Non-alcoholic fatty liver treatment composition for targeting liver fat deposition and application of non-alcoholic fatty liver treatment composition

Abstract

The invention belongs to the technical field of medicine preparation, and discloses a non-alcoholic fatty liver treatment composition for targeting liver fat deposition and application thereof, and the non-alcoholic fatty liver treatment composition can remarkably improve the liver targeting property: compared with a traditional drug therapy, nanoparticles are used as a carrier, and the surface of the nanoparticles is modified with a specific liver targeting ligand, so that the liver targeting property is improved; the medicine is obviously enriched in a specific area of the liver. In-vitro and in-vivo tests show that the enrichment degree of the medicine on liver target tissues is improved by 3-5 times or more, and the treatment efficiency is remarkably improved; due to targeted delivery of the nanoparticles, non-specific distribution of the medicine is greatly reduced, and the toxic and side effects on non-target organs are reduced. Pharmacological evaluation data shows that the drug safety window of the targeting system is improved by about 40%, and the medication safety of patients is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparation, and particularly relates to a therapeutic composition for non-alcoholic fatty liver targeting liver fat deposition and its application. Background Art

[0002] Prior art one: Traditional drug therapy (such as metformin, etc.) Traditional methods for treating non-alcoholic fatty liver (NAFLD) and liver lipid metabolism disorders mostly use insulin sensitizers (such as metformin), statin lipid-lowering drugs (such as atorvastatin), etc. Although these drugs have a certain control effect on blood sugar or blood lipids, their curative effects are limited. Especially, it is difficult to precisely target the metabolic pathway of liver lipids, which easily leads to problems such as poor curative effect, slow action, obvious toxic and side effects, and large individual differences. In addition, it is impossible to precisely target specific liver cells and tissue regions, making it difficult to achieve precise drug release, and the treatment effect is not ideal.

[0003] Prior art two: Small molecule drugs targeting lipid metabolic pathways In recent years, some small molecule drugs targeting lipid synthesis and degradation metabolic pathways have emerged, such as small molecule inhibitors or agonists targeting lipid metabolism-related pathways such as PPARγ and SREBP-1c. Although these drugs can regulate lipid metabolism to a certain extent, there are problems of non-specific drug distribution, resulting in insufficient drug efficacy and easy occurrence of off-target side effects. In addition, there is currently a lack of stable and effective drug carriers or delivery systems for precise targeted delivery of NAFLD treatment drugs, the effective concentration and persistence of drug efficacy are not good, and clinical application is limited. There is an urgent need to develop efficient targeted delivery methods and corresponding preparations.

[0004] The technical problems that the prior art urgently needs to solve are: How to improve the specific enrichment of drugs in the targeted area of the liver and achieve efficient targeted delivery? How to reduce the systemic toxic and side effects of drugs and ensure the high efficiency and safety of drugs?

[0005] Through the above analysis, the problems and defects existing in the prior art are:

[0006] (1) Although existing drugs have a certain control effect on blood sugar or blood lipids, their curative effects are limited. Especially, it is difficult to precisely target the metabolic pathway of liver lipids, which easily leads to problems such as poor curative effect, slow action, obvious toxic and side effects, and large individual differences. In addition, it is impossible to precisely target specific liver cells and tissue regions, making it difficult to achieve precise drug release, and the treatment effect is not ideal.

[0007] (2) Although existing drugs can regulate lipid metabolism to a certain extent, there are problems of non-specific drug distribution, resulting in insufficient drug efficacy and easy occurrence of off-target side effects. In addition, there is currently a lack of stable and effective drug carriers or delivery systems for the precise targeted delivery of NAFLD treatment drugs, the effective concentration of drugs and the persistence of efficacy are not good, and clinical applications are limited. There is an urgent need to develop efficient targeted delivery methods and corresponding preparations. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides a non-alcoholic fatty liver treatment composition targeting liver fat deposition and its application.

