Anti-loss type bionic spherical high-density lipoprotein preparation as well as preparation method and application thereof

Abstract

The invention discloses an anti-loss type bionic spherical high-density lipoprotein preparation as well as a preparation method and application thereof. The bionic spherical high-density lipoprotein preparation mainly comprises the following components: phospholipid, auxiliary lipid, apolipoprotein, an anti-loss functional module and a protein stabilizer, and the particle size of the preparation is 7-50nm. The bionic spherical high-density lipoprotein preparation disclosed by the invention is simple in preparation process, has the particle size and appearance close to those of natural high-density lipoprotein, has an enzyme-like antioxidant property, can protect own lipid and protein components in inflammation and oxidative stress pathological environments and maintain the structure stability, maintains the function of the high-density lipoprotein, and has a good application prospect. The medicine leakage can be reduced when the medicine is used as a medicine carrier. When being applied to treatment of inflammatory diseases such as sepsis, atherosclerosis and the like, the compound can remarkably reduce the ROS level in cells, change the phenotype of inflammatory cells and improve the survival rate of animals, and a feasible treatment scheme is provided for treatment of the inflammatory diseases.

Description

Technical Field

[0001] The invention relates to an anti-disfunction type bionic spherical high-density lipoprotein preparation and a preparation method and application thereof, belonging to the field of pharmaceutical preparations. Background Art

[0002] High-density lipoprotein (HDL) is an endogenous plasma protein. It is a complex lipoprotein composed of lipids and proteins and various regulatory factors carried by them. Among all endogenous lipoproteins, HDL has the smallest particle size (7-14nm) and can be divided into two types: nascent (disc-shaped) and mature (globular). Disc-shaped HDL gradually changes into globular HDL under enzyme mediation. They have apolipoproteins such as apoA-I, apoA-II, apoE, and apoM as the main proteins, phospholipids, free cholesterol, and cholesterol esters as the main lipid components, and contain a variety of proteomes, such as phosphatidylcholine cholesterol acyltransferase (LCAT), lipopolysaccharide transfer protein (LBP), and paraoxonase-1 (PON-1), which play important roles in reverse cholesterol transport (RCT), endotoxin removal, and anti-oxidation. These characteristics give HDL the therapeutic potential in various inflammatory diseases such as atherosclerosis, sepsis, and Alzheimer's disease.

[0003] However, extracting HDL from plasma for clinical application has certain challenges, including poor controllability of components, poor structural and functional stability, and low lipoprotein purity. In order to overcome the above shortcomings, researchers have developed recombinant HDL (rHDL). rHDL retains the basic structure of natural HDL phospholipids and apolipoproteins, aims to simulate the structure and function of natural HDL, and has higher purity and controllable properties. At present, a variety of rHDL have been developed, such as CSL-112 developed by CSL Behring and CER-001 developed by Cerenis Therapeutics, which are used to treat diseases such as atherosclerosis, congenital HDL deficiency, and sepsis-induced renal damage. In addition, by simulating the characteristics of natural HDL lipid transport, rHDL can also be used as a drug carrier to improve the targeting and bioavailability of drugs, such as patents CN110123761A and WO2023243865A1.

[0004] However, with the failure of the Phase III clinical trial of CSL-112 in acute myocardial infarction, researchers gradually realized that the function of HDL is closely related to its structure, and the lipid type, protein type and disease microenvironment can affect its physiological function. In fact, under pathological conditions, HDL can easily undergo structural changes and lose its function. For example, the pathological environment of sepsis is usually accompanied by the generation of a large number of reactive oxygen species (ROS) and acute phase proteins, which can lead to lipid and protein oxidation and aggravate oxidative stress. Acute phase proteins can also cause apoA-I to fall off, resulting in decreased HDL function. In addition, rHDL preparations lack the complex proteome and various regulatory factors of natural HDL, and it cannot fully simulate all the functional characteristics of natural HDL. For example, PON-1 is a calcium-dependent HDL-related enzyme that can hinder the peroxidation of HDL. Its absence will further affect the antioxidant and anti-inflammatory capabilities of rHDL.

[0005] Taking advantage of the properties of rHDL drug carriers, encapsulating small molecule antioxidants in its membrane is a simple antioxidant strategy. For example, the formula of CSL-112 contains α-tocopherol to prevent the oxidation of soy lecithin. However, this traditional antioxidant will continue to consume its own antioxidant structure under inflammatory conditions, and the effect is often not ideal. Antioxidant enzymes can remove ROS in a cyclic manner, which is more efficient and durable than traditional oxidants. Direct modification of PON-1 during rHDL preparation is the antioxidant method closest to endogenous HDL, but this method is costly, and PON-1 activity is reduced or shed under inflammatory conditions, losing its protective effect on HDL. Therefore, screening antioxidant enzymes with long-lasting activity is of great significance for enhancing the anti-inflammatory and antioxidant functions of HDL.

[0006] As mentioned above, endogenous HDL has two configurations, discoid and spherical. Discoid rHDL preparations will continuously leak drugs in the core and shed surface proteins during allosteric behavior, ultimately resulting in a reduction in the expected efficacy. Spherical rHDL preparations are expected to have better stability, but the preparation of spherical rHDL still faces challenges. Patent US20040266662A1 discloses a biomimetic spherical rHDL with cholesterol ester as the core mediated by LCAT enzyme, but cholesterol ester will be oxidized by excessive ROS in the inflammatory environment, thereby aggravating oxidative stress and inflammatory damage. The literature also reports spherical rHDL with polylactic acid-glycolic acid copolymer (PLGA) as the core through extrusion. This method does not need to consider enzyme-related harsh conditions such as temperature, activity, and reaction time, making industrial production more economical and convenient. However, this rHDL often has a larger particle size (~100nm), which is difficult to reach the particle size range of natural HDL.

