CN112891326B - A kind of alginic acid gel drug film loaded with natamycin and its preparation method - Google Patents

Abstract

The invention relates to the technical field of medical materials, and relates to a natamycin-loaded alginic acid gel medicinal membrane and a preparation method thereof, wherein the natamycin-loaded alginic acid gel medicinal membrane comprises the following components in percentage by weight: each 1mL of the medicinal membrane contains 0.01-0.03g of sodium alginate, 0.0025-0.0075g of polyoxyethylene and 0.005-0.015g of natamycin; the alginic acid gel is used as a carrier, the natamycin is loaded, and the ethanol solution containing calcium ions is used for crosslinking, so that the spatial structure of the material is more cohesive, the medicine-carrying gel is more stable after film formation, the size of pores in the medicine film is reduced, the medicine release is delayed, and the action time of the natamycin is prolonged; the alginic acid has good histocompatibility, and reduces eye irritation and discomfort of natamycin; the medicine can be better attached to the corneal ulcer surface, has long action time, good effect, no irritation and discomfort and high patient acceptance; is a novel eye medicine film, and has simple preparation method, low cost and wide market prospect.

Description

A kind of alginic acid gel drug film loaded with natamycin and its preparation method

Technical field:

The invention relates to the technical field of medical materials, and relates to a natamycin-loaded alginic acid gel film and a preparation method thereof. The alginic acid gel is used as a carrier and the natamycin is loaded into a therapeutic ophthalmic film.

technical background:

Fungal keratitis (FK) is a serious corneal disease. Common pathogenic fungi include Aspergillus and Fusarium. FK is one of the main causes of blindness in my country. The treatment effect of the disease is poor, and most of the patients are young adults working in the front line, which has a great impact on life and production.

As an avascular organ, the cornea has unique defense mechanisms and anatomical and physiological barriers. Most topical antifungal eye drops have poor corneal penetration, and most of them are washed out by tears, blinking and drainage through the nasolacrimal duct after dripping, with little local absorption, which makes drug treatment very difficult. When drug treatment alone is difficult to control, the combination of therapeutic keratoplasty and drug treatment is a common method for the treatment of fungal keratitis. Surgical treatment has the shortage of corneal donors, high cost, recurrence of fungal infection after surgery, corneal graft rejection, subsequent Issues such as glaucoma. Lead to the poor treatment effect of the disease and unsatisfactory recovery of vision. Therefore, the innovative treatment of fungal keratitis is the focus and difficulty that needs to be solved at present, and there is an urgent need for a drug delivery method that can maintain the effectiveness and continuity of drug concentration.

Natamycin is a tetraene antibiotic extracted from Streptomyces. Its mechanism of action is through the combination of drug molecules with the sterol part of the fungal cell membrane to form a polyene sterol complex, which changes the permeability of the cell membrane and causes the basic cell components in the fungal cell to flow out, resulting in the death of the fungus. Clinically used natamycin suspension can be used to treat fungal keratitis, but natamycin has a large particle size, poor solubility, and is affected by tear secretion, so it is difficult to achieve a sustained release effect. Researchers have been working on improving the administration effect of natamycin. For example, Chinese patent CN200810140337.7 discloses a cation-adhesive natamycin nano eye drop, including natamycin. agent and pH regulator, also including chitosan, Pluronic F-68 and phospholipids, wherein the mass percent concentration of phospholipids in eye drops is 0.01%-2%, the concentration of Pluronic F-68 in eye drops The mass percent concentration is 0.1%-20%, the mass percent concentration of chitosan in eye drops is 0.01-4%, the molecular weight of chitosan is 5000-300000, and the degree of deacetylation is >85%; Natamycin is usually suspended Suspension granules with a diameter of 10-1000nm are prepared from the preparation to increase the solubility of the drug, improve safety and effectiveness, and modify the natamycin drug particles of the common suspension with phospholipids and chitosan to make it With a positive charge, it helps to improve the corneal absorption of drugs, enhance drug efficacy and prolong drug action time, and chitosan has a bioadhesive effect, which enhances the corneal adhesion of drug particles; however, there are many types of raw materials and complicated preparation processes; China The patent application CN201911030779.0 discloses a natamycin polymer micelle eye drop, including a certain mass volume ratio of natamycin polymer micelle and phosphate buffer, wherein the natamycin polymer micelle The mass percentage of natamycin is 10-30%, and the balance is polyethylene glycol-polyglyceryl methacrylate block copolymer; the polyethylene glycol-polyglyceryl methacrylate block copolymer The copolymer is placed in ethanol to self-assemble cross-linked micelles, and the natamycin polymer micelle eye drops obtained after entrapping natamycin can prolong the release time of natamycin and reduce the number of medications, but its treatment The effect is not improved.

