Activatable semiconductor polymer as well as preparation method and application thereof

Abstract

The invention provides an activatable semiconductor polymer as well as a preparation method and application thereof. The activatable semiconductor polymer disclosed by the invention can be efficiently accumulated at a tumor site, due to the existence of a side chain electron withdrawing group, the fluorescence of the polymer is in a quenched state, the fluorescence of the polymer is recovered after tumor markers such as biological mercaptans, active oxygen and cathepsin B are respectively responded, and accurate imaging and sonodynamic therapy can be carried out at the tumor site. Meanwhile, the activatable semiconductor polymer can mark immune cells (such as dendritic cells and macrophages) and track the process of transferring the immune cells from tumors to tumor drainage lymph nodes.

Description

Technical Field

[0001] The present invention relates to the technical field of new biomedical materials, and in particular to an activatable semiconductor polymer and a preparation method and application thereof. Background Art

[0002] Semiconducting polymers (SP), as a class of organic materials with π-conjugated aromatic main chains, have attracted extensive attention due to their excellent photophysical properties, good biosafety, adjustable chemical flexibility, easy preparation and high stability. With these unique advantages, SP has been widely used in an emerging non-invasive cancer treatment method: sonodynamic therapy (SDT), which activates sonosensitizers by ultrasound to produce reactive oxygen species (ROS) to kill tumors, and can also induce immunogenic cell death (ICD) of cancer cells for immunotherapy.

[0003] In addition, the development of anti-cancer immunity involves multiple immune cell populations, which are often highly dynamic and need to move from the tumor to the draining lymph nodes and back again. Antigen presenting cells, including dendritic cells (DCs) and macrophages, play a key role in the initiation of cancer-specific adaptive immunity and are also an important component of current cancer immunity research. The migration of immune cells between tumors and tumor-draining lymph nodes determines the immune status of the tumor. Tracking immune cell migration is mainly used to observe and study the movement and distribution of immune cells in the body, thereby helping scientists understand immune responses and disease progression.

[0004] However, the materials currently used for both technologies often have problems such as poor stability, low sensitivity, and difficulty in synthesis, which limits their widespread application. Therefore, the development of an activatable semiconductor polymer material for tracking immune cell migration and ultrasound-controlled ICD immunotherapy will have certain guiding significance for achieving effective, convenient, and deep tumor cell clearance immunotherapy.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide an activatable semiconductor polymer and a preparation method and application thereof. The activatable semiconductor polymer provided by the present invention can be efficiently enriched at the tumor site. After responding to tumor markers: biothiols, ROS and cathepsin B respectively, the polymer fluorescence is restored, and precise fluorescence imaging and sonodynamic therapy can be performed at the tumor site. Secondly, the activatable semiconductor polymer can mark immune cells (such as dendritic cells, macrophages) and track their process of metastasis from the tumor to the tumor-draining lymph nodes. It can significantly improve the accuracy of immune cell tracking and the efficacy of sonodynamic therapy.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] In a first aspect, the present invention provides an activatable semiconductor polymer having a structure as shown in the following formula I:

[0009]

[0010] Wherein, the R group represents a substituent group having a structure as shown in the following formula II:

[0011]

[0012] in, represents the group connection position, group A represents an electron-withdrawing quenching group, m≥20, n≥1.

[0013] Preferably, the A group is selected from any one of the following A-1, A-2 or A-3 groups; wherein, Representative group connection position:

[0014]

[0015] Preferably, the activatable semiconductor polymer comprises any one of the following polymer I-1, polymer I-2 or polymer I-3:

[0016]

[0017] Preferably, the maximum absorption wavelength of the activatable semiconductor polymer represented by Formula I is 558-562 nm, preferably 560 nm.

[0018] Preferably, the polymer I-1 specifically responds to biothiol substances and then recovers fluorescence; wherein the biothiol substances include any one of glutathione, cysteine ​​or homocysteine, or a combination of at least two of them.

[0019] Preferably, the polymer I-2 specifically responds to active oxygen and then recovers fluorescence.

[0020] Preferably, the polymer I-3 specifically responds to cathepsin B and then recovers fluorescence.

[0021] In a second aspect, the present invention provides a method for preparing an activatable semiconductor polymer as described in the first aspect, the preparation method comprising the following steps:

[0022] In the presence of a catalyst, 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene and 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole are used as monomers to carry out a polymerization reaction to obtain a polymer SPBr; the polymer SPBr is subjected to an azide reaction to obtain a polymer SPN3, and the reaction formula is as follows:

[0023]

[0024] The polyethylene glycol compound with terminal modification of alkynyl and amino groups reacts with an electron-withdrawing quencher to obtain the compound DNBS-PEG. The reaction formula is as follows:

[0025]

[0026] Wherein, AX represents an electron-withdrawing quencher, the A group represents an electron-withdrawing quencher group as described in the first aspect, and the X group represents any one of a halogen or a hydroxyl group;

[0027] The polymer SPN3 and the compound DNBS-PEG undergo click chemistry reaction to obtain an activatable semiconductor polymer shown in Formula I. The reaction formula is as follows:

[0028]

[0029] Preferably, the molar ratio of the 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene to 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole is 1:(0.8-1.2).

