Synthesis, Assembled Structures, and DNA Complexation of

Subscriber access provided by ECU Libraries

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Synthesis, Assembled Structures, and DNA Complexation of Thermoresponsive Lysine-Based Zwitterionic and Cationic Block Copolymers Ryosuke Kanto, Yehan Qiao, Kazunori Masuko, Hiroyuki Furusawa, Shigekazu Yano, Kazuhiro Nakabayashi, and Hideharu Mori Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b04303 • Publication Date (Web): 08 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 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

Langmuir

Synthesis, Assembled Structures, and DNA Complexation of Thermoresponsive Lysine-Based Zwitterionic and Cationic Block Copolymers

Ryosuke Kanto,† Yehan Qiao,‡ Kazunori Masuko,† Hiroyuki Furusawa,‡ Shigekazu Yano, ‡ Kazuhiro Nakabayashi,† Hideharu Mori*†‡

†Graduate

School of Organic Materials Science, ‡Graduate School of Science and Engineering, Yamagata University, 4-3-16, Jonan, Yonezawa, 992-8510, Japan

*

To whom correspondence should be addressed. e-mail: [email protected], Phone:+81-238-

26-3765, Fax: +81-238-26-3749

ACS Paragon Plus Environment

1

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

Page 2 of 45

ABSTRACT A series of anionic, zwitterionic, and cationic lysine-based block copolymers with a thermoresponsive segment were synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization of N-acryloyl-N-carbobenzoxy-L-lysine (A-Lys(Cbz)-OH), which contains a carboxylic acid and a protected amine-functionality in the monomer unit. Carboxylic acid-containing homopolymers, poly(A-Lys(Cbz)-OH), with pre-determined molecular weights with relatively low polydispersities were initially synthesized by RAFT polymerization of A-Lys(Cbz)-OH. The chain extension of the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) to N-isopropylacrylamide (NIPAM) via RAFT process and subsequent deprotection afforded the zwitterionic block copolymer composed of thermoresponsive poly(NIPAM) and poly(A-Lys-OH), which exhibited switchability among the zwitterionic, anionic, and cationic states by pH change. The assembled structures, and thermoresponsive and chiroptical properties of these block copolymers were evaluated by dynamic light scattering (DLS), circular dichroism (CD), and turbidity measurements. Finally, the cationic block copolymer, poly(ALys-OMe)-b-poly(NIPAM), was obtained by the methylation of the carboxylic acid group in the zwitterionic poly(A-Lys-OH) segment. Selective interactions of DNA with the cationic poly(A-LysOMe) segment in the lysine-based block copolymer were further evaluated by agarose gel electrophoresis and atomic force microscopy (AFM) measurements, which revealed characteristic assembled structures and temperature-responsive properties of the polyplexes.


2

Page 3 of 45 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

Langmuir

Introduction Stimuli-responsive block polymers respond to external stimuli such as temperature, pH, and ionic strength, and have been extensively studied from both scientific and technological points of view owing to their promising applications in controlled release, biochemical sensing, and smart materials.1-3 In the past few decades, a variety of thermosensitive block copolymers have been developed, which exhibit temperature-induced self-assembling behavior involving normal and reversible micelle formation either above a critical point (lower critical solution temperature, LCST) or below a certain temperature (upper critical solution temperature).1,2,4-6 Among them, N-isopropylacrylamide (NIPAM)-based block copolymers are the most representative examples, as they show an LCST-type soluble–insoluble transition derived from the poly(NIPAM) segment at 32 °C in water, and the transition temperature can be shifted to human body temperature by incorporation of hydrophilic monomers, making it suitable for various biomedical applications.7,8 In recent years, the design and synthesis of thermosensitive block copolymers with zwitterionic segments, which have cationic and anionic ions in the same monomer unit, has received considerable attention.5,9-12 Zwitterionic polymers involving phosphobetaine, sulfobetaine, and carboxybetaine in the side chain have been the subject of continued interest in academia because of their diverse functional features and unique properties, especially their excellent protein resistance.13 Owing to their structural similarity to phospholipids, zwitterionic polymers and materials can be recognized as protein models and bio-interfaces for medical, diagnostic, and biotechnology applications.14,15 Stimuli-responsive block copolymers containing chemically connected zwitterionic and thermoresponsive poly(NIPAM) segments provide a great variety of morphologies and characteristic properties in response to external stimuli, in addition to specific interactions such as hydrogen-bonding, acid-base interactions, and oppositely charged ionic interactions in biomolecules. For example, schizophrenic block copolymers prepared from a nonionic NIPAM and a zwitterionic sulfobetaine methacrylamide exhibited LCST that originated from poly(NIPAM) and upper critical solution temperature that derived from the zwitterionic polymer.5,9 Thermally induced aggregation/switching behaviors10,11 and triple thermoresponsivity/pH sensitivity12 of polysulfobetaine-b-poly(NIPAM)s have also been investigated using various methods. ACS Paragon Plus Environment

3

Langmuir 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

Poly(NIPAM)-b-poly(sulfobetaine

methacrylate)

showed

Page 4 of 45

extremely

high

anticoagulant

and

antihemolytic activities in human blood over a wide range of temperatures.16 Block copolymers with cationic segments are of increasing interest as non-viral vectors for the introduction of therapeutic molecules such as DNA into cells.17-20 Spontaneous formation of DNApolycation complexes (polyplexes) takes place in aqueous solution as a result of electrostatic attraction between the negatively charged phosphate groups of DNA and positive charges in the cationic segment of the block copolymers. Another segment in the block copolymer can contribute to improvement in the properties of the polyplexes, such as their solubility, stability, cell survival, and biodistribution, depending on the nature of the segment. Cationic block copolymers with stimuli-responsive segment have been studied as promising candidates for stimuli-responsive gene delivery vectors, in which DNA uptake and gene release ability can be manipulated by external stimuli.21,22 Several block copolymers consisting of cationic and thermoresponsive segments have been developed, which include quaternized poly(4-vinylpyridine)-b-poly(oligoethyleneglycol

methyl

ether

methacrylate),23

poly(2-

(dimethylamino)ethyl acrylate)-b-poly(NIPAM),24 poly(ethyleneimine)-b-poly(NIPAM),25 and poly((3acrylamidopropyl)trimethylammonium chloride)-b-poly(NIPAM).26-28 Poly(NIPAM)-based random copolymers with cationic species have been also investigated as stimuli-responsive gene delivery vectors.29 Other examples involve a triblock copolymer consisting of hydrophilic poly(2-ethyl-2oxazoline), thermoswitchable amphiphilic poly(2-n-propyl-2-oxazoline), and cationic poly(L-lysine),30 and double thermoresponsive polybetaine-based ABA triblock copolymers.31 A noteworthy aspect of the thermoresponsive block copolymers with cationic sites is that their suitability for polyplex formation with DNA is governed by their chemical structures, comonomer composition, charge-to-charge stoichiometry, and charge densities of the cationic segment, in addition to the temperature.


4

Page 5 of 45 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

Langmuir Anionic-thermoresponsive BC S n O

O O

NH

N

O

O

O NIPAM

O

AIBN

O

m

n

S OH

N H

b

NH

NH

O

NH

OH

N H

O

Poly(A-Lys(Cbz)-OH)-b-poly(NIPAM)

Poly(A-Lys(Cbz)-OH)

TFA, HBr Cationic-thermoresponsive BC b

m

n O

NH

O

NH

OCH3

H2N

Zwitterionic-thermoresponsive BC b

m

n

(CH3)3SiCHN2

O

MeOH

NH

O

NH

OH

H2N O

O Poly(A-Lys-OMe)-b-poly(NIPAM)

Poly(A-Lys-OH)-b-poly(NIPAM)

Scheme 1. Synthesis of lysine-based anionic, zwitterionic, and cationic block copolymers (BCs) with thermoresponsive poly(NIPAM) segment by RAFT polymerization of N-acryloyl-N-carbobenzoxy-Llysine (A-Lys(Cbz)-OH) and subsequent chemical modifications.

Driven by their ability to form highly ordered hierarchical structures through intra- and interchain associations involving noncovalent forces of the amino acid appendages and numerous potential applications, there has been a growing interest in amino acid-based polymers/block copolymers.32-36 A variety of amino acid-based block copolymers have been synthesized by reversible additionfragmentation chain transfer (RAFT) polymerization, in which their pH-responsive, thermoresponsive, and dual stimuli-responsive properties are governed by the nature of the amino acid components with non-ionic species and/or free carboxylic functionality.33 Amino acid-based cationic polymers, particularly primary amine-containing polymers, are also of interest, because of their ability to react/interact with bioactive molecules, drugs, and biological signaling molecules, leading to their potential applications in biomedicine.37 Owing to attractive properties such as low protein adsorption, good biocompatibility, and antifouling properties, zwitterionic polymers with amino acids in the side ACS Paragon Plus Environment

5

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

Page 6 of 45

chains are one of the other key developments in this field. Various amino acid-based zwitterionic polymers have been developed, such as poly(cysteine methacrylate) brush showing excellent resistance to biofouling and negligible cytotoxicity,38 poly(serine methacrylate) as an antifouling material,39 serine, lysine, ornithine, glutamic acid, and aspartic acid-based polymers with antifouling properties and low cytotoxicity40 and high resistance to long-term bacterial adhesion,41 amphiphilic polymers containing glutamic acid with long alkyl side chains for pH-responsive and selective protein adsorption,42 poly(lysine methacrylamide) and poly(ornithine methacrylamide) as superlow fouling polymers,43 aspartic acid and glutamic acid-based polymers for antifouling gold surfaces,44 poly(histidine methacrylamide) with pH-dependent antifouling properties and chelation capability,45 and poly(-Llysinyl acrylamide) for Cu(II)-induced aggregation into nanostructures.46 Schmidt et al. demonstrated that poly(ε-N-methacryloyl-L-lysine) exhibited low cytotoxicity and could be converted upon addition of Zn

2+

ions into polycations that formed complexes with DNA.47 Most of these amino acid-based

zwitterionic polymers have been typically synthesized by conventional radical polymerization or surface-initiated polymerization, while instances of controlled radical polymerization are limited. Recently, Mandal et al. reported the RAFT synthesis of dual-stimuli-responsive poly(L-serinyl acrylate) as a zwitterionic upper critical solution temperature-type homopolymer with tunable thermosensitivity.48 Stenzel et al. demonstrated the synthesis of zwitterionic guanidine-based block copolymers by RAFT polymerization and their cellular uptake.49 In contrast, thermoresponsive block copolymers bearing zwitterionic and switchable amino acid-based segment in the side chain have received lesser attention. In this study, to enrich the family of amino acid-based smart polymeric materials, a series of lysinebased block copolymers comprising thermoresponsive poly(NIPAM) and anionic, zwitterionic, and cationic lysine-based segments have been synthesized by RAFT polymerization of N-acryloyl-Ncarbobenzoxy-L-lysine (A-Lys(Cbz)-OH) and subsequent chemical modifications (Scheme 1). Of particular interest are those systems in which the lysine-based segment can switch between the zwitterionic, anionic, and cationic states by pH change (temporally) and chemical modifications (permanently), which affect the assembled structures, thermoresponsive property, and specific


