Targeted drug-loaded nano-bubble as well as preparation method and application thereof

Abstract

The invention relates to the technical field of targeted drug-loaded nanobubbles, and discloses a targeted drug-loaded nanobubble and a preparation method and application thereof, and the targeted drug-loaded nanobubble is prepared by chemically connecting GEM and cholesterol through an amide reaction. According to the scheme, the characteristic that a cholesterol shell of the nano-bubble can be combined with NPC1L1 with ultrahigh expression of pancreatic cancer cells in a targeting manner is utilized, and a chemotherapeutic drug GEM is carried on the shell of the nano-bubble, so that the targeting GEM-loaded nano-bubble is constructed. The ultrasonic molecular imaging characteristics of the nano-bubble after penetrating through tumor blood vessels and being combined with pancreatic cancer cells in a targeted manner and the effect of the nano-bubble on relieving CD8 + T cell immunosuppression in a tumor microenvironment are researched and discussed, and the effect of ultrasonic irradiation nano-bubble burst to release GEM to accurately kill pancreatic cancer cells and improve the synergistic treatment effect of the tumor immune microenvironment is clarified; a safe and effective brand-new strategy for synergistic chemotherapy and immune combined treatment is provided for diagnosis and treatment of pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted drug-loaded nanobubbles, and in particular to a targeted drug-loaded nanobubble and a preparation method and application thereof. Background Art

[0002] Pancreatic cancer is a highly malignant digestive system tumor. Its early symptoms are hidden and lack specific clinical manifestations, making early diagnosis extremely difficult. Most patients are already in the late stage of the disease when diagnosed, having missed the best time for radical surgical resection. This makes the search for effective early diagnosis and treatment methods a major problem that needs to be overcome in the current clinical diagnosis and treatment of pancreatic cancer. At the same time, recent studies have found that abnormal cholesterol metabolism plays a key role in the occurrence and development of pancreatic cancer. The cholesterol transporter Niemann-pick-c1-like 1 (NPC1L1) in pancreatic cancer tissue is abnormally overexpressed, and its expression level in pancreatic cancer tissue is nearly 100 times higher than that in normal pancreatic tissue. NPC1L1 can not only specifically bind to cholesterol in pancreatic cancer tissue, but also prompt pancreatic cancer cells to competitively plunder cholesterol from the tumor microenvironment, and even "hijack" CD8 + Cholesterol in T cells provides a new direction for the research of therapeutic targets for pancreatic cancer.

[0003] Currently, chemotherapy plays an increasingly important role in the comprehensive treatment of pancreatic cancer. It can provide some tumors that were previously incompletely resectable with the opportunity for radical resection, thereby improving patient prognosis. Pancreatic cancer chemotherapy drugs, represented by gemcitabine (GEM), can prolong the survival of some pancreatic cancer patients in clinical applications. However, their long-term treatment effects are still unsatisfactory, seriously restricting the improvement of patients' prognosis. In-depth analysis of the reasons for this is mainly due to the dense interstitial structure and lack of blood supply of pancreatic cancer tissue, which seriously hinders the effective delivery of drugs to the tumor site. At the same time, the immunosuppressive microenvironment of pancreatic cancer also greatly weakens the effect of chemotherapy.

[0004] In view of this, the development of a targeted drug-loaded nanobubble and its preparation method and application not only effectively makes up for the shortcomings of existing technologies, but also has important significance for enhancing the chemotherapy effect and improving the prognosis of pancreatic cancer patients. Summary of the Invention

[0005] The present invention aims to provide a targeted drug-loaded nanobubble and its preparation method and application, in order to solve the technical problem that the existing GEM chemotherapy has poor drug delivery, resulting in unsatisfactory effect in controlling pancreatic cancer through chemotherapy and seriously restricting the improvement of prognosis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing targeted drug-loaded nanobubbles, comprising chemically linking GEM and cholesterol through an amide reaction to prepare targeted GEM-loaded nanobubbles.

[0007] Preferably, as an improvement, the method comprises the following steps: Step 1: Preparation of CHOL-PEG2000-GEM: S1. Add CHOL-PEG2000-COOH and GEM to dimethyl sulfoxide and shake to dissolve to obtain mixed solution I; S2. Dissolve dicyclohexylcarbodiimide and 4-dimethylaminopyridine in dimethyl sulfoxide, and then slowly add dropwise to the mixed solution I. Rotate under nitrogen to cause an amide reaction, and then add methanol to quench the reaction to obtain a reaction solution. S3. The reaction solution was transferred to a dialysis bag to purify the compound. Unreacted compounds and organic solvents were removed by magnetic stirring at 4°C for 24 hours, and then freeze-dried to obtain CHOL-PEG2000-GEM. Step 2: Preparation of targeted drug-loaded nanobubbles CHOL@GEM-NBs: S4, preparing a mixed lipid molecule, the mixed lipid molecule including DPPA, DPPC, DPPE, DPPG and CHOL-PEG2000-GEM prepared in S3; S5. Dissolve the mixed lipid molecules in glycerol:PBS hydration solution and shake thoroughly in an ice-water bath at 0°C to obtain mixed solution II. S6. Transfer the mixed solution II to a vial and introduce perfluoropropane gas to replace the air in the vial. After sufficient shaking, let it stand at low temperature, then centrifuge and wash for purification. The middle emulsion is the cholesterol-shelled nanobubbles CHOL@GEM-NBs loaded with GEM.

[0008] Preferably, as an improvement, in S1, the mixing molar ratio of CHOL-PEG2000-COOH and GEM is 1:1 to 1:3.

[0009] Preferably, as an improvement, in S2, the molar ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:0.05-1:0.1; and the amide reaction is carried out by rotating at 25-40° C. for 12-24 hours.

[0010] Preferably, as an improvement, in S4, the mixed lipid molecules include DPPA, DPPC, DPPE, DPPG, and CHOL-PEG2000-GEM, which are mixed in a mass ratio of 1:3~4:3~4:3~4:1~2.