Sodium alginate is a by-product of extracting iodine and mannitol from brown algae kelp or sargassum, and its molecules are composed of β-D-mannuronic acid (β-D-mannuronic, M) and α-L-gulo Uronic acid (α-L-guluronic, G) is connected by (1→4) bonds, is a natural polysaccharide, and has the stability, solubility, viscosity and safety required for pharmaceutical excipients. Sodium alginate has been widely used in food industry and medicine. Therefore it is necessary to develop a drug carrier with alginic acid gel for the treatment of fungal keratitis containing natamycin drug film, reduce the eye irritation and discomfort of natamycin, delay drug release , enhanced the action time of natamycin. There is no report about the alginate gel film loaded with natamycin at present.

Invention content:

Aiming at the shortcomings of the prior art, the present invention provides a natamycin-loaded alginic acid gel drug film and a preparation method thereof, using alginic acid gel as a carrier, and natamycin-loaded therapeutic ophthalmic medicine membrane.

In order to achieve the above object, the present invention provides a natamycin-loaded alginic acid gel film, its composition and content are: every 1mL volume of the film contains 0.01-0.03g sodium alginate, 0.0025-0.0075g polyethylene oxide and 0.005-0.015g of natamycin.

The present invention also provides the preparation method of the alginic acid gel film loaded with natamycin, the specific preparation process is as follows:

(1) Dissolve 0.256-0.768g of sodium alginate powder and 0.065-0.195g of polyethylene oxide powder into 20mL and 5mL of deionized water respectively to ensure that sodium alginate and polyethylene oxide are not attached to the bottom and side walls of the beaker, and then The two solutions are mixed and stirred gently;

(2) Place the above solution on a magnetic stirrer, stir at room temperature for 3 to 5 hours to obtain a uniform gel, add natamycin to the uniform gel, and the mass volume ratio of natamycin to gel is 0.005 ～0.015g: 1mL, then stir for 30 minutes at 50°C in a dark environment until uniform without precipitation;

(3) Take 3-10 g of the hydrogel containing natamycin in step (2) and spread it on a petri dish with a diameter of 10 cm, and place it in the dark for defoaming;

(4) Prepare ethanol solution with absolute ethanol and deionized water, wherein the volume ratio of absolute ethanol to deionized water is 0-2:1-2, then add calcium chloride dihydrate, calcium chloride dihydrate and ethanol The mass volume ratio of the solution is 0.662g:9mL, and the ethanol-water crosslinking solution containing calcium ions is obtained, wherein the mass volume ratio concentration of calcium ions is 0.02g/mL, and is filtered once with a 0.22 μm filter membrane;

(5) Pour 9mL of ethanol-water cross-linking solution into a petri dish covered with natamycin-containing hydrogel, mix and cross-link the drug-loaded hydrogel and cross-linking solution evenly, let stand for 1 hour, and then use Rinse the drug-loaded gel membrane with ionized water for 3 times;

(6) Put the drug-loaded gel film in a drying oven at 50° C. and dry it in the dark, and then the natamycin-loaded alginate gel film will be obtained.

Compared with prior art, the beneficial effect of the present invention is:

1) Alginic acid gel is used as the drug carrier. The properties of alginic acid are similar to human extracellular matrix, and it has good tissue compatibility. It can be degraded into polysaccharides that are non-toxic and do not participate in metabolism, reducing the eye irritation of natamycin sex and discomfort.

