Nanolipid particles containing cationic lipids and their use in self-replicating mrna delivery systems

Abstract

The application discloses a kind of nanolipid particles containing cationic lipid and its application in self-replicating mRNA delivery system, nanolipid particles containing cationic lipid are prepared by microflow using lipid phase, water phase, the lipid phase includes the compound containing cationic lipid, DSPC, CHO-HP, DMP-PEG2000.The polyethylene glycol linker and tail of the present application are retained, amino acid residues are introduced in the head, the pKa of the lipid is adjusted by changing the type of head group, a new JK-0045 structural formula is obtained.The change of pKa makes the ionizable lipid and the composition of other several components encapsulated mRNA have better transfection efficiency, higher in vivo release and better cell activity, can effectively express target protein in non-self-replicating mRNA system, and can better maintain long-term expression of target protein in self-replicating mRNA system.

Description

Technical Field

[0001] This invention belongs to the field of nanolipid particle technology, specifically relating to a nanolipid particle containing cationic lipids and its application in a self-replicating mRNA delivery system. Background Technology

[0002] mRNA (messenger RNA) drugs represent an emerging field of drug development. These drugs deliver synthesized mRNA molecules into specific cells within the body, utilizing the cell's transcription and translation mechanisms to synthesize the proteins encoded by the mRNA, thereby achieving therapeutic effects. mRNA drugs offer several advantages over traditional drugs. First, they can be designed, synthesized, and produced rapidly, aiding in responses to emergencies and emerging pathogens. Second, mRNA drugs offer greater flexibility, allowing for customization for different diseases and individuals. Furthermore, mRNA drugs can activate multiple immune responses, contributing to immunotherapy and vaccine development. However, the development and application of mRNA drugs also face several challenges. One major challenge is effectively delivering mRNA into target cells, overcoming limitations in stability and cell permeability. Additionally, for long-term treatment and multiple-dose applications, issues related to immune responses and adverse events need to be addressed.

[0003] Regarding self-replicating and non-self-replicating mRNAs: Self-replicating mRNAs and non-self-replicating mRNAs are two different types of mRNAs in two gene expression systems. Self-replicating mRNAs contain an embedded replication system, enabling them to produce more mRNA molecules through replication. Self-replicating mRNAs have longer sequences than non-self-replicating mRNAs, requiring additional information for replication. Due to their self-replicating ability, self-replicating mRNAs can continuously replicate within the cell and produce large amounts of protein, resulting in long-term therapeutic effects. Because of their ability to produce large amounts of protein, self-replicating mRNAs exhibit higher resistance to immune responses. Non-self-replicating mRNAs lack an embedded replication system and cannot replicate themselves. They only contain information for encoding proteins and have shorter sequences. Once transcribed and translated within the cell, non-self-replicating mRNAs cannot be produced again. Due to their simplicity and stability, non-self-replicating mRNAs are widely used in gene expression systems, particularly in vaccine development.

[0004] mRNA drug delivery systems are technologies used to deliver mRNA molecules into specific cells within the body. These systems protect mRNA molecules from degradation by both in vivo and in vitro environments and facilitate their entry into target cells, enabling gene expression and protein production. Common mRNA drug delivery systems include lipid nanoparticles (LNPs), nanopolymers, and viral vectors. These delivery systems each possess specific characteristics and advantages, such as high delivery efficiency, stability, and biocompatibility. Different delivery systems can be selected and optimized based on specific needs and application areas. When using these systems, factors such as safety, immune responses, and side effects must be considered, and detailed evaluations and studies are necessary.

[0005] Lipid nanoparticle (LNP) technology is a key technology commonly used for mRNA drug delivery. It utilizes lipid nanoparticles as carriers to encapsulate mRNA, enabling efficient delivery into cells. LNP lipid nanoparticles are primarily composed of lipid components, including phospholipids, cholesterol, and PEG (polyethylene glycol). These components can self-assemble into nanoscale particles, are stable in aqueous environments, and exhibit good biocompatibility. Under appropriate conditions, the lipid components and mRNA mix to form lipid-nucleic acid complexes containing mRNA. These complexes stably encapsulate the mRNA within the lipid nanoparticles through electrostatic interactions, hydrophobic effects, and other interactions, forming LNPs. LNPs deliver mRNA to target cells by targeting receptors or other recognition mechanisms on specific cell surfaces. Once inside the cell, the lipid nanoparticle fuses with the cell membrane, releasing the mRNA into the cytoplasm. Cellular biological mechanisms then convert the mRNA into proteins through translation and transcription. While LNP lipid nanoparticle technology plays a crucial role in mRNA drug delivery, relatively few lipids are applicable to self-replicating mRNA delivery systems.

