Targeted acid responsive liposome as well as preparation method and application thereof
By preparing targeted acid-responsive liposomes, and utilizing Anti-SFTPC to target type II alveolar epithelial cells and combine it with the acid-responsive mechanism, the controllability and targeting issues of drug release in the alveolar cavity of traditional liposomes were solved, achieving efficient drug delivery and therapeutic effects in the lungs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-09
- Publication Date
- 2026-03-24
AI Technical Summary
Traditional liposomes have limitations in terms of targeting and controllable drug release, especially in achieving precise drug release and targeted efficacy within the alveolar cavity.
Acid-responsive liposomes are used to target type II alveolar epithelial cells via Anti-SFTPC, and drug release is controlled by an acid-responsive mechanism. The liposomes are composed of DPPC, DOPE and DSPE-PEG2000-COOH, with DSPE-PEG2000-COOH linked to Anti-SFTPC molecules, enabling controlled drug release in the alveolar cavity.
It improves the targeting and efficacy of drugs in the lungs, enables controlled release of drugs in an acidic environment, reduces the impact on non-target areas, and improves drug delivery efficiency and therapeutic effect.
Smart Images
Figure CN121714722A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of drug carrier technology, specifically relating to a targeted acid-responsive liposome, its preparation method, and its application. Background Technology
[0002] Liposomes are artificial membranes, consisting of closed vesicles with a bilayer structure formed by dispersing lipids such as phospholipids and cholesterol in water. This structure is similar to that of skin cell membranes, giving them good biocompatibility and moisturizing properties. In particular, liposomes coated with moisturizing substances such as hyaluronic acid and polyglucosides exhibit even better moisturizing performance. The diameter of liposomes is usually between 25 and 1000 nanometers. Because their structure is similar to that of biological membranes, they are also called artificial biological membranes.
[0003] Currently, liposomes are widely used as a drug delivery system, but traditional liposomes still have certain limitations in terms of targeting and controllability of drug release. Although some liposomes have been combined with targeting antibodies for targeted action, how to achieve precise drug release and targeted efficacy within the alveolar cavity remains an urgent problem to be solved. Therefore, how to effectively target type II alveolar epithelial cells and control drug release through acid response mechanisms to enable drugs to exert therapeutic effects within the alveolar cavity has significant scientific research and clinical application value. Summary of the Invention
[0004] To address the problems in the prior art, this invention provides a targeted acid-responsive liposome, its preparation method, and its application. Anti-SFTPC targets type II alveolar epithelial cells and utilizes an acid-responsive mechanism to control drug release, thereby exerting its effect within the alveolar cavity and improving the drug's targeting and efficacy in the lungs.
[0005] The technical problem solved by this invention is achieved by the following technical solution:
[0006] The present invention aims to provide a targeted acid-responsive liposome, comprising DPPC and DOPE, wherein the surface of the liposome is coated with DSPE-PEG. 2000 -COOH is linked to an Anti-SFTPC molecule.
[0007] A method for preparing acid-targeting responsive liposomes includes the following steps:
[0008] (1) Preparation of liposome suspension: DPPC, DOPE and cholesterol are mixed and dispersed in a solvent, ultrasonically treated, then evaporated and dried, and then a buffer solution is added and stirred to hydrate and form a liposome suspension.
[0009] (2) DSPE-PEG 2000 Preparation of SFTPC antibody conjugate: EDC solution, DSPE-PEG2000 After mixing and stirring the COOH and NHS solutions, the pH of the solution was adjusted to alkaline. SFTPC antibody was then added, the mixture was stirred, and the concentrate was collected after centrifugation; this concentrate is DSPE-PEG. 2000 -SFTPC antibody conjugate;
[0010] (3) Preparation of liposomes: The liposome suspension was mixed with DSPE-PEG 2000 - The SFTPC antibody conjugate was mixed and stirred, incubated, and cooled to obtain liposomes.
[0011] Furthermore, the molar ratio of DPPC, DOPE, and cholesterol is 5:3:2.
[0012] Further, in step (1), the patient is sonicated for 10-15 minutes (20kHz, 100W), evaporated at a speed of 50-60 rpm under a vacuum of 0.08 MPa, dried at 25-30°C for 12 hours, and then 5 ml of phosphate buffer solution is added. The mixture is stirred at a constant temperature of 45°C at a speed of 170 rpm for 45 minutes to form a liposome suspension.
[0013] Furthermore, step (1) also includes passing the liposome suspension through a liposome extruder and extruding it at 45°C using 0.2µm and 0.1µm polycarbonate membranes respectively to obtain liposomes with uniform particle size; the average particle size of the liposomes is in the range of 100-200nm.