[0007] In addition to maintaining enzyme activity, it is also necessary to ensure the overall stability of the preparation, which poses a challenge to the formulation and structural design of rHDL. The combination of antioxidant enzymes with suitable phospholipids and / or auxiliary lipids is conducive to improving the stability of the preparation and the enzyme encapsulation rate, and achieving the expected function; in addition, by enhancing the stability of the apolipoprotein itself, it can further prevent its shedding, and by enhancing its binding force with lipids through protein stabilizers or coupling it with the core through linkers, it is expected that apolipoprotein can be protected.

[0008] The present invention screened a series of antioxidant enzymes with long-lasting activity, used them as hard templates and matched them with different phospholipids, auxiliary lipids or protein stabilizers / linkers to prepare a bionic spherical high-density lipoprotein preparation with a particle size equivalent to that of natural HDL. The preparation has excellent antioxidant effect, can protect its own lipid and protein components, and significantly reduce protein shedding or drug leakage. The preparation has excellent therapeutic effect when applied to the treatment of inflammatory diseases. Summary of the invention

[0009] The purpose of the present invention is to provide a bionic spherical high-density lipoprotein preparation that resists loss of function in view of the above-mentioned deficiencies in the prior art.

[0010] Another object of the present invention is to provide a method for preparing the anti-disfunction type bionic spherical high-density lipoprotein preparation.

[0011] Another object of the present invention is to provide the application of the anti-loss of function type bionic spherical high-density lipoprotein preparation.

[0012] The purpose of the present invention can be achieved through the following technical solutions:

[0013] A biomimetic spherical high-density lipoprotein preparation that resists functional loss, comprising any one of the following components:

[0014] (I) phospholipids, auxiliary lipids, apolipoproteins, anti-disfunction modules and protein stabilizers;

[0015] (II) phospholipids, helper lipids, apolipoproteins coupled to the anti-disfunction module via a linker, and protein stabilizers;

[0016] The anti-defunctionalization template is a nanocarrier with antioxidant enzyme properties, selected from any one or more of the following: an organic monomer polymer template, a metal template composed of a combination of metal ions and organic ligands, a carbon-based template composed of a carbon core, or a material doped or modified as needed, and still having antioxidant enzyme activity;

[0017] The apolipoprotein is selected from apolipoprotein or a mimetic peptide thereof;

[0018] The particle size of the anti-disfunction type bionic spherical high-density lipoprotein preparation is 7-50nm.

[0019] The mass ratio of auxiliary lipids to phospholipids is 1:1-40, the mass ratio of apolipoprotein to total lipids is 1:1-10; the mass ratio of anti-defunctionalization template to total lipids is 1:2-10, and the mass ratio of protein stabilizer to total lipids is 1:1-100. The total lipids are the general term for auxiliary lipids and phospholipids.

[0020] The bionic spherical high-density lipoprotein preparation has a nearly spherical structure and a particle size comparable to that of natural HDL. The preparation can be used alone as a therapeutic drug or as a drug delivery carrier.

[0021] The phospholipids described in the invention include one or more of the following, such as soybean lecithin (SPC), hydrogenated soybean lecithin (HSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), sphingomyelin (SM), lysophospholipids, etc.

[0022] The auxiliary lipids described in the invention are auxiliary lipids used to improve the stability or encapsulation efficiency of the preparation, and include one or more of the following, such as cholesterol, cholesterol ester, triglyceride, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), etc.

[0023] The apolipoprotein described in the invention includes one or more of apoA-I, apoA-II, apoA-IV, apoCs, apoB, apoE, apoJ, apoM and their mimetic peptides, wherein the mimetic peptides include apoA-I mimetic peptide 22A, apoA-I mimetic peptide 4F, apoE mimetic peptide COG1410, etc.

[0024] The anti-destructive function template is a nanoscale composition with antioxidant enzyme properties, including one or more of the following: an organic monomer polymer template, such as polydopamine (PDA) and polytannic acid (PTA); the metal template composed of a combination of metal ions and organic ligands is selected from iron nanoparticles modified with dodecyl mercaptan, manganese nanoparticles modified with oleic acid, and cerium nanoparticles modified with oleylamine; a carbon-based template composed of a carbon core, such as carbon dots; they can be doped and modified as needed and still have antioxidant enzyme activity.

[0025] The present invention provides a blank and drug-loaded preparation of an anti-disfunction bionic spherical high-density lipoprotein preparation, which is prepared by the following method: (1) dissolving phospholipids, auxiliary lipids, anti-disfunction modules and fat-soluble drugs (if any) in chloroform or chloroform-methanol organic solution, stirring for 1 to 6 hours, rotary evaporating at 37 to 60° C. to form a film, hydrating with an aqueous solution containing a protein stabilizer for 0.5 to 3 hours, ultrasonically activating the particle size to make it uniform, extruding with nitrogen, adding apolipoprotein or a mimetic peptide, and incubating at 4° C. in the dark for 8 hours. (2) dissolving phospholipids, auxiliary lipids and lipophilic drugs (if any) in chloroform or chloroform-methanol organic solution, stirring for 1-6 hours, rotary evaporation at 37-60°C to form a film, hydrating with an aqueous solution of apolipoprotein or a mimetic peptide containing a coupled anti-disfunction module for 0.5-3 hours, using probe ultrasound to make the particle size uniform, and extruding with nitrogen to obtain a spherical high-density lipoprotein preparation with chemically cross-linked apolipoprotein.

