Liposome with radio frequency thermal sensitization effect as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of radio-frequency thermal ablation liposomes, and particularly discloses a liposome with a radio-frequency thermal sensitization effect and a preparation method and application thereof, and the liposome comprises a phospholipid bilayer formed by phospholipid and a surface functionally modified by a temperature-sensitive polymer; and the temperature-sensitive polymer is distearoyl phosphatidyl ethanolamine-poly (N-isopropylacrylamide). According to the lipidosome with the radio-frequency thermal sensitization effect and the preparation method and application thereof, the prepared lipidosome has the radio-frequency thermal sensitization characteristic, non-invasive radio-frequency thermal therapy can be achieved, synergistic anti-tumor application can be achieved after treatment substances are loaded in the lipidosome, and the lipidosome has great clinical transformation prospects.

Description

Technical Field

[0001] This invention belongs to the field of radiofrequency thermal ablation liposome technology, specifically relating to a liposome with radiofrequency thermal sensitization effect, its preparation method and application. Background Technology

[0002] Radiofrequency ablation (RFA) is a therapeutic technique that uses high-frequency current to generate heat and precisely destroy tumor tissue. It has been extensively used clinically, and is one of the most commonly used treatments for primary and metastatic liver cancer. With the continuous development of RFA, various types of radiofrequency thermosensitizers have been prepared and researched. Among these, the development and preparation of radiofrequency-responsive radiofrequency thermosensitizers is currently a research hotspot. These nanosensitizers utilize the thermal responsiveness of radiofrequency fields to heat tumor tissue treated with nanomaterials, thereby effectively killing tumor cells at low radiation doses. Currently, many inorganic nanomaterials have been found to have excellent radiofrequency thermosensitizing effects; however, these inorganic radiofrequency thermosensitizers lack excellent drug loading capacity and usually need to be constructed into relatively complex treatment systems with other therapeutic substances to achieve synergistic therapy. Furthermore, biosafety is also a significant issue restricting their further development.

[0003] Furthermore, local tumor recurrence is common when using radiofrequency ablation (RFA) to treat liver tumors larger than 3 cm. These recurrences may be caused by lesion areas at the lesion periphery that were not effectively killed. Based on this, some researchers have proposed that combining RFA with other treatment methods may improve therapeutic efficacy. Among numerous nanocarriers, liposomes have attracted widespread attention due to their excellent drug loading capacity. Duke University developed a thermosensitive liposome for doxorubicin (ThermoDox), which exhibits thermoresponsive drug release behavior. ThermoDox achieved encouraging results in Phase I and II clinical trials due to its ability to release drugs at specific points by heating the target site. However, the results of its Phase III clinical trial were unsatisfactory. Hijnen et al. analyzed the reasons for the failure of ThermoDox's clinical trials and pointed out that blood flow absorbs heat energy, thereby lowering the temperature of the tumor parenchyma, leading to a potential thermal deposition effect. This results in a lower temperature in the central region far from the RFA needle, insufficient to kill tumor cells, thus leading to the unsatisfactory clinical efficacy of ThermoDox. Therefore, developing a liposomal nanomedicine with radiofrequency thermosensitizing effect is of significant practical importance for enhancing tumor thermal ablation.

[0004] In summary, given the current challenges faced by radiofrequency ablation and thermosensitive liposomes in the field of radiofrequency ablation (RFA), it is urgent to address the need to develop a liposome with both radiofrequency thermal sensitization and thermosensitivity that can be used in RFA to enhance the RFA effect while simultaneously achieving RF-responsive drug release. Summary of the Invention

[0005] The purpose of this invention is to provide a liposome with radiofrequency thermosensitization effect, its preparation method and application. The prepared liposome has radiofrequency thermosensitization characteristics, which can realize non-invasive radiofrequency hyperthermia. After loading therapeutic substances into the liposome, it can achieve synergistic anti-tumor application, which has great clinical translation prospects.

[0006] To achieve the above objectives, the present invention provides a liposome with radio frequency thermal sensitization effect, comprising a phospholipid bilayer composed of phospholipids and a surface functionalized by a thermosensitive polymer; the liposome with radio frequency thermal sensitization effect exhibits radio frequency responsive drug release behavior and radio frequency thermal sensitization effect, and the radio frequency thermal effect is positively correlated with power.

[0007] Thermosensitive polymer is distearylphosphatidylethanolamine-poly(N-isopropylacrylamide) (DSPE- p NIPAM).

[0008] This invention also provides a method for preparing liposomes with radiofrequency thermosensitization effect, comprising the following steps: Step S1: Prepare initiator 1,2-distearate-sn-glycerol-3-phosphoethanolamine-2'-bromoisobutyrate (DSPE-Br). Step S2: Preparation of the thermosensitive polymer distearylphosphatidylethanolamine-poly(N-isopropylacrylamide) (DSPE- p NIPAM); Step S3: Prepare liposomes (TSLIP) with radio frequency thermal sensitization effect using thin film hydration method.

