Application and method of flavonoid compound in enhancing nucleic acid molecule delivery efficiency of lipid nanoparticles

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application and a method of flavonoid compounds in enhancing nucleic acid molecule delivery efficiency of lipid nanoparticles. The invention aims to solve the problems of limited LNP in-vivo and in-vitro delivery efficiency, toxic risk and the like. According to the invention, seven low-toxicity and biocompatible natural compounds such as genistein, glabridin, rhoifolin, luteolin, hesperetin, galangin or pinocembrin are used as an LNP high-efficiency expression enhancer, the compounds and target cells are co-incubated, and then LNP containing nucleic acid molecules is added. According to the scheme, the LNP-mediated gene expression efficiency can be remarkably improved, the cytotoxicity can be reduced, and in-vivo experiments prove that LNP biological distribution can be optimized, target tissue expression can be enhanced, and collaborative optimization of LNP safety and high efficiency is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application and method of flavonoids in enhancing the efficiency of lipid nanoparticles in delivering nucleic acid molecules. Background Technology

[0002] Lipid nanoparticles (LNPs), as an advanced drug delivery system in the biopharmaceutical field, have played a revolutionary role in the clinical translation of nucleic acid drugs. Their core consists of ionizable lipids, cholesterol, auxiliary lipids, and polyethylene glycol (PEG)-lipid conjugates. With their highly efficient encapsulation and protection capabilities for nucleic acid molecules such as messenger RNA (mRNA) and small interfering RNA (siRNA), LNPs can precisely deliver nucleic acid drugs into target cells, thereby achieving the expression of exogenous proteins or the silencing of specific genes. Currently, the superior delivery efficiency and good biocompatibility of LNP technology have been validated in multiple approved drugs, especially in the successful application of COVID-19 mRNA vaccines, fully demonstrating the enormous potential and broad application prospects of this technology in the biopharmaceutical field.

[0003] However, the in vivo delivery efficiency of LNPs remains significantly limited by their inherent immunogenicity, a key bottleneck hindering their further development. Numerous studies have confirmed that LNPs are easily recognized by the innate immune system after entering the body, thereby activating immune signaling pathways such as Toll-like receptors (TLRs) and triggering the production of type I interferon and various pro-inflammatory cytokines. This unexpected innate immune response can not only cause transient inflammatory side effects but also activate immune cells to clear transfected target cells or inhibit the translation of exogenous mRNA through intracellular signaling pathways, ultimately leading to a significant decrease in the expression level of target proteins and severely impacting the efficacy of LNP-mediated nucleic acid drugs. Therefore, effectively inhibiting the immunogenicity induced by LNPs has become a core challenge in overcoming their delivery efficiency limitations and improving efficacy and safety.

[0004] The natural product library contains a wealth of compounds with anti-inflammatory and antioxidant activities, providing a novel research direction for addressing the immunogenicity of plant-derived nutrients (LNPs). These natural compounds can intervene in immune signaling pathways through multi-target regulatory mechanisms, effectively mitigating excessive immune activation induced by LNP components. Previous studies have shown that natural products such as flavonoids, phenolic acids, and terpenes extracted from plants can not only directly neutralize free radicals to reduce oxidative stress damage, but also regulate key signaling pathways such as NF-κB and Nrf2, inhibiting the release of inflammatory factors at the gene expression level. If these natural products can be intelligently combined with the LNP system, it is hoped that its biocompatibility and safety can be further improved while retaining the efficient delivery capability of LNPs.

