Lactoferrin nanoparticles, method for preparing same, and use thereof

Abstract

The application provides a lactoferrin nanoparticle and a preparation method and application thereof, and adopts tannic acid, polyethylene glycol and lactoferrin to self-assemble to form tannic lactoferrin nanoparticles; a multiple non-covalent cross-linking network formed between the polyphenol TA and the lactoferrin greatly enhances the structural integrity of the nanoparticles, so that the nanoparticles can effectively resist blood dilution and protein adsorption, and prolong the in-vivo circulation time; the efficient drug loading can be realized by utilizing the pi-pi stacking effect of the TA and the lactoferrin and the efficient capture of the protein drugs in the self-assembly process; and the TA component itself has strong antioxidant activity, and can produce a significant synergistic effect with the loaded lactoferrin drug, so that the high efficient treatment effect is realized.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to lactoferrin nanoparticles, their preparation method, and applications. Background Technology

[0002] Acute myocardial infarction (AMI) is characterized by a sudden reduction in blood supply to the heart. Despite substantial progress in diagnosis and treatment, it remains a leading cause of death worldwide. Persistent ischemia leads to irreversible, progressive changes in myocardial structure and function, with cardiomyocyte death being the core form of damage. Timely restoration of coronary blood flow is the most effective strategy for mitigating ischemic damage and limiting infarct size; however, the reperfusion process itself can cause severe myocardial dysfunction and structural damage, a phenomenon known as myocardial ischemia-reperfusion injury (MIRI). Due to the extremely limited regenerative capacity of adult mammalian cardiomyocytes, significant ventricular fibrotic remodeling is frequently observed in acute cardiomyocyte death events such as MIRI. These progressive fibrotic changes can destroy the normal tissue structure of the myocardium, thereby impairing the heart's mechanical contractile and diastolic functions and electrical conduction system, ultimately leading to fatal heart failure and malignant arrhythmias. Currently available drugs for intervening in MIRI, such as antioxidants (e.g., vitamin C and N-acetylcysteine) and antifibrotic therapies (e.g., TGF-β inhibitors and β-blockers), have limited overall efficacy, are difficult to halt disease progression, and long-term treatment increases the risk of hepatotoxicity. Therefore, there is an urgent need to develop new strategies that simultaneously inhibit cardiomyocyte death and regulate abnormal fibrosis repair in order to effectively treat MIRI.

[0003] Mounting evidence suggests that iron-dependent lipid peroxidation (LPO)-mediated cell death (known as ferroptosis) is a primary cause of myocardial ischemia-reperfusion injury (MIRI). The heart, as a highly metabolic organ, is rich in mitochondria to meet its energy demands. Mitochondria play a central role in iron metabolism and are the primary site of reactive oxygen species (ROS) production. These two key processes drive ferroptosis in the pathology of MIRI. Ischemic-hypoxic stress disrupts cardiomyocyte iron homeostasis, reducing the mitochondrial capacity to process and store iron, leading to the release of free iron. 2+ Abnormal accumulation. These excess Fe... 2+ Lipid peroxidation catalyzes the Fenton reaction, generating large amounts of lipid ROS and hydroxyl radicals (•OH), which directly damage cells and induce oxidative stress, ultimately leading to ferroptosis in cardiomyocytes. This oxidative damage simultaneously inhibits the GSH / GPX4 antioxidant axis, promoting the accumulation of lipid peroxides. The sudden oxygen supply during reperfusion induces mitochondrial reverse electron transport, resulting in the production of large amounts of mitochondrial reactive oxygen species (mtROS), exacerbating this damage and creating a vicious cycle of iron imbalance, oxidative stress, and antioxidant depletion. Therefore, targeting ferroptosis and oxidative stress provides an important cardioprotective strategy for improving MIRI.

