Electroresponsive silk fibroin microneedles and their preparation method

Abstract

This invention discloses a method for preparing electroresponsive silk fibroin microneedles, comprising the following steps: adding cysteine ​​hydrochloride solution dropwise to an activated silk fibroin solution to achieve a final cysteine ​​hydrochloride concentration of 20-80 mmol / L; adjusting the pH of the final solution to 5-6; stirring the reaction mixture, then allowing it to stand to obtain a thiolated silk fibroin solution; dialyzing the reacted silk fibroin solution in a dialysis bag; after dialysis, centrifuging to obtain the supernatant and then purifying the thiolated silk fibroin solution; pouring the purified thiolated silk fibroin solution into a microneedle mold and removing air bubbles by vacuuming in a vacuum drying oven; then drying the degassed mold system under a constant temperature and humidity environment; and finally, after drying and demolding, obtaining the electroresponsive silk fibroin microneedles. The electroresponsive silk fibroin microneedles of this invention can achieve changes in swelling degree under low voltage stimulation and exhibit high swelling characteristics under energized conditions, enabling controlled drug release.

Description

[0001] This application is a divisional application of the invention patent application filed on March 16, 2022, with application number 202210258571X and invention title "An electroresponsive silk fibroin material and its preparation method and electroresponsive silk fibroin microneedles". Technical Field

[0002] This invention relates to the field of silk fibroin microneedle patches and medical and aesthetic materials, specifically to a method for preparing silk fibroin microneedles with increased internal pore size after being energized, and an electroresponsive silk fibroin microneedle prepared using this method. Background Technology

[0003] Smart responsive drug delivery is a current research hotspot in materials science and biomedicine. Stimuli such as changes in pH and temperature, triggered by electric fields, electric currents, magnetic fields, light, and mechanical forces, can induce drug delivery into the body in a responsive manner. However, controlling the delivery method to ensure convenient use, effective delivery, and precise dosage control remains a challenge.

[0004] Microneedle transdermal drug delivery is a method that uses an array of microneedles less than 1 mm in length to puncture the epidermis and create micropores, thereby improving the efficiency of transdermal drug delivery. It allows for easy, painless, safe, and convenient drug delivery and is playing an important role in the field of intelligent drug delivery. Furthermore, because the microneedle array adheres to the skin during use, it can effectively receive external stimuli, especially electric currents, electric fields, and magnetic fields, to achieve controlled drug release.

[0005] Silk fibroin is a green, natural, bio-based material with low immunogenicity, excellent biocompatibility, and outstanding mechanical properties. With the development of modern science and technology, silk is no longer limited to the textile field but has been widely used in drug delivery. However, drug-loaded silk fibroin microneedles lack intelligent responsiveness, which is determined by the structure of silk fibroin. This is because the silk fibroin molecule lacks corresponding stimulus-responsive groups or has too few responsive groups. Overall, pure silk fibroin does not exhibit stimulus-responsive changes and therefore cannot be used for intelligent drug delivery.

[0006] To address the aforementioned issues, Chinese invention patent CN108047466A discloses a method for preparing silk microneedles, using glutaraldehyde as the chemical cross-linking agent. The chemically cross-linked silk fibroin solution is then used to form microneedles. After forming the microneedles, steam treatment is applied to obtain high-strength silk fibroin microneedles. These microneedles can easily penetrate the skin and can incorporate large amounts of medication. However, after steam treatment, the internal structure of these microneedles is predominantly β-sheet, making them less prone to water absorption and swelling, resulting in slow drug release, low release rate, and lack of responsiveness. Furthermore, glutaraldehyde, as a cross-linking agent, poses significant health risks, irritating both the skin and digestive system. Similarly, Chinese invention patent CN102580232B discloses a silk fibroin microneedle system, silk fibroin nanoparticles, and their preparation method. The silk fibroin microneedles prepared by this method dissolve rapidly upon skin penetration, releasing nanoparticles. However, this microneedle system releases the drug into the body in a single dose, making it impossible to control the intelligent release and dosage of the drug.

