Naphthalocyanine copper liposome as well as construction method and application thereof

Abstract

The invention discloses a copper naphthalocyanine liposome as well as a construction method and application thereof, and belongs to the technical field of liposome preparations. According to the copper naphthalocyanine liposome disclosed by the invention, the copper naphthalocyanine and the catalase are wrapped in the liposome, so that the problems of poor water solubility, insufficient stability and the like of an existing sound-sensitive agent can be improved; meanwhile, the nanomaterial has the effect of catalyzing excessive H2O2 in a brain glioma microenvironment to generate O2 to supply the O2 to a sonodynamic therapy (SDT) to continuously generate ROS, the sonodynamic performance of the nanomaterial is enhanced, the treatment effect of the brain glioma is improved, the nanomaterial has the advantages of good biological safety, low immunogenicity and in-vivo long circularity, and the nanomaterial can effectively penetrate through a blood brain barrier and is delivered to a tumor site, so that the nanomaterial has a good application prospect. The problems that in a traditional brain glioma treatment method, medicine cannot penetrate through the blood brain barrier easily and cannot reach the effective concentration are solved, and a new strategy is provided for treatment of brain glioma.

Description

Technical Field

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

[0002] Glioblastoma (GBM) is one of the most common primary tumors of the central nervous system (CNS). It originates from the malignant proliferation of glial cells such as astrocytes and oligodendrocytes. It rapidly proliferates, infiltrates and invades normal brain tissue, and has a blurred boundary with normal tissue. Furthermore, the unique immune environment of the CNS endows glioma cells with immune evasion capabilities, making them difficult for the immune system to recognize and eliminate. Furthermore, the presence of the blood-brain barrier (BBB) ​​makes it difficult for most drug molecules and chemicals to penetrate this natural barrier and reach the lesions, presenting a significant obstacle to glioma treatment.

[0003] Currently, surgical resection, radiotherapy, and chemotherapy remain the main clinical treatments for gliomas. However, for malignant gliomas, complete resection is extremely difficult due to the unclear boundary between the lesions and normal brain tissue, and the surgical risk and postoperative recurrence rate are high. During radiotherapy and chemotherapy, drug resistance is a frequent problem; chemotherapy drugs such as temozolomide and bevacizumab are restricted by the blood-brain barrier and are difficult to reach effective therapeutic concentrations in the brain; radiotherapy may also cause side effects such as cognitive dysfunction. These factors lead to poor prognosis and short survival for patients with malignant gliomas, seriously threatening the quality of life of patients.

[0004] In recent years, new treatment strategies have continued to emerge. Among them, sonodynamic therapy (SDT), with its advantages of non-invasiveness and high tissue penetration, offers new hope for overcoming the limitations of the blood-brain barrier and precisely treating gliomas. SDT uses sonosensitizers to generate reactive oxygen species (ROS) under the action of ultrasound, inducing tumor cell apoptosis. SDT has the advantages of deep tissue penetration, precise focusing, safety, non-invasiveness, and repeatability. Its application in the treatment of gliomas can improve the shortcomings of current traditional therapies. However, due to the existence of the blood-brain barrier, the sonosensitizers required for SDT are hindered from entering brain tumor tissue. Existing sonosensitizer delivery systems are inefficient, greatly affecting the treatment effect. Summary of the Invention

[0005] The purpose of the present invention is to provide a copper naphthalocyanine liposome and its construction method and application to solve the problems existing in the above-mentioned prior art. The present invention has developed a liposome drug delivery system. By encapsulating copper naphthalocyanine and catalase in liposomes, it can improve the problems of poor water solubility and insufficient stability of existing sonosensitizers; at the same time, it has the function of catalyzing the excessive H2O2 in the microenvironment of brain glioma to produce O2 to supply SDT to continuously produce ROS, thereby enhancing its sonodynamic performance and improving the therapeutic effect of brain glioma. It has the advantages of good biosafety, low immunogenicity, and long circulation in the body. It can effectively penetrate the blood-brain barrier and be delivered to the tumor site, solving the problems of traditional methods for treating brain gliomas, such as the difficulty of drugs in crossing the blood-brain barrier and the inability to reach effective concentrations, and provides a new strategy for the treatment of brain gliomas.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a copper naphthalocyanine liposome with the effect of treating brain glioma. The copper naphthalocyanine liposome is obtained by encapsulating copper naphthalocyanine and catalase in the liposome by a thin film dispersion method.

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

[0009] Furthermore, the components of the liposome include distearoyl phosphatidylcholine, polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine and cholesterol.

