Liposomes encapsulating lbp, methods of making and use thereof

Abstract

The application discloses a liposome encapsulating LBP, a preparation method and application thereof, and belongs to the technical field of biological medicines. The liposome comprises a phospholipid bilayer, the inner cavity of the phospholipid bilayer encapsulates LBP, the phospholipid bilayer is formed by mixed lipids, the mixed lipids are composed of cationic lipids, neutral auxiliary lipids and solid sterols, and the neutral auxiliary lipids are dipalmitoyl lecithin. The liposome realizes synergistic protection in the treatment of acute lung injury through the strategy of DPPC in the lipid membrane and encapsulating LBP, has excellent lung targeting and immunological safety, and thus realizes the treatment of acute lung injury, and provides a new scheme for the application of LBP protein in the treatment of acute lung injury and the improvement of the treatment effect of acute lung injury.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to liposomes encapsulating LBP, their preparation methods, and applications. Background Technology

[0002] Lipopolysaccharide-binding protein (LBP) is an acute-phase protein synthesized by hepatocytes and secreted into the bloodstream. Its core biological function lies in specifically recognizing and binding lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria. By regulating the interaction between LPS and the CD14 / TLR4 receptor complex on the surface of immune cells, LBP plays a dual regulatory role in the innate immune response: on the one hand, it can promote the presentation of LPS to immune cells to initiate an appropriate inflammatory response; on the other hand, it can also inhibit excessive inflammatory signal transduction by forming an LBP-LPS complex, thereby playing a key homeostatic regulatory role in pulmonary inflammatory pathologies such as endotoxin-driven acute lung injury (ALI).

[0003] In recent years, replacement therapy strategies based on endogenous immune regulatory proteins have received widespread attention. Studies have shown that exogenous supplementation of recombinant LBP can significantly reduce LPS-induced pulmonary inflammatory infiltration, increased capillary permeability, and oxidative stress damage in animal models. However, there are currently no drugs for treating acute lung injury using LBP protein, mainly because: firstly, direct supplementation of exogenous LBP may not control its direction of action and may instead promote systemic inflammatory response syndrome or multiple organ failure; secondly, direct administration of LBP can deliver LPS to immune receptors (such as CD14 / TLR4), amplifying inflammatory signals and leading to more severe tissue damage; furthermore, as a water-soluble large molecular weight glycoprotein (molecular weight approximately 50 kDa), LBP has inherent drawbacks such as rapid protease degradation, high pulmonary clearance, and short in vivo half-life after systemic administration, and it is difficult to effectively accumulate in lung parenchymal targets, resulting in highly unstable actual effective delivery doses. These reasons limit the application of LBP protein in the treatment of acute lung injury.

[0004] Liposomes, as a mature nanoscale drug carrier, have been widely used for drug encapsulation and delivery. However, existing liposome delivery technologies still face key technical bottlenecks in lung-targeting applications against structure-sensitive endogenous immunomodulatory proteins like LBP: The drugs encapsulated in liposome delivery systems are mainly small molecule compounds such as doxorubicin and paclitaxel, which are relatively stable in physicochemical properties due to their low molecular weight. In contrast, LBP has multiple disulfide bonds and a complex tertiary structure, making it highly sensitive to physicochemical factors such as pH, temperature, organic solvents, and mechanical shearing. Conventional liposome composition and preparation methods easily lead to LBP adsorption inactivation, aggregation, or covalent cross-linking, significantly reducing encapsulation efficiency and functional integrity.

[0005] In summary, developing a liposome that can stably encapsulate LBP, achieving excellent lung targeting and immune safety, to enable the application of LBP in the treatment of acute lung injury and provide a new drug for the treatment of acute lung injury is of great significance. Summary of the Invention

[0006] In view of this, the primary objective of this application is to provide a liposome encapsulating LBP, which achieves a synergistic protective effect in the treatment of acute lung injury through the combination of DPPC in the lipid membrane and LBP encapsulation strategy, and has lung targeting and immune safety, thereby successfully applying LBP to the treatment of acute lung injury and breaking through the current technical barrier that LBP cannot be directly used to treat acute lung injury.

