Naphthalocyanine copper liposome and construction method and application thereof

Abstract

The application discloses a naphthalene phthalocyanine copper liposome and a construction method and application thereof, and belongs to the technical field of liposome preparations. The naphthalene phthalocyanine copper liposome can improve the problems of poor water solubility and insufficient stability of existing sonosensitizers by wrapping naphthalene phthalocyanine copper and catalase in the liposome. Meanwhile, the naphthalene phthalocyanine copper liposome has the function of catalyzing the production of O2 by excessive H2O2 in the microenvironment of brain glioma to supply the continuous production of ROS in the sonodynamic therapy (SDT), enhances the sonodynamic performance, improves the treatment effect of brain glioma, has the advantages of good biological safety, low immunogenicity and long circulation in the body, can effectively penetrate the blood-brain barrier and be delivered to the tumor site, solves the problems that drugs are difficult to penetrate the blood-brain barrier and cannot reach an effective concentration in the traditional treatment method of brain glioma, and provides a new strategy for the treatment of brain glioma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of liposome preparation, in particular to a naphthalocyanine copper liposome and a construction method and application thereof. BACKGROUND

[0002] Glioblastoma (GBM) is one of the most common primary tumors in the central nervous system (CNS). It is derived from the malignant proliferation of neural glial cells such as astrocytes and oligodendrocytes, has the characteristics of rapid proliferation and infiltration of normal brain tissue, and has a blurred boundary with normal tissue. More troublesome is that the special immune environment of the central nervous system endows glioblastoma cells with immune escape ability, making them difficult to be recognized and cleared by the body's immune system. In addition, the existence of the blood brain barrier (BBB) makes it difficult for most drug molecules and chemicals to penetrate this natural barrier to reach the lesion, which is a great obstacle to the treatment of glioblastoma.

[0003] Currently, surgical resection, radiotherapy and chemotherapy are still the main clinical treatment methods for glioblastoma. However, for malignant glioblastoma, it is extremely difficult to achieve complete resection due to the unclear boundary between the lesion and normal brain tissue, and the surgical risk is high and the postoperative recurrence rate is high. Drug resistance problems occur frequently during radiotherapy and chemotherapy; chemotherapy drugs such as temozolomide and bevacizumab are limited by the blood brain barrier and are difficult to reach effective therapeutic concentrations in the brain; radiotherapy can also cause side effects such as cognitive dysfunction. These factors lead to poor prognosis and short survival time for patients with malignant glioblastoma, seriously threatening the quality of life of patients.

[0004] In recent years, new treatment strategies have emerged, among which sonodynamic therapy (SDT) has brought new hope for breaking through the blood brain barrier and precisely treating glioblastoma due to its non-invasive and high tissue penetration. SDT induces tumor cell apoptosis by generating reactive oxygen species (ROS) under the action of ultrasound through a sound sensitizer. SDT has the advantages of deep tissue penetration, precise focusing, safety and non-invasiveness, and repeatability, and its application in the treatment of glioblastoma can improve the shortcomings of current traditional therapies. However, due to the existence of the blood brain barrier, the sound sensitizer required by SDT is hindered from entering the brain tumor tissue, and the existing sound sensitizer delivery system is inefficient, which greatly affects the treatment effect. SUMMARY

[0005] The present application aims to provide a naphthalocyanine copper liposome, a construction method and application thereof, so as to solve the problems of the prior art. The present application develops a liposome drug delivery system, which can improve the poor water solubility and insufficient stability of the existing photosensitizer by encapsulating naphthalocyanine copper and catalase in the liposome; at the same time, it can catalyze the excess H2O2 in the brain glioma microenvironment to produce O2 to supply SDT to continuously generate ROS, enhance the sonodynamic performance, improve the treatment effect of brain glioma, and has the advantages of good biological safety, low immunogenicity, long circulation in vivo, can effectively penetrate the blood-brain barrier and deliver to the tumor site, solve the problems that the drug is difficult to penetrate the blood-brain barrier and cannot reach the effective concentration in the traditional treatment of brain glioma, and provide a new strategy for the treatment of brain glioma.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0007] The present application provides a naphthalocyanine copper liposome having a therapeutic effect on brain glioma, which is obtained by encapsulating naphthalocyanine copper and catalase in the liposome by using a film dispersion method.

