Preparation method of selenium-rich polymer nano photosensitizer and application of selenium-rich polymer nano photosensitizer in tumor photodynamic therapy

Abstract

The invention discloses a preparation method of a selenium-rich polymer nano photosensitizer and application of the selenium-rich polymer nano photosensitizer in tumor photodynamic therapy, and belongs to the technical field of fine chemical engineering. The polymer photosensitizer (PEG-b-PSeMet) is obtained by directly polymerizing an n-phenoxycarbonyl functionalized amino acid (NPC) derivative. The polymer photosensitizer can be self-assembled into nano particles (PolySe NPs) in an aqueous solution, and shows good tumor targeting property. Wherein the PolySe NPs remarkably improves the intersystem crossing efficiency of the photosensitizer pyropheophorbide-a (PPa) through the action of intermolecular heavy atoms, and compared with a polymer nano photosensitizer (PolyC NPs) without heavy atoms, the PolySe NPs shows that the ROS generation is enhanced by 1.6 times, and the killing power on various cancer cells is improved by about 1.4 times. Compared with small molecule PPa, the polymer nano photosensitizer can obviously improve the tumor targeting property of PPa and greatly enhance the concentration of the photosensitizer at a tumor part, so that the solid anti-tumor effect of photodynamic therapy is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fine chemical industry, and particularly relates to a kind of selenium-rich polymer nano photosensitizer for enhancing photodynamic therapy of tumor by intermolecular heavy atom effect. Through intermolecular heavy atom effect, the intersystem crossing rate of photosensitizer fragments can be significantly enhanced, thereby effectively improving the efficiency of active oxygen generation. In addition, the nano drug can realize effective targeting of tumor through the high permeability and retention effect of tumor, greatly improve the photosensitizer concentration at the tumor site, and show excellent inhibition performance in solid tumors. BACKGROUND

[0002] Photodynamic therapy (PDT) is a treatment technology based on photosensitizer generating singlet oxygen (¹O2) under the excitation of a specific light source to kill tumor cells. Its advantages lie in precise targeting, low toxicity and side effects, and repeatable treatment. However, the clinical efficacy of this therapy is restricted by the singlet oxygen generation efficiency of photosensitizer and the insufficient oxygen content in tumor microenvironment. To break through this technical bottleneck, researchers have developed various efficiency strategies: on the one hand, through molecular engineering means to modify photosensitizer with heavy atoms, using intramolecular heavy atom effect to enhance intersystem crossing process, thereby improving singlet oxygen yield, but this method is limited due to complex synthesis process and significant individual differences in effect; on the other hand, functional nano drug delivery systems are constructed, and through the structural partition design of polymer carriers, multifunctional integration is achieved, which can not only serve as a photosensitizer targeted delivery carrier, but also can synergistically enhance the treatment effect. It is worth noting that the heavy atom element is innovatively integrated into the polymer skeleton to construct a multifunctional delivery platform, which forms a nano composite system by co-assembly with photosensitizer, not only can avoid the complex synthesis process of traditional photosensitizer molecular modification, but also can significantly improve the photosensitivity through external heavy atom effect. This strategy based on polymer-heavy atom collaborative platform opens up a new path for PDT technology innovation, especially for simplifying the existing photosensitizer development process has important application value. SUMMARY

[0003] The present application is directed to the problems of insufficient targeting and limited photosensitive activity of small molecule photosensitizers in the treatment of solid tumors, and innovatively develops a selenium-rich polymer nano-photosensitizer (PolySe NPs) by using a direct polymerization strategy, and systematically explores the enhancement mechanism of intermolecular heavy atom effect on photosensitive performance. The nano system takes polyethylene glycolated polypeptide as the skeleton, and the end is covalently modified with pyropheophorbide-a (PPa) units with near-infrared absorption characteristics, which not only serves as the photosensitive active core, but also realizes the integration of diagnosis and treatment through fluorescence signal. The specially designed polypeptide chain is rich in carbon, sulfur and selenium atoms, which can serve as an efficient promoter for reactive oxygen species (ROS) generation. Due to the relative hydrophobicity of PPa, the photosensitizer PPa is tightly wrapped in the nano-hydrophobic core by polyethylene glycol. Experimental data show that the ROS yield of PolySe NPs containing selenium is increased by 1.56 times compared with PolyC NPs without heavy atoms, and the phototoxicity effect on various cancer cells is enhanced by about 1.4 times. Theoretical calculation further reveals that the energy level difference (ΔE S-T ) between the first excited singlet state (S1) and the triplet state (T1) of PPa in PolySe NPs is reduced by 0.349 eV compared with PolyC NPs, which significantly promotes the intersystem crossing efficiency. Under the guidance of near-infrared fluorescence imaging, PolySe NPs exhibit the most significant tumor inhibition effect due to their excellent photosensitive activity and tumor-targeting enrichment ability. This study provides an innovative solution for the precision and efficiency of tumor photodynamic therapy by constructing polymer nano-photosensitizers with intermolecular heavy atom synergistic effect.