[0007] Electrically assisted drug delivery is inexpensive and easy to perform, helping drugs overcome tissue barriers and enter the body. Its drug release mechanisms mainly include electroporation and iontophoresis. However, these methods all utilize relatively large currents or electric fields, posing certain safety risks. Therefore, it is of great significance to achieve intelligent responsive drug delivery in silk fibroin microneedles under low current or electric field conditions, which can greatly expand the precise, convenient, and safe application of intelligent drug delivery in the human body.

[0008] Gu et al. [Nature Biomedical Engineering, 2020, 4(7): 1-8.] developed a method for creating glucose-responsive N-vinylpyrrolidone-based microneedle patches containing copolymers designed for glucose-triggered insulin delivery. These microneedles have a high drug loading capacity and intelligent responsiveness, making them suitable for closed-loop insulin delivery. However, the microneedle substrate is made from polymer materials, which have insufficient biosafety and sustainability, and the glucose intelligent response is slow, hindering rapid release. Moonjeong et al. [DOI: 10.1038 / s41598-020-58822-w] developed a multifunctional system composed of hyaluronic acid microneedles for rapid transdermal drug delivery. Under ultrasound, the acoustic pressure vibrations of the microneedles induce hyaluronic acid dissolution, while alternating current iontophoresis improves the electrostatically driven diffusion of hyaluronic acid ions and rhodamine. This method provides rapid drug release and enables fast local drug delivery. However, since the high-voltage electric field is applied directly to the drug or human skin, it will bring certain risks of drug deterioration and harm to the human body, and has certain limitations in its use.

[0009] Developing an electroresponsive material that can be applied to microneedles to achieve rapid and intelligent responsive changes in the material through low-voltage electrical stimulation, thereby controlling the rapid release of drugs and achieving electroresponsive intelligent drug release, is of paramount importance. Summary of the Invention

[0010] In view of this, in order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing an electroresponsive silk fibroin microneedle, wherein the prepared silk fibroin microneedle can achieve the purpose of controlling the pore size in the material and controlling the drug release rate by means of switching electricity.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A method for preparing an electroresponsive silk fibroin material includes the following steps:

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

[0014] (2) Thioylation of silk fibroin: Add cysteine ​​hydrochloride solution with a concentration of 40-60 mg / mL to the activated silk fibroin solution to make the final concentration of cysteine ​​hydrochloride in the solution 20-80 mmol / L. Then adjust the pH of the final solution to 5-6 using a buffer solution. Stir the reaction at 0-4℃ for 2-5 h, and then let it stand at 2-8℃ for 8-10 h to obtain a thiolated silk fibroin solution.

[0015] (3) Purification of thiolized silk fibroin: The silk fibroin solution after the reaction was completed was placed into a dialysis bag for dialysis; after dialysis, the supernatant was collected by centrifugation to obtain the purified thiolized silk fibroin solution.

[0016] (4) Preparation of electroresponsive silk fibroin material: Take the purified thiolized silk fibroin solution from the above steps, pour it into a mold, and remove air bubbles by vacuuming in a vacuum drying oven; then place the degassed mold in a constant temperature and humidity environment for drying, and obtain the electroresponsive silk fibroin material after drying and demolding. As for the specific shape of the electroresponsive silk fibroin material, the mold can be designed according to actual needs, and then the corresponding shape of silk fibroin material can be prepared, such as preparing an electroresponsive silk fibroin microneedle patch.

[0017] According to some preferred embodiments of the present invention, the electroresponsive silk fibroin material contains silk fibroin and thiol groups grafted onto the silk fibroin; the thiol content is 20-100 μmol / g. If the thiol content is too low, the electroresponsiveness is small, and the swelling degree does not change significantly. On the other hand, the carboxyl content in the silk fibroin molecule is limited; therefore, the control of the above reaction conditions aims to graft thiol groups onto the reactive carboxyl groups to increase the thiol content, thereby achieving better electroresponsiveness and swelling degree control.

