CN112891326B

CN112891326B - Natamycin-loaded alginic acid gel medicine film and preparation method thereof

Info

Publication number: CN112891326B
Application number: CN202110386922.0A
Authority: CN
Inventors: 李翠; 赵桂秋; 李道浩; 彭旭东; 尹晓妮; 贾文妍; 张晓萍; 张乐园
Original assignee: Affiliated Hospital of University of Qingdao
Current assignee: Affiliated Hospital of University of Qingdao
Priority date: 2021-04-12
Filing date: 2021-04-12
Publication date: 2023-04-14
Anticipated expiration: 2041-04-12
Also published as: CN112891326A

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

Natamycin-loaded alginic acid gel medicine film and preparation method thereof

The technical field is as follows:

the invention relates to the technical field of medical materials, and relates to a natamycin-loaded alginic acid gel medicine film and a preparation method thereof.

Technical background:

fungal Keratitis (FK) is a serious corneal disease, common pathogenic fungi include aspergillus and fusarium, and FK is one of the main causes of blindness in our country. The disease has poor treatment effect, and most of patients are young and strong in first-line work, which causes great influence on life and production.

The cornea acts as an avascular organ with a unique defense mechanism and anatomical physiological barrier. Most of the local antifungal eye drops have poor corneal permeability, most of the eye drops are flushed by tears, transient eyes and nasolacrimal ducts after the eye drops are used, and the local absorption is low, so that the drug treatment is very difficult. When the simple drug therapy is difficult to control, the combination of the therapeutic corneal transplantation and the drug therapy is a common method for treating the fungal keratitis, and the surgical therapy has the problems of lack of corneal donors, high cost, relapse of postoperative fungal infection, corneal graft rejection, secondary glaucoma and the like. Resulting in poor therapeutic effect and unsatisfactory recovery of vision. Therefore, innovative treatment of fungal keratitis is a key and difficult point to be solved at present, and a drug delivery method capable of maintaining the effectiveness and continuity of drug concentration is urgently needed.

Natamycin is a tetraene antibiotic extracted from streptomyces. The action mechanism is that the medicine molecule is combined with sterol part of fungus cell membrane to form polyene sterol compound, so as to change permeability of cell membrane and make the basic cell component in fungus cell flow out to kill fungus. The natamycin suspension used clinically can be used for treating fungal keratitis, but natamycin has larger particle size and poorer solubility, and is influenced by lacrimal secretion, so that the effect of sustained release is difficult to achieve. Researchers are always dedicated to improving the administration effect of natamycin, for example, a cationic adhesion type natamycin nano eye drop disclosed in chinese patent CN200810140337.7 comprises natamycin, a proper amount of osmotic pressure regulator and pH regulator, and further comprises chitosan, pluronic F-68 and phospholipid, wherein the mass percent concentration of the phospholipid in the eye drop is 0.01% -2%, the mass percent concentration of the pluronic F-68 in the eye drop is 0.1% -20%, the mass percent concentration of the chitosan in the eye drop is 0.01% -4%, the molecular weight of the chitosan is 5000-300000, and the degree of deacetylation is >85%; the common natamycin suspension is prepared into suspension particles with the diameter of 10-1000nm, the solubility of the medicine is increased, the safety and the effectiveness are improved, the natamycin medicine particles of the common suspension are modified by phospholipid and chitosan, so that the natamycin medicine particles are positively charged, the corneal absorption of the medicine is improved, the medicine effect is enhanced, the medicine action time is prolonged, the chitosan has a biological adhesion effect, and the corneal adhesion of the medicine particles is enhanced; but the raw materials are various, and the preparation process is complex; chinese application patent CN201911030779.0 discloses natamycin polymer micelle eye drops, which comprise natamycin polymer micelles and phosphate buffer solution with a certain mass-volume ratio, wherein the natamycin in the natamycin polymer micelles accounts for 10-30% by mass, and the balance is polyethylene glycol-poly (glyceryl methacrylate) block copolymer; the natamycin polymer micelle eye drops obtained by putting the polyethylene glycol-poly (glyceryl methacrylate) segmented copolymer into ethanol to self-assemble cross-linked micelles and entrapping natamycin can prolong the release time of natamycin and reduce the times of medication, but the treatment effect is not improved.

