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Amphiphilic Poly[poly(ethylene glycol) methacrylate]s with OH Groups in the PEG Side Chains for Controlling Solution/Rheological Properties and toward Bioapplication Yuta Koda, Daiki Takahashi, Yoshihiro Sasaki, and Kazunari Akiyoshi ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00836 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 20, 2019

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ACS Applied Bio Materials

Amphiphilic Poly[poly(ethylene glycol) methacrylate]s with OH Groups in the PEG Side Chains for Controlling Solution/Rheological Properties and toward Bioapplication Yuta Koda,1,2 Daiki Takahashi,1 Yoshihiro Sasaki,1 and Kazunari Akiyoshi*1,2

1) Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University Katsura, Nishikyo-ku, Kyoto 615-8510, Japan Tel: +81-75-383-2590, Fax: +81-75-383-2589, E-mail: [email protected] 2) ERATO Akiyoshi Bio-Nanotransporter Project, Japan Science and Technology Agency (JST), Katsura IntÕtech center, Katsura, Nishikyo-ku, Kyoto 615-8530, Japan Tel:+81-75-383-2152, Fax: +81-75-383-2154

Key Words: Poly(ethylene glycol) / PEGylation / Amphiphilic Copolymers / Living Radical Polymerization / Protein-Polymer Conjugation / Bioapplication

Present Address Yuta Koda: Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138, Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan

Conflict of Interest The authors declare no conflict of interest.

ORCID Yuta Koda: 0000-0003-1724-2359 Daiki Takahashi: 0000-0002-4890-6827 Yoshihiro Sasaki: Kazunari Akiyoshi:

Abstract Poly[poly(ethylene glycol) methacrylate]s

with OH groups

on the PEG side

chains

[poly(PEGOHMA)s] were synthesized using ruthenium-catalyzed living radical polymerization (Ru-LRP) to diversify the polymer design of PEGylated methacrylate-based copolymers. Poly(PEGOHMA)s could not be prepared using the approach previously reported for synthesis of poly[poly(ethylene glycol) methyl ether methacrylate [poly(PEGMA)], therefore the polymerization was adapted for poly(PEGOHMA)s. As a result, both homopolymerization and random and block copolymerization of PEGOHMA with other hydrophobic

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ACS Applied Bio Materials 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

monomers were successfully achieved, resulting in the preparation of amphiphilic random block and star polymers. The solution and bulk properties of PEGOHMA-based (co)polymers were markedly different from those of PEGMA-based (co)polymers. By reacting the OH groups with biotin, protein-poly(PEGOHMA) conjugates were successfully prepared, however it was not possible to prepare protein-polymer conjugates using terminal biotinylated PEGMA-based copolymers owing to the steric hindrance of the unreactive PEG side chains.


Page 2 of 26

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Introduction The use of poly(ethylene glycol)-functionalized (PEGylated) polymers has been growing in a considerable number of fields, with PEGylated polymers being among the most important polymeric materials in both industry and academic research.1Ð28 For example, PEGylated diacrylates have been applied in tissue sealant development and the resulting materials such as FocalSeal¨ are commercially available owing to their biocompatibility.1Ð4 In addition, PEGylated polymers have been used in construction materials (e.g., concretes, sealants), cosmetics and detergents, and biomaterials (e.g., DOXIL¨, CAELYX¨).5Ð8 PEG also shows lower critical solution temperature (LCST)-type phase separation in water,9Ð14 therefore PEGylated copolymers have been used to develop a variety of functionalized materials such as thermoresponsive micelles and polymersomes in water,15Ð17 polymeric catalysts and scavengers,18Ð20 protein stabilizers,21Ð24 and nanocarriers in drug delivery systems (DDS).25Ð28 In particular, poly(ethylene glycol) methyl ether methacrylate (PEGMA) can be polymerized via living radical polymerization (LRP) with narrow molecular weight distribution (MWD), resulting in the preparation of well-controlled random, block, and star copolymers.29Ð31 Owing to the high degree of control possible, PEGMA-based copolymers with well-controlled architectures have been applied in many different fields, for example poly(PEGMA)-based copolymers have been used in nanocarriers for DDS.25Ð28,32,33 The poly(PEGMA)-based copolymers were found to interact more efficiently with cells compared with liner PEG-based polymers.32Ð35 The results suggested that diversifying the molecular design of PEGylated (meth)acrylate-based copolymers would allow control of both the cellular and tissue distribution of therapeutic carriers, and would contribute to the further development of other bioapplications such as cell engineering.36 In spite of the advantages of PEGMA-based copolymers obtained by LRP, such copolymers are limited in terms of post-functionalization because poly(PEGMA) has no reactive sites in the polymer chains unless reactive initiators are used. In contrast, PEGMA with an OH terminal end on the PEG side chain (PEGOHMA) offers significant promise for diversifying the molecular design of PEGylated (meth)acrylate based copolymers through post-functionalization of the hydroxyl group. PEGOHMA was polymerized via coppercatalyzed LRP (Cu-LRP), however it is difficult to synthesize amphiphilic copolymers based on PEGOHMA because polar solvents [e.g., dimethyl sulfoxide (DMSO) and pure water] are required for efficient catalysis, however hydrophobic monomers can not then be used owing to their low solubility.37Ð42 In contrast, SawamotoÕs LRP system that uses ruthenium catalysts (Ru-LRP) can be conducted in a variety of solvents [e.g., toluene, N,N-dimethylformamide (DMF), ethanol, and H2O], which enables the preparation of a range of amphiphilic copolymers.29Ð31 However, the radical polymerization of PEGOHMA via Ru-LRP can not be controlled to prepare well-controlled amphiphilic random, block, and star copolymers based on PEGOHMA. Based on the previous work described, in this work PEGOHMA was polymerized via Ru-LRP to introduce functionalizable groups into the molecular design of PEGylated (meth)acrylate-based amphiphilic