[0008] Further, the mass ratio of the naphthalocyanine copper, the catalase and the liposome is 2-2.5:1.5-2:30-40.

[0009] Further, the components of the liposome include distearoyl lecithin, polyethylene glycol monomethyl ether-2000-dioctadecyl phosphatidyl ethanolamine and cholesterol.

[0010] Further, the mass ratio of the distearoyl lecithin, the polyethylene glycol monomethyl ether-2000-dioctadecyl phosphatidyl ethanolamine and the cholesterol is 25-30:10-15:4-5.

[0011] The present application also provides a construction method of the above-mentioned naphthalocyanine copper liposome, which comprises the following steps:

[0012] The naphthalocyanine copper solution and the liposome solution are mixed, a film is obtained by rotary evaporation, the catalase solution is added, and the water bath ultrasonic is performed, and then the filtration is performed to obtain the naphthalocyanine copper liposome.

[0013] Further, the temperature of the water bath ultrasonic is 42-46℃; the power of the water bath ultrasonic is 300W / hour, the frequency is 40KHz, and the time is 5min.

[0014] Further, the filtration is sequentially performed through 0.45 μm filter membrane and 0.22 μm filter membrane.

[0015] Optionally, the solvent of the naphthalocyanine copper solution and the liposome solution is chloroform; and the solvent of the catalase solution is phosphate buffer.

[0016] The application also provides application of the above naphthalocyanine copper liposome or the naphthalocyanine copper liposome constructed by the above construction method in preparation of a drug for treating brain glioma.

[0017] The application also provides a drug for treating brain glioma, wherein an effective component of the drug is the above naphthalocyanine copper liposome or the naphthalocyanine copper liposome constructed by the above construction method.

[0018] The application discloses the following technical effects:

[0019] The application develops a liposome drug delivery system, wherein naphthalocyanine copper and catalase are wrapped in liposomes, so as to improve the problems of poor water solubility and insufficient stability of existing sonosensitizers; meanwhile, the liposome drug delivery system has the function of catalyzing H2O2 in the microenvironment of brain glioma to generate O2, supplying SDT to continuously generate ROS, enhancing the sonodynamic performance, and improving the treatment effect on brain glioma.

[0020] The naphthalocyanine copper liposome has the advantages of good biological safety, low immunogenicity and long circulation in vivo, can effectively penetrate the blood-brain barrier and be delivered to the tumor site, solves the problems that drugs are difficult to penetrate the blood-brain barrier and cannot reach an effective concentration in the traditional treatment method of brain glioma, and provides a new strategy for the treatment of brain glioma. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0022] Figure 1 The figure is a detection result graph of particle size and Zeta potential of each liposome, wherein A is a particle size distribution graph, and B is a Zeta potential measurement result graph.

[0023] Figure 2 The figure is a standard curve drawing result graph of the CuNPc solution, wherein A is an ultraviolet absorption spectrum graph, and B is a standard curve graph.

[0024] Figure 3 The figure is a stability detection result graph of the CuNPc@CAT liposome, wherein A is a graph showing the change trend of the particle size of the liposome with time in different solvents, and B is a graph showing the change trend of the PDI of the liposome with time in different solvents.

[0025] Figure 4 The figure is a blood compatibility detection result graph of distilled water, PBS and CuNPc@CAT liposomes with different concentrations.

[0026] Figure 5 Figure for affinity detection results of CuNPc@CAT liposome and tumor cells;

[0027] Figure 6 Figure for ROS production performance of each liposome in solution level; wherein, A is the absorption spectrum of CuNPc@CAT liposome after H2O2 co-incubation and ultrasonic treatment for different lengths of time, the curves at the position indicated by the arrow, from top to bottom are 0, 1, 2, 3, 4, 5, 6, 7, 8 min; B is the statistical chart of absorbance decrease rate of DPBF at 420 nm under different treatment conditions;

[0028] Figure 7 Figure for ROS production performance of each liposome in cell level (scale: 50 μm);

[0029] Figure 8 Figure for cytotoxicity experiment results of each liposome on C6 cells; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0030] Figure 9 Figure for live / dead cell staining results of each liposome on C6 cells (scale: 50 μm). DETAILED DESCRIPTION

[0031] The following detailed description is provided to understand certain aspects, features and embodiments of the present application. It should be understood that the detailed description and specific examples, while indicating certain embodiments of the application, are given by way of illustration only, and should not be construed in any manner to limit the application.

