Hydrogel sustained-release material loaded with melatonin and mecobalamin as well as preparation method and application of hydrogel sustained-release material

Abstract

The invention provides a hydrogel sustained-release material loaded with melatonin and mecobalamin as well as a preparation method and application of the hydrogel sustained-release material, and belongs to the technical field of medicine synthesis. According to the invention, HyStem-HP and thiolated gelatin are taken as hydrogel carriers, melatonin and mecobalamin are loaded, and the hydrogel sustained-release material loaded with melatonin and mecobalamin is prepared. A three-dimensional network structure is constructed through a physical or chemical cross-linking technology, synergistic slow release of melatonin and mecobalamin is achieved, the drug action time is prolonged, frequent drug administration requirements are reduced, oxidation resistance and apoptosis resistance of melatonin are combined with the nerve repairing function of mecobalamin, photoreceptor cell degeneration is doubly delayed, and the melatonin-mecobalamin-melatonin-mecobalamin-melatonin-mecobalamin-melatonin-mecobalamin-melatonin-mecobalamin- meanwhile, high-concentration accumulation of the medicine at the retinopathy part can be realized through local injection of the hydrogel, and the medicine is continuously released for several weeks, so that the blood retina barrier limitation is broken through.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical synthesis, and particularly relates to a hydrogel sustained-release material loaded with melatonin and mecobalamin, and a preparation method and application thereof. Background Art

[0002] Retinitis pigmentosa is a hereditary and degenerative disease characterized by the progressive loss of photoreceptor cells and pigment epithelium function. According to statistics, there are 1.5 million patients globally. In the United States and Europe, the incidence of RP is 1 / 4000 - 1 / 3000, and in China it is about 1:3500. RP patients show night blindness and progressive narrowing of the visual field in the early stage, and gradually develop visual acuity decline. There are obvious pigment abnormalities in the mid-peripheral part of the fundus, and typical osteocyte-like pigmentation can be seen. In the late stage of the disease, the visual field of the patient progressively shrinks to a tubular visual field, and the visual acuity drops severely or even completely loses sight, and only a few patients can maintain partial vision for a long time. Currently, there is no effective method to cure or prevent the progression of RP.

[0003] Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine hormone secreted by the pineal gland and has the function of regulating the circadian rhythm. It can be detected in almost all tissues, indicating its general importance in the human body. Research shows that melatonin has a series of biological functions such as regulating aging, anti-inflammatory, antioxidant, and inhibiting the activity of tumor cells. Recent research results show that melatonin can promote the healing of gastric ulcer mucosal epithelium and the healing of rat skin wounds. In addition, in vitro experimental studies show that melatonin can improve the proliferation and migration activity of keratinocytes stimulated by high glucose and reduce the apoptosis of keratinocytes.

[0004] Mecobalamin is a coenzyme form of vitamin B12. Its structure is similar to that of vitamin B12, and it is essentially a biologically active alkylcobalamin. Currently, the research on mecobalamin mainly focuses on facial nerve paralysis, tumors, diabetic neuropathy, visual degradation, improving heart rate variability, inhibiting HIV-1 infection, male infertility, sleep disorders, etc. And mecobalamin has been used clinically to deal with hyperhomocysteinemia and peripheral neuropathy caused by diabetes. However, it has been found through research that there is no report on the treatment of diabetic skin with mecobalamin.

[0005] Therefore, how to construct a melatonin-mecobalamin system to treat retinitis pigmentosa has become an urgent problem to be solved at present.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The first technical problem to be solved by the present invention is to provide a preparation method of a hydrogel sustained-release material loaded with melatonin and mecobalamin. This method constructs a three-dimensional network structure through physical or chemical cross-linking technology to achieve the synergistic sustained release of melatonin and mecobalamin, extend the drug action time, and reduce the need for frequent drug administration. The second technical problem to be solved by the present invention is to provide a hydrogel sustained-release material loaded with melatonin and mecobalamin, which can achieve high-concentration accumulation of the drug at the site of retinal lesions, release continuously for several weeks, and break through the limitation of the blood-retinal barrier. The third technical problem to be solved by the present invention is to provide the application of a hydrogel sustained-release material loaded with melatonin and mecobalamin as a drug reagent in the treatment of retinitis pigmentosa diseases.

