Liposome nanoparticle in-vitro release medium and in-vitro release test method

Abstract

The invention discloses a liposome nanoparticle in-vitro release medium and an in-vitro release test method. The liposome nanoparticle in-vitro release medium comprises the following components: a buffer solution and lipid, wherein the lipid is one or more of DOPE and at least one of DOPS, POPS, DOPC, DSPC, Egg PC, Lyso PC, DPPC, DPPE, DMPE, POPE, DOPG and DGPG. The liposome nanoparticle in-vitro release medium simulates an in-vivo environment in vitro, and an in-vitro release curve is drawn by detecting the contents of nucleic acid, polypeptide and antibody, so that the liposome nanoparticle in-vitro release medium has important significance on prescription development and quality research of liposome nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid drug technology, specifically relating to an in vitro release medium for liposome nanoparticles and an in vitro release testing method. Background Technology

[0002] In recent years, lipid nanoparticles (LNPs) have been successfully used as a delivery platform for mRNA vaccines and therapies. Naked mRNA is a negatively charged hydrophilic macromolecule that is difficult to enter cells due to electrostatic repulsion of the cell membrane and is easily degraded and hydrolyzed by nucleases. Therefore, a protective shell is needed for cell entry. Encapsulating mRNA in LNPs can protect it from extracellular ribonucleases and facilitate intracellular mRNA delivery.

[0003] Given the increasing number of mRNA-LNP products being registered, drug regulatory agencies in the United States, the European Union, and China have all issued reference opinions on LNP development. These reference opinions all regard in vitro release behavior as a quality research indicator for lipid nanoparticles and require thorough research during the LNP development process, because the drug release, absorption, in vivo safety, efficacy, and in vitro and in vivo stability of mRNA-LNP can all be reflected in in vitro release behavior.

[0004] The key factors affecting in vitro release are the research methods, the release medium, and the detection methods. Existing technologies include:

[0005] (1) In vitro release study methods, mainly including:

[0006] ①Dialysis bag method

[0007] This is currently the most widely used and classic method. A certain amount of LNP sample is placed in a dialysis bag (with a molecular weight cutoff much larger than the drug but much smaller than the LNP), and then the dialysis bag is immersed in a large release medium. Once the drug is released from the LNP, it diffuses through the dialysis bag into the external medium, thus achieving a "sink condition." This method is simple, low-cost, easy to operate, and can maintain the sink condition well. However, sampling is relatively cumbersome, the dialysis membrane itself may adsorb the drug, and the stirring efficiency of the external medium can affect the diffusion rate.

[0008] ② Sample cell method (Franz diffusion cell)

[0009] The sample cell consists of a supply chamber (for the LNP sample) and a receiving chamber (filled with the release medium), separated by a semi-permeable membrane. Magnetic stirring ensures uniformity in the receiving chamber, and samples are taken periodically through the sampling port. The advantages of this method are that the leakage conditions are easier to maintain and control, sampling is convenient, it does not affect the system volume, and it has a high degree of automation (commercially available automated sampling systems exist). However, the equipment cost is higher than that of the dialysis bag method, and there may be problems such as membrane adsorption.

[0010] ③ Ultracentrifugation

[0011] By centrifuging liposome samples at high speeds (e.g., 10,000-15,000 rpm) at different time points, unreleased liposomes are precipitated, and the supernatant is then used to determine the amount of free drug. This method is direct and rapid, does not require a dialysis membrane, and avoids membrane adsorption problems. However, the centrifugation process may disrupt the stability of liposomes or alter the release equilibrium; the operation is cumbersome and not suitable for continuous monitoring; and it is not applicable to drugs with densities similar to liposomes.

[0012] (2) The in vitro release medium is usually determined according to the research objective:

[0013] PBS buffer (pH 7.4): It simulates the basic environment of blood circulation and is the most fundamental medium.

[0014] Serum / plasma media: This is crucial for evaluating the in vivo stability of liposomes. Components in serum, such as lipoproteins and proteins, can interact with liposomes, disrupting their structure and leading to rapid drug leakage (often referred to as serum-induced instability). This is essential for predicting the circulation time of liposomes in the bloodstream.

[0015] Acidic buffer solutions (e.g., pH 5.0 or 6.0): These mimic the tumor tissue microenvironment or the intracellular endosome / lysosomal environment. They are commonly used to study the triggered release behavior of pH-sensitive liposomes.

