Reductively Responsive Gel Capsules Prepared Using a Water

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

Reductively Responsive Gel Capsules Prepared Using a Water-soluble Zwitterionic Block Copolymer Emulsifier Hiroshi Nakaura, Akifumi Kawamura, and Takashi Miyata Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01608 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 25, 2018

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Reductively Responsive Gel Capsules Prepared Using a Water-soluble Zwitterionic Block Copolymer Emulsifier Hiroshi Nakaura,† Akifumi Kawamura,*,†,‡ Takashi Miyata*,†,‡ †Department of Chemistry and Materials Engineering and ‡Organization for Research and Development of Innovative Science and Technology, Kansai University, 3-3-35, Yamate-cho, Suita, Osaka 564-8680, Japan.

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ABSTRACT

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Utilizing the unique solubility of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC),

3

which is soluble in only water and alcohol, a water-soluble block copolymer emulsifier

4

composed of a hydrophilic PMPC block and an amphiphilic poly[oligo(ethylene glycol)

5

methacrylate] (POEGMA) block was synthesized via reversible addition-fragmentation chain

6

transfer (RAFT) polymerization. Water-in-oil (W/O) emulsions were successfully formed in the

7

presence of the resulting PMPC-b-POEGMA, which acted as a stabilizer of water droplets in a

8

chloroform continuous phase because the PMPC and POEGMA blocks were distributed to the

9

water and chloroform phases, respectively. Next, the amphiphilic poly[poly(ethylene glycol)

10

methacrylate] (PPEGMA) gel layer, which contained bis(2-methacryloyl)oxyethyl disulfide as a

11

reducing-environment-responsive cross-linker, was prepared by inverse miniemulsion periphery

12

RAFT polymerization from the PMPC-b-POEGMA that stabilized the W/O emulsions. The

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resulting PPEGMA gel capsules were colloidally stable in not only chloroform but also water

14

without additional hydrophilic surface modification. The drug-release behavior from the

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PPEGMA gel capsules in response to dithiothreitol (DTT), which is a reducing agent, was

16

investigated using fluorescein-conjugated dextran (FITC-Dex) as a model drug. The FITC-Dex

17

release rate from the gel capsules in a phosphate buffer solution (pH 7.4, 20 mM) with DTT was

18

fast compared to that without DTT. The reducing-environment-responsive FITC-Dex release is

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attributed to the cleavage of disulfide bonds that act as cross-links in the PPEGMA gel layer. The

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fascinating properties of the PPEGMA gel capsules suggest that they can provide a useful

21

platform for designing drug carriers for protein and gene delivery and nanobioreactors.

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INTRODUCTION

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Zwitterionic polymers are of great interest because they show various fascinating properties,

3

such as high water-solubility, low protein adsorption, and high lubrication. The unique properties

4

of zwitterionic polymers enable synthesis of biocompatible materials1-2 and low-friction

5

materials3-4. Utilizing the extreme water solubility of zwitterionic polymers, various polymer

6

surfactants have been designed via copolymerization of zwitterionic monomers with

7

hydrophobic monomers. For example, an amphiphilic random copolymer, poly(MPC-co-n-

8

butylmethacrylate) (PMB-30) with 0.30 unit mole fraction of MPC, can solubilize paclitaxel,

9

which is a hydrophobic anticancer drug, because amphiphilic PMB-30 acts as a surfactant5.

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Amphiphilic block copolymers having a zwitterionic block as a hydrophilic block were also

11

synthesized for the formation of polymer assemblies, such as micelles and vesicles

12

(polymersomes)6-7. The micelles and polymersomes formed from the amphiphilic block

13

copolymers have been extensively studied for constructing carriers for drug delivery systems8-9.

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Recently, hollow polymeric nanocapsules having an inner water phase have attracted

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considerable attention because they can encapsulate and deliver water-soluble drugs and

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biocatalysts. The hollow polymeric nanocapsules are prepared by the colloidal template method,

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which involves the coating of sacrificial colloidal templates with polymers through the layer-by-

18

layer method and surface-initiated polymerization, followed by the stabilization of the polymer

19

shell via a cross-linking reaction10-11. Subsequently, the colloidal templates are removed to obtain

20

hollow polymeric nanoparticles. Whereas this is a popular method for preparing hollow

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polymeric nanocapsules, the encapsulated compounds are extremely limited because

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preservation of their structures and functions are required even after the template removal

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process. The cross-linking of the polymersome is another popular method for preparing hollow


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polymeric nanocapsules12-13. However, the low encapsulation efficiency of drugs is a barrier to

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the practical use of cross-linked polymersomes as drug carriers and nanobioreactors.

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On the other hand, Zetterlund and Martina et al. have reported the novel design of hollow

4

polymeric nanocapsules having an inner water phase in which water-soluble drugs, such as

5

proteins and anticancer drugs, were encapsulated14-16. The hollow polymeric nanoparticles were

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prepared using inverse miniemulsion periphery reversible addition fragmentation chain transfer

7

polymerization (IMEPP). After the formation of W/O emulsions using an amphiphilic block

8

copolymer, which was synthesized via reversible addition fragmentation chain transfer (RAFT)

9

polymerization, the monomers and cross-links were copolymerized in the organic continuous

10

phase from the surface of the W/O emulsion to obtain hollow polymeric nanocapsules. The

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preparative method for hollow polymeric nanocapsules via IMEPP is an outstanding strategy

12

because it can encapsulate water-soluble drugs into the nanocapsules with high efficiency.

