Research and Application Progress of Silk Fibroin Membranes

Chan ZHOU, Jinfeng LU*, Jieping WANG, Yao ZENG, Qunzhong MA, Shanlin GU

1. Chongqing Academy of Animal Sciences, Chongqing 402460, China; 2. Silk & Related Biomaterials Research Center, Chongqing Academy of Animal Sciences, Chongqing 402460, China

Abstract This paper mainly introduced the preparation of silk fibroin membranes and their structural change characteristics. Silk fibroin membranes can be used as tissue engineering materials, enzyme-immobilizing membranes, biosensors and drug controlled-release membranes and other different materials. They have excellent characteristics such as non-toxic, non-polluting and degradable, and thus have broad application prospects.

Key words Silk fibroin membrane, Tissue engineering, Biosensor, Polymer blend membrane, Immobilized enzyme carrier, Controlled release

1 Introduction

Silk has brought excellent clothing enjoyment to mankind because of its unique performance and style, and is known as "fiber queen". With the advancement of science and technology, the research of silk in non-clothing fields has become more and more attractive. The main structure of silk is the inner layer of silk fibroin and the outer layer of sericin. Silk fibroin accounts for 70%-80% of the mass of silk. It is the basic material used by people. The inner silk fibroin is a natural protein composed of 18 amino acids such as aminoacetic acid, alanine, serine, and tyrosine. Studies have shown that it has good biocompatibility and is non-toxic, non-polluting, and degradable. Furthermore, the research of silk fibroin membranes made of silk fibroin is very active in the biological field.

2 Composition and structure of silk fibroin

2.1 Composition

Silk is composed of two kinds of proteins. The outer layer of silk is sericin, and the inner layer is silk fibroin. In different types of silk, the content of silk fibroin is not the same, but in general, silk fibroin accounts for 70%-80% of the total mass of silk. Silk fibroin is composed of 18 kinds of amino acids, of which 8 are essential amino acids for the human body.Due to different types, tussah silk and mulberry silk differ obviously in amino acid content of silk fibroin. In addition to carbon, hydrogen and nitrogen, silk fibroin also contains a variety of other elements such as potassium, calcium, silicon, strontium, phosphorus, iron, copper,

etc.

These elements are directly related to the properties of silk fibroin and the mechanism of silkworm spinning.

2.2 Structure

Silk fibroin is a kind of fibrous protein, which is composed of an amorphous area on the surface and an inner crystalline area. The crystalline area includes two crystal forms, which are called silk I type and silk II type, respectively. The three-dimensional conformation of the silk I crystals is of a crank shape, which is an intermediate form between β2 fold and α2 helix, and has following unit cell parameters: a=4 149, b=7 120, c=9 108; and the silk II crystals have an anti-parallel β2 folded layer structure, which belongs to the monoclinic crystal system, and the unit cell parameters are as follows: a=9 144, b=6 197, c=9 120, β=90°. Peptide chains first form the folded layer, which then forms entire silk II crystals.

In the silkⅡcrystals, peptide chain segments are arranged neatly, and hydrogen bonds between adjacent segments and intermolecular gravitational forces make them very tightly combined, so the silkⅡcrystals are strongly resistant to external stretching and low flexibility, difficult to dissolve in water and strongly resistant to acids, alkalis, salts, enzymes and heat. The chain segments of peptide chains in amorphous silk fibroin are not neatly arranged, and the binding force between the chain segments is weak, so amorphous silk fibroin is easily soluble in water, and has high flexibility, weak ability to resist external force stretching, strong moisture absorption, and weak resistance to salt, enzymes and heat.

Observed under a transmission electron microscope and a scanning electron microscope, the most basic structural unit of fibroin fibers is microfibrils with a diameter of 10-50 nm. The microfibrils aggregate into fibrils of tens of thousands of nanometers, and about 100 fibrils aggregate into macrofibrils on the order of 100 000 nanometers, and the macrofibrils aggregate into fibroin fibers of several million nanometers.

In recent years, Regina1V and others have discovered a new fibroin crystal morphology that exists on the air interface of fibroin solution 2 and is called silk III. Its crystal structure is similar to that of polyglycine II and belongs to the hexagonal crystal system. The three-dimensional conformation of its peptide chains is β2 folded helix.

