Curcumin-loaded cerium-based nanoszyme, preparation method and application thereof

Abstract

The application belongs to the technical field of medicine preparation, and particularly relates to a curcumin-loaded cerium-based nanoenzyme as well as a preparation method and application thereof. The curcumin-loaded cerium-based nanoenzyme comprises a core carrier, a drug loaded on the core carrier and a shell; the core carrier is a cerium metal organic framework; the drug is curcumin; and the shell is hyaluronic acid. The application provides a super-small nanoenzyme HC@CeMOF with strong antioxidant and targeting functions, and data results of the application show that the HC@CeMOF can be used for treating chronic pancreatitis. Specifically, the HC@CeMOF can be used for treating chronic pancreatitis by utilizing the inflammation targeting ability of hyaluronic acid, so that the HC@CeMOF is preferentially gathered at a chronic pancreatitis inflammation site, then through the synergistic effect of curcumin and CeMOF, active oxygen (ROS) at the lesion site is effectively removed, and the expression of NF-kappa B is down-regulated, so as to regulate the local inflammatory cytokine microenvironment, and finally significantly improve the pancreas fibrosis.

Description

Technical Field

[0001] This application belongs to the field of drug preparation technology, specifically relating to a curcumin-loaded cerium-based nanozyme, its preparation method, and its application. Background Technology

[0002] Chronic pancreatitis (CP) is a lifelong, progressive fibrotic inflammatory disease caused by the interaction of genetic and environmental factors. Clinically, CP can lead to pancreatic exocrine and endocrine insufficiency, accompanied by recurrent pain, severely impacting patients' quality of life. Furthermore, it significantly increases the risk of pancreatic cancer. However, current treatments for CP mainly rely on palliative measures such as enzyme replacement and nutritional support, and a cure is not yet possible. Therefore, developing safe and effective treatment strategies for CP is of great significance for maintaining human health.

[0003] Pancreatic fibrosis is a hallmark pathological feature of pancreatic fibrosis (CP) and a fundamental obstacle to its incurability. Persistent and recurrent inflammatory stimulation is a major driver of fibrosis, while reactive oxygen species (ROS) act as a key mediator linking inflammation and fibrosis in CP. Under persistent inflammatory stress, mechanisms such as mitochondrial dysfunction, abnormal activation of pancreatic enzymes, and immune cell respiratory bursts lead to the excessive generation and accumulation of ROS in the pancreas. These ROS directly induce oxidative damage in pancreatic cells and activate pro-inflammatory signaling pathways such as NF-κB, thus forming a vicious cycle between inflammation and oxidative stress. Simultaneously, excessive ROS promotes the activation of pancreatic stellate cells (PSCs) into fibrotic myofibroblasts, driving collagen deposition and pancreatic fibrosis, ultimately leading to irreversible tissue destruction. Therefore, ROS acts as both an "amplifier" of inflammation and a direct mediator of fibrosis; targeting oxidative stress may provide a promising strategy for alleviating the pathological progression of CP. However, despite decades of research and numerous clinical trials, the efficacy of antioxidant therapy in CP remains controversial, with some studies even questioning its clinical benefit. This inconsistency may stem from the pancreas's anatomical characteristics: located retroperitoneally and surrounded by vital organs and blood vessels, it makes it difficult for drugs to penetrate and directly reach the lesion site. Furthermore, although the pancreas has a rich blood supply, its uneven distribution may limit the effective concentration of therapeutic drugs throughout the organ. Therefore, developing efficient drug delivery strategies to improve the bioavailability of antioxidants may be a feasible solution to address these challenges. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies, at least to solve the problem of poor clinical efficacy caused by the difficulty in targeting the pancreas, easy degradation, and low bioavailability of antioxidants in existing technologies. The solution of this invention, through nano-sizing and targeted modification, significantly improves the solubility and stability of the hydrophobic drug curcumin and increases its concentration at the lesion site, enhancing the therapeutic effect while potentially reducing systemic side effects. Specifically, it provides a curcumin-loaded cerium-based nanozyme, its preparation method, and its application, which specifically adopts the following technical solution:

[0005] In a first aspect, the present invention provides a curcumin-loaded cerium-based nanozyme, wherein the curcumin-loaded cerium-based nanozyme comprises a core carrier, a drug loaded on the core carrier, and a shell;

[0006] The core carrier is a cerium-based metal-organic framework; the drug is curcumin; and the outer shell is hyaluronic acid.

