A novel fullerene hydrogel and a preparation method and application thereof

Abstract

The application discloses a novel fullerene hydrogel and a preparation method and application thereof. The fullerene hydrogel is formed by chemical cross-linking of a fullerene derivative shown in formula I and oxidized hyaluronic acid (oxHA). The fullerene-hyaluronic acid hydrogel material provided by the application has a porous framework, a large pore size and a cell loading potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a novel fullerene hydrogel, its preparation method, and its application. Background Technology

[0002] Currently, drug delivery devices made of biocompatible materials, placed at the intended target site, can provide controlled local delivery of immunomodulators. Hydrogels are typically three-dimensional cross-linked molecular networks of polymers formed by covalent or non-covalent interactions, capable of holding large amounts of water. Injectable and implantable hydrogels, in particular, are attracting increasing attention due to their ability to precisely control pharmacological and cellular / drug delivery while avoiding systemic exposure.

[0003] Fullerenes and their derivatives have been extensively studied over the past decade due to their excellent performance in modulating immunotherapy for major diseases. However, currently reported hydrogel-loaded fullerene delivery systems typically load fullerenes and their derivatives into the three-dimensional network pores of a hydrogel formed by polymers such as polysaccharides and cross-linking agents. Limited by the porosity and gelling effect of the hydrogel, these traditional hydrogel-loaded fullerene delivery systems struggle to avoid the rapid and large-volume release of fullerenes in the early stages of drug administration, making it difficult to precisely control the pharmacokinetics of pre-loaded fullerenes and their derivatives, and thus failing to meet the requirements for sustained immunomodulatory effects in the mid-to-late stages of chronic diseases. Therefore, there is an urgent need to develop a novel fullerene hydrogel material to achieve precise control of the pharmacokinetics of fullerenes and their derivatives, and to meet the requirements for sustained high-concentration release of fullerene nanomedicines in the mid-to-late stages of chronic diseases. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a fullerene derivative and a novel fullerene hydrogel prepared based on the fullerene derivative.

[0005] To achieve the above objectives, the following technical solution is adopted:

[0006] In a first aspect, the present invention provides a fullerene derivative.

[0007] The structural formula of the fullerene derivative is shown in Formula I:

[0008]

[0009] In Formula I, n is 12-450, and can be 12, 23, 45, 114, or 228; X is 1-7, and can be 1, 3, or 7; the cage-like structure represents a hollow fullerene or a metal-intercalated fullerene; the hollow fullerene is composed of C 2m The carbon atom composition, 30≤m≤60, can be C. 60 C 70 C 82Or C 84 The metal-intercalated fullerene includes M@C 2n M2@C 2n MA@C 2n M3N@C 2n M2C2@C 2n M2S@C 2n M2O@C 2n and MxA3-xN@C 2n One or more of the following, wherein: M and A both represent metallic elements and M and A are selected from any one of Sc, Y and lanthanide elements, 30≤n≤60; 0≤x≤3.

[0010] According to one embodiment of the present invention, the fullerene derivative shown in Formula I is specifically C 60 -4arm-PEG-HZ, its structural formula is shown below:

[0011]

[0012] The C 60 -4arm-PEG-HZ has a spherical shape and a particle size of approximately 15nm.

[0013] Secondly, the present invention provides a method for preparing the fullerene derivative shown in Formula I.

[0014] The method for preparing the fullerene derivative of Formula I provided by the present invention includes the following steps: in the presence of an amidation reaction condensing agent and an organic base, a dicarboxylic acid pyrrolidine fullerene (structural formula as shown in Formula a) is subjected to an amidation reaction with X-arm-polyethylene glycol-hydrazide (X-Arm-PEG-HZ, structural formula as shown in Formula b, where X is 2-8, optionally 2, 3, 4, 6, 8) to obtain the fullerene derivative of Formula I.

[0015]

[0016] In the above method, the amidation reaction condensing agent is selected from at least one of the following: HATU, HBTU, HCTU, TBTU, TSTU, TNTU.

[0017] In the above method, the organic base is selected from at least one of the following: triethylamine, N,N-diisopropylethylamine.

