Drug-loaded recombinant high-density lipoprotein nanoparticles for treating obesity, oral drug delivery system and preparation method and application thereof

Abstract

The present application relates to a kind of drug-loaded recombinant high-density lipoprotein nanoparticles for treating obesity, oral drug delivery system and its preparation method and application, the drug-loaded recombinant high-density lipoprotein nanoparticles include the following ingredients: apolipoprotein, phospholipid, cholesterol, fatty acid, cholesteryl ester and drug.It utilizes the advantages of recombinant high-density lipoprotein nanoparticles, such as stable nature, uniform size, has the advantage of regulating adipocyte metabolism and the natural targeting advantage of adipocyte, can be completely degraded without causing immune response, and can avoid the recognition and removal of reticuloendothelial system;And the fat-reducing drug loaded therein can realize the controlled release and long-acting sustained release of drug, improve the bioavailability of drug, and can reduce the accumulation of fat-reducing drug in non-target tissue, reduce the toxic and side effects of drug, and ultimately achieve safe and effective fat-reducing effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a drug-loaded recombinant high-density lipoprotein nanoparticle for treating obesity and its preparation method, an oral drug delivery system for treating obesity and its preparation method, and its application in the preparation of fat-reducing drugs. Background Technology

[0002] Obesity is a chronic and complex global epidemic that can lead to a variety of chronic and metabolic diseases. Clinically, the drug treatment options available to obese patients are very limited, and there are currently no particularly safe and effective weight-loss drugs. Traditional treatments such as lifestyle interventions, drug therapy, and liposuction have several drawbacks, including limited effectiveness, significant side effects, infection risks, and difficulty in maintaining the treatment. Therefore, developing novel drug delivery strategies to replace traditional weight-loss methods and achieve safer and more effective obesity treatment is of paramount importance. In recent years, the browning of white adipocytes (WAT) has attracted increasing attention as an alternative method for stimulating energy dissipation in weight loss. Promoting the formation of brown adipocytes can effectively reduce energy storage and control weight, and holds promise as a new approach to treating obesity.

[0003] Rosiglitazone (Rosi) belongs to the thiazolidinedione (TZD) family and is an effective inducer for the conversion of white adipocytes into brown adipocytes, with existing technologies reporting related applications. For example, CN108350423A reports that rosiglitazone can be used to produce brown / beige adipocytes from subcutaneous white adipose tissue cells. However, due to off-target effects of rosiglitazone, which can have adverse effects on other organs (including the liver, kidneys, and brain), its clinical application is limited.

[0004] High-density lipoprotein (HDL), as a natural nanoparticle, can load and transport cholesterol in the body, playing an important role in reducing lipid accumulation in tissues. However, natural HDL is difficult to synthesize, purify, and isolate, severely limiting its applications. In recent years, various peptide mimics have been developed to replace ApoA-1, a key component in its preparation process, to prepare recombinant high-density lipoprotein nanoparticles (rHDL). These rHDL nanoparticles can transport various substances (drugs, nucleic acids, peptides, etc.) in vivo, without easily inducing immune responses and reducing drug toxicity. Their ultra-small size facilitates tissue penetration and accumulation, and they can target cells and tissues with high expression of type B scavenger receptor (SR-BI), such as adipocytes and other tissues. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a drug-loaded recombinant high-density lipoprotein nanoparticle for treating obesity and its preparation method, an oral drug delivery system for treating obesity and its preparation method, and its application in the preparation of fat-reducing drugs.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a drug-loaded recombinant high-density lipoprotein nanoparticle for treating obesity, wherein the drug-loaded recombinant high-density lipoprotein nanoparticle comprises the following components: apolipoprotein, phospholipid, cholesterol, fatty acid, cholesterol ester and drug.

[0008] The drug-loaded recombinant high-density lipoprotein nanoparticles involved in this invention are a synergistic fat-reducing drug delivery system. They utilize the stable and uniform size of recombinant high-density lipoprotein nanoparticles, their advantages in regulating adipocyte metabolism and their natural targeting advantage to adipocytes. These nanoparticles can be completely degraded without triggering an immune response, are non-toxic to the human body, safe and reliable, and can avoid recognition and clearance by reticuloendothelial tissue, exhibiting a longer half-life. Loading drugs into these nanoparticles enables controlled and sustained drug release, improves drug bioavailability, reduces the frequency of administration, and reduces drug accumulation in non-target tissues, thus lowering drug toxicity and ultimately achieving a safe and effective fat-reducing effect.

