Characterization of Silver Nanoparticle In Situ Synthesis on Porous Sericin Gel for Antibacterial Application

1State Key Laboratory of Silkworm Genome Biology, Southwest University, Beibei, Chongqing 400715, China 2Chongqing Engineering and Technology Research Center for Novel Silk Materials, College of Biotechnology, Southwest University, Beibei, Chongqing 400715, China 3College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing 400715, China 4National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China


Introduction
Sericin is a protein which is coated on the surface of fibroin fiber when Bombyx mori silkworm spins cocoon for protective and adhesive effects [1].Sericin could be extracted by boiling cocoon in an alkaline solution or through high temperature and high pressure extraction process [2].Processing of raw silk cocoon produces about 50,000 tons of sericin worldwide each year.However, sericin is usually discarded in silk processing wastewater [3], resulting in a high chemical oxygen demand level of the degumming wastewater [4], which greatly caused the environmental pollution and the waste of resources.Sericin consists of 18 kinds of amino acids.Most of them have strong polar side groups such as hydroxyl, carboxyl, and amino groups.Sericin is characterized by a high serine content which is about one-third of the total amino acids [5].Because of its good hydrophilicity, reaction activity, biocompatibility, and biodegradability, sericin has been applied widely in biomedical materials such as cell culture scaffolds, drug carriers, and tissue engineering scaffolds [6][7][8][9].
In natural conditions, sericin could spontaneously form hydrogel, but its mechanical property is relatively poor.Though the mechanical property of sericin hydrogel could be improved by adding functional cross-linker, the toxicity of the hydrogel may be introduced by the additives.Thus, it is highly desired to develop a green synthesis method without any cross-linking reagent to produce sericin hydrogel with porous structure from aqueous Bombyx mori sericin.According to a previous report, sericin gel could be prepared with certain mechanical properties from soluble sericin through freeze-drying without adding any chemical reagent [10].
Surface immobilization with antibacterial material is a kind of common method to make antibacterial materials.Silver nanoparticle is a regular and effective antibacterial material, which has shown a broad spectrum of antibacterial activity against fungus, viruses, and Gram-positive and Gram-negative bacteria [11][12][13].With the development of nanotechnology, numerous nanomaterials with multifarious particular characteristics have been developed [14][15][16][17][18].Recently, the immobilization of fibroin fiber with nanomaterials for multifunctional textiles has been developed [19,20].Some natural polymers such as gelatin, chitosan, and starch immobilized with silver nanoparticle are also developed for antibacterial purpose [21][22][23].Sericin gel has the characteristics of porous and good water absorbability, which shows great potentials as scaffold materials, drug carriers, and mouldable wound dressing [24,25].Although sericin gel has a lot of unique properties, the protein nature makes it a matrix for bacterial adhesion and thriving, further resulting in its deformation and degradation [26].Thus, it is necessary to produce a kind of sericin with antibacterial activity for broad application.
Two types of methods have been developed to produce AgNPs-functionalized natural polymers.One is to coat with presynthesized AgNPs on the surface of natural polymer.This method needs the synthesis of silver nanoparticles and composite beforehand on the surface of the polymer, which has been abandoned gradually by researchers due to its complexity and redundancy [8,27].Another one is to grow AgNPs in situ directly on the surface of natural polymer without the presynthesis step.Silver ions attach on the surface of polymer materials via electrostatic adsorption or ion exchange, followed by a reduction step to form AgNPs in situ.Natural polymers with small specific surface area and less active group are frequently required to cross-link polymers such as polyamide network polymer and polyacrylic acid to functionalize the surface [28], which will increase the density of AgNPs immobilized on the surface of natural polymers.However, cross-linking functional materials on the surface of natural polymers not only increase the cost, but also likely introduce potential contaminations to environment.Sericin gel has a porous structure, large surface area, and lots of active groups, which may facilitate sericin gel to adsorb more silver nanoparticles on its surface than other natural polymers without cross-linking.Therefore, it is worth developing a facile and green approach to fabricate porous sericin gel modified with silver nanoparticles for antibacterial application.
In previous reports, ultraviolet (UV) light has been applied to assist in the reduction of silver ions to immobilize AgNPs on the surface of polymer [29].In this study, we developed a synthesis approach to immobilize AgNPs in situ on the surface of sericin gel with the assistance of UV.Scanning electron microscopy (SEM), X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC) were applied to characterize the surface tomography and structure of the AgNPs-sericin material.The antimicrobial tests, including growth curve and inhibition zone, were carried out to investigate the antibacterial activities of AgNPs-sericin gel against Escherichia coli (E.coli) and Staphylococcus aureus (S. aureus).Our research suggested the AgNPs-sericin gel with porous structure could effectively inhibit the growth of both Gram-negative and Gram-positive bacteria.This novel antibacterial material may be regarded as potential biomedical materials such as wound dressing material.

Materials and Chemicals. Silkworm cocoon was kindly provided by the State Key Laboratory of Silkworm Genome
Biology, Southwest University, China.Silver nitrate (AgNO 3 ) (AR, 99.99%) was purchased from Aladdin Corporation (Shanghai, China).MilliQ water was used in the experiments.All other chemicals in this study are of analytical grade and directly used without further purification.

