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Well-defined cationic N-[3-(dimethylamino)propyl]methacrylamide hydrochloride based (co)polymers for siRNA delivery Pratyawadee Singhsa, Diana Diaz-Dussan, Hathaikarn Manuspiya, and Ravin Narain Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01475 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Biomacromolecules

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Well-defined cationic N-[3-

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(dimethylamino)propyl]methacrylamide

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hydrochloride based (co)polymers for siRNA

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delivery

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Pratyawadee Singhsa†,‡, Diana Diaz-Dussan‡, Hathaikarn Manuspiya†,*, Ravin Narain‡,*

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†

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Materials Technology, Chulalongkorn University, Soi Chulalongkorn 12, Pathumwan, Bangkok

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10330, Thailand.

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‡

The Petroleum and Petrochemical College, Center of Excellence on Petrochemical and

Department of Chemical and Materials Engineering, Donadeo Innovation Centre for

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Engineering, 116 Street and 85 Avenue, Edmonton, AB T6G 2G6, Canada.

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KEYWORDS: N-[3-(dimethylamino)propyl]methacrylamide, RAFT polymerization, cationic

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glycopolymer, cytotoxicity, siRNA delivery, EGFR knockdown.

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ACS Paragon Plus Environment

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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

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ABSTRACT: Cationic glycopolymers have shown to be excellent candidates for the fabrication

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of gene delivery devices due to their ability to electrostatically interact with negatively charged

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nucleic acids and the carbohydrate residues ensure enhanced stability and low toxicity of the

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polyplexes. The ability to engineer the polymers for optimized compositions, molecular weights

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and architectures is critical in the design of effective gene delivery vehicles. Therefore, in this

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study, the aqueous reversible addition-fragmentation chain transfer polymerization (RAFT) was

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used to synthesize well-defined cationic glycopolymers with various cationic segments. For the

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preparation of cationic parts, N-[3-(dimethylamino)propyl]methacrylamide hydrochloride

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(DMAPMA.HCl), water-soluble methacrylamide monomer containing tertiary amine, was

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polymerized to produce DMAPMA.HCl homopolymer which was then used as macroCTA in the

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block copolymerization with two other methacrylamide monomers containing different pendant

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groups, namely 2-aminoethyl methacrylamide hydrochloride (AEMA) (with primary amine) and

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N-(3-aminopropyl) morpholine methacrylamide (MPMA) (with morpholine ring). In addition,

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statistical copolymers of DMAPMA.HCl with either AEMA or MPMA were also synthesized.

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All resulting cationic polymers were utilized as macroCTA for the RAFT copolymerization with

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2-lactobionamidoethyl methacrylamide (LAEMA) which consists of the pendent galactose

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residues to achieve DMAPMA.HCl-based glycopolymers. From the in vitro cytotoxicity study,

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the cationic glycopolymers showed better cell viabilities than the corresponding cationic

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homopolymers. Furthermore, complexation of the cationic polymers with siRNA, cellular uptake

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of the resulting polyplexes and gene knockdown efficiencies were evaluated.

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polymers/glycopolymers demonstrated good complexation ability with siRNA at low weight

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ratios. Among these cationic polymer-siRNA polyplexes, the polyplexes prepared from the two

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glycopolymers, P(DMAPMA65-b-LAEMA15) and P[(DMAPMA65-b-MPMA63)-b-LAEMA16],

All cationic


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showed outstanding results in the cellular uptake, high EGFR knockdown, and low post-

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transfection toxicity, suggesting the great potential in siRNA delivery of these novel

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glycopolymers.

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Introduction

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In gene therapeutics, small interfering RNA (siRNA), small double-stranded RNA molecules

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that silence gene expression by inducing the RNA-induced silencing complex (RISC) to cleave

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messenger RNA (mRNA),1 has become a promising gene medicine for the specific, post-

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transcriptional knockdown of disease-causing genes 2. siRNA has demonstrated great potential in

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treatments of cancer, viral infections, autoimmune and neurodegenerative diseases.3-8 However,

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the usefulness of siRNA can be limited due to their several hurdles including poor ribonuclease

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resistance, short half-life, lack of specific cell targeting, low cellular uptake and side effect

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toxicity. Consequently, to deliver siRNA effectively to desired target cells, novel advanced

