Multi-stage liver-targeted drug delivery system based on ionizable amino lipid and preparation method of multi-stage liver-targeted drug delivery system

Abstract

The invention discloses a multilevel liver targeting drug delivery system based on ionizable amino lipid and a preparation method thereof, the system comprises the following components: target modified ionizable amino lipid, ionizable amino lipid, auxiliary lipid and structural lipid in a molar ratio of (10-30): (30-60): (5-40): (15-55), according to the system, nano lipid particles exist in a water-in-oil-in-water nano emulsion form; wherein the ionizable ammonia lipid is a compound with a structure as shown in a formula (I). The system is suitable for delivering nucleic acid, protein and other active pharmaceutical ingredients to the liver so as to improve the accuracy and treatment effect of drug delivery and reduce the side effects of non-target organs.

Description

Technical Field

[0001] This invention relates to drug delivery technology and nanomedicine, and in particular to a multi-level liver-targeting drug delivery system based on ionizable amino lipids and its preparation method. Background Technology

[0002] Lipid nanoparticles currently exhibit high encapsulation efficiency and intracellular release capacity in the delivery of nucleic acids, proteins, and small molecule drugs. However, insufficient particle size control and surface functionalization often lead to imprecise in vivo distribution and toxicity due to accumulation in non-target organs. In recent years, combining various liver-targeting modification molecules with engineered cell membrane coating and near-infrared tracing technologies has provided new approaches for enhanced targeting and dynamic monitoring. Furthermore, in the field of gene delivery, non-viral vectors are easily mass-produced, but traditional cationic polymers are often limited by low transfection efficiency or high cytotoxicity.

[0003] In recent years, nanomedicine delivery systems have gradually become a hot topic in research on the treatment of liver diseases (such as liver cancer, viral hepatitis, and liver fibrosis). Liposomes, as drug delivery carriers, are widely used for the delivery of various drugs and genes due to their high encapsulation efficiency, biocompatibility, and controlled release capabilities. However, traditional liposomes suffer from problems such as short circulation time, non-specific distribution, and difficulty in overcoming intracellular barriers, which affect their clinical efficacy. Summary of the Invention

[0004] Objective of the Invention: The objective of this invention is to provide a multi-stage liver-targeted drug delivery system and its preparation method. The core idea lies in constructing drug-loaded nanolipid particles using ionizable aminolipids, and then achieving targeted molecule access through a special PEG linking technology. Specifically, the ionizable aminolipids exhibit excellent drug encapsulation and release properties in vivo. They maintain good stability in neutral or weakly alkaline environments, while rapidly converting to a positive charge in acidic environments (such as intracellular endosomes or lysosomes), thereby promoting liposome rupture and timely drug release, ensuring the delivery system can efficiently release the active pharmaceutical ingredient.

[0005] Technical solution: The multi-stage liver-targeted drug delivery system of the present invention comprises components in the following molar ratio:

[0006] Targeted modified ionizable amino lipids: Ionizable amino lipids: Auxiliary lipids: Structural lipids = 10-30: 30-60: 5-40: 15-55. This system consists of nanolipid particles in the form of a water-in-oil-in-water nanoemulsion.

[0007] Preferably, the system comprises components in the following molar ratio:

[0008] Targeted modified ionizable amino lipids: Ionizable amino lipids: Auxiliary lipids: Structural lipids = 10-30: 30-60: 15-20: 15-55. The system consists of nanolipid particles in the form of a water-in-oil-in-water nanoemulsion.

[0009] Among them, the ionizable amino lipids are compounds having the structure shown in formula (I):

[0010]

[0011] L1 and L2 may be the same or different, and each can be independently selected from O or C;

[0012] When L1 or L2 is C, it has an R1 substituent.

[0013] R1 and R2 may be the same or different, and each can be independently selected from H or one of the following structures:

[0014]

[0015] R3 and R4 may be the same or different, and each can be independently selected from H or OH;

[0016] The targeted modified ionizable amino lipid: the hydroxyl groups in the ionizable amino lipid are linked to a polyethylene glycol derivative to form an ionizable amino lipid-PEG, wherein the polyethylene glycol derivative is selected from one of the following two types:

[0017] (i) HO-PEG-COOH, which is polyethylene glycol with a carboxyl group at the end;

[0018] (ii) HO-PEG-NH2, i.e., polyethylene glycol with an amino group at the end; and the linkage reaction adopts an SN2 reaction mechanism to generate a stable ether bond, while retaining the functional group at the end of the polyethylene glycol; the functional group is selected from COOH or NH2;

[0019] The polyethylene glycol used is selected from PEG-500, PEG-1000, and PEG-2000;

[0020] The terminal functional group of the polyethylene glycol is then linked to at least one target molecule via a chemical coupling reaction, the target molecule comprising:

[0021] Galactose; used to bind to ASGPR receptors on the surface of hepatocytes;

[0022] Bile acids, deoxycholic acid, or their derivatives; they are used to bind to bile acid receptors;

[0023] Tracer molecules are used for real-time dynamic monitoring of drug delivery. Preferably, the tracer molecules include near-infrared fluorescent molecules and fluorescent dyes. More preferably, the tracer molecules include Cy5, DAPI, Merge, and AIE.

[0024] The system described above, wherein the targeted modified ionizable amino lipid is prepared by the following method: (a) the hydroxyl groups of the ionizable amino lipid are first treated with p-toluenesulfonyl chloride (TsCl) to generate an ionizable amino lipid-tosylate intermediate; (b) the polyethylene glycol derivative is subjected to anhydrous conditions using sodium hydride (NaH) to generate an ion from its internal alcohol groups; (c) the anion solution obtained in the previous step is reacted with the ionizable amino lipid-tosylate at 60-80°C for 4-12 hours, and the hydroxyl groups in the ionizable amino lipid react with the hydroxyl groups in the polyethylene glycol derivative through an SN2 reaction to generate stable ether bonds; (d) the product obtained in the previous step can be further reacted with the target molecule through EDC / NHS chemical coupling.

