A magnesium chelated vitamin C nanomaterial, a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine materials, and particularly relates to a magnesium chelated vitamin C nanomaterial, a preparation method and application thereof. The preparation raw material of the nanomaterial comprises VCMg, a lipid modification material and a solvent I. The preparation raw material of the VCMg comprises an oil phase emulsion and an aqueous phase emulsion. The oil phase emulsion contains a magnesium source, a non-ionic surfactant, a stabilizer and a solvent II. The aqueous phase emulsion contains vitamin C, a dispersing agent and water. The research results of the present application establish that the VMNP is a unique nanometer platform integrating coordination chemistry and biological regulation, reveal a new biochemical mechanism of stabilizing unstable nutrients through materials, promote the anti-fibrosis treatment based on vitamin C, and provide a new technical idea for the cross research of nanotherapy and molecular biology mechanism.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a magnesium-chelated vitamin C nanomaterial, its preparation method, and its application. Background Technology

[0002] Chronic liver injury induces a pathological wound repair process in liver fibrosis, characterized by abnormal accumulation of extracellular matrix (ECM) and a gradual decrease in the number of hepatocytes. As a precursor to cirrhosis and hepatocellular carcinoma, this disease significantly contributes to global mortality, accounting for approximately 3.5% of annual deaths worldwide. A hallmark of fibrosis is the persistent excessive production of reactive oxygen species (ROS) by damaged hepatocytes. As potent signaling molecules, ROS can function as second messengers by activating multiple pro-fibrotic pathways. In particular, ROS mediates the activation and sustained survival of hepatic stellate cells (HSCs) by stimulating core signaling pathways such as TGF-β / Smad, NF-κB, and MAPK. Although evidence suggests that ROS is a core mediator of liver fibrosis, the regulatory mechanisms between oxidative stress and hepatic metabolic disorders remain unclear.

[0003] Vitamin C (VC), an essential water-soluble micronutrient, plays a central role in maintaining redox homeostasis and supporting key enzymatic processes in cellular function. However, its therapeutic potential is currently limited by oxidative instability—under conditions of high reactive oxygen species (ROS), VC is rapidly oxidized to dehydroascorbic acid (DHA), leading to loss of biological activity. Recent epidemiological studies have shown a negative correlation between serum VC levels and the severity of liver fibrosis in patients with non-alcoholic fatty liver disease. Furthermore, experimental evidence suggests that liver injury and iron overload exacerbate VC depletion, further amplifying oxidative stress and fibrosis progression. These findings highlight the need to develop strategies that can both stabilize VC and achieve functional delivery within the fibrotic microenvironment.

[0004] Studies have shown that the inherent oxidative instability of vitamin C mainly stems from the enediol group in its molecular structure. This group has a strong electron-donating ability and can be rapidly converted into dehydroascorbic acid (DHA) via ascorbic acid radical intermediates. Certain metal ions can stabilize the enediol structure and reduce its redox activity by forming chelates with the adjacent hydroxyl groups of the enediol group; however, the application of transition metals and lanthanides may pose significant safety risks due to potential toxicity and off-target biological effects.

[0005] Chinese invention patent application CN110546133A discloses an anti-fibrotic compound that addresses the problem of tranilast hyperbilirubinemia through novel amide compounds, such as (E)-N-(2-fluorophenyl)-3-(3-methoxy-4-(prop-2-yn-1-yloxy)phenyl)acrylamide, providing effective inhibition of fibrosis and inflammation, suitable for the prevention and treatment of fibrosis-related diseases, particularly diabetic cardiomyopathy. Chinese invention patent CN110958882B discloses a tricyclic compound as a glycogen synthase kinase 3 (GSK3) inhibitor and its uses, providing a compound that selectively inhibits GSK3, solving the problem of effectively inhibiting protein kinases in existing technologies, and achieving effective treatment of related diseases. However, vitamin C in these existing technologies only exerts conventional antioxidant effects, and its poor stability remains unresolved.

[0006] Against this backdrop, it is imperative to develop an anti-liver fibrosis strategy based on nanomaterial-mediated, targeted regulation of p38 MAPK. Summary of the Invention

[0007] To address the aforementioned technical challenges, this invention designs a magnesium-chelated vitamin C nanomaterial as a nanotherapeutic platform. Magnesium ions coordinate with the enediol groups of vitamin C to form a redox-stable complex, which can resist oxidative degradation in the liver environment rich in reactive oxygen species (ROS). Encapsulation with a self-assembled biocompatible liposome shell further enhances the liver's accumulation capacity and achieves sustained release, ensuring that vitamin C remains stable and exerts its biological activity persistently in fibrotic tissue. Unlike traditional antioxidant strategies that only use vitamin C as a ROS scavenger, VMNP utilizes vitamin C to modify the lysine residues of p38 MAPK (a mitogen-activated protein kinase, an intracellular signaling protein molecule), exerting a unique regulatory role that has not been previously reported. This post-translational modification can disrupt MAPK signaling and induce hepatic stellate cell cycle arrest, thereby delaying the fibrosis process. The results of this invention establish VMNP as a unique nanoplatform integrating coordination chemistry and biological regulation, revealing a biochemical mechanism for stabilizing unstable nutrients through materials. This study not only advances vitamin C-based antifibrotic therapy but also elucidates the universal concept of material-driven nutrient regulation in disease treatment.

[0008] The first aspect of this invention provides a magnesium chelated vitamin C nanomaterial (VMNP), wherein the raw materials for preparing the nanomaterial include: VCMg (magnesium chelated vitamin C precursor material), lipid modification material, and solvent one; The raw materials for preparing VCMg include oil-phase emulsion and aqueous-phase emulsion; The oil phase emulsion contains a magnesium source, a nonionic surfactant, a stabilizer, and solvent II. The aqueous emulsion contains vitamin C, a dispersant, and water.

[0009] Magnesium, as the second most abundant cation in cells, preferentially coordinates with oxygen-containing ligands and plays a crucial role in various physiological processes. This invention unexpectedly discovered that Mg... 2+ Coordination with vitamin C not only protects vitamin C from oxidative degradation but also forms a local vitamin C reservoir in fibrotic liver tissue to maintain its sustained activity. This materials-centric research strategy provides a rational approach for preserving the biological function of vitamin C and regulating reactive oxygen species-driven fibrosis.

