An organic photosensitive nanomaterial, its preparation method and application

Abstract

The present invention belongs to the field of biomedical technology, and discloses an organic photosensitive nanomaterial, a preparation method thereof and an application thereof. The organic photosensitive nanomaterial of the present invention has a core-shell structure; the shell structure includes an amphiphilic polymer, and the core structure includes an organic conjugate and an electron sacrificial agent; the preparation method of the organic photosensitive nanomaterial of the present invention includes the following steps: self-assembling the organic conjugate, the electron sacrificial agent and the amphiphilic polymer. Due to its unique structural design and excellent GSH consumption performance, the organic photosensitive nanomaterial of the present invention can consume endogenous GSH in tumor cells, reduce its loss of ROS, and further enhance the oxidative stress in tumor cells; and can jointly regulate the level of GSH in tumor cells and increase the utilization rate of ROS in hypoxic tumors, and is used as a drug regimen for realizing GSH consumption to promote the treatment effect of hypoxic tumors in PDT.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an organic photosensitive nanomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Tumor is a chronic and non-communicable disease with a relatively high mortality rate in the world. Traditional therapies such as chemotherapy, radiotherapy, and surgical treatment are used in the clinical treatment of tumors. However, the disadvantages of traditional therapies, such as poor selectivity, low efficacy, and large toxic and side effects, limit their wider clinical application and treatment effects. As a novel treatment method, a highly spatiotemporally selective specific site-directed treatment strategy - photodynamic therapy (PDT) has attracted extensive attention in the field of tumor treatment. PDT is that a photosensitizer interacts with molecular oxygen under the irradiation of light with a specific wavelength to generate reactive oxygen species (ROS) with strong oxidizing properties, which further destroys various biomolecules in cells, such as proteins, DNA, and cell membranes, triggering apoptosis or necrosis of cells. At the same time, research shows that PDT does have a significant killing effect on solid tumors and has been officially approved by relevant government departments in many countries and has become a conventional means of tumor treatment. So far, this therapy has been successfully used to treat a number of malignant tumors.

[0003] However, due to the rapid growth of tumor cells resulting in insufficient blood supply, the oxygen content in tumor tissues is lower than that in normal tissues. This hypoxic environment is not conducive to the generation of ROS in PDT, and the high oxygen consumption of PDT will further exacerbate the hypoxia of tumor cells, thus limiting the treatment effect of PDT. Although many new methods have been proposed in the prior art to reverse the tumor hypoxia problem, whether it is to provide additional oxygen supply for tumors or to "throttle" oxygen, the treatment effect of PDT will be enhanced to a certain extent. However, on the one hand, these methods increase the complexity of treatment, and on the other hand, they cannot fundamentally solve the problem of the dependence of PDT on O2. Type I PDT has a lower oxygen dependence, but the overexpression of glutathione (GSH) in the tumor cell microenvironment is another key factor restricting its efficacy. GSH can directly scavenge ROS as a reducing agent, thereby reducing the treatment efficiency of ROS.

[0004] Currently, inorganic nanomaterials are used as photosensitizers to directly interact with GSH through their glutathione peroxidase-like activity to reduce the GSH level in tumor cells. However, the biosafety of inorganic nanomaterials remains the main factor restricting their development. Compared with inorganic semiconductor materials, organic semiconductor materials have the advantages of flexible preparation, adjustable properties, good biosafety, and reproducibility. These properties make them suitable for type I PDT, but due to the chemical inertness of organic semiconductor materials, it is difficult to eliminate the negative effect of GSH on PDT. Therefore, there is an urgent need to develop a safe and efficient organic photosensitive nanomaterial that can be used for hypoxic tumor photodynamic therapy and consume GSH during light irradiation. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide an organic photosensitive nanomaterial, its preparation method and application.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides an organic photosensitive nanomaterial, and the organic photosensitive nanomaterial has a core-shell structure; the shell structure includes an amphiphilic polymer, and the core structure includes an organic conjugate and an electron sacrificial agent.

[0008] In the organic photosensitive nanomaterial of the present invention, the organic conjugate can absorb light energy to generate electron-hole pairs under light irradiation, and these electron-hole pairs are then separated to form photo-generated electrons (e - ) and photo-generated holes (h + ). At the same time, the electron sacrificial agent consumes photo-generated e - during light irradiation, enhances the photo-oxidation of reducing substances in tumor cells by photo-generated h + , and further cooperates with the organic conjugate to promote the diffusion and accumulation of ROS in tumor cells, improve the killing effect of ROS on tumor cells, and significantly enhance the effect of photodynamic therapy. Especially under hypoxic conditions, the nanomaterial of the present invention still generates highly efficient ROS, which mainly benefits from its unique structural design, making the separation and utilization of photo-generated e - and h + more efficient. Even in the tumor microenvironment with low oxygen content, the nanomaterial of the present invention can inhibit the overexpression of GSH and continuously generate ROS through the synergistic effect of the electron sacrificial agent and the organic conjugate in the nuclear material, realizing the effective treatment of hypoxic tumors.

