FA-ICG-NBs nanobubbles, preparation method and application

Abstract

This invention discloses a FA-ICG-NBs nanobubble, its preparation method, and its application in the preparation of tumor-targeted therapeutic agents. The FA-ICG-NBs nanobubble employs a composite phospholipid shell, with folic acid (FA) covalently bonded to the surface and indocyanine green (ICG) loaded onto it, encapsulating a mixture of oxygen and sulfur hexafluoride. The preparation method includes: preparing a lipid solution for forming the composite phospholipid shell; mixing the lipid solution with the oxygen and sulfur hexafluoride mixture, and then performing a reciprocating shearing gas-liquid interface self-assembly to obtain the FA-ICG-NBs nanobubble. The FA-ICG-NBs nanobubble prepared by this invention utilizes dual-wavelength photoacoustic imaging for non-invasive assessment of tumor blood oxygenation and hypoxia improvement, combined with ICG fluorescence timing for screening the optimal therapeutic window, and synergistic oxygen-enhanced sonodynamic therapy. It exhibits good biocompatibility, structural stability, and integrates targeted enrichment, blood oxygenation assessment, hypoxia monitoring, timing regulation, and enhanced therapeutic effects.

Description

Technical Field

[0001] This invention relates to a FA-ICG-NBs nanobubble, its preparation method, and its application in the preparation of tumor-targeted diagnostic and therapeutic agents. Specifically, it relates to a lipid nanobubble FA-ICG-NBs that is covalently modified with folic acid (FA), covalently loaded with indocyanine green (ICG), and co-encapsulated with an oxygen-fluorine mixed gas, belonging to the field of biomedical nanomaterials. Background Technology

[0002] The core technological challenge of precision oncology lies in achieving a complete closed loop of efficient targeted drug delivery, rational timing of treatment, dynamic non-invasive monitoring of the tumor hypoxic microenvironment, and synergistic improvement in treatment efficacy. Tumor hypoxia is a unique microenvironmental characteristic commonly found in solid tumors and a key factor contributing to drug resistance and significant attenuation of therapeutic efficacy in sonodynamic therapy, radiotherapy, chemotherapy, and targeted therapy. Therefore, real-time non-invasive quantitative assessment of tumor blood oxygen saturation and visual dynamic monitoring of the degree of improvement in tumor hypoxia are crucial prerequisites and important guarantees for optimizing tumor treatment plans and improving clinical outcomes.

[0003] Indocyanine green (ICG) is a clinically available near-infrared multifunctional biomedical reagent with excellent near-infrared fluorescence imaging and tracing performance. It can also serve as a highly efficient photosensitizer for mediating sonodynamic therapy of tumors, integrating intracellular targeted accumulation tracing and therapeutic mediation, making it an ideal functional component for constructing integrated tumor diagnostic and therapeutic agents. However, free ICG has inherent drawbacks such as a short circulating half-life, rapid metabolic clearance, low tumor-targeted enrichment concentration, and difficulty in achieving long-term sustained functional effects, making it impossible to achieve stable imaging tracing and highly efficient sonodynamic therapy synergistically on its own. Existing conventional phospholipid nanobubble structures are simple in design and single in function, with difficulties in effectively loading targeting molecules and photosensitizing agents. Furthermore, their preparation processes are crude, resulting in large bubble sizes and poor structural stability, failing to meet the requirements for tumor targeted enrichment and long-term imaging therapy, and making it difficult to reverse tumor hypoxia and improve the efficacy of sonodynamic therapy. Summary of the Invention

[0004] To address the shortcomings of existing diagnostic and therapeutic agents, such as insufficient targeting, fragmented imaging and therapeutic functions, poor bubble stability due to simple preparation processes, limited improvement of hypoxia, and difficulty in controlling the timing of treatment, this invention employs a quantitative lipid scientific ratio and a proprietary low-pressure reciprocating shear perturbation preparation process to construct FA-ICG-NBs targeted multifunctional nanobubbles. This integrates FA targeting, ICG fluorescence tracing and photosensitized therapy, and oxygen-fluorine mixed bubble stabilization for enhanced efficacy, thus meeting the practical application needs of precision tumor diagnosis and treatment.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a FA-ICG-NBs nanobubble, wherein the FA-ICG-NBs nanobubble adopts a composite phospholipid membrane shell, the surface of the composite phospholipid membrane shell is covalently modified with folic acid (FA) and loaded with indocyanine green (ICG), and the interior is encapsulated with a mixture of oxygen and sulfur hexafluoride.

