Multiple targeting biomimetic nanodelivery system, and preparation method and application thereof

Abstract

The application discloses a multiple-targeting biomimetic nano drug delivery system, a preparation method and application thereof, and belongs to the field of drug delivery systems. The drug delivery system is a drug-loaded liposome composed of paeonol, metformin, phospholipid and cholesterol, and is obtained by modifying a platelet membrane and a 4T1 tumor cell membrane on the surface of the drug-loaded liposome. The drug delivery system can effectively cross the blood-brain barrier of a rat, selectively gather in an ischemic area of the brain, and deliver PAE and PolyMet into damaged cells through membrane fusion or pinocytosis, so that the area of cerebral infarction is significantly reduced, the inflammation level is reduced, neurons are protected, the symptoms of ischemic stroke are relieved, and the survival quality is improved.

Description

Technical Field

[0001] This invention relates to a nano-drug delivery system, particularly a multi-targeted biomimetic nano-drug delivery system, and also to the preparation method and application of the above-mentioned drug delivery system. Background Technology

[0002] Currently, the main treatments for ischemic stroke are intravenous thrombolysis or arterial thrombectomy, which restore blood flow to the ischemic site, reconstruct substrate supply, and improve patient survival and quality of life. However, both cerebral ischemia and reperfusion lead to the production of large amounts of reactive oxygen species (ROS) and exacerbation of inflammatory responses in the ischemic area, causing secondary brain injury, reducing patients' quality of life, and even threatening their lives. Therefore, timely delivery of neuroprotective agents and anti-inflammatory substances to the ischemic site within a limited time is an effective strategy for treating cerebral ischemia and alleviating reperfusion injury.

[0003] The blood-brain barrier (BBB) ​​is a major obstacle to drug delivery to ischemic areas of the brain. Although the activation of immune cells and the inflammatory response induced by cerebral ischemia / reperfusion can disrupt the BBB to some extent, it is still insufficient to achieve selective accumulation of drugs in ischemic areas and thus fail to exert a therapeutic effect.

[0004] In recent years, biomimetic nanomedicine delivery systems have gradually become a research hotspot due to their ability to retain the natural recognition capabilities of organisms, high biocompatibility, and long circulation time. Nanomedicine carriers constructed using natural cells and biomolecules are an effective means to address the problem of drugs not effectively penetrating the blood-brain barrier (BBB), achieving targeted accumulation at the site of brain injury, and improving drug bioavailability. Currently, some studies have used platelet membrane-based biomimetic nanomedicine delivery systems to treat ischemic stroke; however, relying solely on the single targeting effect of the platelet membrane limits its ability to cross the BBB, making it difficult to achieve effective accumulation of therapeutic drugs in the brain. Therefore, how to overcome the BBB and achieve efficient targeted accumulation of drugs at the ischemic site has become a key research focus. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a multi-targeting biomimetic nanodelivery system that has multiple targeting properties, specific aggregation in damaged brain tissue, high biocompatibility, long in vivo circulation time, and anti-inflammatory and neuroprotective effects, and to provide the preparation method and application of the above-mentioned drug delivery system.

[0006] Technical solution: The multi-targeted biomimetic nanodelivery system of the present invention is a drug-loaded liposome (PP@lip) composed of paeonol, polymethyl guanidine, phospholipids and cholesterol, and a biomimetic nanodelivery system (PP@PCL) formed by modifying the surface of the drug-loaded liposome with platelet membrane and 4T1 tumor cell membrane.

[0007] Preferably, the mass ratio of paeonol, metformin, phospholipids and cholesterol in the drug-loaded liposomes is 10:1-6:50-150:10-50.

[0008] Preferably, the mass ratio of platelet membrane to 4T1 tumor cell membrane in the drug-loaded liposome is 1:3 to 3:1.

[0009] Preferably, the drug-loaded liposome is a drug-loaded liposome formed by loading paeonol between phospholipid bilayers and encapsulating polymethyl guanidine in a water-soluble cavity formed by a hydrophilic head of phospholipid; the biomimetic nanodelivery system is formed by simultaneously coating the above-mentioned drug-loaded liposomes with 4T1 tumor cell membranes and platelet membranes.