[0014] Furthermore, EDC solution, DSPE-PEG 2000 The molar ratio of COOH solution to NHS solution is 20:2:40.
[0015] Furthermore, in step (2), the EDC solution and DSPE-PEG... 2000 - Mix COOH solution and NHS solution and react at room temperature for 15 minutes; then use 85.5 mg / ml sodium bicarbonate solution to adjust the pH of the solution to 8-8.5, add SFTPC antibody and stir to disperse, react at room temperature for 2 hours, and then centrifuge at 4℃ and 5000g for 10-15 minutes using an ultrafiltration centrifuge.
[0016] Furthermore, the liposome suspension with DSPE-PEG 2000 -SFTPC antibody-conjugate products were mixed at a molar ratio of 10:1.
[0017] Further, in step (3), the liposomes were incubated in a metal bath at 37°C for 1 hour and then cooled to room temperature. The resulting liposomes had a particle size in the range of 100-200 nm and a PDI < 0.2.
[0018] Application of a targeted acid-responsive liposome or a method for preparing a targeted acid-responsive liposome in the preparation of a drug delivery system targeting type II alveolar epithelial cells.
[0019] Compared with the prior art, the beneficial technical effects of the present invention are as follows:
[0020] 1. The targeted acid-responsive liposomes of the present invention can achieve controlled drug release in the acidic environment of the alveolar cavity.
[0021] High targeting: Through the targeting action of Anti-SFTPC, liposomes can be precisely delivered to type II alveolar epithelial cells, reducing the impact of drugs on non-target areas, improving the targeted delivery efficiency of drugs, and thus enhancing the therapeutic effect.
[0022] Acid responsiveness: Liposomes can rapidly release drugs in the acidic environment of the alveolar cavity, achieving controlled drug release and improving therapeutic efficacy.
[0023] Stability: Liposomes have good stability, which can effectively prevent drug leakage and improve drug delivery efficiency.
[0024] Clinical application prospects: The targeted acid-responsive liposomes provided by this invention can be widely used in the treatment of lung diseases such as pneumonia, ARDS, and COPD, and have important clinical application value.
[0025] 2. The specific proportions of components and preparation method of this invention further improve the stability, acid responsiveness and targeting of liposomes.
[0026] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, it can be implemented according to the contents of the specification. Furthermore, in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description
[0027] Figure 1 This is a schematic diagram of the structure of the acid-responsive liposomes of the present invention.
[0028] Figure 2 This is a flowchart of the thin-film hydration method for preparing drug-loaded acid-responsive liposomes according to the present invention.
[0029] Figure 3 This invention describes the process for synthesizing and applying targeted acid-responsive liposomes.
[0030] Figure 4 The images show the distribution of liposomes with particle sizes of 88-150 nm and PDI < 0.2, along with their transmission electron microscopy (TEM) images.
[0031] Figure 5This is a Zeta potential diagram of different liposomes in this invention.
[0032] Figure 6 This is a distribution map of drugs, liposomes, and targeting antibodies in a drug delivery system.
[0033] Figure 7 This diagram shows the release of the acid-responsive targeted liposome (Lipo-DOPE@Anti-SFTPC) of the present invention in different environments.
[0034] Figure 8 This is a graph showing the cumulative drug release curves of different types of liposome-encapsulated drugs in different pH environments according to the present invention.
[0035] Figure 9 This is a fluorescence distribution map in mice used in this invention. Detailed Implementation
[0036] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.
[0037] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.
