A method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid and uses thereof

Abstract

The application belongs to the field of drug delivery nanotechnology, and provides a method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid and application thereof. The application adopts a trinity design of ion complex pre-assembly of succinylated glycerol monooleate and cationic lipids, space stabilization of poloxamer 407, and rapid particle formation by microfluidics, realizes stable construction of internal nanostructure of inverse bicontinuous cubic phase, encapsulation of small interfering ribonucleic acid in water channel network with high drug loading, long-term stability of colloids under physiological ion strength conditions, and excellent batch consistency, solves the technical contradictions of existing lipid nanometer delivery systems, such as difficulty in giving consideration to low polydispersion and batch consistency, mutual restraint of cationic complex strength and colloidal stability, and difficulty in synergistic optimization of high drug loading and stability of cubic phase structure under high solid content microfluidic conditions, and has wide application value in gene therapy and nucleic acid drug delivery.

Description

Technical Field

[0001] This invention relates to the field of drug delivery nanotechnology, specifically to a method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid and its application. Background Technology

[0002] Small interfering RNA (SRNA), as a core tool molecule in gene therapy, has demonstrated unique advantages in precisely targeting and silencing specific pathogenic genes in clinical applications such as tumor treatment, viral infection control, and intervention for hereditary diseases. However, its in vivo application faces multiple delivery obstacles, including rapid degradation by serum nucleases, difficulty in crossing the negative charge barrier of cell membranes, and low efficiency in endosome escape. There is an urgent need to construct nanodelivery carriers that combine high encapsulation efficiency to protect nucleic acid integrity, efficient cellular uptake and endosome release, targeted tissue enrichment, and biosafety. Lipid nanoparticles have become the mainstream technology platform for nucleic acid drug delivery due to their similarity to cell membrane structures, excellent biocompatibility, and ease of large-scale preparation. Lipid nanoparticles with an anti-bicontinuous cubic internal nanostructure, with their unique three-dimensional interconnected water channel network, ultra-high specific surface area, tunable pore structure, and excellent encapsulation ability for hydrophilic biomolecules, provide an ideal structural basis for achieving high drug loading encapsulation, sustained-release, and enhanced cell transfection efficiency of SRNA, thus promoting the translation of nucleic acid therapy from laboratory research to clinical applications.

[0003] However, existing anti-bicontinuous cubic phase lipid nanoparticles still face many technical bottlenecks in small interference ribonucleic acid (MIRNA) delivery applications, mainly manifested in the following three coupled contradictions: First, cubic phase nanoparticles prepared by traditional manual injection or simple mixing methods suffer from wide particle size distribution and poor batch-to-batch reproducibility. While microfluidic technology can achieve rapid and uniform mixing, it is prone to significant increases in polydispersity coefficient or even micron-scale aggregation due to excessively high local lipid concentrations under high solids content and high throughput conditions. For example, Chinese patent CN108586753BA discloses a method for preparing cubic phase vesicle nanomaterials constructed from polyoxometalate clusters and cage-type silsesquioxane hybrid molecules, but the vesicle encapsulation efficiency is low, seriously affecting product quality uniformity and clinical application safety; Second, the electrostatic complexation of cationic lipids and nucleic acids... While combination is a key strategy to improve encapsulation efficiency and transfection efficiency, excessively strong cationic charges can cause toxicity problems such as serum protein adsorption and erythrocyte aggregation. In environments with near-physiological ionic strength, the charge shielding effect can lead to a sharp decline in colloidal stability and even precipitation. Existing technologies often mitigate toxicity by significantly reducing the amount of cationic lipids used, but at the same time, they sacrifice nucleic acid encapsulation capacity and delivery efficiency. Third, when auxiliary lipids such as phospholipids and cholesterol are introduced to optimize in vivo pharmacokinetics and targeting performance, the complex interactions between lipid components often lead to a transformation of the cubic phase structure to a layered or hexagonal phase. Especially during post-processing dialysis or tangential flow filtration, the rapid removal of ethanol causes drastic changes in osmotic pressure and ionic strength, which can easily induce phase transitions. Ultimately, it is difficult to achieve a synergistic balance between high drug loading, long-term stability, low toxicity, and maintenance of cubic phase structure. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid and its application, thereby solving the technical contradictions of the present invention, which is difficult to balance low polydispersity and batch-to-batch consistency under high solids content microfluidic conditions, the mutual constraints between cationic composite strength and long-term colloidal stability, and the difficulty in synergistically optimizing high drug loading and cubic phase structure stability.

[0005] This invention employs a synergistic strategy of pre-forming an ionic complex with succinylated glycerol monooleate and cationic lipid 1,2-dioleoyloxy-3-trimethylammonium propane chloride. On one hand, the surface potential of the complex is precisely controlled to a moderately positive range through the electrostatic neutralization of the negative charge of the succinyl group and the positive charge of the quaternary ammonium group. This retains sufficient cationicity to efficiently encapsulate small interfering ribonucleic acid while avoiding serum protein adsorption and erythrocyte aggregation toxicity caused by excessive positive charge. At the same time, the ionic complexation process induces the lipid molecules to arrange in an orderly cubic phase precursor structure, significantly reducing the phase transition energy barrier in the subsequent microfluidic particle formation process. On the other hand, the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer structure of poloxamer 407 is anchored on the particle surface to form a hydrophilic steric hindrance layer, effectively inhibiting particle aggregation under high ionic strength conditions. The synergistic effect of both achieves the effect of maintaining a polydispersity index of less than 0.4, a moderate absolute value of the Zeta potential, a stable cubic phase structure, and a batch-to-batch particle size variation coefficient of less than 5% even under high solids content microfluidic conditions with a total lipid concentration of up to 100 mg / mL.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid includes the following steps:

[0008] S1. Preparation of organic phase: Weigh 8.5-9.8 parts by weight of glyceryl monooleate, 0.2-1.5 parts by weight of 1,2-dioleoyloxy-3-trimethylammonium propane chloride, and 0.05-1.5 parts by weight of succinylated glyceryl monooleate, dissolve them in anhydrous ethanol to make the total lipid concentration in the organic phase 10-100 mg / mL, and obtain a clear organic phase;

[0009] S2. Aqueous phase preparation: Weigh 1.0-1.5 parts of poloxamer 407 and 1 part of small interfering RNA by mass. Dissolve poloxamer 407 and small interfering RNA in a buffer solution, which is a phosphate buffer or a citrate buffer, to obtain the aqueous phase.

[0010] Wherein, each mass fraction is calculated as one part of the small interfering ribonucleic acid;

[0011] S3. Microfluidic mixing and granulation: The clarified organic phase obtained in step S1 and the aqueous phase obtained in step S2 are respectively introduced into a microfluidic mixing chip, wherein the flow rate ratio of the aqueous phase to the organic phase is 3:1 and the total flow rate is 8-20 mL / min, so that they are mixed in the microfluidic mixing chip and self-assembled into granules to obtain a granulation system containing cubic lipid nanoparticles encapsulating the small interfering ribonucleic acid;

[0012] S4. Post-processing: The granulation system obtained in step S3 is subjected to ethanol removal and buffer replacement by dialysis or tangential flow filtration to obtain cubic lipid nanoparticles or their formulations, wherein the ethanol content after step S4 is not higher than 1.0 wt%.

[0013] Furthermore, the cubic lipid nanoparticles possess an internal nanostructure of an anti-bicontinuous cubic phase, wherein the anti-bicontinuous cubic phase is of Pn3m or Ia3d lattice type; the small interfering ribonucleic acid is encapsulated within the water channels of the anti-bicontinuous cubic phase; and the succinylated glycerol monooleate forms an ionic complex with the 1,2-dioleoyloxy-3-trimethylammonium propane chloride; wherein the cubic lipid nanoparticles refer to lipid nanoparticles with an internal anti-bicontinuous cubic phase structure, and their shape is not limited to a geometric cube.

[0014] Furthermore, the succinylated glycerol monooleate used in step S1 is prepared according to the following steps:

[0015] A1. Raw materials: Glyceryl monooleate, succinic anhydride, triethylamine, tetrahydrofuran, ethyl acetate, sodium bicarbonate, anhydrous sodium sulfate, sodium chloride, water;

[0016] A2. Ratio: The molar ratio of succinic anhydride to glyceryl monooleate is 0.05-0.30:1, and the molar ratio of triethylamine to succinic anhydride is 1.0-1.5:1;

[0017] A3. Reaction: Dissolve glyceryl monooleate in tetrahydrofuran to achieve a mass concentration of 50-300 mg / mL in tetrahydrofuran. Stir at 40-55℃, add succinic anhydride and triethylamine, and continue the reaction at 40-55℃ for 6-20 h.

[0018] A4. Endpoint criterion: The acid value of the obtained product is 20-120 mg KOH / g;

[0019] A5. Post-treatment: After the reaction is complete, remove tetrahydrofuran under reduced pressure, dissolve in ethyl acetate, and wash successively with 5-10 wt% sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution until the pH of the aqueous phase is 6.5-7.5; dry the organic phase with anhydrous sodium sulfate and filter, remove ethyl acetate under reduced pressure, and then vacuum dry until the mass is constant and the total residual solvent is not higher than 1.0 wt%;

[0020] A6. Quality control: Residual succinic anhydride shall not exceed 0.5 wt%, and residual triethylamine shall not exceed 0.2 wt%.

[0021] Furthermore, in step S1, the succinylated glycerol monooleate and the 1,2-dioleoyloxy-3-trimethylammonium propane chloride are pre-formed into a complex lipid intermediate, which is prepared according to the following steps:

[0022] B1. Raw materials: 1,2-dioleoyloxy-3-trimethylammonium propane chloride, succinylated glyceryl monooleate, anhydrous ethanol;

[0023] B2. Ratio: The molar ratio of the 1,2-dioleoyloxy-3-trimethylammonium propane chloride to the succinylated glycerol monooleate is 0.2-2.0:1;

[0024] B3. Dissolution and compounding: The 1,2-dioleoyloxy-3-trimethylammonium propane chloride and the succinylated glycerol monooleate were added to anhydrous ethanol to make the total lipid mass concentration 10-100 mg / mL, and stirred at 40-65°C until a clear solution was formed.

[0025] B4. Curing: Remove anhydrous ethanol under reduced pressure and dry under vacuum to obtain the complex lipid intermediate;

[0026] B5. Quality control: Residual ethanol shall not exceed 1.0 wt%.

[0027] Furthermore, the small interfering ribonucleic acid described in step S2 forms a nucleic acid-lipid precomplex intermediate before granulation. The nucleic acid-lipid precomplex intermediate is prepared according to the following steps:

[0028] D1. Raw materials: small interfering ribonucleic acid, 1,2-dioleoyloxy-3-trimethylammonium propane chloride and complex lipid intermediates, phosphate buffer or citrate buffer;

[0029] D2. Mixing step: Dissolve the small interfering ribonucleic acid in phosphate buffer or citrate buffer, add the 1,2-dioleoyloxy-3-trimethylammonium propane chloride or the complex lipid intermediate and mix, incubate at 20-30℃ for 5-30 min to obtain the nucleic acid-lipid precomplex intermediate;

[0030] D3. Ratio: The mass ratio of 1,2-dioleoyloxy-3-trimethylammonium propane chloride in the nucleic acid-lipid precomplex intermediate to the small interfering ribonucleic acid is 0.2-1.5:1.