[0009] Preferably, the apolipoprotein sequence contains DWFKAFYDKVAEKFKEAF, which can target adipose tissue with high expression of SR-BI receptor to achieve a fat reduction therapeutic effect.

[0010] Preferably, the phospholipid includes any one or a combination of at least two of the following: myristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), 1,2-tetradecanoyl phosphatidylethanolamine (DMPE), dioleoyl phosphatidylethanolamine (DOPE), lecithin, and hydrogenated soybean phospholipid (HSPC).

[0011] Preferably, the fatty acid includes palmitic acid.

[0012] Preferably, the cholesterol ester includes cholesterol oleate.

[0013] Preferably, the drug is hydrophobic and has the effects of reducing fat and promoting the browning of white fat, including any one or a combination of at least two of rosiglitazone, resveratrol, or capsaicin.

[0014] Preferably, the mass ratio of the phospholipids, cholesterol esters, fatty acids and cholesterol is (45-55):1:(2-8):(4-6).

[0015] The specific point values ​​in (45-55) can be 45, 47.5, 50, 52.5, 55, etc.; the specific point values ​​in (2-8) can be 2, 4, 6, 8, etc.; the specific point values ​​in (4-6) can be 4, 4.5, 5, 5.5, 6, etc.; other specific point values ​​within the above ranges can be selected, and will not be elaborated on here.

[0016] In a second aspect, the present invention provides a method for preparing drug-loaded recombinant high-density lipoprotein nanoparticles for treating obesity according to the first aspect, the preparation method comprising:

[0017] (1) Phospholipids, cholesterol, fatty acids, cholesterol esters and drugs are mixed and dissolved in an organic solvent, and the mixture is rotary evaporated to form a lipid film. After adding deionized water, the mixture is sonicated and filtered to obtain a drug-loaded liposome solution.

[0018] (2) Then the aqueous solution of apolipoprotein is added dropwise to the drug-loaded liposome solution and stirred to obtain the drug-loaded recombinant high-density lipoprotein nanoparticles.

[0019] The preparation method of the drug-loaded recombinant high-density lipoprotein nanoparticles involved in this invention is simple and easy to operate, and is suitable for industrial production.

[0020] Preferably, the ratio of the amount of drug to the total mass of the raw materials for liposome preparation is 1:(13-20), such as 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0021] The ratio of the drug dosage to the total mass of the liposome preparation raw materials meets the above-mentioned specific numerical range, which can ensure both a high drug loading rate and the overall stability of the nanoparticles.

[0022] Preferably, the organic solvent is a mixture of methanol and chloroform.

[0023] Preferably, the power of the ultrasound is 200-400W, such as 200W, 220W, 250W, 280W, 300W, 320W, 350W, 380W, 400W, etc.; the time is 10-20min, such as 10min, 12min, 15min, 18min, 20min, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0024] Preferably, the ratio of the amount of apolipoprotein fed to the total mass of the raw materials for preparing drug-loaded liposomes is 1:(10-18), such as 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0025] The ratio of the amount of apolipoprotein fed to the total mass of the raw materials for preparing drug-loaded liposomes meets the above-mentioned specific numerical range, so that the recombinant high-density lipoprotein prepared can better mimic natural high-density lipoprotein particles, and has the advantages of better regulating adipocyte metabolism and natural targeting advantages to adipocytes.

[0026] Preferably, the stirring speed is 500-1000 rpm, such as 500 rpm, 700 rpm, 800 rpm, 1000 rpm, etc.; the time is 3-6 h, such as 3 h, 4 h, 5 h, 6 h, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0027] Thirdly, the present invention provides an oral drug delivery system for treating obesity, the oral drug delivery system comprising sodium alginate microspheres and drug-loaded recombinant high-density lipoprotein nanoparticles for treating obesity as described in the first aspect, coated within the sodium alginate microspheres.

[0028] Furthermore, the aforementioned drug-loaded recombinant high-density lipoprotein nanoparticles can be used to prepare oral drug formulations by encapsulating them in sodium alginate microspheres. The nanoparticles are stable and uniformly dispersed within the sodium alginate microspheres, and the microspheres achieve a high encapsulation rate of nearly 100%. The sodium alginate microspheres protect the nanoparticles from gastric acid degradation. During intestinal degradation, the sodium alginate microspheres release the drug-loaded recombinant high-density lipoprotein nanoparticles, which are then absorbed into the bloodstream through the intestines. This promotes adipocyte browning, treats fatty liver, improves the intestinal environment of obese patients, and achieves systemic fat reduction.