[0018] In the above method, the amidation reaction is carried out in a solvent, which may be DMF.

[0019] In the above method, the reaction conditions for the amidation reaction are: stirring at room temperature for 24-72 hours.

[0020] Thirdly, the present invention provides applications of the fullerene derivatives shown in Formula I.

[0021] Applications of the fullerene derivatives of Formula I provided by this invention, wherein the applications are selected from 1) and / or 2) below:

[0022] 1) Application in the preparation of ROS scavengers;

[0023] 2) Application in the preparation of products that inhibit macrophage inflammation.

[0024] Fourthly, the present invention provides a novel fullerene hydrogel based on the fullerene derivative shown in Formula I.

[0025] The novel fullerene hydrogel provided by this invention is formed by chemical cross-linking of the fullerene derivative shown in Formula I with oxidized hyaluronic acid (oxHA).

[0026] Furthermore, the chemical cross-linking reaction is a reaction between the hydrazine bond on the fullerene derivative shown in Formula I and the aldehyde group on oxHA, forming a fullerene-hyaluronic acid hydrogel.

[0027] Furthermore, the fullerene derivative shown in Formula I and oxidized hyaluronic acid (oxHA) are both added in the form of a solution (such as an aqueous solution).

[0028] The volume ratio of the fullerene derivative solution to the oxidized hyaluronic acid (oxHA) solution shown in Formula I is 1:(0.5-2), specifically 1:1.

[0029] Wherein, the concentration of the fullerene derivative solution shown in Formula I is 20-80 mg / mL, specifically 80 mg / mL; the concentration of the oxidized hyaluronic acid solution is 3-10%, specifically 7%.

[0030] Furthermore, the oxidized hyaluronic acid (oxHA) is obtained by oxidizing hyaluronic acid with sodium periodate.

[0031] Furthermore, the degree of oxidation of the oxidized hyaluronic acid is 18-55%, specifically 12%. The molecular weight is 5000-1000000, specifically 300000.

[0032] The oxidation degree of oxHA (oxidation degree = integral of aldehyde / integral of -CH3 on polymer backbone) was determined to be 21% for the synthesized oxHA.

[0033] Fifthly, the present invention provides the application of novel fullerene hydrogels based on fullerene derivatives shown in Formula I.

[0034] The applications provided by this invention are selected from the following a) and / or b):

[0035] a) Application in the preparation of products loaded with and / or delivered bioactive ingredients;

[0036] b) Application in the preparation of loaded and / or delivered bioactive ingredients.

[0037] In this invention, the bioactive components include cells, active factors, drugs, proteins, exosomes, etc.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] Compared to existing hydrogel delivery materials for fullerene derivatives, the novel fullerene-hyaluronic acid hydrogel provided by this invention significantly delays the peak release rate of the fullerene derivative release curve, avoiding the large-scale release of the fullerene derivative in the early stages, and better meeting the demand for sustained high-concentration release of fullerene nanomedicines in the middle and late stages of chronic diseases. Furthermore, this novel fullerene-hyaluronic acid hydrogel exhibits excellent biocompatibility, releasing C... 60 -4arm-PEG-HZ exhibits excellent broad-spectrum scavenging effect on excess ROS and significantly inhibits macrophage inflammation. Attached Figure Description

[0040] Figure 1 The reaction equation for the synthesis of oxHA is shown;

[0041] Figure 2 The nuclear magnetic resonance spectra of HA and oxHA are shown;

[0042] Figure 3 C is shown 60 The reaction equation for the synthesis of -4arm-PEG-HZ;

[0043] Figure 4 C is shown 60 Image (left) and surface zeta potential (right) of aqueous solution of -4arm-PEG-HZ;

[0044] Figure 5 C is shown 60 TEM image of -4arm-PEG-HZ nanoparticles;

[0045] Figure 6 Different concentrations of C were shown 60 -4arm-PEG-HZ treatment of HUVEC (left) and NIH3T3 (right) cell viability;

[0046] Figure 7 C is shown 60 O2 before and after -4arm-PEG-HZ treatment ·- EPR spectrum (left) and quantitative statistical results (right);

[0047] Figure 8 C is shown 60 EPR spectra of ·OH before and after treatment with -4arm-PEG-HZ;

[0048] Figure 9 The levels of TNF-α, IL-6, and IL-1β in the culture supernatant of inflammatory macrophages under different treatments are shown.