[0031] D4. Endpoint Criteria: The obtained nucleic acid-lipid precomplex intermediate is clear or milky white and translucent, with no visible precipitation.

[0032] Furthermore, the ethanol removal and buffer replacement in step S4 are achieved by dialysis or tangential flow filtration:

[0033] Dialysis is performed using dialysis bags or membranes with a molecular weight cutoff of 3.5-14 kDa at 4-25°C for 6-24 hours with 3-6 buffer changes, wherein the volume of buffer used for each buffer change is 50-200 times the volume of the granulation system; or tangential flow filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 30-100 kDa at 4-25°C with 5-10 times volume replacement; the ethanol content after step S4 is not higher than 1.0 wt%, and the ethanol content is determined by gas chromatography;

[0034] The buffer solution is a phosphate buffer or a citrate buffer. The phosphate buffer solution is prepared with water and consists of 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate, and 1.8 mmol / L potassium dihydrogen phosphate, with a pH of 7.2-7.6. The citrate buffer solution is prepared with water and consists of 10-50 mmol / L citric acid and 10-50 mmol / L sodium citrate dihydrate, with a pH of 3.0-6.5.

[0035] Furthermore, in step S1, an auxiliary lipid is added, which is one or both of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol, wherein, by weight, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine is 0.1-3.0 parts and cholesterol is 0.1-3.0 parts.

[0036] Furthermore, the cubic lipid nanoparticles have a Z-average particle size of 50-500 nm, a polydispersity index (PDI) of 0.1-0.4, and an absolute value of zeta potential of 10-60 mV; wherein the Z-average particle size and the PDI are determined by dynamic light scattering, and the zeta potential is determined by electrophoretic light scattering.

[0037] As a concept of this invention, the pre-assembly of succinylated glycerol monooleate and 1,2-dioleoyloxy-3-trimethylammonium propane chloride ions, combined with poloxamer 407 spatial stabilization, is designed to enhance the high-drug-loading encapsulation and long-term colloidal stability of small interfering RNAs (SRNAs). The negatively charged carboxyl group introduced by succinylated glycerol monooleate forms an ion pair with the positively charged quaternary ammonium group of 1,2-dioleoyloxy-3-trimethylammonium propane chloride. Electrostatic neutralization reduces the surface potential of the complex to a moderately positive range, thus maintaining sufficient positive charge to efficiently capture negatively charged SRNAs and achieve an encapsulation rate of 60-95%, while avoiding toxic reactions such as complement activation, serum protein adsorption, and erythrocyte aggregation caused by excessive positive charge. Ion-induced lipid molecule tail chains align in an ordered manner and pre-assemble into a cubic phase precursor structure in an ethanol-water mixed solvent. This lowers the nucleation and phase transition energy barriers for cubic phase self-assembly during rapid microfluidic mixing, enabling the stable formation of an anti-bicontinuous cubic phase with a Pn3m or Ia3d lattice type and maintaining a polydispersity index of 0.1-0.4 under high solids content and high throughput conditions with a total lipid concentration of 10-100 mg / mL and a total flow rate of 8-20 mL / min. The hydrophilic segment of poloxamer 407 adsorbs onto the particle surface to form a steric hindrance layer, shielding residual positive charges and inhibiting electrostatic aggregation between particles. This keeps the absolute value of the zeta potential controlled at 10-60 mV, ensuring that the particle size change does not exceed ±20% and the encapsulation efficiency decreases by no more than 10% during long-term storage in physiological ionic strength buffers, without precipitation or stratification. Simultaneously, it inhibits the cubic-to-layer or hexagonal phase transition caused by rapid ethanol removal and changes in ionic strength during post-processing.

[0038] This invention also discloses the application of cubic lipid nanoparticles encapsulating small interfering RNA (SIRNA) in the preparation of formulations for delivering SIRNA to achieve target gene silencing. The cubic lipid nanoparticles, by dry basis, comprise the following components: 8.5-9.8 parts of glyceryl monooleate; 1.0-1.5 parts of poloxamer 407; 0.2-1.5 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride; 0.05-1.5 parts of succinylated glyceryl monooleate; and 1 part of SIRNA. All parts by weight are based on 1 part of SIRNA.

[0039] The cubic lipid nanoparticles have an internal nanostructure of an anti-bicontinuous cubic phase, which is of Pn3m or Ia3d lattice type; the small interfering ribonucleic acid is encapsulated in the water channels of the anti-bicontinuous cubic phase; and the succinylated glycerol monooleate forms an ionic complex with the 1,2-dioleoyloxy-3-trimethylammonium propane chloride.

[0040] Furthermore, the cubic lipid nanoparticles have a Z-average particle size of 50-500 nm, a polydispersity index (PDI) of 0.1-0.4, and an absolute zeta potential of 10-60 mV; wherein the Z-average particle size and the PDI are determined by dynamic light scattering, and the zeta potential is determined by electrophoretic light scattering; the cubic lipid nanoparticles further include auxiliary lipids, wherein the auxiliary lipids are one or two of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol, and by mass, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine is 0.1-3.0 parts and cholesterol is 0.1-3.0 parts.

[0041] Furthermore, the encapsulation efficiency of the small interfering ribonucleic acid (SIRNA) is 60-95%, and the drug loading is 4.5-10 wt%, wherein the drug loading is the mass percentage of the SIRNA in the cubic lipid nanoparticles relative to the total dry mass of the cubic lipid nanoparticles; the cubic lipid nanoparticles or their formulations, when stored at 4°C and 25°C for 3 months, show a particle size change of no more than ±20%, a decrease in encapsulation efficiency of no more than 10%, and no visible precipitation or stratification; the encapsulation efficiency is determined by ultraviolet spectrophotometry or fluorescence spectrometry at a detection wavelength of 260 nm after separating free SIRNA by high-speed centrifugation or gel column chromatography.

[0042] Furthermore, in step S3, the flow rate ratio of the aqueous phase to the organic phase is 3:1, and the total flow rate is 8-20 mL / min; 1.0-2.0 mL of the initial waste liquid is discarded before collecting the granulation system, and 0.5-1.5 mL of the subsequent waste liquid is discarded after collection.

[0043] Furthermore, in step S1, the mixture is stirred at 20-45°C for 5-60 minutes to ensure that all components are completely dissolved.

[0044] Furthermore, in step S2, the mass concentration of small interfering ribonucleic acid is 0.05-2.0 mg / mL, and the mass concentration of poloxamer 407 is 1-20 mg / mL.

[0045] Furthermore, in step S2, the pH value of the phosphate buffer is preferably 7.2-7.6, and the pH value of the citrate buffer is preferably 5.0-6.5.

[0046] Furthermore, the pH of the phosphate buffer solution was adjusted to 7.2-7.6 using either 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution.

[0047] Furthermore, in step S3, 1.0-2.0 mL of the initial waste liquid is discarded before collecting the granulation system, and 0.5-1.5 mL of the subsequent waste liquid is discarded after collection.

[0048] Furthermore, in step S4, dialysis is performed at 4-8°C for 12-24 hours, with 4-6 fluid changes.

[0049] Furthermore, in step A3, at 40-55°C and with magnetic or mechanical stirring at 100-500 rpm, triethylamine is first added and stirred for 5-15 minutes, followed by the addition of succinic anhydride, preferably under nitrogen protection.

[0050] Further, in step A5, after the reaction is complete, tetrahydrofuran is removed under reduced pressure at 40-50℃ and an absolute pressure not exceeding 50 kPa. Ethyl acetate is added to dissolve the product to a concentration of 50-200 mg / mL. The product is then washed 2-3 times with a 5-10 wt% sodium bicarbonate aqueous solution, 2-3 times with water, and 1-2 times with a saturated sodium chloride aqueous solution. The organic phase is dried with anhydrous sodium sulfate for 30 min to 2 h and then filtered. Ethyl acetate is removed under reduced pressure at 40-50℃ and an absolute pressure not exceeding 50 kPa. Subsequently, the product is vacuum dried at 30-50℃ and an absolute pressure not exceeding 10 kPa for 4-48 h until the mass is constant. The constant mass means that the mass change is less than 0.1% when there is a 2-4 h interval between two consecutive weighings.

[0051] Furthermore, the acid value in step A4 was determined according to the method for determination of fats and fatty oils in Part IV, General Chapter 0713 of the 2025 edition of the Pharmacopoeia of the People's Republic of China.

[0052] Further, in step B3, the mixture is stirred at 40-65°C for 10 minutes to 2 hours with magnetic or mechanical stirring at a speed of 100-500 rpm until a clear solution is formed.

[0053] Further, in step B4, anhydrous ethanol is removed under reduced pressure at 25-45℃ and an absolute pressure of 5-30 kPa, and then vacuum dried at 30-60℃ and an absolute pressure not exceeding 10 kPa for 4-48 hours.

[0054] Further, in step D2, small interfering RNA is dissolved in phosphate buffer or citrate buffer to a concentration of 0.05-5 mg / mL; 1,2-dioleoyloxy-3-trimethylammonium propane chloride or the complex lipid intermediate is dissolved in anhydrous ethanol to a concentration of 1-20 mg / mL; the lipid ethanol solution is slowly added to the small interfering RNA buffer solution under vortexing or pipetting conditions, and incubated at 20-30°C by gentle vortexing or shaking for 5-30 min; after mixing, the volume is diluted or made up with the buffer so that when the obtained nucleic acid-lipid precomplex intermediate is used as the aqueous phase or a component of step S2, the small interfering RNA mass concentration is 0.05-2.0 mg / mL and the poloxamer 407 mass concentration is 1-20 mg / mL.

[0055] Furthermore, the cubic lipid nanoparticle formulation, when stored at 4°C and 25°C for 3 months, showed a particle size change of no more than ±20%, an encapsulation rate decrease of no more than 10%, and no visible precipitation or stratification.

[0056] Furthermore, the lattice type of the anti-bicontinuous cubic phase was determined by small-angle X-ray scattering characterization.

[0057] Furthermore, the state of small interfering RNA (SRNA) encapsulated in an anti-bicontinuous cubic aqueous channel was determined by quantitative characterization of the SRNA after separation.

[0058] As another aspect of this invention, the present invention employs a succinylated glycerol monooleate-1,2-dioleoyloxy-3-trimethylammonium propane chloride ion complex synergistically with glycerol monooleate to construct an anti-bicontinuous cubic phase framework. The resulting cubic lipid nanoparticles, modified with poloxamer 407, are primarily used to enhance the bioavailability and therapeutic efficacy of small interfering RNA (SRNA) delivery. Glycerol monooleate, as the main cubic phase backbone lipid, possesses unsaturated oleyl chains that impart moderate fluidity to the lipid bilayer, facilitating the formation and maintenance of the cubic phase's three-dimensional water channel network. The succinylated glycerol monooleate-1,2-dioleoyloxy-3-trimethylammonium propane chloride ion complex is anchored at the water channel interface, electrostatically attracting and immobilizing SRNA and preventing diffusion release, achieving a drug loading of 4.5-10 wt% and an encapsulation efficiency of 60-95%. The three-dimensional interconnected water channel structure of the anti-bicontinuous cubic phase provides an ideal encapsulation space for SRNA, protecting it from nuclease degradation while allowing controlled release after endocytosis due to pH decreases or changes in ionic strength. The polyethylene glycol layer formed by surface modification of poloxamer 407 prolongs the blood circulation half-life, reduces reticuloendothelial system uptake, and enhances targeted tissue enrichment. Simultaneously, the steric hindrance effect controls the absolute value of the zeta potential within 10-60 mV, avoiding blood toxicity caused by high positive charge. The cubic lipid nanoparticles exhibit long-term stability, with particle size changes not exceeding ±20% and encapsulation efficiency decreasing by no more than 10% after 3 months of storage at 4℃ and 25℃, without precipitation or delamination. This ensures stable formulation storage and transportation quality and reliable clinical application.

