Preparation method and application of an injectable probucol-loaded hydrogel

Abstract

The present invention relates to the technical field of ophthalmic preparations, and specifically relates to a preparation method and application of an injectable hydrogel loaded with probucol. PEG-PLGA and PB are dissolved in 1 ml of dichloromethane, and then the solution is dropped into an aqueous phase containing 1 wt% polyvinyl alcohol. Then, it is ultrasonically treated in an ice bath for 10 minutes. Next, the emulsion is added to a 0.3 wt% PVA solution, and magnetically stirred for 12 hours to evaporate the organic solvent. The emulsion is centrifuged at 13,500 rpm for 10 minutes, and rinsed three times with PBS to remove PVA. Finally, the washed NPs are freeze-dried and collected to obtain probucol nanoparticles; NaIO4 is added to an aqueous solution of 8 wt% polysaccharide, and the reaction is stirred at room temperature for 6 hours. After quenching with ethylene glycol for 1 hour, the mixture is dialyzed against deionized water for 4 days, and then freeze-dried to obtain oxidized polysaccharide; the oxidized polysaccharide is dissolved in 0.1 M borax, and then mixed with a gelatin solution containing PB NPs to prepare a hydrogel.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic preparations, and particularly relates to a preparation method and application of an injectable hydrogel loaded with probucol. Background Art

[0002] Pathological corneal neovascularization not only destroys the normal physiological structure of the cornea, but also seriously affects the vision of patients. At present, there is still a lack of effective countermeasures for this disease.

[0003] Probucol has strong antioxidant and anti-inflammatory properties, but its bioavailability through intravenous injection or oral administration is extremely low. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of an injectable hydrogel loaded with probucol, which solves the technical problem of extremely low bioavailability of probucol.

[0005] To achieve the above purpose, the present invention provides a preparation method of an injectable hydrogel loaded with probucol, including the following steps:

[0006] Step 1: Preparation of probucol (PB)-loaded nanoparticles (NPs). Dissolve PEG-PLGA and PB in 1 ml of dichloromethane, then drop the solution into an aqueous phase containing 1 wt% polyvinyl alcohol (PVA). Then, ultrasonicate for 10 minutes in an ice bath. Next, add the emulsion to a 0.3 wt% PVA solution, and stir magnetically for 12 hours to evaporate the organic solvent. Centrifuge the emulsion at 13500 rpm for 10 minutes, and wash three times with PBS to remove PVA. Finally, lyophilize and collect the washed NPs;

[0007] Step 2: Synthesis of oxidized polysaccharide. Add NaIO4 to an 8 wt% aqueous solution of polysaccharide, and stir and react at room temperature for 6 hours. After quenching with ethylene glycol for 1 hour, dialyze the mixture against deionized water for 4 days, and then lyophilize to obtain oxidized polysaccharide;

[0008] Step 3: Preparation of injectable hydrogel. Dissolve the oxidized polysaccharide in 0.1 M borax, and then mix it with a gelatin (Gel) solution containing PBNPs to prepare a hydrogel.

[0009] Among them, in Step 1, the mass of PEG-PLGA is 10 - 30 mg, and PB is 1 - 5 mg.

[0010] Among them, in Step 2, the polysaccharide is a natural polymer such as hyaluronic acid, dextran, cellulose, chitosan, etc., and the mass ratio of sodium periodate to polysaccharide is 0.5 - 3.

[0011] Among them, in step three, the mass fraction of the oxidized polysaccharide is 5-20 wt%, and the mass fraction of gelatin is 10-25 wt%.

[0012] Among them, the injectable hydrogel loaded with probucol is used as an injection for regressing corneal neovascularization.

[0013] A preparation method and application of an injectable hydrogel loaded with probucol of the present invention. Dissolve PEG-PLGA and PB in 1 ml of dichloromethane, then drop the solution into an aqueous phase containing 1 wt% polyvinyl alcohol, then ultrasonicate in an ice bath for 10 minutes, and then add the emulsion to a 0.3 wt% PVA solution, stir magnetically for 12 hours to evaporate the organic solvent, centrifuge the emulsion at 13500 rpm for 10 minutes, and wash three times with PBS to remove PVA. Finally, freeze-dry and collect the washed NPs to obtain probucol nanoparticles; add NaIO4 to an aqueous solution of 8 wt% polysaccharide, stir and react at room temperature for 6 hours, quench with ethylene glycol for 1 hour, then dialyze the mixture with deionized water for 4 days, and then freeze-dry to obtain oxidized polysaccharide; dissolve the oxidized polysaccharide in 0.1 M borax, and then mix it with a gelatin solution containing PB NPs to prepare a hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0015] Figure 1 It is a flowchart of the preparation method of the injectable hydrogel loaded with probucol according to the first embodiment of the present invention.

