A two-component multifunctional nanofilm and a preparation method and application thereof

Abstract

The application provides a kind of two-component multifunctional nanometer membrane and its preparation method and application, it is related to biological medical material technical field, the nanometer membrane, by including the following mass fraction of component is prepared: high molecular matrix component one 50~75%, high molecular matrix component two 24~49%, functional component 1%;The high molecular matrix component one is hyaluronic acid or hyaluronic acid derivative, the high molecular matrix component two is one of collagen, gelatin, methacrylated gelatin or acellular matrix, the functional component includes drug and active factor;The drug is loaded on high molecular matrix component one, and the active factor is loaded on high molecular matrix component two;The nanometer membrane is formed by chemical crosslinking.This application overcomes the shortcomings and deficiencies of existing corneal alkali burn and infection treatment means, provides a kind of two-component multifunctional nanometer membrane with good biological safety, effective control infection and inhibits inflammatory response, for treating corneal injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a two-component multifunctional nanofilm and a preparation method and application thereof. Background Art

[0002] Severe corneal injuries, such as corneal alkali burns and bacterial or fungal corneal infections, are associated with significant tissue damage, difficulty in wound repair, and poor clinical treatment outcomes, leading to blindness and severely compromising patients' vision and quality of life. Immune inflammation and persistent infection are the primary pathological mechanisms of blindness caused by corneal burns and infections. Currently, there are no proven treatments for corneal alkali burns. The clinical use of steroids for inflammatory bowel movements is limited by steroid-related complications, such as inhibition of epithelial healing and subsequent corneal thinning and perforation. Amniotic membrane coverage is limited by its poor fixation, the need for sutures that are easily absorbed, and the need for repeated surgeries. Bacterial or fungal corneal infections require combined systemic and topical antibiotics or antifungals. However, topical eye drops of antibiotics or antifungals are associated with poor ocular penetration, the development of resistance, low bioavailability, the need for multiple drugs, and toxic side effects due to high-concentration and frequent administration. Therefore, further research is needed to optimize ocular drug delivery and improve the therapeutic efficacy of corneal alkali burns and infections. Summary of the Invention

[0003] The purpose of the present invention is to provide a two-component multifunctional nanomembrane and its preparation method and application, to overcome the shortcomings and deficiencies of existing corneal alkali burn and infection treatment methods, and to provide a two-component multifunctional nanomembrane and its preparation method that has good biosafety, effectively controls infection and inhibits inflammatory response, and can be used to treat corneal injuries such as infection and alkali burns.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a two-component multifunctional nanomembrane, which is prepared from the following components in mass fraction: 50-75% of a polymer matrix component one, 24-49% of a polymer matrix component two, and 1% of a functional component; the polymer matrix component one is hyaluronic acid or a hyaluronic acid derivative, the polymer matrix component two is one of collagen, gelatin, methacrylated gelatin or acellular matrix, and the functional component includes a drug and an active factor; the drug is loaded on the polymer matrix component one, and the active factor is loaded on the polymer matrix component two; the nanomembrane is formed by chemical cross-linking.

[0006] Preferably, the hyaluronic acid derivative is a derivative obtained by functionally modifying 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 β-cyclodextrin; the drug is one or more of dexamethasone, dexamethasone sodium phosphate, doxycycline or doxycycline hydrochloride; the active factor is one of antibacterial polypeptides, anti-angiogenic polypeptides and epithelial growth factor.

[0007] Preferably, the thickness of the nanofilm is 20 to 500 nm.

[0008] The present invention also provides a method for preparing the dual-component multifunctional nanofilm, comprising the following steps: 1) mixing the polymer matrix component 1, the polymer matrix component 2, and water, and shaking to obtain a mixed solution; dissolving the drug in the mixed solution; and centrifuging to obtain a supernatant as the raw material mixed solution;

[0009] 2) adding the raw material mixed solution obtained in step 1) dropwise to a silicon wafer for a spin coating, first air-drying, then cross-linking, and first vacuum freeze-drying to obtain a pretreated nanofilm;

[0010] 3) The pretreated nanomembrane is immersed in a solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt, then washed with an ethylenediaminetetraacetic acid solution, then immersed in a PBS solution of the active factor, washed with deionized water, and vacuum freeze-dried a second time to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0011] 4) The PVA solution is added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and spin-coated for a second time, air-dried for a second time, and then peeled off from the silicon wafer to obtain the nanofilm.

[0012] Preferably, in step 1), the mass ratio of the polymer matrix component one and the polymer matrix component two is 1 to 3:1, and the mass ratio of the total mass of the polymer matrix component one and the polymer matrix component two to water is 1:90 to 110; the oscillation temperature is 40 to 60°C, the oscillation frequency is 500 to 1000 Hz, and the oscillation time is 90 to 120 min.

[0013] Preferably, in step 1), the ratio of the drug to the mixed solution is 10-200 mg:1 mL; the centrifugal speed is 2500-3500 rpm, and the centrifugal time is 5-15 min.

[0014] Preferably, the acceleration of the spin coating in step 2) is 1400-1600 rad / s 2The rotation speed of the first spin coating is 5000-8000 rpm, and the time of the first spin coating is 20-40 s; the temperature of the first air drying is 20-30°C; the temperature of the first vacuum freeze drying is -70--80°C, 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.

[0015] Preferably, the concentration of the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester in step 3) is 1 to 3 mg / mL, and the immersion time in the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester is 0.5 to 1.5 h; the concentration of the ethylenediaminetetraacetic acid solution is 0.2 to 0.4 mg / mL; the concentration of the PBS solution in step 3) is 0.5 to 3 mg / mL, the immersion temperature in the PBS solution is 0 to 8°C, and the immersion time in the PBS solution is 8 to 12 h; the temperature of the second vacuum freeze-drying is -60 to -80°C, the vacuum degree of the second vacuum freeze-drying is 10 to 100 Pa, and the time of the second vacuum freeze-drying is 12 to 24 h.

[0016] Preferably, the mass concentration of the PVA solution in step 4) is 15-25%, the amount of the PVA solution is 0.5-1.5 mL; the acceleration of the secondary spin coating is 100-500 rad / s 2 The rotation speed of the secondary spin coating is 2000-3000 rpm, the time of the secondary spin coating is 20-40 s; the temperature of the second air drying is 15-25°C.

[0017] The present invention also provides the use of the dual-component multifunctional nanofilm or the nanofilm obtained by the preparation method in preparing products for treating corneal damage.

[0018] The present invention provides a two-component multifunctional nanomembrane and its preparation method and application. Guided by clinical needs, based on the excellent drug-loading performance, modifiable performance and adhesion performance of the nanomembrane, it is proposed to use the nanomembrane for the repair of corneal alkali burns and infections. According to the pathological damage characteristics and clinical treatment needs of corneal alkali burns and infections, two components, i.e. two different biomacromolecules, are used as base materials to load active factors and anti-inflammatory drugs, providing a new way to more safely and effectively treat corneal injuries such as alkali burns and infections, prevent serious complications, and prevent blindness. The two-component multifunctional nanomembrane spontaneously adheres to the damaged cornea through van der Waals forces or hydrogen bonding forces. The membrane continuously releases the loaded drugs and active factors at the wound, which greatly increases the residence time of the drugs on the ocular surface compared with traditional eye drops. In addition, the selection of natural biomacromolecules as the base material is conducive to promoting the healing of damaged corneal epithelium. The present invention prepares a nano-membrane with good biocompatibility, simple and fast operation, close fit to the damaged cornea, effective antibacterial or anti-vascular, inhibition of inflammatory response of corneal burns and infection, and promotion of damaged corneal repair. It breaks through the treatment limitations of frequent and multiple administration of local eye drops in clinical practice, poor eye permeability of antibacterial drugs, and the need for sutures to suture the amniotic membrane carrier, and has great practical promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The operation steps and thickness changes of the nanofilm adhesion on the rabbit cornea in vivo (A is the optical photograph and OCT structural layer map of the normal rabbit cornea; B is the optical photograph and OCT structural layer map of the cornea after scraping the corneal epithelium; C is the optical photograph and OCT structural layer map of the nanofilm attached to the corneal surface; D is the optical photograph and OCT structural layer map after the sacrificial layer is dissolved);

