AMD3100 nucleus-immobilizing lipid nanoparticle and application thereof

Abstract

The invention discloses an AMD3100 nucleus-immobilizing lipid nanoparticle and an application of the AMD3100 nucleus-immobilizing lipid nanoparticle. The invention provides a nucleus-immobilizing lipid nanoparticle. The nucleus-immobilizing lipid nanoparticle is prepared from a DOPA (Dioctyl-Polyamide) modified calcium phosphate core, DSPE-PEG2000-AMD3100, DSPE-PEG2000, cholesterol and DOTAP (Dioctyl-Polyamide), the structure of the DSPE-PEG2000-AMD3100 is as shown in a formula I which is described in the specification; in the DOPA modified calcium phosphate core, the DOPA is loaded on the surface of the calcium phosphate core. According to the invention, AMD3100 is used as a target head of the drug delivery carrier to be modified on the solid nucleus lipid nanoparticle carrier, so that a large amount of therapeutic drugs can be delivered to damaged cells and newborn cells in an ischemic area, targeted drug delivery in the ischemic brain area is realized, and the drug treatment effect is improved. # imgabs0 #

Description

[0001] This application claims the priority of Chinese Patent Application No. 2024101284220 with an application date of January 30, 2024, and Chinese Patent Application No. 2024101284216 with an application date of January 30, 2024. This application incorporates the entire texts of the above-mentioned Chinese patent applications by reference. Technical Field

[0002] The present invention relates to an AMD3100 solid-core lipid nanoparticle and its application. Background Art

[0003] Stroke is the leading cause of disease-related death in China, and approximately 70% of which are ischemic strokes (IS). The most effective treatment for IS is timely thrombolysis within 3 - 4.5 hours. However, due to the strict limitation of the thrombolysis time window, the thrombolysis rate in China is less than 20%. In addition to the high incidence, IS is characterized by high disability rate, high fatality rate, and high recurrence rate. In China, about 30% of the patients die, and 70% of the survivors have severe disabilities such as hemiplegia and aphasia. Even in the United States, where the incidence has been decreasing year by year in the past decade, the disability and fatality rates have been on the rise. Therefore, the treatment of IS has become a major medical problem worldwide.

[0004] According to the pathological characteristics of IS, its course can be roughly divided into two stages: one is the ischemic stage, where cerebral blood flow supply is severely insufficient, and the brain tissue is in a "no blood flow" state, making it difficult for drugs to enter the "no blood flow" brain tissue; then comes the reperfusion stage and the recovery stage, where blood flow recovers to a certain extent, and the permeability of the blood-brain barrier (BBB) increases. However, during reperfusion, a large number of white blood cells in the blood accumulate in the ischemic area and adhere to the vascular endothelial cells, blocking the cerebral microvessels, resulting in microvascular occlusion and the inability of blood flow to continue to recover, triggering the "no-reflow" phenomenon, and drugs still cannot enter the "no-reflow" brain tissue. Therefore, the pathological characteristics of "no blood flow" and "no reflow" have become the biggest barriers to drug delivery in the brain for IS. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the bottleneck problem of difficult drug delivery in the brain during the onset of IS. The present invention provides an AMD3100 solid-core lipid nanoparticle and its application. The present invention modifies the targeting head of AMD3100 as a drug delivery carrier on the solid-core lipid nanoparticle carrier, which helps to deliver a large amount of therapeutic drugs to damaged cells and newly generated cells in the ischemic area, achieving targeted drug delivery in the ischemic brain region and improving the drug treatment effect.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] In the present invention, the application of AMD3100 as a targeting head molecule in targeted drug delivery for ischemic stroke.

[0008] In the present invention, the ways in which AMD3100 is applied to the drug delivery system include but are not limited to: coupling with any common functional material in the drug delivery system.

[0009] In the present invention, the functional materials include but are not limited to: dendrimeric poly-L-lysine, functional PEG, functional phospholipids, functional cholesterol, functional polymers, etc.

[0010] In the present invention, the drug delivery systems include but are not limited to: nanoparticles, liposomes, phospholipid complexes, microspheres, polymers, nanospheres, nanoemulsions, self-microemulsions, inclusion complexes, and any other common clinical dosage forms.

[0011] The present invention also provides a solid-core lipid nanoparticle, which comprises a DOPA-modified calcium phosphate core (CaP), DSPE-PEG2000-AMD3100, DSPE-PEG2000, cholesterol, and DOTAP; the structure of the DSPE-PEG2000-AMD3100 is shown in Formula I; in the DOPA-modified calcium phosphate core, the DOPA is loaded on the surface of the calcium phosphate core;

[0012]

[0013] wherein, n is 40 - 45.

[0014] In a certain embodiment of the present invention, the structure of the DSPE-PEG2000-AMD3100 is shown in Formula I-1:

[0015]

[0016] In a certain embodiment of the present invention, the DOPA-modified calcium phosphate core is preferably prepared by an oil-in-water type reverse microemulsion method.

[0017] In a certain embodiment of the present invention, the molar ratio of the DSPE-PEG2000-AMD3100 to the DSPE-PEG2000 is (0.1 - 0.3):1000; for example, 0.24:1000.

[0018] In a certain embodiment of the present invention, in the DOPA-modified calcium phosphate core, the molar ratio of the DOPA to the calcium phosphate core is (4 - 6):1, for example, 5:1.

[0019] In a certain embodiment of the present invention, the molar ratio of DSPE-PEG2000, DOTAP, and cholesterol is (0.8 - 1.2):1:1, preferably 0.9:1:1.

[0020] In a certain embodiment of the present invention, the molar ratio of DOPA and DSPE-PEG2000 is 1:(0.7 - 0.8), preferably 1:0.8.

[0021] In a certain embodiment of the present invention, the solid-core lipid nanoparticles are composed of the following components: a DOPA-modified calcium phosphate core (CaP), DSPE-PEG2000, DSPE-PEG2000-AMD3100, cholesterol, and DOTAP.

[0022] In a certain embodiment of the present invention, the solid-core lipid nanoparticles are composed of 4% - 6% calcium phosphate core, 20% - 30% DOPA, 20% - 30% DOTAP, 20% - 30% cholesterol, 20% - 30% DSPE-PEG2000, and 0.004% - 0.006% DSPE-PEG2000-AMD3100;

[0023] Preferably, the solid-core lipid nanoparticles are composed of 4.310% calcium phosphate core, 21.550% DOPA, 25.861% DOTAP, 25.861% cholesterol, 22.413% DSPE-PEG2000, and 0.005% DSPE-PEG2000-AMD3100; wherein, "%" refers to the percentage of the molar amount of each component in the total molar amount of all components of the solid-core lipid nanoparticles.