[0006] Therefore, a nanolipid particles containing cationic lipids and their application in self-replicating mRNA delivery systems urgently need to be proposed. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides nanolipid particles containing cationic lipids and their application in a self-replicating mRNA delivery system.

[0008] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0009] The first objective of this invention is to provide a compound containing cationic lipids, the structure of which is shown in JK-0045, and the molecular formula of JK-0045 is: C 49 H 97 N3O6, its preparation route is as follows:

[0010]

[0011] The second objective of this invention is to provide a cationic lipid nanoparticle, which is prepared by microfluidic control using a lipid phase and an aqueous phase. The lipid phase includes a compound containing cationic lipids, DSPC, CHO-HP, and DMP-PEG2000, and the molar concentration ratio of cationic, DSPC, CHO-HP, and DMP-PEG2000 in the compound containing cationic lipids is 50:7:40:3.

[0012] Preferably, the volume ratio of the aqueous phase is 1:3.

[0013] A third objective of the present invention is to provide a self-replicating mRNA delivery system comprising nanolipid particles containing cationic lipids.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] This invention retains the polyethylene glycol linker and tail, introduces amino acid residues into the head, and adjusts the pKa of the lipid by changing the type of head group, resulting in the novel JK-0045 structure. This change in pKa allows the combination of this ionizable lipid with several other components to encapsulate mRNA with better transfection efficiency, higher in vivo release, and better cellular activity. It effectively expresses the target protein in non-self-replicating mRNA systems and maintains longer-term expression of the target protein in self-replicating mRNA systems. Attached Figure Description

[0016] Figure 1 This is the mass spectrum of compound JK-0045 in Example 1 of this invention;

[0017] Figure 2 This is the NMR spectrum of compound JK-0045 in Example 1 of this invention;

[0018] Figure 3 This is G2 in vivo imaging 12 hours after taking the medicine, as described in Example 3 of this invention.

[0019] Figure 4 This is G8 in vivo imaging 12 hours after taking the medicine, as described in Example 3 of this invention.

[0020] Figure 5This is G9 in vivo imaging 12 hours after taking the medicine, as described in Example 3 of this invention.

[0021] Figure 6 This is G2 in vivo imaging 24 hours after taking the medicine, as described in Example 3 of this invention.

[0022] Figure 7 This is G8 in vivo imaging 24 hours after taking the medicine, as described in Example 3 of this invention.

[0023] Figure 8 This is G9 in vivo imaging 24 hours after taking the medicine, as described in Example 3 of this invention.

[0024] Figure 9 This is G2 in vivo imaging 48 hours after taking the medicine, as described in Example 3 of this invention.

[0025] Figure 10 This is G8 in vivo imaging 48 hours after taking the medicine, as described in Example 3 of this invention.

[0026] Figure 11 This is G9 in vivo imaging 48 hours after taking the medicine, as described in Example 3 of this invention.

[0027] Figure 12 This is G2 in vivo imaging 96 hours after taking the medicine, as described in Example 3 of this invention.

[0028] Figure 13 This is G8 in vivo imaging 96 hours after taking the medicine, as described in Example 3 of this invention.

[0029] Figure 14 This is G9 in vivo imaging 96 hours after taking the medicine, as described in Example 3 of this invention.

[0030] Figure 15 The changes in in vivo fluorescence at G2, G8, and G9 96 hours after administration of the traditional Chinese medicine in Example 3 of this invention;

[0031] Figure 16 This is G1 in vivo imaging 12 hours after taking the medicine, as described in Example 3 of this invention.

[0032] Figure 17 This is G7 in vivo imaging 12 hours after taking the medicine, as described in Example 3 of this invention.

[0033] Figure 18 This is G1 in vivo imaging 24 hours after taking the medicine, as described in Example 3 of this invention.

