Near-infrared photoinduced semiconductor polymer and hydrolysate thereof, side chain modified polymer, drug delivery system, photostimulation response nano-delivery system, preparation method and application

Abstract

The invention provides a near-infrared photoinduced semiconductor polymer and a hydrolysate thereof, a side chain modified polymer, a drug delivery system, a photostimulation response nano-delivery system and a preparation method and application thereof, and relates to the technical field of biological medicines. The near-infrared photoinduced semiconductor polymer and the hydrolysate thereof, the side chain modified polymer, the drug delivery system (OPTC) and the photostimulation response nano-delivery system (OPTC-RNP) provided by the invention all have lower spontaneous background fluorescence, deeper tissue penetrability and higher signal-to-noise ratio during near-infrared second-region imaging, the near-infrared second-region imaging effect is excellent, and the near-infrared photoinduced semiconductor polymer and the hydrolysate thereof, the side chain modified polymer, the drug delivery system (OPTC) and the photostimulation response nano-delivery system (OPTC-RNP) have good application prospects. Accumulation of the nano-delivery system at the tumor site can be observed. Moreover, the photosensitizer in the delivery system can generate singlet oxygen under irradiation of near-infrared light, so that a chemical bond connected between OPTC and RNP can be broken to release a ribonucleoprotein complex (RNP) while tumor photodynamic therapy is realized, and light-controllable gene therapy is realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a near-infrared photoinduced semiconductor polymer and a preparation method and application thereof, a hydrolyzate of a near-infrared photoinduced 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 has significant tumor heterogeneity. Common treatments include surgery and chemotherapy. These standard treatments are severely limited in their efficacy due to significant side effects and adverse effects on the host immune system. In particular, the unique immunosuppressive tumor immune microenvironment of TNBC further limits the effectiveness of immunotherapy. Immune system checkpoint blockade (ICB) has great 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 duration of immunotherapy and improve the effectiveness of immunotherapy is of great significance in cancer treatment.

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

[0004] Semiconductor polymer nanomaterials composed of polymers have become a versatile near-infrared (NIR) absorbing / emitting biomaterial for molecular imaging, phototherapy, and biological regulation. Existing near-infrared emitting materials mainly include inorganic near-infrared quantum dots and halide perovskites, as well as various organic near-infrared materials including traditional fluorescent small molecule materials, conjugated polymers, stable luminescent free radicals, thermally activated delayed fluorescence (TADF) materials, and metal organic complex phosphorescent materials. Among the many near-infrared emitting materials in molecular imaging applications, the fluorescence emission of most materials is mainly concentrated in the I region (650~900nm). However, due to the large number of background interference factors in the I region imaging, weak penetration, and low signal-to-noise ratio, the imaging effect of the material is poor, which limits the application of near-infrared emitting 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 photoinduced semiconductor polymer and its hydrolyzate, side chain modified polymer, drug delivery system, light stimulus responsive nano delivery system and 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 zone II imaging, and has excellent near-infrared zone II imaging effect.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a near-infrared photoinduced semiconductor polymer having a structure shown in Formula I: Formula I; Among them, n is 45~65.

[0007] The present invention also provides a method for preparing the near-infrared photoinduced semiconductor polymer described in the above technical solution, comprising the following steps: Performing Suzuki reaction on monomer B and monomer C to obtain the near-infrared photoinduced semiconductor polymer; ; Wherein, X is a halogen.

[0008] The present invention also provides a hydrolysis product of a near-infrared photoinduced semiconductor polymer, wherein the near-infrared photoinduced semiconductor polymer is the near-infrared photoinduced semiconductor polymer described in the above technical solution or the near-infrared photoinduced semiconductor polymer prepared by the preparation method described in the above technical solution, and has the structure shown in OSP: ; Among them, n is 45~65.

[0009] The present invention also provides a side chain modified polymer having a structure shown in OSP-PEG or OPT: ; Among them, n is 45~65, and m is 30~60.

[0010] The present invention also provides a method for preparing the side chain modified polymer described in the above technical solution. The preparation method of the side chain modified polymer having the structure shown in OSP-PEG comprises the following steps: subjecting the hydrolyzate of the infrared light-induced semiconductor polymer described in the above technical solution to an amidation reaction with bisamino polyethylene glycol to obtain the side chain modified polymer having the structure shown in OSP-PEG; the degree of polymerization of the bisamino polyethylene glycol is m; The preparation method of the side chain modified polymer having the structure shown in OPT comprises the following steps: subjecting the side chain modified polymer having the structure shown in OSP-PEG to an amidation reaction with 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to obtain a side chain modified polymer having the structure shown in OPT.

[0011] 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 modified substance is the side chain modified polymer having the structure shown in OPT as described in the above technical scheme or the side chain modified polymer having the structure shown in OPT obtained by the preparation method described in the above technical scheme.

[0012] The present invention also provides a method for preparing the drug delivery system described in the above technical solution, comprising the following steps: mixing a side chain modified polymer having a structure shown in OPT, a photosensitizer, an organic solvent and water, and performing self-assembly to obtain the drug delivery system.

[0013] The present invention also provides a light stimulus responsive nano delivery 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.

[0014] The present invention also provides a method for preparing the light stimulus responsive nano delivery system described in the above technical solution, comprising the following steps: subjecting the drug delivery system and the gene editor to an amide reaction to obtain the light stimulus responsive nano delivery system.

[0015] The present invention also provides the use of the near-infrared photoinduced semiconductor polymer described in the above technical scheme, the near-infrared photoinduced semiconductor polymer obtained by the preparation method described in the above technical scheme, the hydrolyzate of the near-infrared photoinduced semiconductor polymer described in the above technical scheme, the side chain modified polymer described in the above technical scheme, the side chain modified polymer obtained by the preparation method described in the above technical scheme, the drug delivery system described in the above technical scheme, the drug delivery system obtained by the preparation method described in the above technical scheme, the light stimulus responsive nano delivery system described in the above technical scheme, or the light stimulus responsive drug delivery system obtained by the preparation method described in the above technical scheme in biological imaging, preparation of gene therapy drugs, preparation of photodynamic therapy drugs for tumors, or preparation of anti-inflammatory drugs.

