Double-drug-loading recombinant high-density lipoprotein nanoparticles for improving or treating intestinal inflammation as well as preparation method and application of double-drug-loading recombinant high-density lipoprotein nanoparticles

Abstract

The invention relates to a double-drug-loading recombinant high-density lipoprotein nanoparticle for improving or treating intestinal inflammation and application thereof. The double-drug-loading recombinant high-density lipoprotein nanoparticle is prepared from the following raw materials: apolipoprotein, phospholipid, cholesterol, fatty acid, cholesteryl ester and a drug, the medicine is hydrophobic and has antibacterial, anti-inflammatory or intestinal epithelial tissue barrier repair promoting effects, and comprises ciprofloxacin or enrofloxacin, and also comprises paeonol or cinnamyl aldehyde. The product uses five specific components as raw materials for preparing recombinant high density lipoprotein (rHDL), uses the rHDL as a drug delivery carrier, has uniform particle size, very stable structure and property, high biocompatibility and degradability and longer half-life period, and naturally targets macrophages and intestinal epithelial cells, so that the preparation method is simple and easy to implement, and the application prospect is wide. Targeted controllable long-acting sustained release can be realized through drug loading, and accumulation of drugs in non-target tissues is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a dual-drug-loaded recombinant high-density lipoprotein nanoparticle for improving or treating intestinal inflammation, and a preparation method and application thereof. Background Art

[0002] Bacteria can protect themselves from antibiotics through a variety of pathways, including bacterial membrane protection (downregulation of outer membrane porins, decreased membrane permeability), drug target mutations (gyrA, gyrB, parC and parE, etc.), horizontal transfer of drug resistance genes (pilus conjugation, plasmid transfer, etc.), drug efflux (upregulation of AcrAB-TolC, etc.) and intracellular parasitic strategies. Especially those bacteria that can survive and reproduce in host cells, that is, intracellular parasitic bacteria, such as Mycobacterium tuberculosis, Salmonella and Listeria. Long-term evolution has enabled them to acquire the ability to survive and reproduce in cells, and their position in cells provides them with a superior ecological niche, allowing these bacteria to not only evade the host's immune defense mechanism, but also avoid the killing effect of traditional antibiotics through complex resistance strategies.

[0003] At present, drugs used as the first-line treatment of Salmonella infection in the clinic, such as water-soluble drugs (such as β-lactams and aminoglycosides), are difficult to penetrate the lipid bilayer membrane of the hydrophobic structure to enter the cell, so they cannot effectively kill intracellular bacteria. Although hydrophobic drugs (such as fluoroquinolones and macrolides) can enter the cell, they are often rapidly discharged by the cell efflux pump and cannot accumulate to an effective therapeutic concentration in the cell. This makes it difficult for the drug in the host cell to reach and maintain an effective bactericidal concentration, thereby limiting its killing effect on these intracellular parasitic bacteria. And the strategy of high dose and repeated administration will promote the production of drug-resistant bacteria while causing various side effects in the body. This is not only not conducive to treatment, but also causes secondary infection and chronic inflammation. Therefore, for intracellular bacterial infection, it is necessary to develop a new drug delivery system to deliver the drug directly into the cell.

[0004] High-density lipoprotein (HDL) is a dynamic natural nanoparticle composed of a variety of biomacromolecules, which is biocompatible, non-immunogenic and completely biodegradable. These endogenous particles can circulate for a long time and transport lipids, proteins and microRNA from donor cells to recipient cells. Based on its inherent targeting properties, HDL is regarded as a very promising drug delivery system. In order to mass produce and avoid blood-borne contamination, recombinant high-density lipoprotein (rHDL) with the biological characteristics of HDL has been developed. rHDL is a new type of biomimetic nanocarrier. Compared with other lipoproteins, they have higher density and smaller size, with a diameter between 8 and 12 nanometers, and are mainly composed of proteins and phospholipids. It can transport antibiotics, nucleic acids and peptides in the body, and its ultra-small size is conducive to tissue penetration and enrichment. Therefore, it is very meaningful to develop a drug delivery system with rHDL as a carrier that can effectively inhibit intracellular bacteria. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a dual-drug-loaded recombinant high-density lipoprotein nanoparticle for improving or treating intestinal inflammation and a preparation method and application thereof, specifically to provide a dual-drug-loaded recombinant high-density lipoprotein nanoparticle for improving or treating intestinal inflammation and a preparation method thereof, an oral drug-loaded composite microsphere for improving or treating intestinal inflammation and a preparation method thereof, and application thereof in the preparation of a drug for improving or treating intestinal inflammation, especially intestinal inflammation caused by Salmonella infection.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a dual-drug-loaded recombinant high-density lipoprotein nanoparticle for improving or treating intestinal inflammation, wherein the raw materials for preparing the dual-drug-loaded recombinant high-density lipoprotein nanoparticle include the following components: apolipoprotein, phospholipids, cholesterol, fatty acids, cholesterol esters and drugs;

[0008] The drug is hydrophobic and has antibacterial, anti-inflammatory or intestinal epithelial tissue barrier repair promoting effects, including ciprofloxacin or enrofloxacin, and also includes paeonol or cinnamaldehyde.

[0009] The dual-drug-loaded recombinant high-density lipoprotein nanoparticles involved in the present invention are a drug delivery system in which each component can improve or treat intestinal inflammation, especially intracellular parasitic Salmonella infection, through synergistic action. The product uses five specific components, namely apolipoprotein, phospholipids, cholesterol, fatty acids, and cholesterol esters as raw materials for preparing rHDL, and uses them as drug delivery carriers, with uniform particle size and very stable structure and properties. At the same time, the rHDL has high biocompatibility and degradability, and can avoid being recognized and cleared by reticuloendothelial tissue, and has a longer half-life. At the same time, the rHDL naturally targets macrophages and intestinal epithelial cells that highly express SR-BI receptors, and the drug load can effectively achieve targeted release, controlled release, and long-term sustained release of drugs, reduce the number of drug administrations, promote effective intracellular accumulation of drugs, reduce the accumulation of drugs in non-target tissues, and reduce drug toxicity and side effects. Finally, it can simultaneously meet the needs of removing bacteria in the intestinal cavity, intracellular parasites, anti-inflammatory, and promoting intestinal epithelial cell barrier repair.

[0010] Preferably, the sequence of the apolipoprotein contains DWFKAFYDKVAEKFKEAF, which targets phagocytic immune cells and intestinal epithelial cells with high expression of SR-BI receptor to achieve improvement or therapeutic effects.

[0011] Preferably, the phospholipid comprises any one of dimyristoylphosphatidylcholine (DMPC), eicosylphosphatidylcholine (DPPC), dodecylphosphatidylcholine (DPhPC), eicosylphosphatidylinositol (DPPCI), phosphatidylethanolamine (PE), eicosylphosphatidylethanolamine (DPE), dipalmitoylphosphatidylcholine (DPPC), 1,2-tetradecanoylphosphatidylethanolamine (DMPE), dioleoylphosphatidylethanolamine (DOPE), lecithin or hydrogenated soybean lecithin (HSPC) or a combination of at least two thereof.

[0012] Preferably, the fatty acid comprises any one of palmitic acid, stearic acid, arachidic acid or linoleic acid, or a combination of at least two thereof.