2) Polyethylene oxide increases the viscosity of the drug-loaded gel, delays the release of the drug, enhances the flexibility and ductility of the drug-loaded film, reduces the brittleness of the drug-loaded film, and strengthens its plasticity, so that the drug film can be better attached to the cornea Ulcer surface.

3) Using the calcium ion cross-linking method to replace the sodium ions in sodium alginate, making the spatial structure of the material more cohesive, and making the drug-loaded gel more stable after forming a film, reducing the pore size inside the drug film, and delaying drug release. Enhanced duration of action of natamycin.

4) Ethanol has the ability to destroy the cohesive force and hydrogen bond, improve the wettability of sodium alginate gel, reduce the surface tension, and make it easier for cross-linked ions to enter the deep part of the gel. The volume ratio of ethanol and water affects the infiltration amount of calcium ions and their diffusion depth in alginate. The higher the ethanol content, the deeper the degree of cross-linking, the smaller the pores, and the slower the drug release.

In summary, the present invention uses alginic acid gel as a drug carrier, loads natamycin, cross-links with calcium ions and controls the cross-linking strength with the ratio of ethanol to water, and the prepared gel drug film can be better attached to corneal ulcers On the one hand, the drug has long action time, good effect, no irritation and discomfort, and high patient acceptance; it is a new type of ophthalmic film with simple preparation method, low cost and broad market prospect.

Description of drawings:

Figure 1 is a schematic diagram of the elastic modulus test results of the alginic acid gel film loaded with natamycin in Example 5 of the present invention.

Fig. 2 is a schematic diagram of the drug release curve of the alginic acid gel drug film loaded with natamycin according to Example 6 of the present invention.

Fig. 3 is a schematic diagram of the cytotoxicity test results of the alginic acid gel film loaded with natamycin in Example 7 of the present invention.

Fig. 4 is a schematic diagram of the experimental results of the antibacterial effect of the alginic acid gel film loaded with natamycin in Example 8 of the present invention.

Fig. 5 is a schematic diagram of the experimental results of the therapeutic effect of the alginic acid gel film loaded with natamycin in the animal model of fungal keratitis in mice according to Example 9 of the present invention.

Detailed ways:

The specific embodiment of the present invention is as follows:

Example 1:

This embodiment relates to the alginic acid gel film loaded with natamycin, and its composition and content are: every 1mL volume of the film contains 0.01-0.03g of sodium alginate, 0.0025-0.00755g of polyethylene oxide and 0.005 The natamycin of -0.015g; Concrete preparation process is as follows:

(1) Dissolve 0.256-0.768g of sodium alginate powder and 0.065-0.195g of polyethylene oxide powder into 20mL and 5mL of deionized water respectively to ensure that sodium alginate and polyethylene oxide are not attached to the bottom and side walls of the beaker, and then The two solutions are mixed and stirred gently;

(2) Place the above solution on a magnetic stirrer, stir at room temperature for 3 to 5 hours to obtain a uniform gel, add natamycin to the uniform gel, and the mass volume ratio of natamycin to gel is 0.005 ～0.015g: 1mL, then stir for 30 minutes at 50°C in a dark environment until uniform without precipitation;

(3) Take 3-10 g of the hydrogel containing natamycin in step (2) and spread it on a petri dish with a diameter of 10 cm, and place it in the dark for defoaming;

(4) Prepare ethanol solution with absolute ethanol and deionized water, wherein the volume ratio of absolute ethanol to deionized water is 0-2:1-2, then add calcium chloride dihydrate, calcium chloride dihydrate and ethanol The mass volume ratio of the solution is 0.662g:9mL, and the ethanol-water crosslinking solution containing calcium ions is obtained, wherein the mass volume ratio concentration of calcium ions is 0.02g/mL, and is filtered once with a 0.22 μm filter membrane;

(5) Pour 9mL of ethanol-water cross-linking solution into a petri dish covered with natamycin-containing hydrogel, mix and cross-link the drug-loaded hydrogel and cross-linking solution evenly, let stand for 1 hour, and then use Rinse the drug-loaded gel membrane with ionized water for 3 times;

(6) Put the drug-loaded gel film in a drying oven at 50° C. and dry it in the dark, and then the natamycin-loaded alginate gel film will be obtained.