[0010] The method for manufacturing the above-mentioned drug delivery system includes the following steps:

[0011] (1) Paeonol, phospholipids and cholesterol were dissolved in an organic solvent by ultrasonication, rotary evaporated and dried at room temperature to obtain a drug-loaded phospholipid bilayer;

[0012] (2) Dissolve polymethyl methacrylate and hyaluronic acid separately in water, mix them, pipette and let stand at room temperature to obtain a water-soluble core;

[0013] (3) Mix the water-soluble core and the drug-loaded phospholipid bilayer, rotary evaporate and sonicate to obtain a uniform liposome dispersion;

[0014] (4) Take platelet membrane and 4T1 tumor cell membrane, and sonicate to obtain composite membrane dispersion; mix composite membrane dispersion with drug-loaded liposome dispersion at a mass ratio of 1 to 2:1, sonicate, and then extrude several times in a liposome extruder covered with polycarbonate membrane to obtain composite membrane biomimetic liposome dispersion, which is a multi-targeted biomimetic nano-drug delivery system.

[0015] Preferably, it includes the following steps:

[0016] (1) Preparation of drug-loaded phospholipid bilayer: PAE, phospholipids and cholesterol were dissolved in methanol by ultrasonication. The mixed dispersion was placed in a 100 mL eggplant-shaped flask and evaporated by rotary evaporation for 15 min. The eggplant-shaped flask was then placed in a vacuum desiccator and dried at room temperature overnight.

[0017] (2) Preparation of water-soluble core: PolyMet and hyaluronic acid (HA) were dissolved in deionized water to prepare a stock solution of appropriate concentration for later use; the PolyMet and HA solutions were mixed, pipetted 10-20 times, allowed to stand at room temperature, and deionized water was added to complete the preparation of water-soluble core.

[0018] (3) Add the prepared water-soluble core solution to the eggplant-shaped flask that forms the drug-loaded phospholipid bilayer, place the eggplant-shaped flask on a rotary evaporator, and water bath for 30 minutes until the phospholipid bilayer is completely detached from the inner wall; place the liquid in the eggplant-shaped flask on a cell sonicator and sonicate to form a clear and transparent homogeneous drug-loaded liposome dispersion (PP@lip).

[0019] (4) Fresh blood was collected from SD rats, and blood cells and white blood cells were separated by centrifugation at room temperature to obtain platelet-rich plasma (PRP). PBS buffer containing prostaglandin E1 (PGE1) and EDTA was added to the obtained PRP. After centrifugation at room temperature, the precipitate was resuspended in PBS and repeatedly frozen and thawed at -80°C. After centrifugation of the frozen and thawed solution, the supernatant was collected, washed several times with PBS solution, resuspended, and then sonicated for an appropriate time using a probe sonicator. Centrifugation was continued, and the supernatant was collected to obtain the pure platelet membrane. The obtained platelet membrane was pre-frozen and then freeze-dried using a freeze dryer for storage.

[0020] (5) 4T1 tumor cells were cultured in a complete culture medium prepared with RPMI-1640 medium, 10% fetal bovine serum, and 1% penicillin-streptomycin at 37°C and 5% CO2. The cells were digested with trypsin and centrifuged to obtain a 4T1 tumor cell pellet. The pellet was resuspended in Tris-magnesium sulfate buffer (TM Buffer), the cell density was adjusted to a suitable level, and the cells were centrifuged at 4°C. The resulting pellet was resuspended in a mixture of TM Buffer and sucrose solution and repeatedly centrifuged to obtain purified 4T1 tumor cell membranes.

[0021] (6) The purified platelet membrane and 4T1 tumor cell membrane were mixed at a certain mass ratio and sonicated for an appropriate time with a specific power probe to obtain a composite membrane dispersion. The above dispersion was mixed with PP@lip dispersion and sonicated for a certain time to ensure uniform mixing. Then, it was extruded back and forth several times in a liposome extruder covered with a polycarbonate membrane to obtain a composite membrane biomimetic drug-loaded liposome dispersion (PP@PCL), which is a multi-targeted biomimetic nano-drug delivery system.

[0022] Preferably, in step (1), the mass ratio of paeonol to phospholipid is 1:5 to 15, and the mass ratio of paeonol to cholesterol is 1:1 to 5.

[0023] Preferably, in step (2), the mass ratio of PolyMet to HA is 1-3:3-1, the number of blows is 10-20, and the settling time is 5-20 minutes.

[0024] Preferably, in step (3), the water bath temperature is 20-40℃, the probe ultrasonic power is 100-300W, and the ultrasonic time is 10-20min.

