WO2023174176A1 - 一种电响应型丝素蛋白材料、电响应型丝素蛋白微针、具有电致响应性的丝素蛋白胰岛素微针贴片及其制备方法、透皮给药装置 - Google Patents

一种电响应型丝素蛋白材料、电响应型丝素蛋白微针、具有电致响应性的丝素蛋白胰岛素微针贴片及其制备方法、透皮给药装置 Download PDF

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Publication number
WO2023174176A1
WO2023174176A1 PCT/CN2023/080804 CN2023080804W WO2023174176A1 WO 2023174176 A1 WO2023174176 A1 WO 2023174176A1 CN 2023080804 W CN2023080804 W CN 2023080804W WO 2023174176 A1 WO2023174176 A1 WO 2023174176A1
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WIPO (PCT)
Prior art keywords
silk fibroin
solution
insulin
preparation
thiolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/CN2023/080804
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English (en)
French (fr)
Inventor
卢神州
祁珍珍
陶晓晟
郭仕杰
严正
李玉剑
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Pharsun Medical Biotechnics Shanghai Co Ltd
Suzhou University
Original Assignee
Pharsun Medical Biotechnics Shanghai Co Ltd
Suzhou University
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Priority claimed from CN202210263237.3A external-priority patent/CN114699511B/zh
Priority claimed from CN202210258571.XA external-priority patent/CN114921105B/zh
Application filed by Pharsun Medical Biotechnics Shanghai Co Ltd, Suzhou University filed Critical Pharsun Medical Biotechnics Shanghai Co Ltd
Publication of WO2023174176A1 publication Critical patent/WO2023174176A1/zh
Priority to US18/886,923 priority Critical patent/US12350460B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
    • A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
    • A61M2037/0053—Methods for producing microneedles

Definitions

  • the invention relates to the technical fields of silk fibroin microneedle patches and medical materials and medical aesthetic materials. Specifically, it relates to a preparation method of a silk fibroin material whose internal pore diameter increases after being energized, and an electrically responsive silk fibroin prepared by the preparation method. Protein material, electro-responsive silk fibroin microneedle based on the electro-responsive silk fibroin material, electro-responsive silk fibroin insulin microneedle patch and preparation method thereof, a transdermal drug delivery system based on the above-mentioned material medicine device.
  • the field of intelligent responsive drug delivery is a current research hotspot in materials science and biomedicine. Based on the effects of electric fields, currents, magnetic fields, light, mechanical external forces, changes in pH value, temperature and other stimuli, drugs can be delivered responsively into the body. However, how to control the drug delivery method to make intelligent responsive drug delivery convenient to use, effective delivery and precise control of the dosage is a challenge.
  • Microneedle transdermal drug delivery is a method that uses a microneedle array less than 1mm in length to pierce the epidermal layer of the skin and form micropores to improve the efficiency of transdermal drug delivery.
  • Drug delivery can be achieved easily, painlessly, safely and conveniently. It is playing an important role in the field of smart drug delivery.
  • the microneedle array is attached to the skin during use, it can effectively accept external stimuli, especially current, electric fields and magnetic fields, to achieve controlled release of drugs.
  • 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 is no longer limited to the textile field, but has 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. Because the silk fibroin molecules lack corresponding stimulus-responsive groups or have too few responsive groups. Overall, pure silk fibroin does not have stimulus-responsive changes and cannot be used for smart drug delivery.
  • Chinese invention patent CN108047466A discloses a method for preparing silk microneedles.
  • the chemical cross-linking agent used is glutaraldehyde, and then the chemically cross-linked silk fibroin solution is made into microneedles; after the microneedles are made, After the needle is steamed, high-strength silk fibroin microneedles are obtained. These microneedles can penetrate the skin more easily and can be mixed with a large amount of medicine.
  • this kind of microneedle has been treated with water vapor, and its internal structure is mainly ⁇ -sheet, which is not easy to absorb water and swell, resulting in slow drug release, low drug release rate, and lack of responsiveness; at the same time, glutaraldehyde, as a cross-linking agent, It is very harmful to the human body and can cause irritation to the skin and digestive system.
  • Another example is the Chinese invention patent CN102580232B, which discloses a silk fibroin microneedle system, silk fibroin nanoparticles and a preparation method thereof.
  • the silk fibroin microneedles prepared by this method dissolve quickly after being inserted into the skin, thereby releasing the nanoparticles.
  • this microneedle system releases drugs into the body at one time and cannot control the intelligent release and amount of drugs.
  • one of the purposes of the present invention is to provide a method for preparing an electrically responsive silk fibroin material.
  • the prepared silk fibroin material and silk fibroin microneedles can realize the use of switches. Electrically control the pore size in the material and achieve the purpose of controlling the drug release rate.
  • a method for preparing electrically responsive silk fibroin materials including the following steps:
  • 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°C, and use a buffer solution to dilute the silk fibroin solution. Adjust the pH value to 5-6; add 1-10wt% N-hydroxysuccinimide relative to the silk fibroin mass to the above silk fibroin solution, and then add 2-20wt% 1-hydroxysuccinimide relative to the silk fibroin mass. (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, mix evenly and react for 0.4-1h to obtain an activated silk fibroin solution;
  • NHS and EDC are used to activate the carboxyl groups on silk fibroin, making it easier to react with amino groups for subsequent grafting of cysteamine and silk fibroin, while maintaining the biocompatibility of silk fibroin;
  • the electroresponsive silk fibroin material contains silk fibroin and thiol groups grafted on the silk fibroin; the content of the thiol groups is 20-100 ⁇ mol/g.
  • the sulfhydryl content is too low, the electrical responsiveness is small, and the swelling degree does not change much.
  • the carboxyl group content in silk fibroin molecules is limited.
  • the control of the above reaction conditions is also to add thiol groups to the carboxyl groups that can react as much as possible to increase the content of thiol groups to achieve better electrical response and control of swelling degree. .
  • the silk fibroin material has current responsiveness, with a swelling rate of 50-120% for 1 hour without electricity, and a swelling rate of 120 after 1 hour of electricity at a voltage of 0.6V. -250%.
  • the redox potential between the thiol group and the disulfide bond is about 0.6 volts.
  • the voltage cannot be higher than 1 volt to prevent electrolysis of water. Below 0.6 volts, disulfide bonds cannot be reduced. In actual application, the voltage can be set between 0.6-0.9 volts.
  • the current responsiveness of the present invention refers to the preparation of The performance (swelling rate) of the silk fibroin material has changed before and after electricity is applied. The swelling rate of the material after electricity is significantly improved compared to the swelling rate without electricity.
  • the mass ratio of N-hydroxysuccinimide to silk fibroin is 1:100-1:10; 1-(3-dimethylaminopropyl)-3-ethyl
  • the mass ratio of carbodiimide hydrochloride to silk fibroin is 1:50-1:5; the mass ratio of cysteamine hydrochloride to silk fibroin is 1:100-1:2.5.
  • sodium thiosulfate is added to the deionized water for dialysis and inert gas is introduced for protection; the deionized water with sodium thiosulfate added is replaced every 2-4 hours, and repeated
  • the dialysis procedure was carried out for 2 days and the inert gas protection was maintained; after that, deionized water without sodium thiosulfate was used for dialysis for 1 day.
  • the inert gas is preferably nitrogen N 2 .
  • the concentration of sodium thiosulfate in deionized water is 0.001-0.0015 mol/L.
  • the molecular weight cutoff of the dialysis bag used in dialysis is 8-14 kDa.
  • the constant temperature and humidity conditions are a temperature of 20-30°C and a relative humidity of 55-65%.
  • the buffer solution is selected from the group consisting of 2-(N-morpholino)ethanesulfonic acid, glycine-hydrochloric acid, citric acid-sodium citrate, acetic acid-sodium acetate, hydrogen phthalate One of potassium-sodium hydroxide and Tris-hydrochloride buffer.
  • the silk fibroin aqueous solution is obtained from domestic silkworm silk through degumming, dissolution, and dialysis.
  • the second object of the present invention is to provide an electrically responsive silk fibroin material prepared by the above preparation method.
  • the third object of the present invention is to provide an electrically responsive silk fibroin microneedle, which uses the thiolated silk fibroin as described above, pours it into a microneedle mold, and vacuums it in a vacuum drying box to remove air bubbles. ;Then, the degassed mold system is placed in a constant temperature and humidity environment for drying. After drying and demoulding, the electrically responsive silk fibroin microneedle patch is obtained.
  • the present invention provides a method for preparing a silk fibroin insulin microneedle patch with electroresponsiveness.
  • the prepared silk fibroin insulin microneedle patch can control the electrolyte in the material by switching electricity.
  • the pore size can be controlled to control the release rate of insulin in the microneedle, achieving intelligent release of insulin.
  • a method for preparing an electroresponsive silk fibroin insulin microneedle patch includes the following steps:
  • the pancreatic islets in the silk fibroin insulin microneedle patch The element content is 10-100mg/g, and the sulfhydryl content is 10-100 ⁇ mol/g.
  • the sulfhydryl content is too low, the electrical responsiveness is small, and the swelling degree does not change much.
  • the carboxyl group content in silk fibroin molecules is limited.
  • the control of the above reaction conditions is also to add thiol groups to the carboxyl groups that can react as much as possible to increase the content of thiol groups to achieve better electrical response and control of swelling degree. .
  • the fourth object of the present invention is to provide an electroresponsive silk fibroin insulin microneedle patch prepared by the above preparation method.
  • the mass ratio of silk fibroin and insulin is 4 to 20:1.
  • the electroresponsiveness is achieved by switching the DC power supply; by energizing the silk fibroin insulin microneedle patch, the swelling degree of the silk fibroin insulin microneedle patch increases, accelerating the release of insulin.
  • the electroresponsive silk fibroin insulin microneedle patch can achieve changes in swelling degree under low voltage (voltage below 1V) stimulation. It has high swelling characteristics under energized conditions and low swelling characteristics when not energized. , to achieve controlled release of insulin.
  • the microneedle transdermal patch avoids the first-pass effect of the liver and can improve the bioavailability of drugs.