[0049] Figure 10 The levels of TGF-β1 in the culture supernatant of inflammatory macrophages under different treatments are shown.

[0050] Figure 11 The expression levels of TNF-α and iNOS in inflammatory macrophages under different treatments were shown.

[0051] Figure 12 The flow state of substances under different formulations before and after the bottle pouring method is shown;

[0052] Figure 13 SEM images and pore diameter statistics of fullerene-hyaluronic acid hydrogel materials are shown;

[0053] Figure 14 The shear strain scan curve (left) and viscosity shear frequency scan curve (right) of the fullerene-hyaluronic acid hydrogel are shown.

[0054] Figure 15 Images of a fullerene-hyaluronic acid hydrogel self-healing are shown.

[0055] Figure 16 The shear strain cycle curve of the fullerene-hyaluronic acid hydrogel is shown.

[0056] Figure 17 C is shown 60 Absorbance standard curve of -4arm-PEG-HZ at 550nm (left) and C in fullerene-hyaluronic acid hydrogel 60 -4arm-PEG-HZ release curve (right);

[0057] Figure 18 The diagram shows a fullerene-hyaluronic acid hydrogel co-culture system with cells in transwell wells (left) and the cell viability of HUVEC and NIH3T3 cells after treatment with fullerene-hyaluronic acid hydrogel at different times (right). Detailed Implementation

[0058] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0059] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0060] Example 1: Preparation of oxidized hyaluronic acid (oxHA)

[0061] The reaction equation is as follows Figure 1 As shown, the specific synthesis method is as follows: 2g of hyaluronic acid (HA, Mw: 300000) was dissolved in 200mL of distilled water, and then 16mL of sodium periodate (NaIO4, 0.5M) was added dropwise with stirring. The reaction was carried out at room temperature in the dark for 2.5h. After the reaction, 12mL of ethylene glycol was added to the system to terminate the reaction and quench unreacted NaIO4, and the reaction continued for 1h. The obtained product was dialyzed with distilled water for 3 days to remove byproducts. Finally, the oxidized hyaluronic acid solution was lyophilized to a powder state for further characterization and use.

[0062] Structural characterization: To verify the successful synthesis of oxHA, the obtained product was dissolved in heavy water (D2O), and the degree of oxidation of oxHA was characterized by nuclear magnetic resonance spectroscopy (NMR, Avance III HD-400, Bruker, Germany).

[0063] Conclusion: Sodium periodate oxidizes the vicinal diol on the hyaluronic acid glycan ring to form an aldehyde group. Nuclear magnetic resonance (¹H NMR) spectroscopy was used to verify the formation of the aldehyde and calculate the degree of oxidation. Based on the NMR spectrum (… Figure 2 The results showed that new chemical shifts of 4.82, 4.98, and 5.08 ppm were generated in oxHA due to the presence of aldehyde groups. The oxidation degree of oxHA was calculated by comparing it with the -CH3-specific signal on the hyaluronic acid polymer backbone (oxidation degree = integral of aldehyde / integral of -CH3 on polymer backbone), and the oxidation degree of the synthesized oxHA was determined to be 12%.

[0064] Example 2: C 60 Preparation of -4arm-PEG-HZ

[0065]

[0066] 2.92 g of 1,3-propanone dicarboxylic acid was dissolved in 100 mL of dichloromethane. 20 mL of tert-butanol and 500 mg of 4-dimethylaminopyridine were added, and the mixture was stirred in an ice bath for 5 min to pre-cool the system. Then, 6.683 mL of N,N-dicyclohexylcarbodiimide was added, and the mixture was stirred in an ice bath for 5 min before removing the ice bath and continuing the reaction for 3 h. After the reaction was complete, a large amount of dichloromethane was added, and the mixture was filtered under reduced pressure. The filtrate was collected. The filtrate was washed successively with dilute hydrochloric acid (1 mol / L) and saturated sodium bicarbonate solution. Finally, water in the organic phase was removed with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation using silica gel powder (300-500 mesh). Compound a was obtained after separation and purification using silica gel column chromatography with a mobile phase of n-hexane:ethyl acetate = 1:6.