[0029] Fourthly, the present invention provides a method for preparing an oral drug delivery system for treating obesity according to the third aspect, the method comprising:

[0030] Sodium alginate was mixed and dissolved with drug-loaded recombinant high-density lipoprotein nanoparticles in water. The solution was then sprayed into an aqueous calcium chloride solution through an injection needle. The solution was stirred to solidify the sodium alginate gel microspheres. The microspheres were collected by pouring out the calcium chloride solution and washing with deionized water to obtain the oral drug delivery system for treating obesity.

[0031] The preparation method of the drug-loaded recombinant high-density lipoprotein nanoparticles involved in this invention is simple and easy to operate, and is suitable for industrial production.

[0032] Preferably, the injection flow rate is 1.0-1.5 mL / min, such as 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0033] Preferably, the injection needle is a 32G injection needle.

[0034] Preferably, the concentration of the calcium chloride aqueous solution is 4-6% (w / v), such as 4%, 4.5%, 5%, 5.5%, 6%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0035] Preferably, the stirring time is 1.5-3 hours, such as 1.5 hours, 1.7 hours, 1.8 hours, 2 hours, 2.2 hours, 2.5 hours, 2.7 hours, 2.8 hours, 3 hours, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0036] Fifthly, the present invention provides the application of the drug-loaded recombinant high-density lipoprotein nanoparticles described in the first aspect or the oral drug delivery system described in the third aspect in the preparation of weight-loss drugs.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The drug-loaded recombinant high-density lipoprotein nanoparticles involved in this invention are a synergistic fat-reducing drug delivery system. They utilize the stable and uniform size of the recombinant high-density lipoprotein nanoparticles, their advantages in regulating adipocyte metabolism and their natural targeting advantage to adipocytes. These nanoparticles can be completely degraded without triggering an immune response, are non-toxic to the human body, safe and reliable, and can avoid recognition and clearance by reticuloendothelial tissue, exhibiting a longer half-life. Loading rosiglitazone or its pharmaceutically acceptable salts into these nanoparticles enables controlled and sustained drug release, improves drug bioavailability, reduces the frequency of administration, and reduces the accumulation of rosiglitazone in non-target tissues, thus reducing drug toxicity and ultimately achieving a safe and effective fat-reducing effect.

[0039] Furthermore, the aforementioned drug-loaded recombinant high-density lipoprotein nanoparticles can be used to prepare oral drug formulations by encapsulating them in sodium alginate microspheres. The nanoparticles are stable and uniformly dispersed within the sodium alginate microspheres, and the microspheres achieve a high encapsulation rate of nearly 100%. The sodium alginate microspheres protect the nanoparticles from gastric acid degradation. During intestinal degradation, the sodium alginate microspheres release the drug-loaded recombinant high-density lipoprotein nanoparticles, which are then absorbed into the bloodstream through the intestines. This promotes adipocyte browning, treats fatty liver, improves the intestinal environment of obese patients, and achieves systemic fat reduction. Attached Figure Description

[0040] Figure 1 This is a transmission electron microscopy (TEM) characterization image of the recombinant high-density lipoprotein nanoparticles (rHDL) prepared in Example 1-1;

[0041] Figure 2 This is a particle size characterization diagram of the recombinant high-density lipoprotein nanoparticles (rHDL) prepared in Example 1-1;

[0042] Figure 3 This is a potential characterization diagram of the recombinant high-density lipoprotein nanoparticles (rHDL) prepared in Example 1-1;

[0043] Figure 4 This is a particle size characterization diagram of the drug-loaded recombinant high-density lipoprotein nanoparticles (rHDL@Rosi) prepared in Example 2-1;

[0044] Figure 5 This is a potential characterization diagram of the drug-loaded recombinant high-density lipoprotein nanoparticles (rHDL@Rosi) prepared in Example 2-1;

[0045] Figure 6 This is an optical microscope image of the drug-loaded recombinant high-density lipoprotein oral sodium alginate microspheres (rHDL@Rosi / MS) prepared in Example 4;

[0046] Figure 7 This is a graph showing the statistical results of intracellular fluorescence intensity in each group of the endocytosis assay;

[0047] Figure 8 This is a diagram showing the results of oil red staining of lipid droplets in adipocytes of each group;

[0048] Figure 9 This is a statistical chart showing the results of qPCR detection of RNA levels of (A) Ucp1, (B) PGC-1α, and (C) Fgf21 genes in adipocytes of each group;

