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Synthesis and Properties of Upper Critical Solution Temperature (UCST) Responsive Nanogels Maho Ohshio, Kazuhiko Ishihara, Atsushi Maruyama, Naohiko Shimada, and Shin-ichi Yusa Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b00849 • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Manuscript for Langmuir

Synthesis and Properties of Upper Critical Solution Temperature (UCST) Responsive Nanogels

Maho Ohshio†, Kazuhiko Ishihara‡, Atsushi Maruyama§, Naohiko Shimada§, Shin-ichi Yusa*,† †Department

of Applied Chemistry, Graduate School of Engineering, University of Hyogo,

2167 Shosha, Himeji, Hyogo 671-2280, Japan ‡Department

of Materials Engineering, School of Engineering, The University of Tokyo,

7-3-1 Hongo, Bunkyo-ku Tokyo 113-8656, Japan §Department

of Biomolecular Engineering, Tokyo Institute of Technology, 4259 B-57,

Nagatsuta, Midori, Yokohama 226-8501, Japan

ABSTRACT: A random copolymer ((U/A10)165) bearing pendent ureido groups and a small amount (10 mol%) of primary amino groups exhibits an upper critical solution temperature (UCST). We prepared a diblock copolymer (PMPC20P(U/A10)165) composed of water-soluble poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and (U/A10)165 blocks via reversible

addition-fragmentation

chain

transfer

radical

polymerization

with

post-modification reaction. The sub-numbers are the degrees of polymerization of each block. Although in water PMPC20P(U/A10)165 dissolves as a unimer above the UCST phase transition temperature (Tp), it forms polymer micelles composed of dehydrated (U/A10)165 cores and hydrophilic PMPC shells. A nanogel was prepared by cross-linking the pendent primary amines in the micelle core using (hydroxymethyl)phosphonium chloride below Tp. 1

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NMR and light scattering data indicated that the nanogel core shrinks upon dehydrated below Tp and swells upon hydration above Tp. The nanogel can encapsulate guest molecules such as hydrophobic fluorescence probes and bovine serum albumin (BSA) below Tp mainly owing to hydrophobic interactions in the core. Encapsulated BSA can be held in the nanogel core below Tp and subsequently released above Tp.

Graphical Abstract

INTRODUCTION Physical, chemical, and biological properties of certain polymers can be tuned by external-stimuli, such as changes in pH, temperature, magnetic force, and concentration of chemical compound by enzymatic reaction.1-5 Thus, the aggregation and dissociation behavior of block copolymers designed to contain stimuli-responsive polymers can be tuned by external-stimuli. Guest molecules encapsulated into stimuli-responsive polymer aggregates can be released by specific external stimuli. Size-controlled nanocarriers can potentially be used as drug delivery systems (DDSs) for cancer treatment by exploiting the enhanced permeation and retention effect.6 Akiyoshi et al. have reported that hydrophobic cholesteryl groups-bearing water-soluble pullulan (CHP) forms a nanogel due to the hydrophobic interactions of the cholesteryl groups.7,8 Proteins can be encapsulated by CHP through hydrophobic interactions between proteins and its cholesteryl groups. Furthermore, cationic-group-bearing CHP can 2


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encapsulate anionic proteins effectively through hydrophobic and electrostatic interactions.9 The encapsulated proteins can be released from CHP upon hydrolysis of the polysaccharide backbone by enzymes. Encapsulated proteins can be released in HeLa cells by CHP over 18 h upon decomposition of its polysaccharide backbone by hydrolysis. Lower critical solution temperature (LCST)-type thermoresponsive polymers that dissolve in water below the LCST but not above the LCST have been widely studied by a large number of research groups.10 Typical examples are poly(N,N-diethylacrylamide) and poly(N-isopropylacrylamide) (PNIPAM), which show LCST behavior.11-13 Conversely, upper critical solution temperature (UCST)-type polymers are able to dissolve in water above the UCST but not below the UCST. However, there are a fewer reports on the aqueous behavior of UCST-type polymers compared to those on LCST polymers.14,15 Recently, water-soluble UCST polymers bearing pendent ureido groups such as poly(allylurea)16-18 and poly(2-ureidoethyl methacrylate) (PUEM) have been reported.19 The immune system has difficulty in recognizing poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC) as a foreign material because PMPC has the same structure as phosphorylcholine, the hydrophilic part of phospholipids in cell membranes.20 The diblock copolymer consisting of hydrophilic PMPC and UCST-type thermoresponsive PUEM blocks forms spherical polymer micelles composed of PUEM cores and PMPC shells below the UCST of the PUEM block in water.21 Above the UCST, the polymer micelle dissociates into a unimer state. Therefore, the diblock copolymer may potentially be applied to controlled release of encapsulated drugs in DDS. However, a serious problem is that the polymer micelle structure dissociates independently of temperature when the polymer concentration (Cp) is lower than the critical association concentration (CAC). Maintaining the polymer micelle structure below the CAC is one of the necessary condition to deliver physiologically active substances in blood circulation. When the micelle core is cross-linked below the UCST, the 3


