A nano photosensitizer and its preparation method and application

Abstract

The present invention discloses a nanophotosensitizer and its preparation method and application. The nanophotosensitizer uses a dihydrochlorin e6 photosensitizer as a parent core and constructs a targeted near-infrared photosensitizer by introducing a hydrophobic targeting molecule PARP inhibitor into the Ce6 photosensitizer structure. This fully exerts the photodynamic therapy effect of Ce6 while breaking the limitations of carrier-assisted nanomaterials. It does not have side effects such as carrier toxicity or immunogenicity, has better biosafety, and can exert anti-tumor effects with high efficiency and low toxicity. The present invention combines a targeted PARP inhibitor with an efficient photosensitizer and uses a carrier-free self-assembly strategy to prepare nanomedicines. A self-assembled carrier-free nanomedicine with a high drug loading capacity can be obtained. Under 660nm LED illumination conditions, singlet oxygen can be generated to induce tumor cell apoptosis while reducing toxic side effects. The nanomedicine can be used to prepare photodynamic therapy drugs for inhibiting the growth of tumor cells such as ovarian cancer SKOV3 cells and breast cancer 4T1 cells.

Description

Technical Field

[0001] The present invention relates to the technical field of photosensitive materials, and in particular to a nano photosensitizer and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy is one of the most promising non-invasive methods for treating malignant tumors. This therapy mainly destroys tumor cells by producing reactive oxygen species (such as singlet oxygen) through the photodynamic reaction of photosensitizers under the excitation of a light source of a specific wavelength. In the past 50 years, the first generation of photosensitizers represented by hematoporphyrin (HpD) have been used in the clinical treatment of bladder cancer, esophageal cancer, and lung cancer (Cancers 2020, 12, 2793). However, the first generation of photosensitizers generally have shortcomings such as complex composition, poor tissue selectivity, shallow treatment depth, and obvious skin phototoxicity. In view of this, the second generation of photosensitizers represented by dihydrochlorin e6 (Chlorin e6, Ce6) has attracted much attention in recent years. It has the advantages of a single and clear structure, high biosafety, and superior photodynamic activity (Photochem.Photobiol 2023, 99, 469-497). Despite this, there are still many problems that need to be solved in the clinical treatment of tumors with photosensitizers, such as poor tumor targeting, low delivery efficiency, and limited intratumoral drug distribution.

[0003] In recent years, a large number of studies have utilized polymers, peptides, and inorganic nanomaterials as delivery vehicles to improve the tumor targeting of photosensitizers and enhance their intratumoral distribution, thereby enhancing the efficacy of tumor photodynamic therapy. The poly(adenosine diphosphate-ribose) polymerase (PARP) family is an important factor in the cellular DNA damage response, with its expression upregulated in many malignant tumors. Therefore, PARP-based DNA damage repair inhibitors, such as olaparib and niraparib, have shown promising therapeutic potential in recent years. Prior art discloses methods for preparing and applying tumor-targeted drug-loaded nanoformulations based on polyglutamic acid-conjugated photosensitizers. By conjugating the polymer to the photosensitizer, hydrophobic drugs such as PARP inhibitors are encapsulated and coated on the tumor cell membrane, achieving tumor targeting while enhancing the efficacy of photodynamic therapy. While the use of nanotechnology to construct targeted nanodrug delivery systems can improve the solubility and delivery efficiency of traditional drugs, enhance their bioavailability, and reduce adverse reactions, the efficacy, safety, pharmacokinetics, and other drugability characteristics of nanodrug carriers still require in-depth and systematic research. In addition, there are still some "bottleneck" problems in the future large-scale production and application of nanocarrier drugs, such as low drug loading capacity, complex preparation of carrier materials, high production costs, potential systemic toxicity and immunogenicity, etc., which greatly limit their clinical application.