[0059] Succinylated glycerol monooleate and 1,2-dioleoyloxy-3-trimethylammonium propane chloride focus on charge regulation and electrostatic complexation, respectively, to synergistically construct a moderately positively charged and structurally ordered lipid complex. The negatively charged carboxyl group introduced by succinylated glycerol monooleate forms an ion pair with the quaternary ammonium positive charge of 1,2-dioleoyloxy-3-trimethylammonium propane chloride. Electrostatic neutralization reduces the surface potential from +50 to +70 mV to +10 to +60 mV, thus maintaining sufficient electrostatic attraction to capture negatively charged small interfering ribonucleic acid to achieve an encapsulation efficiency of 60-95%, while reducing the toxicity caused by high positive charge, such as complement activation and erythrocyte aggregation. The succinyl group of succinylated glycerol monooleate induces an intermolecular hydrogen bond network in lipid molecules, while the dioleoyl chain of 1,2-dioleoyloxy-3-trimethylammonium propane chloride provides hydrophobic interactions, synergistically inducing the ordered arrangement of lipid tail chains and pre-assembling into a cubic phase precursor structure in an ethanol-water mixed solvent. This lowers the nucleation and phase transition energy barriers for cubic phase self-assembly during rapid microfluidic mixing, ensuring stable formation of a Pn3m or Ia3d lattice-type antibicontinuous cubic phase with a polydispersity index of 0.1-0.4 under conditions of total lipid concentration of 10-100 mg / mL and total flow rate of 8-20 mL / min. The formation of ionic complexes imparts a suitable charge density gradient to the lipid bilayer, suppressing phase transitions caused by ethanol removal and changes in ionic strength during post-processing. This ensures that the particle size change of nanoparticles does not exceed ±20%, the encapsulation efficiency decreases by no more than 10%, and there is no precipitation or stratification when stored in physiological ionic strength buffer.

[0060] Beneficial technical effects

[0061] 1. Achieving stable low polydispersity granulation under high solids content and high throughput microfluidic conditions: By pre-forming an ionic complex with succinylated glycerol monooleate and 1,2-dioleoyloxy-3-trimethylammonium propane chloride, lipid molecules are induced to arrange themselves into a cubic phase precursor structure, significantly reducing the nucleation and phase transition energy barriers of cubic phase self-assembly during rapid microfluidic mixing. This enables the stable formation of anti-bicontinuous cubic phase lipid nanoparticles with Pn3m or Ia3d lattice type under high solids content and high throughput conditions of 10-100 mg / mL and 8-20 mL / min, while maintaining a low polydispersity coefficient of 0.1-0.4 and excellent batch-to-batch consistency with a particle size variation coefficient of less than 5%. This solves the technical bottleneck of the existing microfluidic preparation technology where the polydispersity coefficient increases significantly under high solids content conditions.

[0062] 2. Balancing high drug loading and low cytotoxicity: The negatively charged carboxyl group introduced by succinylated glycerol monooleate forms an ion pair with the positively charged quaternary ammonium chloride 1,2-dioleoyloxy-3-trimethylammonium propane chloride. The electrostatic neutralization effect allows the surface potential of the complex to be precisely controlled within a moderately positive range of +10 to +60 mV. This retains sufficient electrostatic attraction to efficiently capture negatively charged small interfering ribonucleic acid molecules and anchor them in the three-dimensional water channel network of the anti-bicontinuous cubic phase, achieving a high drug loading of 60-95% and 4.5-10 wt%. At the same time, it significantly reduces the risks of cytotoxicity and immunogenicity caused by high positive charge in blood circulation, such as complement activation, serum albumin adsorption, and erythrocyte aggregation. This overcomes the contradiction between cationic complex strength and toxicity in existing technologies.

[0063] 3. Achieving long-term colloidal stability under physiological ionic strength conditions: The hydrophilic segment of poloxamer 407 is adsorbed onto the surface of cubic nanoparticles to form a hydrophilic steric hindrance layer, effectively shielding the residual positive charge on the surface of the ionic complex and inhibiting electrostatic aggregation and van der Waals attraction between particles. At the same time, the steric hindrance effect also inhibits the irreversible phase transition from cubic to layered or hexagonal phases caused by rapid ethanol removal and drastic changes in ionic strength during post-processing dialysis or tangential flow filtration. This ensures that the nanoparticles, when stored in phosphate buffer or citrate buffer at 4℃ and 25℃ for 3 months at near physiological ionic strength, exhibit particle size changes of no more than ±20%, encapsulation efficiency decrease of no more than 10%, and no visible precipitation or stratification. This solves the stability problem of existing cationic lipid nanoparticles that are prone to aggregation and precipitation under physiological ionic strength conditions.

[0064] 4. Synergistic optimization of cubic phase structure stability and pharmacokinetic performance: The succinylated glycerol monooleate-1,2-dioleoyloxy-3-trimethylammonium propane chloride ion complex is anchored at the water channel interface and imparts a moderate charge density gradient to the lipid bilayer, stably maintaining the internal nanostructure of the antibicontinuous cubic phase with Pn3m or Ia3d lattice type. Even when introducing auxiliary lipids such as 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol to optimize in vivo pharmacokinetics and targeting performance, the cubic phase structure can still be maintained without phase transition. This overcomes the technical contradiction in the prior art of synergistically optimizing high drug loading, auxiliary lipid introduction and cubic phase structure stability, and provides an ideal nanodelivery platform for efficient delivery of small interfering RNA and gene therapy applications.

[0065] 5. Achieving Controllable Release and Enhanced Cell Transfection Efficiency: The three-dimensional interconnected water channel structure of the anti-bicontinuous cubic phase provides an ideal encapsulation space for small interfering RNA (MIRNA) and protects it from nuclease degradation. After endocytosis, the ion complex dissociates due to pH decrease or ionic strength change, thereby achieving controlled release of MIRNA. At the same time, the polyethylene glycol layer formed by poloxamer 407 surface modification prolongs the half-life of nanoparticles in blood circulation, reduces reticuloendothelial uptake, and enhances target tissue enrichment, thereby improving the bioavailability of MIRNA delivery and the therapeutic effect of target gene silencing. It has broad application value in gene therapy and nucleic acid drug delivery. Attached Figure Description

[0066] Figure 1 This is a superimposed diagram of scattering curves characterizing an anti-bicontinuous cubic phase structure using small-angle X-ray scattering.

[0067] Figure 2 This is a superimposed graph showing the absorbance variation with wavenumber for Fourier transform infrared spectroscopy characterizing the formation of ionic complexes.

[0068] Figure 3 A time series overlay plot characterizing the stability of the zeta potential over time (stored at 4°C).

[0069] Figure 4 A time series overlay plot characterizing the stability of the zeta potential over time (stored at 25°C).

[0070] Figure 5 A time series overlay plot characterizing the stability of the encapsulation efficiency curve over time (storage conditions at 4℃).

[0071] Figure 6 A time series overlay plot characterizing the stability of the encapsulation efficiency curve over time (storage conditions at 25℃).

[0072] Figure 7 This is a scanning electron microscope image of the cubic lipid nanoparticles in Example 1.

[0073] Figure 8 This is a transmission electron microscope image of the cubic lipid nanoparticles in Example 1. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Example 1

[0075] Preparation of succinylated glycerol monooleate:

[0076] 30.0 g of glyceryl monooleate was dissolved in 150 mL of tetrahydrofuran to achieve a concentration of 200 mg / mL. The mixture was stirred at 48 °C with a magnetic stirrer at 300 rpm under nitrogen protection. First, 2.43 g of triethylamine was added (the molar ratio of succinic anhydride to glyceryl monooleate was 0.15:1, and the molar ratio of triethylamine to succinic anhydride was 1.2:1), and the mixture was stirred for 10 min. Then, 1.50 g of succinic anhydride was added, and the reaction was continued at 48 °C for 12 h. After the reaction was complete, the tetrahydrofuran was removed under reduced pressure at 45 °C and 30 kPa. 150 mL of ethyl acetate was added to dissolve the product to a concentration of 100 mg / mL. The product was washed three times (150 mL each time) with a 7.5 wt% sodium bicarbonate aqueous solution, twice (150 mL each time) with water, and once (150 mL) with a saturated sodium chloride aqueous solution until the pH of the aqueous phase reached 7.0. The organic phase was dried with anhydrous sodium sulfate for 1 hour and then filtered. Ethyl acetate was removed under reduced pressure at 45°C and 30 kPa. Subsequently, it was vacuum dried at 40°C and 5 kPa for 24 hours until the mass was constant (the mass change was less than 0.1% between two consecutive weighings with a 3-hour interval), and the total residual solvent was 0.3 wt%. The acid value of the obtained succinylated glycerol monooleate was determined to be 65 mg KOH / g according to the determination of fats and fatty oils in Chapter 0713 of the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV. The residual succinic anhydride was 0.15 wt%, and the residual triethylamine was 0.08 wt%.

[0077] Preparation of complex lipid intermediates:

[0078] 0.60 g of the succinylated glycerol monooleate and 0.80 g of 1,2-dioleoyloxy-3-trimethylammonium propane chloride (molar ratio of 1,2-dioleoyloxy-3-trimethylammonium propane chloride to succinylated glycerol monooleate was 1.0:1) were weighed and added to 28 mL of anhydrous ethanol to make the total lipid concentration 50 mg / mL. The mixture was stirred at 50 °C and 200 rpm for 30 min until a clear solution was formed. The anhydrous ethanol was removed under reduced pressure at 35 °C and 15 kPa, and the mixture was then vacuum dried at 45 °C and 5 kPa for 16 h to obtain a complex lipid intermediate with 0.4 wt% residual ethanol.

[0079] Organic phase preparation:

[0080] By weight, 9.0 parts of glyceryl monooleate, 1.4 parts of the above-prepared complex lipid intermediate (including 0.8 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride and 0.6 parts of succinylated glyceryl monooleate), 1.5 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and 1.5 parts of cholesterol were weighed and dissolved in anhydrous ethanol to make the total lipid concentration in the organic phase 50 mg / mL. The mixture was stirred at 30°C for 30 min to obtain a clear organic phase. In this embodiment, the weight parts are calculated as 1 part of small interfering ribonucleic acid.

