Porphyra polysaccharide-cerium self-assembled nano-enzyme, and preparation method and application thereof

Abstract

This invention discloses a self-assembled nanozyme of porphyra polysaccharide-Ce, its preparation method, and its application. The nanozyme is obtained by the self-assembly of porphyra polysaccharide and Ce ions. The preparation method is as follows: Pyra polysaccharide (PYP) and Ce(NO3)3·6H2O are weighed at a mass ratio of (0.9–1.1):1 and dissolved in ultrapure water. The solution is filtered using filter paper with a pore size of 15–20 μm, and the filtrate is mixed under ultrasonic conditions at a frequency of 38–42 kHz in an ultrasonic cleaner to obtain a mixed liquid. The mixture is then stirred using a magnetic stirrer at a speed of 800–1200 rpm for 30 min to obtain the Ce-PYP nanozyme. The porphyra polysaccharide-Ce self-assembled nanozyme of this invention can effectively improve the in vivo stability of porphyra polysaccharide, enhance its ability to antagonize calcium oxalate crystal formation in vitro, enhance its antioxidant properties, enhance its ability to antagonize cellular oxidative damage under high oxalate conditions, and enhance its ability to antagonize kidney damage under high glyoxylate conditions. It has good application prospects in the preparation of products for the treatment of calcium oxalate stones and kidney damage.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a self-assembled nanozyme of porphyra polysaccharide-Ce, its preparation method, and its application. Background Technology

[0002] Kidney stones are one of the most common clinical diseases in urology, and their incidence is on the rise globally, posing a significant public health problem. In China, the prevalence of kidney stones is 5.8%, with a recurrence rate as high as 50% within 5-10 years. Kidney stones can cause serious complications such as urinary tract infections, lower back pain, gastrointestinal reactions, hydronephrosis, and decreased renal function. The high recurrence rate increases the need for surgical intervention. This not only brings physical pain and psychological and social distress to patients but also imposes a heavy economic burden on individuals, families, and society. Types of kidney stones include calcium oxalate stones, uric acid stones, calcium phosphate stones, cystine stones, and magnesium ammonium phosphate stones. Approximately 80% of kidney stones are composed of calcium oxalate (CaOx) crystals, making it the hardest type of stone. Among CaOx stones, the COM crystal form is the most common.

[0003] Currently, the main drugs used clinically to treat CaOx stones include thiazide diuretics, calcium citrate, and potassium citrate. These drugs increase the soluble portion of urinary calcium and inhibit crystal growth through chelation. However, recurrence is common after surgery, and this phenomenon has not been effectively improved, possibly because the currently used drugs do not mitigate the kidney damage caused during the procedure. Damaged cells can exacerbate the nucleation, growth, and aggregation of urinary microcrystals, processes that can be inhibited after the damaged cells are repaired. Natural plant polysaccharides can repair damaged cells and may be more effective than commonly used drugs in inhibiting stone formation and recurrence.

[0004] Porphyra yezoensis polysaccharide (PYP) possesses a wide range of biological activities, exhibits minimal toxicity to normal cells, and demonstrates significant nephroprotective effects. PYP promotes cell adhesion and endocytosis of microcomplex crystals on the cell surface, reducing crystal nucleation sites. Simultaneously, PYP mimics various enzyme activities, exerting antioxidant effects to counteract oxidative stress damage to cells caused by a high-oxalate environment, reducing the nucleation, growth, and aggregation of microcomplex crystals on the cell surface, thereby reducing the recurrence of urinary tract stones. However, as a natural polysaccharide, PYP is easily metabolized in vivo, has a short circulation time, and carries the potential risk of insufficient bioavailability.

[0005] Therefore, developing drugs that retain the advantages of PYP while having better stability and bioavailability is of great significance for clinical application in the treatment of recurrent stones.

[0006] Self-assembled nanozymes generally refer to nanostructures that spontaneously assemble from molecules and ions (such as polysaccharides, peptides, or metal ions) through non-covalent interactions (such as electrostatic interactions, hydrogen bonds, and π–π interactions). These structures can form enzyme-like active sites on their surface or inside, thereby mimicking the catalytic function of natural enzymes. Compared to traditional nanozymes, self-assembled nanozymes have advantages such as simple construction process, strong structural tunability, and good biocompatibility. Furthermore, their catalytic performance can be optimized by controlling the assembly units and microstructure. Relying on the high specific surface area and multifunctional synergistic effect brought about by ordered nanostructures, they not only exhibit various enzyme-like activities (such as peroxidase, superoxide dismutase, and catalase-like activities), but also show promising application prospects in areas such as regulating oxidative stress, anti-inflammation, and disease treatment, becoming an important candidate nanomaterial in biomedical research in recent years. In summary, self-assembled nanomaterials have advantages such as structural stability, low cost, engineerability, multi-enzyme-like activity, and small particle size, making them better suited for the treatment of kidney stones.