[0004] To address these mechanisms, researchers have designed small-molecule iron chelators to bind intracellular free iron and prevent Fenton reaction-driven ROS production. Compounds such as dexrazoxane (DXZ) and iron statin-1 (Fer-1) can improve cardiac function by alleviating myocardial iron overload and nuclear oxidative stress after pretreatment. Despite their promising prospects, these traditional chelators face significant limitations: short half-life, insufficient tissue penetration, disruption of metal ion homeostasis, and systemic toxicity risks. These drawbacks highlight the need for novel drugs with optimized delivery and safety. Summary of the Invention

[0005] To overcome at least one of the problems existing in the prior art, one objective of the present invention is to provide lactoferrin nanoparticles. A second objective of the present invention is to provide a method for preparing the aforementioned lactoferrin nanoparticles. A third objective of the present invention is to provide a pharmaceutical composition. A fourth objective of the present invention is to provide applications of the aforementioned lactoferrin nanoparticles or pharmaceutical composition. A fifth objective of the present invention is to provide applications of the aforementioned lactoferrin nanoparticles or pharmaceutical composition.

[0006] Lactoferrin (Ltf) is an iron-binding glycoprotein with higher iron affinity, higher specificity, and fewer side effects than conventional iron chelators. Evidence suggests that Ltf downregulation promotes ferroptosis, while exogenous supplementation inhibits this process. Despite these properties, the therapeutic potential of Ltf is limited by its short half-life, insufficient tissue penetration, low bioavailability, and demanding storage / transport conditions. Nanotechnology has facilitated various nanoparticle-based protein delivery systems, including lipid-based nanosystems, polymer-based nanosystems, and nanozymes. However, the efficacy of cardiac drug delivery is hampered by shear stress, leading to poor bioavailability, limited therapeutic effects, and potential systemic toxicity. Therefore, enhancing the retention and accumulation of nanomedicines in the high-flow cardiac environment is crucial. While invasive methods such as intramyocardial injection can achieve precise delivery, they carry risks of tissue damage and infection. Notably, tannins (TA) exhibit high affinity for cardiac extracellular matrix components such as elastin and collagen. TA modification significantly enhances protein accumulation at cardiac lesions. Its abundant phenolic hydroxyl groups enable proteins to bind and confer potent antioxidant activity, protecting proteins from oxidative damage. Furthermore, TA-modified proteins can selectively scavenge mtROS by binding to mitochondrial outer membrane proteins, restoring mitochondrial integrity and function. Leveraging this, this invention synthesizes tannin-modified lactoferrin nanoparticles (PTL) via a one-pot method. PTLs efficiently accumulate in ischemic myocardium, are internalized by cardiomyocytes, and escape from endosomal tissues through the proton sponge effect. Crucially, PTLs can chelate intracellular iron, inhibit lipid peroxidation, specifically scavenge mtROS, and restore mitochondrial function, thereby inhibiting mitochondrial-driven ferroptosis by breaking the vicious cycle of iron overload-oxidative stress.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides lactoferrin nanoparticles comprising the following raw materials: tannic acid (TA), polyethylene glycol (PEG) and lactoferrin (Ltf).

[0008] Preferably, the mass ratio of tannic acid to polyethylene glycol is 1:(0.5-2).

[0009] More preferably, the mass ratio of tannic acid to polyethylene glycol is any one of the following, or any two of the following ranges: 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0.

[0010] Preferably, the mass ratio of tannic acid to lactoferrin is 1:(0.5-2).

[0011] More preferably, the mass ratio of tannic acid to lactoferrin is any one of the following or any two values: 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0.

[0012] Preferably, the lactoferrin nanoparticles are spherical, cubic, conical, cylindrical, prismatic, pyramidal, or other regular or irregular shapes.

[0013] More preferably, the lactoferrin nanoparticles are spherical in shape, and the particle size of the lactoferrin nanoparticles is 10-500 nm.

[0014] More preferably, the particle size of the lactoferrin nanoparticles is any value or any two values ​​of 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 nm.

[0015] A second aspect of this invention provides a method for preparing the lactoferrin nanoparticles described in the first aspect, comprising the following steps: Tannic acid, polyethylene glycol, and lactoferrin were mixed in water and self-assembled to obtain the lactoferrin nanoparticles.

[0016] Preferably, the process includes the following steps: mixing polyethylene glycol aqueous solution, lactoferrin aqueous solution and tannic acid aqueous solution in sequence, and performing self-assembly to obtain the lactoferrin nanoparticles.

[0017] More preferably, the concentration of polyethylene glycol in the polyethylene glycol aqueous solution is 1-100 mg / mL.

[0018] More preferably, the concentration of polyethylene glycol in the polyethylene glycol aqueous solution is 1-20 mg / mL.

[0019] More preferably, the concentration of lactoferrin in the lactoferrin aqueous solution is 1-100 mg / mL.

