Introduction

Recent advancement in intelligent polymers aroused increasing interest in preparation of stimuli-responsive hydrogels, which undergo volume phase transition as the external stimuli such as pH, temperature, or ionic strength changes [1]. Because of these unique properties, the so-called “smart” hydrogels have been extensively investigated for their potential applications as drug delivery vehicles [2]. As compared with the bulk hydrogels, microgels exhibit faster response to external changes due to their colloidal particle nature and are more suitable for subcutaneous administration [3, 4]. Microgels that are sensitive to pH and/or temperature are the most commonly studied types, which are prepared using N-isopropylacrylamide (NIPAM) [5], acrylic acid (AAc) [6], methacrylic acid [7], N-vinylcaprolactam [8], and 2-(diethylamino)ethyl methacrylate [9] as monomers or comonomers [10–13] and N,N′-methylene bisacrylamide as cross-linker. However, most of these microgels are not biodegradable, which limits their in vivo application to a certain extent. To overcome this limitation, many natural polymers including dextrans [14], starch [15], chitosan [16], and sodium alginate [17] are employed to prepare microgels because of their excellent biodegradability and biocompatibility. For example, Smedt et al. reported microgels synthesized from dextran hydroxyethyl methacrylate and methacrylic acid, which are biodegradable through the hydrolysis of the carbonate esters in the crosslinks that connect the dextran chains [14]. Whereas, there are still some drawbacks to this approach: (1) most of these microgels are prepared using complex emulsion technique; (2) it is hard to control the molecular weight and polydispersity of the natural polymers as synthetic polymers.

In this report, a facile strategy was developed to prepare thermo- and pH-responsive microgels. Poly(N-isopropylacrylamide) (PNIPAM) was chosen as the thermosensitive moiety, which exhibits a reversible phase transition at about 32 °C (defined as lower critical solution temperature, LCST) [18]. The microgels containing PNIPAM are fabricated at temperature above its LCST, under which condition it becomes insoluble and forms aggregates. The pH-responsive component is poly(L-glutamic acid) (PGA). As a synthetic polypeptide, its molecular weight can be well tailored via the N-carboxyanhydride ring-opening polymerization technique [19–22]. More importantly, it has been demonstrated to be generally biodegradable and biocompatible in vitro and in vivo [23, 24]. As a polypeptide, it also exhibits unique pH-induced secondary structure changes, which can mimic the conformation change of natural protein that did not exist in other synthetic polymers [25].

In our synthetic routes, 2-hydroxyethyl methacrylate (HEMA) was firstly conjugated onto PGA backbones by the coupling reaction between the pendant carboxyl group of PGA and hydroxyl group of HEMA. The resultant polymer was copolymerized with NIPAM at 60 °C to produce a thermo- and pH-responsive microgel. Here, the HEMA-modified PGA acts not only as the pH-responsive part of the microgels but also is employed as a biodegradable cross-linker. To the best of our knowledge, biodegradable microgel composed of polypeptide has not been reported yet.

Experimental section

Materials

N-Isopropylacrylamide (NIPAM, 99%, Sigma) was recrystallized from hexane and dried under vacuum for 24 h prior to use. PGA was prepared according to our previous method [26]. 4-Dimethylaminopyridine (DMAP, 98%, Fluka) and N,N′-dicyclohexyl carbodiimide (DCC, Sigma) were used as received. HEMA (96%, Acros) was distilled under reduced pressure before use. Dimethyl sulfoxide (DMSO) was purified by vacuum distillation over CaH2.

Synthesis of poly(L-glutamic acid)-g-2-hydroxyethyl methacrylate

The poly(L-glutamic acid)-g-2-hydroxyethyl methacrylate (PGH) was prepared by the coupling reaction between the pendant carboxyl group of PGA and hydroxyl group of HEMA, as shown in Scheme 1. Typically, PGA (1.0 g) and HEMA (0.26 mL) were dissolved in dry DMSO (40 mL) in a flask at room temperature. The esterification of PGA with HEMA was started by adding 5 mL DMSO solution of DCC (0.2 g, 1.2:1 in molar ratio to HEMA) and DMAP (10 mg, 1:10 in molar ratio to HEMA) to the flask. After 24 h, the mixture was filtered to remove the precipitated dicyclohexylurea. Then the solution was poured into excess chloroform to precipitate the PGH. The product was isolated by filtration, repeatedly washed by chloroform, and dried under vacuum at 35 °C for 48 h.

