Synthesis of PEGylated polypeptides bearing thioether pendants for injectable ROS-responsive hydrogels

2.1 Materials and characterization

The starting material, 1-tert-butyl N-(tert-butoxycarbonyl)-l-glutamate (Boc-Glu-OTBu), was supplied by Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China). 2-(Methylthio)ethanol was acquired from Energy Chemical (Shanghai, China). Methoxy (polyethylene glycol) (mPEG_2k, M _n = 2,000) was purchased from Ponsure Biotechnology Co., Ltd (Shanghai, China), and mPEG_2k-NH₂ was synthesized following our previous procedure. Sodium hydroxide and tetrahydrofuran were purchased from Sinopharm Co., Ltd., and N,N-dimethylformamide (DMF) was obtained from Shanghai Qiangshun Chemical Reagent Co., Ltd. Dulbecco's modified eagle medium (DMEM) cell culture medium was purchased from Biotechnology Co., Ltd. Trypsin was purchased from Wuhan Seville Biotechnology Co., Ltd., and penicillin–streptomycin solution was purchased from Beijing Sevin Innovation Biology Co., Ltd. MTT was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. Dichloromethane, sodium chloride, trimethylamine, ethyl acetate, anhydrous magnesium sulfate, sodium bicarbonate, and other chemicals were all bought from Xilong Scientific Co., Ltd. (Guangzhou, China).

2.2 Synthesis of mPEG_2k-b-PMLG

The monomer, N-carboxyanhydride from methylthioethyl l-glutamate (MLG-NCA), was synthesized following our previous protocols (24). The di-block copolymer was synthesized through the ring-opening polymerization of MLG-NCA with mPEG_2k-NH₂ serving as a macroinitiator (Figure S1). Briefly, MLG-NCA and mPEG_2k-NH₂ macroinitiator (molar ratio 17:1) were added in a round-bottom flask under nitrogen atmosphere and dissolved by dry DMF. Following a 72 h reaction period at 25°C, the solution was subsequently subjected to dialysis and lyophilization. The product mPEG_2k-b-PMLG was obtained as white solid.

2.3 Preparation of mPEG_2k-b-PMLG hydrogels

The mPEG_2k-b-PMLG was dissolved in a 0.01 M PBS with a pH of 7.4 at 4°C. Different concentrations of polymer solutions were prepared. Subsequently, 0.5 mL of each polymer solution was transferred to a small glass vial and placed in a water bath with a programmed warming process of 1°C per minute. And each temperature was maintained for 5 min. At a specific temperature, the glass vial was inverted. If the polymer solution did not flow within 30 s, it was considered the hydrogels have formed. Each sample was subjected to this test in triplicate for accuracy.

2.4 Dynamic rheological analysis

Dynamic rheological analysis was conducted using a US 302 (Anton Paar rheometer) with 25 mm diameter plates, operated in oscillation mode, and a gap distance of 0.5 mm. For testing, a 300 μL polymer solution was placed on the sample table, and the edges of the sample were sealed with silicone oil to prevent moisture evaporation. The changes of energy storage modulus (G′) with temperature were recorded with testing parameters of a strain (λ) of 1% and a frequency (ω) of 1 Hz. The heating rate was 0.5°C·min⁻¹, and the temperature range of the analysis was from 20°C to 60°C.

2.5 Temperature sensitivity of the polymers

The polymer was dissolved in deionized water at a concentration of 0.05 mg·mL⁻¹. The size and size distribution of the self-assembled particles were measured by DLS at 20°C and 60°C, respectively. CD measurement was performed over a temperature range of 10°C–60°C to monitor the secondary structure of the polypeptide mPEG_2k-b-PMLG. Prior to testing, the samples were equilibrated at each temperature for 10 min. ¹³C NMR was performed in the temperature range of 20°C–50°C to assess the polymer structure.

2.6 Oxidative sensitivity of the polymers

The mPEG_2k-b-PMLG was dissolved in 0.01 M PBS with 12.0 wt% concentration, and hydrogel was formed at 35°C. Then, the hydrogel was incubated with PBS solution containing 10 mM or 100 mM H₂O₂. After 6 h or 12 h, the degradation medium was removed by dialysis, and the buck products after degradation were collected, freeze–dried, and submitted for ¹H NMR and Fourier transform infrared spectroscopy test.