6

Page 7 of 45 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

Langmuir

interactions with DNA. Initially, the anionic block copolymer, poly(A-Lys(Cbz)-OH)-b-poly(NIPAM), was synthesized by RAFT polymerization of A-Lys(Cbz)-OH, which contains a carboxylic acid and a protected amine-functionality in the monomer unit, followed by the polymerization of NIPAM using poly(A-Lys(Cbz)-OH) as the macro-chain transfer agent (CTA). In the next step, the deprotection of the N-carbobenzoxy group in the anionic block copolymer afforded zwitterionic block copolymer, poly(ALys-OH)-b-poly(NIPAM), in which the zwitterionic, anionic, and cationic states of the poly(A-Lys-OH) segment could be tuned by pH change. The chemical nature of the switchable block and its composition could be manipulated by RAFT polymerization of the rationally designed lysine-based monomer (ALys(Cbz)-OH), providing a great opportunity for tuning their properties and functions, as well as achieving highly ordered structures. The aggregation behavior, thermoresponsiveness, and chiroptical properties of these block copolymers were evaluated by dynamic light scattering (DLS), UV-Vis spectroscopy, and circular dichroism (CD) measurements. Finally, the cationic block copolymer, poly(A-Lys-OMe)-b-poly(NIPAM), was obtained by the methylation of the carboxylic acid group in the zwitterionic poly(A-Lys-OH) segment. The poly(A-Lys-OMe) segment in the resulting cationic block copolymer is recognized as a cationic polyelectrolyte with primary amino groups that act as protonaccepting sites for complexation with DNA. The simple mixing procedure of two transparent aqueous solutions of the block copolymers and DNA led to the formation of DNA-block copolymer nanoparticles through the selective interaction of DNA and the cationic poly(A-Lys-OMe) segment. Thus, the aim of this study was to understand how cationic block copolymers having a thermoresponsive segment would interact with DNA, in terms of the cation/anion charge ratio in the feed, the cationic/non-ionic composition of the block copolymer, and temperature change.

Experimental Section Materials.

2,2′-Azobis(isobutyronitrile)

(AIBN,

Kanto

Chemical,

97%)

was

purified

by

recrystallization from methanol. N-Isopropylacrylamide (NIPAM, TCI, >98%) was recrystallized twice from hexane. Benzyl 1-pyrrolecarbodithioate was used as a dithiocarbamate-type chain transfer agent (CTA) and synthesized by following a previously reported procedure.50,51 The methylation agent, ACS Paragon Plus Environment

7

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

Page 8 of 45

trimethylsilyldiazomethane (TCI, 10% solution in hexane), N-carbobenzoxy-L-lysine (TCI, 98%), 1,4dioxane (dehydrated, Kanto Chemical, 99.5%), and all other materials were purchased from commercial suppliers and used as received.

N-Acryloyl-N-carbobenzoxy-L-lysine (A-Lys(Cbz)-OH). The monomer was prepared by the reaction of acryloyl chloride with N-carbobenzoxy-L-lysine according to a slightly modified literature method used for other amino acid-containing acrylamides.52 N-Carbobenzoxy-L-lysine (6.0 g, 0.02 mol) and 214 mL of 1 N NaOH were placed in a three-necked round-bottom flask, and the mixture was stirred continuously. This was followed by the addition of 108 mL of 1,4-dioxane and 21.4 mL of 1 N NaOH to the flask. Acryloyl chloride (2.42 mL, 0.03 mol) and then 1,4-dioxane (24 mL) were added dropwise under nitrogen to the aqueous solution over 1 h, which was kept at 0 °C by cooling with an external ice bath. After complete addition, the mixture was stirred for 16 h while the temperature was allowed to rise to room temperature. After the mixture was acidified to pH = 4 with 1 N HCl, the product was extracted from the reaction mixture using methylene chloride and then washed three times with a distilled water. The extract was dried over anhydrous MgSO4, filtered, and the solvent was evaporated to give a crude solid. The crude product was purified by reprecipitation from the chloroform solution into hexane, followed by decantation. After the product was dissolved in chloroform, most of solvent was removed by evaporation, and it was finally freeze-dried under vacuum to afford a white solid, yield: 4.48 g, 68%. 1H

NMR (400 MHz, DMSO-d6): δ1.2-1.5 (4H, -CHCH2CH2CH2), 1.5-1.8 (2H, -NHCHCH2), 2.9-3.0

(2H, -NHCH2), 4.1-4.3 (1H, -NHCHCOOH), 4.9-5.1 (2H, -OCH2), 5.5-6.4 (2H, -CH=CH2) 6.3-6.4(1H, -CH=CH2), 7.1-7.5 (6H, -CH2NHCOO and -C6H5), and 8.3-8.4 (1H, -CONHCH) ppm. 13C NMR (400 MHz, DMSO-d6): δ24.0 (-CHCH2CH2CH2), 29.6 and 31.3 (-CHCH2CH2CH2), 52.0 (-CH2CH2NH), 65.8 (-NHCHCOOH), 66.8 (-OCH2C), 126.2-128.9 (-CH=CH2 and -C5H5), 131.8 (-CH=CH2), 137.8 (CH2CC5H5), 156.8 (-NHCOOCH2), 165.1 (CH2=CHCO), and 174.0 (-CHCOOH) ppm. 1H and

13C

NMR spectra of A-Lys(Cbz)-OH are shown in Figure S1 (see Supporting Information). Anal. Calcd for C17H22N2O5: C, 61.1; H, 6.63; N, 8.38. Found: C, 60.9; H, 6.88; N, 8.16.


8

Page 9 of 45 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

Langmuir

RAFT Polymerization of A-Lys(Cbz)-OH. All polymerizations were carried out using AIBN as the initiator in a degassed sealed tube. A representative example for the synthesis of poly(A-Lys(Cbz)-OH) is as follows: A-Lys(Cbz)-OH (0.835 g, 2.5 mmol), benzyl 1-pyrrolecarbodithioate (22.4 mg, 0.10 mmol), AIBN (8.0 mg, 0.05 mmol), and dehydrated 1,4-dioxane (2.5 mL) were placed in a dry glass ampoule equipped with a magnetic stirring bar, and the solution was degassed by three freeze-evacuatethaw cycles. After sealing the ampule by flame under vacuum, the mixture was stirred at 60 °C for 24 h. The monomer conversion was determined to be 99% by the integration of the monomer C=C-H resonance at 5.6 ppm and comparison with the sum of the NH and aromatic peak intensities of the polymer and monomer at 6.9–8.2 ppm. The resulting polymer was purified by reprecipitation in a large excess of diethyl ether and filtration. The product was dried under vacuum at room temperature to afford a pale yellow solid: yield 0.648 g, 76%. The resulting poly(A-Lys(Cbz)-OH) was soluble in basic aqueous solution (pH = 12), DMSO, DMF, CHCl3, THF, and 1,4-dioxane, while it was insoluble in neutral water (ca. pH = 7), acidic water (pH = 2), diethylether, and hexane. 1H NMR (400 MHz, DMSO-d6): δ1.0-2.3 (9H, CH2-CHCO-, -CHCH2CH2CH2), 2.8-3.1 (2H, -NHCH2CH2), 4.0-4.5 (1H, NHCHCOOH), 4.9-5.1 (2H, -OCH2C6H5), 7.0-8.2 (7H, -CH2NHCOO, -C6H5, and -CONHCH) ppm. For size-exclusion chromatography (SEC) measurements, crude poly(A-Lys(Cbz)-OH) was modified by methylation of the carboxylic acid groups using trimethylsilyldiazomethane according to a slightly modified literature method.53,54 The 1H NMR spectrum of poly(A-Lys(Cbz)-OH) is shown in Figure S2 (see Supporting Information). The solubility data of the monomer and homopolymers are summarized in Table S1 (Supporting Information).

Synthesis of Anionic Block Copolymer Using Poly(A-Lys(Cbz)-OH) as the Macro-CTA. A representative synthetic procedure of the block copolymer employing poly(A-Lys(Cbz)-OH) as the macro-CTA is as follows: the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) (Mn = 8200, Mw/Mn = 1.28, 0.82 g, 0.10 mmol), AIBN (8.0 mg, 0.05 mmol), NIPAM (0.565 g, 5.0 mmol), and 1,4-dioxane (10 mL) were placed in a dry ampoule. After degassing the solution by three freeze-thaw cycles, ACS Paragon Plus Environment

9

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

Page 10 of 45

polymerization was conducted at 60 °C for 24 h. The conversion of the double bonds, as detected by 1H NMR, was 99%. The reaction mixture was precipitated into diethylether and isolated by filtration. The product was finally dried under vacuum at room temperature to obtain the anionic block copolymer as a pale yellow solid: yield 0.989 g, 63%. 1H NMR (400 MHz, DMSO-d6): δ 0.9-1.2 (6H, CH3CHCH3), 1.0-2.3 (CH2-CH in the main chain and -CHCH2CH2CH2), 2.8-3.1 (2H, -NHCH2), 3.4-3.9 (1H, CH3CHCH3) 4.0-4.5 (1H, -NHCHCOOH), 4.9-5.1 (2H, -OCH2C), 7.0-8.2 (-CH2NHCOO, -C6H5, and CONHCH) ppm. The comonomer composition was calculated by comparing the peak at 0.9–1.2 ppm, attributed to the methyl protons of the NIPAM unit (-CH(CH3)2), and the peak at 4.9–5.1 ppm, attributed to the methylene protons of the A-Lys(Cbz)-OH unit (-OCH2C). Thus, the comonomer composition can be calculated using the equation 1, 2(x) 6(1-x)

=

Integral at 4.9-5.1 ppm Integral at 0.9-1.2 ppm

(1)

where x is the fraction of the A-Lys(Cbz)-OH and 1-x is the fraction of NIPAM in the block copolymer. Similar to the homopolymer, the methylated sample obtained by the modification of the carboxylic acid groups of the anionic block copolymer using trimethylsilyldiazomethane53,54 was employed for SEC measurement. Typically, 25 mg of poly(A-Lys(Cbz)-OH)-b-poly(NIPAM) was dissolved in a mixture of chloroform/methanol (2/1 vol-%, to achieve solubilization at room temperature), overall volume 3.0 mL. The yellow solution of trimethylsilyldiazomethane (0.60 mL, 3.0 mmol) was added dropwise at room temperature into the polymer solution, and then the mixture was stirred at room temperature overnight. After the product was precipitated into diethyl ether and separated by filtration, it was dried at room temperature in vacuo, and the resulting product was employed for SEC measurement.

Synthesis of Zwitterionic Block Copolymer, Poly(A-Lys-OH)-b-poly(NIPAM). The deprotection of the N-carbobenzoxy group in the poly(A-Lys(Cbz)-OH) segment in the block copolymer was conducted using trifluoroacetic acid/HBr according to a previously reported method with slight modifications.55 ACS Paragon Plus Environment

10

Page 11 of 45 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

Langmuir

Typically, 500 mg of poly(A-Lys(Cbz)-OH)-b-poly(NIPAM) (Cbz content = 0.74 mmol) and 5 mL of trifluoroacetic acid were placed in a glass vial, and the mixture was stirred at room temperature for 1 h. Then, a four-fold molar excess of a 33 wt% solution of HBr in acetic acid (0.6 mL, 3.0 mmol) was added dropwise to the polymer mixture. After the solution was stirred for 1.5 h, the product was precipitated into diethyl ether and separated by filtration. The product was then purified by dialysis (Spectra Pore; MWCO 1000Da) with distilled water for two days. After the solvent was removed by reduced pressure, the product was dried overnight under vacuum at room temperature to yield zwitterionic poly(A-Lys-OH)-b-poly(NIPAM) as a white solid (0.409 g, 97%). The zwitterionic block copolymer obtained after the deprotection was soluble in methanol and water over wide pH ranges (pH = 2, 7, 12), as shown in Table S2 (Supporting Information). 1H NMR (400 MHz, D2O): δ 0.9-1.1 (6H, CH3CHCH3), 1.1-2.3 (CH2-CH in the main chain and -CHCH2CH2CH2), 2.8-3.0 (2H, -CH2NH2), 3.64.1 (-CH3CHCH3, and -NHCHCOOH) ppm. The deprotection of the anionic homopolymer, poly(A-Lys(Cbz)-OH), was carried out under the same conditions. The resulting zwitterionic homopolymer, poly(A-Lys-OH), was also soluble over a wide pH range (pH = 2, 7, and 12) and in methanol, while it was insoluble in most organic solvents (Table S1, Supporting Information). The 1H NMR spectra of poly(A-Lys(Cbz)-OH) and deprotected poly(A-LysOH) are shown in Figure S3 (see Supporting Information).

