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Silk Fibroin/Polyvinyl Pyrrolidone Interpenetrating Polymer Network Hydrogels Dajiang Kuang 1 , Feng Wu 1 , Zhuping Yin 1 , Tian Zhu 1 , Tieling Xing 1 , Subhas C. Kundu 2 and Shenzhou Lu 1, * ID 1

2

*

ID

National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China; [email protected] (D.K.); [email protected] (F.W.); [email protected] (Z.Y.); [email protected] (T.Z.); [email protected] (T.X.) 3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, University of Minho, AvePark, Barco, 4805-017 Guimaraes, Portugal; [email protected] Correspondence: [email protected]; Tel.: +86-512-6706-1152

Received: 25 December 2017; Accepted: 4 February 2018; Published: 6 February 2018

Abstract: Silk fibroin hydrogel is an ideal model as biomaterial matrix due to its excellent biocompatibility and used in the field of medical polymer materials. Nevertheless, native fibroin hydrogels show poor transparency and resilience. To settle these drawbacks, an interpenetrating network (IPN) of hydrogels are synthesized with changing ratios of silk fibroin/N-Vinyl-2pyrrolidonemixtures that crosslink by H2 O2 and horseradish peroxidase. Interpenetrating polymer network structure can shorten the gel time and the pure fibroin solution gel time for more than a week. This is mainly due to conformation from the random coil to the β-sheet structure changes of fibroin. Moreover, the light transmittance of IPN hydrogel can be as high as more than 97% and maintain a level of 90% within a week. The hydrogel, which mainly consists of random coil, the apertures inside can be up to 200 µm. Elastic modulus increases during the process of gelation. The gel has nearly 95% resilience under the compression of 70% eventually, which is much higher than native fibroin gel. The results suggest that the present IPN hydrogels have excellent mechanical properties and excellent transparency. Keywords: silk fibroin; PVP; interpenetrating polymer network; hydrogel; protein biopolymer

1. Introduction Hydrogels are of special soft and wet material with a three-dimensional network structure and high water content, the interior presents a porous, water-dispersed system, a certain strength and soft nature. They can be prepared by physical and chemical crosslinking [1–3]. Due to their porous structure, good biocompatibility and mechanical properties, the hydrogels are used as cell culture, drug delivery, biosensors and tissue engineering [4–6]. Silk fibroin (SF), derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications [7]. Silk fibroin has remarkable mechanical properties when formed into different materials, which demonstrate biocompatibility [8–10]. It can be processed into different kinds of materials under different conditions, mainly including solution, film, microsphere, gel, filament, nanotube and scaffold [11–15]. Silk hydrogels are widely used as artificial skin, drug delivery carriers, microneedle systems and, biological sensors and tissue repairing [1,13,16–18]. Previous studies find that the hydrogel formation and sol-gel transition of the hydrogels depend on the protein concentration, temperature and pH [19]. In order to further improve the properties of fibroin hydrogel, silk fibroin can be mixed with other various polymers or macromolecules, such as chitosan, polyvinyl alcohol, Polymers 2018, 10, 153; doi:10.3390/polym10020153