[0016] The near-infrared photoinduced semiconductor polymer provided by the present invention has a conjugated polymer main chain synthesized by alternatingly connecting fluorene and bithiophene, so that it has near-infrared second zone (NIR-II, 1.0~1.7μm) fluorescence emission performance, and has lower spontaneous background fluorescence, deeper tissue penetration and higher signal-to-noise ratio during second zone imaging. The near-infrared second zone imaging effect is excellent, and the accumulation of the nanodelivery system at the tumor site can be observed in a second zone fluorescence imaging instrument.

[0017] The polymer backbone of the infrared photoinduced semiconductor polymer having the structure shown in OSP provided by the present invention has excellent near-infrared two-zone imaging effect, and the accumulation of the nano-delivery system at the tumor site can be observed in a two-zone fluorescence imaging instrument. Moreover, the presence of carboxyl groups in the hydrolyzate increases the water solubility, chemical resistance, mechanical strength, thermal stability and biocompatibility of the polymer. Moreover, the carboxyl group is a highly active functional group that can undergo a variety of chemical reactions with other compounds to achieve the functionalization of the polymer, which provides more possibilities for further modification and application of polymer materials.

[0018] The polymer main chain of the side chain modified polymer with the structure shown in OSP-PEG provided by the present invention has excellent near-infrared two-zone imaging effect, and the accumulation of the nano delivery system at the tumor site can be observed in the two-zone fluorescence imaging instrument. Moreover, bisamino polyethylene glycol has good water solubility, and water solubility is achieved by introducing it into the side chain of the polymer and the self-assembly of the amphiphilic polymer. Bisamino polyethylene glycol has a high chemical stability, and by introducing it into the side chain of the polymer, the polymer can maintain good stability under various environments. Bisamino polyethylene glycol has good biocompatibility, and by introducing it into the side chain of the polymer, the interaction between the polymer and the organism can be improved, and adverse reactions can be reduced. The amino groups at both ends of bisamino polyethylene glycol can be coupled with molecules with carboxyl, aldehyde or other reactive groups to form covalent bonds, thereby obtaining a bioconjugate with a specific structure and function, and this characteristic makes it have a wide range of application prospects in the construction of complex biomaterials and drug delivery systems, and expands its application range.

[0019] The polymer backbone of the side chain modified polymer with the structure shown in OPT provided by the present invention has excellent near-infrared second-zone imaging effect, and the accumulation of the nano-delivery system at the tumor site can be observed in a second-zone fluorescence imaging instrument. At the same time, the polymer can self-assemble to form nanoparticles and encapsulate photosensitizers (such as Ce6), and produce a large amount of singlet oxygen ( 1 O2) to achieve photodynamic therapy. Moreover, the singlet oxygen responsive group in the side chain modified polymer with the structure shown in OPT can be connected to the ribonucleoprotein complex (RNP) to form a nano-delivery system.

[0020] The polymer backbone of the drug delivery system (denoted as OPTC) provided by the present invention has excellent near-infrared second-zone imaging effect, and the accumulation of the nano-delivery system in the tumor site can be observed in a second-zone fluorescence imaging instrument. Moreover, the photosensitizer can produce a large amount of 1 O2, to achieve photodynamic therapy of tumors.

[0021] The polymer backbone of the light-stimulated responsive nano-delivery system (denoted as OPTC-RNP) provided by the present invention has excellent near-infrared second-zone imaging effect, and the accumulation of the nano-delivery system in the tumor site can be observed in a second-zone fluorescence imaging instrument. Moreover, the photosensitizer can produce 1 O2 can achieve photodynamic therapy for tumors while breaking the chemical bond between OPTC and RNP, thereby releasing RNP to achieve light-controlled gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The fluorescence emission spectrum of OPTC-RNP prepared in Example 1; Figure 2 UV-visible absorption spectra of OSP, OPT and OPTC, as well as TK and Ce6 prepared in Example 1; Figure 3 TEM image of OPTC-RNP prepared in Example 1; Figure 4 The dynamic light scattering (DLS) curves of OPTC and OPTC-RNP prepared in Example 1; Figure 5 Zeta potential diagram of OSP, OPT, OPTC and OPTC-RNP prepared in Example 1; Figure 6 is a graph showing the relative absorbance of 1,3-diphenylisobenzofuran (DPBF) after adding OPTC-RNP prepared in Example 1 under and without NIR irradiation (660 nm); Figure 7 The SDS-PAGE images of the residual amount of RNP in the OPTC-RNP prepared in Example 1 after NIR irradiation for different time periods; Figure 8 The RNP release graphs corresponding to the OPTC-RNP prepared in Example 1 under different NIR irradiation times; Fig. 9 Confocal laser scanning microscopy (CLSM) images of OPTC-RNP prepared in Example 1 labeled with enhanced green fluorescent protein (EGFP) and incubated with 4T1 cells for 6 hours; Fig.10The distribution diagram of OPTC-RNP prepared in Example 1 in mice was detected at different time points by near-infrared zone II in vivo imaging instrument; Fig.11 The graphs of the changes in the signal intensity of OPTC-RNP prepared in Example 1 in the mouse liver and tumor at different time points; Fig.12 The two-region imaging analysis diagram of the main organs and tumor tissues of mice 48 hours after administration (OPTC-RNP prepared in Example 1); Fig.13 2′,7′-dichlorofluorescein diacetate (DCFH-DA, detection) was used for 4T1 cells in different treatment groups. 1 O2) Fluorescence staining images; Fig.14 Live and dead fluorescence staining images of 4T1 cells in different treatment groups; Fig.15 Figure 2 shows the apoptosis of 4T1 cells detected by flow cytometry under different treatment conditions; Fig.16 This is the frequency diagram of PD-L1 gene mutation in 4T1 cells after different treatments detected by T7EI method; Fig.17 This is the WB detection of PD-L1 in 4T1 cells treated with different methods; in, Figure 13~Figure 17 The conditions of each group are as follows: I: PBS / NIR, II: OPTC prepared in Example 1, III: OPTC prepared in Example 1 / NIR, IV: OPTC-RNP prepared in Example 1, V: OPTC-RNP prepared in Example 1 / NIR. DETAILED DESCRIPTION

[0023] The present invention provides a near-infrared photoinduced semiconductor polymer having a structure shown in Formula I: Formula I; Wherein, n is 45-65, and in a specific embodiment can be 45, 50, 53, 55, 60 or 65.