[0025] Preferably, in step (4), the centrifugation conditions for separating leukocytes and blood cells are: centrifugation at 100g for 20min, concentrations of EDTA and PGE1 of 1mM and 2mM respectively, freezing at -80℃ for 10-24h, repeated freeze-thaw cycles of 0-5 times, ultrasonic disruption power of the probe of 50-100W, and disruption time of 5-10min.

[0026] Preferably, in step (5), the suitable cell density is 1×10⁻⁶. 7 ~3×10 7 per mL.

[0027] Preferably, in step (6), the mass ratio of platelet membrane to 4T1 tumor cell membrane is 1-3:3-1, the ultrasonic power is 5-100W, the ultrasonic time is 5-10min, the polycarbonate membrane pore size is 200-400μm, and the number of extrusions is 10-40 times.

[0028] The above-mentioned drug delivery system is used in the preparation of drugs for treating ischemic stroke.

[0029] Invention Principle: The multi-targeted biomimetic nanodrug delivery system of this invention loads PAE between phospholipid bilayers using a thin-film dispersion method, with PolyMet encapsulated within the water cavity formed by the hydrophilic heads of the phospholipids, forming drug-loaded liposomes. This system is expected to simultaneously address the problems of poor water solubility and low in vivo bioavailability of PAE, and the large molecular weight, low BBB penetration efficiency, and low biocompatibility of PolyMet, thereby maximizing the efficacy of both. Utilizing the morphological characteristics of liposomes—composed of phospholipid molecules with hydrophilic heads and hydrophobic tails—the amphiphilic nature of phospholipid molecules endows liposomes with the ability to simultaneously deliver both hydrophobic and hydrophilic drugs. The phospholipid bilayer formed by the hydrophobic tails of the phospholipid molecules can effectively encapsulate poorly water-soluble drugs, forming highly stable and leak-resistant nanoparticles. The core formed by the hydrophilic heads of the phospholipids can encapsulate hydrophilic drug molecules, reducing their biotoxicity. Liposomes modified with polymers, peptides, or other functional substances can serve as nano-drug delivery carriers, which can not only improve the bioavailability of poorly water-soluble drugs and prolong their circulation time in vivo, but also achieve temporal and spatial response release of drugs and reduce systemic toxicity.

[0030] Paeonol (PAE), the main active ingredient in the dried root bark (Paeonia suffruticosa) of the Ranunculaceae family and the dried root, rhizome, or whole herb of Cynanchum paniculatum (Cynanchum paniculatum) of the Asclepiadaceae family, possesses sedative, antibacterial, antipyretic, and analgesic effects. Recent studies have found that PAE also has anti-inflammatory effects on the nervous system, inhibits the production of oxygen free radicals, and has anticoagulant properties. Under conditions of glucose and hypoxia, PAE can inhibit mitochondrial oxidative stress, upregulate mitochondrial membrane potential, reduce mitochondrial damage, and thus inhibit neuronal apoptosis, exhibiting a protective function against neurons. By loading PAE between phospholipid bilayers, the drawbacks of poor water solubility, low melting point, volatility, and poor stability of PAE have been improved.

[0031] Metformin (MET) is a biguanide drug widely used in the treatment of type 2 diabetes and metabolic syndrome. Studies have shown that metformin possesses various pharmacological activities, such as hypoglycemic, anti-infective, antibacterial, anti-apoptotic, and anti-tumor effects. Polymetformin (PolyMet) is a novel polymer in which the biguanide group in dicyandiamine is covalently linked to the high molecular weight material chitosan hydrochloride through an addition reaction. This polymer is rich in biguanide groups and has the advantages of good stability, strong water solubility, and high pharmacological activity. PolyMet inherits many of the pharmacological properties of MET: it can reduce ROS levels, alleviate oxidative stress in cells, and reduce cell death; it can inhibit the TLR4 / NF-κB pathway, reduce the inflammatory response after ischemic stroke reperfusion, and exert a neuroprotective effect; it can improve mitochondrial metabolic levels, restore normal mitochondrial function, and repair damaged cells; it can reduce apoptosis genes, weaken autophagosome formation, and thus reduce cell apoptosis and autophagy. By encapsulating PolyMet within a water cavity formed by a hydrophilic phospholipid head, the problems of PolyMet's positive charge and low biocompatibility are improved, and its large molecular weight and difficulty in penetrating the BBB are solved, making it effective in the treatment of ischemic stroke.

