formation of high molecular weight polymer, which confirmed the formation of core cross-linked star polymers, but also some residual arms (Figures S6−S8). After additional purification by precipitation in petroleum ether/diethyl ether mixture (the composition is optimized according to the star) to remove the unreacted arm, the different star-POEGMA polymers were further characterized by NMR, FTIR, and GPC to confirm their structures. Three different star polymers [Star 1 (48 mol % POEGMA with short-cationic side arms), Star 2 (51 mol % POEGMA with long-cationic side arms), and Star 3 (12.5 mol % POEGMA with long-cationic side arms)] were obtained (Figure 1). GPC demonstrated the synthesis of star-POEGMA polymers with a low molecular weight distribution (PDI < 1.35) (Figure 2A and Table 1), while NMR confirmed the presence of (CH3)2N (DMAEMA) and OCH3 (POEGMA) groups at 2.3 and 3.3 ppm, respectively (Figures S9−S11). The molar composition was determined using the following equation: f DMAEMA = (I(CH3)2/6)/[(IOCH3/3) + (I(CH3)2/6)], where I(CH3)2 and IOCH3 correspond to the dimethylamino group (before quaternization) at 2.3 ppm and methoxy groups (OCH3) at ∼3.4 ppm, respectively (Figures S9−S11). FTIR

showed the incorporation of cross-linker by the characteristic amide band at 1,660 cm−1, while ether (O−C) and tertiary amine (N−C) bands appeared at 1150 and 1230 cm−1 (Figure S12). Subsequently, the star-POEGMA polymers were quaternized using an acidic aqueous solution ([HCl] = 10−3 M, pH = 3.0). After purification by dialysis to remove unreacted HCl, the star-POEGMA polymers were freeze-dried and analyzed by NMR using deuterium oxide (Figure 2B and Figures S13−S15) and by FTIR (Figure 2C). Figure 2B showed the presence of quaternized tertiary amine at 2.9 ppm, while FTIR confirmed the presence of ester [(CO)O], amide [(CO)N], quaternized tertiary amine (N−C) and ether (O−C) bands at 1,730, 1,650, 1,230 and 1,150 cm−1 (Figure 2C). After redispersion in water, we measured the size of the star-POEGMA polymers using dynamic light scattering. All three star-POEGMA polymers were soluble in water and displayed sizes ranging from 25 to 35 nm (±5−10 nm) (Table 1). Figure 2D displays the number distribution, while Figure S16 shows the volume and intensity distribution, and the presence of small aggregates. Transmission electron microscopy confirmed the formation of small spherical nanoparticles (Figure 2E). Collectively, these results demonstrated the successful synthesis of three star polymers with different structural properties, including length of cationic side-arms and percentage incorporation of POEGMA. 2343


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Figure 4. Cytotoxicity profile and gene silencing activity of star-POEGMA-siRNA in pancreatic cancer cells. (A-C) Graphs showing cell viability of MiaPaCa-2 cells transfected with star 1, star 2, star 3 or star polymers without POEGMA (star) [8:1−30:1 (w/w) ratio] complexed to nonfunctional siRNA (100 nM). Cell viability was expressed as a fraction of control (cells incubated in culture media only). Each value represents the mean ± standard error of triplicate wells (n = 3 individual experiments; *p < 0.001, #p < 0.05). (D-F) Graphs demonstrating βIII-tubulin mRNA expression in MiaPaCa-2 cells transfected with increasing amounts of star 1, star 2 or star 3 [8:1−30:1 (w/w)] complexed to βIII-tubulin siRNA (100 nM). Cells incubated in culture medium served as controls. Each value represents the mean ± standard error from three independent experiments (*p < 0.001). All data were normalized to the housekeeping gene 18S. (G-I) Representative Western blots showing βIII-tubulin protein expression in MiaPaCa-2 cells transfected with increasing amounts of star 1, star 2 or star 3 [(8:1−30:1 (w/w)] complexed to βIII-tubulin siRNA (100 nM). GAPDH was used as a protein loading control; three independent experiments were performed.

siRNA Binding Efficiency of Star-POEGMA Polymers. To examine how the length of the cationic side-arms and percentage of POEGMA would effect star polymer capacity to bind siRNA, increasing amounts of star 1 (short cationic side arm and 48 mol % POEGMA), star 2 (long cationic side arm and 51 mol % POEGMA), or star 3 (long cationic side arm and 12.5 mol % POEGMA) were mixed with siRNA. The complexes of star-POEGMA-siRNA were then separated on an agarose gel. siRNA alone traveled to the cathode (bottom of gel) due to its high negative charge (Figure 3A−C). siRNA complexed to either star 1 or star 2 [18:1 (w/w) ratio] did not migrate, suggesting that complexation between the siRNA and the star-POEGMA polymers had been achieved (Figure 3A and 3B). Star 3 complexed with siRNA [10:1 (w/w) ratio] more readily compared to star 1 and star 2 (Figure 3C). Indeed, the amount of star 3 needed to complex siRNA was not too dissimilar to star polymers without POEGMA (star) (Figure 3C). Previously, we had shown that star complexed siRNA at an 8:1 (w/w) ratio.18 Hence, the difference in siRNA binding efficiency between the POEGMA star polymers (star 1, star 2, star 3) and non-POEGMA star polymers (star) was most likely

due to steric hindrance of POEGMA and overall reduction in net positive surface charge due to the presence of the POEGMA. For example, star polymers without POEGMA had a zeta-potential of +50 mV,18 while, star 1 had a zeta potential of +18 mV (±8 mV), star 2 and star 3 + 25 mV (±10 mV) (Table 1). After complexation with siRNA, the zeta potential of all three star-POEGMA polymers decreased to +9 mV (±4 mV) for star 1, + 18 mV (±8 mV) for star 2, and +12 mV (±5 mV) for star 3, indicating that the siRNA and starPOEGMA polymers formed a complex via an electrostatic interaction (results not shown). Notably, the size of each starPOEGMA nanoparticle when complexed to siRNA did not significantly change. Star 3 before complexing siRNA had an overall size of 35 nm (±10 nm) (Table 1), and after complexing siRNA, the size was slightly increased to 38 nm (±10 nm) (results not shown). The size and surface charge of the star-POEGMA-siRNA complexes are ideal for taking advantage of the disrupted tumor vasculature observed in many different types of solid tumors. Most recently, studies have reported that nanoparticle−siRNA complexes with a size between 50 and 100 nm, and which contain a slight positive 2344


