A sterile, injectable, and robust sericin hydrogel prepared by degraded sericin

doi:10.21203/rs.3.rs-3324975/v1

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A sterile, injectable, and robust sericin hydrogel prepared by degraded sericin

https://doi.org/10.21203/rs.3.rs-3324975/v1

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Sericin hydrogels have been widely studied and applied in tissue engineering, drug delivery and clinical medicine due to their unique properties. However, their poor mechanical strength, fragility behavior and inconvenient sterilization are the key hurdles for applications. To address these challenges, a sericin hydrogel with excellent physical and chemical properties and cytocompatibility was prepared through crosslinking genipin with degraded sericin extracted from fibroin deficient silkworm cocoons by the high temperature and pressure method. Our reported sericin hydrogels possess good elasticity, injectability, and robust behaviors. The sericin hydrogel formed by sericin solution at concentration 8% (w/v) can be smoothly through 16 G needle. The 12% sericin hydrogel was unbroken until its compression ratio reached 70%, and accompanied with the compression strength of 674 kPa. The maximum stretch of 12% sericin hydrogel was 740%, and the breaking strength was 375 kPa in the tensile test. The tensile modulus of the 12% hydrogel is 477 kPa. Besides that, the hydrogel system has excellent cell-adhesive capability, effectively promoting cell attachment, proliferation. The swelling and degradation behaviors of the hydrogels are pH responsiveness. Sericin hydrogel releases drugs in a sustained manner. Furthermore, this study overcomes the problem that sericin hydrogels are difficult to be sterilized (sterilization will inevitably lead to the destruction of their structures). In addition, it breaks the previous idea that sericin extracted at high temperature and pressure is difficult to be directly cross-linked into a stable hydrogel. This hydrogel system developed in this study is promising to be a new multifunctional platform for extending the application area of sericin.

Sericin

Hydrogel

High temperature and pressure

Degraded sericin

Robustness

Polymer hydrogels, a type of polymers with three-dimensional network structure containing a large amount of water, have attracted persistent attention in biomedical fields by acting as multifunctional platforms for tissue engineering [1], drug delivery [2], cell modulation [3], optical devices[4], and biosensors [5]. Hydrogels formed by natural biological materials were favored in the biomedical field due to their excellent biocompatibility and bioactivities[6]. Recently, sericin, a natural non-immunogenic protein with multiple biological activities, has received great attention and is considered as a new promising candidate for fabricating hydrogel [1].

Sericin, a bioactive protein containing many polypeptides, is produced from the middle silk gland of silkworms. Sericin is abundant in resources and yield. It is estimated that there are more than thousands of silkworm strains and the annual output of sericin is over 50, 000 tons in the world [7, 8]. Silkworms are divided into wild-type and fibroin-deficient mutant according to whether they synthesize fibroin[9, 10]. The cocoons of wild-type silkworms contain high amounts of fibroin (approximately 75%) [11]. Currently, the existing methods for extracting sericin from wild-type cocoons are harsh degumming. Unfortunately, such extraction method inevitably destroys the structure of sericin, resulting in degraded highly water-soluble sericin. This degraded sericin is difficult to form stable hydrogel network [1]. Generally, the degraded sericin is mixed into other polymers (polyacrylamide[12], poly(N-isopropylacrylamide)[13], chitosan[14–16], Poly(vinyl alcohol)[17, 18], sodium alginate[19, 20], agar[21], poly(ethylene glycol) dimethacrylate[21], collagen[22], gelatin[23]) hydrogel systems to improve their physical-chemical properties and biological activities.