[0007] This invention provides a core-shell structured curcumin-loaded cerium-based nanoenzyme material; wherein the core is an ultra-small cerium-based metal-organic framework (CeMOF) synthesized by hydrothermal method; this CeMOF can not only serve as a drug carrier, but also has enzyme-like antioxidant activity (mimicking SOD, CAT, etc.), and can efficiently scavenge ROS, which is the first functional core of this invention for treating chronic pancreatitis; secondly, loading curcumin (Cur) into CeMOF can provide strong anti-inflammatory and anti-fibrotic effects, and can inhibit PSC activation and pro-inflammatory signaling pathways such as NF-κB, which is the second functional core of this invention.

[0008] This invention employs CeMOF as a core to remove excess ROS, breaking the vicious cycle of "oxidative stress-inflammation" and creating a favorable microenvironment for Cur to function. On the other hand, it uses Cur to inhibit the process of inflammation and fibrosis, reducing the production of ROS from the root. The two work together to efficiently regulate the spectrum of inflammatory factors and inhibit the NF-κB signaling pathway, thereby achieving the ultimate goal of alleviating CP.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned curcumin-supported cerium-based nanozyme, comprising the following steps:

[0010] Ce-MOF nanozyme powder was dispersed in ethanol to obtain a CeMOF suspension;

[0011] Curcumin was dissolved in ethanol to obtain a curcumin solution;

[0012] Hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution;

[0013] CeMOF suspension was mixed with curcumin solution and reacted. After the reaction was completed, the mixture was centrifuged and freeze-dried to obtain Cur@CeMOF nanozyme.

[0014] The hyaluronic acid solution was dispersed in the suspension of the Cur@CeMOF nanozyme, mixed evenly, reacted, centrifuged, and freeze-dried to obtain the curcumin-loaded cerium-based nanozyme.

[0015] This invention constructs an ultrasmall cerium-based metal-organic framework (CeMOF) loaded with curcumin (Cur) and possessing intrinsic antioxidant activity. Further encapsulation with a hyaluronic acid (HA) shell creates a targeted nanotherapy platform (named HC@CeMOF) for treating chronic pancreatitis and effectively alleviating the progression of pancreatic fibrosis. The nanomaterial is composed of Ce-MOF nanozymes, curcumin (Cur), and hyaluronic acid (HA).

[0016] Among them, cerium-based nanozymes can utilize Ce 3+ and Ce 4+ Reversible redox cycles between oxidized states selectively scavenge reactive oxygen species (ROS). Among these, cerium-based nanozymes derived from metal-organic frameworks (MOFs) exhibit additional advantages, such as ultra-small particle size and superior ROS scavenging ability, making them highly valuable for antioxidant therapy. Curcumin (Cur), a polyphenolic compound extracted from turmeric rhizomes, has well-known anti-inflammatory and antioxidant properties. Cur can reduce the synthesis and secretion of extracellular matrix (ECM)-related proteins by inhibiting the activation of cytotoxic cells (PSCs) and the production of monocyte chemoattractant protein-1 (MCP-1) induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), thereby playing an anti-fibrotic role in cytotoxicity cerebrovascular disease (CP). These findings highlight the significant potential of Cur in CP treatment. Hyaluronic acid (HA), an endogenous polysaccharide, has a high affinity for the CD44 receptor highly expressed on activated macrophages, endowing nanomaterials with the ability to precisely target inflammatory sites.

[0017] This invention leverages the CD44 receptor, highly expressed on infiltrating macrophages in inflamed pancreatic tissue, to promote the preferential accumulation of HC@CeMOF at the site of inflammation. Once localized to the lesion area, HC@CeMOF exerts a synergistic therapeutic effect through CeMOF's efficient scavenging of reactive oxygen species (ROS) and Cur's potent anti-inflammatory and anti-fibrotic effects. This dual mechanism of action modulates the inflammatory cytokine spectrum and inhibits the NF-κB signaling pathway, thereby effectively alleviating the progression of chronic pancreatitis.

[0018] As a further preferred embodiment, the Ce-MOF nanozyme powder is prepared by the following steps:

[0019] Terephthalic acid was dissolved in an organic solvent, and then an aqueous solution of (NH4)2Ce(NO3)6 was added. The mixture was stirred until homogeneous, then transferred to a high-pressure reactor and heated to react. After the reaction was completed, the mixture was cooled to room temperature, dialyzed, and freeze-dried to obtain the Ce-MOF nanozyme powder.

[0020] As a further preferred embodiment, the ratio of (NH4)2Ce(NO3)6 to terephthalic acid in the aqueous solution of (NH4)2Ce(NO3)6 is 1.16 g: 0.354 g.

[0021] As a further preferred embodiment, the heating reaction temperature is 90℃-110℃; the reaction time is 0.5h-1.5h.

[0022] As a further preferred embodiment, the ratio of Ce-MOF nanozyme powder, curcumin, and hyaluronic acid is 5 mg: 20 mg: 1 mg.