[0011] Preferably, as an improvement, in S5, the volume ratio of glycerol to PBS in the glycerol:PBS hydration solution is 1:9.

[0012] Preferably, as an improvement, in S6, the low-temperature standing is standing in a 4°C refrigerator for 2 to 12 hours; and the centrifugation is centrifugation at 300 to 500 rpm / min for 3 to 5 minutes.

[0013] Preferably, as an improvement, the method further comprises S7, preparing blank nanobubbles Blank NBs: replacing CHOL-PEG2000-GEM with CHOL-PEG2000-GEM, repeating S4 to S6, and replacing CHOL-PEG2000-GEM in the CHOL@GEM-NBs with DSPE-PEG2000 to prepare blank nanobubbles Blank NBs; The method further includes S8, preparing cholesterol nanobubbles CHOL@NBs: replacing CHOL-PEG2000-GEM with DSPE-PEG2000 and CHOL, repeating S4 to S6, replacing CHOL-PEG2000-GEM in the above CHOL@GEM-NBs with DSPE-PEG2000 and CHOL, and preparing cholesterol nanobubbles CHOL@NBs.

[0014] Preferably, as an improvement, the present solution also provides a targeted drug-loaded nanobubble prepared by the above preparation method.

[0015] Preferably, as an improvement, the present solution also provides a use of targeted drug-loaded nanobubbles in the preparation of a pancreatic cancer chemotherapy drug composition.

[0016] The principles and advantages of this solution are: Pancreatic cancer cell subpopulations that overexpress NPC1L1 are the dominant cell population in the tumor microenvironment that suppresses anti-tumor immunity. This protocol exploits the ability of the nanobubble's cholesterol shell to bind to hyperexpressed NPC1L1 in pancreatic cancer cells. The chemotherapeutic drug GEM (gemcitabine) is then loaded onto the nanobubble's shell to construct targeted GEM-loaded nanobubbles. These targeted, drug-loaded nanobubbles efficiently accumulate in pancreatic cancer cells with high NPC1L1 expression. Ultrasound imaging significantly enhances the contrast between tumor tissue and surrounding normal tissue, clearly demonstrating the location, size, and morphology of the tumor, significantly improving the accuracy of early diagnosis of pancreatic cancer. Ultrasound-induced nanobubble bursting also enables visual therapy, enabling targeted and precise destruction of pancreatic cancer cells with high NPC1L1 expression, effectively killing the cell subpopulation that contributes to the malignant phenotype of pancreatic cancer. The multifunctional, integrated nanobubbles prepared in this protocol combine diagnostic and therapeutic functions, achieving integrated diagnosis and treatment for pancreatic cancer.

[0017] 1. Compared to existing direct chemotherapy technologies, which are generally less effective, this approach innovatively targets the cholesterol metabolism target NPC1L1 in pancreatic cancer cells, constructing targeted drug-loaded nanobubbles (CHOL@GEM-NBs). Combined with ultrasound irradiation, this approach achieves precise drug release and effective killing of pancreatic cancer cells with high NPC1L1 expression. This effectively reverses the suppressive immune microenvironment of pancreatic cancer, enhances chemotherapy efficacy, promotes anti-tumor immunity in pancreatic cancer, and achieves integrated ultrasound molecular diagnosis and treatment of pancreatic cancer.

[0018] 2. The targeted drug-loaded nanobubbles in this approach exhibit excellent stability and penetrability. They can bind to NPC1L1, a protein highly expressed in pancreatic cancer cells. Combined with ultrasound irradiation and cavitation, they effectively increase the permeability of tumor blood vessels and cells, overcoming the hypovascular microenvironment of pancreatic cancer and ensuring the local concentration of CHOL@GEM-NBs in the tumor, thereby effectively enhancing chemotherapy efficacy and improving prognosis.

[0019] 3. The lipid material used to prepare the targeted drug-loaded nanobubbles and the perfluoropropane gas within the nanobubbles have been shown to exhibit excellent biosafety and promising clinical application prospects. They can serve as highly efficient drug delivery vehicles, effectively carrying GEM, a commonly used chemotherapy drug for pancreatic cancer. These drugs are targeted to tumor tissues, and upon ultrasound irradiation, the nanobubbles rupture and release the GEM, precisely killing tumor cells. This approach can effectively reduce the toxic side effects of systemic GEM chemotherapy, laying the foundation for further clinical translation and application.

[0020] 4. This scheme constructs targeted nanobubbles for pancreatic cancer. Compared with traditional blood pool contrast agents, they have stronger penetration and targeting specificity. They can pass through tumor blood vessels into tumor tissue and bind to relevant targets on the surface of tumor cells to achieve specific ultrasound molecular imaging of the tumor. The imaging effect is stable and is expected to solve the current problems in early diagnosis and efficacy evaluation of pancreatic cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the chemical synthesis of CHOL-PEG2000-GEM in an embodiment of the present invention (a, synthesis flow chart of CHOL-PEG2000-GEM; b, composition and structure diagram of CHOL@GEM-NBs).

[0022] Figure 2 1H NMR spectrum of CHOL-PEG2000-GEM in the embodiment of the present invention.

[0023] Figure 3 Characteristics of CHOL@GEM-NBs in an embodiment of the present invention (a, CHOL@GEM-NBs observed under a light microscope; b, CHOL@GEM-NBs observed under an electron microscope).

[0024] Figure 4Characteristics of CHOL@GEM-NBs in the examples of the present invention (a, particle size distribution of CHOL@GEM-NBs and Blank NBs; b, Zeta potential of CHOL@GEM-NBs and Blank NBs).

[0025] Figure 5 This is the stability evaluation of the particle size and polydispersity index (PDI) of CHOL@GEM-NBs in the examples of the present invention.