Silk fibroin is composed of heavy and light chains, the molecular weights of which are 350 and 25 KD, respectively. The heavy chains and light chains are connected by disulfide bonds, and there is a segment called P25 that is connected to the heavy and light chains by non-covalent bonds. The molecular weight of P25 is 27KD.

The subunits of silk fibroin are composed of three subunits a, b, and c, with molecular weights of 280, 230, and 25 KD, respectively, and a and c among them account for more than 90% of the total protein. The amino acid sequence of

Bombyx

mori

silk fibroin has been determined. The amorphous region of silk fibroin is mainly composed of a large number of repetitive sequences: T GSSGF GP YVAD GGYSRREGYEAWSS KSDFET, and the crystalline region is mainly composed of repetitive sequences: GASSGS. The non-repetitive region at the carboxyl end of

B.

mori

silk fibroin molecules contains more basic amino acids, especially arginine. Tussah silk fibroin not only contains basic amino acids, but also contains a tripeptide sequence known as a cell adhesion site: arginine 2 glycine 2 aspartic acid.

3 Properties of silk fibroin

Silk fibroin is porous and has a high moisture regain rate. It is insoluble in water and soluble in high concentrations of certain inorganic salts. Silk fibroin begins to be dehydrated at 100 ℃, and gradually loses weight from 175 ℃, the color changes from white to yellow, to completely black at 280 ℃, and decomposition occurs at 305 ℃. Silk fibroin molecules contain phenolic hydroxyl groups and other structures, so they are easily denatured by absorbing ultraviolet light. As the irradiation time increases, the degree of yellowing of silk fibroin also increases, especially in the presence of water, with which the degree of yellowing increases.

The conformation of silk fibroin in a silk fibroin solution is a random coil structure, and a silk fibroin membrane containing silk I structure can be obtained after it is dried and solidified at a moderate speed at 45 ℃; and when the temperature is higher than 45 ℃, silk I is transformed to silk II. The structure after drying and solidification of a silk fibroin solution is not only affected by temperature, but also closely related to the drying speed, pH and composition of the solution. Silk I is formed in the process of drying at room temperature for a long time, but it will eventually form a stable silk II; when the drying speed is too fast, even if it is above 50 ℃, the amorphous structure is still dominant; if the pH value of a solution is greater than 5 at the initial stage of drying, an amorphous structure will be formed; if the pH value of a solution at the initial stage of drying is less than 5, silk II will be formed; and if solvents such as methanol and ethanol are added to a silk fibroin aqueous solution, silk II will be formed. In addition, when a silk fibroin aqueous solution is stirred or solid silk fibroin is rapidly stretched, silk II is formed; and when a silk fibroin aqueous solution is placed in a 7 kV electric field, part of the polar chain segments are aligned under the action of the electric field to make the silk fibroin conformation to be silk II. From this we can see that when a silk fibroin solution is stimulated by the external environment, it is easy to form its relatively stable structure, silk II.

Some metal ions have a certain effect on the conformation of silk fibroin. Under certain pH conditions, Niions induce the formation of silk II through four-coordinate chelation. Viney speculates that the increase of Cacan accelerate the formation of silk II according to inductively coupled plasma (ICP) technology. The results of quantitative fitting by Li Guiyang

et

al.

using NMR spectra showed that the presence of Caand Cuwas beneficial to the formation of silk II. Paola Taddei found that the coordination of Coalso had a certain impact on the structure of silk fibroin.

4 Silk fibroin membranes

4.1 Preparation of silk fibroin membranes

Silk fibroin membranes are divided into natural silk fibroin membranes and regenerated silk fibroin membranes. For the preparation of natural silk fibroin membranes, liquid silk fibroin is collected from the rear silk gland the day before silkworm or tussah silk is spun, then diluted to about 1% with water, and then cast on a polyethylene film and dried in an environment at 25 ℃ with a relative humidity of 65%. The silk fibroin molecules of the obtained natural silk fibroin membranes can be cross-linked by appropriate chemical or physical methods, so that the membranes are not easy to dissolve in water. For regenerated silk fibroin membranes, different scholars have different preparation methods.Li Qun

et

al.