[0025] The system described herein, wherein the hydroxyl groups in the ionizable amino lipid react with the hydroxyl groups in the polyethylene glycol derivative to form stable ether bonds, resulting in the following structure:

[0026]

[0027] L1 and L2 may be the same or different, and each is independently selected from O or C;

[0028] When L1 or L2 is C, it has an R1 substituent.

[0029] R5 and R6 may be the same or different, and each is independently selected from H or one of the following structures;

[0030]

[0031] n is selected from an integer between 20 and 30. Preferably, n is selected from 22.

[0032] In the system described above, the auxiliary lipids include dioleoylphosphatidylethanolamine (DOPE); the structural lipids include cholesterol.

[0033] The liver-targeted modified amino PEG lipids of the system are prepared by one or more of the following methods: (a) galactose-modified PEG lipids: 6-aminogalactose is reacted with a carboxyl-terminated PEG lipid (e.g., LAL-PEG-COOH) via EDC / NHS coupling method to generate an ionizable amino lipid -PEG-Gal; (b) cholic acid-modified PEG lipids: deoxycholic acid or its derivatives are reacted with an amino-terminated PEG lipid (e.g., ionizable amino lipid -PEG-NH2) via EDC / NHS chemical activation method to generate an ionizable amino lipid -PEG-Bile; (c) fluorescent dye-modified PEG lipids: fluorescent dyes retain carboxyl or amino groups and are reacted with amino or carboxyl-terminated PEG lipids via EDC / NHS chemical activation method to generate PEG lipids.

[0034] The preparation method of the multi-stage liver-targeted drug delivery system includes the following steps:

[0035] a. Preparation of targeted modified ionizable amino lipids (i) Dissolve ionizable amino lipids in anhydrous dichloromethane, add p-toluenesulfonyl chloride and triethylamine to convert their hydroxyl groups into ionizable amino lipid-tosylate intermediates; (ii) Use polyethylene glycol derivatives (HO-PEG-COOH or HO-PEG-NH2) containing terminal COOH or terminal NH2, respectively, and add NaH in anhydrous tetrahydrofuran at 0-4℃ to generate PEG anions, wherein the PEG used only utilizes its unprotected internal hydroxyl groups to participate in the reaction and is retained. Terminal function; (iii) Under strictly anhydrous conditions, the PEG anion solution generated in step (ii) is added dropwise to the ionizable amino lipid-tosylate solution in step (i), heated to 60-80°C and reacted for 4-12 hours to generate ionizable amino lipid-PEG through SN2 reaction, wherein a stable ether bond (-COC-) is formed, while the terminal COOH or NH2 of PEG is retained for subsequent targeting molecule coupling; (iv) The terminal COOH or NH2 of the polyethylene glycol is then connected to the targeting molecule through a chemical coupling reaction;

[0036] b. Assembling nanolipid particles (i) Dissolve ionizable amino lipids, auxiliary lipids and structural lipids with the targeted modified polyethylene glycol lipids (ionizable amino lipids-PEG-COOH and / or ionizable amino lipids-PEG-NH2) prepared in step a in anhydrous ethanol to form a homogeneous organic phase; (ii) Mix the organic phase with an aqueous phase containing the target drug (such as siRNA, mRNA or small molecule drug) at a volume ratio of 1:1 to 5 and prepare an oil-in-water emulsion by ultrasonic emulsification or microfluidic technology, and then form a water-in-oil-in-water nanoemulsion with water;

[0037] The average particle size of the resulting multi-stage liver-targeted drug delivery system was 40–80 nm.

[0038] The system is applicable to the treatment of liver-related diseases, including liver cancer, viral hepatitis, liver fibrosis, and drug-induced liver injury.

[0039] The system further engineeres the obtained lipid nanoparticles by coating them with cell membranes or by combining them with oleic acid-modified superparamagnetic nanoparticles.

[0040] The preparation method described herein, wherein the target drug comprises: siRNA, mRNA, or a chemotherapeutic drug. Preferably, the chemotherapeutic drug comprises 5-FU.

[0041] The system is used in animal experiments for the following purposes: to treat liver tumors in animal models; to study the mechanisms of liver fibrosis and the effects of anti-fibrotic treatment; and to verify drug delivery and gene editing efficiency, with delivery efficiency monitored by near-infrared in vivo imaging.

[0042] In this invention, ionizable amino lipids (LAL, i.e., compound (I)) are first converted into LAL-tosylate intermediates by treatment with p-toluenesulfonyl chloride (TsCl). Subsequently, unprotected internal hydroxyl groups in polyethylene glycol (HO-PEG-COOH or HO-PEG-NH2) containing terminal COOH or NH2 are used to generate anions under the action of sodium hydride (NaH). Then, the PEG anions are coupled to LAL-tosylate via an SN2 reaction to form stable ether bonds (-COC-), thereby constructing a stable LAL-PEG linker unit. This method not only ensures the stability of the linker bond but also preserves the functional groups at the PEG ends, providing sufficient chemically active sites for subsequent targeted modification.

[0043] In particular, the liposome platform used in this invention has extremely high flexibility.

[0044] Among them, ionizable amino lipids, as an important component of liposomes, have advantages mainly reflected in drug delivery. Ionizable amino lipids exhibit a weak charge under neutral or alkaline conditions, thus demonstrating better stability in vivo and reducing non-specific binding to plasma proteins, resulting in a longer half-life of the drug delivery system in the bloodstream. When the system enters an acidic environment (such as intracellular endosomes or lysosomes), ionizable amino lipids rapidly convert to a positively charged state, which not only promotes cell membrane disruption and endosome escape but also facilitates timely drug release, thereby improving drug efficacy.

[0045] This invention utilizes ionizable amino lipids to construct drug-loaded lipid nanoparticles, and its advantages are:

[0046] Furthermore, this invention employs different targeting methods—galactose modification and bile acid modification—to achieve active targeting of the liver by binding to specific receptors on the surface of hepatocytes (ASGPR or bile acid receptors), ensuring that the delivery system has efficient drug release and cellular uptake functions in the liver.

