Reversible pKa Modulation of Carboxylic Acids in Temperature

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Biological and Medical Applications of Materials and Interfaces

Reversible pKa Modulation of Carboxylic Acids in TemperatureResponsive Nanoparticles through Imprinted Electrostatic Interactions Yu Hoshino, Toshiki Jibiki, Masahiko Nakamoto, and Yoshiko Miura ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11397 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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ACS Applied Materials & Interfaces

Reversible pKa Modulation of Carboxylic Acids in TemperatureResponsive Nano-particles through Imprinted Electrostatic Interactions Yu Hoshino*, Toshiki Jibiki, Masahiko Nakamoto, and Yoshiko Miura Department of Chemical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan ABSTRACT: The acid dissociation constants (pKa values) of Brønsted acids at the active sites of proteins are reversibly modulated by intra-molecular electrostatic interactions with neighboring ions in a reaction cycle. The resulting pKa shift is crucial for the proteins to capture, transfer, and release target ions. On the other hand, reversible pKa modulation through electrostatic interactions in synthetic polymer materials has seldom been realized because the interactions are strongly shielded by solvation water molecules in aqueous media. Here, we prepared hydrogel nano-particles (NPs) bearing carboxylic acid groups whose pKa values can be reversibly modulated by electrostatic interactions with counterions in the particles. We found that the deprotonated states of the acids were stabilized by electrostatic interactions with countercations only when the acids and cations were both imprinted in hydrophobic microdomains in the NPs during polymerization. Cationic monomers, like primary amine- and guanidium group-containing monomers, which interacted strongly with growing NPs showed greater pKa modulation than monomers that did not interact with the NPs, such as quaternary ammonium group-containing monomers. Modulation was enhanced when the guanidium moieties were protected with hydrophobic groups during polymerization so that the guanidium ions were imprinted in the hydrophobic microdomains; the lowest pKa of ~4.0 was achieved as a result. The pKa modulation of the acids could be reversibly removed by inducing a temperature-dependent volume phase transition of the gel NPs. These design principles are applicable to other stimuli-responsive materials and integral to the development of synthetic materials that can be used to capture, transport, and separate target ions. KEYWORDS: Ion Imprinting, pKa modulation, electrostatic interaction, temperature responsive, proton pump, hydrogel, nanoparticles, nanogel

Introduction The acid dissociation constants (pKa values) of Brønsted acids at the active sites of proteins, such as ion transporters1-3 and enzymes,3–6 vary widely and shift reversibly in response to protein conformational changes (Figure 1A). Recently, it was shown that ionized states of the carboxylic acids are stabilized by intramolecular electrostatic interactions with neighboring counterions, resulting in low pKa values. 7–10 For example, the pKa values of carboxylic acids (Asp 85) on the proton pathway of bacteriorhodopsin, a light-driven proton pump, are decreased to as low as 2.5 by electrostatic interactions with Schiff bases and/or guanidium ions (Figure 1A). Another acid on the pathway (Asp 96) are increased to as high as 11 because of hydrophobic microenvironment around the acids (Figure 1A). Furthermore, the pKa value of each carboxylic acid in the pathway increase and decrease reversibly by more than four log units in response to protein conformational changes induced by photoisomerization of retinal (Figure 1A).1,2,9,10 Such pKa modulation is crucial for the functions of these proteins.1–10

Synthetic materials with sophisticated functions like those of proteins are of great interest as low-cost robust materials for molecular/ion separation, chemical conversion, and medical applications. Synthetic polymers containing Brønsted acids that show reversible pKa shifts have been synthesized and used for reversible capture of target molecules,11 ions,12–16 and gases. 17–20 However, all the pKa modulation of these acids are achieved as a response to the hydrophilic-to-hydrophobic transition of the microenvironment around the acids12-20 and/or by designing structure monomers bearing the acids12,13,15,19. Electrostatic attractions and repulsion have been widely used in polymer materials to aid recognition of target molecules11,14–22 and to produce hydrogels with desired mechanical properties and self-healing capability.23–27 However, few synthetic materials with Brønsted acids whose pKa values can be reversibly modulated by electrostatic interactions have been reported. Although modulation of pKa values by electrostatic interactions with counterions is a viable strategy, the interactions are strongly shielded by water molecules in aqueous media.28–30 Recently, Chen et al.30 reported that elec-

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trostatic interactions can be enhanced if the ions are localized at hydrophobic interfaces even in aqueous media. It is therefore expected that the pKa values of carboxylic acids in aqueous media could be strongly modulated by imprinting both countercations and the acids into the hydrophobic microdomains in a material. Here, we prepare temperature-responsive hydrogel nanoparticles (NPs) containing carboxylic acids whose pKa values can be reversibly modulated by electrostatic interactions with countercations in the particles. We use the collapsed-to-swollen (dehydrated-to-hydrated) phase transition of hydrogel NPs, which leads to large volume increases at temperatures below the volume phase transition temperature (VPTT),31 to reversibly tune the microenvironments and polymer structure around the acids in the NPs. We hypothesize that the pKa values of carboxylic acids in the collapsed NPs could be modulated by electrostatic interactions with countercations if the cations can be imprinted into hydrophobic microdomains around the acids.32–36 In addition, the removal of the ability to modulate pKa upon swelling and hydrating the NPs by lowering the temperature is investigated.37 Reversible pKa modulation may be achieved if the imprinted electrostatic interactions can be regenerated by collapsing the NPs after swelling (Figure 1B).