[0020] More preferably, the concentration of lactoferrin in the lactoferrin aqueous solution is 1-20 mg / mL.

[0021] More preferably, the concentration of tannic acid in the tannic acid aqueous solution is 1-100 mg / mL.

[0022] More preferably, the concentration of tannic acid in the tannic acid aqueous solution is 1-20 mg / mL.

[0023] More preferably, the volume ratio of the polyethylene glycol aqueous solution to the lactoferrin aqueous solution is 1:(0.5-2).

[0024] More preferably, the volume ratio of the polyethylene glycol aqueous solution to the lactoferrin aqueous solution is any value or a range formed by any two values ​​from 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, to 1:2.0.

[0025] More preferably, the volume ratio of the polyethylene glycol aqueous solution to the tannic acid aqueous solution is 1:(0.5-2).

[0026] More preferably, the volume ratio of the polyethylene glycol aqueous solution to the tannic acid aqueous solution is any value or a range formed by any two of the following: 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, and 1:2.0.

[0027] More preferably, the method further includes the following steps: mixing and then adding water to quantify the mixture, wherein the concentrations of polyethylene glycol, lactoferrin, and tannic acid in the quantified mixed solution are each independently 0.1-1 mg / mL.

[0028] Preferably, the self-assembly is carried out under stirring conditions.

[0029] Preferably, the self-assembly is carried out at 15-40°C.

[0030] More preferably, the experiment is conducted at 23-26°C.

[0031] A third aspect of the present invention provides a pharmaceutical composition comprising the lactoferrin nanoparticles described in the first aspect.

[0032] Preferably, it also includes one or more pharmaceutically acceptable excipients.

[0033] More preferably, the pharmaceutically acceptable excipient refers to a drug carrier conventional in the pharmaceutical field, including at least one of fillers, binders, disintegrants, solubilizers, suspending agents, wetting agents, pigments, fragrances, solvents, surfactants, or flavoring agents.

[0034] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances such as phosphates; glycine; sorbic acid; potassium sorbate; mixtures of partial glycerides of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blocking polymers; lanolin; sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives. Biological agents such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; Ringer's solution; ethanol; phosphate buffer solutions; and other non-toxic and suitable lubricants such as sodium lauryl sulfate and magnesium stearate; colorants; release agents; preservatives and antioxidants.

[0035] Preferably, the dosage form of the composition includes any one of oral formulations, injectable formulations, respiratory formulations, and transdermal formulations.

[0036] More preferably, the dosage forms of oral preparations include granules, tablets, powders, capsules, oral solutions, oral suspensions, dry suspensions, oral emulsions, gels, oral liquids, emulsions, emulsions, colloidal solutions, mixtures, tinctures, drops, suspension drops, syrups, sustained-release tablets, sustained-release coated tablets, controlled-release tablets, sustained-release capsules, and controlled-release capsules.

[0037] More preferably, the dosage form of the injectable preparation includes injections, injection solutions, solutions for injection, injection solutions for intravenous infusion, suspensions for injection, sterile powders for injection, intravenous injections, emulsions for injection, emulsion injections, powder injections, injections, sterile powder injections, lyophilized powder injections, and concentrated solutions for injection.

[0038] The fourth aspect of the present invention provides the use of the lactoferrin nanoparticles described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for treating myocardial damage.

[0039] Preferably, the drug is used to treat myocardial ischemia-reperfusion injury.

[0040] The fifth aspect of this invention provides the use of the lactoferrin nanoparticles described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of targeted drugs that inhibit ferroptosis.

[0041] Preferably, the lactoferrin nanoparticles or pharmaceutical composition are used in the preparation of a therapeutic drug for cardiomyocyte ferroptosis-related diseases.

[0042] More preferably, the cardiomyocyte ferroptosis-related diseases include myocardial ischemia-reperfusion injury, drug-induced heart failure, and septic cardiomyopathy.

