Recombinant lipid nanoparticle as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a recombinant lipid nanoparticle for treating cerebral arterial thrombosis as well as a preparation method and application of the recombinant lipid nanoparticle. Celery seeds (CS) are used as a raw material, lipid is extracted by adopting an organic solvent extraction method, and then the celery seed recombinant lipid nanoparticles (CS-rLNPs) are successfully prepared by adopting an ethanol injection method. The extraction process disclosed by the invention is simple, the raw materials are easy to obtain, and the prepared nano-drug CS-rLNPs has the advantages of high biological safety, high stability and the like; in-vivo and in-vitro experiments show that CS-rLNPs can effectively penetrate through a blood brain barrier, is preferentially distributed to an ischemic semi-dark region, can be combined with neurons in a cerebral ischemia region, and shows unique neuronal targeting; in addition, the nanoparticles have practical value in treatment of cerebral arterial thrombosis. Therefore, the recombinant lipid nanoparticles provided by the invention have good clinical application value and prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a recombinant lipid nanoparticle for treating ischemic stroke, and a preparation method and application thereof. Background Art

[0002] Ischemic stroke (IS) is caused by cerebral vasoconstriction or occlusion and is one of the leading determinants of mortality worldwide. The incidence of IS is increasing due to the aging of the global population. The current gold standard for the treatment of IS is the rapid recanalization of the occluded artery, which usually requires the help of neuroprotective agents to achieve better therapeutic effects. Therefore, the development of effective neuroprotective agents and their effective delivery are of great significance for the better management of IS.

[0003] The blood-brain barrier (BBB) ​​is a semi-permeable biological barrier between brain tissue and plasma. The effectiveness of many drugs is often limited because they cannot cross the BBB and thus cannot reach the ischemic penumbra, which prevents the drugs from reaching therapeutic concentrations and affects the effective treatment of IS. In order to improve the therapeutic effect of drugs related to IS, nanomedicines have emerged. Previous studies usually use targeting ligands or coatings to modify nanoparticles so that they can specifically bind to neurons, but this usually requires a complex preparation process, is prone to poor reproducibility, and neuronal targeting (the main therapeutic target of neuroprotectants) is often overlooked. Therefore, it is still a considerable challenge to develop effective neuroprotectants that can simultaneously overcome the BBB barrier and achieve neuron-targeted delivery.

[0004] Lipid nanoparticles (LNPs) offer a promising platform for delivering neuroprotectants due to their good biocompatibility, degradability, and targeted delivery potential. They are easy to prepare and modify, making them a promising approach for delivering neuroprotectants. In addition, the lipid composition can be customized as needed, and the biophysical properties of LNPs help to expand the potential of nanomedicine in neuron targeting. The reorganization of LNPs is expected to lead to changes in the field of drug delivery, enhance the ability to penetrate the BBB, and achieve precise neuron targeting.

[0005] Seed oils are rich in bioactive components, especially lipids, which have neuroprotective properties and can reduce oxidative damage and inflammation associated with IS. Therefore, lipids derived from seeds may be potential neuroprotectants for IS. Reinfusing these lipids into lipid nanoparticles (LNPs) has a dual advantage: fully utilizing natural neuroprotectants while applying all-in-one nanomedicine-based therapeutic LNPs, which may help overcome the blood-brain barrier and precisely target neurons. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a recombinant lipid nanoparticle and a preparation method and application thereof in view of the deficiencies in the prior art.

[0007] In order to solve the above technical problems, the present invention discloses a recombinant lipid nanoparticle and its preparation method and application. The specific technical scheme is as follows:

[0008] A method for preparing recombinant lipid nanoparticles, characterized in that it comprises the following steps:

[0009] (1) extracting celery seed lipids from celery seeds by an organic solvent extraction method, wherein the celery seed lipids include triglycerides, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, diglycerol, phosphatidylinositol, monoglycerol, ceramide and (O-acyl)-1-hydroxy fatty acids;

[0010] (2) The celery seed lipids extracted in step (1) are injected into the celery seed lipids by an ethanol method to prepare recombinant lipid nanoparticles.