[0067] Compound a (2.22 g) was dissolved in methanol, and glycine benzyl ester hydrochloride (1.7617 g) and sodium cyanoborohydride (401.4 mg) were added. The mixture was sonicated until the solid was completely dissolved, and reacted at room temperature under a nitrogen atmosphere for 48 hours. After the reaction was completed, saturated sodium bicarbonate solution was added dropwise to adjust the pH to 7. The mixture was then extracted with dichloromethane, washed with saturated brine, dehydrated with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain a colorless oily crude product b.

[0068] Crude product b (1.24 g) was dissolved in a mixed solvent of ethyl acetate (210 mL) and methanol (50 mL), and Pd / C (10%, 488.5 mg) was added. The entire system was purged with nitrogen and then purged with H2. The reaction was carried out under H2 atmosphere with vigorous stirring at room temperature for 24 h. After the reaction, the mixture was filtered through diatomaceous earth, and the solvent was removed using a rotary evaporator. Hexane was added as a poor solvent to precipitate the colorless crude product c.

[0069] Use toluene (200 mL) to C 60 (227.05mg) was completely dissolved, product c (181.4mg) and paraformaldehyde (27.21mg) were added, the reactants were heated and refluxed to 110℃ and the temperature was maintained for 1h. The system was cooled to room temperature, the mixture was taken out, most of the solvent was removed by rotary evaporation, and then product d was obtained by silica gel column chromatography.

[0070] Product d (100 mg) was dissolved in chloroform (20 mL), and trifluoroacetic acid (7 mL) was added. The mixture was stirred at room temperature for 18 h, and then the solvent was removed by rotary evaporation. After drying, the product, namely dicarboxylic acid pyrrolidine fullerene (product e), was obtained.

[0071] The above-mentioned dicarboxylic acid pyrrolidine fullerene (10 mg) was dissolved in 2 mL of DMF, and 88 mg of 4-arm-PEG-hydrazide (4-arm-PEG-HZ, Mw: 2000, Yuanye, Shanghai) was added and stirred until dissolved. Then, HBTU (23 mg) was added as a condensing agent for the amidation reaction, and N,N-diisopropylethylamine (DIPEA, 10 μL) was added to provide an alkaline environment. The mixture was stirred at room temperature for 24 h. After the reaction was complete, a large amount of diethyl ether was added to precipitate the product, the precipitate was collected by centrifugation, and vacuum dried overnight. After redissolving the precipitate in water, the product was purified using a dialysis bag with a molecular weight cutoff of 10000 Da, with the water changed twice daily. After dialysis purification and lyophilization, C was obtained. 60 -4arm-PEG-HZ solid.

[0072] Basic characterization: The obtained product was dispersed in water at an appropriate concentration and characterized by dynamic light scattering (DLS, NanoZSZEN3600, Malvern, UK). 60 The surface zeta potential of -4arm-PEG-HZ nanoparticles was determined. The C4 nanoparticles were characterized using transmission electron microscopy (TEM, HT 7700, Hitachi, Japan). 60 Morphology and size of -4arm-PEG-HZ nanoparticles.

[0073] Conclusion: Product C 60 -4arm-PEG-HZ is a brownish-yellow transparent solution with good water solubility. Figure 4 C 60 The zeta potential on the surface of the -4arm-PEG-HZ nanoparticles is 17.73 ± 1.63 mV. TEM ( Figure 5 ) Display C 60 -4arm-PEG-HZ nanoparticles are spherical with a particle size of approximately 15 nm.

[0074] Example 3: C 60 -4arm-PEG-HZ cytotoxicity assay

[0075] Cells: Human venous endothelial cells (HUVEC), mouse embryonic fibroblasts (NIH3T3).

[0076] Grouping: The blank control group was prepared in high-glucose DMEM medium (Gibco, USA, C11995500BT) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) (complete medium). The experimental groups were prepared in different concentrations of C... 60 Complete culture medium of 4-arm-PEG-HZ (20, 50, 100, 200, 500, 800, 1000 mg / mL).

