Preparation method and application of IR780 and gambogic acid prodrug loaded liposome

Abstract

The invention discloses a preparation method of IR780 and gambogic acid prodrug-loaded lipidosome, which comprises the following steps: dissolving mPEG-TK-NH2 in dimethylformamide, adding gambogic acid, benzoyl peroxide and triethylamine, stirring for reaction, concentrating and recrystallizing to obtain mPEG-TK-GA, adding mPEG-TK-GA, soybean phosphatidylcholine, cholesterol and IR780 into an organic solvent, and carrying out rotary evaporation to remove the organic solvent, thereby obtaining the IR780 and gambogic acid prodrug-loaded lipidosome. The SIRP-alpha gene engineering exosome preparation method comprises the following steps: preparing a lipid membrane, adding a phosphate buffer solution, heating, carrying out ultrasonic treatment, granulating to obtain a liposome solution, mixing an SIRP-alpha gene engineering exosome and IG Lipo, and carrying out membrane fusion by adopting a freeze-thaw method to obtain the SIRP-alpha gene engineering exosome preparation. Compared with the prior art, the liposome prepared by the method has a responsive drug release effect, can relieve toxic and side effects while improving the treatment effect, is also loaded with IR780, and can reduce the dosage and improve the treatment effect in combination with photothermal therapy.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and nanotechnology intersection, and specifically relates to a preparation method and application of liposomes loaded with IR780 and garcinia acid prodrug. Background Art

[0002] Garcinia, derived from the dried resin secreted by Garcinia hanbaryi Hook.f., a plant of the Garcinia family, is mainly produced in Cambodia, India, Thailand and Vietnam, and is also cultivated in Guangdong and Hainan provinces in China. In ancient Chinese medical books, Garcinia is recorded to have multiple effects such as breaking blood and dispersing knots, detoxifying, stopping bleeding, and killing insects because of its cold nature, sour taste, pungent, astringent, and toxicity. Gambogic acid (GA), as an active ingredient extracted from the traditional Chinese medicine Garcinia, is a broad-spectrum anti-tumor drug that has a significant inhibitory effect on a variety of tumor cells. Studies have shown that the anti-tumor mechanism of GA is complex and diverse, including inducing tumor cell apoptosis, inhibiting cell cycle progression, affecting the expression of oncogenes and tumor suppressor genes and their related proteins, and inhibiting tumor cell metastasis, etc. These characteristics are not possessed by many current chemotherapy drugs.

[0003] However, in practical applications, garcinia acid often has poor effects due to its own defects, such as poor solubility. During intravenous injection, it is often necessary to add excipients to aid solubility, which causes irritation to the body; free garcinia acid has low bioavailability and short half-life in the body; and garcinia acid also has a narrow safe therapeutic window during treatment. These problems not only limit the clinical application of garcinia acid, but also affect the full play of its therapeutic effect. Therefore, it is particularly important to modify the structure of garcinia acid or develop new dosage forms. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a method for preparing liposomes loaded with IR780 and gambogic acid prodrugs. The liposomes prepared by the method have a responsive drug release effect and can be responsively released at the tumor site, thereby improving the therapeutic effect while reducing toxic side effects. The liposomes are also loaded with IR780, and combined with photothermal therapy, the dosage can be reduced and the therapeutic effect can be improved. The liposomes also have biological camouflage and tumor targeting, can avoid the phagocytic effect of the mononuclear macrophage system, and are efficiently enriched at the tumor site.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A method for preparing liposomes loaded with IR780 and gambogic acid prodrug comprises the following steps:

[0007] S1, dissolving mPEG-TK-NH2 (methoxy polyethylene glycol-thioketal-amino) in dimethylformamide (DMF), then adding gambogic acid (GA), benzoyl peroxide (BOP) and triethylamine for stirring reaction, concentrating and recrystallizing the obtained reaction solution, filtering and collecting the product to obtain mPEG-TK-GA;

[0008] S2, soybean phosphatidylcholine (S 100 PC), cholesterol, IR780 and mPEG-TK-GA were added to an organic solvent, and after being completely dissolved, the organic solvent was removed from the mixed solution by rotary evaporation to obtain a lipid film, and a phosphate buffer solution (PBS) was added, heated and ultrasonically treated to obtain a crude liposome solution, and after granulation, a liposome solution (IG Lipo) was obtained;

[0009] S3. Gene transfection of 4T1 cells to prepare SIRP-α genetically engineered exosomes, the SIRP-α genetically engineered exosomes and liposome solution were mixed, and then membrane fusion was performed using the freeze-thaw method to prepare liposomes loaded with IR780 and garcinia acid prodrug (IG Exo-Lipo).

[0010] Furthermore, in step S1, the molecular weight of PEG in the mPEG-TK-NH2 is 2000.

[0011] Furthermore, in step S1, the molar ratio of mPEG-TK-NH2, gambogic acid, benzoyl peroxide and triethylamine is 1:1.1:1.3:5.0.

[0012] Furthermore, in step S2, the mass ratio of IR780, mPEG-TK-GA, soybean phosphatidylcholine and cholesterol is 15:(1-5):150:75.

[0013] Furthermore, in step S2, the whole particle is processed by an ultrasonic cell disruptor with an ultrasonic frequency of 100 KHz.