[0008] To solve the above problems, the technical solutions adopted by the present invention are as follows:

[0009] A preparation method of a hydrogel sustained-release material loaded with melatonin and mecobalamin, comprising the following steps:

[0010] 1) Prepare a melatonin stock solution and a mecobalamin stock solution respectively;

[0011] 2) Mix carboxymethyl hyaluronic acid-thiopropionyl hydrazide and heparin-thiopropionyl hydrazide and stir to form a HyStem-HP matrix. Mix HyStem-HP with thiolated gelatin, add phosphate buffer solution, and prepare a matrix stock solution;

[0012] 3) Mix the melatonin and mecobalamin stock solutions obtained in step 1) with the matrix stock solution obtained in step 2), add a cross-linking agent, pour it into a mold and seal it;

[0013] 4) Rinse the hydrogel obtained in step 3) with sterile PBS to remove uncrosslinked substances, and finally obtain a hydrogel loaded with melatonin and mecobalamin.

[0014] Further, in step 1), during the preparation process of the melatonin stock solution: dissolve melatonin in absolute ethanol, magnetically stir at 800 rpm and 25 °C until completely dissolved to obtain a mother liquor, aliquot it into brown cryopreservation tubes, and store it in the dark at -20 °C.

[0015] Further, in step 1), during the preparation process of the mecobalamin stock solution: dissolve mecobalamin in sterile physiological saline, vortex at 2000 rpm for 30 s until completely dissolved to obtain a mother liquor, aliquot it and store it in the dark at -20 °C.

[0016] Further, in step 2), the mass ratio of carboxymethyl hyaluronic acid-thiopropionyl hydrazide to heparin-thiopropionyl hydrazide is 99.7:0.3.

[0017] Further, in step 2), the mass ratio of HyStem-HP to thiolated gelatin is 1:1.

[0018] Further, in the step 3), the volume ratio of melatonin to mecobalamin stock solution is 1:1.

[0019] Further, in the step 3), the concentrations of melatonin and mecobalamin stock solution are 50 mmol / L.

[0020] Further, in the step 3), the crosslinking agent is polyethylene glycol diacrylate, and the dropping rate is 50 μL / s.

[0021] Further, for the preparation method of the hydrogel sustained-release material loaded with melatonin and mecobalamin, the hydrogel sustained-release material loaded with melatonin and mecobalamin is obtained.

[0022] Further, the application of the hydrogel sustained-release material loaded with melatonin and mecobalamin as a drug for treating retinitis pigmentosa.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] (1) The present invention constructs a three-dimensional network structure through physical or chemical crosslinking technology to achieve the synergistic sustained release of melatonin and mecobalamin, extend the drug action time, and reduce the need for frequent drug administration; and adopts processes such as low-temperature or photoinitiated polymerization to avoid the destruction of drug activity by high temperature or organic solvents and ensure the stability of the two components.

[0025] (2) The present invention selects natural polymers (such as hyaluronic acid) or biodegradable synthetic materials to reduce the risk of immune rejection and adapt to the ocular environment. At the same time, by adjusting the crosslinking degree or material ratio, the mechanical strength of the gel and the drug release rate can be precisely controlled to meet the personalized treatment needs.

[0026] (3) The hydrogel prepared by the present invention can be locally injected (such as intravitreally) to achieve high-concentration accumulation of the drug at the retinal lesion site, release continuously for several weeks, and break through the limitation of the blood-retinal barrier; the antioxidant and anti-apoptotic properties of melatonin, combined with the nerve repair function of mecobalamin, can double-delay the degeneration of photoreceptor cells, which is superior to single-drug treatment.

[0027] (4) The present invention can locally slow down the risk of systemic absorption, avoid adverse reactions such as drowsiness and gastrointestinal discomfort caused by oral or injection administration, and achieve long-term efficacy with a single injection, improving patient compliance, especially suitable for chronic disease management. Description of the Drawings

[0028] Figure 1 It is the surface electron micrograph (left) and cross-sectional electron micrograph (right) of the melatonin-mecobalamin hydrogel prepared in this application;

[0029] Figure 2Mitochondrial membrane potential map of melatonin - mecobalamin hydrogel prepared for this application;

[0030] Figure 3 Application of melatonin - mecobalamin hydrogel prepared for this application in animals; wherein, A is the flow chart of animal experiment; B is the OCT result map of rat retina; C is the statistical result map of retina thickness shown by OCT; D is the HE staining map of retina section. Detailed implementation manners

[0031] To make the above objects, features and advantages of the present invention more obvious and understandable, the following specific embodiments are used to describe the detailed implementation manners of the present invention in detail.

