SiRNA targeting neutrophil HMGB2, delivery system and construction method and application thereof

Abstract

The invention discloses siRNA of targeted neutrophil HMGB2, a delivery system and a construction method and application of the delivery system, and belongs to the technical field of biological medicine. The siRNA of the targeted neutrophil HMGB2 comprises a positive-sense strand and an antisense strand, and the sequences of the positive-sense strand and the antisense strand are respectively SEQ ID NO: 1 and SEQ ID NO: 2. The siRNA expressed by HMGB2 target genes is prepared, lipid nanoparticles with a neutrophil targeting function are further provided to wrap the siRNA to serve as a delivery system, HMGB2 gene expression in neutrophil in the brain of a mouse after stroke is remarkably inhibited, brain injury of the mouse after stroke is relieved, and a new potential medicine is provided for treatment of stroke.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to siRNA targeting neutrophil HMGB2, delivery system and construction method and application thereof. BACKGROUND

[0002] A number of studies have confirmed that opening the occluded blood vessels and rescuing the ischemic penumbra is the key treatment for AIS patients with large vessel occlusion. Although the current endovascular treatment can achieve a recanalization rate of about 80%, however, even if the occluded blood vessels are opened in time, nearly half of the patients still have poor functional recovery later, that is, "ineffective recanalization". Ineffective recanalization consumes a lot of resources and brings heavy burden to individuals, families and society, and how to reduce the occurrence of ineffective recanalization after endovascular treatment of AIS is an important scientific problem in the field of neurointervention. Therefore, it is of great significance to further study the pathogenesis of ineffective recanalization, screen potential new targets and develop new neuroprotective drugs to further improve the efficacy of endovascular treatment for AIS patients.

[0003] Studies have shown that the core mechanism of ineffective recanalization is closely related to neutrophil-mediated microcirculation thrombus inflammation. In recent years, developing drugs targeting neutrophils to intervene in ischemic stroke is a research hotspot in the field. At present, the drug development strategies targeting neutrophils mainly include inhibiting their infiltration and activation, promoting their apoptosis, etc. However, due to the lack of target specificity and other reasons, the effect of many drugs in clinical trials is limited, and even there are toxic side effects. So far, there is no clinical drug targeting neutrophils that can precisely intervene in ischemic stroke. Therefore, how to precisely inhibit the harmful effects of neutrophils without affecting their normal immune function is the key and difficulty faced by drug development targeting neutrophils. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides siRNA targeting neutrophil HMGB2, a delivery system and a construction method and application thereof.

[0005] The object of the present application can be achieved by the following technical solutions:

[0006] In a first aspect of the present application, siRNA targeting neutrophil HMGB2 is provided, which comprises a sense strand and an antisense strand, and the sequences are SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0007] Optionally, the 3' end of the sense strand and / or the antisense strand is overhanging and substituted with deoxyribonucleotides; or the 3' end of the sense strand and / or the antisense strand is overhanging and added with deoxyribonucleic acid dTdT or added with ribonucleotides UU

[0008] Optionally, the sense strand and / or antisense strand nucleotide is modified by fluorine substitution, methoxy modification or thio backbone modification.

[0009] In a second aspect, the present application relates to a delivery system, comprising:

[0010] The siRNA targeting neutrophil HMGB2 as described above;

[0011] The lipid component encapsulating the siRNA comprises at least one of ionizable lipid, DSPC, cholesterol, PEG lipid and PEG lipid targeting neutrophil formyl peptide receptor.

[0012] Optionally, the amino acid sequence of the neutrophil-targeting short peptide is cFLFLFK, wherein c represents cinnamoyl peptide modification.

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

[0014] Optionally, the ionizable lipid is at least one of DLin-MC3-DMA, ALC0315, SM-102.

[0015] In a third aspect, the present application relates to a preparation method of the delivery system, comprising the following steps:

[0016] The siRNA targeting neutrophil HMGB2 as described above is configured into siRNA sodium acetate buffer solution with sodium acetate buffer.

[0017] The ionizable lipid, DSPC, cholesterol, PEG lipid and cinnamoyl peptide-modified PEG lipid are configured into a lipid mixture solution.