[0030] Preferably, the catalyst comprises bis(triphenylphosphine)palladium dichloride and tri(o-methylphenyl)phosphine.

[0031] Preferably, the polymerization reaction temperature is 90-110° C., and the polymerization reaction time is 6-10 min.

[0032] Preferably, the polymerization reaction is carried out in a solvent comprising chlorobenzene.

[0033] Preferably, the azide reagent used in the azide reaction is sodium azide.

[0034] Preferably, the molar ratio of the polymer SPBr to sodium azide is 1:(2-2.5).

[0035] Preferably, the temperature of the azidation reaction is 20-30° C., and the time of the azidation reaction is 18-30 h.

[0036] Preferably, the azide reaction is carried out in a solvent comprising tetrahydrofuran and / or N,N-dimethylformamide.

[0037] Preferably, the molar ratio of the polyethylene glycol compound to the electron-withdrawing quencher is 1:(1-1.5).

[0038] Preferably, the temperature for the reaction of the polyethylene glycol compound and the electron-withdrawing quencher is 20 to 30° C. and the time is 6 to 30 hours.

[0039] Preferably, the electron-withdrawing quencher comprises wherein the X group represents any one of halogen or hydroxy.

[0040] Preferably, the molar ratio of the polymer SPN3 to the compound DNBS-PEG is 1:(2-2.5);

[0041] Preferably, the click chemistry reaction is carried out in a solvent, and the solvent comprises tetrahydrofuran;

[0042] Preferably, the temperature of the click chemistry reaction is 20 to 30° C., and the time of the click chemistry reaction is 8 to 16 hours.

[0043] In a second aspect, the present invention provides a use of the activatable semiconductor polymer as described in the first aspect in preparing a tumor imaging detection agent.

[0044] Furthermore, the activatable semiconductor polymer is efficiently accumulated in a mouse subcutaneous breast cancer tumor model.

[0045] In a third aspect, the present invention provides a use of the activatable semiconductor polymer as described in the first aspect in preparing a tracer for marking immune cell migration.

[0046] Furthermore, the immune cell migration is the migration process of immune cells from the tumor site to the tumor-draining lymph nodes.

[0047] In a fourth aspect, the present invention provides a use of the activatable semiconductor polymer as described in the first aspect in the preparation of tumor therapeutic drugs.

[0048] Furthermore, the tumor therapeutic drug is a tumor in situ sonodynamic therapy drug.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The activatable semiconductor polymer of the present invention can be efficiently accumulated at the tumor site. Due to the presence of electron-withdrawing groups on the side chain, the fluorescence of the polymer is in a quenched state. After responding to tumor markers: biothiols, ROS and cathepsin B, the fluorescence of the polymer is restored, and precise imaging and sonodynamic therapy can be performed at the tumor site.

[0051] (2) The activatable semiconductor polymer of the present invention can mark immune cells (such as dendritic cells and macrophages) and track their migration from tumors to tumor-draining lymph nodes, thereby achieving effective monitoring and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0053] Figure 1A This is the ultraviolet absorption spectrum of the activatable semiconductor polymer I-1 provided in Example 1.

[0054] Figure 1B This is the ultraviolet absorption spectrum of the activatable semiconductor polymer I-2 provided in Example 2.

[0055] Figure 1C This is the ultraviolet absorption spectrum of the activatable semiconductor polymer I-3 provided in Example 3.

[0056] Figure 2A This is the H-NMR spectrum of the activatable semiconductor polymer I-1 provided in Example 1.

[0057] Figure 2B This is the H-NMR spectrum of the activatable semiconductor polymer I-2 provided in Example 2.

[0058] Figure 2C This is the H-NMR spectrum of the activatable semiconductor polymer I-3 provided in Example 3.

[0059] Figure 3 This is a near-infrared fluorescence imaging intensity diagram of the activatable semiconductor polymer I-1 provided in Example 1 in the presence of amino acids such as GSH, Cys, and Hcy (5 mM).

[0060] Figure 4 These are near-infrared fluorescence images of mouse 4T1 tumors and draining lymph nodes (dLN) at different times after injection of the activatable semiconductor polymer I-1 provided in Example 1.

[0061] Figure 5 These are near-infrared fluorescence images of the tumor site 12h and 24h after different ultrasound treatment times of the activatable semiconductor polymer I-1 provided in Example 1.

[0062] Figure 6This is a flow cytometric graph of the migration of immune cells (dendritic cells and macrophages) labeled by I-1 in the tumor to the draining lymph nodes after different ultrasound treatment times of the activatable semiconductor polymer I-1 provided in Example 1.

[0063] Figure 7 This is a graph showing the tumor growth curve of tumor-bearing mice after different treatments with the activatable semiconductor polymer I-1 provided in Example 1.

[0064] Figure 8 This is the fluorescence spectrum of the activatable semiconductor polymer I-2 provided in Example 2.

