Cationic Polypeptoids with Optimized Molecular Characteristics

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Cationic Polypeptoids with Optimized Molecular Characteristics toward Efficient Non-Viral Gene Delivery Lipeng Zhu, Jessica M. Simpson, Xin Xu, Hua He, Donghui Zhang, and Lichen Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06031 • Publication Date (Web): 27 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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ACS Applied Materials & Interfaces

Cationic Polypeptoids with Optimized Molecular Characteristics toward Efficient Non-Viral Gene Delivery Lipeng Zhu1,§, Jessica M. Simpson2,§, Xin Xu1, Hua He1, Donghui Zhang2,*, Lichen Yin1,*

1

Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices,

Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou 215123, P.R. China 2

Department of Chemistry and Macromolecular Studies Group, Louisiana State

University, Baton Rouge, LA 70803, USA

§

These authors contributed equally.

* Corresponding authors: [email protected] (Yin L.), Tel: +86-512-65882039; [email protected] (Zhang D.), Tel: +1- 225-5784893

1

ACS Paragon Plus Environment


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ABSTRACT: The rational design of gene vectors relies on the understanding of their structure-property

relationship.

Polypeptoids,

structural

isomers

of

natural

polypeptides, hold great potentials as gene delivery vectors due to their facial preparation, structural tunability, and most importantly, desired proteolytic stability. We herein designed a library of polypeptoids with different cationic side-chain terminal groups, degrees of polymerization (DP), side-chain lengths, and incorporated aliphatic side chains, attempting to unravel the structure-property relationship toward maximized gene delivery efficiency while minimized cytotoxicity. In HeLa cells, polypeptoid bearing primary amine side-chain terminal group exhibited remarkably higher transfection efficiency than its analogues containing secondary, tertiary, or quaternary amine groups. Elongation of the polypeptoid backbone length (from 28 to 251 mer) led to enhanced DNA condensation level as well as cellular uptake level, while at the meantime caused higher cytotoxicity. Upon a proper balance between DNA uptake and cytotoxicity, polypeptoid with DP of 46 afforded the highest transfection efficiency. Elongating the aliphatic spacer between backbone and side amine groups enhanced the hydrophobicity of side chains, which resulted in notably increased membrane activities and transfection efficiency. Further incorporation of hydrophobic decyl side chains led to an improvement of transfection efficiency by ~6 fold. The top-performing material identified, P11, mediated successful gene transfection

under

serum-containing

conditions,

outperforming

commercial

transfection reagent PEI by nearly 4 orders of magnitude. In consistence with its 2


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serum-resistant properties, P11 further enabled effective transfection in vivo following intratumoral injection to melanoma-bearing mice. This study will help the rational design of polypeptoid-based gene delivery materials, and the best-performing material identified may provide a potential supplement to existing gene vectors.

KEYWORDS: non-viral gene delivery, polypeptoid, structure-property relationship, hydrophobicity, transfection efficiency, cytotoxicity

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INTRODUCTION Gene therapy holds great potentials to treat human diseases, including cancer,

infectious diseases, and immunodeficiency.1-7 High transfection efficiency and low toxicity are the two basic requirements for gene vectors.8-12 Cationic polymers (also referred as polycations) feature potent gene condensation capabilities to promote the cellular internalization, and thus have received wide applications as gene transfection materials.13-19 However, the relatively low transfection efficiency still remains as a critical issue for polycations, which is mainly attributed to the various biological barriers that pose different or even conflicting requirements for the molecular design of polycations. Therefore, it is highly demanded that the structure-property relationship of polycations be systemically unraveled, such that their molecular characteristics could be optimized to synergistically overcome the multiple cellular barriers against gene transfection. Polypeptides, exemplified by poly-L-lysine20-21 and poly-L-arginine,22-23 are the first generation of materials developed and utilized for gene delivery, mainly because of their capability to condense the anionic plasmid DNA. However, polypeptides are easily degraded by the proteases in vivo, and the metabolic instability of peptide-based delivery vector serves as a critical hurdle against the delivery efficiency of associated or encapsulated cargoes, including nucleic acids.24-26 Polypeptoids are structural isomers of polypeptides, wherein the substituent position is shifted from the α-carbon to the amide nitrogen.27-28 Because of such substitution, polypeptoids lack extensive hydrogen bonding and main-chain 4