[0018] The lipid mixture solution is injected into the siRNA sodium acetate buffer solution to obtain the LNP solution encapsulating the siRNA targeting neutrophil HMGB2 as described above.

[0019] Optionally, the amino acid sequence of the neutrophil-targeting short peptide is cFLFLFK, wherein c represents cinnamoyl peptide modification.

[0020] The nitrogen to phosphorus ratio of the lipid component to siRNA is (3-10):1.

[0021] Optionally, the solvent in the LNP solution is removed by ultrafiltration to obtain the siRNA-loaded lipid nanoparticle.

[0022] In a fourth aspect, the present application relates to the siRNA targeting neutrophil HMGB2 as described above or the delivery system as described above, for use in the preparation of a medicament for treating acute ischemic stroke.

[0023] Optionally, the sodium acetate concentration is 25 mM, and the pH of the prepared siRNA buffer is 5.0.

[0024] Optionally, the total lipid concentration in the lipid mixture is 8-10 mg / mL.

[0025] Optionally, the volume ratio of the lipid mixture to the buffer solution is 1:3.

[0026] Optionally, the pH value of the prepared siRNA-loaded lipid nanoparticles is approximately 7.4.

[0027] The beneficial effects of this invention are:

[0028] 1. This invention utilizes siRNA targeting the HMGB2 target gene expression in neutrophils, administered via tail vein injection to mice in a single dose, significantly inhibiting HMGB2 expression in neutrophils in the periinfarct area after stroke in mice. Therefore, the si-HMGB2 delivery system provided by this invention can serve as a neutrophil HMGB2 inhibitor, formulated into a drug with one or more pharmaceutically acceptable excipients, and applied to treat indications related to HMGB2 gene expression, such as cardiovascular diseases, neurodegenerative diseases, cancer, and autoimmune diseases.

[0029] 2. In some embodiments, the pharmaceutical composition of the present invention can be administered via tail vein injection.

[0030] 3. In some embodiments, in a mouse model of ischemic stroke, mice were administered a single dose via tail vein injection. Brains were then removed 1 day and 3 days after the initial administration for Western blotting and PCR analysis. The results showed that the siRNA drug of this invention significantly inhibited HMGB2 gene expression in the mouse brain, alleviating post-stroke brain injury, and this effect was statistically significant. These experimental results indicate that siRNA targeting the HMGB2 gene expression in neutrophils holds promise as a treatment for stroke. Attached Figure Description

[0031] The invention will now be further described with reference to the accompanying drawings.

[0032] Figure 1 The preparation and characterization of lipid nanoparticles (pepLNP) are shown in Figure a. The preparation process of pepLNP is shown in Figure b. The microstructure of pepLNP under transmission electron microscopy is shown in Figure cd. The particle size distribution of LNP and pepLNP is shown in Figure e. The PDI of LNP and pepLNP is shown in Figure f. The zeta potential of LNP and pepLNP is shown in Figure g. The encapsulation efficiency of LNP and pepLNP is shown in Figure g.

[0033] Figure 2To verify the in vivo delivery efficiency of pepLNP-encapsulated si-HMGB2 to the mouse brain. Figure a is a near-infrared fluorescence image of isolated brain tissue, Figure b is a quantitative analysis of Figure a, Figure c is a near-infrared fluorescence image of the brain at different time points at both in vivo and isolated levels, and Figures de are quantitative analyses of Figure c.

[0034] Figure 3 To verify the in vivo efficacy of pepLNP-encapsulated si-HMGB2 knockdown of HMGB2 in brain tissue. Figures ab show the expression and quantitative analysis of HMGB2 in the peri-infarct area of ​​mice taken 1 day after tMCAO modeling using Western blotting (WB); Figure cd shows the expression and quantitative analysis of HMGB2 in the peri-infarct area of ​​mice taken 3 days after tMCAO modeling using WB; Figure ef shows the expression and quantitative analysis of HMGB2 in peripheral blood taken 3 days after tMCAO modeling using WB; Figures ef and ef show the HMGB2 level detected by PCR in the peri-infarct area of ​​mice taken 1 and 3 days after tMCAO modeling. These experimental results all demonstrate that HMGB2 expression was significantly knocked down.

