Wet spinning is employed to produce spider silk with high elasticity

Araneus ventricosus, a species of orb-web spider, could produce seven different types of silk and viscous liquid, each with a different structure and corresponding biological function.¹ Pyriform glands produced up to two types of silk and glue. PySp1 acts as an anchor for the web, creating disks for attachment to various substrates,² while PySp2 is a glue-like material produced deeper in the gland, which coats the pyriform silk fiber during spinning.³ The structure of the pyriform silk protein contains both nonrepetitive and repetitive domains, similar to the other six spider silk types.⁴ The repetitive domain of pyriform silk consists of APAP (Ala-Pro-Ala-Pro), QQS (Gln-Gln-Ser), and SS (Ser-Ser) sequences, flanked by N-terminal and C-terminal non-repetitive domains.⁵ 

The process of spider silk spinning in vitro started from a high concentrated soluble protein solution stored in the gland^6–8 sac, which is connected to a long and complex duct that leads to the spigot.⁹ Initially, the protein in the funnel solution forms a liquid crystal structure, allowing it to flow smoothly through the narrow conduits.¹⁰ The high concentration of the protein undergoes biochemical changes influenced by shear force, ion concentration, and the liquid crystal structure, ultimately forming fibrils.¹¹ The presence of water throughout the duct and spigot suggests that water content may also play a role in the spinning process.¹² 

Various techniques have been employed to form fibers from silk solutions, including solvent extrusion, wet-spinning using a coagulation bath, electrospinning, microfluidic approaches, and organic solvents.¹³ Industrial-scale fibers produced via melt-extrusion and wet-spinning tend to have larger diameters, while electrospinning and microfluidic spinning can achieve micrometer-scale fibers in laboratory settings.¹⁴ Wet spinning, a traditional method, involves immersing the spinneret in a coagulation bath, where the fibers solidify and separate from the polymer solution.¹⁵ The advantages of wet spinning include low cost, simple composition, ease of operation, minimal environmental impact, and less equipment compared to electrospinning.¹⁶ The drawback involved the wastage of material during the spinning process until a suitable spinning solvent and a compatible coagulation bath were identified.¹⁵ 

Previous studies have used recombinant spidroin silk with polycaprolactone (PCL) in electrospinning to improve the strength of composite membranes, but the elasticity of pure protein membranes was not satisfactory.⁵ It has been reported that recombinant spidroin spinning dope can be spun using formic acid and hexafluoroisopropanol (HFIP).²² In this experiment, HFIP was selected as the solvent to dissolve purified spider silk protein powder, while methanol was chosen as the coagulation bath. The resulting stable continuous fibers were then subjected to secondary stretching in both air and methanol to investigate how the concentration of methanol and secondary stretching affect the physical and chemical properties of the fibers.

The amino acid combination of this protein is shown in Fig. S2, the N-terminal domain and C-terminal domain were from the minor ampullate spider protein (Araneus ventricosus), and the repeat domain was from the pyriform spider protein (Araneus ventricosus). The protein contained 526 amino acids, and the molecular weight was 51.9 kDa.

Both at 16 and 20 °C, the recombinant spidroin was expressed in inclusion bodies. Therefore, urea was chosen for the purification process. As shown in Fig. 1, lanes 7 and 8 depict the recombinant spidroin washed with 2M urea, indicating no significant difference between the supernatants obtained at 16 and 20 °C. After dissolution in 8M urea, the majority of the target protein was found in the supernatant. The purified protein exhibited a purity of ∼95% after dialysis in pure water, as observed in lane 10 of Fig. 1. The dialyzed protein was frozen at −80 °C for ∼24 h, followed by lyophilization. The resulting target protein powder yielded ∼80 mg per 1 l of bacterial fluid and was stored at −20 °C prior to fiber spinning.

This recombinant spidroin powder is insoluble in acetic acid, formic acid, and isopropanol, but it was soluble in HFIP. The spinning dope, with a concentration of 10% (w/v), formed a white mass at the end of the needle when immersed in pure methanol, and when deionized water (10% v/v) was added each time, it resulted in fiber formation until the ratio reached 5/9 (V_methanol/V_{total volume}). However, continuous fiber formation could not be achieved. As the concentration of methanol decreased, the spinning dope showed better filament continuity. In pure deionized water, the spinning dope appeared to dissolve rather than forming fibers. For secondary stretching, fibers were subjected to either air stretching in a 1% methanol coagulation bath (Fig. S1-B) or solution stretching in the coagulation bath (Fig. S1-C). The secondary stretched fibers were collected by rotating them in a scroll with a diameter of 10 mm at a speed of 90 rpm. The distance between the coagulation bath and the scroll for fiber collection was ∼10 cm.