[0049] Figure 10 This is a statistical graph showing the changes in body weight of mice in each group;

[0050] Figure 11 The graph shows the weight statistics of the following areas in each group of mice: (B) inguinal white fat (iWAT), (C) epididymal white fat (eWAT), (D) perirenal white fat (pWAT), (E) scapular brown fat (sBAT), and (A) liver. Detailed Implementation

[0051] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0052] The sodium alginate used in the following examples was purchased from Maclean's, product number S875037; the drug rosiglitazone was purchased from Maclean's, product number R832516; dimyristoyl phosphatidylcholine was purchased from Sgima, product number 850345P; cholesterol oleate was purchased from Sigma, product number C9253; palmitic acid was purchased from Sigma, product number P0500; cholesterol was purchased from Sigma, product number C8667; and apolipoprotein ApoA-I mimic peptide (4F peptide, DWFKAFYDKVAEKFKEAF) was purchased from Jier Biochemical (Shanghai) Co., Ltd.

[0053] Example 1

[0054] This embodiment provides a recombinant high-density lipoprotein nanoparticle (rHDL), the preparation method of which includes the following steps:

[0055] (1) Weigh 12.5 mg of myristoyl phosphatidylcholine, 0.25 mg of cholesterol oleate, 0.5 mg of palmitic acid, and 1 mg of cholesterol, dissolve them in 5 mL of a methanol and chloroform mixture (1:1, v / v), and transfer the mixture to a 100 mL round-bottom flask. Under the conditions of a 37 °C water bath and a rotation speed of 100 r / min, remove the solvent by rotary evaporation. The remaining components form a thin film on the flask wall. Add 3 mL of deionized water to the round-bottom flask, sonicate at 25 °C for 15 min at 300 W power, and filter through a 0.22 μm aqueous membrane to obtain the lipid components in rHDL.

[0056] (2) Weigh 3 mg of apolipoprotein ApoA-I mimic peptide (4F peptide), dissolve it in 3 mL of water, and then centrifuge at 6000 rpm for 10 min to obtain the 4F peptide supernatant. Transfer the prepared liposome aqueous solution to a flat-bottomed glass bottle, and add the 4F peptide supernatant dropwise while stirring at 1000 rpm. The solution gradually changes from light milky white to clear and transparent. Continue stirring for 4 h to obtain rHDL nanoparticles.

[0057] The prepared rHDL nanoparticles were characterized by transmission electron microscopy, such as... Figure 1 As shown, by Figure 1 It can be seen that rHDL nanoparticles have uniform particle size and disk-like structure, good dispersibility, and stable properties. Then, their particle size and potential were characterized, such as... Figure 2 and Figure 3 As shown in the figure, the average particle size of rHDL nanoparticles is 24.36±3.28 nm, and the zeta potential is -12.14±0.75 mV.

[0058] Example 2-1

[0059] This embodiment provides a drug-loaded recombinant high-density lipoprotein nanoparticle (rHDL@Rosi), the preparation method of which includes the following steps:

[0060] (1) Weigh 12.5 mg of myristoyl phosphatidylcholine, 0.25 mg of cholesterol oleate, 0.5 mg of palmitic acid, 1 mg of cholesterol, and 1 mg of rosiglitazone, dissolve them in 5 mL of a methanol and chloroform mixture (1:1, v / v), and transfer the mixture to a 100 mL round-bottom flask. Under the conditions of a 37 °C water bath and a rotation speed of 100 r / min, remove the solvent by rotary evaporation. The remaining components form a thin film on the flask wall. Add 3 mL of deionized water to the round-bottom flask, sonicate at 25 °C for 15 min at 300 W power, and filter through a 0.22 μm aqueous membrane to obtain the drug-loaded liposome component.

[0061] (2) Weigh 3 mg of apolipoprotein ApoA-I mimic peptide (4F peptide), dissolve it in 3 mL of water, and then centrifuge at 6000 rpm for 10 min to obtain the 4F peptide supernatant. Transfer the prepared drug-loaded liposome aqueous solution to a flat-bottomed glass bottle, and add the 4F peptide supernatant dropwise while stirring at 1000 rpm. The solution gradually changes from light milky white to clear and transparent. Continue stirring for 4 h to obtain rHDL@Rosi nanoparticles.