[0005] In summary, there is an urgent need in this field to develop a technical solution based on natural product pretreatment, which can effectively reduce the immunogenicity of LNPs while synergistically enhancing their nucleic acid delivery and protein expression performance by creating a suitable microenvironment for cells, thereby providing key tools and core components for the development of next-generation high-performance LNP drug delivery systems. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a scheme for enhancing the delivery efficiency of lipid nanoparticles (LNPs) based on pretreatment with natural compounds. Specifically, seven natural compounds with low toxicity and biocompatibility—genistein, glycyrrhizin, robinin, luteolin, hesperidin, galangin, and pinosin—are used as high-efficiency LNP expression enhancers. A "compound pretreatment of cells + subsequent LNP transfection" approach is adopted. This approach significantly improves the delivery efficiency of LNPs to nucleic acid molecules and the gene expression level of target cells, while reducing the immunogenicity and inflammatory side effects of LNPs, thus achieving a synergistic optimization of safety and efficiency.

[0007] The technical solution of this invention is as follows: On the one hand, the present invention provides the application of flavonoids in enhancing the efficiency of lipid nanoparticles in delivering nucleic acid molecules, wherein the flavonoids are selected from at least one of genistein, glycyrrhizin, roxithrin, luteolin, hesperidin, galangin, or pinosin.

[0008] Specifically, the application involves pretreating target cells with flavonoids and then adding lipid nanoparticles encapsulating nucleic acid molecules for further culture.

[0009] Specifically, the concentration of the flavonoid compound used can be 1 μmol / L to 10 μmol / L.

[0010] Preferably, in some embodiments, the concentration of the flavonoid compound used may be 1 μmol / L.

[0011] Preferably, in other embodiments, the concentration of the flavonoid compound used may be 5 μmol / L.

[0012] Preferably, in other embodiments, the concentration of the flavonoid compound used may be 10 μmol / L.

[0013] Specifically, the pretreatment time is 4-8 hours; the continued culture time is 20-30 hours.

[0014] Preferably, the pretreatment time is 6 hours; the continued culturing time is 24 hours.

[0015] 5. A method for enhancing the efficiency of lipid nanoparticles in delivering nucleic acid molecules, characterized by comprising the following steps: (1) Preparation of lipid nanoparticles encapsulating nucleic acid molecules; (2) The flavonoids were co-incubated with the target cells, and then lipid nanoparticles were added and cultured for a longer period of time. The flavonoids are selected from at least one of genistein, glycyrrhizin, arbutin, luteolin, hesperidin, galangin, or pinosin.

[0016] Specifically, the concentration of the flavonoids used in step (2) is 1 μmol / L-10 μmol / L.

[0017] Preferably, the concentration of the flavonoid compound used in step (2) is 1 μmol / L, 5 μmol / L or 10 μmol / L.

[0018] Specifically, the co-incubation time in step (2) is 4-8 hours; the continued culture time is 20-30 hours.

[0019] Preferably, the co-incubation time is 6 hours; the continued culture time is 24 hours.

[0020] Specifically, the lipid nanoparticle preparation method in step (1) is as follows: ionizable lipids, auxiliary lipids, cholesterol and phospholipids are dissolved in ethanol and mixed to obtain a total lipid organic phase; mRNA is dissolved in a citrate-sodium citrate buffer solution to obtain an mRNA aqueous solution; the total lipid organic phase and the mRNA aqueous solution are rapidly mixed by microfluidic control to prepare lipid nanoparticles; the lipid nanoparticles are dialyzed to obtain the final product.

[0021] More specifically, the ionizable lipid is selected from at least one of ALC-0315, MC3, or DLin-MC3-DMA; the neutral auxiliary lipid is selected from at least one of DOPE, DSPC, or DPPC; and the polyethylene glycol lipid is selected from at least one of DMG-PEG2000, DSPE-PEG2000, or DSPE-PEG5000.

[0022] Preferably, the ionizable lipid is ALC-0315; the neutral auxiliary lipid is DOPE; and the polyethylene glycol lipid is DMG-PEG2000.

[0023] More preferably, the molar ratio of ionizable lipids, DOPE, cholesterol, and DMG-PEG2000 can be 50:38:10.5:1.5.

[0024] Preferably, the mRNA sequence is as shown in SEQ ID NO.1.