[0024] In a certain embodiment of the present invention, the average particle size of the solid-core lipid nanoparticles is 70 - 100 nm, such as 75.92 ± 1.54 nm.

[0025] In a certain embodiment of the present invention, the PDI of the solid-core lipid nanoparticles is 0.2 - 0.3, such as 0.286 ± 0.01.

[0026] The present invention also provides a pharmaceutical composition, which comprises a drug molecule and the solid-core lipid nanoparticles according to any one of the present invention.

[0027] In a certain embodiment of the present invention, the drug molecule can be a gene drug, and the gene drug can be a nucleic acid drug, such as mRNA, or miRNA-210; most preferably, the sequence of the miRNA-210 is SEQ ID NO: 1.

[0028] In one embodiment of the present invention, in the pharmaceutical composition, the ratio of the drug molecule to the calcium phosphate core is (15 μg - 20 μg): 40 μg, preferably 18 μg: 40 μg.

[0029] In one embodiment of the present invention, the average particle size of the pharmaceutical composition is 110 - 125 nm, such as 117.97 ± 2.91 nm.

[0030] In one embodiment of the present invention, the PDI of the pharmaceutical composition is 0.2 - 0.3, such as 0.222 ± 0.02.

[0031] In one embodiment of the present invention, the encapsulation efficiency of the pharmaceutical composition is 55 - 60%, such as 56.1%.

[0032] The present invention also provides a method for preparing the solid-core lipid nanoparticles as described in any one of the present invention, which comprises the following steps: mixing the DOPA-modified calcium phosphate core, DSPE-PEG2000, DSPE-PEG2000-AMD3100, cholesterol, DOTAP and an organic solvent, and evaporating to form a film.

[0033] In one embodiment of the present invention, the organic solvent can be a conventional solvent in the art, preferably one or more of alcohol solvents and halogenated alkyl solvents; the alcohol solvent is preferably ethanol; the halogenated alkyl solvent is preferably chloroform.

[0034] In one embodiment of the present invention, the evaporation can be carried out by a conventional evaporation method in the art, such as rotary evaporation; the evaporation is preferably carried out at room temperature, such as rotary evaporation at 25°C.

[0035] In one embodiment of the present invention, the preparation method further includes a method for preparing DSPE-PEG2000-AMD3100, which comprises the following steps: mixing and reacting an organic solvent, AMD3100 and DSPE-PEG2000-NHS, and then dialyzing to obtain DSPE-PEG2000-AMD3100.

[0036] In one embodiment of the present invention, in the method for preparing DSPE-PEG2000-AMD3100, the organic solvent can be an amide solvent, such as DMF.

[0037] In one embodiment of the present invention, in the method for preparing DSPE-PEG2000-AMD3100, the molar ratio of AMD3100 to DSPE-PEG2000-NHS is (1 - 5): 1, such as (1 - 3): 1, preferably 1.5: 1, 2: 1 or 3: 1.

[0038] In a certain embodiment of the present invention, in the preparation method of DSPE-PEG2000-AMD3100, the reaction temperature of the reaction can be 20°C - 32°C, preferably 25°C.

[0039] In a certain embodiment of the present invention, in the preparation method of DSPE-PEG2000-AMD3100, the reaction time of the reaction can be 12h - 48h, such as 20h - 30h, preferably 24h.

[0040] The present invention also provides an application of the solid lipid nanoparticles as described in any embodiment of the present invention in the preparation of a targeted delivery system.

[0041] In a certain embodiment of the present invention, the targeted cells of the targeted delivery system can be neonatal vascular endothelial cells, mature vascular endothelial cells, neurons in the cerebral ischemic region, astrocytes or microglia.

[0042] The present invention provides an application of the pharmaceutical composition as described in any embodiment of the present invention in the preparation of a drug for treating and / or preventing ischemic stroke.

[0043] In a certain embodiment of the present invention, the drug can target neonatal vascular endothelial cells, mature vascular endothelial cells, neurons in the cerebral ischemic region, astrocytes or microglia.

[0044] The present invention provides a compound as shown in Formula I,

[0045]

[0046] wherein, n is 40 - 45.

[0047] On the basis of not violating the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0048] The reagents and raw materials used in the present invention are all commercially available.

[0049] The positive and progressive effects of the present invention are as follows:

[0050] The targeted preparation prepared by the present invention can significantly accumulate in the ischemic brain region and different damaged cells in the ischemic brain region, thereby realizing targeted drug delivery to the ischemic brain region and different cells. Compared with free drugs and ordinary liposome preparations, it can significantly improve the therapeutic effect of drugs and improve the recovery of nerve function. Brief Description of the Drawings

[0051] Figure 1 It is a schematic diagram of the synthesis route of DSPE-PEG2000-AMD3100;

[0052] Figure 21H NMR spectra of the product (A) after 12 h of reaction and the product (B) after 24 h of reaction;

[0053] Figure 3 1H NMR spectra of DSPE-PEG2000-NHS (A) and the product (B) under the condition of 37 °C;

[0054] Figure 4 1H NMR spectrum of AMD3100;

[0055] Figure 5 1H NMR spectrum of DSPE-PEG2000-NHS;

[0056] Figure 6 1H NMR spectrum of DSPE-PEG2000-AMD3100;

[0057] Figure 7 MALDI-TOF spectra of reactants and products (A) DSPE-PEG2000-NHS, (B) DSPE-PEG2000-AMD3100;

[0058] Figure 8 TEM images of calcium phosphate core (A), LCP (B) and T-LCP (C);

[0059] Figure 9 In vivo imaging results at different times after intravenous injection of nanocarriers into rats with MCAO for 12 h;

[0060] Figure 10 In vivo imaging and semi-quantitative statistical results of major organ tissues after intravenous injection of different nanocarriers into rats with MCAO for 12 h; where ns: no statistical difference; *: p < 0.05;

[0061] Figure 11 In vivo imaging results at different times after intravenous injection of nanocarriers into rats with MCAO for 24 h;

[0062] Figure 12 In vivo imaging and semi-quantitative statistical results of major organ tissues after intravenous injection of different nanocarriers into rats with MCAO for 24 h; where ns: no statistical difference; *: p < 0.05