[0065] Fig. 9 This is the in vivo fluorescence imaging image of the activatable semiconductor polymer I-3 provided in Example 3. DETAILED DESCRIPTION

[0066] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0067] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0068] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] In a first aspect, the present invention provides an activatable semiconductor polymer having a structure as shown in the following formula I:

[0070]

[0071] Wherein, the R group represents a substituent group having a structure as shown in the following formula II:

[0072]

[0073] in, represents the group connection position, group A represents an electron-withdrawing quenching group, m≥20, n≥1.

[0074] As an optional implementation, m≥20, for example, it can be 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 70, 80, 100, etc.

[0075] As an optional implementation, n≥1, for example, it can be 1, 2, 4, 5, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.

[0076] It should be noted that the present invention does not specifically limit the degree of polymerization n, and real-time monitoring is performed during the polymerization reaction. The reaction is stopped when the maximum absorption wavelength of the activatable semiconductor polymer shown in Formula I reaches a specified value (such as a maximum absorption wavelength of 560 nm) to obtain the activatable semiconductor polymer shown in Formula I with a suitable molecular weight and degree of polymerization.

[0077] As an optional embodiment, the A group is selected from any one of the following A-1, A-2 or A-3 groups; wherein, Representative group connection position:

[0078]

[0079] As an optional embodiment, the activatable semiconductor polymer includes any one of the following polymer I-1, polymer I-2 or polymer I-3:

[0080]

[0081] As an optional embodiment, the maximum absorption wavelength of the activatable semiconductor polymer shown in Formula I is 558-562 nm, for example, it can be 558 nm, 559 nm, 560 nm, 561 nm, 562 nm, etc., preferably 560 nm.

[0082] It should be noted that the maximum absorption wavelengths of polymers I-1, I-2 and I-3 of the present invention are all around 560 nm. This is because the groups that affect the maximum absorption wavelength are mainly Polymer I-1, polymer I-2 and polymer I-3 have the same main ring structure, and the side chain R group does not affect the shift of the maximum absorption wavelength.

[0083] As an optional embodiment, the polymer I-1 specifically responds to biothiol substances and then recovers fluorescence; wherein the biothiol substances include any one of glutathione, cysteine ​​or homocysteine, or a combination of at least two of them.

[0084] As an optional embodiment, the polymer I-2 recovers fluorescence after specifically responding to active oxygen.

[0085] As an optional embodiment, the polymer I-3 specifically responds to cathepsin B and then recovers fluorescence.

[0086] In a second aspect, the present invention provides a method for preparing an activatable semiconductor polymer as described in the first aspect, the preparation method comprising the following steps:

[0087] In the presence of a catalyst, 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene and 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole are used as monomers to carry out a polymerization reaction to obtain a polymer SPBr; the polymer SPBr is subjected to an azide reaction to obtain a polymer SPN3, and the reaction formula is as follows:

[0088]

[0089] The polyethylene glycol compound with terminal modification of alkynyl and amino groups reacts with an electron-withdrawing quencher to obtain the compound DNBS-PEG. The reaction formula is as follows:

[0090]

[0091] Wherein, AX represents an electron-withdrawing quencher, the A group represents an electron-withdrawing quencher group as described in the first aspect, and the X group represents any one of a halogen or a hydroxyl group;

[0092] The polymer SPN3 and the compound DNBS-PEG undergo click chemistry reaction to obtain an activatable semiconductor polymer shown in Formula I. The reaction formula is as follows:

[0093]

[0094] As an optional embodiment, the molar ratio of 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene to 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole is 1:(0.8-1.2), for example, it can be 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, etc.

[0095] As an optional embodiment, the catalyst includes bis(triphenylphosphine)palladium dichloride and tri(o-methylphenyl)phosphine.

[0096] As an optional embodiment, the polymerization reaction temperature is 90-110°C, for example, it can be 90°C, 92°C, 94°C, 95°C, 96°C, 98°C, 100°C, 102°C, 104°C, 105°C, 106°C, 108°C, 110°C, etc., and the polymerization reaction time is 6-10min, for example, it can be 6min, 7min, 8min, 9min, 10min, etc.

[0097] As an optional embodiment, the polymerization reaction is carried out in a solvent, and the solvent includes chlorobenzene.

[0098] As an optional embodiment, the azide reagent used in the azide reaction is sodium azide.

[0099] As an optional embodiment, the molar ratio of the polymer SPBr to sodium azide is 1:(2-2.5), for example, it can be 1:2, 1:2.05, 1:2.1, 1:2.15, 1:2.2, 1:2.25, 1:2.3, 1:2.35, 1:2.4, 1:2.45, 1:2.5, etc.

[0100] As an optional embodiment, the temperature of the azide reaction is 20-30°C, for example, it can be 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the azide reaction is 18-30h, for example, it can be 18h, 20h, 22h, 24h, 26h, 28h, 30h, etc.

[0101] As an optional embodiment, the azide reaction is carried out in a solvent, and the solvent includes tetrahydrofuran and / or N,N-dimethylformamide.

[0102] As an optional embodiment, the molar ratio of the polyethylene glycol compound to the electron-withdrawing quencher is 1:(1-1.5), for example, it can be 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.25, 1:1.3, 1:1.35, 1:1.4, 1:1.45, 1:1.5, etc.

