Photo-acoustic-magnetic three-mode imaging type nano-liposome and preparation method thereof

Abstract

The invention relates to a lipidosome and a preparation method and application thereof, in particular to a photoacoustic-magnetic three-mode imaging type nano lipidosome and a preparation method thereof, a phospholipid bilayer membrane of the nano lipidosome comprises phospholipid, cholesterol, IR-1061, DSPE-PEG2000 and u-CuS QDs, and the phospholipid bilayer membrane of the nano lipidosome comprises phospholipid, cholesterol, IR-1061, DSPE-PEG2000 and u-CuS QDs. The liposome water phase is loaded with indocyanine green. The nano-liposome disclosed by the invention can generate an enhanced NIR-II fluorescence signal, a photoacoustic signal and a magnetic resonance signal at the same time, so that the nano-liposome is used as a multi-mode imaging contrast agent; and the probe can be used as a thermal therapy probe for generating a photo-thermal effect.

Description

Technical Field

[0001] The present invention relates to a liposome and a preparation method and application thereof, and in particular to a photoacoustic-magnetic trimodal imaging nanoliposome and a preparation method thereof. Background Art

[0002] Near-infrared II (NIR-II) fluorescence imaging (FI) has been widely used in clinical procedures requiring real-time, rapid, and long-term monitoring due to its ease of use, high sensitivity, and excellent temporal resolution. However, the limited penetration depth of NIR-II FI in tissues has hampered its further application and development in the biomedical field. To overcome this limitation, recent research has tended to combine NIR-II FI with other imaging modalities to complement each other's strengths and enhance overall imaging quality and diagnostic accuracy.

[0003] In this context, the research and development of multifunctional nanoprobes has become an important direction to solve the above problems. For example, ultra-small copper sulfide (u-Cu 2-x S) Quantum Dots (QDs), as an inorganic nanomaterial, have been proven to be applicable to a variety of imaging technologies such as photoacoustic imaging (PAI), magnetic resonance imaging (MRI), positron emission tomography (PET) and computed tomography (CT), and have good tumor photothermal therapy performance. It is worth noting that although u-Cu 2-x S is an inorganic nanoparticle, but its small size allows for rapid renal excretion and excellent biosafety, resulting in high potential for clinical translation. Combining it with NIR-II fluorescent imaging contrast agents is expected to effectively compensate for the tissue penetration limitations of single fluorescent imaging through multimodal collaborative imaging, thereby obtaining imaging data with higher resolution and contrast.

[0004] Furthermore, liposomes, as closed vesicles formed by the self-assembly of phospholipid molecules in an aqueous environment, have a bilayer structure highly similar to natural biological membranes such as cell membranes, exhibiting excellent biocompatibility and low toxicity, thus showing broad application prospects in drug delivery and molecular imaging. The core aqueous region of liposomes can be used to load water-soluble substances, while the hydrophobic bilayer can stably encapsulate hydrophobic molecules. This property makes liposomes an ideal carrier platform for the integration of a variety of imaging probes and therapeutic drugs. Specifically, the hydrophobic layer of liposomes can immobilize hydrophobic NIR-II fluorescent dyes (such as IR-1061) and oleylamine-ligand-modified copper sulfide quantum dots through intermolecular forces, thereby improving the stability and biocompatibility of these probes in physiological environments. Furthermore, the central aqueous space can be used to load indocyanine green (ICG), a clinically approved small molecule near-infrared (NIR-I) fluorescent dye. However, free ICG tends to aggregate and form dimers in aqueous solution, resulting in reduced fluorescence signal and even self-quenching. By encapsulating ICG in liposomes, not only can its fluorescence bleaching process be effectively inhibited, but also the fluorescence lifetime can be extended and the fluorescence intensity can be enhanced, thereby significantly improving the imaging depth and sensitivity.

[0005] In summary, liposomes were used as multifunctional carriers to integrate NIR-II fluorescent dyes, ICG and u-Cu 2-x Imaging probes such as S QDs can be used to construct liposome nanosystems with multimodal imaging capabilities, which can provide new strategies and technical paths for integrated precision diagnosis and treatment.