[0020] Figure 2 The therapeutic effect of the nanofilm in the New Zealand rabbit corneal alkali burn model (A and C show the corneal condition 7 days after alkali burn modeling; B and D show the corneal epithelial repair condition 7 days after alkali burn modeling);

[0021] Figure 3 Figure 3 shows the therapeutic effect of the nanofilm on Pseudomonas aeruginosa keratitis in New Zealand rabbits (A and C show the conditions 7 days after Pseudomonas aeruginosa keratitis modeling; B and D show the corneal epithelial repair conditions 7 days after Pseudomonas aeruginosa keratitis modeling; E shows the comparison of corneal transparency after sampling in each group; F and G show the corneal homogenates of each group followed by culture and CFU counting). DETAILED DESCRIPTION

[0022] The present invention provides a two-component multifunctional nanomembrane, which is prepared from the following components in mass fraction: 50-75% of a polymer matrix component one, 24-49% of a polymer matrix component two, and 1% of a functional component; the polymer matrix component one is hyaluronic acid or a hyaluronic acid derivative, the polymer matrix component two is one of collagen, gelatin, methacrylated gelatin or acellular matrix, and the functional component is a drug and an active factor; the drug is loaded on the polymer matrix component one, and the active factor is loaded on the polymer matrix component two; the nanomembrane is formed by chemical cross-linking.

[0023] In the nanomembrane of the present invention, the mass fraction of the polymer matrix component 1 is 50-75%, more preferably 55-70%, and further preferably 60-65%; the mass fraction of the polymer matrix component 2 is 24-49%, more preferably 28-45%, and further preferably 32-41%; the mass fraction of the functional component is 1%.

[0024] In the nanomembrane of the present invention, the polymer matrix component 1 is hyaluronic acid or a hyaluronic acid derivative, more preferably a hyaluronic acid derivative, the hyaluronic acid derivative is preferably a derivative obtained by functionally modifying 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 β-cyclodextrin; specifically, methacryloyl hyaluronic acid, hyaluronic acid cyclodextrin derivative or aldehyde-modified hyaluronic acid.

[0025] In the nanomembrane of the present invention, the second polymer matrix component is one of collagen, gelatin, methacrylated gelatin or acellular matrix, and more preferably one of gelatin, methacrylated gelatin or acellular matrix.

[0026] In the nanomembrane of the present invention, the functional components include drugs and active factors. The drugs are preferably one or more of dexamethasone, dexamethasone sodium phosphate, doxycycline or doxycycline hydrochloride, and the active factors are preferably one of antibacterial polypeptides, anti-angiogenic polypeptides and epidermal growth factor.

[0027] In the present invention, the thickness of the nanofilm is preferably 20 to 500 nm, more preferably 100 to 400 nm, and even more preferably 200 to 300 nm.

[0028] The present invention also provides a method for preparing the dual-component multifunctional nanofilm, comprising the following steps: 1) mixing the polymer matrix component 1, the polymer matrix component 2, and water, and shaking to obtain a mixed solution; dissolving the drug in the mixed solution; and centrifuging to obtain a supernatant as the raw material mixed solution;

[0029] 2) adding the raw material mixed solution obtained in step 1) dropwise to a silicon wafer for a spin coating, first air-drying, then cross-linking, and first vacuum freeze-drying to obtain a pretreated nanofilm;

[0030] 3) The pretreated nanomembrane is immersed in a solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt, then washed with an ethylenediaminetetraacetic acid solution, then immersed in a PBS solution of the active factor, washed with deionized water, and vacuum freeze-dried a second time to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0031] 4) The PVA solution is added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and spin-coated for a second time, air-dried for a second time, and then peeled off from the silicon wafer to obtain the nanofilm.

[0032] In the preparation method of the present invention, step 1) the polymer matrix component one, the polymer matrix component two and water are mixed and shaken to obtain a mixed solution; the mass ratio of the polymer matrix component one to the polymer matrix component two is preferably 1-3:1, more preferably 1.5-2.5:1, and further preferably 2:1; the mass ratio of the total mass of the polymer matrix component one and the polymer matrix component two to water is preferably 1:90-110, more preferably 1:95-105, and further preferably 1:98-102; the shaking temperature is preferably 40-60°C, more preferably 45-55°C, and further preferably 48-52°C; the shaking frequency is preferably 500-1000Hz, more preferably 700-900Hz, and further preferably 750-800Hz; the shaking time is preferably 90-120min, more preferably 95-115min, and further preferably 100-110min.

[0033] In the preparation method of the present invention, in step 1), after dissolving the drug in the mixed solution, centrifugation is performed to obtain the supernatant, which is the raw material mixed solution; the ratio of the drug to the mixed solution is preferably 10-200 mg:1 mL, more preferably 20-180 mg:1 mL, and further preferably 40-160 mg / mL; the dissolution is preferably stirred, and the stirring is preferably carried out under light-proof conditions, and the stirring speed is preferably 1000-5000 r / min, more preferably 2000-4000 r / min, and further preferably 2500-3000 r / min; the centrifugal speed is preferably 2500-3500 rpm, more preferably 2600-3400 rpm, and further preferably 2800-3200 rpm, and the centrifugal time is preferably 5-15 min, more preferably 6-14 min, and further preferably 8-12 min.

[0034] In the preparation method of the present invention, step 2) the raw material mixed solution obtained in step 1) is added dropwise to a silicon wafer for a spin coating, first air-dried, then cross-linked, and first vacuum freeze-dried to obtain a pretreated nanofilm; the silicon wafer is preferably a silicon wafer hydrophobically treated with trichloro(1H,1H,2H,2H-tridecafluorooctyl)silane, and the acceleration of the spin coating is preferably 1400 to 1600 rad / s 2 , more preferably 1450~1550rad / s 2 , and further preferably 1480~1520rad / s 2 The rotation speed of a spin coating is preferably 5000-8000 rpm, more preferably 6000-7000 rpm, and further preferably 6200-6800 rpm. The time of a spin coating is preferably 20-40 s, more preferably 25-35 s, and further preferably 28-32 s. The spin coating is preferably completed by a spin coater, which is commercially available, model KW-4C (Beijing Said Case Electronics Co., Ltd.); the temperature of the first air drying is preferably 20-30 ° C, more preferably 22-28 ° C, and further preferably 24-2 6°C, the air-drying is preferably carried out in a clean bench; the cross-linking is a polymerization reaction initiated by EDC-NHS or ultraviolet light; the temperature of the first vacuum freeze-drying is preferably -70 to -80°C, more preferably -72 to -78°C, and further preferably -74 to -76°C, the vacuum degree of the first vacuum freeze-drying is preferably 10 to 100 Pa, more preferably 15 to 40 Pa, and further preferably 18 to 20 Pa, the time of the first vacuum freeze-drying is preferably 6 to 12 h, more preferably 7 to 11 h, and further preferably 8 to 10 h.