[0024] The present invention also provides a method for preparing the near-infrared photoinduced semiconductor polymer described in the above technical solution, comprising the following steps: subjecting monomer B and monomer C to Suzuki reaction to obtain the near-infrared photoinduced semiconductor polymer; ; Wherein, X is a halogen, and in a specific embodiment, it can be fluorine, chlorine, bromine or iodine.

[0025] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0026] In the present invention, the molar ratio of the monomer B to the monomer C is preferably 1:0.8-1, and in specific embodiments may be 1:0.8, 1:0.9 or 1:1.

[0027] In the present invention, the Suzuki reaction is preferably carried out under the conditions of a catalyst, an alkaline agent and a solvent. Specifically, monomer B, monomer C, a catalyst, an alkaline agent and a solvent are mixed to carry out the Suzuki reaction.

[0028] 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 the monomer B to the catalyst is preferably 1:0.05-0.1, and in specific embodiments can be 1:0.05, 1:0.06, 1:0.07, 1:0.08, 1:0.09 or 1:0.1.

[0029] In the present invention, the alkaline agent preferably includes an alkali metal carbonate, and more preferably includes at least one of Cs2CO3, K2CO3, Na2CO3 and Li2CO3. In the present invention, the molar ratio of the monomer B to the alkaline agent is preferably 1:8-9, and in specific embodiments 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.

[0030] 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 organic solvent is preferably 1:2-3, and in a specific embodiment, it can be 1:2, 1:2.5 or 1:3. In the present invention, the amount ratio of monomer B to solvent is preferably 1mmol:30-40mL, and in a specific embodiment, it can be 1mmol:30mL, 1mmol:35mL or 1mmol:40mL.

[0031] 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.

[0032] After completing the Suzuki reaction, the present invention preferably further comprises cooling the reaction system obtained by the Suzuki reaction to room temperature, settling methanol, separating solid and liquid, concentrating the obtained liquid component to constant weight, and obtaining the near-infrared photoinduced semiconductor polymer. In the present invention, the temperature of the methanol settling is preferably room temperature, and the time is preferably 12 to 48 hours, and in specific embodiments, it can be 12 hours, 15 hours, 20 hours, 24 hours, 30 hours, 35 hours, 40 hours, 45 hours or 48 hours; the volume fraction of methanol in the system during the methanol settling is preferably 85 to 95%, and in specific embodiments, it can be 85%, 88%, 90%, 92% or 95%. In the present invention, the concentration preferably comprises rotary evaporation.

[0033] In the present invention, the preparation method of the monomer B preferably comprises the following steps: mixing monomer A, bispinacol diborate, a catalyst, an alkaline reagent and an organic solvent, and performing a borate esterification reaction to obtain monomer B. The preparation route is as follows: ; Wherein, X is a halogen, and in a specific embodiment, it can be fluorine, chlorine, bromine or iodine.

[0034] In the present invention, the molar ratio of the monomer A to the bispinacol diborate is preferably 1:2.5-3.5, and in specific embodiments may be 1:2.5, 1:3 or 1:3.5.

[0035] In the present invention, the catalyst preferably includes at least one of Pd(dppf)2Cl2 (1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride) 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 can be 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.64 or 1:0.7.

[0036] In the present invention, the alkaline agent preferably includes one or more of alkali metal acetate, alkali metal phosphate, and alkali metal carbonate, 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 alkaline agent is preferably 1:2.5-7, 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.

[0037] In the present invention, the organic solvent preferably includes at least one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and butanone. In the present invention, the usage ratio of the monomer A to the organic solvent is preferably 1mmol:2-10mL, and in specific embodiments, it can be 1mmol:2mL, 1mmol:3mL, 1mmol:4mL, 1mmol:5mL, 1mmol:6mL, 1mmol:7mL, 1mmol:8mL, 1mmol:9mL or 1mmol:10mL.

[0038] In the present invention, the temperature of the borate esterification reaction is preferably 90-120°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 borate esterification reaction is preferably 12-24h, and in specific embodiments it can be 12h, 15h, 18h, 20h, 22h or 24h; the borate esterification reaction is preferably carried out under a protective atmosphere, and the protective atmosphere preferably includes nitrogen, argon or helium.

[0039] After the borate esterification reaction, the present invention preferably further comprises: filtering the reaction system obtained by the borate esterification reaction, concentrating and drying the obtained filtrate, and then purifying it on a silica gel column to obtain monomer B. In the present invention, the eluent used in the silica gel column purification preferably includes petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 1 to 5:1, and in specific embodiments can be 1:1, 2:1, 3:1, 4:1 or 5:1.

[0040] In the present invention, the preparation method of the monomer A preferably comprises the following steps: mixing 2,7-dihalofluorene, tert-butyl acrylate, a catalyst, an alkaline reagent and an organic solvent, and performing a nucleophilic substitution reaction to obtain the monomer A. The preparation route is as follows: .

[0041] In the present invention, the molar ratio of the 2,7-dihalogenated fluorene to tert-butyl acrylate is preferably 1:4-4.5, and in 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-dihalogenated fluorene preferably includes fluorine, chlorine, bromine or iodine. In the present invention, the tert-butyl acrylate is used as an alkylating agent.

[0042] 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 1:0.01~0.1, 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.