  • the fifth object of the present invention is to provide a transdermal drug delivery device that can reduce components while ensuring perfect functions, and make the voltage loaded on the microneedle patch controllable to achieve intelligent transdermal control of macromolecular drugs.
  • the transdermal drug delivery device includes a power supply, a switch, a fixed resistor, an adjustable resistor, and a microneedle patch containing the drug.
  • the power supply, switch, and adjustable resistor constitute a main circuit; the fixed resistor and the microneedle The patches are connected in parallel and then connected in series to the main circuit.
  • the microneedle patch is a conductive microneedle patch; the microneedle patch is used to release drugs when electricity is applied; the microneedle patch It is an electroresponsive silk fibroin insulin microneedle patch as described above.
  • an adjustable resistor By setting an adjustable resistor, the voltage loaded on the microneedle patch can be adjusted, while a fixed resistor is set to ensure safe use.
  • Microneedle patches are drug carriers and can conduct electricity.
  • the power supply is a direct current power supply.
  • the voltage of the power supply is 3V-3.6V.
  • the power source is a rechargeable button-type lithium-ion battery of 3V to 3.6V, or a non-rechargeable 3V button-type lithium manganese battery.
  • the resistance of the fixed resistor is 200-600 ⁇ , preferably 500 ⁇ .
  • the resistance adjustment range of the adjustable resistor is 200-2000 ⁇ .
  • the fixed resistor and the adjustable resistor are used to maintain the voltage loaded on the microneedle patch within a safe voltage.
  • the safety voltage is 0.6-1V.
  • the safety voltage needs to ensure safe use, and on the other hand, it needs to cooperate with the microneedle patch to achieve response.
  • an alarm component is provided on the main circuit, and the alarm component is an indicator light and/or a buzzer.
  • the above-mentioned transdermal drug delivery device uses fewer components and occupies less space on the terminal. It can realize the miniaturization and portability of the corresponding product. At the same time, it can make the voltage loaded on the microneedle patch adjustable. Combined with the micro The needle patch enables controlled release of drugs.
  • the reaction principle of the present invention is as follows: the thiolation modification of silk fibroin is by combining silk fibroin and cysteamine hydrochloride using N-hydroxysuccinimide/1-(3-dimethylaminopropyl)- It is prepared by coupling reaction of 3-ethylcarbodiimide hydrochloride system. In the initial stage of the reaction, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) reacts with aspartic acid and glutamic acid in the molecular structure of silk fibroin.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • the carboxyl group on the residue reacts to form a promoter, an unstable urea derivative, which then reacts with N-hydroxysuccinimide (NHS) to form a more stable ester to enhance the carbodiimide cross-linked product water stability, and at the same time, the carboxyl group is in an activated state.
  • NHS N-hydroxysuccinimide
  • the amino group on the cysteamine hydrochloride reacts with the activated carboxyl group to form an amide bond.
  • the cysteamine is successfully grafted to the silk fibroin fraction. Above the child.
  • the redox potential of the disulfide bond is relatively low, with an apparent reduction potential of about 0.6 volts, and reversible redox reactions are prone to occur.
  • the degree of cross-linking between silk fibroin molecular chains is high, resulting in a smaller swelling rate of silk fibroin microneedles; in the electricity state, the current provides a reducing environment, and the disulfide bonds obtain electrons to undergo electrochemistry. During the reduction reaction, the disulfide bond is broken to form sulfhydryl groups, the covalent cross-linking points between silk fibroin molecular chains are reduced, and the swelling rate of silk fibroin microneedles is increased.
  • This change in swelling degree can be controlled by switching the power supply on and off. Therefore, an electrically responsive silk fibroin material is obtained, which can control the change in swelling degree by responding to electric current, thereby controlling the release rate of drugs in microneedles.
  • the present invention has the following benefits compared with the prior art: with the preparation method of the electrically responsive silk fibroin material of the present invention, the prepared electrically responsive silk fibroin material can achieve low
  • the change in swelling degree under voltage stimulation has high swelling properties under energized conditions and low swelling properties under non-energized conditions, thereby achieving controlled release of drugs.
  • Figure 1 is a microscope photo of the microneedle patch prepared in the preferred embodiment 2 of the present invention.
  • Figure 2 is a diagram showing the change in swelling degree of the current-responsive silk fibroin microneedle patch prepared in Examples 2-7 of the present invention with and without electricity;
  • Figure 3 is a scanning electron microscope image of the internal pore size of the current-responsive silk fibroin microneedle patch prepared in Example 2 of the present invention, with and without power supply;
  • Figure 3 (a-b) is the internal pore size of the microneedle before power supply.
  • Electron microscope image, Figure 3 (c-d) is an electron microscope image of the internal pore size of the microneedle after energization. It can be seen that after energization, the pore size inside the microneedle increases, which is conducive to the passage of drugs;
  • Figure 4 is a schematic diagram of the conversion of sulfhydryl groups and disulfide bonds in the material before and after the current-responsive silk fibroin microneedles are energized in the embodiment of the present invention
  • FIG. 5 is a schematic structural diagram of a transdermal drug delivery device in Embodiment 8 of the present invention.
  • R1 is a fixed resistor
  • R2 is an adjustable resistor.
  • the concentration of the silk fibroin aqueous solution was diluted to 30 mg/mL, and then the solution beaker was stabilized to 2°C in an ice bath, and the pH value of the silk fibroin solution was adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution.
  • the reaction-completed silk fibroin solution into a dialysis bag (molecular weight cutoff: 8-14kDa) for dialysis.
  • the dialysis environment is to add a small amount of sodium thiosulfate (0.001mol/L) to deionized water and pass inert gas N 2 Protect. Change the deionized water with sodium thiosulfate added every 4 hours, repeat the dialysis procedure for 2 days, and then switch to deionized water without adding sodium thiosulfate for 1 day.
  • the supernatant was centrifuged to obtain the graft-modified thiolated silk fibroin solution. Then measure the mass concentration (wt%) of the silk fibroin solution and store it in a 4°C refrigerator for later use.
  • the sulfhydryl group content in the modified silk fibroin solution was 92.9 ⁇ 5.7 ⁇ mol/g.
  • the preparation method of the electroresponsive silk fibroin insulin microneedle patch specifically includes the following steps:
  • Example 2 The steps and parameters described in Example 2 were used to carry out 1) the preparation of the thiolated silk fibroin solution and 2) the dialysis of the thiolated silk fibroin solution.
  • the concentration of the silk fibroin aqueous solution was diluted to 20 mg/mL, and then the solution beaker was stabilized to 3°C in an ice bath, and the pH value of the silk fibroin solution was adjusted to 5 using 2-(N-morpholino)ethanesulfonic acid solution.
  • cysteamine hydrochloride solution Slowly add 60 mg/mL cysteamine hydrochloride solution to the above silk fibroin solution, so that the concentration of cysteamine hydrochloride in the final solution is 40 mmol/L and the concentration of silk fibroin is 20 mg/mL.
  • the pH value of the final solution was adjusted and stabilized to 5 using 2-(N-morpholino)ethanesulfonic acid solution. Stir the reaction in an ice bath for 4 hours, then take it out and let it stand overnight in a 4°C low-temperature refrigerator.
  • the reaction-completed silk fibroin solution into a dialysis bag (molecular weight cutoff: 8-14kDa) for dialysis.
  • the dialysis environment is to add a small amount of sodium thiosulfate (0.0015mol/L) to deionized water and pass inert gas N 2 Protect. Change the deionized water with sodium thiosulfate added every 4 hours, repeat the dialysis procedure for 2 days, and then switch to deionized water without adding sodium thiosulfate for 1 day.
  • the supernatant was centrifuged to obtain the graft-modified thiolated silk fibroin solution. Then measure the mass concentration (wt%) of the silk fibroin solution and store it in a 4°C refrigerator for later use.
  • the sulfhydryl group content in the modified silk fibroin solution was 23.3 ⁇ 2.2 ⁇ mol/g.
  • the preparation method of the electroresponsive silk fibroin insulin microneedle patch specifically includes Includes the following steps:
  • Example 3 The steps and parameters described in Example 3 were used to carry out 1) the preparation of the thiolated silk fibroin solution and 2) the dialysis of the thiolated silk fibroin solution.
  • the concentration of the silk fibroin aqueous solution was diluted to 20 mg/mL, and then the solution beaker was stabilized to 2°C in an ice bath, and the pH value of the silk fibroin solution was adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution.
  • the dialysis environment is to add a small amount of sodium thiosulfate (0.0015mol/L) to deionized water and pass inert gas N 2 Protect. Change the deionized water with sodium thiosulfate added every 4 hours, repeat the dialysis procedure for 2 days, and then switch to deionized water without adding sodium thiosulfate for 1 day. After dialysis, the supernatant was centrifuged to obtain the graft-modified thiolated silk fibroin solution. Then measure the mass concentration (wt%) of the silk fibroin solution and store it in a 4°C refrigerator for later use. The sulfhydryl group content in the modified silk fibroin solution is 48.3 ⁇ 2.9 ⁇ mol/g.
  • the preparation method of the electroresponsive silk fibroin insulin microneedle patch specifically includes the following steps:
  • Example 4 The steps and parameters described in Example 4 were used to carry out 1) the preparation of the thiolated silk fibroin solution and 2) the dialysis of the thiolated silk fibroin solution.
  • the concentration of the silk fibroin aqueous solution was diluted to 25 mg/mL, and then the solution beaker was stabilized to 2°C in an ice bath, and the pH value of the silk fibroin solution was adjusted to 6 using 2-(N-morpholino)ethanesulfonic acid solution.
  • cysteamine hydrochloride solution Slowly add 60 mg/mL cysteamine hydrochloride solution to the above silk fibroin solution, so that the concentration of cysteamine hydrochloride in the final solution is 20 mmol/L and the concentration of silk fibroin is 20 mg/mL.
  • the pH value of the final solution was adjusted and stabilized to 6 using 2-(N-morpholino)ethanesulfonic acid solution. Stir the reaction in an ice bath for 4 hours, then take it out and let it stand overnight in a 4°C low-temperature refrigerator.