Sodium alginate is a byproduct after iodine and mannitol are extracted from brown algae such as kelp or gulfweed, the molecule of the sodium alginate is formed by connecting beta-D-mannuronic acid (beta-D-mannuronic acid, M) and alpha-L-guluronic acid (alpha-L-guluronic acid, G) according to a (1 → 4) bond, and the sodium alginate is a natural polysaccharide and has stability, solubility, viscosity and safety required by pharmaceutical preparation auxiliary materials. Sodium alginate has been widely used in the food industry and in the pharmaceutical field. Therefore, it is necessary to develop a natamycin-carrying drug film for treating fungal keratitis by taking alginic acid gel as a drug carrier, which reduces the eye irritation and discomfort of natamycin, delays the drug release and prolongs the action time of natamycin. No report on the natamycin-loaded alginic acid gel drug membrane exists at present.

The invention content is as follows:

aiming at the defects in the prior art, the invention provides a natamycin-loaded alginic acid gel medicine film and a preparation method thereof.

In order to achieve the purpose, the invention provides an alginic acid gel drug film loaded with natamycin, which comprises the following components by weight: each 1mL volume of the drug film contains 0.01-0.03g of sodium alginate, 0.0025-0.0075g of polyoxyethylene and 0.005-0.015g of natamycin.

The invention also provides a preparation method of the natamycin-loaded alginic acid gel medicinal film, which comprises the following specific preparation processes:

(1) Respectively dissolving 0.256-0.768g of sodium alginate powder and 0.065-0.195g 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 a beaker, mixing the two solutions, and then gently stirring;

(2) Placing the solution in a magnetic stirrer, stirring for 3-5 hours at room temperature to obtain uniform gel, adding natamycin into the uniform gel, wherein the mass-volume ratio of the natamycin to the gel is 0.005-0.015 g:1mL, and then stirring for 30 minutes in a dark environment at 50 ℃ until the mixture is uniform and does not precipitate;

(3) Spreading 3-10 g of the natamycin-containing hydrogel obtained in the step (2) in a culture dish with the diameter of 10cm, and standing and defoaming in a dark place;

(4) Preparing an ethanol solution by using absolute ethyl alcohol and deionized water, wherein the volume ratio of the absolute ethyl alcohol to the deionized water is 0-2 and is 1-2, then adding calcium chloride dihydrate, and the mass volume ratio of the calcium chloride dihydrate to the ethanol solution is 0.662g/9mL to obtain an ethanol-water crosslinking solution containing calcium ions, wherein the mass-volume ratio concentration of the calcium ions is 0.02g/mL, and filtering for 1 time by using a 0.22 mu m filtering membrane;

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

(6) And (3) placing the drug-loaded gel membrane in a drying box, and drying the drug-loaded gel membrane in a dark condition at 50 ℃ to obtain the natamycin-loaded alginic acid gel membrane.

Compared with the prior art, the invention has the beneficial effects that:

1) The alginic acid gel is used as a drug carrier, has the property similar to human extracellular matrix, has good histocompatibility, can be degraded into non-toxic polysaccharide which does not participate in metabolism, and reduces the eye irritation and discomfort of natamycin.

2) The polyethylene oxide increases the viscosity of the drug-loaded gel, delays the release of the drug, enhances the flexibility and the ductility of the drug-loaded membrane, reduces the brittleness of the drug-loaded membrane, strengthens the plasticity of the drug-loaded membrane, and enables the drug-loaded membrane to be better attached to the corneal ulcer surface.

3) The calcium ion crosslinking method is used for replacing sodium ions in sodium alginate, so that the space structure of the material is more cohesive, the drug-loaded gel is more stable after film formation, the size of pores in the drug film is reduced, the drug release is delayed, and the action time of natamycin is prolonged.