[0007] Oxygen is used to improve tumor hypoxia and relieve inhibition of sonodynamic therapy; sulfur hexafluoride is highly inert, has low water solubility, and is not easily diffused, which helps maintain the stability of the bubble structure and reduce gas leakage; the surface of the composite phospholipid membrane is covalently modified with folic acid (FA) targeting molecules to achieve active targeted enrichment of the nanobubbles into the tumor; the composite phospholipid membrane is loaded with indocyanine green (ICG), which has near-infrared fluorescence imaging tracer and can also act as a photosensitizer to mediate sonodynamic therapy; the average hydrodynamic particle size of the FA-ICG-NBs nanobubbles is 292 nm. By using 757 nm and 855 nm dual-wavelength photoacoustic imaging combined with endogenous hemoglobin detection of tumor blood oxygenation, the improvement of hypoxia can be monitored in real time, and the optimal treatment time can be determined by ICG fluorescence timing, synergistically enhancing sonodynamic therapy with oxygen.

[0008] In some embodiments, the FA-ICG-NBs nanobubbles have a spherical structure and an average hydrodynamic particle size of 280–305 nm, preferably 292 nm.

[0009] The FA-ICG-NBs described in this application have an average hydrodynamic particle size of 292 nm, which is adapted to the permeability requirements of the endothelial space of tumor tissue and the standard of normal blood circulation in vivo. They have both good tumor enrichment and penetration capabilities and in vivo biosafety. Furthermore, they can release internally encapsulated oxygen under ultrasound stimulation, thereby improving the effect of tumor hypoxia and synergistically enhancing the therapeutic effect of ICG-mediated sonodynamic therapy.

[0010] In some embodiments, the composite phospholipid membrane shell is prepared by mixing DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k, and DSPC in a certain proportion.

[0011] In some embodiments, under normal temperature (25 degrees Celsius) and normal oxygen (normal atmospheric) conditions, the oxygen content of the FA-ICG-NBs nanobubble aqueous solution diluted 5 times is 10~20 mg / L, and in this embodiment it is 14.41 mg / L.

[0012] Secondly, the present invention provides a method for preparing FA-ICG-NBs nanobubbles, comprising:

[0013] Prepare a lipid solution for forming a composite phospholipid membrane shell;

[0014] FA-ICG-NBs nanobubbles were obtained by mixing a lipid solution with a mixture of oxygen and sulfur hexafluoride gas and then self-assembling the gas-liquid interface through reciprocating shearing.

[0015] In some embodiments, a lipid solution for forming a composite phospholipid membrane shell is prepared, comprising:

[0016] The lipid materials DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k and DSPC were dissolved in an hydrated solution to obtain a lipid solution;

[0017] In the hydrated solution, the mass ratio of ultrapure water, glycerol, anhydrous ethanol and sodium chloride is 1:0.1386:0.0456:0.009.

[0018] The mass ratio of the lipid materials DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k, and DSPC is (0.10~0.16):(0.10~0.16):(0.60~0.90):(0.08~0.12), preferably 0.13:0.13:0.75:0.10;

[0019] The mass-to-volume ratio of lipid material to hydrated solution is (0.90~1.30) mg:2 mL, preferably 1.11 mg:2 mL.

[0020] In some embodiments, the volume ratio of oxygen to sulfur hexafluoride in the mixed gas of oxygen and sulfur hexafluoride is (40~52):(4~8), preferably 44:6.

[0021] Oxygen in the mixed gas is used to reverse tumor hypoxia and ensure treatment efficacy; SF6 is highly inert, has low water solubility, and does not diffuse easily, maintaining the stability of the bubble structure and reducing gas leakage.

