A Rationally Optimized Nanoparticle System for the Delivery of RNA

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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 Marie Sagnella, Hien T.T Duong, Jie Liu, George Sharbeen, David Goldstein, Thomas P. Davis, Maria Kavallaris, and Phoebe A. Phillips Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00185 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 16, 2016

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A rationally optimized nanoparticle system for the delivery of RNA

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interference therapeutics into pancreatic tumors in vivo.

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Joann Teo 1,2#, Joshua A. McCarroll 1,2#, Cyrille Boyer 2,3, Janet Youkhana 4, Sharon M. Sagnella

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1,2

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Maria Kavallaris 1,2,6*, Phoebe A. Phillips 2,4*

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1

Tumour Biology and Targeting Program, Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, NSW, Australia.

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2

9 10 11 12

, Jie Liu 4, George Sharbeen 4, David Goldstein

4,5

, Thomas P. Davis

7,8

,

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Centre for Advanced Macromolecular Design, School of Chemical Engineering, UNSW Australia, NSW, Australia. 4

Pancreatic Cancer Translational Research Group, Lowy Cancer Research Centre, Prince of Wales Clinical School, UNSW Australia, NSW, Australia. 5

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6

18

2,3

Australian Centre for NanoMedicine, UNSW Australia, NSW, Australia.

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16 17

, Hien T. T Duong

Prince of Wales Hospital, Prince of Wales Clinical School, Sydney, NSW, Australia.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology UNSW Australia, NSW, Australia. 7

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia. 8

Department of Chemistry University of Warwick, Coventry, United Kingdom.

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# Contributed equally to the study.

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*Corresponding authors: Phoebe. A. Phillips (P.P): Phone: 612-9385-2785; E-mail:

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[email protected];

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[email protected] (M.K.). For P.P: Pancreatic Cancer Translational Research

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Group, Lowy Cancer Research Centre, UNSW Australia. M.K: Tumour Biology and Targeting

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Program, Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Australia, NSW,

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Australia; Australian Centre for NanoMedicine, UNSW Australia, NSW Australia.

Maria

Kavallaris

(M.K):

Phone:

1 ACS Paragon Plus Environment

612-9385-1556;

Email:

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Keywords: Star polymers, RAFT Polymerization, siRNA, pancreatic cancer cells, orthotopic

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pancreatic cancer mouse model, βIII-tubulin. Running title: Star polymer nanoparticles as

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effective delivery vehicles for siRNA to pancreatic cancer cells.

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Conflict of interest disclosure: The authors declare no competing financial interest.

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ABSTRACT

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Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA

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(siRNA) based therapeutics hold promise for the treatment of cancer. However, development of

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efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the

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synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using

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reversible addition-fragmentation chain transfer polymerization (RAFT) for the delivery of

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siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths

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of cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts

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of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-

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POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-

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POEGMA polymers were non-toxic to normal cells and delivered siRNA with high efficiency to

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pancreatic cancer cells to silence a gene (TUBB3/βIII-tubulin) which is currently undruggable

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using chemical agents, and is involved in regulating tumor growth and metastases. Notably,

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systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to

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orthotopic pancreatic tumors in mice and silenced βIII-tubulin expression by 80% at the gene and

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protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for

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the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors.

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INTRODUCTION

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Pancreatic cancer is ranked as the 4th leading cause of cancer-related deaths in Western societies,

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with a dismal 5-year survival rate of 6% 1. This poor prognosis is due to its resistance to

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chemotherapy drugs and aggressive growth/metastases. Hence, there is an urgent need to develop

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novel therapeutic strategies to treat this disease.

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RNA interference (RNAi) is a powerful gene-silencing mechanism that occurs in mammals 2. In

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brief, this mechanism involves short-interfering RNA (siRNA) assembling within a multi-protein

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RNA induced silencing complex (RISC)

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complementary to target mRNA, which results in its degradation and reduced translation 2. RNAi

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can be manipulated by the introduction of chemically synthesized siRNA to suppress genes

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which are involved in regulating human disease 3. An attractive feature for exploiting the

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therapeutic potential of RNAi is its ability to silence any gene, including those difficult to inhibit

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using chemical inhibitors

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therapeutics for the treatment of many types of human disease, including cancer.

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Microtubules are cytoskeletal proteins that comprise α- and β-tubulin heterodimers which

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contribute to essential cellular processes such as maintenance of cell shape, intracellular

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transport and mitosis 6. β-tubulins’ have been extensively studied in cancer given they are the

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target site for tubulin-binding chemotherapy drugs such as taxanes, vinca alkaloids and

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epothilones 6. However, development of drug resistance and potential off-target toxicity has

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limited their efficacy 6. β-tubulin has 7 different isotypes (βI, βII, βIII, βIVa, βIVb, βV and βVI)

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which share high sequence homology, encoded by different genes and are distinguished by their

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unique carboxy terminal tail 6. The different isotypes have tissue-specific expression 6. High βIII-

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tubulin (encoded by the TUBB3 gene) expression has been reported in different tumor types and

3-5

2

. Together, siRNA-RISC binds with perfect

. This has led to an intense research effort to design RNAi


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is associated with poor patient survival and increased chemoresistance 6. Recently, we

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demonstrated that human pancreatic tumors and pancreatic cancer cells expressed high levels of

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βIII-tubulin 7. Importantly, stable suppression of βIII-tubulin using shRNA in pancreatic cancer

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cells reduced tumor growth and metastases in a clinically-relevant orthotopic pancreatic cancer

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xenograft mouse model 7. Moreover, inhibition of βIII-tubulin expression in pancreatic cancer

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cells increased their sensitivity to chemotherapy drugs in vitro 7. Despite the promise of βIII-

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tubulin as a novel therapeutic target for pancreatic cancer, there are no pharmacological agents

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which can specifically inhibit βIII-tubulin and design of such inhibitors is problematic due to its

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high sequence homology with the other β-tubulin proteins. Therefore, a unique opportunity

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exists to develop siRNA-based therapies to selectively silence βIII-tubulin expression in

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pancreatic cancer cells.