[0004] The latest research shows that carrier-free self-assembled nanomedicines have attracted widespread attention due to their simple and flexible preparation methods, high drug loading capacity and delivery efficiency, long blood circulation half-life, and the ability to avoid side effects such as carrier-related toxicity and immunogenicity (Progress in Biochemistry and Biophysics 2022, 49 (12), 2278-2291). More and more carrier-free nanomedicines are being used in biomedical fields such as anti-tumor, antibacterial, anti-inflammatory and antioxidant. For example, Zhu et al. self-assembled the ferroptosis inducer Erastin and the photosensitizer Ce6 into carrier-free nanoparticles through π-π stacking and hydrogen bonding, thereby achieving a higher tumor cell uptake (Theranostics 2019, 9, 3293-3307). However, the current development and utilization of carrier-free self-assembled nanophotosensitizers is still very limited and urgently needs to be expanded. In addition, more importantly, the vast majority of clinically available photosensitizers have poor targeting to tumor cells, and their photodynamic therapy effects are not ideal. Therefore, how to develop a nanophotosensitizer with high delivery efficiency and good tumor targeting is the core of solving problems in the field of photodynamic therapy. Summary of the Invention

[0005] To address the aforementioned technical issues, the present invention aims to provide a nanophotosensitizer, its preparation method, and its application for tumor-targeted photodynamic therapy, effectively exerting photodynamic activity while reducing systemic toxic side effects. This invention combines a targeted PARP inhibitor with a highly effective photosensitizer, utilizing a carrier-free self-assembly strategy to prepare the nanomedicine, offering a new approach for more precise and effective anti-tumor treatment.

[0006] The present invention is achieved through the following technical solutions:

[0007] The first object of the present invention is to provide a nano photosensitizer having the structural formula shown in formula (I):

[0008]

[0009] The nano photosensitizer PARP-Ps provided by the present invention is a carrier-free targeted nano photosensitizer used to achieve tumor-targeted photodynamic therapy, which can effectively exert photodynamic activity while reducing systemic toxic side effects.

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned nanophotosensitizer, comprising the following steps: reacting a compound represented by formula (II) with dihydrochlorin e6 in a solvent in the presence of a catalyst and an acid-binding agent to obtain the compound represented by formula (I);

[0011] Wherein, the structural formula of formula (II) is as follows:

[0012]

[0013] The compound represented by formula (II) of the present invention is 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, which is a precursor of poly (adenosine diphosphate) ribose polymerase (PARP) inhibitor. PARP inhibitors have the following advantages: first, the hydrophobic structure of PARP inhibitors is conducive to the self-assembly process of nanomedicines; second, PARP inhibitors are overexpressed in tumor cells and can achieve tumor targeting; third, PARP inhibitors, as DNA damage repair inhibitors, have the effect of enhancing photodynamic therapy when used in combination with photosensitizers having DNA damaging effects.

[0014] The present invention uses chlorine e6 (Chlorine6, Ce6) photosensitizer as the mother core, and introduces a hydrophobic targeted molecule PARP inhibitor into the Ce6 photosensitizer structure. First, the carboxylic acid group in the Ce6 structure reacts with the catalyst 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to generate an active intermediate, and then the amino group in the compound represented by formula (II) attacks the carbonyl group in the active intermediate to obtain the compound represented by formula (I), and then the targeted near-infrared photosensitizer is formed by self-assembly, which fully exerts the photodynamic therapy effect of Ce6 while overcoming the limitations of the existing technology, and the synthetic preparation route is simple, with certain transformation prospects. The preparation method of the present invention is simple to operate and does not require any chemical modification. More importantly, this method can break the limitations of carrier-assisted nanomaterials, does not have side effects such as carrier toxicity or immunogenicity, has better biosafety, and can exert anti-tumor effects with high efficiency and low toxicity.

[0015] Furthermore, the catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), 1-hydroxybenzotriazole, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or 4-dimethylaminopyridine.

[0016] Furthermore, the acid binding agent is N,N-diisopropylethylamine (DIPEA), triethylamine (Et3N) or pyridine.

[0017] Furthermore, the solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran and dimethyl sulfoxide.

[0018] Furthermore, the molar ratio of the compound represented by (II), dihydrochlorin e6, catalyst and acid binding agent is 1:(1-2):(1.1-1.5):(0.2-2.5).

