Method for quickly loading RNA (Ribonucleic Acid)
By mixing the pegylated no-loaded liposomes with RNA in an acidic solution and adjusting the pH to neutral, the existing LNP delivery RNA problems are solved, and fast, low-cost RNA loading and efficient RNA transfection are achieved.
Patent Information
- Application Number
- CN202411980857.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-05-06
AI Technical Summary
The steps for delivering RNA by existing lipid nanoparticles (LNPs) are cumbersome and costly, resulting in excessive RNA screening working time and material costs.
A method of rapid RNA loading is achieved by mixing the pegylated no-loaded liposomes with the target RNA in an acidic solution and adjusting the solution pH to neutral.
This method simplifies the RNA loading process, reduces costs, and improves the efficiency and reliability of RNA transfection.
Smart Images
Figure CN119932118A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology and medical technology, and in particular to a method for rapidly loading RNA. Background Art
[0002] Delivery carriers are essential for RNA molecules to function. Currently, the delivery carriers are mainly lipid nanoparticles (LNPs). The lipid nanoparticle delivery system is one of the important technologies in the lipid carrier drug delivery system. It can be used as an effective transport carrier to deliver various bioactive molecules to target cells and tissues, including small molecule drugs and nucleic acid substances. Compared with traditional drug delivery methods, lipid carrier technology has many advantages, such as increasing the stability and bioavailability of drugs. In addition, the application scope of LNP has also expanded to other fields such as cosmetics and food industry.
[0003] Generally, RNA molecules are mixed with lipid molecules through microfluidic chips to form lipid nanoparticles, which are then dialyzed before use. However, RNA drugs prepared using this method have a minimum feed amount limit, which brings huge time and material costs to RNA screening. Therefore, there is an urgent need to develop nucleic acid delivery technologies that are easy to use, have reliable transfection effects, and are low-cost. Summary of the invention
[0004] The purpose of the present invention is to provide a method for rapid RNA loading in order to overcome the limitations of the existing LNP delivery of RNA, which is cumbersome and costly. The mode of rapid RNA loading described in the present invention is to mix the PEGylated empty liposomes with the target RNA in an acidic solution, and then adjust the solution to a neutral pH to complete the rapid loading.
[0005] The purpose of the present invention can be achieved by the following technical solutions:
[0006] A method for rapid RNA loading, the specific steps are as follows:
[0007] S1. preparing PEGylated empty liposomes;
[0008] S2, mixing the target RNA with the PEGylated empty liposomes obtained in step S1 at an acidic pH to obtain a mixed solution;
[0009] S3. The pH of the mixed solution obtained in step S2 is adjusted to neutral, and the target RNA can be quickly loaded into the liposome, which can be used for RNA transfection experiments in cells and living organisms.
[0010] Furthermore, in step S1, the specific method for preparing PEGylated empty liposomes is as follows:
[0011] S1-1, preparing an organic phase solution containing ionizable cationic lipids, auxiliary phospholipids, cholesterol and PEGylated phospholipids;
[0012] S1-2, preparing a metal ion aqueous solution;
[0013] S1-3, adding the metal ion solution obtained in step S1-2 to the organic phase solution obtained in step S1-1, mixing evenly to obtain a mixed solution;
[0014] S1-4, using a shear mixer, an ultrasonic crusher, a high-pressure homogenizer or a liposome extruder to process the mixed solution obtained in step S1-3 to obtain a metallized lipid nanoparticle solution with a particle size distribution of 50-500 nm;
[0015] S1-5, removing free metal ions and ethanol from the metallized lipid nanoparticle solution obtained in step S1-4 by ultrafiltration or dialysis to obtain a metallized liposome mother solution;
[0016] S1-6, mixing the metallized liposome mother solution obtained in step S1-5 with a lyophilization protective agent, and lyophilizing to obtain PEGylated empty liposomes.
[0017] As a preferred technical solution, the ionizable cationic lipid contains functional groups that can be protonated under acidic conditions, such as amino, imidazole, pyridyl and phenolic hydroxyl groups.
