Nanolipid particles for efficient delivery of RNA into cells

Abstract

The application belongs to the technical field of medicine, and particularly relates to a kind of nanolipid particles for efficiently delivering RNA into cells.In order to develop safer and more effective RNA delivery materials, the application provides a kind of nanolipid particle containing fatty acid or cholesterol coupled polypeptide, which can quickly encapsulate in vitro synthesized circular RNA in a microfluidic nanomedicine preparation system, and the average particle size is 100-200 nm.Meanwhile, the nanolipid particle encapsulating circular RNA can efficiently deliver circular RNA into cells and promote the expression of exogenous genes.Compared with the classic nanolipid particle without lipid polypeptide, the nanolipid particle containing fatty acid or cholesterol coupled polypeptide of the application can significantly improve the expression amount of exogenous genes, and has good application prospect in the field of nucleic acid delivery.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a nanolipid particle for efficiently delivering RNA into cells. Background Art

[0002] Protein-coding RNA (such as mRNA) has become a new class of therapeutic drugs and is used in the prevention and treatment of various diseases in areas such as viral vaccines, protein replacement therapy, cancer immunotherapy, cell reprogramming and genome editing.

[0003] Because RNA is unstable in both in vitro and in vivo environments and is easily degraded and inactivated, conventional drug delivery systems are unable to meet the delivery needs of RNA. Therefore, there is an urgent need for a safe, effective, and stable delivery system that can avoid RNA degradation and allow cellular uptake and RNA release, allowing RNA to exert its therapeutic effects in vivo.

[0004] To date, a variety of RNA delivery materials (including lipids, lipid-like materials, polymers, and protein derivatives, etc.) have been developed, and various corresponding RNA delivery systems have been developed. Among these delivery systems, nanolipid particles (LNPs) have the advantages of high encapsulation efficiency, good stability, and low cytotoxicity. At the same time, LNPs can also improve immune efficacy and produce a strong immune response. The above advantages ultimately make LNP the most common RNA delivery system at present, and it has successfully entered the clinic for the delivery of mRNA. For example: the Moderna COVID-19 vaccine approved by the US FDA, as well as the Biotech and Pfizer COVID-19 vaccines, all use LNP as an mRNA delivery system.

[0005] However, current RNA delivery systems still have deficiencies in targeted delivery and endosomal escape. Therefore, it is necessary to develop safer and more effective RNA delivery materials to further meet clinical needs. Summary of the Invention

[0006] In order to overcome the shortcomings of the above-mentioned prior art, the present invention proposes a nanolipid particle (LNP) containing a fatty acid or cholesterol-coupled polypeptide, which can deliver RNA to cells more effectively than classic LNPs and has good application prospects in the fields of nucleic acid delivery and so on.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a nanolipid particle for efficiently delivering RNA into cells. The nanolipid particle comprises an oil phase and an aqueous phase, wherein the volume ratio of the oil phase to the aqueous phase is 1:2-5; the oil phase comprises a cationic lipid SM-102, distearoylphosphatidylcholine (DSPC), cholesterol (Cholestrol), PEG lipid and a lipid polypeptide, wherein the lipid polypeptide is a palmitic acid (Pal) or cholesterol (Chol) coupled polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID No. 1 (GGRWVKVNGOWIKQ); the aqueous phase comprises a citric acid buffer and circular RNA.

[0009] Preferably, the aqueous phase further comprises ATP; and the mass-to-volume ratio of the ATP to the circular RNA is 1.8-2.2:0.2-0.3 (mg / mL). More preferably, the mass-to-volume ratio of the ATP to the circular RNA is 2 mg / mL:0.26 mg / mL.

