Dual-drug co-loaded intelligent liposome and preparation method and use thereof

Abstract

The application provides a kind of double drug co-loading intelligent liposome and its preparation method and purposes, belong to the field of biological medicine.The double drug co-loading intelligent liposome includes the following raw materials: curcumin, near-infrared type photosensitizer, phospholipid A, sterol compound, polyethylene glycol modified targeting lipid;The polyethylene glycol modified targeting lipid is sequentially connected by phospholipid B, responsive link, polyethylene glycol and tumor targeting ligand.The application constructs double drug co-loading liposome system with active targeting and tumor microenvironment responsiveness, forms the efficient synergistic treatment strategy of "vascular normalization+PDT", realizes drug precise delivery and enrichment, has excellent ability to inhibit tumor growth, induce tumor vascular normalization and relieve tumor hypoxia, has synergistic effect in inhibiting HIF-1 alpha expression, significantly improves the treatment effect, and provides a new scheme for efficient and safe treatment of tumor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dual-drug co-loaded smart liposome, its preparation method, and its uses. Background Technology

[0002] Cancer is a major malignant disease threatening human life and health, with its incidence and mortality rates continuing to rise. Traditional cancer treatments (surgery, radiotherapy, chemotherapy) have limitations such as high invasiveness, strong side effects, and insufficient targeting, which has driven the development of new treatment technologies. Photodynamic therapy (PDT) is an emerging cancer treatment technology that has shown significant potential in cancer treatment due to its advantages such as high selectivity, minimally invasiveness, repeatability, and low toxicity, becoming an important alternative to traditional treatments.

[0003] The core of PDT treatment is the synergistic effect of photosensitizers, light of specific wavelengths, and molecular oxygen. Its mechanism of action is clear: In the tumor, after the photosensitizer is irradiated by a specific light source with a wavelength that matches its absorption, it is excited to an excited state. The excited photosensitizer transfers its energy to the surrounding molecular oxygen, converting the molecular oxygen into reactive oxygen species (ROS) with strong oxidizing activity. ROS can rapidly oxidize and damage the cell membranes, proteins, nucleic acids, and other biomolecules of tumor cells, thereby inducing tumor cell apoptosis and necrosis. At the same time, it can also destroy the vascular structure of tumor tissue, block the tumor's nutrient supply, and achieve the purpose of inhibiting tumor growth and eliminating tumor lesions.

[0004] However, the therapeutic efficiency of phototherapy (PDT) in clinical applications remains severely limited, with the core bottleneck lying in the unique characteristics of the tumor microenvironment. The tumor microenvironment is a complex internal environment for the survival and development of tumor cells, predominantly characterized by hypoxia and abnormal vascular structures. These two factors directly constrain the therapeutic effect of PDT. To address the challenges of hypoxia and abnormal vascular structures in PDT, vascular normalization strategies have received widespread attention. Vascular normalization strategies work by regulating tumor angiogenesis-related factors, restoring angiogenesis balance, and normalizing structurally abnormal and functionally dysfunctional tumor vessels. This increases blood perfusion and oxygen concentration in tumor tissue, effectively alleviating local tumor hypoxia. Simultaneously, it improves tumor tissue permeability, promoting the efficient delivery of photosensitizers, drugs, and other therapeutic factors to the tumor lesion.

[0005] Currently, the main strategy for inducing vascular normalization is to use anti-angiogenic targeted drugs. However, this strategy has a narrow effective window, and excessive anti-angiogenic therapy can lead to excessive pruning of tumor vessels, which can further reduce blood perfusion in tumor tissues and exacerbate tumor hypoxia. This not only fails to improve the efficacy of PDT (prophylactic tumor treatment) but may also accelerate tumor progression and metastasis, thus limiting its clinical application value.

[0006] In summary, while photodynamic therapy (PDT) is a promising novel cancer treatment technology, its clinical application is limited by the challenges of hypoxia and abnormal vascular structures in the tumor microenvironment. Existing strategies for inducing vascular normalization with anti-angiogenic targeted drugs suffer from inherent limitations, such as a narrow vascular normalization window and the potential to exacerbate tumor hypoxia, making it difficult to effectively overcome the treatment bottlenecks of PDT and fully realize its therapeutic advantages. Therefore, developing a safe, efficient, and controllable vascular normalization induction protocol to address the clinical treatment challenges of PDT and improve its cancer treatment efficacy has become a critical technical issue urgently needing to be solved in the field of cancer treatment. Summary of the Invention

[0007] The purpose of this invention is to provide a dual-drug co-loaded smart liposome, its preparation method, and its uses.

[0008] This invention provides a dual-drug co-loaded smart liposome, comprising the following raw materials: curcumin, near-infrared photosensitizer, phospholipid A, sterol compound, and polyethylene glycol-modified targeting lipid; wherein the polyethylene glycol-modified targeting lipid is formed by sequentially linking phospholipid B, responsive linker, polyethylene glycol, and tumor-targeting ligand.

[0009] Further, the mass ratio of curcumin to near-infrared photosensitizer is 1:2 to 1:5; the total mass ratio of curcumin and near-infrared photosensitizer to the total mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeting lipids is 1:2 to 1:5; and the mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeting lipids is 11 to 22: 1 to 5: 0.5 to 1.5.

