Cis-platinum-indocyanine green composite liposome for synergistically inducing pyroptosis of tumor cells as well as preparation and application of cisplatin-indocyanine green composite liposome

Abstract

The invention discloses a cisplatin-indocyanine green composite liposome for synergistically inducing pyroptosis of tumor cells as well as preparation and application of the cisplatin-indocyanine green composite liposome. According to the cisplatin-indocyanine green composite liposome, a unique bilayer structure of the liposome is utilized, hydrophilic cisplatin is innovatively encapsulated in an inner water phase, and hydrophobic / amphiphilic ICG is embedded into a lipid hydrophobic layer. The inside and outside separated medicine carrying mode is high in medicine carrying efficiency, and more importantly, differentiated release kinetics and a synergistic effect mode can be realized. Compared with the prior art, the preparation method disclosed by the invention has the advantages that the biocompatibility and the deep tumor permeability are obviously improved, the efficiency of specifically inducing pyroptosis and the immune activation intensity are obviously enhanced, finally, the prior art is surpassed, and particularly, the outstanding clinical transformation potential is shown in the aspects of activating anti-tumor immunity and inhibiting far-end metastasis.

Description

(I) Technical Field

[0002] This invention belongs to the field of biomedical technology, and in particular relates to a cisplatin-indocyanine green complex liposome for synergistic induction of tumor cell pyroptosis, its preparation method and application. (II) Background Technology

[0004] Head and neck squamous cell carcinoma (HNSCC) is one of the major challenges in clinical oncology treatment. It is predicted that by 2030, the global annual incidence will exceed one million cases. As a major type of head and neck cancer, HNSCC still has a local recurrence and distant metastasis rate exceeding 50% even with current treatments, highlighting a critical weakness in the current treatment arsenal. Conventional treatments have limitations in many aspects: platinum-based chemotherapy, represented by cisplatin, has some efficacy but is limited by severe systemic toxicity and acquired resistance developed by tumor cells; radiotherapy often fails to prevent tumor metastasis; even emerging immune checkpoint inhibitor therapies suffer from uneven response rates. This grim clinical situation urgently requires innovative treatment methods that can simultaneously achieve highly efficient local tumor clearance and effective activation of the systemic immune system, while minimizing toxic side effects.

[0005] Indocyanine green (ICG), an FDA-approved near-infrared photosensitizer, combines photodynamic and photothermal therapy functions, and its clinical safety has been widely validated. In terms of treatment strategy, conventional intravenous injection combined with minimally invasive local laser irradiation offers high clinical compatibility and operability, particularly suitable for head and neck malignancies with complex anatomical structures requiring precise targeting. However, ICG alone has key drawbacks such as limited targeting, rapid metabolism, insufficient photostability, uneven laser penetration depth in complex tissues, and a narrow treatment window. Improvements in dosage form and optimization of irradiation techniques are needed to enhance its efficacy and reliability.

[0006] Recent breakthroughs in programmed cell death research suggest that pyroptosis is a promising new target for cancer therapy. Unlike apoptosis, pyroptosis is a form of cell death mediated by Gasdermin family proteins and accompanied by a strong inflammatory response. This process initiates a series of chain immune responses: NLRP3 inflammasome activation subsequently activates caspase-1; activated caspase-1 cleaves Gasdermin D (GSDMD) to form pores in the cell membrane and converts precursors of interleukin-1β (IL-1β) and IL-18 into mature inflammatory factors. Subsequently, damage-associated molecular patterns (DAMPs) released by the cell, such as ATP, high-mobility group box 1 (HMGB1), and calreticulin, collectively create a pro-inflammatory microenvironment that effectively promotes dendritic cell (DC) activation and recruits cytotoxic T lymphocytes (CTLs), thereby significantly enhancing the anti-tumor immune response. Preclinical studies have even shown that pyroptosis in only a small number of tumor cells can significantly improve the tumor immune microenvironment, suggesting that it may play a key role as an immune "amplifier" in cancer treatment.

[0007] However, there are still key bottlenecks in translating the powerful mechanism of pyroptosis into clinical therapy: existing delivery technologies are unable to achieve tumor-targeted delivery and controllable release of pyroptosis inducers, resulting in high off-target toxicity risks and limited efficacy. Common problems include (1) lack of tumor specificity, which easily induces excessive systemic inflammation; (2) low delivery efficiency, with insufficient accumulation of active ingredients at the tumor site; and (3) inability to respond to the tumor microenvironment to achieve intelligent activation, resulting in poor controllability.

[0008] From a translational medicine perspective, the ideal design of novel antitumor agents should integrate multiple clinical advantages. Liposomes, as classic drug carriers, can effectively overcome the pharmacokinetic challenges faced by free cisplatin, significantly prolonging its blood circulation time while substantially reducing its dose-limiting nephrotoxicity. Existing nanospheres typically encapsulate two drugs in a hydrophobic polymer matrix. Drug release depends on material degradation or matrix disintegration caused by photothermal processes. The drug release behavior is synchronous but difficult to precisely control, and the therapeutic mechanism is limited to direct cell killing, failing to systematically activate antitumor immunity.