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upon conformation changes of the protein. (B) Preparation of nanoparticles (NPs) bearing carboxylic acids whose pKa values is modulated by electrostatic interactions with counterions. pKa of the acids can be reversibly modulated if the imprinted electrostatic interaction can be removed and regenerated upon swell and collapse of the NPs. Hydrophobic and hydrophilic microdomains/environments are shown in gray and sky blue, respectively.

RESULTS AND DISCUSSION Effect of hydrophobicity around acids on pKa of the acids We selected an N-isopropylacrylamide (NIPAAm) polymer (polyNIPAAm) as the base material for the NPs. PolyNIPAAm has a lower critical solution temperature (LCST) of 32 °C in water.38 PolyNIPAAm NPs can be prepared by pseudo-precipitation polymerization in water at temperatures higher than the LCST.39,40 NPs of poly(AAcco-NIPAAm) (AAc NPs) were prepared by polymerization of NIPAAm (95 mol%), acrylic acid (AAc) (3 mol%) as a monomer with a carboxylic acid group, and a crosslinker [N,N'-methylenebisacrylamide (BIS) (2 mol%)] in water (pH 3) at 70 °C (Table S1, A3.0D0.0).14,15,40 The hydrodynamic diameter of the NPs determined by dynamic light scattering (DLS) measurements indicated that the polymerized NPs had a VPTT of around 40 °C (Figure S1, A3.0D0.0). The apparent pKa value of the carboxylic acid groups in the NPs determined by acid–base titration and the Henderson–Hasselbalch equation was 7.3 in the dehydrated collapsed-phase NPs at 75 °C (above the VPTT). This pKa value is 2.1 log units higher than that of the hydrated swollen-phase NPs at 10 °C (below the VPTT, pKa = 5.2) (Figure 2A and S2, and Table S2, A3.0D0.0). The titration results also indicated that amount of incorporated carboxylic acids in the NPs are almost equal to the feed of acrylic acids (Table S2, A3.0D0.0). We replaced NIPAAm with more hydrophilic dimethylacrylamide (DMAAm) to prepare a control polymer that does not show volume phase transition (VPT) behavior. Poly(AAc-co-DMAAm) was prepared by polymerization of DMAAm and AAc in water (pH 3) at 70 °C [pDMAAm (AAc), Table S3]. We confirmed that pDMAAm (AAc) did not show temperature-dependent dehydration and the pKa values of the carboxylic acid in the copolymer were around 5 at both 75 and 10 °C [Table S4, Figures S3 and S4, black diamonds].

Figure 1. (A) pKa modulation of carboxylic acids on the proton pathway of bacteriorhodopsin by electrostatic interaction with counterions (Asp 85) and hydrophobic microenvironment (Asp 96). The modulation can be reversibly removed

These results confirm that the exceptionally high pKa values for aliphatic carboxylic acids in dehydrated AAc NPs are attributable to a proton-imprinting effect14,15: the NPs were polymerized at pH 3, which is lower than the pKa of AAc in water; therefore, AAc was fully protonated during polymerization. A collapsed polyNIPAAm particle has both hydrophobic and hydrophilic microdomains,41 but the protonated monomer and/or growing NIPAAm


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oligomers containing protonated AAc can easily be incorporated into hydrophobic microdomains formed in the dehydrated particles during polymerization. The bonds between the carboxylate anions and protons are stabilized by hydrogen bonding with oxygen atoms of the carbonyl groups of amides and acids in the hydrophobic microdomains, resulting in the high pKa values of the acids (Figure 2B)14,15,42. Effect of incorporation of countercations on pKa of the acids To modulate the pKa values of the acids in NPs using electrostatic interactions, various amounts of N-[3(dimethylammonium)propyl]acrylamide chloride (DMA), which has a tertiary ammonium ion on its side chain, were incorporated into NPs during copolymerization. NPs of poly(AAc-co-DMA-co-NIPAAm) [(AAc + DMA) NPs] were prepared under the conditions used to synthesize the AAc NPs (Table S1). All the NPs showed a VPT at temperatures between 40 and 60 °C (Figure S1). The titration curves and pKa values of the carboxylic acids in NPs that were copolymerized with various amounts of AAc and DMA are summarized in Figure S2 and 2C, and Table S2. For hydrophilic swollen NPs (at 10 °C), pKa of the carboxylic acids decreased only slightly upon ammonium group incorporation (Figure 2A and 2C). The pKa values are almost the same as that for the case without countercations (pKa = 5), indicating that electrostatic interactions between carboxylate anions and ammonium cations are weak in the hydrated NPs because charges are shielded by solvation water molecules on the ions (Figure 2C). The pKa values of acids in the collapsed phase (at 75 °C) strongly depended on the amount of ammonium ions incorporated into the NPs (Figure 2A and 2C). The pKa value of the carboxylic acids in the hydrophobic collapsed NPs was around 7 when the amount of ammonium groups was smaller than that of carboxylic acids. The pKa value dropped by 1.5–2 log units when the amount of incorporated ammonium ions was the same or larger than that of carboxylic acids (Figure 2C). The titration results also indicated that amount of incorporated ammonium ions in the NPs are slightly smaller than the feed of DMA, although incorporation of acids are almost equal or slightly higher than the feed of AAc (Table S2). The relatively low incorporation of DMA might be due to ionization of the DMA during polymerization process. Ionized monomer and/or growing oligo(NIPAAm-co-DMA) would show relatively low hydrophobic interaction with growing NPs, preventing the monomers and the oligomers to be completely incorporated into the NPs.14,15