[0006] As in the prior art, the liposome-based nano-diagnostic and therapeutic agent for multimodal imaging and photothermal therapy and its preparation method with patent application number CN201510080017.7, through the hydrophobic nano-copper sulfide encapsulated between the bilayers of the liposome and the organic liquid-gas phase transition cutoff fluorocarbon compound encapsulated in the inner core of the liposome, can be used as an ultrasound, near-infrared heat, photoacoustic multimodal imaging contrast agent and a photothermal therapeutic agent under the induction of near-infrared light. However, the fluorescence performance and optical performance are relatively weak.

[0007] There are also existing technologies, such as a NIR-II fluorescence-enhanced J-aggregated indocyanine green cationic liposome and its preparation method and application with patent application number CN202311059315.9. The components of the phospholipid bilayer membrane of the liposome include phospholipids and cholesterol, and the components of the phospholipids contain positively charged phospholipids. The liposome aqueous phase is loaded with indocyanine green, which is adsorbed on the phospholipid bilayer membrane and exists in the form of a J-aggregate state. Although it can enhance the imaging ability of indocyanine green in NIR-II, it can only achieve fluorescence imaging. Summary of the Invention

[0008] The present invention aims to provide a photoacoustic-magnetic trimodal imaging nanoliposome and its preparation method, which can generate photoacoustic and magnetic resonance signals while enhancing NIR-II fluorescence signals, and can be produced and applied on a large scale as a multimodal imaging contrast agent; at the same time, it can also be applied to thermotherapy probes to generate photothermal effects while imaging. In order to achieve the above object, the present invention adopts the following technical solutions: The first scheme is a photoacoustic-magnetic trimodal imaging nanoliposome, wherein the phospholipid bilayer membrane of the nanoliposome includes phospholipids, cholesterol, IR-1061, DSPE-PEG2000 and u-CuS QDs; the liposome aqueous phase is loaded with indocyanine green.

[0009] Preferably, the molar ratio of the phospholipid to indocyanine green is 20-200:1.

[0010] Preferably, the molar ratio of the phospholipid to IR-1061 is 20-200:1.

[0011] Preferably, the mass ratio of the phospholipid to u-CuS QDs is 20-200:1.

[0012] Preferably, the molar ratio of phospholipid to cholesterol is 2-4:1.

[0013] Preferably, the phospholipids include electrically neutral phospholipid molecules and DSPE-PEG2000.

[0014] Preferably, the molar ratio of the electrically neutral phospholipid molecules to DSPE-PEG2000 is 8-10:1.

[0015] Preferably, the electrically neutral phospholipid molecule includes any one of dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine and distearoylphosphatidylcholine.

[0016] The second solution is a method for preparing a photoacoustic-magnetic trimodal imaging nanoliposome, which is used to prepare the photoacoustic-magnetic trimodal imaging nanoliposome described in the first solution, comprising: S101, sulfur and oleylamine are mixed to form a sulfur-oleylamine complex, and copper acetylacetonate is mixed with oleylamine and chloroform to form a copper-oleylamine complex; S102, the copper-oleylamine complex and the sulfur-oleylamine complex are slowly mixed, mechanically dispersed, and washed with an organic solvent to obtain u-CuS QDs.

[0017] Preferably, the method further includes: S201, mixing materials for preparing a phospholipid bilayer to form a uniform solution, removing the organic solvent by rotary evaporation, and obtaining a phospholipid film; S202, preparing indocyanine green into an indocyanine green solution using PBS to obtain an aqueous phase; S203, adding the aqueous phase to the phospholipid film for hydration, and dispersing by mechanical force to obtain indocyanine green liposomes.

[0018] Description of the drawings Figure 1 Schematic diagram of the structure of a photoacoustic-magnetic trimodal imaging nanoliposome according to Example 1 (a); Figure 2 (b) is a schematic structural diagram of a photoacoustic-magnetic trimodal imaging nanoliposome according to Example 1; Figure 3 This is a transmission electron microscope image of a photoacoustic-magnetic trimodal imaging nanoliposome of Example 1, wherein sub-image (b) is a two-dimensional imaging image of the nanoliposome under the transmission electron microscope image, sub-image (b); Figure 4 Schematic diagram of the DLS test results of Experimental Example 1; Figure 5 The electron microscope image display diagram of Experimental Example 2, wherein sub-image (a) is the electron microscope image display diagram of BLP, and sub-image (b) is the electron microscope image of IR-1061-ICG-QD-LP.

