Brain-targeted stem cell membrane bionic cationic liposome as well as preparation method and application thereof

Abstract

The invention discloses brain-targeted stem cell membrane bionic cationic liposome as well as a preparation method and application thereof. The preparation method comprises the following steps: extracting and purifying to obtain a stem cell membrane; preparing a cationic liposome; the stem cell membrane and the cationic liposome are fused to obtain the stem cell membrane bionic cationic liposome, and the relative dosage of the stem cell membrane and the cationic liposome is that the mass ratio of the total protein mass of the stem cell membrane to the total fat mass of the cationic liposome is 1: (2-24). The stem cell membrane bionic cationic liposome constructed by using the purified stem cell membrane has high brain targeting ability, low immune irritation and efficient nucleic acid drug carrying ability, can break through the blood brain barrier, realizes efficient targeting delivery of nucleic acid drugs to cells of the brain including neurons, and has good application prospects. Particularly, the polypeptide can be used as a targeted delivery platform of nucleic acid drugs for targeted therapy of cerebral diseases, especially cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid drug targeted delivery systems, and in particular to a brain-targeted stem cell membrane biomimetic cationic liposome and a preparation method and application thereof. Background Art

[0002] With the rapid development of society, brain diseases such as stroke, Alzheimer's disease, and epilepsy have become major diseases that endanger people's lives and health and greatly affect people's living standards. Among them, ischemic stroke is characterized by high morbidity, high disability rate, high mortality rate, and high recurrence rate. Ischemic stroke cases account for more than 80% of stroke. In clinical treatment, thrombolytic agents such as tissue plasminogen activator can be used to restore blood flow, thereby reducing ischemic damage to brain tissue and improving patients' survival rate and the possibility of neurological function recovery. However, reperfusion can also trigger oxidative stress response and activate inflammatory pathways, which affect the structural integrity of cerebral blood vessels through oxidative damage of cell molecules, infiltration of inflammatory cells and release of inflammatory mediators, upregulation of matrix metalloproteinases, and regulation of tight junction proteins, causing secondary damage to the blood-brain barrier and aggravating nerve damage.

[0003] Gene drugs have gradually become the focus of stroke research due to their advantages such as wide range of target selection, high specificity, and rapid production. Among them, small interfering RNA (siRNA), microRNA (miRNA), and circular RNA (circRNA) have shown great therapeutic potential in the field of stroke treatment, providing new directions for future neuroprotection and functional recovery. For example, siRNA targeting the adaptor protein p66 (Shc) maintains the structural integrity of the blood-brain barrier after ischemia-reperfusion by silencing p66 (Shc); miR-124 specifically expressed in brain tissue protects damaged nerves after stroke through its anti-inflammatory, anti-apoptotic, and neuroregeneration-promoting effects; circSCMH1 promotes the recovery of motor function after stroke by enhancing neuronal plasticity, inhibiting glial cell activation, and peripheral immune cell infiltration.

[0004] Gene drugs face many challenges in the treatment of brain diseases, including the difficulty of penetrating the blood-brain barrier, instability due to nuclease degradation, and poor cell membrane permeability caused by negative charge. The development of nanocarrier technology provides a new strategy to solve these problems. Nanocarriers have the functions of improving drug stability, enhancing blood-brain barrier penetration, and promoting cell uptake. Among them, liposomes, as a classic and highly commercialized nanocarrier, not only have high drug loading capacity and good biocompatibility, but also can effectively carry nucleic acid drugs through their cationic surface and promote cellular internalization. However, the lack of targeting of cationic liposomes limits their application in gene therapy of brain diseases.