[0010] Optionally, the magnesium source is an aqueous solution of magnesium sulfate; the concentration of the aqueous solution of magnesium sulfate is 0.2-0.4 mol / L.

[0011] The nonionic surfactants may include Tween (such as Tween-20, Tween-40, Tween-60, Tween-80), Span (such as Span-20, Span-40, Span-60, Span-80), polyglycerol fatty acid esters (PGFE), poloxamer (such as F68, F127), etc.

[0012] Optionally, the nonionic surfactant is Tween-80 or Span-80.

[0013] Optionally, the stabilizer includes at least one selected from 1-hexanol, n-pentanol, n-heptanol, isoamyl alcohol, and ethylene glycol monobutyl ether; further optionally, it is 1-hexanol.

[0014] Optionally, the second solvent is an oil-phase solvent, which may include cyclohexane, n-hexane, n-heptane, ethyl acetate, liquid paraffin, etc.; it may further be cyclohexane.

[0015] The volume ratio of the magnesium sulfate aqueous solution, Tween-80, Span-80, 1-hexanol and cyclohexane is (0.5-2):(2-5):(1-4):(0.5-3):(10-40); the most preferred ratio is 1.2:4:2:1.5:20.

[0016] Optionally, the dispersant includes at least one of sodium tripolyphosphate, sodium hexametaphosphate, sodium pyrophosphate, sodium carboxymethyl cellulose (CMC-Na), and polyvinylpyrrolidone (PVP); more preferably, it is sodium tripolyphosphate.

[0017] Optionally, the molar concentration of vitamin C in the aqueous emulsion is 0.5-1.2 mol / L; more preferably (0.6-0.9) mol / L.

[0018] Optionally, the mass concentration of sodium tripolyphosphate in the aqueous emulsion is 10-30 g / L; more preferably, it is 14-20 g / L.

[0019] Optionally, the lipid-modifying material includes phospholipid compounds and cholesterol.

[0020] Optionally, the phospholipid compound includes at least one of DPPC (dispalmitoylphosphatidylcholine), distearylphosphatidylcholine (DSPC), myristoylphosphatidylcholine (DMPC), soybean lecithin, egg yolk lecithin, DPPE-PEG (dispalmitoylphosphatidylethanolamine-polyethylene glycol), DMPE-PEG (distearylphosphatidylethanolamine-polyethylene glycol), DSPE-PEG (distearylphosphatidylethanolamine-polyethylene glycol), or their derivatives (such as DSPE-PEG-COOH, DSPE-PEG-NH2).

[0021] Further optionally, the phospholipid compounds include DPPC (dispalmitoylphosphatidylcholine) and DSPE-PEG (distearate-phosphatidylethanolamine-polyethylene glycol).

[0022] Optionally, the mass ratio of DSPE-PEG, DPPC and cholesterol is 1:(1.5-3):(0.5-2); further optionally, it is 1:2:1.

[0023] The solvent can include, for example, chloroform, dichloromethane, methanol, ethanol, tert-butanol, diethyl ether, etc.; chloroform can also be selected as a further alternative.

[0024] Optionally, the ratio of VCMg, lipid-modifying material, and solvent is (80-120) mg:(5-30) mg:(5-20) mL. A second aspect of the present invention provides a method for preparing magnesium-chelated vitamin C nanomaterials, wherein the preparation steps of the nanomaterials include: S1. Prepare oil phase emulsion and aqueous phase emulsion separately. Add the aqueous phase emulsion to the oil phase emulsion and stir evenly. After centrifugation and resuspending, VCMg is obtained. S2. Mix the VCMg with solvent one, then add the lipid modification material, mix and stir, then remove solvent one to obtain lipid-coated VMNP.

[0025] The hepatic reticuloendothelial system possesses a strong ability to capture natural nanomaterials, providing a unique opportunity to design nanoparticles for targeted therapy of liver fibrosis. Based on this physiological advantage, this invention synthesized VCMg nanoparticles via a reverse microemulsion polymerization strategy. Experimental results show that VC and Mg... 2+Coordination forms uniform nanoscale assemblies. Further to improve the biocompatibility and oxidative stability of the nanomaterials, this invention utilizes the self-assembly of DPPC / DSPE-PEG / cholesterol lipids onto the surface of VCMg nanoparticles, successfully preparing lipid-stable VMNP nanoparticles.

[0026] Optionally, after mixing the VCMg with solvent one, DSPE-PEG, DPPC and cholesterol are added sequentially.

[0027] In some embodiments, the oil phase emulsion is prepared by adding an aqueous solution of magnesium sulfate dropwise to a mixture of Tween-80, Span-80, 1-hexanol and cyclohexane under continuous magnetic stirring, and stirring until homogeneous to obtain an oil phase emulsion.

[0028] In some embodiments, the aqueous emulsion is prepared by adding 1 mmol of vitamin C and 20 mg of sodium tripolyphosphate to water under continuous magnetic stirring, and stirring until homogeneous to obtain an aqueous emulsion.

[0029] In some embodiments, the aqueous emulsion is added to the oil emulsion using a syringe pump, stirred for 1-3 hours, centrifuged at 8000-20000 rpm for 5-30 minutes, and the precipitate is resuspended in a mixture of methanol and water. After multiple washing / centrifugation cycles, VCMg is obtained.

[0030] In some embodiments, step S2 includes: dispersing VCMg in chloroform, adding DSPE-PEG, DPPC and cholesterol sequentially, stirring the mixture in an ice-salt bath for 10-60 min, removing chloroform by rotary evaporation, and obtaining lipid-coated VMNP.

[0031] A third aspect of the present invention provides an application of a magnesium-chelated vitamin C nanomaterial, wherein the nanomaterial is used to induce vitamin C modification of p38 MAPK.

[0032] The fourth aspect of this invention provides an application of a magnesium-chelated vitamin C nanomaterial, wherein the nanomaterial is used in the preparation of a drug for treating liver fibrosis.