[0047] In summary, this invention leverages the unique advantages of ionizable amino lipids in drug delivery and constructs a multi-level liver-targeted drug delivery system through stable LAL-PEG linkage technology and single-target ligand modification. This system not only improves the enrichment rate of drugs in the liver but also allows for real-time monitoring of drug delivery dynamics, significantly improving the treatment efficacy for related diseases.

[0048] Beneficial Effects: The technical solution of the present invention has the following advantages over the prior art: (1) Efficient drug encapsulation and release: Ionizable amino lipids achieve stable encapsulation and precise drug release during delivery by changing the charge state. This characteristic provides a good platform for the delivery of nucleic acids, proteins and small molecule drugs. (2) Active targeting and endosome escape: In this system, the stable liposomes formed by the SN2 reaction of ionizable amino lipids and PEG derivatives can retain the PEG terminal function (facilitating the modification of targeting ligands) and exert a positive charge effect in an acidic environment, increasing the uptake and release efficiency of drugs in hepatocytes. (3) Reduced non-specific toxicity: By regulating the ratio of ionizable amino lipids and auxiliary lipids, the present invention significantly reduces the accumulation of drugs in non-target organs while ensuring efficient drug delivery, thereby reducing toxic reactions and improving treatment safety. Attached Figure Description

[0049] Figure 1 DLS results for galactose-modified amino liposomes;

[0050] Figure 2 TEM image of galactose-modified amino liposomes;

[0051] Figure 3 This is a graph showing the results of a pH-responsive drug release test.

[0052] Figure 4 This is a diagram showing the results of a cell uptake experiment. Specific Implementation

[0053] The following is an example of an aminolipid molecule used for targeted modification; other aminolipids are also applicable to the following synthetic route.

[0054]

[0055] The synthetic route for amino lipids—PEG-COOH / NH2 is as follows:

[0056]

[0057] The synthetic route for carboxyamino PEG lipids is similar, except that NH2 is converted to COOH.

[0058] Preparation of LAL-A:

[0059]

[0060] In a 250 mL reaction flask, starter A (22.3 g, 100 mmol) and starter B (25.6 g, 100 mmol) were added sequentially, followed by 100 mL of dichloromethane. After stirring and dissolving, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (23.0 g, 120 mmol), 4-dimethylaminopyridine (0.61 g, 5 mmol), and N,N-diisopropylethylamine (25.8 g, 200 mmol) were added. The mixture was reacted at room temperature for 2 h. After washing three times with water, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified using a rapid column chromatography system (petroleum ether: ethyl acetate = 10:1 to 2:1) to give intermediate I-1 (41.5 g, 90% yield).

[0061] 1H NMR (500MHz, Chloroform-d) δ4.66, 3.45, 2.29, 1.85, 1.56, 1.54, 1.52, 1.42, 1. 42, 1.39, 1.31, 1.31, 1.29, 1.29, 1.28, 1.27, 1.27, 1.27, 1.26, 1.25, 1.25, 0.89.

[0062]

[0063] In a 100 mL reaction flask, starter D (3.50 g, 10 mmol) and starter F (13.1 g, 100 mmol) were added sequentially, along with 30 mL of ethanol. After stirring and dissolving, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added. The mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain intermediate III-1 (3.26 g, yield 68%).

[0064] 1H NMR (500MHz, Chloroform-d) δ 4.14-4.00 (m, 2H), 3.77-3.63 (m, 2H), 3.63-3.54 (m, 3H), 3.20-3.10 (m, 1H), 3.03-2.91 (m, 3H), 2.85 (dt, J=9. 0, 6.0Hz, 1H), 2.31 (td, J=7.1, 0.8Hz, 2H), 1.85-1.74 (m, 1H), 1.77-1.66 (m, 3H), 1.66-1.41 (m, 7H), 1.43-1.22 (m, 17H), 0.94-0.84 (m, 3H).

[0065] Intermediate I-1 (355 mg, 1 mmol), intermediate III-1 (556 mg, 1.2 mmol), and 20 mL of acetonitrile were added sequentially to a 100 mL reaction flask. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h. Then, 100 mL of dichloromethane was added, and the mixture was washed three times with water. After drying with anhydrous sodium sulfate, the mixture was concentrated and purified using a rapid column chromatography system (dichloromethane:methanol = 20:1 to 5:1) to obtain compound 3 (510 mg, yield 67%), which is LAL-A.

[0066]

[0067] 1H NMR (500MHz, Chloroform-d) δ4.14-3.99 (m, 0H), 3.74-3.53 (m, 1H), 2.67-2.51 (m, 1H), 2.35-2.18 (m, 1H), 1.85-1.66 (m, 1H), 1.70-1.59 (m, 1H), 1. 63-1.38 (m, 3H), 1.42-1.35 (m, 1H), 1.35 (ddd, J=5.5, 4.0, 1.1Hz, 1H), 1. 35-1.28 (m, 1H), 1.31-1.24 (m, 3H), 1.28-1.18 (m, 4H), 0.94-0.83 (m, 2H).

[0068] Example 1

[0069] Preparation and in vitro testing of a multi-stage liver-targeted drug delivery system based on ionizable amino lipids

[0070] certificate

[0071] 1. Preparation of LAL-A-tosylate intermediates

[0072] 1.1 Preparation of raw materials and solutions

[0073] Weigh 100 mg (0.13 mmol) of LAL-A and dissolve it in 50 mL of anhydrous dichloromethane;

[0074] • p-Toluenesulfonyl chloride (TsCl, 0.15 mmol) and triethylamine (TEA, 0.15 mmol) were added as catalysts and neutralizing agents.

[0075] 1.2 Reaction and Post-treatment

[0076] • Stirring at room temperature for 4 hours to convert LAL hydroxyl groups to LAL-A-tosylate;

[0077] • After the reaction is complete, wash with saturated NaCl solution to separate the organic layer, and dry with anhydrous sodium sulfate;

[0078] • The solvent is removed by rotary evaporation to obtain the LAL-A-tosylate intermediate.

