A triple-targeting tumor microenvironment cationic liposome and a preparation method and application thereof

Abstract

The application discloses a kind of triple targeting tumor microenvironment cationic liposome and preparation method and application thereof.The cationic liposome includes: cationic lipid, auxiliary phospholipid, antitumor drug, hyaluronic acid-lipid derivative and sialic acid derivative.The cationic liposome utilizes sialic acid and can specifically bind to the surface Siglecs receptor of TAM, hyaluronic acid can specifically target the high expression CD44 receptor of CSC, and cationic lipid can be combined with the negative tumor vascular endothelial cell by electrostatic adsorption, to realize triple targeting delivery to tumor vascular endothelial cell, tumor-associated macrophage and tumor stem cell.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more particularly to a cationic liposome that triple-targets the tumor microenvironment, its preparation method, and its application. Background Technology

[0002] The occurrence and development of solid tumors such as melanoma are highly dependent on the tumor microenvironment (TME). Its key components include tumor vascular endothelial cells (TECs), tumor-associated macrophages (TAMs), and tumor stem cells (CSCs). These key components jointly mediate tumor proliferation, immune escape, drug resistance, and recurrence, which are the core obstacles to tumor treatment.

[0003] Cationic liposomes, with their positive surface charge, can bind to negatively charged TECs, inducing endothelial cell apoptosis and effectively inhibiting tumor growth. However, unmodified cationic liposomes have significant drawbacks: their exposed positive charge makes them easily adsorbed by plasma proteins, rapidly cleared by the mononuclear phagocyte system (MPS), have a short circulating half-life, and limited tumor accumulation; furthermore, they lack active targeting specificity, making it difficult to accurately identify tumor-specific biomarkers, with weak targeting of TAMs and CSCs, resulting in limited overall antitumor efficacy and a higher risk of toxic side effects.

[0004] Current cationic liposome delivery strategies are mostly based on single-modification designs, which suffer from problems such as uneven modification, particle size dispersion, unstable encapsulation efficiency, and poor formulation stability. In particular, their functions are relatively limited, unable to simultaneously target multiple key targets of the tumor microenvironment (TME), and cannot effectively and actively target tumor tissue, resulting in limited therapeutic effects and high recurrence rates. Therefore, there is an urgent need to develop a cationic liposome that combines multi-target targeting performance with a simple preparation process. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a cationic liposome that triple-targets the tumor microenvironment. This cationic liposome utilizes sialic acid (SA) to specifically bind to Siglecs receptors on the surface of tumor endothelial cells (TAMs), and hyaluronic acid (HA) to specifically target CD44 receptors highly expressed in tumor sclerosis cells (CSCs). Triple-targeted delivery to TECs (cationic lipid electrostatic adsorption), TAMs (SA-Siglecs axis), and CSCs (HA-CD44) is achieved through co-modification with hyaluronic acid and sialic acid.

[0006] Another objective of this invention is to provide a method for preparing cationic liposomes that triple-target the tumor microenvironment. This method involves modifying the surface of the cationic liposomes with sialic acid, and then modifying the surface with hyaluronic acid (HA) via a post-insertion method to achieve co-modification of SA and HA.

[0007] Another object of the present invention is to provide the use of the above-mentioned cationic liposomes in the preparation of tumor drugs.

[0008] To achieve the above objectives, the present invention is accomplished through the following technical solutions.

[0009] A cationic liposome that triple-targets the tumor microenvironment, the cationic liposome comprising: cationic lipids, helper phospholipids, antitumor drugs, hyaluronic acid-lipid derivatives, and sialic acid derivatives.

[0010] In the above technical solution, the cationic lipids include DOTAP, DOTMA, DODAB, DDAB, DOTIM, DOSPA, DOGS, DC-Chol, and DLin-MC3-DMA.

[0011] In the above technical solution, the auxiliary phospholipids include disqualyl phosphatidylcholine (DEPC), lecithin, distearate phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, distearate phosphatidylglycerol, and egg yolk phosphatidylglycerol.

[0012] In the above technical solution, the anti-tumor drugs include docetaxel (DOC), cabazitaxel, carmustine, idarubicin, anlotinib, bortezomib, vincristine, irinotecan, etoposide, mitoxantrone, paclitaxel, doxorubicin, and epirubicin.

[0013] In the above technical solution, the hyaluronic acid-lipid derivative includes DSPE-HA.

[0014] In the above technical solution, the molecular weight of HA in DSPE-HA is 1k~50k.

[0015] In the above technical solution, the sialic acid derivative includes sialic acid. Cholesterol conjugates, sialic acid glutaric acid Stearyl alcohol, sialic acid polyethylene glycol Distearate phosphatidylethanolamine, sialic acid Octadecylamine, Methyl sialic acid stearic acid and sialic acid Eighteen kinds of acid.

[0016] In the above technical solution, the cationic liposomes are spherical or near-spherical vesicle structures.

[0017] In the above technical solution, the cationic liposomes have a particle size of 150~170 nm, a polydispersity index (PDI) ≤0.2, a zeta potential of -40~-30 mV, and an encapsulation efficiency ≥90%.

[0018] In the above technical solution, the cationic lipid is preferably DOTAP, the auxiliary phospholipid is preferably disorhodophosphatidylcholine, the hyaluronic acid-lipid derivative is preferably DSPE-HA, and the sialic acid derivative is preferably sialic acid. Cholesterol conjugate, wherein the hyaluronic acid-lipid derivative is preferably DSPE-HA. 5k .

[0019] In the above technical solution, the ratio of the cationic lipid, cofactor phospholipid, antitumor drug, and sialic acid derivative, based on the molar amounts, is (1 / 2) * ... 50): (40) 89): 5: 5.

