Introduction

Smart or stimuli-responsive polymers are a new generation of polymers that exhibit reversible physicochemical changes in response to changes in their environment, such as temperature, pH, light, and magnetic field variations or the addition of other molecules [1, 2]. Among such materials, temperature-responsive polymers undergo changes in response to the solution temperature. This ability, especially in aqueous solutions (i.e., reversible hydrophilic/hydrophobic changes), has been applied in a wide range of fields, such as cell cultures, drug carriers, chromatography, microchannels, protein preservation, and detections in cell biology [3,4,5,6,7,8]. These polymers typically contain a temperature-responsive block, and the temperature responsivity is limited to a single structural feature (i.e., hydrophilicity and hydrophobicity). To fine tune the structural properties, block copolymers with two (or more) different temperature-responsive blocks have been reported [9,10,11]. Three kinds of structural features, i.e., hydrophilic/hydrophilic, amphiphilic, and hydrophobic/hydrophobic structures, can be prepared from dual temperature-responsive block copolymers via modification of the individual block properties. Sugihara et al. prepared a triple temperature-responsive block copolymer by living cationic copolymerization [12]. Weiss and Laschewsky prepared tri-block copolymers with three temperature-responsive blocks, and 12 types of structures were obtained by hydrophilicity/hydrophobicity variations and using different combinations of the three blocks in different sequences [13]. To the best of our knowledge, no reports exists on temperature-responsive block copolymers with more than four different temperature-responsive blocks. There are two main reasons for this: (1) it is difficult to synthesize block copolymers using the available synthesis techniques and (2) it is difficult to obtain more than four types of monomers with different LCSTs. With recent developments in polymerization techniques, block copolymers consisting of more than 10 blocks have been reported [14]. Zhang et al. prepared multi-block glycopolymers for the inhibition of lectin and the gp120 proteins in human immunodeficiency virus (HIV) [15].

In this study, a hexa-block copolymer consisting of six types of temperature-responsive blocks was prepared using two ethylene glycol-based monomers (Fig. 1). The homo-polymers of (2-(2-methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA)) exhibit lower critical solution temperatures (LCSTs) of 28 and 90 °C, respectively [16]. Therefore, the LCSTs of the P(MEO2MA-co-OEGMA) copolymers can be designed to be between 28 and 90 °C by varying the monomer composition [17]. The hexa-block copolymer was prepared by simple syringe-based method in 12 h, and each block was prepared with a different OEGMA content to obtain different LCSTs. To the best of our knowledge, this is the first report of a block copolymer consisting of more than four kinds of temperature-responsive blocks. The physicochemical properties of the block copolymers were investigated using 1H nuclear magnetic resonance (1H NMR) spectroscopy, gel permeation chromatography (GPC), UV-vis spectroscopy, micro-differential scanning calorimetry (micro-DSC), and scanning electron microscopy (SEM).

Fig. 1
figure 1

a Structures of the hexa-block copolymer with different OEGMA contents. b Preparation methods of the block copolymers

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Experimental section

Materials and methods

2-(2-Methoxyethoxy)ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA, Mn = 475 g/mol) were purchased from Sigma-Aldrich and purified by passage through a basic alumina column. All other chemicals and solvents were used as received. The distilled water used in this study was purified with a Millipore Milli-Q system.