[0026] The power of the probe ultrasound in the above steps is 100-400W, and the ultrasound duration is 2-10min; the pore size of the nitrogen extrusion membrane is 50-800nm, and the extrusion times are 1-20 times.

[0027] The particle size of the anti-disfunction bionic spherical high-density lipoprotein preparation prepared above is about 7-50nm, optimally 10-20nm, and has an appearance similar to natural HDL. The antioxidant enzyme encapsulation rate is more than 80%, and the drug loading is more than 1.0%.

[0028] The application refers to the anti-loss of function type bionic spherical high-density lipoprotein preparation having enzyme-like antioxidant activity, inhibiting inflammatory response; preventing its own lipids and proteins from being oxidized and maintaining a stable structure under the pathological environment of inflammation and oxidative stress; and reducing drug leakage when used to prepare drug carriers.

[0029] The experiment showed that the preparation had obvious antioxidant enzyme activity, and its own lipids and proteins did not undergo obvious oxidation within 24 hours under the Fenton reaction conditions, proving its antioxidant properties; the preparation was incubated with acute phase proteins, and it was found that it could prevent the shedding of apolipoproteins; the drug release test further proved that the preparation had a stable structure and reduced drug leakage. Mouse mononuclear macrophage RAW264.7 was used as a model cell, and the cells were stimulated with hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) to construct an inflammatory model, proving the therapeutic potential of the preparation in inflammatory diseases. The animal survival rate further proved that it has a good therapeutic effect.

[0030] Application of the anti-disfunction type bionic spherical high-density lipoprotein preparation in the preparation of medicines for treating inflammatory diseases.

[0031] The inflammatory disease is preferably sepsis or atherosclerosis.

[0032] When the anti-disability type bionic spherical high-density lipoprotein preparation of the present invention is used to prepare drugs for treating inflammatory diseases, known fat-soluble drugs for treating inflammatory diseases such as curcumin, quercetin, atorvastatin, triptolide, resveratrol, rapamycin, epicatechin gallate, artesunate, triptolide, capsaicin, ginsenosides, baicalin, cinnamaldehyde, gibberellin, shikonin, azithromycin, etc. can be prepared into anti-disability type bionic spherical high-density lipoprotein preparations using the method of the present invention.

[0033] Compared with the prior art, the present invention has the following advantages:

[0034] (1) The anti-disability bionic spherical high-density lipoprotein preparation of the present invention has good enzyme-like activity, and its antioxidant effect is better than that of natural HDL, and it can protect its own lipids and proteins under inflammatory and oxidative conditions;

[0035] (2) The present invention achieves high enzyme encapsulation rate and high protein stability by screening a series of antioxidant enzymes, lipids, and auxiliary lipid combinations, and further enhances the binding of apolipoproteins through protein stabilizers or protein linkers, thereby stabilizing its own structure and preventing apolipoproteins from falling off;

[0036] (3) The particle size and appearance of the anti-disability bionic spherical high-density lipoprotein blank preparation or drug-loaded preparation of the present invention are close to those of natural HDL, and when used as a carrier, they can reduce drug leakage caused by shape change;

[0037] (4) The preparation process of the anti-disability bionic spherical high-density lipoprotein preparation of the present invention is simple. Its application in the treatment of inflammatory diseases can significantly reduce the intracellular ROS level and change the phenotype of inflammatory cells, thereby improving the survival rate of animals and providing ideas for the treatment of inflammatory diseases. (5) BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Changes in the content of malondialdehyde (left figure) and protein carbonyl (right figure) in the anti-loss of function bionic spherical high-density lipoprotein preparation under Fenton reaction conditions in Example 10;

[0040] Figure 2 : The change in apolipoprotein content after the loss-of-function bionic spherical high-density lipoprotein preparation and acute phase protein are co-incubated in Example 11;

[0041] Figure 3 : Transmission electron microscopy images of the blank (left) or drug-loaded (right) anti-disability biomimetic spherical high-density lipoprotein preparation in Example 13, with a scale bar of 50 nm;

[0042] Figure 4: This is a drug release test of the anti-disability bionic spherical high-density lipoprotein drug-loaded preparation in Example 14;

[0043] Figure 5 : The therapeutic effect of the anti-disfunction bionic spherical high-density lipoprotein preparation in Example 15 in the inflammatory disease cell model, the cells are RAW264.7, and they are stimulated with H2O2 or LPS respectively;

[0044] Figure 6 : The intracellular ROS level after the atherosclerotic immune cells were treated with the anti-disfunction bionic spherical high-density lipoprotein preparation in Example 16;

[0045] Figure 7 : is the ratio of inflammatory cells M1 / M2 after the anti-disability bionic spherical high-density lipoprotein preparation in Example 17 treats the sepsis cell model;

[0046] Figure 8 : The animal survival rate after the sepsis animal model was treated with the anti-disfunction type bionic spherical high-density lipoprotein preparation in Example 18. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

[0048] Embodiment 1:

[0049] 0.75g polyvinyl pyrrolidone and 0.0275g tannic acid were accurately weighed and dissolved in 10mL 75% ethanol solution (containing 0.01M HCl), mixed well and placed in a water bath at 80°C for 3 hours. After cooling to room temperature, the mixture was purified by centrifugation at 1000×g for 10min using an ultrafiltration tube (MWCO: 100kDa), washed twice with deionized water, and freeze-dried to obtain a PTA anti-defunctionalization template.

[0050] Accurately weigh 100 mg of PTA, fully dissolve it in 10 mL of deionized water, adjust the solution to a pH of 7.4, add 5 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS), stir and incubate at room temperature for 30 min, then add 10 mg of apoA-I, stir and incubate for 3 h, and use size exclusion chromatography to remove uncross-linked PTA and apoA-I to obtain apoA-I coupled with PTA.