[0023] As a further preferred embodiment, the concentration of the CeMOF suspension is 5 mg / mL;

[0024] The concentration of the curcumin solution is 2 mg / mL; the concentration of the hyaluronic acid solution is 1 mg / mL.

[0025] Thirdly, the present invention provides the application of the above-mentioned curcumin-loaded cerium-based nanozyme in the preparation of a drug for treating chronic pancreatitis.

[0026] Fourthly, the present invention provides a drug for treating chronic pancreatitis, wherein the drug has the above-mentioned curcumin-loaded cerium-based nanozyme as the main active ingredient.

[0027] As a further preferred embodiment, the drug also includes a pharmaceutically acceptable carrier.

[0028] The beneficial effects of this invention are as follows:

[0029] This invention provides an ultrasmall nanozyme HC@CeMOF with potent antioxidant and targeting functions, and the data results show that this HC@CeMOF can be used to treat chronic pancreatitis. Specifically, by utilizing the inflammation-targeting ability of hyaluronic acid, HC@CeMOF preferentially accumulates at sites of chronic pancreatic inflammation. Then, through the synergistic effect of curcumin and CeMOF, it effectively scavenges reactive oxygen species (ROS) at the lesion site and downregulates NF-κB expression, thereby regulating the local inflammatory cytokine microenvironment and ultimately significantly improving pancreatic fibrosis. This invention combines targeted drug delivery with powerful ROS scavenging function, showing good therapeutic effects on chronic pancreatitis and providing a potential strategy for future intervention and treatment of this intractable disease. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The TEM and SEM images of the materials prepared in Example 1 are shown; wherein, A in the figure is a TEM image of CeMOF nanozyme; B in the figure is a SEM image of CeMOF nanozyme; C in the figure is a TEM image of HC@CeMOF; and D in the figure is a SEM image of HC@CeMOF.

[0032] Figure 2 The figures show the XPS and XRD patterns of the materials prepared in Example 1; where A is the XPS pattern of CeMOF; B is the XPS spectrum of C1s; C is the XPS spectrum of N1s; D is the XPS spectrum of O1s; E is the XPS spectrum of Ce; and F is the XRD pattern of CeMOF and HC@CeMOF.

[0033] Figure 3 The figures show the particle size distribution and zeta potential of the material prepared in Example 1; where A in the figure represents the particle size distribution of HC@CeMOF; B in the figure represents the zeta potential of HA, Cur, CeMOF and HC@CeMOF; and C in the figure represents the particle size and zeta potential of HC@CeMOF within 14 days.

[0034] Figure 4 The figures show the cell viability after 1, 2, and 3 days of co-culturing HC@CeMOF prepared in Example 1 (Figure A); the live / dead cell staining experiment after 3 days of co-culturing HC@CeMOF (Figure B); and the hemolysis experiment of HC@CeMOF (Figure C).

[0035] Figure 5 The figure shows the H2O2 removal rate (A) of the HC@CeMOF scavenging process prepared in Example 1; the O2 removal rate of the HC@CeMOF scavenging process is shown in Figure 1. 2- Scavenging rate (B in the figure); ABTS radical scavenging rate and scavenging process of HC@CeMOF (C in the figure); DPPH radical scavenging rate and scavenging process of HC@CeMOF (D in the figure).

[0036] Figure 6The diagram shows the interaction between HA and CD44 protein (A in the figure); the free energy landscape and interaction conformation of CD44 protein and hyaluronic acid small molecules in molecular dynamics simulation (B in the figure); the molecular dynamics changes of CD44 protein RMSF before and after binding (C in the figure); the table of MM / PBSA binding free energy calculation (D in the figure); where ΔvdW: van der Waals interaction energy; ΔEEL: electrostatic interaction energy between charges; ΔEPB: polar solvation energy; ΔESA: nonpolar solvation energy; ΔEtot: total binding free energy; and the energy analysis of the unit residue decomposition of binding free energy (E in the figure).

[0037] Figure 7 The figures show the dynamic changes of Cy5.5-labeled HC@CeMOF accumulation over time in the pancreatic tissue of CP mice after tail vein injection (A); the biodistribution of Cy5.5-labeled HC@CeMOF and Cy5.5-labeled Cur@CeMOF in CP mice and healthy mice (B); and fluorescence imaging of pancreatic tissue of healthy and CP mice after incubation with Cy5.5-labeled nanomaterials (C). In the figures, red indicates nanomaterials and blue indicates cell nuclei.

[0038] Figure 8 The images show optical images of mouse pancreas after different treatments (A in the figure); Western blotting analysis of the expression levels of pancreatic fibrosis protein after different treatments (B in the figure); Western blotting quantitative analysis of the expression levels of pancreatic fibrosis protein after different treatments (C in the figure); and pancreatic H&E staining, Masson staining, and Sirius red staining (D in the figure).