[0004] The technical scheme adopted by the present application is as follows: a kind of nano-photosensitizer has the following molecular structure formula:

[0005] .

[0006] Preferably, the nano-photosensitizer is:

[0007] .

[0008] The present application also provides a preparation method of the selenium-rich nano-photosensitizer, and the steps are as follows:

[0009]

[0010] (1) L-selenomethionine and NaHCO3 are dissolved in water. Then, methyl tert-butyl ether (MTBE) is added to the solution, and then phenyl chloroformate (PCF) is added at 0°C. The reaction mixture is stirred at room temperature for 3 hours. After the reaction, the aqueous phase is washed with MTBE and dehydrated. Then, the pH of the aqueous phase is adjusted to 2-3. The resulting mixture is extracted with ethyl acetate, and the product NPCSeMet in the form of pure white powder is obtained after concentration. The synthesis of NPC-Nle and NPC-Met follows the same procedure

[0011] (2) In a glove box, NPCSeMet and PEG2000-NH2 were mixed in dimethylacetamide. After stirring overnight at 80°C, the mixture was precipitated with cold diethyl ether and dried to obtain the target PEG-b-PSeMet. Block copolymers PEG-b-PNle and PEG-b-PMet were synthesized using a similar method.

[0012] (3) PEG-b-PSeMet, PPa, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDC, and N-hydroxysuccinimide NHS were dissolved in DMF. They were then thoroughly mixed and stirred at room temperature. After 48 hours, the reaction mixture was filtered to remove insoluble byproducts. The mixture was then dialyzed twice with deionized water and freeze-dried to obtain PEG-b-PSeMet-PPa powder. PEG-b-PNle-PPa and PEG-b-PMet-PPa were synthesized using the same method.

[0013] (4) Selenium-enriched polymer nanophotosensitizers PolySe NPs were prepared by reacting PEG-b-PSeMet in aqueous solution. PolyC NPs and PolyS NPs were prepared using the same method.

[0014] In step (1), the molar ratio of L-selenomethionine, NaHCO3 and PCF is 1:3:1.2.

[0015] In step (2), the molar ratio of NPCSeMet to PEG2000-NH2 is 10:1;

[0016] In step (3), the molar ratio of PEG-b-PSeMet, PPa, EDC, and NHS is 1:1:2:2;

[0017] In step (4), a dialysis bag with a molecular weight of 3500 Da is used.

[0018] The method is as follows:

[0019] (1) L-selenomethionine (12.1 g, 62.0 mmol) and NaHCO3 (15.7 g, 186.1 mmol) were dissolved in water (130 mL). Then, 130 mL of methyl tert-butyl ether (MTBE) was added to the solution, followed by the gradual addition of 9.1 mL (73.2 mmol) of PCF to the biphase mixture maintained at 0°C. The reaction mixture was then stirred at room temperature for 3 hours. After the reaction, the alkaline aqueous phase was removed by washing with MTBE (3 × 150 mL). The aqueous phase was then acidified with saturated hydrochloric acid until the pH reached 2–3. The mixture was then transferred to a solution of 10% citric acid (150 mL). The resulting mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to give NPC-SeMet as a pure white powder. The synthesis of NPC-Nle and NPC-Met followed the same procedure.

[0020] (2) In a glove box, NPC-SeMet (1.58 g, 5.0 mmol) and PEG2000-NH2 (1 g, 0.5 mmol) were mixed in dimethylacetamide (DMAc, 20 mL). After stirring at 80 °C for 20 h, the solution mixture was precipitated into excess cold diethyl ether and dried in a vacuum oven to obtain the target PEG-b-PSeMet. The synthesis of PEG-b-PNle and PEG-b-PMet followed the same procedure.

[0021] (3) PEG-b-PSeMet (213 mg, 0.056 mmol), PPa (30 mg, 0.056 mmol), EDC (20 mg, 0.12 mmol), and NHS (14 mg, 0.12 mmol) were dissolved in 5 ml of DMF at a molar ratio of 1:1:2:2. The mixture was then thoroughly mixed and stirred at room temperature. After 48 hours, the reaction mixture was filtered to remove insoluble byproducts. The mixture was then dialyzed twice with deionized water and freeze-dried to obtain PEG-b-PSeMet-PPa powder. The synthesis of PEG-b-PNle-PPa followed the same procedure as that of PEG-b-PMet-PPa.