[0062] The particle size and potential of the prepared rHDL@Rosi nanoparticles were characterized, such as... Figure 4 and Figure 5 As shown in the figure, the average particle size of the rHDL nanoparticles is 28.21 ± 5.27 nm, and the zeta potential is -12.97 ± 0.57 mV. The drug-loaded recombinant high-density lipoprotein prepared by this method has uniform particle size and distribution, and the solution is clear.

[0063] Example 2-2

[0064] This embodiment provides a drug-loaded recombinant high-density lipoprotein nanoparticle (rHDL@Rosi). The only difference between this preparation method and that of Example 2-1 is the removal of cholesterol oleate component in step (1), while all other conditions remain unchanged.

[0065] The particle size of the prepared rHDL nanoparticles was characterized, as shown in Table 1. The average particle size of the rHDL nanoparticles was 1000±23.56 nm, indicating that the drug-loaded recombinant high-density lipoprotein prepared by this method aggregated, had a non-uniform particle size distribution, and did not form nanoparticles. Furthermore, particle sedimentation occurred during the preparation process.

[0066] Example 2-3

[0067] This embodiment provides a drug-loaded recombinant high-density lipoprotein nanoparticle (rHDL@Rosi). The only difference between this preparation method and that of Example 2-1 is the removal of cholesterol components in step (1), while all other conditions remain unchanged.

[0068] The particle size of the prepared rHDL nanoparticles was characterized, as shown in Table 1. The average particle size of the rHDL nanoparticles was 756±48.75 nm, indicating that the drug-loaded recombinant high-density lipoprotein prepared by this method aggregated, had a non-uniform particle size distribution, and did not form nanoparticles. Furthermore, flocculent precipitates were observed in the solution during the preparation process.

[0069] Examples 2-4

[0070] This embodiment provides a drug-loaded recombinant high-density lipoprotein nanoparticle (rHDL@Rosi). The preparation method differs from that of Example 2-1 only in the addition ratio of myristoyl phosphatidylcholine, cholesterol oleate, palmitic acid, cholesterol, and rosiglitazone. The ratio is 12.5 mg myristoyl phosphatidylcholine, 0.25 mg cholesterol oleate, 0.5 mg palmitic acid, 0.5 mg cholesterol, and 0.5 mg rosiglitazone. All other conditions remain unchanged.

[0071] The particle size of the prepared rHDL nanoparticles was characterized, as shown in Table 1. The average particle size of the rHDL nanoparticles was 122 ± 20.28 nm, and the particle size distribution was non-uniform, indicating that the drug-loaded recombinant high-density lipoprotein prepared by this method aggregated. Furthermore, flocculent precipitates appeared in the solution during the preparation process.

[0072] Examples 2-5

[0073] This embodiment provides a drug-loaded recombinant high-density lipoprotein nanoparticle (rHDL@Rosi). The only difference between this preparation method and that of Example 2-1 is the addition ratio of myristoyl phosphatidylcholine, cholesterol oleate, palmitic acid, cholesterol, and rosiglitazone. The ratio is 12.5 mg myristoyl phosphatidylcholine, 0.25 mg cholesterol oleate, 20.5 mg palmitic acid, 1 mg cholesterol, and 6 mg rosiglitazone. All other conditions remain unchanged.

[0074] The particle size of the prepared rHDL nanoparticles was characterized, as shown in Table 1. The average particle size of the rHDL nanoparticles was 578±12.83 nm, and the particle size distribution was not uniform. In addition, the solution was turbid during the preparation process, indicating that a large amount of rosiglitazone was precipitated and not completely encapsulated in the recombinant high-density lipoprotein.

[0075] Table 1

[0076]

[0077]

[0078] Example 3

[0079] This embodiment provides an oral sodium alginate microsphere (MS), the preparation method of which includes the following steps:

[0080] Sodium alginate was dissolved in deionized water (final concentration 3% (w / v)). Using an ISPLab01 syringe pump, the flow rate was adjusted to 1.20 mL / min in perfusion mode, with a circulation interval of 2 s. The solution was sprayed into 50 mL of 5% (w / v) calcium chloride aqueous solution through a 32G syringe needle while gently stirring. The mixture was stirred slowly for 2 hours to solidify sodium alginate gel microspheres. The microspheres were collected by slowly pouring out the calcium chloride solution and washed with deionized water to obtain sodium alginate microspheres.