The measurement of the fiber diameter started from the point where the fiber could be picked out from the surface of the coagulation bath until it broke. Consequently, the range of the fiber diameter was relatively high. However, it was observed that the average diameter of the fibers decreased as the concentration of the coagulation bath decreased. For example, the average diameter decreased from about 21 µm at a ratio of 1:2 to about 14 µm at a ratio of 1:100 (V_methanol/V_{total volume}), as shown in Table I. The fibers subjected to secondary stretching exhibited a more stable diameter under 0.1% methanol compared to air stretching. This indicates that the concentration of methanol in the coagulation bath has a limited effect on the thickness of the fibers.

The selection of methanol as the coagulation bath was based on its ability to induce a rapid conformation transition of the silk protein and promote the formation of β-sheet.¹⁷ Methanol, as a monohydric alcohol, possesses strong properties that facilitate the desired structural changes in the silk protein during the spinning process. On the other hand, ethanol has also been studied for its effects on protein secondary structure transitions. However, it induces a different kind of conformational change, leading to alterations in the protein's secondary structure.¹⁸ This difference in effect makes methanol a more suitable choice for promoting the desired conformational changes and β-sheet formation in the recombinant spidroin during the spinning process compared to ethanol.

Under light microscopy, the fibers displayed a smooth morphology, as shown in Fig. 2(a). As the concentration of methanol decreased, the spinning fiber became thinner. Conversely, scanning electron microscope (SEM) analysis [Fig. 2(b)] indicated that fibers produced with methanol concentrations of 1:2, 1:5, 1:10, and 1:50 (V_methanol/V_{total volume}) exhibited axial orientation fissures and grooves resulting from the spinning process and methanol concentration. However, at a methanol concentration of 1:100, the fiber morphology appeared smooth without fissures when magnified at 2000×. This smooth morphology was consistent during secondary stretching both in air and in a 0.1% methanol environment. In the coagulation bath, the 1:2 concentration yielded a rough and irregular surface compared to the 1:5 concentration. Notably, fibers formed in the 1:2 methanol coagulation bath displayed clear micro-voids and grooves. As the methanol concentration in the coagulation bath decreased, the size of these grooves and fissures also diminished. This can be attributed to the accelerated water removal from the fibers during the coagulation process and the conformational transition of the silk fiber. After post-drawing, whether in air or in a 0.1% methanol solution, the fiber surface exhibited enhanced regularity and smoothness. This can be attributed to the uniform alignment of molecular chains during the post-drawing process. However, the delicate nature of silk fiber often resulted in solidification and breakage at the edge of the tank when immersed in a methanol solution.

Fractures were observed in the fibers spun in methanol coagulation baths with concentrations of 1:2, 1:5, 1:10, and 1:50 during sample preparation, as shown in Fig. S3. However, no fractures were found in the fibers spun in the 1:100 methanol concentration coagulation bath or in the fibers subjected to secondary stretching. Both high and low methanol concentration coagulation baths did not exhibit significant voids. The irregular shape and surface observed in the SEM images may be a result of fiber breakage.

The morphology of wet spinning fibers is also influenced by the kinetics of coagulation. Generally, slow solvent diffusion results in a uniform porous structure. The solidification kinetics are significantly affected by various factors, such as the solvent system, temperature, polymer concentration and molecular weight, presence of additives in the solution and coagulation bath, nozzle diameter, and injection rate.¹⁵ 

The chemical structure of a spinning dope was determined by measuring the circular dichroism spectrum. When the spidroin powder was dissolved in 10% HFIP, a predominant secondary structure observed was α-helix.⁵ When the spidroin was transformed into spinning fibers, there was only a slight difference in the secondary structure of the protein within the wavelength range of 1600–1700 cm⁻¹, as observed in the amide I band [Fig. 3(b)]. Similarly, the amide II band [Fig. 3(c)] also showed minimal variation. In contrast, there was no significant difference in the secondary structure at the amide III and IV bands [Figs. 3(d) and 3(e)] among the coagulation baths with concentrations of 50%, 20%, and 10%. However, both the 2% and 1% concentration baths, as well as the 1%-air and 1%-methanol conditions, displayed some differences, which were more pronounced.