[0018] According to some preferred embodiments of the present invention, the silk fibroin material exhibits current responsiveness, with a swelling rate of 50-120% after 1 hour without energization, and a swelling rate of 120-250% after 1 hour of energization at 0.6V. The redox potential between the thiol group and the disulfide bond is approximately 0.6 volts. The voltage cannot exceed 1 volt to prevent water electrolysis. Below 0.6 volts, the disulfide bond cannot be reduced. In practical applications, the voltage can be set between 0.6 and 0.9 volts. The current responsiveness of the present invention refers to the change in the properties (swelling rate) of the prepared silk fibroin material before and after energization, with the swelling rate of the material after energization significantly higher than that without energization.

[0019] According to some preferred embodiments of the present invention, 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 cysteine ​​hydrochloride to silk fibroin is 1:100-1:2.5.

[0020] According to some preferred embodiments of the invention, during dialysis, sodium thiosulfate is added to the deionized water used for dialysis and an inert gas is introduced for protection; the deionized water with added sodium thiosulfate is replaced every 2-4 hours, and the dialysis procedure is repeated for 2 days while maintaining inert gas protection; then, dialysis is performed for 1 day using deionized water without added sodium thiosulfate. Nitrogen (N2) is preferably used as the inert gas.

[0021] According to some preferred embodiments of the invention, 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 invention, the dialysis bag used during dialysis has a molecular weight cutoff of 8-14 kDa.

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

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

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

[0026] The present invention also provides an electroresponsive silk fibroin material prepared by the preparation method described above.

[0027] The present invention also provides an electroresponsive silk fibroin microneedle, which uses thiolated silk fibroin as described above, which is poured into a microneedle mold and vacuumed in a vacuum drying oven to remove air bubbles; then the degassed mold system is placed in a constant temperature and humidity environment for drying, and after drying and demolding, an electroresponsive silk fibroin microneedle patch is obtained.

[0028] The reaction principle of this invention is as follows: The thiolization modification of silk fibroin is prepared by coupling silk fibroin with cysteine ​​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 silk fibroin molecule to form a promoter—an unstable urea derivative. This derivative then reacts with N-hydroxysuccinimide (NHS) to form a more stable ester, thereby enhancing the water stability of the carbodiimide crosslinked product. Simultaneously, the carboxyl groups are in an activated state. After the carboxyl groups are activated, the amino groups on the cysteine ​​hydrochloride react with the activated carboxyl groups to form amide bonds, successfully grafting cysteine ​​onto the silk fibroin molecule. Simultaneously, during the reaction, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are converted into water-soluble urea derivatives, which can be removed during subsequent dialysis to preserve the good biocompatibility of silk fibroin.

[0029] Disulfide bonds have a relatively low redox potential, with an apparent reduction potential of around 0.6 volts, making them prone to reversible redox reactions. After thiol groups are grafted onto silk fibroin, in the presence of oxygen in the air, these thiol groups are oxidized, forming disulfide cross-linking points between the silk fibroin molecular chains. These disulfide cross-linking points are reversible; under reducing conditions, they break back to thiol groups, and the cross-linking points between the molecular chains break. In the absence of electricity, the degree of cross-linking between silk fibroin molecular chains is high, resulting in a low swelling rate for silk fibroin microneedles. However, under energized conditions, the current provides a reducing environment, causing the disulfide bonds to gain electrons and undergo electrochemical reduction, breaking the disulfide bonds to form thiol groups. This reduces the number of covalent cross-linking points between silk fibroin molecular chains, increasing the swelling rate of the silk fibroin microneedles. This change in swelling can be controlled by switching the power supply on and off. Therefore, by obtaining electroresponsive silk fibroin materials and controlling the change in swelling through current response, the release rate of drugs from microneedles can be controlled.

