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Highly Branched Poly(#-amino esters) for Non-Viral Gene Delivery: High Transfection Efficiency and Low Toxicity Achieved by Increasing Molecular Weight Yongsheng Gao, Jian-Yuan Huang, Jonathan O'Keeffe Ahern, Lara Cutlar, Dezhong Zhou, Feng-Huei Lin, and Wenxin Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01120 • Publication Date (Web): 17 Sep 2016 Downloaded from http://pubs.acs.org on September 22, 2016

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Highly Branched Poly(β-amino esters) for Non-Viral

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Gene Delivery: High Transfection Efficiency and

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Low Toxicity Achieved by Increasing Molecular

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Weight

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Yongsheng Gao,

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Dezhong Zhou, † Feng-Huei Lin, ‡ and Wenxin Wang,* †

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†

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Ireland

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‡ Institute

†, §

Jian-Yuan Huang,

†, ‡, §

Jonathan O’Keeffe Ahern,

†, §

Lara Cutlar,

†

Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin 4,

of Biomedical Engineering, National Taiwan University, Taipei, China

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ABSTRACT

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A successful polymeric gene delivery vector is denoted by both transfection efficiency and

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biocompatibility. However, the existing vectors with combined high efficacy and minimal

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toxicity still fall short. The most widely used polyethylene imine (PEI), polyamidoamine

14

(PAMAM) and poly(dimethylaminoethyl methacrylate) (PDMAEMA) suffer from the

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correlation: either too toxic or little effective. Here, we demonstrate that with highly branched

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poly(β-amino esters) (HPAEs), a type of recently developed gene delivery vector, the high gene

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transfection efficiency and low cytotoxicity can be achieved simultaneously at high molecular

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weight (MW). The interactions of HPAE/DNA polyplexes with cell membrane account for the

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favorable correlation between molecular weight and biocompatibility. In addition to the effect of

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molecular weight, the molecular configuration of linear and branched segments in HPAEs is also

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pivotal to endow high transfection efficiency and low cytotoxicity. These findings provide

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renewed perspective for the further development of clinically viable gene delivery vectors.

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INTRODUCTION

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Gene therapy has long been identified as the optimal corrective therapy for a wide range of

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inherited diseases and genetic disorders1, 2. Potential gene delivery systems must exhibit both

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high transfection efficiency and biocompatibility for consideration for their development from

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bench to clinical bedside3-5. Viral vectors facilitate excellent transfection efficiency. Although

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many AAVs have been demonstrated to be non-toxic, but possible concerns with their usage

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remain due to severe host immune response, and costly and complex large scale production

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restricting their clinical application to date4. In contrast, non-viral vectors can easily deliver large

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genetic payloads and their production is both cheap and facile. However they are unable to match

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viral vectors for transfection efficiency6, 7. There exists a strong association between efficacy and

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cytotoxicity for non-viral gene delivery vectors. The “efficacy-toxicity” paradox - high gene

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transfection efficiency induces significant cytotoxicity while low transfection efficiency elicits

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minimal cytotoxicity - acting as a substantial bottleneck hindering the translation of non-viral

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delivery vectors into widespread use8-10. As such there exists a substantial need to gain a more


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comprehensive understanding of the gene delivery vectors properties that contribute to

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transfection efficacy and toxicity.

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One established factor in determining transfection performance, is molecular weight (MW), for

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cationic polymers this been shown to play a particularly key role in influencing both gene

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delivery efficiency and biocompatibility11-13. For polyethylene imine (PEI), the original gold

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standard for polymer-based gene carriers, the effect that molecular weight has on cytotoxicity

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and transfection efficiency has been repeatedly reported12. In general, PEI of a higher molecular

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weight produces superior transfection efficiency, but also significant associated levels of

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cytotoxicity thus limiting practical applications to date14, 15.Taking the findings of Godbey et al.