Preparation of Sericin Gel.
Sericin was prepared according to a previous reported procedure with minor modification [30].The details on the extraction of sericin and preparation of sericin gel were described in Figure 1.Bombyx mori cocoons were first cut into small pieces and placed into a beaker containing milliQ water.The beaker was autoclaved with a temperature of 121 ∘ C and a pressure of 0.1 Mpa for 30 min to dissolve sericin.The aqueous sericin solution was collected and then used to produce sericin powder by freezedrying.Sericin powder and milliQ water were mixed with appropriate proportion and heated until the powder was completely dissolved.Sericin solution was freezed under −20 ∘ C for 2 hours to form sericin hydrogel.Sericin hydrogel was further dehydrated by freeze-drying to become sericin gel.

UV-Assisted
In Situ Synthesis of AgNPs.Sericin gel was cut into small pieces of sheets and then soaked into 50 mM AgNO 3 solution.At the same time, sericin gel sheets were irradiated with a 365 nm UV light lamp (24 W) for 10 min, 30 min, and 60 min to make AgNPs on the surface of sericin gel, respectively.Sericin hydrogel, sericin gel, and AgNPs modified sericin gel were shown in Figure 2. AgNPs modified sericin gel was collected and dried at room temperature, which will be used for the following experiments to characterize its surface tomography, structure, and antimicrobial activity.

Materials Characterization.
The surface morphologies of sericin gel and AgNPs modified sericin gel were imaged using SEM (JCM-5000, JEOL, Tokyo, Japan).Sericin gel and AgNPs modified sericin gel were pressed to form compact pellets for FT-IR measurements (Nicolet iz10, Thermo Electronic Corporation, USA).DSC curves were recorded on a differential scanning calorimetry instrument (DSC 200PC, Nestal, Germany) in the temperature range of 50-400 ∘ C. The heating rate for the test was kept at 10 ∘ C/min and the flow rate of nitrogen gas was controlled at 50 mL/min.XRD spectra of the modified and unmodified sericin gel were examined with a 2 range of 10-70 ∘ (X'Pert Powder, PANalytical, Netherlands).

Analysis of Growth
Curve.E. coli and S. aureus at lag phase were inoculated into 10 mL Luria-Bertani (LB) medium (pH 7.4) in the absence and presence of the modified or unmodified sericin gel and then cultured with constant shaking speed (220 rpm) at 37 ∘ C. The optical density at 600 nm (OD 600 ) was measured for 0.5 mL bacterial collected at different intervals.All growth curve tests were made in triplicate to ensure the reproducibility of the tests.

Inhibition Zone
Assay.E. coli and S. aureus were inoculated in 100 mL LB medium (pH 7.4) and cultured with constant shaking speed (220 rpm) at 37 ∘ C for 12 hours, respectively.Bacteria at lag phase were spread uniformly on agar medium plates; then a circular sericin or AgNPs modified sericin gel with diameter of about 1.05 cm was introduced into the plate.After incubation at 37 ∘ C overnight, the antibacterial activities of the samples were evaluated based on the diameter of the bacterial inhibition zones.All antibacterial activity tests were done in triplicate to ensure that the assay could be reproduced.