[0033] Beneficial effects: This invention provides a magnesium-chelated vitamin C nanomaterial, its preparation method, and its application, which have the following advantages: (1) In this invention, magnesium-chelated vitamin C precursor material VCMg was prepared by Mg-VC coordination-driven reverse microemulsion method, and VMNP with anti-fibrotic function was obtained by specific lipid coating. (2) This invention confirms that the prepared VMNP can selectively inhibit the proliferation of hepatic stellate cells by stabilizing vitamin C and inducing vitamin C modification of p38 MAPK, effectively delaying the process of liver fibrosis, and clarifying the anti-fibrotic therapeutic efficacy of the nanomaterial. (3) This invention reveals a new mechanism by which vitamin C-derived protein modifications regulate the cell cycle, filling a gap in the study of post-translational modification functions of proteins in related fields; (4) This invention has discovered the vitamin C modification mode of lysine residues on the p38 MAPK protein molecule, which expands the scope of redox-sensitive signal regulation network and reveals an optimization mechanism that has not been fully considered in the regulation of MAPK signaling pathway. (5) This invention establishes the link between nanomaterial design and protein post-translational modification mechanism, providing a new technical approach for the cross-study of nanotherapy and molecular biological mechanisms; it provides a promising new strategy for anti-fibrotic treatment, and promotes the development of the emerging field of vitamin C-mediated protein chemistry, which has important scientific research and clinical translation value. Attached Figure Description

[0034] Figure 1 A schematic diagram illustrating the technical principle of this invention; Figure 2 Characterization results of VCMg and VMNP prepared in Example 1; Figure 2 In the figure, a is the transmission electron microscope (TEM) image of VCMg, b is the elemental distribution map of VCMg, c is the X-ray photoelectron spectroscopy (XPS) spectrum of VCMg, d is the nuclear magnetic resonance (NMR) spectrum of VCMg, e is the Fourier transform infrared (FTIR) spectrum of VCMg, f is the ultraviolet-visible (UV-vis) spectrum of VCMg, g is the hydrodynamic dimensions of VCMg and VMNP, h is the zeta potential of VCMg and VMNP, and i is the TGA (thermogravimetric analysis) curve of VCMg and VMNP. Figure 3 In vitro functional characterization results of VMNP prepared in Example 1; Figure 3 In the figure, a represents the hydrodynamic size change of VMNP in PBS (phosphate buffer) over time; b represents the Mg accumulation of VMNP over time under different conditions. 2+Release percentage; c is the 1H NMR spectrum of the VC-Mg complexation experiment (temperature 298K, solvent D2O); d is the UV-vis spectrum of VC and VMNP cultured in hydrogen peroxide solution with gradually increasing concentration (VC on the left, VMNP on the right); e is the kinetic change of VC and VMNP after culture in hydrogen peroxide; f is the UV-vis spectrum of VC and VMNP cultured in hydrogen peroxide solution with gradually increasing concentration in the presence of FeCl2 (VC on the left, VMNP on the right); f is the kinetic change of VC and VMNP after culture in hydrogen peroxide in the presence of FeCl2. Figure 4 The in vivo distribution and biocompatibility test results of VMNP prepared in Example 1; Figure 4 In Figure 'a', the concentration of Mg in the major organs of C57 mice after tail vein injection of VMNP (20 mg / kg, n=3) is [data missing]. 2+ a) Biodistribution of mice; b) Changes in body weight of mice within 30 days after tail vein injection of PBS and VMNP (n=3); c) Blood biochemical parameters of mice 3 days and 30 days after intravenous injection of PBS and VMNP (n=3); d) H&E staining images of tissue sections of major organs (heart, liver, spleen, lung and kidney) of mice treated with PBS and VMNP (n=3). Figure 5 Results of in vivo anti-fibrotic assays of VMNP in a CCl4-induced liver fibrosis model; Figure 5 Table a shows a schematic diagram of the CCl4-induced liver fibrosis model and the treatment timeline of PBS, VC, MgCl2, or VMNP; table b shows the characterization of liver injury in CCl4-induced mice by serum ALT and AST in different treatment groups (n=6); table c shows immunofluorescence staining images of H&E, Sirius red, and α-SMA (α-smooth muscle actin) in CCl4-induced fibrotic livers in different treatment groups (scale bar 50 μm); table d shows the quantitative analysis results of the severity of liver fibrosis by the positive area of ​​collagen and α-SMA (n=3); and table e shows the relative mRNA expression results of α-SMA, COL1A1, TGF-β, IL-1β, and TNF-α in CCl4-induced fibrotic livers in different treatment groups (n=3). Figure 6 Transcriptomic response of VMNP in a CCl4-induced liver fibrosis mouse model; Figure 6Table a shows the Venn diagram results for the control and treatment groups; table b shows the volcano plot of differentially expressed genes (DEGs) between the VMNP and PBS groups, where each dot represents a gene: red indicates upregulated genes and blue indicates downregulated genes; table c shows the heatmap of expression levels of target gene categories (cell cycle, collagen, and inflammation) obtained after treatment screening; table d shows the bar chart of GO (Gene Ontology) pathway enrichment analysis of DEGs in the VMNP and PBS groups, where the bar length represents the number of genes; table e shows the KEGG (Encyclopedia of Genomics) enrichment analysis of DEGs to characterize their enrichment features in cell biological processes and to show the most significant enrichment categories; each group has 3 biologically independent samples (n=3). Figure 7 VMNP's test results showed that vitamin C modification at the K53 / K54 sites blocked p38 nuclear translocation; Figure 7Image a shows a confocal microscopy image of Lx-2 cells treated with FITC-labeled VMNP (1 mM) for 24 hours, with the nucleus and cytoskeleton stained with DAPI (fluorescent dye 4',6-diamino-2-phenylindole) and phalloidin, respectively. Scale bar: 20 μm. Image b shows Western blotting analysis of CDK1 and Cyclin B1 expression levels in Lx-2 cells after the above treatment. The data shown are representative results from three independent replicate experiments. Image c shows the frequency of each cell cycle stage (n=3) obtained by flow cytometry after 24 hours of treatment with the specified VMNP concentrations in Lx-2 cells. Image d shows the cytotoxicity of VMNP at different concentrations on Lx-2 cells after 24 hours (n=6). Image e shows the expression levels of CDK1 and Cyclin B1 in Raw264.7 and Lx-2 cells after treatment. Western blot analysis of B1 expression; the experimental data shown is a representative set of results from three independent replicate experiments; f is the result of flow cytometry quantitative analysis of the cell proportion of each cell cycle phase in a specific cell line, detected 24 hours after VMNP (1mM) treatment (n=3); g is the Western blot analysis of phosphorylated p38MAPK, p38 MAPK, phosphorylated ERK1 / 2, and ERK in Lx-2 cells after treatment; three independent experiments were conducted and consistent results were obtained, and the data shown is from one of the experiments (n=3); h is the HPLC-MS / MS (high performance liquid chromatography-mass spectrometry tandem) analysis spectrum of vitamin C-modified peptides (K53 and K54 sites of human p38 MAPK) extracted from Lx-2 cell peptides (cell peptides); i is the temperature-dependent solubility level of p38 MAPK in Lx-2 cells after treatment as shown by the Western blot results; j is the immunofluorescence assay of P-p38 in Lx-2 cells after 24 hours of treatment with 1mM VMNP. Nuclear translocation results of MAPK, scale bar at 20 μm; k represents the stability test results of p38 MAPK and ERK1 / 2 in Lx-2 cells after different times of culture with 100 mg / mL cyclohexylimide, with the experimental group set as control with / without 1 mM VMNP treatment; three independent experiments were conducted and consistent results were obtained, and the data in the figure comes from one of the experiments (n=3). Figure 8 TEM images of nanoparticles prepared by the reverse microemulsion method under different precursor conditions provided in Example 1; Figure 8 In Figure a, particle samples were prepared using different molar ratios of MgSO4 and VC; in Figure b, particle samples were prepared by changing the concentration of VC while keeping the molar ratio of MgSO4 to VC fixed at 0.4:1. Detailed Implementation