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cross-linked polymer micelle can maintain its core-shell micelle structure below the CAC.22 A core cross-linked polymer micelle is termed a nanogel, and such nanogels can encapsulate hydrophobic guest molecules in their cores below the UCST. Above the UCST, the encapsulated guest molecules can be released to the aqueous phase because the core becomes hydrophilic and swollen (Figure 1). Thermoresponsive nanogels do not dissociate upon blood circulation in the body, allowing them to deliver guest molecules to tumor sites. Furthermore, it is expected that encapsulated guest molecules such as drugs are released by heating the area of the body intended for treatment.

Figure 1. (a) Chemical structure of PMPC20P(U/A10)165 and the temperature-responsive behavior of (b) PMPC20P(U/A10)165 and (c) its nanogel in water.

RESULTS AND DISCUSSION 4


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A nanogel that exhibits UCST behavior possessing a hydrophilic biocompatible PMPC shell and cross-linked core was prepared following the procedure illustrated in Scheme S1.

The

diblock

copolymer,

PMPC20PAEM165

was

synthesized

via

reversible

addition-fragmentation chain transfer (RAFT) polymerization of AEM, which contains the pendant primary amine, using PMPC-macro CTA. Then, 90 mol% of the pendent primary amino groups were changed to ureido groups to prepare PMPC20P(U/A10)165. PMPC20P(U/A10)165 shows UCST-type thermoresponsive behavior in aqueous solution. At 20 °C, PMPC20P(U/A10)165 forms micelles comprising of hydrophilic PMPC shells and hydrophobic (U/A10)165 core. The pendant primary amines in the micelle core were cross-linked using tetrakis(hydroxymethyl)phosphonium chloride (THPC) to prepare the nanogel.

PMPC20P(U/A10)165

was

dissolved

in

2-(4-(2-hydroxyethyl)piperazin-1-yl)

ethanesulfonic acid (HEPES) buffer (pH 7.6) and stirred at 60 °C overnight. THPC was added to the solution to give (number of amino groups in the pendant AEM in the polymer)/THPC = 4/1. The solution was stirred at 20 °C for 20 h. The reaction mixture was dialyzed against pure water at 60 °C for one day. The nanogel was recovered by freeze-drying. 1H

NMR analysis of PMPC macro-CTA was performed (Figure S1a). Degree of

polymerization (DP) of PMPC macro-CTA was 20 as estimated from the integrated intensity for the terminal phenyl protons attributed to CTA at 7.4–8.0 ppm and the pendent methylene protons at 3.4 ppm. The DP of PAEM block in PMPC20PAEM165 was 165 as estimated from the signal intensities for the pendent methylene protons at 3.4 ppm in the PMPC block and those at 2.6 ppm in the PAEM block (Figure S1b). We performed GPC measurements (Figure S2) for PMPC macro-CTA and PMPC20PAEM165 using 0.3 M Na2SO4 and 0.5 M acetic acid containing aqueous solution as the eluent. The GPC elution curve of PMPC20PAEM165 was shifted to higher molecular weight compared to that of PMPC macro-CTA. The Mw/Mn values of PMPC20PAEM165 and PMPC are 1.12 and 1.23, respectively, which indicates that the 5


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polymers have well-controlled structute (Table 1).

Table 1. Number-average Molecular Weight (Mn), Degree of Polymerization (DP), and Molecular Weight Distribution (Mw/Mn) of the Polymers Mn(NMR)

Mn(GPC)

Mn(theory)

(g/mol)

(g/mol)

(g/mol)

PMPC20

6,130

11,900

PMPC20PAEM165

33,400

PMPC20P(U/A10)165

34,400

aDP

of PMPC macro-CTA.

bDP

DP

Mw/Mn

6,190

20a

1.12

24,100

33,900

165b

1.23

-c

34,400

165

-c

of the PAEM block in the polymer.

cGPC

of

PMPC20P(U/A10)165 cannot be measured due to unexpected interactions between the column and polymer.

