A near-infrared light-induced semiconductor polymer and its hydrolysis product, side-chain modified polymer, drug delivery system, light-stimulus-responsive nano-delivery system, and preparation method and application thereof

Abstract

The present invention provides a near-infrared light-induced semiconductor polymer and its hydrolysis product, side-chain modified polymer, drug delivery system, light-stimulated responsive nanodelivery system, and preparation methods and applications, relating to the field of biomedical technologies. The near-infrared light-induced semiconductor polymer and its hydrolysis product, side-chain modified polymer, drug delivery system (OPTC), and light-stimulated responsive nanodelivery system (OPTC-RNP) provided by the present invention all have low spontaneous background fluorescence, deep tissue penetration, and high signal-to-noise ratio during near-infrared second-window imaging, with excellent near-infrared second-window imaging effects, and the accumulation of the nanodelivery system at the tumor site can be observed. Moreover, the photosensitizer in the delivery system can generate singlet oxygen under the irradiation of near-infrared light, realizing tumor photodynamic therapy while breaking the chemical bond connecting OPTC and RNP to release ribonucleoprotein complex (RNP), thus realizing light-controllable gene therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a near-infrared light-induced semiconductor polymer and a preparation method and application thereof, a hydrolysis product of the near-infrared light-induced semiconductor polymer and an application thereof, a side-chain modified polymer and a preparation method and application thereof, a drug delivery system and a preparation method and application thereof, and a light-stimulus-responsive nano-delivery system and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) lacks clear molecular targets and significant tumor heterogeneity. Common treatment methods include surgery and chemotherapy. These standard treatment methods severely limit their efficacy due to significant side effects and their adverse effects on the host immune system. In particular, the unique immunosuppressive tumor immune microenvironment of TNBC further limits the effect of immunotherapy. The immune system checkpoint blockade (ICB) method has good application prospects in permanently modifying target gene expression and stimulating the patient's immune system to fight cancer cells. Therefore, exploring and developing effective strategies to stimulate and prolong the immunotherapy time and improve the immunotherapy effect is of great significance in cancer treatment.

[0003] Immunogenic cell death (ICD) releases damage-associated molecules, including adenosine-5'-triphosphate (ATP), surface-exposed calreticulin (CALR), etc., which induce immune responses and play a key role in activating the tumor microenvironment. In particular, when combined with ICB, ICD can effectively activate and significantly enhance tumor-specific immune responses. During the treatment process of the commonly used photothermal therapy (PTT), the high temperature generated will cause irreversible damage to normal tissues and tissue edema. In contrast, photodynamic therapy (PDT) has more controllable spatiotemporality and safety. PDT shows great potential in highly efficient tumor treatment by inducing immunogenic cell death in tumors and combining gene editing technology to amplify the immune response.

[0004] Semiconductor polymer nanomaterials composed of polymers have become a versatile near-infrared (NIR) absorption / emission biomaterial for molecular imaging, phototherapy, and biological regulation, etc. Existing near-infrared emission materials mainly include inorganic near-infrared quantum dots, halide perovskites and other materials, as well as various organic near-infrared materials including traditional fluorescent small molecule materials, conjugated polymers, stable luminescent radicals, thermally activated delayed fluorescence (TADF) materials, and metal-organic complex phosphorescent materials, etc. Among many near-infrared emission materials in molecular imaging applications, the fluorescence emission of most materials mainly focuses on region I (650 - 900 nm). However, due to more imaging background interference factors, weak penetration power, and low signal-to-noise ratio in region I, the imaging effect of the materials is poor, which limits the application of near-infrared emission materials in the field of biological imaging. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a near-infrared light-induced semiconductor polymer, its hydrolysis product, side-chain modified polymer, drug delivery system, light-stimulated response nano-delivery system, preparation method and application. Each polymer and nano-delivery system provided by the present invention has low spontaneous background fluorescence, deep tissue penetration and high signal-to-noise ratio during near-infrared second-region imaging, and the near-infrared second-region imaging effect is excellent.

[0006] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a near-infrared light-induced semiconductor polymer having the structure shown in Formula I:

[0008] Formula I;

[0009] Wherein, n is 45-65.

[0010] The present invention also provides a preparation method of the near-infrared light-induced semiconductor polymer described in the above technical solution, including the following steps:

[0011] Performing a Suzuki reaction on monomer B and monomer C to obtain the near-infrared light-induced semiconductor polymer;

[0012] ;

[0013] Wherein, X is a halogen.

[0014] The present invention also provides a hydrolysis product of the near-infrared light-induced semiconductor polymer, and the near-infrared light-induced semiconductor polymer is the near-infrared light-induced semiconductor polymer described in the above technical solution or the near-infrared light-induced semiconductor polymer prepared by the preparation method described in the above technical solution, having the structure shown in OSP:

[0015] ;

[0016] Wherein, n is 45-65.

[0017] The present invention also provides a side-chain modified polymer having the structure of OSP-PEG or OPT:

[0018] ;

[0019] Wherein, n is 45-65 and m is 30-60.

[0020] The present invention also provides a preparation method of the side-chain modified polymer described in the above technical solution,

[0021] The preparation method of the side-chain modified polymer with the structure shown by OSP-PEG comprises the following steps: subjecting the hydrolysis product of the infrared light-induced semiconductor polymer described in the above technical solution to an amidation reaction with diamino polyethylene glycol to obtain the side-chain modified polymer with the structure shown by OSP-PEG; the degree of polymerization of the diamino polyethylene glycol is m;

[0022] The preparation method of the side-chain modified polymer with the structure shown by OPT comprises the following steps: subjecting the side-chain modified polymer with the structure shown by OSP-PEG to an amidation reaction with 3,3'-(propane-2,2-diylbis(thioanediyl))dipropanoic acid to obtain the side-chain modified polymer with the structure shown by OPT.

[0023] The present invention also provides a drug delivery system, comprising a side-chain modified polymer and a photosensitizer encapsulated in the side-chain modified polymer; the side-chain modifier is the side-chain modified polymer with the structure shown by OPT described in the above technical solution or the side-chain modified polymer with the structure shown by OPT prepared by the preparation method described in the above technical solution.

[0024] The present invention also provides a preparation method of the drug delivery system described in the above technical solution, comprising the following steps: mixing the side-chain modified polymer with the structure shown by OPT, a photosensitizer, an organic solvent and water, and performing self-assembly to obtain the drug delivery system.

[0025] The present invention also provides a near-infrared light-stimulated response nanodelivery system, comprising a drug delivery system and a gene editor; the drug delivery system is the drug delivery system described in the above technical solution or the drug delivery system prepared by the preparation method described in the above technical solution.

[0026] The present invention also provides a preparation method of the near-infrared light-stimulated response nanodelivery system described in the above technical solution, comprising the following steps: performing an amidation reaction on the drug delivery system and the gene editor to obtain the near-infrared light-stimulated response nanodelivery system.