  • the dialysis environment is to add a small amount of sodium thiosulfate (0.001mol/L) to deionized water and pass inert gas N 2 Protect. Change the deionized water with sodium thiosulfate added every 4 hours, repeat the dialysis procedure for 2 days, and then switch to deionized water without adding sodium thiosulfate for 1 day. After dialysis, the supernatant was centrifuged to obtain the graft-modified thiolated silk fibroin solution. Then measure the mass concentration (wt%) of the silk fibroin solution and store it in a 4°C refrigerator for later use. The sulfhydryl group content in the modified silk fibroin solution is 41.2 ⁇ 3.1 ⁇ mol/g.
  • the preparation method of the electroresponsive silk fibroin insulin microneedle patch specifically includes the following steps:
  • Example 5 The steps and parameters described in Example 5 were used to carry out 1) the preparation of the thiolated silk fibroin solution and 2) the dialysis of the thiolated silk fibroin solution.
  • the concentration of the silk fibroin aqueous solution was diluted to 30 mg/mL, and then the solution beaker was stabilized to 2°C in an ice bath, and the pH value of the silk fibroin solution was adjusted to 5 using 2-(N-morpholino)ethanesulfonic acid solution.
  • cysteamine hydrochloride solution Slowly add 40 mg/mL cysteamine hydrochloride solution to the above silk fibroin solution, so that the concentration of cysteamine hydrochloride in the final solution is 60 mmol/L and the concentration of silk fibroin is 30 mg/mL.
  • the pH value of the final solution was adjusted and stabilized to 5 using 2-(N-morpholino)ethanesulfonic acid solution. Stir the reaction in an ice bath for 4 hours, then take it out and let it stand overnight in a 4°C low-temperature refrigerator.
  • the reaction-completed silk fibroin solution into a dialysis bag (molecular weight cutoff: 8-14kDa) for dialysis.
  • the dialysis environment is to add a small amount of sodium thiosulfate (0.0015mol/L) to deionized water and pass inert gas N 2 Protect. Change the deionized water with sodium thiosulfate added every 4 hours, repeat the dialysis procedure for 2 days, and then switch to deionized water without adding sodium thiosulfate for 1 day.
  • the supernatant was centrifuged to obtain the graft-modified thiolated silk fibroin solution. Then measure the mass concentration (wt%) of the silk fibroin solution and store it in a 4°C refrigerator for later use.
  • the sulfhydryl group content in the modified silk fibroin solution is 53.3 ⁇ 2.6 ⁇ mol/g.
  • the preparation method of the electroresponsive silk fibroin insulin microneedle patch specifically includes the following steps:
  • Example 6 The steps and parameters described in Example 6 were used to carry out 1) the preparation of the thiolated silk fibroin solution and 2) the dialysis of the thiolated silk fibroin solution.
  • the concentration of the silk fibroin aqueous solution was diluted to 20 mg/mL, and then the solution beaker was stabilized to 2°C in an ice bath, and the pH value of the silk fibroin solution was adjusted to 5.5 using 2-(N-morpholino)ethanesulfonic acid solution.
  • the reaction-completed silk fibroin solution into a dialysis bag (molecular weight cutoff: 8-14kDa) for dialysis.
  • the dialysis environment is to add a small amount of sodium thiosulfate (0.001mol/L) to deionized water and pass inert gas N 2 Protect. Change the deionized water with sodium thiosulfate added every 4 hours, repeat the dialysis procedure for 2 days, and then switch to deionized water without adding sodium thiosulfate for 1 day.
  • the supernatant was centrifuged to obtain the graft-modified thiolated silk fibroin solution. Then measure the mass concentration (wt%) of the silk fibroin solution and store it in a 4°C refrigerator for later use.
  • the sulfhydryl group content in the modified silk fibroin solution is 63.3 ⁇ 4.5 ⁇ mol/g.
  • the preparation method of the electroresponsive silk fibroin insulin microneedle patch specifically includes the following steps:
  • Example 7 The steps and parameters described in Example 7 were used to carry out 1) the preparation of the thiolated silk fibroin solution and 2) the dialysis of the thiolated silk fibroin solution.
  • the transdermal drug delivery device in this embodiment includes a power supply, a switch, an alarm component, a fixed resistor R1, an adjustable resistor R2, a microneedle patch containing the drug, and wires connected between various components.
  • the power supply, switch, alarm component and adjustable resistor R2 form the main loop, and the fixed resistor R1 is connected to the main loop in parallel with the microneedle patch. That is, the fixed resistor R1 is connected in parallel with the microneedle patch and then connected in series with the power supply, the adjustable resistor R2 and the switch.
  • the alarm component in this embodiment is an indicator light.
  • the power supply is a DC power supply with a voltage range of 3 ⁇ 3.6V.
  • Various small energy output products can be selected, such as: 3.6V rechargeable lithium-ion button battery (LIR series), 3V rechargeable lithium-ion button battery (ML or VL series), non-rechargeable ones include 3V lithium manganese button batteries (CR series), etc.
  • the resistance of the fixed resistor R1 is 500 ⁇ ; the resistance adjustment range of the adjustable resistor R2 is 200-2000 ⁇ .
  • the fixed resistor R1 and the adjustable resistor R2 are used together to maintain the voltage loaded on the microneedle patch within the safe voltage of 0.6-1V.
  • the safety voltage needs to ensure safe use, and on the other hand, it needs to cooperate with the microneedle patch to achieve response.
  • the microneedle patch used in this embodiment contains medicine, which is a drug carrier and can conduct electricity to release the medicine when electricity is applied. It is preferably an electrically responsive microneedle patch to adjust the matching voltage. Achieve controlled release of drugs. By adjusting the resistance value of the adjustable resistor R2, the voltage loaded on the microneedle patch can be adjusted to achieve controlled release of the drug. At the same time, a fixed resistor R1 is set to ensure safe use.
  • the microneedle patch can be in the form of a conductive polymer microneedle patch containing drugs or a microneedle patch of polymer hydrogel that responds to electrical stimulation, preferably the silk fibroin insulin microneedle described in the above embodiment. patch.
  • Table 1 shows the sulfhydryl group content in the silk fibroin microneedles prepared in Examples 2-7 of the present invention and the corresponding swelling rate before and after electrification.
  • Table 2 shows the changes in the sulfhydryl content and insulin release rate in the silk fibroin insulin microneedle patch in Examples 2-1 to 7-1 with and without electricity.
  • the release rate of insulin was measured and calculated as follows: a Franz diffusion cell was used as the in vitro drug receiving pool, 0.01M PBS buffer solution at 32°C was used as the simulated body fluid, and silk fibroin insulin was taken.
  • the microneedle patch the microneedles are inserted into the skin of the rat by hand. Cover the skin flatly on the round mouth of the receiving chamber of the Franz transdermal diffusion cell. The dermal layer is attached to the receiving chamber. Use stainless steel iron clips to clamp the donor chamber and the receiving chamber to make them stable. The solution in the transdermal drug release cell is taken. Samples were taken regularly every hour, the concentration of insulin was detected by HPLC, and the insulin release rate was calculated.
  • the insulin release rate of the silk fibroin insulin microneedle patch prepared in the above-mentioned Examples 2-1 to 7-1 is significantly higher than that in the non-energized state, that is, the above implementation
  • the microneedle patch containing insulin prepared in this example can achieve controlled release of insulin.
  • a thiolated silk fibroin is obtained through thiolation of silk fibroin molecules, and then cast to form microneedles.
  • This kind of silk fibroin containing sulfhydryl groups will undergo a redox reaction of the sulfhydryl groups when current passes through, causing changes in the degree of disulfide bond cross-linking, 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, and can achieve controlled drug release under current switching. It can be used in scenarios where the dosage needs to be controlled in medical or aesthetic medicine.
  • a thiolated silk fibroin is obtained through thiolation of silk fibroin molecules, the modified silk fibroin solution is mixed with the insulin solution, and then poured into a silicone rubber microneedle mold After degassing and drying, an electroresponsive silk fibroin insulin microneedle transdermal patch was obtained.
  • This kind of silk fibroin containing sulfhydryl groups will undergo a redox reaction of sulfhydryl groups when current passes through, causing changes in the degree of disulfide bond cross-linking. Thereby, in response to the current, different swelling states of silk fibroin are formed, changing the release rate of insulin.
  • the insulin microneedle transdermal patch prepared by this method has good mechanical properties, biocompatibility and excellent current responsiveness, and can achieve electro-responsive controlled release of insulin to meet the patient's blood sugar control before and after eating.