[0020] In the above technical solution, the ratio of the antitumor drug to the hyaluronic acid-lipid derivative by mass is 1:(0.5~10).

[0021] A method for preparing cationic liposomes that target the tumor microenvironment in three ways includes the following steps:

[0022] Step 1: Sialic acid-modified cationic liposomes are prepared by using cationic lipids, auxiliary phospholipids, antitumor drugs and sialic acid derivatives. The method includes, but is not limited to, any one or more of the following: ethanol injection method, modified ethanol injection method, thin film dispersion method, reverse evaporation method, high pressure homogenization method, ultrasonic dispersion method, microfluidic method and freeze-drying and reconstitution method. Step 2: Hyaluronic acid-lipid derivatives are inserted into sialic acid-modified cationic liposomes by incubation at 55-70℃ using a post-insertion method to obtain cationic liposomes that triple-target the tumor microenvironment.

[0023] In step 1, the modified ethanol injection method involves dissolving cationic lipids, cofactor phospholipids, antitumor drugs, and sialic acid derivatives in ethanol, removing the ethanol, hydrating, extruding and granulating to obtain sialic acid-modified cationic liposomes.

[0024] In step 2, the post-insertion method involves adding a hyaluronic acid-lipid derivative solution to sialic acid-modified cationic liposomes, incubating at 55-70°C for 5-20 minutes to sterilize, and obtaining cationic liposomes that triple-target the tumor microenvironment.

[0025] In the above technical solution, the operation of removing ethanol includes: stirring at a constant temperature of 20~50℃ until the ethanol evaporates and is removed.

[0026] In the above technical solution, the hydration treatment operation includes: injecting sterile water for injection at 20~50℃ and stirring for 5~30 minutes.

[0027] In the above technical solution, the granulation operation includes: passing the granules sequentially through polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm under nitrogen pressure in a constant temperature water bath at 20~50℃.

[0028] In step 2, the hyaluronic acid-lipid derivative solution is a mixture of hyaluronic acid-lipid derivative and water, and the concentration of the hyaluronic acid-lipid derivative is 1~20 mg·mL. -1 .

[0029] In step 2, the sterilization is performed by filtration using a 0.22 μm aqueous microporous membrane.

[0030] The above-mentioned cationic liposomes with triple targeting of the tumor microenvironment are used in the preparation of tumor drugs.

[0031] In the above technical solution, the cationic liposomes that target the tumor microenvironment triple achieve triple targeted delivery of TECs (cationic lipid-electrostatic adsorption), TAMs (SA-Siglecs axis), and CSCs (HA-CD44).

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The cationic liposomes of the present invention that target the tumor microenvironment in a triple manner can specifically target the Siglecs receptor of tumor-associated macrophages and CD44-positive tumor stem cells. At the same time, the cationic liposomes can bind to negatively charged tumor vascular endothelial cells through electrostatic adsorption, thereby achieving triple synergistic anti-tumor effect and overcoming the defects of existing delivery systems that target only one and have limited anti-tumor effect.

[0033] (2) The cationic liposomes of the present invention, which target the tumor microenvironment, use hyaluronic acid to shield the positive charge of the cations during delivery, forming a hydration layer, which significantly prolongs the in vivo circulation time, reduces non-specific recognition and clearance, and solves the problem that traditional cationic liposomes are easily cleared by the mononuclear phagocytic system and have a short in vivo circulation time.

[0034] (3) The cationic liposomes of the present invention targeting the tumor microenvironment encapsulate docetaxel with uniform particle size, low PDI, high encapsulation efficiency, and pH-sensitive release. It is stable in the physiological environment and rapidly releases the drug in the acidic environment of the tumor, thus taking into account both stability and drug release efficiency.

[0035] (4) The cationic liposomes of the present invention targeting the tumor microenvironment significantly reduce the toxicity of docetaxel and cationic charge toxicity, improve biosafety, overcome the defects of traditional chemotherapy drugs with high toxicity, and achieve a tumor volume inhibition rate of 89.30% and a comprehensive tumor inhibition index of 50.69. It has no cytotoxicity and reduces the degree of damage to liver, spleen and lung tissue.

[0036] (5) The preparation method of the present invention is simple and easy to scale up, and has strong batch consistency, overcoming the defects of existing formulations that have multiple modifications, complex operations, and poor formulation stability. Attached Figure Description

