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Comparing gene silencing and physiochemical properties in siRNA bound cationic star-polymer complexes Megan Dearnley, Nicholas P. Reynolds, Peter Cass, Xiaohu Wei, Shuning Shi, A. Aalam Mohammed, Tam Le, Pathiraja Gunatillake, Mark L. Tizard, San H. Thang, and Tracey M. Hinton Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01029 • Publication Date (Web): 06 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016

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Biomacromolecules

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Comparing gene silencing and physiochemical

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properties in siRNA bound cationic star-polymer

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complexes

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Megan Dearnley1‡, Nicholas P. Reynolds2‡, Peter Cass3, Xiaohu Wei 3,4, Shuning Shi1, A. Aalam

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Mohammed1, Tam Le3, Pathiraja Gunatillake3*, Mark L. Tizard1, San H. Thang3*, Tracey M.

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Hinton1*

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‡

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*Corresponding Authors

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1

These authors contributed equally

CSIRO-Health and Biosecurity Business Unit, Australian Animal Health Laboratory, 5

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Portarlington Road, Geelong, Vic 3220 Australia.

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2

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3122, Australia.

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3

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4

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Beijing 100029, China

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Keywords Gene-Silencing, siRNA, quantitative nanoscale mechanical-AFM, Nanoparticle

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Tracking Analysis, siRNA-polymer complex stiffness, siRNA-polymer complex, Cell Uptake,

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Protein Corona

ARC Training Centre for Biodevices, Swinburne University of Technology, Hawthorn, Vic

CSIRO-Manufacturing Business Unit, Bayview Avenue, Clayton, Vic 3168 Australia. College of Materials Science and Engineering, Beijing University of Chemical Technology,

ACS Paragon Plus Environment

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ABSTRACT

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The translation of siRNA into clinical therapies has been significantly delayed by issues

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surrounding the delivery of naked siRNA to target cells. Here we investigate siRNA delivery by

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cationic acrylic polymers developed by Reversible Addition Fragmentation chain Transfer

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(RAFT) mediated free radical polymerization. We investigated cell uptake and gene silencing of

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a series of siRNA-star polymer complexes both in the presence and absence of a protein

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“corona”. Using a multidisciplinary approach including quantitative nanoscale mechanical-

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atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis we have

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characterized the nanoscale morphology, stiffness and surface charge of the complexes with and

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without the protein corona. This is one of the first examples of a comprehensive physiochemical

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analysis of siRNA-polymer complexes being performed alongside in vitro biological assays,

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allowing us to describe a set of desirable physical features that promote gene silencing in

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cationic polymer complexes. Multi-faceted studies such as this will improve our understanding

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of structure-function relationships in nano-therapeutics, facilitating the rational design of

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polymer-mediated siRNA delivery systems for novel treatment strategies.

35 36 37 38 39 40


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Introduction

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Therapies based on gene silencing through small interfering (si)RNAs have the potential to

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revolutionize the treatment of a number of genetic and viral diseases, including influenza,1-3

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respiratory syncytial virus,4 and dengue virus.5 However, the development of clinically viable

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therapeutics is limited due to a lack of reliable methods for the safe and effective delivery of

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siRNA to specific tissues and target cells. Several key problems surrounding robust siRNA

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delivery are yet be overcome, including protection of the siRNA from RNAase degradation in

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body fluids, efficient cell uptake and proficient release of siRNA within the cell cytoplasm.

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In an effort to address these issues, there has been a rapid development around technologies that

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function to protect siRNA and enhance cellular delivery. Conjugation of siRNA to nano-carriers,

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such as polymer complexes, has led to reports of successful delivery and siRNA-mediated

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treatment in human cancer studies.6 Similarly, siRNA therapeutic approaches delivered within

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‘smart’ lipoplexes7 and linear, cationic cyclodextrin-based polymers8,9 have also entered clinical

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trials. Cationic polymers show promise for siRNA delivery10 as they can assemble into

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complexes through interactions with anionic phosphates on the siRNA. The resultant polymer

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complex offers physical protection of the siRNA from RNAse degradation and enhances cellular

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uptake.11

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siRNA uptake efficiency in relation to polymer architecture has been investigated in studies

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including di-blocks,12 linear ABA triblocks,13 polymeric micelles,14 dendrimers15 and star

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polymers.16-22 The efficiency by which a siRNA-polymer complex will achieve cellular uptake

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has been shown to depend on a combination of factors, including the surface chemistry, polymer

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architecture and physical properties of the complexes.23 Additional studies have indicated that


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star polymers are more efficient at nucleic acid delivery than equivalent linear polymers due to a

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combination of factors including a smaller hydrodynamic radius, increased molecular mass, low

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solution viscosity, high charge density, and abundant internal and peripheral groups for

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functionalization.19,24 For example, Pafiti et al.25 showed that star homopolymers of poly 2-

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dimethlyamino ethyl methacrylate (pDMAEMA) with the cross-linker

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bis(methacryloyloxyethyl)methylamine (BMEMA) performed better than equivalent linear

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polymers, with greater gene silencing and lower toxicity due to the formation of more compact,

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stable complexes.

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Oligoethylene based star polymer systems have been used to demonstrate that an increasing

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molecular mass improves serum stability, prolongs blood circulation times and enhances

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distribution of the polymer to multiple organs.26-2822,23 For polymer-mediated DNA delivery, the

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optimal molecular mass of the delivery vector was assessed to be between 50 and 100 kDa.21

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However, increasing molecular mass can also increase cellular toxicity.29 Therefore there is a

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fine balance between optimal transfection efficiency and cytotoxicity.