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stereogenic center that are typical for polypeptides. It thus allows facial tunability of the physicochemical properties of polypeptoids (e.g., conformation, solubility, thermal property, and crystallinity) by controlling the sequence, side-chain chemistry, and molecular architecture.29-32 As such, polypeptoids have been widely applied for non-fouling coating,33 antimicrobial therapy,34-36 drug delivery,37-38 and etc.39 More importantly, polypeptoids feature desired stability against proteolytic degradation, which makes them better-suited for biomedical applications in vivo.40-41 There is evidence that polypeptoids are more membrane permeable than structurally analogous peptides,42-43 which is related to the enhanced backbone stability. However, as far as we know, studies on polypeptoids as gene vectors are still lacking.44 While oligopeptoids have been previously explored for gene delivery,45 they were synthesized using the solid-phase method which suffered from tedious synthesis, low yields

(less

than

20%),

and

difficulty

in

manipulating

the

molecular

structures/functionalities.46 Recently, the controlled synthesis of polypeptoids via living polymerization of N-carboxy anhydride (NCA) monomers has been reported, which allows facile preparation of polypeptoids with high molecular weights (MW), low dispersities, large scales, precise control over MW, and diverse architectures.28, 31 With the aid of the highly effective azide-alkyne Huisgen cycloaddition reaction (so-called “click” chemistry), the structures and functions of polypeptoid backbone or side chain can be easily modulated, which greatly expands their applications. In this study, a library of polypeptoids with different molecular structures was developed via the ring-opening polymerization (ROP) of NCA and subsequent 5



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side-chain functionalization using the “click” chemistry (Scheme 1), and their structure-property relationship toward gene delivery was mechanistically explored. Particularly, polypeptoids with different cationic side-chain terminal groups, degrees of polymerization (DP), side-chain lengths, and incorporated aliphatic side chains were synthesized, and their DNA condensation capability, DNA release, cellular uptake, intracellular kinetics, cytotoxicity, in vitro transfection efficiencies in mammalian cells, and in vivo transfection efficiencies in melanoma tumors were comprehensively evaluated, in attempt to identify the optimal polypeptoid structure with maximized transfection efficiency yet minimized cytotoxicity.

EXPERIMENTAL SECTION Materials, Cell Lines, and Animals. All chemicals and solvents were

purchased from Sigma-Aldrich (USA) and used without further purification unless otherwise specified. Dimethyl formamide (DMF) and tetrahydrofuran (THF) used for polymerization and click reactions were purified by passing through alumina columns under argon. Branched polyethylenimine (PEI, average Mw ∼25 kDa by LS, average Mn ∼10 kDa by GPC) was also purchased from Sigma-Aldrich (USA). N-propargyl N-carboxy anhydride (Pg-NCA) and N-decyl N-carboxy anhydride (De-NCA) were synthesized according to published procedures.47 Poly(N-propargyl glycine) homopolymers (PNPgG) were synthesized by adapting a reported procedure.48 All ω-azido alkyl ammonium compounds used in the synthesis of cationic polypeptoids were prepared by adapting previously reported procedures.49-51

6


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HeLa (human cervix adenocarcinoma), B16F10 (mouse melanoma), and COS-7 (African green monkey kidney fibroblast) were purchased from the ATCC (Rockville, MD, USA). Cells were grown in DMEM containing 1% L-glutamine, 1% non-essential amino acids, and 10% fetal bovine serum (FBS) (hereafter referred to as complete medium), at 37 °C in a 5% CO2 humidified atmosphere. Male C57/BL6 mice (6−8 wk, 18-22 g) were obtained from Shanghai Slaccas Experimental Animal Co., Ltd. (Shanghai, China) and were housed in an SPF room. The animal protocols were approved by the Institutional Animal Care and Use Committee, Soochow University. Instrumentation.