[0077] Experimental Methods: Cells were cultured in high-glucose DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS). HUVEC or NIH3T3 cells were treated for 24 hours after reaching appropriate density, according to the grouping requirements. Then, cells were treated with a CCK-8 assay kit (Bai Rui Ji, China) in the dark for 30 minutes. Subsequently, the OD value was measured at 450 nm using a multi-well microplate reader.

[0078] Calculation method: Cell viability = OD of treated cells / OD of control cells. The final cell viability result was normalized relative to the mean of the blank control group.

[0079] Conclusion: C 60 -4arm-PEG-HZ is safe and non-toxic within a wide concentration range of 0-1000 μg / mL, and has good biocompatibility. Figure 6 ).

[0080] Example 4: C 60 -4arm-PEG-HZ free radical scavenging ability

[0081] Experimental methods:

[0082] Evaluation of C using electron paramagnetic resonance (EPR, E500, Bruker, Germany) 60 O2 removal by -4arm-PEG-HZ ·- Activity: The KO2-crown ether-DMSO mixture generates O2. ·- BMPO was used as a spin trapping agent, and BMPO (final concentration 50 mM), DTPA (final concentration 100 μM), KO2-crown ether-DMSO (final concentration 10 mM), and various concentrations of C were combined. 60 -4arm-PEG-HZ or water was mixed, and the EPR signal was detected after 2 minutes of reaction. The 100 mM KO2-crown ether-DMSO working solution was prepared as follows: 71.1 mg KO2 and 600 mg 18-crown-6 were dissolved in 10 mL ultra-dry DMSO until a clear yellow solution was formed.

[0083] Use EPR to evaluate C 60 -4arm-PEG-HZ hydroxyl radical scavenging activity: DMPO was used as a spin trapping agent. 25 μL of H2O2 (100 mM), 10 μL of DMPO (200 mM), and 25 μL of C at different concentrations were used to scavenge hydroxyl radicals. 60 -4arm-PEG-HZ was mixed, and then the sample was irradiated with ultraviolet light. After 15 seconds, the EPR signal was collected.

[0084] Conclusion: EPR results show C 60-4arm-PEG-HZ exhibits excellent O2 performance in a concentration-dependent manner. ·- Clearance ability ( Figure 7 C 60 -4arm-PEG-HZ significantly scavenges ·OH in a concentration-dependent manner. Figure 8 In short, C 60 -4arm-PEG-HZ itself has excellent broad-spectrum scavenging effect on excess ROS.

[0085] Example 5: C 60 -4arm-PEG-HZ inhibits inflammation in macrophages.

[0086] Cells: Mouse mononuclear macrophages (RAW 264.7).

[0087] Grouping: The blank control group received high-glucose DMEM medium (Gibco, USA, C11995500BT) containing 10% fetal bovine serum (Gibco, USA, 10099141C) (complete medium); the model control group received complete medium containing 100 ng / mL LPS; and the experimental group received complete medium containing 1 mg / mL C. 60 -4arm-PEG-HZ, complete culture medium of 100ng / mL LPS.

[0088] Experimental Methods: RAW 264.7 cells were cultured in high-glucose DMEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS). After reaching appropriate cell density, RAW 264.7 cells were treated for 24 hours according to grouping requirements. Intracellular proteins and cell culture supernatant were collected. Intracellular protein expression levels of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were analyzed using Western blotting (WB). The concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), TNF-α, and transforming growth factor-β1 (TGF-β1) in the cell culture suspension were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jianglai, China).

[0089] Conclusion: Compared with the control group, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the cell culture supernatant of the model group were significantly increased. 60 -4arm-PEG-HZ treatment reduced the levels of pro-inflammatory cytokines in inflammatory macrophages ( Figure 9 C 60 -4arm-PEG-HZ inhibited the increase of pro-fibrotic factor TGF-β1 levels in inflammatory macrophages ( Figure 10 Compared to the control group, the levels of iNOS and TNF-α in macrophages were significantly increased in the model group, C 60-4arm-PEG-HZ inhibits this tendency ( Figure 11 In short, C 60 -4arm-PEG-HZ exhibits excellent inhibitory effects on macrophage inflammation.