[0007] To achieve the above objectives, this application adopts the following technical solution: One aspect of this application discloses a liposome encapsulating LBP, the liposome comprising a phospholipid bilayer, the lumen of the phospholipid bilayer encapsulating LBP, the phospholipid bilayer being formed of a mixture of lipids, the mixture of lipids being composed of cationic lipids, neutral auxiliary lipids and sterols, wherein the neutral auxiliary lipid is dipalmitoyl lecithin.

[0008] Another aspect of this application discloses a method for preparing liposomes encapsulating LBP as described above, comprising the following steps: Neutral auxiliary lipids, sterols and cationic lipids are dissolved in an organic solvent according to the specified ratio to form a homogeneous lipid organic solution, which is then vacuum rotary evaporated to form a uniform lipid film. After mixing the lipid film with the hydration solution, the mixture is continuously stirred at a constant temperature to hydrate and detach the lipid film, forming a milky white and uniform liposome suspension. The hydration solution is a protein solution containing LBP preheated to temperature T, where temperature T is greater than the phase transition temperature of the mixed lipids. The liposome suspension was subjected to multiple extrusion operations using a liposome extruder to obtain an aqueous liposome dispersion encapsulating LBP.

[0009] Another aspect of this application discloses the use of the liposomes encapsulating LBP described above in the preparation of a medicament for treating acute lung injury.

[0010] Another aspect of this application discloses a medicament for treating acute lung injury, comprising liposomes encapsulating LBP as described above.

[0011] This application has at least the following beneficial effects: In this application, LBP protein was successfully encapsulated in a phospholipid bilayer to form stable LBP-encapsulated liposomes. The neutral auxiliary lipid in the phospholipid bilayer is dipalmitoyl lecithin (DPPC). DPPC is not only a component of the lipid membrane but also a therapeutic agent. As a major component of pulmonary surfactant, it can replenish depleted surfactant and improve lung compliance. At the same time, DPPC synergistically with pulmonary surfactant proteins (SP-B, SP-C) can instantly spread into a monolayer at the alveolar air-fluid interface. Through this spreading behavior, LBP is efficiently, uniformly, and persistently fixed at the alveolar air-fluid interface. Thus, through physiological reabsorption pathways, LBP is delivered into alveolar macrophages in a non-inflammatory manner. Subsequently, LBP is degraded and released in the acidic environment of lysosomes or slowly leaked, achieving precise local release of LBP. This effectively avoids the pro-inflammatory effect of LBP, allowing it to precisely inhibit excessive inflammatory response in the lungs and reduce lung tissue damage.

[0012] In summary, this application achieves a synergistic protective effect in the treatment of acute lung injury by combining DPPC in the lipid membrane with internally encapsulated LBP. This results in liposomes with excellent lung targeting and immune safety, effectively treating acute lung injury and providing a new approach for the application of LBP protein in the treatment of acute lung injury and improving the therapeutic effect of acute lung injury. Attached Figure Description

[0013] Figure 1 The lipid film formed under rotary evaporation conditions of 35°C and 150 rpm (left 4) is compared with the blank round-bottom flask (right 1).

[0014] Figure 2 The particle size analysis results are for liposomes with a raw material ratio of DPPC:cholesterol:DOTAP = 14:6:5 (molar ratio).

[0015] Figure 3 Coomassie Brilliant Blue staining was used to detect whether proteins were degraded during liposome synthesis.

[0016] Figure 4 The results of particle size analysis (NTA) of liposomes (unextruded) synthesized using the optimized preparation process are shown.

[0017] Figure 5 Particle size analysis (NTA) results of liposomes synthesized without the optimized preparation process (extruded 20 times).

[0018] Figure 6 To observe the morphology and localization of LBP liposomes under light and fluorescence microscopy.

[0019] Figure 7 The one-week survival curves of NC mice and mice after intratracheal instillation of liposomes are shown.

[0020] Figure 8 The results show the relative expression levels of inflammatory factor mRNA in lung tissues of NC mice and mice after intratracheal instillation of liposomes.

[0021] Figure 9 WT mice were infused with 20 mg / kg body weight of LPS, and after 3 hours, they were infused with empty LBP liposomes via tracheal infusion. One-week survival curves were obtained.