Theoretical value of Mn (Mn(theory)) can be estimated by the following formula:

(2)

where [M]0 and [CTA]0 are the initial monomer and CTA concentrations, p is the percent monomer conversion, MM and MCTA are the molecular weights of monomer and CTA. The degree of conversion from the primary amine to the ureido group in PMPC20P(U/A10)165 was estimated using 1H NMR measurements (Figure S1c). When the primary amine changes to the ureido group, the 1H NMR signal attributed to methylene protons at 2.8 ppm is shifted to 3.2 ppm. Conversion form the amine to ureido group is 89.6 mol%, as estimated from the decrease in the peak intensity at 2.8 ppm after the ureido reaction. This means that ca. 10 mol% primary amine remains in the (U/A10)165 block. The remaining primary amino groups 6


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can be cross-linked in the micelle core to prepare a nanogel below the UCST. GPC measurement of PMPC20P(U/A10)165 could not be performed due to unexpected interactions between the diblock copolymer and the columns.19 We obtained 1H NMR data for PMPC20P(U/A10)165 and the nanogel in D2O at 60 °C. The decrease of the signal attributed to the pendent methylene protons in the AEM unit in PMPC20P(U/A10)165 after core cross-linking indicated that 94.0 mol% primary amine was used in the cross-linking reaction to prepare the nanogel (Figure S1e).

Figure 2. 1H NMR integrated area intensity ratio (IUEM/IMPC) of peaks at 3.2 ppm (IUEM) and 3.4 ppm (IMPC) attributed to the UEM unit and the PMPC block at Cp = 5.0 g/L as a function of temperature for PMPC20P(U/A10)165 (〇) and the nanogel (◇) in D2O containing 0.1 M NaCl.

1H

NMR spectra of PMPC20P(U/A10)165 and the nanogel in D2O containing 0.1 M

NaCl were recorded with increasing temperature. The NMR peak intensity ratio (IUEM/IMPC) was estimated from the intensity (IUEM) of the UEM pendent methylene protons at 3.2 ppm and the intensity (IMPC) of the PMPC pendent methylene protons at 3.4 ppm. The values of IUEM/IMPC for PMPC20P(U/A10)165 and the nanogel are plotted as a function of temperature (Figure 2). The phase transition temperatures (Tp) were estimated from the intersection of the tangent lines drawn from the gentle and steep slopes in the low temperature region. The Tp 7


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values for PMPC20P(U/A10)165 and the nanogel are 24 and 26 °C, respectively. The IUEM/IMPC values of PMPC20P(U/A10)165 and the nanogel are low below Tp. Generally, the NMR signal intensity decreases due to decreasing spin-spin relaxation time when the motion of protons is restricted.23 Therefore, the small IUEM/IMPC values below Tp indicate the restricted motion of protons in the UEM units in the core due to dehydration. Above Tp, PMPC20P(U/A10)165 dissociates into the unimer state. As the motion of protons in the UEM units increased above Tp, the IUEM/IMPC value of PMPC20P(U/A10)165 increased. In the case of nanogel, above Tp the IUEM/IMPC value increased, however the IUEM/IMPC value is smaller than that of PMPC20P(U/A10)165. The motion of the nanogel core is lower than that of PMPC20P(U/A10)165 above Tp because the core of the nanogel is cross-linked.

Figure 3. Temperature dependence on hydrodynamic radii (Rh) for PMPC20P(U/A10)165 (〇) and the nanogel (◇) at Cp = 5 g/L in 0.1 M NaCl.

Temperature dependence on percent transmittance (%T) was measured to determine Tp. For both PMPC20P(U/A10)165 and the nanogel in water, the %T values are almost constant at 100%, irrespective of temperature. Therefore, we could not determine Tp from the %T vs. temperature plot. The values of Rh for the pre-cross-linking PMPC20P(U/A10)165 and the nanogel in 0.1 M NaCl are plotted as a function of temperature (Figure 3). The Tp value for PMPC20P(U/A10)165 is 24 °C, as estimated from the Rh vs. temperature plot, which is 8