[0027] The present invention also provides the applications of the near-infrared light-induced semiconductor polymer described in the above technical solution, the near-infrared light-induced semiconductor polymer prepared by the preparation method described in the above technical solution, the hydrolysis product of the near-infrared light-induced semiconductor polymer described in the above technical solution, the side-chain modified polymer described in the above technical solution, the side-chain modified polymer prepared by the preparation method described in the above technical solution, the drug delivery system described in the above technical solution, the drug delivery system prepared by the preparation method described in the above technical solution, the near-infrared light-stimulated response nanodelivery system described in the above technical solution or the near-infrared light-stimulated response drug delivery system prepared by the preparation method described in the above technical solution in biological imaging, preparing gene therapy drugs, preparing photodynamic therapy drugs for tumors or preparing anti-inflammatory drugs.

[0028] In the near-infrared light-induced semiconductor polymer provided by the present invention, fluorene-bithiophene is alternately connected to synthesize a conjugated polymer main chain, endowing it with fluorescence emission properties in the second near-infrared region (NIR-II, 1.0 - 1.7 μm). Moreover, during imaging in this region, it has low autofluorescence background, deep tissue penetration, and high signal-to-noise ratio, with excellent NIR-II imaging effect. The accumulation of the nano-delivery system at the tumor site can be observed in a NIR-II fluorescence imaging instrument.

[0029] The polymer main chain of the infrared light-induced semiconductor polymer with the structure shown by OSP provided by the present invention has excellent NIR-II imaging effect. The accumulation of the nano-delivery system at the tumor site can be observed in a NIR-II fluorescence imaging instrument. Moreover, the presence of carboxyl groups in the hydrolysis products increases the water solubility, chemical resistance, mechanical strength, thermal stability, and biocompatibility of the polymer. Moreover, carboxyl is a highly reactive functional group that can undergo various chemical reactions with other compounds, thereby realizing the functionalization of the polymer, which provides more possibilities for the further modification and application of polymer materials.

[0030] The polymer main chain of the side-chain modified polymer with the structure shown by OSP-PEG provided by the present invention has excellent NIR-II imaging effect. The accumulation of the nano-delivery system at the tumor site can be observed in a NIR-II fluorescence imaging instrument. Moreover, diamino polyethylene glycol has good water solubility. By introducing it into the side chain of the polymer, water solubility is achieved through the self-assembly of amphiphilic polymers. Diamino polyethylene glycol has high chemical stability. By introducing it into the side chain of the polymer, the polymer can maintain good stability in various environments. Diamino polyethylene glycol has good biocompatibility. By introducing it into the side chain of the polymer, the interaction between the polymer and the organism can be improved, reducing adverse reactions. The amino groups at both ends of diamino polyethylene glycol can undergo coupling reactions with molecules carrying carboxyl, aldehyde, or other reactive groups to form covalent bonds, thereby obtaining bio-conjugates with specific structures and functions. This property makes it have broad application prospects in constructing complex biomaterials and drug delivery systems, expanding its application scope.

[0031] The polymer main chain of the side-chain modified polymer with the structure shown by OPT provided by the present invention has excellent NIR-II imaging effect. The accumulation of the nano-delivery system at the tumor site can be observed in a NIR-II fluorescence imaging instrument. Meanwhile, the polymer can self-assemble into nanoparticles and encapsulate photosensitizers (such as Ce6). Under the excitation of near-infrared light, a large amount of singlet oxygen ( 1 1O2) is generated to achieve photodynamic therapy. Moreover, the singlet oxygen-responsive group in the side-chain modified polymer with the structure shown by OPT can be connected to ribonucleoprotein complex (RNP) to form a nano-delivery system.

[0032] The polymer backbone of the drug delivery system (denoted as OPTC) provided by the present invention has excellent second near-infrared imaging effect, and the accumulation of the nano-delivery system at the tumor site can be observed in a second-region fluorescence imaging instrument. Moreover, the photosensitizer can generate a large amount of 1 O2 under the irradiation of near-infrared light to achieve photodynamic therapy of tumors.

[0033] The polymer backbone of the light-stimulated responsive nano-delivery system (denoted as OPTC-RNP) provided by the present invention has excellent second near-infrared imaging effect, and the accumulation of the nano-delivery system at the tumor site can be observed in a second-region fluorescence imaging instrument. Moreover, the photosensitizer can generate 1 O2 under the irradiation of near-infrared light. While achieving photodynamic therapy of tumors, the chemical bond connecting OPTC and RNP can be broken, thereby releasing RNP to achieve light-controllable gene therapy. Description of the Drawings

[0034] Figure 1 Fluorescence emission spectrum of OPTC-RNP prepared in Example 1;

[0035] Figure 2 UV-visible absorption spectra of OSP, OPT, OPTC, TK and Ce6 prepared in Example 1;

[0036] Figure 3 TEM image of OPTC-RNP prepared in Example 1;

[0037] Figure 4 Dynamic light scattering (DLS) curves of OPTC and OPTC-RNP prepared in Example 1;

[0038] Figure 5 Zeta potential diagrams of OSP, OPT, OPTC and OPTC-RNP prepared in Example 1;

[0039] Figure 6 Relative absorbance diagram of 1,3-diphenylisobenzofuran (DPBF) after adding OPTC-RNP prepared in Example 1 with or without NIR irradiation (660 nm);

[0040] Figure 7 SDS-PAGE diagram of the remaining amount of RNP in OPTC-RNP prepared in Example 1 after NIR irradiation for different times;

[0041] Figure 8 RNP release diagram corresponding to OPTC-RNP prepared in Example 1 under NIR irradiation for different times;

[0042] Figure 9Confocal laser scanning microscopy (CLSM) images of OPTC-RNP prepared for Example 1 of labeled enhanced green fluorescent protein (EGFP) incubated with 4T1 cells for 6 h respectively;

[0043] Figure 10 Distribution maps of OPTC-RNP prepared in Example 1 in mice detected by a second near-infrared in vivo imager at different time points;

[0044] Figure 11 Graphs of the changes in the signal intensities of OPTC-RNP prepared in Example 1 in the liver and tumors of mice at different time points;

[0045] Figure 12 Two-region imaging analysis diagrams of the major organs and tumor tissues of mice 48 h after administration (OPTC-RNP prepared in Example 1);

[0046] Figure 13 Fluorescent staining images of 2′,7′-dichlorofluorescein diacetate (DCFH-DA, detecting 1 O2) of 4T1 cells in different treatment groups;

[0047] Figure 14 Live-dead fluorescent staining images of 4T1 cells in different treatment groups;

[0048] Figure 15 Diagrams of the apoptosis of 4T1 cells detected by flow cytometry under different treatment conditions;

[0049] Figure 16 Diagrams of the gene mutation frequencies of PD-L1 in 4T1 cells detected by the T7EI method after different treatments;

[0050] Figure 17 Diagrams of PD-L1 in 4T1 cells detected by WB after different treatments;

[0051] Among them, Figures 13 to 17 The conditions of each group are as follows, I: PBS / NIR, II: OPTC prepared in Example 1, III: OPTC / NIR prepared in Example 1, IV: OPTC-RNP prepared in Example 1, V: OPTC-RNP / NIR prepared in Example 1. Detailed implementation mode

[0052] The present invention provides a near-infrared light-induced semiconductor polymer having the structure shown in Formula I:

[0053] Formula I;

[0054] Wherein, n is 45 - 65, and in specific embodiments, it can be 45, 50, 53, 55, 60 or 65.

