Protein-based oral fast-dissolving nanofiber films and preparation and use thereof

Abstract

Disclosed is a protein-based oral dissolving nanofiber film, comprising the following components: 20-35 wt% protein, 40-60 wt% hydroxypropyl methylcellulose, and 15-30 wt% polyethylene oxide, based on the total mass of the nanofiber film. The oral dissolving nanofiber film of the present application can load natural polyphenol active ingredients, and improve the stability of the active ingredients under light, heat and oxidation conditions through protein-polyphenol interaction. The nanofiber film can quickly disintegrate and release the active ingredients in a simulated saliva environment, has good mechanical properties, flexibility and biocompatibility, and can be used in the fields of oral health maintenance, local drug delivery and functional care film.

Description

Technical Field

[0001] This invention belongs to the field of functional oral membrane materials technology, specifically, it relates to a protein-based oral fast-dissolving nanofiber membrane, and further relates to its preparation method and application. Background Technology

[0002] Oral instant films are thin-film formulations that rapidly disintegrate and release active ingredients in the oral environment. They are convenient to take, have a rapid onset of action, and good patient compliance, and are widely used in oral care, functional foods, and topical drug delivery. Currently, most oral instant films use high-molecular materials such as polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), and sodium carboxymethyl cellulose as film-forming substrates, and are prepared through solution casting or hot pressing. Their film structures are mostly dense planar structures with limited specific surface area, resulting in insufficient ability to regulate the release rate of active ingredients.

[0003] In recent years, oral fast-dissolving nanofiber membranes have been prepared by electrospinning synthetic polymer materials. However, most existing electrospinned oral membrane systems are based on single synthetic polymers, which have limitations in terms of biocompatibility.

[0004] On the other hand, milk proteins or casein are used as carrier materials for the encapsulation and stabilization of polyphenols or functional components. Casein micelle structures are used to improve polyphenol stability, but these applications are mostly concentrated in emulsion systems or powder carriers, and have not formed nanofiber structures suitable for rapid dissolution and release in the oral cavity. Natural polyphenols are prone to degradation under light, heat, and oxidation, resulting in uneven release or insufficient stability.

[0005] Furthermore, the dispersibility of casein in solvent systems and its fiber-forming stability after blending with other polymers still present certain challenges. The impact of differences in molecular structure, flexibility, and interfacial activity among different protein types on fiber formation behavior, fiber diameter distribution, and membrane mechanical properties during electrospinning lacks systematic research, especially in isopropanol-water mixed solvent systems, where the influence of protein content and polymer ratio on the fiber-forming window needs further clarification.

[0006] CN116211833A discloses an imidazolidin oral quick-dissolving film and its preparation method. However, the film has a dense layered planar film structure and does not have high specific surface area and interconnected porous characteristics. It can be used for small molecule chemical drugs with good stability, but it is not suitable for natural active substances (such as polyphenols) that are sensitive to light, heat and oxygen.

[0007] CN112996487A discloses a rapidly disintegrating oral membrane matrix, which uses hydroxypropyl methylcellulose as the film-forming polymer, combined with a rapidly dissolving binder and a moisture-proof polymer, and is prepared by slurry extrusion curing. This membrane matrix is ​​still a traditional dense cast membrane, and the protection of sensitive active ingredients relies on the moisture-proof coating material. It does not improve the loading stability of active ingredients at the carrier structure level, and it is difficult to balance biocompatibility with natural components with rapid disintegration and uniform release performance.

[0008] To address the aforementioned issues, this invention provides a protein-based oral fast-dissolving nanofiber membrane prepared by electrospinning, which has a stable structure, good biocompatibility, improved stability of active substance loading, and improved release performance. Summary of the Invention

[0009] The purpose of this invention is to provide a protein-based oral fast-dissolving nanofiber membrane, which has improved stability of active substance loading and release performance, and is suitable for rapid dissolution and release in the oral cavity.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A protein-based oral fast-dissolving nanofiber membrane comprises: protein, polyethylene oxide, and hydroxypropyl methylcellulose, wherein the protein, polyethylene oxide, and hydroxypropyl methylcellulose are 20-35 wt%, 15-30 wt%, and 40-60 wt% respectively by mass percentage, based on the total mass of the nanofiber membrane.