Preparation of the ethylene glycol-based hexa-block copolymer

A reversible addition-fragmentation chain transfer (RAFT) polymerization was employed to synthesize block copolymers with a narrow molecular weight distribution. 4-Cyanopentanoic acid dithiobenzoate (CTP) was selected as the RAFT agent. The hexa-block copolymer was prepared from blocks containing different contents of OEGMA. The first block, i.e., P(MEO2MA), was polymerized in a polymerization tube. MEO2MA (2.26 g, 12.0 mmol), CTP (13.97 mg, 0.05 mmol), and 4,4′-azobis-4-cyanovaleric acid (ACVA, 5.5 mg, 0.022 mmol) ([MEO2MA]0/[CTP]0/[ACVA]0 = 240/1/0.44) were dissolved in 5 mL of methanol. After degassing with nitrogen for 30 min, the mixture was allowed to polymerize for 2 h at 60 °C. Then, one quarter of the polymerization solution was removed as P(MEO2MA) with a syringe, and an OEGMA solution (602.8 mg in 0.85 mL of methanol) was added to the polymerization tube to synthesize the di-block copolymer (P(MEO2MA)-b-P(MEO2MA-co-OEGMA)). The extracted P(MEO2MA) was purified by dialysis against ethanol and acetone and dried under reduced pressure. After 2 h, one quarter of the polymerized solution (i.e., P(MEO2MA)-b-P(MEO2MA-co-OEGMA)) was again extracted, and an OEGMA solution (532.0 mg in 0.92 mL of methanol) was added in the same way for the polymerization of the tri-block copolymer. After six steps of such polymerizations, a hexa-block copolymer made of blocks with different OEGMA contents was synthesized. Different OEGMA solutions were added to the polymerization tube to obtain the tetra-block (578.0 mg in 0.87 mL of methanol), penta-block (670.0 mg in 0.78 mL of methanol), and hexa-block (822.0 mg in 0.63 mL of methanol) copolymers. The composition of each block was calculated using 1H NMR and GPC analyses. For example, the monomer units in P(M13) (i.e., P(MEO2MA)) were calculated as shown below.

Units = ((Mn of P(MEO2MA) from GPC) − (Mn of RAFT agent))/(Mn of MEO2MA)

The composition of the di-block copolymer (i.e., P(M13)-b-P(M17-co-O3)) was calculated as shown below.

The molar ratio of MEO2MA (fM) and OEGMA (fO) (fM + fO = 1) in the di-block copolymer was measured by 1H NMR. Therefore, the total units can be calculated using the Mn (from GPC) and the ratio.

The total units:

((Mn of di-block copolymer) − (Mn of RAFT agent))/(Mn of MEO2MA) × (fM) + (Mn of OEGMA) × (fO)) = 33 units (MEO2MA: 30 units, and OEGMA: 3 units).

For the first block of P(MEO2MA), 13 units of MEO2MA were used. Therefore, the composition of the di-block copolymer was calculated based on P(M13)-b-P(M17-co-O3). In addition, the OEGMA content (16 mol%) in the P(M17-co-O3) block was calculated using the numbers of units. The compositions of the tri-, tetra-, penta-, and hexa-block copolymers were calculated using same methods.

Characterizations

1H NMR spectra of copolymers were recorded on a JNM-GSX300 spectrometer (300 MHz, JEOL, Tokyo, Japan) to confirm the successful synthesis and determine the chemical composition of the synthesized copolymers.

The molecular weight and polydispersity of the synthesized copolymers were determined by GPC at 40 °C (DMF containing 10 mM LiBr, 1 mL/min) with a TOSOH TSK-GEL α-2500 and α-4000 columns (Tosoh, Tokyo, Japan) connected to an RI-2031 refractive index detector (JASCO International Co., Ltd., Tokyo, Japan). Poly(ethylene glycol)/poly(ethylene oxide) standards were used to prepare a calibration curve.

The transmittance of the copolymer solution at 500 nm was continuously recorded at a heating rate of 1.0 °C/min on a UV-Vis spectrometer V-550 or V-650 (JASCO International Co., Ltd., Tokyo, Japan) to determine the LCST. The synthesized copolymers were dissolved in an aqueous solution at a given concentration. The LCSTs of the copolymers were measured at 50% transmittance.

The specific heat capacity (Cp) of the hexa-block copolymer solution (0.1 wt% in water) was determined by differential scanning calorimetry (MicroCal VP-DSC). The baseline was stabilized for 12 h with milliQ water prior to sample measurement. The heating rate was 1.0 °C/min.

The morphology of the nanoparticles was observed by SEM (JSM-IT 100, JEOL, Tokyo, Japan) at an accelerating voltage of 15.0 kV. A drop of the sample solution (0.1 wt% in water at the desired temperature) was placed on a silicon wafer by spin coating and allowed to dry under atmospheric pressure. The sample was then sputter-coated with Au.