13

C and 1H NMR spectra were recorded on AVIII-400 and

Bruker AV-400 spectrometers, respectively. Size-exclusion chromatography (SEC) was recorded on an Agilent 1200 system equipped with three Phenomenex columns (pore size 5 µm, 300 × 7.8 mm), a differential refractive index (DRI) detector (Wyatt OptilabrEX) with a 690-nm light source, and a multiangle light scattering (MALS) detector (Wyatt DAWN EOS, GaAs 30mW laser at λ = 690 nm). DMF containing 0.1 M LiBr was used as the eluent (flow rate = 0.5 mL/min). The temperature of the detector and column was set at 25 °C.48 Mn and polydispersity index (PDI) of PNPgG were obtained using a dn/dc of 0.1012 ± 0.0007 g/mol. Copper content was measured on Varian SpectrAA 220 Atomic Absorption Spectrometer. The flame absorption spectrometry method was used with acetylene/air, lamp current of 4 mA, and slit width of 0.5 nm. Synthetic Procedure for PNPgG. In a glovebox, a predetermined volume of 7



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BnNH2/THF stock solution (120 µL, 200 mM) was added to a THF solution of Pg-NCA (350 mg, 2.52 mmol, 0.5 M). After stirring for 18 h at 50 °C, the conversion was determined by the FT-IR spectroscopic analysis of an aliquot of the reaction mixture. The final product was dissociated by precipitation into hexane and dried at room temperature to collect as white solid (230 mg, 96% yield). Mn and PDI of the polymer were determined by SEC. PNPgG with various DPs (28-250) and low PDI were obtained (Table 1). 1H NMR (400 MHz, CD2Cl2), δ ppm: 7.23-7.19 ppm (br. m, −C6H5, Ha), 4.67-4.04 ppm (br. m, −COCH2N−, −CCH2N−, Hb, Hc), 2.66-2.30 ppm (br. m, −CHCCH2−, Hd) 13C NMR (100 MHz, CD2Cl2), δ ppm: 168.9 (−COCH2N−), 128.7-127.5 (C6H5−), 77.5 (−NCH2CC−), 73.0 (−NCH2CC−), 47.1 (C6H5CH2N−), 37.6 (−COCH2N−), 36.6 (−NCH2CC−). Synthetic Procedure for the Poly[(N-propargyl glycine)-ran-(N-decyl glycine)] Random Copolymer P(NPgG-r-NDeG). In a glovebox, a predetermined volume of BnNH2/THF stock solution (200 mM, 116 µL) was added to a THF solution of Pg-NCA (129 mg, 0.93 mmol, 0.4 M) and De-NCA (56 mg, 0.23 mmol, 0.1 M). After stirring for 18 h at 50 °C, the conversion was determined by FT-IR analysis of an aliquot of the reaction mixture. The final product was dissociated by precipitation into hexane and dried at room temperature to collect as white solid (120 mg, 85.7% yield). Copolymer composition (Table 1) was determined using 1H NMR. For example, the integration of terminal alkyne proton of the propargyl side chain at 2.36-2.87 ppm and the terminal methyl proton of the decyl side chain at 0.83 ppm relative to the integration of the aromatic protons of the benzyl end group at 7.28 ppm 8


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were used to determine the DP of the PNPgG and PNDG segments, respectively. 1H NMR (400 MHz, CD2Cl2), δ ppm: 7.23-7.19 ppm (br. m, −C6H5, Ha), 4.43-4.15 ppm (br. m,

Pg/De

−COCH2N−, CHCCH2N−, Hb, Hm, Hn), 3.26 ppm (br. m,

CH3(CH2)8CH2N−, Hc), 2.43-2.28 ppm (br. m, CHCCH2−, Ho), 1.63-1.50 ppm (br. m, CH3(CH2)6(CH2)2CH2N−, Hd, He), 1.37 ppm (br. d, CH3(CH2)5CH2(CH2)3N−, Hf), 1.18 ppm (br. s, CH3(CH2)5(CH2)4N−, Hg, Hh, Hi, Hj, Hk), 0.79 ppm (br. s, CH3CH2(CH2)8N−,

