I-type photosensitizer with near-infrared two-region photoresponsiveness as well as preparation method and application of I-type photosensitizer

Abstract

The invention discloses an I-type photosensitizer with near-infrared second-region photoresponsiveness and a preparation method and application thereof, and relates to the technical field of photosensitizers, the preparation method of the I-type photosensitizer comprises the following steps: (1) mixing melamine and potassium chloride, and calcining at high temperature to obtain a precursor; and (2) reacting the precursor with copper chloride to obtain the I-type photosensitizer. The I-type photosensitizer with near-infrared two-region photoresponsiveness prepared by the invention shows relatively good biocompatibility and relatively low dark toxicity, and can efficiently realize photodynamic therapy so as to more efficiently induce tumor cell death.

Description

Technical Field

[0001] The present invention relates to the technical field of photosensitizers, and in particular to a type I photosensitizer with near-infrared second-region light responsiveness, and a preparation method and application thereof. Background Art

[0002] Malignant tumors pose a significant challenge to global public health, and traditional treatments face significant bottlenecks. While widely used in clinical practice, conventional approaches such as surgical resection, radiotherapy, and chemotherapy often suffer from limitations such as poor selectivity, significant systemic toxicity, and poor adaptability to the tumor microenvironment.

[0003] Photodynamic therapy (PDT) has shown unique value in precision tumor treatment due to its precise spatiotemporal selectivity, controllable toxicity and side effects, and synergistic properties. However, most of the existing clinical photosensitizers follow the type II photodynamic mechanism, which generates 1 The oxygen-dependent nature of O2 limits the efficacy of this therapy in the hypoxic microenvironment commonly found in solid tumors. In contrast, type I photodynamic reactions directly generate O2 through electron transfer. - , ·OH and other reactive oxygen species, whose low oxygen adaptability and strong oxidizing properties provide a new idea for breaking through the bottleneck of PDT efficacy.

[0004] Current development of type I photosensitizers focuses primarily on metal-organic frameworks, transition metal complexes, and inorganic nanocomposite systems. Within this context, graphitic carbon nitride (GCN) has attracted significant attention due to its unique non-metallic properties, excellent photostability, and favorable biosafety profile. As a two-dimensional multiphoton absorbing material, GCN exhibits a typical large π-conjugated structure. Despite its approximately 2.7 eV bandgap, it can generate various reactive oxygen species upon near-infrared excitation, demonstrating significant potential for use in type I PDT applications. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a type I photosensitizer with near-infrared second-region light responsiveness, thereby obtaining graphite-phase carbon nitride nanoparticles loaded with potassium and copper elements, which can realize multiphoton absorption process under near-infrared light excitation, reach a high-energy excited state and generate O2 through type I electron transfer pathway. - , inducing tumor cell death.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] The first object of the present invention is to provide a method for preparing a type I photosensitizer having near-infrared second region light responsiveness, comprising the following steps:

[0008] (1) mixing melamine and potassium chloride and calcining at high temperature to obtain a precursor;

[0009] (2) The precursor is reacted with copper chloride to obtain a type I photosensitizer.

[0010] In the present invention, melamine is used as a carbon and nitrogen source, and graphite phase carbon nitride (g-C3N4) is formed after high-temperature calcination. At the same time, potassium ions are loaded on g-C3N4 using a potassium source. The potassium ions are inserted into the interlayer of g-C3N4, coordinated with nitrogen atoms, and change the interlayer distance and charge distribution. Subsequently, copper ions are loaded on g-C3N4 through the reaction of a precursor with copper chloride, and the copper ions replace part of the potassium ions and coordinate with nitrogen atoms. Finally, the molar ratio of potassium and copper is close to 1:1, forming a potassium-copper co-modified structure.

[0011] Furthermore, the mass ratio of melamine to potassium chloride is 1: 1. By controlling the mass ratio of melamine to potassium chloride, not only can the loading amount of potassium ions on g-C3N4 be guaranteed, but also the utilization rate of carbon, nitrogen and potassium atoms can be improved.

[0012] Furthermore, the high-temperature calcination is carried out in a crucible with a lid, preferably an alumina crucible with a lid. The crucible with a lid can maintain the temperature, prevent dust and impurities from entering, and improve safety.

[0013] Furthermore, the high-temperature calcination temperature is 550-650° C., the calcination time is 3-5 hours, and the heating rate is 5-25° C. / min. Melamine and potassium chloride react during the high-temperature calcination process to obtain potassium-doped g-C 3 N 4 .

[0014] Furthermore, the mass ratio of the precursor to copper chloride is 20: 3. By controlling the mass ratio of the precursor to copper chloride, not only can the loading amount of potassium and copper on g-C3N4 be controlled, but also the utilization rate of copper atoms can be improved.

