Platelet biomimetic diselenide liposome loaded deferoxamine, and preparation method and application thereof

Abstract

The present application relates to a kind of platelet biomimetic double selenium liposome carries deferoxamine and its preparation method and application, belong to biomedical engineering and nano drug delivery technical field.Deferoxamine is loaded in the hybrid membrane formed by the fusion of lipid and platelet membrane, and the lipid includes lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 with mass ratio of (7-9) : (1-2) : 1.By introducing ROS-responsive double selenium bond, intelligent control release of drug in lesion site is realized, and treatment efficiency is greatly improved;By synergistically integrating the iron chelating ability of DFO and the antioxidant capacity of selenium, multi-target, full-path inhibition of iron death pathway after ICH is realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering and nanomedicine delivery technology, specifically relating to a platelet biomimetic diselenyl liposome carrying deferroamine, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Intracerebral hemorrhage (ICH) is a highly fatal and disabling subtype of stroke. Its pathological process includes primary and secondary injury. Secondary brain injury involves the lysis of red blood cells within the hematoma, releasing large amounts of hemoglobin, which then degrades to produce free ferrous ions (Fe²⁺). + These iron ions catalyze the production of reactive oxygen species (ROS) through the Fenton reaction, leading to lipid peroxidation, mitochondrial dysfunction, and inactivation of glutathione peroxidase 4 (GPX4), ultimately triggering an iron-dependent programmed cell death process—ferroptosis. This cell death process, driven by iron ion toxicity and oxidative stress, is one of the core mechanisms leading to nerve cell death and neurological deficits.

[0004] To address iron toxicity following ischemic stroke (ICH), iron chelators, particularly desferrioxamine (DFO), have been extensively studied as a treatment strategy. However, traditional DFOs suffer from drawbacks such as short plasma half-life, poor blood-brain barrier penetration, and lack of targeting, and they do not directly scavenge reactive oxygen species (ROS) and lipid peroxides. Nanodelivery systems can improve drug stability and brain delivery efficiency; however, they are easily recognized and cleared by the human mononuclear phagocyte system, affecting therapeutic efficacy. On the other hand, selenium is a key cofactor of GPX4 and can directly reduce toxic lipid peroxides, fundamentally blocking ferroptosis. Although some studies have attempted to construct ROS-responsive platelet liposomes for the treatment of brain diseases, they mainly target reperfusion injury after thrombolysis in ischemic stroke. The core pathology of reperfusion injury is the oxidative burst and inflammatory response triggered by the restoration of blood flow to the ischemic area, and its microenvironment and cell death mechanisms are fundamentally different from the formation and development of hematomas after ICH. Following incision and hematoma (ICH), the hematoma area experiences massive lysis of erythrocytes, releasing hemoglobin and iron ions. This leads to the continuous generation of reactive oxygen species (ROS) and activation of ferroptosis via the Fenton reaction. This pathological chain necessitates treatment strategies that possess multiple synergistic functions, including active targeting and hemostasis at the bleeding site, efficient iron chelation, and targeted antioxidant effects. Therefore, in the pathological environment of ICH, how to intelligently release selenium at the bleeding site while simultaneously exerting multiple functions such as hemostasis, antioxidant effects, and inhibition of ferroptosis remains an unsolved technical challenge. Summary of the Invention

[0005] In view of the current state of technology, the purpose of this invention is to provide a platelet biomimetic diselenyl liposome loaded with deferroamine, its preparation method and application, and to provide a platelet membrane-doped, deferroamine-loaded diselenyl bond liposome (P-Lip-DFO). This platform integrates the natural targeting and long circulation capabilities of platelets, the iron chelating ability of DFO, the antioxidant capacity of selenium, and the ROS-responsive release capability of diselenyl bonds, thereby achieving synergistic, efficient and safe neuroprotective therapy after ICH.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, a platelet biomimetic diselenyl liposome carrying deferoxamine, wherein the deferoxamine is loaded in a hybrid membrane formed by the fusion of lipids and platelet membranes; wherein the lipids include lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 in a mass ratio of (7~9):(1~2):1.

[0007] Secondly, the preparation method of the above-mentioned platelet biomimetic diselenyl liposomes loaded with deferoxamine includes the following steps: S1. Dissolve lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 in a mixed solvent in a predetermined mass ratio, and evaporate under reduced pressure to obtain a lipid film; S2. The lipid film is hydrated with deferoxamine solution, ultrasonically vibrated, and then extruded to encapsulate deferoxamine in the lipid film to obtain DFO-loaded liposomes. S3. The suspension of DFO-loaded liposomes was mixed with the platelet membrane suspension, and after ultrasonic oscillation, the platelet membrane was doped with DFO-loaded liposomes by extrusion to obtain platelet biomimetic diselenyl liposomes loaded with deferroamine.

[0008] Thirdly, the above-mentioned platelet biomimetic double selenium liposomes loaded with deferroamine are used in at least one of the following (a1) to (a3); (a1) Preparation of products that inhibit oxidative stress and ferroptosis after ICH; (a2) Prepare products that reduce hematoma after ICH; (a3) Prepare products that improve motor function after ICH.

[0009] The beneficial effects of this invention are as follows: 1. This invention successfully solves the challenges of targeted drug delivery and long-term circulation in ICH treatment by constructing a platelet membrane biomimetic nanoplatform; by introducing ROS-responsive double selenium bonds, it achieves intelligent controlled release of drugs at the lesion site, greatly improving treatment efficiency; by synergistically integrating the iron chelating ability of DFO and the antioxidant ability of selenium, it achieves multi-target, full-pathway inhibition of the ferroptosis pathway after ICH, with significantly better efficacy than single therapy.