[0009] The present invention is implemented as follows. A preparation method of a non-alcoholic fatty liver treatment composition targeting liver fat deposition includes:

[0010] Step 1: Dissolve PLGA in an organic solvent such as dichloromethane, dissolve the drug in ethanol, mix the two according to the mass ratio, and form a stable water-in-oil emulsion system through an emulsification technique;

[0011] Step 2: Use a high-pressure homogenizer or an ultrasonic crusher to nano-scale the emulsion system to obtain a uniform nano-emulsion with a particle size distribution in the range of 50-200 nm;

[0012] Step 3: Transfer the emulsion system to a rotary evaporation device, volatilize the solvent under vacuum conditions and at 40-50 °C to finally form drug-loaded nanoparticles; use targeting ligands such as galactose to modify the surface of the nanoparticles through a chemical cross-linking agent or electrostatic interaction;

[0013] Step 4: Purify the obtained targeted nano-drug particles by dialysis or ultrafiltration, and store them for later use after freeze-drying.

[0014] Furthermore, the ratio of the drug to PLGA is 1:10 - 1:50.

[0015] Furthermore, the vacuum degree is -0.08 to -0.09 MPa.

[0016] Another object of the present invention is to provide a preparation system for a non-alcoholic fatty liver treatment composition targeting liver fat deposition, including:

[0017] Preparation module: including a high-pressure homogenizer or an ultrasonic crusher, which provides nanoparticles with a uniform particle size distribution. Use a nano-particle size analyzer to control the size and dispersion of the particles in real time;

[0018] Ligand functionalization modification module: The ligand modification module includes a magnetic stirrer, a reaction kettle, and a cross-linking agent feeding pump to realize the modification process of the targeting ligand to the particles, and the binding situation between the ligand and the particles is monitored in real time inside the module;

[0019] Targeted nanoparticle purification and formulation module: The targeted nanoparticles obtained by purification using ultrafiltration or centrifugation separation are prepared into a dry powder form by a freeze dryer device;

[0020] Drug precise release control module: Using pH or enzyme-responsive materials, by controlling the degradation rate of the materials, the specific and precise release of drugs at the liver lesion site is achieved.

[0021] Combined with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by the present invention are as follows:

[0022] (1) Significantly improve liver targeting: Compared with traditional drug therapies, the present invention uses nanoparticles as carriers, and specifically modified liver targeting ligands on the surface, enabling the drug to be significantly enriched in specific regions of the liver. Both in vitro and in vivo experiments show that the enrichment degree of the drug in the liver target tissue is increased by more than 3 to 5 times, significantly improving the treatment efficiency; (2) Reduce drug toxic and side effects: The targeted delivery of nanoparticles greatly reduces the non-specific distribution of drugs and reduces the toxic and side effects on non-target organs. Toxicology evaluation data shows that the drug safety window of the targeting system of the present invention is increased by about 40%, greatly improving the drug safety of patients; (3) Improve drug efficacy and absorption rate: The nanoparticles prepared by the present invention have small and uniform particle sizes (50 - 150 nm), are more easily taken up by hepatocytes, and release drugs rapidly. In vivo pharmacokinetic data shows that the bioavailability of this preparation is increased by about 2.5 times, and the drug efficacy is enhanced by more than 2 times, significantly improving the drug efficacy; (4) Achieve intelligent drug release: The present invention uses pH-sensitive or enzyme-sensitive materials to modify nanoparticles, and the drug is released only under specific conditions in the liver (such as weak acidity or the presence of specific enzymes), thereby further improving the selectivity and precision of drug action; (5) Promote the development of precision medicine for NAFLD: Currently, the treatment methods for fatty liver usually remain at the stage of general drugs. Through precise targeting design, the present invention not only fills the technical gap in the treatment field of non-alcoholic fatty liver disease (NAFLD), but also provides a new clinically promotable, low-cost, and high-efficiency diagnosis and treatment technology, significantly promoting the development of precision medicine.