[0015] Furthermore, the reaction temperature of the precursor and copper chloride is 15-30° C. The reaction of the precursor and copper chloride at room temperature can reduce energy consumption.

[0016] The second object of the present invention is to provide a type I photosensitizer having near-infrared second region light responsiveness prepared by the aforementioned preparation method.

[0017] Furthermore, the type I photosensitizer is in the form of nanoparticles with a size of 100 to 200 nm. The type I photosensitizer powder obtained by reacting the precursor with copper chloride can be converted into type I photosensitizer nanoparticles by ultrasound and filtration.

[0018] The third object of the present invention is to provide the use of the aforementioned type I photosensitizer having near-infrared second region light responsiveness in the preparation of near-infrared excitation type tumor therapeutic drugs.

[0019] The fourth object of the present invention is to provide the use of the aforementioned type I photosensitizer with near-infrared second-region light responsiveness in the preparation of a tumor photodynamic-chemotherapy combined therapy agent.

[0020] The beneficial effects of the present invention are as follows: the photosensitizer preparation method provided by the present invention has the characteristics of readily available raw materials, low cost, and easy operation, and the prepared type I photosensitizer with near-infrared second-region light responsiveness shows good biocompatibility and low dark toxicity, and can efficiently achieve photodynamic therapy, thereby more efficiently inducing tumor cell death. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 X-ray diffraction patterns of M-gCN-Cu and M-gCN prepared in Example 1 and gCN-Cu and gCN prepared in Comparative Example 1;

[0022] Figure 2 X-ray photoelectron spectra of M-gCN-Cu and M-gCN prepared in Example 1 and gCN-Cu and gCN prepared in Comparative Example 1;

[0023] Figure 3 Band gap calculations for M-gCN-Cu and M-gCN prepared in Example 1 and gCN-Cu and gCN prepared in Comparative Example 1;

[0024] Figure 4 Mott-Schottky plots of M-gCN-Cu and M-gCN prepared in Example 1 and gCN-Cu and gCN prepared in Comparative Example 1 at different frequencies;

[0025] Figure 5 The energy band structures of M-gCN-Cu and M-gCN prepared in Example 1 and gCN-Cu and gCN prepared in Comparative Example 1;

[0026] Figure 6 Electron paramagnetic resonance spectra of M-gCN-Cu and M-gCN prepared in Example 1 and gCN-Cu and gCN prepared in Comparative Example 1;

[0027] Figure 7 This is a scanning electron micrograph of the M-gCN-Cu NPs prepared in Example 1;

[0028] Figure 8 This is the uptake of M-gCN-Cu NPs prepared in Example 1 by HepG2 cells;

[0029] Figure 9The MTT method was used to determine the dark toxicity (a) and phototoxicity (b) of the M-gCN-Cu NPs prepared in Example 1 on HepG2 cells and 293T cells. DETAILED DESCRIPTION

[0030] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments and illustrations.

[0031] Example 1

[0032] (1) 10 g of melamine and 10 g of potassium chloride were ground in an agate mortar, mixed thoroughly, and transferred into a 50 mL alumina crucible with a lid. The mixture was then heated to 600 °C at a heating rate of 20 °C / min and calcined for 4 h. After cooling naturally to room temperature, the mixture was ground into powder, washed with deionized water and anhydrous ethanol in sequence, centrifuged at 10,000 r / min for 2 min, and dried in an oven at 60 °C for 10 h to obtain the precursor (M-gCN).

[0033] (2) 100 mg of M-gCN prepared in step (1) was dispersed in 100 mL of deionized water, and 15 mg of anhydrous copper chloride was added. The mixture was stirred at 20°C for 10 h, and then centrifuged at a speed of 10,000 r / min for 5 min. The resulting solid was washed with deionized water and anhydrous ethanol in sequence, and then centrifuged at a speed of 10,000 r / min for 2 min. It was dried in an oven at 60°C for 10 h to obtain a powdered type I photosensitizer (M-gCN-Cu).

[0034] (3) 50 mg of the M-gCN-Cu prepared in step (2) was dispersed in 100 mL of deionized water, ultrasonically treated at 20°C for 10 h, the ultrasonic frequency was 40 kHz, the ultrasonic power was 240 W, and the mixture was centrifuged at a speed of 10,000 r / min for 5 min. The supernatant collected through a 0.22 μm microporous filter membrane was centrifuged at a speed of 12,000 r / min for 15 min. The resulting solid was dried in an oven at 60°C for 10 h to obtain type I photosensitizer nanoparticles (M-gCN-Cu NPs).

[0035] Comparative Example 1

[0036] The method of Example 1 was followed, except that potassium chloride was not added, and gCN was prepared by step (1) and gCN-Cu was prepared by step (2).

