plasmid DNA micelles postmodified with functional

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Block copolymer/plasmid DNA micelles postmodified with functional peptides via thiol-maleimide conjugation for efficient gene delivery into plants Takaaki Miyamoto, Kousuke Tsuchiya, and Keiji Numata Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01304 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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Biomacromolecules

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Block copolymer/plasmid DNA micelles

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postmodified with functional peptides via thiol-

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maleimide conjugation for efficient gene delivery

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into plants

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Takaaki Miyamoto, Kousuke Tsuchiya*, Keiji Numata*

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Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1

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Hirosawa, Wako-shi, Saitama 351-0198, Japan

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KEYWORDS: micelle postmodification, thiol-maleimide conjugation, cell-penetrating peptide, nuclear localization signal, plant gene delivery

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1 ACS Paragon Plus Environment

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ABSTRACT

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Introducing exogenous genes into plant cells is essential for a wide range of applications in

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agriculture and plant biotechnology fields. Cationic peptide carriers with cell-penetrating and

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DNA-binding domains successfully deliver exogenous genes into plants. However, their cell-

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penetrating activity may be attenuated by undesired electrostatic interactions between the cell

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penetrating peptide (CPP) domain and DNA cargo, resulting in a limited gene delivery efficiency.

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Here, we developed a block copolymer, maleimide-conjugated tetra(ethylene glycol) and poly(L-

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lysine) (MAL-TEG-PLL). Through electrostatic interactions with plasmid DNA (pDNA), MAL-

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TEG-PLL formed a micelle that presented maleimide groups on its surface. The micelle enabled

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postmodification with cysteine-containing functional peptides, including a CPP (BP100-Cys) and

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nuclear localization signal (Cys-NLS), via thiol-maleimide conjugation, thereby avoiding

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undesired interactions. According to a comparison of gene delivery efficiencies among the peptide-

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postmodified micelles, the amount of BP100-Cys on the micelle surface was key for efficient gene

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delivery. The BP100-postmodified micelle showed a more efficient delivery compared with that

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of the BP100-premodified micelle. Thus, postmodification of polymeric micelles with functional

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peptides opens a door to designing highly efficient plant gene delivery systems.

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Biomacromolecules

INTRODUCTION

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Gene transfer methods applicable to plant systems have been gaining interest along with the

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growing demand for genetically modified plants for various applications in agriculture,1

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biorefinery,2 and the energy industry.3 However, the current gene transfer methods, such as particle

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bombardment,4 agrotransfection,5 and poly(ethylene glycol) (PEG)-mediated gene transfer,6 are

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limited with respect to applicable plant types, transgene sizes, or target organelles. As an

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alternative approach, peptide-based gene delivery systems have emerged, and several groups have

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demonstrated peptide-mediated delivery of various sizes of nucleic acids into various types of

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plant cells.7–16 Many studies on peptide-based gene delivery into plant cells utilized cell-

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penetrating peptides (CPPs), including Tat2,8,11,13,14 Pep-1,13 and arginine-rich CPPs7,10,15, as gene

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carriers because of the excellent permeation ability of CPPs into a wide variety of plant species

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and tissues.17

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We have developed fusion peptides by linking a CPP with a polycationic peptide that functions

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as a DNA binding domain.18–20 The fusion peptides delivered plasmid DNA (pDNA) into plant

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nuclei more efficiently than did CPPs alone.18 In addition to pDNA, double-strand DNA

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(dsDNA)19 and double-strand RNA (dsRNA)20 were transferred into intact plants by the fusion

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peptide. More recently, we successfully demonstrated pDNA release in response to a cytosolic

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environment by a fusion peptide composed of a CPP and reducible DNA binding domain.21 Other

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fusion peptides were designed by joining an organelle targeting peptide, such as a chloroplast or

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mitochondria targeting signal, to a polycationic peptide, and successfully used for selective gene

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delivery into target organelles, such as mitochondria and plastids.22,23 The high selectivity against

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target organelles is a remarkable advantage of our fusion peptide-based system over the currently

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existing methods. However, the plant gene delivery mediated by fusion peptides is still at an early



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stage in terms of efficiency. A possible problem of fusion peptides is an undesired interaction

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between anionic DNA and cationic functional peptide sequence, such as CPP. Although the

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polycationic moiety is intended to interact with DNA in fusion peptides, the cationic CPP moiety

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also can electrostatically interact with DNA, leading to attenuated cell-penetrating activity and a

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subsequent decrease in the gene delivery efficiency.

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In the present study, to avoid potential undesired interactions between cationic functional

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domains of carriers and DNA cargos, we developed a block copolymer consisting of maleimide-

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modified tetra(ethylene glycol) and poly(L-lysine), referred to as MAL-TEG-PLL (Figure 1). We

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envisaged that MAL-TEG-PLL would condense pDNA and form a core shell micelle that displays

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maleimide groups on the surface (Figure 1), as well-established block copolymers composed of

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PEG and poly(L-lysine) (PLL) segments form a micelle structure with the PEG layer surrounding

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the core, where DNA is condensed by PLL.24 The maleimide group displayed on the micelle

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surface is expected to enable postmodification with cysteine-containing cationic functional

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peptides via thiol-maleimide conjugation (Figure 1). Thiol-maleimide click chemistry has been

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widely used to prepare functionalized nanoparticles with high conjugation efficiency in mild

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conditions.25,26 We hypothesized that cationic functional peptides, such as CPPs, conjugated on

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the micelle surface would not electrostatically interact with the pDNA cargo, since the PLL

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segment is highly cationic and able to neutralize the negative charge of the pDNA in the micelle

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core. Thus, the postmodification strategy was expected to avoid undesired interactions between

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cationic functional peptides and anionic pDNA, leading to enhanced gene delivery in plants.

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To prove the proposed concept, we postmodified MAL-TEG-PLL/pDNA micelles with a CPP

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and nuclear localization signal (NLS), a cationic peptide enhancing nuclear import, using thiol-

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maleimide conjugation. The comparison of the gene delivery efficiency in plants among several


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peptide-postmodified micelles revealed that the amount of CPP on the micelle surface was a

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critical factor affecting the efficiency. Furthermore, the higher gene delivery efficiency of a CPP-

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postmodified micelle than of a CPP-premodified micelle clearly demonstrated the utility of the

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present strategy for efficient gene delivery into plants.

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Figure 1. Schematic representation of MAL-TEG-PLL/pDNA micelle formation and micelle

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postmodification with functional peptide via thiol-maleimide conjugation.

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EXPERIMENTAL METHODS

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Materials. Papain (EC No. 3.4.22.2) was purchased from Tokyo Chemical Industry Co., Ltd.

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(Tokyo, Japan) and used as received. Peptides including BP100-Cys (KKLFKKILKYLC) and

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Cys-NLS (CPKKKRKV) were obtained from RIKEN BSI (Wako, Japan). Hoechst 33258 was

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purchased from Thermo Fisher Scientific (Waltham, MA, U.S.A.). The other chemicals were

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purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), FUJIFILM Wako Pure

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Chemical Corporation (Osaka, Japan), or Watanabe Chemical Industries, Ltd. (Hiroshima, Japan),

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and used as received without purification unless otherwise noted. The pDNA used in this study

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encoded an engineered Oplophorus luciferase (Nluc) or a green fluorescent protein (GFP) gene



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with the cauliflower mosaic virus 35S promoter and the Agrobacterium tumefaciens nopaline

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synthase gene terminator (p35S-Nluc-tNOS or p35S-GFP-tNOS).