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In addition to architecture, high cellular uptake is dependent on a complicated relationship

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between complex size, shape, stiffness and nonspecific interactions with the cell (i.e.

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hydrophobic, electrostatic and Van der Waals forces).23 Yi et. al.28 developed a theoretical model

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that predicts that the endocytosis of a nanoscale particle is highly sensitive to the ratio of

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stiffness between the particle and the cell membrane. That is, as the particle becomes softer

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endocytosis becomes more difficult to complete. This effect can be explained by the fact that

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softer particles will undergo increased deformation and spread to a greater extent on the

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extracellular side of the cell membrane, creating a large bending energy barrier that must be

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overcome for the particle to complete endocytosis. The tendency for stiffer particles to be more


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successfully endocytosed has also been demonstrated experimentally for complexes with a lipid

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core-shell structure,30 hydrogel nanopartices30 and by comparing solid particles with fluid

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vesicular particles.31

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Complex size and surface chemistry can also be strongly affected by the adsorption of proteins

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and other biomolecules from the biological milieu.32,33 The protein “corona”, which forms

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around the complex upon contact with biological fluid, can also affect cellular uptake. In some

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instances, adsorbed biomolecules interact with receptor proteins on the cell membrane to

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enhance cell uptake,34 whilst in instances uptake is reduced.35 Thus, it is important to consider

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the effect of the protein corona when performing in vitro measurements of cellular

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internalisation.

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Whilst the chemistry and physical properties of a range of complexes has been investigated,23

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there are few studies which perform a detailed comparison between the physical parameters of a

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complex, such as size and stiffness, and its ability to elicit the targeted action of a cargo

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therapeutic agent. Our laboratory recently reported the synthesis and characterization of mikto

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star polymers prepared by combining hydrophobic [poly(n-butylmethacrylate), P(BMA)],

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cationic [poly(2-dimethlyamino ethyl methacrylate), P(DMAEMA)], and hydrophilic

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[poly(oligoethylene glycol methacrylates), P(OEGMA)] arms using Bis(2-methacryloyl)oxyethyl

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disulfide (DSDMA) cross linker to form the core of the star polymer.36 This formulation was

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designed to include disulfide bonds in the cross-linking bridge, allowing for polymer

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degradability.36 Similarly, a 4 arm star containing disulfide bonds in the RAFT core was

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produced by the core first approach and characterized by our laboratory.17


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Here we compare the in vitro gene silencing ability of the mikto star and 4-arm star polymers

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with and without the hydrophobic BMA block present. Complex stability, cellular uptake and

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gene silencing ability of the siRNA cargo was compared with the nanoscale morphology, surface

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charge and stiffness of the polymer complexes. All experiments were performed both in the

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presence and absence of foetal bovine serum (FBS) to model protein corona forming conditions

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in vitro.

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From our detailed quantitative nanoscale analysis we have identified the best performing

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complex and suggest a combination of physiochemical properties that are likely to promote gene

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silencing in similar cationic polymer-nucleic acid complexes. To our knowledge this is the first

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time that both gene silencing and a comprehensive physiochemical analysis of a range of

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polymeric siRNA complexes derived from RAFT polymerization has been investigated

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simultaneously, allowing us to identify a subset of physiochemical characteristics important for

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efficient siRNA delivery.

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Materials and Methods

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Materials:

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Oligo(ethylene glycol) methacrylate (OEGMA8-9, Mn ~0.475 kDa ), 2-(dimethylamino)ethyl

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methacrylate (DMAEMA), n-butyl methacrylate (BMA), monomers were purchased from Sigma

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Aldrich and purified by stirring in the presence of inhibitor-remover (for hydroquinone or

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hydroquinone monomethyl ether for 30 min prior to use. Methacryloxyethyl thiocarbamoyl

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rhodamine B (PolyFluor 570, PF) monomer was purchased from Polysciences and used as

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received. 4-Cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (DTTCP),37

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disulfide dimethacrylate (DSDMA) cross-linker were prepared according to the previously


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published methods. 1,1’-azobis(cyclohexanecarbonitrile) (ACHN, DuPont Vazo 88), 4,4'-

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azobis(4-cyanovaleric acid) (ACVA) initiator, tributylphosphine (Bu3P, Aldrich) reducing agent

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and methyl iodide (Sigma Aldrich) were used as received. N,N-dimethylformamide (DMF),

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dichloromethane (DCM), n-heptane, diisopropyl ether, methanol, and other chemicals were

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purchased as commercial reagents and used without further purification. Spectra/Por Dialysis

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membrane MM cutoff of 3.5 kDa and 25 kDa were obtained from Spectrumlabs.

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Polymer characterization:

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Proton nuclear magnetic resonance (1H NMR) spectra were obtained with a Bruker Avance 400

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MHz spectrometer (1H 400 MHz). Gel permeation chromatography (GPC) measurements of

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polymers were performed on a Shimadzu system equipped with a CMB-20A controller system, a

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SIL-20A HT autosampler, a LC-20AT tandem pump system, a DGU-20A degasser unit, a CTO-

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20AC column oven, a RDI-10A refractive index detector with 4 Waters Styragel columns (HT2,

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HT3, HT4, HT5 each 300 × 7.8 mm) providing an effective molar mass range of 100−4×106, and

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with N,N-dimethylacetamide (DMAc) containing 2.1 gL−1 of lithium chloride (LiCl) as eluent

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with a flow rate of 1 mLmin−1 at 80°C. The molar masses in poly(methyl methacrylate) (PMMA)

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equivalents were obtained from a calibration curve constructed with low dispersity PMMA

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standards (Polymer Laboratories). A third-order polynomial was used to fit the log Mp versus

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time calibration curve, which was approximately linear across the molar mass range from 1.020

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to 1.94 kDa.

