5-amino-ketoglutaric acid metal complex, liposome and preparation method thereof

Abstract

The present application relates to the field of pharmaceutical preparations, in particular to 5-amino-ketoglutaric acid metal complex, liposome and preparation method thereof. The 5-amino-ketoglutaric acid metal complex of the present application is formed by 5-amino-ketoglutaric acid and metal ion through coordination bond, and is a photosensitizer which can be used for photodynamic therapy of tumor. After ALA forms complex with metal ion, the reaction activity of amino and ketone groups in ALA molecule is reduced, the adverse influence of external environment on the molecule is delayed, and the stability is enhanced. Moreover, the co-aggregation of ALA and copper ion (or iron ion) to target tissue, the two means of ALA photodynamic therapy of tumor and copper (iron) ion promoting tumor cell apoptosis play drug efficacy, and have synergistic anti-tumor effect. The present application further provides liposome loaded with 5-amino-ketoglutaric acid metal complex, and preparation containing 5-amino-ketoglutaric acid metal complex or liposome.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical formulations, in particular to 5-aminolevulinic acid metal complexes, liposomes and methods for their preparation. BACKGROUND

[0002] 5-aminolevulinic acid (ALA), also known as 5-aminolevulinic acid, is a naturally occurring compound in the synthesis of hemoglobin. It is known for its role as a photosensitizer prodrug in photodynamic therapy (PDT). It can be converted into protoporphyrin IX (PpIX) with significant photosensitivity, which can generate singlet oxygen and other reactive oxygen species under specific wavelength light, triggering apoptosis and necrosis, providing a safe and economical method for treating tumor types such as basal cell carcinoma, glioma, bladder cancer, etc.

[0003] Although ALA has shown great therapeutic potential in tumor photodynamic therapy, the ALA molecule contains amino and keto functional groups, which are relatively active and can be degraded through oxidation, decarboxylation and polymerization, etc. Especially in the presence of oxygen, it can be oxidized to form the corresponding ketoxime or aldehyde compound. At the same time, due to the presence of a conjugated double bond system, ALA is sensitive to light and is prone to photochemical reactions such as photooxidation or photolysis under light, thereby reducing its stability.

[0004] Good stability of a drug is a prerequisite for its clinical application. The poor stability of ALA limits its application in tumor photodynamic therapy. Currently, the strategies to improve the stability of 5-aminolevulinic acid (ALA) mainly include the following: (1) The stability of ALA is best in an acidic environment with a pH value of 4 to 6, so adjusting the pH value can enhance its resistance to environmental changes; (2) Store ALA in the dark at 2 to 8℃ to slow down its degradation rate; (3) Use freeze-drying technology to remove water, which not only improves the stability of ALA, but also extends its shelf life; (4) Add antioxidants, metal chelating agents and cyclodextrin auxiliary ingredients, which can effectively prevent the degradation of ALA; (5) Encapsulate ALA in nano-carriers to provide a protective barrier, which protects it from external environmental factors, thereby significantly enhancing its stability.

[0005] The above measures mainly improve the stability of ALA by controlling pH, avoiding light, removing moisture and nano-wrapping, etc. These measures focus on controlling external factors affecting the stability of ALA, and do not fundamentally solve the problem of the stability of ALA. ALA contains amino and ketone groups that can provide electron pairs, and has the ability to form complexes with other substances. The formation of complexes helps to protect ALA molecules from the influence of external environment (such as temperature, oxygen, light), thereby reducing the degradation rate.