[0032] It should be understood that the terms used in the specification are merely for the purpose of describing particular embodiments and are not intended to limit the application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is specifically disclosed. Each intermediate value between any stated value or stated range, as well as any other stated value or intermediate value in the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between any document incorporated by reference and the present specification, the present specification will control.

[0034] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0035] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or list of materials.

[0036] The reagents and consumables involved in the following examples are shown in Table 1, and the instruments and equipment are shown in Table 2; the experimental cells are rat glioma cells C6, which are from Wuhan Punsai Life Science and Technology Co., Ltd.

[0037] Table 1 Reagents and consumables

[0038]

[0039] Table 2 Main instruments and equipment

[0040]

[0041] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0042] Example 1 Preparation of copper naphthalocyanine liposome

[0043] Thin film dispersion method was used to prepare copper naphthalocyanine liposome. 3.0 mg of CuNPc was accurately weighed and dissolved in a certain amount of chloroform to prepare a 12 mg / mL mother liquor. 25.8 mg of DSPC, 11.8 mg of DSPE-MPEG2000, and 4.3 mg of cholesterol were accurately weighed and dissolved in a certain amount of chloroform to prepare a 20 mg / mL mother liquor. 1.9 mg of CAT was accurately weighed and dissolved in 5 mL of PBS to prepare a hydration solution. 175 μL of CuNPc mother liquor and 1750 μL of lipid mother liquor were mixed uniformly and then transferred to a 50 mL round-bottom flask. The solvent was evaporated completely at 30°C under reduced pressure for 1 h (vacuum degree 0.1 MPa, rotation speed 40 rpm). Then, the pre-prepared hydration solution was added, and the resulting solution was ultrasonically treated for 5 min (45°C, 300W / time, 40KHz) in a water bath, and then filtered through 0.45 μm and 0.22 μm filter membranes to prepare CuNPc@CAT liposome (CuNPc@CAT).

[0044] CuNPc liposome without CAT (CuNPc) was prepared in the same way, and blank liposome without CuNPc and CAT was prepared as well.

[0045] Example 2 Evaluation of physicochemical properties of naphthalocyanine copper liposome

[0046] 1. Particle size and Zeta potential

[0047] The particle size and Zeta potential of the liposome were determined by dynamic light scattering method (DLS). 200 μL of the sample prepared in Example 1 was diluted 5 times with PBS, and then the particle size and Zeta potential were determined using a nanoparticle size and Zeta potential analyzer.

[0048] The results are shown in Table 1. Figure 1 Among them, the particle size of the blank liposome was 109.97 ± 0.54 nm, and the Zeta potential was -2.00 ± 0.20 mV; the particle size of the CuNPc liposome was 114.27 ± 0.95 nm, and the Zeta potential was -2.14 ± 0.16 mV; the particle size of the CuNPc@CAT liposome was 123.93 ± 0.93 nm, and the Zeta potential was -3.37 ± 0.30 mV. The particle size of the liposome containing CuNPc and CAT was slightly larger than that of the blank liposome, which could preliminarily indicate that it was successfully loaded into the liposome.

[0049] 2. Drug loading and encapsulation efficiency

[0050] The drug loading (Drug loading content, DL) and encapsulation efficiency (Encapsulation efficiency, EE) of CuNPc@CAT liposome were determined by ultraviolet-visible spectrophotometry.

[0051] CuNPc standard curve preparation: 3.4 mg of CuNPc was accurately weighed and dissolved in 2125 μL of chloroform to prepare a CuNPc stock solution of 1.6 mg / mL. The stock solution was then diluted to 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL, respectively. 100 μL of each solution was added to a 96-well plate, and the full wavelength absorption spectrum (200-1000 nm) and the absorbance value at 860 nm (A860) were determined using a multifunctional enzyme marker. The CuNPc solution concentration was taken as the abscissa, and the A 860 was taken as the ordinate to draw the CuNPc standard curve. The ultraviolet absorption spectrum and standard curve are shown in Figure 1. Figure 2 As shown by A Figure 2 in the range of 450-1000 nm, CuNPc molecules have multiple absorption peaks. As shown by A Figure 2As shown in Figure B, the equation obtained after linear regression analysis is Y = 0.03076 * X + 0.05497, wherein R 2 is 0.9997, indicating that the regression equation has a high degree of fitting with the linear model.