[0010] 2. This invention is specifically designed for the pathological microenvironment following ICH, particularly targeting specific pathological mechanisms such as the massive release of iron ions, severe oxidative stress, and activation of ferroptosis in the hematoma region, resulting in enhanced disease adaptability and therapeutic precision. The introduction of DSPE-Se-Se-PEG-NH2 not only provides ROS-responsive diselenium bonds, but its terminal amino groups (-NH2) also form locally functionalized microdomains on the nanoparticle surface. In the acidic microenvironment unique to hematomas after ICH, these amino groups can undergo protonation, enhancing the interaction between the nanoparticles and negatively charged components in the damaged tissue, thereby promoting their retention and accumulation in the hemorrhagic lesion. Simultaneously, this structure helps optimize the fracture kinetics of diselenium bonds under oxidative stress and the release efficiency of selenium, ensuring that selenium can be effectively utilized by cells as a key cofactor of GPX4, synergistically interacting with the iron chelation of DFO, thus more precisely and efficiently inhibiting ferroptosis. Furthermore, it fully utilizes the specific binding ability of natural proteins on the platelet membrane surface (such as CD41, CD61, GPVI, etc.) to collagen and von Willebrand factor at the site of vascular injury in bleeding sites, achieving active targeting that is closer to the physiological mechanism. At the same time, it preserves the natural hemostatic function of platelets, which helps control hematoma expansion. Through the fusion of platelet membrane and double selenium liposomes, it achieves multi-functional integration of targeting, hemostasis, long circulation, and ROS-responsive drug release, making it more suitable for clinical treatment scenarios of ICH.

[0011] 3. This invention can protect HT22 cells from oxidative damage and inhibit ferroptosis, significantly reduce hematoma volume in the collagenase ICH model, and significantly alleviate ferroptosis and oxidative stress, improve neurological function scores, and restore motor coordination in both ICH models. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 These are detection diagrams for different prepared products in specific implementation methods. A represents the hydrated particle size diagram of P-Lip-DFO with different lipid-to-platelet membrane ratios; B represents the Zeta potential diagram of P-Lip-DFO with different lipid-to-platelet membrane ratios; C represents the particle size diagram of Lip-DFO; D represents the particle size diagram of P-Lip-DFO; E represents the transmission electron microscopy detection diagram; F represents the morphological transformation diagram of Lip-DFO and P-Lip-DFO in the release medium; G represents the fluorescence microscopy results of platelet membrane and liposome co-localization; H represents the immunoblotting results of different prepared products; I represents the quantitative proteomics analysis results; J represents the KEGG pathway bubble diagram; and K represents the drug release curves of Lip-DFO and P-Lip-DFO.

[0014] Figure 2 These are detection graphs for antiferroptosis of different prepared products in specific implementation methods. A represents the ultraviolet absorption spectrum, B represents the detection result graph of ABTS method, C represents the detection result graph of MDA in biochemical detection, D represents the detection result graph of GSH in biochemical detection, E represents the result graph of Calcein-AM / PI staining, F represents the data analysis result graph corresponding to E, G represents the detection result graph of DCFH-DA fluorescent probe, H represents the data analysis result graph corresponding to G, I represents the result graph of FerroOrange staining, J represents the data analysis result graph corresponding to I, K represents the detection result graph of Western blot, L represents the data analysis result graph of TFR1, M represents the data analysis result graph of GPX4, N represents the data analysis result graph of PRDX3, and O represents the data analysis result graph of 4-HNE.

[0015] Figure 3These are test graphs of hemostatic function of different prepared products in specific implementation methods. Among them, A represents the in vitro coagulation effect graph, B represents the data analysis result graph corresponding to A, C represents the mouse tail truncation model, D represents the bleeding time test result graph, E represents the bleeding area test result graph, F represents the bleeding weight test result graph, G represents the lesion volume graph 2 h after ICH, H represents the lesion volume graph 6 h after ICH, I represents the lesion volume statistics graph 2 and 6 h after ICH, J represents the lesion volume increase statistics graph 6 h after ICH compared to 2 h after ICH, and K represents the percentage increase statistics graph of lesion volume 6 h after ICH compared to 2 h after ICH.

[0016] Figure 4 This is a diagram showing the in vivo pharmacodynamic evaluation of different prepared products in the specific implementation method. In this diagram, A represents the experimental flowchart, B represents the behavioral test diagram, C represents the neurological deficit scores of different groups, D represents the tensile test results of different groups, E represents the cornering test results of different groups, F represents the grid test results of different groups, G represents the right forelimb stride results of different groups, H represents the right limb coordination results of different groups, I represents the right hindlimb stride results of different groups, J represents the hematoma volume results on day 7, and K represents the data analysis results of J.

[0017] Figure 5 This is a graph showing the effect of different prepared products on ferrodeogenesis after ICH in a specific implementation method. In this graph, A represents the DHE staining fluorescence image of brain tissue from each group of mice; B represents the data analysis results of A; C represents the MDA content graph; D represents the GSH content graph; E represents the H2O2 content graph; F represents the total iron content graph; G represents the Prussian blue staining result graph; H represents the data analysis results of G; I represents the mitochondrial damage graph; J represents the mitochondrial atrophy percentage graph; K represents the average mitochondrial size graph; L represents the mitochondrial aspect ratio graph; M represents the Western blot results of TFR1, GPX4, and PRDX3 in brain tissue; N represents the Western blot results of 4-HNE in brain tissue; O represents the TFR1 content graph; P represents the GPX4 content graph; Q represents the PRDX3 content graph; and R represents the 4-HNE content graph.