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Synthesis of Nε-Boc-protected poly(L-lysine) [PLL(Boc)] by Chemoenzymatic

4

Polymerization. Chemoenzymatic polymerization of lysine was performed using a method

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modified from a previous report.27–29 Nε-(tert-Butoxycarbonyl)-L-lysine methyl ester hydrochloride

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(5 g; 16.8 mmol) was added into phosphate buffer (9 mL, 1 M, pH 8.0) and stirred in a 10 mL

7

glass tube until the substrate was completely dissolved. Papain (1.0 g) suspended in phosphate

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buffer (5 mL; 1 M; pH 8.0) was added into the substrate solution. The mixture was stirred at 40 °C

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and 800 rpm for 4 h with an EYELA ChemiStation (Tokyo, Japan). After the reaction, the mixture

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was transferred into a 50 mL Falcon tube and centrifuged at 8,000 rpm for 15 min. The resulting

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precipitate was washed twice with MilliQ and lyophilized to afford PLL(Boc) as a white powder

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(1.09 g, 28% yield). The degree of polymerization of PLL(Boc) was analyzed by MALDI-TOF

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MS (Figure S1). The chemical structure of PLL(Boc) was confirmed by 1H NMR (Figure S2).

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Synthesis of MAL-TEG-PLL. PLL(Boc) (0.235 g, 0.168 mmol), 19-maleimido-17-oxo-

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4,7,10,13-tetraoxa-16-azanonadecanoic acid (MAL-TEG-COOH) (0.115 g, 0.276 mmol), 1-ethyl-

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3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.106 g, 0.552 mmol), and chloroform (1

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mL) were mixed in a flask equipped with a magnetic stir bar. After stirring at 25 °C for 24 h, the

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solvents were removed by a rotary evaporator. The residue was suspended in tetrahydrofuran (5

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mL) and gradually added into MilliQ (40 mL) in a 50 mL Falcon tube. A white precipitate was

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collected by centrifugation at 8,000 rpm for 15 min and lyophilized to obtain MAL-TEG-

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PLL(Boc) as a white powder. MAL-TEG-PLL(Boc) (0.276 g) and trifluoroacetic acid (3 mL) were

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mixed in a flask equipped with a magnetic stir bar. The mixture was stirred at 25 °C for 24 h. After

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removing the solvents under the reduced pressure, the crude product was washed three times with


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diethyl ether (30 mL) and dried in vacuo overnight. The obtained hygroscopic solid was

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lyophilized to afford MAL-TEG-PLL as a brown sticky powder (0.216 g, 68% yield). The obtained

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MAL-TEG-PLL was analyzed by proton nuclear magnetic resonance (1H NMR), matrix assisted

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laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and reversed

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phase-high performance liquid chromatography (RP-HPLC).

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Preparation and Characterization of MAL-TEG-PLL/pDNA Micelles. MAL-TEG-PLL

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powder was dissolved in Milli-Q water. The concentration of MAL-TEG-PLL was adjusted to 0.2

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mg/mL and confirmed by measuring the absorbance at 220 nm with a spectrophotometer (V-750;

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Jasco, Tokyo, Japan). Different amounts of MAL-TEG-PLL solution (0.2 mg/mL) were added into

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Milli-Q water containing pDNA (p35S-GFP-tNOS) (20 µg) at various N/P ratios (defined as the

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number of amino groups from MAL-TEG-PLL/the number of phosphate groups from pDNA). The

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final volume and pDNA concentration were fixed to 800 µL and 25 µg/mL, respectively. The

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solution was vortexed and incubated at least for 1 h at 4 °C to obtain a MAL-TEG-PLL/pDNA

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micelle. MAL-TEG-PLL/pDNA micelle solutions (800 µL), prepared at various N/P ratios, were

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transferred to a folded capillary cell (DTS1070; Malvern Panalytical, Worcestershire, U.K.) and

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used for dynamic light scattering (DLS) and zeta potential measurements with a zeta potentiometer

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(Zetasizer Nano-ZS; Malvern Panalytical, Worcestershire, U.K.). DLS measurements were

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performed three times at 25 °C, and the obtained hydrophobic diameter and polydispersity index

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(PDI) values were averaged. Zeta potential measurements were performed three times at 25 °C to

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obtain an average value of the zeta potential.

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Preparation and Characterization of Micelles Postmodified with Either BP100-Cys or

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Cys-NLS. The MAL-TEG-PLL/pDNA (p35S-Nluc-tNOS) micelle (prepared at N/P 6) was

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postmodified with either BP100-Cys or Cys-NLS in HEPES buffer (3 mM, pH 7.0). The final



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concentration of MAL-TEG-PLL was fixed to 60 µM, whereas that of the peptide was adjusted to

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24 or 48 µM. The mixture was stirred at 25 °C and 1,200 rpm for 1 h with a mixer (Thermomixer

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comfort, Eppendorf, Hamburg, Germany), and then used for MALDI-TOF MS, DLS, and RP-

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HPLC analyses.

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MALDI-TOF MS spectra were recorded on a MALDI-TOF spectrometer (Autoflex speed

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LRF; Bruker Daltonics, Billerica, MA, U.S.A) operating in a positive ion reflection mode at an

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accelerating voltage of 15 kV. Samples for MALDI-TOF MS analyses were prepared by mixing

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micelle solutions after modification (2 µL) and water/acetonitrile (20/80, 2 µL) containing α-

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cyani-4-hydroxycinnamic acid (10 mg/mL) and trifluoroacetic acid (TFA) (0.1%). The samples (2

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µL) were deposited on an MTP 384 ground steel BC target plate and dried under reduced pressure.

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DLS measurements were performed using the modified micelle solutions (200 µL) in a similar

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way as described above. The RP-HPLC system consisted of an auto sampler (AS-2055, JASCO,

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Tokyo, Japan), a gradient pump (PU2089, JASCO), a column oven (CO-4060, JASCO), and a C18

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column (YMC-Triart C18, particle size 5 µm, 150 × 4.6 mm i.d., YMC, Kyoto, Japan). Boc-Gly-

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OH and tris(2-carboxyethyl)phosphine hydrochloride solutions (TCEP) were used for an internal

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standard and a reducing agent, respectively. Boc-Gly-OH and TCEP were added into the modified

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micelle solutions (160 µL), and their final concentrations were adjusted to 1 mg/mL and 100 mM,

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respectively. The mixtures were incubated for 5 min to reduce a disulfide bond between the

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peptides. Then, the mixtures (100 µL) were injected into the RP-HPLC system with the mobile

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phase composed of Milli-Q water (eluent A), acetonitrile (eluent B), and 1% (v/v) TFA aqueous

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solution (eluent C). For analyses of BP100-modified micelles, the composition of the mobile phase

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changed from 85% A, 5% B, and 10% C to 35% A, 55% B, and 10% C over 20 min in a linear

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gradient mode at a flow rate of 1 mL/min. Meanwhile, for analyses of NLS-modified micelles, the


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Biomacromolecules

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composition altered gradually from 85% A, 5% B, and 10% C to 55% A, 35% B, and 10% C over

2

30 min at 1 mL/min. The column temperature was kept at 25 °C and elution of the various

3

compounds was monitored by UV absorbance at 220 nm. Peak areas and retention times of eluting

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compounds were determined by chromatography software (ChromNAV, JASCO, Tokyo, Japan).