[0006] Copper is an essential trace element for human body, which acts as a cofactor in a variety of enzymes and plays a crucial role in maintaining copper ion homeostasis in cells. In tumor cells, this copper homeostasis is meticulously regulated by specific proteins such as STEAP, COX17, and SLC25A3, which are responsible for the transport and reduction of copper ions, ensuring normal cell function. Once the copper homeostasis is disrupted, copper ions can accumulate abnormally in cells, leading to metabolic chaos. This accumulation can trigger protein toxicity stress response by inhibiting the synthesis of iron-sulfur cluster proteins in the mitochondrial respiratory chain, activate specific cell death programs, and ultimately lead to cell death. Targeting excess copper ions to tumor cells to promote apoptosis has become an innovative strategy in the field of tumor treatment. Similarly, iron ions also participate in the killing process of tumor cells, which are involved in key physiological activities such as cell proliferation, metabolism, and differentiation. In recent years, the relationship between iron ions and tumor cells has attracted widespread attention, especially the iron-dependent programmed cell death—ferroptosis. This mode of death is related to the imbalance of intracellular reactive oxygen species (ROS) and lipid peroxidation, which is significantly different from traditional apoptosis. Ferroptosis plays an important role in the occurrence, development, and metastasis of tumors, and the in-depth study of its mechanism opens up new horizons and methods for cancer treatment.

[0007] Based on the above technical problems, the present application is proposed, which improves the stability of ALA by metal complexes and obtains synergistic anti-tumor effect. SUMMARY

[0008] To solve the above technical problems, the first aspect of the present application provides a 5-aminolevulinic acid metal complex formed by 5-aminolevulinic acid and metal ions through coordination bond, which is a photosensitizer and can be used for tumor photodynamic therapy. After ALA forms a complex with metal ions, the reactivity of amino and ketone groups in ALA molecules is reduced, the adverse effects of external environment (such as oxygen, light) on the molecules are delayed, and the stability is enhanced.

[0009] Further, the metal ion is selected from copper ion and / or iron ion. Copper ion is targeted to enrich in tumor cells, which can promote apoptosis; iron ion is involved in iron-dependent programmed cell death. Copper ion or iron ion complex can achieve the co-accumulation of ALA and copper ion (or iron ion) in target tissues, and the two means of ALA photodynamic therapy of tumor and copper (iron) ion promoting tumor cell apoptosis play drug efficacy, which has synergistic anti-tumor effect.

[0010] The present application also provides a preparation method of the above-mentioned 5-aminolevulinic acid metal complex, comprising the following steps:

[0011] (1) adding 5-aminolevulinic acid or its salt into an organic solution with acidic pH, dissolving and cooling to below 10℃ to obtain a 5-aminolevulinic acid solution;

[0012] (2) dissolving a metal salt in an organic solvent and adjusting to acidic pH, and then cooling to below 10℃ to obtain a metal salt solution;

[0013] (3) slowly adding the metal salt solution into the 5-aminolevulinic acid solution, uniformly mixing and keeping the reaction below 10℃ for 2-5h; after the reaction is completed, adding an organic solvent, centrifuging to precipitate, washing the precipitate and drying to obtain the 5-aminolevulinic acid metal complex.

[0014] Further, the 5-aminolevulinic acid or its salt is 5-aminolevulinic acid hydrochloride.

[0015] Further, the metal salt is selected from one or more of copper chloride, copper sulfate, copper acetate, copper nitrate, iron chloride, iron acetate, iron sulfate and iron nitrate, preferably copper chloride and / or iron chloride.

[0016] The present application also provides a liposome for photodynamic therapy, which comprises a blank liposome and the 5-aminolevulinic acid metal complex loaded in the blank liposome; the blank liposome comprises phospholipid molecules, cholesterol and octadecanol suberate; the mass ratio of the phospholipid molecules, cholesterol and octadecanol suberate is 10:0.1-1:0.5-2, preferably 10:0.1:1. The structural schematic diagram is shown in Figure 1 .

[0017] Further, the mass ratio of the blank liposome and the 5-aminolevulinic acid metal complex is 45-85:1, preferably 60:1.