[0043] In the present invention, the alkaline agent 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 alkaline agent is preferably used in the form of an alkaline agent aqueous solution, and the concentration of the alkaline agent aqueous solution is preferably 1~2M (mol / L), and in specific embodiments, it can be 1M, 1.5M or 2M. In the present invention, the molar ratio of the 2,7-dihalofluorene to the alkaline agent is preferably 1:0.5~1.5, and in specific embodiments, it can be 1:0.5, 1:0.8, 1:1, 1:1.2 or 1:1.5.

[0044] In the present invention, the organic solvent preferably includes at least one of toluene and acetone. In the present invention, the amount 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.

[0045] 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 hours, and in specific embodiments it can be 10, 11, 12, 13 or 14 hours; the nucleophilic substitution reaction is preferably carried out under a protective atmosphere, and the protective atmosphere preferably includes nitrogen, argon or helium.

[0046] After the nucleophilic substitution reaction, the present invention preferably further comprises: washing the reaction system obtained by the nucleophilic substitution reaction with water, washing with a 0.1-1M HCl aqueous solution, extracting with dichloromethane, drying the obtained organic phase with Na2SO4, filtering, rotary evaporating the filtrate to dryness, and purifying with a silica gel column to obtain monomer A. In the present invention, the eluent used for the silica gel column purification preferably comprises petroleum ether and ethyl acetate, and the volume ratio of the petroleum ether to ethyl acetate is preferably 1-5:1, and in specific embodiments can be 1:1, 2:1, 3:1, 4:1 or 5:1.

[0047] The present invention also provides a hydrolysis product of a near-infrared photoinduced semiconductor polymer, wherein the near-infrared photoinduced semiconductor polymer is the near-infrared photoinduced semiconductor polymer described in the above technical solution or the near-infrared photoinduced semiconductor polymer prepared by the preparation method described in the above technical solution, and has the structure shown in OSP: ; Wherein, n is 45 to 65, and in specific embodiments can be 45, 50, 53, 55, 60 or 65, and the definition of n is the same as that of n in Formula I.

[0048] The present invention also provides a method for preparing the hydrolysis product of the near-infrared photoinduced semiconductor polymer described in the above technical solution, comprising the following steps: hydrolyzing the semiconductor polymer described in the above technical solution to obtain the hydrolysis product of the near-infrared photoinduced semiconductor polymer.

[0049] In the present invention, the hydrolysis is preferably carried out under acid and organic solvent conditions. In the present invention, the acid preferably includes trifluoroacetic acid; the dosage ratio of the near-infrared photoinduced semiconductor polymer to the acid is preferably 1mmol:10~15mL, and in a specific embodiment, it can be 1mmol:10mL, 1mmol:11mL, 1mmol:12mL, 1mmol:13mL, 1mmol:14mL or 1mmol:15mL. In the present invention, the organic solvent is preferably a halogenated alkane, and more preferably includes at least one of dichloromethane (DCM) and methyl bromide (CH3Br). In the present invention, the dosage ratio of the near-infrared photoinduced semiconductor polymer to the solvent is preferably 1mmol:100~150mL, and in a specific embodiment, it can be 1mmol:100mL, 1mmol:110mL, 1mmol:120mL, 1mmol:130mL, 1mmol:140mL or 1mmol:150mL.

[0050] In the present invention, the hydrolysis temperature 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.

[0051] The present invention also provides a side chain modified polymer having a structure shown in OSP-PEG or OPT: ; Wherein, n is 45-65, and in specific embodiments can be 45, 50, 53, 55, 60 or 65, and the definition of n in OSP-PEG and OPT is the same as that of n in Formula I; m is 30-60, and in specific embodiments can be 30, 35, 40, 45, 50, 55 or 60.

[0052] The present invention also provides a method for preparing a side chain modified polymer having a structure shown by OSP-PEG as described in the above technical scheme, comprising the following steps: subjecting the hydrolysis product of the infrared photoinduced semiconductor polymer described in the above technical scheme to an amidation reaction with bisamino polyethylene glycol to obtain the side chain modified polymer having a structure shown by OSP-PEG; the degree of polymerization of the bisamino polyethylene glycol is m.

[0053] In the present invention, the molar ratio of the hydrolyzate of the infrared photoinduced semiconductor polymer (calculated in terms of the amount of structural units) to bisamino polyethylene glycol (NH2-PEG-NH2) is preferably 1:4-5, and in specific embodiments may be 1:4, 1:4.2, 1:4.5, 1:4.8 or 1:5.

[0054] In the present invention, the amidation reaction is carried out under the conditions of a preferred catalyst. 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 hydrolyzate of the infrared light-induced semiconductor polymer to the first catalyst is preferably 1:40-50, and in a specific embodiment, it can be 1:40, 1:42, 1:45, 1:48 or 1:50. In the present invention, the molar ratio of the hydrolyzate of the infrared light-induced semiconductor polymer to the second catalyst is preferably 1:20-30, and in a specific embodiment, it can be 1:20, 1:22, 1:25, 1:28 or 1:30.

[0055] 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 hydrolyzate of the infrared photoinduced semiconductor polymer (calculated in terms of the amount of structural units) to the organic solvent is preferably 1mmol:60~100mL, and in specific embodiments it can be 1mmol:60mL, 1mmol:70mL, 1mmol:80mL, 1mmol:90mL or 1mmol:100mL.

[0056] In the present invention, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 48 to 72 hours, and in specific embodiments, it can be 48 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours or 72 hours. Taking EDC and NHS as an example, in the present invention, during the amidation reaction, EDC first reacts with a carboxyl group to generate an active intermediate, and the active intermediate then reacts with NHS to generate an NHS ester intermediate, which is relatively stable and helps to improve the yield of the reaction.

[0057] After the amidation reaction, the present invention preferably further comprises: subjecting the reaction system obtained by the amidation reaction to water dialysis and then freeze-drying to obtain the side chain modified polymer having the structure shown in OSP-PEG. In the present invention, the temperature of the water dialysis is preferably room temperature, the time of the water dialysis is preferably 3 to 4 days, and in a specific embodiment, it can be 3 days, 3.5 days or 4 days; the molecular weight cutoff of the dialysis bag used for the water dialysis is preferably 8 to 14 kDa, and in a specific embodiment, 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 used.

