Drug delivery system crossing blood brain barrier and preparation method thereof

Abstract

The invention belongs to the technical field of biological materials, and relates to a blood brain barrier crossing drug delivery system and a preparation method thereof, and the drug delivery system is a liposome prepared from 1-stearoyl-sn-glycerol-3-phosphorylcholine (LPC). The LPC is selected as an important component of the drug delivery system with the blood brain barrier crossing capacity for the first time, and the drug delivery system has the advantages of being good in stability, high in brain entering efficiency, long in circulation time and the like. Different from the traditional non-specific passive transportation and invasive brain entering modes, the method has the capability of efficiently delivering target molecules such as drugs to a brain area in a non-invasive targeting manner, and has extremely strong clinical transformation significance; and the synthesis route is simple, the reaction condition is mild, and the damage to the possibly carried target compound is small.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a drug delivery system that crosses the blood-brain barrier and a preparation method thereof. Background Art

[0002] Central nervous system (CNS) diseases include brain infections, brain tumors, neurodevelopmental diseases such as autism, psychiatric diseases such as anxiety and depression, and neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's syndrome (PD), and Huntington's disease (HD). These diseases seriously endanger human health and cause a heavy social burden.

[0003] The blood-brain barrier (BBB) ​​is a protective barrier between brain blood vessels, cells and other components of brain tissues, mainly composed of pericytes, brain capillary endothelial cells, basement membranes, astrocyte foot processes and tight junctions between brain vascular endothelial cells. On the one hand, the BBB maintains CNS homeostasis by strictly controlling specific nutrients and limiting the passage of harmful molecules; on the other hand, the existence of the blood-brain barrier prevents 98% of small molecule therapeutics and almost all macromolecular drugs (including monoclonal antibodies, proteins and gene therapies) from effectively entering the brain, which not only reduces the therapeutic effect of the drugs, but also the retention of drugs that have not entered the brain in other organs and tissues aggravates the toxic side effects of the drugs. Therefore, there is an urgent need for a safe and effective drug delivery system that targets the blood-brain barrier to increase the utilization rate of drugs and reduce their toxic side effects.

[0004] Nanodiscs (NDs) are disk-shaped nanoparticles composed of a self-assembled phospholipid membrane and apolipoprotein. NDs assembled with human apolipoprotein have been used in a variety of biotechnological applications, including membrane protein solubilization, drug delivery, and diagnostic imaging.

[0005] Currently, the clinically available intracerebral drug delivery routes mainly include: intraventricular or intraparenchymal injection, intracranial delivery using micropumps, catheter infusion, focused ultrasound to open the blood-brain barrier, etc., but they often rely on certain invasive operations and have the risk of infection. For oral drugs, although chemical modification of the structure can increase its lipid solubility, thereby increasing the ability of the drug to pass through the blood-brain barrier, the retention time and action time of highly lipid-soluble therapeutic drugs in non-target organs are longer, resulting in considerable side effects, and increasing lipophilicity is not a universal strategy because it may also inhibit the biological activity of the target drug. If the drug is administered through surgery, its scope of use is limited due to the high technical requirements and the susceptibility to infection. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the present invention provides a drug delivery system across the blood-brain barrier and a preparation method thereof. The specific technical scheme is as follows:

[0007] The first object of the present invention is to provide a drug delivery system across the blood-brain barrier, which is a liposome prepared from 1-stearoyl-sn-glycero-3-phosphorylcholine (LPC).

[0008] The system of the present invention can load the second zone fluorescent dye, and has the potential to load various hydrophobic large and small molecular drugs, and can be used as a delivery system for diagnosis and treatment. The present invention uses LPC to target the choline receptors on endothelial cells where the blood-brain barrier may exist. LPC increases the ability of materials to enter the blood-brain barrier, can efficiently penetrate the blood-brain barrier, and has the ability to deliver target molecules into the brain in a non-invasive manner, and has clinical transformation value.

