Topology Effect of AIEgen-Appended Poly(acrylic acid) with

Jun 4, 2018 - We recently reported that tetraphenylethene-appended poly(acrylic acid) derivatives (e.g., PAA-TPE0.02) can serve as fluorescent Ca2+ ...
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Letter Cite This: ACS Macro Lett. 2018, 7, 711−715

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Topology Effect of AIEgen-Appended Poly(acrylic acid) with Biocompatible Segments on Ca2+-Sensing and Protein-AdsorptionResistance Properties Fumitaka Ishiwari, Minami Sakamoto, Satoko Matsumura, and Takanori Fukushima* Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan S Supporting Information *

ABSTRACT: We recently reported that tetraphenyletheneappended poly(acrylic acid) derivatives (e.g., PAA-TPE0.02) can serve as fluorescent Ca2+ sensors in the presence of physiological concentrations of biologically relevant ions, amino acids, and sugars. However, in the presence of basic proteins such as albumins, the Ca2+-sensing property of the polymer is significantly impaired due to the nonspecific adsorption of protein molecules, which competes with binding to Ca2+. To solve this problem, we explored new designs by focusing on the polymer-chain topology of PAA-TPE0.02 with biocompatible segments. Here, we report the Ca2+-sensing and protein-adsorption-resistance properties of various types of PAATPE0.02 copolymers with a poly(oligoethylene glycol acrylate) (polyOEGA) segment, featuring a random, diblock, triblock, or 4armed-star-block structure. Through this study, we show an interesting topology effect; i.e., a branch-shaped PAA-TPE0.02-copolyOEGA with biocompatible segments at every terminal (i.e., 4-armed-star-block copolymer) exhibits both good Ca2+-sensing and protein-adsorption-resistance properties. he development of fluorescent sensors that can detect biologically meaningful chemical or physical signals is an important subject in physiology and pathology. For this purpose, polymer-based sensors1−24 have attracted increasing attention because their sensing properties can be readily tuned by, for example, changing the composition ratios of the polymer segments.1−18 Another striking feature of polymer sensors, distinct from small molecular sensors, is that they can be used not only in a solution state but also in various forms of insoluble states1−6 such as thin-films,1−5 nano and microparticles,6,9,11,19−24 gels,10,14,17 and devices.1−5,16 Uchiyama and co-workers developed copolymer-based fluorescent thermometers by incorporating an N-{2-[(7-N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazol-4-yl](methyl)amino}ethyl-N-methylacrylamide (DBD-AA) unit into the side chains of thermally responsive poly-N-n-propylacrylamide (NNPAM), which allows in vivo imaging of intracellular temperature.7,8 The temperature-sensitivity of the copolymers can be finely tuned by changing the composition ratio of NNPAM and DBD-AA units. Takeuchi and co-workers reported long-term in vivo glucose-monitoring using a fiber-shaped copolymer gel composed of biocompatible polyacrylamide with a glucoseresponsive fluorescent unit.10 These examples reflect the advantages of polymer-based sensors. Inspired by the structure of an extracellular Ca2+-sensing receptor (CaSR) with a particular cluster-like arrangement of carboxylic acids for Ca2+-binding, we recently developed a new polymer-based Ca2+ sensor (PAA-TPEx, Figure 1a) using

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© XXXX American Chemical Society