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Unreacted peptides (BP100-Cys or Cys-NLS) were quantified from the peak area corrected by that

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of Boc-Gly on the basis of a calibration curve, which was obtained by plotting the peak area of the

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peptide against the concentration. The conversion rate of MAL-TEG-PLL was estimated from the

8

amount of unreacted peptide.

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Preparation and Characterization of Micelles Postmodified with Both BP100-Cys and

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Cys-NLS. The MAL-TEG-PLL/pDNA (p35S-Nluc-tNOS) micelle (prepared at N/P 6) was

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concurrently postmodified with BP100-Cys and Cys-NLS in HEPES buffer (3 mM, pH 7.0), where

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the final concentration of MAL-TEG-PLL was 60 µM. Three combinations of peptide

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concentrations were used: BP100-Cys/Cys-NLS, 24 µM/24 µM, 48 µM/24 µM, or 24 µM/48 µM.

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The mixture was stirred at 25 °C and 1,200 rpm for 1 h with the mixer and analyzed by MALDI-

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TOF MS, DLS, and RP-HPLC per the above described method.

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Plant Preparation Nicotiana benthamiana seeds were germinated in pots with planting

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medium consisting of soil and vermiculite in a ratio of 2:1. The plant was grown in a plant

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incubator (Biotron NK System, Japan) under 16 h-day/8 h-night cycles at 30 °C and 70% relative

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humidity with light intensity of 50,000 lux. After incubation for 4 weeks from germination, the

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grown plants with several expanded leaves were used for experiments.

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Evaluation of Gene Delivery Efficiency in Plants. N. benthamiana leaves were infiltrated

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with solutions (~100 µL) containing naked pDNA (25 µg/mL, p35S-Nluc-tNOS), MAL-TEG-

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PLL/pDNA micelle (prepared at N/P 6), or peptide-modified micelles (prepared in the same way



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as described above) using a needleless syringe; that is, we gently pressed the syringe containing

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each solution onto the abaxial (backside) surface of the leaves, as reported previously.18 The

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infiltrated plants were incubated for 16 h in the plant incubator described above. The infiltrated

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regions in the leaves were cut into 1 cm-diameter disks with a cork borer. Each leaf disk was

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homogenized in Renilla Luciferase Assay Lysis Buffer (100 µL, Promega, Madison, WI) and

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incubated at 25 °C for 6 h to ensure complete lysis. The lysate was centrifuged at 13,000 rpm for

7

1 min, and the supernatant (50 µL) was added into a mixture (50 µL) of Nano-Glo® Luciferase

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Assay Substrate (Promega) and Nano-Glo® Luciferase Assay Buffer (Promega). Immediately

9

after vortexing, the Nluc expression level in the mixture was evaluated by measuring the

10

luminescence intensity in relative light units (RLU) with a luminometer (Glo®Max 20/20,

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Promega). The supernatant obtained from the lysate was diluted with Milli-Q water and mixed

12

with a Bradford reagent (APRO SCIENCE, Tokushima, Japan) to quantify the protein amount

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from the absorbance at 595 nm. The RLU was divided by the protein amount to obtain the RLU/mg

14

value. The background correction was performed by subtracting an average RLU/mg value of non-

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infiltrated leaves from each RLU/mg value of the infiltrated leaf. The corrected RLU/mg values

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obtained from four samples were averaged and used for quantitative evaluation of pDNA delivery

17

efficiency.

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Evaluation of Intracellular Gene Expression and Distribution of BP100-postmodified

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Micelles. The MAL-TEG-PLL/pDNA (p35S-GFP-tNOS) micelle (prepared at N/P 6) was

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modified with BP100-Cys (48 µM) as described above. The BP100-modified micelle was

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infiltrated into N. benthamiana leaves with a needleless syringe. After 16 h of incubation in the

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plant incubator, GFP expression in the infiltrated leaves was confirmed by confocal laser scanning


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Biomacromolecules

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microscopy (CLSM, Leica Microsystems, Wetzlar, Germany). GFP fluorescence was excited at

2

480 nm, and observed in an emission range from 490 to 550 nm.

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Cy3-labeled pDNA was prepared with a Label IT Nucleic Acid Labeling Kit (Mirus Bio,

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LLC, Madison, WI, U.S.A). Micelles of Cy3-labeled pDNA (25 µg/mL) and MAL-TEG-PLL (N/P

5

6) were postmodified with 48 µM BP100-Cys using the above-described method. The resulting

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BP100-modified micelle was infiltrated into N. benthamiana leaves. After 16 h of incubation, the

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infiltrated region of the leaf was cut and dipped into solution containing Hoechst (8 µM) for 10

8

min, followed by washing with Milli-Q. The treated leaf was then used for CLSM observation:

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Hoechst, excitation (405 nm), emission (430–515 nm); Cy3, excitation (514 nm), emission (545–

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615 nm); chlorophyll, excitation (514 nm), emission (650–730 nm).

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Synthesis of BP100-MAL-TEG-PLL and Preparation of BP100-premodified Micelle.

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MAL-TEG-PLL (final concentration, 2.14 mM) and BP100-Cys (2.6 mM) were reacted in 500 µL

13

of HEPES buffer (50 mM, pH 7.3). After incubation at 25 °C and 1200 rpm for 4 h, the reaction

14

mixture was subjected to purification by RP-HPLC. The conditions were same as for the above-

15

described RP-HPLC analysis of the BP100-postmodified micelle. A fraction containing BP100-

16

MAL-TEG-PLL was collected and then lyophilized. The obtained powder was analyzed by

17

MALDI-TOF MS and RP-HPLC (Figure S5). To prepare a BP100-premodified micelle, BP100-

18

MAL-TEG-PLL and MAL-TEG-PLL solutions were added into Milli-Q containing pDNA (20

19

µg), and the final volume was adjusted to 832 µL. The final concentrations of BP100-MAL-TEG-

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PLL, MAL-TEG-PLL, and pDNA were 48 µM, 24 µM, and 24 µg/mL, respectively. The BP100-

21

premodified micelle was characterized by DLS and zeta potential analyses in the same method as

22

described before. The Nluc assay of the BP100-premodified micelle was performed using the

23

above-mentioned method.



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Statistical Analysis. Significant differences in the Nluc assay were evaluated by a Mann-

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Whitney U test. All data obtained from the Nluc assay were presented as the means ± standard

3

deviation (n = 4). Differences between two means were considered statistically significant when

4

P values were less than 0.05.