Example 2:

This embodiment relates to the preparation process of the alginic acid gel film loaded with natamycin, and the specific steps are as follows:

(1) Dissolve 0.512g of sodium alginate powder and 0.127g of polyethylene oxide powder into 20mL and 5mL of deionized water respectively to ensure that sodium alginate and polyethylene oxide are not attached to the bottom and side walls of the beaker, and then mix the two solutions then stir gently;

(2) Place the above solution on a magnetic stirrer at room temperature and stir for 3 hours to obtain a uniform gel, add natamycin to the uniform gel, the mass volume ratio of natamycin to gel is 0.01g: 1mL , Stir for 30 minutes at 50°C in a dark environment until uniform and without precipitation;

(3) Spread 5 g of hydrogel containing natamycin on a petri dish with a diameter of 10 cm, and place it in the dark for defoaming;

(4) Mix 6 mL of absolute ethanol with 3 mL of deionized water to make an ethanol solution, then add 0.662 g of calcium chloride dihydrate to obtain an ethanol-water crosslinking solution containing calcium ions, wherein the mass-volume ratio of calcium ions is 0.02g/mL, and filter once with a 0.22μm filter membrane;

(5) Pour the obtained 9mL cross-linking solution into a petri dish covered with natamycin-containing hydrogel, mix and cross-link the drug-loaded hydrogel and cross-linking solution evenly, let it stand for 1 hour, and then use deionized Wash the drug-loaded gel film with water for 3 times;

(6) Put the drug-loaded gel in a drying oven at 50° C. and dry it in the dark, and the natamycin-loaded alginate gel drug film will be obtained.

Example 3:

This embodiment relates to the alginic acid gel film loaded with natamycin, and its specific preparation process is as follows:

(1) Dissolve 0.256g of sodium alginate powder and 0.065g of polyethylene oxide powder into 20mL and 5mL of deionized water respectively to ensure that sodium alginate and polyethylene oxide are not attached to the bottom and side walls of the beaker, and then mix the two solutions then stir gently;

(2) Place the above solution on a magnetic stirrer, stir at room temperature for 5 hours to obtain a uniform gel, add natamycin to the uniform gel, the mass volume ratio of natamycin to gel is 0.005g: 1mL, then stirred at 50°C for 30 minutes in a dark environment until uniform and without precipitation;

(3) Take 3 g of the hydrogel containing natamycin in step (2) and spread it on a petri dish with a diameter of 10 cm, and place it in the dark for defoaming;

(4) Get deionized water 9mL, then add calcium chloride dihydrate, the mass volume ratio of calcium chloride dihydrate and deionized water is 0.662g:9mL, obtain the cross-linking liquid containing calcium ion, wherein the mass of calcium ion The volume specific concentration is 0.02g/mL, and filtered once with a 0.22μm filter membrane;

(5) Pour 9mL of cross-linking solution into a petri dish covered with natamycin-containing hydrogel, mix and cross-link the drug-loaded hydrogel and cross-linking solution evenly, let it stand for 1 hour, and then rinse with deionized water Rinse the drug-loaded gel film 3 times;

(6) Put the drug-loaded gel film in a drying oven at 50° C. and dry it in the dark, and then the natamycin-loaded alginate gel film will be obtained.

Example 4:

This embodiment relates to the alginic acid gel film loaded with natamycin, and its specific preparation process is as follows:

(1) Dissolve 0.768g of sodium alginate powder and 0.195g of polyethylene oxide powder into 20mL and 5mL of deionized water respectively to ensure that sodium alginate and polyethylene oxide are not attached to the bottom and side walls of the beaker, and then mix the two solutions then stir gently;

(2) Place the above solution on a magnetic stirrer, stir at room temperature for 5 hours to obtain a uniform gel, add natamycin to the uniform gel, and the mass volume ratio of natamycin to gel is 0.015g: 1mL, then stirred at 50°C for 30 minutes in a dark environment until uniform and without precipitation;