poly(acrylic acid)(PAA) derivatives carrying a few mol % of tetraphenylethene (TPE) units as an aggregation induced emission luminogen (AIEgen).17 In the presence of millimolarorder Ca2+, PAA-TPEx undergoes single-chain aggregation,18 which in turn triggers the aggregation of TPE units, resulting in the enhancement of fluorescence intensity (Figure 1b). By changing the TPE composition ratio (x) of PAA-TPEx, the dissociation constant (Kd) for Ca2+ can be tuned in a range of millimolar order. For instance, PAA-TPE0.02 acts as an excellent fluorescent Ca2+ sensor in the presence of physiological concentrations of biologically relevant ions, amino acids, and sugars. Importantly, cross-linking of PAA-TPE0.02 gave rise to a gel that allows the imaging of extracellular Ca2+, which has recently been recognized as a first messenger.17 However, there remains a problem to solve; in the presence of basic proteins such as albumins, PAA-TPE0.02 becomes fluorescent, presumably due to single-chain aggregation through the formation of polyion complexes with proteins.17 To date, although random or diblock copolymers7−13,15,19−24 with sensing and biocompatible segments25−30 have been prepared for use in physiological conditions, polymer topology has scarcely been a focus of attention in the development of polymer-based biosensors. This situation is in contrast to the case of protein functionalization using biocompatible polymers Received: April 18, 2018 Accepted: May 31, 2018

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DOI: 10.1021/acsmacrolett.8b00291 ACS Macro Lett. 2018, 7, 711−715

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ACS Macro Letters

GA), featuring a random (1), diblock (2), triblock (3), or 4armed-star-block (4) structure (Figure 1c−f), and investigated their Ca2+-sensing and protein-adsorption-resistance properties, in terms of topology. Through this paper, we show an interesting topology effect that branch-shaped PAA-TPE0.02-copolyOEGA with biocompatible segments at every terminal (i.e., 4-armed-star-block copolymer 4) exhibits both good Ca2+sensing and protein-adsorption-resistance properties. Among biocompatible monomers, we chose oligoethylene glycol acrylate (OEGA, 9, see also Figure S1a) with a numberaveraged molecular weight (Mn) of 480 Da. OEGA exhibits good copolymerizability for acrylic monomers, and its nonionic nature is advantageous for precise structural analysis of the resulting copolymers by size-exclusion chromatography (SEC). To discuss the topology effect of PAA-TPE0.02-co-polyOEGA, we designed random (1), diblock (2), triblock (3), and 4armed-star-block (4) copolymers with a similar molecular weight and degree of OEGA content (Figure 1c−f). The synthesis of these polymers was achieved by reversible addition−fragmentation chain-transfer (RAFT) polymerization.37−40 As shown in Figure S1a,41 copolymerization of acrylate monomers 7, 8, and 9 having TPE, t-butyl, and OEGA groups, respectively, in the presence of bifunctional RAFT reagent 1039 gave 11, the precursor of random copolymer 1. The terminal trithiocarbonate groups and t-butyl groups in the main chain were successively removed by radical-induced reduction using N-ethylpiperidine hypophosphite (EPHP)40 and hydrolysis with trifluoroacetic acid (TFA) to afford random copolymer 1. For the synthesis of diblock copolymer 2 (Figure S2a), monomer 9 was first polymerized using monofunctional RAFT reagent 13 to give macro RAFT initiator 14.41 RAFT copolymerization of 7 and 8 in the presence of 14 afforded precursor diblock copolymer 15, which was converted into 2 by procedures similar to those for the synthesis of random copolymer 1 via successive treatments with EPHP and TFA (Figure S2a).41 Copolymerization of 7 and 8 with bifunctional RAFT reagent 10 gave polymer 17 (Figure S3a), which provided a macro initiator as well as the central segment for ABA-type triblock copolymer 3.41 RAFT polymerization of 9 with 17 afforded 18 having side segments of poly(OEGA). Removal of the terminal trithiocarbonate groups, followed by acid hydrolysis of the t-butyl groups, resulted in triblock copolymer 3. Using procedures essentially identical to those for the synthesis of 6, core−shell-type, 4-armed-star-block copolymer 4 was obtained, except that tetrafunctional RAFT agent 10 was used (Figure S4a).41 We also prepared a linear (5) and a 4-armed-star-block polymer (6) of PAA-TPE0.02 as reference polymers to evaluate protein-adsorption-resistance