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However, further modification is required for rendering the capsule membranes hydrophilic,

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because the capsule membrane prepared in an oil phase has a hydrophobic surface that prevents

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nanocapsules from stable dispersion in an aqueous medium.

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To design water-dispersible hollow gel capsules, we have focused on the characteristic

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dissolution properties of PMPC and poly(ethylene glycol) (PEG). PEG is a well-known polymer

18

with many applications, such as surfactants, pharmaceutics, and biomaterials. PEG is dissolved

19

in not only water but also various organic solvents, such as alcohol, chloroform, tetrahydrofuran,

20

benzene, and toluene. In particular, PEG is preferentially distributed in the organic phase when

21

PEG is added to a water-organic solvent two-phase system, such as a water-chloroform system.

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On the other hand, PMPC does not dissolve in most organic solvents but dissolves in water and

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alcohol. Based on the unique solubility of PMPC and PEG, we designed novel emulsifier


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composed of water-soluble block copolymer containing a PMPC block and a poly[oligo(ethylene

2

glycol) methacrylate] (POEGMA) block with an oligo(ethylene glycol) side chain. The block

3

copolymer, PMPC-b-POEGMA, is expected to act as an emulsifier in a water-organic solvent

4

two-phase system because PMPC and POEGMA are distributed to the water phase and organic

5

phase, respectively. In this study, we investigated the formation of W/O emulsions using PMPC-

6

b-POEGMA and prepared the water-dispersible gel capsules via IMEPP of poly(ethylene glycol)

7

methacrylate (PEGMA) and disulfide cross-links from the surface of the W/O emulsion (Scheme

8

1). The gel capsules can be highly dispersed in not only the organic phase but also water, without

9

additional modification for rendering their surface hydrophilic, owing to the amphiphilicity of

10

poly(PEGMA) gel layer. This paper also describes the release of encapsulated model drugs from

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the gel capsules in response to a reducing environment. The fundamental research on the

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preparation of water-dispersible reducing-environment-responsive gel capsules with an inner

13

water phase will contribute significantly to creating smart drug carriers for protein delivery

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systems and nanobioreactors.

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EXPERIMENTAL METHODS

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Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was kindly provided by NOF

18

Corporation (Tokyo, Japan). Oligo(ethylene glycol) methacrylate (OEGMA, Mn: 300),

19

poly(ethylene glycol) methacrylate (PEGMA, Mn: 1020), 4-cyano-4-(thiobenzoylthio) pentanoic

20

acid (CTPA), bis(2-methacryloyl)oxyethyl disulfide (BMOD), and fluorescein-conjugated

21

dextran (FITC-Dex, Mw: 40 kDa) were purchased from Millipore Sigma (St. Louis, MO). 4,4-

22

Azobis(4-cyanovaleric acid) (ACVA), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70),

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and dithiothreitol (DTT) were purchased from WAKO Pure Chemical Industries (Osaka, Japan).


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OEGMA and PEGMA were purified through a column of basic alumina prior to use for removal

2

of the inhibitor. All aqueous solutions were prepared with ultrapure water (Milli-Q, 18.2

3

MΩ·cm). The other solvents were obtained from commercial sources and used without further

4

purification.

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Synthesis of PMPC macroRAFT agent. MPC (2.36 g, 8.0 mmol), CTPA (223.5 mg, 0.8

6

mmol), and ACVA (112 mg, 0.4 mmol) were dissolved in 4 mL of ethanol. The solution was

7

deoxygenated by five cycles of freeze-pump-thaw, followed by stirring at 90 °C for 8 h. Next,

8

the reaction solution was poured into a seamless cellulose tubing (molecular weight cutoff: 1

9

kDa) and was dialyzed against methanol for purification. The solvent was removed under

10

reduced pressure to obtain the PMPC macroRAFT agent.

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Synthesis of PMPC-b-POEGMA. OEGMA (4.05 g, 13.5 mmol), the PMPC macroRAFT

12

agent (650 mg, 0.1 mmol), and ACVA (5.6 mg, 0.02 mmol) were dissolved in 10 mL of ethanol.

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The solution was deoxygenated by five cycles of freeze-pump-thaw, followed by stirring at 90

14

°C for 10 h. Next, the reaction solution was poured into a seamless cellulose tubing (molecular

15

weight cutoff: 3.5 kDa) and was dialyzed against methanol for purification. The solvent was

16

removed under reduced pressure to obtain PMPC-b-POEGMA.

17

Formation of W/O emulsion using PMPC-b-POEGMA as an emulsifier. First, 10, 50, and

18

100 mg of PMPC-b-POEGMA was dissolved in 1 mL of phosphate buffer (PB) solution (pH 7.4,

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20 mM) with 5 mg of fluorescein. Next, 10 mL of chloroform was added to the PB solution

20

containing PMPC-b-POEGMA. The water-chloroform mixture was sonicated using bath-type

21

sonicator (UT-105HS, Output: 100 %, SHARP, Osaka, Japan) for 1 h to obtain W/O emulsions.