[0007] However, different coordinating metals will bring different properties to the self-assembled nanozymes. At present, it is not clear which metal ion can self-assemble with porphyria polysaccharide to form the nanozyme with the best activity. Therefore, the preparation of a self-assembled nanozyme of porphyria polysaccharide and metal ion with excellent calcium oxalate crystal inhibition effect and antioxidant activity is of great significance for the treatment of kidney stones. Summary of the Invention

[0008] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides a self-assembled nanozyme of laver polysaccharide-Ce, its preparation method, and its application.

[0009] The first objective of this invention is to provide a nanozyme.

[0010] A second objective of this invention is to provide a method for preparing nanozymes.

[0011] A third objective of this invention is to provide the application of the nanozyme and the nanozyme prepared by the method in antioxidant applications.

[0012] A fourth objective of this invention is to provide the application of the nanozyme and the nanozyme prepared by the preparation method in alleviating kidney injury.

[0013] The fifth objective of this invention is to provide the application of the nanozyme and the nanozyme prepared by the preparation method in the preparation of calcium oxalate stone treatment products.

[0014] The sixth objective of this invention is to provide a calcium oxalate crystal inhibitor.

[0015] To achieve the above objectives, the present invention is implemented through the following solution: This invention claims protection for the following: A nanozyme, wherein the nanozyme is obtained by self-assembly of porphyra polysaccharide and Ce ions.

[0016] Preferably, the mass ratio of the laver polysaccharide to Ce ions is 0.9 to 1.1:1.

[0017] The present invention also claims a method for preparing a nanozyme, comprising the following steps: dissolving laver polysaccharide and Ce(NO3)3·6H2O in water, filtering with filter paper to obtain the filtrate, mixing under ultrasonic conditions, stirring for 20-40 min, and obtaining the nanozyme.

[0018] Preferably, the filter paper has a pore size of 15–20 μm.

[0019] Preferably, the frequency of the ultrasound is 38–42 kHz.

[0020] Preferably, the stirring speed is 800-1200 rpm.

[0021] And the following applications: Application of the nanozyme or the nanozyme prepared by the preparation method in antioxidation.

[0022] Application of the nanozyme or the nanozyme prepared by the preparation method in alleviating kidney damage.

[0023] Application of the nanozyme or the nanozyme prepared by the preparation method in the preparation of calcium oxalate stone treatment products.

[0024] And a calcium oxalate crystal inhibitor, wherein the calcium oxalate crystal inhibitor comprises the nanozyme or the nanozyme prepared by the preparation method.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The self-assembled nanozyme of porphyra polysaccharide-Ce of the present invention can effectively improve the in vivo stability of porphyra polysaccharide; enhance the ability to antagonize calcium oxalate crystal formation in vitro (the effect is better than that of Ce(NO3)3·6H2O+PYP at the same concentration); enhance antioxidant properties; enhance the ability to antagonize cellular oxidative damage under high oxalate environment (the effect is better than that of Ce(NO3)3·6H2O+PYP at the same concentration); and enhance the ability to antagonize kidney damage under high glyoxylate environment (the effect is better than that of Ce(NO3)3·6H2O+PYP at the same concentration). It has good application prospects for the preparation of calcium oxalate stones and kidney damage treatment products. Attached Figure Description

[0026] Figure 1The results are TEM analysis results; where A to D are the TEM analysis results of Ce-PYP, Cu-PYP, Fe-PYP, and Mn-PYP nanozymes prepared by Ce(NO3)3·6H2O and CuSO4, FeCl3, (CH3COO)2Mn and PYP (mass ratio of 0.9 to 1.1:1), respectively.

[0027] Figure 2 The results of experiments on the antagonistic formation of calcium oxalate crystals of different concentrations of PYP, Cu-PYP, Fe-PYP, Mn-PYP, Ce-PYP, Ce(NO3)3·6H2O, and Ce(NO3)3·6H2O+PYP are presented.

[0028] Figure 3 The results are TEM, elemental analysis, XPS, and ESR analyses of Ce-PYP; where A is the TEM detection result, B is the elemental analysis detection result, C-D are the XPS detection results, and E is the ESR result.

[0029] Figure 4 Experimental results on the regulation of calcium oxalate crystal morphology by different concentrations of Ce-PYP.

[0030] Figure 5 The results of Ce-PYP simulated antioxidant enzyme activity experiments are shown. Among them, A to D are the antioxidant experiments of Ce-PYP at different concentrations for DPPH, ABTS, MB, and TMB, respectively, and E to H are the absorbance results of the antioxidant experiments for DPPH, ABTS, MB, and TMB, respectively.

