CN116327742A

CN116327742A - Double-component multifunctional nano film and preparation method and application thereof

Info

Publication number: CN116327742A
Application number: CN202310404946.3A
Authority: CN
Inventors: 施雪涛; 梁丹; 黄海香; 武培敏; 柴牧原
Original assignee: South China University of Technology SCUT; Zhongshan Ophthalmic Center
Current assignee: South China University of Technology SCUT; Zhongshan Ophthalmic Center
Priority date: 2023-04-17
Filing date: 2023-04-17
Publication date: 2023-06-27

Abstract

The invention provides a double-component multifunctional nano film, a preparation method and application thereof, and relates to the technical field of biomedical materials, wherein the nano film is prepared from the following components in percentage by mass: 50-75% of a first polymer matrix component, 24-49% of a second polymer matrix component and 1% of a functional component; the first polymer matrix component is hyaluronic acid or a hyaluronic acid derivative, the second polymer matrix component is collagen, gelatin, methacryloylated gelatin or acellular matrix, and the functional components comprise drugs and active factors; the drug is loaded on the first polymer matrix component, and the active factor is loaded on the second polymer matrix component; the nano film is formed by chemical crosslinking. The invention overcomes the defects and the shortcomings of the existing cornea alkali burn and infection treatment means, and provides the bi-component multifunctional nano-film which has good biological safety, effectively controls infection and inhibits inflammatory reaction and is used for treating cornea injury.

Description

Double-component multifunctional nano film and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a double-component multifunctional nano film and a preparation method and application thereof.

Background

Serious cornea injury, such as cornea alkali burn and cornea infection caused by bacteria or fungi, has the difficult problems of serious tissue injury degree, large wound repair difficulty, poor clinical treatment effect and the like, and seriously damages the eyesight and life quality of patients. Immune inflammation and persistent infection are the main pathological mechanisms of corneal burns and infection blindness. Currently, there is no therapeutic agent for treating corneal alkali burn with definite curative effect, and clinically using hormone anti-inflammatory is limited by hormone-related complications such as inhibition of epithelial healing and secondary corneal thinning perforation. The amniotic membrane covering treatment has the limitations of poor amniotic membrane fixation, need for suture suturing, easy absorption and need for repeated operation. Bacterial or fungal corneal infection requires combined systemic and local antibiotic or antifungal drug treatment, however, local eye drops of antibiotic or antifungal drug have the problems of poor ocular penetration, easy drug resistance, low bioavailability, multiple drug combination, toxic and side effects caused by high-concentration and high-frequency administration, and the like. Therefore, effective drug carriers and novel materials are needed to be further researched for optimizing the ocular administration mode and improving the therapeutic effect of cornea alkali burn and infection.

Disclosure of Invention

The invention aims to provide a double-component multifunctional nano-film and a preparation method and application thereof, overcomes the defects and shortcomings of the existing cornea alkali burn and infection treatment means, has good biological safety, effectively controls infection and inhibits inflammatory reaction, and can be used for treating cornea injury such as infection and alkali burn, and a preparation method thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a double-component multifunctional nano film which is prepared from the following components in percentage by mass: 50-75% of a first polymer matrix component, 24-49% of a second polymer matrix component and 1% of a functional component; the first polymer matrix component is hyaluronic acid or a hyaluronic acid derivative, the second polymer matrix component is collagen, gelatin, methacryloylated gelatin or acellular matrix, and the functional components comprise drugs and active factors; the drug is loaded on the first polymer matrix component, and the active factor is loaded on the second polymer matrix component; the nano film is formed by chemical crosslinking.

Preferably, the hyaluronic acid derivative is a derivative obtained by performing functional modification on one or more side chain groups of hyaluronic acid, wherein the one or more side chain groups are one or more of carboxyl, aldehyde group, amide group and grafted beta-cyclodextrin; the medicine is one or more of dexamethasone, dexamethasone sodium phosphate, doxycycline or doxycycline hydrochloride; the active factor is one of antibacterial polypeptide, anti-angiogenesis polypeptide and epithelia promoting growth factor.

Preferably, the thickness of the nano film is 20-500 nm.

The invention also provides a preparation method of the double-component multifunctional nano film, which comprises the following steps: 1) Mixing the first polymer matrix component, the second polymer matrix component and water, oscillating to obtain a mixed solution, dissolving the medicine in the mixed solution, and centrifuging to obtain a supernatant as a raw material mixed solution;

2) Dripping the raw material mixed solution obtained in the step 1) into a silicon wafer for one-time spin coating, performing first air drying, then performing cross-linking, and performing first vacuum freeze drying to obtain a pretreated nano film;

3) The pretreated nano film is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution, then is washed by ethylenediamine tetraacetic acid solution, is soaked in PBS solution of active factors, is washed by deionized water, and is subjected to second vacuum freeze drying, so that the pretreated nano film loaded with the drugs and the active factors is obtained;

4) And (3) dropwise adding the PVA solution to the surface of the pretreated nano film loaded with the drug and the active factors obtained in the step (3) for secondary spin coating, performing secondary air drying, and then stripping from a silicon wafer to obtain the nano film.

Preferably, in the step 1), the mass ratio of the mixture of the first polymer matrix component and the second polymer matrix component is 1-3:1, and the mass ratio of the total mass of the first polymer matrix component and the second polymer matrix component to water is 1:90-110; the temperature of the oscillation is 40-60 ℃, the frequency of the oscillation is 500-1000 Hz, and the time of the oscillation is 90-120 min.

Preferably, the ratio of the drug to the mixed solution in step 1) is 10-200 mg:1mL; the rotating speed of the centrifugation is 2500-3500 rpm, and the time of the centrifugation is 5-15 min.

Preferably, the acceleration of the one spin coating in step 2) is 1400-1600 rad/s ² The rotating speed of the primary spin coating is 5000-8000 rpm, and the time of the primary spin coating is 20-40 s; the temperature of the first air drying is 20-30 ℃; the temperature of the first vacuum freeze drying is-70 to-80 ℃, the vacuum degree of the first vacuum freeze drying is 10-100 Pa, and the time of the first vacuum freeze drying is 6-12 h.

Preferably, the concentration of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution in the step 3) is 1-3 mg/mL, and the soaking time in the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution is 0.5-1.5 h; the concentration of the ethylenediamine tetraacetic acid solution is 0.2-0.4 mg/mL; the concentration of the PBS solution in the step 3) is 0.5-3 mg/mL, the soaking temperature in the PBS solution is 0-8 ℃, and the soaking time in the PBS solution is 8-12 h; the temperature of the second vacuum freeze drying is-60 to-80 ℃, the vacuum degree of the second vacuum freeze drying is 10-100 Pa, and the time of the second vacuum freeze drying is 12-24 h.

Preferably, the mass concentration of the PVA solution in the step 4) is 15-25%, and the dosage of the PVA solution is 0.5-1.5 mL; the acceleration of the secondary spin coating is 100-500 rad/s ² The rotating speed of the secondary spin coating is 2000-3000 rpm, and the time of the secondary spin coating is 20-40 s; the temperature of the second air drying is 15-25 ℃.