[0009] Furthermore, the liposome also includes phospholipids, cell membrane far-infrared fluorescent probe (DiR), dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) and cholesterol;

[0010] The molar ratio of the phospholipid, LPC, cell membrane far-infrared fluorescent probe (DiR), dimyristoylglycerol-polyethylene glycol 2000 (DMG-PEG 2000) and cholesterol is (65-75): (10-20): (10-14): 1: (5-10).

[0011] Furthermore, the drug delivery system also includes apolipoprotein, and the apolipoprotein forms a nanodisc with the liposome; the molar ratio of the apolipoprotein to the liposome is 1:(25-100), preferably 1:60.

[0012] Furthermore, the phospholipid is dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylserine (DOPS) or the like phospholipids, preferably DOPC.

[0013] Furthermore, the apolipoproteins are membrane scaffold proteins (MSPs).

[0014] Furthermore, the apolipoprotein is MSP1E3D1, MSP1D1, MSP1D1-dH5 or MSP2N2, preferably MSP1E3D1, and the nanodiscs made from the apolipoprotein are uniform in size.

[0015] The second object of the present invention is to provide a method for preparing the above-mentioned drug delivery system across the blood-brain barrier, comprising the following steps:

[0016] (1) dissolving each component of the liposome in chloroform and mixing well to obtain a liposome mixed system;

[0017] (2) taking the liposome mixture obtained in step (1) into a nitrogen blowpipe, and blowing the lipids dry with a nitrogen blowpipe to form a blue lipid film;

[0018] (3) dissolving the lipid film and the apolipoprotein in a nanodisc buffer solution (ND Buffer) respectively, and then mixing the two to obtain a mixed system;

[0019] (4) Transfer the mixed system to SM2 Bio-Beads moistened with ultrapure water

[0020] (5) The product of (4) is incubated in a shaking incubator at 3-5° C. for 12-16 h to obtain an incubation solution, and the incubation solution is purified to prepare a nanodisk.

[0021] Furthermore, the lipid content in the system is 300 nmol.

[0022] Furthermore, the purification method comprises the following steps: centrifuging the incubation solution in a centrifuge tube to remove unbound lipids; separating the nanodiscs containing proteins by molecular sieves; and concentrating the nanodiscs by centrifuge tubes.

[0023] Furthermore, the nanodisk buffer solution is a nanodisk buffer solution containing 1% n-octyl-β-D-pyranoglucoside (OG).

[0024] Furthermore, the molecular weight cut-off of the centrifuge tube is 30 kDa, the rotation speed is 10000 rpm, and the centrifugation time is 5 min.

[0025] Furthermore, the molecular sieve is Superdex 200 molecular sieve.

[0026] The beneficial effects of the present invention are:

[0027] The present invention selects LPC for the first time as an important component of a drug delivery system capable of crossing the blood-brain barrier. The drug delivery system of the present invention has the advantages of good stability, high brain entry efficiency, long circulation time, etc. Different from the traditional non-specific passive transport and invasive ways of entering the brain, the present invention can deliver target molecules such as drugs to the brain region in a non-invasive, targeted and efficient manner, which has a strong clinical transformation significance. In addition, the present invention has a simple synthesis route, mild reaction conditions, and little damage to target compounds (drugs, diagnostic reagents, etc.) that may be carried. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The preparation process of the drug delivery system of the present invention;

[0029] Figure 2 This is a transmission electron microscope characterization image of the LPC nanodisk of the present invention;

[0030] Figure 3 This is a comparison of the in vivo brain imaging of LPC nanodisks and MPC nanodisks of the present invention, with the left mouse injected with MPC nanodisks and the right mouse injected with LPC nanodisks;

[0031] Figure 4 A graph showing the fluorescence intensity of the brain region of mice after injection of LPC nanodisks and MPC nanodisks of the present invention. DETAILED DESCRIPTION

[0032] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention.