[0019] In a specific embodiment, the compound represented by formula (II) is reacted with dihydrochlorin e6 in the presence of a catalyst and an acid-binding agent in a solvent, and the reaction further comprises the steps of extraction with an organic solvent, washing, drying, removing the organic solvent, and purification.

[0020] In a specific embodiment, chlorin e6 is dissolved in an organic solvent, and a compound represented by formula (II), a catalyst, and an acid-binding agent are added. The reaction is allowed to proceed at room temperature for 5-6 hours. After the reaction is completed, the solution is diluted with water and the pH is adjusted to 3 with a weak acid. The solution is extracted with an organic solvent, and the organic solvent layer is washed and dried. The organic solvent is removed using a rotary evaporator to obtain a crude product, which is then purified by silica gel column chromatography to obtain a dark green solid product, the nanophotosensitizer PARP-Ps.

[0021] Furthermore, the weak acid is a 5% citric acid aqueous solution, the organic solvent is preferably CH2Cl2, the washing operation uses a saturated sodium chloride solution to wash the organic solvent layer, the drying operation uses anhydrous Na2SO4 for drying, and the purification eluents are methanol and dichloromethane.

[0022] The third object of the present invention is to provide a nano-micelle, wherein the nano-micelle is formed by molecular self-assembly of the above-mentioned nano-photosensitizer in water.

[0023] Due to the amphiphilic structure of the nano photosensitizer of the present invention, a carrier-free self-assembled nanodrug with a high drug loading capacity can be obtained by dialysis to form nanomicelle PARP-Ps NPs. Under the condition of 660nm LED light, the singlet oxygen generated can induce tumor cell apoptosis while reducing toxic side effects.

[0024] A fourth object of the present invention is to provide a method for preparing the above-mentioned nanomicelles, comprising the following steps:

[0025] (1) dissolving the nano photosensitizer in an organic solvent to form a dispersion;

[0026] (2) adding water to the dispersion to prepare the nanomicelles by dialysis.

[0027] Furthermore, the organic solvent is dimethyl sulfoxide, tetrahydrofuran or N,N-dimethylformamide.

[0028] The use of different organic solvents may lead to differences in nanoparticle formation efficiency, morphology and size due to the different solubility of PARP-Ps in different solvents.

[0029] The fifth object of the present invention is to provide the use of the above-mentioned nano-photosensitizer or the above-mentioned nano-micelle in the preparation of photodynamic therapy drugs, which can be used to inhibit the growth of tumor cells such as ovarian cancer SKOV3 cells and breast cancer 4T1 cells.

[0030] Beneficial effects of the present invention:

[0031] 1. The present invention uses the chlorin e6 (Chlorine6, Ce6) photosensitizer as the parent core and introduces a hydrophobic targeting molecule PARP inhibitor into the Ce6 photosensitizer structure to construct a targeted near-infrared photosensitizer. This fully utilizes the photodynamic therapy effect of Ce6 while overcoming the limitations of existing technologies. The synthetic preparation route is simple and has certain transformation prospects. The preparation method of the present invention is simple to operate and does not require any chemical modification. More importantly, this method can overcome the limitations of carrier-assisted nanomaterials, does not have side effects such as carrier toxicity or immunogenicity, has better biosafety, and can exert anti-tumor effects with high efficiency and low toxicity.

[0032] 2. The nanophotosensitizer PARP-Ps provided by the present invention is a carrier-free targeted nanophotosensitizer used to achieve tumor-targeted photodynamic therapy, effectively exerting photodynamic activity while reducing systemic toxic side effects. Due to its amphiphilic structure, dialysis can be used to obtain self-assembled, carrier-free nanomedicines with high drug loading, forming nanomicelle PARP-Ps NPs. Under 660nm LED illumination, the singlet oxygen produced can induce tumor cell apoptosis while reducing toxic side effects. The nanophotosensitizer PARP-Ps and nanomicelle PARP-Ps NPs can be used to prepare photodynamic therapy drugs for inhibiting the growth of tumor cells such as ovarian cancer SKOV3 cells and breast cancer 4T1 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the synthetic route of PARP-Ps.