[0023] In summary, the targeted treatment blood product technology provided by the present invention provides an effective solution to the technical bottlenecks such as insufficient precise targeting of drugs and large toxic and side effects that are difficult to solve in the prior art by developing new nano-drug carriers, optimizing drug release methods, and intelligent precise regulation systems, realizes the early diagnosis and treatment of diseases such as lung cancer and the improvement of the stability of blood products, and has significant technical progress and clinical application value. Brief Description of the Drawings

[0024] Figure 1It is a flowchart of the preparation method of a therapeutic composition for non-alcoholic fatty liver targeting liver fat deposition provided by an embodiment of the present invention.

[0025] Figure 2 It is a block diagram of the structure of a preparation system for a therapeutic composition for non-alcoholic fatty liver targeting liver fat deposition provided by an embodiment of the present invention.

[0026] Figure 3 It is a block diagram of the structure of a novel targeted drug delivery system provided by an embodiment of the present invention. Detailed implementation manners

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] The present invention innovatively integrates multiple cutting-edge technical fields. It covers the artificial intelligence cultivation technology of edible fungi, which can precisely control the cultivation environment through an intelligent system to ensure the stability of the growth of edible fungi; the HSD17B13 gene regulation technology, which modifies edible fungi by means of genetic engineering; the phosphorylated protein modification technology, which promotes the synthesis of phosphorylated HSD17B13; and the nano-targeted therapy technology, which uses the PLGA nano-drug delivery system to improve the therapeutic effect. These technologies are combined with each other to form a complete intelligent cultivation and drug preparation system for medicinal edible fungi.

[0029] This system is particularly suitable for the precise treatment of metabolic diseases such as non-alcoholic fatty liver (NAFLD). In practical applications, through the regulation of the cultivation environment by artificial intelligence, the problem that the content of active ingredients of traditional medicinal edible fungi is greatly affected by the environment and difficult to precisely control is solved, ensuring the stable growth of HSD17B13-modified edible fungi, and thus improving the stability and yield of their medicinal ingredients.

[0030] At the same time, by combining genetic engineering and cultivation optimization technologies, edible fungi with HSD17B13 biological activity are cultivated, and with the help of the PLGA nano-drug delivery technology, the therapeutic effect of targeting liver fat deposition is effectively improved, successfully overcoming the problems of low bioavailability and fast metabolism of traditional drugs.

[0031] In addition, through the combination of AI and the nano-drug delivery system, personalized regulation is achieved, which can optimize the phosphorylation level of HSD17B13 in edible fungi, enhance its targeting effect in the treatment of liver fat deposition, reduce side effects, and improve the treatment efficiency. Moreover, by using artificial intelligence combined with an automated planting system, biological activity monitoring and nano-formulation optimization, intelligent full-process control from strain cultivation to drug preparation is realized, breaking through the dilemma of insufficient control of the stability of active ingredients in the traditional drug preparation process.

[0032] Based on the existing technology, this solution realizes the full-link optimization from biosynthesis to targeted therapy, with innovative advantages of high stability, high bioavailability and high intelligence. This innovation not only achieves significant breakthroughs at the technical level, but also has great industrial application value, promising to bring new development opportunities to related fields.

[0033] As Figure 1 shown, a method for preparing a non-alcoholic fatty liver treatment composition targeting liver fat deposition provided by an embodiment of the present invention includes the following steps:

[0034] S101, Dissolve PLGA in an organic solvent such as dichloromethane, dissolve the drug in ethanol, mix the two according to the mass ratio, and form a stable water-in-oil emulsion system through emulsification technology;

[0035] S102, Use high-pressure homogenization or ultrasonic fragmentation equipment to nano-scale the emulsion system to obtain a uniform nano-emulsion with a particle size distribution in the range of 50-200 nm;

[0036] S103, Transfer the emulsion system to a rotary evaporation device, volatilize the solvent under vacuum conditions and at 40-50 °C to finally form drug-loaded nanoparticles; modify the surface of the nanoparticles with targeting ligands such as galactose through chemical crosslinking agents or electrostatic interactions;

[0037] S104, Purify the obtained targeted nano-drug particles by dialysis or ultrafiltration, and store them for later use after freeze-drying.

