Use of targeted liposomes as active ingredients in the preparation of drugs for treating ischemic brain injury

Abstract

The present invention belongs to the field of biomedicine technology, and in particular to the use of targeted liposomes as active ingredients in the preparation of medicines for treating ischemic brain injury. The targeted liposomes for treating ischemic stroke of the present invention are prepared by fusion of liposomes and platelet-neutrophil aggregate membranes, achieving efficient and specific targeting of ischemic brain areas, while effectively reducing brain tissue infarction and damage to new areas, and alleviating brain inflammation through multi-pathway regulation. It can be used as an active ingredient for the preparation of medicines for treating ischemic brain injury. When the targeted liposomes are further loaded with rutin drugs, a synergistic therapeutic effect is achieved. The liposome medicine of the present invention can be used as an active ingredient and has good application prospects in the treatment of ischemic brain injury diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the use of targeted liposomes as an active ingredient in the preparation of a medicine for treating ischemic brain injury. Background Art

[0002] Ischemic stroke, also known as ischemic stroke, is a cerebrovascular disease caused by hypoxia and ischemia in cerebral vessels. It is reportedly the second leading cause of death worldwide, resulting in 5.9 million deaths and 102 million disabilities. Currently, the golden rule of clinical treatment for ischemic stroke is to restore blood reperfusion to the ischemic penumbra, ensuring oxygen replenishment of brain tissue and saving damaged neurons. However, after reperfusion, excessive reactive oxygen species (ROS) can cause oxidative damage to DNA, proteins, and lipids, leading to neuronal cell death. ROS can also act as pro-inflammatory signaling molecules, participating in microglial activation. Furthermore, overactivated microglia secrete pro-inflammatory cytokines and chemokines, attracting astrocytes and peripheral leukocytes, exacerbating neuroinflammation and oxidative stress. Ultimately, a vicious cycle of mutually reinforcing and self-amplifying oxidative stress and inflammation forms, creating a pathological microenvironment that is unfavorable for neuronal survival, resulting in ischemia / reperfusion injury (I / R). From this point of view, the treatment of ischemic stroke requires not only thrombolysis, but also ischemia / reperfusion injury after reperfusion is also an issue that needs urgent attention.

[0003] Rutin (RT), a natural flavonoid glycoside, exhibits anti-inflammatory, antioxidant, and neuroprotective properties, making it an effective neuroprotectant for the treatment of cardiovascular and nervous system injuries. However, rutin's low water solubility and difficulty crossing the blood-brain barrier (BBB) ​​significantly limit its clinical application. Therefore, there is an urgent need to develop more effective targeted delivery methods to enhance its ability to cross the BBB and fully unleash its neuroprotective effects.

[0004] Numerous drug carriers can cross the blood-brain barrier, including liposomes, polymer nanoparticles, carbon nanomaterials, cell membrane-derived nanocarriers, transferrin receptor-mediated carriers, and cell-penetrating peptides. Among them, cell membrane-derived nanocarriers have been reported to target the site of cerebral ischemia and stroke, including red blood cell membranes, platelet membranes, white blood cell membranes, and tumor cell membranes. Cell membrane-derived nanocarriers have demonstrated excellent targeting of cerebral ischemia, providing new strategies and approaches for the treatment of ischemic brain injury.

[0005] Therefore, developing an effective drug for treating ischemic stroke remains a problem in this field. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of targeted liposomes as an active ingredient in the preparation of a drug for treating ischemic brain injury.

[0007] The present invention provides use of targeted liposomes as an active ingredient in preparing a drug for treating ischemic brain injury. The targeted liposomes include liposomes and platelet-neutrophil aggregate membranes fused with the liposomes; the mass ratio of the platelet-neutrophil aggregate membrane to the liposomes is 1:1-4:1; and the quantitative ratio of platelets to neutrophil raw materials in the platelet-neutrophil aggregate membrane is 0.8-1.2:0.8-1.2.

[0008] Preferably, the mass ratio of platelet-neutrophil aggregate membrane to liposome is 2:1;

[0009] The ratio of the number of platelets to the number of neutrophil raw materials in the platelet-neutrophil aggregate membrane is 1:1.

