Organic photosensitive nano material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses an organic photosensitive nano material as well as a preparation method and application thereof. The organic photosensitive nano material is of a shell-core structure; the shell structure comprises an amphiphilic polymer, and the core structure comprises an organic conjugate and an electron sacrificial agent; the preparation method of the organic photosensitive nano-material comprises the following steps: self-assembling the organic conjugate, the electron sacrificial agent and the amphiphilic polymer to obtain the organic photosensitive nano-material. Due to the unique structural design and excellent GSH consumption performance of the organic photosensitive nano material, endogenous GSH in tumor cells can be consumed, loss of ROS by the endogenous GSH is reduced, and oxidative stress in the tumor cells is further enhanced; the level of GSH in tumor cells can be jointly regulated and controlled, the utilization rate of ROS in hypoxic tumors is increased, and as a medication scheme for achieving GSH consumption, the compound is applied to PDT to promote the treatment effect of the hypoxic tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an organic photosensitive nanomaterial and a preparation method and application thereof. Background Art

[0002] Cancer is a chronic and non-communicable disease with a high mortality rate in the world. The clinical treatment of tumors adopts traditional therapies, such as chemotherapy, radiotherapy and surgery. However, the disadvantages of traditional therapies, such as poor selectivity, low efficacy and large toxic side effects, limit their wider clinical application and therapeutic effects. Photodynamic therapy (PDT), a specific site-specific treatment strategy with high spatiotemporal selectivity, has attracted widespread attention in the field of tumor treatment as an emerging treatment method. PDT is a process in which photosensitizers interact with molecular oxygen under light irradiation of a specific wavelength to produce highly oxidizing reactive oxygen species (ROS), which in turn destroys a variety of biological molecules in cells, such as proteins, DNA and cell membranes, triggering cell apoptosis or necrosis. At the same time, studies have shown that PDT does have a significant killing effect on solid tumors, and this therapy has been successfully used to treat a number of malignant tumors.

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

[0004] At present, inorganic nanomaterials are used as photosensitizers to reduce the level of GSH in tumor cells by directly interacting with GSH through their glutathione oxidase-like activity. However, the biosafety of inorganic nanomaterials is still the main factor restricting their development. Compared with inorganic semiconductor materials, organic semiconductor materials have the advantages of flexible preparation, adjustable performance, good biosafety and repeatability. 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 effects of GSH on PDT. Therefore, it is urgent to develop a safe and efficient organic photosensitive nanomaterial that can be used for photodynamic therapy of hypoxic tumors and can consume GSH during illumination. Summary of the invention

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

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

[0007] In a first aspect, the present invention provides an organic photosensitive nanomaterial, wherein the organic photosensitive nanomaterial is 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] The organic conjugate in the organic photosensitive nanomaterial of the present invention can absorb light energy under light irradiation to generate electron-hole pairs, which are then separated to form photogenerated electrons (e - ) and photogenerated holes (h + ), and at the same time, the electron sacrificial agent consumes the photogenerated electrons during the illumination process. - , enhanced photogeneration + The photooxidation of reducing substances in tumor cells, and further synergistically with organic conjugates, promotes the diffusion and accumulation of ROS in tumor cells, improves the killing effect of ROS on tumor cells, and significantly improves the effect of photodynamic therapy. Especially under hypoxic conditions, the nanomaterials of the present invention still produce efficient ROS, which is mainly due to its unique structural design, making the photogenerated ROS - and h + Even in a 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 core material, thereby achieving effective treatment of hypoxic tumors.

[0009] The organic photosensitive nanomaterial of the present invention has an inhibition rate of 80% on the survival of tumor cells under hypoxic conditions, which can significantly reduce the volume and weight of tumor cells in living organisms, and even completely eliminate the tumor without obvious toxic side effects on living organisms.

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

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

[0012] The organic conjugate in the organic photosensitive nanomaterial of the present invention has an ADA molecular structure composed of a molten ladder core of an electron donor (D) and two electron acceptor (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, and improving the separation efficiency of photogenerated electrons and photogenerated holes; it is also beneficial to the delocalization of electrons along the molecular skeleton, resulting in a decrease in the molecular band gap and strong absorption in the near-infrared region. At the same time, it is used in combination with an electron sacrificial agent, so that the organic photosensitive nanomaterial can reduce the loss of GSH for ROS in photodynamic therapy, increase the utilization efficiency of ROS, and thus achieve 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 diphenyl ether and 2-ethylhexyl-4-trimethoxycinnamate; preferably, the electron sacrificial agent is benzophenone.