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as an example, they demonstrated that using a number of branched PEI with molecular weights

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ranging from 0.6 to 70 kDa, that higher molecular weight variants mediated orders-of-magnitude

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greater in-vitro gene transfection efficiency was speculated to be as a result of a superior

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capacity for endosomal escape16. Similarly, this molecular weight dependent transfection

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performance was also well observed in polyamidoamine (PAMAM)17 dendrimers and poly(2-

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(dimethylamino)ethyl methacrylate (PDMAEMA)9. For example, Cheng et al. reported that the

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fifth generation (G5, Mw ~ 28 kDa) of PAMAM dendrimers surpassed their lower generation

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equivalent (G2, Mw ~ 3 kDa) for transfection efficiency but induced substantial cytotoxicity17, 18.

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Likewise, higher molecular weight PDMAEMA (Mw > 300 kDa) achieved superior in-vitro

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gene transfection efficiency compared to its lower molecular weight (Mw < 60 kDa)

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counterparts, but again, as has come to be expected, displayed high levels of cell cytotoxicity9.

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Likewise for other cationic polymers such as poly(l-lysine) (PLL)13 and chitosan (CS)14, the

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improved efficiency seen with using a higher molecular weighted polymer all suffered from


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notably increased levels of cytotoxicity. Therefore, it has become the expected causality in

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polymer synthesis that the higher molecular weight the greater the toxicity.

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Over the past decade, a class of cationic polymers, poly(β-amino esters) (PAEs), have shown

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great promise as gene delivery agents compared to the established PEI , PDMAEMA, etc.,

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owing to their facile synthesis, structural adaptability, widespread availability of monomers,

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biodegradability and high transfection efficiency19, 20. Previously more than 2,350 linear PAEs

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(LPAEs) were designed and systematically screened on a multitude of cell lines to evaluate the

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transfection efficiency and biocompatibility by Anderson and Langer et al19, 21, 22. The identified

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lead candidate achieved transfection efficiency results on human primary cells even comparable

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to that of an adenovirus21. LPAEs to date have demonstrated their superiority over previous

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established gene delivery polymers such as PEI23 and Lipofectamine 200019,

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highly branched poly (β-amino ester)s (HPAEs) have rarely been developed as gene delivery

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vectors owing to significant challenges in their synthesis26-28. In comparison to their linear

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counterparts, highly branched polymers are superior in gene transfection as a result of their well-

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defined three-dimensional (3D) structure and multiple terminal groups9, 18, 29, 30. Recently, we

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proposed a controllable and flexible “A2+B3+C2” strategy for HPAE synthesis26, 29, 31, 32. The

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developed HPAEs show higher gene transfection capability compared with LPAEs26,

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Importantly,

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biocompatibility still remains unresolved. While LPAE’s topological structure yields a

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homogenous backbone of linear chains, HPAEs introduce branching junctions to give a

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heterogeneous backbone consisting of linear and branched segments coexisting and distributed

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randomly. We hypothesized that the branched topology would lead to a different association

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between the molecular weight and cytotoxicity in HPAEs.

the correlation

between

molecular weight,

transfection

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, however,

31, 32

.

efficiency and


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MATERIALS AND METHODS

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Materials. TMPTA, BE, MPA, S4, dimethyl sulfoxide (DMSO), FITC were purchased from

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Sigma-Aldrich. Diethyl ether (99%) and DMF were purchased from Fisher Chemical. Gaussia

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Princeps luciferase plasmid (pCMV-GLuc) was obtained from New England Biolabs UK. Green

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Fluorescent Protein plasmid (gWiz-GFP) was obtained from Aldevron. Branched PEI (1.8 kDa

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and 25 kDa), PAMAM dendrimer (G2 and G5) were purchased from Sigma-Aldrich and

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LipofectamineTM 2000 was obtained from Life Technologies as positive controls for transfection

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studies. Linear PDMAEMA was synthesized according to our previous work9.