The invention also provides application of the bi-component multifunctional nano film or the nano film obtained by the preparation method in preparation of products for treating cornea injury.

The invention provides a bi-component multifunctional nano film and a preparation method and application thereof, which take clinical requirements as guidance, and based on excellent drug carrying performance, modifiable performance and adhesive performance of the nano film, the nano film is used for repairing cornea alkali burn and infection, and according to the pathological damage characteristics of the cornea alkali burn and infection and clinical treatment requirements, bi-components, namely two different biological macromolecules are used as base materials to carry active factors and anti-inflammatory drugs, thereby providing a new way for safely and effectively treating cornea damage such as alkali burn and infection, preventing serious complications and preventing blindness; the bi-component multifunctional nano-film is spontaneously adhered to the damaged cornea through Van der Waals force or hydrogen bond acting force, and the loaded medicine and active factors are continuously released at the wound, so that the residence time of the medicine on the ocular surface is greatly prolonged compared with the traditional eye drop treatment; in addition, natural biological macromolecules are selected as the base material, so that the healing of damaged cornea epithelium is promoted; the nano-membrane prepared by the invention has good biocompatibility, simple and rapid operation, is tightly attached to the damaged cornea, effectively resists bacteria or blood vessels, inhibits inflammatory reaction of cornea burn and infection, and promotes repair of the damaged cornea, breaks through the treatment limitations of frequent multiple administrations of local eye drops, poor ocular permeability of antibacterial drugs and suture of amniotic membrane carriers in clinic, and has larger practical popularization value.

Drawings

FIG. 1 shows the steps and thickness variations of nanomembrane adhesion on rabbit cornea in vivo (A is the photomicrograph and OCT structure level diagram of normal rabbit cornea; B is the photomicrograph and OCT structure level diagram of cornea after scraping off cornea epithelium; C is the photomicrograph and OCT structure level diagram of nanomembrane attached to cornea surface; D is the photomicrograph and OCT structure level diagram after dissolving sacrificial layer);

FIG. 2 shows the therapeutic effect of nanomembranes in New Zealand rabbit corneal alkali burn model (A and C are the cases of cornea 7 days after alkali burn molding; B and D are the cases of corneal epithelium repair 7 days after alkali burn molding);

FIG. 3 shows the therapeutic effect of nanomembranes in New Zealand rabbit Pseudomonas aeruginosa keratitis (A and C are 7 days after molding Pseudomonas aeruginosa keratitis; B and D are 7 days after molding Pseudomonas aeruginosa keratitis; E is a transparency comparison after taking the cornea of each group; F and G are culture and CFU counts after homogenizing cornea of each group).

Detailed Description

The invention provides a double-component multifunctional nano film which is prepared from the following components in percentage by mass: 50-75% of a first polymer matrix component, 24-49% of a second polymer matrix component and 1% of a functional component; the first polymer matrix component is hyaluronic acid or a hyaluronic acid derivative, the second polymer matrix component is collagen, gelatin, methacryloylated gelatin or acellular matrix, and the functional component is a drug and an active factor; the drug is loaded on the first polymer matrix component, and the active factor is loaded on the second polymer matrix component; the nano film is formed by chemical crosslinking.

In the nano film of the invention, the mass fraction of the first polymer matrix component is 50-75%, more preferably 55-70%, still more preferably 60-65%; the mass fraction of the second polymer matrix component is 24-49%, more preferably 28-45%, still more preferably 32-41%; the mass fraction of the functional component is 1%.

In the nano-film of the invention, the first polymer matrix component is hyaluronic acid or a hyaluronic acid derivative, more preferably a hyaluronic acid derivative, the hyaluronic acid derivative is preferably a derivative obtained by carrying out functional modification on one or more side chain groups of hyaluronic acid, and the one or more side chain groups are preferably one or more of aldehyde groups, amide groups and grafted beta-cyclodextrin; specifically, the hyaluronic acid is methacryloylated hyaluronic acid, a hyaluronic acid cyclodextrin derivative or an aldehyde hyaluronic acid.

In the nanomembrane of the present invention, the polymer matrix component II is one of collagen, gelatin, methacryloylated gelatin, or acellular matrix, and more preferably one of gelatin, methacryloylated gelatin, or acellular matrix.

In the nano-membrane, the functional component comprises a medicament and an active factor, wherein the medicament is preferably one or more of dexamethasone, dexamethasone sodium phosphate, doxycycline or doxycycline hydrochloride, and the active factor is preferably one of antibacterial polypeptide, anti-angiogenic polypeptide and epitheliogenic growth factor.

In the present invention, the thickness of the nano-film is preferably 20 to 500nm, more preferably 100 to 400nm, still more preferably 200 to 300nm.

In the preparation method, step 1) the polymer matrix component I, the polymer matrix component II and water are mixed and vibrated to obtain a mixed solution; the mass ratio of the first polymer matrix component to the second polymer matrix component is preferably 1-3:1, more preferably 1.5-2.5:1, and even more preferably 2:1; the mass ratio of the total mass of the first polymer matrix component and the second polymer matrix component to water is preferably 1:90-110, more preferably 1:95-105, and even more preferably 1:98-102; the temperature of the oscillation is preferably 40-60 ℃, more preferably 45-55 ℃, still more preferably 48-52 ℃, and the frequency of the oscillation is preferably 500-1000 Hz, more preferably 700-900 Hz, still more preferably 750-800 Hz; the time of the shaking is preferably 90 to 120 minutes, more preferably 95 to 115 minutes, still more preferably 100 to 110 minutes.

In the preparation method of the invention, step 1) after dissolving the medicine in the mixed solution, centrifuging to obtain supernatant fluid, namely the raw material mixed solution; the ratio of the medicine to the mixed solution is preferably 10-200 mg:1mL, more preferably 20 to 180mg:1mL, still more preferably 40 to 160mg/mL; the dissolving is preferably carried out by stirring, the stirring is preferably carried out under a light-shielding condition, and the stirring rotating speed is preferably 1000-5000 r/min, more preferably 2000-4000 r/min, still more preferably 2500-3000 r/min; the rotational speed of the centrifugation is preferably 2500 to 3500rpm, more preferably 2600 to 3400rpm, still more preferably 2800 to 3200rpm, and the time of the centrifugation is preferably 5 to 15 minutes, more preferably 6 to 14 minutes, still more preferably 8 to 12 minutes.

