蚕丝蛋白基高强度水凝胶的制备及其生物应用

马跃; 胡艳磊; 刘亮; 俞娟; 陈美娟; 范一民

doi:10.13801/j.cnki.fhclxb.20230425.003

蚕丝蛋白基高强度水凝胶的制备及其生物应用

doi: 10.13801/j.cnki.fhclxb.20230425.003

马跃^1,,
胡艳磊¹,
刘亮¹,
俞娟¹,
陈美娟²,
范一民^1, ,

1.
南京林业大学　化学工程学院，南京 210037
2.
江苏奥普莱医疗用品有限公司，高邮 225600

基金项目: 国家自然科学基金(32271812)

详细信息

通讯作者:
范一民，博士，教授，博士生导师，研究方向为生物质纳米材料　E-mail: fanyimin@njfu.edu.cn

中图分类号: O636.9；TB332
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出版历程
- 收稿日期: 2023-02-24
- 修回日期: 2023-04-09
- 录用日期: 2023-04-15
- 网络出版日期: 2023-04-26
- 刊出日期: 2023-09-15

Preparation and biological application of high strength hydrogels based on silk fibroin

MA Yue^1
,,
HU Yanlei¹,
LIU Liang¹,
YU Juan¹,
CHEN Meijuan²,
FAN Yimin^{1
, ,}

1.
College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China
2.
Jiangsu Opera Medical Supplies CO., LTD., Gaoyou 225600, China

Funds: National Natural Science Foundation of China (32271812)

摘要

摘要: 水凝胶具有亲水三维网络结构，因其结构和功能与生物组织相似的特性，使其在生物医学等领域中得以广泛应用。蚕丝蛋白(Silk fibroin，SF)由于其资源丰富，具有良好的生物降解性和生物相容性，成为一种极具潜力的水凝胶基材。然而，由于在蚕丝蛋白制备过程中对蚕丝纤维天然层级结构的溶解和破坏，丧失了蚕丝纤维机械强度高的天然优势，导致力学性能差成为限制蚕丝蛋白基水凝胶广泛应用的主要原因之一，因此，研究者不断寻求策略制备蚕丝蛋白基高强度水凝胶(SF-high strength hydrogels，SF-HSHs)。本文首先介绍了SF的基本结构；然后阐述了SF水凝胶的制备方法和凝胶化机制；进而详细讨论了物理交联、双交联、双网络和复合SF-HSHs；最后简要分析了SF-HSHs的生物应用及其前景与挑战。
- 蚕丝蛋白 /
- 水凝胶 /
- 凝胶化机制 /
- 高强度 /
- 生物应用
Abstract: Hydrogels have hydrophilic three-dimensional network structure, which are widely used in biomedicine and other fields because of its similar structure and function to biological tissue. Silk fibroin (SF) has become one of potential hydrogel substrates due to its abundant resources, good biodegradability and biocompatibility. However, due to the dissolution and destruction of the natural hierarchical structure of silk fibers in the process of preparing silk fibroin, the natural advantage of high mechanical strength of silk fibers is lost, and the poor mechanical properties become one of the main reasons limiting the wide application of silk fibroin-based hydrogels. Therefore, researchers are constantly seeking strategies to prepare silk fibroin-based high strength hydrogels (SF-HSHs). This review first introduced the basic structure of SF. Then the preparation methods and gelation mechanisms of SF hydrogel were described. Furthermore, physical cross-linking, dual cross-linking, dual network and composite SF-HSHs were discussed in detail. Finally, the biological applications, prospects and challenges of SF-HSHs were briefly analyzed.
- silk fibroin /
- hydrogel /
- high strength /
- gelation mechanism /
- biological applications

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图 1 蚕丝纤维(SF)的五级结构示意图^[10]

Figure 1. Levels of hierarchical structures of silk fibers (SF)^[10]

G—Glycine; A—Alanine; Y—Tyrosine; S—Serine; V—Valine

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图 2 蚕丝蛋白提取和水溶液制备过程示意图^[23]

Figure 2. Schematic of silk fibroin extraction and aqueous solution preparation procedure^[23]

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图 3 蚕丝蛋白纳米纤维分散液的制备：(a) 氯化钙/乙醇/水三元体系^[13]；(b) 硫酸水解体系^[28]；(c) 碱处理体系^[29]；(d) 低共熔溶剂(DES)体系预处理^[30]

Figure 3. Preparation of silk nanofiber dispersion: (a) Ternary system of CaCl₂/ethanol/water^[13]; (b) Sulfuric acid hydrolysis treatment^[28]; (c) Alkali treatment system^[29]; (d) Pretreatment of deep eutectic solvents (DES) system^[30]

SMFs—Silk millimeter/microfibers

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图 4 (a) 不同凝胶化阶段纳米纤维网络的形成图解^[33]；(b) 丝蛋白浓度对二级结构和凝胶化的影响^[34]；(c) SF凝胶化阶段与pH对凝胶化的调控^[35]