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Synthesis of linear triblock (LB-C&D)

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The linear RAFT agent and disulfide linked linear triblock copolymers used in this study were

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synthesised as described previously in Hinton et al.2 using the bis RAFT agent (1). Refer to


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supplementary information section for details of the procedures to synthesize the RAFT agent,

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bis-macro-RAFT agent and triblock copolymer LB-C&D. S C12H25 S

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O CH3 O CH3 C S CH2CH2 C O CH2CH2S-SCH2CH2 O CH2CH2 S CN CN

S S C12H25

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Disulfide linked linear RAFT agent (1)

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Synthesis of 4-arm star block copolymers (4-arm star + BMA and 4-arm star –BMA):

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Synthesis of 4 -arm star block copolymers PDMAEME -b-(POEGMA8-9)4: The disulfide linked

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4-arm RAFT agent (2) was synthesized using the procedure described by Rosselgong et. al.17

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disulfide linked 4-arm RAFT agent (2)

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Refer to the supplementary section for details of the procedures used to prepare 4-arm RAFT

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agent (2), macro-RAFT DMAEMA, 4-arm star + BMA and 4-arm star – BMA. Representative

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NMR spectra are shown in Figure 1 and SI Figure S2.

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Synthesis of mikto star polymer (mikto star + BMA and mikto star – BMA):

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Synthesis of the dansyl-RAFT agent:The precursor dansyl ethylenediamine (3) for making the

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title dansyl-RAFT agent was prepared in 84.8% yield after recrystallization in


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dichloromethane:n-hexane (1:1) solvent mixture according to a published procedure by Schrader

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et. al.38

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Scheme 1. Synthesis of dansyl-RAFT (3): a) DIC, DMAP, CH2Cl2, room temperature, 4 h.

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To a solution of dansyl ethylenediamine (1) (293 mg, 1.0 mmol), 4-cyano-4-

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[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (2) (403 mg, 1.0 mmol) and catalytic N,N-

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dimethylaminopyridine (DMAP) in dichloromethane (5 mL) was added diisopropyl

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carbodiimide (DIC) (140 mg, 1.1 mmol), and the reaction mixture was allowed to stir at room

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temperature for 4 h. The DIC-urea by-product was filtered, volatiles removed in vacuum and the

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crude reaction mixture (790 mg) was purified by column chromatography (ethyl acetate: n-

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hexane 3:2 v/v as eluent) to give the title product dansyl-RAFT (3) agent as a yellow liquid (460

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mg, 67.8%). 1H NMR (CDCl3) δ(ppm) 0.88 (t, 3H, CH3), 1.21 – 1. 40 (br.s, 18H, 9xCH2), 1.70

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(m, 2H, CH2), 1.85 (s, 3H, CH3), 2.30-2.42 (m, 4H, CH2CH2), 2.89 (s, 6H, N(CH3)2), 3.05 (m,

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2H, C(=O)NH CH2), 3.30 (m, 2H, S(O)2NHCH2), 3.33 (dd, 2H, CH2S), 5.49 (t, 1H, NH), 5.99 (t,

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1H, NH), 7.21 (d, 1H, Ar-H), 7.53 (dd, 1H, Ar-H), 7.60 (dd, 1H, Ar-H), 8.24 (dd, 1H, Ar-H),

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8.55 (d, 1H, Ar-H).

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Synthesis of dansyl functional macroRAFT agent: A typical procedure for RAFT polymerization

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is as follows. Stock solution I of ACHN (25 mg) in DMF (2.5 g) was prepared in a flask. A

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mixture of dansyl-RAFT (0.113 g), OEGMA8-9 (3.0 g), stock solution I (0.406 g) and DMF (2.7

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g) was prepared in a second flask. This stock solution was transferred to an ampoule which was

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degassed by three consecutive freeze-evacuate-thaw cycles and sealed under vacuum. The


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ampoule was heated at 90°C for 4.5 h and then subjected to GPC and 1H NMR analysis. Dansyl-

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P(OEGMA8-9)32 (M-CTA) was purified by two precipitations into pentane and dried under

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vacuum to give 60% yield by weight.

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The dansyl- P(DMAEMA)94 and dansyl-P(BMA)104 were also prepared using a similar

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procedure, except the respective monomers DMAEMA and BMA were used in place of the

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OEGMA8-9.

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The optimal experimental conditions to form the mikto star polymer + BMA in using

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macroRAFT agents based on hydrophilic P[(OEGMA8-9)], cationizable P(DMAEMA) and

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hydrophobic P(BMA) were established in the previous study.36 The experimental detials for the

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synthesis of macro RAFT agents and mikto star + BMA and mikto star – BMA are provided in

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the supplementary section.

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Quaternization of copolymers:

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Previous research has shown that quaternization of the polymer complexes significantly

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improves cell uptake.2,13 Thus, in this study all the purified copolymers were quaternized by

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redissolving in either acetonitrile (LB(C&D), 4-Arm star-BMA, 4-arm star + BMA), or DMF

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(mikto star + BMA and mikto star-BMA). An excess of methyl iodide with respect to the

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PDMAEMA portion in the block copolymer was then added. The mixture was stirred at room

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temperature overnight, and excess solvents and methyl iodide were removed at 40°C in vacuo.