[0079] In this step, the fully generated and purified LAL-A-tosylate provides a highly active leaving group for subsequent reactions, and its conversion effect directly affects the efficiency of subsequent PEG coupling.

[0080] 2. Preparation of LAL-A-PEG1000-COOH (via SN2 reaction)

[0081] 2.1 Generation of PEG negative ions

[0082] • Take 200 mg of HO-PEG1000-COOH (molecular weight 1000, with COOH at the end) and dissolve it in 20 mL of anhydrous tetrahydrofuran (THF);

[0083] • Place the solution in an ice bath (0°C) and slowly add an appropriate amount of sodium hydride (NaH, 0.5 mmol of 60% oil solution), stirring for 1 hour to deprotonate the internal hydroxyl groups (note to retain the COOH at the PEG end).

[0084] Importantly, this process only encourages the hydroxyl groups within PEG1000 to participate in the reaction, thereby ensuring that the functional groups at the PEG ends are preserved (which is crucial for subsequent targeted coupling).

[0085] 2.2 SN2 linkage reaction

[0086] • Under strictly anhydrous conditions, the PEG anion solution generated in step 2.1 is added dropwise to the LAL-A-tosylate solution obtained in step 1;

[0087] • The mixture was heated to 80°C and the reaction was continued for 8 hours to induce PEG-O- to replace the tosyl group to form a stable ether bond (-COC-), thereby forming LAL-A-PEG1000-COOH;

[0088] After the reaction was completed, the sample was extracted with ethyl acetate and washed to remove low molecular weight impurities. Finally, the sample was purified by silica gel column chromatography to obtain pure LAL-A-PEG1000-COOH.

[0089] This reaction not only completed the connection between LAL-A and PEG1000, but also ensured the complete retention of the COOH group at the end of the PEG, which provides the necessary active site for the next step of targeted molecular chemical coupling.

[0090] 3. Targeted modification of galactose access

[0091] The synthetic route is as follows:

[0092]

[0093] 3.1 Carboxyl group activation

[0094] • Dissolve the purified LAL-A-PEG1000-COOH (100 mg) in anhydrous DMF (10 mg / mL);

[0095] • Add equimolar amounts of EDC·HCl and NHS (0.1 mmol each), activate the terminal carboxyl group of PEG at room temperature, and stir for 1 hour.

[0096] 3.2 Coupling Reaction

[0097] • Dissolve 0.4 equivalents of 6-aminogalactose in a suitable solvent (DMSO / water mixture, while adjusting the pH to 7.5), and slowly add it to the activation solution;

[0098] • The reaction was carried out at room temperature for 4 hours until the coupling was complete;

[0099] • The product was purified by dialysis and then freeze-dried under vacuum to obtain LAL-A-PEG1000-Gal (galactose-modified PEG lipid).

[0100] In this step, the PEG terminal active groups retained in the previous step are the key factor to ensure the smooth progress of the EDC / NHS reaction.

[0101] The above steps constitute a coherent modular process: First, the preparation of the LAL-A-tosylate intermediate from LAL-A is the basis for the subsequent SN2 reaction between the PEG1000 anion and it; second, the generation of the PEG1000 anion under anhydrous conditions using NaH ensures that the internal reaction of PEG1000 is carried out without loss of terminal activity; third, through the SN2 reaction between the PEG1000 anion and LAL-A-tosylate, not only is a stable LAL-A-PEG1000 structure constructed, but also functional groups are retained for subsequent targeted molecule coupling; finally, through EDC / NHS chemical coupling, the targeted molecule (galactose) is introduced, thereby obtaining a final product with targeted properties.

[0102] 4. Liposome assembly

[0103] 4.1.1 Component Configuration

[0104] • The components used and the recommended molar ratios are as follows: (a) Ionizable amino lipids (LAL-A): 40%; (b) DOPE (Drug-O-Peptide): 15%; (c) Structural lipids (cholesterol): 30%; (d) Galactose-modified lipids (LAL-A-PEG1000-Gal): 15%.

[0105] 4.1.2 Preparation process

[0106] • The above lipid components are dissolved in anhydrous ethanol in a predetermined ratio to form a homogeneous organic phase;

[0107] • Dissolve the target drug (5-FU) in the aqueous phase of 50 mM sodium acetate buffer (pH 4.0);

[0108] • The organic phase and the aqueous phase are mixed at a volume ratio of 1:3, and a preliminary water-in-oil (W / O) emulsion is prepared by ultrasonic emulsification.

[0109] • The initial emulsion was then mixed with an aqueous phase (50 mM sodium acetate buffer, pH 4.0) to form a water-in-oil-in-water (W / O / W) nanoemulsion;

[0110] • After removing ethanol by dialysis, the medium is replaced with PBS buffer, and finally sterilized by 0.22μm filtration to obtain the final product.

[0111] 4.1.3 Particle size and morphology characterization

[0112] • The average particle size (target 40–80 nm) and PDI (less than 0.2) of liposomes were determined using dynamic light scattering (DLS).

[0113] • The morphology and distribution of liposomes were observed using transmission electron microscopy (TEM), and the results are shown in [Figure number missing]. Figure 1 .

[0114] 4.2.1 Following the assembly of liposomes described above, missing molecules were added to the component configuration, and the other steps were the same to obtain liposomes.

[0115] The components used and their recommended molar ratios are as follows: (a) Ionizable amino lipids (LAL-A): 39%; (b) DOPE (Drug-Oxygenated Lipids): 15%; (c) Structural lipids (cholesterol): 30%; (d) Galactose-modified lipids (LAL-A-PEG1000-Gal): 15%. For ease of subsequent observation, 1% tracer molecule Cy5 was added.

[0116] 4.2.2 The assembly of liposomes is the same as in 4.2.1, except that 1% tracer molecule DAPI is added to prepare liposomes.

[0117] 4.2.3 The assembly of liposomes is the same as in 4.2.1, except that 1% tracer molecule Merge is added to prepare liposomes.

