Preparation method and application of dual-targeting antioxidant nano-drug

Abstract

The invention discloses a preparation method and application of a dual-targeting antioxidant nano-drug, and the preparation method comprises the following steps: 1) mixing a chondroitin sulfate solution with a CTGFVAVR solution, and reacting for 0.5-1.5 h under a stirring condition to obtain a CS / CTGFVAVR solution; (2) a low-molecular-weight chitosan solution is added into the CS / CTGFVAVR solution, stirring continues to be conducted for 24-48 h at the room temperature, a CS-coated LMWC-coated CTGFVAVR nano-composite is prepared, and the molar ratio of chondroitin sulfate to low-molecular-weight chitosan to CTGFVAVR in the nano-composite is 1: 1: 1; according to the invention, on the basis of a nano-composite of chondroitin sulfate (CS) and low-molecular-weight chitosan (LMWC), the dual-targeting delivery of the CTGFVAVR is realized through electrostatic self-assembly, and the limitation of traditional antioxidant therapy is broken through.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of a dual-targeted antioxidant nanomedicine. Background Art

[0002] Acute kidney injury (AKI) is a common clinical critical illness, and its high morbidity and mortality have become a global public health burden. However, there is currently a lack of specific therapeutic drugs, and existing therapies are mainly supportive, which cannot effectively block the oxidative stress and inflammatory cascade reactions. Traditional nanodelivery systems rely on passive targeting, but are limited by the unique filtration barrier and hemodynamic characteristics of the kidney, resulting in low targeting efficiency. At the same time, synthetic polymer materials (such as PLGA) have potential immunogenicity, and existing technologies mostly focus on single receptors (such as Megalin or KIM-1), which cannot cover the multi-stage injury markers of renal tubular epithelial cells (RTEC) during the course of AKI (such as CD44, which is specifically upregulated after injury). In addition, although small molecule antioxidants and gene therapy drugs (such as siRNA) have potential, they face challenges such as poor stability, off-target toxicity, and low targeted delivery efficiency. Therefore, the development of delivery systems based on natural materials with dual targeting and dynamic responsiveness has become a key need to break through the existing treatment bottleneck. Summary of the Invention

[0003] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a preparation method and application of a dual-targeted antioxidant nanomedicine based on natural materials and having both dual targeting and dynamic responsiveness.

[0004] The technical solution of the present invention is as follows: a method for preparing a dual-targeted antioxidant nanomedicine, specifically comprising the following steps:

[0005] 1) Mixing the chondroitin sulfate solution and the CTGFVAVR solution, reacting for 0.5-1.5 hours under stirring conditions to obtain a CS / CTGFVAVR solution;

[0006] 2) adding a low molecular weight chitosan solution to the CS / CTGFVAVR solution and continuing stirring at room temperature for 24-48 hours to prepare a CS@LMWC@CTGFVAVR nanocomposite, wherein the molar ratio of chondroitin sulfate, low molecular weight chitosan and CTGFVAVR in the nanocomposite is 1:1:1.

[0007] Wherein, the chemical structural formula of the CTGFVAVR is as follows:

[0008]

[0009] Preferably, the molecular weight of the chondroitin sulfate is 0.5 to 3.0 kDa.

[0010] Preferably, the molecular weight of the low molecular weight chitosan is 0.8-5.0 kDa.

[0011] Preferably, the solvent of the chondroitin sulfate solution and the CTGFVAVR solution is water.

[0012] The present invention also provides an application of a dual-targeted antioxidant nanomedicine for preparing a medicine for treating acute kidney injury.

[0013] The beneficial effects of the present invention are as follows: the nanocomposite of chondroitin sulfate (CS) and low molecular weight chitosan (LMWC) of the present invention realizes dual targeted delivery of antioxidant peptide (CTGFVAVR) through electrostatic self-assembly, and has the following characteristics:

[0014] 1) Receptor synergy and dynamic response: LMWC binds to the megalin receptor (expressed in physiological state), and CS targets the CD44 receptor (upregulated after injury), forming a "physiological-pathological" dual-stage targeting, accurately covering different stages of AKI; compared with existing single-target delivery (such as siRNA systems that rely solely on megalin or biomimetic cell membrane strategies), it significantly improves targeting efficiency and microenvironmental responsiveness.

