Method for efficiently preparing nucleic acid lipid nanoparticle

Abstract

The present disclosure relates to a method for preparing a lipid nanoparticle. In this method, a mechanical pump with multiple pump heads, such as a peristaltic pump or a plunger pump, is used, and a lipid-containing organic phase and a nucleic acid-containing aqueous phase for forming the lipid nanoparticle are mixed. The method further relates to the preparation of the lipid nanoparticle using a mechanical apparatus in an A configuration or a B configuration.

Description

A method for efficient preparation of nucleic acid lipid nanoparticles

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202510106861.6, filed on January 23, 2025. The entire contents of the earlier application are considered to be the disclosure of this application and are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of medicine, specifically relating to a method for preparing lipid nanoparticles. Background Technology

[0004] In nucleic acid drugs, lipid nanoparticle delivery systems play a crucial role in delivering drug molecules to specific targets. However, current methods for preparing lipid nanoparticles suffer from problems such as low flow rates, easy channel blockage, and uneven mixing. Therefore, developing a method for preparing stable lipid nanoparticles is of paramount importance. Summary of the Invention

[0005] This invention provides a method for preparing lipid nanoparticles and the mechanical equipment used in this method. The lipid nanoparticles prepared by this method exhibit better stability and overcome problems such as low flow rate, easy channel blockage, and uneven mixing found in traditional methods.

[0006] In a first aspect of this disclosure, a method for preparing lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids is disclosed. This method employs a mechanical pump with multiple pump heads, such as a peristaltic pump or a plunger pump, to mix the lipid-containing organic phase and the nucleic acid-containing aqueous phase. In one embodiment, the flow rate of any one pump head is independently 0-500 mL / min. In one embodiment, the flow rate ratio of the organic phase to the aqueous phase is 1:3. In one embodiment, the temperature for preparing the lipid nanoparticles is 4-65°C, 4-50°C, preferably 15-25°C. In one embodiment, the mass ratio of nucleic acids to lipids is 1:2-1:40, preferably 1:3-1:29, preferably 1:3-1:27, preferably 1:4-1:20, preferably 1:5-1:18, more preferably 1:6-1:15, for example 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15.

[0007] A second aspect of this disclosure discloses an apparatus for preparing lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids, comprising a mechanical pump with multiple pump heads and multiple tee fittings. In one embodiment, the multiple pump heads are linked via multiple tee fittings in an A-configuration or a B-configuration. In one embodiment, the organic phase and the aqueous phase are mixed through the apparatus at a flow rate ratio of 1:3.

[0008] A third aspect of this disclosure discloses lipid nanoparticles prepared using the method of the first aspect of this disclosure or the apparatus of the second aspect of this disclosure.

[0009] A fourth aspect of this disclosure discloses the use of lipid nanoparticles from the third aspect of this disclosure for drug delivery.

[0010] A fifth aspect of this disclosure discloses a method for delivering a drug, which includes using lipid nanoparticles of a third aspect of this disclosure. Attached Figure Description

[0011] Figure 1 shows a schematic diagram of peristaltic pump configuration A.

[0012] Figure 2 shows a schematic diagram of the peristaltic pump B configuration.

[0013] Figure 3A shows the liquid chromatogram of HC016 at 25°C in Example 7.

[0014] Figure 3B shows the liquid chromatogram of HC016 encapsulated in the lipid complex at 50°C in Example 7.

[0015] Figure 3C shows the liquid chromatogram of HC016 encapsulated in the lipid combination at 65°C in Example 7. Detailed Implementation

[0016] The following specific embodiments illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification.

[0017] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0018] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0019] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0020] In this document, unless otherwise stated, the singular and plural forms of terms may be used interchangeably.

[0021] In this article, the term "mechanical pump" refers to a mechanical device that moves different fluids (gas or liquid) from one location to another by applying mechanical force. The most common type of pump converts electrical energy into hydraulic energy to transport fluids.

[0022] In this article, the term "peristaltic pump" refers to a mechanical pump that uses the periodic compression of a flexible tube wall to transport fluids. It features one or more rotating rollers that compress a flexible tube to form a sealed chamber. As the rollers rotate, the sealed chamber moves along the tube, thus transporting the fluid. A seal is formed between the rollers and the tube wall, ensuring that the transported fluid does not come into contact with the pump's mechanical components. This makes peristaltic pumps suitable for applications requiring the avoidance of contamination or the maintenance of fluid purity. The flow rate and pressure of the fluid can be precisely controlled by adjusting the roller rotation speed. By changing the direction of roller rotation, the peristaltic pump can achieve forward or reverse flow of the fluid.