[0031] Figure 6 Experiments were conducted to assess biosafety and the effects of materials on cell viability under high oxalate conditions. Specifically, A was a blood compatibility experiment with different concentrations of Ce-PYP; B was an evaluation of the effects of different concentrations of Ce-PYP on cell viability using the CCK-8 assay; C was an evaluation of the effects of different concentrations of Ce-PYP on cell viability under high oxalate conditions using the CCK-8 assay; and D was an evaluation of the effects of Ce(NO3)3·6H2O, PYP, and Ce(NO3)3·6H2O+PYP on cell viability under high oxalate conditions using the CCK-8 assay.

[0032] Figure 7 This is a cellular localization map of Ce-PYP.

[0033] Figure 8 The DCFH-DA probe was used to detect changes in ROS fluorescence intensity distribution after NRK-52E cells were treated under different conditions.

[0034] Figure 9 To detect cell death in NRK-52E cells after treatment with different conditions using the Calcein-AM / PI double staining method.

[0035] Figure 10 The results of different drug treatment groups after constructing a C57 mouse kidney stone model using glyoxylic acid are shown. Among them, A is the result of H&E staining of kidney tissue, and B and C are the results of blood Cr and BUN, respectively. Detailed Implementation

[0036] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0039] Example 1: Preparation and characterization of nanozymes self-assembled from laver polysaccharides and different metal ions I. Experimental Methods 1. Preparation of Ce-PYP nanozymes (1) Weigh PYP and Ce(NO3)3·6H2O according to a mass ratio of (0.9~1.1):1 and dissolve them in an appropriate amount of ultrapure water; (2) Filter the solution using filter paper with a pore size of 15-20 μm, and mix the solution under the ultrasonic cleaning instrument with a frequency of 38-42 kHz to obtain a mixed liquid. Stir the solution for 30 min at a speed of 800-1200 rpm using a magnetic stirrer to obtain the self-assembled nanozyme Ce-PYP.

[0040] 2. Preparation of Cu-PYP, Fe-PYP, and Mn-PYP nanozymes The same method as the preparation of Ce-PYP self-assembled nanozymes was used, except that Ce(NO3)3·6H2O was replaced with CuSO4, FeCl3 and (CH3COO)2Mn, respectively.

[0041] 3. Morphological characterization of Ce-PYP, Cu-PYP, Fe-PYP and Mn-PYP nanozymes The morphology of Ce-PYP, Cu-PYP, Fe-PYP, and Mn-PYP nanozymes was characterized by transmission electron microscopy (TEM). The specific methods are as follows: Small amounts of Ce-PYP, Cu-PYP, Fe-PYP, and Mn-PYP were taken separately and added to deionized water. The mixtures were ultrasonically dispersed for 5–10 min to obtain homogeneous suspensions. These suspensions were then pipetted onto an ultrathin carbon film copper grid and dried under an infrared lamp or by natural air drying. The microstructure and particle size of the samples were observed using a transmission electron microscope at an accelerating voltage of 200 kV.

[0042] II. Experimental Results like Figure 1 As shown, Ce-PYP nanoparticles are uniformly dispersed and have a particle size of less than 20 nm, which is smaller than that of Cu-PYP, Fe-PYP, and Mn-PYP.

[0043] Example 2: The role of seaweed polysaccharide and nanozymes self-assembled with different metal ions in regulating the formation of calcium oxalate crystals. I. Experimental Methods Different concentrations of Ce-PYP, Cu-PYP, Fe-PYP, and Mn-PYP nanozymes prepared in Example 1 were formulated to investigate their role in regulating calcium oxalate crystal formation. The specific methods are as follows: (1) Prepare Tris-HCl buffer solution Dissolve 2.4228 g Tris and 10.53 g NaCl in 2 L of ultrapure water, adjust the pH with HCl to prepare a buffer solution with a pH of 7.38–7.42, a Tris concentration of 10 mM, and a NaCl concentration of 90 mM. Divide the 2 L buffer solution into two 1 L portions in large beakers.

[0044] (2) Preparation of CaCl2 solution and Na2C2O4 solution Weigh 0.222 g of CaCl2 and 0.134 g of Na2C2O4, dissolve them separately in 1 L of buffer solution, stir to dissolve, and prepare 1 mmol / L CaCl2 solution and 0.5 mmol / L Na2C2O4 solution.

[0045] (3) Crystal regulation experiment Take a 6-well plate and add the prepared CaCl2 and Na2C2O4 solutions in a 1:1 volume ratio to each well. At the same time, prepare PYP, Cu-PYP, Fe-PYP, Mn-PYP, Ce-PYP, Ce(NO3)3·6H2O and Ce(NO3)3·6H2O+PYP solutions with concentrations of 0, 60, 100, 200, and 400 μg / mL, respectively. In the Ce(NO3)3·6H2O+PYP experimental group, Ce(NO3)3·6H2O and PYP of equal concentration are added to the well simultaneously, and its concentration is the same as that of Ce-PYP in the same concentration group. Each well is adjusted to a volume of 5 mL with an equal volume of CaCl2 and Na2C2O4 solution. After standing for 24 h, observe under a microscope.