[0043] The beneficial effects of this invention are: This invention provides lactoferrin nanoparticles, which are formed by the self-assembly of tannic acid, polyethylene glycol, and lactoferrin to form tannic lactoferrin nanoparticles. The multiple non-covalent cross-linked networks formed between the polyphenol TA and lactoferrin greatly enhance the structural integrity of the nanoparticles, enabling them to effectively resist blood dilution and protein adsorption, and prolonging in vivo circulation time. Utilizing the π-π stacking effect between TA and lactoferrin, and the efficient capture of protein drugs during the self-assembly process, highly efficient drug loading can be achieved. Furthermore, the TA component itself has strong antioxidant activity, which can produce a significant synergistic effect with the loaded lactoferrin drug, jointly achieving a highly effective therapeutic effect.

[0044] Further explanation reveals that in this nanoparticle system, polyethylene glycol (PEG) is not an inert carrier, but rather a key hub for maintaining the stability and functionality of the nanoparticles through synergistic effects with proteins and tannins. Firstly, the ether oxygen atoms in the PEG molecular chain act as strong hydrogen bond acceptors, forming a more stable hydrogen bond network with the phenolic hydroxyl groups of TA than the protein-TA complex. This competitive binding not only finely regulates the crosslinking density between TA and lactoferrin, preventing excessive aggregation that could lead to particle instability, but also forms a dynamic "protective shell" under physiological conditions, effectively shielding non-specific adsorption sites on the protein surface, thus significantly resisting blood dilution and protein crown formation, and prolonging in vivo circulation time. Furthermore, PEG plays a crucial role in regulating "molecular crowding" during this self-assembly process. The exclusion volume effect generated by its long-chain structure effectively increases the effective collision frequency between lactoferrin and tannins in the local system and weakens the hydration layer on their surfaces, lowering the energy barrier for self-assembly nucleation. Simultaneously, PEG effectively inhibits the disordered aggregation between newly formed particles through steric hindrance, guiding more precursor substances towards the target nanostructure. This synergy between kinetics and thermodynamics significantly improves the synthesis yield of nanoparticles, providing a strong guarantee for subsequent industrial production.

[0045] The lactoferrin nanoparticles of the present invention can be used to treat myocardial damage. They target mitochondria by affinity with mitochondrial outer membrane proteins, independent of mitochondrial membrane potential, and can further scavenge mitochondrial reactive oxygen species (mtROS), reduce oxidative stress and iron accumulation, block the vicious cycle of mitochondrial damage-ferroptosis, ultimately reduce cardiomyocyte death, restore mitochondrial function, and promote myocardial repair. Attached Figure Description