[0005] Cell membrane biomimetic technology provides an innovative solution to improve the brain-targeted delivery efficiency of nanocarriers. Cell membrane biomimetic technology not only simplifies the functional preparation process of carriers by integrating the biological functionality of cell membranes into nanocarriers, but also significantly improves the multifunctionality and targeting of carriers. The choice of cell membrane determines the functional type of biomimetic carriers. Among them, stem cells have the characteristics of natural inflammation targeting and low immunogenicity, which enable them to have the potential to deliver drugs to the brain. Specific receptors and ligands on the surface of stem cell membranes, such as CXCR4 and VLA-4, can play a role in mediating targeted delivery on the surface of biomimetic carriers. In addition, the purity of the cell membrane used for biomimetic modification will also affect the function of the carrier. If the cell membrane is mixed with cellular components such as nucleoproteins that can stimulate endogenous immune responses, the immunostimulatory properties of the biomimetic carriers prepared by them will increase, and ultimately affect the targeting efficiency of the carriers. At present, the methods used to separate and purify cell membranes mainly include differential centrifugation, two-phase distribution method and density gradient centrifugation, but there is no unified standard yet. Among them, density gradient centrifugation can achieve detailed separation of different cell components through sophisticated density gradient design, and has the potential to further improve the purity of cell membranes. However, the operation process of this method is relatively complicated, and its separation effect is affected by many parameters, including dispersion medium gradient setting, rotation speed, and centrifugation time. Therefore, establishing a set of standardized operating methods is crucial to ensure the repeatability of the experiment, improve the separation purity, and the reliability of the results. Summary of the invention

[0006] The purpose of the present invention is to provide a stem cell membrane biomimetic cationic liposome capable of targeted brain delivery of nucleic acids, and a preparation method and application thereof, which can be used as a nucleic acid drug targeted delivery platform for brain diseases, especially the treatment of ischemic stroke, to solve the problems faced by the clinical application of nucleic acid drugs, such as weak targeting and difficulty in breaking through the blood-brain barrier.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing stem cell membrane biomimetic cationic liposomes, comprising the following steps:

[0009] (1) Extracting and purifying to obtain stem cell membrane;

[0010] (2) preparing cationic liposomes;

[0011] (3) fusing the stem cell membrane obtained in step (1) with the cationic liposome obtained in step (2) to obtain a stem cell membrane biomimetic cationic liposome,

[0012] The relative amounts of stem cell membrane and cationic liposome are as follows: the mass ratio of total protein amount of stem cell membrane to total lipid amount of cationic liposome is 1:2-24.

[0013] If the ratio of stem cell membrane is too large, the positive potential of the carrier surface will be reduced, reducing its carrying capacity for negatively charged nucleic acid drugs; if the ratio is too small, insufficient membrane proteins will be transferred to the cationic liposomes along with the stem cell membrane, greatly reducing the brain targeting of the carrier.

[0014] Preferably, the relative amount of stem cell membrane and cationic liposome is: the mass ratio of total protein of stem cell membrane to total lipid of cationic liposome is 1:12. In the embodiment of the present invention, the mass ratio of total protein of stem cell membrane to total lipid of cationic liposome is preferably 1:12 as an example, but it is not limited thereto.

[0015] Specifically, the stem cell membrane is selected from at least one of umbilical cord-derived mesenchymal stem cell membranes, placenta-derived mesenchymal stem cell membranes, bone marrow-derived mesenchymal stem cell membranes, neural stem cell membranes and induced pluripotent stem cell membranes.

[0016] Specifically, the cationic liposome is prepared by mixing cationic lipids and neutral lipids, wherein the mass ratio of the cationic lipids to the neutral lipids is 1:1-5.

[0017] Specifically, the cationic lipid is selected from at least one of DOTAP ((2,3-dioleyloxypropyl)trimethylammonium chloride), DOTMA (trimethyl-2,3-dioleyloxypropylammonium chloride), DOSPA (dimethyl-2,3-dioleyloxypropyl-2-(2-sperminecarboxamido)ethylammonium trifluoroacetate), DDAB (dioctadecylammonium bromide), DOGS (N-(2-sperminecarboxyl)-N',N'-dioctadecylglycinamide) or DC-Chol (3β-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol).