[0037] Figure 1 The left image shows the appearance of the cationic liposomes obtained in Example 1 and Comparative Examples 1-3, and the right image shows their transmission electron microscopy (TEM) images. Figure 2 Schematic diagrams of DOC-HA-SAL prepared under different post-insertion stirring temperature conditions under natural light and laser irradiation; Figure 3 The in vitro release curves of the drug cationic liposomes under (A) pH=7.4 and (B) pH=5.0 conditions are shown. Figure 4 The image shows a laser confocal microscope image of B16F10 cells taking up DiD cationic liposomes in Example 4-1. The first row shows the fluorescence distribution of the cell nucleus, the second row shows the fluorescence distribution of the formulation, and the third row shows the fluorescence distribution of the formulation taken up by the cells. Figure 5 The image shows a laser confocal microscope image of RAW264.7 cells taking up DiD cationic liposomes in Example 4-1. The first row shows the fluorescence distribution of the cell nucleus, the second row shows the fluorescence distribution of the formulation, and the third row shows the fluorescence distribution of the formulation taken up by the cells. Figure 6 The first column shows a general flow cytometry plot and the second column shows the general flow cytometry plots (first column) and bar charts (second column) of the quantitative analysis of the uptake of DiD cationic liposomes by (A~B) B16F10 cells and (C~D) RAW264.7 cells using flow cytometry in Examples 4-2. Figure 7 (A) shows the in vivo tumor fluorescence imaging of the four DiR cationic liposomes in Example 5 in a tumor-bearing mouse model; Figure 7 (B) is an in vitro fluorescence image of tumor tissue and major organs of tumor-bearing mice after treatment with four types of DiR cationic liposomes in Example 5; Figure 8 This is a bar chart showing the average fluorescence intensity of tumor tissues from tumor-bearing mice after treatment with four types of DiR cationic liposomes in Example 5. Figure 9 The MTT assay was used to investigate the inhibitory effects of the cationic liposomes obtained in Example 1 and Comparative Examples 1-3, as well as the corresponding blank cationic liposomes, on RAW264.7 and B16F10 cells; (A-B) cell viability of the drug group and (C-D) blank group; (A) and (C) cell inhibition of RAW264.7 cells, and (B) and (D) cell inhibition of B16F10 cells; Figure 10 The figures for (A) tumor volume growth curves and (B) tumor volume inhibition rate of B16F10 tumor-bearing mice after treatment in the drug administration group and the Control group in Example 7-1 are shown. Figure 11 The following are the (A) body weight, (B) net body weight, (C) relative tumor inhibition index and (D) tumor inhibition index of B16F10 tumor-bearing mice after treatment in the drug administration group and the Control group in Example 7-1; Figure 12 This is a survival analysis curve of B16F10 tumor-bearing mice in Example 7-1 after treatment in the drug administration group and the Control group; Figure 13 Images of hematoxylin-eosin stained sections of liver, spleen, lungs, and tumors from mice in the treatment group and control group in Example 7-1; Figure 14 (A) shows the results of F4 / 80, iNOS and CD206 immunofluorescence staining of tumor tissues after treatment with cationic liposomes obtained in Example 1 and Comparative Examples 1-3; Figure 14 (B) is Figure 14 The average red fluorescence density of iNOS in (A); Figure 14 (C) is Figure 14 The average yellow fluorescence density of CD206 in (A); Figure 14 (D) is Figure 14 (B) and Figure 14 The fluorescence intensity ratio of (C); Figure 15 Immunofluorescence colocalization maps of CD44 receptor (green fluorescence) and DiR-labeled liposomes (red fluorescence) in tumor tissues treated with cationic liposomes obtained in Example 1 and Comparative Examples 1-3; Figure 16 The results show the determination of the content of relevant cytokines in tumor tissues after treatment with cationic liposomes obtained in Example 1 and Comparative Examples 1-3. Among them, (A) is the result of TNF-α content determination, (B) is the result of IL-10 content determination, and (C) is the result of TGF-β content determination. Detailed Implementation

[0038] The following examples are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0039] The reagents used in the following examples and their manufacturers are as follows: Docetaxel (DOC), purity ≥98%, purchased from Dalian Meilun Biotechnology Co., Ltd. Trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) and disorhodophosphatidylcholine (DEPC) were both purchased from Shanghai Aivito Pharmaceutical Technology Co., Ltd. Sialic acid-cholesterol conjugate (SA-CH) was purchased from Jiaxing Hanchun Biotechnology Co., Ltd. 5% glucose injection (5% Glu) was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0040] In the following embodiments, DSPE-HA 5k The structural formula is n=13~16; Preparation of DSPE-HA 5k The method involved dissolving hyaluronic acid (0.2 mmol) and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (0.2 mmol) in 3 mL of formamide. The reaction was carried out under nitrogen protection in the dark for 1 h. DSPE solution was added dropwise, and the pH was adjusted to 7.5 with triethylamine to obtain the reaction solution. The reaction solution was then incubated at 65 °C for 24 h. After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 2 kDa, and dialyzed with 1000 mL of pure water for 48 h, changing the dialysate every 8 h. After 48 h, the liquid in the dialysis bag was collected and freeze-dried to obtain DSPE-HA. 5k The DSPE solution was prepared by mixing 150 mg of 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE) with 5 mL of THF at 65 °C.

[0041] In the following examples, the B16F10 mouse melanoma cell line (B16F10 cells) and RAW264.7 mouse mononuclear macrophages (RAW264.7 cells) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; unless otherwise specified, when the cells in the cell suspension were RAW264.7 cells, the density was 2 × 10⁻⁶ cells / mL. 5 cells·mL -1 The corresponding serum-free culture medium used was RPMI-1640 medium; when the cells in the cell suspension were B16F10 cells, the density was 5 × 10⁻⁶ cells / mL. 5 cells·mL -1 The corresponding serum-free culture medium used was DMEM high glucose medium.

[0042] In the following examples, C57BL / 6 mice, weighing 18-20 g, male, were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0043] Example 1 A cationic liposome co-modified with hyaluronic acid and sialic acid (designated: DOC-HA-SAL), comprising: DOTAP, DEPC, DOC, SA-CH, and DSPE-HA. 5k Of these, by molar amount, the ratio of DOTAP, DEPC, DOC, and SA-CH is 5:85:5:5; by mass, the ratio of DOC and DSPE-HA is... 5k The ratio is 1:4.