[0035] Vitamin C is a key substance for maintaining physiological health, but its potential biological benefits are limited by the technical bottleneck of maintaining high local concentrations under oxidative conditions. For example... Figure 1 As shown, this invention develops a redox-stabilized magnesium-vitamin C coordination self-assembly material encapsulated in a biomimetic liposome shell. This nanomaterial accumulates in the liver, protecting vitamin C from degradation caused by reactive oxygen species. This local enrichment leads to covalent vitamin C modification of p38 MAPK at the K53 / K54 sites in hepatic stellate cells, thereby blocking its nuclear translocation and triggering G2 / M phase cell cycle arrest, thus limiting the proliferation of pro-fibrotic cells. Further identification revealed this modification as a vitamin C-derived post-translational modification that directly regulates the MAPK signaling pathway in hepatic stellate cells. This mechanism distinguishes the role of vitamin C from the traditional antioxidant model and reveals its function as a covalent regulator of signaling pathways. This invention establishes vitamin C modification as a biochemical principle connecting nutritional chemistry and cell cycle regulation, while providing a potential new strategy for the treatment of liver fibrosis.

[0036] Vitamin C, magnesium sulfate, carbon tetrachloride (CCl4), hydrogen peroxide, FeCl2, Tween-80, Span-80, cholesterol, 1-hexanol, and cyclohexane used in the embodiments of the present invention were all purchased from Macklin Company; DPPC, DSPE-PEG, and FITC (fluorescein isothiocyanate) were all purchased from Adamas Company.

[0037] Unless otherwise specified, all raw materials, equipment and other consumables used in this invention are commercially available; the solvents of all solutions are water (e.g., 1 mM VMPN means 1 mmol VMPN dissolved in 1 L of water).

[0038] Furthermore, all experimental procedures involving animals in this invention comply with the relevant regulations of the Fudan University Laboratory Animal Welfare and Ethics Committee (Approval No.: 2025-CLX-003).

[0039] Example 1 This embodiment provides a magnesium-chelated vitamin C nanomaterial and its preparation method. The raw materials for preparing the nanomaterial include: VCMg, lipid modification material and solvent 1 (chloroform).

[0040] The raw materials for preparing VCMg include oil-phase emulsion and aqueous-phase emulsion; The oil phase emulsion is composed of a magnesium source (aqueous magnesium sulfate solution), nonionic surfactants (Tween-80 and Span-80), a stabilizer (1-hexanol), and solvent 2 (cyclohexane).

[0041] The aqueous emulsion consists of vitamin C, a dispersant (sodium tripolyphosphate), and water.

[0042] The lipid-modifying materials include phospholipid compounds (DPPC and DSPE-PEG) and cholesterol.

[0043] The dosage of each raw material is described in the preparation steps.

[0044] The preparation steps of the nanomaterial include: S1. Synthesis of VCMg: 1.2 mL of magnesium sulfate aqueous solution (containing 0.4 mmol magnesium sulfate) was added dropwise to a mixture of Tween-80 (4 mL), Span-80 (2 mL), 1-hexanol (1.5 mL) and cyclohexane (20 mL) under continuous magnetic stirring to obtain an oil phase emulsion. 1 mmol of vitamin C and 20 mg of sodium tripolyphosphate were added to 1.2 mL of water under continuous magnetic stirring and stirred until homogeneous to obtain an aqueous emulsion. The aqueous emulsion was added to the oil emulsion at a rate of 300 μL / min using a syringe pump. After stirring for 2 h, the mixture was centrifuged at 12000 rpm for 10 min. The precipitate was resuspended in a mixture of methanol and water (volume ratio 1:1). The resuspended solid was washed three times with ethanol to obtain VCMg.

[0045] S2. Synthesizing VMNP: 100 mg VCMg was dispersed in 10 mL of chloroform, and then 5 mg DSPE-PEG, 10 mg DPPC and 5 mg cholesterol were added in sequence at a mass ratio of 1:2:1. The mixture was placed in an ice-salt bath and stirred for 30 min. The chloroform was removed by rotary evaporation to obtain lipid-coated VMNP.

[0046] Performance testing 1. Particle characterization test Microscopic characterization of VCMg and VMNP nanoparticles was performed, and the results are shown in the figure. Figure 2 .