Figure 2. (A) The pKa values of carboxylic acids in AAc NPs (black) and (AAc + DMA) NPs (green) as a function of temperature. The content of AAc in AAc NPs was 3 mol% and those of AAc and DMA in (AAc + DMA) NPs were 3 and 4 mol%, respectively. (B) Proposed mechanism of formation and the temperature-responsive pKa shift of AAc NPs. Hydrophobic and hydrophilic microdomains/environments are shown in gray and sky blue, respectively. (C) Effect of stoichiometric ratios of AAc and DMA on pKa values of carboxylic acids in NPs at 75 °C (red) and 10 °C (blue).

Poly(AAc-co-DMA-co-DMAAm) [pDMAAm (AAc + DMA), Table S3], a control polymer that does not show VPT behavior, did not display a marked pKa decrease on incorporation of cationic monomers at both high and low temperatures (pKa ~5) (Table S4 and Figure S4). These results indicate that pKa of the carboxylic acids can be modulated by electrostatic interactions with a stoichiometric amount of countercations when the acids are incorporated into dehydrated collapsed NPs. Effect of structure of countercation on pKa of the acids The pKa modulation of carboxylic acids by countercations was enhanced by incorporating cationic monomers with various chemical structures into AAc NPs. N-(3Ammoniumpropyl)methacrylamide chloride (APM), DMA, (3-acrylamidopropyl)trimethylammonium chloride (ATA), and N-(3-methacrylamidopropyl)guanidinium chloride (GUA) were used as monomers with primary ammonium, tertiary ammonium, quaternary ammonium, and guanidinium groups, respectively (Figure 3A). NPs of poly(AAc-co-cations-co-NIPAAm) [(AAc + cations) NPs] were prepared under the conditions used to synthesize AAc NPs (Table S5). The pH of the polymerization solution was adjusted to 3 with HCl so that AAc was protonated and could be incorporated into hydrophobic microdomains. The incorporated amounts of acid and base were almost equal in each NP sample (Table S6). The VPT behavior of the NPs was investigated by DLS. All the NPs showed a VPTT of 35–55 °C (Figure S5).



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the pKa values were 5.1 and 5.0, respectively (Figure 3B). These results suggest that the pKa modulation of carboxylic acids can be enhanced by a combination of electrostatic interactions and hydrogen-bonding ability of countercations. 43-46 The interactions between each cationic monomer in an aqueous solution and AAc NPs were investigated to study the mechanism of the pKa modulation. ATA, DMA, APM, and GUA were incubated with AAc NPs at 70 °C and the amount of each monomer adsorbed on the NPs was quantified after ultrafiltration. ATA, which is not a hydrogenbond donor, was not adsorbed on the NPs, whereas 55% of APM and 60% of GUA, which are good hydrogen-bond donors, were adsorbed on the NPs (Figure 3C). These results indicate that the quaternary ammonium ions on ATA and/or on the growing oligo(ATA-coNIPAAm) did not interact with growing (AAc + cations) NPs and were localized and immobilized on the hydrophilic exterior of the NPs.15,47 Therefore, electrostatic interactions between ATA and acids were shielded by solvation water molecules. In contrast, guanidinium groups on GUA and/or the growing oligo(GUA-co-NIPAAm) interacted with growing (AAc + cations) NPs and were immobilized inside the NPs near the hydrophobic domains, where the acids were incorporated, through hydrogen bonding.15,47 Therefore, pKa modulation of carboxylic acids was enhanced by countercations with groups that were good hydrogen donors (Figure 3D).

Figure 3. (A) Chemical structures of basic monomers copolymerized in NPs as countercations to carboxylic acids. (B) Effect of chemical structure of the countercation on pKa values of carboxylic acids in NPs at 75 °C (red) and 10 °C (blue). (C) Amounts of adsorbed cationic monomers on collapsed AAc NPs. Measurements were performed three times, and the average values and standard deviations are shown. (D) Proposed mechanism of formation and the temperatureresponsive pKa shift of (AAc + GUA) NPs. Hydrophobic and hydrophilic microdomains/environments are shown in gray and sky blue, respectively.

The titration results in Figure 3B show that the pKa values of the carboxylic acids in the hydrophilic swollen NPs (10 °C) were almost the same regardless of the structure of the countercation (pKa 4.8–4.9; Figure 3B). In contrast, the pKa values of the acids in collapsed NPs depended on the countercation structure. When quaternary ammonium ions were incorporated into the NPs, pKa of the carboxylic acids was 5.7. When primary ammonium and guanidinium ions, which are good hydrogen donors for hydrogen bonding, were incorporated into the NPs,

