An electro-responsive silk fibroin material

Abstract

The application discloses an electric response type silk fibroin material, which contains silk fibroin and a thiol group grafted on the silk fibroin; the content of the thiol group is 20-100 mu mol / g. The electric response type silk fibroin material can realize the change of swelling degree under low-voltage stimulation, has high swelling characteristics under the condition of power-on, has low swelling characteristics under the condition of power-off, and realizes the controllable release of drugs.

Description

[0001] This application is a divisional application of the invention patent application with the application date of March 16, 2022, the application number of 202210258571X, and the invention name of "an electrically responsive silk fibroin material, its preparation method and electrically responsive silk fibroin microneedle". TECHNICAL FIELD

[0002] The present application relates to the technical field of silk fibroin microneedle patches and medical and medical materials, and specifically relates to a silk fibroin material with increased internal pore size after electrification. BACKGROUND

[0003] The field of intelligent responsive drug delivery is a research hotspot in current materials science and biomedical science. Based on the effects of electric field, electric current, magnetic field, light and mechanical force, changes in pH value and temperature can stimulate the responsive delivery of drugs to the body. However, how to control the drug delivery method to make the intelligent responsive drug delivery convenient to use, effective to deliver and accurately control the drug dosage is a challenge.

[0004] Microneedle transdermal drug delivery is a method that uses a microneedle array with a length of less than 1mm to pierce the skin epidermis layer and form micropores to improve the transdermal drug delivery efficiency. It can easily, painlessly, safely and conveniently achieve drug delivery and is playing an important role in the field of intelligent drug delivery. At the same time, since the microneedle array is attached to the skin during use, it can effectively receive external stimuli, especially electric current, electric field and magnetic field, to achieve controlled release of drugs.

[0005] Silk fibroin is a green natural bio-based material with low immunogenicity, excellent biocompatibility and excellent mechanical properties. With the development of modern science and technology, silk has not only been limited to the field of textiles, but has also been widely used in the field of drug delivery. However, drug-loaded silk fibroin microneedles do not have intelligent responsiveness, which is determined by the structure of silk fibroin. Due to the lack of corresponding stimulus-responsive groups or too few responsive groups on the silk fibroin molecules. Overall, pure silk fibroin does not have stimulus-responsive changes, so it cannot be used for intelligent drug delivery.

[0006] To solve the above problems, Chinese invention patent CN108047466A discloses a preparation method of silk microneedle, which uses glutaraldehyde as a chemical crosslinking agent, and then the silk fibroin solution after chemical crosslinking is made into a microneedle; after the microneedle is made, water vapor treatment is carried out to obtain a high-strength silk fibroin microneedle. This microneedle can easily penetrate the skin and can mix a large amount of drugs. However, this microneedle is treated by water vapor, and the internal structure is mainly β-sheet, which is not easy to swell by water absorption, thereby causing slow drug release, low drug release rate, and lack of responsiveness; at the same time, glutaraldehyde as a crosslinking agent is harmful to the human body and can stimulate the skin and digestive system. For example, Chinese invention patent CN102580232B discloses a silk fibroin microneedle system and a preparation method of silk fibroin nanoparticles. The silk fibroin protein microneedle prepared by this method dissolves rapidly after penetrating the skin, thereby releasing nanoparticles. However, this microneedle system releases the drug into the body at one time, and cannot control the intelligent release and release amount of the drug.

[0007] Electrically assisted drug delivery, which is cheap and easy to perform, can help drugs overcome tissue barriers and enter the body. The drug release mechanism mainly includes electroporation and iontophoresis, etc. However, these methods all use some large current or electric field, which can cause certain safety hazards. It is very meaningful to complete the intelligent responsive delivery of drugs in silk fibroin microneedles under low current or electric field, which can greatly expand the application of intelligent drug delivery in the human body in terms of precision, convenience and safety.

[0008] Gu et al. [Nature Biomedical Engineering, 2020, 4(7): 1-8.] developed a method of glucose-responsive N-vinylpyrrolidone-based microneedle patch, which contains a copolymer designed for glucose-triggered insulin delivery. This microneedle has a large drug loading capacity and an intelligent response effect, and can be used for insulin closed-loop delivery. However, the microneedle substrate is prepared using a polymer material, which has insufficient biological safety and green sustainability. At the same time, the glucose intelligent response speed is slow and cannot respond quickly.