[0018] The present application also provides a preparation method of the liposome, comprising the following steps:

[0019] (a) dissolving phospholipid molecules, cholesterol and octadecanol suberate in an organic solvent to obtain an oil phase for ice bath standby;

[0020] (b) dissolving 5-aminolevulinic acid metal complex in water, and preparing water phase in ice bath;

[0021] (c) adding water phase into oil phase under high speed stirring, and continuing to stir, and controlling system temperature below 10℃ in ice bath;

[0022] (d) after stirring, removing volatiles to obtain liposome suspension, and obtaining liposome by ultrasonic disruption.

[0023] The third aspect of the present application provides application of the 5-aminolevulinic acid metal complex or liposome in preparation of anti-tumor drugs.

[0024] Further, the tumor is esophageal cancer, gastric cancer, lung cancer, rectal cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, uterine cancer or endometrial cancer, etc.

[0025] The fourth aspect of the present application further provides a pharmaceutical composition for photodynamic therapy, comprising the 5-aminolevulinic acid metal complex or liposome, and pharmaceutically acceptable adjuvant.

[0026] Further, the adjuvant is selected from solvent, solubilizer, emulsifier, filler, stabilizer, buffer, plasticizer, thickener, etc.

[0027] The present application further provides a preparation for photodynamic therapy, comprising the pharmaceutical composition.

[0028] Further, the dosage form of the preparation is gel, emulsion, ointment, powder, etc.

[0029] Compared with the prior art, the present application has at least the following beneficial effects:

[0030] 1. Improved stability of ALA. After ALA forms a complex with copper ion compound and iron ion compound, the reaction activity of amino group and ketone group in ALA molecule is reduced, the adverse effects of external environment (such as oxygen, light) on the molecule are delayed, and the stability is enhanced.

[0031] 2. Synergistic killing of tumor cells, and better anti-tumor effect. ALA copper ion or iron ion complex can realize the co-aggregation of ALA and copper ion (or iron ion) to target tissue, and the two means of ALA photodynamic therapy of tumor and copper (iron) ion promoting tumor cell apoptosis play drug effects, and have synergistic anti-tumor effect.

[0032] 3. Loading ALA copper ion or iron ion complex into the inside of liposome. Since the liposome has good biocompatibility, small particle size, easy fusion with cells, good tissue permeability and targeting, it realizes high permeability of ALA and copper ion (or iron ion) to tumor tissue and targeting of tumor tissue. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of liposomes loaded with 5-aminolevulinic acid metal complex.

[0034] Figure 2 The results are the single-crystal analysis results of the ALA-CUCl2 complex.

[0035] Figure 3 The results are XRD analysis of ALA, CuCl2, and ALA-CUCl2 complex powders.

[0036] Figure 4 The results are high-resolution mass spectrometry values ​​of the ALA-CUCl2 complex in methanol.

[0037] Figure 5 The results of the 1H NMR spectrum of the ALA-CUCl2 complex after incubation in heavy water at 37°C for 0 and 7 days are shown.

[0038] Figure 6 The results show the cytotoxicity of different samples under dark conditions.

[0039] Figure 7 The results show the cytotoxicity of the samples after culturing for 3 hours and then irradiating them with light (630 nm, 5 min).

[0040] Figure 8 The results show the cytotoxicity after culturing the sample for 5 minutes and then irradiating it with light (630 nm, 5 min). Detailed Implementation

[0041] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the invention are merely illustrative examples of specific implementations of the invention and are intended to explain the invention, but do not constitute a limitation thereof.

[0042] The endpoints of the ranges and any values ​​disclosed herein are not limited to the exact ranges or values, which should be understood to include those close to them.

