A method for preparing and applying copper porphyrin merocyanine nanoparticles switchable from photodynamic, photothermal effect to chemical dynamic effect

Abstract

The application discloses a preparation method and application of copper porphyrin merocyanine nanoparticles converted from photodynamic and photothermal effects to chemical dynamic effects. The copper porphyrin merocyanine molecule is prepared through a meso-substitution reaction of hydroxyl copper porphyrin and heptamethine merocyanine, and nanoparticles are formed through co-assembly with distearoyl phosphatidylcholine and distearoyl phosphatidyl ethanolamine-polyethylene glycol. Under light conditions, the copper porphyrin merocyanine nanoparticles have good photodynamic and photothermal effects. During the light process, the gradual degradation of merocyanine leads to the depolymerization of nanoparticles, and the loose structure promotes the opening of the chemical dynamic performance, so that the switching from photodynamic / photothermal effects to chemical dynamic effects is realized. The copper porphyrin merocyanine nanoparticles prepared by the application have good application prospects in the fields of biological tracing, efficient tumor killing and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical synthesis, molecular self-assembly and anti-cancer drugs, and particularly relates to a kind of copper porphyrin cyanine molecules from photodynamic and photothermal effect to chemical dynamic effect switchable synthesis, J aggregation nanoparticle preparation method and application in the preparation of intelligent anti-cancer drugs. BACKGROUND

[0002] Photosensitizers play an important role in cancer treatment, life safety and health protection. Photosensitizers can kill tumor cells through active oxygen species (ROS) generated by light, with high efficiency and high selectivity. However, among the generated ROS, singlet oxygen is easily limited by oxygen concentration, and hydroxyl radical and superoxide anion have short half-lives, making it difficult to completely kill tumor cells. The chemical dynamic therapy developed in 2016 does not depend on light time and oxygen concentration, and can continuously generate hydroxyl radicals through Fenton reaction between chemical dynamic reagent and hydrogen peroxide to kill tumor cells. Therefore, the combination of photosensitizers and chemical dynamic reagents can overcome their own defects and achieve a treatment effect of 'one plus one greater than two'.

[0003] The chemical dynamic process requires the participation of metal ions such as iron, copper, cobalt, manganese, etc., so there is a certain risk of heavy metal leakage. In addition, the separate effect of chemical dynamic reagent is poor, and the Fenton reaction rate is fast during treatment, and the generated hydroxyl radicals are easy to inactivate. Therefore, it is necessary to control the chemical dynamic effect before the light treatment takes effect, prolong the duration of the chemical dynamic effect, and ensure effective tumor cell killing. SUMMARY

[0004] The present application provides a preparation method and application of copper porphyrin cyanine nanoparticles that can switch from photodynamic and photothermal effect to chemical dynamics. The present application prepares copper porphyrin cyanine molecules through the meso-substitution reaction of hydroxyl copper porphyrin and heptamethine cyanine, and forms nanoparticles through co-assembly with distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). Due to the steric hindrance of copper porphyrin and the polarizable large conjugated structure of cyanine molecules, copper porphyrin can promote the dislocation arrangement of cyanine molecules, making copper porphyrin cyanine easily form J-aggregate structure in the hydrophobic layer of DSPC, effectively increasing the photodynamic performance. Under light conditions, copper porphyrin cyanine nanoparticles have good photodynamic and photothermal effect. During the light process, the degradation of cyanine leads to the depolymerization of nanoparticles, and the loose structure promotes the opening of the chemical dynamic performance, thereby realizing the switching from photodynamic / photothermal effect to chemical dynamic effect. The copper porphyrin cyanine nanoparticles prepared by the present application have near-infrared absorption and uniform nanosize, and the preparation method is simple and convenient, which has good application prospect in the fields of biological tracing, efficient tumor killing, etc.

[0005] The chemical structural formula of the copper porphyrin merocyanine molecule is:

[0006]

[0007] wherein R1=H, Br or CH3; R2=methyl, ethyl or propyl.

[0008] The preparation method of the copper porphyrin merocyanine nanoparticle is: the copper porphyrin merocyanine molecule is co-assembled with distearoyl phosphatidylcholine and distearoyl phosphatidyl ethanolamine-polyethylene glycol to form J aggregate nanoparticle.

[0009] The specific operation of the preparation method of the copper porphyrin merocyanine nanoparticle is: the methanol solution of the copper porphyrin merocyanine molecule is mixed with the methanol solution of DSPC and DSPE-PEG, the mixed solution is dried, and then a phosphate buffer solution is added to ultrasonically assemble at 60-80°C to obtain a J aggregate nanoparticle dispersion liquid, and the dispersion liquid is centrifuged to obtain the copper porphyrin merocyanine nanoparticle.