[0013] Preferably, the cholesterol ester includes any one of cholesterol oleate, cholesterol palmitate or cholesterol stearate, or a combination of at least two thereof.

[0014] Preferably, the mass ratio of the phospholipid, cholesterol ester, fatty acid and cholesterol is (40-55):1:(2-10):(3-6).

[0015] The specific point values ​​in (40-55) can be selected from 40, 42, 44, 45, 47, 50, 52, 55, etc.; the specific point values ​​in (2-10) can be selected from 2, 4, 6, 8, 9, 10, etc.; the specific point values ​​in (3-6) can be selected from 3, 3.5, 4, 4.5, 5, 5.5, 6, etc.; other specific point values ​​within the above numerical ranges can be selected, and they will not be listed here one by one.

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

[0017] (1) dissolving phospholipids, cholesterol, fatty acids, cholesterol esters and drugs in an organic solvent, rotary evaporating to form a lipid film, adding deionized water and ultrasonicating to obtain a dual-drug-loaded liposome solution;

[0018] (2) Then, the saturated apolipoprotein aqueous solution is added dropwise into the stirring dual-drug-loaded liposome solution, and stirring is continued to obtain the drug-loaded recombinant high-density lipoprotein nanoparticles.

[0019] The preparation method of the dual-drug-loaded recombinant high-density lipoprotein nanoparticles of the present invention is simple and easy to operate and is easy for industrial production.

[0020] Preferably, the ratio of the feed mass of the drug to the total mass of phospholipids, cholesterol, fatty acids and cholesterol esters is 1:(10-20), for example 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0021] The ratio of the drug feed mass to the total mass of the raw material for preparing rHDL satisfies 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 mixed solution of methanol and chloroform.

[0023] Preferably, the power of the ultrasound is 300-500W, for example 300W, 320W, 350W, 380W, 400W, 420W, 450W, 480W, 500W, etc.; the time is 10-30min, for example 10min, 15min, 20min, 25min, 30min, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0024] Preferably, the ratio of the mass of the apolipoprotein to the total mass of the raw materials for preparing the dual-drug loaded liposomes in step (1) is 1:(8-20), such as 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0025] The ratio of the feed mass of the apolipoprotein to the total mass of the raw materials for preparing the dual-drug-loaded liposomes satisfies the above-mentioned specific numerical range so that the prepared rHDL can be closer to the function of natural high-density lipoprotein particles, enabling it to have a better advantage in targeting phagocytic immune cells and intestinal epithelial cells.

[0026] Preferably, the stirring speed is 200-800rpm, for example, 200rpm, 500rpm, 700rpm, 800rpm, etc.; the time is 1-6h, for example, 1h, 2h, 3h, 4h, 5h, 6h, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be described one by one here.

[0027] In a third aspect, the present invention provides an oral drug-loaded composite microsphere for improving or treating intestinal inflammation, wherein the raw materials for preparing the oral drug-loaded composite microsphere include an oil phase material, an aqueous phase material, and a cross-linking agent;

[0028] The oil phase material includes an oil phase and an emulsifier;

[0029] The aqueous phase material comprises gelatin, sodium alginate, chitosan, carboxymethyl cellulose salt, the dual-drug-loaded recombinant high-density lipoprotein nanoparticles described in the first aspect, and water.

[0030] The present invention further uses the above-mentioned dual-drug-loaded recombinant high-density lipoprotein nanoparticles to prepare oral pharmaceutical preparations, and embeds them into composite microspheres. The properties of the composite microspheres are stable, the dispersion is uniform, and the microspheres have a high encapsulation rate for nanoparticles, which can basically reach 100% encapsulation. In the composite microspheres, gelatin and sodium alginate are the core components of the composite microspheres. The further addition of chitosan can increase the intestinal sustained-release ability of the microspheres, and the further addition of carboxymethyl cellulose salt can avoid the additional swelling of the composite microspheres in the acidic environment of the stomach due to the addition of chitosan. Therefore, the composite microspheres can simultaneously achieve intragastric protection and intestinal sustained release. In addition, it has excellent intestinal pH response and infection microenvironment MMP9 protease response, and can release drugs at specific locations.

[0031] Preferably, the oil phase comprises any one of liquid paraffin, olive oil, peanut oil, triglyceride, fish oil or linseed oil, or a combination of at least two thereof.

[0032] Preferably, the emulsifier comprises Tween (eg, Tween-80, etc.) and / or Span (eg, Span-80, etc.).

[0033] Preferably, the carboxymethylcellulose salt comprises sodium carboxymethylcellulose.

[0034] Preferably, the cross-linking agent includes any one of glutaraldehyde, calcium chloride, polyethyleneimine, aluminum sulfate or polycarboxylic acid, or a combination of at least two thereof.

[0035] Preferably, the raw materials for preparing the oral drug-loaded composite microspheres also include prebiotics.

[0036] Preferably, the prebiotics include any one of xylooligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, soybean oligosaccharides or inulin, or a combination of at least two of them.

[0037] Preferably, the mass ratio of gelatin, sodium alginate, chitosan, carboxymethyl cellulose salt and dual-drug loaded recombinant high-density lipoprotein nanoparticles is (0.1-4):1:(0.1-2):(0.1-2):(0.01-0.1).

[0038] The specific point values ​​in (0.1-4) can be selected from 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.; the specific point values ​​in (0.1-2) can be selected from 0.1, 0.5, 1, 1.5, 2, etc.; the specific point values ​​in (0.01-0.1) can be selected from 0.01, 0.03, 0.05, 0.08, 0.1, etc. Other specific point values ​​within the above numerical ranges can be selected, and they will not be repeated here.

[0039] In a fourth aspect, the present invention provides a method for preparing the oral drug-loaded composite microspheres according to the third aspect, the preparation method comprising the following steps:

[0040] (1) mixing an oil phase and an emulsifier to obtain an oil phase material; mixing gelatin, sodium alginate, dual-drug-loaded recombinant high-density lipoprotein nanoparticles, optional prebiotics, and water to obtain an aqueous phase material;

[0041] (2) dropping the aqueous phase material into the oil phase material and stirring to obtain a suspension;

[0042] (3) placing the suspension in an ice bath and stirring to shape the suspension;

[0043] (4) then adding the crosslinking agent solution, stirring to carry out the crosslinking reaction, and centrifuging to obtain gelatin-sodium alginate oral drug-loaded composite microspheres;

[0044] (5) dropping the chitosan solution into the above-mentioned gelatin-sodium alginate oral drug-loaded composite microspheres, stirring, and centrifuging to obtain gelatin-sodium alginate / chitosan oral drug-loaded composite microspheres;

[0045] (6) dripping the carboxymethyl cellulose salt solution into the above-mentioned gelatin-sodium alginate / chitosan oral drug-loaded composite microspheres, stirring, and centrifuging to obtain gelatin-sodium alginate / chitosan / carboxymethyl cellulose salt oral drug-loaded composite microspheres, i.e., the oral drug-loaded composite microspheres.

[0046] The preparation method of the oral drug-loaded composite microspheres of the present invention is simple and easy to operate and is easy for industrial production.