[0022] In some embodiments, a lipid solution is mixed with a mixture of oxygen and sulfur hexafluoride gas, and FA-ICG-NBs nanobubbles are obtained by reciprocating shearing of the gas-liquid interface. The mixture includes:

[0023] A lipid solution was sealed in a container and combined with a syringe filled with a mixture of oxygen and sulfur hexafluoride to form a sealed system. After the air in the container was expelled, the syringe piston was repeatedly pushed under a water bath at 58℃~65℃ to control the reduction of the gas volume inside the container between 0% and 67%. This process was repeated several times. The fluid disturbance and shear stress field generated when the syringe needle expelled the gas promoted the thorough homogeneous mixing of the lipid phase and the gas phase, thereby enabling the lipid material to form a fusion film. Free bubbles were generated during the repeated pressurization and depressurization of the system, and the fusion film self-assembled at the gas-liquid interface to prepare FA-ICG-NBs nanobubbles.

[0024] It should be noted that the composite phospholipid membrane shell described in this application is prepared by scientifically proportioning multiple lipid materials, including DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k, and DSPC. The proportions of each component meet the requirements for membrane shell preparation. The membrane shell is prepared by water bath ultrasonic dissolution, low-pressure reciprocating shearing, and gas-liquid interface self-assembly, forming a composite membrane structure with excellent biocompatibility. The membrane shell achieves simultaneous folic acid targeted modification and stable ICG loading through phospholipid covalent linkage, avoiding rapid metabolic clearance of ICG in vivo, while effectively prolonging the in vivo circulation time of nanobubbles and stably maintaining the physicochemical structure and targeted diagnostic and therapeutic functions of nanobubbles.

[0025] In DSPE-PEG2k-ICG, ICG is stably covalently bonded to the end of the DSPE-PEG2k phospholipid chain via amide bonds, resulting in a strong bond that is not easily dissociated. The intensity of the near-infrared fluorescence signal of ICG is positively correlated with the amount of nanobubbles accumulated at the tumor site. The time point when the fluorescence signal reaches its peak corresponds to the optimal intervention window for sonodynamic therapy, providing a reliable basis for rationally determining the timing of treatment.

[0026] Thirdly, the present invention provides the application of the FA-ICG-NBs nanobubbles in the preparation of tumor-targeted diagnostic and therapeutic agents, photoacoustic imaging tumor blood oxygenation assessment reagents, and tumor hypoxia dynamic monitoring reagents.

[0027] The tumor-targeted diagnostic and therapeutic agent utilizes FA active targeting to achieve precise tumor enrichment, relies on dual-wavelength photoacoustic imaging to complete non-invasive blood oxygen quantification and hypoxia monitoring, and uses ICG near-infrared fluorescence imaging to trace the accumulation of nanobubbles in vivo, simultaneously carrying out diagnostic and therapeutic work.

[0028] Fourthly, this invention also provides the application of the aforementioned FA-ICG-NBs nanobubbles in the preparation of tumor sonodynamic therapy drugs that combine fluorescence timing regulation and photoacoustic visualization monitoring. The tumor sonodynamic therapy drug utilizes the excellent photosensitizer function of ICG to mediate highly efficient sonodynamic therapy, while simultaneously monitoring the tumor's blood oxygenation and hypoxia improvement status in real time through dual-wavelength photoacoustic imaging. The timing of treatment intervention is precisely controlled by changes in ICG fluorescence timing, achieving an integrated synergistic effect of precise tumor monitoring and highly efficient treatment.

[0029] This application's FA-ICG-NBs nanobubbles utilize endogenous hemoglobin in vivo to achieve non-invasive, quantitative, and dynamic tumor blood oxygen saturation detection, accurately reflecting the degree of improvement in the tumor hypoxic microenvironment; at the same time, the ICG core acts as a photosensitizer, efficiently generating reactive oxygen species under ultrasound triggering, which, together with the oxygen in the nanobubbles, synergistically enhances the tumor cell killing effect, achieving highly efficient sonodynamic therapy.