[0014] Further, in step S3, the method for preparing the SIRP-α genetically engineered exosomes is as follows: 4T1 cells are incubated in a 6-well culture plate overnight, and then a lentivirus encapsulating a SIRP-α plasmid is added to the cells. After 24 hours, the cells are cultured with fresh DMEM medium to remove the lentivirus, 4T1 cells transfected with the SIRP-α gene are selected with streptomycin, and the culture supernatant is collected, and separated and extracted by ultracentrifugation to obtain SIRP-α genetically engineered exosomes.

[0015] Furthermore, in step S3, the mass ratio of the SIRP-α genetically engineered exosomes to the liposome solution is 1:1.

[0016] Furthermore, in step S3, the freeze-thaw method for membrane fusion is as follows: the mixture is frozen at -80°C for 15 minutes, then incubated at 37°C for 15 minutes, and the freeze-thaw cycle is repeated 3 times.

[0017] The liposome loaded with IR780 and gambogic acid prodrug prepared by the above method is used in the preparation of therapeutic drugs for breast cancer, melanoma, lung cancer, prostate cancer and liver cancer.

[0018] Beneficial effects of the present invention: The present invention develops a multifunctional nanoplatform IG Exo-Lipo disguised by genetically engineered exosomes by fusing them with responsive drug-releasing liposomes, which is used for triple-negative breast cancer therapy combined with low-dose chemotherapy and low-temperature photothermal therapy (PTT). First, 4T1 cells are transfected with genes to obtain exosomes overexpressing SIRPα, and CD47-SIRPα is blocked to achieve the purpose of prolonging circulation time, escaping phagocytosis of the mononuclear phagocyte system, and enhancing homologous targeting in vivo; then, the genetically engineered exosomes are fused with liposomes to form Exo-Lipo, and the ROS-responsive prodrug of gambogic acid (mPEG-TK-GA) and the photothermal agent IR780 iodide are encapsulated into Exo-Lipo to prepare an engineered exosome-disguised multifunctional nanoplatform IG Exo-Lipo for the treatment of triple-negative breast cancer. In this system, IG Exo-Lipo can prolong blood circulation time and increase drug accumulation at the tumor site through SIRPα genetically engineered exosome modification and PEGylation. After IR780 is taken up by tumor cells, it exhibits low-temperature PTT under near-infrared radiation and triggers the generation and release of ROS. In addition, the generated ROS can trigger the cleavage of mPEG-TK-GA and release GA from IG Exo-Lipo as needed. The released GA exerts a low-dose chemotherapeutic effect in tumor cells and inhibits the expression of HSP90, improving the therapeutic effect of low-temperature PTT by reversing the photothermal resistance feedback. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the H NMR spectrum of mPEG-TK-GA prepared in Example 1;

[0020] Figure 2 The results of testing the content of SIRPα-positive cells in 4T1 cells after gene transfection in Example 1;

[0021] Figure 3 The UV absorption spectra of IG Exo-Lipo prepared in Example 1, IR780 Exo-Lipo prepared in Comparative Example 1, and GA Exo-Lipo prepared in Comparative Example 2;

[0022] Figure 4The particle size distribution and zeta potential diagram of IG Exo-Lipo prepared in Examples 1-4;

[0023] Figure 5 TEM image of IG Exo-Lipo prepared in Example 1;

[0024] Figure 6 This is a ROS-responsive drug release curve of IG Exo-Lipo prepared in Example 1;

[0025] Figure 7 The results of cytotoxicity tests for different treatment groups;

[0026] Figure 8 The results of the cellular uptake test of free IR780 and IG Exo-Lipo;

[0027] Fig. 9 The results of the apoptotic cell percentage test after treatment with different treatment groups;

[0028] Fig.10 The test results of ROS content of IG Exo-Lipo in vitro;

[0029] Fig.11 Western-Blot diagram of IG Exo-Lipo regulating HSP90 expression;

[0030] Fig.12 The highest temperature test results of the tumor site of mice in each treatment group;

[0031] Fig.13 The fluorescence intensity test results of free IR780 and IG Exo-Lipo in the tumor site at 2h and 6h;

[0032] Fig.14 The results of the volume and weight tests of the tumors in mice in different treatment groups;

[0033] Fig.15 is the body weight of tumor-bearing mice in each treatment group during the treatment period;

[0034] Fig.16 H&E staining results of visceral tissue sections of each treatment group;

[0035] Fig.17 The H&E, TUNEL, Ki-67, and heat shock protein 90 (HSP90) staining results of tumor tissue sections in each treatment group. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is clearly and completely described below in conjunction with the accompanying drawings and specific embodiments.

[0037] It should be noted that in the following examples, mPEG2000-TK-NH2 was purchased from Xi'an Ruixi Biological Co., Ltd.; Garcinia acid was purchased from Aladdin Biochemical Technology Co., Ltd.; IR780 was purchased from Sigma-Aldrich; 4T1 cells were purchased from Guangzhou Saiku Biotechnology Co., Ltd.; and the exosome rapid extraction kit was purchased from Yuyisheng Biological Co., Ltd.; but they are not limited to these.