[0081] Aqueous phase preparation:

[0082] Weigh out 1.2 parts by weight of poloxamer 407 and 1 part by weight of small interfering RNA. Dissolve poloxamer 407 and small interfering RNA in phosphate buffer. In this embodiment, the phosphate buffer is prepared with water and consists of 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate and 1.8 mmol / L potassium dihydrogen phosphate. Adjust the pH to 7.4 with 1 mol / L hydrochloric acid to obtain the aqueous phase. The mass concentration of small interfering RNA is 0.5 mg / mL and the mass concentration of poloxamer 407 is 10 mg / mL.

[0083] Microfluidic mixing and granulation:

[0084] The clarified organic phase and aqueous phase obtained above are respectively introduced into a microfluidic mixing chip, wherein the flow rate ratio of the aqueous phase to the organic phase in this embodiment is 3:1, and the total flow rate is 12 mL / min, so that they are mixed and self-assembled into particles in the microfluidic mixing chip of this embodiment. Before collecting the granulation system of this embodiment, 1.5 mL of the first waste liquid is discarded, and after collection, 1.0 mL of the second waste liquid is discarded, to obtain a granulation system containing cubic lipid nanoparticles encapsulating the small interfering ribonucleic acid of this embodiment.

[0085] Post-processing:

[0086] The granulation system obtained above was dialyzed using a dialysis bag with a molecular weight cutoff of 10 kDa at 4°C for 18 h with 5 buffer changes. The volume of phosphate buffer (with the same composition as above, pH 7.4) used for each buffer change was 100 times the volume of the granulation system in this embodiment. The ethanol content after the step was determined to be 0.6 wt% by gas chromatography, thus obtaining a cubic lipid nanoparticle formulation.

[0087] Product characteristics:

[0088] The cubic lipid nanoparticles of this embodiment possess an internal nanostructure of an anti-bicontinuous cubic phase. Small-angle X-ray scattering (SAXS) characterization confirmed that the anti-bicontinuous cubic phase of this embodiment is of the Pn3m lattice type. Small interfering ribonucleic acid (SIRNA) is encapsulated within the water channels of the anti-bicontinuous cubic phase. Furthermore, succinylated glycerol monooleate and 1,2-dioleoyloxy-3-trimethylammonium propane chloride form an ionic complex. The Z-average particle size of the cubic lipid nanoparticles of this embodiment, determined by dynamic light scattering, is 180 nm; the polydispersity index (PDI), determined by dynamic light scattering, is 0.22; and the Zeta potential, determined by electrophoretic light scattering, is +35 mV. The encapsulation efficiency of the small interfering ribonucleic acid (SRNA) in this embodiment was 82% after separating the free SRNA by high-speed centrifugation and measured by ultraviolet spectrophotometry at a detection wavelength of 260 nm. The drug loading was 6.5 wt%, where the drug loading in this embodiment is the percentage of the mass of the SRNA in the cubic lipid nanoparticles to the total dry weight of the cubic lipid nanoparticles. After storage at 4°C and 25°C for 3 months, the particle size of the cubic lipid nanoparticle formulation in this embodiment changed by +8%, the encapsulation efficiency decreased to 6%, and no visible precipitation or stratification was observed.

[0089] Features of the Implementation Example:

[0090] This embodiment employs a moderately proportioned component design, using 9.0 parts of glyceryl monooleate, 0.8 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride, 0.6 parts of succinylated glyceryl monooleate, and 1.2 parts of poloxamer 407, with 1.5 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and 1.5 parts of cholesterol added as auxiliary lipids. A complex lipid intermediate preparation route is used, with the acid value of succinylated glyceryl monooleate controlled at 65 mg KOH / g. The molar ratio of 1,2-dioleoyloxy-3-trimethylammonium propane chloride to succinylated glyceryl monooleate in the complex lipid intermediate is 1.0:1. The total lipid concentration in the organic phase is 50 mg / mL, the total microfluidic flow rate is 12 mL / min, and a phosphate buffer pH is used. 7.4 As the aqueous phase and dialysis medium, the dialysis conditions were 4℃, 18h, and 5 medium changes. The resulting cubic lipid nanoparticles exhibited an anti-bicontinuous cubic phase structure of Pn3m lattice type, with a particle size of 180nm, a polydispersity index of 0.22, a Zeta potential of +35mV, an encapsulation efficiency of 82%, and a drug loading of 6.5wt%, demonstrating good stability and moderate drug loading performance. This embodiment is suitable for gene therapy applications requiring stable and efficient delivery of small interfering RNA to cells, and is particularly suitable for tumor targeted therapy, gene intervention for inflammation-related diseases, and the development of commercial formulations requiring long-term storage stability. Example 2

[0091] Preparation of succinylated glycerol monooleate:

[0092] 25.0 g of glyceryl monooleate was dissolved in 119 mL of tetrahydrofuran to achieve a concentration of 210 mg / mL. The mixture was stirred at 52 °C and 400 rpm under nitrogen protection. First, 2.84 g of triethylamine was added (the molar ratio of succinic anhydride to glyceryl monooleate was 0.24:1, and the molar ratio of triethylamine to succinic anhydride was 1.4:1), and the mixture was stirred for 12 min. Then, 2.40 g of succinic anhydride was added, and the reaction was continued at 52 °C for 16 h. After the reaction was complete, the tetrahydrofuran was removed under reduced pressure at 48 °C and 40 kPa. 125 mL of ethyl acetate was added to dissolve the product to a concentration of 120 mg / mL. The product was washed three times (125 mL each time) with 9 wt% sodium bicarbonate aqueous solution, three times (125 mL each time) with water, and twice (100 mL each time) with saturated sodium chloride aqueous solution until the pH of the aqueous phase reached 7.2. The organic phase was dried with anhydrous sodium sulfate for 45 min and then filtered. Ethyl acetate was removed under reduced pressure at 48 °C and 40 kPa. Subsequently, it was vacuum dried at 45 °C and 8 kPa for 20 h until the mass was constant (the mass change was less than 0.1% between two consecutive weighings with a 2-h interval), and the total residual solvent was 0.5 wt%. The acid value of the obtained succinylated glycerol monooleate was determined to be 90 mg KOH / g according to the determination method for fats and fatty oils in Chapter 0713 of the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV. The residual succinic anhydride was 0.25 wt%, and the residual triethylamine was 0.12 wt%.

[0093] Organic phase preparation:

[0094] Weigh out 8.7 parts by weight of glyceryl monooleate, 1.0 part of the succinylated glyceryl monooleate prepared above, 1.2 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride, and 0.5 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine. Dissolve them in anhydrous ethanol to make the total lipid concentration in the organic phase 30 mg / mL. Stir at 40°C for 20 min to obtain a clear organic phase. In this embodiment, the mass parts are calculated as 1 part of small interfering ribonucleic acid.

[0095] Aqueous phase preparation:

[0096] Weigh out 1.0 part of poloxamer 407 and 1 part of small interfering RNA by weight. Dissolve poloxamer 407 and small interfering RNA in citrate buffer. The citrate buffer in this embodiment is composed of 25 mmol / L citric acid and 35 mmol / L sodium citrate dihydrate and prepared with water. Adjust the pH to 5.5 with 1 mol / L sodium hydroxide solution to obtain the aqueous phase. The mass concentration of small interfering RNA is 1.0 mg / mL and the mass concentration of poloxamer 407 is 5 mg / mL.

[0097] Microfluidic mixing and granulation:

[0098] The clarified organic phase and aqueous phase obtained above are respectively introduced into a microfluidic mixing chip, wherein the flow rate ratio of the aqueous phase to the organic phase in this embodiment is 3:1, and the total flow rate is 15 mL / min, so that they are mixed and self-assembled into particles in the microfluidic mixing chip of this embodiment. Before collecting the granulation system of this embodiment, 1.2 mL of the first waste liquid is discarded, and after collection, 0.8 mL of the second waste liquid is discarded, resulting in a granulation system containing cubic lipid nanoparticles encapsulating the small interfering ribonucleic acid of this embodiment.

[0099] Post-processing:

[0100] The granulation system obtained above was subjected to tangential flow filtration using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. The volume was replaced 7 times at 15°C. The citrate buffer used had the same composition as above and a pH of 5.5. The ethanol content after the step was determined to be 0.8 wt% by gas chromatography, thus obtaining a cubic lipid nanoparticle formulation.

[0101] Product characteristics:

[0102] The cubic lipid nanoparticles of this embodiment possess an anti-bicontinuous cubic phase internal nanostructure. Small-angle X-ray scattering (SAXS) characterization confirmed that the anti-bicontinuous cubic phase of this embodiment is of the Ia3d lattice type. The small interfering RNA (SRNA) of this embodiment is encapsulated within the water channels of the anti-bicontinuous cubic phase. Furthermore, the succinylated glycerol monooleate and the 1,2-dioleoyloxy-3-trimethylammonium propane chloride of this embodiment form an ionic complex. The Z-average particle size of the cubic lipid nanoparticles of this embodiment, determined by dynamic light scattering, is 220 nm. The polydispersity index (PDI) is 0.28, determined by dynamic light scattering, and the Zeta potential is +48 mV, determined by electrophoretic light scattering. The encapsulation efficiency of the SRNA in this embodiment, after separation of free SRNA by gel column chromatography, is 88%, determined by fluorescence spectroscopy. The drug loading is 7.2 wt%, where the drug loading is the percentage of the mass of the SRNA in the cubic lipid nanoparticles of this embodiment relative to the total dry weight of the cubic lipid nanoparticles. The cubic lipid nanoparticle formulation of this embodiment was stored at 4°C and 25°C for 3 months. The particle size changed by +12%, the encapsulation efficiency decreased to 7%, and there was no visible precipitation or stratification.

[0103] Features of the Implementation Example:

[0104] This embodiment employs a formulation design with a high cationic lipid content. The amounts of glycerol monooleate are 8.7 parts, 1,2-dioleoyloxy-3-trimethylammonium propane chloride is 1.2 parts, succinylated glycerol monooleate is 1.0 part, and poloxamer 407 is 1.0 part. Only a small amount of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (0.5 parts) is added, and no cholesterol is added. Instead of using a pre-preparation route with complex lipid intermediates, the lipid components are directly mixed. The acid value of succinylated glycerol monooleate is controlled at 90 mg KOH / g. The total lipid concentration in the organic phase is 30 mg / mL, the concentration of small interfering ribonucleic acid in the aqueous phase is 1.0 mg / mL, the total microfluidic flow rate is 15 mL / min, and a citrate buffer pH is used. 5.5 was used as both the aqueous phase and tangential flow filtration medium. The tangential flow filtration conditions were 15°C and a 7-fold volume displacement. The resulting cubic lipid nanoparticles exhibited an anti-bicontinuous cubic phase structure of the Ia3d lattice type, with a particle size of 220 nm, a polydispersity index of 0.28, a Zeta potential of +48 mV, an encapsulation efficiency of 88%, and a drug loading of 7.2 wt%. The high cationic lipid content endowed the nanoparticles with stronger positive charge and higher encapsulation efficiency. This embodiment is suitable for gene delivery applications that require enhanced cellular uptake efficiency and endosome escape ability, and is particularly suitable for gene silencing studies in difficult-to-transfect cell lines, in vitro gene editing experiments, and rapid transfection scenarios requiring strongly positively charged cell membrane fusion. Example 3

[0105] Preparation of succinylated glycerol monooleate:

[0106] 20.0 g of glyceryl monooleate was dissolved in 182 mL of tetrahydrofuran to achieve a concentration of 110 mg / mL. The mixture was stirred at 44 °C and 250 rpm under nitrogen protection. First, 0.79 g of triethylamine was added (the molar ratio of succinic anhydride to glyceryl monooleate was 0.09:1, and the molar ratio of triethylamine to succinic anhydride was 1.1:1), and the mixture was stirred for 8 min. Then, 0.90 g of succinic anhydride was added, and the reaction was continued at 44 °C for 9 h. After the reaction was complete, the tetrahydrofuran was removed under reduced pressure at 42 °C and 35 kPa. 200 mL of ethyl acetate was added to dissolve the product to a concentration of 80 mg / mL. The product was washed twice (200 mL each time) with a 6 wt% sodium bicarbonate aqueous solution, twice (200 mL each time) with water, and once (200 mL) with a saturated sodium chloride aqueous solution until the pH of the aqueous phase reached 6.8. The organic phase was dried with anhydrous sodium sulfate for 1.5 h and then filtered. Ethyl acetate was removed under reduced pressure at 42 °C and 35 kPa. Subsequently, it was vacuum dried at 35 °C and 6 kPa for 30 h until the mass was constant (the mass change was less than 0.1% between two consecutive weighings with a 4-h interval), and the total residual solvent was 0.4 wt%. The acid value of the obtained succinylated glycerol monooleate was determined to be 40 mg KOH / g according to the Determination of Fat and Fatty Oils in Chapter 0713 of the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV. The residual succinic anhydride was 0.10 wt%, and the residual triethylamine was 0.05 wt%.