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Synthesis of HPAEs. HPAEs were synthesized via Michael addition reaction. The monomer

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feed ratios of TMPTA : BE : MPA were set at 0.5 : 1 : 1.46 respectively. The [vinyl] : [NH] of

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these reactions were set at 1.2 : 1. For polymerization, monomers were pre-dissolved in DMSO

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and added into a round bottom flask along with a magnetic stirring bar. Reactions were set at

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90oC and monitored for molecular weight by GPC. Once the desired molecular weight was

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achieved, reactions were terminated and polymers were end-capped with 0.12 M of the S4 end-

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capping reagent in DMSO, and the mixture was stirred for 24 hrs at room temperature. After

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end-capping, polymers were purified by precipitation in diethyl ether and dried under vacuum.

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Final polymer products were stored at -20°C as 100 mg/mL solutions in DMSO.

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Two-step Approach to Synthesis Structural Variation HPAEs. Three different structural

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variation HPAEs were synthesis via two-step Michael addition reaction. Initially, monomer feed

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ratios of TMPTA : BE were set as 1 : 1, 1 : 2 and 1 : 3. All [vinyl] : [NH] of these reactions were

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set as 1.2 : 1. Once the molecular weight of polymers reached above 12 kDa, a second monomer

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mixture (TMPTA, BE and MPA) were added into the reaction vessel at TPMTA : BE (1 : 3, 1 :


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2, and 1 : 1) ratios. The [vinyl] : [NH] of these mixtures were also set as 1.2 : 1. Monomers were

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pre-dissolved in DMSO and reacted in a flask at 90°C under stirring with a magnetic bar.

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Reactions were terminated once the polymer reached over 27 kDa, following this polymers were

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end-capped with S4 for 24hrs. Purification and storage of polymers was performed as per section

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above.

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GPC Characterization. Number average molecular weight (Mn), weight average molecular

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weight (Mw), polydispersity index (PDI), and alpha-value of the Mark-Houwink plots of the

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polymers were performed using a GPC (Agilent Technologies, PL-GPC 50) equipped with a

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refractive index detector (RI), a viscometer detector (VS DP) and a dual angle light scattering

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detector (LS 15° and LS 90°). The columns (30 cm PLgel Mixed-C, two in series) were eluted by

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dimethylformamide (DMF) with 0.1% LiBr. The flow rate was 1 mL/min at 50°C. Poly(methyl

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methacrylate) (PMMA) standards were used for calibration. Before analysis, samples were

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dissolved in DMF at a concentration of 5 mg/ml and passed through a 0.2 µm filter.

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Nuclear Magnetic Resonance (NMR) Analysis. 1H NMR was performed on a 400 MHz Varian

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NMR system spectrometer. The spectra were analyzed using MestReNova processing software.

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The chemical shifts were referenced to the lock chloroform-d (7.26 ppm).

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Particle Size Distribution and Zeta Potential Measurements. HPAEs and plasmids were

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diluted in 25 mM sodium acetate buffer (NaOAc) with various concentrations and then mixed at

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a 1 : 1 volume ratio with a polymer : DNA w/w ratio ranging from 5 : 1, 10 : 1, 15 : 1, to 20 : 1.

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After incubating for 10 min at room temperature, 1.5 mL of phosphate buffered saline (PBS) was

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added to the mixture in a disposable cuvette. Zeta potential and particle size distribution were


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analyzed using a Malvern Zetasizer Nano ZS. Particle size was measured by dynamic light

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scattering (DLS), and the zeta potential was analyzed by electrophoretic light scattering. The

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measured range of particle sizes were from 0.3 nm to 10 µm at 25°C with each sample

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measurement repeated in quadruplicate.