[0063] Figure 13 Distribution of different formulations in the ipsilateral and contralateral newly formed blood vessels of the ischemic brain in MCAO rats;

[0064] Figure 14 Distribution of different formulations in the ipsilateral and contralateral vascular endothelial cells of the ischemic brain in MCAO rats;

[0065] Figure 15 For the distribution of newborn neurons in the ipsilateral and contralateral subventricular zones of the ischemic brain in MCAO rats for different preparations;

[0066] Figure 16 For the distribution of neurons in the ipsilateral and contralateral ischemic brains of MCAO rats for different preparations;

[0067] Figure 17 For the distribution of astrocytes in the ipsilateral and contralateral ischemic brains of MCAO rats for different preparations;

[0068] Figure 18 For the distribution of microglia in the ipsilateral and contralateral ischemic brains of MCAO rats for different preparations;

[0069] Figure 19 For the expression of newborn neurons in the ischemic area and its statistical results (scale = 20 μm, n = 5); where, *: p < 0.05; **: p < 0.01; ***: p < 0.001;

[0070] Figure 20 For the expression of Iba-1+ / CD206+ cells in the ischemic area and its statistical results (scale = 50 μm, n = 5); where, *: p < 0.05; **: p < 0.01; ***: p < 0.001;

[0071] Figure 21 For the TTC staining and semi-quantitative statistical results (n = 5); where, *: p < 0.05; **: p < 0.01; ***: p < 0.001;

[0072] Figure 22 For the neurological deficit scores of MCAO rats at different times (n = 10); where, *: p < 0.05; **: p < 0.01; ***: p < 0.001. Detailed implementation manners

[0073] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0074] The reagents involved in the embodiments of the present invention are specifically as follows:

[0075] Cyclohexane, EDTA: Reagents from Sinopharm Chemical Reagent Co., Ltd., China

[0076] Igepal CO-520: sigma, USA

[0077] miRNA-210, Cy5-miRNA-210, miRNA-Negative control: GenePharma, China (Sequence of miRNA210: 66-CUGUGCGUGUGACAGCGGCUGA–87, SEQ ID NO: 1; Sequence of miRNA-Negative control: 5’-UUCUCCGAACGUGUCACG UTT-3, SEQ ID NO: 2)

[0078] DOPA, DOTAP, DSPE-PEG2000-NHS: Avanti Polar Lipids, USA

[0079] Cholesterol, Triton X-100: Meilunbio, China

[0080] Cholesterol (Analytical Reagent Grade): Sinopharm Chemical Reagent Co., Ltd.

[0081] AMD3100: Macklin Biochemical Co., Ltd., China

[0082] N,N-Dimethylformamide (DMF): Sinopharm Reagent, China

[0083] mPEG2000-NHS: Pengsheng Bio, China

[0084] Deuterated DMSO, Deuterium Oxide: Cambridge Isotope Laboratorics, USA

[0085] The specific experimental instruments are as follows:

[0086] TP-350E + Magnetic Stirrer: Mio Instruments, China

[0087] RV 3eco Rotary Evaporator: IKA, Germany

[0088] SHB-IIIT Vacuum Pump: Huxi Industry Co., Ltd., China

[0089] Zetasizer Nano-ZS Laser Particle Size Analyzer: Malvern, UK

[0090] One in One Hundred Thousandths Balance: Sartorius, Germany

[0091] Milli-Q Water Purification System: Millipore, USA

[0092] TUC-10H Multi-functional Touch Screen Ultrasonic Cleaner: Titan Scientific Co., Ltd., China

[0093] 3-18R Medical Centrifuge: Hengnuo Instruments, China

[0094] CF1524R Desktop High-Speed Refrigerated Microcentrifuge: Scilogex, USA

[0095] DHG Series Electrothermal Blast Drying Oven: Yiheng Scientific Instruments, China

[0096] Tecnai G2 F20 200kV Field Emission Transmission Electron Microscope: FEI, USA

[0097] Cary Eclipse Fluorescence Spectrophotometer: Agilent, USA

[0098] MV-100 Vortexer: Huiling Biology, China

[0099] Mini MR Magnetic Stirrer: IKA, Germany

[0100] Regenerated Cellulose Dialysis Membrane (2000D): Yuanye Bio-Technology, China

[0101] FreeZone 2.5 Freeze Dryer: LABCONCO, Germany

[0102] 400MHz Nuclear Magnetic Resonance Spectrometer: Varian, USA

[0103] High-Resolution Tandem Multistage Mass Spectrometry System: Thermo, USA

[0104] Coupling of AMD3100 with Functional Phospholipids

[0105]

Example 1, Comparative Example 1

[0106] 1. Effect of Different Reaction Times on the Synthesis Yield:

[0107] Precisely weigh the raw materials AMD3100 and DSPE-PEG2000-NHS according to 2:1 (molar ratio), dissolve them separately in 10 mL of anhydrous DMF and mix. Stir the reaction mixture in the dark at room temperature for 12 h or 24 h. After the stirring is completed, transfer the reaction solution completely into a dialysis membrane (MWCO = 2000D), use pure water as the dialysis medium, and stir and dialyze at room temperature for 48 h, replacing the release medium in a timely manner. After dialysis, transfer the reaction solution to a -80 °C refrigerator and pre-freeze overnight, then freeze-dry to obtain the product DSPE-PEG2000-AMD3100 as a white fluffy solid (the synthetic structure is shown in Figure 1 ), and set aside.

[0108] As Figure 2As shown, A is the 1H NMR spectrum of the product AMD3100-PEG2000 obtained under the condition of reacting for 12 h, and B is the 1H NMR spectrum of the product AMD3100-PEG2000 obtained under the condition of reacting for 24 h. It can be seen from A and B that both have the characteristic peaks of the benzene ring in AMD3100, with a chemical shift of about 7.3 ppm; both have the characteristic peaks of the methylene group of PEG in mPEG2000-NHS, with a chemical shift of 3.5 ppm, and the characteristic peaks of the NHS group are at about 2.8 ppm. We set the integral of the hydrogen on the benzene ring of A and B to 1, then the integrals of the hydrogen in the NHS group of A and B are 35.94 and 28.96 respectively, (35.94 - 28.96) / 35.94 * 100% = 19.42%. The reaction time of 24 h (B) consumes 19.42% more NHS groups than 12 h (A), so it is inferred that the synthesis yield increases by 19.42%. From Figure 2 it can be seen that the reaction time of 24 h can consume more NHS than 12 h, and the yield is higher. Therefore, we choose 24 h as the reaction time for synthesis.