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The resulting W/O emulsions were observed using a fluorescence microscope (BX51, Olympus

2

corp., Tokyo, Japan).

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Preparation of gel capsules via IMEPP. PMPC-b-POEGMA (100 mg, 1.7×10-7 mol) was

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dissolved in 1 mL of PB solution as a water phase. OEGMA (Mn: 300) (24 mg, 8.0×10-5 mol) or

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PEGMA (Mn: 1020) (76 mg, 8.0×10-5 mol) and BMOD (4.9 mg, 1.7×10-5 mol) were dissolved in

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9 mL of chloroform. The chloroform solution was deoxygenated by bubbling Ar gas for 30 min.

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Next, this solution was added to the PB solution containing PMPC-b-POEGMA. The water-

8

chloroform mixture was sonicated (output: 100 %) for 1 h to form a W/O emulsion. The

9

resulting emulsion was transferred into a two-necked flask filled with Ar gas. Next, 1 mL of

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chloroform solution containing V-70 (8.5×10-5 M), which was deoxygenated by bubbling Ar gas

11

for 30 min, was added to the emulsion. Polymerization was performed at 40 °C for 6 h. After the

12

addition of 10 mL of water to the reaction mixture, the chloroform was removed under reduced

13

pressure to obtain a gel capsule dispersion. The FITC-Dex-loaded gel capsules were prepared via

14

the same method, using a PB solution containing FITC-Dex as the dispersed water phase of the

15

W/O emulsion.

16

Release of FITC-Dex from gel capsules. The FITC-Dex release from the gel capsules was

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investigated using the dialysis method. A PB solution containing FITC-Dex-loaded gel capsules

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was transferred into a cellulose tubing (molecular weight cutoff: 1,000 kDa). The FITC-Dex-

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loaded gel capsules were dialyzed against 100 mL of PB solution with and without dithiothreitol

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(DTT). The FITC-Dex release were carried out at 25 °C in a thermostatic chamber. The

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concentration of FITC-Dex in the PB solution was determined using the calibration curve

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prepared using a standard FITC-Dex solution.


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Size exclusion chromatography (SEC) measurements. SEC measurements were conducted

2

using a TOSOH modular system (Tosoh corp., Tokyo, Japan) equipped with TSKgel

3

G6000PWXL and TSKgel G3000PWXL (Tosoh Bioscience, Tokyo, Japan) at 30 °C under a flow

4

rate of 0.5 mL/min using a refractive index detector. A PB solution (20 mM, pH 7.4) was used as

5

an eluent. The Mn, Mw, and Mw/Mn (Ð) were calculated using near-monodisperse poly(ethylene

6

glycol) standards.

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Dynamic light scattering (DLS) measurements. DLS measurements were conducted using

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an ELS-Z1000 spectrometer (Otsuka Electronics Co., Ltd., Osaka, Japan) equipped with a He-Ne

9

laser (λ = 633.8 nm) at 25 °C. The detection angle was fixed at 165°. The diameter and

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polydispersity index were calculated using the cumulant method, and the size distribution was

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obtained via the Marquart analysis.

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RESULTS AND DISCUSSION

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Synthesis of PMPC-b-POEGMA as an emulsifier via RAFT polymerization. The

15

preparation of gel capsules via miniemulsion periphery RAFT polymerization from the surface

16

of the template W/O emulsion requires the design of the block copolymer emulsifier having a

17

RAFT agent at the terminus of the lipophilic block that is distributed to a continuous oil phase.

18

Therefore, a PMPC macroRAFT agent was synthesized through RAFT polymerization of MPC,

19

followed by the polymerization of OEGMA using the resulting PMPC macroRAFT agent to

20

obtain PMPC-b-POEGMA. The PMPC macroRAFT agent with a narrow molecular weight

21

distribution was successfully synthesized via RAFT polymerization of MPC (Figure 1).

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Furthermore, the diblock copolymer, PMPC-b-POEGMA, was synthesized via RAFT


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polymerization of OEGMA in the presence of the resulting PMPC macroRAFT agent. The

2

molecular weight distribution of the PMPC macroRAFT agent was shifted toward a higher

3

molecular weight through the RAFT polymerization of OEGMA using the PMPC macroRAFT

4

agent. In addition, a unimodal molecular size distribution of PMPC-b-POEGMA without a

5

significant peak of the PMPC macroRAFT agent indicates the successful chain extension of the

6

POEGMA block from the PMPC macroRAFT agent. The synthetic results of the PMPC

7

macroRAFT agent and PMPC-b-POEGMA are summarized in Table 1. The synthesized PMPC-

8

b-POEGMA had 22 repeating units of MPC and 171 repeating units of POEGMA.

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Preparation of W/O emulsions using PMPC-b-POEGMA emulsifier. PEG is used as a

11

hydrophilic block in conventional nonionic emulsifiers, such as Tween 20 and Span 80.