considered that to prepare a more ideal silk fibroin membrane, a CaClsolution was used instead of a LiBr solution to dissolve silk, and methanol or a glutaraldehyde solution was used for β treatment, so that the membrane had good stability in aqueous solutions and was economically feasible. Li Mingzhong

et

al.

believed that using low-grade polyols as a crosslinking agent for silk fibroin membranes could significantly increase the strength, elongation and water permeability of fibroin membranes, and reduce the solubility and vapor permeability of silk fibroin membranes. The application of reinforcing materials can prepare high-strength composite silk fibroin membranes, and evenly distributed pits are formed on the surface of membranes. The pits are a good place for cell proliferation, and thus create conditions for cell culture when the silk fibroin membranes are applied to artificial skin and other biomedical materials. Natural and regenerated silk fibroin membranes treated with pure methanol will produce different structures. The differences can be discussed based on TMA (thermomechanical curve) data. They have slight differences in physical properties and chemical structures. The molecular weight of natural silk fibroin membranes is relatively large, and they are not easily affected by methanol aqueous solutions, indicating that the degree of interaction of their molecular chains is relatively high. Natural and regenerated silk fibroin membranes have different displacements when being transferred from room temperature to 250 ℃, which is caused by the differences in their physical structures, which may indicate that the low-molecular-weight polypeptide chains in the regenerated silk fibroin membranes have higher thermal mobility. Therefore, in practical applications, attention should be paid to the characteristics of silk fibroin membrane starting materials and the treatment conditions for their crystallization treatment, so as to meet the needs of special application technologies.

4.2 Application research of silk fibroin membranes

4.2.1

Application of silk fibroin membranes in tissue engineering. Sun Tao

et

al.

observed the growth of human salivary gland cells on several biological materials. In the experiment, human submandibular gland epithelial cell line (U5G) cells were respectively inoculated on biomaterials including silk fibroin, polylactic acid, and polyether ester (polyethylene terephthalate polybutylene terephthalate block copolymer), and it was observed that the number of U5G cells growing on silk fibroin membranes was the largest, and the cells had good adherence and grew well. It is believed that silk fibroin is more suitable for the growth of U5G cells and can be used as a surface coating material for tissue engineering artificial salivary gland scaffolds. Scholars also discussed the

in

vitro

culture of human liver cells on silk fibroin membranes. At 24 h after seeding hepatocytes on silk fibroin membranes, the cells were observed to adhere along the surface and the inner network of the matrix, forming multiple structures. The test results of glutamic-oxaloacetic transaminase (GOT) and glutamate pyruvic transaminase (GPT) showed that the GOT and GPT activity values of the hepatocytes attached to silk fibroin membranes hardly decreased during 1-7 d after inoculation, and the ammonia processing capacity and hepatocyte albumin secretion capacity of hepatocytes cultured on silk fibroin membranes were not lower than the best hepatocyte culture medium—collagen.Minoura Norihiko

et

al.

observed and compared the attachment and growth of mouse fibroblasts (L-929) on silk fibroin membranes and collagen. The basic cell matrix material used in the experiment was a polystyrene film, which was a protein-coated film obtained by immersing a polystyrene film in a silk fibroin solution or a collagen solution. It was found that fibroblasts adhered tightly to the silk fibroin-coated film, pseudopodia could be seen, and they were spindle-shaped; and the adhesion rate and proliferation rate were equivalent to collagen.Inouge Kuniyo

et

al.

studied the growth of mammalian cells on silk fibroin-coated membranes (polystyrene membranes) and compared them with collagen-coated membranes and polystyrene membranes. In the experiment, human cancer cells were compared with mouse hybrid cells and insect cell lines, and it was found that silk fibroin had the effect of promoting the growth of mammalian cells, which was equivalent to collagen and better than polystyrene membrane, while for hybrid cells and insect cells, there were no obvious differences between the three.Chiarini Anna

et

al.

observed four kinds of adult fibroblasts on silk fibroin-coated membranes (polyurethane, SF-PCU), and compared them with blank PCU. They found that the cell adhesion rate on SF-PCU was 2.2 times that on PCU in the first 3 h; and after 30 d, it was 2.5 times, the number of cells was 13.3 times, and the proliferation rate was 16.5 times.