[0044] The preparation method of the above-mentioned cationic liposomes co-modified with hyaluronic acid and sialic acid (No.: DOC-HA-SAL) includes the following steps: Step 1: Prepare sialic acid-modified cationic liposomes using a modified ethanol injection method: Weigh DOC, DOTAP, DEPC, and SA-CH into a 10 mL vial, and add anhydrous ethanol (volume equal to 10% of the volume of sterile water for injection). v / v The mixture was stirred continuously at a constant temperature of 35 °C until the anhydrous ethanol evaporated and was removed. Sterile water for injection at 35 °C was slowly injected and stirred for 15 min for incubation to obtain the initial drug-loaded liposome product. The initial drug-loaded liposome product was transferred to a liposome extruder and extruded and granulated under nitrogen pressure in a constant temperature water bath at 35 °C, sequentially through polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm. Each pore size was extruded 8 times to obtain sialic acid modified cationic liposomes. Step 2, use the post-insertion method to insert DSPE-HA 5k Modification onto sialic acid-modified cationic liposomes: A concentration of 10 mg / mL was added dropwise to the sialic acid-modified cationic liposomes. -1 DSPE-HA 5k Solution (DSPE-HA) 5k The solution is (DSPE-HA) 5k The mixture of hyaluronic acid and water was stirred at a constant temperature of 60℃ for 20 min for incubation, and then filtered through a 0.22 μm aqueous microporous membrane for sterilization to obtain cationic liposomes co-modified with hyaluronic acid and sialic acid (number: DOC-HA-SAL).

[0045] Comparative Example 1 An unmodified cationic liposome (designated: DOC-CL) comprises DOTAP, DEPC, and DOC. The ratio of DOTAP, DEPC, and DOC, by molar amount, is 5:85:5.

[0046] The preparation method of the above-mentioned unmodified cationic liposomes (number: DOC-CL) is basically the same as the preparation method in step 1 of Example 1, except that SA-CH is not added.

[0047] Comparative Example 2 A sialic acid-modified cationic liposome (designated: DOC-SAL) comprises DOTAP, DEPC, DOC, and SA-CH. The molar ratio of DOTAP, DEPC, DOC, and SA-CH is 5:85:5:5.

[0048] The preparation method of the above-mentioned sialic acid modified cationic liposomes (number: DOC-SAL) is consistent with the preparation method in step 1 of Example 1.

[0049] Comparative Example 3 A hyaluronic acid-modified cationic liposome (designated: DOC-HA-CL), comprising: DOTAP, DEPC, DOC, and DSPE-HA. 5k Of these, the ratio of DOTAP, DEPC, and DOC by molar amount is 5:85:5; and the ratio of DOC and DSPE-HA by mass is... 5k The ratio is 1:4.

[0050] The preparation method of the above-mentioned hyaluronic acid-modified cationic liposomes (No.: DOC-HA-CL) includes the following steps: To the unmodified cationic liposomes obtained in Comparative Example 1, a concentration of 10 mg·mL was added. -1 DSPE-HA 5k The solution was stirred at a constant temperature of 60℃ for 20 min for incubation, and then filtered through a 0.22 μm aqueous microporous membrane for sterilization to obtain hyaluronic acid modified cationic liposomes (number: DOC-HA-CL).

[0051] Example 2 Example 2-1 The morphology of the cationic liposomes obtained in Example 1 and Comparative Examples 1-3 was observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown, by Figure 1 It can be seen that the cationic liposomes obtained in Example 1 and Comparative Examples 1-3 all have spherical or near-spherical vesicle structures and are uniformly dispersed in aqueous media.

[0052] Example 2-2 To investigate the effect of post-insertion stirring temperature on DSPE-HA 5kThe effect of sialic acid-modified cationic liposomes was investigated. Following the preparation method of Example 1, the stirring temperature in step 2 was replaced with 35°C and 50°C, respectively, to obtain DOC-HA-SAL prepared under different post-insertion stirring temperature conditions. Their apparent morphology was observed, and their particle size, polydispersity index (PDI), and Zeta potential were determined using dynamic light scattering. The apparent morphologies at 60°C and 35°C (under natural light and laser irradiation) are as follows: Figure 2 As shown in Table 1. Particle size, PDI, and Zeta potential are shown in Table 1.

[0053] Table 1

[0054] Depend on Figure 2 Based on the data in Table 1, it can be seen that DOC-HA-SAL obtained at a stirring temperature of 35℃ appears as a white, turbid state in aqueous medium, while DSPE-HA... 5k It failed to effectively insert into sialic acid-modified ionic liposomes, only undergoing electrostatic adsorption leading to aggregation; while DOC-HA-SAL obtained at 50℃ stirring temperature still showed partial aggregation in aqueous medium; DOC-HA-SAL obtained at 60℃ stirring temperature was pale blue, clear and transparent in aqueous medium with obvious opalescence, DSPE-HA 5k It has been stably embedded in the liposome bilayer and HA modification has been completed.

[0055] Examples 2-3 0.2 mL of the DOC-HA-SAL obtained in Example 1 was measured and diluted to 10 mL with methanol (demulsifier). The docetaxel content was determined by reversed-phase high-performance liquid chromatography (RP-HPLC), and the encapsulation efficiency of the DOC-HA-SAL obtained in Example 1 was calculated. EE The percentage (%) was 96.5 ± 2.4%, indicating that DOC-HA-SAL was successfully prepared.

[0056] In Examples 3-7 below, the drug cationic liposomes are one of the following: DOC-HA-SAL obtained in Example 1, DOC-CL obtained in Comparative Example 1, DOC-SAL obtained in Comparative Example 2, and DOC-HA-CL obtained in Comparative Example 3.

[0057] Example 3 Example 3-1 To assess storage stability, the DOC-HA-SAL obtained in Example 1 was placed in a brown vial, filled with nitrogen, sealed, and stored in a low-temperature environment at 4±2℃ in the dark for 15 days. The particle size and encapsulation efficiency of DOC-HA-SAL were measured every 5 days. It was found that the particle size and encapsulation efficiency of DOC-HA-SAL did not change significantly, indicating that DOC-HA-SAL has good short-term storage stability.

[0058] Example 3-2 The in vitro release behavior of drug cationic liposomes was investigated using dialysis. Drug cationic liposomes were placed in dialysis bags (molecular weight cutoff 8000–14000 Da) and incubated at 37°C with shaking at 100 rpm for 48 h using either phosphate buffer (pH=7.4) or phosphate buffer (pH=5.0) containing 0.5 wt% Tween 80 as the release medium. The cumulative release of the drug cationic liposomes was measured at 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h. The results are shown below. Figure 3 As shown.