Although the pKa of carboxylic acid groups in the collapsed-phase NPs changed from 7 to 5 as a result of electrostatic interactions with guanidinium ions, the pKa value of the collapsed NPs was not lower than that of the swollen NPs and isobutyric acid (pKa ~5),14,15 a smallmolecule analog of AAc in the polymer chains, in water. The pKa values of carboxylic acids in bacteriorhodopsin can be modulated to as low as 2.5 by countercations via hydrogen bonding in a hydrophobic microdomain (Figure 1A).1,2,9,10 The relatively poor pKa modulation effect of carboxylic acids by guanidinium ions in the (AAc + GUA) NPs might result from the acid and cation distributions in the NPs: AAc can be incorporated into the hydrophobic microdomains because the polymerization pH is lower than the pKa values of AAc and AAc in the growing polymer chains.14,15 However, although GUA can be immobilized inside the NPs near the hydrophobic domains, GUA incorporation in the hydrophobic microdomains may not be preferred because the guanidinium groups are fully ionized at the polymerization pH. The pKa of the carboxylic acid groups can therefore only be modulated by hydrated cations and possibly through an indirect hydrogen-bonding network by countercations with good hydrogen donors imprinted in the hydrophilic microdomains (Figure 3C). Protection of counterions during polymerization To incorporate guanidinium ions into the hydrophobic microdomains in which the acids are incorporated, we protected the guanidine groups in GUA with hydrophobic


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tert-butoxycarbonyl (Boc) groups (BGUA) and then removed the Boc protecting groups after copolymerization of NPs of poly(AAc-co-BGUA-co-NIPAAm) [(AAc + BGUA) NPs] (Figure 4A and Table S7). The NPs were polymerized under weakly acidic conditions (pH 4) so that AAc could be incorporated into the hydrophobic microdomains and to prevent decomposition of the Boc protecting groups via acid-catalyzed hydrolysis during polymerization.48 The guanidinium groups were deprotected with HCl after NP purification. The incorporation of BGUA and removal of Boc groups were confirmed by 1H NMR spectroscopy (Figure S6). The pKa of the carboxylic acid groups in the deprotected (AAc + BGUA) NPs was determined after deprotection. In the collapsed state (75 °C), the cations incorporated into the NPs via Boc protection substantially modulated the pKa value of the carboxylic acids. The pKa values of the acids in the NPs were about 0.5 log units lower than those of the acids in NPs polymerized with ionized countercations (Figure 4B and Table S5). Importantly, when counter cations were incorporated via Boc protection, the pKa vaue of the collapsed NPs (75 °C) was lower than that of the swollen NPs (10 °C). This suggests that the protected guanidinium groups were incorporated and imprinted in the hydrophobic microdomians near the carboxylic acids during polymerization and modulated the pKa value of the carboxylic acid groups by formation of salt bridges with GUA after deprotection (Figure 4A).43,49 To confirm this mechanism, we examined interactions between BGUA and the NPs. Almost 100% of BGUA was adsorbed on collapsed NPs (Figure 4C), indicating that BGUA can be effectively incorporated into the hydrophobic domains of growing NPs and imprinted in the hydrophobic microdomains, presumably through hydrophobic and hydrogen-bonding interactions (Figure 4A). Preservation of imprinted microstructure The VPT behavior of NPs prepared via Boc deprotection differed from that of the other NPs including the ones before deprotection. The NPs after deprotection did not shrink sufficiently, even at temperatures above the VPTT (75 °C) (Figure 4D and S7). This might be a result of the increased osmotic pressure in the collapsed NPs after deprotection, because salt bridges formed in the hydrophobic microdomains in the NPs (Figure 4A). The swelling of NPs at high temperature may disrupt preservation of imprinted hydrophobic microstructure around the acids and BGUA in the NPs, preventing ideal pKa modulation of the acids through electrostatic interactions. To further enhance pKa modulation by guanidinium groups, we attempted to maintain the hydrophobic microstructure around the acids and BGUA, which was constructed during polymerization, by replacing a certain amount of NIPAAm with tert-butylacrylamide (TBAAm), which is more hydrophobic than NIPAAm [(AAc + BGUA + TBAAm) NPs, Table S7]. The incorporation of TBAAm prevented NP swelling at high temperatures (Figures 4D

and S7). As expected, the pKa value of the carboxylic acids in the collapsed NPs at 75 °C decreased to about 4.0 (Figure 4B and Table S8).

Figure 4. (A) Proposed mechanism of formation and the temperature-responsive pKa shift of (AAc + BGUA + TBAAm) NPs. Hydrophobic and hydrophilic microdomains/environments are shown in gray and sky blue, respectively. (B) Effects of Boc protection, TBAAm, and BIS on the pKa values of carboxylic acids in NPs with GUA as a counter cation at 75 °C (red) and 10 °C (blue). (C) Amounts of adsorbed GUA and BGUA on collapsed AAc NPs. (D) Hydrodynamic diameters of NPs normalized by NP diameters at 10 °C as a function of temperature. Measurements were performed three times, and the average values and standard deviations are shown.

