5-fluorouracil carbon dot freeze-dried film as well as preparation method and application thereof

Abstract

The invention provides a 5-fluorouracil carbon dot freeze-dried film as well as a preparation method and application thereof, and relates to the technical field of glaucoma surgical implant materials. The preparation method of the 5-fluorouracil carbon dot freeze-dried film comprises the following steps: dissolving saccharides, carbon-carbon double bond unsaturated molecules and 5-fluorouracil in water to obtain a mixed raw material; putting the mixed raw material into a hydrothermal reaction kettle, and carrying out hydrothermal carbonization reaction to obtain a 5-fluorouracil carbon dot solution; the 5-fluorouracil carbon dot solution is subjected to freeze drying, and the 5-fluorouracil carbon dot freeze-dried film is obtained. The 5-fluorouracil carbon dot freeze-dried film is good in biocompatibility due to addition of the saccharides, slow release of the 5-fluorouracil anti-metabolism medicine can be achieved, the medicine toxicity effect caused by early-stage sudden release is reduced, the long-term anti-fibrosis effect is achieved, and therefore the scarring of a filtration area after a glaucoma drainage valve operation can be reduced, and the success rate of the operation is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of glaucoma surgery implant materials, and in particular to a 5-fluorouracil carbon dot freeze-dried film and a preparation method and application thereof. Background Art

[0002] Refractory glaucoma refers to a type of patient who still has difficulty controlling intraocular pressure within the normal range after repeated anti-glaucoma surgeries and combined use of anti-metabolites and ocular hypotensive drugs. The effective treatment of this type of glaucoma is a difficult problem and hot topic that needs to be solved urgently. The Ahmed glaucoma valve (AGV) has opened up a new path for the treatment of refractory glaucoma. However, as an implant, the AGV will inevitably cause a characteristic wound healing response, leading to scarring of the subconjunctival tissue of the drainage valve body in some patients, inhibiting the outflow of aqueous humor, causing the intraocular pressure to rise again, and bringing the risk of surgical failure. Therefore, it is crucial to prevent scarring of the anti-filtration area. 5-Fluorouracil (5-FU) is an antimetabolite drug commonly used during and after glaucoma drainage valve surgery. 5-FU is clinically used to reduce subconjunctival tissue scarring. However, the instantaneous high-concentration drug release caused by intraoperative and postoperative use is prone to tissue toxicity and has a short duration of action. Therefore, multiple invasive procedures are required for drug administration. Multiple injections are prone to cause serious complications such as infection, bleb leakage, and endophthalmitis. Summary of the invention

[0003] The problem solved by the present invention is how to solve the problem of tissue toxicity and short effect caused by instantaneous high concentration drug release when 5-fluorouracil is used as an anti-metabolite drug during and after glaucoma drainage valve surgery.

[0004] In order to solve the above problems, the present invention provides a 5-fluorouracil carbon dot freeze-dried film and a preparation method and application thereof.

[0005] In a first aspect, the present invention provides a method for preparing a 5-fluorouracil carbon dot freeze-dried film, comprising the following steps:

[0006] S1: dissolving sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil in water to obtain a mixed raw material;

[0007] S2: placing the mixed raw materials in a hydrothermal reactor to perform a hydrothermal carbonization reaction to obtain a 5-fluorouracil carbon dot solution;

[0008] S3: freeze-drying the 5-fluorouracil carbon dot solution to obtain a 5-fluorouracil carbon dot freeze-dried film.

[0009] Optionally, the sugar is chitosan, fructose, sucrose, mannitol or glucose.

[0010] Optionally, the carbon-carbon double bond unsaturated molecule is acrylamide or olefin.

[0011] Optionally, step S2 specifically includes: placing the mixed raw materials in a hydrothermal reactor to perform a hydrothermal carbonization reaction, cooling after the reaction is completed, removing the precipitate by centrifugation, filtering the filtrate again and dialyzing to obtain a 5-fluorouracil carbon dot solution.

[0012] Optionally, during the hydrothermal carbonization reaction in step S2, the temperature is controlled at 100-300 degrees Celsius and the reaction time is 1-10 hours.

[0013] In a second aspect, the present invention provides a 5-fluorouracil carbon dot freeze-dried film, which is prepared by the preparation method of the 5-fluorouracil carbon dot freeze-dried film as described above.