[0009] The inhibition rate of the organic photosensitive nanomaterial of the present invention on the survival of tumor cells under hypoxic conditions has reached 80%, which can significantly reduce the volume and weight of tumor cells in living organisms, and even has no obvious toxic and side effects on living organisms while making the tumor completely disappear.

[0010] As a preferred embodiment of the organic photosensitive nanomaterial of the present invention, the organic conjugate is an organic conjugate small molecule with type I photodynamic performance.

[0011] Preferably, the organic conjugate is the organic conjugate small molecule Y6.

[0012] In the organic photosensitive nanomaterial of the present invention, the organic conjugate has an A-D-A molecular structure composed of a fused ladder core with one electron-donating (D) group and two electron-accepting (A) end groups. This structure not only enhances the electron push-pull effect between the donor (D) and the acceptor (A), making it easier for electrons to transfer between the donor and the acceptor, improving the separation efficiency of photo-generated electrons and photo-generated holes; but also facilitates the delocalization of electrons along the molecular backbone, resulting in a decrease in the molecular band gap and strong absorption in the near-infrared region. At the same time, when used in combination with an electron sacrificial agent, the organic photosensitive nanomaterial can reduce the loss of GSH for ROS in photodynamic therapy and increase the utilization efficiency of ROS, thereby achieving an efficient type I photodynamic therapy effect.

[0013] As a preferred embodiment of the organic photosensitive nanomaterial of the present invention, the electron sacrificial agent is at least one of benzophenone, benzoin dimethyl ether, and 2-ethylhexyl-4-trimethoxycinnamate; preferably, the electron sacrificial agent is benzophenone.

[0014] The organic photosensitive nanomaterial of the present invention selects a simple-structured electron sacrificial agent and an organic conjugate for combined use, which can reduce the loss of GSH for ROS and increase the utilization efficiency of ROS, thereby achieving an efficient type I photodynamic therapy effect.

[0015] As a preferred embodiment of the organic photosensitive nanomaterial of the present invention, the mass ratio of the organic conjugate to the electron sacrificial agent is 1:(0.5 - 5).

[0016] Preferably, the mass ratio of the organic conjugate to the electron sacrificial agent is 1:1.

[0017] The present invention has found through a large number of experiments that when the mass ratio of the organic conjugate to the electron sacrificial agent is 1:1, the organic photosensitive nanomaterial has better ability to consume GSH and inhibit hypoxic tumors.

[0018] As a preferred embodiment of the organic photosensitive nanomaterial of the present invention, the amphiphilic polymer is at least one of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), poloxamer F127 (Pluronic F127), bovine serum albumin BSA, and polystyrene 5000-b-polyethylene glycol 5000 (PS5000-b-PEG5000).

[0019] Preferably, the amphiphilic polymer is distearoyl phosphatidylethanolamine - polyethylene glycol 2000 (DSPE - PEG2000).

[0020] As a preferred embodiment of the organic photosensitive nanomaterial of the present invention, the average particle size of the organic photosensitive nanomaterial is 134 nm - 152 nm; the excitation wavelength of the organic photosensitive nanomaterial is 650 nm - 900 nm.

[0021] In a second aspect, the present invention provides an application of the organic photosensitive nanomaterial in preparing a photosensitizer for photodynamic therapy.

[0022] The organic photosensitive nanomaterial of the present invention can be used to prepare a photosensitizer for use in photodynamic therapy to reduce the loss of GSH for ROS and increase the utilization efficiency of ROS, thereby achieving an efficient type I photodynamic therapy effect, especially suitable for preparing a photosensitizer for use in photodynamic therapy to promote the treatment effect of hypoxic tumors.

[0023] In a third aspect, the present invention provides a preparation method of the organic photosensitive nanomaterial, comprising the following steps: self - assembling an organic conjugate, an electron sacrificial agent and an amphiphilic polymer, and that's it.

[0024] The preparation method of the organic photosensitive nanomaterial of the present invention prepares an organic photosensitive nanomaterial with reduced loss of GSH for ROS and increased utilization efficiency of ROS to achieve an efficient type I photodynamic therapy effect by introducing a specific electron sacrificial agent, and can be used to promote the treatment effect of hypoxic tumors in photodynamic therapy.

[0025] As a preferred embodiment of the preparation method of the organic photosensitive nanomaterial of the present invention, the mass ratio of the organic conjugate to the amphiphilic polymer is 1:(10 - 50).

[0026] Preferably, the mass ratio of the organic conjugate to the amphiphilic polymer is 1:20.

[0027] As a preferred embodiment of the preparation method of the organic photosensitive nanomaterial of the present invention, it comprises the following steps: (1) dissolving the amphiphilic polymer, the electron sacrificial agent and the organic conjugate in a good solvent to obtain a mixed solution; (2) adding the mixed solution in step (1) to a poor solvent and ultrasonicating, and that's it.

