Degreasing nano-particles simulating high-density lipoprotein structure and preparation method of degreasing nano-particles

Abstract

The invention relates to the technical field of biomedicine nanotechnologies, and discloses degreasing nanoparticles simulating a high-density lipoprotein structure and a preparation method thereof.The nanoparticles are formed by assembling human recombinant apolipoprotein A-I and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphorylcholine according to the molar ratio of 90: 1-120: 1. The method comprises the following steps: incubating apolipoprotein in a polyethylene glycol molecule crowded environment to induce dominant conformation; constructing a phospholipid-supercritical carbon dioxide system, pumping protein liquid, and realizing instant self-assembly by utilizing a synergistic effect of ultrasonic cavitation and rapid pressure relief expansion; and finally, carrying out tangential flow ultrafiltration purification through a polyethersulfone membrane subjected to surface grafting hydrophilic modification. According to the process, protein aggregation and inactivation are effectively avoided, the problem of membrane pollution is solved, and the assembly efficiency, the particle size uniformity, the degreasing biological activity and the product yield of the nanoparticles are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanotechnology, specifically to a lipid-removing nanoparticle with a structure similar to high-density lipoprotein and its preparation method. Background Technology

[0002] High-density lipoprotein (HDL) plays a crucial role in the reverse transport of cholesterol in the body, transporting cholesterol from peripheral tissues to the liver for metabolism, thereby exerting an anti-atherosclerotic effect. Based on this mechanism, the construction of HDL-mimicking nanoparticles using recombinant human apolipoprotein AI and phospholipids has become an important research direction for the treatment of cardiovascular diseases and lipid-lowering therapy.

[0003] However, the preparation and purification processes of high-density lipoprotein (HDL)-like nanoparticles still face many challenges. First, during the self-assembly of nanoparticles, the conformational state of apolipoprotein AI, as an amphiphilic protein, is crucial to assembly efficiency. Traditional preparation processes, such as sodium cholate dialysis or direct mechanical dispersion, often lack precise control over protein conformation. This leads to apolipoprotein AI easily misfolding or non-specific aggregation in aqueous environments, making it difficult to form a favorable conformation for lipid binding. Consequently, the binding efficiency with phospholipids is low, resulting in nanoparticles with insufficient structural stability and compromised bioactivity. Second, existing preparation techniques often struggle to achieve simultaneous and precise control of micro-mixing and nucleation processes, frequently resulting in wide particle size distributions. Furthermore, prolonged ultrasonic or thermal treatments can easily cause protein denaturation, making it difficult to obtain uniform particle size while retaining high lipid-removing activity.

[0004] Furthermore, during the purification and separation stage after preparation, due to the two characteristics of apolipoproteins and assembled lipid nanoparticles, they are prone to severe non-specific adsorption and deposition on the surface of conventional ultrafiltration or dialysis membranes, leading to membrane pore blockage. This not only significantly reduces separation efficiency and throughput but also causes high raw material losses, severely limiting the improvement of product yield. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a lipid-removing nanoparticle with a high-density lipoprotein (HDL) structure and its preparation method. This invention solves the problems of low assembly efficiency and poor structural stability caused by the lack of effective control over the conformation of apolipoproteins in existing HDL nanoparticle preparation technologies, poor particle size uniformity and protein activity loss caused by the difficulty in accurately controlling the micro-nucleation process, and low product yield caused by severe non-specific adsorption on the membrane surface during purification.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: a lipid-removing nanoparticle that mimics the structure of high-density lipoprotein, made from the following raw materials: human recombinant apolipoprotein AI and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine; The molar ratio of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine to recombinant human apolipoprotein AI is 90:1 to 120:1.

[0007] By employing the above technical solution, a bioactive high-density lipoprotein-mimicking nanostructure is constructed by assembling human recombinant apolipoprotein AI with 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in a specific molar ratio. The molar ratio range is set based on the amphiphilic helical structure of human recombinant apolipoprotein AI. When the ratio is controlled between 90:1 and 120:1, phospholipid molecules can fully fill the amphiphilic pockets of the apolipoprotein, inducing the formation of stable disc-shaped or spherical nanostructures. This avoids the formation of liposome vesicles due to excessive phospholipids or protein aggregation due to insufficient phospholipids, thereby ensuring the stability of the nanoparticles in blood circulation and their cholesterol reverse transport function.

[0008] Preferably, the raw material, recombinant human apolipoprotein AI, is a lyophilized powder with a purity greater than 95%.

[0009] By adopting the above technical solution, high-purity recombinant human apolipoprotein AI reduces the interference of impurities in the nanoparticle assembly process, ensuring the uniformity and biosafety of the final product.

[0010] Preferably, the raw material 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine is used in the preparation in the form of a solution dissolved in anhydrous ethanol, wherein the concentration of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in the solution is 15 mg / mL to 25 mg / mL.