[0014] Optionally, the 5-fluorouracil carbon dot freeze-dried film is composed of sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil.

[0015] Optionally, the sugar is chitosan, fructose, sucrose, mannitol or glucose.

[0016] Optionally, the carbon-carbon double bond unsaturated molecule is acrylamide or olefin.

[0017] In a third aspect, the present invention provides an application of the 5-fluorouracil carbon dot freeze-dried film as described above, wherein the 5-fluorouracil carbon dot freeze-dried film is applied to the surface of a glaucoma drainage valve to prepare a drainage valve loaded with the 5-fluorouracil carbon dot freeze-dried film.

[0018] The beneficial effects of the 5-fluorouracil carbon dot freeze-dried film and the preparation method and application thereof of the present invention are as follows: carbon-carbon double bond unsaturated molecules are used as fluorescent precursors of carbon dots, polymerized into polymerized carbon-carbon double bond chains, the polymerized carbon-carbon double bond chains are transferred to the surface of sugar molecules and 5-fluorouracil, the polymerized carbon-carbon double bond chains are cross-linked with sugar molecules and 5-fluorouracil for dehydration and hydrothermal carbonization reaction, thereby obtaining a 5-fluorouracil carbon dot solution, and freeze-dried to obtain a 5-fluorouracil carbon dot freeze-dried film. The addition of sugars makes the 5-fluorouracil carbon dot freeze-dried film have good biocompatibility, can achieve slow release of 5-fluorouracil anti-metabolite drugs, reduce drug toxicity caused by early burst release, and achieve long-term anti-fibrosis effects, thereby reducing scarring of the filtration area after glaucoma drainage valve surgery and improving the success rate of surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the in vitro release curve of 5-FU-CDs in PBS of Example 1;

[0020] Figure 2 It is a bar graph of cell viability of 5-FU-CDs solutions with different concentrations in Example 1;

[0021] Figure 3 Live / dead cell staining images of the four groups of solution cultured cells in Example 1 after 1, 3, and 5 days;

[0022] Figure 4 The bar graphs are of the cell survival numbers of the four groups of solution cultured cells in Example 1 after 1, 3, and 5 days;

[0023] Figure 5 This is a schematic diagram of the 5-FU-CDs flake carbon dot solid coated on the surface of the glaucoma drainage valve of this Example 1;

[0024] Figure 6 The graphs of intraocular pressure before implantation and on the 10th, 20th and 30th days after implantation of the three groups of drainage devices in Example 1 are shown;

[0025] Figure 7 Schematic diagram of the thickness of the top and bottom collagen layers of scar tissue in the filtration area of ​​the eye after three sets of drainage devices are implanted in Example 1;

[0026] Figure 8 This is a bar graph of the top and bottom collagen layer thickness values ​​of the scar tissue in the filtration area of ​​the three groups of drainage devices implanted in the eyes of Example 1. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Although certain embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be interpreted as being limited to the embodiments described herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present invention. It should be understood that the drawings and embodiments of the present invention are only for exemplary purposes and are not intended to limit the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as those commonly understood by those skilled in the art to which this application belongs; the terms used in the specification of this application are only for the purpose of describing specific implementation methods and are not intended to limit this application;

[0029] The term "including" and its variations used in this article are open inclusions, that is, "including but not limited to"; the term "based on" is "based at least in part on"; the term "one embodiment" means "at least one embodiment"; the term "another embodiment" means "at least one other embodiment"; the term "some embodiments" means "at least some embodiments"; the term "optionally" means "optional embodiments". The relevant definitions of other terms will be given in the following description. It should be noted that the concepts of "first" and "second" mentioned in the present invention are used to distinguish different objects, rather than to describe a specific order or a primary and secondary relationship. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, the meaning of "multiple" is two or more.

[0030] In view of the problems existing in the above-mentioned related technologies, this embodiment provides a 5-fluorouracil carbon dot freeze-dried film and a preparation method and application thereof.

[0031] The present invention provides a method for preparing a 5-fluorouracil carbon dot freeze-dried film, comprising the following steps:

[0032] S1: dissolving sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil in water to obtain a mixed raw material;

[0033] S2: placing the mixed raw materials in a hydrothermal reactor to perform a hydrothermal carbonization reaction to obtain a 5-fluorouracil carbon dot solution;

[0034] S3: freeze-drying the 5-fluorouracil carbon dot solution to obtain a 5-fluorouracil carbon dot freeze-dried film.