[0018] Furthermore, in step S1-1, the ionizable cationic lipid is selected from 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-DMA-MC3), ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate) (SM-102), 1,2-dioleyl-3- Molecules such as methylamino-propane (DODMA), 3-(dimethylamino)propane-1,2-diyl dioleate (DODAP), 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid heptadecan-9-yl ester, ((3-hydroxypropyl)azadiyl)bis(nonane-9,1-diyl)bis(octanoic acid dibutyl ester), L319, Dlin-KC2-DMA, YSK12-C4, CL4H6, Arcturus10q or ssPalmO-Phe.
[0019] Furthermore, in step S1-1, the auxiliary phospholipid is selected from distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearate-3-phosphatidylethanolamine polyethylene glycol (DSPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidic acid (DPPA), 1-oleoyl-2-european-SN-glycero-phosphoethanolamine (OEPE) or 1-oleoyl-2-palmitoleoyl-SN-glycero-phosphoethanolamine (OPPE), etc.
[0020] Furthermore, in step S1-1, the molar percentage of the ionizable cationic lipid, auxiliary phospholipid and cholesterol is 40% to 60%: 8% to 12%: 35% to 40%, preferably 50%: 10%: 38.5%.
[0021] Furthermore, in step S1-1, the ionizable cationic lipids in the organic phase solution contain PEGylated phospholipids, and calculated by molar amount, the PEGylated phospholipids account for 0.3%-15% of the organic phase solution, preferably 1.5%.
[0022] Furthermore, in step S1-1, the organic solvent in the organic phase solution is selected from ethanol, methanol, chloroform or tetrahydrofuran.
[0023] Furthermore, in step S1-2, the metal ions are selected from any one or more of Ca, Mg, Zn, Ba, Mn, Ni, Co, Fe, Cu, Al, Bi or Ti.
[0024] Furthermore, in step S1-3, the mixing method of the metal ion solution and the organic phase solution is selected from a microfluidic LNP preparation method, a solvent injection method, a thin film dispersion method or a high-pressure homogenization method.
[0025] Furthermore, in step S1-3, the concentration range of the metal ions in the mixed solution is 0.1-30 mM.
[0026] Furthermore, in step S1-6, the lyoprotectant is selected from sucrose or trehalose.
[0027] Furthermore, in step S1-6, the mass ratio of the metallized liposome mother solution to the lyophilization protective agent is 8 to 10:1, preferably 9:1.
[0028] Furthermore, in step S2, the acidic pH range is 2.0 to 7.0.
[0029] Furthermore, in step S2, the target RNA is dissolved in an acidic buffer, and then PEGylated empty liposomes are added and dissolved to obtain a mixed solution.
[0030] In the above, the target RNA: acidic buffer: PEGylated empty liposomes = 1-5 μg: 25-500 μL: 25-500 μL;
[0031] Furthermore, in step S2, the target RNA is dissolved in an acidic buffer, the PEGylated empty liposomes are dissolved in an acidic buffer, the target RNA in the acidic environment and the PEGylated empty liposomes are mixed, suspended evenly, and a mixed solution is obtained after waiting for a period of time.
[0032] Furthermore, in the above, the concentration of the PEGylated empty liposomes is 1 to 10 mg / mL, preferably 1.5 mg / mL.
[0033] In the above, the acidic buffer is selected from molecules such as formic acid, acetic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, tartaric acid, isotartaric acid, malic acid, citric acid or isocitric acid, and the concentration range of the acidic buffer is 0.1mM to 10mM.
[0034] Furthermore, in step S3, the neutral pH range is 7-8.
[0035] Furthermore, in step S3, an alkaline buffer is added to the mixed solution obtained in step S2 to adjust its pH to neutral, so that the target RNA can be quickly loaded into the liposome, which can be used for RNA transfection experiments in cells and living organisms.
[0036] In the above further aspect, the alkaline buffer is Tris base.
[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0038] The incorporation of PEG in the present invention solves the problem of increased liposome particle size and reduced transfection efficiency after RNA is loaded in a mix-and-use mode.