[0010] The lipid polypeptide can be a linear polypeptide coupled with a fatty acid and a polypeptide, or a bi-branched peptide formed by coupling two linear lipid polypeptides. Preferably, the lipid polypeptide includes a palmitic acid-coupled linear lipid polypeptide, a palmitic acid-coupled bi-branched peptide, and a cholesterol-coupled bi-branched peptide; the palmitic acid-coupled linear lipid polypeptide is formed by modifying a palmitic acid molecule at the N-terminus of the polypeptide (abbreviated as Pal), the palmitic acid-coupled bi-branched peptide is formed by modifying a palmitic acid molecule at the N-terminus of the polypeptide, and a lysine couples the two polypeptide C-termini (abbreviated as di-Pal), and the cholesterol-coupled bi-branched peptide is formed by modifying a cholesterol molecule at the N-terminus of the polypeptide, and a lysine couples the two polypeptide C-termini (abbreviated as di-Chol).

[0011] Research has shown that LNP formulations containing lipid peptides and ATP can more effectively deliver RNA to cells than classic LNP formulations. Specifically, bifurcated lipid peptides are more effective at promoting RNA delivery than monomeric lipid peptides, demonstrating promising application prospects in areas such as nucleic acid delivery.

[0012] Preferably, the molar ratio of the lipid polypeptide to the circular RNA base is 1:2.5-20. More preferably, the molar ratio of the lipid polypeptide to the circular RNA base is 1:2.5, 1:5, 1:10, or 1:20.

[0013] Preferably, the pH of the citric acid buffer is 3.5-4.5, more preferably 4.0.

[0014] Preferably, the molar ratio of the cationic lipid SM-102, distearoylphosphatidylcholine, cholesterol, and PEG lipid is 45-55:9-11:35-40:1-2. More preferably, the molar ratio of the cationic lipid SM-102, distearoylphosphatidylcholine, cholesterol, and PEG lipid is 50:10:38.5:1.5.

[0015] Preferably, the nitrogen to phosphorus ratio of the circular RNA to the cationic lipid SM-102 is 5-7: 1. The nitrogen to phosphorus ratio of the circular RNA to the cationic lipid SM-102 is preferably 6:1.

[0016] Preferably, the PEG lipid includes (but is not limited to) DMG-PEG2000.

[0017] Preferably, the solvent of the oil phase includes (but is not limited to) ethanol. More preferably, the solvent of the oil phase is anhydrous ethanol.

[0018] Preferably, the lipid polypeptide is dissolved in 45-55% ethanol and then added to the oil phase.

[0019] The second aspect of the present invention provides a method for preparing nanolipid particles for efficiently delivering RNA into cells as described in the first aspect, specifically: on a rapid nanodrug preparation system (Micro&Nano INano E), a microfluidic chip with a channel pore size of 200-300 μm is used to prepare nanolipid particles together with the oil phase and the aqueous phase.

[0020] Preferably, the microfluidic program is set to a total flow rate of 10-15 mL / min, a flow rate of the oil phase:water phase of 1:2-5; and the front waste is set to 0.2 mL and the rear waste is set to 0.05 mL.

[0021] Preferably, after the nanolipid particles are prepared, ultrafiltration is performed using a 100 kDa ultrafiltration tube, and the solution in the nanolipid particles is replaced with PBS.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The RNA encoding gene can be used as a vaccine or a drug for treating diseases. However, how to efficiently deliver the intact RNA molecule into cells is a problem in the development of nucleic acid drugs. To this end, the present application provides a nano-lipid particle containing a fatty acid or cholesterol coupled polypeptide, which can quickly encapsulate the circular RNA synthesized in vitro in a microfluidic nano-drug preparation system, and the average particle size thereof is 100-200 nm. At the same time, the nano-lipid particle encapsulating the circular RNA of the present application can efficiently deliver the circular RNA into cells and promote the expression of exogenous genes. Compared with the classic nano-lipid particle without lipid polypeptide, the nano-lipid particle containing the fatty acid or cholesterol coupled polypeptide of the present application can significantly improve the expression amount of exogenous genes, and has good application prospect in the field of nucleic acid delivery. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A map of T4 bacteriophage type I intron self-cleavage system plasmid (T4 PIE);

[0025] Figure 2 A backbone mode of circular RNA, mainly containing two short fragments of exons (E1 and E2) and intron fragments (3'intron and 5'intron) in the type I intron of T4 bacteriophage thymidylate synthase (Td) gene, encephalomyocarditis virus internal ribosome entry site (EMCV IRES), gene coding region (CDS), spacer sequence (spacer1 and spacer2);

