Asymmetric liposome with targeting function, its preparation method and application

Abstract

This invention discloses asymmetric liposomes with targeting function, their preparation method, and applications. The asymmetric liposomes are formed from saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine, unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine, functionalized phospholipids, cholesterol, and paclitaxel. The functionalized phospholipid is 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine-polyethylene glycol-folic acid. The molar ratio of the saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine to the unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine is 2:1 to 1:2. This invention improves the targeting of drug delivery and reduces drug toxicity by locally modifying the liposome surface with a targeting ligand and by allowing the asymmetric liposomes to undergo phase separation at physiological temperatures to form an asymmetric structure.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to an asymmetric liposome with targeting function, its preparation method, and its application in drug delivery. Background Technology

[0002] Liposomes, as a traditional drug delivery system, are formed by the self-assembly of amphiphilic phospholipids and possess a structure similar to biological membranes, making them a focus of attention in the field of drug delivery. In the process of drug development and treatment, many drugs, due to their inherent physicochemical properties such as low solubility, poor stability, and insufficient targeting, struggle to effectively reach lesion sites and exert therapeutic effects. Liposomes, with their unique structure, can encapsulate drugs internally or embed them within a phospholipid bilayer. In this way, liposomes can improve the pharmacokinetic properties of drugs, enhance drug stability, and reduce toxic side effects on normal tissues. Furthermore, by modifying the surface of liposomes, targeted delivery to specific tissues or cells can be achieved, thereby improving the therapeutic effect. Currently, most liposome carriers are homogeneous, while research on Janus-type liposome carriers is relatively limited. These asymmetric liposomes can regulate the spatial distribution of functional components, enhance the aggregation of specific components (such as ligands) on the membrane surface, and thus improve the targeting of liposome carriers. Therefore, designing Janus-type liposome carriers for targeted drug delivery can enhance the application value of liposome carriers in the field of drug delivery.

[0003] Paclitaxel (PTX) is a broad-spectrum anticancer drug approved by the U.S. Food and Drug Administration (FDA). However, this drug suffers from problems such as poor water solubility, reliance on solubilizers leading to severe allergic reactions, intravenous administration only, rapid elimination from the body, and a short half-life. Therefore, using asymmetric liposomes to load paclitaxel may improve the drug's targeting and reduce its toxicity. Summary of the Invention

[0004] This invention provides a method for preparing asymmetric drug-loaded liposomes based on phase separation and their applications. This method is simple to prepare and has strong versatility. These liposomes can efficiently load drugs that are poorly soluble in water, which is beneficial for drug dissolution and delivery. Furthermore, the liposomes possess a controllable asymmetric structure, allowing for the regulation of the spatial distribution of different functional components and enhancing the localized enrichment of specific components. By modifying their surface with locally targeted ligands, the targeting accuracy of liposome-delivered drugs can be improved, and drug toxicity can be reduced, demonstrating promising applications in the field of drug delivery.

[0005] This invention relates to an asymmetric liposome loaded with paclitaxel, which is formed from saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine, unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine, functionalized phospholipids, cholesterol, and paclitaxel. The functionalized phospholipid is modified with folic acid ligand to form 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA), and the molar ratio of the saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine to the unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine is 2:1 to 1:2. The asymmetric liposome of this invention undergoes phase separation at physiological temperatures to form an asymmetric structure, and the asymmetry of the liposome structure can be controlled by adjusting the ratio of phospholipids. By adjusting the drug concentration and the ratio of phospholipid components, the encapsulation efficiency and sustained-release efficiency of drugs by liposomes can be controlled. Local modification of the liposome surface with targeting ligands can improve the targeting of drug delivery and reduce drug toxicity.

[0006] In a preferred embodiment of the present invention, the average particle size of the asymmetric liposomes is 100 nm-500 nm.

[0007] In a preferred embodiment of the invention, the molar ratio of the saturated phospholipid 1,2-distearate-sn-glycerol-3-phosphocholine to the unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine is 1:0.8 to 0.8:1; particularly preferably 1:1. Within this preferred molar ratio range, the liposome structure exhibits greater asymmetry, which is more conducive to improving the targeting of drug delivery.