[0055] The present invention also provides a method for preparing the near-infrared light-induced semiconductor polymer described in the above technical solution, which includes the following steps: performing a Suzuki reaction on monomer B and monomer C to obtain the near-infrared light-induced semiconductor polymer;

[0056] ;

[0057] Wherein, X is a halogen, and in specific embodiments, it can be fluorine, chlorine, bromine or iodine.

[0058] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0059] In the present invention, the molar ratio of monomer B to monomer C is preferably 1:0.8 - 1, and in specific embodiments, it can be 1:0.8, 1:0.9 or 1:1.

[0060] In the present invention, the Suzuki reaction is preferably carried out under the conditions of a catalyst, a basic reagent and a solvent. Specifically, monomer B, monomer C, the catalyst, the basic reagent and the solvent are mixed to carry out the Suzuki reaction.

[0061] In the present invention, the catalyst preferably includes at least one of tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) and bis(triphenylphosphine)palladium dichloride; the molar ratio of monomer B to the catalyst is preferably 1:0.05 - 0.1, and in specific embodiments, it can be 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09 or 1:0.1.

[0062] In the present invention, the basic reagent preferably includes alkali metal carbonates, and more preferably includes at least one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3. In the present invention, the molar ratio of monomer B to the basic reagent is preferably 1:8 - 9, and in specific embodiments, it can be 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:8.9 or 1:9.

[0063] In the present invention, the solvent preferably includes an organic solvent and water, and the organic solvent preferably includes at least one of toluene and acetone; the volume ratio of water to the organic solvent is preferably 1:2 - 3, and in specific embodiments, it can be 1:2, 1:2.5 or 1:3. In the present invention, the dosage ratio of monomer B to the solvent is preferably 1 mmol:30 - 40 mL, and in specific embodiments, it can be 1 mmol:30 mL, 1 mmol:35 mL or 1 mmol:40 mL.

[0064] In the present invention, the temperature of the Suzuki reaction is preferably 65 - 95 °C, and in specific embodiments, it can be 65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C or 95 °C; the time of the Suzuki reaction is preferably 20 - 40 min, and in specific embodiments, it can be 20 min, 25 min, 30 min, 35 min or 40 min; the Suzuki reaction is preferably carried out under a protective atmosphere, and the protective atmosphere preferably includes nitrogen, argon or helium.

[0065] After completing the Suzuki reaction, the present invention preferably further includes cooling the reaction system obtained from the Suzuki reaction to room temperature, sedimenting with methanol, separating the solid and liquid, and concentrating the obtained liquid component to a constant weight to obtain the near-infrared light-induced semiconductor polymer. In the present invention, the temperature of the methanol sedimentation is preferably room temperature, and the time is preferably 12 - 48 h, and in specific embodiments, it can be 12 h, 15 h, 20 h, 24 h, 30 h, 35 h, 40 h, 45 h or 48 h; the volume fraction of methanol in the system during the methanol sedimentation is preferably 85 - 95%, and in specific embodiments, it can be 85%, 88%, 90%, 92% or 95%. In the present invention, the concentration preferably includes rotary evaporation.

[0066] In the present invention, the preparation method of the monomer B preferably includes the following steps: mixing the monomer A, bis(pinacolato)diboron, a catalyst, a basic reagent and an organic solvent, and carrying out a borylation reaction to obtain the monomer B. The preparation route is as follows:

[0067] ;

[0068] Among them, X is a halogen, and in specific embodiments, it can be fluorine, chlorine, bromine or iodine.

[0069] In the present invention, the molar ratio of the monomer A to bis(pinacolato)diboron is preferably 1:2.5 - 3.5, and in specific embodiments, it can be 1:2.5, 1:3 or 1:3.5.

[0070] In the present invention, the catalyst preferably includes at least one of Pd(dppf)2Cl2 (1,1'-bis(diphenylphosphino)ferrocene dichloropalladium) and Pd(PPh3)4 (tetrakis(triphenylphosphine)palladium); the molar ratio of the monomer A to the catalyst is preferably 1:0.3 - 0.7, and in specific embodiments, it can be 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.64 or 1:0.7.

[0071] In the present invention, the basic reagent preferably includes one or more of alkali metal acetates, alkali metal phosphates, and alkali metal carbonates, and more preferably includes at least one of KOAc, NaOAc, K3PO4, Na3PO4, Na2CO3, and K2CO3. In the present invention, the molar ratio of the monomer A to the basic reagent is preferably 1:2.5 to 7, and in specific embodiments, it can be 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, or 1:7.

[0072] In the present invention, the organic solvent preferably includes at least one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and methyl ethyl ketone. In the present invention, the dosage ratio of the monomer A to the organic solvent is preferably 1 mmol: 2 to 10 mL, and in specific embodiments, it can be 1 mmol: 2 mL, 1 mmol: 3 mL, 1 mmol: 4 mL, 1 mmol: 5 mL, 1 mmol: 6 mL, 1 mmol: 7 mL, 1 mmol: 8 mL, 1 mmol: 9 mL, or 1 mmol: 10 mL.

[0073] In the present invention, the temperature of the borylation reaction is preferably 90 to 120 °C, and in specific embodiments, it can be 90 °C, 95 °C, 100 °C, 105 °C, 110 °C, 115 °C, or 120 °C; the time of the borylation reaction is preferably 12 to 24 h, and in specific embodiments, it can be 12 h, 15 h, 18 h, 20 h, 22 h, or 24 h; the borylation reaction is preferably carried out under a protective atmosphere, and the protective atmosphere preferably includes nitrogen, argon, or helium.

[0074] After the borylation reaction, the present invention preferably further includes: filtering the reaction system obtained from the borylation reaction, concentrating and drying the obtained filtrate, and purifying it by a silica gel column to obtain the monomer B. In the present invention, the eluent used for the silica gel column purification preferably includes petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is preferably 1 to 5:1, and in specific embodiments, it can be 1:1, 2:1, 3:1, 4:1, or 5:1.

[0075] In the present invention, the preparation method of the monomer A preferably includes the following steps: mixing 2,7-dihalofluorene, tert-butyl acrylate, a catalyst, a basic reagent, and an organic solvent, and carrying out a nucleophilic substitution reaction to obtain the monomer A. The preparation route is as follows:

[0076] 。

[0077] In the present invention, the molar ratio of the 2,7-dihalofluorene to tert-butyl acrylate is preferably 1:4 to 4.5, and in specific embodiments, it can be 1:4, 1:4.1, 1:4.2, 1:4.3, 1:4.4, or 1:4.5. In the present invention, the halogen in the 2,7-dihalofluorene preferably includes fluorine, chlorine, bromine, or iodine. In the present invention, the tert-butyl acrylate is used as an alkylating agent.

[0078] In the present invention, the catalyst preferably includes at least one of tetrabutylammonium bromide and bis(triphenylphosphine)palladium dichloride; the molar ratio of the 2,7-dihalofluorene to the catalyst is preferably 1:0.01 to 0.1, and in specific embodiments, it can be 1:0.01, 1:0.02, 1:0.03, 1:0.04, 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09, or 1:0.1.