[0030]

[0031] Due to the adoption of the above technical solutions, the present invention has the following advantages compared with the prior art: The preparation method of the electroresponsive silk fibroin microneedles of the present invention can achieve changes in swelling degree under low voltage stimulation, have high swelling characteristics under energized conditions, and have low swelling characteristics under non-energized conditions, thereby realizing the controllable release of drugs. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a microscope image of the microneedle patch prepared in the preferred embodiment 2 of the present invention;

[0034] Figure 2 The graph shows the swelling degree of the current-responsive silk fibroin microneedle patches prepared in Examples 2-7 of this invention under conditions of energization and de-energization.

[0035] Combination Figure 2 As can be seen from Examples 2-7, excessive addition of N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride will reduce the current response and swelling performance of silk fibroin microneedle patches, while increasing the amount of cysteine ​​hydrochloride can improve the swelling degree of microneedle patches.

[0036] Figure 3The images show scanning electron microscope (SEM) images of the internal pore size of the current-responsive silk fibroin microneedle patch prepared in Example 2 of this invention under both energized and de-energized conditions. Figure 3 (ab) is an electron microscope image of the internal aperture of the microneedle before energization. Figure 3 (cd) is an electron microscope image of the internal pore size of the microneedle after being energized. It can be seen that after being energized, the pore size inside the microneedle increases, which is conducive to the passage of drugs.

[0037] Figure 4 This is a schematic diagram illustrating the transformation of thiol groups and disulfide bonds within the material of the current-responsive silk fibroin microneedles before and after energization in an embodiment of the present invention. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0039] Example 1: Preparation of silk fibroin aqueous solution

[0040] Weigh 3g of NaHCO3 and 1g of Na2CO3 and dissolve them in 4000mL of deionized water. Heat to boiling, add 80g of silk, and maintain a gentle boil at 98℃ for 30 minutes. Remove and rinse with deionized water. Repeat the above steps three times and then dry in a 60℃ oven to obtain degummed silk fibroin fibers.

[0041] Prepare a 9.3 mol / L LiBr solution. Take 100 mL of the LiBr solution and heat it to 65°C in a water bath. Add 15 g of degummed silk fibroin in several portions, stirring to dissolve it. Continue heating and stirring for 40 min. Transfer the liquid to a dialysis bag and dialyze it in deionized water for 72 h, changing the deionized water every 2 h. After dialysis, filter the solution to obtain an aqueous solution of silk fibroin.

[0042] Example 2

[0043] The preparation method of the electroresponsive silk fibroin microneedles in this embodiment specifically includes the following steps:

[0044] 1) Preparation of thiolized silk fibroin solution

[0045] The concentration of the silk fibroin aqueous solution was diluted to 30 mg / mL, and then the solution beaker was stabilized at 2°C in an ice bath. The pH of the silk fibroin solution was adjusted to 5.5 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 10 wt% of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing thoroughly, the mixture was reacted for 0.5 h to activate the carboxyl groups on the silk fibroin.

[0046] A 50 mg / mL cysteine ​​hydrochloride solution was slowly added dropwise to the silk fibroin solution to achieve a final cysteine ​​hydrochloride concentration of 60 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution. The reaction was carried out with stirring in an ice bath for 4 hours, and then allowed to stand overnight at 4°C.

[0047] 2) Dialysis of thiolized silk fibroin solution

[0048] 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 (0.001 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 92.9 ± 5.7 μmol / g.

[0049] 3) Preparation of silk fibroin microneedles

[0050] 1 mL of thiolized silk fibroin solution was poured into a single PDMS (dimethylsiloxane) microneedle mold. The mold was then vacuum-dried in a vacuum drying oven to remove air bubbles, repeating this process three times. Afterward, the degassed mold system was placed in a constant temperature and humidity chamber (25℃, 55%RH) with air circulation for 8 hours to dry. Silk fibroin microneedles were obtained after demolding. The swelling rate of the microneedles after 1 hour without power was 72±3.63%, and the swelling rate after 1 hour with power at 0.6V was 227±13.86%.