[0018] Specifically, the neutral lipid is selected from at least one of SPC (sphingosine phosphorylcholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or Chol (cholesterol).

[0019] Preferably, cationic lipid DOTAP and neutral lipid SPC are selected and mixed in a mass ratio of 2:3 to prepare cationic liposomes.

[0020] Preferably, in step (1), the stem cell membrane is first extracted by repeated freezing and thawing, and then the stem cell membrane is purified by density gradient centrifugation;

[0021] In step (2), the cationic liposomes are prepared by a thin film dispersion method.

[0022] The purified stem cell membrane is obtained by two consecutive sucrose density gradient centrifugation separations and purifications, contains less cell nuclear components, and has lower immunostimulation when used for bionic modification.

[0023] By modifying cationic liposomes with high-purity stem cell membranes, the resulting bionic carrier inherits the natural inflammatory targeting of stem cells without increasing immunostimulation, while retaining the ability of cationic liposomes to efficiently carry nucleic acid drugs. At the same time, the vesicle structure helps the bionic carrier to deform, pass through the narrow cell gaps in the body, and break through biological barriers.

[0024] Specifically, in step (3), when the stem cell membrane is fused with the cationic liposome, the fusion is carried out in nuclease-free water or an isotonic solution;

[0025] The isotonic solution is selected from at least one of a sorbitol isotonic solution, a glucose isotonic solution or a mannitol isotonic solution.

[0026] The parameters for the fusion of the stem cell membrane and the cationic liposome are as follows: the fusion is performed by ultrasound, the ultrasound power of the ultrasound method is 15-30W, the ultrasound time is 3min (working 2s, resting 3s), and the number of ultrasounds is 1-3 times. In the embodiment of the present invention, the ultrasound power of the ultrasound method is 20W, the ultrasound time is 3min (working 2s, resting 3s), and the number of ultrasounds is 3 times as an example, but it is not limited thereto.

[0027] The ultrasound method was used to prepare stem cell membrane biomimetic cationic liposomes. This method is simple to operate and has a higher yield than the extrusion method.

[0028] The present invention also provides stem cell membrane bionic cationic liposomes prepared by the preparation method.

[0029] The present invention also provides the use of the stem cell membrane biomimetic cationic liposome in preparing a drug carrier for delivering a nucleic acid drug targeted to the brain, wherein the nucleic acid drug is used to treat brain diseases.

[0030] The nucleic acid drug is selected from small nucleic acid drugs including siRNA, miRNA, antisense oligonucleotide and pDNA.

[0031] Preferably, the brain disease is cerebral stroke (cerebral ischemia-reperfusion injury), Alzheimer's disease, Parkinson's disease, traumatic brain injury, epilepsy or brain tumor.

[0032] The present invention also provides a gene drug targeting the brain, comprising a carrier and a nucleic acid drug, wherein the carrier is the stem cell membrane biomimetic cationic liposome, and the nucleic acid drug is used for treating brain diseases.

[0033] Preferably, the nitrogen-phosphorus ratio of the gene drug is 2-10. A nitrogen-phosphorus ratio that is too small is likely to exceed the drug loading limit of the carrier, causing aggregation between carriers; a nitrogen-phosphorus ratio that is too large will reduce the drug loading capacity of the carrier. More preferably, the nitrogen-phosphorus ratio of the gene drug is 8. In the embodiment of the present invention, the nitrogen-phosphorus ratio of the gene drug is selected as 8 as an example, but it is not limited thereto.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] (1) Membrane-coated nanoparticles require that their cores must be negatively charged, otherwise they are prone to form a cross-linked network structure, making it impossible to coat the cell membrane on the surface of the nanoparticles, so it is difficult to achieve the carrying and delivery of nucleic acid drugs. The present invention realizes the biomimetic modification of positively charged materials by cell membranes, and constructs a stem cell membrane biomimetic cationic liposome with a positively charged surface by fusing and assembling stem cell membranes with cationic liposomes, thereby achieving efficient carrying and brain-targeted delivery of nucleic acid drugs.