[0028] Preferably, the volume ratio of the good solvent to the poor solvent is 1:(3 - 10).

[0029] More preferably, the volume ratio of the good solvent to the poor solvent is 1:5.

[0030] Preferably, the ultrasonic power is 500W - 1000W, and the ultrasonic time is 0.5h - 3h.

[0031] More preferably, the ultrasonic power is 600W, and the ultrasonic time is 0.5h.

[0032] Preferably, after the ultrasonic step is completed, it further includes a step of dialyzing the ultrasonicated solution in water to obtain an organic photosensitive nano-aqueous solution.

[0033] More preferably, the molecular weight cut-off value of the dialysis bag used in the dialysis step is 5000 - 15000Da.

[0034] Even more preferably, the molecular weight cut-off value of the dialysis bag used in the dialysis step is 14000Da.

[0035] More preferably, the dialysis time is 10h - 48h.

[0036] More preferably, after the dialysis step is completed, it further includes a step of ultrafiltering the organic photosensitive nano-aqueous solution to obtain organic photosensitive nanoparticles.

[0037] Even more preferably, the molecular weight cut-off value of the ultrafiltration membrane used in the ultrafiltration step is 10000Da.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows: First, the organic photosensitive nanomaterials of the present invention can consume endogenous GSH in tumor cells, reduce its loss of ROS, and further enhance the oxidative stress in tumor cells; Second, the organic photosensitive nanomaterials of the present invention jointly regulate the level of GSH in tumor cells and increase the utilization rate of ROS in hypoxic tumors, overcoming the defect of poor single treatment effect in the prior art and promoting the clinical transformation of the drug use plan for GSH consumption in PDT; Finally, the preparation method of the organic photosensitive nanomaterials of the present invention has mild conditions, simple preparation steps, and is easy to repeat operations and quantitative production. Description of the Drawings

[0039] Figure 1 It is a (a) reaction flow schematic diagram; (b) 1H NMR spectrum; (c) 13C NMR spectrum of the organic conjugated small molecule Y6 of the present invention;

[0040] Figure 2 It is the absorption spectrum of the organic conjugated small molecule Y6 of the present invention in CHCl3 solution and thin film;

[0041] Figure 3 It is the preparation flow chart of the organic photosensitive nanomaterials in Example 1 of the present invention;

[0042] Figure 4 It is the absorption spectrum of the organic photosensitive nanomaterials in Verification Example 1 of the present invention;

[0043] Figure 5 This is the particle size diagram of the organic photosensitive nanomaterial in Verification Example 1 of the present invention;

[0044] Figure 6 This is the absorption spectrum diagram of the organic photosensitive nanomaterial in Verification Example 2 of the present invention; (a)-(b) Y6 NPs; (c)-(d) YB NPs;

[0045] Figure 7 This is the electron paramagnetic resonance spectrum diagram of the organic photosensitive nanomaterial in Verification Example 2 of the present invention;

[0046] Figure 8 This is the GSH consumption capacity diagram of the organic photosensitive nanomaterial in Verification Example 2 of the present invention: (a) Y6 NPs under aerobic conditions; (b) Y6 NPs under anaerobic conditions; (c) YB NPs under aerobic conditions; (d) YB NPs under anaerobic conditions;

[0047] Figure 9 This is the diagram of the effect of the MTT method for detecting the organic photosensitive nanomaterial on cell viability in Verification Example 3 of the present invention;

[0048] Figure 10 This is the diagram of the effect of the Calcein AM / PI probe for detecting the organic photosensitive nanomaterial on cell viability in Verification Example 3 of the present invention;

[0049] Figure 11 This is the diagram of the consumption of intracellular GSH by the organic photosensitive nanomaterial detected by the kit in Verification Example 3 of the present invention;

[0050] Figure 12 This is the effect diagram of detecting the photodynamic therapy of the organic photosensitive nanomaterial on living mice in Verification Example 4 of the present invention: (a) Diagram of the growth of mouse tumor volume; (b) Physical picture of mouse tumor; (c) Diagram of mouse tumor weight; (d) Diagram of the change in mouse body weight. Detailed implementation manners

[0051] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0052] The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents, equipment, etc. used are all commercially available unless otherwise specified.