[0025] The beneficial effects of this invention are as follows: (1) The technical solution of the present invention can significantly improve the delivery efficiency of LNP to mRNA and siRNA, and enhance the expression of target tissue genes at both the cellular and animal levels.

[0026] (2) The technical solution of the present invention can downregulate immune activation genes, reduce the inflammatory response induced by LNP, and reduce immunogenicity.

[0027] (3) The technical solution of the present invention can upregulate antioxidant genes and downregulate nucleic acid degradation genes, reduce mRNA damage, and ensure expression effect.

[0028] (4) The technical solution of the present invention has high cell survival rate, good biocompatibility and low toxicity.

[0029] (5) The technical solution of the present invention is adaptable to a variety of LNP preparation processes and nucleic acid drug types, and is flexible in application. Attached Figure Description

[0030] Figure 1 This is a flowchart of Embodiment 1 of the present invention.

[0031] Figure 2 This is a heatmap showing the screening results in three cell types in Example 1 of the present invention.

[0032] Figure 3 This is a statistical chart showing the fold increase in expression of LNP by the high-efficiency expression enhancers selected in Example 1 of the present invention.

[0033] Figure 4 This is the structural formula of the LNP high-efficiency expression enhancer screened in Example 1 of the present invention.

[0034] Figure 5 The morphology of Luciferase mRNA LNP in Example 3 of this invention is shown under a transmission electron microscope.

[0035] Figure 6 The particle size distribution results of Luciferase mRNA LNP in Example 3 of this invention are shown.

[0036] Figure 7 The toxicity of the LNP expression enhancer screened in Example 3 of this invention to cells.

[0037] Figure 8 This is a flowchart of Embodiment 8 of the present invention.

[0038] Figure 9 The results are fluorescence imaging results of small animals used in Example 8 of this invention for screening LNP expression enhancers in vivo.

[0039] Figure 10This is a statistical graph of in vitro organ Luciferase expression of LNP high-efficiency expression enhancers screened in vivo in Example 8 of the present invention.

[0040] Figure 11 This is a statistical graph of in vivo Luciferase expression for LNP high-efficiency expression enhancers screened in vivo in Example 8 of the present invention.

[0041] Figure 12 The volcano plot highlights the genistein-treated DEG compared to the PBS-treated DEG.

[0042] Figure 13 Selected DEGs related to immune activation.

[0043] Figure 14 Selected DEGs for antioxidant purposes.

[0044] Figure 15 Selected DEGs related to protein degradation.

[0045] Figure 16 Selected DEGs related to RNA degradation. Detailed Implementation

[0046] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0047] The 240 natural compounds described in this embodiment of the invention (Table 1) were purchased from Shanghai Taoshu Biotechnology Co., Ltd.; ALC-0315 was purchased from MedChemExpres, catalog number: HY-138170; DOPE, cholesterol, and DMG-PEG2000 were purchased from Avanti® Polar Lipids.

[0048] Table 1. Library of 240 natural compounds

[0049] The mice used in this embodiment of the invention are wild-type C57BL / 6 mice, 6-8 weeks old, female, weighing approximately 20g, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0050] ALC-0315 represents an ionizable lipid, with the structural formula shown in Formula 1; DOPE represents a neutral auxiliary lipid, with the structural formula shown in Formula 3; cholesterol represents a sterol lipid, with the structural formula shown in Formula 3; DMG-PEG2000 represents a polyethylene glycol-modified lipid derivative, with the structural formula shown in Formula 4; and genistein has the structural formula shown in Formula 5.

[0051]

[0052] Formula 1

[0053] Formula 2

[0054] Formula 3

[0055] Formula 4

[0056] Formula 5 Example 1 This embodiment provides a method for preliminary screening of compounds that can enhance the delivery efficiency of lipid nanoparticles. The lipid nanoparticles (LNPs) used for screening are prepared by the following steps: (1) Total lipid ethanol solution. ALC-0315, DOPE, Cholesterol and DMG-PEG2000 were respectively prepared into solutions with a concentration of 5 mg / mL by anhydrous ethanol solution.