[0035] In the preparation method of the present invention, step 3) the pre-treated nanofilm is immersed in a solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt, then washed with ethylenediaminetetraacetic acid solution, and then immersed in a PBS solution of active factors, washed with deionized water, and vacuum freeze-dried for the second time to obtain a pre-treated nanofilm loaded with drugs and active factors; the 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt solution The concentration is preferably 1 to 3 mg / mL, more preferably 1.5 to 2.5 mg / mL, and further preferably 1.8 to 2.2 mg / mL, and the immersion time in the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester is preferably 0.5 to 1.5 h, more preferably 0.6 to 1.4 h, and further preferably 0.8 to 1.2 h; the concentration of the ethylenediaminetetraacetic acid solution is preferably 0.2 to 0.4 mg / mL, and further preferably The first step is preferably 0.25-0.35 mg / mL, and further preferably 0.28-0.32 mg / mL; the concentration of the PBS solution is preferably 0.5-3 mg / mL, more preferably 1-2.5 mg / mL, and further preferably 1.5-2 mg / mL, the temperature for soaking in the PBS solution is preferably 0-8°C, more preferably 2-6°C, and further preferably 3-5°C, and the soaking time in the PBS solution is preferably 8-12 h, more preferably 9-11 h, and further preferably 9.5-10.5 h; the temperature for the second vacuum freeze-drying is preferably -60--80°C, more preferably -65--75°C, and further preferably -68--72°C, the vacuum degree of the second vacuum freeze-drying is preferably 10-100 Pa, more preferably 12-40 Pa, and further preferably 15-20 Pa, and the time for the second vacuum freeze-drying is preferably 6-12 h, more preferably 8-12 h, and further preferably 10-12 h.

[0036] In the preparation method of the present invention, in step 4), a PVA solution is added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3), and a secondary spin coating is performed, followed by air drying and then peeling from the silicon wafer to obtain the nanofilm; the mass concentration of the PVA solution is preferably 15-25%, more preferably 16-24%, and further preferably 18-22%, and the amount of the PVA solution is preferably 0.5-1.5 mL, more preferably 0.6-1.4 mL, and further preferably 0.8-1.2 mL; the acceleration of the secondary spin coating is preferably 100-500 rad / s 2 , more preferably 150~350rad / s 2 , and further preferably 200 to 300 rad / s2 The rotation speed of the secondary spin coating is preferably 2000-3000 rpm, more preferably 2200-2800 rpm, and further preferably 2400-2600 rpm. The time of the secondary spin coating is preferably 20-40 s, more preferably 25-35 s, and further preferably 26-34 s. The spin coating is preferably carried out in a spin coater, which is commercially available. The model of the spin coater is KW-4C (Beijing Saidcase Electronics Co., Ltd.); the temperature of the second air-drying is preferably 15-25 ° C, more preferably 16-24 ° C, and further preferably 18-22 ° C. The second air-drying is preferably carried out in a clean bench.

[0037] The present invention also provides a method for preparing the dual-component multifunctional nanofilm or the use of the nanofilm in preparing a product for treating corneal damage. When used, the nanofilm is bonded to the cornea and the sacrificial layer is removed using deionized water.

[0038] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] A two-component multifunctional nano-membrane, the mass fractions of the components are: 50% of a polymer matrix component 1, 49% of a polymer matrix component 2, and 1% of a functional component.

[0041] 1) Methacryloylated hyaluronic acid (EFL, Product No. EFL-HAMA-400K) and methacryloylated gelatin (EFL, Product No. EFL-GM-90) were mixed in a mass ratio of 1:1 and dissolved in deionized water. The total mass concentration of the methacryloylated hyaluronic acid and methacryloylated gelatin was 1%. The mixture was shaken at 40°C, the frequency of the shaken ...

[0042] 2) The raw material mixed solution obtained in step 1) is added dropwise to the single crystal silicon wafer for one spin coating, and the acceleration of one spin coating is 1500 rad / s 2The first spin coating speed was 5500 r / min, the first spin coating time was 20 s, the temperature was 25 ° C for the first air drying, and then photocrosslinking was performed, crosslinking was performed under ultraviolet irradiation for 10 min, and the first vacuum freeze drying was performed at a temperature of -75 ° C, the vacuum degree was 10 Pa, and the time was 6 h to obtain a pretreated nanofilm;

[0043] 3) The pretreated nanomembrane was immersed in a 2 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt (ACMEC, Product No. N65560) for 1 hour, then washed with a 0.3 mg / mL solution of ethylenediaminetetraacetic acid (ACMEC, Product No. SA03307), and then immersed in a 0.5 mg / mL solution of antimicrobial peptide (Qiangyao Biotechnology Co., Ltd.) in PBS at 4°C for 10 hours; washed with deionized water, and freeze-dried at -80°C for a second time under vacuum at a vacuum degree of 10 Pa for 12 hours to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0044] 4) 1 mL of 20% polyvinyl alcohol (PVA, Macklin, Product No. P815725) solution was added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and spin-coated for a second time. The acceleration of the second spin-coating was 500 rad / s. 2 The second spin coating was performed at a speed of 2500 rpm for 30 seconds. A second air-drying step was performed at a temperature of 20°C to form a film. After drying, the nanofilm was peeled from the single-crystalline silicon wafer with the support of a protective layer, cut into the desired shape and size, and then encapsulated. This resulted in a dual-component multifunctional nanofilm with a protective layer, loaded with dexamethasone sodium phosphate, and an antimicrobial peptide.

[0045] Example 2

[0046] A two-component multifunctional nano-membrane, the mass fractions of the components are: polymer matrix component 1 60%, polymer matrix component 2 39%, and functional component 1%.

[0047] Cyclodextrin was grafted onto the side chain carboxyl groups of hyaluronic acid. The specific preparation method is as follows: 3g of sodium hyaluronate (Yuanye Bio, Product No. S24592) was dissolved in 60mL of PBS solution and stirred to dissolve. 1.75mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, MacLean, Product No. N808856) and 1.75mmol of N-hydroxysuccinimide (NHS, MacLean, Product No. H6231) were added and stirred at room temperature for 30 minutes. 0.25mmol of β-CD-EDA (MacLean, Product No. C6289) was first dissolved in 10mL of PBS. The two solutions were mixed and stirred at room temperature for 24 hours. The resulting solution was dialyzed against deionized water for 5 days. The solution was pre-frozen at -20°C and freeze-dried in a freeze dryer to obtain a white powder, which is the hyaluronic acid-cyclodextrin derivative.

[0048] 1) Hyaluronic acid cyclodextrin derivative and collagen (Yuanye Bio, product number S25686) were mixed in a mass ratio of 2:1, and the resulting mixture was configured into an aqueous solution with deionized water. The total mass concentration of the hyaluronic acid cyclodextrin derivative and collagen in the aqueous solution was 1%. The mixture was shaken at a temperature of 50°C, a frequency of 800 Hz, and a shaking time of 100 min to obtain a mixed solution. Dexamethasone sodium phosphate was added at a drug-to-mixture ratio of 10 mg / mL; the mixture was stirred at a speed of 2500 rpm for 24 h; and the supernatant was then centrifuged to obtain the raw material mixed solution at a speed of 2500 rpm for 15 min.

[0049] 2) The raw material mixed solution obtained in step 1) was added dropwise to the silicon wafer for one spin coating, and the acceleration of the one spin coating was 1400 rad / s 2 The spin coating speed is 5000 rpm, the spin coating time is 20 s, the first air drying is performed, and the first air drying temperature is 20° C.; then chemical cross-linking is performed, and the spin-coated silicon wafer is placed in an EDC / NHS ethanol solution for chemical cross-linking for 24 hours, wherein the EDC concentration is 14.4 mM and the NHS concentration is 5.6 mM; the first vacuum freeze drying is performed, and the first vacuum freeze drying temperature is -70° C., the first vacuum freeze drying degree is 40 Pa, and the first vacuum freeze drying time is 8 hours; thus, a pretreated nanofilm is obtained;

[0050] 3) The pretreated nanomembrane was immersed in a 1 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt for 0.5 h, then washed with a 0.2 mg / mL ethylenediaminetetraacetic acid solution, and then immersed in a 2 mg / mL PBS solution of an anti-angiogenic polypeptide at 0° C. for 8 h; washed with deionized water, and subjected to a second vacuum freeze-drying at a temperature of -65° C., a vacuum degree of 40 Pa, and a second vacuum freeze-drying time of 18 h to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0051] 4) 0.5 mL of 15% PVA aqueous solution was added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and subjected to secondary spin coating at an acceleration of 300 rad / s. 2 The rotation speed of the second spin coating is 2000 rpm, and the time of the second spin coating is 20 s; the second air drying, the temperature of the second air drying is 15 ° C, and then it is peeled off from the silicon wafer and cut to obtain a nano film.