[0037] The structures of the M-gCN-Cu and M-gCN prepared in Example 1 and the gCN-Cu and gCN samples prepared in Comparative Example 1 were analyzed by X-ray diffractometer. Figure 1 As shown. Figure 1It can be seen that the (002) peaks of M-gCN-Cu and M-gCN have shifted significantly. This indicates that the introduction of potassium has led to changes in the extended in-plane structure and interlayer π-π stacking of M-gCN, but the introduction of copper has not had a significant effect on the crystal structure. The M-gCN-Cu and M-gCN prepared in Example 1 and the gCN-Cu and gCN prepared in Control Example 1 were analyzed by photoelectron spectrometer. The results are shown in Figure 2. Figure 2 and Table 1.

[0038] Table 1

[0039]

[0040]

[0041] from Figure 2 As can be seen from Table 1, the M-gCN-Cu prepared in Example 1 contains both potassium and copper elements, accounting for 2.33% and 2.51% respectively, which is close to 1:1; while the copper element in the gCN-Cu prepared in Control Example 1 accounts for only 0.30%, indicating that the addition of potassium chloride will also affect the loading amount of copper on g-C3N4.

[0042] UV-visible diffuse reflectance spectroscopy (UV-visible diffuse reflectance spectroscopy) was performed on the M-gCN-Cu and M-gCN prepared in Example 1 and the gCN-Cu and gCN samples prepared in Comparative Example 1. Figure 3 ) and electrochemical performance ( Figure 4 ) test, and the band structures of these samples were obtained, such as Figure 5 As shown. Figure 5 It can be seen that the conduction band positions of all samples can thermodynamically reduce O2 to O2 - .

[0043] The electrochemical test was performed using an electrochemical workstation CHI 760E (Shanghai Chenhua Instrument Co., Ltd.) based on a standard three-electrode system. 2 mg of sample was thoroughly mixed with 200 μL of Nafion solution and then evenly coated on a tin-indium oxide (ITO) electrode. The ITO electrode coated with the sample (size: 20 mm × 10 mm × 1.1 mm, coating area: 10 mm × 10 mm) was used as the working electrode, platinum wire as the counter electrode, silver / silver chloride as the reference electrode, and a sodium sulfate solution with a concentration of 0.5 mol / L as the electrolyte. Mott-Schottky measurements were performed at 1000 Hz, 2000 Hz, and 3000 Hz, respectively, to obtain the results. Figure 4 .

[0044] Example 2

[0045] M-gCN-Cu NPs were prepared according to the method of Example 1, except that the high-temperature calcination conditions were changed to 550°C, calcination time was 5 h, and heating rate was 10°C / min.

[0046] Example 3

[0047] M-gCN-Cu NPs were prepared according to the method of Example 1, except that the high-temperature calcination conditions were changed to 650°C, calcination time was 3 h, and heating rate was 15°C / min.

[0048] The generation of active oxygen species was detected for the M-gCN-Cu and M-gCN prepared in Example 1 and the gCN-Cu and gCN samples prepared in Comparative Example 1:

[0049] 1 O2 detection:

[0050] Using deionized water as the solvent, the M-gCN and M-gCN-Cu prepared in Example 1 and the gCN and gCN-Cu samples prepared in Control Example 1 were respectively prepared into a dispersion with a concentration of 1 mg / mL, and then 10 μL of a 100 mmol / L 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) aqueous solution was added. The mixture was irradiated with visible light of a 300 W xenon lamp for 30 seconds, and then 40 μL of the resulting mixture was injected into a quartz capillary tube and immediately subjected to electron spin resonance (ESR) spectroscopy analysis. The results are shown in FIG. Figure 6 (a).

[0051] O2 - Detection:

[0052] Using methanol as solvent, the M-gCN and M-gCN-Cu prepared in Example 1 and the gCN and gCN-Cu samples prepared in Control Example 1 were respectively prepared into a dispersion with a concentration of 1 mg / mL, and then 10 μL of a 100 mmol / L 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) aqueous solution was added. The mixture was irradiated with visible light of a 300 W xenon lamp for 30 seconds, and then 40 μL of the resulting mixture was injected into a quartz capillary and immediately subjected to ESR spectroscopy analysis. The results are shown in FIG. Figure 6 (b).

[0053] from Figure 6 It can be seen that M-gCN-Cu is better than M-gCN. 1 The generation of O2 was significantly inhibited, and O2 - The generation capacity of ROS was significantly enhanced, which revealed that the ROS generation pathway of M-gCN-Cu was dominated by the type II mechanism (mainly producing 1O2) into electron transfer-dominated type I mechanism (mainly producing O2 - ).