Figure 4. (a) Illustration of the formation of nanofibrillar network at different gelation stages^[33]; (b) Effect of silk protein concentration on secondary structure and gelation^[34];(c) The gelation stage of SF and the regulation of pH on gelation^[35]

C_g—Critical concentration; DW—Drained mass

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图 5 (a) 京尼平化学交联SF水凝胶^[44]；(b) HRP/H₂O₂酶交联SF水凝胶^[45]；(c) 漆酶介导交联mHA-SF水凝胶^[46]

Figure 5. (a) Genipin chemical crosslinked SF hydrogel^[44]; (b) HRP/H₂O₂ enzyme cross-linked SF hydrogel^[45];(c) Laccase mediated cross-linked mHA-SF hydrogel^[46]

HRP—Horseradish peroxidase; mHA—Modified HA

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图 6 (a) 光聚合大豆分离蛋白(SPI)/SF杂化水凝胶^[52]；(b) 核黄素光固化SF水凝胶^[53]

Figure 6. (a) Photopolymerized soy protein isolate (SPI)/SF hybrid hydrogel^[52]; (b) Riboflavin photocured SF hydrogel^[53]

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图 7 (a) 二元溶剂诱导构象转变(BSICT)策略制备具有高力学性能SF水凝胶^[61]；(b) SDS调节SF物理凝胶化^[62]

Figure 7. (a) Preparation of SF hydrogels with high mechanical properties by binary solvent Induced conformational transformation (BSICT) strategy^[61]; (b) SDS regulates the physical gelation of SF^[62]

DI—Deionized water; DS—Negatively charged ionic groups ionized from SDS in water

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图 8 (a) 双交联SF-HSHs双交联凝胶化示意图^[70]；(b) 辣根过氧化物酶(HRP)/H₂O₂+乙醇交联^[63]；(c) γ射线+乙醇诱导双交联^[55]

Figure 8. (a) Double-crosslinked SF-HSHs schematic diagram of double-crosslinked gelation^[70]; (b) Horse radish peroxidase (HRP)/H₂O₂+ ethanol crosslinking^[63]; (c) Double cross-linking induced by γ-ray and ethanol^[55]

RSF—Regenerated silk fibroin; EE—Enzyme-electric field; N—Naturally formed physical crosslinking; EE—Enzyme-electric field-alcohol; EA—Enzyme and Alcohol-treated; E—Enzymatic; SF-E—Silk fibroin-ethanol crosslinking; SF-S—Silk fibroin-γ-ray radiation crosslinking; SF-D—Silk fibroin- γ-ray radiation and ethanol crosslinking

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图 9 (a) 制备复合SF水凝胶的反向透析示意图；(b) SF水凝胶和其他基于SF的水凝胶的断裂应力与杨氏模量对比图；(c) 断裂应变与杨氏模量的关系^[65]

Figure 9. (a) Scheme of reverse dialysis procedure to fabricate composite SF hydrogels; (b) Ashby plot of fracture stress versus Young’s modulus; (c) Fracture strain versus Young’s modulus of SF hydrogels and other reported SF-based hydrogels^[65]

PAA—Polyacrylic acid; PVA—Polyvinyl alcohol; PAMPS—Poly(2-acrylamido-2-methylpropanesulfonic acid)

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图 10 (a) SF-聚丙烯酰胺(HPAAm)双网络(DN)水凝胶^[66]；(b) 明胶-SF互穿网络(IPN)水凝胶^[67]

Figure 10. (a) SF-polyacrylamide (HPAAm) dual network (DN) hydrogel^[66]; (b) Gelatin-SF interpenetrating network (IPN) hydrogel^[67]

SN—Single network; UV—Ultraviolet light; AAm—Acrylamide; mTG—Microbial transglutaminase

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图 11 SF-HSHs应用于组织工程：(a) 细菌纤维素与明胶-丝蛋白水凝胶混合，增强3D打印水凝胶支架力学性能^[78]；(b) 内源性骨髓干细胞(BMSC)与明胶/丝蛋白结合，用于关节软骨修复^[79]；(c) 两种丝纳米纤维与HRP和电场诱导交联增强水凝胶，用于骨组织再生^[80]；(d) 光化学交联SPI/SF水凝胶，提高力学性能和细胞附着效果^[81]

Figure 11. Application of SF-HSHs in tissue engineering: (a) Mixing bacterial cellulose with gelatin-silk hydrogel to enhance the mechanical properties of 3D printed hydrogel scaffolds^[78]; (b) Endogenous bone marrow stem cells (BMSC) bound to gelatin/silk protein scaffold for articular cartilage repair^[79]; (c) Two kinds of silk nanofibers were crosslinked with HRP and electric field induced hydrogel for bone regeneration^[80]; (d) Photochemically cross-linked SPI/SF hydrogels to improve mechanical properties and cell attachment^[81]

SFG—Silk fibroin gelatin; MF—Microfracture; E7—EPLQLKM molecular; SANS—Small angle neutron scattering

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图 12 SF-HSHs用作黏合剂：(a) 丝素蛋白-聚丙烯酰胺双网络水凝胶在汗湿条件下对人体皮肤有强附着力^[83]；(b) SF@单宁酸(TA)@羟基磷灰石(HA)提高了水凝胶的韧性和黏附强度，可作为骨黏合剂^[84]