[0011] Wherein, in step (1), the organic solvent extraction method is specifically as follows: celery seeds are mixed with methanol and then methyl tert-butyl ether (MTBE) is added to mix, followed by adding water and standing to separate into two phases, collecting the supernatant by centrifugation, adding dichloromethane, centrifuging and drying to obtain the product. Preferably, the volume ratio of the added water to the volume of methanol is 7:12, the standing time is 10 min, and the centrifugation conditions involved are all 14000 rpm, 4°C, and 10 min.

[0012] The methanol is a methanol aqueous solution with a concentration of 75% v / v; the amount of the methanol is calculated by mixing 2 to 6 mL of methanol per gram of celery seeds; preferably, 4 mL of methanol is mixed per gram of celery seeds.

[0013] The dosage of the methyl tert-butyl ether is calculated by mixing 1 to 20 mL of methyl tert-butyl ether per gram of celery seeds. Preferably, 4 mL of methyl tert-butyl ether is mixed per gram of celery seeds.

[0014] The amount of dichloromethane used is calculated as 0.1-2 mL of dichloromethane per gram of celery seeds. Preferably, 0.5-0.7 mL of dichloromethane is used per gram of celery seeds.

[0015] Wherein, the mixing temperature is 4-30°C and the mixing time is 30-120 minutes. Preferably, the mixing temperature is 4°C, the mixing time with methanol is 30 minutes, and the mixing time after adding methyl tert-butyl ether is 120 minutes.

[0016] Wherein, in step (1), the celery seed lipid includes lipids with the following mass contents: 15-20% triglycerides, 10-12% phosphatidylcholine, 5-9% phosphatidylglycerol, 5-9% phosphatidylethanolamine, 5-9% phosphatidylserine, 3-6% diglycerol, 3-6% phosphatidylinositol, 3-6% monoglyceride, 3-6% ceramide and 1-3% (O-acyl)-1-hydroxy fatty acid. Preferably, the triglycerides are 16-17%, the phosphatidylcholine is 10-12%, the phosphatidylglycerol is 7-9%, the phosphatidylethanolamine is 7-8%, the phosphatidylserine is 6-7%, the diglycerols are 5-6%, the phosphatidylinositol is 4-4.5%, the monoglycerols are 3-5%, the ceramides are 3-4%, and the (O-acyl)-1-hydroxy fatty acids are 2-3%; further preferably, the triglycerides are 16.6%, the phosphatidylcholine is 11%, the phosphatidylglycerol is 8.2%, the phosphatidylethanolamine is 7.2%, the phosphatidylserine is 6.7%, the diglycerols are 5.3%, the phosphatidylinositol is 4.1%, the monoglycerols are 4%, the ceramides are 3.5%, the (O-acyl)-1-hydroxy fatty acids are 2.9%, and other lipids are 30.5%.

[0017] Wherein, in step (2), the ethanol injection method is specifically as follows: the lipid prepared in step (1) is mixed with ethanol, filtered to obtain a lipid ethanol solution, and then quickly injected into water while stirring, and dialyzed. Wherein, 30 to 50 mg of the celery seed lipid is mixed in each mL of ethanol; the pore size of the filter membrane used in the filtration is 0.22 μm; the stirring speed is 1000 to 2000 rpm; the volume ratio of the lipid ethanol solution to water is 1:15 to 20; the molecular weight cutoff of the dialysis membrane is 3000 Da. Preferably, 40 mg of the lipid is mixed per mL of ethanol; the volume ratio of the lipid ethanol solution to purified water is 1:19.

[0018] Preferably, the preparation method comprises the following steps:

[0019] (1) 3 g of celery seeds were crushed, soaked in 12 mL of 75% v / v methanol aqueous solution, stirred in an ice bath for 30 min, 30 mL of methyl tert-butyl ether (MTBE) was added, and stirring was continued in an ice bath for 120 min; 7 mL of ultrapure water was added, and the mixture was allowed to stand for 10 min to separate the phases; the supernatant was collected by centrifugation, and a crude product was obtained by rotary evaporation; the crude product was redispersed with 2 mL of dichloromethane; centrifuged, and the lower layer was collected; the lower layer was dried by nitrogen blowing, and the extracted lipids were stored at -80°C for later use.