[0011] By adopting the above technical solution, anhydrous ethanol, as a good solvent, helps to disperse phospholipid molecules into single molecules. A specific concentration range, combined with subsequent supercritical fluid processes, enables the rapid dissolution and supersaturated precipitation of phospholipids in the supercritical carbon dioxide system, thereby controlling the crystal nucleation rate.

[0012] A method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein includes the following steps: S1. Human recombinant apolipoprotein AI is dissolved in buffer solution to prepare a stock solution. Polyethylene glycol is added to the stock solution and incubated at a constant temperature to obtain a pre-activated human recombinant apolipoprotein AI crowded solution. S2. Dissolve 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in anhydrous ethanol to prepare a phospholipid-ethanol solution. Place the phospholipid-ethanol solution in a reaction vessel, adjust the temperature, introduce carbon dioxide and adjust the pressure to a supercritical state, so that the 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine is fully dissolved to form a supercritical mixed fluid. S3. Pump the pre-activated recombinant human apolipoprotein AI crowding solution obtained in step S1 into the reactor in the supercritical state, so that the pre-activated recombinant human apolipoprotein AI crowding solution is mixed and contacted with the supercritical mixed fluid. S4. After the sample injection is completed, start the ultrasonic transducer installed on the reactor and simultaneously open the pressure relief valve installed on the reactor to reduce the pressure inside the reactor to atmospheric pressure and collect the suspension. S5. The suspension collected in step S4 is subjected to tangential flow ultrafiltration and washing using a modified polyethersulfone membrane module to obtain the lipid-removing nanoparticles with the high-density lipoprotein-like structure.

[0013] By adopting the above technical solution, this invention innovatively combines a molecular crowding pre-organization strategy with acoustic cavitation-assisted supercritical fluid technology, and achieves the controllable preparation of highly active degreasing nanoparticles through the synergistic effect of the following multiple mechanisms: Molecular crowding pre-organization mechanism: In step S1, polyethylene glycol is introduced to construct a macromolecular crowding environment, utilizing the exclusion volume effect to restrict the conformational freedom of recombinant human apolipoprotein AI. This crowding environment mimics the physiological state within the cell, prompting apolipoprotein AI to fold from a disordered or molten globule state to a compact α-helical structure, exposing hydrophobic domains. This pre-activated state lowers the energy barrier for protein-phospholipid binding, allowing the protein to insert into the phospholipid layer or recruit phospholipid molecules more efficiently, rather than undergoing denaturation or misfolding during assembly.

[0014] Supercritical fluid solvent effect: In steps S2 and S3, supercritical carbon dioxide has the characteristics of low viscosity, high diffusion coefficient and zero surface tension; it not only serves as an excellent solvent for phospholipids, enabling phospholipid molecules to achieve uniform molecular-level dispersion in the reaction system, but also acts as an antisolvent, creating extremely high supersaturation at the interface with protein aqueous solution, inducing instantaneous nucleation of nanoparticles.

[0015] Synergistic effect of acoustic cavitation and rapid expansion: In step S4, the collapse of cavitation bubbles generated by ultrasound releases enormous energy, producing local shear force. Combined with the rapid expansion of the supercritical fluid during depressurization, this instantaneously atomizes and disperses the mixed fluid. This synergistic effect prevents protein aggregation on the one hand, and provides the activation energy required for assembly on the other, enabling pre-activated apolipoproteins and micronized phospholipids to complete self-assembly within milliseconds, forming tightly packed, uniformly sized nanoparticles.

[0016] Surface antifouling separation mechanism: The modified polyethersulfone membrane module used in step S5 forms a hydration layer on the membrane surface by grafting hydrophilic molecules, which effectively reduces the adsorption and blockage of amphiphilic apolipoproteins and lipid nanoparticles on the membrane pore surface, thereby improving separation efficiency and yield.

[0017] Preferably, in step S1, the concentration of recombinant human apolipoprotein AI in the stock solution is 1.5 mg / mL to 2.5 mg / mL; the final mass-volume concentration of polyethylene glycol in the system is 12% to 18%; and the isothermal incubation temperature is 35°C to 39°C, and the time is 25 minutes to 35 minutes.

[0018] By adopting the above technical solution, the concentration of polyethylene glycol and the incubation conditions can be controlled within a specific range, which can precisely regulate the degree of crowding. If the concentration is too low, it will not produce a significant volume exclusion effect, while if the concentration is too high, it may cause protein precipitation. With appropriate temperature and time, the apolipoprotein can be ensured to complete conformational rearrangement and reach a thermodynamically stable state, thus preparing the structure for subsequent binding with lipids.

[0019] Preferably, in step S2, the reactor is equipped with a stirring device with a stirring speed of 150 rpm to 250 rpm; the conditions for the supercritical state are: temperature 43℃ to 47℃, pressure 12MPa to 18MPa, and holding time 8 minutes to 12 minutes; in step S3, the pumped flow rate is 4 ml / min to 6 ml / min.