[0023] In this article, the term "plunger pump" refers to an important working component in a hydraulic system, belonging to both positive displacement and reciprocating pumps. It achieves oil suction and pressure by the reciprocating motion of a plunger within a cylinder, causing changes in the volume of the sealed working chamber. Plunger pumps offer advantages such as high rated pressure, compact structure, high efficiency, and convenient flow rate adjustment.

[0024] In this article, the term "lipid" refers to the collective term for esters and their derivatives formed by the reaction of fatty acids and alcohols, including oils (triglycerides) and lipids (phospholipids, sterols), and is also a general term for oils, fats, and lipids.

[0025] In this article, "A configuration" represents dendritic convergence. An exemplary A configuration is shown in Figure 1, where pump head 1 and pump head 2 are connected via pump tubing by tee 1, pump head 3 and pump head 4 are connected via pump tubing by tee 2, and tee 1 and tee 2 are connected via pump tubing by tee 3, ultimately converging into a single pump tubing. Similarly, all similar configurations belong to the A configuration. The A configuration can be connected to one or more mechanical pumps, such as peristaltic pumps or plunger pumps.

[0026] In this article, "B configuration" represents a river-like confluence. An exemplary B configuration is shown in Figure 2, where pump head 1 and pump head 2 are connected via pump pipe by tee No. 1, pump head 3 is connected to tee No. 1 via pump pipe by tee No. 2, and pump head 4 is connected to tee No. 2 via pump pipe by tee No. 3, ultimately converging into a single pump pipe. Similarly, similar configurations all belong to the B configuration. The B configuration can be connected to one or more mechanical pumps, such as peristaltic pumps or plunger pumps.

[0027] In this article, the terms "te-fitting," "te," "te connector," and "te-pipe" refer to a pipe fitting with three ports that can connect three pipes. It is used to control the flow direction of fluids and to achieve mixing, splitting, or switching of fluid flow between different pipes. The main characteristic of a T-type tee fitting is its "T"-shaped structure, with one end being the main pipe and the other two ends being side pipes perpendicular to the main pipe. A Y-type tee fitting has two parallel inlet ports and a common outlet port, resembling the letter "Y." The two parallel side pipes converge at a point and then merge into a single main pipe.

[0028] Preparation method of lipid nanoparticles

[0029] This disclosure provides a method for preparing lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids. The method employs a mechanical pump with multiple pump heads, such as a peristaltic pump or a plunger pump, to mix the lipid-containing organic phase and the nucleic acid-containing aqueous phase. In some embodiments, the flow rate of any one pump head is independently 0-500 mL / min. In some embodiments, the flow rate ratio of the organic phase to the aqueous phase is 1:3. In some embodiments, the temperature for preparing the lipid nanoparticles is 4-65°C, 4-50°C, preferably 15-25°C. In some embodiments, the mass ratio of nucleic acid to lipids in this disclosure is 1:2-1:40, preferably 1:3-1:29, preferably 1:3-1:27, preferably 1:4-1:20, preferably 1:5-1:18, more preferably 1:6-1:15, for example 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15.

[0030] The lipid nanoparticles prepared by the method provided in this disclosure have better stability and overcome the problems of low flow rate, easy channel blockage, and uneven mixing in traditional methods.

[0031] In some embodiments, the flow rate of any pump head in this disclosure is independently 0-500 mL / min, preferably 150-500 mL / min, more preferably 300-400 mL / min, for example 300 mL / min, 350 mL / min, 400 mL / min.

[0032] In some embodiments, multiple pump heads are connected in an A configuration or a B configuration via multiple tee fittings; preferably, there are 4 pump heads and 3 tee fittings.

[0033] In some embodiments, the multiple pump heads of this disclosure are connected by multiple tee fittings, and any one of the tee fittings is independently selected from T-type or Y-type. In some preferred embodiments, any one of the tee fittings of this disclosure is independently selected from one of the following: T-032, T-024, PEEK internal thread T-type, stainless steel T-type, Y-024, Y-type PEEK, Y-032.

[0034] In some embodiments, the inner diameter of any tee fitting disclosed herein is independently 0.5-3 mm, preferably 0.8-2.36 mm, for example 0.8, 1, 1.25, 1.58, 2, 2.36 mm.

[0035] In some embodiments, the nucleic acid disclosed herein is selected from one or more of mRNA, DNA, siRNA, sgRNA, shRNA, microRNA, and antisense nucleic acid. In some preferred embodiments, the nucleic acid disclosed herein is HC016 or RX-0201.

[0036] In this article, the term "siRNA," short for Small Interfering RNA, refers to a class of double-stranded RNA molecules, 20-25 base pairs in length. In the RNA interference (RNAi) pathway, siRNA interferes with gene expression by hybridizing with complementary mRNA molecules. This interference triggers mRNA degradation, thereby inhibiting the expression of specific genes.