[0043] Example 1, Preparation of ALA-CUCl2 Complex

[0044] 5-Aminolevulinic acid hydrochloride (ALA-HCl) 100 mg, 50% ethanol 2 ml and glacial acetic acid 0.5 ml were placed in a 50 ml test tube, sonicated for 1 hour and gradually cooled to room temperature, and then to 0°C to obtain solution ①. 50 mg of copper acetate was dissolved in 2.5 ml of ethanol, and after adding 0.5 ml of 3M hydrochloric acid, it was sonicated for 1 hour and cooled to room temperature, and then cooled to 0°C to obtain solution ②. Solution ② was slowly added to the pre-cooled solution ①, and after mixing, it was sonicated for 3 hours, and the temperature was controlled by replacing the water in time. Then, 5 ml of tetrahydrofuran and 20 ml of ethyl acetate were added to the resulting mixture, stirred for 30 to 90 minutes, the precipitate was separated by centrifugation, and washed twice with 10 ml of ethyl acetate, and the precipitate was dried at room temperature to obtain the ALA-CUCl2 complex.

[0045] Example 2, Preparation of ALA-FeCl3 complex

[0046] 5-Aminolevulinic acid hydrochloride (ALA-HCl) 100 mg, 50% ethanol 2 ml and glacial acetic acid 0.5 ml were placed in a 50 ml test tube, sonicated for 1 hour and gradually cooled to room temperature, and then to 0°C to obtain solution ①. 50 mg of copper acetate was dissolved in 2.5 ml of ethanol, and after adding 0.5 ml of 3M hydrochloric acid, it was sonicated for 1 hour and cooled to room temperature, and then cooled to 0°C to obtain solution ②. Solution ② was slowly added to the pre-cooled solution ①, and after mixing, it was sonicated for 3 hours, and the temperature was controlled by replacing the water in time. Then, 5 ml of tetrahydrofuran and 20 ml of ethyl acetate were added to the resulting mixture, stirred for 30 to 90 minutes, the precipitate was separated by centrifugation, and washed twice with 10 ml of ethyl acetate, and the precipitate was dried at room temperature to obtain the ALA-CUCl2 complex.

[0047] Example 3, Preparation of ALA complex liposome

[0048] 1. Preparation of octadecyl alcohol azelate

[0049] 4. 9 g of octadecyl alcohol, 3.5 g of azelaic acid, 0.05 g of p-toluenesulfonic acid, and 50 ml of toluene were placed in a round-bottom flask, and the flask was connected to a reflux system equipped with a condenser. The reaction system was protected by nitrogen, and the reaction temperature was controlled at 110-120°C. The reaction was carried out for 3 hours with constant stirring using a magnetic stirrer, and water generated during the reaction was continuously removed through a water separator. After the reaction was completed, the mixture was cooled to room temperature, 50 ml of purified water was added, and the organic phase was separated by a separatory funnel. The organic phase was then dried over anhydrous magnesium sulfate, and the solvent was evaporated at 60°C using a rotary evaporator to obtain a crude product. The crude product was recrystallized to obtain octadecyl alcohol azelate.

[0050] 2. Preparation of ALA-CUCl2 complex liposome

[0051] Octadecanol azelate 0.1 g, phosphatidylcholine 1 g, cholesterol 0.1 g were dissolved in 20 ml dichloromethane and kept in ice bath for standby, to obtain oil phase. ALA-CUCl2 complex 20 mg was dissolved in 10 ml deionized water and kept in ice bath for standby, to obtain water phase. The water phase was added to the oil phase under high speed stirring at 1000 rpm, and the stirring was continued for 1 min after the addition was completed. The temperature of the system was controlled below 10 °C by ice bath during the process. After the stirring was completed, the temperature was gradually increased to 30 °C, and the dichloromethane was removed by magnetic stirring to obtain the initial suspension of liposomes. The initial suspension of liposomes was sonicated to obtain azelate-modified ALA-CUCl2 complex liposomes.