Page 4 of 26

copolymers for biomedical applications (Scheme 1). We achieved well-controlled poly(PEGOHMA) with a ruthenium catalytic system [RuCp*Cl(PPh3)2 / HO(CH2)4N(CH3)2] in EtOH/H2O (= 3/1, v/v) at 25 ¡C. The EtOH mixed solvent enabled the copolymerization of PEGOHMA and a hydrophobic methacrylate (BMA, butyl methacrylate; DMA, dodecyl methacrylate) resulting in amphiphilic copolymers. In particular, AB-, ABA- and ABC-type di- or triblock and star polymers were successfully synthesized by in situ addition of 2nd and 3rd monomers owing to the high controllability of the system. Unlike poly(PEGMA), poly(PEGOHMA) showed no LCST-type phase separation in water. The viscoelastic properties of the polymers in the bulk state were also investigated to show the effect of the OH groups on the PEG side chains. In addition, PEGOHMA-based amphiphilic block copolymers were easily conjugated with model protein streptavidin (SAv) following post-functionalization of the OH groups with biotin; unlike end-functionalized PEGMA-based amphiphilic block copolymers which did not bind SAv. Therefore, our molecular design strategy for PEGylated (meth)acrylate-based copolymers with OH groups will provide new possibilities for PEGylated functional materials, particularly contributing to the development of biomedical applications such as cell engineering and DDS nanocarrier design.

Homo/Random Polymers PEGOHMA Ru Cl O

O O H k

(k = 4.5 or 9)

! Random

! Homo

P14, P15

PPh3 PPh3 Cl

R

N

HO

in EtOH/H2O (25

Cl

R O m O H

O

O

O m O H

4.5

oC)

O

p

O

4.5

Block Polymers ! AB-Type

! Gel-Core Star

! AB-Type (Random Block) P18, P19

P16, P17

P33 Ð P35 Arm

Core Cl

R

Cl

R O m O H

O

O

Cl

R

O

p

O

O

O m O H

4.5

O

O

O n O

9

O m O H

O

p

O

p

O

4.5

O

9

O

! ABC-Type P29 Ð P32

! ABA-Type P20 Ð P22 Cl

R O

O m O H k

O

O

O n O

p

O

O RÕ

O m O H 4.5

2

9

Cl

R O O

p

O

O n O 4.5

RÕ = ÐC4H9 (BMA), ÐC12H25 (DMA)

Scheme 1. Synthesis of amphiphilic homo-, random, block, and star (co)polymers based on PEGOHMA via ruthenium-catalyzed living radical polymerization


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Results and Discussion Precision Synthesis Homo- and Random Polymerization In the classical system, PEG9MA [PEGkMA; CH2=C(CH3)COO(CH2CH2O)kCH3, k = 4.5 or 9] was polymerized with a chloride initiator (ECPA, ethyl-2-chloro-2-phenylacetate) and a ruthenium catalyst system [RuCp*Cl(PPh3)2 / 4-DMAB; Cp*, pentamethylcyclopentadienyl; 4-DMAB, HO(CH2)4N(CH3)2] in EtOH at 40 ¡C (P1; Figure 1a; Table 1).30 PEG9MA was smoothly consumed up to 84%, and well-controlled poly(PEG9MA) was achieved with a narrow MWD [P1; Mn (SEC) = 7500, Mw/Mn (SEC) = 1.11]. Owing to the high functionality-tolerance of the catalytic system, PEG9MA and 2-hydroxyethyl methacrylate (HEMA) were copolymerized to yield well-controlled PEG9MA/HEMA random copolymers [P3; Figure 1b; Mn (SEC) = 6900, Mw/Mn (SEC) = 1.19].

Nevertheless, the polymerization of PEG4.5OHMA [PEGkOHMA;

CH2=C(CH3)COO(CH2CH2O)kH, k = 4.5 or 9] could not be controlled using the similar system (P4 Ð P8; Figure 1c; Table 1; Mn (SEC) = 20700 Ð 28700, Mw/Mn (SEC) = 1.43 Ð 1.91), indicating that the OH group on the PEG side chain behaved differently to the OH group of HEMA.44 This is thought to be because the OH groups of PEGOHMA interacted with the Ru catalyst in EtOH making the catalyst inappropriate for the polymerization.

;?>-!"

;@>-!#

!"#$ %&'() !' !*+!'