[0010] The molar ratio of the copper porphyrin merocyanine molecule, DSPC and DSPE-PEG is 1:5-40:0.17-2.67, preferably 1:10-20:0.33-1.33.

[0011] The concentration of the copper porphyrin merocyanine molecule in the solution formed by adding the phosphate buffer solution is 20-50 μM.

[0012] The synthesis method of the copper porphyrin merocyanine molecule is:

[0013] (1) 2-methyl indoline derivative and quaternary amination reagent with a molar ratio of 1:2-5 are dissolved in acetonitrile, and refluxed under nitrogen protection for 8-15 hours, and then precipitated with methyl tert-butyl ether or ethyl ether, washed and dried to obtain a quaternary aminated indoline derivative;

[0014] (2) The quaternary aminated indoline derivative and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-formaldehyde with a molar ratio of 1:2-2.5 are dissolved in a mixed solvent of toluene and n-butanol, and refluxed under nitrogen protection for 6-10 hours, and then the reaction liquid is cooled and filtered, and the obtained solid is washed with methyl tert-butyl ether or ethyl ether, and chromatographically purified to obtain a heptamethine merocyanine compound;

[0015] (3) The monohydroxy copper porphyrin is dissolved in N,N-dimethylformamide, and a catalyst triethylamine is added, and reacted at room temperature under nitrogen protection for 10-15 minutes, and then the heptamethine merocyanine compound is added, and the molar ratio of the heptamethine merocyanine compound to the monohydroxy copper porphyrin is 1:1-1.5, and reacted at 80-90°C under nitrogen protection for 4-6 hours, and then water and dichloromethane or trichloromethane are added to the reaction liquid for extraction, and the organic phase is rotary dried, and the obtained crude product is chromatographically purified to obtain the copper porphyrin merocyanine molecule.

[0016] The 2-methylindoline derivative is one or more of 2,3,3-trimethyl-3H-indole, 2,3,3,5-tetramethylindole, 2,3,3-trimethyl-5-bromo-3H-indole.

[0017] The quaternary amination reagent is one or more of methyl iodide, ethyl iodide, and propyl iodide.

[0018] The dispersion of the copper porphyrin chlorin nanoparticle prepared above is irradiated by a 750-850 nm laser, the photodynamic and photothermal effects are turned off, and the chemodynamic effect is turned on.

[0019] The copper porphyrin chlorin nanoparticle prepared above is applied in the preparation of an intelligent anticancer drug.

[0020] The copper porphyrin chlorin molecule prepared in the application has good aggregation performance, can be assembled to form regular J aggregates under the hydrophilic and hydrophobic interaction provided by DSPC, has more red-shifted absorption and emission. In addition, the chlorin J aggregation can improve the photodynamic performance of the molecule. Compared with the traditional chemical complexation means, the strategy of regulating the chemodynamic effect through molecular aggregation is simple in design and more widely applied. Molecular aggregation can limit the rate of Fenton reaction, and through light-triggered induction of depolymerization, the switching from photodynamic / photothermal effect to chemodynamic effect is realized, which can fully exert the advantages of various performances and improve the killing effect on tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a synthesis flow chart of the copper porphyrin chlorin molecule in the embodiment 1 of the application.

[0022] Figure 2 It is a nuclear magnetic resonance spectrum of the heptamethine chlorin compound molecule in the embodiment 1 of the application.

[0023] Figure 3 It is a nuclear magnetic resonance spectrum of the Cu-PCy molecule in the embodiment 1 of the application.

[0024] Figure 4 It is a transmission electron microscope image of (A) Cu-PCy JNPs and (B) disassembled body after light irradiation in the embodiment 2 of the application.

[0025] Figure 5 It is an absorption spectrum of Cu-PCy JNPs in PBS and Cu-PCy molecules in methanol in the embodiment 2 of the application.

[0026] Figure 6Figure for the change of absorbance value of water-soluble 1,3-diphenyl isobenzofuran at 412 nm under the photodynamic effect of Cu-PCy JNPs and Cu-PCy Self-Assemblies in Example 1 of the present application.

[0027] Figure 7 Figure for the photothermal heating effect of Cu-PCy JNPs at different concentrations in Example 2 of the present application.

[0028] Figure 8 Figure for the degradation ratio of methylene blue under the photodynamic effect of Cu-PCy JNPs before and after light irradiation in Example 3 of the present application.