[0051] Example 3

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

[0053] 1) Preparation of thiolized 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 silk fibroin aqueous solution was diluted to 20 mg / mL, and then the solution beaker was stabilized at 2°C in an ice bath. The pH of the silk fibroin solution was adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution. First, 2.5 wt% (relative to the weight of silk fibroin) of N-hydroxysuccinimide was slowly added to the above silk fibroin solution, followed by 4 wt% of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing thoroughly, the mixture was reacted for 0.8 h to activate the carboxyl groups on the silk fibroin.

[0064] A 55 mg / mL cysteine ​​hydrochloride solution was slowly added dropwise to the silk fibroin solution to achieve a final cysteine ​​hydrochloride concentration of 80 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution. The reaction was carried out with stirring in an ice bath for 4 hours, and then allowed to stand overnight at 4°C.

[0065] 2) Dialysis of thiolized silk fibroin solution

[0066] 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 48.3 ± 2.9 μmol / g.

[0067] 3) Preparation of silk fibroin microneedles

[0068] 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 105±5.53%, and the swelling rate after 1 hour with a voltage of 0.6V is 182±8.32%.

[0069] Example 5

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

[0071] 1) Preparation of thiolized silk fibroin solution

[0072] The concentration of the silk fibroin aqueous solution was diluted to 25 mg / mL, and the solution beaker was stabilized at 2°C in an ice bath. The pH of the silk fibroin solution was adjusted to 6 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 7.5 wt% of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After mixing thoroughly, the mixture was reacted for 0.5 h to activate the carboxyl groups on the silk fibroin.

[0073] A 60 mg / mL cysteine ​​hydrochloride solution was slowly added dropwise to the silk fibroin solution to achieve a final cysteine ​​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 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.

[0074] 2) Dialysis of thiolized silk fibroin solution

[0075] 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 (0.001 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 41.2 ± 3.1 μmol / g.

[0076] 3) Preparation of silk fibroin microneedles

[0077] 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 97±3.87%, and the swelling rate after 1 hour with a voltage of 0.6V is 132±6.21%.

[0078] Example 6

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

[0080] 1) Preparation of thiolized silk fibroin solution

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

[0082] Cysteine ​​hydrochloride solution (40 mg / mL) was slowly added dropwise to the silk fibroin solution to achieve a final concentration of 60 mmol / L for cysteine ​​hydrochloride and 30 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.

[0083] 2) Dialysis of thiolized silk fibroin solution

[0084] 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 53.3 ± 2.6 μmol / g.

[0085] 3) Preparation of silk fibroin microneedles

[0086] 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 115±6.27%, and the swelling rate after 1 hour with a voltage of 0.6V is 176±7.53%.

[0087] Example 7

[0088] The preparation method of the electroresponsive silk fibroin microneedles in this embodiment specifically includes the following steps:

[0089] 1) Preparation of thiolized silk fibroin solution

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

[0091] A 50 mg / mL cysteine ​​hydrochloride solution was slowly added dropwise to the silk fibroin solution to achieve a final cysteine ​​hydrochloride concentration of 60 mmol / L and a silk fibroin concentration of 20 mg / mL. The pH of the final solution was then adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution. The reaction was carried out with stirring in an ice bath for 4 hours, and then allowed to stand overnight at 4°C.

[0092] 2) Dialysis of thiolized silk fibroin solution

[0093] 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 (0.001 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 63.3 ± 4.5 μmol / g.

[0094] 3) Preparation of silk fibroin microneedles

[0095] 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 55±6.49%, and the swelling rate after 1 hour with a voltage of 0.6V is 123±7.18%.

[0096] Table 1 shows the thiol content in the silk fibroin microneedles prepared in Examples 2-7 of this invention and the corresponding swelling rates before and after electrolysis. The swelling rate was tested as follows: the microneedles were soaked in deionized water (37°C) for 1 hour, and the mass increase rate before and after soaking was compared. Swelling rate = (mass after soaking - mass before soaking) / mass before soaking.

