Method for preparing organic molecular cage hydrogel and application thereof

Abstract

The application belongs to the technical field of organic molecular cage materials and corneal injury repair, and provides a preparation method and application of an organic molecular cage hydrogel, wherein the organic molecular cage hydrogel is prepared from an organic molecular cage containing a secondary amino group and oxidized gellan gum as precursors, and the preparation method is simple in operation and fast in reaction speed. The material has the effects of antibiosis, active oxygen scavenging, and promotion of corneal epithelial injury repair.

Description

Technical Field

[0001] This invention belongs to the field of organic molecular cage materials and corneal damage repair technology, specifically relating to the preparation method and application of organic molecular cage hydrogels. Background Technology

[0002] Commonly used hydrogel types in clinical practice include collagen-based hydrogels, hyaluronic acid (HA)-based hydrogels, chitosan-based hydrogels, fibrin gels, and polyethylene glycol (PEG)-based hydrogels. Corneal repair hydrogel materials with both antibacterial and antioxidant functions need to be achieved through loading active ingredients or material modification. Currently, the most promising types of gels in clinical practice and research include: (1) Chitosan-based hydrogels, which have good biocompatibility and low cost, used to treat infectious corneal ulcers; the disadvantage is that the hydrogel may irritate the ocular surface, requiring pH adjustment to neutral. (2) Collagen / hyaluronic acid gels loaded with antioxidants, with high bioactivity, used to treat chronic inflammatory damage; the disadvantage is that the antioxidants are easily degraded. (3) Polyphenol self-assembled gels, with all-natural ingredients and high safety; the disadvantage is that the color is dark, which may affect corneal transparency. (4) Metal nanoparticle composite gels, with long-lasting antibacterial / antioxidant effects, used to treat drug-resistant ocular infections; the disadvantage is that the potential toxicity of the nanoparticles needs strict evaluation.

[0003] Organic molecular cage materials are discrete structures with defined three-dimensional cavities and windows, formed by the self-assembly of organic molecules through covalent bonds or supramolecular interactions. Their cavity size and chemical environment can be precisely controlled for host-guest interactions or functional applications. Organic molecular cage materials obtained through the [4+6]imine condensation reaction of trimesin and an aliphatic vicinal diamine are composed of independent, discontinuous tetrahedral molecular cage molecules. Besides possessing the high specific surface area found in other microporous materials such as metal-organic frameworks (MOFs), covalent organic frameworks (COFs), and conjugated microporous polymers (CMPs), these materials are also soluble in a few organic solvents. Currently, these materials are widely used in gas adsorption, storage, and catalysis. However, due to the presence of only benzene rings, ethylene, and Schiff base imine structures in their molecular structure, these materials are difficult to modify through post-structural modification. To further investigate the functionalization reactions of these organic molecular cages, Cooper et al. [1,2]Organic molecular cages (CC1) prepared from trimesin and ethylenediamine were reduced to obtain organic molecular cages (RCC1) containing twelve secondary amino groups. The reduced product RCC1 can not only prepare MOF materials through coordination with metals, but also undergo post-modification reactions with acyl halides. Furthermore, patent document CN108148205A discloses the use of a mixed pore-forming system of methanol, 1,4-butanediol, and polyethylene glycol 10000 to perform a ring-opening polymerization reaction between the functional monomer RCC1 containing secondary amino groups and the epoxy crosslinking agent ethylene glycol diglycidyl ether under heating conditions to form an organic monolithic material. Patent document CN112999145 A discloses a method for preparing a triamcinolone-loaded hydrogel composite: 1) Organic molecular cage RCC1, glycidyl methacrylate, methanol, and polyethylene glycol 10000 are mixed uniformly to prepare a solution; 2) The prepared solution is reacted at 50-55℃ for 4-6 hours, and then a photoinitiator is added and mixed uniformly to continue the reaction; 3) The mixed solution from step 2) is irradiated with ultraviolet light for 6-8 minutes, and the white reaction product is washed with ethanol to obtain an unmodified monolithic material; 4) A 10% DMPA solution is added to an acryloyl-modified gelatin solution to prepare a mixed solution. The mixed solution is then passed through the unmodified monolithic material prepared in step 3), and then irradiated with ultraviolet light to obtain the matrix. To date, there are no reports of organic molecular cage RCC1 reacting directly with aldehyde-functionalized molecules to prepare organic molecular cage hydrogels.

