Preparation method and application of targeting nanoparticles for efficiently delivering and locally controlling oxygen release to improve HER-2 positive gastric cancer sonodynamic and antibody treatment effect

Abstract

The invention discloses targeted nano-particles TPPO NPs capable of efficiently delivering and locally controlling oxygen release to improve the curative effect of sonodynamic and antibody therapy, and relates to a preparation method of multifunctional nano-particles and application of the multifunctional nano-particles in tumor diagnosis and treatment. According to the preparation method disclosed by the invention, targeting nanoparticles capable of efficiently carrying O2 are prepared by using a lipidated sound-sensitive agent, namely pyropheophorbide a lipid, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-NHS, perfluorobromooctane (PFOB) and trastuzumab (Tra). Under the targeting effect of trastuzumab, the nanoparticles recognize HER-2 positive gastric cancer cells, and efficient enrichment of the nanoparticles at the HER-2 positive gastric cancer cells is achieved together with permeation enhancing and retention enhancing effects. Under the ultrasonic action, on one hand, the sonosensitizer generates reactive oxygen species (ROS), and the sonodynamic therapy effect is achieved; on the other hand, O2 carried by perfluorobromooctane PFOB in the nanoparticles can be controllably released, so that the hypoxia of tumors is relieved, and the sonodynamic and antibody treatment effects are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing and applying targeted nanoparticles for efficiently delivering and locally controlling the release of oxygen to improve the sonodynamics and antibody therapeutic efficacy of HER-2 positive gastric cancer. Background Art

[0002] Gastric cancer is one of the common malignant tumors of the digestive tract, ranking fifth in morbidity and mortality among all malignant tumors. Since the early symptoms of gastric cancer are not obvious, most patients are in the middle and late stages when diagnosed. Therefore, there is still an urgent need to find effective treatments for gastric cancer to prolong the survival of gastric cancer patients. HER-2 positive gastric cancer is a special type of gastric cancer that was first discovered in 1986. HER-2 is a transmembrane glycoprotein that belongs to the epidermal growth factor receptor family. It phosphorylates the tyrosine kinase domain through homodimerization or heterodimerization to induce cell proliferation, differentiation, and signal transduction. HER-2 positive gastric cancer cells grow faster, are more invasive, and patients have a worse prognosis.

[0003] Trastuzumab is an anti-HER-2 monoclonal antibody and a targeted drug for the treatment of HER-2 positive gastric cancer. At present, trastuzumab combined with chemotherapy is the first-line treatment for HER-2 positive gastric cancer, but chemotherapy has obvious toxic side effects and is prone to drug resistance. Trastuzumab may also have toxic side effects on normal tissues due to off-target effects. Compared with chemotherapy, sonodynamic therapy has attracted more and more attention as a minimally invasive therapy with fewer side effects. It uses ultrasound to activate sonosensitizers to produce reactive oxygen species to kill tumor cells. It has the advantages of no drug resistance, repeatable treatment, and few side effects. However, sonosensitizers have poor water solubility and are difficult to specifically identify tumor cells. Therefore, we envision that by using sonodynamic therapy instead of chemotherapy combined with trastuzumab, we can avoid the shortcomings of chemotherapy and improve the effect of sonodynamic therapy. Therefore, it is urgent to develop an efficient drug delivery system combining sonodynamics with trastuzumab to improve the treatment of gastric cancer.

[0004] Another important issue related to treatment is hypoxia, which is an inherent characteristic of gastric cancer and promotes the proliferation, invasion and metastasis of tumor cells. Under hypoxic conditions, tumor cells adapt to the hypoxic environment through a variety of mechanisms, including upregulating the expression of the HER-2 gene and the expression of the hypoxia-inducible factor-1α (HIF-1α) protein, activating the PI3K / AKT signaling pathway, etc. HER-2 overexpression directly regulates the synthesis of hypoxia-inducible factor-1α, which is one of the key factors leading to resistance to trastuzumab antibody-dependent cellular cytotoxicity. In addition, the efficacy of sonodynamic therapy requires the consumption of O2, which further aggravates tumor hypoxia. Therefore, local self-oxygen supply at the tumor site is important for both antibody therapy and sonodynamic therapy. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing and using targeted nanoparticles that can efficiently deliver and locally control the release of O2 to enhance the efficacy of sonodynamic therapy and antibody therapy. The targeted nanoparticles provided by the present invention can achieve efficient enrichment of sonosensitizers, trastuzumab and O2 in tumors, and release O2 locally in tumors under the action of ultrasound, thereby improving the effects of sonodynamic therapy and antibody therapy.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] A targeted nanoparticle for efficient delivery and local controlled release of oxygen, wherein the nanoparticle is composed of lipidated sonosensitizer pyropheophorbide a lipid, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 , Distearoylphosphatidylethanolamine-polyethylene glycol 2000 -NHS and perfluorooctyl bromide PFOB, trastuzumab is coupled to the surface of the nanoparticles through amide reaction, and the mass ratio of nanoparticles to trastuzumab is 2:1. Pyropheophorbide a lipid, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 , Distearoylphosphatidylethanolamine-polyethylene glycol 2000 The molar ratio of PFOB to NHS was 8:1:1. 1 mg of nanoparticles contained 10 μl of perfluorooctyl bromide (PFOB).