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and star 2 complexed to siRNA displayed no toxicity at the 20:1 (w/w) ratio (Figure 4-B). At 30:1 (w/w) there was no toxicity in MiaPaCa-2 cells transfected with star 1-siRNA, and only a very slight decrease (≈15%) in cell viability in cells transfected with star 2-siRNA (Figure 4A,B). By contrast, a 90% decrease in cell viability was observed in cells treated with the star polymers without POEGMA complexed to siRNA (Figure 4A,B). Star 3 was also nontoxic at all of the examined concentrations [8:1−18:1 (w/w)] (Figure 4C). Similar results were obtained using a colorimetric cell proliferation assay (results not shown). These results are in accordance with a previous study reported by Cho et al.12 which showed that star polymers synthesized using atom transfer radical copolymerization (ATRP) and containing a short PEG block (2KDa) were also less toxic in cells when compared to cationic nanoparticles. The increased toxicity observed with star polymers without POEGMA at the higher concentrations is not unexpected and is typical for most highly charged cationic nanoparticles. Both poly(amidoamine) (PAMAM) dendrimers and highly branched polyethylenimine (PEI) polymers possess positively charged surface groups, and have been reported to destabilize cell surface membranes leading to mitochondrial-mediated cell death.36,37 Together, these results demonstrate that the incorporation of POEGMA into star polymers significantly improves their cytotoxic profile. Star-POEGMA-siRNA Gene Silencing Activity in Pancreatic Cancer Cells. To determine whether star-POEGMA nanoparticles were able to release siRNA into the cytosol of pancreatic cancer cells and induce post-transcriptional gene silencing, we transfected MiaPaCa-2 cells with increasing amounts of star 1, star 2, and star 3 complexed with siRNA (100 nM) targeted against the microtubule protein βIII-tubulin. At an 8:1 (w/w) ratio with βIII-tubulin siRNA both star 1 and star 2 did not silence βIII-tubulin expression (Figure 4D,E). This result is expected based on the fact that both nanoparticles failed to complex with siRNA at the 8:1 (w/w) ratio (Figure 3A,B). However, at the 30:1 (w/w) ratio, star 1-βIII-tubulin siRNA significantly silenced βIII-tubulin gene expression in MiaPaCa-2 cells by 44% (p < 0.05) when compared to control cells (Figure 4D). This led to a reduction in βIII-tubulin protein expression (Figure 4G). Transfection of MiaPaCa-2 cells with higher amounts of star 1-βIII-tubulin siRNA did not improve gene silencing activity (results not shown). Interestingly, MiaPaCa-2 cells transfected with star 2-βIII-tubulin siRNA showed a significant decrease in βIII-tubulin mRNA and protein expression at the lower ratio of 20:1 (w/w) when compared to control cells (Figure 4E−H). At 30:1 (w/w), there was a > 50% (p < 0.05) decrease in βIII-tubulin mRNA and protein levels (Figure 4E−H). This result suggests that the longer cationic side-arms present on star 2 may provide an advantage for the binding of siRNA to the star polymer and/or its release once inside the cell cytoplasm. Star 3-βIII-tubulin siRNA was able to potently silence βIII-tubulin expression at much lower ratios. For example, at 8:1 (w/w) ratio, star 3-βIIItubulin siRNA silenced βIII-tubulin gene and protein levels by 50% (p < 0.05) compared to control cells (Figure 4F−I). At the 16:1 and 18:1 (w/w) ratios, star 3-βIII-tubulin siRNA silenced βIII-tubulin expression by 80% (p < 0.001) at the gene and protein levels compared to control cells (Figure 4F and 4I). In fact, gene silencing activity of star 3 was the same as star polymers without POEGMA complexed to siRNA (star) (Figure 4F−I). The potent gene silencing activity of star 3βIII-tubulin siRNA was confirmed using another pancreatic

surface charge are able to penetrate solid tumors via passive delivery and the ‘enhanced permeability and retention effect’ in preclinical animal models.33−35 Together, these results show that star-POEGMA polymers are able to effectively selfassemble with siRNA to form small uniform nanoparticles, and while the length of the cationic side arm does not influence the ability of star polymers to interact with siRNA, the amount of POEGMA does have an impact on their ability to form a complex with siRNA. Pancreatic Cancer Cell Uptake of Star-POEGMA Polymers. To assess the ability of star-POEGMA polymers to deliver siRNA to pancreatic cancer cells, we transfected MiaPaCa-2 cells with star 1, star 2, or star 3 complexed to fluorescently labeled (AlexaFlour 488, Green) siRNA, and examined its pattern of distribution 24 h post-transfection using confocal microscopy. Additionally, to assess whether siRNA could be released into the cell cytosol, we also examined the colocalization of siRNA with endosomes/lysosomes using the LysoTracker-Red stain 24 h post-transfection. Confocal microscopy demonstrated that all three star-POEGMA nanoparticles could deliver fluorescent siRNA into the cytoplasm of pancreatic cancer cells (Figure 3D; panels I, II and III). Interestingly, lysoTracker staining and high power magnification confocal microscope images of MiaPaCa-2 cells transfected with star 3-siRNA showed that siRNA which were internalized into the cells had markedly less colocalization [as indicated by free siRNA (Green)] with endosomes [(Red), indicating endosomal release)] (Figure 3D panel VI) when compared to siRNA delivered by star 1 or star 2 (evidenced by yellow punctate dots in the cytoplasm which indicate colocalization of siRNA with endosomes) (Figure 3D panels IV, V). This result suggests that the physicochemical properties of star 3 (long cationic side-arms and 12.5 mol % POEGMA) may allow for more siRNA to be released from within endosomes allowing it to enter the cell cytoplasm. However, it is also possible that star 3-siRNA complexes may utilize different intracellular trafficking pathways compared to star 1 and 2 within MiaPaCa-2 cells that allow the complexes to be rapidly disassembled and/or degraded or recycled by endosomal membrane receptors. Future work in our laboratory will further characterize the internalization and trafficking processes of star 3 in pancreatic cancer cells. Cell Toxicity Profile of Star-POEGMA Polymers. Based on our data showing that the incorporation of POEGMA did not adversely affect the ability of star polymers to interact with siRNA and deliver it to pancreatic cancer cells, we next examined whether the presence of POEGMA could influence their cytotoxicity profile. MiaPaCa-2 cells were transfected with increasing amounts of star 1, star 2 or star 3 complexed to nonfunctional siRNA (100 nM) [8:1−30:1 (w/w) with siRNA]. As a comparison, cells were transfected with increasing concentrations (w/w ratio with siRNA) of non-POEGMA star polymers bound to siRNA (star). The viability of the cells was assessed 24 h post-transfection by phase contrast microscopy (data not shown) and by analysis of cell counting using trypan blue. Previously, we had shown that star polymers without POEGMA when complexed to siRNA were nontoxic in cancer cells at an 8:1 (w/w) ratio.18 We confirmed this result showing that, at 8:1 (w/w) ratio, the star was nontoxic to MiaPaCa-2 cells (Figure 4A-C). However, at the higher concentrations, star polymers without POEGMA (star) showed marked toxicity in the cancer cells with a 70% decrease in cell viability at the 20:1 (w/w) ratio after 24 h (Figure 4A,B). By contrast, both star 1 2345