[0058] The present invention also provides a method for preparing a side chain modified polymer having a structure shown in OPT as described in the above technical scheme, comprising the following steps: subjecting the side chain modified polymer having a structure shown in OSP-PEG to an amidation reaction with 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to obtain a side chain modified polymer having a structure shown in OPT.

[0059] In the present invention, the usage ratio of the side chain modified polymer having the structure shown in OSP-PEG to 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid (TK) is preferably 32 mg: 0.1-0.15 mmol, and 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.

[0060] 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 amount ratio of the side chain modified polymer having the structure shown in OSP-PEG to the first catalyst is preferably 32mg: 0.8~1mmol, and in a specific embodiment, it can be 32mg: 0.8mmol, 32mg: 0.9mmol or 32mg: 1mmol. In the present invention, the amount ratio of the side chain modified polymer having the structure shown in OSP-PEG to the second catalyst is preferably 32mg: 0.8~1mmol, and in a specific embodiment, it can be 32mg: 0.8mmol, 32mg: 0.9mmol or 32mg: 1mmol.

[0061] 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 embodiments it can be 32 mg: 8 mL, 32 mg: 9 mL or 32 mg: 10 mL.

[0062] In the present invention, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 48 to 72 hours, and in specific embodiments it can be 48 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours or 72 hours.

[0063] After the amidation reaction, the present invention preferably further comprises: subjecting the reaction system obtained by the amidation reaction to water dialysis and then freeze-drying to obtain the side chain modified polymer having the structure shown in OPT. In the present invention, the temperature of the water dialysis is preferably room temperature, the time of the water dialysis is preferably 3 to 4 days, and in a specific embodiment, it can be 3 days, 3.5 days or 4 days; the molecular weight cutoff of the dialysis bag used for the water dialysis is preferably 8 to 14 kDa, and in a specific embodiment, 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 used.

[0064] 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 modified substance is the side chain modified polymer having the structure shown in OPT as described in the above technical scheme or the side chain modified polymer having the structure shown in OPT obtained by the preparation method described in the above technical scheme.

[0065] In the present invention, the photosensitizer preferably includes a photosensitizer that can generate 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 0.5-1 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.

[0066] The present invention also provides a method for preparing the drug delivery system described in the above technical solution, comprising the following steps: mixing a side chain modified polymer having a structure shown in OPT, a photosensitizer, an organic solvent and water, and performing self-assembly to obtain the drug delivery system.

[0067] In the present invention, the mass ratio of the side chain modified polymer having the structure shown in OPT to the photosensitizer is preferably 1:1-2, and in specific embodiments it can be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8 or 1:2.

[0068] In the present invention, the organic solvent preferably includes at least one of dimethyl sulfoxide (DMSO) and tetrahydrofuran. In the present invention, the amount ratio of the side chain modified polymer having the structure shown in OPT to the organic solvent is preferably 1 mg: 0.8~1 mL, and in specific embodiments it can be 1 mg: 0.8 mL, 1 mg: 0.9 mL or 1 mg: 1 mL.

[0069] In the present invention, the volume ratio of the organic solvent to water is preferably 1:1-9, and in specific embodiments can be 1:1, 1:2, 1:3, 1:4, 1:56, 1:7, 1:8 or 1:9.

[0070] In the present invention, the mixing preferably includes: dissolving the side chain modified polymer having the structure shown in OPT and the photosensitizer in an organic solvent, and adding the obtained mixed solution into water.

[0071] In the present invention, the self-assembly temperature is preferably room temperature, the self-assembly time is preferably 12 to 24 hours, and in specific embodiments it can be 12 hours, 15 hours, 18 hours, 20 hours, 22 hours or 24 hours; the self-assembly is preferably carried out under ultrasonic conditions, and the ultrasonic power is preferably 150 to 200 W, and in specific embodiments it can be 150 W, 160 W, 170 W, 180 W, 190 W or 200 W.

[0072] After the self-assembly, the present invention preferably further comprises: subjecting the self-assembled system to water dialysis and then freeze-drying to obtain the drug delivery system (OPTC). In the present invention, the water dialysis temperature is preferably room temperature, and the water dialysis time is preferably 2 to 24 hours, and in a specific embodiment, it can be 2 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours, 23 hours or 24 hours. 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 used.

[0073] The present invention also provides a light stimulus responsive nano delivery system (denoted as OPTC-RNP), comprising a drug delivery system and a gene editor; the drug delivery system is the drug delivery system described in the above technical scheme or the drug delivery system prepared by the preparation method described in the above technical scheme.

[0074] 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 25-50%, and in specific embodiments it can be 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0075] In the present invention, taking the Cas9 / sgRNA complex as an example, the TK bond ( 1 O2-breaking bond) connected (Cas9-P) to form OPTC-RNP, realizing the release of RNP on demand.

[0076] In the present invention, the preparation method of the Cas9 / sgRNA complex preferably comprises the following steps: mixing the Cas9 protein, sgRNA and a buffer solution to obtain a Cas9 / sgRNA complex. In the present invention, the mass ratio of the Cas9 protein to the sgRNA is preferably 1:1~3, and in a specific embodiment, 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 a Tris-HCl buffer; the pH value of the Tris-HCl buffer is preferably 7~7.5, and in a specific embodiment, it can be 7, 7.2 or 7.5. In the present invention, the mixing temperature is preferably room temperature, the mixing time is preferably 10~12min, and in a specific embodiment, it can be 10min, 11min or 12min; the mixing is preferably carried out under stirring conditions.

[0077] The present invention also provides a method for preparing the light stimulus responsive nano delivery system described in the above technical solution, comprising the following steps: subjecting the drug delivery system and the gene editor to an amide reaction to obtain the light stimulus responsive nano delivery system.