[0053] The raw materials used in the following examples and comparative examples are described as follows, but are not limited to these materials:

[0054] TPBT-CHO, CAS No.: 2304444-53-7, purchased from Shenzhen Ruixun Optoelectronic Materials Technology Co., Ltd.;

[0055] 2FIC, CAS No.: 2083617-82-5, purchased from Shenzhen Ruixun Optoelectronic Materials Technology Co., Ltd.;

[0056] DSPE-PEG2000, purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd.;

[0057] Benzophenone (BP), purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0058] The preparation method of the organic conjugated small molecule Y6 described in the following examples and comparative examples: Under N2 atmosphere, 123 mg (0.12 mmol) of TPBT-CHO, 110 mg (0.3 mmol) of 2FIC and 0.5 mL of pyridine were added to 20 mL of CHCl3, and the reaction was carried out at room temperature. The reaction progress of TPBT-CHO was monitored by thin layer chromatography (TLC) until it completely disappeared, that is, the reaction ended. After removing the solvent in the reaction system, the crude product was purified by column chromatography (silica gel) with a mixed solvent of petroleum ether and dichloromethane (volume ratio 1:1), and finally 113 mg of brownish-black solid Y6 (yield 65%) was obtained. The composition of the product was determined by nuclear magnetic resonance spectroscopy.

[0059] Schematic diagram of the reaction process of the organic conjugated small molecule Y6 and 1 1H-NMR and 13 13C-NMR are as Figure 1 shown.

[0060] As Figure 2 shown, the absorbance of the solid film or chloroform solution of the synthesized organic conjugated small molecule Y6 was measured using ultraviolet-visible spectrophotometry.

[0061] Example 1: Preparation of organic photosensitive nanomaterials YB NPs

[0062] As Figure 3As shown, 10 mg of DSPE-PEG2000 was dissolved in 10 mL of tetrahydrofuran (THF). Then, 0.5 mg of the above-synthesized organic conjugated small molecule Y6 and 0.5 mg of benzophenone (BP) were dissolved in 10 mL of tetrahydrofuran (THF) and uniformly mixed with the tetrahydrofuran solution of DSPE-PEG2000. Subsequently, the mixed solution and 2 mL of CHCl3 were quickly added to 100 mL of ultrapure water, and ultrasonic treatment (600 W, 40 KHz) was carried out for 30 min. Then, THF and CHCl3 were removed using a rotary evaporator. The solution was transferred to a dialysis bag (MWCO = 14000) and dialyzed against deionized water for 24 h. The deionized water in the nanoparticles was removed by ultrafiltration (MWCO = 10000) to obtain YB NPs particles, which were stored at 4 °C.

[0063] Example 2: Preparation of organic photosensitive nanomaterial YB NPs

[0064] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs described in this example and that in Example 1 is that the mass ratio of the organic conjugated small molecule Y6, benzophenone (BP), and DSPE-PEG2000 is 1:2:20.

[0065] Example 3: Preparation of organic photosensitive nanomaterial YB NPs

[0066] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs described in this example and that in Example 1 is that the mass ratio of the organic conjugated small molecule Y6, benzophenone (BP), and DSPE-PEG2000 is 1:4:20.

[0067] Example 4: Preparation of organic photosensitive nanomaterial YB NPs

[0068] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs described in this example and that in Example 1 is that the mass ratio of the organic conjugated small molecule Y6, benzophenone (BP), and DSPE-PEG2000 is 1:0.5:10.

[0069] Example 5: Preparation of organic photosensitive nanomaterial YB NPs

[0070] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs described in this example and that in Example 1 is that the mass ratio of the organic conjugated small molecule Y6, benzophenone (BP), and DSPE-PEG2000 is 1:5:50.

[0071] Example 6: Preparation of organic photosensitive nanomaterial YB NPs

[0072] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs in this example and that in Example 1 is that the volume ratio of the good solvent to the poor solvent is 1:3.

[0073] Example 7: Preparation of organic photosensitive nanomaterial YB NPs

[0074] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs in this example and that in Example 1 is that the volume ratio of the good solvent to the poor solvent is 1:10.

[0075] Example 8: Preparation of organic photosensitive nanomaterial YB NPs

[0076] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs in this example and that in Example 1 is that the ultrasonic power is 500 W.

[0077] Example 9: Preparation of organic photosensitive nanomaterial YB NPs

[0078] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs in this example and that in Example 1 is that the ultrasonic power is 1000 W.

[0079] Comparative Example 1: Preparation of organic photosensitive nanomaterial Y6 NPs

[0080] Dissolve 1 mg of the above-prepared Y6 and 10 mg of DSPE-PEG2000 in 10 mL of tetrahydrofuran (THF) respectively and mix them evenly. Then quickly add this solution and 2 mL of CHCl3 to 100 mL of ultrapure water, and ultrasonically treat (600 W, 40 KHz) for 30 min. Then remove THF and CHCl3 with a rotary evaporator. Transfer the solution to a dialysis bag (MWCO = 14000) and dialyze it against deionized water for 24 h. Then remove the deionized water in the nanoparticles by ultrafiltration (MWCO = 10000) to obtain Y6 NPs particles and store them at 4 °C.

[0081] Comparative Example 2: Preparation of organic photosensitive nanomaterial YB NPs

[0082] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs in this comparative example and that in Example 1 is that the mass ratio of the organic conjugated small molecule Y6 to benzophenone (BP) is 1:0.2.