[0051] Embodiment 2:

[0052] 0.360g polyethyleneimine and 0.108g p-phenylenediamine were dissolved in 40mL ethanol, and the mixture was added into a polytetrafluoroethylene reactor and reacted at 200°C for 12h. The reaction mixture was purified by centrifugation at 1000×g for 10min using an ultrafiltration tube (MWCO: 1kDa), washed twice with deionized water, and freeze-dried to obtain a carbon dot anti-loss functional module.

[0053] 100 mg of carbon dots were accurately weighed and fully dissolved in 10 mL of deionized water. The pH of the solution was adjusted to 6.5. 5 mM EDC and NHS were added. The mixture was stirred and incubated at room temperature for 30 min. Then 10 mg of apoE was added. After stirring and incubating for 2 h, the uncross-linked carbon dots and apoE were removed by size exclusion chromatography to obtain apoE coupled to carbon dots.

[0054] Embodiment 3:

[0055] Accurately weigh 0.43g of cerium (III) acetate hydrate and 3.25g of oleylamine, and then add 15mL of xylene. The mixture is heated at 90℃ for 24h under argon protection. Quickly inject 1mL of deionized water and heat age at 90℃ for 3h. After the solution is cooled to room temperature, add 50mL of ethanol and mix, centrifuge at 4000rpm for 10min and remove the upper liquid, repeat washing twice, and dry to obtain the cerium oxide anti-destruction function module.

[0056] 100 mg of apoA-I mimetic peptide 4F was accurately weighed and fully dissolved in PBS solution (pH = 7.4), 10 mM 2-iminothiolane was added, and the mixture was stirred and incubated at room temperature for 1 h. Unreacted 2-iminothiolane was removed by ultrafiltration to obtain thiolated apoA-I. 10 mg of thiolated apoA-I was accurately weighed and dissolved in 1 mL of DMSO, 100 mg of cerium nanoparticle anti-loss function module was added, and the mixture was stirred at room temperature for 12 h. Uncoupled nanoparticles and 4F were removed by size exclusion chromatography to obtain 4F coupled to cerium nanoparticles.

[0057] Embodiment 4:

[0058] Accurately weigh 5 mg PTA, 6 mg DSPC and 4 mg DSPE-PEG and dissolve them in 3 mL chloroform-methanol (3:1) solution, stir for 4 h, and rotary evaporate at 55 °C to form a film. Then, add an aqueous solution containing 2 mg sodium cholate to hydrate for 2 h, ultrasonicate at 400 W for 10 min, extrude nitrogen at 800 nm for 3 times, add 4 mg apoA-I mimetic peptide 22A, incubate at 4 °C for 12 h, and extrude nitrogen at 400 nm for 3 times to obtain a loss-of-function bionic spherical high-density lipoprotein preparation.

[0059] Embodiment 5:

[0060] Accurately weigh 4 mg of carbon dots, 9 mg of DPPC and 1 mg of cholesterol and dissolve them in 2 mL of chloroform-methanol (2:1) solution. Stir for 1 h, and rotary evaporate at 40 °C to form a film. Then add an aqueous solution containing 5 mg of sodium deoxycholate to hydrate for 1 h, ultrasonicate at 100 W for 2 min, extrude nitrogen gas at 200 nm twice, add 10 mg of apoE mimetic peptide COG1410, incubate at 4 °C for 24 h, and extrude nitrogen gas at 100 nm twice to obtain a loss-of-function bionic spherical high-density lipoprotein preparation.

[0061] Embodiment 6:

[0062] Accurately weigh 2 mg of cerium nanoparticles, 6 mg of DMPC, 3 mg of DOPE and 1 mg of DOTAP and dissolve them in 3 mL of chloroform solution. Stir for 3 h, and rotary evaporate at 50 °C to form a film. Then, add PBS solution containing 4 mg of sodium cholate to hydrate for 1.5 h, ultrasonicate at 200 W for 10 min, extrude nitrogen at 400 nm for 5 times, add 2.5 mg of apoE and 2.5 mg of apoA-I, incubate at 4 °C for 8 h, and extrude nitrogen at 200 nm for 5 times to obtain a loss-of-function bionic spherical high-density lipoprotein preparation.

[0063] Embodiment 7:

[0064] Accurately weigh 5 mg SPC and 5 mg DSPE-PEG and dissolve them in 3 mL chloroform solution. Stir for 1 h. Rotary evaporate at 45 °C to form a film. Then add PBS solution containing 4 mg cerium oxide-coupled 4F and hydrate for 1 h. Ultrasonic probe at 400 W for 5 min and 200 nm nitrogen extrusion for 10 times to obtain a bionic spherical high-density lipoprotein preparation that is resistant to loss of function.

[0065] Embodiment 8:

[0066] Accurately weigh 9 mg of lysolecithin and 1 mg of cholesterol and dissolve them in 2 mL of chloroform solution. Stir for 1 h, and rotary evaporate at 37 °C to form a film. Then add an aqueous solution containing 2 mg of PTA-coupled apoA-I and hydrate for 3 h. Ultrasonic probe ultrasound at 300 W for 5 min, and 100 nm nitrogen extrusion for 15 times to obtain a loss-of-function bionic spherical high-density lipoprotein preparation.