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Abstract

本发明公开了一种电响应型丝素蛋白材料的制备方法，包括以下步骤：（1）丝素蛋白的活化：将丝素蛋白水溶液稀释，之后将温度稳定至0-4℃，将pH值调节至5-6；加入相对于丝素蛋白质量1-10wt％的N-羟基琥珀酰亚胺，然后加入相对于丝素蛋白质量2-20wt％的1-（3-二甲基氨基丙基）-3-乙基碳二亚胺盐酸盐，混合均匀后反应，获得活化的丝素蛋白溶液；（2）丝素蛋白的巯基化：向活化的丝素蛋白溶液中滴加半胱胺盐酸盐溶液，将最终溶液pH值调节至5-6；0-4℃下搅拌反应，取出后在2-8℃温度下静置反应，获得巯基化丝素蛋白；（3）巯基化丝素蛋白的纯化；（4）电响应型丝素蛋白材料的制备：取纯化的巯基化丝素蛋白溶液，浇注在模具中，脱泡后并进行干燥，脱模后获得电响应型丝素蛋白材料。

Description

一种电响应型丝素蛋白材料、电响应型丝素蛋白微针、具有电致响应性的丝素蛋白胰岛素微针贴片及其制备方法、透皮给药装置 技术领域
本发明涉及丝素蛋白微针贴以及医疗材料和医美材料技术领域，具体涉及一种通电后内部孔径增大的丝素蛋白材料的制备方法、采用该制备方法制备得到的电响应型丝素蛋白材料、基于该电响应型丝素蛋白材料的电响应型丝素蛋白微针、具有电致响应性的丝素蛋白胰岛素微针贴片及其制备方法、一种基于上述材料的透皮给药装置。
背景技术
智能响应性给药领域是当前的材料学以及生物医学的研究热点，基于电场、电流、磁场、光照和机械外力的作用，pH值、温度的改变等刺激可以使得药物响应性递送至体内。然而如何控制给药方式以使得智能响应性给药便捷使用、有效递送以及精确控制给药量面临挑战。
微针透皮给药是一种通过长度小于1mm的微针阵列刺破皮肤表皮层并形成微孔以提高透皮给药效率的方法，可以轻松、无痛、安全且方便地实现药物递送，在智能药物递送领域正在发挥着重要的作用。同时，由于微针阵列在使用过程中附着于皮肤之上，可有效接受外部刺激尤其是电流、电场以及磁场以达到药物的控制释放。
丝素蛋白是一种具备低免疫原性，生物相容性优异且力学性能出色的绿色天然生物基材料。随着现代科学技术的发展，蚕丝已经不仅仅局限于纺织领域，并且已经广泛用于药物递送领域。然而，负载药物的丝素蛋白微针并不具备智能响应性，这是由丝素蛋白的结构所决定的。由于丝素蛋白分子上缺乏相应的刺激响应性基团或响应性基团过少。整体上，纯丝素蛋白并不具备刺激响应性变化，从而无法用来进行智能药物递送。
为了解决上述问题，中国发明专利CN108047466A公开了一种蚕丝微针的制备方法，采用的化学交联剂为戊二醛，然后将化学交联后的丝素溶液制成微针；在制成微针后进行水蒸气处理得到高强度的丝素微针，此微针能够较为容易的刺入皮肤，并可混入大量药物。但是这种微针经过水蒸气处理，内部结构以β-折叠为主，不易发生吸水溶胀行为，从而导致药物释放缓慢，释药率低，缺乏响应性；同时，戊二醛作为交联剂，对人体危害较大，对皮肤和消化系统均会产生刺激。又如中国发明专利CN102580232B公开了一种丝素微针系统和丝素纳米颗粒及其制备方法。此方法制备得到的丝素蛋白微针刺入皮肤后迅速溶解，从而释放纳米颗粒。但是这种微针系统将药物一次性释放至体内，无法控制药物的智能释放和释放量。
电辅助给药，价格便宜且易于执行，可帮助药物克服组织屏障进入体内，其释药机制主要是电穿孔以及离子电渗疗法等，然而这些方法均会使用到一些较大的电流或电场，会带来一定的安全隐患。如何在低电流或电场下在丝素蛋白微针中完成药物的智能响应性传递十分有意义，可以极大地拓展智能给药在人体上精准、便捷及安全化应用。
Gu等人[Nature Biomedical Engineering,2020,4(7):1-8.]开发了一种具有葡萄糖响应性的N-乙烯基吡咯烷酮基微针贴片的方法，所述微针贴片包含经设计用于葡萄糖触发的胰岛素递送的共聚物。这种微针载药量大且具备智能响应效 果，可用于胰岛素闭环递送。然而微针基材使用聚合物材料进行制备，其生物安全性及绿色可持续性较为不足，同时葡萄糖智能响应速度较慢，无法快速响应释放。
又如，Moonjeong等[DOI:10.1038/s41598-020-58822-w]开发了由透明质酸微针组成的多功能系统，作为用于快速局部药物的有效透皮递送。该微针在超声波作用下，声压振动将诱导透明质酸溶解，而交流电离子电渗疗法可改善静电力驱动的透明质酸离子和若丹明的扩散。这种方法药物释放速度快，可以进行快速局部药物递送。然而由于使用较高压电场直接作用于药物或是人体皮肤，这将会带来一定的药物变质风险及人体危害，具有一定的使用局限性。
因此，如何开发一种可通过作用于微针的电响应材料，通过低压电刺激实现材料的快速智能响应性变化，进而控制药物的快速响应释放，完成电响应智能药物释放至关重要。
发明内容
有鉴于此，为了克服现有技术的缺陷，本发明的目的之一是提供一种电响应型丝素蛋白材料的制备方法，制备得到的丝素蛋白材料和丝素蛋白微针能够实现采用开关电的方式来控制材料中的孔径并达到控制药物释放速度的目的。
为达到上述目的，本发明采用以下的技术方案：
一种电响应型丝素蛋白材料的制备方法，包括以下步骤：
(1)丝素蛋白的活化：将丝素蛋白水溶液稀释至浓度为20-30mg/mL，之后将溶液放入冰浴，使温度稳定至0-4℃，使用缓冲溶液将丝素蛋白溶液的pH值调节至5-6；向上述丝素蛋白溶液中加入相对于丝素蛋白质量1-10wt％的N-羟基琥珀酰亚胺，然后加入相对于丝素蛋白质量2-20wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.4-1h，获得活化的丝素蛋白溶液；
采用NHS和EDC对丝素蛋白上的羧基进行活化，使其更加容易与氨基反应，用于后续半胱胺与丝素蛋白的接枝，并保持丝素蛋白的生物相容性；
(2)丝素蛋白的巯基化：向上述活化的丝素蛋白溶液中滴加浓度为40-60mg/mL的半胱胺盐酸盐溶液，并使得最终溶液中的半胱胺盐酸盐浓度为20-80mmol/L，然后使用缓冲溶液将最终溶液pH值调节至5-6；0-4℃下搅拌反应2-5h，取出后在2-8℃温度下静置反应8-10h，获得巯基化丝素蛋白溶液；
(3)巯基化丝素蛋白的纯化：将反应完成的丝素蛋白溶液装入透析袋中进行透析；透析完毕后，离心取上清液获得纯化后的巯基化丝素蛋白溶液；
(4)电响应型丝素蛋白材料的制备：取上述步骤中纯化的巯基化丝素蛋白溶液，浇注在模具中，在真空干燥箱中抽真空去除气泡；之后将脱泡后的模具置于恒温恒湿环境下进行干燥，干燥脱模后即获得电响应型丝素蛋白材料。至于将电响应型丝素蛋白材料具体做成什么形状，可以根据实际需求设计模具，进而制备得到对应形状的丝素蛋白材料，如制备成电响应型丝素蛋白微针贴片。
根据本发明的一些优选实施方面，所述电响应型丝素蛋白材料含有丝素蛋白以及接枝于所述丝素蛋白上的巯基；巯基的含量为20-100μmol/g。巯基含量太低，电响应性较小，溶胀度没有太大的变化。另一方面，丝素蛋白分子中的羧基含量是有限的，上述反应条件的控制也是尽量在能够反应的羧基上接入巯基以增加巯基的含量，达到更好的电响应效果和溶胀度的控制。
根据本发明的一些优选实施方面，所述丝素蛋白材料具有电流响应性，在未通电情况下1小时的溶胀率为50-120％，在电压0.6V下通电1小时后的溶胀率为120-250％。巯基与二硫键之间的氧化还原电位是0.6伏特左右。电压不能高于1伏特，防止水的电解。低于0.6伏特，二硫键不能被还原。在实际应用过程中，可以设定电压在0.6-0.9伏特之间。本发明的电流响应性指的是制备得到 的丝素蛋白材料在通电前后的性能(溶胀率)有所改变，通电后材料的溶胀率比未通电情况下的溶胀率有明显的提升。
根据本发明的一些优选实施方面，所述N-羟基琥珀酰亚胺与丝素蛋白的质量比为1:100-1:10；1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐与丝素蛋白的质量比为1:50-1:5；半胱胺盐酸盐与丝素蛋白的质量比为1:100-1:2.5。
根据本发明的一些优选实施方面，所述透析时在透析用去离子水中加入硫代硫酸钠并通入惰性气体进行保护；每2-4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d和保持惰性气体保护；之后再换用未添加有硫代硫酸钠的去离子水进行透析1d。惰性气体优选氮气N2。
根据本发明的一些优选实施方面，去离子水中硫代硫酸钠的浓度为0.001-0.0015mol/L。
根据本发明的一些优选实施方面，透析时采用的透析袋的截留分子量为8-14kDa。
根据本发明的一些优选实施方面，所述恒温恒湿的条件为温度20-30℃、相对湿度55-65％。
根据本发明的一些优选实施方面，所述缓冲溶液为选自2-(N-吗啉代)乙磺酸、甘氨酸-盐酸、柠檬酸-柠檬酸钠、乙酸-乙酸钠、邻苯二甲酸氢钾-氢氧化钠、Tris-盐酸缓冲液中的一种。
根据本发明的一些优选实施方面，所述丝素蛋白水溶液以家蚕丝为原料，通过脱胶、溶解、透析后获得。
本发明的目的之二是提供了一种采用如上所述的制备方法制备得到的电响应型丝素蛋白材料。
本发明的目的之三是提供了一种电响应型丝素蛋白微针，其采用如上所述的巯基化丝素蛋白，将其浇注在微针模具中，在真空干燥箱中抽真空去除气泡；之后将脱泡后的模具系统置于恒温恒湿环境下进行干燥，干燥脱模后即获得电响应型丝素蛋白微针贴片。
优选地，本发明提供了一种具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法，制备得到的丝素蛋白胰岛素微针贴片能够实现采用开关电的方式来控制材料中的孔径并达到控制微针中胰岛素释放速度的目的，实现了胰岛素的智能释药。
在一些实施例中，一种具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法，包括以下步骤：
(1)丝素蛋白的活化、(2)丝素蛋白的巯基化、(3)巯基化丝素蛋白的纯化；这三个步骤与前述电响应型丝素蛋白材料的制备方法中的步骤相同，在此不再赘述；