[0078] In the present invention, the mass ratio of the drug delivery system to the gene editor is preferably 1:0.01-0.02, and in specific embodiments may be 1:0.01, 1:0.012, 1:0.015, 1:0.018 or 1:0.02.

[0079] 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, and activating the mixture to obtain an activated drug delivery system.

[0080] In the present invention, the activator preferably includes a first activator and a second activator; the first activator preferably includes at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N,N'-diisopropylcarbodiimide (DIC); the second activator preferably includes at least one of N-hydroxysuccinimide (NHS) and 1-hydroxybenzotriazole (HOBT).

[0081] 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.

[0082] 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 subjecting the aforementioned self-assembled system to water dialysis.

[0083] In the present invention, the activation temperature is preferably room temperature, and the activation time is preferably 24 to 48 hours, and in specific embodiments, it can be 24 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours or 48 hours. In the present invention, taking EDC and NHS as examples, during the activation process, the side chain carboxyl group in the drug delivery system is activated, and EDC first reacts with the carboxyl group to generate an active intermediate, and the active intermediate then reacts with NHS to generate an NHS ester intermediate, which is relatively stable and helps to improve the yield of the reaction.

[0084] After the activation, the present invention preferably further comprises: subjecting the activated system to water dialysis to obtain an activated drug delivery system. In the present invention, the water dialysis temperature is preferably room temperature, and the water dialysis time is preferably 1 to 3 days, and in specific embodiments, it can be 1 day, 1.5 days, 2 days, 2.5 days or 3 days.

[0085] In the present invention, the temperature of the amide reaction is preferably 1-4°C, and in a specific embodiment it can be 1°C, 2°C, 3°C or 4°C; the time of the amide reaction is preferably 10-12h, and in a specific embodiment it can be 10h, 11h or 12h; 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.

[0086] The present invention also provides the use of the near-infrared photoinduced semiconductor polymer described in the above technical solution, the near-infrared photoinduced semiconductor polymer obtained by the preparation method described in the above technical solution, the hydrolyzate of the near-infrared photoinduced semiconductor polymer described in the above technical solution, the side-chain modified polymer described in the above technical solution, the side-chain modified polymer obtained by the preparation method described in the above technical solution, the drug delivery system described in the above technical solution, the drug delivery system obtained by the preparation method described in the above technical solution, the light stimulus responsive nano delivery system described in the above technical solution, or the light stimulus responsive drug delivery system obtained by the preparation method described in the above technical solution in biological imaging, preparation of gene therapy drugs, preparation of photodynamic therapy drugs for tumors, or preparation of anti-inflammatory drugs. The light stimulus 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 two-zone fluorescence imaging.

[0087] In order to further illustrate the present invention, the near-infrared photoinduced semiconductor polymer and its hydrolyzate, side chain modified polymer, drug delivery system, light stimulus responsive nano delivery system, preparation method and application provided by the present invention are described in detail below in combination with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0088] Example 1 1. Preparation of Semiconducting Polymers 2,7-dibromofluorene, tert-butyl acrylate and tetrabutylammonium bromide were mixed in a molar ratio of 10.2:41:0.78, protected by nitrogen, and toluene and 2M KOH aqueous solution were added, stirred at room temperature for 12 hours, washed with water, washed with 0.5M HCl aqueous solution, extracted with dichloromethane, and the obtained organic phase was dried with Na2SO4, filtered, and the filtrate was rotary evaporated to dryness and purified by silica gel column (eluent was petroleum ether and ethyl acetate, volume ratio was 4:1), to obtain monomer A (light yellow, yield was 75%). Among them, the molar ratio of 2,7-dibromofluorene and KOH was 10.2:10, and the amount ratio of 2,7-dibromofluorene to toluene was 10.2mmol:50mL.

[0089] Monomer A, bispinacol diboronate, Pd(dppf)2Cl2 and KOAc were mixed in a molar ratio of 3.7:9.5:2:17, and DMF was added to dissolve, and stirred and refluxed at 90°C for 12 hours under nitrogen protection, filtered, and the obtained filtrate was concentrated and purified on a silica gel column (the eluent was petroleum ether and ethyl acetate, the volume ratio was 5:1) to obtain monomer B (white, the yield was 60%). The usage ratio of monomer A and DMF was 3.7mmol:10mL.

[0090] Monomer B and monomer C (4,9-bis(5-bromothienyl)-6,7-bis(4-hexyloxyphenyl)-2-thia-1,3,5,8-tetraaza-cyclopentadienyl[b]naphthalene) were mixed in a molar ratio of 1:1, tetrakis(triphenylphosphine)palladium and K2CO3 were added, and finally toluene and water were added to dissolve, reacted at 90°C for 20 minutes, cooled to room temperature, and methanol (the volume fraction of methanol in the system was 95%) was precipitated for 24 hours, filtered, and the obtained liquid component was rotary evaporated to dryness to obtain a near-infrared photoinduced 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 amount ratio of monomer B, toluene and water was 1mmol:30mL:10mL.

[0091] The near-infrared photoinduced semiconductor polymer was dissolved in DCM and trifluoroacetic acid for hydrolysis at room temperature for 24 hours, and then rotary evaporated to dryness to obtain the hydrolysis product of the near-infrared photoinduced semiconductor polymer (OSP, n=53). The usage ratio of monomer B, DCM and trifluoroacetic acid was 1mmol:150mL:15mL.

[0092] 2. Side chain modification of semiconductor polymers OSP, NH2-PEG-NH2, NHS, EDC and DMSO were mixed in a molar ratio of 1:5:20:50, reacted at room temperature for 48 hours, dialyzed in water for 4 days, and freeze-dried to obtain OSP-PEG (dark green). The amount of OSP is calculated based on the structural unit in OSP, n=53, the molar ratio of OSP, NH2-PEG-NH2, NHS and EDC is 1:5:20:50, and the amount ratio of OSP to DMSO is 1mmol:100mL. The molecular weight cutoff of the dialysis bag used for water dialysis is 14kDa.