[0034] Figure 19 This is G7 in vivo imaging 24 hours after taking the traditional Chinese medicine in Example 3 of this invention;

[0035] Figure 20 This is G1 in vivo imaging 48 hours after taking the medicine, as described in Example 3 of this invention.

[0036] Figure 21This is G7 in vivo imaging 48 hours after taking the medicine, as described in Example 3 of this invention.

[0037] Figure 22 This is G1 in vivo imaging 96 hours after taking the medicine, as described in Example 3 of this invention.

[0038] Figure 23 This is G7 in vivo imaging 96 hours after taking the medicine, as described in Example 3 of this invention.

[0039] Figure 24 This is G1 in vivo imaging 168 hours after taking the medicine, as described in Example 3 of this invention.

[0040] Figure 25 This is G7 in vivo imaging 168 hours after taking the medicine, as described in Example 3 of this invention.

[0041] Figure 26 This is G9 in vivo imaging 168 hours after taking the medicine, as described in Example 3 of this invention.

[0042] Figure 27 This is G1 in vivo imaging 336 hours after taking the medicine, as described in Example 3 of this invention.

[0043] Figure 28 This is G7 in vivo imaging 336 hours after taking the medicine, as described in Example 3 of this invention.

[0044] Figure 29 This is G9 in vivo imaging 336 hours after taking the medicine, as described in Example 3 of this invention.

[0045] Figure 30 This is G1 in vivo imaging 504 hours after taking the medicine, as described in Example 3 of this invention.

[0046] Figure 31 This is G7 in vivo imaging 504 hours after taking the medicine, as described in Example 3 of this invention.

[0047] Figure 32 This is G9 in vivo imaging 504 hours after taking the medicine, as described in Example 3 of this invention.

[0048] Figure 33 The changes in in vivo fluorescence of G1, G7, and G9 504 hours after administration of the traditional Chinese medicine in Example 3 of this invention;

[0049] Figure 34 These are the G1 to G9 in vivo liver coefficients in Example 3 of this invention;

[0050] Figure 35 These are the in vivo spleen coefficients G1 to G9 in Example 3 of this invention;

[0051] Figure 36 The ratio of G1 to G9 live original spleens and livers in Example 3 of this invention. Detailed Implementation

[0052] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0053] Example 1

[0054] The structure of the compound is shown in JK-0045 (hereinafter referred to as compound JK-0045). The molecular formula of JK-0045 is: C 49 H 97 N3O6, its preparation route is as follows:

[0055]

[0056] The preparation process of compound JK-0045 is as follows:

[0057] N-tert-Butoxycarbonyl-1,3-propanediamine (20 g, 114.8 mmol) was dissolved in 75 mL of a mixed solvent of acetonitrile and tetrahydrofuran (2:1 v / v). Potassium carbonate (10.56 g, 76.5 mmol), potassium iodide (0.64 g, 3.9 mmol), and heptadecan-9-yl 8-bromooctanoate (17.6 g, 38.3 mmol) were added and stirred until dissolved. The mixture was stirred at 50 °C for 3 hours. After the reaction was complete as indicated by TLC, the mixture was filtered, and the filtrate was evaporated by rotary evaporation. The solution was dissolved in 50 mL of dichloromethane, washed twice with 50 mL of water, dried over anhydrous sodium sulfate, and evaporated by rotary evaporation. Column chromatography (dichloromethane:methanol:triethylamine = 100:1:0.1) yielded compound JK-0044-1 (9.2 g, pale yellow oil), with a yield of 22%.

[0058] Compound JK-0044-1 (9.2 g, 16.6 mmol) was dissolved in 50 mL of a mixed solvent of acetonitrile and tetrahydrofuran (volume ratio 2:1) at room temperature. Potassium carbonate (4.6 g, 33.2 mmol), potassium iodide (1.4 g, 8.3 mmol), and undecanoate 6-bromohexanoate (5.8 g, 16.6 mmol) were added and stirred until dissolved. The mixture was stirred at 80 °C for 12 hours. After the reaction was complete as indicated by TLC, the mixture was filtered, and the filtrate was evaporated by rotary evaporation. The solution was dissolved in 50 mL of dichloromethane, washed twice with 50 mL of water, dried over anhydrous sodium sulfate, and evaporated by rotary evaporation. The solution was then obtained by column chromatography (dichloromethane:methanol:triethylamine = 100:1:0.1) to give compound JK-0044-2 (8.9 g, pale yellow oil), with a yield of 65%.