[0008] The average particle size of the above-mentioned targeted nanoparticles TPPO NPs is about 249±3.41 nanometers.

[0009] The targeted nanoparticles TPPO NPs improve the delivery efficiency of sonosensitizer pyropheophorbide a and trastuzumab, and release O2 locally under the action of ultrasound, thereby improving the efficacy of sonodynamic therapy and antibody therapy for HER-2 positive gastric cancer tumors. The frequency range of ultrasound action is 1 MHz, 0.5-2 W / cm2, and the duty cycle is 10%-50%.

[0010] The present invention also provides a method for preparing targeted nanoparticles for efficient delivery and local controlled release of O2, comprising the following steps:

[0011] Step 1: Distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Dissolve in anhydrous ethanol to obtain solution A.

[0012] Step 2: Distearoyl phosphatidylethanolamine-polyethylene glycol 2000 -NHS was dissolved in anhydrous ethanol to obtain solution B.

[0013] Step 3: Dissolve the lipidated sonosensitizer (pyropheophorbide a lipid) in tetrahydrofuran to obtain solution C.

[0014] Step 4: Solutions A, B, and C are mixed, and the organic reagent is removed by rotary evaporation. Phosphate buffer or physiological saline is added under water bath ultrasound conditions to form nanoparticles PNPs containing sonosensitizers.

[0015] Step 5: The obtained sonosensitizer nanoparticles PNPs were mixed with trastuzumab in phosphate buffer or physiological saline, and gently stirred for 6 hours under light-proof conditions. Unattached trastuzumab was removed by dialysis (molecular weight = 500 kDa) to obtain sonosensitizer-trastuzumab nanoparticles TPNPs.

[0016] Step 6: Under ice bath ultrasound conditions, perfluorooctyl bromide PFOB is loaded into sonosensitizer-trastuzumab nanoparticles to obtain TPPNPs.

[0017] Step 7: Place the TPP NPs dispersion in an oxygen chamber and saturate with a constant airflow for 30 minutes to obtain oxygen-carrying sonosensitizer-trastuzumab nanoparticles TPPO NPs.

[0018] In step 4, the molar ratio of solution A, solution B, and solution C is 1:1:8.

[0019] In step five, the mass ratio of the sonosensitizer-containing nanoparticles PNPs to trastuzumab is 2:1.

[0020] In step six, 10 μl of PFOB was added for every 1 mg of sonosensitizer-trastuzumab nanoparticles TPNPs.

[0021] The present invention also provides the use of the targeted nanoparticles prepared according to the above technical scheme in the treatment of HER-2 positive gastric cancer.

[0022] In the present invention, distearoyl phosphatidylethanolamine-polyethylene glycol is used. 2000 , Distearoylphosphatidylethanolamine-polyethylene glycol 2000 -NHS and pyropheophorbide a lipid were used to prepare sonosensitizer nanoparticles, which were then coupled to trastuzumab through an amide reaction, loaded with perfluorooctyl bromide PFOB under probe ultrasound conditions, and saturated with oxygen in an oxygen chamber with a constant airflow to obtain oxygen-carrying sonosensitizer-trastuzumab nanoparticles TPPO NPs. Oxygen-carrying sonosensitizer-trastuzumab nanoparticles TPPO NPs achieve efficient enrichment in tumor sites under the enhanced permeation and retention effects and the active targeting of trastuzumab, improving the delivery efficiency of sonosensitizer pyropheophorbide a, trastuzumab and O2. Under the action of ultrasound, oxygen-carrying sonosensitizer-trastuzumab nanoparticles TPPO NPs release O2 locally in the tumor, improve tumor hypoxia, and at the same time improve the therapeutic effects of sonodynamics and trastuzumab, thereby improving the treatment outcome of HER-2 positive gastric cancer.

[0023] The beneficial effects of the present invention are:

[0024] The present invention uses lipidated sonosensitizer and trastuzumab to prepare targeted nanoparticles, effectively avoiding the poor water solubility of free sonosensitizer and the toxic side effects caused by premature leakage of sonosensitizer and trastuzumab, and improving the bioavailability of both. The oxygen affinity of perfluorooctyl bromide PFOB is used to improve the delivery efficiency of O2, and O2 is released locally in the tumor under the action of exogenous ultrasound, improving tumor hypoxia, and simultaneously improving the therapeutic effects of sonodynamics and trastuzumab, significantly improving the treatment outcome of HER-2 positive gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention. Figure 1 Schematic diagram of the preparation process and mechanism of action of targeted nanoparticles for efficient delivery and local controlled release of O2 as described in Example 1; Figure 2 This is an electron micrograph of the targeted nanoparticles described in Example 2, with a scale bar of 200 nm; Figure 3 This is an elemental analysis diagram of the targeted nanoparticles described in Example 3; Figure 4 The ultrasound-responsive O2 release and its concentration dependence of the targeted nanoparticles described in Example 4; Figure 5 This is a test of the generation of reactive oxygen species by the targeted nanoparticles described in Example 5 under ultrasound; Figure 6 This is an investigation of the uptake of targeted nanoparticles by HER-2 positive cells as described in Example 6; Figure 7 This is the detection of reactive oxygen species produced in cells by the targeted nanoparticles described in Example 7, the scale bar is 200 μm; Figure 8 The detection of targeted nanoparticles as described in Example 8, which are locally released by ultrasound to improve hypoxia, has a scale bar of 200 μm; Fig. 9 This is the live-dead staining of the targeted nanoparticles described in Example 9 that exert tumor killing effects under ultrasound, and the scale is 200 μm; the numbers in the figure correspond to the groups: 1. control group; 2. ultrasound group; 3. trastuzumab group; 4. oxygen-containing sonosensitizer-trastuzumab nanoparticle group; 5. non-oxygen-containing sonosensitizer-trastuzumab nanoparticle + ultrasound group; 6. oxygen-containing sonosensitizer nanoparticle + ultrasound group; 7. oxygen-containing sonosensitizer-trastuzumab nanoparticle group + ultrasound group; Fig.10This is an investigation of the targeted nanoparticles described in Example 10 in improving hypoxia, enhancing trastuzumab and sonodynamic therapy through ultrasound; the numbers in the figure correspond to the groups: 1. control group; 2. ultrasound group; 3. trastuzumab group; 4. oxygen-containing sonosensitizer-trastuzumab nanoparticle group; 5. non-oxygen-containing sonosensitizer-trastuzumab nanoparticle + ultrasound group; 6. oxygen-containing sonosensitizer nanoparticle + ultrasound group; 7. oxygen-containing sonosensitizer-trastuzumab nanoparticle group + ultrasound group; Fig.11 The tissue distribution of the targeted nanoparticles described in Example 11 in an animal body; Fig.12 This is the tumor growth curve after the targeted nanoparticles described in Example 12 were used to treat the animals. The numbers in the figure correspond to the groups: 1. control group; 2. ultrasound group; 3. trastuzumab group; 4. oxygen-containing sonosensitizer-trastuzumab nanoparticle group; 5. non-oxygen-containing sonosensitizer-trastuzumab nanoparticle + ultrasound group; 6. oxygen-containing sonosensitizer nanoparticle + ultrasound group; 7. oxygen-containing sonosensitizer-trastuzumab nanoparticle + ultrasound group. DETAILED DESCRIPTION

[0026] The following specific embodiments will help to understand the present invention, but do not limit the content of the present invention. Unless otherwise specified, the reagents and materials used in the following implementations are purchased from the company.

[0027] Example 1

[0028] Distearoylphosphatidylethanolamine-polyethylene glycol 2000 , Distearoylphosphatidylethanolamine-polyethylene glycol 2000 -NHS and lipidated sonosensitizer were mixed in a molar ratio of 1:1:8, and then the organic reagent was removed by rotary evaporation, and 600 microliters of phosphate buffer PBS was added under water bath ultrasound to obtain nanoparticles PNPs containing sonosensitizer. The obtained sonosensitizer nanoparticles PNPs were mixed with trastuzumab in a mass ratio of 2:1, gently stirred for 6 hours under light-proof conditions, and unattached trastuzumab was removed by dialysis (molecular weight = 500 kDa) to obtain sonosensitizer-trastuzumab nanoparticles TP NPs. Then 10 microliters of perfluorooctyl bromide PFOB was added to the sonosensitizer-trastuzumab nanoparticles TP NPs dispersion under ice bath probe ultrasound conditions, and then placed in an oxygen chamber and saturated with oxygen at a constant gas flow rate of 7.5 liters / minute for 30 minutes to obtain oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs (see Figure 1 ).

[0029] Example 2

[0030] The morphology of oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs was analyzed by electron microscopy. The prepared oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs were diluted with deionized water, and the diluted sample was dropped onto a 300-mesh polyvinyl acetate-coated copper grid, left to stand for 10 minutes, and the residual water was absorbed with filter paper, and then negatively stained with a 4% uranyl acetate aqueous solution for 10 minutes, washed with deionized water 3 times, air-dried at room temperature, and observed and photographed using a 100 kV transmission electron microscope. Figure 2 As shown, it is an electron microscope image of the oxygen-containing sonosensitizer-trastuzumab nanoparticles obtained in this example. The results show that the nanoparticles are spherical and about 200 nm in size.

[0031] Example 3

[0032] To evaluate the effective loading of perfluorooctyl bromide PFOB on TPP NPs, electron microscopy elemental analysis was performed. The same preparation method as in Example 2 was used, and observation and photography were performed using a Jem-2100F electron microscope at 200 kV. Figure 3 As shown, the fluorine and bromine elements are obviously surrounded by carbon and nitrogen elements, further proving that perfluorooctyl bromide PFOB is effectively encapsulated into the sonosensitizer-trastuzumab nanoparticles TPP NPs.

[0033] Example 4

[0034] Investigation of ultrasound-responsive O2 release of targeted nanoparticles and its concentration dependence. The oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs of perfluorooctyl bromide PFOB with different concentrations (0, 20, 40, 80 μL / mL) were placed in a vial. The unencapsulated oxygen O2 in the solution was first removed by nitrogen N2, and then the dissolved oxygen meter probe was placed and the bottle mouth was sealed. After that, ultrasonic irradiation (1 MHz, 1 W / cm2, 50% duty cycle) was performed to compare the O2 content in the sample solution treated under the same ultrasonic irradiation conditions at different time points. Figure 4 As shown, the O2 concentration in degassed water is proportional to the concentration of perfluorooctyl bromide PFOB. When the concentration of perfluorooctyl bromide PFOB is 20, 40, and 80 μL / mL aqueous solution, the O2 release of oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs is 3.24 mg / L, 6.89 mg / L, and 12.72 mg / L, respectively, while in the absence of ultrasound irradiation, there is no obvious O2 release.