[0067] Comparative Example 1:

[0068] Accurately weigh 0.05 mg of small molecule antioxidant α-tocopherol and 10 mg of DMPC and dissolve them in 2 mL of chloroform solution. Stir for 1 h, and rotary evaporate at 37°C to form a film. Then add an aqueous solution containing 4 mg of sodium cholate to hydrate for 3 h. Ultrasonicate at 300 W for 5 min, extrude 10 times with 400 nm nitrogen, add 4 mg of apoA-I powder, incubate at 4°C for 8 h, and extrude 5 times with 200 nm nitrogen to obtain an antioxidant disc-shaped rHDL control preparation.

[0069] Comparative Example 2:

[0070] Accurately weigh 0.05 mg of small molecule antioxidant α-tocopherol, 8 mg of DMPC, 1 mg of triglyceride and 1 mg of cholesterol ester and dissolve them in 2 mL of chloroform solution. Stir for 2 h, and rotary evaporate at 37 °C to form a film. Then, add an aqueous solution containing 4 mg of sodium cholate to hydrate for 3 h, ultrasonicate at 400 W for 5 min, extrude 10 times with 200 nm nitrogen, add 4 mg of apoA-I powder, incubate at 4 °C for 8 h, and extrude 10 times with 100 nm nitrogen to obtain the antioxidant spherical rHDL control preparation.

[0071] Comparative Example 3:

[0072] Accurately weigh 2 mg of cerium nanoparticles, 8 mg of DPPC and 2 mg of DSPE-PEG and dissolve them in 2 mL of chloroform solution. Stir for 3 h, and perform rotary evaporation at 50 °C to form a film. Then add deionized water for hydration for 3 h, perform probe ultrasound at 400 W for 5 min, extrude nitrogen gas at 200 nm for 10 times, add 4 mg of apoA-I powder, incubate at 4 °C for 8 h, and extrude nitrogen gas at 100 nm for 10 times to obtain a spherical rHDL control preparation without added protein stabilizer.

[0073] Comparative Example 4:

[0074] Accurately weigh 8 mg DPPC and 2 mg DSPE-PEG and dissolve them in 2 mL chloroform solution. Stir for 3 h, rotary evaporate at 50 °C to form a film, add deionized water for hydration for 3 h, ultrasonicate at 400 W for 5 min, extrude 10 times with 200 nm nitrogen, add 4 mg apoA-I powder, incubate at 4 °C for 8 h, and extrude 10 times with 100 nm nitrogen to obtain a disc-shaped rHDL control preparation without added protein stabilizer.

[0075] Comparative Example 5:

[0076] Accurately weigh 2 mg of cerium nanoparticles and 10 mg of DPPC and dissolve them in 2 mL of chloroform solution. Stir for 3 h, and perform rotary evaporation at 50 °C to form a film. Then add deionized water for hydration for 3 h, perform probe ultrasound at 400 W for 5 min, and extrude 10 times with 200 nm nitrogen. Then add 4 mg of apoA-I powder, incubate at 4 °C for 8 h, and extrude 10 times with 100 nm nitrogen to obtain a spherical rHDL control preparation without the addition of auxiliary lipids.

[0077] Example 9: Particle size, PDI, antioxidant enzyme encapsulation efficiency and drug loading of anti-disfunction biomimetic spherical high-density lipoprotein preparation

[0078] The four parameters of hydrated particle size, polydispersity index (PDI), antioxidant enzyme encapsulation efficiency (EE, %) and drug loading (DL, %) were used to evaluate the loss-of-function bionic spherical high-density lipoprotein preparations prepared in Examples 4, 5, 6, 7, and 8, and compared with natural HDL. The results are shown in Table 1. The particle sizes of the loss-of-function bionic spherical high-density lipoprotein preparations prepared in Examples 4, 5, 6, 7, and 8 are uniform and consistent, which is comparable to the particle size of natural HDL. In addition, they all have high encapsulation efficiency and drug loading of the loss-of-function module.

[0079] Table 1. Parameters of high density lipoprotein preparations prepared in different embodiments

[0080]

[0081]

[0082] Example 10: Protective ability of anti-disfunction bionic spherical high-density lipoprotein preparation on lipids and proteins

[0083] The anti-lipid peroxidation ability of each preparation was evaluated by a malondialdehyde kit containing thiobarbituric acid. In the experiment, 50 μM CuSO4 and 150 μM H2O2 were mixed with the rHDL preparations in Examples 4, 5, 6, 7, 8, and 9, the rHDL preparations in Comparative Examples 1 and 2, or natural HDL, and incubated in a constant temperature oscillator at 37°C and 120 rpm. Samples were taken at 0 and 24 hours and the MDA change level in the system was calculated.

[0084] After the protein is oxidized, the carbonyl content increases, and the carbonyl group can react with 2,4-dinitrophenylhydrazine to generate a reddish brown precipitate. After the precipitate is dissolved, the carbonyl content of the protein can be measured and calculated on a spectrophotometer. According to the same experimental steps as above, the protein carbonyl content changes of the rHDL preparations in Examples 4, 5, 6, 7, 8, and 9, the rHDL preparations in Comparative Examples 1 and 2, or natural HDL are measured and calculated using a protein carbonyl content kit.

[0085] The results are as follows Figure 2 As shown, the anti-disability bionic spherical high-density lipoprotein preparations prepared in Examples 4, 5, 6, 7, and 8 were 2+ Under the strong oxidative conditions of H2O2, the MDA content did not increase significantly at 24 hours, and the MDA level was significantly reduced compared to Comparative Example 1 using traditional small molecules as antioxidants, Comparative Example 2 without antioxidant protection, or natural HDL. In addition, each anti-disfunction bionic spherical high-density lipoprotein preparation significantly reduced the protein carbonyl content compared to Comparative Examples 1, 2 or natural HDL, further proving that the anti-disfunction bionic spherical high-density lipoprotein preparation of the present invention has excellent antioxidant properties, which is conducive to its expected therapeutic effect in inflammatory diseases.