[0016] Enzyme-modifying media: For example, adding phospholipase to mimic biomembrane degradation, or adding esterase to study the release of precursor lipids containing hydrolyzable bonds.

[0017] (3) Common methods for detecting drugs released in vitro:

[0018] HPLC (High Performance Liquid Chromatography): The gold standard. It has high specificity and accuracy, and is suitable for the analysis of most drugs, especially for samples in complex media (such as those containing serum).

[0019] UV-Vis or fluorescence spectroscopy: If the drug has characteristic absorption or fluorescence, the method is rapid and simple. However, it is easily affected by the medium, especially in serum-containing media, where background absorption is very high.

[0020] Radiolabeling: It has extremely high sensitivity and can be used to track trace amounts of drug release, but it is complex to operate and has safety limitations.

[0021] ④ Other methods: such as mass spectrometry, etc.

[0022] In vitro release studies of nucleic acid-based drugs (LNPs) are a core evaluation step in their formulation development. Many existing methods suffer from limitations, such as the inability to release nucleic acid drugs or the degradation of these drugs during testing, failing to fully meet the objectives of in vitro release studies of nucleic acid-LNPs. Therefore, there is an urgent need to develop a new in vitro release study method. Crucially, the experiment must be conducted in a medium containing cell membrane mimicry; otherwise, the data obtained will not accurately reflect the fate of LNPs in vivo. The ultimate goal is to optimize LNP formulations, predict in vivo performance, and ensure batch-to-batch consistency through in vitro release behavior. Summary of the Invention

[0023] The purpose of this invention is to provide an in vitro release medium for liposome nanoparticles and an in vitro release test method, which is applicable to the in vitro release test of LNPs and has important significance and value for LNP formulation development and quality control.

[0024] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0025] An in vitro release medium for liposome nanoparticles, the in vitro release medium for liposome nanoparticles comprising the following components: buffer solution and lipids;

[0026] The lipid is DOPE and one or more of at least DOPS, POPS, DOPC, DSPC, Egg PC, Lyso PC, DPPC, DPPE, DMPE, POPE, DOPG, and DGPG.

[0027] In one or more embodiments of the present invention, the concentration of the lipid is 1.5 mg / mL to 4.5 mg / mL.

[0028] In one or more embodiments of the present invention, the lipids include DOPE, Lyso PC, and DOPS.

[0029] In one or more embodiments of the present invention, the molar ratio of DOPE, Lyso PC, and DOPS is (30~70):(10~40):(10~40).

[0030] In one or more embodiments of the present invention, the molar ratio of DOPE, Lyso PC, and DOPS is (40~60):(20~30):(20~30).

[0031] In one or more embodiments of the present invention, the pH of the in vitro release medium of the liposome nanoparticles is 4.0-8.0.

[0032] In one or more embodiments of the present invention, the buffer is at least one selected from PBS buffer, Tris buffer, HEPES buffer, phosphate buffer, borate buffer, acetate buffer, carbonate buffer, and citrate buffer.

[0033] Another specific embodiment of the present invention provides the following technical solution:

[0034] A method for preparing an in vitro release medium for liposome nanoparticles, the method comprising the following steps:

[0035] The lipids and organic solvents are mixed to obtain a mixed solution;

[0036] The mixed solution was evaporated to remove the organic solvent, resulting in a lipid film.

[0037] The lipid film and buffer solution were mixed, hydrated, and broken down to obtain an in vitro release medium for liposome nanoparticles.

[0038] In one or more embodiments of the present invention, the hydration operation is: ultrasonic hydration at 30℃-60℃ with a power of 150W-225W for 0.5h-1h; and / or,

[0039] The crushing operation is as follows: crush at 200W-500W for 10-30 minutes.

[0040] Another specific embodiment of the present invention provides the following technical solution:

[0041] A method for in vitro release assay of liposome nanoparticles includes the following steps:

[0042] The liposome nanoparticles and the above-mentioned in vitro release medium for the liposome nanoparticles were mixed to obtain a mixed system;

[0043] The content of liposome nanoparticles released in the mixed system was detected at different time points, and in vitro release curves were plotted.