[0011] The protein-based oral fast-dissolving nanofiber membrane has a three-dimensional network structure, formed by continuous and uniform nanofibers interwoven together. The average diameter of the nanofibers is 100–800 nm.

[0012] The protein may be selected from one or more of casein, β-casein, collagen, silk fibroin, and milk protein. Preferably, the protein is casein or β-casein; most preferably, it is β-casein.

[0013] The protein-based oral fast-dissolving nanofiber membrane of this invention exhibits a complete disintegration time of 12–45 s under simulated saliva conditions, thereby rapidly releasing the active ingredients. For polyphenolic active substances loaded onto this membrane, such as epigallocatechin gallate, quercetin, and rutin, it demonstrates good loading stability and rapid oral release performance.

[0014] This invention further relates to a method for preparing the protein-based oral fast-dissolving nanofiber membrane, comprising the following steps: S1. Dissolve the protein components in deionized water and stir at room temperature until fully dissolved to form a protein aqueous solution; S2. Dissolve polyethylene oxide and hydroxypropyl methylcellulose in isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. S3. Slowly add the polymer solution obtained in step S2 to the protein aqueous solution obtained in step S1, and continue stirring at room temperature until a uniform transparent or semi-transparent spinning solution is formed. S4. The spinning solution obtained in step S3 is loaded into an electrospinning device for electrospinning and collected by a collector to obtain a protein-based oral fast-dissolving nanofiber membrane. S5. The obtained nanofiber membrane is naturally dried at room temperature to obtain the finished product of protein-based oral fast-dissolving nanofiber membrane.

[0015] In step S1, the stirring time is approximately 1–2 hours to ensure complete dissolution of the protein. The protein content is 1%–8% (w / v), based on the volume of deionized water.

[0016] The protein is selected from casein or β-casein, preferably β-casein. There are no restrictions on the source of casein and β-casein; they can be obtained commercially or prepared by conventional membrane filtration.

[0017] In step S2, the content of polyethylene oxide is 2% to 7% (w / v), and the content of hydroxypropyl methylcellulose is 2% to 7% (w / v), based on the volume of the solvent isopropanol.

[0018] In step S3, the volume ratio of the polymer solution to the protein aqueous solution is 1:1. The stirring time is approximately 12–20 hours.

[0019] In step S4, the electrospinning equipment can be a single-needle electrospinning machine or a multi-nozzle electrospinning machine. The electrospinning is carried out under the following conditions: ambient humidity 18%–22%, ambient temperature 20–30℃, and voltage 20–30 kV; the distance from the nozzle to the receiving device is 10–20 cm, and the feed rate (i.e., the liquid pushing speed) is 0.2–1.0 mL / h. The collector can be an aluminum foil plate or a rotary drum collector.

[0020] In the method of this invention, the polyphenolic active substance to be loaded can be added in step S3 and uniformly dispersed in the spinning solution, so that after the subsequent spinning step, the active substance is loaded onto the oral instant nanofiber membrane. The amount of the polyphenolic active substance can be 0.5 to 2 wt%, based on the mass of the oral instant nanofiber membrane.

[0021] The present invention further relates to the use of the protein-based oral fast-dissolving nanofiber membrane, which can be well applied in the fields of oral health maintenance, local drug delivery, oral antibacterial, antioxidant and oral microecological improvement, for example as an oral health maintenance membrane, a local drug delivery membrane, an oral antibacterial membrane, an antioxidant membrane and an oral microecological improvement membrane.