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siRNA requires a delivery vehicle to protect it from degradation within the circulatory system

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and allow it to enter cells

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encapsulate or complex siRNA and deliver it to a host of different cell types 5. Nanoparticle-

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siRNA therapies are in clinical trial to treat a number of diseases and have been shown to be safe

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8-10

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to produce; can be synthesized in large-scale amounts with high reproducibility for clinical

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translation; are non-toxic; can self-assemble with siRNA; and have high tumor bioavailability

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and retention time.

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Star polymers have received increased attention over the last several years as delivery vehicles

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for different therapeutic agents

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large-scale quantities, and importantly their structure is well-defined and can be easily tailored

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for a desired application 16. For example, their internal core and /or peripheral side-arms can be

3-5

. To overcome this problem nanotechnology has been used to

. However, despite their potential there is still a need to develop nanomaterials which are easy

11-17

. They are cost-effective to produce, can be synthesized in


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modified to control their size or to incorporate biodegradable components that allow them to be

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processed within a cell

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contain moieties to actively target specific cell types 16. Notably, studies have demonstrated that

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star polymers can be designed to self-assemble DNA or siRNA and deliver it to different cell

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types in vitro. Cho et al.

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. Their side-arms can also be manipulated to increase their stability or

11, 12

showed that star polymers synthesized using atom transfer radical

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copolymerization (ATRP) can complex siRNA or DNA and deliver them to different non-tumor

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cell types in vitro. A study reported by Pafiti et al.

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using “living” polymerization and containing dimethylaminoethyl methacrylate (DMAEMA)

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were able to deliver siRNA to mouse myoblasts and silence a gene with greater efficiency

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compared to linear DMAEMA homopolymers. However, while these studies provided proof-of-

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principle for the use of star-shaped nanoparticles as carriers for siRNA, they did not examine

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their ability to deliver siRNA and silence target gene expression in vivo. Moreover, there have

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been no studies to determine whether star polymers could be used as a delivery vehicle for

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siRNA to solid tumors in mouse models which closely mimic the human setting.

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Recently, we reported on the design and synthesis of cationic star polymers using DMAEMA as

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the monomer via reversible addition-fragmentation chain transfer (RAFT) polymerization, which

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complexed siRNA resulting in nanoparticles with a size of ~ 35 nm

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siRNA complexes were able to deliver siRNA and silence target gene expression in lung and

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pancreatic cancer cells

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(intratumoral) delivery of siRNA to silence gene expression in tumor cells in vivo

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despite their clinical potential for local delivery of RNAi agents, most cancer therapeutics require

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systemic administration in an effort to target both primary and metastatic tumor sites

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limitation of highly charged cationic nanoparticles for systemic delivery of siRNA is their

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showed that star polymers synthesized

18

. These nanoparticle-

. We also showed that star polymers may be used for local


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. However,

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. A

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toxicity and potential to interact with serum proteins present in the bloodstream, which can lead

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to the formation of large aggregates and poor gene-silencing activity. To overcome this problem

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researchers have utilized PEGylation [incorporation of poly(ethylene glycol) (PEG) or

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poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA)] as a strategy to shield the

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positive charge of cationic nanoparticles, thereby reducing toxicity and masking serum protein

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aggregation

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silencing activity by preventing or retarding the ability of siRNA to complex with nanoparticles,

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as well as inhibit cellular uptake and endosomal escape within the cell

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describe the synthesis and characterization of a series of core crosslinked star polymers with

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various compositions. We demonstrate that star-POEGMA-siRNA nanocomplexes are non-toxic

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and act as highly effective delivery vehicles for siRNA to pancreatic cancer cells. We also show

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for the first time that star-POEGMA-siRNA nanoparticles accumulate at high levels in pancreatic

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tumors in mice and potently silence βIII-tubulin expression and decrease tumor growth.

20, 21

. However, it is also recognized that PEGylation can impede siRNA gene

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22

. In this study, we

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MATERIALS AND METHODS

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Materials. Dimethylaminoethyl methacrylate (DMAEMA, 99%, Sigma-Aldrich), oligoethylene

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glycol methylether methacrylate (OEGMA, Sigma-Aldrich) and dimethylaminoethyl acrylate

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(DMAEA, Sigma-Aldrich) were de-inhibited by passing through a basic alumina column. N,N’-

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methylenebisacrylamide (Sigma-Aldrich, 98%) and N,N’-bis(acryloyl)cystamine (Sigma-

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Aldrich, 99%) were used without additional purification. 4-cyanopentanoic acid dithiobenzoate

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was prepared as previously described

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supplementary information). The initiator, 2,2′-azobisisobutyronitrile (AIBN), was crystallized

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twice using methanol. High purity N2 (Linde gases) was used for reaction solution purging. All

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other reactants were used as received without further purification. Dulbecco’s Modified Eagle’s

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Medium (DMEM, Lonza, USA), heat inactivated fetal calf serum (FCS), horse serum and 2 nM

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L-glutamine were purchased from SAFC Biosciences (Lexena, KS, USA). Cell culture

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consumables were obtained from Thermo Fisher Scientific (Lafayette, CO, USA). Lipofectamine

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2000TM was purchased from Life Technologies (Carlsbad, CA, USA). All reagents used to

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measure gene-silencing activity using real-time PCR (qPCR) were purchased from Life

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Technologies (Carlsbad, CA, USA). 18S Quantitect housekeeping gene primer assay was

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purchased from Qiagen (Valencia, CA, USA). BCA protein assay kit was purchased from Pierce

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(Danvers, MA. USA). siRNAs were purchased from GE Lifesciences (Lafayette, CO, USA).