[0026] Figure 6 The drug loading rate of GEM in CHOL@GEM-NBs in the examples of the present invention (a, UV-Vis standard curve of GEM; b, UV-Vis absorption spectra of GEM, CHOL@GEM-NBs and Blank NBs at a wavelength of 270 nm).

[0027] Figure 7 The results of the hemolysis experiment of NBs in the examples of the present invention are shown (a, hemolysis image of CHOL@GEM-NBs co-incubated with red blood cells, PBS and 0.1% Triton X-100 were used as negative and positive controls, respectively; b, hemolysis rate statistics of CHOL@GEM-NBs and Blank NBs).

[0028] Figure 8 In vitro CEUS of CHOL@GEM-NBs in an embodiment of the present invention (a, CEUS images before and after blasting of CHOL@GEM-NBs with different concentrations; b, quantitative analysis of CEUS intensity (**P<0.01, ***P<0.001, n=3)).

[0029] Figure 9 In vitro CEUS of CHOL@GEM-NBs in an embodiment of the present invention (a, in vitro CEUS images of CHOL@GEM-NBs, Blank NBs, and PBS; b, CEUS intensity statistics of CHOL@GEM-NBs, Blank NBs, and PBS; ns indicates no statistical difference (***P < 0.001, n = 3)).

[0030] Figure 10 is the attenuation rate of CHOL@GEM-NBs and Blank NBs in vitro CEUS in the examples of the present invention.

[0031] Figure 11NPC1L1 expression in pancreatic cancer cells according to an embodiment of the present invention (a, images of NPC1L1 expression in NPC1L1 OE and OE control cells (scale bar = 100 μm); b, quantitative analysis of NPC1L1 expression in NPC1L1 OE and OE control cells (***P < 0.001, n = 5)).

[0032] Figure 12 NPC1L1 expression in pancreatic cancer cells according to an embodiment of the present invention (a, images of NPC1L1 expression in NPC1L1 KO and KO control cells (scale bar = 100 μm); b, quantitative analysis of NPC1L1 expression in NPC1L1 KO and KO control cells (***P < 0.001, n = 5)).

[0033] Figure 13 Verification of the targeting activity of CHOL@GEM-NBs in the examples of the present invention (a, immunofluorescence image of CHOL@GEM-NBs after co-incubation with NPC1L1 OE and OE Control cells for 1 h (scale bar = 50 μm); b, quantitative analysis of the uptake of CHOL@GEM-NBs into NPC1L1 OE and OE Control cells (***P < 0.001, n = 5)).

[0034] Figure 14 Verification of the targeting ability of CHOL@GEM-NBs in the examples of the present invention (a, immunofluorescence image of CHOL@GEM-NBs after co-incubation with NPC1L1 KO and KO Control cells for 1 h (scale bar = 50 μm); b, quantitative analysis of CHOL@GEM-NBs uptake by NPC1L1 KO and KO Control cells (***P < 0.001, n = 5)).

[0035] Figure 15 Affinity detection of CHOL@GEM-NBs in the examples of the present invention (a, flow cytometry images of affinity detection of NPC1L1 OE and OE control cells for CHOL@GEM-NBs or Blank NBs cells; b, quantitative analysis of affinity of NPC1L1 OE and OE control cells for CHOL@GEM-NBs or Blank NBs cells (***P < 0.001, n = 3)).

[0036] Figure 16Affinity detection of CHOL@GEM-NBs in the examples of the present invention (a, flow cytometry images of affinity detection of NPC1L1 KO and KO control cells for CHOL@GEM-NBs or Blank NBs cells; b, quantitative analysis of affinity of NPC1L1 KO and KO control cells for CHOL@GEM-NBs or Blank NBs cells (***P < 0.001, n = 3)).

[0037] Figure 17 Figure 2 shows tumor-targeted enhanced imaging of tumor-bearing mice in the NPC1L1 OE group and NPC1L1 KO group according to the examples of the present invention (a, enhanced CEUS images of CHOL@GEM-NBs and Blank NBs in NPC1L1 OE and OE Control tumors; b, enhanced CEUS time-intensity curves of CHOL@GEM-NBs and Blank NBs in NPC1L1 OE tumors; c, enhanced CEUS time-intensity curves of CHOL@GEM-NBs and Blank NBs in OE Control tumors; d, AUC values ​​under the enhanced CEUS time-intensity curves of NPC1L1 OE and OE Control tumors using CHOL@GEM-NBs and Blank NBs (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3)).

[0038] Figure 18 Figure 2 shows tumor-targeted enhanced imaging of tumor-bearing mice in the NPC1L1 KO group according to an embodiment of the present invention (a, enhanced CEUS images of CHOL@GEM-NBs and Blank NBs in NPC1L1 KO and KO Control tumors; b, enhanced CEUS time-intensity curves of CHOL@GEM-NBs and Blank NBs in NPC1L1 KO tumors; c, enhanced CEUS time-intensity curves of CHOL@GEM-NBs and BlankNBs in KO Control tumors; d, AUC under the enhanced CEUS time-intensity curves of CHOL@GEM-NBs and BlankNBs in NPC1L1 KO and KO Control tumors (*P < 0.05, **P < 0.01, ***P < 0.001, n = 3)).

[0039] Figure 19 The results of ultrasound-irradiated CHOL@GEM-NBs anti-tumor treatment in vivo according to the present invention (a is a schematic diagram of the treatment scheme of CHOL@GEM-NBs combined with UTND in a mouse pancreatic tumor model; b is an image of in vitro tumors in different treatment groups; c is the in vitro tumor weight and inhibition rate in different treatment groups; d is an immunopathological image of tumor tissue, where CD8 +Immunofluorescence images of tumor tissues detected by fluorescence microscopy for T (scale bar = 50 μm), CRT (scale bar = 100 μm), HMGB1 (scale bar = 50 μm), and DAPI (blue). DETAILED DESCRIPTION

[0040] The inventive concept of this proposal: During the research and development process, the applicant also attempted to use traditional nanobubble technology to prepare drug-loaded nanobubbles. However, the targeting modification and drug loading of the nanobubbles prepared by traditional technology are separated from each other. As a result, the hydrophilic drugs encapsulated in the nanobubbles can only reach tumor cells through passive transport after release in the body. This leads to problems such as insufficient targeting, limited drug loading capacity, and poor stability. In addition, the nanobubbles are prone to drug leakage and structural disintegration during the preparation and storage process, making it impossible to ensure that the drug maintains effective activity and structural integrity before reaching the lesion site, making it difficult to fully meet the clinical needs for early, accurate diagnosis and efficient treatment of pancreatic cancer.