[0083] Comparative Example 3: Preparation of organic photosensitive nanomaterial YB NPs

[0084] The only difference between the preparation method of the organic photosensitive nanomaterial YB NPs in this comparative example and that in Example 1 is that the mass ratio of the organic conjugated small molecule Y6 to benzophenone (BP) is 1:8.

[0085] Verification Example 1: Morphological Study of Organic Photosensitive Nanomaterials

[0086] As Figure 4 shown, the light absorption intensities of the organic photosensitive nanomaterials of Examples 1-3 and Comparative Example 1 at different wavelengths were measured using ultraviolet-visible spectrophotometry. It can be seen from the figure that Y6 and BP in Examples 1-3 have been successfully assembled into nanoparticles, indicating the successful synthesis of the nanoparticle YB NPs.

[0087] As Figure 5 shown, the particle sizes of the organic photosensitive nanomaterials of Example 1 and Comparative Example 1 were measured.

[0088] Verification Example 2: Photodynamic Performance Study of Organic Photosensitive Nanomaterials

[0089] This verification example verified the photodynamic performance of the organic photosensitive nanomaterials prepared in Example 1 and Comparative Example 1 at the solution level.

[0090] (1) Hydroxyl radical (·OH) detection: o-Phenylenediamine (OPD) was used as an indicator to examine the ability of the organic photosensitive materials Y6 NPs and YB NPs to generate ·OH at the solution level, and to judge their type I photodynamic performance.

[0091] The nanoparticles of Example 1 and Comparative Example 1 were respectively dissolved in deionized water to make their concentrations 100 μg / mL. 3 mL was taken and mixed evenly with 30 μL of a DMF solution of OPD (100 mM). Then, the mixture was irradiated with an 808 nm (0.3 W / cm 2 ) laser for different times (0 min, 3 min, 6 min, 9 min, 12 min). The absorbance change of the solution at 520 nm was detected using an ultraviolet / visible spectrophotometer (Shimadzu UV-3600plus, Japan) to verify the type I photodynamic performance of the organic photosensitive nanomaterials.

[0092] From Figure 6 it can be seen that under normoxic conditions, the organic photosensitive nanomaterials Y6 NPs and YB NPs can generate ·OH to oxidize OPD, and its oxidation product has a characteristic absorption peak around 420 nm. And from Figure 6 -a and 6-c, it can be seen that the ability of the two to generate hydroxyl radicals is not much different, indicating that the introduction of BP has little effect on the photodynamic performance of Y6 under normoxic conditions.

[0093] (2) EPR Test: Qualitatively detect ·OH through electron paramagnetic resonance spectrometer (ESR / EPR), and use the specific ROS scavenger DMPO to detect the generated ·OH in the reaction system. After mixing the nanomaterials of Example 1 and Comparative Example 1 at 75 μg / mL with the DMPO scavenger at 25 mM respectively, irradiate with 808 nm (0.3 W / cm 2 ) laser for 10 min, and then detect the generation of ·OH with an electron paramagnetic resonance spectrometer.

[0094] It can be seen from Figure 7 that characteristic signals can be detected for the organic photosensitive nanomaterials under the action of the specific ROS scavenger, indicating that under 808 nm laser irradiation, the organic photosensitive nanomaterials Y6 NPs and YB NPs can generate ·OH, further demonstrating their type I photodynamic performance.

[0095] Verification Example 3: Study on the glutathione (GSH) consumption ability of organic photosensitive nanomaterials

[0096] This verification example verifies the glutathione (GSH) consumption ability of the organic photosensitive nanomaterials prepared in Example 1 and Comparative Example 1 at the solution level, mainly verifying the unique oxidation ability of the organic photosensitive nanomaterial YB NPs for glutathione (GSH) under anaerobic conditions.

[0097] Glutathione (GSH) consumption detection: Use 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as an indicator probe to detect the consumption of glutathione by the organic photosensitive nanomaterials under light.

[0098] (1) Normoxia: Mix 1 mg of the nanomaterial samples of Example 1 and Comparative Example 1 and GSH (60 μL, 10 mM) in PBS solution (3.5 mL, pH = 6.5) respectively to obtain a mixture, and then irradiate the mixture with 808 nm (0.3 W / cm 2 ) laser for different times (0 min, 5 min, 10 min, 15 min, 20 min) to simulate the consumption process of GSH by the nanomaterials under light. Then filter the nanomaterials through an ultrafiltration tube, take out 2.5 ml of the filtrate and mix it with 400 μL of PBS (pH = 6.5) solution of DTNB (0.3 mg / mL), and then measure the absorbance of the mixture with a UV-visible-near-infrared spectrometer.

[0099] (2) Anaerobic: To detect the consumption of glutathione in a low-oxygen environment, load the mixture into a vial and perform three vacuum nitrogen operations to strictly remove oxygen. Subsequently, the light treatment, filtration, reaction with DTNB, and absorbance measurement operations are the same as those in the normoxic environment.