(3) Take 8 g of the hydrogel containing natamycin in step (2) and spread it on a petri dish with a diameter of 10 cm, and place it in the dark for defoaming;

(4) Configure ethanol solution with absolute ethanol and deionized water, wherein the volume ratio of absolute ethanol to deionized water is 2:1, then add calcium chloride dihydrate, the mass volume of calcium chloride dihydrate and ethanol solution The ratio is 0.662g:9mL to obtain the ethanol-water cross-linked liquid containing calcium ions, wherein the mass volume ratio concentration of calcium ions is 0.02g/mL, and filter once with a 0.22 μm filter membrane;

(5) Pour 9mL of ethanol-water cross-linking solution into a petri dish covered with natamycin-containing hydrogel, mix and cross-link the drug-loaded hydrogel and cross-linking solution evenly, let stand for 1 hour, and then use Rinse the drug-loaded gel membrane with ionized water for 3 times;

(6) Put the drug-loaded gel film in a drying oven at 50° C. and dry it in the dark, and then the natamycin-loaded alginate gel film will be obtained.

Example 5:

This embodiment is the elastic modulus detection test of the alginic acid gel drug film loaded with natamycin prepared in Example 2, and the specific steps are as follows:

Use a tensile testing machine to test the mechanical strength of the drug film, stretch at a speed of 10mm/min, and test the elastic modulus of the stress-strain curve under the condition of 1% strain force, and the results are shown in Figure 1.

It can be seen from Figure 1 that the drug-loaded film was found to bear a maximum tensile force of 6.392N, a strength of 0.66MPa, an elastic modulus of 0.57MPa, and a ductility of 100% when measured at a speed of 10mm/min. The alginic acid gel film loaded with natamycin has good mechanical strength and ductility.

Embodiment 6:

This embodiment is an in vitro drug sustained-release experiment of the alginic acid gel film loaded with natamycin prepared in Example 2, and the specific steps are:

1. To establish the absorbance-concentration standard curve of natamycin, first configure natamycin standard solutions with different gradient concentrations, then measure the absorbance of each standard solution at λmax=304nm, and draw the absorbance-concentration of natamycin standard curve line;

2. Cut the alginate gel film loaded with natamycin and place it in PBS buffer solution, stir gently at 37°C in the dark, and take out 2ml samples at different time intervals (see Table 1). According to the standard curve, the released natamycin concentration was measured at λmax=304nm and the percentage of drug release was calculated. The results are shown in Table 1 and Figure 2.

It can be seen from Figure 2 that the entire drug release stage presents a 3-stage pattern, releasing 10.9% of the total drug amount in the first 3 hours; the drug release in the second stage is rapid, reaching 86.5% of the total drug load, of which Released 59.2% of the total drug load in the 9th hour; the drug release of the third stage was slow, and after the 12th hour, it was released to the maximum release of 95.4% from 86.5% of the total drug load (natamycin in the test film The concentration after the theoretical complete release is 50 μg/ml).

Table 1 The in vitro drug sustained release test results of the alginate gel film loaded with natamycin

Embodiment 7:

This example is the cytotoxicity experiment of the alginic acid gel drug film loaded with natamycin prepared in Example 2, specifically the CCK8 cell proliferation-toxicity detection, the steps are as follows:

Inoculate the suspension of immortalized human corneal epithelial cells in a 96-well plate, and place the culture plate in an incubator for pre-cultivation for 24 hours. The experiment was divided into three groups, namely the alginic acid gel film group without natamycin (2:1N-NATA), and the alginic acid gel film group with natamycin (2:1NATA) and the control group, the control group is the pure culture solution group; the gel film of the first two groups was cut to the same size and added to 150 μl of culture solution, and stood at 37 °C for 24 hours under dark conditions; then the 96-well Add 150 μl of culture solution containing gel film components to the plate culture solution, add equal volume of culture solution to the control group, 32 wells in each group; incubate the culture plate in the incubator for 24 hours, replace the culture solution and then add the same volume of culture solution to each well. Add 10 microliters of CCK8 solution; place the culture plate in an incubator and incubate for 4 hours; measure the absorbance at 450 nm with a microplate reader, and the results are shown in FIG. 3 .