[0030] Compared with existing technologies, the present invention has the following advantages: The FA-ICG-NBs phospholipid nanobubbles of the present invention are lipid nanobubbles FA-ICG-NBs covalently modified with FA, covalently loaded with ICG, and co-encapsulated with an oxygen-fluorine mixed gas. The membrane shell is prepared by scientifically quantitatively compounding DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k, and DSPC, and then undergoing water bath ultrasonic dissolution, low-pressure reciprocating shearing, and gas-liquid interface self-assembly. It has the ability to actively target tumors and stably load ICG. ICG is used for near-infrared fluorescence imaging tracing and acoustic-dynamic photosensitivity. The interior is filled with a 44:6 mixture of oxygen and sulfur hexafluoride. Oxygen improves tumor hypoxia, while sulfur hexafluoride is highly inert, has low water solubility, and does not diffuse easily, maintaining the stability of the bubble structure and reducing gas leakage. The average particle size of the nanobubbles is 292 nm, and they have good biocompatibility. Dual-wavelength photoacoustic imaging is used to assess blood oxygenation, and ICG fluorescence is used to determine treatment time, synergistically achieving integrated targeted diagnosis and treatment. FA modification enables precise tumor targeting and reduces toxic side effects; dual-wavelength photoacoustic combined with ICG fluorescence timing allows for rational determination of treatment timing; the combined use of oxygen and fluorine mixed carrier gas improves hypoxia with oxygen, while sulfur hexafluoride stabilizes the bubbles and prevents leakage, ensuring long-term bubble stability; ICG integrates near-infrared fluorescence imaging tracing and photosensitive therapy, achieving integrated diagnosis and treatment with significant synergistic effects; and the use of a quantitative ratio and low-pressure shearing proprietary preparation process results in stable bubble structure and great potential for clinical translation. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the electron microscopy structure of FA-ICG-NBs nanobubbles obtained in an embodiment of the present invention;

[0032] Figure 2 A shows the change in average particle size of the FA-ICG-NBs solution over time. A stable particle size indicates good dispersion and excellent colloidal stability and morphological uniformity. B shows the concentration of FA-ICG-NBs (1.8 x 10⁻⁶) measured using nanoparticle tracking technology. 10 A high bubble concentration of 1 bubble per mL provides a favorable basis for improving tumor hypoxia and sonodynamic therapy.

[0033] Figure 3 Under normal temperature and oxygen conditions, the oxygen content of 1 mL of FA-ICG-NBs nanobubble aqueous solution (diluted 5 times) added to 4 mL of ultrapure water gradually increases over time, reaching a maximum of 14.41 mg / L.

[0034] Figure 4A shows the changes in near-infrared fluorescence intensity of FA-ICG-NBs and Free ICG solutions over time; B shows the quantification of near-infrared fluorescence intensity of FA-ICG-NBs and Free ICG solutions over time. Under the same ICG concentration, the near-infrared fluorescence intensity and fluorescence stability of the FA-ICG-NBs aqueous solution are significantly enhanced; after 48 h, the fluorescence intensity of the FA-ICG-NBs aqueous solution is approximately 1.77 times that of the Free ICG aqueous solution.

[0035] Figure 5 A shows the change in near-infrared fluorescence intensity at the tumor site in mice over time after tail vein injection of FA-ICG-NBs and Free ICG solution (equivalent ICG concentration 20 μg / mL, 80 μL); B shows the fluorescence intensity quantification corresponding to A. Thanks to the tumor-targeting effect of FA and the fluorescence stabilizing effect of nanobubbles, 8 h after drug injection, the fluorescence intensity of the FA-ICG-NBs group was 17.0 times that of the Free ICG group; 24 h later, the fluorescence intensity of the FA-ICG-NBs group was 13.6 times that of the Free ICG group.

[0036] Figure 6 A shows the change in blood oxygen saturation at the tumor site of mice over time after tail vein injection of FA-ICG-NBs and Free ICG solution; B shows the photoacoustic imaging intensity quantification corresponding to A, where the difference in blood oxygen saturation intensity between groups gradually increases over time, reaching the maximum difference at 4 hours, at which point the blood oxygen saturation intensity of the FA-ICG-NBs group is 18.2% higher than that of the PBS group.