[0097] Table 1 shows the thiol content of the silk fibroin microneedles prepared in the examples.

[0098]

[0099]

[0100] Combining Table 1 and Figure 2 The results show that the swelling rate of the electroresponsive silk fibroin microneedles prepared in the examples increased significantly after energization, and the higher the thiol content, the better the swelling effect.

[0101] For ease of description and understanding, the steps have been differentiated and numbered. In actual preparation, these steps can be performed simultaneously or in any order. All raw materials not specifically mentioned in the examples were commercially available. Operations without a specified temperature were performed at room temperature. Operating methods and conditions not specifically mentioned can be performed using methods and conditions known or conventional in the art.

[0102] This invention obtains a thiolized silk fibroin by thiolizing silk fibroin molecules, and then casts it into microneedles. This thiol-containing silk fibroin undergoes a redox reaction when an electric current passes through it, causing a change in the degree of disulfide bond crosslinking, thus responding to the current and forming different swelling states of the silk fibroin. The microneedle transdermal patch prepared by this method has good mechanical properties, biocompatibility, and excellent current-responsive swelling changes, enabling controlled drug release under current switching. It can be applied in medical or aesthetic settings where dosage control is required.

[0103] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing electroresponsive silk fibroin microneedles, characterized in that, Includes the following steps: Add cysteine ​​hydrochloride solution dropwise to the activated silk fibroin solution to make the final cysteine ​​hydrochloride concentration 20-80 mmol / L; adjust the pH of the final solution to 5-6; stir the reaction, remove and let stand to obtain thiolized silk fibroin solution; The reacted silk fibroin solution was placed in a dialysis bag for dialysis; after dialysis, the supernatant was collected by centrifugation to obtain the purified thiolized silk fibroin solution. Take the purified thiolized silk fibroin solution, pour it into a microneedle mold, and remove air bubbles by vacuuming in a vacuum drying oven; then place the degassed mold system in a constant temperature and humidity environment for drying, and obtain the electroresponsive silk fibroin microneedles after drying and demolding; the thiol content in the electroresponsive silk fibroin microneedles is 20-100 μmol / g.

2. The preparation method according to claim 1, characterized in that, The electroresponsive silk fibroin microneedles contain silk fibroin and thiol groups grafted onto the silk fibroin.

3. The preparation method according to claim 1, characterized in that, The electroresponsive silk fibroin microneedles have current responsiveness, wherein the swelling rate of the silk fibroin microneedles is greater when energized than when not energized.

4. The preparation method according to claim 1, 2, or 3, characterized in that, The electroresponsive silk fibroin microneedles exhibit a swelling rate of 50-120% after 1 hour without power, and a swelling rate of 120-250% after 1 hour of power application at 0.6V.

5. The preparation method according to claim 1, 2, or 3, characterized in that, The current-responsive voltage of the electroresponsive silk fibroin microneedles is 0.6-0.9V.

6. The preparation method according to claim 1, characterized in that, During dialysis, sodium thiosulfate is added to the deionized water used for dialysis and an inert gas is introduced for protection. The deionized water with added sodium thiosulfate is replaced every 2-4 hours, and the dialysis procedure is repeated while maintaining inert gas protection. After that, dialysis is performed using deionized water without added sodium thiosulfate.

7. The preparation method according to claim 6, characterized in that, The concentration of sodium thiosulfate in the deionized water is 0.001-0.0015 mol / L.

8. The preparation method according to claim 6, characterized in that, The dialysis bag used during dialysis has a molecular weight cutoff of 8-14 kDa.

9. The preparation method according to claim 1, characterized in that, The activation of the silk fibroin solution is carried out according to the following steps: Add 1-10 wt% N-hydroxysuccinimide relative to the amount of silk fibroin to the silk fibroin solution, then add 2-20 wt% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride relative to the amount of silk fibroin, mix well and react for 0.4-1 h to obtain an activated silk fibroin solution.

10. An electroresponsive silk fibroin microneedle prepared by the preparation method according to any one of claims 1-9.

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