[0015] 2) Material safety and process advantages: CS and LMWC are both FDA-approved natural materials, avoiding the metabolic toxicity of synthetic carriers (such as PLGA), and the electrostatic self-assembly process simplifies the production process, meeting the needs of clinical transformation.

[0016] 3) Multi-mechanism synergistic therapy: CTGFVAVR blocks oxidative stress by clearing ROS and synergizes with dual targeted delivery. Compared with single gene silencing (such as p53 siRNA) or immune modulation (such as STING inhibitors), it breaks through the limitations of traditional antioxidant therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Particle size and distribution of CS@LMWC@CTGFVAVR nanocomposites.

[0018] Figure 2 Transmission electron microscopy image of CS@LMWC@CTGFVAVR nanocomposite.

[0019] Figure 3 Release behavior of CS@LMWC@CTGFVAVR nanocomplex under different pH conditions (pH 7.4, 6.0 and 5.0).

[0020] Figure 4 Fluorescence imaging results of active transport of CS@LMWC@CTGFVAVR(F) nanocomplex in renal tubular epithelial cells.

[0021] Figure 5 Fluorescence imaging results of the distribution of CS@LMWC@CTGFVAVR(F) nanocomplex in the major organs of mice with acute kidney injury model.

[0022] Figure 6 Changes in blood creatinine and urea nitrogen levels in mice with acute kidney injury model.

[0023] Figure 7 Changes of superoxide dismutase and malondialdehyde levels in renal tissue of mice with acute kidney injury model.

[0024] Figure 8 Changes in inflammatory factor levels in renal tissue of mice with acute kidney injury model.

[0025] Figure 9 These are the results of kidney tissue pathology sections in mice with acute kidney injury model. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments.

[0027] Example

[0028] The dual-targeted antioxidant nanoparticles (CS@LMWC@CTGFVAVR nanocomplexes) were prepared by the following steps:

[0029] Chondroitin sulfate (CS, molecular weight 1.0 kDa) solution was mixed with CTGFVAVR solution and stirred for 1 hour to obtain a uniform solution of CS / CTGFVAVR; when the feeding amounts of CS and CTGFVAVR were determined, low molecular weight chitosan (LMWC, molecular weight 1.2 kDa) solution was added and stirred at room temperature for 24 hours to obtain a CS@LMWC@CTGFVAVR nanocomposite (the molar ratio of the three components was 1:1:1).

[0030] The particle size distribution and polydispersity index (PDI) of CS@LMWC@CTGFVAVR nanocomposites with different formulations were measured using a particle size and surface potential analyzer. 1 mL (1 mg / mL) of the nanocomposites was added to a disposable sample cell without dilution to measure the particle size distribution and PDI. Separately, 0.5 mL of CS@LMWC@CTGFVAVR was added to a zeta potential cell to measure the zeta potential of the CS@LMWC@CTGFVAVR.

[0031] The morphology of CS@LMWC@CTGFVAVR was observed using a transmission electron microscope. The nanocomposite was diluted with ultrapure water to a CS@LMWC@CTGFVAVR dispersion concentration of 1.0 mg / mL. 10 μL was pipetted with a pipette and dropped onto a copper mesh covered with a carbon film. The mesh was then placed in a cool place to evaporate naturally. 2% (w / v) phosphotungstic acid was added for staining for 30 seconds, the staining solution was aspirated, and the mesh was dried at room temperature. The morphology and size of CS@LMWC@CTGFVAVR were observed using a transmission electron microscope.