[0036] (2) The present invention establishes a set of standardized operating methods for cell membrane separation and purification based on density gradient centrifugation, which can effectively remove the nuclear components in the cell membrane. The purified stem cell membrane obtained according to this method has low immunostimulation when used for biomimetic modification.

[0037] (3) The present invention uses stem cell membrane biomimetic cationic liposomes constructed using purified stem cell membranes, which have high brain targeting ability, low immunostimulation and high efficiency nucleic acid drug carrying capacity, can break through the blood-brain barrier, and achieve efficient targeted delivery of nucleic acid drugs to brain cells including neurons.

[0038] (4) The preparation method of the present invention is simple and can be used as a targeted delivery platform for nucleic acid drugs for brain diseases, especially targeted treatment of ischemic stroke, to solve the problems faced by nucleic acid drugs in clinical applications, such as weak targeting and difficulty in breaking through the blood-brain barrier, and has good prospects for clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The expression levels of nuclear marker protein Histone H3 and cell membrane marker protein ATP1A1 in stem cell membranes of different purities (A), and their relative expression levels (B); ** indicates p < 0.01.

[0040] Figure 2 Uptake of different carriers by Raw 264.7 macrophages (A) and the proportion of M1 macrophages (B); ** indicates p<0.01.

[0041] Figure 3 This is a transmission electron microscopy image of stem cell membrane biomimetic cationic liposomes (M-pLIPO).

[0042] Figure 4 Particle size (A) and potential (B) of cationic liposomes (pLIPO) and stem cell membrane biomimetic cationic liposomes (M-pLIPO).

[0043] Figure 5 To investigate the degree of fusion between lipid materials and stem cell membranes in stem cell membrane biomimetic cationic liposomes (M-pLIPO).

[0044] Figure 6 These are the qualitative detection results of the total protein composition on the surface of cationic liposomes (pLIPO) and stem cell membrane biomimetic cationic liposomes (M-pLIPO).

[0045] Figure 7 The figure shows the results of the determination of RNA encapsulation efficiency of stem cell membrane biomimetic neutral liposomes (M-LIPO) and stem cell membrane biomimetic cationic liposomes (M-pLIPO); where **** indicates p<0.0001.

[0046] Figure 8 This is a graph showing the results of efficient delivery of RNA to PC12 neuronal cells by stem cell membrane biomimetic cationic liposomes (M-pLIPO) in an in vitro cell experiment.

[0047] Fig. 9 Stem cell membrane-mimetic cationic liposomes carrying RNA (RNA-M-pLIPO) retain specific targeting to the hemisphere of cerebral ischemia-reperfusion injury after intravenous injection.

[0048] Fig.10 The distribution of RNA carried by stem cell membrane biomimetic cationic liposomes (RNA-M-pLIPO) and free RNA (Free RNA) in hemispheric neurons injured by cerebral ischemia-reperfusion. DETAILED DESCRIPTION

[0049] Example 1: Extraction and purification of stem cell membranes

[0050] 1) Take stem cells in the logarithmic growth phase, digest the cells, collect the cell pellet after centrifugation, add an appropriate amount of phosphate buffer to resuspend, place the obtained stem cell suspension in liquid nitrogen for rapid freezing, then quickly thaw in a 37°C water bath, repeat the freeze-thaw cycle operation 3 times to break the cells, centrifuge at 2000g for 15 minutes at 4°C, collect the pellet, and resuspend it with an appropriate amount of 0.154 mol / L sucrose solution; pre-spread 0.833 mol / L sucrose solution in a centrifuge tube, then spread the pellet suspension on the liquid surface of the centrifuge tube, centrifuge at 200000g for 45 minutes at 4°C, collect the supernatant, and retain the pellet A here; after the supernatant is diluted by half with pure water, centrifuge at 200000g for 45 minutes at 4°C, take the pellet and resuspend it with an appropriate amount of pure water to obtain component A. The precipitate A was resuspended with an appropriate amount of 0.154 mol / L sucrose solution, 0.925 mol / L sucrose solution was pre-laid in the centrifuge tube, and the precipitate suspension was spread on the liquid surface of the centrifuge tube, centrifuged at 200000g for 45 min at 4°C, the supernatant was collected, diluted once with pure water, centrifuged at 200000g for 45 min at 4°C, and the precipitate was resuspended with an appropriate amount of pure water to obtain component B. Components A and B were combined to obtain purified stem cell membranes.