[0019] Figure 6 Schematic diagram of wavelength detection results of different comparative examples of Experimental Example 2 and Example 1; Figure 7 Schematic diagram of the fluorescence spectrum signal detection results of Experimental Example 2; Figure 8 Schematic diagram of the MRI performance test results of Experimental Example 3; Figure 9 Schematic diagram of the PA imaging performance test results of Experimental Example 4; Figure 10 Schematic diagram of the PA imaging performance test results of Experimental Example 5; Figure 11 This is a schematic diagram of the detection results of the particle size change of nanoliposomes over time using the photoacoustic-magnetic trimodal imaging method of Experimental Example 6. Specific embodiments Example 1 like Figure 1 and Figure 2As shown, this embodiment provides a nanoliposome capable of photoacoustic and magnetic trimodal imaging. The liposome is composed of a phospholipid bilayer containing DPPC, cholesterol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000), IR-1061, and u-CuS QDs. Indocyanine green molecules are encapsulated in the aqueous phase of the nanoliposome. Together, the indocyanine green and IR-1061 create a FRET effect, enhancing the fluorescence intensity of IR-1061. Furthermore, the u-CuS QDs embedded within the phospholipid bilayer enable laser-induced photothermal and photoacoustic imaging, while also enabling magnetic resonance imaging.

[0021] Specifically, the molar ratio of phospholipid to indocyanine green is 20-200:1, the molar ratio of phospholipid to IR-1061 is 20-200:1, the mass ratio of phospholipid to u-CuS QDs is 20-200:1, and the molar ratio of phospholipid to cholesterol is 2-4:1.

[0022] The phospholipids include electrically neutral phospholipid molecules and DSPE-PEG2000. The molar ratio of electrically neutral phospholipid molecules to DSPE-PEG2000 is 8-10:1. The electrically neutral phospholipid molecules include any one of dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0023] like Figure 3 As shown in sub-figure (a), the transmission electron microscopy image shows that the quantum dots of nanoliposomes are spherical, with regular morphology, narrow size distribution and uniform particle size distribution. Figure 3 In sub-figure (b), the particle size distribution of nanoliposomes can be seen. The quantum dots have a narrow size distribution and an average particle size of 2.55 nm, which can be loaded by liposomes.

[0024] Beneficial effects of this embodiment First, a relatively low concentration of IR-1061 combines with the hydrophilicity of the phospholipid bilayer, with a molar ratio of phospholipid to IR-1061 of 20-200:1. This achieves enhanced fluorescence intensity while maintaining high stability and ease of preparation. This embodiment utilizes a hydrophilic-hydrophobic layered co-loading strategy of hydrophilic ICG and the hydrophobic IR-1061 dye. Through the fluorescence resonance energy transfer (FRET) mechanism, this effectively reduces the IR-1061 dosage while maintaining excellent fluorescence imaging performance. In contrast, existing liposomes typically only load high doses of the hydrophobic IR-1061 dye, which can easily induce self-quenching at excessively high concentrations, leading to a significant decrease in fluorescence intensity and affecting imaging results.

[0025] Secondly, it can be put into large-scale production and application. This embodiment utilizes the synergistic photoacoustic enhancement effect between IR-1061 and copper sulfide quantum dots to maintain excellent photoacoustic imaging while reducing the dosage of copper sulfide quantum dots, thereby achieving good structural stability and drug encapsulation efficiency of the liposomes. To achieve good structural stability and drug encapsulation efficiency of liposomes, existing liposomes are easily destabilized when loaded with copper sulfide quantum dots at high doses, which can cause leakage of the contents and affect encapsulation efficiency and imaging performance.

[0026] Finally, the unique multi-component compatibility and distribution control. Through the synergistic low-concentration combination of DPPC, DSPE-mPEG2000, IR-1061, and u-CuS QDs, the fluorescence imaging performance and imaging intensity can be further improved, while the production cost is low and the liposome stability is high. Existing multimodal liposomes do not have synergistic effects and usually use a single material to further improve the strength. This not only requires a large amount of materials and high production costs, but also makes the liposome stability or uniformity difficult to control, making it unsuitable for widespread application in large-scale production.

[0027] Example 2 In this example, a method for preparing photoacoustic-magnetic trimodal imaging nanoliposomes is provided, focusing on the preparation of ultrasmall copper sulfide quantum dots (u-Cu2-xS QDs) and indocyanine green.