4) The ethanol has the capability of destroying the adhesive force and the hydrogen bond, improves the wettability of the sodium alginate gel, reduces the surface tension and enables cross-linking ions to enter the deep part of the gel more easily. The volume ratio of ethanol and water influences the infiltration amount of calcium ions and the diffusion depth of the calcium ions in alginate, and the higher the content of ethanol is, the deeper the crosslinking degree is, the smaller the pores are, and the slower the drug release is.

In conclusion, the invention takes the alginic acid gel as a medicine carrier, loads natamycin, uses calcium ions for crosslinking, and uses the proportion of ethanol and water for regulating and controlling the crosslinking strength, and the prepared gel medicine film can be better attached to the corneal ulcer surface, and has the advantages of long medicine 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 of the drawings:

FIG. 1 is a schematic diagram showing the results of the elastic modulus test experiment of the natamycin loaded alginic acid gel film of example 5 in accordance with the present invention.

FIG. 2 is a schematic diagram showing the drug release profile of the natamycin loaded alginic acid gel membrane of example 6 according to the present invention.

FIG. 3 is a schematic diagram showing the results of the cytotoxicity test of the natamycin-loaded alginate gel membrane of example 7 according to the present invention.

FIG. 4 is a schematic diagram of the experimental results of the bacteriostatic effect of the alginic acid gel membrane loaded with natamycin in example 8.

FIG. 5 is a schematic diagram showing the experimental results of the therapeutic effect of the alginic acid gel film loaded with natamycin of example 9 in the animal model of fungal keratitis in mice.

The specific implementation mode is as follows:

the specific embodiment of the invention is as follows:

example 1:

the embodiment relates to a natamycin-loaded alginic acid gel drug film, which comprises the following components in percentage by weight: each 1mL of the medicinal membrane contains 0.01-0.03g of sodium alginate, 0.0025-0.00755g of polyoxyethylene and 0.005-0.015g of natamycin; the specific preparation process is as follows:

(1) Respectively dissolving 0.256-0.768g of sodium alginate powder and 0.065-0.195g 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 a beaker, mixing the two solutions, and then stirring the two solutions lightly;

(3) Spreading 3-10 g of the natamycin-containing hydrogel obtained in the step (2) in a culture dish with the diameter of 10cm, and standing in a dark place for defoaming;

(4) Preparing an ethanol solution by using absolute ethyl alcohol and deionized water, wherein the volume ratio of the absolute ethyl alcohol to the deionized water is (0-2) - (1-2), then adding calcium chloride dihydrate, and the mass volume ratio of the calcium chloride dihydrate to the ethanol solution is (0.662g) 9mL to obtain an ethanol water cross-linked solution containing calcium ions, wherein the mass volume ratio concentration of the calcium ions is 0.02g/mL, and filtering for 1 time by using a 0.22 mu m filtering membrane;

(6) And (3) placing the drug-loaded gel film in a drying box, drying at 50 ℃ in a dark condition to obtain the natamycin-loaded alginic acid gel drug film.

Example 2:

the embodiment relates to a preparation process of an alginic acid gel drug membrane loaded with natamycin, which comprises the following specific steps:

(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 and defoaming in a dark place;

(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) And (3) placing the drug-loaded gel in a drying box, and drying the drug-loaded gel in a dark condition at 50 ℃ to obtain the natamycin-loaded alginic acid gel drug membrane.

Example 3:

the embodiment relates to an alginic acid gel drug membrane loaded with natamycin, which comprises the following specific preparation processes:

(1) Respectively dissolving 0.256g of sodium alginate powder and 0.065g 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 mixed solution;

(2) And (2) placing the solution in a magnetic stirrer, stirring for 5 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 0.005g:1mL, and then stirring for 30 minutes in a dark environment at 50 ℃ until the mixture is uniform and does not precipitate;

(3) Spreading 3g of the natamycin-containing hydrogel obtained in the step (2) in a culture dish with the diameter of 10cm, and standing and defoaming in a dark place;

(4) Taking 9mL of deionized water, then adding 9mL of calcium chloride dihydrate, wherein the mass-volume ratio of the calcium chloride dihydrate to the deionized water is 0.662g/mL, so as to obtain a cross-linking solution containing calcium ions, wherein the mass-volume ratio concentration of the calcium ions is 0.02g/mL, and filtering for 1 time by using a 0.22 mu m filtering membrane;