[0103] As an optional embodiment, the reaction temperature of the polyethylene glycol compound and the electron-withdrawing quencher is 20-30°C, for example, it can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, etc., and the time is 6-30h, for example, it can be 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, 26h, 28h, 30h, etc.

[0104] As an optional embodiment, the electron-withdrawing quencher comprises wherein the X group represents a halogen (eg, -Cl, -Br, -I, etc.) or a hydroxyl group.

[0105] As an optional embodiment, the molar ratio of the polymer SPN3 and the compound DNBS-PEG is 1:(2-2.5), for example, it can be 1:2, 1:2.05, 1:2.1, 1:2.15, 1:2.2, 1:2.25, 1:2.3, 1:2.35, 1:2.4, 1:2.45, 1:2.5, etc.

[0106] As an optional embodiment, the click chemistry reaction is carried out in a solvent, and the solvent includes tetrahydrofuran.

[0107] As an optional embodiment, the temperature of the click chemistry reaction is 20-30°C, for example, it can be 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the click chemistry reaction is 8-16h, for example, it can be 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, etc.

[0108] In a third aspect, the present invention provides a use of the activatable semiconductor polymer as described in the first aspect in preparing a tumor imaging detection agent.

[0109] In a fourth aspect, the present invention provides a use of the activatable semiconductor polymer as described in the first aspect in preparing a tracer for marking immune cell migration.

[0110] As an optional embodiment, the immune cell migration is the migration process of immune cells from the tumor site to the tumor-draining lymph nodes.

[0111] In a fifth aspect, the present invention provides a use of the activatable semiconductor polymer as described in the first aspect in the preparation of tumor therapeutic drugs.

[0112] As an optional embodiment, the tumor therapeutic drug is an in situ tumor sonodynamic therapy drug.

[0113] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0114] Example 1

[0115] This embodiment provides an activatable semiconductor polymer I-1, and the structural formula of the activatable semiconductor polymer I-1 is as follows:

[0116]

[0117] The preparation method of the activatable semiconductor polymer I-1 comprises the following steps:

[0118] (a) Preparation of polymer SPN3, the reaction formula is as follows:

[0119]

[0120] 2,7-Dibromo-9,9-bis(6-bromohexyl)fluorene (40 mg, 0.062 mmol), 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole (38.8 mg, 0.062 mmol), PdCl2(PPh3)2 (6 mg, 0.007 mmol) and tri-o-methylphosphine (3 mg, 0.010 mmol) were dissolved in 6 mL of chlorobenzene under nitrogen atmosphere. Degassing was performed by three freeze pump-thaw cycles, and the polymerization reaction was vigorously stirred at 100 ° C for 8 min. After the reaction was completed, the mixture was precipitated and purified in methanol to obtain polymer SPBr.

[0121] 20 mg SPBr and 6 mg NaN3 were dissolved in 10 mL tetrahydrofuran and N,N-dimethylformamide (v / v=2:1), stirred at room temperature in the dark for 24 h, and THF and DMF were removed under reduced pressure. The crude product was dissolved in dichloromethane, washed with saturated sodium chloride three times, then collected in a flask and dried under vacuum to obtain polymer SPN3.

[0122] (b) Preparation of DNBS-PEG-1, the reaction formula is as follows:

[0123]

[0124] 80 mg of alkynyl-PEG-amino and 28 μL of triethylamine were added to 3 mL of ultra-dry dichloromethane to dissolve, and 53 mg of 2,4-dinitrobenzenesulfonyl chloride was added on ice. The mixture was stirred at room temperature for 8 h under nitrogen protection. After the reaction, the mixture was washed with water, precipitated in ether, and centrifuged to obtain a solid product, DNBS-PEG-1.

[0125] (c) Preparation of activatable semiconductor polymer I-1, the reaction formula is as follows:

[0126]

[0127] 9 mg of polymer SPN3 and 25 mg of solid product DNBS-PEG-1 were dissolved in 5 mL of tetrahydrofuran, stirred at room temperature in the dark for 12 h under a nitrogen environment, and the tetrahydrofuran was removed under reduced pressure. The crude product was dissolved in water and dialyzed against water to obtain activatable semiconductor polymer Ⅰ-1.

[0128] Characterization of activatable semiconductor polymer Ⅰ-1:

[0129] (1) UV absorption spectrum test: Test the UV absorption spectrum of the activated semiconductor polymer I-1 (20 μg / mL). The results are shown in Figure 1A , Figure 1A is the ultraviolet absorption spectrum of the activated semiconductor polymer Ⅰ-1. Figure 1A As shown, the maximum absorption wavelength of the semiconductor polymer provided in this application is 560nm.

[0130] (2) Nuclear magnetic resonance spectrum test: The activatable semiconductor polymer I-1 was characterized by hydrogen spectrum nuclear magnetic resonance. Figure 2A This is the NMR spectrum test of the activated semiconductor polymer Ⅰ-1.