[0109] Table 1

[0110] Reaction time Example 1 24 hours Comparative Example 1 12 hours

[0111]

Example 2, Comparative Example 2

[0112] 2. Effect of different reaction temperatures on the synthesis yield

[0113] Precisely weigh the raw materials AMD3100 and DSPE-PEG2000-NHS according to 1.5:1 (molar ratio), dissolve them separately in 10 mL of anhydrous DMF and mix. Stir the reaction in the dark and airtight at room temperature (25 °C) or 37 °C for 24 h. After the stirring is completed, transfer the reaction solution completely into a dialysis bag (MWCO = 2000 D), use pure water as the dialysis medium, and stir and dialyze at room temperature for 48 h, and replace the release medium in time during this period. After the dialysis is completed, transfer the reaction solution to a -80 °C refrigerator for pre-freezing overnight, and then freeze-dry to obtain a white fluffy solid and DSPE-PEG2000-AMD3100 for standby.

[0114] Table 2

[0115] Reaction temperature Example 2 25℃ Comparative Example 2 37℃

[0116] Investigate the effect of different reaction temperatures on the synthesis of the product. The results are as Figure 3As shown, where A is the 1H-NMR spectrum of DSPE-PEG2000-NHS and B is the 1H-NMR spectrum of the product obtained under the condition of 37 °C. It can be seen from A that the characteristic peak of the methylene group of PEG in mPEG2000-NHS is at 3.5 ppm, and the characteristic peak of the methylene group of DSPE is around 1.2 ppm. Comparing B with A, it can be seen that the peak of the methylene group of DSPE in B is missing, which may be due to the cleavage of the chemical bond between DSPE and PEG caused by heating. Therefore, the synthesis reaction is still carried out at room temperature.

[0117]

Example 3

[0118] Precisely weigh the raw materials AMD3100 and DSPE-PEG2000-NHS according to 3:1 (molar ratio), dissolve them separately in 10 mL of anhydrous DMF and mix, and stir the reaction in the dark at room temperature for 24 h. After the stirring is completed, transfer the reaction solution completely into a dialysis bag (MWCO = 2000 D), use pure water as the dialysis medium, and stir and dialyze at room temperature for 48 h, and replace the release medium in time during this period. After the dialysis is completed, transfer the reaction solution to a -80 °C refrigerator for pre-freezing overnight, and then freeze-dry to obtain a white fluffy solid, weigh and record, and store it in a -20 °C refrigerator.

[0119] The 1H-NMR of the reactants AMD3100, DSPE-PEG2000-NHS and the product DSPE-PEG2000-AMD3100 1 is as Figures 4 - 6 shown, where Figure 4 and Figure 5 are the 1H-NMR spectra of AMD3100 and DSPE-PEG2000-NHS respectively, Figure 6 is the 1H-NMR spectrum of the product DSPE-PEG2000-AMD3100. Among them, the characteristic peak of the benzene ring in AMD3100 is at 7.9 ppm, the characteristic peak of the methylene group in the PEG repeating unit is at 3.5 ppm, and the characteristic peaks of the methylene group and methyl group in DSPE are at 1.22 ppm and 0.84 ppm respectively, indicating that DSPE-PEG2000-AMD3100 has been successfully synthesized.

[0120] As Figure 7 shown, A is the reactant DSPE-PEG2000-NHS, and B is the synthetic product DSPE-PEG2000-AMD3100 conjugated with AMD3100. It can be seen that the molecular weight of the reactant A is about 3028 Da, which is relatively close to the theoretical value of 2990 Da, and the molecular weight of the product B is about 3314 Da, which is close to the theoretical molecular weight of the product of 3393 Da, indicating that most of AMD3100 and DSPE-PEG2000-NHS are conjugated.

[0121]

Example 4, Comparative Example 3

[0122] Coupling of AMD3100 with Functional Phospholipids

[0123] Precisely weigh the raw materials AMD3100 and DSPE-PEG2000-NHS at a molar ratio of 3:1, dissolve them separately in 10 mL of anhydrous DMF and mix. Stir the reaction in the dark at room temperature for 24 h. After stirring, transfer the reaction solution completely into a dialysis bag (MWCO = 2000D), use pure water as the dialysis medium, and stir and dialyze at room temperature for 48 h, replacing the release medium in a timely manner. After dialysis, transfer the reaction solution to a -80 °C refrigerator for pre-freezing overnight, and then freeze-dry to obtain a white fluffy solid and DSPE-PEG2000-AMD3100 for standby.

[0124] Preparation of Targeting Lipid calciumphosphate (T-LCP) Modified with AMD3100:

[0125] Prepare calcium phosphate core (CaP) by using the water-in-oil type reverse microemulsion method. Measure 20 mL of cyclohexane and the non-ionic surfactant Igepal CO-520 (cyclohexane:Igepal CO-520 = 71:29, volume ratio), transfer them to a 100 mL round-bottom flask, and mix evenly by magnetic stirring as the oil phase. Prepare another oil phase in the same way. Add 600 μl of CaCl 2 solution (12.5 mM) to one oil phase, and mix evenly by magnetic stirring for 20 min to prepare the Ca 2+ phase; add 600 μl of Na 2 HPO 4 solution (2.5 mM) to the other oil phase, and mix evenly by magnetic stirring for 20 min to prepare the HPO 4 2- phase. Mix the two-phase solutions, and mix evenly by magnetic stirring for 20 min to obtain calcium phosphate complex (CaP). Add 250 μL of DOPA chloroform solution (20 mM) to it, continue to stir and mix for 30 min, then add 40 mL of absolute ethanol to break the emulsion, and stir and mix for 30 min to obtain DOPA-modified calcium phosphate core (CaP). Transfer it to a centrifuge tube, centrifuge at 12 000 g and 4 °C for 20 min, pour off the supernatant, add absolute ethanol, vortex and mix evenly, repeat centrifugation and washing twice, evaporate the absolute ethanol, dissolve the obtained calcium phosphate precipitate in chloroform, centrifuge at 10 000 rpm and 4 °C for 15 min, and take the supernatant to obtain calcium phosphate core CaP for standby.

