Ergothioneine-entrapped fullerene hollow nanosphere aggregate with pH responsiveness and preparation method and application thereof
By preparing fullerene hollow nanosphere aggregates containing ergothionine with pH responsiveness, the problem of difficulty in controlling drug release and low drug loading rate in the prior art is solved, and the effectiveness of the drug is achieved Controlled release and high drug loading rate have significant anti-inflammatory effects.
Patent Information
- Application Number
- CN202411599150.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-11
- Publication Date
- 2025-02-14
AI Technical Summary
The existing fullerene nanodrug carriers are difficult to effectively control drug release, and the drug loading rate is low, which limits its application in the field of biomedicine.
By preparing a fullerene hollow nanosphere aggregate with pH-responsive ergothionine, ergothionine is encapsulated in fullerene hollow nanospheres by a simple method, and the release of drugs is controlled through pH response.
It realizes effective controlled release of drugs, improves drug loading rate, enhances the bioavailability and treatment efficiency of drugs, and reduces side effects and has anti-inflammatory effects.
Smart Images
Figure CN119424682A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of nanomaterials, and in particular to a pH-responsive ergothioneine-encapsulated fullerene hollow nanosphere aggregate, a preparation method and application thereof. Background Art
[0002] In the health field, nanotechnology research has made significant progress, especially in the development of drug delivery systems and diagnostic tools. Nanocarrier drug delivery systems (NDDS) can be prepared from a variety of materials (such as lipids, polymers, proteins, metals and inorganic materials, etc.), and achieve transport across biological membrane barriers through a variety of mechanisms. Nanocarriers improve drug solubility and stability, synergistically deliver multiple drugs, improve bioavailability, mediate sustained or controlled drug release, improve treatment efficiency, and reduce side effects.
[0003] Fullerene C 60 It is a hollow molecule composed entirely of carbon. It is a unique allotrope among many carbon nanomaterials. Based on its special p-π conjugated structure, fullerene can adsorb free radicals through carbon-carbon double bonds, thus having good antioxidant and cell protection capabilities, and can be used to load drugs and treat inflammation. However, the hydrophobic nature of fullerene limits its application in the biomedical field because it is only soluble in a few organic solvents, such as toluene and carbon tetrachloride. In order to overcome these limitations, researchers have developed a variety of modification methods, including amination, carboxylation and hydroxylation, to improve the water solubility of fullerene. Based on the easy aggregation of fullerene, researchers can simply synthesize fullerene aggregates with different sizes, morphologies and surface reactivity through the liquid-liquid interface precipitation (LLIP) method. This method provides new possibilities for the application of fullerene in the field of nanocarriers and expands its application prospects in the biomedical field. However, it is necessary to improve the drug loading rate of fullerene aggregates and control drug release to improve treatment efficiency and reduce side effects.
[0004] Therefore, it remains a challenge to develop a simple and effective method to prepare fullerene nanoparticle drug carriers that can responsively control the release of drugs while maintaining a high drug loading rate. Solving this challenge is crucial to improving the drug loading effect of fullerene nanoparticle carriers and is also one of the key issues in promoting the further development of nanotechnology in the health field. Summary of the invention
[0005] In view of the above problems, an object of the present invention is to provide a method for preparing a pH-responsive fullerene hollow nanosphere aggregate containing ergothioneine. The drug ergothioneine is encapsulated in the fullerene hollow nanosphere aggregate by simple means, and the drug can be released by pH response control.
[0006] The second object of the present invention is to provide a pH-responsive fullerene hollow nanosphere aggregate encapsulating ergothioneine, which can control the release of drugs and scavenge free radicals.
[0007] The third object of the present invention is to provide an application of a pH-responsive fullerene hollow nanosphere aggregate loaded with ergothioneine. The pH-responsive fullerene hollow nanosphere aggregate loaded with ergothioneine can control the release of drugs, remove free radicals, protect cells, and thus play an anti-inflammatory role, and can be applied to anti-inflammatory aspects.
[0008] The first technical solution adopted by the present invention is: a method for preparing a pH-responsive ergothioneine-loaded fullerene hollow nanosphere aggregate, comprising the following steps:
[0009] Step 1: preparing a fullerene organic solution and an ergothioneine mixed solution respectively;
[0010] Step 2: adding the thioneine mixed solution to the fullerene organic solution, shaking and keeping it still until the end of the reaction to obtain a turbid solution;
[0011] Step 3: post-treating the turbid liquid to obtain pH-responsive fullerene hollow nanosphere aggregates loaded with ergothioneine.