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hyaluronic acid, sodium alginate, gelatin and others [16,20–22]. The major drawbacks of these silk fibroin hydrogels are poor mechanical strength and bad biomedical applications [1,3,19]. To overcome this drawback, in this study silk hydrogels properties are improved upon by forming interpenetrating networks of silk blended with N-Vinyl-2-pyrrolidone, IPN (Interpenetrating Polymer Network). The polymer composites can be fabricated by thoroughly mixing the two or more types of molecular chains [23,24]. The most important feature of IPN polymer network is the thermodynamically incompatible polymer can be mixed to form at least the kinetic stability of the composites properties of the material in order to achieve the performance between the complementary components [25]. At the same time, characteristics about IPN like special cell structure, interfacial interpenetration and biphasic continuous structural morphological make them in the performance or function to produce a special synergistic effect. It is known that polymer of N-Vinyl-2-pyrrolidone (PVP) has excellent biocompatibility and inert nature. This makes them suitable for applications in medical and pharmacy, human metabolism. Besides, PVP holds outstanding hydrophilicity and lubricity, which make it suitable to composite with other hydrophobic polymer and then increase the mixing system’s hydrophilicity. Hosaka et al. mix and crosslink polyurethane with PVP monomer to produce high strength PVP hydrogel film [26–29]. Polyvinylpyrrolidone is used extensively with synthetic polymers such as polyvinyl alcohol and methyl methacrylate to prepare composite hydrogels to improve performances [30–32]. In this report, keeping in mind the past work and based on the excellent biosafety of hydrogel, we fabricate silk fibroin and N-Vinyl-2-pyrrolidone interactive network hydrogel with high mechanical strength and elasticity as well as high transparency. This may be applied to contact lens and artificial vitreous materials. 2. Materials and Methods 2.1. Materials The fresh silk cocoons from domestic mulberry silkworm Bombyx mori were purchased from Suzhou Sirui Biological Technology Co., Ltd., hydrogen Peroxide 30% (Sinopharm Chemical Reagent Chemical Group Co., Ltd., Shanghai, China). Lithium bromide, N-vinyl-2-pyrrolidone (NVP), peroxidase from horseradish (HRP) (Aladdin Industrial Corporation, Shanghai, China). Anhydrous sodium carbonate, sodium bicarbonate, sodium chloride and all other chemicals (Sigma Chemicals Co., St. Louis, MO, USA,) used in this experiment were purchased. 2.2. Preparation of Regenerated Silk Fibroin The silk cocoons (80 g) were cut into small pieces and degummed by boiling three times, in 4000 mL solution of 0.01 M Na2 CO3 /NaHCO3 in order to remove silk sericin [33]. The silk was then washed, air dried and dissolved in 9.3 M LiBr at 65 ◦ C for 1 h followed by dialysis against deionized water for 3 days at 4 ◦ C using cellulose dialysis membrane (MWCO 8–10 kDa) with frequent water changes. The solution was finally centrifuged at 6000 rpm for 15 min to remove impurities and precipitated matter [34]. 2.3. Preparation of IPN Hydrogels IPN hydrogels were prepared by free radical reaction. Briefly, silk fibroin (SF) protein concentration was adjusted to 40 mg/mL and NVP adjusted to 50 mg/mL with deionized water. Hydrogels were prepared by adding 2 mL of blended solution in centrifuge tube including SF/NVP (10:0, 9:1, 8:2, 7:3, 6:4, 5:5), 1 mM H2 O2 and 10 unit/mL HRP. To this mixture, SF and NVP were added according to the blending ratios (Table 1). The solutions were allowed to gel in an incubator at 37 ◦ C. The samples were finally removed from the mold, washed and immersed in deionized water for 24 h to remove unreacted monomers.

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Table 1. Indicates the content of each component in the mixed system. Composition

40 mg/mL SF (µL)

50 mg/mL NVP (µL)

Deionized Water (µL)

SF/NVP: 10/0 SF/NVP: 9/1 SF/NVP: 8/2 SF/NVP: 7/3 SF/NVP: 6/4 SF/NVP: 5/5

500 450 400 350 300 250

0 40 80 120 160 200

1298.6.6 1308.6 1318.6 1328.6 1338.6 1348.6

All components were added H2 O2 68 µL (1 mg/mL), HRP133.4 µL (0.25 mg/mL). The final volume of the mixed solution was 2 mL. The solid content was 10 mg/mL, the temperature was 25 ◦ C and the relative humidity was 65%.

2.4. Morphology of PVP-SF IPN Hydrogels To characterize the internal structure of IPN, the hydrogel samples were observed using a scanning electron microscope (Hitachi TM3030, Hitachi Ltd., Tokyo, Japan). The samples were frozen by liquid nitrogen and then the sample were placed in a freezing dryer (Christ) and dried for 24 h to remove water. SEM images were acquired after gold sputtering at operating voltage of 15 kV [35]. 2.5. FTIR Measurement All infrared spectra were recorded in the range of 600–1800 cm−1 using Thermo Nicolet 570 FTIR spectrometer (Nicolet Co., Madison, WI, USA). Each spectrum was acquired by accumulation of 16 scans with a resolution of 4 cm. Background measurements were subtracted from sample readings. 2.6. Wide-Angle X-ray Diffraction Measurement The crystalline state of Freezing-dried IPN hydrogels was tested by XRD. Test conditions: tube voltage 40 kV, tube current 35 mA, scanning rate of 2◦ /min and using Cu Kα rays. XRD patterns were recorded in the 2θ region from 10◦ to 40◦ . 2.7. Light Transmittance Characterization SF pure hydrogels and IPN hydrogels were equilibrated in 24-well plates and equilibrated in a 37 ◦ C incubator for 24 h. The transmittance values were measured at 492, 550, 700 nm using a multifunctional microplate reader [36]. The thickness of the hydrogel in the experiment is controlled below 5 mm and the temperature is maintained at 37 ◦ C. 2.8. Mechanical Properties 2.8.1. The Compressive Stress—Strain Curve of IPN Gels In order to compare the compressive properties of hydrogels with different mass ratios, SF pure hydrogels and IPN hydrogels with different ratios were prepared. Cylindrical hydrogel samples with flat and parallel surfaces were prepared and the initial diameter and height of samples were 10 ± 0.5 mm and 8 ± 0.5 mm respectively. Compressive strength was determined by using Universal Testing Machine (Instron-3365, Instron Co., Norwood, MA, USA) at 25◦ and 65% relative humidity. The test speed was set at 10 mm/min. All data were collected six samples (n = 6). 2.8.2. Resilience Test of IPN Gels SF pure hydrogel and IPN hydrogel with different ratios were made into cylinders with a diameter of 10 ± 0.5 mm and a height of 8 ± 0.5 mm. The samples were compressed twice and the compression displacement was 70% of the original height of the samples. The variation curve was analyzed in the system software and the parameter values are obtained and the variation law was analyzed [37]. Test conditions: control the lifting arm speed of 1 mm/s, the trigger force of 10 g, the test used in the circular probe model P/2, detection accuracy ≥0.015%.