2.7 Cytotoxicity evaluation

The in vitro cytotoxicity was assessed by using MTT assay. First, L929 cells were cultivated in 96-well plates with a seeding density of 8,000 cells per well with 200 μL DMEM medium and incubated at 37°C with 5% CO₂ for 24 h. Afterward, the culture medium was removed, and 200 μL of oxidized polymer solution by H₂O₂ or raw polymer solution in DMEM was added, with concentrations ranging from 0.063 to 2 mg·mL⁻¹. It should be noted that the raw mPEG_2k-b-PMLG solution formed hydrogel at 37°C. The control group was cultured with DMEM without polymer. Following an additional 24 h incubation, 20 μL MTT solution was added and incubated for 4 h. Then, the culture medium was discarded, and 150 μL of DMSO was added. The absorbance or optical density (OD) at 490 nm for each well was measured using a microplate reader. The cell viability rate (cell viability) was calculated using the following formula:

2.8 Cellular protective effect of gels in oxidative environment

The L929 cells were cultured (8,000 cells/well, 200 μL DMEM/well) at 37°C for 24 h. The culture medium was removed, and 50 μL polymer solution (12 wt%) was added to each well to form a stable hydrogel on the cell surface. Subsequently, 180 μL of DMEM medium containing H₂O₂ (0.125, 0.25, 0.5 or 1 mM) was added. After 24 h, the medium and gel were removed. Cells were washed with PBS for 3 times to remove the remaining small amount of gel. Then, 150 μL DMEM culture solution was added. The cells that did not contain the gel were treated as the control group in the same protocols. MTT assay was used to detect the cell viability.

2.9 In vivo test

Female Sprague-Dawley (SD) rats (∼200 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal experimental protocols were approved by the Animal Care and Use Committee of Changchun Institute of Applied Chemistry (No. 2022-0082). The mPEG_2k-b-PMLG solution (12.0 wt% in PBS) was subcutaneously injected into the back of SD rats through a syringe. The rats were killed 15 min later. The gel formation was checked, and photographs were taken.

3.1 Characterization of mPEG_2k-b-PMLG₁₅

The synthesis pathway for mPEG_2k-b-PMLG, which is sensitive to both oxidation and temperature, is depicted in Figure S1 and detailed in the Experimental section. Initially, the thioether group is attached to glutamate through the esterification of Boc-MLG-OTBu with 2-(methylthio)ethanol. The MLG-NCA was then synthesized according to protocols in our previous report (24). The desired product, mPEG_2k-b-PMLG, is ultimately obtained through the ring-opening polymerization of MLG-NCA utilizing mPEG_2k-NH₂ as a macromolecular initiator in anhydrous DMF. By controlling the molar ratio of MLG-NCA and mPEG_2k-NH₂ macromolecular initiator (17:1), the DP of PMLG is finally determined to be 15 by integrating the area of proton peaks f and i in Figure 1. GPC profiles showed that the average molecular weight of mPEG_2k-b-PMLG₁₅ is 6.1 kDa.

Figure 1

¹H NMR spectrum of mPEG_2k-b-PMLG₁₅ in mixture solvent of trifluoroacetic acid-d and CDCl₃.

3.2 Self-assembly of mPEG2k-b-PMLG₁₅

Some mPEG_2k-based block copolymers exhibit an interesting behavior. As the temperature increases, the enhanced interaction between the hydrophobic fragments and the partial dehydration of the mPEG_2k contributes to micelle accumulation. Eventually, this leads to a phase transition in the polymer solution, resulting in the formation of a gel (26,27). Our amphiphilic mPEG_2k-b-PMLG₁₅ could self-assemble into micelles or nanoparticles (NPs) in water. The critical micelle concentration (CMC) was determined to be 0.00083 mg·mL⁻¹ by using pyrene as fluorescence probe (Figure 2a). DLS tests have confirmed that these micelles maintain a stable 272 nm size at room temperature (Figure 2b).

Figure 2

(a) CMC measurement determined by fluorescence assay of pyrene probe incubation with different concentrations of mPEG-b-PMLG₁₅ solution and (b) DLS profiles of the self-assembled NPs at 1 mg·mL⁻¹.

3.3 Thermal-induced gelling and the mechanism

Dynamic rheological analysis of the aqueous solution of mPEG_2k-b-PMLG₁₅ was performed at the concentration ranging from 10 to 14 wt%. As shown in Figure 3a, when the temperature is lower than the phase transition temperature, the storage modulus G′ of the polymer solution remains basically unchanged; At the vicinity of the phase transition temperature, G′ suddenly increases, indicating that there is a solution transition to gel state, and with the increase of temperature, G′ continues to increase. Specifically, the phase transition temperature was determined to be 33°C for 12 wt% polymer solution.

Figure 3

(a) The storage modulus (G′) profiles of the mPEG_2k-b-PMLG₁₅ solution (10, 12, and 14 wt%) from 10°C to 60°C, (b) ¹³C NMR spectra of polymer solution at temperature of 20°C–50°C, and (c) CD spectra of the mPEG_2k-b-PMLG₁₅ solution (0.05 mg·mL⁻¹, 10°C–60°C).

As depicted in Figure 3b, the resonance absorption peak of carbon in the mPEG methylene was observed at 70.3 ppm at 20°C. As the temperature gradually increased, this peak shifted toward the lower field and tends to broaden. This shift suggests that the mPEG segment experienced gradual dehydration as the temperature increased, accompanied by partial molecular immobilization. The positive absorption peak at 195 nm and negative absorption peak at 225 nm in the CD spectra indicated a β-sheet structure of the PMLG₁₅ polypeptide (Figure 3c). The β-sheet structure became less at higher temperature as the intensity of peak at 225 nm gradually decreased when the temperature increased from 10°C to 60°C (Figure S2). Based on the aforementioned results, it can be inferred that the changes occurring in the mPEG segment and the secondary structure of the polyamino acid chain segments played a significant role in promoting the transition from a solution to a gel state as the temperature increased (25,26,27,28).