[0046] II. Experimental Results like Figure 2 As shown, the number of calcium oxalate crystals in the microscopic field of Ce-PYP at a concentration of 60 μg / mL is less than that at a concentration of 0 μg / mL, and is also less than that in PYP, Cu-PYP, Fe-PYP, and Mn-PYP at concentrations of 60, 100, 200, and 400 μg / mL. In contrast, the crystals in the Ce(NO3)3·6H2O and Ce(NO3)3·6H2O+PYP groups become needle-shaped, and the crystals continue to increase in size as the concentration increases.

[0047] In summary, Ce-PYP can regulate the formation of calcium oxalate crystals, reducing crystal nucleation, and its effect is superior to PYP, Cu-PYP, Fe-PYP, Mn-PYP, Ce(NO3)3·6H2O and Ce(NO3)3·6H2O+PYP.

[0048] Example 3 Characterization of Ce-PYP I. Experimental Methods The Ce-PYP prepared in Example 1 was characterized as follows: (1) Observation by transmission electron microscopy (TEM) Take a small amount of Ce-PYP, add it to deionized water, and ultrasonically disperse for 5–10 min to obtain a uniform suspension. Use a pipette to drop the suspension onto an ultrathin carbon film copper grid, and dry it under an infrared lamp or allow it to air dry naturally. Observe the microstructure and particle size of the sample using a transmission electron microscope at an accelerating voltage of 200 kV.

[0049] (2) Energy-dispersive X-ray spectroscopy (EDS) analysis A small amount of Ce-PYP was added to deionized water and ultrasonically dispersed for 5–10 min to obtain a homogeneous suspension. This suspension was then pipetted onto an ultrathin carbon film copper grid and dried under an infrared lamp or by air drying. Energy dispersive X-ray spectroscopy (EDS) was used to perform elemental surface scanning and point-to-point analysis on the selected area, acquiring characteristic X-ray signals of C, N, O, and Ce to obtain the distribution of each element in the nanostructure.

[0050] (3) X-ray photoelectron spectroscopy (XPS) analysis Ce-PYP was freeze-dried into powder, and an appropriate amount was pressed onto double-sided conductive adhesive. X-ray photoelectron spectroscopy was used with monochromatic Al Kα rays (hν = 1486.6 eV) as the excitation source, with a power of approximately 150 W. The full-spectrum scan pass energy was 100 eV with a step size of 1.0 eV; the high-resolution narrow-spectrum scan pass energy was 30 eV with a step size of 0.05 eV. The vacuum level of the analytical chamber was better than 1 × 10⁻⁶. - 8 mbar. All binding energies were charge-corrected using the C 1s (284.8 eV) of surface-contaminated carbon. The characteristic peaks of Ce metal were subjected to peak fitting to analyze its chemical valence state and possible coordination forms with polysaccharide groups.

[0051] (4) Electron Spin Resonance (ESR) Analysis Weigh an appropriate amount of lyophilized Ce-PYP powder, pack it into a quartz capillary tube, compact it, and then place it in a resonant cavity. Tests were performed using an electron spin resonance spectrometer at room temperature. Typical parameter settings were as follows: microwave frequency approximately 9.4 GHz (X-band), central magnetic field 3360 G, sweep width 100–500 G, microwave power 2 mW, modulation amplitude 1.0 G, modulation frequency 100 kHz, and time constant 40.96 ms. The obtained spectra were used to detect the presence of unpaired electrons (such as signals from free radicals or specific paramagnetic metal ions) in Ce-PYP.

[0052] II. Experimental Results like Figure 3 As shown in Figure A, Ce-PYP nanoparticles were observed to be uniformly dispersed under transmission electron microscopy; as shown in Figure A. Figure 3 As shown in Figure B, the EDS elemental analysis results revealed the presence of C, N, O, and Ce elements; Figure 3 As shown in C and D, XPS detection results show that Ce-PYP retains the characteristic peaks of PYP (such as O1s and C1s), and also exhibits a new characteristic peak (Ce3d) brought about by Ce ions; Figure 3As shown in Figure E, the ESR detection results showed that no obvious signal was observed in the Background group, only baseline noise, indicating that there were almost no unpaired electrons or free radicals in the system. The PYP group showed a clear ESR characteristic signal, with multiple splitting peaks visible in the range of approximately 3480–3520 G, suggesting the presence of a certain concentration of free radicals in the system. The ESR signal intensity of the Ce-PYP group was stronger than that of the PYP group, and the spectral morphology was basically the same, indicating that the introduction of Ce did not change the type of free radicals, but affected the number of free radicals. These results prove the successful synthesis of the self-assembled nanozyme Ce-PYP.