[0044] In one or more embodiments of the present invention, the mixture is placed under shaking conditions at 36°C-38°C and 50rpm-150rpm, and then the content of liposome nanoparticle release in the mixture is detected at different time points.

[0045] In one or more embodiments of the present invention, the liposome nanoparticles are liposome nanoparticles comprising at least one of nucleic acids, peptides, and antibodies; and / or,

[0046] The method for detecting the content of liposome nanoparticle release in the mixed system is at least one of RiboGreen, UV, HPLC, and UPLC.

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] 1. This invention preferably uses three lipids: DOPE, Lyso PC, and DOPS. The release medium is prepared by combining membrane hydration method with ultrasonic disruption method. Under neutral environment, the lipid bilayer composed of DOPE, Lyso PC, and DOPS simulates the bimolecular structure of cell membrane; under acidic environment, DOPS carries a negative charge to simulate endosomal structures, and Lyso PC and DOPE can promote the release of mRNA from LNP.

[0049] 2. This invention uses phosphate buffer as the release medium and adjusts the pH to simulate the in vivo pH environment, wherein pH 4.0-pH 6.0 simulates the endosomal environment and pH 6.8-pH 8.0 simulates the pH of muscle tissue fluid.

[0050] 3. This invention uses a constant-temperature shaker to study the in vitro drug release behavior of LNP. The mRNA-LNP is directly added to the in vitro release medium and shaken, eliminating the need for dialysis bags and flow cells. The equipment is readily available and easy to operate.

[0051] 4. The liposome nanoparticle in vitro release medium of the present invention is used for nucleic acid-LNP formulation research, which effectively avoids the degradation of total nucleic acid drug during the experiment and detects a release curve with discriminative power.

[0052] 5. This invention uses methods such as RiboGreen to detect the content of free mRNA in LNP at different time points, which is more convenient, faster, and has high sensitivity and specificity.

[0053] 6. This invention utilizes a self-made liposome nanoparticle in vitro release medium to successfully generate a release curve with discriminative power, achieving a cumulative release rate of over 80%, and verifying the pH sensitivity of the liposome nanoparticles, thus overcoming the shortcomings of existing in vitro release studies. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 This is a particle size distribution diagram of medium A (pH 5.5) in Example 1 of the present invention;

[0056] Figure 2 This is a particle size distribution diagram of medium B (pH 7.4) in Example 1 of the present invention;

[0057] Figure 3 This is a potential distribution diagram of medium A (pH 5.5) in Example 1 of the present invention;

[0058] Figure 4 This is a potential distribution diagram of medium B (pH 7.4) in Example 1 of the present invention;

[0059] Figure 5 This is a graph showing the effect of different amounts of in vitro release media added to liposome nanoparticles on the cumulative release amount in this invention;

[0060] Figure 6 This is the release curve of the mRNA-LNP sample in an in vitro simulated environment in Example 2 of the present invention;

[0061] Figure 7 This is a standard curve of mRNA concentration versus RiboGreen absorbance values ​​in one embodiment of the present invention;

[0062] Figure 8 This refers to the change in total mRNA content in an in vitro simulated environment in one embodiment of the present invention;

[0063] Figure 9 This is a flowchart of an in vitro release test method for liposome nanoparticles in one embodiment of the present invention.

[0064] illustrate: Figure 6 In this context, pH 5.5 refers to the release curve of the mRNA-LNP sample in pH 5.5 buffer A, pH 5.5+ simulated solution refers to the release curve of the mRNA-LNP sample in pH 5.5 medium A, pH 7.4 refers to the release curve of the mRNA-LNP sample in pH 7.4 buffer B, and pH 7.4+ simulated solution refers to the release curve of the mRNA-LNP sample in pH 7.4 medium B.

[0065] Figure 8In this context, pH 5.5 refers to the change in total mRNA content in pH 5.5 buffer A, pH 5.5+ simulated solution refers to the change in total mRNA content in pH 5.5 medium A, pH 7.4 refers to the change in total mRNA content in pH 7.4 buffer B, and pH 7.4+ simulated solution refers to the change in total mRNA content in pH 7.4 medium B. Detailed Implementation

[0066] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0067] A specific embodiment of the present invention provides an in vitro release medium for liposome nanoparticles, comprising the following components: buffer solution and lipids; wherein the lipids are DOPE and one or more of at least DOPS, POPS, DOPC, DSPC, Egg PC, Lyso PC, DPPC, DPPE, DMPE, POPE, DOPG, and DGPG.