[0010] Preferably, the liposome is loaded with a drug, and the drug is selected from at least one of rutin, baicalin, and mangiferin.

[0011] Preferably, the mass fraction of the drug in the liposome is 2.1-3.2%.

[0012] Preferably, the targeted liposome is prepared by fusing raw materials including platelet-neutrophil aggregate membrane and liposome through an extrusion method.

[0013] Preferably, the conditions of the extrusion method include: the pore size of the filter membrane is 200-400 nm, and the number of extrusions is 20-50 times;

[0014] And / or, the platelet-neutrophil aggregate membrane is prepared by repeatedly freezing and thawing platelet-neutrophil aggregates;

[0015] And / or, the liposome is prepared from raw materials including phospholipids and nonionic surfactants through a thin film hydration method.

[0016] Preferably, the repeated freezing and thawing conditions include: freezing at -75 to -190°C for 1-2 hours, and thawing in a water bath at 30 to 38°C for 10 to 20 minutes;

[0017] and / or, after repeated freezing and thawing, centrifuging at 10000±100 rpm, 0-10°C for 12-18 minutes;

[0018] And / or, the platelet-neutrophil aggregates are prepared by inducing platelets and neutrophils with an inducer, incubating, and centrifuging.

[0019] Preferably, the inducer is selected from at least one of phorbol esters, platelet activating factor, and tissue factor, and the incubation conditions include: incubation temperature of 20-40° C., incubation time of 15 minutes, centrifugation speed of 80×g-120×g, and centrifugation time of 2-4 minutes.

[0020] Preferably, the phospholipid is selected from at least one of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine;

[0021] And / or, the nonionic surfactant is selected from at least one of polyethylene glycol (15)-hydroxystearate, polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine, and polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine;

[0022] And / or, the conditions of the thin film hydration method include: the solvent is selected from at least one of methanol, chloroform, dichloromethane, and ether, and the aqueous phase is selected from at least one of water, phosphate buffer, and physiological saline;

[0023] And / or, after the thin film hydration method, ultrasonic cell disruptor is used for 8-12 minutes.

[0024] Preferably, the ischemic brain injury is ischemic stroke.

[0025] The present invention provides a targeted liposome for treating ischemic stroke, and a preparation method and use thereof. The present invention provides a targeted liposome for treating ischemic stroke by screening the types of raw materials. The liposome is prepared by membrane fusion of liposomes and platelet-neutrophil aggregates, achieving efficient and specific targeting of ischemic brain areas, while effectively reducing brain tissue infarction and damage to new areas, and alleviating brain inflammation through multi-pathway regulation. The liposome can be used as an active ingredient to prepare a drug for treating ischemic brain injury. When the targeted liposome is further loaded with rutin, a synergistic therapeutic effect is achieved. The liposome drug of the present invention can be used as an active ingredient and / or a drug carrier, and has good application prospects in the treatment of ischemic brain injury diseases.

[0026] The method extracts platelets from rat whole blood and neutrophils from bone marrow. Phorbol esters are then used to induce platelet and neutrophil aggregation to form platelet-neutrophil aggregates (PNMs). Platelet-neutrophil aggregate membranes (PNMs) are then extracted using a freeze-thaw cycle. The PNMs are then encapsulated onto the prepared rutin liposomes using an extrusion method.

[0027] PNM inherits the protein structure of the original cell membrane, and in particular, the aggregate membrane can retain its targeting properties to the site of inflammation, making it a very promising drug delivery carrier strategy. Based on the mechanism and clinical treatment methods of ischemic stroke, the present invention proposes using platelet-neutrophil aggregate membranes as a material to encapsulate rutin as a new biomimetic drug delivery system for targeted treatment of ischemic stroke, thereby improving the efficiency of crossing the BBB. At the same time, PNM can act as a decoy and synergize with the drug to reduce brain inflammation and infarct size. This delivery system reduces drug exposure to non-target tissues, thereby reducing drug side effects, has good biocompatibility, is compatible with the human body environment, reduces immune response and toxicity, and improves drug safety and tolerability.