[0037] Figure 7 A shows a comparison of tumor growth effects among the experimental groups; B shows the tumor size measurement corresponding to A. The tumor size of the FA-ICG-NBs group was only 6.3% of that of the control group and 17.0% of that of the Free ICG group.

[0038] Figure 8 This is a schematic diagram illustrating the preparation method of FA-ICG-NBs nanobubbles in the embodiments of this application. Detailed Implementation

[0039] The following description, in conjunction with the accompanying drawings and specific embodiments, provides a further detailed explanation of the above-described content of this application. However, this should not be construed as limiting the scope of the subject matter of this application to the following examples. Any modifications made without departing from the spirit and principles of this application, as well as equivalent substitutions or improvements made based on ordinary technical knowledge and common practice in the art, should be included within the scope of protection of this application.

[0040] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] In this embodiment, DSPE-PEG2k-FA and DSPE-PEG2k-ICG were purchased from Xi'an Ruixi Biotechnology Co., Ltd., DSPE-PEG2k was purchased from Jiangsu Southeast Nanomaterials Co., Ltd., and DSPC was purchased from Aivito (Shanghai) Pharmaceutical Technology Co., Ltd.

[0043] Example 1: A method for preparing FA-ICG-NBs nanobubbles, comprising the following steps:

[0044] Weigh the following lipid materials: DSPE-PEG2k-FA (0.13 mg), DSPE-PEG2k-ICG (0.13 mg), DSPE-PEG2k (0.75 mg), and DSPC (0.1 mg). Add the above lipid materials to 2 mL of hydrated solution composed of ultrapure water, glycerol, anhydrous ethanol, and sodium chloride in a mass ratio of 1:0.1386:0.0456:0.009. Sonicate in a water bath for 30 minutes to fully dissolve and mix the lipid materials to obtain a lipid solution.

[0045] Take 2 mL of the above lipid solution and seal it in a 3 mL reagent bottle. Combine this with a 5 mL syringe filled with O2 and SF6 (volume ratio 44:6) to form a sealed system. After expelling the air from the bottle, leave 3 mL of mixed gas in the syringe. Under a 60℃ water bath, repeatedly push the syringe piston to control the volume reduction ratio of the gas inside the reagent bottle between 0% and 67%, and repeat this cycle 20 times. Relying on the disturbance of the syringe and high shear stress, the mixed gas of O2 and SF6 is fully mixed with the lipid solution, promoting the formation of a fusion film of the lipid material. Free bubbles are generated during the repeated pressurization and depressurization of the system, and the fusion film self-assembles at the gas-liquid interface to prepare FA-ICG-NBs. Finally, ultrafiltration is used to remove unassembled free lipid impurities to obtain the purified FA-ICG-NBs nanobubbles.

[0046] The FA-ICG-NBs prepared in Example 1 were tested below:

[0047] I. Physicochemical characterization:

[0048] Take 1 mL of the nanobubble solution prepared in Example 1, and determine its particle size and particle size distribution using dynamic light scattering. Figure 1As shown, the prepared FA-ICG-NBs nanobubbles have an average hydrodynamic particle size of 292 nm and are stable when stored at 4℃. The particle size is suitable for the permeability requirements of tumor tissue endothelial spaces and the standards of normal blood circulation in vivo, combining good tumor enrichment and penetration capabilities with in vivo biocompatibility. Furthermore, they can release encapsulated oxygen under ultrasound stimulation, enhancing the improvement of tumor hypoxia and synergistically improving ICG-mediated sonodynamic therapy.

[0049] Figure 2 In the middle: A represents the change in average particle size of the FA-ICG-NBs solution over time. A stable particle size indicates good dispersion and excellent colloidal stability and morphological uniformity. B represents the concentration of FA-ICG-NBs (1.8 x 10⁻⁶) measured using nanoparticle tracking technology. 10 A high bubble concentration of 1 bubble per mL provides a favorable basis for improving tumor hypoxia and sonodynamic therapy.

[0050] 10 μL of the nanobubble solution prepared in Example 1 was dropped onto a 300-mesh copper grid, allowed to stand for 10 minutes, and after removing excess sample with filter paper, it was vacuum dried for 12 hours. The sample was then examined using a transmission electron microscope (accelerating voltage 100 kV). Figure 1 As shown, the microstructure of FA-ICG-NBs nanobubbles is a spherical structure with a particle size of about 200 nm.