[0046] Figure 1 For the preparation and characterization of PTL, (A) a schematic diagram of the PTL synthesis system; (B) the binding affinity of PTL; (C) the Ltf loading of PTL prepared with different TA to Ltf volume ratios; (D) the size and polydispersity index of PTL prepared with different TA to Ltf volume ratios; (E) the Zeta potential of PTL prepared with different TA to Ltf volume ratios; (F) the XPS image of PTL; (G) the SEM image of PTL. Figure 2 The results of mitochondrial binding and endosome escape experiments of PTL are shown below. (A) Representative images of co-localization of Cy5-PTL (red) with endosomes (green) after different incubation times in H9C2 cells; (B) Verification of the endosome escape mechanism of PTL using a calcein release assay; (C) Subcellular localization of Cy5-PTL, with organelles stained with a specific tracking agent (green); cell nuclei are shown in blue; (D) Quantitative analysis of co-localization (R) between Cy5-PTL and specific organelles; (E) Mitochondrial binding capacity of Cy5-Ltf, Cy5-TA, and Cy5-PTL after incubation with isolated mitochondria by flow cytometry; (F) SDS-PAGE analysis of the precipitate and supernatant of PTL after incubation with various biomolecules for 24 hours; (G) Schematic diagram of intracellular kinetics of PTL. Figure 3 The results of PTL's antioxidant experiments are shown in Figure (A), which is a schematic diagram of the H2O2-induced oxidative stress cell model; (B,K) intracellular ROS labeled with DCFH-DA (B,C), mitochondrial ROS labeled with MitoSOXRed (mtROS) (D,E), mitochondrial membrane potential (MMP) labeled with TMRM (F,G), and intracellular Fe²⁺ labeled with FerroOrange. + (H,I) Representative fluorescence images and corresponding quantifications of lipid peroxidation (J,K) labeled with BODIPY™ 581 / 591C11; (LM) Flow cytometry analysis of total ROS (L) and mtROS (M) levels; (N) Viability of H9C2 cells in an oxidative stress model after treatment with different concentrations of PTL; (O) Intracellular ATP levels in different treatment groups; (P) Cardioprotective mechanism of PTL by inhibiting ferroptosis and restoring mitochondrial function through reducing iron accumulation and oxidative stress. Figure 4The results of the cardiac accumulation experiment of PTL are shown in the following: (A) Protein coating experiment demonstrates the strong affinity of PTL for the main components of the myocardial extramedullary matrix (collagen and elastin); (B) Schematic diagram of the experimental design for intravenous administration and in vivo tracking of PTL; (C) Representative fluorescence images showing the retention of Ltf and PTL in rat myocardial tissue; (D) Heart samples were collected from MIRI rats 6 hours after intravenous injection of Cy5-PTL. Subsequently, the collected hearts and their sections were subjected to fluorescence imaging, and the corresponding sections were stained with TTC to explore the colocalization of fluorescence with ischemic sites. Figure 5 The results of PTL treatment experiments in mice include: (A) a timeline of rat MIRI model establishment and PTL treatment regimens; (B) representative M-mode echocardiograms of different treatment groups at 14 and 28 days after MIRI; (C) quantitative assessment of cardiac function parameters in MIRI rats receiving different treatments at 14 and 28 days after model establishment, including ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic volume (LVESV), and left ventricular systolic diameter (LVID); (D) thermographic analysis of short-axis cardiac function parameters from echocardiograms in Figure C; and (EF) using bio-based... Serum levels of myocardial enzymes CK and CK-MB were measured using a chemical analyzer; (G) Representative results of H&E and Masson trichrome staining of myocardial tissue; (H,L) Immunofluorescence analysis of type I collagen (red) and type III collagen (green) levels in the MIRI region of rats after different treatments; (I) Representative H&E stained sections of the left ventricle (left) and corresponding segmental analysis of ventricular wall thickness (right); the left ventricle was divided into 6 segments for quantitative assessment using ImageJ; (J) Immunohistochemical staining of GPX4 and 4-HNE on day 28 after MIRI modeling; Figure 6 The diagram shows the PTL treatment for MIRI, where (A) is a schematic diagram of PTL synthesis and mouse experiments; and (B) is a schematic diagram of the treatment for MIRI. Detailed Implementation

[0047] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial channels or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0048] Example 1 This embodiment provides lactoferrin nanoparticles (PTL), the preparation process of which is as follows: Figure 1 PTL was prepared using a one-pot self-assembly strategy for component A. The specific preparation method is as follows: Polyethylene glycol (PEG, Sigma-Aldrich, Mw = 10 kDa), lactoferrin (LTF, Aladdin), and TA were dissolved in deionized water to prepare a stock solution with a concentration of 5 mg / mL. 200 μL of PEG and Ltf were added sequentially to a vial containing deionized water. Then, 200 μL of TA was added to the vial to initiate the self-assembly process. The final liquid volume was 2 mL. The solution was stirred at room temperature for 30 min to obtain PTL, and the solution was observed to gradually change from clear to turbid. After the reaction was complete, the mixture was transferred to a centrifuge tube and centrifuged at high speed (8000 g, 10 min) to remove unbound free molecules. The purified PTL was dispersed in deionized water or a buffer solution for later use.

[0049] Characterization of lactoferrin nanoparticles Characterization of lactoferrin nanoparticles as follows: Figure 1 The volume ratio of TA to Ltf was systematically varied in the BH model. The Ltf loading was investigated by quantifying the Ltf content in the supernatant and precipitate (PTL) using Coomassie Brilliant Blue staining. The size, polydispersity index (PDI), and zeta potential of the nanoparticles were evaluated using a Malvern particle size analyzer to determine the optimal formulation ratio. After comprehensive optimization, the optimal PTL preparation conditions were determined to be 10 kDa PEG and a Ltf to TA volume ratio of 1:1:1, which were used for subsequent experiments. Molecular simulations showed that the binding free energy between Ltf and TA was -9.8 kcal / mol, indicating that Ltf and TA can undergo a self-assembly reaction (Figure 1B). XPS elemental analysis verified the successful loading of Ltf. Figure 1 F). When the PTL solution was irradiated with a laser pointer, the Tyndall effect experiment showed a clear beam path, confirming the formation of nanoparticles (Fig. 1G). Transmission electron microscopy (TEM) showed that the PTL exhibited a uniform spherical morphology, good dispersion, and an average size of approximately 150 nm. Figure 1 G).