[0038] Example 1

[0039] A method for preparing liposomes loaded with IR780 and gambogic acid prodrug comprises the following steps:

[0040] S1. Dissolve 1 mM mPEG2000-TK-NH2 (methoxypolyethylene glycol-ketalthiocarboxylate-amino) in 10 mL dimethylformamide (DMF), then add gambogic acid (GA, 1.1 mM), benzoyl peroxide (BOP, 1.3 mM) and triethylamine (5.0 mM), stir and react for 0.5 hours after dissolution, concentrate the obtained reaction solution by rotary and reduced pressure evaporation, recrystallize with ethanol, collect the product by filtration, and then concentrate in vacuo to obtain mPEG-TK-GA. Use hydrogen spectrum nuclear magnetic resonance ( 1 H NMR) spectroscopy;

[0041] S2, 150mg soybean phosphatidylcholine (S 100 PC), 75 mg cholesterol, 15 mg IR780 and 4 mg mPEG-TK-GA were added to 20 mL dichloromethane. After complete dissolution, the mixed solution was transferred to a round-bottom flask, and dichloromethane was removed by rotary evaporation. A lipid film was formed at 40°C, and a phosphate buffer solution (PBS) with a pH of 7.4 was added. The mixture was heated to 60°C and sonicated with a probe sonicator to obtain a crude liposome solution. The free IR780 and mPEG-TK-GA were removed by filtration through a 0.22 μm microporous filter, and the granules were sized using an ultrasonic cell disruptor (ultrasonic frequency of 100 KHz) to obtain a liposome solution (IGLipo);

[0042] S3. Gene transfection of 4T1 cells to prepare SIRP-α genetically engineered exosomes. The specific method is as follows: 4T1 cells were incubated in a 6-well culture plate overnight, and then a lentivirus (constructed by Fubaiao Biotechnology Co., Ltd.) encapsulating SIRP-α plasmid was added to the cells. After 24 hours, the cells were cultured with fresh DMEM medium to remove the lentivirus, and the 4T1 cells transfected with SIRP-α gene were selected with streptomycin, and the culture supernatant was collected and separated and extracted by ultracentrifugation to obtain SIRP-α genetically engineered exosomes.

[0043] SIRP-α genetically engineered exosomes and IG Lipo were mixed at a mass ratio of 1:1, and then the mixture was frozen at -80°C for 15 min and incubated at 37°C for 15 min. After 3 cycles, liposomes loaded with IR780 and gambogic acid prodrugs were obtained through lipid membrane fusion between liposomes and exosomes, which were recorded as IG Exo-Lipo.

[0044] Example 2

[0045] In step (2), the amount of mPEG-TK-GA used is 1 mg, and the rest is the same as in Example 1.

[0046] Example 3

[0047] In step (2), the amount of mPEG-TK-GA used is 2 mg, and the rest is the same as in Example 1.

[0048] Example 4

[0049] In step (2), the amount of mPEG-TK-GA used is 3 mg, and the rest is the same as in Example 1.

[0050] Comparative Example 1

[0051] Preparation of IR780 Exo-Lipo, comprising the following steps:

[0052] S1, 150mg soybean phosphatidylcholine (S 100 PC), 75 mg cholesterol, and 15 mg IR780 were added to 20 mL dichloromethane. After complete dissolution, the mixed solution was transferred to a round-bottom flask, and dichloromethane was removed by rotary evaporation to form a lipid film at 40°C. A phosphate buffer solution (PBS) with a pH of 7.4 was added, and the mixture was heated to 60°C and sonicated with a probe sonicator to obtain a crude liposome solution. The free IR780 was removed by filtration through a 0.22 μm microporous filter, and the granules were sized using an ultrasonic cell disruptor (ultrasonic frequency of 100 KHz) to obtain a liposome solution (IR780 Lipo);

[0053] S2. Gene transfection of 4T1 cells to prepare SIRP-α genetically engineered exosomes. The specific method is as follows: 4T1 cells were incubated in a 6-well culture plate overnight, and then a lentivirus (constructed by Fubaiao Biotechnology Co., Ltd.) encapsulating SIRP-α plasmid was added to the cells. After 24 hours, the cells were cultured with fresh DMEM medium to remove the lentivirus, and the 4T1 cells transfected with SIRP-α gene were selected with streptomycin, and the culture supernatant was collected and separated and extracted by ultracentrifugation to obtain SIRP-α genetically engineered exosomes.

[0054] SIRP-α genetically engineered exosomes and IR780 Lipo were mixed at a mass ratio of 1:1, and then the mixture was frozen at -80°C for 15 min and incubated at 37°C for 15 min. After 3 cycles, the lipid membranes between liposomes and exosomes were fused to obtain exosomes-liposomes loaded with IR780, which were recorded as IR780Exo-Lipo.