[0047] Preferably, the volume ratio of the emulsifier, oil phase and water phase material is (1-6):(80-120):(15-30).

[0048] The specific point values ​​in (1-6) can be selected from 1, 2, 3, 4, 5, 6, etc.; the specific point values ​​in (80-120) can be selected from 80, 90, 100, 110, 120, etc.; the specific point values ​​in (15-30) can be selected from 15, 20, 25, 30, etc.; other specific point values ​​within the above numerical ranges can be selected, and they will not be listed here one by one.

[0049] Preferably, the oil phase and the emulsifier are mixed by stirring at a speed of 300-800rpm, for example, 300rpm, 400rpm, 500rpm, 600rpm, 700rpm, 800rpm, etc.; at 40-55°C, for example, 40°C, 45°C, 50°C, 55°C, etc.; other specific point values ​​within the above numerical ranges can be selected, which will not be described one by one here.

[0050] Preferably, the water phase material is dripped into the oil phase material, and the flow rate of the water phase material is 0.5-1.5 mL / min, such as 0.5 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min, 1.5 mL / min, etc.

[0051] Preferably, the stirring in step (2) is carried out at 300-800 rpm (e.g. 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, etc.) and at 35-55°C (e.g. 35°C, 40°C, 45°C, 50°C, 55°C, etc.) for 10-50 min (e.g. 10 min, 20 min, 30 min, 40 min, 50 min, etc.).

[0052] Preferably, the stirring in step (3) is carried out at 300-800 rpm (e.g. 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, etc.) and 35-55°C (e.g. 35°C, 40°C, 45°C, 50°C, 55°C, etc.) for 10-50 min (e.g. 10 min, 20 min, 30 min, 40 min, 50 min, etc.).

[0053] Preferably, the addition rate of the cross-linking agent solution in step (4) is 0.5-1.5 mL / min, for example, 0.5 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min, 1.5 mL / min, etc.

[0054] Preferably, the stirring in step (4) is carried out at 300-800 rpm (e.g. 300 rpm, 400 rpm, 500 rpm, 600 rpm, 700 rpm, 800 rpm, etc.) and 35-55°C (e.g. 35°C, 40°C, 45°C, 50°C, 55°C, etc.) for 30 min-180 min, for example 30 min, 50 min, 70 min, 80 min, 90 min, 100 min, 150 min, 180 min, etc.

[0055] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0056] Preferably, the microspheres are washed after the centrifugation in step (4).

[0057] In a fifth aspect, the present invention provides the use of the dual-drug-loaded recombinant high-density lipoprotein nanoparticles described in the first aspect or the oral drug-loaded composite microspheres described in the third aspect in the preparation of drugs for improving or treating intestinal inflammation.

[0058] Preferably, the intestinal inflammation includes intestinal inflammation caused by infection with intracellular parasitic bacteria.

[0059] Preferably, the intestinal inflammation includes intestinal inflammation caused by Salmonella infection.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The dual-drug-loaded recombinant high-density lipoprotein nanoparticles involved in the present invention are a drug delivery system in which each component can improve or treat intestinal inflammation, especially intracellular parasitic Salmonella infection, through synergistic action. The product uses five specific components, namely apolipoprotein, phospholipids, cholesterol, fatty acids, and cholesterol esters as raw materials for preparing rHDL, and uses them as drug delivery carriers, with uniform particle size and very stable structure and properties. At the same time, the rHDL has high biocompatibility and degradability, and has a longer half-life. At the same time, the rHDL naturally targets macrophages and intestinal epithelial cells that highly express SR-BI receptors, and the drug load can effectively achieve targeted release, controlled release, and long-term sustained release of drugs, reduce the number of drug administrations, and promote effective intracellular accumulation of drugs, reduce the accumulation of drugs in non-target tissues, reduce drug toxicity and side effects, and ultimately can simultaneously meet the needs of removing bacteria in the intestinal cavity, intracellular parasites, anti-inflammatory, and promoting intestinal epithelial cell barrier repair.

[0062] The oral drug-loaded composite microspheres involved in the present invention have stable properties and high nanoparticle encapsulation efficiency. The composite microspheres can achieve intragastric protection and intestinal sustained release, and have excellent intestinal pH response and infection microenvironment MMP9 protease response, excellent intracellular parasitic bacteria clearance effect, control inflammatory response, promote intestinal barrier repair and optimize intestinal flora structure. It is a very promising drug delivery platform for the treatment of intracellular bacterial infections, and provides a comprehensive and efficient treatment strategy for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is a transmission electron microscopy characterization image of recombinant high-density lipoprotein nanoparticles (rHDL);

[0064] Figure 2 is the particle size distribution diagram of recombinant high-density lipoprotein nanoparticles (rHDL);

[0065] Figure 3 is the potential characterization diagram of recombinant high-density lipoprotein nanoparticles (rHDL);

[0066] Figure 4 It is the particle size distribution diagram of dual-drug loaded recombinant high-density lipoprotein nanoparticles (rHDL@Pae / Cip);

[0067] Figure 5 It is the potential characterization diagram of dual-drug loaded recombinant high-density lipoprotein nanoparticles (rHDL@Pae / Cip);

[0068] Figure 6 This is an optical microscope observation of the oral drug-loaded composite microspheres (Gel-Alg / Cs / CMC@rHDL@Pae / Cip);

[0069] Figure 7 This is the statistical result of the intracellular fluorescence intensity of intestinal epithelial cells Caco-2 after treatment in each group of endocytosis test;

[0070] Figure 8 This is the statistical result of the fluorescence intensity inside the Raw264.7 macrophages after treatment in each group of the endocytosis test;

[0071] Fig. 9 This is the plate coating result diagram after each group treated the macrophage Raw264.7 cells in the intracellular bacteria clearance test;

[0072] Fig.10 This is the plate coating result after treating intestinal epithelial cells Caco-2 in each group of intracellular bacteria elimination test;

[0073] Fig.11 This is the fluorescence microscopy observation result after each group treated the macrophage Raw264.7 cells in the intracellular bacteria clearance test;

[0074] Fig.12 This is the fluorescence microscopy observation result of each group in the intracellular bacteria clearance test after treating intestinal epithelial cells Caco-2;

[0075] Fig.13 This is the result of ELISA method to detect the effect of each group of treatment on the expression of IL-6 and IL-1β in macrophage Raw264.7 after LPS stimulation;

[0076] Fig.14 This is the result of Western blot analysis of the effects of each group of treatments on the expression of inflammatory factors in the macrophage Raw264.7 cells stimulated by LPS;

[0077] Fig.15 This is the result of ELISA method to detect the effect of each group of treatment on the expression of tight junction protein Claudin-1 in macrophage Raw264.7 after LPS stimulation;

[0078] Fig.16 This is the result of Western blot analysis of the effects of each group of treatments on the expression of tight junction protein Claudin-1 in macrophage Raw264.7 cells stimulated by LPS;

[0079] Fig.17 is a graph showing the cecum and colon atrophy of mice in each group;

[0080] Fig.18 is a picture of spleen edema in each group of mice;

[0081] Fig.19 This is a statistical graph of the bacterial load in each tissue and organ of each group of mice;

[0082] Fig. 20 It is the HE staining result of the cecum section of each group of mice;