[0100] It can be seen from Figure 8-a and 8-c show that under aerobic conditions, both organic photosensitive nanomaterials Y6 NPs and YB NPs exhibit excellent GSH consumption ability. This is mainly because under aerobic conditions, oxygen acts as an electron acceptor, promoting the generation of ROS by organic photosensitive nanomaterials under light irradiation. These ROS have strong oxidizing properties and can quickly react with GSH, resulting in the consumption of GSH. And from Figure 8 -b, it can be seen that under anaerobic conditions, Y6 NPs do not show the ability to consume GSH because the lack of oxygen as an electron acceptor prevents Y6 NPs from effectively generating ROS; and from Figure 8 -d, it can be seen that due to the presence of the electron sacrificial agent BP in YB NPs, the photogenerated electrons produced by them can be quickly consumed, and the remaining photogenerated holes can oxidize GSH, leading to the consumption of GSH, thus showing a unique GSH consumption ability. Verification Example 3: Study on the promoting effect of organic photosensitive nanomaterials on the photodynamic therapy of tumor cells

[0101] This verification example verifies the promoting effect of the organic photosensitive nanomaterials prepared in Example 1 and Comparative Example 1 on tumor photodynamic therapy at the cellular level, mainly by reflecting the cell survival situation through the MTT method and the live / dead cell staining method, and detecting the GSH consumption in cells through a kit.

[0102] The organic photosensitive nanomaterials prepared in Example 1 and Comparative Example 1 are directly dissolved in the complete medium to make their concentration in the complete medium 50 μg / mL.

[0103] Experimental steps:

[0104] (1) Detection of cell viability by the MTT method: CT26 cells (from the American Type Culture Collection (ATCC)) are inoculated into 96-well plates, a total of six groups, with five replicates in each group, and 6×10 3 cells in each well. After inoculation, they are placed in a sterile incubator (Thermo Fisher) (under normoxic conditions, the incubator is set at 37 °C, 21% O2, 5% CO2; under hypoxic conditions, the incubator is set at 37 °C, 1% O2, 5% CO2) and incubated overnight; after the cells adhere and reach a density of 70%, they are treated with the samples grouped as follows:

[0105] Normoxic conditions:

[0106] (i) Control: Incubate the cells with the complete medium without any drugs for 7 h and do not perform any treatment;

[0107] (ii) Y6 NPs (50 μg / mL): Incubate the cells with the complete medium containing 50 μg / mL of Y6 NPs for 7 h;

[0108] (iii) YB NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of YB NPs for 7 h;

[0109] (iv) Y6 NPs (50 μg / mL) + NIR (44.5 °C, 10 min): After incubating the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h, irradiate the cells with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min;

[0110] (v) YB NPs (50 μg / mL): After incubating the cells with complete medium containing 50 μg / mL of YB NPs for 7 h, irradiate the cells with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min;

[0111] Hypoxic condition:

[0112] (vi) Control: Incubate the cells with complete medium without any drugs for 7 h and do not perform any treatment;

[0113] (vii) Y6 NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h;

[0114] (viii) YB NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of YB NPs for 7 h;

[0115] (ix) Y6 NPs (50 μg / mL) + NIR (44.5 °C, 10 min): After incubating the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h, irradiate the cells with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min;

[0116] (x) YB NPs (Y6: 50 μg / mL, BP: 50 μg / mL) After incubating the cells with complete medium containing 50 μg / mL of YB NPs for 7 h, irradiate the cells with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min.

[0117] After the treatment, the 96-well plate was placed back into the sterile incubator (under normoxic conditions, the incubator was set at 37 °C, 21% O2, 5% CO2; under hypoxic conditions, the incubator was set at 37 °C, 1% O2, 5% CO2) and incubated for 17 h; an MTT solution at 5 mg / mL was prepared and pre-cooled at 4 °C, then 10 μL of the MTT solution was added to each well, gently shaken evenly, and then continued to be incubated in the sterile incubator (under normoxic conditions, the incubator was set at 37 °C, 21% O2, 5% CO2; under hypoxic conditions, the incubator was set at 37 °C, 1% O2, 5% CO2) for 4 h. After incubation, the supernatant was aspirated, 150 μL of DMSO was added to each well, shaken on a shaker in the dark for 15 min, and the absorbance values of the cells in each group at 490 nm were detected by a microplate reader (BioTek), and the cell survival rate was calculated.

[0118] Survival rate = absorbance value of each experimental group / absorbance value of the Control group × 100%.

[0119] From Figure 9 it can be seen that regardless of normoxic or hypoxic conditions, only using the 808 nm laser has no killing effect on tumor cells, while under 808 nm (0.3 W / cm 2 ) laser irradiation, the photosensitive material YB NPs has excellent killing effect on tumor cells. Especially for tumor cells under hypoxic conditions, after being treated with the photosensitive material YB NPs, their cell survival rate has dropped to 20%, which further proves the effective promotion of the photodynamic therapy effect of the organic photosensitive material of the present invention on hypoxic tumors.