[0020] (2) Dissolve 40 mg of the lipid obtained in step (1) in 1 mL of ethanol, filter with a 0.22 μm filter membrane to remove insoluble impurities, inject the lipid ethanol solution into purified water under vigorous stirring, and dialyze (MWCO: 3000Da) to remove ethanol to obtain the product.

[0021] In a second aspect, the present invention provides recombinant lipid nanoparticles prepared by the preparation method described in the first aspect.

[0022] In a third aspect, the present invention provides use of the recombinant lipid nanoparticles described in the second aspect in crossing the blood-brain barrier or targeting neurons.

[0023] In a fourth aspect, the present invention provides use of the recombinant lipid nanoparticles described in the second aspect in the preparation of a drug for treating ischemic stroke.

[0024] Beneficial effects:

[0025] The present invention successfully extracts celery seed lipids by organic solvent extraction, and prepares stable celery seed recombinant lipid nanoparticles (CS-rLNPs) by ethanol injection. The CS-rLNPs prepared by the present invention have good biosafety and stability, and have unique neuron targeting, and have practical value for the treatment of ischemic stroke, and have good clinical application value and prospects. Specifically, it has the following beneficial effects:

[0026] (1) The extraction process disclosed in the present invention is simple, the raw materials are easily available, and the prepared nanomedicine CS-rLNPs has the advantages of biosafety and good stability;

[0027] (2) The recombinant lipid nanoparticles (CS-rLNPs) prepared by the present invention can effectively penetrate the blood-brain barrier and preferentially distribute to the ischemic penumbra, and can bind to neurons in the ischemic area of ​​the brain, showing unique neuron targeting;

[0028] (3) The recombinant lipid nanoparticles (CS-rLNPs) prepared by the present invention have practical value in the treatment of ischemic stroke and have good clinical application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0030] Figure 1 is the sample solution diagram of CS-rLNPs;

[0031] Figure 2 The particle size distribution and Zeta potential distribution of CS-rLNPs;

[0032] Figure 3 is the transmission electron microscopy image of CS-rLNPs;

[0033] Figure 4 is the stability diagram of CS-rLNPs in water;

[0034] Figure 5 is the stability diagram of CS-rLNPs in PBS;

[0035] Figure 6 The figure is the result of in vitro cytotoxicity experiment of CS-rLNPs;

[0036] Figure 7 This is the imaging picture of labeled CS-rLNPs in the mouse brain;

[0037] Figure 8 This is the fluorescence imaging distribution diagram of labeled CS-rLNPs in the mouse brain;

[0038] Fig. 9 This is the colocalization diagram of CS-rLNPs and cortical neurons;

[0039] Fig.10 Representative images of TTC-stained brain sections and quantification of cerebral infarction volume. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to the examples, but the present invention is not limited to these examples. In the following examples, the experimental methods described are all commonly used methods in the art unless otherwise specified, and the experimental materials and reagents described are all conventional materials and reagents in the art unless otherwise specified.

[0041] Example 1 Extraction of lipids

[0042] The celery seed lipid extraction method of the present invention comprises the following steps:

[0043] (1) 3 g of celery seeds were crushed and soaked in 12 mL of 75% v / v methanol aqueous solution. The mixture was stirred in an ice bath for 30 min. 30 mL of methyl tert-butyl ether (MTBE) was added and the mixture was stirred in an ice bath for 120 min.

[0044] (2) Add 7 mL of ultrapure water and let stand for 10 min to allow phase separation; centrifuge at 14,000 rpm and 4°C for 10 min, collect the supernatant, and rotary evaporate to obtain a crude product.

[0045] (3) Re-dispersing the crude product of step (2) with 2 mL of dichloromethane; centrifuging at 14,000 rpm and 4° C. for 10 min, collecting the lower layer; drying the lower layer with nitrogen blown, and storing the extracted celery seed lipid at -80° C. for later use.

[0046] The specific components of the extracted celery seed lipids were detected by liquid chromatography-mass spectrometry. The specific detection method is as follows:

[0047] Chromatographic conditions:

[0048] Column temperature: 55°C; mobile phase A: acetonitrile: water = (60:40, v / v), containing 0.1% formic acid (FA) and 10 mM NH4COOH; mobile phase B: acetonitrile: isopropanol = (10:90, v / v), containing 0.1% FA and 10 mM NH4COOH; flow rate: 0.35 mL / min; injection volume: 2 μL.