[0022] (4) 1 mg of the polymer photosensitizer PEG-b-PSeMet-PPa was dissolved in 200 μL of DMF. While sonicating at 300 W, the solution was gradually introduced into 5 ml of ultrapure water. The resulting dispersion was further sonicated for 10 minutes, and then dialyzed in ultrapure water for 24 hours to remove the organic solvent. During dialysis, the ultrapure water was changed every 6 hours, using a regenerated cellulose dialysis bag with a molecular weight of 3500 Da. Finally, the solution taken from the dialysis bag constituted the mother liquor for obtaining the polymer nanophotosensitizer PolySe NPs, which was then stored at 4°C. The preparation of PolyC NPs and PolyS NPs followed the same procedure.

[0023] Furthermore, the photosensitizer nanoparticles are used to prepare drugs for anti-tumor therapy.

[0024] Furthermore, the photosensitizer is used to prepare a drug for anti-tumor therapy targeting tumor tissue.

[0025] Furthermore, it targets lysosomes in tumor tissue.

[0026] Furthermore, the photosensitizer is used in antitumor therapy drugs to improve photosensitivity efficiency.

[0027] Furthermore, the polymer photosensitizer self-assembles into nanoparticles in ultrapure water, with the nanoparticle size ranging from 10³ to 10⁹ nm.

[0028] Furthermore, tumors treated with nanophotosensitizers include, but are not limited to, breast cancer.

[0029] The selenium-enriched polymer photosensitizer PolySe NPs in this invention exhibits significantly improved ISC efficiency and reactive oxygen species yield compared to heavy atom-free polymer photosensitizer PolyC NPs. This effectively avoids the synthetic difficulties caused by directly introducing heavy atoms into the photosensitizer structure. Furthermore, it greatly enhances the tumor-targeting ability of the photosensitizer, making it more valuable for anti-tumor treatment of solid tumors.

[0030] The detection process for the absorption and fluorescence of the polymer nanophotosensitizer is as follows: the absorption spectrum is detected by a UV-Vis spectrophotometer, and the emission spectrum is detected by a fluorescence spectrometer.

[0031] The detection process for the photosensitizer to self-assemble into nanoparticles is as follows: the particle size of the nanoparticles is detected by a particle size analyzer, and the morphology of the nanoparticles is observed by transmission electron microscopy.

[0032] The accelerated simulation of the intersystem crossing process of the selenium-enriched polymer nanophotosensitizer PolySe NPs is as follows: In an implicit aqueous solvent model, six polymer molecules are placed into nanoparticles using molecular dynamics and semi-empirical methods. The structure is optimized by simulating the self-assembly process through structural optimization for subsequent intersystem crossing (ISC) calculations. The KISC between S1 and T1, and ΔE are used as the basis for the calculations. S-T As an indicator for evaluating inter-system crossing processes.

[0033] The enhanced reactive oxygen species (ROS) generation detection process of the polymer nanophotosensitizer is as follows: 9,10-anthratridiyl-bis(methylene)dimalonic acid (ABDA) is used as a singlet oxygen indicator, and changes in ABDA absorption are detected at equal time intervals. 2′,7′-dichlorofluorescein (DCF) is used as an ROS indicator, and changes in DCF fluorescence are detected at equal time intervals. The rate of decrease in ABDA absorption at 400 nm is used as an evaluation index of singlet oxygen generation capacity. The rate of increase in the F / F0 ratio of DCF at 529 nm is used as an evaluation index of ROS generation capacity.

[0034] The process for enhancing the detection of lysosomal damage using the polymer nanophotosensitizer involves using acridine orange (AO) as a lysosomal damage detection probe. Using C... PPa = 5 μm, then co-incubated with MCF-7 for a period of time, and then subjected to 10 mW cm -2 Images of lysosomal damage were captured by confocal microscopy after irradiation with a 700 nm laser for 3 minutes. The decrease in red fluorescence within the lysosomes was used as an evaluation index for enhanced lysosomal damage.

[0035] The detection process for the enhanced cytotoxicity of the polymer nanophotosensitizer in vitro is as follows: cell viability is tested using the MTT assay, and different concentration gradients C are set. PPa = 0 – 10 μm, and then after different treatments with MCF-7 and 4T1 cells, the absorbance was measured at 490 nm using a microplate reader, and cell viability was calculated. The half-maximal inhibitory concentration (IC50) obtained from the data is the first evaluation indicator of enhanced phototoxicity.