[0012] Preferably, the volume ratio of the thioneine mixed solution to the fullerene organic solution is 3:7.5-8.5.
[0013] Preferably, the method for preparing the fullerene organic solution is: dispersing fullerene in an organic solvent to obtain a fullerene organic solution;
[0014] The organic solvent is a toluene solution, and the mass volume ratio of the fullerene to the toluene solution is 1:1.9-2.1.
[0015] Preferably, the preparation method of the thioneine mixed solution is: dissolving thioneine in deionized water to obtain an thioneine aqueous solution, sequentially adding ethylenediamine and isopropanol to the thioneine aqueous solution, and shaking well to obtain the thioneine mixed solution.
[0016] Preferably, the concentration of ergothioneine in the ergothioneine aqueous solution is 5-40 mg / mL.
[0017] Preferably, the volume ratio of the thioneine aqueous solution, isopropyl alcohol and ethylenediamine is 1:19:2-10.
[0018] Preferably, the reaction endpoint is the presence of a brown precipitate in the turbid liquid.
[0019] Preferably, the post-treatment includes: separating the turbid liquid from the solid-liquid, collecting the solid, washing, and drying.
[0020] The second technical solution adopted by the present invention is: a pH-responsive fullerene hollow nanosphere aggregate encapsulating ergothioneine.
[0021] The third technical solution adopted by the present invention is: application of pH-responsive fullerene hollow nanosphere aggregates encapsulating ergothioneine in the field of nano drug carriers and anti-inflammatory fields.
[0022] Beneficial effects of the above technical solution:
[0023] (1) The preparation method provided by the present invention can encapsulate the drug ergothioneine in fullerene hollow nanosphere aggregates by simple means, and can control the release of the drug by pH response.
[0024] (2) The pH-responsive fullerene hollow nanosphere aggregates loaded with ergothioneine provided by the present invention can control the release of drugs, scavenge free radicals, protect cells and thus play an anti-inflammatory role, and can be used in anti-inflammatory aspects. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the infrared spectrum (FT-IR) of the amino-loaded fullerene nanospheres;
[0026] Figure 2 This is a scanning electron microscope (SEM) image of amino-encapsulated fullerene nanospheres;
[0027] Figure 3 This is a transmission electron microscope (TEM) image of amino-encapsulated fullerene nanospheres;
[0028] Figure 4 The particle size distribution histogram of the amino-containing fullerene nanospheres was obtained by TEM.
[0029] Figure 5 The wall thickness distribution histogram of the amino-containing fullerene nanospheres by TEM statistics;
[0030] Figure 6 The inner diameter distribution histogram of the amino-containing fullerene nanospheres by TEM statistics;
[0031] Figure 7 This is the elemental analysis diagram of the amino-loaded fullerene nanospheres;
[0032] Figure 8 is the release curve of the amino-encapsulated fullerene nanospheres in simulated gastric fluid;
[0033] Fig. 9 The figure is a cytotoxicity test diagram of amino-loaded fullerene nanospheres;
[0034] Fig.10 This is a diagram for the cytotoxicity test of hydrogen peroxide;
[0035] Fig.11 A diagram showing the protection of amino-loaded fullerene nanospheres against cells damaged by hydrogen peroxide. DETAILED DESCRIPTION
[0036] The following is a further detailed description of the implementation methods of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than an exhaustive list of all the embodiments. It should be noted that the embodiments and features in the embodiments of the present application can be combined with each other without conflict.
[0037] The terms "first", "second", etc. (if any) in the specification and claims are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the terms used in this way are interchangeable where appropriate so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products, or devices.
[0038] It should be understood that the term "and / or" used in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.
[0039] Embodiment 1
[0040] Preparation of fullerene toluene solution and ergothioneine mixture:
[0041] 2 mg of fullerene (C 60 ) was dispersed in 4 mL of toluene to prepare a 0.5 mg / mL fullerene toluene solution.
[0042] 20 mg of thioneine was dissolved in 1 mL of water to prepare a 20 mg / mL thioneine aqueous solution. 0.05 mL of the thioneine aqueous solution was taken, and 0.5 mL of ethylenediamine and 0.95 mL of isopropanol were added in turn, and the thioneine mixed solution was obtained by shaking.