The characteristic bands within the ranges of 1600–1700 cm⁻¹, 1480–1580 cm⁻¹, 1220–1310 cm⁻¹, and 680–820 cm⁻¹ mainly correspond to amide I (C=O stretching), amide II (N–H-in-plane bending), amide III (C–N stretching and N–H bending), and amide IV (C–H bending out-of-plane), respectively.¹⁹ These results indicate that the change in methanol concentration affects the interaction of the peptide chain but has only a slight impact on the secondary structure of the protein.

Thermogravimetric analysis (TGA) was conducted to evaluate the thermal properties of the silk fiber. Figure 4 illustrates the weight loss rate curve obtained from the analysis. The curve exhibits a single-stage degradation pattern. Initially, as the temperature increased from room temperature to 100 °C, there was a weight loss of ∼5%. This can be attributed to the evaporation of moisture or water content present in the fiber. The second stage occurred between 100 and 240 °C, which showed a slow thermal decomposition. During this stage, a weight loss of around 10% was observed. This suggests that the silk fiber possesses thermal stability, with no significant thermal degradation occurring before 200 °C. A distinct change was observed in the third stage, spanning from 240 to 363 °C, where a rapid weight loss occurred. This could be attributed to the breakage of peptide bonds and side groups within the protein structure of the fiber.¹⁹ Beyond 363 °C, the weight loss rate gradually decreased, indicating a gentle decline in weight. This stage persisted until 600 °C.

In summary, the thermogravimetric analysis indicates that the silk fiber exhibits a thermal stability of up to 200 °C, with significant weight loss and structural changes occurring at higher temperatures, particularly above 240 °C.

When the fiber was just continuously spinning from the coagulation bath, the engineering strain of the fiber was relatively small. The maximum strain of fibers with methanol concentrations of 50%, 20%, and 10% is less than 4%, with no significant difference observed, as shown in Figs. 5 and 6(c). Secondary stretching under different conditions, such as in air or 0.1% methanol, results in different strain changes. Stretching in air shows no significant difference (p > 0.05) compared to spinning directly in 1% methanol. However, stretching in 0.1% methanol leads to a significant difference in strain compared to spinning in 1% methanol and stretching in air (p < 0.01). Notably, the strain is significantly improved with 1% methanol and further stretching, especially in 0.1% methanol. As shown in Fig. 5, the strain in regions I, II, and III is lower than 0.04 mm/mm. In region IV, where the methanol concentration is 2%, the strain mostly increases to around 0.75 mm/mm, with some values reaching 1.5 mm/mm. Fibers spun in a coagulation bath with 1% methanol exhibit further improved strain, mostly around 1 mm/mm, with a maximum achieved strain of 2 mm/mm (region V). The maximum strain reaches up to 2 mm/mm in air stretching (V-A) and 3 mm/mm in 0.1% methanol stretching (V-M), indicating an overall improvement in strain through secondary stretching.

Figure 6(b) and Table II demonstrate that fibers spun in 2% methanol have the lowest modulus, ∼1.2507 GPa. The modulus of these fibers is significantly different from the others (0.01 < p < 0.05). However, there is no significant difference in the stress at which the fibers break among the different types (p > 0.05), as shown in Fig. 6(b). Therefore, the conclusion is that the concentration of methanol in the coagulation bath has a deep influence on the fiber’s strain, a slight influence on the modulus, and little effect on stress. The strain remains smooth and steady when stretched in air but is further improved when stretched in 0.1% methanol.

Analyzing the relationship between diameter and strain [Fig. 6(d)], fibers spun with methanol concentrations of 50%, 20%, and 10% have diameters that decrease from 20.9136 to 15.387 µm, and there is no significant difference in strain among them. However, as the diameter of fibers in regions IV and V becomes thinner, the strain increases. Moreover, when spinning fibers are stretched in air (V-A) or in 0.1% methanol (V-M), the strain increases further. This indicates that as the spinning fibers become thinner, the strain increases. Previous studies have reported strains of recombinant spider silk protein (RSSP) in coagulation solvents, such as MeOH/FA 90/10, 85/15, and 80/20, to be ∼30%–40% (0.3–0.4 mm/mm), while strains in MeOH/water, EtOH, and MeOH are below 40% (0.4 mm/mm).²⁰ Additionally, the best strain reported for recombinant Aciniform silk pulled from 20 mM Tris (pH 8.0) by hand-drawing is 0.34 mm/mm.²¹ The average strains for recombinant pyriform silk are reported as 0.76, 0.70, and 0.70 mm/mm for NRC, N2RC, and N3RC, respectively.²² 