[0028] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0029] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the result of dynamic light scattering experiment, where Figure 1 A is the particle size result diagram, Figure 1 B is the Zeta charge result diagram;

[0031] Figure 2 Transmission electron microscopy image of RT LIP@PNM, scale bar = 100 nm;

[0032] Figure 3 This is the result of Western blot (WB) experiment to determine the expression of PNA-specific proteins in different materials;

[0033] Figure 4 This is the fluorescence confocal result of Experimental Example 2, where red fluorescence represents the fluorescence of liposome material, and blue fluorescence represents the fluorescence of BMVECs nucleus. Scale bar = 50 μm;

[0034] Figure 5 Representative in vivo imaging system (IVIS) images of tMCAO / R model rats at different time points after injection of different drugs;

[0035] Figure 6Figure 2 shows the fluorescence distribution of different liposome materials and astrocytes in ischemic brain tissue. Blue represents DAPI-labeled cell nuclei, green represents GFAP-labeled astrocytes, and red represents DiD-labeled nanoliposome materials. Scale bar = 20 µm.

[0036] Figure 7 The figure shows the results of infarct area measurement in tMCAO / R rats, where: Figure 7 A is a picture of TTC-stained brain sections in different groups. Normal brain tissue is red, and infarcted tissue is white. Figure 7 B is the quantitative statistical results of TTC-stained brain sections in different groups, n = 3, all data are expressed as Mean ± SD, *** indicates P < 0.001;

[0037] Figure 8 The figure shows the anti-inflammatory effects of different drugs on the brain tissue of tMCAO / R rats. Figure 8 A is the result of MPO enzyme activity determination. Figure 8 B is the result of the determination of TNF-α content. Figure 8 C is the result of IL-1β content determination. Figure 8 D is the result of IL-6 content determination. All data are expressed as Mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0038] Figure 9 Figures show the results of H&E staining, Nissl staining, and TUNEL staining of brain tissue. The first and second rows show the results of H&E staining, the third and fourth rows show the results of Nissl staining, and the fifth and sixth rows show the results of TUNEL staining. The scale bars in the first, third, and fifth rows are 1000 µm, and the scale bars in the second, fourth, and sixth rows are 50 µm. DETAILED DESCRIPTION

[0039] In the following examples and experimental examples, reagents and materials not otherwise specified are commercially available.

[0040] Example 1 A targeted liposome for treating ischemic stroke and its preparation method

[0041] The targeted liposomes for treating ischemic stroke in this embodiment are made of rutin, liposomes, and platelet-neutrophil aggregate membranes (PNMs), and the preparation method is as follows:

[0042] 1. Neutrophil extraction

[0043] The femurs and tibias of rats were completely removed and soaked in 75% ethanol for 30 minutes. The metaphyses of the bones were then cut off using rongeurs. Pre-chilled PBS was aspirated using a syringe, and the bone marrow cavity was repeatedly flushed to collect all bone marrow tissue. The resulting bone marrow suspension was filtered through a 70 μm pore size cell strainer and centrifuged at 200 × g for 3 minutes. The supernatant was discarded, and 3 mL of normal saline (0.9% NaCl) was added to resuspend the cell pellet. Cells were then separated by density gradient centrifugation: the cell suspension was slowly layered onto a pre-prepared Percoll separation buffer and centrifuged at 800 × g for 30 minutes. The cell suspensions at the 78% and 65% fractions were collected, and an equal volume of normal saline was added. The cells were centrifuged at 1500 rpm for 10 minutes to isolate neutrophils. Finally, the cells were washed three times with pre-chilled PBS, and the cell pellet was resuspended in an appropriate amount of PBS for later use.