[0093] OSP-PEG, TK, NHS and EDC were mixed in a ratio of 32 mg: 0.12 mmol: 0.9 mmol: 0.9 mmol, reacted at room temperature for 48 h, dialyzed in water for 4 days, and freeze-dried to obtain OPT (green, OSP-PEG-TK). The amount of OSP-PEG and DMSO used was 32 mg: 10 mL, and the molecular weight cutoff of the dialysis bag used for water dialysis was 14 kDa.

[0094] 3. Preparation of drug delivery system (OPTC) OPT and free Ce6 were dissolved in DMSO at a mass ratio of 1:2 under ultrasound, and then injected into H2O for self-assembly at room temperature and 150-200W ultrasound conditions for 24 hours, dialyzed in water for 24 hours, and freeze-dried to obtain OPTC (green, OSP-PEG-TK-Ce6). The dosage ratio of OPT, DMSO, and H2O injected under ultrasound was 320 mg: 10 mL: 90 mL.

[0095] 4. Preparation of light stimulus responsive nanodelivery system (OPTC-RNP) The 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 a Cas9 / sgRNA complex (RNP).

[0096] 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 days to obtain activated OPTC.

[0097] The activated OPTC was added dropwise to the RNP, and the mixture was refrigerated and shaken at 4° C. for 12 h to obtain a nano-delivery system (OPTC-RNP).

[0098] Test Case Performance test of OSP, OPT, OPTC and OPTC-RNP prepared in Example 1 The OPTC-RNP prepared in Example 1 was measured by a second-zone fluorescence emission spectrometer. Figure 1 It is a fluorescence emission spectrum diagram of OPTC-RNP. By measuring the fluorescence spectrum, it can be known that the fluorescence emission of OPTC-RNP prepared by the present invention is in the second zone position, which meets the second zone imaging condition.

[0099] Figure 2 The UV-visible absorption spectra of OSP, OPT, OPTC, TK and Ce6 show that OPTC-RNP has near-infrared absorption characteristics.

[0100] Figure 3 This is a transmission electron microscope (TEM) image of OPTC-RNP. It can be seen that OPTC-RNP successfully self-assembles to form nanoparticles.

[0101] The surface charge (zeta potential, mV) of OSP, OPT, OPTC, and OPTC-RNP was measured by dynamic light scattering.

[0102] Figure 4 The dynamic light scattering (DLS) curves of OPTC and OPTC-RNP show that the number of nanoparticles formed by OPTC slightly increases after connecting to RNP.

[0103] Figure 5 Zeta potential diagrams of OSP, OPT, OPTC and OPTC-RNP. It can be seen that the charge of OPT modified with NH2-PEG-NH2 and TK side chains changes from positive (OSP) to negative, and the charge value is stable after encapsulating Ce6 and connecting RNP.

[0104] The generation of singlet oxygen was detected by measuring the absorption intensity at 420 nm in the UV-visible absorption spectrum. Figure 6 The relative absorbance of 1,3-diphenylisobenzofuran (DPBF) after adding OPTC-RNP under NIR irradiation (660 nm) and without NIR irradiation (660 nm) indicates that OPTC-RNP successfully produced single oxygen 1 O2.

[0105] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the residual amount of RNP in OPTC-RNP. Figure 7 The SDS-PAGE diagram of the residual amount of RNP in OPTC-RNP after NIR irradiation for different times shows that the response group TK between OPTC and RNP was successfully cut off under near-infrared light conditions.

[0106] Figure 8 The RNP release diagram corresponding to OPTC-RNP under NIR irradiation at different times was calculated by subtracting the residual amount of OPTC-RNP from the original amount using Image J. It can be seen that under near-infrared light conditions, the group between OPTC and RNP was successfully cut off and RNP was effectively released.

[0107] Confocal fluorescence microscopy (CLSM) was used to investigate the cellular internalization of OPTC-RNP@CM. Fig. 9 The CLSM images of OPTC-RNP labeled with EGFP (enhanced green fluorescent protein) and 4T1 cells incubated for 6 hours. 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.

[0108] OPTC-RNP@CM nanodrug (1 mg / mL, 200 μL) was intravenously injected into breast cancer tumor-bearing mice. Fig.10 The distribution of OPTC-RNP in mice was detected by near-infrared zone II in vivo imaging at different time points. It was found that OPTC-RNP was mainly accumulated in the tumors and liver of breast cancer-bearing mice after intravenous injection.

[0109] Fig.11 The figure shows the changes in OPTC-RNP signal intensity in mouse liver and tumor at different time points. It can be seen that 24 hours after intravenous injection of OPTC-RNP into breast cancer-bearing mice, the fluorescence intensity at the mouse tumor site was the strongest.

[0110] The mice were euthanized 48 hours after administration (OPTC-RNP), and the main organs and tumor tissues were collected for two-region imaging analysis. Fig.12It can be seen that OPTC-RNP accumulated in the tumors, spleen, and liver of breast cancer-bearing mice after intravenous injection.

[0111] 4T1 cells were pre-seeded in 24-well plates and cultured for 24 hours, and then co-incubated with different treatment groups in a 37 ℃ incubator for 6 hours. The cells were washed with PBS to remove the nanoparticles that did not enter the cells. The culture medium was then 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. 4T1 cells were irradiated with a 660 nm laser. After illumination, the cells were washed with PBS. The cells were observed with the Thunder imaging system, and DCFH-DA could be oxidized by free radicals to highly fluorescent DCF, and the green fluorescence in 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, Fig.13 DCF of 4T1 cells in different treatment groups (detection 1 O2) fluorescence staining image. It can be seen that OPTC-RNP successfully produced single oxygen after NIR irradiation. 1 O2.

[0112] The cells were pre-seeded in a 24-well plate and incubated for 24 hours. They were treated with different nanomedicines and then irradiated with a 660nm laser. After washing with PBS, 100 μL of Calcein AM / PI detection working solution was added to each well. After incubation at 37°C in the dark for 30 minutes, the staining effect was observed under Thunder. Fig.14 Live and dead fluorescence staining images of 4T1 cells in different treatment groups. Green, Calcein-AM (calcein); Red, PI (propidium iodide), scale bar: 300μm, same for each group Fig.13 It can be seen that OPTC-RNP killed a large number of 4T1 cells after being irradiated by NIR.