[0131] Example 2

[0132] This embodiment provides an activatable semiconductor polymer I-2, and the structural formula of the activatable semiconductor polymer I-2 is as follows:

[0133]

[0134] The preparation method of the activatable semiconductor polymer I-2 comprises the following steps:

[0135] (a) Preparation of polymer SPN3, the reaction formula is as follows:

[0136]

[0137] 2,7-Dibromo-9,9-bis(6-bromohexyl)fluorene (40 mg, 0.062 mmol), 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole (38.8 mg, 0.062 mmol), PdCl2(PPh3)2 (6 mg, 0.007 mmol) and tri-o-methylphosphine (3 mg, 0.010 mmol) were dissolved in 6 mL of chlorobenzene under nitrogen atmosphere. Degassing was performed by three freeze pump-thaw cycles, and the polymerization reaction was vigorously stirred at 100 ° C for 8 min. After the reaction was completed, the mixture was precipitated and purified in methanol to obtain polymer SPBr.

[0138] 20 mg SPBr and 6 mg NaN3 were dissolved in 10 mL tetrahydrofuran and N,N-dimethylformamide (v / v=2:1), stirred at room temperature in the dark for 24 h, and THF and DMF were removed under reduced pressure. The crude product was dissolved in dichloromethane, washed with saturated sodium chloride three times, then collected in a flask and dried under vacuum to obtain polymer SPN3.

[0139] (b) Preparation of DNBS-PEG-2, the reaction formula is as follows:

[0140]

[0141] R1 (300.00 mg, 1.19 mmol, 1.0 equiv.) and HOBt (40.16 mg, 0.5 equiv.) were dissolved in DCM (5.00 mL), and ice-bathed for 10 min under nitrogen protection. EDCI (15.17 mg, 0.700 equiv.) was added to continue the reaction for 2 h, and 3,5-dinitrophenylhydrazine (117.78 mg, 594.43 μmol, 0.5 equiv.) was added to move the reaction to room temperature 45 ° C and stir for 24 h. TLC (DCM / MeOH=10 / 1) was used to detect until there was no obvious change in the reaction, and the reaction was terminated. Concentrate under reduced pressure, wash with saturated NaHCO3 and H2O respectively, dry, concentrate under reduced pressure, and purify by 200-300 mesh silica gel column chromatography to collect compound R2. Take 20 mg of alkynyl-PEG-amino and 10 mg of the target product R2, stir at room temperature for 12 h, remove the solvent by rotary evaporation, and precipitate with ether to obtain a solid product DNBS-PEG-2.

[0142] (c) Preparation of activatable semiconductor polymer I-2, the reaction formula is as follows:

[0143]

[0144] 20 mg of polymer SPN3 and 82 mg of solid product DNBS-PEG-2 were dissolved in 10 mL of tetrahydrofuran, stirred at room temperature in the dark for 12 h under a nitrogen environment, and the tetrahydrofuran was removed under reduced pressure. The crude product was dissolved in water and dialyzed against water to obtain activatable semiconductor polymer I-2.

[0145] Characterization of activatable semiconductor polymer Ⅰ-2:

[0146] (1) UV absorption spectrum test: Test the UV absorption spectrum of the activated semiconductor polymer I-2 (20 μg / mL). The results are shown in Figure 1B , Figure 1B is the ultraviolet absorption spectrum of the activated semiconductor polymer Ⅰ-2. Figure 1B As shown, the maximum absorption wavelength of the semiconductor polymer provided in this application is 560nm.

[0147] (2) Nuclear magnetic resonance spectrum test: The activatable semiconductor polymer I-2 was characterized by hydrogen spectrum nuclear magnetic resonance. Figure 2B This is the NMR spectrum test of the activated semiconductor polymer Ⅰ-1.

[0148] Example 3

[0149] This embodiment provides an activatable semiconductor polymer I-3, the structural formula of the activatable semiconductor polymer I-3 is as follows:

[0150]

[0151] The preparation method of the activatable semiconductor polymer I-1 comprises the following steps:

[0152] (a) Preparation of polymer SPN3, the reaction formula is as follows:

[0153]

[0154] 2,7-Dibromo-9,9-bis(6-bromohexyl)fluorene (40 mg, 0.062 mmol), 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole (38.8 mg, 0.062 mmol), PdCl2(PPh3)2 (6 mg, 0.007 mmol) and tri-o-methylphosphine (3 mg, 0.010 mmol) were dissolved in 6 mL of chlorobenzene under nitrogen atmosphere. Degassing was performed by three freeze pump-thaw cycles, and the polymerization reaction was vigorously stirred at 100 ° C for 8 min. After the reaction was completed, the mixture was precipitated and purified in methanol to obtain polymer SPBr.

[0155] 20 mg SPBr and 6 mg NaN3 were dissolved in 10 mL tetrahydrofuran and N,N-dimethylformamide (v / v=2:1), stirred at room temperature in the dark for 24 h, and THF and DMF were removed under reduced pressure. The crude product was dissolved in dichloromethane, washed with saturated sodium chloride three times, then collected in a flask and dried under vacuum to obtain polymer SPN3.

[0156] (b) Preparation of DNBS-PEG-3, the reaction formula is as follows:

[0157]

[0158] Add 500 mg of 2-chlorotriphenyl resin to a solid phase synthesis tube, activate with DMF for 5 min, add Fmoc-Lys-OH (1.18 g, 2 mmol) and DIPEA (417 μL, 2.5 mmol) and react for 3 h, wash three times with DMF. Add 20% piperidine in DMF solution and react for 15 min, repeat once, and wash three times with DMF.