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However, PEG is dissolved in not only water but also various polar and apolar organic solvents,

13

such as alcohol, chloroform, tetrahydrofuran, dimethylsulfoxide, dimethylformamide, benzene,

14

and toluene17. When PEG is added to a water-chloroform mixture as a water-organic solvent

15

two-phase system, PEG is preferentially distributed in the chloroform phase. Such unique

16

amphiphilicity of PEG indicates that it acts as not only a hydrophilic component but also a

17

lipophilic component of the emulsifier. On the other hand, zwitterionic PMPC shows extremely

18

limited solubility. PMPC is easily dissolved in water and alcohol but insoluble in most organic

19

solvents, such as acetone, acetonitrile, and tetrahydrofuran18-19. These unique properties of PEG

20

and PMPC led us to consider that the block copolymer composed of PMPC and POEGMA

21

blocks with a PEG side chain acts as an emulsifier in a water-organic solvent two-phase system

22

because the PMPC and POEGMA blocks are preferentially distributed in the water and organic

23

phases, respectively. Therefore, we formed the W/O emulsion using PMPC-b-POEGMA as an


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emulsifier in the water-chloroform mixture. Upon sonication of the water-chloroform mixture

2

containing PMPC-b-POEGMA for 1 h, the mixture became milky in appearance. Figure 2 shows

3

the microscope images of the resulting milky mixture prepared using 10 % (w/v) of PMPC-b-

4

POEGMA in the dispersed phase. The submicron-sized droplets were observed on phase contrast

5

images. In addition, fluorescence of the fluorescein for staining the water phase was observed in

6

the inner portion of the droplets. These results indicate that W/O emulsions were successfully

7

formed by the sonication of the water-chloroform mixture containing PMPC-b-POEGMA

8

because PMPC-b-POEGMA stabilized the water droplets as an emulsifier at the water-

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chloroform interfaces.

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In general, the concentration of the emulsifier strongly affects the droplet size and stability of

11

emulsions. Optimization of the preparative condition is necessary for forming stable W/O

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emulsions that act as a template for the preparation of gel capsules via IMEPP. Therefore, we

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investigated the effect of the PMPC-b-POEGMA emulsifier concentration on the droplet size and

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stability of the resulting W/O emulsion. Figure 3 shows the photographs of the W/O emulsions

15

prepared using 1, 5, and 10 % (w/v) of the PMPC-b-POEGMA emulsifier. The diameter of the

16

initial W/O-emulsion droplets was measured using DLS after sonication for 1 h. Increasing the

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PMPC-b-POEGMA emulsifier concentration from 1 to 10 % (w/v) (relative to dispersed phase)

18

resulted in a decrease in the cumulant diameter of the W/O-emulsion droplets from 635 to 261

19

nm. Observation of no phase separation for 1 week demonstrates that the W/O emulsions

20

prepared using 10 % (w/v) of the PMPC-b-POEGMA emulsifier were stable. On the other hand,

21

an obvious phase separation and creaming were observed in W/O emulsions prepared using 1

22

and 5 % (w/v) of the PMPC-b-POEGMA emulsifier. The stability of the W/O emulsions were

23

evaluated using DLS equipped with backscatter optics. The backscatter optics enables the


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determination of a diameter under high concentration because it minimizes the multiple

2

scattering effect. DLS measurements of the W/O emulsions in the continuous phase also

3

indicated the high stability of W/O emulsions prepared using 10 % (w/v) of the PMPC-b-

4

POEGMA emulsifier because the diameter of the W/O-emulsion droplets remained unchanged

5

with an increase in the incubation time (Figure 4). On the other hand, the diameters of the W/O-

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emulsion droplets prepared using 1 and 5 % (w/v) of PMPC-b-POEGMA emulsifier in the

7

continuous phase decreased with increasing incubation time. In general, large droplet in an

8

emulsion leads to creaming because of their strong buoyant force20. The initial emulsions with

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large droplet sizes formed by 1 and 5 % (w/v) of PMPC-b-POEGMA were separated from the

10

continuous chloroform phase by creaming. On the other hand, the emulsions with small droplet

11

sizes formed through sonication remained stably dispersed in the continuous chloroform phase.

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Thus, the cumulant diameter of the W/O-emulsion droplets decreased with an increase in the

13

incubation time. These results confirmed that 10 % (w/v) of the PMPC-b-POEGMA emulsifier

14

was suitable for the formation of stable W/O emulsions when 10 mL of chloroform and 1 mL of

15

water were used as a continuous and a dispersed phase, respectively.

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Synthesis of gel capsules via IMEPP. The PMPC-b-POEGMA emulsifier possessed a

18

dithiobenzoate group acting as a chain transfer agent of the RAFT polymerization at the terminus

19

of POEGMA block. When a W/O emulsion was prepared using the PMPC-b-POEGMA

20

emulsifier in a water-chloroform mixture, the POEGMA block with the dithiobenzoate terminus

21

was located in the continuous chloroform phase. Therefore, a gel layer with disulfide cross-links

22

can be formed through copolymerization of OEGMA as an amphiphilic monomer and BMOD as

23

a cross-linker dissolved in the continuous chloroform phase from the PMPC-b-POEGMA


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emulsifier stabilizing the W/O emulsions via IMEPP. First, POEGMA and PPEGMA were

2

copolymerized without using BMOD as a cross-linker via IMEPP (Table 2, run 1, 2). Figure 5

3

shows the molecular weight distributions of the PMPC-b-POEGMA emulsifier, PMPC-b-

4

POEGMA-b-POEGMA and PMPC-b-POEGMA-b-PPEGMA synthesized via IMEPP. The W/O

5

emulsions were maintained after a chain extension reaction by IMEPP. The resulting PMPC-b-