[0008] The present invention also provides a method for preparing the above-mentioned asymmetric plasmid, which includes the following steps: (1) Take saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine, unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol, 1%-3% DSPE-PEG-FA and the drug paclitaxel, and dissolve them together in chloroform. The final phospholipid concentration is 10 mg / mL - 60 mg / mL, and the paclitaxel concentration is 50 μg / mL - 2 mg / mL; (2) The phospholipid solution in (1) is evaporated in a rotary evaporator to form a film. The evaporation temperature is 35℃-40℃, and the film is placed under vacuum for 10 h-12 h. (3) Add an equal volume of deionized water to (2) equal to the volume of chloroform in (1), and hydrate at 55℃-70℃ for 1 h-3 h; (4) The liposome dispersion obtained in (3) is placed in a phospholipid extruder and repeatedly pushed. The temperature of the extruder is 55℃-70℃ to obtain folic acid ligand modified drug-loaded liposomes.

[0009] Another aspect of the present invention relates to the use of the above-mentioned asymmetric liposomes loaded with paclitaxel in the preparation of antitumor drugs.

[0010] In a preferred embodiment of the present invention, the antitumor drug is used to treat human breast cancer; preferably, the antitumor drug targets human breast cancer.

[0011] This invention utilizes the phase separation properties of phospholipids to prepare Janus-type drug-loaded liposomes with an asymmetric structure. By controlling the proportion of phospholipids, the asymmetry of the liposome structure is regulated, and an asymmetric distribution of its surface functionalized components is achieved, thereby improving the targeted delivery capability of the liposomes. By controlling the drug concentration and the proportion of phospholipid components, the encapsulation efficiency of the liposomes is not less than 80%, which can improve the targeting of drug delivery and reduce drug toxicity. Attached Figure Description

[0012] Figure 1 Schematic diagram of the preparation of paclitaxel encapsulated in Janus-type liposomes; Figure 2 Confocal micrographs of liposomes with different phospholipid component ratios; Figure 3 (a) Drug loading and encapsulation efficiency of Janus liposomes at different paclitaxel drug concentrations; (b) Drug loading and encapsulation efficiency of paclitaxel in liposomes with different phospholipid ratios; (c) Cumulative in vitro drug release curves of free paclitaxel and liposomes with different phospholipid ratios; Figure 4 (a) Confocal plot of cellular uptake of Janus liposomes and non-Janus liposomes; (b) Flow cytometry plot; (c) Corresponding fluorescence intensity plot. Detailed Implementation

[0013] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments represent only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0014] Example 1 This embodiment uses a thin-film hydration method to prepare Janus-type liposomes loaded with paclitaxel (PTX). The preparation process is as follows: Figure 1 As shown, it includes the following steps: a. Take different molar amounts of 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), cholesterol, 1% of 23-(dipyrrometheneborondifluoride)-24-norcholesterol (Bodipy-Chol), 1% of Rhodamine B-1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (Rhod-PE), 1% of 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA), and different concentrations of paclitaxel, and dissolve them together in chloroform. The composition included DSPC (1 part), DOPC (1 part), and cholesterol (1 part); the final phospholipid concentration was 10 mg / mL, the paclitaxel concentration was 50 μg / mL - 2 mg / mL, and Bodipy-Chol and Rhod-PE were used for fluorescent labeling of the liposomes. b. The phospholipid solution in a was evaporated to form a film by rotary evaporation at 37°C and then placed under vacuum for 12 h. c. Add an equal volume of deionized water to chloroform in a to b, and hydrate at 65°C for 2 hours. d. The liposome dispersion obtained in c is placed in a phospholipid extruder and repeatedly extruded at a temperature of 60°C to obtain folic acid ligand-modified Janus-type liposomes (Janus PTX@FA-Liposome).