[0039] The present invention utilizes acidic pH to regulate the surface charges of PEGylated empty liposomes and RNA, and the RNA can be loaded into the liposomes by simply mixing the two, thereby achieving cell transfection capability.
[0040] The PEGylated empty liposomes prepared in the present invention need to be prepared in advance and are generally stored at 2-8° C. The target RNA stock solution is stored at -20° C. to 70° C. Before use, the two are mixed under acidic conditions and the pH is adjusted. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Figure 1 Schematic diagram of the effect of PEG lipids on the particle size of liposomes loaded with RNA;
[0042] Figure 2 This is a statistical graph of the average fluorescence intensity of flow cytometry analysis after Hela cells were transfected with mRNA loaded under different types and concentrations of carboxylic acid conditions. DETAILED DESCRIPTION
[0043] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.
[0044] In the following examples, the sources of the reagents are as follows:
[0045] SM-102, DSPC, cholesterol and polyethylene glycol phospholipids were purchased from Avituo Company, and anhydrous ethanol, zinc nitrate and citric acid were purchased from Sinopharm Company.
[0046] In conjunction with the accompanying drawings, some embodiments of the present invention are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.
[0047] Example 1
[0048] This embodiment provides a method for preparing PEGylated empty liposomes. Taking the microfluidic preparation of LNP as an example, metal ions are incorporated into the preparation of PEGylated empty liposomes. The specific steps are as follows:
[0049] S1. Prepare a single-component lipid solution: dissolve SM-102, DSPC, cholesterol and polyethylene glycol phospholipid in anhydrous ethanol to obtain four single-component lipid solutions with a concentration of 10 mg / mL;
[0050] S2, the four single-component lipid solutions obtained in step S1, including 142 μL of SM-102, 31.6 μL of DSPC, 59 μL of cholesterol, and 15 μL of polyethylene glycol, were mixed, and then 500 μL of anhydrous ethanol was added to form an organic phase solution;
[0051] S3, preparing a 5 mM zinc nitrate aqueous solution as the aqueous phase;
[0052] S4, the zinc nitrate aqueous solution obtained in step S3 and the organic phase solution obtained in step S2 are mixed uniformly at a volume ratio of aqueous phase: organic phase = 3:1, and subjected to microfluidics (total flow rate of 10 mL / min) to form a metallized lipid nanoparticle solution. After standing for 5 minutes, an equal volume of ultrapure water is added to obtain a mixed solution;
[0053] S5. Removing free metal ions from the metallized lipid nanoparticle solution obtained in step S4 by ultrafiltration or dialysis system: The metallized lipid nanoparticle solution obtained in steps S1-4 is passed through a 10 kDa ultrafiltration tube or dialysis box, and free metal ions in the metallized lipid nanoparticle solution obtained in step S4 are removed by repeated rinsing or dialysis; for larger volumes, consider using a hollow fiber membrane ultrafiltration system to remove free metal ions to obtain a metallized liposome mother solution.
[0054] S6. Mix the metallized liposome mother solution obtained in step S5 with 20% sucrose solution in equal volumes, dispense into 1.5 mL EP tubes, 300 μL per tube, and place them in a freeze dryer with the opening opened for freeze drying to obtain PEGylated empty liposomes.
[0055] Example 2
[0056] This embodiment provides a method for rapid RNA loading, and the specific steps are as follows:
[0057] S1. Take 100 μL of 1 mM citric acid solution, add 4.5 μg of eGFP mRNA, mix well, and obtain an acidic RNA solution;
[0058] S2, directly adding the acidic RNA solution obtained in S1 to 100 μL of the PEGylated empty liposomes prepared in Example 1 to dissolve the lyophilized liposomes to obtain a mixed solution;
[0059] S3. After standing for 30 seconds, add 8 μL of 0.1 M Tris base solution to the mixed solution obtained in step S2 and adjust its pH to neutral, so as to quickly load the target RNA into the liposome.