Alternatively, swelling of NPs at temperatures above the VPTT (75 °C) can be prevented by increasing the amount of crosslinker from 2 to 5 mol% [(AAc + BGUA + BIS) NPs, Table S7].14 Strong pKa modulation (pKa = 4.0),comparable to that for (AAc + BGUA + TBAAm) NPs (pKa = 4.0), was observed for the highly crosslinked NPs, indicating that the imprinted hydrophobic microstructures in the NPs can be stabilized by crosslinking the structure (Figure 4B and Table S8). However, the pKa values of acids in the NPs at low temperatures (10 °C) also decreased from 4.7 to about 4.4 upon increasing the amount of crosslinker (Figure 4B and Table S8). The pKa was much lower than that of isobutyric acid in water (pKa ~5),14,15 which indicates that the imprinted microstructures were not fully denatured because of high crosslinking and that the pKa values of the acids were still modulated by guanidinium ions even at temperatures below the VPTT (Figure S7 and S8).50 Reversibility of pKa modulation



To confirm the reversibility of pKa modulation by electrostatic interaction with counterions, the pKa values of the NPs were measured several times at temperatures below (10 °C) and above (75 °C) their VPTTs. Figure 5 shows that pKa modulation by countercations imprinted during polymerization was completely reversible. This suggests that structural information imprinted in the NPs during polymerization process is memorized, maybe in the form of a monomer sequence, configuration and entanglement of polymer chains, crosslinking structure, and/or interactions between side chains, even after particle swelling (denaturation).14,15,37 The electrostatic interactions between carboxylate ions and countercations can therefore be accurately regenerated by heating. A time course of reversible pKa shift during the heating and cooling cycle is shown in Figure S8. The pKa shifted up and down in a few minutes. The kinetics of the pKa shift (τ < min) was much slower than the reported kinetics of VPT of hydrogel NPs (τ < µs)50 because heating and cooling process limits the kinetics of VPT and pKa shift process in this experiment.

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ues of carboxylic acids in the NPs because they interacted strongly with growing NPs. The greatest pKa modulation ∆pKa of −3.3 log units was achieved when guanidium monomers were protected by hydrophobic groups prior to polymerization. This indicates the importance of imprinting countercations in the hydrophobic microdomains in the dehydrated NPs. The lowest pKa value of 4.0, which is one log units lower than that of carboxylic acids in swollen NPs and a small-molecule analog of the acids (isobutyric acid, pKa = 5.0), was achieved as a result of strong electrostatic interactions with guanidinium ions in the hydrophobic microdomains in the NPs. The pKa modulation of the acids could be reversibly and completely removed by inducing a temperature-dependent dehydrated-to-hydrated phase transition of the NPs. The greatest pKa modulation achieved in this study was approximately 3.3 log units (from 7.3 to 4.0) at 75 °C. The modulation range was smaller than that of carboxylic acids at active sites of bacteriorhodopsin (from 11 to 2.5, ∆≈ = −8.5 log units) (Figure 1A),1,2 but comparable to that observed for imidazolium groups of hemoglobin (from 8.0 to 4.7, ∆pKa = −3.3 log units).5,7,8 These principles of imprinting functional groups that facilitate electrostatic interactions to realize reversible pKa modulation are broadly applicable to other stimuliresponsive materials, such as photo-, electro-, pressure-, and ligand-responsive materials, and represent an integral step in the development of synthetic materials containing Brønsted acids whose pKa values can be reversibly modulated to a desired level. Such materials can be used to capture, transport, and separate target ions, gases, and molecules in response to external stimuli, and can therefore be used for separation media and to regulate acid–base equilibria.

Figure 5. Reversible shifts of pKa values of NPs in response to repeatedly switching the temperature between 10 and 75 °C.

CONCLUSION Temperature-responsive hydrogel NPs containing carboxylic acids whose pKa values can be reversibly modulated by intra-particle electrostatic interactions were prepared. We found that the pKa values of carboxylic acids in the NPs can be strongly modulated by countercations in the NPs only when localization of the cations in the particles is carefully controlled during polymerization. When the NPs were polymerized with acrylic acids in the absence of countercations, protonated acrylic acids were incorporated into hydrophobic microdomains in the dehydrated NPs, and the acids showed high pKa values. The pKa values were lowered when a stoichiometric amount of cationic monomers was incorporated into the dehydrated NPs. Cationic monomers with primary ammonium and guanidinium ions, which are good hydrogen donors for hydrogen bonding, effectively modulated the pKa val-

Experimental Section General. N-Isopropylacrylamide (NIPAAm; Wako Pure Chemical Industries, Ltd.) was purified by recrystallization from benzene/n-hexane and then dried in vacuo at room temperature. 2,2'-Azobis(isobutyronitrile) (AIBN; Wako Pure Chemical Industries, Ltd.) was purified by recrystallization from methanol and then dried in vacuo at room temperature. The polymerization inhibitor in N[3-(dimethylamino)propyl]acrylamide (DMA; Tokyo Chemical Industry Co., Ltd.) and (3acrylamidopropyl)trimethylammonium chloride solution (ATA; Sigma-Aldrich Co., LLC) was removed using an activated alumina column (Merck, Ltd.). N,N'Methylenebis(acrylamide) (BIS; Tokyo Chemical Industry Co., Ltd.), cetyltrimethylammonium bromide (CTAB; Wako Pure Chemical Industries, Ltd.), N,N'dimethylacrylamide (DMAAm; Tokyo Chemical Industry Co., Ltd.), N-tert-butylacrylamide (TBAAm; MRC Unitec Co., Ltd.), acrylic acid (AAc; Wako Pure Chemical Industries, Ltd.), N-(3-aminopropyl)methacrylamide hydrochloride (APM; Polyscience, Inc.), N,N'-di-Boc-1Hpyrazole-1-carboxamidine (Boc-Py; Wako Pure Chemical