[0086] Example 11: Anti-loss of function biomimetic spherical high-density lipoprotein preparation prevents self-protein shedding

[0087] Apolipoproteins were labeled with rhodamine isothiocyanate B (RBITC), and RBITC-labeled rHDL preparations were prepared according to the preparation methods in Examples 4, 5, 6, 7, 8, 9, and Comparative Examples 3, 4, and 5. 200 μL of the above preparation was mixed with the acute phase protein serum amyloid A (SAA, final concentration 100 μg / mL), and incubated at 37°C in the dark for 0 and 60 min. Free RBITC-labeled apolipoproteins were removed by centrifugation at 10000 rpm for 10 min through a 100 kDa ultrafiltration tube, and the fluorescence intensity of the remaining RBITC-labeled apolipoproteins in the preparation was measured (Ex: 466; Em: 576), which were recorded as F 0 min and F 60 min .

[0088] The remaining apolipoprotein content (%) was calculated according to the following formula:

[0089]

[0090] The results are as follows Figure 2 As shown, after incubation with SAA for 60 minutes, only about 43%, 21% and 33% of apolipoprotein were retained in comparative examples 3, 4 and 5, respectively, while the remaining apolipoprotein content of the anti-disability bionic spherical high-density lipoprotein preparations prepared in Examples 4, 5, 6, 7 and 8 was between 58% and 85%, and the remaining protein content was significantly increased, indicating that the anti-disability bionic spherical high-density lipoprotein preparation can stabilize its own structure and prevent protein shedding under the inflammatory simulation environment. This time, the rHDL preparation coupled with the anti-disability template has the strongest anti-protein shedding effect, while the control spherical rHDL preparation and discoid rHDL preparation without adding protein stabilizer or auxiliary lipid have poor effects, which also shows that the appropriate prescription and structure have a significant impact on enhancing the stability of rHDL protein.

[0091] Example 12: Preparation of anti-disfunction biomimetic spherical high-density lipoprotein drug-loaded preparation

[0092] Taking the rHDL preparation prepared in Example 7 as an example, curcumin, quercetin, atorvastatin, triptolide, resveratrol or rapamycin are model drugs. 5 mg SPC, 5 mg DSPE-PEG, and 0.5 mg drug powder are accurately weighed and dissolved in 3 mL chloroform solution, stirred for 1 hour, and rotary evaporated at 45 ° C to form a film, and then 4 mg cerium oxide-coupled 4F PBS solution is added for hydration for 1 hour, and the probe is ultrasonicated at 400W for 5 minutes, and 200nm nitrogen is extruded 10 times, that is, the anti-defunctional bionic spherical high-density lipoprotein preparation is obtained.

[0093] The particle size, encapsulation efficiency and drug loading of the anti-destructive bionic spherical high-density lipoprotein drug-loaded preparation were tested, and the results are shown in Table 2. The particle size of the preparation did not change much after drug loading, and it had a high encapsulation efficiency and drug loading, proving that it can be used as a universal fat-soluble drug carrier.

[0094] Table 2. Parameters of the drug-loaded biomimetic spherical high-density lipoprotein preparations that resist loss of function

[0095]

[0096] Example 13: Microstructure of anti-loss of function biomimetic spherical high-density lipoprotein blank or drug-loaded preparation

[0097] The microstructure of the anti-destructive bionic high-density lipoprotein blank or drug-loaded preparation in Examples 7 and 12 was observed by transmission electron microscopy. The results are as follows: Figure 3 The blank and drug-loaded preparations were nearly spherical, with particle sizes of approximately 10 to 15 nm, which was comparable to natural HDL.

[0098] Example 14: Drug release of anti-disability biomimetic spherical high-density lipoprotein drug-loaded preparation

[0099] Using rapamycin enzyme model drug, 1 mL of the rHDL drug-loaded preparation (rapamycin final concentration 0.1 mg / mL) prepared in Example 12 (rapamycin final concentration 0.1 mg / mL) and Comparative Examples 1 and 2 loaded with rapamycin was precisely drawn and transferred to a dialysis bag with a molecular weight cutoff of 8-14 kDa. The dialysis bag was placed in a 50 mL centrifuge tube. The dialysis medium was 30 mL 0.01 M PBS (pH 7.4, containing 0.1% SDS). The centrifuge tube was placed in a constant temperature oscillator at 37°C and 120 rpm. 0.2 ml of the dialysis medium was taken at 0, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h to prepare samples, and the same volume of dialysis medium was added. The samples were injected by HPLC to measure the drug concentration. C i and C k are the drug concentrations in the dialysis medium at different time points; i = 1, 2, 3, ...; k = 1, 2, ..., i-1; V is the volume of the dialysis medium; C I is the initial drug concentration of the preparation before dialysis.

[0100] The cumulative release rate (%) of the drug was calculated according to the following formula:

[0101]

[0102] The experimental results are as follows Figure 4As shown, it can be seen that compared with the disc-shaped drug-loaded preparation in Comparative Example 1 and the spherical drug-loaded preparation in Comparative Example 2, the anti-defunctionalization bionic spherical high-density lipoprotein drug-loaded preparation prepared in Example 12 has less cumulative drug release within 48 hours, indicating that it has good stability in plasma and reduces drug leakage caused by conformational changes.