[0059] Depend on Figure 3 It was found that in the release medium simulating neutral physiological conditions (pH=7.4), the cumulative release amount of the drug cationic liposomes within 48 h was less than 40%. Among them, the DOC-HA-SAL obtained in Example 1, due to the presence of both a dense rigid barrier of SA and a thick hydration layer of HA on its surface, formed a double barrier, thus exhibiting the slowest release at pH=7.4. In the release medium simulating the weakly acidic environment of a tumor (pH=5.0), the cumulative release amount of the liposome aqueous dispersions obtained in Examples 1 and Comparative Examples 1-3 was greater than 50% within 48 h, and the drug release rate was significantly accelerated. Among them, the DOC-HA-CL obtained in Comparative Example 3 and the DOC-HA-SAL obtained in Example 1 showed obvious pH-responsive drug release characteristics. In summary, DOC-HA-SAL can maintain relatively stable structure in physiological environments, avoiding premature drug leakage; and can rapidly release drugs in the acidic environment of a tumor, improving therapeutic efficacy.

[0060] Example 4 Sialic acid (SA) can specifically bind to the Siglecs receptor on the surface of TAMs, while hyaluronic acid (HA) can bind to the CD44 receptor highly expressed in CSCs. Based on this, we evaluated whether co-modification with SA and HA could enhance the uptake efficiency of cationic liposomes on B16F10 and RAW264.7 cells through an active targeting mechanism. Cells were pre-saturated with free SA, HA, or SA+HA to pre-occupy the binding sites of SA and HA on the cell surface, and the uptake mechanism of HA and SA co-modified cationic liposomes by B16F10 and RAW264.7 cells was explored.

[0061] However, docetaxel (DOC) lacks fluorescence properties, making direct tracking difficult. In contrast, the lipophilic cell membrane fluorescent probe 1,1'-bis(octadecyl-3,3,3',3'-tetramethylaminocyanoperchlorate) (DiD) can be encapsulated in the lipid bilayer of cationic liposomes via hydrophobic interactions, similar to the DOC encapsulation method. Therefore, DiD (at a concentration of 1 mg / mL) was used for direct tracking. -1 The DiD solution (comprising DiD powder and anhydrous ethanol) was introduced to replace the drug DOC. The mass ratio of DiD to DOTAP was 0.20:0.82. Following the preparation methods of Comparative Examples 1-3 and Example 1, four types of DiD cationic liposomes, namely DiD-CL, DiD-SAL, DiD-HA-CL and DiD-HA-SAL, were prepared sequentially.

[0062] Example 4-1 500 μL of cell suspension was seeded into 12-well plates containing climbing slides and cultured overnight. The original solution was then removed from the wells. Experimental group, SA pre-saturation group, HA pre-saturation group, and HA+SA pre-saturation group were set up, with the following group configurations: Experimental group: The first solution was added to the wells and incubated at 37°C for 3 h; the first solution was obtained by diluting DiD cationic liposomes with serum-free culture medium, and the DiD concentration in the first solution was 0.2 μg·mL. -1 DiD cationic liposomes are one of DiD-CL, DiD-SAL, DiD-HA-CL and DiD-HA-SAL; SA pre-saturated group: SA was added to the wells to a final concentration of 10 mg·mL. -1 The SA solution (SA solution is a mixture of SA and serum-free culture medium) was incubated for 1 h, the liquid in the well was removed, and then the first solution was added and incubated at 37°C for 3 h. The DiD cationic liposomes in the first solution were either DiD-SAL or DiD-HA-SAL, and the corresponding test results were labeled as DiD-SAL(SA) and DiD-HA-SAL(SA), respectively.

[0063] HA presaturated group: basically the same as SA presaturated group, except that the final HA concentration was 10 mg·mL. -1 The HA solution (HA solution is a mixture of HA and serum-free culture medium) was used to replace the SA solution. In the first solution, the DiD cationic liposomes were either DiD-HA-CL or DiD-HA-SAL, and the corresponding test results were labeled as DiD-HA-CL (HA) and DiD-HA-SAL (HA), respectively.

[0064] HA+SA presaturated group: basically the same as the SA presaturated group, except that the final concentrations of HA and SA are each 10 mg·mL. -1 The HA+SA solution (HA+SA solution is a mixture of HA, SA and serum-free culture medium) was used to replace the SA solution. In the first solution, the DiD cationic liposomes were DiD-HA-SAL, and the corresponding test results were labeled as DiD-HA-SAL (HA+SA).

[0065] Cells from the experimental group, SA pre-saturation group, HA pre-saturation group, and HA+SA pre-saturation group were washed with PBS after 3 h of incubation, fixed with 4% paraformaldehyde for 30 min, and stained with DAPI. Blue fluorescence (DAPI nuclear staining), red fluorescence (DiD preparation), and the combined fluorescence of both were observed under a laser confocal microscope, as shown in Figures 4-5. Figures 4-5 It was found that, compared to DiD-CL, RAW264.7 and B16F10 cells treated with HA-modified DiD-HA-CL or SA-modified DiD-SAL exhibited stronger overall red fluorescence, indicating that SA and HA modification enhanced cellular uptake of cationic liposomes through receptor-mediated active targeting. In the SA-presaturated and HA-presaturated groups, cellular uptake of DiD-SAL or DiD-HA-CL was significantly decreased, while uptake by cells co-modified with SA and HA in DiD-HA-SAL was further decreased. This suggests that SA and HA co-modification synergistically improves the cell targeting efficiency of cationic liposomes. This synergistic effect depends on the co-mediation of Siglecs receptor and CD44 receptor, with no significant antagonistic effect between the two.