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RESULTS

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Synthesis of MAL-TEG-PLL. We synthesized MAL-TEG-PLL by condensing MAL-TEG-

8

COOH and PLL(Boc), which was obtained by the papain-catalyzed polymerization of Nε-Boc-

9

lysine methyl ester, followed by deprotection of the Nε-Boc groups. The chemical structure of

10

MAL-TEG-PLL was confirmed by 1H NMR analysis (Figure 2A). The MALDI-TOF MS

11

spectrum indicated two major peaks that corresponded to the molecular weights of the block

12

copolymer containing penta(L-lysine) and hexa(L-lysine) segments (Figure 2B). In the RP-HPLC

13

chromatogram (Figure 2C), a major peak assignable to MAL-TEG-PLL was observed at 7.7 min.

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Biomacromolecules

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Figure 2. Characterization of MAL-TEG-PLL. (A) 1H NMR spectrum. Peaks observed in the

3

spectrum were attributed to protons of MAL-TEG-PLL. (B) MALDI-TOF MS spectrum. Peaks

4

appearing at m/z values of 1074 and 1200 corresponded to the block copolymers containing penta

5

and hexa(L-lysine) segments, respectively. (C) RP-HPLC chromatogram. The major peak eluting

6

at 7.7 min corresponded to MAL-TEG-PLL.



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Micelle Formation of MAL-TEG-PLL with pDNA. We performed DLS and Zeta potential

2

analyses of MAL-TEG-PLL/pDNA (p35S-Nluc-tNOS) micelles prepared at various N/P ratios (1–

3

7) to determine their sizes, size distributions, and surface charges. The mean hydrodynamic

4

diameters of the micelles decreased from ~210 to ~80 nm with increasing N/P ratios from 2 to 4,

5

whereas the diameters remained 80–90 nm at N/P 4–7 (Figure 3A), which suggests that pDNA

6

condensation was induced by micelle formation with MAL-TEG-PLL and was completed at an

7

N/P ratio of 4. The PDI values less than 0.19 at all N/P ratios were indicative of the monodispersity

8

of the micelles (Figure 3B). The zeta potentials increased from −30 to 30 mV accompanied by the

9

increase of N/P from 1 to 7 (Figure 3C).

10

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Figure 3. Physicochemical properties of MAL-TEG-PLL/pDNA (p35S-Nluc-tNOS) micelles.

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(A) Hydrodynamic diameters, (B) PDI values, and (C) zeta potentials of MAL-TEG-PLL/pDNA

14

micelles prepared at N/P ratios ranging from 1 to 7. The hydrodynamic diameters and PDI values

15

were determined by DLS measurements, whereas the zeta potentials were obtained from zeta

16

potential analyses. All data represent the means ± standard deviations (n = 3).

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Postmodification of MAL-TEG-PLL/pDNA Micelles with Functional Peptides by Thiol-

3

maleimide Conjugation. To postmodify MAL-TEG-PLL/pDNA micelles with either a CPP or a

4

NLS via thiol-maleimide conjugation, a cysteine residue was introduced into the C-terminus of

5

BP100, an antimicrobial peptide with efficient cell-penetrating activity,30,31 and the N-terminus of

6

NLS derived from Simian virus 40 large T antigen.32 The resulting peptides, BP100-Cys and Cys-

7

NLS, were used to postmodify the micelles prepared at an N/P ratio of 6, in which the maleimide

8

concentration corresponded to 72 µM. The conjugation was performed in 3 mM HEPES buffer

9

(pH 7.0) for 1 h at 25°C and confirmed by MALDI-TOF MS analysis (Figure S3). We

10

characterized the peptide-postmodified micelles by RP-HPLC (Figure S4) and DLS analyses to

11

determine the percentages of maleimide groups conjugated to functional peptides, hydrodynamic

12

diameters, and PDI values of the micelles. When the micelle was postmodified with 24 and 48 µM

13

BP100-Cys, the percentage of maleimide groups conjugated to BP100-Cys was 29 ± 0 and 57 ±

14

1%, respectively (Table 1). Similar to the BP100-postmodified micelles, the micelle reacted with

15

24 and 48 µM Cys-NLS possessed 26 ± 1 and 51 ± 2% of peptide-conjugated maleimide groups,

16

respectively (Table 1). The hydrodynamic diameters (76–85 nm) and PDI values (0.136–0.201)

17

of the BP100- or NLS-postmodified micelles were similar to those of the micelle before

18

modification (N/P 6, 90 ± 3 nm, 0.168 ± 0.024).

19

Next, we performed concurrent postmodification of the micelle with BP100-Cys and Cys-

20

NLS. The micelle was reacted with both the peptides using three different combinations of peptide

21

concentrations: (i) 24 µM BP100-Cys and Cys-NLS, (ii) 48 µM BP100-Cys and 24 µM Cys-NLS,

22

(iii) 24 µM BP100-Cys and 48 µM Cys-NLS. The peptide-modified micelles were characterized

23

by MALDI-TOF MS (Figure S3), RP-HPLC (Figure S4) and DLS analyses. In the MALDI-TOF



Page 16 of 33

1

MS spectrum (Figure S3), we observed peaks corresponding to the molecular weights of MAL-

2

TEG-PLL conjugated to BP100-Cys and Cys-NLS, showing that the micelle was simultaneously

3

modified with both functional peptides. Based on the RP-HPLC analysis of the peptide-modified

4

micelles (Figure S4), we calculated the percentages of maleimide group conjugated to BP100-Cys

5

and Cys-NLS. For the micelle modified with 24 µM BP100-Cys and Cys-NLS, the percentage of

6

maleimide groups conjugated to Cys-NLS (29 ± 0%) was twice those conjugated to BP100-Cys

7

(14 ± 1%) (Table 1). The micelle modification with the same BP100-Cys (24 µM) and an increased

8

Cys-NLS (48 µM) concentration resulted in a percentage of maleimide conjugated to Cys-NLS

9

(37 ± 2%) that was three times greater than that conjugated to BP100-Cys (13 ± 3%) (Table 1). In

10

contrast, when 48 µM BP100-Cys and 24 µM Cys-NLS were used for conjugation, the percentage

11

of maleimide conjugated to BP100-Cys (28 ± 2%) was larger than that of maleimide conjugated

12

to Cys-NLS (19 ± 0%) (Table 1). Similar to the micelles modified with the single peptide, all three

13

micelles, which were concurrently modified with BP100-Cys and Cys-NLS, exhibited similar

14

hydrodynamic diameters (71–84 nm) and size distributions (PDI, 0.114–0.181) to those of the

15

unmodified micelle (Table 1).

16 17 18 19 20 21 22 23


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Biomacromolecules

1 2

3 4 5

Table 1. Characterization of MAL-TEG-PLL micelles modified with functional peptides Maleimide Maleimide group Hydrodynamic group conjugated to conjugated to diameter (d. NLS-Cys nm) b BP100-Cys a a (%) (%)

PDI b

[BP100-Cys] (µM)

[Cys-NLS] (µM)

24

−

29 ± 0

−

77 ± 3

0.180 ± 0.011

48

−

57 ± 1

−

76 ± 4

0.136 ± 0.026

−

24

−

26 ± 1

83 ± 3

0.147 ± 0.012

−

48

−

51 ± 2

85 ± 5

0.201 ± 0.024

24

24

14 ± 1

29 ± 0

71 ± 0

0.181 ± 0.025

48

24

28 ± 2

19 ± 0

71 ± 2

0.114 ± 0.028

24

48

13 ± 3

37 ± 2

84 ± 2

0.147 ± 0.022

a

Values were determined by RP-HPLC analysis and shown as the means ± standard deviation (n = 3). b Values were determined by DLS measurements and shown as the means ± standard deviation (n = 3).