[0055] Comparative Example 2

[0056] Preparation of GA Exo-Lipo, comprising the following steps:

[0057] S1, dissolving 1mM mPEG2000-TK-NH2 (methoxypolyethylene glycol-ketalthiocarboxylate-amino) in 10mL dimethylformamide (DMF), then adding gambogic acid (GA, 1.1mM), benzoyl peroxide (BOP, 1.3mM) and triethylamine (5.0mM), stirring and reacting for 0.5 hours after dissolution, concentrating the obtained reaction solution by rotary and reduced pressure evaporation, recrystallizing with ethanol, collecting the product by filtration, and then concentrating in vacuo to obtain mPEG-TK-GA;

[0058] S2, 150mg soybean phosphatidylcholine (S 100 PC), 75 mg cholesterol and 4 mg mPEG-TK-GA were added to 20 mL dichloromethane. After complete dissolution, the mixed solution was transferred to a round-bottom flask, and dichloromethane was removed by rotary evaporation. A lipid film was formed at 40°C, and a phosphate buffer solution (PBS) with a pH of 7.4 was added. The mixture was heated to 60°C and sonicated with a probe sonicator to obtain a crude liposome solution. The free mPEG-TK-GA was removed by filtration through a 0.22 μm microporous filter, and the granules were sized using an ultrasonic cell disruptor (ultrasonic frequency of 100 KHz) to obtain a liposome solution (GA Lipo);

[0059] S3. Gene transfection of 4T1 cells to prepare SIRP-α genetically engineered exosomes. The specific method is as follows: 4T1 cells were incubated in a 6-well culture plate overnight, and then a lentivirus (constructed by Fubaiao Biotechnology Co., Ltd.) encapsulating SIRP-α plasmid was added to the cells. After 24 hours, the cells were cultured with fresh DMEM medium to remove the lentivirus, and the 4T1 cells transfected with SIRP-α gene were selected with streptomycin, and the culture supernatant was collected and separated and extracted by ultracentrifugation to obtain SIRP-α genetically engineered exosomes.

[0060] SIRP-α genetically engineered exosomes and GA Lipo were mixed at a mass ratio of 1:1, and then the mixture was frozen at -80°C for 15 min and incubated at 37°C for 15 min. After 3 cycles, exosomes-liposomes loaded with garcinia acid prodrug were obtained through lipid membrane fusion between liposomes and exosomes, which were recorded as GA Exo-Lipo.

[0061] Comparative Example 3

[0062] Preparation of Blank Exo-Lipo, comprising the following steps:

[0063] S1, 150mg soybean phosphatidylcholine (S 100 PC), 75 mg cholesterol were added into 20 mL dichloromethane, and after being completely dissolved, the mixed solution was transferred into a round-bottom flask, and dichloromethane was removed by rotary evaporation, and a lipid film was formed at 40°C, and a phosphate buffer solution (PBS) with a pH of 7.4 was added, and the mixture was heated to 60°C and ultrasonicated with a probe sonicator to obtain a crude liposome solution, which was granulated with an ultrasonic cell disruptor (ultrasonic frequency of 100 KHz) to obtain a liposome solution (BlankLipo);

[0064] S2. Gene transfection of 4T1 cells to prepare SIRP-α genetically engineered exosomes. The specific method is as follows: 4T1 cells were incubated in a 6-well culture plate overnight, and then a lentivirus (constructed by Fubaiao Biotechnology Co., Ltd.) encapsulating SIRP-α plasmid was added to the cells. After 24 hours, the cells were cultured with fresh DMEM medium to remove the lentivirus, and the 4T1 cells transfected with SIRP-α gene were selected with streptomycin, and the culture supernatant was collected and separated and extracted by ultracentrifugation to obtain SIRP-α genetically engineered exosomes.

[0065] SIRP-α genetically engineered exosomes and Blank Lipo were mixed at a mass ratio of 1:1, and then the mixture was frozen at -80°C for 15 min and incubated at 37°C for 15 min. After 3 cycles, blank exosome-liposomes were obtained through lipid membrane fusion between liposomes and exosomes, recorded as Blank Exo-Lipo.

[0066] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the mPEG-TK-GA prepared in Example 1. The chemical structure of mPEG-TK-GA was characterized by 1H NMR. As shown in the figure, the typical peak at about 1.10 ppm corresponds to the proton of the CH3 group in the TK-containing linker. The peak at about 3.70 ppm corresponds to the proton of the CH2 group in the TK-containing linker, and the results show that mPEG-TK-GA was successfully synthesized.

[0067] Figure 2The results of the test of the content of SIRPα-positive cells in 4T1 cells after gene transfection in Example 1 are shown in the figure. As shown in the figure, stronger green fluorescence (SIRPα-positive cells) was observed in the cells transfected with SIRPα gene, and flow cytometric analysis showed that the percentage of SIRPα-positive cells in the gene-transfected 4T1 cells was 66.57±1.52%, which was much higher than that of wild-type 4T1 cells. The results show that successful gene transfection increases the expression of SIRPα on the surface of 4T1 cells.

[0068] Figure 3 The UV absorption spectra of IG Exo-Lipo prepared in Example 1, IR780 Exo-Lipo prepared in Comparative Example 1, and GA Exo-Lipo prepared in Comparative Example 2 show that IG Exo-Lipo can successfully co-load IR780 and GA. These results confirm that IG Exo-Lipo is a stable and uniform nanoformulation that successfully co-encapsulates IR780 and GA, which has the potential to be used in subsequent experiments.

[0069] 1. The particle size and zeta potential of the IG Exo-Lipo prepared in Examples 1-4 were determined by Malvern particle size analyzer, the morphology of the IG Exo-Lipo was observed by transmission electron microscopy, and the contents of IR780 and GA in the IG Exo-Lipo were determined by high performance liquid chromatography to determine the encapsulation efficiency and drug loading.