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Dialysis:

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Further purification was carried out by dialysis against de-ionized water for 3 days using dialysis

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membrane MM cut off of 3.5 kDa for copolymers of linear triblock and 4-Arm star. Molecular


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cut off of 25 kDa was used for mikto star. After dialysis, the water was removed from the

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polymer solution by lyophilization.

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Cells:

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Chinese Hamster Ovary cells constitutively expressing Green Fluorescent Protein (CHO-GFP)

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(kindly donated from K. Wark; CSIRO Australia) were grown in MEMα modification

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supplemented with 10% foetal bovine serum, 10 mM Hepes, 0.01% penicillin and 0.01%

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streptomycin at 37 °C with 5% CO2 and subcultured twice weekly.

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Adenocarcinomic human alveolar basal epithelial cells (A549; ATCC No.CCL-185) and human

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hepatocarcinoma cells (Huh7; kindly received from VIDRL, Victoria Australia) were grown in

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DMEM supplemented with 10% foetal bovine serum, 10 mM Hepes, 2 mM glutamine, 0.01%

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penicillin and 0.01% streptomycin at 37 °C with 5% CO2 and subcultured twice weekly.

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Synthetic siRNA oligonucleotides:

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The anti-GFP siRNA was obtained from QIAGEN (USA). The anti-GFP siRNA sequence is

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sense 5’- GCAAGCUGACCCUGAAGUUCAU [dT][dT] -3’ and antisense 5’-

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GAACUUCAGGGUCAGCUUGCCG [dT][dT] -3’ and is referred to as si22. Fluorescently

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labelled [6 FAM] si22 was purchased from Sigma Aldrich (USA) with the FAM label on the 5’

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end of the sense strand.

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The anti coatomer protein complex, subunit alpha (COPA) siRNA pool was purchased from

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Sigma Aldrich (USA). The four siRNA sequences are 1; 5’-

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ACUCAGAUCUGGUGUAAUA[dT][dT]-3’ 2; 5’-GCAAUAUGCUACACUAUGU[dT][dT]-3’


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3; 5’-GAUCAGACCAUCCGAGUGU[dT][dT]-3’ 4; 5’-

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GAGUUGAUCCUCAGCAAUU[dT][dT]-3’.

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Formation of polymer-siRNA complexes:

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Nitrogen: Phosphate (N:P) ratios of polymer to siRNA were calculated at required

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concentrations. Complexes were formed by the addition of media with or without 5% FBS

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(Invitrogen, USA) to Eppendorf tubes. The required amount of polymer resuspended in water

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was added to the tubes and the mixture vortexed. siRNA was then added to the tubes and the

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sample vortexed. Complexation was allowed to continue for 1 h at room temperature.

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Agarose gel electrophoresis:

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Samples at molar ratios of polymer relative to 50 pmol siRNA were electrophoresed on a 2%

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agarose gel in TBE at 100V for 40 min. siRNA was visualized by gel red (Jomar Bioscience) on

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a UV transilluminator with camera, the image was recorded by the GeneSnap program (Syngene,

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USA).

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In-vitro disulfide bond reduction assay:

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TCEP solution (50 mM) was prepared using deoxygenated water and stored at -20°C. Polymer-

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si22 complexes (50 pmol) were assembled as described above. These polyplexes were subjected

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to 50 mM TCEP reduction in the presence of 30 mM NaCl in pH 5 sodium acetate buffer.

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Reactions were incubated at 37°C for 4 h and analysed for si22 release by electrophoresis on a

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2% agarose gel as described above.

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Heparin displacement assay:


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10 units of heparin were added to the polymer-siRNA complexes. Competition by the heparin for

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binding with the polymer was assessed by siRNA release through electrophoresis as described

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above.

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Quantitative Nanoscale Mechanical-Atomic Force Microscopy (QNM-AFM):

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QNM-AFM was performed on a Multimode 8 AFM with a nanoscope V controller (Bruker). All

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imaging was performed using the fluid cell either in PBS or PBS containing 5% FBS. Scanasyst-

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Fluid (Bruker) cantilevers were used with an approximate spring constant of 0.7 N/m and a

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resonant frequency of 150 kHz. To produce quantitative nano-mechanical maps all imaging was

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performed in peak force tapping mode where the cantilever is oscillated at a frequency much

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lower than its resonant frequency. Before imaging each cantilever was calibrated using the

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absolute method outlined by Bruker to accurately determine the deflection sensitivity, spring

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constant, and the tip radius. Deflection sensitivity was calculated from a force curve generated by

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indenting the tip into a hard sapphire surface. The spring constant was determined in both air and

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the imaging fluid using the thermal tune procedure. The tip radius was calculated by analysis of a

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1.5 µm scan of the roughened Ti control sample using the tip analysis tool in the NanoScope

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Analysis software (Bruker). Nanoscale force maps were generated by analyzing force–

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indentation curves by using the Derjaguin–Muller–Toporov (DMT) model,39 and analysed using

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the section tool in the software without any additional processing. After absolute calibration 1

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µm2 scans of a polycaprolactone film were captured and found to possess a mean DMT modulus

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typically within 10% of the known elasticity (200 MPa).

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Topographic images were analysed using the NanoScope Analysis software. Images were

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flattened using the first order flattening algorithm and subjected to no further processing.


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Complex diameters were calculated from datasets of 200-300 complexes by analysis of multiple

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images using the particle analysis tool in the software. Errors quoted are the standard error of

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mean from the average value of at least 3 separate experiments.