20-. /51 0833 4)44

0/-. /41+/71 7633 4)46

4,-. 851 8233 4)47

438 435 9:-;

438 435 9:-;

43,

;A>-!$

2,-. 581 ,/33 4)47 43,

;B>-!"%

,-. /01 23033 4)55

4-. 501 43633 4)25

438 435 9:-;

43,

46-. /21 25333 4),5 438 435 9:-;

43-. 731 4/433 4)20 43,

Figure 1. SEC curves of (a) P1, (b) P3, (c) P5, and (d) P12 prepared by Ru-LRP. Conditions: (a, b) PEG9MA / HEMA / ECPA / RuCp*Cl(PPh3)2 / 4-DMAB = 500 or 250 / 0 or 250 / 20 / 2.0 / 40 mM in EtOH at 40 ¡C;30 (c) PEG4.5OHMA / ECPA / RuCp*Cl(PPh3)2 / 4-DMAB = 500 / 20 / 2.0 / 40 mM in EtOH/H2O = 3/1 (v/v) at 40 ¡C;44 (d) PEG4.5OHMA / ECPA / RuCp*Cl(PPh3)2 / 4-DMAB = 250 / 10 / 1.0 / 5.0 mM in EtOH/H2O = 3/1 (v/v) at 25 ¡C.



Table 1.

Page 6 of 26

Synthesis of PEGylated Homo and Random Copolymers via Ru-LRPa [PEG Temperature

Code

PEG monomer

RMA

m / n / pb

[4-DMAB]

Time

Conversion

Mnd

Mw/Mnd

(mM)

(h)

(%)c

(SEC)

(SEC)

monomer]

Solvent (¡C)

(mM) P1

PEG9MA

P2

PEG4.5MA

P3

PEG9MA

P4

Ð

0/25/0

EtOH

40

500

40

27

84

7500

1.11

0/25/0

EtOH

40

500

40

27

86

7500

1.15

HEMA

0/12.5/12.5

EtOH

40

250

40

78

81/86

6900

1.19

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

40

500

0

26

26

N/Ae

N/Ae

P5

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

40

500

10

3

87

20700

1.44

P6

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

40

500

20

3

87

24300

1.48

P7

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

40

500

40

2

94

22500

1.72

P8

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

40

500

100

2

92

28000

1.91

P9

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

40

250

20

2

82

28700

1.41

P10

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

0

250

5

3

15

N/Ae

N/Ae

P11

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

0

250

20

3

19

N/Ae

N/Ae

P12

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

25

250

5

19

82

24000

1.34

P13

PEG4.5OHMA

Ð

25/0/0

EtOH/H2O

25

250

20

19

86

29400

1.34

P14

PEG4.5OHMA

BMA

25/0/10

EtOH/H2O

25

200

5

3.5

53/63

11000

1.49

P15

PEG4.5OHMA

BMA

25/0/20

EtOH/H2O

25

200

5

5

53/66

13900

1.49

a

P1 Ð P3: [PEG9MA] / [PEG4.5MA] / [HEMA] / [ECPA] / [RuCp*Cl(PPh3)2] / [4-DMAB] = 0, 250, or 500 / 0 or 500 / 0 or 250 / 20 / 2.0 / 40 mM in EtOH at 40 ¡C.30 P4 Ð P8: [PEG4.5OHMA] / [ECPA] / [RuCp*Cl(PPh3)2] / [4-DMAB] = 500 / 20 / 2.0 / 0 Ð 100 mM in EtOH/H2O (= 3/1, v/v) at 40 ¡C.45 P9 Ð P13: [PEG4.5OHMA] / [ECPA] / [RuCp*Cl(PPh3)2] / [4-DMAB] = 250 / 10 / 1.0 / 5 Ð 20 mM in EtOH/H2O (= 3/1, v/v) at 0 or 25 ¡C. P14 Ð P15: [PEG4.5OHMA] / [BMA] / [ECPA] / [RuCp*Cl(PPh3)2] / [4-DMAB] = 250 / 100 or 250 / 5.0 / 0.50 / 5 mM in EtOH/H2O (= 3/1, v/v) at 25 ¡C. PEGkMA, poly(ethylene glycol) methyl ether methacrylate [CH2=C(CH3)COO(CH2CH2O)kCH3; k = 4.5 or 9]; PEG4.5OHMA, poly(ethylene glycol) methacrylate [CH2=C(CH3)COO(CH2CH2O)4.5H]; HEMA, 2hydroxyethyl methacrylate [CH2=C(CH3)COO(CH2)2OH]; BMA, buthyl methacrylate; 4-DMAB, N,N-dimethylamino4-butanol [HO(CH2)4N(CH3)2]. b Targeted degree of polymerization at 100% (P1ÐP13) or 50% (P14, P15) monomer conversion: m = [PEG4.5OHMA]/[ECPA] (P1ÐP13), 0.5 ! [PEG4.5OHMA]/[ECPA] (P14, P15), n = [PEG9MA]/[ECPA], p = [HEMA]/[ECPA] (P3), 0.5 ! [BMA]/[ECPA] (P14, P15). ECPA, ethyl-2-chloro-2phenylacetate. c Monomer conversion determined by 1H NMR. d Number-average molecular weight (Mn) and distribution (Mw/Mn) determined by size-exclusion chromatography (SEC) in DMF ([LiBr] = 10 mM) with PMMA standards. e Could not be characterized by SEC because the polymerization did not proceed and stopped at low monomer conversion.