[0059] Compound JK-0044-2 (8.9 g, 10.8 mmol) was dissolved in 30 mL of dichloromethane, and 5 mL of 1,4-dioxane hydrogen chloride solution (4 M) was added. The mixture was stirred at room temperature for 1 h. After the reaction was completed by TLC, the solvent was removed by rotary evaporation to obtain product JK-0044-3 (7.8 g, pale yellow solid), with a yield of 95%.

[0060] Fmoc-L-threonine (1.9 g, 5.5 mmol) was dissolved in 30 mL of dichloromethane, and 2 mL of acetonitrile, compound JK-0044-3 (3.3 g, 4.5 mmol), HATU (2.6 g, 6.8 mmol), and DIPEA (1.8 g, 13.9 mmol) were added. The mixture was stirred at room temperature for 3 h. After the reaction was complete as indicated by TLC, 30 mL of water was added to quench the reaction. The mixture was separated, and the organic phase was retained. The aqueous phase was extracted once more with 30 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to give compound JK-0045-1 (2.3 g, pale yellow oil), with a yield of 50%.

[0061] Compound JK-0045-1 (2.0 g, 1.9 mmol) was dissolved in 20 mL of dichloromethane, and 20 mL of diethylamine was added. The mixture was stirred at room temperature for 12 h. After the reaction was complete as shown by TLC, the solution was concentrated and separated by column chromatography (dichloromethane:methanol:triethylamine = 20:1:0.1) to give product JK-0045 (1 g, pale yellow oil), with a yield of 65%. The mass spectrum and NMR spectrum of JK-0045 are shown below. Figure 1 and Figure 2 As shown.

[0062] Example 2: Encapsulation of luciferase mRNA and negative blank control PBS was achieved using compound JK-0045 and positive control MC3 cationic lipid.

[0063] Microfluidic LNP Encapsulation: Microfluidic devices utilize tiny channels and microvalves to control the flow of minute liquids. By precisely adjusting the opening and closing states and timing of these valves, precise manipulation and control of the liquid are achieved. Two inlet valves respectively introduce the organic and aqueous phases. By controlling the flow of these two liquid phases within the microfluidic microchannel grid, the two phases meet and mix, forming lipid and mRNA encapsulation. Finally, the mixed liquid forms uniformly encapsulated LNP nanoparticles that flow out from the outlet.

[0064] Different cationic lipid grouping designs: different cations were used to encapsulate self-replicating and non-self-replicating mRNAs. Among them, cation MC3 was used as the control cation and was numbered in groups G1 and G2. Compound JK-0045 was encapsulated and numbered in groups G7 and G8. Group G9 was the blank control group and cation MC3 was used as the negative control (Table 1).

[0065] Table 1:

[0066] G1 Dlin-MC3 JJ0023-AU-FLUCQEGFP-Sap-2 Self-replicating G2 Dlin-MC3 JJ0011-FLUCQEGFP-PJJJ-SalI Non-self-replicating G7 JK-0045 JJ0023-AU-FLUCQEGFP-Sap-2 Self-replicating G8 JK-0045 JJ0011-FLUCQEGFP-PJJJ-SalI Non-self-replicating G9 PBS replacement PBS replacement /

[0067] The formulation used in the organic phase: self-replicating and non-self-replicating mRNAs were encapsulated in LNPs. The molar ratio of cations (DLin-MC3-DMA, JK-0045):DSPC:CHO-HP:DMP-PEG2000 in the lipid phase was 50:7:40:3. The total molar concentration of the lipid phase was 19.5 mmol / L, and the N:P ratio was 5.3.

[0068] Aqueous mRNA stock solution: The stock solution was thawed on ice and diluted with citrate buffer to a concentration of about 0.2 mg / ml. The citrate buffer contains 130 mM NaCl and is referred to as the RNA working solution.

[0069] The organic phase and aqueous phase (mRNA working solution) were mixed at a volume ratio of 1:3 and prepared into LNP nanoparticles using a microfluidic preparation instrument. The flow rates of the aqueous phase and the lipid phase were 24 mL / min and 8 mL / min, respectively.