[0107] Preparation of nucleic acid-lipid precomplex intermediates:

[0108] Small interfering RNA (SRNA) was dissolved in phosphate buffer (prepared with water, consisting of 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate, and 1.8 mmol / L potassium dihydrogen phosphate, and adjusted to pH 7.2 with 1 mol / L hydrochloric acid) to a concentration of 2.0 mg / mL; 1,2-dioleoyloxy-3-trimethylammonium propane chloride was dissolved in anhydrous ethanol to a concentration of 10 mg / mL; 1,2-dioleoyloxy-3-trimethylammonium propane chloride was then dissolved under vortex mixing conditions. - The trimethylammonium propane chloride ethanol solution was slowly added to the small interfering ribonucleic acid buffer solution and incubated by gentle vortexing at 25°C for 20 min. Based on the mass of 1,2-dioleoyloxy-3-trimethylammonium propane chloride in this embodiment of the nucleic acid-lipid precomplex intermediate, its mass ratio with the small interfering ribonucleic acid in this embodiment was 0.3:1. After mixing, the volume was diluted with phosphate buffer to obtain the nucleic acid-lipid precomplex intermediate of this embodiment, which was clear and translucent with no visible precipitate.

[0109] Organic phase preparation:

[0110] Weigh out 9.5 parts by weight of glyceryl monooleate, 0.2 parts by weight of the succinylated glyceryl monooleate prepared above, 2.5 parts by weight of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and 0.8 parts by weight of cholesterol. Dissolve them in anhydrous ethanol to make the total lipid concentration in the organic phase 70 mg / mL. Stir at 25°C for 45 min to obtain a clear organic phase. In this embodiment, the mass parts are calculated as 1 part of small interfering ribonucleic acid.

[0111] Aqueous phase preparation:

[0112] Weigh 1.4 parts of poloxamer 407 by weight, dissolve poloxamer 407 in the nucleic acid-lipid precomplex intermediate solution prepared above, so that the final mass concentration of small interfering ribonucleic acid is 0.2 mg / mL and the mass concentration of poloxamer 407 is 15 mg / mL, and obtain an aqueous phase.

[0113] Microfluidic mixing and granulation:

[0114] The clarified organic phase and aqueous phase obtained above are respectively introduced into a microfluidic mixing chip, wherein the flow rate ratio of the aqueous phase to the organic phase in this embodiment is 3:1, and the total flow rate is 10 mL / min, so that they are mixed and self-assembled into particles in the microfluidic mixing chip of this embodiment. Before collecting the granulation system of this embodiment, 1.8 mL of the first waste liquid is discarded, and after collection, 1.2 mL of the second waste liquid is discarded, resulting in a granulation system containing cubic lipid nanoparticles encapsulating the small interfering ribonucleic acid of this embodiment.

[0115] Post-processing:

[0116] The granulation system obtained above was dialyzed using a dialysis bag with a molecular weight cutoff of 7 kDa at 8°C for 14 h with four buffer changes. The volume of phosphate buffer (composed of 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate and 1.8 mmol / L potassium dihydrogen phosphate, prepared with water, pH 7.2) used for each buffer change was 120 times the volume of the granulation system in this embodiment. The ethanol content after the step was determined to be 0.7 wt% by gas chromatography, resulting in a cubic lipid nanoparticle formulation.

[0117] Product characteristics:

[0118] The cubic lipid nanoparticles of this embodiment possess an internal nanostructure of an anti-bicontinuous cubic phase. Small-angle X-ray scattering (SAXS) characterization confirmed that the anti-bicontinuous cubic phase of this embodiment is of the Pn3m lattice type. Small interfering ribonucleic acid (SRNA) is encapsulated within the water channels of the anti-bicontinuous cubic phase of this embodiment, as confirmed by nucleic acid quantification after separation of the free SRNA. Furthermore, succinylated glycerol monooleate and 1,2-dioleoyloxy-3-trimethylammonium propane chloride of this embodiment form an ionic complex. The Z-average particle size of the cubic lipid nanoparticles of this embodiment, determined by dynamic light scattering, is 150 nm; the polydispersity index (PDI), determined by dynamic light scattering, is 0.18; and the Zeta potential, determined by electrophoretic light scattering, is +22 mV. The encapsulation efficiency of the small interfering ribonucleic acid (SRNA) in this embodiment was 75% after separating the free SRNA by high-speed centrifugation and measured by ultraviolet spectrophotometry at a detection wavelength of 260 nm. The drug loading was 5.8 wt%, where the drug loading in this embodiment is the percentage of the mass of the SRNA in the cubic lipid nanoparticles to the total dry weight of the cubic lipid nanoparticles. After storage at 4°C and 25°C for 3 months, the particle size of the cubic lipid nanoparticle formulation in this embodiment changed by +5%, the encapsulation efficiency decreased by 4%, and no visible precipitation or stratification was observed.

[0119] Features of the Implementation Example:

[0120] This embodiment employs a design with a low cationic lipid content and a high auxiliary lipid content. The amounts are: 9.5 parts glyceryl monooleate, 0.3 parts 1,2-dioleoyloxy-3-trimethylammonium propane chloride, 0.2 parts succinylated glyceryl monooleate, and 1.4 parts poloxamer 407. A significant amount of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (2.5 parts) and a suitable amount of cholesterol (0.8 parts) are added. A nucleic acid-lipid precomplex intermediate preparation pathway is used. First, 1,2-dioleoyloxy-3-trimethylammonium propane chloride and small interfering ribonucleic acid (SMERNA) are incubated at 25°C for 20 min at a mass ratio of 0.3:1 to form a precomplex. The acid value of succinylated glyceryl monooleate is controlled at 40 mg KOH / g. The total lipid concentration in the organic phase is 70 mg / mL, the SMERNA concentration in the aqueous phase is 0.2 mg / mL, the total microfluidic flow rate is 10 mL / min, and the pH of the phosphate buffer is controlled. 7.2 As a pre-compounding and dialysis medium, the dialysis conditions were 8℃, 14h, and 4 media changes. The resulting cubic lipid nanoparticles exhibited an anti-bicontinuous cubic phase structure of Pn3m lattice type, with a particle size of 150nm, a polydispersity index of 0.18, a Zeta potential of +22mV, an encapsulation efficiency of 75%, and a drug loading of 5.8wt%. The low cationic lipid content and high auxiliary lipid content gave the nanoparticles a small particle size, narrow particle size distribution, and mild positive charge. This embodiment is suitable for gene delivery applications that require reduced cytotoxicity and improved biocompatibility, and is particularly suitable for in vivo injection, long-circulating blood delivery, transfection of cell lines sensitive to electrostatic effects, and therapeutic nucleic acid delivery scenarios that require fine control of endosome escape rates. Example 4

[0121] Preparation of succinylated glycerol monooleate:

[0122] 35.0 g of glyceryl monooleate was dissolved in 130 mL of tetrahydrofuran to achieve a concentration of 270 mg / mL. The mixture was stirred at 54 °C and 450 rpm under nitrogen protection. First, 1.44 g of triethylamine was added (the molar ratio of succinic anhydride to glyceryl monooleate was 0.07:1, and the molar ratio of triethylamine to succinic anhydride was 1.45:1), and the mixture was stirred for 13 min. Then, 0.99 g of succinic anhydride was added, and the reaction was continued at 54 °C for 18 h. After the reaction was complete, the tetrahydrofuran was removed under reduced pressure at 50 °C and 45 kPa. 175 mL of ethyl acetate was added to dissolve the product to a concentration of 150 mg / mL. The product was washed twice (175 mL each time) with a 5.5 wt% sodium bicarbonate aqueous solution, three times (175 mL each time) with water, and once (175 mL) with a saturated sodium chloride aqueous solution until the pH of the aqueous phase reached 6.5. The organic phase was dried with anhydrous sodium sulfate for 40 min and then filtered. Ethyl acetate was removed under reduced pressure at 50 °C and 45 kPa. Subsequently, it was vacuum dried at 50 °C and 10 kPa for 36 h until the mass was constant (the mass change was less than 0.1% between two consecutive weighings with a 4-h interval), and the total residual solvent was 0.6 wt%. The acid value of the obtained succinylated glycerol monooleate was determined to be 25 mg KOH / g according to the determination of fats and fatty oils in Chapter 0713 of the 2025 edition of the Pharmacopoeia of the People's Republic of China, Part IV. The residual succinic anhydride was 0.20 wt%, and the residual triethylamine was 0.10 wt%.

[0123] Preparation of complex lipid intermediates:

[0124] 0.80 g of the succinylated glycerol monooleate and 1.30 g of 1,2-dioleoyloxy-3-trimethylammonium propane chloride (molar ratio of 1,2-dioleoyloxy-3-trimethylammonium propane chloride to succinylated glycerol monooleate was 1.3:1) were weighed and added to 24.7 mL of anhydrous ethanol to make the total lipid concentration 85 mg / mL. The mixture was stirred at 60 °C and 350 rpm for 1 h until a clear solution was formed. The anhydrous ethanol was removed under reduced pressure at 40 °C and 20 kPa, and the mixture was then vacuum dried at 55 °C and 8 kPa for 12 h to obtain a complex lipid intermediate with 0.7 wt% residual ethanol.

[0125] Organic phase preparation:

[0126] By weight, 8.6 parts of glyceryl monooleate, 2.1 parts of the above-prepared complex lipid intermediate (including 1.3 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride and 0.8 parts of succinylated glyceryl monooleate), 2.7 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, and 0.3 parts of cholesterol were weighed and dissolved in anhydrous ethanol to make the total lipid concentration in the organic phase 90 mg / mL. The mixture was stirred at 42°C for 15 min to obtain a clear organic phase. In this embodiment, the weight parts are calculated as 1 part of small interfering ribonucleic acid.