[0159] Fmoc-Phe-OH (0.774 g, 2 mmol), DIPEA (417 μL, 2.5 mmol), HoBt (0.338 g, 2.5 mmol) and HBTU (0.947 g, 2.5 mmol) were added and reacted for 2 h, and washed with DMF three times. A 20% piperidine solution in DMF was added and reacted for 15 min, and the reaction was repeated once, and washed with DMF three times.

[0160] DNP-Gly (0.774 g, 2 mmol), DIPEA (417 μL, 2.5 mmol), HoBt (0.338 g, 2.5 mmol) and HBTU (0.947 g, 2.5 mmol) were added to react for 2 h, and washed with DCM three times. A 1% TFA solution in DCM was added, and the mixture was blown for 2-3 min, and the DCM was collected, and the mixture was vortexed to dryness, and ether was precipitated, and vacuum dried at 40°C for 1 h to obtain compound 1. The compound was dissolved in 95% trifluoroacetic acid, stirred at room temperature for 1 h, and ether was precipitated to obtain compound 2.

[0161] Compound 2 (20 mg, 0.033 mmol, 1.0 equiv.) was dissolved in V CHCl3 / V CH3OH =3 / 1, add EDCI (10 mg, 0.05 mmol, 1.5 equiv.) and NHS (6 mg, 0.05 mmol, 1.5 equiv.) and react at room temperature for 1 h, add alkynyl-PEG-amino (67 mg, 0.033 mmol, 1.0 equiv.) and react for 24 h, ether precipitation several times to obtain the product DNBS-PEG-3.

[0162] (c) Preparation of activatable semiconductor polymer I-3, the reaction formula is as follows:

[0163]

[0164] 20 mg of polymer SPN3 and 50 mg of solid product DNBS-PEG-3 were dissolved in 10 mL of tetrahydrofuran, stirred at room temperature in the dark for 12 h under a nitrogen environment, and the tetrahydrofuran was removed under reduced pressure. The crude product was dissolved in water and dialyzed against water to obtain activatable semiconductor polymer I-3.

[0165] Characterization of activatable semiconductor polymer I-3:

[0166] (1) UV absorption spectrum test: Test the UV absorption spectrum of the activated semiconductor polymer I-3 (20 μg / mL). The results are shown in Figure 1C , Figure 1C is the ultraviolet absorption spectrum of the activated semiconductor polymer Ⅰ-2. Figure 1CAs shown, the maximum absorption wavelength of the semiconductor polymer provided in this application is 560nm.

[0167] (2) Nuclear magnetic resonance spectrum test: The activatable semiconductor polymer I-3 was characterized by hydrogen spectrum nuclear magnetic resonance. Figure 2C This is the NMR spectrum test of the activated semiconductor polymer Ⅰ-1.

[0168] Test Example 1

[0169] Test of response ability to biological thiols

[0170] Test sample: the activatable semiconductor polymer I-1 provided in Example 1;

[0171] Test method: Semiconducting polymer I-1 (40 μg / mL) was incubated with different amino acids such as GSH, Cys, and Hcy at 37°C for 1 h, and detected by fluorescent in vivo imaging;

[0172] Test results:

[0173] Figure 3 The responsiveness of the semiconductor polymer prepared in Example 1 to biothiol substances is Figure 3 It can be seen that the semiconductor polymer I-1 provided in the present application can respond to tumor markers: biothiol substances (GSH, Cys, Hcy).

[0174] Test Example 2

[0175] 4T1 tumor model was used for in vivo fluorescence imaging experiments

[0176] Test sample: the activatable semiconductor polymer I-1 provided in Example 1.

[0177] Cell culture: In the present invention, cell lines such as 4T1 are selected, and the cell culture method is a method well known to those skilled in the art and is not particularly limited. In the present invention, cells are preferably cultured in 1640 medium containing 10% fetal bovine serum, and the culture conditions are preferably continuous culture in an incubator with a volume fraction of 5% carbon dioxide, and the culture temperature is preferably 37°C.

[0178] Animal model: In the present invention, BALB / c mice were used for tumor model experiments. BALB / c mice were subcutaneously injected with 4T1 tumor cells to establish a subcutaneous breast cancer tumor model. When the tumor volume reached 100 mm 3 When , start the experiment.

[0179] In vivo imaging detection: When the volume of subcutaneous breast cancer tumors in BALB / c mice reached 100mm 3 At the same time, polymer I-1 was injected through the tail vein, and fluorescent in vivo imaging was performed on the tumor site and lymph node site, respectively.

[0180] Test method: Three BALB / c mice weighing about 20 g were used, and 1.0×10 6 The cells were inoculated subcutaneously in mice with 4T1 tumors. When the tumor volume reached about 100 mm 3 When the above-mentioned agent was injected into the tail vein (the material concentration was 200 μg / mL), the mice were imaged in vivo at different time points.

