CN117899000A - Drug-loaded composite gel lacrimal duct plug for dry eye treatment and preparation method thereof - Google Patents

Abstract

The invention discloses a drug-loaded composite gel lacrimal duct plug for dry eye treatment and a preparation method thereof. The drug-loaded composite gel lacrimal plug mainly comprises an anti-inflammatory drug and a hydrogel material. The anti-inflammatory drug can be glucocorticoid drugs, immunosuppressant drugs and the like for dry eye treatment, and specifically can be dexamethasone, cyclosporins, tacrolimus and the like. The hydrogel material may be selected from at least one of the following: chitosan, PEG hydrogels, silMA, gelMA (methacryloylated gelatin), and the like. The drug-loaded composite gel provided by the invention has good swelling performance, biocompatibility and degradability, and can prolong the release time of the anti-inflammatory drug while slowing down tear loss, thereby realizing the dual treatment effects of increasing the tear volume of the ocular surface and resisting inflammation.

Description

Drug-loaded composite gel lacrimal duct plug for dry eye treatment and preparation method thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a drug-loaded composite gel lacrimal duct plug for treating xerophthalmia and a preparation method thereof.

Background

Dry eye, a multifactorial chronic ocular surface disease, is a tear film instability or ocular surface microenvironment imbalance caused by abnormalities in tear quality, volume and dynamics, which may be accompanied by ocular surface inflammatory response, tissue damage and neurological abnormalities, resulting in various ocular discomfort symptoms and/or visual dysfunction [ chinese conference of asia dry eye association, the institute of the national institutes of medicine and health, the institute of the specialized committee of the eye surface and tear chemistry, and the department of the ophthalmologist of chinese physician association, the group of the eye surface and dry eye chemistry, chinese dry eye expert consensus: definition and classification journal of chinese ophthalmology, 2020.56 (6): 418-422 ]. With the development of society and the progress of scientific technology in recent years, the burden of life pressure of people and the appearance of more and more electronic products, the prevalence of dry eye is obviously increased. Analysis of published dry eye prevalence data reported in 2017 DEWS II indicated that DED (Dry Eye Disease) had a prevalence ranging from 5-50% and that signs were more prevalence than symptoms and more variability and increased linearly with age 【Craig,J.P.,K.K.Nichols,E.K.Akpek,B.Caffery,H.S.Dua,C.K.Joo,Z.Liu,J.D.Nelson,J.J.Nichols,K.Tsubota and F.Stapleton(2017)."TFOS DEWS II Definition and Classification Report."Ocul Surf 15(3):276-283】.

The clinic treatment means for xerophthalmia mainly comprise prescription drugs, lacrimal passage embolism and operation treatment. Dry eye is largely due to insufficient tear fluid, and thus artificial tears and the like are often used clinically to increase the tear fluid volume of the eye; dry stress causes ocular surface damage and induces congenital and adaptive immune responses, which in turn cause ocular surface damage and inflammation to form a long-term malignant cycle; for moderately severe dry eye patients, discomfort symptoms such as pain cannot be relieved only by supplementing artificial tears, tear can be reserved to a greater extent by using lacrimal embolism, wet atrial mirror wearing or other methods, and in addition, local anti-inflammatory treatment purposes can be achieved by using corticosteroids or non-glucocorticoid immunomodulatory drugs and the like locally [ Chinese conference of Asian dry eye society, eye surface and tear chemistry group of the specialized committee of the medical and health society of the strait and eye surface and dry eye chemistry group of the ophthalmologist of the Chinese society of doctors, and the consensus of Chinese dry eye specialist: treatment (2020), journal of Chinese ophthalmology, 2020 (12): 907-913. However, the separate use of drugs that slow down ocular surface tear loss and anti-inflammatory drugs remains the current mainstay of combination therapy, and in order to further improve patient compliance in therapy, developing a drug-loaded tear plug that has both anti-inflammatory and increased tear fluid volume functions has practical significance in the field of dry eye treatment.