(5) Pouring 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 with deionized water for 3 times;

Example 4:

(1) Respectively dissolving 0.768g of sodium alginate powder and 0.195g 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 mixed solution;

(2) Placing the solution in a magnetic stirrer, stirring for 5 hours at room temperature to obtain uniform gel, adding natamycin into the uniform gel, wherein the mass volume ratio of the natamycin to the gel is 0.015g:1mL, and then stirring for 30 minutes in a dark environment at 50 ℃ until the mixture is uniform and does not precipitate;

(3) Spreading 8g of the natamycin-containing hydrogel obtained in the step (2) in a culture dish with the diameter of 10cm, and standing and defoaming in a dark place;

(4) Preparing an ethanol solution by using absolute ethyl alcohol and deionized water, wherein the volume ratio of the absolute ethyl alcohol to the deionized water is 2:1, then adding calcium chloride dihydrate, wherein the mass-to-volume ratio of the calcium chloride dihydrate to the ethanol solution is 0.662g/mL, so as to obtain ethanol-water crosslinking solution containing calcium ions, wherein the mass-to-volume ratio concentration of the calcium ions is 0.02g/mL, and filtering for 1 time by using a 0.22 mu m filtering membrane;

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

Example 5:

this example is an elastic modulus test of the natamycin loaded alginate gel membrane prepared in example 2, and the specific steps are as follows:

the mechanical strength of the film was measured using a tensile tester, and the elastic modulus was measured using a stress-strain curve under a condition of 10mm/min tensile force and 1% strain force, and the results are shown in FIG. 1.

As can be seen from FIG. 1, the maximum tensile force of the loaded film is 6.392N, the strength is 0.66MPa, the elastic modulus is 0.57MPa, and the ductility reaches 100% when the loaded film is measured under the speed condition of 10mm/min, which shows that the alginic acid gel loaded with natamycin of the invention has better mechanical strength and ductility.

Example 6:

this example is an in vitro drug release experiment of the natamycin loaded alginate gel membrane prepared in example 2, and the specific steps are as follows:

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 natamycin loaded alginate gel membranes were cut out and placed in PBS buffer, gently stirred at 37 ℃ in the dark, 2ml samples were taken at different time intervals (see table 1), the released natamycin concentration was measured at λ max =304nm and the percentage of drug released was calculated according to the standard curve, the results are shown in table 1 and fig. 2.

As can be seen from fig. 2, the entire drug release period is shown in 3 steps, releasing 10.9% of the total drug amount 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 during the third phase was slow, from 86.5% of the total drug loading to a maximum release of 95.4% after 12 hours (concentration of 50 μ g/ml after theoretical total release of natamycin in the test membrane).

TABLE 1 slow release test result of natamycin loaded alginic acid gel membrane

Example 7:

this example is a cytotoxicity experiment of the natamycin loaded alginate gel membrane prepared in example 2, specifically a CCK8 cell proliferation-toxicity assay, and includes the following steps:

immortalized human corneal epithelial cell suspensions were inoculated into 96-well plates, and the plates were pre-cultured in an incubator for 24 hours. The experiment is divided into three groups, namely a alginic acid gel drug membrane group (2; cutting the gel membranes of the first two groups to the same size, adding into 150 μ l culture solution, and standing at 37 deg.C in dark for 24 hr; then adding 150 mul of two groups of culture solution containing gel drug membrane components into the 96-well plate culture solution, adding equal volume of culture solution into a control group, and adding 32 wells in each group; incubating the culture plate in an incubator for 24 hours, replacing culture solution, and adding 10 microliters of CCK8 solution into each hole; placing the culture plate in an incubator for incubation for 4 hours; the absorbance at 450nm was measured by a microplate reader, and the results are shown in FIG. 3.

As can be seen from FIG. 3, the medium of the natamycin loaded alginate gel membrane component has no obvious toxic effect on human corneal epithelial cells.

The alginic acid gel film without natamycin loading of this example was prepared by the method of example 2, except that natamycin was not added.