[0009] How to develop a kind of electric response material by acting on micro-needle, realize the quick intelligent response change of material by low voltage electric stimulation, and then control the quick response release of drug, which is very important to complete the electric response intelligent drug release. SUMMARY

[0010] Therefore, in order to overcome the defects of the prior art, the purpose of the present application is to provide an electric response silk fibroin material, which can realize the control of the pore size in the material by using switch electric and achieve the purpose of controlling the drug release speed.

[0011] In order to achieve the above purpose, the present application adopts the following technical scheme:

[0012] A preparation method of an electric response silk fibroin material, comprising the following steps:

[0013] (1) Activation of silk fibroin: dilute the silk fibroin aqueous solution to a concentration of 20-30 mg / mL, then put the solution into an ice bath to stabilize the temperature to 0-4℃, and adjust the pH value of the silk fibroin solution to 5-6 using a buffer solution; add 1-10 wt% of N-hydroxysuccinimide to the above-mentioned silk fibroin solution relative to the mass of silk fibroin, then add 2-20 wt% of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride relative to the mass of silk fibroin, mix uniformly, and react for 0.4-1 h to obtain an activated silk fibroin solution;

[0014] (2) Thiolation of silk fibroin: add a cysteamine hydrochloride solution with a concentration of 40-60 mg / mL dropwise to the above-mentioned activated silk fibroin solution, and make the final concentration of cysteamine hydrochloride in the solution 20-80 mmol / L, then adjust the pH value of the final solution to 5-6 using a buffer solution; stir and react at 0-4℃ for 2-5 h, then take out and react at a temperature of 2-8℃ for 8-10 h to obtain a thiolated silk fibroin solution;

[0015] (3) Purification of thiolated silk fibroin: put the reacted silk fibroin solution into a dialysis bag for dialysis; after dialysis, centrifuge to obtain the supernatant to obtain a purified thiolated silk fibroin solution;

[0016] (4) Preparation of electric response silk fibroin material: take the purified thiolated silk fibroin solution in the above-mentioned step, pour it into a mold, and remove air bubbles in a vacuum drying box; then place the defoamed mold in a constant temperature and humidity environment for drying, and after drying and demolding, an electric response silk fibroin material is obtained. As for the specific shape of the electric response silk fibroin material, a mold can be designed according to actual needs, and then a silk fibroin material with a corresponding shape can be prepared, such as an electric response silk fibroin microneedle patch.

[0017] According to some preferred embodiments of the present application, the electrically responsive silk fibroin material contains silk fibroin and thiol groups grafted onto the silk fibroin; the content of thiol groups is 20-100 μmol / g. If the content of thiol groups is too low, the electric response is small and the swelling degree does not change much. On the other hand, the content of carboxyl groups in silk fibroin molecules is limited, and the control of the above reaction conditions is also to graft thiol groups onto the available carboxyl groups to increase the content of thiol groups, so as to achieve better electric response and control of the swelling degree.

[0018] According to some preferred embodiments of the present application, the silk fibroin material has current response, and the swelling rate in 1 hour without power supply is 50-120%, and the swelling rate after power supply for 1 hour under a voltage of 0.6 V is 120-250%. The redox potential between thiol groups and disulfide bonds is about 0.6 V. The voltage cannot be higher than 1 V to prevent water electrolysis. If the voltage is lower than 0.6 V, the disulfide bonds cannot be reduced. In actual application, the voltage can be set to be between 0.6 V and 0.9 V. The current response of the present application refers to the change of the performance (swelling rate) of the prepared silk fibroin material before and after power supply. After power supply, the swelling rate of the material is obviously improved compared with the swelling rate without power supply.

[0019] According to some preferred embodiments of the present application, the mass ratio of N-hydroxysuccinimide to silk fibroin is 1:100-1:10; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to silk fibroin is 1:50-1:5; and the mass ratio of cysteamine hydrochloride to silk fibroin is 1:100-1:2.5.

[0020] According to some preferred embodiments of the present application, during dialysis, sodium thiosulfate is added to the dialysis deionized water and inert gas is supplied for protection; the deionized water with added sodium thiosulfate is replaced every 2-4 h, and the dialysis procedure is repeated for 2 d and the inert gas protection is maintained; then the dialysis is performed for 1 d using deionized water without added sodium thiosulfate. The inert gas is preferably nitrogen N2.

[0021] According to some preferred embodiments of the present application, the concentration of sodium thiosulfate in the deionized water is 0.001-0.0015 mol / L.