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Nanoparticle Tracking Analysis (NTA) by NanoSight:

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Nanoparticle tracking analysis (NTA) was performed with a Nanosight NS300 (NanoSight,

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Amesbury, United Kingdom) to determine the size distribution of the polymer-siRNA complexes

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in solution. Samples were prepared at the optimal N:P ratio in nuclease free water, vortexed and

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incubated for a minimum of 4 h at room temperature. Immediately prior to analysis, samples

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were diluted in pre-filtered water to a concentration range between 1x108 - 1x109 particles/ml.

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All samples were vortexed thoroughly before loading on the sample chamber using a sterile

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syringe. Triplicate measurements were taken with 405 nm laser at 25°C over a 60 second period

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using a CMOS camera. Automated analysis was performed using the NTA 3.0 software package

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using a constant detection threshold between samples.

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Dynamic Light scattering and zetapotential:

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Dynamic light scattering (DLS) experiments were used to calculate the mean hydrodynamic

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diameters of the polymer-siRNA complexes by converting fluctuations of scattered light based

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on Brownian motion to the hydrodynamic diameter of the particle according to the Stokes–

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Einstein equation. Measurements were taken on a Malvern Zetasizer Nano Series DLS detector

288

with a 22 mW He–Ne laser operating at 632.8 nm, an avalanche photo-diode detector with high

289

quantum efficiency and an ALV/LSE-5003 multiple digital correlate electronics system. The

290

samples were prepared at 0.1 mgml-1 concentration. All measurements were performed in

291

triplicate at 25°C. The zeta potential of the complexes were obtained by the same instrument in


14

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Biomacromolecules

292

different buffered solutions. 1 mL samples of the polyplexes were prepared at approximately 300

293

mgml-1 concentration in 10 mM NaCl. All measurements were performed via three separate

294

measurements averaging approximately 10 scans at 25°C.

295

FAM labelled siRNA uptake:

296

Polymer/si22-FAM complexes at required concentration were assembled as described above

297

using the [6FAM] labelled si22.

298

Flow cytometry:

299

A549 and CHO-WT cells were seeded at 3x104 cells per well in 96-well tissue culture plates and

300

grown overnight at 37°C with 5% CO2. Polymer/FAM siRNA complexes at optimal N:P ratios

301

were prepared, cell media was removed and replaced with 200 µl media with or without 10%

302

FBS. The siRNA-polymer complexes at appropriate concentrations were added in triplicate for

303

each sample and incubated for 5 h. Cells were then trypsinized and analysed by a Becton

304

Dickinson LSRII flow cytometer. All experiments were performed as biological replicates.

305

Confocal microscopy and immunofluorescence analysis:

306

A549 and CHO-WT cells were seeded at 1x105 cells on 13 mm round glass coverslips (Menzel,

307

Germany) in 24 well plates (Nunc, USA) and grown overnight at 37°C with 5% CO2. Polymer

308

and [6FAM] labelled siRNA complexes were produced as described above and added to the cells

309

for 4 h at 37°C with or without 10% FBS. Following incubation, cells were washed in 0.5 mgml-

310

1

311

interacting with the cell membrane. Cells were fixed in 4% paraformaldehyde (Sigma, USA) in

312

PBS and processed as required.

Heparin Sulfate (Sigma, USA) in PBS to remove residual polymer that may be weakly


15

Biomacromolecules

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Page 16 of 47

313

For immunofluorescence analysis, cells were permeabilized with 0.1% (v/v) Triton X-100 in

314

PBS for 10 min and blocked in 0.5% bovine serum albumin in PBS (PBS/BSA) for 30 min.

315

EEA-1 and LAMP-2 antibodies (DSHB, U.S.A) were diluted 1:200 in PBS/BSA and incubated

316

at room temperature for 2 h. After 3 × 5 min washes, primary antibodies were detected with

317

fluorescent-conjugated species-specific secondary antibodies diluted 1:200 in PBS/BSA (Life

318

Technologies) for 1 h. Following 3 × 5 min washes in PBSA and one wash with dH20, nuclei

319

were stained with a 1:4000 4’,6’-Diamidino-2-Phenylindole (DAPI) (Sigma). Coverslips were

320

mounted onto glass microscope slides in Vectashield mounting media (Vector Laboratories,

321

USA) and images acquired on a Leica SP5 confocal microscope (Leica Microsystems,

322

Germany).

323

Silencing assay:

324

CHO-GFP, and A549 cells were seeded at 1x104 or 2x104 cells per well respectively in 96-well

325

tissue culture plates and grown overnight at 37°C with 5% CO2. As controls samples siRNAs

326

were transfected into cells using Lipofectamine 2000 (Invitrogen, USA) as per manufacturer’s

327

instructions. Briefly, the appropriate concentration of the relevant siRNA were mixed with 0.5 µl

328

of Lipofectamine 2000 both diluted in 50 µl OPTI-MEM (Invitrogen, USA) and incubated at

329

room temperature for 20 min. The siRNA: lipofectamine mix was added to cells and incubated

330

for 4 h. Cell media was replaced and incubated for 72 h. For polymer/siRNA complexes

331

prepared cell media was removed and replaced with 200 µl media with or without 10% FBS.

332

siRNA:polymer complexes was added in a volume of 10µl to 3 wells of cells per sample and

333

incubated for 5 h. Cell media was replaced and cells incubated for a further 72 h. All

334

experiments performed as biological replicates.