[0083] Fig.21 It is the HE staining result of colon sections of mice in each group;

[0084] Fig. 22 It is the HE staining result of liver sections of mice in each group;

[0085] Fig.23 The following are the HE staining results of spleen sections of mice in each group. DETAILED DESCRIPTION

[0086] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0087] The sodium alginate involved in the following examples is the product purchased from McLean with model number S875037; gelatin is the product purchased from McLean with model number G6317; chitosan is the product purchased from Sigma with model number 448869; sodium carboxymethyl cellulose is the product purchased from Sigma with model number 9004-32-4; ​​prebiotic oligoxylose is the product purchased from Source Leaf Biotechnology with model number S11137; dimyristoyl phosphatidylcholine is the product purchased from Sgima with model number 850345P; cholesterol oleate is the product purchased from Sigma with model number C9253; palmitic acid is the product purchased from Sigma with model number P0500; cholesterol is the product purchased from Sigma with model number C8667; apolipoprotein ApoA-Ⅰ mimetic peptide (4F peptide, DWFKAFYDKVAEKFKEAF) was purchased from Yujier Biochemical (Shanghai) Co., Ltd.

[0088] Example 1

[0089] This embodiment provides a recombinant high-density lipoprotein nanoparticle (rHDL), and the preparation method thereof comprises the following steps:

[0090] (1) Weigh 12.5 mg of dimyristoylphosphatidylcholine, 0.25 mg of cholesterol oleate, 0.5 mg of palmitic acid, and 1 mg of cholesterol, dissolve them in 4 mL of a mixed solution of methanol and chloroform (1:1, v / v), and transfer the mixed solution to a 100 mL round-bottom flask. Remove the solvent by rotary evaporation for 30 min at a speed of 100 r / min and a 37°C water bath. The remaining components form a lipid film on the wall of the flask. Add 3 mL of deionized water to the round-bottom flask, sonicate at 300 W and 25°C for 15 min, and filter with a 0.22 μm aqueous membrane to obtain an aqueous solution of rHDL precursor liposomes.

[0091] (2) Weigh 3 mg of apolipoprotein ApoA-Ⅰ mimetic peptide (4F peptide) and dissolve it in 2.4 mL of water, then centrifuge at 8000 rpm for 10 min to obtain a saturated 4F peptide aqueous solution. Transfer the above-prepared precursor liposome aqueous solution to a flat-bottomed glass bottle, and add the saturated 4F peptide aqueous solution drop by drop while stirring at 200 r / min. The solution first becomes turbid (milky white) and then becomes clear (clear like water). Keep stirring at the same speed for 3 h to obtain recombinant high-density lipoprotein nanoparticles (rHDL).

[0092] The prepared rHDL nanoparticles were characterized by transmission electron microscopy. Figure 1 As shown by Figure 1 It can be seen that rHDL nanoparticles have uniform particle size and disc-shaped structure, good dispersibility and stable properties. Then the particle size and potential were characterized, such as Figure 2 and Figure 3 As shown in the figure, it can be seen that the average particle size of rHDL nanoparticles is 32.67±4.5nm, and the zeta potential is -13.25±0.75mV.

[0093] Example 2-1

[0094] This embodiment provides a dual-drug loaded recombinant high-density lipoprotein nanoparticle (rHDL@Pae / Cip), the preparation method of which comprises the following steps:

[0095] (1) Weigh 12.5 mg of dimyristoylphosphatidylcholine, 0.25 mg of cholesterol oleate, 0.5 mg of palmitic acid, 1 mg of cholesterol, 1 mg of paeonol, and 0.5 mg of ciprofloxacin, dissolve them in 4 mL of a mixed solution of methanol and chloroform (1:1, v / v), and transfer the mixed solution to a 100 mL round-bottom flask. Remove the solvent by rotary evaporation for 30 min at a speed of 100 r / min and a 37°C water bath. The remaining components form a thin film on the wall of the flask. Add 3 mL of deionized water to the round-bottom flask, sonicate at 300 W for 15 min at 25°C, and filter with a 0.22 μm aqueous membrane to obtain the drug-loaded liposome component.

[0096] (2) Weigh 3 mg of apolipoprotein ApoA-Ⅰ mimetic peptide (4F peptide) and dissolve it in 3 mL of water, then centrifuge at 6000 rpm for 10 min to obtain a saturated 4F peptide aqueous solution. Transfer the above-prepared precursor liposome aqueous solution to a flat-bottomed glass bottle, and add the saturated 4F peptide aqueous solution drop by drop while stirring at 1000 r / min. The solution first becomes turbid (milky white) and then becomes clear (clear like water). Keep stirring at the same speed for 4 h to obtain dual-drug recombinant high-density lipoprotein nanoparticles (rHDL@Pae / Cip).

[0097] The particle size and potential of the prepared rHDL@Pae / Cip nanoparticles were characterized. Figure 4 and Figure 5 As shown in the figure, it can be seen that the average particle size of rHDL@Pae / Cip nanoparticles is 36.65±3.62nm, and the zeta potential is -17.33±0.64mV.

[0098] Example 2-2

[0099] This embodiment provides a dual-drug loaded recombinant high-density lipoprotein nanoparticle (rHDL@Pae / Cip), the preparation method of which is different from that of Example 2-1 only in that the cholesterol oleate component is removed in step (1), and other conditions remain unchanged.

[0100] The particle size of the prepared rHDL@Pae / Cip nanoparticles was characterized. As shown in Table 1, the average particle size of the nanoparticles was 950±33.88nm, indicating that the drug-loaded recombinant high-density lipoprotein prepared by this method was aggregated, the particle size distribution was uneven, and no nanoparticles were formed. In addition, particle sedimentation occurred during the preparation process.

[0101] Example 2-3

[0102] This embodiment provides a dual-drug loaded recombinant high-density lipoprotein nanoparticle (rHDL@Pae / Cip), the preparation method of which is different from that of Example 2-1 only in that the cholesterol component is removed in step (1), and other conditions remain unchanged.

[0103] The particle size of the prepared rHDL@Pae / Cip nanoparticles was characterized. As shown in Table 1, the average particle size of the nanoparticles was 667±63.15nm, indicating that the drug-loaded recombinant high-density lipoprotein prepared by this method was aggregated, the particle size distribution was uneven, and no nanoparticles were formed. In addition, flocculent precipitation occurred in the solution during the preparation process.

[0104] Embodiment 2-4

[0105] This embodiment provides a dual-drug loaded recombinant high-density lipoprotein nanoparticle (rHDL@Pae / Cip), the preparation method of which is different from that of Example 2-1 only in that the addition ratio of dimyristoyl phosphatidylcholine, cholesterol oleate, palmitic acid, cholesterol, paeonol and ciprofloxacin is different, namely 12.5 mg dimyristoyl phosphatidylcholine, 0.25 mg cholesterol oleate, 0.5 mg palmitic acid, 0.5 mg cholesterol, 0.3 mg paeonol and 0.2 mg ciprofloxacin, and other conditions remain unchanged.

[0106] The particle size of the prepared rHDL@Pae / Cip nanoparticles was characterized. As shown in Table 1, the average particle size of the nanoparticles was 136±45.18nm, and the particle size distribution was uneven, indicating that the drug-loaded recombinant high-density lipoprotein prepared by this method was aggregated. In addition, flocculent precipitation occurred in the solution during the preparation process.