[0037] In this article, the term "sgRNA," short for "small guide RNA," refers to a small non-coding RNA that guides the insertion or deletion of uridine residues into the kinetoplastid during RNA editing. It can pair with pre-mRNA and guide Cas9 to perform site-specific DNA editing.

[0038] In this article, the term "shRNA," short for short hairpin ribonucleic acid, refers to two short inverted repeat sequences. The shRNA cloned into the shRNA expression vector consists of two short inverted repeat sequences separated by a stem-loop sequence, forming a hairpin structure controlled by the polIII promoter. Subsequently, 5-6 T molecules are added as a transcription terminator for RNA polymerase III.

[0039] In this article, the term "microRNA" refers to a class of non-coding single-stranded RNA molecules, approximately 22 nucleotides in length, encoded by endogenous genes. These molecules participate in post-transcriptional gene expression regulation in plants and animals. miRNAs can regulate target mRNAs by disrupting their stability and inhibiting their translation.

[0040] In this article, the term "antisense nucleic acid" refers to an RNA or DNA molecule that can precisely complement a specific mRNA and specifically block its translation. Antisense nucleic acids are used to specifically block the expression of certain genes, resulting in low or no expression. This involves three main technologies: antisense RNA, antisense DNA, and ribozymes.

[0041] In this article, "HC016" has the following chemical structural formula:

[0042] Molecular formula: C 254 H 295 N 97 Na 25 O 129 P 25 S 25 (Sodium salt), C 254 H 320 N 97 O 129 P 25 S 2s (Free radicals).

[0043] In this article, "RX-0201" has the following chemical formula.

[0044] Abbreviations: dA = adenine deoxyribonucleoside; dC = cytosine deoxyribonucleoside; dG = guanine deoxyribonucleoside; dT = thymine deoxyribonucleoside.

[0045] Molecular formula: C 194 H 247 N 70 Na 19 O 103 P 19 S 19 (Sodium salt), C 194 H 247 N70O 103 P 19 S 19 (Free radicals).

[0046] In some embodiments, the lipids provided in this disclosure comprise one or more of ionizable lipids, cationic lipids, neutral lipids, auxiliary lipids, and PEGylated lipids.

[0047] In this article, the term "ionizable lipids" refers to a special class of lipids that can carry an electric charge at a specific pH value. This property makes ionized lipids very important in the field of drug delivery, especially in the delivery of nucleic acid drugs (such as small interfering RNA, mRNA, etc.). Appropriate ionized lipids can help improve the delivery efficiency and therapeutic effect of nucleic acid drugs.

[0048] Ionizable lipids include, but are not limited to, tertiary amines and their derivatives, pyrrolidines and their derivatives, piperazines and their derivatives, and piperidines and their derivatives, such as DODMA (1,2-dioleyloxy-3-dimethylaminopropane, 1,2-dioleyl-3-dimethylaminopropane, CAS: 104162-47-2), ALC-0159, A066(Z016)(1-(2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine), L-319 (CAS: 1351586-50-9), DODAP, C12-200, 5A2-SC8, 3060i10, Moderna Lipid5, Acuitas A9, ALC-0315, SM-102 (or HUO, Moderna Lipid H; 1-octylnonyl) 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate), DLin-MC3-DMA((6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate), DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, other DLinDMA classes, other DLin-K-DMA classes, lipid 5A2-SC8.

[0049] In some embodiments, the electrolytically ionizable lipids are selected from one or more of the following: DODMA, ALC-0159, A066, L-319, DODAP, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, ALC-0315, SM-102, DLin-MC3-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, and lipids 5A2-SC8.

[0050] In some embodiments, the electrolytically ionizable lipids are selected from one or more of the following: DODMA, ALC-0159, A066, L-319, DODAP, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, ALC-0315, SM-102, DLin-MC3-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, and lipids 5A2-SC8.

[0051] In this article, the term "cationic lipids" refers to a class of positively charged lipid molecules with wide applications in drug delivery, gene therapy, and bioimaging. Cationic lipid molecules typically consist of three main parts: one or more cationic heads, a linker bond, and a hydrophobic tail.

[0052] The cationic lipids are selected from one or more of quaternary ammonium compounds and their derivatives, tertiary amines and their derivatives, compounds containing guanidin groups, polyethyleneimine and its derivatives, alcoholic amines and their derivatives, meglumine and its derivatives, lysine and its derivatives, histidine and its derivatives, arginine and its derivatives, and protamine and its derivatives. In some embodiments, the cationic lipids include, but are not limited to, DOTAP (CAS: 132172-61-3, (2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride), DOTMA (CAS No. 104872-42-6), DDBA, DMRIE, DOTIM, SAINT, DC-Chol (DC cholesterol, 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol). Hydrochloride, BGTC, CTAP, DOSPA, DORIE, DODAB, DOIC, DMEPC, DOGS, DIMRI, DC-6-14, CLIP1, DORIE, DOSPA, CLIP6, CLIP9, and those disclosed in US Patent 5,049,386, International Publications WO91 / 16024, WO97 / 019675, WO2005 / 121348, WO2009 / 086558 and WO2011 / 13636.