[0036] The detection process for the enhanced cytotoxicity of the polymer nanophotosensitizer in vitro involves simultaneous dual labeling of live and dead cells using a Calcein AM / PI double staining kit. The increase in red fluorescence intensity and the decrease in green fluorescence intensity are used as the second evaluation index for the phototoxicity of the photosensitizer.

[0037] The detection process for the targeting ability of the polymer nanophotosensitizer to solid tumors is as follows: PolySe NPs are reacted with the small molecule photosensitizer PPa (C PPaThe photosensitizer (50 μm) was injected intravenously into mice, and fluorescence images of the mice at different time points and organ distribution fluorescence images at the maximum enrichment time were recorded using a small animal imaging system. Fluorescence intensity within the tumor region was used as an evaluation index of the photosensitizer's tumor-targeting ability.

[0038] The detection process for the inhibitory ability of the polymer nanophotosensitizer on solid tumors is as follows: when the tumor volume in mice reaches 100 mm... 3 Mice were randomly divided into seven groups: G1 (Control + Light), G2 (PolyC NPs), G3 (PolyS NPs), G4 (PolySe NPs), G5 (PolyC NPs + Light), G6 (PolyS NPs + Light), and G7 (PolySe NPs + Light). The antitumor therapeutic application of the photosensitizer was evaluated based on the results of photodynamic therapy experiments in tumor-bearing mice (tumor volume, tumor inhibition rate, and ex vivo tumor mass).

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] (1) High targeting: By adopting the photosensitizer nano-strategy, the selenium-rich polymer nano-photosensitizer PolySe NPs can greatly enhance the tumor targeting of small molecule photosensitizers through the high penetration and long retention effect of tumors, thereby increasing the concentration of photosensitizers at the tumor site, reducing systemic toxic side effects, and improving the photodynamic therapy effect;

[0041] (2) High intersystem crossing efficiency: ΔE between the S1 and T1 states of PPa in selenium-enriched polymer nanophotosensitizer PolySe NPs S-T The value was 1.6164 eV, significantly lower than the 1.9654 eV of PolyC NPs. Furthermore, the PPa of PolySe NPs was 8.69 × 10⁻⁶ eV. 8 s -1 K from state S1 to state T1 ISC It is significantly higher than PolyC NPs, which is conducive to efficient intersystem crossing processes.

[0042] (3) High reactive oxygen yield: The singlet oxygen yield of selenium-enriched polymer nanophotosensitizer PolySe NPs in water is 1.42 times that of PolyC NPs, and the reactive oxygen yield is 1.56 times that of PolyC NPs.

[0043] (4) Significant anti-tumor effect: Compared with PolyC NPs, the selenium-enriched polymer nanophotosensitizer PolySe NPs has about 1.4 times the killing ability of various cancer cells in vitro, and has achieved a more obvious tumor-suppressing effect in solid tumors. Attached Figure Description

[0044] Figure 1 The absorption and emission spectra of polymer nanophotosensitizers PolySe NPs and comparative nanophotosensitizers PolyC NPs and PolyS NPs are shown.

[0045] Figure 2 These are the particle size and transmission electron microscopy images of polymer nanophotosensitizers PolySe NPs and comparative nanoparticles PolyC NPs and PolyS NPs.

[0046] Figure 3 This involves testing the singlet oxygen generation capacity of polymer nanophotosensitizers PolySe NPs and comparative nanopolymers PolyC NPs and PolyS NPs.

[0047] Figure 4 The test focused on the reactive oxygen species generation capacity of polymer nanophotosensitizers PolySe NPs and comparative nanopolymers PolyC NPs and PolyS NPs.

[0048] Figure 5 The images show electron-hole diagrams of polymer nanophotosensitizers PolySe NPs and contrasting fragments in nano-PolyC NPs.

[0049] Figure 6 This is a summary of theoretical calculation data for polymer nanophotosensitizers PolySe NPs and comparative nanophotosensitizers PolyC NPs.

[0050] Figure 7 This is a time-dependent fluorescence imaging of polymer nanophotosensitizers PolySe NPs in MCF-7 cells.

[0051] Figure 8 This is a cell colocalization experiment of the polymer nanophotosensitizer PolySe NPs in MCF-7 cells.

[0052] Figure 9 These are the MTT test results for the polymer nanophotosensitizer PolySe NPs.

[0053] Figure 10 The IC50 curves of polymer nanophotosensitizer PolySe NPs and comparative nanophotosensitizers PolyC NPs and PolyS NPs under light irradiation are shown for different cancer cells.