[0126] Dissolve the obtained DOPA-modified calcium phosphate core CaP and the outer layer phospholipids 300 μL DOTAP (20 mM), 300 μL cholesterol (20 mM), 260 μL DSPE-PEG2000 (20 mM), and the synthesized 84 μL DSPE-PEG2000-AMD3100 (50 μg / mL) in 3 mL of chloroform, mix well by ultrasound, and then transfer to a 50 mL round-bottom flask. Rotate and evaporate at room temperature for 30 min. After evaporating the chloroform, a uniform lipid film is obtained at the bottom of the flask. Add an appropriate amount of normal saline to the flask and mix well by ultrasound in an ice-water bath for 10 min to form the targeted modified solid-core lipid calcium phosphate nanocarrier T-LCP.

[0127] Preparation of ordinary LCP: Dissolve the obtained DOPA-modified calcium phosphate core CaP and the outer layer phospholipids 300 μL DOTAP (20 mM), 300 μL cholesterol (20 mM), and 260 μL DSPE-PEG2000 (20 mM) in 3 mL of chloroform and mix well. Transfer to a 50 mL round-bottom flask and rotate and evaporate at room temperature for 30 min. After evaporating the chloroform, a uniform lipid film is obtained at the bottom of the flask. Add an appropriate amount of normal saline to the flask and mix well by ultrasound in an ice-water bath for 10 min to form ordinary LCP.

[0128] Table 3

[0129]

[0130] Perform transmission electron microscopy observation on the prepared calcium phosphate core CaP, LCP, and T-LCP. The results are shown in Figure 8 as follows. Among them, it is shown in A that the particle size of the calcium phosphate core is about 25 nm and the appearance is spherical; it is shown in B that the particle size of the LCP nanocarrier is about 80 nm, the appearance is round, and the morphology is spherical; it can be seen from C that the particle size of T-LCP is within 100 nm and the morphology is spherical. There is no significant difference in the morphology between LCP and T-LCP.

[0131]

Example 5, Comparative Example 4

[0132] Preparation of gene-loaded drug miRNA-210 LCP

[0133] Measure 20 mL of cyclohexane and the nonionic surfactant Igepal CO-520 (cyclohexane:Igepal CO-520 = 70:30, volume ratio), transfer to a 100 mL round-bottom flask, and mix well by magnetic stirring as the oil phase. Take a centrifuge tube containing 1 OD 260 of miRNA-210 dry powder, first centrifuge at a speed of 3500 rpm for 1 min, then add 125 μl of DEPC water to it, and then add 600 μL of CaCl2 The solution was mixed evenly. Then it was mixed with the oil phase to form a Ca 2+ phase. The remaining steps were the same as those in Example 4, and the operation was carried out in the dark throughout the process. Ordinary or targeted lipid calcium phosphate nanocarriers (miR-LCP or miR-T-LCP) loaded with gene drugs were prepared. The results of measuring the particle size, zeta potential and encapsulation efficiency of blank LCP, miR-LCP or miR-T-LCP are shown in Table 5. The results showed that the particle size of LCP increased after loading the gene drug, and the particle size of targeted miR-T-LCP was significantly larger than that of LCP and miR-LCP. There was no significant difference in the encapsulation efficiency between drug-loaded LCP and T-LCP.

[0134] Table 4

[0135]

[0136] Table 5

[0137] LCP miR-LCP miR-T-LCP Average particle size (nm) 75.92±1.54 89.20±1.09 117.97±2.91 PDI 0.286±0.01 0.271±0.01 0.222±0.02 Entrapment efficiency % — 55.7 56.1 Zeta potential (mV) 12.40±0.78 5.97±0.42 4.63±0.28

[0138]

Effect Examples 1, 2, Comparative Examples 5, 6

[0139] Targeting evaluation of the targeted preparation in the ischemic brain region

[0140] The modified calcium phosphate core CaP was prepared according to Example 4. 5 mg of DiR powder was accurately weighed and dissolved in 5 mL of chloroform to prepare a 1 mg / mL DiR chloroform solution. CaP was dissolved in 3 mL of chloroform together with 300 μL of outer layer phospholipid DOTAP (20 mM), 300 μL of cholesterol (20 mM), 260 μL of DSPE-PEG2000 (20 mM), and 84 μL of DSPE-PEG2000-AMD3100 (50 μg / mL) synthesized (prepared according to Example 4). Then 1 mL of DiR chloroform solution was added, and the mixture was ultrasonicated and mixed evenly. After mixing evenly, it was transferred to a 50 mL round-bottom flask, and the chloroform was evaporated at room temperature for 30 min. After the chloroform was evaporated to dryness, a uniform lipid film was obtained at the bottom of the flask. An appropriate amount of physiological saline was added to the flask, and the mixture was ultrasonicated and mixed evenly in an ice-water bath for 10 min to prepare fluorescently labeled T-LCP. Fluorescently labeled LCP was prepared in the same way.

[0141] Table 6

[0142]

[0143]

[0144] An animal model of ischemia-reperfusion was established by the middle cerebral artery occlusion method (MCAO). At 12 and 24 hours after ischemia-reperfusion, two groups of preparations, LCP and T-LCP labeled with DiR, were injected via the tail vein (the injection dose was 100 μg / kg in terms of DiR). Then, a small animal in vivo imaging system was used to observe the distribution of LCP and T-LCP in ischemic rats at 1 h, 2 h, 4 h, 10 h, and 24 h after administration.

[0145] When administered 12 hours after reperfusion, as Figure 9 shown, obvious fluorescence aggregation could be observed in the brain 1 h after the tail vein injection of the DiR-labeled LCP group and T-LCP group preparations. Compared with the LCP group, the T-LCP group had more obvious brain targeting, and the fluorescence signal of T-LCP lasted until 24 h.

[0146] The rats were anesthetized 24 h after administration. After perfusion, the rat brains and major organs were removed, and the fluorescence distribution was observed by small animal in vivo imaging. As Figure 10 shown in A in, among the major organs, both the LCP group and the T-LCP group had more fluorescence distribution in the liver and spleen. Moreover, compared with the LCP group, the T-LCP group had more fluorescence distribution in the liver, which might be related to the large amount of SDF-1 in the liver. As can be seen from B and C, both groups of preparations were also distributed in the brain, and the T-LCP group had better brain targeting and could target the ischemic hemisphere, with strong targeting to the ischemic brain region. At 24 h after administration, the T-LCP group could more significantly increase the accumulation of the preparation in the ischemic brain region.