[0009] Therefore, strategically combining mature clinical components with innovative nanotechnology platforms provides a promising translational pathway for developing novel therapies for head and neck squamous cell carcinoma. (III) Summary of the Invention

[0011] The purpose of this invention is to provide a cisplatin-indocyanine green complex liposome for synergistic induction of tumor cell pyroptosis, its preparation method, and its applications. This invention provides an innovative formulation that utilizes a liposome with a specific composition to synergistically deliver cisplatin and ICG, specifically and efficiently activating the NLRP3 / Caspase-1 / GSDMD pathway through a three-mode synergistic effect (chemotherapy / photothermal / photodynamic therapy), inducing tumor cell pyroptosis, and thereby triggering a sustained anti-tumor immune response. This liposome aims to overcome the limitations of existing cisplatin-indocyanine green nanodelivery systems (such as polymer nanospheres), which mainly rely on physical controlled release and receptor targeting, have therapeutic mechanisms limited to direct cell killing, and cannot systematically activate anti-tumor immunity. It addresses the technical bottlenecks in the treatment of head and neck squamous cell carcinoma, such as poor local control, difficulty in effectively activating systemic anti-tumor immunity, significant toxic side effects of chemotherapy drugs, and easy tumor recurrence and metastasis.

[0012] The technical solution adopted in this invention is:

[0013] To address the core shortcomings of existing pyroptosis induction strategies, such as lack of targeting and low delivery efficiency, this invention provides a cisplatin-indocyanine green (ICG) composite liposome for synergistic induction of tumor cell pyroptosis. The composite liposome comprises a lipid bilayer consisting of phospholipids, cholesterol, and polyethylene glycol, with cisplatin as the hydrophilic layer and indocyanine green (ICG) as the hydrophobic layer. Cisplatin serves as both a chemotherapeutic drug and a pyroptosis initiator, while indocyanine green acts as a photosensitizer, simultaneously endowing the liposome with photodynamic and photothermal therapeutic functions. This invention's composite liposome co-loads cisplatin and indocyanine green (ICG). Building upon the pharmacokinetic advantages of classic liposome carriers, it employs a co-loading strategy with hydrophilic and hydrophobic spatial partitioning, encapsulating cisplatin in a hydrophilic core and embedding ICG within the phospholipid hydrophobic layer. This approach achieves efficient tumor accumulation through active targeted modification and leverages the weakly acidic tumor microenvironment for initial drug release. Cisplatin initiates pyroptosis-related pathways, while ICG acts as a built-in photosensitive "switch," generating photothermal / photodynamic effects under local near-infrared laser irradiation. This not only directly kills tumor cells but also amplifies the pyroptosis process in situ and precisely through heat and ROS, while simultaneously releasing a large number of damage-associated molecular patterns (DAMPs). Cisplatin preferentially induces immunogenic cell death, followed by enhanced immune response through ICG-mediated photothermal effects. The two work synergistically to construct a strong and controllable inflammatory microenvironment in the tumor, thereby efficiently activating dendritic cells and recruiting cytotoxic T lymphocytes. Ultimately, this maximizes the immune "amplifier" effect of pyroptosis, while effectively avoiding systemic toxicity through precise spatiotemporal control. This overcomes the limitations of simple direct killing, synergistically activating systemic anti-tumor immunity and establishing long-term immune memory. This provides a novel targeted induction protocol for tumor immunotherapy and offers a more promising new strategy for clinical translation.

[0014] Furthermore, the mass ratio of phospholipids to cholesterol and polyethylene glycol is 7-9:1-2:3-5 (preferably 8:1.5:4); the mass ratio of indocyanine green to the total mass of phospholipids, cholesterol and polyethylene glycol is 1:10-15 (preferably 1:13.5); and the mass ratio of indocyanine green to cisplatin is 1:1-5 (preferably 1:1).

[0015] Furthermore, the phospholipid is L-α-phosphatidylcholine, and the polyethylene glycol is DSPE-PEG. 2000 .

[0016] The present invention also provides a method for preparing the cisplatin-indocyanine green complex liposomes, the method comprising the following steps:

[0017] (1) Preparation of indocyanine green solution: Under light-protected conditions, indocyanine green is added to methanol and dissolved by sonication to obtain ICG-methanol solution;

[0018] (2) Preparation of lipid mixture solution: Phospholipids (preferably L-α-phosphatidylcholine), cholesterol and stabilizer polyethylene glycol (preferably DSPE-PEG) are mixed together. 2000 The lipid mixture was added to methanol, sonicated to dissolve and mix thoroughly to obtain a lipid mixture solution.

[0019] (3) Preliminary mixing: Under continuous magnetic stirring, the ICG-methanol solution obtained in step (1) is slowly and evenly added (using a micro-injection pump or dropper) to the lipid mixture obtained in step (2). After the addition is complete, continue stirring to mix evenly so that ICG and lipid molecules are initially combined to obtain the initial solution of ICG-loaded liposomes.

