Delivery system of saRNA medicine

Abstract

The invention discloses a saRNA drug delivery system, which is composed of a saRNA drug and lipid nanoparticles, and the lipid nanoparticles are composed of ionizable cationic lipid, neutral phospholipid, sterol lipid and PEGylated phospholipid. The delivery system is stable in structure and good in dispersity, and can be prepared into an injection-grade nano preparation; meanwhile, saRNA medicine can be effectively delivered to a cell nucleus, the mRNA level of a target gene FXR is remarkably up-regulated, and a definite treatment effect is achieved.

Description

Technical Field

[0001] The present invention relates to a delivery system for nucleic acid drugs, and in particular to a delivery system for saRNA drugs. Background Art

[0002] Nucleic acid drugs refer to a class of small molecule drugs that can play a role by regulating gene transcription and expression. Currently, miRNA, siRNA, saRNA and ASO are widely used. Although compared with traditional small molecule drugs and antibody drugs, nucleic acid drugs have the advantages of simple design, high therapeutic efficiency, low drug toxicity and strong specificity, nucleic acid drugs still face problems such as poor stability in the body, easy degradation by nucleases, difficult transmembrane transport due to carrying negative charges, and easy hydrolysis by lysosomes after entering cells.

[0003] Encapsulating nucleic acid drugs in delivery carriers can, to a certain extent, isolate them from direct contact with nucleases in the body, thereby improving their stability in the body. At the same time, after loading the drug, the delivery carrier can assist the nucleic acid drug to cross the body barrier and escape from the lysosomes to reach the target, thus achieving the purpose of precise treatment of nucleic acid drugs. At present, there are relatively mature delivery systems for nucleic acid drugs such as ASO and siRNA. For example, related drugs based on the GalNac system include Givosiran, Lumasiran, and Inclisiran; nucleic acid drugs based on the LNP system include Patisiran, etc. However, ASO and siRNA nucleic acid drugs only work in the cytoplasm, while saRNA nucleic acid drugs need to pass through the cytoplasm and enter the nucleus to work. At present, there is no effective delivery carrier for saRNA nucleic acid drugs. Summary of the invention

[0004] Purpose of the invention: The present invention aims to provide a delivery system that can stably carry saRNA drugs into the cell nucleus.

[0005] Technical solution: The delivery system of the saRNA drug of the present invention comprises a saRNA drug and lipid nanoparticles, wherein the saRNA drug is a sense chain with a 5'-AAGGUUCCUUUCUAUGUUU-3' sequence and an antisense chain with a 5'-AAACAUAGAAAGGAACCUU-3' sequence (hdsFXR-103) having an RNAs effect, or a sense chain with a 5'-GGGAAGUGAUAGAGCUAUU-3' sequence and an antisense chain with a 5'-AAUAGCUCUAUCACUUCCC-3' sequence (hdsFXR-319); the lipid nanoparticles comprise ionizable cationic lipids, neutral phospholipids, sterol lipids and PEGylated phospholipids, and the ionizable cationic lipids are selected from ALC-0315, ALC-0315anlogue-2, ALC-0159, M-DTDA-2000, and M-DMG-2000.

[0006] The present invention designs a specific delivery saRNA-LNP, whose core lipid adopts ionizable cationic lipid ALC-0315, presents near electrical neutrality and has good stability. In addition to ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol and polyethylene glycol (PEG)-phospholipids are also included. By optimizing the structural lipids (neutral auxiliary phospholipids and cholesterol), LNP can form a solid structure and maintain the stability of LNP; by optimizing PEG-phospholipids, LNP has a suitable particle size to ensure that the drug can be delivered to the cell nucleus and give full play to the therapeutic effect of the drug.

[0007] Preferably, the neutral phospholipid is selected from DSPC, DSPE, DEPC, DPPE, and DPPC.

[0008] Preferably, the sterol lipid is selected from cholesterol and sitosterol.

[0009] Preferably, the PEGylated phospholipid is selected from one or more of DMG-PEG2000, DSPE-PEG2000-Galactose, DSPE-MPEG2000, and DSPE-PEG-Glucose.

[0010] More preferably, the PEGylated phospholipid is selected from a combination of DMG-PEG2000 and DSPE-PEG2000-Galactose.

[0011] Preferably, the mass ratio between the lipid nanoparticles and the saRNA drug is 20:1.