In the current research, collagen, silica gel, acrylic acid polymer and other gel materials are mainly used as the preparation materials 【N.Jehangir,G.Bever,S.M.J.Mahmood,M.Moshirfar,Comprehensive review of the literature on existing punctal plugs for the management of dry eye disease J.Ophthalmol.2016,9312340.】. of lacrimal embolism products, and the main administration route for clinically treating dry eye is a local eye dropping mode, however, due to the hydrophobicity of dexamethasone, the medicine dropped into conjunctival sac is rapidly discharged through nasolacrimal duct through blinking, so that the bioavailability of the medicine is greatly reduced. In recent years, drug crystals have been widely used in drug delivery system research due to their advantages of high drug content and high stability. However, studies have not been reported on the use of regularly shaped drug crystals in combination with gel materials for ocular local drug delivery, particularly dry eye lacrimal embolism therapy.

Disclosure of Invention

The invention aims to provide a degradable drug-carrying composite gel for treating xerophthalmia.

The drug-loaded composite gel provided by the invention has good swelling performance, biocompatibility and degradability, and can prolong the release time of the anti-inflammatory drug while slowing down tear loss, thereby realizing the dual treatment effects of increasing the tear volume of the ocular surface and resisting inflammation.

The drug-loaded composite gel provided by the invention mainly comprises an anti-inflammatory drug and a hydrogel material.

The anti-inflammatory drug can be glucocorticoid drugs, immunosuppressant drugs and the like for dry eye treatment, and specifically can be dexamethasone, cyclosporins, tacrolimus and the like.

According to one embodiment of the invention, the anti-inflammatory drug is dexamethasone, which takes the form of dexamethasone crystallites.

Further, the apparent shape of the dexamethasone microcrystal is quadrilateral. The side length of the dexamethasone microcrystal is 4-15 mu m; the average diameter was 10.71. Mu.m.

The dexamethasone microcrystals can be prepared according to the existing method.

The preparation method comprises the following steps: recrystallizing the dexamethasone crystals serving as the raw material to obtain dexamethasone microcrystals; the recrystallization includes: dripping the absolute ethanol solution of the dexamethasone crystal serving as the raw material into PVA water solution, stirring to obtain suspension after dripping, standing overnight, and centrifuging to remove supernatant; the PVA represents polyvinyl alcohol; specifically, the concentration of the anhydrous ethanol solution of the dexamethasone crystal is 2.5-10mg/mL; in the dripping step, the dripping speed is 0.5-2mL/min; the concentration of the PVA aqueous solution is 0-1g/L and is not 0; specifically 1g/L; the volume ratio of the absolute ethanol solution of the dexamethasone crystal to the PVA aqueous solution is 1:10-20 parts of a base; the stirring is high-speed stirring; the stirring speed is specifically 600-10800rpm; in the standing overnight step, the temperature is 4-25 ℃.

The hydrogel material may be selected from at least one of the following: chitosan, PEG hydrogels, silMA, gelMA (methacryloylated gelatin), and the like.

According to one embodiment of the invention, the hydrogel material is GelMA. The MA substitution degree of the GelMA is 90+/-5%. The GelMA gel is directly available from commercial sources.

According to one embodiment of the invention, the drug-loaded composite gel consists of dexamethasone microcrystals and GelMA hydrogel; wherein, the mixing concentration of dexamethasone microcrystal and GelMA hydrogel solution is 5-80mg/mL.

The maximum swelling rate of the drug-loaded composite gel provided by the invention is 510-900%; the degradation time is 21-36h.