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Industries, Ltd.), and N,N-diisopropylethylamine (DIEA; Tokyo Chemical Industry Co., Ltd.) were used as received. A dialysis membrane with a molecular weight cut-off (MWCO) of 12–14 kDa was purchased from Thermo Fisher Scientific. A cation-exchange resin (Muromac C1002-H, Muromachi Technos Co., Ltd.) was pretreated with 1 M NaOH and then washed with deionized water. An anionexchange resin (A2004-OH, Muromachi Technos Co., Ltd.) was pretreated with 1 M NaOH and then washed with deionized water. An Amicon Ultra 0.5-mL centrifugal filter (MWCO: 100 kDa) was purchased from Merck KGaA. Water was purified using a Direct-Q Ultrapure Water System (Merck, Ltd.). 1H NMR spectra were recorded using a JNM-ECZ400 (JEOL Ltd.) spectrometer at 400 MHz. Synthesis of Boc-protected guanidinium monomer (BGUA). BGUA was synthesized using a slightly modified version of the procedure reported by Lu and colleagues (Scheme S1).52 APM (800 mg, 4.47 mmol), Boc-Py (1.18 g, 3.80 mmol), and DIEA (1.40 mL, 8.00 mmol) were dissolved in a mixture of dichloromethane (10 mL) and acetonitrile (6 mL). The mixture was stirred at room temperature for 25 h under reflux in a nitrogen atmosphere. The reaction was quenched by adding dichloromethane (20 mL) and water (30 mL). The aqueous fractions were discarded and the organic fractions were collected and washed twice with brine (30 mL each time). The organic fractions were dried over MgSO4 and then the solvent was removed in vacuo. The crude product was purified by silica-gel column chromatography using ethyl acetate as the eluent; the pure product was obtained as a white solid. Yield: 1.15 g (79%). 1H NMR (CD3Cl) δ 11.53 (s, 1H), 8.50 (t, 1H), 7.38 (t, 1H), 5.81 (s, 1H), 5.30 (s, 1H), 3.50 (dt, 2H), 3.33 (dt, 2H), 2.00 (s, 3H), 1.71 (m, 2H), 1.51 (s, 9H), 1.47 (s, 9H) (Figure S10). Synthesis of guanidinium monomer (GUA). GUA was synthesized by a modified version of a reported procedure (Scheme S2).53 BGUA (600 mg, 1.56 mmol) was dissolved in 4 M HCl in dioxane (6 mL). The mixture was stirred for 20 h at room temperature. The product precipitated from the solution as a white solid, which was purified via filtration and washing with anhydrous dioxane. Yield: 357 mg (104%). 1H NMR (DMSO-d6) δ 8.05 (t, 1H), 7.70 (t, 1H), 7.65–6.80 (br, 4H), 5.68 (s, 1H), 5.33 (s, 1H), 3.16 (m, 4H), 1.85 (s, 3H), 1.65 (quin, 2H) (Figure S11). Preparation of poly(AAc-co-DMA-co-NIPAAm) (AxDy-NPs). A number of papers have been published about preparation of temperature responsive hydrogel particles containing both positive and negative charges.5456 In this study, polymerization condition, such as choice and concentration of surfactant and monomers, and polymerization pH, has been optimized to prepare stable NPs without yielding any aggregates during polymerization and after purification process. NIPAm [98 to (x – y) mol%], BIS (2.0 mol%), AAc (x mol%), and DMAPA (y mol%) were dissolved in water; the total monomer concentration was 312 mM. CTAB (6.21 mM), a surfactant, was added to the monomer solution. The solution pH was adjusted with HCl solution and monitored with a pH me-

ter. Nitrogen was bubbled through the reaction mixture for 30 min at 70 °C. AIBN (0.69 mM/2 vol% DMSO) was added and then polymerization was performed at 70 °C for 3 h in a nitrogen atmosphere (Table S1). The polymerized solution was purified by dialysis against an excess amount of water for 2 days and 1 mM HCl for 1 day (changed more than three times per day). Traces of counteranions and countercations were removed with a strong cation-exchange resin and strong anion-exchange resin, respectively. The resins were filtered out after exchange for 30 min. The concentration of the NP solution was determined from the weight of NPs obtained by lyophilization of 1 mL of the purified solution. Synthesis of poly(AAc-co-cations-co-NIPAAm) [(AAc + cations) NPs]. NIPAm (90.5 mol%), BIS (2.0 mol%), AAc (2.5 mol%), and various cationic monomers (5.0 mol%) were dissolved in water; the total monomer concentration was 312 mM. Monomers containing cationic groups, i.e., ATA, DMA, APM, and GUA, were used in this study. CTAB (6.21 mM) was added to the monomer solution. The solution pH was adjusted with HCl solution and monitored with a pH meter. Nitrogen was bubbled through the reaction mixture for 30 min at 70 °C. AIBN (0.69 mM/2 vol% DMSO) was added and then polymerization was performed at 70 °C for 3 h in a nitrogen atmosphere (Table S5). The polymerized solution was purified by dialysis against an excess amount of water (changed more than three times per day) for 2 days and 1 mM HCl for 1 day. Traces of counteranions and countercations were removed with a strong cation-exchange resin and strong anion-exchange resin, respectively. The resins were filtered out after exchange for 30 min. The concentration of the NP solution was determined from the weight of NPs obtained by lyophilization of 1 mL of the purified solution. Synthesis of poly(AAc-co-BGUA-co-NIPAAm) [(AAc + BGUA) NPs]. NIPAm [90.5 to (a – b) mol%], BIS (a mol%, a = 2 or 5), and AAc (2.5 mol%) were dissolved in water. TBAAm (b mol%, b = 0 or 20) dissolved in methanol (4 vol%) and BGUA (5.0 mol%) dissolved in methanol (3 vol%) were slowly added at room temperature; the total monomer concentration was 156 mM. CTAB (3.11 mM) was added to the monomer solution. The solution pH was adjusted with NaOH solution and monitored with a pH meter. Nitrogen was bubbled through the reaction mixture for 30 min at 70 °C. AIBN (0.35 mM/2 vol% DMSO) was added and then polymerization was performed at 70 °C for 3 h in a nitrogen atmosphere (Table S7). The polymerized solution was purified by dialysis against an excess amount of water (changed more than three times per day) for 3 days. Traces of counteranions and countercations were removed with a strong cation-exchange resin and strong anion-exchange resin, respectively. The resins were filtered out after exchange for 30 min. The Boc group was removed by adding 1 M HCl (10 mL) to the NP solution (90 mL); the reaction was performed for 12 h at 70 °C. The reaction solution was purified by dialysis against an excess amount of water for 1 day. Traces of counteranions and countercations were removed with a