6

POEGMA-b-POEGMA and PMPC-b-POEGMA-b-PPEGMA had high molecular weights

7

compared to the PMPC-b-POEGMA emulsifier acting as a macroRAFT agent of IMEPP. The

8

clear shift to a higher molecular weight indicates the chain extensions from the PMPC-b-

9

POEGMA emulsifier stabilizing the W/O emulsions. However, shoulders are observed in the

10

molecular weight distribution of PMPC-b-POEGMA-b-POEGMA and PMPC-b-POEGMA-b-

11

PPEGMA. In this system, the RAFT polymerization proceeded in the chloroform phase, which

12

was an undesired solvent in radical polymerization because chloroform can act as a chain

13

transfer agent. Therefore, IMEPP in the chloroform phase offers the possibility of uncontrolled

14

chain extensions owing to the chain transfer reaction to the chloroform.

15

When the continuous chloroform phase was exchanged for water, the scattering intensity of the

16

resulting W/O emulsions after IMEPP drastically decreased, and the diameters of the droplets

17

could not be determined (Table 2, run 1 and 2). The decrease in the scattering intensity indicates

18

the decomposition of the W/O emulsions in the water. The decomposition of W/O emulsions is

19

attributed to the fact that the triblock copolymers synthesized via IMEPP using the PMPC-b-

20

POEGMA emulsifier stabilizing the W/O emulsions are soluble in an aqueous solution because

21

the PMPC, POEGMA and PPEGMA blocks are soluble in water. On the other hand, the stable

22

hollow polymeric nanoparticles composed of water-soluble polymers can be prepared by

23

introducing cross-linking in the polymer shell16. We attempted to prepare stable gel capsules


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using IMEPP of OEGMA or PEGMA with a cross-linker (Table 2, run 3 and 4). W/O emulsions

2

after the IMEPP of OEGMA and PEGMA in the presence of the cross-linker were colloidally

3

stable in both chloroform and water. Therefore, the cross-linking of the amphiphilic POEGMA

4

and PPEGMA layer of W/O emulsions prevented triblock copolymers from being dissolved in

5

water. In addition, the monomers for the IMEPP (i.e., OEGMA and PEGMA) were dissolved in

6

a continuous chloroform phase because the PEG side chain was preferentially distributed in the

7

chloroform phase in a water-chloroform system. Therefore, the polymerization proceeded in not

8

a dispersed water phase but a continuous chloroform phase, followed by the formation of a gel

9

layer on the surface of the W/O emulsions stabilized by PMPC-b-POEGMA. Furthermore,

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spherical structure with small concave surface was observed in the scanning electron microscopy

11

image of the dried PPEGMA gel capsule (Figure S1). Thus, gel capsules having an inner water

12

phase were successfully prepared through IMEPP using a BMOD cross-linker. The size

13

distribution of the resulting gel capsules is shown in Figure 6. The gel capsules prepared using

14

OEGMA and PEGMA as monomers were stably dispersed with a monomodal size distribution in

15

chloroform. When the gel capsules prepared using PEGMA (PPEGMA gel capsules) were

16

redispersed in water, the PPEGMA gel capsules maintained a monomodal size distribution. In

17

contrast, the gel capsules prepared using OEGMA (POEGMA gel capsules) showed a bimodal

18

size distribution with a large diameter in water. The large size of the POEGMA gel capsules

19

indicates aggregation of the POEGMA gel capsules in water. These results imply that the

20

POEGMA gel capsules are not colloidally stable in water owing to the relatively hydrophobic

21

BMOD cross-linker in the gel layer. On the other hand, PPEGMA in the gel layer is relatively

22

hydrophilic enough for the gel capsules to be stably dispersed in water even though the

23

hydrophobic BMOD cross-linker is copolymerized with PPEGMA for forming the gel layer.


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Page 14 of 43

1

From these results, we concluded that water-dispersible gel capsules with an inner water phase

2

were successfully prepared using PEGMA as a hydrophilic monomer and BMOD as a disulfide

3

cross-linker for the gel capsule layer.

4 5

Reducing-environment-responsive release of FITC-Dex from PPEGMA gel capsules. A

6

variety of polymer capsules have been designed for the development of DDS carriers and

7

nanobioreactors because they can encapsulate various compounds. In particular, stimuli-

8

responsive polymer capsules that release encapsulated compounds in response to external stimuli

9

have been extensively studied for constructing smart DDS carriers. Because the polymer

10

capsules prepared via IMEPP have an inner water phase, they are candidates as DDS carriers that

11

deliver hydrophilic drugs, such as water-soluble low-molecular-weight drugs, proteins, and

12

DNAs. The PPEGMA gel capsules prepared in this study have disulfide cross-links that are

13

cleavable under reducing environments. The PPEGMA gel capsules can be decomposed under

14

reducing environments because the cleaved PPEGMA gel capsules can dissolve in an aqueous

15

solution. When drug-loaded PPEGMA gel capsules are immersed in a buffer solution containing

16

a reducing agent, the drugs can be substantially released from the PPEGMA gel capsules owing

17

to the decomposition of the PPEGMA gel capsules. Therefore, we investigated the release

18

behavior of FITC-Dex (Mw: 40 kDa) as a model drug from PPEGMA gel capsules. The FITC-