[0022] Compared with existing technologies, the nanofiber membrane of this invention improves the loading stability of active substances at the carrier structure level, especially for the effective loading of polyphenolic natural active substances. The nanofiber membrane of this invention uses protein as a carrier material, leveraging its excellent biocompatibility to effectively improve the loading stability of active substances. For example, casein has good compatibility with oral mucosa, reducing immune rejection reactions. Furthermore, the method of this invention uses an isopropanol-water mixed solvent system, which improves the fiber-forming stability of natural proteins during electrospinning, forming a stable nanostructure and obtaining a uniformly structured nanofiber membrane, thus improving the loading stability of polyphenolic active substances and the rapid oral release performance. The protein-based oral fast-dissolving nanofiber membrane of this invention can rapidly disintegrate in a simulated saliva environment, with a complete disintegration time as fast as 14 seconds, thereby rapidly releasing the loaded active ingredients. For example, EGCG exhibits explosive release within 0–15 minutes and diffusion-controlled release within 5–120 minutes. Furthermore, the nanofiber membrane of this invention possesses good mechanical properties and flexibility; for example, its tensile strength can reach 31 and its elongation at break can reach 21. Detailed Implementation

[0023] To more clearly illustrate the purpose, technical solution, and advantages of the present invention, the following embodiments are provided for further detailed explanation. These embodiments are merely exemplary and do not constitute any limitation on the present invention. The equipment and raw materials used in the embodiments are commercially available. The electrospinning machine used in the embodiments was purchased from Beijing Xinrui Baina Technology Co., Ltd. as a TEADFS-100.

[0024] Example 1: Preparation of protein-based oral fast-dissolving nanofiber membrane Add 4 grams of β-casein to 100 ml of deionized water and stir magnetically for 30 minutes at room temperature to fully dissolve the β-casein and form a homogeneous protein aqueous solution. Dissolve 6 g of hydroxypropyl methylcellulose and 4 g of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; the mixture was stirred continuously at room temperature for 20 h to obtain a uniform spinning solution. The obtained spinning solution was loaded into an electrospinning apparatus, and the distance from the nozzle to the collection device was set to 15 cm. Electrospinning was carried out at a push rate of 0.5 mL / h under a voltage of 25 kV and an ambient humidity of 20%. The solution was collected with an aluminum foil plate to obtain a nanofiber membrane. The obtained nanofiber membrane was naturally dried at room temperature to obtain a protein-based oral fast-dissolving nanofiber membrane M1.

[0025] Example 2: Preparation of protein-based oral fast-dissolving nanofiber membrane Add 4 grams of β-casein to 100 ml of deionized water and stir at room temperature for 30 minutes to fully dissolve the β-casein and form a homogeneous protein aqueous solution. Dissolve 5 g of hydroxypropyl methylcellulose and 5 g of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; stirring was continued at room temperature for 20 h to obtain a uniform spinning solution. The obtained spinning solution was loaded into a multi-nozzle electrospinning apparatus, and the distance between the nozzle and the collection device was set to 15 cm. Electrospinning was carried out at a push rate of 0.5 mL / h under a voltage of 25 kV and an ambient humidity of 20%. The solution was collected with an aluminum foil plate to obtain a nanofiber membrane. The resulting nanofiber membrane was naturally dried at room temperature to obtain a protein-based oral fast-dissolving nanofiber membrane M2.

[0026] Example 3: Preparation of protein-based oral fast-dissolving nanofiber membranes loaded with EGCG Add 4 grams of β-casein to 100 ml of deionized water and stir at room temperature for 30 minutes to fully dissolve it and form a homogeneous protein aqueous solution. Dissolve 6 g of hydroxypropyl methylcellulose and 4 g of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; stirring was continued at room temperature for 20 h to form a uniform spinning solution. Add 0.15 g of epigallocatechin gallate (EGCG) to the spinning solution and stir until homogeneous; The obtained spinning solution was loaded into a multi-nozzle electrospinning apparatus, the distance between the nozzle and the collection device was set to 15 cm, and electrospinning was carried out at a push rate of 0.5 mL / h under a voltage of 25 kV and an ambient humidity of 20%. The solution was collected with an aluminum foil plate to obtain a nanofiber membrane. The obtained nanofiber membrane was naturally dried at room temperature to obtain EGCG-loaded protein-based oral fast-dissolving nanofiber membrane M3.