[0010] Furthermore, the mass ratio of the distearoyl phosphatidylcholine, the polyethylene glycol monomethyl ether-2000-dioctadecyl phosphatidylethanolamine and the cholesterol is 25-30:10-15:4-5.

[0011] The present invention also provides a method for constructing the above-mentioned copper naphthalocyanine liposome, comprising the following steps:

[0012] The copper naphthalocyanine solution and the liposome solution are mixed, and a film is obtained by rotary evaporation. A catalase solution is added, and the mixture is ultrasonicated in a water bath and filtered to obtain the copper naphthalocyanine liposome.

[0013] Furthermore, the temperature of the water bath ultrasound is 42-46° C.; the power of the water bath ultrasound is 300 W / h, the frequency is 40 KHz, and the time is 5 min.

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

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

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

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

[0018] The present invention discloses the following technical effects:

[0019] The present invention develops a liposome drug delivery system that encapsulates copper naphthalocyanine and catalase in liposomes to improve the problems of poor water solubility and insufficient stability of existing sonosensitizers. At the same time, it catalyzes the excessive H2O2 in the microenvironment of brain gliomas to produce O2 to supply SDT to continuously produce ROS, thereby enhancing its sonodynamic performance and improving the therapeutic effect of brain gliomas.

[0020] The naphthalocyanine copper liposomes of the present invention have the advantages of good biosafety, low immunogenicity, and long circulation in the body. They can effectively penetrate the blood-brain barrier and be delivered to the tumor site, solving the problems of traditional methods for treating gliomas, such as the difficulty of drugs crossing the blood-brain barrier and the inability to reach effective concentrations, and provide a new strategy for the treatment of gliomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Figures 1 and 2 are the particle size and Zeta potential test results of each liposome; A is the particle size distribution diagram; B is the Zeta potential test result diagram;

[0023] Figure 2 The result graph of the standard curve of CuNPc solution is drawn; wherein A is the ultraviolet absorption spectrum graph; B is the standard curve graph;

[0024] Figure 3 Figure 2 is the stability test result of CuNPc@CAT liposomes; A is the trend of liposome particle size change over time in different solvents; B is the trend of liposome PDI change over time in different solvents;

[0025] Figure 4 Figure 2 is the blood compatibility test results of distilled water, PBS and CuNPc@CAT liposomes with different concentrations;

[0026] Figure 5 This is the affinity test result of CuNPc@CAT liposomes and tumor cells;

[0027] Figure 6 Figure 1 shows the results of the study on the ROS production performance of each liposome at the solution level; A is the absorption spectrum of H2O2 and CuNPc@CAT liposomes after co-incubation with ultrasonic treatment for different lengths of time. The curves indicated by the arrows are 0, 1, 2, 3, 4, 5, 6, 7, and 8 minutes from top to bottom; B is a statistical graph of the decrease rate of DPBF absorbance at 420 nm under different treatment conditions;

[0028] Figure 7 Figure 2 shows the results of the study on the ROS production performance of each liposome at the cellular level (scale: 50 μm);

[0029] Figure 8 The results of the cytotoxicity test of each liposome on C6 cells are shown in the figure; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001;

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

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Some of 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 brain glioma cells C6, which are obtained from Wuhan Punosai Life Science 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, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0042] Example 1 Preparation of copper naphthalocyanine liposomes

[0043] Copper naphthalocyanine liposomes were prepared using a thin film dispersion method. 3.0 mg of CuNPc was accurately weighed and dissolved in a certain amount of chloroform to prepare a 12 mg / mL stock solution. 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 stock solution. 1.9 mg of CAT was accurately weighed and thoroughly dissolved in 5 mL of PBS to prepare a hydration solution. 175 μL of the CuNPc stock solution and 1750 μL of the lipid stock solution were thoroughly mixed and transferred to a 50 mL round-bottom flask. The solvent was completely evaporated by rotary evaporation at 30°C for 1 h (vacuum 0.1 MPa, rotation speed 40 rpm). Subsequently, the pre-prepared hydration solution was added thereto, and water bath sonication was performed for 5 min (45°C, 300W / h, 40KHz). The obtained solution was sequentially passed through 0.45 μm and 0.22 μm filter membranes to prepare CuNPc@CAT liposomes (CuNPc@CAT).

[0044] CuNPc liposomes without CAT (CuNPc) and blank liposomes without CuNPc and CAT were prepared in the same way.