[0029] Figure 9 Figure for the cell fluorescence imaging of dichlorofluorescein diacetate (DCFH-DA) under light irradiation and without light irradiation by Cu-PCy JNPs and the control group in Example 4 of the present application.

[0030] Figure 10 Figure for (A) dark toxicity and (B) cell killing effect under light irradiation of Cu-PCy JNPs in Example 5 of the present application. DETAILED DESCRIPTION

[0031] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content described in the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

[0032] Example 1

[0033] 2,3,3-trimethyl-5-bromo-3H-indole and iodomethane molecules were selected for quaternary amination reaction, and further synthesis of a cyanine molecule and a copper porphyrin cyanine molecule, and the preparation method is as follows:

[0034] (1) 0.5 g (2.1 mmol) of 2,3,3-trimethyl-5-bromo-3H-indole was dissolved in acetonitrile (10 mL), 0.7 g (4.2 mmol) of iodopropane was added, and the reaction was refluxed under nitrogen protection for 8 hours. After the mixture was cooled to room temperature, it was precipitated with methyl tert-butyl ether and washed and dried to obtain a propyl quaternary aminated bromoindole derivative.

[0035] (2) 480 mg (1.0 mmol) of propyl quaternary aminated bromoindole derivative and 86 mg (0.5 mmol) of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1- carboxaldehyde were dissolved in 10 mL of a mixed solvent of toluene and n-butanol (7 mL of toluene and 3 mL of n-butanol), and the mixture was refluxed for 8 hours under nitrogen atmosphere. After the mixture was cooled to room temperature, it was precipitated with methyl tert-butyl ether and washed and dried. The crude product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 30:1) to obtain a heptamethine cyanine compound (176 mg) as a green solid at a yield of 35%. The nuclear magnetic resonance hydrogen spectrum is shown in FIG. 2. Figure 2 .

[0036] (3) 50 mg (0.072 mmol) of monohydroxycopper porphyrin was dissolved in 3 mL of N,N-dimethylformamide, and after triethylamine (0.2 mL) was added thereto, the mixture was stirred at room temperature for 10 minutes, and then 50 mg (0.058 mmol) of the heptamethine cyanine compound was added thereto. The mixture was reacted at 85°C for 5 hours under nitrogen atmosphere. After the mixture was extracted with water and dichloromethane, N,N-dimethylformamide was removed, and then dichloromethane was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 20:1) to obtain 35 mg of a green solid copper porphyrin cyanine molecule (Cu-PCy) at a yield of 37%. The nuclear magnetic resonance hydrogen spectrum is shown in FIG. 3. Figure 3 .

[0037] Example 2

[0038] The Cu-PCy prepared in Example 1 was prepared as a methanol solution (1 mg / mL), and DSPC (10.5 mg) and DSPE-PEG2000 (1 mg) were prepared as a mixed solution of methanol (1 mL). 0.1 mL of each of the two methanol solutions was mixed, and after the solution was dried, 2 mL of a PBS buffer solution (pH 7.4) was added, and the solution was ultrasonically assembled at 60°C to obtain J-aggregated nanoparticle Cu-PCy JNPs.

[0039] Application Example 1

[0040] Cu-PCy methanol solution was dropped into water (water:methanol = 20:1) to prepare Cu-PCy self-assemblies without J-aggregation structure. A Cu-PCy self-assemblies solution and a PBS solution of Cu-PCy JNPs were each prepared at the same concentration, and a water-soluble 1,3-diphenyl isobenzofuran was added as a ROS indicator, and the absorbance values at different light irradiation times (808 nm, 10 mW / cm 2 ) were measured by a UV-visible spectrophotometer.

[0041] Application Example 2

[0042] PBS solutions (2 mL) of Cu-PCy JNPs with different concentrations were prepared and irradiated using an 808 nm laser (1.0 W / cm 2 ) and the temperature at different time points was recorded by an infrared camera. The photothermal conversion efficiency of Cu-PCy JNPs was calculated to be 32.6% by a photothermal calculation formula.

[0043] Application Example 3

[0044] A PBS solution (2 mL) of Cu-PCy JNPs was placed in a cuvette, glutathione (10 mM) was added, and the reaction was allowed to stand for 10 minutes. Then, methylene blue was added as a hydroxyl radical indicator, and hydrogen peroxide (10 mM) was added, and the methylene blue absorption value at different time points was recorded by a UV-Vis spectrophotometer. The Cu-PCy JNPs solution was mixed with glutathione, irradiated for 10 minutes using an 808 nm laser (1.0 W / cm 2 ), the same dose of hydrogen peroxide was added, and the methylene blue absorption value at different time points was recorded. The methylene blue degradation rate of the nanoparticles after irradiation was doubled, indicating that the kinetic process was turned on.