[0014] The organic photosensitive nanomaterial of the present invention selects an electron sacrificial agent with a simple structure and uses it in combination with an organic conjugate, which can reduce the loss of GSH to 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 the mass ratio of the organic conjugate to the electron sacrificial agent is 1:1, and the organic photosensitive nanomaterial has better GSH consumption capacity and hypoxic tumor inhibition capacity.

[0018] As a preferred embodiment of the organic photosensitive nanomaterial described in the present invention, the amphiphilic polymer is at least one of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000), poloxamer F127 (PluronicF127), bovine serum albumin BSA, and polystyrene 5000-b-polyethylene glycol 5000 (PS5000-b-PEG5000).

[0019] Preferably, the amphiphilic polymer is distearoylphosphatidylethanolamine-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 134nm-152nm; the excitation wavelength of the organic photosensitive nanomaterial is 650nm-900nm.

[0021] In a second aspect, the present invention provides the use 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 photosensitizers for use in photodynamic therapy to reduce the loss of ROS by GSH and increase the utilization efficiency of ROS, thereby achieving an efficient type I photodynamic therapy effect. It is particularly suitable for preparing photosensitizers for use in photodynamic therapy to promote the therapeutic effect of hypoxic tumors.

[0023] In a third aspect, the present invention provides a method for preparing the organic photosensitive nanomaterial, comprising the following steps: self-assembling an organic conjugate, an electron sacrificial agent and an amphiphilic polymer to obtain the obtained product.

[0024] The preparation method of the organic photosensitive nanomaterial of the present invention introduces a specific electron sacrificial agent to prepare an organic photosensitive nanomaterial that reduces the loss of GSH to ROS, increases the utilization efficiency of ROS, and achieves an efficient type I photodynamic therapy effect. The organic photosensitive nanomaterial can be used in photodynamic therapy to promote the therapeutic effect of hypoxic tumors.

[0025] As a preferred embodiment of the method for preparing 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 method for preparing the organic photosensitive nanomaterial of the present invention, the method comprises the following steps: (1) dissolving an amphiphilic polymer, an electron sacrificial agent, and an organic conjugate in a good solvent to obtain a mixed solution; and (2) adding the mixed solution described in step (1) to a poor solvent and sonicating the mixture.

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

[0029] Further 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] Further preferably, the ultrasonic power is 600 W and the ultrasonic time is 0.5 h.

[0032] Preferably, after the ultrasonic step is completed, the method further comprises dialyzing the ultrasonicated solution in water to obtain an organic photosensitive nano-water solution.

[0033] Further preferably, the dialysis step uses a dialysis bag with a molecular weight cutoff of 5000-15000Da.

[0034] More preferably, the dialysis step uses a dialysis bag with a molecular weight cutoff of 14000 Da.

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

[0036] Further preferably, after the dialysis step is completed, the method further comprises the step of ultrafiltration of the organic photosensitive nano-water solution to obtain organic photosensitive nano-particles.

[0037] More preferably, the molecular weight cutoff 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 reduce the loss of ROS by consuming endogenous GSH in tumor cells, thereby further enhancing 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, thereby overcoming the defect of poor single treatment effect of the prior art and promoting the clinical transformation of the medication regimen for achieving 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 and quantitatively produce. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0040] Figure 2 The absorption spectra of the organic conjugated small molecule Y6 of the present invention in CHCl3 solution and film;

[0041] Figure 3 This is a flow chart of the preparation of the organic photosensitive nanomaterial according to Example 1 of the present invention;

[0042] Figure 4 This is the absorption spectrum of the organic photosensitive nanomaterial of Verification Example 1 of the present invention;

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

[0044] Figure 6Absorption spectra of organic photosensitive nanomaterials of Verification Example 2 of the present invention; (a)-(b) Y6 NPs; (c)-(d) YB NPs;