[0009] Preferably, step S1 specifically includes: Step S11: Add 5-10 mL of chloroform and 100-150 μL of triethylamine to 0.1-0.2 g of distearate phosphatidylethanolamine (DSPE) and stir until dissolved; Step S12: Place the mixture in an ice bath at 0-4℃ and add 30-40 μL of 2-bromoisobutyryl bromide at a rate of 2 μL / min. After all the 2-bromoisobutyryl bromide has been added, stir at 15-25℃ for 30 min, and then react in a constant temperature oil bath at 35-45℃ for 20-30 h to obtain the reaction product. Step S13: Remove the solvent from the reaction product by rotary evaporation at a pressure reduced to 10 mbar in a water bath at 33°C, and dissolve the reaction product in 45-55 mL of dichloromethane. Then wash the product successively with saturated sodium chloride solution, saturated sodium bicarbonate solution, 1% dilute hydrochloric acid, and deionized water. Step S14: After removing the solvent by rotary evaporation at a pressure of 10 mbar in a water bath at 31°C, the product is dried in a vacuum drying oven at 0.1 mbar and a temperature of 15-25°C for 24 hours to remove the residual solvent, thus obtaining 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-2'-bromoisobutyrate.

[0010] Preferably, step S2 specifically involves synthesizing the linear polymer DSPE-Br using DSPE-Br as an initiator via atom transfer radical polymerization (ATRP) of N-isopropylacrylamide. p NIPAM: Step S21: Add the mixed solvent of isopropanol and tetrahydrofuran to 2-3g of monomer N-isopropylacrylamide, stir at 15-25℃ until N-isopropylacrylamide is completely dissolved, freeze with liquid nitrogen and then remove oxygen by vacuum. Step S22: Add 35-50 mg of initiator 1,2-distearate-sn-glycerol-3-phosphoethanolamine-2'-bromoisobutyrate and 15-30 μL of ligand tris(2-dimethylaminoethyl). Repeat the liquid nitrogen freezing-vacuuming-water bath melting cycle twice. Then add 5-10 mg of catalyst cuprous chloride. Repeat the liquid nitrogen freezing-vacuuming-water bath melting cycle three more times. Finally, introduce argon gas. Step S23: After reacting in an oil bath at 25-35℃ for 30-40 hours, transfer the mixture to a 3500Da dialysis bag and dialyze it in deionized water for 3-4 days. Step S24: After freeze-drying for 3 days, the thermosensitive polymer distearate phosphatidylethanolamine-poly(N-isopropylacrylamide) is obtained.

[0011] Preferably, in step S21, the volume ratio of isopropanol to tetrahydrofuran in the mixed solvent is 1-3:1; In steps S21 and S22, the liquid nitrogen freezing time is 5 minutes and the vacuuming time is 5 minutes. In step S23, the water bath melting specifically refers to thawing in a water bath at a temperature of 15-25℃.

[0012] Preferably, step S3 specifically includes: Step S31: The temperature-sensitive polymer distearate phosphatidylethanolamine-poly(N-isopropylacrylamide) (DSPE-) is added to the product. p NIPAM), cholesterol, and phospholipids are mixed in a molar ratio of 1:1-10:1-15 and then dissolved in 5-15 mL of chloroform. The mixture is stirred at 15-25°C until all components are completely dissolved. Step S32: Remove the solvent chloroform by rotary evaporation at 10 mbar in a water bath at 33°C. Dry in a vacuum drying oven at 0.1 mbar and 15-25°C for 24 h to remove residual organic solvent. Then add 5-15 mL of deionized water and stir at 200 rpm for 30 min in an oil bath at 40-55°C. Then stir at 200 rpm for 30 min at 15-25°C. After ultrasonic treatment with a probe-type ultrasonic instrument, liposomes with radio frequency thermal sensitization effect (TSLIP) are obtained.

[0013] Preferably, in step S31, the phospholipid is one or more of (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), distearate phosphatidylcholine (DSPC), and dioleoyl phosphatidylethanolamine (DOPE).

[0014] Preferably, in step S32, the ultrasonic treatment power is 200-400W, the ultrasonic treatment frequency is 20-25kHz, and the ultrasonic treatment time is 1-5min.

[0015] The present invention also provides an application of liposomes with radiofrequency thermosensitization effect, which is used in non-invasive radiofrequency ablation antitumor therapy.

[0016] Preferably, the tumor is a subcutaneous liver cancer tumor in mice; the non-invasive radiofrequency ablation treatment time is 10 minutes and the power is 300W.

[0017] The present invention utilizes the above-mentioned liposome with radio frequency thermosensitizing effect, its preparation method, and its application, with the following beneficial effects: 1. This invention utilizes a thin-film hydration method with DSPE- p NIPAM replaces DSPE-PEG in liposomes, endowing liposomes with thermosensitivity, and successfully prepared liposomes TSLIP with radio frequency thermosensitization effect. 2. The TSLIP prepared by this invention has good radio frequency responsiveness and can generate excellent radio frequency thermal effect under non-contact radio frequency field irradiation. The radio frequency thermal effect is positively correlated with radio frequency power.