[0045] Application Example 4

[0046] Mouse breast cancer cells (4T1) were selected as a model. During the experiment, the 4T1 cells were in the logarithmic growth phase. Dichlorofluorescein diacetate (DCFH-DA) was used as a probe to detect intracellular ROS production. The medium containing Cu-PCy JNPs was co-cultured with cells for 4 hours, and irradiated using an 808 nm laser (1.0 W / cm 2 ) for 7 minutes. Then, after incubation with DCFH-DA-containing medium for 30 minutes, PBS was used for washing 3 times, and the fluorescence change was observed under a microscope.

[0047] Application Example 5

[0048] The medium containing Cu-PCy JNPs was co-cultured with 4T1 cells for 24 hours. The medium was replaced with a medium containing 10% CCK-8 and incubated for 0.5 to 2 hours. Then, the relative cell activity was detected by a microplate reader to test the cytotoxicity of Cu-PCy JNPs. Further, the medium containing Cu-PCy JNPs was co-cultured with cells in a 96-well plate for 4 hours, and then irradiated using an 808 nm laser (1.0 W / cm2) for 7 minutes. The same CCK-8 experiment was used to detect cell activity to test the tumor cell killing effect of Cu-PCy JNPs.

Claims

1. A copper porphyrin cyanine molecule, characterized in that: The chemical structural formula of the copper porphyrin cyanine molecule is: ， Wherein, R1=H, Br or CH3; R2=methyl, ethyl or propyl.

2. A method for preparing copper porphyrin cyanine nanoparticles, characterized in that: The preparation method comprises the following steps: co-assembling the copper porphyrin cyanine molecule according to claim 1 with distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol to form J aggregate nanoparticles.

3. A method for preparing copper porphyrin cyanine nanoparticles, characterized in that: The specific operation of the preparation method is: mixing the methanol solution of the copper porphyrin cyanine molecule according to claim 1 with the methanol solution of DSPC and DSPE-PEG, drying the mixed solution, adding phosphate buffer solution and ultrasonically assembling at 60-80°C to obtain a J-aggregated nanoparticle dispersion, and centrifuging the dispersion to obtain copper porphyrin cyanine nanoparticles.

4. The preparation method according to claim 3, characterized in that The molar ratio of the copper porphyrin cyanine molecule, DSPC, and DSPE-PEG is 1:5-40:0.17-2.

67.

5. The preparation method according to claim 3, characterized in that The concentration of the copper porphyrin cyanine molecules in the solution formed by adding the phosphate buffer solution is 20-50 μM.

6. The preparation method according to claim 3, characterized in that The synthesis method of the copper porphyrin cyanine molecule is: (1) dissolving a 2-methylindoline derivative and a quaternary ammonium reagent in acetonitrile at a molar ratio of 1:2-5, refluxing for 8-15 hours under nitrogen protection, precipitating with methyl tert-butyl ether or diethyl ether, washing, and drying to obtain a quaternary indoline derivative; (2) A quaternized indoline derivative and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde at a molar ratio of 1:2-2.5 are dissolved in a mixed solvent of toluene and n-butanol, and the mixture is refluxed for 6-10 hours under nitrogen protection. The reaction solution is cooled and filtered, and the resulting solid is washed with methyl tert-butyl ether or diethyl ether, and purified by chromatography to obtain a heptamethine cyanine compound; (3) Dissolve monohydroxy copper porphyrin in N,N-dimethylformamide, add catalyst triethylamine, react for 10-15 minutes at room temperature under nitrogen protection, then add heptamethine cyanine compound, the molar ratio of heptamethine cyanine compound to monohydroxy copper porphyrin is 1:1-1.5, react at 80-90℃ under nitrogen protection for 4-6 hours, add water and dichloromethane or chloroform to the reaction solution for extraction, take the organic phase and spin dry, and purify the crude product by chromatography to obtain copper porphyrin cyanine molecule; The 2-methylindoline derivative is one or more of 2,3,3-trimethyl-3H-indole, 2,3,3,5-tetramethylindole, and 2,3,3-trimethyl-5-bromo-3H-indole; The quaternizing agent is one or more of methyl iodide, ethyl iodide and propyl iodide.

7. The preparation method according to claim 3, characterized in that The dispersion of copper porphyrin cyanine nanoparticles is irradiated with a 750-850nm laser, the photodynamic and photothermal effects are turned off, and the chemical dynamics are turned on.

8. Use of copper porphyrin cyanine nanoparticles prepared according to the method according to any one of claims 3 to 6 in the preparation of intelligent anticancer drugs.

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