[0070] Figure 4 The particle size distribution and zeta potential diagram of IG Exo-Lipo prepared in Examples 1-4, wherein: Figure 4 A is the particle size distribution diagram, Figure 4 B is the zeta potential. It can be seen that the average particle size of IG Exo-Lipo is 156±2nm and the average zeta potential is 3±1mv, indicating that the surface of IG Exo-Lipo has a slight positive charge.

[0071] The particle size and EEs test results of IG Exo-Lipo prepared in Examples 1-4 are shown in Table 1:

[0072] Table 1

[0073]

[0074]

[0075] It can be concluded from Table 1 that the IG Exo-Lipo prepared in Examples 1-4 of the present invention all have good encapsulation efficiency and particle size distribution.

[0076] Figure 5This is the transmission electron microscopy image of IG Exo-Lipo prepared in Example 1. It can be seen that IG Exo-Lipo has a nearly spherical structure.

[0077] 2. The ROS-responsive drug release behavior of IG Exo-Lipo prepared in Example 1 was detected by dialysis, and H2O2 was used to simulate the generation of ROS in vivo. In detail, IG Exo-Lipo was placed in dialysis bags containing PBS solution (Control group) and PBS solution containing 0.1mM H2O2 (0.1mM H2O2 group), respectively. At different time points, all released dissolution media (PBS solution containing 0.5% Tween 80) were collected and replaced with fresh dissolution media. After the collection was completed, GA in the dissolution medium was detected by HPLC, and the release curves of GA under different conditions were calculated.

[0078] Figure 6 The ROS-responsive drug release curve of IG Exo-Lipo prepared in Example 1 shows that the release rate of GA in the Control group is very slow, with only 7.92±0.85% of GA released in the external medium at 24 hours, compared with 37.78±6.74% of GA released in the 0.1mM H2O2 group at the same time. During the entire release process, GA in the control group was released slowly, with only 27.03±4.72% of GA released in the in vitro release medium at 336h, while GA in the 0.1mM H2O2 group was almost completely released with a faster release rate, with a cumulative release of 94.19±10.14% at 168h and 98.9±9.99% at 336h.

[0079] 3. Cytotoxicity test:

[0080] The experiment set up the following treatment groups: NS, NS+L, Blank Exo-Lipo, GA Exo-Lipo, IR780Exo-Lipo, IR780 Exo-Lipo+L, IG Exo-Lipo, IG Exo-Lipo+L, among which the NS group used normal saline, the NS+L used normal saline plus light treatment, the Blank Exo-Lipo group used the blank exosome-liposome BlankExo-Lipo prepared in Comparative Example 3, the GA Exo-Lipo group used the GA Exo-Lipo prepared in Comparative Example 2, the IR780 Exo-Lipo group used the IR780 Exo-Lipo prepared in Comparative Example 1, the IR780 Exo-Lipo+L group used the IR780 Exo-Lipo prepared in Comparative Example 1 plus light treatment, the IG Exo-Lipo group used the IG Exo-Lipo prepared in Example 1, and the IG Exo-Lipo+L group used the IG Exo-Lipo prepared in Example 1 plus light treatment.

[0081] First, 4T1 cells were seeded into 96-well plates and incubated for 24 hours. Different treatment group preparations were added and incubated at 37°C for 6 hours. DMEM medium was replaced with fresh DMEM medium, and the groups that needed light treatment were irradiated with NIR laser for 5 minutes (808nm, 1.0W / cm 2 ), and then incubated for another 24 hours, and then 20 μL of MTT (5 mg / mL) was added to each well, and the cells were incubated for 4 hours at 37° C. Finally, 200 μL of DMSO was added to dissolve the precipitated formazan, and the absorbance at 570 nm was measured with a microplate reader to calculate the cell activity.

[0082] Figure 7 The results of cytotoxicity tests for different treatment groups show that the cell survival rate of the NS+L group was as high as 90.95±4.28%, indicating that only the 808nm near-infrared laser had no obvious cytotoxic effect on 4T1 cells; while the GAExo-Lipo, IR780 Exo-Lipo+L, IG Exo-Lipo and IG Exo-Lipo+L groups showed obvious cytotoxicity, indicating that the therapeutic preparation has a certain anti-tumor effect. Among them, the IG Exo-Lipo+L group showed the strongest cytotoxic effect (33.92±2.37%) compared with the IG Exo-Lipo group (48.41±4.33%) and other groups.

[0083] 4. Cellular uptake experiment:

[0084] 4T1 cells were seeded in culture flasks and divided into the following groups: NS group, free IR780 1h, 2h and 4h groups, and IG Exo-Lipo 1h, 2h and 4h groups. Free IR780 and IG Exo-Lipo with the same IR780 concentration were added and incubated for 2h. Subsequently, cells in each group were collected and detected by flow cytometry.

[0085] Figure 8 The results of the cell uptake test of free IR780 and IG Exo-Lipo are shown in the figure. The percentage of cells containing IR780 in the free IR780 group was 15.27±1.23% at 1h, 19.89±1.27% at 2h, and 21.79±0.92% at 4h; the percentage of cells containing IR780 in the IG Exo-Lipo group was 53.05±5.91% at 1h, 71.76±2.08% at 2h, and 85.54±1.89% at 4h. The percentage of cells containing IR780 in the IGExo-Lipo group was significantly higher than that in the free IR 780 group at all three time points. The experimental results show that the engineered exosome-camouflaged biodegradable nanoplatform enhances the active targeting function and the uptake of drugs by tumor cells, which is beneficial to increase the accumulation of drugs in tumor cells.