[0022] Figure 10 WT mice were infused with 20 mg / kg body weight of LPS. At 3 hours, they were infused with empty LBP liposomes via tracheal infusion. Body weight was recorded after one week. Detailed Implementation

[0023] The embodiments of this application will be clearly and completely described below. The technical solutions in the embodiments described below are exemplary and only possible technical implementations of this application, not all possible implementations. Those skilled in the art can combine the embodiments of this application to obtain other embodiments without creative effort, and these embodiments are also within the protection scope of this application.

[0024] This application first discloses a liposome encapsulating LBP, the liposome comprising a phospholipid bilayer, the lumen of which encapsulates LBP, the phospholipid bilayer being formed of a mixture of lipids, the mixture of lipids being composed of cationic lipids, neutral auxiliary lipids and sterols, wherein the neutral auxiliary lipid is dipalmitoyl lecithin.

[0025] In the examples of this application, the mass ratio of LBP to neutral adjuvant lipids is 1:2 to 1:10, for example, it can be any mass ratio or a range between any two ratios of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. If the mass of LBP is too high, protein aggregation is likely to occur, reducing the encapsulation efficiency and leading to protein waste. If the amount of neutral adjuvant lipids is relatively high, the liposomes will encapsulate less protein, resulting in poor therapeutic effect. Preferably, the mass ratio of LBP to neutral adjuvant lipids is 1:5.

[0026] This invention utilizes a closed luminal environment constructed from a phospholipid bilayer to effectively encapsulate and deliver the bioactive macromolecular protein LBP, encapsulating LBP within the liposomes and avoiding its systemic pro-inflammatory side effects. The neutral helper lipid in the phospholipid bilayer is dipalmitoyl lecithin (DPPC), which also functions as a therapeutic agent in this application. Supplementation with DPPC improves alveolar tension, increases lung compliance, and reduces alveolar collapse. Furthermore, through synergy with pulmonary surfactant proteins (SP-B and SP-C), DPPC delivers LBP into alveolar macrophages, achieving precise release of LBP at the lesion site. This allows LBP to clear LPS in the lungs, inhibit excessive inflammatory responses, and reduce lung tissue damage. The synergistic protective effect of DPPC and LBP in the treatment of acute lung injury is achieved through this liposome, which exhibits excellent lung targeting and immune safety, making it highly effective in treating acute lung injury.

[0027] In this application, liposomes refer to ultrafine particles with a double-membrane closed structure formed spontaneously by amphiphilic lipid molecules in an aqueous environment, typically with a particle size in the nanometer to micrometer range. The liposome structure in this application comprises an outer shell composed of a phospholipid bilayer and one or more internal aqueous chambers defined by the outer shell, exhibiting a vesicle morphology.

[0028] The phospholipid bilayer refers to the aggregate of all lipid molecules capable of forming a bilayer membrane structure. It is amphiphilic, meaning the hydrophilic head faces outward (towards the external aqueous phase and internal water cavity), and the hydrophobic tail faces inward (aggregated in the middle hydrophobic region of the bilayer). In this application, the phospholipid bilayer is self-assembled from mixed lipids through intermolecular forces (such as hydrophobic interactions, van der Waals forces, etc.), wherein the mixed lipids consist of cationic lipids, neutral accessory lipids, and sterols.

[0029] In the mixed lipids of this application, except for the neutral auxiliary lipid which must be dipalmitoyl lecithin (DPPC) to achieve synergistic cooperation with the encapsulated LBP protein, other lipid components, such as cationic lipids and sterols, can be conventional compositions in the art without any particular restrictions or requirements. Those skilled in the art can make specific selections based on research needs through experiments.

[0030] The cationic lipids mentioned above refer to amphiphilic lipid molecules whose molecular structure contains at least one polar head group that can be protonated and carry a positive charge under physiological pH conditions (usually around pH 7.4), and one or more hydrophobic tail chains. Specific examples include, but are not limited to: 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol (DC-Chol), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-distearyl-3-trimethylammonium propane (DSTAP), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dilauroyl-3-trimethylammonium propane (DLTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), and N-1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium sulfate methyl ester (DOTAP). -MS), dimethyl dioctadecylammonium (DDAB), 2,3-dioleoyloxy-N-[2-(sperminecarbamoyl)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), O,O'-dimyristoyl-N-(α-trimethylaminoacetyl)diethanolamine chloride (DC-6-14), 1,2-dilinoleoyl-3-trimethylammonium propane (DLinTAP), 1,2-dilinolenoyl-3-trimethylammonium propane (DLenTAP), 1,2-diarachidonicoyl-3-trimethylammonium propane (DATAP), 1-palmitoyl-2-oleoyl-3-trimethylammonium propane (POTAP), 1-stearoyl-2-oleoyl-3-trimethylammonium propane (POTAP) or a combination of two or more of these.