[0018] 3. The TSLIP prepared by this invention can achieve responsive drug release under a non-contact radio frequency field.

[0019] 4. This invention studied the thermal response and radiofrequency pulsed drug release behavior of the prepared TSLIP under RF field irradiation. In the H22 tumor-bearing mouse model, it was confirmed that TSLIP can effectively inhibit tumor growth through RFA under non-invasive RF irradiation. After loading anti-tumor drugs, it can inhibit tumor growth more effectively, which can realize the application of multimodal tumor treatment and thus has great clinical translation prospects.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This invention relates to a liposome with radiofrequency thermal sensitization effect, its preparation method, and its application. Example 1 shows DSPE-Br and DSPE- p NIPAM's proton NMR spectrum; Figure 2 This invention relates to a liposome with radio frequency thermal sensitization effect, its preparation method, and its application. Example 1 illustrates DSPE-. p NIPAM gel permeation chromatogram; Figure 3 This invention relates to a liposome with radio frequency thermal sensitization effect, its preparation method, and its application. Example 1 illustrates DSPE-. p Transmittance of NIPAM in different media as temperature changes; Figure 4 This invention relates to a liposome with radio frequency thermal sensitization effect, its preparation method, and application. Example 2 shows the hydrated particle size values ​​of TSLIP in different media over 15 days. Figure 5 The TEM image of TSLIP in Experiment Example 2 of the present invention is a liposome with radio frequency thermal sensitization effect, its preparation method and application. Figure 6 The temperature rise curves of TSLIP under different power RF field irradiation in Experiment Example 2 of the present invention are shown for a liposome with radio frequency thermal sensitization effect, its preparation method and application. Figure 7 The figures show the heating curves of TSLIP and gold nanoclusters of the same mass concentration under different power RF fields in Experiment 2 of the present invention, which describes a liposome with radio frequency thermal sensitization effect, its preparation method, and its application. Wherein, A is TSLIP and B is gold nanoclusters. Figure 8 This invention relates to a liposome with radiofrequency thermal sensitization effect, its preparation method, and application. In Experiment Example 3, the drug loading rate and encapsulation efficiency of TSLIP loaded with 3BP in the form of DOTAP-3BP (3BP-TSLIP) and directly loaded with 3BP (3BP@TSLIP) were measured. Figure 9 The images show the hemolysis rates of different concentrations of TSLIP and 3BP-TSLIP in Experiment 4 of the present invention, which describes a liposome with radiofrequency thermosensitization effect, its preparation method, and its application. The images also show the results of microscopic observation of erythrocytes after co-incubation with different substances. In the images, A represents the hemolysis rates of different concentrations of TSLIP and 3BP-TSLIP, and B represents the results of microscopic observation of erythrocytes after co-incubation with different substances. Figure 10 This invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 5, which describes the drug release behavior of 3BP-TSLIP in different pH environments and under RF irradiation; wherein, A represents the drug release behavior of 3BP-TSLIP in different pH environments, and B represents the drug release behavior under RF irradiation. Figure 11 The present invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application example 6, showing the flow cytometry plots and corresponding quantitative statistical results of H22 cells taking up free Cy5 and Cy5-TSLIP; wherein, A is the flow cytometry plot and B is the corresponding quantitative statistical result; Figure 12 The present invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application. In Experiment Example 6, H22 cells were treated with different methods for 24 hours to obtain cell viability results. Figure 13 This invention presents a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 6, showing the apoptosis flow cytometry of H22 cells after different treatments and the corresponding quantitative statistical results; where A is the apoptosis flow cytometry and B is the corresponding quantitative statistical result. Figure 14 This invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 7, where H22 cells were treated in different ways to obtain ICD effect indicators; where A is CRT, B is HMGB1, and C is ATP. Figure 15 The present invention relates to a liposome with radiofrequency thermal sensitization effect, its preparation method, and application example 7, showing the DC maturation flow cytometry plot and corresponding quantitative statistical results; wherein, A is the DC maturation flow cytometry plot and B is the corresponding quantitative statistical results; Figure 16 The above is an infrared thermographic image of the tumor in tumor-bearing mice treated with TSLIP and 3BP-TSLIP at different time points under RF field irradiation, which is an example of the preparation method and application of a liposome with radiofrequency thermosensitization effect of the present invention. Figure 17 This invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 8, showing tumor growth curves and tumor images of tumor-bearing mice in each group during treatment; where A is the growth curve, B is the tumor image, G1: saline group; G2: TSLIP group; G3: free 3BP group; G4: TSLIP+RF group; G5: 3BP-TSLIP group; G6: 3BP-TSLIP+RF group; Figure 18This invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and the tumor weight of each group of tumor-bearing mice after treatment in Experiment Example 8. Figure 19 The images show the H&E pathology and TUNEL immunohistochemical sections of tumors in each treatment group of tumor-bearing mice after treatment in Experiment Example 8 of the present invention, which describes a liposome with radiofrequency thermosensitization effect, its preparation method, and its application. Figure 20 This invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 8, showing the weight change curves of mice in each treatment group during the treatment process. Figure 21 The image shows the H&E pathological sections of major organs of mice in each treatment group after treatment in Experiment Example 8 of the present invention, which describes a liposome with radiofrequency thermosensitization effect, its preparation method, and its application. Figure 22 This invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 8. The results show the proportion of mature dendritic cells (DCs) and the expression rate of MHC-II in the adjacent lymph nodes of tumor-bearing mice after 14 days of treatment with different methods. Wherein, A represents the proportion of mature DCs in the lymph nodes, and B represents the expression rate of MHC-II. Figure 23 The present invention relates to a liposome with radiofrequency thermosensitization effect, its preparation method, and application experiment example 8. After tumor-bearing mice were treated in different ways for 14 days, the proportions of Treg and MDSC in the tumor and cytotoxic T cells in the spleen were observed. Among them, A represents Treg, B represents MDSC, and C represents the proportion of cytotoxic T cells in the spleen. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Example A method for preparing liposomes with radiofrequency thermosensitization effect includes the following steps: Step S1: Prepare the initiator DSPE-Br.