[0057] (2) Take the corresponding volumes of ALC-0315 solution, DOPE solution, Cholesterol solution and DMG-PEG2000 solution in sequence according to the molar ratio of 50:38:10.5:1.5, and mix them thoroughly to obtain the organic phase.

[0058] (3) Preparation of mRNA (SEQ ID NO.1) solution. Prepare a solution of mRNA with a concentration of 70 μg / mL using citrate-sodium citrate buffer solution, which is the aqueous phase.

[0059] (4) Microfluidic preparation of lipid nanoparticles. The tubing of the microfluidic dual-channel syringe pump was cleaned with a citric acid-sodium citrate buffer solution (pH 4.0) and anhydrous ethanol to fill the microfluidic chip tubing with citric acid and anhydrous ethanol. 150 μL of organic phase and 450 μL of aqueous phase were drawn up using a 1 mL syringe. The flow rates of the two channels were set on the microfluidic dual-channel syringe pump interface to 9 mL / min for the aqueous phase and 3 mL / min for the organic phase, for a total flow rate of 12 mL / min. After setting, the program was saved, the positions of each component were adjusted, and then the program was run to collect the crude LNP product solution.

[0060] (5) Dialysis of LNP solution. The prepared crude LNP product solution was added to a 20kDa dialysis bag, and dialysis was performed at 25°C for 6 hours with 1× phosphate buffer (PBS, pH=7.4) as the external aqueous phase to obtain LNP solution.

[0061] The mRNA sequence is shown in SEQ ID NO.1:

[0062] In the screening process, HeLa, 3T3, and HepG2 cells (manufacturer: Yuanjing Biotechnology, catalog numbers: HeLa: YC-A012, 3T3: YC-A013, HepG2: YC-C001) were seeded into 96-well plates at a cell density of 10,000 cells / well and cultured until the cell density reached 80-90%. 240 compounds from the natural product library were diluted in the culture medium at 1 μmol / L, 5 μmol / L, and 10 μmol / L and co-incubated with the cells for 6 h. (Preparation of compound solutions: Dissolve the purchased dry powders of each compound (240 compounds) in DMSO to prepare a 10 mmol / L stock solution, and aliquot and store at -20°C. Before use, serially dilute the stock solution with complete culture medium to prepare working concentrations of 10 μmol / L, 5 μmol / L and 1 μmol / L, ensuring that the final concentration of DMSO in all experimental groups is below 0.05% (v / v)).

[0063] After 6 hours of pretreatment, 3.4 μL of freshly synthesized mRNA-LNP (synthesis route see Example 1, which encapsulates 20 μg mRNA) was added directly to each well. After gently shaking to mix, the cell plate was cultured for another 24 hours.

[0064] After transfection, the culture medium was aspirated, and the cells were gently washed once with PBS. Then, an appropriate amount of lysis buffer was added, or the fluorescence intensity of the reporter protein was detected directly using a fluorescence microplate reader. Transfection efficiency was expressed as an increase in fluorescence intensity relative to the control group.

[0065] The results are as follows Figures 2-3 As shown, Figure 2 This study statistically analyzed the fold increase in mRNA transfection efficiency of 240 compounds compared to the control group. Figure 2 The analysis revealed that seven compounds significantly enhanced mRNA transfection capacity compared to the control group. The results for these seven compounds are presented in a bar chart. Figure 3 Moreover, all seven compounds that enhance transfection efficiency are flavonoids.