[0041] The inventors then stumbled upon the idea of ​​leveraging cholesterol's unique activity in pancreatic cancer cells to achieve targeted drug delivery and enhance drug efficacy. Specifically, this approach uses polyethylene glycol to simultaneously link GEMs and cholesterol. The linked cholesterol acts as a targeting ligand, specifically targeting NPC1L1. Furthermore, cholesterol enhances the affinity of the nanobubbles because, as a component of the metabolically active surface of tumor cells, cholesterol readily binds to the surface. Due to this unique connection structure, the nanobubbles release the PEGylated GEMs upon ultrasound irradiation. Unlike traditional methods that encapsulate hydrophilic drugs and passively transport them to tumor cells after release, the GEMs in this approach utilize the hydrophilic polyethylene glycol chains to create steric hindrance, reducing the likelihood of enzymatic degradation or immune clearance, effectively ensuring the drug's effective activity and structural integrity before reaching the lesion. Furthermore, the targeting properties of cholesterol also enable active drug delivery to cancer cells, achieving target specificity and sustained release. This method of chemically connecting GEM and cholesterol through amide reaction to form targeted drug-loaded nanobubbles has a stable reaction, is conducive to clinical transformation, and provides a basis for subsequent research.

[0042] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0043] Example This protocol provides a method for preparing targeted drug-loaded nanobubbles, which chemically connects GEM and cholesterol via an amide reaction to prepare targeted GEM-loaded nanobubbles. The method includes the following steps: Step 1: Preparation of CHOL-PEG2000-GEM: S1. Place CHOL-PEG2000-COOH and gemcitabine (GEM) in a round-bottom flask at a molar ratio of 1:1 to 1:3. Add dimethylsulfoxide (DMSO) and shake at room temperature until completely dissolved to obtain mixed solution I.

[0044] In this stage, an amidation reaction will occur, and the reaction equation is as follows: Figure 1 As shown in a.

[0045] S2. Weigh dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) in a molar ratio of 1:0.05 to 1:0.1 and dissolve them in DMSO. After complete dissolution, slowly add the solution dropwise to the mixed solution I of CHOL-PEG2000-COOH and GEM. Under nitrogen protection, rotate and react at 25-40°C for 12-24 hours. After the reaction is completed, add excess methanol to the mixed solution to quench the reaction to obtain a reaction solution.

[0046] S3. Transfer the reaction solution to a dialysis bag to purify the compound. Magnetic stirring was performed at 4° C. for 12 to 24 hours to remove unreacted compounds and organic solvents. The CHOL-PEG2000-GEM was obtained by freeze-drying.

[0047] Step 2: Preparation of targeted drug-loaded nanobubbles CHOL@GEM-NBs: S4. Prepare a mixed lipid molecule, which includes DPPA (diphenylphosphoazide), DPPC (dipalmitoylphosphatidylcholine), DPPE (1,2-bis(diphenylphosphino)ethane), DPPG (1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol), and CHOL-PEG2000-GEM, mixed in a mass ratio of 1:3~4:3~4:3~4:1~2; in this example, DPPA, DPPC, DPPE, DPPG, CHOL, and CHOL-PEG2000-GEM are specifically mixed in a ratio of 1:3:3:3:1, totaling 11 mg.

[0048] S5. Dissolve the above mixed lipid molecules in 1 mL of glycerol:PBS (V / V=1:9) hydration solution, and shake thoroughly in an ice-water bath at 0°C to obtain mixed solution II.

[0049] S6. Transfer the mixed solution II to a vial and introduce perfluoropropane gas (C3F8) to replace the air in the bottle. Use an HL-AH silver-mercury blender to shake the solution back and forth horizontally. Place the solution in a refrigerator at 4°C for 2 hours, then centrifuge at 300 rpm / min for 3 minutes for washing and purification. The middle emulsion is the cholesterol-shelled nanobubbles CHOL@GEM-NBs loaded with GEM.

[0050] S7. Preparation of Blank NBs: Replace CHOL-PEG2000-GEM with CHOL-PEG2000-GEM, repeat S4 to S6, and replace CHOL-PEG2000-GEM in the above CHOL@GEM-NBs with DSPE-PEG2000 to prepare Blank NBs; S8. Preparation of cholesterol nanobubbles CHOL@NBs: Replace CHOL-PEG2000-GEM with DSPE-PEG2000 and CHOL (mass ratio 1:1), repeat S4 to S6, and replace CHOL-PEG2000-GEM in the above CHOL@GEM-NBs with DSPE-PEG2000 and CHOL to prepare cholesterol nanobubbles CHOL@NBs.

[0051] This solution also provides an application of targeted drug-loaded nanobubbles in the preparation of pancreatic cancer chemotherapy drugs.

[0052] Experimental Example 1: Physical Performance Characterization CHOL-PEG2000-COOH and GEM were synthesized by amidation reaction (synthesis flow chart as shown in Figure 1 As shown in a, the composition and structure diagram of the synthesized CHOL@GEM-NBs are shown in Figure 1 b). CHOL-PEG2000-GEM was detected by nuclear magnetic hydrogen spectrum, as shown in Figure 2 As shown, the methylene peak of PEG2000 is at about 3.6 ppm, the characteristic peak of GEM is at about 3.3 ppm, and the characteristic peak of CHOL is at about 1.1 ppm.