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Plasmid DNA Binding Assay. A PicoGreen assay was performed to determine polymer : DNA

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complexation efficiency. HPAEs were firstly diluted in 25 mM NaOAc and then mixed with

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DNA (60 µg/mL in NaOAc) at a 1 : 1 volume ratio. Polymer/DNA w/w ratio ranged from 5 : 1,

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10 : 1, 15 : 1, to 20 : 1.The solutions were mixed vigorously and allowed to incubate for 10 min

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to allow for polymer/DNA polyplex formation. PicoGreen working solution (diluting 10 µL of

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the purchased stock in to 1.9 mL NaOAc) was added into polyplex solution at a 1 : 1 volume

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ratio. After 5-min incubation, 30 µL of polymer-DNA-PicoGreen solution was mixed with 200

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µL DMEM medium in a black 96-well plate. Fluorescence was measured on a plate reader

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(SpectraMax® M3) at excitation of 490 nm and emission of 530 nm. The relative DNA binding

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efficiency (relative fluorescence (RF)), was calculation by the following relationship:

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where FDNA is the fluorescence value of a sample with DNA-PicoGreen without polymer as a

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control group, sample is the fluorescence value of the polymer-DNA-PicoGreen sample and

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Fblank is the fluorescence value of a sample with DNA-PicoGreen without polymer or DNA as a

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blank group (PicoGreen only).

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TEM Characterization of HPAE-M5/DNA and HPAE-M21/DNA Polyplexes. Polyplexes

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were prepared as above. 10 µL of polyplex solution was applied to holey carbon films on 200


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mesh copper grids and dried in air for 2 hours. The copper grids were then washed with

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deionized water to remove the crystals of buffer salts. Samples were stained with uranium

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acetates (0.5%) for 2 minutes. TEM (FEI Tecnai 120) was operated at 120 kV at UCD Conway

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Core Technology Center.

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HPAE Labeling with FITC. 100 mg of HPAE-M5 or HPAE-21 were dissolved in 1 ml DMSO,

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and then FITC DMSO stock solution was added, the mixture was stirred overnight in dark. The

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unreacted FITC was removed by dialysis in acetone for 2 days.

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Evaluation of Transfection Activity. HeLa cells and SHSY-5Y astrocytes were purchased from

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ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) with sodium pyruvate and

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L-glutamine. Culture media was further supplemented with 10% Fetal Bovine Serum (FBS) and

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1% penicillin-streptomycin. Cells in culture were maintained in 5% CO2 at 37°C. In vitro

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transfection ability of the HPAE : DNA polyplexes were assessed via GFP expression and

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Gaussia (G)-luciferase activity. Cells were seeded at a density of 10,000 cells/well in a 96-well

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plate and grown for 24 hrs. Both synthetic polymers and plasmids were diluted in 25 mM

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NaOAc (pH 5.2) and then mixed at a 1 : 1 volume ratio for a HPAE : DNA w/w ratio of 5 : 1, 10

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: 1, 15 : 1, or 20 before undergoing a 10-min incubation. Afterwards 20µL of polyplexes were

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added into 100µL cell culture medium containing serum. The final amount of plasmid DNA was

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0.5 µg/well. After 4-hr incubation, cells were washed by HBSS, replenished with fresh serum

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containing medium and incubated for 48 hrs. Transfected cells were analyzed for G-luciferase

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activity and GFP expression using the Gaussia Luciferase Assay Kit (BioLux®) and a

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fluorescence microscope (Olympus) respectively.


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Determination of Cell Viability. The biocompatibility of HPAEs was evaluated by

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alamarBlue™ assay (Thermo scientific), which assessed metabolic activity of transfected cells.

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48-hr post-transfection, cell viability was assessed by following the alamarBlue™ assay

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manufacturers’ protocol. After a 2-hr incubation with 100 µL alamarBlue reagent, absorbance at

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570 and 600 nm was recorded on a multi-plate reader (SpectraMax® M3) and alamarBlue

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reduction percentage was calculated.

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Statistical Analysis. G-luciferase activity and cell viability in HeLa cells after transfection with

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HPAE: DNA polyplexes underwent one-way ANOVA compared to controls. A p-value of < 0.05

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was considered statistically significant.