[0079] In the present invention, the basic reagent preferably includes one or more of alkali metal hydroxides, alkali metal acetates, alkali metal bicarbonates, and alkali metal carbonates, and more preferably includes one or more of KOH, NaOH, KOAc, NaOAc, KHCO3, NaHCO3, K2CO3, and Na2CO3; the basic reagent is preferably used in the form of an aqueous solution of the basic reagent, and the concentration of the aqueous solution of the basic reagent is preferably 1 to 2 M (mol / L), and in specific embodiments, it can be 1 M, 1.5 M, or 2 M. In the present invention, the molar ratio of the 2,7-dihalofluorene to the basic reagent is preferably 1:0.5 to 1.5, and in specific embodiments, it can be 1:0.5, 1:0.8, 1:1, 1:1.2, or 1:1.5.

[0080] In the present invention, the organic solvent preferably includes at least one of toluene and acetone. In the present invention, the dosage ratio of the 2,7-dihalofluorene to the organic solvent is preferably 1 mmol:4 to 6 mL, and in specific embodiments, it can be 1 mmol:4 mL, 1 mmol:4.5 mL, 1 mmol:5 mL, 1 mmol:5.5 mL, or 1 mmol:6 mL.

[0081] In the present invention, the temperature of the nucleophilic substitution reaction is preferably room temperature; the time of the nucleophilic substitution reaction is preferably 10 to 14 h, and in specific embodiments, it can be 10, 11 h, 12 h, 13 h, or 14 h; the nucleophilic substitution reaction is preferably carried out under a protective atmosphere, and the protective atmosphere preferably includes nitrogen, argon, or helium.

[0082] After the nucleophilic substitution reaction, the present invention preferably further includes: washing the reaction system obtained from the nucleophilic substitution reaction with water, washing with 0.1 - 1M HCl aqueous solution, extracting with dichloromethane, drying the obtained organic phase with Na2SO4, filtering, evaporating the filtrate to dryness by rotary evaporation, and purifying by silica gel column to obtain monomer A. In the present invention, the eluent used for the silica gel column purification preferably includes petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to the ethyl acetate is preferably 1 - 5:1, and in specific embodiments, it can be 1:1, 2:1, 3:1, 4:1, or 5:1.

[0083] The present invention also provides a hydrolysis product of a near-infrared light-induced semiconductor polymer, where the near-infrared light-induced semiconductor polymer is the near-infrared light-induced semiconductor polymer described in the above technical solution or the near-infrared light-induced semiconductor polymer prepared by the preparation method described in the above technical solution, and has the structure shown by OSP:

[0084] ;

[0085] Among them, n is 45 - 65, and in specific embodiments, it can be 45, 50, 53, 55, 60, or 65, and the definition of n is the same as that of n in the formula I.

[0086] The present invention also provides a preparation method of the hydrolysis product of the near-infrared light-induced semiconductor polymer described in the above technical solution, including the following steps: hydrolyzing the semiconductor polymer described in the above technical solution to obtain the hydrolysis product of the near-infrared light-induced semiconductor polymer.

[0087] In the present invention, the hydrolysis is preferably carried out under acidic and organic solvent conditions. In the present invention, the acid preferably includes trifluoroacetic acid; the dosage ratio of the near-infrared light-induced semiconductor polymer to the acid is preferably 1 mmol: 10 - 15 mL, and in specific embodiments, it can be 1 mmol: 10 mL, 1 mmol: 11 mL, 1 mmol: 12 mL, 1 mmol: 13 mL, 1 mmol: 14 mL, or 1 mmol: 15 mL. In the present invention, the organic solvent is preferably a halogenated alkane, and more preferably includes at least one of dichloromethane (DCM) and bromomethane (CH3Br). In the present invention, the dosage ratio of the near-infrared light-induced semiconductor polymer to the solvent is preferably 1 mmol: 100 - 150 mL, and in specific embodiments, it can be 1 mmol: 100 mL, 1 mmol: 110 mL, 1 mmol: 120 mL, 1 mmol: 130 mL, 1 mmol: 140 mL, or 1 mmol: 150 mL.

[0088] In the present invention, the temperature of the hydrolysis is preferably room temperature (20 - 25 °C), and the time is preferably 20 - 24 h, and in specific embodiments, it can be 20 h, 21 h, 22 h, 23 h, or 24 h.

[0089] The present invention also provides a side-chain modified polymer having a structure represented by OSP-PEG or OPT:

[0090] ;

[0091] Wherein, n is 45-65, and in specific embodiments, it can be 45, 50, 53, 55, 60 or 65. The definition of n in OSP-PEG and OPT is the same as that of n in the formula I; m is 30-60, and in specific embodiments, it can be 30, 35, 40, 45, 50, 55 or 60.

[0092] The present invention also provides a preparation method of the side-chain modified polymer having a structure represented by OSP-PEG as described in the above technical solution, including the following steps: subjecting the hydrolysis product of the infrared light-induced semiconductor polymer described in the above technical solution to an amidation reaction with diamino polyethylene glycol to obtain the side-chain modified polymer having a structure represented by OSP-PEG; the degree of polymerization of the diamino polyethylene glycol is m.

[0093] In the present invention, the molar ratio of the hydrolysis product of the infrared light-induced semiconductor polymer (calculated by the amount of structural units) to diamino polyethylene glycol (NH2-PEG-NH2) is preferably 1:4-5, and in specific embodiments, it can be 1:4, 1:4.2, 1:4.5, 1:4.8 or 1:5.

[0094] In the present invention, the amidation reaction is carried out under preferably catalytic conditions. In the present invention, the catalyst preferably includes a first catalyst and a second catalyst. The first catalyst preferably includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N,N'-diisopropylcarbodiimide (DIC); the second catalyst preferably includes at least one of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBT). In the present invention, the molar ratio of the hydrolysis product of the infrared light-induced semiconductor polymer to the first catalyst is preferably 1:40-50, and in specific embodiments, it can be 1:40, 1:42, 1:45, 1:48 or 1:50. In the present invention, the molar ratio of the hydrolysis product of the infrared light-induced semiconductor polymer to the second catalyst is preferably 1:20-30, and in specific embodiments, it can be 1:20, 1:22, 1:25, 1:28 or 1:30.

[0095] In the present invention, the organic solvent used in the amidation reaction preferably includes at least one of dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); the dosage ratio of the hydrolysis product of the infrared light-induced semiconductor polymer (calculated by the amount of structural units) to the organic solvent is preferably 1 mmol: 60-100 mL, and in specific embodiments, it can be 1 mmol: 60 mL, 1 mmol: 70 mL, 1 mmol: 80 mL, 1 mmol: 90 mL or 1 mmol: 100 mL.

[0096] In the present invention, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 48-72 h. In specific embodiments, it can be 48 h, 50 h, 55 h, 60 h, 65 h, 70 h or 72 h. Taking EDC and NHS as examples, in the present invention, during the amidation reaction, EDC first reacts with the carboxyl group to generate a reactive intermediate, and the reactive intermediate then reacts with NHS to generate an NHS ester intermediate. This NHS ester intermediate is relatively stable, which helps to improve the yield of the reaction.