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AlexaFlour-488 conjugated and AlexaFlour-647 conjugated siRNAs for nanoparticle cell uptake

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studies were purchased from Qiagen (Valencia, CA, USA). Primary antibodies: anti-βIII-tubulin

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(clone TUJ1) was purchased from Covance (Richmond, CA, USA), anti-GAPDH (clone 6C5)

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was purchased from Sapphire Bioscience (Waterloo, NSW, Australia) and anti-CD31 was

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purchased from AbCam, Ltd, Australia. Horseradish peroxidase (HRP)-conjugated IgG goat

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anti-mouse secondary antibody was purchased from GE Healthcare (Buckinghamshire, UK).

23

(a brief description of its preparation is provided in the


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Eight well chamber slides were purchased from Ibidi (Martinsried, Germany). ProLong® Gold

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Antifade Reagent with DAPI, LysoTracker® Red DND-99, Hoechst 33342 were purchased from

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Life Technologies (Carlsbad, CA, USA), D-Luciferin potassium salt was purchased from Gold

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Biotechnology (St Louis, MO, USA) and growth-factor reduced matrigel from BD Biosciences

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(San Jose, CA, USA).

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Synthesis of star-POEGMA polymers.

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Synthesis of the POEGMA arm homopolymer. Oligo(ethylene glycol) methyl ether

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methacrylate [OEGMA (MW 300 g/mol), 15.0 g, 0.05 mol], 2,2’-azobisisobutyronitrile (AIBN,

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33.0 mg, 2 × 10-4 mol), 4-cyanopentanoic acid dithiobenzoate (CPADB, 0.310 g, 1.11 × 10-3

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mol) and dry toluene (50 ml) were added to a round bottom flask, (100 ml), and stirred. The

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mixture was then degassed with nitrogen at 0 oC for 1 hour. The solution was stirred at 65 °C for

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14 hours and quenched. An aliquot was then collected and analyzed using GPC and 1H NMR.

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The monomer conversion was ~70%. The mixture was concentrated by rotary evaporation and

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the polymer precipitated in petroleum ether (low boiling point) to remove trace amounts of

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monomer. The purified POEGMA was analyzed by NMR, GPC and UV-visible spectroscopy.

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The molecular weight was calculated by NMR spectroscopy (Mn, NMR = 10,600 g/mol) to be close

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to the theoretical value (Mn, theor. = 10,100 g/mol) and corresponded with GPC results (Mn, GPC =

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11,500 g/mol, PDI = 1.14).

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Synthesis of PDMAEMA homopolymers. Two homopolymers of PDMAEMA were prepared

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by RAFT polymerization with a molecular weight of ~ 5,000 and 10,000 g/mol.

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Dimethylaminoethyl methacrylate (DMAEMA, Mw = 157 g/mol, 7.78 g, 0.05 mol), 2,2’-

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azobisisobutyronitrile (AIBN, 31.8 mg, 1.94 × 10-4 mol), 4-cyanopentanoic acid dithiobenzoate 8 ACS Paragon Plus Environment

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(CPADB, 0.271 g, 9.7 × 10-4 mol) and dry acetonitrile (30 ml) were added to a round bottom

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flask (100 ml), and stirred. The reaction mixture was cooled and degassed using nitrogen at 0 oC

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for 1 hour. The solution was stirred at 65 °C for 14 hours and then quenched. An aliquot was

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collected for GPC and 1H NMR analyses (monomer conversion ~ 70%). The solvent was

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removed using rotary evaporation and the red polymer mixture was dissolved in acetone. The

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polymer was precipitated twice in petroleum ether to remove any traces of monomer. The

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polymer was collected and dried under vacuum. The purified PDMAEMA was analyzed by

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NMR, UV-vis and GPC. The molecular weight was determined using NMR spectroscopy (Mn,

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NMR

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with GPC results (Mn, GPC = 5,100 g/mol, PDI = 1.09).

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A similar procedure was employed for the synthesis of PDMAEMA (Mn = 10,700 g/mol),

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dimethylaminoethyl methacrylate [DMAEMA (Mw =157 g/mol), 7.78 g, 0.05 mol], 2,2’-

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azobisisobutyronitrile (AIBN, 17.0 mg, 1.03 × 10-4 mol), 4-cyanopentanoic acid dithiobenzoate

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(CPADB, 0.145 g, 5.19 × 10-4 mol) and dry acetonitrile (30 ml) were added to a round bottom

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flask (100 ml) and stirred. The reaction was cooled and degassed using nitrogen at 0 oC for 1

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hour. This solution was then stirred for 14 hours at 65 °C. Following this the reaction was

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quenched and an aliquot assessed by GPC and 1H NMR. The monomer conversion was ~70%.

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Rotary evaporation was used to concentrate the mixture and the polymer precipitated twice in

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petroleum ether to remove any traces of monomer. The purified PDMAEMA was analyzed by

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NMR, UV-vis and GPC. The molecular weight was calculated using NMR spectroscopy (Mn,

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NMR

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agreement with the GPC results (Mn, GPC = 10,700 g/mol, PDI = 1.09).

= 5,000 g/mol) was close to the theoretical value (Mn, theor. = 5,600 g/mol) and correlated

= 10,000 g/mol) and was close to the theoretical value (Mn, theor. = 10,500 g/mol) and in good


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Synthesis of PDMAEMA/POEGMA core crosslinked star via the arm first methodology

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(Star 1). PDMAEMA (Mn,

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11,500 g/mol, 5.1 g, 4.43 × 10-4 mol) were added to a vial containing a magnetic stirrer, together

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with AIBN (78 mg, 4.76 × 10-4 mol) and toluene (100 ml). Crosslinker [(N,N’-

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bisacryloyl(cystamine), 3.0 g, 1.15 × 10-2 mol] and dimethylaminoethyl acrylate (DMAEA, 0.80

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g, 5.59 × 10-3 mol) were then added and the vials sealed and purged with nitrogen for 1 hour at 0

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o

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polymerization process, aliquots were collected and analyzed by 1H NMR and GPC. 1H NMR

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analysis confirmed the high conversion of crosslinker and DMAEA. Next, the reaction mixture