[0068] Specifically, an in vitro release medium was prepared by selecting DOPE (dioleoylphosphatidylethanolamine), DOPS (dioleoylphosphatidylserine), POPS (palmitoyloleoylphosphatidylserine), DOPC (dioleoylphosphatidylcholine), DSPC (distearylphosphatidylcholine), EggPC (egg lecithin), LysoPC (lysophosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DMPE (dimyristoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), DOPG (dioleoylphosphatidylglycerol), and DGPG (1,2-di-O-octadecenyl-sn-glycerol-3-phosphate-racemic-(1-glycerol)) and a buffer solution. This in vitro release medium can simulate the normal internal environment of the body, such as cell membranes, endosomes, and body fluids, and can more accurately test the release behavior of liposome nanoparticles.

[0069] Furthermore, the lipid concentration ranges from 1.5 mg / mL to 4.5 mg / mL, specifically 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, and 4.5 mg / mL. If the lipid concentration is too low, the release rate of the liposome nanoparticles will be too slow, leading to longer testing times or even preventing release altogether; if the concentration is too high, the release rate of the liposome nanoparticles will be too fast, which is not conducive to sampling, detection, and plotting of release curves.

[0070] Furthermore, the lipids preferably include DOPE, Lyso PC, and DOPS, with a molar ratio of (30~70):(10~40):(10~40), preferably (40~60):(20~30):(20~30).

[0071] Phosphatidylserine (PS) is an endogenous phospholipid found in all polyanionic macromolecules. DOPS is an excellent alternative to natural Brain PS, with similar physical properties and greater stability against oxidation. LysoPC, a product of the hydrolysis of a fatty acid chain of phosphatidylcholine (PC), can be used as a model molecule to study the physical and biological properties of cell membranes and participate in the structural and functional regulation of cell membranes. Dilinoleoylphosphatidylethanolamine (DOPE) is a commonly used, neutral accessory lipid. Neutral lipids are often used as structural lipids in LNP formulations because they can spontaneously organize into a lipid bilayer, and their higher phase transition temperatures enhance the membrane stability of LNPs. Conversely, LNPs can disrupt endosome stability during endosome escape, thereby improving nucleic acid delivery efficiency.

[0072] When used in combination with LysoPC and DOPE in a lipid mixture, DOPS can better mimic cell membrane states in vitro for in vitro release assays. Under neutral conditions, the lipid bilayer composed of DOPS, LysoPC, and DOPE simulates the bimolecular structure of the cell membrane; under acidic conditions, DOPS, carrying a negative charge, simulates endosomes, while LysoPC and DOPE can promote the release of mRNA from LNPs.

[0073] Furthermore, the pH of the in vitro release medium for liposome nanoparticles is 4.0-8.0, with a pH range of 4.0-6.0, to simulate the endosome environment; and the pH range is 6.8-8.0, to simulate the pH of muscle tissue fluid.

[0074] Furthermore, the buffer is at least one of PBS buffer, Tris buffer, HEPES buffer, phosphate buffer, borate buffer, acetate buffer, carbonate buffer, and citrate buffer, preferably potassium phosphate buffer, with a molar concentration of 0.1M-1M, preferably 0.2M-0.6M.

[0075] Another specific embodiment of the present invention provides a method for preparing an in vitro release medium for liposome nanoparticles, comprising the following steps:

[0076] Step 1: Prepare the buffer solution.

[0077] Specifically, first prepare a buffer solution with a molar concentration of 0.1M-1M, then adjust the pH of the buffer solution to 4.0-8.0 using NaOH with a molar concentration of 1M-20M, then filter it through a 0.22μm filter membrane to obtain the buffer solution, and store it in a refrigerator at 2℃-8℃ for later use.

[0078] Step 2: Prepare an in vitro release medium for liposome nanoparticles using a thin-film hydration method.