[0103] Example 15: Therapeutic effect of anti-disfunction biomimetic spherical high-density lipoprotein preparation in inflammatory cell model

[0104] Convert RAW.264.7 to 2×10 4 The cells were inoculated at a density of 100 μg / mL in each well of a 96-well plate and placed in a 37°C, 5% CO2 constant temperature incubator until the cell density in each well was about 50-60%. After discarding the supernatant, 800 μM H2O2 or 2.5 μg / mL LPS was added and incubated for 2 h, followed by addition of 0.63 μM, 0.125 μM and 0.25 μM high, medium and low doses of free drug, blank formulation Example 7, and drug-loaded formulation Example 13 containing rapamycin, and continued to be cultured for 24 h. The cell activity of each group was then measured by the MTT method.

[0105] The experimental results are as follows Figure 5 As shown, the cell viability of RAW264.7 after H2O2 or LPS injury was approximately 40% and 50%, respectively. Both the blank formulation Example 7 and the drug-loaded formulation Example 13 had good therapeutic effects. The cell viability was greater than 70% under treatment with all doses of formulations, and even greater than 85% with the medium-dose drug-loaded formulation, demonstrating that the anti-disability bionic spherical high-density lipoprotein formulation has an excellent therapeutic effect in the inflammatory cell model and can greatly improve cell activity.

[0106] Example 17: Loss-of-function biomimetic spherical high-density lipoprotein formulation reduces ROS levels in inflammatory cells in atherosclerosis

[0107] Convert RAW.264.7 to 2×10 4The cells were inoculated at a density of 100 μM in each well in a 96-well plate and placed in a constant temperature incubator at 37°C and 5% CO2 until the cell density in each well was about 50-60%. After discarding the supernatant, 800 μM H2O2 was added and incubated for 2 hours, and then free drug, blank preparation Example 7, and drug-loaded preparation Example 13 containing rapamycin (rapamycin concentration was 0.125 μM) were added and cultured for 24 hours. At the same time, the cell wells with only H2O2 added were used as positive controls, and the cell wells with only complete culture medium added were used as negative controls. After incubation for 24 hours, the supernatant was discarded, and 1 mL PBS was added to wash the cells 3 times. 0.5 mL of 10 μM DCFH-DA was added under light-proof conditions and incubated for 30 minutes. The supernatant was discarded, washed twice with PBS, and the cells were resuspended with 0.5 mL PBS, and the fluorescence signal in each sample was detected by BD flow cytometer.

[0108] The experimental results are as follows Figure 6 As shown, DCF + The higher the cell ratio, the higher the overall ROS level of the cells. + The cell ratio increased significantly. Both the free drug and blank preparation Example 7 had a certain anti-ROS effect. After the combination of the two, the drug-loaded preparation Example 13DCF + The cell ratio was further reduced and had no significant difference with the negative control group, demonstrating its antioxidant effect in atherosclerosis.

[0109] Example 18: Anti-disfunction biomimetic spherical high-density lipoprotein preparation changes the phenotype of sepsis inflammatory cells

[0110] Convert RAW.264.7 to 2×10 4The cells were inoculated at a density of 100 wells in a 96-well plate and placed in a constant temperature incubator at 37°C and 5% CO2 until the cell density in each well was about 50-60%. After discarding the supernatant, 1 μg / mL LPS was added and incubated for 2 hours, followed by the addition of free drug, blank preparation Example 7, and drug-loaded preparation Example 13 containing rapamycin (rapamycin concentration was 0.125 μM), and continued to be cultured for 24 hours. At the same time, the cell wells with only LPS added were used as positive controls, and the cell wells with only complete culture medium added were used as negative controls. After 24 hours, the culture medium was discarded, PBS was added to resuspend the collected cells, and the supernatant was discarded after centrifugation. The cells were resuspended with 3% BSA, blocked at 37°C for 15 minutes, and the supernatant was discarded after centrifugation. PBS was added to resuspend the cells, and then 5 μL APC-CD86 antibody was added. After mixing evenly, the cells were incubated at 4°C in the dark for 30 minutes, and washed once with PBS. Subsequently, 4% paraformaldehyde was added, fixed at room temperature in the dark for 15 minutes, and washed once with PBS. Add 0.1% Triton X-100, permeabilize the cell membrane for 15 minutes at room temperature, and wash once with PBS. Add PBS to resuspend the cells and add 5μL PE-CD206 antibody. Mix well and incubate at 4℃ in the dark for 30 minutes, and wash once with PBS. Finally, add PBS to resuspend the cells, and detect the fluorescence signal in each sample by BD flow cytometer. Among them, CD86+CD206- cells are determined as M1 macrophages, and CD86-CD206+ cells are determined as M2 macrophages.

[0111] The experimental results are as follows Figure 7 As shown, both the free drug and blank preparation Example 7 significantly reduced the proportion of M1 macrophages in sepsis and increased the proportion of M2. After the combination of the two, the M1 / M2 cell ratio of the drug-loaded preparation Example 13 was further reduced, proving that the anti-disability bionic spherical high-density lipoprotein preparation can change the phenotype of macrophages in sepsis, significantly reduce the proportion of pro-inflammatory cells and increase the proportion of anti-inflammatory cells, which is beneficial to the resolution of inflammation and tissue repair.

[0112] Example 19: Anti-disfunction bionic spherical high-density lipoprotein preparation improves the survival rate of septic animals

[0113] 40 ICR mice were evenly divided into 4 groups (n=10), and a mouse sepsis model was established by tail vein injection of 15 mg / kg LPS. PBS, free drug, blank preparation Example 7, and drug-loaded preparation Example 13 encapsulating rapamycin were injected 30 minutes after modeling, and the 7-day survival rate of mice was recorded.