[0052] Example 3

[0053] A two-component multifunctional nano-membrane, the mass fractions of the components are: polymer matrix component 1 75%, polymer matrix component 2 24%, and functional component 1%.

[0054] The decellularized matrix derived from bovine Achilles tendon was prepared by the laboratory. The specific preparation method is as follows: fresh bovine Achilles tendon was collected from a local slaughterhouse and cut into small pieces of 1-2 mm in size using tissue scissors. The pieces were first soaked in a 1% sodium dodecyl sulfate (BioReagent, Product No. L3771) aqueous solution for 72 hours, and then soaked in a 1% TritonX-100 (Sigma-Aldrich, Product No. X100) solution for 2 hours. The decellularized tissue treated above was further soaked in isopropanol for 2 hours, and then soaked and washed with PBS at a mass ratio of 1:1000 for 3 days, with new PBS changed twice a day. The tissue was sterilized using a 0.1% peracetic acid solution for 4 hours, then washed with sterile deionized water for 6 hours. The sterilized decellularized tissue was pre-frozen at -20°C and placed in a freeze dryer for freeze drying to obtain white block decellularized tissue. Grind the decellularized tissue using a mortar and pestle. Pepsin (Yuanye Bio, Catalog No. S10027) was added to the pulverized tissue sample at a 10:1 mass ratio and digested in a 1:10 mass ratio of acetic acid for 72 hours. The digested solution was filtered through a 40 μm mesh (Corning) to remove undigested impurities. The resulting solution was pre-frozen at -20°C and freeze-dried in a freeze-dryer. The resulting white powder was the decellularized matrix.

[0055] 1) Hyaluronic acid and acellular matrix were mixed in a mass ratio of 3:1, dissolved in water, and the total mass concentration of hyaluronic acid and acellular matrix in the water was 1%. The mixture was shaken at 60° C., a frequency of 1000 Hz, and a time of 90 minutes to obtain a mixed solution. Doxycycline hydrochloride was added at a ratio of 100 mg / mL to the mixed solution. The mixture was stirred at 5000 rpm. The supernatant was then centrifuged at 3500 rpm for 15 minutes to obtain the raw material mixed solution.

[0056] 2) The raw material mixed solution obtained in step 1) was added dropwise to the silicon wafer for one spin coating, and the acceleration of the one spin coating was 1600 rad / s 2 , the rotation speed of the first spin coating is 8000 rpm, the time of the first spin coating is 40 s, the first air drying, the temperature of the first air drying is 30 ° C; then chemical cross-linking is carried out, immersing in an ethanol solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) for cross-linking for 24 hours, wherein the EDC concentration is 14.4 mM and the NHS concentration is 5.6 mM; the first vacuum freeze drying, the temperature of the first vacuum freeze drying is -80 ° C, the vacuum degree of the first vacuum freeze drying is 100 Pa, and the time of the first vacuum freeze drying is 12 hours; to obtain a pretreated nanofilm;

[0057] 3) The pretreated nanomembrane was immersed in a 3 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt for 1.5 h, then washed with a 0.4 mg / mL ethylenediaminetetraacetic acid solution, and then immersed in a 3 mg / mL PBS solution of epithelial growth factor at 8° C. for 12 h; washed with deionized water, and subjected to a second vacuum freeze-drying at a temperature of -75° C., a vacuum degree of 100 Pa, and a time of 24 h to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0058] 4) 1.5 mL of 25% PVA solution was added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and subjected to secondary spin coating at an acceleration of 100 rad / s. 2 The rotation speed of the second spin coating is 3000 rpm, and the time of the second spin coating is 40 s; the second air drying, the temperature of the second air drying is 25 ° C, and then it is peeled off from the silicon wafer and cut to obtain a nano film.

[0059] Example 4

[0060] A two-component multifunctional nano-membrane, the mass fractions of the components are: polymer matrix component 1 75%, polymer matrix component 2 24%, and functional component 1%.

[0061] 1) Aldehyde-modified hyaluronic acid (CreativePEGWorks, Product No. ZCR-HA-311) and gelatin (Sigma-Aldrich, Product No. V900863) were mixed in a mass ratio of 3:1 and dissolved in water to a total mass concentration of 1% for aldehyde-modified hyaluronic acid and gelatin. The mixture was shaken at 60°C, a frequency of 1000 Hz, and a time of 90 minutes to obtain a mixed solution. Doxycycline hydrochloride (Sigma-Aldrich, Product No. D3072) was added at a ratio of 100 mg / mL to the mixed solution. The mixture was stirred at 3000 rpm and centrifuged at 3500 rpm for 15 minutes to obtain the supernatant, which was the raw material mixed solution.

[0062] 2) The raw material mixed solution obtained in step 1) was added dropwise to the silicon wafer for one spin coating, and the acceleration of the one spin coating was 1600 rad / s 2 , the rotation speed of the first spin coating is 8000 rpm, the time of the first spin coating is 40 s, the first air drying, the temperature of the first air drying is 30 ° C; then chemical cross-linking is carried out, immersing in an ethanol solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) for cross-linking for 24 hours, wherein the EDC concentration is 14.4 mM and the NHS concentration is 5.6 mM; the first vacuum freeze drying is carried out, the temperature of the first vacuum freeze drying is -80 ° C, the vacuum degree of the first vacuum freeze drying is 50 Pa, and the time of the first vacuum freeze drying is 10 hours; to obtain a pretreated nanofilm;

[0063] 3) The pretreated nanomembrane was immersed in a 3 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt for 1.5 h, then washed with a 0.4 mg / mL ethylenediaminetetraacetic acid solution, and then immersed in a 3 mg / mL antimicrobial peptide solution in PBS at 8° C. for 12 h; washed with deionized water, and subjected to a second vacuum freeze-drying process at -75° C., a vacuum degree of 50 Pa, and a second vacuum freeze-drying time of 24 h to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0064] 4) 1.5 mL of 25% PVA solution was added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and subjected to secondary spin coating at an acceleration of 100 rad / s. 2 The rotation speed of the second spin coating is 3000 rpm, and the time of the second spin coating is 20 s; the second air drying, the temperature of the second air drying is 15 ° C, and then it is peeled off from the silicon wafer and cut to obtain a nano film.

[0065] Example 5

[0066] A two-component multifunctional nano-membrane, the mass fractions of the components are: 50% of a polymer matrix component 1, 49% of a polymer matrix component 2, and 1% of a functional component.

[0067] 1) Methacrylated hyaluronic acid and methacrylated gelatin were mixed in a mass ratio of 1:1 and dissolved in deionized water. The total mass concentration of the methacrylated hyaluronic acid and methacrylated gelatin was 1%. The mixture was shaken at 40°C, the frequency of the shaken frequency was 1000 Hz, and the shaken time was 120 min to obtain a mixed solution. Dexamethasone sodium phosphate (ACMEC, product number D32140) was mixed with the mixed solution at a ratio of 1 mg:100 mL. At the same time, a photoinitiator I2959 (Sigma-Aldrich, product number 410896) accounting for 0.5% of the total mass fraction of the solution was added. The mixture was stirred at a speed of 1000 rpm, and then centrifuged at 3000 rpm for 10 min. The supernatant was obtained as the raw material mixed solution.