[0027] Example 4: Preparation of protein-based oral fast-dissolving nanofiber membrane Add 2 grams of β-casein to 100 ml of deionized water and stir magnetically for 30 minutes at room temperature to fully dissolve the β-casein and form a homogeneous protein aqueous solution. Dissolve 6 g of hydroxypropyl methylcellulose and 2 g of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; the mixture was stirred continuously at room temperature for 20 h to obtain a homogeneous spinning solution. The obtained spinning solution was loaded into an electrospinning apparatus, and the distance from the nozzle to the collection device was set to 15 cm. Electrospinning was carried out at a push rate of 0.5 mL / h under a voltage of 25 kV and an ambient humidity of 20%. The solution was collected with an aluminum foil plate to obtain a nanofiber membrane. The obtained nanofiber membrane was naturally dried at room temperature to obtain protein-based oral fast-dissolving nanofiber membrane M4.

[0028] Example 5: Preparation of protein-based oral fast-dissolving nanofiber membrane Add 3 grams of β-casein to 100 ml of deionized water and stir magnetically for 30 minutes at room temperature to fully dissolve the β-casein and form a homogeneous protein aqueous solution. Dissolve 5 g of hydroxypropyl methylcellulose and 2.5 g of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; the mixture was stirred continuously at room temperature for 20 h to obtain a homogeneous spinning solution. The obtained spinning solution was loaded into an electrospinning apparatus, and the distance from the nozzle to the collection device was set to 15 cm. Electrospinning was carried out at a push rate of 0.5 mL / h under a voltage of 25 kV and an ambient humidity of 20%. The solution was collected with an aluminum foil plate to obtain a nanofiber membrane. The obtained nanofiber membrane was naturally dried at room temperature to obtain a protein-based oral fast-dissolving nanofiber membrane M5. Example 6: Preparation of a protein-based oral fast-dissolving nanofiber membrane:

[0029] Add 3.5 g of β-casein to 100 ml of deionized water and stir magnetically for 30 min at room temperature to fully dissolve the β-casein and form a homogeneous protein aqueous solution. Dissolve 4 g of hydroxypropyl methylcellulose and 2.5 g of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; the mixture was stirred continuously at room temperature for 20 h to obtain a homogeneous spinning solution. The obtained spinning solution was loaded into an electrospinning apparatus, and the distance from the nozzle to the collection device was set to 15 cm. Electrospinning was carried out at a push rate of 0.5 mL / h under a voltage of 25 kV and an ambient humidity of 20%. The solution was collected with an aluminum foil plate to obtain a nanofiber membrane. The obtained nanofiber membrane was naturally dried at room temperature to obtain protein-based oral fast-dissolving nanofiber membrane M6.

[0030] Comparative Example 1 Add 4 grams of casein to 100 ml of deionized water and stir at room temperature until fully dissolved to form a homogeneous protein solution. Dissolve 4 grams of hydroxypropyl methylcellulose in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; the mixture was stirred at room temperature for 20 h to form a homogeneous solution. The obtained solution was electrospun in a multi-nozzle electrospinning apparatus under the following conditions: voltage 25 kV, nozzle-to-collector distance 15 cm, and push rate 0.5 mL / h. The solution was collected using an aluminum foil plate to prepare a comparative nanofiber membrane. Experimental results showed that the solution in this comparative example had low viscosity, an unstable jet flow, and could not form a continuous fiber structure; therefore, no fiber membrane was obtained. Comparative Example 2:

[0031] Add 4 grams of casein to 100 ml of deionized water and stir at room temperature until fully dissolved to form a homogeneous protein solution. Dissolve 3 grams of polyethylene oxide in 100 ml of isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. The polymer solution was slowly added to the protein aqueous solution at a volume ratio of 1:1; the mixture was stirred at room temperature for 12 hours to form a homogeneous solution. The resulting solution was electrospun in a multi-nozzle electrospinning apparatus under the following conditions: voltage 25 kV, nozzle-to-collector distance 15 cm, and push rate 0.5 mL / h. The solution was collected using an aluminum foil plate to prepare a comparative nanofiber membrane.

[0032] Experimental results show that the solution in this comparative example can form fibers, but the film-forming stability on the collection substrate is poor.

[0033] The oral fast-dissolving nanofiber membranes prepared in the above examples and comparative examples were cut into small pieces of 2 cm × 5 cm with smooth and flat edges. The sample membranes were placed in the upper and lower clamps of a TA.XT Plus C texture analyzer, with the center line of the sample membrane coinciding with the center lines of the upper and lower clamps, and an initial distance of 25 mm. The tensile speed was 50 mm / min. From the start of the test until the sample membrane broke, the tensile strength and elongation at break were obtained. All samples were measured three times, and the average value was taken. The results are shown in Table 1.