[0022] According to some preferred embodiments of the present application, the dialysis bag used during dialysis has a molecular weight cut-off of 8-14 kDa.

[0023] According to some preferred embodiments of the present application, the constant temperature and humidity conditions are a temperature of 20-30 ℃ and a relative humidity of 55-65%.

[0024] According to some preferred embodiments of the present application, the buffer solution is one selected from 2-(N-morpholino)ethanesulfonic acid, glycine-hydrochloric acid, citric acid-sodium citrate, acetic acid-sodium acetate, potassium hydrogen phthalate-sodium hydroxide, Tris-hydrochloric acid buffer.

[0025] According to some preferred embodiments of the present application, the aqueous silk fibroin solution is prepared from domestic silkworm silk, and is obtained through degumming, dissolving and dialysis.

[0026] The present application also provides an electro-responsive silk fibroin material prepared by the preparation method described above.

[0027] The present application also provides an electro-responsive silk fibroin microneedle, which is prepared by pouring the thiolated silk fibroin described above into a microneedle mold, removing air bubbles in a vacuum drying oven, and then drying the defoamed mold system in a constant temperature and humidity environment, to obtain an electro-responsive silk fibroin microneedle patch after drying and demolding.

[0028] The reaction principle of the present application is as follows: the thiolation modification of silk fibroin is prepared by coupling reaction of silk fibroin and cysteamine hydrochloride using an N-hydroxysuccinimide / 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride system. In the initial stage of the reaction, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) reacts with the carboxyl groups on the aspartic acid and glutamic acid residues in the molecular structure of silk fibroin to form an initiator, unstable urea derivative, and then reacts with N-hydroxysuccinimide (NHS) to form a more stable ester, thereby enhancing the water stability of the carbodiimide crosslinking product, and at the same time, the carboxyl group is in an activated state. After the carboxyl group is activated, the amino group on the cysteamine hydrochloride reacts with the activated carboxyl group to form an amide bond, and the cysteamine is successfully grafted onto the silk fibroin molecule. At the same time, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are converted into water-soluble urea derivatives during the reaction process, which can be removed in the subsequent dialysis process to retain the good biocompatibility of silk fibroin.

[0029] The redox potential of the disulfide bond is relatively low, and the apparent reduction potential is about 0.6 volts, and the reversible redox reaction is easy to occur. After the grafting of the sulfydryl on the silk fibroin, in the presence of oxygen in the air, the sulfydryl is oxidized to form a disulfide bond crosslinking point between the silk fibroin molecular chains. The crosslinking point formed by the disulfide bond is reversible, and will be broken into sulfydryl under the reducing condition, and the crosslinking point between the molecular chains will be broken. In the case of no electricity, the crosslinking degree between the silk fibroin molecular chains is high, so that the silk fibroin microneedle has a small swelling rate; and in the case of electricity, the current provides a reducing environment, the disulfide bond is electrochemically reduced by electrons, the disulfide bond is broken to form sulfydryl, and the covalent crosslinking point between the silk fibroin molecular chains is reduced, so that the silk fibroin microneedle has an increased swelling rate. The change in the swelling degree can be controlled by means of the switching power supply. Therefore, the electroresponsive silk fibroin material is obtained, the change in the swelling degree is controlled by the response to the current, and the release speed of the drug in the microneedle can be controlled.

[0030]

[0031] Due to the adoption of the above technical scheme, the electroresponsive silk fibroin material of the present application has the following advantages compared with the prior art: the electroresponsive silk fibroin material of the present application can realize the change in the swelling degree under low-voltage stimulation, has high swelling characteristics under the condition of electricity, has low swelling characteristics in the case of no electricity, and realizes the controllable release of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0033] Figure 1 A microscope photo of the microneedle patch prepared in the preferred embodiment 2 of the present application;

[0034] Figure 2 A swelling degree change graph of the current-responsive silk fibroin microneedle patch prepared in the embodiments 2-7 of the present application under the conditions of electricity and no electricity;

[0035] Combination Figure 2 As can be seen from the embodiments 2-7, the excessive addition of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride can reduce the current responsiveness and swelling performance of the silk fibroin microneedle patch, and the increase of the addition amount of cysteamine hydrochloride can improve the swelling degree of the microneedle patch.