[0051] Example 2: In vitro performance characterization of FA-ICG-NBs

[0052] Figure 4 A represents the change in near-infrared fluorescence intensity of FA-ICG-NBs, ICG-NBs, and Free ICG solutions over time; B represents the quantification of near-infrared fluorescence intensity of FA-ICG-NBs, ICG-NBs, and Free ICG solutions over time.

[0053] Preparation method of Free ICG solution: Weigh 10 μg (20 μg) of ICG powder and dissolve it in 1 mL of ultrapure water to prepare a 10 μg / mL (20 μg / mL) Free ICG solution.

[0054] Physicochemical properties were tested. The prepared FA-ICG-NBs solution was stored at 4℃ in the dark for one week, and its average particle size was measured continuously. The change in particle size with storage time was analyzed. Stable particle size indicates that the system has good dispersion and excellent colloidal stability and morphological uniformity. The concentration of FA-ICG-NBs measured by nanoparticle tracking technology was 1.8*10. 10 A high bubble concentration of [number] bubbles / mL provides a favorable basis for improving tumor hypoxia and sonodynamic therapy. Figure 2 ).

[0055] Under normal temperature and oxygen conditions, the oxygen content in 4 mL of ultrapure water after adding 1 mL of FA-ICG-NBs nanobubble aqueous solution (diluted 5 times) gradually increased over time, reaching a maximum of 14.41 mg / L. (See [reference needed]). Figure 3 Take 1 mL of the prepared FA-ICG-NBs and free ICG solution, and add them to each of the 12-well culture plates. Use a small animal in vivo imaging system for fluorescence imaging detection. Figure 4 As shown, A is a schematic diagram of the change of near-infrared fluorescence intensity of FA-ICG-NBs and Free ICG solutions over time; B is a schematic diagram of the quantification of near-infrared fluorescence intensity of FA-ICG-NBs and Free ICG solutions over time. Under the same ICG concentration, the near-infrared fluorescence intensity and fluorescence stability of the FA-ICG-NBs aqueous solution are significantly enhanced; after 48 h, the fluorescence intensity of the FA-ICG-NBs aqueous solution is about 1.77 times that of the Free ICG aqueous solution.

[0056] Example 3: Validation of the in vivo tumor diagnosis and treatment efficacy of FA-ICG-NBs

[0057] A mouse subcutaneous solid tumor model was constructed, and in vivo experiments were conducted by tail vein injection of FA-ICG-NBs; for example... Figure 5 As shown, ICG fluorescence time-series monitoring showed that FA-ICG-NBs had a good targeted enrichment effect, and the tumor accumulation reached its peak 8 hours after administration, which was determined to be the optimal treatment window.

[0058] Figure 5 The near-infrared fluorescence intensity of mouse tumor sites was measured over time after tail vein injection of FA-ICG-NBs and Free ICG solution (equivalent ICG concentration 20 μg / mL, 80 μL). Eight h after injection, the fluorescence intensity of the FA-ICG-NBs group was 17.0 times that of the Free ICG group; 24 h later, the fluorescence intensity of the FA-ICG-NBs group was 13.6 times that of the Free ICG group.

[0059] Figure 6 In case A, FA-ICG-NBs (ICG concentration 20 μg / mL) and 80 μL of PBS solution were injected via the tail vein. The difference in blood oxygen saturation intensity between the groups gradually increased over time, reaching the maximum difference at 4 h. At this time, the blood oxygen saturation intensity of the FA-ICG-NBs group was 18.2% higher than that of the PBS group.

[0060] Figure 7 The diagram shows the comparison of tumor growth effects among the experimental groups. The tumor volume in the FA-ICG-NBs group was only 6.3% of that in the control group and 17.0% of that in the free ICG group, with a corresponding tumor inhibition rate as high as 93.7%, demonstrating a significant tumor inhibition effect.

[0061] In summary, the FA-ICG-NBs prepared in this application possess good structural stability, fluorescence / photoacoustic dual-modal imaging performance, and excellent tumor targeting and tumor suppression effects, providing an ideal nanoplatform for precise tumor imaging monitoring and targeted therapy.