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

[0046] Figure 8 This is a graph of the GSH consumption capacity of organic photosensitive nanomaterials in 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] Fig. 9 This is a diagram showing the effect of organic photosensitive nanomaterials on cell viability detected by MTT method in Example 3 of the present invention;

[0048] Fig.10 This is a graph showing the effect of organic photosensitive nanomaterials on cell survival detected by Calcein AM / PI probe in Verification Example 3 of the present invention;

[0049] Fig.11 This is a diagram showing the consumption of intracellular GSH by organic photosensitive nanomaterials detected by the kit in Verification Example 3 of the present invention;

[0050] Fig.12 The following are graphs showing the effect of organic photosensitive nanomaterials on photodynamic therapy of living mice in Verification Example 4 of the present invention: (a) graph of mouse tumor volume growth; (b) graph of mouse tumor in real life; (c) graph of mouse tumor weight; and (d) graph of mouse weight change. DETAILED DESCRIPTION

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

[0052] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, equipment, etc. used, unless otherwise specified, can be obtained from commercial sources.

[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) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0058] The following examples and comparative examples are prepared by adding 123 mg (0.12 mmol) of TPBT-CHO, 110 mg (0.3 mmol) of 2FIC and 0.5 mL of pyridine to 20 mL of CHCl3 under N2 atmosphere, reacting at room temperature, and monitoring the reaction progress of TPBT-CHO by thin layer chromatography (TLC) until it completely disappears, i.e., the reaction is completed. After removing the solvent in the reaction system, the crude product is purified by column chromatography (silica gel) with a mixed solvent of petroleum ether and dichloromethane (volume ratio of 1: 1), and finally 113 mg of brown-black solid Y6 (yield of 65%) is obtained, and the composition of the product is determined by nuclear magnetic resonance spectroscopy.

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

[0060] like Figure 2 As shown, the absorbance of the solid film or chloroform solution of the synthesized organic conjugated small molecule Y6 was tested by UV-visible spectrophotometry.

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

[0062] like Figure 3 As shown, 10 mg of DSPE-PEG2000 was dissolved in 10 mL of tetrahydrofuran (THF), and then 0.5 mg of the organic conjugated small molecule Y6 synthesized above 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. Then the mixed solution and 2 mL of CHCl3 were quickly added to 100 mL of ultrapure water, and ultrasonic (600 W, 40 KHz) was applied for 30 min, and then THF and CHCl3 were removed by rotary evaporator. The solution was transferred to a dialysis bag (MWCO = 14000) and dialyzed with deionized water for 24 h. The deionized water in the nanoparticles was removed by ultrafiltration (MWCO = 10000) to obtain YB NPs particles and 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 method for preparing the organic photosensitive nanomaterial YB NPs described in this embodiment and that in Embodiment 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 method for preparing the organic photosensitive nanomaterial YB NPs described in this embodiment and that in Embodiment 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 described in this embodiment and that in Embodiment 1 is that the ultrasonic power is 500W.

[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 described in this embodiment and that in Embodiment 1 is that the ultrasonic power is 1000W.

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

[0080] 1 mg of the prepared Y6 and 10 mg of DSPE-PEG2000 were dissolved in 10 mL of tetrahydrofuran (THF) and mixed uniformly. Then the solution and 2 mL of CHCl3 were quickly added to 100 mL of ultrapure water, and ultrasound (600 W, 40 KHz) was applied for 30 min. Then, THF and CHCl3 were removed by rotary evaporator. The solution was transferred to a dialysis bag (MWCO = 14000) and dialyzed with deionized water for 24 h. The deionized water in the nanoparticles was then removed by ultrafiltration (MWCO = 10000) to obtain Y6 NPs particles and stored 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 described 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 described 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] like Figure 4 As shown, the light absorption intensity of the organic photosensitive nanomaterials of Examples 1-3 and Comparative Example 1 at different wavelengths was measured using UV-visible spectrophotometry. It can be seen from the figure that Y6 and BP in Examples 1-3 have been successfully assembled into nanoparticles, thereby indicating the successful synthesis of nanoparticles YB NPs.

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

[0088] Verification Example 2: Study on the Photodynamic Properties of Organic Photosensitive Nanomaterials

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

[0090] (1) Hydroxyl radical (·OH) detection: Orthophenylenediamine (OPD) was used as an indicator to test the ability of organic photosensitive materials Y6 NPs and YB NPs to produce ·OH at the solution level to determine their type I photodynamic properties.