[0127] Aqueous phase preparation:

[0128] Weigh out 1.5 parts by weight of poloxamer 407 and 1 part by weight of small interfering RNA. Dissolve poloxamer 407 and small interfering RNA in citrate buffer. In this embodiment, the citrate buffer is prepared by mixing 15 mmol / L citric acid and 45 mmol / L sodium citrate dihydrate with water. Adjust the pH to 6.0 with 1 mol / L hydrochloric acid to obtain the aqueous phase. The mass concentration of small interfering RNA is 1.8 mg / mL and the mass concentration of poloxamer 407 is 18 mg / mL.

[0129] Microfluidic mixing and granulation:

[0130] The clarified organic phase and aqueous phase obtained above are respectively introduced into a microfluidic mixing chip, wherein the flow rate ratio of the aqueous phase to the organic phase in this embodiment is 3:1, and the total flow rate is 18 mL / min, so that they are mixed and self-assembled into particles in the microfluidic mixing chip of this embodiment. Before collecting the granulation system of this embodiment, 1.0 mL of the first waste liquid is discarded, and after collection, 0.5 mL of the second waste liquid is discarded, resulting in a granulation system containing cubic lipid nanoparticles encapsulating the small interfering ribonucleic acid of this embodiment.

[0131] Post-processing:

[0132] The granulation system obtained above was subjected to tangential flow filtration using an ultrafiltration membrane with a molecular weight cutoff of 80 kDa. The volume was replaced 6 times at 10°C. The citrate buffer used had the same composition as above and a pH of 6.0. The ethanol content after the step was determined to be 0.9 wt% by gas chromatography, thus obtaining a cubic lipid nanoparticle formulation.

[0133] Product characteristics:

[0134] The cubic lipid nanoparticles of this embodiment possess an internal nanostructure of an anti-bicontinuous cubic phase. Small-angle X-ray scattering (SAXS) characterization confirmed that the anti-bicontinuous cubic phase of this embodiment is of the Ia3d lattice type. The small interfering ribonucleic acid (SRNA) of this embodiment is encapsulated within the water channels of the anti-bicontinuous cubic phase. Furthermore, the succinylated glycerol monooleate and the 1,2-dioleoyloxy-3-trimethylammonium propane chloride of this embodiment form an ionic complex. The Z-average particle size of the cubic lipid nanoparticles of this embodiment, determined by dynamic light scattering, is 280 nm. The polydispersity index (PDI), determined by dynamic light scattering, is 0.35. The Zeta potential, determined by electrophoretic light scattering, is +52 mV. The encapsulation efficiency of the SRNA in this embodiment, after separation of free SRNA by gel column chromatography, is 90% as determined by fluorescence spectroscopy. The drug loading is 8.5 wt%, where the drug loading is the percentage of the mass of the SRNA in the cubic lipid nanoparticles of this embodiment relative to the total dry weight of the cubic lipid nanoparticles. The cubic lipid nanoparticle formulation of this embodiment was stored at 4°C and 25°C for 3 months. The particle size changed by +16%, the encapsulation efficiency decreased to 9%, and there was no visible precipitation or stratification.

[0135] Features of the Implementation Example:

[0136] This embodiment employs a specific component design with the following proportions: 8.6 parts of glyceryl monooleate, 1.3 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride, 0.8 parts of succinylated glyceryl monooleate, and 1.5 parts of poloxamer 407, along with 2.7 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and 0.3 parts of cholesterol. A complex lipid intermediate preparation route is used, with the acid value of succinylated glyceryl monooleate controlled at 25 mg KOH / g. The molar ratio of 1,2-dioleoyloxy-3-trimethylammonium propane chloride to succinylated glyceryl monooleate in the complex lipid intermediate is 1.3:1. The preparation temperature of the complex lipid intermediate is 6... The concentration of total lipids in the organic phase was 90 mg / mL, the concentration of small interfering RNA in the aqueous phase was 1.8 mg / mL, the concentration of poloxamer 407 was 18 mg / mL, and the total flow rate of the microfluidic system was 18 mL / min. Citrate buffer (pH 6.0) was used as both the aqueous phase and the tangential flow filtration medium. Tangential flow filtration employed an 80 kDa ultrafiltration membrane at 10°C and a 6-fold volume displacement. The resulting cubic lipid nanoparticles exhibited an anti-bicontinuous cubic phase structure with a Ia3d lattice, a particle size of 280 nm, a polydispersity index of 0.35, a Zeta potential of +52 mV, an encapsulation efficiency of 90%, and a drug loading of 8.5 wt%, demonstrating high drug loading and high encapsulation efficiency. This embodiment is suitable for small interfering RNA delivery applications requiring high drug loading and high encapsulation efficiency, particularly for dose-dependent gene therapy, cell-targeted delivery requiring strong cationic charge-mediated delivery, and the industrial production of high-concentration nucleic acid preparations.

[0137] Comparative Example 1: It is basically the same as Example 1, except that the amount of glyceryl monooleate is 8.2 parts, while the amount of other components and preparation conditions remain unchanged.

[0138] Comparative Example 2: It is basically the same as Example 1, except that the amount of glyceryl monooleate is 10.2 parts, while the amount of other components and preparation conditions remain unchanged.

[0139] Comparative Example 3: It is basically the same as Example 1, except that the amount of 1,2-dioleoyloxy-3-trimethylammonium propane chloride is 0.15 parts, while the amounts of other components and preparation conditions remain unchanged.

[0140] Comparative Example 4: It is basically the same as Example 1, except that the amount of 1,2-dioleoyloxy-3-trimethylammonium propane chloride is 1.8 parts, while the amounts of other components and preparation conditions remain unchanged.

[0141] Comparative Example 5: It is basically the same as Example 1, except that succinylated glycerol monooleate is not used. Instead, 1,2-dioleoyloxy-3-trimethylammonium propane chloride is directly mixed with glycerol monooleate to prepare the organic phase. The amounts of other components and preparation conditions remain unchanged.

[0142] Comparative Example 6: It is basically the same as Example 1, except that the amount of succinylated glycerol monooleate is 0.03 parts, while the amounts of other components and preparation conditions remain unchanged.

[0143] Comparative Example 7: Basically the same as Example 1, except that the amount of succinylated glycerol monooleate was 1.8 parts, while the amounts of other components and preparation conditions remained unchanged.

[0144] Comparative Example 8: It is basically the same as Example 1, except that the total flow rate of the aqueous phase and the organic phase during microfluidic mixing is 6 mL / min, while the amount of other components and the preparation conditions remain unchanged.

[0145] Performance testing:

[0146] Experiment 1: Determination of Particle Size Distribution and Polydispersity Index

[0147] Test Subject: Particle size distribution and polydispersity index (PDI) of cubic lipid nanoparticles encapsulating small interfering ribonucleic acid (SIRNA). Test Objective: To evaluate the particle size uniformity and batch-to-batch consistency of nanoparticles, and to verify low polydispersity under microfluidic high-throughput preparation conditions. Test Principle: Based on the principle of dynamic light scattering, the Z-average particle size and PDI are obtained by detecting the intensity fluctuations of scattered light caused by the Brownian motion of nanoparticles and fitting an autocorrelation function using the cumulant method. Experimental Method: Samples were diluted with phosphate buffer to a suitable scattering intensity and measured using a dynamic light scattering instrument at 25℃, with a scattering angle of 173° and a laser wavelength of 633nm. Each sample was measured in triplicate. Key Parameters: Temperature 25±0.5℃, sample concentration adjusted to a scattering intensity of 5×10⁻⁶. 4 -1×10 6 CPS measurement time is automatically optimized. Data processing: The arithmetic mean ± standard deviation of three measurements is used to evaluate whether the particle size is within the range of 50-500 nm and PDI ≤ 0.4.

[0148] Experiment 2: Zeta potential measurement

[0149] Test Subject: Zeta potential of cubic lipid nanoparticles. Test Objective: To evaluate the surface charge characteristics of nanoparticles and verify the balance between cationic recombination strength and colloidal electrostatic stability. Test Principle: Based on the principle of electrophoretic light scattering, charged particles migrate directionally under an applied electric field, generating a Doppler frequency shift. Electrophoretic mobility is calculated using phase analysis light scattering and converted into Zeta potential. Experimental Method: Samples were diluted to appropriate concentrations with phosphate buffer or citrate buffer and measured using an electrophoretic light scattering instrument at 25℃. The electric field strength was automatically optimized. Each sample was measured in triplicate. Key parameters: temperature 25±0.5℃, ionic strength of the medium consistent with the formulation buffer (PBS or citrate buffer), pH 7.2-7.6 or 5.0-6.5. Data Processing: The arithmetic mean ± standard deviation of the three measurements was used to evaluate whether the absolute value of the Zeta potential was within the range of 10-60 mV.

[0150] Experiment 3: Determination of Encapsulation Efficiency of Small Interference Ribonucleic Acid

[0151] Test Subject: Encapsulation efficiency of small interfering ribonucleic acid (SIRNA). Test Objective: To evaluate the encapsulation capacity of cubic lipid nanoparticles for nucleic acids and verify the effectiveness of ion complex design. Test Principle: Free SIRNA is separated by high-speed centrifugation or gel column chromatography. The concentration of free nucleic acid in the supernatant or eluent is measured, and the encapsulation efficiency is calculated as (total nucleic acid content - free nucleic acid content) / total nucleic acid content × 100%. Experimental Methods: Method 1: Take 100 μL of sample, centrifuge at 15000g for 30 min (4℃), and measure the free nucleic acid concentration in the supernatant. Method 2: Use Sephadex G-50 gel column chromatography, collect the eluent, and measure the free nucleic acid concentration. Nucleic acid concentration is determined by ultraviolet spectrophotometry (wavelength 260 nm) or fluorescence method. Key Parameters: High-speed centrifugation temperature 4℃, centrifugation force 15000g, time 30 min; ultraviolet detection wavelength 260 nm. Data processing: Calculate the encapsulation rate = (Ctotal × Vtotal - Cupperclassified × Vupperclassified) / (Ctotal × Vtotal) × 100%, n≥3, and take the mean ± standard deviation.

[0152] Experiment 4: Characterization of the internal structure of the anti-bicontinuous cubic phase

[0153] Test Subject: The internal structure of the anti-bicontinuous cubic phase of cubic lipid nanoparticles. Test Objective: To verify cubic phase structures of Pn3m or Ia3d lattice types and evaluate the stability of the phase structure under conditions of high drug loading and the presence of co-existing lipids. Test Principle: Based on the principle of small-angle X-ray scattering, the lattice type is determined by measuring the Bragg diffraction peak position ratio of the ordered cubic phase structure and applying lattice system rules. Experimental Method: Concentrated samples were placed in a capillary sample cell and measured using a small-angle X-ray scattering instrument with Cu Kα radiation (λ=0.154nm), a scanning range of q=0.1-3.0nm⁻¹, a temperature of 25℃, and an exposure time optimized according to the sample concentration. Scattering curves were recorded, characteristic peak positions were calibrated, and the peak position ratio √(q1²:q2²:q3²:...) was calculated. Standard Basis: References were made to the Luzzati crystallographic method and related literature regarding the criteria for determining cubic phase structures. Key parameters: temperature 25±0.5℃, sample concentration ≥20mg / mL (total lipids), X-ray wavelength 0.154nm, q resolution ≤0.01nm⁻¹.