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RESULTS AND DISCUSSION

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To validate our hypothesis, two HPAEs of relatively low and high molecular weights were

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synthesized via the “A2+B3+C2” type Michael addition reaction as shown in Scheme 1. 3-

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Morpholinopropylamine (MPA, A2) and Bisphenol A ethoxylate diacrylate (BE, C2) were

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chosen as effective monomers for efficient transfection according to their high performance in

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previous literature26, 29, 31, 32. TMPTA (B3) was chosen as the branching monomer to facilitate the

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HPAEs 3D branched architecture, allowing for multiple functional terminal groups26,

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Amino-1-butanol (S4) was used as the end-capping agent. The molecular weight of polymers

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were monitored using gel permeation chromatography (GPC), and upon approaching 6 and 12

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kDa respectively, polymers were end-capped by adding an excess of S4 into the reaction vessel

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to produce end-capped HPAEs with molecular weight of 6.8 and 12.4 kDa. To reinforce the

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comparison among gene transfection efficiency and cytotoxicity, commercially available gene

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transfection agents branched PEI (1.8 kDa and 25 kDa), PAMAM dendrimer (3.5 kDa and 13.9

31

. 4-


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kDa) along with synthesized linear PDMAEMA (5.8 kDa and 16.7 kDa)9 were used as controls.

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Figure. 1 outlines the G-luciferase activity and viability of HeLa cells post transfection. Quite

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noticeably the 4 high molecular weight polymers exhibit 1~3 orders-of-magnitude superior G-

4

luciferase activity compared to their low molecular weight equivalents. In accordance with

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previous reports, the high molecular weight PEI14, PAMAM dendrimer18 and PDMAEMA9

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induced substantially higher cytotoxicity than their low molecular weight counterparts. In stark

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contrast, the high molecular weight HPAE exhibited much lower cytotoxicity than its low

8

molecular weight counterpart (91.2±1.13% versus 57.6±2.34%). This interesting observation is

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in sharp contrast to the well-established causality viewpoint that higher molecular weight

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cationic polymers induce higher cytotoxicity.

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Scheme 1. Synthesis of HPAEs for gene delivery via an “A2+B3+C2” type Michael addition

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reaction

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To further validate our initial findings, a second library of HPAEs with 6 different molecular

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weight ranging from 5.4 kDa to 21 kDa was synthesized (Table 1 and Figure. S1), and gene


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transfection was carried out on both robust/malleable HeLa cells and relatively fragile/sensitive

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human SHSY-5Y astrocytes33-36. G-luciferase activity demonstrated that HPAE-M10, HPAE-

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M16 and HPAE-M21, having molecular weight of 10.62 kDa, 16.51 kDa and 21.01 kDa

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respectively, mediated higher gene transfection efficiency compared to their lower molecular

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weight equivalents HPAE-M5, HPAE-M7 and HPAE-M9 (of molecular weight 5.43 kDa, 7.45

6

kDa and 9.23 kDa, respectively), as shown in Figure. 2a and Figure. S2. Specifically, in HeLa

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cells at w/w = 20:1, HPAE-M21 displayed 1.85, 6.69, 10.7, 86.6, 1723 fold higher G-luciferase

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activity compared to the lower molecular weight HPAE analogues. Remarkably, HPAE-M21

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maintained 94.1% cell viability, up to 60% higher compared to the other counterparts (Figure.

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2c). In the human SHSY-5Y astrocytes, similar results further substantiated our findings

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(Figure. 2b and 2d). These results demonstrate that in these HPAEs, molecular weight can

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circumvent the conventional correlation between efficacy and cytotoxicity in HeLa cells and

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SHSY-5Y astrocytes. This finding provides a new opportunity to overcome one of the key

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hurdles for non-viral delivery vectors – compromising biocompatibility for efficiency.