Results and Discussion
Sericin gel has lots of hydroxyl and active groups on its surface, which may react with silver ions under the irritation of UV to form AgNPs on the surface of sericin gel [31,32].
In this work, we developed a facile, green, efficient, and fast approach to fabricate the AgNPs modified sericin gel without addition of any chemicals.The procedures were shown in Figure 1.Sericin concentration may affect the formation of sericin hydrogel from sericin solution.Here, we tested different sericin concentrations and finally determined an appropriate sericin concentration.Sericin gel has a porous structure and good water absorbency, as shown in Figure 2. Figure 2(a) shows the morphology of sericin hydrogel prepared from 8% sericin solution (wt).After freeze-drying, sericin hydrogel was dehydrated to form dry sericin gel with porous structure (Figure 2(b)).The dry gel reformed hydrogel once absorbing water (Figure 2(c)).Figure 2(d) showed a small piece of sericin gel with porous structure.The AgNPs modified sericin gel was shown in Figure 2(e).This result suggested that sericin could form sericin gel with certain mechanical property without addition of any chemicals, and this kind of sericin gel had porous structure, good permeability, moisture retention, and hygroscopicity.In addition, it was environmentally friendly, biocompatible, and biodegradable.In wound healing process, infection, dehydration, and secondary trauma are key barriers.Therefore, sericin gel is regarded as a potential material for biomedical purpose such as wound dressing material.
SEM was carried out to observe the surface morphologies of the sericin gel before and after the immobilization of AgNPs (Figure 3).As shown in Figure 3(a), sericin gel has a porous structure, which may favor effective nutrient or gas exchange for cells in the process of wound healing [33].Some small dots on the surface of sericin gel had been observed after an irradiation for 10 min in a silver nitrate solution (Figure 3(b)), indicating a high-efficiency in situ synthesis of AgNPs on the surface of sericin gel.As the irradiation time increases to 30 minutes and 60 minutes, the amount of AgNPs on the surface of sericin gel greatly increases (Figures 3(c) and 3(d)).The results showed that in situ synthesis of AgNPs on the surface of sericin gel could be regulated by the irradiation time.
Further, XRD was applied to verify in situ synthesis of AgNPs on the surface of sericin gel.XRD patterns of unmodified and AgNPs modified sericin gel were shown in Figure 4.In a previous study, two characteristic peaks located at 2 = 19.2∘ and 2 = 23.2∘ are observed [5].There was no significant change observed on the peaks of sericin gel with the modification of AgNPs, which suggested that UV irradiation and AgNPs modification did not alter the structure of sericin gel.For the sericin gel modified with AgNPs, one peak was observed at the position of 2 = 37.5 ∘ , which may be attributed to the (111) plane of the face-centered cubic structure of AgNPs [34].
FT-IR was widely used to characterize the structure of materials [35].Here, it was applied to investigate the effect of AgNPs modification on the structure of sericin gel.As shown in Figure 5, there were four characteristic peaks at 3280 cm −1 , 1620 cm −1 , 1520 cm −1 , and 1228 cm −1 on both the unmodified sericin gel and the AgNPs modified sericin gel, which may be attributed to the N-H stretching absorption, Amid III, Amid II, and Amid I peaks of sericin [36,37].Since the growth of AgNPs on the surface of sericin gel had no influence on the amid peaks of sericin gel, it may suggest that the synthesis procedures did not affect the structure of sericin gel.Amino acid groups on the surface of sericin gel may play an important role in the adsorption and reduction of silver ions.The coordination of nitrogen atom interaction with silver ions may promote the reduction of silver ions [38].The porous structure of sericin gel will favor the interaction of amino acid residues with silver ions, thus promoting more and more AgNPs to be hybridized on the surface of sericin gel.To further investigate the effects of silver nanoparticles on the structure of sericin gel, the thermal behavior of sericin gel with and without the modification of AgNPs was analyzed by DSC. Figure 6 showed the DSC curves of unmodified and modified sericin gel after freeze-drying.In the case of sericin gel, there were two endothermic peaks at 218 ∘ C and 316 ∘ C, respectively, which were corresponding to the thermally induced molecular motion of an amorphous region and thermal decomposition of sericin [39].The DSC curve of sericin gel modified with AgNPs showed that the first endothermic peak slightly shifted to about 223 ∘ C, and the second peak shifted to about 381 ∘ C.This result suggested that AgNPs modification on the sericin gel may increase the thermostability of sericin gel.
Bacterial growth curves were used to assess the antimicrobial activities of sericin gel and AgNPs modified sericin gel.In this study, E. coli and S. aureus were chosen as a model of Gram-positive bacteria and Gram-negative bacteria to assess the antibacterial activities of AgNPs modified sericin gel, respectively [40].As shown in Figure 7, the unmodified sericin gel did not affect the growth of bacteria.However, the AgNPs modified sericin gel showed significantly antibacterial activities against E. coli and S. aureus.In the presence of AgNPs modified sericin gel, the lag phase of bacteria Besides the growth curves, inhibition zone is also regarded as an important indicator for antibacterial activities.The inhibition zone of sericin gel and AgNPs modified sericin gel was shown in Figure 8.No inhibition zone was observed for unmodified sericin gel on both E. coli and S. aureus agar plates, indicating that sericin gel did not affect the growth of bacteria on the plate.On the contrary, obvious inhibition zone could be observed in the presence of sericin gel modified with AgNPs for 10 min, 30 min, and 60 min.The diameters of the inhibition zones are summarized in Table 1.The result suggested that increasing the reaction time could increase the antibacterial activities of AgNPs modified sericin gel, which was consistent with the assay of the growth curves.

Conclusions
In this study, we developed a green and efficient approach to synthesize silver nanoparticles in situ on the surface of sericin gel.The porous structure of sericin gel will not be affected by the modification of AgNPs, as confirmed by SEM, XRD, and FT-IR.DSC analysis showed that the modification of AgNPs also increased the thermal stability  of sericin gel.Sericin gel immobilized with AgNPs showed good antimicrobial activities against both Gram-negative and Gram-positive bacteria.This novel material shows a great potential for biomedical application.

Figure 3 :
Figure 3: SEM images of sericin gel (a) and sericin gel in silver nitrate solution irradiated with a UV light for 10 min (b), 30 min (c), and 60 min (d).

Figure 7 :
Figure 7: Growth curves of E. coli (a) and S. aureus (b) in the presence of sericin gel and sericin gel modified with AgNPs for 10 min, 30 min, and 60 min.

Table 1 :
Diameters of inhibition zones of sericin gel and AgNPssericin gel against E. coli and S. aureus.Zone of inhibition is expressed as the diameter in centimeters.Values represent means ± standard deviations of inhibition zones from three individual experiments.