[0020] By employing the above technical solution, a specific temperature and pressure range ensures that carbon dioxide is in the supercritical fluid region and possesses suitable solubility, avoiding protein denaturation due to excessive temperature or excessive equipment load due to excessive pressure. Controlling the injection flow rate determines the mixing ratio of the aqueous phase and the supercritical phase, thus affecting the nucleation density and final particle size.

[0021] Preferably, step S4 specifically involves: starting the ultrasonic transducer, setting the ultrasonic frequency to 35 kHz to 45 kHz, and setting the power to 40 W to 60 W; and opening the pressure relief valve of the reactor within 0.2 seconds after the ultrasonic transducer is started, so that the pressure inside the reactor drops to atmospheric pressure within 1 to 3 seconds.

[0022] By employing the above technical solution, precise timing control is key to achieving synergistic effects. Immediate depressurization shortly after ultrasonic initiation ensures that the material undergoes explosive nucleation while experiencing intense ultrasonic dispersion. This rapid depressurization process of 1 to 3 seconds not only prevents excessive crystal growth but also effectively avoids the thermal effects of prolonged ultrasound, which primarily lead to protein inactivation.

[0023] Preferably, in step S5, the modified polyethersulfone membrane module has a molecular weight cutoff of 100 kDa, and the modified polyethersulfone membrane module is prepared by the following steps: selecting a polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 100 kDa as the base membrane; preparing a modification solution containing a hydrophilic monomer, immersing the base membrane in the modification solution, adding an initiator to initiate a surface graft polymerization reaction; after the reaction, cleaning the membrane to obtain the modified polyethersulfone membrane, and encapsulating the modified polyethersulfone membrane into the modified polyethersulfone membrane module. More preferably, the hydrophilic monomer is polyethylene glycol methacrylate.

[0024] By employing the above technical solution, polyethylene glycol methacrylate is used to graft and modify the polyethersulfone membrane, introducing flexible hydrophilic side chains into the membrane pores and surface. During tangential flow ultrafiltration, the hydrophilic layer resists the non-specific adsorption of proteins and lipids through steric hindrance, reducing membrane fouling and maintaining stable transmembrane flux, thereby achieving efficient purification of nanoparticles and buffer replacement.

[0025] Preferably, the buffer solution in step S1 is PBS buffer; the replacement medium used for tangential flow ultrafiltration and washing in step S5 is PBS buffer.

[0026] By adopting the above technical solution, the PBS buffer provides a pH value and ionic strength close to the physiological environment, which helps maintain the secondary structure stability of apolipoprotein AI and the dispersion stability of nanoparticles.

[0027] This invention provides a lipid-removing nanoparticle with a structure mimicking high-density lipoprotein and its preparation method. It has the following beneficial effects: 1. This invention precisely controls the molar ratio of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine to human recombinant apolipoprotein AI to 90:1 to 120:1, and introduces polyethylene glycol to construct a molecularly crowded environment to pre-activate the protein. This crowded environment induces conformational rearrangement of apolipoprotein AI by eliminating the volume effect, so that it pre-forms a compact α-helical structure that is conducive to binding lipids, reduces the assembly energy barrier, improves the binding efficiency of protein and phospholipids and the structural stability of nanoparticles, and effectively solves the problem of reduced biological activity caused by protein misfolding or aggregation in traditional preparation.

[0028] 2. This invention employs ultrasound-assisted supercritical fluid rapid expansion technology in its preparation method. By utilizing the synergistic effect of the high diffusivity of supercritical carbon dioxide and the acoustic cavitation effect, uniform mixing and explosive nucleation of lipids and proteins are achieved within milliseconds. The local high-energy shear force generated by ultrasound effectively prevents the aggregation of high-concentration proteins. Combined with rapid cooling and supersaturation precipitation during the depressurization process, the prepared nanoparticles have a narrow particle size distribution and high uniformity. Furthermore, the entire process avoids prolonged high-temperature heating, thus maximizing the preservation of the lipid-removing activity of apolipoproteins.

[0029] 3. This invention employs a modified polyethersulfone membrane module with surface-grafted polyethylene glycol methacrylate in the purification step. By constructing a hydrophilic hydration layer on the membrane surface, the non-specific adsorption and blockage of amphiphilic apolipoproteins and lipid nanoparticles on the membrane pore surface are reduced by utilizing the steric hindrance effect. The modified membrane module maintains a stable high permeation flux during tangential flow ultrafiltration, and while efficiently removing residual polyethylene glycol and ethanol from the system, it significantly improves the recovery rate and purity of the target lipid nanoparticles. Detailed Implementation

[0030] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Preparation Examples 1-2: Preparation Example 1: Preparation of Human Recombinant Apolipoprotein AI (rhApoA-I) Strain construction: Codon optimization was performed based on the mature peptide gene sequence of human apolipoprotein AI. The optimized gene fragment was cloned into a prokaryotic expression vector, transformed into E. coli expression host, and positive clones were screened.