[0028] Specifically, in step S101, 8 mg of sulfur and 1.5 mL of oleylamine were added to a 25 mL flask. The mixture was ultrasonically cleaned in an ultrasonic bath until the sulfur was completely dissolved, forming a sulfur-oleylamine complex. The solution was then orange-red. The reaction system was then placed in an oil bath at 70°C.

[0029] In step S102, add 65 mg of copper acetylacetonate to 0.5 mL of oleylamine and 5 mL of chloroform at room temperature and mix thoroughly to form a copper-oleylamine complex. The solution is now transparent green. Add the copper-oleylamine complex to the reactants in step S101 and stir rapidly to mix thoroughly.

[0030] In step S201, materials for preparing a phospholipid bilayer are mixed to form a uniform solution, and the organic solvent is removed by rotary evaporation to obtain a phospholipid film. In step S202, indocyanine green is prepared in PBS to form an indocyanine green solution to obtain an aqueous phase. In step S203, the aqueous phase is added to the phospholipid film for hydration, and mechanical dispersion is performed to obtain indocyanine green liposomes.

[0031] The solution gradually changed from orange-red to dark green and reacted at 70°C with stirring for 30 minutes. Oleylamine served as a surfactant during this process. After the reaction was completed, the chloroform was partially removed by vacuum rotary evaporation at 40°C. The reaction mixture was then placed in a 15 mL centrifuge tube, ethanol was added, and the tube was centrifuged at 11,000 rpm for 10 minutes. After three centrifugations, the tube was redispersed in chloroform for later use.

[0032] Example 3 Different from Example 2, this example focuses on the preparation of photoacoustic-magnetic trimodal imaging liposomes (IR-1061-ICG-QD-LP).

[0033] Specifically, in step 1, 35 mg of dipalmitoylphosphatidylcholine, 15 mg of cholesterol, 6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, 200 μg of IR-1061 and 6 mg of Tween-80 were weighed, placed in a 150 mL round-bottom flask, and shaken and dissolved with 10 mL of dichloromethane.

[0034] Step 2: Add 5 mL of 200 μg / mL chloroform-dispersed u-Cu 2-x S QDs were placed in a round-bottom flask and thoroughly mixed on a rotary evaporator at 45 °C and 60 rpm for 5 min.

[0035] Step 3: Add 10 mL of dichloromethane and 2 mL of anhydrous ethanol, and remove the organic solvent by rotary evaporation at 45 °C and 100 rpm. A uniform film will form on the bottom of the round-bottom flask, and place it on a vacuum pump for 6 h to remove the remaining organic solvent.

[0036] Step 4: Prepare 10 mL of ICG solution at a concentration of 10 μg / mL using PBS solution, preheat at 55°C, add to a round-bottom flask, and place in a thermostatic oscillator for hydration at 37°C and 140 rpm for 2.5 hours. Step 5: Ultrasonicate the liposomes for 1 minute using an ultrasonic cleaner in an ice bath, and filter the solution three times using 0.45 μm and 0.22 μm microporous membranes. Then dialyze overnight using a 100,000 molecular weight dialysis membrane, transfer to a 5 mL brown glass bottle, set the liposome phospholipid concentration to 5 mg / mL, and store in a refrigerator at 0-4°C.

[0037] Comparative Example 1 This comparative example provides a preparation of a blank liposome B-LP.

[0038] Specifically, in step 1, 35 mg of dipalmitoylphosphatidylcholine, 15 mg of cholesterol, 6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, and 6 mg of Tween-80 were weighed and placed in a 150 mL round-bottom flask, shaken and dissolved with 10 mL of dichloromethane, and rotated on a rotary evaporator at 45 ° C and 60 rpm for 5 min to fully mix.

[0039] Step 2: 8 mL of ultrapure water was preheated at 55°C and added to the round-bottom flask, which was then placed in a thermostatic oscillator at 37°C and 140 rpm for hydration for 2.5 hours.

[0040] Step 3: Ultrasonicate the liposomes for 1 minute using an ultrasonic cleaner in an ice bath. Filter the solution three times using 0.45 μm and 0.22 μm microporous membranes. Then, dialyze overnight using a 100,000 molecular weight dialysis membrane. Transfer the solution to a 5 mL brown glass bottle and set the liposome phospholipid concentration to 5 mg / mL. Store in a refrigerator at 0–4°C.