[0026] Figure 3 A agarose gel electrophoresis map before and after the circular RNA is formed and before and after the Rnase R digestion treatment;

[0027] Figure 4 A structure schematic diagram of the lipid coupled polypeptide;

[0028] Figure 5 A molecular structure diagram of di-Pal (A), Pal (B) and di-Chol (C);

[0029] Figure 6 An ESI-MS map of di-Pal;

[0030] Figure 7 An ESI-MS map of Pal;

[0031] Figure 8 An ESI-MS map of di-Chol;

[0032] Figure 9FIG. 6 shows the particle size distribution of LNP encapsulating circular RNA encoding GFP protein with the addition of di-Pal to the classic LNP formulation, where the base molar ratio of di-Pal to RNA is 1 :2.5, 1 :5, 1 :10, 1 :20, or 1 :40;

[0033] Figure 10 FIG. 7 shows the fluorescence intensity distribution of cells transfected with LNP encapsulating circular RNA encoding GFP protein with the addition of di-Pal to the classic LNP formulation (A), and the comparison of the mean fluorescence intensity of different groups of cells (B), where the data difference analysis is compared with the LNP group as control, ns indicates no statistical difference, *** indicates p < 0.001, **** indicates p < 0.0001;

[0034] Figure 11 FIG. 8 shows the particle size distribution of LNP encapsulating circular RNA encoding GFP protein with the addition of di-Pal, Pal, or di-Chol polypeptide to the classic LNP formulation, where the base molar ratio of di-Pal, Pal, or di-Chol polypeptide to RNA is 1 :10;

[0035] Figure 12 FIG. 9 shows the fluorescence intensity distribution of cells transfected with LNP delivering circular RNA encoding GFP to the cells, where A-D are the LNP formulations without lipid polypeptide, and the LNP formulations with the addition of di-Pal, Pal, or di-Chol polypeptide, where the base molar ratio of lipid polypeptide to RNA is 1 :10, and the transfection amount of RNA in each group of cells is 5-160 ng;

[0036] Figure 13 FIG. 10 shows the particle size distribution of LNP encapsulating circular RNA encoding Renilla luciferase with the addition of di-Pal, Pal, or di-Chol polypeptide to the classic LNP formulation, where the base molar ratio of di-Pal, Pal, or di-Chol polypeptide to RNA is 1 :10;

[0037] Figure 14 FIG. 11 shows the transfection efficiency of LNP encapsulating circular RNA encoding Renilla luciferase with the addition of di-Pal, Pal, or di-Chol polypeptide to the classic LNP formulation, where the base molar ratio of di-Pal, Pal, or di-Chol polypeptide to RNA is 1 :10, and the transfection amount of circular RNA encoding Renilla luciferase is 5-320 ng, where the firefly luciferase encapsulated by the classic LNP is the internal reference, and the increase in the expression of Renilla luciferase is shown in panel D, where the data difference analysis is compared with the LNP group as control, ns indicates no statistical difference, * indicates p < 0.05, **** indicates p < 0.0001. DETAILED DESCRIPTION

[0038] The following is a further description of specific embodiments of the present invention. It should be noted that the description of these embodiments is intended to facilitate understanding of the present invention and does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0040] Example 1 Preparation of protein-encoding circular RNA

[0041] RNA circularization was performed using the T4 phage type I intron self-cleavage method. The vector skeleton (hereinafter referred to as T4PIE) was synthesized by Guangzhou Ruibo Technology Co., Ltd., and the firefly luciferase (hereinafter referred to as Fluci), Renilla luciferase gene (hereinafter referred to as Rluci), and green fluorescent protein gene (hereinafter referred to as GFP) were inserted between BstXⅠ and SalⅠ of the vector by PCR cloning. The constructed vectors were named T4 PIE-Fluci, T4 PIE-Rluci, and T4 PIE-GFP, respectively. Figure 1 The following is a map of the plasmid backbone, which primarily consists of two short exon segments (E1 and E2) and intron segments (3' intron and 5' intron) within the group I intron of the T4 phage thymidylate synthase (Td) gene, the encephalomyocarditis virus internal ribosome entry site (EMCV IRES), the gene coding region (CDS) (GFP or Rluci or GFP), and spacer sequences (spacer1 and spacer2). The sequences of the components of the T4PIE vector are shown in Table 1:

[0042] Table 1 Sequence information of each component of T4PIE vector

[0043]

[0044]

[0045]

[0046]

[0047] The bacterial solution of T4 PIE-Fluci, T4 PIE-Rluci and T4 PIE-GFP plasmids with correct sequencing (the bacterial solution of Escherichia coli DH5a was commissioned by Guangzhou Ruibo Company to complete the process, and the bacterial solution OD260 =0.6-0.8) was inoculated into liquid LB medium containing ampicillin (0.05 mg / mL) at a volume ratio of 1:100 and cultured in a shaking incubator at 37°C for 12-16 hours. Plasmid DNA was then extracted using a plasmid mini-extraction kit (Meiji Bio). After obtaining the plasmid, the quality and concentration of the plasmid DNA (A260 / A260) were detected using a Nanodrop 2000. 280 =1.8-2.0, with a concentration of approximately 100 ng / μL). The extracted plasmid was then linearized using the EcoRV endonuclease at 37°C for 1 hour. 0.1 mg / mL proteinase K was then added to the reaction system and digested at 55°C for 40-60 minutes to remove residual protein from the plasmid DNA. Following the reaction, the DNA was recovered using the Wizard DNA Clean-Up System (Promega), and the resulting DNA was dissolved in RNase-free deionized water.

[0048] Next, an in vitro transcription reaction was performed using the linearized plasmid DNA obtained above as a template using T7 RNA polymerase. The reaction system was as follows: 40 mM Tris-HCl (pH 8.0), 8 mM MgCl2, 5 mM KCl, 100 mM DTT, 2 mM spermidine, 2 mM NTP, 1 U / μL Ribolock RNase Inhibitor, 0.001 U / μL inorganic pyrophosphatase, 2 U / μL T7 RNA polymerase, and 20 ng / μL plasmid DNA. The reaction was incubated at 37°C for 2 hours, followed by the addition of 1 μL DNase I (2 U / μL) and an additional 15 minutes at 37°C. The reaction was terminated by the addition of EDTA to a final concentration of 10 mM, and RNA was recovered using an RNA purification kit (Meiji Biotechnology).

[0049] Finally, the resulting RNA was circularized using the following reaction system: 0.5 μg / μL RNA, 1 mM DTT, 2 mM GTP, 50 mM Tris-HCl (pH 7.0), 150 mM NaCl, and 20% glycerol. The sample was incubated at 70°C for 5 minutes, followed by the addition of MgCl2 to a final concentration of 20 mM. The reaction was then continued at 55°C for 8 minutes to complete the circularization reaction. RNase R was then added to 1 U of RNase R (200 ng of linear RNA) for 15 minutes at 37°C to digest any uncircularized linear RNA. The reaction was terminated by the addition of EDTA to a final concentration of 25 mM. Finally, the circular RNA was recovered using an RNA purification kit (MeiGen Biotech) and stored at -80°C. Figure 2 A schematic diagram of circularized RNA.

[0050] The RNA samples before and after the cyclization reaction were digested with RNase R and then subjected to agarose gel electrophoresis to detect whether the RNA cyclization was successful. Figure 3 As shown in the figure, the RNA after the circularization reaction showed the characteristics of resistance to RNase R exo-cleavage compared with the RNA without circularization treatment, indicating the successful preparation of circular RNA.