[0107] Embodiment 2-5

[0108] This embodiment provides a dual-drug loaded recombinant high-density lipoprotein nanoparticle (rHDL@Pae / Cip), the preparation method of which is different from that of Example 2-1 only in that the addition ratio of dimyristoyl phosphatidylcholine, cholesterol oleate, palmitic acid, cholesterol, paeonol and ciprofloxacin is different, namely 12.5 mg dimyristoyl phosphatidylcholine, 0.25 mg cholesterol oleate, 20.5 mg palmitic acid, 1 mg cholesterol, 4 mg paeonol and 2 mg ciprofloxacin, and other conditions remain unchanged.

[0109] The particle size of the prepared rHDL@Pae / Cip nanoparticles was characterized. As shown in Table 1, the average particle size of the nanoparticles was 546±15.35 nm, the particle size distribution was uneven, and white turbidity appeared in the solution during the preparation process, indicating that a large amount of paeonol and ciprofloxacin were precipitated and were not completely encapsulated in the recombinant high-density lipoprotein.

[0110] Table 1

[0111]

[0112] Example 3

[0113] This embodiment provides an oral blank composite microsphere (denoted as Gel-Alg / Cs / CMC), and the preparation method thereof comprises the following steps:

[0114] (1) Dissolving gelatin and sodium alginate in deionized water (final concentrations of gelatin: 2% (w / v), sodium alginate: 1% (w / v)) to form an aqueous phase;

[0115] (2) Add 450 μL of Span-80 to 45 mL of liquid paraffin, stir at 450 rpm at 45°C to form an oil phase;

[0116] (3) Using a syringe, add 10 mL of the aqueous phase to the oil phase at a flow rate of 1 mL / min and continue stirring at 450 rpm for 30 min at 45 °C;

[0117] (4) Transfer to an ice water bath and stir at 450 rpm at 45 °C for 30 min to complete shaping;

[0118] (5) Add 2 mL of 20% glutaraldehyde and 10 mL of 500 mM calcium chloride at a flow rate of 1 mL / min and stir at 450 rpm at 45°C for 30 min to complete crosslinking;

[0119] (6) collecting the microspheres by centrifugation at 3000 rpm for 5 min, washing them three times with anhydrous ether, isopropanol, and deionized water, respectively, and centrifuging to remove water to obtain gelatin-sodium alginate microspheres;

[0120] (7) Dissolve chitosan in water (final concentration: 1% (w / v)), take 5 mL, add it dropwise to the gelatin-sodium alginate microspheres at a flow rate of 1 mL / min, and stir at 450 rpm at 45°C for 30 min, wash three times with deionized water to remove excess chitosan, and remove water by centrifugation to obtain gelatin-sodium alginate / chitosan microspheres;

[0121] (8) Dissolve carboxymethyl cellulose salt in water (final concentration: 1% (w / v)), take 5 mL, and add it dropwise to the above-mentioned gelatin-sodium alginate / chitosan microspheres at a flow rate of 1 mL / min, and continue stirring at 450 rpm at 45°C for 30 min, and wash three times with deionized water to remove excess carboxymethyl cellulose salt. Finally, resuspend the microspheres in deionized water to obtain the oral blank composite microspheres (Gel-Alg / Cs / CMC).

[0122] Example 4

[0123] This embodiment provides an oral dual-drug loaded composite microsphere (denoted as Gel-Alg / Cs / CMC@rHDL@Pae / Cip), the preparation method of which comprises the following steps:

[0124] (1) Dissolving gelatin, sodium alginate, xylo-oligosaccharide, and rHDL@Pae / Cip prepared in Example 2-1 in water (final concentrations of gelatin: 2% (w / v), sodium alginate: 1% (w / v), xylo-oligosaccharide: 1% (w / v), and rHDL@Pae / Cip: 0.1% (w / v)) to form an aqueous phase;

[0125] (2) Add 450 μL of Span-80 to 45 mL of liquid paraffin, stir at 450 rpm at 45°C to form an oil phase;

[0126] (3) Using a syringe, add 10 mL of the aqueous phase to the oil phase at a flow rate of 1 mL / min and continue stirring at 450 rpm for 30 min at 45 °C;

[0127] (4) Transfer to an ice water bath and stir at 450 rpm at 45 °C for 30 min to complete shaping;

[0128] (5) Add 2 mL of 20% glutaraldehyde and 10 mL of 500 mM calcium chloride at a flow rate of 1 mL / min and stir at 450 rpm at 45°C for 30 min to complete crosslinking;

[0129] (6) collecting the microspheres by centrifugation at 3000 rpm for 5 min, washing them three times with anhydrous ether, isopropanol, and deionized water, respectively, and centrifuging to remove water to obtain gelatin-sodium alginate microspheres;

[0130] (7) Dissolve chitosan in water (final concentration: 1% (w / v)), take 5 mL, add it dropwise to the gelatin-sodium alginate microspheres at a flow rate of 1 mL / min, and stir at 450 rpm at 45°C for 30 min. Wash three times with deionized water to remove excess chitosan, and remove water by centrifugation to obtain gelatin-sodium alginate / chitosan microspheres;

[0131] (8) Dissolve carboxymethyl cellulose salt in water (final concentration: 1% (w / v)), take 5 mL, and add it dropwise to the above-mentioned gelatin-sodium alginate / chitosan microspheres at a flow rate of 1 mL / min, and stir continuously at 450 rpm at 45°C for 30 min, and wash three times with deionized water to remove excess carboxymethyl cellulose salt. Finally, resuspend the microspheres in deionized water to obtain the oral dual-drug composite microspheres (Gel-Alg / Cs / CMC@rHDL@Pae / Cip).

[0132] The prepared drug-loaded composite microspheres were observed under an optical microscope. Figure 6 As shown by Figure 6 It can be seen that the composite microspheres are evenly distributed and have an average particle size of 33.0±5.7 μm.

[0133] Test Example 1

[0134] This test example detects the endocytosis effect of rHDL@Pae / Cip nanoparticles in macrophages RAW264.7 and intestinal epithelial cells Caco-2:

[0135] (1) Cell culture:

[0136] Macrophages RAW264.7 and intestinal epithelial cells Caco-2 (within 20 generations) were cultured in 10% and 20% newborn calf serum (NCS) DMEM complete culture medium, respectively, in 5% CO2, 37°C, and passaged at a ratio of 1:3 when the density reached 80% to 90%.

[0137] (2) Cell plating:

[0138] 6-well cell culture plate, Raw264.7 cell density is 2×10 6After 1 day of culture in the cell culture plate, the cell density reached 100%; in a 6-well cell culture plate, the Caco-2 cell density was 0.5×10 6 After culturing for 2 days in the cell culture plate, the cell density reached 100%.