[0033] Example:

[0034] like Figure 1 As shown, a preparation of a drug delivery system that crosses the blood-brain barrier comprises the following steps:

[0035] (1) Dissolving DOPC, LPC, DiR, DMG-PEG 2000 and cholesterol Cholesterol liposome raw materials in chloroform, mixing, and then adding a certain amount of chloroform to make the volume 1 mL, so that the total amount of lipids in the system is 3000 nmol, to obtain a liposome mixed system; wherein the mass fractions of DOPC, LPC, DiR, DMG-PEG 2000 and cholesterol Cholesterol are 69%, 15%, 10%, 1% and 5%;

[0036] (2) Take out 100 μL of the prepared liposome mixture system and place it in a nitrogen blowpipe, and use a nitrogen blower to blow dry the lipids to form a blue lipid film;

[0037] (3) The lipid film and MSP1E3D1 protein were dissolved in nanodisc buffer solution (nanodisc buffer solution containing 1% n-octyl-β-D-pyranoglucoside (OG)) respectively; the two were then mixed to obtain a mixed system; the amount of lipid film: MSP1E3D1 protein was 5 nmol, the molar ratio of MSP1E3D1 protein was 60:1, and the amphiphilicity of MSP1E3D1 protein could constrain phospholipids to form nanodiscs; the nanodisc buffer solution was then used to fill the final volume to 300 μL;

[0038] (4) Vortexing to dissolve the lipid film components into the nanodisc buffer solution. After sufficient dissolution, the mixed system is transferred to 300 mg of SM2 Bio-Beads moistened with ultrapure water to remove the OG in the system;

[0039] (5) Incubate on a shaker at 4°C overnight to form nanomaterial-nanodisc.

[0040] (6) After incubation overnight, the nanomaterials were taken out and centrifuged at 10,000 rpm for 5 min using a centrifuge tube with a 30 kDa molecular weight cutoff to remove unbound lipids;

[0041] (7) To further purify the nanomaterials, the protein-containing nanomaterials were separated using Superdex 200 molecular sieves;

[0042] (8) The product was concentrated by centrifugation at 10,000 rpm for 5 min using a centrifuge tube with a molecular weight cutoff of 30 kDa, and finally the drug delivery system across the blood-brain barrier, LPC nanodisc, was obtained.

[0043] Comparative Example:

[0044] A drug delivery system - preparation of MPC (2-methacryloyloxyethyl phosphorylcholine) nanodiscs, comprising the following steps:

[0045] The preparation method of the MPC nanodisk is the same as that of Example 1, that is, the LPC in step (1) of Example 1 is replaced by MPC, which will not be described in detail here.

[0046] MPC was purchased from Aladdin.

[0047] Experimental testing:

[0048] 1. Characterization of LPC Nanodisks

[0049] (1) Dilute the LPC nanomaterials 5-10 times with ND Buffer, pipette 10 μL and drop it on the sealing film, place the copper mesh in contact with the nanomaterial droplet and suspend it, then incubate for 10 min;

[0050] (2) Use filter paper to absorb the excess solution on the copper mesh, draw 10 μL of uranium dye solution and drop it on the copper mesh on the sealing film, put the front of the copper mesh in contact with the uranium dye solution droplet, and incubate for 1 min;

[0051] (3) Use filter paper to absorb excess dye solution on the copper screen and fully evaporate the dye solution;

[0052] (4) LPC nanodisks were characterized using transmission electron microscopy (TEM).

[0053] like Figure 2 From the transmission electron microscope characterization image of the LPC nanodisk shown, it can be seen that the nanodisk produced by the method of the present invention has a relatively uniform size of about 10 nm.

[0054] 2. In vivo animal imaging (characterization of LPC nanodiscs’ function across the blood-brain barrier):

[0055] (1) MPC nanodisks were used as the control group. The fluorescence intensity of MPC and LPC nanodisks was measured using a microplate reader and fluorescence spectroscopy. The fluorescence intensity of the nanomaterial injections in different groups was adjusted to be the same according to the fluorescence size.