[0004] [1]Culshaw, JL; Cheng, G.; Schmidtmann, M.; Hasell, T.; Liu, M.; Adams, DJ; Cooper, AIDodecaamide cages: organic 12-arm building blocks for supramolecular chemistry.J Am Chem Soc2013,135(27),10007-10010.DOI:10.1021 / ja403987j.

[0005] [2] Liu, M.; Little, MA; Jelfs, KE; Jones, JT; Schmidtmann, M.; Chong, SY; Hasell, T.; Cooper, AIAcid-and base-stable porous organic cages: shapepersistence and pH stability via post-synthetic "tying" of a flexible aminecage. J Am Chem Soc 2014,136(21),7583-7586.DOI:10.1021 / ja503223j. Summary of the Invention

[0006] This invention provides a method for preparing an organic molecular cage hydrogel that promotes corneal epithelial repair and its application. The organic molecular cage hydrogel is prepared through an aldehyde-amine condensation reaction, and the preparation method is simple and fast. This material has antibacterial properties, scavenges reactive oxygen species, and promotes the repair of corneal epithelial damage.

[0007] This invention is achieved through the following technical solution:

[0008] This invention provides a method for preparing an organic molecular cage hydrogel, which uses an organic molecular cage (RCC1) containing twelve secondary amino groups and oxidized gellan gum (OGG) as precursors, and obtains the organic molecular cage hydrogel through an aldehyde-amine condensation reaction.

[0009] The above-mentioned oxidized gellan gum was prepared by the following method: gellan gum was dispersed in water, and NaIO4 was added to oxidize a pair of vicinal diols on each repeating unit of gellan gum into aldehyde groups, thus maintaining the water solubility and skeletal stability of gellan gum; then excess ethylene glycol was added dropwise to terminate the reaction; the liquid was collected and dialyzed in distilled water, with a molecular weight cutoff of 12-14 kD for the dialysis membrane; the retained solution in the dialysis membrane was then freeze-dried under vacuum to obtain oxidized gellan gum.

[0010] The above-mentioned organic molecular cage containing twelve secondary amino groups was prepared by the following method: ethylenediamine and trimesin were reacted with a Schiff base to form an imine bond intermediate, and then the imine bond was selectively reduced to a secondary amino bond with NaBH4 to form a stable secondary amino precursor.

[0011] Specifically: Ethylenediamine (0.52 g) and trimethylolpropionate (TFB) (0.94 g) were dissolved in 213 mL and 287 mL of methanol, respectively. The ethylenediamine solution was placed in a 1 L round-bottom flask and cooled in an ice bath. The TFB solution was added dropwise through a dropping funnel with continuous stirring at 300 rpm for 12 h. Then, NaBH4 (0.76 g) was added and stirring continued for 12 h, followed by the addition of deionized water (5 mL) and stirring for another 12 h. Finally, the mixture was concentrated to dryness under vacuum. The resulting white powder was extracted three times with dichloromethane (50 mL), and the filtrates were combined and rotary evaporated, then dried under vacuum at 60 °C for 12 h to obtain a white solid, RCC1.

[0012] Furthermore, under heating conditions, the secondary amino group of RCC1 undergoes a condensation reaction with the aldehyde group in the oxidized gellan gum.

[0013] Furthermore, the heating temperature is 60–70°C.

[0014] Furthermore, the organic molecular cage is dissolved in deionized water, the oxidized gellan gum is uniformly dispersed in deionized water, and the two liquids are mixed and heated to react.

[0015] Furthermore, the concentration of the RCC1 solution is 2.5–15.0 mg / mL, and the mass fraction of the oxidized gellan gum (OGG) dispersion is 0.5%–1.5% (w / w).

[0016] Furthermore, the reaction is heated for 1–3 hours.