[0066] Example 4-2 Cells from the experimental group, SA pre-saturation group, HA pre-saturation group, and HA+SA pre-saturation group in Example 4-1 were incubated for 3 h, washed three times with PBS, and 0.5 mL of trypsin was added to each well to digest the cells. The cell suspension was centrifuged at 1200 rpm for 4 min, and the cell pellet was collected. The cell pellet was resuspended in 200 μL of 4% paraformaldehyde solution, and quantitative analysis was performed by flow cytometry. The results are as follows: Figure 6 As shown, by Figure 6 It can be seen that, compared with DiD-HA-CL, RAW264.7 or B16F10 cells had the highest average fluorescence intensity after co-incubation with DiD-HA-SAL, which was about 3.4 times the average fluorescence intensity of DiD-CL in B16F10 cells. This indicates that the addition of HA and SA can significantly enhance the targeting ability of DiD-HA-SAL to tumor cells and macrophages, which is basically consistent with the results of laser confocal microscopy imaging experiments.

[0067] Example 5 To assess the effect of SA and HA co-modification on the tissue distribution of liposomes in B16F10 tumor-bearing mice, cationic liposomes were labeled with the fluorescent probe 1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole) tricarbonyanine iodide (DiR). This avoids the cytotoxic effects of DOC on the health status of experimental animals, thus allowing for a more objective evaluation of the in vivo distribution and metabolic processes of the delivery system. DiR (at a concentration of 5 mg / mL) was used... -1 The DiR solution (comprising DiR powder and anhydrous ethanol) was introduced to replace the drug DOC. The mass ratio of DiR to DOTAP was 0.20:0.82. Following the preparation methods of Comparative Examples 1-3 and Example 1, four types of DiR cationic liposomes, namely DiR-CL, DiR-SAL, DiR-HA-CL and DiR-HA-SAL, were prepared sequentially.

[0068] Establishment of a B16F10 tumor-bearing mouse model: The hair on the right back of 36 healthy C57BL / 6 mice was shaved, and after local disinfection with 75% alcohol, 0.2 mL of tumor-bearing cell suspension (tumor-bearing cell count 5 × 10⁻⁶) was injected subcutaneously. 5 (cells), after 10 days the tumor volume was approximately 300-400 mm. 3 DiR cationic liposomes were injected via the tail vein. In vivo fluorescence images were acquired at 1 h, 4 h, 8 h, 12 h, 24 h, and 48 h post-injection. Figure 7 As shown in (a), after imaging was completed, the mouse was euthanized by cervical dislocation, and then tumor tissue and major organs (heart, lungs, liver, spleen, and kidneys) were removed. In vitro fluorescence images were then acquired on black cardstock as shown in (a). Figure 7 As shown in (b), by Figure 7 It can be seen that in the early stage, the lung and liver signals treated with DiR-HA-SAL were weak, indicating that co-modification with HA and SA can reduce non-specific accumulation in the lung and liver. As time goes on, the fluorescence intensity of the spleen drops to a low level, indicating that DiR-HA-SAL can be gradually cleared from the spleen without long-term accumulation, which is conducive to the targeted delivery of drugs to the tumor site.

[0069] Using the ROI tool of a live in vivo imaging system, the average fluorescence intensity of tumor tissue was calculated, such as... Figure 8As shown, within 48 hours after injection, the fluorescence intensity of DiR at the tumor site in mice exhibited a trend of first increasing and then decreasing. Within 48 hours after DiR-HA-SAL treatment, the fluorescence intensity at the tumor site was higher than that of single-ligand modified and unmodified tumors at all time points, with a peak at 8 hours. This is because HA modification achieves active and effective targeting by specifically binding to the CD44 receptor overexpressed on the surface of B16F10 tumor cells. The addition of SA can promote the uptake by tumor-associated macrophages, delivering cationic liposomes to the tumor site, thereby achieving efficient accumulation at the tumor site.

[0070] Example 6 Following the preparation methods of Comparative Examples 1-3 and Example 1 respectively, without adding DOC during the preparation process, four types of blank liposomes were successively prepared: blank cationic liposome (No.: Blank-CL), blank sialic acid modified cationic liposome (No.: Blank-SAL), blank hyaluronic acid modified cationic liposome (No.: Blank-HA-CL), and blank hyaluronic acid and sialic acid co-modified cationic liposome (No.: Blank-HA-SAL).

[0071] The MTT assay was used to perform in vitro cytotoxicity experiments on four blank liposomes and DOC-HA-SAL, DOC-CL, DOC-SAL, and DOC-HA-CL obtained in Example 1 and Comparative Examples 1-3. The experimental groups were set up as follows: Drug group: B16F10 cells or RAW264.7 cells were injected at a rate of 2 × 10⁻⁶. 5 cells·mL -1 The samples were seeded at a density of [missing information] in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. Then, 100 μL of a second solution was added, and the plates were incubated for another 48 h. Finally, MTT solution (5 mg / mL) was added. -1 Incubate with 10 μL / well of the triple solution for 4 h, then add 100 μL / well of the triple solution and continue incubation for 24 h. Measure the OD value at 570 nm and calculate cell viability and half-maximal inhibitory concentration (IC50). 50 The second solution was obtained by diluting the drug cationic liposomes with serum-free culture medium, with a DOC concentration gradient of 0.1 μg·mL⁻¹. -1 1 μg·mL -1 5 μg·mL -1 25 μg·mL -1 50 μg·mL -1 100 μg·mL -1 and 250 μg·mL -1 .

