Glucoside sterol component lipid nanoparticle for regulating mRNA transfection as well as preparation method and application of glycoside sterol component lipid nanoparticle
By using components such as stigmasterol to replace cholesterol in lipid nanoparticles, the problem of LNP accumulation in the liver and enhanced immune response in vivo was solved, achieving targeted delivery to the spleen and improving anti-tumor immune effects, which has the potential for industrial application.
Patent Information
- Application Number
- CN202511946352.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-23
- Publication Date
- 2026-04-17
AI Technical Summary
Existing lipid nanoparticles (LNPs) tend to accumulate in the liver when applied in vivo, making it difficult to achieve targeted delivery to other organs. Furthermore, they may lead to enhanced immune responses and reduced therapeutic efficacy in non-target tissues. Current research mainly focuses on ionizable cationic lipids, while there is relatively little research on other lipid components such as cholesterol.
Lipid nanoparticles were constructed by using stigmasterol instead of cholesterol and combining it with amygdalin, β-sitosterol, ginsenoside Rg1, and other components. By regulating the lipid composition, the enrichment effect in the spleen was improved and the immunomodulatory effect was enhanced. The preparation method is simple and can be mass-produced.
It improves the enrichment effect of lipid nanoparticles in the spleen, enhances the anti-tumor immune effect, simplifies the preparation process, and has good industrial application value.
Smart Images
Figure CN121868247A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to a lipid nanoparticle containing glycoside sterol components that regulates mRNA transfection, its preparation method, and its application. Background Technology
[0002] LNPs are the most prevalent nonviral gene delivery vectors in clinical practice, and the U.S. Food and Drug Administration (FDA) has approved three nucleic acid drugs using LNP technology. LNPs are modular nanoparticles containing ionizable cationic lipids, cofactor lipids, cholesterol, and polyethylene glycol lipids. However, LNPs have some limitations in in vivo application, such as primarily accumulating in the liver, making targeted delivery to other organs difficult, and distribution in non-target tissues may lead to enhanced immune responses and reduced therapeutic efficacy. Current research on LNPs focuses on ionizable cationic lipids, with limited research on other lipid components. Cholesterol typically constitutes 30%–40% of LNP formulations and plays a crucial role in maintaining the LNP structure. Studies have demonstrated that adjusting the cholesterol ratio can improve LNP transfection efficiency.
[0003] Amygdalin is a common cyanogenic glycoside and an active ingredient in the traditional Chinese medicine bitter almond. It is a derivative of α-hydroxynitrogenides and has become a commonly used expectorant, antitussive, and adjuvant anticancer drug in medicine. However, there are currently no reports of amygdalin acting as a cholesterol substitute in LNP formation. The incorporation of β-sitosterol can promote the escape of LNPs from the body and improve their transfection efficiency. Stigmasterol is an orally effective immunomodulator that can cross the blood-brain barrier and has anti-inflammatory and neuroprotective effects. It can activate AMPK, thereby inhibiting the NF-κB and NLRP3 signaling pathways, reducing microglia-mediated neuroinflammation, alleviating cognitive impairment and Alzheimer's disease, and therefore can be used for neurodegenerative diseases, inflammatory diseases, and pain management. Ginsenoside Rg1 has the effects of promoting hippocampal neurogenesis, improving neuroplasticity, adjuvant antitumor activity, and repairing sexual function, and has broad application prospects in the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides lipid nanoparticles containing glycoside sterols that regulate mRNA transfection, along with their preparation method and applications. Specifically, replacing cholesterol with stigmasterol in the construction of lipid nanoparticles enhances their accumulation in the spleen.
[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:
[0006] This invention provides lipid nanoparticles containing glycoside sterol components for regulating mRNA transfection, which are composed of DLin-MC3-DMA, DOPE, various glycosides or sterols, and DSPE-PEG-GSH. The various glycosides or sterols include: amygdalin (Amy), β-sitosterol, stigmasterol (Sti), ginsenoside Rg1, and rhodioloside (Sal).
[0007] The molar ratio of DLin-MC3-DMA, DOPE, glycoside or sterol, and DSPE-PEG-GSH is 35-40:10-15:45-50:2-4.