[0015] The product prepared above was characterized, as detailed in the following figures. Figure 2 To facilitate observation of the liposome structure, a polycarbonate membrane with a micron-sized pore was used during liposome extrusion to obtain micron-sized liposomes. The saturated and unsaturated regions of the liposomes were fluorescently labeled with the green fluorescent dye 23-(dipyrrometheneborondifluoride)-24-norcholesterol (Bodipy-Chol) and the red fluorescent dye rhodamine B-1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (Rhod-PE), respectively. Figure 2 The images show laser confocal scanning images of the resulting liposomes. In the asymmetric liposomes, DSPC and cholesterol form ordered regions, while DOPC and other unsaturated phospholipids form disordered regions. The asymmetry of the liposome structure can be controlled by adjusting the molar ratio of DSPC to DOPC to achieve ratios of 1:0, 2:1, 1:1, 1:2, and 0:1.

[0016] Example 2 By loading paclitaxel onto Janus-type liposomes as described in Example 1, and adjusting the drug concentration and the ratio of phospholipid components, the drug encapsulation efficiency of the liposomes can be controlled. Figure 3 As shown in ab. Since paclitaxel is insoluble in water, the drug-loaded liposomes were centrifuged at low speed (3000 rpm for 15 min) to remove unencapsulated paclitaxel. 200 μL of the supernatant liposomes was then added to 2 mL of methanol and sonicated for 10 min to break the emulsion. The UV absorption wavelength of the drug was measured to be 227 nm. The drug concentration before and after encapsulation was determined by high-performance liquid chromatography (HPLC), and the drug loading rate and encapsulation efficiency of the liposomes were calculated. With increasing drug concentration, the encapsulation efficiency of Janus-type liposomes decreased. The highest encapsulation efficiency of paclitaxel (over 80%) was observed at a drug concentration of 100 μg / ml. The encapsulation efficiency of the liposomes also increased with increasing unsaturated phospholipid content. However, excessively high membrane fluidity may be detrimental to sustained drug release; therefore, further investigation of the drug release behavior of liposomes with different proportions is needed.

[0017] One mL of Janus-type liposomes encapsulating paclitaxel (as described in Example 1) and one mL of free paclitaxel were placed separately in dialysis bags (MWCO = 8000 Da). The bags were incubated at 37°C with constant shaking. At regular intervals, 1 mL samples were taken, and 1 mL of fresh release medium was added simultaneously. The release medium was 30 mL of PBS buffer (pH 7.4) containing 1% Tween 80. The drug concentration was determined by high-performance liquid chromatography (HPLC), and the cumulative release was calculated. Figure 3 As shown in Figure c. To determine the concentration of lipid-soluble drugs in PBS, surfactants were used to promote drug diffusion from the membrane phase to the aqueous phase and increase the apparent solubility of the drug in the release medium. Free paclitaxel was rapidly released in the release medium, with a cumulative release rate of approximately 68% after 48 h. The cumulative drug release rates of asymmetric liposomes with different phospholipid ratios (1:0, 1:1, and 0:1) within 48 h were 18%, 37%, and 48%, respectively. The results indicate that the asymmetric liposome structure has good stability and exhibits certain sustained-release characteristics, and the drug release rate increases accordingly with the increase of the unsaturated phospholipid ratio. Considering both the encapsulation efficiency and drug release behavior, when the phospholipid ratio is 1:1, the Janus-type liposomes achieve the most ideal sustained-release effect while ensuring high drug loading, reaching the optimal balance between drug sustained-release performance and release efficiency, and demonstrating the best drug delivery capability.

[0018] Example 3 Janus-type (1:1) and non-Janus-type liposomes prepared in Example 1 were co-incubated with breast cancer cells for 2 hours. Cell uptake efficiency was quantitatively analyzed by confocal laser scanning microscopy and flow cytometry, as follows: a. First, fluorescently labeled Janus-type and non-Janus-type liposomes were prepared. 1,2-Distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), and cholesterol were mixed in a molar ratio of 1:1:1. 1%-3% of 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA) and 1%-3% of rhodamine B-labeled 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (Rhod-PE) were added and dissolved together in chloroform. Rhodamine B-labeled Janus-type liposomes were obtained through film hydration and extrusion. 1,2-Distearyl-sn-glycerol-3-phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), and cholesterol were mixed in a molar ratio of 2:0:1. 1%-3% of 1,2-distearyl-sn-propyltriyl-3-phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA) and 1%-3% of rhodamine B-labeled 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (Rhod-PE) were added and dissolved together in chloroform. Rhodamine B-labeled non-Janus type liposomes were obtained by film hydration and extrusion.