[0020] (4) Preparation of cisplatin aqueous solution: Add cisplatin (DDP) to phosphate buffer (PBS, pH 7.4), sonicate to dissolve completely, and obtain a homogeneous cisplatin solution;

[0021] (5) Formation of liposome film and hydration: The initial solution of ICG-loaded liposomes obtained in step (3) is evaporated under reduced pressure to remove the organic solvent methanol until a uniform ICG liposome composite film is formed on the bottle wall; then, the cisplatin solution in step (4) is added and hydration is carried out under rotation conditions so that the liposome film is completely eluted and spontaneously forms liposomes encapsulating cisplatin, and crude cisplatin-indocyanine green composite liposome solution is obtained.

[0022] (6) Post-processing and purification: The crude cisplatin-indocyanine green complex liposome solution obtained in step (5) is extruded through a microporous filter membrane to obtain cisplatin-indocyanine green complex liposomes (DI@Lipo) with uniform particle size.

[0023] Preferably, the ultrasonic power in steps (1), (2) and (4) is 100-200W and the ultrasonic time is 5-10 minutes.

[0024] Preferably, the volume of methanol used in step (1) is 0.5-1.5 mL / mg based on the mass of indocyanine green, and more preferably 1 mL / mg.

[0025] Preferably, the mass ratio of phospholipids, cholesterol and polyethylene glycol in step (2) is (7-9):(1-2):(3-5), preferably 8:1.5:4; the volume of methanol used is 0.7-1.8 mL / mg based on the mass of phospholipids, preferably 0.8 mL / mg.

[0026] Preferably, the magnetic stirring speed in step (3) is controlled at 1000-1200 rpm, and the total time for dripping and mixing is 5-10 minutes; the amount of indocyanine green methanol solution is based on the mass of indocyanine green, the amount of lipid mixture solution is based on the total mass of lipids, and the mass ratio of indocyanine green to total lipids is 1:10-15 (preferably 1:13.5).

[0027] Preferably, the concentration of the cisplatin solution in step (4) is 0.2-1 mg / mL.

[0028] Preferably, in step (5), the amount of the initial liposome solution loaded with ICG is based on the mass of ICG, the amount of the cisplatin solution is based on the mass of cisplatin, and the mass ratio of ICG to cisplatin is 1:1-5, preferably 1:1.

[0029] Preferably, the water bath temperature for step (5) of vacuum evaporation is controlled at 38-40 ℃, the rotation speed is 55-65 rpm, and the vacuum degree is maintained at -0.08 to -0.1 MPa.

[0030] Preferably, the hydration temperature in step (5) is controlled at 44-46℃, the rotation speed is 55-65 rpm, and the hydration time is 30-45 minutes.

[0031] Preferably, the pore size of the microporous filter membrane in step (6) is 0.45 μm and 0.22 μm.

[0032] The present invention also provides the application of the cisplatin-indocyanine green complex liposome in the preparation of a drug for treating head and neck squamous cell carcinoma.

[0033] Preferably, the drug is a combination therapy that induces pyroptosis of tumor cells and activates an anti-tumor immune response.

[0034] Preferably, the drug dosage form includes injections and topical administration preparations, with injections being the preferred option.

[0035] Preferably, the drug is administered via intravenous injection or peritumoral injection.

[0036] Preferably, the drug further includes pharmaceutically acceptable excipients, including polyethylene glycol (PEG) as a stabilizer and phosphate-buffered saline (PBS) as a buffer system.

[0037] The composite liposomes described in this invention target and accumulate in tumor tissue through enhanced permeation and retention (EPR) effects. Under near-infrared laser irradiation, the local hyperthermia (photothermal therapy) and reactive oxygen species (photodynamic therapy) generated by ICG can directly kill tumor cells and enhance cell membrane permeability. Simultaneously, cisplatin is rapidly released, and the DNA damage and mitochondrial stress it causes synergistically with the oxidative stress generated by ICG to strongly activate the NLRP3 / Caspase-1 / Gasdermin D pyroptosis signaling pathway. The pyroptosis of tumor cells induced by this composite liposome can release large amounts of tumor-associated antigens and damage-related molecules such as HMGB1 and ATP, thereby efficiently activating dendritic cells and promoting the infiltration and killing of cytotoxic T lymphocytes into the tumor site, ultimately establishing a positive feedback loop of systemic anti-tumor immune response triggered by local pyroptosis.

[0038] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0039] 1. Fundamental Improvement in Drug Delivery Method and Stability: This invention innovatively encapsulates hydrophilic cisplatin within the inner aqueous phase and embeds hydrophobic / amphiphilic ICG into the hydrophobic lipid layer using the unique bilayer structure of liposomes. This "internal and external separation" drug delivery method not only achieves high drug delivery efficiency but, more importantly, enables differentiated release kinetics and synergistic effects. Under near-infrared irradiation, ICG first responds by generating heat / ROS, disrupting the local lipid membrane and enhancing cell membrane permeability. At this point, cisplatin can be released more efficiently and enter the cell nucleus. The two drugs form a perfect synergy in space and time, which is difficult to achieve by simply mixing and embedding drugs in a polymer matrix. This solves the problem that existing nanospheres typically embed two drugs together in a hydrophobic polymer matrix, where drug release depends on material degradation or matrix disintegration caused by photothermal effects, resulting in synchronous but difficult-to-precise drug release behavior.