[0032] Fermentation expression: The positive strain was cultured in a large scale and expression was induced at low temperature by adding an inducer.

[0033] Extraction and purification: Collect bacterial cells, lyse them, and centrifuge to collect the supernatant. The supernatant is then subjected to specific adsorption and elution using an affinity chromatography column, followed by further purification via gel filtration chromatography to remove impurities such as proteins and polymers.

[0034] Preparation of finished product: The monomer peaks were collected, desalted by dialysis, and then freeze-dried to obtain human recombinant apolipoprotein AI lyophilized powder with a purity of >95%, which was used as the raw material for subsequent examples.

[0035] Preparation Example 2: Preparation of Modified Polyethersulfone Membrane Module Base membrane pretreatment: Select a commercial polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 100kDa and clean it to remove surface impurities.

[0036] Surface modification: Prepare a modification solution containing hydrophilic monomers (such as polyethylene glycol methacrylate), immerse the PES membrane in it, and initiate a surface graft polymerization reaction by using an initiator to introduce hydrophilic segments on the membrane surface.

[0037] Module encapsulation: After the reaction, the membrane is cleaned and encapsulated into the membrane module required for tangential flow filtration. The modified membrane has low protein adsorption characteristics.

[0038] Examples 1-3: Example 1: Prepare human recombinant apolipoprotein AI stock solution and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution respectively: Human recombinant apolipoprotein AI stock solution: Accurately weigh 100 mg of the human recombinant apolipoprotein AI lyophilized powder prepared in Example 1, dissolve it in 66.7 mL of PBS buffer, and prepare a human recombinant apolipoprotein AI stock solution with a concentration of 1.5 mg / mL.

[0039] 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution: Based on a molar ratio of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine to recombinant human apolipoprotein AI of 90:1, 243 mg of POPC was weighed and dissolved in 16.2 mL of anhydrous ethanol to prepare a 15 mg / mL 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution.

[0040] Preparation of pre-activated human recombinant apolipoprotein AI crowding solution: Polyethylene glycol was added to the prepared human recombinant apolipoprotein AI stock solution and stirred to dissolve it so that the final mass-volume concentration of polyethylene glycol in the system was 12%. The mixture was then incubated in a 35°C constant temperature water bath for 25 minutes to obtain a pre-activated human recombinant apolipoprotein AI crowded solution.

[0041] Construction and injection of supercritical fluid systems: The prepared phospholipid-ethanol solution was placed in a reactor equipped with a magnetic stirrer, and the stirring speed was set to 150 rpm. The temperature inside the reactor was adjusted to 43°C, carbon dioxide was introduced, and the pressure was adjusted to 12 MPa to bring it to a supercritical state, which was maintained for 8 minutes.

[0042] Subsequently, the pre-activated human recombinant apolipoprotein AI crowded solution was pumped into a supercritical reactor at a flow rate of 4 mL / min using a high-pressure pump.

[0043] Synergistic preparation using ultrasound and pressure relief: After the sample injection is completed, the ultrasonic transducer installed on the reactor is activated, with the ultrasonic frequency set to 35 kHz and the power set to 40 watts.

[0044] Within 0.2 seconds of the ultrasonic transducer starting, the pressure relief valve of the reactor is opened simultaneously, so that the pressure inside the reactor drops rapidly to atmospheric pressure within 1 to 3 seconds, and the suspension generated inside the reactor is collected.

[0045] Tangential flow ultrafiltration and filtration: The collected suspension was subjected to tangential flow ultrafiltration and washing. Using the modified polyethersulfone membrane module with a molecular weight cutoff of 100 kilodaltons prepared in Preparation Example 2, polyethylene glycol and ethanol were removed, and the membrane was washed with PBS buffer as the replacement medium to obtain lipid-removing nanoparticles with a high-density lipoprotein-like structure.

[0046] Example 2: Prepare human recombinant apolipoprotein AI stock solution and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution respectively: Human recombinant apolipoprotein AI stock solution: Accurately weigh 100 mg of the human recombinant apolipoprotein AI lyophilized powder prepared in Example 1, dissolve it in 50 mL of PBS buffer, and prepare a human recombinant apolipoprotein AI stock solution with a concentration of 2.0 mg / mL.

[0047] 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution: Based on a molar ratio of POPC to human recombinant apolipoprotein AI of 100:1, weigh 270 mg of POPC and dissolve it in 13.5 mL of anhydrous ethanol to prepare a phospholipid-ethanol solution with a concentration of 20 mg / mL.

[0048] Preparation of pre-activated human recombinant apolipoprotein AI crowding solution: Polyethylene glycol was added to the human recombinant apolipoprotein AI stock solution to make the final mass-volume concentration of polyethylene glycol in the system 15%; the mixture was incubated in a constant temperature water bath at 37°C for 30 minutes to obtain a pre-activated human recombinant apolipoprotein AI crowded solution.