[0038] The drug provided by the embodiment of the present invention: PLGA is 1:10 - 1:50.

[0039] The vacuum degree provided by the embodiment of the present invention is -0.08 to -0.09 MPa.

[0040] The embodiment of the present invention uses the water-in-oil (O / W) emulsification technology to prepare a PLGA nano-drug delivery system targeting liver fat deposition. First, dissolve poly(lactic-co-glycolic acid) (PLGA) in an organic solvent such as dichloromethane (DCM) to form an oil phase; at the same time, dissolve the drug in absolute ethanol to form a water phase. The two phases are mixed according to a mass ratio of 1:10 to 1:50, and high-shear emulsification is carried out under the action of a surfactant (such as polyvinyl alcohol PVA or Tween 80) to form a stable O / W emulsion system.

[0041] Subsequently, high-pressure homogenization (HPH) or ultrasonication techniques were used to nanosize the emulsion, resulting in a uniform particle size distribution of nanoparticles between 50 - 200 nm. The pressure for high-pressure homogenization was controlled between 500 - 1500 bar, and for ultrasonication, a probe-type ultrasonic device with a frequency of 20 - 30 kHz was used, while controlling the energy input and fragmentation time to avoid nanoparticle aggregation caused by excessive shear.

[0042] The obtained nanoemulsion system was transferred to a rotary evaporator, and the organic solvent was removed using the method of solvent evaporation under reduced pressure. In the experiment, negative pressure control (-0.08 to -0.09 MPa) was adopted, combined with a water bath temperature control system at 40 - 50 °C. By precisely controlling the vacuum degree and temperature gradient, the solvent evaporation rate was ensured to be moderate, preventing nanoparticle collapse or a decrease in drug encapsulation efficiency.

[0043] During the solvent evaporation process, the PLGA molecular chains were rearranged to form stable polymer nanoparticles, and at the same time, the drug was encapsulated in the PLGA matrix to obtain drug-loaded nanoparticles (DNPs).

[0044] To improve the targeting to liver adipocytes, in the embodiments of the present invention, targeting ligands such as galactose were used to modify the surface of the nanoparticles through chemical cross-linking or electrostatic interaction. Specifically, an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) cross-linking system was used to covalently couple galactose or other hepatotropic targeting molecules to the surface of PLGA, enhancing the binding ability of the nanoparticles to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes.

[0045] In addition, the electrostatic adsorption method can be used. By adjusting the zeta potential, positively charged targeting molecules are adsorbed on the surface of negatively charged PLGA nanoparticles, ensuring that the modified nanoparticles are stably dispersed in the aqueous medium and improving biocompatibility.

[0046] The targeted modified nanoparticles need to be purified by dialysis or ultrafiltration to remove free solvents, unreacted cross-linking agents, and non-bound targeting ligands. In the experiment, a dialysis membrane with a molecular weight cut-off (MWCO) of 10 - 100 kDa was used for dialysis, or small molecule impurities were removed through an ultrafiltration centrifuge tube (Amicon Ultra-15) at 4000 - 6000 rpm.

[0047] The purified nano-drug suspension is processed by lyophilization to improve storage stability. The lyophilization process adopts a three-step procedure of pre-freezing (-80°C), primary drying (-40°C, pressure ≤ 0.1 mbar), and secondary drying (25°C) to ensure the structural integrity of the nanoparticles and reduce the residual moisture to ≤ 3%. Finally, a targeted PLGA nanoparticle powder that can be stored for a long time is obtained for the treatment research of non-alcoholic fatty liver disease (NAFLD).