[0051] 2) The expression of nuclear marker protein Histone H3 and cell membrane marker protein ATP1A1 in stem cell membranes of different purities was detected by Western blotting, and the relative expression levels of the two were calculated. The results are as follows Figure 1 As shown. Compared with the unpurified stem cell membrane, the cell nuclear components in the purified stem cell membrane obtained by two consecutive sucrose density gradient centrifugation are significantly reduced. This shows that the standardized operation method established by the present invention can effectively remove the cell nuclear components in the cell membrane extract and obtain a stem cell membrane with higher purity.

[0052] 3) Neutral liposomes were prepared by thin film dispersion method. About 5 mg of neutral phospholipid material soybean lecithin was weighed and dissolved in 20 mL of anhydrous ethanol. The phospholipid components were labeled with fluorescent dye DiI. The mixture was evaporated by rotary evaporation in a 40°C water bath under reduced pressure (vacuum degree of 1) until a thin film formed on the inner wall of the eggplant-shaped bottle. An appropriate amount of phosphate buffer was added and the neutral liposome suspension was obtained by repeated blowing. Stem cell membranes of different purities were mixed with the liposome suspension at a mass ratio of total cell membrane protein to total phospholipid of 1:12, and probe ultrasonic treatment was performed in an ice bath, and the parameters were set as follows: ultrasonic power, 20 W; ultrasonic time: 3 min (working 2 s, resting 3 s); ultrasonic number, 3 times. Liposomes without bionic modification (LIPO), bionic liposomes prepared with unpurified stem cell membranes (UM-LIPO), and bionic liposomes prepared with purified stem cell membranes (M-LIPO) were prepared.

[0053] 4) The three DiI-labeled vectors were incubated with Raw 264.7 macrophages in vitro for 3 hours. After the incubation, the cell samples were collected, and the M1 macrophages and M2 macrophages in the samples were labeled with fluorescently labeled anti-CD86 antibodies and fluorescently labeled anti-CD206 antibodies. M1 and M2 represent two different activation states of macrophages, among which M1 macrophages are involved in proinflammatory responses. The uptake of different vectors by macrophages and the proportion of M1 macrophages after uptake of different vectors were detected by flow cytometry. The results are as follows: Figure 2 As shown. Compared with UM-LIPO, the uptake of M-LIPO by macrophages increased significantly, but the proportion of M1 cells decreased significantly. The cellular uptake of the carrier and the proportion of M1 cells in the M-LIPO group were similar to those in the LIPO group. This indicates that the purified stem cell membrane used for biomimetic modification has low immunostimulation.

[0054] Example 2: Preparation of stem cell membrane biomimetic cationic liposomes

[0055] 1) Prepare cationic liposomes by thin film dispersion method. Weigh about 2 mg of cationic lipid material DOTAP and 3 mg of neutral lipid material SPC, dissolve them in 20 mL of anhydrous ethanol, and rotary evaporate in a 40°C water bath under reduced pressure conditions (vacuum degree is 1) until a thin film forms on the inner wall of the eggplant-shaped bottle. Add an appropriate amount of nuclease-free water or isotonic solution, and repeatedly blow to obtain a cationic liposome suspension. According to the mass ratio of total cell membrane protein to total lipid of 1:12, the purified stem cell membrane is mixed with the liposome suspension to obtain a biomimetic cationic liposome suspension.

