CircSCMH1 delivery system and preparation method and application thereof

Abstract

The invention discloses a circSCMH1 delivery system as well as a preparation method and application thereof, and belongs to the field of biological medicines. The method comprises the following steps: taking circSCMH1, and preparing a circSCMH1 sodium acetate solution by using a sodium acetate buffer solution; taking ionizable lipid, DSPC, cholesterol and PEG lipid, and preparing a lipid ethanol solution; injecting the lipid ethanol solution into the buffer solution to obtain a no-load nanoparticle suspension; the circSCMH1 sodium acetate solution and the no-load nanoparticle suspension are stirred and mixed, and an LNP initial solution loaded with circSCMH1 is obtained; and filtering to remove ethanol in the LNP initial solution, so as to obtain the circSCMH1-loaded lipid nanoparticles. The delivery system has high stability, and the circSCMH1 delivery pharmaceutical composition formed by the delivery system and the circSCMH1 delivery system has potential application value in treatment of cerebral apoplexy and can remarkably improve the cognitive function of mice after cerebral apoplexy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a circSCMH1 delivery system and a preparation method thereof, and applications thereof in improving cognitive function in ischemic stroke. Background Art

[0002] Stroke is a neurological disease characterized by high morbidity, high mortality, and high disability rate. It is the main cause of permanent disability in adults worldwide, seriously affecting the quality of life of patients and placing a heavy burden on patients' families and the social economy. Ischemic stroke accounts for 85% of all strokes, with brain damage caused by occlusion of cerebral blood vessels as the main pathological feature. Currently, clinical treatments for ischemic stroke include drug thrombolysis and mechanical thrombectomy. However, the treatment time window is narrow (only within 4-6 hours after onset), and only a very small number of patients can benefit. Mechanical thrombectomy is the most direct and effective measure, but due to its high requirements for the operating environment, patients often cannot receive timely treatment. Therefore, there is an urgent need to develop new treatment options that can benefit more patients.

[0003] circRNA is a non-coding RNA produced by reverse splicing of precursor RNA. In the past, it was believed that circRNA was only a byproduct of RNA splicing and did not have important biological functions. However, in recent years, many studies have shown that circRNA can act as a "miRNA sponge" to bind to miRNA, making the latter unable to bind to the target mRNA and blocking its RNA interference effect, thereby regulating the occurrence and development of various diseases. Previous studies have shown that multiple circRNAs are involved in the pathological process of acute ischemic stroke. In the brain tissue of tMCAO rats, the expression levels of 283 circRNAs changed, suggesting that circRNA is related to acute ischemic stroke and may play an important role in the disease process. In the plasma of AIS patients, 3 circRNAs were significantly upregulated, and the combination of the three had good diagnostic ability for AIS. In the peripheral blood mononuclear cells of AIS patients, 13 circRNAs were involved in the complex physiological and pathological processes after ischemia, such as neuroinflammatory and immune responses. The above characteristics suggest that circRNA has the potential to be used as a drug target and biomarker for acute ischemic stroke. The latest research shows that circSCMH1 can increase neuroplasticity after ischemic stroke and improve motor function recovery in rhesus monkeys and mice. However, there is currently no lipid nanoparticle-based drug delivery system developed to deliver circSCMH1, and the improvement of cognitive function of mice after stroke by circSCMH1 remains unknown. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention proposes a circSCMH1 delivery system and a preparation method and application thereof.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention relates to a method for preparing lipid nanoparticles for delivering circSCMH1, comprising the following steps:

[0007] Take circSCMH1 and prepare circSCMH1 sodium acetate solution with sodium acetate buffer;

[0008] Take ionizable lipids, DSPC, cholesterol and PEG lipids and prepare a lipid ethanol solution;

[0009] The lipid ethanol solution was injected into the buffer to obtain an empty nanoparticle suspension;

[0010] The circSCMH1 sodium acetate solution and the empty nanoparticle suspension are stirred and mixed to obtain an initial LNP solution loaded with circSCMH1;

[0011] The ethanol in the LNP initial solution was removed by filtration to obtain the lipid nanoparticles carrying circSCMH1.

[0012] Optionally, the ionizable lipid is one of DLin-MC3-DMA, NT1-O14B, and SM-102.