[0039] Figure 9 The diagram shows the in vivo anti-inflammatory mechanism of HC@CeMOF (A in the figure); the expression levels of inflammatory factors in mice after different treatments were determined by ELISA (B-J in the figure). Detailed Implementation

[0040] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0041] Example 1

[0042] A hyaluronic acid-modified curcumin-supported cerium-based nanozyme (HC@CeMOF) is prepared by the following steps:

[0043] (1) Hydrothermal synthesis of Ce-MOF nanozymes:

[0044] 0.354 g of terephthalic acid (H2BDC) was dissolved in 24 mL of the organic solvent N,N-dimethylformamide (DMF), followed by the addition of 8 mL of deionized water containing 1.16 g of (NH4)2Ce(NO3)6. The mixture was stirred continuously for 10 minutes to ensure thorough mixing of the reactants. The homogeneous reaction solution was transferred to a polytetrafluoroethylene-lined high-pressure reactor, which was then placed in an oven at 100°C for 1 hour. After the reaction system cooled naturally to room temperature, the precipitate was collected. The precipitate was dialyzed in deionized water for 2 days using a dialysis bag with a molecular cutoff of 14000 Da. The dialyzed solution was then freeze-dried to obtain Ce-MOF nanozymes.

[0045] (2) Curcumin loading:

[0046] 20 mg Cur was dissolved in 10 mL of ethanol; 5 mg of dried Ce-MOF nanozyme powder was redispersed in 1 mL of ethanol to prepare a 5 mg / mL CeMOF suspension; the ethanol solution of Cur was slowly added dropwise to the CeMOF suspension; the mixture was stirred overnight to allow the drug to be fully loaded onto the nanocarrier. After the reaction was completed, the drug-loaded precipitate was collected by centrifugation, and the precipitate was freeze-dried to obtain Cur@CeMOF nanozyme.

[0047] (3) Surface modification of hyaluronic acid (HA):

[0048] 1 mg of HA was dissolved in 1 mL of deionized water to prepare a 1 mg / mL HA solution; 1 mg of Cur@CeMOF nanozyme was dissolved in 1 mL of ethanol to obtain a 1 mg / mL Cur@CeMOF suspension; the HA solution was added to the redispersed Cur@CeMOF suspension; the mixture was stirred continuously for 24 hours to ensure that HA was fully coated on the surface of the nanoparticles. After the reaction was completed, the final product was collected by centrifugation and freeze-dried to obtain hyaluronic acid modified curcumin-supported cerium nanozyme (HC@CeMOF).

[0049] The hyaluronic acid-modified curcumin-supported cerium-based nanozyme prepared above was characterized and tested, and the results are as follows:

[0050] like Figure 1 As shown, the TEM image of CeMOF nanozymes reveals a polyhedral cubic morphology with an average size of approximately 60 nm. Figure 1 (A in the image), which is consistent with SEM observations ( Figure 1Consistent with (B) in [the original text]. TEM images of HC@CeMOF show that, compared to pure CeMOF, HC@CeMOF exhibits an additional organic coating on the surface (…). Figure 1 C in the image). SEM images also show a slight increase in particle size after HA encapsulation. Figure 1 (D in the middle).

[0051] like Figure 2 As shown, this invention investigated the oxidation state of cerium in CeMOF nanoparticles. XPS analysis revealed that the material mainly contains carbon (C), nitrogen (N), oxygen (O), and cerium (Ce). Figure 2 A in the figure). Charge correction was performed using the sp² hybridized C–C signal at 284.8 eV in the high-resolution C 1s spectrum. Figure 2 In the B spectrum: the peaks at 286.7 eV and 289 eV are attributed to C–OH and C=O groups, respectively, reflecting a rich carbon-oxidative environment; N 1s spectrum ( Figure 2 The C in the spectrum shows a weak peak at 400.5 eV, which is a characteristic peak of the –NH bond; the O 1s spectrum ( Figure 2 The D in the figure shows a main peak at 531.7 eV, corresponding to surface chemisorbed oxygen species (such as O). - O 2- and O2 2- The shoulder peak near 533.5 eV is attributed to lattice oxygen within the CeMOF lattice framework. High-resolution Ce 3d spectrum ( Figure 2 The E-value shows a complex structure formed by the hybridization of Ce 4f orbitals and oxygen orbitals, as well as the presence of partially occupied electronic states. Corresponding low-binding-energy 3d-wave orbitals can be identified. 5 / 2 Orbital and high binding energy 3D 3 / 2 The two distinct spin-orbit splitting peaks in the orbit confirm the presence of Ce in the material. 4+ With Ce 3+ Coexistence. Further XRD analysis was used to characterize the crystal phases of CeMOF and HC@CeMOF. Figure 2 The results show that the synthesized CeMOF has a cubic crystal structure, and its diffraction peaks are consistent with Ce-UIO-66-BDC, proving the successful synthesis of this material. Compared with the uncoated CeMOF, the overall diffraction intensity of HC@CeMOF is slightly reduced, which may be due to the amorphous coating layer formed by curcumin (Cur) and hyaluronic acid (HA) partially masking the diffraction signal. However, the characteristic diffraction peaks of CeMOF are still preserved, indicating that its framework structure remains intact.