[0040] 2. Significantly Enhanced Biocompatibility and Deep Tumor Permeability: Degradation of polymer nanoparticles may cause local pH changes or mild inflammation, and their rigid structure may also limit penetration into dense tumor tissue. The liposomes of this invention use L-α-phosphatidylcholine and cholesterol as the main membrane materials, whose composition is highly similar to that of cell membranes, exhibiting inherently excellent biocompatibility and biodegradability, and lower systemic toxicity risk (see Appendix). Figure 4 (Hemolysis experiment results). Meanwhile, liposomes (especially those with a particle size of ~100 nm and PEGylated) have good flexibility and can be more effectively enriched in tumor tissue through the EPR effect and penetrate deep into the tumor, overcoming the shortcomings of insufficient penetration of some rigid nanoparticles.

[0041] 3. Significantly Enhanced Efficacy and Immune Activation Induction for Specific Pyroptosis: In existing technologies, nanospheres primarily function for targeted delivery and photothermal controlled release, inducing cell death mainly through apoptosis or necrosis, without clearly and efficiently guiding the specific immunogenic cell death pathway of pyroptosis. This invention, through the aforementioned drug loading and release design, enables the DNA damage stress from cisplatin and the ROS / thermal stress generated by ICG light to highly overlap and amplify each other in time and space, thereby generating a unique and intense integrated stress signal in cells. Experimental data (see appendix) Figure 5 , 6 Surprisingly, this liposome-mediated synergistic effect demonstrates an exceptionally high and specific activation of the NLRP3 / Caspase-1 / GasderminD pyroptosis signaling pathway, with a significantly stronger activation level than free drug combinations or single-modality treatments. This is not merely a simple synergistic effect, but rather an enhancement of the therapeutic mechanism—from direct killing to highly efficient immunogenic death triggering.

[0042] 4. Advantages of the preparation process in terms of drug loading efficiency: Polymer nanospheres are commonly prepared using emulsification-solvent evaporation methods, which achieve high encapsulation efficiency for hydrophobic drugs but often have lower encapsulation efficiency for hydrophilic cisplatin, and the processes are complex. This invention achieves high encapsulation efficiency for the hydrophilic drug cisplatin by optimizing the thin-film dispersion-active hydration method, while ensuring stable ICG embedding in the lipid film. The high efficiency and simplicity of this one-step method for co-loading two drugs with vastly different properties are difficult to achieve simultaneously in the preparation of polymer nanospheres.

[0043] In summary, the core improvement of this invention lies in the creative selection and optimization of the "all-liposome" specific carrier platform and its supporting preparation process. This choice brings a series of synergistic advantages and unexpected superior effects in terms of drug delivery method, biocompatibility, tumor penetration, and most importantly, drug efficacy (efficient induction of pyroptosis and triggering of a sustained anti-tumor immune response), ultimately surpassing existing technologies, especially in activating anti-tumor immunity and inhibiting distant metastasis, demonstrating outstanding clinical translational potential. (iv) Description of the attached drawings

[0045] Figure 1 This is a flowchart illustrating the preparation process of cisplatin-indocyanine green complex liposomes (DI@Lipo).

[0046] Figure 2 This is a transmission electron microscope (TEM) image of DI@Lipo.

[0047] Figure 3 This is a particle size distribution diagram of dynamic light scattering (DLS) of DI@Lipo.

[0048] Figure 4 The hemolysis rate of DI@Lipo at different concentrations.

[0049] Figure 5 Cytotoxicity (CCK-8) of different treatment groups against head and neck squamous cell carcinoma cells.

[0050] Figure 6 Western blot plot of expression levels of key pyroptosis proteins such as GSDMD-N and NLRP3 in tumor cells after treatment in different treatment groups.

[0051] Figure 7 Microscopic images of tumor cells after treatment in different treatment groups.

[0052] Figure 8 A statistical graph showing the growth curves of head and neck squamous cell carcinoma tumors in mice from different treatment groups.

[0053] Figure 9 Immunofluorescence images showing the inhibitory effects of different treatment groups on distant metastatic tumors in mice.

[0054] Figure 10 Statistical graph showing the effects of flow cytometry analysis on the spleens of mice in different treatment groups. (V) Detailed Implementation Methods

[0056] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: the room temperature in the embodiments of the present invention refers to 25-30℃.

[0057] Example 1: Preparation of cisplatin-indocyanine green complex liposomes (DI@Lipo)

[0058] Reference Figure 1 The cisplatin-indocyanine green complex liposomes were prepared according to the following procedure:

[0059] (1) Preparation of ICG methanol solution: At room temperature, accurately weigh 1.0 mg of indocyanine green (ICG) powder, dissolve it in 1 mL of methanol, and place it in a brown bottle. Then, sonicate it in an ultrasonic cleaner with a power of 150 W and a frequency of 40 kHz for 5 minutes in the dark until the powder is completely dissolved, to obtain 1 mL of ICG stock solution with a concentration of 1 mg / mL, which is stored in the dark and refrigerated for later use.