[0056] 3) The cationic liposome suspension and the biomimetic cationic liposome suspension were placed in an ice bath for probe ultrasonic treatment, and the parameters were set as follows: ultrasonic power, 20W; ultrasonic time: 3min (working 2s, resting 3s); ultrasonic times, 3 times. Cationic liposomes (pLIPO) and stem cell membrane biomimetic cationic liposomes (M-pLIPO) were prepared.

[0057] Example 3: Characterization of stem cell membrane biomimetic cationic liposomes

[0058] 1) The morphology of stem cell membrane biomimetic cationic liposomes (M-pLIPO) was observed by transmission electron microscopy. Figure 3 As shown, M-pLIPO has a complete spherical vesicle-like structure.

[0059] 2) The particle size and surface potential of the prepared cationic liposomes (pLIPO) and stem cell membrane biomimetic cationic liposomes (M-pLIPO) were tested using a particle size potential meter. The test results are as follows: Figure 4 shown.

[0060] 3) Using red fluorescent dye DiD to label phospholipid components and green fluorescent dye DiO to label purified stem cell membranes, as in Example 2, fluorescently labeled stem cell membrane biomimetic cationic liposomes (M-pLIPO) were prepared. After incubating with BV2 cells for 3 h, the cells were fixed and the cell nuclei were stained with blue fluorescent dye DAPI. The fusion of phospholipid components in M-pLIPO and stem cell membranes was observed by laser scanning confocal microscopy, and fluorescence co-localization analysis was performed using ImageJ software to generate a co-localization pixel map (white pixels represent co-localization points). The results are shown in Figure 2. Figure 5 The red fluorescence signal marking the phospholipid component and the green fluorescence signal marking the stem cell membrane mostly overlap, thus presenting an obvious yellow signal. In the co-localization pixel map obtained by software analysis, there are also large-area co-localization points, indicating that the fusion degree of the liposome membrane and the stem cell membrane is high.

[0061] 4) The total protein composition of the surface of cationic liposomes (pLIPO) and stem cell membrane biomimetic cationic liposomes (M-pLIPO) was characterized by Coomassie brilliant blue staining after SDS-PAGE gel electrophoresis. Figure 6 As shown, the membrane protein components from stem cell membranes were retained on the surface of M-pLIPO.

[0062] Example 4: High drug loading capacity of stem cell membrane biomimetic cationic liposomes for RNA

[0063] 1) Prepare neutral liposomes by thin film dispersion method. Weigh about 5 mg of neutral lipid material SPC, dissolve it in 20 mL of anhydrous ethanol, and rotary evaporate in a 40°C water bath and reduced pressure conditions (vacuum degree is 1) until a thin film is formed on the inner wall of the eggplant-shaped bottle. Add an appropriate amount of nuclease-free water and repeatedly blow and beat to obtain a neutral liposome suspension. Mix the purified stem cell membrane with the liposome suspension in a mass ratio of total cell membrane protein to total lipid of 1:12 to obtain a bionic neutral liposome suspension. As in Example 2, stem cell membrane bionic neutral liposomes (M-LIPO) and stem cell membrane bionic cationic liposomes (M-pLIPO) are prepared.

[0064] 2) Dilute the FAM-labeled microRNA mimics single-stranded negative control (Gene Gene, B04004) with nuclease-free water to an appropriate concentration, slowly add it dropwise to an equal volume of M-LIPO or M-pLIPO solution at a concentration ratio of 8 for nitrogen to phosphorus, and incubate at room temperature for 30 minutes to obtain M-LIPO or M-pLIPO preparations loaded with RNA.

[0065] 3) The encapsulation efficiency of the above preparation was detected by ultrafiltration. First, an ultrafiltration tube with a molecular weight cutoff of 300 kDa was used to separate the free RNA in the preparation solution. The centrifugation conditions were: speed 4000 rpm; duration 15 min. The FAM-labeled free RNA in the filtrate was detected by a fluorescence detection microplate reader. The encapsulation efficiency calculation formula is:

[0066] Encapsulation efficiency = (total RNA amount - free RNA not carried) / total RNA amount × 100%.