Figure 1. (a) Chemical structure of PAA-TPEx. (b) Schematic illustration of Ca2+-sensing with PAA-TPEx. Chemical structures and schematic illustrations of (c) random (1), (d) diblock (2), (e) triblock (3), (f) 4-armed-star-block (4) copolymers of PAA-TPE0.02 and polyOEGA investigated in this work, and chemical structure and schematic illustrations of (g) linear (5) and (h) 4-armed-star (6) PAATPE0.02 for reference polymers.

such as poly(ethylene glycol) (PEG), in which the topology of PEG is known to largely affect the suppression of immunogenicity and aggregation behavior.31−36 For instance, the attachment of a branched-shaped PEG to proteins leads to enhancement of the stability of proteins toward a change in pH and temperature as well as proteolytic digestion.31−35 We hypothesized that, similar to the case of protein PEGylation, the topology of copolymer-based biosensors with biocompatible segments should be important for achieving both excellent sensing and biocompatibility. In this study, we synthesized various types of copolymers of PAA-TPE0.02 and biocompatible poly(oligoethylene glycol acrylate) (polyOE-

Table 1. Structures and Properties of Copolymers (1−4) and Reference Polymers (5 and 6) polymer

topology of polymers

1 2 3 4

random copolymer diblock copolymer triblock copolymer 4-armed-star-block copolymer linear polymer 4-armed-star polymer

5 6

molar ratio of PAA-TPE0.02a

molar ratio of OEGAa

Mn (kDa)b,c

Mw/ Mnb,c

apparent Kd for Ca2+ (mM)d

dynamic range for Ca2+ d

apparent Kd for BSA (mg/L)d

0.70 0.69 0.63 0.65

0.30 0.31 0.37 0.35

44.8 29.3 35.6 31.8

2.52 2.11 2.68 2.23

e 2.31 7.91 4.43

1.4 21 7.1 24

53 14 31 35

1.00 1.00

0 0

33.1 34.7

1.62 1.47

1.62 1.21

41 57

14 11

a

Determined by 1H NMR spectroscopy. bObserved for the t-Bu protected precursors. cDetermined by SEC measurements. dDetermined by titration experiments in an aqueous buffer solution ([HEPES] = 70 mM, pH = 7.4).41 eCould not be determined due to the slight response of 1 to Ca2+. 712

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that the dynamic range in the PAA-TPE-based sensors correlates with the inner density of the polymer chain upon aggregation.18 The observed difference between linear (5) and branched (6) reference polymers can be accounted for by the same scenario. Furthermore, the fact that the block copolymers exhibited a rather lower dynamic range than reference polymers seems reasonable, since the highly water-soluble polyOEGA segments should prevent the single-chain aggregation of the PAA-TPE segments. In particular, triblock copolymer 3 having polyOEGA segments at both sides of the PAA-TPE segment should have the largest impact among the block copolymers. The results of Ca2+-titration experiments (Figure 2 and Table 1) suggest that branched star-shape polymer 4 can maintain its internal density to some extent, upon functionalization with hydrophilic segments. Dynamic light scattering (DLS) experiments showed that the hydrodynamic radius (Rh) of 4 does not change significantly in the presence of various amounts of Ca2+ (Figure S14).41 Thus, as in the case of PAA-TPE0.02,17,18 the Ca2+-sensing property of 4 arises from single-chain folding rather than interpolymer aggregation. We evaluated the protein-adsorption-resistance properties of the copolymers (1−4) and reference polymers (5 and 6) based on bovine serum albumin (BSA)-titration experiments (Figures S7−S12b)41 in an aqueous buffer solution ([HEPES] = 70 mM, pH = 7.4). Fitting the titration curves (Figure 3) using Hill’s