[0018] Figure 6 This is a schematic diagram of the principle in a specific implementation method, where A is a schematic diagram of the preparation process and B is a schematic diagram of the mechanism of action of the pathological chain of hematoma after ICH. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] DSPE-Se-Se-PEG-NH2, distearate phosphatidylethanolamine-diselement bond-polyethylene glycol-amino, is produced by Huabio, catalog number WH001820301.

[0022] One or more embodiments of the present invention provide a platelet biomimetic diselenyl liposome loaded with deferoxamine, comprising deferoxamine, wherein the deferoxamine is loaded in a hybrid membrane formed by the fusion of lipids and platelet membrane; wherein the lipids comprise lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 in a mass ratio of (7~9):(1~2):1.

[0023] In the above structure, proteins retained on the platelet membrane surface (such as CD41, CD61, and GPVI) can specifically bind to collagen and von Willebrand factor exposed at the site of vascular injury, achieving active targeting; at the same time, "don't eat me" signaling proteins such as CD47 can help nanoparticles evade immune clearance and significantly prolong the circulation time in vivo; the selenium contained in the lipid membrane is a key cofactor of GPX4, which can directly reduce toxic lipid peroxides and fundamentally block the execution of ferroptosis; combined with the ability of DFO to chelate iron ions, it can effectively achieve multi-target, full-pathway inhibition of the ferroptosis pathway after ICH.

[0024] Optionally, the mass ratio of the lipid film to deferoxamine is 10:(1~10); the encapsulation efficiency and drug loading rate are high, and the liposomes exhibit significantly accelerated drug release characteristics when stimulated by reactive oxygen species (ROS, H2O2).

[0025] Optionally, the mass ratio of lipids to platelet membrane proteins is (2~3):1; the surface charge is -22.1 ±0.2mV, which is highly consistent with the platelet membrane surface charge.

[0026] Optionally, the hydrated particle size of the platelet biomimetic diselenyl liposomes loaded with deferroamine is 100~200 nm.

[0027] One or more embodiments of the present invention provide a method for preparing the above-mentioned platelet biomimetic diselenyl liposomes loaded with deferoxamine, comprising the following steps: S1. Dissolve lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 in a mixed solvent in a predetermined mass ratio, and evaporate under reduced pressure to obtain a lipid film; S2. The lipid film is hydrated with deferoxamine solution, ultrasonically vibrated, and then extruded to encapsulate deferoxamine in the lipid film to obtain DFO-loaded liposomes. S3. The suspension of DFO-loaded liposomes was mixed with the platelet membrane suspension, and after ultrasonic oscillation, the platelet membrane was doped with DFO-loaded liposomes by extrusion to obtain platelet biomimetic diselenyl liposomes loaded with deferroamine.

[0028] Optionally, in S1, the mixed solvent includes methanol and chloroform in a volume ratio of 1:(0.5~1.5).

[0029] Optionally, in S1, the rotary evaporation under reduced pressure is heated in a 45°C water bath.

[0030] Optionally, in S2, the deferoxamine solution is a deferoxamine solution in phosphate buffer with a concentration of 1.5~2.5 mg / mL.

[0031] Optionally, in S2, the extrusion method includes: extruding a set number of times sequentially through a 400 nm membrane and a 200 nm membrane of a liposome extruder.

[0032] Optionally, in S2, after extrusion, the suspension containing DFO-loaded liposomes is centrifuged at 10,000-15,000 rpm for 10-20 min, the supernatant is discarded, and the precipitate is resuspended with PBS to obtain purified DFO-loaded liposomes.

[0033] Optionally, in S3, the concentration of the suspension carrying DFO liposomes is 2 mg / mL.

[0034] Optionally, in S3, the extrusion method includes: extruding a set number of times through a 200 nm membrane via a liposome extruder.

[0035] One or more embodiments of the present invention provide the application of the above-mentioned platelet biomimetic diselenyl liposome loaded with deferroamine in at least one of the following (a1) to (a3); (a1) Preparation of products that inhibit oxidative stress and ferroptosis after ICH; (a2) Preparation of products that reduce hematoma volume after ICH; (a3) Prepare products that improve motor function after ICH.

[0036] The present invention will be further described below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions. Unless otherwise specified, all raw materials and reagents used in the following embodiments are commercially available.

[0037] Preparation Example Platelet membrane suspension, preparation includes: Whole blood was centrifuged at 100 ×g for 20 min to obtain platelet-rich plasma (PRP). PRP was centrifuged at 800 ×g for 20 min to obtain platelet precipitate. The platelet precipitate was resuspended in pre-cooled Tyrode's buffer (containing 1% PMSF and PGE1) to obtain a suspension. The suspension was frozen at -80℃ and then thawed in a 37℃ water bath, and the freeze-thaw cycle was repeated 5 times. The lysate after freeze-thaw was centrifuged at 12,000 rpm for 30 min at 4℃, and the supernatant (cytoplasmic proteins) was discarded to obtain the precipitate, which was the purified platelet membrane fragment. PBS buffer was added to the precipitate, and the mixture was sonicated at 25℃ for 5 min using an ultrasonic cell disruptor (power 100 Hz) to obtain a homogeneous platelet membrane suspension, which was aliquoted and stored at -80℃ for later use.