16

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Biomacromolecules

335

GFP silencing read out; Following incubation with siRNA polymer complexes, cells were

336

washed twice with PBS, and read on a Fluoroskan Ascent FL (Thermo Scientific, USA) and

337

EGFP silencing was analysed as a percentage of the siDNA or polymer/siDNA complexes mean

338

EGFP (ex 488 nm em 516 nm) fluorescence.

339

COPA silencing read out; Cells were washed twice with PBS, toxicity was measured using the

340

Alamar Blue reagent (Invitrogen USA) according to manufacturer’s instructions and described

341

previously.13

342

Haemolysis assay:

343

Mouse blood in EDTA was obtained from C57/Blk6 mice from the AAHL small animal facility

344

according to AEC approval. The blood was washed in PBS three times and resuspended at a

345

concentration of approximately 7.5 x 106 cells/ml. Polymer complexes at the required

346

concentration were added to triplicate wells in a 96 well plate in 100 µl of PBS. An aliquot of

347

100 µl of diluted mouse blood was added to the materials and incubated for 1 h at 37°C with

348

constant shaking. After removal of the un-lysed erythrocytes by centrifugation (1000 x g, 5 min),

349

150 µl of the supernatant were transferred to a new microtiter plate, and haemoglobin absorption

350

was determined at 450 nm on an EL808 absorbance microplate reader (BIOTEK, USA) with

351

background correction at 750 nm. 100% lysis was determined by adding 5 µ1 of a 0.1% Triton

352

X-100 solution prior to centrifugation. Results are presented as percentage haemoglobin release

353

compared to the PBS control. All experiments performed as biological replicates.

354

Statistics:


17

Biomacromolecules

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Page 18 of 47

355

The difference between two groups was statistically analysed by one way repeated measures

356

ANOVA, parametric, with Dunnett post analysis. ***p LB(C&D)) (SI Table S3). However, here the

654

complexes were larger in size; presumably due to the formation of a protein corona on the

655

complex surface. The effect was most dramatic for the mikto star + BMA complex with the


34

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Biomacromolecules

656

mean diameter increasing to 94.4 ± 13.2 nm. The large increase in diameter observed for the

657

mikto star + BMA in FBS is likely due to the hydrophobicity of the BMA polymer chains, as it is

658

generally accepted that protein adsorption on surfaces and interfaces increases with increasing

659

hydrophobicity.58 Further support for this hypothesis is seen by the fact that the increase in

660

diameter of the polar surfaces of the lipofectamine siRNA lipoplexes (L2000) is only modest

661

(65.13 ± 14.9 nm in PBS and 80.3 ± 11.4 nm in 5% FBS).

662

The 4 arm + BMA complex does not exhibit this dramatic increase in diameter in the presence of

663

FBS. This is explained by differences in composition between the 4 arm and mikto star

664

complexes. NMR analysis (Table 1) showed that the molar percentage of BMA in the 4 arm

665

complex was only 15%, whereas in the mikto star complex it was 49%. This considerable

666

increase in hydrophobicity of the mikto star complexes would conceivably promote the

667

adsorption of serum proteins, resulting in the larger protein corona seen via AFM (Figure 7).


35

Biomacromolecules

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Page 36 of 47

668 669

Figure 7. AFM imaging of complex morphology and elastic modulus, ai-ei) Topographical

670

images with z-scale optimised to each image (ai = 10 nm, bi = 20 nm, ci = 6 nm, di = 10 nm, ei =

671

5 nm), aii – eii). QNM-AFM images showing quantitative maps of elastic modulus with z-scale

672

optimised to each image (ai = 1000 MPa, bii = 200 MPa, cii-eii = 40 MPa), insets show higher

673

magnification images at the same z-scale, f) particle diameter analysis of all complexes and


36

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Biomacromolecules

674

lipofectamine siRNA lipoplexes (L2000) was performed for > 300 complexes for AFM images

675

taken either in PBS (black bars) or 5% FBS (red bars), g) elastic modulus analysis of > 50

676

complexes for AFM images taken either in PBS (black bars) or 5% FBS (red bars). * *, p < 0.01,

677

* * *, p < 0.001. Error bars show ± standard error of mean.

678

In addition to AFM, complex size was quantified from much larger data sets containing over 106

679

complexes using DLS and NTA. As expected the measured diameters quantified by DLS and

680

NTA were larger than those measured by AFM, as AFM measures the true diameter of the

681

polymer complex whilst the other techniques measure the hydrodynamic diameter.39,54,55

682

Encouragingly the trends measured by all 3 techniques were similar. Both DLS and NTA

683

showed the mikto star complexes to be largest (average particle size > 100 nm), and the 4 arm

684

and LB(C&D) to display an average size between 58-90 nm (Table 2 and Figure 8). Objects

685

between 50-200 nm are believed to be preferential for cellular endocytosis and therefore all of

686

the complexes fall within the ideal range for cellular uptake and delivery of siRNA cargo.59 DLS

687

experiments were also performed on the polymers prior to siRNA complexation (not shown). At

688

0.1 mgml-1 no complexes were detected for the triblock or 4 arm polymers. Conversely the mikto

689

stars formed particles with diameters only slightly smaller than the siRNA complexes

690

(approximately 140 nm for both mikto stars).