[0013] Optionally, the nitrogen-to-phosphorus ratio of the lipid component to circSCMH1 is (3-10):1.

[0014] Optionally, the molar ratio of the ionizable lipid, DSPC, cholesterol and PEG lipid is (40-65):(5-15):(30-50):(1-5).

[0015] The second aspect of the present invention relates to a delivery system for delivering circSCMH1, comprising a lipid component for carrying circSCMH1, wherein the lipid component comprises ionizable lipids, DSPC, cholesterol and PEG lipids.

[0016] Optionally, the ionizable lipid is one of DLin-MC3-DMA, NT1-O14B, and SM-102.

[0017] Optionally, the nitrogen-to-phosphorus ratio of the lipid component to circSCMH1 is (3-10):1.

[0018] Optionally, the molar ratio of the ionizable lipid, DSPC, cholesterol and PEG lipid is (40-65):(5-15):(30-50):(1-5).

[0019] Optionally, it also includes one or more of cationic polymers, lipid nanoparticles, inorganic nanoparticles, and small molecule compounds with targeted delivery function.

[0020] The third aspect of the present invention relates to a drug for treating ischemic stroke, comprising:

[0021] The above-mentioned delivery system for delivering circSCMH1, and a pharmaceutically acceptable carrier;

[0022] The pharmaceutically acceptable carrier includes: one or more of cationic polymers, lipid nanoparticles, inorganic nanoparticles, and small molecule compounds with targeted delivery function.

[0023] The fourth aspect of the present invention relates to the use of the above-mentioned delivery system or drug in the preparation of a drug for treating ischemic stroke, or for improving motor dysfunction or cognitive dysfunction caused by ischemic stroke.

[0024] In some embodiments, the pharmaceutical composition of the present invention can be administered via nasal, oral, intravenous, intraperitoneal, subcutaneous or brain microinjection routes.

[0025] In some embodiments, after the ischemic stroke model is established in mice, a single nasal administration is performed, and behavioral testing is performed until 28 days after the end of the administration. The brain, heart, liver, spleen, lungs, and kidneys are removed for RT-qPCR and flow cytometry analysis, and blood is collected from the orbits to prepare serum for ELISA testing.

[0026] Beneficial effects:

[0027] 1. The circSCMH1 of the present invention can effectively improve the recovery of neurological function after stroke and significantly improve the cognitive function of mice with statistical significance. The experimental results show that circSCMH1 is expected to be a drug for treating ischemic stroke and improving cognitive impairment after stroke.

[0028] 2. The delivery system of the present invention has the characteristics of high load and high stability, and can efficiently deliver circSCMH1, escape from endosomes after entering cells, and has higher brain targeting ability compared with ordinary lipid nanoparticles. A single nasal administration after delivery by lipid nanoparticles can effectively replenish the decreased expression level of circSCMH1 in brain tissue after ischemic stroke, and compared with tail vein administration, the non-target distribution of circSCMH1 in normal tissues and organs is significantly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings.

[0030] Figure 1Characterization of the physical and chemical properties of circSCMH1@LNP. Figure A is a schematic diagram of the preparation process of circSCMH1@LNP, Figure B is the particle size distribution of circSCMH1@LNP, Figure C is the TEM result potential of circSCMH1@LNP, Figure D is the particle size stability test result of circSCMH1@LNP, and Figure E is a summary of the various properties of circSCMH1@LNP.

[0031] Figure 2 The distribution of circSCMH1 in the brain after nasal and tail vein administration was detected by in vivo fluorescence imaging after cy5-labeled circSCMH1 was coated on LNP. A is the fluorescence imaging of the brain in vivo of mice, B is the fluorescence intensity of the isolated brain tissue of mice at each time point, C is the quantitative analysis of the fluorescence intensity of the mouse brain, D is the co-labeling of neurons and circSCMH1, and E is the co-labeling of astrocytes and circSCMH1.

[0032] Figure 3 Fluorescence imaging of cy5-labeled circSCMH1 after LNP encapsulation detected the accumulation in normal organs after nasal administration and tail vein administration. A is the whole-body fluorescence imaging of mice in vivo, B is the fluorescence intensity of peripheral tissues of mice in vitro at each time point after nasal administration, C is the fluorescence intensity of peripheral tissues of mice in vitro at each time point after intravenous administration, and DF is the quantitative analysis of the relative fluorescence intensity of peripheral tissues after two administration methods.