[0008] All lipids, except amygdalin, were dissolved in anhydrous ethanol. Amygdalin was dissolved in a mixed solution of ethanol and DMSO to prepare lipid stock solutions with concentrations ranging from 3 to 25 mg / mL. The lipid phase solutions were then mixed according to the above molar ratio to obtain lipid phase solutions.
[0009] Nucleic acid was dissolved in a buffer solution as the aqueous phase, specifically a 10 mM pH 4.0 citrate buffer solution;
[0010] The volume ratio of the lipid phase to the aqueous phase is 1:3;
[0011] The purification steps include: allowing the obtained mixed solution to stand for 10 min, then diluting it with 1×PBS buffer, and then dialyzing it in 1 L of 1×PBS buffer.
[0012] The LNPs prepared by the above method have a particle size of about 130 nm.
[0013] The beneficial technical effects of one or more of the above technical solutions are as follows: stigmasterol can enhance the accumulation of LNP in the spleen, ginsenosides can regulate immunity and enhance anti-tumor immune effects, the preparation method is simple, the required equipment is all conventional equipment, and large-scale production can be achieved. Therefore, it has good industrial application value and market prospects. Attached Figure Description
[0014] Figure 1 These are the DLS characterization results of LNP delivery systems for different glycosides or sterols after the addition of Dir dye;
[0015] Figure 2 The results are DLS characterizations of mRNA-LNP delivery systems containing different glycosides or sterols.
[0016] Figure 3 These are Dir in vivo imaging results from LNP delivery systems containing different glycosides or sterols;
[0017] Figure 4 These are Dir in vitro imaging results from LNP delivery systems containing different glycosides or sterols;
[0018] Figure 5 It is a characterization of Dir ex vivo brain imaging data of LNP delivery systems containing different glycosides or sterols;
[0019] Figure 6 These are in vitro fluorescence imaging results of mRNA-LNP delivery systems containing different glycosides or sterols;
[0020] Figure 7 It is a fluorescent lung and brain data characterization of mRNA-LNP delivery systems containing different glycosides or sterols. Detailed Implementation
[0021] Example 1
[0022] This embodiment provides a lipid nanoparticle delivery system for regulating mRNA transfection using glycoside sterol components, which is prepared using the following method:
[0023] Luciferase mRNA (Luc mRNA) was prepared into an aqueous stock solution of 1 μg / μL using 10 mM pH 4.0 citrate buffer.
[0024] D-Lin-MC3-DMA, DOPE, and DSPE-PEG-GSH were dissolved in anhydrous ethanol at concentrations of 12.94 mg / mL, 12.96 mg / mL, and 21.59 mg / mL, respectively, to prepare stock solutions. β-sitosterol was dissolved in anhydrous ethanol at a concentration of 3.8 mg / mL, stigmasterol (Sti) at a concentration of 6 mg / mL, and ginsenoside Rg1 at a concentration of 10 mg / mL. Amygdalin (Amy) was dissolved in a mixed solution of ethanol and DMSO at a concentration of 10 mg / mL, with a volume ratio of ethanol to DMSO of 166:60. These stock solutions were then used for lipid-phase mixing. Amygdalin (Amy), β-sitosterol, stigmasterol (Sti), ginsenoside Rg1, and rhodioloside (Sal) were used as substitutes for different glycosides or sterols in the LNP preparation.
[0025] The formulation used was a D-Lin-MC3-DMA:DOPE:glycoside or sterol:DSPE-PEG-GSH molar ratio of 39:11:47.3:2.7, resulting in lipid concentrations between 12-20 mg / mL. The lipid and aqueous phases were mixed at a 1:3 volume ratio and incubated for 10 min, followed by the addition of 1×PBS to achieve a final mRNA concentration of 10 ng / μL. The prepared mRNA-LNP was then dialyzed through a dialysis membrane (WMCO3.5KD) in 1 L of 1×PBS solution for 2 h. In the control group, the glycoside or sterol was replaced with cholesterol, and DSPE-PEG-GSH was replaced with a 16:0 PEG2000 PE.
[0026] After preparation, dynamic light scattering (DLS) was used to determine the particle size and polydispersity index (PDI) of the LNPs.
[0027] Add 1 mL of ultrapure water and 20 μL of sample to a plastic dish, and measure the particle size using a Malvern particle size analyzer (model: MAL1299478) with a refractive index of 1.45 and an absorbance of 0.001.