[0053] Example 4: Effect of different concentrations of Ce-PYP on the regulation of calcium oxalate crystal morphology I. Experimental Methods Same as Example 2, except that step (3) is changed to: take a 6-well plate, add the prepared CaCl2 and Na2C2O4 solutions in a 1:1 ratio to each well, and add Ce-PYP at concentrations of 0, 15, 16, 17, 18, and 19 μg / mL respectively. Make up the volume of each well with an equal volume of CaCl2 and Na2C2O4 solutions to 5 mL, let stand for 24 h, and observe under a microscope.

[0054] II. Experimental Results like Figure 4 As shown, at a concentration of 0 μg / mL, the calcium oxalate crystals under a microscope exhibit the classic COM crystal form, with a uniform and relatively dense distribution. At a concentration of 15 μg / mL, the calcium oxalate crystals become relatively rounded in the

[001] crystal direction. At a concentration of 16 μg / mL, the calcium oxalate crystals become even rounder, and the distribution density decreases. At a concentration of 17 μg / mL, the calcium oxalate crystals transform into the COD crystal form, and the distribution density further decreases. At a concentration of 18 μg / mL, the calcium oxalate crystals shrink further, and the distribution density further decreases. At a concentration of 19 μg / mL, the calcium oxalate crystals transform into an amorphous state.

[0055] In summary, Ce-PYP can regulate the formation of calcium oxalate crystals, altering the crystal morphology in a concentration-dependent manner and reducing crystal nucleation.

[0056] Example 5: Ce-PYP mimics the activity of antioxidant enzymes I. Experimental Methods The simulated antioxidant enzyme activity of Ce-PYP prepared in Example 1 was detected using the following experimental method: (1) DPPH free radical scavenging activity Different concentrations of Ce-PYP were mixed with an equal volume of 250 μM DPPH ethanol solution and reacted for 30 min. The final concentrations of Ce-PYP were 0, 25, 50, 100, 200, and 400 μg / mL, and the final concentration of DPPH was 125 μM. The absorption spectra of the reaction system in the range of 400–700 nm were detected using a microplate reader.

[0057] (2) ABTS free radical scavenging activity 7 mM ABTS solution was mixed with 2.45 mM potassium persulfate solution and incubated in the dark at room temperature for 12 h to activate ABTS+· free radicals. Different concentrations of Ce-PYP were mixed with equal volumes of ABTS+· solution and reacted for 20 min, resulting in final Ce-PYP concentrations of 0, 25, 50, 100, 200, and 400 μg / mL. The absorption spectra of the reaction system in the range of 400–900 nm were detected using a microplate reader.

[0058] (3) MB (methylene blue) determination The ·OH radicals generated by the Fenton reaction can degrade MB; therefore, the residual OD value of MB in solution reflects the ability of Ce-PYP to scavenge ·OH. Different concentrations of Ce-PYP were mixed with MB, and then Fe was added... 2+ / H2O2(Fe 2+ The final concentrations of Ce-PYP were 0, 25, 50, 100, 200, and 400 μg / mL, respectively, and the final concentration of MB was 100 μM. The absorption spectra of the reaction system in the range of 500–900 nm were detected using an ELISA reader.

[0059] (2) TMB experiment Ce-PYP solutions of different concentrations were mixed with 125 μM TMB and Fenton solution (Fe2+). 2+ Mix 1 mM H2O2 (10 mM) with an equal volume of HAc / NaAc buffer (pH 4.5) to obtain final Ce-PYP concentrations of 0, 25, 50, 100, 200, and 400 μg / mL. After reacting for 5 min, the absorption spectrum of the reaction system in the range of 500–800 nm was detected using a microplate reader.

[0060] II. Experimental Results like Figure 5As shown, A to D are solution photographs of Ce-PYP at different concentrations for DPPH, ABTS, MB, and TMB antioxidant experiments, respectively. It can be seen that Ce-PYP concentration-dependently changes the solution color. E to F are the absorbance results of DPPH, ABTS, MB, and TMB antioxidant experiments, respectively. It can be seen that Ce-PYP concentration-dependently shifts the absorbance curve downward in the Y-axis direction, indicating that Ce-PYP concentration-dependently scavenge DPPH, ABTS, and ·OH free radicals.

[0061] Example 6: Biocompatibility of Ce-PYP I. Experimental Methods The blood compatibility and cell viability of the Ce-PYP nanozyme prepared in Example 1 were tested to assess its safety. The specific methods are as follows: 1. Blood compatibility Fresh whole blood was collected from healthy animals (SD rats), and an anticoagulant (heparin sodium) was added to prevent clotting. The blood was centrifuged at 3000 rpm for 10 min, and the supernatant plasma was discarded. Red blood cells were washed three times with PBS buffer (pH 7.4) to prepare a 2% (v / v) red blood cell suspension. Ce-PYP was dispersed in PBS buffer to prepare sample solutions of different concentrations (25, 50, 100, 200, 400 μg / mL). Equal volumes of red blood cell suspension were mixed with different concentrations of Ce-PYP nanozyme solution (0.5 mL each), gently mixed, and incubated at 37°C for 2 h.