[0056] (2) Each C57 mouse was injected with 100 μL of nanomaterials via tail vein injection;

[0057] (3) After administration, mice were anesthetized by gas, and small animal imaging was performed using the Newton 7.0 in vivo imaging system. The imaging time points were determined to be 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 24h, 36h, 48h, 72h, and 96h, and fluorescent images of the mouse brain and liver were taken at each time point.

[0058] Depend on Figure 3 It can be seen that the LPC nanodiscs prepared by the present invention with LPC as a component can cross the blood-brain barrier to reach the brain area of ​​mice, and the effect is better than that of MPC nanodiscs, as shown by the stronger fluorescence intensity in the brain area of ​​mice injected with LPC nanodiscs, indicating that the LPC nanodiscs prepared by the present invention can efficiently cross the BBB. Figure 4 This is the trend of the data curve of the fluorescence intensity of the brain area of ​​mice 1-72 hours after the injection of the nanomaterial. It can be seen that the LPC nanomaterial of the present invention can exist stably in the body for a long time.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A drug delivery system across the blood-brain barrier, characterized in that: Liposomes prepared from 1-stearoyl-sn-glycero-3-phosphocholine LPC.

2. The drug delivery system across the blood-brain barrier according to claim 1, characterized in that: The liposome further comprises phospholipids, cell membrane far-infrared fluorescent probe, dimyristoylglycerol-polyethylene glycol 2000 and cholesterol; The molar ratio of the phospholipid, LPC, cell membrane far-infrared fluorescent probe, dimyristoylglycerol-polyethylene glycol 2000 and cholesterol is (65-75): (10-20): (10-14): 1: (5-10).

3. The drug delivery system across the blood-brain barrier according to claim 2, characterized in that: The phospholipid is dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine or dioleoylphosphatidylserine.

4. The drug delivery system across the blood-brain barrier according to claim 1, characterized in that: It also includes apolipoprotein, which forms nanodiscs with the liposome; the molar ratio of the apolipoprotein to the liposome is 1:(25-100).

5. The drug delivery system across the blood-brain barrier according to claim 4, characterized in that: The apolipoprotein is a membrane scaffold protein.

6. The drug delivery system across the blood-brain barrier according to claim 4, characterized in that: The apolipoprotein is MSP1E3D1, MSP1D1, MSP1D1-dH5 or MSP2N2.

7. A method for preparing a drug delivery system across the blood-brain barrier as claimed in any one of claims 4 to 6, characterized in that: The steps include: (1) dissolving each component of the liposome in chloroform and mixing well to obtain a liposome mixed system; (2) taking the liposome mixture obtained in step (1) into a nitrogen blowpipe, and blowing the lipids dry with a nitrogen blowpipe to form a blue lipid film; (3) dissolving the lipid film and the apolipoprotein in the nanodisc buffer solution respectively, and then mixing the two to obtain a mixed system; (4) Transfer the mixed system to SM2 Bio-beads moistened with ultrapure water; (5) Incubate the product obtained in step (4) on a shaking platform at 3-5° C. for 12-16 hours to obtain an incubation solution, purify the incubation solution, and prepare a nanodisk.

8. The method for preparing the drug delivery system across the blood-brain barrier according to claim 7, characterized in that: In the step (1), the lipid content in the system is 300 nmol.

9. The method for preparing a drug delivery system across the blood-brain barrier according to claim 7, characterized in that: The purification method in step (5) is to remove unbound lipids from the incubation solution by centrifugation in a centrifuge tube; then separate the nanodiscs containing proteins by molecular sieve; and then concentrate by centrifugation in a centrifuge tube.

10. The method for preparing a drug delivery system across the blood-brain barrier according to claim 7, characterized in that: The nanodisk buffer solution is a nanodisk buffer containing 1% n-octyl-β-D-pyranoglucoside; the molecular weight cutoff of the centrifuge tube is 30 kDa, the rotation speed is 10000 rpm, and the centrifugation time is 5 min; and the molecular sieve is Superdex 200 molecular sieve.

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