[0032] In the following embodiments, mecobalamin, melatonin, hydrogel, carboxymethyl hyaluronic acid - thio - propionyl hydrazide, heparin - thio - propionyl hydrazide, thiolated gelatin, and polyethylene glycol diacrylate are all purchased from Sigma Company, USA.

[0033] All operations in the following embodiments need to be completed in a biosafety cabinet or a clean workbench, and experimental instruments and solutions need to be sterilized. Ultra - pure water (18.2 MΩ·cm) is used to prepare solutions to avoid impurity interference. The drug working solution needs to be prepared and used immediately to avoid degradation by light.

[0034] In the following embodiments, the chromatographic conditions for detecting melatonin and mecobalamin by high - performance liquid chromatography (HPLC) are as follows: For melatonin, a C18 chromatographic column (4.6 mm × 250 mm, 5 μm) is used, the mobile phase is methanol - water - trifluoroacetic acid (45:55:0.05), the detection wavelength is 222 nm, the flow rate is 0.8 mL / min, the column temperature is recommended to be 30 °C, and the injection volume is 10 μL. For mecobalamin, a C18 chromatographic column (such as Luna C18) is also used, the mobile phase is 0.03 mol / L potassium dihydrogen phosphate (pH 4.5) - acetonitrile (84:16), the detection wavelength is 342 nm, the flow rate needs to be adjusted to make the retention time about 12 minutes, the column temperature is recommended to be room temperature, and the injection volume is 20 μL. Both use reverse - phase chromatography, but the mobile phase and detection wavelength are significantly different, and the conditions need to be optimized separately to ensure the separation effect and sensitivity.

[0035] In the following embodiments, the calculation formula for the drug sustained - release rate is as follows:

[0036] Drug sustained - release rate (%) = (cumulative release amount / total drug loading amount) × 100%

[0037] In the formula, cumulative release amount: the drug release amount measured at each time point by timed sampling, and the total amount accumulated to the target time point (such as 72 hours);

[0038] Total drug loading amount: the total amount of melatonin and mecobalamin initially loaded in the hydrogel, which can be determined by complete dissolution or destructive extraction method.

[0039] Example 1

[0040] A preparation method of a hydrogel sustained-release material loaded with melatonin and mecobalamin comprises the following steps:

[0041] (1) Preparation of drug stock solutions

[0042] Melatonin stock solution: Accurately weigh 50 mg of melatonin powder, dissolve it in 2.15 mL of absolute ethanol, and stir magnetically (800 rpm, 25 °C) until completely dissolved to obtain a mother liquor with a concentration of 100 mmol / L. Aliquot it into brown cryotubes and store it in the dark at -20 °C. Dilute it to a 50 mmol / L working solution with sterile normal saline before use.

[0043] Mecobalamin stock solution: Precisely weigh 50 mg of mecobalamin powder, dissolve it in 3.20 mL of sterile normal saline, and vortex (2000 rpm, 30 s) until completely dissolved to obtain a mother liquor with a concentration of 100 mmol / L. Aliquot it and store it in the dark at -20 °C. Dilute it to a 50 mmol / L working solution with sterile normal saline before use.

[0044] (2) Preparation of hydrogel matrix: Weigh carboxymethyl hyaluronic acid-thiopropionyl hydrazide (CMHA-DTPH) and heparin-thiopropionyl hydrazide (HP-DTPH) according to a mass ratio of 99.7:0.3, place them in a sterile centrifuge tube, and vortex and mix (2000 rpm, 5 min) to form a HyStem-HP matrix. Mix HyStem-HP with thiolated gelatin (GTN-DTPH) according to a mass ratio of 1:1, add pre-cooled phosphate buffer solution (PBS, pH 7.4, 4 °C), and prepare a 1× matrix stock solution with a final concentration of 4% (w / v). Let it stand and swell at 4 °C for 30 min for standby.