[0114] The results are as follows Figure 8As shown, compared with PBS, both the free drug and blank preparation Example 7 increased the survival rate of mice, and after treatment with the combined drug-loaded preparation Example 13, the survival rate of septic mice was significantly increased to 90%, further demonstrating its potential for application in inflammatory diseases.

[0115] The present invention combines rHDL with antioxidant enzymes to prepare a loss-of-function bionic spherical high-density lipoprotein preparation with enzyme-like antioxidant activity. The preparation can protect its own lipids and proteins and maintain the stability of the spherical structure under inflammatory and stress environments. When used as a carrier, it can effectively reduce the leakage of fat-soluble drugs, has good therapeutic effects in inflammation-related cell and animal models, can significantly reduce intracellular ROS levels and change the phenotype of inflammatory cells, and improve animal survival rates.

Claims

1. A biomimetic spherical high-density lipoprotein preparation that resists functional loss, characterized in that: Any combination of the following ingredients: (I) phospholipids, auxiliary lipids, apolipoproteins, anti-disfunction modules and protein stabilizers; (II) phospholipids, helper lipids, apolipoproteins coupled to the anti-disfunction module via a linker, and protein stabilizers; The anti-defunctionalization template is a nanocarrier with antioxidant enzyme properties, selected from any one or more of the following: an organic monomer polymer template, a metal template composed of a combination of metal ions and organic ligands, a carbon-based template composed of a carbon core, or a material doped or modified as needed, and still having antioxidant enzyme activity; The apolipoprotein is selected from apolipoprotein or a mimetic peptide thereof; The particle size of the anti-disfunction type bionic spherical high-density lipoprotein preparation is 7-50nm.

2. The anti-disfunction bionic spherical high-density lipoprotein preparation according to claim 1, characterized in that: The phospholipids are selected from any one or more of the following: soybean lecithin, hydrogenated soybean lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl lecithin, distearoyl phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylglycerol, sphingomyelin, and lysolecithin.

3. The anti-disfunction bionic spherical high-density lipoprotein preparation according to claim 1, characterized in that: The auxiliary lipid is used to improve the structural stability and encapsulation rate of the preparation, and is selected from any one or more of the following: cholesterol, cholesterol ester, triglyceride, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-poly(ethylene glycol), (2,3-dioleoyl-propyl)-trimethylamine; the mass ratio of the auxiliary lipid to the phospholipid is 1:1-40.

4. The anti-disfunction bionic spherical high-density lipoprotein preparation according to claim 1, characterized in that: The apolipoprotein is selected from one or more of apoA-I, apoA-II, apoA-IV, apoCs, apoB, apoE, apoJ, apoM or their mimetic peptides, wherein the mimetic peptide is selected from apoA-I mimetic peptide 22A, apoA-I mimetic peptide 4F, and apoE mimetic peptide COG1410; the mass ratio of the apolipoprotein to the total lipid is 1:1 to 10.

5. The anti-disability bionic spherical high-density lipoprotein preparation according to claim 1, characterized in that: The organic monomer polymer template is selected from polydopamine and polytannic acid; the metal template composed of metal ions and organic ligands is selected from iron nanoparticles modified with dodecyl mercaptan, manganese nanoparticles modified with oleic acid, and cerium nanoparticles modified with oleylamine; the carbon-based template composed of the carbon core is selected from carbon quantum dots; and the mass ratio of the anti-defunctionalization template to the total lipids is 1:2 to 10.

6. The anti-disfunction bionic spherical high-density lipoprotein preparation according to claim 1, characterized in that: The protein stabilizer is used to enhance the stability of apolipoprotein and is selected from any one or more of the following: sodium cholate, sodium deoxycholate, Tween, tetradiaminetetraacetic acid, and phosphate; the mass ratio of the protein stabilizer to the total lipid is 1:1-100.

7. The method for preparing the anti-disfunction type biomimetic spherical high-density lipoprotein preparation according to any one of claims 1 to 6, characterized in that: Choose any of the following methods: (I) dissolving phospholipids, auxiliary lipids and anti-disfunction modules, or phospholipids, auxiliary lipids, anti-disfunction modules and fat-soluble drugs in chloroform or chloroform-methanol organic solution, stirring for 1 to 6 hours, rotary evaporating at 37 to 60° C. to form a film, hydrating with an aqueous solution containing a protein stabilizer for 0.5 to 3 hours, ultrasonically balancing the particle size, extruding with nitrogen, adding apolipoprotein, incubating at 4° C. in the dark for 8 to 24 hours, and extruding with nitrogen to obtain a spherical anti-disfunction bionic spherical high-density lipoprotein preparation physically embedded with apolipoprotein; (II) Dissolve phospholipids and auxiliary lipids, or phospholipids, auxiliary lipids and fat-soluble drugs in chloroform or chloroform-methanol organic solution, stir for 1 to 6 hours, and rotary evaporate at 37 to 60° C. to form a film. Then, use an aqueous solution of apolipoprotein containing a coupled anti-disordered functional module to hydrate for 0.5 to 3 hours, use probe ultrasound to make the particle size uniform, and extrude with nitrogen to obtain a spherical anti-disordered functional biomimetic spherical high-density lipoprotein preparation with chemically cross-linked apolipoprotein.

8. The preparation method according to claim 7, characterized in that: The power of the probe ultrasound is 100-400W, and the ultrasound duration is 2-10min; The pore size of the nitrogen extrusion membrane is 50 to 800 nm, and the number of times is 1 to 20 times.

9. Use of the anti-disfunction type bionic spherical high-density lipoprotein preparation according to any one of claims 1 to 6 in the preparation of a drug for treating inflammatory diseases.

10. The use according to claim 9, characterized in that: The inflammatory disease is sepsis or atherosclerosis.

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