In the preparation method of the invention, the step 2) is carried outDripping the raw material mixed solution obtained in the step 1) into a silicon wafer for one-time spin coating, performing first air drying, then performing cross-linking, and performing first vacuum freeze drying to obtain a pretreated nano film; the silicon wafer is preferably a silicon wafer which is subjected to hydrophobic treatment by trichloro (1H, 2H-tridecafluoron-octyl) silane, and the acceleration of the primary spin coating is preferably 1400-1600 rad/s ² More preferably 1450 to 1550rad/s ² Still more preferably 1480 to 1520rad/s ² The rotation speed of one spin coating is preferably 5000-8000 rpm, more preferably 6000-7000 rpm, still more preferably 6200-6800 rpm, the time of one spin coating is preferably 20-40 s, more preferably 25-35 s, still more preferably 28-32 s, the spin coating is preferably completed by a spin coater which is commercially available and has the model KW-4C (Beijing SeideKasi electronic Co., ltd); the temperature of the first air drying is preferably 20-30 ℃, more preferably 22-28 ℃, still more preferably 24-26 ℃, and the air drying is preferably performed in an ultra clean bench; the crosslinking is polymerization initiated by EDC-NHS or ultraviolet light; the temperature of the first vacuum freeze-drying is preferably-70 to-80 ℃, more preferably-72 to-78 ℃, still more preferably-74 to-76 ℃, the vacuum degree of the first vacuum freeze-drying is preferably 10 to 100Pa, more preferably 15 to 40Pa, still more preferably 18 to 20Pa, and the time of the first vacuum freeze-drying is preferably 6 to 12 hours, more preferably 7 to 11 hours, still more preferably 8 to 10 hours.

In the preparation method, step 3) pre-treating the nano film, soaking the nano film in a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution, then cleaning the nano film by using an ethylenediamine tetraacetic acid solution, soaking the nano film in an active factor PBS solution, cleaning the nano film by using deionized water, and carrying out second vacuum freeze drying to obtain the pre-treating nano film loaded with the medicine and the active factor; the concentration of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution is preferably 1 to 3mg/mL, more preferably 1.5 to 2.5mg/mL, still more preferably 1.8 to 2.2mg/mL, and the soaking time in the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution is preferably 0.5 to 1.5 hours, more preferably 0.6 to 1.4 hours, still more preferably 0.8 to 1.2 hours; the concentration of the ethylenediamine tetraacetic acid solution is preferably 0.2 to 0.4mg/mL, more preferably 0.25 to 0.35mg/mL, still more preferably 0.28 to 0.32mg/mL; the concentration of the PBS solution is preferably 0.5-3 mg/mL, more preferably 1-2.5 mg/mL, even more preferably 1.5-2 mg/mL, the soaking temperature in the PBS solution is preferably 0-8 ℃, more preferably 2-6 ℃, even more preferably 3-5 ℃, the soaking time in the PBS solution is preferably 8-12 h, more preferably 9-11 h, even more preferably 9.5-10.5 h; the temperature of the second vacuum freeze-drying is preferably-60 to-80 ℃, more preferably-65 to-75 ℃, still more preferably-68 to-72 ℃, the vacuum degree of the second vacuum freeze-drying is preferably 10 to 100Pa, more preferably 12 to 40Pa, still more preferably 15 to 20Pa, and the time of the second vacuum freeze-drying is preferably 6 to 12 hours, more preferably 8 to 12 hours, still more preferably 10 to 12 hours.

In the preparation method, step 4) dripping PVA solution on the surface of the pretreated nano film loaded with the drug and the active factors obtained in step 3) for secondary spin coating, performing secondary air drying, and then stripping from a silicon wafer to obtain the nano film; the mass concentration of the PVA solution is preferably 15 to 25%, more preferably 16 to 24%, even more preferably 18 to 22%, and the amount of the PVA solution is preferably 0.5 to 1.5mL, more preferably 0.6 to 1.4mL, even more preferably 0.8 to 1.2mL; the acceleration of the secondary spin coating is preferably 100-500 rad/s ² More preferably 150 to 350rad/s ² Even more preferably 200 to 300rad/s ² The rotation speed of the secondary spin coating is preferably 2000-3000 rpm, more preferably 2200-2800 rpm, still more preferably 2400-2600 rpm, the time of the secondary spin coating is preferably 20-40 s, more preferably 25-35 s, still more preferably 26-34 s, the spin coating is preferably performed in a spin coater, the spin coater is commercially available, and the model of the spin coater is KW-4C (Beijing Saidelkes electronic Co., ltd.); the temperature of the second air drying is preferably 15 to 25 ℃, more preferably 16 to 24 ℃, still more preferably 18 to the upper The second air drying is preferably carried out in an ultra clean bench at 22 ℃.

The invention also provides a preparation method of the double-component multifunctional nano film or application of the nano film in preparing a product for treating cornea damage, and the nano film is adhered to cornea when in use, and the sacrificial layer is removed by using deionized water.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A double-component multifunctional nano-film comprises the following components in parts by mass: 50% of a polymer matrix component, 49% of a polymer matrix component and 1% of a functional component.

1) Mixing the methacryloyl hyaluronic acid (EFL, product No. EFL-HAMA-400K) and the methacryloyl gelatin (EFL, product No. EFL-GM-90) according to a mass ratio of 1:1, dissolving the mixture in deionized water, wherein the total mass concentration of the methacryloyl hyaluronic acid and the methacryloyl gelatin is 1%, oscillating at 40 ℃, the oscillating frequency is 1000Hz, the oscillating time is 120min, and obtaining a mixed solution according to a ratio of dexamethasone sodium phosphate to the mixed solution of 1mg:100mL of the mixture was mixed while adding 0.5% by weight of photoinitiator I2959 (Sigma-Aldrich, cat. No. 410896) based on the total mass of the solution, (only a portion of the starting materials needed to be used were photoinitiators, some starting materials used were other crosslinking methods); stirring at 1000r/min, centrifuging at 3000rpm for 10min, and collecting supernatant to obtain a raw material mixed solution;

2) Dripping the raw material mixed solution obtained in the step 1) into a monocrystalline silicon wafer to carry out primary spin coating, wherein the acceleration of the primary spin coating is 1500rad/s ² The rotational speed of the primary spin coating is 5500r/min, the time of the primary spin coating is 20s, the temperature is 25 ℃ for the first air drying, then photo-crosslinking is carried out, the crosslinking is carried out for 10min under ultraviolet irradiation, the first vacuum freeze drying is carried out at the temperature of minus 75 ℃, the vacuum degree is 10Pa, and the time is 6h, thus obtaining the pretreated nano film;

3) The pretreated nanomembrane was immersed in a 2mg/mL solution of 4- (N-maleimidomethyl) cyclohexane-1-carboxylate-3-thio-N-succinimidyl ester sodium salt (ACMEC, cat# N65560) for 1h, then washed with a 0.3mg/mL solution of ethylenediamine tetraacetic acid (ACMEC, cat# SA 03307), and then immersed in a 0.5mg/mL solution of antimicrobial polypeptide (strong light Biotech Co., ltd.) in PBS at a temperature of 4℃for 10h; washing with deionized water, and freeze-drying at-80deg.C under vacuum degree of 10Pa for 12 hr; obtaining a pretreated nano film loaded with drugs and active factors;

4) Dripping 1mL of 20% polyvinyl alcohol (PVA, michelin, product number P815725) solution on the surface of the pretreated nano film loaded with the drug and the active factors obtained in the step 3 for secondary spin coating, wherein the acceleration of the secondary spin coating is 500rad/s ² The rotation speed of the secondary spin coating is 2500rpm, and the time of the secondary spin coating is 30s; and (3) drying the nano film to form a film, wherein the temperature of the second air drying is 20 ℃, peeling the nano film from the monocrystalline silicon piece under the support of the protective layer after drying, cutting the nano film into a required shape and size, and packaging. The double-component multifunctional nano-film with the protective layer, the dexamethasone sodium phosphate and the antibacterial polypeptide is obtained.