For a long time, it is a challenge to fabricate pure sericin hydrogels with stable networks. Until the early 21st century, the fibroin-deficient mutant cocoons (e.g.,Sericin hope, 140 Nd-s) without fibroin were developed to obtain intact and non-degraded sericin by a gentle extraction method (for example, LiBr) [8, 24]. This type of sericin was prone to form stable hydrogel networks by chemical crosslinking agents or physical factors. A series of sericin hydrogels were successfully developed based on such sericin by treating using glutaraldehyde[8], genipin[25], and physical sonication[2]. However, it is difficult to obtain high concentration of native sericin from cocoons through the mild extracting process at present. These sericin hydrogels are poor in elastic modulus and inconvenient for sterilization (the structure of sericin hydrogels would be unavoidably destroyed under existing sterilization methods[26] ), resulting in seriously restricting their practical applications. To address these problems, the high temperature and pressure method is employed to extract high-content sericin solution from fibroin-deficient cocoons powder for fabricating a sterile hydrogel with high hardness, avoiding the subsequent complex sterilization processes. Depressingly, this hydrogel is brittle and fragile [26]. Furthermore, we fabricated a robust sericin hydrogel based on a high concentration native sericin isolated from fibroin-deficient silkworm bodies [27]. But some problems in this hydrogel system still exist, including laborious sericin extraction processes and inconvenient sterilization, thus limiting its application. Now, it is still a challenge to fabricate a sterile robust sericin hydrogel.

In this work, a new sericin hydrogel with excellent physical-chemical properties and cytocompatibility was fabricated by a simple process. Once the sericin gelling reaction system is placed in a bio-clean environment, a sterile and robust sericin hydrogel can be fabricated directly. Our new hydrogel system overcomes the problem that sericin hydrogels are difficult to be sterilized. Moreover, this sericin hydrogel possesses good elasticity, injectability, and robust behaviors.

2.1 Materials

The fibroin-deficient mutant silk cocoons were produced by 180 Nd-s silkworms that were preserved in our lab. Genipin (98%) was purchased from Linchuan Zhixin Biotechnology Co., Ltd, China. Horseradish peroxidase (HRP) and ninhydrin were obtained from BBI life sciences (Shanghai, China).

2.2 Extraction of sericin solution

Sericin was isolated from the natural fibroin-deficient mutant silk cocoons (Bombyx mori, 180 Nd-s) according to the method as our previous report [26]. Briefly, the cocoons were ground into powder, and then the powder was mixed with ultrapure water thoroughly. The mixture was treated at 121^oC for 30 minutes. The obtained soup was centrifuged at 5,000 rpm for 5min to collect the supernatant sericin solution. The concentration of sericin solution was determined by the oven drying method. The molecular weight distribution of sericin was detected by SDS-page method.

2.3 Free amino group content detection

The content of free amino groups in the sericin was determined by ninhydrin colorimetry method according to the previous report with slight modifications [28]. Glycine was employed as a reference standard. Firstly, glycine was dissolved and diluted into different concentrations (0.01 mg/ml, 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml, and 0.05 mg/ml) of solution using ultra-pure water. The glycine solution, ninhydrin ethanol solution (2%, w/v,) and PBS (pH 6.8) were mixed at a constant volume ratio of 1:2:2. Then the mixture was bathed in a hot water at 100℃ for 15 min. After that, the samples were taken out and cooled at room temperature. The absorbance value of samples was measured at 570 nm using a spectrophotometer. A linear standard curve was drawn, plotting the average absorbance values obtained for the mixture triplicate values against the standard glycine concentration. Then the glycine solution was replaced by sericin solution in the above reaction system and the absorbance values of sericin solution samples were obtained. At last, these absorbance values were brought into standard curve equation and then the free amino group content in the sericin solution can be worked out.

2.4 Amino Acid Analysis

The amino acid composition of sericin protein was determined by using high-performance liquid chromatography. Briefly, 1 g freeze-dried sericin sample was hydrolyzed by 20 ml HCl (6 M) at 110℃ for 24 h in a blast drying oven. The hydrolyzed sample was transferred into a colorimetric tube and diluted with deionized water to 25 ml when its temperature drops to the environment. Next, 1 ml clear solution was collected and blow-dried in the water bath at 85℃. After that, 1 ml water was added and blow-dried again. The 10 ml HCl (0.02 M) was added and shaken well. Then, 500 µl solution was taken out and mixed with 250 µl phenyl isothiocyanate acetonitrile (0.1 M) and 250 µl triethylamine acetonitrile (1 M). 1 hour later, 2 ml n-hexane was added, and mixed well. The reacted sample was then placed statically for a period. The lower solution was tested on a high-performance liquid chromatograph (Agilent 1260) after it was filtered through 0.45 µm organic membranes.