[0054] Figure 11 Images show lysosomal damage to polymer photosensitizers PolySe NPs and, in contrast, nanopolymers PolyC NPs and PolyS NPs.

[0055] Figure 12These are double-stained images of live / dead cells of polymer photosensitizer PolySe NPs and contrasting nanopolymers PolyC NPs and PolyS NPs.

[0056] Figure 13 This includes tumor enrichment images and organ enrichment images of polymer nanophotosensitizers PolySe NPs and small molecule PPa, as well as their quantification.

[0057] Figure 14 The images show solid images of the polymer photosensitizer PolySe NPs, as well as comparative images of the tumor volume in mice treated with PolyC NPs and PolyS NPs, and records of changes over 14 days. Detailed Implementation

[0058] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0059] Example 1

[0060] The selenium-enriched polymer nanophotosensitizer of this invention is prepared by the following method:

[0061]

[0062] (1) L-selenomethionine (12.1 g, 62.0 mmol) and NaHCO3 (15.7 g, 186.1 mmol) were dissolved in water (130 mL). Then, 130 mL of methyl tert-butyl ether (MTBE) was added to the solution, followed by the gradual addition of 9.1 mL (73.2 mmol) of phenyl chloroformate (PCF) to the biphase mixture maintained at 0°C. The reaction mixture was then stirred at room temperature for 3 hours. After the reaction, the alkaline aqueous phase was removed by washing with MTBE (3 × 150 mL). The aqueous phase was then acidified with saturated hydrochloric acid until the pH reached 2–3. The mixture was then transferred to a solution of 10% citric acid (150 mL). The resulting mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated to give NPCSeMet as a pure white powder.

[0063] Replacing L-selenomethionine with ortholeucine and methionine yields NPC-Nle and NPC-Met, respectively.

[0064] (2) In a glove box, NPC-SeMet (1.58 g, 5.0 mmol) and PEG2000-NH2 (1 g, 0.5 mmol) were mixed in dimethylacetamide (DMAc, 20 mL). After stirring at 80 °C for 20 h, the solution mixture was precipitated into excess cold diethyl ether and dried in a vacuum oven to obtain the target PEG-b-PSeMet. The synthesis of PEG-b-PNle and PEG-b-PMet followed the same procedure.

[0065]

[0066] (3) PEG-b-PSeMet (213 mg, 0.056 mmol), PPa (30 mg, 0.056 mmol), EDC (20 mg, 0.12 mmol), and NHS (14 mg, 0.12 mmol) were dissolved in 5 ml of DMF at a molar ratio of 1:1:2:2. They were then thoroughly mixed and stirred at room temperature. After 48 hours, the reaction mixture was filtered to remove insoluble byproducts. The mixture was then dialyzed twice with deionized water and freeze-dried to obtain PEG-b-PSeMet-PPa powder. The synthesis of PEG-b-PNle-PPa and PEG-b-PSeMet followed the same procedure.

[0067] (4) 1 mg of the polymer photosensitizer PEG-b-PSeMet-PPa was dissolved in 200 μL of DMF. While sonicating at 300 W, the solution was gradually introduced into 5 ml of ultrapure water. The resulting dispersion was further sonicated for 10 minutes, and then dialyzed in ultrapure water for 24 hours to remove the organic solvent. During dialysis, the ultrapure water was changed every 6 hours, using a regenerated cellulose dialysis bag with a molecular weight of 3500 Da. Finally, the solution taken from the dialysis bag constituted the mother liquor for obtaining the polymer nanophotosensitizer PolySe NPs, which were then stored at 4°C. The preparation of PolyC NPs and PolyS NPs followed the same procedure.

[0068] Example 2: Absorption and emission spectra of PolySe NPs, PolyS NPs and PolyC NPs

[0069] Prepare 3 ml solutions of PolyC NPs, PolyS NPs, and PolySe NPs. The testing temperature is 25℃, the testing range is 200-800 nm, and the fluorescence emission spectrum testing range is 650 nm-850 nm. Figure 1As shown, (a) is the absorption spectrum and (b) is the emission spectrum. PolyC NPs, PolyS NPs, and PolySe NPs all show absorption peaks around 700 nm.

[0070] Example 3: Particle size and morphology testing of PolySe NPs, PolyS NPs and PolyC NPs

[0071] A certain volume of nanoparticle solution was added to the sample cell. The hydrated particle size was measured by dynamic light scattering particle size analyzer (DLS), and the nanomorphology was measured by transmission electron microscopy (TEM). Figure 2 As shown in the figure, (a) is the particle size and (b) is the transmission electron microscope image. In the figure, the particle sizes of PolySe NPs, PolyS NPs and PolyC NPs are 103.2 nm, 109.2 nm and 107.9 nm, respectively, and they are nanospheres.