[0066] Example 2 Characterization of LNP Particle size testing, the testing method is as follows: Particle size was determined using a Zetasizer Nano (Malvern). The prepared LNP stock solution was diluted (10 μL of stock solution was added to 1 mL of ultrapure water) and added to the particle size sample cell. The particle size of the LNP was measured by dynamic light scattering (DLS). The test conditions were: equilibration time 2 minutes, measurement interval 10 seconds, and temperature 25°C.

[0067] The results show that the particle size is stable. Figure 6 ,from Figure 5 As can be seen, the LNP structure is stable and intact, indicating that the LNP prepared in this invention has good stability. Furthermore, the LNP used is a commercially available lipid: ALC-0315 (purchased from MedChemExpres, catalog number: HY-138170), which has good stability and is only used as characterization data.

[0068] Example 3 Cytotoxicity Assay The cytotoxicity of seven pretreatment compounds—genistein, glabridin, rhoifolin, luteolin, hesperetin, galangin, and pinocemrin—was determined. The test methods are as follows: Cytotoxicity of seven drugs—genistein, glabridin, rhoifolin, luteolin, hesperetin, galangin, and pinocembrin—was assessed in HeLa cells using a CCK-8 assay kit (manufacturer: abcam, catalog number: ab228554). Cells were grown at 5 × 10⁶ cells per well. 3 The microplates were seeded at a density of 10 μmol / L in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow complete adhesion. The original culture medium was then discarded, and fresh culture medium containing the corresponding concentrations (10 μmol / L, 5 μmol / L, and 1 μmol / L) of the seven compounds was added. The blank control group was treated with complete culture medium without any compounds. Multiple replicates were set for each concentration. After another 24 hours of incubation, 10 μL of CCK-8 solution was added to each well, and after incubation for 0.5–1 hour, the absorbance of each well was immediately measured at 450 nm using a microplate reader.

[0069] The results showed that ( Figure 7 The cell viability was >80% for all seven compounds at three different concentrations, indicating that none of the compounds produced effective toxicity to the cells, thus ruling out any impact on cell activity. Figure 2 The impact of the results.

[0070] Example 4: In vivo efficacy verification test The method is as follows: To evaluate seven substances—genistein, glabridin, rhoifolin, luteolin, hesperetin, galangin, and pinocemrin—as drugs, they were administered via pre-gastric gavage. Six hours later, ionizable lipid nanoparticles were injected intramuscularly to determine their biodistribution and delivery efficiency in vivo. This invention also conducted bioluminescence imaging experiments in female C57BL / 6 mice (6-8 weeks old).

[0071] The experiment was divided into two groups. One group used seven compounds, including genistein, as drugs for pre-gavage administration of ionizable lipid nanoparticles. The other group used lipid nanoparticles (ALC-0315). The prepared drug solutions were administered via gavage. (Compound preparation: The purchased dry powder of each compound (1 mg) (7 compounds) was first dissolved in DMSO to prepare a 10 mmol / L stock solution, which was then aliquoted and stored at -20°C. Before use, the stock solution was serially diluted with complete culture medium to prepare working concentrations of 10 μmol / L, 5 μmol / L, and 1 μmol / L, ensuring that the final concentration of DMSO in all experimental groups was below 0.05% (v / v)).

[0072] Six hours after administration (dosage: the stock solution was serially diluted with complete culture medium to prepare working concentrations of 10 μmol / L, 5 μmol / L, and 1 μmol / L, and 156 μL of each concentration was added to the drug), D-fluorescein potassium substrate (150 mg / kg) was administered intraperitoneally. After a 5-minute wait, whole-body imaging of mice anesthetized with isoflurane was performed using a small animal in vivo optical imaging system (IVIS) to acquire bioluminescent signals from the dorsal and ventral sides and major organs.

[0073] The results are as follows Figures 8-11 As shown, the results were the same as those in cell experiments, and the transfection effect was also improved in animals. Moreover, genistein showed the most significant improvement, just like in cell experiments. Subsequent RNA sequencing focused on genistein as the main research object.