2.5 Preparation of sterile robust sericin hydrogel

Genipin was diluted with ultra-water and filtered with a 0.22 µm filter membrane. Then the genipin solution was dried at 25^oC in a sterile environment. The dried genipin was added into sericin solution according to genipin and free amino groups of sericin solution with a constant mole ratio of 1:2. Then, the reaction buffer was mixed sufficiently and formed into a hydrogel in a clean environment at room temperature. The hydrogel was defined as n% sericin hydrogel according to the concentration of sericin solution (n%, w/v) that was mixed with genipin.

2.6 Gelation time of sericin hydrogel

Gelation time was determined according to a previously described method [8]. The sericin solution and genipin solution were mixed fully, followed by loading them into a vial tube. The gelation time was obtained by timing from solution mixing to the time point when the reaction buffer became viscous and couldn’t move downwards along the vertical tube wall.

2.7 Scanning electron microscopy (SEM)

The sericin hydrogel samples were frozen at -196^oC in liquid nitrogen. Then, the hydrogel samples were moved into a vacuum drying equipment (FD-1A-80, Beijing Boyikang Experiment Instrument Co.,Ltd)and dried at -196°C. The freeze-dried specimens were observed by using a scanning electron microscopy (JSM-IT300, JEOL Ltd, Japan) with working voltage of 25 kV. The pore sizes of hydrogel scaffolds were calculated by averaging 30 randomly selected pores using Image J software.

2.8 Porosity analyses

The porosity of the 8% sericin hydrogel samples was evaluated by a conventional liquid displacement method as reported previously[26]. The lyophilized sample was immersed in a known volume (V_K) of ultra-pure water. One hour later, the volume of water containing hydrogel was determined and denoted as V_T. The hydrogel sample was then removed and the remaining water was recorded as V_R. The porosity of the lyophilized hydrogel sample was then determined according to the following equation:

$\text{p}\text{o}\text{r}\text{o}\text{s}\text{i}\text{t}\text{y} \left(\text{%}\right)=\frac{\text{V}\text{K}-\text{V}\text{R}}{\text{V}\text{T}-\text{V}\text{R}}\times 100$ %

2.9 Evaluation of swelling behaviors and degradation dynamics

The swelling behavior of 8% sericin hydrogel was evaluated by a gravimetric method [29]. Lyophilized hydrogels were weighed and then immersed in phosphate-buffered saline (PBS) at different pH (pH 3, pH 7.4, and pH 11) at 37°C atmosphere. The hydrogel samples were taken out and weighted at predetermined intervals. The swelling ratios of the lyophilized hydrogel scaffolds were calculated by:

$Swelling\left(\%\right)=\frac{\text{M}1-\text{M}0}{\text{M}0}$ ×100%

Where M₀ is the dry weight of the scaffolds, and M₁ is the swollen weight of the scaffolds.

The degradation behavior of 8% sericin hydrogel in vitro was evaluated by a gravimetric method. Briefly, three randomly selected hydrogel samples were weighted and dried. Then, their wet weight (M_w0) and dry weight (M_d0) were noted. Then other dried hydrogels were immersed in PBS (pH 7.4) at 37°C and replaced with fresh PBS (pH 7.4) every day. The hydrogel samples were taken out, washed, and dried at determined time points. The degradation ratios of hydrogels were calculated by:

$$Degradation=\frac{M0*R-Md}{\text{M}0\text{*}\text{R}}$$

Where, M$0$ is the initial wet weight of sample, and Md is the dry weight of sample. R is the ratio of M_d0 and M_w0.

The degradation behavior of sericin hydrogel in vivo was evaluated according to a method as previously reported with slight modifications [30]. Female C57BL/6 mice (8 weeks old) were first anesthetized by intraperitoneal administration of 10% chloral hydrate (5 µl/g•BW). Next, 8% hydrogel samples (0.2 g per sample) were placed subcutaneously in mice. According to the pre-set time points, the mice were euthanized, followed by resecting the implanted hydrogels and washing carefully with ultra-pure water. Then, the resected samples were dried for further analysis. All the animal experiments were carried out according to the guidelines and authorized by the Ethics Committee of Jiangsu University of Science & Technology (Zhenjiang, China).