[0045] (3) Preparation of drug-loaded hydrogel: Under light-shielded conditions, take 500 μL of 50 mmol / L melatonin working solution and 500 μL of 50 mmol / L mecobalamin working solution, and dropwise add them into 2 mL of pre-cooled HyStem-HP / GTN-DTPH mixed solution, and gently pipette and mix well (avoid generating bubbles). Slowly add 0.5 mL of polyethylene glycol diacrylate (PEGDA, molecular weight 3400) cross-linking agent, control the dropping rate at 50 μL / s, and at the same time stir magnetically in an "8" shape (200 rpm, 3 min) to make the cross-linking agent evenly dispersed. Transfer the mixture to a polytetrafluoroethylene mold, and let it stand in the dark at 25 °C for 20 min to complete gelation.

[0046] (4) Hydrogel post-treatment: The formed hydrogel was rinsed 3 times with sterile PBS (pH 7.4) to remove uncrosslinked substances, immersed in sterile physiological saline, and stored in the dark at 4°C with a validity period of 7 days. When lyophilization was required, the hydrogel was quickly frozen in liquid nitrogen for 5 min and then transferred to a lyophilizer (-50°C, 0.1 mbar) for drying for 24 h to obtain a solid gel.

[0047] The hydrogel sustained-release material loaded with melatonin and mecobalamin prepared in Example 1 was characterized.

[0048] (1) Quality control and characterization: The freeze-dried hydrogel sample was sputter-coated with gold (ion sputtering instrument, gold coating thickness 5 nm), and the surface and cross-sectional morphology were observed using a scanning electron microscope (SEM, acceleration voltage 5 kV). As Figure 1 shown, it can be seen that the hydrogel presented a uniform porous structure.

[0049] (2) Drug release test: The hydrogel was immersed in PBS (37°C, pH 7.4), samples were taken at regular intervals, and the cumulative release amounts of melatonin and mecobalamin were detected by high-performance liquid chromatography (HPLC). The results showed that the drug sustained-release rate reached over 85% within 72 h.

[0050] (3) Detection of cell mitochondrial membrane potential

[0051] The JC-1 kit (Beyotime, Shanghai, China) was used to determine mitochondrial membrane disruption and potential. The cells were cultured in an incubator containing 5% CO2 at 37°C for 48 h, then the cells were incubated with the JC-1 probe at 37°C for 20 min, washed with ice-cold assay buffer, and detected by fluorescence microscopy. The fluorescence microscopy setting parameters were: λex nm; λem 530 nm (green) (JC-1 monomer) and λex 525 nm; λem (red) (JC-1 aggregate).

[0052] For the JC-1 MMP detection kit (Shanghai Yisen Biotechnology Co., Ltd.), the culture medium of each group of cells was discarded, the cells were washed once with PBS, an equal amount of culture medium and JC-1 staining working solution were added, and mixed well. Then, the cells were cultured in a 37°C cell incubator for 20 min. Then the supernatant was discarded, the cells were washed twice with the prepared 1×JC-1 staining buffer, and cell culture medium was added. Thereafter, the cells were washed with PBS and observed under an inverted fluorescence microscope. The average fluorescence ratio of JC-1 aggregates (red 590 nm) to monomers (green 490 nm) was used to represent early cell apoptosis.

[0053] From Figure 2From the mitochondrial membrane potential diagram of the prepared melatonin-methylcobalamin hydrogel, it can be seen that the mitochondrial membrane potential in ARPE-19 cells induced by H2O2 with MT+MB was significantly increased, thereby reducing apoptosis.

[0054] Example 2

[0055] Application of a hydrogel sustained-release material loaded with melatonin and methylcobalamin in the treatment of retinitis pigmentosa, the method is as follows:

[0056] (1) Preparation of retinitis pigmentosa animal model and drug administration treatment

[0057] Select 7-week-old SD rats, and induce a retinitis pigmentosa model by injecting sodium iodate (40 mg / kg) into the tail vein. Feed adaptively for 1 week before modeling. Administer drugs by single-eye intravitreal injection, the administration dose is 5 μm, once every 3 days, for 3 times. The control eye is given the same volume of PBS. The animal experiment flow chart is as Figure 3 shown in A.