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analyzed [37]. Test conditions: control the lifting arm speed of 1 mm/s, the trigger force of 10 g, the test used in 10, the153 circular probe model P/2, detection accuracy ≥0.015%. Polymers 2018, 4 of 11 2.9. Enzymatic Degradation of IPN Gels In Vitro 2.9. Enzymatic Degradation of IPN Gels In Vitro IPN hydrogels were immersed in PBS (phosphate buffer saline) (37 °C, pH: 7.4) containing IPN XIV hydrogels were immersed inSt.PBS (phosphate buffer saline) (37of◦ C, pH:(w/v). 7.4) containing protease (2 U/mL, Sigma-Aldrich, Louis, MO, USA) at a bath ratio 1/100 Hydrogels protease XIV (2 U/mL, Sigma-Aldrich, St. Louis, MO, USA) at a bath ratio of 1/100 (w/v). Hydrogels peptide content derived from enzymatic degradation was measured by sampling at designated time peptide4content from enzymatic degradation was measuredIPN by sampling designated time points, parallelderived samples each group [38]. Percentage enzymatic hydrogelsatdegradation was points, 4 parallel samples each group [38]. Percentage enzymatic IPN hydrogels degradation was calculated using Equation (1) mentioned above. calculated using Equation (1) mentioned above.

m WeightRemain% =m2 2 ×100% WeightRemain% = m× 100% m1 1

(1) (1)

where m1 and m2 are the mass of hydrogels before and after immersion in PBS. where m 1 and m2 are the mass of hydrogels before and after immersion in PBS. 3. Results 3.1. Preparation of IPN Hydrogels Interpenetrating network network hydrogel hydrogel were were fabricated fabricated mixing mixing solutions solutions of of silk silk fibroin with NVP Interpenetrating solutions, HRP and H2O O22.. During Duringthis thisprocess, process,NVP NVP was was used used to to generate generate free free radicals radicals under the solutions, catalysis of initiator H2O O22 and and horseradish horseradish peroxidase peroxidase (HRP), (HRP), which which initiated the polymerization of polyvinylpyrrolidone and reacted silk fibroin macromolecule entanglement, monomers totoproduce produce polyvinylpyrrolidone and with reacted with silk fibroin macromolecule ultimately formed the interpenetrating network (IPN) network hydrogel(IPN) (Scheme 1). entanglement, ultimately formed the interpenetrating hydrogel (Scheme 1).

Scheme 1. Formation of PVP–SF IPN hydrogel using HRP-H system. HRP-H22O22 system.

3.2. Morphology of IPN Hydrogels The IPN hydrogels performed dense and uniform porous structures (SEM, Figure 1). The SEM images also revealed that regular pore structure with clear pores and high porosity within PVP-SF IPN hydrogels. The The pore pore sizes sizes ranged ranged from from 80 80 to 200 μm, µm, which were much larger than the internal pore sizes of pure silk hydrogels. The larger pores allow for the efficient delivery of the water and The nutrients inside the gel. This This provides sufficient space and suitable environment for cell growth and proliferation. Decrease in silk fibroin content, the internal porosity of the hydrogel became larger and uniform. uniform. With With the the increase increase of NVP content, the conversion rate decreased. This This would reduce the entanglement points of polymer and silk fibroin macromolecules. This results in the internal internal structure structure of the hydrogel loose and the pores became larger. larger.

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(A)

(B)

(C)

(D)

(E)

(F)

Figure 1. SEM images of cross-section morphology with different ratios of SF/NVP: (A) 90/10, (B)

Figure 1. SEM images of cross-section morphology with different ratios of SF/NVP: (A) 90/10, 80/20, (C) 70/30, (D) 60/40, (E) 50/50 and (F) pure SF hydrogel. (B) 80/20, (C) 70/30, (D) 60/40, (E) 50/50 and (F) pure SF hydrogel.

3.3. Conformation and Aggregation Structure of Fibroin Hydrogels

3.3. Conformation and Aggregation Structure of Fibroin Hydrogels

The FITR spectra of the freeze-dried IPN hydrogel and pure SF hydrogel are shown in Figure 2.