[0017] Another aspect of the present invention provides the application of the organic molecular cage hydrogel prepared by the present invention in the preparation of corneal epithelial repair products.

[0018] Furthermore, the organic molecular cage hydrogel serves as an antibacterial, reactive oxygen species scavenging, and corneal epithelial repair component.

[0019] Furthermore, the organic molecular cage hydrogel is used in the preparation of drugs that promote the proliferation of corneal epithelial cells.

[0020] Furthermore, the organic molecular cage hydrogel is used in the preparation of drugs that scavenge reactive oxygen species in corneal epithelial cells.

[0021] Furthermore, the organic molecular cage hydrogel is used in the preparation of drugs that reduce the incidence of corneal ulcers.

[0022] Furthermore, the organic molecular cage hydrogel is used in the preparation of drugs that reduce corneal epithelial cell inflammation.

[0023] Furthermore, the corneal epithelial repair product is a gel eye drop.

[0024] The beneficial effects of this invention are:

[0025] This invention utilizes an organic molecular cage with 12 secondary amino groups to react with oxidized polysaccharides to prepare a novel organic molecular cage hydrogel material. The preparation method is simple and the reaction is rapid. This material exhibits excellent antibacterial, reactive oxygen species scavenging, and corneal epithelial damage repair effects. Attached Figure Description

[0026] Figure 1 Visual and injectable images of organic molecular cage hydrogels.

[0027] Figure 2 This is a scanning electron microscope image of an organic molecular cage hydrogel.

[0028] Figure 3 A shows the infrared spectra of RCC1, OGG, and organic molecular cage hydrogels; Figure 3 B is the XPS elemental analysis diagram of the organic molecular cage hydrogel.

[0029] Figure 4 The catalytic activity of organic molecular cage hydrogels.

[0030] Figure 5 Photographs and statistical graphs of the antifungal and antibacterial activity of organic molecular cage hydrogels.

[0031] Figure 6 Photographs of corneal fluorescein-stained corneas and statistical charts of healing rates in the hydrogel group and control group at 0h, 12h, 24h, 36h and 48h after corneal epithelial injury repair experiment.

[0032] Figure 7 This image shows corneal nerve staining photographs and nerve density statistics of the hydrogel group and the control group 7 days after corneal epithelial injury in a corneal epithelial injury repair experiment. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0035] Example 1

[0036] (1) Add 7.5 mg of RCC1, an organic molecular cage functional monomer containing a secondary amino group, to a centrifuge tube;

[0037] (2) Add 500.0 μL of deionized water to the centrifuge tube from step 1), and mix by inverting to dissolve it completely.

[0038] (3) Add 10.0 mg of oxidized gellan gum to another centrifuge tube;

[0039] (4) Add 500.0 μL of deionized water to the centrifuge tube from step (3) and mix by sonication.

[0040] (5) Transfer the liquid in the centrifuge tube in step (2) to the centrifuge tube in step (4) and mix it by ultrasonication;

[0041] (6) Place the centrifuge tube containing the mixed solution obtained in step (5) in a 65°C water bath for 2 hours to obtain the organic molecular cage hydrogel.

[0042] Example 2

[0043] 1) Add 5.0 mg of RCC1, an organic molecular cage functional monomer containing a secondary amino group, to a centrifuge tube;

[0044] 2) Add 500.0 μL of deionized water to the centrifuge tube from step 1), invert and mix well to dissolve completely;

[0045] 3) Add 10.0 mg of oxidized gellan gum (OGG) to another centrifuge tube;

[0046] 4) Add 500.0 μL of deionized water to the centrifuge tube from step 3) and mix by sonication;

[0047] 5) Transfer the liquid in the centrifuge tube from step 2) to the centrifuge tube from step 4), and mix thoroughly by ultrasonication;

[0048] 6) Place the centrifuge tube containing the mixed solution obtained in step 5) in a 60°C water bath for 3 hours to obtain the organic molecular cage hydrogel.