[0010] Furthermore, the mass ratio of curcumin to near-infrared photosensitizer is 1:4; the ratio of the total mass of curcumin and near-infrared photosensitizer to the total mass of phospholipid A, sterol compounds and polyethylene glycol-modified targeting lipids is 1:3; and the mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeting lipids is 16.5:3.3:1.

[0011] Further, the near-infrared photosensitizer includes at least one of indocyanine green, IR780, dihydroporphyrin E6, sulfonated phthalocyanine, zinc phthalocyanine, and aluminum phthalocyanine; the phospholipid A and phospholipid B are independently selected from at least one of synthetic phospholipids and natural phospholipids; the sterol compound is selected from at least one of cholesterol, sitosterol, and stigmasterol; the polyethylene glycol has a molecular weight of 1000-5000; the responsive linker is selected from one of disulfide bonds, hydrazone bonds, borate ester bonds, and matrix metalloproteinase-sensitive peptide bonds; the tumor-targeting ligand is selected from one of folic acid, transferrin, RGD peptide, and nucleic acid aptamers.

[0012] Further, phospholipid A is a mixture of synthetic and natural phospholipids, wherein the mass ratio of synthetic to natural phospholipids is 8-15:3-7; phospholipid B is a synthetic phospholipid; the synthetic phospholipid is selected from at least one of 1,2-distearyl-sn-glycerol-3-phosphocholine, 1,2-distearyl-sn-glycerol-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, or 1,2-distearyl-sn-glycerol-3-phosphoethanolamine; the natural phospholipid is lecithin; and the polyethylene glycol has a molecular weight of 2000.

[0013] Furthermore, the phospholipid A is composed of synthetic phospholipids and natural phospholipids, wherein the mass ratio of synthetic phospholipids to natural phospholipids is 11:5.5; the polyethylene glycol-modified targeted lipid is composed of 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, a disulfide bond, polyethylene glycol with a molecular weight of 2000, and folic acid linked sequentially, and is DSPE-SS-PEG2000-FA.

[0014] This invention also provides a method for preparing dual-drug co-loaded smart liposomes, comprising the following steps: (1) Curcumin, near-infrared photosensitizer, phospholipid, sterol compounds, and polyethylene glycol-modified targeted lipids were mixed and added to an organic solvent, and a homogeneous oil phase solution was obtained after dissolution. (2) After evaporating the oil phase solution to remove the organic solvent, add water and stir to obtain the primary emulsion solution; (3) The colostrum solution was broken to obtain a uniform liposome suspension. The suspension was then filtered to obtain a dual-drug co-loaded smart liposome containing curcumin and near-infrared photosensitizer.

[0015] The present invention also provides the use of dual-drug co-loaded smart liposomes combined with a laser irradiation device in the preparation of a photodynamic therapy device for the prevention and / or treatment of tumors.

[0016] Furthermore, the photodynamic therapy device for preventing and / or treating tumors is a photodynamic therapy device for inhibiting tumor growth.

[0017] The present invention also provides the use of dual-drug co-loaded smart liposomes combined with a laser irradiation device in the preparation of photodynamic therapy devices for anti-tumor angiogenesis and / or induction of tumor angiogenesis normalization.

[0018] Furthermore, the laser is near-infrared light, and the wavelength of the near-infrared light is 700~900nm.

[0019] Furthermore, the tumors include breast cancer, lung cancer, stomach cancer, colorectal cancer, liver cancer, melanoma, oral cancer, or pancreatic cancer.

[0020] This invention constructs a dual-drug co-loaded liposome system with both active targeting and tumor microenvironment responsiveness, achieving precise delivery and enrichment of curcumin and a near-infrared photosensitizer (indocyanine green) at the tumor site, improving drug utilization efficiency. Furthermore, curcumin induces tumor angiogenesis normalization, synergistically enhancing anticancer efficacy through indocyanine green-mediated photodynamic therapy (PDT). Raw material ratio screening experiments demonstrated that the optimal mass ratio of curcumin to indocyanine green is 1:4, and the optimal ratio of the total mass of curcumin and indocyanine green to the total mass of phospholipid A, sterol compounds, and polyethylene glycol-modified targeting lipids is 1:3. This optimal ratio yields liposomes with the smallest average particle size and the highest encapsulation efficiency of curcumin and indocyanine green.

[0021] In vitro and in vivo experiments have demonstrated that curcumin-indocyanine green co-loaded liposomes exhibit slow drug release in normal physiological environments, but rapid drug release under high GSH conditions in the tumor microenvironment, enabling precise and controllable drug release and increasing local drug concentration. Simultaneously, the curcumin-indocyanine green co-loaded liposomes possess active targeting properties, significantly improving drug accumulation efficiency at tumor sites and reducing drug toxicity. This addresses the shortcomings of curcumin, such as poor water solubility, low bioavailability, and rapid in vivo metabolism, as well as the insufficient targeting and easy accumulation of indocyanine green in normal tissues, thus improving the safety and efficacy of curcumin and indocyanine green administration. Furthermore, in combination with phototherapy, the curcumin-indocyanine green co-loaded liposomes demonstrated excellent abilities to inhibit tumor growth, suppress tumor angiogenesis, induce tumor angiogenesis normalization, and alleviate tumor hypoxia, exhibiting a synergistic effect in reducing the expression level of the hypoxia / angiogenesis-related protein HIF-1α.