[0097] After the amidation reaction, the present invention preferably further includes: subjecting the reaction system obtained from the amidation reaction to dialysis in water and then freeze-drying to obtain the side-chain modified polymer having the structure shown by OSP-PEG. In the present invention, the temperature of the dialysis in water is preferably room temperature, and the time of the dialysis in water is preferably 3-4 days. In specific embodiments, it can be 3 days, 3.5 days or 4 days; the cut-off molecular weight of the dialysis bag used for the dialysis in water is preferably 8-14 kDa, and in specific embodiments, it can be 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa or 14 kDa. The present invention has no special limitation on the freeze-drying, and the freeze-drying conditions well-known to those skilled in the art can be adopted.

[0098] The present invention also provides a preparation method of the side-chain modified polymer having the structure shown by OPT according to the above technical solution, including the following steps: subjecting the side-chain modified polymer having the structure shown by OSP-PEG to an amidation reaction with 3,3'-(propane-2,2-diylbis(thioalkandiyl))dipropionic acid to obtain a side-chain modified polymer having the structure shown by OPT.

[0099] In the present invention, the dosage ratio of the side-chain modified polymer having the structure shown by OSP-PEG to 3,3'-(propane-2,2-diylbis(thioalkandiyl))dipropionic acid (TK) is preferably 32 mg: 0.1-0.15 mmol. In specific embodiments, it can be 32 mg: 0.1 mmol, 32 mg: 0.11 mmol, 32 mg: 0.12 mmol, 32 mg: 0.13 mmol, 32 mg: 0.14 or 32 mg: 0.15 mmol.

[0100] In the present invention, the amidation reaction is preferably carried out under catalyst conditions. In the present invention, the catalyst preferably includes a first catalyst and a second catalyst; the first catalyst preferably includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N,N'-diisopropylcarbodiimide (DIC); the second catalyst preferably includes at least one of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBT). In the present invention, the dosage ratio of the side-chain modified polymer having the structure shown by OSP-PEG to the first catalyst is preferably 32 mg: 0.8-1 mmol, and in specific examples, it can be 32 mg: 0.8 mmol, 32 mg: 0.9 mmol, or 32 mg: 1 mmol. In the present invention, the dosage ratio of the side-chain modified polymer having the structure shown by OSP-PEG to the second catalyst is preferably 32 mg: 0.8-1 mmol, and in specific examples, it can be 32 mg: 0.8 mmol, 32 mg: 0.9 mmol, or 32 mg: 1 mmol.

[0101] In the present invention, the organic solvent used in the amidation reaction preferably includes at least one of dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); the dosage ratio of the side-chain modified polymer having the structure shown by OSP-PEG to the organic solvent is preferably 32 mg: 8-10 mL, and in specific examples, it can be 32 mg: 8 mL, 32 mg: 9 mL, or 32 mg: 10 mL.

[0102] In the present invention, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 48-72 h. In specific examples, it can be 48 h, 50 h, 55 h, 60 h, 65 h, 70 h, or 72 h.

[0103] After the amidation reaction, the present invention preferably further includes: subjecting the reaction system obtained from the amidation reaction to dialysis in water and then freeze-drying to obtain the side-chain modified polymer having the structure shown by OPT. In the present invention, the temperature of the dialysis in water is preferably room temperature, and the time of the dialysis in water is preferably 3-4 days. In specific examples, it can be 3 days, 3.5 days, or 4 days; the cut-off molecular weight of the dialysis bag used for the dialysis in water is preferably 8-14 kDa. In specific examples, it can be 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, or 14 kDa. The present invention has no special limitation on the freeze-drying, and the freeze-drying conditions well-known to those skilled in the art can be adopted.

[0104] The present invention also provides a drug delivery system (denoted as OPTC), comprising a side-chain modified polymer and a photosensitizer encapsulated in the side-chain modified polymer; the side-chain modifier is the side-chain modified polymer having the structure shown by OPT in the above technical solution or the side-chain modified polymer having the structure shown by OPT prepared by the preparation method in the above technical solution.

[0105] In the present invention, the photosensitizer preferably includes a photosensitizer capable of generating reactive oxygen species (ROS), and in specific embodiments, it can be one or more of chlorin e6 (Ce6), hematoporphyrin, hematoporphyrin monomethyl ether, and zinc phthalocyanine. In the present invention, the mass fraction of the photosensitizer in the drug delivery system is preferably 0.5 to 1 mg / mL, and in specific embodiments, it can be 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL.

[0106] The present invention also provides a preparation method of the drug delivery system in the above technical solution, comprising the following steps: mixing a side-chain modified polymer having the structure shown by OPT, a photosensitizer, an organic solvent, and water, and performing self-assembly to obtain the drug delivery system.

[0107] In the present invention, the mass ratio of the side-chain modified polymer having the structure shown by OPT to the photosensitizer is preferably 1:1 to 2, and in specific embodiments, it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, or 1:2.

[0108] In the present invention, the organic solvent preferably includes at least one of dimethyl sulfoxide (DMSO) and tetrahydrofuran. In the present invention, the dosage ratio of the side-chain modified polymer having the structure shown by OPT to the organic solvent is preferably 1 mg:0.8 to 1 mL, and in specific embodiments, it can be 1 mg:0.8 mL, 1 mg:0.9 mL, or 1 mg:1 mL.

[0109] In the present invention, the volume ratio of the organic solvent to water is preferably 1:1 to 9, and in specific embodiments, it can be 1:1, 1:2, 1:3, 1:4, 1:56, 1:7, 1:8, or 1:9.

[0110] In the present invention, the mixing preferably includes: dissolving a side-chain modified polymer having the structure shown by OPT and a photosensitizer in an organic solvent to obtain a mixed solution, and adding the mixed solution to water.

[0111] In the present invention, the temperature for self-assembly is preferably room temperature, and the time for self-assembly is preferably 12 to 24 h, which can be 12 h, 15 h, 18 h, 20 h, 22 h or 24 h in specific embodiments; the self-assembly is preferably carried out under ultrasonic conditions, and the power of the ultrasonic wave is preferably 150 to 200 W, which can be 150 W, 160 W, 170 W, 180 W, 190 W or 200 W in specific embodiments.

[0112] After the self-assembly, the present invention preferably further includes: subjecting the system obtained by self-assembly to water dialysis and then freeze-drying to obtain the drug delivery system (OPTC). In the present invention, the temperature for water dialysis is preferably room temperature, and the time for water dialysis is preferably 2 to 24 h, which can be 2 h, 5 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, 23 h or 24 h in specific embodiments. The present invention has no special limitation on the freeze-drying, and the freeze-drying conditions well-known to those skilled in the art can be adopted.

[0113] The present invention also provides a light-stimulus-responsive nano-delivery system (denoted as OPTC-RNP), which includes a drug delivery system and a gene editor; the drug delivery system is the drug delivery system described in the above technical solution or the drug delivery system prepared by the preparation method described in the above technical solution.