[0050] Performance analysis of lactoferrin nanoparticles 1. Analysis of mitochondrial binding and endosome escape of PTL To evaluate the mitochondrial binding capacity of PTL, mitochondria were isolated from H9C2 cells and co-incubated with Cy5-labeled TA, PTL, and Ltf for 1 h. The binding efficiency was then quantitatively analyzed by flow cytometry. The results showed... Figure 2Compared to free Ltf, PTL exhibits significantly enhanced mitochondrial binding capacity. To clarify the subcellular localization of PTL in living cells, Cy5-labeled PTL was co-incubated with H9C2 cells for 4 h, and the results were observed using organelle-specific probe staining. The results showed that the co-localization effect of PTL with mitochondria was significantly better than that of other organelles (Pearson correlation coefficient R = 0.78), indicating that it has mitochondrial-preferred localization characteristics.

[0051] To investigate the endosome escape and mitochondrial targeting capabilities of PTL, this invention co-incubated H9C2 cardiomyocytes with Cy5-labeled PTL and systematically analyzed its intracellular localization and transport processes using flow cytometry and confocal microscopy (CLSM). The endosome escape capability of PTL was assessed through co-localization experiments, observing the distribution characteristics of Cy5-PTL (red fluorescence) and the endosome marker (green fluorescence): In the early stages of incubation, a large number of yellow spots were formed by the co-localization of red and green fluorescence, indicating that PTL was successfully captured in the endosome compartment. As the incubation time increased, the number of yellow spots gradually decreased, and the overlap of red and green fluorescence significantly decreased, indicating that PTL exhibited time-dependent endosome escape.

[0052] To elucidate the endosome escape mechanism of PTL, a "label-free" validation was performed using calcein combined with bafloxacin A1 (using unlabeled PTL). Calcein is a membrane-impermeable dye; when the endosome membrane is intact, it is confined to the endosome compartment and exhibits punctate fluorescence. When the endosome membrane ruptures and its contents are released, calcein diffusely distributes throughout the cytoplasm. The results showed that cells treated with PTL exhibited diffuse calcein fluorescence, suggesting that it promotes endosome membrane rupture and contents release. Pretreatment with bafloxacin A1 (a V-ATPase inhibitor) resulted in punctate calcein fluorescence in the PTL-treated cells, indicating that the proton sponge effect is one of the important mechanisms mediating PTL endosome escape. In summary, PTL possesses highly efficient endosome escape characteristics, successfully escaping the endosome compartment and targeting mitochondria, laying an important foundation for its subsequent functions in intracellular antioxidation, iron chelation, and regulation of ferroptosis.

[0053] 2. Analysis of the antioxidant properties of PTL To verify its antioxidant effect, an H2O2-induced oxidative damage cell model was constructed. Figure 3 In section A), the levels of total ROS and mtROS in cells were detected using DCFH-DA and MitoSOX Red fluorescent probes, respectively. The results of CLSM and flow cytometry are shown below. Figure 3The iron chelating and anti-LPO effects of the iron-containing compound were significantly increased after H2O2 induction. To verify its iron chelating and anti-LPO effects at the cellular level, H2O2-stimulated and oxygen-glucose deprivation (OGD / R) cell models were constructed, and intracellular iron was detected using FerroOrange and BODIPY™ 581 / 591 C11 fluorescent probes, respectively. 2+ and LPO levels. CLSM test results are as follows: Figure 3 HK cells, after H2O2 stimulation and OGD / R treatment, showed increased intracellular Fe. 2+ Both LPO levels were significantly elevated; Ltf treatment alone slightly reduced both levels, but the reduction was more significant in the PTL group, confirming that PTL exerts a cytoprotective effect by chelating iron and inhibiting LPO. Compared with the Ltf treatment alone group, the PTL treatment group significantly reduced total ROS and mtROS levels (e.g., Ltf alone). Figure 3 The antioxidant effect of PTL is more prominent, which confirms that PTL can effectively reduce cellular oxidative stress damage.