[0053] Then the morphology of nanobubbles was observed using optical microscope and transmission electron microscope. Figure 3 As shown, light microscopy ( Figure 3 a) and transmission electron microscopy ( Figure 3 b) CHOL@GEM-NBs are uniformly dispersed and have a spherical, smooth, hollow structure.

[0054] Subsequently, the nanobubbles were diluted to 10,000 times using PBS gradients. The concentration of the nanobubbles was calculated after counting with a hemocytometer. The particle size, polydispersity index (PDI), and zeta potential of the nanobubbles were measured using a Zetasizer nano ZS90 particle size detector. The results are shown in Tables 1 and Figure 4 The relevant parameters of CHOL@GEM-NBs and Blank NBs were detected (Table 1). The concentration of CHOL@GEM-NBs was 9.84±0.77×10 10 / mL, Blank NBs concentration was 1.35±0.34×10 11 / mL. The particle sizes of blank NBs and CHOL@GEM-NBs were 380.01±13.27 nm and 419.52±20.34 nm, respectively, and the PDIs were 0.51±0.07 and 0.37±0.03, respectively. Figure 4 a). Zeta potentials were -5.33±0.65 mV and -11.93±1.07 mV ( Figure 4 b).

[0055] Table 1 Parameters related to CHOL@GEM-NBs and Blank NBs 𝑥̅±𝑠

[0056] Experimental Example 2: Testing the Stability of CHOL@GEM-NBs Considering that nanobubbles have a certain physical stability during the experimental period to meet the requirements of drug delivery system for carrier integrity and uniformity, the prepared nanobubbles were stored at 4°C, and the particle size and PDI of CHOL@GEM-NBs were measured at 0 days, 1 day, 2 days, 4 days, and 7 days to analyze the stability of CHOL@GEM-NBs. The results are detailed in Figure 5 The results showed that the particle sizes of CHOL@GEM-NBs at different time points (0, 1, 2, 4, and 7 days) were 396.7±17.06, 405.7±26.11, 396.1±12.66, and 402.8±11.41 nm, respectively, indicating that the particle size of the nanobubbles did not change significantly within a week (P>0.05), and the PDI of the nanobubbles changed significantly on the 7th day (P<0.05).

[0057] Experimental Example 3: Calculation of encapsulation efficiency and drug loading of CHOL@GEM-NBs In order to detect the loading efficiency of GEM in CHOL@GEM-NBs, the absorbance of GEM methanol mixed solutions at different concentrations (200 ug / mL, 100 ug / mL, 50 ug / mL, 25 ug / mL, 12.5 ug / mL, 6.25 ug / mL, 3.125 ug / mL) was measured by UV-visible spectrophotometer (UV-Vis). A GEM standard curve was drawn, and the UV absorbance value spectrum of CHOL@GEM-NBs and Blank NBs methanol solutions at different concentrations was plotted. The results are shown in Figure 2. Figure 6 The encapsulation efficiency and drug loading rate of GEM in CHOL@GEM-NBs were calculated according to the following formula.

[0058]

[0059]

[0060] The results showed that the standard curve was prepared for the peak absorbance value at 270 nm ( Figure 6 a) As the concentration of GEM increases, there is a linear correlation between concentration and absorbance (R 2 =0.9991). Compared with the UV-Vis spectrum of GEM, CHOL@GEM-NBs has an absorption peak at 270nm ( Figure 6 b), while Blank NBs showed a flat straight line, proving that GEM was successfully loaded into CHOL@GEM-NBs. The loading rate was calculated using the GEM standard curve, and the results showed that the GEM encapsulation efficiency was 53.81±1.07% and the drug loading efficiency was 4.44±0.08%.

[0061] Experimental Example 4: CHOL@GEM-NBs hemolysis experiment Hemolysis is assessed by the amount of hemoglobin released during incubation of the nanobubbles with red blood cells. A hemolysis rate below 5% is considered an important indicator of biomaterial safety. A low hemolysis rate indicates minimal damage to the red blood cell membrane and good hemocompatibility. The testing steps are as follows: (1) Use a siphon to collect blood from the mouse eyeball and collect it in an anticoagulant tube. Transfer it to a 10 mL centrifuge tube and mix it thoroughly. Add 3 times the amount of PBS and centrifuge it at 2000 rpm / min for 10 min. Remove the supernatant and repeat the washing process until the supernatant becomes transparent.

[0062] (2) Take an appropriate amount of red blood cells and prepare a 2% red blood cell suspension in PBS, and add different concentrations of CHOL@GEM-NBs or Blank NBs (1×10 7 / mL, 5×107 / mL, 1×10 8 / mL, 5×10 8 / mL, 1×10 9 / mL) were incubated in a centrifuge tube for 1 h. Double-distilled water 0.1% TritonX-100 was used as a positive control, and PBS was used as a negative control.

[0063] (3) Take 100 μL of supernatant from each well and use a multifunctional microplate reader to measure the absorbance of each well at 545 nm. Calculate the hemolysis rate according to the following formula.

[0064]

[0065] Experimental data show that if Figure 7 As shown in a, in the positive control group, the solution was clear red, indicating that the red blood cells were completely destroyed and a large amount of hemoglobin was released. The supernatant of CHOL@GEM-NBs and the negative control was colorless and transparent. The quantitative results showed that ( Figure 7 b) Compared with the positive control, the hemolysis rates of CHOL@GEM-NBs and Blank NBs at different concentrations were all less than 5%.

[0066] Experimental Example 5: In vitro CEUS of CHOL@GEM-NBs The concentration was 1×10 8 / mL, 5×10 7 / mL, 1×10 7 / mL, 5×10 6 / mL, 1×10 6 CHOL@GEM-NBs (1000 mL / mL) were placed in a 1% agarose gel pore model. Ultrasound images in B-mode and CEUS mode were acquired using a Vevo 2100 small animal ultrasound imager at a center frequency of 18 MHz and a gain of 35 dB. Ten mechanical bursts were performed on the CHOL@GEM-NBs using the "Flash" function key for high-intensity mechanical index ultrasound, followed by acquisition of CEUS ultrasound images before and after the bursts. Images of CHOL@GEM-NBs and Blank NBs were acquired within 15 minutes. Image intensity was quantitatively analyzed using ImageJ software. The results are detailed in [ 15 ]. Figures 8 to 10 .