Example 8:

this example is an experiment on the in vitro bacteriostatic effect of the natamycin loaded alginate film prepared in example 2.

Adding fungal spore with concentration of 1 × 10 into Sabouraud's medium ⁶ CFU/ml spore, cutting the natamycin-loaded alginic acid gel drug film (SA-PEO-NATA), natamycin-free alginic acid gel drug film (SA-PEO), natamycin-containing drug sensitive paper sheet and natamycin-free drug sensitive paper sheet into standard size of bacteriostatic ring experiment, placing in culture medium, culturing in incubator for 48 hours, recording the size of fungus-inhibiting region, and the result is shown in FIG. 4.

The results showed that after 48 hours of incubation, the mean diameter of the zone of inhibition of the drug-sensitive paper containing the same amount of natamycin was 10. + -. 0.4mm (FIG. 4A white), the mean diameter of the zone of inhibition of the alginate gel membrane loaded with natamycin was 12. + -. 0.3mm (FIG. 4B white), while no significant zone of inhibition (black) was seen after 48 hours of incubation for both the groups of drug membrane and paper without natamycin.

The natamycin content in the drug sensitive paper containing the same amount of natamycin in this example was the same as that in the natamycin loaded alginic acid gel drug film prepared in example 2.

Example 9:

this example is an application experiment of the natamycin loaded alginate film prepared in example 2 in the treatment of fungal keratitis in mice.

30 healthy C57BL/6 female mice of 8 weeks old are taken and randomly divided into five groups, one group is a blank control group (Normal) without any treatment, and the other four groups are used for establishing mouse aspergillus fumigatus keratitis animal models, and the right eye is used as an experimental eye. After successful model building, one of the groups was fungal infection (AF) and no treatment was given; one group is an alginic acid medicine membrane group (AF + SA-PEO) without natamycin, the alginic acid medicine membrane without natamycin is implanted into the eyes after modeling, and the medicine membrane is replaced for 1 time every 24 hours; one group is a alginic acid gel drug film group (AF + SA-PEO-NATA) loaded with natamycin, alginic acid gel drug film loaded with natamycin is implanted into the modeled eyes, and the drug film is replaced for 1 time every 24 hours; one group was a Natamycin treatment group (AF + Natamycin) to which Natamycin was administered in eye drops 6 times per day. According to the O' Day standard, the keratitis severity scoring standard is carried out, the scoring condition of the corneal ulcer of the mouse is observed and recorded under a slit lamp, and the experimental result is shown in figure 5.

As can be seen from fig. 5: the cornea of the blank control group is transparent and has no pathological changes; after the model is established for 3 days, the turbid areas of the corneas of the mouse with the fungus infection group and the alginic acid membrane group without natamycin reach 70-90 percent, the turbid degrees are not uniform, irregular edema exists, and a part of the cornea shows niche-shaped ulcer or the ulcer area is larger; the corneal opacity of mice in the natamycin treatment group was 50%, the opacity was reduced, and irregular edema was observed; the mouse cornea turbid area of the alginic acid gel drug film group loaded with the natamycin is 26-40 percent, the turbid degree is light, and irregular ulcer and mild corneal edema exist. Clinical scores showed that the Natamycin treatment group (AF + Natamycin) scored lower than the non-loaded gel membrane group (AF + SA-PEO) and the fungal infection group (AF), and higher than the loaded gel membrane group (AF + SA-PEO-NATA). The drug-loaded gel membrane group (AF + SA-PEO-NATA) was scored lower than the non-drug-loaded gel membrane group (AF + SA-PEO) and the Natamycin treatment group (AF + Natamycin).

Example 10:

this example is an application of natamycin loaded alginate gel film in treating patients with fungal keratitis.

40 patients with fungal keratitis treated at the affiliated hospital of Qingdao university from 12 months 2017 to 12 months 2020 were collected, wherein 20 of the patients were 20 of the men and 20 of the women, and the ages of the patients were 21 to 75 years, and the average (48.95 +/-5.93) of the ages. Randomly dividing into a medicine film group and a control group, wherein each group comprises 20 cases, wherein the medicine film group comprises 11 cases of men and 9 cases of women, the age is 22-73 years, and the average (47.96 +/-5.25) years is; 9 men and 11 women in the control group had ages of 21-75 years, with the average (49.82 + -4.39) years. The difference between gender and age was not statistically significant.