[0036] Figure 3The scanning electron microscope images of the internal aperture of the current response type fibroin microneedle patch prepared in embodiment 2 of the present application under the conditions of power on and power off; wherein Figure 3 (a-b) are the internal aperture electron microscope images of the microneedle before power on, Figure 3 (c-d) are the internal aperture electron microscope images of the microneedle after power on, it can be seen that the internal aperture of the microneedle is increased after power on, which is beneficial to the passage of the drug;

[0037] Figure 4 The principle diagram of the conversion of the material internal sulfhydryl and disulfide bond of the current response type fibroin microneedle before and after power on in the embodiment of the present application. DETAILED DESCRIPTION

[0038] In order to enable the personnel in the technical field to better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the protection scope of the present application.

[0039] Embodiment 1 preparation of fibroin aqueous solution

[0040] Weigh 3g of NaHCO3 and 1g of Na2CO3 and dissolve them in 4000mL of deionized water, heat to boiling, put in 80g of silk, keep 98℃ micro boiling for 30min, take out and wash with deionized water. Repeat the above steps three times and place in a 60℃ oven to dry to obtain degummed silk fibroin.

[0041] Prepare a 9.3mol / L LiBr solution, take 100mL of LiBr solution and heat to 65℃ in a water bath, put in 15g of degummed silk in multiple times, stir to dissolve, continue to heat and stir for 40min. The liquid is loaded into a dialysis bag and placed in deionized water for dialysis for 72h, and the deionized water is replaced every 2h. After dialysis, the solution is filtered to obtain a fibroin aqueous solution.

[0042] Embodiment 2

[0043] The preparation method of the electric response type fibroin microneedle in this embodiment specifically includes the following steps:

[0044] 1) Preparation of thiolated fibroin solution

[0045] The concentration of the silk fibroin aqueous solution was diluted to 30 mg / mL, and then the solution was stabilized in an ice bath at 2°C. The pH of the silk fibroin solution was adjusted to 5.5 using a 2-(N-morpholino)ethanesulfonic acid solution. First, 5 wt% (relative to the mass of the silk fibroin) N-hydroxysuccinimide was slowly added to the above-mentioned silk fibroin solution, followed by the addition of 10 wt% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing well, the reaction was carried out for 0.5 h to activate the carboxyl groups on the silk fibroin.

[0046] A 50 mg / mL cysteamine hydrochloride solution was slowly added dropwise to the above-mentioned silk fibroin solution, so that the final solution had a cysteamine hydrochloride concentration of 60 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted and stabilized to 5.5 using a 2-(N-morpholino)ethanesulfonic acid solution. The reaction was stirred in an ice bath for 4 h, and then the solution was removed and allowed to stand in a 4°C refrigerator overnight.

[0047] 2) Dialysis of the thiolated silk fibroin solution

[0048] The reaction-completed silk fibroin solution was placed in a dialysis bag (with a molecular weight cut-off of 8-14 kDa) for dialysis. The dialysis environment was deionized water to which a small amount of sodium thiosulfate (0.001 mol / L) was added, and the dialysis was protected by the introduction of inert gas N2. The deionized water containing sodium thiosulfate was replaced every 4 h, and the dialysis procedure was repeated for 2 d. After that, the dialysis was performed using deionized water without the addition of sodium thiosulfate for 1 d. After dialysis, the supernatant was obtained by centrifugation to obtain the grafted and modified thiolated silk solution. The mass concentration (wt%) of the silk fibroin solution was then measured, and the solution was stored in a 4°C refrigerator for later use. The thiol content in the modified silk fibroin solution was 92.9 ± 5.7 μmol / g.

[0049] 3) Preparation of silk fibroin microneedles

[0050] 1 mL of the thiolated silk fibroin solution was cast into a single-piece PDMS (dimethylsiloxane) microneedle mold, and vacuum was applied in a vacuum drying oven to remove air bubbles, which was repeated three times. The degassed mold system was then placed in a constant temperature and humidity chamber (25°C, 55% RH) and dried for 8 h under air flow. The silk fibroin microneedles were obtained after demolding. The swelling rate of the microneedles was 72 ± 3.63% without electricity for 1 h, and the swelling rate was 227 ± 13.86% with a voltage of 0.6 V for 1 h.