[0067] The acoustic impedance difference between the gas core and the lipid shell inside the nanobubble gives the nanobubble a strong CEUS echo signal and ultrasound responsiveness. The experimental results show that in the agarose in vitro imaging model, the CEUS intensity of CHOL@GEM-NBs is high ( Figure 8a). After high mechanical index ultrasound exposure, the ultrasound imaging intensity of CHOL@GEM-NBs decreased significantly, indicating that CHOL@GEM-NBs can be blasted by high mechanical index ultrasound ( Figure 8 b) CHOL@GEM-NBs and Blank NBs at the same concentration can significantly enhance CEUS intensity compared with PBS ( Figure 9 a). There is no difference in ultrasound contrast enhancement intensity between Blank NBs and CHOL@GEM-NBs at the same concentration ( Figure 9 b). During the in vitro stability imaging process, the imaging intensity of CHOL@GEM-NBs and Blank NBs decreased with time, and there was no significant difference in the ultrasound imaging intensity decay rate between the two ( Figure 10 ).

[0068] Experimental Example 6: Detection of NPC1L1 expression levels in pancreatic cancer cell lines Immunofluorescence was used to detect the expression level of NPC1L1 in Panc02 NPC1L1 KO, KO control, NPC1L1 OE, and OE control cells. The steps are as follows: (1) Logarithmically growing NPC1L1 KO, KO control, NPC1L1 OE and OE control cells were inoculated into a 24-well plate with a cell slide in advance, with a cell density of 1×10 per well. 5 / mL, and discard the supernatant after overnight culture.

[0069] (2) After fixing with 4% paraformaldehyde for 15 min, add 5% bovine serum albumin in PBS solution and block at 37°C for 1 h.

[0070] (3) Incubate with 1:200 rabbit anti-mouse NPC1L1 antibody at 4°C overnight, then add 1:500 diluted FITC-labeled goat anti-rabbit secondary antibody and incubate in the dark at room temperature for 1 h.

[0071] (4) After rinsing the surface with PBS three times, the cell nuclei were stained with 4'6-diamidino-2-phenylindole (DAPI) anti-fluorescence quenching mounting medium, and the expression of NPC1L1 in tumor cells was observed using a laser confocal microscope.

[0072] (5) Quantitative analysis was performed using ImageJ and GraphPad Prism software. Figure 11-12 .

[0073] like Figure 11 a. Figure 12As shown in a, obvious green fluorescence signals were observed in NPC1L1 KO control cells, NPC1L1 OE cells, and OE control cells, while no obvious green fluorescence signals were observed in NPC1L1 KO cells. Figure 11 b. Figure 12 As shown in b, the expression levels of NPC1L1 in the NPC1L1 KO and NPC1L1 OE groups were significantly different.

[0074] Experimental Example 7: In vitro binding assay of CHOL@GEM-NBs The nanoplatform's targeted and efficient accumulation are key factors in ensuring its accurate diagnostic and therapeutic efficacy. The binding ability of CHOL@GEM-NBs and Blank NBs to Panc 02 NPC1L1 KO, KO control, NPC1L1 OE, and OE control cells was observed using laser confocal microscopy. The following steps were performed: (1) NPC1L1 KO, KO control, NPC1L1 OE and OE control cells were cultured at a rate of 1×10 5 / mL was inoculated into 24-well plates and cultured overnight.

[0075] (2) The supernatant was discarded and the cells were fixed in 4% paraformaldehyde for 15 min. The cells were blocked with 5% bovine serum albumin in PBS at 37°C for 1 h and incubated with DiI-labeled CHOL@GEM-NBs and Blank NBs for 1 h, respectively.

[0076] (3) After rinsing with PBS, the cell nuclei were stained with DAPI for 5 min, and the binding of CHOL@GEM-NBs and Blank NBs to the cells was observed under a laser confocal microscope.

[0077] The results are shown in Figure 13-14 .like Figure 13 a. Figure 14 As shown in a, a large number of DiI-labeled CHOL@GEM-NBs were observed around pancreatic cancer NPC1L1-positive cells. The number of CHOL@GEM-NBs targeted binding was proportional to the expression level of NPC1L1 on the cell membrane surface ( Figure 13 b. Figure 14 b).

[0078] Experimental Example 8: In vitro internalization experiment of CHOL@GEM-NBs To further verify the affinity of NPC1L1 KO, KO control, NPC1L1 OE and OE control cells to NBs (CHOL@GEM-NBs or Blank NBs), flow cytometry was used to detect the fluorescence intensity after co-incubation of tumor cells with NBs. The steps were as follows: NPC1L1 KO, KO control, NPC1L1 OE and OE control cells were divided into three groups in a 6-well plate. The first group was the untreated control group. The second group was added with 5×10 DiI-labeled 7 / mL targeted nanobubbles CHOL@GEM-NBs, and the third group was added with the same concentration of DiI-labeled Blank NBs. After incubation at 37°C for 1 h, the supernatant was discarded, the surface was washed with PBS, and the cells in each well were digested. The DiI fluorescence intensity of the cells in each group was detected by flow cytometry.

[0079] The results are shown in Figure 15 and Figure 16 The results showed that in NPC1L1-positive cells, CHOL@GEM-NBs shifted more significantly to the right than Blank NBs, indicating that NPC1L1-positive cells took up more CHOL@GEM-NBs than Blank NBs (e.g. Figure 15 a. Figure 16 a), and as NPC1L1 expression increased, the uptake of CHOL@GEM-NBs increased (as shown in Figure 15 b. Figure 16 (b) The binding rates of CHOL@GEM-NBs and Blank NBs were 67.0±1.4% and 6.3±0.3% in NPC1L1 OE cells, 37.9±1.4% and 2.3±0.3% in NPC1L1 Control cells, and 51.4±6.2% and 6.0±1.4% in NPC1L1 KO Control cells, respectively. These differences were statistically significant between the two groups. In contrast, the results showed no significant difference in the affinity of CHOL@GEM-NBs and Blank NBs for NPC1L1 KO cells.