[0147] When administered 24 hours after reperfusion, as Figure 11 shown, a small amount of fluorescence aggregation could be seen 2 h after the tail vein injection of DiR-labeled LCP, and the fluorescence gradually increased within 24 h; obvious fluorescence signals could be observed 1 h after the tail vein injection of DiR-labeled T-LCP, and it continuously increased within 24 h and was significantly higher than the LCP group, indicating that T-LCP administration could rapidly aggregate towards the brain and continuously aggregate towards the brain, indicating that its targeting effect was rapid in onset and long-lasting.

[0148] The rats were anesthetized 24 h after administration. After perfusion, the rat brains and major organs were removed. As Figure 12 shown in A of, among the major organs, both the LCP group and the T-LCP group had more distribution in the liver. Furthermore, as can be seen from B and C, compared with the LCP group, T-LCP aggregated more in the ipsilateral hemisphere of the ischemic brain, indicating that at 24 h after administration, the T-LCP group could significantly increase the accumulation of the preparation in the ischemic brain region.

[0149] It can be seen from the in vivo imaging results that when administered within 24 hours after MCAO in rats, T-LCP has strong brain targeting ability, can target the cerebral ischemic area, and accumulates in the ischemic area for at least 24 hours.

[0150]

Effect Examples 3 and 4, Comparative Examples 7 and 8

[0151] Evaluation of the targeted drug delivery effect of the targeted preparation on neonatal vascular endothelial cells and mature vascular endothelial cells in the cerebral ischemic area

[0152] Prepare ordinary or targeted lipid calcium phosphate nanocarriers loaded with fluorescently labeled Cy5-miRNA-210 according to Example 5, where miRNA-210 is replaced with Cy5-miRAN210 labeled with the same amount of fluorescent label Cy5, and Cy5-miR-LCP or Cy5-miR-T-LCP is prepared to evaluate the binding effect of the preparation with vascular endothelial cells in the ischemic area.

[0153] An ischemia-reperfusion animal model was established by the MCAO method. At 12 hours after ischemia-reperfusion, two groups of preparations, Cy5-labeled LCP and T-LCP (the injection dose was calculated based on Cy5-miRAN210 and was 0.4 OD / kg), were injected into the tail vein. At 24 hours after administration, the rat brain tissues were taken for sectioning, and the neonatal vascular endothelial cells and vascular endothelial cells in the brain were labeled with CD34 antibody and vWF antibody respectively, and the immunofluorescence staining results of the ipsilateral and contralateral regions of the rat cerebral ischemia were observed. Red represents Cy5-labeled LCP and T-LCP, and green represents neonatal vascular endothelial cells labeled with CD34 antibody or mature endothelial cells labeled with vWF antibody.

[0154] The results are as Figure 13 shown. After administration of the Cy5-labeled T-LCP nanocarrier, compared with the ischemic contralateral region, the preparation can target the neonatal vascular endothelial cells in the ischemic ipsilateral region, which is significantly higher than the LCP group. At the same time, the binding of the T-LCP nanocarrier to neonatal vascular endothelial cells can facilitate the role of miRNA-210 in promoting angiogenesis, and thus treat ischemic stroke diseases.

[0155] As Figure 14 shown, red represents Cy5-labeled LCP and T-LCP, and green represents mature vascular endothelial cells labeled with vWF antibody. After administration of the T-LCP nanocarrier, compared with the ischemic contralateral region, the preparation can target the vascular endothelial cells in the ischemic ipsilateral region, and the T-LCP group is significantly higher than the LCP group, which is beneficial to reducing the damage of mature vascular endothelial cells.

[0156] Table 7

[0157]

[0158]

Examples of Effects 5 - 8, Comparative Examples 9 - 12

[0159] Evaluation of the Targeted Drug Delivery Effect of the Targeted Preparation on Neurons, Astrocytes, and Microglia in the Cerebral Ischemia Region

[0160] Prepare ordinary or targeted lipid calcium phosphate nanocarriers loaded with fluorescently labeled Cy5 - miRNA - 210 according to Example 5, where miRNA - 210 is replaced with Cy5 - miRAN210 labeled with the same amount of fluorescent label Cy5 to obtain Cy5 - miR - LCP or Cy5 - miR - T - LCP, which is used to evaluate the effect of the preparation on binding to nerve cells in the ischemic region.

[0161] Use the MCAO method to establish an ischemia - reperfusion animal model. At 12 hours after ischemia - reperfusion, inject the two groups of preparations of Cy5 - labeled LCP and T - LCP once via the tail vein (the injection dose is calculated based on Cy5 - miRAN210 and is 0.4 OD / kg). At 24 hours after administration, take the rat brain tissue for sectioning. Use DCX antibody to label newborn neurons in the brain (Example of Effect 5, Comparative Example 9), MAP2 antibody to label neurons in the brain (Example of Effect 6, Comparative Example 10), GFAP antibody to label astrocytes (Example of Effect 7, Comparative Example 11), and Iba - 1 antibody to label microglia (Example of Effect 8, Comparative Example 12).

[0162] Table 8

[0163]

[0164]

[0165] As Figure 15 shown, after administering the T - LCP nanocarrier, the preparation can target newborn neurons in the ischemic subventricular zone (SVZ), which is significantly higher than the LCP group, and can be beneficial for exerting the role of miRNA - 210 in promoting nerve regeneration. There is no significant difference in their distribution in the non - ischemic contralateral brain region.

[0166] As Figure 16 shown, T - LCP can target mature damaged neurons in the ischemic side, and is significantly higher than the LCP group. It can be beneficial for exerting the role of miRNA - 210 in reducing ischemic injury. There is no significant difference in their distribution in the non - ischemic contralateral brain region.

[0167] As Figure 17As shown, after administration of the T-LCP nanocarrier, compared with the ischemic contralateral region, the targeted preparation can target astrocytes in the ischemic side, which is significantly higher than that of the LCP group, facilitating the drug to reduce astrocyte damage.

[0168] As Figure 18 shown, after administration of the T-LCP nanocarrier, compared with the ischemic contralateral region, the preparation can target microglia in the ipsilateral ischemic region, which is significantly higher than that of the LCP group, facilitating the drug to play a role in regulating microglial polarization.

[0169]

Effect Examples 9 and 10, Comparative Examples 13 and 14

[0170] Effect of the gene-loaded drug targeted preparation on newborn neurons and microglial polarization in the cerebral ischemic region

[0171] LCP-miRNA-210 and T-LCP-miRNA-210 loaded with the gene-loaded drug miR210 were prepared according to Example 5. For the preparation loaded with the negative control, miRNA-210 was replaced with an equal amount of miRNA-Negative, and the negative control preparation LCP-miR-NC was prepared by the above method.