Scheme 1
scheme 1

Synthetic routes of PGH and microgel

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Synthesis of microgels

The microgels were synthesized by the free radical precipitation polymerization. Briefly, 1.4 g of NIPAM and 0.6 g of PGH were dissolved in 100 mL of 0.05 M phosphate buffer (pH = 7.0). The solution was filtered to remove any possible precipitates. Then, the reaction mixture was transferred to a three-necked round-bottom flask equipped with a condenser and a nitrogen inlet and heated to 60 °C under a gentle stream of nitrogen. After 1 h, 1 mL of ammonium persulfate (0.06 g) solution was added to initiate the reaction. The reaction was allowed to proceed for 6 h. The resultant microgels were purified by dialysis (MWCO = 100,000) against water for 1 week at room temperature with daily changing of water. Then, the product was collected by lyophilization.

Characterization

Proton nuclear magnetic resonance (1H NMR) spectra were recorded by a Bruker 300 MHz spectrometer. ZetaPALS (Brookhaven Instruments) was used to determine the size and size distribution of the microgels (laser wavelength was 659 nm). The solution pH was adjusted by either 0.1 N NaOH or HCl. Each sample was equilibrated at the preset temperature for 15 min. The results obtained were an average of triplicate measurements. Turbidimetric measurements were carried out on a UV–vis spectrometer (Shimadzu UV-2401PC) equipped with a temperature controller (Shimadzu S-1700) at a wavelength of 500 nm with a heating rate of 0.5 °C/min. The solution was prepared by dispersing the microgels in 0.03 M phosphate-buffered saline (PBS) with a concentration of 8 mg/mL at different pH. The ionic strength of all solutions was adjusted to 0.15 mol/L by NaCl. The molecular weight of the PGA was estimated by viscosity measurement in 0.4 M NaCl and 0.01 M NaH2PO4 solution at pH 6.8 and 25.5 °C using the intrinsic viscosity–molecular weight relationship derived from Haekins et al. [27]: [η] = 2.93 × 10−5  M 0.923

Results and discussion

Synthesis of microgels

Our strategy to prepare thermo- and pH-responsive biodegradable microgels involves first the synthesis of vinyl groups functionalized PGA, followed by the crosslinking of NIPAM with PGH. From the relationship reported by Haekins et al. [27], the molecular weight of PGA was determined to be 4.4 × 104 g/mol. To modify PGA with reactive double bonds, HEMA was chemically coupled to its pendant carboxyl groups using DCC and DMAP. The chemical structures of PGH are shown in Scheme 1. 1H NMR spectrum of PGH presented in Fig. 1a confirmed the successful synthesis of PGH. The mole content of HEMA in PGH was calculated from the integration ratio of the characteristic proton resonance of PGA at δ = 4.4 ppm (–NHCHCO–) to the typical proton resonance of HEMA appearing at δ = 5.7 ppm (H–CH═CCH3–), which was determined to be 24.6%. The microgels were prepared by the free radical copolymerization of PGH and NIPAM. Their structures are confirmed by the 1H NMR, which is shown in Fig. 1b. The characteristic resonance peak of vinyl groups of HEMA at δ = 5.7 and 5.4 ppm disappeared. Moreover, the methylene signal of HEMA was weakened because its motility was restricted after the crosslinking. At the same time, the resonance signals ascribed to PNIPAM appeared, indicating the successful incorporation of PNIPAM into the microgels. The contents of PNIPAM in microgels were determined by the integration ratio of proton resonance of PGA at δ = 4.2 ppm (–NHCHCO–) and the proton resonance of PNIPAM at δ = 3.7 [–NHCH(CH3)2]. As shown in Table 1, the contents of PNIPAM in microgels are in good accordance with the feeding ratios, indicating the crosslinking reaction is effective.