arising from the poly(OEGA) segments (Figures 1g,h, S5a, and S6a).41 As confirmed by 1H NMR (Figures S1−S6b)41 and SEC (Figures S1−S6c) 41 analysis, the reaction steps, after copolymerization and block copolymerization, proceeded successfully without undesirable side reactions. The numberaveraged molecular weight (Mn) and polydispersity (Mw/Mn) of all the copolymers were evaluated by SEC analysis of the corresponding precursor polymers with tert-butyl protecting groups using polystyrene standards. The composition ratios of TPE, tert-butyl, and OEGA groups in the copolymers were determined based on the integration ratio of each group in their 1 H NMR spectra. The structural parameters thus obtained are summarized in Table 1. Note that a series of copolymers (1−4) of PAA-TPE0.02 and polyOEGA have a similar molecular weight (Mn = 29−45 kDa) and degree of OEGA content (30−37 mol %) for a reasonable comparison (Table 1). Further details of the synthesis of the copolymers with different topology are described in the Supporting Information. Figure 2 shows plots of the change in fluorescence-intensity of the copolymers (Figures S7−S12, a)41,42 upon titration of

Figure 2. Ca2+-titration curves of 1−6 in a HEPES buffer solution (70 mM, pH = 7.4, [polymers] = 10 mg/L based on PAA-TPE0.02).

Ca2+ in an aqueous buffer solution ([HEPES] = 70 mM, pH = 7.4). Table 1 summarizes the apparent dissociation constants (K d ) and dynamic ranges of the copolymers for Ca 2+ determined by fitting the titration curves (Figure 2) using Hill’s equation. As expected from the previous study,17 the fluorescence intensities of reference polymers 5 and 6 were greatly enhanced as the Ca2+ concentration was increased. Upon addition of Ca2+, diblock (2), triblock (3), and 4-armedstar-block (4) copolymers likewise showed a clear increase in fluorescence intensity, whereas random copolymer 1 remained nonfluorescent. The difference between the three block copolymers and the random copolymer is most likely related to the density of carboxylic acid functionality involved in the Ca2+-sensing PAA-TPE segments; the density for the random copolymer is much lower than those for the block copolymers as well as the original PAA-TPE0.02 polymer. Although all the block copolymers showed a millimolar-order apparent Kd for Ca2+ (Table 1), suitable for extracellular Ca2+sensing, the value were larger than those for reference polymers 5 and 6, indicating that the polyOEGA segment somewhat lowers the affinity of the block copolymers for Ca2+. The dynamic ranges of block copolymers 2 (21), 3 (7.1), and 4 (24) were also decreased (Table 1), compared to those of reference polymers 5 (41) and 6 (57). In the previous study, we showed

Figure 3. BSA−titration curves of 1−6 in a HEPES buffer solution (70 mM, pH = 7.4, [polymers] = 10 mg/L based on PAA-TPE0.02). The relative fluorescence intensity is defined as (F − Fmin)/(Fmax − Fmin), where F, Fmax, and Fmin represent observed, maximum and minimum fluorescence intensities, respectively.

equation gave apparent Kd values (Table 1). A larger Kd value for BSA represents a better protein-adsorption resistance. Both reference polymers displayed high affinity for BSA (apparent Kd value ∼10 mg/L). In the presence of BSA, random copolymer 1 without Ca2+-sensing ability became fluorescent and provided an apparent Kd value of 53 mg/mL, which was much larger than those obtained for the reference polymers. Thus, the polyOEGA segment certainly suppresses protein adsorption. However, despite the presence of the polyOEGA segment, diblock copolymer 2 showed an apparent Kd value that was almost identical to those obtained for the reference polymers. In contrast, the BSA-titration curve of triblock copolymer 3 obviously shifted to a higher BSA concentration region, and the apparent Kd was increased to 31 mg/L. A further shift of the titration-curve to a higher concentration region and a slight increase in apparent Kd (35 mg/mL) were observed for 4armed-star-block copolymer 4. Clearly, the attachment of the 713

DOI: 10.1021/acsmacrolett.8b00291 ACS Macro Lett. 2018, 7, 711−715

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the present study, which is the first to shed light on a topology effect, namely, how to incorporate biocompatible segments into polymer-based biosensors, may provide new insights into the design of polymer-based biosensors for use in vivo.