ThereFITR wasspectra no significant difference between differentand proportions of interpenetrating network The of the freeze-dried IPN hydrogel pure SF hydrogel are shown in Figure 2. the FITR spectra (Figurebetween 2A). The different pure SF hydrogel exhibits absorption Therehydrogels was no ofsignificant difference proportions ofcharacteristic interpenetrating network −1 which are the characteristic peaks of the β-sheet structure [39]. SF/PVP peaks at 1233, 1532, 1635 cm hydrogels of the FITR spectra (Figure 2A). The pure SF hydrogel exhibits characteristic absorption IPN hydrogels had obvious absorption peaks at 1530, 1630 cm−1 which are the characteristic peaks of peaks at 1233, 1532, 1635 cm−1 which are the characteristic peaks of the β-sheet structure [39]. SF/PVP random coil of SF [39]. The results indicate that the main structure of SF in IPN hydrogels is random IPN hydrogels had obvious absorption peaks at 1530, 1630 cm−1 which are the characteristic peaks coil. of random coil 2B of shows SF [39]. indicate thehydrogel main structure SF in IPN hydrogels is Figure theThe XRDresults diffraction curvethat of IPN material.of X-ray diffraction is an random coil. effective method for studying crystal materials and amorphous material structures [40]. There was Figure 2B shows the XRD diffraction curvediffraction of IPN hydrogel diffraction is no significant difference between the XRD curve ofmaterial. different X-ray proportions of an effective method for studying crystal materials and amorphous material structures [40]. There was no significant difference between the XRD diffraction curve of different proportions of interpenetrating network hydrogels (Figure 2B). It can be observed from the figure that the IPN hydrogel had a large

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interpenetrating network hydrogels (Figure 2B). It can be observed from the figure that the 6IPN Polymers 2018, 10, x FOR PEER REVIEW of 11

bunted peak at 20a◦large , which waspeak characterized bywas a typical amorphous structure. That was consistent hydrogel had bunted at 20°, which characterized by a typical amorphous structure. ◦ and 24.3◦ with a sharp absorption peak. withThat the FTIR spectrum. Whereas the pure silk gel at 20.3 interpenetrating network hydrogels (FigureWhereas 2B). It can be observed from that the IPN was consistent with the FTIR spectrum. the pure silk gel at 20.3°the andfigure 24.3° with a sharp This absorption indicated that pure silkpeak fibroin had a certain crystal (Figure 2B). (Figure hydrogel had a the large bunted atthe 20°, which characterized by acrystal typical amorphous structure. peak. This indicated that pure silkwas fibroin had astructure certain structure 2B). That was consistent with the FTIR spectrum. Whereas the pure silk gel at 20.3° and 24.3° with a sharp absorption peak. This indicated that the pure silk fibroin had a certain crystal structure (Figure 2B). 5 to 5 6 to 4 7 to 3 8 to 2 to15 95to 6 to 4 SF 7 to 3 8 to 2 9 to 1 SF

Diffraction Intensity Diffraction Intensity

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-1 (A) Wavenumber(/cm )

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(B)

Diffraction angle/2θ(°)

Figure 2. Structure of fibroin hydrogels. (A) FTIR; (B) X-ray diffraction. The solid content of hydrogel

Figure 2. Structure of fibroin solid content of hydrogel (A) hydrogels. (A) FTIR; (B) X-ray diffraction. The(B) was 10 g/L. was 10 g/L. Figure 2. Structure of fibroin hydrogels. (A) FTIR; (B) X-ray diffraction. The solid content of hydrogel