[0034] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of PARP-Ps.

[0035] Figure 3 This is the mass spectrum of PARP-Ps.

[0036] Figure 4 These are the hydrated particle size images and TEM images of PARP-Ps NPs; (a) is the hydrated particle size image, and (b) is the TEM image.

[0037] Figure 5 is the Zeta potential diagram of PARP-Ps NPs and Ce6 NPs.

[0038] Figure 6 This is the particle size stability test results of PARP-Ps NPs and Ce6 NPs.

[0039] Figure 7This is the photostability test results of PARP-Ps NPs and Ce6 NPs.

[0040] Figure 8 UV-visible absorption spectra of PARP-Ps, PARP-Ps NPs, Ce6, and Ce6 NPs.

[0041] Figure 9 are the fluorescence emission spectra of PARP-Ps, PARP-Ps NPs, Ce6, and Ce6 NPs.

[0042] Figure 10 This is a diagram showing the quenching of DPBF by PARP-Ps NPs, Ce6, and Ce6 NPs under light conditions.

[0043] Figure 11 This is a graph showing the toxicity test results of PARP-Ps, PARP-Ps NPs, Ce6 and Ce6 NPs on SKOV3 cells.

[0044] Figure 12 This is a graph showing the toxicity test results of PARP-Ps, PARP-Ps NPs, Ce6 and Ce6 NPs on 4T1 cells.

[0045] Figure 13 This is a curve of mouse tumor changes in the PARP-Ps NPs and PBS groups within 15 days. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0049] The present invention provides a nano photosensitizer PARP-Ps having the structural formula shown in formula (I):

[0050]

[0051] The present invention provides a method for preparing the above-mentioned nano photosensitizer, comprising the following steps: reacting a compound represented by formula (II) with dihydrochlorin e6 in a solvent in the presence of a catalyst and an acid-binding agent to obtain the compound represented by formula (I);

[0052] Among them, the compound represented by formula (II) is 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, and its structural formula is shown below:

[0053]

[0054] Synthesis route diagram Figure 1 As shown, the present invention uses Ce6 photosensitizer as the mother core and constructs a targeted near-infrared photosensitizer by introducing a hydrophobic targeting molecule PARP inhibitor into the Ce6 photosensitizer structure.

[0055] The present invention provides a nano micelle PARP-Ps NPs. The nano micelle is formed by molecular self-assembly of the nano photosensitizer in water.

[0056] Example 1

[0057] A nano photosensitizer and a preparation method thereof, comprising the following steps:

[0058] 50 mg of Ce₆ was dissolved in 3 mL of N,N-dimethylformamide (DMF) solution. Then, 31 mg of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 24.3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and 27.4 mg of N,N-diisopropylethylamine (DIPEA) were added. The molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, Ce₆, EDCl, and DIPEA was 1:1:1.5:2.5. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the reaction mixture was diluted with water, the pH was adjusted to 3 with 5% aqueous citric acid, and the mixture was extracted three times with CHCl₂. The CHCl₂ layer was washed with saturated sodium chloride solution and dried over anhydrous Na₂SO₄. CH2Cl2 was removed by rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography (eluents: methanol and dichloromethane) to obtain a dark green solid product PARP-Ps with a yield of 40%.

[0059] Weigh 0.5 mg of PARP-Ps and dissolve it in 1 mL of dimethyl sulfoxide and ultrasonically disperse it for 20 minutes. Then, slowly add 3 mL of deionized water during ultrasonication, mix well, and continue ultrasonication for 20 minutes. Place the above solution in a 3500Da dialysis bag and dialyze it for 24 hours to obtain PARP-Ps NPs.