It can be seen from Figure 3 that the culture medium loaded with natamycin-based alginate gel drug film components has no obvious toxic effect on human corneal epithelial cells.

The alginic acid gel drug film not loaded with natamycin involved in this example was prepared by the method in Example 2, except that no natamycin was added.

Embodiment 8:

This example is an in vitro bacteriostatic effect test of the alginic acid film loaded with natamycin prepared in Example 2.

Add the fungal spore concentration of 1×10 ⁶ CFU/ml spores to the Sabouraud medium, and the alginate gel film (SA-PEO-NATA) loaded with natamycin, seaweed without natamycin The acid gel drug film (SA-PEO), the drug-sensitive paper sheet containing the same amount of natamycin and the drug-sensitive paper sheet without natamycin were cut into the standard size of the inhibition zone test and placed in the culture medium. In , after 48 hours of incubation in the incubator, the size of the fungus-inhibiting area was recorded, and the results are shown in Figure 4.

The results showed that after culturing for 48 hours, the average diameter of the antibacterial zone of the drug-sensitive paper sheet containing the same amount of natamycin was 10 ± 0.4mm (white in Fig. The average diameter of the bacteriostatic zone was 12±0.3mm (white in Figure 4B), while no obvious bacteriostatic zone (black) was found in the two groups of drug films and paper sheets without natamycin after 48 hours of incubation.

The natamycin content in the drug-sensitive paper sheet containing the same amount of natamycin involved in this embodiment is the same as the natamycin content in the alginic acid gel drug film loaded with natamycin prepared in Example 2 same.

Embodiment 9:

This example is an application experiment of the alginic acid film loaded with natamycin prepared in Example 2 in the treatment of fungal keratitis in mice.

Take 30 C57BL/6 healthy female mice aged 8 weeks and divide them into five groups at random. Take the right eye as the experimental eye. After the model was successfully established, one group was the fungal infection group (AF), without any treatment; the other group was the alginic acid film group without natamycin (AF+SA-PEO), after the modeling A sodium alginate film without natamycin was implanted in the eyes, and the film was replaced every 24 hours; one group was the alginate gel film group loaded with natamycin (AF+SA-PEO-NATA ), the alginate gel film loaded with natamycin was implanted in the modeled eyes, and the film was replaced every 24 hours; one group was the natamycin treatment group (AF+Natamycin), given Natamycin was instilled 6 times a day. According to the O'Day standard for scoring the severity of keratitis, the scoring of corneal ulcers in mice was observed and recorded under the slit lamp. The experimental results are shown in Figure 5.

As can be seen from Figure 5: the corneas of the blank control group were transparent and had no lesions; after 3 days of model establishment, the turbid area of the corneas of the mice in the fungal infection group and the alginic acid film group not loaded with natamycin reached 70%-90%. %, the degree of turbidity is uneven, with irregular edema and part of the performance niche-like ulcer or ulcer area is larger; the corneal turbid area of mice in the natamycin treatment group is 50%, the degree of turbidity is alleviated, and there is irregular edema; The area of corneal turbidity of mice in the alginic acid gel film group of tamycin was 26%-40%, and the degree of turbidity was relatively mild, with irregular ulcers and mild corneal edema. The clinical score showed that the score of natamycin treatment group (AF+Natamycin) was lower than that of non-drug-loaded gel film group (AF+SA-PEO) and fungal infection group (AF), and higher than that of drug-loaded gel film group group (AF+SA-PEO-NATA). The scores of drug-loaded gel film group (AF+SA-PEO-NATA) were lower than those of non-drug-loaded gel film group (AF+SA-PEO) and natamycin-treated group (AF+Natamycin).

Example 10:

This example is an application case of alginic acid gel film loaded with natamycin in the treatment of patients with fungal keratitis.

A total of 40 patients with fungal keratitis treated in the Affiliated Hospital of Qingdao University from December 2017 to December 2020 were collected, including 20 males and 20 females, aged 21-75 years, with an average (48.95±5.93) years old. They were randomly divided into a drug film group and a control group, 20 cases in each group, including 11 males and 9 females in the drug film group, aged 22 to 73 years, with an average (47.96±5.25) years old; 9 males and 11 females in the control group. For example, the age ranged from 21 to 75 years old, with an average of (49.82±4.39) years old. There was no statistically significant difference in gender and age between the two groups.