[0079] Specifically, lipids are dissolved using an organic solvent such as chloroform, and then the organic solvent is removed by rotary evaporation to form a lipid film. The lipid film is mixed with the buffer solution prepared in step 1 and ultrasonically hydrated at 30°C-60°C on a rotary evaporator (power 150W-225W, time 0.5h-1h), then transferred to centrifuge tubes; subsequently, it is ultrasonically disrupted on ice for 10 min-30 min using an ultrasonic disruptor at 200 W-500 W to obtain a clear solution, which is the in vitro release medium for liposome nanoparticles, and stored at 2°C-8°C for later use.

[0080] Another specific embodiment of the present invention provides a method for in vitro release testing of liposome nanoparticles, such as... Figure 9 As shown, it includes the following steps:

[0081] Step S1: Mix the liposome nanoparticles and the in vitro release medium of the liposome nanoparticles to obtain a mixed system.

[0082] Specifically, for experimental convenience, a high-concentration in vitro release medium for liposome nanoparticles was first prepared for use. Liposome nanoparticles were taken and diluted with a certain amount of buffer solution, then incubated at 50-150 rpm and 37±1℃ for 10-20 minutes. Then, the high-concentration in vitro release medium for liposome nanoparticles was added to a certain volume to dilute the medium, achieving a lipid concentration of 1.5 mg / mL-4.5 mg / mL. The mixture was then shaken at 50-150 rpm and 37±1℃. The equipment used for incubation and shaking could be a constant-temperature shaker, a Wave reactor, a constant-temperature water bath, or a magnetic stirrer.

[0083] Liposome nanoparticles are liposome nanoparticles containing at least one of nucleic acid, peptide, and antibody, wherein the nucleic acid can be DNA, siRNA, mRNA, circRNA, dsRNA, saRNA, ASO, or miRNA.

[0084] Step S2: Detect the content of liposome nanoparticle release in the mixed system at different time points and plot the in vitro release curve.

[0085] Specifically, samples were taken at 0 min, 15 min, 30 min, 60 min, 240 min, and 420 min to detect the mRNA free rate in the extracted sample solution, and an in vitro release curve was plotted accordingly. The specific detection method was at least one of RiboGreen (RiboGreen fluorescent dye method), UV (ultraviolet spectrophotometry), HPLC (high performance liquid chromatography), and UPLC (ultra-high performance liquid chromatography). These methods are highly sensitive and specific, suitable for mRNA detection, and can ensure the accuracy of the detection.

[0086] The present invention will be further described in detail below with reference to specific embodiments.

[0087] Unless otherwise specified, all raw materials used in this invention are commercially available. The following raw materials are sourced from: DOPE, AVANTI; Lyso PC, AVANTI, DOPS, AVANTI.

[0088] The mRNA-LNP samples were prepared in-house, and the preparation process is as follows:

[0089] ① Preparation of the lipid organic phase: Dlin-DMA-MC3 (4-(N,N-dimethylamino)butyric acid), DSPC (1,2-distearyl-sn-glycerol-3-phosphocholine), PEG-DMG (polyethylene glycol-distearylglycerol), and cholesterol (CHOL) were dissolved in ethanol to prepare a lipid solution. The lipid concentration was 12 mg / mL. Dlin-DMA-MC3 accounted for 50 mol% of the total lipids in the nucleic acid lipid nanoparticles, DSPC accounted for 10 mol%, PEG-DMG accounted for 1.5 mol%, and cholesterol accounted for 38.5 mol%.

[0090] ②Preparation of mRNA aqueous solution: Dissolve the mRNA to be encapsulated in 50mM citrate buffer (pH 4.0) to form a 0.2mg / mL nucleic acid solution with a nitrogen-to-phosphorus ratio of 6:1.

[0091] ③ Rapid mixing: The organic phase and aqueous phase are mixed instantaneously under high pressure using a microfluidic device at a volume ratio of 1:3, with a total flow rate of 24-80 mL / min. The ionizable lipids are positively charged in an acidic environment and bind to the negatively charged mRNA. Simultaneously, all lipids self-assemble into a spherical nanoparticle (LNP) containing the mRNA.

[0092] ④ Purification and Replacement Buffer: Use tangential flow filtration to remove ethanol, free mRNA and lipids from the reaction, and replace the solution with the final storage buffer Tris to obtain a pure mRNA-LNP product.

[0093] ⑤ Characterization: The prepared LNPs were characterized by determining their particle size, encapsulation efficiency, and polydispersity index (PDI). Particle size potential was measured using a particle size analyzer, and encapsulation efficiency was determined using the Quant-iT™ RiboGreen RNA Quantification Kit.