[0051] Example 2 Preparation of circular RNA-loaded nanolipid particles (LNPs)

[0052] Cationic lipid SM-102, distearoylphosphatidylcholine (DSPC), Cholestrol, and PEG lipid (DMG-PEG2000) were purchased from MCE. Palmitic acid (Pal) or cholesterol (Chol) coupled peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. The structural model is shown in Figure 2. Figure 4 As shown, the amino acid sequence of the polypeptide is GGRWVKVNGOWIKQ. The linear lipid polypeptide formed by modifying a palmitic acid molecule at the N-terminus of the polypeptide is abbreviated as Pal; the bifurcated lipid polypeptide formed by modifying a palmitic acid molecule at the N-terminus of the polypeptide and coupling two polypeptide C-termini with a lysine is abbreviated as di-Pal; and the bifurcated lipid polypeptide formed by modifying a cholesterol molecule at the N-terminus of the polypeptide and coupling two polypeptide C-termini with a lysine is abbreviated as di-Chol. The polypeptide portion of the lipid polypeptide contains multiple positively charged amino acids (K, R), which are speculated to have charge attraction with RNA; the fatty acid or cholesterol portion of the lipid polypeptide can then interact with other lipids. Figure 5 It is the molecular structure of three lipid polypeptides (di-Pal, Pal and di-Chol). Figure 6-8 The following is a mass spectrometry analysis of three lipid peptides (di-Pal, Pal, and di-Chol). The mass spectrometry results show that the purity of these three lipid peptides is greater than or equal to 95%, and their molecular weights are 3871.05, 1800.44, and 4331.65, respectively.

[0053] To prepare circular RNA-encapsulated nanolipid particles, the nitrogen-to-phosphorus ratio (RNA nitrogen content / cationic lipid phosphorus content) was controlled at 6:1. The oil phase solvent was anhydrous ethanol, and the molar ratio of the components was: cationic lipid: DSPC: Cholestrol: PEG lipid = 50:10:38.5:1.5, i.e., cationic lipid (50 mM), DSPC (10 mM), cholesterol (38.5 mM), and PEG lipid (1.5 mM). The lipid peptide was dissolved in 50% ethanol (typically to a 10 mg / mL stock solution) and then added to the oil phase. The aqueous phase contained citrate buffer (pH 4.0) and 0.0895 mg / mL of circular RNA. Nanolipid particles were prepared using a rapid nanodrug preparation system (Micro&Nano INano E) using a microfluidic chip with a channel pore size of 200-300 μm. The microfluidic program was set to a total flow rate of 12 mL / min (oil phase:water phase ratio = 1:3), with a pre-waste volume of 0.2 mL and a post-waste volume of 0.05 mL. The prepared nanolipid particles were added to a 100 kDa ultrafiltration tube and ultrafiltered at 1000 g for 10 min. The solution in the nanolipid particles was then replaced with PBS at least 40 times the sample volume. Approximately 25 μL of the ultrafiltered sample was removed for particle size measurement. The remaining sample was diluted with PBS containing 50% sucrose, mixed, and filtered through a 0.22 μM filter membrane in a clean hood. The aliquoted nanolipid particles were stored at -80°C.

[0054] (1) Palmitic acid-coupled di-branched peptide (di-Pal) improves the efficiency of RNA delivery in classic LNP formulations

[0055] Circular RNA encoding green fluorescent protein (GFP) was prepared into RNA-encapsulated nanolipid particles according to the above method. The formulations of the oil phase components, di-Pal (added to the oil phase), and RNA are shown in Table 2.

[0056] The RNA-encapsulated nanolipid particles were characterized using a Malvern particle sizer. The results were as follows: Figure 9 As shown, when the di-Pal to RNA base ratio (calculated based on the number of RNA bases that can be bound by a molecular weight of coupled polypeptide) was 1 / 10, 1 / 20, and 1 / 40, the particle size of the nanolipid particles was close to that without lipid polypeptide, at approximately 100 nm. When the di-Pal to RNA base ratio was 1 / 2.5 and 1 / 5, the particle size of the nanolipid particles increased significantly, approaching 200 nm.