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was concentrated under vacuum. Core crosslinked star polymers were precipitated twice in cold

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petroleum ether/diethyl ether mixture (05/95 v/v) to remove the residual arms to yield a yellow

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product (yield ~60%). The purified core crosslinked star polymer was analyzed by GPC (Mn, GPC

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= 125,000 g/mol and PDI = 1.19), FTIR and NMR spectroscopy to determine its composition

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(feed ratio: fOEGMA/fDMAEMA 34/66 mol-% (or 50/50 w-%), final composition: fOEGMA/fDMAEMA

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48/52 mol-% (or 64/36 w-%). The purified core crosslinked star polymer was solubilized in

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methanol and then stored in fridge to avoid irreversible crosslinking. Core crosslinked star

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polymers were dissolved in methanol and dialyzed with water/HCl (pH = 3.0) for 24 hours. A

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further dialysis against water was then carried out for 48 hours. Finally, the solution was freeze

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dried to yield POEGMA/PDMAEMA star polymers and characterized by NMR, FTIR

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spectroscopy, DLS and TEM.

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Synthesis of PDMAEMA/POEGMA star using an arm first approach (Star 2). The

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synthesis of star 2 was carried out using similar techniques as described above for star 1. In brief,

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PDMAEMA (Mn = 10,700 g/mol, 5.0 g, 4.67 × 10-4 mol) and POEGMA (Mn = 11,500 g/mol, 5.0

GPC

= 5,100 g/mol, 5.1 g, 1 × 10-3 mol) and POEGMA (Mn

GPC

=

C. The vials were then incubated in an oil bath for 24 hours at 70 °C. At the end of the


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g, 4.34 × 10-4 mol) were added to a vial containing a magnetic stir bar, together with AIBN (50

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mg, 3.06 × 10-4 mol) and toluene (100 ml). Crosslinker (N,N’-bisacryloyl(cystamine) (1.910 g,

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7.34 × 10-3 mol) and DMAEA (0.500 g, 3.49 × 10-3 mol) were added. The mixture was

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polymerized according to the previous condition. The core crosslinked star polymer was

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precipitated twice in cold mixture petroleum ether/diethyl ether (5/95 v/v) to remove the residual

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arms to yield a yellow product (yield ~65%). The purified core crosslinked star polymer was

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analyzed by GPC (Mn,

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purification to determine its composition (feed ratio: fOEGMA/fDMAEMA 34/66 mol-% (or 50/50 w-

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%), final composition: fOEGMA/fDMAEMA 51/49 mol-% (or 66/34 w-%). The purified core

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crosslinked star polymer was dissolved in methanol and stored in the fridge to avoid irreversible

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crosslinking. Core crosslinked star polymers were solubilized in methanol and dialyzed with

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water/HCl (pH = 3.0) for 24 hours. A further dialysis against water was then carried out for 48

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hours. The resulting solution was freeze dried to yield PDMAEMA/POEGMA core crosslinked

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star polymers and characterized by NMR, FTIR spectroscopy, DLS and TEM.

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Synthesis of PDMAEMA/POEGMA star via the arm-first methodology (Star 3). The

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synthesis of star 3 was carried out using similar techniques as above. In brief, PDMAEMA (Mn =

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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

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mol) were added into a vial containing a magnetic stir bar along with AIBN (33 mg, 2× 10-4 mol)

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and toluene (100 ml). Crosslinker (N,N’-bisacryloyl(cystamine) (1.139 g, 4.38 × 10-3 mol) and

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DMAEA (0.308 g, 2.15 × 10-3 mol) were then added. The mixture was polymerized according to

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the previous conditions. The core crosslinked star polymer was precipitated twice in cold diethyl

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ether/petroleum spirit (90/10 v/v) to remove unreacted arm to yield a yellow product (yield

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~80%). The purified core crosslinked star polymer was analyzed by GPC (Mn,

GPC

= 145,000 g/mol and PDI = 1.29), and by NMR and FTIR after


GPC

= 145,000

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g/mol and PDI = 1.29), and by NMR and FTIR after purification to determine its composition

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(feed ratio: fOEGMA/fDMAEMA 7.7/92.3 mol-% (or 14/86 w-%), final composition: fOEGMA/fDMAEMA

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12.5/87.5 mol-% (or 21.5/78.5 w-%). The purified core crosslinked star polymer was then

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analyzed by GPC (Mn,

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solubilized in methanol and dialyzed with acidic water (pH = 3.0) for 24 hours, and then further

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dialyzed using water (pH = 6.5) for 48 hours. Finally, the freeze dried polymer was analyzed by

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NMR and FTIR.

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Physicochemical characterization of star-POEGMA polymers.

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Gel permeation chromatography (GPC) measurements. DMAc GPC analyses of the

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polymers were performed in N,N-dimethylacetamide [DMAc; 0.03% w/v LiBr, 0.05% 2, 6–di-

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butyl-4-methylphenol (BHT)] at 50 °C (flow rate = 1 mL.min-1) using a Shimadzu modular

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system comprised of an SIL-10AD auto-injector, a PL 5.0-mm bead-size guard column (50 × 7.8

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mm) followed by four linear PL (Styragel) columns (105, 104 and 103). A RID-10A differential

260

refractive-index detector was used. Calibration was achieved using commercial poly(methyl

261

methacrylate) and polystyrene standards ranging from 500 to 106 g/mol. The star polymer

262

molecular weight was determined using poly(methyl methacrylate) calibration. The star polymer

263

solutions were filtered before analysis using 0.45 µm filter.

264

Nuclear magnetic resonance (NMR). The structures of the polymers which were synthesized

265

were analyzed by NMR spectroscopy as described previously by Boyer et al. 18.

266

UV-visible spectroscopy. UV-visible spectra were recorded using methods described previously

267

by Boyer et al. 18.

GPC

= 120,000 g/mol and PDI = 1.35) and by NMR. The polymer was


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268

Dynamic light scattering (DLS). DLS measurements were performed as described previously

269

by Boyer et al.