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Table 1. Polymerization time, composition and structural information of HPAEs. Polymer HPAE-M5 HPAE-M7 HPAE-M9 HPAE-M10 HPAE-M16 HPAE-M21

Reaction Time (h) 2.5 3.5 4.5 5.0 6.5 8.5

Mw (Da) 5400 7400 9200 10600 16500 21000

PDI 2.95 2.42 2.32 3.03 2.78 3.48

α 0.17 0.26 0.23 0.26 0.19 0.24

16 17


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Figure 1. G-luciferase activity and viability of Hela cells after transfection with PEI,

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PAMAM dendrimer, PDMAEMA and HPAE with relatively low and high molecular

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weights. One-way ANOVA, mean ± SD, n = 4,*P < 0.05 superior luciferase activity compared

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with their lower molecular weighted counterparts/DNA polyplex;

a

b

c

d

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Figure 2. Correlation between molecular weight, G-luciferase activity and cell viability

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on Hela and SHSY-5Y cells. (a) and (b) G-luciferase activity vs increasing molecular weight in


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HeLa cells and SHSY-5Y astrocytes post transfection; (c) and (d) Cell viability vs increasing

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molecular weight on HeLa cells and SHSY-5Y astrocytes. One-way ANOVA, mean ± SD, n =

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4,*P < 0.05 superior luciferase activity compared with PEI/DNA polyplex; #P < 0.05 superior

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luciferase activity compared with Lipo 2000/DNA polyplex.

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To decipher the possible mechanisms behind the positive correlation between molecular weight

6

and gene transfection in HPAEs, multiple investigations were conducted. 1H-NMR analysis

7

confirmed that the 6 HPAEs have almost identical chemical compositions (Figure. S3 and S4),

8

excluding the possibility that observed differences in gene transfection efficiency and

9

cytotoxicity are as a result of different chemical compositions. Key determinants of vector

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cytotoxicity and gene transfection efficiency were further investigated for HPAE’s including:

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DNA binding efficiency, polyplex size, surface charge and morphology. DNA binding efficiency

12

which is related to the ability of HPAEs to condense and shield plasmid DNA was first examined

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for all HPAEs (Figure. S5a). At high HPAE/DNA weight (w/w) ratios, e.g. 10:1, 15:1 and 20:1,

14

all the HPAEs achieved a high and equal DNA binding efficiency, whereas at w/w = 5:1, binding

15

efficiency decreased with each decreasing molecular weight significantly. HPAE-M5 produced

16

an insufficient binding efficiency of 24%. However, here DNA binding affinity did not correlate

17

well with transfection efficiency and cytotoxicity as HPAE-M7 at 10:1 w/w produced lower

18

DNA binding capability than 15:1 w/w, yet achieved greater transfection efficiency (Figure. 2).

19

Irrespective of molecular weight, once w/w ratios were above 10:1, all polyplexes had positive

20

zeta potentials (+ 5 - 11 mV) as shown in Figure. S5b. However, at w/w = 5:1, HPAE-M5/DNA

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and HPAE-M7/DNA polyplexes had much lower zeta potentials (- 7 and - 6 mV, respectively),

22

in contrast to that of the HPAE-M16/DNA and HPAE-M21/DNA polyplexes. With respect to

23

polyplex sizes, HPAE/DNA sizes decreased significantly with an increasing molecular weight


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and w/w ratio (Figure. S5c), with the w/w = 20:1, size of HPAE-M5/DNA polyplexes almost

2

twice as big as that of the HPAE-M21/DNA counterparts. TEM images further supplement our

3

findings with molecular weight having a substantial influence on polyplex morphology. At the

4

same w/w ratio, HPAE-M5/DNA polyplexes manifest large and irregular aggregate

5

morphologies, while HPAE-M21/DNA polyplexes exhibit small and relatively uniform spherical

6

particles (Figure. 3).