19

Dex-loaded gel capsules were prepared via IMEPP of PPEGMA and BMOD from the W/O

20

emulsions formed using PB solution containing FITC-Dex as a dispersed water phase. Figure 7

21

exhibits the FITC-Dex release behavior from the FITC-Dex-loaded gel capsules in a PB solution

22

with and without DTT as a reducing agent at 25 °C. The release rate of FITC-Dex from the

23

PPEGMA gel capsules in a PB solution with DTT was fast compared to that without DTT. These


14

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1

results indicate that the release behavior of FITC-Dex from the gel capsules was strongly

2

affected by reducing environments. However, the PPEGMA gel capsules did not show a burst

3

release of FITC-Dex under a reducing environment. The hollow polymer capsules with disulfide

4

cross-links reported previously showed a burst release of encapsulated drugs in response to

5

reducing environment because cleavage of the disulfide cross-links under reducing environment

6

resulted in the disintegration of capsules16, 21-23. The PPEGMA gel capsules are expected to be

7

completely decomposed and to substantially release FITC-Dex under reducing environments

8

because the PPEGMA gel capsule layer contains BMOD-based cross-links that are cleaved in

9

response to reducing environments. In fact, the diameter of the PPEGMA gel capsules slightly

10

increased under a reducing environment compared to that in a PB solution without DTT (Figure

11

S2). The slight increase in the diameter of the PPEGMA gel capsules is attributed to the fact that

12

the gel capsules swelled owing to the decrease in cross-linking density resulting from the

13

dissociation of the BMOD-based cross-links. The preservation of the gel capsule structure under

14

a reducing environment implies that 10 mM DTT was not enough for the complete cleavage of

15

the disulfide cross-links. There is also a possibility that noncleavable cross-links were formed

16

during the IMEPP owing to the chain transfer reaction to chloroform. Detailed studies on the

17

structure of the gel capsule layer are under investigation.

18

The reducing-environment-responsive release behavior of FITC-Dex from the PPEGMA gel

19

capsules can be explained through a tentative model schematically illustrated in Figure 8. In

20

general, the diffusion coefficient of a probe molecule in the gel depends on the correlation length

21

of the polymer network and the hydrodynamic diameter of the probe molecule24. For example,

22

our previous studies revealed that an antigen-responsive hydrogel with antigen-antibody complex

23

cross-links controlled the permeation of drugs by changing their network cross-linking density in


15

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Page 16 of 43

1

response to a target antigen25-26. The release of the FITC-Dex in the absence of DTT implies that

2

the network size of the PPEGMA gel capsule layer is larger than the hydrodynamic diameter of

3

FITC-Dex. When the BMOD-based cross-links, which contain disulfide bonds, in the PPEGMA

4

gel capsules layer are cleaved in a PB solution with DTT, a decrease in the cross-linking density

5

of the layer induced an increase in the diffusion coefficient of FITC-Dex within the gel capsule

6

layer. Therefore, the FITC-Dex release from the PPEGMA gel capsules was enhanced under a

7

reducing environment. Although the application of PPEGMA gel capsules as smart DDS carriers

8

require further research regarding their structural change in response to reducing environments

9

and controlling the drug release rate, the smart functions of the gel capsules can provide useful

10

platforms for constructing a drug carrier that can deliver water-soluble drugs, such as low-

11

molecular-weight drugs, proteins, and DNAs into the cytosol, which has a reducing environment.

12 13

CONCLUSIONS

14

The novel water-soluble emulsifier was composed of a block copolymer that contained a

15

PMPC block as a hydrophilic component and an amphiphilic POEGMA block as a lipophilic

16

component and was synthesized via RAFT polymerization. W/O emulsions were successfully

17

formed upon sonication of a water-chloroform mixture containing the resulting PMPC-b-

18

POEGMA. The W/O emulsion formation is attributed to the fact that the PMPC-b-POEGMA

19

acts as an emulsifier that stabilizes water droplets in a continuous chloroform phase. The

20

concentration of the PMPC-b-POEGMA emulsifier affected the droplet size and colloidal

21

stability of the W/O emulsions, which was a similar attribute of conventional surfactants.

22

Furthermore, the PPEGMA gel capsules were successfully prepared via IMEPP of PPEGMA and

23

BMOD from the PMPC-b-POEGMA emulsifier that stabilized the W/O emulsions. The


16

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1

PPEGMA gel capsules were stably dispersed in not only chloroform as a solvent in IMEPP but

2

also water because of the amphiphilicity of their gel capsule layer. Furthermore, FITC-Dex-

3

loaded PPEGMA gel capsules were prepared via IMEPP from W/O emulsions formed using an

4

aqueous FITC-Dex solution as a dispersed phase. FITC-Dex was slowly released from FITC-

5

Dex-loaded PPEGMA gel capsules in a PB solution without DTT as a reducing agent; however,

6

the release was effectively enhanced in a solution with DTT, i.e., under a reducing environment,

7

because the cross-linking density of the gel capsule layer decreased through the cleavage of the

8

disulfide bonds in the BMOD-based cross-links. Our method for the synthesis of gel capsules

9

using IMEPP of amphiphilic PEGMA from the water-soluble zwitterionic block copolymer

10

stabilizing the W/O emulsion will contribute significantly to the development of smart carriers

11

for drug delivery systems and nanobioreactors.