[0118] 5. In vitro functional verification

[0119] 5.1 Cell uptake experiment

[0120] • Using the HepG2 hepatocyte line, liposomes labeled with near-infrared tracer molecules such as Cy5 were added to the cell culture medium;

[0121] • Cellular uptake of liposomes was observed using fluorescence microscopy, and the differences in absorption between targeted modified liposomes and unmodified liposomes were compared. Results are as follows: Figure 4 As shown, targeted modified liposomes are better taken up by hepatocytes than unmodified liposomes.

[0122] 5.2 Drug Release Experiment

[0123] • Drug release experiments can be conducted in buffer solutions with different pH values ​​(pH 7.4 and pH 5.5) to verify the response characteristics of ionizable ammonia lipids triggering liposome structure rupture and rapid drug release under acidic conditions.

[0124]

[0125] Example 2

[0126] 1. Preparation of LAL-A-tosylate intermediates

[0127] • Procedure Overview: Dissolve 100 mg (0.13 mmol) of LAL-A in 50 mL of anhydrous dichloromethane, add p-toluenesulfonyl chloride (0.15 mmol) and triethylamine (0.15 mmol), and stir at room temperature for 6 hours. After the reaction, wash the organic layer with saturated NaCl solution, dry (using anhydrous sodium sulfate), and rotary evaporate to obtain the LAL-A-tosylate intermediate.

[0128] 2. Preparation of LAL-A-PEG1000-NH2 (SN2 reaction)

[0129] • PEG negative ion generation: Take 200 mg of HO-PEG1000-NH2 (molecular weight 1000, terminal NH2) and dissolve it in 20 mL of anhydrous tetrahydrofuran (THF). Place it in an ice bath (0℃) and slowly add an appropriate amount of sodium hydride (NaH, 0.5 mmol) while stirring for 1 hour to deprotonate the internal hydroxyl groups (while keeping the terminal NH2 unaffected).

[0130] The SN2 linkage reaction was carried out under strictly anhydrous conditions by adding the PEG anion solution dropwise to the LAL-A-tosylate solution obtained in step 1, followed by heating to 60°C and reacting for 12 hours to allow the PEG-O to bond. - The tosyl group is replaced to form a stable ether bond (-COC-), thus forming LAL-A-PEG1000-NH2. After the reaction is complete, the product is extracted with ethyl acetate, washed, dried, and purified by dialysis to obtain a pure product.

[0131] 3. Deoxycholic acid coupling modification

[0132] The synthetic route is as follows:

[0133]

[0134] • Pretreatment and activation: The purified LAL-A-PEG1000-NH2 (100 mg) was dissolved in anhydrous DMF (concentration 20 mg / mL). Deoxycholic acid (0.1 mmol) was dissolved in a small amount of DMF, and the pH was adjusted to 8.5.

[0135] For EDC / NHS coupling, deoxycholic acid was first activated by adding equimolar amounts of EDC·HCl and NHS to its carboxyl group. After 1 hour of activation, the activated solution was slowly added to a LAL-A-PEG1000-NH2 solution. The reaction was carried out at 40°C for 24 hours until cholic acid-modified LAL-A-PEG1000 (denoted as LAL-A-PEG1000-Bile) was formed via an amide bond. Finally, the product was purified by column chromatography and lyophilized to obtain a dried product.

[0136] 4. Liposome assembly

[0137] • Recommended molar ratios of each component in the liposome formulation:

[0138] O-ionizable amino lipids (LAL-A): 40%.

[0139] ο DOPE (O-diol-dependent lipids): 20%.

[0140] οStructural lipids (cholesterol): 30%.

[0141] ο Targeted modified lipids (LAL-A-PEG1000-Bile): 10%.

[0142] • Preparation process: ① Dissolve all lipid components in anhydrous ethanol according to the specified ratio to form a homogeneous organic phase; ② The target drug, RNA, is dissolved in an aqueous phase of 50 mM sodium acetate buffer (pH 4.0); ③ Mix the organic phase and aqueous phase at a volume ratio of 1:3 and prepare a preliminary W / O type emulsion using microfluidic technology; ④ Mix the preliminary emulsion with the aqueous phase (50 mM sodium acetate buffer, pH 4.0) to form a W / O / W type nanoemulsion; ⑤ Remove ethanol by ultrafiltration, replace with PBS buffer, and finally sterilize by 0.22 μm filtration to obtain bile acid modified liposomes.

[0143] 5. In vitro functional verification

[0144] • Particle size and morphology were determined by dynamic light scattering (DLS) to measure the average particle size of the carrier (target 40–80 nm, PDI < 0.2), and the morphology of liposomes was observed by TEM.

[0145] • Cellular uptake assays were conducted in a HepG2 hepatocyte model using confocal microscopy and flow cytometry to compare the cellular uptake of bile acid-modified liposomes versus unmodified liposomes. The results showed that targeted modified liposomes were taken up by hepatocytes more effectively than unmodified liposomes.

[0146]

[0147] Example 3: Preparation and Validation of Dual-Targeted Modified Liposomes

[0148] 1. Preparation of LAL-A-tosylate intermediates

[0149] • Procedure Overview: LAL-A (100 mg, 0.13 mmol) was dissolved in anhydrous dichloromethane, p-toluenesulfonyl chloride (0.15 mmol) and triethylamine (0.15 mmol) were added, and the mixture was reacted at room temperature for 5 hours. The mixture was then washed, dried and rotary evaporated to obtain the LAL-A-tosylate intermediate.

[0150] 2. Two types of LAL-PEG substrates were prepared separately.

[0151] 2.1 Preparation of LAL-A-PEG1000-COOH

[0152] • Dissolve HO-PEG1000-COOH (molecular weight 1000, with COOH at the end) in anhydrous THF, and add NaH in an ice bath to generate PEG anions;

[0153] • Under anhydrous conditions, PEG anions were added dropwise to a LAL-tosylate solution and heated to 70°C for 8 hours to generate LAL-A-PEG1000-COOH, which was then extracted and purified to obtain the pure product.