[0032] Meanwhile, the pathogenesis of ischemic stroke is closely related to platelet (PLT) activity. Under normal circumstances, platelet activation is inhibited by cytokines such as prostaglandin PDI2, nitric oxide, and CD39. In healthy blood vessels, platelets slowly roll along the inner wall without aggregation. When a blood vessel is damaged, membrane proteins on the slowly rolling platelets recognize the exposed endothelial matrix at the injury site. Platelet adhesion at the injury site is enhanced, forming pseudopodia to cover the damaged area. This triggers intraplatelet signal transduction, inducing the release of clotting substances, which in turn recruits more platelets to aggregate at the injury site, forming a thrombus network to resist blood flow and promote healing. During this process, abnormal platelet activation or thrombus detachment can lead to vascular occlusion, obstructing blood circulation to the brain. For hours or days after cerebral blood circulation is obstructed, cellular energy and material supply are blocked. Mitochondria in cells become dysfunctional due to lack of substrate supply, leading to the efflux and extracellular accumulation of excitatory neurotransmitters, as well as a large influx of calcium ions. High intracellular calcium ion levels can cause the degradation of enzymes that maintain cell structure and function, and the excessive production of reactive oxygen species and reactive nitrogen free radicals, leading to irreversible cell death. Simultaneously, the active migration of immune cells to the site of vascular injury triggers an inflammatory response at the stroke site, further expanding the infarct area and exacerbating the stroke damage. Therefore, platelets play a crucial role in the development of ischemic stroke. Based on this principle, the inventors designed a biomimetic nanoparticle drug delivery system using platelet membranes. A sophisticated platelet membrane extraction process preserves functional proteins on the membrane surface, endowing the modified nanoparticles with natural vascular injury and thrombus targeting properties. This allows for targeted accumulation of nanoparticles in the ischemic stroke region, precisely delivering therapeutic or neuroprotective drugs loaded within the nanoparticles, improving the treatment efficacy of ischemic stroke and enhancing patient prognosis.

[0033] Another key to the successful delivery of neuroprotective drugs to ischemic sites is their ability to cross the blood-brain barrier (BBB). The inventors' research revealed that 4T1 tumor cells easily cross the BBB, colonize the brain, and possess a high capacity for brain metastasis. CD138 on the 4T1 tumor cell membrane can bind to platelet-endothelial cell adhesion molecule 1 (CD31) on platelets, endothelial cells, and leukocytes surrounding cerebral blood vessels, facilitating the tumor cells' crossing of the BBB and achieving adhesion within the brain. Furthermore, the highly expressed vascular cell adhesion molecule 1 (VCAM-1) on the 4T1 tumor cell membrane has a high affinity for late-appearing antigen 4 (VLA-4) on leukocytes, allowing it to recognize leukocytes that preferentially accumulate at brain injury sites, thus targeting and accumulating at ischemic sites. In addition to these excellent brain-targeting properties, the 4T1 tumor cell membrane also highly expresses the CD47 "don't eat me" signal, enabling it to evade recognition by the immune system and achieve long-term circulation in the body.

[0034] This invention utilizes a fusion membrane of platelet membrane and 4T1 tumor cell membrane as a biomimetic membrane, which is modified onto the surface of PAE and PolyMet liposomes to construct a biomimetic nanodelivery system for treating ischemic stroke. This biomimetic nanodelivery system leverages the crucial role of platelets in the development of ischemic stroke, naturally targeting damaged blood vessels or thrombi under the recruitment of cytokines and inflammatory factors secreted by cells at the ischemic site. Utilizing the specific binding of receptors on the 4T1 tumor cell membrane to ligands on hematopoietic cells and leukocytes aggregated in damaged brain tissue, and the strong bile duct permeability of 4T1 tumor cells, PAE and PolyMet are precisely delivered to the stroke area, exhibiting good anti-inflammatory effects and excellent neuroprotective capabilities. It significantly reduces the infarct area and pathological features in rats with ischemic stroke, improves the survival rate of MCAO / R model rats, and enhances their quality of life.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) It solves the problem of poor water solubility and low bioavailability of PAE in vivo; (2) PolyMet is applied to the treatment of ischemic stroke. By utilizing the pharmacological activity of PolyMet, it can significantly alleviate the reperfusion injury of ischemic stroke and play a neuroprotective role; (3) A biomimetic nano-drug delivery system is constructed by using a fusion membrane of platelet membrane and 4T1 tumor cell membrane to achieve multiple targeting functions, improve the selective accumulation of drugs in the stroke area, and show a strong therapeutic effect, which can effectively alleviate brain damage. Attached Figure Description

[0036] Figure 1 This is a particle size distribution diagram of PP@PCL in Example 2;

[0037] Figure 2 This is a graph showing the Zeta potential results of PP@PCL in Example 2;

[0038] Figure 3 The following is a Western blot result of PM, CM, and PP@PCL in Example 2;

[0039] Figure 4 This is a graph showing the serum stability results of PP@PCL in Example 2;

[0040] Figure 5 This is a diagram showing the results of the MTT assay in Example 3 for detecting the reversal and restoration of neuronal cell function by PP@PCL.