6 7

Plant Gene Delivery of Peptide-postmodified MAL-TEG-PLL/pDNA Micelles. To prove

8

the benefits of postmodification of MAL-TEG-PLL/pDNA micelles with functional peptides for

9

gene delivery in intact plants, we compared the peptide-postmodified micelles with an unmodified

10

micelle in gene delivery efficiency. According to the above-described conditions, we prepared the

11

micelles at an N/P ratio of 6 without modification (referred to as unmodified) and several micelles

12

were postmodified with different combinations and amounts of functional peptides: a micelle



Page 18 of 33

1

(prepared at N/P 6) modified with 48 µM BP100-Cys (referred to as BP100-48), a micelle modified

2

with 24 µM BP100-Cys (BP100-24), a micelle modified with 48 µM Cys-NLS (NLS-48), a micelle

3

comodified with 24 µM BP100-Cys and Cys-NLS (BP100-24/NLS-24), and a micelle comodified

4

with 48 µM BP100-Cys and 24 µM Cys-NLS (BP100-48/NLS-24). The pDNA (p35S-Nluc-tNOS)

5

was used for micelle preparation. A solution containing each micelle or a naked pDNA (negative

6

control) was infiltrated into leaves of N. benthamiana, which served as a model plant system.

7

Expression of Nluc in the infiltrated leaves was quantitatively evaluated by Nluc assay and

8

considered as gene delivery efficiency. The gene delivery efficiency of unmodified micelles (794

9

± 249 RLU/mg) was significantly higher than that of naked pDNA (5 ± 82 RLU/mg) (Figure 4A,

10

green and black bars), indicating that micelle formation of pDNA with MAL-TEG-PLL efficiently

11

promoted gene delivery in plants. In the comparison of two BP100-modified micelles with the

12

unmodified micelle, there was no significant difference in delivery efficiencies between BP100-

13

24 (578 ± 196 RLU/mg) and unmodified (794 ± 249 RLU/mg) (Figure 4A, yellow and green bars).

14

In contrast, the efficiency of BP100-48 (2144 ± 420 RLU/mg) was significantly higher than that

15

of unmodified (Figure 4A, red and green bars).

16

To investigate the effect of micelle modification with NLS, the gene delivery efficiency of

17

NLS-48 was compared with that of unmodified. No significant difference was observed between

18

NLS-48 (437 ± 158 RLU/mg) and unmodified (794 ± 249 RLU/mg) (Figure 4A, cyan and green

19

bars). In addition, we could not observe significant difference among BP100-24/NLS-24 (738 ±

20

284 RLU/mg), BP100-48/NLS-24 (726 ± 260 RLU/mg), and unmodified (794 ± 249 RLU/mg)

21

(Figure 4A, purple, pink and green bars). Interestingly, the efficiencies of both BP100-48/NLS-

22

24 and BP100-24/NLS-24 were similar to that of BP100-24 (578 ± 196 RLU/mg), but lower than

23

that of BP100-48 (2144 ± 420 RLU/mg). Taking these results into account, the gene delivery


Page 19 of 33 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

Biomacromolecules

1

efficiency of micelles was affected by the amount of maleimide group conjugated to BP100-Cys

2

rather than that conjugated to Cys-NLS.

3

We focused on BP100-48, which exhibited the highest gene delivery efficiency among the

4

investigated micelles, and further examined its gene delivery ability by fluorescence imaging with

5

CLSM. Using a GFP-encoding pDNA (p35S-GFP-tNOS), BP100-48 was prepared in the above-

6

described condition and then infiltrated into N. benthamiana leaves. At 16 h after the infiltration,

7

green fluorescence originating from GFP was detected in the cytosolic region of epidermal cells

8

of the infiltrated leaf (Figure 4B), showing the successful transgene expression. To observe the

9

subcellular localization, we performed CLSM observation of N. benthamiana leaves infiltrated

10

with the BP100-48 micelle containing Cy3-labeled pDNA and subsequently treated with Hoechst.

11

In the CLSM images of the treated leaf, we visually confirmed that the fluorescent signal of Cy3-

12

pDNA overlapped with that of the stained nucleus (Figure 4C).



Page 20 of 33

1 2

Figure 4. Plant gene delivery mediated by peptide-postmodified MAL-TEG-PLL/pDNA micelles.

3

(A) Efficiencies of gene delivery into the leaves of N. benthamiana with naked pDNA, unmodified

4

and peptide-postmodified micelles determined by Nluc assay. A micelle prepared at N/P 6 without

5

modification is represented as unmodified. Micelles (prepared at N/P 6) modified with 24 and 48


Page 21 of 33 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

Biomacromolecules

1

µM BP100-Cys are represented as BP100-24 and BP100-48, respectively, whereas a micelle

2

(prepared at N/P 6) modified with 24 µM Cys-NLS is represented as NLS-48. A micelle (prepared

3

at N/P 6) concurrently modified with 48 µM BP100-Cys and 24 µM Cys-NLS is represented as

4

BP100-24/NLS-24, while that concurrently modified with 48 µM BP100-Cys and 24 µM Cys-

5

NLS is represented as BP100-48/NLS-24. A sterisks (*) and error bars represent significant

6

differences (P < 0.05, Mann–Whitney U-test) and standard deviations (n = 4), respectively. (B)

7

GFP expression in epidermal cells of an N. benthamiana leaf at 16 h after infiltration with BP100-

8

48 containing the pDNA, which encoded GFP. (C) Intracellular distribution of BP100-48 in an N.

9

benthamiana leaf at 16 h after infiltration. For fluorescence observation, BP100-48 was prepared

10

with Cy3-labeled pDNA, while the nuclei were stained with Hoechst. Chlorophylls were

11

visualized by their autofluorescence, and the cells were displayed in brightfield. Merge is the

12

composite of Cy3, Hoechst, chlorophyll, and brightfield, and the yellow arrow indicates an overlap

13

fluorescence of Cy3 and Hoechst.

14 15

Comparison of Gene Delivery Efficiencies between Postmodified and Premodified

16

Micelles. To verify the advantages of the postmodification strategy for gene delivery in plants, we

17

compared BP100-48 prepared by the postmodification method with another BP100-modified

18

micelle (referred to as Pre-BP100-48) prepared by a pre-modification method. In the

19

premodification method, the micelle was prepared by mixing BP100-Cys conjugated to MAL-

20

TEG-PLL (final concentration, 48 µM), MAL-TEG-PLL (24 µM), and pDNA (25 µg/mL) (Figure

21

5A). According to DLS and zeta potential analyses, the hydrodynamic diameter of Pre-BP100-48

22

(103 ± 10 nm) was slightly larger than that of BP100-48 (76 ± 4 nm), although their PDI and zeta

23

potential values were similar (Pre-BP100-48, 0.161 ± 0.032, 39 ± 1 mV; BP100-48, 0.136 ± 0.026,



Page 22 of 33

1

32 ± 0 mV). Nluc assays using N. benthamiana showed that the pDNA delivery efficiency of

2

BP100-48 (2144 ± 420 RLU/mg) was significantly higher than that of Pre-BP100-48 (999 ± 535

3

RLU/mg) (Figure 5B).