3.4. Compression was 10 g/L. Mechanical Properties

3.4. Compression Mechanical Properties

In the stress-strain curve, both the hydrogel samples showed some nonlinear behaviors. With 3.4. Compression Mechanical Properties the increase of strain, the compressive stress on the material increased and the pure silk fibroin In the stress-strain curve, both the hydrogel samples showed some nonlinear behaviors. With the In the stress-strain curve, both the hydrogel samples showed some nonlinear behaviors. hydrogel appeared at about 50% compression compressive rupture phenomenon (Figure 3A).With The increase of strain, the compressive stress on the material increased and the pure silk fibroin hydrogel the increase strain, the stress on theupmaterial increased and pure silk fibroin IPN hydrogel material in thecompressive maximum compression to 70% of the stress wasthe still increased. The appeared at aboutof 50% compression compressive rupture phenomenon (Figure 3A). The IPN hydrogel hydrogelremained appearedintact at about compressive (Figure 3A). The The material and50% the compression material quickly rebound rupture after thephenomenon pressure was removed. material in the maximum compression up to 70% of the stress was still increased. The material IPN hydrogel in internal the maximum compression to 70% were of theregular, stress was still increased. The results showedmaterial that the structures of IPN up hydrogel cross-linked closely, remained intact and the material quickly rebound after the pressure was removed. The results showed material compressive remained intact and and the material reboundsignificantly after the pressure was The excellent strength resilience.quickly IPN hydrogel enhanced theremoved. compressive that the internal structures IPN hydrogel were regular, cross-linked closely, excellent compressive results showed that theof internal of because IPN hydrogel were regular, cross-linked closely, strength and compressive resilience ofstructures the hydrogel of its macromolecule network entanglement. strength and resilience. IPN hydrogel enhanced compressive strength compressive excellent compressive strength and significantly resilience. IPN hydrogelthe significantly enhanced the and compressive resilience of the hydrogel because of its macromolecule network entanglement. strength and compressive resilience of the hydrogel because of its macromolecule network entanglement. 96.1 94.9 5 to 5 6 to 4 7 to 3 to25 85to to14 96to 7 to 3 SF 8 to 2 9 to 1 SF

Stress Stress (Kpa) (Kpa)

20 25 15 20 10 15

100

86.5

84.7 94.9

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96.1 86.5

84.7

82.1

82.1

Resilience(%) Resilience(%)

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0 9/1

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7/3

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12.2 SF gel

SF gel

Ratio Figure 3. Compression mechanical properties. (A) Stress-strain curves of PVP-SF IPN hydrogels. (B) (A) (B) Stress resilience of PVP-SF IPN hydrogels. The solid content was 10 g/L, the temperature was 25 °C and the 3. relative humidity was 65%. properties. (A) Stress-strain curves of PVP-SF IPN hydrogels. (B) Figure Compression mechanical

Figure 3. Compression mechanical properties. (A) Stress-strain curves of PVP-SF IPN hydrogels. Stress resilience of PVP-SF IPN hydrogels. The solid content was 10 g/L, the temperature was 25 °C (B) Stress resilience of PVP-SF IPN hydrogels. The solid content was 10 g/L, the temperature was 25 ◦ C and the relative humidity was 65%. and the relative humidity was 65%.

The sample of IPN hydrogel was compressed by TPA (Test process analysis) test for consecutive compressions. Resilience is the ability of the sample to rebound during the first compression,

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Polymers 10, of x FOR REVIEW 7 of 11 which is the2018, ratio thePEER elastic energy released by the sample upon return during the first compression cycle to the energy dissipated by the probe during compression. The sample of IPN hydrogel was The sample of IPN hydrogel was compressed by TPA (Test process analysis) test for consecutive compressed by TPA test for two consecutive compressions. IPN hydrogel materials have a high compressions. Resilience is the ability of the sample to rebound during the first compression, which rebound rate of up to 95% resilience, compared with only about 12% of pure silk fibroin hydrogel. is the ratio of the elastic energy released by the sample upon return during the first compression cycle Due to interpenetrating structure within the material, entanglement and the was physical to the the network energy dissipated by the probe during compression. Thethe sample of IPN hydrogel crosslink densitybyofTPA the test molecular were compressions. greatly increased. The material underwent elastic compressed for two chain consecutive IPN hydrogel materials have a high deformation when being compressed without any damaged. A texture analyzer was used to measure rebound rate of up to 95% resilience, compared with only about 12% of pure silk fibroin hydrogel. and evaluated the mechanical differences between different ratios PVP-SF IPNand hydrogel and pure Due to the network interpenetrating structure within the material, theofentanglement the physical crosslink density of the molecular wereshowed greatly non-linear increased. The materialStress underwent silk hydrogel. Notably, both the gel chain samples behavior. of theelastic hydrogel deformation when being withoutin any damaged. texture analyzer was used to measure showed an increasing trendcompressed with an increase strain. The A compression fracture occurred when the and evaluated the mechanical differences between different ratios of PVP-SF IPN hydrogel and pure silk gel was compressed by about 50%. IPN hydrogel at the maximum compression up pure to 70% of silk hydrogel. Notably, both the gel samples showed non-linear behavior. Stress of the hydrogel the stress was still increased. The shape was complete and the rapid removal of hydrogel after the showed an increasing trend with an increase in strain. The compression fracture occurred when the pressure rebound, the IPN hydrogel showed higher mechanical properties compared with the pure pure silk gel was compressed by about 50%. IPN hydrogel at the maximum compression up to 70% silk hydrogel. This compressive compressive resilience of the stress wassignificantly still increased.increased The shapethe was complete andstrength the rapidand removal of hydrogel after theof the hydrogel by virtue of the regular cross-linking of the internal structure of the macromolecule network. pressure rebound, the IPN hydrogel showed higher mechanical properties compared with the pure This is summarized insignificantly Figure 3A,B. silk hydrogel. This increased the compressive strength and compressive resilience of the

hydrogel by virtue of the regular cross-linking of the internal structure of the macromolecule network.