[0047] like Figure 2 As shown, transmission electron microscopy images reveal that the VCMg prepared in Example 1 has a distinct spherical morphology. Figure 2 a) The elemental distribution diagram confirms the uniform distribution of oxygen (O) and magnesium (Mg) inside the nanoparticles. Figure 2 b). XPS further verified VC and Mg 2+ The chemical coordination of Mg and O exhibits characteristic signals. Figure 2 c) Compared with the spectrum of free VC, the C1s peak (286.28 eV, COC) and C=O peak (288.68 eV) shifted after Mg doping, confirming that the VC olefin group interacts with Mg. 2+ Coordination function ( Figure 2c). The 1H NMR spectrum showed a significant change in the mass spectrum signal of VCMg compared to free VC, particularly the disappearance of the proton signal associated with the enediol group; this observation indicates that Mg 2+ Coordination with the hydroxyl groups of the enediol moiety confirms the coordination-driven assembly of VCMg. Figure 2 d). FTIR spectra further validated the coordination results. Figure 2 e shows C=O (1754cm) -1 ) and -OH (3000-3600cm) -1 The intensity of stretching vibrations changes. UV-Vis spectroscopy also revealed a characteristic absorption peak at 260 nm for VC. These characterization results collectively confirm the successful coordination-driven assembly of VCMg nanoparticles. Figure 2 f).

[0048] Dynamic light scattering analysis confirmed that VMNPs have a uniform hydrodynamic particle size (approximately 100 nm) and a narrow particle size distribution. Figure 2 g). Zeta potential testing results showed that the surface potential of the nanoparticles increased after lipid coating, confirming successful surface modification and the formation of a more stable colloidal interface. Figure 2 Furthermore, TGA analysis revealed the introduction of additional organic components into the lipid layer, further validating the surface modification effect and improved stability. Figure 2 i). The results showed that lipid modification transformed VCMg into VMNP with enhanced biocompatibility and resistance to reactive oxygen species, providing a reliable nanoplatform for subsequent biological evaluation of liver fibrosis treatment.

[0049] like Figure 8 As shown, this invention also evaluated the adjustment of the molar ratio of MgSO4 and VC ( Figure 8 a) or changing the VC concentration while maintaining a fixed molar ratio ( Figure 8 b) Differences in material properties. TEM results showed that in the absence of MgSO4, a clear nanostructure could not be formed, indicating that Mg-VC coordination is essential for nanoparticle formation. At low MgSO4 content, the particles exhibited a shallower internal region, possibly due to insufficient coordination-induced solidification, resulting in partially hollow or loose structures. In contrast, higher MgSO4 concentrations increased particle size and produced a darker internal region, which can be attributed to droplet fusion at higher ionic strengths and the formation of Mg-rich inorganic aggregates within the particles.

[0050] Secondly, when the molar ratio of MgSO4 to VC is fixed at 0.4:1, changing the VC concentration also affects the formation of nanoparticles. At low VC concentrations (0.5 mmol), incomplete coordination leads to secondary nucleation, resulting in many small particles coexisting with larger particles. At high VC concentrations (2 mmol), the increased ionic strength destabilizes the droplets and leads to excessive aggregation, forming a paste-like structure rather than dispersed particles.

[0051] 2. In vitro functional characterization test The physicochemical stability of the VMNPs prepared in Example 1 was tested under a physiological simulation environment. TEM images were obtained using a Hitachi HT-7800. For TEM sample preparation, VMNP nanoparticles were dissolved in anhydrous ethanol to obtain a suspension, which was then dropped onto a carbon-coated copper grid and dried at room temperature before testing. Elemental distribution maps were acquired using an AZtec X-Max Extreme EDS system. FTIR spectra were measured using a Nicolet 6700 spectrometer (Thermo Fisher Scientific) via potassium bromide precipitation. UV-Vis spectra were measured using a Lambd A750 spectrophotometer. XPS was measured using a PHI Genesis 500, with the C1s peak at a binding energy of 284.8 eV used as the calibration benchmark. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Malvern Zetasizer Nano ZS90. TGA curves were measured using a Pyris 1 analyzer. pH changes in the VMNP hydrolysate were monitored using a calibrated pH meter. Element concentrations were quantitatively measured using inductively coupled plasma atomic emission spectrometry (ICP-OES, iCAP7400, Thermo Fisher Scientific).

[0052] Dynamic light scattering analysis showed that the hydrodynamic dimensions of VMNP remained nearly constant after several days in PBS (phosphate buffer), indicating its excellent colloidal stability in an aqueous environment. Figure 3 a). To further evaluate its responsiveness, VMNP was placed under different pH conditions to monitor its Mg content. 2+ Release status: The amount of ion release detected in neutral PBS was extremely low, while acidic conditions significantly accelerated the release of Mg. 2+ Release reflects the pH-sensitive dissociation of VMNP ( Figure 3 b). Furthermore, the VC-Mg complexation reaction results showed that the 1H NMR spectrum of free VC exhibited a characteristic resonance peak at δ≈4.85 ppm, which could be attributed to the cyclomethylene group adjacent to the olefinic group; the introduction of Mg... 2+ The signal then broadened and disappeared, accompanied by linear perturbations in the range of 3.5-4.8 ppm, reflecting Mg 2+Chelation at the enediol site leads to a rapid exchange between the free and bound states, and the reproducibility of this signal under acidic conditions is achieved by quenching the resonance of adjacent CH molecules through chemical shift. Figure 3 c) This confirmed the acid instability of VC-Mg coordination and the reversible dissociation of VC in low pH environments.

[0053] Given that oxidative stress is a key pathological feature of liver fibrosis, this invention further investigated whether VMNP can protect VC from oxidative degradation. The test method involved adding VMNP to aqueous solutions of different concentrations of hydrogen peroxide and culturing them for a period of time, followed by UV-vis (ultraviolet-visible absorption) spectroscopy testing; FeCl2 was also introduced into the system as a control.