[0035] Figure 4 To verify in vivo effects of pepLNP-coated si-HMGB2 on sensorimotor function in mice after stroke and its effect on cerebral thrombosis and inflammation in stroke-affected mice. Figure a shows the timeline of the mouse pharmacodynamic experiment. Figures b and c show the significant reduction in infarct volume in tMCAO mice after treatment with pepLNP-coated si-HMGB2, as shown by TTC staining. Figures de show the significant reduction in brain atrophy volume in tMCAO mice after treatment with pepLNP-coated si-HMGB2, as shown by Nissl staining. Figures f and h show the mouse stepping, cylinder, and adhesion removal behavioral experiments, respectively. The results indicate that sensorimotor function was significantly improved in tMCAO mice after treatment with pepLNP-coated si-HMGB2. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] In some embodiments of the present invention, a research process is disclosed for a siRNA targeting HMGB2, a delivery vector, and its therapeutic effect on stroke, including the following steps:

[0038] 1. HMGB2 sequence and siRNA design

[0039] HMGB2 was sequenced and analyzed to obtain a human-mouse homologous HMGB2 sequence fragment. Based on this fragment, DICER substrate sequence was designed and chemically modified, and siRNA structure was synthesized. Specific information is shown below.

[0040] The sense strand of the HMGB2 target has a nucleotide sequence of 5'-GAGCGACAAAGCUCGUUAUTT-3' or its homologous sequence, and the antisense strand has a nucleotide sequence of 5'-AUAACGAGCUUUGUCGCUCTT-3' or its homologous sequence.

[0041] 2. Preparation process of pepLNP coated Si-HMGB2

[0042] (1) Take si-HMGB2 and prepare a si-HMGB2 sodium acetate solution with pH=5.0, si-HMGB2 concentration of 0.2mg / mL and sodium acetate concentration of 25mM using sodium acetate buffer (pH=5.0);

[0043] (2) Take ionizable lipids (DLin-MC3-DMA in this embodiment), DSPC, cholesterol, and PEG lipids, and prepare a lipid ethanol solution with a total lipid concentration of 8-10 mg / mL using anhydrous ethanol; inject 0.1 mL of the lipid ethanol solution into 0.3 mL of sodium acetate buffer using a 1 mL BD Reliable insulin syringe at a volume ratio of 1:3 to prepare empty lipid nanoparticles (LNPs); wherein the PEG lipids are loaded with neutrophil-targeting short peptides, the specific amino acid sequence of which is cFLFLFK, where c represents cinnamyl peptide modification, used to target neutrophil formyl peptide receptors.

[0044] (3) Inject 0.1 mL of the si-HMGB2 sodium acetate solution obtained in step (1) into 0.3 mL of the pepLNP obtained in step (2) using a 1 mL BD Shurui insulin syringe, and incubate for 1 minute under vortex stirring to obtain pepLNP (si-HMGB2@pepLNP) loaded with si-HMGB2.

[0045] The above steps (1) to (3) can be repeated to merge and obtain more si-HMGB2@pepLNP.

[0046] If anhydrous ethanol is present during the preparation process, the ethanol in pepLNP is removed by ultrafiltration with a molecular weight cutoff of 100kDa using a centrifugal force of 13000g. The pH of pepLNP is then adjusted to 7.4 using PBS buffer to obtain the si-HMGB2@pepLNP suspension.

[0047] 3. Detect the brain delivery efficiency of si-HMGB2@pepLNP

[0048] C57BL / 6 mice were divided into 3 groups: sham+ Cy5 si-HMGB2@pepLNP group, tMCAO+ Cy5 si-HMGB2@LNP group and tMCAO+ Cy5 Five mice per group (si-HMGB2@pepLNP) were administered the drug via tail vein injection to C57BL / 6 mice at a dose of 0.8 mg / kg. Mice were anesthetized with isoflurane at 3, 6, 12, and 24 hours post-injection, and fluorescence intensity was observed using an IVIS Spectrum imaging system. Mice were sacrificed after imaging, and their brains were harvested for further fluorescence imaging. tMCAO+ Cy5 si-HMGB2@LNP group and tMCAO+ Cy5 The experimental results of the si-HMGB2@pepLNP group show that, after targeting neutrophils, the drug can be precisely delivered to the brain.