[0060] Example 2

[0061] A nano photosensitizer and a preparation method thereof, comprising the following steps:

[0062] 25 mg of Ce6 was weighed and dissolved in 3 mL of N,N-dimethylformamide (DMF). Then, 15.4 mg of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 17.6 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 13.6 mg of N,N-diisopropylethylamine (DIPEA) were added. The molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, Ce6, HATU, and DIPEA was 1:1:1.1:2.5. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the reaction mixture was diluted with water, the pH was adjusted to 3 with 5% aqueous citric acid, and the mixture was extracted three times with CHCl. ​​The CHCl layer was washed with saturated sodium chloride solution and dried over anhydrous NaSO. CH2Cl2 was removed by rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography (eluents: methanol and dichloromethane) to obtain a dark green solid product with a yield of 54%.

[0063] 1.0 mg of PARP-Ps was dissolved in 2 mL of dimethyl sulfoxide and ultrasonically dispersed for 20 min. Then, 5 mL of deionized water was slowly added dropwise during ultrasonication. After mixing, ultrasonication was continued for 20 min. The above solution was placed in a 3500 Da dialysis bag and dialyzed for 48 h to obtain PARP-Ps NPs.

[0064] Example 3

[0065] A nano photosensitizer and a preparation method thereof, comprising the following steps:

[0066] 97.8 mg of Ce₆ was dissolved in 4 mL of N,N-dimethylformamide (DMF) solution, followed by the addition of 30 mg of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 23.6 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and 26.5 mg of N,N-diisopropylethylamine (DIPEA). The molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, Ce₆, EDCl, and DIPEA was 1:2:1.5:2.5. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the reaction mixture was diluted with water, the pH adjusted to 3 with 5% aqueous citric acid, and extracted three times with CHCl₂. The CHCl₂ layer was washed with saturated sodium chloride solution and dried over anhydrous Na₂SO₄. CH2Cl2 was removed by rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography (eluent: methanol and dichloromethane) to obtain a dark green solid product with a yield of 30%.

[0067] Weigh 0.5 mg of PARP-Ps and dissolve it in 1 mL of tetrahydrofuran and ultrasonically disperse it for 20 minutes. Then, slowly add 5 mL of deionized water during ultrasonication, mix well, and continue ultrasonication for 20 minutes. Place the above solution in a 3500Da dialysis bag and dialyze it for 24 hours to obtain PARP-Ps NPs.

[0068] Example 4

[0069] A nano photosensitizer and a preparation method thereof, comprising the following steps:

[0070] 50 mg of Ce₆ was dissolved in 4 mL of N,N-dimethylformamide (DMF) solution, followed by the addition of 31 mg of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 24.3 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and 1.7 mg of triethylamine (Et₃N). The molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, Ce₆, EDCl, and Et₃N was 1:1:1.5:0.2. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the reaction mixture was diluted with water, the pH adjusted to 3 with 5% aqueous citric acid, and extracted three times with CHCl₂. The CHCl₂ layer was washed with saturated sodium chloride solution and dried over anhydrous Na₂SO₄. CH2Cl2 was removed by rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography (eluent: methanol and dichloromethane) to obtain a dark green solid product with a yield of 40%.

[0071] 1.0 mg of PARP-Ps was dissolved in 2 mL of tetrahydrofuran and ultrasonically dispersed for 20 min. Then, 5 mL of deionized water was slowly added dropwise during ultrasonication. After mixing, ultrasonication was continued for 20 min. The above solution was placed in a 3500 Da dialysis bag and dialyzed for 48 h to obtain PARP-Ps NPs.

[0072] Example 5

[0073] A nano photosensitizer and a preparation method thereof, comprising the following steps:

[0074] 100 mg of Ce₆ was dissolved in 5 mL of N,N-dimethylformamide (DMF) solution, followed by the addition of 55.8 mg of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, 43.8 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl), and 49.2 mg of N,N-diisopropylethylamine (DIPEA). The molar ratio of 4-(4-fluoro-3-(piperazine-1-carbonyl)benzyl)phthalazin-1(2H)-one, Ce₆, EDCl, and DIPEA was 1:1.1:1.5:2.5. The mixture was allowed to react at room temperature for 5 hours. After completion of the reaction, the reaction mixture was diluted with water, the pH adjusted to 3 with 5% aqueous citric acid, and extracted three times with CHCl₂. The CHCl₂ layer was washed with saturated sodium chloride solution and dried over anhydrous Na₂SO₄. CH2Cl2 was removed by rotary evaporator to obtain a crude product, which was purified by silica gel column chromatography (eluent: methanol and dichloromethane) to obtain a dark green solid product with a yield of 50%.