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consistent with that estimated from the IUEM/IMPC vs. temperature plot. Below Tp, the Rh value for PMPC20P(U/A10)165 is 30 nm, which suggests that the bock copolymer forms polymer micelles composed of (U/A10)165 cores and hydrophilic PMPC shells. Above 40 °C the Rh value for PMPC20P(U/A10)165 is 7 nm, which suggests that the diblock copolymer dissolves in aqueous solution in the unimer state. Although the Tp value for the nanogel is ca. 19 °C, as estimated from the Rh vs. temperature plot, the Tp value is not entirely clear due to the gradual Rh change. The nanogel does not dissociate above Tp because of the cross-linked core. The Rh value for the nanogel increases upon swelling with increasing temperature because the pendent ureido groups in the core are hydrated above Tp. No hysteresis in the heating and cooling process for PMPC20P(U/A10)165 or the nanogel is observed (Figure S3). We performed SLS measurements for the nanogel at 20 °C, which is lower than Tp (Figure S4, Table 2). The apparent weight-average molecular weight (Mw) value for the nanogel was estimated from extrapolating both Cp and θ to zero. Aggregation number (Nagg) was estimated from the apparent Mw of the nanogel and the molecular weight of a single PMPC20P(U/A10)165 polymer chain before cross-linking, as estimated from NMR data. The Nagg value for the nanogel is 341. From the slope of θ at a Cp of zero (from extrapolation), the radius of gyration (Rg) of the nanogel can be obtained (= 41.0 nm). The Rg/Rh ratio estimated from DLS at 20 °C is 1.40 for the nanogel. It is known that Rg/Rh is 0.775 for monodisperse hard spheres, 1 for spherical shapes, and greater than 2 for rod-like shapes.24 The Rg/Rh value of the nanogel is 1.40, indicating that its shape may be generally spherical.

Table 2. Light Scattering Data for the Nanogel in 0.1 M NaCl at 20 °C Sample nanogel

Mwa (SLS)

Rhb

Rga

RTEMc

(g/mol)

(nm)

(nm)

(nm)

1.41 × 107

29.2

41.0

21.9

9


Rg/Rh

Naggd

1.40

341

dn/dCp (mL/g) 0.12

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aMeasrued

with SLS in 0.1 M NaCl. bDetermined by DLS in 0.1 M NaCl at 20 °C. cEstimated

from TEM observations. dAggregation number of nanogel calculated from the apparent Mw of the nanogel as determined by SLS and the molecular weight of the corresponding unimers as determined by NMR.

Figure 4. TEM images of the nanogel at different magnifications.

We performed TEM observation for the nanogel at 20 °C (Figure 4). The average radius (RTME) as estimated from the TEM images is 21.9 nm. The RTME is smaller than the Rh value (= 29.2 nm) estimated using DLS. This is because the nanogel shrinks prior to TEM observation due to the samples drying under reduced pressure.

10


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Figure 5. Temperature dependence on hydrodynamic radii (Rh) for (a) PMPC20P(U/A10)165 before cross-linking and (b) the nanogel at Cp = 1 (◇), 2.5 (△), and 5 (○) g/L in 0.1 M NaCl.

For poly(N-acryloylglycinamide) in water, the UCST increases with increasing Cp.25 Also, the UCST for poly(2-ureidoethylmethacrylate) in water increases with increasing Cp.19 We evaluated the dependence of Cp on Tp for the nanogel in 0.1 M NaCl aqueous solution. The values of Rh for the nanogel at Cp = 1, 2.5, and 5 g/L are plotted as a function of temperature in Figure 5. The Tp values for PMPC20P(U/A10)165 at Cp = 1, 2.5, and 5 g/L are 10, 16, and 24 °C, respectively. Hydrogen bonding interactions between the pendent ureido groups form more readily with increasing Cp owing to increasing collision frequency. To break the hydrogen bonding interactions of the ureido groups at high Cp, larger energy input is required. Therefore, the Tp values for PMPC20P(U/A10)165 increase with increasing Cp. Conversely, the Tp values for the nanogel are largely constant and thus independent of Cp. The local concentration of the pendent ureido groups in the nanogel core is constant irrespective of Cp. Therefore, the Tp values for the nanogel are constant at ca. 19 °C. The core-shell structure 11


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of the nanogel can be maintained at low Cp. However, below Tp, the Rh values for the nanogel are low due to dehydration of the core, while above Tp, the Rh values increase due to swelling of the core upon hydration. The Rh value of the nanogel upon maintaining the temperature at 60 °C for one month at Cp = 1 g/L was found to be 34 nm in 0.1 M NaCl. This Rh value is similar to that recorded immediately after preparation of the nanogel in aqueous solution. Thus, the time dependence of the size of the nanogel can be neglected. We studied the NaCl concentration ([NaCl]) dependence of the Tp values for PMPC20P(U/A10)165 and the nanogel. The Rh values were plotted as a function of temperature at [NaCl] = 0.1, 0.2, and 0.5 M at Cp = 5 g/L with heating (Figure S5). The Tp values for PMPC20P(U/A10)165 and the nanogel are 24 and 19 °C. The Tp values are almost constant irrespective of [NaCl].