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

[0092] from Figure 6 It can be seen from the figure that under normal oxygen conditions, organic photosensitive nanomaterials Y6 NPs and YB NPs can produce OH to oxidize OPD, and the oxidation product has a characteristic absorption peak around 420nm. Figure 6 It can be seen from 6-a and 6-c that there is little difference in the ability of the two to produce hydroxyl radicals, indicating that the introduction of BP has little effect on the photodynamic performance of Y6 under normoxic conditions.

[0093] (2) EPR test: The ·OH was qualitatively detected by ESR / EPR spectrometer, and the ·OH generated in the reaction system was detected by using a specific ROS scavenger DMPO. 75 μg / mL of the nanomaterials of Example 1 and Comparative Example 1 were mixed with 25 mM DMPO scavenger, respectively, and the ·OH was detected by 808 nm (0.3 W / cm 2 ) After laser irradiation for 10 min, the generation of ·OH was detected by paramagnetic resonance spectrometer.

[0094] from Figure 7 It can be seen that the characteristic signals of organic photosensitive nanomaterials can be detected under the action of specific ROS capture agents, indicating that under 808nm laser irradiation, organic photosensitive nanomaterials Y6 NPs and YB NPs can produce ·OH, which further proves that they have type I photodynamic properties.

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

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

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

[0098] (1) Normal oxygen: 1 mg of the nanomaterial samples of Example 1 and Comparative Example 1 and GSH (60 μL, 10 mM) were mixed in PBS solution (3.5 mL, pH = 6.5) to obtain a mixture, and then the mixture was heated at 808 nm (0.3 W / cm 2 ) The mixture was irradiated with laser for different time periods (0 min, 5 min, 10 min, 15 min, 20 min) to simulate the consumption process of GSH by the nanomaterials under light. The nanomaterials were then filtered through an ultrafiltration tube, and 2.5 ml of the filtrate was taken out and mixed with 400 μL of DTNB (0.3 mg / mL) in PBS (pH = 6.5) solution. The absorbance of the mixture was then measured using a UV-Vis-NIR spectrometer.

[0099] (2) Anaerobic: To detect the consumption of glutathione under hypoxic conditions, the mixture was placed in a vial and subjected to three vacuum nitrogen operations to strictly deoxygenate. The subsequent illumination treatment, filtration, reaction with DTNB, and absorbance measurement were consistent with those under normoxic conditions.

[0100] from Figure 8 -a, 8-c show that under aerobic conditions, both organic photosensitive nanomaterials Y6 NPs and YBNPs show excellent GSH consumption ability. This is mainly because oxygen as an electron acceptor promotes the generation of ROS by organic photosensitive nanomaterials under light. These ROS have strong oxidizing properties and can react quickly with GSH, resulting in the consumption of GSH. Figure 8 -b, it can be seen that under anaerobic conditions, Y6 NPs did not show the ability to consume GSH. This is because Y6 NPs cannot effectively generate ROS due to the lack of oxygen as an electron acceptor. Figure 8 -d, it can be seen that due to the presence of the electron sacrificial agent BP, the photogenerated electrons generated by YB NPs can be quickly consumed, and the remaining photogenerated holes can oxidize GSH, resulting in the consumption of GSH, thus showing a unique GSH consumption ability. Verification Example 3: Study on the 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 using the MTT method and live-dead cell staining method to reflect cell survival, and using a kit to detect intracellular GSH consumption.

[0102] The organic photosensitive nanomaterials prepared in Example 1 and Comparative Example 1 were directly dissolved in complete culture medium to a concentration of 50 μg / mL in the complete culture medium.