[0054] Oxidative stress and ferroptosis can damage mitochondrial function, leading to decreased mitochondrial membrane potential (MMP) and reduced ATP production. Oxidative stress during reperfusion further exacerbates mitochondrial damage, resulting in persistent respiratory chain dysfunction and ATP depletion. TMRM staining results show that oxidative stress induces MMP depolarization, while PTL treatment can significantly reverse this damage. ATP assays indicate that H2O2 significantly inhibits ATP synthesis, and PTL can restore it to near-normal levels (e.g., ...). Figure 3 The above results indicate that PTL can effectively protect mitochondrial function, restore MMP and ATP production, thereby exerting a cardioprotective effect (e.g., MMP and ATP production). Figure 3 (P in the middle).

[0055] 3. Analysis of PTL accumulation in ischemic myocardial tissue To investigate whether intravenously injected PTL can effectively accumulate in ischemic myocardial tissue, PTL and free Ltf were labeled with Cy5, and their in vivo distribution was dynamically tracked using a small animal in vivo fluorescence imaging system. The results showed that in MIRI-induced rat models, the fluorescence intensity in the heart region of the Cy5-PTL group was significantly higher than that of the Cy5-Ltf group, indicating that PTL has superior targeting ability to ischemic myocardium compared to free Ltf (e.g., Figure 4 (A in the text). Further observation revealed that the fluorescence intensity of Cy5-PTL in the infarcted area was significantly higher than that in the non-infarcted area (e.g., in the infarct area). Figure 4 The C value suggests that PTL can specifically accumulate in ischemic myocardial tissue. This targeted enrichment characteristic may be closely related to the high affinity of PTL for components such as elastin and collagen in the myocardial extramural matrix. Protein coating experiment results are shown below. Figure 4The D in the sample showed a high affinity for the main components of the extramyocardial matrix (collagen Col and elastin Ela), suggesting that its myocardial targeting ability may be due to the binding of TA to the extramyocardial matrix.

[0056] 4. Analysis of PTL's role in inhibiting ferroptosis and improving cardiac function Timeline of rat MIRI model establishment and PTL treatment regimen as follows: Figure 5 Male SD rats were randomly divided into three groups: sham-operated group (Sham), model group (PBS), and experimental group (Ltf, PTL). A MIRI model was established by transient ligation of the left anterior descending coronary artery (LAD). The drug administration regimen was as follows: the corresponding drug was injected via the tail vein 10 minutes before reperfusion, followed by once-daily administration for one week. Cardiac function was monitored by echocardiography on days 14 and 28 post-treatment. Figure 5 B). M-mode ultrasound quantitative analysis such as Figure 5 CD results showed that the ejection fraction (EF) and fractional shortening (FS) were significantly decreased in the PBS group, indicating severe cardiac dysfunction. Ltf treatment could partially restore cardiac function in MIRI rats; while the PTL group significantly improved EF and FS levels. Left ventricular size measurements showed that the left ventricular end-systolic diameter (LVIDs) and left ventricular end-systolic volume (LVESV) were significantly increased in the PBS group, indicating progressive ventricular dilatation. PTL treatment effectively inhibited ventricular remodeling, restoring LVIDs and LVESV to levels close to those in the Sham group. In addition, serum myocardial injury markers on day 28 showed ( Figure 5 In the PBS group, CK and CK-MB levels were significantly elevated, while PTL treatment reduced them to the levels seen in the Sham group. In conclusion, PTL treatment can significantly improve cardiac function after MIRI, reduce ventricular remodeling, and lower the levels of myocardial injury markers. Its protective effect is superior to free Ltf, demonstrating good potential for clinical application.

[0057] Twenty-eight days after treatment, rats were euthanized and their heart tissues were harvested. The intervention effect of PTL on myocardial pathological remodeling after MIRI was systematically evaluated using H&E staining, Masson staining, and immunofluorescence staining. A clock face model was used to divide the left ventricle into six segments, with color coding reflecting the thickness gradient. Results showed that the PBS group exhibited compensatory septal thickening, while the PTL treatment group showed a more uniform distribution of ventricular wall thickness across all segments. H&E staining showed (… Figure 5In normal myocardial tissue, cardiomyocytes are regularly arranged, connected by intercalated discs, and striations are clearly visible. After MIRI, a large number of cardiomyocytes are lost in the damaged area, and the remaining cells are disordered. The cardiomyocyte density in the Ltf and PTL treatment groups was significantly restored, and the tissue structure tended to be regular. Masson staining showed that the myocardial tissue in the Sham group was mainly red, with very little blue collagen deposition; in the PBS group, necrotic myocardium was extensively replaced by loose collagen fibers, and blue collagen staining was significantly enhanced; the fibrosis deposition in the Ltf treatment group was moderately reduced; and the collagen content in the PTL treatment group was significantly reduced. Immunofluorescence staining was used to detect type I and type III collagen to further assess the degree of cardiac fibrosis. The results showed that PTL treatment significantly reduced the deposition of both types of collagen ( Figure 5 The above results indicate that PTL can effectively reduce pathological remodeling and compensatory left ventricular hypertrophy after MIRI.