Figure 6. Hydrodynamic radius (Rh) for the nanogel at 10 (○) and 60 °C (△) in 0.1 M NaCl at Cp = 5 g/L over five heating and cooling cycles.

We studied the repeated nanogel swelling and shrinking upon successive heating and cooling cycles. The Rh of the nanogel in 0.1 M NaCl was measured over five cycles with heating from 10 to 60 °C and cooling from 60 to 10 °C (Figure 6). The Rh values are ca. 29 and 35 nm at 10 and 60 °C, respectively. Thus, the nanogel is capable of repeated swelling and shrinking with temperature change. 12


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To confirm the polarity changes of the nanogel core depending on temperature, we used PNA as a fluorescence hydrophobicity probe. In hydrophilic environments like water, the fluorescence intensity decreases and its maximum fluorescence wavelength (λmax) shifts to longer wavelengths.26 The PNA fluorescence intensity and λmax (= 456 nm) are largely constant independently of temperature in the range 10–60 °C in 0.1 M NaCl. PNA fluorescence was measured in the presence of the nanogel at 10 and 60 °C in 0.1 M NaCl (Figure S6). At 10 °C in the presence of the nanogel, λmax shifts to the shorter wavelength of 419 nm and has strong fluorescence intensity. At 10 °C, which is lower than Tp, PNA is encapsulated into the dehydrated hydrophobic core of the nanogel. Conversely, at 60 °C, λmax shifts the longer wavelength of 431 nm and has weak fluorescence intensity. At 60 °C, which is higher than Tp, PNA is released from the hydrated nanogel core to the aqueous phase. Some PNA molecules remain inside the core, as indicated by the fact that, at 60 °C, the λmax is 431 nm, which is shorter than (λmax = 456 nm) without the nanogel at 60 °C. Fluorescence-labeled BSA was encapsulated into the nanogel core by stirring a mixture of the nanogel and fluorescence-labeled BSA in phosphate buffered saline (PBS) buffer for 1 h at 10 °C. This solution was dialyzed against PBS at 10 °C, which is lower than Tp. A dialysis membrane with a pore size of 50 nm was used. The Rh value of BSA in PBS was 5.1 nm.27 The Rh value of the nanogel before incorporation of BSA was 27.6 nm in PBS at 10 °C. When BSA was encapsulated into the nanogel, the Rh increased to be 35.1 nm. BSA can pass through the membrane, but the nanogel cannot. As a reference experiment, dialysis of fluorescence-labeled BSA in PBS solution without the nanogel was also performed at 10 °C as the same manner. After dialysis, fluorescence measurements of the PBS solutions inside in the dialysis bags were taken (Figure S7). BSA can be trapped into the nanogel core presumably because of hydrophobic interactions. There is possibility that the residual cross-linking agent, THPC was reacted with the primary amine residues in BSA, and thus the 13


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BSA was covalently attached inside the nanogels. Fluorescence is not observed for the PBS solution without the nanogel after dialysis because the BSA leaks from the dialysis membrane. Conversely, fluorescence with λmax = 520 nm from the PBS solution with the nanogel is observed after dialysis. BSA remained in the dialysis bag because it was encapsulated in the dehydrated nanogel core at 10 °C. The nanogel cannot pass through the membrane owing to its particle size. BSA does not adsorb to the PMPC shells on the outermost layer of the nanogel, it penetrated into the dehydrated nanogel core.28 The loading rate (LR) for BSA can be calculated by the following equation: Loading rate (LR) = (total protein – free protein)/total protein, where ‘total protein’ is the total molar amount of BSA used in the experiment and ‘free protein’ is the molar amount of BSA that is not encapsulated into the nanogel as determined by fluorescence intensity. The LR and loading content for BSA are 14.3% and 12.5 μg, respectively.