[0053] In some embodiments, the cationic lipid is selected from one or more of the following: DOTAP, DOTMA, DDBA, DMRIE, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOSPA, DORIE, DODAB, DOIC, DMEPC, DOGS, DIMRI, DC-6-14, CLIP1, DORIE, DOSPA, CLIP6, and CLIP9.

[0054] In this article, the term "neutral lipids" refers to a class of hydrophobic molecules lacking charged groups, primarily including triacylglycerol (TAG) and cholesterol and its esters. Triacylglycerol, also known as triglycerides, is a glycerol ester formed by the esterification of the three hydroxyl groups of glycerol with three fatty acid molecules.

[0055] Neutral lipids include, but are not limited to, phosphatidylcholine and / or phosphatidylethanolamine, such as distearylphosphatidylcholine (DSPC), egg yolk lecithin (EPC), soybean lecithin, hydrogenated soybean lecithin (HSPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoylphosphatidylcholine (DOPC), arachidoylphosphatidylcholine (DAPC), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), distearylphosphatidylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoylethanolamine (POPE), 1-hexadecane-2-(cis-9-octadecenoyl)-sn-glycerol-3-phosphate choline (POPC), and cholesterol.

[0056] In some embodiments, the neutral lipids are selected from one or more of the following: cholesterol, DSPC, EPC, soybean lecithin, HSPC, DOPE, DLPC, DMPC, DPPC, DOPC, DAPC, DMPE, DLPE, DSPE, DPPE, POPE, and POPC.

[0057] In this article, the term "auxiliary lipids" refers to a class of small-molecule phospholipids used in conjunction with primary phospholipids (such as ionized lipids and ionizable lipids) to optimize nanoparticle formation and improve drug delivery efficiency. Auxiliary lipids (such as DSPC, DOPC, and DOPE) with different alkyl chain saturation, chain lengths, and polar head groups can assemble to form lipid delivery carriers with different structures.

[0058] In some embodiments, the auxiliary lipids include, but are not limited to, one or more of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1,2-distearate-sn-glycero-3-phosphatidylethanolamine (DSPE), and 1,2-dispalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE).

[0059] In this article, the term "PEGylated lipids," also known as "polyethylene glycol-modified lipids," refers to a nanomedicine delivery system that achieves functionalization by modifying the surface of liposomes with polyethylene glycol (PEG). PEG can prolong the circulation time of liposomes, prevent the binding of plasma proteins, and also control the size of nanoparticles. This technology is widely used for targeted drug delivery.

[0060] PEGylated lipids include, but are not limited to, DSPE-PEG, DMPE-PEG, DOPE-PEG, DMG-PEG, and DSG-PEG.

[0061] In some embodiments, PEGylated lipids include, but are not limited to, 1,2-dimyristoyl-rac-glycero-3-methoxy polyethylene glycol (DMG-PEG or PEG-DMG), 1,2-distearate-rac-glycero-3-methoxy polyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-rac-glycero-3-methoxy polyethylene glycol (DPG-PEG), 1,2-distearate-sn-glycero-3-phosphatidylethanolamine-methoxy polyethylene glycol (DSPE-PEG), 14:0 polyethylene glycol-polyethylene (DMPE-PEG), and DOPE-PEG (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine-polyethylene glycol).

[0062] Equipment for preparing lipid nanoparticles

[0063] This disclosure provides an apparatus for preparing lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids, comprising a mechanical pump with multiple pump heads and multiple tee fittings, the multiple pump heads being linked in an A-configuration or B-configuration via the multiple tee fittings. In some embodiments, the organic phase and aqueous phase disclosed herein are mixed using the apparatus disclosed herein at a flow rate ratio of 1:3. In some embodiments, there are one or more mechanical pumps.

[0064] In some embodiments, the present disclosure includes four pump heads and three tee fittings, with one pump head conveying an organic phase containing lipids and three pump heads conveying an aqueous phase containing nucleic acids.

[0065] In some embodiments, the flow rate of any pump head of this disclosure is independently 0-500 mL / min, preferably 150-500 mL / min, more preferably 300-400 mL / min, for example 300 mL / min, 350 mL / min, 400 mL / min.

[0066] In some embodiments, any tee fitting of this disclosure is independently selected from T-type or Y-type. In some preferred embodiments, any tee fitting of this disclosure is independently selected from one of the following: T-032, T-024, PEEK internal thread T-type, stainless steel T-type, Y-024, Y-type PEEK, Y-032.