[0035] Example 5

[0036] In order to investigate whether targeted nanoparticles produce reactive oxygen species under ultrasound, and that the release of O2 can enhance the generation of reactive oxygen species, the singlet oxygen green fluorescent probe SOSG probe was used to detect the generation of reactive oxygen species ROS. The specific experimental steps are as follows: ① Take a powdered singlet oxygen green fluorescent probe, add 33 μL of anhydrous methanol to prepare a mother solution with a concentration of 5 mmol / L, and then add 297 μL of deionized water to dilute it to 0.5 mmol / L; ② Take 60 μL of 0.5 mmol / L singlet oxygen green fluorescent probe liquid and add it to 2940 μL of the sample aqueous solution to be tested, so that the working concentration of the singlet oxygen green fluorescent probe reaches 5 μM, and then the solution to be tested is subjected to ultrasonic irradiation; ③ Use a fluorescence spectrophotometer to measure the absorption spectrum (excitation wavelength: 480 nm, emission wavelength: 525 nm). The experimental groups were phosphate buffer + ultrasound, oxygen-free sonosensitizer-trastuzumab nanoparticles + ultrasound, oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound (lipid sonosensitizer concentration was 20 μg / ml), and the ultrasound parameters were 1 MHz, 1 W / cm2, and 50% duty cycle. After ultrasound irradiation, the fluorescence intensity of each group was measured using a fluorescence spectrophotometer. Figure 5 As shown in the figure, the fluorescence intensity of the singlet oxygen green fluorescent probe at 525 nanometers changes with the extension of ultrasound irradiation time. Within 3 minutes, the fluorescence intensity of the singlet oxygen green fluorescent probe in the oxygen-containing sonosensitizer-trastuzumab nanoparticle group increased by about 5.3 times, and the fluorescence intensity of the singlet oxygen green fluorescent probe in the non-oxygen sonosensitizer-trastuzumab nanoparticle group increased by about 3.8 times, while the fluorescence intensity of the singlet oxygen green fluorescent probe in the simple phosphate buffer group did not change significantly after ultrasound, indicating that reactive oxygen species are produced by ultrasound acting on the sonosensitizer, and the supply of O2 increases the yield of reactive oxygen species.

[0037] Example 6

[0038] To investigate the effect of oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs on the delivery efficiency of sonosensitizer, the uptake of sonosensitizer by HER-2 positive NCI-N87 gastric cancer cells was observed by confocal microscopy. The specific operation steps are as follows: ① NCI-N87 cells were cultured at 6×10 4The cells / wells were inoculated into confocal culture dishes and incubated for 24 hours at 37°C and 5% CO2 to allow the cells to adhere. ② The original culture medium was replaced with 1640RPMI complete culture medium containing free sonosensitizer (pyropheophorbide a lipid), non-targeted oxygen-containing sonosensitizer nanoparticles and targeted oxygen-containing sonosensitizer-trastuzumab nanoparticles (equivalent concentration of pyropheophorbide a lipid: 20 μg / ml). ③ After continuing to culture for 4 hours, wash three times with phosphate buffer, fix with 4% paraformaldehyde at room temperature for 30 minutes, and stain with DAPI for 5 minutes. ④ Then wash three times with phosphate buffer, place under a laser confocal microscope, and observe the fluorescence of the sonosensitizer (excitation wavelength: 633 nm; emission wavelength: 650-720 nm) and DAPI fluorescence (excitation wavelength: 405 nm; emission wavelength: 410-490 nm). The specific results are as follows Figure 6 As shown, the red fluorescence intensity of the targeted oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs group was significantly higher than that of the non-targeted oxygen-containing sonosensitizer nanoparticle PPO NPs group and the pyropheophorbide a lipid PL group, indicating that nanoparticle-mediated endocytosis and the targeting effect of trastuzumab can effectively enhance the uptake of sonosensitizers by NCI-N87 cells.

[0039] Example 7

[0040] The ability of oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs to generate reactive oxygen species in cells under ultrasound was investigated. The specific operation steps are as follows: ① NCI-N87 cells were inoculated into 24-well cell culture plates (8×10 4 ② Replace the original cell culture medium with fresh culture medium containing different drugs (oxygenated sonosensitizer-trastuzumab nanoparticles TPPO NPs, oxygenated sonosensitizer nanoparticles PPO NPs, oxygen-free sonosensitizer-trastuzumab nanoparticles TPP NPs, pyropheophorbide a lipid PL concentration: 20 μg / ml) and continue to culture for 8 hours. ③ Dilute the DCFH-DA fluorescent probe 1000 times with RPMI 1640 complete culture medium to obtain the DCFH-DA staining working solution. After washing the original culture medium with phosphate buffer, add 500 μL of DCFH-DA staining working solution and load the probe in a cell culture incubator at 37°C and 5% CO2. ④ The cells were irradiated with ultrasound (1 min, 1.0 MHz, 50% duty cycle, 1.0 W / cm2), washed 3 times with phosphate buffer, and then observed and photographed under a fluorescence microscope (FITC channel). Figure 7As shown: There was almost no green fluorescence in the control group, ultrasound group and oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs group, while green fluorescence was observed in the oxygen-containing sonosensitizer nanoparticle + ultrasound group, the oxygen-free sonosensitizer-trastuzumab nanoparticle + ultrasound group, and the oxygen-containing sonosensitizer-trastuzumab nanoparticle + ultrasound group, indicating that ultrasound irradiation of sonosensitizers can produce reactive oxygen species in cells and achieve sonodynamic therapy of tumors. In addition, the green fluorescence of the oxygen-containing sonosensitizer-trastuzumab nanoparticle + ultrasound group was significantly stronger than that of the oxygen-containing sonosensitizer nanoparticle + ultrasound and the oxygen-free sonosensitizer-trastuzumab nanoparticle + ultrasound groups, which was attributed to the targeting effect of trastuzumab increasing the uptake of sonosensitizers, and the O2 delivered by perfluorooctyl bromide PFOB provided the necessary O2 source for the generation of reactive oxygen species.

