Preparation of ROS responsive liposome and application of ROS responsive liposome in anti-pancreatic cancer drugs

Abstract

The invention provides preparation of ROS responsive lipidosome and application of the ROS responsive lipidosome in anti-pancreatic cancer drugs, and belongs to the technical field of ROS responsive lipidosome preparation. The liposome can synergistically deliver formononetin and salvianolic acid B, overcomes the defects of formononetin and salvianolic acid B in the aspects of solubility, stability and bioavailability, and improves the curative effect of formononetin and salvianolic acid B in pancreatic cancer treatment. The ROS-responsive formononetin liposome and the salvianolic acid B liposome are wrapped by the cell-derived nano-vesicles through co-extrusion with the nano-vesicles on the outer layer, so that the ROS-responsive liposome is prepared. The liposome can be used for preparing anti-pancreatic cancer drugs, realizes targeted treatment of pancreatic cancer, inhibition of pancreatic cancer cell proliferation, promotion of pancreatic cancer cell apoptosis and improvement of pancreatic cancer tumor microenvironment energy metabolism disorder, and has the advantages of high biological safety, controllable drug release and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of ROS-responsive liposome preparation, and in particular relates to the preparation of ROS-responsive liposome and the application of the liposome in anti-pancreatic cancer drugs. Background Art

[0002] Pancreatic cancer is a highly malignant tumor, often diagnosed at an advanced stage and poorly treated. Formononetin, a flavonoid extracted from the traditional Chinese medicine Astragalus membranaceus, exhibits antioxidant and antitumor properties, while salvianolic acid B, a polyphenol extracted from the traditional Chinese medicine Salvia miltiorrhiza, exhibits anti-inflammatory and antitumor effects. Although these two ingredients have shown promising anticancer potential in pharmacological studies, their clinical application is limited by various factors.

[0003] Formononetin has poor water solubility, resulting in low bioavailability, impaired absorption and distribution in the body, and a prolonged duration of effectiveness. Salvianolic acid B also has poor chemical stability and is susceptible to oxidation and discoloration, reducing its effectiveness in pharmaceutical formulations. These properties make these two ingredients less effective in the treatment of pancreatic cancer.

[0004] Drug delivery systems help improve the bioavailability and stability of delivered drug ingredients. Existing drug delivery systems are unable to effectively address the shortcomings of formononetin and salvianolic acid B. For example, while traditional liposomes can improve drug bioavailability, they lack responsiveness in drug release within the tumor microenvironment, preventing precise release and effectively addressing the high concentration of ROS in tumor tissue. Furthermore, existing drug combination regimens fail to fully consider drug interactions, resulting in poor therapeutic efficacy. Therefore, developing a novel liposome capable of responsive drug release in the high ROS environment of tumor tissue is key to addressing these issues. Summary of the Invention

[0005] In response to the above problems, the present invention proposes a preparation method of ROS-responsive liposomes and their application in anti-pancreatic cancer drugs.

[0006] The technical solution adopted in the present invention is as follows:

[0007] In a first aspect, the present invention provides a method for preparing ROS-responsive liposomes, comprising the following steps:

[0008] 1) Mixing a neutral phospholipid, a cationic lipid, and a PEGylated lipid to form a liposome precursor; the PEGylated lipid is modified with a ROS-responsive group;

[0009] 2) dissolving the liposome precursor mixture in an organic solvent to obtain a uniform lipid solution;

[0010] 3) adding the formononetin solution to the lipid solution and stirring to obtain a lipid solution with a mass ratio of total lipid to formononetin of (5-20):1;

[0011] 4) evaporating the solvent in the lipid solution obtained in step 3), adding phosphate buffer containing salvianolic acid B to the lipid film after desolvation, and stirring to obtain a lipid solution with a mass ratio of total lipids to salvianolic acid B of (5-20):1;

[0012] 5) Extruding the liposomes using a polycarbonate membrane filter to obtain small-sized liposomes, and dialysis purification to remove unbound drug and solvent; 200 nm and 100 nm polycarbonate membrane filters are preferred;

[0013] 6) The purified liposomes were modified with four-arm polyethylene glycol-o-diphenol and stored in an argon environment at 4° C. for subsequent use.