For the controlled polymerization, H2O was mixed with the EtOH solvent to weaken the interaction between the OH groups on PEGOHMA and the Ru catalyst, as well as to activate the Ru catalyst.44 Pure water was not used as a solvent owing to the strong hydrophobicity of monomers such as BMA and DMA, which


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means water would not support the preparation of amphiphilic copolymers. As anticipated, PEGOHMA could be polymerized by the ruthenium catalytic system in EtOH/H2O (= 3/1, v/v) at 25 ¡C to yield well-controlled poly(PEGOHMA) [P12; Figure 1d; Table 1; Mn(SEC) = 24000, Mw/Mn = 1.34]. The H2O content of the mixed solvent was an important factor: low H2O content could not weaken the interaction between PEGOHMA and the Ru catalyst sufficiently, and high H2O content excessively accelerated the polymerization so that there was no control over the catalytic cycle.44 In addition, reducing both the concentration of the monomers and the temperatureÑcompared with the polymerization conditions for PEGMAÑand adding aminoalcohol, enhanced the activity of the Ru catalyst (P4 Ð P13; Figure 1c, 1d; Table 1; [monomer] = 500 to 250 mM; temperature = 40 to 25 ¡C). These results suggest that PEGOHMA interacts more strongly than PEGMA with Ru catalyst, and that the interaction must be considered in the polymerization of PEGOHMA. As a result of our refinements, PEGOHMA was copolymerized with BMA using the Ru catalyst system. The ratio was set as m = PEGOHMA/chlorine (in ECPA) = 25 and p = BMA/chlorine = 10 or 20 to confirm the versatility of that system. Both monomers were smoothly consumed up to 53% (PEGOHMA) and 63% (BMA) (Figure S1). The time-conversion curves indicated that the polymerization was random copolymerization, and well-controlled amphiphilic random copolymers based on PEGOHMA were successfully synthesized [P14, P15; Figure S1; Table 1; Mn(SEC) = 11000 (P14), 13900 (P15); Mw/Mn (SEC) = 1.49 (P14), 1.49 (P15)]. 1H nuclear magnetic resonance (NMR) confirmed the success of the random copolymerization. The spectra clearly showed proton signals attributed to PEGOHMA and BMA, and the Mn(NMR) and DPs were determined and were almost the same as the targeted Mn and DPs [Figure S2; Mn(NMR) = 10500 (P14), 10600 (P15); m = 23 (P14), 23 (P15); p = 13 (P14), 22 (P15)].

Block Polymerization: Linear Diblock, Triblock, and Star-Shaped Copolymers To vary the polymer design based on PEGOHMA, amphiphilic block copolymers were synthesized. Following the polymerization of PEGOHMA as described (conv. ~87%, m = 25) for 16 h, BMA was added to the polymerization solution in situ. An excess of BMA, twice the targeted DP, was added (p = 5 or 25) and the polymerization was stopped when the conversion of BMA reached approximately 50% so as not to reduce the end-functionality through side reactions. BMA was smoothly consumed up to 53% to yield well-controlled AB-type amphiphilic diblock copolymers [P16, P17; Figure 2a, 2b; Table 2; Mw/Mn (SEC) = 1.38 (P16), 1.44 (P17)]. BMA was also polymerized in the same way following the random copolymerization of PEG9OHMA and PEG9MA [P18, P19; Figure 2c, 2d; Table 2; n = PEGMA/chlorine; Mw/Mn (SEC) = 1.27 (P18), 1.24 (P19)]. Using dichloroacetophenone (DCAP) as an initiator in lieu of ECPA, ABA-type amphiphilic triblock copolymers were also successfully synthesized [P20 Ð P22; Figure 2e, 2f; Table 2; RMA, BMA (P20, P21), DMA (P22); Mw/Mn (SEC) = 1.28 (P20), 1.25 (P21), 1.58 (P22)].



(b) P16

(a) P16 PEG4.5OHMA

Conversion, %

100 80

BMA

60

0 0

5

10 15 Time, h

20

Mn Mw/Mn

14 h 94%

9500 1.36

14 + 5 h 97% 55%

18400 1.38

106

105 104 MW (PMMA)

103

(d) P19

(c) P19 100 Conversion, %

Time Conv.

40 20

PEG9OHMA + PEG9MA

80 60

BMA

Time Conv.

Mn Mw/Mn

9.5 h 86%

16400 1.40

9.5 + 2.5 h 91% 49%

17300 1.24

40 20 0 0

2

4 6 8 Time, h

10 12

106

105 104 MW (PMMA)

103

(f) P21

(e) P21 PEG9OHMA + PEG9MA

100 Conversion, %

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 26

80 BMA

60

Time Conv.