[0114] In the present invention, the gene editor preferably includes a ribonucleoprotein complex (RNP); the ribonucleoprotein complex preferably includes Cas9 / sgRNA, and the mass fraction of Cas9 in the Cas9 / sgRNA is preferably 25 to 50%, which can be 20%, 25%, 30%, 35%, 40%, 45% or 50% in specific embodiments.

[0115] In the present invention, taking the Cas9 / sgRNA complex as an example, the TK bond ( 1 O2-cleavage bond) in the drug delivery system (OPTC) is connected to (Cas9-P) to form OPTC-RNP, realizing the on-demand release of RNP.

[0116] In the present invention, the preparation method of the Cas9 / sgRNA complex preferably comprises the following steps: mixing Cas9 protein, sgRNA and a buffer solution to obtain the Cas9 / sgRNA complex. In the present invention, the mass ratio of the Cas9 protein to the sgRNA is preferably 1:1 to 3, and in specific embodiments, it can be 1:1, 1:1.5, 1:2, 1:2.5 or 1:3. In the present invention, the buffer solution preferably comprises Tris-HCl buffer; the pH value of the Tris-HCl buffer is preferably 7 to 7.5, and in specific embodiments, it can be 7, 7.2 or 7.5. In the present invention, the temperature of the mixing is preferably room temperature, the mixing time is preferably 10 to 12 min, and in specific embodiments, it can be 10 min, 11 min or 12 min; the mixing is preferably carried out under stirring conditions.

[0117] The present invention also provides a preparation method of the photo-responsive nanodelivery system described in the above technical solution, comprising the following steps: performing an amide reaction on a drug delivery system and a gene editor to obtain the photo-responsive nanodelivery system.

[0118] In the present invention, the mass ratio of the drug delivery system to the gene editor is preferably 1:0.01 to 0.02, and in specific embodiments, it can be 1:0.01, 1:0.012, 1:0.015, 1:0.018 or 1:0.02.

[0119] In the present invention, the drug delivery system is preferably activated before use, and the activation preferably comprises: mixing the drug delivery system and an activator to carry out activation to obtain an activated drug delivery system.

[0120] In the present invention, the activator preferably comprises a first activator and a second activator; the first activator preferably comprises at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N,N'-diisopropylcarbodiimide (DIC); the second activator preferably comprises at least one of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBT).

[0121] In the present invention, the dosage ratio of the drug delivery system (dry weight) to the first activator is preferably 30 mg: 0.5 - 1 mmol, and in specific embodiments, it can be 30 mg: 0.5 mmol, 30 mg: 0.6 mmol, 30 mg: 0.7 mmol, 30 mg: 0.8 mmol, 30 mg: 0.9 mmol, or 30 mg: 1 mmol. In the present invention, the dosage ratio of the drug delivery system to the second activator is preferably 30 mg: 0.5 - 1 mmol, and in specific embodiments, it can be 30 mg: 0.5 mmol, 30 mg: 0.6 mmol, 30 mg: 0.7 mmol, 30 mg: 0.8 mmol, 30 mg: 0.9 mmol, or 30 mg: 1 mmol.

[0122] In the present invention, the drug delivery system is preferably used in the form of an aqueous solution of the drug delivery system, and the aqueous solution of the drug delivery system is preferably obtained by performing hemodialysis on the system self-assembled as described above.

[0123] In the present invention, the activation temperature is preferably room temperature, and the activation time is preferably 24 - 48 h. In specific embodiments, it can be 24 h, 25 h, 30 h, 35 h, 40 h, 45 h, or 48 h. In the present invention, taking EDC and NHS as examples, during the activation process, the side-chain carboxyl groups in the drug delivery system are activated. EDC first reacts with the carboxyl group to generate a reactive intermediate, and the reactive intermediate then reacts with NHS to generate an NHS ester intermediate. This NHS ester intermediate is relatively stable, which helps to improve the reaction yield.

[0124] After the activation, the present invention preferably further includes: performing hemodialysis on the activated system to obtain an activated drug delivery system. In the present invention, the temperature of the hemodialysis is preferably room temperature, and the time of the hemodialysis is preferably 1 - 3 d. In specific embodiments, it can be 1 d, 1.5 d, 2 d, 2.5 d, or 3 d.

[0125] In the present invention, the temperature of the amide reaction is preferably 1 - 4 °C, and in specific embodiments, it can be 1 °C, 2 °C, 3 °C, or 4 °C; the time of the amide reaction is preferably 10 - 12 h, and in specific embodiments, it can be 10 h, 11 h, or 12 h; the amide reaction is preferably carried out under shaking conditions; the amide reaction is an amide reaction between a carboxyl group and an amino group.

[0126] The present invention also provides the application of the near-infrared light-induced semiconductor polymer described in the above technical solution, the near-infrared light-induced semiconductor polymer prepared by the preparation method described in the above technical solution, the hydrolysis product of the near-infrared light-induced semiconductor polymer described in the above technical solution, the side-chain modified polymer described in the above technical solution, the side-chain modified polymer prepared by the preparation method described in the above technical solution, the drug delivery system described in the above technical solution, the drug delivery system prepared by the preparation method described in the above technical solution, the light-stimulated responsive nano-delivery system described in the above technical solution or the light-stimulated responsive drug delivery system prepared by the preparation method described in the above technical solution in biological imaging, preparing gene therapy drugs, preparing photodynamic therapy drugs for tumors or preparing anti-inflammatory drugs. The light-stimulated responsive drug delivery system provided by the present invention can observe the accumulation of drugs at the tumor site under the guidance of high-resolution second-region fluorescence imaging.

[0127] To further illustrate the present invention, the following examples are used to describe in detail the near-infrared light-induced semiconductor polymer provided by the present invention, its hydrolysis product, side-chain modified polymer, drug delivery system, light-stimulated responsive nano-delivery system and the preparation method and application, but they should not be construed as limiting the protection scope of the present invention.

[0128] Example 1

[0129] 1. Preparation of semiconductor polymer

[0130] Mix 2,7-dibromofluorene, tert-butyl acrylate and tetrabutylammonium bromide in a molar ratio of 10.2:41:0.78. Under nitrogen protection, add toluene and 2M KOH aqueous solution, stir at room temperature for 12 h, wash with water, wash with 0.5M HCl aqueous solution, extract with dichloromethane, dry the obtained organic phase with Na2SO4, filter, evaporate the filtrate to dryness by rotary evaporation, and purify by silica gel column (the eluent is petroleum ether and ethyl acetate, with a volume ratio of 4:1) to obtain monomer A (light yellow, yield 75%). Among them, the molar ratio of 2,7-dibromofluorene to KOH is 10.2:10, and the dosage ratio of 2,7-dibromofluorene to toluene is 10.2 mmol:50 mL.

[0131] Mix monomer A, bis(pinacolato)diboron, Pd(dppf)2Cl2 and KOAc in a molar ratio of 3.7:9.5:2:17, dissolve in DMF, stir and reflux at 90 °C for 12 h under nitrogen protection, filter, concentrate the obtained filtrate and purify by silica gel column (the eluent is petroleum ether and ethyl acetate, with a volume ratio of 5:1) to obtain monomer B (white, yield 60%). Among them, the dosage ratio of monomer A to DMF is 3.7 mmol:10 mL.