[0072] Example 4: Enhanced generation test of singlet oxygen and reactive oxygen species in PolySe NPs

[0073] Using ABDA as a singlet oxygen indicator, C PPa = 20 μm, with 10 mW cm -2 The absorption change of ABDA at 400 nm was detected at the same time interval under 700 nm laser irradiation. DCF was used as a reactive oxygen species indicator. PPa = 5 μm, at 10 mWcm -2 Irradiation with a 700 nm laser was performed, and the fluorescence change of the DCF at 520 nm was detected at the same time interval. For example... Figure 3 As shown in the figure, (ac) represents the ABDA absorption decrease curves under different treatments, and (d) represents the quantitative ABDA absorption decrease under different treatments. The fastest ABDA decrease rate in the PolySe NPs group indicates that its singlet oxygen yield is 1.21 times that of PolyS NPs and 1.42 times that of PolyC NPs. Figure 4 As shown in the figure, (ac) is the DCF fluorescence increase curve under different treatments, and (d) is the F / F0 increase rate under different treatments. PolySe NPs showed the fastest F / F0 increase rate, indicating that PolySe NPs have a 1.35 times greater ability to generate reactive oxygen species than PolyS NPs and 1.56 times greater ability than PolyC NPs.

[0074] Example 5: TD-DFT calculation results of PolySe NPs and PolyC NPs

[0075] In an implicit aqueous solvent model, six polymer molecules were incorporated into nanoparticles using molecular dynamics and semi-empirical methods. Since the PPa end is hydrophobic and polyethylene glycol is hydrophilic, the structure was optimized by simulating the self-assembly process for subsequent intersystem crossing (ISC) calculations. Molecular dynamics simulations were performed using molecular dynamics and a semi-empirical method (PM7). Molecular fragments of PolyC and PolySe NPs were extracted and optimized at the B3LYP / 6-31G* level. The frequencies of the S1 and T1 states were calculated at the ORCA B3LYP / def2-tzvp level, resulting in a HESS file. The ISC rates of the fragment molecules were calculated using ORCA's ESD module. The solvent model used was SMD (water). Figure 5 As shown, (a) is the electron-hole density map of the PolyC NPs fragment, where the ΔE between the S1 and T1 states of PPa in PolySe NPs is shown. S-T The value is 1.6164 eV, lower than the 1.9654 eV of PolyC NPs. (b) is the electron-hole density map of the PolySe NPs fragment. Figure 6 As shown, the calculated data are summarized, where PolySeNPs PPa (8.69 × 10⁻⁶) 8 s -1 K from state S1 to state T1 ISC The significantly higher rate than PolyC NPs indicates a significantly increased intersystem crossing rate of photosensitizers within PolySe NPs.

[0076] Example 6: Cellular uptake and subcellular organelle localization assay of PolySe NPs

[0077] MCF-7 cells were seeded in confocal culture dishes at a density of 5 × 10⁶ cells / mL. 4 Cells / mL, incubated for 24 hours for adhesion. Combined with PolyC NPs, PolyS NPs and PolySe NPs (C... PPa = 10 μM). Fluorescence emission intensity of the nanoparticles was detected at equal time intervals. Intracellular distribution of PolySe NPs was determined by cell localization assays. MCF-7 cells were incubated with PolySe NPs (C = 10 μM). PPa= 10 μM) incubated for 8 h, then stained with different commercial dyes for 30 min, and scanned under a confocal microscope. The green channels of MitoTracker green, Lyso tracker, and ER tracker green were excited at 488 nm and collected at 500–550 nm. The Hoechst 33342 channel was excited at 405 nm and collected at 420–480 nm. The red channel of PPa was excited at 640 nm and collected at 700–800 nm. (The text abruptly ends here.) Figure 7 The image shows the uptake of PolySe NPs in MCF-7 cells. Figure 8 The image shows the subcellular organelle localization of PolySe NPs within MCF-7 cells. This indicates that PolySe NPs can be effectively taken up by cells and accumulated in lysosomes.