Synthesis of Cationic Block Copolymer, Poly(A-Lys-OMe)-b-poly(NIPAM). The methylation of the poly(A-Lys-OH) segment in the block copolymer was conducted using trimethylsilyldiazomethane according to a method reported previously with a slight modification.53,54 Briefly, 0.20 g of poly(A-LysOH)-b-poly(NIPAM) (carboxylic acid content = 0.4 mmol) was dissolved in 8 mL of methanol, and then the yellow solution of trimethylsilyldiazomethane (4.7 mL, 2.8 mmol, (CH3)3SiCHN2/COOH unit in poly(A-Lys-OH) = 7/1 molar ratio) was added dropwise at room temperature into the polymer solution. After the complete addition of the methylation agent, the solution was stirred overnight at room temperature. Subsequently, the product was precipitated into diethyl ether, separated by filtration, ACS Paragon Plus Environment

11

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

Page 12 of 45

and dried at room temperature in vacuo to afford poly(A-Lys-OMe)-b-poly(NIPAM) as a white solid (0.201 g, 99%). 1H NMR (400 MHz, D2O): δ 0.9-1.1 (6H, CH3CHCH3), 1.1-2.3 (CH2-CH in the main chain and -CHCH2CH2CH2), 2.8-3.0 (2H, -CH2NH2), 3.6-4.1 (-CH3CHCH3 and -NHCHCOOCH3) ppm.

Preparation of Block Copolymer/DNA Polyplexes. Polyplex formation was conducted by mixing the stock solutions of water-soluble DNA and the lysine-based block copolymer in potassium phosphate buffer (0.05 mg/mL) under various conditions. The cation/anion charge ratio (N/P) was the number ratio of the primary amine-based nitrogen atoms of the A-Lys-OMe units in the block copolymer to the phosphate groups in DNA. The stock solutions of DNA (DNA sodium salt from salmon testes, SigmaAldrich (D1626) (2000 bp), 0.2 mg/mL) and the polymer (1.0 mg/mL) were prepared independently by dissolving in 10 mM potassium phosphate buffer (pH = 7.4) at room temperature. The proper amount of the polymer solution was added gradually to the DNA solution, in order to adjust the N/P ratio, followed by the addition of potassium phosphate buffer to reach a constant DNA concentration (0.05 mg/mL). The DNA concentration in the solution is given in molarity units of the negatively charged phosphate groups in DNA.

Instrumentation. The 1H (400 MHz) and

13C

NMR (100 MHz) spectra were recorded using a JEOL

JNM-ECX400. The number-average molecular weight (Mn) and molecular weight distribution (Mw/Mn) were estimated by size-exclusion chromatography (SEC) using a Tosoh HPLC HLC-8220 system equipped with refractive index and ultraviolet detectors at 40 oC. The column set was as follows: four consecutive hydrophilic vinyl polymer-based gel columns [TSK-GELs (bead size, exclusion limited molecular weight): α-M (13 μm, > 1 x 107), α-4000 (10 μm, 4 x 105), α-3000 (7 μm, 9 x 104), α-2500 (7 μm, 5 x 103), 30 cm each] and a guard column [TSK-guardcolumn α, 4.0 cm]. The system was operated at the flow rate of 1.0 mL/min using DMF containing 10 mM LiBr as the eluent. Polystyrene standards (Tosoh) ranging from 1050 to 1090000 were employed for calibration. The phase-separation temperatures of the polymers were measured by monitoring the transmittance of a 500 nm light beam through their aqueous solutions (2.0 mg/mL) in a quartz sample cell. The ACS Paragon Plus Environment

12

Page 13 of 45 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

Langmuir

transmittance was recorded on a JASCO V-630BIO UV-vis spectrophotometer equipped with a temperature controller (JASCO EHC-716 and EHC-717, respectively). The temperature was increased at a rate of 1.0 ºC/min in heating scans between 10 and 70 ºC. The circular dichroism (CD) was measured using a JASCO J-720 spectropolarimeter. Dynamic Light Scattering (DLS) was performed using a Zetasizer Nano (Sysmex) with a He-Ne laser (633 nm). Scattered light signals were collected at detection angle of 173°. The intensity-averaged hydrodynamic diameters were calculated by the manufacturer’s software, utilizing the Stokes–Einstein equation. Prior to the measurements, the aqueous solutions were allowed to stand at least 5 min at targeted temperature. Zeta potential data was also obtained on a Zetasiser Nano-ZS in a distilled water. The pH was adjusted using sodium hydroxide and hydrogen chloride. For atomic force microscopy (AFM) measurements, the polymer (1 mg/mL) and DNA (0.2 mg/mL) solutions

were

prepared

independently

by

dissolving

in

2-[4-(2-hydroxyethyl)-1-

piperazinyl]ethanesulfonic acid (HEPES) buffer (10 mM, pH = 7.4). The proper amount of the polymer solution was added gradually to the DNA solution in order to adjust the N/P ratio, followed by addition of HEPES buffer (10 mM, pH = 7.4) to reach a constant DNA concentration (0.05 mg/mL). After the buffer solution was allowed to stand for 1 h to reach equilibrium, it was diluted into [DNA] = 0.001 mg/mL using HEPES buffer (10 mM, pH = 7.4). The surface-modified substrate was prepared from a freshly cleaved mica surface, which was treated immediately with 30 µL of MgCl2 aqueous solution (5 mM) for 5 min and then sponged up using Kimwipe®. Next, 30 µL of the polyplex solution ([DNA] = 0.001 mg/mL, temperature = 25 °C or 50 °C) was dropped on the MgCl2-treated mica, allowed to stay for 5 min, and then sponged up using Kimwipe®. Similarly, 30 µL of distilled water was dropped on the substrate, allowed to remain for 5 min, and then sponged up. The washing process was repeated twice to remove the non-absorbed DNA and polyplex from the substrate. After drying the substrate in vacuo, the height and phase images were observed by AFM operated in the tapping mode. All measurements were conducted using Nanoscope IIIa (Veeco), and the images were acquired in ambient conditions at room temperature. Transmission electron microscopy (TEM) measurements were performed on a JEOL TEM-


13

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

Page 14 of 45

2100F field emission electron microscope at an accelerating voltage of 200 kV. The sample for TEM observation was prepared by mounting a drop of potassium phosphate buffer (0.05 mg/mL) on carboncoated Cu grids. After 1 min, the excess of the solution was removed out by using Kimwipe®, followed by drying in air. The electrophoretic mobility of the DNA-block copolymer polyplexes at different N/P ratios was studied by gel electrophoresis using 0.8 wt% agarose gel containing tris acetate EDTA (TAE) buffer solution (pH = 8). After mixing 8 µL of the polyplex solution and 2 µL of loading buffer, each complex solution was loaded onto the agarose gel. Naked DNA treated under the same conditions without the polymer was used as a control sample. Experiments were conducted at 50 V for 1 h. DNA was illuminated by ethidium bromide and visualized under UV illumination (312 nm) using ATTO Printgraph (AE-6905 CF CCD Camera Controller).

Results and Discussion Synthesis of Anionic Block Copolymer by RAFT Polymerization of A-Lys(Cbz)-OH. Initially, for the synthesis of the lysine-based block copolymers with thermoresponsive poly(NIPAM), the conditions for controlled radical polymerization via RAFT of A-Lys(Cbz)-OH, which contains a carboxylic acid and a protected amine-functionality in the monomer unit, were optimized (Scheme 1). Polymerization of A-Lys(Cbz)-OH was examined with six different CTAs at [M]0/[CTA]0/[AIBN]0 = 100/2/1 in 1,4dioxane at 60 °C for 24 h. The resulting poly(A-Lys(Cbz)-OH) was converted to the methyl ester by treating the carboxylic acid groups with trimethylsilyldiazomethane for SEC measurements.53,54 These preliminary investigations suggested that the dithiocarbamate-type CTA was a suitable mediating agent to achieve poly(A-Lys(Cbz)-OH) with pre-determined molecular weight and low dispersity, as shown in Table S3 (Supporting Information). Since the dithiocarbamate-type CTA was known to be an efficient RAFT agent for the controlled polymerization of NIPAM56 and synthesis of poly(NIPAM)-based block copolymers,57,58 it was also selected for the synthesis of the lysine-based thermoresponsive block copolymers.


14

Page 15 of 45 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

Langmuir

< insert Table 1>

In an effort to manipulate the molecular weights of resulting lysine-based polymers, in the next stage, RAFT polymerization of A-Lys(Cbz)-OH with the dithiocarbamate-type CTA was conducted at different [M]0/[CTA]0 molar ratios ranging from 25 to 100, with a constant CTA/initiator ratio at [CTA]0/[AIBN]0 = 2. As shown in Table 1, almost quantitative monomer conversions were obtained in 1,4-dioxane at 60 °C for 24 h in all cases, and a unimodal SEC peak was clearly seen for the resulting poly(A-Lys(Cbz)-OMe), which shifted towards higher molecular weights with increasing [M]0/[CTA]0 ratio (Figure S4a, Supporting Information). Slight increase in the polydispersity index was also detected with increasing [M]0/[CTA]0 ratio. Nevertheless, the increase in the Mn of the poly(A-Lys(Cbz)-OMe)s with increasing [M]/[CTA] indicated the feasibility of exerting control over the molecular weights of the weak anionic polyelectrolyte, poly(A-Lys(Cbz)-OH). The deviation between the molecular weights determined by SEC and theoretical calculations was attributed to the polystyrene SEC calibrations. The chemical structures of the resulting poly(A-Lys(Cbz)-OH) and chain-end group were confirmed by 1H NMR spectroscopy (Figure S2, Supporting Information). The molecular weights of poly(A-Lys(Cbz)OH) were also evaluated by comparison of the area of the peak at 4.9–5.1 ppm corresponding to the methylene protons of the benzoxy group in A-Lys(Cbz)-OH repeating units to the peak at 6.2–6.3 ppm, which corresponded to the two protons of the pyrrole-end group. The molecular weights of the polymers calculated by comparing the integrals of the peaks for the chain-end protons to those of the main-chain protons were comparable to the observed values by SEC and the theoretical values for relatively low monomer-to-CTA ratios ([M]0/[CTA]0 = 25 and 50). These results suggest that the majority of the chain end of poly(A-Lys(Cbz)-OH)s were functionalized with the dithiocarbamate end group, serving as macro-CTAs for further chain extension. When designing a block copolymer by RAFT, the order of the preparation of the block is crucial. For the syntheses of lysine-based block copolymers, in this work, the chain extension of the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) macro-CTA to NIPAM was conducted by RAFT polymerization, as shown in Scheme 1. The reaction conditions, monomer conversions, molecular ACS Paragon Plus Environment