[0080] Experimental Example 9: In vivo ultrasound-targeted enhanced imaging of CHOL@GEM-NBs Given that CHOL@GEM-NBs have been shown to target NPC1L1 at the cellular level, the targeting-enhanced imaging capabilities of CHOL@GEM-NBs and blank NBs were further analyzed in mice bearing NPC1L1 KO, KO control, NPC1L1 OE, and OE control cells. The following steps were performed: C57BL / 6 mice bearing Panc02 NPC1L1 KO, KO control, NPC1L1 OE, and OE control cells were anesthetized and immobilized with 3% isoflurane inhalation. The maximum cross-section of the tumor was scanned using a Vevo 2100 small animal ultrasound imager with a high-frequency probe (MS-250, center frequency 18 MHz, gain 35 dB). After the probe was fixed, the ultrasound imager continuously acquired CEUS mode. CHOL@GEM-NBs (volume 100 μL; concentration 5×10 7 / mL) injection. After 15 minutes of imaging, the high-intensity ultrasound "Flash" button was pressed to continuously blast the residual nanobubbles in the mouse tumor. The CEUS intensity value at the tumor site was basically consistent with the intensity before CHOL@GEM-NBs contrast imaging. BlankNBs (volume 100 μL; concentration 5×10 7 / mL), and continuously acquire images. Using the built-in CEUS measurement module of the ultrasound imager, the time-intensity curves of CHOL@GEM-NBs and Blank NBs were obtained, and the ultrasound-enhanced imaging parameters of targeted and non-targeted nanobubbles in various tumors were compared, including the time to peak (TTP) represented by the highest point of the time-intensity curve, the peak intensity (PI), and the area under the ultrasound-enhanced imaging curve (AUC) within 15 minutes. The results are detailed in the Figure 17-18 , Table 2~Table 3.

[0081] Table 2 TTP values, PI values, and AUC values ​​of time-intensity curves of different nanobubbles in NPC1L1 OE and OE Control tumors (𝑥̅±𝑠)

[0082] Note: ***P<0.001 compared with Blank NBs in the same group.

[0083] Table 3 TTP values, PI values, and time-intensity AUC values ​​(𝑥̅±𝑠) of different nanobubbles in NPC1L1 KO and KO Control tumors

[0084] Note: **P<0.01 compared with Blank NBs in the same group.

[0085] The experimental results show that the acquired enhanced CEUS images show ( Figure 17 a. Figure 18 a) In OE group tumors, CHOL@GEM-NBs showed higher contrast intensity than Blank NBs. According to the time-intensity curve ( Figure 17 b. Figure 17 c, Table 2), CHOL@GEM-NBs and Blank NBs of the same concentration rose rapidly to the highest point of the time-intensity curve within about 10 s. As shown in Table 2, there was no significant difference in PI value. Subsequently, the contrast intensity began to decline, and showed a gradual weakening trend within 900 s (15 min). Among them, CHOL@GEM-NBs showed higher ultrasound imaging intensity than Blank NBs starting from 100 s, and its downward trend was more gentle. In the tumors of the OE control group, the ultrasound intensity value of CHOL@GEM-NBs showed significant difference from that of Blank NBs from 400 s. In contrast, the Blank NBs signal was weak and almost unobservable at 15 min. The time-intensity AUC value showed ( Figure 17 d), there were statistical differences between CHOL@GEM-NBs and Blank NBs in both groups. The time-intensity curve results showed that ( Figure 18 b. Figure 18 c, Table 3). In the KO control group, the ultrasound imaging intensity of CHOL@GEM-NBs differed from that of BlankNBs from 100 s onwards and maintained a relatively gentle downward trend within 15 min. In the KO group, there was no statistical difference in CEUS intensity between CHOL@GEM-NBs and BlankNBs in the time-intensity curve. Time-intensity AUC value ( Figure 18 d, Table 3). In KO control tumors, the CHOL@GEM-NBs group showed statistically significant differences compared to blank NBs. KO tumors showed no difference and had lower time-intensity AUC values ​​than KO control tumors.

[0086] Experimental Example 10: Ultrasound irradiation enhances the targeting and penetration of CHOL@GEM-NBs Panc02 cell-bearing C57BL / 6 mice were randomly divided into five groups, each consisting of five mice: PBS, GEM, CHOL@GEM-NBs, CHOL@NBs+US, and CHOL@GEM-NBs+US. All treatment groups received GEM via tail vein injection every three days. The total dose of GEM in the GEM, CHOL@GEM-NBs, and CHOL@GEM-NBs+US groups was 2 mg / kg. The CHOL@NBs+US and CHOL@GEM-NBs+US groups were also irradiated with ultrasound (2 min, 1 W / cm², 60% duty cycle). After three doses, the mice were observed for 12 days. At the end of the experiment, the in vitro tumor weight of each treatment group was weighed, and the tumor inhibition rate was calculated using the formula: (1 - average tumor weight in the treatment group / average tumor weight in the control group) × 100%. Tumor tissues were obtained and routinely fixed, embedded, and sectioned before CD8 + T, CRT, and HMGB1 immunofluorescence staining were performed to analyze immune effect-related indicators.

[0087] like Figure 19 a~ Figure 19 As shown in Figure c, compared with the control group, the CHOL@GEM-NBs+US group had the smallest tumor mass and a tumor inhibition rate of 86%, which was statistically different from the GEM group and CHOL@GEM-NBs group, while CHOL@NBs+US showed a suboptimal antitumor effect.