[0078] Example 7: In vitro cytotoxicity assay of PolySe NPs

[0079] MCF-7, HeLa, and 4T1 cells were seeded in 96-well plates (1 × 10⁶ cells per well). 4 Cells were incubated in 100 μL DMEM. When the cell density approached 80%, they were incubated for 8 h with nanoparticle solutions of different concentrations. Light irradiation groups (700 nm, 10 mW cm⁻¹) were then established. -2 After treatment with the dark group (5 min), the mixture was incubated for another 24 h, and then MTT solution (100 μL, 0.5 mg / mL) was added to each well. -1 The culture medium was then added to each well in DMEM (dimethylformamide) and incubated at 37°C for 4 h. Afterward, the culture medium was removed, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. Absorbance was measured at 490 nm using a Bio-Rad microplate reader, and cell viability was calculated.

[0080]

[0081] The experimental group consisted of cell culture groups treated with different concentrations of nanoparticles; the blank group consisted of cell culture groups treated with culture medium only; and the control group consisted of cell culture groups without nanoparticles. OD was the absorbance value of formazan in DMSO solution at 490 nm. Each experiment was performed in 5 replicates.

[0082] like Figure 9 As shown, PolySe NPs, PolyS NPs, and PolyC NPs exhibit phototoxicity and dark toxicity in 4T1 and MCF-7 cells. Figure 10The figure shows the IC50 curves of PolySe NPs, PolyS NPs, and PolyC NPs under light irradiation on various cells. The IC50 value of PolySe NPs is significantly lower than that of PolyC NPs and PolyS NPs, indicating that PolySe NPs significantly enhance phototoxicity at the same photosensitizer concentration.

[0083] Example 8: PolySe NPs Lysosomal Damage Test

[0084] MCF-7 cells were seeded in 34 mm confocal culture dishes and cultured for 24 h. Then, they were co-incubated with specific concentrations of PolyC NPs, PolyS NPs, and PolySe NPs for 8 h. Following this, they were co-incubated with AO working solution for 0.5 h. Cells were then subjected to different light irradiation groups (700 nm, 10 mW cm⁻¹). -2 After treatment with the dark group (5 min), the samples were observed using a laser confocal microscope. The samples were washed three times with PBS before imaging. The excitation / emission ratio for the AO green channel was 488 / 530 nm, and for the red channel it was 488 / 640 nm. Figure 11 As shown, the images and fluorescence quantification of lysosome damage in different treatment groups under illumination show that the PolySe NPs treatment group had the lowest relative intensity of red fluorescence, indicating that it caused the greatest degree of lysosome damage.

[0085] Example 9: PolySe NPs live / dead cell imaging assay

[0086] MCF-7 cells were seeded in 34 mm confocal culture dishes and cultured for 24 h. Then, they were co-exposed to PolyC NPs, PolyS NPs, and PolySe NPs at the same concentration (10 μM) for 8 h. After exposure to light (700 nm, 10 mW / cm²), cells were cultured separately. -2 The samples were treated with a dark group (5 min) and then incubated with Calcein AM / PI working solution for 0.5 h using a confocal microscope, followed by imaging using an Olympus FV-3000 laser confocal microscope. Calcein excitation wavelength: 488 nm, emission wavelength: 500-540 nm. PI excitation wavelength: 488 nm, emission wavelength: 650-690 nm. Figure 12 As shown, the images of live / dead cells stained under different treatment groups and their fluorescence quantification are displayed. The PolySe NPs treatment group has the lowest relative intensity of red fluorescence, indicating the greatest cytotoxicity.

[0087] Example 10: Tumor targeting ability and organ distribution test of PolySe NPs

[0088] All animal research protocols complied with the "Guidelines for the Care and Use of Laboratory Animals." All animal experiments were conducted in accordance with the guidelines approved by the Ethics Committee of Dalian University of Technology (DUT20210902). 4T1 tumor-bearing mice were established in BALB / c mice, with 100 μL injected subcutaneously at a concentration of 1 × 10⁻⁶ μL / mL. 6 4 T1 cells per cell. When the mouse tumor reached approximately 200 mm... 3 At that time, 100 μL of nanoparticle solution (100 μM) was injected intratumorally. Fluorescence images of mice at different time points and organ distribution fluorescence images at the time of maximum enrichment were recorded using the NightOWL II LB983 small animal imaging system. Figure 13 As shown, (a) fluorescence imaging of mice after tail vein injection of PolySe NPs, (b) organ imaging of mice 12 h after PolySe NPs injection, (c) fluorescence imaging of mice after tail vein injection of PPa, and (d) fluorescence quantification of image (b). The tail vein injection of PolySe NPs showed a significantly increased enrichment in the tumor region compared to PPa. Organ fluorescence quantification revealed that the nanoparticles were mainly distributed in the liver and tumor regions.