[0086] 5. Annexin V-PI double staining was used to detect cell apoptosis induced by IG Exo-Lipo, and finally flow cytometry analysis was performed. The following treatment groups were set up in the experiment: NS, NS+L, Blank Exo-Lipo, GA Exo-Lipo, IR780 Exo-Lipo, IR780 Exo-Lipo+L, IG Exo-Lipo and IG Exo-Lipo+L. Among them, the NS group used normal saline, the NS+L group used normal saline plus light treatment, the Blank Exo-Lipo group used Blank Exo-Lipo prepared in Comparative Example 3, the GAExo-Lipo group used GA Exo-Lipo prepared in Comparative Example 2, the IR780 Exo-Lipo group used IR780Exo-Lipo prepared in Comparative Example 1, the IR780 Exo-Lipo+L group used IR780 Exo-Lipo prepared in Comparative Example 1 plus light treatment, the IGExo-Lipo group used IG Exo-Lipo prepared in Example 1, and the IG Exo-Lipo+L group used IGExo-Lipo prepared in Example 1 plus light treatment.

[0087] 4T1 cells were inoculated into 6-well plates and incubated for 24 hours. Then, different treatment group agents (1 mL each) were added for treatment and incubated with cells for 6 hours respectively. The culture medium in each well was replaced with fresh culture medium, and NS+L, IR780 Exo-Lipo+L, and IG Exo-Lipo+L groups were irradiated with 808nm NIR light for 5 minutes each, and then incubated for 24 hours. Finally, cells were collected and tested by flow cytometry, and the ratio of early and late apoptotic cells was counted as the total apoptosis ratio.

[0088] Fig. 9 The results of the apoptotic cell percentage test after treatment with different treatment groups are shown in the figure. The apoptotic cell percentage of the IG Exo-Lipo+L group (23.52±1.21%) is much higher than that of the IG Exo-Lipo group (18.1±0.19%) and other groups. The experimental results show that the IG Exo-Lipo+L group has a strong apoptosis-inducing effect on triple-negative breast cancer cells.

[0089] 6. The production of intracellular ROS was detected by ROS assay kit. The fluorescent probe DCFH-DA can be stimulated by intracellular ROS to produce fluorescent DCF. 4T1 cells were seeded in a 12-well plate, and 1 mL of each treatment group (NS, NS+L, Blank Exo-Lipo, GA Exo-Lipo, IR780 Exo-Lipo, IR780 Exo-Lipo+L, IG Exo-Lipo, and IG Exo-Lipo+L) was added to each well and illuminated with an 808 nm near-infrared laser (1 W / cm 2 ) irradiated NS+L, IR780 Exo-Lipo+L and IG Exo-Lipo+L groups for 5 minutes each. Afterwards, the cells were collected and incubated with 10 μM / L fluorescent probe DCFH-DA for 20 minutes. Subsequently, the cells were washed three times with serum-free medium to remove DCFH-DA that did not enter the cells. Finally, the production of intracellular ROS was further detected by flow cytometry.

[0090] Fig.10 The test results of ROS content of IG Exo-Lipo in vitro show that ROS production of 4T1 cells increased significantly under NIR laser, and IG Exo-Lipo showed higher efficiency of ROS production under NIR irradiation compared with IR780 Exo-Lipo and other groups. The photostability of IG Exo-Lipo and the enhancing effect of GA on ROS production, where more GA was associated with more ROS production, may have contributed to this result.

[0091] 7. The expression of HSP90 in 4T1 cells was detected by western blotting. 4T1 cells were inoculated into 6-well plates for 24 hours, and then 1 mL of each treatment group (NS, GA and IG Exo-Lipo) was added to each well and incubated for 24 hours. Subsequently, RIPA lysate and PAGE protein loading buffer were added to prepare protein samples. The concentration of each protein sample was accurately determined by the BCA protein assay kit (Biyuntian) to ensure the standardization of subsequent experiments. The proteins were then electrophoresed with a 10% SDS-PAGE gel and transferred to a nitrocellulose filter (NC membrane). The NC membrane was then fixed in 5% skim milk for 2 hours, washed three times with TBST, incubated with the primary antibody for HSP90, diluted, incubated overnight, and then incubated with the secondary antibody. The in vitro expression level of HSP90 was detected by the enhanced chemiluminescence detection system.

[0092] Fig.11 The figure is a Western-Blot diagram of IG Exo-Lipo regulating HSP90 expression. As shown in the figure, compared with the GA group, the gray value of the IG Exo-Lipo group was significantly reduced, indicating that IG Exo-Lipo further reduced the expression of HSP90 and enhanced the anti-tumor effect of low-temperature PTT. The reason may be that Exo-Lipo increased the accumulation of GA in 4T1 cells, thereby further exerting the effect of GA on downregulating HSP90.

[0093] 8. The photothermal effect of near-infrared laser irradiation on the tumor site of mice was recorded by using a thermal imager.