[0139] (3) Endocytosis experiment:

[0140] The cell endocytosis experiment was carried out by the method reported in the literature (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.), and the RAW264.7 cells and Caco-2 cells cultured as above were used as experimental cell lines, PBS without any drug was used as the control group (NC), fluorescein coumarin 6 (C6) was used as the small molecule drug group, and recombinant high-density lipoprotein nanoparticles loaded with coumarin 6 (rHDL@C6) were used as the nanoparticle group (refer to the preparation method of Example 2-1), and finally, rHDL@C6 and SR-B1 receptor-specific inhibitor fucoidan (Fuc) were added as the control group (rHDL@C6+Fuc). After different groups of drugs were co-incubated with intestinal epithelial cells and macrophages, the cells were washed with PBS to remove the fluorescent drugs that failed to be internalized into the cells. The mean fluorescence intensity of the cells was detected by flow cytometry to evaluate the endocytosis of macrophages.

[0141] The statistical results are as follows Figure 7 and Figure 8 As shown in the figure, compared with the PBS group and the group with fluorescent coumarin 6 (C6) as a small molecule drug, the recombinant high-density lipoprotein nanoparticles loaded with coumarin 6 (rHDL@C6) as the nanoparticle group can significantly increase the concentration of C6 in intestinal epithelial cells and macrophages. In contrast, since the C6 small molecule does not have the function of targeting intestinal epithelial cells and macrophages, and small molecule drugs can only enter the cell through passive transport, the cumulative concentration of intracellular drugs is low, which is manifested as a lower fluorescence intensity. In the group with both Fuc and rHDL@C6 added, the concentration of C6 entering the cell decreased significantly because the SR-B1 receptor was inhibited.

[0142] This suggests that rHDL can indeed specifically target intestinal epithelial cells and macrophages with high expression of SR-B1, and enhance the endocytosis of rHDL@C6 by intestinal epithelial cells and macrophages, which is manifested as enhanced intracellular fluorescence intensity. This process improves the bioavailability of the drug, while reducing drug accumulation in non-target tissues, reducing the toxic side effects of the drug, and improving biosafety.

[0143] Test Example 2

[0144] This test investigated the clearance effect of rHDL@Pae / Cip nanoparticles on intracellular parasitic Salmonella in intestinal epithelial cells and macrophages.

[0145] (1) Cell culture:

[0146] Macrophages RAW264.7 and intestinal epithelial cells Caco-2 (within 20 generations) were cultured in 10% and 20% newborn calf serum (NCS) DMEM complete culture medium, respectively, in 5% CO2, 37°C, and passaged at a ratio of 1:3 when the density reached 80% to 90%.

[0147] (2) Cell plating:

[0148] In the confocal microplate, the density of Raw264.7 cells was 1.2×10 6 After 1 day of culture in the cell culture plate, the cell density reached 100%; in the confocal dish, the Caco-2 cell density was 0.5×10 6 After culturing for 2 days in the cell culture plate, the cell density reached 100%.

[0149] (3) Salmonella invades cells:

[0150] The fluorescent strain GFP14028 of Salmonella typhimurium was cultured to the logarithmic growth phase and the concentration was adjusted to McFarland turbidimetric value = 0.5 (1.5×10 8 CFU / mL). 10 times the amount of bacteria as the number of cells was added to the confocal dish and cultured in a 37°C 5% constant temperature incubator for 2 h. Subsequently, DMEM containing 1% FBS was used to wash away bacteria that did not invade the cells, and 100 μg / mL of gentamicin was added for 30 min to eliminate extracellular bacteria.

[0151] (4) Effect of drug group in removing intracellular bacteria:

[0152] The cell endocytosis experiment was carried out according to the method reported in reference (Junhua H, yingxian C, et al. A Comparative Analysis of the Antibacterial Spectrum of Ultrasmall Manganese Ferrite Nanozymes with Varied Surface Modifications [J]. ACS Applied Materials & Interfaces, 2024, 16 (12): 14385-14404.). The above mouse macrophages and intestinal epithelial cells invaded by the fluorescent strain GFP14028 of Salmonella typhimurium were used as experimental cell lines, and PBS without any drug was used as the control group. The drug treatment groups were paeonol (Pae) ​​treatment group (using concentration of 250 μg / mL), ciprofloxacin (Cip) treatment group (using concentration of 0.125 μg / mL), paeonol / ciprofloxacin (Pae / Cip) double drug treatment group (paeonol and ciprofloxacin used concentrations of 250 μg / mL and 0.125 μg / mL, respectively) and rHDL@Pae / Cip treatment group prepared in Example 2-1 (paeonol and ciprofloxacin used concentrations of 250 μg / mL and 0.125 μg / mL, respectively). The above mouse macrophages and intestinal epithelial cells invaded by the fluorescent strain GFP14028 of Salmonella typhimurium were treated for 4 hours. After the cells were broken, the bacteria were collected and plate counted to determine the clearance effects of different treatment groups on intracellular parasitic Salmonella typhimurium.

[0153] In addition, the effect of rHDL@Pae / Cip in removing intracellular bacteria was analyzed by fluorescence microscopy. The data of surviving fluorescent strains in the cells were observed and counted under an inverted fluorescence microscope.

[0154] The statistical results are as follows Figure 9-12 As shown in the figure, the clearing effect of each treatment group on intracellular bacteria is rHDL@Pae / Cip>Pae / Cip=Cip>Pae>PBS from strong to weak. Compared with other treatment groups, after rHDL@Pae / Cip treatment, rHDL naturally targets macrophages and intestinal epithelial cells that highly express SR-B1 receptors, enabling them to efficiently transport Pae and Cip inside them into cells, thereby improving bioavailability and effectively clearing Salmonella typhimurium parasitic in cells.

[0155] Test Example 3

[0156] This test investigates the evaluation of the anti-inflammatory effect of rHDL@Pae / Cip nanoparticles on infected macrophages:

[0157] (1) Cell culture:

[0158] Macrophage RAW264.7 cells were cultured in DMEM complete culture medium containing 10% newborn calf serum (NCS), 5% CO2, and 37°C, and passaged at a ratio of 1:3 when the density reached 80% to 90%.

[0159] (2) Cell plating:

[0160] 6-well cell culture plate, Raw264.7 cell density is 2×10 6 After culturing for 1 day in the cell culture plate, the cell density reached 100%.

[0161] (3) Salmonella typhimurium LPS stimulated cells:

[0162] 5 μg / mL LPS derived from Salmonella typhimurium was used to stimulate macrophages in the cell culture plate to secrete inflammatory factors.

[0163] (4) Anti-inflammatory effects of different drug treatment groups on macrophages:

[0164] The above LPS-stimulated macrophages were treated with drugs. Among them, PBS without any drug was used as the control group. The drug-treated groups were paeonol (Pae) ​​treatment group (using concentration of 250μg / mL), ciprofloxacin (Cip) treatment group (using concentration of 0.125μg / mL), paeonol / ciprofloxacin (Pae / Cip) double-drug treatment group (paeonol and ciprofloxacin used at concentrations of 250μg / mL and 0.125μg / mL, respectively) and rHDL@Pae / Cip treatment group prepared in Example 2-1 (paeonol and ciprofloxacin used at concentrations of 250μg / mL and 0.125μg / mL, respectively). The above LPS-stimulated macrophages were treated for 8h.