Example 2

A double-component multifunctional nano-film comprises the following components in parts by mass: 60% of polymer matrix component one, 39% of polymer matrix component two and 1% of functional component.

The preparation method comprises the steps of grafting cyclodextrin on the side chain carboxyl of hyaluronic acid: 3g of sodium hyaluronate (Source leaf organism, cat# S24592) were dissolved in 60mL of PBS solution, after stirring and dissolution 1.75mmoL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (abbreviated as EDC, microphone, cat# N808856) and 1.75mmoL of N-hydroxysuccinimide (abbreviated as NHS, microphone, cat# H6231) were added and stirred at room temperature for 30min.0.25mmoLβ -CD-EDA (Michelin, cat. C6289) was first dissolved in 10mL PBS, and after mixing the two solutions, stirred at room temperature for 24 hours; the resulting solution was dialyzed against deionized water for 5 days. Pre-freezing at-20 deg.c, freeze drying in freeze dryer to obtain white powder, i.e. cyclodextrin hyaluronic acid derivative.

1) Mixing the hyaluronic acid cyclodextrin derivative and collagen (source leaf organism, product number S25686) according to a mass ratio of 2:1, preparing an aqueous solution of the obtained mixture by deionized water, oscillating at 50 ℃ and with the frequency of 800Hz for 100min, obtaining a mixed solution, adding dexamethasone sodium phosphate, and the ratio of the medicine to the mixed solution is 10mg/mL; stirring at 2500r/min for 24h; centrifuging to obtain supernatant as raw material mixed solution, wherein the rotation speed of centrifugation is 2500rpm, and the centrifugation time is 15min;

2) Dripping the raw material mixed solution obtained in the step 1) into a silicon wafer to perform primary spin coating, wherein the acceleration of the primary spin coating is 1400rad/s ² The rotating speed of the primary spin coating is 5000rpm, the time of the primary spin coating is 20s, the primary spin coating is air-dried, and the temperature of the primary air-drying is 20 ℃; then carrying out chemical crosslinking, and placing the spin-coated silicon wafer in an ethanol solution of EDC/NHS for chemical crosslinking for 24 hours, wherein the EDC concentration is 14.4mM, and the NH S concentration is 5.6mM; the first vacuum freeze drying is carried out, the temperature of the first vacuum freeze drying is-70 ℃, the vacuum degree of the first vacuum freeze drying is 40Pa, and the time of the first vacuum freeze drying is 8 hours; obtaining a pretreated nano film;

3) The pretreated nano-membrane is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 1mg/mL for 0.5h, then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.2mg/mL, and then is soaked in PB S solution of anti-angiogenesis polypeptide with the concentration of 2mg/mL for 8h at the temperature of 0 ℃; washing with deionized water, performing second vacuum freeze drying at-65deg.C, wherein the vacuum degree of the second vacuum freeze drying is 40Pa, and the second vacuum freeze drying time is 18h; obtaining a pretreated nano film loaded with drugs and active factors;

4) Dripping 0.5mL of PVA water solution with the mass concentration of 15% on the surface of the pretreated nano film loaded with the drug and the active factors obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 300rad/s ² The rotation speed of the secondary spin coating is 2000rpm, and the time of the secondary spin coating is 20s; and (3) performing second air drying, wherein the temperature of the second air drying is 15 ℃, then stripping and cutting from the silicon wafer to obtain the nano film.

Example 3

A double-component multifunctional nano-film comprises the following components in parts by mass: 75% of polymer matrix component one, 24% of polymer matrix component two and 1% of functional component.

The acellular matrix derived from bovine Achilles tendon is prepared by a laboratory, and the specific preparation method comprises the following steps: fresh bovine achilles tendon was collected from a local slaughterhouse, chopped into 1-2 mm-sized pieces using tissue scissors, soaked with 1% by mass aqueous sodium dodecyl sulfate (BioReagent, cat No. L3771) for 72h, and then soaked with 1% by mass triton X-100 (Sigma-Aldrich, cat No. X100) for 2h. The decellularized tissue treated as described above was further soaked with isopropanol for 2 hours, then soaked with 1:1000 mass ratio of PBS for 3 days, and replaced with fresh PBS 2 times per day. Sterilizing the tissue with 0.1% peracetic acid solution for 4h, washing with sterile deionized water for 6h, pre-freezing the sterilized decellularized tissue at-20deg.C, and lyophilizing in a lyophilizer to obtain white block decellularized tissue. The decellularized tissue was ground with a mortar and pestle and the ground tissue sample was prepared at a ratio of 10: pepsin (source leaf organism, cat. Number S10027) was added in a mass ratio of 1 and digested in acetic acid solution in a mass ratio of 1:10 for 72h. Filtering the digested solution with 40 μm sieve (Corning) to remove undigested impurities, pre-freezing the obtained solution at-20deg.C, and lyophilizing in a lyophilizer to obtain white powder as acellular matrix.

1) Mixing hyaluronic acid and acellular matrix according to a mass ratio of 3:1, dissolving with water, wherein the total mass concentration of hyaluronic acid and acellular matrix in water is 1%, oscillating at 60 ℃, the oscillating frequency is 1000Hz, the oscillating time is 90min, obtaining a mixed solution, adding doxycycline hydrochloride, and the ratio of doxycycline hydrochloride to the mixed solution is 100mg/mL; stirring at a rotation speed of 5000r/min; centrifuging to obtain supernatant which is the raw material mixed solution, wherein the rotation speed of the centrifugation is 3500rpm, and the centrifugation time is 15min;

2) Dripping the raw material mixed solution obtained in the step 1) into a silicon wafer to carry out primary spin coating, wherein the acceleration of the primary spin coating is 1600rad/s ² The rotating speed of one-time spin coating is 8000rpm, the time of one-time spin coating is 40s, the first air drying is carried out, and the first air is carried outThe dry temperature was 30 ℃; then chemical crosslinking is carried out, and the mixture is soaked in ethanol solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) for crosslinking for 24 hours, wherein the EDC concentration is 14.4mM and the NHS concentration is 5.6mM; the first vacuum freeze drying is carried out, the temperature of the first vacuum freeze drying is-80 ℃, the vacuum degree of the first vacuum freeze drying is 100Pa, and the time of the first vacuum freeze drying is 12h; obtaining a pretreated nano film;

3) The pretreated nano-film is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 3mg/mL for 1.5 hours, then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.4mg/mL, and then is soaked in PB S solution of epitheliotropic growth factor with the concentration of 3mg/mL for 12 hours at the temperature of 8 ℃; washing with deionized water, performing second vacuum freeze drying at-75deg.C, wherein the vacuum degree of the second vacuum freeze drying is 100Pa, and the second vacuum freeze drying time is 24h; obtaining a pretreated nano film loaded with drugs and active factors;

4) Dripping 1.5mL of PVA solution with mass concentration of 25% on the surface of the pretreated nano film loaded with the drug and the active factors obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 100ra d/s ² The rotating speed of the secondary spin coating is 3000rpm, and the time of the secondary spin coating is 40s; and (3) performing second air drying, wherein the temperature of the second air drying is 25 ℃, then stripping and cutting from the silicon wafer to obtain the nano film.