[0075] 1.0 mg of PARP-Ps was dissolved in 2 mL of N,N-dimethylformamide and ultrasonically dispersed for 20 min. Then, 5 mL of deionized water was slowly added dropwise during ultrasonication. After mixing, ultrasonication was continued for 20 min. The above solution was placed in a 3500 Da dialysis bag and dialyzed for 48 h to obtain PARP-Ps NPs.

[0076] Test Example 1 Structural Characterization

[0077] The structures of the PARP-Ps prepared in Examples 1 to 5 were characterized by 1H NMR nuclear magnetic resonance and mass spectrometry.

[0078] The test results are as follows Figure 2 、 Figure 3 As shown, Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of PARP-Ps, Figure 3 This is the mass spectrum of PARP-Ps, Figure 2 、 Figure 3 This shows that the examples of the present invention all prepared PARP-Ps having the structural formula shown in formula (I).

[0079] Test Example 2 Morphology Characterization

[0080] The particle size distribution and Zeta potential of the PARP-Ps NPs prepared in Example 1 were measured using a Malvern dynamic light scattering instrument, and the NPs were dropped onto a carbon support film, and the morphology of the nanoparticles was observed using a transmission electron microscope.

[0081] The test results are as follows Figure 4 、 Figure 5 As shown, Figure 4 These are the hydration particle size diagram and TEM diagram of PARP-Ps NPs. It can be seen from the diagram that the hydration kinetic size of the PARP-Ps NPs prepared by the present invention is 143.2 nm, and the polydispersity coefficient is 0.202, indicating that the prepared PARP-Ps NPs are uniform in size, suitable in particle size and well dispersed. At the same time, transmission scanning electron microscopy shows that the particle size is about 63 nm. Figure 5 is the Zeta potential diagram of PARP-Ps NPs and Ce6 NPs. Among them, the preparation method of Ce6 NPs is the same as that of PARP-Ps NPs. The Zeta potential of PARP-Ps NPs is shown to be -31.5 mV, which is higher than that of Ce6 NPs, indicating that PARP-Ps NPs has higher stability than Ce6 NPs.

[0082] Test Example 3: Long-term stability test

[0083] Under storage conditions of 4° C., the particle size stability of the PARP-Ps NPs and Ce6 NPs prepared in Example 1 was tested within 15 days.

[0084] The test results are as follows Figure 6 As shown, Figure 6 Figure 3 is the particle size stability test results of PARP-Ps NPs and Ce6 NPs. Compared with Ce6 NPs, PARP-Ps NPs showed good particle size stability within 15 days.

[0085] Test Example 4: Light Stability Test

[0086] Under illumination of a 660 nm LED lamp, the absorbance changes of the PARP-Ps NPs and Ce6 NPs prepared in Example 1 at 660 nm were monitored to test the photostability of the PARP-Ps NPs and Ce6 NPs.

[0087] The test results are as follows Figure 7 As shown, Figure 7This is the photostability test results of PARP-Ps NPs and Ce6 NPs. Compared with Ce6NPs, PARP-Ps NPs have good photostability under 660nm light conditions.

[0088] Test Example 5: UV-Vis Absorption Spectrum and Fluorescence Emission Spectrum Test

[0089] The PARP-Ps, PARP-Ps NPs, Ce6, and Ce6 NPs prepared in Example 1 were subjected to ultraviolet-visible absorption spectroscopy (UV-vis) and fluorescence emission spectroscopy (FL) tests. The test method is as follows:

[0090] PARP-Ps N,N-dimethylformamide solution, PARP-Ps NPs and Ce6 N,N-dimethylformamide solution, Ce6 NPs were prepared into 20 μM solutions, placed in cuvettes, and tested using a UV-visible spectrophotometer and a fluorescence spectrophotometer.