[0041] Figure 6 This is a graph showing the results of extracellular LDH release levels in Example 3;

[0042] Figure 7 The following are the in vivo pharmacodynamic results of PP@PCL in Example 4, where Figure A shows the results of TTC staining to examine the area of ​​cerebral infarction, and Figure B shows the results of the percentage of infarction.

[0043] Figure 8 The images show the H&E staining results of ischemic brain tissue sections from rats in each group in Example 4.

[0044] Figure 9 The images shown are the neurological scoring results in Example 4, where Figure A is the mNSS scoring result, Figure B is the Bederson method neurological scoring result, and Figure C is the Longa method neurological scoring result. Detailed Implementation

[0045] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0046] Example 1

[0047] Preparation of PP@lip

[0048] Preparation of drug-loaded phospholipid bilayer: Accurately weigh 1 mg of PAE, soybean phospholipids, and cholesterol, and sonicate them in 5 mL of methanol. Transfer the resulting liquid to a 100 mL round-bottom flask. Place the flask on a rotary evaporator and evaporate under reduced pressure at 40 °C for 15 min. A uniform honeycomb-like film is obtained on the inner wall of the flask, which is the PAE-loaded phospholipid bilayer. Dry the flask in a vacuum desiccator overnight.

[0049] Preparation of the water-soluble core: Accurately weigh 100 μg PolyMet and 300 μg HA into beakers, dissolve them in PBS to prepare a stock solution, and transfer 1 mL of each into a 10 mL EP tube. Gently pipette the solution 20 times and allow it to stand at room temperature for 20 min. Add deionized water to the EP tube until a certain volume is reached to form the water-soluble core. PolyMet, as a high molecular weight polymer, has a linear structure, making direct encapsulation of PolyMet within the aqueous cavity of liposomes challenging. Through electrostatic binding, PolyMet and HA become entangled, forming polymer microspheres, which are more easily encapsulated within the aqueous cavity of liposomes, greatly improving the encapsulation efficiency and drug loading of PolyMet in liposomes.

[0050] A water-soluble core was added to a phospholipid bilayer loaded with PAE. The flask was placed on a rotary evaporator and rotary evaporated in a water bath at a suitable temperature for 30 minutes until the film was completely detached. The liquid in the flask was transferred to an EP tube and sonicated at a suitable power for 20 minutes using a probe-type ultrasonic disruptor to obtain a clear, transparent, and homogeneous liposome dispersion (PP@lip).

[0051] Example 2

[0052] Preparation and characterization of PP@PCL

[0053] (1) Extraction and purification of platelet membrane (PM)

[0054] Platelet-rich plasma (PM) was extracted using a repeated freeze-thaw method. First, fresh blood from SD rats was collected and placed in EP tubes containing sodium heparin. The collected rat blood was centrifuged at 100g for 20 min at room temperature. The resulting precipitate consisted of red blood cells and white blood cells, while the supernatant was platelet-rich plasma (PRP). The supernatant was collected, and the above steps were repeated to obtain purified PRP. Appropriate concentrations of EDTA and prostaglandin E1 (PGE1) were added to the purified PRP, and the mixture was thoroughly mixed. The solution was centrifuged at 800g for 20 min at room temperature, and the resulting precipitate was platelets. The platelets were resuspended in PBS and frozen at -80℃ for 12 h. After thawing at room temperature, this process was repeated three times. The freeze-thawed solution was centrifuged at 3000 rpm for 30 min, and platelet fragments and supernatant were collected. After washing three times with PBS, the platelets were sonicated for 5 min using a probe-type ultrasonic disruptor and centrifuged at 3000 rpm at room temperature. The resulting supernatant was the purified PM. The PM solution was pre-frozen in a -80℃ freezer for 24 hours, and then freeze-dried using a freeze dryer to obtain red solid PM, which was then stored for later use.