[0113] Take a portion of the tumor tissue collected from each group, cut it into small pieces, add collagenase for treatment according to the instructions, and then grind it into a homogenate and filter it with a filter. Use pre-cooled PBS to wash the filtered single cell suspension, centrifuge and discard the wash to remove cell debris, then resuspend the washed cells with 2% BSA, then add the corresponding antibodies for staining, and finally use a multi-color analytical flow cytometer for analysis. Fig.15 In order to detect the apoptosis of 4T1 cells under different treatment conditions by flow cytometry, the cells were divided into two groups and Fig.13 It can be seen that OPTC-RNP caused apoptosis of 4T1 cells after NIR irradiation.

[0114] Genomic DNA of 4T1 cells was collected using a universal genomic DNA extraction kit. The DNA fragment targeted by sgRNA was amplified using a high-fidelity DNA polymerase with the following primers: AGTACACCACTAACGCAAGC. To reduce nonspecific amplification, a decremental polymerase chain reaction (PCR) program was used [98°C for 30 s, (98°C for 10 s, 60°C for 20 s, 72°C for 20 s) for 35 cycles, and 72°C for 120 s]. After gel recovery and purification using a DNA purification kit, the frequency of indel generation was detected according to the T7 endonuclease kit. The purified DNA was analyzed by 2% agarose gel electrophoresis. The frequency of indel formation was calculated by ImageJ. Fig.16 The T7EI method was used to detect the gene mutation frequency of PD-L1 in 4T1 cells after different treatments. Fig.13 , it can be seen that RNP was successfully released after NIR irradiation, and the PD-L1 gene of a large number of 4T1 cells was knocked out.

[0115] Take a portion of the tumor tissue collected from each group, cut the tissue into small pieces, and then use the Western and IP cell lysate instructions to process and extract proteins. Load and separate equal amounts of total protein in SDS / PAGE gel and transfer to polyvinylidene difluoride (PVDF) membrane. The sample was blocked with a blocking solution containing 5% skim milk powder for 2 hours at room temperature. After blocking, the membrane was washed 3 times with TBST for 10 minutes each time, and then incubated with the corresponding primary antibody. Incubate overnight at 4°C, wash 3 times with TBST after incubation, 10 minutes each time. Then incubate with secondary antibody at room temperature for 1 hour. After incubation, wash the membrane 5 times in TBST buffer for 10 minutes each time. Finally, the target protein bands were visualized by ECL (enhanced chemiluminescence reagent), and the blot images were obtained and analyzed using an imaging system. Fig.17 To detect the expression level of PD-L1 in 4T1 cells after different treatments by WB, the cells were divided into groups with the same Fig.13 It can be seen that the expression level of 4T1 cells treated with OPTC-RNP / NIR was significantly reduced.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A near-infrared photoinduced semiconductor polymer, characterized in that: It has the structure shown in Formula I: Formula I; Among them, n is 45~65.

2. The method for preparing the near-infrared photoinduced semiconductor polymer according to claim 1, characterized in that: The following steps are involved: Performing Suzuki reaction on monomer B and monomer C to obtain the near-infrared photoinduced semiconductor polymer; ； Wherein, X is a halogen.

3. A hydrolysis product of a near-infrared photoinduced semiconductor polymer, wherein the near-infrared photoinduced semiconductor polymer is the near-infrared photoinduced semiconductor polymer according to claim 1 or the near-infrared photoinduced semiconductor polymer prepared by the preparation method according to claim 2, and has the structure shown in OSP: ； in, n is 45~65.

4. A side chain modified polymer, characterized in that Having the structure shown in OSP-PEG or OPT: ； Among them, n is 45~65, and m is 30~60.

5. The method for preparing the side chain modified polymer according to claim 4, characterized in that: The method for preparing a side chain modified polymer having a structure represented by OSP-PEG comprises the following steps: subjecting the hydrolyzate of the infrared light-induced semiconductor polymer according to claim 3 to an amidation reaction with bisamino polyethylene glycol to obtain the side chain modified polymer having a structure represented by OSP-PEG; the degree of polymerization of the bisamino polyethylene glycol is m; The preparation method of the side chain modified polymer having the structure shown in OPT comprises the following steps: subjecting the side chain modified polymer having the structure shown in OSP-PEG to an amidation reaction with 3,3'-(propane-2,2-diylbis(sulfanediyl))dipropionic acid to obtain a side chain modified polymer having the structure shown in OPT.

6. A drug delivery system, characterized in that It comprises a side chain modified polymer and a photosensitizer encapsulated in the side chain modified polymer; the side chain modified product is the side chain modified polymer having the structure shown in OPT in claim 4 or the side chain modified polymer having the structure shown in OPT obtained by the preparation method described in claim 5.

7. The method for preparing the drug delivery system according to claim 6, 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.

8. 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 6 or the drug delivery system prepared by the preparation method according to claim 7.

9. The method for preparing the light stimulus responsive nano delivery system according to claim 8, 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.

10. Use of the near-infrared photoinduced semiconductor polymer according to claim 1, the near-infrared photoinduced semiconductor polymer obtained by the preparation method according to claim 2, the hydrolyzate of the near-infrared photoinduced semiconductor polymer according to claim 3, the side-chain modified polymer according to claim 4, the side-chain modified polymer obtained by the preparation method according to claim 5, the drug delivery system according to claim 6, the drug delivery system obtained by the preparation method according to claim 7, the light-stimulation-responsive nano-delivery system according to claim 8 or the light-stimulation-responsive nano-delivery system obtained by the preparation method according to claim 9 in bioimaging, preparation of gene therapy drugs, preparation of photodynamic therapy drugs for tumors or preparation of anti-inflammatory drugs.

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