[0058] 4-Hydroxynonenal (4-HNE) is a highly toxic aldehyde byproduct produced during lipid peroxidation. It can modify intracellular proteins and damage DNA, ultimately leading to cell damage and ferroptosis. GPX4, as a key GSH-dependent antioxidant enzyme, forms the core antioxidant system together with GSH. Under physiological conditions, functional GPX4 can effectively inhibit the generation of lipid peroxidation byproducts such as 4-HNE, reducing cell damage. However, oxidative stress during MIRI consumes intracellular GSH and inhibits GPX4 activity, leading to the accumulation of lipid peroxides. Simultaneously, the accumulated 4-HNE further accelerates GPX4 degradation, creating a vicious cycle. To investigate the effect of mPPN on this antioxidant axis, immunohistochemical verification was performed on myocardial tissue 28 days after MIRI modeling. The results showed ( Figure 5 Compared with the Sham group, the PBS group showed significantly downregulated GPX4 expression and significantly upregulated 4-HNE expression; while mPPN treatment alleviated this trend, significantly improved oxidative stress and lipid peroxidation, and ultimately inhibited ferroptosis and protected the myocardium.

[0059] In summary, the PTL of the present invention is synthesized through one-step self-assembly. A schematic diagram of PTL treatment for MIRI is shown below. Figure 6 As shown, PTL nanoparticles can specifically scavenge mtROS, effectively chelate intracellular iron, inhibit lipid peroxidation, and restore mitochondrial function, breaking the vicious cycle of ferroptosis and mitochondrial dysfunction. This effectively inhibits cardiomyocyte death and reduces pathological fibrosis without affecting natural repair, showing broad application potential in the treatment of restoring myocardial function.

[0060] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A lactoferrin nanoparticle, characterized in that, The raw materials used in its preparation include tannic acid, polyethylene glycol, and lactoferrin.

2. The lactoferrin nanoparticles according to claim 1, characterized in that, The mass ratio of tannic acid to polyethylene glycol is 1:(0.5-2); And / or, the mass ratio of tannic acid to lactoferrin is 1:(0.5-2).

3. The lactoferrin nanoparticles according to claim 1, characterized in that, The lactoferrin nanoparticles are spherical, cubic, conical, cylindrical, prismatic, pyramidal, or other regular or irregular shapes. Preferably, the lactoferrin nanoparticles are spherical in shape and have a particle size of 10-500 nm.

4. A method for preparing lactoferrin nanoparticles according to any one of claims 1-3, comprising the following steps: Tannic acid, polyethylene glycol, and lactoferrin were mixed in water and self-assembled to obtain the lactoferrin nanoparticles.

5. The method for preparing lactoferrin nanoparticles according to claim 4, characterized in that, Specifically, the process includes the following steps: mixing polyethylene glycol aqueous solution, lactoferrin aqueous solution and tannic acid aqueous solution in sequence, and then performing self-assembly to obtain the lactoferrin nanoparticles.

6. A pharmaceutical composition, characterized in that, Including the lactoferrin nanoparticles according to any one of claims 1-3.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition includes any one of oral preparations, injectable preparations, respiratory preparations, and transdermal preparations.

8. The use of the lactoferrin nanoparticles according to any one of claims 1-3 and the pharmaceutical composition according to any one of claims 6-7 in the preparation of a medicament for treating myocardial damage.

9. The application according to claim 8, characterized in that, The drug is used to treat myocardial ischemia-reperfusion injury.

10. The use of the lactoferrin nanoparticles according to any one of claims 1-3 and the pharmaceutical composition according to any one of claims 6-7 in the preparation of targeted drugs that inhibit ferroptosis.

Images