[0025] Step S11: Add 0.15g of DSPE to a round-bottom flask, evacuate the flask for 5 minutes using an oil pump, then introduce argon gas, add 10mL of chloroform and 144μL of triethylamine, and stir until completely dissolved.

[0026] Step S12: Place the container in an ice bath at 0-4℃ and slowly add 33 μL of 2-bromoisobutyryl bromide at a rate of 2 μL / min. After all the 2-bromoisobutyryl bromide has been added, stir at 20℃ for 30 min, then transfer the reaction apparatus to a constant temperature oil bath at 40℃ and react for 24 h to obtain the reaction product.

[0027] Step S13: The solvent in the reaction product is removed by rotary evaporation in a water bath at 33°C under reduced pressure to 10 mbar. The reaction product is then dissolved in 50 mL of dichloromethane and washed successively with saturated sodium chloride solution, saturated sodium bicarbonate solution, 1% dilute hydrochloric acid, and deionized water.

[0028] Step S14: After removing the solvent by rotary evaporation in a water bath at 31°C under reduced pressure to 10 mbar, the product is dried in a vacuum drying oven at 0.1 mbar and 20°C for 24 h to remove residual solvent, thus obtaining the pale yellow product DSPE-Br.

[0029] Step S2: Thermosensitive polymer DSPE- is prepared using atom transfer radical polymerization (ATRP). p NIPAM.

[0030] Step S21: Weigh 2.26g of monomer N-isopropylacrylamide and place it in a tetrafluoroethylene reaction tube. Add 3mL of isopropanol and 1.5mL of tetrahydrofuran as a mixed solvent. Stir at 20°C until the N-isopropylacrylamide is completely dissolved, then transfer to liquid nitrogen and freeze for 5min. Evacuate the tube for 5min, then place it in a water bath at 20°C until thawed and argon gas is introduced.

[0031] Step S22: Add 45 mg of initiator DSPE-Br and 23.5 μL of ligand tris(2-dimethylaminoethyl), repeat the liquid nitrogen freezing-vacuuming-water bath melting cycle 2 times, add 7.5 mg of catalyst cuprous chloride, repeat the liquid nitrogen freezing-vacuuming-water bath melting cycle 3 times, and then introduce argon gas.

[0032] The liquid nitrogen freezing time is 5 minutes, and the vacuuming time is 5 minutes.

[0033] Water bath thawing specifically refers to thawing in a water bath at a temperature of 20°C.

[0034] Step S23: After reacting in an oil bath at 30°C for 35 hours, transfer the mixture to a 3500 Da dialysis bag and dialyze it in deionized water for 4 days.

[0035] Step S24: After freeze-drying for 3 days, the temperature-sensitive polymer DSPE- is obtained. p NIPAM.

[0036] Step S3: Prepare liposomes (TSLIP) with radio frequency thermosensitization effect.

[0037] Step S31, DSPE- p NIPAM, cholesterol, dioleoylphosphatidylethanolamine (DOPE), and (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP) were dissolved in 10 mL of chloroform in a molar ratio of 1:7:4:6, and stirred at 20 °C until all components were completely dissolved.

[0038] Step S32: The solvent chloroform is removed by rotary evaporation at 10 mbar in a water bath at 33°C. The mixture is then dried in a vacuum drying oven at 0.1 mbar and 20°C for 24 hours to remove residual organic solvent. Subsequently, 10 mL of deionized water is added, and the mixture is stirred at 200 rpm for 30 minutes in an oil bath at 50°C, followed by stirring at 200 rpm for 30 minutes at 20°C. The mixture is then sonicated using a probe-type ultrasonic instrument at a power of 400 W, a frequency of 20-25 kHz, and a duration of 3 minutes. This yields liposomes (TSLIP) with radiofrequency thermosensitization effect.