2.10 The release of drug from sericin hydrogels

The controlled drug release behavior of sericin hydrogels was evaluated using HRP as a model drug. HRP-loaded hydrogel samples were prepared by mixing HRP (2 mg/ml) with sericin solution (8%, w/v) containing genipin at a constant volume ratio of 1:175. The samples were immersed in fresh PBS (pH 7.4) and kept at 37°C in a constant temperature incubator. The HRP content in the PBS was measured by ELISA kit according to the manufacturer's instructions.

2.11 Fourier Transform Infrared (FTIR) Spectroscopy and x-ray diffraction

The FTIR spectra of the specimens were carried out on a Fourier transform infrared spectroscopy (Nexus, Thermal Nicolet, USA) for the spectral region of 4000 − 400 cm^− 1. The software OPUS 5.5 (Bruker Optics, GmbH) was used to analyze the spectrum. The X-ray diffraction (XRD) pattern of the specimens was evaluated on a D8-Advance X-ray diffractometer (Bruker, Germany). The diffraction patterns run from 5° to 70° of 2 θ angles.

2.12 Mechanical analysis

The compression test was performed on a universal testing machine (AUST, China, Zhuhai) equipped with a 20 N load cell. The hydrogel specimens for the compression test were molded into a cylindrical structure with a 7-mm height and 8-mm diameter for stress-strain analysis. While the samples for the tension test were molded into dumbbell shapes (15 mm gauge length and 10 mm width) and performed on a universal testing machine (QLW-5E, Xiamen Qunlong Scientific Instrument Co., Ltd., China). All the tests were carried out at a constant velocity of 5 mm/min.

2.13 Injectable property

The 8% sericin hydrogel was formed in a syringe. Then, the injectability of the sericin hydrogel was analyzed by a syringe with a 16G needle.

2.14 In vitro cell test

Cell culture assays were performed on the sericin hydrogels, with the uncoated cell-culture plates set as controls. Briefly, the 8% hydrogel samples gelled in 24-well or 96-well culture clusters, followed by 5 rinses with fresh PBS (pH 7.4). Then, the hydrogel samples were rinsed 3 times with fresh high glucose DMEM medium containing 10% fetal bovine serum before further use. Mouse embryonic fibroblasts cells (NIH3T3) were cultured with high glucose DMEM medium in a cell incubator (37°C, 5% CO₂, 100% humidity). The cell adhesion behavior of sericin hydrogels was determined according to a reported method [2]. NIH3T3 cells cultured in 24-well cell culture plates with 1×10⁴ cells /well of initial cell density. Furthermore, the CCK-8 kit was employed to assess the viability of NIH3T3 cells which cultured in the 96-well cell culture plates at a density of 4×10³ cells/well.

2.15 Statistical analysis

All the experiments were performed for n = 3 samples unless otherwise specified. All the values were expressed as mean ± SD. Data analysis was done using statistical software GraphPad Prism 8. The significance level was measured by comparing the data between groups and within groups by performing one-way analysis of variance (ANOVA) followed by Tukey's test. Microscopic images were analyzed using Image J software. P < 0.05 was regarded as significant difference.