[0012] Preferably, the molar ratio of the ionizable cationic lipid, neutral phospholipid, sterol lipid and PEGylated phospholipid is (73.3-78):(14.6-15):(56.4-60):(2.1-2.2).

[0013] More preferably, the molar ratio of the ionizable cationic lipid, neutral phospholipid, sterol lipid and PEGylated phospholipid is selected from 73.3:14.6:56.4:2.1, 78:15:60:2.2.

[0014] More preferably, the molar ratio between the DMG-PEG2000 and DSPE-PEG2000-Galactose is selected from (1.1-1.2):1.

[0015] More preferably, the molar ratio between the DMG-PEG2000 and DSPE-PEG2000-Galactose is selected from 1.1:1 and 1.2:1.

[0016] Specifically, the saRNA drug delivery system of the present invention comprises a sense strand having a 5'-AAGGUUCCUUUCUAUGUUU-3' sequence and an antisense strand having a 5'-AAACAUAGAAAGGAACCUU-3' sequence (hdsFXR-103) having an RNAs effect, or a sense strand having a 5'-GGGAAGUGAUAGAGCUAUU-3' sequence and an antisense strand having a 5'-AAUAGCUCUAUCACUUCCC-3' sequence (hdsFXR-319). NA drug and lipid nanoparticles composed of ALC-0315, DSPC, cholesterol, DMG-PEG2000, and DSPE-PEG2000-Galactose; the mass ratio between the lipid nanoparticles and the saRNA drug is 20:1; the molar ratio between ALC-0315, DSPC, cholesterol, DMG-PEG2000, and DSPE-PEG2000-Galactose in the lipid nanoparticles is selected from 73.3:14.6:56.4:1.1:1, 78:15:60:1.2:1.

[0017] Preferably, the particle size of the saRNA drug delivery system of the present invention is 100±10 nm.

[0018] The preparation method of the saRNA drug delivery system of the present invention comprises the following steps:

[0019] Cationic lipids, neutral phospholipids, sterol lipids and PEGylated phospholipids are dissolved in an ethanol solution to generate an oil phase, the saRNA drug is dissolved in a citric acid-sodium citrate solution at pH = 4 to prepare an aqueous phase, the oil phase and the aqueous phase are quickly mixed into a citric acid-sodium citrate solution at pH = 6, and the saRNA drug delivery system is obtained by mixing.

[0020] Use of the saRNA drug delivery system of the present invention in the preparation of drugs for related diseases caused by down-regulation of FXR expression.

[0021] Further preferred is the use of the saRNA drug delivery system in the preparation of drugs for anti-hepatocyte apoptosis.

[0022] More preferably, the drug is a drug for treating liver disease.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0024] The saRNA-LNP designed in the present invention has a stable structure and good dispersibility, and can be prepared as an injection-grade nanoformulation; at the same time, it can effectively deliver saRNA drugs to the cell nucleus, significantly upregulate the mRNA level of the target gene FXR, and has a clear therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the particle size distribution diagram of the saRNA drug delivery system;

[0026] Figure 2 This is the zeta potential diagram of the saRNA drug delivery system;

[0027] Figure 3 TEM image of saRNA drug delivery system;

[0028] Figure 4 The results of the solution stability test of the saRNA drug delivery system are as follows;

[0029] Figure 5 The regulatory effect of saRNA drug delivery system on FXR mRNA level ( *** P<0.001). DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with embodiments.

[0031] Example 1: Preparation of saRNA-LNP

[0032] 1. Experimental Materials

[0033] The saRNA drug was obtained by referring to the method disclosed in CN112941076B, and the remaining biological materials / reagents can be obtained from commercial sources.