[0035] In this embodiment, carbon-carbon double bond unsaturated molecules are used as fluorescent precursors of carbon dots, polymerized into polymerized carbon-carbon double bond chains, which are transferred to the surface of sugar molecules and 5-fluorouracil. The polymerized carbon-carbon double bond chains are cross-linked with sugar molecules and 5-fluorouracil, dehydrated and subjected to hydrothermal carbonization reaction to obtain a 5-fluorouracil carbon dot solution, which is freeze-dried to obtain a 5-fluorouracil carbon dot freeze-dried film (5-FU-CDs). The addition of sugars makes the 5-fluorouracil carbon dot freeze-dried film have good biocompatibility, which can achieve the slow release of 5-fluorouracil anti-metabolite drugs, reduce the drug toxicity caused by early burst release, and achieve long-term anti-fibrosis effects. Therefore, it can reduce scarring of the filtration area after glaucoma drainage valve surgery and improve the success rate of surgery.

[0036] Specifically, the total concentration of sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil in the mixed raw material is 0.5wt%-5wt%.

[0037] Optionally, the sugar is chitosan, fructose, sucrose, mannitol or glucose.

[0038] In this optional embodiment, sugars can improve biocompatibility.

[0039] Optionally, the carbon-carbon double bond unsaturated molecule is acrylamide or olefin.

[0040] Specifically, when the carbon-carbon double bond unsaturated molecule used as the fluorescent precursor is acrylamide, the acrylamide monomer is polymerized into a polyacrylamide chain, and the polyacrylamide chain is transferred to chitosan and 5-fluorouracil (5-FU). Then, the polyacrylamide chain is cross-linked with the 5-FU and chitosan segments, dehydrated, and hydrothermally carbonized.

[0041] Optionally, step S2 specifically includes: placing the mixed raw materials in a hydrothermal reactor to perform a hydrothermal carbonization reaction, cooling after the reaction is completed, removing the precipitate by centrifugation, filtering the filtrate again and dialyzing to obtain a 5-fluorouracil carbon dot solution.

[0042] Specifically, after the hydrothermal carbonization reaction is completed, an unpurified carbon dot solution is obtained, the precipitate is removed by centrifugation, and the filtrate is filtered again and dialyzed to obtain a clear 5-fluorouracil carbon dot solution.

[0043] Optionally, during the hydrothermal carbonization reaction in step S2, the temperature is controlled at 100-300 degrees Celsius and the reaction time is 1-10 hours.

[0044] The embodiment of the present invention provides a 5-fluorouracil carbon dot freeze-dried film, which is prepared by the preparation method of the 5-fluorouracil carbon dot freeze-dried film as described above.

[0045] In this embodiment, the 5-fluorouracil anti-metabolite drug is slowly released, with a cumulative release rate of 20%-40% on the 5th day, 40%-60% on the 10th day, and 60%-80% on the 15th day, thereby reducing drug toxicity caused by early burst release and achieving long-term anti-fibrosis effects. Therefore, it is possible to reduce scarring of the filtration area after glaucoma drainage valve surgery and improve the success rate of the operation.

[0046] Optionally, the 5-fluorouracil carbon dot freeze-dried film is composed of sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil.

[0047] Optionally, the sugar is chitosan, fructose, sucrose, mannitol or glucose.

[0048] In this optional embodiment, sugars can improve biocompatibility.

[0049] Optionally, the carbon-carbon double bond unsaturated molecule is acrylamide or olefin.

[0050] Specifically, when the carbon-carbon double bond unsaturated molecule used as the fluorescent precursor is acrylamide, the acrylamide monomer is polymerized into a polyacrylamide chain, and the polyacrylamide chain is transferred to chitosan and 5-fluorouracil (5-FU). Then, the polyacrylamide chain is cross-linked with the 5-FU and chitosan segments, dehydrated, and hydrothermally carbonized.

[0051] An embodiment of the present invention provides an application of the above-mentioned 5-fluorouracil carbon dot freeze-dried film, in which the 5-fluorouracil carbon dot freeze-dried film is applied to the surface of a glaucoma drainage valve to prepare a drainage valve loaded with the 5-fluorouracil carbon dot freeze-dried film.