[0041] In summary, the preparation of B-LP is completed from step 1 to step 3.

[0042] Comparative Example 2 This comparative example provides a preparation method of IR-1061 liposomes (IR-1061-LP).

[0043] Specifically, in step 1, 35 mg of dipalmitoylphosphatidylcholine, 15 mg of cholesterol, 6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, 200 μg of IR-1061 and 6 mg of Tween-80 were weighed and placed in a 150 mL round-bottom flask, shaken and dissolved with 10 mL of dichloromethane, and rotated on a rotary evaporator at 45°C and 60 rpm for 5 min to fully mix.

[0044] Step 2: 8 mL of ultrapure water was preheated to 55°C and added to the round-bottom flask, which was then placed in a thermostatic oscillator at 37°C and 140 rpm for hydration for 2.5 hours.

[0045] Step 3: Ultrasonicate the liposomes for 1 minute using an ultrasonic cleaner in an ice bath. Filter the solution three times using 0.45 μm and 0.22 μm microporous membranes. Then, dialyze overnight using a 100,000 molecular weight dialysis membrane. Transfer the solution to a 5 mL brown glass bottle and set the liposome phospholipid concentration to 5 mg / mL. Store in a refrigerator at 0–4°C.

[0046] In summary, the preparation of IR-1061-LP is completed from step 1 to step 3.

[0047] Comparative Example 3 This comparative example provides a preparation method of ICG liposomes (ICG-LP).

[0048] Specifically, in step 1, 35 mg of dipalmitoylphosphatidylcholine, 15 mg of cholesterol, 6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, and 6 mg of Tween-80 were weighed and placed in a 150 mL round-bottom flask, shaken and dissolved with 10 mL of dichloromethane, and rotated on a rotary evaporator at 45 ° C and 60 rpm for 5 min to fully mix.

[0049] Step 2: Prepare 10 mL of ICG solution with a concentration of 10 μg / mL using PBS solution, preheat at 55°C, add to a round-bottom flask, place in a constant temperature oscillator, and hydrate at 37°C and 140 rpm for 2.5 hours.

[0050] Step 3: Ultrasonicate the liposomes for 1 minute using an ultrasonic cleaner in an ice bath. Filter the solution three times using 0.45 μm and 0.22 μm microporous membranes. Then, dialyze overnight using a 100,000 molecular weight dialysis membrane. Transfer the solution to a 5 mL brown glass bottle and set the liposome phospholipid concentration to 5 mg / mL. Store in a refrigerator at 0–4°C.

[0051] In summary, the preparation of ICG-LP is completed from step 1 to step 3.

[0052] Comparative Example 4 This comparative example provides a preparation method of u-CuS QDs liposomes (QD-LP).

[0053] Specifically, in step 1, 35 mg of dipalmitoylphosphatidylcholine, 15 mg of cholesterol, 6 mg of distearate phosphatidylethanolamine-polyethylene glycol 2000, and 6 mg of Tween-80 were weighed, placed in a 150 mL round-bottom flask, and shaken with 10 mL of dichloromethane to dissolve.

[0054] Step 2: Add 5 mL of 200 μg / mL chloroform-dispersed u-Cu2-xS QDs into a round-bottom flask and rotate on a rotary evaporator at 45 °C and 60 rpm for 5 min to mix thoroughly.

[0055] Step 3: After step 2, add 10 mL of dichloromethane and 2 mL of anhydrous ethanol, and remove the organic solvent by rotary evaporation at 45 °C and 100 rpm to form a uniform film at the bottom of the round-bottom flask. Place it on a vacuum pump for 6 h to remove as much residual organic solvent as possible.

[0056] Step 4: 8 mL of ultrapure water preheated to 55°C was added to the round-bottom flask, and the mixture was placed in a thermostatic oscillator at 37°C and 140 rpm for hydration for 2.5 hours.

[0057] Step 5: Ultrasonicate the liposomes for 1 minute in an ice bath using an ultrasonic cleaner. Filter the solution three times using 0.45 μm and 0.22 μm microporous membranes. Then, dialyze overnight using a 100,000 molecular weight dialysis membrane. Transfer the solution to a 5 mL brown glass bottle and set the liposome phospholipid concentration to 5 mg / mL. Store in a refrigerator at 0–4°C.