[0060] Example 3
[0061] This embodiment provides a method for rapid RNA loading, and the specific steps are as follows:
[0062] S1. Take 15 μL of 10 mM acidic buffer, add 4.5 μg of eGFP mRNA, mix well, and obtain an acidic RNA solution;
[0063] S2, taking 135 μL of 0-5 mM acidic buffer to dissolve 100 μL of the PEGylated empty liposomes prepared in Example 1 to obtain an acidic metallized liposome solution;
[0064] S3, adding the acidic RNA solution obtained in step S1 to the acidic metallized liposome solution obtained in step S2, mixing well to obtain a mixed solution;
[0065] S4. After standing for 30 seconds, add 6 μL of 0.1 M Tris base solution to the mixed solution obtained in step S3 and adjust its pH to neutral, so as to quickly load the target RNA into the liposome.
[0066] In this embodiment, in step S1 and step S2, the acidic buffer solution includes citric acid solution, malonic acid solution, succinic acid solution, tartaric acid solution and malic acid solution.
[0067] Comparative Example 1
[0068] This comparative example provides an existing method for loading RNA, except that polyethylene glycol is not added when preparing empty liposomes, the remaining steps are the same as those of Example 1 and Example 2 for loading eGFP mRNA.
[0069] Comparative Example 2
[0070] This comparative example provides an existing method for loading RNA. Except for replacing carboxylate with PBS during the loading process, the remaining steps are the same as those of Example 1 and Example 3 for loading eGFP mRNA.
[0071] Test Example 1
[0072] This test example uses a particle size potentiometer to measure the particle size of the liposomes during RNA loading in Example 2 and Comparative Example 1. The experimental results are shown in Figure 1 As shown, compared with the metallized liposomes without PEG prepared in Comparative Example 1, the metallized liposomes containing PEG prepared in Example 1 can maintain the same liposome particle size after RNA loading, which is beneficial to mRNA delivery, thus proving that polyethylene glycol can stabilize the particle size of liposomes during RNA loading.
[0073] Test Example 2
[0074] This test example tests the transfection efficiency of cells loaded with mRNA under acidic conditions. The specific steps are as follows:
[0075] Hela cells were used as target cells for transfection experiments. 50,000 cells were plated in 24 wells. After 24 hours, eGFP mRNA was loaded according to the RNA loading steps of Example 3 and Comparative Example 2. 500 ng of mRNA was added to each well, and cells were collected for flow cytometry analysis after 24 hours.
[0076] The statistical graph of the average fluorescence intensity of flow cytometry analysis after Hela cells were transfected with mRNA under different types and concentrations of carboxylic acid is shown in the figure. Figure 2 As shown, when the concentrations of citric acid solution, malonic acid solution, succinic acid solution, tartaric acid solution and malic acid solution are 0.5 mM and 1 mM, the average fluorescence intensity is high, and the loaded mRNA is suitable for transfecting Hela cells.
[0077] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.
Claims
1. A method for rapid RNA loading, characterized in that: The specific steps are as follows: S1. preparing PEGylated empty liposomes; S2, mixing the target RNA with the PEGylated empty liposomes obtained in step S1 at an acidic pH to obtain a mixed solution; S3. The pH of the mixed solution obtained in step S2 is adjusted to neutral, and the target RNA can be quickly loaded into the liposome, which can be used for RNA transfection experiments in cells and living organisms.
2. A method for rapid RNA loading according to claim 1, characterized in that: In step S1, the specific method for preparing PEGylated empty liposomes is as follows: S1-1, preparing an organic phase solution containing ionizable cationic lipids, auxiliary phospholipids, cholesterol and PEGylated phospholipids; S1-2, preparing a metal ion aqueous solution; S1-3, adding the metal ion solution obtained in step S1-2 to the organic phase solution obtained in step S1-1, mixing evenly to obtain a mixed solution; S1-4, using a shear mixer, an ultrasonic crusher, a high-pressure homogenizer or a liposome extruder to process the mixed solution obtained in step S1-3 to obtain a metallized lipid nanoparticle solution with a particle size distribution of 50-500 nm; S1-5, removing free metal ions and ethanol from the metallized lipid nanoparticle solution obtained in step S1-4 by ultrafiltration or dialysis to obtain a metallized liposome mother solution; S1-6, mixing the metallized liposome mother solution obtained in step S1-5 with a lyophilization protective agent, and lyophilizing to obtain PEGylated empty liposomes.