[0055] (2) Extraction and purification of 4T1 tumor cell membrane (CM)

[0056] 4T1 tumor cell culture conditions: RMPI-1640 medium, 10% fetal bovine serum, 1% penicillin-streptomycin; incubator conditions: 37℃, 5% CO2. When cell confluence reached 90%, cells were digested with trypsin at 37℃, centrifuged, precisely counted, resuspended in TM Buffer, and the cell density adjusted to 1×10⁻⁶ cells / year. 7 ~3×10 7 / mL. The obtained cell suspension was lysed overnight at 4°C, and then extruded several times in a liposome extruder without a polycarbonate membrane to further disrupt the cells. 1M sucrose solution was then added to the cell suspension, and the mixture was centrifuged at 2000g for 30 min at 4°C. The supernatant obtained was the 4T1 tumor cell membrane. The supernatant was further centrifuged at 3000g for 30 min at 4°C, and the resulting precipitate was the 4T1 tumor cell membrane. To obtain a purer 4T1 tumor cell membrane, the above steps were repeated once. The resulting precipitate was pre-frozen at -80°C for 24 h and then freeze-dried. The resulting white solid was the 4T1 tumor cell membrane, which was stored for later use.

[0057] (3) Preparation of PP@PCL

[0058] Platelet membranes and 4T1 tumor cell membranes were mixed at a 1:1 mass ratio and dissolved in PBS solution by sonication. The mixed cell membrane suspension was then mixed with PP@lip dispersion at a 2:1 mass ratio and sonicated for 5 minutes to ensure homogeneous mixing of the cell membranes and liposomes. The sonicated dispersion was then extruded several times using a liposome extruder coated with 200μm and 400μm polycarbonate membranes to obtain the PP@PCL dispersion.

[0059] (4) Characterization of PP@PCL

[0060] The PP@PCL prepared with the optimal formulation was characterized. The hydrated particle size of the PP@PCL was determined, and the results are as follows: Figure 1 As shown, the PP@PCL particles have a diameter of 131 nm and are uniformly distributed. The Zeta potential of the PP@PCL was measured, as shown... Figure 2 As shown, the potential is -18mV, indicating that the biomimetic nanocomposite is negatively charged and has good biocompatibility after entering the body.

[0061] Western blot experiments were used to examine the retention of characteristic proteins on PM, CM, and PP@PCL. The results are as follows: Figure 3As shown, the PM extracted by the inventors retains the specific proteins P-selectin and CD47, while the CM retains the characteristic protein VCAM-1, indicating successful extraction of PM and CM. PP@PCL simultaneously retains all three proteins, indicating that PM and CM can fuse and be encapsulated on PP@lip. These results demonstrate the successful preparation of PP@PCL, and the resulting biomimetic nanomedicine delivery system possesses characteristics that mimic the functions of platelets and 4T1 tumor cells. After entering the body, it can specifically target ischemic areas and exert neuroprotective effects.

[0062] The stability of PP@PCL in complete culture medium was investigated. The results are as follows: Figure 4 As shown, under storage conditions of 4℃, the particle size change of PP@PCL within 120h was less than 20%, and the PDI remained less than 0.3, indicating that PP@PCL has good stability under suitable storage conditions and can be stored for a long time.

[0063] Example 3

[0064] In vitro pharmacodynamic study of PP@PCL

[0065] (1) MTT assay for cytotoxicity

[0066] The MTT assay was used to determine whether different drugs could reverse and restore neuronal function in an OGD / R cell model. After cell modeling, cells were treated with PAE solution, PAE@lip dispersion, PP@lip dispersion, PP@PCL dispersion, and culture medium (Control) at the same PAE concentration (15 μg / mL). The drug-containing culture medium was discarded, and after washing with PBS, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated for 4 h. After incubation, the MTT solution was discarded, and 150 μL of LDMSO solution was added to each well. The 96-well plate was shaken on a microplate reader, and the absorbance of each well was measured at 490 nm. Cell viability was calculated using the following formula.

[0067] Cell viability = (A Sample -A PBS ) / (A Control -A PBS )×100%

[0068] A Sample —Absorbance of the drug-treated cell group

[0069] A PBS —Absorbance of PBS solution

[0070] A Control —Absorbance of untreated cell groups

[0071] The results are as follows Figure 5 As shown, after OGD / R treatment, the survival rate of PC12 cells decreased to about 35%. After treatment in different groups, the viability of PC12 cells recovered to varying degrees. Among them, the PP@PCL treatment group had the highest cell survival rate, which was close to 100%, indicating that the viability of PC12 cells could be significantly restored after PP@PCL treatment.