Results and discussion

The block copolymers were synthesized by RAFT polymerization. MEO2MA and OEGMA were selected as the monomers for the target block copolymers. The homo-polymers of P(MEO2MA) and P(OEGMA) present lower critical solution temperatures (LCSTs) of 28 and 90 °C, respectively [17]. The LCSTs of P(MEO2MA-co-OEGMA) copolymers can be controlled by varying the monomer composition. Copolymers polymerized using Z-C( = S)-S-R-type RAFT agents have Z and R groups at the chain ends [18]. The end groups of the copolymers can thus be controlled by careful selection of the RAFT agent, which in this work was 4-cyanopentanoic acid dithiobenzoate (CTP). The hexa-block copolymer was prepared from blocks containing different OEGMA contents. The first block, i.e., P(MEO2MA), was polymerized in a polymerization tube for 2 h (Fig. 1b). Then, one quarter of the polymerized solution was removed as P(MEO2MA) via a syringe, and an OEGMA solution was added to the polymerization tube to synthesize the di-block copolymer (P(MEO2MA)-b-P(MEO2MA-co-OEGMA)). After 2 h, another quarter of the polymerized solution (i.e., P(MEO2MA)-b-P(MEO2MA-co-OEGMA)) was removed, and an OEGMA solution was again added in the same way for the polymerization of the tri-block copolymer. Six such polymerization steps afforded a hexa-block copolymer composed of blocks with different OEGMA contents.

Figure 2 shows the GPC chromatograms for each block copolymer. The retention time of the GPC peaks decreased as the number of blocks increased from the P(MEO2MA) (the first-block) to the hexa-block copolymer. Table 1 shows the OEGMA composition, molecular weight (Mn), and molecular weight distribution (Mw/Mn) of each block copolymer. The OEGMA compositions were calculated using the 1H NMR and GPC data. The Mn and Mw/Mn of P(MEO2MA) were 2800 g/mol and 1.18, respectively. The Mn of the di-block copolymer P(MEO2MA)-b-P(MEO2MA-co-OEGMA) was 7400 g/mol and its Mw/Mn was 1.18. The OEGMA content in the P(MEO2MA-co-OEGMA) block of the di-block copolymer was 16 mol%. The Mn of the tri-block copolymer was 12,000 g/mol, and the OEGMA content of the third block of the P(MEO2MA-co-OEGMA) fragment was 30 mol%. The Mn of the tetra- (18,500 g/mol), penta- (26,800 g/mol), and hexa-block (40,400 g/mol) copolymers increased with the number of blocks, and the OEGMA contents of the fourth, fifth, and sixth block were 39, 64, and 91 mol%, respectively. The Mw/Mn values of all the block copolymers were relatively narrow (1.18–1.42). The degree of polymerization of the hexa-block copolymer was P(M13)-b-p(M17-co-O3)-b-p(M12-co-O5)-b-p(M13-co-O8)-b-p(M8-co-O14)-b-p(M3-co-O28), where the numbers denote the number of monomer units (M: MEO2MA and O: OEGMA) calculated from the molecular weight and OEGMA content. Moreover, another hexa-block copolymer was synthesized to confirm the polymerization method (Table S1, Figures S1 and S2). To increase the molecular weight of the first block (P(MEO2MA)), the polymerization time was increased from 2 to 3 h. The polymerization for the other five blocks was 2 h. The Mn of the P(MEO2MA) was 4200 g/mol, which is higher than that of P(MEO2MA) in P(M13)-b-p(M17-co-O3)-b-p(M12-co-O5)-b-p(M13-co-O8)-b-p(M8-co-O14)-b-p(M3-co-O28). The hexa-block copolymer was thus identified as P(M21)-b-p(M13-co-O3)-b-p(M13-co-O5)-b-p(M9-co-O7)-b-p(M6-co-O12)-b-p(M2-co-O21).