[0054] Test results showed that free VC exhibited a gradual loss of its characteristic UV-Vis absorption peak with increasing hydrogen peroxide concentration, confirming its susceptibility to oxidation. Figure 3 (d, e). In contrast, VMNP retained the spectral characteristics of VC under the same conditions, with a significantly slower decay rate, indicating stronger resistance to hydrogen peroxide-induced oxidation. This protective effect was even more pronounced under Fenton reaction conditions: Fe in the system 2+ It catalyzes the generation of highly reactive hydroxyl radicals; free VC degrades rapidly, manifested as a sharp decrease in absorbance, while VMNP significantly slows down this process and maintains more stable spectral characteristics. Figure 3 These results collectively indicate that VC and Mg 2+ Coordination forms stable nanoparticles, which, combined with lipid surface modification, not only stabilize VC in aqueous solution but also enhance its resistance to oxidative damage, thereby maintaining its structural and functional integrity under conditions simulating the oxidative microenvironment of liver fibrosis.

[0055] 3. In vivo biodistribution and biocompatibility study of VMNP The in vivo metabolism and biosafety of VMNP were systematically evaluated in mice after intravenous administration. Results are shown in [Table / Reference]. Figure 4 The method for assessing in vivo circulation was as follows: fibrotic mice were injected intravenously with VMNP (20 mg / kg) via the tail vein. Mice were sacrificed at 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours post-injection. Organs were collected, and the magnesium levels in each organ were determined using ICP-OES (iCAP7400, Thermo Fisher Scientific). 2+ content.

[0056] Inductively coupled plasma analysis results showed that Mg²⁺ derived from VMNP + It shows significant accumulation in the liver, which is consistent with the physiological function of the hepatic reticuloendothelial system and also aligns with its intended use in the treatment of liver fibrosis. Figure 4a) In contrast, only trace amounts of Mg were detected in the spleen and other organs. 2+ Notably, although VMNPs exhibited relatively high distribution in the heart at an early time point (3 hours), these nanoparticles were rapidly cleared from myocardial tissue and subsequently accumulated persistently in the liver. This dynamic distribution characteristic highlights the strong hepatic tropism of VMNPs, confirming their suitability as a nanoplatform for liver-targeted therapy.

[0057] To further evaluate the systemic tolerability of VMNP, body weight changes in mice were continuously monitored for 30 days after repeated intravenous injections. No significant weight loss or abnormal fluctuations were observed in either the VMNP group or the PBS control group, indicating good overall tolerability. Figure 4 b). Meanwhile, on days 3 and 30 post-injection, blood biochemical parameters related to liver function (ALT, AST, and ALP) and kidney function (creatinine, uric acid, and urea) were all within the normal physiological range, showing no significant difference compared to the PBS-treated control group. Figure 4 c). Serum markers of cardiac function (CK, CK-MB, and LDH-L) did not show abnormal fluctuations after VMNP administration, indicating that the transient cardiac accumulation observed earlier did not lead to functional impairment. Figure 4 c). Similarly, routine blood indicators remained normal throughout the treatment period. Figure 4 c).

[0058] Organ histological examination based on hematoxylin and eosin (H&E) staining provided further evidence for biocompatibility. Representative sections of the heart, liver, spleen, lungs, and kidneys of VMNP-treated mice showed no pathological abnormalities, including necrosis, inflammatory infiltration, or structural damage, and were not significantly different from the PBS-treated control group sections. Figure 4 d). These test results all indicate that VMNP exhibits preferential accumulation characteristics in the liver and spleen, and is safe and non-toxic with excellent biocompatibility in vivo. These results support its applicability for further therapeutic studies.

[0059] 4. In vivo anti-fibrotic effect of VMNP The therapeutic effect of VMNP on liver fibrosis was evaluated in a CCl4-induced liver fibrosis mouse model. Results are shown below. Figure 5 A schematic diagram of the construction of a mouse model of liver fibrosis and a timeline of treatment with PBS, VC, MgCl2, or VMNP are shown below. Figure 5 a.

[0060] Test methods: Six-week-old male C57 mice were injected with CCl4 (4 mL / kg) to establish a liver fibrosis model. Two weeks later, the mice were randomly divided into four groups (n=6 per group): (1) PBS (control group), (2) MgCl2 (20 mg / kg), (3) Vitamin C (20 mg / kg), and (4) VMNP (20 mg / kg); all treatments were administered via tail vein injection. Blood samples were collected for the quantitative detection of two serum aminotransferases (ALT and AST), and liver tissue was taken for histological analysis; the analysis methods included hematoxylin-eosin (H&E) staining, Sirius red staining, and α-SMA immunofluorescence staining.

[0061] Compared with the PBS, VC, and MgCl2 groups, VMNP treatment resulted in a significant decrease in serum ALT and AST levels, indicating a significant reduction in liver injury. Figure 5 b). Histological examination further confirmed its anti-fibrotic effect: H&E staining showed significant hepatocyte necrosis, inflammatory infiltration, and structural destruction in the PBS group, while these pathological changes gradually improved in the VC and MgCl2 groups, with the most significant improvement in the VMNP treatment group. Sirius red staining showed a large amount of collagen deposition in the fibrotic liver of the control group mice, while this phenomenon was significantly reduced after VMNP administration. Figure 5 c). The test results also showed that immunofluorescence staining of α-SMA (a marker of activated hepatic stellate cells) showed a strong positive signal in PBS-treated fibrotic livers, while the staining intensity in VMNP-treated mice was significantly reduced. Quantitative analysis confirmed that, compared with other treatment groups, VMNP produced the least amount of collagen and α-SMA-positive areas. Figure 5 d).

[0062] Analysis of fibrotic (α-SMA, COL1A1, TGF-β) and pro-inflammatory (IL-1β, TNF-α) transcripts in CCl4-damaged liver based on molecular-level changes showed that the transcriptional levels in the VMNP group were significantly lower than those in the PBS group, with a decrease even greater than that in the VC or MgCl2 groups. Figure 5 e). Combining biochemical and histological data, these results demonstrate that VMNP can alleviate liver injury, limit extracellular matrix deposition, and inhibit inflammatory signaling in a carbon tetrachloride model, validating its therapeutic potential against liver fibrosis.