3.5. Light Transmittance Characterization This is summarized in Figure 3A,B.

The contact lens material of contact optical system consists, corneal related part, contact lens 3.5. Light Transmittance Characterization and the formation of tears in the cornea [41]. The copolymer hydrogels are used in the application contactlens lens materials, material of contact systemto consists, corneallight related part, contact lens and light of cornealThe contact whichoptical is required have good transmission. When the formation of tears in the cornea [41]. The copolymer hydrogels are used in the application of propagates through a polymeric material, it causes a material-valence electron transition that converts corneal contact lens materials, which is required to have good light transmission. When light part of the light energy into heat energy. This causes the light to decay. Based on the thickness of the propagates through a polymeric material, it causes a material-valence electron transition that artificial cornea and contact lens, the actual thickness of the hydrogel (4.4 mm) was corrected to the converts part of the light energy into heat energy. This causes the light to decay. Based on the light thickness transmittance. of the artificial cornea and contact lens, the actual thickness of the hydrogel (4.4 mm) was IPN hydrogels formed for 24 h and the transmissivity reached more than 97%. This is more corrected to the light transmittance. than national standard.formed The unstinted lens transmission ratio97%. is greater than than 92% [42]. IPN hydrogels for 24 h andsoft the contact transmissivity reached more than This is more national standard. The silk unstinted soft contact transmission ratio is greater than 92% showed [42]. The good The transmittance of pure hydrogel is only lens about 21% (Figure 4A,B). IPN hydrogel pure silk hydrogel is onlyindicate about 21% (Figure 4A,B). IPN of hydrogel showed material good lightchain light transmittance transmissionofproperties. The results that the structure the hydrogel transmission properties. results and indicate that the of substantially the hydrogel material chain is is interpenetrating networkThe structure the pure silkstructure hydrogel crystalline structure. interpenetrating network structure and the pure silk hydrogel substantially crystalline structure. This This hinders the light pass through, so the light transmittance is low. IPN hydrogels still retain good hinders the light pass through, so the light transmittance is low. IPN hydrogels still retain good light light transmission over time. The light transmission remains as high as 80% over a week. This is transmission over time. The light transmission remains as high as 80% over a week. This is mainly mainly due to the high degree of entanglement of the molecular chains endow the hydrogel stable due to the high degree of entanglement of the molecular chains endow the hydrogel stable internal internal non-crystalline structure. This insures its excellent light transmission non-crystalline structure. This insures its excellent light transmission properties. properties. 105 100

97

492nm 550nm 700nm

Transimittance (%)

80 60 40 21 20 0

Transmittance (%)

100 95 90 85 80

IPN gel

SF gel Sample

(A)

75 0h

1/6h

1/2

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Figure 4. Light transmittance (A) at 550 nm. (B) IPN hydrogel transmittance at different times; The

Figure 4. Light transmittance (A) at 550 nm. (B) IPN hydrogel transmittance at different times; The solid solid content of IPN hydrogel was 10 g/L, SF:NVP was 8:2 and the thickness of material was 4.4 mm. content of IPN hydrogel was 10 g/L, SF:NVP was 8:2 and the thickness of material was 4.4 mm.

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3.6. Enzymatic Degradation of Hydrogels In Vitro 3.6. Enzymatic Degradation of Hydrogels In Vitro Interpenetrating network structure in the loss of a macromolecule, the degree of crosslinking Interpenetrating network structure in the loss of a macromolecule, the degree of crosslinking decreased rapidly dissolved in water. The rate of degradation of the hydrogel was up to over 80% in decreased rapidly dissolved in water. The rate of degradation of the hydrogel was up to over 80% in 24 h, whereas pure silk fibroin hydrogel was degraded only about 40% (Figure 5). The main reason 24 h, whereas pure silk fibroin hydrogel was degraded only about 40% (Figure 5). The main reason might be due to the enzymatic action, the silk fibroin in the IPN gel was degraded. This resulted in a might be due to the enzymatic action, the silk fibroin in the IPN gel was degraded. This resulted decrease in the degree of crosslinking of the gel and rapid degradation. Thus, the IPN gel was a crossin a decrease in the degree of crosslinking of the gel and rapid degradation. Thus, the IPN gel was linked network of hydrogels formed by intertwining and cross-linking of silk fibroin molecules and a cross-linked network of hydrogels formed by intertwining and cross-linking of silk fibroin molecules PVP molecules. and PVP molecules. IPN gel SF gel

Weight Remain (%)

100

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2

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24

Time (h) ◦ C, Figure pH: Figure5.5.Degradation Degradationofofsilk silkfibroin fibroinhydrogels. hydrogels.InIn22U/mL U/mLprotease proteaseXIV XIVPBS PBSsolution solution(37 (37°C, pH:7.4). 7.4). The solid content of SF hydrogel was 10 g/L, SF:NVP was 8:2 and the thickness of material was 4.4 The solid content of SF hydrogel was 10 g/L, SF:NVP was 8:2 and the thickness of material was 4.4 mm. mm.