[0028] Example 2: Evaluation of the in vitro delivery effect of lipid nanoparticles containing glycoside sterols
[0029] In Example 1, DiR dye (DiR Iodide) was added to the lipid phase at a rate of 10 μg per 250 μL volume, followed by microfluidic mixing with sterile, enzyme-free water using a Myanna microfluidic device (INano L+). Balb / c mice underwent hair removal treatment, and the LNP prepared in Example 1 was injected intravenously via the tail vein at a dose of 150 μL per mouse. In vivo imaging was performed at 3, 6, 9, 14, 25, and 36 h, and ex vivo imaging was performed at 6, 12, 18, and 24 h. Three mice were used in each group, with the control group receiving the same treatment.
[0030] Balb / c mice underwent hair removal treatment. LNP prepared in Example 1 was injected intravenously at a dose of 200 μL / mouse. At 6 h and 15 h later, mice were intraperitoneally injected with 200 μL of a 15 mg / mL D-fluorescein potassium solution. Three minutes after injection of the D-fluorescein potassium solution, in vivo and in vitro imaging were used to evaluate the transfection regulation of different glycoside or sterol component lipid nanoparticle delivery systems regulating Luc-mRNA transfection. Three mice were in each group. The control group underwent the same treatment.
Claims
1. Nucleic acid lipid nanoparticles based on different glycosides or sterols, characterized in that... The common components of the nucleic acid lipid nanoparticles based on different glycosides or sterols are DLin-MC3-DMA (4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), phospholipid-polyethylene glycol-glutathione (DSPE-PEG-GSH), and different glycosides or sterols; the glycosides or sterols include: amygdalin (Amy), β-sitosterol, stigmasterol (Sti), ginsenoside Rg1, and rhodioloside (Sal); The molar ratio of DLin-MC3-DMA, DOPE, the above-mentioned glycosides or sterols, and DSPE-PEG-GSH is 35-40:10-15:45-50:2-4.
2. The glycoside or sterol-based nucleic acid lipid nanoparticles as described in claim 1, characterized in that, The nanoparticles also contain a pharmaceutically active ingredient, which is a nucleic acid or a nucleic acid analogue.
3. The method for preparing nucleic acid lipid nanoparticles based on different glycosides or sterols as described in claim 1, characterized in that, Includes the following steps: (1) Prepare an aqueous stock solution of 1 μg / μL by using 10 mM pH 4.0 citrate buffer solution to prepare Luc mRNA; (2) D-Lin-MC3-DMA, DOPE, and DSPE-PEG-GSH were dissolved in anhydrous ethanol at concentrations of 12.94 mg / mL, 12.96 mg / mL, and 21.59 mg / mL, respectively, to serve as the stock solution; β-Sitosterol was dissolved in anhydrous ethanol at a concentration of 3.8 mg / mL; stigmasterol (Sti) was dissolved in acetone at a concentration of 6 mg / mL; ginsenoside Rg1 was dissolved in anhydrous ethanol at a concentration of 10 mg / mL; amygdalin (Amy) was dissolved in a mixed solution of ethanol and DMSO at a concentration of 10 mg / mL, with a volume ratio of ethanol to DMSO of 166:60, and the resulting mother liquors were used for lipid phase mixing; rhodioloside (Sal) was dissolved in anhydrous ethanol at a concentration of 6 mg / mL. Among them, amygdalin, β-sitosterol, stigmasterol, ginsenoside Rg1, and rhodioloside Sal are different glycosides or sterols used in LNP formulation. (3) The formula used is D-Lin-MC3-DMA:DOPE:glycoside or sterol:DSPE-PEG-GSH with a molar ratio of 35-40:10-15:45-50:2-4, and finally a mixed solution with a total mass concentration between 12-20 mg / mL is obtained as the lipid phase; The lipid phase and aqueous phase were mixed at a volume ratio of 1:3 and incubated for 10 min. Then, 1×PBS was added to make the final mRNA concentration 10 ng / μL. Finally, the mixture was dialyzed through a dialysis membrane for 2 h to obtain mRNA-LNP.
4. The use of the nucleic acid lipid nanoparticles based on different glycosides or sterols as described in claim 1 or 2 in the preparation of drugs.
5. A drug for promoting the delivery of nucleic acids by lipid nanoparticles, characterized in that, It includes nucleic acid lipid nanoparticles based on different glycosides or sterols as described in claim 1 or 2.