Figure 12. SF-HSHs used as adhesive: (a) SF-PAAm DN hydrogel has strong adhesion to human skin under the condition of sweat^[83]; (b) SF@tannic acid (TA)@hydroxyapatite (HA) improves the toughness and adhesion strength of hydrogel and can be used as bionic bone adhesive^[84]

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图 13 SF-HSHs用作伤口愈合：(a) SF-TA水凝胶作为伤口敷料促进伤口愈合并预防细菌感染^[87]；(b) SF-Ag-甘草酸(GA)水凝胶用于抗菌和伤口愈合^[88]

Figure 13. SF-HSHs used for wound healing: (a) Use of SF-TA hydrogel as a wound dressing to promote wound healing and prevent bacterial infection^[87]; (b) SF-Ag-glycyrrhizic acid (GA) hydrogels are used for antibacterial and wound healing^[88]

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图 14 SF-HSHs用作药物缓释：(a) 丝纳米纤维与去铁胺共混形成持续的药物递送系统^[89]；(b) 京尼平交联热敏水凝胶修饰的丝质医用敷料，用于对乙酰氨基酚的缓释^[90]

Figure 14. SF-HSHs is used for sustained drug release: (a) A continuous drug delivery system formed by blending silk nanofibers with desferrioxamine^[89]; (b) Genipin cross-linked thermosensitive hydrogel modified silk medical dressing for sustained release of paracetamol^[90]

DFO—Desferrioxamine; CGG—Chitosan/glycerol-phosphate disodium salt/genipin; VEGF—vascular endothelial growth factor; SDF-1α—Stromal cell-derived factor-1alpha

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表 1 蚕丝蛋白的氨基酸组成^[15]

Table 1. Amino acid composition of silk protein^[15]

Amino acid content/mol%	Native silk	Regenerated silk	Amino acid content/mol%	Native silk	Regenerated silk
Aspartic acid	2.4	1.5	Methionine	0.1	0.1
Threonine	1.6	0.8	Isoleucine	0.6	0.6
Serine	12.3	10.8	Leucine	0.5	0.4
Glutamic acid	1.2	0.9	Tyrosine	5	4.9
Proline	0.7	0.5	Phenylalanine	0.7	0.6
Glycine	43.5	46.2	Histidine	0.2	0.2
Alanine	28	29.7	Lysine	0.5	0.3
Cysteine	0.1	0	Arginine	0.6	0.4
Valine	2.3	2.1

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表 2 SF基高强度水凝胶(SF-HSHs)交联方式、力学性能对比

Table 2. Comparison of crosslinking methods and mechanical properties of SF-high strength hydrogels (SF-HSHs)

Type of gelation	Hydrogel systems	Mechanical properties	Applications	Ref.
Physical crosslinked	SF/HFIP/water	Water content: 85%-90%, tensile strength: (0.7±0.04) MPa, Young's modulus: (6.5±0.2) MPa	Cell culture	[60-61,62]
	SF/SDS	Tensile modulus: 3.0 MPa, tensile strength: (0.7±0.12) MPa, elongation: (134±21)%	Tissue engineering
	SF/EMImAc/EtOH	Water content: 90%, tensile modulus: 0.5-3.68 MPa, compressive modulus: 0.59-4.6 MPa	Conductor material
Dual crosslinked	SF/HRP/H₂O_2/EtOH	Water content: 90%, tensile modulus: 2.5-3.0 MPa, compressive strength: 0.14-0.7 MPa	Tissue engineering	[55,63]
Dual crosslinked	SF/γ-ray /EtOH	Water content: 85%, compressive modulus: 1.2-2.41 MPa, compressive strength: (1.37±0.1) MPa	Tissue engineering	[55,63]
SF composite gel	SF/PEG/HPMC	Water content: 51%-58%, Young's modulus: 30-91 MPa, tensile strength: 3.42-4.1 MPa	Biomedical science	[64-65]
SF composite gel	SF/cellulose	Young's modulus: 9.45-14 MPa, ultimate stress: 0.94-1.1 MPa, toughness: 84.73-108.3 KJ·m⁻³	Cell adhesion	[64-65]
Dual/IPN network	SF/SDS/HPAAm	Water content: 67%-81%, tensile strength: 0.3-1.17 MPa, toughness: 1.98-11.25 MJ·m⁻³	Biosensor	[66-67]
Dual/IPN network	SF/Gelatin/EtOH	Water content: 50%-75%, compressive modulus: 0.85-11.6 MPa	Tissue regeneration	[66-67]
Notes: HFIP—Hexafluoroisopropanol; SDS—Sodium Dodecyl Sulfonate; EMImAc—1-ethyl-3-methylimidazole acetate; EtOH—Ethyl alcohol; PEG—Polyethylene glycol; HPMC—Hydroxyl propyl methyl cellulose; HPAAm—Hydrophobically associated acrylamide; HRP—Horse radish peroxidase.

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