Fig. 2
figure 2

GPC chromatograms of the block copolymers

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Table 1 Characterization of the block copolymers
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Next, the temperature-responsive properties of the block copolymers were determined. Figure 3a shows the changes in the transmittance of the block copolymers with changing temperature. All the block copolymers were dissolved in pH 7.4 phosphate-buffered saline (PBS) at 0.1 wt%. The LCST of the first block of P(MEO2MA) was ~34 °C. The temperature of the strong decrease in transmittance increased with the number of blocks (tri < tetra < penta < hexa). In the di-block copolymer, the transmittance drop was observed at ~39 °C. The di-block copolymer contains two different temperature responsive blocks, and thus it is expected to exhibit two transmittance reductions via the dehydrations of both P(M13) and P(M17-co-O3) blocks. Indeed, the dual temperature-responsive block copolymer showed two different transmittance changes due to conformational changes, i.e., water-soluble hydrophilic/hydrophilic block copolymers, amphiphilic block copolymers (nanoparticles), and hydrophobic/hydrophobic block copolymers (nanoparticle aggregates/precipitates) [19,20,21,22]. The di-block copolymer exhibits a single transmittance drop up to 1 wt% (Figure S3). In the dual temperature-responsive block copolymer, the temperature-responsive properties of one block are strongly affected by the presence of the other block. The chain length of the P(M13) block is shorter than that of P(M17-co-O3). The temperature-responsive behavior of the P(M13) block might be coupled to that of the P(M17-co-O3) block, or the behavior might be due to the gradient-like structure of the copolymers. The di-block copolymer of P(M21)-b-p(M13-co-O3) (Table S1) contains a longer P(M21) block because of the longer polymerization time employed in its synthesis. The transmittance change of P(M21)-b-p(M13-co-O3) is shown in Figure S4. A transmittance drop was observed at approximately 33 °C and 1 wt%. These result made us wonder if each block exhibits temperature-responsive properties. Figure 3b shows the micro-DSC results for the hexa-block copolymer in milliQ water. DSC presents the temperature-responsive properties in the form of endothermic peaks [23]. First, the endothermic peak of the hexa-block copolymer increased from 35 to 65 °C with a broad curve. The starting temperature (35 °C) was similar to the LCST of the first block of P(MEO2MA). The temperature range of the broad curve includes the LCSTs of the second, third, and fourth blocks. Moreover, the endothermic peak also increased from 65 °C, and a sharp peak was observed at 73 °C, which is similar to the LCST value of the sixth block. These results suggest that each block of the hexa-block copolymer exhibits observable temperature-responsive properties. However, it should be noted that the chain lengths of each block are relatively short. In addition, it is difficult to fix the boundaries between adjacent blocks. Moreover, the LCSTs of each block consisting of several tens of OEGMA/MEO2MA units will be strongly affected by slight changes in the number of OEGMA units. The slight variations will be observed in each block from a probabilistic viewpoint of polymerization.

Fig. 3
figure 3

a Transmittance change of 0.1 wt% solutions of block copolymers in pH 7.4 PBS as a function of temperature (○: P(MEO2MA), ∆: di-block copolymer, □: tri-block copolymer, ●: tetra-block copolymer, ▲: penta-block copolymer, and ■: hexa-block copolymer). b Micro-DSC curve of 0.1 wt% solution of the hexa-block copolymer in milliQ water as a function of temperature. c Transmittance change of a 0.5 wt% solution of the hexa-block copolymer in pH 7.4 PBS as a function of temperature