[0039] Experimental Example 1 The initiator DSPE-Br and the thermosensitive polymer DSPE-Br prepared in the examples p NIPAM was structurally characterized by 1H-NMR and gel permeation chromatography (GPC).

[0040] like Figures 1-2 As shown, the temperature-sensitive polymer DSPE- p NIPAM has lower critical solubility temperatures of 35°C in deionized water and 32°C in PBS, both below human body temperature. Therefore, if... Figure 3 As shown, it can change from a hydrophilic state to a hydrophobic state in vivo.

[0041] Experimental Example 2 The liposomes (TSLIP) with radiofrequency thermal sensitization effect prepared in the examples were characterized.

[0042] 1. Perform structural characterization on TSLIP.

[0043] TSLIP was characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. 5 mg / mL TSLIP was dispersed in different media, and its hydrated particle size at room temperature in different media was measured using DLS over 15 days. Figure 4 As shown, DLS measurements of the hydrated particle size revealed that TSLIP maintained good stability in different media over 15 days. Subsequently... Figure 5 As shown, TEM revealed that TSLIP particles have a relatively uniform size distribution, with a particle size of approximately 131 nm, exhibiting a typical phospholipid bilayer structure.

[0044] 2. Characterize the radio frequency thermal effects of TSLIP.

[0045] 1 mL of 5 mg / mL TSLIP was placed in a radio frequency field and irradiated for 5 min. The radio frequency irradiation distance was set to 1.5 cm. The real-time temperature of the sample was recorded every 30 s using an infrared thermal imager to characterize the radio frequency thermal effect of the sample.

[0046] The irradiation powers of the radio frequency fields were 75W, 100W, and 125W, respectively. For example... Figure 6 As shown, the RF thermal effect of TSLIP gradually increases with increasing RF power, indicating that the RF thermal effect of TSLIP is power-dependent. Furthermore, as... Figure 7 As shown, when TSLIP and gold nanoclusters of the same mass concentration were irradiated with radio frequency fields of different powers, TSLIP showed a more significant radio frequency thermal effect than gold nanoclusters, indicating that TSLIP is an excellent radio frequency thermal sensitizer.

[0047] Experimental Example 3 The therapeutic substance 3-bromopyruvic acid was loaded onto a liposome (TSLIP) with a radiofrequency thermosensitizing effect prepared in the example.

[0048] The preparation of liposomes (3BP-TSLIP) loaded with the therapeutic substance 3-bromopyruvic acid and exhibiting radiofrequency thermosensitization effect, with steps S1-S2 being the same as in the previous example, and step S3 specifically being: The total mass of 50 mg of 3-bromopyruvic acid (3BP)-DOTAP ion complex, DOTAP, cholesterol, DOPE, and DSPE- was added. p NIPAM was dissolved in 10 mL of chloroform at a molar ratio of 6:4:7:1:1, and stirred at 20 °C until all components were completely dissolved. The solvent chloroform was removed by rotary evaporation at 10 mbar in a water bath at 33 °C. The solution was then dried in a vacuum drying oven at 0.1 mbar and 20 °C for 24 h to remove residual organic solvent. Subsequently, 10 mL of deionized water was added, and hydration was performed in a 50 °C water bath and at 20 °C. The solution was then sonicated using a probe-type sonicator at a power of 400 W, a frequency of 20-25 kHz, and a duration of 3 min, yielding 3BP-loaded thermosensitive liposomes (3BP-TSLIP).

[0049] The obtained 3BP-TSLIP was subjected to ultrafiltration to remove unloaded drug. The ultrafiltration-treated 3BP-TSLIP was demulsified with 0.5% Triton X-100, and the drug loading and encapsulation efficiency were calculated using a UV-Vis spectrophotometer. Figure 8 As shown, loading 3BP in the form of 3BP-TSLIP can significantly improve the drug encapsulation efficiency of TSLIP. Compared with directly loading free 3BP, the drug encapsulation efficiency is increased by 5.79 times, demonstrating excellent drug loading capacity.

[0050] Experiment Example 4 Hemolysis was evaluated for the TSLIP prepared in the examples and the 3BP-TSLIP prepared in Experimental Example 3.

[0051] Whole blood containing the anticoagulant sodium heparin was centrifuged (2000 rpm, 10 min). 200 μL of the whole blood precipitate was dissolved in 10 mL of physiological saline. 0.5 mL of this solution was added to the same volume of the sample to be tested, mixed well, and incubated at 37°C for 1 h. Subsequently, the sample was centrifuged (2000 rpm, 10 min), and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance was measured using a microplate reader. Figure 9 As shown, the hemolysis rate of each group of samples was calculated. The hemolysis results showed that both TSLIP and 3BP-TSLIP had good biocompatibility.