[0033] Figure 4 The results of behavioral tests to verify the in vivo level of LNP-coated circSCMH1 in the treatment of ischemic stroke in PT mice. A is a schematic diagram of the behavioral experimental process, B is stepping into the air, C is adhesion removal, and D is the cylinder result. The results showed that LNP-coated circSCMH1 significantly improved the sensorimotor dysfunction of stroke mice.

[0034] Figure 5 The results of cognitive function test for in vivo verification of LNP-encapsulated circSCMH1 in the treatment of ischemic stroke in PT mice. A is the result of water maze experimental training, B is the water maze trajectory diagram, and CF is the quantitative analysis of water maze trajectory results. The results showed that LNP-encapsulated circSCMH1 significantly improved cognitive dysfunction in stroke mice. DETAILED DESCRIPTION

[0035] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the raw materials and reagents used in the examples are conventional products that can be obtained commercially; experimental methods without specific conditions specified in the examples are usually carried out according to conventional conditions in the art or according to conditions recommended by manufacturers.

[0037] The information of some components involved in the examples of this application is as follows:

[0038] 1,2dilinoleyloxy3dimethylaminopropane(DLin-MC3-DMA,CAS No.1224606-06-7),

[0039] NT1-O14B(CAS No.2739805-64-0),

[0040] SM-102 (CAS No. 2089251-47-6),

[0041] 1,2-distaroyl-sn-glycero-3-phosphocholine (DSPC, CAS No.816-94-4),

[0042] 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000, CAS No. 160743-62-4).

[0043] LNP (Lipid Nanoparticle) is a lipid nanoparticle with the coexistence of multiple charged lipids. This delivery system has the characteristics of high load and high stability. It can efficiently deliver circSCMH1, escape from the endosome after entering the cell, and has a higher brain targeting ability compared with ordinary lipid nanoparticles.

[0044] Example 1

[0045] Preparation of 1-LNP-circSCMH1

[0046] Take circSCMH1 and use sodium acetate buffer (pH = 5.0) to prepare a circSCMH1 sodium acetate solution with pH = 5.0, circSCMH1 concentration of 10uM, and sodium acetate concentration of 25mM;

[0047] Ionizable lipids, DSPC, cholesterol and DMG-PEG2000 were prepared into a lipid ethanol solution with a total lipid concentration of 4 mg / mL using anhydrous ethanol; wherein, in terms of molar percentage, the lipid components contained DOTAP 1.5%, SM-102 50%, DSPC 8.5%, cholesterol 38.5%, DMG-PEG2000

[0048] 1.5%. 0.1 mL of lipid ethanol solution was injected into 1 mL of sodium acetate buffer using a 1 mL BD Shurui insulin syringe to prepare an empty nanoparticle suspension (LNP3). SM-102 can be replaced by DLin-MC3-DMA or NT1-O14B and prepared into empty nanoparticle suspensions with different processes (DLin-MC3-DMA is LNP1, NT1-O14B is LNP2);

[0049] 0.2 mL of the sodium acetate solution of circSCMH1 prepared in step (1) was injected into 0.2 mL of the empty nanoparticle solution prepared in step (2) using a 1 mL BD Shurui insulin syringe, and incubated for 1 minute under vortex stirring to obtain the initial LNP solution containing circSCMH1. The above steps (1) to (3) can be repeated to obtain more initial LNP solutions containing circSCMH1;

[0050] Use an ultrafiltration tube with a molecular weight cutoff of 100 kDa to ultrafilter at a centrifugal force of 13,000 g to remove ethanol in the LNP initial solution, adjust the circSCMH1 concentration to 300 uM with denuclease water, and adjust the pH to 7.0 with 0.1 M sodium hydroxide or hydrochloric acid solution to obtain a LNP suspension encapsulating circSCMH1.

[0051] 2-In vivo level verification showed that LNP-circSCMH1 had a more significant distribution in the peri-infarct area after nasal administration compared with tail vein administration, while effectively reducing its distribution in normal tissues and organs.