[0067] In some embodiments, the inner diameter of any tee fitting disclosed herein is independently 0.5-3 mm, preferably 0.8-2.36 mm, for example 0.8, 1, 1.25, 1.58, 2, 2.36 mm.

[0068] Lipid nanoparticles and their pharmaceutical applications

[0069] This disclosure provides lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids, prepared by the methods or apparatus of this disclosure.

[0070] This disclosure also provides the use of lipid nanoparticles prepared by the methods or apparatus of this disclosure for drug delivery.

[0071] This disclosure also provides methods for delivering drugs, including delivering drugs using lipid nanoparticles prepared using the methods or devices of this disclosure.

[0072] In some implementations, the drug is selected from anti-tumor drugs, anti-infective (e.g., antiviral, antibacterial, antifungal) drugs, immunotherapeutic drugs, vaccines, etc.

[0073] Example

[0074] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0075] The information on the tee fittings used in the embodiments is summarized in Table 1.

[0076] Table 1. Information on Tee Fittings

[0077] Example 1: Preparation methods of sodium citrate solution, lipid ethanol solution, and HClO16 buffer solution

[0078] Preparation method of sodium citrate solution: Weigh 14.42g of citric acid and 12.9g of sodium citrate, place them in a 5L screw bottle, add 5kg of pure water, stir and dissolve completely at room temperature (15-25℃) to prepare a 5L sodium citrate buffer solution.

[0079] Preparation method of HC016 buffer solution: Based on the HC016 content (calculated as 78.8%), weigh 0.75g of HC016 and add it to a 3L screw bottle. Add 1.5Kg of 25mM citrate buffer, stir and dissolve at room temperature to prepare a pH 4.0, 25mM citrate buffer solution containing 0.5mg / ml of HC016, i.e., 1.5L of HC016 buffer.

[0080] Lipid solution preparation method: Add each lipid component to a 1L screw bottle according to the information in Table 2, add 395g of anhydrous ethanol (0.5L of ethanol), dissolve at 60℃, and prepare a 1L lipid ethanol solution of 15mg / ml.

[0081] Table 2. Lipid composition ratio

[0082] Example 2: The effect of peristaltic pump mixing method on the preparation of lipid nanoparticles

[0083] The flow rate ratio of the lipid ethanol solution and the HC016 buffer solution prepared in Example 1 was set to 1:3. A peristaltic pump with four pump heads connected in series was selected, with one pump head connected to the lipid ethanol solution and the other three pump heads connected to the HC016 buffer solution.

[0084] There are two configurations for the pump head and pipe connections: Configuration A, as shown in Figure 1, connects the silicone tubing of pump head 1 and pump head 2 with a T-shaped tube with an inner diameter of 2.36 mm, and connects the silicone tubing of pump head 3 and pump head 4 with the same T-shaped tube. These two sets are then connected with a single T-shaped tube, forming a "tree-like confluence" hybrid configuration. Configuration B, as shown in Figure 2, connects the silicone tubing of pump head 1 and pump head 2 with a T-shaped tube with an inner diameter of 2.36 mm, then connects it to the silicone tubing of pump head 3 with a T-shaped tube, and finally connects to pump head 4, forming a "river-like confluence" hybrid configuration.

[0085] For the two configurations shown in Figures 1 and 2, the particles were encapsulated in sodium citrate buffer and lipid ethanol solution, and the particle size and particle size distribution (PDI) were determined using a Malvern nanoparticle size analyzer. PDI is a dimensionless value for the particle size distribution width. The results are shown in Table 3 below.

[0086] Table 3. Particle size and PDI of lipid nanoparticles after different mixing methods

[0087] The screening criteria were: particle size between 50 nm and 150 nm, especially between 40 and 100 nm, and PDI less than 0.2, with lower PDI being better. As shown in Table 3, compared with configuration A, the lipid nanoparticles prepared by configuration B under both low and high flow rate conditions had smaller particle size and PDI than configuration A, with particle size between 50 nm and 150 nm and PDI less than 0.2. Therefore, configuration B will be used as the application configuration in the subsequent examples.

[0088] Example 3: Effects of T-fitting and Flow Rate on the Preparation of Lipid Nanoparticles

[0089] Based on the B-configuration mixing method, the flow rate and the internal structure of the tee have a significant impact on the prepared lipid nanoparticles. By setting various types of tees and flow rates, and using citrate buffer and lipid ethanol solution for encapsulation, the particle size and PDI were measured to conduct the following experiments, and the experimental results are shown in Table 4.