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Figure 5. Inhibition of clathrin-dependent and -independent endocytosis and star-POEGMA-siRNA internalization into pancreatic cancer cells. (A− C) Panel I shows representative live cell confocal microscope images of MiaPaCa-2 cells transfected with star 1, star 2, or star 3 complexed to fluorescently labeled siRNA (red) in the absence of endocytosis inhibitors [nuclear DNA (blue, DAPI stained)]. Panels II and III, show live cell confocal microscope images of MiaPaCa-2 cells pretreated with methyl-β-cyclodextrin (MβCD) or genestein (clathrin-independent inhibitors) prior to transfection with star 1, star 2, or star 3 complexed to fluorescently labeled siRNA (red) [nuclear DNA (blue, DAPI stained)]. Panel IV, shows live cell confocal microscope images of MiaPaCa-2 cells pretreated with chlorpromazine (clathrin-dependent inhibitor) prior to transfection with star 1, star 2, or star 3 complexed to fluorescently labeled siRNA (red) [nuclear DNA (blue, DAPI stained)]. Representative images were taken from n = 3 independent experiments.

shown). MiaPaCa-2 cells transfected with star-POEGMAfluorescent siRNA in the absence of the inhibitors served as controls (Figure 5A−C, panel I). Inhibition of clathrinindependent endocytosis using MβCD or genistein completely blocked the uptake of star 1-siRNA into MiaPaCa-2 cells (Figure 5A, panels II−III). Interestingly, uptake of star 3-siRNA was not blocked when the cells were treated with the above inhibitors (Figure 5C, panels II−III). Similar results were observed for star 2-siRNA when treated with MβCD; however, the uptake of siRNA in the presence of genestein appeared to be reduced in the cancer cells when compared to control cells (Figure 5B, panels I and III). This suggests that the caveolaemediated endocytosis pathway may in part play a role in the internalization of star 2-siRNA into cancer cells. Finally, inhibition of clathrin-dependent endocytosis using chlorpromazine failed to block uptake of star 1-siRNA, but almost completely abolished uptake of star 2- and star 3-siRNA in MiaPaCa-2 cells (Figure 5A-C, panels IV). These results suggest that the different star-POEGMA polymers when complexed to siRNA are taken up by pancreatic cancer cells by different endocytic pathways. For example, star 1, which contains short cationic side-arms and 48% POEGMA appeared to primarily use a clathrin-independent pathway; star 2, which contains long cationic side-arms and 51% POEGMA appeared to use both clathrin-dependent and -independent endocytosis pathways; while star 3 which also contains long cationic sidearms but a reduced amount of POEGMA (12.5%) appeared to primarily utilize the clathrin-dependent endocytosis pathway for entry into the cancer cells. The physicochemical properties of nanoparticles including, size, shape, and surface charge can have a major influence on the mode of internalization and intracellular trafficking.5,40

cancer cell line (HPAF-II) which was derived from a metastatic site. Star 3-βIII-tubulin siRNA silenced βIII-tubulin gene expression by 80% (p < 0.001) in HPAF-II cells (Figure S17A). This led to a > 80% reduction in βIII-tubulin protein expression (Figure S17B−C). Notably, the gene silencing activity of star 3 was comparable to the commercial delivery vehicle lipofectamine 2000 (L2K) (Figure S17A−C). Furthermore, star 3 by itself or when complexed to siRNA was nontoxic in HPAF-II cells, and nontumorigenic human pancreatic ductal epithelial cells (cells of origin for pancreatic adenocarcinoma) (Figure S18A,B). These results show for the first time that star polymers containing differing lengths of cationic side-arms and amounts of POEGMA when complexed to siRNA possess potent gene silencing activity in pancreatic cancer cells. Mode of Cellular Internalization of Star-POEGMAsiRNA in Pancreatic Cancer Cells. To explore how starPOEGMA-siRNA complexes are internalized into pancreatic cancer cells, we treated MiaPaCa-2 cells with small molecule endocytosis inhibitors that block either the clathrin-dependent (Chlorpromazine) or independent [Methyl β-cyclodextrin (MβCD) and Genistein] endocytosis pathways.29 Both pathways have been well-characterized in the literature and can carry their cargo into early endosomes. where they are subjected to further processing into the lysosomal pathway, trans-Golgi network or recycled to the cell surface.38,39 Prior to transfection with star-POEGMA-siRNAs the specificity and concentration for each inhibitor needed to block their respective endocytic pathways was determined using methods recently described by our group29 (results not shown). The inhibitor concentrations used to inhibit endocytosis were confirmed to be nontoxic to MiaPaCa-2 cells (results not 2346