Mn Mw/Mn

6h 85%

12800 1.22

6+3h 95% 55%

14200 1.25

40 20 0 0

2

4 6 8 Time, h

10 12

106

105 104 MW (PMMA)

103

Figure 2. Time-conversion (PEGOHMA, light blue; PEGMA, blue; BMA, orange) and SEC curves of (a, b) P16, (c, d) P19, and (e, f) P21 prepared by Ru-LRP in EtOH/H2O = 3/1 (v/v) at 25 ¡C. Conditions (ratio): (a, b) PEG4.5OHMA / ECPA / RuCp*Cl(PPh3)2 / 4-DMAB // BMAadd / 4-DMABadd = 25 / 1 / 0.1 / 0.5 // 10 / 0.5; (c, d) PEG9OHMA / PEG9MA / ECPA / RuCp*Cl(PPh3)2 / 4-DMAB // BMAadd / 4DMABadd = 5 / 20 / 1 / 0.1 / 0.5 // 10 / 0.5; (e, f) PEG9OHMA / PEG9MA / DCAP / RuCp*Cl(PPh3)2 / 4DMAB // BMAadd / 4-DMABadd = 5 / 20 / 1 / 0.1 / 0.5 // 20 / 0.5.


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Table 2.

Synthesis and Characterization of Amphiphilic Block and Nanogel-Core Star Polymersa Time

Conversion

Mnd

Mw/Mnd

m/n/pobsde

Mne

(h)

(%)c

(SEC)

(SEC)

(NMR)

(NMR)