[0052] 3. Preparation of ALA-FeCl3 complex liposomes

[0053] Octadecanol azelate 0.1 g, phosphatidylcholine 1 g, cholesterol 0.1 g were dissolved in 20 ml dichloromethane and kept in ice bath for standby, to obtain oil phase. ALA-FeCl3 complex 20 mg was dissolved in 10 ml deionized water and kept in ice bath for standby, to obtain water phase. The water phase was added to the oil phase under high speed stirring at 1000 rpm, and the stirring was continued for 1 min after the addition was completed. The temperature of the system was controlled below 10 °C by ice bath during the process. After the stirring was completed, the temperature was gradually increased to 30 °C, and the dichloromethane was removed by magnetic stirring to obtain the initial suspension of liposomes. The initial suspension of liposomes was sonicated to obtain azelate-modified ALA-FeCl3 complex liposomes.

[0054] Example 4, Preparation of ALA, ALA-CUCl2 complex, ALA-CUCl2 gel dosage form

[0055] 1 g xanthan gum, 1 g konjac glucomannan were added to 100 ml deionized water to swell uniformly, and after high pressure sterilization at 121 °C for 30 minutes, the temperature was reduced to 37 °C to form a xanthan hydrogel. ALA, ALA-CUCl2 complex, ALA-CUCl2 complex liposomes were respectively diluted with appropriate amount of 1% methylcellulose gel to a gel agent containing ALA at a concentration of 2.5 μmol / mL.

[0056] Example 5, Performance evaluation of 5-aminolevulinic acid metal complex

[0057] 1. Structural characterization

[0058] The structure of the 5-aminolevulinic acid (ALA) copper chloride (CuCl2) complex of Example 1 was confirmed and characterized. Crystal structure analysis techniques revealed the presence of new coordination bonds in the ALA-CUCl2 complex, which provided direct evidence for understanding its unique molecular configuration and electronic distribution. By precisely measuring the atomic distances and angles, a three-dimensional structural model of the complex was constructed, revealing the precise coordination mode between the ALA molecule and the copper chloride ion (e.g., Figure 2(As shown). Powder X-ray diffraction (XRD) analysis further confirmed the crystal structure of the ALA-CUCl2 complex. The XRD pattern showed a series of characteristic diffraction peaks (such as...). Figure 3 As shown in the figure, the diffraction peaks are significantly different from those of pure ALA or CuCl2, indicating that the crystalline powder obtained in this patent has a novel structure. High-resolution mass spectrometry (HRMS) analysis was performed in methanol solvent, and no individual ALA molecular peaks were observed (e.g., as shown in the figure). Figure 4 As shown in the figure, this further confirms that ALA and copper chloride form a completely new structural compound, rather than a simple physical mixture.

[0059] 2. Stability of ALA-CUCl2 complex

[0060] 2.1 Content Changes:

[0061] Chromatographic conditions:

[0062] C 18 Column (150 mm × 4.6 mm, 5 μm); Mobile phase: Acetonitrile (A) - 20 mmol·L⁻¹ potassium dihydrogen phosphate (B) (pH 2.0, containing 2.0 mmol·L⁻¹ sodium octanesulfonate); Gradient elution program: 0–5 min 18% A, 5–10 min 18% A–55% A, 10–15 min 55% A, 15–20 min 55% A–18% A; Flow rate: 0.5 mL·min⁻¹; Detection wavelength: 205 nm; Column temperature: 25 °C; Injection volume: 20 μL.

[0063] Measurement method:

[0064] ALA and the ALA-CUCl2 complex were dissolved in pure water and incubated at 37°C for 10 days. The ALA content was measured at 0, 5, and 10 days. The results are shown in Table 1. Compared with ALA, the ALA-CUCl2 complex increased the stability of ALA.

[0065] Table 1. Stability test results

[0066]

[0067] 2.2 Proton NMR spectrum

[0068] ALA (copper chloride) was dissolved in heavy water, and its proton NMR spectrum was measured. The sample was then incubated at 37°C for 7 days before further testing. Results are shown below. Figure 5 The experimental results show that the ALA copper chloride complex has good stability in water at 37℃, and the chemical structure shift peak of ALA has not changed significantly, indicating that it has not decomposed or been oxidized.