[0090] Table 4. Particle size and PDI of lipid nanoparticles loaded with different T-junctions and flow rates

[0091] Based on the experimental results, it can be observed that different tee types and flow rates have a certain impact on lipid nanoparticles. Generally speaking, T-type tee fittings produce smaller particle sizes than Y-type fittings at the same flow rate. For the same type of tee, the smaller the inner diameter, the smaller the particle size. Higher flow rates also result in smaller particle sizes. This is because the collision between the two liquid streams is most intense and mixing is most thorough in T-type tee fittings. A smaller inner diameter and higher flow rate lead to greater pressure during collision, resulting in more thorough mixing and thus smaller particle sizes. However, intense collision and mixing also increase the PDI (lipid density index). Further optimization of the process will be conducted in the future.

[0092] Example 4: Effect of flow rate on the preparation of loaded lipid nanoparticles

[0093] To further investigate the feasibility of various batch combinations, based on the use of Y-type tee fittings, RX-0201 was used as a model drug to encapsulate drug molecules for flow rate studies, and HC016 was used for preliminary process verification. The buffer solution for dissolving RX-0201 raw material was the same as that for HC016. The experimental results are shown in Table 5.

[0094] Table 5. Particle size and PDI of lipid nanoparticles loaded at different flow rates

[0095] The experimental results show that when using a buffer containing nucleic acid, the smaller the inner diameter of the No. 1 tee, the better the particle size and PDI of the prepared lipid nanoparticles. The lipid nanoparticles prepared by using a Y-type PEEK tee with an inner diameter of 1.25 mm at a flow rate of 350 mL / min have suitable particle size and PDI.

[0096] Example 5: Effect of lipid concentration on the preparation of lipid nanoparticles

[0097] The concentrations of lipids and nucleic acids not only affect the preparation time but also the subsequent concentration and ultrafiltration replacement times. Cholesterol will precipitate in high-concentration lipid ethanol solutions. Process screening was conducted using different lipid concentrations (15 mg / mL, 30 mg / mL, and 60 mg / mL), and high-concentration lipid ethanol solutions were incubated at different water bath temperatures. The experimental results are shown in Table 6.

[0098] Table 6. Particle size and PDI of lipid nanoparticles prepared with different lipid concentrations

[0099] The results showed that increasing the lipid concentration to 60 mg / mL increased the particle size and PDI of the prepared lipid nanoparticles, and even increasing the flow rate did not improve the situation.

[0100] Example 6: Effect of lipid to nucleic acid ratio on the preparation of lipid nanoparticles

[0101] The ratio of lipids to nucleic acids not only affects the mixing effect during preparation but also influences the size and stability of the prepared lipid nanoparticles. Lipid nanoparticles were prepared using different lipid-to-nucleic acid mass ratios, with the same three-way valve type as batch 09. Particle size and PDI were measured. The experimental results are shown in Table 7.

[0102] Table 7. Particle size and PDI of lipid nanoparticles prepared with different lipid-nucleic acid ratios

[0103] The results showed that during the preparation of lipid nanoparticles, both excessively high and low mass ratios of nucleic acids and lipids would cause uneven mixing of lipid nanoparticles, with a PDI greater than 0.3.

[0104] The method for calculating the mass ratio of nucleic acid to lipid is as follows: [(concentration of nucleic acid buffer) x (3 pump heads) x (flow rate of each pump head)]: [(concentration of lipid ethanol) x (1 pump head) x (flow rate of each pump head)].

[0105] Example 7: Effect of temperature on the preparation of lipid nanoparticles

[0106] The loading temperature affects the frequency and intensity of intermolecular collisions, thus influencing the preparation of lipid nanoparticles. Three batches of lipid nanoparticles were prepared at different temperatures (25℃, 50℃, and 65℃), with the same three-way connector as the 09 batch. The effect of loading temperature on lipid nanoparticles was investigated. Particle size and PDI were measured using a Malvern nanoparticle size analyzer, and the encapsulation efficiency was calculated using a fluorescence method measured by an ELISA reader. This method includes the following steps: 1) Detection of free nucleic acid: In the nucleic acid lipid nanoparticle solution, free nucleic acid not encapsulated by lipid nanoparticles can directly contact and bind to the dye. After appropriately diluting the sample containing nucleic acid lipid nanoparticles, RiboGreen was added, which binds to the free nucleic acid and generates a fluorescence signal. The intensity of this fluorescence signal was detected by an ELISA reader (excitation wavelength 485nm, emission wavelength 535nm), and the concentration of free nucleic acid in the sample could be calculated based on a pre-prepared standard curve. 2) Total Nucleic Acid Detection: The nucleic acid lipid nanoparticle sample is treated with a demulsifier (such as Triton X-100). The structure of the lipid nanoparticles is disrupted, releasing the encapsulated nucleic acids. At this point, a nucleic acid dye is added, which binds to all nucleic acids (including both the originally encapsulated and free nucleic acids), generating a fluorescent signal. The fluorescence signal intensity is then detected using a microplate reader (excitation wavelength 485nm, emission wavelength 535nm), and the total nucleic acid concentration in the sample is calculated based on a standard curve. 3) Encapsulation Efficiency Calculation: After obtaining the free nucleic acid concentration and total nucleic acid concentration, the encapsulation efficiency is calculated using the formula: Encapsulation Efficiency = (Total Nucleic Acid Concentration - Free Nucleic Acid Concentration) / Total Nucleic Acid Concentration x 100%.