3. A method for rapid RNA loading according to claim 2, characterized in that: In step S1-1, the ionizable cationic lipid is selected from 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecanyloxy)hexyl)amino)octanoate), 1,2-dioleyl-3-dimethylamino-propane, 3-(di methylamino)propane-1,2-diyl dioleate, 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoic acid heptadecan-9-yl ester, ((3-hydroxypropyl)azadiyl)bis(nonane-9,1-diyl)bis(octanoic acid dibutyl ester), L319, Dlin-KC2-DMA, YSK12-C4, CL4H6, Arcturus10q, or ssPalmO-Phe molecules; The auxiliary phospholipid is selected from distearoylphosphatidylcholine, 1,2-dioleoyl-SN-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-distearic acid-3-phosphatidylethanolamine polyethylene glycol, dipalmitoyl phosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidic acid, 1-oleoyl-2-monocetyl-SN-glycero-phosphoethanolamine or 1-oleoyl-2-palmitoleoyl-SN-glycero-phosphoethanolamine; The molar percentages of the ionizable cationic lipid, auxiliary phospholipid and cholesterol are 40% to 60%: 8% to 12%: 35% to 40%. The ionizable cationic lipids in the organic phase solution contain PEGylated phospholipids, and the PEGylated phospholipids account for 0.3%-15% of the organic phase solution in terms of molar weight. The organic solvent in the organic phase solution is selected from ethanol, methanol, chloroform or tetrahydrofuran.
4. A method for rapid RNA loading according to claim 2, characterized in that: In step S1-2, the metal ions are selected from any one or more of Ca, Mg, Zn, Ba, Mn, Ni, Co, Fe, Cu, Al, Bi or Ti.
5. A method for rapid RNA loading according to claim 2, characterized in that: In step S1-3, the mixing method of the metal ion solution and the organic phase solution is selected from a microfluidic LNP preparation method, a solvent injection method, a thin film dispersion method or a high-pressure homogenization method; The concentration range of metal ions in the mixed solution is 0.1-30 mM; In step S1-6, the lyoprotectant is selected from sucrose or trehalose; The mass ratio of the metallized liposome mother solution to the freeze-drying protective agent is 8-10:
1.
6. A method for rapid RNA loading according to claim 1, characterized in that: In step S2, the acidic pH range is 2.0 to 7.0; In step S2, the target RNA is dissolved in an acidic buffer, and then PEGylated empty liposomes are added and dissolved to obtain a mixed solution.
7. A method for rapid RNA loading according to claim 6, characterized in that: The target RNA: acidic buffer: PEGylated empty liposomes = 1-5 μg: 25-500 μL: 25-500 μL.
8. A method for rapid RNA loading according to claim 1, characterized in that: In step S2, the target RNA is dissolved in an acidic buffer, the PEGylated empty liposomes are dissolved in an acidic buffer, the target RNA in the acidic environment and the PEGylated empty liposomes are mixed, suspended evenly, and a mixed solution is obtained after waiting for a period of time.
9. A method for rapid RNA loading according to claim 8, characterized in that: The concentration of PEGylated empty liposomes is 1-10 mg / mL. The acidic buffer is selected from molecules such as formic acid, acetic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, tartaric acid, isotartaric acid, malic acid, citric acid or isocitric acid, and the concentration range of the acidic buffer is 0.1mM to 10mM.
10. A method for rapid RNA loading according to claim 1, characterized in that: In step S3, the neutral pH range is 7-8; In step S3, an alkaline buffer is added to the mixed solution obtained in step S2 to adjust its pH to neutral, so that the target RNA can be quickly loaded into the liposome, which can be used for RNA transfection experiments in cells and living organisms.