[0038] Example 1 A platelet-inspired diselenyl liposome loaded with deferroamine, the preparation process is as follows: Figure 6 As shown in A, it includes: S1. Accurately weigh 8.75 mg of lecithin, 1.25 mg of cholesterol, and 1.0 mg of DSPE-Se-Se-PEG-NH2 (for ease of calculation, the lipids in this example only include lecithin and cholesterol, amounting to 10 mg; the mixture of lecithin, cholesterol, and DSPE-Se-Se-PEG-NH2 is the total lipids), dissolve them in 10 mL of methanol / chloroform (1:1, v / v) mixed solvent, and then transfer them to a 100 mL round-bottom flask. Under reduced pressure, evaporate the mixture to dryness in a rotary evaporator at 45°C in a water bath to form a uniform lipid film on the flask wall.

[0039] S2. Prepare a 2 mg / mL DFO solution using DFO and phosphate buffer (pH 7.4). Add 1 mL of the DFO solution (2 mg DFO) to the flask in S1. Sonicate the solution to completely hydrate and detach the lipid membrane, forming a liposome suspension. Extrude the liposome suspension through a 400 nm and a 200 nm polycarbonate membrane 11 times each using a liposome extruder to obtain a suspension containing DFO-loaded liposomes of uniform particle size (Lip-DFO). Centrifuge the Lip-DFO suspension at 12,000 rpm for 15 min at 4 °C. Discard the supernatant (to remove unencapsulated free DFO). Resuspend the precipitate in PBS to obtain a purified Lip-DFO suspension.

[0040] S3. The purified Lip-DFO suspension (containing 11 mg of total lipids) was mixed with the platelet membrane suspension (containing 4 mg of membrane proteins), and after ultrasonic oscillation for 30 min, the platelet biomimetic diselenyl liposomes loaded with deferroamine, namely P-Lip-DFO nanoparticles, were obtained by extrusion using a liposome extruder through a 200 nm polycarbonate membrane 11 times. The nanoparticles were stored at 4℃ for later use.

[0041] S4. Adjust the amount of DFO solution added in step S2, keeping the total lipids constant at 11 mg, and prepare P-Lip-DFO with DFO additions of 0.5 mg, 1.0 mg and 10.0 mg respectively.

[0042] S5. Adjust the mass ratio of lipids and membrane proteins in step S3 to 5:1, 5:2, 5:3, 5:4 and 5:5 to obtain P-Lip-DFO with different lipid-to-platelet membrane (for ease of calculation, the mass of platelet membrane is calculated according to membrane proteins) ratios.

[0043] The hydrated particle size of P-Lip-DFO with different lipid-to-platelet membrane ratios was determined by dynamic light scattering (DLS) as follows: Figure 1 As shown in A, the Zeta potential is as follows Figure 1 As shown in B, the hydrated particle size of P-Lip-DFO with a mass ratio of 5:2 is 154.3 ± 9.1 nm, and the Zeta potential is -22.1 ± 0.2 mV, which is highly consistent with the surface charge of platelet membrane.

[0044] 10 μL of sample was added to a copper mesh and deposited for 10 min. After removal, 10 μL of 2% uranium oxyacetate was added and stained for 8 min. After removing the stain, an electron microscope sample was prepared. The Lip-DFO particle size was as follows: Figure 1 As shown in C, the particle size of P-Lip-DFO is as follows: Figure 1 As shown in D, both supports were successfully prepared and have uniform particle size.

[0045] Transmission electron microscopy (TEM) such as Figure 1 As shown in E, the platelet membrane separated by extrusion exhibits a homogeneous morphology, and liposomes with uniform particle size were successfully prepared. After fusion with the platelet membrane, they form hybrid nanoparticles with consistent morphology.

[0046] Liposomes and platelet membranes were labeled with Cy5.5 (red) and the fluorescent membrane dye Dil (orange-red; pseudo-color: green), respectively; these labeled components were co-extruded to obtain dual-labeled platelet membrane hybrid liposomes (P-Lip), as shown below. Figure 1 As shown in G, fluorescence microscopy confirmed that the platelet membrane and liposomes were successfully integrated into the P-Lip carrier; Western blotting assays determined whether P-Lip-DFO contained platelet membrane-specific proteins (such as CD41, CD61, and GPVI, which are closely related to platelet adhesion and aggregation), and the results are shown in G. Figure 1 As shown in H, P-Lip-DFO retains key functional proteins such as CD41, CD61, and GPVI, which lays the foundation for the targeted enrichment of P-Lip-DFO at in vivo hemorrhage sites.

[0047] Quantitative proteomics analysis identified 50 proteins associated with cell adhesion in P-Lip-DFO and revealed that P-Lip-DFO contains key platelet components, such as... Figure 1 As shown in Figure I, its protein composition highly overlaps with and is significantly enriched in platelet characteristic functional proteins, as shown in the heatmap of protein abundance in platelet samples and P-Lip-DFO samples. This high similarity highlights the biological significance of P-Lip-DFO.

[0048] Bubble diagram showing significant enrichment of the KEGG pathway, as shown in the figure. Figure 1 As shown in J, P-Lip-DFO participates in focal adhesion and platelet activation processes, indicating its ability to target bleeding sites and play an important role in hemostasis.

[0049] The encapsulation efficiency and drug loading were determined by high performance liquid chromatography (HPLC). As shown in Table 1, with a DFO content of 1 mg, both the encapsulation efficiency and drug loading showed a decreasing trend as the hydration volume increased. Therefore, a fixed hydration volume of 1 mL was used in the experiment.

[0050] Table 1. Encapsulation efficiency and drug loading rate of DFO liposomes at different hydration volumes

[0051] Furthermore, the encapsulation efficiency and drug loading rate of DFO-loaded liposomes at different DFO doses are shown in Table 2, and the encapsulation efficiency and drug loading rate of P-Lip-DFO at different DFO doses are shown in Table 3. When the DFO dosage is 2 mg (lipid:DFO = 5:1), the encapsulation efficiency (58.89%) and drug loading rate (9.66%) reach a better balance, so this ratio of P-Lip-DFO was selected for subsequent in vitro and in vivo experiments.