Hl);

13

C

NMR

(100

MHz,

CD2Cl2),

δ

ppm:

168.9

(Pg/De−COCH2N−), 127.6 (−C6H5), 78.1 (−CHCCH2−), 73.3 (−CHCCH2−), 48.5 (CH3(CH2)8CH2N−),

47.3

(CH3(CH2)7CH2CH2N−),

(Pg/De−NCOCH2−), 31.8

37.7

(Pg−CCH2N−),

(C6H5CH2N−), 29.6

and

36.6 29.3

(CH3(CH2)3(CH2)4(CH2)2N−), 27.4 and 26.9 (CH3CH2(CH2)2(CH2)6N−), 22.6 (CH3CH2(CH2)7CH2N−), 13.8 (CH3(CH2)8CH2N−). Synthetic Procedure for the Cationic Polypeptoid Homopolymers (P1-P10). In a glovebox, a measured amount of ω-azido hexyl ammonium salt (101 mg, 0.56 mmol) ([N3]0:[propargyl]0 = 2:1) was added into a DMF solution of the PNPgG polymer (27.2 mg, 4 µmol, 0.28 mmol propargyl groups), followed by the addition of a measured volume of DMF solution of CuBr/PMDETA (250 mM, 564 µL, [Cu]0:[PMDETA]0:[propargyl]0 = 33:33:100). After stirring for 18 h at 50 °C and addition of EDTA aqueous solution, the mixture was further dialyzed. The reaction mixture was then lyophilized to yield a light greenish blue powder (70 mg, 91% yield). Synthetic Procedure for the Cationic Polypeptoid Random Copolymer 9



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(P11). In a glovebox, a measured amount of ω-azido hexyl ammonium salt (82 mg, 0.46 mmol,

[N3]0:[propargyl]0 = 2:1) was added into a DMF solution of

P(NPgG39-r-NDeG9) (33 mg, 6 µmol, 0.23 mmol propargyl groups), followed by the addition of a measured volume of DMF solution of CuBr/PMDETA (250 mM, 460 µL [Cu]0:[PMDETA]0:[propargyl]0 = 33:33:100). After stirring for 18 h at 50 °C and addition of EDTA aqueous solution, the mixture was further dialyzed. The reaction mixture was then lyophilized to yield a light green powder (85 mg, 91% yield). Determination of Copper Ion Content in the Cationic Polymers by Flame Atomic Absorption Spectrometry (FAAS). Polypeptoids (10 mg) were dissolved in DI water, and then analyzed by FAAS. The copper ion content was determined by using the copper (II) absorption at 324.8 nm against a calibration curve constructed using a CuSO4 standard. Preparation and Characterization of Polypeptoid/DNA Polyplexes. Polypeptoids and pCMV-Luc were dissolved in DEPC-treated water at 1 and 0.1 mg/mL, respectively, and they were mixed at various polypeptoid/DNA weight ratios (the N/P ratios of each polypeptoid at various weight ratios were shown in Table S1). PEI as the control was dissolved in DEPC-treated water at 0.1 mg/mL, and mixed with DNA (0.1 mg/mL) at the PEI/DNA weight ratio of 1 (N/P = 10). The polyplexes were formed after vortex for 10 s and incubation at 37 oC for 30 min. The DNA condensation level was first assessed by using a qualitative gel retardation assay as previously reported.52 An ethidium bromide (EB) exclusion assay was further used to quantitatively monitor the DNA condensation level as previously reported.53 10