[0081] Example 4

[0082] This embodiment provides a drug-loaded recombinant high-density lipoprotein oral sodium alginate microsphere (rHDL@Rosi / MS), the preparation method of which includes the following steps:

[0083] Sodium alginate was dissolved in the rHDL@Rosi aqueous solution prepared in Example 2 (final concentration 3% (w / v)). Using an ISPLab01 injection pump, the flow rate was adjusted to 1.20 mL / min in perfusion mode, with a circulation interval of 2 s. The solution was sprayed into 50 mL of 5% (w / v) calcium chloride aqueous solution through a 32G injection needle while gently stirring. The mixture was stirred slowly for 2 h to solidify the sodium alginate gel microspheres. The microspheres were collected by slowly pouring out the calcium chloride solution and washed with deionized water to obtain drug-loaded recombinant high-density lipoprotein oral sodium alginate microspheres.

[0084] The obtained product is observed under an optical microscope, such as... Figure 6 As shown, by Figure 6 It can be seen that the drug-loaded recombinant high-density lipoprotein oral sodium alginate microspheres are uniformly distributed, with an average particle size of 72±10.16μm.

[0085] Test Example 1

[0086] This test case investigates the endocytosis of rHDL@Rosi nanoparticles by adipocytes:

[0087] (1) Adipogenic differentiation of 3T3-L1 preadipocytes

[0088] (1.1) Preparation of mother liquor:

[0089]

[0090] (1.2) Cell culture:

[0091] 3T3-L1 cells (within 20 passages) were cultured in complete medium containing 10% newborn calf serum (NCS) at 37°C with 5% CO2, and passaged at a 1:3 ratio when the cell density reached 80% to 90%.

[0092] Complete culture medium formulation for mouse embryonic fibroblast 3T3-L1 cells (100 mL):

[0093] Newborn calf serum (Aubian) 10mL Glutamax (Invitrogen 35050061) 1mL Non-essential Amino Acids,100×(Invitrogen 11140050) 1mL Sodium pyruvate 100mM Solution(Invitrogen 11360070) 1mL

[0094] (1.3) Cell plating:

[0095] The cell density of the 6-well cell culture plate is 4 × 10⁶. 5 Cells were cultured per well in cell culture plates until the cell density reached 100% after 1 day. Cultured for another 4 days to allow complete contact inhibition. Drug induction was then performed.

[0096] (1.4) First stage induction:

[0097] Add 1 mL of induction medium to each well of a 6-well plate and induce for 72 hours. Change the induction medium every 24 hours, and prepare the induction medium fresh each time. Preparation of the first stage induction medium: Take 10 μL of 1000×IBMX, 4 μL of 2500×DEX, 1 μL of 10000×Rosiglitazone, and 28.6 μL of Insulin (Novolin R), and add 10% NCS complete culture medium to a final volume of 10 mL.

[0098] (1.5) Second stage induction:

[0099] Before changing the induction medium, wash the cells twice with complete culture medium. Induce the cells continuously for 72 hours in the second stage. Change the induction medium every 12 hours, and prepare the induction medium fresh each time. Preparation of the second-stage induction medium: Take 42.9 μL of Insulin (Novolin R) and add 10% NCS complete culture medium to a final volume of 10 mL.

[0100] (1.6) Replace the entire culture medium:

[0101] After the second stage of induction, the medium was replaced with 10% NCS complete culture medium and cultured again, with the medium changed every day to observe changes in lipid droplets. On the 4th day after replacing the complete culture medium, small lipid droplets began to aggregate into large lipid droplets, and by the 6th-7th day, the distribution of large lipid droplets was the largest. Under the microscope, the adipocyte morphology was observed to be monovesicular adipocytes. Within a single cell, 90% of the cell volume was occupied by lipid droplets, pushing the cytoplasm to the edge of the cell and forming a "ring"-like intracellular lipid droplet, thus inducing the transformation into white adipocytes.

[0102] (2) Cell endocytosis assay:

[0103] Cell endocytosis experiments were conducted using the method reported in Zhao Q, Liu J, Liu S, et al. Multipronged Micelles–Hydrogel for Targeted and Prolonged Drug Delivery in Chronic Wound Infections[J].ACS Applied Materials & Interfaces, 2022, 14(41):46224-46238. White adipocytes induced to differentiate as described above were used as the experimental cell line. PBS without any drugs was used as the control group. Luciferin 6 (C6) was used as the small molecule drug group, and recombinant high-density lipoprotein nanoparticles loaded with luciferin 6 (rHDL@C6) were used as the nanoparticle group (prepared according to the method in Example 2-1). After co-incubating the different drug groups with adipocytes, the cells were washed with PBS to remove the fluorescent drugs that failed to enter the cells. Flow cytometry was used to detect the average fluorescence intensity of the cells to evaluate the endocytosis of adipocytes.