[0052] Table 2. Encapsulation efficiency and drug loading rate of DFO liposomes at different DFO doses

[0053] Table 3. Encapsulation efficiency and drug loading of P-Lip-DFO at different DFO doses

[0054] Comparative Example 1 Free DFO is prepared by dissolving commercially available deferoxamine methanesulfonate powder in physiological saline and preparing an injection solution of the required concentration.

[0055] Comparative Example 2 DFO-loaded liposomes were obtained according to steps S1-S2 in Example 1 and were used as ordinary liposomes (Lip-DFO); the difference from Example 1 is that the platelet membrane doping step was not performed.

[0056] Experimental Example 1: ROS Responsive Release Experiment One of Lip-DFO and P-Lip-DFO was placed in a release medium containing 100 μM H2O2 (simulating a high ROS environment) or pure water (normal environment), respectively, and kept at a constant temperature of 37℃ with shaking. Samples were taken at time points of 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h. The supernatant was collected by centrifugation, and the DFO concentration in the supernatant was determined by HPLC. The cumulative release rate was calculated. The results are as follows: Figure 1 As shown in Figure F, the selenium-selenium bonds in the liposome structure break under hydrogen peroxide stimulation, triggering a time-dependent morphological transformation of the nanoparticles within 24 hours. Under H2O2 stimulation, both types of liposomes achieved a rapid release of nearly 50% within 0.5 hours, far faster than the release rate in pure water, demonstrating their excellent ROS response characteristics. This process lays the foundation for scavenging reactive oxygen species under subsequent physiological conditions. The corresponding drug release curves of Lip-DFO and P-Lip-DFO both exhibit time-dependent and H2O2-dependent characteristics, as shown in Figure F. Figure 1As shown in J, they all exhibit release patterns that change over time. When stimulated by reactive oxygen species (ROS, H2O2), liposomes show significantly accelerated drug release characteristics, laying a theoretical foundation for the dual function of clearing oxidative stress and achieving controlled drug release under pathophysiological conditions after ICH.

[0057] Experiment Example 2, Iron Death Experiment The iron scavenging and free radical scavenging abilities of the nanoparticles were determined by ultraviolet-visible spectroscopy (UV-VIS) and 2,2'-azacyclopropane bis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS). The UV absorption spectroscopy results showed that... Figure 2 As shown in Figure A, DFO, Lip-DFO, and P-Lip-DFO all exhibited significant absorption peaks at 430 nm, confirming their iron chelation capabilities. To comprehensively evaluate the free radical scavenging capabilities of each sample, the ABTS method was used for further detection, and the results are shown below. Figure 2 As shown in B, Lip-DFO and P-Lip-DFO exhibit superior free radical scavenging efficiency due to their lipid-intercalated double selenium bond structure; while the limited scavenging ability of DFO is mainly attributed to its isohydroxamic acid group—a type of group with hydrogen / electron donor properties.

[0058] Hippocampal neurons from HT22 mice were used as experimental material. HT22 cells were purchased from Shanghai Anwei Biotechnology Co., Ltd. and cultured in a humid environment containing 5% carbon dioxide in high-glucose DMEM medium at 37°C. The medium was supplemented with 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. Ferrocytes of HT22 cells were induced by Erastin (500 nM), and DFO, Lip-DFO or P-Lip-DFO were added at 100 μM equivalent concentrations, respectively, for intervention. After 16 h, Calcein-AM / PI staining, FerroOrange staining or DCFH-DA fluorescent probe and biochemical detection were performed.

[0059] Biochemical test results such as Figure 2 As shown in C and D, DFO, Lip-DFO, and P-Lip-DFO significantly reduced Erastin-induced malondialdehyde (MDA) and glutathione peroxidase (GSH) oxidative stress indicators. P-Lip-DFO most significantly reduced ROS levels and MDA content, and increased GSH levels.

[0060] Calcein-AM / PI staining, as shown in Figure 2 The E in the figure shows the data analysis results as follows: Figure 2As shown in F, Erastin treatment significantly increased cell death, while DFO, Lip-DFO, and P-Lip-DFO significantly reduced the proportion of dead cells in the Erastin group from 47% to 12-16%, with the P-Lip-DFO group having the lowest proportion of dead cells.

[0061] The detection results of the DCFH-DA fluorescent probe are as follows: Figure 2 As shown in G, the data analysis results are as follows: Figure 2 As shown in H, DFO, Lip-DFO, and P-Lip-DFO significantly reduced intracellular reactive oxygen species levels.

[0062] FerroOrange staining results are as follows: Figure 2 As shown in Figure I, the data analysis results are as follows: Figure 2 As shown in J, P-Lip-DFO most effectively reduces Erastin-induced intracellular iron deposition.

[0063] Key proteins associated with oxidative stress and ferroptosis, including transferrin receptor 1 (TFR1), the lipid peroxidation marker 4-hydroxynonenal (4-HNE), and the antioxidant proteins GPX4 and peroxidase 3 (PRDX3), were detected using Western blotting. The results are as follows: Figure 2 As shown in K, the data analysis results are as follows: Figure 2 K~ Figure 2 As shown in the figure, these results confirm that P-Lip-DFO has a significant effect on alleviating oxidative stress damage and inhibiting ferroptosis at the cellular level.