[0091] The test results are as follows Figure 8 、 9 As shown, Figure 8 is the UV-visible absorption spectra of PARP-Ps, PARP-Ps NPs, Ce6 and Ce6 NPs, Figure 9 The fluorescence emission spectra of PARP-Ps, PARP-Ps NPs, Ce6 and Ce6 NPs. It can be seen from the figure that the maximum absorption wavelengths of PARP-Ps are at 404nm and 661nm. When the excitation wavelength is fixed at 600nm, the maximum emission wavelength of PARP-Ps is at 665nm. PARP-Ps NPs have obvious absorption and emission in the near-infrared region. The maximum absorption wavelengths of PARP-Ps NPs are at 408nm and 669nm. When the excitation wavelength is fixed at 600nm, the maximum emission wavelengths of PARP-Ps NPs are at 655 and 722nm.

[0092] Test Example 6 Singlet Oxygen Quantum Yield Test

[0093] The singlet oxygen quantum yields of the PARP-Ps NPs, Ce6, and Ce6 NPs prepared in Example 1 were tested under light conditions. The testing method was as follows:

[0094] Ce6, Ce6 NPs and PARP-Ps NPs solutions were prepared respectively. 2.97 mL of each sample solution was taken and 30 μL of 800.0 μg mL -1 The singlet oxygen probe 1,3-diphenylisobenzofuran (DPBF) solution was mixed well and placed in a quartz cuvette. ... -2) were used to irradiate the above samples respectively, and the absorbance at 415 nm was recorded at 0, 10, 20, 30, 40, 50, 60, and 70 seconds after irradiation.

[0095] The line graph is obtained by plotting the absorbance change. The test results are as follows: Figure 10 As shown, Figure 10 The figure shows the quenching of DPBF by PARP-Ps NPs, Ce6 and Ce6 NPs under light conditions. PARP-Ps NPs showed a strong ability to produce singlet oxygen under light conditions. Ce6 (Φ Δ＝0.26 ) as a reference, the singlet oxygen quantum yield of PARP-Ps NPs was calculated to be 0.26, which is higher than the 0.09 of Ce6 NPs. These experimental results show that PARP-Ps NPs have the ability to generate singlet oxygen and show great potential in photodynamic therapy.

[0096] Test Example 7 Cytotoxicity Test

[0097] The toxicity of PARP-Ps, PARP-Ps NPs, Ce6, and Ce6 NPs prepared in Example 1 to ovarian cancer SKOV3 cells and breast cancer 4T1 cells under illumination and non-illumination conditions was tested using CCK8 assay. The test method was as follows:

[0098] Ovarian cancer SKOV3 and breast cancer 4T1 cells in the logarithmic growth phase were inoculated into 96-well plates and cultured, with the number of cells per well being 8.0×10 3 The cells were placed in a cell culture incubator (37°C, 5% CO2) and incubated for 24 hours. The illumination group and the non-illumination group were set up, and PARP-Ps, PARP-Ps NPs and Ce6, Ce6 NPs solutions were added respectively. The concentration gradient was set to 0, 0.002, 0.02, 0.1, 0.2, 1, 2, 5, 10, 20 μM, and five replicate wells were set for each concentration (100.0 μL per well). The cells were placed in the incubator and incubated for another 24 hours. After incubation, the culture medium was replaced. The illumination group was illuminated with LED light (660 nm, 50 mW·cm -2 ) for 10 minutes (the non-illumination group received no treatment) and then incubated for 24 hours. After incubation, CCK8 solution (10 μL per well) was added to each well and incubated in an incubator for another hour. Finally, the absorbance (OD) of the cell samples at 450 nm was measured using a microplate reader and calculated.