[0034] Table 1: Tensile property test results of oral fast-dissolving nanofiber membranes

[0035] Preparation of phosphate-buffered saline (PBS): Dissolve 8.0 g sodium chloride, 0.2 g potassium chloride, 3.63 g disodium hydrogen phosphate dodecahydrate, and 0.24 g potassium dihydrogen phosphate in 1 L of ultrapure water, and adjust the pH to 7.4 with 0.1 mol / L NaOH. Place a 1 cm × 1 cm orally dissolving nanofiber membrane in a petri dish containing 20 mL of PBS and shake repeatedly at 37 ± 0.5 °C. Record the disintegration time when the orally dissolving membrane begins to disintegrate. Test each sample at least three times and take the average value. The results are shown in Table 2. It can be seen that the disintegration time of the oral orally dissolving nanofiber membrane of the present invention is much shorter than that of the comparative example.

[0036] Table 2: Results of Disintegration Time Measurement of Oral Fast-Dissolving Nanofiber Membranes .

Claims

1. A protein-based oral fast-dissolving nanofiber membrane comprising the following components: 20-35 wt% protein, 40-60 wt% hydroxypropyl methylcellulose and 15-30 wt% polyethylene oxide, based on the total mass of the fiber membrane.

2. The protein-based oral fast-dissolving nanofiber membrane according to claim 1, characterized in that: The protein is selected from one or more of casein, β-casein, collagen, silk fibroin, and milk protein.

3. The oral fast-dissolving nanofiber membrane according to claim 1, characterized in that: The nanofiber membrane is a three-dimensional network structure formed by continuous and uniform interwoven nanofibers; the average diameter of the nanofibers is 100–800 nm.

4. The method for preparing the protein-based oral fast-dissolving nanofiber membrane according to any one of claims 1-3, comprising the following steps: S1. Dissolve the protein components in deionized water and stir at room temperature until fully dissolved to form a protein aqueous solution; S2. Dissolve polyethylene oxide and hydroxypropyl methylcellulose in isopropanol and stir at room temperature until fully dissolved to form a homogeneous polymer solution. S3. Slowly add the polymer solution obtained in step S2 to the protein aqueous solution obtained in step S1, and continue stirring at room temperature until a uniform spinning solution is formed. S4. The spinning solution obtained in step S3 is loaded into an electrospinning device for electrospinning and collected by a collector to obtain a protein-based oral fast-dissolving nanofiber membrane. S5. The obtained nanofiber membrane is dried naturally at room temperature to obtain the protein-based oral fast-dissolving nanofiber membrane product.

5. The preparation method according to claim 4, characterized in that: In step S1, the protein content is 1% to 8% (w / v), based on the volume of deionized water.

6. The preparation method according to claim 4, characterized in that: In step S2, the content of polyethylene oxide is 2% to 7% (w / v), and the content of hydroxypropyl methylcellulose is 2% to 7% (w / v), based on the volume of isopropanol.

7. The preparation method according to claim 4, characterized in that: In step S3, the volume ratio of the polymer solution to the protein aqueous solution is 1:

1.

8. The preparation method according to claim 4, characterized in that: In step S4, the electrospinning is carried out under the conditions of ambient humidity of 18% to 22%, ambient temperature of 20 to 30°C and voltage of 20 to 30 kV; the distance from the nozzle to the receiving device is 10 to 20 cm and the feeding speed is 0.2 to 1.0 mL / h.

9. The use of the protein-based oral fast-dissolving nanofiber membrane according to any one of claims 1 to 3 in the fields of oral health maintenance, local drug delivery, oral antibacterial, antioxidant and oral microecological improvement.

10. The use according to claim 9, characterized in that: The protein-based oral fast-dissolving nanofiber membrane is used as an oral health maintenance membrane, a topical drug delivery membrane, an oral antibacterial membrane, an antioxidant membrane, and a membrane for improving the oral microecology.

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