3.1 Synthesis and mechanism of sericin hydrogels

The sericin hydrogel was formed by sericin which was isolated from yellow fibroin-deficient mutant silkworm cocoons (180 Nd-s) using a high temperature and pressure method. The detailed preparation process of sericin hydrogel was depicted in Fig. 1. In agreement with previously reported [8], the sericin extracted by high temperature and pressure resulted in protein degradation as evidenced by a smear with the vague bands ranging from 10 ~ > 180 kDa (Fig. 2A). However, in our previous study, we found that the solubility of sericin of fibroin-deficient mutant silkworm cocoons in hot water is far higher than that of wild-type silkworm cocoons [26]. Only a small amount of sericin protein can be dissolved in hot water in the wild-type silkworm cocoons [31]. Intriguingly, the sericin in the cocoon powder could be almost completely dissolved in our study. The results indicated that the compositions of sericin solution in this study are different from that extracted from wild-type silkworm cocoons. Previous reports argued that the sericin isolated from silkworm cocoons by harsh extracting methods, including high temperature and pressure, is hard to crosslink into stable sericin hydrogel due to its high degradation and water solubility [1]. To be excited, we found such degraded sericin could form a robust hydrogel with excellent elasticity (Supplementary Video S1, S2). Why the degraded sericin can be crosslinked into a stable hydrogel by genipin? As we know, genipin is a popular and biocompatible crosslinker for fabricating hydrogels or scaffolds by reacting with free amino groups of polysaccharides or peptides [32]. To address this confusion, we determined the content of free amino end group and the amino acid composition of the sericin. The results showed that the content of free amino end groups in the sericin isolated from 180 Nd-s was as high as 12 times that in the sericin extracted from wild-type silkworms (Jingsong A) (Table 1). Sericin with higher content of free amino end groups provides more opportunities to crosslink with genipin. Moreover, the higher content of free amino end groups would raise the crosslink density between sericin and genipin. However, there is little difference in amino acid composition of sericins between the wild-type silkworm cocoons and fibroin-deficient mutant silkworm cocoons (Table 1).

Table 1

Free amino groups and amino acid composition of sericins isolated from wild-type silkworm cocoons (Jingsong A) and fibroin deficient cocoons (*180 Nd-s*)
Free amino groups(in mol/kg)/amino acids (in mole %)	Sericin isolated from Jingsong A	Sericin isolated from 180 Nd-s
Free amino groups	0.09	1.05
Serine	32.81	31.93
Aspartic acid	15.89	15.47
Glycine	10.28	9.68
Threonine	9.13	8.86
Glutamic acid	6.27	7.43
Tyrosine	5.03	5.64
Arginine	4.92	4.73
Lysine	3.49	3.62
Alanine	3.26	3.40
Valine	3.15	3.31
Histidine	1.79	1.72
Leucine	1.64	1.70
Isoleucine	0.87	0.87
Proline	0.73	0.81
Phenylalanine	0.63	0.71
Methionine	0.11	0.11
Cystine	not detected	not detected

3.2 Gelation kinetics of sericin hydrogels

The influence of sericin concentrations on the gelation rate of sericin hydrogels was investigated. The results in c exhibited that the gelation time was shorted as the sericin concentration increased. The average gelation times of the hydrogels can be adjusted from 13 to 80 minutes according to the sericin concentration.

3.3 The micro-architecture of sericin hydrogel

The micro-architecture of lyophilized sericin hydrogel scaffolds was revealed by scanning electron microscopy (SEM). We analyzed the microstructure of 4%, 8%, 10%, and 12% sericin hydrogels, followed by the calculation of pore size, porosity and pore wall thickness. The results showed that the pore size of these hydrogel scaffolds is distributed between 4.9 µm to 7.1 µm. Porosity was decreased as the concentrations of sericin solutions was increased. The porosities of 4%, 8%, 10%, and 12% sericin hydrogel are 79%, 70%, 67%, and 66%. respectively (Table 2). It is interesting that pore wall thickness of sericin hydrogel is increased with the concentration of sericin solution increased.

Table 2

The pore size, porosity and pore wall thickness of the different lyophilized sericin hydrogel scaffolds were frozen at -196^oC.
hydrogels	4% sericin hydrogel	8% sericin hydrogel	10% sericin hydrogel	12% sericin hydrogel
Pore size (µm)	4.9 ± 0.6	6.0 ± 1.9	6.7 ± 0.8	7.1 ± 1.5
Porosity (%)	79 ± 5	70 ± 4	67 ± 1	66 ± 3
Pore wall thickness(µm)	0.31 ± 0.1	0.68 ± 0.2	0.96 ± 0.3	1.39 ± 0.4