[0062] ddH2O was used as a positive control group, and PBS buffer was used as a negative control group. After incubation, each group of samples was centrifuged at 3000 rpm for 10 min, the supernatant was collected, and the absorbance (OD value) was measured at 540 nm.

[0063] Calculate the hemolysis rate using the following formula: Hemolysis rate (%) = [(OD_sample)] OD_PBS) / (OD_ddH2O [OD_PBS]×100%; In the formula, OD_sample represents the absorbance of the experimental group, OD_PBS represents the absorbance of the negative control group, and OD_ddH2O represents the absorbance of the positive control group. Each concentration was repeated three times, and the average value was taken.

[0064] 2. Cell viability assay (1) Cell culture NRK-52E (mouse-derived renal tubular epithelial cells): cultured in DMEM medium containing 10% fetal bovine serum at pH 7.4, 37°C, and 5% CO2 humidification; cells in the logarithmic growth phase were used for cell experiments.

[0065] (2) CCK-8 experiment The effect of Ce-PYP on cell viability was evaluated using the CCK-8 assay. A density of 6 × 10⁶ cells was used during the logarithmic growth phase. 3 100 μL of NRK-52E cells per mL were cultured in 96-well plates until adherence. Different concentrations of Ce-PYP (25, 50, 100, 150, 200, 250, 300 μg / mL) were added and incubated for 24 h. Then, 10 μL of CCK-8 was added to each well and incubated for 2 h. Changes in cell viability were detected in each group. Each experiment was repeated in 3 parallel wells. The absorbance (OD value) was measured at 450 nm using an enzyme labeling instrument to observe the cell viability of each group.

[0066] II. Experimental Results 1. Blood compatibility like Figure 6 As shown in Figure A, the hemolysis rate of Ce-PYP increases in a concentration-dependent manner, and is below 5% at a concentration of 200 μg / mL.

[0067] 2. Cell viability assay like Figure 6 As shown in Figure B, compared with the control group, NRK-52E cells exposed to 300 μg / ml Ce-PYP for 24 h did not show a decrease in cell viability, which remained at 101.01%. The results indicate that Ce-PYP has no significant effect on the viability of NRK-52E cells within the range of 0–300 μg / mL.

[0068] Example 7: The ability of Ce-PYP to resist cell damage under high oxalate conditions I. Experimental Methods The effects of Ce-PYP prepared in Example 1 on cell viability, resistance to oxidative stress damage, and anti-cell death activity under a high oxalate environment were determined using the following specific experimental methods: (1) Cell culture Same as Example 6.

[0069] (2) Cell viability The effect of high oxalate levels on cell viability in Ce-PYP cells was assessed using the CCK-8 assay. A density of 4 × 10⁻⁶ cells was used during the logarithmic growth phase. 3100 μL of NRK-52E cells per mL were cultured in 96-well plates until adherence. In groups other than the control group, 125 mM oxalate was added to simulate a high-oxalate environment in vivo as a model group. Different concentrations of Ce-PYP (0, 50, 75, 100, 150, 200, 300 μg / mL) were added to the model group and incubated for 24 h. Then, 10 μL of CCK-8 was added to each well, and the cells were incubated for 2 h. Changes in cell viability were detected in each group, and each experiment was repeated in three parallel wells. Cell viability was observed by measuring absorbance (OD value) at 450 nm using an enzyme labeling instrument.

[0070] The effects of PYP, Ce(NO3)3·6H2O, and Ce(NO3)3·6H2O+PYP on cell viability under high oxalate conditions were evaluated using the CCK-8 assay. The density during the logarithmic growth phase was set at 4 × 10⁻⁶ cells / year. 3 100 μL of NRK-52E cells per mL were cultured in 96-well plates until adherence. In addition to the control group, 125 mM oxalate was added to simulate a high-oxalate environment in vivo as a model group. 200 μg / mL of PYP, Ce(NO3)3·6H2O, and Ce(NO3)3·6H2O+PYP were added to each model group. In the Ce(NO3)3·6H2O+PYP experimental group, 200 μg / mL of Ce(NO3)3·6H2O and PYP were added simultaneously to the wells, with the concentrations identical to those in the Ce-PYP group. After 24 h of incubation, 10 μL of CCK-8 was added to each well, and the cells were incubated for 2 h. Cell viability changes were detected in each group, and each experiment was repeated in three parallel wells. Cell viability was observed by measuring absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay (ELISA) labeler.

[0071] (3) Cellular localization of Ce-PYP Preparation of coumarin 6-labeled Ce-PYP to quantify drug behavior in NRK-52E cells: NRK-52E cells in logarithmic growth phase (density 1×10⁻⁶) were used. 5 Lysosomes (cells / mL) were cultured in 6-well plates until they adhered to the plate. Lysosomes and nuclei were stained with lysotracker (red) and DAPI (blue), respectively. 10 μL of 1 mg / mL Ce-PYP was added and the cells were incubated for 0, 2, 4 and 8 h. The fluorescence signal of the drug in the cells was observed under a fluorescence microscope.