25/0/5

19

97/Ð/55

18400

1.38

35/0/8.2

13800

25/0/25

19

96/0/44

20800

1.44

35/0/29

16900

2nd Code

Structure

1st monomer

3rd monomer

m/n/pb

monomer P16

diblock

PEG4.5OHMA

BMA

Ð

P17

diblock

PEG4.5OHMA

BMA

P18

diblock

PEG9OHMA/PEG9MA

BMA

Ð

40/10/5

9.5

95/95/36f

22800

1.27

34/10/6.4

23000

P19

diblock

PEG9OHMA/PEG9MA

BMA

Ð

20/5/5

12

91/91/49f

17300

1.24

24/6.2/5.4

16100

P20

triblock

PEG9OHMA/PEG9MA

BMA

Ð

40/10/5

9

91/91/44f

23700

1.28

28/11/8.5

18300

P21

triblock

PEG9OHMA/PEG9MA

BMA

Ð

20/5/5

9

95/95/55f

14200

1.25

18/4.4/10

12500

P22

triblock

PEG4.5OHMA

DMA

Ð

75/0/5

15

94/0/67.4

29300

1.58

60/0/5.8

23400

P23

triblock

PEG4.5MA

BMA

PEG4.5OHMA

25/25/10

50

55/100/100

36300

9.28

Ð

Ð

P24

triblock

PEG4.5MA

BMA

PEG4.5OHMA

25/25/25

50

48/87/100

40400

11

Ð

Ð

P25

triblock

PEG4.5MA

BMA

PEG4.5OHMA

25/25/10

53

53/100/82

23000

3.01

Ð

Ð

P26

triblock

PEG4.5MA

BMA

PEG4.5OHMA

25/25/25

53

45/100/89

22000

1.80

Ð

Ð

P27

triblock

PEG4.5OHMA

BMA

PEG4.5MA

25/25/20

39

100/0/69

N/A

N/A

Ð

Ð

P28

triblock

PEG4.5OHMA

BMA

PEG4.5MA

25/25/20

36

100/0/80

N/A

N/A

Ð

Ð

P29

triblock

PEG4.5OHMA

BMA

PEG4.5MA

25/10/12.5

14

100/54/86

20900

1.43

35/12/13

18300

P30

triblock

PEG4.5OHMA

BMA

PEG4.5MA

25/10/25

14

100/60/88

24100

1.44

34/11/29

20000

P31

triblock

PEG4.5OHMA

BMA

PEG4.5MA

25/25/12.5

22

100/50/79

27500

1.49

35/31/14

24400

P32

triblock

PEG4.5OHMA

BMA

PEG4.5MA

25/25/25

19

100/50/79

30800

1.55

35/29/31

25900

P33

star

PEG4.5OHMA

EGDMA

Ð

25/0/5

21

100/0/90

30100

4.06

Ð

Ð

P34

star

PEG4.5OHMA

EGDMA

Ð

25/0/10

21

100/0/89

41400

2.23

Ð

Ð

P35

star

PEG4.5OHMA

EGDMA

Ð

25/0/25

22

100/0/93

88000

14.8

Ð

Ð

P36g

diblock

PEG9MA

Ð

0/25/5

59

0/88/65

13700

1.09

0/25/6.4

13600

Ð

0/25/25

58

0/96/49

11600

1.16

0/26/19

15700

Ð

0/25/5

40

0/85/36

18000

1.13

0/39/5.3

20700

BMA/RhB MA BMA/RhB P37g

diblock

PEG9MA MA

P38h

Biotinylated

PEG9MA

BMA

a

The detailed procedures are shown in the supplementary information, and the representative conditions were as follows. P16 (ratio): PEG4.5OHMA1st / ECPA1st / RuCp*Cl(PPh3)2,1st / 4-DMAB1st // BMAfinal / 4-DMABfinal = 25 / 1 / 0.1 / 0.5 // 50 / 0.5 in EtOH/H2O (= 3/1, v/v) at 25 ¡C. P18 (ratio): PEG9OHMA1st / PEG9MA1st / ECPA1st / RuCp*Cl(PPh3)2,1st / 4DMAB1st // BMAfinal = 40 / 10 / 1 / 0.1 / 0.5 // 5 in EtOH/H2O (= 3/1, v/v) at 25 ¡C. P20 (ratio): PEG9OHMA1st / PEG9MA1st / DCAP1st / RuCp*Cl(PPh3)2,1st / 4-DMAB1st // BMAfinal = 40 / 10 / 1 / 0.2 / 1 // 20 in EtOH/H2O (= 3/1, v/v) at 25 ¡C. P26 (ratio): PEG4.5MA1st / ECPA1st / RuCp*Cl(PPh3)2,1st / 4-DMAB1st // BMA2nd / 4-DMAB2nd // PEG4.5OHMAfinal = 25 / 1 / 0.1 / 2 // 25 / 40 // 50 in EtOH (1st, 2nd polymn.) and EtOH/H2O (= 3/1, v/v; final polymn.) at 25 ¡C. P32 (ratio): PEG4.5OHMA1st / ECPA1st / RuCp*Cl(PPh3)2,1st / 4-DMAB1st // BMA2nd / 4-DMAB2nd // PEGMAfinal = 25 / 1 / 0.5 / 0.2 / 4 // 20 // 50 in EtOH/H2O (= 3/1, v/v) at 25 ¡C. P33 (ratio): PEG4.5OHMA1st / ECPA1st



Page 10 of 26

/ RuCp*Cl(PPh3)2,1st / 4-DMAB1st // EGDMAfinal / 4-DMABfinal = 25 / 1 / 0.1 / 0.5 // 5 / 0.5 in EtOH/H2O (= 3/1, v/v) at 25 ¡C. b Targeted degree of polymerization at 100% (1st or 2nd) or 50% (final) monomer conversion: DP = [monomer]/[chlorine] (1st or 2nd monomer), 0.5 ! [final monomer]/[chlorine] (final monomer; 2nd or 3rd monomer). c Monomer conversion determined by 1H NMR (PEGkOHMA / PEGkMA / RMA). d Number-average molecular weight (Mn) and distribution (Mw/Mn) determined by size-exclusion chromatography (SEC) in DMF ([LiBr] = 10 mM) with PMMA standards. e Observed DPs and absolute number-average molecular weight [Mn (NMR)] determined by 1H NMR. f Total conversion of PEGOHMA and PEGMA. g RhBMA, a methacrylate monomer bearing RhB (see Scheme S2). h see Scheme S3.

In general, it is difficult to synthesize block copolymers that comprise more than three segments by sequential in situ addition of monomers and the order of monomer addition is an important factor. Indeed, PEG4.5MAn-block-BMAp-block-PEG4.5OHMAmÐCl could not be well controlled, and the 3rd block polymerization of PEG4.5OHMA did not proceed efficiently (P23 Ð P26; Figure 3a, 3b; Table 2). In comparison,

well-controlled

ABC-type

triblock

copolymers

(PEG4.5OHMAm-block-BMAp-block-

PEG4.5MAnÐCl) were efficiently prepared: BMA and PEG4.5MA were sequentially added to the reaction mixture in the stated order following the polymerization of PEG4.5OHMA with the catalyst system in EtOH/H2O = 3/1 at 25 ¡C [P29 Ð P32; Figure 3c, 3d; Table 2; Mw/Mn (SEC) = 1.43 Ð 1.55]. This was thought to be because in situ addition of water at the time of PEG4.5OHMA addition could affect the Ru catalytic system. In contrast, water in the reaction mixture from the polymerization of the 1st monomer (PEG4.5OHMA) would afford the appropriate catalyst for the block polymerization. When analyzed by 1H NMR, the block copolymers exhibited proton signals that could be attributed to PEGOHMA, PEGMA, and BMA. The Mn(NMR) and DPs were determined from the area of each peak against the phenyl end group and corresponded to the targeted Mn and DPs [Figure 4; Table 2; Mn(NMR) = 20900 Ð 30800; m = 34 Ð 35; n = 11 Ð 31; p = 13 Ð 31].


Page 11 of 26

(a) P26

(b) P26 Cl

R O

O 25 O

O

25

O

O

4.5

O 25 O H

PEG4.5MA

Conversion, %

Time Conv.

Mn Mw/Mn

27 h 86%

7500 1.11

4.5

100 80

BMA

27 + 24 h 95% PEG4.5OHMA 73%

60

10000 1.11

40 51 + 2 h 100%, 89% 45%

20 0 40 20 Time, h

0

60

(c) P32

106

22000 1.80

105 104 103 MW (PMMA)

(d) P32 Cl

R O 25 O H

O

O O

25

O

4.5

O 25 O

Time Conv.