[0049] 4. In vivo verification of the therapeutic effect of si-HMGB2@pepLNP on ischemic stroke.

[0050] First, mice included in the behavioral experiment were trained, and then a tMCAO model of ischemic stroke was established in mice, followed by randomization into groups. C57BL / 6 stroke model mice were divided into three groups (n=20 per group): the sham group, the tMCAO+si-NC@pepLNP group, and the tMCAO+si-HMGB2@pepLNP group. Drug administration was administered one hour after reperfusion, with a total of one dose. On the third day, five mice from each group were randomly selected for TTC staining. The remaining mice underwent behavioral tests after treatment to verify the therapeutic effect of si-HMGB2@pepLNP on ischemic stroke. Specific behavioral tests included the cylinder test, grid-walking test, and adhesive removal test. After the behavioral tests, mouse brain tissue was collected for Nissl staining.

[0051] The results are shown in the figure.

[0052] As shown in the figure, compared with the si-NC group, the expression of HMGB2 after si-HMGB2@pepLNP intervention was significantly reduced, indicating that the si-HMGB2@pepLNP constructed in this application had a significant knockdown effect on HMGB2. Figure 4 The results showed that, in comparison, intervention with si-HMGB2@pepLNP significantly reduced brain atrophy volume. Figure 4Behavioral experiments on mouse tripping, cylinder movement, and adhesion removal showed that si-HMGB2@pepLNP significantly improved sensorimotor function after stroke in mice, and this improvement was statistically significant.

[0053] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0054] The foregoing has shown and described 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. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A siRNA targeting HMGB2 in neutrophils, characterized in that, It includes a justice chain and an antisense chain, with sequences SEQ ID NO:1 and SEQ ID NO:2, respectively.

2. The siRNA targeting HMGB2 in neutrophils according to claim 1, characterized in that, The 3' end overhangs of the sense and / or antisense strands are substituted with deoxyribonucleotides; or, the 3' end overhangs of the sense and / or antisense strands are supplemented with deoxyribonucleic acid dTdT or ribonucleotide UU.

3. The siRNA targeting HMGB2 in neutrophils according to claim 1, characterized in that, The sense and / or antisense nucleotides are modified with fluorine substitution, methoxylation, or thiosclerosis.

4. A delivery system, characterized in that, include: The siRNA targeting HMGB2 neutrophils as described in any one of claims 1 to 3; In addition, the lipid components encapsulating the siRNA include ionizable lipids, DSPC, cholesterol, PEG lipids, and PEG lipids loaded with neutrophil-targeting short peptides.

5. The delivery system according to claim 4, characterized in that, The amino acid sequence of the neutrophil-targeting short peptide is cFLFLFK; the nitrogen-to-phosphorus ratio of the lipid component to the siRNA is (3-10):

1.

6. The delivery system according to claim 4, characterized in that, The ionizable lipid is at least one of DLin-MC3-DMA, ALC0315, and SM-102.

7. A method for preparing a delivery system, characterized in that, Includes the following steps: S1. Prepare a sodium acetate buffer solution for the siRNA targeting HMGB2 neutrophils as described in any one of claims 1 to 3 using sodium acetate buffer. Ionizable lipids, DSPC, cholesterol, PEG lipids, and PEG lipids modified with neutrophil-targeting short peptides were prepared into a lipid mixture solution. S3. Inject the lipid mixture into the siRNA sodium acetate buffer to obtain an LNP solution containing the siRNA targeting neutrophil HMGB2 as described in any one of claims 1 to 3.

8. The method for preparing the delivery system according to claim 7, characterized in that, The amino acid sequence of the neutrophil-targeting short peptide is cFLFLFK, where c represents cinnamic yl peptide modification.

9. The method for preparing the delivery system according to claim 7, characterized in that, The solvent in the LNP solution is removed by ultrafiltration to obtain lipid nanoparticles loaded with siRNA.

10. The use of the siRNA targeting neutrophil HMGB2 as described in any one of claims 1 to 3 or the delivery system as described in any one of claims 4 to 6 in the preparation of a medicament for treating acute ischemic stroke.

Images