[0154] 2.2 Preparation of LAL-A-PEG1000-NH2

[0155] Using the same method, HO-PEG1000-NH2 (terminus NH2) was dissolved in anhydrous THF and reacted in an ice bath. After SN2 reaction, LAL-A-PEG1000-NH2 was generated, and the product was obtained after purification.

[0156] 3. Targeted molecular access and dual modification

[0157] 3.1 Galactose Modification

[0158] • Dissolve LAL-A-PEG1000-COOH in anhydrous DMF, add EDC / NHS to activate the PEG terminal carboxyl group, then add 6-aminogalactose, react at room temperature to 40℃ for 15 hours, and purify by column chromatography to obtain galactose-modified lipid (denoted as LAL-A-PEG1000-Gal).

[0159] 3.2 Bile acid modification

[0160] • Dissolve LAL-A-PEG1000-NH2 in anhydrous DMF, and activate the carboxyl group in deoxycholic acid (dissolved in DMF and adjusted to pH 7.0) using the EDC / NHS method, so that it couples with the terminal amino group of LAL-A-PEG1000-NH2.

[0161] • After the reaction, the lipid was purified to obtain bile acid modified lipid (denoted as LAL-A-PEG1000-Bile).

[0162] 3.3 Mixing ratio

[0163] • According to the preset modification ratio, LAL-A-PEG1000-Gal and LAL-A-PEG1000-Bile are mixed in a 1:1 ratio to be used as dual-targeted modified lipids.

[0164] 4. Liposome assembly

[0165] • Component configuration:

[0166] ο Ionizable amino lipids (LAL-A): 40%,

[0167] ο DOPE (O-diol-dependent lipids): 17%,

[0168] οStructural lipids (cholesterol): 30%,

[0169] ο Targeted modified lipids (a mixture of LAL-A-PEG1000-Gal and LAL-A-PEG1000-Bile): 13%,

[0170] Preparation process: Each lipid component was dissolved in anhydrous ethanol in a set ratio to form a homogeneous organic phase; the target drug was dissolved in an aqueous phase of 50 mM sodium acetate buffer (pH 4.0); the organic phase and the aqueous phase were mixed at a volume ratio of 1:3, and a W / O emulsion was prepared by ultrasonic emulsification, which was then mixed with the aqueous phase (50 mM sodium acetate buffer, pH 4.0) to form a W / O / W type nanoemulsion; after removing ethanol by ultrafiltration, the emulsion was replaced with PBS, and finally sterilized by 0.22 μm filtration to obtain the final dual-modified liposomes.

[0171] • Following the assembly of liposomes described above, missing molecules were added to the component configuration, and the other steps were the same to obtain liposomes.

[0172] The components used and their recommended molar ratios are as follows: Ionizable amino lipids (LAL-A): 40%,

[0173] ο DOPE (O-diol-dependent lipids): 16%,

[0174] οStructural lipids (cholesterol): 30%,

[0175] ο Targeted modified lipids (a mixture of LAL-A-PEG1000-Gal and LAL-A-PEG1000-Bile): 13%,

[0176] Add 1% tracer molecule Cy5.

[0177] 5. Characterization and Verification

[0178] • Particle size and morphology determination: Dynamic light scattering (DLS) was used to ensure that the average particle size of liposomes was controlled between 40 and 80 nm (PDI < 0.2), and the morphology was observed by transmission electron microscopy (TEM).

[0179] • Cellular uptake assay: In HepG2 cells, the uptake of dual-modified liposomes and single-modified liposomes was compared. After being coated with the near-infrared fluorescent dye Cy5, the uptake was observed using a fluorescence microscope. The dual modification showed a synergistic targeting effect. Figure 4 ).

[0180] • Drug release response test: Simple release tests can be performed under different pH conditions to verify that the drug release rate is significantly accelerated in an acidic environment (pH 5.5), thus proving the pH responsiveness of the carrier.

[0181]

[0182] Additional Implementation Plan - Cell Membrane Coating Technology

[0183] 1. Cell membrane extraction

[0184] 1.1 Cell source and culture

[0185] • Selected from HepG2 hepatocytes

[0186] • After the cells were proliferated in DMEM containing 10% FBS to a confluence of 80%–90%, the cells were collected.

[0187] 1.2 Cell membrane extraction steps

[0188] • Wash the collected cells thoroughly with PBS to remove any culture medium residue.

[0189] • Resuspend the cells in 10mM Tris-HCl, pH 7.4, containing protease inhibitors, and let them stand for 10 minutes to allow the cells to swell and lyse.

[0190] • Repeated freeze-thaw cycles were used to mechanically disrupt the cell suspension, further promoting membrane structure rupture.

[0191] • Remove large cell nuclei and unlysed cells by heating the lysed cell suspension at 1000×g for 10 minutes.

[0192] • Transfer the supernatant to a new centrifuge tube and centrifuge at 20,000 g for 15 minutes to precipitate cell membrane fragments.

[0193] • After collecting the precipitate, resuspend it with PBS and wash it multiple times until other intracellular components are removed.

[0194] 1.3 Cell membrane nanofiber processing

[0195] • The washed cell membrane suspension was placed in a homogenizer and repeatedly squeezed through a 200nm polycarbonate membrane (using a micro extruder) to form uniform membrane vesicles and cell membrane nanofragments.

[0196] • The squeezed cell membrane is centrifuged and concentrated to obtain a cell membrane solution for coating.

[0197] 2. Liposomes and cell membrane encapsulation

[0198] 2.1 Pre-prepared liposome suspension

[0199] • Multilevel liver-targeting liposomes (containing ionizable amino lipids and targeted modified lipids, with a particle size controlled at 40-80 nm) prepared in Examples 1-3 above were used as the coating basis.

[0200] 2.2 Coating process

[0201] • Mix the pre-prepared liposome suspension with a certain amount of cell membrane suspension at a mass ratio of 1:1.

[0202] • Place the mixture in a 37°C water bath and let it stand for 30 minutes to allow the cell membrane to come into full contact with the liposome surface.