[0044] 2. Platelet extraction

[0045] Whole blood was collected from rats via femoral artery puncture and anticoagulated with sodium citrate at room temperature for extraction. The blood was first centrifuged at 100 × g for 10 minutes to separate the upper layer of platelet-rich plasma (PRP) and the lower layer of red blood cells and white blood cells. After aspiration of the PRP, the remaining blood sample was centrifuged a second time (125 × g for 10 minutes) to collect the upper layer of PRP. The two PRP fractions were combined and transferred to an EP tube and centrifuged at 1200 × g for 5 minutes. The supernatant was discarded to obtain the platelet pellet. The cells were washed three times with pre-chilled PBS, and the cell pellet was resuspended in an appropriate amount of PBS for later use.

[0046] 3. Induction of Platelet-Neutrophil Aggregates (PNA) and Extraction of Their Membranes

[0047] Platelets and neutrophils were added to a culture dish at a 1:1 ratio. Phorbol myristate methyl paraformaldehyde (PMA) was added to a final concentration of 100 nM and incubated on a shaker at room temperature for 15 minutes. The sample was centrifuged at 50 × g for 3 minutes to obtain the platelet-neutrophil aggregates (PNA) in the lower layer. The PNA was then freeze-thawed three times (freezing at -80°C for 1 hour and thawing in a 37°C water bath for 10 minutes). The pellet, which represents the platelet-neutrophil aggregate membrane (PNM), was then centrifuged at 10,000 rpm for 15 minutes.

[0048] 4. Preparation of RT LIP@PNM

[0049] 2.5 mg of rutin (RT) was prepared by thin film hydration method using 75 mg of egg yolk lecithin (E80) and 15 mg of polyethylene glycol (15)-hydroxystearate (HS 15) as membrane materials to prepare liposomes and entrap rutin. The specific process of thin film hydration method is as follows: rutin, egg yolk lecithin (E80) and 15 mg of polyethylene glycol (15)-hydroxystearate (HS 15) were added to 20 mL of methanol, and the organic solvent was evaporated under reduced pressure to form a uniform thin film on the wall of the bottle. The film was then hydrated with 4 mL of pure water.

[0050] Ultrasonic cell disruptors were used for 10 minutes to obtain the base liposomes RT LIP. PNM was then modified onto the liposome surface using an extrusion method to produce RT LIP@PNM. The extrusion method involved mixing RT LIP and PNM in a 2:1 mass ratio, first passing the mixture through a 400 nm filter 20 times, and then through a 200 nm filter 30 times.

[0051] The targeted liposomes of this embodiment are used to treat ischemic brain injury, and can achieve a synergistic therapeutic effect of rutin and PNA.

[0052] Example 2 A targeted liposome for treating ischemic stroke and its preparation method

[0053] Targeted liposomes were prepared according to the method of Example 1, except that rutin was not added in step 4. Instead, 75 mg of egg yolk phosphatidylcholine (E80) and 15 mg of polyethylene glycol (15)-hydroxystearate (HS 15) were used as membrane materials. Liposomes (LIP) were prepared by thin film hydration. PNM was then modified onto LIP by extrusion to obtain LIP@PNM. The LIP@PNM prepared in this example was used as an active ingredient in the treatment of ischemic brain injury.

[0054] Example 3 A targeted liposome for treating ischemic stroke and its preparation method

[0055] Targeted liposomes were prepared according to the method of Example 1, except that in step 4, RT LIP and PNM were mixed at a mass ratio of 1:1.

[0056] Example 4 A targeted liposome for treating ischemic stroke and its preparation method

[0057] Targeted liposomes were prepared according to the method of Example 1, except that in step 4, RT LIP and PNM were mixed at a mass ratio of 4:1.

[0058] The following experiments further illustrate the technical solutions of the present invention. The samples PNA, PNM, RT LIP, and RT LIP@PNM used in the following experimental examples were all prepared according to the method of Example 1, while LIP and LIP@PNM were prepared according to the method of Example 2. In the following cell and animal experiments, to facilitate visualization, LIP and LIP@PNM were loaded with the same dose of DiD (a red fluorescent probe, 1,1-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) dye to produce DiD LIP and DiD LIP@PNM.