[0060] (2) Preparation of lipid mixture solution: At room temperature, accurately weigh 8.0 mg of L-α-phosphatidylcholine, 1.5 mg of cholesterol, and 4.0 mg of DSPE-PEG2000 (mass ratio of 8:1.5:4) and place them together in a brown bottle. Add 10 mL of methanol and sonicate in an ultrasonic cleaner with a power of 150 W and a frequency of 40 kHz for 5 min in the dark until all lipid components are completely dissolved, to obtain 10 mL of clear and transparent lipid mixture solution.

[0061] (3) Preliminary mixing: At room temperature, place the above 10 mL lipid mixture on a magnetic stirrer and set the speed to 1100 rpm. Then, use a dropper to slowly and evenly add 1 mL of the ICG stock solution prepared in step (1) to the stirred lipid mixture. After the addition is complete, continue stirring under these conditions for 5 minutes to obtain 11 mL of ICG-loaded liposome initial solution (ICG@Lipo).

[0062] (4) Preparation of cisplatin aqueous solution: At room temperature, accurately weigh 1.0 mg of cisplatin (DDP), dissolve it in 5 mL of phosphate buffer (PBS) with pH 7.4, and sonicate (100W) for 5 minutes to obtain 5 mL of cisplatin solution with a concentration of 0.2 mg / mL.

[0063] (5) Film formation and hydration: Transfer the 11 mL initial liposome solution obtained in step (3) to a rotary evaporator. Set the water bath temperature to 39 °C and the rotation speed to 60 rpm. Rotate and evaporate under a vacuum of -0.08 MPa for 30-40 minutes until a uniform, blue-green liposome composite film is formed on the bottle wall.

[0064] Subsequently, 5 mL of the cisplatin solution prepared in step (4) was added to the round-bottom flask. The flask was placed on a magnetic metal stirrer, and the temperature was set to 45°C and the rotation speed to 60 rpm. The mixture was hydrated for 30 minutes to completely elute the lipid film and form a suspension.

[0065] (6) Granulation and purification: The suspension from step (5) was extruded and granulated sequentially through 0.45 μm and 0.22 μm polycarbonate microporous membranes, for a total of 10 cycles, to obtain liposomes with uniform particle size. Finally, the suspension was filtered through a 0.22 μm sterile membrane, and the filtrate was collected to obtain a cisplatin-indocyanine green composite liposome (DI@Lipo) suspension with a concentration of 200 μg / mL (for easy comparison between systems, this is the theoretical concentration calculated based on the amount of raw materials added, i.e., the theoretical concentration of ICG and cisplatin is 200 μg / mL each), and stored at 4℃ in the dark.

[0066] Preparation of indocyanine green liposome (ICG@Lipo) suspension: Replace 5 mL of cisplatin solution in step (5) above with 5 mL of PBS, and perform the same other operations to obtain indocyanine green liposome (ICG@Lipo) suspension with a concentration of 200 μg / mL (for easy comparison between systems, this is the theoretical concentration calculated based on the amount of raw materials added, that is, the theoretical concentration of ICG is 200 μg / mL), and store at 4℃ in the dark.

[0067] Preparation of cisplatin liposome (DDP@Lipo) suspension: steps (1) and (3) are omitted. In step (2), the lipid mixture solution is hydrated and formed into a thin film using the method in step (3), and cisplatin liposome (DDP@Lipo) suspension is prepared using the same method as in step (6). The concentration is 200 μg / mL (for easy comparison between systems, this is the theoretical concentration calculated based on the amount of raw material added, i.e., the theoretical concentration of DDP is 200 μg / mL). It is stored at 4℃ in the dark.

[0068] Example 2: Characterization of the composite liposome DI@Lipo

[0069] (1) Morphology and particle size: 0.25 mL of the DI@Lipo liposome suspension prepared in Example 1 was diluted with 0.75 mL of deionized water and dropped onto a 200-mesh copper grid. The grid was negatively stained with phosphotungstic acid aqueous solution and observed under a transmission electron microscope. The results showed that ( Figure 2 (Used to demonstrate its morphology and particle size distribution), DI@Lipo liposomes exhibit regular spherical or near-spherical shapes with a clear lipid bilayer structure and uniform particle size distribution. Dynamic light scattering analysis showed that the hydrated particle size was less than 100 nm, and the dispersion index was 0.12, indicating good monodispersity of the system. Figure 3 The zeta potential was measured to be -26.3 mV using a Malvern nanoparticle size and zeta potential analyzer (MalvernZetasizer Nano ZS).