[0072] (2) Lactate dehydrogenase (LDH) method for determining cytotoxicity

[0073] After brain cell damage, intracellular LDH is released into the extracellular space, leading to a significant increase in extracellular LDH activity. Therefore, by measuring the extracellular LDH level, it is possible to reflect whether the prepared PP@PCL has a reversal repair effect on the OGD / R cell model. First, PC12 cells after modeling were treated with PAE, PAE@lip, PP@lip, and PP@PCL at the same final concentration. After incubation at 37℃ for 24 h, 60 μL of the detection working solution was added, mixed, and incubated at room temperature in the dark for 30 min. Then, the OD value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) machine.

[0074] The results are as follows Figure 6 As shown, the extracellular LDH level was significantly increased after OGD / R, and the cell membrane was obviously damaged. After treatment with different drugs, LDH release was significantly reduced. Among them, the cells treated with PP@PCL had the lowest LDH release, indicating that PP@PCL has a significant reversal and repair effect on the OGD / R cell model and can effectively protect PC12 cells after OGD / R.

[0075] Example 4

[0076] PP@PCL in vivo pharmacodynamic study

[0077] (1) Assessment of cerebral infarction level

[0078] The level of cerebral infarction in MCAO model rats under different treatment groups was evaluated using TTC staining. Rats were injected with different drugs after MCAO surgery and divided into five groups: sham-operated group (Sham), saline group (Saline), PAE@lip group, PP@lip group, and PP@PCL group, with five rats in each group. Rats were decapitated 24 hours after drug administration, and their brains were collected and frozen at -20℃ for 30 min. After freezing, coronal sections were prepared approximately 2 mm anterior and posterior to the optic chiasm, with each brain section divided into five slices. The brain slices were stained in 2% TTC staining solution at 37℃ in the dark for 30 min, turning the slices every 5 min to ensure uniform staining. Brain slices from different treatment groups were photographed, and the percentage of cerebral infarction volume was calculated using ImageJ.

[0079] Experimental results are as follows Figure 7 As shown in Figure A, normal brain tissue appears red after TTC staining, while infarcted areas appear white. After treatment with different drugs, the area of ​​the white region decreased to varying degrees in each treatment group, with the PP@PCL group showing the smallest white area, indicating that PP@PCL treatment is most effective in treating MCAO model rats.

[0080] like Figure 7 As shown in Figure B, the infarction percentage in the model group was approximately 50%. After treatment with different drugs, the infarct area decreased in all groups of mice. The infarction percentage in the PP@lip group was significantly different from that in the PAE@lip group, indicating that PolyMet can effectively alleviate cell damage caused by cerebral ischemia. The infarction percentage in the PP@PCL group was also significantly different from that in the PP@lip group, indicating that liposomes modified with PM and CM can enhance drug accumulation at the ischemic site, thereby significantly reducing the infarct area and alleviating brain damage. The infarction percentage results demonstrate that PP@PCL significantly increases accumulation at the ischemic site, significantly enhancing the concentration of PAE and PolyMet at the ischemic site, improving efficacy, and ameliorheic cerebral infarction levels in MCAO model rats.

[0081] (2) Hematoxylin and eosin staining of ischemic brain tissue (H&E Staining)

[0082] Hematoxylin and eosin (H&E) staining was used to evaluate the level of ischemic brain tissue cell damage in MCAO model rats under different treatment groups. MCAO model rats were sacrificed 24 hours after drug administration, and their brains were harvested, fixed in 4% paraformaldehyde, embedded in paraffin, and then stained with H&E to observe the cell damage in each group.

[0083] The results are as follows Figure 8 As shown, the cells in the Sham group had intact morphology and no obvious cell damage was found; in the Saline group, a large number of cell nuclei were shrunken or even disappeared, and the gaps between cells were significantly increased; after treatment with PAE@lip and PP@lip, cell damage was alleviated; and after treatment with PP@PCL, cell damage was significantly reversed, and the cell morphology was close to that of the Sham group, with intact nuclei without shrunkenness, and the cells were tightly arranged and intact. This indicates that the multi-targeted biomimetic nanomedicine delivery system can effectively reverse cell damage caused by ischemia and shows excellent potential for treating ischemic stroke.