[0052] CuNPc content determination: an appropriate amount of CuNPc liposomes and CuNPc@CAT liposomes prepared in Example 1 were mixed with ethanol at a volume ratio of 1:1, and after water bath ultrasonic treatment for a period of time, 100 μL of the mixed solution was taken in a 96-well plate, and the absorbance value at 860 nm was detected by a multifunctional enzyme label instrument. The content of CuNPc encapsulated in the liposomes was calculated according to the CuNPc standard curve. The drug loading and encapsulation efficiency were calculated according to Formula 1 and Formula 2, respectively:

[0053] DL (%) = C × V / (C × V + W) × 100% Formula 1

[0054] EE (%) = C × V / M × 100% Formula 2

[0055] In the formula, C is the measured CuNPc concentration in the liposomes; V is the volume of the recovered sample after hydration and filtration; M is the total mass of CuNPc; and W is the total mass of the lipids.

[0056] The results are shown in Table 3. It can be seen that the encapsulation efficiency and drug loading of CuNPc@CAT liposomes prepared by the same method are not lower than those of CuNPc liposomes, indicating that the introduction of CAT does not hinder the loading of CuNPc.

[0057] Table 3 Drug loading and encapsulation efficiency of CuNPc-containing liposomes

[0058]

[0059] 3. Stability

[0060] CuNPc molecules have strong hydrophobicity, resulting in poor biocompatibility. Therefore, improving the hydrophilicity of CuNPc molecules is the key to promoting its application. In practical applications, charged substances such as inorganic salts and proteins in biological fluids can cause nanocarriers to aggregate or precipitate, and then be quickly cleared by the immune system; and serum proteins in the culture medium can also change the physicochemical properties and biological behavior of nanocarriers, leading to premature disintegration and increasing potential side effects. Therefore, it is of great significance to ensure the long-term stability of CuNPc series liposomes in PBS and culture medium. The stability of nanocarriers in PBS and culture medium is also the basis for predicting the key indicators of nanocarriers, such as in vivo circulation time and biological safety.

[0061] Dynamic light scattering method (DLS) was used to evaluate the particle size stability of CuNPc@CAT liposome. 200 μL of the liposome sample prepared in Example 1 was diluted 5 times with ultrapure water, PBS and Ham's F-12K respectively, and the changes of particle size and polydispersity index (PDI) within 9 days at room temperature were determined using a nanoparticle size and Zeta potential analyzer. The results are shown in Table 1. Figure 3 As can be seen, the particle size fluctuation range of CuNPc@CAT liposome in water is the smallest, indicating that charged ions and proteins and other substances have a slight degree of interference on its physicochemical properties, but the particle size change range of CuNPc@CAT liposome in the three systems within 9 days is within ±10 nm, and the polydispersity index is less than 0.3, indicating that the CuNPc@CAT liposome prepared by the present application has good dispersibility and stability in water, PBS and Ham's F-12K.

[0062] 4. Blood compatibility

[0063] An in vitro hemolysis experiment was used to evaluate the blood compatibility of CuNPc@CAT liposome, and the safe dose range of the liposome was determined by the hemolysis rate at different concentrations, providing a reference for subsequent experiments. The blood compatibility of CuNPc@CAT liposome was evaluated by hemolysis experiment. An appropriate amount of anticoagulated mouse blood was centrifuged at 3000 rpm for 5 min, and the supernatant was discarded and washed with PBS and then centrifuged according to the above parameters until the supernatant was colorless. An appropriate amount of PBS was added to the erythrocyte precipitate to prepare a 4% erythrocyte suspension. This experiment was divided into a positive control group, a negative control group and an experimental group. 2 mL centrifuge tubes were taken, 500 μL of the prepared 4% erythrocyte suspension was added to each tube, 500 μL of distilled water was added to each tube in the positive control group, 500 μL of PBS was added to each tube in the negative control group, and 500 μL of CuNPc@CAT liposome sample with concentrations of 2 μg / mL, 6 μg / mL, 10 μg / mL, 14 μg / mL, 18 μg / mL and 22 μg / mL was added to each tube in the experimental group, respectively, and then mixed gently and incubated at 37°C for 1 h. After centrifugation at 3000 rpm for 5 min, 100 μL of supernatant was taken to a 96-well plate, and the absorbance value (A540) at 540 nm was detected using a multifunctional enzyme marker. The hemolysis rate (Hemolysis ratio, HR) of each group was calculated according to formula 3:

[0064] HR (%) = (A s -A p ) / (A w -A p )×100% Formula 3

[0065] In the formula, A s is the absorbance value of the experimental group, A w is the absorbance value of the positive control group, and Ap The absorbance value of the negative control group.

[0066] The results, as shown in Figure 4 Table 2, show that no obvious hemolysis was observed in the negative control group and the different concentration CuNPc@CAT liposome groups, while obvious hemolysis was observed in the positive control group. The supernatant of each group was detected by a multifunctional enzyme label instrument, and the hemolysis rate was calculated. The results show that the hemolysis rate of CuNPc@CAT liposomes at different concentrations is less than 3%, which proves that CuNPc@CAT liposomes in this concentration range have good hemocompatibility and can be used for subsequent in vitro anti-glioma pharmacodynamics research.

[0067] 5、Tumor cell affinity

[0068] To explore the affinity of CuNPc@CAT liposomes with C6 cells, a cell uptake experiment was used to evaluate the affinity of CuNPc@CAT liposomes for tumor cells. A proper amount of Nile Red was dissolved in DMSO to prepare a 1 mM storage solution. The CuNPc@CAT liposomes prepared in Example 1 were mixed with the Nile Red storage solution at a volume ratio of 1000:1 at room temperature for 10 min to prepare Nile Red-labeled CuNPc@CAT liposomes. 5×10 4

[0069] The results, as shown in Figure 5 Table 3, show that the Hoechst 33342-labeled C6 cells and the Nile Red-labeled CuNPc@CAT liposomes showed different degrees of colocalization, indicating that C6 cells and liposomes have certain affinity. The uptake of CuNPc@CAT liposomes by C6 cells showed time dependence, with the highest uptake at 12 h, which provides an important reference for the selection of liposome and cell co-incubation time in the subsequent experiments.

[0070] Example 3 Evaluation of the sonodynamic performance of naphthalocyanine copper liposomes

[0071] 1、Solution level ROS production performance

[0072] ​1,3-Diphenylisobenzofuran (DPBF) was used as a probe to detect 1 O2 production. 1 O2 can react with DPBF to form unstable endoperoxide, which leads to the breakage of the conjugated structure of DPBF and a significant decrease in the absorbance of the characteristic peak at 420 nm. By detecting the change in absorbance over time using a UV-visible spectrophotometer, the content of O2 can be indirectly reflected. 1

[0073] 2.5 mg of DPBF was precisely weighed and completely dissolved in DMSO to prepare a working solution of 5 mg / mL. To detect the effect of CAT on the production of O2 generated by the decomposition of H2O2 to enhance SDT, 80 μL of the CuNPc@CAT liposome sample prepared in Example 1 was mixed with 20 μL of H2O2 solution (100 mM) and incubated for 10 min, then transferred into a 96-well plate and added with 2 μL of the DPBF working solution, and ultrasonically treated (1 W / cm 2 , 1 min each time), and the 96-well plate was transferred into a multifunctional enzyme marker every 1 min to detect the absorption spectrum of 300-900 nm. Under the same conditions, the PBS+H2O2 (100 mM) group was used as a control.