a

b

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Figure 3. TEM images of polyplexes. (a)HPAE-M5/DNA (N/P=20:1) and (b)HPAE-M21/DNA

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(20:1).

a

b

c

d

10


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Figure 4. Morphology of HeLa cells before and after incubation. (a, c) Incubated with

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HPAE-M5/DNA polyplexes and (b,d) incubated with HPAE-M21/DNA polyplexes for 4 hours

3

at w/w = 20:1 (N/P).

a

b

4 5

Figure 5. Fluorescent images of HeLa cells after incubation. (a) Incubated with HPAE-

6

M5/DNA polyplexs, (b) incubated with HPAE-M21/DNA polyplexes. Both are incubated for 4

7

hours at w/w = 20:1(N/P) labeled with FITC.

8

As shown in Figure. 4a and c, after 4 hours incubation with HPAE-M5/DNA polyplexes, the

9

majority of cells display abnormal morphology. In contrast, cell morphology was largely

10

preserved after incubation with HPAE-21/DNA polyplexes (Figure. 4b and d). At the same w/w

11

ratio (20:1), HPAE-5/DNA and HPAE- M21/DNA polyplexes have similar zeta potentials

12

(around + 9 mV), but markedly different sizes and particle shape (Figure. 3 and S5c). Previous

13

reports indicated that the over-enhanced interactions of polycations with cells would induce the

14

formation of nanoscale holes in the cell membrane bilayer leading to severe cytotoxicity37. We

15

speculate that the larger size and aggregated morphology of HPAE-5/DNA would possibly result

16

in excessive interactions of the polyplexes with the cell membrane, one possibly reason

17

accounting for the observed cytotoxicity. To confirm this, HPAE-M5 and HPAE-M21 were

18

labelled with a green fluorescence dye fluorescein isothiocyanate (FITC). As postulated, cells


15

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

Page 16 of 25

1

incubated with labelled HPAE-5/DNA polyplexes showed 2.82-folder stronger fluorescence

2

compared with those incubated with labelled HPAE-M21/DNA polyplexes, which indicates

3

there are more HPAE-M5/DNA polyplexes adsorbed to the cell membrane (Figure. 5). These

4

results support our hypothesis that the different interactions of polyplexes with the cells lipid

5

bilayer dictate the cytotoxicity and ultimately mediate different levels of gene transfection

6

efficiency by the polyplexes. Similar molecular weight/transfection efficiency/cytotoxicity

7

correlations were also observed in HPAEs synthesized from 1,4-butanol diacrylate (B4), 5-

8

amino-1-pentanol (S5) and TMPTA, or poly(ethylene glcol) diacrylate (PEGDA700), 4-amino-

9

1-butanol (S4) and TMPTA, however, the trend was not as obvious as the HPAEs we reported

10

here.

11

In addition to molecular weight, different molecular topological structures may also contribute to

12

varied polyplex interactions with cell membranes, thus affecting gene transfection efficiency and

13

cytotoxicity of HPAEs7. To investigate this assumption, three HPAE variations of differing

14

distributions of the linear (generated by the conjugate addition of MPA to BE) and branched

15

segments (generated by the conjugate addition of MPA to TMPTA) were synthesized via a two-

16

step reaction strategy as shown in Figure. 6a: HPAE-A with a larger fraction of external linear

17

segments; HPAE-B with a random distribution of linear and branched segments; while HPAE-C

18

with larger amounts of external branched segments. 1H NMR and GPC confirmed that these

19

three HPAEs have similar chemical composition and molecular weight (Table S1 and Figure.

20

S6).