[0052] Experimental Example 5 The controlled drug release behavior of 3BP-TSLIP prepared in Experimental Example 3 in a non-invasive RF field was studied.

[0053] The 3BP-TSLIP was first placed in a 37°C water bath, and then irradiated for 1 minute every 10 minutes under a non-contact RF field. The supernatant was collected at each time point, and the concentration of 3BP in the supernatant was determined using a UV-Vis spectrophotometer. Figure 10 As shown, the results indicate that 3BP-TSLIP exhibits non-invasive RF field-triggered pulsed drug release behavior.

[0054] Experimental Example 6 The effects of 3BP-TSLIP+RF on tumor cell apoptosis were investigated using cell uptake assays, CCK-8 cell viability assays, and apoptosis assays.

[0055] 1. Cell uptake experiment.

[0056] H22 cells in logarithmic growth phase were seeded into 6-well plates, 300,000 cells per well, and cultured overnight at 37°C. Then, equal concentrations (0.00004 mg / mL) of Cy5 and Cy5-loaded TSLIP (Cy5-TSLIP) were added to each group of cells, and the cells were co-incubated for 2 h, 4 h, and 6 h, respectively. Cells were then collected, washed with PBS, and the mean fluorescence intensity of Cy5 in each treatment group was detected by flow cytometry. Figure 11 As shown, with the increase of co-incubation time, the uptake of Cy5 and Cy5-TSLIP by tumor cells gradually increased. However, at different incubation times, tumor cells showed a better uptake capacity for TSLIP than for free Cy5.

[0057] 2. CCK-8 cell viability assay.

[0058] H22 cells in the growth phase were seeded into 96-well plates at a rate of 1 × 10⁶ cells per well. 4 Cells were cultured overnight at 37°C. Then, free 3BP and 3BP-TSLIP at the same concentration were added to 96-well plates. After incubation for 6 hours, some of the 3BP-TSLIP-treated cells were irradiated with a non-invasive RF field for 10 minutes. After further incubation for 18 hours, the absorbance of each well was measured at 450 nm using a microplate reader. Figure 12 As shown, after RF field irradiation, 3BP-TSLIP can induce more tumor cell death.

[0059] 3. Apoptosis experiment.

[0060] H22 cells in logarithmic growth phase will be treated with 3 × 10⁻⁶ cells. 5 Tumor cells were seeded at a density of 100 cells / well in 6-well plates and cultured overnight in a cell culture incubator. Free 3BP, TSLIP, 3BP-TSLIP, TSLIP+RF, and 3BP-TSLIP+RF were prepared at a concentration of 80 µM, with a culture medium group serving as a control. The culture medium containing these materials was added to the wells, and the plates were incubated for 6 hours. The RF group was then treated with non-invasive RF field irradiation and cultured for another 6 hours. Tumor cells were collected from the wells and stained according to an apoptosis detection kit. Flow cytometry was used to analyze the apoptosis rate of tumor cells in each treatment group. Figure 13 As shown, tumor cell apoptosis was significantly increased under the combined effects of 3BP-TSLIP and RF field irradiation.

[0061] Experimental Example 7 Detecting immunogenic cell death (ICD) markers and dendritic cell (DC) maturation status to evaluate the synergistic activation of antitumor immune responses in vivo by TSLIP-loaded drugs in conjunction with RF (3BP-TSLIP+RF).

[0062] 1. ICD marker detection experiment.

[0063] H22 cells in logarithmic growth phase were seeded into 6-well plates, 300,000 cells per well, and cultured overnight in a cell culture incubator. Free 3BP, TSLIP, 3BP-TSLIP, TSLIP+RF, and 3BP-TSLIP+RF were prepared at a concentration of 80 µM, with a medium group serving as a control. The medium containing these materials was added to the wells, and the plates were incubated for 6 hours. The RF group was then treated with a non-invasive RF field for 10 minutes, followed by another 6 hours of incubation. Cells and supernatant were collected, and the expression of calreticulin (CRT) in tumor cells was detected using flow cytometry. The levels of ATP and HMGB1 released by tumor cells were measured using an ATP and HMGB1 assay kit. Figure 14 As shown, after treatment with 3BP-TSLIP+RF, the expression level of CRT in tumor cells increased significantly, and the release of HMGB1 was also significantly higher than that in the other groups. Due to the inhibition of tumor cell glycolysis by 3BP, the ATP release in the 3BP-TSLIP+RF treatment group was lower than that in the TSLIP+RF group, but significantly higher than that in the 3BP-TSLIP group. This indicates that under the synergistic effect of 3BP-TSLIP and RF irradiation, damage-associated molecular patterns (DAMPs) increased significantly, and the ICD effect was effectively activated.

[0064] 2. DCs maturity status detection.