The drug film group received natamycin-loaded alginate gel film treatment, once a day, and the control group received natamycin eye drops, 6 times a day, for 21 consecutive days.

According to the relevant standards, the clinical effect of the patient after treatment was evaluated. Cure: after treatment, the corneal infiltration and edema subsided, the conjunctival hyperemia disappeared, the fungal culture was negative, and the corneal ulcer healed. After the fluorescein sodium staining examination, it showed Negative; markedly effective: after treatment, the relevant examination results of the patient were negative, and all indicators were significantly improved, with only conjunctival hyperemia; effective: after treatment, the relevant examination results of the patient were negative, and only 1-2 Items were improved; Ineffective: All indicators did not change before and after treatment, or there was an aggravation. Total effective rate of treatment=(cure+marked effect+effective)/total number of cases×100%. The symptoms of the patients were evaluated according to the relevant standards, and the maximum score for each symptom was 5 points, and the higher the score, the more severe the symptom.

After statistics, after 20 patients in the drug film group received treatment, 19 patients had significant therapeutic effects, including 4 cases cured, 13 cases markedly effective, 2 cases effective, and 1 case ineffective, and the treatment effective rate was 95.0% (19/20); After 20 patients in the control group received treatment, 13 patients had significant therapeutic effects, including 2 cases cured, 10 cases markedly effective, 2 cases effective, and 6 cases ineffective. The effective rate of treatment was 65.0% (13/20). The effect parameters of the drug film group before and after treatment are shown in Table 2.

Table 2 Comparison of effect parameters before and after treatment of the drug film group

Clinical case application

Case 1: Wang ××, female, 37 years old, was scratched by a tree branch half a month ago in her right eye, she had red eyes, tearing, and foreign body sensation, and was not treated. During this period, she rubbed her eyes many times, accompanied by aggravated blurred vision. , Diagnosed as "keratitis (right)", given Colabito eye drops treatment, and then went to our hospital, ophthalmological examination: right eye vision 0.3, intraocular pressure 20mmHg, conjunctival mixed hyperemia, cornea near the limbus at 5 o'clock See 2mm×3mm ulcer, toothpaste-like, deep into the deep stroma, pseudopodia and satellite lesions around, surrounding corneal edema, aqueous humor cells (+). Diagnosed as "fungal keratitis (right)", the right eye was treated with corneal debridement combined with natamycin eye drops q2h. The patient reported poor compliance with the application of eye drops, obvious eye discomfort after instillation, and poor therapeutic effect, so he switched to alginic acid film containing natamycin once a day for treatment. After 7 days, the ophthalmic examination showed that the visual acuity of the right eye was 0.5, The intraocular pressure was 18mmHg, the area of corneal opacity was reduced, and the depth of ulcer became shallower.

Case 2: Liu ×, male, 27 years old, his left eye was scratched by a "bamboo stick at a barbecue stand" due to "drunk" arguing with others a week ago, and he immediately developed eye pain, photophobia, and tears in his left eye, and no special treatment was given. One week later, he still had red eyes, sore eyes, and decreased vision. He came to our hospital for treatment. Ophthalmological examination: left eye visual acuity 0.1, intraocular pressure 15mmHg, irregular ulcer of 4mm×4mm in the center of the cornea, peripheral edema, irregular border, and visible satellite lesions . Aqueous humor cells (+), round pupil, good response to light, transparent lens. Diagnosed as "fungal keratitis (left)", corneal debridement combined with natamycin-loaded sodium alginate drape was administered once daily. Four days later, the visual acuity of the left eye was 0.3, the intraocular pressure was 12mmHg, the cornea was slightly edema, the opacity area was lighter than before, and the ulcer became shallower.