[0107] Based on the above experiments, the experimental results are shown in Table 8.

[0108] Table 8. Encapsulation efficiency of lipid nanoparticles prepared at different temperatures

[0109] The total preparation time for lipid nanoparticles, including incubation, was approximately 1 hour. When measuring the content and encapsulation efficiency of lipid nanoparticles prepared at 50℃, a slight change in the liquid phase peak shape of the nucleic acid was observed compared to that of the active pharmaceutical ingredient HC016 at 25℃. However, the liquid phase peak shape of the nucleic acid encapsulated in lipid nanoparticles prepared at 65℃ was completely altered, indicating that excessively high temperatures can lead to nucleic acid degradation. Therefore, the preparation temperature for lipid nanoparticles should be below 65℃, ideally below 50℃, or maintained between 15℃ and 25℃. The above liquid phase peak shape data are shown in Figures 3A-C. In Table 8, " / " indicates that the encapsulation efficiency was not measured because nucleic acid degradation occurred at 65℃.

[0110] The reagents, solutions, and standards used in the above-mentioned liquid chromatography (LC) determination of nucleic acids were selected from ultrapure water, hexafluoroisopropanol, acetonitrile, triethylamine, 50×TAE buffer, Triton X-100, and HC016 working standard. The instruments used included a high-performance liquid chromatograph (HPLC) and an electronic balance. The preparation of relevant solutions for LC determination of nucleic acids included: 1) Constructing mobile phase A: Accurately measure 1000 mL of water, add 2 mL of triethylamine, mix well, then add 10 mL of hexafluoroisopropanol, mix well, and degas by sonication. 2) Constructing mobile phase B: Accurately measure 750 mL of water and 250 mL of acetonitrile, mix well, add 1 mL of triethylamine, mix well, then add 5 mL of hexafluoroisopropanol, mix well, and degas by sonication. 3) Preparing diluent A: Measure 2 mL of 50×TAE buffer, place it in a 100 mL volumetric flask, dilute with water to the 100 mL mark, and mix well. 4) Prepare diluent B: Measure 20 mL of Triton X-100 and 20 mL of ethanol, shake to dissolve the Triton X-100 as much as possible, then measure 20 mL of 50×TAE buffer, add 980 mL of water, and mix well. 5) Prepare reference solution: Accurately weigh approximately 40 mg of reference standard (approximately equivalent to 32 mg of HClO16-DS free acid), place in a 20 mL volumetric flask, add diluent A to dilute to the 20 mL mark, and mix well. This is the reference standard stock solution. Accurately measure 1 mL of the reference standard stock solution, place in a 100 mL volumetric flask, add diluent B to dilute to the 100 mL mark, and mix well. This is the reference solution. Repeat this step to prepare two parallel reference solutions. 5) Prepare test solution: Accurately measure 1 mL of this product, place in a 100 mL volumetric flask, add diluent B to dilute to the 100 mL mark, and mix well. Repeat this step to prepare two parallel test solutions.

[0111] In the liquid chromatography determination, the chromatographic conditions were configured and the chromatogram was adjusted according to the parameters shown in Table 9, which served as the experimental parameters for liquid chromatography required for the experiment.

[0112] Table 9. Configuration parameters of liquid chromatograph

[0113] In liquid chromatography determination, the injection sequence should be prepared according to the injection sequence and corresponding number of injections shown in Table 10.

[0114] Table 10. Injection sequence and corresponding number of injections

[0115] To verify the system's suitability, the following parameters need to be determined for the relevant experimental results: the RSD of the main peak area of ​​the standard solution 1 for 5 consecutive tests should be ≤2.0%, the minimum theoretical plate number should be ≥5000, and the maximum tailing factor should be ≤2.0; the recovery rate of STD2 calculated based on STD1 should be between 98.0% and 102.0%; the RSD of the peak area of ​​the standard solution 1 compared with the peak area of ​​the standard solution 1 for 5 consecutive tests should be ≤2.0%.

[0116] Based on the experimental data, the external standard method was applied to calculate the experimental results. The external standard method calculation formula is shown below:

[0117] Wherein, Cs1 is the concentration of reference standard 1; Cs2 is the concentration of reference standard 2; As1 is the average peak area of ​​reference standard 1 across 5 injections; As2 is the average peak area of ​​reference standard 2 across 2 injections; Au is the peak area of ​​the test sample; and D is the diluent factor for the test sample.