[0052] like Figure 3 As shown, the particle size of HC@CeMOF exhibits a normal distribution, with an average particle size of 68.42 nm. Figure 3 (A in the middle). Figure 3Figure B shows the Zeta potentials of each component: due to electrostatic interactions (HA: -23.93 mV, CeMOF: 39.53 mV, curcumin: -10.56 mV), hyaluronic acid and curcumin can spontaneously bind to the CeMOF surface, resulting in a final Zeta potential of approximately -5.73 mV for HC@CeMOF. Colloidal stability was assessed using dynamic light scattering (DLS) and Zeta potential measurements. During the 14-day monitoring period, the particle size fluctuated slightly within the range of ~70 nm to ~80 nm, without any sustained or significant increase; and the overall Zeta potential remained stable. These results collectively confirm the successful preparation of the HC@CeMOF ultrasmall nanozyme.

[0053] Example 2

[0054] The hyaluronic acid-modified curcumin-loaded cerium-based nanozyme (HC@CeMOF) prepared in Example 1 was subjected to CCK-8 cell viability assays, live / dead cell staining assays, and blood compatibility tests. The specific procedures are as follows:

[0055] CCK-8 cell viability assay: Human pancreatic stellate cells (HPSCs) were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 96-well plates. After cell attachment, the medium was replaced with 100 μL of fresh medium containing different concentrations (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL) of HC@CeMOF. The control group received medium without HC@CeMOF. Cells were cultured for 24, 48, or 72 hours, respectively. After the predetermined incubation time, 100 μL of 10% CCK-8 reagent working solution was added to each well, and the cells were incubated at 37°C for 30 minutes. The absorbance of the water-soluble formazan product was measured at 450 nm using a microplate reader. Each experimental group was configured with three replicates. Cell viability was calculated using the following formula:

[0056] Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0057] Live / dead cell staining assay: After CCK-8 assay, 10 μL of a mixed dye of calcein-AM and propidium iodide (PI) was directly added to the culture medium of HPSCs for incubation. The cells were then kept at 37°C in the dark for 30 minutes, and fluorescence imaging was performed using an inverted fluorescence microscope.

[0058] Blood compatibility test: First, fresh whole blood was collected from healthy rats and centrifuged at 3000 rpm for 15 minutes to separate cell components. The collected red blood cells were diluted with physiological saline. Then, 300 μL of the diluted red blood cell suspension was added to each test tube. Positive control group (H2O): 1.2 mL of deionized water was added; negative control group (PBS): 1.2 mL of PBS was added; experimental group: 1.2 mL of physiological saline solution containing different concentrations (20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL) of HC@CeMOF was added. All samples were incubated at 37°C for 2 hours, centrifuged, and the absorbance (OD value) of the supernatant was measured at 540 nm using an ELISA reader. The hemolysis rate was calculated using the following formula:

[0059] Hemolysis rate (%) = (OD value of experimental group - OD value of negative control group) / (OD value of positive control group - OD value of negative control group) × 100%

[0060] The results are as follows Figure 4 As shown, the CCK-8 assay indicated that at a concentration of 60 µg / mL, HC@CeMOF and HPSCs were co-cultured for 3 days, and cell viability remained above 90%. Figure 4 (A) Live / dead cell staining was used to observe HPSCs co-cultured for 3 days. Fluorescence microscopy images showed that at a concentration of 60 µg / mL, the experimental group exhibited good cell compatibility compared to the control group. Figure 4 (B in the text). Additionally, its blood compatibility was assessed, such as... Figure 4 As shown in C, with H2O as the positive control and PBS as the negative control, the hemolysis rates of HC@CeMOF at concentrations of 20 µg / mL, 40 µg / mL, 60 µg / mL and 80 µg / mL were 0.2%, 0.7%, 1% and 1.2%, respectively. All values ​​were far below the 5% hemolysis threshold, indicating that it has excellent blood compatibility.