15

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

Page 16 of 45

weights, and polydispersities of the block copolymers are summarized in Table 1. When the polymerization was carried out at [NIPAM]0/[macro-CTA]0 molar ratios ranging from 25 to 200 in 1,4dioxane at 60 °C for 24 h, the conversions as determined by 1H NMR were almost quantitative. 1H NMR spectrum of the block copolymer in DMSO-d6 clearly showed the peaks corresponding to both blocks (Figure 1a), which allowed the determination of the comonomer compositions and molecular weights. Figure S4b (Supporting Information) showed the SEC chromatograms of the starting macroCTA and block copolymers, after the derivatization of the carboxylic acid groups by methylation. A shift in the SEC trace toward higher molecular weights was observed until a ratio of [A-Lys(Cbz)OH]0/[macro-CTA]0 = 50, with the polydispersity remaining below 1.35, demonstrating efficient block formation. The block copolymers obtained at higher monomer-to-CTA ratios ([M]0/[macro-CTA]0 = 100 and 200) afforded broad polydispersities with the shoulder at low-molecular-weight region, corresponding to the macro-CTA. Under the conditions, fragmentation from the intermediate radical to dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) radical, as well as the reinitiation process, may occur incompletely, which is probably due to the change of the solubility of the macro-CTA through hydrogen bonding with the second monomer (NIPAM) in the reaction mixture. Nevertheless, the molecular weights of the resulting poly(A-Lys(Cbz)-OH)-b-poly(NIPAM)s determined by 1H NMR were in reasonably good agreement with the theoretical values calculated from the conversion and feed ratio of the comonomer. The results clearly demonstrated that the chain extension of the poly(A-Lys(Cbz)-OH) macro-CTA with NIPAM was well-controlled at relatively low monomer/macro-CTA ratios ([ALys(Cbz)-OH]0/[macro-CTA]0 = 25 and 50), providing anionic block copolymers with as-designed chain structures.


16

Page 17 of 45 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

Langmuir

a

(a)

b

a'

b

O

j k

O f

h

O

N H i

g

m O

NH c'

OH

e' d' e'

NH c d

e

H O DMSO 2

b'

n

O

c, c', i, k

j d, d'

a, b a', b' e~g

h

e'

DO 2

(b)

a

b

b

a'

b' m

n O f

h H2N

NH e d

g

O OH

NH c' e' d' e'

O

d, d' h

(c)

a

b

b

a'

h H2N

g

NH e d

O

DO 2

b' m

n O f

O

NH c'

OMe d' e' i e'

e' i, d, d' h

8

e'

a, b a', b' e~g

7

6

5

4

3

a, b a', b' e~g

2

1

0 ppm

Figure 1. 1H NMR spectra of (a) poly(A-Lys(Cbz)-OH)-b-poly(NIPAM), which was obtained by the polymerization at [NIPAM]0/[macro-CTA]0/[AIBN]0 = 100/2/1 (BC2 in Table 1), and (b) poly(A-LysOH)-b-poly(NIPAM) and (c) poly(A-Lys-OMe)-b-poly(NIPAM) in (a) DMSO-d6 and (b, c) D2O. The comonomer composition was calculated by comparing the peak at 0.9–1.2 ppm (-CH(CH3)2 in NIPAM) and the peak at 4.9–5.1 ppm (-OCH2C in A-Lys(Cbz)-OH), shown in (a), using equation 1 (see experimental section).


17

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

Page 18 of 45

Synthesis and Self-assembly of Lysine-based Zwitterionic Block Copolymers The lysine-based zwitterionic block copolymers, poly(A-Lys-OH)-b-poly(NIPAM)s, were prepared by quantitative deprotection of the poly(A-Lys(Cbz)-OH) segment in the anionic block copolymers obtained by RAFT polymerization (Scheme 1). Removal of the N-carbobenzoxy group in the poly(ALys(Cbz)-OH) segment was conducted using trifluoroacetic acid/HBr and the product was purified by dialysis against water. Successful deprotection of the carbobenzoxy group was confirmed by the disappearance of the phenyl signal at 7.0–8.2 ppm in the 1H NMR spectra of the block copolymer (Figure 1b) and homopolymer (Figure S3, Supporting Information). After the deprotection, the anionic block copolymers, poly(A-Lys(Cbz)-OH)-b-poly(NIPAM)s, were converted into double hydrophilic block copolymers composed of zwitterionic poly(A-Lys-OH) and thermoresponsive poly(NIPAM) segments. Consequently, they were readily soluble in aqueous solution, independent of the pH values. Since A-Lys-OH has carboxylic acid and primary amino groups in the monomer unit, the zwitterionic poly(A-Lys-OH) was soluble in water over a wide pH range (pH = 2, 7, and 12) and methanol (Table S2, Supporting Information). The solubility was apparently distinct from that of the anionic poly(ALys(Cbz)-OH), which exhibited good solubility in basic aqueous solution (pH = 12) and many organic solvents, while being insoluble in neutral (pH = 7) and acidic (pH = 2) water. As shown in Table S3 (Supporting Information), the solubility of the zwitterionic block copolymer composed of poly(A-LysOH) and poly(NIPAM) was almost the same as that of zwitterionic poly(A-Lys-OH) homopolymer, except for the insolubility in a neutral water (pH = 7) at 50 °C. The pH-dependent zeta potential measurement of the zwitterionic block copolymer and homopolymer were evaluated to determine isoelectronic points and charge status in water (conc. = 2.0 mg/mL) at 25 °C. As shown in Figure 2a, the zeta potential of the zwitterionic block copolymer having ~30% of ALys-OH content, poly(A-Lys-OH)24-b-poly(NIPAM)53, was almost the zero at pH = 4.0–9.0. These values were in good agreement with the general tendency of zwitterionic polymers, in which the zeta potential should be zero at the isoelectronic point. The zeta potential varied from +2 to –20 mV on increasing the pH from 9 to 12, suggesting the presence of a negatively charged chain derived from the deprotonation of the carboxylic group of poly(A-Lys-OH) segment. In contrast, the value increased ACS Paragon Plus Environment

18

Page 19 of 45 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

Langmuir

from +5 to +40 mV upon decreasing the pH from 4 to 2, which indicated the existence of a positively charged chain owing to the ammonium cation. Similar tendency was observed in the zwitterionic homopolymer (Figure S5, Supporting Information). These results suggest that the poly(A-Lys-OH) segment in the block copolymer exists predominantly in the zwitterionic form in the pH range of 4.0– 9.0, and intramolecular electrostatic interactions between the positively charged ammonium and negatively charged carboxylate group are predominant in the monomer unit, as shown in Figure 3a. In the basic region (pH < 4), the nitrogen atoms are protonated by HCl instead of the carboxylic acid in the A-Lys-OH unit, whereas the negatively charged carboxylate group are surrounded by oppositely charged counterion in a higher pH region (pH >9). These two transition points are related to the pK values of lysine (2.18 for -carboxyl group and 10.53 for the -amino group, respectively

59).

It has

been reported that the distance between the carboxylate and amine groups affects their acidity and basicity, respectively, and the carbon spacer between them has a significant inﬂuence on their properties and characteristics.45,60 In the present system, a relatively long spacer between the cationic amine group and anionic carboxylic group in the monomer unit and efficient hydration derived from the amide group in poly(A-Lys-OH) were likely responsible for the good water solubility, regardless of pH range. The resulting block copolymer was composed of the zwitterionic poly(A-Lys-OH) and thermoresponsive poly(NIPAM), which formed temperature-responsive assembled structures in aqueous solutions. In this study, the design approach for lysine-based smart materials focused on the creation of chiral micelles with multi-responsive properties, in which the zwitterionic poly(A-Lys-OH) segment corresponded to the shell, whereas the core part consisted of the dehydrated poly(NIPAM) segment, on increasing temperature. The solution and chiroptical properties of the zwitterionic block copolymer having ~30% of A-Lys-OH content were investigated under various conditions. Initially, the solution property in neutral water (pH = 7.4), being a good solvent for both components, poly(A-Lys-OH) and poly(NIPAM), was studied by turbidity and DLS measurements. As can be seen in Figure 2b, the transmittance of poly(A-Lys-OH)24-b-poly(NIPAM)53 decreased slightly ranging from 100% at 30 °C to 90% at 50 °C, which corresponded to the LCST of poly(NIPAM) chain with a heat-induced phase transition from the coiled to globular state in water. The heating and cooling curves showed negligible ACS Paragon Plus Environment

19

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

Page 20 of 45

hysteresis, indicating no significant intrachain and interchain associations in the dehydrated poly(NIPAM) segment in the core. As shown in Figure 2c, the DLS traces of the block copolymer in neutral water (pH = 7.4) exhibited a monomodal distribution of the hydrodynamic diameter above the LCST (Dh = 59 nm at 50 °C), which was attributed to the existence of spherical micelles consisting of a hydrophobic core of dehydrated poly(NIPAM) and a hydrophilic shell of poly(A-Lys-OH) (Figure 3b). At 25 °C, bimodal distributions were observed, in which the smaller species (Dh < 10 nm) presumably corresponded to unimers. The larger species were likely aggregates and/or assembled products having partially insoluble parts because of the formation of hydrogen bonds between the amide moiety in both units and/or acid-based ionic interactions derived from the A-Lys-OH unit. Similar bimodal distribution was observed in the DLS traces of poly(A-Lys-OH) in water under various conditions (Figure S6, Supporting Information). Hence, the aggregates and/or assembled products observed as larger species were attributed to the specific interactions between the poly(A-Lys-OH) component. As shown in Table 2, the zeta potentials of the zwitterionic block copolymer were almost zero at 25 and 50 °C, suggesting that the existence of the zwitterionic form of the poly(A-Lys-OH) segment was independent of the temperature.


20

Page 21 of 45

(a) Zeta potential (mV)

20 10 0 -10 -20 2

4

6

pH

8

10

80 60 40 20 0

12

(e)

80 60 40 20 0

(c)

35 16 14 30 12

25

10

20

8

15 6

30

40 50 o60 Temperature ( C)

70

5000

2

Transmittance (%)

100

Molar elipiticity deg (cm /dmol)

(b)

24:117 24:216

100 Transmittance (%)

30

-30

24:26 24:53

(d)

40

f (Is) (%) Intensity

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

Langmuir

Heating Cooling 30

40 50 o60 Temperature ( C)

70

0 -5000 4

-1x10

o

25 C o

50 C 4

-1.5x10

180 190 200 210 220 230 240 250 Wavelength (nm)

250 C o

0.5 o h 501 h C 2h 3h 4h

4 10 2

5

0

01 10 100 1000 0 1 (nm) 2 3 D 1x10 1x10 h 1x10 1x10 D (nm) h

Figure 2. (a) pH-Dependent zeta potential in water (concentration = 2.0 mg/mL) at 25 °C, (b, d) temperature-dependent turbidity and (c) DLS traces in water (concentration = 2.0 mg/mL) at pH = 7.0, and (e) CD spectra in water (concentration = 0.03 mg/mL) at pH = 7.0 of the zwitterionic block copolymers, poly(A-Lys-OH)n-b-poly(NIPAM)m (n:m = 24:53 for a-c and e, and different n:m ratios for d).