4. Discussion 4. Discussion Reports over the past decades have stated that silk hydrogel was prepared by ultrasonic induction, Reports over thecrosslinking past decades have stated that prepared by ultrasonic temperature, pH or agent [21,43–45]. Thesilk silkhydrogel hydrogelwas prepared by these methods, induction, temperature, pH or crosslinking agent [21,43–45]. The silk hydrogel prepared by these which exhibit a weak resilience and low light transmission, because of β-sheet structure. Meanwhile methods, which exhibit a weak resilience and low light transmission, because of β-sheet structure. the toxicity of chemical crosslinking agents was present, which may limit its practical use. To improve Meanwhile the and toxicity chemical crosslinking was present, may PVP-SF limit its practical use. the resilience lightoftransmission as well asagents the biosecurity, wewhich prepared IPN hydrogel To improve the resilience and light transmission as well as the biosecurity, we prepared PVP-SF IPN using HRP-H2 O2 systems. hydrogel using HRP-H 2O2 systems. As shown in Scheme 2, the SF molecules form a loose globular structure in the solution with As shown in Scheme 2, the SFadded molecules form a loose structure in the solution with random coil structure. When NVP into SF solution, theglobular small NVP molecules disperse in water random coil structure. When NVP added into SF solution, the small NVP molecules disperse in water and in loose globular formed by SF macromolecules. When PVP macromolecules synthesized by the and loose globular formedof byNVP SF macromolecules. When PVP macromoleculesoccurs synthesized by the freeinradical polymerization in HRP-H2 O2 systems, the polymerization in the solvent free radical polymerization in HRP-H2O2 systems, theThen polymerization occurs in the (water), as well the interfaceofofNVP SF macromolecules and water. the crosslinking points of solvent physical (water), as well the interface of SF macromolecules and water. Then the crosslinking points of entanglement are formed by segments of SF macromolecules and segments of PVP macromolecules, physical entanglement are formed network. by segments macromolecules of value, PVP which can be called interpenetrating WhenoftheSFcrosslinking density and rises segments to the critical macromolecules, which can be called interpenetrating network. When the crosslinking density rises the viscosity of the solution rises to infinity and the IPN hydrogel is formed. The movement of silk tofibroin the critical thedue viscosity the solution rises to infinity andThus, the IPN formed. The chain value, is limited to the of existence of physical crosslinking. the hydrogel silk fibroinischain segment movement fibroin chain is limited due to the structure. existence of physical crosslinking. Thus, is difficult of to silk rotate freely to form a stable β-sheet That is why the structure of SFthe in silk IPN fibroin chain segment is difficult to rotate freely to form a stable β-sheet structure. That is why the hydrogel is mainly random coil, while the structure of pure SF hydrogel is mainly β-sheet (Figure 2A). structure SF in IPN is mainly random coil, while structure of pure SF hydrogel is The of formation of hydrogel larger particles or silk II crystals, whichthe may make hydrogel opaque, is also mainly (Figure 2A). limitedβ-sheet by the interpenetrating network structure formed by SF and PVP. That is the reason why PVP-SF The formation of larger particles or silk II remain crystals,for which may make opaque, is also IPN hydrogel has good transparency and can a long time. Thehydrogel transmittance of PVP-SF limited by the (8:2) interpenetrating networkhigher structure by SF and PVP.hydrogel That is the reason why3). IPN hydrogel is 97% significantly than formed that of pure silk fibroin (21%) (Figure PVP-SF IPN hydrogel has good transparency and can remain for a long time. The transmittance When PVP-SF IPN hydrogel was immersed into protease XIV PBS solution, the chain of of SF PVP-SF IPNwas hydrogel is pieces 97% significantly higher than hydrogel (21%) molecular cut into(8:2) small by the enzyme while the that chainofofpure PVPsilk willfibroin not be cut and still keep (Figure 3). However, due to SF molecular was cut from long chain to small fragments, the crosslinking long chain. point of physical entanglement will be destroyed. And then the interpenetrating network structure was

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break up. The PVP and small fragments of SF was dissolved in water. The degradation process is very fast due to the structure of SF is random coil but not β-sheet and It is no need to completely degrade SF molecular while only need cut the point of physical entanglement. That is why the degradation rate of PVP-SF IPN so far faster than that of pure silk hydrogel (Figure 5). Polymers 2018, 10, xhydrogel FOR PEERis REVIEW 9 of 11