[0090] Example 6: Synthesis of Fullerene-Hyaluronic Acid Hydrogel

[0091] Experimental method: C 60 The -4arm-PEG-HZ solution was concentrated to 80 mg / mL using an ultrafiltration tube with a molecular weight cutoff of 10000 Da. A 7% (m / m, mass percentage) aqueous solution of oxHA was prepared. An equal volume of C... 60 The -4arm-PEG-HZ solution was rapidly mixed with the oxHA aqueous solution and then thoroughly mixed using a vortex mixer. 60 -4arm-PEG-HZ reacts rapidly with the aldehyde group on oxHA to form a fullerene-hyaluronic acid hydrogel through chemical cross-linking.

[0092] Characterization: The changes before and after the formation of fullerene-hyaluronic acid hydrogel were demonstrated using the vial pouring method.

[0093] Conclusion: Combining oxHA with C 60 When 4arm-PEG-HZ is simply mixed in the above proportions, chemical cross-linking is achieved, the solution stops flowing, and a solid hydrogel is formed. Figure 12 ).

[0094] Example 7: Morphology and Mechanical Properties Characterization of Fullerene-Hyaluronic Acid Hydrogel

[0095] Experimental methods:

[0096] Scanning electron microscopy characterization of fullerene-hyaluronic acid hydrogel: An equal volume of C 60 -4arm-PEG-HZ solution and oxHA aqueous solution were rapidly mixed in a centrifuge tube to form a fullerene-hyaluronic acid hydrogel. The hydrogel skeleton was obtained by freeze-drying the fullerene-hyaluronic acid hydrogel. The hydrogel was cut open with a knife, and the surface morphology of the cut surface of the fullerene-hyaluronic acid hydrogel was observed using a field emission scanning electron microscope (SEM, SU8020, Hitachi, Japan).

[0097] Rheological testing of fullerene-hyaluronic acid hydrogel: Equal volumes of C 60 -4arm-PEG-HZ solution and oxHA aqueous solution were rapidly mixed and molded in a 3cm diameter circular mold. The gel was then placed in the parallel clamps of a rheometer (ARES G2, TA Instruments, USA), and the storage modulus (G'), loss modulus (G”), and viscosity of the gel were monitored by changing the magnitude or frequency of shear strain.

[0098] Qualitative photographs of the self-healing properties of fullerene-hyaluronic acid hydrogel: equal volumes of C 60 -4arm-PEG-HZ solution and oxHA aqueous solution were respectively loaded into both ends of a dual syringe. The mixed solution was squeezed into a round mold and a rectangular mold. After the gel solidified, the fullerene-hyaluronic acid hydrogel was removed. The round and rectangular fullerene-hyaluronic acid hydrogels were cut in half lengthwise, and the cut edges of the semi-circular and square gels were tightly adhered together. After standing for 10 minutes, the healing process was assessed with tweezers.

[0099] Quantitative characterization of the self-healing rheology of fullerene hydrogels: Equal volumes of C 60 -4arm-PEG-HZ solution and oxHA aqueous solution were rapidly mixed and molded in a 3cm diameter circular mold. The gel was then placed in the parallel clamp of a rheometer and maintained at 1% shear strain for 100s, followed by a rapid increase in shear strain to 200% and maintenance for 100s. This cycle was repeated three times between 1% and 200% shear strain, and the changes in the storage modulus (G') and loss modulus (G”) of the gel were monitored.

[0100] Conclusion: SEM images show that the fullerene-hyaluronic acid hydrogel material has a porous framework with relatively large pore sizes, averaging 28 μm, and exhibits a hydrogel cross-linked network porous structure. Figure 13 The storage modulus (G') of the fullerene-hyaluronic acid hydrogel material is greater than its loss modulus (G”) within the shear strain range of 0.1%–100%, indicating that the gel material is mechanically stable. Figure 14 (Left). Furthermore, the viscosity of the fullerene-hyaluronic acid hydrogel decreases as the shear frequency increases from 0.1 to 1000 rad / s, indicating that this hydrogel exhibits shear-thinning characteristics. Figure 14 (Right). The self-healing photo shows that after two gel blocks of different shapes were joined together, the fullerene-hyaluronic acid hydrogel could re-heal after a period of time. Figure 15 The self-healing properties of the hydrogel were quantitatively evaluated by cycling it between 1% and 200% shear strain using a rheometer. Both G' and G” exhibited rapid responses to changes in shear strain, recovering to their previous values ​​when the shear strain returned to 1%. After three cycles, G' and G” still showed rapid recovery. These rheological curves quantitatively demonstrate the self-healing properties of the fullerene-hyaluronic acid hydrogel material. Figure 16 ).