[0072] (4) Ce-PYP resists cellular oxidative stress NRK-52E cells in logarithmic growth phase were used at a concentration of 1×10⁻⁶. 5Cells were cultured at a density of 10 cells / mL in 6-well plates. After cell adhesion, 125 mM oxalic acid was added to each well except for the control group. Four wells were then cultured with 50 μg / mL PYP, 200 μg / mL PYP, 50 μg / mL Ce-PYP, and 200 μg / mL Ce-PYP, respectively, for 4 h. After removing the culture medium from the wells, the cells were washed three times with PBS. 1 mL of DCFH-DA diluted 1:1000 with serum-free medium was added, and the cells were incubated in the dark for 30 min. The cells were then washed twice with serum-free medium to remove excess DCFH-DA. The ROS fluorescence intensity distribution of each group was observed and photographed under a fluorescence microscope. ROS showed green fluorescence.

[0073] (5) Ce-PYP anti-cell death NRK-52E cells in logarithmic growth phase were used at a concentration of 1×10⁻⁶. 5 Cells were cultured at a density of 10 cells / mL in 6-well plates. After the cells adhered, 125 mM oxalic acid was added to each well except for the control group. Four wells were then cultured with 50 μg / mL PYP, 200 μg / mL PYP, 50 μg / mL Ce-PYP, and 200 μg / mL Ce-PYP, respectively, for 4 h. The culture medium was then removed from the wells and the cells were washed three times with PBS.

[0074] Preparation of Calcein-AM / PI staining working solution: Take Calcein-AM stock solution (1 mM) and dilute it with serum-free culture medium at a ratio of 1:1000 to a final concentration of 1 μM. At the same time, add PI to make a final concentration of 2 μg / mL. Mix well and operate in the dark.

[0075] Add 1 mL of the above staining working solution to each well and incubate at 37°C in the dark for 20 min. After incubation, discard the staining solution, gently wash once with PBS (2 mL), and add an appropriate amount of PBS to keep the cells moist. Then observe and photograph under a fluorescence microscope. Live cells stained with Calcein-AM show green fluorescence, and dead cells stained with PI show red fluorescence.

[0076] II. Experimental Results (1) Cell activity like Figure 6 As shown in Figure C, in the experimental group with 125 mM oxalic acid and 0 μg / mL Ce-PYP, cell viability decreased to 60.57%. In the range of 0–200 μg / mL, cell viability gradually increased with the increase of Ce-PYP concentration. In the experimental group with 125 mM oxalic acid and 200 μg / mL Ce-PYP, cell viability was 79.84%.

[0077] like Figure 6As shown in Figures C and D, in the experimental group with 125 mM oxalic acid, cell viability decreased to 59.28%; in the experimental group with oxalic acid and Ce(NO3)3·6H2O, cell viability was 56.14%; in the experimental group with oxalic acid and PYP, cell viability was 70.59%; and in the experimental group with oxalic acid and Ce(NO3)3·6H2O+PYP, cell viability was 59.80%. The results indicate that Ce-PYP, under a high oxalic acid environment, concentration-dependently increased the viability of NRK-52E cells in the range of 0–200 μg / mL, and the effect at 200 μg / mL was superior to that of the experimental groups with 200 μg / mL PYP, Ce(NO3)3·6H2O, and Ce(NO3)3·6H2O+PYP, respectively.

[0078] (2) Cellular localization of Ce-PYP like Figure 7 As shown, after 2 hours of drug incubation, obvious green fluorescence was observed in the cytoplasm of NRK-52E cells, indicating that the drug had entered the cells; after 4 hours, the intensity of overlapping red and green fluorescence increased, indicating that the drug had entered the lysosomes; after 8 hours, the intensity of green fluorescence decreased, and after the red and green fluorescence overlapped, red became dominant, indicating that Ce-PYP gradually detached from the lysosomes. The results indicate that Ce-PYP enters NRK-52E cells through endocytosis, and this endocytosis is time-dependent.

[0079] (3) Ce-PYP resists cellular oxidative stress like Figure 8 As shown, compared with the oxalic acid modeling (ox) group, both PYP and Ce-PYP reduced the fluorescence intensity of ROS in a concentration-dependent manner, and Ce-PYP was more effective than PYP in reducing the fluorescence intensity of ROS at the same concentration.

[0080] (4) Ce-PYP anti-cell death like Figure 9 As shown, compared with the oxalate modeling (ox) group, both PYP and Ce-PYP reduced the distribution intensity of red fluorescence in a concentration-dependent manner, that is, reduced cell death in a concentration-dependent manner, and Ce-PYP was more effective than PYP in reducing cell death at the same concentration.