Mn Mw/Mn

9h 88%

17200 1.44

9+7h 94% 76%

21400 1.43

16 + 3 h 100%, 79% 50%

30800 1.55

4.5

PEG4.5OHMA 100 BMA Conversion, %

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


80 PEG4.5MA

60 40 20 0 0

5

10 15 Time, h

20

106

105 104 103 MW (PMMA)

Figure 3. Time-conversion (PEGOHMA, light blue; PEGMA, blue; BMA, orange) and SEC curves of (a, b) P26 and (c, d) P32 prepared by Ru-LRP at 25 ¡C. Conditions (ratio): (a, b) PEG4.5MA1st / ECPA1st / RuCp*Cl(PPh3)2,1st / 4-DMAB1st // BMA2nd / 4-DMAB2nd // PEG4.5OHMAfinal = 25 / 1 / 0.1 / 2 // 25 / 3 // 50 in EtOH (1st, 2nd polymn.) and EtOH/H2O (= 3/1, v/v; final polymn.); (c, d) PEG4.5OHMA1st / ECPA1st / RuCp*Cl(PPh3)2,1st / 4-DMAB1st // BMA2nd / 4-DMAB2nd // PEG4.5MAfinal = 25 / 1 / 0.5 / 0.2 / 4 // 20 // 50 in EtOH/H2O (= 3/1, v/v).



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Page 12 of 26

Page 13 of 26 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


Finally, gel-core star polymers (P33 Ð P35) were synthesized to vary the polymer design based on PEGOHMA.18Ð20,45 PEG4.5OHMA was first polymerized with RuCp*Cl(PPh3)2, 4-DMAB, and ECPA in EtOH/H2O = 3/1 (v/v) at 25 ¡C (m = 25). After the polymerization of PEG4.5OHMA (conv. ~ 88%, 10 h), ethylene glycol dimethacrylate (EGDMA) was added to the solution in situ (p = EGDMA/chlorine = 5, 10, or 25), and the cross-linking reaction proceeded in accordance with the consumption of EGDMA (Figure S3, S4). The best ratio of EGDMA was p = 5 to yield star polymers in high yield (82%) calculated from the area of the SEC curve (P33), however the best ratio was p " 10 in the case of poly(PEG9MA) arm polymers.45 This is thought to be because the steric hindrance of poly(PEG4.5OHMA)25 arm polymers was larger than that of poly(PEG9MA) arm polymers and because the hydration of poly(PEG4.5OHMA) affected the star polymer synthesis. In summary, precision polymerization of amphiphilic random, block, and star copolymers as well as the homopolymer based on PEGOHMA, were successfully achieved by our refinement of the system. Owing to the high block efficiency, ABC-type triblock, as well as AB-type and ABA-type block copolymers were efficiently synthesized, and the amphiphilic copolymers based on PEGOHMA will contribute to the further development of PEGylated polymeric materials.

LCST-Type Phase Separation in Water PEGylated polymeric materials are generally soluble in water owing to their hydrophilicity and they often exhibit LCST-type phase separation. Indeed, poly(PEG4.5MA)25 (P2) showed phase separation in water, with a cloud point (Cp) of 77 ¡C, which depended on the length of PEG side chains (Figure 5a).13,14 Cps were determined from the transmittance of the solution monitored by UV/Vis spectroscopy at ! = 670 nm (heating/cooling rate = 1 ¡C/min), and Cp was defined as the temperature at which the transmittance became 90%.13,14,46 Poly(PEG4.5OHMA)25 (P12) did not exhibit any LCST-type phase separation in water, however poly(PEG4.5MA)25 did because the hydrophilicity of PEGOHMA was strong as a result of the OH groups on the PEG side chains. In addition, amphiphilic random (P14) and block (P16) copolymers based on PEGOHMA exhibited LCST-type phase separation in water, and the Cps were 74 and 98 ¡C, respectively (Figure 5b). These results suggest that the solution properties could be controlled not only by the PEG length, but also by the OH groups on the PEG side chains.



Transmittance, %

(a) Homopolymers PEG4.5OHMA25 (P12) 100 80 60 40

PEG4.5MA25 (P2)

20 0

Transmittance, %

20 30 40 50 60 70 80 90 100 Temperature, oC (b) Copolymers P16 100 80 60 40 P14 20 0 20 30 40 50 60 70 80 90 100 Temperature, oC (c) Solvents PBS 100 Pure H2O HEPES 80 Transmittance, %

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 26

60 40 20 0 20 30 40 50 60 70 80 90 100 Temperature, oC

Figure 5. Transmittance of the aqueous solutions of (a) poly(PEG4.5MA) (black, P2) and poly(PEG4.5OHMA) (red, P12), (b) P14 (black) and P16 (red), and (c) the aqueous (black), PBS (blue) and HEPES (red) buffer solutions of P14 monitored at 670 nm by changing temperature (heating/cooling rate = 1 ¡C/min) from 20 to 100 ¡C; [polymer] = 1.0 mg/mL.


Page 15 of 26 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


Finally, the effect of buffering agents on LCST-type phase separation was investigated using P14. As shown in Figure 5c, the PBS (phosphate buffered saline; pH = 7.4) and HEPES buffer solution [HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; pH = 7.5] of P14 showed LCST-type phase separation, and the Cps were 65 ¡C (PBS) and 59 ¡C (HEPES), respectively. Therefore, Cp depended on the buffering agent, and the Cps were lower in the buffers tested than in pure water (Figure 5c). This is thought to be because the structure of the hydration water surrounding P14 on the molecular scale was different, owing primarily to the interaction of the buffering agents with the PEGOH side chains.