[0014] Preferably, the neutral phospholipid is selected from DMPC (1,2-eicosanoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine); DMPC is further preferred.

[0015] Preferably, the cationic lipid is selected from DOTAP (1,2-eicosanoyl-sn-glycero-3-phosphodialkylamino), DC-Chol ((2,3-dioxyethyl)trimethylammonium formyloxycholesterol), DODAB (1,2-distearoyloxy-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide) or DOTMA ((2,3-dimyristoyloxy)propyl-N,N,N-trimethylammonium chloride); DOTAP is further preferred.

[0016] Preferably, the PEGylated lipid is selected from DSPE-PEG-HPBA (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-p-hydroxymethylphenylboronic acid), DSPE-PEG-Sel (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-selenoether), and DSPE-PEG-TE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-thioether); DSPE-PEG-HPBA is further preferred, which has high ROS responsiveness and is a typical ROS responsive group; in addition, DSPE-PEG-Sel has medium to high ROS responsiveness, and the seleno group is sensitive to the redox environment; DSPE-PEG-TE has low or weak ROS responsiveness, and the thioether can be oxidized but the response is weak.

[0017] Preferably, in the liposome precursor obtained in step 1), the molar ratio of neutral phospholipid, cationic lipid and PEGylated lipid is (50-90):(5-30):10; more preferably 78:12:10.

[0018] Preferably, the concentration of the lipid solution obtained in step 4) is 1-2 mg / ml; more preferably 1 mg / ml.

[0019] Preferably, in step 5), multiple extrusions are performed using 200 nm and 100 nm polycarbonate membrane filters. The extrusion process gradually controls the size of the liposomes and improves their uniformity through the polycarbonate membrane (e.g., 200 nm and 100 nm), significantly enhancing their biocompatibility and targeting. Smaller liposome sizes help reduce recognition and clearance by the immune system, thereby increasing their circulation time in the body. At the same time, uniform size distribution reduces uncertainty in biological responses. Small liposomes are more likely to penetrate tumor tissue (using the EPR effect) and deliver targeted drugs through endocytosis. Reduced size also accelerates drug release (high surface area to volume ratio) and improves stability.

[0020] Preferably, in step 6), the volume ratio of the ROS responsive groups on the surface of the purified liposomes to the four-arm polyethylene glycol-o-diphenol to be modified is (15-1):1.

[0021] In a second aspect, the present invention provides a ROS-responsive liposome, which maintains a non-fusogenic state during blood circulation and BBB crossing, and is activated to restore fusogenicity in high-ROS tumor tissue, thereby achieving targeted fusion and drug release.

[0022] In a third aspect, the present invention provides a use of the above-mentioned ROS-responsive liposome in the preparation of an anti-pancreatic cancer drug.

[0023] The beneficial effects of the present invention are:

[0024] (1) In terms of liposome composition: the present invention adopts a certain proportion of neutral phospholipids, cationic lipids, and PEGylated lipids modified with ROS-responsive groups to form a lipid mixture, wherein the neutral phospholipids (such as DMPC) provide structural stability, the cationic lipids (such as DOTAP) are positively charged and form electrostatic interactions with the negatively charged flavonoids to enhance the drug loading capacity, and the PEGylated lipids modified with ROS-responsive groups (such as DSPE-PEG-HPBA) are chemically bonded to four-arm polyethylene glycol-o-diphenol to form a non-fusogenic state. Under ROS stimulation, the ROS-responsive groups undergo oxidation and hydrolysis reactions, resulting in the dissociation of 4-arm PEG-oDP from the liposomes, thereby achieving responsive release of the drug in the high ROS environment of tumor tissue.