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Figure 6. Gene silencing activity of star 3-βIII-tubulin siRNA in the presence of serum proteins. (A) Graph showing βIII-tubulin mRNA expression in MiaPaCa-2 cells transfected with star polymers without POEGMA complexed to βIII-tubulin siRNA or star 3-βIII-tubulin siRNA (star) in culture medium containing 10% fetal bovine serum. Cells incubated in culture medium served as controls. Each value represents the mean ± standard error from three independent experiments (*p < 0.01 vs control). All data were normalized to the housekeeping gene 18S. (B) Western blot and corresponding densitometry graph showing βIII-tubulin protein expression in MiaPaCa-2 cells transfected with star polymers without POEGMA complexed to βIII-tubulin siRNA (star) or star 3-βIII-tubulin siRNA in culture medium containing 10% fetal bovine serum. Cells incubated in culture medium served as controls. GAPDH was used a protein loading control. Each value represents the mean ± standard error from three independent experiments (*p < 0.01 vs control). (C) Graph showing βIII-tubulin mRNA expression in subcutaneous pancreatic tumors in mice, 48 h after local administration of star 3-βIII-tubulin siRNA (2 mg/kg), n = 4−5 mice/group, *p < 0.05. Mice treated with star 3-nonfunctional siRNA served as controls. (D) Western blot and densitometry graph showing βIII-tubulin protein expression in subcutaneous pancreatic tumors in mice 48h after local administration of star 3-control (Ctrl) siRNA or star 3-βIII-tubulin siRNA (2 mg/kg), n = 4−5 mice/group, *p < 0.05. (E) Immunohistochemical images of subcutaneous pancreatic tumors collected from two individual mice treated with star 3-βIII-tubulin siRNA (panels III and IV) showing reduced βIII-tubulin expression compared to 2 individual mice treated with star 3-control siRNA (panels I and II). (F) Graph showing a reduction in tumor growth in mice treated with star 3-βIII-tubulin siRNA compared to mice treated with star 3-control siRNA (n = 4−5 mice/group), *p < 0.05.

Accumulating evidence shows that different modes of endocytosis can lead to different intracellular trafficking of nanoparticles within the cell, which can impact transfection efficiency.5,40 Intracellular trafficking is a dynamic process and is responsible for transporting internalized molecules to different subcellular locations.38,39 For example, nanoparticles that are internalized via a clathrin-dependent endocytic pathway can be transported from early endosomes to lysosomes. For this mode of trafficking, it was thought that when cells were exposed to nanoparticles carrying siRNA that following endosomal acidification the result was lysosomal osmotic swelling and rupture, leading to the release of its contents into the cytosol (protonsponge effect).5,41 Several studies have reported this mode of internalization and trafficking to be an efficient route for nanoparticles to release siRNA into the cytoplasm.40−42

However, recent studies have suggested that siRNA release from nanoparticles may be more complex and involve multiple internalization and trafficking processes. Gilleron et al.43 demonstrated that the internalization and trafficking of lipid nanoparticles is cell-type dependent and can use both clathrinmediated endocytosis and micropinocytosis. Notably, they also demonstrated that siRNA escapes from endosomes into the cytosol at a very low efficiency and only during a defined stage of endosomal progression. A study by Sahay et al.44 showed that multiple proteins and signaling processes are needed for nanoparticles to enter cells via macropinocytosis, and that a major proportion of the internalized siRNA undergoes exocytosis via a recycling process in the late endosomes/ lysosomes. 2347


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Figure 7. Biodistribution and gene-silencing activity of star 3-βIII-tubulin siRNA in orthotopic pancreatic tumors in mice. (A) Ex vivo fluorescent images of heart, lungs, liver, spleen, kidney and orthotopic pancreatic tumors from mice injected (via the tail vein) with PBS, fluorescent siRNA (AlexaFluor-647), or star 3-fluorescent siRNA (AlexaFluor-647). Organs were collected and imaged at 1, 4, and 24 h postinjection. (B,C) Confocal images of frozen sections of orthotopic pancreatic tumors showing the presence of fluorescent siRNA (red) in tumor tissue in mice administered (via the tail vein) with star 3-fluorescent siRNA (AlexaFluor-647), 24 h postinjection. Fluorescent siRNA was extravasated from the tumor vasculature into the surrounding tumor cells. Mice injected with fluorescent siRNA (AlexaFluor-647) alone served as controls. Fluorescent siRNA (red) and nuclear DNA (blue), tumor vessels (green) (white arrows indicate the location of siRNA). (D) Graph showing knockdown of βIII-tubulin mRNA expression in orthotopic pancreatic tumors in mice 24 h after the final injection with star 3-βIII-tubulin siRNA (4 mg/kg). Mice injected with star 3control siRNA served as controls (n = 3−4 mice/group, *p < 0.01). (E,F) Western blot and immunohistochemistry staining from 2 individual mice showing reduced βIII-tubulin protein expression in orthotopic pancreatic tumors in mice injected with star 3-βIII-tubulin siRNA (4 mg/kg) 24 h after the final injection. Mice injected with star 3-control siRNA served as controls. GAPDH was used as a protein loading control.

transfected with star 3-βIII-tubulin siRNA in cell culture medium containing serum proteins (10% fetal bovine serum). As a comparison, cells were also transfected with star polymers without POEGMA complexes to βIII-tubulin siRNA (star). Cells transfected with star 3-βIII-tubulin siRNA had a 50% decrease (p < 0.001) in βIII-tubulin gene expression, and a 60% decrease (p < 0.001) in βIII-tubulin protein expression when compared to control cells (Figure 6A,B). By contrast, star-βIIItubulin siRNA did not silence βIII-tubulin gene or protein expression in the presence of serum proteins (Figure 6A,B). This is most likely due to the high cationic charge of the star polymers without POEGMA, which may result in the binding of serum proteins to its surface, thereby preventing or retarding its cellular internalization and/or ability to release siRNA into the cytosol. To our knowledge, this is the first study to report the ability of star polymers containing POEGMA to deliver siRNA and silence a target gene in tumor cells in the presence of serum proteins. The ability of star 3-βIII-tubulin siRNA to silence βIII-tubulin was further illustrated in a tumor