[0068] 2) The raw material mixed solution obtained in step 1) is added dropwise to the single crystal silicon wafer for one spin coating, and the acceleration of one spin coating is 1500 rad / s 2 The first spin coating speed was 5500 r / min, the first spin coating time was 20 s, the temperature was 25 ° C for the first air drying, and then photocrosslinking was performed, crosslinking was performed under ultraviolet irradiation for 10 min, and the first vacuum freeze drying was performed at a temperature of -75 ° C, the vacuum degree was 10 Pa, and the time was 6 h to obtain a pretreated nanofilm;

[0069] 3) The pretreated nanomembrane was immersed in a 2 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt for 1 hour, then washed with a 0.3 mg / mL ethylenediaminetetraacetic acid solution, and then immersed in a 0.5 mg / mL PBS solution of an anti-angiogenic peptide (Qiangyao Biotechnology Co., Ltd.) at 4°C for 10 hours; washed with deionized water, and freeze-dried at -80°C for a second time with a vacuum degree of 10 Pa for 12 hours to obtain a pretreated nanomembrane loaded with drugs and active factors;

[0070] 4) 1 mL of 20% PVA solution was added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and subjected to secondary spin coating at an acceleration of 500 rad / s. 2The second spin coating was performed at a speed of 2500 rpm for 30 seconds. A second air-drying step was performed at 20°C to form a film. After drying, the nanofilm was peeled from the single-crystalline silicon wafer with the support of a protective layer, cut into the desired shape and size, and then encapsulated. This resulted in a dual-component multifunctional nanofilm with a protective layer, loaded with dexamethasone sodium phosphate, and an anti-angiogenic peptide.

[0071] Comparative Example 1

[0072] A two-component multifunctional nano-membrane, the mass fractions of the components are: 50% of a polymer matrix component 1, 49% of a polymer matrix component 2, and 1% of a functional component.

[0073] 1) Methacrylated hyaluronic acid and methacrylated gelatin were mixed in a mass ratio of 1:1 and dissolved in deionized water. The total mass concentration of the methacrylated hyaluronic acid and methacrylated gelatin was 1%. The mixture was shaken at 40°C, with a frequency of 1000 Hz, and a shaking time of 120 min to obtain a mixed solution. Dexamethasone sodium phosphate was mixed with the mixed solution in a ratio of 1 mg:100 mL, and a photoinitiator I2959 (Sigma-Aldrich, Catalog No. 410896) accounting for 0.5% of the total mass fraction of the solution was added. The mixture was stirred at a speed of 1000 rpm, and then centrifuged at 3000 rpm for 10 min. The supernatant was obtained as the raw material mixed solution.

[0074] 2) The raw material mixed solution obtained in step 1) is added dropwise to the single crystal silicon wafer for one spin coating, and the acceleration of one spin coating is 1500 rad / s 2 The first spin coating speed was 5500 r / min, the first spin coating time was 20 s, the temperature was 25 ° C for the first air drying, and then photocrosslinking was performed, crosslinking was performed under ultraviolet irradiation for 10 min, and the first vacuum freeze drying was performed at a temperature of -75 ° C, the vacuum degree was 10 Pa, and the time was 6 h to obtain a pretreated nanofilm;

[0075] 3) The pretreated nanofilm was immersed in a 2 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt for 1 hour, then washed with a 0.3 mg / mL ethylenediaminetetraacetic acid solution; washed with deionized water, and freeze-dried at -80°C under a vacuum of 10 Pa for 12 hours to obtain a drug-loaded pretreated nanofilm;

[0076] 4) 1 mL of 20% PVA solution was added dropwise to the surface of the drug-loaded pretreated nanofilm obtained in step 3) and subjected to secondary spin coating at an acceleration of 500 rad / s. 2The second spin coating is performed at a speed of 2500 rpm and a time of 30 seconds. The second air drying is performed at a temperature of 20°C to form a film. After drying, the nanofilm is peeled off from the single crystal silicon wafer with the support of a protective layer, cut into the desired shape and size, and packaged to obtain the nanofilm.

[0077] Comparative Example 2

[0078] A two-component multifunctional nano-membrane, the mass fractions of the components are: 50% of a polymer matrix component 1, 49% of a polymer matrix component 2, and 1% of a functional component.

[0079] 1) Methacrylated hyaluronic acid and methacrylated gelatin were mixed in a mass ratio of 1:1 and dissolved in deionized water. The total mass concentration of the methacrylated hyaluronic acid and methacrylated gelatin was 1%. The mixture was shaken at 40°C, a frequency of 1000 Hz, and a time of 120 minutes to obtain a mixed solution. A photoinitiator, I2959 (Sigma-Aldrich, Catalog No. 410896), accounting for 0.5% of the total mass fraction of the solution, was added. The mixture was stirred at 1000 rpm, then centrifuged at 3000 rpm for 10 minutes. The supernatant was obtained as the raw material mixed solution.

[0080] 2) The raw material mixed solution obtained in step 1) is added dropwise to the single crystal silicon wafer for one spin coating, and the acceleration of one spin coating is 1500 rad / s 2 The first spin coating speed was 5500 r / min, the first spin coating time was 20 s, the temperature was 25 ° C for the first air drying, and then photocrosslinking was performed, crosslinking was performed under ultraviolet irradiation for 10 min, and the first vacuum freeze drying was performed at a temperature of -75 ° C, the vacuum degree was 10 Pa, and the time was 6 h to obtain a pretreated nanofilm;

[0081] 3) The pretreated nanomembrane was immersed in a 2 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt for 1 hour, then washed with a 0.3 mg / mL ethylenediaminetetraacetic acid solution, and then immersed in a 0.5 mg / mL PBS solution of an anti-angiogenic peptide at 4°C for 10 hours; washed with deionized water, and freeze-dried at -80°C for a second time under vacuum at a vacuum degree of 10 Pa for 12 hours to obtain a pretreated nanomembrane loaded with active factors;

[0082] 4) 1 mL of 20% PVA solution was added dropwise to the surface of the pretreated nanofilm loaded with active factors obtained in step 3) and subjected to secondary spin coating at an acceleration of 500 rad / s. 2The second spin coating is performed at a speed of 2500 rpm and a time of 30 seconds. The second air drying is performed at a temperature of 20°C to form a film. After drying, the nanofilm is peeled off from the single crystal silicon wafer with the support of a protective layer, cut into the desired shape and size, and packaged to obtain the nanofilm.

[0083] Comparative Example 3

[0084] A two-component multifunctional nano-membrane, the mass fractions of the components are: polymer matrix component one 50%, polymer matrix component two 49%.

[0085] 1) Methacrylated hyaluronic acid and methacrylated gelatin were mixed in a mass ratio of 1:1 and dissolved in deionized water. The total mass concentration of the methacrylated hyaluronic acid and methacrylated gelatin was 1%. The mixture was shaken at 40°C, a frequency of 1000 Hz, and a time of 120 minutes to obtain a mixed solution. A photoinitiator, I2959 (Sigma-Aldrich, Catalog No. 410896), accounting for 0.5% of the total mass fraction of the solution, was added. The mixture was stirred at 1000 rpm, then centrifuged at 3000 rpm for 10 minutes. The supernatant was obtained as the raw material mixed solution.