[0031] The sterols described are a class of steroidal compounds with a cyclopentanoperhydrophenanthrene backbone, which act as physiological regulators of membrane fluidity in liposome formulations, embedded in the hydrophobic regions of the phospholipid bilayer. Specific examples include, but are not limited to: cholesterol, cholesterol sulfate, cholesterol hemisuccinate, dihydrocholesterol, 7-dehydrocholesterol, lanosterol, ergosterol, 7-dehydroergosterol, stigmasterol, sitosterol, campesterol, fucosterol, spinachosterol, stigmasterol, sitosterol, and any pharmaceutically acceptable derivatives, esters, salts, or mixtures thereof in any proportion of the above sterols.

[0032] In some specific embodiments of this application, the cationic lipid is preferably 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); and / or, the sterol is preferably cholesterol. By optimizing the lipid composition, the liposomes are endowed with suitable phase transition temperatures and membrane fluidity, promoting fusion with alveolar macrophages while maintaining structural stability, further facilitating the controllable release of LBP protein. Specifically, DOTAP, as a cationic lipid, can enhance the interaction between protein and lipid membranes through electrostatic interactions, while moderately increasing the fluidity and flexibility of the lipid membrane, thereby further improving the encapsulation efficiency of water-soluble proteins. In liposomes co-formulated with neutral phospholipids (such as DPPC) and cholesterol, the addition of an appropriate amount of DOTAP not only helps to improve the protein encapsulation rate but also maintains the structural stability of the liposomes, achieving a balance between encapsulation efficiency and membrane stability. Cholesterol can regulate lipid membrane fluidity, reduce particle aggregation, and improve particle size uniformity. It is particularly noteworthy that this application omits the polyethylene glycol-modified lipids commonly used in liposomes, thereby significantly increasing the particle size of the liposomes and improving the encapsulation efficiency, drug loading, and post-encapsulation stability of LBP protein. It is understood that the above are merely preferred examples, and those skilled in the art can select appropriate lipid components and determine suitable ratios according to research needs.

[0033] Furthermore, the proportions of the components in the mixed lipids and the particle size of the liposomes are not particularly limited. Those skilled in the art can adjust or optimize them as needed. A suitable lipid ratio and liposome particle size can provide a larger encapsulation space for the LBP protein, thereby improving the protein encapsulation efficiency and enhancing lung-targeted delivery. In some specific embodiments, the molar ratio of neutral auxiliary lipids, sterols, and cationic lipids is 14:6:5. This specific ratio imparts suitable phase transition temperature, membrane fluidity, and liposome particle size to the liposomes, enhancing their fusion ability in the lung microenvironment and the controllability of drug release. The liposome particle size is 110nm-130nm, for example, any particle size among 110nm, 115nm, 120nm, 125nm, and 130nm, or a range between any two particle sizes.

[0034] This application further discloses a method for preparing liposomes encapsulated with LBP, which employs a combination of thin-film hydration and extrusion granulation, specifically including the following steps: S1. A homogeneous lipid organic solution is formed and rotary evaporated to form a film. Neutral auxiliary lipids (DPPC), sterols (such as cholesterol), and cationic lipids (such as 1,2-dioleoyl-3-trimethylammonium propane) are dissolved in an organic solvent according to the specified ratio to form a homogeneous lipid organic solution. Vacuum rotary evaporation is then used to form a uniform lipid film.

[0035] In this step, the organic solvent is typically chosen to be volatile and have good lipid solubility, preferably a classic thin-film hydration solvent. As a preferred example, a mixed solvent of chloroform and methanol (e.g., a volume ratio of 2:1 or 3:1) can be used to ensure the complete dissolution of all lipid components and guarantee the homogeneity of the lipid film.

[0036] In practice, the above-mentioned mixed lipid solution is placed in a round-bottom flask and connected to a rotary evaporator. Rotary evaporation is carried out under vacuum conditions. As the organic solvent is slowly removed, a uniform, transparent or translucent lipid film, visible to the naked eye, will be deposited on the flask wall. This effectively avoids damage to the LBP protein structure caused by residual organic solvent and improves protein stability during the encapsulation process.