691

Time resolved DLS studies were performed to check the stability of the complexes over time

692

(Figure S10). After 48 h, little complex aggregation was observed. Only the 4 arm - BMA and 4

693

arm + BMA complexes showed a small increase in observed particle diameter compared to t = 0

694

(26 ± 9% and 34.7 ± 9% increases for the 4 arm and 4 arm + BMA respectively). The increased

695

aggregation of the 4 arm complexes is likely due to their lower zeta potential (Table 2) resulting

696

in less electrostatic repulsion between the complexes. To investigate if complex aggregation was


37

Biomacromolecules

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Page 38 of 47

697

affected in protein corona forming conditions, the DLS time course experiment was repeated in

698

5% FBS (Figure S11). Under these circumstances no significant complex aggregation was

699

observed after 48 h.

700

In summary, the results of the particle size analysis showed a large increase in particle size for

701

the mikto + BMA complex in the presence of serum proteins is likely due to the increased

702

hydrophobicity of the complex that promotes the adsorption of a large corona around the

703

complex. Surprisingly, cell uptake (Figure 3) and gene silencing (Figure 6) of the mikto + BMA

704

with a large protein corona, is inhibited to a lesser degree than the other 4 polymer complexes.

705

The physical basis of this seemingly counterintuitive effect of corona formation, is further

706

explored below.

707

Improved cell uptake and gene silencing in hydrophobic mikto star complexes may be

708

aided by increased stiffness

709

In addition to size and shape, elasticity of each complex plays an important role in determining

710

uptake efficiency. Quantitative Nanomechanical Atomic Force Microscopy (QNM-AFM) is able

711

to provide nanoscale maps of the elastic modulus of polymeric materials with minimal tip

712

indentation depth (< 2 nm). This is particularly important as complex stiffness has been shown to

713

be important in governing cell uptake.23 Simultaneous recordings of topography and elastic

714

modulus were performed on complexes immobilized on mica substrates (Figure 7, and SI Figure

715

S6).39 From these maps we quantified a mean value for the stiffness of each complex, imaged in

716

both PBS and 5% FBS. All complexes investigated possessed an elastic modulus of less than 150

717

MPa, within the same order of magnitude as previously investigated polymer complexes30 and

718

the L2000 controls (Figure 7).


38

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Biomacromolecules

719

In PBS, the mikto star complexes were significantly stiffer than the 4 Arm or LB(C&D)

720

polymers. In FBS the same trend is observed. However, as with particle diameter, the increase in

721

stiffness for the mikto + BMA complexes is disproportionally large. This is likely due to the

722

greater concentration of hydrophobic BMA units which encourage the adsorption of densely

723

packed serum proteins to form an extensive protein corona encapsulating the softer complex. The

724

increase in both complex size and stiffness of the mikto + BMA complex is in agreement with

725

the fact that no significant increase in stiffness was seen in the presence of FBS for the

726

lipofectamine control, presumably due to the polar nature of the lipoplex surface. It is interesting

727

to note that there appears to be a linear correlation between the measured diameter of the

728

complexes and their stiffness (Figure S12). This correlation is stronger for the complexes imaged

729

in FBS (R2 = 9.1) than PBS (R2 = 8.6), giving support to the hypothesis that a large protein

730

corona increases the stiffness of the complexes.

731

Table 2: Complex characterisation in PBS via QNM-AFM, DLS, Zeta potential and

732

nanoparticle tracking analysis (NTA). Polymer

Diameter

Stiffness

Diameter

PDI

AFM

QNM-AFM

DLS

(nm)

(DMT MPa)

(ZAve d.nm)

LB(C&D)

28.7 ± 4.6

10.4 ± 3.4

58.16

0.5

4 Arm -BMA

34.4 ± 5.3

5.9 ± 0.4

40.30

4 Arm +BMA

30.1 ± 7.6

19.5 ± 1.3

Mikto Arm BMA

39.7 ± 8.3

38.7 ± 1.6

Zeta Potential (mV)

Diameter

Diameter

NTA (Mode nm)

NTA (Mean nm)

41.6 ± 1.72

77.1 ± 3.2

81.0 ± 2.6

0.4

10.7 ± 0.8

75.1 ± 14.5

92.0 ± 12.1

43.57

0.4

18.0 ±1.7

73.9 ± 6.6

98.1 ± 9.6

151.2

0.5

41.7 ± 1.9

87.4 ± 3.8

102.1 ± 1.9


39

Biomacromolecules

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Mikto Arm +BMA

46.2 ± 16.4

41.2 ± 2.8

163.40

0.5

Page 40 of 47

41.9 ± 1.0

118.5 ± 15.8

134.0 ± 2.4

733

734


40

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Biomacromolecules

735

Figure 8. Nanoparticle tracking analysis summary of polymer/siRNA complexes size range

736

profiles. Complexes were prepared at the optimal N:P ratio in nuclease free water at a

737

concentration range between 1x108 - 1x109 particles/ml. Triplicate measurements were taken

738

with 405 nm laser at 25°C for 60 s using a CMOS camera. Automated analysis was performed

739

using the NTA 3.0 software package using a constant detection threshold between samples. Red

740

error bars = ± 1 standard deviation.

741

Our hypothesis that a large protein corona surrounding the mikto + BMA complex permits a

742

more efficient cell uptake than complexes coated with smaller coronas, may be partially

743

explained by the large increase in stiffness observed by QNM-AFM. Increased stiffness has been

744

theoretically proven to promote endocytosis, as the energy barrier to the membrane wrapping

745

required for endocytosis is lower for stiffer complexes due to reduced complex deformation.23

746

Similarly, the increase in stiffness of the mikto + BMA complexes may also explain the more

747

efficient membrane rupture in the haemolysis assay (Figure 5) as the stiffer complexes will

748

deform less and exert a greater bending force that is sufficient to damage the lipid bilayer of the

749

RBC. This physical property of the polymer may in turn may play a role in promoting endosome

750

escape.