[0101] Example 8: C of fullerene-hyaluronic acid hydrogel 60 -4arm-PEG-HZ release curve

[0102] Experimental method: 75 μL of C was used for each... 60-4arm-PEG-HZ solution and oxHA aqueous solution were rapidly mixed in a centrifuge tube to form a gel. The gel was then transferred to a glass bottle, and 4 mL of PBS buffer was added. The bottle was placed on a 37°C shaker and continuously shaken. 600 μL of the soaking solution was collected on days 0, 1, 3, 7, 14, 19, and 21, and then an equal volume of fresh PBS was added, followed by further shaking. The C in the release solution... 60 The concentration of -4arm-PEG-HZ was measured using a UV spectrophotometer at a wavelength of 550 nm. The C content in the hydrogel... 60 The release rate of -4arm-PEG-HZ is calculated using the following formula:

[0103]

[0104] A x C represents the cumulative release rate on day x. g This indicates the initial state of C in the hydrogel. 60 The concentration of -4arm-PEG-HZ in this experiment, C g 40 mg / mL; V g V represents the total volume of the hydrogel. In this experiment, V g 150 μL; C x C in the soaking solution on day x 60 -4arm-PEG-HZ concentration; V0 represents the total volume of the soaking solution, which is 4 mL in this experiment; v represents the total volume of the soaking solution collected at each time point, which is 0.6 mL in this experiment; x-1 represents the predetermined time point for collecting the soaking solution on day x; C t C in the soaking solution on day t 60 The concentration of -4arm-PEG-HZ.

[0105] Conclusion: C 60 The total release rate of -4arm-PEG-HZ increased from 1.52% to 12.66% from day 1 to day 7, exhibiting a slow release initially. Over the next 14 days, the drug release increased to 53.93%, demonstrating a good sustained-release effect. By day 19, C... 60 The drug release rate of -4arm-PEG-HZ was 77.88%, after which the release rate gradually decreased. This curve proves that C 60 -4arm-PEG-HZ is primarily released into the surrounding environment through material degradation. During the first 7 days, the degradation rate of the fullerene-hyaluronic acid hydrogel is relatively slow, avoiding rapid early drug release and its impact on subsequent sustained release. After day 7, the degradation rate of the fullerene-hyaluronic acid hydrogel accelerates significantly, leading to increased cross-linking of C-terminal molecules within the hydrogel. 60-4arm-PEG-HZ was rapidly released into the environment. After day 19, as the material's continued degradation ceased, C 60 The release rate of -4arm-PEG-HZ also gradually slowed down. Figure 17 ).

[0106] Example 9: Biosafety of Fullerene-Hyaluronic Acid Hydrogel

[0107] Cells: Human venous endothelial cells (HUVEC), mouse embryonic fibroblasts (NIH3T3).

[0108] Grouping: The blank control group was a high-glucose DMEM medium (Gibco, USA, C11995500BT) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) (complete medium), while the experimental group was a complete medium supplemented with fullerene-hyaluronic acid hydrogel.

[0109] Experimental methods: HUVEC and NIH3T3 cells were cultured at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at a density of 1 cell / well in the lower chamber of a 24-well Transwell plate (Corning Hyaluronic Acid Biotech, USA) with 0.4 μm pore size and cultured overnight. Subsequently, 50 μL of fullerene-hyaluronic acid hydrogel was added to the upper chamber of the Transwell plate. Wells without hydrogel served as blank controls. After incubation at 37°C for 24 or 72 hours, the upper chamber was removed. Cell viability was assessed using a CCK-8 assay kit (Bai Rui Ji, China) according to the manufacturer's instructions, treating cells for 30 minutes. OD values ​​were then measured at 450 nm using a multi-well plate reader.