[0051] Example 3

[0052] The preparation method of the electroresponsive silk fibroin microneedles in this example specifically includes the following steps:

[0053] 1) Preparation of a thiolated silk fibroin solution

[0054] The concentration of the silk fibroin aqueous solution was diluted to 20 mg / mL, and then the solution beaker was stabilized at 3°C ​​in an ice bath. The pH of the silk fibroin solution was adjusted to 5 using 2-(N-morpholino)ethanesulfonic acid solution. First, 5 wt% (relative to the weight of silk fibroin) of N-hydroxysuccinimide was slowly added to the above silk fibroin solution, followed by 8 wt% of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing thoroughly, the mixture was reacted for 0.6 h to activate the carboxyl groups on the silk fibroin.

[0055] Cysteine ​​hydrochloride solution (60 mg / mL) was slowly added dropwise to the silk fibroin solution to achieve a final concentration of 40 mmol / L for cysteine ​​hydrochloride and 20 mg / mL for silk fibroin. The pH of the final solution was then adjusted to 5 using 2-(N-morpholino)ethanesulfonic acid solution. The reaction was stirred in an ice bath for 4 hours, and then allowed to stand overnight at 4°C.

[0056] 2) Dialysis of thiolized silk fibroin solution

[0057] The reacted silk fibroin solution was placed in a dialysis bag (molecular weight cutoff 8-14 kDa) for dialysis. The dialysis environment consisted of deionized water with a small amount of sodium thiosulfate (0.0015 mol / L) and protected by an inert gas N2 atmosphere. The deionized water with added sodium thiosulfate was replaced every 4 hours, and the dialysis procedure was repeated for 2 days, followed by dialysis with deionized water without added sodium thiosulfate for 1 day. After dialysis, the supernatant was collected by centrifugation to obtain the grafted and modified thiolized silk fibroin solution. The mass concentration (wt%) of the silk fibroin solution was then measured and stored at 4°C for later use. The thiol content in the modified silk fibroin solution was 23.3 ± 2.2 μmol / g.

[0058] 3) Preparation of silk fibroin microneedles

[0059] Take 1 mL of the thiolized silk fibroin solution prepared in (2) and pour it into a single PDMS microneedle mold. Remove air bubbles by vacuuming in a vacuum drying oven, repeating this process three times. Then, place the degassed mold system in a constant temperature and humidity chamber (25℃, 55%RH) and dry it under air circulation for 6 hours. After demolding, silk fibroin microneedles are obtained. The swelling rate of the microneedles after 1 hour without power is 84±5.76%, and the swelling rate after 1 hour with a voltage of 0.6V is 163±7.81%.

[0060] Example 4

[0061] The preparation method of the electroresponsive silk fibroin microneedles in this embodiment specifically includes the following steps:

[0062] 1) Preparation of thiolized silk fibroin solution

[0063] The concentration of the aqueous solution of silk fibroin was diluted to 20 mg / mL, and then the solution was stabilized in an ice bath at 2°C. The pH of the silk fibroin solution was adjusted to 5.5 using a 2-(N-morpholino)ethanesulfonic acid solution. First, 2.5 wt% (relative to the mass of silk fibroin) N-hydroxysuccinimide was slowly added to the above-mentioned silk fibroin solution, followed by the addition of 4 wt% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing, the reaction was carried out for 0.8 h to activate the carboxyl groups on the silk fibroin.

[0064] A 55 mg / mL cysteamine hydrochloride solution was slowly added dropwise to the above-mentioned silk fibroin solution, so that the final solution had a cysteamine hydrochloride concentration of 80 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted and stabilized to 5.5 using a 2-(N-morpholino)ethanesulfonic acid solution. The reaction was stirred in an ice bath for 4 h, and then the solution was removed and allowed to stand in a 4°C refrigerator overnight.

[0065] 2) Dialysis of the thiolated silk fibroin solution

[0066] The reaction-completed silk fibroin solution was placed in a dialysis bag (with a molecular weight cut-off of 8-14 kDa) for dialysis. The dialysis environment was deionized water to which a small amount of sodium thiosulfate (0.0015 mol / L) was added, and the system was protected by the introduction of inert gas N2. The deionized water containing sodium thiosulfate was replaced every 4 h, and the dialysis procedure was repeated for 2 d. After that, the dialysis was performed using deionized water without the addition of sodium thiosulfate for 1 d. After dialysis, the supernatant was obtained by centrifugation to obtain the grafted and modified thiolated silk solution. The mass concentration (wt%) of the silk fibroin solution was then measured, and the solution was stored in a 4°C refrigerator for later use. The thiol content in the modified silk fibroin solution was 48.3 ± 2.9 μmol / g.