[0016] Figure 2 It is the shear thinning behavior of the injectable hydrogel loaded with probucol of the present invention.

[0017] Figure 3 It is the frequency sweep of the injectable hydrogel loaded with probucol of the present invention.

[0018] Figure 4 It is the continuous step strain diagram of the injectable hydrogel loaded with probucol of the present invention.

[0019] Figure 5 It is the scanning electron microscope image of the injectable hydrogel loaded with probucol of the present invention.

[0020] Figure 6 It is the digital photo of the rat corneal alkali burn after treatment with the hydrogel for different times. DETAILED DESCRIPTION OF THE INVENTION

[0021] Embodiments of the present invention will be described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0022] The first embodiment of this application is as follows:

[0023] Please refer to Figure 1 , Figure 1 which is a flowchart of the preparation method of the injectable hydrogel loaded with probucol according to the first embodiment of the present invention. By the foregoing solution, the problem of low bioavailability of probucol is solved. It can be understood that the foregoing solution can be used in the case of corneal neovascularization and can also be used for the solution of controlled release.

[0024] A preparation method of the injectable hydrogel loaded with probucol includes the following steps:

[0025] S101: Preparation of probucol (PB) nanoparticles (NPs). Dissolve PEG-PLGA and PB in 1 ml of dichloromethane, then drop the solution into an aqueous phase containing 1 wt% polyvinyl alcohol (PVA). Then, ultrasonicate in an ice bath for 10 minutes. Next, add the emulsion to a 0.3 wt% PVA solution and stir magnetically for 12 hours to evaporate the organic solvent. Centrifuge the emulsion at 13,500 rpm for 10 minutes and wash three times with PBS to remove PVA. Finally, lyophilize and collect the washed NPs;

[0026] S102: Synthesis of oxidized polysaccharide. Add NaIO4 to an 8 wt% aqueous solution of polysaccharide and stir and react at room temperature for 6 hours. After quenching with ethylene glycol for 1 hour, dialyze the mixture against deionized water for 4 days, and then lyophilize to obtain the oxidized polysaccharide;

[0027] S103: Preparation of injectable hydrogel. Dissolve the oxidized polysaccharide in 0.1 M borax, and then mix it with a gelatin (Gel) solution containing PB NPs to prepare the hydrogel.

[0028] Among them, in step one, the mass of PEG-PLGA is 10 - 30 mg, and PB is 1 - 5 mg.

[0029] Secondly, in step two, the polysaccharide is a natural polymer such as hyaluronic acid, dextran, cellulose, chitosan, etc., and the mass ratio of sodium periodate to polysaccharide is 0.5 - 3.

[0030] At the same time, in step three, the mass fraction of the oxidized polysaccharide is 5 - 20 wt%, and the mass fraction of gelatin is 10 - 25 wt%.

[0031] The prepared hydrogels were detected, and the specific operation process and results are as follows:

[0032] Characterization of the prepared hydrogels

[0033] For the rheological behavior of the Gel / ODex / Bo hydrogel, the strain in the frequency sweep experiment of the rheometer used was 1%, and the dynamic oscillation frequency range was 1 - 100 rad / s. The strain range in the strain sweep experiment was 0.01% - 500%, and the frequency was set at 1 Hz.

[0034] The rheological properties of the gel / ODex / Bo hydrogel were detected by dynamic frequency sweep rheological tests. The viscosity of the hydrogel was measured at 37°C. Please refer to Figure 2 , Figure 2 which is the shear thinning behavior of the injectable aqueous hydrogel loaded with probucol of the present invention, Figure 2 indicating that the viscosity decreases with the increase of the shear rate.

[0035] Please refer to Figure 3 , Figure 3 which is the frequency sweep of the injectable aqueous hydrogel loaded with probucol of the present invention, showing the functions of G′ and G″ of the hydrogels with or without PB NPs varying with frequency. Within the test frequency range, G′ of all hydrogels is greater than G″, indicating that all hydrogels behave like viscoelastic solids.

[0036] Subsequently, the rheological recovery behavior of the prepared hydrogel was demonstrated by the continuous step strain method. As shown in the following figure, under the condition of low oscillatory shear strain (1%), neither modulus changed, and G′ was always greater than G″ within the first interval, indicating the formation of a stable hydrogel network. When the strain was switched to 400%, G′ decreased sharply and was lower than G″, meaning that the hydrogel network ruptured. Interestingly, when the strain was reduced to 1%, the ruptured network could be rapidly rebuilt, as evidenced by the rapid recovery of G′ and G″ to their initial values. In addition, this destruction and recovery process could also be alternately repeated under oscillatory strain. These results indicate that the hydrogel network has the property of rapid self - recovery after destruction.