Claims

1. A method for preparing lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids, wherein a mechanical pump with multiple pump heads, such as a peristaltic pump or a plunger pump, is used to mix the organic phase containing lipids and the aqueous phase containing nucleic acids, wherein... The flow rate of any one of the pump heads is independently 0-500 mL / min; The flow rate ratio of the organic phase to the aqueous phase is 1:

3. The temperature for preparing lipid nanoparticles is 4-65℃, preferably 15-25℃; The mass ratio of nucleic acid to lipid is 1:2-1:40, preferably 1:3-1:27, and more preferably 1:6-1:

15.

2. The method according to claim 1, wherein the flow rate of any one of the pump heads is independently 150-500 mL / min, preferably 300-400 mL / min.

3. The method according to claim 1, wherein the plurality of pump heads are connected in configuration A or configuration B via a plurality of tee fittings; preferably, there are 4 pump heads and 3 tee fittings.

4. The method according to any one of claims 1-3, wherein the plurality of pump heads are connected by a plurality of tee fittings, and any one of the tee fittings is independently selected from T-type or Y-type; preferably, any one of the tee fittings is independently selected from one of the following: T-032, T-024, PEEK internal thread T-type, stainless steel T-type, Y-024, Y-type PEEK, Y-032.

5. The method according to claim 3 or 4, wherein the inner diameter of any of the tee fittings is independently 0.5-3 mm, preferably 0.8-2.36 mm.

6. The method according to any one of claims 1-5, wherein the nucleic acid is selected from one or more of mRNA, DNA, siRNA, sgRNA, shRNA, microRNA, and antisense nucleic acid; preferably, the nucleic acid is HC016 or RX-0201.

7. The method according to any one of claims 1-6, wherein the lipid comprises one or more of ionizable lipids, cationic lipids, neutral lipids, auxiliary lipids, and PEGylated lipids; preferably, the lipid comprises DOTAP, DODMA, DSPC, cholesterol, and PEG-DMG.

8. The method according to claim 7, wherein the cationic lipid is selected from one or more of the following: DOTAP, DOTMA, DDBA, DMRIE, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOSPA, DORIE, DODAB, DOIC, DMEPC, DOGS, DIMRI, DC-6-14, CLIP1, DORIE, DOSPA, CLIP6, and CLIP9.

9. The method according to claim 7, wherein the PEGylated lipid is selected from one or more of: DSPE-PEG, DMPE-PEG, DOPE-PEG, DMG-PEG, DPG-PEG, and DSG-PEG.

10. The method according to claim 7, wherein the electrolytically ionizable lipid is selected from one or more of the following: DODMA, ALC-0159, A066, L-319, DODAP, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, ALC-0315, SM-102, DLin-MC3-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, and lipid 5A2-SC8.

11. The method according to claim 7, wherein the neutral lipid is selected from one or more of cholesterol, DSPC, EPC, soybean lecithin, HSPC, DOPE, DLPC, DMPC, DPPC, DOPC, DAPC, DMPE, DLPE, DSPE, DPPE, POPE, and POPC.

12. An apparatus for preparing lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids, comprising a mechanical pump with multiple pump heads and multiple tee fittings, the multiple pump heads being linked in an A configuration or a B configuration via the multiple tee fittings, wherein the organic phase and the aqueous phase are mixed through the apparatus at a flow rate ratio of 1:

3.

13. The device according to claim 12, wherein the plurality of pump heads comprises four units, the plurality of tee fittings comprises three units, and one pump head transports an organic phase containing lipids and the three pump heads transport an aqueous phase containing nucleic acids.

14. The device according to claim 12 or 13, wherein the flow rate of any one of the pump heads is independently 0-500 mL / min, preferably 150-500 mL / min, more preferably 300-400 mL / min.

15. The device according to any one of claims 12-14, wherein any one of the tee fittings is independently selected from T-type or Y-type; preferably, any one of the following is independently selected from: T-032, T-024, PEEK internal thread T-type, stainless steel T-type, Y-024, Y-type PEEK, Y-032.

16. The device according to any one of claims 12-14, wherein the inner diameter of any one of the tee fittings is independently 0.5-3 mm, preferably 0.8-2.36 mm.

17. Lipid nanoparticles comprising an organic phase containing lipids and an aqueous phase containing nucleic acids, prepared by the method of any one of claims 1-11 or by the apparatus of any one of claims 12-16.

18. Use of the lipid nanoparticles of claim 17 for drug delivery.

19. A method of delivering a drug, comprising delivering the drug using the lipid nanoparticles of claim 17.

Images