[0049] Example 3

[0050] 1) Add 10.0 mg of RCC1, an organic molecular cage functional monomer containing a secondary amino group, to a centrifuge tube;

[0051] 2) Add 500.0 μL of deionized water to the centrifuge tube from step 1), invert and mix well to dissolve completely;

[0052] 3) Add 10.0 mg of oxidized gellan gum (OGG) to another centrifuge tube;

[0053] 4) Add 500.0 μL of deionized water to the centrifuge tube from step 3) and mix by sonication;

[0054] 5) Transfer the liquid in the centrifuge tube from step 2) to the centrifuge tube from step 4), and mix thoroughly by ultrasonication;

[0055] 6) Place the centrifuge tube containing the mixed solution obtained in step 5) in a 70°C water bath for 1 hour to obtain the organic molecular cage hydrogel.

[0056] Example 4

[0057] The prepared organic molecular cage hydrogel was observed, verifying the successful preparation of the target product and demonstrating its injectability.

[0058] The organic molecular cage hydrogel material prepared in Example 1 was observed macroscopically and microscopically, and its injectability was verified by injection with a syringe. Then, its morphology was observed under an electron microscope, and infrared spectroscopy analysis was performed to verify that the target product was obtained. Figure 1 The images show a direct view of the organic molecular cage hydrogel (within the dashed box) and an injectable image. The hydrogel is a transparent, pale yellow, flowable colloid. Figure 2 The image shows a scanning electron microscope (SEM) image of an organic molecular cage hydrogel, revealing a typical macroporous structure. Figure 3 A represents the infrared spectral characterization of RCC1, OGG, and organic molecular cage hydrogels, at 2894 and 2822 cm⁻¹. -1 The methylene stretching vibration peak of RCC1 can be observed at 2949 cm⁻¹. -1 The peak at 2921 cm⁻¹ corresponds to the methylene stretching vibration of OGG, while the peaks at 2921 and 2851 cm⁻¹ are... -1 At this point, mixed vibrational signals of organic molecular cage hydrogels appeared. Of particular note is the 1604 cm⁻¹... -1 (In-plane bending vibration of secondary amine in RCC1) and 1728 cm -1 The weakening peak intensity at (stretching vibration of aldehyde group in OGG) indicates that the secondary amine reacted with the aldehyde group during the formation of the organic molecular cage hydrogel. Figure 3 B is the XPS elemental analysis diagram of the organic molecular cage hydrogel, which shows the presence of both N from RCC1 and O from OGG, proving that RCC1 and oxidized gellan gel underwent a condensation reaction, and the organic molecular cage hydrogel was successfully synthesized.

[0059] Example 5

[0060] The organic molecular cage hydrogel material prepared in Example 1 was used in experiments on antibacterial activity, scavenging of reactive oxygen species, and promotion of corneal epithelial damage repair.

[0061] Antibacterial test: Prepare 2ml centrifuge tubes, adding 900.0μL of PBS or organic molecular cage hydrogel to each tube. Collect Fusarium spores of Solanum rot and adjust the concentration to 1×10⁻⁶. 8Add 100.0 μL of fungal suspension to each centrifuge tube at CFU / mL. After incubating the sample at 28°C for 24 h, dilute the sample 10,000 times, take 20.0 μL and inoculate it on potato dextrose agar medium, then continue incubation at 28°C for another 48 h to record the number of colonies formed, take pictures and calculate the inhibition rate.

[0062] Reactive oxygen species (ROS) scavenging assays: The ·OH scavenging capacity of the organic molecular cage hydrogel was determined using a hydroxyl radical assay kit (Nanjing Jiancheng, China). The superoxide anion scavenging capacity of the organic molecular cage hydrogel was determined using a superoxide anion assay kit (Nanjing Jiancheng, China). The total antioxidant capacity (T-AOC) of the organic molecular cage hydrogel was determined using an ABTS microplate method (Beijing Regen Biotechnology Co., Ltd.).