[0022] This invention forms a highly efficient synergistic treatment strategy of "vascular normalization + PDT", which significantly improves the treatment effect and provides a brand-new solution for efficient and safe treatment of tumors.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 The images show the particle size (A) and potential (B) of Blank lip and CI lip detected by dynamic light scattering; and the TEM image of CI lip (C).

[0026] Figure 2 The responsive release results of CI lip are: (A) the release result of Cur; (B) the release result of ICG.

[0027] Figure 3 It is a fluorescence detection method for the distribution of free ICG, ICG lip, and ICG lip-FA in the heart, liver, spleen, lung, kidney, and tumors of mice.

[0028] Figure 4 These are the results of CCK-8 assay for the toxicity of L, Cur lip, ICG lip, ICG lip+L, CI lip, and CI lip+L to 4T1 cells, where L represents light exposure.

[0029] Figure 5 The results of the in vitro angiogenesis experiment of HUVEC cells are as follows: (A) Fluorescent image of capillary tubular structure formation (bar=50μm); (B) Quantitative statistical graph of tubular structure formation based on image analysis (n=3).

[0030] Figure 6 The effects of Control, Curlip, ICGlip, CIlip, and CIlip+L on HIF-1α expression are as follows: (A) Western Blot analysis of HIF-1α expression level; (B) Relative expression level of HIF-1α protein.

[0031] Figure 7 The results of Western Blot analysis of vascular-related factors are as follows: (A) Statistical results of gray values ​​of VEGFA protein; (B) Statistical results of gray values ​​of VEGFR2 protein; (C) WB bands of vascular-related factors (n=3). p<0.05, p<0.01, p<0.001.

[0032] Figure 8 The results of HUVEC cell migration experiments are as follows: (A) Cell migration results at 0h and 24h (bar=200μm); (B) Quantitative statistical graph of cell migration rate based on image analysis (n=3). p<0.05, p<0.01, p<0.001.

[0033] Figure 9 The results are the immunofluorescence results (bar=50μm) of NO generation for Control, Cur lip, ICG lip+L, CI lip, and CI lip+L.

[0034] Figure 10The results are immunofluorescence results of vascular normalization-related factors (MVD, α-SMA / CD31, NG2 / CD31, Collagen Ⅳ / CD31) of Control, Cur lip, ICG lip+L, CI lip, and CI lip+L.

[0035] Figure 11 This is a graph showing the changes in body weight of mice in the Control, L, Cur lip, ICG lip, ICG lip+L, CI lip, and CI lip+L groups.

[0036] Figure 12 These are the blood biochemical results of mice in the Control, L, Cur lip, ICG lip, ICG lip+L, CI lip, and CI lip+L groups.

[0037] Figure 13 The results are H&E staining of various organs (heart, liver, spleen, lung, and kidney) of mice in the Control, L, Cur lip, ICG lip, ICG lip+L, CI lip, and CI lip+L groups. Detailed Implementation

[0038] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0039] Example 1: Preparation and characterization of curcumin-indocyanine green co-loaded liposomes 1. Preparation of curcumin-indocyanine green co-loaded liposomes (1) Weigh 1.47 mg curcumin (Cur, purity ≥ 98%), 6.2 mg indocyanine green (ICG, purity ≥ 95%), 11 mg 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 5.5 mg lecithin, 3.3 mg cholesterol and 1 mg DSPE-SS-PEG2000-FA (DSPE: 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine; PEG2000: polyethylene glycol with a molecular weight of 2000; FA: folic acid), and place them in a 50 mL round-bottom flask; add 2 mL of a mixed solution of chloroform and 4 mL of methanol, and sonicate (power 100 W, time 5 min) until completely dissolved to obtain a homogeneous oil phase solution; (2) Place the round-bottom flask containing the oil phase solution on a rotary evaporator and evaporate it at 40°C and 60 rpm for 2-3 hours. When the organic solvent has completely evaporated and the product forms a uniform film on the inner wall of the round-bottom flask, add 3 mL of ultrapure water to the round-bottom flask and stir and hydrate it in a water bath at 40°C for 30 minutes to obtain the initial emulsion solution. (3) Place the colostrum solution in an ultrasonic cell disruptor and sonicate at 195W for 5 min (5 s sonication, 1 s pause) to obtain a uniform liposome suspension; then filter the suspension through 0.45 μm and 0.22 μm polycarbonate membranes 2-3 times to remove unloaded free drugs, thus obtaining the target-responsive curcumin-indocyanine green co-loaded liposome (CI lip), which is stored at 4℃ protected from light for later use.

[0040] 2. Characterization of curcumin-indocyanine green co-loaded liposomes (1) Characterization methods The particle size and potential of CI lip were determined by dynamic light scattering (DLS); the surface morphology of CI lip-FA was observed by transmission electron microscopy (TEM).