strong cation-exchange resin and strong anion-exchange resin, respectively. The resins were filtered out after exchange for 30 min. The concentration of the NP solution was determined from the weight of NPs obtained by lyophilization of 1 mL of the purified solution. The introduction and removal of BGUA to/from the NPs were confirmed by 1H NMR spectroscopy (Figure S6). Synthesis of poly(AAc-co-DMA-co-DMAAm) (pDMAAm). DMAAm [(98 – c) mol%], AAc (2 mol%), and DMA (c mol%, c = 0 or 3.5) were dissolved in water (50 mL); the total monomer concentration was 312 mM. The solution pH was adjusted with HCl solution and monitored with a pH meter. Nitrogen was bubbled through the reaction mixture for 30 min at 70 °C. AIBN (0.69 mM/2 vol% DMSO) was added and then polymerization was performed at 70 °C for 3 h in a nitrogen atmosphere (Table S3). The polymerized solution was purified by dialysis against an excess amount of water for 1 day (changed more than three times per day). Traces of counteranions and countercations were removed with a strong cation-exchange resin and strong anion-exchange resin, respectively. The resins were filtered out after exchange for 30 min. The concentration of the polymer solution was determined from the weight of polymer obtained by lyophilization of 1 mL of the purified polymer. pH measurements. A SevenMultiTM pH meter equipped with pH probes (InLab® Routine Pro, MettlerToedo) was calibrated with three standard buffers (pH 4.0, 7.0, and 9.2) prior to use. The solution temperature was controlled with a water bath. The solution temperature and pH were measured every 3 s and monitored in situ with a personal computer. Quantification of ionic groups and the pKa values of carboxylic acid. The amounts of carboxylic acid groups and countercations in the NPs were determined by titration (Figure S0, S2, S3, S12 and S13). An NP solution (20 mg/mL) was titrated with 0.1 M NaOH at 30 °C while stirring at 500 rpm. NPs containing TBAAm were titrated at 0 °C. The temperature of the polymer solution was controlled using a water bath. The carboxylic acid groups and countercations in the NPs were protonated by adjusting the solution pH to 2.0–2.5 with HCl before the measurements. The pKa values of the carboxylic acids in collapsed and swollen NPs were determined using the Henderson–Hasselbalch equation. The pH values of the NP solution (4 mg/mL) at 10 and 75 °C were measured after adding HCl (1.0 equiv with respect to the countercations in the NPs) and NaOH (0.5 equiv with respect to the carboxylic acid groups in the NPs). The pH values of the NP solutions at 10 and 75 °C were defined as the pKa values of the carboxylic acids in the swollen and collapsed NPs, respectively. Similar experiments were performed for pDMAAm. Quantification of hydrodynamic diameters. The hydrodynamic diameters and polydispersity indexes of the NPs were measured using a DLS instrument (Zetasizer Nano, Malvern Instruments Ltd.). The ion-exchanged solutions were sealed in quartz cells filled with argon. The

Page 8 of 17

solutions were equilibrated at each temperature for 2 min prior to the measurements. Measurements were performed on the NP solution (0.1 mg/mL) after adding HCl (1.0 equiv with respect to the countercations in the NPs) and NaOH (0.5 equiv with respect to the carboxylic acid groups in the NPs) (Figure S1, S5, S7 and S8). Quantification of the amount of adsorbed cationic monomers on NPs. A3.0D0.0 NPs were dissolved in water at a concentration of 30 mg/mL. Various monomers, i.e., ATA, DMA, APM, GUA, and BGUA [2.78 mM (moladd)] were added to the NP solution. The solution was adjusted to the same pH as that used for the polymerization with HCl or NaOH solution, and then the pH was monitored with a pH meter. After incubation for 1 h at 70 °C, the NP solution (400 µL) was filtered by centrifugal ultrafiltration (Amicon Ultra 0.5-mL centrifugal filter, Merck KGaA, MWCO: 100 kDa) for 15 min at 40 °C and 14.0 kG. The amount of monomer in the filtrate (molfiltrate) was quantified by 1H NMR spectroscopy. After removal of the water in the filtrate by lyophilization, the monomer was dissolved in DMSO-d6 containing 7.75 mM of chloroform; molfiltrate was determined from the ratio of the integrated values for monomers and chloroform obtained by 1 H NMR spectroscopy. The amount of adsorbed cationic monomers on NPs was calculated as amount of adsorbed cationic monomers (%) = (moladd – molfiltrate)/moladd × 100 Measurements were performed three times.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *[email protected]