[0072] Blank group: Essentially the same as the drug group, except that the second solution was replaced with the third solution. The third solution was prepared by diluting the blank liposomes with the same volume ratio as the DOC concentration gradient in the experimental group's drug cationic liposomes, sequentially preparing the corresponding concentration gradient: 0.1 μg·mL⁻¹. -1 1 μg·mL -1 5 μg·mL -1 25 μg·mL -1 50 μg·mL -1 100 μg·mL -1 and 250 μg·mL -1 .

[0073] Cell viability in the drug group and the control group as follows Figure 9 As shown, IC 50 As shown in Table 2.

[0074] Table 2

[0075] Depend on Figure 9 As shown in Table 2, DOC-HA-SAL exhibited dose-dependent inhibitory effects on both cell types; both SA and HA single modifications significantly reduced IC50. 50 The blank hyaluronic acid and sialic acid co-modified cationic liposomes (Blank-HA-SAL) showed a cell survival rate greater than 80% at the corresponding concentrations and no significant cytotoxicity, demonstrating that the drug delivery system has good biosafety.

[0076] Example 7 Example 7-1 A B16F10 tumor-bearing mouse model was established according to the method in Example 5. After 7 days, the tumor volume was approximately 100 mm. 3 At that time, the mice were randomly divided into 6 groups (6 mice in each group), and the grouping was set as follows: The treated group (i.e., the treated group): The drug solution was injected via tail vein on day 7, followed by a tail vein injection every 2 days. The dosage of DOC in the liposome was 10 mg per kg of mouse body weight, for a total of 5 administrations. Tumor volume and mouse body weight (g) were measured every 1 day after day 7. The drug solution was either liposomes or docetaxel solution (e.g., Figures 10-12 (As shown in "DOC-S"), docetaxel solution consists of DOC, 2 wt% Tween 80 and 2 wt% anhydrous ethanol.

[0077] Control group: basically the same as the drug administration group, except that the injected drug solution was replaced with 5% glucose injection.

[0078] During the experiment, mouse mortality events were recorded daily for each group. Tumor volume inhibition rate, tumor inhibition index, relative tumor inhibition index, and overall tumor inhibition index were calculated at each interval after day 7, using the following formulas: Tumor volume (V, mm) 3 = 0.5 × a × b 2 (a: long diameter of the tumor, b: short diameter of the tumor); Tumor volume inhibition rate TIR V ): TIR V = ( V Control - V Treated ) / V Control × 100%; Net body weight (g) = Body weight - Tumor mass (assuming tumor density is 1 g·cm³ during the experiment) -3 (Tumor mass equals tumor volume multiplied by tumor density). Tumor inhibition index TI index , g / g) = Body weight / Tumorweight; The relative tumor-inhibition index (RTI) RTI index ): TI indexTreated / TI indexControl ; Comprehensive tumor inhibition index CTI index ): TI index ×Survival rate final ).

[0079] Plot tumor volume growth curves, tumor volume inhibition rate, body weight, tumor inhibition index, and relative tumor inhibition index for the treatment group and control group, such as... Figures 10-11 As shown, TI on day 31 index RTI index and CTI index The results are shown in Table 3.

[0080] Table 3

[0081] Depend on Figure 10 It can be seen that the cationic liposomes of the drug can effectively control tumor growth during administration. Among them, DOC-HA-SAL has the best tumor-suppressing effect, with a tumor volume inhibition rate of 89.30% on day 31, which is significantly higher than that of DOC-CL (60.63%) and DOC-S (48.42%). Figure 11 It can be seen that after the DOC-HA-SAL administration, the net body weight of the mice steadily recovered, the survival quality of the mice was good, and the comprehensive tumor inhibition index was 50.69.

[0082] Survival analysis curves were plotted based on the mortality events of mice in each group, such as... Figure 12 As shown. By Figure 12 It was found that all mice in the Control group died on day 34 of tumor onset. Mice injected with docetaxel solution via tail vein had a survival rate of 50% on day 32 of tumor onset, while mice injected with liposome solution via tail vein had a survival rate of over 80% on day 32 of tumor onset. This indicates that cationic liposomes reduced the non-specific toxicity of DOC, resulting in longer survival times and better quality of life for the mice. Furthermore, mice injected with DOC-HA-SAL via tail vein had a 100% survival rate on day 38 of tumor onset and exhibited good health, demonstrating that encapsulating DOC within cationic liposomes co-modified with HA and SA can reduce the toxic side effects of the drug and the cationic membrane material.

[0083] Example 7-2 To further evaluate the biosafety of the mice in each experimental group, major organ (liver, spleen, lung) and tumor samples from the mice in Example 7-1 were taken, fixed with 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), stained with hematoxylin and eosin (H&E), and observed and photographed under an inverted microscope. The results are as follows. Figure 13 As shown, by Figure 13 It was found that after administration of DOC-S, DOC-CL, and DOC-HA-CL, the alveolar walls thickened significantly, the alveolar structure partially disappeared, and a certain amount of inflammatory cell infiltration was observed. After administration of DOC-S, most hepatocytes showed enlargement and edema (indicated by the box in the figure), with loose and pale cytoplasm, indicating drug-induced damage. In addition, after administration of DOC-S and DOC-CL, some central venous congestion was observed (indicated by the arrow in the figure), and after administration of DOC-HA-CL, a certain amount of focal inflammatory cells (mainly lymphocytes and monocytes) aggregated (indicated by the arrow in the figure), indicating that there may be some systemic inflammatory damage. After administration of DOC-HA-SAL, large areas of necrotic areas and cavity formation were observed in the tumor tissue, and the degree of damage to the liver, spleen, and lung tissues was significantly reduced compared with the administration of DOC-S and DOC-CL.