[0104] Statistical results are as follows Figure 7 As shown, Figure 7 As shown, rHDL can target leukocytes with high SR-BI receptor expression, increasing their concentration in adipocytes and further enhancing the endocytosis of rHDL@C6 by adipocytes, resulting in increased intracellular fluorescence intensity. In contrast, the C6 small molecule does not have targeting function; the small molecule drug enters the cell via passive transport, resulting in a lower intracellular drug accumulation concentration and exhibiting lower fluorescence intensity. This demonstrates that the rHDL drug delivery carrier can enhance the concentration of the loaded drug in adipocytes, improve bioavailability, and simultaneously reduce drug accumulation in non-target tissues, thereby reducing drug toxicity and improving biosafety.

[0105] Test Example 2

[0106] This test case investigates the in vitro adipocyte browning effect of rHDL@Rosi nanoparticles:

[0107] (1) The method reported by Jung YJ, Kim HK, Cho Y, et al. Cell reprogramming using extracellular vesicles from differentiating stem cells into white / beigeadipocytes[J]. Science advances, 2020, 6(13): eaay6721. was used to observe the changes in cell browning genes by Oil Red staining of adipocytes. The white adipocytes induced by the above were used as experimental cell lines. PBS without any added fat-reducing drugs was used as the control group. Rosiglitazone (Rosi) was used as the drug treatment group. Example 1 was the recombinant high-density lipoprotein nanoparticle (rHDL) group, and Example 2-1 was the recombinant high-density lipoprotein nanoparticle (rHDL@Rosi) group loaded with rosiglitazone. The adipocytes of different groups were co-incubated with the drugs. The concentration of rosiglitazone in each group was the same.

[0108] (2) Oil red staining was used to stain lipid droplets in adipocytes. The size and number of lipid droplets were observed under a microscope to determine the degree of adipocyte browning. The results are as follows: Figure 8 As shown in the figure, rHDL enhances endocytosis, carrying rosiglitazone into adipocytes and promoting its accumulation. This results in a strong adipocyte browning effect, manifested as a decrease in the number and size of lipid droplets within the adipocytes. Rosiglitazone alone does not have cell targeting; at the same drug concentration, compared to rHDL@Rosi, rosiglitazone exhibits a poorer white adipocyte browning effect.

[0109] (3) The RNA levels of Ucp1, PGC-1α, and Fgf21 genes in adipocytes were detected by qPCR to comprehensively assess the degree of adipocyte browning. Results are as follows: Figure 9 As shown in the figure, rosiglitazone is a peroxisome proliferator-activated protein γ (PPARγ) agonist, which can effectively activate the PPARγ signaling pathway, induce the expression of brown adipose tissue-related genes, regulate mitochondrial function, increase cellular thermoproduction, and further promote the browning of white adipocytes. rHDL has enhanced endocytosis, carrying rosiglitazone into adipocytes and promoting its accumulation, resulting in a strong browning effect. Brown adipose tissue markers Ucp1, PGC-1α, and Fgf21 genes are significantly upregulated.

[0110] Test Example 3

[0111] This test case investigates the in vivo fat-reducing effect of rHDL@Rosi / MS oral microspheres:

[0112] (1) High-fat diet-induced obesity and fatty liver (DIO) model in mice: Six-week-old male C57BL / 6J mice were selected as experimental animals. They were kept in a 25℃ constant temperature laminar flow chamber with alternating 12-hour light and 12-hour dark cycles, and were allowed free access to standard pelleted feed and drinking water for one week. After fasting for 24 hours, the mice were fed a high-fat diet with 60% fat content. The feed and water were changed every three days to ensure freshness. After 12 weeks of high-fat diet, the average body weight was about 40g, and the liver showed obvious fatty degeneration. The liver was brownish-yellow and significantly enlarged in volume, with a sticky texture, proving that the model was successful.

[0113] (2) After the DIO mouse model was established using the above method, PBS without any added fat-reducing drugs was used as the control group. Example 3 was the blank microsphere (MS) group, rosiglitazone was dispersed in sodium alginate microspheres of Example 3 as rosiglitazone oral microspheres (Rosi / MS) group, Example 1 was dispersed in sodium alginate microspheres of Example 3 as recombinant high-density lipoprotein oral microspheres (rHDL / MS) group, and Example 4 was the recombinant high-density lipoprotein oral microsphere group loaded with rosiglitazone (rHDL@Rosi / MS) group.