270

influence of larger aggregates (Table 1). Intensity and volume distributions are also presented in

271

Figure S16. The polydispersity index (PDI) was used to describe the width and size distribution

272

of star polymers with and without siRNA using the DTS software along with a Cumulants

273

analysis of the measured intensity autocorrelation function; it is related to the standard deviation

274

of the hypothetical Gaussian distribution (i.e. PDI = s2/ZD2, where s is the standard deviation and

275

ZD is the Z average mean size).

276

Transmission electron microscopy (TEM). The size and shape of the star-POEGMA polymers

277

were examined using a transmission electron microscope (TEM, JEOL1400) at an accelerating

278

voltage of 100 kV. The star-POEGMA polymers were dispersed in water (1 mg/mL). The

279

solution was deposited onto 200 mesh, holey film, copper grid (ProSciTech) and dried at room

280

temperature for 4 hours before analysis.

281

siRNA binding to star-POEGMA polymers. Agarose gel electrophoresis was used to assess

282

the ability of star polymers (star 1, star 2, star 3) to complex with siRNA as described 18.

283

Biological characterization of star-POEGMA polymers.

284

Cell culture. The human pancreatic cancer cell line MiaPaCa-2 was maintained in DMEM

285

culture medium supplemented with 10% fetal calf serum, 2.5% horse serum, and 2 mM of L-

286

glutamine as described 24-26. Human HPAF-II pancreatic cancer cells were grown in DMEM with

287

10% fetal calf serum and 2mM of L-glutamine as described

288

human pancreatic ductal epithelial (HPDE) cells (a kind gift from Ming Tsao, Ontario Cancer

289

Institute) were grown in keratinocyte-serum-free (KSF) medium supplemented with 50 mg/ml

18

. The number-average hydrodynamic particle size was reported to reduce the


24-26

. Normal non-tumorigenic

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290

bovine pituitary extract (BPE) and 5 ng/ml epidermal growth factor (EGF) as previously

291

described 24, 27. All cells were regularly tested for mycoplasma and were negative.

292

Toxicity analysis. The toxicity profile of star POEGMA-siRNA complexes were examined in

293

MiaPaCa-2 (1.1×105) cells. In brief, cells were plated into 6-well plates the day (16 hours) before

294

transfection. The next day, cells were transfected with star, star 1, star 2 or star 3 at increasing

295

w/w ratios with siRNA (100nM). Cells incubated in cell culture media alone served as controls.

296

Cells were trypsinized, collected, and trypan blue exclusion studies were performed to determine

297

the number of viable cells 24 hours post-transfection.

298

siRNA transfection. To examine star-POEGMA-siRNA gene silencing activity, MiaPaCa-2 and

299

HPAF-II cells were transfected with siRNA targeted against the microtubule protein, βIII-tubulin

300

(encoded by the TUBB3 gene) which is expressed at high levels in pancreatic cancer cells

301

Briefly, cells were seeded into 6-well plates (1.1×105) the day prior to transfection. Cells were

302

then transfected with increasing amounts of star-POEGMA-siRNA complexes [8:1 – 30:1 (w/w)

303

ratio

304

GUACGUGCCUCGAGCCAUUUU-3′, antisense: 5′-UUCAUGCACGGAGAGCUCGGUAA-

305

3′.

306

GCUAUGGCUGAAUACAAAUUUU-3′, antisense: 5′-UUCGAUACCGACUUAUGUUUAA-

307

3′ or cells transfected with TUBB3 siRNA (also referred to as βIII-tubulin siRNA) complexed to

308

Lipofectamine 2000 (L2K) served as positive and negative controls respectively. Total RNA and

309

whole cell lysates were collected 72 hours post-transfection, and βIII-tubulin mRNA and protein

310

levels measured using qPCR and western blotting as described 24.

with

Cells

siRNA].

treated

with

TUBB3

siRNA

non-silencing

(100nM)

(Control)

siRNA


sense

sense

strand:

strand:

24, 28

.

5′-

5′-

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311

Intracellular uptake. To observe the cellular uptake and pattern of distribution of star-

312

POEGMA-siRNA complexes, MiaPaCa-2 cells were seeded into 8 well chambers slides

313

(1.2×103 cells) the day prior to transfection. The next day, cells were transfected with

314

fluorescently labeled (AlexaFlour-488, green) siRNA (100 nM) complexed to star-POEGMA

315

polymers (star 1, star 2, star 3) for 4 hours at 37°C. Cells transfected with fluorescent siRNA

316

complexed to L2K or fluorescent siRNA by itself served as positive and negative controls

317

respectively. Twenty four hours post-transfection, cells were incubated with 1 µM

318

LysoTracker® (diluted with culture medium) for 1 hour. The cells were washed 3 times in warm

319

PBS for 5 minutes, and then fixed with 4% paraformaldehyde for 3 minutes. Slides were

320

mounted using ProLong® Gold Antifade Reagent with DAPI. Uptake of siRNA was visualized

321

as previously described by Boyer et al. 18.

322

Real-time PCR (qPCR). βIII-tubulin (TUBB3) mRNA expression in MiaPaCa-2 and HPAF-II

323

cells was measured using qPCR as described

324

gene 18S (18S Quantitect primer assays, Qiagen).

325

Western blotting. Seventy two hours post-transfection with star-POEGMA-βIII-tubulin siRNA,

326

MiaPaCa-2 and HPAF-II cells were harvested and βIII-tubulin and GAPDH expression was

327

measured in whole cell lysates as previously described 24.