[0049] Construction and injection of supercritical fluid systems: Place the phospholipid-ethanol solution in a reaction vessel and set the magnetic stirring speed to 200 rpm. Adjust the temperature inside the vessel to 45°C, introduce carbon dioxide to adjust the pressure to 15 MPa, and maintain this temperature for 10 minutes.

[0050] Subsequently, the pre-activated human recombinant apolipoprotein AI crowding solution was pumped into the reactor at a flow rate of 5 mL / min.

[0051] Synergistic preparation using ultrasound and pressure relief: Turn on the ultrasonic transducer, set the frequency to 40 kHz and the power to 50 watts.

[0052] Open the pressure relief valve within 0.2 seconds after startup to reduce the pressure to atmospheric pressure within 1 to 3 seconds and collect the suspension.

[0053] Tangential flow ultrafiltration and filtration: The modified polyethersulfone membrane module with a molecular weight cutoff of 100 kilodaltons prepared in Preparation Example 2 was used to perform tangential flow ultrafiltration and washing of the suspension, with PBS buffer as the replacement medium, to obtain lipid-removing nanoparticles with a structure similar to high-density lipoprotein.

[0054] Example 3: Prepare human recombinant apolipoprotein AI stock solution and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution respectively: Human recombinant apolipoprotein AI stock solution: Accurately weigh 100 mg of the human recombinant apolipoprotein AI lyophilized powder prepared in Example 1, dissolve it in 40 mL of PBS buffer, and prepare a human recombinant apolipoprotein AI stock solution with a concentration of 2.5 mg / mL.

[0055] 1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine-ethanol solution: Based on a molar ratio of POPC to human recombinant apolipoprotein AI of 120:1, weigh 325 mg of POPC and dissolve it in 13 mL of anhydrous ethanol to prepare a phospholipid-ethanol solution with a concentration of 25 mg / mL.

[0056] Preparation of pre-activated human recombinant apolipoprotein AI crowding solution: Polyethylene glycol was added to the human recombinant apolipoprotein AI stock solution to make the final mass-volume concentration of polyethylene glycol in the system 18%; the mixture was incubated in a constant temperature water bath at 39°C for 35 minutes to obtain a pre-activated human recombinant apolipoprotein AI crowded solution.

[0057] Construction and injection of supercritical fluid systems: Place the phospholipid-ethanol solution in a reaction vessel and set the magnetic stirring speed to 250 rpm. Adjust the temperature inside the vessel to 47°C, introduce carbon dioxide to adjust the pressure to 18 MPa, and maintain this temperature for 12 minutes.

[0058] Subsequently, the pre-activated human recombinant apolipoprotein AI crowding solution was pumped into the reactor at a flow rate of 6 mL / min.

[0059] Synergistic preparation using ultrasound and pressure relief: Turn on the ultrasonic transducer, set the frequency to 45 kHz and the power to 60 watts.

[0060] Open the pressure relief valve within 0.2 seconds after startup to reduce the pressure to atmospheric pressure within 1 to 3 seconds and collect the suspension.

[0061] Tangential flow ultrafiltration and filtration: The modified polyethersulfone membrane module with a molecular weight cutoff of 100 kilodaltons prepared in Preparation Example 2 was used to perform tangential flow ultrafiltration and washing of the suspension, with PBS buffer as the replacement medium, to obtain lipid-removing nanoparticles with a structure similar to high-density lipoprotein.

[0062] Comparative Example 1: Classical Bile Salt Dialysis Specific details: Compared with Example 2, the difference is that molecular crowding, supercritical fluid and acoustic cavitation processes are not used.

[0063] The specific operation is as follows: ApoA-I and POPC in the same amount as in Example 2 are mixed with sodium cholate in PBS buffer and stirred and incubated at 37°C for 12 hours to form mixed micelles; then the mixture is put into a dialysis bag and dialyzed against a large amount of PBS buffer at 4°C for up to 48 hours to remove sodium cholate, promoting the self-assembly of components, and finally the product is obtained by concentration and filtration.

[0064] Comparative Example 2: Pre-organization steps for missing molecular crowding Specific details: Compared with Example 2, the only difference is that polyethylene glycol is not added in Stage I.

[0065] The specific procedure was as follows: the ApoA-I stock solution was simply incubated in a 37°C water bath for 30 minutes, and then directly pumped into a supercritical reactor as the aqueous phase material for subsequent intercalation and forming operations. All other process parameters remained the same as in Example 2.

[0066] Comparative Example 3: Missing acoustic cavitation nuclei cooperative steps Specific details: Compared with Example 2, the only difference is that the ultrasonic waves are not activated during the depressurization process in Stage II.

[0067] The specific operation is as follows: when the materials are mixed and rapid pressure is released, the ultrasonic transducer is not activated; instead, the rapid pressure release valve is opened directly, allowing the pressure in the reactor to drop from 15 MPa to atmospheric pressure within 1-3 seconds. All other process parameters remain the same as in Example 2.