[0081] Example 8: Kidney protective effect of Ce-PYP on a glyoxylic acid-based C57 mouse model of kidney stones. I. Experimental Methods The nephroprotective effect of Ce-PYP prepared in Example 1 on a glyoxylic acid-based C57 mouse model of kidney stones was detected using the following method: (1) Model building Male C57 mice, weighing approximately 20 g. Animals were housed according to SPF standards; all bedding, feed, and drinking water were sterilized, and bedding was changed every 2-3 days. Five mice were placed in each cage, and each mouse was identified and marked using an ear tag.

[0082] Mice were randomly divided into 6 groups, with 5 mice in each group: 1) Control group: 100 μL of normal saline was injected intraperitoneally daily; 2) Model group (Glyoxylic Acid): Each mouse was injected with glyoxylic acid at a dose of 65 mg / kg / d for 7 days; 3) Ce(NO3)3 treatment group (Ce(NO3)3): Glyoxylic acid was injected according to the method of the model group, and Ce(NO3)3 solution at 20 mg / kg / d was injected on days 2, 4 and 6; 4) PYP treatment (PYP): Modeling was performed as described above, and PYP solution was injected at 20 mg / kg / d on days 2, 4, and 6. 5) Ce(NO3)3+PYP treatment group (Ce(NO3)3+PYP): The model was established according to the above method. On days 2, 4 and 6, Ce(NO3)3+PYP solution (Ce(NO3)3 and PYP were both 20 mg / kg / d) was injected into the peritoneum of mice. The Ce(NO3)3·6H2O+PYP experimental group was administered by simultaneously injecting 20 mg / kg Ce(NO3)3·6H2O and 20 mg / kg / d PYP into the peritoneum of mice. The concentrations of Ce(NO3)3·6H2O and PYP were the same as those of Ce ions and PYP in the Ce-PYP experimental group. 6) Ce-PYP treatment group (Ce-PYP): The model was established according to the above method, and Ce-PYP solution was injected at 20 mg / kg / d on days 2, 4 and 6.

[0083] (2) H&E and blood biochemistry tests Mice were euthanized by blood collection from their eyeballs, cervical dislocation, and kidneys were collected and fixed in tissue fixative. Paraffin sections were then prepared, stained with Hematoxylin and eosin (H&E), and the images were uploaded to computer software for processing using a tissue scanner. Blood samples were analyzed for creatinine (Cr) and blood urea nitrogen (BUN).

[0084] II. Experimental Results like Figure 10As shown in Figure A, obvious pathological changes were observed in the kidneys of mice in the glyoxylate model group, including renal tubular dilation, brush border reduction, glomerular atrophy, and increased renal corpuscle area. In the experimental group treated with Ce-PYP, kidney damage was improved, with less significant reduction in brush border, less significant renal tubular dilation, and no significant glomerular atrophy. At the same concentration, Ce-PYP showed better protective effects on the kidneys under glyoxylate model conditions than the Ce(NO3)3, PYP, and Ce(NO3)3+PYP groups. Figure 10 As shown in Figures B and C, the levels of Cr and BUN in the glyoxylic acid model group were higher than those in the control group. In the experimental group treated with Ce-PYP, the levels of Cr and BUN were lower than those in the model group. Moreover, under the same concentration of glyoxylic acid modeling with Ce-PYP, the levels of Cr and BUN in mice were lower than those in the Ce(NO3)3, PYP, and Ce(NO3)3+PYP groups.

Claims

1. A nanozyme, characterized in that, The nanozyme was obtained by the self-assembly of laver polysaccharide and Ce ions.

2. The nanozyme according to claim 1, characterized in that, The mass ratio of the laver polysaccharide to Ce ions is 0.9–1.1:

1.

3. A method for preparing a nanozyme, characterized in that, The process includes the following steps: dissolving laver polysaccharide and Ce(NO3)3·6H2O in water, filtering with filter paper to obtain the filtrate, mixing under ultrasonic conditions, and stirring for 20-40 min to obtain the nanozyme.

4. The preparation method according to claim 3, characterized in that, The filter paper has a pore size of 15–20 μm.

5. The preparation method according to claim 3, characterized in that, The frequency of the ultrasound is 38–42 kHz.

6. The preparation method according to claim 3, characterized in that, The stirring speed is 800-1200 rpm.

7. The application of the nanozyme according to claim 1 or 2, or the nanozyme prepared by the preparation method according to claims 3 to 6, in antioxidation.

8. The application of the nanozyme according to claim 1 or 2, or the nanozyme prepared by the preparation method according to claims 3 to 6, in alleviating kidney injury.

9. The application of the nanozyme according to claim 1 or 2, or the nanozyme prepared by the preparation method according to claims 3 to 6, in the preparation of calcium oxalate stone treatment products.

10. A calcium oxalate crystal inhibitor, characterized in that, The calcium oxalate crystal inhibitor includes the nanozyme of claim 1 or 2, or the nanozyme prepared by the preparation method of claims 3 to 6.

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