(4)胰岛素溶液的配置：取胰岛素于离心管中，先加入0.01M-0.05M的盐酸溶液，混匀后充分溶解胰岛素，使溶液中的胰岛素浓度为10-50mg/mL；再加入0.01M-0.05M的缓冲溶液，调整溶液的pH为6.7-7.4，形成胰岛素溶液；
(5)混合溶液的制备：取上述步骤中制备的胰岛素溶液加入到巯基化丝素蛋白溶液中，搅拌均匀，使得混合溶液中巯基化丝素蛋白的浓度为10-40mg/mL，胰岛素的浓度为1-10mg/mL；控制微针的载药量，防止载药量过多胰岛素析出且降低微针强度；
(6)胰岛素微针贴片的制备：取混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡，将脱泡后的模具系统置于恒温恒湿环境下干燥，干燥脱模后即获得具有电致响应性的丝素蛋白胰岛素微针贴片。
根据本发明的一些优选实施方面，所述丝素蛋白胰岛素微针贴片中的胰岛 素含量为10-100mg/g，巯基含量为10-100μmol/g。巯基含量太低，电响应性较小，溶胀度没有太大的变化。另一方面，丝素蛋白分子中的羧基含量是有限的，上述反应条件的控制也是尽量在能够反应的羧基上接入巯基以增加巯基的含量，达到更好的电响应效果和溶胀度的控制。
本发明的目的之四是提供了一种采用如上所述的制备方法制备得到的具有电致响应性的丝素蛋白胰岛素微针贴片。丝素蛋白胰岛素微针贴片中，丝素蛋白和胰岛素的质量比为4～20:1。电致响应性通过直流电源的开关来实现；通过对丝素蛋白胰岛素微针贴片进行通电，使得丝素蛋白胰岛素微针贴片的溶胀度增加，加快了胰岛素的释放。具有电致响应性的丝素蛋白胰岛素微针贴片能够实现低电压(电压在1V以下)刺激下溶胀度的变化，在通电条件下具备高溶胀特性，在不通电的情况下具备低溶胀特性，实现胰岛素的可控释放。同时，微针透皮贴片避免了肝脏首过效应，能够提高药物的生物利用率。
本发明的目的之五是提供了一种透皮给药装置，能够在保证功能完善的同时减少元器件，且使得加载在微针贴片上的电压可控，达到控制大分子药物智能透皮释药的目的。具体的，透皮给药装置包括电源、开关、固定电阻、可调节电阻以及含有药物的微针贴片，所述电源、开关以及可调节电阻构成主回路；所述固定电阻与所述微针贴片并联后串联连接在所述主回路上，所述微针贴片为能够导电的微针贴片；所述微针贴片用于在通电时向外释放药物；所述微针贴片为如上所述的具有电致响应性的丝素蛋白胰岛素微针贴片。通过设置可调节电阻能够调节加载在微针贴片上的电压，同时设置固定电阻保证使用安全。微针贴片为药物载体，同时能够实现导电。
根据本发明的一些优选实施方面，所述电源为直流电源。
根据本发明的一些优选实施方面，所述电源的电压为3V～3.6V。
根据本发明的一些优选实施方面，所述电源为3V～3.6V的可充电扣式锂离子电池，或不可充电的3V扣式锂锰电池。
根据本发明的一些优选实施方面，所述固定电阻的电阻大小为200-600Ω，优选为500Ω。
根据本发明的一些优选实施方面，所述可调节电阻的电阻调节范围为200-2000Ω。
根据本发明的一些优选实施方面，所述固定电阻和可调节电阻用于维持加载在所述微针贴片上的电压位于安全电压内。
根据本发明的一些优选实施方面，所述安全电压为0.6～1V。安全电压一方面需要保证使用安全，另一方面需要与微针贴片配合实现响应。
根据本发明的一些优选实施方面，所述主回路上设置有报警组件，所述报警组件为指示灯和/或蜂鸣器。
上述的透皮给药装置，使用的元器件少，结合在终端上占用空间少，能够实现相应产品的小型化和便携化，同时能够使得加载在微针贴片上的电压可调，结合微针贴片实现药物的可控释放。
本发明的反应原理如下：丝素蛋白的巯基化改性是通过将丝素蛋白与半胱胺盐酸盐采用N-羟基琥珀酰亚胺/1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐体系偶联反应所制备得到的。在反应起始阶段，1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)通过与丝素蛋白分子结构中的天冬氨酸和谷氨酸残基上的羧基进行反应形成一种促发剂——不稳定的脲衍生物，然后与N-羟基琥珀酰亚胺(NHS)反应，形成更稳定的酯从而增强碳二亚胺交联产物的水稳定性，同时此时羧基处于被活化状态。在羧基被活化后，半胱胺盐酸盐上的氨基会与此时被活化的羧基发生反应从而形成酰胺键，半胱胺被成功接枝于丝素蛋白分 子之上。同时反应过程中1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺转变成水溶性的脲衍生物，可以在后面的透析过程中取出，以保留丝素蛋白良好的生物相容性。
二硫键的氧化还原电位比较低，表观还原电位在0.6伏特左右，容易发生可逆的氧化还原反应。丝素蛋白接枝上巯基以后，在空气中氧气的存在下，巯基被氧化从而在丝素蛋白分子链间形成二硫键交联点。这种二硫键形成的交联点是可逆的，在还原性条件下会被断开变回巯基，分子链间交联点会断开。在未通电情况下，丝素蛋白分子链间交联程度较高，从而使丝素微针溶胀率较小；而在通电状态下，电流提供了还原性环境，二硫键得到电子发生电化学还原反应，二硫键断开形成巯基，丝素蛋白分子链间共价交联点减少，丝素微针溶胀率增大。这种溶胀度的变化可以通过开关电源的方式来控制。因此获得电响应性丝素蛋白材料，通过对电流的响应，达到控制溶胀度变化的目的，进而可以控制微针中药物的释放速度。
由于采用了以上的技术方案，本发明与现有技术相比具有如下的有益之处：本发明的电响应型丝素蛋白材料的制备方法，制备得到的电响应型丝素蛋白材料能够实现低电压刺激下溶胀度的变化，在通电条件下具备高溶胀特性，在不通电的情况下具备低溶胀特性，进而实现药物的可控释放。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明优选实施例2中制备得到的微针贴片的显微镜照片；
图2为本发明实施例2-7所制备的电流响应型丝素蛋白微针贴片在通电与不通电的情况下溶胀度变化图；
结合图2和实施例2-7可以看出：N-羟基琥珀酰亚胺和1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐的过多加入会降低丝素蛋白微针贴片的电流响应性及溶胀性能，半胱胺盐酸盐添加量的增加可以提高微针贴片的溶胀度；
图3为本发明实施例2所制备的电流响应型丝素蛋白微针贴片在通电与不通电的情况下内部孔径的扫描电镜图；其中图3(a-b)是通电前微针的内部孔径电镜图，图3(c-d)是通电后微针的内部孔径电镜图，可以看到通电后，微针内部的孔径加大，有利于药物的通过；
图4为本发明实施例中电流响应型丝素蛋白微针通电前后的材料内巯基和二硫键转化的原理图；
图5为本发明实施例8中透皮给药装置的结构示意图；附图中，R1为固定电阻，R2为可调节电阻。
具体实施方式
为了使本技术领域的人员更好地理解本发明的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
实施例1丝素蛋白水溶液的制备
称取3g NaHCO3和1g Na2CO3溶于4000mL去离子水中，加热至沸腾，放入80g蚕丝，保持98℃微沸30min，取出后用去离子水清洗。重复上述步骤三次后放置于60℃烘箱中干燥得到脱胶的丝素纤维。
配制9.3mol/L的LiBr溶液，取100mL的LiBr溶液水浴加热至65℃，分多次放入脱胶丝素15g搅拌溶解后，继续加热并搅拌40min。液体装入透析袋中放置于去离子水中透析72h，每隔2h更换去离子水。透析后溶液经过过滤获得丝素蛋白水溶液。
实施例2
本实施例中电响应型丝素蛋白微针的制备方法具体包括以下步骤：
1)巯基化丝素蛋白溶液的制备
将丝素蛋白水溶液浓度稀释至30mg/mL，之后将溶液烧杯在冰浴中稳定至2℃，使用2-(N-吗啉代)乙磺酸溶液将丝素蛋白溶液pH值调至5.5。先分别向上述丝素蛋白溶液中缓慢加入5wt％(相对于丝素蛋白质量)的N-羟基琥珀酰亚胺，然后加入10wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.5h以活化丝素蛋白上的羧基基团。
分别向上述丝素蛋白溶液中缓慢滴加50mg/mL的半胱胺盐酸盐溶液，使得最终溶液的半胱胺盐酸盐浓度为60mmol/L，丝素蛋白的浓度为20mg/mL，后使用2-(N-吗啉代)乙磺酸溶液将最终溶液pH值调节稳定至5.5。冰浴中搅拌反应4h，取出后在4℃低温冰箱静置反应过夜。
2)巯基化丝素蛋白溶液的透析
将反应完成的丝素蛋白溶液装入透析袋(截留分子量为8-14kDa)中进行透析，透析环境为在去离子水中加入少量硫代硫酸钠(0.001mol/L)并通入惰性气体N2保护。每4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d后换用未添加有硫代硫酸钠的去离子水透析1d。透析完毕后，离心取上清液获得接枝改性后的巯基化丝素溶液。然后测量丝素蛋白溶液的质量浓度(wt％)，置于4℃冰箱保存备用。改性后丝素蛋白溶液中的巯基含量为92.9±5.7μmol/g。
3)丝素蛋白微针的制备
取1mL巯基化丝素蛋白溶液，浇注在单块PDMS(二甲基硅氧烷)微针模具中，在真空干燥箱中抽真空去除气泡，重复三次。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)，空气流通处下干燥8h，脱模后即获得丝素蛋白微针。微针在未通电情况下1小时的溶胀率为72±3.63％，在电压0.6V下通 电1小时的溶胀率为227±13.86％。
实施例2-1
本实施例中具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法具体包括以下步骤：
采用实施例2中记载的步骤和参数进行1)、巯基化丝素蛋白溶液的制备和2)、巯基化丝素蛋白溶液的透析。
3)胰岛素溶液的制备
称取胰岛素于离心管中，先加入0.01M的盐酸溶液，震荡混匀，充分溶解胰岛素。再加入0.02M的Tris缓冲溶液，使溶液的pH为7.1，胰岛素浓度为10mg/mL。