[0084] Example 7-3 Tumor tissue sections from Examples 7-2 were stained with DAPI and then subjected to F4 / 80 (pan-macrophage marker, green fluorescence), iNOS (M1 macrophage marker, red fluorescence), and CD206 (M2 macrophage marker, yellow fluorescence) immunofluorescence staining. ImageJ software was used to calculate the positive fluorescence density of iNOS and CD206 in each field of view, and the fluorescence intensity ratio of M1 macrophage marker to M2 macrophage marker (M1 / M2) was calculated. Figure 14 As shown, by Figure 14 It was found that after DOC-HA-SAL administration, the CD206 positive fluorescence in tumor tissue decreased most significantly, while the iNOS positive fluorescence density increased significantly. The M1 / M2 ratio, from high to low, was DOC-HA-SAL > DOC-SAL > DOC-HA-CL > DOC-CL > DOC-S > Control, indicating that DOC-HA-SAL can effectively kill the M2 phenotype, effectively inhibit tumor growth, activate the body's anti-immune response, increase M1 phenotype expression, and improve the immunosuppressive state of the tumor microenvironment.

[0085] Example 7-4 Tumor tissue sections stained with DAPI in Examples 7-2 were used to examine the co-localization of DiR-labeled liposomes (red fluorescence) and positive CD44 receptors (green fluorescence) in the tumor site using immunofluorescence. DiR-labeled liposomes (red fluorescence) were also used. The degree of co-localization between DiR fluorescence and CD44 fluorescence was quantitatively evaluated using Pearson correlation coefficients (PCCs). The results are as follows: Figure 15 As shown, by Figure 15 It was found that in tumor tissues after DOC-CL and DOC-SAL administration, the DiR fluorescence signal and CD44 signal did not significantly overlap; however, in tumor tissues after DOC-HA-CL and DOC-HA-SAL administration, red and green fluorescence showed obvious spatial co-localization, forming a yellow overlapping area. The Pearson correlation coefficient quantitative results were consistent with the fluorescence images, indicating that HA modification successfully mediated the specific binding of cationic liposomes to the CD44 receptor on the surface of tumor cells, effectively killing tumor cells and tumor stem cells, inhibiting tumor recurrence, and overcoming the shortcomings of existing agents in inhibiting recurrence.

[0086] Examples 7-5 Tumor tissue homogenates from Examples 7-2 were used, and the levels of immunosuppressive cytokines IL-10, TGF-β, and pro-inflammatory cytokine TNF-α were detected using an ELISA kit. The results are as follows: Figure 16 As shown, by Figure 16It was found that in tumor tissues after DOC-HA-SAL administration, the levels of IL-10 and TGF-β were the lowest in the administration group, while TNF-α was significantly higher than that of DOC-CL and DOC-SA-CL, indicating that DOC-SA-CL can most effectively activate the anti-tumor immune response and improve the local immunosuppressive microenvironment.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the implementation method. The scope of protection of the present invention should be determined by the scope defined in the claims. Other variations or modifications can be made based on the above description. Obvious variations or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A cationic liposome that triple-targets the tumor microenvironment, characterized in that, The cationic liposomes include: cationic lipids, cofactor phospholipids, antitumor drugs, hyaluronic acid-lipid derivatives, and sialic acid derivatives.

2. The cationic liposomes according to claim 1, characterized in that, The cationic lipids include DOTAP, DOTMA, DODAB, DDAB, DOTIM, DOSPA, DOGS, DC-Chol, and DLin-MC3-DMA.

3. The cationic liposomes according to claim 1, characterized in that, The auxiliary phospholipids include disqualyl phosphatidylcholine, lecithin, distearyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, distearyl phosphatidylglycerol, and egg yolk phosphatidylglycerol.

4. The cationic liposomes according to claim 1, characterized in that, The antitumor drugs include docetaxel, cabazitaxel, carmustine, idarubicin, anlotinib, bortezomib, vincristine, irinotecan, etoposide, mitoxantrone, paclitaxel, doxorubicin, and epirubicin.

5. The cationic liposome according to claim 1, characterized in that, The hyaluronic acid-lipid derivative includes DSPE-HA; The molecular weight of HA in the DSPE-HA is 1k~50k.

6. The cationic liposomes according to claim 1, characterized in that, The sialic acid derivative includes sialic acid. Cholesterol conjugates, sialic acid glutaric acid Stearyl alcohol, sialic acid polyethylene glycol Distearate phosphatidylethanolamine, sialic acid Octadecylamine, Methyl sialic acid stearic acid and sialic acid Eighteen kinds of acid.

7. The cationic liposomes according to claim 1, characterized in that, The cationic liposomes are spherical or near-spherical vesicle structures.

8. The cationic liposomes according to claim 1, characterized in that, The ratio of the cationic lipid, cofactor phospholipid, antitumor drug, and sialic acid derivative, by molar amount, is (1 / 2) * ... 50): (40) 89): 5: 5; The ratio of the antitumor drug to the hyaluronic acid-lipid derivative is 1:(0.5~10) by mass.

9. A method for preparing cationic liposomes as described in any one of claims 1 to 8, characterized in that, Includes the following steps: Step 1: Sialic acid-modified cationic liposomes are prepared by using cationic lipids, auxiliary phospholipids, antitumor drugs and sialic acid derivatives. The method includes, but is not limited to, any one or more of the following: ethanol injection method, modified ethanol injection method, thin film dispersion method, reverse evaporation method, high pressure homogenization method, ultrasonic dispersion method, microfluidic method and freeze-drying and reconstitution method. Step 2: Hyaluronic acid-lipid derivatives are inserted into sialic acid-modified cationic liposomes by incubation at 55-70℃ using a post-insertion method to obtain cationic liposomes that triple-target the tumor microenvironment.

10. The use of the cationic liposomes as described in any one of claims 1 to 8 in the preparation of tumor drugs.

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