[0049] Mass spectrometry conditions:

[0050] The mass spectrometry system used was the Qtrap6500 plus mass spectrometry detection system from AB Sciex, USA, equipped with an electrospray (ESI) ion source and an Analyst 1.7 workstation. The optimized mass spectrometry analysis conditions were as follows: curtain gas 40psi; ion spray voltage -4500 / 5500V; source temperature 400℃; nebulizer gas 50psi; auxiliary heating gas: 55psi, and the schedule-MRM mode was used to perform high-throughput analysis of more than 1,000 lipid substances.

[0051] The test results showed that the proportions of each lipid in the prepared celery seed lipids were as follows: the mass proportion of triglycerides was 16.6%, the mass proportion of phosphatidylcholine was 11%, the mass proportion of phosphatidylglycerol was 8.2%, the mass proportion of phosphatidylethanolamine was 7.2%, the mass proportion of phosphatidylserine was 6.7%, the mass proportion of diglycerol was 5.3%, the mass proportion of phosphatidylinositol was 4.1%, the mass proportion of monoglycerol was 4%, the mass proportion of ceramide was 3.5%, the mass proportion of (O-acyl)-1-hydroxy fatty acids was 2.9%, and the mass proportion of other lipids was 30.5%.

[0052] Example 2 Lipid Extraction Method Optimization

[0053] This example optimizes the conditions involved in the celery seed lipid extraction method, such as the solid-liquid ratio, stirring time, centrifugation conditions, and dichloromethane dosage, as follows:

[0054] 1. Optimization of material-liquid ratio

[0055] The solid-liquid ratio optimization experiment includes the solid-liquid ratio of celery seeds and 75% v / v methanol aqueous solution in step (1) of the extraction method described in Example 1, and the solid-liquid ratio of celery seeds and methyl tert-butyl ether. The extraction method is similar to Example 1, except that the solid-liquid ratios involved are different. The extraction results obtained with different solid-liquid ratios are shown in Tables 1 and 2. The solid-liquid ratios of celery seeds and 75% v / v methanol aqueous solution are optimized in Table 1, which are 1:2, 1:3, 1:4, 1:5 and 1:6 (w / vg / mL), respectively. By fixing the addition amount of celery seeds at 3g, the addition volume of 75% v / v methanol aqueous solution is changed to achieve different addition amounts of solid-liquid ratios. The solid-liquid ratios of celery seeds and methyl tert-butyl ether are optimized in Table 2, which are 1:1, 1:5, 1:10, 1:15 and 1:20 (w / vg / mL), respectively. The addition amount of celery seeds is fixed at 3 g and the addition volume of methyl tert-butyl ether is changed to achieve different solid-liquid ratios.

[0056] Table 1 Effect of the solid-liquid ratio of celery seeds and methanol on lipid extraction rate

[0057]

[0058] Table 2 Effect of the solid-liquid ratio of celery seeds and methyl tert-butyl ether on lipid extraction rate

[0059]

[0060] As can be seen from Tables 1 and 2, changes in the solid-liquid ratio may affect the interaction between the sample and the solvent, and thus affect the extraction rate of lipids. In the experiment to optimize the ratio of celery seeds to methanol, when the solid-liquid ratio is ≤1:4 (including 1:4, 1:5, 1:6), it is conducive to the effective dissolution and extraction of lipids, so that the lipid extraction amount reaches a high level. When the solid-liquid ratio is greater than 1:4 (including 1:2, 1:3), the plant material may not be completely covered, and some plant components cannot be fully dissolved and extracted, resulting in an unsatisfactory extraction rate (see Table 1 for specific data).

[0061] In the experiment of celery seeds and methyl tert-butyl ether, the ratio of 1:10 and 1:20 can provide more extraction solvent, which is conducive to the efficient extraction of lipids. High solid-liquid ratios (including 1:1 and 1:5) lead to solution saturation, which limits the lipid extraction rate (see Table 2 for specific data). In summary, the solid-liquid ratio of celery seeds to 75% v / v methanol aqueous solution is selected as 1:4, and the solid-liquid ratio of celery seeds to methyl tert-butyl ether is 1:10, that is, 3g celery seeds, 12mL 75% v / v methanol aqueous solution, and 30mL methyl tert-butyl ether.