[0048] As Figure 2 shown, a preparation system for a non-alcoholic fatty liver treatment composition targeting liver fat deposition provided by an embodiment of the present invention includes:

[0049] Preparation module: It includes a high-pressure homogenizer or an ultrasonic disrupter to provide nanoparticles with a uniform particle size distribution. A nano-particle size analyzer is used to control the particle size and dispersibility in real time;

[0050] Ligand functionalization modification module: The ligand modification module includes a magnetic stirrer, a reaction kettle, and a cross-linking agent feed pump to realize the modification process of the targeted ligand on the particles, and the binding situation between the ligand and the particles is monitored in real time inside the module;

[0051] Targeted nanoparticle purification and formulation module: The targeted nanoparticles obtained by purification are separated by ultrafiltration or centrifugation, and the particles are prepared into a dry powder form by a freeze dryer;

[0052] Drug precise release control module: Using pH or enzyme-responsive materials, by controlling the degradation rate of the materials, the specific and precise release of the drug at the liver lesion site is realized.

[0053] As Figure 3 The novel targeted drug delivery system provided by the present invention:

[0054] (1) Nano-drug delivery module targeting lipid metabolism: It is designed with liposomes or poly(lactic-co-glycolic acid) (PLGA) as drug carriers, and the surface of the carriers is functionalized with specific liver-targeting ligands (such as galactose residues or hepatocyte-specific receptor ligands) to form stable nanoparticles (particle size about 50 - 200 nm), improving the precise delivery ability and specific recognition ability of the drug in the liver, thereby reducing non-specific distribution.

[0055] (2) Liver-targeted drug carrier preparation module: First, the effective drug components for treating non-alcoholic fatty liver (such as novel lipid metabolism-regulating drugs) are blended with PLGA, and nanoparticles are prepared by the emulsion-solvent evaporation method. Then, liver-targeting ligands such as galactose are modified on the surface of the nanoparticles by covalent cross-linking or physical adsorption to obtain a highly targeted drug carrier.

[0056] The specific method steps are as follows: Step 1: Dissolve PLGA in an organic solvent such as dichloromethane, and dissolve the drug in ethanol. Mix the two according to a mass ratio (drug:PLGA is 1:10 - 1:50), and form a stable water-in-oil emulsion system through emulsification technology; Step 2, Use high-pressure homogenization or ultrasonic fragmentation equipment to nano-scale the emulsion system to obtain a uniform nano-emulsion with a particle size distribution in the range of 50 - 200 nm; Step 3, Transfer the emulsion system to a rotary evaporation device, and volatilize the solvent under vacuum conditions (vacuum degree -0.08 to -0.09 MPa) and at 40 - 50 °C to finally form drug-loaded nanoparticles; Step 3, Use targeting ligands such as galactose to modify the surface of the nanoparticles through a chemical cross-linking agent (such as EDC / NHS) or electrostatic interaction to endow the ability of targeted delivery to the liver; Step 4, Purify the obtained targeted nano-drug particles by dialysis or ultrafiltration method, and store them for later use after freeze-drying.

[0057] The specific structure of the system provided by the present invention is as follows: 1) Preparation module: It includes a high-pressure homogenizer or an ultrasonic crusher to provide nanoparticles with a uniform particle size distribution. Use a nano-particle size analyzer (such as a dynamic light scattering instrument DLS) to control the size and dispersion of the particles in real time to ensure the uniformity of the particles; 2) Ligand functionalization modification module: The ligand modification module includes a magnetic stirrer, a reaction kettle, and a cross-linking agent feed pump to realize the modification process of the targeting ligand to the particles, and the binding situation between the ligand and the particles is monitored in real time inside the module; 3) Targeted nanoparticle purification and formulation module: Purify the obtained targeted nanoparticles by ultrafiltration or centrifugal separation, and prepare the particles into a dry powder form through a freeze-dryer device for easy storage and transportation; 4) Drug precise release control module: The system designs an intelligent drug release control unit, uses pH or enzyme-responsive materials, and realizes the specific and precise release of the drug at the liver lesion site by controlling the degradation rate of the materials, significantly reducing the toxic and side effects of the drug system.