[0041] Example 8

[0042] Ultrasound acts on oxygen-containing sonosensitizer-trastuzumab nanoparticles to locally release O2 to improve tumor hypoxia. The specific operation steps are as follows: NCI-N87 cells were inoculated in a 24-well cell culture plate (8×10 4 Cells) were cultured in a 37°C, 1% O2 hypoxic incubator for 24 hours to allow them to fully adhere to the wall. The original cell culture medium was replaced with fresh culture medium containing different drugs (oxygenated sonosensitizer-trastuzumab nanoparticles TPPO NPs, oxygenated sonosensitizer nanoparticles PPO NPs, non-oxygenated sonosensitizer-trastuzumab nanoparticles TPP NPs, pyropheophorbide a lipid PL concentration: 20 μg / ml) (the culture medium was deoxygenated in advance). After 8 hours, the ultrasound treatment group was irradiated with ultrasound (1 minute, 1.0 MHz, 50% duty cycle, 1.0 W / cm2) and then cultured for 8 hours. [Ru(dpp)3]Cl2 was prepared to 10 μg / ml using deoxygenated RPMI 1640 culture medium and continued to be cultured in a 1% O2 hypoxic incubator for 8 hours. The normoxic condition (37°C, 21% O2 incubator) was used as a negative control. After incubation, the cells were washed three times with deoxygenated phosphate buffer and then placed under an inverted fluorescence microscope for observation and photography (red fluorescence channel) to evaluate the release of O2 in the cells. Figure 7 As shown in the figure, strong red fluorescence signals were observed in NCI-N87 cells in both the control group and the ultrasound group, indicating that the cells were in a hypoxic state. Due to the dual effects of cellular hypoxia and oxygen consumption, a stronger red fluorescence signal was observed in the oxygen-free sonosensitizer-trastuzumab nanoparticles + ultrasound group. Compared with the oxygen-containing sonosensitizer-trastuzumab nanoparticles and the oxygen-free sonosensitizer-trastuzumab nanoparticles + ultrasound group, the red fluorescence signal of the oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group was significantly reduced, proving that the oxygen-containing sonosensitizer-trastuzumab nanoparticles can effectively deliver O2 into cells and release it in a controllable manner under ultrasound to improve hypoxia.

[0043] Example 9

[0044] Live-dead staining was used to investigate the tumor killing effect of targeted nanoparticles under ultrasound. The specific operation was as follows: ① NCI-N87 cells were inoculated into 24-well cell culture plates (8×10 4 ② Replace the original culture medium with fresh culture medium containing different drugs (trastuzumab, oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs, oxygen-containing sonosensitizer nanoparticles PPO NPs and non-oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPNPs, pyropheophorbide a lipid PL concentration: 50 μg / ml), and then continue incubation for 8 hours. ③ Treat the cells with ultrasound (1.0 MHz, 50% duty cycle, 1.0 W / cm2) for 3 minutes and continue to culture for 16 hours. ④ Dilute calcein AM and propidium iodide PI 1000 times with phosphate buffer, then add to the cells and stain in a 5% CO2 cell culture incubator at 37°C for 25 minutes. Gently wash the cells 3 times with phosphate buffer and observe under a fluorescence microscope. The results are as shown in Fig. 9 As shown in the figure, both the control group and the ultrasound group had strong green fluorescence signals and no obvious red fluorescence. Under hypoxic conditions, the red fluorescence signals of the trastuzumab, oxygen-containing sonosensitizer-trastuzumab nanoparticles, non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound, and oxygen-containing sonosensitizer nanoparticles + ultrasound groups were weaker than those of the corresponding groups under normoxic conditions, indicating that hypoxic conditions would weaken the efficacy of trastuzumab and sonodynamics. In contrast, even under hypoxic conditions, obvious red fluorescence signals could be observed in the oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group, while the green fluorescence signal was negligible, further indicating that oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs improved the efficacy of sonodynamics and antibody therapy due to the active targeting of trastuzumab and ultrasound-mediated O2 release. Fig. 9 The numbers in the middle correspond to the groups: 1. control group; 2. ultrasound group; 3. trastuzumab group; 4. oxygen-containing sonosensitizer-trastuzumab nanoparticles group; 5. non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group; 6. oxygen-containing sonosensitizer nanoparticles + ultrasound group; 7. oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group.