Author Contributions All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT This research was supported by JSPS KAKENHI Grant Number JP15H05486, Japan, MEXT Innovative Areas of “Fusion Materials”, Grant Number 25107726, Japan, and JST-ALCA Grant Number JPMJAL1403, Japan.

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Page 11 of 17 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


TOC figure


11


A

Page 12 of 17

hv

Asp 96

Asp 96 HO pKa = 11

H

O

Schiff Base

pKa < 6

NO

O

NH O

HO

O

O

6

Asp 85 pKa = 2.5

B

Asp 85 pKa = 6.9

O O HN

+

HN HN

O

O

+

OH

+

O HN NH

N-isopropylacrylamide (NIPAAm)

N,N’-methylene bisacrylamide (BIS) Collapsed state

Free Radical Polymerization

Imprinted Electrostatic Interaction

Acrylic acid (AAc)

Cationic monomer

Swollen state Phase Transition

Reversible pKa Shift


Hydration of Ions

Page 13 of 17

pK a of carboxylic acid

A

8

B

AAc AAc + DMA

O

7

O HN

6

HN

+

HN

4 10

C

O

+

O OH

5

NI PAAm

pK a of carboxylic acid

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


30 50 Temperature (ºC)

8

6

AAc

70

75ºC (AAc - 2.0 mol%) 10ºC (AAc - 2.0 mol%) 75ºC (AAc - 3.0 mol%) 10ºC (AAc - 3.0 mol%) 75ºC (AAc - 5.0 mol%) 10ºC (AAc - 5.0 mol%)

7

BIS

O

Polymerization

O OH

at 70ºC at pH 3

Phase transition

N

H

HN

O O O

5

4

+ H T = 75ºC 0

1 2 DMA (mol) / AAc (mol)


T = 10ºC


A

O

O

HN

O

O HN

HN

HN

H

N

N H

Cl

B

Cl

N-[3-(dimethyl ammonium)propyl] acrylamide chloride (DMA)

(3-acrylamido propyl) trimethyl ammonium chloride (ATA)

N

H

H

75ºC

5.5

10ºC

5.0 4.5 4.0

D

AAc + DMA

AAc + APM

AAc + GUA

O O

HN

BIS

H

(3-methacrylamido amidopropyl) guanidnium chloride (GUA)

80 60 40 20 0

ATA

O

+

O

+

DMA APM GUA

HN

OH

H HN

NIPAAm

N

H Cl

O

HN

+

HN

H

N

100

Amounts of adsorbed monomers (%)

7.0

AAc + ATA

HN

Cl

methacrylamide chloride (APM)

C

AAc

H

N-(3-ammonium propyl

8.0

pKa of carboxylic acid

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 17

AAc

GUA

Cl

H

N N

H

H

HN HN

H H N H O

Polymerization Cl

at 70ºC at pH 3

H HN HN

H NH

O OH

O

HN

Purification

H HN

by dialysis and ion exchange

HN

H NH

O O

H N

Phase transition HN

O O O N H


O


O O

HN

+

HN

O

HN

+

O

O

+

HN

OH

Boc

HN

NIPAAm

BIS

AAc

N

BGUA

N

Boc

H NH

H

HN

H N H O

O

Polymerization

OH

at 70ºC at pH 4

HN

Boc

N Boc O

H N

O

Deprotection

O

HCl aq.

HN H

H N

Phase transition

H NH H N

O

O

N H

N H

O

O

N H

T = 75ºC 5.5

75ºC 10ºC

5.0

4.5

4.0 AAc + GUA

AAc + BGUA

AAc AAc + BGUA + BGUA + TBAAm + BIS

C

100 80 60 40 20

0 ACS Paragon Plus Environment GUA BGUA

T = 10ºC

D

1.2

Normalized diameter (nm/nm)

B

Amounts of adsorbed monomers (%)

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

A

pKa of carboxylic acid

Page 15 of 17

1.0

AAc + GUA AAc + BGUA AAc + BGUA + TBAAm

0.5

0.0 10

30 50 Temperature (ºC)

70


8.0

AAc

7.0 pKa of carboxylic acid

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

Page 16 of 17

5.5

AAc + DMA

5.0

AAc + GUA

4.5 4.0 3.5

AAc + BGUA + TBAAm 10

75

10

75

10

75

10

75

Temperature (ºC)


10

75

Page 17 of 17 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


Imprinted Electrostatic Interaction

H NH HN

H N H O

O

Low pKa O

H N

H NH HN H

H N

O

OH

High pKa

N H

O N H

Reversible pKa Modulation


O

Reversible pKa Modulation of Carboxylic Acids in Temperature

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