[0067] The encapsulation efficiency was determined as Figure 7 As shown, the M-pLIPO constructed by the present invention has an encapsulation rate of RNA of more than 80%, and has a higher loading efficiency for RNA than M-LIPO, indicating that M-pLIPO has a high drug loading capacity for RNA.

[0068] Example 5: Stem cell membrane biomimetic cationic liposomes for efficient delivery of RNA to neuron-like cells in vitro

[0069] 1) As in Example 2, a stem cell membrane biomimetic cationic liposome (M-pLIPO) was prepared in a sorbitol isotonic solution. The Cy5-labeled microRNA mimics single-stranded negative control (Jima Gene, B02030) was diluted to an appropriate concentration using a sorbitol isotonic solution, and was slowly added dropwise to an equal volume of M-pLIPO solution at a concentration ratio of 8 nitrogen to phosphorus, and incubated at room temperature for 30 minutes to obtain an M-pLIPO preparation loaded with RNA.

[0070] 2) The RNA-loaded M-pLIPO preparation or an equal amount of free Cy5-labeled RNA was co-incubated with PC12 neuron cells in vitro for 3 h, and the final concentration of RNA in the culture system was controlled to be 50 nM. After the co-incubation, the cells were fixed, and the cell nuclei were stained with the blue fluorescent dye DAPI. The RNA uptake by PC12 neuron cells was observed by laser scanning confocal microscopy. The results were as follows: Figure 8 As shown. Among them, Cy5-labeled RNA is shown as a green signal in the figure. The M-pLIPO constructed using the present invention can effectively improve the delivery of RNA to cells.

[0071] Example 6: Use of stem cell membrane biomimetic cationic liposomes to deliver RNA to neuronal cells in the hemisphere of cerebral ischemia-reperfusion injury

[0072] 1) Establishment of mouse middle cerebral artery occlusion / reperfusion model: 18-20g male C57BL / 6 mice were selected for surgery. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution, the common carotid artery was ligated, and the thread plug was inserted and pushed to about 8-10mm into the internal carotid artery to block the blood flow of the middle carotid artery. The wound was sutured and the mice were placed in an incubator at 37℃ to maintain body temperature. After 1h of ischemia, the thread plug was pulled out to restore collateral circulation blood supply and achieve blood reperfusion. After the mice regained consciousness, they were placed back in the breeding environment.

[0073] 2) Using fluorescent dye DiI to label phospholipid components, as in Example 5, DiI-labeled M-pLIPO preparations and DiI-labeled M-pLIPO preparations carrying Cy5-labeled RNA (RNA-M-pLIPO) were prepared. When the model mice were reperfused for 6 hours, DiI-labeled M-pLIPO and RNA-M-pLIPO were injected through the tail vein, respectively. At 8 hours after intravenous injection, the brains of the model mice were taken and the distribution of the DiI-labeled vector in the brain was observed using a small animal in vivo imaging system. The results are shown in FIG. Fig. 9 Both M-pLIPO and RNA-M-pLIPO can be distributed more in the hemisphere of cerebral ischemia-reperfusion injury and accumulate at the injury site, indicating that M-pLIPO carrying RNA retains its specific inflammatory targeting ability.

[0074] 3) When the model mice were reperfused for 6 hours, the above RNA-M-pLIPO preparation or an equal amount of free Cy5-labeled RNA (Free RNA) was injected through the tail vein, and at 8 hours after intravenous injection, the brain of the model mice was taken, fixed, dehydrated, and frozen and embedded, and further prepared into coronal sections for convenient observation of the cortex and hippocampus, and the frozen sections were immunofluorescently stained. Among them, the primary antibody was NeuN rabbit antibody, which was used to mark neurons; the secondary antibody was Alexa Fluor 488-labeled donkey anti-rabbit IgG (Yisheng Biological, 34206ES60). Finally, the cell nucleus was stained with DAPI, and the sections were scanned by the virtual digital slice scanning system.