4)混合溶液的制备
取上述步骤中制备的胰岛素溶液缓慢加入到巯基化丝素蛋白溶液中，使得混合溶液中巯基化丝素蛋白的浓度为20mg/mL，胰岛素的浓度为5mg/mL。
5)胰岛素微针贴片的制备
取3.5mL混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)干燥，干燥后脱模后即获得电致响应性的丝素蛋白胰岛素微针。在0.6V电压通电的情况下，胰岛素的最大释放速度为89.03±7.34μg/h，未通电下的胰岛素最大释放速度为46.34±7.98μg/h。
实施例3
本实施例中电响应型丝素蛋白微针的制备方法具体包括以下步骤：
1)巯基化丝素蛋白溶液的制备
将丝素蛋白水溶液浓度稀释至20mg/mL，之后将溶液烧杯在冰浴中稳定至3℃，使用2-(N-吗啉代)乙磺酸溶液将丝素蛋白溶液pH值调至5。先分别向上述丝素蛋白溶液中缓慢加入5wt％(相对于丝素蛋白质量)的N-羟基琥珀酰亚胺，然后加入8wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.6h以活化丝素蛋白上的羧基基团。
分别向上述丝素蛋白溶液中缓慢滴加60mg/mL的半胱胺盐酸盐溶液，使得最终溶液的半胱胺盐酸盐浓度为40mmol/L，丝素蛋白的浓度为20mg/mL，后使用2-(N-吗啉代)乙磺酸溶液将最终溶液pH值调节稳定至5。冰浴中搅拌反应4h，取出后在4℃低温冰箱静置反应过夜。
2)巯基化丝素蛋白溶液的透析
将反应完成的丝素蛋白溶液装入透析袋(截留分子量为8-14kDa)中进行透析，透析环境为在去离子水中加入少量硫代硫酸钠(0.0015mol/L)并通入惰性气体N2保护。每4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d后换用未添加有硫代硫酸钠的去离子水透析1d。透析完毕后，离心取上清液获得接枝改性后的巯基化丝素溶液。然后测量丝素蛋白溶液的质量浓度(wt％)，置于4℃冰箱保存备用。改性后丝素蛋白溶液中的巯基含量为23.3±2.2μmol/g。
3)丝素蛋白微针的制备
取1mL的(2)中制备的巯基化丝素蛋白溶液，浇注在单块PDMS微针模具中，在真空干燥箱中抽真空去除气泡，重复三次。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)，空气流通处下干燥6h，脱模后即得丝素蛋白微针。微针在未通电情况下1小时的溶胀率为84±5.76％，在电压0.6V通电下1小时的溶胀率为163±7.81％。
实施例3-1
本实施例中具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法具体包 括以下步骤：
采用实施例3中记载的步骤和参数进行1)、巯基化丝素蛋白溶液的制备和2)、巯基化丝素蛋白溶液的透析。
3)胰岛素溶液的制备
称取胰岛素于离心管中，先加入0.05M的盐酸溶液，震荡混匀，充分溶解胰岛素。再加入0.01M的Tris缓冲溶液，使溶液的pH为6.7，胰岛素浓度为15mg/mL。
4)混合溶液的制备
取上述步骤中制备的胰岛素溶液缓慢加入到巯基化丝素蛋白溶液中，使得混合溶液中巯基化丝素蛋白的浓度为20mg/mL，胰岛素的浓度为3mg/mL。
5)胰岛素微针贴片的制备
取3.5mL混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)干燥，干燥后脱模后即获得电致响应性的丝素蛋白胰岛素微针。在0.6V电压通电的情况下，胰岛素的最大释放速度为43.08±1.99μg/h，未通电下的胰岛素最大释放速度为27.74±2.98μg/h。
实施例4
本实施例中电响应型丝素蛋白微针的制备方法具体包括以下步骤：
1)巯基化丝素蛋白溶液的制备
将丝素蛋白水溶液浓度稀释至20mg/mL，之后将溶液烧杯在冰浴中稳定至2℃，使用2-(N-吗啉代)乙磺酸溶液将丝素蛋白溶液pH值调至5.5。先分别向上述丝素蛋白溶液中缓慢加入2.5wt％(相对于丝素蛋白质量)的N-羟基琥珀酰亚胺，然后加入4wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.8h以活化丝素蛋白上的羧基基团。
分别向上述丝素蛋白溶液中缓慢滴加55mg/mL的半胱胺盐酸盐溶液，使得最终溶液的半胱胺盐酸盐浓度为80mmol/L，丝素蛋白的浓度为20mg/mL，后使用2-(N-吗啉代)乙磺酸溶液将最终溶液pH值调节稳定至5.5。冰浴中搅拌反应4h，取出后在4℃低温冰箱静置反应过夜。
2)巯基化丝素蛋白溶液的透析
将反应完成的丝素蛋白溶液装入透析袋(截留分子量为8-14kDa)中进行透析，透析环境为在去离子水中加入少量硫代硫酸钠(0.0015mol/L)并通入惰性气体N2保护。每4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d后换用未添加有硫代硫酸钠的去离子水透析1d。透析完毕后，离心取上清液获得接枝改性后的巯基化丝素溶液。然后测量丝素蛋白溶液的质量浓度(wt％)，置于4℃冰箱保存备用。改性后丝素蛋白溶液中的巯基含量为48.3±2.9μmol/g。
3)丝素蛋白微针的制备
取1mL的(2)中制备的巯基化丝素蛋白溶液，浇注在单块PDMS微针模具中，在真空干燥箱中抽真空去除气泡，重复三次。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)，空气流通处下干燥6h，脱模后即得丝素蛋白微针。微针在未通电情况下1小时的溶胀率为105±5.53％，在电压0.6V通电下1小时的溶胀率为182±8.32％。
实施例4-1
本实施例中具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法具体包括以下步骤：
采用实施例4中记载的步骤和参数进行1)、巯基化丝素蛋白溶液的制备和2)、巯基化丝素蛋白溶液的透析。
3)胰岛素溶液的制备
称取胰岛素于离心管中，先加入0.01M的盐酸溶液，震荡混匀，充分溶解胰岛素。再加入0.01M的Tris缓冲溶液，使溶液的pH为7.0，胰岛素浓度为15mg/mL。
4)混合溶液的制备
取上述步骤中制备的胰岛素溶液缓慢加入到巯基化丝素蛋白溶液中，使得混合溶液中巯基化丝素蛋白的浓度为30mg/mL，胰岛素的浓度为2.5mg/mL。
5)胰岛素微针贴片的制备
取3.5mL混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)干燥，干燥后脱模后即获得电致响应性的丝素蛋白胰岛素微针。在0.6V电压通电的情况下，胰岛素的最大释放速度为34.38±2.74μg/h，未通电下的胰岛素最大释放速度为26.64±1.78μg/h。
实施例5
本实施例中电响应型丝素蛋白微针的制备方法具体包括以下步骤：
1)巯基化丝素蛋白溶液的制备
将丝素蛋白水溶液浓度稀释至25mg/mL，之后将溶液烧杯在冰浴中稳定至2℃，使用2-(N-吗啉代)乙磺酸溶液将丝素蛋白溶液pH值调至6。先分别向上述丝素蛋白溶液中缓慢加入5wt％(相对于丝素蛋白质量)的N-羟基琥珀酰亚胺，然后加入7.5wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.5h以活化丝素蛋白上的羧基基团。
分别向上述丝素蛋白溶液中缓慢滴加60mg/mL的半胱胺盐酸盐溶液，使得最终溶液的半胱胺盐酸盐浓度为20mmol/L，丝素蛋白的浓度为20mg/mL，后使用2-(N-吗啉代)乙磺酸溶液将最终溶液pH值调节稳定至6。冰浴中搅拌反应4h，取出后在4℃低温冰箱静置反应过夜。
2)巯基化丝素蛋白溶液的透析
将反应完成的丝素蛋白溶液装入透析袋(截留分子量为8-14kDa)中进行透析，透析环境为在去离子水中加入少量硫代硫酸钠(0.001mol/L)并通入惰性气体N2保护。每4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d后换用未添加有硫代硫酸钠的去离子水透析1d。透析完毕后，离心取上清液获得接枝改性后的巯基化丝素溶液。然后测量丝素蛋白溶液的质量浓度(wt％)，置于4℃冰箱保存备用。改性后丝素蛋白溶液中的巯基含量为41.2±3.1μmol/g。
3)丝素蛋白微针的制备
取1mL的(2)中制备的巯基化丝素蛋白溶液，浇注在单块PDMS微针模具中，在真空干燥箱中抽真空去除气泡，重复三次。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)，空气流通处下干燥6h，脱模后即得丝素蛋白微针。微针在未通电情况下1小时的溶胀率为97±3.87％，在电压0.6V通电下1小时的溶胀率为132±6.21％。
实施例5-1
本实施例中具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法具体包括以下步骤：
采用实施例5中记载的步骤和参数进行1)、巯基化丝素蛋白溶液的制备和2)、巯基化丝素蛋白溶液的透析。
3)胰岛素溶液的制备
称取胰岛素于离心管中，先加入0.01M的盐酸溶液，震荡混匀，充分溶解胰岛素。再加入0.04M的Tris缓冲溶液，使溶液的pH为7.2，胰岛素浓度为 25mg/mL。
4)混合溶液的制备
取上述步骤中制备的胰岛素溶液缓慢加入到巯基化丝素蛋白溶液中，使得混合溶液中巯基化丝素蛋白的浓度为25mg/mL，胰岛素的浓度为4.5mg/mL。
5)胰岛素微针贴片的制备
取3.5mL混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)干燥，干燥后脱模后即获得电致响应性的丝素蛋白胰岛素微针。在0.6V电压通电的情况下，胰岛素的最大释放速度为42.94±5.21μg/h，未通电下的胰岛素最大释放速度为33.78±0.86μg/h。
实施例6
本实施例中电响应型丝素蛋白微针的制备方法具体包括以下步骤：
1)巯基化丝素蛋白溶液的制备