[0067] 3) Preparation of silk fibroin microneedles

[0068] 1 mL of the thiolated silk fibroin solution prepared in (2) was cast into a single-piece PDMS microneedle mold, and vacuum was applied in a vacuum drying oven to remove air bubbles. This process was repeated three times. The degassed mold system was then placed in a constant temperature and humidity chamber (25°C, 55% RH) and dried for 6 h in an air flow. The silk fibroin microneedles were obtained after demolding. The swelling rate of the microneedles was 105 ± 5.53% in 1 h without electricity, and the swelling rate was 182 ± 8.32% in 1 h under a voltage of 0.6 V.

[0069] Example 5

[0070] The preparation method of the electroresponsive silk fibroin microneedles in this example specifically includes the following steps:

[0071] 1) Preparation of a thiolated silk fibroin solution

[0072] The concentration of the aqueous solution of silk fibroin was diluted to 25 mg / mL, and then the solution was stabilized in an ice bath at 2°C. The pH of the silk fibroin solution was adjusted to 6 using a 2-(N-morpholino)ethanesulfonic acid solution. First, 5 wt% (relative to the mass of silk fibroin) N-hydroxysuccinimide was slowly added to the above-mentioned silk fibroin solution, followed by the addition of 7.5 wt% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing, the reaction was carried out for 0.5 h to activate the carboxyl groups on the silk fibroin.

[0073] A 60 mg / mL solution of cysteamine hydrochloride was slowly added dropwise to the above-mentioned silk fibroin solution, so that the final solution had a cysteamine hydrochloride concentration of 20 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted to 6 using a 2-(N-morpholino)ethanesulfonic acid solution. The reaction was stirred in an ice bath for 4 h, and then the solution was left to stand in a 4°C refrigerator overnight.

[0074] 2) Dialysis of the thiolated silk fibroin solution

[0075] The reaction-completed silk fibroin solution was placed in a dialysis bag (with a molecular weight cut-off of 8-14 kDa) for dialysis. The dialysis environment was deionized water to which a small amount of sodium thiosulfate (0.001 mol / L) was added, and the dialysis was protected by the introduction of inert gas N2. The deionized water containing sodium thiosulfate was replaced every 4 h, and the dialysis procedure was repeated for 2 d. After that, the dialysis was performed using deionized water without the addition of sodium thiosulfate for 1 d. After dialysis, the supernatant was obtained by centrifugation to obtain the grafted and modified thiolated silk solution. The mass concentration (wt%) of the silk fibroin solution was then measured, and the solution was stored in a 4°C refrigerator for later use. The thiol content in the modified silk fibroin solution was 41.2 ± 3.1 μmol / g.

[0076] 3) Preparation of silk fibroin microneedles

[0077] 1 mL of the thiolated silk fibroin solution prepared in (2) was cast into a single-piece PDMS microneedle mold, and vacuum was applied in a vacuum drying oven to remove air bubbles, which was repeated three times. The degassed mold system was then placed in a constant temperature and humidity chamber (25°C, 55% RH) and dried for 6 h in an air flow. The silk fibroin microneedles were obtained after demolding. The swelling rate of the microneedles was 97 ± 3.87% in 1 h without electricity, and the swelling rate was 132 ± 6.21% in 1 h under a voltage of 0.6 V.

[0078] Example 6

[0079] The preparation method of the electroresponsive silk fibroin microneedles in this example specifically includes the following steps:

[0080] 1) Preparation of a thiolated silk fibroin solution

[0081] The concentration of the aqueous solution of silk fibroin was diluted to 30 mg / mL, and then the solution was stabilized in an ice bath at 2°C. The pH of the silk fibroin solution was adjusted to 5 using a 2-(N-morpholino)ethanesulfonic acid solution. First, 10 wt% (relative to the mass of silk fibroin) N-hydroxysuccinimide was slowly added to the above-mentioned silk fibroin solution, followed by the addition of 2.5 wt% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing well, the reaction was carried out for 0.5 h to activate the carboxyl groups on the silk fibroin.

[0082] To the above-mentioned silk fibroin solution, 40 mg / mL of a cysteamine hydrochloride solution was slowly added dropwise, so that the final solution had a cysteamine hydrochloride concentration of 60 mmol / L and a silk fibroin concentration of 30 mg / mL. The pH of the final solution was then adjusted to 5 using a 2-(N-morpholino)ethanesulfonic acid solution. The reaction was stirred in an ice bath for 4 h, and then the solution was left to stand in a 4°C refrigerator overnight.