[0038] 1. Preparation of acid-responsive liposomes
[0039] (1) Preparation of liposome suspension: DPPC, DOPE and cholesterol were mixed in a molar ratio of 5:3:2 (10 mg of DPPC, 6 mg of DOPE and 4 mg of cholesterol were accurately weighed using an electronic balance with an accuracy of 0.1 mg) in a flask. 3 ml of chloroform was slowly added, and the mouth of the flask was covered with a ground glass stopper to prevent solvent evaporation. The flask was gently shaken to initially disperse the lipids in the solvent. The flask was placed in an ultrasonic instrument and ultrasonically treated for 10-15 minutes until the lipids were completely dissolved and a uniform and transparent solution was formed. The flask was connected to a rotary evaporator and evaporated under reduced pressure (vacuum degree of 0.08 MPa) at a speed of 50-60 rpm to gradually remove the chloroform. The tilt angle of the flask was adjusted so that a uniform and transparent film was formed on the inner wall of the container. After evaporation, the flask was placed in a vacuum drying oven and dried at 25-30℃ for 12 hours (overnight) to completely remove the residual chloroform. Remove the flask from the vacuum drying oven and add 5 ml of phosphate buffered saline (PBS, pH 7.4) preheated to above the phase transition temperature (e.g., 45°C). If hydrophilic drugs need to be encapsulated, the drugs can be dissolved in PBS and added together. Place the flask in a 45°C constant-temperature magnetic stirrer, shake by hand, then add a stir bar and stir at 170 rpm for 45 minutes to fully hydrate the membrane and form a liposome suspension. Pass the liposome suspension through a liposome extruder using 0.2 μm and 0.1 μm polycarbonate membranes, extruding 20 times at 45°C to obtain liposomes with uniform particle size. Use dynamic light scattering (DLS) to detect the liposome particle size, ensuring that the average particle size is within the range of 100-200 nm and that the distribution is uniform (PDI < 0.2). Store the prepared liposome suspension in a 4°C refrigerator, avoiding light and vigorous shaking, for later use.
[0040] (2) Weigh an appropriate amount of EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide], dissolve it in MES buffer [2-(N-morpholino)ethylenesulfonic acid, pH 5.5], and prepare a 20 mM EDC solution. Weigh an appropriate amount of DSPE-PEG. 2000 -COOH, dissolved in MES buffer, dissolved by heating in a 37°C metal bath for 5-10 minutes to accelerate dissolution, to prepare a 2mM DSPE-PEG solution. 2000 -COOH solution, weigh an appropriate amount of NHS (N-hydroxysuccinimide), dissolve it in MES buffer, and prepare a 40mM NHS solution; massage the EDC solution and DSPE-PEG in a 20:2:40 ratio. 2000 -COOH solution and NHS solution were added to the reactor, and the reaction components were thoroughly mixed using a magnetic stirrer to ensure homogeneity. The reaction was carried out at room temperature (25°C) for 15 minutes to activate DSPE-PEG. 2000The carboxyl group on the -COOH group; add 85.5 mg / ml sodium bicarbonate solution (NaHCO3) dropwise to the reaction solution to adjust the pH to 8-8.5; add an appropriate amount of SFTPC antibody to the activated DSPE-PEG. 2000 In the COOH solution, the solution was thoroughly stirred with a magnetic stirrer to ensure uniform antibody dispersion. The reaction was carried out at room temperature (25°C) for 2 hours to promote DSPE-PEG. 2000 The -COOH was coupled with the SFTPC antibody; the reaction solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 10 kDa), and centrifuged at 4°C and 5000g for 10-15 minutes to remove unreacted EDC and DSPE-PEG. 2000 -COOH, NHS and other small molecule impurities; collect the concentrate in the ultrafiltration tube, which is DSPE-PEG. 2000 -SFTPC antibody conjugate; store the product in a 4°C freezer, avoiding light and repeated freeze-thaw cycles, for later use.
[0041] (3) Mix the liposome suspension with DSPE-PEG 2000 The SFTPC antibody-conjugate product was mixed in a reaction tube at a molar ratio of 1:10. The mixture was gently stirred with a magnetic stirrer to ensure thorough mixing. The reaction tube was then placed in a 37°C metal bath and incubated for 1 hour to allow the DSPE-PEG to mature. 2000 - The SFTPC antibody was fully inserted into the liposome membrane. The reaction solution was removed from the metal bath and cooled to room temperature. 100 μL of the reaction solution was diluted to 1 mL with PBS. The liposome particle size distribution and polydispersity index (PDI) were detected using a dynamic light scattering instrument to ensure that the liposome particle size was within the range of 100-200 nm and the PDI < 0.2, indicating uniform particle size distribution. The qualified liposome suspension was stored in a 4°C refrigerator, avoiding light and violent shaking, for subsequent use.
[0042] Figure 2 This is a flowchart of the thin-film hydration method for preparing drug-loaded acid-responsive liposomes according to the present invention. Figure 1 This is a schematic diagram of the structure of the acid-responsive liposomes prepared according to the present invention. The liposomes of the present invention have a spherical structure, and drugs can be encapsulated in both their hydrophilic interior and hydrophobic bilayer. The liposome membrane is mainly composed of DPPC, DOPE, and cholesterol, and DSPE-PEG is applied to the membrane surface. 2000 -COOH is linked to an Anti-SFTPC molecule, which specifically extends to the outside of the liposome and can bind to SFTPC on the surface of type II alveolar epithelial cells.