[0094] Example 1

[0095] The in vitro release media for liposome nanoparticles in the embodiments of this invention were all prepared using the following methods:

[0096] (1) Prepare mRNA-LNP release buffer

[0097] Phosphate buffer was used as the release medium to simulate the pH environment in vivo. A 0.4 M KH2PO4 buffer was prepared and the pH was adjusted to 5.5 (simulating the pH of the endosome environment, denoted as buffer A) and 7.4 (simulating the pH of muscle tissue fluid, denoted as buffer B) using 10 M NaOH. The buffers were then filtered through a 0.22 μm filter membrane to obtain two buffers with different pH values, which were stored at 2°C for later use.

[0098] (2) Preparation of in vitro release media for liposome nanoparticles by thin-film hydration method

[0099] ①Accurately weigh approximately 450 mg of DOPE, LysoPC, and DOPS according to the molar ratio of DOPE : LysoPC : DOPS = 50 : 25 : 25, add 10 mL of chloroform to dissolve them; remove the organic solvent by rotary evaporation to form a transparent film.

[0100] ② Hydration: Mix the transparent film from ① with approximately 30 mL of buffer A, and sonicate it at 50°C on a rotary evaporator (power 200 W, hydration time 0.5 h), then transfer it to a centrifuge tube; subsequently, use an ultrasonic homogenizer at 300 W on ice for 20 min to obtain the in vitro release medium for liposome nanoparticles, denoted as medium A; similarly, mix the transparent film from ① with approximately 30 mL of buffer B, and sonicate it at 50°C on a rotary evaporator (power 200 W, hydration time 0.5 h), then transfer it to a centrifuge tube; subsequently, use an ultrasonic homogenizer at 300 W on ice for 20 min to obtain the in vitro release medium for liposome nanoparticles, denoted as medium B.

[0101] ③ Both medium A and medium B obtained were clear solutions with a particle size of < 120 nm and PDI < 0.3. They were stored at 2℃ for later use.

[0102] In this embodiment, the lipid concentration in both medium A and medium B is 15 mg / mL. When medium A and medium B are used in the in vitro release test of liposome nanoparticles, they need to be diluted to a concentration of 1.5 mg / mL-4.5 mg / mL.

[0103] Table 1. Particle size distribution and potential data of media A and media B

[0104]

[0105] Example 2

[0106] The in vitro release tests of liposome nanoparticles in the embodiments of this invention were all performed using the following methods:

[0107] (1) Add the mRNA-LNP sample to the in vitro release medium of liposome nanoparticles.

[0108] ① Take 2.25 mL of LNP sample containing 0.5 mg / mL mRNA, dilute with buffer A at pH 5.5, and incubate at 37℃ for 15 min using a constant temperature shaker at 100 rpm. This sample is designated as sample A. ② Take another 2.25 mL of LNP sample, dilute with buffer B at pH 7.4, and incubate at 37℃ for 15 min using a constant temperature shaker at 100 rpm. This sample is designated as sample B.

[0109] ② Add 3 mL of pH 5.5 medium A to sample A to make a final volume of 10 mL; add 3 mL of pH 7.4 medium B to sample B to make a final volume of 10 mL; shake at 37℃ and 100 rpm, and take samples at 0 min, 15 min, 30 min, 60 min, 240 min and 420 min respectively to detect the mRNA free rate of the extracted sample solution. The results are shown in Table 3.

[0110] (2) Comparison of mRNA-LNP in vitro release data in buffer solution without additional liposome nanoparticle in vitro release medium:

[0111] Take 2.25 mL of LNP sample containing 0.5 mg / mL mRNA, and dilute it to the same volume as in (1) using buffer A (pH 5.5) and buffer B (pH 7.4), respectively. Shake at 37℃ and 100 rpm. According to the sampling time points of the in vitro release medium of liposome nanoparticles added in ②, samples were taken at 0 min, 60 min, 240 min, and 420 min, respectively, and the mRNA free rate of the extracted sample solution was detected. The results are shown in Table 2.

[0112] In the above testing methods, .

[0113] LNP was added to different pH buffers and the mixture was incubated at 37°C and 100 rpm to simulate the in vivo environment. The mRNA release rate was measured at different time points, and release curves were plotted. (Details are as follows...) Figure 6 As shown.