[0041] (2) Characterization results like Figure 1 As shown in A~B, the average particle size of CI lip is 131.7±2.53nm, the polydispersity index (PDI) is ≤0.2, and the surface potential is -50.17±0.51 mV, indicating that the formulation is uniformly dispersed and has good stability.

[0042] like Figure 1 The TEM image shown in C shows that CI lip has a uniform spherical structure with a particle size of 80 nm. The membrane structure is clear and there is no obvious aggregation.

[0043] Comparative Example 1: Preparation of Curcumin Liposomes Referring to Example 1, the only difference is that indocyanine green is not added, resulting in curcumin liposomes.

[0044] Comparative Example 2: Preparation of Indocyanine Green Liposomes Referring to Example 1, the only difference is that curcumin is not added, resulting in indocyanine green liposomes (ICG lip).

[0045] Experiment Example 1: Raw Material Proportion Screening Experiment 1. Experimental Methods Referring to Example 1, firstly, with the total mass ratio of curcumin and indocyanine green to the total mass of lipids fixed at 1:2, curcumin-indocyanine green co-loaded liposomes with mass ratios of 1:2, 1:3, 1:4, and 1:5 were prepared, and their particle size and encapsulation efficiency were tested. Then, with the mass ratio of curcumin to indocyanine green fixed at 1:4, curcumin-indocyanine green co-loaded liposomes with total mass ratios of 1:3, 1:4, and 1:5 were prepared, and their particle size and encapsulation efficiency were tested.

[0046] The total lipid mass includes the total mass of DPPC, lecithin, cholesterol, and DSPE-SS-PEG2000-FA.

[0047] 2. Experimental Results As shown in Table 1, when the ratio of the total mass of curcumin and indocyanine green to the total mass of lipids is 1:2, the particle size of the resulting liposomes first decreases and then increases as the mass ratio of curcumin and indocyanine green decreases. The encapsulation efficiency of curcumin and indocyanine green also shows a trend of first increasing and then decreasing. Among them, when the mass ratio of curcumin and indocyanine green is 1:4, the particle size of the resulting curcumin-indocyanine green co-loaded liposomes is the smallest, at 142.6 nm, and the encapsulation efficiency of curcumin and indocyanine green is the largest, at 94.5% and 90.2%, respectively.

[0048] When the mass ratio of curcumin to indocyanine green was 1:4, the particle size and encapsulation efficiency of curcumin and indocyanine green showed a trend of first increasing and then decreasing as the ratio of the total mass of curcumin and indocyanine green to the total mass of lipids decreased. Among them, when the ratio of the total mass of curcumin and indocyanine green to the total mass of lipids was 1:3, the particle size of the obtained curcumin-indocyanine green co-loaded liposomes was the smallest, at 131.7 nm, and the encapsulation efficiency of curcumin and indocyanine green was the largest, at 97.3% and 93.8%, respectively.

[0049] Table 1. Particle size and encapsulation efficiency of different curcumin-indocyanine green co-loaded liposomes Based on the particle size and encapsulation efficiency test results, the preferred mass ratio of curcumin to indocyanine green was determined to be 1:4, and the preferred ratio of the total mass of curcumin and indocyanine green to the total mass of lipids was 1:3. Under this preferred ratio, the average particle size of liposomes was the smallest, and the encapsulation efficiency of curcumin and indocyanine green was the highest.

[0050] Experimental Example 2: In vitro responsive drug release experiment 1. Experimental Methods Drug release test: (1) Inner aqueous phase: The prepared CI lip nanoparticles were dispersed in ultrapure water to prepare a CI lip solution with a Cur concentration of 1 mg / mL; outer aqueous phase: water with pH=7.4 (blank group) and an aqueous solution containing 5 mM glutathione (GSH) with pH=7.4 (GSH group), wherein both the water and the aqueous solution containing GSH contained 0.5 wt% Tween 80; (2) Put 1 mL of the inner aqueous phase into the prepared dialysis bag (1000 Da), seal both ends of the dialysis bag with a rope, put it into a centrifuge tube (15 mL capacity) containing 10 mL of the outer aqueous phase, and place it in a shaker at 37°C and shake at 80 r / min. (3) At 2h, 4h, 6h, 8h, 10h and 24h, 1mL of external aqueous phase was taken from each group and 1mL of the same external aqueous phase was added. The taken external aqueous phase was freeze-dried, then reconstituted with 200μL methanol and centrifuged at 12000r / min for 10min. The supernatant was taken and its absorbance value at 427nm and 780nm wavelength was measured using an enzyme-linked immunosorbent assay (ELISA) reader. (4) Calculate its cumulative release rate according to formula (1): Cumulative release rate (%) = (C n ×V1+∑(C n-1 ×V)) / M×100% (1; Where, C n (mg / mL): Mass concentration of Cur / ICG in the release solution at the nth sampling; V1 (mL): Total volume of buffer solution; V (mL): Volume of replacement solution; M (mg): Drug loading of nanoparticles.

[0051] 2. Experimental Results like Figure 2 As shown, the cumulative release rates of Cur and ICG in the blank group were 28.6% and 31.2% in 24 hours, respectively; while the cumulative release rates of Cur and ICG in the GSH group were 82.5% and 85.7% in 24 hours, respectively, and the release rate accelerated significantly within 6 hours. This indicates that CI lip releases drugs slowly in the normal physiological environment, which can avoid premature drug leakage, and can release drugs rapidly under the high GSH conditions of the tumor microenvironment, achieving precise and controllable release and increasing local drug concentration.