[0074] Example 5 RNA Sequencing RNA sequencing was performed by seeding HepG2 cells (manufacturer: Yuanjing Biotechnology, catalog number: YC-C001) in 6-well plates (5 × 10⁶ cells per well). 6Cells were treated with PBS and diluted with complete culture medium to a 10 μmol / L genistein solution for 5 hours (see Example 1 for detailed preparation steps). Cells were then washed three times with PBS, and total RNA was extracted. Sequencing was performed in Novogene, and differentially expressed genes (DEGs) were analyzed using the DEGSeq R software package (1.20.0). P-values ​​were adjusted using the Benjamini and Hochberg methods. A threshold of 0.05 corrected for P-value and log2 (fold change) 1 was selected to determine significant DEGs.

[0075] Sequencing results revealed that drug treatment led to the upregulation of 896 genes and the downregulation of 1313 genes. Figure 12 Treatment with genistein reduced intracellular oxidative stress, characterized by upregulation of antioxidant genes TXNRD1, SRXN1, UGT1A1, AKR1B10, and GSTZ1. Figure 14 Furthermore, pathways related to innate immune activation are also significantly reduced. Figure 13 Furthermore, genistein treatment was found to lead to increased expression of genes associated with protein and RNA degradation. Figure 15 , Figure 16 This is consistent with previous results, indicating that intracellular oxidative stress and innate immune activation inhibit RNA translation and trigger RNA and protein degradation. In contrast, the expression of genes related to oxidative stress, innate immune activation, and RNA and protein degradation was significantly lower in genistein-treated cells than in PBS-treated cells, thus explaining the transfection results in cells and animals. This suggests that providing an antioxidant environment reduces inflammation caused by the innate immune response to LNP in vivo, thereby reducing RNA and protein degradation.

[0076] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. Use of a flavonoid compound for enhancing the efficiency of lipid nanoparticle delivery of a nucleic acid molecule, characterized in that, The flavonoids are at least one of genistein, glabridin, ruscogenin, luteolin, hesperetin, galangin or pinocembrin.

2. Use according to claim 1, characterized in that, The application is to pre-treat the target cells with the flavonoids, and then add the lipid nanoparticles encapsulating the nucleic acid molecules for further culture.

3. Use according to claim 2, characterized in that, The concentration of the flavonoids is 1-10 μmol / L.

4. Use according to claim 3, characterized in that, The pre-treatment time is 4-8 hours, and the further culture time is 20-30 hours.

5. A method of enhancing the efficiency of delivery of a nucleic acid molecule by a lipid nanoparticle, characterized in that, The method comprises the following steps: (1) preparing the lipid nanoparticles encapsulating the nucleic acid molecules; (2) co-incubating the flavonoids with the target cells, then adding the lipid nanoparticles for further culture. The flavonoids are at least one of genistein, glabridin, ruscogenin, luteolin, hesperetin, galangin or pinocembrin.

6. The method of claim 5, wherein, The concentration of the flavonoids in step (2) is 1-10 μmol / L.

7. The method of claim 6, wherein, The concentration of the flavonoids in step (2) is 1, 5 or 10 μmol / L.

8. The method of claim 5, wherein, The co-incubation time in step (2) is 4-8 hours, and the further culture time is 20-30 hours.

9. The method of claim 5, wherein, The method for preparing the lipid nanoparticles in step (1) is as follows: ionizable lipids, auxiliary lipids, cholesterol and phospholipids are dissolved in ethanol respectively, and mixed to obtain a total lipid organic phase; mRNA is dissolved in a citric acid-sodium citrate buffer solution to obtain an mRNA aqueous solution; the total lipid organic phase and the mRNA aqueous solution are mixed by microfluidics to prepare the lipid nanoparticles; and the lipid nanoparticles are dialyzed to obtain the lipid nanoparticles.

10. The method of claim 9, wherein, The mRNA sequence is shown in SEQ ID NO. 1.

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