[0062] 2. Optimization of mixing time

[0063] The ratio of the first stirring time of celery seeds in 75% v / v methanol aqueous solution and the second stirring time after adding methyl tert-butyl ether in step (1) of the extraction method described in Example 1 was optimized. The extraction method is similar to that of Example 1, except that the time ratio is different. By fixing the first stirring time to 30 minutes, the ratio of the first stirring time to the second stirring time is optimized (including 1:1, 1:2, 1:3, 1:4 and 1:5). The results are shown in Table 3.

[0064] Table 3 Effect of first stirring time: second stirring time on lipid extraction rate

[0065]

[0066] As can be seen from Table 3, increasing the second stirring time helps to increase the contact time between the plant sample and the solvent and accelerate the dissolution and migration of lipids. As the stirring time increases, the lipid content increases. When the time ratio is 1:4, the lipid extraction amount reaches a peak, and further extension of the time does not significantly increase, so the preferred time ratio is 1:4, specifically, the first stirring time is 30 minutes, and the second stirring time is 120 minutes.

[0067] 3. Optimization of centrifugation conditions

[0068] The extraction method is similar to the method described in Example 1, except that the centrifugation conditions in steps (2) and (3) are different, and the centrifugation conditions in steps (2) and (3) are optimized simultaneously. The results are shown in Table 4.

[0069] Table 4 Effect of different centrifugation conditions on lipid extraction rate

[0070]

[0071] It can be seen from Table 4 that for different centrifugation conditions, high-speed centrifugation is helpful for effectively precipitating solid particles and separating the solution, and a higher extraction rate can be achieved when the time is more than 10 min, while temperature has no significant effect on the extraction amount. In order to prevent lipid oxidation, low temperature and relatively short centrifugation time are selected, i.e., 14000 rpm, 4°C, 10 min.

[0072] 4. Optimization of dichloromethane dosage

[0073] The extraction method is similar to that described in Example 1, except that the amount of dichloromethane used in step (3) is different. The results are shown in Table 5.

[0074] Table 5 Effect of dichloromethane dosage on lipid extraction efficiency

[0075]

[0076] As can be seen from Table 5, increasing the amount of dichloromethane helps to fully extract lipids from plant materials. The amount of dichloromethane is positively correlated with the amount of lipid extraction, and saturation is reached after 2 mL. In order to save solvent, 2 mL was finally selected. Example 3 Preparation of recombinant lipid nanoparticles CS-rLNPs

[0077] 40 mg of the celery seed lipid prepared in Example 1 was dissolved in 1 mL of ethanol and filtered with a 0.22 μm filter membrane to remove insoluble impurities. Under vigorous stirring at 1500 rpm, the lipid ethanol solution was rapidly injected into purified water at a volume ratio of 1:19, and dialyzed (MWCO: 3000 Da) to remove ethanol.

[0078] After dialysis, the sample was concentrated by rotary evaporation, and the final concentration of the prepared CS-rLNPs was 2 mg / mL. Figure 1 The sample picture shows that the prepared CS-rLNPs have good dispersion, no precipitation and precipitation, and are opalescent, proving that the recombinant lipid nanoparticle CS-rLNPs solution is successfully prepared.

[0079] Example 4 Characterization of CS-rLNPs

[0080] (1) Determination of hydrated particle size and zeta potential of CS-rLNPs

[0081] Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) were used to measure the particle size and surface charge of the CS-rLNPs nanoparticles prepared in Example 3. Figure 2 The results showed that the prepared CS-rLNPs had good dispersibility, with an average hydrated particle size of 58.8 nm, a polydispersity index of 0.134, and a Zeta potential of -11.57 mV, also showing a uniform distribution.

[0082] (2) Transmission electron microscopy experiment of CS-rLNPs

[0083] The diluted sample was dropped onto a carbon-supported copper mesh, and the mesh was placed in an electron drying oven to dry overnight to prepare a transmission electron microscope (TEM) sample. Figure 3 As shown (scale bar is 100 nm), CS-rLNPs are spherical nanoparticles, and their actual diameters are similar to the diameter range determined by DLS.