Scheme 2. 2. Formation and degradation degradation of of PVP–SF PVP–SF IPN IPN hydrogel. hydrogel. Scheme

When PVP-SF IPN hydrogel was immersed into protease XIV PBS solution, the chain of SF In summary, the PVP-SF IPN hydrogels fabricated are superior to the pure silk fibroin hydrogel molecular was cut into small pieces by the enzyme while the chain of PVP will not be cut and still in terms of mechanical properties, light transmittance and biodegradability due to the mutual keep long chain. However, due to SF molecular was cut from long chain to small fragments, the network structure. crosslinking point of physical entanglement will be destroyed. And then the interpenetrating network structure was break up. The PVP and small fragments of SF was dissolved in water. The 5. Conclusions degradation process is very fast due to the structure of SF is random coil but not β-sheet and It is no We prepared a series of PVP–SF IPN hydrogels with high desirable and tunable features need to completely degrade SF molecular while only need cut the point of physical entanglement. for biomedical applications. The PVP–SF IPN hydrogels have an excellent translucency (97%) That is why the degradation rate of PVP-SF IPN hydrogel is so far faster than that of pure silk degree, higher transmittance, higher compressive strength and compressive resilience and rapid hydrogel (Figure 5). protease degradation as compared to pure SF hydrogels. We also demonstrated that, by varying In summary, the PVP-SF IPN hydrogels fabricated are superior to the pure silk fibroin hydrogel the concentration of silk fibroin in the IPN hydrogel, the structural, pore size and Compression in terms of mechanical properties, light transmittance and biodegradability due to the mutual mechanical properties of the resulting IPN hydrogels could be tuned. It is expected that this type of network structure. hydrogel can be used in cell migration investigation and also where transparency films are needed as biomedical materials. 5. Conclusions Acknowledgments: work of wasPVP–SF supported by The Nationalwith Key Research and Development Program of China We preparedThis a series IPN hydrogels high desirable and tunable features for (Grant No. 2017YFC1103602), National Natural Science Foundation of China (Grant No. 51373114, 51741301), biomedical applications. The PVP–SF IPN hydrogels have an excellent translucency (97%) degree, PAPD and Nature Science Foundation of Jiangsu, China (Grant No. BK20171239, BK20151242).

higher transmittance, higher compressive strength and compressive resilience and rapid protease

Author Contributions: Shenzhou Lu, Zhuping Yin and Tieling Xing conceived and designed the experiments; degradation as compared pure SF hydrogels. We also demonstrated that, varying the Tian Zhu, Dajiang Kuang and to Feng Wu performed the experiments and analyzed the data;byDajiang Kuang, concentration of silk fibroin in the IPN hydrogel, the structural, pore size and Compression Subhas C. Kundu and Feng Wu wrote the paper. All authors discussed the results and improved the final text of the paper. mechanical properties of the resulting IPN hydrogels could be tuned. It is expected that this type of

hydrogelofcan be used cell migration and also where transparency films are needed Conflicts Interest: Thein authors declare no investigation conflict of interest. as biomedical materials. References Acknowledgments: This work was supported by The National Key Research and Development Program of 1. Hamidi, M.; Azadi, A.; Rafiei, P. Hydrogel nanoparticles in drug delivery. Adv. Drug Deliv. Rev. 2008, 60, China (Grant No. 2017YFC1103602), National Natural Science Foundation of China (Grant No. 51373114, 1638–1649. [PubMed] 51741301), PAPD[CrossRef] and Nature Science Foundation of Jiangsu, China (Grant No. BK20171239, BK20151242). 2. Numata, K.; Katashima, T.; Sakai, T. State of water, molecular structure and cytotoxicity of silk hydrogels. Author Contributions:2011, Shenzhou Lu, Zhuping Yin and Tieling Xing conceived and designed the experiments; Biomacromolecules 12, 2137–2144. [CrossRef] [PubMed] Tian Zhu, Dajiang Kuang and Feng Wu performed the experiments and analyzed the data; Dajiang Kuang, 3. Peppas, N.A.; Bures, P.; Leobandung, W.; Ichikawa, H. Hydrogels in pharmaceutical formulations. Eur. J. Subhas C. Kundu and Feng Wu wrote the paper. All authors discussed the results and improved the final text Pharm. Biopharm. 2000, 50, 27–46. [CrossRef] of the paper. 4. Tomme, S.R.V.; Storm, G.; Hennink, W.E. In situ gelling hydrogels for pharmaceutical and biomedical Conflicts of Interest: authors declare no conflict of interest. applications. Int.The J. Pharm. 2008, 355, 1–18. [CrossRef] [PubMed]

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