[0037] Please refer to Figure 5 , Figure 5 which is the scanning electron micrograph of the injectable aqueous hydrogel loaded with probucol of the present invention. The surface morphology of the freeze - dried hydrogel was observed with a scanning electron microscope (Nanosem 430). It was found that the surface of H - PB had a porous structure, and the surface pore size was about 115 μm.

[0038] Please refer to Figure 6 , Figure 6These are digital photos of rat corneas at different times after alkali burn and treated with hydrogels. To study the efficacy of PB hydrogels on corneal healing, we used an alkali burn model. Rats were randomly divided into four groups and received PBS, PB NPs, H-C, or H-PB treatment respectively. After continuous examination, bright-field corneal images under a slit lamp microscope showed that subconjunctival injection of PB NPs or H-PB could significantly relieve edema and rapidly restore the transparency of the injured cornea to normal levels. On the 9th day, corneal opacity in all groups (including the PBS group and the H-C group) was further alleviated. These effects were most obvious in the H-PB group, indicating that PB-HA could accelerate corneal healing and neovascular regression.

[0039] When using the preparation method of an injectable hydrogel loaded with probucol of this embodiment,

[0040] The injectable hydrogel loaded with probucol prepared by the preparation method of the injectable hydrogel loaded with probucol is used as an injection for regressing corneal neovascularization.

[0041] The injectable hydrogel loaded with probucol prepared by the preparation method of the injectable hydrogel loaded with probucol is used to accelerate corneal healing.

[0042] The preparation method of the injectable hydrogel loaded with probucol can be used for controlled release by changing the drug concentration and soaking time.

[0043] The above-disclosed are only one or more preferred embodiments of this application, and the scope of rights of this application cannot be limited thereby. Those of ordinary skill in the art can understand all or part of the processes of implementing the above embodiments, and the equivalent changes made according to the claims of this application still fall within the scope covered by this application.

Claims

1. Use of an injectable probucol-loaded hydrogel in the preparation of a drug for treating corneal alkali burns, characterized in that, The preparation method of the injectable hydrogel loaded with probucol comprises the following steps: Step 1: Preparation of probucol PB nanoparticles NP: Dissolve PEG-PLGA and PB in 1 ml of dichloromethane, then drop the solution into an aqueous phase containing 1 wt% polyvinyl alcohol PVA. Then, ultrasonicate for 10 minutes in an ice bath. Next, add the emulsion to a 0.3 wt% PVA solution, and stir magnetically for 12 hours to evaporate the organic solvent. Centrifuge the emulsion at 13500 rpm for 10 minutes, and wash it three times with PBS to remove PVA. Finally, lyophilize and collect the washed probucol PB nanoparticles NP; Step 2: Synthesis of oxidized polysaccharide: Add NaIO4 to an 8 wt% aqueous solution of polysaccharide, stir and react at room temperature for 6 hours. After quenching with ethylene glycol for 1 hour, dialyze the mixture against deionized water for 4 days, and then lyophilize to obtain the oxidized polysaccharide, wherein the polysaccharide is a natural polymer such as hyaluronic acid, dextran, cellulose or chitosan; Step 3: Preparation of injectable hydrogel: Dissolve the oxidized polysaccharide in 0.1 M borax, and then mix it with a gelatin Gel solution containing probucol PB nanoparticles NP to prepare the hydrogel; Characterization and determination of the prepared hydrogel: The strain of the frequency sweep experiment of the rheometer used for the rheological behavior of the hydrogel is 1%, and the dynamic oscillation frequency range is 1-100 rad / s; the strain range of the strain sweep experiment is 0.01%-500%, and the frequency is set to 1 Hz.

2. The application of the injectable hydrogel loaded with probucol according to claim 1 in the preparation of a drug for treating corneal alkali burn, characterized in that In step 1, the mass of PEG-PLGA is 10-30 mg, and the mass of PB is 1-5 mg.

3. The application of the injectable hydrogel loaded with probucol according to claim 1 in the preparation of a drug for treating corneal alkali burn, characterized in that In step 2, the mass ratio of sodium periodate to polysaccharide is 0.5-3.

4. The application of the injectable hydrogel loaded with probucol according to claim 1 in the preparation of a drug for treating corneal alkali burn, characterized in that In step 3, the mass fraction of the oxidized polysaccharide is 5-20 wt%, and the mass fraction of gelatin is 10-25 wt%.

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