4.2.2

Silk fibroin membranes as a carrier for immobilizing enzymes. Miyairi Sachio

et

al.

first used silk fibroin membranes as an enzyme carrier in 1978 to immobilize β2 glucase. After immobilization, the enzyme activity was significantly enhanced, and its stability to heat, electrodialysis and enzyme treatment increased. When the thickness of the silk fibroin membranes increased, their affinity to the enzyme decreased and the enzyme activity could only be restored by half. Kuzuhara and Asakuraalso immobilized glucose oxidase on silk fibroin membranes using the same procedure as above, and used silk fibroin membranes treated with methanol and glutaraldehyde as controls. The results showed that the silk fibroin membranes treated with 80% methanol had stronger enzymatic activity than the silk fibroin membranes treated with glutaraldehyde, and the enzyme activity could be maintained by more than 98%; and the thermostability and pH stability of the enzyme were both better than those in free state. The infrared spectra of the silk fibroin membranes showed that the structure of the silk fibroin membranes changed after glucose oxidase (GOD) was fixed. The structure of the silk fibroin membranes before methanol treatment contained 80% random coils and 20% anti-parallel β-sheet structures, and these two structures were in and on the surface of the silk fibroin membranes, respectively. According to nuclear magnetic resonance and Fourier infrared spectroscopy, the silk fibroin membranes had a non-uniform structure, which could prevent the dissolution of enzymes. This structure allowed the silk fibroin membranes to maintain enzyme activity for more than one month, and the enzyme properties were stable at 40 ℃. Moreover, due to the higher diffusibility of glucose in the silk fibroin membranes, the enzyme also had a higher activity recovery rate.Asakura Tetsuo

et

al.

studied the interaction between silk fibroin and GOD by 13 C-NMR and ESR methods, and the results showed that the interaction between them was minimal. Demura

et

al.

measured the membrane potential that should be formed between silk fibroin membranes and immobilized enzymes, and the results showed that the response range of the membrane potential depended on the concentration of glucose. It proves that silk fibroin is an excellent biological material for immobilized enzymes. Some scholars used other enzymes such as: POD and lipase, pectinase, uricase, penicillin acylase to make enzyme-immobilized membranes. These enzyme-immobilized membranes have a variety of high-level enzyme activities, and their storage time is longer. They can be stored for more than two years without enzyme inactivation, so that the shortcomings of enzyme membranes that are difficult to preserve can be overcome. Chen Jianyong

et

al.

once studied that silk fibroin membranes had the special property of amphoteric charge, and its safety was better than synthetic polymer membranes. In a membrane-solution system, silk fibroin membranes had the characteristic of being negative when the pH value was greater than their isoelectric point and being positive when the pH was less than their isoelectric point. Therefore, the charging characteristics of silk fibroin membranes can be used to control the penetration rate of certain concentrations of ions and ionizable drugs.

4.2.3

Application of silk fibroin membranes in biosensors. Biosensors are a system of two biochemical transducers and a physical transducer. The most researched and applied biosensors are enzyme sensors, which detect the physical and chemical quantities produced by the outside world and convert them into electrical signals. The concept of using enzymes as binding agents to electrodes was proposed by Clark and Lyons. The immobilization of biologically active enzymes is a necessary step in the preparation of almost all types of biosensors. When enzyme-immobilized silk fibroin membranes are used on electrodes to make sensors, the enzyme activity and the electrode repetition rate are significantly improved. Demura and Asakura

et

al.

first applied glucose oxidase-immobilized silk fibroin membrane biosensors to an analysis system in 1989. When the linear response range was 0-5 mmol/L, as the amount of glucose oxidase in the silk fibroin membranes increased, the linear response range of the membranes to substrate glucose decreased. Zhang Yuqing

et

al.

developed a flow injection analysis current-type glucose biosensor based on glucose oxidase-immobilized silk fibroin membranes and oxygen electrodes. It had relatively stable performance, and a wide linear response range of glucose (0.5-15.0 mmol/L), with correlation coefficient

r

=0.999, and it could measure glucose repeatedly for more than 1 000 times. Furthermore, Zhang Yuqing

et

al.