[0089] Example 11: PolySe NPs were administered intratumorally to tumor-bearing mice, and the changes in tumor volume over time were recorded to assess their in vivo antitumor ability.

[0090] When the tumor volume in mice reaches approximately 100 mm 3 Mice were randomly divided into seven groups and treated with the following intratumoral injection: G1: PBS (100 μL) + light radiation (700 nm, 150 mW / cm²) 12 h after injection. -2 , 15 min), G2: PolyC NPs (100 μL, 50 μM in PBS), G3: PolyS NPs (100 μL, 50 μM in PBS), G4: PolySe NPs (100 μL, 50 μM in PBS), G5: PolyC NPs (100 μL, 50 μM in PBS) + light radiation (700 nm,150mWcm -2 , 15 min), G6: PolyS NPs (100 μL, 50 μM in PBS) + light radiation (700 nm, 150 mWcm) 12 h after injection -2 , 15 min), G7; PolySe NPs (100 μL, 50 μM in PBS) + light radiation (700 nm, 150 mW cm⁻¹) 12 h after injection -2(15 min) Each group consisted of five mice, and tumor volume changes were measured every two days over 14 days. Figure 14 As shown, (a) tumor volume monitoring of mice in different treatment groups within 14 days, (b) tumor inhibition rate of different treatment groups, and (c) tumor mass of ex vivo tumors in different treatment groups. Among them, the tumor volume and tumor mass of the PolySe NPs group were significantly lower than those of other groups, while the tumor inhibition rate was significantly higher than that of other groups.

[0091] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solution and inventive concept, should be covered within the scope of protection of the present invention. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, and substitutions and selections of auxiliary components, etc., all fall within the scope of protection and disclosure of the present invention.

Claims

1. A class of nano-photosensitizers characterized in that, The nano photosensitizer has a structural formula as shown in the following formula (I) : 。 2. The nano-photosensitizer according to claim 1, wherein: The nano photosensitizer has a particle size ranging from 103 nm to 109 nm.

3. The nano-photosensitizer according to claim 1, wherein, The nano photosensitizer has a structure as shown in the following formula (I) : 。 4. The preparation method of the nano photosensitizer according to claim 1 or 2, wherein: ； The synthesis steps are as follows: (1) Dissolve the amino acid raw material and NaHCO3 in water, add methyl tert-butyl ether to the solution, and then add phenyl chloroformate; stir the reaction mixture at room temperature; after the reaction, wash, extract, and concentrate to obtain the intermediate in the form of a pure white powder; The amino acid raw material is L-selenomethionine, norleucine, and L-methionine, and the corresponding obtained intermediates are NPC-SeMet, NPC-Nle, and NPC-Met, respectively; (2) In a glove box, mix the first intermediate and PEG2000-NH2 in dimethylacetamide; after stirring overnight, precipitate with cold ether, and dry to obtain the polyethylene intermediate; The polyethylene intermediate is PEG-b-PSeMet, PEG-b-PNle, and Peg-b-PMet; (3) Dissolve the polyethylene intermediate, PPa, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, and N-hydroxysuccinimide in DMF, mix thoroughly, and stir at room temperature after the reaction, and then filter the reaction mixture; dialyze with deionized water, and freeze-dry to obtain the polymer photosensitizer; The polymer photosensitizer is PEG-b-PSeMet-PPa, PEG-b-PNle-PPa, and PEG-b-PMet-PPa; (4) Dissolve the polymer photosensitizer in DMF, disperse in ultrapure water by ultrasonic treatment; after dialysis, the polymer nano photosensitizer is obtained; The polymer nano photosensitizer is PolySe NPs, PolyC NPs, and PolyS NPs.

5. The preparation method according to claim 4, wherein: In step (1), the molar ratio of the amino acid raw material, NaHCO3, and PCF is 1:3:1.2; In step (2), the molar ratio of the intermediate and PEG2000-NH2 is 10:1; In step (3), the molar ratio of the polyethylene intermediate, PPa, EDC, and NHS is 1:1:2:

2.

6. The preparation method according to claim 4, wherein: In step (4), a dialysis bag with a molecular weight of 3500 Da is used.

7. Use of a nano-photosensitizer according to any one of claims 1-3, characterized in that: The nano photosensitizer is used for preparing an anti-tumor treatment drug.

8. Use according to claim 7, characterized in that: The tumor is breast cancer.

9. Use according to claim 7, characterized in that: The nano photosensitizer is used for preparing an anti-tumor treatment drug targeting tumor tissue.

10. Use according to claim 9, characterized in that: The nano photosensitizer is used for preparing an anti-tumor treatment drug targeting lysosomes in tumor tissue.

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