328

Inhibition of endocytosis. To examine which endocytic pathways may be involved in the

329

internalization of star-POEGMA-siRNA, MiaPaCa-2 cells were plated into 8 well chambers

330

slides (1.2×103 cells) the day before transfection. The next day, MiaPaCa-2 cells were incubated

331

with a clathrin-dependent inhibitor chlorpromazine (5 µg/ml) or two different clathrin-

332

independent inhibitors, methyl-β-cyclodextrin (MβCD, 5 mM) and genestein (200 µM) for 2

24

. All data were normalized to the housekeeping


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Page 16 of 51

333

hours. The specificity and effectiveness of each inhibitor was confirmed as previously described

334

by our laboratory

335

siRNA (red) complexed to star-POEGMA polymers (star 1, star 2, star 3) for 1 hour at 37°C. The

336

nucleus was then labeled with Hoechst 33342 (blue). Live cell images were taken to visualize

337

siRNA uptake using a Zeiss LSM 780 confocal microscope.

338

Pancreatic cancer mouse models. i) Subcutaneous model: Eight-week old BALB/c nude mice

339

were used. All animal experiments were approved by the Animal ethics committee, UNSW

340

Australia (ACEC 13/130B). In brief, 4x106 MiaPaCa-2 cells were injected subcutaneously into

341

the flank of mice as previously described

342

were randomized into treatment groups and administered intratumorally with star 3-βIII-tubulin

343

siRNA (2mg/kg) twice weekly for 2 weeks. Mice injected with star 3-control siRNA served as

344

controls. Tumor size was measured twice weekly as previously described

345

experiment, tumors were harvested and RNA and protein lysates prepared as previously

346

described

347

Bioanalyzer with an RNA integrity number (RIN) a value >7. βIII-tubulin expression was

348

measured by qPCR, western blotting and immunohistochemistry as described

349

model: Eight-week old BALB/c nude mice were used. In brief, human pancreatic cancer cells

350

(MiaPaCa-2 and HPAF-II) were xenografted orthotopically into the tail of the pancreas as we

351

have described previously 24-26.

352

Star 3-siRNA biodistribution in Vivo. To examine the biodistribution of siRNA complexed to

353

star 3 in vivo, mice with orthotopic pancreatic MiaPaCa-2 (6 weeks post-tumor cell implantation)

354

or HPAF-II (3 weeks post-tumor implantation) tumors were administered (via the tail vein) with

355

star 3 complexed to fluorescently-labeled siRNA (AlexaFluor-647, Red). Mice injected with

24

29

. Subsequently, the cells were transfected with AlexaFlour-647 labeled

18

. Once tumors reached the size of ≥50 mm3, mice

18

. At the end of the

. Total RNA integrity was confirmed using an Agilent Technologies 2100


24

. ii) Orthotopic

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356

PBS or siRNA alone served as controls. One, four and twenty-fours post-injection mice were

357

humanely euthanized and hearts, lungs, livers, spleens, kidneys and pancreatic tumors were

358

collected and infrared fluorescence measured using the IVIS imaging system as described

359

After imaging, pancreatic tumor tissue was snap-frozen in OCT embedding media and siRNA

360

uptake

361

immunofluorescence staining and confocal microscopy.

362

Gene-silencing activity of star 3 in vivo. To examine the gene-silencing activity of star 3-βIII-

363

tubulin in vivo, MiaPaCa-2 pancreatic tumors were established as described above. Six-weeks

364

post-tumor cell injection mice were randomized into treatment groups and administered

365

systemically (via the tail vein) with star 3-βIII-tubulin siRNA (4mg/kg) once daily for 3 days.

366

Mice treated with control siRNA served as controls. Twenty four hours after the final injection

367

pancreatic tumors were harvested and βIII-tubulin expression measured by qPCR, western

368

blotting and immunohistochemistry as described 24.

369

Statistics. Data are presented as the means of at least three independent experiments ± standard

370

error of the mean (SEM) (error bars). Graphpad Prism-6 was used to perform ANOVA or paired

371

two-tailed Student’s t tests followed by Tukey’s or Dunnet post tests were used to measure

372

statistical differences between experimental and control groups, with p < 0.05 considered

373

statistically significant.

and

extravasation

from

tumor

vessels

in

tissue

374


sections

visualized

24

.

by

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375

RESULTS AND DISCUSSION

376

Synthesis and characterization of star polymers. The core crosslinked star-POEGMA

377

polymers were prepared using an arm-first approach and living radical polymerization

378

(reversible addition fragmentation chain transfer polymerization, RAFT) 30-32. In a first step, the

379

synthesis of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA) and

380

poly(dimethylaminoethyl methacrylate), (PDMAEMA) was carried out using 4-cyanopentanoic

381

acid dithiobenzoate as the chain transfer agent and 2,2’-azobisisobutyronitrile (AIBN) as the

382

radical initiator. The chosen POEGMA polymer is closely related to poly(ethylene glycol) (PEG)

383

and has similar low-fouling properties. The polymerizations were performed for 14 hours at 65oC

384

to yield a monomer conversion of around ~70%. The ratio between the monomer and RAFT

385

agent was varied to control the molecular weight of polymers. PDMAEMA with two different

386

molecular weights, 5,100 and 10,700 g/mol and POEGMA with a molecular weight of 11,500

387

g/mol were prepared with a narrow molecular weight distribution (PDI < 1.20) (Figure S1-S3).

388

After several rounds of purification by precipitation, homopolymer chains were extended using a

389

diacrylamide monomer [(N,N'-bis(acryloyl)cystamine)] as a crosslinker, AIBN as the initiator

390

and dimethylaminoethyl acrylate (DMAEA) as a co-monomer at 70oC in toluene for 24 hours

391

(Figure S4). To obtain the greatest incorporation of arm into the star polymers with the lowest

392

molecular weight distribution, we used a [Arm]:[DMAEA]:[crosslinker] ratio of ~ 1:4:8. High

393

monomer and crosslinker conversion (≥85% for both monomers) were observed by NMR

394

analysis after 24 hours (Figure S5 exhibits a typical NMR of unpurified star polymer). GPC

395

analysis showed the formation of high molecular weight polymer, which confirmed the

396

formation of core crosslinked star polymers, but also some residual arms (Figures S6-S8). After

397

additional purification by precipitation in petroleum ether/diethyl ether mixture (the composition