[0062] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A FA-ICG-NBs nanobubble, characterized in that, The FA-ICG-NBs nanobubbles are made of composite phospholipid membrane shells. The surface of the composite phospholipid membrane shells is covalently modified with folic acid (FA) and loaded with indocyanine green (ICG), and the interior is filled with a mixture of oxygen and sulfur hexafluoride.

2. The FA-ICG-NBs nanobubbles according to claim 1, characterized in that, The FA-ICG-NBs nanobubbles have a spherical structure and an average hydrodynamic particle size of 280–305 nm, preferably 292 nm. And / or, the composite phospholipid membrane shell is prepared by mixing DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k, and DSPC in a certain proportion.

3. The FA-ICG-NBs nanobubbles according to claim 1, characterized in that, The oxygen content of the FA-ICG-NBs nanobubble aqueous solution after being diluted 5 times reaches 10~20 mg / L.

4. The method for preparing FA-ICG-NBs nanobubbles according to any one of claims 1-3, characterized in that, include: Prepare a lipid solution for forming a composite phospholipid membrane shell; FA-ICG-NBs nanobubbles were obtained by mixing a lipid solution with a mixture of oxygen and sulfur hexafluoride gas and then self-assembling the gas-liquid interface through reciprocating shearing.

5. The method for preparing FA-ICG-NBs nanobubbles according to claim 4, characterized in that, Preparation of a lipid solution for forming a composite phospholipid membrane shell includes: The lipid materials DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k and DSPC were dissolved in an hydrated solution to obtain a lipid solution.

6. The method for preparing FA-ICG-NBs nanobubbles according to claim 5, characterized in that, Meet at least one of the following: In the hydrated solution, the mass ratio of ultrapure water, glycerol, anhydrous ethanol and sodium chloride is 1:0.1386:0.0456:0.

009. In the lipid solution, the mass ratio of lipid materials DSPE-PEG2k-FA, DSPE-PEG2k-ICG, DSPE-PEG2k, and DSPC is (0.10~0.16):(0.10~0.16):(0.60~0.90):(0.08~0.12), preferably 0.13:0.13:0.75:0.10; The mass-to-volume ratio of lipid material to hydrated solution is (0.90~1.30) mg:2 mL, preferably 1.11 mg:2 mL.

7. The method for preparing FA-ICG-NBs nanobubbles according to claim 4, characterized in that, In the mixture of oxygen and sulfur hexafluoride, the volume ratio of oxygen to sulfur hexafluoride is (40~52):(4~8), preferably 44:

6.

8. The method for preparing FA-ICG-NBs nanobubbles according to claim 4, characterized in that, Lipid solutions were mixed with a mixture of oxygen and sulfur hexafluoride gas, and FA-ICG-NBs nanobubbles were obtained by reciprocating shearing of the gas-liquid interface. These nanobubbles included: A lipid solution was sealed in a container and combined with a syringe filled with a mixture of oxygen and sulfur hexafluoride to form a sealed system. After the air in the container was expelled, the syringe piston was repeatedly pushed under a water bath at 58℃~65℃ to control the reduction of the gas volume inside the container between 0% and 67%. This process was repeated several times. The fluid disturbance and shear stress field generated when the syringe needle expelled the gas promoted the thorough homogeneous mixing of the lipid phase and the gas phase, thereby enabling the lipid material to form a fusion film. Free bubbles were generated during the repeated pressurization and depressurization of the system, and the fusion film self-assembled at the gas-liquid interface to prepare FA-ICG-NBs nanobubbles.

9. The application of FA-ICG-NBs nanobubbles according to any one of claims 1-8 in the preparation of tumor-targeted diagnostic and therapeutic agents, photoacoustic imaging tumor blood oxygenation assessment reagents, and tumor hypoxia dynamic monitoring reagents.

10. The application of FA-ICG-NBs nanobubbles according to any one of claims 1-8 in the preparation of tumor acoustic dynamic therapy drugs with both fluorescence timing regulation and photoacoustic visualization monitoring.

Images