[0084] (3) Stability experiment of CS-rLNPs

[0085] The prepared CS-rLNPs were dispersed in water or PBS (0.01 M, pH = 7.4), and their stability was evaluated by monitoring the changes in particle size and polydispersity (PDI) of CS-rLNPs in different media for one week. Figure 4As shown, CS-rLNPs showed good water stability over 7 days. Figure 5 The results showed that CS-rLNPs also had good particle size stability in PBS, indicating that CS-rLNPs were stable under physiological conditions.

[0086] Example 5 Biocompatibility experiment of CS-rLNPs

[0087] In this experiment, the MTT method was used to investigate the cytotoxicity of CS-rLNPs prepared in Example 3 on mouse brain endothelial cells (bEnd.3) and human normal liver cells (L02). First, 3×10 3 The cells were cultured to adhere to the wall and the prepared CS-rLNPs (diluted with serum-free medium) were added to make the final concentration in the 96-well plate 20, 50, 100, 150, and 200 μg / mL. The cells were incubated for 24 h and then the cell viability was determined by MTT method. Figure 6 As shown, the cell viability of both cell lines remained above 89% at concentrations up to 200 μg / mL for 24 h, indicating that the cytotoxicity of CS-rLNPs was low.

[0088] Example 6 CS-rLNPs can effectively penetrate the blood-brain barrier and preferentially distribute to the ischemic penumbra

[0089] The mice used in this embodiment are male C57BL / 6 mice, 20-22g, 11-12 weeks old. The establishment method of cerebral ischemia reperfusion model mice (MCAO / R) is as follows: MCAO is induced by silicon sutures, and mice are anesthetized at the same time. After MCAO1h, the sutures are removed for reperfusion (R). The sham-operated mice undergo the same operation, but the artery is not ligated. During the operation, the respiratory rate and state of the mice are carefully tracked and observed, and the infarct area is confirmed by magnetic resonance imaging (MRI) scanning. 24h after surgery, CS-rLNPs (injection volume of 100 μL, concentration of 2mg / mL) labeled by IR780 are injected into the tail vein to give the model mice (MCAO / R). 48h after injection, the mice are killed, their brains are collected, and ex vivo imaging is performed by an in vivo imaging system. DiI-labeled CS-rLNPs (injection volume of 100 μL, concentration of 2 mg / mL) were injected into the tail vein of model mice (MCAO / R), and their brains were removed and frozen at -80°C. Coronal sections of the brain were prepared using a cryostat microtome, and wide-angle images of brain sections were captured using a THUNDER microscopy system.

[0090] IR780 signal imaging in the isolated brain 48 hours after injection Figure 7As shown in Figure 2, after a single intravenous injection, IR780-labeled CS-rLNPs showed significant brain accumulation and showed that CS-rLNPs were better distributed in the brain than free dye IR780. At the same time, the distribution of DiI signals was further monitored by fluorescence imaging, and the DiI signals showed the location of CS-rLNPs in the brain. Figure 8 As shown, compared with the free dye DiL ( Figure 8 Left), CS-rLNPs( Figure 8 Right) Efficiently crosses the BBB with the ability to selectively distribute to the ischemic penumbra.

[0091] Example 7 Neuron targeting ability of CS-rLNPs

[0092] The colocalization method was used to further study the selective accumulation of CS-rLNPs in the cerebral ischemic area. Similarly, DiO-labeled CS-rLNPs (injection volume of 100 μL, concentration of 2 mg / mL) were injected into the model mice (MCAO / R) through the tail vein, and the mouse brain tissue was collected for cryomicrosectioning. Immunofluorescence (IF) staining NeuN antibody was used to label cortical neuronal cells. First, brain tissue sections were embedded in paraffin, dewaxed with xylene, and hydrated with gradient ethanol. Afterwards, the sections were mounted on slides. Immunofluorescence of NeuN was stained with NeuN primary anti-polyclonal antibody (Proteintech, 26975-1-AP), and then stained with AlexaFluor 555-labeled secondary antibody (Invitrogen, A-31572). After rinsing the slides in DAPI solution, NeuN-positive cells were imaged using a fluorescence microscope.