[0074] The results are shown in Figure 6 . The CuNPc liposomes and CuNPc@CAT liposomes were treated with ultrasonic waves of 1 W / cm 2 , and the results showed that the absorbance of the characteristic peak of DPBF at 420 nm gradually decreased with the increase of ultrasonic time, indicating that both of them can produce O2 with high efficiency under the action of ultrasonic waves. 1 The blank control group treated under the same ultrasonic parameters showed a lower degree of degradation of DPBF than the liposome group, further indicating that CuNPc is an effective sonosensitizer. After the CuNPc@CAT liposomes were incubated with H2O2 for 10 min, the efficiency of O2 production under the action of ultrasonic waves was 1.54 times higher than that of the CuNPc liposome group, indicating that the loading of CAT can effectively decompose H2O2 to produce O2 to supply the continuous production of O2 by SDT, further improving the sonodynamic performance of CuNPc@CAT liposomes. 1 1

[0075] 2. ROS production performance at the cell level

[0076] ​​​DCFH-DA was used as a probe to detect the intracellular ROS production. After being taken up by cells, DCFH-DA was hydrolyzed by esterase into DCFH, which could not permeate the cell membrane. ROS in cells could oxidize DCFH into DCF, which emitted strong green fluorescence. The green fluorescence production was observed by fluorescence microscopy to evaluate the intracellular ROS production.

[0077] 1 x 10 5 C6 cells were inoculated in 12-well plates and cultured in a cell incubator (37°C, 5% CO2) for 24 h. A total of 6 groups were set up: control group, CuNPc liposome group, CuNPc@CAT liposome group, ultrasound group, CuNPc liposome + ultrasound group, and CuNPc@CAT liposome + ultrasound group. The ultrasound parameter was 1 W / cm 2 Ultrasound was performed for 5 min. After the cells in the liposome-containing groups were incubated with 10 μg / mL of CuNPc or CuNPc@CAT liposome for 12 h, the cells in the control group and ultrasound group were cultured with blank medium for 12 h, and then the cells were gently washed with PBS for 3 times. 1 mL of DCFH-DA working solution (10 μM) was added to each group of cells, which were incubated at 37°C for 30 min. After the cells were treated with ultrasound, they were gently washed with PBS for 3 times, and the green fluorescence production was observed under a fluorescence microscope.

[0078] The results are shown in Table 1. Figure 7 No obvious green fluorescence was observed in the three groups without ultrasound, indicating that CuNPc liposome and CuNPc@CAT liposome did not activate the production of intracellular ROS after being incubated with cells. After the cells in each group were treated with ultrasound at 1 W / cm 2 After the cells in each group were treated with ultrasound at 1 W / cm

[0079] Example 4 In Vitro Anti-Glioma Efficacy Evaluation of CuNPc Liposome

[0080] 1. Cytotoxicity experiment

[0081] CCK-8 was used to characterize the cell survival after SDT. 5 x 10 3C6 cells were cultured in 96-well plates in a cell incubator (37°C, 5% CO2) for 24 h. CCK-8 stock solution was diluted with C6 cell culture medium at a volume ratio of 1:10 to prepare CCK-8 working solution. A total of 6 groups were set up: Control group, CuNPc group, CuNPc@CAT group, ultrasound control group, ultrasound group, and CuNPc@CAT + ultrasound group. The ultrasound parameter was 1 W / cm 2 2 for 5 min. The cells in the liposome-containing groups were incubated with 10 μg / mL CuNPc liposomes or CuNPc@CAT liposomes for 12 h, and the cells in the control and ultrasound groups were cultured with blank medium for 12 h, followed by gentle PBS rinsing 3 times. After ultrasound treatment, 100 μL of CCK-8 working solution was added to each well, and incubation was performed at 37°C for 2 h. The absorbance at 450 nm was detected using a multifunctional enzyme label meter.

[0082] The results are shown in Figure 8 Without ultrasound, the survival rate of the cells incubated with CuNPc liposomes or CuNPc@CAT liposomes decreased significantly. This phenomenon may be caused by two reasons: first, the organic solvent may not have been completely removed during the preparation of the liposome sample, which may have caused certain toxicity to the cells; second, although the tumor cell affinity experiment in Example 2 showed that the optimal accumulation of liposomes in cells could be achieved after 12 h of incubation, the long-term accumulation of CuNPc in cells may have caused certain damage to the cells, thereby leading to a decrease in cell survival rate. After 5 min of ultrasound treatment at 1 W / cm 2 The survival rate of the cells in the CuNPc liposome group was 35%, and the survival rate of the cells in the CuNPc@CAT liposome group was only 27%, which showed a certain statistical difference between the two groups, indicating that the introduction of CAT could improve the killing effect of SDT on brain glioma cells to a certain extent; compared with the control group, there was a significant statistical difference, indicating that CuNPc series liposomes combined with SDT had good anti-brain glioma effect.