The invention also provides a preparation method of the degradable drug-loaded composite gel lacrimal duct plug, which comprises the following steps:

1) The anti-inflammatory drug and the hydrogel material solution are physically blended to obtain an anti-inflammatory composite gel solution;

2) Pouring the anti-inflammatory composite gel solution into a custom-made lacrimal plug mold;

3) Using visible light with 365-405 wave bands to irradiate and solidify the anti-inflammatory composite gel solution in the mould into gel;

4) And (3) carrying out vacuum freeze drying on the medicine-carrying composite gel which is solidified and formed in the mould, and then demoulding to obtain the medicine-carrying composite gel.

In the step 1), the anti-inflammatory drug may be specifically dexamethasone microcrystals, and the hydrogel material solution may be specifically sterile GelMA solution;

the preparation method of the dexamethasone microcrystalline composite gel solution comprises the following steps:

a) Adding the LAP photoinitiator into sterile PBS liquid, oscillating, and heating for 15-30mins to obtain LAP photoinitiator standard solution;

b) Adding GelMA into LAP photoinitiator standard solution, oscillating, and heating for 15-30mins to obtain GelMA gel precursor solution; passing the GelMA gel precursor solution through a 0.22 μm sterile filter to obtain a sterile GelMA gel solution;

c) Adding dexamethasone microcrystal into sterile GelMA gel solution, and mixing by vortex to obtain the dexamethasone microcrystal composite gel solution.

In the above method step a), the concentration of LAP in the LAP initiator standard solution is 0.25% (w/v); the pH value of the PBS is 7.2-7.4; the heating temperature is 40-50 ℃ and the heating time is 15-30 minutes.

In the above method step b), the mass-to-volume ratio of GelMA to LAP photoinitiator standard solution is (0.05 g-0.2 g): 1ml. The concentration of the GelMA gel solution can be 10-20% (w/v); the heating temperature is 60-70 ℃ and the heating time is 20-30 minutes.

In the method step c), the dosage ratio of the dexamethasone microcrystals to the sterile GelMA gel solution is (5-80) mg:100mL.

In the step 2), the specification of the lacrimal plug mold is 1mm in diameter and 1.5mm in length of each hole in the mold.

In the step 3), the irradiation time of the visible light with the wavelength of 405nm may be 15-240s, specifically, for example, 120s.

In addition, the application of the drug-loaded composite gel provided by the invention in at least one aspect of the following aspects also belongs to the protection scope of the invention: 1) Use in the preparation of an ocular drug delivery system; 2) Application in preparing lacrimal duct embolism products; 3) The application in preparing tissue repair products.

Further, the tissue repair is mainly ocular tissue repair.

Compared with the prior art, the invention has the following beneficial technical effects:

The invention selects GelMA gel and dexamethasone microcrystal as preparation materials of drug-loaded composite gel lacrimal plug. GelMA is widely used in the biomedical field due to its suitable biological properties and physical adjustability; gelMA-based hydrogels find wide application in tissue engineering applications, such as cartilage, bone and vascular tissue engineering studies; in addition to tissue engineering, other applications of GelMA hydrogels include cell research, drug delivery, and the like. The drug-loaded composite gel lacrimal plug prepared by combining dexamethasone microcrystal and GelMA gel not only ensures that the lacrimal plug has dual treatment effects of slowing down the flow of tear fluid on the ocular surface and resisting inflammation, but also can achieve the purpose of slowing down the release speed of drugs, and the combination has important application value in the field of lacrimal embolism materials.

Drawings

FIG. 1 is a scanning electron microscope image of a GelMA gel (a), dexamethasone microcrystals (b), drug-loaded composite gel (c), and a drug-loaded composite gel high-magnification image (d);

FIG. 2 is the swelling properties of GelMA gel (GelMA 90) and drug loaded composite gel (GelMA 90/DEX);

FIG. 3 is an in vitro degradation experimental result of GelMA gel (GelMA 90) and drug loaded composite gel (GelMA 90/DEX);

FIG. 4 shows the results of cytotoxicity experiments on GelMA gel (GelMA 90) and drug loaded composite gel (GelMA 90/DEX);

FIG. 5 is a graph of drug release profile of dexamethasone microcrystals (DEX MCs) and drug loaded composite gel (GelMA 90/DEX) in vitro.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

GelMA used in the examples below was purchased from the Suzhou Intelligent Credit institute under the trade designation EFL-GM-90 and had a MA substitution of 85% to 95%.