398

is optimized according to the star) to remove unreacted arm, the different star-POEGMA 18 ACS Paragon Plus Environment

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Biomacromolecules

399

polymers were further characterized by NMR, FTIR and GPC to confirm their structures. Three

400

different star polymers [Star 1 (48 mol-% POEGMA with short-cationic side arms), Star 2 (51

401

mol-% POEGMA with long-cationic side arms) and Star 3 (12.5 mol-% POEGMA with long-

402

cationic side arms)] were obtained (Figure 1). GPC demonstrated the synthesis of star-POEGMA

403

polymers with a low molecular weight distribution (PDI < 1.35) (Figure 2A and Table 1), whilst

404

NMR confirmed the presence of (CH3)2N (DMAEMA) and OCH3 (POEGMA) groups at 2.3

405

ppm and 3.3 ppm, respectively (Figures S9-S11). The molar composition was determined using

406

the following equation: f

407

IOCH3correspond to the dimethylamino group (before quaternization) at 2.3 ppm and methoxy

408

groups (OCH3) at ~3.4 ppm, respectively (Figures S9-S11). FTIR showed the incorporation of

409

crosslinker by the characteristic amide band at 1,660 cm-1, while ether (O-C) and tertiary amine

410

(N-C) bands appeared at 1,150 and 1,230 cm-1 (Figure S12). Subsequently, the star-POEGMA

411

polymers were quaternized using an acidic aqueous solution ([HCl] = 10-3M, pH = 3.0). After

412

purification by dialysis to remove unreacted HCl, the star-POEGMA polymers were freeze dried

413

and analyzed by NMR using deuterium oxide (Figure 2B and Figures S13-S15) and by FTIR

414

(Figure 2C). Figure 2B showed the presence of quaternized tertiary amine at 2.9 ppm, while

415

FTIR confirmed the presence of ester [(C=O)O], amide [(C=O)N], quaternized tertiary amine

416

(N-C) and ether (O-C) bands at 1,730, 1,650, 1,230 and 1,150 cm-1 (Figure 2C). After re-

417

dispersion in water, we measured the size of the star-POEGMA polymers using dynamic light

418

scattering. All three star-POEGMA polymers were soluble in water and displayed sizes ranging

419

from 25-35 nm (±5-10 nm) (Table 1). Figure 2D displays the number distribution, while Figure

420

S16 shows the volume and intensity distribution, and the presence of small aggregates.

421

Transmission electron microscopy confirmed the formation of small spherical nanoparticles

DMAEMA

= (I(CH3)2/6)/ [(IOCH3 /3) + (I(CH3)2/6)], where I(CH3)2 and


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Page 20 of 51

422

(Figure 2E). Collectively, these results demonstrated the successful synthesis of three star

423

polymers with different structural properties including, length of cationic side-arms and

424

percentage incorporation of POEGMA.

425

siRNA binding efficiency of star-POEGMA polymers. To examine how the length of the

426

cationic side-arms and percentage of POEGMA would effect star polymer capacity to bind

427

siRNA, increasing amounts of star 1 (short cationic side-arm and 48 mol-% POEGMA), star 2

428

(long cationic side-arm and 51 mol-% POEGMA) or star 3 (long cationic side-arm and 12.5 mol-

429

% POEGMA) were mixed with siRNA. The complexes of star-POEGMA-siRNA were then

430

separated on an agarose gel. siRNA alone travelled to the cathode (bottom of gel) due to its high

431

negative charge (Figure 3A-C). siRNA complexed to either star 1 or star 2 [18:1 (w/w) ratio] did

432

not migrate suggesting that complexation between the siRNA and the star-POEGMA polymers

433

had been achieved (Figure 3A and 3B). Star 3 complexed with siRNA [10:1 (w/w) ratio] more

434

readily compared to star 1 and star 2 (Figure 3C). Indeed, the amount of star 3 needed to

435

complex siRNA was not too dissimilar to star polymers without POEGMA (star) (Figure 3C).

436

Previously, we had shown that star complexed siRNA at an 8:1 (w/w) ratio

437

difference in siRNA binding efficiency between the POEGMA star polymers (star 1, star 2, star

438

3) and non-POEGMA star polymers (star) was most likely due to steric hindrance of POEGMA

439

and overall reduction in net positive surface charge due to the presence of the POEGMA. For

440

example, star polymers without POEGMA had a zeta-potential of +50 mV 18, while, star 1 had a

441

zeta potential of +18 mV (±8 mV), star 2 and star 3 +25 mV (±10 mV) (Table 1). After

442

complexation with siRNA, the zeta potential of all three star-POEGMA polymers decreased to

443

+9 mV (±4 mV) for star 1, +18 mV (±8 mV) for star 2, and +12 mV (±5 mV) for star 3,

444

indicating that the siRNA and star-POEGMA polymers formed a complex via an electrostatic 20 ACS Paragon Plus Environment

18

. Hence, the

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Biomacromolecules

445

interaction (results not shown). Notably, the size of each star-POEGMA nanoparticle when

446

complexed to siRNA did not significantly change. Star 3 before complexing siRNA had an

447

overall size of 35 nm (±10 nm) (Table 1), and after complexing siRNA the size was slightly

448

increased to 38 nm (±10 nm) (results not shown). The size and surface charge of the star-

449

POEGMA-siRNA complexes are ideal for taking advantage of the disrupted tumor vasculature

450

observed in many different types of solid tumors. Most recently, studies have reported that

451

nanoparticle-siRNA complexes with a size between 50-100 nm, and which contain a slight

452

positive surface charge are able to penetrate solid tumors via passive delivery and the ‘enhanced

453

permeability and retention effect’ in pre-clinical animal models

454

show that star-POEGMA polymers are able to effectively self-assemble with siRNA to form

455

small uniform nanoparticles, and whilst the length of the cationic side-arm does not influence the

456

ability of star polymers to interact with siRNA, the amount of POEGMA does have an impact on

457

their ability to form a complex with siRNA.