[0099] The test results are as follows Figure 11 、 Figure 12 As shown, Figure 11 This is the toxicity test results of PARP-Ps, PARP-Ps NPs, Ce6 and Ce6 NPs on SKOV3 cells. Figure 12Figure 1 shows the toxicity test results of PARP-Ps, PARP-Ps NPs, Ce6, and Ce6 NPs on 4T1 cells. Under non-illumination conditions, PARP-Ps, PARP-Ps NPs, Ce6, and Ce6 NPs showed no significant toxicity to SKOV3 cells and 4T1 cells. Under illumination conditions, PARP-Ps NPs showed stronger phototoxicity than Ce6 NPs in both SKOV3 cells and 4T1 cells. The IC of PARP-Ps NPs in SKOV3 cells was 2.34, which was 0.03. 50 The IC value of PARP-Ps NPs in 4T1 cells was 0.1 μM. 50 The value is 0.26 μM. The above results show that PARP-Ps has a significant photodynamic therapy effect, can achieve tumor molecular targeting while effectively exerting photodynamic therapy, and after being self-assembled into nanoparticles, it has a significant inhibitory effect on tumor cells.

[0100] Test Example 7 Animal Tumor Inhibition Effect Test

[0101] The PARP-Ps NPs prepared in Example 1 and phosphate buffered saline (PBS) were tested for their tumor inhibition effects in animals. The test method was as follows:

[0102] Construct a subcutaneous 4T1 tumor model with a tumor volume of 60 mm 3 Female BALB / c tumor-bearing mice were divided into PARP-Ps NPs group and PBS group (3 mice in each group). The PARP-Ps NPs were injected into the tail vein (10 mg kg -1 ), and an LED light (660 nm, 50 mW·cm -2 ) The tumor site of the mouse was irradiated for 30 minutes, and the changes in tumor volume within 15 days after illumination were recorded. A curve of tumor volume change over time was drawn to evaluate the anti-tumor effect of the preparation.

[0103] The test results are as follows Figure 13 As shown, Figure 13 This is a curve of mouse tumor changes in the PARP-Ps NPs and PBS groups within 15 days. It can be seen from the figure that the PARP-Ps NPs illumination group showed a significant tumor inhibition effect and has the potential for photodynamic therapy of tumors.

[0104] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A nano photosensitizer, characterized in that: It has the structural formula shown in formula (I): (Ⅰ)。 2. A method for preparing the nano photosensitizer according to claim 1, characterized in that: The following steps are involved: The compound represented by formula (II) is reacted with dihydrochlorin e6 in the presence of a catalyst and an acid-binding agent in a solvent to obtain the compound represented by formula (I); Wherein, the structural formula of formula (II) is as follows: (Ⅱ)。 3. The preparation method according to claim 2, wherein The catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-hydroxybenzotriazole, 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate or 4-dimethylaminopyridine.

4. The preparation method according to claim 2, wherein The acid binding agent is N, N-diisopropylethylamine, triethylamine or pyridine.

5. The preparation method according to claim 2, wherein The solvent is selected from one or more of N,N-dimethylformamide, tetrahydrofuran and dimethyl sulfoxide.

6. The preparation method according to claim 2, characterized in that The molar ratio of the compound represented by formula (II), dihydrochlorin e6, catalyst and acid binding agent is 1:(1-2):(1.1-1.5):(0.2-2.5).

7. A nanomicelle, characterized in that: Prepared by the following method: (1) dissolving the nano photosensitizer according to claim 1 in an organic solvent to form a dispersion; (2) Water is added to the dispersion to prepare the nanomicelles by dialysis.

8. A method for preparing the nanomicelles according to claim 7, characterized in that: The following steps are involved: (1) dissolving the nano photosensitizer according to claim 1 in an organic solvent to form a dispersion; (2) Water is added to the dispersion to prepare the nanomicelles by dialysis.

9. The method for preparing nanomicelles according to claim 8, wherein The organic solvent is selected from dimethyl sulfoxide, tetrahydrofuran or N, N-dimethylformamide.

10. Use of the nanophotosensitizer according to claim 1 or the nanomicelle according to claim 7 in the preparation of photodynamic therapy drugs.

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