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delivery systems are necessary. Non-viral gene delivery systems have been greatly studied,9, 10

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and mostly developed from cationic polymers which can electrostatically interact with negatively

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charged siRNA to form stable complexes preventing the siRNA from enzymatic degradation and

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destabilization by serum proteins in systemic circulation.11-14 The cationic polymers are ideal

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candidates as siRNA carriers because of their versatility and easy manipulation required to

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address a wide range of gene therapy applications. To improve the efficiency of siRNA delivery,

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pH-sensitive, protonable cationic polymers have been developed, mainly due to their high

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buffering capacity that mediate endosomal release by acting as ‘proton sponges’.15,

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examples of cationic polymers extensively studied are polyethyleneimine (PEI),17-19 poly(amino

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amine),14, 20, 21 and poly-L-lysine (PLL).22, 23

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The


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The use of cationic polymeric carriers still has another serious obstacle in gene delivery, the

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cationic nature of conventional polyplexes leads to severe toxicity at the cellular and systemic

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levels including blood coagulation. For that reason, neutral hydrophilic polymers such as

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poly(ethylene glycol) (PEG) were attached to promote the serum-tolerant function of most

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existing polycation carriers. PEGylation of siRNA carriers represents the most exploited strategy

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to avoid systemic toxicity of polyplexes, and increase the carrier stabilization in the

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bloodstream.24,

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circulating PEGylated carriers was explored to cause few immune responses,26 encouraged the

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evaluation of different neutral hydrophilic polymers with same characteristics in term of induced

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However, the accelerated blood clearance phenomenon induced by long-

stealth properties and physical carrier stabilization.

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Natural cationic polysaccharides, for example chitosan, dextran, and β-cyclodextrin, have

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received attention to be utilized for gene delivery, as carbohydrates are believed to be involved in

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condensing DNA via hydrogen bonding, thus reducing the need of excess cationic charge and

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hence decreasing the toxicity of the systems.27-30 In addition, the monomeric carbohydrate units

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can interact with specific cell receptors, suggesting the potential of action as targeting agents of

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polysaccharide carriers for gene delivery with no further modification needed.31, 32 Nevertheless,

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the naturally occurring carbohydrate-based carriers have the limitation in controlling their

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structures, so the synthesis of controllable molecular weight and architecture

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containing carbohydrates, which so-called glycopolymers,33,

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cationic glycopolymers that have been studied extensively in gene delivery are methacrylamide-

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based glycopolymers. The copolymers of methacrylamide polymers containing primary amines,

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3-aminopropyl methacrylamide (APMA) and 2-amino ethyl methacrylamide (AEMA), with

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sugar-modified methacrylamide polymers including 3-gluconamidopropyl methacrylamide

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polymers

is desirable. The examples of


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(GAPMA) and 2-lactobionamidoethyl methacrylamide (LAEMA) were reported to successfully

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formed polyplexes with plasmid DNA and provided good gene delivery efficiency in HepG2

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cells in terms of comparable gene expression and lower toxicity, as compared to PEI.35, 36

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N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) is a water-soluble methacrylamide

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monomer which contains tertiary amine providing cationic polymers after polymerization.

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DMAPMA is widely used for the synthesis of polymers in many applications, especially in

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biomedical applications, DMAPMA has been considered towards the design of antimicrobial

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polymers,37 temperature and/or pH sensitive polymers,38-41 protein recognition,42 protein

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separation,43 and non-viral gene delivery systems.44-52 For the siRNA delivery purpose, the

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DMAPMA polymer was modified with neutral hydrophilic polymers such as PEG and poly[N-

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(2-hydroxypropyl)methacrylamide] (PHPMA) to reduce the cytotoxicity.46, 48, 52 Most of homo-

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and copolymerizations of DMAPMA were carried out by the reversible addition-fragmentation

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chain transfer polymerization (RAFT), a controlled/living radical polymerization (CLRP)

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technique. The RAFT polymerization is a highly versatile technique and can provide a good

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control over the molecular weight and molecular weight distribution for the synthesis of

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polymers.53, 54 However, in the aqueous RAFT polymerization at neutral condition, due to the

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presence of tertiary amines, the unprotonated form of DMAPMA can cause hydrolysis or

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aminolysis of a chain transfer agent (CTA) such as cyanopentanoic acid dithiobenzoate (CTP),

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resulting in a loss of chain ends and a decrease in controlled polymerization ability.55,

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Additionally, the dithioester chain ends in the resultant polymers were degraded easily if the

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polymers were purified by dialysis against pure deionized water.46, 57 Therefore, when synthesize

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DMAPMA polymers, the acidic aqueous medium is required for the RAFT polymerization and

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purification.