4

5 6

Figure 5. Comparison between postmodified and premodified micelles. (A) Schematic

7

representation of the micelle preparation by a premodification method. The micelle was prepared

8

by mixing BP100-Cys conjugated to MAL-TEG-PLL (final concentration, 48 µM), MAL-TEG-

9

PLL (24 µM), and pDNA (25 µg/mL). The resulting micelle is represented as Pre-BP100-48. (B)

10

Gene delivery efficiencies of BP100-48 and Pre-BP100-48 determined by Nluc assay using the

11

leaves of N. benthamiana. Asterisks (*) and error bars represent significant differences (P < 0.05,

12

Mann–Whitney U-test) and standard deviations (n = 4), respectively.

13 14


Page 23 of 33 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

1

Biomacromolecules

DISCUSSION

2

This study demonstrated the utility of MAL-TEG-PLL as a carrier for efficient plant gene

3

delivery. The MAL-TEG-PLL/pDNA micelle system enabled a modular postmodification strategy

4

using thiol-maleimide click chemistry (Figure 1). The micelle was successfully postmodified with

5

cysteine-inserted functional peptides, such as BP100-Cys and Cys-NLS. The enhanced gene

6

delivery efficiency of a BP100-postmodified micelle was indicative of the successful micelle

7

functionalization for efficient gene delivery. Additionally, higher delivery efficiency of the BP100-

8

postmodified micelle than of the BP100-premodified one verified the benefits of the present

9

postmodification strategy in gene delivery into plants.

10

MAL-TEG-PLL, composed of tetra(ethylene glycol) and short poly(L-lysine) (DP: mainly 5

11

or 6) segments (Figure 2), is a relatively short block copolymer compared to well-studied PEG-

12

PLL, which consists of a polyethylene glycol (12 kDa) segment and various lengths (DP: 7–48) of

13

poly(L-lysine) segments.33,34 The MAL-TEG-PLL/pDNA micelles, prepared at N/P 4 or above,

14

exhibited similar diameters (80–90 nm) and PDI values (0.162–0.186) to those of reported PEG-

15

PLL/pDNA micelles (Figure 3A and 3B).33,34 This similarity may suggest that the MAL-TEG-

16

PLL/pDNA complex could adopt a core-shell micelle structure. However, the zeta potentials of

17

the MAL-TEG-PLL/pDNA micelles were positive (12–30 mV) above N/P 1 (Figure 3C), whereas

18

those of reported PEG-PLL/pDNA micelles were approximately neutral at the corresponding N/P

19

region.33,34 Their different zeta potentials could be explained by the different chain lengths between

20

PEG and TEG segments; that is, the tetra(ethylene glycol) segment of MAL-TEG-PLL may be too

21

short to shield the positive charge of the cationic core. Positive surface charges and sizes less than

22

200 nm of nanoparticles are known to be suitable for electrostatic interactions with cell

23

membrane35 and cellular uptake by clathrin-mediated endocytosis.36 This might contribute to



Page 24 of 33

1

higher gene delivery efficiency of the MAL-TEG-PLL/pDNA prepared at N/P 6 than naked pDNA

2

(Figure 4A).

3

Although thiol-maleimide conjugation has been widely used for functionalization of

4

nanoparticles,37–40 reports on postmodification with multiple compounds using this chemistry are

5

limited. The MAL-TEG-PLL/pDNA micelle enabled simultaneous postmodification with two

6

functional peptides (BP100-Cys and Cys-NLS) without disrupting its micelle formation (Table 1).

7

Notably, the present postmodification strategy is modular, that is, various compounds with a thiol

8

group are potentially available for micelle functionalization. For effective gene delivery into plants,

9

several different functions must be imparted to gene carrier systems because plants possess many

10

barriers, such as cell walls and plasma membranes, and several target organelles, including nuclei,

11

chloroplasts, and mitochondria. In this point of view, the present strategy could be useful to provide

12

various micelles with optimal functions for different targets in plants.

13

The highest gene delivery efficiency of the micelle postmodified with 48 µM BP100-Cys

14

(BP100-48) among the investigated micelles emphasized that the BP100-Cys amount on the

15

micelle surface was a key factor for efficient plant gene delivery. Interestingly, the efficiency of

16

BP100-48 was higher than that of the micelles co-modified with BP100-Cys and Cys-NLS

17

(BP100-24/NLS-24 and BP100-48/NLS-24) (Figure 4A) in accordance with the higher percentage

18

of the BP100-conjugated maleimide group of BP100-48 than those of BP100-24/NLS-24 and

19

BP100-48/NLS-24 (Table 1). We ensured the pDNA transfer into the plant nucleus with BP100-

20

48 by the CLSM observations of the GFP expression in the transfected leaf (Figure 4B) and the

21

colocalization of Cy3-labeled pDNA with the Hoechst-stained nucleus in the leaf treated with

22

BP100-48 (Figure 4C). These findings suggest that the cellular internalization may be more


Page 25 of 33 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

Biomacromolecules

1

cumbersome process than the nuclear import in plant gene delivery mediated by the present micelle

2

system, which might highlight the benefits of BP100-conjugation rather than NLS-conjugation.

3

We demonstrated the advantage of our postmodification strategy over premodification by

4

comparing the gene delivery efficiency between BP100-48 and Pre-BP100-48 (Figure 5B). Higher

5

efficiency of BP100-48 than that of Pre-BP100-48 can be explained by undesired interactions

6

between BP100-Cys conjugated to the micelle and pDNA. The undesired interaction occurred

7

concomitantly with Pre-BP100-48 formation, resulting in a decrease in the amount of BP100-Cys

8

on the Pre-BP100-48 surface, which could attenuate the cell-penetrating function of Pre-BP100-

9

48, leading to less-efficient pDNA delivery by Pre-BP100-48 compared to that by BP100-48.

10

Accordingly, the present postmodification strategy based on thiol-maleimide conjugation can be

11

useful to provide functional micelles modified by cationic peptides, owing to fewer undesired

12

interactions between the cationic peptides and anionic pDNA. In addition, the postmodification

13

may be available for an effective screening of functional peptides to test their utility as parts of

14

gene carriers, since this strategy can readily attach the peptides to micelles without disrupting

15

peptide activities and micelle formation.

16

The present micelle system was successfully applied to transient transgene expression in

17

living plants. Previously, we achieved the integration of exogenous DNA into mitochondrial and

18

chloroplast genomes in plants using peptide-based gene carriers, which contain organelle targeting

19

signals, and the DNA integration vectors, which possess homologous recombination sites.23 The

20

present micelle system is expected to allow the functionalization with organelle targeting signals

21

and the delivery of the vector with homologous recombination sites. Thus, the present micelle

22

system might be used for the exogenous DNA integration into plant organelle genomes, which is

23

necessary for stable transgene expression in plants.