[0114] Table 2. Release rate (%) of mRNA in buffer A at pH 5.5 and buffer B at pH 7.4

[0115]

[0116] Table 3. Release rate (%) of mRNA in medium A at pH 5.5 and medium B at pH 7.4

[0117]

[0118] The test results showed that, in an environment without the addition of liposome nanoparticles as an in vitro release medium, the mRNA-LNP exhibited good stability, with the free rate consistently remaining below 10%, and no significant nucleic acid leakage or release behavior. In a neutral environment with the addition of liposome nanoparticles as an in vitro release medium, a slight release of mRNA occurred, but it was not significant, with the free rate increasing only to 15.2% at 7 h; while in an acidic environment, the free rate of mRNA reached over 80% at 7 h. This indicates that the LNP sample has good pH sensitivity, and the mRNA-LNP sample remains relatively intact before reaching the acidic endosome environment, making nucleic acid leakage unlikely.

[0119] The above test results demonstrate that the liposome nanoparticle in vitro release medium prepared by this invention can well simulate the cell membrane in the body's internal environment, and by adjusting the pH value, it can simulate the environment of different parts of the body, successfully producing a discriminative in vitro release curve.

[0120] Further determination of total mRNA content to assess whether degradation occurred during the testing process, as detailed below:

[0121] First, a standard curve was plotted using the mRNA stock solution standard, as shown in Table 4. Figure 7 As shown:

[0122] Table 4 Standard Curve of mRNA Concentration and RiboGreen Absorbance Value

[0123]

[0124] Table 5. Total mRNA content (μg) in pH 5.5 buffer A and pH 7.4 buffer B

[0125]

[0126] Table 6. Total mRNA content (μg) in pH 5.5 medium A and pH 7.4 medium B

[0127]

[0128] From Table 5, Table 6 and Figure 8 The test results show that the total mRNA content did not change significantly in the pH 5.5 / pH 7.4 buffer solution with or without liposome nanoparticles as the in vitro release medium, indicating no significant degradation. This proves that the mRNA had not been degraded under the experimental conditions of in vitro release, thus eliminating factors that could affect degradation.

[0129] Screening was conducted based on Examples 1 and 2:

[0130] (1) Screening lipid types

[0131] Based on Example 1, with a fixed total lipid content of 20 mM, liposome nanoparticles with pH 5.5 were prepared using different lipid ratios (molar ratios), as shown in Table 7 below.

[0132] Table 7. Combinations of different types of lipids

[0133]

[0134] The physicochemical properties of the in vitro release media of liposome nanoparticles with different lipid ratios were detected, including particle size and PDI. The in vitro release test of liposome nanoparticles was performed using the method in Example 2 to verify the effect of the in vitro release media of liposome nanoparticles with different lipid ratios on the cumulative release of RNA-LNP, as shown in Table 8.

[0135] Table 8 Physicochemical Indicators and Cumulative Release Results

[0136]

[0137] As shown in Tables 7 and 8, the release media prepared by the combined thin-film hydration and ultrasonic disruption methods for different lipid types exhibited similar particle size and PDI distribution, demonstrating uniformity and stability. Cumulative release results indicated that DOPS is essential for mRNA release, and the combination of the three lipids (DOPE, Lyso PC, and DOPS) was more advantageous in generating discriminative release profiles.

[0138] (2) Screening of lipid molar ratios

[0139] The release medium was further prepared using three lipids (DOPE, Lyso PC, and DOPS). DOPE, as an important component of the cell membrane, had the highest proportion and was more conducive to simulating the cell membrane state. Based on Example 1, the total lipid concentration was kept constant (20 mM), and an in vitro release medium of liposome nanoparticles with pH 5.5 was prepared. The in vitro release test was carried out based on the method of Example 2, and the molar ratio was optimized.

[0140] Table 9. Effect of different molar ratios of release media on cumulative release amount

[0141]

[0142] The results are shown in Table 9. When the molar ratio of DOPE : Lyso PC : DOPS = (30~70): (10~40): (10~40), the release curves generated based on the cumulative release amounts at 1 h and 7 h showed good differentiation. Among them, the release curve generated when the molar ratio of DOPE : Lyso PC : DOPS = (40~60): (20~30): (20~30) was more in line with expectations. Therefore, this experiment adopted a molar ratio of DOPE : Lyso PC : DOPS = 50 : 25 : 25 for the in vitro release study of mRNA-LNP.