Figure 7. (a) Release of BSA from the nanogel in PBS buffer at 10 °C (〇) and 40 °C (□) and a blank solution without the nanogel at 10 °C ( ◇ ) and 40 °C ( △ ). (b) ln(I/I0) plotted 14


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against dialysis time (t), where I is fluorescence intensity at t and I0 is the initial fluorescence intensity at t = 0 h.

We then investigated the release of the encapsulated BSA from the nanogel. A BSA-encapsulated nanogel PBS solution was dialyzed against PBS at 10 and 40 °C, and the fluorescence intensity at 520 nm was monitored at varying dialysis times to calculate the cumulative release rate of BSA (Figure 7a). A dialysis membrane with a pore size of 50 nm was used. The BSA release rates after 312 h in the presence of nanogel at 40 and 10 °C are 55.6 and 21.5%, respectively. The BSA release is higher at 40 °C than that at 10 °C. This suggests that the interior of the nanogel becomes hydrophilic at 40 °C, which is above Tp, making it easier to release BSA. However, the BSA release rate was only 55.6% at 40 °C after 312 h, which indicates that some BSA molecules remain inside the nanogel core. Thus, there may be hydrophobic and other interactions between BSA and nanogel. Without nanogel, the BSA release amounts at 40 and 10 °C after 72 h are 95.3 and 74.7%, respectively, the difference being due to the decrease in thermal motion at 10 °C compared to that at 40 °C. Thus, in the presence of the nanogel, BSA release is less than that without the nanogel because the BSA is encapsulated into the hydrophobic nanogel core. If the release of BSA from the dialysis membrane follows the first-order kinetics, the rate constant (k) can be determined by the following equation: ln(I/I0) = –kt, where t is the dialysis time, I is the fluorescence intensity of BSA at t, and I0 is the fluorescence intensity of BSA before dialysis.29,30 Thus, from the slope of ln(I/I0) vs. t, the k value can be determined (Figure 7b). The k values without the nanogel at 40 and 10 °C are 1.23 and 0.492 h−1, respectively. Thus, the k value without the nanogel at 10 °C is 0.40-times that at 40 °C. The release rate of BSA from dialysis bag at 10 °C is lower due to the decreased thermal motion of the molecules with cooling. The k values in the presence of the nanogel at 40 and 10 °C are 15


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0.230 and 0.0421 h–1, respectively. Thus, the k value with the nanogel at 10 °C is 0.183-times that at 40 °C. This observation indicates that molecular motion is decreased at 10 °C and that BSA is encapsulated into the dehydrated nanogel core. The encapsulated BSA can be released at 40 °C, because the nanogel core changes to be hydrophilic.

CONCLUSIONS The diblock copolymer, PMPC20P(U/A10)165 comprising a UCST-type random copolymer block containing small amount of pendent primary amino groups and hydrophilic PMPC block was synthesized via RAFT and post-reaction treatment. The Tp value for PMPC20P(U/A10)165 is 24 °C at Cp = 5 g/L, as estimated from 1H NMR and DLS measurements. Below Tp, PMPC20P(U/A10)165 forms c spherical micelles composed of PMPC shells and (U/A10)165 core. Above Tp, PMPC20P(U/A10)165 micelles dissociated to be in a unimer state. A nanogel was prepared by cross-linking the core below Tp. With increasing temperature, the nanogel core swells upon hydration while retaining its core-shell structure. The Tp value of PMPC20P(U/A10)165 increases with increasing Cp, whereas the Tp value of the nanogel is independent of Cp. The local concentration of the pendent ureido groups in the core of the nanogel is also independent of Cp. The nanogel remained as a core-shell structure even at low Cp. The nanogel core is hydrophobic below Tp, and BSA can be encapsulated into the core. Above Tp the nanogel core becomes hydrophilic, and the encapsulated guest molecules, in this case BSA, can be released to the aqueous phase.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.xxxxxxx. 16


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Experimental section, synthesis scheme; 1H NMR; GPC; Rh vs temperature; Zimm plot fluorescence spectra (PDF)

AUTHOR INFORMATION Corresponding Author E-mail: [email protected] ORCID Shin-ichi Yusa: 0000-0002-2838-5200 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was funded by a Grant-in-Aid for Scientific Research, KAKENHI (17H03071 and 16K14008) from the Japan Society for the Promotion of Science (JSPS), JSPS Bilateral Joint Research Projects, and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices (20184035).”

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