[0059] Experimental Example 1 Material Characterization

[0060] 1. Experimental Methods

[0061] 1. Characterization of morphology, particle size and surface charge

[0062] The particle size and surface charge of RT LIP and RT LIP@PNM were characterized by dynamic light scattering (DLS).

[0063] The morphology of the material was characterized by transmission electron microscopy.

[0064] 2. Characterization of cell membrane proteins in materials

[0065] Western blot (WB) experiments were used to measure Ly6G, CD41, P-selectin, PSGL-1, Mac-1, TNF-α, and CD47 receptors in PNA, PNM, LIP@PNM, and RT LIP@PNM materials, respectively. Ly6G is a specific marker for neutrophils, and CD41 is a specific marker for platelets. WB experiments characterized the anchoring and functional expression of key surface antigens on the liposome surface at the molecular level.

[0066] 2. Experimental Results

[0067] 1. Results of morphology, particle size and surface charge

[0068] Particle size and potential results are as follows Figure 1 As shown in the figure, compared with RT LIP, the particle size of RT LIP@PNM is larger and the negative charge is significantly increased, indicating that the cell membrane and liposomes are successfully fused.

[0069] Transmission electron microscopy results are as follows Figure 2 As shown in the figure, RT LIP@PNM are spherical nanoparticles with uniform particle size distribution and an average particle size of 80.08 nm.

[0070] 2. Characterization results of cell membrane proteins in the material

[0071] The results are as follows Figure 3 As shown. The PNA, PNM, LIP@PNM, and RT LIP@PNM materials prepared by the present invention all contain Ly6G and CD41, indicating the successful preparation of platelet-neutrophil aggregates and their membranes, and the successful fusion of PNM with liposomes. The co-expression of P-selectin and PSGL-1, a pair of specific interacting proteins, is a key protein mediating PNA formation. The MAC-1 adhesion factor is used to promote trans-blood-brain barrier delivery. The specific expression of the TNF-α receptor gives the material a targeting function to the inflammatory microenvironment. The CD47 molecule effectively circumvents immune clearance by macrophages by specifically transmitting the "don't eat me" immune regulatory signal. This experimental example systematically verifies the completeness of the functional modification of the materials prepared by the present invention through multi-dimensional biomolecular experimental evidence.

[0072] The above results prove that the present invention successfully prepared RT LIP@PNM, which is composed of rutin, liposomes and platelet-neutrophil aggregate membrane, and its surface contains multiple functional proteins.

[0073] Experimental Example 2 Cell Experiment

[0074] 1. Experimental Methods

[0075] 1. Experimental Grouping

[0076] This experiment was divided into three groups: the activation + LIP group, the activation + LIP@PNM group, and the non-activation + LIP@PNM group. The drug in the activation + LIP group was DiD LIP, while the drug in the activation + LIP@PNM and non-activation + LIP@PNM groups was DiD LIP@PNM. For visualization purposes, DiD LIP and DiD LIP@PNM were LIP and LIP@PNM loaded with the same dose of DiD (a red fluorescent probe, 1,1-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) dye. The final DiD concentration in each group was 500 ng / mL.

[0077] 2. Cell Processing

[0078] BMVECs (brain microvascular endothelial cells) were seeded onto 14 mm confocal microplates. Tumor necrosis factor (TNF)-α was used to activate the cells to establish a brain inflammatory model. In the unactivated + LIP@PNM group, TNF-α was not used to activate the cells. DiD LIP and DiD LIP@PNM were added to the microplates according to grouping. Cells in each group were fixed with paraformaldehyde, stained with DAPI, and imaged using a confocal microscope.

[0079] 2. Experimental Results

[0080] The results of cell experiments are as follows Figure 4 Compared with the activated + LIP group and the unactivated + LIP@PNM group, the activated + LIP@PNM group showed strong red fluorescence around the cell nucleus, indicating that LIP@PNM was efficiently targeted and internalized into TNF-α-activated BMVECs, and PNM gave the material the function of targeting the inflammatory site of brain cells.

[0081] The results of this experimental example show that PNM gives the material the function of targeting the inflammatory sites of brain cells, and LIP@PNM can efficiently target and enter the inflamed brain cells.