[0045] Example 10

[0046] Immunoblotting was performed to investigate the effects of ultrasound on targeted nanoparticles in improving hypoxia, enhancing trastuzumab and sonodynamic therapy. The specific operations are as follows: ① Protein extraction: NCI-N87 cells were inoculated in a 6-well cell culture plate and cultured for 24 hours at 37°C and 5% CO2 to allow the cells to fully adhere to the wall. The experimental groups were as follows: control, ultrasound, trastuzumab, oxygen-containing sonosensitizer-trastuzumab nanoparticles, non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound, oxygen-containing sonosensitizer nanoparticles + ultrasound, oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound. The previous culture medium was replaced with a culture medium containing the above-mentioned drugs, and after continuing to culture for 8 hours, ultrasound treatment (1.0 MHz, 50% duty cycle, 1.0 W / cm2) was performed for 3 minutes. After ultrasound treatment, the cells were cultured for 16 hours, and the cells were digested with trypsin and collected by centrifugation. After washing with phosphate buffer, transfer to a 1.5 ml centrifuge tube, centrifuge at 1000 rpm for 5 minutes to obtain a cell pellet, add 75 μl of protein lysis solution containing 1% protease inhibitor, and then perform ultrasonic treatment (power 200W, working time 6 seconds, interval time 6 seconds, repeat 3 times) to break the cells, after ice bathing for 30 minutes, centrifuge at 4°C, 12000 rpm for 10 minutes, and take the cell supernatant. ② Protein concentration determination: According to the BCA protein quantification kit, the protein concentration of each group was measured, and the standard protein sample in the kit was prepared into 2000 μg / ml, and then diluted into 500, 400, 300, 200, 100, 50 μg / ml and 25 μg / ml protein standards. Take 2 microliters of protein standard and the supernatant of the protein to be tested and add them to a 96-well cell culture plate, then add 18 microliters of phosphate buffer, three replicates for each sample, then add 200 microliters of BCA working solution (the A solution and B solution provided in the BCA kit are prepared at a ratio of 1:50), incubate in a 37°C incubator for 30 minutes, then use an enzyme reader to measure the absorbance at 563 nanometers and calculate the protein concentration based on the standard curve obtained from the standard. ③ Sample denaturation: According to the sample with the lowest concentration, dilute the other samples with phosphate buffer to the same concentration. Add 5×SDS protein loading buffer to each protein sample at a ratio of 1:4, boil in a 95°C metal bath for 5 minutes, and then centrifuge at 12,000 rpm for 1 minute. ④ Protein electrophoresis: Place the precast gel in the electrophoresis tank, pull out the comb and add 8 microliters of protein sample, and store the remaining protein sample in a -80°C refrigerator for use. Add protein markers on both sides of the sample, 8 microliters on the left and 4 microliters on the right, respectively. First, perform protein electrophoresis at 80 volts. When the protein standard bands begin to separate, adjust the voltage to 100 volts until the bromophenol blue bands run to the bottom, and then terminate the electrophoresis.⑤ Transfer: Prepare the transfer solution and place it in the refrigerator to pre-cool. Gently pry open the glass plate, take out the protein gel, cut off the upper gel and the separation gel under the bromophenol blue band, wash it with transfer solution, put it on the transfer filter paper, and then cut out the NC membrane of the same size according to the size, put the NC membrane on the gel, put the transfer filter paper on it and put it in the transfer tank. Put the transfer device in a 4℃ refrigerator and transfer the membrane at 100 volts for 1 hour. ⑥ Blocking: Take out the NC membrane, rinse it with 1×TBST, and then block it with 5% milk for 1h. ⑦ Incubate the primary antibody: After blocking, rinse with 1×TBST until the solution is clear, add the primary antibodies of β-Actin, HIF-1α, P-AKT, Cle-caspase3, and Bcl2 respectively (the dilution ratio of β-Actin is 1:1000, and the dilution ratio of HIF-1α, P-AKT, Cle-caspase3, and Bcl2 is 1:500), and incubate overnight at 4℃. ⑧ Incubate with secondary antibody: After rinsing the NC membrane with 1×TBST for 3 times, add the corresponding secondary antibody (dilution ratio is 1:1000), incubate for 1 hour, and then repeat rinsing with 1×TBST for 3 times. ⑨ Exposure: Mix exposure solution A and B in a 1:1 ratio, dry the NC membrane with filter paper and place it in a clean culture dish, rinse the protein surface of the membrane evenly with exposure solution, then absorb the excess exposure solution and expose it. Fig.10 As shown in the figure, due to ultrasound-mediated O2 release, the expression of HIF-1α in the oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group was significantly lower than that in the non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group and the oxygen-containing sonosensitizer nanoparticles + ultrasound group. Compared with the control group, the expression of HIF-1α in the non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group was upregulated, which was related to the consumption of O2 by sonodynamic therapy. Trastuzumab inhibits the PI3K / AKT signaling pathway by inhibiting the phosphorylation of HER-2. The results of immunoblotting showed that the expression of pAKT in trastuzumab and oxygen-sonosensitizer-trastuzumab nanoparticles was slightly lower than that in the control group and ultrasound group, indicating that trastuzumab inhibited the phosphorylation of AKT. The pAKT in the oxygen-sonosensitizer-trastuzumab nanoparticles + ultrasound group and the non-oxygen-sonosensitizer-trastuzumab nanoparticles + ultrasound group was downregulated, but the oxygen-sonosensitizer-trastuzumab nanoparticles + ultrasound group was more significantly downregulated, further indicating that ultrasound-mediated O2 release improved the therapeutic effect of trastuzumab. Cle-caspase3 was increased in the non-oxygen-sonosensitizer-trastuzumab nanoparticles + ultrasound group, the oxygen-sonosensitizer nanoparticles + ultrasound group, and the oxygen-sonosensitizer-trastuzumab nanoparticles + ultrasound group, among which the oxygen-sonosensitizer-trastuzumab nanoparticles + ultrasound group was more significantly increased. Bcl2 was downregulated in the non-oxygen sonosensitizer-trastuzumab nanoparticles + ultrasound, oxygen-containing sonosensitizer nanoparticles + ultrasound, and oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound groups, and the downregulation was more obvious in the oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group. Fig.10 The numbers in the middle correspond to the groups: 1. control group; 2. ultrasound group; 3. trastuzumab group; 4. oxygen-containing sonosensitizer-trastuzumab nanoparticles group; 5. non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group; 6. oxygen-containing sonosensitizer nanoparticles + ultrasound group; 7. oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group.