[0052] Experiment Example 3: In vivo biodistribution experiment 1. Experimental Methods ICG was used instead of Cur to prepare ICG lip' (without DSPE-SS-PEG2000-FA) and ICG lip-FA according to the preparation method of CI lip.

[0053] Establishment of Balb / c mouse orthotopic mammary gland model: After purchasing Balb / c mice, they were acclimatized and fed for one week. The mice were then anesthetized, and the hair near the left second mammary pad was shaved. Healthy 4T1 cells in logarithmic growth phase were digested, resuspended in serum-free culture medium, and counted. Cells were then cultured at a concentration of 3 × 10⁻⁶ cells / mL. 5 100 μL of each sample was injected subcutaneously into the second mammary gland pad of mice.

[0054] When the tumor grows to 200mm 3Mice were randomly divided into three groups and injected with free ICG, ICGlip', and ICGlip-FA via the tail vein, respectively. The concentration of ICG was 100 μg / mL, and each mouse was injected with 100 μL. The mice were sacrificed at 10 h, and the heart, liver, spleen, lungs, kidneys, and tumors were dissected. After being gently rinsed with PBS, the organs and tumor tissues were fixed with 4% paraformaldehyde and placed in a small animal in vivo imaging system for imaging. The fluorescence intensity of ICG in each tissue was analyzed.

[0055] 2. Experimental Results like Figure 3 As shown, the fluorescence intensity of tumor tissue in the ICG lip-FA group was 2.1 times that of the free ICG group, and the fluorescence intensity in the heart, liver, spleen, lung, and kidney tissues was significantly lower than that in the free ICG group. This indicates that the FA-modified liposome CI lip of the present invention can significantly improve the enrichment efficiency of drugs in tumor sites, reduce the distribution in normal tissues, and reduce the toxic side effects of drugs through receptor-mediated active targeting.

[0056] Experiment Example 4: In vitro cytotoxicity experiment 1. Experimental Methods Collect 4T1 cells in good growth condition and prepare 5×10⁻⁶ cells. 4 Cell suspension at 100 μL / mL was seeded into 96-well plates. After 24 h, a series of concentrations of Cur lip (4 μM, 8 μM, 12 μM, 16 μM, 20 μM based on Cur concentration), ICG lip (8 μM, 16 μM, 24 μM, 32 μM, 40 μM based on ICG concentration), ICG lip+L (8 μM, 16 μM, 24 μM, 32 μM, 40 μM based on ICG concentration), CI lip (4 μM, 8 μM, 12 μM, 16 μM, 20 μM based on Cur concentration), and CI lip+L (4 μM, 8 μM, 12 μM, 16 μM, 20 μM based on Cur concentration) were diluted with fresh culture medium. After 24 h, the culture medium was discarded, and 100 μL of the light-exposed (L) experimental group was added. After PBS, near-infrared laser irradiation (808nm, 0.75W•cm) was performed. -2 After 5 min), 100 μL of fresh culture medium was added to each group and incubated in an incubator for 4 h. The CCK-8 test solution was diluted 10 times with fresh culture medium, and 100 µL of diluent was added to each well. The cells were incubated in a cell culture incubator for another 2 h. The absorbance value of each well at 450 nm was then detected by an ELISA reader, and the cell viability was calculated.

[0057] 2. Experimental Results like Figure 4 As shown in Table 2, the CI lip+L group showed an effect on the IC50 of 4T1 cells. 50The concentration was 6.56 μM, and the 24-hour cell survival rate was only 17.01 ± 1.41%, significantly lower than other groups; indicating that Curlip combined with PDT (CIlip+L) can significantly enhance the killing ability against breast cancer cells.

[0058] Table 2. Cell viability of each experimental group at 24 h Experiment Example 5: In vivo anti-tumor experiment 1. Experimental Methods A Balb / c mouse orthotopic breast cancer model was constructed (same as in Example 3), and the tumor was allowed to grow to 100 mm. 3 Mice were randomly divided into 7 groups (Control group, L group, Cur lip group, ICG lip group, ICG lip+L group, CI lip group, and CI lip+L group), with 5 mice in each group. The drugs were administered via tail vein injection. The Cur dose was 5 mg / kg, and the ICG dose was 21 mg / kg. Administration was repeated every 2 days for 16 consecutive days. In the light-exposed (L) experimental groups, the tumor site was irradiated with an 808 nm near-infrared laser at a power of 0.75 W·cm² 2 hours after each administration. -2 Irradiation time was 5 minutes (PDT treatment); tumor volume was measured every 2 days, and mice were sacrificed after 16 days, tumor weight was measured, and tumor inhibition rate was calculated.

[0059] 2. Experimental Results As shown in Table 3, the tumor inhibition rate of the CI lip+L group was 64.92±10.66%, which was significantly higher than that of other treatment groups, indicating that the combined treatment of Curlip and PDT can significantly inhibit tumor growth in vivo.