[0165] The ELISA method was used to detect the expression of IL-6 and IL-1β inflammatory factors in the supernatant of the cell culture medium after stimulation with LPS from Salmonella typhimurium. Fig.13 As shown in the figure, there is no significant difference in the expression levels of inflammatory cytokines IL-6 and IL-1β between the ciprofloxacin group and the PBS group, indicating that the application of Cip alone cannot have an anti-inflammatory effect; the expression levels of IL-6 and IL-1β in the Pae group and the Pae / Cip group decreased significantly, with obvious anti-inflammatory effects; and the anti-inflammatory effect of Pae / Cip was slightly higher than that of the Pae group; the rHDL@Pae / Cip group had the best anti-inflammatory effect, which was significantly better than other treatment groups.

[0166] Western-blot was used to detect the expression of IL-6 and IL-1β inflammatory factors in cells after stimulation with LPS from Salmonella typhimurium. Fig.14 As shown in the figure, the anti-inflammatory effects are as follows: rHDL@Pae / Cip>Pae / Cip>Pae>Cip>PBS. This is consistent with the results of ELISA test. This shows that rHDL@Pae / Cip targets the SR-B1 receptor highly expressed in intestinal epithelial cells and macrophages, enabling it to effectively deliver anti-inflammatory drugs into cells and significantly improve its anti-inflammatory effect.

[0167] Test Example 4

[0168] This test investigates the evaluation of the effect of rHDL@Pae / Cip nanoparticles on the repair of tight junction proteins in infected macrophages:

[0169] (1) Cell culture:

[0170] Macrophage RAW264.7 cells were cultured in DMEM complete culture medium containing 10% newborn calf serum (NCS), 5% CO2, and 37°C, and passaged at a ratio of 1:3 when the density reached 80% to 90%.

[0171] (2) Cell plating:

[0172] 6-well cell culture plate, Raw264.7 cell density is 2×10 6 After culturing for 1 day in the cell culture plate, the cell density reached 100%.

[0173] (3) Salmonella typhimurium LPS stimulated cells:

[0174] Macrophages in cell culture plates were stimulated with 5 μg / mL LPS derived from Salmonella typhimurium to destroy their tight junction barrier.

[0175] (4) Effects of different drug treatment groups on the repair of tight junction proteins in macrophages:

[0176] The above LPS-stimulated macrophages were treated with drugs. Among them, PBS without any drug was used as the control group. The drug-treated groups were paeonol (Pae) ​​treatment group (using concentration of 250μg / mL), ciprofloxacin (Cip) treatment group (using concentration of 0.125μg / mL), paeonol / ciprofloxacin (Pae / Cip) double-drug treatment group (paeonol and ciprofloxacin used at concentrations of 250μg / mL and 0.125μg / mL, respectively) and rHDL@Pae / Cip treatment group prepared in Example 2-1 (paeonol and ciprofloxacin used at concentrations of 250μg / mL and 0.125μg / mL, respectively). The above LPS-stimulated macrophages were treated for 8h.

[0177] The expression of tight junction protein Claudin-1 in the supernatant of cell culture medium after stimulation with LPS from Salmonella typhimurium was detected by ELISA. Fig.15 As shown in the figure, there is no significant difference in the expression level of tight junction protein Claudin-1 between the ciprofloxacin group and the PBS group, indicating that the application of Cip alone cannot promote the repair of intestinal epithelial cell barrier; the expression level of tight junction protein Claudin-1 in the Pae group and the Pae / Cip group was significantly upregulated, indicating that Pae has a significant effect on promoting the repair of intestinal epithelial tissue barrier; and the effect of Pae / Cip is slightly higher than that of the Pae group, indicating that there is a little synergistic effect between the two; the rHDL@Pae / Cip group has the best repair-promoting effect, which is significantly better than other treatment groups.

[0178] Western-blot was used to detect the expression of tight junction protein Claudin-1 in cells after stimulation with LPS from Salmonella typhimurium. Fig.16 As shown in the figure, the expression of tight junction protein Claudin-1 is from high to low: rHDL@Pae / Cip>Pae / Cip>Pae>Cip>PBS. This is consistent with the results of ELISA test. This shows that rHDL@Pae / Cip targets the SR-B1 receptor highly expressed in intestinal epithelial cells and macrophages, enabling it to effectively deliver drugs into cells and significantly improve its effect of promoting intestinal epithelial cell tissue repair.

[0179] Test Example 5

[0180] This test investigates the therapeutic effect of oral composite microspheres loaded with rHDL@Pae / Cip on the mouse enteritis infection model:

[0181] (1) Establishment of enteritis model in mice infected with Salmonella typhimurium:

[0182] Six-week-old C57BL / 6J male mice were selected as experimental animals. They were adaptively raised for one week in a constant temperature laminar flow chamber at 25°C with alternating 12-hour light and 12-hour darkness, with free access to standard pelleted feed and drinking water. After fasting for 12 hours, they were pretreated with antibiotics by gavage with 100 μL of 200 mg / mL streptomycin, and continued to fast for 24 hours with only sterile water provided.

[0183] After 24 hours (drinking sterile water during this period), gavage for 10 7 The fresh feces were collected at 24h, 48h, 72h and 96h after infection with Salmonella typhimurium ATCC 14028 strain with a dose of CFUs, and weighed one by one. The colonization number (CFU / g) was calculated using the Salmonella chromogenic medium (drug-resistant bacteria can use drug plates). If the colonization number does not drop sharply but shows a gentle trend, the infection model is considered to be successfully established.

[0184] (2) After the mouse enteritis model was established using the above method, PBS without any antibacterial or anti-inflammatory drugs was used as the control group (denoted as Group G1), and the drug treatment groups were paeonol treatment group (denoted as Group G2), ciprofloxacin treatment group (denoted as Group G3), paeonol / ciprofloxacin dual-drug treatment group (denoted as Group G4) and Gel-Alg / Cs / CMC@rHDL@Pae / Cip treatment group prepared in Example 4 (denoted as Group G5), with 5 mice in each group.

[0185] (3) Intervention method: The mice in each group were gavaged with a dose of 50 mg / kg body weight of paeonol and 10 mg / kg body weight of ciprofloxacin, twice a day for 7 consecutive days, and the changes in the weight and fecal bacterial load of the mice were recorded.

[0186] (4) 12 hours after the last administration, the mice were dissected, and the cecum and colon atrophy, spleen edema, and bacterial load in various tissues and organs were removed and counted. Figure 17-19 As shown, the degree of cecal atrophy, the degree of spleen edema and the bacterial load of each tissue and organ all showed a trend of PBS>Pae>Cip=Pae / Cip>Gel-Alg / Cs / CMC@rHDL@Pae / Cip.

[0187] (5) In addition, the cecum, colon, liver, and spleen were sectioned and stained with HE to observe pathological changes. Figure 20-23As shown, the pathological changes in the sections of these tissues were analyzed. First, in the cecum and colon tissues, all treatment groups showed varying degrees of inflammatory cell infiltration, edema, and structural damage. In the liver and spleen tissues, the PBS and Pae treatment groups showed obvious inflammatory cell infiltration, increased cytoplasmic eosinophilia, and nuclear condensation or fragmentation, while the pathological changes in Cip and Pae / Cip were slightly milder, and there was no obvious pathological change in the Gel-Alg / Cs / CMC@rHDL@Pae / Cip group. Therefore, the severity of damage to these tissues and organs was PBS>Pae>Cip=Pae / Cip>Gel-Alg / Cs / CMC@rHDL@Pae / Cip.