replaced the glucose oxidase-immobilized silk fibroin membranes with uricase-immobilized silk fibroin membranes to make a uric acid sensor, which not only had the same physical properties as the immobilized glucose silk fibroin membranes, but also had good reproducibility. It could divide more than 60 serum samples per hour, and a piece of enzyme membrane could repeatedly measure 1 000 to 2 000 times. Furthermore, correlation analysis with the enzymatic method commonly used in hospitals and practical research were also carried out. The measurement principle of non-labeled immunosensors is the immunochemical reaction between detected substances and antibodies that are fixed on the surface of fixing membranes and are not inactivated. The immunochemical reaction causes changes in membrane charge status, and then changes in transmembrane potential can be determined. Peng Tuzhi

et

al.

immobilized alpha-fetoprotein (AFP) on silk fibroin membranes by hydrochloric acid activation-chemical cross-linking method after comparison. The membranes prepared by this method were fixed on self-made silver chloride electrodes, which could measure the membrane potential of silk fibroin antibodies, and the membrane potential of sample serum membranes and the membrane potential of negative serum membranes had a logarithmic linear positive correlation with the concentration of AFP, indicating that it is a kind of good non-calibrated immunosensor.

4.2.4

Silk fibroin membranes as a controlled release material. A variety of wound coverings for burns have been studied at home and abroad, including biological and synthetic dressings. They can relieve pain, reduce the loss of body fluids and protein, and prevent bacteria from invading wounds. Zhang Youzhu

et

al.

added a pore-forming agent to silk fibroin membranes, and they used the embedding method or covalent method to dissolve a broad-spectrum antibacterial drug with table drug activity, good compatibility with silk fibroin and low bactericidal concentration with a silk fibroin solution, which was then added into a mold to form a drug silk fibroin membrane by evaporating water at a certain temperature. The drug membranes are more flexible than silk fibroin membranes, and have better conformability, strength and elongation, and water permeability when covering the human body surface. In terms of biological properties, they have strong bactericidal properties and good adhesion with the body surface, and meet the requirements for wound coverings. Moreover, the drug membranes have no toxicity, no irritation to the skin and a cytotoxicity of class 1, and are suitable for the protection and treatment of skin injury wounds such as deep second-degree burns and skin injury wounds such as plastic-surgery skin removal areas, so they have outstanding advantages in clinical trials. Wu Zhengyu

et

al.

used silk fibroin membranes to make "artificial skin" wound protective membranes, which were compared with fresh pig skin by doing a comparative test on rabbits. The results showed that the performance of silk fibroin membranes was even better than that of pig skin. In clinical trials of deep second-degree wounds and shallow second-degree wounds, they had good moisture permeability and adhesion to the wounds, and promoted wound healing.

4.2.5

Silk fibroin blend membranes. Because silk fibroin membranes are still weaker in toughness than polymer material membranes, many scholars use polymer materials and silk fibroin to make blended membranes, so as to improve the water absorption and mechanical properties of silk fibroin membranes. Polyvinyl alcohol-silk fibroin membranes and blended fibers and silk fibroin-cellulose blend membranes have been reported, as well as silk fibroin-sodium alginate, silk fibroin-polyacrylamide, chitosan-silk fibroin membranesand other blended membranes. Parkand Chenrespectively studied the properties of chitosan-silk fibroin blend membranes. The results showed that in the chitosan-silk fibroin blend membranes, there was a strong hydrogen bond interaction between silk fibroin and chitosan, and the crystallinity and density increased, and they were compatible. The internal structure of silk fibroin changed from random coil to β-sheet structure, and when the content of chitosan was 40%, its breaking strength was about 7 times that of pure silk fibroin membranes. When the content of chitosan was 55%, the water absorption of the membranes was 3 times that of pure silk fibroin membranes. When the content of silk fibroin in the chitosan-silk fibroin blend membranes was less than 40%, the separation ability to ethanol-water blend systems was greater than that of pure chitosan. Super-absorbent natural polymer membranes are excellent materials for making artificial skin. Membranes required under different conditions can be obtained by adjusting the ratio of components in blended membrane systems. Research in this field has aroused the interest of many scholars.