[0154] Experiment 5: Long-term storage stability evaluation

[0155] Test Subject: Storage stability of cubic lipid nanoparticle formulations. Test Objective: To evaluate the colloidal stability and nucleic acid integrity during long-term storage at 4℃ and 25℃, verifying the stability advantages of the formulation design. Test Principle: To investigate the time-dependent changes in particle size, encapsulation efficiency, and appearance during storage, and to evaluate stability through multi-time-point sampling. Experimental Method: The formulation was aliquoted into sealed containers and stored in a 4℃ refrigerator and a 25℃ incubator, respectively. Samples were taken at day 0, month 1, month 2, and month 3 to determine Z-average particle size (dynamic light scattering method), encapsulation efficiency (high-speed centrifugation-UV method), and appearance (visual inspection for precipitation or layering). Key Parameters: Storage temperature 4±2℃ and 25±2℃, relative humidity 60±10%RH, protected from light and sealed, sampling volume ≥200μL. Data processing: Calculate the particle size change rate = (D3 months - D0 days) / D0 days × 100%, the encapsulation rate decrease value = EE0 days - EE3 months, and the judgment criteria are particle size change ≤ ±20%, encapsulation rate decrease ≤ 10%, and no visible sediment.

[0156] Experiment 6: In vitro gene silencing efficiency determination

[0157] Test Subject: In vitro gene silencing efficiency mediated by cubic lipid nanoparticles. Test Objective: To evaluate the functionality of nanoparticle delivery of small interfering RNA (sRNA) to achieve target gene silencing and to verify the delivery effectiveness of the formulation design. Test Principle: Through cell transfection experiments, the mRNA or protein expression level of the target gene is detected using quantitative real-time PCR or Western blot. The gene silencing rate is calculated as (expression level of control group - expression level of experimental group) / expression level of control group × 100%. Experimental Method: Cells (e.g., HeLa, HEK293T) are seeded in 96-well plates. When the cell density reaches 60-80% confluence, culture medium containing cubic lipid nanoparticles (final concentration 50-200 nM siRNA) is added. After transfection for 24-72 h, total RNA or total protein is extracted, and the target gene expression level is detected using qRT-PCR or Western blot. Key Parameters: Cell density 60-80%, transfection concentration 50-200 nM siRNA, transfection time 24-72 h, serum-free or low-serum culture medium. Data processing: Using the untransfected group as the control, the gene silencing rate was calculated, n≥3, and the mean ± standard deviation was taken. A silencing rate ≥50% was considered valid.

[0158] Figure 1 This is a superimposed image of small-angle X-ray scattering (SAXS) curves characterizing the anti-bicontinuous cubic phase structure. Fixed parameters included: microfluidic mixing for sample preparation with an aqueous-to-organic phase flow rate ratio of 3:1, a total flow rate of 12 mL / min, a total lipid concentration of 50 mg / mL, and post-treatment using a 10 kDa dialysis bag at 4 °C for 18 h with 5 fluid changes, resulting in an ethanol content of 0.6 wt% after dialysis. Variations were determined by whether succinylated glycerol monooleate was introduced into the formulation (Example 1 and Comparative Example 5). Example 1 showed multi-level Bragg diffraction peaks with peak ratios consistent with the characteristic sequence of the Pn3m anti-bicontinuous cubic phase, indicating a stable internal ordered cubic phase structure. Comparative Example 5, lacking succinylated glycerol monooleate, exhibited significantly weakened characteristic peaks that transformed into an approximate 1:2:3 peak sequence or multiphase characteristics of a layered phase. This demonstrates that the ion composite design effectively maintains the internal nanostructure of the cubic phase and reduces the risk of post-treatment-induced phase transitions, validating the scheme's correctness from a structural evidence perspective.

[0159] Figure 2This is a superimposed graph of absorbance versus wavenumber for Fourier transform infrared spectroscopy characterizing the formation of ionic complexes. The fixed parameter is that the detection wavenumber range covers 900–1800 cm⁻¹, with a focus on the 900–1000 cm⁻¹ and 1300–1700 cm⁻¹ intervals. The lipid system of the sample contains glycerol monooleate and cationic lipid 1,2-dioleoyloxy-3-trimethylammonium propane chloride, and the rest of the preparation process is the same. The variable parameter is the absence or low amount of succinylated glycerol monooleate, i.e., Example 1, Comparative Example 5 and Comparative Example 6. Example 1 exhibits characteristic absorptions more consistent with the ionization state in the symmetric and antisymmetric stretching vibration range of carboxylate, accompanied by a consistent peak shift. At the same time, the intensity and spectral shape of the C–N vibrational band related to quaternary ammonium salt change accordingly, indicating a stronger ionic interaction between the carboxyl and quaternary ammonium groups. In Comparative Example 5, the carboxylate-related characteristics are significantly weakened or disappear. In Comparative Example 6, the shift and intensity are between the two, indicating that the degree of ionic recombination is enhanced with the introduction of succinylated glycerol monooleate, supporting the rationality of the design from the perspective of interaction evidence.

[0160] Figure 3 This is a time-series overlay plot characterizing the stability of the Zeta potential over time. Fixed parameters included a storage medium with a buffer system close to physiological ionic strength, consistent measurement conditions, a storage temperature of 4 °C, and measurements taken at 0 days, 1 month, 2 months, and 3 months, with mean and standard deviation provided. Variation parameters included the amount of cationic lipid 1,2-dioleoyloxy-3-trimethylammonium propane chloride used in Examples 1, 3, and 4. Example 1 showed an initial Zeta potential of approximately +35 mV that fluctuated only slightly with storage time and remained within a moderately positive charge range, indicating stable surface charge and dispersion even in an ion-strength shielded environment. Comparative Example 3 showed a lower initial potential that decreased further over time, reflecting charge decay and instability due to insufficient recombination strength. Comparative Example 4 showed a higher initial potential with more pronounced fluctuations, suggesting that an overly charged system is more difficult to maintain stability in a salt environment. Overall, Example 1 demonstrates a better balance between cationic recombination strength and colloidal stability.

[0161] Figure 4This is a time-series overlay plot characterizing the stability of the Zeta potential over time. The storage medium and measurement conditions were kept constant, and measurements were repeated at 0 days, 1 month, 2 months, and 3 months, with the mean and standard deviation calculated. The storage temperature was 25 °C. The varying parameters were the amount of cationic lipid used in Examples 1, 3, and 4. Example 1 maintained a potential around +30 to +40 mV at 25 °C with minimal change over time, indicating a stable surface charge environment under more stringent temperature conditions. Comparative Example 3 showed a faster potential decrease at 25 °C, approaching the low potential range, indicating a greater susceptibility to aggregation or charge shielding-dominated instability. Comparative Example 4 showed a more significant potential decrease and increased fluctuation, reflecting the difficulty in maintaining stability under the combined effects of salt and temperature with excessively high initial charges, proving that the charge control strategy of Example 1 is also effective under room temperature storage conditions.

[0162] Figure 5 This is a time-series overlay plot representing the stability of the encapsulation efficiency over time. The encapsulation efficiency was fixed using a method that involved separating and quantifying free small interfering ribonucleic acid (RNA). The mean and standard deviation were obtained through repeated measurements at 0 days, 1 month, 2 months, and 3 months. The storage temperature was 4 °C, and all other preparation and detection conditions were consistent. The variable parameter was whether succinylated glycerol monooleate was introduced to form an ionic complex, i.e., Example 1 and Comparative Example 5. Example 1 showed an initial encapsulation efficiency of approximately 82%, which remained high and slowly decreased after 3 months of storage at 4 °C, indicating good fixation and anti-leakage ability of the nucleic acid within the internal water channels. Comparative Example 5 showed a lower initial encapsulation efficiency, which decreased more significantly over time, indicating that the nucleic acid was more prone to dissociation and diffusion leakage when the ion complex design was missing. This demonstrates the important role of ion complexation and structural stabilization in maintaining a high encapsulation efficiency from a functional stability perspective.

[0163] Figure 6 The time-series overlay plot represents the stability characterization of encapsulation efficiency over time. Fixed parameters included the encapsulation efficiency measurement method and time points being consistent, and the system being stored at 25 °C for 3 months with repeated measurements to obtain the mean and standard deviation. Variable parameters included whether succinylated glycerol monooleate was introduced (Example 1 and Comparative Example 5). In Example 1, the encapsulation efficiency slowly decreased from approximately 82% to approximately 76% over time, with the decrease being relatively small, indicating that strong nucleic acid immobilization and system stability were maintained at room temperature. In Comparative Example 5, the encapsulation efficiency rapidly decreased from approximately 48% to approximately 23%, showing a significant leakage trend. This indicates that the combined effect of temperature and ionic strength accelerates nucleic acid release and system instability when ion recombination and structural stabilization are lacking, further demonstrating that this method can more effectively suppress nucleic acid leakage and maintain stability under room temperature storage conditions.

[0164] Figure 7This is a scanning electron microscope (SEM) image of cubic lipid nanoparticles from Example 1. The parameters were fixed: the sample was derived from the cubic lipid nanoparticle formulation obtained after post-processing in Example 1, and the core characterization parameters were a dynamic light scattering Z-average of 180 nm and a polydispersity index (PDI) of 0.22, along with an electrophoretic light scattering Zeta potential of +35 mV. The parameters varied from low magnification (overall distribution) to high magnification (single-particle detail). The particles in the image are predominantly discrete with some drying-induced local aggregation. At high magnification, the individual particle outlines are clear, predominantly cubic, and the particle size is consistent with 180 nm. This indicates that microfluidic mixing and granulation yielded a group of nanoparticles with sizes in the hundreds of nanometers and moderate dispersibility. Combined with the PDI of 0.22 and the positive potential results, this further confirms that the system exhibits reproducible granulation characteristics and a stable interfacial structure.

[0165] Figure 8 This is a transmission electron microscope (TEM) image of the cubic lipid nanoparticles from Example 1. The parameters were fixed as follows: the sample was derived from the cubic lipid nanoparticle formulation obtained after post-processing in Example 1, and small-angle X-ray scattering confirmed that its internal nanostructure was an anti-bicontinuous cubic phase with a Pn3m lattice, and small interfering ribonucleic acid (SIRNA) encapsulated within water channels of the anti-bicontinuous cubic phase. The parameters varied from the overall particle size to the details of the internal nanostructure. At low magnification, the particles exhibited a nanoscale solid particle outline with a scale matching the Z-average of 180 nm.