3.4 Swelling characteristics of the sericin hydrogels

The swelling properties of hydrogels suggest the capacity to absorb water, which is vital as it affects many aspects of a hydrogel (such as absorb water, volume and so on) [8, 33]. Usually, the water absorption of hydrogels can be indicated by swelling properties. To characterize the swelling properties of sericin hydrogels, we determined the swelling dynamics of lyophilized sericin hydrogel at different pH (pH 3.0, pH 7.4, pH 11.0). The results in Fig. 4A exhibited the expansibility of 8% sericin hydrogel under different pH conditions. During the first 12 hours, the burst swelling appeared no matter what kind of pH conditions. The reason for this phenomenon is that abundant hydrophilic groups contained in sericin are easily combined with water molecules [34] The first phase was followed by a slowly increasing trend of swelling rate. The peaks of lyophilized sericin hydrogel samples appeared within 100 hours under acidic (pH 3.0) or neutral conditions (pH 7.4). Under basic conditions (pH 7.4), the swelling peak occurred relatively late at 225 hours. After a period of water absorption and swelling, the swelling ratios of samples reached 400, 700, and 800 at pH 3.0, pH 7.4, and pH 11.0, respectively. The results showed that swelling behaviors of the sericin hydrogels are responsive to pH, which is consistent with that of sericin hydrogels in our earlier reports [2, 26, 27].

3.5 Degradability of the sericin hydrogels

Controlling the degradation of a hydrogel is important for drug release in vivo application. A degradable hydrogel can release encapsulated bioactive molecules to exert its functions. Here, the in vitro degradation behaviors of sericin hydrogel under different pH conditions were evaluated. As presented in Fig. 4B, the degradation of sericin hydrogel was the fastest at alkaline conditions (pH 11.0). The cumulative degradation rate was 85.2% within 3 weeks. While the cumulative degradation rates of sericin hydrogel under neutral conditions (pH 7.4) and acidic conditions (pH3.0) were 63.7% and 24.5% within 5 weeks, respectively. In agreement with the sericin hydrogels previously reported, the sericin hydrogel had an apparent response to pH. The cause was ascribed to the sericin with pI of 4 and containing abundant acidic amino acids [35]. Compared with self-assembled sericin hydrogel [26], the hydrogel formed by covalent crosslinking in this study has a longer life and better stability in aqueous solution. Further, degradation dynamics of the hydrogel in vivo were carried out in C57BL/6 mice. The life span of hydrogel was over 5 weeks in vivo(Fig. 4C). The results gave evidence that the sericin hydrogel has a stable network for further evaluating the controlled drug release.

3.6 Drug release of sericin hydrogels

The controlled release delivery systems could avoid the sudden release of drugs and prolong the drugs’ therapeutic effects at targeted sites, thus improving drug utilization [36]. Hydrogels are a type of most important biomaterials for drug delivery. In this study, we employed horseradish peroxidase (HRP) as a model drug to evaluate the drug release kinetics of the sericin hydrogel. Then the drug release profile of HRP from the sericin hydrogel was monitored. As shown in Fig. 4D, HRP burst release appeared within the beginning of 6 hours, which was consistent with previous reports [2, 8]. Then the release rate of HRP slowed down over time. The cumulative release rate of HRP reached approximately 60% within 192 hours. The results indicated that sericin hydrogel has great potential in drug delivery.

3.7 Secondary structural of sericin hydrogels

The Fourier transformation infrared spectroscopy (FTIR) and X-ray diffraction (XRD) are used to evaluate the structure of proteins or polypeptides. The structure of a protein was revealed in terms of its characteristic absorption band based on stretching vibrations of the structure repeat. The absorption bands used for analyzing protein secondary structure include amide I, amide II, amide III, and amide V. The amide I (1700 − 1600 cm^− 1) represents the C = O stretching vibration of peptide bonds. The amide II (1600 − 1500 cm^− 1) is mainly generated by N-H stretching vibration and C-N stretching vibration of amino acids. The absorption peak of amide III (1330 − 1220 cm^− 1) mainly comes from C-N stretching vibration coupled with amino acid N-H in-plane bending vibration. The amide V (800 − 640 cm^− 1) is mainly determined by the N-H stretching vibration outside the plane. Among them, amide I is most sensitive to change in the secondary structure of proteins. Hence, amide I is most useful to analyze the secondary structure of proteins [37–42]. Generally, the characteristic absorption peaks of β -folding, random coil and α -helix in the amide I are located at 1630 cm^− 1, 1645 cm^− 1, and 1655 cm^− 1, respectively. In order to clarify the composition of secondary structure in sericin before and after gel, amide Ⅰ of samples was analyzed. As shown in Fig. 5A-C, the peak position of the amide I band shifted from 1655 cm^− 1 to 1654 cm^− 1 after gel by comparing the spectra of sericin solution with sericin hydrogel. According to the fitting curve values, the secondary structure of sericin before and after gel formation was shown in Table 3. Notably, both α-helix and β-sheet in sericin were increased greatly after gel, indicating that sericin polypeptides were transformed into more stable networks by crosslinking with genipin [26].