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Sequential dehydrations of the hexa-block copolymer lead to the formation of self-assembled particles owing to the amphiphilic structure of the copolymer. For example, dual temperature-responsive block copolymers become amphiphilic at temperatures between the LCSTs of each block and form nanoparticles via self-assembly. Therefore, the hexa-block copolymer is also expected to form nanoparticles in a specific temperature range. Figure 3c shows the transmittance change of 0.5 wt% hexa-block copolymer in PBS. A small transmittance decay (of ~5%) was observed at 45 °C. Then, the transmittance gradually decreased, and a drastic drop was observed at 70 °C. Interestingly, the onset temperature of 45 °C was similar to the response temperature of the third block (P(M12-co-O5)). These results suggest that the hexa-block copolymer forms nanoparticles between 45 and 70 °C. Figure. 4 shows the SEM images of the nanoparticles formed by the hexa-block copolymer. The nanoparticles were kept at 50, 60, and 70 °C for 20 min before sample preparation for SEM measurements. The size of the nanoparticles was thus found to be influenced by temperature. Such sizes changes may arise from the ratio of hydrophobic and hydrophilic fragments in the hexa-block copolymer. Wei et al. prepared an LCST-LCST-type PEG-b-P(NIPAAm)-b-P(NIPAAm-co-HEAAm) tri-block copolymer [24]. The tri-block copolymer formed micelles (37–45 °C) and vesicles (>50 °C) depending on the proportion of hydrophobic fragments. Figure. 5 shows the reversibility of the dispersion and aggregation/precipitation of the nanoparticles in suspension after heating–cooling cycles. A hysteresis of ~2 °C was observed for each heating-cooling cycle. The ethylene glycol-type temperature-responsive copolymers P(MEO2MA), P(OEGMA), and their copolymers are well known to show a relatively small hysteresis [25], and the hexa-block copolymer contains a sixth p(M3-co-O28) block located on the surface of the nanoparticles. The chain length of each block is relatively short, and the nanoparticles also bear dithioester groups, originating from the RAFT agent, on the surface. The surface properties strongly affect the dispersion/aggregation of the nanoparticles. Nakayama et al. reported the effect of pH-responsive sulfadimethoxine (SD) end groups on temperature-responsive nanoparticles, and the LCSTs were modified by the end group [26]. A nanogel with a shell of P(MEO2MA92-co-OEGMA24) with COOH groups on the surface was found to show high dispersion via electrostatic repulsion [27]. The relatively short polymer chain and their surface properties might have led to the observed small hysteresis of the temperature-responsive properties.

Fig. 4
figure 4

SEM images of the hexa-block copolymer. The polymer solution (0.1 wt% in pH 7.4 PBS) was maintained at a 50 °C, b 60 °C, and c 70 °C for 15 min before the sample preparation

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Fig. 5
figure 5

Transmittance change during heating–cooling cycles of 0.1 wt% solution hexa-block copolymers in pH 7.4 PBS as a function of temperature

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Conclusion

In conclusion, an ethylene glycol-based hexa-block copolymer was prepared via RAFT polymerization. The molecular weight (Mn) and OEGMA content increased with the number of blocks (from the first block to the hexa-block copolymer). The Mn and Mw/Mn of the hexa-block copolymer were 40,400 g/mol and 1.41, respectively, and the OEGMA content was zero for the first block (P(MEO2MA)) and 16, 30, 39, 64, and 91 mol% for each of the additional blocks. Each block of the hexa-block copolymer exhibited its own temperature-responsive behavior upon sequential dehydration, and the size of the resulting polymer nanoparticles was influenced by the temperature. Such a size variation may arise from the ratio of hydrophobic to hydrophilic fragments in the hexa-block copolymer. The nanoparticles in suspension displayed reversible dispersion and aggregation/precipitation under heating-cooling cycles with a hysteresis of ~2 °C. The structure of the hexa-block copolymer is similar to that of gradient-copolymers [28]. In the preparation of the hexa-block copolymer, the required OEGMA monomer was added for the polymerization of each block. The OEGMA concentration in the polymerization tube was increased in batches (not gradually). Therefore, the OEGMA content in each block was expected to be clearly different. However, because of the relatively short chain length of each block, it was difficult to fix the boundaries between adjacent blocks. Very recently, Liu et al. successfully prepared 10-block copolymers (97,000 g/mol) using a combination of enzymatic cascade catalysis and RAFT polymerization [29]. We would like to synthesize a temperature-responsive hexa-block copolymer with longer chain lengths in future. The present multi-temperature-responsive block copolymers exhibit multiple structural changes and can potentially be used as biomimetic materials.