16

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Biomacromolecules

a

c

b

d 5:1

10:1

15:1

20:1

1 2

Figure 6. Modulation of the spatial configuration of HPAEs by two step synthesis. (a) A

3

scheme of two-step Michael addition to modulate the spatial configuration of HPAE-A, HPAE-B

4

and HPAE-C; (b) and (c) correlation between HPAE structures and relative cell viability and G-

5

luciferase activity; (d) GFP expression of HeLa cell after transfection with the three different

6

structured HPAEs. One-way ANOVA, mean ± SD, n = 4,*P < 0.05 superior luciferase activity

7

compared with PEI/DNA polyplex; #P < 0.05 superior luciferase activity compared with Lipo

8

2000/DNA polyplex.

9

For both HeLa cells and SHSY-5Y astrocytes, HPAE-A/DNA polyplexes induced severe

10

cytotoxicity and correspondingly, lower gene transfection efficiency (Figure. 6b, c and Figure.

11

S7). Comparatively, HPAE-B/DNA and HPAE-C/DNA polyplexes preserved at least 85% of the

12

cells viability even at high w/w ratios. Remarkably, HPAE-C exhibited up to a 17 fold gene

13

transfection enhancement in comparison with HPAE-A. These results demonstrate that by


17

Biomacromolecules

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Page 18 of 25

1

modulating the configuration, high gene transfection efficiency and biocompatibility can be

2

achieved simultaneously with HPAEs of high molecular weight. To date, most polymeric

3

delivery system platforms such as PEI14, PAMAM dendrimer17, 28 and PDMAEMA9 demonstrate

4

higher gene transfection capabilities at higher molecular weight, however the severe cytotoxicity

5

limits their usage as safe gene delivery vehicles13. In contrast, our studies demonstrate that for

6

HPAEs, a modulation of molecular weight and molecular configuration can lead to superior

7

biocompatibility and gene transfection efficiency simultaneously. These findings provide new

8

insight for the understanding of vector structure-function relationship.

9

CONCLUSION

10

In summary, the effects of molecular weight and configuration of HPAEs on gene transfection

11

biocompatibility and efficiency were investigated. Differing from the conventional PEI,

12

PAMAM dendrimer and PDMAEMA, higher gene transfection efficiency and low cytotoxicity

13

were achieved by HPAEs with high molecular weight simultaneously. The interaction between

14

HPAE/DNA polyplexes and a cells lipid bilayer dictates the overall cytotoxicity and gene

15

transfection efficiency of HPAEs. Furthermore, by utilizing the HPAEs highly branched nature

16

to modulate their molecular configuration, high levels of cytotoxicity induced by a high

17

molecular weight can be circumvented. Taken together these results provide a new

18

understanding as to the effects of both polymer molecular weight and configuration on gene

19

transfection capability and cytotoxicity, which will serve as a valuable tool for the development

20

of next generation gene delivery vectors.

21

ASSOCIATED CONTENT


18

Page 19 of 25

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Biomacromolecules

1

Supplemental Information includes Experimental Procedures, seven figures, and one table. This

2

material is available free of charge via the Internet at http://pubs.acs.org.

3

AUTHOR INFORMATION

4

Corresponding Author

5

*E-mail: [email protected]

6

Author Contributions

7

§These authors contributed equally. The manuscript was written through contributions of all

8

authors. All authors have given approval to the final version of the manuscript.

9

ACKNOWLEDGMENT

10

The authors acknowledge Prof. Dimitri Scholz of the Conway EM Core, UCD, for TEM support.

11

This work was funded by SFI (14/TIDA/2367, 15/IFA/3037, 10/IN.1/B2981 (T), 12/IP/1688)

12

and HRB (HRA-POR-2013-412). University College Dublin is acknowleged for scholarship of

13

Y.G.

14

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1 2 3 4 5 6 7

Table of Contents Graphic

8 9

The high gene transfection efficiency and low cytotoxicity can be achieved simultaneously at

10

high molecular weight (MW), highly branched poly(β-amino esters). This not only refreshes our

11

traditional recognition of structure-function properties (i.e. branched vs linear) but also provides

12

new optimism for the use of cationic polymers.


25

Highly Branched Poly(Î²-amino esters) - ACS Publications - American

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