[0037] S2, Constant temperature stirring hydration After mixing the lipid film with the hydration solution, the mixture is continuously stirred at a constant temperature to ensure complete hydration and detachment of the lipid film, forming a milky white, homogeneous liposome suspension. The hydration solution is a protein solution containing LBP preheated to temperature T, where temperature T is higher than the phase transition temperature of the mixed lipids.

[0038] In this step, the phase transition temperature refers to the critical temperature at which the lipid bilayer membrane transforms from an ordered gel phase to a disordered liquid crystal phase. For the mixed lipid system involved in this application (containing unsaturated phospholipids and cholesterol), its phase transition temperature is typically low. However, in practice, to ensure sufficient hydration, the temperature T must be significantly higher than this temperature. At this temperature, the lipid molecular chains are highly mobile, and the film easily curls, peels off, hydrates, and self-assembles from the glass wall to form vesicles encapsulating the hydration solution (containing LBP). Furthermore, preheating the hydration solution avoids the brittle rupture of the lipid membrane or the inactivation of LBP caused by a sudden drop in local temperature, ensuring a gentle and efficient encapsulation process. The specific temperature T can be appropriately adjusted or optimized according to the different mixed lipid components. In some specific examples, the temperature T is 55-65℃, for example, any temperature or a range between any two of 55℃, 58℃, 60℃, 62℃, and 65℃.

[0039] During the specific stirring hydration process, the stirring speed and time can be optimized as needed to ensure that the LBP protein is fully embedded in the lipid membrane gap and to reduce the tendency to aggregate, thereby improving the initial encapsulation efficiency. In some specific examples, hydration is carried out continuously at 450-550 rpm for 55-65 minutes. Within this range, the speed and time can be arbitrarily adapted, such as hydration at 450 rpm for 65 minutes, 500 rpm for 60 minutes, or 550 rpm for 55 minutes; preferably, hydration at 500 rpm for 60 minutes is preferred.

[0040] S3, liposome extruder multiple extrusions The liposome suspension was subjected to multiple extrusion operations through a liposome extruder to obtain an aqueous dispersion of liposomes encapsulating LBP.

[0041] The liposome extruder described in this application is a device that uses external pressure (such as nitrogen cylinder pressure) to force a coarse liposome suspension through a polycarbonate filter membrane with a specific pore size. The specific pore size of the filter membrane and the number of extrusions can be adjusted or optimized as needed, as long as a suitable liposome particle size can be obtained. In some specific examples of this application, a polycarbonate membrane with a pore size of 200 nm is used, and the membrane is repeatedly extruded 20 times in a high-pressure extrusion device to obtain liposomes with an average particle size distribution of 110-130 nm. Within this particle size range, it is beneficial for efficient retention in the alveolar region and preferential uptake by alveolar macrophages, significantly enhancing lung targeting and local anti-inflammatory efficacy.

[0042] This application further discloses the use of liposomes encapsulating LBP in the preparation of medicaments for treating acute lung injury. It also further discloses medicaments for treating acute lung injury.

[0043] As is well known to those skilled in the art, acute lung injury (ALI) is an acute, progressive hypoxic respiratory failure caused by various intrapulmonary and extrapulmonary pathogenic factors. Its pathological features include damage to the alveolar epithelium and capillary endothelial barrier, diffuse interstitial pulmonary edema, and uncontrolled inflammatory responses. Encapsulating LBP in the liposomes of this application, utilizing the unique form of DPPC in the lipid membrane as a major component of pulmonary surfactant, significantly enhances the lung targeting of LBP and the drug exposure in lung tissue, achieving excellent therapeutic effects for ALI. This addresses the problems of free LBP being easily and rapidly degraded in the blood, having a short half-life, poor lung targeting, uncontrollable therapeutic effects, and easily inducing systemic inflammatory effects. Therefore, this application, by introducing DPPC into the phospholipid bilayer and encapsulating LBP, achieves effective treatment of ALI.

[0044] The active ingredient in the drug is a liposome encapsulating LBP, and further includes at least one pharmaceutically acceptable excipient.