[0058] In summary, the preparation of QD-LP is completed from step 1 to step 5.

[0059] Test Example 1 like Figure 4 As shown, after DLS detection of the photoacoustic-magnetic trimodal imaging nanoliposome described in Example 1 and Comparative Examples 1 to 4, the average particle size of B-LP, IR-1061-LP, ICG-LP, QD-LP, and IR-1061-ICG-QD-LP was between 120 and 170 nm, showing a unimodal distribution.

[0060] Table 1 DLS test results for Example 1 and Test Examples 1 to 4

[0061] As shown in Table 1, the B-LP liposome had the smallest average particle size, at 118 ± 3.1 nm, indicating that the loading of IR-1061, ICG, and QDs had an impact on the liposome size. The IR-1061-LP liposome had a particle size of 139.5 ± 1.4 nm, smaller than the QD-LP liposome (145.8 ± 1.1 nm). The IR-1061-ICG-QD-LP liposome had an average particle size of 161.6 ± 5.5 nm, with a polydispersity index (PDI) of 0.15 ± 0.005, indicating uniform particle size distribution in aqueous solution.

[0062] Beneficial effects of this test example DLS analysis revealed that the average particle size of the photoacoustic-magnetic trimodal imaging nanoliposomes (IR-1061-ICG-QD-LP) in Example 1 was 161.6 ± 5.5 nm, and the polydispersity index (PDI) was 0.15 ± 0.005, indicating a uniform particle size distribution. Compared with blank liposomes (B-LP) and other liposomes containing only a single component, the successful loading of IR-1061, ICG, and u-CuS QDs resulted in a slightly increased liposome size, but the size remained within a reasonable range and was uniformly distributed. This demonstrates that the nanoliposomes, even after integrating multiple imaging probes, can maintain good particle size characteristics and achieve multimodal imaging, potentially paving the way for widespread clinical application.

[0063] Test Example 2 like Figure 5 As shown in (a), it can be seen that BLP is spherical and evenly distributed in the electron microscope image. Figure 5 (b) An electron microscopy image of IR-1061-ICG-QD-LP shows that the liposomes are spherical, well-dispersed, and free of aggregation in water. Zooming in on the liposomes in the lower right corner reveals u-CuS QDs (indicated by red arrows), demonstrating successful liposome loading. Compared to B-LP, the particle size of IR-1061-ICG-QD-LP is significantly larger, consistent with the DLS results.

[0064] like Figure 6 As shown, the liposomes loaded with ICG and IR-1061 exhibit characteristic peaks for both ICG and IR-1061 between 600 and 1200 nm, whereas the corresponding B-LP liposomes lack these peaks. The characteristic peak for IR-1061 in IR-1061-LP and IR-1061-ICG-QD-LP is at 1010 nm, which is somewhat blue-shifted compared to the standard IR-1061 absorption spectrum.

[0065] like Figure 7 As shown in the figure, fluorescence spectroscopy results show that B-LP exhibits no fluorescence signal. ICG-LP exhibits no emission peak beyond 1000 nm. IR-1061-LP exhibits an emission peak near 1061 nm. IR-1061-ICG-QD-LP, on the other hand, exhibits a strong emission peak at 1125 nm, with a fluorescence intensity approaching 1000, twice that of IR-1061-LP. Furthermore, fluorescence signal analysis using the NIR-II in vivo imaging system revealed that the fluorescence intensity of IR-1061-ICG-QD-LP was significantly higher than that of both ICG-LP and IR-1061-LP, consistent with the fluorescence spectroscopy results.