[0025] (2) Improved drug loading and stability: The lipid mixture is first dissolved in an organic solvent, and then formononetin is dissolved therein. After the organic solvent evaporates, the mixture is stirred with a PBS solution containing salvianolic acid B to obtain a lipid solution. This method not only increases the drug loading capacity, but also significantly enhances the chemical stability of salvianolic acid B, preventing it from oxidative discoloration.

[0026] (3) Liposome size control: Small-sized liposomes are obtained by extrusion through a polycarbonate membrane filter, which improves the stability and bioavailability of liposomes and also facilitates the penetration and accumulation of liposomes in tumor tissues.

[0027] (4) ROS-responsive release mechanism: Liposomes are mixed with four-arm polyethylene glycol-o-diphenol (4-arm PEG-oDP), and 4-arm PEG-oDP is modified on the liposome surface by vigorous vortexing to achieve ROS-responsive release. This mechanism enables liposomes to rapidly release drugs in the high ROS environment of tumor tissue, improving the drug's targeting and efficacy.

[0028] The ROS-responsive liposomes of the present invention can significantly improve the bioavailability and stability of drugs used in pancreatic cancer treatment, overcoming the shortcomings of traditional chemotherapy drugs in pancreatic cancer treatment. By optimizing the liposome preparation and drug loading process, the effectiveness of formononetin and salvianolic acid B is ensured. Furthermore, the liposomes can achieve specific release in the tumor microenvironment, responding to high ROS concentrations, thereby enhancing the therapeutic efficacy of the drugs and showing promising clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a transmission electron microscopy (TEM) image of the ROS-responsive DMPC / DOTAP / DSPE-PEG-HPBA liposomes prepared in Example 1;

[0030] Figure 2 The results of in vitro stability tests of the ROS-responsive DMPC / DOTAP / DSPE-PEG-HPBA liposomes prepared in Example 1 in PBS at pH 7.4 at 37°C incubator were presented;

[0031] Figure 3 These are the results of the effect of the ROS-responsive DMPC / DOTAP / DSPE-PEG-HPBA liposomes prepared in Example 1 on the viability of pancreatic tumor cells in vitro. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0033] Example 1

[0034] Preparation of ROS-responsive DMPC / DOTAP / DSPE-PEG-HPBA liposomes:

[0035] 1. Prepare the lipid mixture: Prepare three lipid components: DMPC (1,2-eicosanoyl-sn-glycero-3-phosphocholine), DOTAP (1,2-eicosanoyl-sn-glycero-3-phosphodialkylamino) and DSPE-PEG-HPBA (polyethylene glycol-modified distearoylphosphatidylcholine). Mix these three components in a molar ratio of 78:12:10 (n / n) to form the precursor of the basic liposome.

[0036] 2. Dissolve the lipid mixture: Dissolve the lipid mixture in a mixture of anhydrous chloroform and methanol at a volume ratio of 4:1 (v / v) to obtain a uniform lipid solution. This step ensures that the lipid components are fully dissolved, preparing for subsequent drug loading.

[0037] 3. Loading Formononetin: Formononetin (at a concentration of 1 mg / ml) was added to the lipid solution at a total lipid to formononetin ratio of 10:1 (w / w). The solution was gently stirred to ensure that the formononetin was evenly dispersed throughout the liposomes. This process aims to increase the bioavailability of formononetin, enabling it to more effectively exert its anti-cancer effects.

[0038] 4. Evaporation of organic solvent: In a well-ventilated environment, use a rotary evaporator to evaporate the organic solvent until the solvent is completely removed. Subsequently, add a PBS (phosphate buffer) solution containing salvianolic acid B to the residual lipid film, with a mass ratio of total lipids to salvianolic acid B of 10:1 (w / w), and stir at 37°C until a homogeneous lipid solution is formed (concentration of 1 mg / ml). This step ensures the effective loading of salvianolic acid B. In the lipid solution obtained at this time, each 1 ml of lipid solution contains 1 mg of liposomes and the drug entrapped therein.