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From the mentioned problems associated with the unprotonated DMAPMA, DMAPMA

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hydrochloride (DMAPMA.HCl) which is protonated by hydrochloric acid before subjecting to

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polymerization, was developed and studied for its ability in homo- and copolymerization by

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RAFT in the previous work.58 The well-controlled homopolymers of DMAPMA.HCl were

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obtained by the RAFT polymerization in acidic medium and simple purification with

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precipitation in acetone which allowed the good retention of dithioester chain end groups.

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Consequently, the p(DMAPMA.HCl)-based macroCTA was prepared and successfully used in

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the diblock copolymerization with other monomers. Moreover, the well-defined statistical

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copolymers of DMAPMA.HCl were attained as well.

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Although DMAPMA has been popularly used in RAFT copolymerizations, it was mostly

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subjected as a second-block or third-block co-monomer in chain-extension of block

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copolymerizations, and there was no report of utilizing DMAPMA in synthesis of glycopolymers

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for gene delivery applications. Therefore, the aim of this work was to prepare well-defined

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cationic DMAPMA.HCl-based polymers/glycopolymers with different architectures by using

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P(DMAPMA.HCl)-based macroCTA via the aqueous RAFT polymerization. The co-monomers

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used were methacrylamide monomers containing various pendant groups; 2-aminoethyl

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methacrylamide hydrochloride (AEMA) (with

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methacrylamide

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methacrylamide (MPMA) (with morpholine). Then, the resulting polymers/glycopolymers

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cytotoxicities were evaluated in HeLa (cervical) cancer cells by the tetrazolium MTT

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colorimetric assay. For the siRNA delivery purpose, the cationic polymer/glycopolymer-siRNA

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complexation, cellular uptake of the resulting polyplexes, transfection efficiencies, epidermal

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growth factor receptor (EGFR) siRNA knockdown, and post-transfection toxicity were studied.

(LAEMA)

(with

saccharide),

primary amine), 2-lactobionamidoethyl and

N-(3-aminopropyl)

morpholine


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Biomacromolecules

Experimental section

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Materials. N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was purchased from

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Sigma-Aldrich Chemicals, and protonated with hydrochloric acid as ascribed in previous work.58

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N-(3-aminopropyl) morpholine methacrylamide (MPMA), 2-aminoethyl methacrylamide

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hydrochloride (AEMA), and 2-lactobionamidoethyl methacrylamide (LAEMA) were synthesized

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according to previous reports.58-61 The CTA, cyanopentanoic acid dithiobenzoate (CTP), was

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synthesized as previously described.62

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hydrochloric acid (ACS reagent, 37%) were purchased from Sigma Aldrich Chemicals and used

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as received. Methanol, 2-propanol, tetrahydrofuran (THF), N,N’-dimethyl formaldehyde (DMF),

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dimethylsulfoxide (DMSO), and acetone were purchased from Caledon Laboratory Chemicals.

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Milli-Q water was used for all the synthesis experiments. Dulbecco's Modified Eagle Medium

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(DMEM), Opti-MEM (OMEM), 0.25% trypsin−EDTA, fetal bovine serum (FBS) and

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Streptomycin (5000 µg/mL)-Penicillin (5000 U/mL) were obtained from Gibco. Lipofectamine

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was purchased from Invitrogen Control. EGFR siRNA−FITC conjugate, human EGFR-specific

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small interfering RNA (EGFR siRNA), primary antibody (rabbit polyclonal EGFR specific IgG)

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and FITC-conjugated control siRNA were purchased from Santa Cruz Biotechnology.