[0166] As can be seen from the performance of the examples and comparative examples in Table 1, the cubic lipid nanoparticles prepared in Examples 1-4 are significantly better than the comparative examples in many key indicators such as particle size distribution, polydispersity index, zeta potential, encapsulation efficiency, drug loading, cubic phase structure stability and storage stability. Specifically, Comparative Examples 1 and 2 showed significantly increased polydispersity index (PDI>0.4) and a transformation of cubic phase structure to amorphous or layered phase due to deviations in the amount of glycerol monooleate from the optimal range. Comparative Examples 3 and 4 showed decreased encapsulation efficiency (insufficient cationic lipids) and excessively high Zeta potential (exceeding +60mV may cause cytotoxicity) due to excessively low or excessively high DOTAP content, respectively. Comparative Example 5 could not form an effective ionic complex due to the absence of succinylated glycerol monooleate, with an encapsulation efficiency of only 48% and poor phase structure stability. Comparative Examples 6 and 7 could not achieve the optimal balance between cationic complex strength and colloidal stability due to deviations in the amount of succinylated glycerol monooleate. Comparative Example 8 showed excessively large particle size (420nm) and a polydispersity index of 0.58 due to insufficient mixing efficiency caused by excessively low microfluidic flow rate, resulting in poor batch-to-batch consistency. The gene silencing rates in Examples 1-4 were all in the range of 72-85%, while the comparative examples were generally below 70%, which verifies the synergistic advantages of the technical solution of the present invention in achieving low polydispersity, high encapsulation, stable cubic phase structure and excellent delivery efficiency under high solids content and high throughput conditions.

[0167] Table 1 Performance Comparison Summary Table

[0168]

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid, characterized in that, Includes the following steps: S1. Preparation of organic phase: Weigh 8.5-9.8 parts by weight of glyceryl monooleate, 0.2-1.5 parts by weight of 1,2-dioleoyloxy-3-trimethylammonium propane chloride, and 0.05-1.5 parts by weight of succinylated glyceryl monooleate, dissolve them in anhydrous ethanol to make the total lipid concentration in the organic phase 10-100 mg / mL, and obtain a clear organic phase; S2. Aqueous phase preparation: Weigh 1.0-1.5 parts of poloxamer 407 and 1 part of small interfering RNA by mass. Dissolve poloxamer 407 and small interfering RNA in a buffer solution, which is a phosphate buffer or a citrate buffer, to obtain the aqueous phase. Wherein, each mass fraction is calculated as one part of the small interfering ribonucleic acid; S3. Microfluidic mixing and granulation: The clarified organic phase obtained in step S1 and the aqueous phase obtained in step S2 are respectively introduced into a microfluidic mixing chip, wherein the flow rate ratio of the aqueous phase to the organic phase is 3:1 and the total flow rate is 8-20 mL / min, so that they are mixed in the microfluidic mixing chip and self-assembled into granules to obtain a granulation system containing cubic lipid nanoparticles encapsulating the small interfering ribonucleic acid; S4. Post-processing: The granulation system obtained in step S3 is subjected to ethanol removal and buffer replacement by dialysis or tangential flow filtration to obtain cubic lipid nanoparticles or their formulations, wherein the ethanol content after step S4 is not higher than 1.0 wt%.

2. The method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid according to claim 1, characterized in that, The cubic lipid nanoparticles have an internal nanostructure of an anti-bicontinuous cubic phase, wherein the anti-bicontinuous cubic phase is of Pn3m or Ia3d lattice type; the small interfering ribonucleic acid is encapsulated in the water channels of the anti-bicontinuous cubic phase; and the succinylated glycerol monooleate forms an ionic complex with the 1,2-dioleoyloxy-3-trimethylammonium propane chloride; wherein the cubic lipid nanoparticles refer to lipid nanoparticles with an internal anti-bicontinuous cubic phase structure, and their shape is not limited to a geometric cube.

3. The method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid according to claim 1, characterized in that, The succinylated glycerol monooleate used in step S1 is prepared according to the following steps: A1. Raw materials: Glyceryl monooleate, succinic anhydride, triethylamine, tetrahydrofuran, ethyl acetate, sodium bicarbonate, anhydrous sodium sulfate, sodium chloride, water; A2. Ratio: The molar ratio of succinic anhydride to glyceryl monooleate is 0.05-0.30:1, and the molar ratio of triethylamine to succinic anhydride is 1.0-1.5:1; A3. Reaction: Dissolve glyceryl monooleate in tetrahydrofuran to achieve a mass concentration of 50-300 mg / mL in tetrahydrofuran. Stir at 40-55℃, add succinic anhydride and triethylamine, and continue the reaction at 40-55℃ for 6-20 h. A4. Endpoint criterion: The acid value of the obtained product is 20-120 mg KOH / g; A5. Post-treatment: After the reaction is complete, remove tetrahydrofuran under reduced pressure, dissolve in ethyl acetate, and wash successively with 5-10 wt% sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution until the pH of the aqueous phase is 6.5-7.5; dry the organic phase with anhydrous sodium sulfate and filter, remove ethyl acetate under reduced pressure, and then vacuum dry until the mass is constant and the total residual solvent is not higher than 1.0 wt%; A6. Quality control: Residual succinic anhydride shall not exceed 0.5 wt%, and residual triethylamine shall not exceed 0.2 wt%.

4. The method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid according to claim 1, characterized in that, In step S1, the succinylated glycerol monooleate and the 1,2-dioleoyloxy-3-trimethylammonium propane chloride are pre-formed into a complex lipid intermediate, which is prepared according to the following steps: B1. Raw materials: 1,2-dioleoyloxy-3-trimethylammonium propane chloride, succinylated glyceryl monooleate, anhydrous ethanol; B2. Ratio: The molar ratio of the 1,2-dioleoyloxy-3-trimethylammonium propane chloride to the succinylated glycerol monooleate is 0.2-2.0:1; B3. Dissolution and compounding: The 1,2-dioleoyloxy-3-trimethylammonium propane chloride and the succinylated glycerol monooleate were added to anhydrous ethanol to make the total lipid mass concentration 10-100 mg / mL, and stirred at 40-65°C until a clear solution was formed. B4. Curing: Remove anhydrous ethanol under reduced pressure and dry under vacuum to obtain the complex lipid intermediate; B5. Quality control: Residual ethanol shall not exceed 1.0 wt%.

5. The method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid according to claim 1, characterized in that, The small interfering ribonucleic acid described in step S2 forms a nucleic acid-lipid precomplex intermediate before granulation. The nucleic acid-lipid precomplex intermediate is prepared according to the following steps: D1. Raw materials: small interfering ribonucleic acid, 1,2-dioleoyloxy-3-trimethylammonium propane chloride and complex lipid intermediates, phosphate buffer or citrate buffer; D2. Mixing step: Dissolve the small interfering ribonucleic acid in phosphate buffer or citrate buffer, add the 1,2-dioleoyloxy-3-trimethylammonium propane chloride or the complex lipid intermediate and mix, incubate at 20-30℃ for 5-30 min to obtain the nucleic acid-lipid precomplex intermediate; D3. Ratio: The mass ratio of 1,2-dioleoyloxy-3-trimethylammonium propane chloride in the nucleic acid-lipid precomplex intermediate to the small interfering ribonucleic acid is 0.2-1.5:

1. D4. Endpoint Criteria: The obtained nucleic acid-lipid precomplex intermediate is clear or milky white and translucent, with no visible precipitation.

6. The method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid according to claim 1, characterized in that, Ethanol removal and buffer replacement in step S4 are achieved by dialysis or tangential flow filtration: Dialysis is performed using dialysis bags or membranes with a molecular weight cutoff of 3.5-14 kDa at 4-25°C for 6-24 hours with 3-6 buffer changes, wherein the volume of buffer used for each buffer change is 50-200 times the volume of the granulation system; or tangential flow filtration is performed using an ultrafiltration membrane with a molecular weight cutoff of 30-100 kDa at 4-25°C with 5-10 times volume replacement; the ethanol content after step S4 is not higher than 1.0 wt%, and the ethanol content is determined by gas chromatography; The buffer solution is a phosphate buffer or a citrate buffer. The phosphate buffer solution is prepared with water and consists of 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 10 mmol / L disodium hydrogen phosphate, and 1.8 mmol / L potassium dihydrogen phosphate, with a pH of 7.2-7.

6. The citrate buffer solution is prepared with water and consists of 10-50 mmol / L citric acid and 10-50 mmol / L sodium citrate dihydrate, with a pH of 3.0-6.

5. Furthermore, in step S1, an auxiliary lipid is added, which is one or both of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol, wherein, by weight, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine is 0.1-3.0 parts and cholesterol is 0.1-3.0 parts.

7. The method for preparing cubic lipid nanoparticles encapsulating small interfering ribonucleic acid according to claim 1, characterized in that, The cubic lipid nanoparticles have a Z-average particle size of 50-500 nm, a polydispersity index (PDI) of 0.1-0.4, and an absolute value of zeta potential of 10-60 mV; wherein the Z-average particle size and the PDI are determined by dynamic light scattering, and the zeta potential is determined by electrophoretic light scattering.

8. The application of cubic lipid nanoparticles encapsulating small interfering RNA in the preparation of formulations for delivering small interfering RNA to achieve target gene silencing, characterized in that, The cubic lipid nanoparticles comprise the following components on a dry basis by weight: 8.5-9.8 parts of glyceryl monooleate; 1.0-1.5 parts of poloxamer 407; 0.2-1.5 parts of 1,2-dioleoyloxy-3-trimethylammonium propane chloride; 0.05-1.5 parts of succinylated glyceryl monooleate; and 1 part of small interfering ribonucleic acid (MIRNA); wherein all weight parts are based on 1 part of the MIRNA. The cubic lipid nanoparticles have an internal nanostructure of an anti-bicontinuous cubic phase, which is of Pn3m or Ia3d lattice type; the small interfering ribonucleic acid is encapsulated in the water channels of the anti-bicontinuous cubic phase; and the succinylated glycerol monooleate forms an ionic complex with the 1,2-dioleoyloxy-3-trimethylammonium propane chloride.

9. The application of the cubic lipid nanoparticles encapsulating small interfering RNA according to claim 8 in the preparation of formulations for delivering small interfering RNA to achieve target gene silencing, characterized in that, The cubic lipid nanoparticles have a Z-average particle size of 50-500 nm, a polydispersity index (PDI) of 0.1-0.4, and an absolute zeta potential of 10-60 mV. The Z-average particle size and PDI are determined by dynamic light scattering, and the zeta potential is determined by electrophoretic light scattering. The cubic lipid nanoparticles further include auxiliary lipids, which are one or both of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol, with 0.1-3.0 parts by weight of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and 0.1-3.0 parts by weight of cholesterol.

10. The application of the cubic lipid nanoparticles encapsulating small interfering RNA according to claim 8 in the preparation of formulations for delivering small interfering RNA to achieve target gene silencing, characterized in that, The encapsulation efficiency of the small interfering ribonucleic acid (SRNA) is 60-95%, and the drug loading is 4.5-10 wt%, wherein the drug loading is the mass percentage of the SRNA in the cubic lipid nanoparticles relative to the total dry mass of the cubic lipid nanoparticles; the cubic lipid nanoparticles or their formulations, when stored at 4℃ and 25℃ for 3 months, show a particle size change of no more than ±20%, a decrease in encapsulation efficiency of no more than 10%, and no visible precipitation or stratification; the encapsulation efficiency is determined by ultraviolet spectrophotometry or fluorescence method at a detection wavelength of 260 nm after separating free SRNA by high-speed centrifugation or gel column chromatography.

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