12 13

ASSOCIATED CONTENT

14

Supporting Information

15

The supporting information is available free of charge on the ACS publications website.

16

The SEM image of the dried PPEGMA gel capsules, size distributions of PPEGMA gel capsules

17

with and without DTT, 1H NMR spectra of the PMPC and PMPC-b-POEGMA (PDF)

18 19

AUTHOR INFORMATION

20

Corresponding Author

21

*E-mail [email protected]

22

*E-mail [email protected]

23

ORCID


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Page 18 of 43

1

Akifumi Kawamura: 0000-0003-4876-0685

2

Takashi Miyata: 0000-0002-6747-4118

3

Author Contributions

4

The manuscript was written through the contribution of all authors. All authors have given

5

approval to the final version of the manuscript.

6

ACKNOWLEDGMENT

7

This work was partly supported by the MEXT-Supported Program for the Private University

8

Research Branding Project and Takahashi Industrial and Economic Research Foundation.

9 10

REFERENCES

11

(1)

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Matter 2013, 9, 5138-5148.


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defined amphiphilic block copolymers having phospholipid polymer sequences as a novel

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at the nanoscale. Small 2009, 5, 2424-2432.

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switchable nanoparticles based on zwitterionic polymer for enhanced drug delivery to

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Huang, X.; Voit, B., Progress on multi-compartment polymeric capsules. Polym. Chem.

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Cross-linked polymersome membranes: Vesicles with broadly adjustable properties. J.

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Phys. Chem. B 2002, 106, 2848-2854.


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Harada, A.; Ichimura, S.; Yuba, E.; Kono, K., Hollow nanocapsules prepared through

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stabilization of polymer vesicles formed from head–tail type polycations by introducing

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cross-linkages. Soft Matter 2011, 7, 4629–4635.

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Utama, R. H.; Guo, Y.; Zetterlund, P. B.; Stenzel, M. H., Synthesis of hollow polymeric

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nanoparticles for protein delivery via inverse miniemulsion periphery RAFT

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polymerization. Chem. Commun. 2012, 48, 11103-11105.

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Utama, R. H.; Stenzel, M. H.; Zetterlund, P. B., Inverse miniemulsion periphery RAFT

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polymerization: a convenient route to hollow polymeric nanoparticles with an aqueous

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core. Macromolecules 2013, 46, 2118-2127.

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Utama, R. H.; Jiang, Y.; Zetterlund, P. B.; Stenzel, M. H., Biocompatible glycopolymer

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nanocapsules via inverse miniemulsion periphery RAFT polymerization for the delivery

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of gemcitabine. Biomacromolecules 2015, 16, 2144-2156.

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Harris, J. M., Introduction to biotechnical and biomedical applications of poly(ethylene

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glycol). In Poly(ethylene glycol): Biotechnical and biomedical applications, Harris, J. M.,

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methacryloyloxyethyl phosphorylcholine) gel and its strange swelling behavior in

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water/ethanol mixture. J. Biomater. Sci., Polym. Ed. 2002, 13, 213-224.

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alcohol/water mixtures. Langmuir 2010, 26, 7216-7226.

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Gupta, A.; Eral, H. B.; Hatton, T. A.; Doyle, P. S., Nanoemulsions: formation, properties and applications. Soft Matter 2016, 12, 2826-2841.


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Kim, E.; Kim, D.; Jung, H.; Lee, J.; Paul, S.; Selvapalam, N.; Yang, Y.; Lim, N.; Park, C.

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G.; Kim, K., Facile, template-free synthesis of stimuli-responsive polymer nanocapsules

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for targeted drug delivery. Angew. Chem. Int. Ed. Engl. 2010, 49, 4405-4408.

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Teranishi, R.; Matsuki, R.; Yuba, E.; Harada, A.; Kono, K., Doxorubicin Delivery Using

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pH and Redox Dual-Responsive Hollow Nanocapsules with a Cationic Electrostatic

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Barrier. Pharmaceutics 2017, 9, 4.

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Tokita, M.; Miyoshi, T.; Takegoshi, K.; Hikichi, K., Probe diffusion in gels. Phys. Rev. E

Miyata, T.; Asami, N.; Uragami, T., A reversibly antigen-responsive hydrogel. Nature 1999, 399, 766-769.

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Miyata, T.; Asami, N.; Okita, Y.; Uragami, T., Controlled permeation of model drugs

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through a bioconjugated membrane with antigen–antibody complexes as reversible

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crosslinks. Polym. J. 2010, 42, 834-837.

16


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Figure 1. Molecular weight distributions (normalized to peak height) of PMPC (blue line) and PMPC-b-POEGMA (red line).


22

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Figure 2. Phase contrast and fluorescence images of W/O emulsions after sonication for 1 h. The W/O emulsions were prepared using an aqueous fluorescein solution (5 mg/mL) as a water dispersed phase in the presence of PMPC-b-POEGMA (10 wt % relative to the dispersed phase).


23

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Page 24 of 43

Figure 3. Photographs of sonicated water-chloroform system containing PMPC-b-POEGMA after incubation for 0, 8, 24, and 168 h. Polymer concentrations were 1, 5, and 10 % (w/v) (relative to dispersed phase).