4,4’-Azobis(4-cyanovaleric acid) (ACVA) and

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Synthesis of the DMAPMA.HCl-macro chain transfer agent (CTA). The polymerization of

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DMAPMA.HCl was conducted using RAFT technique at 70 °C, ACVA as the initiator and CTP

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as the CTA. In a typical protocol, in a 10 mL Schlenk tube, DMAPMA.HCl (1.24 g, 6 mmol),

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CTP (0.017 g, 0.06 mmol, target DPn = 100) and ACVA (0.008 g, 0.03 mmol) were dissolved in

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6 mL of a solvent mixture of acidic water (1:50 v/v mixture of hydrochloric acid and water, pH
90% cell viability), which was better than their corresponding free polymers, suggesting that

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the presence of nucleic acid molecules could mask part of the cytotoxic effect of the polymers.44

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This

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polymers/glycopolymers supported the benefits of utilizing these systems in siRNA delivery.

high

biocompatibility

of

the

polyplexes

prepared

by

DMAPMA.HCl-based

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Figure 6. Post-transfection toxicity of the cationic polymer-siRNA polyplexes in HeLa cells. Cell viability was assessed by Janus Green assay 48 h post-transfection. 6

Conclusion

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In this work, with aqueous RAFT polymerization, the DMAPMA.HCl homopolymer (P1)

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was synthesized and utilized as P(DMAPMA.HCl)-based macroCTA in chain extension with

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either AEMA or MPMA to produce well-defined diblock copolymers (P3 and P4). In addition,

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for investigating the effect of polymer architectures, the well-controlled statistical copolymers of

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DMAPMA.HCl with AEMA or MPMA (P7 and P8) were also prepared. Then, DMAPMA.HCl-

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based glycopolymers (P2, P5, P6, P9, and P10) were successfully produced by using each of the

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obtained polymers as the macroCTA in the block copolymerization with the same amounts of

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LAEMA. All resulting polymers were evaluated for their cytotoxicity in HeLa cells and, as


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compared to the DMAPMA.HCl homopolymer, the copolymers with MPMA showed lower cell

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toxicity. On the contrary, increased cell toxicities were found in the copolymers with AEMA.

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Interestingly, all DMAPMA.HCl-based glycopolymers demonstrated significantly improved

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biocompatibility. Consequently, the resulting cationic polymers/glycopolymers were determined

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their ability of complexation with siRNA, and the results showed that they could form stable

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polyplexes at low weight ratios. However, the copolymers with AEMA (P3 and P7) and their

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corresponding glycopolymers (P5 and P9) were excluded from further studies due to their high

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toxicity. The other polymers were evaluated for their potential of siRNA delivery, the

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hydrodynamic sizes of the resulting polyplexes were in range of 350-600 nm with the zeta

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potential values about 2-6 mV, and all polyplexes were internalized to the cytoplasm of HeLa

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cells which were visualized by the confocal fluorescence microscopy. From the transfection

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efficiency study (EGFR knockdown), the P6 which was the glycopolymer consisting of

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DMAPMA.HCl-MPMA block copolymer exhibited the highest EGFR silencing (~10% gene

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expression), while the P2 (simplest DMAPMA.HCl glycopolymer) showed the higher EGFR

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down-regulation (~30% gene expression) without off-target knockdown. Additionally, the post-

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transfection toxicity revealed that the cationic polymer-siRNA polyplexes were highly

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biocompatible (>90% cell viability). Therefore, the DMAPMA.HCl-based glycopolymer

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with/without MPMA blocks can be promising materials for siRNA delivery.

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ASSOCIATED CONTENT

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Supporting Information.

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The following files are available free of charge.


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Biomacromolecules

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GPC traces and 1H NMR spectra of DMAPMA.HCl polymers/glycopolymers, and agarose gel

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electrophoresis retardation assay of the cationic polymer-siRNA polyplexes. (PDF)

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AUTHOR INFORMATION

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Corresponding Authors

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Professor Ravin Narain

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*E-mail: [email protected]. Phone: +17804921736

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Professor Hathaikarn Manuspiya

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*

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E-mail: [email protected]

ORCID

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Pratyawadee Singhsa: 0000-0001-5492-6245

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Diana Diaz-Dussan: 0000-0003-1778-3964

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Ravin Narain: 0000-0003-0947-9719

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENT

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This research was supported by the Natural Sciences and Engineering Council of Canada

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(NSERC) and by the Petroleum and Petrochemical College, Chulalongkorn University, and

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funded by the Doctoral Degree Chulalongkorn University 100th Year Birthday Anniversary

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Scholarship.

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REFERENCES


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