4.2.6

Chemically-modified silk fibroin membranes. Pure silk fibroin has a weak regulatory effect on cell growth, so many scholars are working on improving the surface properties of silk fibroin membranes through chemical modification and adding other polymers to improve or control the adhesion and proliferation of silk fibroin membranes to cells.Yang Xin

et

al.

discussed the growth of human vascular endothelial cells (HUVEC) on the surface of silk fibroin membranes after plasma treatment. The results showed that after SOand NHplasma treatment, the surface of silk fibroin membranes was sulfonated and aminated respectively, and both could promote the growth of HUVE cells, and had no obvious effects on cell morphology and the function of producing coagulation factor VIII in cells. It is believed that silk fibroin is a good culture substrate for endothelial cells.Gotoh Yohko

et

al.

made polyvinyl alcohol-silk fibroin membranes (PEG2-SF), which were detected to have increased surface hydrophilicity compared with pure silk fibroin membranes. When L-929 cells were cultured with PEG2-SF, it showed a very low cell attachment rate and proliferation rate for L-929 cells, which were much smaller than pure silk fibroin membranes, and no filopodia of cells were observed. It was believed that the reason might be due to the increase in surface hydrophilicity, and it might also be related to the inhibitory effect of PEG itself. Therefore, Gotoh Yohko

et

al.

believe that PEG2-SF is expected to be used to regulate cell attachment and growth.

4.2.7

Silk fibroin cell culture substrate. Silk fibroin has been used as surgical suture for a long time because of its good affinity with the human body. From the amino acid sequence, silk fibroin does not have the Arg-Gly-Asp chain that can promote cell attachment, so the attachment of cells to silk fibroin cannot be achieved through the unique biochemical effects of the carrier. Since arginine (Arg) contained on silk fibroin is positively charged, when cells indicate negatively charged, they are much easier to attach to substrates with basic groups than to substrates with acidic groups, so the attachment of cells to silk fibroin membranes may be the result of electrostatic interaction between the cells and the basic groups on silk fibroin molecular chains. Minoura Norihiko

et

al.

believe that silk fibroin membranes can be used as a cell culture substrate.

5 Prospects

At present, people’s research and utilization of silk fibroin is still very limited. For example, in tissue engineering, the research on silk fibroin as a support material has just started, and its stability needs further research; in medicine, the use of silk fibroin to make artificial organs and artificial tissues is only the initial stage; and even in cosmetics for which a large number of products are being sold, some mechanisms of action are not very clear. Therefore, the research on the application and development of silk fibroin protein still has a long way to go.

The amino acid sequence of the silk fibroin of silkworms has been determined. If we can start with molecular biology and genetic engineering and cultivate varieties that are more suitable for people’s needs, it will bring a historic revolution to the sericulture industry. Meanwhile, the amino acid composition of silk fibroin is special. Although the amino acid composition of different species of silkworms is quite different, the mass fractions of the four amino acids of glycine, serine, tyrosine and alanine are all as high as about 85%. These amino acids have their own unique physiological functions. For example, glycine has the effect of reducing the concentration of cholesterol in the blood; both serine and glycine have the effect of detoxification and liver protection; alanine has the function of anti-inebriation; and tyrosine has the effect of preventing dementia,

etc.

Therefore, its degradation into silk fibroin peptides or silk fibroin amino acids also has great research value. Researchers obtained a silk fibroin peptide with a blood pressure lowering effect through enzymatic hydrolysis, separation and purification. The enzymatic hydrolysis of silk fibroin by different enzymes will produce different enzymatic hydrolysis products, which greatly increases the possibility that silk fibroin peptides have multiple biological activity after enzymatic hydrolysis.The application of biological technologies in the field of silk, using the structural regions and active sites of silk fibroin, and the electrical property of the amphiphilic dielectric of silk fibroin membranes, makes silk fibroin membranes not only widely used in the medical field, but also in the chemical industry. There is an increasing number of polymer materials made of silk fibroin. Li Mingzhong

et

al.

prepared silk fibroin membranes having a lower surface with a dense structure, an upper surface with a porous structure of a small porosity, and an interior porous structure of a large porosity. For example, contact lenses are relatively good in light and oxygen permeability. Such glasses can be embedded with drugs to achieve the effect of sterilization, eliminating the trouble of frequent immersion. In terms of food, the cling film made of silk fibroin and regenerated fiber has the characteristics of high strength and resistance to stretching. It can maintain the proper humidity of fruits and vegetables without causing excessive moisture, so that the fruits and vegetables can reach the best state of neither decay nor deterioration. The practical prospect of silk fibroin is very broad.