[0070] (2) Biocompatibility: The DI@Lipo liposome suspension prepared in Example 1 was used to prepare dispersions with PBS at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μg / mL. Fresh mouse blood was anticoagulated, centrifuged at 3500 rpm for 30 minutes, washed three times with PBS, and blood cells were collected. Positive control (20 μL blood cells + 1 mL distilled water), negative control (20 μL blood cells + 1 mL PBS), and experimental groups (20 μL blood cells + 1 mL of DI@Lipo dispersions at different concentrations) were set up. The mixtures were incubated at 37°C for 5 hours, centrifuged at 3000 rpm, and 100 μL of the supernatant was transferred to a 96-well plate. The absorbance (A) was measured at 545 nm using a microplate reader. Hemolysis rate (%) = (A experimental group - A negative control group) / (A positive control group - A negative control group) 100%. Figure 4 The results showed that even at the highest drug concentration, the hemolysis rate was still less than 5%, indicating that the material has excellent biocompatibility.

[0071] Example 3: Verification of in vitro antitumor and pyroptosis-inducing effects

[0072] (1) Cell experiments: The mouse head and neck squamous cell carcinoma cell line SCC7, derived from ATCC, was used in the experiments. This cell line was cultured in DMEM / F-12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and incubated at 37°C and 5% C. SCC7 cells were cultured routinely in a constant temperature and humidity incubator. Every 2-3 days, depending on cell growth density, cells were digested and passaged using 0.25% trypsin-EDTA digestion solution. Healthy SCC7 cells in the logarithmic growth phase were digested and prepared into single-cell suspensions, then cultured at an appropriate density (2 × 10⁻⁶ cells / cells). 5 pcs / hole, 8×10 4 Cells were seeded (number per well) into 6-well and 96-well plates. The cells were then returned to the incubator and cultured for 24 hours. Once the cells had fully adhered and entered the exponential growth phase, subsequent drug treatments were performed. The following groups were established: Control group, Free cisplatin (DDP) group, DDP@Lipo group, ICG@Lipo-L (without laser), ICG@Lipo+L (with laser), DI@Lipo-L (without laser), and DI@Lipo+L (with laser).

[0073] The drugs were diluted to different concentration gradients (0, 3.125, 6.25, 12.5, 25, 50 μg / mL) with culture medium and incubated with SCC7 cells at 37℃ and 5% C. After co-incubation for 6 hours under the specified conditions, groups requiring laser irradiation were irradiated using an 808 nm near-infrared laser (power density: 1.0 W / cm², time: 5 minutes). After another 2 hours of culturing, cytotoxicity was assessed using the CCK-8 assay. Figure 5 The results showed that the inhibition rate of SCC7 cells in the DI@Lipo+L group was significantly higher than that in any other single treatment group or the group without laser treatment (p < 0.01), demonstrating a synergistic therapeutic effect.

[0074] (2) Verification of pyroptosis mechanism: Using the same grouping and treatment method as in step (1), after culturing for another 2 hours, the expression of key pyroptosis proteins in the cells was detected by Western blotting. The steps are as follows: Discard the cell culture supernatant and gently wash the cells twice with pre-cooled PBS. Add an appropriate amount of RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor to each well and lyse on ice for 30 minutes. Scrape the lysate with a cell scraper and collect it in a centrifuge tube. Centrifuge at 4℃ and 14400 rpm for 15 minutes. Collect the supernatant, which is the total protein sample. Use the BCA protein quantification kit to determine the protein concentration of each sample, and adjust all samples to the same concentration with lysis buffer. Add 5×SDS-PAGE protein loading buffer, heat in a boiling water bath at 100℃ for 10 minutes to denature the protein, and store at -80℃ for later use. Take 4%-20% of the pre-cast gel, and add an equal amount of protein (30 μg) sample and pre-stained protein marker to the loading wells respectively. In 1×Tris-Glycine electrophoresis buffer, the initial voltage was 80V for concentration. After the sample entered the separating gel, the voltage was increased to 120V, and electrophoresis continued until the target band (based on the molecular weight of the target protein) was fully separated. Using a wet transfer method, the electrophoresed gel and PVDF membrane (activated with methanol before use) were assembled into a "sandwich" structure in transfer buffer (Tris-Glycine buffer containing 20% ​​methanol). Under ice bath conditions, the membrane was transferred at a constant current of 300mA for 60-90 minutes to transfer the protein from the gel to the PVDF membrane. After transfer, the PVDF membrane was blocked for 2 hours at room temperature with TBST buffer (Tris buffered saline solution containing 0.1% Tween-20) containing 5% skim milk to block non-specific binding sites. After blocking, the membrane was washed three times with TBST for 5 minutes each time. Subsequently, the membrane was incubated overnight at 4°C with specific primary antibodies (NLRP3, GSDMD, GSDMD-N, GAPDH). Figure 6 The results showed that only in the DI@Lipo+L group were the expression levels of NLRP3 and the GSDMD-N fragment (the pyroptosis executive protein) significantly upregulated. Meanwhile, Figure 7 Microscopic observation of the cells revealed characteristic pyroptotic vesicles (Pyroptosis vesicles). Figure 7As shown by the middle arrow, a large number of cells in this group exhibited typical pyroptosis morphology, including cell swelling and membrane rupture.