[0120] (2) Live / dead cell staining detection: The cells were stained with Calcein AM / PI probe, and the fluorescence was observed under a Carl Zeiss inverted fluorescence microscope (Carl Zeiss AG).

[0121] The experimental steps are as follows: CT26 cells were seeded in confocal dishes, 5×10 4 cells per dish, and cultured in a sterile incubator (under normoxic conditions, the incubator was set at 37 °C, 21% O2, 5% CO2; under hypoxic conditions, the incubator was set at 37 °C, 1% O2, 5% CO2). After the cell density reached about 80%, the following grouped samples were used to treat them:

[0122] Normoxic conditions:

[0123] (i) Y6 NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h;

[0124] (ii) YB NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of YB NPs for 7 h;

[0125] (iii) Y6 NPs (50 μg / mL) + laser: After incubating the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min of irradiation time;

[0126] (iv) YB NPs (50 μg / mL) + laser: After incubating the cells with complete medium containing 50 μg / mL of YB NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min of irradiation time;

[0127] Hypoxic condition:

[0128] (v) Y6 NPs (50 μg / mL) + laser (44.5 °C, 10 min): After incubating the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min of irradiation time;

[0129] (vi) YB NPs (50 μg / mL) + laser (44.5 °C, 10 min): After incubating the cells with complete medium containing 50 μg / mL of YB NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min of irradiation time.

[0130] After the treatment, aspirate the supernatant, gently rinse once with PBS buffer to remove the excess complete medium and drug reagents, then add the prepared Calcein AM / PI solution and incubate in the dark for 1 h, and finally perform fluorescence imaging using a Zeiss inverted fluorescence microscope (Carl Zeiss AG).

[0131] It can be seen from Figure 10 that the live cells of tumor cells show green fluorescence and the dead cells show red fluorescence after different treatments. Thus, it can be seen that due to the combined use of the electron sacrificial agent BP and the photosensitizing material Y6 in the organic photosensitive nanomaterials of the present invention, the photodynamic therapy effect on hypoxic tumor cells can be effectively improved.

[0132] (3) Detection of intracellular GSH consumption: Seed CT26 cells at 3×10 per well 5Inoculate cells at a certain density into the well plate and place it in a sterile incubator for incubation (under normoxic conditions, the incubator is set at 37°C, 21% O2, 5% CO2; under hypoxic conditions, the incubator is set at 37°C, 1% O2, 5% CO2). After the cell density reaches about 80%, treat them with the samples in the following groups:

[0133] Normoxic conditions:

[0134] (i) Control: Incubate the cells with complete medium without any drugs for 7 h;

[0135] (ii) Y6 NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h;

[0136] (iii) YB NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of YB NPs for 7 h;

[0137] (iv) Y6 NPs (50 μg / mL) + laser: After incubating the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5°C, and the irradiation time is 10 min;

[0138] (v) YB NPs (50 μg / mL) + laser: After incubating the cells with complete medium containing 50 μg / mL of YB NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5°C, and the irradiation time is 10 min;

[0139] Hypoxic conditions:

[0140] (vi) Control: Incubate the cells with complete medium without any drugs for 7 h;

[0141] (vii) Y6 NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h;

[0142] (viii) YB NPs (50 μg / mL): Incubate the cells with complete medium containing 50 μg / mL of YB NPs for 7 h;

[0143] (ix) Y6 NPs (50 μg / mL) + laser: After incubating the cells with complete medium containing 50 μg / mL of Y6 NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min of irradiation time;

[0144] (x) YB NPs (50 μg / mL) + laser: After incubating the cells with complete medium containing 50 μg / mL of YB NPs for 7 h, irradiate them with an 808 nm laser (0.3 W / cm 2 ) to raise the average cell temperature to about 44.5 °C for 10 min of irradiation time.

[0145] After the treatment, aspirate the supernatant, gently rinse the cells once with PBS buffer to remove the excess complete medium and drug reagents, and then treat them with a GSH and GSSG detection kit (Beyotime, catalog number: S0053).

[0146] From Figure 11 the content of GSH in tumor cells CT26 after different treatment methods, it can be seen that YB NPs can also significantly consume the GSH in tumor cells in a lower oxygen environment, indicating that the organic photosensitizing nanomaterials of the present invention can increase the consumption of GSH during the photodynamic process, which is crucial for promoting the photodynamic therapy effect of hypoxic tumors.

[0147] Verification Example 4: Study on the Promoting Effect of Photosensitizing Nanomaterials on Photodynamic Therapy of Tumors in Living Mice

[0148] In this example, the promoting effect of the organic photosensitizing nanomaterials prepared in Example 1 and Comparative Example 1 on the photodynamic therapy of hypoxic tumors was verified at the in vivo level, mainly by reflecting the tumor inhibition situation through in vivo mouse experiments.