polyOEGA segments to both termini of the PAA-TPE segment is effective for improving the protein-absorption-resistance of the copolymer systems (Figure S15).41 On the basis of the above results, 4-armed-star-block copolymer 4 featuring a core−shell-like structure provides the best motif to cope with both Ca2+-sensing and proteinadsorption-resistance properties. The outer shell of the polyOEGA segments should effectively suppress the access of protein molecules to the inner core of the PAA-TPE segments, which are responsible for Ca2+-sensing. With this in mind, we finally tested the Ca2+-sensing property of 4 in the presence of BSA. As shown in Figure 4, even in the presence of a large

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00291. Details of the synthesis and characterization, evaluation of the apparent dissociation constant, NMR, IR, and fluorescence spectra, fluorescence quantum yields, and DLS profiles of copolymers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (T.F.). ORCID

Fumitaka Ishiwari: 0000-0002-0200-4510 Takanori Fukushima: 0000-0001-5586-9238 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by the Dynamic Alliance for Open Innovation Bridging Human, Environment and Materials from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and KAKENHI (Grant-in-Aid for Research Activity Start-up No. 24850008, Challenging Exploratory Research No. 16K14003, Young Scientists B No. 26810067, and Young Scientists A No. 17H04879) to F.I. Japan Science and Technology Agency (JST) Exploratory Research for Advanced Technology (ERATO) Someya Bio-Harmonized Electronics. We thank the Suzukakedai Materials Analysis Division, Technical Department, Tokyo Institute of Technology, for their support in NMR and DLS measurements. We also thank Prof. K. Yamamoto and Dr. K. Albrecht (Tokyo Institute of Technology) for their support in fluorescence quantum yield measurements.

Figure 4. Ca2+-titration curves of 4 in a HEPES buffer solution (70 mM, pH = 7.4) in the presence of 20 mg/L of BSA. Inset is fluorescence spectral changes of 4 in a HEPES buffer solution (70 mM, pH = 7.4) before (blue) and after (green) addition of 20 mg/L of BSA, and after further addition of Ca2+ from 0 mM (black) to 1000 mM (red).

excess of BSA (20 mg/L), 4 served as a fluorescent Ca2+-sensor with an apparent Kd of 5.58 mM and a dynamic range of 15, which may allow extracellular Ca2+-imaging in biological systems.43 In conclusion, to improve the protein-adsorption-resistance properties of AIEgen-appended poly(acrylic acid)-based Ca2+ sensor (PAA-TPE0.02), we developed a series of copolymers (1−4; Figure 1) composed of PAA-TPE and polyOEGA segments with a similar composition ratio of each segment (ratios of PAA-TPE = 63−70 mol %, ratios of polyOEGA = 30−37 mol %) and molecular weight (Mn = 29−45 kDa), while their topologies are different (i.e., random, diblock, triblock, and 4-armed-star-block structure). The random copolymer exhibited a high protein-adsorption-resistance, but hardly responded to Ca2+. Conversely, the diblock copolymer behaved as a Ca2+-sensor with a good dynamic range but did not exhibit protein-adsorption-resistance. The triblock copolymer, in which both end segments are polyOEGA, certainly exhibited Ca2+sensing ability as well as protein-adsorption-resistance, although the dynamic range for Ca2+-sensing was moderate. Among the copolymers examined, the 4-armed-star-block copolymer, featuring a core−shell-type arrangement of the Ca2+-sensing PAA-TPE and biocompatible polyOEGA segments, showed the best performance in terms of both Ca2+-sensing and proteinadsorption-resistance: the dynamic range for Ca2+-sensing was even higher than that of the diblock copolymer, and the protein-adsorption-resistance property was comparable to that of the triblock copolymer (Figure S15).41 More importantly,

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DOI: 10.1021/acsmacrolett.8b00291 ACS Macro Lett. 2018, 7, 711−715