The dexamethasone microcrystals used in the following examples can be prepared as follows:

(1) 100mg of dexamethasone raw material medicine is mixed with the absolute ethanol solution, so that the concentration of the dexamethasone raw material medicine is 10mg/mL, and the dexamethasone absolute ethanol solution is obtained.

(2) The dexamethasone absolute ethanol solution is added into the polyvinyl alcohol aqueous solution with the concentration of 1g/L at the dropping rate of 2mL/min, and the volume ratio of the dexamethasone absolute ethanol solution to the polyvinyl alcohol aqueous solution is 1:10.

(3) Stirring at high speed to obtain suspension. Standing at 4 ℃ overnight, volatilizing absolute ethanol in the solution to obtain microcrystalline suspension.

(4) Centrifuging the microcrystalline suspension with deionized water for 3 times, removing supernatant, packaging, and lyophilizing to obtain dexamethasone microcrystalline powder. FIG. 1 (b) is a scanning electron microscope image of dexamethasone microcrystals; as can be seen from the figure, the side length of the obtained dexamethasone microcrystals is 4-15 μm.

Example 1 preparation of GelMA gel

1) Adding LAP initiator into sterile PBS (pH 7.2-7.4), oscillating, and heating for 30min to obtain LAP initiator standard solution with concentration of 0.25% (w/v);

2) Adding 0.1g of GelMA into 1mL of initiator standard solution, oscillating, and heating for 30mins to obtain GelMA gel precursor solution;

3) Passing the GelMA gel precursor solution through a 0.22 μm sterile filter to obtain a sterile GelMA solution;

4) After 120s of sterile GelMA solution was irradiated with a 405nm visible light source, gelMMA gel was obtained.

Example 2 preparation of drug-loaded composite gel lacrimal plug

1) Adding LAP initiator into sterile PBS (pH 7.2-7.4), oscillating, and heating for 30min to obtain LAP initiator standard solution with concentration of 0.25% (w/v);

2) Adding 0.1g of GelMA into 1mL of LAP initiator standard solution, oscillating, and heating for 30mins to obtain GelMA gel precursor solution;

3) Passing the GelMA gel precursor solution through a 0.22 μm sterile filter to obtain a sterile GelMA solution;

4) Physically blending 20mg of dexamethasone microcrystals with 1mL of sterile GelMA solution to obtain a dexamethasone microcrystal composite gel solution;

5) Pouring the dexamethasone microcrystalline composite gel solution into a custom-made lacrimal plug mold (1 mm diameter and 1.5mm length per hole in the mold);

6) Irradiating and curing the dexamethasone microcrystal composite gel solution in the mold by using visible light with the wave band of 405nm to form gel;

7) Freezing the composite gel solidified and formed in the mold at-80 ℃ for 24 hours, performing vacuum freeze drying (the freezing temperature is-50 ℃) for 48 hours, and then demolding to obtain the drug-carrying composite gel lacrimal duct plug.

In fig. 1, (a) and (c) are scanning electron microscope images of the GelMA gel prepared in example 1 and the drug-loaded composite gel prepared in example 2, and as can be seen from fig. 1 (d), dexamethasone microcrystals can be more uniformly distributed on the inner pore wall of the GelMA gel.

FIG. 2 shows the swelling properties of GelMA gel prepared in example 1 and drug-loaded composite gel prepared in example 2.