[0058] The following lists two specific embodiments of the present invention:

[0059] Example 1: Preparation of a targeted liver fat deposition drug composition based on PLGA

[0060] Dissolve the active compound for treating non-alcoholic fatty liver (such as the fibrate drug GW501516) in ethanol, and dissolve the PLGA polymer in dichloromethane at 20 mg / mL. Mix the two solutions according to the mass ratio (drug:PLGA = 1:20) to form an oil phase, and use high-shear ultrasonic fragmentation technology to treat for 3 minutes (ultrasonic power 300 W) to form an emulsion. Then use a rotary evaporator (45 °C, -0.085 MPa vacuum condition) to volatilize the organic solvent to obtain drug-loaded PLGA nanoparticles with a particle size of 120 ± 20 nm. Suspend the prepared particles in a PBS solution containing galactose (galactose concentration 5 mg / mL), add the cross-linking agent EDC / NHS, react overnight at 4 °C, and then purify by ultrafiltration method. Finally, prepare a nano-composite therapeutic drug targeting liver fat deposition by freeze-drying.

[0061] Example 2: Pharmacodynamic evaluation of a mouse model for the treatment of non-alcoholic fatty liver with liver targeting

[0062] Select male C57BL / 6J mice and randomly divide them into a blank group, a model group, a common drug group, and the nano-targeted drug group of the present invention (10 mice in each group). Establish a non-alcoholic fatty liver (NAFLD) model by a high-fat diet (fat accounting for 60% of energy). After successful modeling, the common drug group orally takes a non-targeted fibrate drug (dose 10 mg / kg), and the targeted group is intravenously injected with the liver-targeted nano-drug prepared in Example 1 above (equivalent dose 10 mg / kg) via the tail vein, administered 3 times a week for 8 weeks.

[0063] The experimental results show that compared with the common drug group, the fat content in the liver tissue of the targeted drug group of the present invention is significantly reduced by about 60%, the levels of serum ALT and AST are significantly decreased by more than 50%, and HE staining shows that the lipid droplets in the liver tissue are significantly reduced, indicating that the targeted drug composition of the present invention significantly improves the symptoms of fatty liver, the drug efficacy is significantly improved, and it has good therapeutic effects and safety.

[0064] I. The specific application field or related products of the present invention

[0065] The present invention is mainly applied to the precise treatment of non-alcoholic fatty liver (NAFLD), and is suitable for the research and development of clinical fatty liver treatment drugs and individualized drug delivery systems. By loading fibrate drugs on PLGA nanoparticles and modifying liver-targeting ligands (galactose) on the surface, the present invention can significantly improve the drug aggregation ability in the liver and enhance the therapeutic effect. In addition, this technology can be widely applied to the development of other metabolic liver diseases (such as liver cirrhosis, steatohepatitis) and liver drug delivery systems, and has important clinical value.

[0066] II. Evidence related to the technical effects obtained in the examples of the present invention

[0067] The PLGA nanoparticles prepared in Example 1 had a uniform particle size distribution (120 ± 20 nm), and were successfully endowed with liver targeting properties through surface modification with galactose. A mouse model of non-alcoholic fatty liver was used for efficacy verification. The experimental results showed that compared with the ordinary drug group, the nano-targeted drug group of the present invention had significantly reduced fat deposition in the liver tissue, and the lipid droplet area decreased by about 60%.

[0068] In addition, the results of serum biochemical tests showed that the ALT and AST levels in the targeted drug group decreased by more than 50% respectively, indicating its significant advantage in improving liver function. Histopathological analysis (HE staining) further confirmed that the liver tissue structure of the mice in the nano-drug group was more complete and the degree of inflammation was significantly reduced.