The drug film group is treated by alginic acid gel drug film loaded with natamycin for 1 time/day, and the control group is treated by natamycin eye drops for 6 times/day for 21 days.

And (3) evaluating the clinical effect of the patient after treatment according to relevant standards, and curing: after treatment, the corneal infiltration and edema of a patient are resolved, the conjunctival congestion condition disappears, the fungus culture is negative, the corneal ulcer is healed, and the cornea ulcer is negative after the examination of fluorescein sodium staining; the effect is shown: after treatment, the relevant examination results of the patients are negative, all indexes are obviously improved, and only the conjunctival congestion condition exists; the method has the following advantages: after treatment, the relevant examination results of the patient are negative, and only 1-2 indexes are improved; and (4) invalidation: before and after treatment, all indexes are unchanged or have aggravation. Total effective rate of treatment = (cure + significant effect + effective)/total number of cases x 100%. The symptoms of the patients are evaluated according to relevant standards, each symptom has a score of 5 at most, and the higher the score is, the more serious the symptom is.

After statistics, after 20 patients in the medicinal membrane group receive treatment, 19 patients have obvious treatment effect, wherein 4 patients are cured, 13 patients have obvious effect, 2 patients have effect, 1 patient has no effect, and the treatment effective rate is 95.0% (19/20); after 20 patients in the control group receive treatment, 13 patients have remarkable treatment effect, wherein 2 patients are cured, 10 patients have remarkable effect, 2 patients have effective effect, 6 patients have ineffective effect, and the treatment effective rate is 65.0% (13/20). The parameters of the effect of the membrane group before and after treatment are shown in table 2.

TABLE 2 comparison of Pre-and post-treatment Effect parameters of the Membrane groups

Clinical case application

Case 1: the eye drops are used for treating the eye drops, namely, the female is aged 37 years old, the right eye is scratched by branches before half a month, red eye, lacrimation and foreign body sensation appear on the eyes, the eyes are kneaded for a plurality of times during the period without treatment, the blurring and the aggravation of visual objects are accompanied, the eye drops are diagnosed in local hospitals and are diagnosed as 'keratitis (right)', the clonbitol eye drops are used for treating, and then the eye drops are diagnosed in our hospital for ophthalmic examination: right eye vision 0.3, intraocular pressure 20mmHg, conjunctival congestion, corneal ulcer of 2mm × 3mm near the limbus at 5 o' clock, toothpaste-like, deep basal layer, pseudopodia and satellite focus around, corneal edema around, and aqueous humor cells (+). The diagnosis is "fungal keratitis (right)", and corneal debridement and natamycin eyedrops are given to treat the right eye for q2 h. The patient can use the alginic acid film with natamycin for 1 treatment every day, and the ophthalmological examination is carried out after 7 days: the vision of right eye is 0.5, the intraocular pressure is 18mmHg, the turbid area of cornea is reduced, and the depth of ulcer is shallow.

Case 2: liu x, male, age 27, the left eye is scratched by a barbeque stall bamboo stick before 1 week because of 'drunk' and the people struggle for, the eye pain, photophobia and lacrimation appear immediately, the eye pain still appears after 1 week without special treatment, the vision is degraded, the doctor visits in our hospital, the eye examination: the left eye vision is 0.1, the intraocular pressure is 15mmHg, irregular ulcer of 4mm multiplied by 4mm is seen in the center of the cornea, the periphery is edematous, the boundary is irregular, and a satellite focus can be seen. Aqueous humor cells (+), round pupil, good response to light, and transparent lens. The diagnosis was "fungal keratitis (left)", and corneal debridement was given once a day in combination with a natamycin-loaded sodium alginate film. The visual acuity of the left eye is 0.3, the intraocular pressure is 12mmHg, the cornea is slightly edematous, the turbid area is reduced compared with the former area, and the ulcer is shallow after the 4-day double diagnosis.

Claims

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

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

(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.