Example 4

1) The preparation method comprises the steps of mixing formylated hyaluronic acid (Creative PEGworks, product number ZCR-HA-311) and gelatin (Sigma-Aldrich, product number V900863) according to a mass ratio of 3:1, dissolving the mixture with water, wherein the total mass concentration of the formylated hyaluronic acid and the gelatin in the water is 1%, oscillating, the temperature of the oscillating is 60 ℃, the frequency of the oscillating is 1000Hz, the time of the oscillating is 90min, obtaining a mixed solution, and adding doxycycline hydrochloride (Sigma-Aldrich, product number D3072), wherein the ratio of the doxycycline hydrochloride to the mixed solution is 100mg/mL; stirring at a rotating speed of 3000r/min; centrifuging to obtain supernatant which is the raw material mixed solution, wherein the rotation speed of the centrifugation is 3500rpm, and the centrifugation time is 15min;

2) Dripping the raw material mixed solution obtained in the step 1) into a silicon wafer to carry out primary spin coating, wherein the acceleration of the primary spin coating is 1600rad/s ² The rotating speed of the primary spin coating is 8000rpm, the time of the primary spin coating is 40s, the primary spin coating is air-dried at the temperature of 30 ℃; then chemical crosslinking is carried out, and the mixture is soaked in ethanol solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) for crosslinking for 24 hours, wherein the EDC concentration is 14.4mM and the NHS concentration is 5.6mM; the first vacuum freeze drying, the temperature of the first vacuum freeze drying is-80 ℃, the vacuum degree of the first vacuum freeze drying is 50Pa, and the time of the first vacuum freeze drying is 10 hours; obtaining a pretreated nano film;

3) The pretreated nano membrane is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 3mg/mL for 1.5 hours, then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.4mg/mL, and then is soaked in PBS solution with the antibacterial polypeptide with the concentration of 3mg/mL for 12 hours at the temperature of 8 ℃; washing with deionized water, performing second vacuum freeze drying at-75deg.C, wherein the vacuum degree of the second vacuum freeze drying is 50Pa, and the second vacuum freeze drying time is 24h; obtaining a pretreated nano film loaded with drugs and active factors;

4) Dripping 1.5mL of PVA solution with mass concentration of 25% on the surface of the pretreated nano film loaded with the drug and the active factors obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 100ra d/s ² The rotating speed of the secondary spin coating is 3000rpm, and the time of the secondary spin coating is 20s; and (3) performing second air drying, wherein the temperature of the second air drying is 15 ℃, then stripping and cutting from the silicon wafer to obtain the nano film.

Example 5

1) The method comprises the steps of mixing the methacryloyl hyaluronic acid and the methacryloyl gelatin according to a mass ratio of 1:1, dissolving the mixture in deionized water, wherein the total mass concentration of the methacryloyl hyaluronic acid and the methacryloyl gelatin is 1%, oscillating at 40 ℃, the oscillating frequency is 1000Hz, the oscillating time is 120min, obtaining a mixed solution, and mixing 1mg of the mixed solution according to dexamethasone sodium phosphate (ACMEC, product number D32140): 100mL of the mixture was mixed while adding 0.5% by weight of photoinitiator I2959 (Sigma-Aldrich, cat. No. 410896) based on the total mass fraction of the solution; stirring at a rotating speed of 1000r/min, centrifuging at 3000rpm for 10min, and taking supernatant as a raw material mixed solution;

3) The pretreated nano-film is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 2mg/mL for 1h, then washed by ethylenediamine tetraacetic acid solution with the concentration of 0.3mg/mL, and soaked in PBS solution of anti-angiogenic peptide (blaze biotechnology Co., ltd.) with the concentration of 0.5mg/mL for 10h at the temperature of 4 ℃; washing with deionized water, and freeze-drying at-80deg.C under vacuum degree of 10Pa for 12 hr; obtaining a pretreated nano film loaded with drugs and active factors;

4) Dripping 1mL of PVA solution with mass concentration of 20% on the surface of the pretreated nano film loaded with the drug and the active factors obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 500rad/s ² The rotation speed of the secondary spin coating is 2500rpm, and the time of the secondary spin coating is 30s; and (3) drying the nano film to form a film, wherein the temperature of the second air drying is 20 ℃, peeling the nano film from the monocrystalline silicon piece under the support of the protective layer after drying, cutting the nano film into a required shape and size, and packaging. The bi-component multifunctional nano-film with a protective layer, dexamethasone sodium phosphate and anti-angiogenesis polypeptide is obtained.

Comparative example 1

1) The method comprises the steps of mixing the methacryloyl hyaluronic acid and the methacryloyl gelatin according to a mass ratio of 1:1, dissolving the mixture in deionized water, oscillating the mixture at a temperature of 40 ℃ with a frequency of 1000Hz and a time of 120min to obtain a mixed solution, and mixing 1mg of dexamethasone sodium phosphate and the mixed solution according to a mass ratio of 1mg:100mL of the mixture was mixed while adding 0.5% by weight of photoinitiator I2959 (Sigma-Aldrich, cat. No. 410896) based on the total mass fraction of the solution; stirring at 1000r/min, centrifuging at 3000rpm for 10min, and collecting supernatant to obtain a raw material mixed solution;

3) The pretreated nano membrane is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 2mg/mL for 1h, and then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.3 mg/mL; washing with deionized water, and freeze-drying at-80deg.C under vacuum degree of 10Pa for 12 hr; obtaining a pretreated nano film loaded with the medicine;

4) Dripping 1mL of PVA solution with mass concentration of 20% on the surface of the drug-loaded pretreated nano film obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 500rad/s ² The rotation speed of the secondary spin coating is 2500rpm, and the time of the secondary spin coating is 30s; and (3) drying the nano film to form a film, wherein the temperature of the second air drying is 20 ℃, peeling the nano film from the monocrystalline silicon piece under the support of the protective layer after drying, cutting the nano film into a required shape and size, and packaging. Obtaining A nano-film.

Comparative example 2

1) Mixing the methacryloyl hyaluronic acid and the methacryloyl gelatin according to a mass ratio of 1:1, dissolving in deionized water, wherein the total mass concentration of the methacryloyl hyaluronic acid and the methacryloyl gelatin is 1%, oscillating at 40 ℃, the oscillating frequency is 1000Hz, the oscillating time is 120min, obtaining a mixed solution, and simultaneously adding a photoinitiator I2959 (Sigma-Aldrich, product number 410896) accounting for 0.5% of the total mass fraction of the solution; stirring at 1000r/min, centrifuging at 3000rpm for 10min, and collecting supernatant to obtain a raw material mixed solution;

3) The pretreated nano-membrane is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 2mg/mL for 1h, then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.3mg/mL, and is soaked in PBS solution of antiangiogenic peptide with the concentration of 0.5mg/mL for 10h at the temperature of 4 ℃; washing with deionized water, and freeze-drying at-80deg.C under vacuum degree of 10Pa for 12 hr; obtaining a pretreatment nano film loaded with active factors;

4) Dripping 1mL of PVA solution with mass concentration of 20% on the surface of the active factor loaded pretreated nano film obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 500rad/s ² The rotation speed of the secondary spin coating is 2500rpm, and the time of the secondary spin coating is 30s; second air drying to form film, the second air drying temperature is 20 ℃, and the nano film is supported by the protective layer after dryingAnd stripping the monocrystalline silicon piece, cutting into the required shape and size, and packaging. Obtaining the nano film.