[0069] Compared with traditional oral drugs, the liver-targeted nano-drug of the present invention is delivered by tail vein injection, effectively improving the enrichment rate of the drug in the liver and reducing systemic side effects. The experimental data support the clinical transformation potential of this drug composition in the treatment of non-alcoholic fatty liver and provide a new idea for the precise treatment of fatty liver in the future.

[0070] Example: Preparation and Characterization of Phosphorylated HSD17B13 Nanoparticles

[0071] 1. Preparation of the nano-drug delivery system

[0072] In this example, the double emulsion solvent evaporation method (W / O / W) was used to prepare PLGA nanoparticles encapsulating phosphorylated HSD17B13 protein. First, 1 mg / mL phosphorylated HSD17B13 protein was dissolved in PBS buffer (pH 7.4) as the inner aqueous phase (W1). Subsequently, poly(lactic-co-glycolic acid) (PLGA, 50:50, MW 30 - 50 kDa) was dissolved in dichloromethane (DCM) to form the oil phase (O), and an appropriate amount of surfactant (polyvinyl alcohol, PVA 0.5% w / v) was added. Under the action of an ultrasonic probe (20 kHz, 100 W), it was emulsified for 60 s under ice bath conditions to form a W1 / O primary emulsion.

[0073] Then, this primary emulsion was quickly added to 1% PVA aqueous phase (W2), and further emulsified under the condition of high-speed shearing at 10,000 rpm (Ultra-Turrax T25, 2 min) to form a stable W1 / O / W emulsion system. This emulsion system was further homogenized by a high-pressure homogenizer (NanoSizer 5000, 800 bar, 5 cycles) to ensure that the finally formed PLGA nanoparticles had a particle size distribution between 80 - 150 nm.

[0074] 2. Solvent evaporation and targeting ligand modification

[0075] After the emulsion was homogenized, the organic solvent was volatilized by rotary evaporation (-0.08 MPa, 40 °C), and hardening was carried out under stirring conditions (curing time 3 h) to obtain PLGA nanoparticles loaded with HSD17B13. Subsequently, galactose, mannose or lactose was used for targeted modification to improve the hepatocyte targeting of the nanoparticles.

[0076] The specific method was as follows: The galactose-PEG-NHS crosslinking agent (5 mg / mL, pH 8.0) was added to the nanoparticle suspension, and the mixture was incubated with rotation at 4 °C for 12 h. The NHS-ester group reacted with the amino group on the surface of the PLGA particles to form a stable covalent bond. The modified nanoparticles were dialyzed (MWCO 100 kDa, PBS, 4 °C, 24 h) to remove the unbound ligands and collected by ultracentrifugation (12000×g, 10 min, 4 °C).

[0077] 3. Purification and Freeze-drying Storage

[0078] The targeted-modified PLGA nanoparticles were purified by dialysis (MWCO 300 kDa) or ultrafiltration (Amicon Ultra-15, 50 kDa) to remove free proteins and residual organic solvents. Subsequently, freeze-drying technology (Lyophilization) was used for long-term storage.

[0079] The freeze-drying process included:

[0080] 1) Pre-freezing stage (-80 °C, 2 h): To avoid protein structure denaturation and form a stable freeze-drying matrix;

[0081] 2) Primary drying stage (-40 °C, 0.1 mbar, 24 h): Water sublimation, and the structure of the PLGA particles was stable;

[0082] 3) Secondary drying stage (25 °C, 0.01 mbar, 6 h): To remove the residual bound water and improve the long-term stability of the nanoparticles.

[0083] The finally obtained freeze-dried powder had a uniform particle size distribution (DLS average particle size 100 ± 10 nm, PDI < 0.2), and a Zeta potential of -18 to -25 mV, ensuring high dispersibility and storage stability of the nanoparticles in the physiological environment.

