Hypromellose-induced conformational transition of silk fibroin

Abstract: A blend film of hydroxypropyl methylcellulose (HPMC) and silk fibroin (SF) was prepared, the structure of the blend film was characterized by FTIR, XRD and DSC methods, and the conformation of HPMC to SF was discussed The results showed that HPMC can effectively induce the conformational transition of SF, and the ratio of HPMC is an important factor affecting the degree of conformational transition of SF. When mixed with 3%-10% HPMC, the conformation of SF changed from random coil or Silk I to Silk II (β-sheet), and when 7% HPMC was added, the proportion of β-sheet conformation was the largest. From the infrared analysis, it can be seen that the conformational transition is caused by the intermolecular hydrogen bond between the mixture of appropriate amount of HPMC and SF. The dissolution rate in water was measured for different proportions of blended membranes, and the results showed that SF was almost insoluble in water when the proportion of HPMC was 7%.

Key words:silk fibroin, conformational transition, hydroxypropyl methylcellulose

Silk fibroin (Bombyx mori) is a natural polymer fibrin extracted from silk, which endows silk with excellent mechanical properties such as strength, elasticity, and flexibility. Due to the good biocompatibility, air permeability and biodegradability of SF in wet state, this polymer has been applied in biotechnology and biomaterials in recent years in addition to traditional textile applications. Typical applications include Enzyme immobilization materials, functional cell culture substrates and drug sustained release carriers, etc. Now people are studying the application of SF in cardiovascular, artificial skin and so on.

In the application of the above-mentioned biomaterials, SF often needs to be made into thin films, porous membranes, powders or gels, so the dissolution of SF is essential. It is generally believed that there are three conformations in SF, Silk I, Silk II (β-sheet) and random coils. The film prepared from SF aqueous solution is mainly in random coil conformation, by changing the film forming conditions such as film forming temperature, initial concentration and drying rate, the conformation of SF can be changed from random coil to β-sheet, this β- The folding transformation can significantly improve the mechanical properties and thermal stability of silk fibroin films.

At present, some literatures have reported that the β-sheet conformation of SF can be increased by mixing natural or synthetic polymers, such as sodium alginate, chitosan, polyvinyl alcohol. Noishiki et al. reported that mixing microcrystalline cellulose can effectively improve the mechanical properties of SF. The physical properties and structure of cellulose/SF blend membranes have been studied. They dissolved SF and cellulose in copper ammonium solution, and then in acetone. Coagulation in the acetic acid bath, and then washed with water, the preparation of the blend film is more complicated.

In order to find a simple and effective way to induce the conformational transition of SF, we noticed a cellulose derivative-hydroxypropylmethylcellulose (HPMC). This polymer has been widely used in the fields of food and medicine, and is soluble in water, which makes the preparation of the blend film simple. In addition, HPMC contains a large number of hydroxyl groups that are easy to form hydrogen bonds, and intermolecular hydrogen may be formed between SF and HPMC key.

This paper reports the conformational transition of SF induced by HPMC. The crystallization transition of the blend film was characterized by FFIR, DSC, XRD and dissolution rate in water, and the effect of the blend ratio on the conformation transition of SF was discussed.

1. Experiment

1.1 Preparation of membrane

Bombyx mori silk was boiled in 0.5% (W) Na2CO3 solution for 1 h to remove sericin, and SF was dissolved in CaCl2/H20/C2H5OH=1:8:2 (molar ratio) solvent at 80°C and used Distilled water was dialyzed for 3 days, filtered to obtain SF aqueous solution, and its concentration was determined by uV-VIS ultraviolet spectrophotometer.

Hydroxypropyl methylcellulose (60RT50) was provided by KIMA CHEMICAL CO.,LTD.

The sF solution (64 g/L) and the HPMC solution (35 g/L) were evenly mixed according to the mass ratio of 10/0, 9.7/0.3, 9.5/0.5, 9.3/0.7, 9.0/1.0, 0/10, and allowed to stand After defoaming, pour it into a polystyrene petri dish and dry it at 50°C to form a film. The serial numbers of the films are a, b, e, d, e, f.

1.2 Structural characterization of the membrane

The infrared spectrum of the film was recorded with a Fourier transform infrared spectrometer of Perkin Company, and the measurement range was 4000-1000 cm-1; (γ=1.54 nm), tube voltage 30 kV, tube current 20 mA, scanning speed 0.5(°)/min, scanning range 5-40°; Elmer—DSC. 7-type thermal analyzer measurement, heating rate 10K/min, nitrogen flow rate 50mL/min.