[0060] Table 3. Tumor inhibition rates of different treatment groups Experiment Example 6: Vascular Normalization Experiment 1. Experimental Methods (1) Tubular vessel formation experiment: Mix the matrix gel and dual-free DMEM-F12 medium at a ratio of 1:10 on ice. Take 100 μL of the mixed liquid and add it to the transwell chamber. Incubate at 37°C for 2-3 h until the matrix gel solidifies. Collect HUVEC cells in good growth condition and resuspend them in DMEM-F12 complete medium containing 10% FBS and 1% antibiotics to a concentration of 3×10⁻⁶. 5Cell suspension at 100 μL / mL was added to the upper chamber of a transwell, followed by complete culture medium containing Curl lip (40 μM Curl), ICG lip+L (80 μM ICG), CI lip (40 μM Curl + 80 μM ICG), and CI lip+L (40 μM Curl + 80 μM ICG), respectively. Complete culture medium without the drug was used as a control. After culturing for 24 h, the cell and drug suspensions in the chamber were aspirated. The light-treated groups were then exposed to 808 nm light (0.75 W·cm²). -2 After 5 min), each group was added to culture medium and cultured for 4 h. Then, the chambers were washed twice with PBS. 0.5 mL of Calcein AM cell viability and cytotoxicity detection working solution was added to the lower chamber and incubated at 37 °C in the dark for 30 min. After washing twice with PBS, the chambers were observed and photographed under a fluorescence microscope.

[0061] (2) Cell migration experiment: using 4×10 5 HUVEC cells in logarithmic growth phase were seeded into 12-well plates at a density of cells / mL. When the cells reached approximately 80% confluence, the old culture medium was discarded. After washing 1-2 times with PBS, a 200 μL pipette tip was used to make a vertical slash from top to bottom in the monolayer of cells in each well. The cells were washed twice with PBS to remove the slashed cells, and then photographed under an inverted microscope, recorded as 0h. Subsequently, different concentrations of 2% FBS medium containing Curlip (40 μM Cur), ICGlip+L (80 μM ICG), CIlip (40 μM Cur + 80 μM ICG), and CIlip+L (40 μM Cur + 80 μM ICG) were added, and the cells were cultured for another 24h. After that, the drug-containing medium was aspirated from each group. The light-treated group was treated with 500 μL PBS and then irradiated with a laser (808 nm, 0.75 W·cm⁻¹). -2 After 5 min), each group was added to 1 mL of culture medium and cultured for 4 h. The cells were then washed twice with PBS and photographed under an inverted microscope to observe cell migration.

[0062] (3) Western Blot detection: HUVEC cells in good growth condition were loaded into each well at a density of 4 × 10⁶ cells / well. 5 Cells were seeded at a density of [number] cells per well in 6-well plates and treated with the corresponding drugs for 24 h: Curl lip (40 μM Curl), ICG lip+L (80 μM ICG), CI lip (40 μM Curl + 80 μM ICG), and CI lip+L (40 μM Curl + 80 μM ICG). After 24 h, the light-treated group was treated with 808 nm light (0.75 W•cm²). -2The membrane was cultured for 4 hours (5 min each time). Proteins were then extracted, and electrophoresis, membrane transfer, and blocking were performed. After blocking, the membrane was washed three times with TBST for 5 min each time. The membrane was then incubated overnight at 4°C with β-actin antibody and hypoxia-inducible factor-1α (HIF-1α) antibody, respectively. The corresponding antibodies were recovered and stored at -20°C. The membrane was washed three times with TBST for 5 min each time, and then incubated with horseradish peroxidase (HRP) at room temperature for 90 min. The antibodies were discarded, and the membrane was washed three times with TBST for 5 min each time. The membrane was then developed. Western blotting was used to detect the expression of VEGFA, VEGFR2, and HIF-1α proteins.

[0063] (4) In vivo experiments: Tumor tissues from mice sacrificed 16 days after Experiment Example 5 were dissected, fixed with 4% paraformaldehyde, embedded in paraffin, and stained with NO fluorescent probe (DAR-1) immunofluorescence. The expression of CD31, α-SMA, and Collagen IV in the tumor tissues was detected by immunofluorescence (DAPI) staining. CD31 labeled tumor vascular endothelial cells, α-SMA labeled smooth muscle cells, NG2 labeled pericytes, and Collagen IV labeled the basement membrane. The microvessel density (MVD) was assessed by CD31 / DAPI, the vascular maturity was assessed by α-SMA / CD31, the pericyte coverage was analyzed by NG2 / CD31, and the basement membrane integrity was assessed by Collagen IV / CD31.

[0064] 2. Experimental Results like Figure 5 As shown in A~B, compared with the blank group (tubular structure formation rate 100.00±5.88%), the Cur lip group significantly reduced the formation of capillary networks, with a tubular structure formation rate of 33.21±4.83%. The ICG lip+L group also showed angiogenesis inhibition, with a tubular structure formation rate of 25.46±3.83%. The CI lip+L group showed the most significant angiogenesis inhibition, with a tubular structure formation rate of only 10.33±2.30%. This indicates that Cur lip combined with PDT treatment can significantly inhibit the formation of vascular tubular structures in HUVEC cells and has a significantly enhanced anti-tumor angiogenesis effect.