[0132] Monomer B and monomer C (4,9-bis(5-bromothienyl)-6,7-bis(4-hexyloxyphenyl)-2-thia-1,3,5,8-tetraazacyclopentadiene[b]naphthalene) were mixed in a molar ratio of 1:1. Tetrakis(triphenylphosphine)palladium and K2CO3 were added. Finally, toluene and water were added for dissolution. The reaction was carried out at 90 °C for 20 min, and then cooled to room temperature. Methanol (the volume fraction of methanol in the system was 95%) was used for sedimentation for 24 h, followed by suction filtration. The obtained liquid component was rotary evaporated to dryness to obtain a near-infrared light-induced semiconductor polymer (Formula I, degree of polymerization n = 53). The molar ratio of monomer B, tetrakis(triphenylphosphine)palladium and K2CO3 was 1:0.1:8.7, and the dosage ratio of monomer B, toluene and water was 1 mmol:30 mL:10 mL.

[0133] The near-infrared light-induced semiconductor polymer was dissolved in DCM and trifluoroacetic acid, and hydrolyzed at room temperature for 24 h, then rotary evaporated to dryness to obtain the hydrolysis product of the near-infrared light-induced semiconductor polymer (OSP, n = 53). Among them, the dosage ratio of monomer B, DCM and trifluoroacetic acid was 1 mmol:150 mL:15 mL.

[0134] 2. Side chain modification of the semiconductor polymer

[0135] OSP, NH2-PEG-NH2, NHS, EDC and DMSO were mixed in a molar ratio of 1:5:20:50 and reacted at room temperature for 48 h, followed by dialysis in water for 4 d and then freeze-dried to obtain OSP-PEG (dark green). Among them, the amount of OSP was calculated based on the structural unit in OSP, n = 53. The molar ratio of OSP, NH2-PEG-NH2, NHS and EDC was 1:5:20:50, and the dosage ratio of OSP and DMSO was 1 mmol:100 mL. Among them, the cut-off molecular weight of the dialysis bag used for water dialysis was 14 kDa.

[0136] OSP-PEG, TK, NHS and EDC were mixed in a ratio of 32 mg:0.12 mmol:0.9 mmol:0.9 mmol and reacted at room temperature for 48 h, followed by dialysis in water for 4 d and then freeze-dried to obtain OPT (green, OSP-PEG-TK). Among them, the dosage of OSP-PEG and DMSO was 32 mg:10 mL, and the cut-off molecular weight of the dialysis bag used for water dialysis was 14 kDa.

[0137] 3. Preparation of the drug delivery system (OPTC)

[0138] OPT and free Ce6 were dissolved in DMSO at a mass ratio of 1:2 by sonication, and then injected into H2O at room temperature under sonication conditions of 150 - 200 W for self-assembly for 24 h, dialyzed in water for 24 h, and freeze-dried to obtain OPTC (green, OSP-PEG-TK-Ce6). Among them, the dosage ratio of OPT, DMSO, and H2O injected under sonication was 320 mg: 10 mL: 90 mL.

[0139] 4. Preparation of photo-responsive nanodelivery system (OPTC-RNP)

[0140] Cas9 protein and sgRNA were incubated at a mass ratio of 1:3 in Tris-HCl buffer (pH = 7.4) at room temperature for 10 min to obtain the Cas9 / sgRNA complex (RNP).

[0141] Activation of OPTC: NHS (0.9 mmol) and EDC (0.9 mmol) were added to the OPTC obtained in step 3, activated at room temperature for 48 h, and dialyzed in water for 4 d to obtain activated OPTC.

[0142] The activated OPTC was dropped into RNP and refrigerated and shaken at 4 °C for 12 h to obtain the nanodelivery system (OPTC-RNP).

[0143] Test examples

[0144] Performance test of OSP, OPT, OPTC, and OPTC-RNP prepared in Example 1

[0145] The OPTC-RNP prepared in Example 1 was measured by a two-region fluorescence emission spectrometer. Figure 1 This is the fluorescence emission spectrum of OPTC-RNP. From the measurement of the fluorescence spectrum, it can be seen that the fluorescence emission of the OPTC-RNP prepared in the present invention is in the two-region position, meeting the two-region imaging conditions.

[0146] Figure 2 This is the UV-visible absorption spectra of OSP, OPT, OPTC, TK, and Ce6. It can be seen that OPTC-RNP has near-infrared absorption characteristics.

[0147] Figure 3 This is the transmission electron microscope (TEM) image of OPTC-RNP. It can be seen that OPTC-RNP successfully self-assembled into nanoparticles.

[0148] The surface charges (zeta potential, mV) of OSP, OPT, OPTC, and OPTC-RNP were measured by dynamic light scattering method.

[0149] Figure 4These are the dynamic light scattering (DLS) curves of OPTC and OPTC-RNP. It can be seen that the nanoparticles formed after OPTC is conjugated with RNP slightly increase.

[0150] Figure 5 These are the Zeta potential diagrams of OSP, OPT, OPTC, and OPTC-RNP. It can be seen that the charge of OPT modified by NH2-PEG-NH2 and the TK side chain changes from positive (OSP) to negative, and after encapsulating Ce6 and conjugating with RNP, the charge value is stable.

[0151] The generation of singlet oxygen is detected by measuring the absorption intensity at 420 nm in the ultraviolet-visible absorption spectrum. Figure 6 These are the relative absorbances of 1,3-diphenylisobenzofuran (DPBF) after adding OPTC-RNP with and without NIR irradiation (660 nm). It can be seen that OPTC-RNP successfully generates singlet oxygen 1 O2.

[0152] The residual amount of RNP in OPTC-RNP is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 7 These are the SDS-PAGE diagrams of the residual amount of RNP in OPTC-RNP after NIR irradiation for different times. It can be seen that the response group TK between OPTC and RNP is successfully cleaved under near-infrared light illumination conditions.

[0153] Figure 8 These are the RNP release diagrams of OPTC-RNP corresponding to different NIR irradiation times. It is calculated by subtracting the original residual amount in OPTC-RNP using Image J. It can be seen that the group between OPTC and RNP is successfully cleaved under near-infrared light illumination conditions, and RNP is effectively released.

[0154] The cellular internalization of OPTC-RNP@CM is studied using confocal laser scanning microscopy (CLSM). Figure 9 These are the CLSM images of OPTC-RNP labeled with EGFP (enhanced green fluorescent protein) incubated with 4T1 cells for 6 h. Blue, DAPI (4',6-diamidino-2-phenylindole); green, EGFP-labeled Cas9 protein, red, CM-Dil (live cell tracer). It can be seen that OPTC-RNP is successfully taken up by 4T1 cells.

[0155] The nanoparticle drug OPTC-RNP@CM (1 mg / mL, 200 μL) is intravenously injected into breast cancer-bearing mice. Figure 10To detect the distribution of OPTC-RNP in mice at different time points using a second near-infrared in vivo imager, it was found that after intravenous injection of OPTC-RNP into breast cancer-bearing mice, it mainly accumulated in the tumors and livers of the mice.