24

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Figure 4. Changes in cumulant diameter of W/O-emulsion droplets in the continuous chloroform phase as a function of time after sonication for 1 h. The concentrations of PMPC-b-POEGMA were 1 (●), 5 (■), and 10 % (w/v) (◆) (relative to dispersed phase).


25

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Page 26 of 43

Figure 5. Molecular weight distributions (normalized to peak height) of PMPC-b-OEGMA (black line), PMPC-b-POEGMA-b-POEGMA (blue line), and PMPC-b-POEGMA-b-PPEGMA (red line) synthesized via IMEPP.


26

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Figure 6. Size distributions of gel capsules with BMOD-based cross-links in chloroform and water. (a) POEGMA gel capsules in chloroform after IMEPP; (b) POEGMA gel capsules after exchanging continuous chloroform phase for water; (c) PPEGMA gel capsules in chloroform after IMEPP; (d) PPEGMA gel capsules after exchanging continuous chloroform phase for water.


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Page 28 of 43

Figure 7. Release profile of FITC-Dex from gel capsules in a 20 mM PB solution with (○) and without 10 mM DTT (●) at 25 °C. .


28

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Figure 8. Schematic illustration for enhancement of the reducing-environment-responsive release of FITC-Dex from PPEGMA gel capsules.


29

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Page 30 of 43

Scheme 1. Schematic illustration for the preparation of water-dispersible gel capsules via IMEPP using template W/O emulsions stabilized by a water-soluble PMPC-b-POEGMA emulsifier.


30

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Table 1. Results of the synthesis of PMPC macroRAFT agent and PMPC-b-POEGMA. Degree of polymerizationa Mn (NMR) (×104) PMPC POEGMA

Mn (SEC)b Mw (SEC)b (×104) (×104)

Ðb

PMPC macroRAFT agent

22

–

0.65

0.50

0.56

1.1

PMPC-b-POEGMA

22

171

5.20

0.66

0.96

1.5

a

calculated from 1H NMR

b

calculated from GPC curves using near-monodisperse poly(ethylene glycol) calibration standards


31

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Page 32 of 43

Table 2. Synthetic condition and diameters of W/O-emulsion droplets and gel capsules in chloroform and water.

Run

Monomer

[M]:[BMOD]:[PMPC-b-POEGMA]:[I]

1

POEGMA

2

Cumulant diameter (nm)a Chloroform

Water

500:0:1:0.5

518

n. d.b

PPEGMA

500:0:1:0.5

315

n. d.b

3

POEGMA

500:100:1:0.5

534

890

4

PPEGMA

500:100:1:0.5

234

217

a

determined from DLS measurements

b

not determined because of low scattering intensity


32

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TOC GRAPHIC


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Figure 1. Molecular weight distributions (normalized to peak height) of PMPC (blue line) and PMPC-bPOEGMA (red line). 74x50mm (300 x 300 DPI)


Page 34 of 43

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Figure 2. Phase contrast and fluorescence images of W/O emulsions after sonication for 1 h. The W/O emulsions were prepared using an aqueous fluorescein solution (5 mg/mL) as a water dispersed phase in the presence of PMPC-b-POEGMA (10 wt % relative to the dispersed phase). 78x60mm (300 x 300 DPI)


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Figure 3. Photographs of sonicated water-chloroform system containing PMPC-b-POEGMA after incubation for 0, 8, 24, and 168 h. Polymer concentrations were 1, 5, and 10 % (w/v) (relative to dispersed phase). 82x70mm (300 x 300 DPI)


Page 36 of 43

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Figure 4. Changes in cumulant diameter of W/O-emulsion droplets in the continuous chloroform phase as a function of time after sonication for 1 h. The concentrations of PMPC-b-POEGMA were 1 (●), 5 (■), and 10 % (w/v) (◆) (relative to dispersed phase). 68x68mm (300 x 300 DPI)


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Figure 5. Molecular weight distributions (normalized to peak height) of PMPC-b-OEGMA (black line), PMPCb-POEGMA-b-POEGMA (blue line), and PMPC-b-POEGMA-b-PPEGMA (red line) synthesized via IMEPP. 76x52mm (300 x 300 DPI)


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Figure 6. Size distributions of gel capsules with BMOD-based cross-links in chloroform and water. (a) POEGMA gel capsules in chloroform after IMEPP; (b) POEGMA gel capsules after exchanging continuous chloroform phase for water; (c) PPEGMA gel capsules in chloroform after IMEPP; (d) PPEGMA gel capsules after exchanging continuous chloroform phase for water. 82x81mm (300 x 300 DPI)


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Figure 7. Release profile of FITC-Dex from gel capsules in a 20 mM PB solution with (○) and without 10 mM DTT (●). 68x68mm (300 x 300 DPI)


Page 40 of 43

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Figure 8. Schematic illustration for enhancement of the reducing-environment-responsive release of FITCDex from PPEGMA gel capsules. 82x51mm (300 x 300 DPI)


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Scheme 1. Schematic illustration for the preparation of water-dispersible gel capsules via IMEPP using template W/O emulsions stabilized by a water-soluble PMPC-b-POEGMA emulsifier. 177x44mm (300 x 300 DPI)


Page 42 of 43

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Table of Contents Graphic 82x44mm (300 x 300 DPI)


Reductively Responsive Gel Capsules Prepared Using a Water

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