[0032] The drug loading capacity and encapsulation efficiency of the CS@LMWC@CTGFVAVR nanocomplex were tested by ultrafiltration centrifugation. CS@LMWC@CTGFVAVR was transferred to the inner tube of a centrifuge tube (molecular weight cutoff: 3 kDa, 1.5 mL specification) and centrifuged at 2000 × g for 5 min. The filtrate in the outer tube was collected and the concentration of CTGFVAVR in the filtrate was determined by high-performance liquid chromatography to calculate the drug loading capacity and encapsulation efficiency.

[0033] The pH of the CS@LMWC@CTGFVAVR dispersion was adjusted to 7.4, 5.0, and 4.5 using sodium hydroxide or hydrochloric acid solutions, respectively. A 1.5 mL centrifuge tube containing the CS@LMWC@CTGFVAVR dispersion was placed in a shaker at 37°C for 10 minutes. The CS@LMWC@CTGFVAVR dispersion was then transferred to an ultrafiltration centrifuge tube (molecular weight cutoff: 3 kDa, 1.5 mL) and centrifuged at 2000 × g for 5 minutes. The CTGFVAVR concentration in the filtrate was determined, and drug release was calculated.

[0034] The results showed that the CS@LMWC@CTGFVAVR nanocomposite has excellent physicochemical properties and functional responsiveness: dynamic light scattering detection Figure 1 As shown in the figure, the average particle size of the nanocomposite is about 70 nm, and the polydispersity coefficient is 0.214±0.085, indicating that the particles are evenly distributed and well dispersed; the Zeta potential is -18.1±2.1 mV, and the strong surface charge can prevent particle aggregation through electrostatic repulsion, thus making CS@LMW C@CTGFVAVR have high stability. Figure 2 As shown, transmission electron microscopy further revealed that the nanoparticles were spherical / quasi-spherical in shape, with a smooth surface and no aggregation. Drug loading performance analysis showed that the electrostatic self-assembly process achieved a drug loading and encapsulation efficiency of CTGFVAVR of 12.6±1.5% and 93.1±3.1%, respectively, demonstrating that this process can efficiently load negatively charged antioxidant peptides.

[0035] In vitro drug release test results Figure 3The results show that the release behavior of CS@LMWC@CTGFVAVR is highly dependent on pH. At pH 7.4, the release of CTGFVAVR from CS@LMWC@CTGFVAVR was 9.2 ± 1.2%. Under acidic conditions, the release rate of CTGFVAVR was significantly accelerated, reaching 62.9 ± 10.7% and 91.3 ± 3.0% at pH 6.0 and 5.0, respectively. CS is an anionic polysaccharide. As the pH decreases, the protonation degree of the carboxylic acid groups on CS increases, reducing the negative charge. LMWC, on the other hand, is a cationic polysaccharide. As the pH decreases, the protonation degree of the amino groups on LMWC increases, increasing the positive charge. These effects weaken the electrostatic attraction between the anionic CS and the cationic LMWC and CTG FVAVR, enhance the electrostatic repulsion between CTGFVAVR and LMWC, disrupting the electrostatic equilibrium and leading to the rapid release of CTGFVAVR at low pH.

[0036] Targeting properties of CS@LMWC@CTGFVAVR nanocomplex

[0037] CS@LMWC@CTGFVAVR(F) nanocomplexes were prepared using the same method, using fluorescein (FAM)-labeled CTGFVAVR(F) as a model drug. Renal tubular epithelial cells (HK-2 cells) were seeded at a density of 1×105 cells / well in 12-well plates. After cell attachment and induction with hydrogen peroxide, the CS@LMWC@CTGFVAVR(F) nanocomplexes or free CTGFVAVR(F) were added and incubated for various times (2 or 4 hours). Normal HK-2 cells served as controls. After incubation, the culture medium was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde, and mounted. The fluorescence distribution in the cells was observed under an inverted fluorescence microscope.