Claims (1)

1. A preparation method of natamycin-loaded alginic acid gel medicine membrane is characterized by comprising the following specific preparation processes:

(1) Respectively dissolving 0.512g of sodium alginate powder and 0.127g of polyoxyethylene powder into 20mL of deionized water and 5mL of deionized water to ensure that the sodium alginate and the polyoxyethylene are not attached to the bottom and the side wall of the beaker, mixing the two solutions, and then slightly stirring the two solutions;

(2) And (2) placing the solution in a magnetic stirrer, stirring for 3 hours at room temperature to obtain uniform gel, and adding natamycin into the uniform gel, wherein the mass volume ratio of the natamycin to the gel is 0.01g:1mL, stirring for 30 minutes in a dark environment at 50 ℃ until the mixture is uniform and no precipitate exists;

(3) Spreading 5g of natamycin-containing hydrogel in a culture dish with the diameter of 10cm, and standing in a dark place for defoaming;

(4) Mixing 6mL of absolute ethyl alcohol with 3mL of deionized water to prepare an ethanol solution, then adding 0.662g of calcium chloride dihydrate to obtain an ethanol-water cross-linking solution containing calcium ions, wherein the mass-volume concentration of the calcium ions is 0.02g/mL, and filtering for 1 time by using a 0.22-micron filtration membrane;

(5) Pouring the obtained 9mL of crosslinking solution into a culture dish paved with natamycin-containing hydrogel, uniformly mixing and crosslinking the drug-loaded hydrogel and the crosslinking solution, standing for 1 hour, and then washing the drug-loaded gel membrane for 3 times by deionized water;

(6) Placing the drug-loaded gel in a drying box, drying at 50 ℃ in a dark condition to obtain the natamycin-loaded alginic acid gel drug membrane;

the mechanical strength of the medicine film is tested by using a tensile testing machine, the medicine film is stretched at the speed of 10mm/min, the elastic modulus is tested by using a stress-strain curve under the condition of 1% strain force, and the result shows that the maximum tensile force borne by the medicine-loaded film is 6.392N, the strength is 0.66MPa, the elastic modulus is 0.57MPa and the ductility reaches 100% in the measurement under the speed condition of 10mm/min, and the natamycin-loaded alginic acid gel medicine film has better mechanical strength and ductility; the method is used for carrying out in-vitro drug sustained release experiments on the natamycin-loaded alginic acid gel drug membrane, and comprises the following specific steps:

(1) Establishing an absorbance-concentration standard curve of natamycin, firstly preparing natamycin standard solutions with different gradient concentrations, then respectively measuring the absorbance of each standard solution at the lambda max =304nm, and drawing the absorbance-concentration standard curve of natamycin;

(2) The alginate gel drug film loaded with natamycin is cut and placed in PBS buffer solution, slightly stirred at 37 ℃ in the dark, 2ml samples are respectively taken out at different time intervals, the concentration of released natamycin is measured at the position of lambada max =304nm according to a standard curve and the percentage of released drug is calculated, and the results are as follows: when the time interval is 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20 and 24h, the concentration of the released medicament is 0.204, 1.236, 2.49, 3.636, 4.767, 5.487, 6.116, 7.078, 9.35, 14.917, 20.134, 23.58, 29.634, 34.553, 40.086, 43.273, 45.707, 47.697 and 47.721 mug/mL respectively, and the percentage of the released medicament is 0.408, 2.472, 4.98, 7.272, 9.534, 10.974, 12.232, 14.156, 18.7, 29.268, 40.268, 47.16, 59.268, 69.106, 80.172, 86.546, 91.442, 91.95, 95.95 and 95 percent respectively;

the whole drug release stage of the drug takes on a 3-stage mode, and 10.9 percent of the total drug is released in the first 3 hours; the drug release in the second stage is rapid, the total drug loading is 86.5 percent, wherein the total drug loading is released at the 9 th hour by 59.2 percent; the drug release in the third stage is slow, after 12 hours, the drug is released from 86.5% of the total drug-loading amount to 95.4% of the maximum release amount, and the concentration of the natamycin in the test membrane after the natamycin is completely released theoretically is 50 mug/mL; the obtained natamycin-loaded alginic acid gel medicinal film is used for preparing the medicine for treating the fungal keratitis.

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