[0075] 4) ImageJ software was used to perform fluorescence co-localization analysis on the neuronal signals (shown in green in the figure) and RNA signals (shown in red in the figure) in the infarcted hemi-lesion area of ​​the brain slices of the RNA-M-pLIPO group or the Free RNA group. The results are shown in Figure 4. Fig.10 As shown. Compared with the FreeRNA group, the RNA carried on the M-pLIPO in the RNA-M-pLIPO group had obvious co-localization points with the neurons in the damaged area. This indicates that the M-pLIPO constructed by the present invention can deliver RNA to the hemisphere of cerebral ischemia-reperfusion injury and increase the uptake of RNA by neurons in the damaged area.

Claims

1. A method for preparing brain-targeted stem cell membrane biomimetic cationic liposomes, characterized in that: The following steps are involved: (1) Extracting and purifying to obtain stem cell membrane; (2) preparing cationic liposomes; (3) fusing the stem cell membrane obtained in step (1) with the cationic liposome obtained in step (2) to obtain a stem cell membrane biomimetic cationic liposome, The relative amounts of stem cell membrane and cationic liposome are as follows: the mass ratio of total protein amount of stem cell membrane to total lipid amount of cationic liposome is 1:2-24.

2. The method for preparing stem cell membrane biomimetic cationic liposomes according to claim 1, characterized in that: The stem cell membrane is selected from at least one of umbilical cord-derived mesenchymal stem cell membranes, placenta-derived mesenchymal stem cell membranes, bone marrow-derived mesenchymal stem cell membranes, neural stem cell membranes and induced pluripotent stem cell membranes.

3. The method for preparing stem cell membrane biomimetic cationic liposomes according to claim 1, characterized in that: The cationic liposomes are prepared by mixing cationic lipids and neutral lipids, wherein the mass ratio of the cationic lipids to the neutral lipids is 1:1-5; The cationic lipid is selected from at least one of DOTAP, DOTMA, DOSPA, DDAB, DOGS and DC-Chol; The neutral lipid is selected from at least one of SPC, DOPE, DSPC, DPPC, DMPC, DSPE, DOPC and Chol.

4. The method for preparing stem cell membrane biomimetic cationic liposomes according to claim 1, characterized in that: In step (1), the stem cell membrane is first extracted by repeated freezing and thawing, and then the stem cell membrane is purified by density gradient centrifugation; In step (2), the cationic liposomes are prepared by a thin film dispersion method.

5. The method for preparing stem cell membrane biomimetic cationic liposomes according to claim 1, characterized in that: In step (3), when the stem cell membrane is fused with the cationic liposome, the fusion is carried out in nuclease-free water or isotonic solution; The isotonic solution is selected from at least one of a sorbitol isotonic solution, a glucose isotonic solution or a mannitol isotonic solution.

6. Stem cell membrane biomimetic cationic liposomes prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the stem cell membrane biomimetic cationic liposome according to claim 6 in the preparation of a drug carrier for targeted brain delivery of a nucleic acid drug, wherein the nucleic acid drug is used to treat brain diseases.

8. The use according to claim 7, characterized in that: The brain disease is stroke, Alzheimer's disease, Parkinson's disease, traumatic brain injury, epilepsy or brain tumor.

9. A gene drug targeting the brain, characterized in that: The invention comprises a carrier and a nucleic acid drug, wherein the carrier is the stem cell membrane biomimetic cationic liposome as claimed in claim 6, and the nucleic acid drug is used for treating brain diseases.

10. The gene drug targeting the brain according to claim 9, characterized in that: The nitrogen-to-phosphorus ratio of the gene medicine is 2-10.

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