[0075] Example 4: Evaluation of in vivo antitumor effect

[0076] (1) Animal model establishment: Female C3H / HeN mice (or C3H mice) aged 4-6 weeks were selected. All mice were housed in an SPF-grade animal facility under the following conditions: constant temperature (22 ± 2℃), constant humidity (50% ± 10%), 12-hour light-dark cycle, and free access to standard irradiated feed and sterile drinking water. All animal experimental procedures were carried out in accordance with laboratory animal welfare and ethical guidelines and were approved by the institution's animal experiment ethics committee.

[0077] Mouse head and neck squamous cell carcinoma cell line SCC7, cultured in the logarithmic growth phase, was harvested. Adherent cells were digested with 0.25% trypsin-EDTA digestion solution, neutralized in DMEM / F-12 medium containing 10% FBS, and a single-cell suspension was prepared. The cell suspension was transferred to centrifuge tubes and centrifuged at 900 rpm for 3 minutes, discarding the supernatant. The cell pellet was resuspended in pre-chilled sterile PBS, and washed twice to thoroughly remove serum components from the culture medium, avoiding inflammatory reactions during inoculation. Finally, the cells were resuspended in PBS and counted, adjusting the cell density to 1 × 10⁶ cells / mL. 7 Cells were stored at a density of 100 μL / mL on ice. Using a 1 mL insulin syringe, 100 μL of the cell suspension was drawn and inoculated into the right axilla of mice to establish a head and neck squamous cell carcinoma tumor model.

[0078] (2) Grouping and administration: When the tumor volume grows to about 100 mm³, the mice are randomly divided into 6 groups (5 mice in each group, n=5): PBS group, free cisplatin (DDP) group, ICG@Lipo-L (without laser) group, ICG@Lipo+L (with laser) group, DI@Lipo-L group, and DI@Lipo+L group. The PBS group received 100 μL of PBS via intraperitoneal injection; the DDP group received 100 μL of cisplatin aqueous solution at a concentration of 50 μg / mL prepared in step (4) of Example 1 via intraperitoneal injection; the ICG@Lipo-L and ICG@Lipo+L groups received 100 μL of ICG@Lipo suspension prepared in step (6) of Example 1, adjusted to a concentration of 50 μg / mL with PBS via tail vein injection; the DI@Lipo-L and DI@Lipo+L groups received 100 μL of DI@Lipo suspension prepared in step (6) of Example 1, adjusted to a concentration of 50 μg / mL with PBS via tail vein injection. Six hours after administration, in groups requiring laser irradiation, the tumor site was irradiated with an 808 nm laser (power density: 1.0 W / cm², time: 10 minutes).

[0079] (3) Results: Tumor volume and mouse weight were measured every 2 days. Figure 8 The results showed that tumors grew rapidly in the PBS group; the free cisplatin group had a certain inhibitory effect, but the mice experienced a significant decrease in body weight and exhibited toxicity; the ICG@Lipo+L group only mildly inhibited tumor growth; while the DI@Lipo+L group showed the strongest tumor growth inhibition effect, and the mice maintained stable body weight throughout the experiment with no obvious toxic side effects. This example fully demonstrates the high efficiency and low toxicity of the DI@Lipo liposomes of the present invention in vivo.

[0080] Example 5: Evaluation of in vivo immune enhancement effect

[0081] (1) Establishment of animal model: SCC7 cell suspension was prepared using the method in step (1) of Example 4 above. 100 μL of the cell suspension was drawn up with a 1mL insulin syringe and SCC7 cells were implanted into two sites: the right neck (primary tumor site) and the right hind limb (metastatic tumor site) to establish a dual tumor model.

[0082] (2) Grouping and administration: When the tumor volume reached approximately 100 mm³, the mice were randomly divided into 4 groups (5 mice per group, n=5): PBS group, free cisplatin (DDP) group, ICG@Lipo group, and DI@Lipo group. The PBS group was administered 100 μL of PBS via tail vein injection; the DDP group was administered 100 μL of cisplatin aqueous solution at 50 μg / mL prepared in step (4) of Example 1 via intraperitoneal injection; the ICG@Lipo group was administered 100 μL of ICG@Lipo suspension prepared in step (6) of Example 1, adjusted to a concentration of 50 μg / mL with PBS via tail vein injection; the DI@Lipo group was administered 100 μL of DI@Lipo suspension prepared in step (6) of Example 1, adjusted to a concentration of 50 μg / mL with PBS via tail vein injection. Six hours after drug administration, the primary tumor sites of the DI@Lipo and ICG@Lipo groups were irradiated with an 808 nm laser (power density: 1.0 W / cm², time: 10 minutes).

[0083] (3) Results: We performed immunofluorescence staining on proximal (primary) and distal (metastatic) tumors. Figure 9The results showed that in proximal tumors (A), the DI@Lipo group exhibited significantly enhanced CD8, CRT, and HMGB1 signals (green fluorescence), indicating strong immune activation. Notably, in distal tumors (B), the CD8 / CRT / HMGB1 signals in the DI@Lipo group were also significantly increased compared to the control group, suggesting that it may inhibit metastatic progression through systemic immune effects. These results collectively demonstrate that DI@Lipo effectively initiates a systemic antitumor immune response by releasing damage-associated molecular patterns (DAMPs) and activating subsequent immune cells.