Comparative example 3

A double-component multifunctional nano-film comprises the following components in parts by mass: 50% of a polymer matrix component I and 49% of a polymer matrix component II.

3) The pretreated nano membrane is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 2mg/mL for 1h, and then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.3 mg/mL; washing with deionized water, and freeze-drying at-80deg.C under vacuum degree of 10Pa for 12 hr; the pretreated nano film which is not loaded with medicines and active factors is obtained;

4) Dripping 1mL of PVA solution with mass concentration of 20% on the surface of the pretreated nano film without carrying the drug and the active factors obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 500ra d/s ² The rotation speed of the secondary spin coating is 2500rpm, and the time of the secondary spin coating is 30s; second air drying to form film, the second air drying temperature is 20 ℃, and the nano film is peeled off from the monocrystalline silicon piece under the support of the protective layer after drying Cutting into required shape and size, packaging, and obtaining nanometer film.

Comparative example 4

1) Mixing the hydroformylation hyaluronic acid and the gelatin according to the mass ratio of 3:1, dissolving with water, wherein the total mass concentration of the hydroformylation hyaluronic acid and the gelatin in the water is 1%, oscillating, the temperature of the oscillating is 60 ℃, the frequency of the oscillating is 1000Hz, the time of the oscillating is 90min, obtaining a mixed solution, and adding doxycycline hydrochloride, wherein the ratio of the doxycycline hydrochloride to the mixed solution is 100mg/mL; stirring at a rotating speed of 3000r/min; centrifuging to obtain supernatant which is the raw material mixed solution, wherein the rotation speed of the centrifugation is 3500rpm, and the centrifugation time is 15min;

3) The pretreated nano-film is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 3mg/mL for 1.5h, and then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.4 mg/mL; washing with deionized water, performing second vacuum freeze drying at-75deg.C, wherein the vacuum degree of the second vacuum freeze drying is 50Pa, and the second vacuum freeze drying time is 24h; obtaining a pretreated nano film loaded with the medicine;

4) Dripping 1.5mL of PVA solution with mass concentration of 25% on the surface of the drug-loaded pretreated nano film obtained in the step 3) for secondary spin coatingAcceleration of the coating is 100rad/s ² The rotating speed of the secondary spin coating is 3000rpm, and the time of the secondary spin coating is 20s; and (3) performing second air drying, wherein the temperature of the second air drying is 15 ℃, then stripping and cutting from the silicon wafer to obtain the nano film.

Comparative example 5

1) Mixing the hydroformylation hyaluronic acid and the gelatin according to a mass ratio of 3:1, dissolving with water, wherein the total mass concentration of the hydroformylation hyaluronic acid and the gelatin in the water is 1%, and oscillating at 60 ℃, wherein the frequency of the oscillating is 1000Hz, and the oscillating time is 90min to obtain a mixed solution; stirring at a rotating speed of 3000r/min; centrifuging to obtain supernatant which is the raw material mixed solution, wherein the rotation speed of the centrifugation is 3500rpm, and the centrifugation time is 15min;

3) The pretreated nano membrane is soaked in 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution with the concentration of 3mg/mL for 1.5 hours, then is washed by ethylenediamine tetraacetic acid solution with the concentration of 0.4mg/mL, and then is soaked in PBS solution with the antibacterial polypeptide with the concentration of 3mg/mL for 12 hours at the temperature of 8 ℃; washing with deionized water, performing second vacuum freeze drying at-75deg.C, wherein the vacuum degree of the second vacuum freeze drying is 50Pa, and the second vacuum freeze drying time is 24h; obtaining a pretreatment nano film loaded with active factors;

4) Dripping 1.5mL of PVA solution with mass concentration of 25% on the surface of the active factor loaded pretreated nano film obtained in the step 3) for secondary spin coating, wherein the acceleration of the secondary spin coating is 100rad/s ² The rotating speed of the secondary spin coating is 3000rpm, and the time of the secondary spin coating is 20s; and (3) performing second air drying, wherein the temperature of the second air drying is 15 ℃, then stripping and cutting from the silicon wafer to obtain the nano film.

Experimental example 1

Eye surface operation and OCT examination for treating cornea diseases by using nano film prepared in example 1 of the invention

The normal cornea of New Zealand white rabbits is adopted to carry out the eye surface operation demonstration of the nano-film, and the cornea hierarchical structure and the nano-film attachment condition are checked by Optical Coherence Tomography (OCT).

As shown in FIG. 1, FIG. 1A is an optical photograph and OCT structure hierarchy of a normal rabbit cornea, showing a normal cornea thickness of about 374 μm and having a 5-layer structure; an epithelial layer 33 μm, a front elastic layer, a matrix layer 320 μm, a rear elastic layer and an endothelial layer; b is the thickness of 410 μm after scraping off the corneal epithelium, the cornea becomes edematous; c is that the nano film is attached to the cornea surface, and the thickness of the functional nano film with the sacrificial layer is about 16 mu m; d is that after the cornea is washed by normal saline, the sacrifice layer is dissolved, the OCT resolution can not detect the nano-level functional nano-film, and the cornea thickness is still 410 mu m; the nano film can be directly attached to the surface of the cornea without a suture, the water-soluble sacrificial layer is convenient to operate, the sacrificial layer can be removed through normal saline after the attachment, and the tight attachment of the nano film and the cornea is reserved.

Experimental example 2

Experiment of nanometer film in treating cornea alkali burn

The new zealand white rabbit cornea alkali burn model is adopted to research the treatment effect of different active ingredients (mainly comprising dexamethasone and/or anti-angiogenesis peptide components) in the nano-film on cornea alkali burn, and the anti-inflammatory, anti-angiogenesis and epithelial repair promotion effects are focused on.

Mature New Zealand white rabbits (2.0-2.5 kg) are subjected to right eye molding, 1M sodium hydroxide solution with equal quantity is taken by a capillary tube to completely wet filter paper with the diameter of 6mm, the filter paper is soaked in the center of a right eye cornea for 30s, and a stable and uniform cornea alkali burn model is established.

After successful molding, the treatment effect on corneal alkali burn was evaluated by randomly dividing the molding into 5 groups of 4 groups, respectively, and observing corneal haze, corneal inflammation, neovascular and epithelial repair conditions, with a blank control group (i.e., model group), dexamethasone nanomembrane (comparative example 1), antiangiogenic peptide nanomembrane (comparative example 2), nanomembrane without drug or active ingredient (comparative example 3), dexamethasone antiangiogenic peptide nanomembrane treatment (example 5).