[0094] Female Balb / C mice (purchased from Jiangsu Huachuang Huayao Technology Co., Ltd.) were raised to establish a subcutaneous tumor model. When the tumor grew to a certain volume, the therapeutic experiment was carried out by tail vein injection of each treatment group agent (NS, NS+L, Blank Exo-Lipo, GAExo-Lipo, IR780 Exo-Lipo, IR780 Exo-Lipo+L, IG Exo-Lipo and IG Exo-Lipo+L). The injection dose of each group was 100 μL, among which the NS and NS+L groups were injected with normal saline, and the Blank Exo-Lipo group was injected with empty exosomes-liposomes; the injection concentration of GA Exo-Lipo group was mPEG-TK-GA 2 mg / kg; the injection concentration of IR780 Exo-Lipo and IR780 Exo-Lipo+L groups was IR7807.5 mg / kg; the injection concentration of IG Exo-Lipo and IG Exo-Lipo+L groups was mPEG-TK-GA 2 mg / kg and IR7807.5 mg / kg. The treatment regimen was: administration was performed via tail vein injection every 3 days, body weight and tumor volume were recorded every 2 days during the treatment, and the survival of the mice was observed. After the treatment, the mice were killed, and tumors and major organs were collected for further analysis.

[0095] After 6 hours, when IG Exo-Lipo accumulated in the tumor site, the mice were anesthetized and the tumor sites of the mice in the irradiation group were irradiated with 808 nm NIR light for 5 min (1 w / cm 2 During the entire treatment period, the temperature of the tumor site was photographed and recorded by a thermal imager. Finally, the highest temperature of the tumor site of each group of mice was recorded and a temperature curve was drawn.

[0096] Fig.12 The maximum temperature test results of the tumor site of mice in each treatment group show that the temperature of the tumor site in the treatment group without NIR irradiation did not change significantly and remained at about 36.5°C. In contrast, the tumor temperature in the IR780 Exo-Lipo+L and IGExo-Lipo+L groups increased to about 46°C after NIR laser irradiation, which was higher than the NS+L group (~40°C) and other treatment groups without NIR irradiation. The maximum temperature of the IG Exo-Lipo+L group was lower than 50°C, which can achieve effective low-temperature PTT and avoid significant damage to adjacent normal tissues.

[0097] 9. The biodistribution of IR780 in mice was detected by in vivo optical imaging technology to evaluate the tumor targeting ability of IG Exo-Lipo.

[0098] Female Balb / C mice were selected to establish a subcutaneous tumor model. IR780 was used as a fluorescent probe in this experiment. The tumor-bearing mice were immediately divided into two groups and given the same concentration (IR780 concentration 7.5 mg / kg) of free IR780 and IG Exo-Lipo solution, respectively. In vivo near-infrared fluorescence imaging was performed 2h and 6h after injection. The fluorescence intensity at the tumor site was analyzed to test the distribution of the drug in vivo and to evaluate the tumor targeting of IG Exo-Lipo.

[0099] Fig.13 The fluorescence intensity test results of free IR780 and IG Exo-Lipo at the tumor site at 2h and 6h are shown in the figure. As shown in the figure, the IR780 Exo-Lipo group showed a much stronger fluorescence signal than free IR780 at the tumor site at 2h and 6h (**, P<0.01). In addition, the fluorescence signal of the IG Exo-Lipo group in normal organs was significantly weaker than that of the free IR780 group. These results indicate that IG Exo-Lipo has effective tumor targeting ability and has the potential to increase the effectiveness of tumor treatment while reducing damage to normal tissues.

[0100] 10. The in vivo antitumor activity of different treatment groups was evaluated in female mice with 4T1 tumors. 3 All mice were randomly divided into eight groups: (1) NS, (2) NS+L, (3) Blank Exo-Lipo, (4) GALipo, (5) IR780 Exo-Lipo, (6) IR780 Exo-Lipo+L, (7) IG Exo-Lipo, and (8) IG Exo-Lipo+L, and given corresponding drug treatments.

[0101] Six hours after administration, the tumors of mice in groups (2), (6), and (8) were irradiated with 808 nm NIR light for 5 minutes (1 w / cm 2 ) The temperature images of the tumor were recorded with a thermal imager. Treatment was performed every 3 days. Body weight and tumor volume were recorded every 2 days during the treatment, and the survival of the mice was observed. After the 21-day treatment course, the mice were killed and the tumors and major organs were collected for further analysis.

[0102] Fig.14 The volume and weight of the tumors in the mice in different treatment groups are shown in Table 1. Fig.14 A is the volume test result of mouse tumor. Fig.14B is the weight test result of mouse tumors. It can be seen that compared with the NS group, each other group showed a moderate tumor growth inhibition effect, among which the IG Exo-Lipo+L group showed a much better anti-tumor effect than any other treatment group. In addition, on day 22, the tumor volume of the IG Exo-Lipo+L group was significantly smaller than that of the IG Exo-Lipo group and other treatment groups. The results of tumor weight in each group showed that the IG Exo-Lipo+L group had the smallest tumor weight among all groups, further revealing the above conclusion. These results show that both PTT and chemotherapy have moderate anti-tumor effects, but the therapeutic benefit needs to be strengthened. In this study, the active tumor targeting conferred by genetic engineering Exo-Lipo modification allows the drug to accumulate more in tumor cells, and ROS generation and HSP90 downregulation increase the therapeutic efficiency of combined therapy. These features designed by us contribute to the IG Exo-Lipo+L group to show excellent tumor growth inhibition effects, and the combination of low-dose chemotherapy and low-temperature PTT can significantly inhibit the growth of triple-negative breast cancer.