[0052] The present invention is further described below in conjunction with specific embodiments.

[0053] Example 1

[0054] 1. Preparation of 5-fluorouracil carbon dots freeze-dried film (5-FU-CDs)

[0055] Weigh sugars, carbon-carbon double bond unsaturated small molecules, and 5-FU respectively and dissolve them in deionized water. After fully dissolving, put them into a hydrothermal reactor for hydrothermal polymerization and carbonization reaction. Take them out and wait for complete cooling to obtain an unpurified carbon dot solution. Put the carbon dot solution into a centrifuge tube, centrifuge to remove the precipitate, filter and dialyze to obtain a clear 5-FU-CDs solution. Take the 5-FU-CDs solution after dialysis in a culture dish and freeze-dry it to finally obtain a 5-FU-CDs freeze-dried film. The thickness of the 5-FU-CDs freeze-dried film can be adjusted according to the shape and size of the container.

[0056] 2. Preparation of drainage valve loaded with 5-fluorouracil carbon dot freeze-dried film

[0057] The surface of the drainage valve was modified, and the 5-FU-CDs freeze-dried film was applied to the surface of the glaucoma drainage valve to prepare a drainage valve loaded with 5-FU-CDs freeze-dried film.

[0058] 3. In vitro drug release experiment of 5-FU-CDs

[0059] The in vitro release performance of 5-FU-CDs prepared in Example 1 was determined by the dialysis bag method. The specific steps are as follows: 2 mL of 5-FU-CDs solution was measured and placed in a dialysis bag with a molecular weight cutoff of 500, placed in a release medium of 50 mL PBS, and shaken at 37°C and 100 rpm. After the release started, 1 mL of sample was taken each day, and the same volume of PBS was added at the same time. Figure 1As shown, the in vitro release curve of 5-FU-CDs in PBS shows that on the 5th day, the cumulative release rate was 20%-40%, on the 10th day, the cumulative release rate was 40%-60%, on the 15th day, the cumulative release rate was 60%-80%, and at 14 days, the release amount of 5-FU was about 62.56%, indicating that 5-FU can be slowly released in PBS, reducing the drug toxicity caused by early burst release, and achieving long-term anti-fibrotic effects.

[0060] IV. Cytotoxicity Experiment of 5-FU-CDs

[0061] The CCK-8 method was used to test the cytotoxicity of CDs at different concentrations. The cells were divided into 8 groups, and each group was set with 0, 12, 25, 50, 100, 200, 400, and 800 μg / mL of 5-FU-CDs solution. 90 μL of culture medium was added, and then 10 μL of 5-FU-CDs solution was added. After incubation for 1d and 3d, the culture medium was removed from each well, and 100 μL of culture medium containing 10% CCK-8 was added after washing with PBS. After incubation at 37°C, the absorbance was measured at 450nm on an enzyme reader to calculate the cell survival rate. Figure 2 As shown, there was no significant difference in the cell survival rate of 5-FU-CDs solutions with different concentrations, and the cell survival rate was all above 90%.

[0062] 5. Inhibition of cell proliferation by 5-FU-CDs

[0063] The inhibitory effect of 5-FU-CDs on cell proliferation in Example 1 was detected using CCK-8 method and live / dead staining. The cells were divided into four groups: 5-FU-CDs group, CDs group, silicone sheet group (Silicon), and blank group (Control). 90 μL of culture medium was added, and then 10 μL of 5-FU-CDs solution, CDs solution and silicone sheet were added respectively. CCK-8 detection and live / dead cell staining were performed after 1, 3, and 5 days of culture. Figure 3 As shown, live / dead staining showed that the number of surviving cells in the 5-FU-CDs group was significantly reduced after 5 days of culture. Figure 4 As shown, Figure 3 and Figure 4 As shown in the results of CCK-8 detection, the cell activity of RTFs in the 5-FU-CDs group was significantly reduced (P<0.01).