[0070] The harvested LNPs were diluted with 1X PBS at a volume eight times their original volume. The concentrate was then centrifuged in a 100 kDa ultrafiltration centrifuge tube at 4°C and 1500 g. When the volume was approximately the same as the initially obtained LNPs, another eight volumes of 1X PBS were added for further dilution, and the concentration was continued until the desired level was reached. The LNP product was obtained by filtration through a 0.2 μm membrane. The particle size, PDI, Zeta potential, and encapsulation efficiency of the prepared samples are shown in Table 2.

[0071] Table 2

[0072]

[0073] Example 3: Animal imaging experiments using nanoparticles encapsulated with different delivery systems.

[0074] 3.1 Animal husbandry:

[0075] After the animals arrive at the facility, they are acclimatized to the facility's feeding conditions for 2-3 days before the experiment begins.

[0076] 3.2 Dosing regimen:

[0077]

[0078] 3.3 Data Acquisition:

[0079] In vivo imaging: G1~G9: 12h, 24h (Day 1), 48h (Day 2), 96h (Day 4) after drug administration Figures 3-23 G1, G7, and G9: Imaging and image capture were performed using an IVIS in vivo imaging system at 168h (7d), 336h (Day 14), and 504h (Day 21) after drug administration. Figures 24-32 Animals were photographed in a supine position, with imaging controlled within 10-15 minutes after injection of fluorescent D-fluorescein potassium salt.

[0080] After 24 hours of in vivo imaging, the animals were euthanized under anesthesia by cervical dislocation. Gross dissection and observation were performed on the experimental animals, and the weights of the liver and spleen were recorded. Figures 34-36 If abnormal organs are found, take photos to record them.

[0081] Statistical analysis: The fluorescent expression regions (ROIs) of each animal were circled, and the total fluorescence intensity (p / s) was measured. Figure 15 , Figure 33 All images were processed using Living Image software (PerkinElmer), and inter-group comparisons were performed using the same fluorescence threshold.

[0082] Mean fluorescence intensity results for each group of animals are expressed as mean ± standard deviation (Mean ± SD). Analysis of variance (one-way ANOVA / two-way ANOVA) was used to compare the differences between the treatment groups and the control group to determine if there were significant differences. Data were analyzed using Graphpad Prism. P < 0.05 was considered statistically significant.

[0083] The results showed that compound JK-0045, along with phospholipids, cholesterol, and PEG, effectively encapsulated luciferase mRNA with an encapsulation rate greater than 95%. The nanoparticles self-assembled into nanoscale particles, exhibiting stability in an aqueous environment and good biocompatibility and transfection efficiency. Animal imaging experiments were conducted using LNPs technology to encapsulate luciferase mRNA nanoparticles with different delivery systems. Animal imaging results showed that the non-self-replicating system mRNA, like the positive control MC3, showed fluorescent expression in animals after 48 hours; the self-replicating system mRNA, like the positive control MC3, still showed strong fluorescent expression after 504 hours. Throughout the experimental period, the animals exhibited normal activity and good health, demonstrating the safety of these LNPs nanolipids, their effective expression of the target protein in non-self-replicating mRNA systems, and their high-efficiency and significant long-term expression of the target protein in self-replicating mRNA systems.

[0084] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound containing cationic lipids, characterized in that, The structure of the compound is shown in JK-0045, and the molecular formula of JK-0045 is: C 49 H 97 N3O6, its preparation route is as follows: ， The compound containing cationic lipids is suitable for the delivery of self-replicating mRNA.

2. A type of nanolipid particles containing cationic lipids, characterized in that, The cationic lipid nanoparticles are prepared by microfluidic control using a lipid phase and an aqueous phase. The lipid phase includes the cationic lipid compound as described in claim 1, DSPC, CHO-HP, DMP-PEG2000, and encapsulates self-replicating mRNA. The molar ratio of cationic, DSPC, CHO-HP, and DMP-PEG2000 in the cationic lipid compound is 50:7:40:

3.

3. The nanolipid particles containing cationic lipids according to claim 2, characterized in that, The volume ratio of the aqueous phase is 1:

3.

4. A self-replicating mRNA delivery system, characterized in that, The self-replicating mRNA delivery system comprises the cationic lipid nanoparticles as described in claim 2 or 3.

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