[0086] 2) The raw material mixed solution obtained in step 1) is added dropwise to the single crystal silicon wafer for one spin coating, and the acceleration of one spin coating is 1500 rad / s 2 The first spin coating speed was 5500 r / min, the first spin coating time was 20 s, the temperature was 25 ° C for the first air drying, and then photocrosslinking was performed, crosslinking was performed under ultraviolet irradiation for 10 min, and the first vacuum freeze drying was performed at a temperature of -75 ° C, the vacuum degree was 10 Pa, and the time was 6 h to obtain a pretreated nanofilm;

[0087] 3) The pretreated nanomembrane was immersed in a 2 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt for 1 hour, then washed with a 0.3 mg / mL solution of ethylenediaminetetraacetic acid; rinsed with deionized water, and freeze-dried at -80°C under a vacuum of 10 Pa for 12 hours to obtain a pretreated nanomembrane without loading of drugs or active factors;

[0088] 4) 1 mL of 20% PVA solution was added dropwise to the surface of the pretreated nanofilm obtained in step 3) without loading drugs and active factors, and the secondary spin coating was performed at an acceleration of 500 rad / s. 2 The rotation speed of the second spin coating is 2500rpm, and the time of the second spin coating is 30s; the second air drying film is formed, and the temperature of the second air drying is 20°C. After drying, the nano film is peeled off from the single crystal silicon wafer under the support of the protective layer, cut into the required shape and size, and packaged to obtain the nano film.

[0089] Comparative Example 4

[0090] A two-component multifunctional nano-membrane, the mass fractions of the components are: polymer matrix component 1 75%, polymer matrix component 2 24%, and functional component 1%.

[0091] 1) Aldehyde-modified hyaluronic acid and gelatin were mixed in a mass ratio of 3:1, dissolved in water, and the total mass concentration of the aldehyde-modified hyaluronic acid and gelatin in the water was 1%. The mixture was shaken at 60° C., a frequency of 1000 Hz, and a shaking time of 90 minutes to obtain a mixed solution. Doxycycline hydrochloride was added at a ratio of 100 mg / mL to the mixed solution. The mixture was stirred at 3000 rpm and centrifuged to obtain the supernatant, which was the raw material mixed solution. The mixture was centrifuged at 3500 rpm for 15 minutes.

[0092] 2) The raw material mixed solution obtained in step 1) was added dropwise to the silicon wafer for one spin coating, and the acceleration of the one spin coating was 1600 rad / s 2 , the rotation speed of the first spin coating is 8000 rpm, the time of the first spin coating is 40 s, the first air drying, the temperature of the first air drying is 30 ° C; then chemical cross-linking is carried out, immersing in an ethanol solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) for cross-linking for 24 hours, wherein the EDC concentration is 14.4 mM and the NHS concentration is 5.6 mM; the first vacuum freeze drying is carried out, the temperature of the first vacuum freeze drying is -80 ° C, the vacuum degree of the first vacuum freeze drying is 50 Pa, and the time of the first vacuum freeze drying is 10 hours; to obtain a pretreated nanofilm;

[0093] 3) The pretreated nanofilm was immersed in a 3 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimidyl ester sodium salt for 1.5 h, then washed with a 0.4 mg / mL solution of ethylenediaminetetraacetic acid; washed with deionized water, and subjected to a second vacuum freeze-drying process at -75°C, a vacuum degree of 50 Pa, and a time of 24 h to obtain a drug-loaded pretreated nanofilm;

[0094] 4) 1.5 mL of 25% PVA solution was added dropwise to the surface of the drug-loaded pretreated nanofilm obtained in step 3) and spin-coated for the second time at an acceleration of 100 rad / s. 2 The rotation speed of the second spin coating is 3000 rpm, and the time of the second spin coating is 20 s; the second air drying, the temperature of the second air drying is 15 ° C, and then it is peeled off from the silicon wafer and cut to obtain a nano film.

[0095] Comparative Example 5

[0096] A two-component multifunctional nano-membrane, the mass fractions of the components are: polymer matrix component 1 75%, polymer matrix component 2 24%, and functional component 1%.

[0097] 1) Aldehydated hyaluronic acid and gelatin were mixed in a mass ratio of 3:1, dissolved in water to a total mass concentration of 1% of the aldehydated hyaluronic acid and gelatin in the water, and shaken at 60° C., a frequency of 1000 Hz, and a shaking time of 90 minutes to obtain a mixed solution; stirred at a speed of 3000 rpm; and then centrifuged at a speed of 3500 rpm for 15 minutes to obtain a supernatant as the raw material mixed solution;

[0098] 2) The raw material mixed solution obtained in step 1) was added dropwise to the silicon wafer for one spin coating, and the acceleration of the one spin coating was 1600 rad / s 2 , the rotation speed of the first spin coating is 8000 rpm, the time of the first spin coating is 40 s, the first air drying, the temperature of the first air drying is 30 ° C; then chemical cross-linking is carried out, immersing in an ethanol solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) for cross-linking for 24 hours, wherein the EDC concentration is 14.4 mM and the NHS concentration is 5.6 mM; the first vacuum freeze drying is carried out, the temperature of the first vacuum freeze drying is -80 ° C, the vacuum degree of the first vacuum freeze drying is 50 Pa, and the time of the first vacuum freeze drying is 10 hours; to obtain a pretreated nanofilm;

[0099] 3) The pretreated nanomembrane was immersed in a 3 mg / mL solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt for 1.5 h, then washed with a 0.4 mg / mL ethylenediaminetetraacetic acid solution, and then immersed in a 3 mg / mL antimicrobial peptide solution in PBS at 8° C. for 12 h; washed with deionized water, and subjected to a second vacuum freeze-drying process at -75° C., a vacuum degree of 50 Pa, and a second vacuum freeze-drying time of 24 h to obtain a pretreated nanomembrane loaded with active factors;

[0100] 4) 1.5 mL of 25% PVA solution was added dropwise to the surface of the pretreated nanofilm loaded with active factors obtained in step 3) and spin-coated for the second time. The acceleration of the second spin-coating was 100 rad / s. 2 The rotation speed of the second spin coating is 3000 rpm, and the time of the second spin coating is 20 s; the second air drying, the temperature of the second air drying is 15 ° C, and then it is peeled off from the silicon wafer and cut to obtain a nano film.

[0101] Experimental Example 1

[0102] Ocular surface manipulation and OCT examination of corneal diseases treated with the nanofilm prepared in Example 1 of the present invention

[0103] The normal cornea of ​​New Zealand white rabbits was used to demonstrate the ocular surface operation of the nanomembrane, and optical coherence tomography (OCT) was used to examine the corneal structure at various levels and the attachment of the nanomembrane.

[0104] like Figure 1 As shown, Figure 1 A in the middle is an optical photograph and OCT structural layer diagram of a normal rabbit cornea, showing that the normal cornea is about 374μm thick and has a 5-layer structure: epithelial layer 33μm, anterior elastic layer, stromal layer 320μm, posterior elastic layer and endothelial layer; B is after scraping the corneal epithelium, the cornea becomes edematous, and the thickness on OCT is 410μm; C is a nanomembrane attached to the corneal surface, and the OCT shows that the thickness of the functional nanomembrane with a sacrificial layer is about 16μm; D is after flushing the cornea with normal saline, the sacrificial layer dissolves, and the OCT resolution cannot detect the nano-level functional nanomembrane, and the corneal thickness is still 410μm; the nanomembrane can be directly attached to the corneal surface without sutures, and the water-soluble sacrificial layer is easy to operate. After attachment, the sacrificial layer can be removed by normal saline, retaining the close fit between the nanomembrane and the cornea.

[0105] Experimental Example 2

[0106] Experimental study on nanofilm treatment of corneal alkali burns

[0107] A New Zealand rabbit corneal alkali burn model was used to study the therapeutic effects of different active ingredients in the nanofilm (mainly containing dexamethasone and / or anti-angiogenic peptide components) on corneal alkali burns, focusing on its anti-inflammatory, anti-angiogenic and epithelial repair effects.