21

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

Page 22 of 45

Figure 3. Schematic illustrations of (a) switchable charged states of the zwitterionic block copolymer and (b) temperature-induced micelle formation of the zwitterionic and cationic block copolymers, and (c) polyplex obtained from DNA and the cationic block copolymer.

The effect of the comonomer composition on the assembled structures and thermoresponsive property was evaluated using four poly(A-Lys-OH)-b-poly(NIPAM)s. Figure 2d exhibits temperature-dependent turbidity of the zwitterionic-thermoresponsive block copolymers having different comonomer compositions. As expected, a clear transition behavior was seen in the aqueous solution of the block copolymer with high NIPAM content (90 %), poly(A-Lys-OH)24-b-poly(NIPAM)216, as shown in Figure 2c. In the case of the poly(A-Lys-OH)24-b-poly(NIPAM)117, there were two transition points. Under these conditions, the zwitterionic-thermoresponsive block copolymers existed in a unimolecular state in water at low temperature, which transformed into micelles and then aggregated structures, depending on the comonomer composition, upon reaching the LCST. In other words, the presence of 10–30% of the


22

Page 23 of 45 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

Langmuir

zwitterionic poly(A-Lys-OH) segment had a significant influence on the thermoresponsive behavior of the poly(NIPAM)-based block copolymers. The effects of the pH value and salt concentration on the thermoresponsive property and assembled structures were also evaluated in aqueous solutions (Figures S7 and S8, Supporting Information). In the case of the block copolymer with ~30% of A-Lys-OH content, poly(A-Lys-OH)24-b-poly(NIPAM)53, there was no remarkable influence of pH on the temperature-dependent turbidity change. The block copolymer with higher NIPAM content, poly(A-Lys-OH)24-b-poly(NIPAM)216, exhibited slight effect of pH on the thermoresponsive behavior. The addition of salt (NaCl) to the aqueous solution of poly(ALys-OH)24-b-poly(NIPAM)53 led to the disappearance of the thermoresponsive behavior in the temperature-dependent transmittance and decrease in the DLS peak corresponding to the assembled/aggregated states (Dh > 100 nm, Figure S8, Supporting Information), which was due to the salt-induced increase in water solubility. Note that bimodal DLS traces of the zwitterionic block copolymer in the potassium phosphate buffer solution at pH = 7.4 were comparable to those in water at pH = 7.0 (Figure S9 and Table S4, Supporting Information). This was consistent with the unique feature of the polyzwitterions that their solubility mostly increases when low-molecular electrolytes are added (salting-in behavior). In contrast, the decrease in the solubility by the addition of salt, which is known as the salting-out behavior observed typically for both polyelectrolytes and nonionic polymers, was seen in the case of the zwitterionic block copolymer with high NIPAM content (90%, Figure S8c,d, Supporting Information), suggesting that the content of the zwitterionic A-Lys-OH unit is related closely to the salt effect on the thermoresponsive behavior and assembled structures of the zwitterionic block copolymers. In this study, the block copolymer with ~30% of A-Lys-OH content was mainly employed towards further investigations, because relatively high NIPAM content was required to afford thermoresponsive property, whereas a suitable chain length of poly(A-Lys-OH) segment was needed to exhibit characteristic zwitterionic behavior. Additionally, a sufficient content of the cationic poly(A-Lys-OMe) segment is crucial for effective complexation of the cationic block copolymer with DNA having reasonable stability.


23

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

Page 24 of 45

The chiroptical behavior of the lysine-based zwitterionic block copolymer was evaluated by CD measurements. In terms of chirality, the poly(A-Lys-OH)-b-poly(NIPAM) prepared in this study was classified as a chiral-achiral type block copolymer. Figure 2e depicts the CD spectra of poly(A-LysOH)-b-poly(NIPAM) in water at pH = 7.0 at 25 and 50 °C. At 25 °C, the zwitterionic block copolymer exhibited a strong negative signal at 198 nm (–1.4 × 104 deg•cm2•dmol–1), which was likely due to the n → π* transition of the carboxy chromophore and π2 → π* or the n → π* transition of the amide group.61 A positive signal was also detected at ~221 nm (2.3 × 103 deg•cm2•dmol–1), which corresponded to the π1 → π* transition of the amide chromophore.61 A slight decrease in the negative peak intensity of the zwitterionic block copolymer was seen at 50 °C, suggesting a temperature-induced conformation change. The strong negative and positive peaks were almost the same as those of the corresponding zwitterionic homopolymer, poly(A-Lys-OH) at pH = 7, as shown in Figure S10 (Supporting Information). The negative peak at ~190 nm observed at pH = 7 shifted toward a higher value of ~200 nm with a slight decrease in the peak intensity at pH = 2, suggesting that the negative peak is related closely to the carboxy chromophore. Similar tendency was observed in poly(A-Lys-OH) at pH = 13, suggesting that the chiroptical property of the zwitterionic homopolymer was affected by the pH. In contrast, the CD spectrum of the anionic poly(A-Lys(Cbz)-OH) at pH = 12 was apparently distinct from that of the zwitterionic poly(A-Lys-OH) under the same condition (Figure S11a, Supporting Information). It means that characteristic chiroptical properties of the lysine-based polymers are also affected by the presence of the hydrophobic Cbz group.

Synthesis and Self-assembly of Lysine-based Cationic Block Copolymers The cationic block copolymer, poly(A-Lys-OMe)-b-poly(NIPAM), was obtained by the methylation of poly(A-Lys-OH) segment of the zwitterionic block copolymer, as shown in Scheme 1. After the methylation of the poly(A-Lys-OH) segment using trimethylsilyldiazomethane,53,54 the product was purified by precipitation into diethyl ether. The degree of esterification was more than 95%, as judged ACS Paragon Plus Environment

24

Page 25 of 45 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

Langmuir

by 1H NMR spectroscopy (Figure 2c) by comparing the integration of the methyl resonance at around 3.6–4.1 ppm (NCH(CH3)2, COOCH3) with the intensity of the methylene resonance at 2.8–3.0 ppm (H2NCH2). Poly(A-Lys-OMe)-b-poly(NIPAM) obtained after the methylation was soluble in most organic solvents, such as chloroform, acetone, THF, 1,4-dioxane, DMF, and DMSO, in addition to methanol and water over a wide pH range (pH = 2, 7, and 12). This solubility behavior is significantly distinct from that of the zwitterionic block copolymer (Table S2, Supporting Information). The cationic lysine-based block copolymer having ~30% of A-Lys-OMe content, poly(A-LysOMe)24-b-poly(NIPAM)53, was investigated in terms of unique self-assembled structures, and thermoresponsive and chiroptical properties. As shown in Figure S12a (Supporting Information), the transmittance of the cationic block copolymer clearly decreased from 100% at 30 °C to 52% at 50 °C in the aqueous solution at pH 7, suggesting that the cationic poly(A-Lys-OMe) segment had lower hydrophilicity as compared to that of zwitterionic poly(A-Lys-OH) in the thermoresponsive block copolymers. Similar to the cases of the zwitterionic block copolymer, the DLS trace of the cationic block copolymer in neutral water (pH = 7.0) exhibited a monomodal distribution at 50 °C, corresponding to spherical micelles having a dehydrated poly(NIPAM) core and hydrophilic poly(ALys-OMe) shell above LCST, whereas multimodal distributions were observed at 25 °C (Figure S12b, Supporting Information). The presence of the cationic poly(A-Lys-OMe) shell at 50 °C was also supported by the increase in the zeta potential with increasing temperature, as shown in Table 2. In the CD spectra, negative and positive peaks at ~200 nm and 220 nm at pH = 7.0 (Figure S12c, Supporting Information) were detected, respectively, in the cationic block copolymer, and the peak positions were comparable to those of the zwitterionic block copolymers. Nevertheless, the difference in the peak intensities between the cationic and zwitterionic block copolymers suggested that a conformational change had occurred on methylation of the carboxylic acid group in the zwitterionic poly(A-Lys-OH) segment in the block copolymers. A slight difference in the peak intensities between the cationic and zwitterionic homopolymers was also detected (Figure S11b, Supporting Information). These results suggested the feasibility of manipulating the thermoresponsive and chiroptical properties and assembled


25

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

Page 26 of 45

structures of the lysine-based block copolymers by the selective transformation of the zwitterionic poly(A-Lys-OH) into the cationic poly(A-Lys-OMe) segment.

Complexation of Lysine-based Cationic Block Copolymers with DNA Lysine-based cationic block copolymer, poly(A-Lys-OMe)-b-poly(NIPAM), was employed for the polyplex formation, in which only the primary amino group in the poly(A-Lys-OMe) segment could interact electrostatically with the negatively charged phosphate groups in DNA. In contrast, it was possible that the thermoresponsive poly(NIAPM) would show no specific interaction with DNA. The advantage of the amino acid-based block copolymers in the application of DNA delivery is considered to be their capability to act as a chiral recognition site, which can support efficient and selective cell uptake and gene release, in addition to the flexibility for the tuning of chirality, amphiphilicity, interactions with biological molecules. The system followed the simple mixing procedure of two transparent aqueous solutions to build up a new smart DNA-block copolymer through the selective interaction of DNA and the cationic poly(A-Lys-OMe) segment. In this regard, the efforts were focused on the design and manipulation of DNA-block copolymer polyplexes using ionic interactions, in addition to the temperature-induced morphological change that originated from the poly(NIPAM)-based segment in the amino acid-based block copolymers with the cationic sites (Figure 3c).


26

Page 27 of 45

25 35

(a)

0 DNA Cationic 0.5 h BC

f (Is)(%) Intensity

30 20

1h 2h 3h 4h

25

15

20

10 15

10 5 5

0

1x10

1

1x10

600

(b)

2

1x10 1x10 D1 (nm) 2

3

1x10

3

1x10 1x10 D (nm) h

10

h

0

400

h

D (nm)

500

-10

300 -20

200

-30

100 0

0

1

2

3

4

5

Zeta potential (mV)

0 0 01x10

-40

N/P

2

(c)


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

Langmuir

6000 4000 2000 0 -2000 -4000 DNA -6000 N/P = 1 -8000 N/P = 2 4 -1x10 220 240 260 280 300 320 Wavelength (nm)

Figure 4. (a) DLS spectra of DNA and DNA-cationic block copolymer polyplex prepared at the cation/anion charge ratio, N/P (the primary amine-based nitrogen atom of the A-Lys-OMe unit/the phosphate group in DNA) = 1, (b) hydrodynamic size and zeta potential of the polyplexes plotted as a function of N/P, and (c) CD spectra of DNA and the polyplexes (N/P = 1 and 2). The polyplexes were prepared using poly(A-Lys-OMe)24-b-poly(NIPAM)53 in potassium phosphate buffer (pH = 7.4, [DNA] = 0.05 mg/mL) at 25 °C.


27

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

Page 28 of 45

Zwitterionic BC/DNA Cationic BC/DNA

N/P

0

0.5

1

2

0.5

1

2

Figure 5. Agarose gel electrophoresis assay of DNA and DNA-block copolymer (poly(A-Lys-OMe)24b-poly(NIPAM)53) polyplexes in TAE buffer at pH = 8.0 with different N/P ratios.