Our results suggest that star 3 has less colocalization with endosomes/lysosomes when compared to siRNA delivered by star 1 or star 2 (Figure 3D; panels IV, V, and VI) and uses different internalization processes to enter pancreatic cancer cells. Future studies will be required to further delineate the mechanisms of internalization and intracellular trafficking of star 3-siRNA complexes in pancreatic cancer cells. Gene Silencing Activity of Star 3-siRNA in the Presence of Serum Proteins. It is documented that serum proteins present in the bloodstream can bind to cationic nanoparticle−siRNA complexes and prevent or retard their cell uptake, thereby dramatically reducing their gene-silencing activity.45 Moreover, it is accepted that the addition of PEG or POEGMA to nanoparticles is a highly efficient strategy to help mask the binding of serum proteins to the surface of nanoparticles. Given that star 3 was nontoxic and had the highest gene silencing activity when complexed to βIII-tubulin, we assessed its ability to deliver siRNA into pancreatic cancer cells in the presence of serum proteins. MiaPaCa-2 cells were 2348


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selectively inhibit βIII-tubulin expression in pancreatic tumor cells, which until now has not been possible with the use of chemical inhibitors. These findings have the potential to not only impact pancreatic cancer, but also other highly malignant tumors which express high levels of βIII-tubulin such as lung, ovarian, breast and prostate. Unique Properties of Star 3 That Maximize Its Ability to Deliver siRNA to Solid Tumors in Vivo. While previous studies11,12,15,17 provided important rationale for the use of star polymers for delivery of siRNA into cells, they did not examine their ability to deliver siRNA to cancer cells, mechanisms of cell uptake, gene silencing activity in the presence of serum proteins (a scenario encountered in vivo), or importantly, siRNA delivery and silencing in vivo. In this study we aimed to generate a star polymer with unique physicochemical properties enabling it to complex with siRNA and maintain a small size. This feature is critical for the delivery of siRNA to solid tumors in vivo.33 Moreover, the star polymer needed to be nontoxic and avoid interactions with serum proteins to deliver siRNA to pancreatic cancer cells in vivo. To address these important challenges, our approach using RAFT polymerization enabled us to easily manipulate the molecular weight and/or amount of PDMAEMA or POEGMA in the side-arms of star polymers by varying the feed ratio. Indeed, we illustrated this by producing three different star polymers (star 1, star 2, star 3) which contain varying amounts and sizes of PDMAEMA and/or POEGMA. Using this approach we are the first to delineate the ideal physicochemical properties needed for star polymers (star 3) to deliver siRNA and silence a target gene with high potency in solid tumors in vivo. This highlights the close relationship between the physicochemical properties of star polymers and their biological activity both in vitro and in vivo. In the previous studies which utilized star polymers as siRNA delivery vehicles, this would have been difficult as these star polymers either did not have any PEG or had a very short PEG block, which would have most likely compromised their stability when exposed to serum proteins in vivo. Moreover, their size and charge would have been problematic for delivering siRNA to solid tumors in vivo. For example, Cho et al.11,12 demonstrated that their star polymers when complexed to siRNA had an overall negative charge with zeta potentials ranging from −6.68 mV to −26.3 mV. This property can compromise the ability of star polymers to enter the tumor cells in vitro and in vivo.47 In contrast, when complexed to siRNA our star polymers had a slight positive charge (+12 mV), which may contribute to their highly efficient cell uptake.

microenvironment setting. Mice implanted with subcutaneous pancreatic tumors were administered star 3-βIII-tubulin siRNA intratumorally, which resulted in a 60% reduction in βIIItubulin gene expression (p < 0.05) in pancreatic tumors when compared to controls (Figure 6C). This corresponded with decreased βIII-tubulin protein expression (Figure 6D,E). Importantly, treatment with star 3-βIII-tubulin siRNA reduced tumor growth by 52% (p < 0.02) at the end of the treatment period compared to mice treated with star 3-control siRNA (Figure 6F). Star 3 Delivers siRNA to Pancreatic Tumors in a Clinically Relevant Orthotopic Mouse Model. To assess the ability of star 3 to deliver siRNA to pancreatic tumors in an in vivo model that closely mimics the clinical setting, we established orthotopic MiaPaCa-2 pancreatic tumors in mice. Tumors in this model grow within the pancreas and possess many of the microenvironmental features which are observed in the clinic.46 Mice were administered systemically with star 3 complexed to near-infrared fluorescent siRNA, and 1, 4, and 24 h postinjection, hearts, lungs, livers, spleens, kidneys and tumors were harvested and ex vivo fluorescent intensity measured. Mice injected with PBS or fluorescent siRNA alone served as controls. No fluorescence was detected in any of the organs in mice injected with PBS or fluorescent siRNA (Figure 7A). By contrast, at 1 h postinjection with star 3-fluorescent siRNA, moderate fluorescence was observed in the lungs, livers, and spleens of the mice. The kidneys showed high fluorescence, while a small amount of fluorescence was detected in pancreatic tumors (Figure 7A). At 4 h postinjection with star 3-fluorescent siRNA, high fluorescence was observed in the liver, and moderate fluorescence in the pancreatic tumor (Figure 7A). Strikingly, at 24 h postinjection with star 3-fluorescent siRNA, there was a large accumulation of fluorescence within the pancreatic tumor and very little to no fluorescence in any of the other organs (Figure 7A). siRNA in the pancreatic tumors was confirmed by confocal microscopy, which showed punctate staining in different areas of the tumor (Figure 7B). Importantly, we confirmed that the siRNA was able to extravasate from the vasculature into the surrounding tumor cells (Figure 7C). Similar results were obtained in mice orthotopically injected with another pancreatic cancer cell line (HPAF-II) into the pancreas. These cells are highly metastatic and form aggressive tumors with a prominent extracellular matrix stroma (Figure S19). Notably, despite star 3-siRNA accumulating at the liver and kidneys at early time points postinjection mice did not show any adverse toxicity. Furthermore, histological analysis of hematoxylin and eosin stained sections of the livers, kidneys, lungs and spleens did not show any gross changes in morphology when compared to control mice (not treated with star 3-siRNA) (Figure S20). Collectively, these results suggest that star 3 can deliver siRNA to orthotopic pancreatic tumors in mice. Star 3-βIII-Tubulin siRNA Silences βIII-Tubulin Expression in Orthotopic Pancreatic Tumors in Mice. Finally, we demonstrated that systemic administration of star 3-βIII-tubulin siRNA significantly silenced βIII-tubulin expression by >80%, (p < 0.001) at the gene level in orthotopic pancreatic tumors (Figure 7D). This led to a marked decrease in βIII-tubulin protein expression (Figure 7E,F). To our knowledge this is the first study to report the use of a siRNA-based therapeutic using nonviral nanoparticles to silence βIII-tubulin in tumor cells. This result is exciting and suggests that star-POEGMA polymers may be a promising novel therapeutic strategy to