[0103] Fig.15 The weight of tumor-bearing mice in each treatment group during the treatment period was not significantly different between the groups. The results of H&E staining of visceral tissues (heart, liver, spleen, lung, and kidney) in each treatment group are shown in Fig.16 As can be seen, there is no obvious substantial damage or abnormality. These results indicate that IG Exo-Lipo+L has no obvious systemic toxicity.

[0104] To verify the antitumor efficacy of the combined treatment, H&E staining of tumor tissue sections, immunochemical staining of Ki-67, TUNEL and HSP90 immunofluorescence were performed. Fig.17 The results of H&E, TUNEL, Ki-67, and heat shock protein 90 (HSP90) staining of tumor tissue sections in each treatment group. As shown in the figure, in the H&E staining of tumor tissue sections, the tumor tissue in the IG Exo-Lipo+L group showed more obvious nuclear condensation, fragmentation, and extensive necrosis than in other groups. Similar to the above study, TUNEL staining showed that the IG Exo-Lipo+L group had the most apoptosis-positive cells among all groups. Ki-67 immunohistochemical staining was used to detect the proliferation of tumor tissue. The Ki-67 positivity rate in the IG Exo-Lipo+L group was significantly lower than that in other groups. These results indicate that the IG Exo-Lipo+L group has stronger inhibition of tumor proliferation, induction of more tumor cell apoptosis, and better antitumor activity. In addition, the immunochemical staining results of HSP90 showed that the expression of HSP90 was significantly reduced in tumor tissues treated with the group containing GA. The above results indicate that the IG Exo-Lipo+L group improves the efficacy of low-temperature PTT by effectively downregulating HSP90 and exhibits the best antitumor activity among all groups.

[0105] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing liposomes loaded with IR780 and gambogic acid prodrug, characterized in that: The steps include: S1, dissolving mPEG-TK-NH2 in dimethylformamide, then adding gambogic acid, benzoyl peroxide and triethylamine to react with stirring, concentrating and recrystallizing the obtained reaction solution, filtering and collecting the product to obtain mPEG-TK-GA; S2, adding soybean phosphatidylcholine, cholesterol, IR780 and mPEG-TK-GA to an organic solvent, and after complete dissolution, removing the organic solvent from the mixed solution by rotary evaporation to obtain a lipid film, adding a phosphate buffer solution, heating and ultrasonic treatment to obtain a liposome crude solution, and obtaining a liposome solution after granulation; S3. Gene transfection of 4T1 cells to prepare SIRP-α genetically engineered exosomes, mixing SIRP-α genetically engineered exosomes with IGLipo, and then performing membrane fusion using the freeze-thaw method to obtain liposomes loaded with IR780 and gambogic acid prodrugs.

2. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug as claimed in claim 1, characterized in that: In step S1, the molecular weight of PEG in the mPEG-TK-NH2 is 2000.

3. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug as claimed in claim 1, characterized in that: In step S1, the molar ratio of mPEG-TK-NH2, gambogic acid, benzoyl peroxide and triethylamine is 1:1.1:1.3:5.

0.

4. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug as claimed in claim 1, characterized in that: In step S2, the mass ratio of IR780, MPEG-TK-GA, soybean phosphatidylcholine and cholesterol is 15:(1-5):150:

75.

5. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug as claimed in claim 1, characterized in that: In step S2, the whole particle is processed by an ultrasonic cell disruptor with an ultrasonic frequency of 100 KHz.

6. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug as claimed in claim 1, characterized in that: In step S3, the preparation method of the SIRP-α genetically engineered exosomes is as follows: 4T1 cells are incubated in a 6-well culture plate overnight, and then the lentivirus encapsulating the plasmid is added to the cells. After 24 hours, the cells are cultured with fresh DMEM medium to remove the lentivirus, and the 4T1 cells transfected with the SIRP-α gene are selected with streptomycin, and the culture supernatant is collected, and separated and extracted by ultracentrifugation to obtain SIRP-α genetically engineered exosomes.

7. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug as claimed in claim 1, characterized in that: In step S3, the mass ratio of the SIRP-α genetically engineered exosomes to the liposome solution is 1:

1.

8. The method for preparing liposomes loaded with IR780 and gambogic acid prodrug according to any one of claims 1 to 7, characterized in that: In step S3, the freeze-thaw method for membrane fusion is as follows: the mixture is frozen at -80°C for 15 minutes, then incubated at 37°C for 15 minutes, and the freeze-thaw cycle is repeated 3 times.

9. Liposome loaded with IR780 and gambogic acid prodrug prepared by the method according to any one of claims 1 to 8.

10. Use of liposomes loaded with IR780 and gambogic acid prodrug prepared by the method according to any one of claims 1 to 8 in the preparation of therapeutic drugs for breast cancer, melanoma, lung cancer, prostate cancer and liver cancer.

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