The complexation of the lysine-based cationic block copolymer (poly(A-Lys-OMe)24-bpoly(NIPAM)53) and DNA was investigated in potassium phosphate buffer (pH = 7.4). The results are summarized in Table 2. The cationic block copolymer, pristine DNA, and polyplexes obtained after mixing the two stock solutions were visually transparent in the buffer solution at relatively low DNA concentration (0.05 mg/mL) at room temperature. The hydrodynamic size and size distribution of DNA/lysine-based block copolymer polyplex and pristine DNA were initially studied by DLS in the phosphate buffer (pH = 7.4) at 25 °C. As shown in Figure 4a, the size of DNA increased after the addition of the block copolymer from 111 nm (pristine DNA) to 228 nm (polyplex obtained at the stoichiometric cation/anion charge ratio, N/P ratio = 1). In the cases of the polyplexes between DNA and block copolymers, zeta potential was measured against pH in order to evaluate the pH-dependent charge status of the polyplexes, which are related closely to the chemical structures and components exited on the outermost surface of the polyplexes. The zeta potential of naked DNA without treatment was –37 mV at pH = 7.4, which changed to nearly zero (–0.1 mV) at N/P ratio = 1 (Figure 4b), suggesting the transformation of negatively charged DNA into electrically neutral polyplexes with no ACS Paragon Plus Environment

28

Page 29 of 45 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

Langmuir

significant deviation of the charge under the conditions employed. At N/P > 1, there was no significant change in the neutral charge of the polyplexes, whereas slight decreases were observed in both the size and size distribution in the DLS histograms (Figure S13, Supporting Information). Beyond N/P = 1 at 25 °C, DNA interacted with the molecularly solubilized poly(A-Lys-OMe) block to form block copolymer/DNA polyplexes, in which DNA was entrapped by the poly(A-Lys-OMe) block chains and the shell part mainly consisted of non-interacted poly(NIPAM). The absence of any significant change in the charge of the polyplexes at higher N/P ratio (i.e., higher content of the A-Lys-OMe unit) with slight decrease in the size and size distribution suggested a preferred presence of the remaining poly(ALys-OMe) in the inside the polyplexes to form tightly interacting cores. The change in the conformation of the DNA-block copolymer polyplexes relative to pristine DNA was evaluated by CD measurements in phosphate buffer (pH = 7.4) at 25 °C. As shown in Figure 4c, pristine DNA exhibited a strong negative peak at 246 nm and a positive peak at 276 nm, which were attributed to the helicity of B-from DNA.62 There was no significant change in the CD spectra after the addition of the cationic block copolymer into DNA solution, suggesting that the interaction with the cationic poly(A-Lys-OMe) segment in the block copolymer did not lead to any significant change in DNA helicity. The increase in the temperature, which leads to the structural change in the cationic block copolymer assembly, also had no remarkable change on the DNA helicity, which was confirmed by CD measurement at 50 °C. The electrophoretic mobilities of the DNA-block copolymer polyplexes at different N/P ratios were studied by gel electrophoresis, which helped to visualize the interaction of DNA with the block copolymers. In addition to the poly(A-Lys-OMe)-b-poly(NIPAM) copolymer with the cationic segment, the zwitterionic block copolymer, poly(A-Lys-OH)-b-poly(NIPAM), was used as a comparison. The　 gel electrophoresis data of the cationic and zwitterionic block copolymers in increasing N/P ratios from 0.5 to 2 is shown in Figure 5. The polyplexes with the zwitterionic block copolymer exhibited migration of DNA across the gel, regardless of the N/P ratio. The migration behavior was similar to that observed for pristine DNA, suggesting that there were either weak or no interactions between DNA and the zwitterionic poly(A-Lys-OH) segment, as expected. In contrast, the polyplex with the cationic block ACS Paragon Plus Environment

29

Langmuir

copolymer showed no significant migration at N/P = 1 and all the bands were located at different positions far from the pristine DNA, suggesting that all DNA molecules were employed for the polyplex formation. A similar tendency was observed in the polyplex with the cationic block copolymer at N/P = 2. The amount of the migration increased at N/P = 0.5, indicating the lesser stability of the ionic polyplexes at this lower N/P ratio.

35

Intensity (%)

(a)

o

30

25 C

25

50 C

o

20 15 10 5

h

6000 4000 2000 0

2

(b)


0 0 1 2 3 1x10 1x10 1x10 1x10 D (nm)

-2000 -4000 o -6000 25 C o -8000 50 C 4 -1x10 220 240 260 280 300 320 Wavelength (nm)

2000

(c)

o

25 C o

50 C

1500 1000

h

D (nm)

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

Page 30 of 45

500 0

0

1

2

3

4

5

N/P ACS Paragon Plus Environment

30

Page 31 of 45 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

Langmuir

Figure 6. (a) DLS spectra and (b) CD spectra of DNA-cationic block copolymer polyplex (N/P = 1), and (c) hydrodynamic size and zeta potential of the polyplexes plotted as a function of N/P, at 25 °C and 50 °C. The polyplexes were prepared using poly(A-Lys-OMe)24-b-poly(NIPAM)53 in potassium phosphate buffer (pH = 7.4, [DNA] = 0.05 mg/mL).

The size and size distribution of DNA/lysine-based block copolymer complex below and above the LCST of poly(NIPAM) segment were also investigated in the potassium phosphate buffer (pH = 7.4). As shown in Figure 6a, there was a drastic increase in the particle size with increasing temperature (Dh = 228 nm at 25 ºC and Dh = 1760 nm at 50 ºC) in the polyplex prepared at a stoichiometric cation/anion charge ratio (N/P = 1). At temperatures (25 °C) below the LCST of poly(NIPAM), the core-shell structures with the core composed of the DNA/poly(A-Lys-OH) complexes and poly(NIPAM) shell were present in the buffer solution. The presence of the extended poly(NIPAM) chains may lead to colloidal-stability in the potassium phosphate buffer (pH = 7.4) at N/P = 1. A temperature-induced rearrangement of the core-shell structure was expected to take place at ~32 ºC because of the dehydration of the poly(NIPAM) shell. At both temperatures, the zeta potentials were almost zero (Table 2), indicating the preservation of electrically neutral polyplexes. The increase in the temperature also led to a slight increase in the positive peak at 276 nm in the CD spectra (Figure 6b), suggesting a secondary structural change of DNA as a result of the condensation of the polyplex at an elevated temperature. Nevertheless, there was no significant CD spectral red- and blue-shift, indicating the stability of DNA helicity in the polyplexes obtained at N/P = 1 under these conditions. In the case of the polyplexes prepared at higher N/P ratios (N/P = 2–5), white insoluble products were obtained on increasing the temperature (50 ºC). Below LCST, DNA was wrapped by the poly(A-Lys-OMe) block chains, while the poly(NIPAM) segment contributed to good solubility of the polyplexes in the buffer solution. In contrast, the intra- and intermolecular collapses via the dehydration of the poly(NIPAM) segments at 50 ºC led to the decrease in the solubility and/or colloidal stability of the polyplexes with tightly interacted cores. In the case of the polyplexes prepared at N/P = 0.5, the polyplex still had a negative charge (–20 mV, as shown in Figure 4b), while raising the temperature from 25 ºC to 50 ºC led to no significant increase in the size (Figure 6c). There are two possible contradictory phenomena, ACS Paragon Plus Environment

31

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

Page 32 of 45

which involve temperature-induced contraction of the polyplexes and temperature-induced increase in the aggregation number, that contribute to the decrease and increase in the size, respectively.28 In this system, the temperature-induced contraction of the polyplexes was preferable as the temperature was raised, and the electrostatic repulsion contributed to maintaining the aggregation number, resulting in unchanged hydrodynamic diameters of the polyplexes.

(a)

DNA only

(b)

Cationic BC/DNA, N/P = 1

(d)


0.5 µm

(c)



32

Page 33 of 45 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

Langmuir

Figure 7. AFM height images of (a) DNA and (b–d) DNA-cationic block copolymer (BC), poly(A-LysOMe)24-b-poly(NIPAM)53, polyplexes at N/P = (b) 1, (c) 5, and (d) 10 on ZnCl2-coated mica ([DNA] = 1 mg/L in HEPES buffer (10 mM, pH = 7.4)) at 25 ºC.

The morphological characteristics of DNA and DNA/block copolymers obtained at different N/P ratios at 25 ºC were studied by AFM. As shown in Figure 7a, the pristine DNA without any treatment exhibited string-like and loose-loop structures that were ~0.4-0.6 nm in height. After complexation with the cationic block copolymer (poly(A-Lys-OMe)24-b-poly(NIPAM)53) at stoichiometric charge ratio (N/P = 1), spherical aggregates having a diameter of 200 nm and height of 1–3 nm, which were composed of a large number of the string-like DNA chains (Figures 7b), were detected. Additionally, isolated string-like DNAs were still visible. Similar aggregated structures were also detected by TEM measurement without staining (Figure S14, Supporting Information). At N/P = 5, a more compact spherical structure with tightly packed DNA chains and lesser number of isolated string-like DNAs were seen (Figures 7c). Further increase in the cation/anion charge ratio (N/P = 10) led to an increase in the size of the aggregates (Figures 7d). The morphological characteristics of the DNA-cationic block copolymer polyplex (N/P = 1) at 50 ºC (above LCST) were further investigated by AFM (Figure S15, Supporting Information), and revealed a significant increase in the height of the polyplexes. The drastic change in the AFM images of DNA on the addition of the cationic block copolymer indicated that DNA was enclosed by the cationic block copolymer, depending on the N/P ratio. Since the controlled release of DNA from the polymer/DNA polyplex is important for gene delivery, we also investigated the stability of the polyplex in the presence of salt (NaCl = 0.1 M), which is known to support the salt-induced destruction of the electrostatic interactions between the cation and anion. At 25 oC, the size of the polyplex obtained at N/P ratio = 1 increased by the addition of NaCl, and broader DLS traces with the additional peak at smaller size region were detected with increasing time (Figure S16, Supporting Information). Whereas the significant decrease in the size of the polyplex was detected after 30 min, and further increase in the time led to no significant change on the size and size


33

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

Page 34 of 45

distribution at 50 oC. These preliminary experimental results suggest the feasibility to achieve temperature-responsive controlled release of DNA from the polymer/DNA polyplex. Finally, the polyplexes prepared using the lysine-based block copolymer with a higher cationic ALys-OMe content, poly(A-Lys-OMe)24-b-poly(NIPAM)26, were evaluated by DLS and agarose gel electrophoresis. As can be seen in Figure S17a (Supporting Information), no detectable migration was seen in the agarose gel electrophoresis experiment at N/P = 1, suggesting the formation of a stable complex via electrostatic interactions. As expected, the assay of the polyplex at N/P = 0.5 exhibited detectable migration, similar to that of pristine DNA. Such tendency of the agarose gel electrophoresis experiments at N/P = 0.5 and 1 was almost the same, regardless of the comonomer composition of poly(A-Lys-OMe)n-b-poly(NIPAM)m (n:m = 24:26 and 24:53, shown in Figure 5). Figure S17b (Supporting Information) showed the bimodal DLS traces of the polyplexes at both 25 °C and 50 °C in the phosphate buffer (pH = 7.4). Interestingly, a slight decrease in the size of the larger species, which may be attributed to the main component of the polyplexes, was detected with increasing temperature. It is worth noting that an appropriate particle size of polymer/DNA nanoparticles and stimuli-triggered change in the size are important parameters for a gene carrier. In the present system, the comonomer composition of the cationic block copolymer affected the temperature-dependent changes of the size and size distribution of the polyplexes, influencing the transfection efficiency.