[0045] Example 2 Evaluation of the physicochemical properties of copper naphthalocyanine liposomes

[0046] 1. Particle size and Zeta potential

[0047] The particle size and zeta potential of the liposomes were determined using dynamic light scattering (DLS). 200 μL of the sample prepared in Example 1 was diluted 5-fold with PBS and then measured using a nanoparticle size and zeta potential analyzer.

[0048] The results are as follows Figure 1 As shown in the figure, the blank liposomes had a particle size of 109.97 ± 0.54 nm and a zeta potential of -2.00 ± 0.20 mV; the CuNPc liposomes had a particle size of 114.27 ± 0.95 nm and a zeta potential of -2.14 ± 0.16 mV; and the CuNPc@CAT liposomes had a particle size of 123.93 ± 0.93 nm and a zeta potential of -3.37 ± 0.30 mV. The CuNPc- and CAT-containing liposomes had a slightly larger particle size than the blank liposomes, preliminarily indicating that they were successfully loaded into the liposomes.

[0049] 2. Drug loading and encapsulation efficiency

[0050] The drug loading content (DL) and encapsulation efficiency (EE) of CuNPc@CAT liposomes were determined by UV-visible spectrophotometry.

[0051] CuNPc standard curve drawing: accurately weigh 3.4 mg CuNPc and dissolve it in 2125 μL chloroform to prepare a 1.6 mg / mL CuNPc stock solution. Then dilute the stock solution to 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL in sequence. Take 100 μL of each solution and add it to a 96-well plate. Use a multifunctional microplate reader to measure its full wavelength absorption spectrum (200-1000 nm) and the absorbance value at 860 nm (A860). The concentration of CuNPc solution is used as the horizontal axis and A is used as the horizontal axis. 860 As the vertical axis, draw the standard curve of CuNPc. Its ultraviolet absorption spectrum and standard curve are as follows Figure 2 As shown. Figure 2 As shown in Figure A, CuNPc molecules have multiple absorption peaks in the range of 450-1000 nm. Figure 2As shown in B, the linear regression analysis equation is Y = 0.03076 * X + 0.05497, where R 2 It is 0.9997, indicating that the regression equation has a high degree of fit with the linear model.

[0052] CuNPc content determination: Take appropriate amounts of the CuNPc liposomes and CuNPc@CAT liposomes prepared in Example 1 and mix them with ethanol at a volume ratio of 1:1. After water bath sonication for a period of time, take 100 μL of the mixed solution into a 96-well plate and measure its absorbance at 860 nm using a multifunctional microplate reader. The content of encapsulated CuNPc in the liposomes is calculated based on the CuNPc standard curve. The drug loading and encapsulation efficiency are calculated according to Formula 1 and Formula 2, respectively:

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

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

[0055] Where: C is the concentration of CuNPc measured in liposomes; V is the volume of sample recovered after hydration of the filtration membrane; M is the total mass of CuNPc added; W is the total mass of lipid added.

[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 Results of drug loading and encapsulation efficiency of CuNPc-containing liposomes

[0058]

[0059] 3. Stability

[0060] CuNPc molecules are highly hydrophobic, resulting in poor biocompatibility. Therefore, improving the hydrophilicity of CuNPc molecules is the key to promoting their application. In practical applications, charged substances such as inorganic salts and proteins in biological fluids may cause nanocarriers to aggregate or precipitate, and then be quickly cleared by the immune system; and serum proteins in the culture medium may also change the physicochemical properties and biological behavior of nanocarriers, causing them to disintegrate prematurely and increase potential side effects. Therefore, ensuring the long-term stability of CuNPc series liposomes in PBS and culture medium is of great significance. The stability of nanocarriers in PBS and culture medium is also the basis for predicting key indicators such as the in vivo circulation time and biosafety of nanocarriers.

[0061] Dynamic light scattering (DLS) was used to evaluate the particle size stability of CuNPc@CAT liposomes. 200 μL of the liposome sample prepared in Example 1 was diluted 5-fold with ultrapure water, PBS, and Ham's F-12K, respectively. The changes in particle size and polydispersity index (PDI) at room temperature over 9 days were measured using a nanoparticle size analyzer and a zeta potential analyzer. The results are shown in Figure 2. Figure 3 As shown in the data, the particle size fluctuation range of CuNPc@CAT liposomes in water is the smallest, indicating that substances such as charged ions and proteins have a slight interference with its physicochemical properties. However, within 9 days, the particle size variation range of CuNPc@CAT liposomes in the three systems is within ±10 nm, and the polydispersity index is lower than 0.3, indicating that the CuNPc@CAT liposomes prepared by the present invention have good dispersibility and stability in water, PBS and Ham's F-12K.