[0143] (3) Screening the amount of liposome nanoparticle in vitro release medium added

[0144] Based on Example 1, a liposome nanoparticle in vitro release medium with a fixed concentration of 20 mM was prepared at pH 5.5. Following the method in Example 2, different amounts of the liposome nanoparticle in vitro release medium were added to a 10 mL total system. The optimal amount was selected by detecting the cumulative release over different time periods, as detailed in Table 10. Figure 5 As shown.

[0145] Table 10 Effect of different amounts of in vitro release media added to liposome nanoparticles on cumulative release amount

[0146]

[0147] As shown in Table 10, good cumulative release can be achieved when the amount of liposome nanoparticle in vitro release medium added is greater than 10%. Among them, the cumulative release can reach more than 80% after 7 h when 30% is added. Finally, 3 mL / 10 mL of liposome nanoparticle in vitro release medium was selected for testing.

[0148] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0149] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An in vitro release medium for liposome nanoparticles, characterized in that, The in vitro release medium for the liposome nanoparticles comprises the following components: buffer solution and lipids; The lipid is DOPE and one or more of at least DOPS, POPS, DOPC, DSPC, Egg PC, Lyso PC, DPPC, DPPE, DMPE, POPE, DOPG, and DGPG.

2. The in vitro release medium for liposome nanoparticles according to claim 1, characterized in that, The concentration of the lipid is 1.5 mg / mL to 4.5 mg / mL.

3. The in vitro release medium for liposome nanoparticles according to claim 1, characterized in that, The lipids include DOPE, Lyso PC, and DOPS.

4. The in vitro release medium for liposome nanoparticles according to claim 3, characterized in that, The molar ratio of DOPE, LysoPC, and DOPS is (30~70):(10~40):(10~40).

5. The in vitro release medium for liposome nanoparticles according to claim 4, characterized in that, The molar ratio of DOPE, LysoPC, and DOPS is (40~60):(20~30):(20~30).

6. The in vitro release medium for liposome nanoparticles according to claim 1, characterized in that, The pH of the in vitro release medium for the liposome nanoparticles is 4.0-8.

0.

7. The in vitro release medium for liposome nanoparticles according to claim 1, characterized in that, The buffer solution is at least one of PBS buffer, Tris buffer, HEPES buffer, phosphate buffer, borate buffer, acetate buffer, carbonate buffer, and citrate buffer.

8. A method for preparing an in vitro release medium for liposome nanoparticles according to any one of claims 1-7, characterized in that, The preparation method of the in vitro release medium of the liposome nanoparticles includes the following steps: The lipids and organic solvents are mixed to obtain a mixed solution; The mixed solution was evaporated to remove the organic solvent, resulting in a lipid film. The lipid film and buffer solution were mixed, hydrated, and broken down to obtain an in vitro release medium for liposome nanoparticles.

9. The method for preparing the in vitro release medium of liposome nanoparticles according to claim 8, characterized in that, The hydration operation is as follows: ultrasonic hydration at 30℃-60℃ with a power of 150W-225W for 0.5h-1h; and / or, The crushing operation is as follows: crush at 200W-500W for 10-30 minutes.

10. A method for in vitro release assay of liposome nanoparticles, characterized in that, Includes the following steps: The liposome nanoparticles and the in vitro release medium of the liposome nanoparticles according to any one of claims 1-7 are mixed to obtain a mixed system; The content of liposome nanoparticles released in the mixed system was detected at different time points, and in vitro release curves were plotted.

11. The in vitro release assay method for liposome nanoparticles according to claim 10, characterized in that, The mixture was placed under shaking conditions at 36℃-38℃ and 50rpm-150rpm, and the content of liposome nanoparticle release in the mixture was then detected at different time points.

12. The in vitro release assay method for liposome nanoparticles according to claim 10, characterized in that, The liposome nanoparticles are liposome nanoparticles containing at least one of nucleic acid, polypeptide, and antibody; and / or, The method for detecting the content of liposome nanoparticle release in the mixed system is at least one of RiboGreen, UV, HPLC, and UPLC.

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