[0065] H22 cells in logarithmic growth phase were seeded into 6-well plates, 300,000 cells per well, and cultured overnight. Free 3BP, TSLIP, 3BP-TSLIP, TSLIP+RF, and 3BP-TSLIP+RF were prepared at 80 µM concentrations, along with a control culture medium. The culture medium containing these materials was added to the wells, and the plates were cultured for 6 hours. The RF group was then treated with a non-invasive RF field for 10 minutes, followed by another 6 hours of culture. The cell supernatant was collected and added to 6-well plates containing DC2.4 cells. After 12 hours of culture, DC2.4 cells were collected, and flow cytometry was used to analyze DC maturation. Figure 15 As shown, the DC maturation results indicate that the DC maturation rate was significantly higher in the 3BP-TSLIP+RF treatment group than in the other groups, which facilitates the activation of the anti-tumor immune response.

[0066] Experimental Example 8 Evaluation of the antitumor and immunomodulatory effects of 3BP-TSLIP+RF.

[0067] Balb / c mice were inoculated with H22 cells to establish an H22 subcutaneous tumor hepatocellular carcinoma model. The tumors were allowed to grow to 190 cm in size. 3 Around day 0, tumor-bearing mice were randomly divided into 6 groups. On day 0, mice in each group received intratumoral injections of different therapeutic substances: physiological saline, TSLIP, and free 3BP at a concentration of 1.75 mg / mL, and 3BP-TSLIP containing an equal concentration of 3BP, with an injection volume of 50 μL. Twelve hours after administration, the tumors were exposed to a 300 W non-invasive radiofrequency field for 10 minutes. Figure 16 As shown, tumor-bearing mice treated with TSLIP and 3BP-TSLIP experienced a rapid increase in tumor temperature under RF irradiation. Tumor volume and body weight were measured every two days after treatment. Mice were sacrificed on day 14 post-treatment, tumors were dissected, weighed, and photographed. H&E and TUNEL staining was performed on the tumor tissue to investigate tumor necrosis and apoptosis in each treatment group. Figures 17-18 As shown, compared with other groups, 3BP-TSLIP and RF synergistically and effectively inhibited tumor growth, significantly reducing tumor volume and weight, with a tumor inhibition rate of 66%.

[0068] In addition, such as Figure 19 As shown, pathological and immunohistochemical section analysis of tumor tissue also indicates that 3BP-TSLIP+RF can significantly kill tumor cells, promote tumor cell apoptosis, and inhibit tumor cell proliferation. Furthermore, as... Figures 20-21 As shown, the weight of mice in each group remained stable during the treatment period, and no major organs were damaged, indicating that the synergistic treatment of TSLIP-based radiofrequency ablation and the therapeutic substance 3BP has good biocompatibility.

[0069] The above experimental results show that TSLIP has good biocompatibility and exhibits significant RFA effect under RF irradiation. Furthermore, after loading therapeutic substances, TSLIP not only has therapeutic efficacy but also achieves further enhancement of RFA therapeutic effect under RF irradiation. This indicates that TSLIP has significant RFA and synergistic therapeutic effects with other treatment methods.

[0070] After treatment, flow cytometry was used to analyze the antitumor immune response in the tumor and major immune organs. For example... Figure 22 As shown, analysis of DC maturation in peritumoral lymph nodes revealed that the DC maturation rate and MHC-II expression rate on the DC surface were higher in the 3BP-TSLIP+RF treatment group than in the other groups, indicating that TSLIP-mediated thermotherapy and glycolysis inhibition have a good ability to activate antitumor immune responses. Furthermore, as... Figure 23 As shown, the proportions of bone marrow-derived immunosuppressive cells (MDSCs) and regulatory T cells (Tregs) in the tumor microenvironment were both downregulated after treatment with 3BP-TSLIP+RF, and the proportion of cytotoxic T lymphocytes in the spleen was also the highest among all treatment groups. Therefore, these results indicate that 3BP-TSLIP+RF-mediated thermotherapy-glycolysis inhibition can induce a significant antitumor immune response.

[0071] Therefore, the present invention employs the above-mentioned liposome with radiofrequency thermosensitization effect, its preparation method and application. The prepared liposome has radiofrequency thermosensitization characteristics, which can realize non-invasive radiofrequency thermotherapy. After loading therapeutic substances into the liposome, it can achieve synergistic anti-tumor application, which has great clinical translation prospects.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A liposome with radio frequency thermosensitization effect, characterized in that: It includes a phospholipid bilayer composed of phospholipids and a surface functionalized by a thermosensitive polymer; the thermosensitive polymer is distearylphosphatidylethanolamine-poly(N-isopropylacrylamide).

2. A method for preparing liposomes with radio frequency thermal sensitization effect as described in claim 1, characterized in that, Includes the following steps: Step S1: Prepare initiator 1,2-distearate-sn-glycerol-3-phosphoethanolamine-2'-bromoisobutyrate; Step S2: Prepare the thermosensitive polymer distearylphosphatidylethanolamine-poly(N-isopropylacrylamide); Step S3: Prepare liposomes with radio frequency thermosensitization effect.