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CONCLUSIONS In summary, we describe the synthesis and characterization of star polymer nanoparticles using RAFT polymerization for a specific purpose of delivering siRNA to pancreatic tumor cells in vivo to silence a gene which is upregulated in pancreatic cancer cells, and which regulates their growth and metastases, and is undruggable using chemical inhibitors. The starPOEGMA polymers were soluble and stable in water and self-assembled with siRNA to form uniform nanoparticles. When complexed to siRNA the star-POEGMA polymers displayed a superior cytotoxicity profile compared to their non-POEGMA counterparts. We showed that the length of cationic side-arms and amount of POEGMA influence how star polymers are internalized into pancreatic cancer cells, as well as impact their gene silencing activity. We demonstrated that the 2349


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Biomacromolecules

Biomedical Imaging Facility, Mark Wainwright Analytical Centre, UNSW Australia.

incorporation of POEGMA is an essential requirement for allowing star polymers to deliver and release siRNA into the cytosol and silence target gene expression in the presence of serum proteins. Finally, we showed that star-POEGMA polymers efficiently delivered siRNA to orthotopic pancreatic tumors in mice and potently silenced βIII-tubulin expression. These studies provide strong rationale for the continued development of star polymers for the therapeutic delivery of siRNA to pancreatic tumors, as well as other solid tumors which have a similar tumor microenvironment such as liver, ovarian, prostate, and breast.

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(1) Hidalgo, M. Pancreatic cancer. N. Engl. J. Med. 2010, 362 (17), 1605−17. (2) Rana, T. M. Illuminating the silence: understanding the structure and function of small RNAs. Nat. Rev. Mol. Cell Biol. 2007, 8 (1), 23− 36. (3) McCarroll, J.; Teo, J.; Boyer, C.; Goldstein, D.; Kavallaris, M.; Phillips, P. A. Potential applications of nanotechnology for the diagnosis and treatment of pancreatic cancer. Front. Physiol. 2014, 5, 2. (4) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discovery 2008, 7 (9), 771−82. (5) Xiang, S.; Tong, H.; Shi, Q.; Fernandes, J. C.; Jin, T.; Dai, K.; Zhang, X. Uptake mechanisms of non-viral gene delivery. J. Controlled Release 2012, 158 (3), 371−8. (6) Parker, A. L.; Kavallaris, M.; McCarroll, J. A. Microtubules and their role in cellular stress in cancer. Front. Oncol. 2014, 4, 153. (7) McCarroll, J. A.; Sharbeen, G.; Liu, J.; Youkhana, J.; Goldstein, D.; McCarthy, N.; Limbri, L. F.; Dischl, D.; Ceyhan, G. O.; Erkan, M.; Johns, A. L.; Biankin, A. V.; Kavallaris, M.; Phillips, P. A. betaIIITubulin: A novel mediator of chemoresistance and metastases in pancreatic cancer. Oncotarget 2015, 6 (4), 2235−49. (8) Coelho, T.; Adams, D.; Silva, A.; Lozeron, P.; Hawkins, P. N.; Mant, T.; Perez, J.; Chiesa, J.; Warrington, S.; Tranter, E.; Munisamy, M.; Falzone, R.; Harrop, J.; Cehelsky, J.; Bettencourt, B. R.; Geissler, M.; Butler, J. S.; Sehgal, A.; Meyers, R. E.; Chen, Q.; Borland, T.; Hutabarat, R. M.; Clausen, V. A.; Alvarez, R.; Fitzgerald, K.; GambaVitalo, C.; Nochur, S. V.; Vaishnaw, A. K.; Sah, D. W.; Gollob, J. A.; Suhr, O. B. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 2013, 369 (9), 819−29. (9) Senzer, N.; Nemunaitis, J.; Nemunaitis, D.; Bedell, C.; Edelman, G.; Barve, M.; Nunan, R.; Pirollo, K. F.; Rait, A.; Chang, E. H. Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Mol. Ther. 2013, 21 (5), 1096−103. (10) Tabernero, J.; Shapiro, G. I.; LoRusso, P. M.; Cervantes, A.; Schwartz, G. K.; Weiss, G. J.; Paz-Ares, L.; Cho, D. C.; Infante, J. R.; Alsina, M.; Gounder, M. M.; Falzone, R.; Harrop, J.; White, A. C.; Toudjarska, I.; Bumcrot, D.; Meyers, R. E.; Hinkle, G.; Svrzikapa, N.; Hutabarat, R. M.; Clausen, V. A.; Cehelsky, J.; Nochur, S. V.; GambaVitalo, C.; Vaishnaw, A. K.; Sah, D. W.; Gollob, J. A.; Burris, H. A., 3rd First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discovery 2013, 3 (4), 406−17. (11) Cho, H. Y.; Averick, S. E.; Paredes, E.; Wegner, K.; Averick, A.; Jurga, S.; Das, S. R.; Matyjaszewski, K. Star polymers with a cationic core prepared by ATRP for cellular nucleic acids delivery. Biomacromolecules 2013, 14 (5), 1262−7. (12) Cho, H. Y.; Srinivasan, A.; Hong, J.; Hsu, E.; Liu, S.; Shrivats, A.; Kwak, D.; Bohaty, A. K.; Paik, H. J.; Hollinger, J. O.; Matyjaszewski, K. Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery. Biomacromolecules 2011, 12 (10), 3478−86. (13) Duong, H. T.; Jung, K.; Kutty, S. K.; Agustina, S.; Adnan, N. N.; Basuki, J. S.; Kumar, N.; Davis, T. P.; Barraud, N.; Boyer, C. Nanoparticle (star polymer) delivery of nitric oxide effectively negates Pseudomonas aeruginosa biofilm formation. Biomacromolecules 2014, 15 (7), 2583−9. (14) Kim, S. J.; Ramsey, D. M.; Boyer, C.; Davis, T. P.; McAlpine, S. R. Effectively delivering a unique hsp90 inhibitor using star polymers. ACS Med. Chem. Lett. 2013, 4 (10), 915. (15) Pafiti, K. S.; Mastroyiannopoulos, N. P.; Phylactou, L. A.; Patrickios, C. S. Hydrophilic cationic star homopolymers based on a novel diethanol-N-methylamine dimethacrylate cross-linker for siRNA transfection: synthesis, characterization, and evaluation. Biomacromolecules 2011, 12 (5), 1468−79.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.6b00185. Additional data is presented describing the physicochemical and biological characterization of the different starPOEGMA polymers with and without siRNA in vitro and in vivo (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Address: Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, UNSW Australia. Phone: 6129385-2785; E-mail: [email protected] (P.P.). *Address: Tumour Biology and Targeting Program, Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, NSW, Australia; Australian Centre for NanoMedicine, UNSW Australia, NSW Australia. Phone: 6129385-1556; Email: [email protected] (M.K.). Author Contributions ◆