[0043] Preparation of pH-responsive fullerene hollow nanosphere aggregates loaded with ergothioneine
[0044] The thioneine mixed solution was added to the fullerene toluene solution, shaken and allowed to stand for three hours, a brown precipitate was precipitated, and the solution was then centrifuged for 6 min (4000 rpm) to fully separate the fullerene hollow nanospheres containing thioneine, and the obtained precipitate was placed in a vacuum oven for drying (vacuum degree: -1 MPa; temperature: 65 ° C) to obtain a pH-responsive fullerene hollow nanosphere aggregate containing thioneine, which was recorded as sample one.
[0045] Embodiment 2
[0046] In the present embodiment, compared with Example 1, only the added volume of ethylenediamine in the thioneine mixed solution was changed to 0.1 ml, and other conditions were unchanged, and the fullerene hollow nanosphere aggregates of thioneine loaded with pH responsiveness were obtained, which was recorded as sample two.
[0047] Embodiment 3
[0048] In the present embodiment, compared with Example 1, only the added volume of ethylenediamine in the thioneine mixed solution was changed to 0.3 ml, and other conditions were unchanged, and the fullerene hollow nanosphere aggregates of thioneine loaded with pH responsiveness were obtained, which was recorded as sample three.
[0049] Embodiment 4
[0050] In the present embodiment, compared with Example 1, only the concentration of the thioneine aqueous solution in the thioneine mixed solution was changed to 5 mg / ml, and other conditions were unchanged, and the fullerene hollow nanosphere aggregates loaded with thioneine having pH responsiveness were obtained, which was recorded as sample four.
[0051] Embodiment 5
[0052] In the present embodiment, compared with Example 1, only the concentration of the thioneine aqueous solution in the thioneine mixed solution was changed to 15 mg / ml, and other conditions were unchanged, and a fullerene hollow nanosphere aggregate of thioneine loaded with pH responsiveness was obtained, which was recorded as sample five.
[0053] Embodiment 6
[0054] In the present embodiment, compared with Example 1, only the concentration of the thioneine aqueous solution in the thioneine mixed solution was changed to 30 mg / ml, and other conditions were unchanged, and the fullerene hollow nanosphere aggregates loaded with thioneine having pH responsiveness were obtained, which was recorded as sample six.
[0055] Embodiment 7
[0056] In the present embodiment, compared with Example 1, only the concentration of the thioneine aqueous solution in the thioneine mixed solution was changed to 40 mg / ml, and other conditions were unchanged, and the fullerene hollow nanosphere aggregates loaded with thioneine having pH responsiveness were obtained, which was recorded as sample seven.
[0057] Performance of pH-responsive fullerene hollow nanosphere aggregates loaded with ergothioneine
[0058] The infrared spectrum of the prepared sample 1 was scanned, and the results were as follows Figure 1 As shown, it can be seen at 3420cm -1 A broad NH band is shown near 1110 cm -1 and 1620cm -1 There are two characteristic bands at C-V and ν C＝C absorption, which further proves that the fullerene was successfully aminated.
[0059] The obtained sample was subjected to scanning electron microscopy (SEM), and the results were as follows: Figure 2 As shown, it can be seen that the prepared fullerene hollow nanospheres encapsulating ergothioneine have a smooth surface and a uniform particle size, and the particle size of the spheres is less than 1 micron, proving that the nanospheres are successfully prepared.
[0060] The obtained sample was subjected to transmission electron microscopy (TEM), and the results were as follows: Figure 3 As shown, the nanospheres have a hollow structure. The particle size, wall thickness, and inner diameter of the hollow spheres are statistically analyzed. The results are as follows Figure 4 , Figure 5 , Figure 6 As shown, the average particle size is 567nm, the average wall thickness is 175nm, and the average inner diameter is 215nm.
[0061] Energy dispersive X-ray spectroscopy (EDS) elemental analysis was performed on the prepared sample 1, and the results were as follows: Figure 7 As shown, it can be seen that carbon element (C) and nitrogen element (N) are distributed in the spherical shell, which proves that the fullerene is successfully aminated, and the sulfur element (S) is well encapsulated in the sphere, proving that ergothioneine is successfully encapsulated in the aminated fullerene hollow nanospheres, and the aminated fullerene hollow nanospheres encapsulating ergothioneine are successfully prepared.