[0181] Test results:

[0182] Figure 4 The near-infrared fluorescence imaging images of the mouse 4T1 tumor and draining lymph nodes (dLN) at different times after injection of the activatable semiconductor polymer I-1 provided in Example 1 are shown in FIG. Figure 4 It can be seen that the fluorescence intensity of the tumor area began to increase 24 hours after the injection of the activatable semiconductor polymer I-1 provided in Example 1, reached a peak at 60 hours, and gradually decreased after 72 hours. At the same time, the draining lymph of the mice was imaged, and it was found that the near-infrared fluorescence intensity of the tumor site was always much higher than that of the draining lymph nodes. At 60 hours, the near-infrared fluorescence intensity of the tumor site was 9.0 times higher than that of the draining lymph nodes. The results show that the activatable semiconductor polymer I-1 provided in Example 1 can be efficiently enriched at the tumor site.

[0183] Test Example 3

[0184] In vivo tracking of immune cell migration assay

[0185] Test sample: the activatable semiconductor polymer I-1 provided in Example 1.

[0186] In vivo immune cell migration assay: Tumor-bearing mice were randomly divided into four groups: (1) no ultrasound group (-US); (2) ultrasound once group (+US-1); (3) ultrasound twice group (+US-2); (4) ultrasound three times group (+US-3). When the volume of subcutaneous breast cancer tumors in BALB / c mice reached 100 mm 3 At 12h and 24h after the treatment, I-1 was injected into the tail vein and the tumor site was treated with ultrasound. Fluorescence in vivo imaging of the lymph node site and tumor site was performed respectively. After the imaging, the mouse lymph nodes were taken for flow cytometry analysis.

[0187] Test method: Twenty BALB / c mice weighing about 20 g were divided into four groups, namely -US group, +US-1 group, +US-2 group and +US-3 group. Each mouse was treated with 1.0×10 6 The cells were inoculated subcutaneously in mice with 4T1 tumors. When the tumor volume reached about 100 mm 3At the same time, the above-mentioned drugs were injected into the tail vein for treatment (the material concentration was 200 μg / mL), and the tumor site was ultrasounded on the 2nd, 3rd and 4th days (-US group did not undergo ultrasound, +US-1 group underwent ultrasound once, +US-2 group underwent ultrasound twice, and +US-3 group underwent ultrasound three times). Fluorescence imaging was performed 12h and 24h after the end of ultrasound. After the imaging, the tumor-draining lymph nodes of the mice were taken for flow cytometry testing.

[0188] Test results:

[0189] Figure 5 The near-infrared fluorescence imaging images of the tumor site 12h and 24h after different ultrasound times of the activatable semiconductor polymer I-1 provided in Example 1. Figure 5 It can be seen that the fluorescence intensity of the draining lymph nodes of mice in the +US-3 group was the highest at 12h and 24h, proving that more I-1 migrated to the draining lymph nodes with the increase in the number of ultrasounds.

[0190] Figure 6 This is a flow cytometric diagram of the migration of immune cells (dendritic cells and macrophages) labeled by I-1 in the tumor to the draining lymph nodes after different ultrasound times of the activatable semiconductor polymer I-1 provided in Example 1. Figure 6 It can be seen that I-1 can label dendritic cells and macrophages.

[0191] Test Example 4

[0192] In vivo therapeutic experiments

[0193] Test sample: the activatable semiconductor polymer I-1 provided in Example 1.

[0194] In vivo sonodynamic therapy: Tumor-bearing mice were randomly divided into five groups: (1) control group; (2) no ultrasound group (-US); (3) ultrasound once group (+US-1); (4) ultrasound twice group (+US-2); (5) ultrasound three times group (+US-3). When the volume of subcutaneous breast cancer tumors in BALB / c mice reached 100 mm 3 On the 2nd, 3rd and 4th days, the +US-1, +US-2 and +US-3 groups were treated with ultrasound (ultrasound conditions: 1.0 MHz, 1.5 W / cm 2 , 50% duty cycle), and the changes in the tumor volume (length × width) of the mice were recorded every other day. 2 / 2).

[0195] Test method: 20 BALB / c mice weighing about 20 g were divided into 5 groups, namely PBS group, -US group, +US-1 group, +US-2 group and +US-3 group. 4T1 tumor cells were subcutaneously inoculated into the mice at a density of 1.0×106 cells per mouse. When the tumor volume reached about 100mm 3 When the above-mentioned drug was injected into the tail vein (the material concentration was 200 μg / mL), the tumor site was ultrasounded on the 2nd, 3rd and 4th days, and the changes in tumor volume were compared after 14 days.

[0196] Test results:

[0197] Figure 7 This is a graph showing the tumor growth curve of tumor-bearing mice after different treatments with the activatable semiconductor polymer I-1 provided in Example 1. Figure 7 It can be seen that -US has almost no inhibitory effect on tumor growth, while the inhibition of tumors in the +US-3 group is more obvious and has a stronger inhibitory effect.

[0198] Test Example 5

[0199] Responsiveness test to active oxygen

[0200] Test sample: the activatable semiconductor polymer I-2 provided in Example 2;

[0201] Test method: Incubate the activatable semiconductor polymer I-2 with H2O2 solution at 37°C for 1 hour, and use the sample without H2O2 solution as a control to perform fluorescence spectrum test;

[0202] Test results: Figure 8 This is the fluorescence spectrum of the activatable semiconductor polymer I-2 provided in Example 2.