[0061] Example 3

[0062] The antioxidant properties of the hyaluronic acid-modified curcumin-supported cerium-based nanozyme (HC@CeMOF) prepared in Example 1 were tested, as follows:

[0063] Using nitrogen-centered free radicals (DPPH·) and cation free radicals (ABTS·) + The scavenging experiments systematically evaluated the scavenging capabilities of HC@CeMOF for reactive nitrogen species (RNS) and reactive oxygen species (ROS).

[0064] DPPH· scavenging assay: Different masses (40 μg, 80 μg, 120 μg, and 160 μg) of HC@CeMOF were added to 2 mL of DPPH· solution. The blank group consisted of only 2 mL of DPPH· solution (no sample).

[0065] ABTS· + Scavenging experiment: HC@CeMOF with the same mass gradient was mixed with 2 mL of ABTS· + Mix the working fluid.

[0066] All the above mixed solutions were incubated at 37°C in the dark for 30 minutes, and then analyzed using a UV spectrophotometer at 519 nm (DPPH·) and 734 nm (ABTS·). + The absorbance was measured at the specified wavelength. The free radical scavenging rate was calculated using the following formula:

[0067] Sweep rate = (A0 - A) ) / A0× 100%

[0068] Where A0 is the absorbance of the blank group, A The absorbance of the supernatant in the experimental group is given.

[0069] The results are as follows Figure 5 As shown, a commercially available detection kit was used to evaluate its ability to remove hydrogen peroxide (H2O2) and superoxide anion (O2). 2- ) ability ( Figure 5 A and Figure 5 (B in the text). The results showed that HC@CeMOF could remove 69.32% of H2O2 and 51.05% of O2 within 30 minutes. 2- In addition, the total antioxidant capacity of HC@CeMOF was evaluated using the ABTS assay. The results showed that the ABTS scavenging efficiency gradually increased with increasing HC@CeMOF concentration. Figure 5 The C in the text is missing. By monitoring the change of the characteristic absorption peak of ABTS at 734 nm, it was found that HC@CeMOF could reduce the absorbance in a concentration-dependent manner, with the highest clearance rate reaching 52.17% at 60 µg / mL. The corresponding optical images of the solution further confirmed that the solution color gradually lightened with increasing nanozyme concentration. Subsequently, DPPH analysis was used (… Figure 5 (D) The ability of HC@CeMOF to scavenge reactive nitrogen species (RNS) was evaluated. Consistent with the results of the ABTS experiment, increasing the concentration of HC@CeMOF led to a decrease in the absorbance of the characteristic peak of DPPH· at 517 nm. In addition, the gradual fading of the solution color also optically confirmed that HC@CeMOF has excellent in vitro antioxidant activity.

[0070] Example 4

[0071] The binding affinity of the hyaluronic acid-modified curcumin-supported cerium-based nanozyme (HC@CeMOF) prepared in Example 1 was tested, and the specific process is as follows:

[0072] A semi-flexible molecular docking strategy was employed to evaluate the binding affinity and conformation between hyaluronic acid (HA) and CD44 protein. The CD44 structure was obtained from the Protein Database (PDB) and preprocessed using PyMOL. Hydrogen atoms were added to both the ligand (HA) and receptor (CD44 protein) using AutoDockVina, and partial charges were calculated. Active binding sites were determined based on existing ligand-receptor complex information in the PDB database. Molecular docking was then performed using AutoDockVina, and binding affinity was evaluated based on docking scores (binding energies). The docking conformation and intermolecular interactions were further visualized and analyzed using Discovery Studio Visualizer to elucidate the binding mode. To better simulate the interaction between HA and CD44 under physiological conditions, molecular dynamics simulations were further performed. The simulations were completed using GROMACS 2022 software. Hydrogen atoms were first added to the protein using the pdb2gmx module, followed by system preparation using the editconf, solvate, and grompp modules, including defining the simulation box (5.979 × 6.599 × 7.250 nm). 3 The system was designed to neutralize its charge by adding solvent water molecules and counterions. Charge distribution of small molecules was determined using the AM1-BCC method in Anttecamber, with ligands using the GAFF force field, proteins using the Amber ff99SB force field, and water molecules using the TIP3P model. The system was first subjected to energy minimization using a steepest descent method over 50,000 steps; then, under conditions of heavy atom position constraints, equilibration was performed for 100 ps each in the NVT and NPT ensembles; finally, a production simulation was conducted for 100 ns at 310 K and 1 bar pressure (using Parrinello-Rahman pressure coupling). Long-range electrostatic interactions were calculated using the particle grid Ewald method, and van der Waals interactions were cut off at a distance of 1 nm. Chemical bonds involving hydrogen atoms were constrained using the SHAKE algorithm. Trajectory data were saved every 10 ps.