1.3 Hot water solubility of the membrane

Accurately weigh 0.2 g (W1) of the film, put it in an oven at 105°C, dry and weigh (W2), and calculate the moisture content (y). Take 0.5 g (W3) sample from the same film, put it into distilled water, wash at (37±1)°C constant temperature shaking for 72 h, then weigh it after drying at 105°C (W4), calculate

Dissolution rate of film (s):

V=(W1-W2)/W3

s=[W3(1-y)-W4]／W3(1-y)

2. Results and Discussion

2.1 Infrared Spectral Analysis

In the infrared spectrum of HPMC film, the peak at 3448 cm-1 is attributed to the stretching vibration of O—H. After blending, a broad absorption peak appears at 3450-3288 cm-1, which is wider than the absorption peaks at N—H of sF and O—H of HPMC, and with the decrease of the proportion of HPMC, the peak goes down The wave number shifts, but the broad peak of sample e (10% HPMC) has no obvious maximum, but a simple superposition of the absorption peaks of sF and HPMC. The above phenomenon shows that when the ratio of HPMC is 3% to 7%, since HPMC contains a large number of hydroxyl groups that are easy to form hydrogen bonds, intermolecular hydrogen bonds are formed between the O—H of HPMC and the N—H of sF, and when the ratio of HPMC is At 10%, the intermolecular force between the two is relatively weakened, this is because HPMC contains a large number of hydroxyl groups, and excess HPMC tends to form intramolecular hydrogen bonds of HPMC, so that the intermolecular hydrogen bond force of SF and HPMC Weaken.

Amide I corresponds to the C—O stretching vibration of amino acid residues. sF has three conformations: random coil, Silk I and Silk II (β-sheet), and the characteristic absorption ranges corresponding to random coil or Silk I and Silk II (β-sheet) are respectively in the range of 1640 to 1660 cm-1 and 1620～1640 cm-1. It can be seen that pure SF has a symmetrical absorption peak at 1641 cm-1, which belongs to random coils or Silk I. When 3%-10% HPMC is mixed in, a broad amide I characteristic absorption peak appears at 1699-1626 cm-1, it can be concluded that the blend film contains β-sheet conformation, and Silk I may also exist, which shows that the addition of HPMC makes The conformation of sF has changed. It is worth noting that the absorption peak at 1699 cm-1 is gradually enhanced when 3% to 7% HPMC is added, and when the amount of HPMC added increases to 10%, the peak intensity weakens, that is, when 7% HPMC is mixed, the absorption peak at 1699 cm The peak intensity at -1 is the largest, and the absorption peak at 1699 cm-1 belongs to the conformation of SilkⅡ (β-sheet), which indicates that the conformational transition of β-sheet is the largest when 7% HPMC is mixed.

Amide II is caused by the deformation vibration of the N—H bond. The absorption peak in the range of 1540~1530 cm-1 belongs to the random coil conformation or Silk I, and the absorption peak at 1522 cm-1 belongs to the β-sheet conformation. The pure sF film has a strong absorption peak at 1514 cm-1 and a weak absorption peak at 1530 cm-1, while the blend films with different proportions have two absorption peaks at 1520 cm-1 and 1541 cm-1, and this The two absorption peaks are superimposed together, indicating that SF has a β-sheet conformation when 3%-10% HPMC is added, which may contain Silk I.

Amide III is the joint action of O—C—N and N—H vibrations, the absorption peak is in the range of 1230—1260 cm-1, and the absorption peak at 1235 cm-1 is random coil or Silk I, 1260 cm-1 The absorption peaks at 1240cm-1 belong to the β-sheet conformation. The pure sF film has an absorption peak at 1234 cm-1, which is a random coil or Silk I. After adding 3% to 10% HPMC, the absorption peak is obviously broadened, ranging from 1230 to 1260 cm-1, and the 3% to 7% HPMC blend film has a maximum value at 1240 cm-1, and at 1260 cm-1 The intensity of the absorption peak increases with the increase of the amount of HPMC added, but the maximum absorption peak of the 10% HPMC blend film is at 1235 cm-1, and the absorption peak at 1260 cm-1 is higher than that of the 7% HPMC blend film at The intensity of the absorption peak here is weak. This further indicated that the addition of HPMC made sF appear in the β-sheet conformation, and it may also contain Silk I. The conformation of sF changed, and when 7% HPMC was mixed, the proportion of β-sheet conformation was the largest.

The above analysis of amide I, amide II and amide III showed that: HPMC can induce the conformational transition of sF. Due to the formation of intermolecular hydrogen bonds between sF and HPMC, the molecular rearrangement of sF results in a change from disordered state to ordered arrangement, so that the conformation of sF changes from random coil or Silk I to β-sheet conformation. Similar conformational transitions also occurred in the blends of sodium alginate/SF, chitosan/SF and cellulose/SF. However, when the blending ratio of HPMC increases to 10%, the intermolecular hydrogen bond between HPMC and sF is weak due to the excess HPMC itself forming intramolecular hydrogen bonds, thus forcing the sF molecules to undergo orderly rearrangement. The degree is greatly reduced.