During the simulation of spider silk tensile strength, it was observed that stretching promoted the rearrangement of peptide chains, leading to their solidification through the reestablishment of hydrogen bonds.¹² The strained state resulting from secondary stretching was found to be greater than that of the unstretched fibers. This can be attributed to the fact that, after stretching, the molecules become more oriented and parallel, resulting in reduced chain slippage under loading. Additionally, as the fiber was stretched, the physical interactions among peptide chains unraveled, allowing for a closer arrangement and adjustment of the secondary structure.¹³ 

In Fig. 7(a), it can be observed that the number of Schwann cells increased on all fibers over time. However, at the second and fourth day, there were no significant differences in cell proliferation between the different fibers and the control. At the sixth day, there were differences in cell growth between fibers spun in the control, II, V, V-A, and V-M (0.05 < p < 0.01), but no significant differences with fibers spun in II, III, and IV (p > 0.05). This suggests that fibers spun in different concentrations of methanol do not have a noticeable effect on Schwann cell growth, but secondary stretching of the fiber can promote cell growth.

The live–dead staining experiment depicted in Figs. 7(b) and 7(c) revealed that Schwann cells were distributed predominantly along the fiber at the sixth day. The count of living cells was consistent with the results obtained from the CCK-8 assay. This indicates that the wet spinning fiber exhibited good biocompatibility and could enhance Schwann cell proliferation.

The recombinant protein was expressed in E. coli BL21 (DE3) cells, induced at 16 °C for 12–15 h. The mycelium was then collected through centrifugation at 4000 rpm for 15 min at 4 °C. A lysis buffer containing 20 mM imidazole was added at a ratio of 50 ml per 1 l bacterial fluid. High-pressure crushing (JN-3000, JNBIO, Guangzhou, China) was performed at 1000 mbar and 4 °C, followed by centrifugation at 4000 rpm for 30 min at 4 °C. The expressed protein was found to be in the form of inclusion bodies. The inclusion bodies were dissolved in 2M urea using ultrasonication with an ON/OFF cycle of 5/8 s, repeated 99 times. The mixture was then centrifuged at 4000 rpm for 15 min at 4 °C. The resulting precipitate was dissolved in 8M urea, followed by ultrasonication and centrifugation as described above. The supernatant was dialyzed in pure water for 3 cycles, each lasting 12 h. It was then frozen at −80 °C for 8 h until the protein solution reached a steady frozen state. The protein solution was lyophilized to obtain protein powder, which was subsequently dissolved in HFIP at a concentration of 10% w/v. The solution was mixed on a magnetic stirrer (Sangon biotech, Shanghai Co., Ltd.) for approximately 8–12 h at room temperature, and it was used as spinning dope.

The spinning process involved transferring the spinning dope, prepared in HFIP, into a 1 ml syringe fitted with a needle with a diameter of 30 µm. The spinning dope was injected into a coagulation bath using a pump. The coagulation bath contained different concentrations of methanol, as specified in Table III. The fibers were spun into the coagulation bath at a spinning rate of 0.2–0.3 ml/h. A digital-controlled device with a scroll mechanism was used to control the spinning process. The spinning process took place in air at room temperature. The fibers were collected on a scroll with a diameter of 30 mm. To prevent entanglement or damage to the fibers, the scroll was covered with a black cardboard cartridge secured by transparent adhesive tape. The scroll rotated at a speed of 30–35 rpm/min. The linear distance between the fiber emerging from the coagulation bath and the reel for fiber collection was ∼10 cm, as illustrated in Fig. S1-A. After the fibers were collected, a post-spin stretching process was employed. The spidroin fibers collected in air and in a 0.1% methanol coagulation bath were subjected to post-spin stretching. The scroll diameter used for post-spin stretching was 10 mm, and the scroll rotated at a speed of 87–90 rpm/min, as depicted in Figs. S1-B and S1-C. Throughout the entire spinning process, the ambient conditions were maintained at a temperature of 25 °C and a humidity level of 30%–50%. Following the spinning process, the fibers were allowed to dry in the same room environment as described above.