The specific experimental method is as follows: performing swelling performance evaluation on the prepared gel sample by adopting a weighing method; accurately weighing the initial mass of the dried gel sample, namely W0, immersing the sample into simulated tear at 37 ℃, respectively taking out the sample at a specific time point, wiping the surface liquid with filter paper, weighing again, and namely Wt;

The calculation formula of the swelling ratio: SR (%) = (Wt-W0)/W0×100%

From the figure, the GelMA gel and the drug-loaded composite gel can reach swelling balance in a short time.

FIG. 3 shows the results of in vitro degradation tests of GelMA gel prepared in example 1 and drug-loaded composite gel prepared in example 2.

The specific experimental method is as follows: performing degradation performance test on the prepared gel sample by adopting a weighing method; weighing the initial mass of the dried gel sample to be W0, immersing the sample in a degradation medium at 37 ℃ and 100rmp; taking out the samples at specific time points respectively, freeze-drying, weighing again, and recording as Wt;

the degradation rate calculation formula: degradation rate = (m 0-mt)/m0×100%

The graph shows that the GelMA pure gel reaches percent degradation at about 24 hours, and the drug-loaded composite gel reaches percent degradation at about 36 hours; both gels show better degradation performance, and the in vitro degradation time of the drug-loaded composite gel is slightly longer than that of the pure gel.

Example 3 cytotoxicity test on GelMA gel and drug-loaded composite gel prepared in examples 1 and 2.

The cells used in this example were P3-P5 Human Corneal Epithelial Cells (HCECs).

The specific experimental method is as follows: forming and solidifying GelMA gel and drug-loaded composite gel in an orifice plate, washing the gel surface in the orifice plate for 3 times by using a basic culture medium DF12, then adding a certain volume of culture medium (DF 12+ serum) into the gel hole, placing the gel into an incubator (37 ℃ and 5% carbon dioxide) for culturing for 24 hours, centrifuging, and taking the supernatant as leaching liquor; the resulting extract was sterilized with a 0.22um filter and stored at 4 ℃ for further use; HCEC cells were added to each well of the 96-well plate at 5×103/well, cultured for 24 hours using complete medium, starved cultured for 12-16 hours by replacing the medium in all wells with basal medium, then aspirated, replaced with leaching media, cultured for 24 hours, 48 hours and 72 hours, and CCK8 solution was added and assayed by an enzyme-labeled instrument at each time point, respectively.

FIG. 4 shows the cytotoxicity test results of GelMA gel and drug-loaded composite gel;

as shown in the figure, the cell viability of HCECs cells cultured for 24h, 48h and 72h by using the extracting solution of GelMA pure gel and drug-loaded composite gel is not significantly different from that of a normal control group, namely the material for preparing the lacrimal plug used in the invention does not influence the normal activity of the cells, which indicates that the gel has good cell compatibility.

Example 4 drug Release Properties of drug-loaded composite gel in vitro

The specific experimental method is as follows: dexamethasone microcrystal with dexamethasone content of 2mg and drug-loaded composite gel sample are respectively weighed, placed in a dialysis bag (MWCO 8k-14 k) and added with dissolution medium, the two ends are fastened, the dialysis bag is placed in a centrifuge tube containing the same medium, vibration is carried out in a 100rmp constant temperature box at 37 ℃,1.0 mL is sampled at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 2d, 3d, 5d, 7d and 9d respectively, the same release medium with the same amount of the same temperature is simultaneously added, and the sample solution under each sampling point is filtered and then subjected to high performance liquid chromatography detection.

Fig. 5 is a graph showing the results of drug release profiles in vitro of the drug-loaded composite gel prepared according to example 2. As can be seen from the figure, the dexamethasone microcrystals observe an initial burst release in the first 8 hours, and substantially complete drug dissolution in 24 hours, with a final dissolution rate of about 85%; the drug-loaded composite gel did not exhibit significant burst release in a short period of time, with a release rate of dexamethasone at 9d of approximately 78%. The drug-loaded composite gel has the controlled release effect on dexamethasone.