[0038] C57BL / 6J mice were used as model animals, and a mouse model of acute kidney injury was established by bilateral renal artery clamping. Mice were injected with either the CS@LMWC@CTGFVAVR(F) nanocomplex or free CTGFVAVR(F). Two hours after administration, the mice were sacrificed by cervical dislocation. The heart, liver, spleen, lung, and kidney were removed and fixed with 4% paraformaldehyde. The sections were mounted and the fluorescence distribution within each organ was observed under an inverted fluorescence microscope.

[0039] The fluorescence experiment results are as follows Figure 4As shown, CS@LMWC@CTGFVAVR(F) exhibits significant targeting advantages in hydrogen peroxide-induced damaged HK-2 cells: compared with normal HK-2 cells, the fluorescence intensity of the nanocomplex in damaged cells increased significantly at both 2h and 4h, suggesting that it is synergistically mediated by Megalin receptors (basal expression) and CD44 (damage-induced upregulation) for efficient endocytosis. Free CTGFVAVR(F), on the other hand, has low fluorescence intensities in both damaged and normal cells, indicating that it cannot effectively penetrate the cell membrane in the absence of a targeting carrier. This comparison confirms that the uptake efficiency of the nanocomplex is highly dependent on the cell damage microenvironment, consistent with the dynamic expression characteristics of the CD44 receptor, validating its "pathologically responsive" targeting mechanism.

[0040] like Figure 5 As shown, in a mouse model of acute kidney injury, CS@LMWC@CTGFVAVR(F) exhibited significant disease-dependent renal accumulation: the fluorescence intensity of CS@LMWC@CTGFVAVR(F) in the kidneys of acute kidney injury mice was significantly higher than that in normal mice. In contrast, the fluorescence intensity of free CTGFVAVR(F) was lower in the kidneys of both acute kidney injury and normal mice, confirming its lack of targeting ability. Furthermore, the fluorescence intensity of the nanocomplex was lower in non-target organs such as the heart and liver of mice with acute kidney injury, further highlighting its dual advantages of "kidney injury-specific accumulation and controllable systemic exposure."

[0041] Pharmacodynamic evaluation of CS@LMWC@CTGFVAVR nanocomplex

[0042] C57BL / 6J mice were used as model animals, and bilateral renal artery clamping was used to construct an acute kidney injury mouse model. The therapeutic effect of CS@LMWC@CTGFVAVR nanocomplex on ischemia-reperfusion acute kidney injury was investigated from the aspects of renal function biochemical indicators, oxidative stress, inflammatory factors, renal injury markers and histopathology.

[0043] 1) Grouping: A, sham surgery + phosphate buffered saline (PBS); B, acute kidney injury + PBS; C, acute kidney injury + CTGFVAVR; D, acute kidney injury + CS@LMWC@CTGFVAVR. Drugs were administered via the tail vein, and relevant indicators were measured 24 hours after administration.

[0044] 2) Use a fully automatic biochemical analyzer to detect blood biochemical indicators (serum creatinine, urea nitrogen) and evaluate the renal function of the model animals.

[0045] 3) Determine the changes in superoxide dismutase (SOD) and malondialdehyde (MDA) levels in renal tissue.

[0046] 4) The levels of tumor necrosis factor-α and interleukin-6 in renal tissue were determined by enzyme-linked immunosorbent assay.

[0047] 5) Pathological examination: Hematoxylin-eosin (H&E) staining was used to observe the morphological changes of renal tissue in each group.

[0048] like Figure 6 As shown in the data, compared with the sham operation group, the serum creatinine (Scr) and blood urea nitrogen (BUN) levels in the acute kidney injury model were significantly increased (Scr: 44.5±6.0μmol / L vs. 229.1±41.3μmol / L; BUN: 8.6±1.3mmol / L vs. 24.7±0.9mmol / L, *p<0.001), indicating that renal function was severely impaired. The Scr and BUN levels in the nanocomplex treatment group were significantly lower than those in the model group (Scr: 83.7±19.7μmol / L, **p<0.01 vs. Group B; BUN: 9.3±1.1mmol / L, ***p<0.001 vs. Model group), and were significantly better than those in the free CTGFVAVR group (Scr: 186.2±7.5μmol / L, **p<0.01; BUN: 18.1±3.0mmol / L, ***p<0.001), suggesting that the CS@LMWC carrier can enhance the kidney-targeted delivery and therapeutic effect of antioxidant peptides.