3. The method for preparing liposomes with radio frequency thermal sensitization effect according to claim 2, characterized in that, Step S1 is as follows: Step S11: Add 5-10 mL of chloroform and 100-150 μL of triethylamine to 0.1-0.2 g of distearate phosphatidylethanolamine and stir until dissolved; Step S12: Place the mixture in an ice bath at 0-4℃ and add 30-40 μL of 2-bromoisobutyryl bromide at a rate of 2 μL / min. After all the 2-bromoisobutyryl bromide has been added, stir at 15-25℃ for 30 min, and then react in a constant temperature oil bath at 35-45℃ for 20-30 h to obtain the reaction product. Step S13: Remove the solvent from the reaction product by rotary evaporation at a pressure reduced to 10 mbar in a water bath at 33°C, and dissolve the reaction product in 45-55 mL of dichloromethane. Then wash the product successively with saturated sodium chloride solution, saturated sodium bicarbonate solution, 1% dilute hydrochloric acid, and deionized water. Step S14: After removing the solvent by rotary evaporation at a pressure of 10 mbar in a water bath at 31°C, the product is dried in a vacuum drying oven at 0.1 mbar and a temperature of 15-25°C for 24 hours to remove the residual solvent, thus obtaining 1,2-distearate-sn-glycerol-3-phosphorylethanolamine-2'-bromoisobutyrate.

4. The method for preparing liposomes with radio frequency thermal sensitization effect according to claim 2, characterized in that, Step S2 is as follows: Step S21: Add the mixed solvent of isopropanol and tetrahydrofuran to 2-3g of monomer N-isopropylacrylamide, stir at 15-25℃ until N-isopropylacrylamide is completely dissolved, freeze with liquid nitrogen and then remove oxygen by vacuum. Step S22: Add 35-50 mg of initiator 1,2-distearate-sn-glycerol-3-phosphoethanolamine-2'-bromoisobutyrate and 15-30 μL of ligand tris(2-dimethylaminoethyl). Repeat the liquid nitrogen freezing-vacuuming-water bath melting cycle twice. Then add 5-10 mg of catalyst cuprous chloride. Repeat the liquid nitrogen freezing-vacuuming-water bath melting cycle three more times. Finally, introduce argon gas. Step S23: After reacting in an oil bath at 25-35℃ for 30-40 hours, transfer the mixture to a 3500Da dialysis bag and dialyze it in deionized water for 3-4 days. Step S24: After freeze-drying for 3 days, the thermosensitive polymer distearate phosphatidylethanolamine-poly(N-isopropylacrylamide) is obtained.

5. The method for preparing liposomes with radio frequency thermal sensitization effect according to claim 4, characterized in that: In step S21, the volume ratio of isopropanol to tetrahydrofuran in the mixed solvent is 1-3:1; In steps S21 and S22, the liquid nitrogen freezing time is 5 minutes and the vacuuming time is 5 minutes. In step S23, the water bath melting specifically refers to thawing in a water bath at a temperature of 15-25℃.

6. The method for preparing liposomes with radio frequency thermal sensitization effect according to claim 2, characterized in that, Step S3 is as follows: Step S31: Mix the thermosensitive polymer distearate phosphatidylethanolamine-poly(N-isopropylacrylamide), cholesterol, and phospholipids in a molar ratio of 1:1-10:1-15 and dissolve them in 5-15 mL of chloroform. Stir at 15-25°C until all components are completely dissolved. Step S32: Remove the solvent chloroform by rotary evaporation at 33℃ and reduced pressure to 10mbar in a water bath. Dry in a vacuum drying oven at 0.1mbar and 15-25℃ for 24h to remove residual organic solvent. Then add 5-15mL of deionized water and stir at 200rpm for 30min in an oil bath at 40-55℃. Stir at 200rpm for 30min at 15-25℃. After ultrasonic treatment with a probe-type ultrasonic instrument, liposomes with radiofrequency thermal sensitization effect are obtained.

7. The method for preparing liposomes with radio frequency thermal sensitization effect according to claim 6, characterized in that: In step S31, the phospholipid is one or more of (2,3-dioleoyl-propyl)-trimethylammonium chloride, distearate phosphatidylcholine, and dioleoyl phosphatidylethanolamine.

8. The method for preparing liposomes with radio frequency thermal sensitization effect according to claim 6, characterized in that: In step S32, the ultrasonic treatment power is 200-400W, the ultrasonic treatment frequency is 20-25kHz, and the ultrasonic treatment time is 1-5min.

9. An application of a liposome with radiofrequency thermal sensitization effect as described in claim 1, characterized in that: Liposomes with radiofrequency thermosensitizing effect are applied to non-invasive radiofrequency ablation antitumor therapy.

10. The application according to claim 9, characterized in that: The tumor was a subcutaneous liver cancer tumor in mice; the non-invasive radiofrequency ablation treatment lasted 10 minutes and the power was 300W.

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