[0043] Figure 4The images show the particle size distribution and transmission electron microscopy (TEM) images of the liposomes of this invention, with a particle size of 88-150 nm and a PDI < 0.2. The narrow distribution indicates a good preparation process. Under TEM, the liposomes appear spherical. Figure 5 The diagram shows the zeta potential of different liposomes in this invention, all of which are negatively charged. Figure 6 This is a distribution diagram of the drug, liposomes, and targeting antibody in the drug delivery system. Green represents the drug, red represents the liposomes, and blue represents the targeting antibody. Successful co-localization was observed by laser confocal microscopy (CLSM), indicating that the drug was successfully encapsulated in the liposomes, the targeting antibody was successfully linked, and the liposome drug delivery system was successfully synthesized.
[0044] 2. In vitro release assay of acid-responsive liposomes
[0045] (1) Drug release under acidic environment
[0046] The targeted acid-responsive liposomes of the present invention were placed in buffer solutions at pH 6.0 (simulating the acidic environment of the alveolar cavity) and pH 7.4 (simulating the physiological neutral environment), respectively; incubated at 37°C, and samples were taken at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours to determine the drug release; the drug concentration was determined using a UV-Vis spectrophotometer or high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated.
[0047] Result: As Figure 7 TEM showed that, compared to a neutral environment, liposomes in an acidic environment (pH 6.0) exhibited irregular shapes, uneven lipid membrane thickness with localized thinning, and discontinuous and ruptured membranes, demonstrating acid-responsiveness. Figure 8 In an acidic environment of pH 6.0, liposomes release >70% of the drug within 48 hours, while in a neutral environment of pH 7.4, the drug release is <20%.
[0048] (2) Control experiment
[0049] Non-acid-responsive targeting liposomes: DOPE-free targeting liposomes (composed of DPPC, cholesterol, and containing Anti-SFTPC) were prepared, and drug release experiments were conducted under the same conditions.
[0050] Results: Drug release was less than 30% at both pH 6.0 and pH 7.4, indicating that the pH sensitivity of the acid-responsive liposomes of the present invention is significantly better than that of the non-acid-responsive liposomes.
[0051] Non-targeted acid-responsive liposomes: Acid-responsive liposomes without Anti-SFTPC were prepared and drug release experiments were conducted under the same conditions.
[0052] Results: The drug release curve was similar to that of acid-responsive liposomes.
[0053] 3. In vivo targeting experiments of acid-responsive liposomes
[0054] (1) Animal model
[0055] A CLP sepsis mouse model was established to simulate the inflammatory environment of the lungs; targeted acid-responsive liposomes were delivered to the mouse lungs via airway administration.
[0056] Control group: non-targeted liposomes (without Anti-SFTPC).
[0057] (2) Fluorescence imaging analysis
[0058] Twenty-four hours after administration, mice were sacrificed, and lung tissue was collected for frozen sections. The distribution of liposomes in the lung tissue was observed using a fluorescence microscope.
[0059] Result: As Figure 9 Fluorescence imaging technology revealed that acid-responsive liposomes exhibited strong fluorescence signals and more extensive fluorescence distribution, primarily accumulating in the lungs, indicating good targeting ability; while non-targeted liposomes showed weak fluorescence signals.
[0060] The liposomes of this invention use DOPE as the acid-responsive component, utilizing its structural changes under acidic conditions to achieve controlled drug release. The liposomes are processed via DSPE-PEG. 2000 -COOH links Anti-SFTPC as a targeting molecule, enabling it to specifically bind to SFTPC on the surface of type II alveolar epithelial cells.
[0061] (1) Composition of liposomes: DPPC (1,2-dipalmitoyl-sn-glycerol-3-phosphocholine) and DOPE (dioleoylphosphatidylethanolamine) were selected as the main lipid components. DOPE has unique phase transition characteristics under acidic conditions, enabling it to respond to acidic environments. DPPC itself can serve as a major component of alveolar surfactant, exhibiting good biocompatibility and biodegradability. Cholesterol can enhance the stability of liposomes, regulate membrane fluidity, improve drug loading capacity, and enhance biocompatibility. Simultaneously, combined with DSPE-PEG... 2000 -COOH (distearate phosphatidylethanolamine polyethylene glycol carboxyl group) assists in modifying liposomes with lipid components, thereby optimizing liposome stability.
[0062] (2) Drug loading: Hydrophilic drugs can be loaded into the aqueous phase inside liposomes using the thin-film hydration method, while lipophilic drugs can be loaded into the lipid bilayer. During the preparation process, the encapsulation efficiency and drug loading can be improved by optimizing conditions such as the ratio of lipid to drug and the buffer system.