[0062] 4. Blood compatibility

[0063] An in vitro hemolysis assay was used to evaluate the blood compatibility of CuNPc@CAT liposomes. The hemolysis rates at different concentrations were used to determine the safe dosage range of the liposomes, providing a reference for subsequent experiments. The hemolysis assay was used to evaluate the blood compatibility of CuNPc@CAT liposomes. An appropriate amount of anticoagulated mouse blood was centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded, washed with PBS, and centrifuged again according to the above parameters until the supernatant was colorless. An appropriate amount of PBS was added to the red blood cell pellet to prepare a 4% red blood cell suspension. This experiment was divided into a positive control group, a negative control group, and an experimental group. Take a 2 mL centrifuge tube and add 500 μL of the prepared 4% red blood cell suspension to each tube. Add 500 μL of distilled water to each tube in the positive control group and 500 μL of PBS to each tube in the negative control group. Add 500 μL of Cu NPc@CAT liposome samples at concentrations of 2 μg / mL, 6 μg / mL, 10 μg / mL, 14 μg / mL, 18 μg / mL, and 22 μg / mL to each tube in the experimental group. Gently shake to mix, incubate at 37°C for 1 hour, centrifuge at 3000 rpm for 5 minutes, and then aspirate 100 μL of the supernatant into a 96-well plate. The absorbance at 540 nm (A540) is measured using a multifunctional microplate reader. The hemolysis ratio (HR) of each group is calculated according to Equation 3:

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

[0065] Where: A s is the absorbance value of the experimental group, A w is the absorbance value of the positive control group, Ap is the absorbance value of the negative control group.

[0066] The results are as follows Figure 4 As shown in the figure, no significant hemolysis was observed in the negative control group and the groups with different concentrations of CuNPc@CAT liposomes, while significant hemolysis was observed in the positive control group. The supernatant of each group was detected using a multifunctional microplate reader and the hemolysis rate was calculated. The results showed that the hemolysis rate of CuNPc@CAT liposomes at different concentrations was less than 3%, demonstrating that CuNPc@CAT liposomes within this concentration range have good hemocompatibility and can be used for subsequent in vitro anti-glioma pharmacodynamic studies.

[0067] 5. Tumor cell affinity

[0068] To investigate the affinity of CuNPc@CAT liposomes for C6 cells, a cell uptake experiment was performed to evaluate the affinity of CuNPc@CAT liposomes for tumor cells. An appropriate amount of Nile Red was dissolved in DMSO to prepare a 1 mM stock solution. The CuNPc@CAT liposomes prepared in Example 1 were incubated with the Nile Red stock solution at a volume ratio of 1000:1 at room temperature for 10 min to prepare NileRed-labeled CuNPc@CAT liposomes. 5×10 4 C6 cells were plated in a 24-well plate and cultured in a cell culture incubator for 24 hours (37°C, 5% CO2). 500 μL of Nile Red-labeled CuNPc@CAT liposomes and 500 μL of serum-free medium were added to each well. The plates were gently shaken to mix thoroughly and then incubated in a cell culture incubator in the dark for 0, 3, 6, 9, and 12 hours (37°C, 5% CO2). The medium was discarded, and the plates were gently rinsed three times with PBS. Hoechst 33342 (10 μM) diluted in serum-free DMEM was added and incubated at 37°C in the dark for 25 minutes. The plates were gently rinsed three times with PBS, and the uptake of CuNPc@CAT liposomes by C6 cells was observed under a fluorescence microscope.

[0069] The results are as follows Figure 5 As shown, Hoechst 33342-labeled C6 cells and Nile Red-labeled CuNPc@CAT liposomes colocalized to varying degrees, indicating a certain affinity between C6 cells and the liposomes. CuNPc@CAT liposome uptake by C6 cells was time-dependent, reaching its highest level at 12 hours. This provides an important reference for selecting the duration of subsequent co-incubation of liposomes with cells.

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

[0071] 1. ROS production performance at the solution level

[0072] 1,3-Diphenylisobenzofuran (DPBF) was used as a probe to detect 1 O2 production situation. 1 O2 can react with DPBF to generate unstable endoperoxides, 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. The change of absorbance over time can be indirectly reflected by the UV-visible spectrophotometer. 1 O2 content.