[0039] 5. Controlling liposome size: To optimize the distribution and release characteristics of liposomes in vivo, liposomes were extruded through 200nm and 100nm polycarbonate membrane filters, repeated 10 times to obtain small liposomes. This process helps improve the biocompatibility and targeting of liposomes.

[0040] 6. Dialysis purification: Transfer the prepared liposome solution to a dialysis bag with a molecular weight cutoff of 10 kDa and perform dialysis at 4°C to remove unbound drug and solvent to ensure the purity and effectiveness of the liposomes.

[0041] 7. Modification of ROS-responsive groups: The purified liposomes were mixed with four-arm polyethylene glycol-o-diphenol (4-arm PEG-oDP) at a volume ratio of HPBA:4-arm PEG-oDP = 3:1 and vortexed vigorously for 1 minute to introduce ROS-responsive groups. This modification enables the liposomes to respond to high concentrations of ROS in the tumor microenvironment, thereby achieving specific release.

[0042] 8. Storage and Use: Finally, the resulting ROS-responsive liposomes were stored in an argon atmosphere at 4°C for subsequent use. This step ensures the stability of the liposomes during storage and maintains their drug release properties.

[0043] In this Example 1, the HPBA group is covalently linked to the liposome surface by reacting the 4-(hydroxymethyl)phenylboronic acid (HPBA) group with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-amine (DSPE-PEG-NH2) on the liposome surface. The HPBA group plays a key role in the subsequent steps and is used to anchor 4-arm PEG-oDP. The HPBA-modified liposomes are mixed with 4-arm PEG-oDP, and the chemical bond between HPBA and the ortho-diphenol groups drives the 4-arm PEG-oDP to be coated on the liposome surface, forming a non-fusogenic state. Under ROS stimulation, the phenylboronic acid group in HPBA undergoes oxidation and hydrolysis reactions, causing the 4-arm PEG-oDP to dissociate from the liposome.

[0044] By integrating 4-arm PEG-oDP as a "lock" into fusogenic liposomes, they maintain a non-fusogenic state during blood circulation and BBB crossing. In tumor tissues with high levels of ROS, the 4-arm PEG-oDP is activated, shedding the liposomes and restoring their fusogenic properties, thereby achieving targeted fusion and drug release.

[0045] Example 2

[0046] The difference from Example 1 is that in step 3, when loading formononetin, the mass ratio of total lipids to formononetin is 15:1 (w / w); in step 4, when loading salvianolic acid B, the mass ratio of total lipids to salvianolic acid B is 15:1 (w / w).

[0047] Example 3

[0048] The difference from Example 1 is that in step 3, the purified liposomes are mixed with four-arm polyethylene glycol-o-diphenol (4-armPEG-oDP) at a volume ratio of HPBA:4-arm PEG-oDP=10:1.

[0049] Test Case

[0050] The DMPC / DOTAP / DSPE-PEG-HPBA liposomes Plo@FCS prepared in Example 1 were observed under a transmission electron microscope. The TEM image is shown in FIG. Figure 1 As shown, the prepared liposomes exhibit regular spherical shapes with uniform particle size distribution, approximately 100 nm in diameter, and are intact in morphology, with a smooth surface and no obvious aggregation. No free drug crystals or crystal precipitation were observed, confirming that the drug was effectively encapsulated within the liposomes.

[0051] The in vitro stability of the DMPC / DOTAP / DSPE-PEG-HPBA liposomes Plo@FCS prepared in Example 1 was tested in PBS at pH 7.4 at 37°C. Figure 2 As shown in the figure, the particle size of Plo@FCS remained stable within 6 days and the PDI was always <0.5, indicating good dispersion uniformity and excellent stability.