The morphology of the spinning fibers was analyzed using both a light microscope and a scanning electron microscope (SEM). For light microscopy, the analysis was performed using a Leica Las 4.12 inverted microscope equipped with a DFC Twain camera from Imaging source Leica, Germany. Ten pictures were taken for each silk sample, and ∼100 measurements were made for each silk in each picture. The spinning fibers were placed on a conductive adhesive, and then, gold spray was applied for 40 s to enhance conductivity and improve imaging quality.

SEM analysis was conducted using a FlexSEM1000 microscope from Hitachi, Japan. The spinning fibers were also prepared by sticking them onto a conductive adhesive. Prior to imaging, the samples were coated with a thin layer of gold to improve electron conductivity. The SEM was operated at an acceleration voltage of 10 kV and at room temperature.

The mechanical structures of different fibers were analyzed using a FTIR spectrometer. Specifically, a NEXUS-670 spectrometer from Thermo, USA, was utilized for this analysis. The measurements were conducted at room temperature. The range of the spectra wavelength examined was set between 500 and 4000 cm⁻¹. A resolution of 4 cm⁻¹ was chosen for the analysis. To ensure accurate measurements, background calibration and normalization techniques were employed to correct for any interference or baseline variations in the obtained spectra.

Thermogravimetric analysis (TGA) of the fibers was carried out using a TA Instruments NETZSCH TG 209F1 instrument from Netzsch, Germany. The weight of the sample V-M fibers was 35 mg. The TGA analysis was conducted within a temperature range of 30–600 °C. The temperature was increased at a rate of 10.0 K/min, allowing for gradual heating of the fibers.

To measure the diameter of the fiber, a series of 2 cm paper frames with a 1 cm square hole were used. The fiber was attached to the paper frame, starting from the coagulation point and extending until it was interrupted or reached the end. The diameter measurements were performed using light microscopy and the Image J software. The images captured through the light microscope were analyzed and processed using the Image J software to determine the diameter of the fibers. After measuring the diameter, the paper frame with the attached fiber was placed on a T150 universal test machine (UTM) manufactured by KLA-Tencor Inc., Oak Ridge, USA. The side of the paper frame was cut to expose the fiber for testing. The tensile testing of the fiber was conducted on the UTM with a strain rate of 10⁻³ mm/s. The testing was performed at room temperature and under a humidity level of 20%–30%. This allowed for the evaluation of the mechanical properties and tensile strength of the fiber.

To assess the biocompatibility of the fiber, Schwann cells from rat stored in laboratory were chosen as the cellular model. These cells were cultured in high glucose Dulbecco’s modified eagle medium (DMEM) obtained from Hyclone, USA. The culture medium was supplemented with 10% fetal bovine serum (FBS) from Gibco, USA, and 1% streptomycin/penicillin solution from Hongsheng Biotechnology, Shanghai. The cell culture was maintained in a CO₂ incubator model Heraeus BB15 from Thermo, USA, at a temperature of 37 °C.

The incubator provides the controlled and optimal conditions required for cell growth and proliferation. The culture medium was refreshed every second day to ensure the availability of nutrients and maintenance of cell viability. In this experiment, the control group consisted of cells cultured on a cell plate without the presence of the fiber. By comparing the behavior and viability of the Schwann cells in contact with the fiber to those in the control group, the biocompatibility of the fiber could be assessed.

The fibers, which were cultured in a 24-well plate, underwent a sterilization process prior to cell seeding. They were immersed in 75% alcohol for 2 h to ensure surface disinfection. Following that, the fibers were subjected to ultraviolet (UV) disinfection in an ultra-clean table for 12 h to eliminate any potential microbial contamination. After the sterilization procedure, Schwann cells were seeded at a density of 10⁴ cells per well and cultured for a period of six days. During this time, the cells proliferated and interacted with the fibers, allowing for assessment of biocompatibility. To evaluate cell viability, a CCK-8 assay was conducted on days 2, 4, and 6 during the culture period. CCK-8 solution was prepared by mixing 1 ml of CCK-8 solution with 9 ml of DMEM. Subsequently, 400 µl of the CCK-8 mixed solution was added to each well containing the cells and fibers. The plate was then incubated in the CO₂ incubator for 1 h. After incubation, the absorbance changes at 450 nm were measured using a Multiskan GO 1.00.40 microplate reader manufactured by Thermo Scientific, USA. These absorbance measurements provided quantitative information about the metabolic activity and cell viability, reflecting the biocompatibility of the fibers with the Schwann cells over time.