[0062] (I) Drug loading rate and encapsulation efficiency of pH-responsive fullerene hollow nanosphere aggregates loaded with ergothioneine
[0063] To prepared sample one, use inductively coupled plasma-mass spectrometry (ICP-MS) test material thioneine drug loading rate and encapsulation efficiency. Weigh 0.15g nano ball, add 3mL nitric acid, microwave digestion after acid etching overnight, dilute constant volume, and the solution of constant volume is gradient diluted again, finally obtain the quality of thioneine in nano ball by inductively coupled plasma-mass spectrometry, the thioneine drug loading rate of sample one calculated by formula (1) is 31.96%; The thioneine encapsulation rate of sample one calculated by formula (2) is 57.53%.
[0064] Drug loading (%) = (mass of drug loaded / total weight of drug loading system) × 100% (1).
[0065] Encapsulation efficiency (%) = (mass of encapsulated drug / total mass of input drug) × 100% (2).
[0066] (II) pH-responsive release of ergothioneine-loaded fullerene hollow nanosphere aggregates
[0067] The prepared sample 1 was subjected to sustained release curve measurement under different pH conditions. The sample was evenly dispersed in pH=7 deionized water and pH=1.2 hydrochloric acid aqueous solution, and the dispersions of the same concentration were loaded into a 500Da dialysis bag. The gastric release was simulated on a shaker at 37°C. 1mL of the release solution was taken at 7 time points, including 10min, 30min, 60min, 90min, 120min, 180min, and 300min. 1ml of the original solution (pH=7 deionized water and pH=1.2 hydrochloric acid aqueous solution) was added. The absorbance of the release solution at 257nm was tested and calculated using an ultraviolet spectrophotometer, and the release curve was drawn. The results are shown in FIG. Figure 8 As shown, it can be seen that the release rate in the environment of pH = 1.2 is much higher than the release rate in deionized water.
[0068] (III) Application of pH-responsive ergothioneine-loaded fullerene hollow nanosphere aggregates in anti-inflammatory applications
[0069] (1) Cytotoxicity
[0070] The CCK-8 method was used to detect the cytotoxicity of the prepared sample 1.
[0071] Sample group: GES-1 (human gastric mucosal cells) were cultured at 1×10 4 The cells were seeded at a density of 1000 cells / well in a 96-well plate and incubated in DMEM ++Culture medium (complete medium, 10% fetal bovine serum and 1% penicillin / streptomycin double antibody solution added to DMEM) for 24 hours. Then remove the medium, add sample dispersions of different concentrations (0.2, 0.15, 0.1, 0.05, 0.01 mg / mL, dispersed in DMEM), and incubate for another 24 hours. Remove the medium and rinse twice with PBS. Finally, add DMEM solution containing 10% CCK-8 and incubate for 1 hour. Measure the absorbance of the solution at 450nm with an enzyme marker. s .
[0072] Control group: The control group used pure DMEM instead of sample solution, and other conditions were the same as those of the sample group. The final absorbance measured at 450nm was recorded as A c .
[0073] Sample (blank) group: No cells were inoculated, and other conditions were the same as those of the sample group. The final absorbance measured at 450nm was recorded as A b .
[0074] Control (blank) group: No cells were inoculated, and pure DMEM was used instead of the sample solution. Other conditions were the same as those of the sample group. The absorbance at 450 nm was recorded as A bc .
[0075] Each experiment was repeated 3 times, and the cell survival rate was calculated as follows:
[0076] Cell survival rate (%) = [(A s -A b ) / (A c -A bc )]×100%(3)
[0077] The results are as follows Fig. 9 As shown, it can be seen that the sample 1 provided by the present invention is above 80% at 25-200 μg / mL and has no obvious cytotoxicity.
[0078] (2) Inflammatory oxidative stress model
[0079] GES-1 cells were plated at 1×10 4 The cells were seeded at a density of 100 μL / well in a 96-well plate. ++ Remove DMEM and culture for 24 hours. ++ Culture medium, add 100 μL DMEM solution containing different concentrations of H2O2 (200-1000 μM) to each well to interfere with the cells for 12 h, then remove the solution, rinse twice with PBS solution, add DMEM solution containing 10% CCK-8, and incubate for 1 h. The interference of hydrogen peroxide with cells (A) was measured using an enzyme marker x), cells without hydrogen peroxide (A0), and cells mixed with simple DMEM / CCK-8 (A b ) at 450 nm. Each experiment was repeated 3 times, and the cell survival rate was calculated according to the following formula:
[0080] Cell survival rate (%) = (A x -A b ) / (A0-A b )×100%(4)
[0081] The results are as follows Fig.10 As shown, it can be seen that the cytotoxicity of 800 μM H2O2 is 61.8%, which meets the cytotoxicity required for modeling. Therefore, the modeling conditions of the inflammatory oxidative stress model are 800 μM H2O2 DMEM solution intervening cells for 12 hours.