将丝素蛋白水溶液浓度稀释至30mg/mL，之后将溶液烧杯在冰浴中稳定至2℃，使用2-(N-吗啉代)乙磺酸溶液将丝素蛋白溶液pH值调至5。先分别向上述丝素蛋白溶液中缓慢加入10wt％(相对于丝素蛋白质量)的N-羟基琥珀酰亚胺，然后加入2.5wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.5h以活化丝素蛋白上的羧基基团。
分别向上述丝素蛋白溶液中缓慢滴加40mg/mL的半胱胺盐酸盐溶液，使得最终溶液的半胱胺盐酸盐浓度为60mmol/L，丝素蛋白的浓度为30mg/mL，后使用2-(N-吗啉代)乙磺酸溶液将最终溶液pH值调节稳定至5。冰浴中搅拌反应4h，取出后在4℃低温冰箱静置反应过夜。
2)巯基化丝素蛋白溶液的透析
将反应完成的丝素蛋白溶液装入透析袋(截留分子量为8-14kDa)中进行透析，透析环境为在去离子水中加入少量硫代硫酸钠(0.0015mol/L)并通入惰性气体N2保护。每4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d后换用未添加有硫代硫酸钠的去离子水透析1d。透析完毕后，离心取上清液获得接枝改性后的巯基化丝素溶液。然后测量丝素蛋白溶液的质量浓度(wt％)，置于4℃冰箱保存备用。改性后丝素蛋白溶液中的巯基含量为53.3±2.6μmol/g。
3)丝素蛋白微针的制备
取1mL的(2)中制备的巯基化丝素蛋白溶液，浇注在单块PDMS微针模具中，在真空干燥箱中抽真空去除气泡，重复三次。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)，空气流通处下干燥6h，脱模后即得丝素蛋白微针。微针在未通电情况下1小时的溶胀率为115±6.27％，在电压0.6V通电下1小时的溶胀率为176±7.53％。
实施例6-1
本实施例中具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法具体包括以下步骤：
采用实施例6中记载的步骤和参数进行1)、巯基化丝素蛋白溶液的制备和2)、巯基化丝素蛋白溶液的透析。
3)胰岛素溶液的制备
称取胰岛素于离心管中，先加入0.05M的盐酸溶液，震荡混匀，充分溶解胰岛素。再加入0.05M的Tris缓冲溶液，使溶液的pH为7.1，胰岛素浓度为40mg/mL。
4)混合溶液的制备
取上述步骤中制备的胰岛素溶液缓慢加入到巯基化丝素蛋白溶液中，使得 混合溶液中巯基化丝素蛋白的浓度为30mg/mL，胰岛素的浓度为7.5mg/mL。
5)胰岛素微针贴片的制备
取3.5mL混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)干燥，干燥后脱模后即获得电致响应性的丝素蛋白胰岛素微针。在0.6V电压通电的情况下，胰岛素的最大释放速度为120.19±11.78μg/h，未通电下的胰岛素最大释放速度为79.93±7.45μg/h。
实施例7
本实施例中电响应型丝素蛋白微针的制备方法具体包括以下步骤：
1)巯基化丝素蛋白溶液的制备
将丝素蛋白水溶液浓度稀释至20mg/mL，之后将溶液烧杯在冰浴中稳定至2℃，使用2-(N-吗啉代)乙磺酸溶液将丝素蛋白溶液pH值调至5.5。先分别向上述丝素蛋白溶液中缓慢加入8wt％(相对于丝素蛋白质量)的N-羟基琥珀酰亚胺，然后加入20wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.5h以活化丝素蛋白上的羧基基团。
分别向上述丝素蛋白溶液中缓慢滴加50mg/mL的半胱胺盐酸盐溶液，使得最终溶液的半胱胺盐酸盐浓度为60mmol/L，丝素蛋白的浓度为20mg/mL，后使用2-(N-吗啉代)乙磺酸溶液将最终溶液pH值调节稳定至5.5。冰浴中搅拌反应4h，取出后在4℃低温冰箱静置反应过夜。
2)巯基化丝素蛋白溶液的透析
将反应完成的丝素蛋白溶液装入透析袋(截留分子量为8-14kDa)中进行透析，透析环境为在去离子水中加入少量硫代硫酸钠(0.001mol/L)并通入惰性气体N2保护。每4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序2d后换用未添加有硫代硫酸钠的去离子水透析1d。透析完毕后，离心取上清液获得接枝改性后的巯基化丝素溶液。然后测量丝素蛋白溶液的质量浓度(wt％)，置于4℃冰箱保存备用。改性后丝素蛋白溶液中的巯基含量为63.3±4.5μmol/g。
3)丝素蛋白微针的制备
取1mL的(2)中制备的巯基化丝素蛋白溶液，浇注在单块PDMS微针模具中，在真空干燥箱中抽真空去除气泡，重复三次。之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)，空气流通处下干燥6h，脱模后即得丝素蛋白微针。微针在未通电情况下1小时的溶胀率为55±6.49％，在电压0.6V通电下1小时的溶胀率为123±7.18％。
实施例7-1
本实施例中具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法具体包括以下步骤：
采用实施例7中记载的步骤和参数进行1)、巯基化丝素蛋白溶液的制备和2)、巯基化丝素蛋白溶液的透析。
3)胰岛素溶液的制备
称取胰岛素于离心管中，先加入0.02M的盐酸溶液，震荡混匀，充分溶解胰岛素。再加入0.01M的Tris缓冲溶液，使溶液的pH为7.3，胰岛素浓度为10mg/mL。
4)混合溶液的制备
取上述步骤中制备的胰岛素溶液缓慢加入到巯基化丝素蛋白溶液中，使得混合溶液中巯基化丝素蛋白的浓度为30mg/mL，胰岛素的浓度为1.5mg/mL。
5)胰岛素微针贴片的制备
取3.5mL混合溶液浇注在微针模具中，在真空干燥箱中抽真空去除气泡。 之后将脱泡后的模具系统置于恒温恒湿间(25℃、55％RH)干燥，干燥后脱模后即获得电致响应性的丝素蛋白胰岛素微针。在0.6V电压通电的情况下，胰岛素的最大释放速度为16.79±0.96μg/h，未通电下的胰岛素最大释放速度为12.62±1.17μg/h。
实施例8
如图5所示，本实施例中的透皮给药装置，包括电源、开关、报警组件、固定电阻R1、可调节电阻R2、含有药物的微针贴片以及连接在各个部件之间的电线。电源、开关、报警组件以及可调节电阻R2构成主回路，固定电阻R1与微针贴片并联后连接在该主回路上。即固定电阻R1与微针贴片并联后与电源、可调节电阻R2和开关串联。本实施例中的报警组件为指示灯。
其中，电源为直流电源，其电压范围为3～3.6V，可以选用各种小型能源输出类产品，如：3.6V可充电锂离子扣式电池(LIR系列)，3V可充锂离子扣式电池(ML或VL系列)，不充电的包括3V锂锰扣式电池(CR系列)等。
固定电阻R1的电阻大小为500Ω；可调节电阻R2的电阻调节范围为200-2000Ω。固定电阻R1和可调节电阻R2配合用于维持加载在微针贴片上的电压位于安全电压0.6～1V内。安全电压一方面需要保证使用安全，另一方面需要与微针贴片配合实现响应。
本实施例中采用的微针贴片内含有药物，其为药物载体，同时能够实现导电，用于在通电时向外释放药物，优选为电响应性微针贴片，以实现匹配电压的调节实现药物的可控释放。通过调节可调节电阻R2的电阻值，能够调节加载在微针贴片上的电压，实现药物的可控释放，同时设置固定电阻R1保证使用安全。具体的，微针贴片可以采用含有药物的导电高分子微针贴片、响应电刺激的聚合物水凝胶的微针贴片形式，优选为上述实施例中记载的丝素蛋白胰岛素微针贴片。
测试与结果
1)表1是本发明实施例2-7所制备的丝素蛋白微针中的巯基含量以及对应的通电前后的溶胀率。
溶胀率的测试方法如下，采用1小时去离子水(37℃)浸泡的方法，比较浸泡前后的质量增加率，溶胀率＝(浸泡后的质量-浸泡前的质量)/浸泡前的质量。
表1实施例2-7中丝素蛋白微针中的巯基含量以及对应的通电前后的溶胀率
结合表1和图2的结果可以看出，实施例中制备得到的电响应型丝素蛋白微针在通电后的溶胀率有明显的增长，且巯基含量越高，溶胀效果越好。
2)表2为实施例2-1至7-1中丝素蛋白胰岛素微针贴片中的巯基含量以及胰岛素释放速度在通电与未通电情况下的变化情况。
胰岛素的释放速度按照如下方法进行测定和计算：采用Franz扩散池作为体外药物接收池，32℃的0.01M PBS缓冲溶液作为模拟体液，取丝素蛋白胰岛素 微针贴片，用手将微针刺入大鼠皮肤。将皮肤平整覆盖在Franz透皮扩散池的接收室圆口处，真皮层贴于接收室，用不锈钢铁夹夹持住供体室和接收室使其稳固，透皮释药池中的溶液采取每小时定时取样，HPLC检测胰岛素验样品浓度，计算胰岛素释药速率。
表2实施例2-1至7-1中微针贴片胰岛素释放速度在通电与未通电情况下的变化
结合表2的结果可以看出，上述实施例2-1至7-1中制备的丝素蛋白胰岛素微针贴片在通电状态的胰岛素释放速度相对于未通电状态下有明显上升，即上述实施例制备得到的含有胰岛素的微针贴片能够实现胰岛素的可控释放。
以上为了便于叙述和方便理解，特将步骤进行了区分和编号，实际制备时，以上步骤可以同时进行或无先后顺序的进行。且实施例中未有特别说明的原料均通过商购获得。没有特别提及温度的操作在室温下进行。未有特别说明的操作方法与条件可采用本领域的公知或常规的手段与条件。
实施例2-7中，通过丝素蛋白分子巯基化获得一种巯基化丝素蛋白，然后浇注形成微针。这种含有巯基的丝素蛋白在电流通过时会发生巯基的氧化还原反应，造成二硫键交联度的变化，从而响应电流，形成丝素蛋白不同溶胀的状态。该方法制备的微针透皮贴片具有良好的力学性能、生物相容性以及优良的电流响应性溶胀变化，可实现电流开关下的药物控制释放。可以应用在医疗或医美中需要控制给药量的场景下。
实施例2-1至7-1中，通过丝素蛋白分子巯基化获得一种巯基化丝素蛋白，并将改性后的丝素蛋白溶液与胰岛素溶液混合，然后浇注在硅橡胶微针模具中，经过脱泡后干燥得到电致响应性的丝素蛋白胰岛素微针透皮贴片。这种含有巯基的丝素蛋白在电流通过时会发生巯基的氧化还原反应，造成二硫键交联度的变化。从而响应电流，形成丝素蛋白不同溶胀的状态，改变胰岛素的释放速度。该方法制备的胰岛素微针透皮贴片具有良好的力学性能、生物相容性以及优良的电流响应性，可实现电致响应性的胰岛素控制释放，满足患者进食前后血糖的控制。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (29)