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The hydrodynamic diameter and zeta potential of polypeptoid/DNA polyplexes were monitored by dynamic light scattering (Zetasizer Nano-ZS, Malvern). The stability of polypeptoid/DNA polyplexes in saline or serum was further evaluated by measuring the particle size after dilution of the polyplexes with PBS or 10% FBS-containing DMEM for 10 fold and incubation for different time. DNA Release. To assess DNA release from polypeptoid/DNA polyplexes, the heparin replacement assay was conducted.54 Basically, heparin at various final concentrations was incubated with polypeptoid/DNA polyplexes for 1 h at 37 °C, and the DNA release level was quantified by using the EB exclusion assay.53 In Vitro DNA Transfection. HeLa cells cultured in 96-well plates (~80% confluence) were incubated with polypeptoid/DNA or PEI/DNA polyplexes in opti-MEM (100 µL/well) at 0.3 µg DNA/well for 4 h. The medium was then replaced by complete medium, and cells were cultured for another 20 h. The Bright-Glo Luciferase assay kit (Promega) was used to determine the luciferase expression level according to the manufacturer’s protocol, and the BCA kit (Pierce) was used to quantify the cellular protein content. Results were represented as relative luminescence unit (RLU) per 1 mg cellular protein. To assess the transfection efficiencies under serum-containing conditions, polyplexes were incubated with cells in medium containing 10%, 30%, or 50% FBS for 4 h. Cells were then cultured in complete medium for another 20 h followed by determination of the transfection efficiency. The transfection capabilities of polypeptoids in B16F10 and COS-7 cells were also determined using the same method to explore their transfection generality. 11



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Intracellular Kinetics. For the purpose of quantifying its cellular uptake level, DNA was labeled with YOYO-1 using the previously reported method,52 and polypeptoid/YOYO-1-DNA polyplexes were form as described above. HeLa cells seeded in 96-well plates (~80% confluence) were treated with polyplexes in opti-MEM (100 µL/well) at 0.1 µg DNA/well for 4 h. Cells were then washed four times with heparin-containing PBS, and were lysed using the RIPA lysis buffer (100 µL/well). The concentration of YOYO-1-DNA in the lysate was measured on a microplate reader (λex = 485 nm, λem = 530 nm), and the protein concentration was monitored by the BCA kit. The cell uptake level was represented as ng YOYO-1-DNA/mg protein. The uptake experiment was also performed at 4 °C or in the presence of endocytic inhibitors. Briefly, cells were treated with polyplexes (polypeptoid/DNA = 2.5, w/w) at 4 °C for 4 h. Otherwise, cells were pre-treated with GNT (100 µg/mL), CPZ (10 µg/mL), WTM (10 µg/mL), or mβCD (5 mM) for 30 min at 37 °C. Polyplexes were then added and incubated with cells for 4 h at 37 °C. The uptake level of YOYO-1-DNA was determined as described above, and results were represented as percentage uptake level of control cells that received polyplexes treatment while no inhibitor treatment at 37 °C for 4 h. The endolysosomal escape of polyplexes was observed by confocal laser scanning microscopy (CLSM, Leica, TCS SP5, Germany). HeLa cells seeded on coverslips

in

24-well

plates

(~20%

confluence)