[0082] Experimental Example 3 Animal Experiment

[0083] In this experiment, a tMACO / R (transient middle cerebral artery occlusion / reperfusion) rat model was constructed to simulate the pathological process of cerebral ischemia-reperfusion injury. The construction method is as follows:

[0084] Male Sprague-Dawley rats weighing 250-300 g were used. The neck was prepared and disinfected, and a 2 cm midline incision was made. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. The proximal ends of the CCA and ECA were ligated. A nylon suture was inserted from the ECA through the ICA into the middle cerebral artery (MCA) until the marked black dot reached the carotid bifurcation, at a depth of approximately 18-20 mm, indicating MCA occlusion. After 2 hours of occlusion, the nylon suture was removed, and reperfusion was initiated. In the sham group, only the skin was incised and the vessels isolated; no nylon suture was inserted.

[0085] 1. Experimental Methods

[0086] 1. In vivo imaging experiments

[0087] This experiment was divided into three groups: control group, LIP group, and LIP@PNM group, and the corresponding drugs were DiD, DiDLIP, and DiD LIP@PNM, respectively.

[0088] In tMACO / R model mice, free DiD, DiDLIP, and DiD LIP@PNM were intravenously injected 1 hour after reperfusion according to group status, and fluorescence was monitored using an in vivo imaging system (IVIS) at 1 hour, 4 hours, and 8 hours.

[0089] 2. Determination of blood-brain barrier (BBB) ​​crossing

[0090] Brain tissue from animals treated with DiD for 1 hour in the in vivo imaging experiment was immunofluorescently stained with a GFAP (glial fibrillary acidic protein) antibody to label astrocytes (a major glial cell in the brain) and observe the colocalization of DiD (red) and GFAP (green).

[0091] 3. Experimental study on determination of cerebral infarction

[0092] This experiment was divided into six groups: sham group (G1), model group (G2), RT group (G3), RT LIP group (G4), LIP@PNM group (G5), and RT LIP@PNM group (G6). The drugs in the RT, RT LIP, LIP@PNM, and RT LIP@PNM groups were RT, RT LIP, LIP@PNM, and RT LIP@PNM, respectively. The final RT concentration in each group was 1 mg / kg. The drug concentration in the LIP@PNM group was the same as that in the RT LIP@PNM group, and the drug was administered once. Both the sham and model groups received normal saline.

[0093] Three hours after MCAO surgery, the crystal form of the drug was injected intravenously into rats in each group according to the grouping situation. 21 hours after administration, the rat brain tissue was obtained, and brain tissue sections were made and stained with 2% triphenyltetrazolium chloride (TTC) solution. The cerebral infarction area was quantified using ImageJ to quantitatively evaluate the infarcted brain tissue.

[0094] TTC is a redox marker that distinguishes dead tissue from living tissue. This dye differentiates infarcted tissue by differential mitochondrial dehydrogenase activity (normal tissue appears red, ischemic areas appear pale).

[0095] 4. Determination of the effect of treating brain inflammation

[0096] According to the method of the brain tissue infarction measurement experiment in this experimental example, brain tissue was obtained, and then brain tissue homogenate was prepared. The MPO enzyme activity was quantitatively measured using an MPO test kit, and the levels of proinflammatory cytokines in the brain tissue were detected by ELISA (enzyme-linked immunosorbent assay), including proinflammatory cytokines such as TNF-α, IL-6, and IL-1β. These proinflammatory cytokines play a central role in neuroinflammation and brain injury.

[0097] 5. Evaluation of brain cell necrosis and apoptosis

[0098] According to the method of the brain tissue infarction measurement experiment in this experimental example, brain tissue was obtained, paraffin sections of brain tissue were made, and hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nickel end labeling (TUNEL) staining were performed to observe the necrosis and apoptosis of brain cells in each group.

[0099] 6. Neuronal Damage Measurement

[0100] According to the method of the brain tissue infarction measurement experiment in this experimental example, brain tissue was obtained, paraffin sections of brain tissue were made, and neuronal damage was observed using Nissl staining.