[0114] Different groups of the drug were administered to mice via gavage at a dose of rosiglitazone 80 mg / kg body weight, twice daily for 28 consecutive days. Mouse body weight was recorded, and fat loss was observed. Results are as follows: Figure 10 As shown in the figure. The weights of inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), perirenal white adipose tissue (eWAT), scapular brown adipose tissue (sBAT), and liver were statistically analyzed in each group of mice. The results are as follows. Figure 11 As shown.

[0115] As shown in the figure above, compared to other groups, after 28 days of continuous oral administration of rHDL@Rosi / MS, the body weight decreased by 11.5%, approaching the body weight of normal healthy mice at 28 days. Thermogenesis in the white adipose tissue increased, white fat cells underwent browning, and the weight of adipose tissue decreased to normal levels. Furthermore, fatty liver was effectively treated, and liver volume and weight returned to normal. Traditional treatment methods such as liposuction, dietary control, and increased exercise have several drawbacks, such as the risk of infection and difficulty in maintaining the regimen. These methods often lead to side effects and are unsuitable for individuals with weak constitutions. Oral microspheres offer a convenient and quick way to reduce fat, minimizing discomfort. The drug can be targeted and released sustainably, reducing toxic side effects while improving bioavailability.

[0116] The applicant declares that this invention illustrates, through the above embodiments, a drug-loaded recombinant high-density lipoprotein nanoparticle for treating obesity, an oral drug delivery system, its preparation method, and its application. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product, addition of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0117] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0118] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An oral drug delivery system for treating obesity, characterized in that, The oral drug delivery system includes sodium alginate microspheres and drug-loaded recombinant high-density lipoprotein nanoparticles embedded in the sodium alginate microspheres. The drug-loaded recombinant high-density lipoprotein nanoparticles are composed of the following components: apolipoprotein, phospholipids, cholesterol, palmitic acid, cholesterol oleate, and the drug rosiglitazone; The sequence of the apolipoprotein is DWFKAFYDKVAEKFKEAF; The phospholipid is dimyristoylphosphatidylcholine; The drug-loaded recombinant high-density lipoprotein nanoparticles were prepared by the following method: (1) Mix 12.5 mg phospholipid, 1 mg cholesterol, 0.5 mg palmitic acid, 0.25 mg cholesterol oleate and 1 mg drug in an organic solvent, rotary evaporate to form a lipid film, add deionized water, sonicate and filter to obtain a drug-loaded liposome solution; (2) Then, the aqueous solution containing 3 mg of apolipoprotein was added dropwise to the drug-loaded liposome solution and stirred to obtain the drug-loaded recombinant high-density lipoprotein nanoparticles. The recombinant high-density lipoprotein nanoparticles have a disk-shaped structure.

2. The oral drug delivery system according to claim 1, characterized in that, The organic solvent is a mixed solution of methanol and chloroform.

3. The oral drug delivery system according to claim 1, characterized in that, The ultrasound power is 200-400W, and the duration is 10-20 minutes.

4. The oral drug delivery system according to claim 1, characterized in that, The stirring speed is 500-1000 rpm, and the stirring time is 3-6 h.

5. A method for preparing an oral drug delivery system for treating obesity according to any one of claims 1-4, characterized in that, The preparation method includes: Sodium alginate was mixed and dissolved with drug-loaded recombinant high-density lipoprotein nanoparticles in water. The solution was then sprayed into an aqueous calcium chloride solution through an injection needle. The solution was stirred to solidify the sodium alginate gel microspheres. The microspheres were collected by pouring out the calcium chloride solution and washing with deionized water to obtain the oral drug delivery system for treating obesity.

6. The method for preparing an oral drug delivery system for treating obesity according to claim 5, characterized in that, The injection flow rate is 1.0-1.5 mL / min.

7. The method for preparing an oral drug delivery system for treating obesity according to claim 5, characterized in that, The injection needle is a 32G injection needle.

8. The method for preparing an oral drug delivery system for treating obesity according to claim 5, characterized in that, The concentration of the calcium chloride aqueous solution is 4-6 w / v.

9. The method for preparing an oral drug delivery system for treating obesity according to claim 5, characterized in that, The stirring time is 1.5-3 hours.

10. The use of the oral drug delivery system according to any one of claims 1-4 in the preparation of a fat-reducing drug.

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