were

incubated

with

polypeptoid/YOYO-1-DNA polyplexes (w/w = 2.5) in opti-MEM (0.5 mL/well, 1 µg 12


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YOYO-1-DNA/well) for 4 h. Cells were then stained with Lysotracker-Red (200 nM, for endosome/lysosome), fixed with paraformaldehyde (4%), stained with DAPI (5 µg/mL, for nuclei), and observed by CLSM. Cytotoxicity. The cytotoxicity of polypeptoids and polypeptoid/DNA polyplexes were determined by the MTT assay as previously reported.53 Briefly, HeLa cells seeded on 96-well plates (~80% confluence) were incubated with free polypeptoids at various concentrations or polyplexes with various polypeptoid/DNA weight ratios (DNA amount maintained constant at 0.3 µg/well) in opti-MEM (100 µL/well). PEI/DNA polyplexes served as a control (N/P = 10, 0.3 µg DNA/well). After treatment for 4 h, the medium was changed to complete medium, and cells were cultured for another 20 h before viability assessment using the MTT assay. Results were denoted as percentage viability of control cells that did not receive treatment with polypeptoids or polyplexes. In Vivo DNA Transfection. B16F10 cells (1 × 107) were s.c. injected to the left flank of mice to establish the melanoma xenograft model. After ~8 days when the tumor reached ~100 mm3, polypeptoid/DNA polyplexes (w/w = 20) or PEI/DNA polyplexes (w/w = 5) were injected intratumorally (20 µg DNA/mouse, 50 µL/injection). HEPES buffer containing 5% glucose (HBG) served as a control. Each group contained four mice. Animals were sacrificed 48 h later, and the luciferase expression level in the tumors was measured using the previously reported method.52 Briefly, tumors were collected, washed with PBS, and homogenized with the passive lysis buffer. The lysate was then frozen-thawed for three cycles, and centrifuged to 13



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isolate the supernatant. The luciferase expression level was then measured and calculated as described above for the in vitro transfection study. Statistical Analysis. Statistical analysis was conducted by the Student’s t-test, and differences were set at p < 0.05 (*p < 0.05, **p < 0.01).

RESULTS Synthesis and Characterization of Polypeptoids. In this study, we

synthesized a library of cationic polypeptoids with varied molecular characteristics (e.g., backbone length, side-chain length, different charged side terminal groups, and incorporated hydrophobicity) (Scheme 1 and Table 1). PNPgG with different Mn were first synthesized via benzylamine-initiated controlled ROP of the corresponding Pg-NCA monomers (Scheme 1). The resulting PNPgG with Mn of 2.8-24 kDa (DP = 28-250) and PDI of 1.08-1.48 were obtained, as characterized by SEC-MALS-DRI and NMR analysis (Figure 1, S2, and S3). A library of polypeptoids was then synthesized by modification of the PNPgG with various ω-azido alkyl ammonium salts (Scheme S1) using the “click” chemistry (Scheme 1). The side-chain derivation was quantitative in all cases, evidenced by the complete disappearance of the terminal alkyne proton of propargyl group at 2.51 ppm and appearance of the triazole proton at 8.0 ppm from 1H NMR analysis (Figure S6-S15). The cationic polypeptoids were purified by treatment with EDTA to remove copper salts followed by extensive dialysis against DI water. Upon lyophilization, all polymers were obtained as white to pale green powders in good yield (60-96%) with copper ion content in the sub-10 ppm 14


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range based on the atomic absorption analysis, which was significantly lower than the concentration threshold that would otherwise cause potential copper ion-induced cytotoxicity. P1-P4 carried different cationic side-chain terminal groups (primary, secondary, tertiary, and quaternary amine) at the fixed DP of 46; P4-P7 possessed primary amine as the side terminal group while had different DP (28, 46, 135, and 250); P8-P10 had the same DP (46) and side terminal group (primary amine), while afforded different spacer lengths between the triazole and amine (3, 4, 5, and 6 methlene groups) (Table 1). Chemical structures of the cationic polypeptoids were verified by 1H NMR spectroscopy. All the resulting cationic polypeptoids afforded desired aqueous solubility, and thus could be readily used to complex DNA and mediate gene transfection in the aqueous medium. P11 bearing a fraction of hydrophobic N-decyl side chains (Figure S16) was similarly synthesized and characterized via modification of the P(NPgG-r-NDeG) random copolymer with ω-azido hexyl ammonium salt using “click” chemistry (Scheme 1). The P(NPgG-r-NDeG) copolymer precursor was synthesized by copolymerization of Pg-NCA and De-NCA using benzylamine as the initiator, and was characterized by SEC-MALS-DRI and NMR analysis (Figure S1, S4 and S5). P11, a random copolypeptoid analogue of P10, contained both primary amine groups (with 6 methylene groups between trizole and amine) and hydrophobic decyl groups on side chains, at the DP of 48 that was similar to P10 (Table 1). Characterization of Polypeptoid/DNA Polyplexes. The gel retardation assay was first conducted to evaluate DNA condensation by the polypeptoids (Figure S17). 15