[0073] 2.5 mg of DPBF was accurately weighed and completely dissolved in DMSO to prepare a 5 mg / mL working solution. To test the effect of CAT on the decomposition of H2O2 to produce O2 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. The mixture was then transferred to a 96-well plate and 2 μL of DPBF working solution was added. The plates were then ultrasonically treated (1 W / cm 2 The 96-well plate was transferred to a multifunctional microplate reader every 1 minute to measure the absorbance spectrum at 300-900 nm. Under the same conditions, the PBS + H2O2 (100 mM) group served as a control.

[0074] The results are as follows Figure 6 As shown. Using 1 W / cm 2 The CuNPc liposomes and CuNPc@CAT liposomes were treated with ultrasound. The results showed that with the increase of ultrasound time, the absorbance of the characteristic peak of DPBF at 420 nm gradually decreased, indicating that the two can produce DPBF more efficiently under ultrasound. 1 O2. The degradation degree of DPBF in the blank control group treated with the same ultrasound parameters was lower than that in the liposome group, which further indicated that CuNPc was an effective sonosensitizer. After CuNPc@CAT liposomes were incubated with H2O2 for 10 min, the DPBF produced by ultrasound was significantly improved. 1 The O2 efficiency 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 SDT. 1 O2, further improving the sonodynamic performance of CuNPc@CAT liposomes.

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

[0076] 2',7'-Dichlorodihydrofluoresceindiacetate (DCFH-DA) was used as a probe to monitor intracellular ROS production. After DCFH-DA is taken up by cells, it is hydrolyzed by esterases to DCFH, which cannot penetrate the cell membrane. Intracellular ROS oxidize DCFH to DCF, which emits strong green fluorescence. Fluorescence microscopy was used to observe the green fluorescence and assess intracellular ROS production.

[0077] Inoculate 1×10 5 C6 cells were plated in 12-well plates and cultured in a cell culture incubator for 24 h (37°C, 5% CO2). Six 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 Ultrasonication was performed for 5 minutes. Cells in the liposome-containing group were incubated with 10 μg / mL CuNPc or CuNPc@CAT liposomes for 12 hours. Cells in the control and ultrasound groups were cultured with blank medium for 12 hours and then gently rinsed three times with PBS. Each cell group was then incubated with 1 mL of DCFH-DA working solution (10 μM) at 37°C for 30 minutes. After ultrasonication, the cells were gently rinsed three times with PBS, and the production of green fluorescence was observed under a fluorescence microscope.

[0078] The results are as follows Figure 7 No obvious green fluorescence was observed in the three groups without ultrasound, indicating that the production of intracellular ROS was not activated after the CuNPc liposomes and CuNPc@CAT liposomes were co-incubated with cells. 2 After 5 minutes of ultrasound treatment of cells in each group, cells co-incubated with CuNPc liposomes and CuNPc@CAT liposomes all displayed significant green fluorescence, indicating that the CuNPc liposomes exhibited high ROS production efficiency under these ultrasound parameters. This is consistent with the results of DPBF probe detection at the solution level, further demonstrating the excellent sonodynamic properties of CuNPc. Weaker green fluorescence was also observed in cells in the ultrasound group, likely due to the ultrasonic cavitation and thermal effects that altered the cellular oxidative stress state, triggering ROS generation.

[0079] Example 4 In vitro evaluation of the anti-glioma pharmacodynamics of copper naphthalocyanine liposomes

[0080] 1. Cytotoxicity assay

[0081] CCK-8 was used to characterize the cell survival after SDT. 3C6 cells were plated in a 96-well plate and cultured in a cell culture incubator for 24 hours (37°C, 5% CO2). CCK-8 stock solution was diluted with C6 cell-specific culture medium at a volume ratio of 1:10 to prepare CCK-8 working solution. Six 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 1W / cm 2 Ultrasonication was performed for 5 minutes. Cells in the liposome-containing groups were incubated with 10 μg / mL CuNPc liposomes or CuNPc@CAT liposomes for 12 hours. Cells in the control and ultrasound groups were cultured with blank medium for 12 hours and then gently rinsed three times with PBS. After ultrasonic treatment, 100 μL of CCK-8 working solution was added to each well and incubated at 37°C for 2 hours. The absorbance of each well was measured at 450 nm using a multi-function microplate reader.