Conclusions In this study, a series of lysine-based block copolymers were synthesized by RAFT polymerization of A-Lys(Cbz)-OH and subsequent chemical modifications. The anionic lysine-based block copolymers with “as-designed” chain structures were synthesized by RAFT polymerization of NIPAM from the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH). Subsequent deprotection of the N-carbobenzoxy group and selective methylation of the carboxylic acid group afforded zwitterionic and cationic block copolymers, respectively. The characteristic thermoresponsive and chiroptical properties, and temperature-induced micelle formation of the lysine-based block copolymers could be tuned by altering the chemical structures and composition of the zwitterionic/cationic segments, which corresponded to a ACS Paragon Plus Environment

34

Page 35 of 45 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

Langmuir

permanent change of the lysine-based unit. The addition of salt had a clear effect on the assembled structures and temperature-responsive properties of the zwitterionic block copolymers, whereas the pHinduced change among the zwitterionic, anionic, and cationic states of the poly(A-Lys-OH) segment (temporally change) had lesser influence. The temperature-responsive behaviors of DNA-block copolymer polyplexes, which are governed by the cation/anion charge ratio (N/P) and the comonomer composition of the cationic block copolymers, were also discussed. The cationic poly(A-Lys-OMe) segment in the block copolymer exhibited electrostatic interaction with DNA to form the polyplexes, exhibiting temperature- and salt-dependent aggregated structures. Since the poly(A-Lys-OH) segment had no interaction with DNA, the zwitterionic amino acid-based polymer can be employed as pHresponsive layer located in the shell part of the polyplexes having attractive properties, such as cytotoxicity, chiral recognition, and good cell penetrating activity. These multi-stimuli responsive properties and switchability (temporally and permanently) of the lysine-based block copolymers make then promising candidates for a variety of applications in the field of biomedicine and in the development of smart surfaces.


35

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

Page 36 of 45

Table 1. RAFT polymerization of N-acryloyl-N-carbobenzoxy-L-lysine (A-Lys(Cbz)-OH) for the syntheses of the anionic homopolymers and block copolymers. Code [M]/[CTA]

(%)

n:m c)

Mn

Conv. c)

Mw/Mn Theoretical

d)

NMR

c)

SEC

e)

e)

P 1 a)

25/1

99

8,300

12,900

8,200

1.28

-

P 2 a)

50/1

99

16,500

16,600

11,300

1.38

-

P 3 a)

100/1

99

33,300

51,200

22,300

1.39

-

BC 1 b)

25/1

96

9,800

9,700

11,200

1.28

24:26

BC 2 b)

50/1

99

12,700

12,600

13,400

1.35

24:53

BC 3 b)

100/1

97

18,100

18,700

20,100

1.45

24:117

BC 4 b)

200/1

95

28,600

28,500

21,400

1.95

24:216

a)

Poly(A-Lys(Cbz)-OH) was synthesized using benzyl 1-pyrrolecarbodithioate as a CTA at

[CTA]/[AIBN] = 2 in 1,4-dioxane (conc. = 1.0 M).

b)

Poly(A-Lys(Cbz)-OH)n-b-poly(NIPAM)m was

synthesized using the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) as a macro-CTA (Mn = 8,200, Mw/Mn = 1.28) at [macro-CTA]/[AIBN] = 2 in 1,4-dioxane (conc. = 0.5 M). c) Calculated by 1H NMR in DMSO-d6. d) Theoretical molecular weight of P1–3 = (MW of A-Lys(Cbz)-OMe) × [A-Lys(Cbz)OH]0/[CTA]0 × conv. + (MW of CTA) and BC1–4 = (MW of NIPAM) × [NIPAM]0/[macro-CTA]0 × conv. + (MW of macro-CTA).

e)

Methylated samples were measured by size-exclusion

chromatography (SEC) using polystyrene standards in N,N-dimethyl formamide (DMF, 10 mM LiBr).


36

Page 37 of 45 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

Langmuir

Table 2. Summary of size and zeta potential of DNA, lysine-based block copolymers, and polyplexes a) Dh (nm)

Zeta potential　(mV)

Sample 25 °C

50 °C

25 °C

50 °C

Cationic BC b)

8.4, 158

119

3.2

12

Zwitterionic BC c)

12, 183

59

-0.45

2.9

Cationic BC/DNA d)

228

1760

-0.1

-1.1

Zwitterionic BC/DNA d)

68, 370

163

-21

-34

DNA

111

19, 106

-37

-41

a) Conditions: b)

solvent = potassium phosphate buffer (pH = 7.4, 2.0 mg/mL), temperature = 25 and 50 °C.

Cationic BC = poly(A-Lys-OMe)24-b-poly(NIPAM)53 (Mn = 17,000, Mw/Mn = 1.37).

c)

Zwitterionic

BC = poly(A-Lys-OH)24-b-poly(NIPAM)53 (Mn = 16,600, Mw/Mn = 1.37). d) Polyplexes obtained at N/P = 1 ([DNA] = 0.05 mg/mL).


37

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

Page 38 of 45

References (1)

Gil, E. S.; Hudson, S. M.: Stimuli-reponsive polymers and their bioconjugates Progress

in Polymer Science 2004, 29, 1173-1222. (2)

Rodríguez-Hernández, J.; Chécot, F.; Gnanou, Y.; Lecommandoux, S.: Toward ‘smart’

nano-objects by self-assembly of block copolymers in solution. Progress in Polymer Science 2005, 30, 691-724. (3)

Dimitrov, I.; Trzebicka, B.; Müller, A. H. E.; Dworak, A.; Tsvetanov, C. B.:

Thermosensitive water-soluble copolymers with doubly responsive reversibly interacting entities Progress in Polymer Science 2007, 32, 1275-1343. (4)

Motokawa, R.; Morishita, K.; Koizumi, S.; Nakahira, T.; Annaka, M.: Thermosensitive

Diblock Copolymer of Poly(N-isopropylacrylamide) and Poly(ethylene glycol) in Water: Polymer Preparation and Solution Behavior. Macromolecules 2005, 38, 5748 - 5760. (5)

Michel Arotçaréna; Bettina Heise, S.; shaya, S. I.; Laschewsky, A.: Switching the Inside

and the Outside of Aggregates of Water-Soluble Block Copolymers with Double Thermoresponsivity. Journal of the American Chemical Society 2002, 3787-3793. (6)

Schilli, C. M.; Zhang, M.; Rizzardo, E.; Thang, S. H.; Chong, Y. K.; Edwards, K.;

Karlsson, G.; Müller, A. H. E.: A New Double-Responsive Block Copolymer Synthesized via RAFT Polymerization: Poly(N-isopropylacrylamide)-block-poly(acrylic acid) Macromolecules 2004, 37, 78617866. (7)

Wei, H.; Cheng, S.-X.; Zhang, X.-Z.; Zhuo, R.-X.: Thermo-sensitive polymeric micelles

based on poly(N-isopropylacrylamide) as drug carriers Progress in Polymer Science 2009, 34, 893-910 (8)

Rzaev, Z. M. O.; Dincer, S.; Piskin, E.: Functional copolymers of N-isopropylacrylamide

for bioengineering applications. Progress in Polymer Science 2007, 32, 534-595. (9)

Virtanen, J.; Arotçaréna, M.; Heise, B.; Ishaya, S.; Laschewsky, A.; Tenhu, H.:

Dissolution and Aggregation of a Poly(NIPA-block-sulfobetaine) Copolymer in Water and Saline Aqueous Solutions. Langmuir 2002, 18, 5360–5365. ACS Paragon Plus Environment

38

Page 39 of 45 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

Langmuir

(10)

Vishnevetskaya, N. S.; Hildebrand, V.; Niebuur, B.-J.; Grillo, I.; Filippov, S. K.;

Laschewsky, A.; Mueller-Buschbaum, P.; Papadakis, C. M.: "Schizophrenic" Micelles from Doubly Thermoresponsive Polysulfobetaine-b-poly(N-isopropylmethacrylamide) Diblock Copolymers Macromolecules 2017, 50, 3985-3999 (11)

Vishnevetskaya, N. S.; Hildebrand, V.; Dyakonova, M. A.; Niebuur, B.-J.; Kyriakos, K.;

Raftopoulos, K. N.; Di, Z.; Mueller-Buschbaum, P.; Laschewsky, A.; Papadakis, C. M.: Dual Orthogonal Switching of the "Schizophrenic" Self-Assembly of Diblock Copolymers. Macromolecules 2018, 51, 2604-2614 (12)

Jimenez, Z. A.; Yoshida, R.: Temperature Driven Self-Assembly of a Zwitterionic Block

Copolymer That Exhibits Triple Thermoresponsivity and pH Sensitivity Macromolecules 2015 48, 4599-4606 (13)

Lowe, A. B.; McCormick, C. L.: Synthesis and Solution Properties of Zwitterionic

Polymers. Chemical Reviews 2002, 102, 4177-4189. (14)

Keefe, A. J.; Jiang, S.: Poly(zwitterionic)protein conjugates offer increased stability

without sacrificing binding affinity or bioactivity. Nature Chemistry 2012, 4, 60-64 (15)

Zhang, L.; Cao, Z.; Bai, T.; Carr, L.; Ella-Menye, J.-R.; Irvin, C.; Ratner, B. D.; Jiang,

S.: Zwitterionic hydrogels implanted in mice resist the foreign-body reaction. Nature Biotechnology 2013, 31, 553-556. (16)

Shih, Y.-J.; Chang, Y.; Deratani, A.; Quemener, D.: "Schizophrenic" Hemocompatible

Copolymers via Switchable Thermoresponsive Transition of Nonionic/Zwitterionic Block SelfAssembly in Human Blood Biomacromolecules 2012, 2849-2858 (17)

Miyata, K.; Nishiyama, N.; Kataoka, K.: Rational design of smart supramolecular

assemblies for gene delivery: chemical challenges in the creation of artificial viruses Chemical Society Reviews 2012, 41, 2562-2574 (18)

Cabral, H.; Miyata, K.; Osada, K.; Kataoka, K.: Block Copolymer Micelles in

Nanomedicine Applications Chemical Reviews 2018, 118, 6844-6892


39

Langmuir 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

(19)

Page 40 of 45

Laechelt, U.; Wagner, E.: Nucleic Acid Therapeutics Using Polyplexes: A Journey of 50

Years (and Beyond) Chemical Reviews 2015, 115, 11043-11078 (20)

Mintzer, M. A.; Simanek, E. E.: Nonviral Vectors for Gene Delivery Chemical Reviews

2009, 109, 259-302 (21)

Zhou, Z.; Liu, X.; Zhu, D.; Wang, Y.; Zhang, Z.; Zhou, X.; Qiu, N.; Chen, X.; Shen, Y.:

Nonviral cancer gene therapy: Delivery cascade and vector nanoproperty integration. Advanced Drug Delivery Reviews 2017, 115, 115-154. (22)

Li, Y.; Gao, J.; Zhang, C.; Cao, Z.; Cheng, D.; Liu, J.; Shuai, X.: Stimuli-Responsive

Polymeric Nanocarriers for Efficient Gene Delivery. Topics in Current Chemistry 2017, 375, 27. (23)

Topuzogullari, M.; Elalmis, Y. B.; Isoglu, S. D.: Thermo-Responsive Complexes of c-

Myc Antisense Oligonucleotide with Block Copolymer of Poly(OEGMA) and Quaternized Poly(4Vinylpyridine) Macromolecular Bioscience 2017, 17, 1600263 (24)

Tran, N. T. D.; Truong, N. P.; Gu, W.; Jia, Z.; Cooper, M. A.; Monteiro, M. J.: Timed-

Release Polymer Nanoparticles Biomacromolecules 2013, 14, 495-502 (25)

Turk, M.; Dincer, S.; Yulug, I.; Piskin, E.: In vitro transfection of HeLa cells with

temperature sensitive polycationic copolymers. Journal of Controlled Release 2004, 96, 325-340 (26)

Tardani, F.; Masci, G.; La Mesa, C.: Block co-polymers undergoing supra-molecular

association Colloid and Surfaces A-Physicochemical and Enginnering Aspects 2011, 384, 374-380 (27)

Calejo, M. T.; Cardoso, A. M. S.; Kjoniksen, A.-L.; Zhu, K.; Morais, C. M.; Sande, S.