[0103] Experimental steps:

[0104] (1) MTT assay for cell viability: CT26 cells (from the American Type Culture Collection (ATCC)) were inoculated into 96-well plates. There were six groups in total, five replicate wells in each group, and 6 × 10 3 After inoculation, the cells were placed in a sterile incubator (Thermo Fisher Scientific) (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 overnight; after the cells adhered to the wall and the density reached 70%, the samples were treated according to the following groups:

[0105] Normoxic conditions:

[0106] (i) Control: cells were incubated with complete medium without any drug for 7 h and no treatment was performed;

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

[0108] (iii) YB NPs (50 μg / mL): cells were incubated 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 cells with complete medium containing 50 μg / mL Y6 NPs for 7 h, an 808 nm laser (0.3 W / cm 2 ) to irradiate the cells until the average cell temperature rises to about 44.5°C, and the irradiation time is 10 min;

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

[0111] Hypoxic conditions:

[0112] (vi) Control: cells were incubated with complete medium without any drug for 7 h and no treatment was performed;

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

[0114] (viii) YB NPs (50 μg / mL): cells were incubated 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 cells with complete medium containing 50 μg / mL Y6 NPs for 7 h, an 808 nm laser (0.3 W / cm 2 ) to irradiate the cells until the average cell temperature rises to about 44.5°C, and the irradiation time is 10 min;

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

[0117] After treatment, the 96-well plate was placed 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) for 17h; a 5mg / mL MTT solution was prepared and precooled at 4°C, and then 10μL MTT solution was added to each well, gently shaken and then continued to incubate 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) for 4h. After incubation, the supernatant was removed, 150μL DMSO was added to each well, and the cells were shaken on a shaker in the dark for 15min. The absorbance of each group of cells at 490nm was detected by an ELISA reader (BioTek) to calculate the cell survival rate.

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

[0119] from Fig. 9 It can be seen that the use of only 808nm laser has no killing effect on tumor cells under normoxic or hypoxic conditions, while at 808nm (0.3W / cm 2) Under laser irradiation, the photosensitive material YB NPs has an excellent killing effect on tumor cells, especially the tumor cells under hypoxic conditions have their cell survival rate reduced to 20% after being treated with the photosensitive material YB NPs, which further proves that the organic photosensitive material of the present invention effectively promotes the photodynamic therapy effect of hypoxic tumors.

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

[0121] The experimental steps are as follows: CT26 cells were seeded in confocal microplates, with 5×10 4 The cells were placed 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) and cultured until the cell density reached about 80% and then treated with the following grouped samples:

[0122] Normoxic conditions:

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

[0124] (ii) YB NPs (50 μg / mL): cells were incubated 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 Y6 NPs for 7 h, an 808 nm laser (0.3 W / cm 2 ) to irradiate the cells until the average cell temperature rises to about 44.5°C, and the irradiation time is 10 min;

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

[0127] Hypoxic conditions:

[0128] (v) Y6 NPs (50 μg / mL) + laser (44.5°C, 10 min): After incubating cells with complete medium containing 50 μg / mL Y6 NPs for 7 h, an 808 nm laser (0.3 W / cm2 ) to irradiate the cells until the average cell temperature rises to about 44.5°C, and the irradiation time is 10 min;

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

[0130] After treatment, the supernatant was removed by suction, and the cells were gently rinsed with PBS buffer to remove excess complete culture medium and drug reagents. Then, the prepared Calcein AM / PI solution was added and incubated in the dark for 1 h. Finally, fluorescence imaging was performed using a Zeiss inverted fluorescence microscope (Carl Zeiss AG).

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

[0132] (3) Detection of intracellular GSH consumption: CT26 cells were plated at 3×10 5 The cells were inoculated into the well plate at a density of 100 μg / cm2 and placed in a sterile incubator for incubation (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). When the cell density reached about 80%, the samples in the following groups were used for treatment:

[0133] Normoxic conditions:

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

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

[0136] (iii) YB NPs (50 μg / mL): cells were incubated 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 Y6 NPs for 7 h, an 808 nm laser (0.3 W / cm2 ) to irradiate the cells until the average cell temperature rises to about 44.5°C, and the irradiation time is 10 min;

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

[0139] Hypoxic conditions:

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

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

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

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

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

[0145] After the treatment, the supernatant was removed by aspiration, and the cells were gently rinsed with PBS buffer to remove excess complete culture medium and drug reagents, and then treated with GSH and GSSG detection kit (Biyuntian, catalog number: S0053).