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.P., M.K., D.G., and J.M. are supported by grants and/or fellowships from the National Health and Medical Research Council (NHMRC, APP1024895), Cancer Council New South Wales program grant (M.K.), Cancer Council New South Wales project grant (P.P., J.M., D.G.; RG16-08), Cancer Council New South Wales project grant (P.P., J.M., D.G.; RG153188), Cure Cancer Australia Foundation Grant (P.P.), Balnaves Young Researcher Award (S.S.), Cancer Institute New South Wales Early Career Fellowship (G.S.) and Career Development Fellowship (J.M.), NHMRC Career Development Fellowship (P.P.; APP1024896), NHMRC Senior Research Fellowship (M.K.; APP1058299) and Australian Research Council (ARC) Laureate Fellowship (T.P.D.; FL140100052). J.T. is supported by a UNSW International Postgraduate Research Scholarship. C.B. holds an ARC-Future Fellowship (FT12010096). T.P.D and M.K.’s research is conducted and funded by the ARC Centre of Excellence in Convergent Bio-Nano Science and Technology (Project Number CE140100036). We would also like to acknowledge Mr. Gino Iori for his valuable consumer involvement in this project, Mr. Leo Carol (leocarol@3d images.com.au) for generating the schematic diagram for the star polymers, and the Biological Resources Imaging Laboratory and the 2350


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Biomacromolecules (16) Wu, W.; Wang, W.; Li, J. star polymers: Advances in biomedical applications. Prog. Polym. Sci. 2015, 46, 55−85. (17) Yin, M.; Ding, K.; Gropeanu, R. A.; Shen, J.; Berger, R.; Weil, T.; Mullen, K. Dendritic star polymers for efficient DNA binding and stimulus-dependent DNA release. Biomacromolecules 2008, 9 (11), 3231−8. (18) Boyer, C.; Teo, J.; Phillips, P.; Erlich, R. B.; Sagnella, S.; Sharbeen, G.; Dwarte, T.; Duong, H. T.; Goldstein, D.; Davis, T. P.; Kavallaris, M.; McCarroll, J. Effective delivery of siRNA into cancer cells and tumors using well-defined biodegradable cationic star polymers. Mol. Pharmaceutics 2013, 10 (6), 2435−44. (19) Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.; Jacks, T.; Anderson, D. G. Treating metastatic cancer with nanotechnology. Nat. Rev. Cancer 2012, 12 (1), 39−50. (20) Rettig, G. R.; Behlke, M. A. Progress Toward In Vivo Use of siRNAs-II. Mol. Ther. 2012, 20 (3), 483−512. (21) Ryan, S. M.; Mantovani, G.; Wang, X.; Haddleton, D. M.; Brayden, D. J. Advances in PEGylation of important biotech molecules: delivery aspects. Expert Opin. Drug Delivery 2008, 5 (4), 371−83. (22) Hatakeyama, H.; Akita, H.; Harashima, H. The polyethyleneglycol dilemma: advantage and disadvantage of PEGylation of liposomes for systemic genes and nucleic acids delivery to tumors. Biol. Pharm. Bull. 2013, 36 (6), 892−9. (23) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Water-Soluble Polymers. 81. Direct Synthesis of Hydrophilic StyrenicBased Homopolymers and Block Copolymers in Aqueous Solution via RAFT. Macromolecules 2001, 34 (7), 2248−2256. (24) McCarroll, J. A.; Dwarte, T.; Baigude, H.; Dang, J.; Yang, L.; Erlich, R. B.; Kimpton, K.; Teo, J.; Sagnella, S. M.; Akerfeldt, M. C.; Liu, J.; Phillips, P. A.; Rana, T. M.; Kavallaris, M. Therapeutic targeting of polo-like kinase 1 using RNA-interfering nanoparticles (iNOPs) for the treatment of non-small cell lung cancer. Oncotarget 2015, 6 (14), 12020−34. (25) Phillips, P. A.; Dudeja, V.; McCarroll, J. A.; Borja-Cacho, D.; Dawra, R. K.; Grizzle, W. E.; Vickers, S. M.; Saluja, A. K. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res. 2007, 67 (19), 9407−16. (26) Phillips, P. A.; Sangwan, V.; Borja-Cacho, D.; Dudeja, V.; Vickers, S. M.; Saluja, A. K. Myricetin induces pancreatic cancer cell death via the induction of apoptosis and inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Cancer Lett. 2011, 308 (2), 181−8. (27) Ouyang, H.; Mou, L.; Luk, C.; Liu, N.; Karaskova, J.; Squire, J.; Tsao, M. S. Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype. Am. J. Pathol. 2000, 157 (5), 1623−31. (28) Lee, K. M.; Cao, D.; Itami, A.; Pour, P. M.; Hruban, R. H.; Maitra, A.; Ouellette, M. M. Class III beta-tubulin, a marker of resistance to paclitaxel, is overexpressed in pancreatic ductal adenocarcinoma and intraepithelial neoplasia. Histopathology 2007, 51 (4), 539−46. (29) Sagnella, S. M.; Duong, H.; MacMillan, A.; Boyer, C.; Whan, R.; McCarroll, J. A.; Davis, T. P.; Kavallaris, M. Dextran-based doxorubicin nanocarriers with improved tumor penetration. Biomacromolecules 2014, 15 (1), 262−75. (30) Keddie, D. J.; Moad, G.; Rizzardo, E.; Thang, S. H. RAFT Agent Design and Synthesis. Macromolecules 2012, 45 (13), 5321−5342. (31) Moad, G.; Rizzardo, E.; Thang, S. H. Toward Living Radical Polymerization. Acc. Chem. Res. 2008, 41 (9), 1133−1142. (32) Moad, G.; Rizzardo, E.; Thang, S. H. RAFT Polymerization and Some of its Applications. Chem. - Asian J. 2013, 8 (8), 1634−1644. (33) Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada, Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size. Nat. Nanotechnol. 2011, 6 (12), 815−23. (34) Eliasof, S.; Lazarus, D.; Peters, C. G.; Case, R. I.; Cole, R. O.; Hwang, J.; Schluep, T.; Chao, J.; Lin, J.; Yen, Y.; Han, H.; Wiley, D. T.;