[0052] After PT (photothrombosis, PT) modeling, mice were divided into nasal administration group and intravenous administration group, and each group was set up with the following time points: 0h group, 2h group, 6h group, 12h group and 24h group. A single administration was given through the nasal cavity or tail vein 24 hours after modeling, and fluorescence imaging and PCR quantitative detection and analysis were performed at the corresponding time points.

[0053] 3-Verify the therapeutic effect of LNP-circSCMH1 on ischemic stroke at the in vivo level.

[0054] After PT (photothrombosis, PT) modeling, mice were divided into five groups, namely sham group, PT group, LNP-circSCMH1 (0.012 mg / kg) group, LNP-circSCMH1 (0.036 mg / kg) group, and LNP-circSCMH1 (0.12 mg / kg) group. A single nasal administration was performed 24 hours after modeling. Specific behavioral test items include: cylinder test, adhesive removal test, and grid-walking test.

[0055] The results are as follows Figure 5 As shown in the figure, it can be seen that LNP-circSCMH1 has a significant improvement on ischemic stroke in mice in the 0.012-0.12 mg / kg dose group, and the effect is statistically significant.

[0056] In other embodiments of the present invention, in addition to the technical contents disclosed in the above embodiments, those skilled in the art can also combine the excipients known in the art and pharmaceutically acceptable to further enhance the therapeutic effect of the drug of the present application or the delivery efficiency of the delivery system. In other words, the present application is not limited to the forms of the drugs and delivery systems disclosed in the above embodiments. These excipients include but are not limited to cationic polymers, lipid nanoparticles, inorganic nanoparticles, small molecule compounds with targeted delivery functions, and other stroke drug components that can be carried on LNP. The above technical contents can be consulted by those skilled in the art in the prior art, and will not be repeated in this application.

[0057] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0058] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing lipid nanoparticles carrying circSCMH1, characterized in that: The following steps are involved: Take circSCMH1 and prepare circSCMH1 sodium acetate solution with sodium acetate buffer; Take ionizable lipids, DSPC, cholesterol and PEG lipids and prepare a lipid ethanol solution; The lipid ethanol solution was injected into the buffer to obtain an empty nanoparticle suspension; The circSCMH1 sodium acetate solution and the empty nanoparticle suspension are stirred and mixed to obtain an initial LNP solution loaded with circSCMH1; The ethanol in the LNP initial solution was removed by filtration to obtain the lipid nanoparticles carrying circSCMH1.

2. The method for preparing lipid nanoparticles carrying circSCMH1 according to claim 1, characterized in that: The ionizable lipid is one of DLin-MC3-DMA, NT1-O14B, and SM-102.

3. The method for preparing lipid nanoparticles carrying circSCMH1 according to claim 1, characterized in that: The nitrogen-to-phosphorus ratio of the lipid component to circSCMH1 was 6:

1.

4. The method for preparing lipid nanoparticles carrying circSCMH1 according to claim 1, characterized in that: The molar ratio of the ionizable lipid, DSPC, cholesterol and PEG lipid is (40-65): (5-15): (30-50): (1-5).

5. A delivery system for delivering circSCMH1, characterized in that: The invention comprises a lipid component for carrying circSCMH1, wherein the lipid component comprises an ionizable lipid, DSPC, cholesterol and PEG lipid.

6. The delivery system for delivering circSCMH1 according to claim 5, characterized in that The ionizable lipid is one of DLin-MC3-DMA, NT1-O14B, and SM-102.

7. The delivery system for delivering circSCMH1 according to claim 5, characterized in that: The nitrogen-phosphorus ratio of the lipid component to circSCMH1 is (3-10):

1.

8. The delivery system for delivering circSCMH1 according to claim 5, characterized in that The molar ratio of the ionizable lipid, DSPC, cholesterol and PEG lipid is (40-65): (5-15): (30-50): (1-5).

9. A drug for treating ischemic stroke, comprising: The delivery system for delivering circSCMH1 according to any one of claims 5 to 8, and a pharmaceutically acceptable carrier; The pharmaceutically acceptable carrier includes: One or more of cationic polymers, lipid nanoparticles, inorganic nanoparticles, and small molecule compounds with targeted delivery function.

10. Use of the delivery system according to any one of claims 5 to 8 or the drug according to claim 9 in the preparation of a drug for treating ischemic stroke, or for improving motor dysfunction or cognitive dysfunction caused by ischemic stroke.

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