[0146] from Fig.11It can be seen from the content of GSH in tumor cells CT26 after different treatment methods that YB NPs can also significantly consume GSH in tumor cells in a lower oxygen environment, indicating that the organic photosensitive nanomaterials of the present invention can increase the consumption of GSH in 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 Photosensitive Nanomaterials on Photodynamic Therapy of Tumors in Living Mice

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

[0149] The organic photosensitive nanomaterials of Example 1 and Comparative Example 1 were dissolved in PBS solution to a concentration of 2 mg / mL.

[0150] SPF-grade female Balb / c mice (4 weeks old, weighing about 20 g) purchased from Guangdong Medicilon Inc. were used, and the experimental unit use license number was 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: CT26 tumor cells resuspended in PBS buffer were subcutaneously injected into the back of mice at a dose of 100 μL / mouse (2×10 6 cells) to establish a mouse xenograft tumor model. When the tumor grew to about 100 mm 3 (tumor volume V = length × width 2 / 2), the mice were randomly divided into 6 groups (5 mice in each group) and the following grouped treatments were repeated on the 3rd and 5th days:

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

[0153] (ii) Control + NIR: Inject PBS solution (50 μL / mouse) into the tumor of 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 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 mice;

[0156] (v) Y6 NPs+NIR: Mice were injected intratumorally with PBS buffer containing nanomaterial Y6 NPs (Y6 NPs concentration 2 mg / mL, 50 μL / mouse), and then irradiated with 808 nm laser (44.5 °C, 10 min) 8 h later.

[0157] (vi) YB NPs + NIR: Mice were injected intratumorally with PBS buffer containing nanomaterial YB NPs (YB NPs concentration 2 mg / mL, 50 μL / mouse) and then irradiated with 808 nm (0.3 W / cm 2 ) Laser irradiation (44.5℃, 10min)

[0158] The above treatment was repeated on the 3rd and 5th days, and the tumor volume and body weight of the mice were measured every other day to evaluate the treatment effect of each group. On the 14th day, the mice in each group were euthanized, and the tumors were removed by dissection, weighed, and photographed.

[0159] from Fig.12 -a and 12-b, it can be seen that at 808nm (0.3W / 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 greatly reduced, and even the tumor disappears completely. Fig.12 -d, it can be seen that the weight of mice showed a stable trend within 14 days, indicating that the organic photosensitive nanomaterial YB NPs and 808nm (0.3W / cm 2 ) Laser irradiation has no obvious toxic 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 of the prior art, and this medication regimen that achieves unique GSH consumption and ROS generation ability 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 solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An organic photosensitive nanomaterial, characterized in that: The organic photosensitive nano material is a core-shell structure; the shell structure comprises an amphiphilic polymer, and the core structure comprises an organic conjugate and an electron sacrificial agent.

2. The organic photosensitive nanomaterial according to claim 1, characterized in that: The organic conjugate is an organic conjugated small molecule substance with type I photodynamic performance.

3. The organic photosensitive nanomaterial according to claim 1 or 2, characterized in that: The organic conjugate is an organic conjugated small molecule Y6.

4. The organic photosensitive nanomaterial according to claim 1, characterized in that: The electron sacrificial agent is at least one of benzophenone, benzoin diphenyl ether and 2-ethylhexyl-4-trimethoxycinnamate.

5. The organic photosensitive nanomaterial according to claim 1, characterized in that: The mass ratio of the organic conjugate to the electron sacrificial agent is 1:(0.5-5).

6. The organic photosensitive nanomaterial according to claim 1, characterized in that: The amphiphilic polymer is at least one of distearoylphosphatidylethanolamine-polyethylene glycol 2000, poloxamer F127, bovine serum albumin BSA, and polystyrene 5000-b-polyethylene glycol 5000.

7. The organic photosensitive nanomaterial according to claim 1, characterized in that: The average particle size of the organic photosensitive nanomaterial is 134nm-152nm; the excitation wavelength of the organic photosensitive nanomaterial is 650nm-900nm.

8. Use of the organic photosensitive nanomaterial according to any one of claims 1 to 7 in the preparation of photosensitizers for photodynamic therapy.

9. The method for preparing the organic photosensitive nanomaterial according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: self-assembling an organic conjugate, an electron sacrificial agent and an amphiphilic polymer to obtain the product.

10. The preparation method according to claim 9, characterized in that: The mass ratio of the organic conjugate to the amphiphilic polymer is 1:(10-50).

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