[0082] (3) Anti-inflammatory effect
[0083] GES-1 cells were plated at 1×10 4 The cells were seeded at a density of 100 μL / well in a 96-well plate. ++ Culture for 24 hours.
[0084] The cells were divided into blank group (C), model group (M), fullerene hollow nanospheres protected group encapsulating ergothioneine (EGT@NFH), and ergothioneine protected group (EGT).
[0085] Remove DMEM ++ Culture medium, 100 μL DMEM solution was added to the C group and the M group, and 50 μg / ml of sample-DMEM solution and ergothioneine DMEM solution were added to the EGT@NFH group and the EGT group, respectively, and protected for 12 hours. Then after removing the solution, the PBS solution was rinsed twice, 100 μL DMEM solution was added to each well of the C group, and 100 μL DMEM solution containing H2O2 (800 μM) was added to each well to intervene in the cells for 12 hours, and then 100 μL DMEM dispersion of different concentrations of 0.05 mg / mL samples was added to each well after removing the solution, and then incubated for 24 hours. Then the sample solution was removed and washed twice with PBS. Finally, a DMEM solution containing 10% CCK-8 was added, incubated for 1 hour, the absorbance was measured with an enzyme marker, and the cell survival rate was calculated using formula (2). Three replicates were set for each group, and the final result was averaged.
[0086] The results are as follows Fig.11 As shown, 50 μg / mL of ergothioneine-encapsulated fullerene hollow nanosphere solution can significantly protect cells from damage by hydrogen peroxide, and the protective effect is significantly improved compared to the use of ergothioneine alone.
[0087] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.
Claims
1. A method for preparing a pH-responsive fullerene hollow nanosphere aggregate loaded with ergothioneine, characterized in that: The steps include: Step 1: preparing a fullerene organic solution and an ergothioneine mixed solution respectively; Step 2: adding the ergothioneine mixed solution to the fullerene organic solution, shaking and keeping it still until the reaction end point, to obtain a turbid solution; Step 3: post-treating the turbid liquid to obtain pH-responsive fullerene hollow nanosphere aggregates loaded with ergothioneine.
2. The method for preparing a pH-responsive fullerene hollow nanosphere aggregate of ergothioneine according to claim 1, wherein The volume ratio of the ergothioneine mixed solution to the fullerene organic solution is 3:7.5-8.
5.
3. The method for preparing the pH-responsive fullerene hollow nanosphere aggregates of thioneine according to claim 1, wherein The preparation method of the fullerene organic solution is: dispersing fullerene in an organic solvent to obtain the fullerene organic solution; The organic solvent is a toluene solution, and the mass volume ratio of the fullerene to the toluene solution is 1:1.9-2.
1.
4. The method for preparing the pH-responsive fullerene hollow nanosphere aggregates of ergothioneine according to claim 1, wherein The preparation method of the thioneine mixed solution comprises: dissolving thioneine in deionized water to obtain an thioneine aqueous solution, sequentially adding ethylenediamine and isopropanol to the thioneine aqueous solution, and shaking well to obtain the thioneine mixed solution.
5. The method for preparing the pH-responsive fullerene hollow nanosphere aggregates of ergothioneine according to claim 4, wherein The concentration of thioneine in the thioneine aqueous solution is 5-40 mg / mL.
6. The method for preparing the pH-responsive fullerene hollow nanosphere aggregates of ergothioneine according to claim 4, wherein The volume ratio of the thioneine aqueous solution, isopropyl alcohol and ethylenediamine is 1:19:2-10.
7. The method for preparing the pH-responsive fullerene hollow nanosphere aggregates of ergothioneine according to claim 1, wherein The reaction endpoint is the presence of a brown precipitate in the turbid liquid.
8. The method for preparing the pH-responsive fullerene hollow nanosphere aggregates of ergothioneine according to claim 1, wherein The post-treatment includes: separating the turbid liquid into solid and liquid, collecting the solid, washing and drying.
9. A pH-responsive ergothioneine-loaded fullerene hollow nanosphere aggregate prepared by the method according to any one of claims 1 to 8.
10. An application of the pH-responsive ergothioneine-loaded fullerene hollow nanosphere aggregates as claimed in claim 9, characterized in that: Application in the field of nano-drug carriers and anti-inflammatory fields.