[0034] 2. Experimental methods

[0035] Ionizable cationic lipid (ALC-0315) was diluted with 90% ethanol to a final concentration of 2 mg / mL solution. Neutral phospholipids (DSPC), sterol lipids (cholesterol) and PEGylated phospholipids (DMG-PEG2000 and DSPE-PEG2000-Galactose) were dissolved in 90% ethanol to prepare a lipid solution with a final concentration of 2 mg / mL for use. Accurately pipette 13.34 μL ALC-0315, 16.5 μL DSPC, 3.93 μL DMG-PEG2000, 31.24 μL cholesterol, 4 μL DSPE-PEG2000-galactose and 30.99 μL 90% ethanol, mix well and set aside. Accurately pipette 8 μL saRNA mother solution, add 92 μL pH = 4 0.01 M citric acid buffer, mix well and set aside. Build a "Y"-shaped pipeline, quickly mix the aqueous and oil phase solutions, and collect the mixed solution into an EP tube containing 200 μL of 0.01M citric acid buffer at pH = 6 to obtain a liposome suspension. The ultrafiltration tube is centrifuged and washed to remove ethanol and uncaptured saRNA to obtain saRNA-LNP. This formula can be used to prepare saRNA-LNP after being scaled up in equal proportions, wherein the mass ratio of the prepared liposome LNP to saRNA is 20:1, and the saRNA drug has a sense strand of the 5'-GGGAAGUGAUAGAGCUAUU-3' sequence and an antisense strand of the 5'-AAUAGCUCUAUCACUUCCC-3' sequence (hdsFXR-319).

[0036] Example 2: Characterization of saRNA-LNPs

[0037] 1. Experimental methods

[0038] (1) Particle size distribution and Zeta potential

[0039] The saRNA-containing LNP solution prepared in Example 1 was injected into a particle size dish, and its particle size and Zeta potential were detected using a Malvern particle size analyzer.

[0040] (2) Morphology observation by transmission electron microscopy

[0041] The saRNA-containing LNP solution prepared in Example 1 was evenly spread on a copper mesh, dried, negatively stained with 2.0% sodium phosphotungstate, dried again, and then the morphology of the nanoparticles was observed under a transmission electron microscope.

[0042] (3) Stability test

[0043] The saRNA-containing LNP solution prepared in Example 1 was resuspended in 1×PBS, and the particle size of the nanoparticles was detected using a Malvern particle size analyzer on days 1, 2, 3, 4, 5, 6, and 7, respectively, and stored at 4° C. The stability of the nanoformulation was determined based on the change in particle size.

[0044] 2. Experimental results

[0045] (1) Particle size, potential and morphology of saRNA-LNP

[0046] like Figure 1 As shown, the particle size of saRNA-LNP is normally distributed, with a particle size of about 100 nm and a narrow distribution, indicating that the nanocarrier system has good monodispersity and meets the requirements of injection; at the same time, carrying different saRNAs does not significantly change the particle size of saRNA-LNP. Therefore, the particle size of the saRNA-LNP designed by the present invention is relatively uniform.

[0047] like Figure 2 As shown in the figure, the absolute value of the Zeta potential of saRNA-LNP is small and the distribution is more uniform; at the same time, there is no significant difference when carrying different saRNAs.

[0048] like Figure 3 As shown, saRNA-LNP is spherical and has a clear phospholipid bilayer structure. The particle size is about 100 nm, which is consistent with the Malvern particle size measurement results.

[0049] (2) Stability of saRNA-LNP

[0050] like Figure 4 As shown, the particle size of LNPs carrying different saRNAs did not change significantly after being placed in PBS buffer or 10% FBS for 7 days, indicating that the saRNA-LNPs designed in the present invention have good stability.

[0051] Example 3: Delivery Applications of LNP

[0052] 1. Experimental methods

[0053] In order to further investigate the application of the LNP designed in the present invention in saRNA delivery, FXR saRNA-LNP was transfected into hepatic parenchymal cells of human FXR transgenic mice for study.

[0054] (1) Cell culture and transfection

[0055] The human FXR gene mouse hepatocytes were isolated and cultured in T25 cell culture flasks at 37°C, 5% CO2 and 90% relative humidity. The culture medium was DMEM containing 10% fetal bovine serum, with penicillin 1 unit / ml and streptomycin 1 μg / ml added. The cells were replaced every other day and subcultured every 3 days. Cells in the logarithmic growth phase were cultured at 2×10 5 Each well was inoculated in a 6-well plate. After the cells adhered for 24 h, 1 μg of saRNA-LNP suspension was added to each well, and the cells were collected after 48 h.

[0056] (2)PCR

[0057] 1) After the cells are stimulated by the drug, take the cell plate out of the cell culture incubator. After washing with pre-cooled PBS, add 1.0 ml of RNAiso Plus. After the cells are completely lysed, add 200 μl of chloroform, shake vigorously for 15 seconds, leave at room temperature for 5 minutes, and centrifuge at 12000g for 15 minutes.