[0083] 2) Dialysis of the thiolated silk fibroin solution

[0084] The reaction-completed silk fibroin solution was placed in a dialysis bag (with a molecular weight cut-off of 8-14 kDa) for dialysis. The dialysis environment was deionized water to which a small amount of sodium thiosulfate (0.0015 mol / L) was added, and the dialysis was protected by the introduction of inert gas N2. The deionized water containing sodium thiosulfate was replaced every 4 h, and the dialysis procedure was repeated for 2 d. After that, the dialysis was performed using deionized water without the addition of sodium thiosulfate for 1 d. After dialysis, the supernatant was obtained by centrifugation to obtain the grafted and modified thiolated silk solution. The mass concentration (wt%) of the silk fibroin solution was then measured, and the solution was stored in a 4°C refrigerator for later use. The thiol content in the modified silk fibroin solution was 53.3 ± 2.6 μmol / g.

[0085] 3) Preparation of silk fibroin microneedles

[0086] 1 mL of the thiolated silk fibroin solution prepared in (2) was cast into a single-piece PDMS microneedle mold, and vacuum was applied in a vacuum drying oven to remove air bubbles, which was repeated three times. The degassed mold system was then placed in a constant temperature and humidity chamber (25°C, 55% RH) and dried for 6 h under air flow. The silk fibroin microneedles were obtained after demolding. The swelling rate of the microneedles was 115 ± 6.27% in 1 h without electricity, and the swelling rate was 176 ± 7.53% in 1 h under a voltage of 0.6 V.

[0087] Example 7

[0088] The preparation method of the electrically responsive silk fibroin microneedles in this example specifically includes the following steps:

[0089] 1) Preparation of a thiolated silk fibroin solution

[0090] The concentration of the aqueous silk fibroin solution was diluted to 20 mg / mL, and then the solution was stabilized in an ice bath at 2°C. The pH of the silk fibroin solution was adjusted to 5.5 using a 2-(N-morpholino)ethanesulfonic acid solution. First, 8 wt% (relative to the mass of the silk fibroin) N-hydroxysuccinimide was slowly added to the above silk fibroin solution, followed by the addition of 20 wt% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing, the carboxyl groups on the silk fibroin were activated by reacting for 0.5 h.

[0091] A 50 mg / mL cysteamine hydrochloride solution was slowly added dropwise to the above silk fibroin solution, so that the final solution had a cysteamine hydrochloride concentration of 60 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted and stabilized to 5.5 using a 2-(N-morpholino)ethanesulfonic acid solution. The reaction was stirred in an ice bath for 4 h, and then the solution was removed and allowed to stand in a 4°C refrigerator overnight.

[0092] 2) Dialysis of the thiolated silk fibroin solution

[0093] The reaction-completed silk fibroin solution was placed in a dialysis bag (with a molecular weight cut-off of 8-14 kDa) for dialysis. The dialysis environment was deionized water with a small amount of sodium thiosulfate (0.001 mol / L) and protection by inert gas N2. The deionized water with sodium thiosulfate was replaced every 4 h, and the dialysis procedure was repeated for 2 d. Then, the dialysis was performed using deionized water without sodium thiosulfate for 1 d. After dialysis, the supernatant was obtained by centrifugation to obtain the grafted and modified thiolated silk solution. The mass concentration (wt%) of the silk fibroin solution was then measured, and the solution was stored in a 4°C refrigerator for later use. The thiol content in the modified silk fibroin solution was 63.3 ± 4.5 μmol / g.

[0094] 3) Preparation of silk fibroin microneedles

[0095] 1 mL of the thiolated silk fibroin solution prepared in (2) was cast into a single-piece PDMS microneedle mold, and vacuum was applied in a vacuum drying oven to remove air bubbles, which was repeated three times. The degassed mold system was then placed in a constant temperature and humidity chamber (25°C, 55% RH) and dried for 6 h in an air flow. After demolding, the silk fibroin microneedles were obtained. The swelling rate of the microneedles was 55 ± 6.49% in 1 h without electricity, and the swelling rate was 123 ± 7.18% in 1 h under a voltage of 0.6 V.

[0096] Table 1 is the thiol content of the silk fibroin microneedles prepared in Examples 2-7 of the present application and the corresponding swelling rate before and after electrification. The test method of the swelling rate is as follows: the method of soaking in deionized water (37℃) for 1 hour is adopted, the mass increase rate before and after soaking is compared, and the swelling rate = (the mass after soaking - the mass before soaking) / the mass before soaking.