[0203] Test Example 6

[0204] Test for responsiveness to cathepsin B

[0205] Test sample: the activatable semiconductor polymer I-3 provided in Example 3;

[0206] Test method: Tumor-bearing mice were randomly divided into three groups: (1) control group; (2) material plus inhibitor group (I-3+lnn.); (3) material group (I-3). When the volume of subcutaneous breast cancer tumor in BALB / c mice reached 100mm 3 I-3 was injected into the tumor at different times. In vivo fluorescence imaging of the tumor site was performed continuously.

[0207] Test results:

[0208] Fig. 9This is a fluorescence imaging result of the tumor site of tumor-bearing mice after different treatments with the activatable semiconductor polymer I-3 provided in Example 3. Fig. 9 It can be seen that the fluorescence intensity of the tumor site in the mice in the group with cathepsin B inhibitor was weaker, while the fluorescence intensity of the tumor site in the material group was more obvious, indicating that I-3 can respond to cathepsin B in the tumor site.

[0209] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An activatable semiconductor polymer, characterized in that: The activatable semiconductor polymer has a structure as shown in the following formula I: Wherein, the R group represents a substituent group having a structure as shown in the following formula II: in, represents the group connection position, group A represents an electron-withdrawing quenching group, m≥20, n≥1.

2. The activatable semiconducting polymer according to claim 1, characterized in that The A group is selected from any one of the following A-1, A-2 or A-3 groups; wherein, Representative group connection position:

3. The activatable semiconducting polymer according to claim 1 or 2, characterized in that: The activatable semiconductor polymer includes any one of the following polymer I-1, polymer I-2 or polymer I-3:

4. The activatable semiconducting polymer according to claim 1, characterized in that The maximum absorption wavelength of the activatable semiconductor polymer represented by Formula I is 558-562 nm, preferably 560 nm.

5. The activatable semiconducting polymer according to claim 3, characterized in that The polymer I-1 specifically responds to biothiol substances and then recovers fluorescence; wherein the biothiol substances include any one of glutathione, cysteine ​​or homocysteine, or a combination of at least two of them; and / or, the polymer I-2 specifically responds to reactive oxygen species and then recovers fluorescence; And / or, the polymer I-3 specifically responds to cathepsin B and then recovers fluorescence.

6. A method for preparing an activatable semiconductor polymer according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: In the presence of a catalyst, 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene and 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole are used as monomers to carry out a polymerization reaction to obtain a polymer SPBr; the polymer SPBr is subjected to an azide reaction to obtain a polymer SPN3; The polyethylene glycol compound with terminal modification of alkynyl and amino groups reacts with an electron-withdrawing quencher to obtain the compound DNBS-PEG; The polymer SPN3 and the compound DNBS-PEG undergo click chemistry reaction to obtain an activatable semiconductor polymer as shown in formula I.

7. The method for preparing an activatable semiconductor polymer according to claim 6, characterized in that: The molar ratio of the 2,7-dibromo-9,9-bis(6-bromohexyl)fluorene to the 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzotriazole is 1:(0.8-1.2); Preferably, the catalyst comprises bis(triphenylphosphine)palladium dichloride and tri(o-methylphenyl)phosphine; Preferably, the polymerization reaction temperature is 90-110° C., and the polymerization reaction time is 6-10 min; Preferably, the polymerization reaction is carried out in a solvent, and the solvent comprises chlorobenzene; Preferably, the azide reagent used in the azide reaction is sodium azide; Preferably, the molar ratio of the polymer SPBr to sodium azide is 1:(2-2.5); Preferably, the temperature of the azidation reaction is 20 to 30° C., and the time of the azidation reaction is 18 to 30 hours; Preferably, the azide reaction is carried out in a solvent, and the solvent comprises tetrahydrofuran and / or N,N-dimethylformamide; Preferably, the molar ratio of the polyethylene glycol compound to the electron-withdrawing quencher is 1:(1-1.5); Preferably, the temperature of the reaction between the polyethylene glycol compound and the electron-withdrawing quencher is 20 to 30° C., and the time is 6 to 30 hours; Preferably, the electron-withdrawing quencher comprises Any one of; wherein the X group represents any one of halogen or hydroxyl; Preferably, the molar ratio of the polymer SPN3 to the compound DNBS-PEG is 1:(2-2.5); Preferably, the click chemistry reaction is carried out in a solvent, and the solvent comprises tetrahydrofuran; Preferably, the temperature of the click chemistry reaction is 20 to 30° C., and the time of the click chemistry reaction is 8 to 16 hours.

8. Use of the activatable semiconductor polymer according to any one of claims 1 to 5 in the preparation of a tumor imaging detection agent.

9. Use of an activatable semiconductor polymer according to any one of claims 1 to 5 in preparing a tracer for marking immune cell migration; And / or, the immune cell migration is the migration process of immune cells from the tumor site to the tumor-draining lymph nodes.

10. Use of an activatable semiconductor polymer according to any one of claims 1 to 5 in the preparation of a tumor therapeutic drug; And / or, the tumor therapeutic drug is a tumor in situ sonodynamic therapy drug.

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