[0057] At the same time, the efficiency of RNA delivery by nanolipid particles was evaluated by detecting the cellular GFP fluorescence intensity. 5Cells were seeded into 24-well plates at a density of 10 cells per well for culture (Gibco DMEM). When the cell density reached 70-90%, RNA-loaded nanolipid particles were added at a dose of 200 ng per well. The cells were then cultured in a 37°C, 5% CO2 incubator for 36 hours. Flow cytometry was then used to detect GFP expression in the cells. Figure 10 Results in A and 10B show that compared to the classic LNP formulation group without di-Pal, the addition of di-Pal to a base ratio of di-Pal to RNA of 1 / 20 or higher significantly enhanced the cellular GFP fluorescence intensity. The fluorescence intensity was highest at a di-Pal to RNA base ratio of 1 / 10, but further increasing the di-Pal to RNA base ratio resulted in a decrease in fluorescence intensity. This may be because the excessive lipid peptide increases the particle size of the nanolipid particles, thereby affecting delivery efficiency.

[0058] Table 2 Formulation of nanolipid particles

[0059]

[0060] (2) ATP enhances the efficiency of LNP delivery of circular RNA encoding green fluorescent protein

[0061] Circular RNA encoding green fluorescent protein (GFP) was prepared as RNA-encapsulated nanolipid particles according to the above method. The formulations of the oil phase components, lipid peptides, ATP (added to the aqueous phase), and RNA are shown in Table 3. The base ratio of Pal, di-Chol, or di-Pal to RNA was 1:10.

[0062] The particle size of RNA-loaded nanolipid particles was characterized using a Malvern particle size analyzer. Figure 11 Results from the study showed that when using Pal and di-Pal peptides, the particle size of the nanolipid particles was close to that of the non-lipid peptides, approximately 100 nm. When using di-Chol peptide, the particle size of the nanolipid particles nearly doubled. However, the addition of ATP had little effect on the particle size of the nanolipid particles.

[0063] At the same time, the efficiency of RNA delivery by nanolipid particles was evaluated by detecting the GFP fluorescence intensity of cells. The transfection amount of GFP circular RNA in each well was 5-160 ng. Figure 12As shown in the cell fluorescence intensity distribution diagram, ATP can increase the efficiency of RNA delivery of the four LNP formulations, especially the formulation with added di-Pal, which has the highest efficiency in RNA delivery. This shows that ATP can strongly promote the delivery of RNA into cells by di-Pal-containing nanolipid particles. In contrast, the monomeric Pal peptide did not significantly improve the transfection efficiency compared to di-Pal, indicating that the bifurcated lipid peptide is more effective than the monomer. Similarly, the bifurcated di-Chol peptide can also significantly improve the transfection efficiency, but because the addition of di-Chol peptide causes the particle size of the nanolipid particles to increase, its transfection efficiency is lower than that of di-Pal.

[0064] Table 3 Formulation 2 of nanolipid particles

[0065]

[0066] (3) ATP enhances the efficiency of LNP delivery of luciferase-encoding circular RNA

[0067] Circular RNA encoding Renilla luciferase (Rluci) and firefly luciferase (Fluci) were prepared into RNA-encapsulated nanolipid particles according to the above method. The formulations of the oil phase components, lipid peptides, and RNA are shown in Table 4. Among them, the base ratio of Pal or di-Chol or di-Pal to RNA was 1:10. At the same time, RNA encoding firefly luciferase (Fluci) was encapsulated using a classic LNP formulation without lipid peptides to serve as an internal reference.

[0068] The particle size of RNA-loaded nanolipid particles was characterized using a Malvern particle size analyzer. Figure 13 As shown, when using Pal and di-Pal peptides, the particle size of the nanolipid particles was close to that of the particles without lipid peptides, approximately 100 nm. When using di-Chol peptide, the particle size of the nanolipid particles nearly doubled. However, the addition of ATP had little effect on the particle size of the nanolipid particles. This result is consistent with the results of the GFP-encapsulated RNA.