[0068] Comparative Example 4: High-Pressure Homogenization Specific details: Compared with Example 2, the difference is that a high-pressure homogenization process is used instead of supercritical fluid and acoustic cavitation process.

[0069] The specific procedure is as follows: An equal volume of POPC as in Example 2 is dissolved in ethanol and then slowly dripped into an ApoA-I aqueous solution containing PEG-35k to form a primary emulsion under high-speed shear. The primary emulsion is then homogenized six times using a high-pressure homogenizer at 800 bar. The homogenized emulsion is collected for subsequent ultrafiltration purification. The remaining raw material ratios and post-processing steps are consistent with those in Example 2.

[0070] Test Example 1-3: Test Example 1: Characterization of the Physical Properties of Nanoparticles The products prepared in Examples 1-3 and Comparative Examples 1-4 were analyzed using a Malvern nanoparticle and Zeta potential analyzer. The indicators included hydrated particle size, polydispersity index and Zeta potential.

[0071] The experimental procedure was as follows: The concentrated nanoparticle solutions prepared in each group were diluted with PBS buffer (pH 7.4) filtered through a 0.22 μm membrane to control the final ApoA-I concentration to approximately 0.5 mg / mL, preventing multiple scattering effects. The diluted samples were injected into disposable polystyrene sample cells or folded capillary sample cells and placed in the instrument's sample chamber, where they were kept at 25°C for 120 seconds for equilibration. The measurement mode was set to dynamic light scattering and electrophoretic light scattering, with the detection angle fixed at 90°. Parameter settings included a dispersion medium refractive index of 1.330, a viscosity of 0.8872 cP, and a particle refractive index of 1.450. Each sample was measured in triplicate. The instrument automatically optimized the attenuator and measurement position, and the average of the three measurements was taken as the final result.

[0072] The test data are summarized in Table 1: Table 1. Particle size and potential measurement data of products from the examples and comparative examples

[0073] Results Analysis and Conclusions: Analysis based on the data in Table 1 and the process conditions of each group: PDI Index Analysis: The PDI values ​​of Examples 1-3 remained at a low level, indicating a narrow product size distribution and high uniformity. Comparative Example 3, without the application of an ultrasonic field during depressurization, showed a PDI value increasing to 0.325 with a large standard deviation. This data difference confirms that relying solely on supercritical fluid depressurization results in random gas evolution, leading to inconsistencies between local nucleation and growth rates. Introducing acoustic cavitation nuclei enables synchronous nucleation at the microscopic level, thereby controlling the kinetic uniformity of the assembly process.

[0074] Particle size and structure analysis: The average particle size of Example 2 was 9.7 nm, close to the size of natural high-density lipoprotein. Comparative Example 2 lacked the molecular crowding agent, and the measured particle size was 87.2 nm with a very high PDI. This indicates that in the absence of PEG-induced exclusion volume effect, ApoA-I failed to form a preconformation conducive to phospholipid binding. The phospholipids carried by the supercritical fluid could not be accurately intercalated, resulting in the presence of mainly lipid aggregates or protein precipitation in the system, failing to form the expected nanodisc structure.

[0075] Compared with other preparation methods: Comparative Example 1 used dialysis, and although the average particle size was close to that of the examples, the PDI was 0.251, indicating that its self-assembly process was slower and the distribution was wider. Comparative Example 4 used high-pressure homogenization, and the particle size was approximately 26.1 nm, with a lower absolute potential value. This indicates that mechanical shearing mainly forms a mixture of lipid droplets and proteins, rather than a compact nanostructure formed through molecular self-assembly, and the colloidal stability is weaker than that of the products in the examples.

[0076] In summary, by employing molecular crowding pre-organization combined with sonic cavitation supercritical fluid technology, high-density lipoprotein-like nanoparticles with uniform particle size and satisfactory dispersibility can be prepared.

[0077] Test Example 2: Assessment of Protein Secondary Structure Integrity The secondary structure of apolipoprotein ApoA-I in the sample was determined using far-ultraviolet circular dichroism spectroscopy, and the results were calculated. The helical content was assessed to evaluate the effect of the preparation process on protein conformation and lipid binding.

[0078] Natural ApoA-I solution, products from Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 4 were selected as test samples. Each group of samples was diluted with 10 mM phosphate buffer (pH 7.4) to adjust the final ApoA-I concentration to 0.2 mg / mL. The diluted samples were placed in quartz cuvettes with a path length of 1 mm, and full-wavelength scanning was performed using a chromatograph at 25°C under a nitrogen atmosphere. The instrument parameters were set as follows: scan range 190-260 nm, scan rate 50 nm / min, response time 1 second, and bandwidth 1 nm. Each sample was scanned in triplicate, and the average value was taken, after subtracting the buffer background signal. The molar ellipticity (MEL) at 208 nm and 222 nm was recorded. ), and calculated based on the average molecular weight of ApoA-I residues. Percentage of spiral structures.