2.2 X-diffraction analysis

From the x-ray diffraction patterns of SF, HPMC and their blend films, it can be seen that the diffraction pattern of pure sF film is a broad amorphous diffraction peak, indicating that the conformation of pure sF film is mainly amorphous, that is, random coils. Weak α and β diffraction peaks appear at 11.0° and 19.3° of HPMC film, respectively. However, the diffraction pattern of the two polymers after blending has changed significantly. When the proportion of HPMC is 3% to 10% (weight ratio), 20 has a strong diffraction peak at 20.0°, and at 11.8°, Two weaker diffraction peaks appear at 24.4°, among which the diffraction peaks at 20° and 24.4° are the characteristic peaks of Silk II (β-sheet) of sF, while 11.8° represents Silk IIII, so It shows that there are Silk I and Silk II in the blend film added with HPMC, and the addition of HPMC causes the conformational transformation of sF. It can be clearly seen from the diffractograms of the blend films at various ratios that the diffraction peak at 20=11.8° appears the minimum when the ratio of HPMC is 7%, which indicates that the β-sheet conformation changes most at this ratio. The above results indicated that adding HPMC made sF appear β-sheet conformation, and the proportion of β-sheet conformation was the highest when 7% HPMC was mixed.

2.3 DSC analysis

In the DSC curve of Figure 4, the pure SF film has an exothermic peak at 224.4C. This peak is the crystallization peak, and the corresponding temperature is the crystallization temperature (T.), which is caused by the temperature rise from the SF conformation to random coil or SilkI Transformed into the Silk II (β-fold conformation) crystal conformation, an endothermic peak appeared at 283.4°C, which was caused by the thermal degradation of SF, and the corresponding temperature was the melting point (Tm). Pure SF film appeared an exothermic peak at 224°C The thermal peak shows that the conformation of pure SF film is mainly random coil or Silk I. After adding 3%~7% HPMC, the exothermic peak at 224°C disappears, which means that the conformation of SF in the blend film is in Silk II That is, the crystalline state is significantly increased. However, when the amount of HPMC added is 10%, the exothermic peak at 224C reappears, indicating that the content of SilkI in the conformation of SF is less, and the intensity of the peak at this position is slightly larger than that of pure SF samples. It may be related to the difference in the amount of samples used in the test. The blended films of 3%~10% HPMC all have endothermic peaks around 283.0°C, and the 7% HPMC blended films have a sharp endothermic peak at 286.3°C , the degree of orientation of SF will affect the melting point temperature to a certain extent, the greater the degree of orientation, the higher the Tm, so it can be explained that the degree of orientation of the 7% HPMC blend film is the largest in each blend ratio, that is, the ordered conformation formed by SF (ilk II) is the most. The above analysis shows that when HPMC is 3%~10%, HPMC can induce the conformational transition of SF. When mixed with 7% HPMC, the proportion of SilkI is the largest. The above results are consistent with the conclusions of FTIR and XRD.

2.4 Hot water dissolution rate

Since the random coils of sF and Silk I are soluble in water, but the β-sheet conformation is insoluble, the crystallinity of the blend film can be judged by measuring the weight loss in water. The weight loss of the blend film decreased with the increase of the HPMC ratio. When the HPMC content was 7%, the blend film was almost insoluble in water, but when the HPMC ratio increased further, the dissolution rate of the blend film increased sharply. This shows that when the amount of HPMC added is 3% to 7%, HPMC effectively induces the conformational transition of sF, and the crystallinity increases with the increase of the proportion of HPMC, but when the proportion of HPMC increases to 10%, due to the The content of converted β-sheet conformation in the sample is less, so its weight loss increases significantly.

Based on the above results, HPMC can effectively induce the conformational transition of sF, and the mixing ratio of HPMC is an important factor affecting the conformational transition. When HPMC is 3% to 7%, the conformation of sF appears β-fold, and the ratio of Lu-fold conformation increases with the increase of HPMC. When HPMC is 7%, the conformational transition is the most complete, which promotes the reason for the conformational transition It is the hydrogen bond formed between HPMC and SF molecules, which makes the SF molecules have to adjust their conformation, thus causing its conformational transformation. However, when the mixing ratio of HPMC increased to 10%, the induction effect of HPMC on the conformational transition of sF was small, because the excess HPMC itself formed intramolecular hydrogen bonds, which weakened the intermolecular hydrogen bond force between sF and HPMC. When HPMC is 7%, the dissolution rate of hot water is the smallest, almost insoluble in water, and because of the small amount of HPMC mixed in, the properties of the blend film mainly reflect the properties of sF, which provides the possibility for the application of sF in artificial blood vessels ．