[0045] In this drug, the content of the active ingredient should be an effective amount. The effective amount refers to the dosage or concentration range of the active ingredient that can achieve the expected therapeutic or preventative effect in the target organism (such as mammals, especially humans) without causing serious toxic side effects. It should be understood that the effective amount is not specifically limited, and its influencing factors are usually multifaceted, such as individual adjustments based on the patient's age, weight, disease severity, liver and kidney function, and tolerance to treatment. This application does not impose specific limitations on this.

[0046] The excipients refer to inactive substances added to pharmaceutical preparations, other than the active ingredient, to impart specific physicochemical properties to the preparation, ensure the stability of the preparation quality, improve drug administration compliance, or facilitate production and processing. Specific examples include: pH adjusters / buffers, osmotic pressure regulators, stabilizers, lyophilization protectants, antibacterial agents, antioxidants / metal ion chelators, etc. The specific selection depends on the route of administration and the drug dosage form. Those skilled in the art are capable of doing so, therefore, there are no particular limitations here.

[0047] In this application, the preferred drug dosage form is lyophilized powder or injection, but it is not limited to these.

[0048] The present application will be further illustrated below with reference to specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present application in any way.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.

[0050] In addition, unless otherwise specified, methods without detailed conditions or steps are conventional methods, and the reagents and materials used are commercially available.

[0051] The hydration solution was a solution containing LBP protein at a concentration of 0.7 mg / ml, and the solvent was DPBS (Dupuy's phosphate buffered saline) that had been autoclaved and filtered through a 0.22 μm filter membrane.

[0052] Example 1: Preparation of liposomes encapsulating LBP Accurately weigh the raw materials (total mass 50 mg) according to the preset ratio and place them together in a clean round-bottom flask. Add 15 mL of organic solvent (chloroform:methanol = 1:1, v / v) to the flask, place the round-bottom flask in a water bath sonicator at room temperature, and sonicate at 100 W power for 10 min, observing the solution state periodically until all solid lipid components are completely dissolved, forming a homogeneous and clear lipid organic solution.

[0053] The above-mentioned lipid organic solution was transferred to a rotary evaporator and the organic solvent was removed by rotary evaporation under reduced pressure (35°C, 150 rpm). The process was continuously observed until a uniform, transparent lipid film formed on the inner wall of the round-bottom flask. Figure 1 Stop rotary evaporation. Place the round-bottom flask in a fume hood overnight (≥12h) to ensure that the residual organic solvents evaporate completely and to avoid affecting the stability of the liposomes.

[0054] Based on the total lipid mass and target concentration, add an appropriate amount of hydration solution (protein solution containing LBP) preheated to 65°C to the round-bottom flask containing the prepared lipid membrane. Place the round-bottom flask in a constant-temperature water bath stirrer, set the water bath temperature to 65°C, and continuously stir at 500 rpm for 60 minutes to allow the lipid membrane to fully hydrate and detach, forming a milky white, homogeneous liposome suspension.

[0055] After hydration, the liposome suspension was transferred from the round-bottom flask to a sterile EP tube to avoid loss of residual lipid membrane. The liposome suspension was then added to a liposome extruder pre-installed with a 200 nm pore size polycarbonate membrane for extrusion, repeating 20 times. After extrusion, the liposome solution was transferred to sterile centrifuge tubes, the caps were sealed, and the tubes were stored at 4°C in the dark for up to one week. Gently mix before use.

[0056] Example 2: Optimization of lipid components and ratios In this embodiment, commercially available BSA protein was used as the model protein. Liposomes encapsulating the protein were prepared following the steps in Example 1 to explore different lipid compositions and ratios. The specific experimental groups are as follows: The liposome raw material is DPPC:cholesterol:DSPE-mPEG2000 = 10:1:2 (mass ratio); The liposome raw material is DPPC:cholesterol:DSPE-mPEG2000:DOTAP = 10:1:2:2 (mass ratio); The liposome raw material is DPPC:cholesterol:DSPE-mPEG2000:DOTAP = 12:5:1:2 (mass ratio); The liposome raw material is DPPC:cholesterol:DSPE-mPEG2000:DOTAP = 12:5:1:2 (molar ratio); The liposome raw materials are DPPC:cholesterol:DOTAP = 14:6:5 (molar ratio).