After the six-day culture period, the cells on the fibers were subjected to staining using the Live–Dead Cytotoxicity Kit from Mesgen, Shanghai, China. This kit allows for the differentiation and visualization of live and dead cells. To perform the staining, a staining buffer was prepared by mixing 1 µl of 1 mM Calcein, AM (which stains live cells) and 1 µl of 1 mg/ml propidium iodide (PI, which stains dead cells) in 1 ml of the staining buffer provided in the kit. The staining solution was then added to the cell-fiber samples and incubated for 30 min at a temperature of 37 °C in a 5% CO₂ environment. This incubation allowed the fluorescent dyes to penetrate the cells and bind to specific intracellular components. After the incubation period, the samples were washed with phosphate-buffered saline (PBS) to remove any excess staining solution and debris. The staining process ensured that live cells would exhibit a green fluorescence when visualized under a fluorescence microscope, whereas dead cells would appear red due to the PI staining. By using a fluorescence microscope equipped with a suitable band-pass filter, it became possible to visualize and distinguish between live (green) and dead (red) cells on the fibers. This staining method provided a qualitative assessment of cell viability and compatibility with the fibers.

In this study, a statistical analysis was performed to compare the silk fibers using an analysis of variance (ANOVA) and a student’s t-test. The p-value was used as a measure of statistical significance.

If the obtained p-value is greater than 0.05 (p > 0.05), it indicates that there is no significant difference between the groups being compared. In other words, the data do not provide enough evidence to conclude that there are any meaningful distinctions between the silk fibers. On the other hand, if the p-value falls between 0.05 and 0.01 (0.05 < p < 0.01), it suggests that there is a difference between the groups. However, this difference may not be considered statistically significant based on conventional criteria. When the p-value is less than 0.01 (p < 0.01), it indicates a significant difference between the groups being compared. This means that the observed differences in the properties or characteristics of the silk fibers are highly unlikely to have occurred by chance alone.

In the wet spinning process of recombinant pyriform repeat domain spidroin, HFIP was chosen as the spinning solvent, and the concentration of methanol in the coagulation bath was investigated. It was found that two-step stretching, specifically stretching in air and in 0.1% methanol, resulted in a higher engineering strain of the fiber. This suggests that the increased water content in the coagulation bath may be responsible for the improved strain of the spun fiber.⁹ 

When examining the chemical structure of the spinning fiber, it was observed that there was little difference in the peak corresponding to the protein secondary structure. However, there were variations in the peaks observed at 1000 to 1500 cm⁻¹, 1750 to 2000 cm⁻¹, and 2750 to 3000 cm⁻¹. These ranges corresponded to the bonds on the surface of the peptide chain. This indicates that the concentration of methanol does have an effect on the peptide chain, but not on the overall protein secondary structure. Lower methanol concentrations were found to induce higher elasticity in the spinning fiber.

Secondary stretching was found to be necessary for improving the mechanical properties of the fiber. However, further optimization of the secondary stretching method is required in order to obtain excellent and unique man-made fibers with desirable mechanical characteristics. Based on the results of this experiment, a simple and easily controllable method was discovered to mimic the natural elasticity of spider silk. This system may also be applicable to other types of recombinant spidroin, and different wet spinning systems may further enhance the strength of these proteins. The fibers obtained in this experiment have the potential to promote Schwann cell growth and can be considered as candidate materials for biomedical applications.

Schematic diagram of wet spinning, amino acid composition of the protein, cross-section of wet spinning fiber are given in the supplementary material.

This work was supported by the Chinese National Natural Science Foundation (Grant No. 31570721) and the Key project of Shanghai Science and Technology Commission (Grant No. 14521100700).

The authors have no conflicts to disclose.

Jie Zhang: Data curation (equal); Writing – original draft (equal). Mengxin Gong: Writing – review & editing (equal). Qing Meng: Funding acquisition (equal); Project administration (equal).

The data that support the findings of this study are available from the corresponding author upon reasonable request.