[0083] 2, Live / dead cell staining experiment

[0084] Calcein-AM / PI dye was used for double staining of cells after SDT. 1×10 5C6 cells were cultured in 12-well plates in a cell incubator (37°C, 5% CO2) for 24 h. Calcein-AM staining solution, PI staining solution and serum-free medium were mixed at a volume ratio of 1:1:1000 to prepare a Calcein / PI working solution. A total of 6 groups were set up: control group, CuNPc liposome group, CuNPc@CAT liposome group, ultrasound group, CuNPc liposome + ultrasound group, CuNPc@CAT liposome + ultrasound group, and the ultrasound parameter was 1 W / cm 2 2 for 5 min. After the cells in the liposome-containing groups were incubated with 10 μg / mL of CuNPc or CuNPc@CAT liposomes for 12 h, they were gently rinsed with PBS for 3 times. After the cells were treated with ultrasound, 500 μL of Calcein-AM / PI working solution was added to each well, and the cells were incubated at 37°C for 35 min. The cell staining was observed under a fluorescence microscope.

[0085] The results are shown in Figure 9 Without ultrasound, the cells incubated with CuNPc liposomes or CuNPc@CAT liposomes for 12 h had a certain degree of death, but most of the live cells emitted green fluorescence. After 1 W / cm 2 2 ultrasound for 5 min, the CuNPc@CAT liposome group showed the highest proportion of dead cells, followed by the CuNPc liposome group, which was consistent with the cell survival rate results of the cytotoxicity experiment, again proving that CuNPc series liposomes combined with SDT had good anti-glioma effect.

[0086] The above-described embodiments are only descriptions of the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A copper naphthalene phthalocyanine liposome with therapeutic effects on glioma, characterized in that, Naphthalene phthalocyanine copper and catalase were encapsulated in liposomes using a thin-film dispersion method to obtain the naphthalene phthalocyanine copper liposomes; The mass ratio of the naphthalene phthalocyanine copper, the catalase, and the liposomes is 2-2.5:1.5-2:30-40; The liposomes are composed of distearate, polyethylene glycol monomethyl ether-2000-octadecylphosphatidylethanolamine and cholesterol. The method for constructing the naphthalene phthalocyanine copper liposomes includes the following steps: A solution of naphthalene phthalocyanine copper was mixed with a liposome solution, and a thin film was obtained by rotary evaporation. Catalase solution was added, and the mixture was subjected to ultrasonication in a water bath and then filtered to obtain the naphthalene phthalocyanine copper liposomes.

2. The naphthalene phthalocyanine copper liposome according to claim 1, characterized in that, The mass ratio of the distearate, the polyethylene glycol monomethyl ether-2000-octadecylphosphatidylethanolamine, and the cholesterol is 25-30:10-15:4-5.

3. The method for constructing naphthalene phthalocyanine copper liposomes according to claim 1 or 2, characterized in that, Includes the following steps: A solution of naphthalene phthalocyanine copper was mixed with a liposome solution, and a thin film was obtained by rotary evaporation. Catalase solution was added, and the mixture was subjected to ultrasonication in a water bath and then filtered to obtain the naphthalene phthalocyanine copper liposomes.

4. The construction method according to claim 3, characterized in that, The temperature of the water bath ultrasound is 42-46℃; the power of the water bath ultrasound is 300W / hour, the frequency is 40KHz, and the duration is 5min.

5. The construction method according to claim 3, characterized in that, The filtration process involves sequentially passing the material through a 0.45 μm filter membrane and a 0.22 μm filter membrane.

6. The construction method according to claim 3, characterized in that, The solvent for the naphthalene phthalocyanine copper solution and the liposome solution is chloroform; the solvent for the catalase solution is phosphate buffer.

7. The use of the naphthalene phthalocyanine copper liposome according to claim 1 or 2, or the naphthalene phthalocyanine copper liposome constructed by the construction method according to any one of claims 3-6, in the preparation of a medicament for treating glioma.

8. A drug for treating glioma, characterized in that, The active ingredient of the drug is the naphthalene phthalocyanine copper liposome as described in claim 1 or 2, or the naphthalene phthalocyanine copper liposome constructed by the construction method described in any one of claims 3-6.

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