[0064] VI. Intraocular pressure at different times after glaucoma drainage valve implantation in three groups

[0065] The 5-FU-CDs carbon dot sheet solid in Example 1 is coated on the surface of the glaucoma drainage valve. Figure 5The experimental animals were healthy New Zealand white rabbits weighing about 2.5-3kg, a total of 18 rabbits, of either sex, and were randomly divided into the following groups: Group A: (6 rabbits) implanted with drainage valve AGV; Group B: (6 rabbits) implanted with drainage valve AGV covered with carbon dots CDs; Group C: (6 rabbits) implanted with drainage valve AGV covered with 5-FU-CDs. The three groups of drainage devices were implanted into the eyes, and the intraocular pressure was measured using a Tono-pen tonometer before implantation and on the 10th, 20th, and 30th days after surgery. Figure 6 As shown, the intraocular pressure of the drainage valve AGV implanted with 5-FU-CDs was the lowest, followed by the drainage valve AGV implanted with carbon dot CDs. The intraocular pressure of the drainage valve AGV implanted with carbon dot CDs was the highest.

[0066] VII. Determination of scar thickness in the filtration zone after glaucoma drainage valve implantation in three groups

[0067] One month after surgery, the animals in step six above were killed, and the eyeballs were removed and fixed in 4% paraformaldehyde for 1 week. The tissues were trimmed to retain the conjunctiva in the surgical area and the scleral tissue at the corresponding position of the filtration bleb. The filtration bleb was cut open from the middle of the drainage tray along the direction of the drainage tube, and the drainage valve was removed. The scar tissue in the filtration area was dehydrated, paraffin-embedded, sliced, HE-stained, and Masson-stained. Image J software was used to measure the thickness of the collagen layer on the top and bottom walls of the filtration bleb after Masson staining. Figure 7 As shown in the figure, after implantation of the drainage valve AGV covered with 5-FU-CDs, the thickness of the top and bottom collagen layers of the scar tissue was significantly reduced, which had a good anti-fibrosis effect. Figure 8 shown.

[0068] Although the present invention is disclosed as above, the protection scope of the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for preparing a 5-fluorouracil carbon dot freeze-dried film, characterized in that: The following steps are involved: S1: dissolving sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil in water to obtain a mixed raw material; S2: placing the mixed raw materials in a hydrothermal reactor to perform a hydrothermal carbonization reaction to obtain a 5-fluorouracil carbon dot solution; S3: freeze-drying the 5-fluorouracil carbon dot solution to obtain a 5-fluorouracil carbon dot freeze-dried film.

2. The method for preparing the 5-fluorouracil carbon dot freeze-dried film according to claim 1, characterized in that: The sugar is chitosan, fructose, sucrose, mannitol or glucose.

3. The method for preparing the 5-fluorouracil carbon dot freeze-dried film according to claim 1, characterized in that: The carbon-carbon double bond unsaturated molecule is acrylamide or olefin.

4. The method for preparing the 5-fluorouracil carbon dot freeze-dried film according to claim 1, characterized in that: The step S2 specifically comprises: placing the mixed raw material in a hydrothermal reactor to perform a hydrothermal carbonization reaction, cooling after the reaction is completed, removing the precipitate by centrifugation, filtering the filtrate again and dialyzing to obtain a 5-fluorouracil carbon dot solution.

5. The method for preparing the 5-fluorouracil carbon dot freeze-dried film according to claim 1, characterized in that: During the hydrothermal carbonization reaction in step S2, the temperature is controlled at 100-300 degrees Celsius and the reaction time is 1-10 hours.

6. A 5-fluorouracil carbon dot freeze-dried film, characterized in that: The 5-fluorouracil carbon dot freeze-dried film is prepared by the preparation method of any one of claims 1 to 5.

7. The 5-fluorouracil carbon dot freeze-dried film according to claim 6, characterized in that: The 5-fluorouracil carbon dot freeze-dried film is composed of sugars, carbon-carbon double bond unsaturated molecules and 5-fluorouracil.

8. The 5-fluorouracil carbon dot freeze-dried film according to claim 7, characterized in that: The sugar is chitosan, fructose, sucrose, mannitol or glucose.

9. The 5-fluorouracil carbon dot freeze-dried film according to claim 7, characterized in that: The carbon-carbon double bond unsaturated molecule is acrylamide or olefin.

10. An application of the 5-fluorouracil carbon dot freeze-dried film according to any one of claims 6 to 9, characterized in that: The 5-fluorouracil carbon dot freeze-dried film is applied to the surface of a glaucoma drainage valve to prepare a drainage valve loaded with the 5-fluorouracil carbon dot freeze-dried film.

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