[0052] CCK8 was used to detect the effect of Plo@FCS on the viability of pancreatic tumor cells in vitro. PANC-1 cells were cultured at a density of 5×10 3 The cells were seeded into a 96-well plate at a density of 100 μL / well and cultured for 24 hours to adhere to the wall. The old culture medium was removed and fresh culture medium containing different concentrations of drugs was added, 100 μL per well, with 3 replicates per group. Culture was continued for 24 hours at 37°C and 5% CO2. 10 μL of CCK8 solution was added to each well and incubated in the dark for 2 hours. The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an enzyme reader. Figure 3 As shown in the figure, the results showed that: with the increase of FCS concentration, the cell survival rate gradually decreased, but the inhibitory effect was weak (for example, the survival rate was still above 50% at 30 μg / ml); at the same concentration of Plo@FCS, the cell survival rate was significantly reduced (for example, the survival rate dropped to below 50% at 30 μg / ml), indicating that liposome encapsulation significantly improved the cytotoxicity or targeted delivery efficiency of the drug.

[0053] The above test results demonstrate that the prepared ROS-responsive DMPC / DOTAP / DSPE-PEG-HPBA liposomes significantly improve the bioavailability and stability of drugs used in pancreatic cancer treatment, overcoming the shortcomings of traditional chemotherapy drugs in pancreatic cancer treatment. The optimized liposome preparation and drug loading process ensures the effectiveness of formononetin and salvianolic acid B, and achieves specific release within the tumor microenvironment, thereby enhancing the therapeutic efficacy of the drugs and showing promising clinical application prospects.

Claims

1. A method for preparing ROS-responsive liposomes, characterized in that: The following steps are involved: 1) Mixing a neutral phospholipid, a cationic lipid, and a PEGylated lipid to form a liposome precursor; the PEGylated lipid is modified with a ROS-responsive group; 2) dissolving the liposome precursor mixture in an organic solvent to obtain a uniform lipid solution; 3) adding the formononetin solution to the lipid solution and stirring to obtain a lipid solution with a mass ratio of total lipid to formononetin of (5-20):1; 4) evaporating the solvent in the lipid solution obtained in step 3), adding phosphate buffer containing salvianolic acid B to the lipid film after desolvation, and stirring to obtain a lipid solution with a mass ratio of total lipids to salvianolic acid B of (5-20):1; 5) Extruding the liposomes using a polycarbonate membrane filter to obtain small-sized liposomes, followed by purification by dialysis to remove unbound drug and solvent; 6) The purified liposomes were modified with four-arm polyethylene glycol-o-diphenol and stored in an argon environment at 4° C. for subsequent use.

2. The preparation method according to claim 1, characterized in that The neutral phospholipid is selected from DMPC, DPPC, DOPC or POPC.

3. The preparation method according to claim 1, characterized in that The cationic lipid is selected from DOTAP, DC-Chol, DODAB or DOTMA.

4. The preparation method according to claim 1, characterized in that The PEGylated lipid is selected from DSPE-PEG-HPBA, DSPE-PEG-Sel or DSPE-PEG-TE.

5. The preparation method according to claim 1, characterized in that In the liposome precursor obtained in step 1), the molar ratio of neutral phospholipid, cationic lipid and PEGylated lipid is (50-90):(5-30):

10.

6. The preparation method according to claim 1, characterized in that The concentration of the lipid solution obtained in step 4) is 1-2 mg / ml.

7. The preparation method according to claim 1, characterized in that In step 5), 200 nm and 100 nm polycarbonate membrane filters were used for multiple extrusions.

8. The preparation method according to claim 1, characterized in that In step 6), the volume ratio of the ROS responsive groups on the surface of the purified liposomes to the four-arm polyethylene glycol-o-diphenol to be modified is (15-1):

1.

9. A ROS-responsive liposome prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The liposome remains in a non-fusogenic state during blood circulation and BBB crossing, and is activated to restore fusogenicity in tumor tissues with high ROS, thereby achieving targeted fusion and drug release.

10. Use of the ROS-responsive liposome according to claim 9 in the preparation of anti-pancreatic cancer drugs.

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