[0064] Experiment Example 3, Hemostasis Function Experiment The in vitro coagulation efficacy of P-Lip-DFO was verified through in vitro clot formation assays, such as... Figure 3 As shown in A, the data analysis results are as follows: Figure 3 As shown in B, P-Lip-DFO retains the physiological function of promoting blood clotting.

[0065] Adopting such Figure 3 The mouse tail truncation model shown in Figure C was used to evaluate the in vivo hemostatic effect of P-Lip-DFO, and compared with physiological saline. Bleeding time was recorded as follows. Figure 3 As shown in D, the bleeding area is as follows: Figure 3 As shown in E, and the weight of bleeding as shown in E. Figure 3 As shown in F; Evaluation of in vivo hemostatic effect: Bleeding time, bleeding area and bleeding weight were significantly shortened in the P-Lip-DFO administration group. These results confirm the strong hemostatic effect of P-Lip-DFO, indicating that its hemostatic effect mainly comes from platelet membrane components, especially the role of key membrane proteins.

[0066] A mouse model of ICH was established (previous studies have shown that lesion volume reaches its maximum 6 hours after ICH onset). Patients were randomly divided into two groups: ICH + Lip-DFO group and ICH + P-Lip-DFO group. MRI images were acquired 2 hours after model establishment, and tail vein administration began. Lesion size was observed at 6 hours. Results showed that the lesion volume in all experimental groups 2 hours after ICH onset was as follows: Figure 3 As shown in G, the data analysis results are as follows: Figure 3 As shown in I, there is no difference; as Figure 3 H~ Figure 3 As shown in K, P-Lip-DFO treatment significantly inhibited the further expansion of the lesion 6 hours after the onset of ICH, demonstrating that P-Lip-DFO has strong hemostatic activity.

[0067] Example 4: In vivo pharmacodynamic evaluation Given that ICH patients are usually admitted to the hospital within hours of symptom onset, and initial treatment is often clinically delayed, a mouse ICH model was established to better simulate real clinical scenarios. Patients were randomly divided into: a normal control group, an ICH+Vehicle group, an ICH+DFO group, an ICH+Lip-DFO group, and an ICH+P-Lip-DFO group. Tail vein administration began 6 hours after model establishment, with a dosage of 30 mg / kg (based on DFO equivalent) once daily for 7 consecutive days. The results are as follows.

[0068] Hematoma volume: A T2-weighted MRI scan was performed on day 7, and the results are as follows. Figure 3 As shown in J in the figure, the data analysis results are as follows: Figure 3 As shown in K, the hematoma volume in the P-Lip-DFO treatment group was the smallest (3.91 ± 0.52 mm³), which was 62.6% smaller than that in the Vehicle group (7.12 ± 1.21 mm³), and the effect was significantly better than that in the DFO group and the Lip-DFO group.

[0069] ICH-induced neuronal damage impairs sensory abilities, motor function, and other basic neural functions in animal models. Given the crucial role of behavioral phenotypic analysis in assessing treatment efficacy, all experimental animals underwent baseline behavioral screening before the experiment to exclude abnormal individuals, followed by behavioral tests, such as... Figure 4 As shown in A, behavioral assessments were conducted on days 1, 3, 5, and 7 after the onset of ICH to observe functional recovery. The assessment process is as follows: Figure 4 As shown in B, it includes neurological deficit scores, tensile tests, cornering tests, grid tests, and gait analysis.

[0070] The neurological deficit score includes the following tests: Mice were placed in a behavioral laboratory for 1 hour to acclimatize under standard indoor conditions, and then subjected to the tests listed in Table 4. The tests consist of 6 items, each with 5 levels, ranging from a maximum of 4 points to a minimum of 0 points. Mice with a total score below 4 points or above 20 points were disqualified.

[0071] Table 4 Neurological deficit scores

[0072] The tensile strength test included: mice were placed in a behavioral laboratory for 1 hour to acclimatize under standard indoor conditions before the experiment. The forelimb strength of the mice was assessed using the Xinruan grip strength testing system: the mouse's forelimb was placed on the grip strength testing device, grasped, and then stretched backward. The average of three grip strength tests was calculated, and the grip strength value was recorded as a grip strength / body weight ratio.

[0073] The corner-turning experiment involved placing mice in a behavioral laboratory for 1 hour to acclimatize them to a standard indoor environment before the experiment. The mice were positioned between two boards forming a 30° angle. While maintaining the angle, the boards were gradually moved towards the mice until they were near the corner, upright, and rotated 180° to face the opening. The direction of the mouse's turn (left or right) was recorded in each trial. Each mouse underwent 10 trials, and the proportion of left turns was recorded.

[0074] The grid test included: Mice were placed in a behavioral laboratory for 1 hour to acclimatize under standard indoor conditions before the experiment. Mice were placed on a 35 cm × 30 cm × 40 cm wire mesh and made to walk for 3 minutes. A camera was used to record the video from below the mesh. The total number of steps and the number of slips on the contralateral hind limb within 3 minutes were counted. The percentage of slips was calculated as (number of slips / total steps) × 100%.

[0075] Gait analysis included: Mice were placed in a behavioral laboratory for 1 hour to acclimatize under standard indoor conditions before the experiment. Gait analysis was used to assess the mice's gait. Mice were placed on a Cyons gait testing board, and cameras were used to record images of the mice walking continuously. Three video clips were recorded for subsequent data analysis.