1

Page 26 of 33

CONCLUSIONS

2

In this study, we demonstrated the modular postmodification of block copolymer/pDNA

3

micelles with cationic functional peptides, such as a cell-penetrating peptide (CPP) and a nuclear

4

localization signal, via thiol-maleimide conjugation. Comparative studies among several peptide-

5

postmodified micelles underscored that a large amount of CPP on the micelle surface was key to

6

enhancing the gene delivery efficiency in plants. As intended, the present postmodification

7

strategy limited undesired interactions between the CPP, conjugated to the micelle, and the pDNA

8

cargo, providing higher efficiency of a CPP-postmodified micelle compared to a CPP-premodified

9

micelle. Although our findings provide useful insights into the development of effective gene

10

delivery systems for plants, the present block copolymer itself has room for improvement,

11

including optimization of the DNA binding sequence, which will further facilitate the gene

12

delivery efficiency in plants.

13 14 15

ASSOCIATED CONTENT

16

Supporting Information. MALDI TOF-MS spectrum of PLL(Boc) obtained by papain-

17

catalyzed polymerization; 1H NMR spectrum of PLL(Boc) obtained by papain-catalyzed

18

polymerization; representative RP-HPLC chromatograms of BP100-48, NLS-48, and BP100-

19

24/NLS-24; representative MALDI-TOF MS spectra of BP100-48, NLS-48, and BP100-

20

24/NLS-24; characterization of BP100-MAL-TEG-PLL; Figures S1–S5 (PDF).

21 22

AUTHOR INFORMATION

23

Corresponding Author


Page 27 of 33 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

Biomacromolecules

1

*E-mail: [email protected]

2

*E-mail: [email protected]

3

Author Contributions

4

T.M., K.T., and K.N. conceived and designed the research. T.M. performed the experiments and

5

analyzed the data. T.M. wrote and K.T. and K.N. edited the manuscript. All authors reviewed the

6

manuscript.

7

Funding Sources

8

This work was supported by Grants-in-Aid from the Japan Science and Technology Agency

9

Exploratory Research for Advanced Technology (JST-ERATO; Grant No., JPMJER1602).

10

Notes

11

The authors declare no competing financial interest.

12 13

ACKNOWLEDGMENTS

14

We are grateful to Ms. Shoko Tsuboyama-Tanaka (Utsunomiya University, Japan) for the kind

15

gift of the p35S-Nluc-tNOS plasmid.

16 17

REFERENCES

18

(1)

19 20

Parisi, C.; Vigani, M.; Rodríguez-Cerezo, E. Agricultural Nanotechnologies: What Are the Current Possibilities? Nano Today 2015, 10, 124–127.

(2)

Wan, S.; Truong-Trieu, V. M.; Ward, T.; Whalen, J. K.; Altosaar, I. Advances in the Use



Page 28 of 33

1

of Genetically Modified Plant Biomass for Biodiesel Generation. Biofuels, Bioprod.

2

Biorefining 2017, 11, 749–764.

3

(3)

4 5

Sticklen, M. B. Plant Genetic Engineering for Biofuel Production: Towards Affordable Cellulosic Ethanol. Nat. Rev. Genet. 2008, 9, 433–443.

(4)

Altpeter, F.; Baisakh, N.; Beachy, R.; Bock, R.; Capell, T.; Christou, P.; Daniell, H.; Datta,

6

K.; Datta, S.; Dix, P. J.; Fauquet, C.; Huang, N.; Kohli, A.; Mooibroek, H.; Nicholson, L.;

7

Nguyen, T. T.; Nugent, G.; Raemakers, K.; Romano, A.; Somers, D. A.; Stoger, E.; Taylor,

8

N.; Visser, R. Particle Bombardment and the Genetic Enhancement of Crops: Myths and

9

Realities. Mol. Breed. 2005, 15, 305–327.

10

(5)

11 12

“Gene-Jockeying” Tool. Microbiol. Mol. Biol. Rev. 2003, 67, 16–37. (6)

13 14

Gelvin, S. B. Agrobacterium-Mediated Plant Transformation: The Biology behind the

Shillito, R. D.; Saul, M. W.; Paszkowski, J.; Müller, M.; Potrykus, I. High Efficiency Direct Gene Transfer to Plants. Nat. Biotechnol. 1985, 3, 1099–1103.

(7)

Chen, C.-P.; Chou, J.-C.; Liu, B. R.; Chang, M.; Lee, H.-J. Transfection and Expression of

15

Plasmid DNA in Plant Cells by an Arginine-Rich Intracellular Delivery Peptide without

16

Protoplast Preparation. FEBS Lett. 2007, 581, 1891–1897.

17

(8)

18 19

Chugh, A.; Eudes, F. Study of Uptake of Cell Penetrating Peptides and Their Cargoes in Permeabilized Wheat Immature Embryos. FEBS J. 2008, 275, 2403–2414.

(9)

Chen, Y.-J.; Liu, B. R.; Dai, Y.-H.; Lee, C.-Y.; Chan, M.-H.; Chen, H.-H.; Chiang, H.-J.;

20

Lee, H.-J. A Gene Delivery System for Insect Cells Mediated by Arginine-Rich Cell-

21

Penetrating Peptides. Gene 2012, 493, 201–210.


Page 29 of 33 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

1

Biomacromolecules

(10)

2 3

Penetrating Peptides in Plant Cells. Adv. Stud. Biol. 2013, 5, 71–88. (11)

4 5

(12)

Qi, X.; Droste, T.; Kao, C. C. Cell-Penetrating Peptides Derived from Viral Capsid Proteins. Mol. Plant-Microbe Interact. 2011, 24, 25–36.

(13)

8 9

Chugh, A.; Amundsen, E.; Eudes, F. Translocation of Cell-Penetrating Peptides and Delivery of Their Cargoes in Triticale Microspores. Plant Cell Rep. 2009, 28, 801–810.

6 7

Liu, M.; Chou, J.; Lee, H. A Gene Delivery Method Mediated by Three Arginine-Rich Cell-

Shim, Y.-S.; Eudes, F.; Kovalchuk, I. dsDNA and Protein Co-Delivery in Triticale Microspores. Vitr. Cell. Dev. Biol. - Plant 2013, 49, 156–165.

(14)

Ziemienowicz, A.; Shim, Y.-S.; Matsuoka, A.; Eudes, F.; Kovalchuk, I. A Novel Method

10

of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-

11

Derived Nano-Complex. Plant Physiol. 2012, 158, 1503–1513.

12

(15)

Unnamalai, N.; Kang, B. G.; Lee, W. S. Cationic Oligopeptide-Mediated Delivery of

13

dsRNA for Post-Transcriptional Gene Silencing in Plant Cells. FEBS Lett. 2004, 566, 307–

14

310.

15

(16)

16 17

Numata, K. Poly(amino Acid)s/polypeptides as Potential Functional and Structural Materials. Polym. J. 2015, 47, 537–545.