[0097] Table 1 Thiol content of the silk fibroin microneedles prepared in the examples

[0098]

[0099] The results of Table 1 and Figure 2 It can be seen from the results that the swelling rate of the electrically responsive silk fibroin microneedles prepared in the examples has a significant increase after electrification, and the higher the thiol content, the better the swelling effect.

[0100] The above steps are distinguished and numbered for the convenience of description and understanding. In actual preparation, the above steps can be carried out simultaneously or without a sequence. And the raw materials not specifically mentioned in the examples are obtained by commercial purchase. The operations without special mention of temperature are carried out at room temperature. The operation methods and conditions without special mention can adopt the known or conventional means and conditions in the art.

[0101] The present application obtains a thiolated silk fibroin by thiolating the silk fibroin molecules, and then pours to form microneedles. The silk fibroin containing thiol will undergo redox reaction of thiol when current passes through, causing the change of disulfide crosslinking degree, thereby responding to the current, forming different swelling states of silk fibroin. The microneedle transdermal patch prepared by the method has good mechanical properties, biocompatibility and excellent current response swelling change, and can realize drug controlled release under the action of current. It can be applied in the scene of medical or medical beauty that needs to control the amount of drug.

[0102] The above examples are only for illustrating the technical concept and characteristics of the present application, the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and it cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. An electro-responsive silk fibroin material, characterized in that, The electric response type silk fibroin material contains silk fibroin and thiol groups grafted on the silk fibroin; the content of thiol groups is 20-100 μmol / g; The electric response type silk fibroin material is prepared by the following method: Cysteamine hydrochloride solution is added dropwise to the activated silk fibroin solution, and the concentration of cysteamine hydrochloride in the final solution is 20-80 mmol / L; the pH value of the final solution is adjusted to 5-6; the reaction is stirred, and after being taken out, it is placed to obtain a thiolated silk fibroin solution; The silk fibroin solution after the reaction is completed is loaded into a dialysis bag for dialysis; after dialysis is completed, the supernatant is obtained by centrifugation to obtain a purified thiolated silk fibroin solution; The purified thiolated silk fibroin solution is poured into a mold, and vacuum is applied to remove air bubbles; then the defoamed mold is placed in a constant temperature and humidity environment for drying, and after drying and demolding, the electric response type silk fibroin material is obtained. The electric response type silk fibroin material has current response, and the current response is that the swelling rate of the silk fibroin material after being electrified is greater than that in the case of not being electrified; the swelling rate of the electric response type silk fibroin material in the case of not being electrified for 1 hour is 50-120%, and the swelling rate after being electrified for 1 hour under a voltage of 0.6 V is 120-250%.

2. The electro-responsive silk fibroin material according to claim 1, wherein, The applied voltage of the current response of the electric response type silk fibroin material is 0.6-0.9 V.

3. The electro-responsive silk fibroin material according to claim 1, wherein, The stirring reaction is stirring reaction at 0-4℃ for 2-5 h; the standing is standing reaction at a temperature of 2-8℃ for 8-10 h.

4. The electro-responsive silk fibroin material of claim 1, wherein, The constant temperature and humidity conditions are temperature 20-30℃ and relative humidity 55-65%.

5. The electro-responsive silk fibroin material of claim 1, wherein, The activation of the silk fibroin solution is carried out according to the following steps: N-hydroxysuccinimide is added to the silk fibroin solution at a mass ratio of 1-10 wt% relative to the mass of silk fibroin, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added at a mass ratio of 2-20 wt% relative to the mass of silk fibroin, the mixture is uniformly mixed, and then reacted for 0.4-1 h to obtain an activated silk fibroin solution.

6. The electro-responsive silk fibroin material according to claim 5, wherein, The mass ratio of N-hydroxysuccinimide to silk fibroin is 1:100-1:10; the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to silk fibroin is 1:50-1:5; the mass ratio of cysteamine hydrochloride to silk fibroin is 1:100-1:2.

5.

7. The electro-responsive silk fibroin material according to claim 5, wherein, The silk fibroin solution is obtained by diluting the silk fibroin aqueous solution to a concentration of 20-30 mg / mL, then stabilizing the temperature of the solution to 0-4℃, and adjusting the pH value of the silk fibroin solution to 5-6 using a buffer solution.

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