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All the positively charged polypeptoids could condense DNA at w/w ratios ≥ 2.5, indicating complete condensation of DNA. In consistence, the EB exclusion assay demonstrated that all the polypeptoids could condense more than 80% of the DNA at w/w ratios ≥ 2.5 (Figure 2). Polypeptoids with different charged groups (P1-P4) or with different side-chain lengths (P4, P8, P9, and P10) showed similar DNA condensation profiles. In comparison, longer polypeptoid backbone correlated to enhanced DNA condensation level, wherein DNA migration in the agarose gel was restricted by P7 (250 mer) at the polymer/DNA w/w ratio of 1 in comparison to the higher weight ratio of 2.5 for P5 (28 mer), P4 (46 mer), and P6 (135 mer). In consistence, the DNA condensation level was represented by the order of P7>P6>P4>P5, mainly due to the facilitated entanglement of longer polypeptoids with DNA molecules (Figure 2B). Additionally, P11 with introduced aliphatic decyl side chains afforded higher DNA condensation level than its un-modified analogue P10 despite its lower content of cationic group (Figure 2C), indicating that the hydrophobic interaction with DNA would provide additional forces to drive the assembly between polymer and DNA. As a result of their DNA condensation capabilities, all polypeptoids formed 100-200 nm polyplexes with DNA at the polypeptoid/DNA weight ratio > 2.5, and the polyplexes displayed positive surface charges that would facilitate binding to cell membranes toward effective cellular internalization (Figure S18). The particle sizes of P10/DNA and P11/DNA polyplexes were unappreciably altered upon dilution with PBS or FBS-containing DMEM, which further indicated their desired stability in the presence of saline or serum (Figure S19). 16


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DNA Release. To simulate DNA release from polyplexes in the cytoplasm, polyplexes were treated with heparin at different concentrations and the DNA unpackaging level was then determined. As shown in Figure 3, DNA could be notably released at the heparin concentration of 0.01 mg/mL, and more DNA was released at further increased heparin concentrations. In consistence with their DNA binding affinities, polypeptoids with longer backbone length achieved lower DNA release level at the same heparin concentration (Figure 3B), presumably due to the enhanced molecular entanglement with DNA. In Vitro Gene Transfection. Polypeptoids containing fixed DP (46) and side-chain length yet different side charged groups (P1, P2, P3 and P4) were first evaluated in terms of the transfection efficiency in HeLa cells in serum-free medium. As shown in Figure 4A, P4 bearing primary amine displayed remarkably higher transfection efficiency than its analogues containing secondary, tertiary, or quaternary amines. As such, based on the structure of P4 with primary amine as the optimal cationic charged group, the DP of polymers was varied (P4, P5, P6 and P7) to explore the effect of backbone length on the gene transfection capabilities. As shown in Figure 4B, an increase of polymer DP from 28 (P5) to 46 (P4) resulted in significantly enhanced transfection efficiency, presumably attributed to the enhanced DNA condensation and interactions with cell membranes. However, a further increment of DP to 135 (P6) and 250 (P7) led to decreased transfection efficiency, which could be due to the excessively long polymer backbone that restricted intracellular DNA release and enabled appreciable toxicity to cells. 17