[0058] 2) Carefully transfer 300 μl of the upper aqueous phase to a new tube, add 300 μl of isopropanol, invert to mix, leave at room temperature for 10 min, then centrifuge at 12,000 g for 10 min and discard the supernatant.

[0059] 3) Wash the RNA precipitate with 1.0 ml of pre-cooled 75% ethanol, then centrifuge at 12000 g for 5 min, discard the supernatant to obtain total RNA, and re-dissolve it with 20 μl of DEPC water, and dilute to 0.5 μg / μl after quantification.

[0060] 4) Reverse transcription, the specific ratio requirements are as follows:

[0061] Reagent Name Dosage 10*RTBuffer 2.0μl 25*dNTPMix(100mM) 0.8μl 10*RTRandomPrimers 2.0μl MultiScribeReverseTransscriptase 1.0μl <![CDATA[RnasefreedH2O]]> 4.2μl Total 10μl Total RNA 10μl

[0062] 5) Real-time fluorescence quantitative PCR, the specific ratio requirements are as follows:

[0063] Reagent Name Dosage SYBRGreen 7.5μl PCRForwardPrimer (10 μM) 1.0μl PCR Reverse Primer (10 μM) 1.0μl cDNA 1.0μl <![CDATA[RnasefreedH2O]]> 4.5μl Total 15.0μl

[0064] The PCR conditions were as follows:

[0065] Stage 1: Pre-denaturation: 95°C, 1 min;

[0066] Stage 2: PCR reaction: 95°C for 15 seconds, 60°C for 15 seconds, 72°C for 30 seconds, 40 cycles;

[0067] Stage 3: Melting curve analysis: 65-95℃.

[0068] 2. Experimental results

[0069] like Figure 5 As shown, the saRNA-LNP designed by the present invention can significantly upregulate the level of FXR mRNA, indicating that it can effectively transfect FXR saRNA into cells and enter the cell nucleus.

Claims

1. A saRNA drug delivery system, characterized in that: The invention comprises a saRNA drug and lipid nanoparticles, wherein the saRNA drug has a sense strand of a 5'-AAGGUUCCUUUCUAUGUUU-3' sequence and an antisense strand of a 5'-AAACAUAGAAAGGAACCUU-3' sequence, or has a sense strand of a 5'-GGGAAGUGAUAGAGCUAUU-3' sequence and an antisense strand of a 5'-AAUAGCUCUAUCACUUCCC-3' sequence; the lipid nanoparticles comprise ionizable cationic lipids, neutral phospholipids, sterol lipids and PEGylated phospholipids, and the ionizable cationic lipids are selected from ALC-0315, ALC-0315anlogue-2, ALC-0159, M-DTDA-2000, and M-DMG-2000.

2. The delivery system according to claim 1, characterized in that The neutral phospholipid is selected from DSPC, DSPE, DEPC, DPPE and DPPC.

3. The delivery system according to claim 1, characterized in that The sterol lipid is selected from cholesterol and sitosterol.

4. The delivery system according to claim 1, characterized in that The PEGylated phospholipid is selected from one or more of DMG-PEG2000, DSPE-PEG2000-Galactose, DSPE-MPEG2000, and DSPE-PEG-Glucose.

5. The delivery system according to claim 4, characterized in that The PEGylated phospholipid is selected from a composition of DMG-PEG2000 and DSPE-PEG2000-Galactose.

6. The delivery system according to claim 1, characterized in that The mass ratio between the lipid nanoparticles and the saRNA drug is 20:

1.

7. The delivery system according to claim 1, characterized in that The molar ratio of the ionizable cationic lipid, neutral phospholipid, sterol lipid and PEGylated phospholipid is (73.3-78):(14.6-15):(56.4-60):(2.1-2.2).

8. The delivery system according to claim 7, characterized in that The molar ratio of the ionizable cationic lipid, the neutral phospholipid, the sterol lipid and the PEGylated phospholipid is selected from 73.3:14.6:56.4:2.1 and 78:15:60:2.

2.

9. The delivery system according to claim 5, characterized in that The molar ratio between the DMG-PEG2000 and the DSPE-PEG2000-Galactose is selected from (1.1-1.2):

1.

10. The delivery system according to claim 9, characterized in that The molar ratio between the DMG-PEG2000 and the DSPE-PEG2000-Galactose is selected from 1.1:1 and 1.2:1.

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