[0172] Thirty SD rats were randomly divided into a sham operation group (SHAM group), an MCAO group, an LCP-miR-NC group, an LCP-miRNA-210 group, and a T-LCP-miRNA-210 group, with 5 rats in each group. The drug administration arrangements were as follows:

[0173] 1) SHAM group: Only the ECA and ICA were separated, and no suture was inserted.

[0174] 2) MCAO group: Normal saline was administered at 12 h and 24 h after ischemia-reperfusion, respectively.

[0175] 3) LCP-miR-NC group: LCP-miR-NC (negative control) was administered at 12 h and 24 h after ischemia-reperfusion, respectively, where miR-NC is a scrambled sequence of miRNA and serves as the negative control of miRNA-210; (the injection dose is calculated as miRNA-Negative and is 0.4 OD / kg)

[0176] 4) LCP-miRNA-210 group: LCP-miRNA-210 was administered at 12 h and 24 h after ischemia-reperfusion, respectively (the injection dose is calculated as miRNA-210 and is 0.4 OD / kg);

[0177] 5) T-LCP-miRNA-210 group: T-LCP-miRNA-210 (the injection dose is calculated based on miRNA-210 and is 0.4 OD / kg) was administered at 12 h and 24 h after ischemia-reperfusion, respectively.

[0178] On the 3rd day after administration, the rats in each group were anesthetized, perfused through the heart, and the brain tissues were taken for sectioning. The DCX antibody was used to label the newly born neurons in the brain, and the immunofluorescence staining results in the subventricular zone (SVZ) of the brain were observed. The brain tissues of the rats were taken for sectioning, and the Iba-1, CD86, and CD206 antibodies were used to label the pro-inflammatory microglia, i.e., M1 microglia (CD86+ / Iba-1+), and anti-inflammatory microglia, i.e., M2 microglia (CD206+ / Iba-1+), in the ischemic area, and the immunofluorescence staining results in the ischemic area of the brain were observed.

[0179] Table 9

[0180]

[0181] As Figure 19 shown, almost no newly born neurons were observed in the SHAM group rats and the MCAO group in the SVZ, and there was no obvious change after intravenous injection of LCP-miR-NC. Compared with the MCAO group and the LCP-NC group, after injection of LCP-210 and T-LCP-210, the number of newly born neurons in the SVZ of MCAO rats increased. And compared with the LCP-210 group, the T-LCP-210 group could more significantly increase the number of newly born neurons in the SVZ, which was more conducive to promoting the regeneration of neurons in the brain of MCAO rats.

[0182] As Figure 20 shown, the number of M1 or M2 microglia in the brain of SHAM group rats was very small. After ischemia-reperfusion (MCAO group) in rats, the number of M1 microglia in the ischemic area of the brain increased significantly, and the number of M2 microglia (CD206+ / Iba-1+) increased slightly; after intravenous injection of LCP-miR-NC, the number of M1 microglia and the number of M2 microglia did not change significantly. Compared with the MCAO group and the LCP-NC group, after injection of LCP-210 and T-LCP-210, the number of M1 microglia in the ischemic area of the brain of MCAO rats decreased, and the number of M2 microglia increased; compared with the LCP-210 group, the T-LCP-210 group could more significantly increase the number of M2 microglia, which was beneficial to promoting the polarization of microglia in the ischemic area of the brain of MCAO rats and reducing the inflammatory response.

[0183]

Effect Example 11, Comparative Example 15

[0184] Effect of Gene-loaded Drug Targeted Preparation on Cerebral Infarct Area

[0185] Prepare LCP-miRNA-210 and T-LCP-miRNA-210 loaded with the gene drug miR210 according to Example 5. For the preparation loaded with the negative control, replace miRNA-210 with an equal amount of miRNA-Negative, and prepare the negative control preparation LCP-miR-NC according to the above method.

[0186] Thirty SD rats were randomly divided into a SHAM group, an MCAO group, an LCP-miR-NC group, an LCP-miRNA-210 group, and a T-LCP-miRNA-210 group, with 5 rats in each group. The rats in each group were administered drugs according to the methods of Effect Examples 9 and 10. Three days after drug administration, the rats in each group were anesthetized, and the brain tissues were taken by cardiac perfusion. After TTC staining, photos were taken, and the cerebral infarct area was calculated using Image J.

[0187] The formula for calculating the cerebral infarct area is: cerebral infarct area = ischemic area / total brain area × 100%

[0188] Table 10

[0189]

[0190] The results and statistics of TTC staining of rat brain sections are as Figure 21 , the red part in the brain is normal brain tissue, and the white part is the infarct area. It can be seen that the cerebral infarct area in the model group (MCAO group) is about 32%. After injecting LCP-NC, the cerebral infarct area was not reduced. However, after administering LCP-miRNA-210 and T-LCP-miRNA-210 T-LCP-210, the infarct area was significantly reduced. Compared with LCP-210, T-LCP-210 can more effectively reduce the infarct area in MCAO rats.

[0191]

Effect Example 12, Comparative Example 16

[0192] Effect of Gene-loaded Drug Targeted Preparation on Nerve Function Injury

[0193] Prepare LCP-miRNA-210 and T-LCP-miRNA-210 loaded with the gene drug miR210 according to Example 5. For the preparation loaded with the negative control, replace miRNA-210 with an equal amount of miRNA-Negative, and prepare the negative control preparation LCP-miR-NC according to the above method.

[0194] Thirty SD rats were randomly divided into a SHAM group, an MCAO group, an LCP-miR-NC group, an LCP-miRNA-210 group, and a T-LCP-miRNA-210 group, with 10 rats in each group. The rats were administered drugs in the same manner as in Effect Examples 9 and 10. A total of three neurological deficit scores were measured during the experiment. The first measurement was performed 12 h after MCAO, i.e., before drug administration; the second measurement was performed 48 h after MCAO, i.e., 1 day after drug administration; and the third measurement was performed 96 h after MCAO, i.e., 3 days after drug administration. The specific criteria for the neurological deficit score (mNSS score) are shown in Table 11. The total score is 18 points. The higher the score, the more severe the neurological impairment of the rats. An mNSS score between 1 and 6 points indicates mild injury, a score between 7 and 12 points indicates moderate injury, and a score between 13 and 18 points indicates severe injury.