[0073] The results are as follows Figure 6As shown, the binding affinity of HA to CD44 on the macrophage surface was first assessed by molecular docking, revealing a strong binding potential with a binding energy of -5.47 kcal / mol. Interface analysis indicated that key residues such as ILE304, SER305, ASP302, GLU37, and ASN25 primarily participate in the HA-CD44 interaction, with hydrogen bonding being the dominant binding mode. Figure 6 Structural analysis of representative MD conformations further confirmed that HA forms hydrogen bonds with CYS77, ILE304, and ARG78, while also undergoing Pi-Alkyl and Alkyl interactions with PHE303. Figure 6 (B in the text). RMSF results showed that the RMSF values ​​of residual Val79, Phe378, and Glu379 fluctuated significantly, with peak RMSF values ​​of 4.017, 5.348, and 6.113, respectively. Figure 6 (C in the text). After removing the periodicity of the MD trajectory, MM / PBSA calculations were performed on the complex within an 80-100 ns window, showing a total binding free energy of -26.81 kcal / mol ( Figure 6 (D in the text). Under physiological conditions, HA mainly interacts with CYS77, ILE304, ARG78, and PHE303 residues. Figure 6 (E in the text).

[0074] Example 5

[0075] The hyaluronic acid-modified curcumin-loaded cerium-based nanozyme (HC@CeMOF) prepared in Example 1 was used to verify its efficacy in treating chronic pancreatitis, as detailed below:

[0076] (1) Modeling: A chronic pancreatitis (CP) model induced by cytosine was successfully established by repeatedly inducing acute pancreatitis. Specifically, mice were randomly divided into a control group, a CP model group, a Cur group, a Cur@CeMOF group, and an HC@CeMOF group. The control group was injected with 200 µL of physiological saline via the tail vein, while the other groups were injected intraperitoneally with cytosine at a dose of 50 µg / kg / hour, 6 times a day for 3 days a week for a total of 4 weeks. The weight of the mice was recorded after the last injection each week, and euthanasia was performed after the last injection.

[0077] (2) HC@CeMOF treatment for chronic pancreatitis: Starting from the second week of administration of cytosine, the CP model group, Cur group, Cur@CeMOF group, and HC@CeMOF group were treated with daily tail vein injections of saline, Cur (6 mg / kg / day), Cur@CeMOF (6 mg / kg / day), or HC@CeMOF (6 mg / kg / day), respectively, for 3 consecutive weeks. After treatment, the mice were euthanized, and serum samples were collected. The expression levels of inflammatory cytokines (including IL-1β, IL-2, IL-4, IL-10, IL-12, IL-17, IL-18, myeloperoxidase activity, and TGF-α) were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Pancreatic tissue was removed, photographed, and weighed, and the ratio of pancreas to body weight was recorded. A portion of the pancreatic tissue was used for Western blot (WB) detection to detect the expression of fibrosis-related proteins and proteins associated with the NF-κB signaling pathway. The remaining pancreatic tissue was embedded and processed for histological evaluation, including H&E staining, Masson staining, and Sirius red staining, to assess the degree of pancreatic fibrosis in mice.

[0078] The results are as follows Figure 7 As shown, the in vivo imaging system (IVIS) revealed that the labeled nanomaterials accumulated in large quantities in the pancreas within 5 minutes of injection and persisted for at least 24 hours. Figure 7 (A) Two hours after injection, the biodistribution of the nanomaterials was further evaluated: In the CP model, unHA-modified Cur@CeMOF was mainly enriched in the liver and kidneys, with very little distribution in the pancreas; while HA-modified HC@CeMOF showed significant accumulation in the pancreas, with reduced distribution in the liver and kidneys. In healthy mice, the accumulation of HC@CeMOF in the pancreas was negligible. Figure 7 (B in the text). Further verification of the above results was achieved by fluorescence microscopy observation of frozen sections of pancreatic tissue: Numerous punctate red fluorescent signals were observed in the pancreas of CP mice treated with HC@CeMOF, while only sporadic signals were observed in the Cur@CeMOF group. Figure 7 (C in the middle).

[0079] like Figure 8 As shown, gross examination of the pancreas revealed significant atrophy and sclerosis in CP mice compared to the control group. Drug treatment partially restored pancreatic morphology, with the HC@CeMOF group exhibiting the most significant improvement. Figure 8 (A) The expression levels of pancreatic fibrosis markers α-SMA and collagen I were detected by Western blotting analysis. The results showed that all drug treatments reduced the expression of these fibrosis proteins in CP mice, with HC@CeMOF expression being the most significantly downregulated. Figure 8 B and Figure 8C). Histopathological evaluation of the pancreatic tissue was performed. Figure 8 The staining methods included H&E staining, Masson staining, and Sirius red staining. The control group showed normal pancreatic tissue structure with no signs of fibrosis. In contrast, CP model mice exhibited widespread acinar atrophy, inflammatory infiltration, and diffuse interstitial fibrosis. Cur or Cur@CeMOF treatment slightly reduced acinar atrophy and fibrosis, while HC@CeMOF treatment significantly reversed CP-related pathological features, including acinar cell atrophy, extracellular matrix (ECM) accumulation, ductal dilation, and immune cell infiltration.