[0082] The results are as follows Figure 8 As shown. Without ultrasound, the survival rate of cells co-incubated with CuNPc liposomes and CuNPc@CAT liposomes decreased significantly. There are two possible reasons for this phenomenon: first, the organic solvent may not be completely removed during the preparation of the liposome sample, which may have a certain toxicity to the cells; second, although the tumor cell affinity experiment results in Example 2 show that the optimal liposome accumulation effect can be achieved by co-incubating the liposome sample with cells for 12 h, the CuNPc that enters the cells may cause a certain degree of damage to the cells after long-term accumulation, thereby leading to a decrease in cell survival rate. Using 1 W / cm 2 After 5 minutes of ultrasonic treatment, the cell survival rate of the CuNPc liposome group was 35%, while that of the CuNPc@CAT liposome group was only 27%. There was a certain statistical difference between the two, indicating that the introduction of CAT can improve the killing effect of SDT on glioma cells to a certain extent; there was a significant statistical difference compared with the control group, indicating that the CuNPc series liposomes combined with SDT have a good anti-glioma effect.

[0083] 2. Live / dead cell staining experiment

[0084] Calcein-AM / PI dye was used to double-stain the cells after SDT. 1×10 5C6 cells were plated in 12-well plates and cultured in a cell culture incubator for 24 h (37°C, 5% CO2). Calcein-AM staining solution, PI staining solution, and serum-free culture medium were mixed at a volume ratio of 1:1:1000 to prepare the Calcein / PI working solution. Six 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 Ultrasonication was performed for 5 minutes. Cells in the liposome-containing group were incubated with 10 μg / mL CuNPc or CuNPc@CAT liposomes for 12 hours and then gently rinsed three times with PBS. After ultrasonic treatment, 500 μL of Calcein-AM / PI working solution was added to each well and incubated at 37°C for 35 minutes. Cell staining was observed under a fluorescence microscope.

[0085] The results are as follows Figure 9 As shown in Figure 3. Without ultrasound, cells incubated with CuNPc liposomes and CuNPc@CAT liposomes for 12 h showed a certain degree of cell death, but most of the living cells emitting green fluorescence were still observed. 2 After 5 minutes of ultrasound, the highest percentage of dead cells was observed in the CuNPc@CAT liposome group, followed by the CuNPc liposome group. This was consistent with the cell survival results characterized by the cytotoxicity experiment, once again proving that the CuNPc series liposomes combined with SDT have a good anti-glioma effect.

[0086] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A copper naphthalocyanine liposome having the effect of treating brain glioma, characterized in that: The copper naphthalocyanine and catalase are encapsulated in liposomes by a thin film dispersion method to obtain the copper naphthalocyanine liposomes.

2. The copper naphthalocyanine liposome according to claim 1, characterized in that The mass ratio of the copper naphthalocyanine, the catalase and the liposome is 2-2.5:1.5-2:30-40.

3. The copper naphthalocyanine liposome according to claim 1, characterized in that The components of the liposome include distearoyl phosphatidylcholine, polyethylene glycol monomethyl ether-2000-dioctadecylphosphatidylethanolamine and cholesterol.

4. The copper naphthalocyanine liposome according to claim 3, characterized in that The mass ratio of the distearoyl phosphatidylcholine, the polyethylene glycol monomethyl ether-2000-dioctadecyl phosphatidylethanolamine and the cholesterol is 25-30:10-15:4-5.

5. The method for constructing the copper naphthalocyanine liposome according to any one of claims 1 to 4, characterized in that: The steps include: The copper naphthalocyanine solution and the liposome solution are mixed, and a film is obtained by rotary evaporation. A catalase solution is added, and the mixture is ultrasonicated in a water bath and filtered to obtain the copper naphthalocyanine liposome.

6. The construction method according to claim 5, characterized in that: The temperature of the water bath ultrasound is 42-46° C.; the power of the water bath ultrasound is 300 W / h, the frequency is 40 KHz, and the time is 5 min.

7. The construction method according to claim 5, characterized in that: The filtration is performed by filtering through a 0.45 μm filter membrane and a 0.22 μm filter membrane in sequence.

8. The construction method according to claim 5, characterized in that: The solvent of the copper naphthalocyanine solution and the liposome solution is chloroform; the solvent of the catalase solution is phosphate buffer.

9. Use of the copper naphthalocyanine liposome according to any one of claims 1 to 4 or the copper naphthalocyanine liposome constructed by the construction method according to any one of claims 5 to 8 in the preparation of a drug for treating brain glioma.

10. A drug for treating brain glioma, characterized in that: The active ingredient of the drug is the copper naphthalocyanine liposome described in any one of claims 1 to 4 or the copper naphthalocyanine liposome constructed by the construction method described in any one of claims 5 to 8.

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