[0058] (2) Observe the changes in the structure and thickness of each layer of the retina of SD rats by OCT

[0059] Use the Reword R640 gas anesthesia machine to anesthetize SD rats with isoflurane gas, dilate the pupils with compound tropicamide eye drops, keep the cornea moist with 0.9 g / 100 mL normal saline, adjust the OCT lens and parameters for observation and photography. The images are collected with a standard of 1 optic disc interval, and the instrument's built-in software is used to measure the thickness of the entire retina. As Figure 3 shown in B for the OCT results of the rat retina and Figure 3 shown in C for the statistical results of the retina thickness shown by OCT. OCT shows that the full-thickness of the retina of retinitis pigmentosa rats increased significantly after intravitreal injection of MT+MB or H+MT+MB.

[0060] (3) HE staining

[0061] Embed the eyeballs of SD rats in wax blocks, and prepare sections axially with the optic nerve and the center of the cornea. Gradient dewax the eyeball sections to water, stain with hematoxylin for 5 min, rinse with running water, differentiate with hydrochloric acid ethanol for 30 s, rinse with running water for 15 min, stain with eosin for 2 min, dehydrate by gradient, and seal with neutral gum for light microscopy observation.

[0062] Figure 3D is a HE staining map of retinal sections. The results show that compared with the NaIO3 group, the thickness of the outer nuclear layer of the retina in the MT+MB group increased (p<0.01), and compared with the NaIO3 group, the thickness of the outer nuclear layer of the retina in the H+MT+MB group increased significantly (p<0.01). The above results indicate that after intravitreal injection of MT+MB or H+MT+MB, the thickness of the outer nuclear layer of the retina in RP rats is higher than that in the NaIO3 group.

[0063] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel sustained-release material loaded with melatonin and methylcobalamin, characterized in that: The following steps are involved: 1) preparing melatonin stock solution and methylcobalamin stock solution respectively; 2) Carboxymethyl hyaluronic acid-thiopropionyl hydrazide and heparin-thiopropionyl hydrazide are mixed and stirred to form a HyStem-HP matrix, HyStem-HP is mixed with thiolated gelatin, and phosphate buffer is added to prepare a matrix stock solution; 3) mixing the melatonin and methylcobalamin stock solutions obtained in step 1) with the matrix stock solution obtained in step 2) and mixing evenly, adding a cross-linking agent, pouring into a mold and sealing; 4) The hydrogel obtained in step 3) is rinsed with sterile PBS to remove uncrosslinked substances, and finally a hydrogel loaded with melatonin and methylcobalamin is obtained.

2. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 1), the preparation process of melatonin stock solution is as follows: melatonin is dissolved in anhydrous ethanol, and magnetically stirred at 800 rpm and 25° C. until completely dissolved to obtain a mother solution, which is then dispensed into brown cryovials and stored at -20° C. in the dark.

3. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 1), the methylcobalamin stock solution is prepared by dissolving methylcobalamin in sterile physiological saline, vortexing at 2000 rpm for 30 seconds until completely dissolved to obtain a mother solution, which is then divided into subpackages and stored at -20°C in the dark.

4. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 2), the mass ratio of carboxymethyl hyaluronic acid-thiopropionyl hydrazide to heparin-thiopropionyl hydrazide is 99.7:0.

3.

5. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 2), the mass ratio of HyStem-HP to thiolated gelatin is 1:

1.

6. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 3), the volume ratio of melatonin to methylcobalamin stock solution is 1:

1.

7. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 3), the concentration of the melatonin and methylcobalamin stock solutions is 50 mmol / L.

8. The method for preparing the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 1, characterized in that: In the step 3), the cross-linking agent is polyethylene glycol diacrylate, and the dropping speed is 50 μL / s.

9. A method for preparing a hydrogel sustained-release material loaded with melatonin and methylcobalamin according to any one of claims 1 to 8, and the prepared hydrogel sustained-release material loaded with melatonin and methylcobalamin.

10. Use of the hydrogel sustained-release material loaded with melatonin and methylcobalamin according to claim 9 as a drug for treating retinitis pigmentosa.

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