[0195] Table 11 Criteria for Neurological Deficit Score

[0196]

[0197]

[0198] The results are as Figure 22 shown. It can be seen that at the first time point, i.e., before drug administration, the neurological deficit scores of the rats in each group were between 7 and 12 points, indicating moderate neurological injury. There was no significant difference in the scores among the groups, indicating that the model was successfully established and stable. The second time point was 1 day after drug administration. From the evaluation results, it can be seen that the scores of the MCAO group and the LCP-NC group were still relatively high, while the neurological deficit scores decreased after treatment with LCP-210 and T-LCP-210 injections, and the neurological function of the rats was improved compared with that before drug administration. However, there was no significant difference between T-LCP-210 and LCP-210. The third time point was 3 days after drug administration. It can be seen that compared with LCP-210, T-LCP-210 could more effectively reduce the neurological deficit score of MCAO rats, improve the behavioral performance, and promote the recovery of their neurological function. Compared with the model group (MCAO group), there was no significant difference in LCP-NC, indicating that when the negative control was given, there was no therapeutic effect, and this therapeutic effect came from miRNA210. Moreover, the targeted delivery of T-LCP significantly increased this therapeutic effect.

Claims

1. A core-fixed lipid nanoparticle, characterized in that: The invention comprises a DOPA-modified calcium phosphate core, DSPE-PEG2000-AMD3100, DSPE-PEG2000, cholesterol and DOTAP; the structure of the DSPE-PEG2000-AMD3100 is shown in Formula I; in the DOPA-modified calcium phosphate core, the DOPA is loaded on the surface of the calcium phosphate core; Among them, n is 40-45.

2. The core-fixed lipid nanoparticle according to claim 1, wherein The structure of the DSPE-PEG2000-AMD3100 is shown in Formula I-1: And / or, the DOPA-modified calcium phosphate core is prepared by a water-in-oil reverse microemulsion method.

3. The core-fixed lipid nanoparticle according to claim 1, wherein It meets one or more of the following conditions: (1) The molar ratio of the DSPE-PEG2000-AMD3100 to the DSPE-PEG2000 is (0.1-0.3):1000; for example, 0.24:1000; (2) In the DOPA-modified calcium phosphate core, the molar ratio of DOPA to the calcium phosphate core is (4-6):1, for example 5:1; (3) the molar ratio of the DSPE-PEG2000, the DOTAP and the cholesterol is (0.8-1.2):1:1, preferably 0.9:1:1; (4) The molar ratio of DOPA to DSPE-PEG2000 is 1:(0.7-0.8), preferably 1:0.8; (5) The average particle size of the core-fixed lipid nanoparticles is 70-100 nm, for example, 75.92±1.54 nm; (6) The PDI of the core-fixed lipid nanoparticles is 0.2-0.3, for example, 0.286±0.

01.

4. The core-fixed lipid nanoparticle according to claim 1, wherein The solid-core lipid nanoparticles are composed of the following components: a DOPA-modified calcium phosphate core, DSPE-PEG2000, DSPE-PEG2000-AMD3100, cholesterol and DOTAP; Preferably, the solid-core lipid nanoparticles are composed of 4%-6% calcium phosphate core, 20%-30% DOPA, 20%-30% DOTAP, 20%-30% cholesterol, 20%-30% DSPE-PEG2000 and 0.004%-0.006% DSPE-PEG2000-AMD3100; More preferably, the solid-core lipid nanoparticles consist of 4.310% calcium phosphate inner core, 21.550% DOPA, 25.861% DOTAP, 25.861% cholesterol, 22.413% DSPE-PEG2000 and 0.005% DSPE-PEG2000-AMD3100.

5. A pharmaceutical composition, characterized in that It comprises a drug molecule and the core-fixed lipid nanoparticles as claimed in any one of claims 1 to 4; Preferably, the pharmaceutical composition satisfies one or more of the following conditions: (1) The drug molecule is a gene drug, and the gene drug is preferably a nucleic acid drug, such as mRNA, and also such as miRNA-210; most preferably, the sequence of the miRNA-210 is SEQ ID NO: 1; (2) The ratio of the drug molecule to the calcium phosphate core is (15 μg-20 μg):40 μg, preferably 18 μg:40 μg; (3) The average particle size of the pharmaceutical composition is 110-125 nm, for example, 117.97±2.91 nm; (4) The PDI of the pharmaceutical composition is 0.2-0.3, for example 0.222±0.02; (5) The encapsulation efficiency of the pharmaceutical composition is 55-60%, for example 56.1%.

6. A method for preparing the core-fixed lipid nanoparticles according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: mixing a DOPA-modified calcium phosphate core, DSPE-PEG2000, DSPE-PEG2000-AMD3100, cholesterol, DOTAP and an organic solvent, and evaporating the mixture to form a film; Preferably, the preparation method satisfies one or more of the following conditions: (1) The organic solvent is one or more of an alcohol solvent and a halogenated alkyl solvent; The alcohol solvent is preferably ethanol; the halogenated alkyl solvent is preferably chloroform; (2) The evaporation is performed by rotary evaporation; the evaporation is preferably performed at room temperature, such as rotary evaporation at 25°C.

7. The preparation method according to claim 6, characterized in that: The preparation method also includes a preparation method of DSPE-PEG2000-AMD3100, which includes the following steps: mixing an organic solvent, AMD3100 and DSPE-PEG2000-NHS for reaction, and dialyzing to obtain DSPE-PEG2000-AMD3100; Preferably, the preparation method of DSPE-PEG2000-AMD3100 satisfies one or more of the following conditions: (1) The organic solvent is an amide solvent, such as DMF; (2) the molar ratio of the AMD3100 to the DSPE-PEG2000-NHS is (1-5):1, for example (1-3):1, preferably 1.5:1, 2:1 or 3:1; (3) The reaction temperature of the reaction is 20°C-32°C, preferably 25°C; (4) The reaction time of the reaction is 12h-48h, for example 20h-30h, preferably 24h.

8. Use of the core-fixed lipid nanoparticles according to any one of claims 1 to 4 in the preparation of a targeted delivery system; Preferably, the target cells of the targeted delivery system are neovascular endothelial cells, mature vascular endothelial cells, neurons in the ischemic area of ​​the brain, astrocytes or microglia.

9. Use of the pharmaceutical composition according to claim 5 in preparing a drug for treating and / or preventing ischemic stroke; Preferably, the drug can target newborn vascular endothelial cells, mature vascular endothelial cells, neurons in the ischemic area of ​​the brain, astrocytes or microglia.

10. A compound as shown in formula I, in, n is 40-45.

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