[0019] b. Human breast cancer cells (MCF-7) were cultured for 36 h in confocal culture dishes using Duchenne modified Eagle (DMEM) medium containing 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin. 1 mL of DMEM solution containing Janus-type and non-Janus-type liposomes was added to the culture dish containing the cells, resulting in a final liposome concentration of 0.2 mg / mL. After co-incubation for 1.5 h, excess liposomes were removed with PBS, and fresh medium was added. The cell nuclei and cell membranes were stained with Hoechst 33342 and Alexa 488 lasers, respectively, for 10 min. Cell fluorescence was observed under a confocal microscope after illumination with lasers at 405 nm, 488 nm, and 561 nm.

[0020] c. Human breast cancer cells (MCF-7) were cultured in 6-well plates, and 0.2 mg / mL of Janus liposomes and non-Janus liposomes were added and incubated for 2 h. After incubation, the cells were washed with PBS, refined with trypsin until detached, and digestion was terminated with culture medium containing fetal bovine serum. The cells were then centrifuged at 1000 rpm for 3 min. The cells were collected from the wells and dispersed in centrifuge tubes with PBS. The intracellular fluorescence intensity was measured by flow cytometry at an excitation wavelength of 561 nm. The control group consisted of cells containing only PBS and no liposomes.

[0021] like Figure 4 As shown, cells in the Janus-type liposome group exhibited significantly enhanced red fluorescence signals, which were markedly higher than those in the non-Janus-type control group, indicating higher cellular uptake efficiency. These results demonstrate that the asymmetric structure of Janus-type liposomes can improve the targeting efficiency of drug delivery, providing a new design approach for developing efficient and low-toxicity targeted drug delivery systems.

[0022] The above description is merely a preferred embodiment of the present invention. It should be noted that this method of asymmetric liposome encapsulation of drugs is applicable to most lipid-soluble drugs. For related systems in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. An asymmetric liposome formed from saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine, unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine, functionalized phospholipid, cholesterol, and paclitaxel, wherein the functionalized phospholipid is modified with folic acid ligand to form 1,2-distearyl-sn-propanetriyl-3-phosphatidylethanolamine-polyethylene glycol-folic acid (DSPE-PEG-FA), and the molar ratio of saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine to unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine is 2:1 to 1:

2.

2. The asymmetric liposomes according to claim 1, wherein the average particle size of the asymmetric liposomes is 100 nm-500 nm.

3. The asymmetric liposome according to claim 1, wherein the molar ratio of the saturated phospholipid 1,2-distearate-sn-glycerol-3-phosphocholine to the unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine is 1:0.8 to 0.8:

1.

4. The asymmetric liposome according to claim 1, wherein the molar ratio of the saturated phospholipid 1,2-distearate-sn-glycerol-3-phosphocholine to the unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine is 1:

1.

5. A method for preparing an asymmetric plasmid according to any one of claims 1-4, comprising the following steps: (1) Take saturated phospholipid 1,2-distearyl-sn-glycerol-3-phosphocholine, unsaturated phospholipid 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol, 1%-3% DSPE-PEG-FA and the drug paclitaxel, and dissolve them together in chloroform. The final phospholipid concentration is 10 mg / mL - 60 mg / mL, and the paclitaxel concentration is 50 μg / mL - 2 mg / mL; (2) The phospholipid solution in (1) is evaporated in a rotary evaporator to form a film. The evaporation temperature is 35℃-40℃, and the film is placed under vacuum for 10 h-12 h. (3) Add an equal volume of deionized water to (2) equal to the volume of chloroform in (1), and hydrate at 55℃-70℃ for 1 h-3 h; (4) The liposome dispersion obtained in (3) is placed in a phospholipid extruder and repeatedly pushed. The temperature of the extruder is 55℃-70℃ to obtain folic acid ligand modified drug-loaded liposomes.

6. The use of the asymmetric liposomes according to any one of claims 1-4 in the preparation of antitumor drugs.

7. The application according to claim 6, wherein the antitumor drug is used to treat human breast cancer.

8. The application according to claim 7, wherein the antitumor drug targets human breast cancer.

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