[0066] Beneficial effects of this test example Electron microscopy images revealed that the IR-1061-ICG-QD-LP liposomes were spherical, uniformly distributed, and well dispersed, exhibiting no aggregation in water. The successful loading of u-CuS QDs into the liposomes was clearly observed. Furthermore, the significant increase in particle size of the IR-1061-ICG-QD-LP compared to the B-LP was consistent with the DLS test results, further verifying the successful loading of each component. In terms of absorption spectra, the liposomes loaded with ICG and IR-1061 exhibited characteristic peaks of both ICG and IR-1061 at 600-1200 nm, with the characteristic peak of IR-1061 in the liposomes exhibiting a certain degree of blue shift. Fluorescence spectroscopy results indicate that IR-1061-ICG-QD-LP exhibits a strong emission peak at 1125 nm, with a fluorescence intensity approaching 1000, twice that of IR-1061-LP. Furthermore, results from the NIR-II in vivo imaging system demonstrate significantly higher fluorescence intensities than both ICG-LP and IR-1061-LP. This demonstrates that the nanoliposomes of the present invention, through a hydrophilic-hydrophobic layered co-loading strategy of the hydrophilic ICG and hydrophobic IR-1061 dyes and leveraging the fluorescence resonance energy transfer (FRET) mechanism, effectively reduce the IR-1061 dosage while maintaining excellent fluorescence imaging performance, significantly enhancing imaging results.

[0067] Test Example 3 like Figure 8 As shown in the figure, the MRI performance of a photoacoustic-magnetic trimodal imaging nanoliposome in Example 1 was investigated, and the results showed that the relaxation rate of IR-1061-ICG-QD-LP was r1 = 0.153 mM -1 s -1 , the linear correlation coefficient between MR signal intensity and Cu concentration was R2 = 0.994.

[0068] Figure 8 The five MRI images above, which gradually brighten with increasing Cu concentration, demonstrate that IR-1061-ICG-QD-LP can induce T1-weighted signal enhancement. Therefore, this experiment demonstrates that IR-1061-ICG-QD-LP nanoparticles can be used as a T1-weighted MRI contrast agent.

[0069] Beneficial effects of this test example This test case investigated the MRI performance of IR-1061-ICG-QD-LP, and the results showed that its relaxivity was r1=0.153 mM -1 s -1 , the linear correlation coefficient R between MR signal intensity and Cu concentration 2= 0.994. As the Cu concentration increased, the MRI images gradually brightened, indicating that IR-1061-ICG-QD-LP can induce T1-weighted signal enhancement and can be used as a T1-weighted MRI contrast agent. This demonstrates that the nanoliposomes of Example 1 successfully incorporated u-CuS QDs, resulting in excellent MRI performance. This lays an important foundation for multimodal imaging and further demonstrates its potential application in biomedical imaging.

[0070] Test Example 4 like Figure 9 As shown, the photoacoustic and magnetic trimodal imaging nanoliposomes of Example 1 were subjected to PA imaging. The results showed that the photoacoustic signal intensity of the IR-1061-ICG-QD-LP aqueous solution increased linearly with the liposome concentration (R 2 =0.9923), with a slope of 0.1816. At the same Cu ion concentration, the QD-LP solution exhibited weaker photoacoustic signal intensity and enhancement trend than the IR-1061-ICG-QD-LP solution (slope of 0.08558). These results indicate that IR-1061 and copper sulfide quantum dots produce a synergistically enhanced photoacoustic signal. This synergistic enhancement of the photoacoustic signal makes the nanoliposomes suitable as contrast agents for photoacoustic imaging.

[0071] Beneficial effects of this test example In photoacoustic imaging, the photoacoustic signal intensity of the IR-1061-ICG-QD-LP aqueous solution increased linearly with liposome concentration (R² = 0.9923), with a slope of 0.1816. At the same Cu ion concentration, the QD-LP solution exhibited weaker photoacoustic signal intensity and enhancement than the IR-1061-ICG-QD-LP solution (slope of 0.08558). This indicates that IR-1061 and copper sulfide quantum dots produce a synergistically enhanced photoacoustic signal, making the nanoliposomes suitable as contrast agents for photoacoustic imaging. This further demonstrates that the integration of multiple imaging probes into the nanoliposomes of the present invention creates a synergistic effect between the components, significantly enhancing photoacoustic imaging and achieving enhanced imaging results while simultaneously achieving photoacoustic and magnetic trimodal imaging.

[0072] Test Example 5 like Figure 10As shown, the photothermal performance of a photoacoustic-magnetic trimodal imaging nanoliposome from Example 1 was studied. Under continuous 650 nm laser irradiation for 2 minutes, the temperature of pure water increased by approximately 2.5°C, and the temperature increased by approximately 5°C at a 1 mg / mL IR-1061-ICG-QD-LP phospholipid concentration. The temperature change of the liposome solution gradually increased with increasing IR-1061-ICG-QD-LP phospholipid concentration, indicating that the photothermal conversion of IR-1061-ICG-QD-LP is concentration-dependent. At a 5 mg / mL IR-1061-ICG-QD-LP phospholipid concentration, the temperature of the liposome solution increased by 30°C within 2 minutes. This demonstrates the good photothermal efficiency of IR-1061-ICG-QD-LP.