[0057] The results are as follows Figure 1 As shown in the figure, after rotary evaporation under the selected conditions, a uniform lipid film was formed on the wall of the round-bottom flask, indicating that the rotary evaporation conditions of this application are feasible and avoid the problem of uneven lipid film formation caused by rotary evaporation, which in turn leads to a decrease in liposome encapsulation efficiency and uneven particle size distribution.

[0058] Meanwhile, the particle size results of liposomes prepared with different lipid compositions and ratios showed that the liposomes prepared using the first four lipid ratios had smaller particle sizes and poor protein encapsulation effects. The liposomes prepared using the fifth ratio had particle sizes of approximately 100-200 nm with almost no secondary peaks, which was in line with expectations. Figure 2 Therefore, this ratio is selected for all lipid raw materials used in subsequent liposome preparations.

[0059] Example 3: Optimization of drug-lipid ratio or hydration time In this embodiment, the mixed lipids were selected as DPPC:cholesterol:DOTAP = 14:6:5 (molar ratio). Following the steps of Example 1, different hydration times (5 min, 10 min, 60 min) and different drug-lipid ratios were set to investigate the effects of hydration time and drug-lipid ratio on encapsulation efficiency.

[0060] The results are shown in Table 1.

[0061] Table 1. Effect of drug-lipid ratio or hydration time

[0062] Table 1 shows that different hydration times have a significant impact on the liposome encapsulation efficiency. Too short a time will lead to a decrease in the liposome encapsulation efficiency. The drug-liposome ratio also has an impact on the liposome encapsulation efficiency. Therefore, a hydration time of 60 min and a drug-liposome ratio of 1:5 (w / w) were selected for the preparation of liposomes.

[0063] Example 4: Liposome Characterization (1) A preliminary liposome loading experiment was conducted using BSA as a model protein. After loading, the protein was demulsified (Triton X-100 reagent was added to the liposome solution to achieve a final concentration of 1% Triton X-100 in the system. The mixture was then pipetted and incubated at room temperature for 15–30 min, with gentle mixing every 5 min). The results were then analyzed by SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining. The staining pattern showed that the protein band corresponding to BSA was clear and sharp, without obvious tailing, and the band migration position was completely consistent with the molecular weight (66 kDa) of the BSA standard. This result confirms that the membrane hydration loading process in this application did not lead to protein degradation or aggregation, and the protein structure showed good stability. Figure 3 ).

[0064] (2) Liposomes encapsulating LBP were prepared according to Example 1, wherein the mixed lipids were DPPC:cholesterol:DOTAP = 14:6:5 (molar ratio), and the mass ratio of LBP to DPPC was 1:5. The particle size of the synthesized LBP-encapsulated lipids was characterized, and the results are shown in Table 2 and... Figure 4 , Figure 5 As shown in the image.

[0065] Table 2. Liposome characterization of LBP encapsulation

[0066] The encapsulation efficiency in Table 2 was quantitatively detected using the BCA method. The total protein content and free protein content after liposome demulsification were measured and calculated using the following formula:

[0067] The test data showed that the encapsulation rate of LBP liposomes without extrusion was 68.88%, while that of LBP liposomes after extrusion was 83.43%. It can be seen that the synthesized liposomes had a higher encapsulation rate and a particle size greater than 100 nm. After 20 extrusion operations, the encapsulation rate of the prepared liposomes was further improved, and the particle size distribution became more uniform.

[0068] (3) The co-localization of LBP and liposomes was observed using confocal laser scanning microscopy to verify the encapsulation effect. Imaging results showed that the spatial localization of the green fluorescence signal and the liposome particles highly overlapped, indicating that LBP was successfully encapsulated into the liposomes via the thin-film hydration method, and no large amount of free protein was observed. Figure 6 ) Example 5: Animal Experiment 5.1 Immunological safety of liposomes encapsulating LBP Healthy male C57BL / 6J mice weighing 20-22g at 6-8 weeks of age were randomly divided into two groups: The control group (NC group) was not treated; the liposome group was prepared by tracheal instillation of LBP liposomes (prepared in Example 4 (2)), 0.1 ml / time, once in total.

[0069] The survival status and survival rate of mice were dynamically monitored for one week after modeling.