[0065] like Figure 6As shown in A~B, the HIF-1α protein level in the CI lip+L group was significantly lower than that in the Cur lip group and the ICG lip+L group. In addition, the HIF-1α protein level in the ICG lip+L group was higher than that in the blank group. This indicates that Cur lip reversed the PDT-induced HIF-1α upregulation effect in combination with PDT (ICG lip+L), which proves that the combination therapy of Cur lip and PDT in the CI lip+L system has a synergistic effect on inhibiting abnormal tumor angiogenesis. At the same time, Cur lip can also improve the therapeutic effect of PDT by regulating the hypoxic microenvironment.

[0066] like Figure 7 As shown in A~C, compared with the blank group, the Cur lip, ICG lip+L, CI lip and CI lip+L groups can significantly downregulate the protein expression of VEGFA and VEGFR2 in HUVEC cells. Figure 8 The results of HUVEC cell migration experiments A-B show that, compared with the blank group, Cur lip, ICG lip+L, CI lip, and CI lip+L groups all significantly inhibited HUVEC cell migration; among them, the scratch healing rate of CI lip+L group was only 15.35±0.07%, which was much lower than that of the blank group, indicating that CI lip can effectively inhibit tumor angiogenesis.

[0067] like Figure 9 As shown, the distribution of NO around tumor vessels was significantly increased in the Cur lip group, CI lip group, and CI lip+L group. CI lip+L had a significant effect on restoring and increasing the NO concentration around the vessels, indicating that CI lip+L can induce tumor angiogenesis normalization by regulating NO concentration.

[0068] like Figure 10 As shown, the tumor microvessel density (MVD) in the CI lip+L group was significantly reduced, the distribution of α-SMA and Collagen IV around the blood vessels was increased, and the vascular structure was more regular. This indicates that the combined treatment of Curlip and PDT can significantly improve the structure and function of tumor blood vessels and induce tumor vascular normalization by reducing MVD, promoting the distribution of smooth muscle cells and pericytes around tumor blood vessels, and enhancing the integrity of the basement membrane.

[0069] The above experimental results indicate that CI lip+L can significantly inhibit the formation of vascular tubular structures in HUVEC cells, significantly downregulate the protein expression of HIF-1α, VEGFA, and VEGFR2 in HUVEC cells, and has a synergistic effect in reducing the protein expression level of HIF-1α. It can effectively inhibit tumor angiogenesis, significantly increase the NO concentration around tumor blood vessels, effectively improve the structure and function of tumor blood vessels, and has a significant effect on inducing tumor angiogenesis normalization.

[0070] Experimental Example 7: In vivo safety evaluation 1. Experimental Methods A Balb / c mouse orthotopic breast cancer model was constructed (same as in Example 3), and the tumor was allowed to grow to 100 mm. 3 Mice were randomly divided into 7 groups (Control group, L group, Cur lip group, ICG lip group, ICG lip+L group, CI lip group, and CI lip+L group), with 5 mice in each group. The drugs were administered via tail vein injection. The Cur dose was 5 mg / kg, and the ICG dose was 21 mg / kg. Administration was repeated every 2 days for 16 consecutive days. In the light-exposed (L) experimental groups, the tumor site was irradiated with an 808 nm near-infrared laser at a power of 0.75 W·cm² 2 hours after each administration. -2 Irradiation time was 5 min (PDT treatment); mice were sacrificed 16 days after treatment; mouse weight changes were monitored during drug administration, and weight was measured every 2 days; after treatment, mouse serum was collected to detect liver function (ALT, AST, ALP) and kidney function (Urea, Crea) indicators; heart, liver, spleen, lung and kidney tissues were separated, H&E staining was performed, and pathological changes were observed.

[0071] 2. Experimental Results like Figure 11 As shown, the body weight of mice in each group did not decrease significantly during treatment; Figure 12 As shown, the serum ALT, AST, ALP, Urea, and Crea levels in mice were all within the normal range; Figure 13 As shown, H&E staining of major organs revealed intact tissue structure with no obvious inflammation, necrosis, or other pathological damage, indicating that CI lip has good biocompatibility and no obvious toxic side effects.