3.4 Oxidation responsiveness of mPEG_2k-b-PMLG₁₅

First, the invert test tube method was applied to confirm the phase transition temperature of mPEG_2k-b-PMLG₁₅, i.e., by observing whether the sample flows within 30 s through the inverted tube, if it does not flow, it is considered to form a gel. As shown in Figure 4a, when the sample was inverted at 35°C, no flow occurred and hydrogel was formed. Scanning electron microscopy image exhibited the irregular pores of the hydrogel formed in vitro (Figure 4b).

Figure 4

(a) Hydrogel formation at high temperature and disassociation in the presence of H₂O₂, (b) scanning electron microscope (SEM) image of the hydrogel formed by mPEG_2k-b-PMLG₁₅ solution at 35°C (scale bar: 20 μm), and (c) infrared spectra of hydrogel incubated with 100 mM H₂O₂ for 0, 6, and 12 h, respectively.

H₂O₂ is one of the key components of ROS generated during organism metabolism and exhibits relatively high stability compared to other ROS species. Therefore, we selected H₂O₂ to create a high-concentration ROS environment to assess the gel’s responsiveness to ROS. In the presence of H₂O₂, the hydrogel was destroyed and turned into liquid again (Figure 4a, right). As illustrated in the ¹H NMR spectra in Figure S3, the three peaks at 2.11, 2.70, and 4.21 ppm corresponding to the methyl and methylene near the thioether groups (labeled as a, b, and c) gradually disappeared after incubation with H₂O₂ for 12 h. Concurrently, new peaks appeared at 2.68 (a′), 3.03 (b′), and 4.41 (c′) ppm, indicating the oxidation of the sulfur atom. Infrared spectra revealed a new absorption peak at 1,035 cm⁻¹ after oxidation, corresponding to the characteristic peak of the sulfoxide group, and the intensity of this characteristic peak significantly increased with prolonged oxidation time (Figure 4c). Therefore, the oxidative response mechanism is that the thioether group in the hydrophobic portion of mPEG_2k-b-PMLG₁₅ side chains is oxidized into hydrophilic sulfoxide groups. As the hydrophilic properties of the polymer increase, its solubility also increases, which, in turn, accelerates the degradation of the hydrogel. This finding established a theoretical foundation for the elimination of ROS at inflammatory sites by mPEG_2k-b-PMLG₁₅ hydrogel and the protection of cells against oxidative damage.

3.5 Cell protection of gel in oxidized environment

The cytotoxicity of mPEG_2k-b-PMLG₁₅ and the hydrogel under oxidative conditions (10 mM H₂O₂) was evaluated using the MTT assay. As illustrated in Figure 5a, after a 24 h co-culture with the polymeric hydrogel and the oxidized products at concentrations ranging from 0.0156 to 1 mg·mL⁻¹, the survival rate of L929 cells in the experimental group remained above 90%. This suggests that mPEG_2k-b-PMLG₁₅ block copolymers and their various oxidation products exhibit excellent cytocompatibility. In the presence of 0.25 and 0.125 mM H₂O₂, the cell survival rates of L929 were more than 80% with gel protection, significantly higher than that without gel protection (Figure 5b).

Figure 5

(a) Cell survival rates of mouse fibroblasts cells (L929) after 24 h incubation with mPEG_2k-b-PMLG₁₅ solution or their oxidation products at different concentrations. It should be noted that the raw mPEG_2k-b-PMLG solution formed hydrogel at 37°C, and (b) cell viability of L929 cells cocultured with 12 wt% hydrogel for 24 h in the presence of 0.125,0.25,0.5, and 1 mM H₂O₂ in DMEM. Data are shown as mean ± SD (n = 3), **P < 0.01, ***P < 0.001.

ROS has high oxidation activity and can cause obvious damage and destruction to cells and tissues. Overexpression of ROS is the root cause of inflammation, including infectious inflammation (bacterial and viral infections) and non-infectious inflammation (such as rheumatoid arthritis), as well as a variety of senile degenerative diseases. Hydrogel formed by mPEG_2k-b-PMLG₁₅ solution could partly eliminate ROS and is potential for the treatment of oxidative stress-related diseases.

3.6 In vivo gelling of mPEG2k-b-PMLG₁₅

A typical feature of temperature-sensitive hydrogels is that the aqueous solution of the polymer is rapidly converted into a hydrogel upon injection into the body. Therefore, SD rats were employed as an animal model to assess the subcutaneous gel process of the mPEG_2k-b-PMLG₁₅ hydrogel. The polymer solution was subcutaneously injected into the rats’ backs, and after 15 min, the rats were euthanized. The back skin was then dissected, revealing the formation of the polymer gel within the subcutaneous tissue (Figure S4).