[0070] Statistical results showed that no mice in either the control group or the liposome group died during the entire observation period, and the survival rate remained at 100%. Furthermore, the liposome group mice did not exhibit any abnormal symptoms such as lethargy, loss of appetite, or respiratory abnormalities, indicating that intratracheal infusion of LBP liposomes did not induce acute toxicity in mice and demonstrated good in vivo tolerability. Figure 7 ).

[0071] Furthermore, lung tissues from both groups of mice were collected, and the mRNA expression levels of inflammation-related factors (IL-6, TNF-α, and IL-1β) were detected using qPCR. The data showed that the mRNA expression levels of IL-6, TNF-α, and IL-1β in the lung tissues of the liposome group were not significantly increased compared to the control group (P>0.05), and no upregulation trend of inflammatory factor expression was observed. Figure 8 This indicates that the liposomes encapsulating LBP prepared in this application have immune safety.

[0072] 5.2 Liposomes encapsulating LBP for the treatment of acute lung injury Healthy 6-8 week old male C57BL / 6J mice weighing 20-22g were randomly divided into two groups (n=11): a model control group (NC group) and an LBP liposome treatment group (LBP group). Both groups of mice underwent intratracheal infusion of LPS (15mg / kg body weight, once). Three hours after modeling, the NC group received no additional intervention, while the LBP group received intratracheal infusion of prepared LBP liposomes (0.1ml, once). Survival rate and body weight changes were dynamically monitored for one week after modeling.

[0073] Survival rate statistics showed that the mortality rate of mice in the NC group increased significantly from 6 hours after modeling, with a survival rate of only 45.5% by the end of the observation period (7 days); while the survival rate of mice in the LBP group was significantly improved, reaching 81.8% by the end of the observation period, and the difference was statistically significant compared with the NC group (P<0.05). Figure 9 ).

[0074] Weight change monitoring results showed that both groups of mice experienced significant weight loss after modeling, but the LBP group recovered weight at a significantly faster rate than the NC group. Furthermore, the LBP group mice exhibited better mental and respiratory status than the NC group after modeling, and did not show obvious signs of severe ALI symptoms such as rapid breathing or lethargy. Figure 10 This demonstrates that the liposomes encapsulating LBP prepared in this application have a good therapeutic effect on acute lung injury.

[0075] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A liposome encapsulating lipopolysaccharide-binding protein (LBP), characterized in that, The liposomes comprise a phospholipid bilayer, the lumen of which is encapsulated with LBP. The phospholipid bilayer is formed from a mixture of lipids, which consists of cationic lipids, neutral accessory lipids, and sterols. The neutral accessory lipid is dipalmitoyl lecithin, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane, and the sterol is cholesterol. The mass ratio of LBP to neutral accessory lipids is 1:2 to 1:10, and the particle size of the liposomes is 110-130 nm.

2. The liposomes encapsulating lipopolysaccharide-binding protein LBP as described in claim 1, characterized in that, In the mixed lipids, the molar ratio of the neutral auxiliary lipids to sterols is 7:

3.

3. The liposomes encapsulating lipopolysaccharide-binding protein LBP as described in claim 1, characterized in that, In the liposomes, the mass ratio of LBP to neutral accessory lipids is 1:

5.

4. A method for preparing liposomes encapsulating lipopolysaccharide-binding protein (LBP) as described in any one of claims 1-3, characterized in that, Includes the following steps: Neutral auxiliary lipids, sterols and cationic lipids are dissolved in an organic solvent according to the specified ratio to form a homogeneous lipid organic solution, which is then vacuum rotary evaporated to form a uniform lipid film. After mixing the lipid film with the hydration solution, the mixture is continuously stirred at a constant temperature to hydrate and detach the lipid film, forming a milky white and uniform liposome suspension. The hydration solution is a protein solution containing LBP preheated to temperature T, where temperature T is greater than the phase transition temperature of the mixed lipids. The liposome suspension was subjected to multiple extrusion operations using a liposome extruder to obtain an aqueous liposome dispersion encapsulating LBP.

5. The use of the liposomes encapsulating lipopolysaccharide-binding protein LBP as described in any one of claims 1-3 in the preparation of a medicament for treating LPS-induced acute lung injury.

6. A drug for treating LPS-induced acute lung injury, characterized in that, Liposomes containing the encapsulated lipopolysaccharide-binding protein LBP as described in any one of claims 1-3.

7. The drug as described in claim 6, characterized in that, The drug dosage form is lyophilized powder or injection.

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