Self-Assembly of Amphiphilic Copolymers in Water The self-assembled structures of the PEGOHMA-based amphiphilic block copolymers were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The hydrodynamic radii (RH) of P16 and P17 in PBS were 15 and 98 nm, respectively (Figure 6a). In comparison, the size of PEGMA-based block copolymers containing the same BMA content (P36, P37; Scheme S1) was smaller than those of P16 and P17, and the RHs were 2.7 and 10 nm, respectively (Figure 6b). P36 in particular existed as a unimer in PBS and did not form assembled structures.36 Their self-assembled structures were also characterized by TEM (Figure 6c, d). Spherical micelle structures were confirmed for P16, P17, and P37, whose sizes approximately corresponded to the DLS results. In contrast, apparent micellar structure was not observed in the case of P36 owing to the unimer structure in water. As a consequence, PEGOHMA-based amphiphilic block copolymers formed spherical micelles in water, and their sizes tended to be larger than the micelles of PEGMA-based amphiphilic block copolymers. This is also thought to be because the hydrated structures of the PEG side chains were different owing to the presence of the OH groups.



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Page 16 of 26

Page 17 of 26

disrupted by the faster shear rate. Compared with P2 and P12, the shear stress and viscosity of P33 gradually increased and decreased, respectively. The intermolecular hydrogen bonding gradually weakened with increasing shear rate. In addition, both shear stress and viscosity discontinuously decreased around at 100 sÐ1, indicating that the aggregate structure in the bulk state collapsed.50 The shear viscosity of P33 decreased over 150 sÐ1 because starÐstar interaction decreased with increasing shear rate.

(a)

5

(b)

5

5

4

P33

), Pa s

2 P12 1 P2

0

0

Ð1

Ð1

Ð2 Ð2

0 1 log d#/dt, sÐ1

2

3 2 2

0

(d)

6

1

1

3

0 Ð1

0

1 log #, %

2

3

Ð2

Ð1

0 log %, rad/s

1

2

3

5 2 )

4 3

log tan $ (

log GÕ (!), log GÕÕ ("), Pa

(c)

Ð1

3

)

1

4

log tan $ (

log ! (

3

3 2

log GÕ (!), log GÕÕ ("), Pa

4

4

log " (!), Pa

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


2 1 1

1

0

0

2

Ð1

Ð1 Ð2

Ð1

0 log %, rad/s

1

2

Figure 7. Viscoelastic properties of P2 (black), P12 (blue), and P33 (green) in the bulk state at 25 ¡C. (a) Shear stress (", filled circle) and viscosity (#, filled square) of the copolymers as a function of shear rate (d$/dt). Shear storage (G%, unfilled circle) and loss (G&, unfilled square) moduli, and loss tangent (tan ', unfilled triangle) of the copolymers as a function of (b) strain ($) and (c, d) frequency (() at 1 Hz.



To investigate the linearity of the viscoelasticity of the copolymers, the shear storage and loss moduli (G% and G&) were characterized by changing the strain (0.1 < $ < 1000%) at 25 ¡C (Figure 7b). G& of P2 was constant over the entire range despite G% not being detected because P2 was too soft. Therefore, the behavior of P2 was linear viscoelasticity over the whole range. P33 also showed linear viscoelasticity under infinitesimal deformation conditions because both G% and G& of P33 were constant. As the strain was increased, both G% and G& of P33 decreased. These results suggested that the local structure collapsed on the molecular scale. In comparison, P12 exhibited non-linear viscoelasticity even under infinitesimal deformation because G% and loss tangent (tan ') decreased and increased over the range, respectively. This supports the observation that the elongation resulting from the intermolecular hydrogen bonding as well as the anisotropic orientation of the polymer chain, must be considered even in the case of infinitesimal deformation, yet the non-linearity of viscoelasticity regarding graft polymers is generally weaker than that of linear polymers. Finally, linear viscoelasticity was characterized by changing the angular frequency (0.01 < ( < 1000 sÐ1) and keeping the strain constant ($ = 1%) at 25 ¡C (Figure 7c, d). P12 exhibits slight non-linear viscoelasticity even under these conditions, however, P12 was characterized under the same conditions to compare the behavior. The slopes of G% and G& for P2 were 2 and 1, respectively, at higher ( (log G% = 1.971 log ( Ð 2.1196; log G& = 1.015 log ( + 0.2624), therefore, the molecular dynamics completely relaxed and the polymer chains did not entangle with each other. From the results of the terminal relaxation, the zero-shear viscosity (#0), the steady-state compliance (Je), and the second-moment average relaxation time (w,G i

(nm)

(País)

(mPaÐ1)

(ms)

f

P2

Homo

PEG4.5MA

25/0

14,500

16,700

Ð

Ð

Ð

1.83

2.27

4.15

P12 j

Homo

PEG4.5OHMA

25/0

14,200

19,000

Ð

Ð

Ð

N/A

N/A

N/A

P33

Star

PEG4.5OHMA

25/5

Ð

Ð

1,420,000

141

42

n.d. k

n.d. k

n.d. k

a

Viscoelastic properties including zero-shear viscosity (#0), steady-state compliance (Je), and second-moment average relaxation time (w,G) determined by #0 and Je:

Amphiphilic Poly[poly(ethylene glycol) methacrylate] - ACS Publications

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