[0073] Beneficial effects of this test example This experimental example studied the photothermal performance of IR-1061-ICG-QD-LP. Under the condition of continuous irradiation of 650nm laser for 2 minutes, the temperature of pure water rose by about 2.5 °C, while the temperature of IR-1061-ICG-QD-LP phospholipid increased by about 5 °C when the concentration of phospholipid was 1 mg / mL. As the liposome concentration increased, the temperature change gradually increased. When the phospholipid concentration was 5 mg / mL, the temperature of the liposome solution rose by 30 °C within 2 minutes, indicating that its photothermal conversion was concentration-dependent and had good photothermal efficiency. This shows that the nanoliposomes of Example 1 not only performed well in imaging, but also had good photothermal therapy performance. It can be used as a thermal therapy probe to produce photothermal effects while imaging, realizing the integration of precise diagnosis and treatment, and can be widely used in clinical practice.

[0074] Test Example 6 like Figure 11 As shown in the figure, by observing the particle size changes of a photoacoustic-magnetic trimodal imaging nanoliposome over time, it can be seen that IR-1061-ICG-QD-LP can maintain a particle size change of <10% within 30 days at 4°C, has good stability, and can be used as a photoacoustic-magnetic trimodal imaging drug used in medical treatment.

[0075] Beneficial effects of this test example Long-term observation of the particle size of IR-1061-ICG-QD-LP over time revealed that the particle size change was maintained at <10% for 30 days at 4°C. This demonstrates that the photoacoustic-magnetic trimodal imaging nanoliposomes of the present invention possess excellent stability and can maintain their physical properties over a long period of time. This suggests that they can maintain long-term stability during storage, transportation, and clinical use, and are suitable for large-scale clinical application.

Claims

1. A photoacoustic-magnetic trimodal imaging nanoliposome, characterized in that: The nano liposome is composed of a phospholipid bilayer, wherein the phospholipid bilayer membrane comprises phospholipid, cholesterol, IR-1061, DSPE-PEG2000 and u-CuS QDs; and the liposome aqueous phase is loaded with indocyanine green.

2. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 1, characterized in that: The molar ratio of the phospholipid to indocyanine green is 20-200:

1.

3. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 1, characterized in that: The molar ratio of the phospholipid to IR-1061 is 20-200:

1.

4. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 1, characterized in that: The mass ratio of the phospholipid to u-CuS QDs is 20-200:

1.

5. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 1, characterized in that: The molar ratio of the phospholipid to cholesterol is 2-4:

1.

6. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 1, characterized in that: The phospholipids include electrically neutral phospholipid molecules and DSPE-PEG2000.

7. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 6, characterized in that: The molar ratio of the electrically neutral phospholipid molecules to DSPE-PEG2000 is 8-10:

1.

8. The photoacoustic-magnetic trimodal imaging nanoliposome according to claim 6, characterized in that: The electrically neutral phospholipid molecules include dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine and distearoylphosphatidylcholine.

9. A method for preparing photoacoustic and magnetic trimodal imaging nanoliposomes, characterized in that: For preparing the photoacoustic-magnetic trimodal imaging nanoliposome according to any one of claims 1 to 8, comprising: S101, mixing sulfur with oleylamine to form a sulfur-oleylamine complex, and mixing copper acetylacetonate with oleylamine and chloroform to form a copper-oleylamine complex; S102, slowly mixing the copper-oleylamine complex and the sulfur-oleylamine complex, dispersing them mechanically, and washing with an organic solvent to obtain u-CuS QDs.

10. A method for preparing photoacoustic and magnetic trimodal imaging nanoliposomes according to claim 9, characterized in that: Also includes: S201, mixing materials for preparing a phospholipid bilayer to form a uniform solution, and removing the organic solvent by rotary evaporation to obtain a phospholipid film; S202, preparing indocyanine green into an indocyanine green solution using PBS to obtain an aqueous phase; S203, adding the aqueous phase into the phospholipid film for hydration, and dispersing by mechanical force to obtain indocyanine green liposomes.

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