[0047] Embodiment 11

[0048] In order to evaluate the targeting effect of trastuzumab and the distribution of nanoparticles in vivo, in vivo near-infrared fluorescence imaging was performed. First, a nude mouse subcutaneous NCI-N87 tumor model was constructed. When the subcutaneous tumor reached 150 cubic millimeters, free sonosensitizer lipids, oxygenated sonosensitizer nanoparticles, and oxygenated sonosensitizer-trastuzumab nanoparticles were injected into the mouse through the tail vein (pyropheophorbide a lipid concentration: 8 mg / kg). At the preset time points, the IVIS small animal in vivo fluorescence imaging system (excitation: 675 nm, emission: 720 nm) was used to observe the enrichment of sonosensitizers in mouse tumors. After the optimal enrichment time point, the main organs (heart, liver, spleen, lung, kidney) and tumor tissues of the mice were collected for in vitro imaging. The results are shown in Figure 2. Fig.11 As shown in the figure, compared with free sonosensitizer lipids, the fluorescence intensity of the oxygenated sonosensitizer nanoparticle group increased, which was related to the enhanced penetration and retention effect. The fluorescence intensity of the oxygenated sonosensitizer-trastuzumab nanoparticles was further enhanced than that of the oxygenated sonosensitizer nanoparticles, indicating that the targeting effect of trastuzumab can further enhance the enrichment of sonosensitizers in tumor sites. Over time, the fluorescence signal at the tumor site gradually increased and reached a peak at 24 hours. The mice were euthanized at 36 hours, and in vitro organ imaging also showed that the fluorescence signal of the tumor tissue was inhibited compared with the in vivo imaging results, with the strongest fluorescence signal in the oxygenated sonosensitizer-trastuzumab nanoparticle group. The tumor fluorescence intensity of the oxygenated sonosensitizer nanoparticles and oxygenated sonosensitizer-trastuzumab nanoparticles groups was approximately 1.52 times and 2.49 times that of the free sonosensitizer lipids, respectively, which again indicated that the enhanced penetration and retention effect mediated by nanoparticles combined with the targeting effect of trastuzumab can allow more sonosensitizer pyropheophorbide a to enter the tumor, thereby improving the therapeutic effect.

[0049] Example 12

[0050] In vivo treatment experiments investigated whether the prepared oxygen-containing sonosensitizer-trastuzumab nanoparticles effectively inhibited tumor growth under ultrasound. When the tumor grew to 150-200 cubic millimeters, nude mice carrying NCI-N87 subcutaneous tumors were randomly divided into 7 groups, and treated with 1. control, 2. ultrasound, 3. trastuzumab, 4. oxygen-containing sonosensitizer-trastuzumab nanoparticles, 5. oxygen-free sonosensitizer-trastuzumab nanoparticles + ultrasound, 6. oxygen-containing sonosensitizer nanoparticles + ultrasound, and 7. oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound. The administration method was 200 microliters (pyropheophorbide a lipid concentration: 8 mg / kg, trastuzumab concentration: 6 mg / kg) in the tail vein. The ultrasound treatment group was subjected to ultrasound irradiation (1.0 MHz, 2 W / cm2, 50% duty cycle, 5 minutes) 24 hours after drug injection. The treatment was carried out on days 1, 4, 7, 10 and 13. The mice were euthanized on day 20 of treatment, and the tumors were removed and weighed. The tumor volume and body weight changes were recorded every two days (tumor volume = 1 / 2 × length × width). 2 ).like Fig.12 As shown in the figure, the tumor volume growth trend of the ultrasound group was basically the same as that of the control group, indicating that ultrasound therapy alone had no effect on tumor growth. The trastuzumab group and the TPPO NPs group were slightly slower than the control group, which was related to the tumor inhibitory effect of trastuzumab. Among them, the oxygen-containing sonosensitizer-trastuzumab nanoparticle group could deliver more trastuzumab into the tumor tissue due to the enhanced penetration and retention effect of the nanoparticles. In sharp contrast, the oxygen-containing sonosensitizer nanoparticles + ultrasound, the oxygen-free sonosensitizer-trastuzumab nanoparticles + ultrasound, and the oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound groups had obvious tumor growth inhibition effects, among which the oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound had the strongest tumor inhibition effect, which was related to the efficient delivery of O2 by targeted nanoparticles, the local release of O2 under ultrasound to improve hypoxia, and the enhanced sonodynamics and antibody therapy. The trend of tumor weight change after the end of treatment was consistent with the change in tumor volume. Fig.12 The numbers in the middle correspond to the groups: 1. control group; 2. ultrasound group; 3. trastuzumab group; 4. oxygen-containing sonosensitizer-trastuzumab nanoparticles group; 5. non-oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group; 6. oxygen-containing sonosensitizer nanoparticles + ultrasound group; 7. oxygen-containing sonosensitizer-trastuzumab nanoparticles + ultrasound group.