The crystalline structure of sericin was determined by X-ray diffraction. The diffraction peaks of sericin powder and sericin hydrogel were at 19.72° and 19.68° (Fig. 5D). This change may be contributed to the conversion of the random coil into β-sheet structure in sericin [26].

Table 3

The secondary structure compositions of sericin protein before and after gel formation.
Secondary structure		α-helix	Random coil	β-sheet	β-turn
Ratio(%)	Sericin powder	21.95	30.50	25.74	21.81
Ratio(%)	Sericin hydrogel	25.74	26.46	32.85	14.95

3.8 Mechanical stability of sericin hydrogels

Mechanical property is one of the key parameters to be evaluated for hydrogel materials. The compression test results as shown in Fig. 6A-C and Supplementary Video S1 (right column), S2. The compressive modulus of hydrogel was increase with increasing the concentration of sericin. The compressive moduli of the 3%, 4%, 8%, 10%, and 12% hydrogels were 25, 75, 289, 311, and 434 kPa respectively (Fig. 6A,B).

High concentration hydrogel with higher compressive moduli may be caused by its lower porosity and thicker pore wall (Fig. 3, Table 2). The 8% hydrogel remained intact when its compression ratio was above 50%. Once the external force was removed, the hydrogel sample could recover to over 95.0% of the original (Fig. 6C and Supplementary Video S1 (right column)). The 12% sericin hydrogel was unbroken until its compression ratio reached 70%, and accompanied with the compression strength of 674 KPa (Fig. 6C and Supplementary Video S2). The above results suggested that the hydrogels formed by degraded sericin own excellent hardness and elasticity. In addition, the maximum stretch of 12% sericin hydrogel could reach 740%, and the breaking strength was 375 kPa in the tensile test. The tensile modulus of the 12% hydrogel is up to 477 kPa (Fig. 6B and Supplementary Video S3). These parameters were much higher than that of pure sericin hydrogel formed by sericin isolated from cocoons reported in previous studies [18], indicating that the sericin hydrogel had excellent mechanical properties.

3.9 Injectable property of sericin hydrogels

Injectable hydrogel systems are attractive in local drug delivery by providing a 3D hydrogel network within the target tissue capable of sustained release of the chemotherapeutics for increasing the therapeutic index [43, 44]. Sericin is widely studied for the synthesis of injectable in situ-forming hydrogels because of ready availability, presence of modifiable functional groups, biocompatibility, and other physiochemical properties [7]. Prior to this study, a few injectable sericin hydrogels were fabricated by low-concentration native sericin isolated from fibroin-deficient cocoons via a mild extracting method (LiBr). In this paper, we propose the problem that whether degraded sericin can be fabricated as an injectable hydrogel. Herein, we used the syringe to evaluate the injectability of the sericin hydrogels formed by sericin with different concentrations. The results showed that the sericin hydrogels formed by sericin solutions at concentration 8% was smoothly through 16 G needles (Fig. 6G, Supplementary Video S4).