[0149] Dissolve the organic photosensitizing nanomaterials of Example 1 and Comparative Example 1 in PBS solution to make their concentration 2 mg / mL.

[0150] Use SPF-grade female Balb / c mice (4 weeks old, body weight about 20 g) purchased from Guangdong Medicilon Inc. The experimental unit's use license number is SYXK (Guangdong) 2021 - 0168. All animal studies were conducted in accordance with the guidelines of the "Regulations for the Administration of Laboratory Animals".

[0151] Experimental procedure: Subcutaneously inject CT26 tumor cells resuspended in PBS buffer into the back of mice at a dose of 100 μL / mouse (2 × 10 6 cells) to establish a mouse transplanted tumor model. When the tumor grows to about 100 mm 3When (tumor volume V = length × width 2 / 2), the mice were randomly divided into 6 groups (5 mice in each group), and the following grouping treatments were repeated on the 3rd and 5th days respectively:

[0152] (i) Control: Inject PBS solution (50 μL / mouse) into the tumor of the mice;

[0153] (ii) Control + NIR: Inject PBS solution (50 μL / mouse) into the tumor of the mice, and perform 808 nm (0.3 W / cm 2 ) laser irradiation for 10 min after 8 h;

[0154] (iii) Y6 NPs: Inject PBS buffer containing nanomaterial Y6 NPs (Y6 NPs concentration 2 mg / mL, 50 μL / mouse) into the tumor of the mice;

[0155] (iv) YB NPs: Inject PBS buffer containing nanomaterial YB NPs (YB BP concentration 2 mg / mL, 50 μL / mouse) into the tumor of the mice;

[0156] (v) Y6 NPs + NIR: Inject PBS buffer containing nanomaterial Y6 NPs (Y6 NPs concentration 2 mg / mL, 50 μL / mouse) into the tumor of the mice, and perform 808 nm laser irradiation (44.5 °C, 10 min) after 8 h

[0157] (vi) YB NPs + NIR: Inject PBS buffer containing nanomaterial YB NPs (YB NPs concentration 2 mg / mL, 50 μL / mouse) into the tumor of the mice, and perform 808 nm (0.3 W / cm 2 ) laser irradiation (44.5 °C, 10 min) after 8 h

[0158] The above treatments were repeated on the 3rd and 5th days respectively, and then the tumor volume and body weight of the mice were measured every other day to evaluate the treatment effects of each group; on the 14th day, the mice in each group were euthanized, and the tumors were dissected, weighed and photographed after removal.

[0159] From Figure 12 -a and 12-b, it can be seen that under 808 nm (0.3 W / cm 2 ) laser irradiation, the organic photosensitive nanomaterial YB NPs of the present invention has excellent inhibitory effect on tumors. After 14 days, the volume and weight of tumor cells are significantly reduced, and even the tumors completely disappear; while from Figure 12 -d, it can be seen that the body weight of the mice shows a stable trend as a whole within 14 days, indicating that the organic photosensitive nanomaterial YB NPs of the present invention and 808 nm (0.3 W / cm 2)The irradiation of the laser has no obvious toxic or side effects on mice.

[0160] From the above experiments, it can be seen that the organic photosensitive nanomaterial YB NPs of the present invention overcomes the defect of poor single treatment effect in the prior art, and this medication scheme that realizes the unique ability to consume GSH and generate ROS is of great significance for promoting the clinical transformation of photodynamic therapy.

[0161] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An organic photosensitive nanomaterial, characterized in that, The organic photosensitive nanomaterial has a core-shell structure; the shell structure includes an amphiphilic polymer, and the core structure includes an organic conjugate and an electron sacrificial agent; the organic conjugate is an organic conjugate small molecule Y6; the electron sacrificial agent is benzophenone; the mass ratio of the organic conjugate to the electron sacrificial agent is 1:0.5-5.

2. The organic photosensitive nanomaterial according to claim 1, wherein The amphiphilic polymer is at least one of distearoyl phosphatidylethanolamine-polyethylene glycol 2000, poloxamer F127, and polystyrene 5000-b-polyethylene glycol 5000.

3. The organic photosensitive nanomaterial according to claim 1, wherein The average particle size of the organic photosensitive nanomaterial is 134 nm - 152 nm; the excitation wavelength of the organic photosensitive nanomaterial is 650 nm - 900 nm.

4. Use of the organic photosensitive nanomaterial according to any one of claims 1-3 in the preparation of a photosensitizer for photodynamic therapy.

5. The preparation method of the organic photosensitive nanomaterial according to any one of claims 1 to 3, characterized in that, It includes the following steps: self-assembling the organic conjugate, the electron sacrificial agent, and the amphiphilic polymer, and that's it.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the organic conjugate to the amphiphilic polymer is 1:10-50.

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