[0063] (3) Acid response mechanism: Under normal physiological pH conditions, DOPE liposomes maintain a stable structure, and the drug is effectively encapsulated within the liposomes. When the liposomes reach the acidic lesion site (such as the acidic environment of the alveolar cavity), DOPE undergoes a phase transition, the liposome structure is destroyed, thereby releasing the encapsulated drug and achieving targeted drug delivery. In this invention, DOPE plays a key role in controlling the timing of drug release.
[0064] (4) Targeted action: DSPE-PEG 2000 After being linked to Anti-SFTPC (anti-alveolar surfactant protein C antibody) with COOH, the liposomes are inserted into the liposomes through co-incubation, giving the liposomes the ability to target type II alveolar epithelial cells, thereby increasing the accumulation of the drug in the alveolar cavity and improving the therapeutic effect.
[0065] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.
[0066] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.
Claims
1. A targeted acid-responsive liposome, characterized in that, Liposomes include DPPC and DOPE, and the surface of the liposomes is PEG-encapsulated. 2000 -COOH is linked to an Anti-SFTPC molecule.
2. The method for preparing a targeted acid-responsive liposome as described in claim 1, characterized in that: Includes the following steps: (1) Preparation of liposome suspension: DPPC, DOPE and cholesterol are mixed and dispersed in a solvent, ultrasonically treated, then evaporated and dried, and then a buffer solution is added and stirred to hydrate and form a liposome suspension. (2) DSPE-PEG 2000 Preparation of SFTPC antibody conjugate: EDC solution, DSPE-PEG 2000 After mixing and stirring the COOH and NHS solutions, the pH of the solution was adjusted to alkaline. SFTPC antibody was then added, the mixture was stirred, and the concentrate was collected after centrifugation; this concentrate is DSPE-PEG. 2000 -SFTPC antibody conjugate; (3) Preparation of liposomes: The liposome suspension was mixed with DSPE-PEG 2000 - The SFTPC antibody conjugate was mixed and stirred, incubated, and cooled to obtain liposomes.
3. The method for preparing a targeted acid-responsive liposome as described in claim 2, characterized in that: The molar ratio of DPPC, DOPE, and cholesterol is 5:3:
2.
4. The method for preparing a targeted acid-responsive liposome as described in claim 2, characterized in that: In step (1), the liposomes are sonicated for 10-15 minutes, evaporated at a speed of 50-60 rpm under a vacuum of 0.08 MPa, dried at 25-30°C for 12 hours, and then 5 ml of phosphate buffer solution is added. The mixture is stirred at a constant temperature of 45°C at a speed of 170 rpm for 45 minutes to form a liposome suspension.
5. The method for preparing a targeted acid-responsive liposome as described in claim 4, characterized in that: Step (1) also includes passing the liposome suspension through a liposome extruder and extruding it at 45°C using 0.2µm and 0.1µm polycarbonate membranes respectively to obtain liposomes with uniform particle size; the average particle size of the liposomes is in the range of 100-200nm.
6. The method for preparing a targeted acid-responsive liposome as described in claim 2, characterized in that: EDC solution, DSPE-PEG 2000 The molar ratio of COOH solution to NHS solution is 20:2:
40.
7. The method for preparing a targeted acid-responsive liposome as described in claim 2, characterized in that: In step (2), EDC solution, DSPE-PEG 2000 -COOH solution and NHS solution were mixed and reacted at room temperature for 15 minutes. Then, using 85.5 mg / ml sodium bicarbonate solution, the pH of the solution was adjusted to 8-8.
5. After adding the SFTPC antibody and stirring to disperse it, the reaction was carried out at room temperature for 2 hours, and then centrifuged at 4℃ and 5000g for 10-15 minutes using an ultrafiltration centrifuge.
8. The method for preparing a targeted acid-responsive liposome as described in claim 2, characterized in that: Liposome suspension with DSPE-PEG 2000 - Mix SFTPC antibody-conjugate products at a molar ratio of 10:
1.
9. The method for preparing a targeted acid-responsive liposome as described in claim 2, characterized in that: Step (3): Incubate in a metal bath at 37°C for 1 hour, then cool to room temperature. The resulting liposomes have a particle size in the range of 100-200 nm and a PDI < 0.
2.
10. The application of the method for preparing an acid-responsive liposome according to claim 1 or any one of claims 2-9 in the preparation of a drug delivery system targeting type II alveolar epithelial cells.