458

Pancreatic cancer cell uptake of star-POEGMA polymers. To assess the ability of star-

459

POEGMA polymers to deliver siRNA to pancreatic cancer cells, we transfected MiaPaCa-2 cells

460

with star 1, star 2 or star 3 complexed to fluorescently labeled (AlexaFlour 488, Green) siRNA,

461

and examined its pattern of distribution 24 hours post-transfection using confocal microscopy.

462

Additionally, to assess whether siRNA could be released into the cell cytosol, we also examined

463

the co-localization of siRNA with endosomes / lysosomes using the LysoTracker®-Red stain 24

464

hours post-transfection. Confocal microscopy demonstrated that all three star-POEGMA

465

nanoparticles could deliver fluorescent siRNA into the cytoplasm of pancreatic cancer cells

466

(Figure 3D; panels I, II and III). Interestingly, lysoTracker® staining and high power

467

magnification confocal microscope images of MiaPaCa-2 cells transfected with star 3-siRNA 21 ACS Paragon Plus Environment

33-35

. Together, these results

Biomacromolecules

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468

showed that siRNA which were internalized into the cells had markedly less co-localization [as

469

indicated by free siRNA (Green)] with endosomes [(Red), indicating endosomal release)] (Figure

470

3D panel VI) when compared to siRNA delivered by star 1 or star 2 (evidenced by yellow

471

punctate dots in the cytoplasm which indicate co-localization of siRNA with endosomes) (Figure

472

3D panels IV, V). This result suggests that the physicochemical properties of star 3 (long

473

cationic side-arms and 12.5 mol-% POEGMA) may allow for more siRNA to be released from

474

within endosomes allowing it to enter the cell cytoplasm. However, it is also possible that star 3-

475

siRNA complexes may utilize different intracellular trafficking pathways compared to star 1 and

476

2 within MiaPaCa-2 cells that allow the complexes to be rapidly disassembled and/or degraded

477

or recycled by endosomal membrane receptors. Future work in our laboratory will further

478

characterize the internalization and trafficking processes of star 3 in pancreatic cancer cells.

479

Cell toxicity profile of star-POEGMA polymers. Based on our data showing that the

480

incorporation of POEGMA did not adversely affect the ability of star polymers to interact with

481

siRNA and deliver it to pancreatic cancer cells, we next examined if the presence of POEGMA

482

could influence their cytotoxicity profile. MiaPaCa-2 cells were transfected with increasing

483

amounts of star 1, star 2 or star 3 complexed to non-functional siRNA (100 nM) [8:1 – 30:1

484

(w/w) with siRNA]. As a comparison, cells were transfected with increasing concentrations (w/w

485

ratio with siRNA) of non-POEGMA star polymers bound to siRNA (star). The viability of the

486

cells was assessed 24 hours post-transfection by phase contrast microscopy (data not shown) and

487

by analysis of cell counting using trypan blue. Previously, we had shown that star polymers

488

without POEGMA when complexed to siRNA were non-toxic in cancer cells at an 8:1 (w/w)

489

ratio

490

cells (Figure 4A-C). However, at the higher concentrations, star polymers without POEGMA

18

. We confirmed this result showing at 8:1 (w/w) ratio, star was non-toxic to MiaPaCa-2


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491

(star) showed marked toxicity in the cancer cells with a 70% decrease in cell viability at the 20:1

492

(w/w) ratio after 24 hours (Figure 4A-B). In contrast, both star 1 and star 2 complexed to siRNA

493

displayed no toxicity at the 20:1 (w/w) ratio (Figure 4-B). At 30:1 (w/w) there was no toxicity in

494

MiaPaCa-2 cells transfected with star 1-siRNA, and only a very slight decrease (≈15%) in cell

495

viability in cells transfected with star 2-siRNA (Figures 4A-B). In contrast, a 90% decrease in

496

cell viability was observed in cells treated with the star polymers without POEGMA complexed

497

to siRNA (Figure 4A-B). Star 3 was also non-toxic at all of the examined concentrations [8:1 –

498

18:1 (w/w)] (Figure 4C). Similar results were obtained using a colorimetric cell proliferation

499

assay (results not shown). These results are in accordance with a previous study reported by Cho

500

et al.

501

copolymerization (ATRP) and containing a short PEG block (2KDa) were also less toxic in cells

502

when compared to cationic nanoparticles. The increased toxicity observed with star polymers

503

without POEGMA at the higher concentrations is not unexpected and is typical for most highly

504

charged cationic nanoparticles. Both poly(amidoamine) (PAMAM) dendrimers and highly

505

branched polyethylenimine (PEI) polymers possess positively charged surface groups, and have

506

been reported to destabilize cell surface membranes leading to mitochondrial-mediated cell death

507

36, 37

508

significantly improves their cytotoxic profile.

509

Star-POEGMA-siRNA gene silencing activity in pancreatic cancer cells. To determine if

510

star-POEGMA nanoparticles were able to release siRNA into the cytosol of pancreatic cancer

511

cells and induce post-transcriptional gene silencing, we transfected MiaPaCa-2 cells with

512

increasing amounts of star 1, star 2 and star 3 complexed with siRNA (100nM) targeted against

513

the microtubule protein βIII-tubulin. At an 8:1 (w/w) ratio with βIII-tubulin siRNA both star 1

12

which showed that star polymers synthesized using atom transfer radical

. Together, these results demonstrate that the incorporation of POEGMA into star polymers


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514

and star 2 did not silence βIII-tubulin expression (Figure 4D-E). This result is expected based on

515

the fact that both nanoparticles failed to complex with siRNA at the 8:1 (w/w) ratio (Figure 3A-

516

B). However, at the 30:1 (w/w) ratio, star 1-βIII-tubulin siRNA significantly silenced βIII-

517

tubulin gene expression in MiaPaCa-2 cells by 44% (p50% (p

A Rationally Optimized Nanoparticle System for the Delivery of RNA

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