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Enhancing hydrophobicity of polymers often facilitates the hydrophobic interaction with cell membranes to promote cellular internalization. To this end, we prepared polypeptide analogues of P4 (DP = 46, 3 methylene groups between triazole and primary amine) with increased hydrophobic side chain lengths (P8, P9, and P10, with 4, 5, and 6 methylene groups between triazole and primary amine, respectively). As expected, the transfection efficiencies of polypeptoids in HeLa cells increased when the hydrophobic side-chain length was prolonged (Figure 4C), presumably due to enhanced membrane activities of the polymers to facilitate intracellular internalization of DNA cargoes. P10 showed the highest transfection efficiency at the low polypeptoid/DNA weight ratio of 2.5, outperforming commercial transfection reagent PEI by ~8 fold at its optimal N/P ratio of 10 that was consistent with previous reports.55 Based on such findings, we were motivated to further modulate the hydrophobicity of polypeptoids, attempting to maximize their transfection capabilities. It have been reported that random copolymers bearing charged side groups and hydrophobic side chains display higher membrane affinities than the non-hydrophobic homopolymers, mainly because of the improved hydrophobic interactions with cell membranes. We thus went on to explore whether such strategy would work to further enhance the gene delivery efficiency of polypeptoids. P11, a random copolymer analogue of P10, was synthesized which contained ~20 mol% of pendent decyl groups. As shown in Figure 4D, P11 displayed significantly improved transfection efficiency over P10 by ~6 fold and it outperformed PEI by ~45 fold, which 18


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substantiated the critical role of introduced hydrophobic aliphatic side chains in promoting intracellular DNA delivery. With P10 and P11 as the best-performing materials identified, we further evaluated their transfection efficiencies in B16F10 and COS-7 cells to explore their generality. Consistent with previous findings in HeLa cells, P11 demonstrated markedly higher transfection efficiencies than its homopolymer analogue P10, and it outperformed PEI by ~10 fold and ~3 fold in B16F10 and COS-7 cells, respectively (Figure 4E and 4F). More importantly, both P10 and P11 achieved the highest transfection efficiency at relatively low polymer/DNA weight ratio of 2.5 (equals to N/P ratio of ~3.6 and ~3, respectively), in comparison to majority of polycation-based gene vectors that often require high N/P ratios to achieve effective gene transfection (e.g. N/P = 10 for PEI,52 N/P = 60 for poly(β-amino ester),56 N/P = 10 for dendrimers57). Such desired property could presumably be attributed to the presence of hydrophobic domains that strengthened the membrane interactions, such that excessive cationic polymers are not necessarily required to impose sufficient binding affinities with negatively charged cell membranes. The low N/P ratio used for gene transfection would reduce the cytotoxicity of the material, and would also render the polyplexes with higher stability in saline or serum (Figure S19). Markedly compromised transfection efficiency in serum serves as a notorious obstacle against polycation-mediated gene transfection. We thus further monitored the transfection efficiencies of the top-performing P10 and P11 in the presence of 10% FBS in HeLa, B16F10, and COS-7 cells. As shown in Figure 5, P10 and P11 suffered 19



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from compromised gene transfection efficiencies at the same polypeptoid/DNA weight ratio of 2.5 when compared to those under serum-free conditions. However, the transfection efficiencies could be largely recovered to the levels under serum-free conditions when the polypeptoid/DNA weight ratio was enhanced to 10, probably because the additional cationic charges as well as hydrophobicity mediated stronger interactions between DNA and polypeptoids that competitively compensated the DNA replacement by serum. When the FBS concentration was further enhanced to 30% and 50%, P11 was still able to mediate high transfection efficiencies that were only several fold lower than those under serum-free conditions (Figure 5C), which indicated their potential utilities toward serum-resistant gene delivery as well as in vivo gene delivery. In direct comparison to PEI, P11 showed 3-4 orders of magnitude higher transfection efficiency in serum-containing media, which further rendered it a promising addition to existing gene delivery materials. Intracellular Kinetics. The gene transfection capability of polyplexes is largely associated with their intracellular kinetics. As such, we explored the intracellular kinetics of the polypeptoid/YOYO-1-DNA polyplexes, including the cellular uptake, internalization mechanism, and endolysosomal escape. As shown in Figure 6A and S20, P4 bearing primary amine side terminal groups displayed higher DNA uptake level than its analogues containing secondary, tertiary, and quaternary amines (P1, P2, and P3). Elongation of the polypeptoid backbone also led to enhanced DNA uptake level in the order of P5 (28 mer)

Cationic Polypeptoids with Optimized Molecular Characteristics

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