[0069] Example 6, Pharmacodynamic Evaluation

[0070] 1. Tumor cell toxicity evaluation: The tumor cell toxicity was evaluated using the ALA-CuCl2 complex of Example 1 as an example.

[0071] Experimental method: 4T1 breast cancer tumor cells were selected to evaluate the cytotoxicity of ALA, CuCl2 and ALA-CUCl2 complex after co-culturing with cells. The cell culture medium was DMEM medium containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin double antibody (Gibco, USA), and the cells were cultured in a 37°C, 5% CO2 concentration incubator. The tumor cells were inoculated in a 96-well plate at a concentration of 1 x 104 per well, and after 24 hours of culture, the cells reached 80%-90% confluence. ALA, ALA-CUCl2 complex powder (Example 1), ALA-CUCl2 complex liposome (Example 2) were dissolved in DMEM respectively, CuCl2 was dissolved in sterile deionized water, and all were diluted with DMEM to prepare sample solutions of different concentrations (0.025, 0.25, 0.5, 1, 2.5, 5 and 10 μmol / mL). Different concentrations of sample solutions were added to the 96-well plate, each sample concentration was repeated 6 times, and cultured in the incubator for 24 hours. After the culture was completed, 100 μL of MTT solution was added to each well, and incubated in the dark for 4 hours. After incubation, the MTT solution was removed, 150 μL of DMSO was added to each well, and after shaking on a shaker for 15 minutes, the absorbance value of each well at 490 nm was determined by an enzyme marker. The cell survival rate calculation formula is as follows.

[0072]

[0073] Where As, Ab and Ac represent the absorbance values of the sample group, the background group and the control group respectively.

[0074] Figure 6 The results of the cell survival rate experiment in the dark (without light) for different concentrations of samples and tumor cells. According to the results, we can know that:

[0075] (1) After ALA was cultured with cells, without light, the sample did not produce toxicity to the cells;

[0076] (2) With the increase of Cu ion concentration, the cytotoxicity increased, and the cytotoxicity was positively correlated with the concentration of copper ions;

[0077] (3) The cytotoxicity of ALA-CUCl2 complex was consistent with that of Cu ions, indicating that ALA-CUCl2 complex showed consistent cytotoxicity with copper ions in the dark.

[0078] (4)ALA-CUCl2 liposome showed higher toxicity at low concentration, and the cytotoxicity was higher than that of ALA-CUCl2 and CUCl2 at the same concentration. It showed that ALA-CUCl2 liposome was easily endocytosed by cells, and more Cu ions could enter the cells to exert cytotoxicity.

[0079] Figure 7 The cell survival rate experiment results after different concentrations of samples were cultured with tumor cells for 3 hours, then irradiated by 630 nm light for 5 minutes, and then the sample solution was removed, fresh culture solution was added, and the cells were cultured for 24 hours.

[0080] According to the experimental results, we can know that:

[0081] (1) ALA was cultured with cells for 3 hours, and then irradiated, which had the highest killing rate of tumor cells of 80% (concentration 1 μmol / mL), indicating that ALA was converted into protoporphyrin IX in tumor cells, and protoporphyrin IX produced active oxygen under light, thereby producing toxicity to tumor cells. With the continuous increase of concentration, the cytotoxicity did not further increase, indicating that the amount of ALA entering the cells was limited;

[0082] (2) With the increase of Cu ion concentration, the cytotoxicity increased, and the cytotoxicity was positively correlated with the copper ion concentration, and the experimental results were basically the same as those without light, indicating that the death of cells caused by Cu ions was not related to light;

[0083] (3) The toxicity of low concentration of ALA-CUCl2 complex to tumor cells was low, but when the concentration of ALA-CUCl2 complex was higher than 0.5 μmol / mL, the killing rate of tumor cells reached 80%, and more importantly, when the concentration continued to increase to 1 μmol / mL, the killing rate of tumor cells reached more than 90%, indicating that the killing rate of tumor cells by ALA-CUCl2 complex was the result of the synergistic effect of copper ions and ALA.