[0076] The results are as follows Figure 4 C~ Figure 4As shown in Figure I, consistent with ICH-induced neurological damage, the solvent control group mice exhibited significant motor dysfunction, while the P-Lip-DFO treatment group showed significant functional recovery, with its neuromotor performance significantly improved compared to all other groups. Specifically, mice treated with P-Lip-DFO showed reduced neurological function scores, increased forelimb strength, and by day 7, motor coordination and balance had essentially recovered to near-normal levels. These findings consistently demonstrate that the P-Lip-DFO treatment group showed the most significant improvement in motor, balance, and coordination abilities, exhibiting a powerful neuroprotective effect and highlighting its therapeutic potential in alleviating ICH-induced functional impairment.

[0077] Experimental Example 5: Effects on oxidative stress and ferroptosis after ICH Intracerebral hemorrhage (ICH) is caused by ruptured blood vessels leading to intracranial hemorrhage. Following ICH, damaged red blood cells release free iron ions, which gradually accumulate around the hematoma over several months. Through the Fenton reaction, excess free iron ions generate hydroxyl radicals, subsequently inducing apoptosis and necrosis, gray matter damage, and blood-brain barrier disruption. Furthermore, iron ions entering neurons drive lipid peroxidation—the core mechanism of ferroptosis, a programmed cell death process regulated by GPX4. Based on the observed improvement in neuromotor function and behavioral recovery, the molecular mechanism by which P-Lip-DFO alleviates ICH damage was further investigated. Brain tissue from mice in Experiment 4 was analyzed. Figure 5 As shown in A, the results are as follows.

[0078] Oxidative stress and ferroptosis biomarkers: Key oxidative stress-related biomarkers such as DHE content, MDA, GSH, and H2O2 in the brain tissue of ICH mice were detected. Figure 5 B in Figure 5 As shown in Figure E, ROS production, MDA, and H2O2 levels were significantly increased in ICH model mice, while GSH content was decreased. The P-Lip-DFO group most effectively reduced iron content, MDA, and H2O2 levels in brain tissue and increased GSH levels. Meanwhile, the tissue iron content detection results were as follows... Figure 5 As shown in F, the iron content in the tissues of ICH mice was approximately twice that of the naive group, while the iron content in the P-Lip-DFO treatment group was significantly reduced; Prussian blue staining results are as follows. Figure 5 As shown in G, the data analysis results are as follows: Figure 5 As shown in H, iron deposition in ICH mouse tissues was significantly reduced after P-Lip-DFO treatment, demonstrating a strong in vivo iron chelation capacity.

[0079] Mitochondrial morphology: TEM observation showed the extent of mitochondrial damage, as follows: Figure 5As shown in Figure I (red arrows indicate atrophied mitochondria, while yellow arrows indicate mitochondria treated with P-Lip-DFO), the mitochondria in the solvent control group showed significant structural damage, manifested as increased outer membrane electron density, marked shrinkage, and severe destruction of cristae structure; while the mitochondrial morphology in the P-Lip-DFO group was significantly improved, showing remarkable morphological preservation characteristics: data analysis results are as follows. Figure 5 J~ Figure 5 As shown by L in the figure, the degree of shrinkage is reduced, the average size is increased, and the aspect ratio is improved.

[0080] Western blot results as follows Figure 5 M~ Figure 5 As shown by R in the figure, the P-Lip-DFO group most significantly downregulated TFR1 and 4-HNE protein expression and upregulated GPX4 and PRDX3 protein expression. The increase in GPX4 expression can be partly attributed to selenium supplementation provided by the liposome formulation, which is a key cofactor for GPX4 biosynthesis and catalytic activity. The increased GPX4 level further enhances cellular antioxidant capacity, thereby strengthening the detoxification of lipid peroxides and conferring resistance to ferroptosis stress. These findings indicate that P-Lip-DFO alleviates ferroptosis stress by inhibiting lipid peroxidation and restoring antioxidant defense mechanisms, thereby inhibiting ferroptosis cell death.

[0081] In summary, the data above indicate that P-Lip-DFO effectively inhibits collagenase-induced ferroptosis in ICH by reducing oxidative stress, regulating iron metabolism, and restoring redox balance. This selenium-enriched liposome formulation not only has iron chelating function but also enhances GPX4 activity, thereby strengthening the antioxidant defense mechanism. Compared with traditional DFO treatment, P-Lip-DFO exhibits superior anti-ferroptosis effects.

[0082] Compared with existing ROS-responsive platelet liposome nanoparticles used for reperfusion injury, this invention has the following key advantages: ① Disease models and pathological treatments are fundamentally different. This invention is specifically designed for the microenvironment of ICH hematoma.

[0083] The core pathology of reperfusion injury is the oxidative burst and inflammatory cascade response following the restoration of blood flow. Treatment focuses on alleviating the widespread oxidative stress caused by reperfusion, and this invention... Figure 6As shown in B, the unique pathological chain of hematoma after ICH is specifically addressed: erythrocyte lysis releases large amounts of hemoglobin and iron ions → continuous local high concentrations of ROS are generated through the Fenton reaction → lipid peroxidation and GPX4 inactivation are triggered → ultimately leading to iron-dependent cell death (ferroptosis). The structural localization and functional synergistic enhancement of selenium not only introduces selenium as a ROS-responsive linker, but also adds a relatively high amount of selenium, clearly functioning as a key cofactor for GPX4 activity. The drug release logic, targeting strategy, and synergistic mechanism are all optimized around the specific pathological process of ICH, thus exhibiting more significant hematoma control and neuroprotective effects in the ICH model, solving the problem of the prior art not targeting the unique pathological mechanism after ICH.

[0084] ②The functional role of platelet membranes has been upgraded from carrier to multifunctional therapeutic component.