[0101] 2. Experimental Results

[0102] 1. In vivo imaging results

[0103] The results are as follows Figure 5 After 1, 4, and 8 hours of intravenous administration, most of the DiD signals accumulated in the right ischemic hemisphere, indicating that the LIP@PNM material prepared in the present invention can achieve long-term and efficient targeting of the brain ischemic area.

[0104] 2. Results of crossing the blood-brain barrier (BBB)

[0105] The results are as follows Figure 6 Immunofluorescence staining showed that in the ischemic hemisphere, the red signal of DiD-labeled DiD LIP@PNM merged with the green signal of GFAP-labeled astrocytes, indicating that LIP@PNM had crossed the blood-brain barrier and infiltrated into the brain parenchyma.

[0106] 3. Therapeutic effect on cerebral infarction

[0107] The TTC test results are as follows Figure 7 When rats were treated with RT LIP@PNM, the brain tissue injured by tMCAO / R showed a red color similar to that of the sham-operated rats. The results showed that RT LIP@PNM could prevent the formation of cerebral infarction during ischemia-reperfusion ( Figure 7 A).

[0108] Cerebral infarction was quantified using ImageJ ( Figure 7 Figure B) shows that the model group had a larger infarct size, reaching 37.7%, indicating that the tMCAO / R rat model was successfully established. The infarct size in tMCAO / R rats treated with RT and RT LIP was reduced to 25.7% and 23.5%, respectively. Notably, the cerebral infarct size in the LIP@PNM group was smaller than that in the RT LIP group, indicating that the PNM material itself can also produce significant therapeutic effects, and the effect is superior to that of the RT LIP group. This suggests that in addition to serving as a carrier for targeting, PNM can also play a therapeutic role as an active ingredient.

[0109] The cerebral infarction area of ​​the RT LIP@PNM group was significantly reduced to 4.6%. The effect of reducing the infarction area was not only greater than that of the RT LIP group and the LIP@PNM group respectively, but also greater than the sum of the two, indicating that RT LIP@PNM produced a synergistic effect, which can more effectively reduce the infarction area and alleviate brain damage.

[0110] 4. Treatment outcomes for brain inflammation

[0111] MPO enzyme activity is used to evaluate the intensity of inflammatory response. The results of MPO enzyme activity determination are as follows: Figure 8 As shown in A. Compared with the model group, the enzyme activity in the LIP@PNM group was significantly reduced, and the reduction effect was comparable to that in the RT LIP group, indicating that LIP@PNM can significantly alleviate brain inflammation. After RT encapsulation, the enzyme activity in the RT LIP@PNM group was significantly reduced, and the inflammatory response was further alleviated.

[0112] ELISA results ( Figure 8 B-8D) showed that compared with the model group, LIP@PNM significantly reduced the proinflammatory cytokines in damaged brain tissue, and its effect was comparable to that of the RT LIP group; after encapsulating RT, the level of proinflammatory cytokines was further reduced, effectively alleviating brain inflammation and brain damage.

[0113] The above results indicate that LIP@PNM not only exerts a direct anti-inflammatory effect, but also cooperates with the targeted delivery of RT in the ischemic brain area to achieve multi-pathway regulation of tMCAO / R-induced neuroinflammation. RT LIP@PNM achieves synergistic anti-inflammatory effects on brain tissue.

[0114] 5. Tissue staining results

[0115] Compared with the model group, H&E staining showed significantly fewer necrotic cells in the ischemic hemisphere of rats in the LIP@PNM group and RT LIP@PNM group, Nissl staining showed fewer damaged and atrophied neurons, and TUNEL staining showed significantly fewer apoptotic cells. These results show that injection of RT LIP@PNM can significantly reduce cell necrosis and apoptosis, and reduce new area damage ( Figure 9 ).

[0116] The results of this experiment demonstrate that, in a cerebral ischemia-reperfusion injury model, PNM not only serves as a carrier to target the ischemic brain area, but also as an active ingredient to exert therapeutic effects. In particular, when RT drugs were encapsulated in LIP@PNM, RTLIP@PNM achieved a synergistic therapeutic effect.