[0108] The right eye of mature New Zealand white rabbits (2.0-2.5 kg) was used for modeling. An equal amount of 1 M sodium hydroxide solution was taken using a capillary tube to completely wet a 6 mm diameter filter paper. The filter paper was then soaked into the center of the right cornea for 30 s to establish a stable and uniform corneal alkali burn model.

[0109] After successful modeling, the mice were randomly divided into 5 groups, with 4 mice in each group, and treated with blank control group (i.e., model group), dexamethasone nanomembrane (Comparative Example 1), anti-angiogenic peptide nanomembrane (Comparative Example 2), nanomembrane without added drugs or active ingredients (Comparative Example 3), and dexamethasone anti-angiogenic peptide nanomembrane (Example 5), respectively. The therapeutic effect on corneal alkali burns was evaluated by observing corneal opacity, corneal inflammation, neovascularization and epithelial repair.

[0110] The results are as follows Figure 2As shown in A and C in Figure 1, A and C are the anterior segment photography observations using a slit lamp microscope 7 days after alkali burn modeling to observe corneal opacity, ocular surface congestion and corneal neovascularization, and score them.

[0111] The scoring standard for the central corneal alkali burn area is 0-5 points: 0 points, the cornea is completely transparent, and no opacity can be seen with any slit lamp examination method. 1 point, very mild corneal opacity, which can only be seen with indirect wide tangential illumination of the slit lamp. 2 points, mild corneal opacity, low-density opacity that can only be seen with careful observation using direct illumination of the slit lamp or diffuse light illumination, and the pupil and iris veins can be seen. 3 points, moderate corneal opacity, medium-density opacity can be directly observed with slit lamp examination, and only the pupil can be seen. 4 points, severe corneal opacity, the opacity obviously blocks the slit lamp light from passing through the cornea, and only the anterior chamber can be seen. 5 points, extremely severe opacity, the opacity completely blocks the slit lamp light from passing through the cornea, and the anterior chamber structure cannot be observed.

[0112] The conjunctival and limbal congestion scoring standard is 0-3 points: 0 point, no congestion; 1 point, mild congestion at the corneal edge; 2 points, moderate congestion at the corneal edge; 3 points, severe congestion at the corneal edge with obvious veins.

[0113] Neovascularization was scored on a scale of 0 to 4: 0, no corneal vessels and normal corneal and scleral limbus; 1, fewer than 5 vascular loops, no larger than 0.3 mm; 2, 5 to 15 vascular loops, no larger than 0.3 mm; 3, more than 15 vessels or vascular loops larger than 0.3 mm; 4, 2 or more vascular loops larger than 0.5 mm.

[0114] The nanofilm group without added drugs or active ingredients (Comparative Example 3) had a central corneal alkali burn area turbidity score of 4.2±0.27, a conjunctival and corneal limbal congestion score of 2.5±0.5, and a neovascularization score of 3.4±0.54. Compared with the blank control group, the central corneal alkali burn area showed a porcelain white circular turbidity, and the intraocular structure in the central area could not be seen. The corneal turbidity score was 4.5±0.0.5, the conjunctival and corneal limbal congestion score was 2.5±0.5, and the neovascularization score was 3.8±0.44. There was no significant difference (P>0.05).

[0115] In the dexamethasone nanomembrane (comparative example 1) group, the opacity score of the central corneal alkali burn area was 3.6±0.41, the conjunctival and corneal limbal congestion score was 1.5±0.61, and the neovascularization score was 1.8±0.83; in the anti-angiogenic peptide nanomembrane (comparative example 2) group, the opacity score of the central corneal alkali burn area was 3.7±0.44, the conjunctival and corneal limbal congestion score was 2.4±0.41, and the neovascularization score was 1.8±0.44, all of which were significantly alleviated; among them, the congestion of the dexamethasone hyaluronic acid nanomembrane group was significantly alleviated; and the neovascularization of both the dexamethasone hyaluronic acid nanomembrane group and the anti-angiogenic peptide hyaluronic acid nanomembrane group was significantly alleviated. The dexamethasone anti-angiogenic peptide nanofilm-treated (Example 5) group had significantly reduced corneal central alkali burn area opacity score of 2.6±0.42, conjunctival and limbal congestion score of 0.6±0.41, and neovascularization score of 0.4±0.55, which were lower than those of the other four groups (P<0.05).

[0116] like Figure 2 As shown in Figures B and D, corneal epithelial repair was observed using sodium fluorescein staining 7 days after alkali burn injury. The corneal epithelial defect was stained with sodium fluorescein as a clearly defined green area. Image J was used to identify the stained area and calculate the area of ​​the defect.

[0117] The central corneal epithelium of the blank control group showed a circular defect. 2 ) and blank control group (14.68±8.85mm 2 ) group (7.87±5.56mm) 2 ) and anti-angiogenic peptide nanofilm (Comparative Example 2) group (7.35±4.64mm 2 ) The corneal epithelial defect area was further reduced and was smaller than that of the control group. 2 ) had no obvious fluorescein staining area, indicating that the corneal epithelium was well repaired; the defect area was smaller than that of the other four groups (P<0.05).

[0118] The above results show that the nanofilm without added drugs or active ingredients (Comparative Example 3) has a promoting effect on epithelial repair of corneal alkali burns. The dexamethasone nanofilm (Comparative Example 1) has an anti-inflammatory effect on corneal alkali burns and reduces ocular surface congestion. The anti-angiogenic peptide nanofilm (Comparative Example 2) has an anti-angiogenic effect on corneal alkali burns and can inhibit the formation of new blood vessels. The dexamethasone anti-angiogenic peptide nanofilm (Example 5) has the multifunctional effects of promoting epithelial repair, anti-inflammation and inhibiting new blood vessels, and can effectively treat corneal alkali burns.

[0119] Experimental Example 3

[0120] Experimental study on nanofilm treatment of Pseudomonas aeruginosa keratitis

[0121] The New Zealand rabbit Pseudomonas aeruginosa keratitis model was used to study the therapeutic effect of nanofilm (loaded with doxycycline hydrochloride and / or antibacterial peptide components) on Pseudomonas aeruginosa keratitis, focusing on its antibacterial effect.

[0122] The right eye of a mature New Zealand white rabbit (2.0-2.5 kg) was used for modeling. The central corneal area was marked with a 6×6 mm trephine drill. A shallow corneal stromal tunnel was made in the marked area of ​​the central cornea with a 30G insulin needle. 10 μL of 10 3 The mice were randomly divided into 4 groups, each with 4 mice, and treated with blank control (i.e., model group), doxycycline hydrochloride nanofilm (Comparative Example 4), antibacterial polypeptide nanofilm (Comparative Example 5), and doxycycline hydrochloride antibacterial polypeptide nanofilm (Example 4), respectively. The therapeutic effect on Pseudomonas aeruginosa keratitis was evaluated by observing the control of corneal infection and performing clinical scoring, extracting and calculating the area of ​​corneal epithelial defect using Image J, and detecting bacterial content in samples.

[0123] The Pseudomonas aeruginosa keratitis model clinical scoring scale ranges from 0 to 4: 0: Transparent cornea, no opacity, and a regular surface. 1: Corneal opacity ranges from 1% to 25%, with mild corneal irregularity and slight corneal opacity, and the iris vessels and pupil are clearly visible. 2: Corneal opacity ranges from 26% to 50%, with corneal stromal edema, bulging or concave surface, and mild corneal opacity, and the iris vessels and pupil are clearly visible. 3: Corneal opacity ranges from 51% to 75%, with significant corneal stromal edema, significant concave surface or Descemet's membrane bulge, uneven corneal opacity, and no visible underlying structures. 4: Corneal opacity ranges from 76% to 100%, with corneal perforation and uniform corneal opacity, and no visible underlying structures.