Results are shown as A, C in fig. 2, and a and C are scored by observing corneal haze, ocular surface hyperemia, and corneal neovascularization 7 days after alkali burn molding using slit lamp microscopy anterior ocular segment photography.

Scoring standard of central alkali burn area of cornea is 0-5 points: at 0 minutes, the cornea was completely transparent and no haze could be seen with any slit lamp examination. 1 minute, very slight corneal haze, was seen only by slit lamp indirect broad tangential illumination. 2 minutes, slight corneal haze, low density haze seen when carefully observed with slit lamp direct illumination or diffuse light illumination, and visible pupils and iris veins. 3 minutes, moderate corneal haze, medium density haze can be directly observed with slit lamp examination, and only pupils can be seen. 4 minutes, severe corneal haze, which clearly obscures slit lamp light from passing through the cornea, is seen only in the anterior chamber. 5 minutes, extremely severe turbidity, which completely blocks slit lamp light from passing through the cornea, and no anterior chamber structure can be observed.

Conjunctival and limbal hyperemia scoring criteria 0-3 points: 0 minutes, no congestion; 1, there was slight hyperemia at the limbus; 2 minutes, moderate hyperemia occurred at the limbus; 3 minutes, severe limbus hyperemia and obvious veins.

Neovascular scoring criteria 0-4 points: 0 minutes, no intracorneal blood vessel exists, and the corneoscleral limbus is normal; 1 minute, less than 5 vascular loops, no more than 0.3mm;2 minutes, 5 to 15 vascular loops, not more than 0.3mm;3 minutes, more than 15 blood vessels or blood vessel loops are more than 0.3mm;4 minutes, 2 or more vascular tabs are greater than 0.5mm.

Nanomembrane without drug or active ingredient (comparative example 3) group central alkali burn area of cornea had a cloudiness score of 4.2±0.27, conjunctival and limbal congestion scores of 2.5±0.5 and neovascular scores of 3.4±0.54 and the central alkali burn area of cornea of the blank group exhibited a porcelain white circular cloudiness, intraocular structures of central area were peeped-free, corneal cloudiness score: 4.5± 0.0.5, conjunctival and limbal hyperemia score: 2.5±0.5, neovascular score: 3.8.+ -. 0.44 no significant difference (P > 0.05).

Dexamethasone nanomembrane (comparative example 1) group central alkali burn area haze score 3.6±0.41, conjunctival and limbal hyperemia score 1.5±0.61, neovascular score 1.8±0.83; the central alkali burn area turbidity score of the cornea of the anti-angiogenic peptide nano-film (comparative example 2) group is 3.7+/-0.44, conjunctival congestion score and limbal congestion score are 2.4+/-0.41, and the neovascular score is 1.8+/-0.44; wherein the congestion of the dexamethasone hyaluronic acid nano-film group is obviously relieved; both the dexamethasone hyaluronic acid nano-membrane group and the anti-angiogenic peptide hyaluronic acid nano-membrane group obviously lighten the new blood vessels. The dexamethasone anti-angiogenic peptide nanomembrane treatment (example 5) group had a clear reduction in corneal central alkali burn area haze score of 2.6.+ -. 0.42, conjunctival and limbal hyperemia scores of 0.6.+ -. 0.41 and neovascular scores of 0.4.+ -. 0.55, which were lower than those of the other 4 groups (P < 0.05).

As shown in B, D of fig. 2, corneal epithelium repair 7 days after alkali burn molding was observed using sodium fluorescein staining. The corneal epithelial defect is stained with sodium fluorescein as a well-defined green area. And identifying the dyed area by using the Image J and calculating the area to obtain a defect area result.

The central epithelium of cornea of the blank group is in circular defect. Comparative example 3 hyaluronic acid nanomembrane group (7.18.+ -. 7.37 mm) ² ) With the blank group (14.68+ -8.85 mm) ² ) Compared with the prior art, the defect area of the cornea epithelium is reduced (P is less than 0.05). Dexamethasone nanomembrane (comparative example 1) group (7.87.+ -. 5.56 mm) ² ) And anti-angiogenic peptide nanomembrane (comparative example 2) group (7.35.+ -. 4.64 mm) ² ) The corneal epithelial defect area was further reduced and both were smaller than the control group. Dexamethasone anti-angiogenic peptide nano-scaleFilm treatment (example 5) group (0.94.+ -. 0.92mm ² ) No obvious fluorescein staining area indicates good cornea epithelial repair; the defect areas are lower than those of the other 4 groups (P is less than 0.05).

The above results indicate that: the nanomembrane (comparative example 3) without added medicine or active ingredient has promoting effect on the repair of epithelium of corneal alkali burn. Dexamethasone nanomembrane (comparative example 1) has anti-inflammatory effect on corneal alkali burn, relieving ocular surface hyperemia. The antiangiogenic peptide nanomembrane (comparative example 2) has effects of preventing angiogenesis on corneal alkali burn, and can inhibit neovascularization. Dexamethasone anti-angiogenic peptide nano-film (example 5) has the multifunctional functions of promoting epithelial repair, resisting inflammation and inhibiting neovascularization, and can effectively treat corneal alkali burn.

Experimental example 3

Experiment of nanomembrane for treating pseudomonas aeruginosa keratitis

The treatment effect of the nano-film (loaded medicine is doxycycline hydrochloride and/or antibacterial polypeptide component) on the pseudomonas aeruginosa keratitis is researched by adopting a new zealand white rabbit pseudomonas aeruginosa keratitis model, and the antibacterial effect is focused.

Mature New Zealand white rabbit (2.0-2.5 kg) right eye mould, central cornea region marked with 6×6 mm trephine light pressure, 30G insulin needle for shallow cornea matrix tunnel in central cornea mark region, 10 μl containing 10 ³ The CFU suspension of pseudomonas aeruginosa was passed into the tunnel. Randomly dividing into 4 groups, and treating each group with blank control (i.e. model group), doxycycline hydrochloride nanometer film (comparative example 4), antibacterial polypeptide nanometer film (comparative example 5) and doxycycline hydrochloride antibacterial polypeptide nanometer film (example 4) respectively. The treatment effect on pseudomonas aeruginosa keratitis is evaluated by observing the cornea infection control condition and carrying out clinical scoring, carrying out area extraction and calculation on the cornea epithelial defect area by using Image J, and obtaining the detected bacterial content.

Clinical scoring standard of pseudomonas aeruginosa keratitis model is 0-4 points: 0 minutes, the cornea is transparent, has no turbidity and has regular surface. 1 minute, the range of corneal haze is 1% -25%, the cornea is slightly irregular, the cornea is slightly turbid, and iris blood vessels and pupils are clearly visible. 2 minutes, the range of corneal opacity is 26% -50%, the corneal stroma is edematous, the surface is raised or depressed, the cornea is slightly turbid, and the iris blood vessel and the pupil are clearly visible. 3 minutes, the range of corneal haze is 51% -75%, the corneal stroma is obviously edema, obvious depression or swelling of the back elastic layer, the cornea is unevenly turbid, and the structure is invisible after that. 4 minutes, the corneal haze ranged from 76% to 100%, the cornea perforated, the cornea uniformly turbid, and the structure was not visible thereafter.