[0054] The M-gCN and M-gCN-Cu prepared in Example 1 and the gCN and gCN-Cu samples prepared in Comparative Example 1 were all calcined at high temperature, making it difficult to obtain nano-scale materials. However, M-gCN-Cu NPs were obtained by the ultrasonic and filtration operations in Example 1, and their morphology was characterized by scanning electron microscopy, as shown in FIG. Figure 7 As shown. Figure 7 It can be seen that the size of M-gCN-CuNPs is about 100-200 nm. Materials in this particle size range have the potential to enter cells through the cell membrane and achieve further therapeutic processes.

[0055] Cellular uptake of M-gCN-Cu NPs:

[0056] HepG2 cells were inoculated into laser confocal microscopy culture dishes and cultured for 48 h. After the cells were completely attached, 200 μL of the aqueous solution of M-gCN-Cu NPs prepared in Example 1 (concentration of 100 μg / mL) was added and incubated for 12 h in the dark. The cells were then gently washed three times with PBS solution. The uptake of M-gCN-Cu NPs by HepG2 cells was observed using a laser confocal microscope, and fluorescence images were collected. The results are shown in FIG. Figure 8 .from Figure 8 It can be seen that M-gCN-Cu NPs were effectively absorbed by HepG2 cells and simultaneously produced fluorescence.

[0057] Cytotoxicity test (MTT method):

[0058] HepG2 cells and 293T cells were seeded in 96-well plates and cultured until the cell monolayers were confluent and adherent. 2 ) conditions, a concentration gradient culture of 0, 25, 50, 100, 150, and 200 μg / mL was performed, with three parallel groups for each concentration. The cells were cultured in a 5% CO2, 37°C incubator for 10 h. After the incubation, 10 μL of MTT reagent was added to each well, and the cells were cultured for another 4 h. After the incubation, 150 μL of dimethyl sulfoxide was added to each well, and the cells were shaken until the crystals were completely dissolved. The absorbance of the solution in each well was measured at 490 nm using a microplate reader, and the survival rate of HepG2 cells was calculated based on the absorbance value. The results are shown in FIG. Figure 9 .

[0059] Depend on Figure 9It can be seen that at a concentration of up to 200μg / mL, M-gCN-Cu NPs also showed extremely low cytotoxicity, and cell viability remained above 90%. After 5 minutes of 1300nm laser irradiation, the cytotoxicity of M-gCN-Cu NPs on HepG2 cells showed a concentration-dependent manner, and cell viability decreased significantly with the increase of M-gCN-Cu NPs concentration. This shows that although M-gCN-Cu NPs themselves have good biocompatibility, laser irradiation can induce their cytotoxicity, and the degree of toxicity is closely related to concentration.

[0060] In summary, the present invention focuses on the field of photodynamic therapy for hypoxic tumor microenvironment. Melamine, potassium chloride and copper chloride are used as raw materials to synthesize g-C3N4 substrate and load potassium and copper elements to prepare a photosensitive material with near-infrared second region and the ability to generate O2 - The photosensitizer is a type I photosensitizer. Nanoparticles of the photosensitizer were obtained by ultrasound and filtration, and were effectively taken up by human liver cancer cells. Under 1300nm (near infrared region II) laser excitation, the photosensitizer can generate a large amount of type I reactive oxygen species·O2 - , inducing tumor cell apoptosis, thereby achieving photodynamic therapy.

[0061] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a type I photosensitizer having near-infrared second region light responsiveness, characterized in that: The following steps are involved: (1) melamine and potassium chloride are mixed and calcined at high temperature to obtain a precursor; (2) The precursor is reacted with copper chloride to obtain a type I photosensitizer.

2. The preparation method according to claim 1, wherein: The mass ratio of the melamine to potassium chloride is 1:

1.

3. The preparation method according to claim 1, wherein: The high-temperature calcination has a calcination temperature of 550-650° C., a calcination time of 3-5 hours, and a heating rate of 5-25° C. / min.

4. The preparation method according to claim 1, wherein: The high-temperature calcination is carried out in a crucible with a lid.

5. The preparation method according to claim 1, wherein: The mass ratio of the precursor to copper chloride is 20:

3.

6. The preparation method according to claim 1, wherein: The reaction temperature of the precursor and copper chloride is 15-30°C.

7. A type I photosensitizer having near-infrared second region light responsiveness prepared by the preparation method according to any one of claims 1 to 6.

8. The type I photosensitizer having near-infrared second region light responsiveness according to claim 7, characterized in that: The type I photosensitizer is a nanoparticle with a size of 100 to 200 nm.

9. Use of the type I photosensitizer with near-infrared second region light responsiveness according to claim 7 or 8 in the preparation of near-infrared excitation-type tumor therapeutic drugs.

10. Use of the type I photosensitizer with near-infrared second region light responsiveness according to claim 7 or 8 in the preparation of a tumor photodynamic-chemotherapy combined therapy agent.

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