[0063] Corneal epithelial injury repair experiment: Mice (n=6) were randomly divided into two groups: a control group and an organic molecular cage hydrogel group, with 3 mice in each group. Mice were anesthetized by intraperitoneal injection of 0.06% sodium pentobarbital. A 2.5 mm trephine was used to gently press and mark the cornea at its center. Subsequently, [the procedure was performed using...]. II. A corneal epithelial scraper removed 2.5 mm of the central corneal epithelium. The control and experimental groups were treated with PBS and hydrogel, respectively, four times daily for 7 days. Corneal fluorescein staining and photography were performed at fixed time points (0, 12, 24, 36, and 48 hours). Mice were euthanized on day 7. Corneas were collected and subjected to corneal patch immunofluorescence (β-III tubulin) staining. Images were captured using a laser scanning confocal microscope (LSM 800, Zeiss, Germany) to assess the effects of different drugs on corneal nerve repair. ImageJ software was used to analyze the corneal epithelial repair area and corneal nerve density.

[0064] Figure 4 The catalytic activity of the organic molecular cage hydrogel indicates that it has the functions of scavenging superoxide anions and hydroxyl radicals, as well as antioxidant functions.

[0065] Figure 5 It exhibits antifungal and antibacterial activity against organic molecular cage hydrogels. Figure 6 Organic molecular cage hydrogel promotes corneal epithelial damage repair; at 36h and 48h after corneal epithelial injury, the corneal epithelial damage healing rate in the organic molecular cage hydrogel eye application group was significantly higher than that in the control group.

[0066] Figure 7 Organic molecular cage hydrogel promotes corneal nerve repair function; 7 days after corneal epithelial injury, the corneal epithelial nerve density in mice treated with organic molecular cage hydrogel was significantly higher than that in the control group.

[0067] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing an organic molecular cage hydrogel, characterized in that, Organic molecular cage hydrogels were prepared by using organic molecular cage RCC1 containing twelve secondary amino groups and oxidized gellan gum as precursors and then undergoing aldehyde-amine condensation reaction. The mass ratio of the organic molecular cage RCC1 containing twelve secondary amino groups to the oxidized gellan gum is 1:1 to 3. The organic molecular cage RCC1 containing twelve secondary amino groups is prepared by the following method: ethylenediamine and pyromellitic aldehyde are reacted with a Schiff base to form an imine bond intermediate, and then the imine bond is selectively reduced to a secondary amino bond with NaBH4 to form a stable secondary amine precursor.

2. The preparation method according to claim 1, characterized in that, The oxidized gellan gum was prepared by the following method: gellan gum was dispersed in water, and NaIO4 was added to oxidize a pair of vicinal diols on each repeating unit of the gellan gum into aldehyde groups; then excess ethylene glycol was added dropwise to terminate the reaction; the liquid was collected and dialyzed in distilled water, with the molecular weight cutoff of the dialysis membrane being 12-14 kD; the retained liquid in the dialysis membrane was freeze-dried under vacuum to obtain the oxidized gellan gum.

3. The preparation method according to claim 1, characterized in that, The organic molecular cage RCC1 containing twelve secondary amino groups was dissolved in deionized water, and the oxidized gellan gum was uniformly dispersed in deionized water. The two liquids were mixed and heated to react at a temperature of 60-70°C for 1-3 hours.

4. The preparation method according to claim 3, characterized in that, The concentration of the organic molecular cage aqueous solution is 2.5 ~ 15.0 mg / mL, and the mass fraction of the oxidized gellan gum dispersion is 0.5% ~ 1.5%.

5. The application of the organic molecular cage hydrogel prepared according to any one of claims 1 to 4 in the preparation of corneal epithelial repair products, characterized in that, The organic molecular cage hydrogel serves as an antibacterial, reactive oxygen species scavenging, and corneal epithelial repair component.

6. The application according to claim 5, characterized in that, Application of the organic molecular cage hydrogel in the preparation of drugs that promote the proliferation of corneal epithelial cells.

7. The application according to claim 5, characterized in that, The application of the organic molecular cage hydrogel in the preparation of drugs that scavenge reactive oxygen species in corneal epithelial cells.

8. The application according to claim 5, characterized in that, Application of the organic molecular cage hydrogel in the preparation of antibacterial drugs.

9. The application according to claim 5, characterized in that, The corneal epithelial repair product is an ophthalmic gel eye drop.

Images