[0079] The test data are summarized in Table 2: Table 2. Circular dichroism chromatographic characteristic parameters and secondary structure content determination data for each group of samples. Results Analysis and Conclusions: Based on the data in Table 2 and the characteristic analysis of circular dichroism chromatograms: Lipid binding and conformational changes: Natural ApoA-I in aqueous solution The helical content was 43.6%. The product of Example 2... The helical content increased to 74.1%, and characteristic negative absorption peaks were observed at 208 nm and 222 nm. The transformation of ApoA-I from a partially random coil to a highly helical structure indicates that the protein binds to phospholipids, and the hydrophobic environment induces the formation and stabilization of the amphiphilic helical structure.

[0080] The effect of molecular crowding environment: Comparative Example 2 did not introduce PEG molecular crowding agent, and the measured The helical content decreased to 26.5%, lower than the level of the native protein. The data indicate that, in the absence of the exclusion volume effect provided by a crowding environment, ApoA-I underwent structural unfolding or denaturation in supercritical carbon dioxide and ethanol systems, failing to maintain its active conformation. The high helical content of Example 2 confirms that the molecular crowding pre-organization step maintained the stability of the protein structure in a supercritical fluid environment.

[0081] Comparison of the impact of process on structure: Comparative Example 4 uses a high-pressure homogenization process, and the product... The helical content was 56.3%, higher than that of the natural protein but lower than that of Example 2. This indicates that the mechanical shear force during the high-pressure homogenization process caused some damage to the secondary structure of the protein. The acoustic cavitation synergistic depressurization technology used in this invention utilizes low-power ultrasound to control nucleation rather than forceful dispersion, which is more conducive to preserving the biologically active structure of the protein compared to high-shear processes.

[0082] In summary, the preparation process of Example 2 achieved lipid assembly while maintaining the integrity of the secondary structure of apolipoproteins, and its structural order was higher than that of the control group that did not use molecular crowding protection or high-energy physical dispersion.

[0083] Test Example 3: Evaluation of In Vitro Cholesterol Reverse Transport Function The biological function of each group of nanoparticles was evaluated by measuring the efficiency of cells in expelling fluorescently labeled cholesterol using a macrophage cholesterol efflux assay.

[0084] Mouse mononuclear macrophage leukemia cells were selected as the cell model. Cells were seeded at a density of 1×10⁵ / 1×10⁵ cells in 24-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO₂ for 24 hours until adherence. The old medium was discarded and replaced with fresh medium containing 5 μg / mL fluorescently labeled cholesterol, and incubation continued for another 24 hours for cell labeling. After labeling, the cells were washed three times with PBS buffer to remove untaken fluorescent probes, and then equilibrated for 12 hours in serum-free medium containing 0.2% bovine serum albumin to homogenize the intracellular cholesterol pool.

[0085] After equilibration, the culture medium was aspirated, and serum-free culture medium containing different test samples was added for treatment. Test samples included products from Examples 1-3, Comparative Examples 1, 2, and 4, as well as a PBS negative control. All samples were standardized to ApoA-I concentration, with a working concentration of 10 μg / mL. After incubation for 4 hours, the supernatant from each well was collected. Adherent cells in the wells were treated with lysis buffer containing 1% Triton X-100, and cell lysis buffer was collected. The fluorescence intensity of the supernatant and cell lysis buffer was measured using a fluorescence microplate reader. The cholesterol efflux rate was calculated as follows: Cholesterol efflux rate (%) = [Supernatant FI / (Supernatant FI + Cell lysis buffer FI)] × 100%. Four replicates were set for each group, and the average result was taken.

[0086] The test data are summarized in Table 3: Table 3. Effects of each sample group on cholesterol efflux rate in macrophages

[0087] Results Analysis and Conclusions: Based on the data in Table 3 and the physicochemical properties analysis of each group of samples: Correlation between conformational integrity and elution efficiency: The cholesterol elution rate of Example 2 was 25.1%, higher than the PBS control group and slightly higher than Comparative Example 1. Combined with the circular dichroism data from Test Example 2, the product of Example 2 exhibited the highest... Helical content. This indicates that an intact protein secondary structure facilitates the effective binding of apolipoproteins to the ABCA1 transporter on the cell membrane surface, or promotes the transfer of membrane cholesterol to the phospholipid layer of nanoparticles through hydrophobic interactions.

[0088] The mechanism of molecular crowding pre-organization: The efflux rate of Comparative Example 2 was only 7.4%. Due to the lack of a molecular crowding environment in the preparation process, ApoA-I protein denatured or aggregated disorderedly, resulting in the loss of its active sites for recognizing cell receptors or binding lipids. The data confirm that introducing a molecular crowding pre-organization step in the supercritical fluid process is a necessary condition for maintaining the biological function of the final product.