Zuckerman, J. E.; Davis, M. E. Correlating preclinical animal studies and human clinical trials of a multifunctional, polymeric nanoparticle. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (37), 15127−32. (35) Zuckerman, J. E.; Gritli, I.; Tolcher, A.; Heidel, J. D.; Lim, D.; Morgan, R.; Chmielowski, B.; Ribas, A.; Davis, M. E.; Yen, Y. Correlating animal and human phase Ia/Ib clinical data with CALAA01, a targeted, polymer-based nanoparticle containing siRNA. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (31), 11449−54. (36) Moghimi, S. M.; Symonds, P.; Murray, J. C.; Hunter, A. C.; Debska, G.; Szewczyk, A. A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Mol. Ther. 2005, 11 (6), 990−5. (37) Mukherjee, S. P.; Byrne, H. J. Polyamidoamine dendrimer nanoparticle cytotoxicity, oxidative stress, caspase activation and inflammatory response: experimental observation and numerical simulation. Nanomedicine 2013, 9 (2), 202−11. (38) Al Soraj, M.; He, L.; Peynshaert, K.; Cousaert, J.; Vercauteren, D.; Braeckmans, K.; De Smedt, S. C.; Jones, A. T. siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat. J. Controlled Release 2012, 161 (1), 132−41. (39) Skotland, T.; Iversen, T. G.; Torgersen, M. L.; Sandvig, K. Cellpenetrating peptides: possibilities and challenges for drug delivery in vitro and in vivo. Molecules 2015, 20 (7), 13313−23. (40) Rappoport, J. Z. Focusing on clathrin-mediated endocytosis. Biochem. J. 2008, 412 (3), 415−23. (41) Malefyt, A. P.; Walton, S. P.; Chan, C. Endocytosis pathways for nucleic acid therapeutics. Nano LIFE 2012, 2 (3), 1241005. (42) Douglas, K. L.; Piccirillo, C. A.; Tabrizian, M. Cell linedependent internalization pathways and intracellular trafficking determine transfection efficiency of nanoparticle vectors. Eur. J. Pharm. Biopharm. 2008, 68 (3), 676−87. (43) Gilleron, J.; Querbes, W.; Zeigerer, A.; Borodovsky, A.; Marsico, G.; Schubert, U.; Manygoats, K.; Seifert, S.; Andree, C.; Stoter, M.; Epstein-Barash, H.; Zhang, L.; Koteliansky, V.; Fitzgerald, K.; Fava, E.; Bickle, M.; Kalaidzidis, Y.; Akinc, A.; Maier, M.; Zerial, M. Imagebased analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31 (7), 638−46. (44) Sahay, G.; Querbes, W.; Alabi, C.; Eltoukhy, A.; Sarkar, S.; Zurenko, C.; Karagiannis, E.; Love, K.; Chen, D.; Zoncu, R.; Buganim, Y.; Schroeder, A.; Langer, R.; Anderson, D. G. Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling. Nat. Biotechnol. 2013, 31 (7), 653−8. (45) Gao, H.; He, Q. The interaction of nanoparticles with plasma proteins and the consequent influence on nanoparticles behavior. Expert Opin. Drug Delivery 2014, 11 (3), 409−20. (46) Vonlaufen, A.; Joshi, S.; Qu, C.; Phillips, P. A.; Xu, Z.; Parker, N. R.; Toi, C. S.; Pirola, R. C.; Wilson, J. S.; Goldstein, D.; Apte, M. V. Pancreatic stellate cells: partners in crime with pancreatic cancer cells. Cancer Res. 2008, 68 (7), 2085−93. (47) Cho, E. C.; Xie, J.; Wurm, P. A.; Xia, Y. Understanding the role of surface charges in cellular adsorption versus internalization by selectively removing gold nanoparticles on the cell surface with a I2/KI etchant. Nano Lett. 2009, 9 (3), 1080−4.

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A Rationally Optimized Nanoparticle System for the Delivery of RNA

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