The results are shown in FIG. 3, and in FIG. 3A and C, the clinical scores of the blank control group (model group) cornea congestion with large-piece gray-white ulcer focus are: 3.65±0.47; doxycycline hydrochloride nanomembrane (comparative example 4, clinical score: 2.25.+ -. 0.28) and antibacterial polypeptide nanomembrane (comparative example 5, clinical score: 1.87.+ -. 0.25) significantly inhibited corneal infection, significantly reduced ulceration lesions, and decreased clinical score (P < 0.05). Doxycycline hydrochloride antibacterial polypeptide nanomembrane (example 4, clinical score: 0.62+ -0.47) effectively inhibits cornea infection, and clinical score is significantly lower than doxycycline hydrochloride nanomembrane and antibacterial polypeptide nanomembrane group (P < 0.05).

In fig. 3B and D show that the repair of the corneal epithelium was observed 7 days after molding of pseudomonas aeruginosa keratitis using sodium fluorescein staining. The corneal epithelial defect is stained with sodium fluorescein as a well-defined green area. And identifying the dyed area by using the Image J and calculating the area to obtain a defect area result.

Blank (model) cornea central epithelium piece staining, cornea epithelium defect area 19.58+ -8.13 mm ² . Doxycycline hydrochloride nanomembrane (comparative example 4) cornea epithelium defect area 5.48+ -4.55 mm ² Antibacterial polypeptide nano-film group (comparative example 5) cornea epithelial defect area 6.04+ -3.53 mm ² Doxycycline hydrochloride nanomembrane (comparative example 4) and antibacterial polypeptide nanomembrane group cornea central boundary clear defect staining region. Doxycycline hydrochloride antibacterial polypeptide nano-membrane group (example 4) cornea epithelial defect area 0.7+/-0.96 mm ² No obvious staining and good cornea epithelial repair (P < 0.05).

In fig. 3, E is a comparison of transparency of the cornea of each group, and doxycycline hydrochloride antimicrobial polypeptide nanomembrane group (example 4) has the best transparency.

In fig. 3, after the cornea of each of the F and G groups was homogenized, the clear colony formation was seen after the culture and CFU counting of the blank control group (model group), the colony formation of doxycycline hydrochloride nanomembrane (comparative example 4) and the colony formation of the antibacterial polypeptide nanomembrane group (comparative example 5) were significantly reduced, the colony formation was hardly seen in the doxycycline hydrochloride antibacterial polypeptide nanomembrane group (example 4), and the CFU counting was minimal.

The above results indicate that: the doxycycline hydrochloride nano-film and the antibacterial polypeptide nano-film can effectively inhibit infection focus of pseudomonas aeruginosa keratitis and promote cornea epithelial repair. The multicomponent nanomembrane (example 4) with doxycycline hydrochloride combined with the antimicrobial polypeptide can treat pseudomonas aeruginosa keratitis more effectively.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The double-component multifunctional nano film is characterized by being prepared from the following components in percentage by mass:

50-75% of a first polymer matrix component, 24-49% of a second polymer matrix component and 1% of a functional component;

the first polymer matrix component is hyaluronic acid or a hyaluronic acid derivative, the second polymer matrix component is collagen, gelatin, methacryloylated gelatin or acellular matrix, and the functional components comprise drugs and active factors; the drug is loaded on the first polymer matrix component, and the active factor is loaded on the second polymer matrix component;

the nano film is formed by chemical crosslinking.

2. The two-component multifunctional nano-film according to claim 1, wherein the hyaluronic acid derivative is a derivative obtained by performing functional modification on one or more side chain groups of hyaluronic acid, and the one or more side chain groups are one or more of carboxyl groups, aldehyde groups, amide groups and grafted beta-cyclodextrin; the medicine is one or more of dexamethasone, dexamethasone sodium phosphate, doxycycline or doxycycline hydrochloride; the active factor is one of antibacterial polypeptide, anti-angiogenesis polypeptide and epithelia promoting growth factor.

3. The two-component multifunctional nanomembrane according to claim 1 or 2, wherein the thickness of the nanomembrane is 20-500 nm.

4. A method for preparing a two-component multifunctional nano-film according to any one of claims 1 to 3, comprising the steps of:

1) Mixing the first polymer matrix component, the second polymer matrix component and water, oscillating to obtain a mixed solution, dissolving the medicine in the mixed solution, and centrifuging to obtain a supernatant as a raw material mixed solution;

5. The method for preparing the two-component multifunctional nano-film according to claim 4, wherein the mass ratio of the polymer matrix component I to the polymer matrix component II in the step 1) is 1-3:1, and the mass ratio of the total mass of the polymer matrix component I and the polymer matrix component II to water is 1:90-110; the temperature of the oscillation is 40-60 ℃, the frequency of the oscillation is 500-1000 Hz, and the time of the oscillation is 90-120 min.

6. The method for preparing the two-component multifunctional nano-film according to claim 4, wherein the ratio of the drug to the mixed solution in the step 1) is 10-200 mg:1mL; the rotating speed of the centrifugation is 2500-3500 rpm, and the time of the centrifugation is 5-15 min.

7. The method for preparing a two-component multifunctional nano-film according to claim 4, wherein the acceleration of the one spin coating in the step 2) is 1400-1600 rad/s ² The rotating speed of the primary spin coating is 5000-8000 rpm, and the time of the primary spin coating is 20-40 s; the temperature of the first air drying is 20-30 ℃; the temperature of the first vacuum freeze drying is-70 to-80 ℃, the vacuum degree of the first vacuum freeze drying is 10-100 Pa, and the time of the first vacuum freeze drying is 6-12 h.

8. The method for preparing a two-component multifunctional nano-film according to claim 4, wherein the concentration of the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution in the step 3) is 1-3 mg/mL, and the soaking time in the 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt solution is 0.5-1.5 h; the concentration of the ethylenediamine tetraacetic acid solution is 0.2-0.4 mg/mL;

the concentration of the PBS solution in the step 3) is 0.5-3 mg/mL, the soaking temperature in the PBS solution is 0-8 ℃, and the soaking time in the PBS solution is 8-12 h; the temperature of the second vacuum freeze drying is-60 to-80 ℃, the vacuum degree of the second vacuum freeze drying is 10-100 Pa, and the time of the second vacuum freeze drying is 12-24 h.

9. The method for preparing the two-component multifunctional nano-film according to claim 4, wherein the mass concentration of the PVA solution in the step 4) is 15-25%, and the dosage of the PVA solution is 0.5-1.5 mL; the acceleration of the secondary spin coating is 100-500 rad/s ² The rotating speed of the secondary spin coating is 2000-3000 rpm, and the time of the secondary spin coating is 20-40 s; the temperature of the second air drying is 15-25 ℃.

10. Use of a bi-component multifunctional nanomembrane according to any one of claims 1 to 3 or a nanomembrane obtained by the preparation method according to any one of claims 4 to 9 for the preparation of a product for the treatment of corneal lesions.