[0110] Calculation method: Cell viability = OD of treated cells / OD of control cells. The final cell viability result was normalized relative to the mean of the blank control group.

[0111] Conclusion: Viability analysis of two cell types co-cultured with the hydrogel in Transwell showed that no cell death was observed after 24 hours of treatment with the fullerene-hyaluronic acid hydrogel. Similarly, CCK-8 assays of cells treated with the hydrogel for 72 hours also indicated that the fullerene-hyaluronic acid hydrogel possesses good cell compatibility. Figure 18 ).

[0112] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A fullerene derivative, the structural formula of which is shown in Formula I: In Formula I, n is 12-450, and can be 12, 23, 45, 114, or 228; X is 1-7, and can be 1, 3, or 7; the cage-like structure represents a hollow fullerene or a metal-intercalated fullerene; the hollow fullerene is composed of C 2m The carbon atom composition, 30≤m≤60, can be C. 60 C 70 C 82 Or C 84 The metal-intercalated fullerene includes M@C 2n M2@C 2n MA@C 2n M3N@C 2n M2C2@C 2n M2S@C 2n M2O@C 2n and MxA3-xN@C 2n One or more of the following, wherein: M and A both represent metallic elements, and M and A are selected from any one of Sc, Y and lanthanide elements, 30≤n≤60; 0≤x≤3.

2. The fullerene derivative according to claim 1, characterized in that: The fullerene derivative shown in Formula I is C 60 -4arm-PEG-HZ, its structural formula is shown below:

3. A method for preparing the fullerene derivative according to claim 1 or 2, comprising the following steps: in the presence of an amidation reaction condensing agent and an organic base, an amidation reaction is carried out between the dicarboxylic acid pyrrolidine fullerene of formula a and the X-arm-polyethylene glycol-hydrazide of formula b to obtain the fullerene derivative of formula I. in, In X-arm-polyethylene glycol-hydrazide, X is 2-8, and can be 2, 3, 4, 6, or 8.

4. The preparation method according to claim 3, characterized in that: And / or, the amidation reaction condensing agent is selected from at least one of the following: HATU, HBTU, HCTU, TBTU, TSTU, TNTU; And / or, the organic base is selected from at least one of the following: triethylamine, N,N-diisopropylethylamine; And / or, the amidation reaction is carried out in a solvent, preferably DMF; And / or, the reaction conditions for the amidation reaction are: stirring at room temperature for 24-72 h.

5. The use of the fullerene derivative according to claim 1 or 2, wherein the use is selected from 1) and / or 2) below: 1) Application in the preparation of ROS scavengers; 2) Application in the preparation of products that inhibit macrophage inflammation.

6. A fullerene hydrogel, formed by chemical crosslinking of the fullerene derivative as described in claim 1 or 2 with oxidized hyaluronic acid.

7. The fullerene hydrogel according to claim 6, characterized in that: The fullerene derivative and oxidized hyaluronic acid (oxHA) shown in Formula I are added in solution form. The volume ratio of the fullerene derivative solution to the oxidized hyaluronic acid solution shown in Formula I is 1:(0.5-2). The concentration of the fullerene derivative solution is 20-80 mg / mL, and the concentration of the oxidized hyaluronic acid solution is 3-10%.

8. The fullerene hydrogel according to claim 6 or 7, characterized in that: The oxidized hyaluronic acid is obtained by oxidizing hyaluronic acid with sodium periodate; And / or, the degree of oxidation of the oxidized hyaluronic acid is 10-55%, and the molecular weight of the hyaluronic acid is 5000-1000000.

9. The application of the fullerene hydrogel according to any one of claims 6-8, wherein the application is selected from a) and / or b): a) Application in the preparation of products loaded with and / or delivered bioactive ingredients; b) Application in the preparation of loaded and / or delivered bioactive ingredients.

10. The application according to claim 9, characterized in that: The bioactive ingredient is selected from at least one of the following: cells, active factors, drugs, proteins, and exosomes.

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