A.; Cardoso, A. L.; Pedroso de Lima, M. C.; Jurado, A.; Nystroem, B.: Temperature-responsive cationic block copolymers as nanocarriers for gene delivery International Journal of Pharmaceutics 2013, 448, 105-114 (28)

Cardoso, A. M.; Teresa Calejo, M.; Morais, C. M.; Cardoso, A. L.; Cruz, R.; Zhu, K.;

Pedroso de Lima, M. C.; Jurado, A. S.; Nystroem, B.: Application of Thermoresponsive PNIPAAM-bPAMPTMA Diblock Copolymers in siRNA Delivery. Molecular Pharmaceutics 2014 11, 819-827


40

Page 41 of 45 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

Langmuir

(29)

Twaites, B.; Alarcon, C.; Cunliffe, D.; Lavigne, M.; Pennadam, S.; Smith, J.; Gorecki,

D.; Alexander, C.: Thermo and pH responsive polymers as gene delivery vectors: effect of polymer architecture on DNA complexation in vitro Journal of Controlled Release 2004, 97, 551-566 (30)

Osawa, S.; Osada, K.; Hiki, S.; Dirisala, A.; Ishii, T.; Kataoka, K.: Polyplex Micelles

with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection Biomacromolecules 2016 17, 354-361 (31)

Dai, F.; Wang, P.; Wang, Y.; Tang, L.; Yang, J.; Liu, W.; Li, H.; Wang, G.: Double

thermoresponsive polybetaine-based ABA triblock copolymers with capability to condense DNA Polymer 2008, 5322-5328 (32)

O'Reilly, R. K.: Using controlled radical polymerisation techniques for the synthesis of

functional polymers containing amino acid moieties Polymer International 2010, 59, 568-573 (33)

Mori, H.; Endo, T.: Amino acid-based block copolymers by RAFT polymerization.

Macromolecular Rapid Communications 2012, 33, 1090−1107. (34)

Roy, S. G.; De, P.: pH Responsive Polymers with Amino Acids in the Side Chains and

Their Potential Applications Journal of Applied Polymer Science 2014, 131, 41084 (35)

Bauri, K.; Nandi, M.; De, P.: Amino acid-derived stimuli-responsive polymers and their

applications Polymer Chemistry 2018, 9, 1257-1287 (36)

Brisson, E. R. L.; Xiao, Z.; Connal, L. A.: Amino Acid Functional Polymers: Biomimetic

Polymer Design Enabling Catalysis, Chiral Materials, and Drug Delivery Australian Journal of Chemistry 2016, 69, 705-716 (37)

Bauri, K.; Roy, S. G.; De, P.: Side-Chain Amino-Acid-Derived Cationic Chiral Polymers

by Controlled Radical Polymerization Macromolecular Chemistry and Physics 2016, 217, 365-379. (38)

Alswieleh, A. M.; Cheng, N.; Canton, I.; Ustbas, B.; Xue, X.; Ladmiral, V.; Xia, S.;

Ducker, R. E.; El Zubir, O.; Cartron, M. L.; Hunter, C. N.; Leggett, G. J.; Armes, S. P.: Zwitterionic Poly(amino acid methacrylate) Brushes Journal of the American Chemical Society 2014, 136, 94049413 ACS Paragon Plus Environment

41

Langmuir 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

(39)

Page 42 of 45

Liu, Q.; Singh, A.; Liu, L.: Amino Acid-Based Zwitterionic Poly(serine methacrylate) as

an Antifouling Material Biomacromolecules 2013, 14, 226-231 (40)

Li, W.; Liu, Q.; Liu, L.: Amino acid-based zwitterionic polymers: antifouling properties

and low cytotoxicity Journal of Biomaterials Science-Polymer Edition 2014, 25, 1730-1742. (41)

Liu, Q.; Li, W.; Wang, H.; Newby, B.-m. Z.; Cheng, F.; Liu, L.: Amino Acid-Based

Zwitterionic Polymer Surfaces Highly Resist Long-Term Bacterial Adhesion. Langmuir 2016, 32, 78667874. (42)

Fujii, S.; Kido, M.; Sato, M.; Higaki, Y.; Hirai, T.; Ohta, N.; Kojio, K.; Takahara, A.:

pH-Responsive and selective protein adsorption on an amino acid-based zwitterionic polymer surface Polymer Chemistry 2015, 6, 7053-7059 (43)

Liu, Q.; Li, W.; Singh, A.; Cheng, G.; Liu, L.: Two amino acid-based superlow fouling

polymers: Poly(lysine methacrylamide) and poly(ornithine methacrylamide) Acta Biomaterialia 2014, 10, 2956-2964. (44)

Li, W.; Liu, Q.; Liu, L.: Antifouling Gold Surfaces Grafted with Aspartic Acid and

Glutamic Acid Based Zwitterionic Polymer Brushes Langmuir 2014, 30, 12619-12626 (45)

Wang, H.; Wu, H.; Lee, C.-J.; Lei, X.; Zhe, J.; Xu, F.; Cheng, F.; Cheng, G.: pH-

Sensitive Poly(histidine methacrylamide) Langmuir 2016, 32, 6544-6550 (46)

Banerjee, S.; Maji, T.; Paira, T. K.; Mandal, T. K.: Amino-Acid-Based Zwitterionic

Polymer and Its Cu(II)-Induced Aggregation into Nanostructures: A Template for CuS and CuO Nanoparticles Macromolecular Rapid Communications 2013, 34, 1480-1486 (47)

Weller, D.; Medina-Oliva, A.; Claus, H.; Gietzen, S.; Mohr, K.; Reuter, A.; Schaeffel,

D.; Schoettler, S.; Koynov, K.; Bros, M.; Grabbe, S.; Fischer, K.; Schmidtt, M.: Solution Properties and Potential Biological Applications of Zwitterionic Poly(epsilon-N-methacryloyl-L-lysine) Macromolecules 2013, 46, 8519-8527 (48)

Maji, T.; Banerjee, S.; Biswas, Y.; Mandal, T. K.: Dual-Stimuli-Responsive L-Serine-

Based Zwitterionic UCST-Type Polymer with Tunable Thermosensitivity Macromolecules 2015 48, 4957-4966 ACS Paragon Plus Environment

42

Page 43 of 45 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

Langmuir

(49)

Kim, Y.; Binauld, S.; Stenzel, M. H.: Zwitterionic Guanidine-Based Oligomers

Mimicking Cell-Penetrating Peptides as a Nontoxic Alternative to Cationic Polymers to Enhance the Cellular Uptake of Micelles Biomacromolecules 2012, 13, 3418-3426 (50)

Chiefari, J.; Mayadunne, R. T. A.; Moad, C. L.; Moad, G.; Rizzardo, E.; Postma, A.;

Skidmore, M. A.; Thang, S. H.: Thiocarbonylthio compounds (S=C(Z)S-R) in free radical polymerization with reversible addition-fragmentation chain transfer (RAFT polymerization). Effect of the activating group Z. Macromolecules 2003, 36, 2273-2283. (51)

Mori, H.; Nakano, S.; Endo, T.: Controlled Synthesis of Poly(N-ethyl-3-vinylcarbazole)

and Block Copolymers via RAFT Polymerization. Macromolecules 2005, 38, 8192-8201. (52)

Bueno, M. P.; Cativiela, C. A.; Mayoral, J. A.; Avenoza, A.: MODELS FOR THE USE

OF ALPHA-AMINO-ACIDS AS CHIRAL AUXILIARIES IN ASYMMETRIC DIELS-ALDER REACTIONS. Journal of Organic Chemistry 1991, 56, 6551-6555. (53)

Couvreur, L.; Lefay, C.; Belleney, J.; Charleux, B.; Guerret, O.; Magnet, S.: First

Nitroxide-Mediated Controlled Free-Radical Polymerization of Acrylic Acid. Macromolecules 2003, 36, 8260-8267. (54)

Mori, H.; Matsuyama, M.; Sutoh, K.; Endo, T.: RAFT Polymerization of Acrylamide

Derivatives Containing L-Phenylalanine Moiety. Macromolecules 2006, 39, 4351-4360. (55)

Rodriguez-Hernandez, J.; Gatti, M.; Klok, H.: Highly branched poly(L-lysine)

Biomacromolecules 2003, 4, 249-258 (56)

Schilli, C.; Lanzendörfer, M. G.; Müller, A. H. E.: Benzyl and cumyl dithiocarbamates as

chain transfer agents in the RAFT polymerization of N-isopropylacrylamide. In situ FT-NIR and MALDI-TOF MS investigation. Macromolecules 2002, 35, 6819-6827. (57)

Abiko, Y.; Matsumura, A.; Nakabayashi, K.; Mori, H.: Thermoresponsive core–shell

nanoparticles with cross-linked π-conjugate core based on amphiphilic block copolymers by RAFT polymerization and palladium-catalyzed coupling reactions. Polymer 2014, 55, 6025-6035.


43

Langmuir 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

(58)

Page 44 of 45

Nakabayashi, K.; Noda, D.; Watanabe, Y.; Mori, H.: Rylene bisimide-based

nanoparticles with cross-linked core and thermoresponsive shell using poly(vinyl amine)-based block copolymers. Polymer 2015, 68, 17-24. (59)

Bailey, J. E.; Ollis, D. F.: Biochemical Engineering Fundamentals; Second Edition ed.;

McGraw-Hill College, 1986. (60)

Lee, C.-J.; Wu, H.; Tang, Q.; Cao, B.; Wang, H.; Cong, H.; Zhe, J.; Xu, F.; Cheng, G.:

Structure-Function Relationships of a Tertiary Amine-Based Polycarboxybetaine Langmuir 2015, 31, 9965-9972. (61)

Morcellet-Sauvage, J.; Morcellet, M.; Loucheux, C.: Poly(methacrylic acid) derivatives.

7. Chiroptical properties of optically active poly(N-methacryloyl-L-alanine) and poly(N-methacryloylL-alanine-co-N-phenylmethacrylamide). Macromolecules 1983, 16, 1564 - 1570. (62)

Vorlickova, M.; Kejnovska, I.; Bednarova, K.; Renciuk, D.; Kypr, J.: Circular Dichroism

Spectroscopy of DNA: From Duplexes to Quadruplexes Chirality 2012, 24, 691-698.


44

Page 45 of 45 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

Langmuir

For Table of Contents Use Only


45

Synthesis, Assembled Structures, and DNA Complexation of

Recommend Documents