751

Whilst the 4 arm and mikto star complexes are all composed of the same basic polymer building

752

blocks (POEGMA, PDMAEMA and PBMA) they do possess different molecular masses and

753

physical properties which we have shown to affect gene silencing efficiency. A number of

754

theories have previously been published to explain the apparent increase in efficacy of star

755

polymers compared to their linear counterparts. Pafity and co-workers cite reduced cytotoxicity

756

and the formation of more compact complexes that can cross the cell membrane more easily, 25

757

whilst others have shown that molecular mass, polymer architecture, degree of branching and


41

Biomacromolecules

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Page 42 of 47

758

charge density can all affect gene delivery.19 The above examples clearly show that the gene

759

silencing efficiency of nucleic acid delivery vehicles depends on multiple physiochemical

760

parameters which should be taken into account during the development and testing of novel

761

complexes. This highlights the importance of studies such as this, where a quantitative

762

physiochemical analysis is undertaken alongside in vitro cell uptake and silencing assays. From

763

our findings we suggest a combination of physical and chemical features that should be

764

considered for polymer design which will enhance siRNA delivery and gene silencing in similar

765

cationic star polymers (Table 3).

766

Table 3: Physiochemical Properties of cationic PDMAEMA/POEGMA/PBMA based star

767

polymers that promote gene silencing Polymer Architecture

Surface Charge

Hydrodynamic Diameter

Absolute Diameter

Stiffness

Hydrophobic/Hydrophilic

~ 40 MPa

More Hydrophobic Preferred

(From AFM) (Linear/Star) Star Polymer

(From DLS) ~40 mV

100-200 nm

40-50 nm

768 769

Following this study, future research in our laboratory will continue to investigate the in vivo

770

gene silencing efficacy of the mikto + BMA complex in mouse models. We will also perform

771

additional studies to systematically enhance some of these key physical parameters governing

772

efficacy of this siRNA polymer complex. This will enable us to develop an improved generation

773

of mikto star complexes tailored towards in vivo delivery applications.

774

Conclusion

775

For the first time we have performed an comprehensive and quantitative physiochemical

776

characterization of a range of siRNA-cationic polymer complexes in conjunction with an in-


42

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Biomacromolecules

777

depth investigation into their gene silencing efficacies. Whilst all the polymer complexes shared

778

similar chemistries they were found to significantly vary in architecture, surface charge, size,

779

stiffness and target gene silencing. The best performing complex was the mikto star polymer

780

containing a hydrophobic poly(butylmethacrylate) block. Not only did it show the best silencing

781

ability both in serum free media, but also retained a significant degree of target gene attenuation

782

in the presence of a foetal bovine serum-induced protein corona.

783

Additional experiments performed to probe the sub-processes required for gene silencing also

784

revealed the hydrophobic mikto star complexes was the best performing complex showing

785

increased cell uptake, endosome escape and siRNA release. These complexes also displayed the

786

largest diameter, elastic modulus, and zeta-potential, suggesting that a combination of large

787

particle size (allowing for greater nucleic acid loading), stiffness (exerting greater mechanical

788

strain on the cell and endosomal membrane), and surface charge (promoting electrostatic

789

interactions with the cell and endosomal membrane) are desirable characteristics for polymer

790

complexes coated with a protein corona.

791

Studies exploring the complex relationships between the physical properties of the delivery

792

vehicles and their cellular uptake will improve our knowledge of structure-function relationships

793

in the field of drug delivery. Approaches such as those presented here are invaluable for future

794

rational design of nano-vehicles in a range of delivery applications and will enhance the

795

manufacture of the next generation of siRNA delivery agents towards therapeutic applications.

796

ASSOCIATED CONTENT

797

Supporting Information. Further details on polymer preparation and characterization. Gene

798

silencing assays to determine the optimal N:P ratio for each polymer. Cell viability data for the


43

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

799

complexes at 125 nM. Further details, images and graphs from complex characterization

800

techniques.

801

AUTHOR INFORMATION

802

Corresponding Author Dr. Tracey Hinton: [email protected]

Page 44 of 47

803 804

Author Contributions

805

The manuscript was written through contributions of all authors. All authors have given approval

806

to the final version of the manuscript. M.D‡. and N.P.R‡. contributed equally.

807

ACKNOWLEDGMENTS

808

N.P.R Would like to thank the ARC Training Centre for Biodevices at Swinburne University of

809

Technology for funding (IC140100023), Dr. Mirren Charnley (Swinburne University of

810

Technology) for valuable discussions and Jay Gilbert (Perdue University) for supplying the

811

polycaprolactone substrates. The authors also acknowledge the scientific and technical assistance

812

from the Pathology and Pathogenesis team and the facilities of the Australian Microscopy &

813

Microanalysis Research Facility linked laboratory at the CSIRO Australian Animal Health

814

laboratories.

815

REFERENCES

816 817 818 819 820 821

(1) Barik, S. Viruses 2010, 2, 1448. (2) Hinton, T. M.; Challagulla, A.; Stewart, C. R.; Guerrero-Sanchez, C.; Grusche, F. A.; Shi, S.; Bean, A. G.; Monaghan, P.; Gunatillake, P. A.; Thang, S. H.; Tizard, M. L. Nanomedicine 2013, 1. (3) Tompkins, S. M.; Lo, C.-Y.; Tumpey, T. M.; Epstein, S. L. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 8682.


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