[0063] 5. Study on the molecular mechanism of VMNP's anti-fibrotic effect To further elucidate the molecular mechanism of VMNP's anti-fibrotic effect, this invention performed RNA-seq analysis on the livers of CCl4-induced liver fibrosis mice treated with PBS, vitamin C, MgCl2, or VMNP to verify the molecular mechanism by which VMNP exerts its anti-fibrotic effect through reprogramming the liver transcriptome. Venn diagrams showed that, in addition to overlapping features with other treatment groups, VMNP also regulates a unique set of genes (…). Figure 6 a). By comparing the transcription results of the VMNP group and the PBS control group, a set of differentially expressed genes (DEGs) can be screened, which covers both upregulated and downregulated transcripts. Figure 6 b).

[0064] The heatmap visualization results showed that the VMNP-treated group exhibited synergistic transcriptome reprogramming characteristics, specifically, the expression of cell cycle-related transcripts was suppressed, while the expression levels of collagen and inflammation-related gene modules were significantly reduced. Figure 6 c). GO enrichment analysis further validated this observation, with the top-ranked gene categories associated with cell cycle progression and mitosis, extracellular matrix organization, and immune response pathways. Figure 6 d), KEGG enrichment analysis further highlighted the significant changes in the MAPK signaling pathway ( Figure 6 e). Comprehensive transcriptome data indicate that VMNP can drive liver tissue to adopt anti-proliferative, anti-fibrotic, and anti-inflammatory phenotypes, with cell cycle inhibition and MAPK pathway regulation being the core mechanisms at play.

[0065] 6. Study on the mechanism by which VMNP induces G2 / M phase arrest in hepatic stellate cells Based on transcriptome enrichment analysis, this invention pioneered the study of the effects of VMNPs on hepatic stellate cells, a major pro-fibrotic cell population in the liver. The detection method is as follows: (1) Cell culture system used in the study: Raw264.7, AML12 and Lx-2 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were used for culture. Raw264.7 and Lx-2 cells were cultured in a solution containing 10% fetal bovine serum (FBS, Gibco, A5256701) and 100 U / mL. -1 Penicillin-streptomycin (PS) were cultured in DMEM (from Gibco, C11995500BT); AML12 cells were cultured in a liquid medium supplemented with 10% FBS, PS, and ITS (from BasalMedia, S450J7) and 40 ng / mL. -1 Cells were cultured in DMEM / F-12 (from BasalMedia, L320KJ) containing dexamethasone (MCE, HY-14648); cells were incubated in a standard incubator at 37°C and 5% CO2.

[0066] (2) Cell uptake capacity detection: LX-2 cells were co-incubated with 1mM FITC-labeled VMNP for 24 hours; the cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, and then stained with phalloidin for 30 minutes; after counterstaining with DAPI for 10 minutes, the samples were imaged and observed by confocal laser scanning microscope.

[0067] (3) Western blot experiment: Cells were lysed using radioimmunoprecipitation (RIPA) lysis buffer containing benzyl sulfonyl fluoride (PMSF). The specific procedure was as follows: equal amounts of protein samples were taken, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were then treated in three steps: blocked with 5% bovine serum albumin (BSA) for 1 hour; primary antibody was added and incubated overnight at 4°C; secondary antibody was added and incubated at room temperature for 1 hour; color development was performed using an enhanced chemiluminescence (ECL) kit, and protein band images were acquired using a Tanon 4600 imaging system; the gray values ​​of the bands were quantitatively analyzed using ImageJ software. The primary antibodies used in the experiment were all from Abways Technology, specifically GAPDH (glyceraldehyde-3-phosphate dehydrogenase, catalog number AB0038), CDK1 (catalog number A12414), Cyclin B1 (catalog number A2056), p38 (catalog number CY5600), P-p38 (phosphorylated p38, catalog number 9216S), ERK (extracellular signal-regulated kinase, catalog number CY5487), and P-ERK (phosphorylated ERK, catalog number CY7165).

[0068] (4) PCR (Polymerase Chain Reaction) Quantitative Analysis: Total RNA was extracted from cultured cells using a cell RNA purification kit (from Adamas Life). 1 μg of RNA was used for reverse transcription using a first-strand cDNA synthesis kit (from Yeasen, Hifair II). Quantitative PCR amplification was performed on a LineGene 9600 system (Bioer) using premixed real-time PCR buffer (from Yeasen, Hieff SYBR Green Master Mix). During the testing process, GAPDH was used as an internal reference gene to standardize and calibrate the expression level of the target gene. Specific primers were used, and the ΔCt method was employed to quantify gene expression. The primer sequences used in this invention are shown in Table 1 below.

[0069] Table 1

[0070] (5) Flow cytometry analysis of cell cycle: Lx-2 cells were seeded in 6-well plates; after adhesion, the medium was replaced with serum-free medium and cultured for 12 hours to synchronize the cell cycle. Then, the cells were treated with different concentrations of VMNP (0, 0.25mM, 0.5mM, 1mM, 2mM or 3mM) for 24 hours; after incubation, the cells were collected and stained with PI (propidium iodide) for 30 minutes under dark conditions. Finally, the cell cycle distribution was analyzed by flow cytometry.

[0071] (6) CETSA (cell heat transfer assay) analysis: To test the temperature-dependent stability of p38 MAPK, Lx-2 cells were treated with PBS, 2 mMVC, 2 mM MgCl2 or 1 mM VMNP for 24 hours, respectively. Protein samples were collected and divided into 10 parallel portions, and heated at 37℃, 41℃, 44℃, 47℃, 50℃, 53℃, 56℃, 59℃, 63℃ and 67℃ for 3 minutes, respectively. The heat-denatured protein samples were analyzed by Western blotting.

[0072] (7) Immunofluorescence experiment: Lx-2 cells were treated in 1mM VMNP for 24 hours, fixed with 4% paraformaldehyde for 15 minutes to maintain cell structure, and permeabilized with 0.5% Triton X-100 for 10 minutes to promote antibody entry; after blocking in PBS buffer containing 5% BSA for 1 hour, P-p38 primary antibody was added and incubated overnight at 4℃; the next day, goat anti-rabbit IgG (H+L) secondary antibody labeled with fluorescent dye Alexa Fluor 488 was added and incubated at room temperature for 1 hour; the cell nuclei were counterstained with DAPI for 10 minutes, and finally the cells were imaged and observed by confocal microscopy.