Claims

1. A targeted nanoparticle for simultaneously delivering a sonosensitizer, O2 and trastuzumab, wherein: The carrier of the targeted nanoparticles is composed of a lipid sonosensitizer, distearoyl phosphatidylethanolamine-polyethylene glycol. 2000 , Distearoylphosphatidylethanolamine-polyethylene glycol 2000 -NHS was used to prepare sonosensitizer nanoparticles PNPs, and trastuzumab was coupled to the nanoparticles through an amide reaction to obtain sonosensitizer-trastuzumab nanoparticles TP NPs, wherein the mass ratio of the sonosensitizer nanoparticles P NPs to trastuzumab was 2:

1. Then, the O2 carrier liquid fluorocarbon was loaded into the TP NPs through probe ultrasound to obtain TPP NPs. The TPP NPs dispersion was placed in an O2 chamber with a constant gas flow rate of 7.5 L / min, and TPPONPs were obtained after oxygen saturation for 30 min.

2. The targeted nanoparticle oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs for simultaneously delivering sonosensitizer, O2 and trastuzumab as claimed in claim 1, characterized in that: The sonosensitizer in the lipidated sonosensitizer is selected from pyropheophorbide a, and the carrier liquid fluorocarbon of O2 is selected from perfluorooctyl bromide PFOB.

3. The targeted nanoparticle oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs for simultaneously delivering sonosensitizer, O2 and trastuzumab as claimed in claim 2, characterized in that: The lipid sonosensitizer is pyropheophorbide a lipid, and its structural formula is as follows:

4. The targeted nanoparticle oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs for simultaneously delivering sonosensitizer, O2 and trastuzumab as claimed in claim 2, characterized in that: The carrier of O2 is perfluorooctyl bromide PFOB, and its structural formula is as follows:

5. The targeted nanoparticle oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs for simultaneously delivering sonosensitizer, O2 and trastuzumab as claimed in claim 1, characterized in that: The average hydrated particle size of the nanoparticles is 249±3.41 nm.

6. The targeted nanoparticle oxygen-containing sonosensitizer-trastuzumab nanoparticle TPPO NPs for simultaneously delivering sonosensitizer, O2 and trastuzumab as claimed in claim 1, characterized in that: Under the action of ultrasound, targeted nanoparticles can release O2 in a controlled manner on the one hand, and play a role in sonodynamic therapy on the other.

7. A method for preparing a targeted nanoparticle for simultaneously delivering a sonosensitizer, O2 and trastuzumab as claimed in any one of claims 1 to 6, comprising the following steps: 1) Distearoyl phosphatidylethanolamine-polyethylene glycol 2000 Dissolve in anhydrous ethanol to obtain solution A. 2) Distearoyl phosphatidylethanolamine-polyethylene glycol 2000 -NHS was dissolved in anhydrous ethanol to obtain solution B. 3) Dissolve the lipidated sonosensitizer in tetrahydrofuran to obtain solution C. 4) Solutions A, B, and C are mixed, and organic reagents are removed by rotary evaporation. Phosphate buffer or physiological saline is added under water bath ultrasound to form nanoparticles PNPs containing sonosensitizers. 5) The obtained sonosensitizer nanoparticles P NPs were mixed with trastuzumab in phosphate buffer or physiological saline, and gently stirred for 6 hours under light-proof conditions. Unattached trastuzumab was removed by dialysis (molecular weight = 500 kDa) to obtain sonosensitizer-trastuzumab nanoparticles TP NPs. 6) Under ice bath ultrasound conditions, perfluorooctyl bromide PFOB was loaded into sonosensitizer-trastuzumab nanoparticles TP NPs to obtain TPPNPs. 7) The TPP NPs dispersion was placed in an oxygen chamber and saturated with a constant airflow for 30 min to obtain the O2-carrying targeted nanoparticle oxygen-containing sonosensitizer-trastuzumab nanoparticles TPPO NPs.

8. A method for preparing targeted nanoparticles for simultaneously delivering sonosensitizer, O2 and trastuzumab as claimed in claim 7, characterized in that The lipidated sonosensitizer in step 3) is pyropheophorbide a lipid; in step 4), the molar ratio of solution A, solution B, and solution C is 1:1:8; in step 5), the mass ratio of sonosensitizer nanoparticles P NPs to trastuzumab is 2:1; in step 6), 10 μl of PFOB is added to 1 mg of sonosensitizer-trastuzumab nanoparticles TP NPs.

9. Use of a targeted nanoparticle for simultaneously delivering a sonosensitizer, O2 and trastuzumab as described in any one of claims 1 to 8 in the diagnosis and treatment of HER-2 positive gastric cancer.

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