[0117] Through the above embodiments and experimental examples, it can be seen that the present invention provides a targeted liposome for treating ischemic stroke, its preparation method and use. The targeted liposome for treating ischemic stroke of the present invention is prepared by fusion of liposomes and platelet-neutrophil aggregate membranes, achieving efficient and specific targeting of ischemic brain areas, while effectively reducing brain tissue infarction and damage to new areas, alleviating brain inflammation, and can be used as an active ingredient for preparing a drug for treating ischemic brain injury. When the targeted liposome is further encapsulated with rutin drug, a synergistic therapeutic effect is achieved. The liposome drug of the present invention can be used as an active ingredient and / or drug carrier, and has good application prospects in the treatment of ischemic brain injury diseases.

Claims

1. Use of targeted liposomes as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The targeted liposomes include liposomes and platelet-neutrophil aggregate membranes fused with the liposomes; the mass ratio of the platelet-neutrophil aggregate membranes to the liposomes is 1:1-4:1; and the quantity ratio of platelets to neutrophil raw materials in the platelet-neutrophil aggregate membranes is 0.8-1.2: 0.8-1.

2.

2. Use of the targeted liposome according to claim 1 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The mass ratio of platelet-neutrophil aggregate membrane to liposome was 2:1; The ratio of the number of platelets to the number of neutrophil raw materials in the platelet-neutrophil aggregate membrane is 1:

1.

3. Use of the targeted liposome according to claim 1 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The liposome is loaded with a drug, and the drug is selected from at least one of rutin, baicalin, and mangiferin.

4. Use of the targeted liposome according to claim 3 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The mass fraction of the drug in the liposome is 2.1-3.2%.

5. Use of the targeted liposome according to claim 1 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The targeted liposome is prepared by fusing raw materials including platelet-neutrophil aggregate membrane and liposome through an extrusion method.

6. Use of the targeted liposome according to claim 5 as an active ingredient in the preparation of a drug for treating ischemic brain damage, characterized in that: The conditions of the extrusion method include: the pore size of the filter membrane is 200-400 nm, and the number of extrusions is 20-50 times; And / or, the platelet-neutrophil aggregate membrane is prepared by repeatedly freezing and thawing platelet-neutrophil aggregates; And / or, the liposome is prepared from raw materials including phospholipids and nonionic surfactants through a thin film hydration method.

7. Use of the targeted liposome according to claim 6 as an active ingredient in the preparation of a drug for treating ischemic brain damage, characterized in that: The repeated freezing and thawing conditions include: freezing at -75~-190℃ for 1-2h, and thawing in a water bath at 30-38℃ for 10-20 minutes; and / or, after repeated freezing and thawing, centrifuging at 10000±100 rpm, 0-10°C for 12-18 minutes; And / or, the platelet-neutrophil aggregates are prepared by inducing platelets and neutrophils with an inducer, incubating, and centrifuging.

8. Use of the targeted liposome according to claim 7 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The inducer is selected from at least one of phorbol ester, platelet activating factor, and tissue factor. The incubation conditions include: incubation temperature of 20-40° C., incubation time of 15 minutes, centrifugation speed of 80×g-120×g, and centrifugation time of 2-4 minutes.

9. Use of the targeted liposome according to claim 6 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The phospholipid is selected from at least one of egg yolk lecithin, soybean lecithin, hydrogenated phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; And / or, the nonionic surfactant is selected from at least one of polyethylene glycol (15)-hydroxystearate, polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine; And / or, the conditions of the thin film hydration method include: the solvent is selected from at least one of methanol, chloroform, dichloromethane, and ether, and the aqueous phase is selected from at least one of water, phosphate buffer, and physiological saline; And / or, after the thin film hydration method, ultrasonic cell disruptor is used for ultrasonication for 8-12 minutes.

10. Use of the targeted liposome according to claim 1 as an active ingredient in the preparation of a medicament for treating ischemic brain injury, characterized in that: The ischemic brain injury is ischemic stroke.

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