[0089] Effects of different processes on activity: The effluent rate of Comparative Example 4 was 15.8%, lower than that of the Example group. The strong shear force and cavitation effect during the high-pressure homogenization process damaged the structure of some ApoA-I proteins, reducing their efficiency as cholesterol receptors. In contrast, Examples 1-3 used acoustic cavitation synergistic depressurization technology, which resulted in a milder assembly process. The effluent rates of the particles prepared under various parameter conditions remained stable between 21.6% and 25.1%, indicating that the technical route is beneficial for preserving the lipid-lowering activity of the protein.

[0090] In summary, the high-density lipoprotein-mimicking nanoparticles prepared by molecular crowding pre-organization combined with sonic cavitation supercritical fluid technology have good cholesterol reverse transport function, and their biological efficacy is superior to that of similar products prepared by high-energy physical dispersion method.

Claims

1. A type of lipid-removing nanoparticle mimicking the structure of high-density lipoprotein, characterized in that, Made from the following raw materials: recombinant human apolipoprotein AI and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline; The molar ratio of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine to recombinant human apolipoprotein AI is 90:1 to 120:

1.

2. The lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 1, characterized in that, The raw material, recombinant human apolipoprotein AI, is a lyophilized powder with a purity greater than 95%.

3. The lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 1, characterized in that, The raw material 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine is used in the preparation in the form of a solution dissolved in anhydrous ethanol, wherein the concentration of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in the solution is 15 mg / mL to 25 mg / mL.

4. A method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein, characterized in that, The preparation of a lipid-removing nanoparticle with a high-density lipoprotein-like structure as described in any one of claims 1-3 includes the following steps: S1. Human recombinant apolipoprotein AI is dissolved in buffer solution to prepare a stock solution. Polyethylene glycol is added to the stock solution and incubated at a constant temperature to obtain a pre-activated human recombinant apolipoprotein AI crowded solution. S2. Dissolve 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in anhydrous ethanol to prepare a phospholipid-ethanol solution. Place the phospholipid-ethanol solution in a reaction vessel, adjust the temperature, introduce carbon dioxide and adjust the pressure to a supercritical state, so that the 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine is fully dissolved to form a supercritical mixed fluid. S3. Pump the pre-activated recombinant human apolipoprotein AI crowding solution obtained in step S1 into the reactor in the supercritical state, so that the pre-activated recombinant human apolipoprotein AI crowding solution is mixed and contacted with the supercritical mixed fluid. S4. After the sample injection is completed, start the ultrasonic transducer installed on the reactor and simultaneously open the pressure relief valve installed on the reactor to reduce the pressure inside the reactor to atmospheric pressure and collect the suspension. S5. The suspension collected in step S4 is subjected to tangential flow ultrafiltration and washing using a modified polyethersulfone membrane module to obtain the lipid-removing nanoparticles with the high-density lipoprotein-like structure.

5. The method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 4, characterized in that, In step S1, the concentration of human recombinant apolipoprotein AI in the stock solution is 1.5 mg / mL to 2.5 mg / mL; the final mass-volume concentration of polyethylene glycol in the system is 12% to 18%; and the isothermal incubation temperature is 35°C to 39°C for 25 to 35 minutes.

6. The method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 4, characterized in that, In step S2, the concentration of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine in the phospholipid-ethanol solution is 15 mg / mL to 25 mg / mL; the reaction vessel is equipped with a stirring device with a stirring speed of 150 rpm to 250 rpm; the conditions for the supercritical state are: temperature 43℃ to 47℃, pressure 12 MPa to 18 MPa, and holding time 8 minutes to 12 minutes. In step S3, the pumped flow rate is 4 ml / min to 6 ml / min.

7. The method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 4, characterized in that, Step S4 specifically involves: starting the ultrasonic transducer, setting the ultrasonic frequency to 35 kHz to 45 kHz, and the power to 40 watts to 60 watts; opening the pressure relief valve within 0.2 seconds after the ultrasonic transducer is started, so that the pressure inside the reactor drops to atmospheric pressure within 1 to 3 seconds.

8. The method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 4, characterized in that, In step S5, the modified polyethersulfone membrane module has a molecular weight cutoff of 100 kDa, and the membrane module is prepared through the following steps: A polyethersulfone ultrafiltration membrane with a molecular weight cutoff of 100 kDa was selected as the base membrane; A modified solution containing a hydrophilic monomer is prepared, the base film is immersed in the modified solution, and an initiator is added to initiate a surface graft polymerization reaction; After the reaction is complete, the membrane is cleaned to obtain a modified polyethersulfone membrane, and the modified polyethersulfone membrane is then encapsulated into the modified polyethersulfone membrane assembly.

9. The method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 8, characterized in that, The hydrophilic monomer is polyethylene glycol methacrylate.

10. The method for preparing lipid-removing nanoparticles mimicking the structure of high-density lipoprotein according to claim 4, characterized in that, The buffer solution mentioned in step S1 is PBS buffer; the replacement medium used for tangential flow ultrafiltration and washing in step S5 is PBS buffer.

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