[0072] In summary, this invention co-loads curcumin and indocyanine green into folic acid-modified GSH-responsive liposomes. Curcumin induces tumor angiogenesis normalization, synergistically enhancing the anti-breast cancer efficacy through ICG-mediated PDT, improving the bioavailability of curcumin and the targeting of ICG, thus achieving controlled drug release. Raw material screening experiments demonstrated that the optimal mass ratio of curcumin to indocyanine green is 1:4, and the optimal ratio of the total mass of curcumin and indocyanine green to the total mass of lipids is 1:3. Under these optimal ratios, the liposomes have the smallest average particle size and the highest encapsulation efficiency of curcumin and indocyanine green. In vitro and in vivo experiments demonstrated that CI lip releases the drug slowly in normal physiological environments but rapidly under high GSH conditions in the tumor microenvironment, achieving precise and controllable drug release and increasing local drug concentration. Simultaneously, CI lip exhibits active targeting, significantly improving drug accumulation efficiency at the tumor site and reducing drug toxicity. In combination therapy, the CI lip+L group significantly reduced the IC50 of 4T1 cells. 50 (6.56 μM) improved the tumor inhibition rate (64.92 ± 10.66%), demonstrating excellent ability to inhibit tumor growth. Tumor angiogenesis normalization experiments showed that CI lip+L significantly inhibited the formation of vascular tubular structures in HUVEC cells, downregulated the protein expression of HIF-1α, VEGFA, and VEGFR2, and had a synergistic effect in reducing HIF-1α protein expression, effectively inhibiting tumor angiogenesis. Simultaneously, it significantly increased the NO concentration around tumor blood vessels, effectively improving the structure and function of tumor blood vessels and playing a significant role in inducing tumor angiogenesis normalization. This invention also addresses the shortcomings of curcumin, such as poor water solubility, low bioavailability, and rapid in vivo metabolism, as well as the problems of insufficient ICG targeting and easy accumulation in normal tissues, improving the safety and efficacy of both drugs.

Claims

1. A dual-drug co-loaded smart liposome, characterized in that, It includes the following raw materials: Curcumin, near-infrared photosensitizer, phospholipid A, sterol compounds, and polyethylene glycol-modified targeted lipids; the polyethylene glycol-modified targeted lipids are composed of phospholipid B, responsive linkages, polyethylene glycol, and tumor-targeting ligands linked sequentially.

2. The dual-drug co-loaded smart liposome according to claim 1, characterized in that, The mass ratio of curcumin to near-infrared photosensitizer is 1:2 to 1:5; the total mass ratio of curcumin and near-infrared photosensitizer to the total mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeting lipids is 1:2 to 1:5; the mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeting lipids is 11 to 22: 1 to 5: 0.5 to 1.

5.

3. The dual-drug co-loaded smart liposome according to claim 2, characterized in that, The mass ratio of curcumin to near-infrared photosensitizer is 1:4; the total mass ratio of curcumin and near-infrared photosensitizer to the total mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeted lipids is 1:3; the mass ratio of phospholipid A, sterol compounds and polyethylene glycol-modified targeted lipids is 16.5:3.3:

1.

4. The dual-drug co-loaded smart liposome according to any one of claims 1 to 3, characterized in that, The near-infrared photosensitizer includes at least one of indocyanine green, IR780, dihydroporphyrin E6, sulfonated phthalocyanine, zinc phthalocyanine, and aluminum phthalocyanine; the phospholipid A and phospholipid B are independently selected from at least one of synthetic phospholipids and natural phospholipids; the sterol compound is selected from at least one of cholesterol, sitosterol, and stigmasterol; the polyethylene glycol has a molecular weight of 1000-5000; the responsive linker is selected from one of disulfide bonds, hydrazone bonds, borate ester bonds, and matrix metalloproteinase-sensitive peptide bonds; the tumor-targeting ligand is selected from one of folic acid, transferrin, RGD peptide, and nucleic acid aptamers.

5. The dual-drug co-loaded smart liposome according to claim 4, characterized in that, Phospholipid A is a mixture of synthetic and natural phospholipids, wherein the mass ratio of synthetic to natural phospholipids is 8-15:3-7; Phospholipid B is a synthetic phospholipid; the synthetic phospholipid is selected from at least one of 1,2-distearyl-sn-glycerol-3-phosphate choline, 1,2-distearyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline, or 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine; the natural phospholipid is lecithin; the polyethylene glycol has a molecular weight of 2000.

6. The dual-drug co-loaded smart liposome according to claim 5, characterized in that, Phospholipid A is composed of synthetic phospholipids and natural phospholipids, with a mass ratio of 11:5.

5. The polyethylene glycol-modified targeted lipid is composed of 1,2-distearate-sn-glycerol-3-phosphatidylethanolamine, a disulfide bond, polyethylene glycol with a molecular weight of 2000, and folic acid linked sequentially, and is DSPE-SS-PEG2000-FA.

7. The method for preparing dual-drug co-loaded smart liposomes according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Curcumin, near-infrared photosensitizer, phospholipid, sterol compounds, and polyethylene glycol-modified targeted lipids were mixed and added to an organic solvent, and a homogeneous oil phase solution was obtained after dissolution. (2) After evaporating the oil phase solution to remove the organic solvent, add water and stir to obtain the primary emulsion solution; (3) The colostrum solution was broken to obtain a uniform liposome suspension. The suspension was then filtered to obtain a dual-drug co-loaded smart liposome containing curcumin and near-infrared photosensitizer.

8. The use of the dual-drug co-loaded smart liposome according to any one of claims 1 to 6 in combination with a laser irradiation device in the preparation of a photodynamic therapy device for the prevention and / or treatment of tumors.

9. The use of the dual-drug co-loaded smart liposome according to any one of claims 1 to 6 in combination with a laser irradiation device in the preparation of a photodynamic therapy device for anti-tumor angiogenesis and / or induction of tumor angiogenesis normalization.

10. The use according to any one of claims 8 to 9, characterized in that, The tumors include breast cancer, lung cancer, stomach cancer, colorectal cancer, liver cancer, melanoma, oral cancer, or pancreatic cancer.

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