[0049] like Figure 7 As shown, superoxide dismutase (SOD) activity in renal tissue of the nanocomplex-treated group recovered to 354.0±53.8 U / mg prot, significantly higher than that of the model group (107.1±53.8 U / mg prot) (***p<0.001) and significantly higher than that of the free drug group (169.5±69.4 U / mg prot, **p<0.01). Malondialdehyde (MDA) levels decreased from 20.1±3.6 nmol / mg prot in the model group to 10.6±1.8 nmol / mg prot (***p<0.001), which was better than that of the free drug group (19.5±1.5 nmol / mg prot, **p<0.01). These results indicate that the nanocomplex more effectively reverses oxidative stress damage in acute kidney injury by enhancing the targeted accumulation of CTGFVAVR.

[0050] like Figure 8As shown, ELISA assays revealed that renal tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in the model group reached 386.1±19.7 pg / mg and 13.2±2.1 pg / mg, respectively, significantly higher than those in the sham-operated group. In the nanocomplex-treated group, TNF-α and IL-6 levels were significantly reduced to 101.9±17.6 pg / mg and 5.6±0.8 pg / mg, respectively. Its therapeutic effect was significantly superior to that of the free drug group (TNF-α: 315.7±75.9 pg / mg, **p<0.01; IL-6: 11.8±1.8 pg / mg, **p<0.01), confirming that its dual-targeting strategy can synergistically inhibit the inflammatory cascade.

[0051] like Figure 9 As shown, the model group showed extensive renal tubular epithelial cell necrosis and brush border loss, with numerous protein casts visible within the tubular lumen. However, the nanocomplex-treated group showed significantly reduced pathological damage, with only focal tubular dilatation and mild vacuolation, and the improvement was superior to that of the free drug group. The sham-operated group maintained intact renal tissue structure. These results morphologically validate the therapeutic advantages of CS@LMWC@CTGFVAVR for AKI.

[0052] The above are only examples of the features of the present invention and do not constitute any limitation on the scope of protection of the present invention. Any technical solution formed by equivalent exchange or equivalent replacement falls within the scope of protection of the present invention.

Claims

1. A method for preparing a dual-targeted antioxidant nanomedicine, characterized in that: The specific steps include: 1) Mixing the chondroitin sulfate solution and the CTGFVAVR solution, reacting for 0.5-1.5 hours under stirring conditions to obtain a CS / CTGFVAVR solution; 2) adding a low molecular weight chitosan solution to the CS / CTGFVAVR solution and continuing stirring at room temperature for 24-48 hours to prepare a CS@LMWC@CTGFVAVR nanocomposite, wherein the molar ratio of chondroitin sulfate, low molecular weight chitosan and CTGFVAVR in the nanocomposite is 1:1:

1.

2. The method for preparing the dual-targeted antioxidant nanomedicine according to claim 1, wherein: The chemical structural formula of the CTGFVAVR is as follows:

3. The method for preparing the dual-targeted antioxidant nanomedicine according to claim 1, wherein: The molecular weight of the chondroitin sulfate is 0.5-3.0 kDa.

4. The method for preparing the dual-targeted antioxidant nanomedicine according to claim 1, wherein: The molecular weight of the low molecular weight chitosan is 0.8-5.0 kDa.

5. The method for preparing the dual-targeted antioxidant nanomedicine according to claim 1, wherein: The solvent of the chondroitin sulfate solution and the CTGFVAVR solution is water.

6. Use of the dual-targeted antioxidant nanomedicine prepared according to any one of claims 1 to 5, characterized in that: Used for preparing drugs for treating acute kidney injury.

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