[0124] The results are as follows Figure 3 As shown, Figure 3 A and C are observations of the anterior segment of the eye using a slit lamp microscope 7 days after modeling of Pseudomonas aeruginosa keratitis. The blank control group (model group) showed corneal congestion with large gray-white ulcer lesions, and the clinical score was 3.65±0.47. The doxycycline hydrochloride nanofilm (Comparative Example 4, clinical score: 2.25±0.28) and the antibacterial polypeptide nanofilm (Comparative Example 5, clinical score: 1.87±0.25) significantly inhibited corneal infection, significantly alleviated ulcer lesions, and reduced clinical scores (P<0.05). The doxycycline hydrochloride antibacterial polypeptide nanofilm (Example 4, clinical score: 0.62±0.47) effectively inhibited corneal infection, and the clinical score was significantly lower than that of the doxycycline hydrochloride nanofilm and antibacterial polypeptide nanofilm groups (P<0.05).

[0125] Figure 3 Images B and D show corneal epithelial repair 7 days after Pseudomonas aeruginosa keratitis modeling using sodium fluorescein staining. The corneal epithelial defect is stained with sodium fluorescein as a clearly defined green area. Image J was used to identify the stained area and calculate the area of ​​the defect.

[0126] In the blank control group (model group), a large area of ​​central corneal epithelium was stained, and the area of ​​corneal epithelial defect was 19.58±8.13mm. 2 The corneal epithelial defect area of ​​doxycycline hydrochloride nanofilm (Comparative Example 4) was 5.48±4.55mm 2 The corneal epithelial defect area of ​​the antibacterial polypeptide nanofilm group (Comparative Example 5) was 6.04±3.53mm 2 The corneal epithelial defect area of ​​the doxycycline hydrochloride nanofilm (Comparative Example 4) and antimicrobial polypeptide nanofilm groups was clearly demarcated in the central cornea. The corneal epithelial defect area of ​​the doxycycline hydrochloride antimicrobial polypeptide nanofilm group (Example 4) was 0.7±0.96 mm 2 , no obvious staining, and the corneal epithelium was well repaired (P<0.05).

[0127] Figure 3 Figure E shows the transparency of the corneas of each group after sampling. The transparency of the doxycycline hydrochloride antibacterial polypeptide nanofilm group (Example 4) is the best.

[0128] Figure 3 The corneas of groups F and G were homogenized and cultured and CFU counted. After culture, obvious colony formation was observed in the blank control group (model group). The colonies in the doxycycline hydrochloride nanofilm (Comparative Example 4) and antibacterial polypeptide nanofilm groups (Comparative Example 5) were significantly reduced. There was almost no obvious colony formation in the doxycycline hydrochloride antibacterial polypeptide nanofilm group (Example 4), and the CFU count was the lowest.

[0129] The above results show that both doxycycline hydrochloride nanofilms and antimicrobial peptide nanofilms can effectively inhibit Pseudomonas aeruginosa keratitis lesions and promote corneal epithelial repair. The multi-component nanofilm combining doxycycline hydrochloride and antimicrobial peptides (Example 4) can more effectively treat Pseudomonas aeruginosa keratitis.

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A two-component multifunctional nanofilm, characterized in that: Prepared from the following components by weight: Polymer matrix component 1 50~75%, polymer matrix component 2 24~49%, functional component 1%; The polymer matrix component 1 is methacryloyl hyaluronic acid or aldehyde hyaluronic acid, the polymer matrix component 2 is gelatin or methacryloyl gelatin, and the functional components include drugs and active factors; the drugs are loaded on the polymer matrix component 1, and the active factors are loaded on the polymer matrix component 2; The nanofilm is formed by chemical cross-linking; The drug is one or more of dexamethasone, dexamethasone sodium phosphate, doxycycline or doxycycline hydrochloride; the active factor is one of antibacterial polypeptide, anti-angiogenic polypeptide and epithelial growth factor; The thickness of the nanofilm is 20 to 500 mm; The method for preparing the dual-component multifunctional nanofilm comprises the following steps: 1) Mixing polymer matrix component 1, polymer matrix component 2 and water, shaking to obtain a mixed solution, dissolving the drug in the mixed solution, and centrifuging to obtain the supernatant as the raw material mixed solution; 2) adding the raw material mixed solution obtained in step 1) dropwise to a silicon wafer for a spin coating, first air-drying, then cross-linking, and first vacuum freeze-drying to obtain a pretreated nanofilm; 3) The pretreated nanomembrane is immersed in a solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt, then washed with an ethylenediaminetetraacetic acid solution, then immersed in a PBS solution of the active factor, washed with deionized water, and vacuum freeze-dried a second time to obtain a pretreated nanomembrane loaded with drugs and active factors; 4) The PVA solution is added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and spin-coated for a second time, air-dried for a second time, and then peeled off from the silicon wafer to obtain the nanofilm.

2. The method for preparing the dual-component multifunctional nanofilm according to claim 1, characterized in that: The following steps are involved: 1) Mixing polymer matrix component 1, polymer matrix component 2 and water, shaking to obtain a mixed solution, dissolving the drug in the mixed solution, and centrifuging to obtain the supernatant as the raw material mixed solution; 2) adding the raw material mixed solution obtained in step 1) dropwise to a silicon wafer for a spin coating, first air-drying, then cross-linking, and first vacuum freeze-drying to obtain a pretreated nanofilm; 3) The pretreated nanomembrane is immersed in a solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester sodium salt, then washed with an ethylenediaminetetraacetic acid solution, then immersed in a PBS solution of the active factor, washed with deionized water, and vacuum freeze-dried a second time to obtain a pretreated nanomembrane loaded with drugs and active factors; 4) The PVA solution is added dropwise to the surface of the pretreated nanofilm loaded with drugs and active factors obtained in step 3) and spin-coated for a second time, air-dried for a second time, and then peeled off from the silicon wafer to obtain the nanofilm.

3. The method for preparing a two-component multifunctional nanofilm according to claim 2, wherein: In step 1), the mass ratio of the polymer matrix component 1 and the polymer matrix component 2 is 1-3:1, and the mass ratio of the total mass of the polymer matrix component 1 and the polymer matrix component 2 to water is 1:90-110; the oscillation temperature is 40-60°C, the oscillation frequency is 500-1000 Hz, and the oscillation time is 90-120 min.

4. The method for preparing a two-component multifunctional nanofilm according to claim 2, wherein: In step 1), the ratio of the drug to the mixed solution is 10-200 mg:1 mL; the centrifugal speed is 2500-3500 rpm, and the centrifugal time is 5-15 min.

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

6. The method for preparing a two-component multifunctional nanofilm according to claim 2, wherein: The concentration of the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester in step 3) is 1 to 3 mg / mL, and the immersion time in the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid-3-thio-N-succinimide ester is 0.5 to 1.5 h; the concentration of the ethylenediaminetetraacetic acid solution is 0.2 to 0.4 mg / mL; The concentration of the PBS solution in step 3) is 0.5-3 mg / mL, the immersion temperature in the PBS solution is 0-8°C, and the immersion time in the PBS solution is 8-12 hours; the temperature of the second vacuum freeze-drying is -60-80°C, the vacuum degree of the second vacuum freeze-drying is 10-100 Pa, and the second vacuum freeze-drying time is 12-24 hours.

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

8. Use of the dual-component multifunctional nanofilm according to claim 1 or the nanofilm obtained by the preparation method according to any one of claims 2 to 7 in the preparation of a product for treating corneal damage.

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