[0203] • To ensure uniform coating of the cell membrane on the liposome surface, a repeated squeezing method can be used. The specific procedure is as follows:

[0204] The mixture was loaded into a polycarbonate membrane extruder with a pore size of 200 nm, and after five repeated extrusions, the cell membrane and liposomes formed a tight fusion structure.

[0205] 2.3 Post-processing

[0206] • After coating is completed, the free uncoated cell membranes and small molecule impurities are removed by dialysis.

[0207] • Finally, the product was sterilized and filtered using a 0.22μm filter membrane to obtain liposome products coated with cell membranes.

[0208] 3. Characterization and Verification

[0209] 3.1 Particle size and morphology detection

[0210] • The particle size of coated liposomes is detected by dynamic light scattering (DLS). The particle size is usually slightly increased due to the coating of the cell membrane, but it should be kept at around 80-120 nm, and the PDI should be kept at a low value.

[0211] • The morphology of the coated liposomes was observed by transmission electron microscopy (TEM), confirming that the liposome surface has a uniform coating layer.

[0212] 3.2 Protein Characterization

[0213] • Western blotting was used to detect the presence and integrity of key target proteins on the cell membrane to verify the functional activity of the cell membrane coating.

[0214] 3.3 Target Function Validation

[0215] In vitro cell uptake experiments were conducted using HepG2 hepatocytes to compare the cell uptake rate of liposomes before and after coating. After coating with the near-infrared fluorescent dye Cy5, the cell uptake of liposomes was significantly increased after cell membrane coating, as observed under a fluorescence microscope. Figure 4 )

[0216] 4. Advantages and Applications

[0217] • Enhanced targeting and stability: The cell membrane coating provides a natural "coat" similar to the surface of the body's own cells, reducing the chances of being recognized and cleared by the body's immune system, while improving the binding efficiency with target cells through natural receptors.

[0218] • Improved circulation performance: The encapsulated cell membrane has good biocompatibility, which helps prolong the circulation time of liposomes in the blood and reduce adsorption and toxicity to non-target organs.

[0219] • Flexible combination strategy: This technology can be used in conjunction with previous targeted modifications (galactose, bile acids) to achieve multi-level and multi-modal targeted delivery, providing a comprehensive advantage for the treatment of liver diseases.

[0220]

[0221]

[0222] Example 4: Drug Release Response Mechanism Experiment

[0223] The purpose of this experiment is to verify the drug release characteristics of the multi-level liver-targeting liposomes prepared in this invention under different pH conditions, and to demonstrate that they remain stable in a neutral environment (pH 7.4) and can be triggered to release rapidly under acidic conditions (pH 5.5), thereby achieving precise control of drug release into target cells.

[0224] Brief description of experimental procedures

[0225] 1. Sample Preparation: The drug-loaded multi-stage liver-targeting liposomes prepared in the foregoing embodiments of this invention were used as experimental samples. 2 mL of the liposome suspension was placed into a dialysis bag with a suitable molecular weight cutoff.

[0226] 2. Preparation of Release Media: Prepare solutions of pH 7.4 PBS and pH 5.5 50mM sodium acetate buffer, respectively. Immerse the dialysis bags containing the liposome samples into their respective buffer solutions, ensuring that the dialysis bags are in full contact with the release media.

[0227] 3. Release Experiment: All samples were placed in a 37°C constant temperature water bath and magnetically stirred to simulate the in vivo environment. Predetermined sampling time points were set (0.5h, 1h, 2h, 4h, 6h, 12h, and 24h). At each time point, an appropriate amount of sample was taken from the buffer solution, and an equal amount of fresh buffer solution was added simultaneously to maintain a constant volume.

[0228] 4. Drug Detection and Data Analysis: The drug concentration of samples collected at each time point was determined using HPLC, and the cumulative release percentage was calculated based on a pre-established standard curve. Release curves were plotted based on the test results, and the release differences under pH 7.4 and pH 5.5 conditions were compared.

[0229] Results and Discussion: Experimental data are as follows Figure 3 The results showed that the drug release rate in the liposomes was low at pH 7.4 (cumulative release rate of about 20% within 12 hours); while in an acidic environment (pH 5.5), the drug release rate was significantly increased (cumulative release rate of over 80% within 12 hours). This result demonstrates the stability and responsiveness of the drug-loaded liposomes of this invention in an acidic environment, which is beneficial for triggering rapid drug release in an intracellular acidic environment, thereby improving therapeutic efficacy and reducing the risk of systemic side effects.

[0230] Results Evaluation

[0231] 1. Product Characterization

[0232] • Particle size and polydispersity were determined using dynamic light scattering (DLS). The average particle size of the final nanoliposomes was controlled between 40 and 80 nm, and the polydispersity index (PDI) was less than 0.2, demonstrating the uniformity of liposome particle size and good dispersibility. Figure 1 ).

[0233] • Morphological observation: Transmission electron microscopy (TEM) revealed that the liposomes exhibited a regular spherical structure with an intact lipid membrane and clear edges, confirming structural stability during self-assembly. Figure 2 ).

[0234] • Surface chemical analysis was performed using infrared spectroscopy to identify LAL-A-PEG1000 and its targeted modifications (galactose and bile acids). The NH stretching vibration at 3300 cm⁻¹ and the amide C=O stretching vibration at 1660 cm⁻¹ both indicated that LAL-A-PEG1000 was successfully modified with galactose. Similar amide characteristic peaks could also be observed in the infrared spectrum of LAL-A-PEG1000-Bile modified with deoxycholic acid.