[0084] (4) Compared with ALA-CUCl2 complex, ALA-CUCl2 liposome showed higher cytotoxicity at low concentration (0.025-0.25 μmol / mL), indicating that ALA-CUCl2 liposome helped cells to uptake more ALA-CUCl2, thereby producing higher cytotoxicity.

[0085] Figure 8 The cell survival rate experiment results after different concentrations of samples were cultured with tumor cells for 5 minutes, then irradiated by 630 nm light for 5 minutes, and then cultured for 24 hours.

[0086] According to the experimental results, we can know that:

[0087] (1) Since ALA needs to be converted into protoporphyrin IX inside tumor cells to exert photodynamic effect, the amount of ALA converted into protoporphyrin IX is small after short-time culture (5 minutes), and therefore the efficiency of killing tumor cells by ALA photodynamic is only about 50%, and even if the concentration is further increased, the cytotoxicity does not increase. These results show that the photodynamic effect of ALA is related to the culture time of cells, and the effect is low;

[0088] (2) The cytotoxicity of CUCl2 is positively correlated with the concentration, and is still independent of light;

[0089] (3) When the concentration of ALA-CUCl2 complex is higher than 0.5 μmol / mL, the chemical and photodynamic synergistic effect is exhibited, and when the concentration is 1 μmol / mL, the killing rate of tumor cells by ALA-CUCl2 complex reaches 99% (chemical and photodynamic synergistic effect), while the killing rate of tumor cells by copper ions is 83% (chemical toxicity), and the killing rate of tumor cells by ALA is 53% (photodynamic toxicity).

[0090] (4) Compared with ALA-CUCl2 complex, even if the culture time is short (5 minutes), ALA-CUCl2 liposome exhibits strong cytotoxicity at low concentration (0.025-0.25 μmol / mL), which again shows that ALA-CUCl2 liposome is more easily endocytosed by cells to exert the synergistic effect of ALA and CU ions.

[0091] By comparing the cytotoxicity of tumor cells with different concentrations of samples, culture time of tumor cells, and light, it is known that the cytotoxicity is positively correlated with the concentration of CU ions, and CUCl2 mainly kills tumor cells by chemical toxicity, and ALA exhibits cytotoxicity only under light, and ALA mainly kills tumor cells by producing reactive oxygen under light, and ALA-CUCl2 complex can produce the synergistic cytotoxic effect of ALA and CU ions under light. ALA-CUCl2 liposome has better cell uptake rate, and can produce better cytotoxic effect even at low concentration (0.025-0.25 μmol / mL) and short-time culture with cells, which shows that ALA-CUCl2 liposome has better advantage in exerting the synergistic cytotoxic effect of ALA and CU ions.

[0092] 2. Evaluation of tumor inhibition rate

[0093] The tumor inhibition rate was evaluated by taking the ALA-CuCl2 complex of Example 1 as an example.

[0094] 4T1 cells in logarithmic growth phase were selected, and were placed in RPMI-1640 medium without serum, and the concentration of the cell suspension was adjusted to 6×10^6 cells per milliliter. Then, 50 μL of the 4T1 breast cancer cell suspension was injected into the fat pad area under the fourth pair of mammary glands of female mice of the BALB / c strain. After one week of examination, 24 mice with tumors formed in the mammary fat pad area were selected to carry out the experiment.