[0188] In summary, it can be seen that the group treated with Gel-Alg / Cs / CMC@rHDL@Pae / Cip has the best recovery effect, indicating that the product involved in the present invention can effectively improve the effect of the drug in the treatment process of mouse enteritis model.

[0189] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0190] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0191] 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 further describe various possible combinations.

Claims

1. A dual-drug-loaded recombinant high-density lipoprotein nanoparticle for improving or treating intestinal inflammation, characterized in that: The raw materials for preparing the dual-drug-loaded recombinant high-density lipoprotein nanoparticles include the following components: apolipoprotein, phospholipids, cholesterol, fatty acids, cholesterol esters and drugs; The drug is hydrophobic and has antibacterial, anti-inflammatory or intestinal epithelial tissue barrier repair promoting effects, including ciprofloxacin or enrofloxacin, and also includes paeonol or cinnamaldehyde.

2. The dual-drug-loaded recombinant high-density lipoprotein nanoparticles for improving or treating intestinal inflammation according to claim 1, characterized in that: The sequence of the apolipoprotein contains DWFKAFYDKVAEKFKEAF, which targets phagocytic immune cells and intestinal epithelial cells with high expression of SR-BI receptor to achieve improvement or therapeutic effects.

3. The dual-drug-loaded recombinant high-density lipoprotein nanoparticles for improving or treating intestinal inflammation according to claim 1 or 2, characterized in that: The phospholipids include any one of dimyristoylphosphatidylcholine, eicosylphosphatidylcholine, dodecylphosphatidylcholine, eicosylphosphatidylinositol, phosphatidylethanolamine, eicosylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, 1,2-tetradecanoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, lecithin or hydrogenated soybean lecithin, or a combination of at least two thereof; Preferably, the fatty acid comprises any one or a combination of at least two of palmitic acid, stearic acid, arachidic acid or linoleic acid; Preferably, the cholesterol ester includes any one or a combination of at least two of cholesterol oleate, cholesterol palmitate or cholesterol stearate; Preferably, the mass ratio of the phospholipid, cholesterol ester, fatty acid and cholesterol is (40-55):1:(2-10):(3-6).

4. The method for preparing dual-drug-loaded recombinant high-density lipoprotein nanoparticles for improving or treating intestinal inflammation according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) dissolving phospholipids, cholesterol, fatty acids, cholesterol esters and drugs in an organic solvent, rotary evaporating to form a lipid film, adding deionized water and ultrasonicating to obtain a dual-drug-loaded liposome solution; (2) Then, the saturated apolipoprotein aqueous solution is added dropwise into the stirring dual-drug-loaded liposome solution, and stirring is continued to obtain the drug-loaded recombinant high-density lipoprotein nanoparticles.

5. The method for preparing dual-drug-loaded recombinant high-density lipoprotein nanoparticles for improving or treating intestinal inflammation according to claim 4, characterized in that: The ratio of the drug feed mass to the total mass of phospholipids, cholesterol, fatty acids and cholesterol esters is 1:(10-20); Preferably, the organic solvent is a mixed solution of methanol and chloroform; Preferably, the power of the ultrasound is 300-500W, and the time is 10-30min; Preferably, the ratio of the mass of the apolipoprotein to the total mass of the raw materials for preparing the dual-drug-loaded liposomes in step (1) is 1:(8-20); Preferably, the stirring speed is 200-800 rpm and the time is 1-6 h.

6. An oral drug-loaded composite microsphere for improving or treating intestinal inflammation, characterized in that: The raw materials for preparing the oral drug-loaded composite microspheres include oil phase materials, water phase materials, and a cross-linking agent; The oil phase material comprises an oil phase and an emulsifier; The aqueous phase material comprises gelatin, sodium alginate, chitosan, carboxymethyl cellulose salt, the dual-drug loaded recombinant high-density lipoprotein nanoparticles according to any one of claims 1 to 3, and water.

7. The oral drug-loaded composite microspheres according to claim 6, characterized in that: The oil phase comprises any one of liquid paraffin, olive oil, peanut oil, triglyceride, fish oil or linseed oil, or a combination of at least two thereof; Preferably, the emulsifier comprises Tween and / or Span; Preferably, the carboxymethyl cellulose salt comprises sodium carboxymethyl cellulose; Preferably, the cross-linking agent comprises any one or a combination of at least two of glutaraldehyde, calcium chloride, polyethyleneimine, aluminum sulfate or polycarboxylic acid; Preferably, the raw materials for preparing the oral drug-loaded composite microspheres also include prebiotics; Preferably, the prebiotics include any one of xylooligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, isomaltooligosaccharides, soybean oligosaccharides or inulin, or a combination of at least two thereof; Preferably, the mass ratio of gelatin, sodium alginate, chitosan, carboxymethyl cellulose salt and dual-drug loaded recombinant high-density lipoprotein nanoparticles is (0.1-4):1:(0.1-2):(0.1-2):(0.01-0.1).

8. The method for preparing oral drug-loaded composite microspheres according to claim 6 or 7, characterized in that: The preparation method comprises the following steps: (1) mixing an oil phase and an emulsifier to obtain an oil phase material; mixing gelatin, sodium alginate, dual-drug-loaded recombinant high-density lipoprotein nanoparticles, optional prebiotics, and water to obtain an aqueous phase material; (2) dropping the aqueous phase material into the oil phase material and stirring to obtain a suspension; (3) placing the suspension in an ice bath and stirring to shape the suspension; (4) then adding the crosslinking agent solution, stirring to carry out the crosslinking reaction, and centrifuging to obtain gelatin-sodium alginate oral drug-loaded composite microspheres; (5) dropping the chitosan solution into the above-mentioned gelatin-sodium alginate oral drug-loaded composite microspheres, stirring, and centrifuging to obtain gelatin-sodium alginate / chitosan oral drug-loaded composite microspheres; (6) dripping the carboxymethyl cellulose salt solution into the above-mentioned gelatin-sodium alginate / chitosan oral drug-loaded composite microspheres, stirring, and centrifuging to obtain gelatin-sodium alginate / chitosan / carboxymethyl cellulose salt oral drug-loaded composite microspheres.

9. The method for preparing oral drug-loaded composite microspheres according to claim 8, characterized in that: The volume ratio of the emulsifier, oil phase and water phase materials is (1-6):(80-120):(15-30); Preferably, the oil phase and the emulsifier are mixed by stirring at a speed of 300-800 rpm at 40-55° C.; Preferably, the water phase material is dripped into the oil phase material, and the flow rate of the water phase material is 0.5-1.5 mL / min; Preferably, the cross-linking agent solution is added at a flow rate of 0.5-1.5 mL / min; Preferably, the microspheres are washed after the centrifugation.

10. Use of the dual-drug-loaded recombinant high-density lipoprotein nanoparticles according to any one of claims 1 to 3 or the oral drug-loaded composite microspheres according to any one of claims 6 to 7 in the preparation of a drug for improving or treating intestinal inflammation; Preferably, the intestinal inflammation includes intestinal inflammation caused by intracellular parasitic bacteria infection; Preferably, the intestinal inflammation includes intestinal inflammation caused by Salmonella infection.

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