[0235] 2. Functional performance testing

[0236] Time (hours) Cumulative release rate (%) at pH 7.4 Cumulative release rate at pH 5.5 (%) 0 0 0 0.5 3 12 1 5 18 2 8 30 4 12 50 6 15 65 12 18 80 24 20 85

Claims

1. A multi-stage liver-targeted drug delivery system, characterized in that, The system comprises components in the following molar ratio: Targeted modified ionizable amino lipids: Ionizable amino lipids: Auxiliary lipids: Structural lipids = 10-30: 30-60: 5-40: 15-55. This system consists of nanolipid particles in the form of a water-in-oil-in-water nanoemulsion. Among them, the ionizable amino lipids are compounds having the structure shown in formula (I): L1 and L2 may be the same or different, and each can be independently selected from O or C; When L1 or L2 is C, it has an R1 substituent. R1 and R2 may be the same or different, and each can be independently selected from H or one of the following structures: R3 and R4 may be the same or different, and each can be independently selected from H or OH; The targeted modified ionizable amino lipid: the hydroxyl groups in the ionizable amino lipid are linked to a polyethylene glycol derivative to form an ionizable amino lipid-PEG, wherein the polyethylene glycol derivative is selected from one of the following two types: (i) HO-PEG-COOH, which is polyethylene glycol with a carboxyl group at the end; (ii) HO-PEG-NH2, i.e., polyethylene glycol with an amino group at the end; and the linkage reaction adopts an SN2 reaction mechanism to generate a stable ether bond, while retaining the functional group at the end of the polyethylene glycol; the functional group is selected from COOH or NH2; The polyethylene glycol used is selected from PEG-500, PEG-1000, and PEG-2000; The terminal functional group of the polyethylene glycol is then linked to at least one target molecule via a chemical coupling reaction, the target molecule comprising: Galactose; Cholic acid, deoxycholic acid or their derivatives; Tracer molecules.

2. The system according to claim 1, characterized in that, The targeted modified ionizable amino lipids are prepared by the following method: (a) the hydroxyl groups of the ionizable amino lipids are first treated with p-toluenesulfonyl chloride to generate an ionizable amino lipid-tosylate intermediate; (b) the polyethylene glycol derivative is subjected to sodium hydride under anhydrous conditions to generate an ion from its internal alcohol groups; (c) the anion solution obtained in the previous step is reacted with the ionizable amino lipid-tosylate at 60-80°C for 4-12 hours, and the hydroxyl groups in the ionizable amino lipids react with the hydroxyl groups in the polyethylene glycol derivative to generate stable ether bonds through an SN2 reaction; (d) the product obtained in the previous step can be further reacted with the target molecule through EDC / NHS chemical coupling.

3. The system according to claims 1-2, characterized in that, The hydroxyl groups in the ionizable amino lipid react with the hydroxyl groups in the polyethylene glycol derivative to form stable ether bonds, resulting in the following structure: L1 and L2 may be the same or different, and each is independently selected from O or C; When L1 or L2 is C, it has an R1 substituent. R5 and R6 may be the same or different, and each is independently selected from H or one of the following structures; n is an integer between 20 and 30.

4. The system according to claims 1-3, characterized in that, The auxiliary lipids include dioleoylphosphatidylethanolamine; the structural lipids include cholesterol.

5. The system according to any one of claims 1-4, characterized in that, The liver-targeted modified amino PEG lipids are prepared by one or more of the following methods: (a) galactose-modified PEG lipids: 6-aminogalactose is reacted with PEG lipids containing carboxyl terminals via EDC / NHS coupling method to generate the ionizable amino lipid -PEG-Gal; (b) cholic acid-modified PEG lipids: deoxycholic acid or its derivatives are reacted with PEG lipids containing amino terminals via EDC / NHS chemical activation method to generate the ionizable amino lipid -PEG-Bile; (c) fluorescent dye-modified PEG lipids: fluorescent dyes retain carboxyl or amino groups and are reacted with PEG lipids containing amino or carboxyl terminals via EDC / NHS chemical activation method to generate the ionizable amino lipid -PEG-Bile.

6. A method for preparing a multi-stage liver-targeted drug delivery system according to any one of claims 1-2, characterized in that, Includes the following steps: a. Preparation of targeted modified ionizable amino lipids (i) Dissolve the ionizable amino lipids in anhydrous dichloromethane, add p-toluenesulfonyl chloride and triethylamine to convert its hydroxyl groups into ionizable amino lipid-tosylate intermediates; (ii) Use polyethylene glycol derivatives containing terminal COOH or terminal NH2, respectively, and add NaH in anhydrous tetrahydrofuran at 0-4°C to generate PEG anions, and the PEG used only utilizes its unprotected internal hydroxyl groups to participate in the reaction and retains the terminal function; (iii) Under strictly anhydrous conditions, add the PEG anion solution generated in step (ii) dropwise to the ionizable amino lipid-tosylate solution in step (i), heat to 60-80°C and react for 4-12 hours to generate ionizable amino lipid-PEG through SN2 reaction, wherein a stable ether bond is formed, while the terminal COOH or NH2 of PEG is retained for subsequent targeting molecule coupling; (iv) The terminal COOH or NH2 of the polyethylene glycol is then connected to the targeting molecule through a chemical coupling reaction; b. Assembling nanolipid particles (i) Dissolve ionizable amino lipids, auxiliary lipids and structural lipids with the targeted modified polyethylene glycol lipids obtained in step a in anhydrous ethanol to form a homogeneous organic phase; (ii) Mix the organic phase with an aqueous phase containing the target drug at a volume ratio of 1:1 to 5 and prepare an oil-in-water emulsion by ultrasonic emulsification or microfluidic technology, and then form a water-in-oil-in-water nanoemulsion with water. The average particle size of the resulting multi-stage liver-targeted drug delivery system was 40–80 nm.

7. The system according to claim 1, characterized in that, The system is applicable to the treatment of liver-related diseases, including liver cancer, viral hepatitis, liver fibrosis, and drug-induced liver injury.

8. The system according to claim 1, characterized in that, The system further engineered the obtained lipid nanoparticles by coating them with cell membranes or by combining them with oleic acid-modified superparamagnetic nanoparticles.

9. The preparation method according to claim 6, characterized in that, The target drugs include: siRNA, mRNA, and chemotherapy drugs.

10. An application of the system according to claim 1 in animal experiments, specifically comprising: Used for liver tumor treatment in animal models, for studying the mechanism of liver fibrosis and the efficacy of anti-fibrotic treatment, and for verifying drug delivery and gene editing efficiency, with delivery efficiency monitored by near-infrared in vivo imaging.

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