[0088] like Figure 19 As shown in d, according to the expression of CD8 + T cell expression analysis showed that more CD8 + T cells were more in the CHOL@NBs+US group and least in the GEM group. The effect of combined treatment on the immunogenic cell death (ICD) effect of primary tumors was further evaluated. CRT fluorescence was weak in the Control group, GEM group, and CHOL@GEM-NBs group, but stronger in the CHOL@NBs+US group, and the CRT expression was strongest in the CHOL@GEM-NBs+US group. In addition, another ICD marker, HMGB1, showed a trend of decreased expression in different groups. These results indicate that CHOL@GEM-NBs+US combined treatment significantly enhances the immunogenicity of tumor cells by inducing CRT externalization and HMGB1 release, and is accompanied by CD8 + T cell infiltration increased, indicating activation of anti-tumor immune responses.

[0089] In summary, this scheme takes advantage of the fact that the cholesterol shell of nanobubbles can target and bind to NPC1L1 which is highly expressed in pancreatic cancer cells, and at the same time carries the chemotherapy drug GEM (Gemcitabine) on its shell to construct targeted GEM-loaded nanobubbles. The aim is to study and explore the ultrasound molecular imaging characteristics of targeted drug-loaded nanobubbles after they pass through tumor blood vessels and target and bind to pancreatic cancer cells (especially the targeted drug-loaded nanobubbles that are efficiently enriched in pancreatic cancer cells with high expression of NPC1L1) and their effect on the elimination of CD8 + The effect of T cell immunosuppression is clarified, and the effect of ultrasound-irradiated nanobubble bursting to release GEM to accurately kill pancreatic cancer cells and improve the synergistic treatment of tumor immune microenvironment is explained, providing a new strategy of safe and effective synergistic chemotherapy and immunotherapy for the diagnosis and treatment of pancreatic cancer.

[0090] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A method for preparing targeted drug-loaded nanobubbles, characterized by: The method includes chemically connecting GEM and cholesterol through an amide reaction to prepare targeted GEM-loaded nanobubbles.

2. The method for preparing targeted drug-loaded nanobubbles according to claim 1, wherein: The steps include: Step 1: Preparation of CHOL-PEG2000-GEM: S1. Add CHOL-PEG2000-COOH and GEM to dimethyl sulfoxide and shake to dissolve to obtain mixed solution I; S2. Dissolve dicyclohexylcarbodiimide and 4-dimethylaminopyridine in dimethyl sulfoxide, and then slowly add dropwise to the mixed solution I. Rotate under nitrogen to cause an amide reaction, and then add methanol to quench the reaction to obtain a reaction solution. S3. The reaction solution was transferred to a dialysis bag to purify the compound. Unreacted compounds and organic solvents were removed by magnetic stirring at 4°C for 24 hours, and then freeze-dried to obtain CHOL-PEG2000-GEM. Step 2: Preparation of targeted drug-loaded nanobubbles CHOL@GEM-NBs: S4, preparing a mixed lipid molecule, the mixed lipid molecule including DPPA, DPPC, DPPE, DPPG and CHOL-PEG2000-GEM prepared in S3; S5. Dissolve the mixed lipid molecules in glycerol:PBS hydration solution and shake thoroughly in an ice-water bath at 0°C to obtain mixed solution II. S6. Transfer the mixed solution II to a vial and introduce perfluoropropane gas to replace the air in the vial. After sufficient shaking, let it stand at low temperature, then centrifuge and wash for purification. The middle emulsion is the cholesterol-shelled nanobubbles CHOL@GEM-NBs loaded with GEM.

3. The method for preparing targeted drug-loaded nanobubbles according to claim 2, wherein: In S1, the mixing molar ratio of CHOL-PEG2000-COOH and GEM is 1:1~1:

3.

4. The method for preparing targeted drug-loaded nanobubbles according to claim 2, wherein: In S2, the molar ratio of dicyclohexylcarbodiimide to 4-dimethylaminopyridine is 1:0.05-0.1; and the amide reaction is carried out by rotating at 25-40° C. for 12-24 hours.

5. The method for preparing targeted drug-loaded nanobubbles according to claim 2, wherein: In S4, the mixed lipid molecules including DPPA, DPPC, DPPE, DPPG, and CHOL-PEG2000-GEM were mixed in a mass ratio of 1:3~4:3~4:3~4:1~2.

6. The method for preparing targeted drug-loaded nanobubbles according to claim 2, wherein: In S5, the volume ratio of glycerol to PBS in the glycerol:PBS hydration solution was 1:

9.

7. The method for preparing targeted drug-loaded nanobubbles according to claim 2, wherein: In S6, low-temperature standing is standing in a 4°C refrigerator for 2 to 12 hours; and centrifugation is centrifugation at 300 to 500 rpm / min for 3 to 5 minutes.

8. The method for preparing targeted drug-loaded nanobubbles according to claim 2, wherein: The process also includes S7, preparing blank nanobubbles (Blank NBs): replacing CHOL-PEG2000-GEM with CHOL-PEG2000-GEM, repeating S4 to S6, and replacing CHOL-PEG2000-GEM in the CHOL@GEM-NBs with DSPE-PEG2000 to prepare blank nanobubbles (Blank NBs); The method further includes S8, preparing cholesterol nanobubbles CHOL@NBs: replacing CHOL-PEG2000-GEM with DSPE-PEG2000 and CHOL, repeating S4 to S6, replacing CHOL-PEG2000-GEM in the above CHOL@GEM-NBs with DSPE-PEG2000 and CHOL, and preparing cholesterol nanobubbles CHOL@NBs.

9. A targeted drug-loaded nanobubble, characterized by: The product is prepared by the preparation method according to claims 1 to 8.

10. An application of targeted drug-loaded nanobubbles, characterized by: This invention includes the use of the targeted drug-loaded nanobubbles according to claim 9 in preparing a pancreatic cancer chemotherapy drug composition.

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