[0156] Figure 11 For the changes in the signal intensity of OPTC-RNP in the livers and tumors of mice at different time points, it was found that 24 hours after intravenous injection of OPTC-RNP into breast cancer-bearing mice, the fluorescence intensity was the strongest at the tumor sites of the mice.

[0157] The mice were euthanized 48 hours after administration (OPTC-RNP), and two-region imaging analysis diagrams of the main organs and tumor tissues were collected. See Figure 12 , it was found that after intravenous injection of OPTC-RNP into breast cancer-bearing mice, it accumulated in the tumors, spleens, and livers of the mice.

[0158] 4T1 cells were pre-seeded in 24-well plates and cultured for 24 hours, then co-incubated with different treatment groups in an incubator at 37 °C for 6 hours. The cells were washed with PBS to remove the nanoparticles that did not enter the cells. Subsequently, the medium was replaced with serum-free DMEM medium, and the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe was added to the wells and incubated in the dark for 30 minutes. The 4T1 cells were irradiated with a 660 nm laser. After irradiation, the cells were washed with PBS. The cells were observed with a Thunder imaging system. DCFH-DA can be oxidized by free radicals to highly fluorescent DCF, and the green fluorescence inside the cells was observed and recorded. Group I: PBS / NIR, Group II: OPTC, Group III: OPTC / NIR, Group IV: OPTC-RNP, Group V: OPTC-RNP / NIR, Figure 13 For the DCF (detecting 1 O2) fluorescence staining images of 4T1 cells in different treatment groups. It was found that singlet oxygen 1 O2 was successfully generated after OPTC-RNP was irradiated with NIR.

[0159] The cells were pre-seeded in 24-well plates and incubated for 24 hours. They were treated with different nano-drugs respectively, and then irradiated with a 660 nm laser. After washing with PBS, 100 μL of calcein acetoxymethyl ester / propidium iodide (Calcein AM / PI) detection working solution was added to each well. After incubating in the dark at 37 °C for 30 minutes, the staining effect was observed under Thunder, Figure 14 For the live / dead fluorescence staining images of 4T1 cells in different treatment groups. Green, Calcein-AM (calcein); red, PI (propidium iodide), scale bar: 300 μm, grouping same as Figure 13 , it was found that a large number of 4T1 cells were killed after OPTC-RNP was irradiated with NIR.

[0160] Take a part of the tumor tissue collected in each group, cut it into small pieces, process it with collagenase according to the instructions, then grind it into a homogenate and filter it through a sieve. Wash the filtered single-cell suspension with pre-cooled PBS, centrifuge to discard the supernatant and wash away cell debris, then resuspend the washed cells with 2% BSA, add the corresponding antibodies for staining, and finally analyze using a multi-color analytical flow cytometer. Figure 15 To detect the apoptosis of 4T1 cells under different treatment conditions by flow cytometry, the grouping is the same as Figure 13 , it can be seen that OPTC-RNP causes apoptosis of 4T1 cells after being irradiated with NIR.

[0161] Collect the genomic DNA of 4T1 cells using a universal genomic DNA extraction kit. Amplify the DNA fragment targeted by sgRNA with high-fidelity DNA polymerase using the following primers: AGTACACCACTAACGCAAGC. To reduce non-specific amplification, use a touchdown polymerase chain reaction (PCR) program [incubate at 98°C for 30 s, (incubate at 98°C for 10 s, 60°C for 20 s, 72°C for 20 s) for 35 cycles, incubate at 72°C for 120 s]. After gel recovery and purification using a DNA purification kit, detect the generation frequency of indels according to the T7 endonuclease kit. Analyze the purified DNA by 2% agarose gel electrophoresis. Calculate the frequency of indel formation through ImageJ. Figure 16 For detecting the gene mutation frequency of PD-L1 in 4T1 cells after different treatments by the T7EI method, the grouping is the same as Figure 13 , it can be seen that RNP is successfully released after NIR irradiation and a large number of PD-L1 genes in 4T1 cells are knocked out.

[0162] Take a part of the tumor tissue collected in each group, cut the tissue into small pieces, and then process it to extract proteins according to the instructions of Western and IP cell lysis buffer. Load and separate equal amounts of total protein on an SDS / PAGE gel and transfer it to a polyvinylidene fluoride (PVDF) membrane. Block the sample with a blocking solution containing 5% non-fat milk powder at room temperature for 2 h. After blocking, wash the membrane 3 times with TBST, 10 min each time, and then incubate with the corresponding primary antibody respectively. Incubate overnight at 4°C. After incubation, wash 3 times with TBST, 10 min each time. Then incubate with the secondary antibody at room temperature for 1 h. After incubation, wash the membrane 5 times in TBST buffer, 10 min each time. Finally, visualize the target protein bands by ECL (enhanced chemiluminescence reagent), and obtain and analyze the blot image using an imaging system. Figure 17 For WB detection of the expression level of PD-L1 in 4T1 cells after different treatments, the grouping is the same as Figure 13, it can be seen that the expression level of 4T1 cells after being treated with OPTC-RNP / NIR is significantly reduced.

[0163] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A drug delivery system, characterized in that It includes a side chain modified polymer and a photosensitizer contained in the side chain modified polymer; the side chain modified substance is a side chain modified polymer having a structure shown in OPT: ； Among them, n is 45~65, and m is 30~60.

2. The drug delivery system according to claim 1, characterized in that The method for preparing the side chain modified polymer having the structure shown in OPT comprises the following steps: The hydrolysis product of the infrared photoinduced semiconductor polymer is subjected to an amidation reaction with bisamino polyethylene glycol to obtain a side chain modified polymer having a structure shown as OSP-PEG; the polymerization degree of the bisamino polyethylene glycol is m; the hydrolysis product of the infrared photoinduced semiconductor polymer has a structure shown as OSP: ； Among them, n is 45~65; The side chain modified polymer having the structure represented by OSP-PEG is subjected to an amidation reaction with 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to obtain a side chain modified polymer having the structure represented by OPT.

3. The method for preparing the drug delivery system according to claim 1 or 2, characterized in that: The following steps are involved: The side chain modified polymer having the structure shown in OPT, a photosensitizer, an organic solvent and water are mixed and self-assembled to obtain the drug delivery system.

4. A light stimulus responsive nano delivery system, characterized in that: It comprises a drug delivery system and a gene editor; the drug delivery system is the drug delivery system according to claim 1 or 2 or the drug delivery system prepared by the preparation method according to claim 3.

5. The method for preparing the light stimulus responsive nano delivery system according to claim 4, characterized in that: The following steps are involved: The drug delivery system and the gene editor are subjected to an amide reaction to obtain the light stimulus responsive nano delivery system.

6. Use of the drug delivery system according to claim 1 or 2, the drug delivery system prepared by the preparation method according to claim 3, the light stimulus responsive nano delivery system according to claim 4, or the light stimulus responsive nano delivery system prepared by the preparation method according to claim 5 in non-therapeutic and non-diagnostic biological imaging, preparation of gene therapy drugs, preparation of photodynamic therapy drugs for tumors, or preparation of anti-inflammatory drugs.

Images