[0084] (3) Neurological scoring test

[0085] Rats were randomly divided into 5 groups: Sham group, Saline group, PAE@lip group, PP@lip group, and PP@PCL group, with 5 rats in each group. Seven days after drug administration, a neurological deficit system was established and scored using the modified Neurological Severity Score (mNSS Score), Bederson method, and Longa method. The neurological scoring results are as follows: Figure 9 As shown in Figures A, B, and C, the mNSS scores in the Saline treatment group ranged from 12 to 17, indicating severe neurological damage. After drug treatment in all groups, the neurological scores decreased. There was a significant difference between the PP@PCL group and the Saline group (p<0.0001), with the PP@PCL group achieving an mNSS score of around 5, indicating mild damage. The Bederson and Longa method scores also showed a reduction in neurological scores after PP@PCL treatment, specifically manifested in reduced pushing resistance on the operated side and bending of the contralateral forelimb after suspension in the MCAO model SD rats. All these results demonstrate that PP@PCL treatment can significantly improve the neurological function defects in model rats, exhibiting excellent therapeutic effects for ischemic stroke.

[0086] This invention encapsulates a PM and CM composite membrane onto the surface of a liposome (PP@lip) simultaneously loaded with paeonol (PAE) and polymethicone (PolyMet), drugs with anti-inflammatory and neuroprotective effects, to form a biomimetic nanodelivery system (PP@PCL) capable of targeting ischemic brain tissue in multiple ways.

[0087] This biomimetic nanodelivery system, based on platelet membranes and 4T1 tumor cell membranes, addresses the issues of poor water solubility of PAE and the large molecular weight and low biosafety of PolyMet. Due to its small size, high biosafety, long in vivo circulation time, ability to improve drug solubility and bioavailability, and alteration of drug spatial distribution in the body, it effectively crosses the blood-brain barrier to deliver PAE and PolyMet to ischemic brain regions, prolongs their half-life, continuously repairs neurons in ischemic stroke, reduces disability rates, and improves patients' quality of life.

Claims

1. A multi-targeted biomimetic nanodelivery system, characterized in that, The drug delivery system is a biomimetic nano-drug delivery system composed of paeonol, polymethyl methacrylate (PMMA), hyaluronic acid, phospholipids, and cholesterol, with platelet membrane and 4T1 tumor cell membrane modified on the surface of the liposomes. The mass ratio of paeonol, PMMA, phospholipids, and cholesterol is 10:1~6:50~150:10~50, the mass ratio of platelet membrane to 4T1 tumor cell membrane is 1~3:3~1, and the mass ratio of PMMA to hyaluronic acid is 1~3:3~1. The drug-loaded liposome is formed by loading paeonol between phospholipid bilayers and encapsulating PMMA in a water-soluble cavity within a hydrophilic head of the phospholipid. The preparation method of the biomimetic nano-drug delivery system includes the following steps: (1) Paeonol, phospholipids and cholesterol were dissolved in an organic solvent by ultrasonication, rotary evaporated and dried at room temperature to obtain a drug-loaded phospholipid bilayer; (2) Dissolve polymethyl methacrylate and hyaluronic acid separately in water, mix them, pipette and let stand at room temperature to obtain a water-soluble core; (3) Mix the water-soluble core and the drug-loaded phospholipid bilayer, rotary evaporate and sonicate to obtain a uniform liposome dispersion; (4) Take platelet membrane and 4T1 tumor cell membrane, and sonicate to obtain composite membrane dispersion; mix composite membrane dispersion and drug-loaded liposome dispersion at a mass ratio of 1~2:1, sonicate, and then extrude several times in a liposome extruder covered with polycarbonate membrane to obtain composite membrane biomimetic liposome dispersion, which is a multi-targeted biomimetic nano-drug delivery system.

2. The multi-targeted biomimetic nanodelivery system according to claim 1, characterized in that, In step (2), the number of blows is 10 to 20, and the settling time is 5 to 20 minutes.

3. The multi-targeted biomimetic nanodelivery system according to claim 1, characterized in that, In step (3), the ultrasound is probe ultrasound with a power of 100~300 W and an ultrasound time of 10~20 min.

4. The multi-targeted biomimetic nanodelivery system according to claim 1, characterized in that, In step (4), the pore size of the polycarbonate membrane is 200~400 μm, and the number of extrusions is 10~40 times.

5. The use of the multi-targeted biomimetic nanodelivery system of claim 1 in the preparation of a drug for treating ischemic stroke.

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