3.10 Cells adhesion and proliferation of the hydrogels

To evaluate the cytocompatibility of the sericin hydrogel, we seeded mouse fibroblast cells (NIH3T3) on the sericin hydrogels. Then, cell adhesion and proliferation of the sericin hydrogel was evaluated. The results showed that there was no significant difference in cell adhesion between sericin hydrogel and polystyrene tissue culture plate (Fig. 7A). The cell viabilities on the sericin hydrogels have no great difference with the cells on polystyrene tissue culture plate (Fig. 7B). Furthermore, a large number of proliferative cells were observed on the sericin hydrogel after 3 days seeding, indicating that the sericin hydrogel could promote the growth and proliferation of NIH3T3 cells (Fig. 7C). Together, the sericin hydrogel possess excellent cell adhesion and biocompatibility, which can serve as a promising cell carrier for supporting cell survival and proliferation.

A sterile sericin hydrogel with excellent physicochemical properties and biocompatibility was successfully fabricated in this study. Prior to our study, various attempts were reported for fabricating pure sericin hydrogels based on sericin isolated from silkworm cocoons or silkworm bodies [2, 8, 25–27]. Compared with these sericin hydrogels, our sericin hydrogel system has several advantages: (1) A sterile sericin hydrogel with injectability, good elasticity, and robust behaviors can be fabricated directly. (2) This hydrogel system breaks the previous idea that sericin extracted by high temperature and pressure is difficult to cross-link directly into a stable hydrogel. (3) This hydrogel system retained the small molecular bioactivity polypeptides of silkworm cocoons. Sericin in silkworm cocoons is comprised of a variety of polypeptides with different molecular weights. Many small polypeptides would be discarded inevitably during extracting sericin process at dialysis. (4) The hydrogel production process is simple and efficient. Besides, the gelation time can be controlled by adjusting sericin concentrations. The hydrogel also owns the satisfactory performance of swelling properties, high porosity, sustained drug release, and promoting cell adhesion and proliferation. The new sericin hydrogel system in this study has guiding significance for further expanding and enriching sericin hydrogel applications.

CRediT authorship contribution statement

Yeshun Zhang: Conceptualization, Supervision, Writing – review & editing. Susu Wang: Methodology, Validation, Investigation. Yurong Li: Writing – original draft. Jingya Zhang: Prepare materials, Investigation. Xiang Li: Drawing. Zhanyan Du: Methodology. Siyu Liu: Investigation. Yushuo Song: Investigation. Yanyan Li Investigation. Meilin Xi Visualization. Guoping Kang: Funding acquisition. Guozheng Zhang: Methodology, Funding acquisition, Writing – review & editing.

Data availability

Data will be made available on request.

Acknowledgements

This work was supported by the Natural Science Foundation of Jiangsu province of China (Grant No. BK20221291), China; China Agriculture Research System (Grant No. CARS-18-ZJ0107), China; Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grant No. KYCX21_3511, KYCX22_3767), China.

The videos including supplementary video s1-4 are used for demonstrating elasticity, robustness and injectability of the hydrogels.

Competing financial interests: The authors declare no competing financial interests.

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A sterile, injectable, and robust sericin hydrogel prepared by degraded sericin

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Materials

2.2 Extraction of sericin solution

2.3 Free amino group content detection

2.4 Amino Acid Analysis

2.5 Preparation of sterile robust sericin hydrogel

2.6 Gelation time of sericin hydrogel

2.7 Scanning electron microscopy (SEM)

2.8 Porosity analyses

2.9 Evaluation of swelling behaviors and degradation dynamics

2.10 The release of drug from sericin hydrogels

2.11 Fourier Transform Infrared (FTIR) Spectroscopy and x-ray diffraction

2.12 Mechanical analysis

2.13 Injectable property

2.14 In vitro cell test

2.15 Statistical analysis

3. Results and discussion

3.1 Synthesis and mechanism of sericin hydrogels

3.2 Gelation kinetics of sericin hydrogels

3.3 The micro-architecture of sericin hydrogel

3.4 Swelling characteristics of the sericin hydrogels

3.5 Degradability of the sericin hydrogels

3.6 Drug release of sericin hydrogels

3.7 Secondary structural of sericin hydrogels

3.8 Mechanical stability of sericin hydrogels

3.9 Injectable property of sericin hydrogels

3.10 Cells adhesion and proliferation of the hydrogels

4. Conclusions

Declarations

References

Supplementary Files

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