[0080] like Figure 9 As shown, based on these results, the mechanism by which HC@CeMOF alleviates CP includes effectively clearing ROS, inhibiting NF-κB / p65 expression, subsequently regulating the inflammatory cytokine spectrum, and ultimately reducing pancreatic fibrosis. Figure 9 In section A), to assess the cytokine expression profile within pancreatic tissue, this invention used ELISA to detect the levels of pro-inflammatory and anti-inflammatory cytokines in CP mice treated with nanomaterials. The results showed that injection of linalool significantly increased the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-2, IL-12, IL-17, and IL-18). Figure 9 B- Figure 9 In addition to reducing the levels of G), it also reduced the levels of anti-inflammatory cytokines (IL-4 and IL-10). Figure 9 H and Figure 9 The study confirmed the presence of a significant inflammatory response during CP induction. Treatment with Cur, Cur@CeMOF, and HC@CeMOF inhibited pro-inflammatory cytokines while upregulating anti-inflammatory cytokine expression, indicating that all three agents modulated the inflammatory response. Among them, HC@CeMOF showed the most significant regulatory effect on cytokine expression, possibly attributed to its enhanced hyaluronic acid-mediated targeting ability, which increased accumulation at CP lesion sites, thereby improving the in vivo bioavailability of the nanomaterials. Furthermore, this invention evaluated myeloperoxidase (MPO) expression—an enzyme primarily secreted by activated neutrophils, whose activity reflects neutrophil proliferation and tissue damage, and is an important indicator of CP inflammation. Results showed that compared to the CP model group, all three treatment regimens (Cur, Cur@CeMOF, and HC@CeMOF) significantly reduced MPO levels, with HC@CeMOF exhibiting the strongest inhibitory effect, superior to the Cur and Cur@CeMOF groups. Figure 9 J in the middle.

[0081] The embodiments of this application have been described above with reference to the accompanying drawings. Specific examples have been used to illustrate the principles and implementation methods of this application. The description of the above embodiments is only for the purpose of helping to understand the core ideas of this application. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. The application of a curcumin-loaded cerium-based nanozyme in the preparation of a drug for treating chronic pancreatitis, characterized in that, The curcumin-loaded cerium-based nanozyme includes a core carrier, a drug loaded on the core carrier, and a shell; The core carrier is a cerium-based metal-organic framework (CeMOF); the drug is curcumin (Cur); and the outer shell is hyaluronic acid.

2. The application according to claim 1, characterized in that, The preparation method of the curcumin-supported cerium-based nanozyme includes the following steps: CeMOF nanozyme powder was dispersed in ethanol to obtain a CeMOF suspension; Curcumin was dissolved in ethanol to obtain a curcumin solution; Hyaluronic acid is dissolved in water to obtain a hyaluronic acid solution; CeMOF suspension was mixed with curcumin solution and reacted. After the reaction was completed, the mixture was centrifuged and freeze-dried to obtain Cur@CeMOF nanozyme. The hyaluronic acid solution was dispersed in the suspension of the Cur@CeMOF nanozyme, mixed evenly, reacted, centrifuged, and freeze-dried to obtain the curcumin-loaded cerium-based nanozyme.

3. The application according to claim 2, characterized in that, The CeMOF nanozyme powder was prepared by the following steps: Terephthalic acid was dissolved in an organic solvent, and then an aqueous solution of (NH4)2Ce(NO3)6 was added. The mixture was stirred until homogeneous, then transferred to a high-pressure reactor and heated to react. After the reaction was completed, the mixture was cooled to room temperature, dialyzed, and freeze-dried to obtain the CeMOF nanozyme powder.

4. The application according to claim 3, characterized in that, The ratio of (NH4)2Ce(NO3)6 to terephthalic acid in the aqueous solution of (NH4)2Ce(NO3)6 is 1.16 g: 0.354 g.

5. The application according to claim 3, characterized in that, The heating reaction temperature is 90℃-110℃; the reaction time is 0.5 h-1.5 h.

6. The application according to claim 2, characterized in that, The ratio of CeMOF nanozyme powder, curcumin, and hyaluronic acid is 5 mg: 20 mg: 1 mg.

7. The application according to claim 6, characterized in that, The concentration of the CeMOF suspension was 5 mg / mL; The concentration of the curcumin solution is 2 mg / mL; the concentration of the hyaluronic acid solution is 1 mg / mL.

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