[0095] The 24 tumor-bearing mice were randomly divided into four groups: the ALA gel group, the ALA-CuCl2 complex gel group, the ALA-CuCl2 complex liposome gel group, and the blank gel group, with 6 mice in each group. 0.1 milliliter of the corresponding gel was applied to the tumor site of the mice every day, and the dosage was 2.5 μmol / mL in terms of the ALA concentration. After 8 hours of administration at 1, 5, 10, 15, 20, 25, and 30 days, the tumors were treated with local irradiation using a semiconductor laser treatment instrument (output power 100 W, wavelength 630 nm, light density 120 J / cm 2 ) for 20 minutes each time. At 24 hours after the last treatment, the mice were sacrificed by cervical dislocation, and the tumor tissues were removed, washed with phosphate buffer solution (PBS), and weighed to calculate the tumor inhibition rate. The results are shown in Table 2.

[0096] Table 2. Tumor inhibition rate experimental results

[0097]

[0098] * represents statistical significance (t<0.05) compared with the blank gel group

[0099] As can be seen from the results, the order of the tumor inhibition rates of the groups is ALA-CuCl2 complex liposome gel group > ALA-CuCl2 complex gel group > ALA gel group, indicating that the ALA-CuCl2 gel group simultaneously plays the photodynamic role of ALA and the chemical toxicity role of CuCl2, which helps to synergistically inhibit tumor growth. The ALA-CuCl2 complex liposome improves the tissue permeability and cell uptake rate of the ALA-CuCl2 complex, and its tumor inhibition effect is the strongest.

[0100] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and do not constitute a limitation on the content of the present application. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A method for preparing a 5-aminolevulinic acid metal complex, characterized in that, Includes the following steps: 100 mg of 5-aminoketovalerate, 2 ml of 50% ethanol, and 0.5 ml of glacial acetic acid were placed in a 50 ml test tube, sonicated for 1 hour, and gradually cooled to room temperature, then cooled to 0°C to obtain solution ①; 50 mg of copper acetate was dissolved in 2.5 ml of ethanol, 0.5 ml of 3M hydrochloric acid was added, and the mixture was sonicated for 1 hour and cooled to room temperature, then cooled to 0°C to obtain solution ②; solution ② was slowly added to the pre-cooled solution ①, mixed thoroughly, and sonicated for 3 hours, changing the water as needed to control the temperature; then, 5 ml of tetrahydrofuran and 20 ml of ethyl acetate were added to the resulting mixture, stirred for 30 to 90 minutes, centrifuged to separate the precipitate, and washed twice with 10 ml of ethyl acetate. The precipitate was then dried at room temperature.

2. A 5-aminolevulinic acid metal complex, characterized in that, Prepared by the preparation method described in claim 1.

3. A liposome for photodynamic therapy, characterized in that, The liposomes comprise blank liposomes and a loaded 5-aminolevulinic acid metal complex as described in claim 2; the blank liposomes contain phospholipid molecules, cholesterol, and octadecanedioate; the mass ratio of the phospholipid molecules, cholesterol, and octadecanedioate is 10:0.1~1:0.5~2.

4. The liposomes as described in claim 3, characterized in that, The mass ratio of the phospholipid molecules, cholesterol, and octadecyl azelate is 10:0.1:

1.

5. The liposomes as described in claim 4, characterized in that, The mass ratio of the blank liposomes to the 5-aminolevulinic acid metal complex is 45~85:

1.

6. The liposomes as described in claim 5, characterized in that, The mass ratio of the blank liposomes to the 5-aminolevulinic acid metal complex was 60:

1.

7. The use of the 5-aminolevulinic acid metal complex of claim 2 or the liposomes of any one of claims 3-6 in the preparation of anti-breast cancer tumor drugs.

8. A pharmaceutical composition for photodynamic therapy, characterized in that, It includes the 5-aminolevulinic acid metal complex of claim 2 or the liposomes of any one of claims 3-6, and pharmaceutically acceptable excipients.

9. A formulation for photodynamic therapy, characterized in that, Includes the pharmaceutical composition according to claim 8.

10. The formulation for photodynamic therapy as described in claim 9, characterized in that, The dosage form of the preparation is a gel, emulsion, ointment, or powder.

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