Claims (10)

1. A composite gel for carrying medicine is prepared from anti-inflammatory medicine and hydrogel material.

2. The drug-loaded composite gel of claim 1, wherein: the anti-inflammatory drug is selected from glucocorticoid drugs and immunosuppressant drugs for treating xerophthalmia, and further, the anti-inflammatory drug is selected from at least one of the following drugs: dexamethasone, cyclosporins, and tacrolimus;

Or, the hydrogel material is selected from at least one of the following: chitosan, PEG hydrogel, silMA, gelMA (methacryloylated gelatin).

3. The drug-loaded composite gel of claim 2, wherein: the anti-inflammatory drug is dexamethasone microcrystal;

or the hydrogel material is GelMA, and the MA substitution degree of the GelMA is 85% -95%.

4. A drug-loaded composite gel according to any one of claims 1-3, wherein: the drug-loaded composite gel comprises dexamethasone microcrystals and GelMA hydrogel; wherein, the mixing concentration of dexamethasone microcrystal and GelMA hydrogel solution is 5-80mg/mL.

5. The drug-loaded composite gel according to any one of claims 1-4, wherein: the maximum swelling rate of the drug-loaded composite gel is 510-900%; the degradation time is 21-36h.

6. Use of a drug-loaded composite gel according to any one of claims 1 to 5 in at least one of the following:

1) Use in the preparation of an ocular drug delivery system;

2) Application in preparing lacrimal duct embolism products;

3) The application in preparing tissue repair products.

7. The use according to claim 6, characterized in that: the tissue repair is ocular tissue repair.

8. A preparation method of a drug-loaded composite gel lacrimal duct plug comprises the following steps:

1) Physically blending the anti-inflammatory drug of any one of claims 1-4 with a hydrogel material solution to obtain an anti-inflammatory composite gel solution;

2) Pouring the anti-inflammatory composite gel solution into a lacrimal plug mold;

3) Using visible light with wave band of 365-405nm to irradiate and solidify the anti-inflammatory composite gel solution in the mould into gel;

4) And (3) carrying out vacuum freeze drying on the medicine-carrying composite gel which is solidified and formed in the mould, and then demoulding to obtain the medicine-carrying composite gel.

9. The method of manufacturing according to claim 8, wherein:

In the step 1), the anti-inflammatory drug is dexamethasone microcrystals, and the hydrogel material solution is a sterile GelMA solution;

the preparation method of the dexamethasone microcrystalline composite gel solution comprises the following steps:

a) Adding the LAP photoinitiator into sterile PBS liquid, oscillating, and heating for 15-30 minutes to obtain LAP photoinitiator standard solution;

b) Adding GelMA into LAP photoinitiator standard solution, oscillating, and heating for 20-30 min to obtain GelMA gel precursor solution; passing the GelMA gel precursor solution through a 0.22 μm sterile filter to obtain a sterile GelMA gel solution;

c) Adding dexamethasone microcrystals into a sterile GelMA gel solution, and then uniformly mixing by vortex to obtain the dexamethasone microcrystal composite gel solution;

Further, in the step a), the concentration of LAP in the LAP initiator standard solution is 0.25% (w/v); the pH value of the PBS is 7.2-7.4; the heating temperature is 40-50 ℃ and the heating time is 15-30 minutes;

or, further, in the step b), the mass-volume ratio of the GelMA to the LAP photoinitiator standard solution is (0.05 g-0.2 g): 1ml; the concentration of the GelMA gel solution is 10-20% (w/v); the heating temperature is 60-70 ℃ and the heating time is 20-30 minutes;

Or, further, in said step c); the dosage ratio of the dexamethasone microcrystal to the sterile GelMA gel solution is (5-80) mg:100mL;

or, in the step 3), the time of irradiation of the visible light with the wave band of 365-405nm is 15-240s.

10. The drug-loaded composite gel lacrimal plug prepared by the method of claim 8 or 9.