[0069] The efficiency of RNA delivery by the nano-lipid particles was evaluated by detecting the expression amount of luciferase in the cells, the transfection amount of the sea-horse luciferase circular RNA in each cell was 5-320 ng, and the amount of the reference firefly luciferase circular RNA was 500 ng. The expression amount of luciferase was evaluated by a dual luciferase reporter assay system (Promega). Specifically, the cell culture medium in the 24-well plate was removed, 100-1000 μL of 1×passive lysis buffer (Promega) was added, and after lysis at room temperature for 15 min, the luminescence signals of the firefly and sea-horse luciferases were detected, respectively. The relative expression amount of luciferase was represented by the luminescence signal generated by the sea-horse luciferase divided by the signal value generated by the firefly luciferase. As shown in Figure 14 As shown in the results of A-C, with the increase of the transfection RNA amount, the expression amount of sea-horse luciferase gradually increased, and in the absence of ATP, the RNA delivery efficiency of the LNP formulation containing the lipid polypeptide was improved compared with the classic LNP formulation, and the expression amount of sea-horse luciferase was increased by 5-6 times Figure 14 D) When ATP was added, the RNA delivery efficiency of other LNP formulations was further improved except for the Pal group, and especially for the di-Pal group, the expression amount of sea-horse luciferase was increased by more than 29 times compared with the classic LNP formulation after the addition of ATP.

[0070] As can be seen from the above, the LNP formulation added with the lipid polypeptide and ATP can more effectively deliver RNA to cells compared with the classic LNP formulation, and has a good application prospect in the field of nucleic acid delivery.

[0071] Table 4 Formulation three of nano-lipid particles

[0072]

[0073] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A nanolipid particle for delivering RNA into cells, characterized in that: The nanolipid particles include an oil phase and an aqueous phase, wherein the volume ratio of the oil phase to the aqueous phase is 1:2-5; the oil phase includes a cationic lipid SM-102, distearoylphosphatidylcholine, cholesterol, PEG lipid, and a lipid polypeptide, wherein the lipid polypeptide is a palmitic acid- or cholesterol-coupled polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID No. 1; the aqueous phase includes a citric acid buffer, circular RNA, and ATP; and the base molar ratio of the lipid polypeptide to the circular RNA is 1:2.5-20. The lipid polypeptide is selected from a palmitic acid-coupled two-branched peptide or a cholesterol-coupled two-branched peptide; the palmitic acid-coupled two-branched peptide is formed by modifying the N-terminus of the polypeptide with a palmitic acid molecule and coupling the two C-termini of the polypeptide with a lysine; the cholesterol-coupled two-branched peptide is formed by modifying the N-terminus of the polypeptide with a cholesterol molecule and coupling the two C-termini of the polypeptide with a lysine.

2. A nanolipid particle for delivering RNA into cells according to claim 1, characterized in that The usage ratio of ATP to circular RNA is 1.8-2.2:0.2-0.

3.

3. A nanolipid particle for delivering RNA into cells according to claim 1, characterized in that The pH of the citric acid buffer is 3.5-4.

5.

4. The nanolipid particle for delivering RNA into cells according to claim 1, characterized in that The molar ratio of the cationic lipid SM-102, distearoylphosphatidylcholine, cholesterol and PEG lipid is 45-55:9-11:35-40:1-2.

5. The nanolipid particle for delivering RNA into cells according to claim 1, characterized in that The nitrogen-phosphorus ratio of the circular RNA to the cationic lipid SM-102 is 5-7:

1.

6. The method for preparing nanolipid particles for delivering RNA into cells according to any one of claims 1 to 5, characterized in that: In the rapid nanomedicine preparation system, a microfluidic chip with a channel pore size of 200-300 μm is used to prepare nanolipid particles together with the oil phase and the water phase.

7. The method for preparing nanolipid particles for delivering RNA into cells according to claim 6, wherein: The microfluidic program was set to a total flow rate of 10-15 mL / min, a flow rate of the oil phase:water phase of 1:2-5; and the front waste was set to 0.2 mL and the rear waste was set to 0.05 mL.

8. The method for preparing nanolipid particles for delivering RNA into cells according to claim 6, wherein: After the nanolipid particles were prepared, they were ultrafiltered using a 100 kDa ultrafiltration tube, and the solution in the nanolipid particles was replaced with PBS.

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