[0084] Figure 10 Flow cytometry analysis of splenic immune cells showed a significantly increased proportion of mature dendritic cells (DCs) in the DI@Lipo group (CD11c+CD80+CD86+, 30.57%), which was 1.68-fold, 1.68-fold, and 1.72-fold higher than the control group, DDP group, and ICG@Lipo group, respectively. This confirms that DI@Lipo combined with laser irradiation can effectively promote DC maturation. The DI@Lipo group showed a higher proportion of tumor-infiltrating CD8+ T cells (CD3+CD8+, 37.98%), which was 1.19-fold, 1.24-fold, and 1.41-fold higher than the control group, DDP group, and ICG@Lipo group, respectively. These results indicate that DI@Lipo-triggered pyroptosis plays a key role in enhancing the body's anti-tumor immune defense, opening up a new avenue for photothermal immunotherapy.

Claims

1. A cisplatin-indocyanine green complex liposome for synergistic induction of pyroptosis in tumor cells, characterized in that, The composite liposome consists of a lipid bilayer composed of phospholipids, cholesterol, and polyethylene glycol, with cisplatin as the hydrophilic layer and indocyanine green as the hydrophobic layer.

2. The composite liposome as described in claim 1, characterized in that, The mass ratio of phospholipids to cholesterol and polyethylene glycol is 7-9:1-2:3-5; the mass ratio of indocyanine green to phospholipids, cholesterol and polyethylene glycol is 1:10-15; and the mass ratio of indocyanine green to cisplatin is 1:1-5.

3. The composite liposome as described in claim 1, characterized in that, The phospholipid is L-α-phosphatidylcholine, and the polyethylene glycol is DSPE-PEG. 2000 .

4. A method for preparing the cisplatin-indocyanine green complex liposomes according to claim 1, characterized in that, The method includes the following steps: (1) Preparation of indocyanine green solution: Under light-protected conditions, indocyanine green is added to methanol and dissolved by sonication to obtain indocyanine green methanol solution; (2) Preparation of lipid mixture solution: Phospholipids, cholesterol and stabilizer polyethylene glycol are added to methanol, ultrasonically dissolved and mixed evenly to obtain lipid mixture solution; (3) Preliminary mixing: Under continuous magnetic stirring, the indocyanine green methanol solution obtained in step (1) is slowly and evenly added dropwise to the lipid mixture obtained in step (2). After the addition is complete, the mixture is stirred and mixed to obtain the initial liposome solution. (4) Preparation of cisplatin aqueous solution: Add cisplatin to pH 7.4 phosphate buffer and sonicate to dissolve completely to obtain cisplatin solution; (5) Formation of liposome film and hydration: The initial liposome solution obtained in step (3) is evaporated under reduced pressure to remove the organic solvent methanol until a liposome composite film is formed; then, the cisplatin solution in step (4) is added and hydration is carried out under rotation conditions so that the film is completely washed away and spontaneously forms liposomes encapsulating cisplatin, and crude cisplatin-indocyanine green composite liposome solution is obtained. (6) Post-processing and purification: The crude cisplatin-indocyanine green complex liposome solution obtained in step (5) is extruded through a microporous filter membrane to obtain cisplatin-indocyanine green complex liposomes with uniform particle size.

5. The preparation method according to claim 4, characterized in that, The ultrasonic power in steps (1), (2) and (4) is 100-200W and the ultrasonic time is 5-10 minutes.

6. The preparation method according to claim 4, characterized in that, The mass ratio of phospholipids, cholesterol and polyethylene glycol in step (2) is (7-9):(1-2):(3-5).

7. The preparation method according to claim 4, characterized in that, In step (3), the amount of indocyanine green methanol solution used is based on the mass of indocyanine green, and the amount of lipid mixed solution used is based on the total mass of lipids. The mass ratio of indocyanine green to total lipids is 1:10-15. In step (5), the amount of initial liposome solution used is based on the mass of indocyanine green, and the amount of cisplatin solution used is based on the mass of cisplatin. The mass ratio of indocyanine green to cisplatin is 1:1-5.

8. The preparation method according to claim 4, characterized in that, Step (5) The water bath temperature for vacuum evaporation is controlled at 38-40 ℃, the rotation speed is 55-65 rpm, and the vacuum degree is maintained at -0.08 to -0.1 MPa; the hydration temperature is controlled at 44-46 ℃, the rotation speed is 55-65 rpm, and the hydration time is 30-45 minutes.

9. The use of the cisplatin-indocyanine green complex liposome according to claim 1 in the preparation of a drug for treating head and neck squamous cell carcinoma.

10. The application as described in claim 9, characterized in that, The drug is a combination therapy that induces pyroptosis in tumor cells and activates an anti-tumor immune response.

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