Fig. 1
figure 1

1H NMR spectra of PGH (a) and MG73 (b) in D2O

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Table 1 Feed ratios of materials and compositions of the microgels
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As the reaction was proceeding, the produced PNIPAM became hydrophobic and formed hydrophobic aggregate which was stabilized by the copolymerized PGH. In addition, the electrostatic repulsion among the ionized carboxyl groups can prevent the macroscopic aggregate of microgels. Therefore, this strategy is suitable to prepare stable colloidal microgels in the absence of surfactant. At the end of the reaction, the solutions of MG73 and MG55 exhibit a blue tinge, which is similar to the solution of micelles. That is quite different from the PNIPAM homopolymer, which underwent macroscopic precipitation at temperature above the LCST. These results further confirmed the formation of microgels.

Phase transition behaviors of microgels

The phase transition behaviors of the microgels in aqueous solutions were characterized by monitoring the change in light transmittance. As shown in Fig. 2, microgels exhibited similar phase transition curves at pH 8.0 ∼ 5.0, and all microgel solutions exhibited >30% lowest transmittance at this pH range. Nevertheless, the solution of the PNIPAM homopolymer was almost light-tight when the temperature was raised above the LCST. This can be attributed to the formation of core–shell structure microgels with hydrophobic PNIPAM as the core and hydrophilic PGA as the shell. It also can be observed that the LCST of the MG73 and MG55 decreased as the pH value from 8.0 to 4.2. At pH = 7.0, the phase transition curves of MG73 and MG55 exhibited a broader transformation. That was because PGA was highly deprotonated in a neutral condition. When the pH value was lowered below pH 4.5, the phase transition curve of all microgels became sharper. Moreover, the original and the lowest transmittance of all microgel solutions decreased remarkably, which can be attributed to the hydrophilic to hydrophobic transition induced by the protonation of PGA. When the pH is reduced to 4.0 or below, the solution becomes turbid at any temperature above 0 °C, due to the aggregation of the PGA backbone.

Fig. 2
figure 2

The phase transition curves of the 0.8 wt.% MG73 (a) and MG55 (b) aqueous solution at different pH values

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The PNIPAM contents in microgels also have a key influence on their phase transition behaviors. It can be seen from Fig. 2 that the phase transition curves became sharper as PNIPAM content increased at all pH ranges investigated. It should be noted that the thermo- and pH-responsive copolymers lost their LCST in very low PAAc content (>10%) [28, 29]. However, in our strategy, microgels with 47 wt.% PGA (MG55) content still had visible phase transition behavior.

Thermo- and pH-responsive size change of microgels

The hydrodynamic diameter (D h) of MG55 was measured by dynamic light scattering. Figure 3a shows the temperature-dependence of D h of the MG55 in aqueous solution at different pH during the heating process. MG55 exhibited thermo-induced volume changes at pH 7.0 ∼ 5.0, undergoing shrinkage due to the collapse of the PNIPAM chains. The transition curves are relatively broader than the PNIPAM homopolymers, which usually exhibit an abrupt decrease of sizes in the narrow temperature range of 31–33 °C. As shown in Fig. 3b, the size distribution of MG55 became narrower at temperature above LCST at pH 5.0, indicating that the hydrophobic interaction among PNIPAM chains made the microgels compacter and more uniform. It can also be observed that the D h of MG55 decreased from 570 to 310 nm as the pH lowered from 7.0 to 5.0 at 25 °C. The result was probably caused by the reduction of electrostatic repulsion among PGA chains induced by the protonation of carboxyl groups.

Fig. 3
figure 3

a The Dh changes of MG55 in aqueous solutions with a concentration of 1 mg/mL at different pH as a function of temperature. b Size distribution profiles for MG55 in aqueous solutions of pH 5.0 at 25 and 45 °C

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Conclusion

A facile route to prepare thermo- and pH-responsive microgel has been developed. Due to the introduction of synthetic polypeptide, poly(L-glutamic acid), the resultant microgels are biodegradable. The microgels exhibited pH-dependent phase transition behaviors in aqueous solutions. Moreover, as the PNIPAM content in microgels increased, their phase transition behaviors became sharper. The pH-sensitivity moiety content in these microgels can reach to 45 wt.% without losing their LCSTs, which is much higher than that of the traditional PNIPAM/PAAc microgels. Because of its unique dual responsive and biodegradable nature, the microgels will have promising applications as drug delivery vehicles.