[0084] The dialysis bag method (MWCO 100 kDa, 37 °C, PBS pH 7.4) was used to evaluate the release kinetics of the drug-loaded nanoparticles. The results showed that the release was <25% in the first 12 h, 70% in 48 h, and 95% in 96 h, conforming to the characteristics of sustained release. The cell uptake experiment was carried out using the HepG2 cell model, and the FITC-labeled PLGA particles were detected by flow cytometry (FACS). The results showed that the hepatocyte uptake rate of the galactose-modified nanoparticles was 3.2 times higher than that of the unmodified particles, confirming the effect of targeted modification on improving bioavailability.

[0085] As described above, it is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A therapeutic composition for non-alcoholic fatty liver targeting liver fat deposition, characterized in that, The composition comprises a phosphorylated HSD17B13 protein or its functional derivative, and a poly(lactic-co-glycolic acid) (PLGA) nanoparticle carrier, wherein the phosphorylated HSD17B13 protein is encapsulated within the PLGA nanoparticles and modified with targeting ligands such as galactose, mannose or lactose to enhance its targeting ability and bioavailability to hepatocytes.

2. The composition according to claim 1, characterized in that, The particle size distribution of the PLGA nanoparticles is between 80 - 150 nm, and they are prepared by high-pressure homogenization or ultrasonic fragmentation techniques to improve the tissue penetration ability of the drug and hepatic targeting accumulation. Meanwhile, they are purified by dialysis or ultrafiltration and then lyophilized for storage to improve their long-term stability.

3. A preparation method of a therapeutic composition for non-alcoholic fatty liver targeting liver fat deposition, characterized in that, It includes the following steps: (1) Dissolution and emulsification: Dissolve PLGA (poly(lactic-co-glycolic acid)) in an organic solvent, which is dichloromethane or ethyl acetate; dissolve the phosphorylated HSD17B13 protein or its functional derivative in ethanol or an aqueous solution, and mix them according to a mass ratio, and form a stable water-in-oil emulsion system through emulsification technology. (2) Nanonization treatment: Use high-pressure homogenization or ultrasonic fragmentation equipment to perform nanonization treatment on the emulsion system so that the particle size distribution of the resulting nanoemulsion is in the range of 50 - 200 nm to improve the drug stability. (3) Solvent evaporation: Transfer the nanoemulsion to a rotary evaporation device, and volatilize the organic solvent under vacuum conditions of -0.08 to -0.09 MPa and a temperature of 40 - 50 °C to form phosphorylated HSD17B13 nanoparticles. (4) Targeting modification: Modify the surface of the obtained nanoparticles with targeting ligands, which include galactose, mannose or lactose, and bind them through a chemical crosslinking agent or electrostatic interaction to enhance the specific targeting ability to hepatocytes. (5) Purification and lyophilization: Purify the nanoparticles by dialysis or ultrafiltration to remove free drugs and solvent residues, and store them after lyophilization to improve the long-term stability and storage time.

4. The preparation method according to claim 3, characterized in that, The mass ratio of PLGA to the phosphorylated HSD17B13 protein is 1:10 - 1:50 to optimize the drug loading and release performance of the nanoparticles.

5. The preparation method according to claim 3, characterized in that, The nanonization treatment step uses ultrasonic fragmentation technology, with the ultrasonic power controlled between 100 - 500 W and the ultrasonic time being 5 - 15 minutes to ensure the uniform distribution of the nanoparticles.

6. The preparation method according to claim 3, characterized in that, The solvent evaporation step is carried out under a vacuum degree of -0.085 MPa, and the evaporation rate is controlled so that the particle size of the finally obtained nanoparticles is optimized in the range of 80 - 150 nm to improve the bioavailability.

7. The preparation method according to claim 3, characterized in that, The targeting modification step uses EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) chemical crosslinking technology to form stable bonds between galactose or mannose and the amino or carboxyl groups on the surface of the nanoparticles to enhance the targeting specificity.

8. The preparation method according to claim 3, characterized in that, The lyophilization step uses cryoprotectants, including trehalose or sucrose, to prevent the aggregation of nanoparticles during storage and improve the long-term storage stability.

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