[0073] (8) Cell viability assay: Lx-2 cells were seeded in 96-well plates and incubated for 24 hours; cells were then treated with different concentrations (0-32 mM) of VMNP for 24 hours. After treatment, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours; the absorbance (A) at 450 nm was measured using a microplate reader (Spark 10M, TECAN) and cell viability was calculated. The formula for calculating cell viability is: Cell viability (%) = A 待测细胞 / A 未处理细胞 ×100%, the absorbance was measured at a wavelength of 450nm.

[0074] (9) Cytotoxicity test: Healthy male C57 mice (approximately 6 weeks old) were randomly divided into three groups (control group, 3-day group and 30-day group, n=3 in each group). The mice were intravenously injected with VMNP (20mg / kg) for 3 days / 30 days. Histological analysis was performed on samples of major organs (heart, liver, spleen, lung and kidney) and blood after injection. The analysis methods included: hematoxylin-eosin (H&E) staining, Sirius red staining and α-SMA immunofluorescence staining.

[0075] The test results showed that FITC-labeled VMNPs were efficiently internalized by Lx-2 cells and distributed throughout the cytoplasm. Figure 7 a); After 24 hours of treatment, VMNP significantly reduced the expression levels of CDK1 and Cyclin B1, thereby causing cell cycle arrest in the G2 / M phase ( Figure 7 b, c). Cell viability experiments showed that this arrest was not caused by nonspecific cytotoxicity. Figure 7 d). Given the important roles of hepatocytes and macrophages in the liver microenvironment, the effects of VMNP on these cells were further evaluated, but no similar effects on cell cycle phases were observed. Figure 7 (e, f) revealed that VMNP has a selective inhibitory effect on hepatic stellate cells.

[0076] KEGG analysis showed that significant enrichment of the MAPK signaling pathway was closely related to cell cycle regulation and stress response. This invention detected changes in key components of the MAPK pathway (including ERK and p38) in Lx-2 cells. The results showed that only p38 exhibited significant phosphorylation changes after VMNP treatment, but this did not translate into enhanced cell proliferation. Figure 7 (g) indicates that VMNP interferes with the functional output of p38 activation. Considering that the reactive lactone structure of vitamin C can covalently modify lysine residues, we further investigated whether VMNP can directly modify p38; by immunoprecipitation combined with protein modification mass spectrometry analysis, vitamin C modification was detected at the K53 and K54 sites of p38MAPK (g). Figure 7 h).

[0077] Corresponding to the aforementioned test results, the CETSA results of Lx-2 cells showed that the thermal stability of p38 was significantly reduced after VMNP treatment (PBS: 45.0°C, Vitamin C: 45.4°C, MgCl2: 46.4°C, VMNP: 42.5°C), proving that direct covalent modification occurred. Figure 7 i). From a functional perspective, this modification impairs the nuclear translocation of phosphorylated p38 (i). Figure 7 Furthermore, the actinomycete ketone tracking experiment confirmed that the half-life of p38 and ERK1 / 2 was shortened in VMNP-treated cells, indicating accelerated turnover of MAPK components. Figure 7 k).

[0078] In summary, the test results indicate that VMNP-induced p38 vitamin C modification can disrupt the MAPK signaling pathway, inhibit p38 nuclear translocation, and accelerate its protein turnover. This mechanism is likely the core reason for G2 / M phase arrest in hepatic stellate cells.

Claims

1. A magnesium-chelated vitamin C nanomaterial, characterized in that, The raw materials for preparing the nanomaterials include: VCMg, lipid modification materials, and solvent one; The raw materials for preparing VCMg include oil-phase emulsion and aqueous-phase emulsion; The oil phase emulsion contains a magnesium source, a nonionic surfactant, a stabilizer, and solvent II. The aqueous emulsion contains vitamin C, a dispersant, and water.

2. The magnesium-chelated vitamin C nanomaterial according to claim 1, characterized in that, The lipid-modifying materials include phospholipid compounds and cholesterol; The phospholipid compounds include DPPC and DSPE-PEG.

3. The magnesium-chelated vitamin C nanomaterial according to claim 2, characterized in that, The mass ratio of DSPE-PEG, DPPC and cholesterol is 1:(1.5-3):(0.5-2).

4. The magnesium-chelated vitamin C nanomaterial according to claim 1, characterized in that, The magnesium source is an aqueous solution of magnesium sulfate; the concentration of the aqueous solution of magnesium sulfate is 0.2-0.4 mol / L.

5. The magnesium-chelated vitamin C nanomaterial according to claim 1, characterized in that, The dispersant includes at least one of sodium tripolyphosphate, sodium hexametaphosphate, sodium pyrophosphate, sodium carboxymethyl cellulose, and polyvinylpyrrolidone.

6. The magnesium-chelated vitamin C nanomaterial according to claim 1, characterized in that, The nonionic surfactants are Tween-80 and Span-80.

7. A method for preparing magnesium-chelated vitamin C nanomaterials according to any one of claims 1-6, characterized in that, The preparation steps of the nanomaterial include: Oil phase emulsion and aqueous phase emulsion were prepared separately. The aqueous phase emulsion was added to the oil phase emulsion and stirred evenly. After centrifugation and resuspending, VCMg was obtained. The VCMg was mixed with solvent one, and then lipid-modified material was added. After mixing and stirring, solvent one was removed to obtain lipid-coated magnesium chelated vitamin C nanomaterials.

8. The method for preparing magnesium-chelated vitamin C nanomaterials according to claim 7, characterized in that, After mixing the VCMg with solvent one, DSPE-PEG, DPPC and cholesterol are added in sequence.

9. An application of the magnesium-chelated vitamin C nanomaterial according to any one of claims 1-6, characterized in that, The nanomaterials are used to induce vitamin C modification of p38 MAPK.

10. An application of the magnesium-chelated vitamin C nanomaterial according to any one of claims 1-6, characterized in that, The nanomaterials are used to prepare drugs for the treatment of liver fibrosis.

Images