[0085] In this invention, the platelet membrane is elevated to a core therapeutic component: such as Figure 6 As shown in Figure B, the natural proteins retained on its surface (such as CD41, CD61, and GPVI) can specifically recognize collagen and von Willebrand factor exposed at the site of vascular injury after ICH, achieving active and precise targeting of the bleeding site. The targeting system matches the bleeding microenvironment, and this technology fully utilizes the natural targeting, aggregation, and hemostatic potential of the platelet membrane at the bleeding site. At the same time, the natural hemostatic function mediated by membrane proteins can directly participate in inhibiting hematoma expansion. This design makes the targeting system a perfect match for the urgent clinical need for hemostasis after ICH, realizing the multiple functions of hemostasis and targeting expected in the background technology.

[0086] ③ The amino group (-NH2) in DSPE-Se-Se-PEG-NH2 endows it with unique adaptability to the hematoma microenvironment. Compared to the inert terminal of DSPE-Se-Se-PEG2000, the amino group at the end of DSPE-Se-Se-PEG-NH2 used in this invention can be protonated (-NH3) in the acidic microenvironment specific to hematomas after ICH. + These locally positively charged microregions enhance the interaction between nanoparticles and negatively charged damaged tissue components at the hematoma site (such as exposed extracellular matrix and debris), thereby promoting the retention and accumulation of nanoparticles in the hemorrhage core. This secondary anchoring effect based on the microenvironment pH is not present in PEG2000, enabling drug delivery to better fit the dynamic pathological environment of the hematoma.

[0087] ④ The preparation process is simplified and the functional integration is high: This invention does not require the modification of exogenous targeting peptides. It can achieve multi-functional integration such as targeting, long circulation, and ROS-responsive drug release simply by fusing platelet membranes with selenium liposomes. The preparation process is simpler and closer to the requirements of clinical translation. At the same time, it avoids the immunogenicity risks that may be brought by exogenous peptides, and has higher clinical translation potential.

[0088] The above examples and experimental cases fully demonstrate that the P-Lip-DFO nanoparticles prepared by this invention have excellent targeting, long-cycle characteristics, ROS-responsive drug release ability and synergistic therapeutic effects. They can effectively reduce oxidative stress and ferroptosis after ICH, promote the recovery of nerve function, and have extremely high biosafety, showing good prospects for clinical translation.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A platelet-bionic diselenyl liposome-loaded with deferoxamine, characterized in that, The deferoxamine is loaded in a hybrid membrane formed by the fusion of lipids and platelet membranes; the lipids include lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 in a mass ratio of (7~9):(1~2):

1.

2. The platelet-bionic diselenoliposome loaded with deferoxamine as described in claim 1, characterized in that, The mass ratio of the lipid film to deferoxamine is 10:(1~10).

3. The platelet-bionic diselenoliposome-loaded deferoxamine as described in claim 1, characterized in that, The mass ratio of lipids to platelet membrane proteins is (2~3):

1.

4. The platelet-bionic diselenoliposome loaded with deferoxamine as described in claim 1, characterized in that, The platelet-inspired diselenium liposomes loaded with deferroamine have a hydrated particle size of 100-200 nm.

5. A method for preparing platelet biomimetic diselenyl liposomes loaded with deferoxamine as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Dissolve lecithin, cholesterol and DSPE-Se-Se-PEG-NH2 in a mixed solvent in a predetermined mass ratio, and evaporate under reduced pressure to obtain a lipid film; S2. The lipid film is hydrated with deferoxamine solution, ultrasonically vibrated, and then extruded to encapsulate deferoxamine in the lipid film to obtain DFO-loaded liposomes. S3. The suspension of DFO-loaded liposomes was mixed with the platelet membrane suspension, and after ultrasonic oscillation, the platelet membrane was doped with DFO-loaded liposomes by extrusion to obtain platelet biomimetic diselenyl liposomes loaded with deferroamine.

6. The method for preparing platelet-bionic diselenoliposomes loaded with deferoxamine as described in claim 5, characterized in that, In S1, the mixed solvent comprises methanol and chloroform in a volume ratio of 1:(0.5~1.5); Alternatively, in S1, the rotary evaporator is heated in a 45°C water bath during vacuum evaporation.

7. The method for preparing platelet-bionic diselenoliposomes loaded with deferoxamine as described in claim 5, characterized in that, In S2, the deferoxamine solution is a solution of deferoxamine in phosphate buffer with a concentration of 1.5~2.5 mg / mL; Alternatively, in S2, the extrusion method includes: sequentially extruding through a 400 nm membrane and a 200 nm membrane of a liposome extruder a set number of times; Alternatively, in S2, after extrusion, the suspension containing DFO-loaded liposomes is centrifuged at 10,000–15,000 rpm for 10–20 min, the supernatant is discarded, and the precipitate is resuspended with PBS to obtain purified DFO-loaded liposomes.

8. The method for preparing platelet-bionic diselenoliposomes loaded with deferoxamine as described in claim 5, characterized in that, In S3, the concentration of the suspension carrying DFO liposomes was 2 mg / mL.

9. The method for preparing platelet-bionic diselenoliposomes loaded with deferoxamine as described in claim 5, characterized in that, In S3, the extrusion method includes: extruding a 200 nm membrane through a liposome extruder a set number of times.

10. The application of platelet biomimetic diselenyl liposomes loaded with deferoxamine as described in any one of claims 1-4, characterized in that, The application includes at least one of the following (a1) to (a3); (a1) Preparation of products that inhibit oxidative stress and ferroptosis after ICH; (a2) Prepare products that reduce hematoma after ICH; (a3) Prepare products that improve motor function after ICH.

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