  1. 一种电响应型丝素蛋白材料的制备方法，其特征在于，包括以下步骤：
    (1)丝素蛋白的活化：将丝素蛋白水溶液稀释至浓度为20-30mg/mL，之后将溶液的温度稳定至0-4℃，使用缓冲溶液将丝素蛋白溶液的pH值调节至5-6；向上述丝素蛋白溶液中加入相对于丝素蛋白质量1-10wt％的N-羟基琥珀酰亚胺，然后加入相对于丝素蛋白质量2-20wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.4-1h，获得活化的丝素蛋白溶液；
    (2)丝素蛋白的巯基化：向上述活化的丝素蛋白溶液中滴加浓度为40-60mg/mL的半胱胺盐酸盐溶液，并使得最终溶液中的半胱胺盐酸盐浓度为20-80mmol/L，然后使用缓冲溶液将最终溶液pH值调节至5-6；0-4℃下搅拌反应2-5h，取出后在2-8℃温度下静置反应8-10h，获得巯基化丝素蛋白溶液；
    (3)巯基化丝素蛋白的纯化：将反应完成的丝素蛋白溶液装入透析袋中进行透析；透析完毕后，离心取上清液获得纯化后的巯基化丝素蛋白溶液；
    (4)电响应型丝素蛋白材料的制备：取上述步骤中纯化的巯基化丝素蛋白溶液，浇注在模具中，并抽真空去除气泡；之后将脱泡后的模具置于恒温恒湿环境下进行干燥，干燥脱模后即获得电响应型丝素蛋白材料。
  2. 一种电响应型丝素蛋白材料的制备方法，其特征在于，包括以下步骤：
    向活化后的丝素蛋白溶液中滴加半胱胺盐酸盐溶液，调节体系pH值至5-6；0-4℃下反应2-5h，取出后在2-8℃温度下静置，获得巯基化丝素蛋白溶液；
    将巯基化丝素蛋白溶液进行透析，获得纯化后的巯基化丝素蛋白溶液；
    将纯化的巯基化丝素蛋白溶液浇注在模具中，干燥脱模后即获得电响应型丝素蛋白材料。
  3. 根据权利要求2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述丝素蛋白的活化通过如下步骤进行：将丝素蛋白水溶液稀释至浓度为20-30mg/mL，之后将溶液的温度稳定至0-4℃，并调节pH值至5-6；向上述丝素蛋白溶液中加入相对于丝素蛋白质量1-10wt％的N-羟基琥珀酰亚胺，然后加入相对于丝素蛋白质量2-20wt％的1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐，混合均匀后反应0.4-1h，获得活化的丝素蛋白溶液。
  4. 根据权利要求2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述半胱胺盐酸盐溶液滴加浓度为40-60mg/mL；滴加完毕后溶液中的半胱胺盐酸盐浓度为20-80mmol/L。
  5. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述电响应型丝素蛋白材料中含有巯基；所述电响应型丝素蛋白材料中的巯基含量为20-100μmol/g。
  6. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述丝素蛋白材料具有电流响应性，在未通电情况下的溶胀率为50-120％，在电压0.6V下通电后的溶胀率为120-250％。
  7. 根据权利要求1或3所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述N-羟基琥珀酰亚胺与丝素蛋白的质量比为1:100-1:10；1-(3-二甲基氨基丙基)-3-乙基碳二亚胺盐酸盐与丝素蛋白的质量比为1:50-1:5；半胱胺盐酸盐与丝素蛋白的质量比为1:100-1:2.5。
  8. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述透析时在透析用去离子水中加入硫代硫酸钠并通入惰性气体进行保护；每2-4h更换一次添加硫代硫酸钠的去离子水，并重复透析程序和保持惰性气体保护；之后再换用未添加有硫代硫酸钠的去离子水进行透析。
  9. 根据权利要求8所述的电响应型丝素蛋白材料的制备方法，其特征在于，去离子水中硫代硫酸钠的浓度为0.001-0.0015mol/L。
  10. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，透析时采用的透析袋的截留分子量为8-14kDa。
  11. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述干燥为恒温恒湿条件，温度20-30℃、相对湿度55-65％。
  12. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，调节pH采用的缓冲溶液为选自2-(N-吗啉代)乙磺酸、甘氨酸-盐酸、柠檬酸-柠檬酸钠、乙酸-乙酸钠、邻苯二甲酸氢钾-氢氧化钠、Tris-盐酸缓冲液中的一种。
  13. 根据权利要求1或2所述的电响应型丝素蛋白材料的制备方法，其特征在于，所述丝素蛋白水溶液以家蚕丝为原料，通过脱胶、溶解、透析后获得。
  14. 一种采用如权利要求1-13任意一项所述的制备方法制备得到的电响应型丝素蛋白材料。
  15. 一种电响应型丝素蛋白微针的制备方法，其特征在于，包括如下步骤：依次采用如权利要求1-13任意一项所述的丝素蛋白的活化、丝素蛋白的巯基化以及巯基化丝素蛋白的纯化步骤后得到纯化的巯基化丝素蛋白，将其浇注在微针模具中，在真空干燥箱中抽真空去除气泡；之后将脱泡后的模具系统置于恒温恒湿环境下进行干燥，干燥脱模后即获得电响应型丝素蛋白微针。
  16. 一种采用如权利要求15所述的制备方法制备得到的电响应型丝素蛋白微针。
  17. 一种具有电致响应性的丝素蛋白胰岛素微针贴片的制备方法，其特征在于，包括以下步骤：
    依次采用如权利要求1-13任意一项所述的丝素蛋白的活化、丝素蛋白的巯基化以及巯基化丝素蛋白的纯化步骤后得到纯化的巯基化丝素蛋白；
    胰岛素溶液的配置：取胰岛素于离心管中，先加入0.01M-0.05M的盐酸溶液，混匀后充分溶解胰岛素，使溶液中的胰岛素浓度为10-50mg/mL；再加入0.01M-0.05M的缓冲溶液，调整溶液的pH为6.7-7.4，形成胰岛素溶液；
    混合溶液的制备：取上述步骤中制备的胰岛素溶液加入到巯基化丝素蛋白溶液中，搅拌均匀，使得混合溶液中巯基化丝素蛋白的浓度为10-40mg/mL，胰岛素的浓度为1-10mg/mL；
    胰岛素微针贴片的制备：取混合溶液浇注在微针模具中，并抽真空去除气泡，将脱泡后的模具系统置于恒温恒湿环境下干燥，干燥脱模后即获得具有电致响应性的丝素蛋白胰岛素微针贴片。
  18. 根据权利要求17所述的丝素蛋白胰岛素微针贴片的制备方法，其特征在于，所述丝素蛋白胰岛素微针贴片中的胰岛素含量为10-100mg/g，巯基含量为10-100μmol/g。
  19. 一种采用如权利要求17或18所述的制备方法制备得到的具有电致响应性的丝素蛋白胰岛素微针贴片。
  20. 根据权利要求19所述的丝素蛋白胰岛素微针贴片，其特征在于，所述丝素蛋白胰岛素微针贴片中的丝素蛋白和胰岛素的质量比为4～20:1。
  21. 一种透皮给药装置，其特征在于，包括电源、开关、固定电阻、可调节电阻以及含有药物的微针贴片，所述电源、开关以及可调节电阻构成主回路；所述固定电阻与所述微针贴片并联后串联连接在所述主回路上，所述微针贴片用于在通电时向外释放药物；所述微针贴片为权利要求16所述的电响应型丝素蛋白微针或权利要求19或20所述的具有电致响应性的丝素蛋白胰岛素微针贴片。
  22. 根据权利要求21所述的一种透皮给药装置，其特征在于：所述电源为 直流电源。
  23. 根据权利要求22所述的一种透皮给药装置，其特征在于：所述电源的电压为3V～3.6V。
  24. 根据权利要求23所述的一种透皮给药装置，其特征在于：所述电源为可充电扣式锂电池或不可充电的扣式锂锰电池。
  25. 根据权利要求21所述的一种透皮给药装置，其特征在于：所述固定电阻的电阻大小为200-600Ω。
  26. 根据权利要求21所述的一种透皮给药装置，其特征在于：所述可调节电阻的电阻调节范围为200-2000Ω。
  27. 根据权利要求21所述的一种透皮给药装置，其特征在于：所述固定电阻和可调节电阻用于维持加载在所述微针贴片上的电压位于安全电压内。
  28. 根据权利要求27所述的一种透皮给药装置，其特征在于：所述安全电压为0.6～1V。
  29. 根据权利要求21-28任意一项所述的一种透皮给药装置，其特征在于：所述主回路上设置有报警组件，所述报警组件为指示灯和/或蜂鸣器。
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US12350460B2 (en) 2022-03-16 2025-07-08 Pharsun Medical Biotechnics (Shanghai) Co., Ltd. Electro-responsive silk fibroin material, electro-responsive silk fibroin microneedle, insulin microneedle patch having electro-responsive silk fibroin and preparation method therefor, and transdermal drug delivery device
CN120501932A (zh) * 2025-04-27 2025-08-19 浙江来益美生物医药有限公司 一种丝素蛋白复合凝胶及其制备方法

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