[0093] Cortical neurons were stained with corresponding antibodies to study the co-localization of DiO-labeled CS-rLNPs and cortical neurons. Fig. 9 The results showed that CS-rLNPs have an innate neuron targeting ability. Unlike previous studies that used specific targeting ligands or coatings to increase neuron affinity, the CS-rLNPs in this study showed an innate targeting preference for neurons without the need for additional modification.

[0094] Example 8 The therapeutic effect of CS-rLNPs on ischemic stroke

[0095] In order to further reveal the therapeutic effect of CS-rLNPs, the protective effect of CS-rLNPs on brain injury was observed 7 days after MCAO / R. The model mice were randomly divided into sham operation group, MCAO / R group and MCAO / R+CS-rLNPs group. 2h after MCAO / R, CS-rLNPs (injection volume of 100μL, concentration of 2mg / mL) were injected into the tail vein of mice, and the injection was repeated every 2 days. 7d after MCAO / R, the mouse brains were quickly collected and frozen at -80℃. After slicing, 2,3,5-triphenyltetrazolium (TTC) with a concentration of 1% (m / v, g / mL) was taken and immersed at 37℃ for 20min for TTC staining.

[0096] The results are as follows Fig.10 As shown, MCAO / R aggravated brain injury, manifested by a significant increase in cerebral infarction area. Compared with the MCAO / R group, the CS-rLNPs group significantly reduced the infarction area, that is, CS-rLNPs alleviated the damage caused by MCAO / R, indicating that celery seed lipids have the potential to treat ischemic stroke.

[0097] The present invention provides a recombinant lipid nanoparticle and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing recombinant lipid nanoparticles, characterized in that: The steps include: (1) extracting celery seed lipids from celery seeds by an organic solvent extraction method, wherein the celery seed lipids include triglycerides, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, diglycerol, phosphatidylinositol, monoglycerol, ceramide and (O-acyl)-1-hydroxy fatty acids; (2) The celery seed lipids extracted in step (1) are injected into the celery seed lipids by an ethanol method to prepare recombinant lipid nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step (1), the organic solvent extraction method is specifically as follows: celery seeds are mixed with methanol and then methyl tert-butyl ether is added to mix, followed by adding water and standing to separate into two phases, centrifuging to collect the supernatant, adding dichloromethane, centrifuging and drying to obtain the celery seed lipid.

3. The preparation method according to claim 2, characterized in that: The methanol is a methanol aqueous solution with a concentration of 75% v / v; the amount of the methanol is calculated by mixing 2 to 6 mL of methanol per gram of celery seeds; The dosage of the methyl tert-butyl ether is calculated by mixing 1 to 20 mL of methyl tert-butyl ether per gram of celery seeds; The amount of dichloromethane used is calculated based on 0.1 to 2 mL of dichloromethane per gram of celery seeds.

4. The preparation method according to claim 2, characterized in that: The mixing temperature is 4-30° C. and the mixing time is 30-120 min.

5. The preparation method according to claim 1, characterized in that: In step (1), the celery seed lipid comprises the following lipids by mass content: Triglycerides 15-20%, phosphatidylcholine 10-12%, phosphatidylglycerol 5-9%, phosphatidylethanolamine 5-9%, phosphatidylserine 5-9%, diglycerol 3-6%, phosphatidylinositol 3-6%, monoglycerol 3-6%, ceramide 3-6% and (O-acyl)-1-hydroxy fatty acid 1-3%.

6. The preparation method according to claim 1, characterized in that: In step (2), the ethanol injection method is specifically as follows: the celery seed lipid prepared in step (1) is mixed with ethanol, filtered to obtain a lipid ethanol solution, and injected into water while stirring, dialyzed, and recombinant lipid nanoparticles are obtained.

7. The preparation method according to claim 6, characterized in that: Mix 30-50 mg of the celery seed lipid in each mL of ethanol; The filtration membrane has a pore size of 0.22 μm; The stirring speed is 1000-2000 rpm; The volume ratio of the lipid ethanol solution to water is 1:15-20; The molecular weight cut-off of the dialysis membrane is 3000Da.

8. The recombinant lipid nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the recombinant lipid nanoparticles according to claim 8 in crossing the blood-brain barrier or targeting neurons.

10. Use of the recombinant lipid nanoparticles according to claim 8 in preparing a drug for treating ischemic stroke.

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