(17)

Numata, K.; Horii, Y.; Oikawa, K.; Miyagi, Y.; Demura, T.; Ohtani, M. Library Screening

18

of Cell-Penetrating Peptide for BY-2 Cells, Leaves of Arabidopsis, Tobacco, Tomato,

19

Poplar, and Rice Callus. Sci. Rep. 2018, 8, 10966.

20

(18)

Lakshmanan, M.; Kodama, Y.; Yoshizumi, T.; Sudesh, K.; Numata, K. Rapid and Efficient



Page 30 of 33

1

Gene Delivery into Plant Cells Using Designed Peptide Carriers. Biomacromolecules 2013,

2

14, 10–16.

3

(19)

Lakshmanan, M.; Yoshizumi, T.; Sudesh, K.; Kodama, Y.; Numata, K. Double-Stranded

4

DNA Introduction into Intact Plants Using Peptide-DNA Complexes. Plant Biotechnol.

5

2015, 32, 39–45.

6

(20)

7 8

Plants via Synthetic dsRNA and Carrier Peptide. Plant Biotechnol. J. 2014, 12, 1027–1034. (21)

9 10

Chuah, J.-A.; Numata, K. Stimulus-Responsive Peptide for Effective Delivery and Release of DNA in Plants. Biomacromolecules 2018, 19, 1154–1163.

(22)

11 12

Numata, K.; Ohtani, M.; Yoshizumi, T.; Demura, T.; Kodama, Y. Local Gene Silencing in

Chuah, J.-A.; Yoshizumi, T.; Kodama, Y.; Numata, K. Gene Introduction into the Mitochondria of Arabidopsis Thaliana via Peptide-Based Carriers. Sci. Rep. 2015, 5, 7751.

(23)

Yoshizumi, T.; Oikawa, K.; Chuah, J.-A.; Kodama, Y.; Numata, K. Selective Gene Delivery

13

for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide–

14

DNA Complexes. Biomacromolecules 2018, 19, 1582–1591.

15

(24)

Kataoka, K.; Harada, A.; Nagasaki, Y. Block Copolymer Micelles for Drug Delivery:

16

Design, Characterization and Biological Significance. Adv. Drug Deliv. Rev. 2001, 47, 113–

17

131.

18

(25)

19 20

Hoyle, C. E.; Lowe, A. B.; Bowman, C. N. Thiol-Click Chemistry: A Multifaceted Toolbox for Small Molecule and Polymer Synthesis. Chem. Soc. Rev. 2010, 39, 1355.

(26)

Nair, D. P.; Podgórski, M.; Chatani, S.; Gong, T.; Xi, W.; Fenoli, C. R.; Bowman, C. N.


Page 31 of 33 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

Biomacromolecules

1

The Thiol-Michael Addition Click Reaction: A Powerful and Widely Used Tool in

2

Materials Chemistry. Chem. Mater. 2014, 26, 724–744.

3

(27)

Fagerland, J.; Finne-Wistrand, A.; Numata, K. Short One-Pot Chemo-Enzymatic Synthesis

4

of L-Lysine and L-Alanine Diblock Co-Oligopeptides. Biomacromolecules 2014, 15, 735–

5

743.

6

(28)

Qin, X.; Xie, W.; Tian, S.; Ali, M. A.; Shirke, A.; Gross, R. A. Influence of Nε‑Protecting

7

Groups on the Protease-Catalyzed Oligomerization of L‑Lysine Methyl Ester. ACS Catal.

8

2014, 4, 1783–1792.

9

(29)

10 11

Tsuchiya, K.; Numata, K. Chemoenzymatic Synthesis of Polypeptides for Use as Functional and Structural Materials. Macromol. Biosci. 2017, 17.

(30)

Badosa, E.; Ferre, R.; Planas, M.; Feliu, L.; Besalú, E.; Cabrefiga, J.; Bardají, E.;

12

Montesinos, E. A Library of Linear Undecapeptides with Bactericidal Activity against

13

Phytopathogenic Bacteria. Peptides 2007, 28, 2276–2285.

14

(31)

Eggenberger, K.; Mink, C.; Wadhwani, P.; Ulrich, A. S.; Nick, P. Using the Peptide Bp100

15

as a Cell-Penetrating Tool for the Chemical Engineering of Actin Filaments within Living

16

Plant Cells. ChemBioChem 2011, 12, 132–137.

17

(32)

18 19

Kalderon, D.; Richardson, W. D.; Markham, A. F.; Smith, A. E. Sequence Requirements for Nuclear Location of Simian Virus 40 Large-T Antigen. Nature 1984, 311, 33–38.

(33)

Itaka, K.; Yamauchi, K.; Harada, A.; Nakamura, K.; Kawaguchi, H.; Kataoka, K. Polyion

20

Complex Micelles from Plasmid DNA and Poly(ethylene Glycol)–poly(L-Lysine) Block

21

Copolymer as Serum-Tolerable Polyplex System: Physicochemical Properties of Micelles



1 2

Page 32 of 33

Relevant to Gene Transfection Efficiency. Biomaterials 2003, 24, 4495–4506. (34)

Katayose, S.; Kataoka, K. Water-Soluble Polyion Complex Associates of DNA and

3

Poly(ethylene glycol)−Poly(L-Lysine) Block Copolymer. Bioconjug. Chem. 1997, 8, 702–

4

707.

5

(35)

6 7

Fröhlich, E. The Role of Surface Charge in Cellular Uptake and Cytotoxicity of Medical Nanoparticles. Int. J. Nanomedicine 2012, 7, 5577.

(36)

Rejman, J.; Oberle, V.; Zuhorn, I. S.; Hoekstra, D. Size-Dependent Internalization of

8

Particles via the Pathways of Clathrin- and Caveolae-Mediated Endocytosis. Biochem. J.

9

2004, 377, 159–169.

10

(37)

Oh, E.; Susumu, K.; Blanco-Canosa, J. B.; Medintz, I. L.; Dawson, P. E.; Mattoussi, H.

11

Preparation of Stable Maleimide-Functionalized Au Nanoparticles and Their Use in

12

Counting Surface Ligands. Small 2010, 6, 1273–1278.

13

(38)

Martínez-Jothar, L.; Doulkeridou, S.; Schiffelers, R. M.; Sastre Torano, J.; Oliveira, S.; van

14

Nostrum, C. F.; Hennink, W. E. Insights into Maleimide-Thiol Conjugation Chemistry:

15

Conditions for Efficient Surface Functionalization of Nanoparticles for Receptor Targeting.

16

J. Control. Release 2018, 282, 101–109.

17

(39)

Yao, W.; Xu, P.; Zhao, J.; Ling, L.; Li, X.; Zhang, B.; Cheng, N.; Pang, Z. RGD

18

Functionalized Polymeric Nanoparticles Targeting Periodontitis Epithelial Cells for the

19

Enhanced Treatment of Periodontitis in Dogs. J. Colloid Interface Sci. 2015, 458, 14–21.

20 21

(40)

Schiffelers, R. Anti-Tumor Efficacy of Tumor Vasculature-Targeted Liposomal Doxorubicin. J. Control. Release 2003, 91, 115–122.


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TOC

CPP plasmid DNA

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plasmid DNA micelles postmodified with functional

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