A light-activated nano-immunoadjuvant drug delivery material and a preparation method and application thereof

Abstract

This invention discloses a photoactivated nanoparticle-based drug delivery material for immunoadjuvants, its preparation method, and its applications. The drug delivery material is constructed from photosensitizer monomers, crosslinking monomers, and immunoadjuvant prodrug monomers within a confined space of a nanoemulsion via a copper-free click polymerization reaction to form crosslinked polymer nanoparticles. The nanoparticle size is controllable, and the composition is precisely defined. Under physiological conditions, the drug delivery material exhibits good colloidal stability and high drug loading. Upon near-infrared light irradiation, the generated ROS can cleave the thioketone bonds, causing the nanoparticles to depolymerize and achieving photo-triggered, controllable release of the immunoadjuvant. Under photodynamic action, this drug delivery material efficiently generates reactive oxygen species to induce immunogenic cell death in tumor cells. Simultaneously, the released immunoadjuvant activates the TLR7 / 8 pathway, promotes dendritic cell maturation, and enhances the systemic anti-tumor immune response, thereby inhibiting the growth of tumors in situ and at distant sites, and reducing the risk of metastasis and recurrence.

Description

Technical Field

[0001] This invention relates to the field of biomedical polymer materials and tumor therapeutic drug delivery technology, specifically to a photoactivated nano-adjuvant drug delivery material, its preparation method, and its application. Background Technology

[0002] Tumor immunotherapy, as an important development in the field of cancer treatment in recent years, has demonstrated significant clinical efficacy in various malignant tumors. Immune checkpoint inhibitors, represented by PD-1 / PD-L1 antibodies and CTLA-4 antibodies, can restore or enhance the body's immune response to tumors, achieving long-term remission in some patients. However, the overall response rate of tumor immunotherapy remains limited, with problems such as a low proportion of beneficiaries, drug resistance, and immune-related adverse reactions. Especially in the context of "cold tumors" and immunosuppressive microenvironments, relying solely on immune checkpoint inhibitors is insufficient to achieve ideal efficacy. How to rationally regulate the tumor immune microenvironment and activate innate and adaptive immune responses is a crucial scientific and technological problem that urgently needs to be solved in the field of tumor immunotherapy.

[0003] Immunoadjuvants, as functional molecules capable of activating innate immunity and enhancing antigen presentation and T-cell responses, hold significant application potential in tumor vaccines and combination immunotherapy. Various small-molecule immunoadjuvants targeting pathways such as TLR7 / 8 and STING have demonstrated significant immune activation and anti-tumor effects in animal experiments. However, free immunoadjuvants generally suffer from problems such as non-accumulation at tumor sites, high systemic exposure levels, significant toxic side effects, and difficulty in precisely controlling dosage and the timing and location of action, limiting their safe and efficient application in tumor immunotherapy. Therefore, achieving targeted enrichment, controlled release, and therapeutic window expansion of immunoadjuvants is one of the key aspects of improving the overall efficacy of tumor immunotherapy.

[0004] To improve the in vivo delivery of immune adjuvants, researchers have developed various nanomedicine delivery materials, including lipid nanoparticles, polymer nanoparticles, inorganic nanocarriers, and multifunctional hybrid nanosystems. These nanodelivery platforms have made progress in improving the accumulation of immune adjuvants at tumor sites, reducing systemic toxicity, and achieving some degree of combination therapy. However, existing drug delivery materials often have complex compositions, and the carriers themselves are mostly pharmaceutically inert components, resulting in problems such as limited drug loading capacity, difficulty in precisely defining structure and composition, sensitivity of preparation processes to conditions, and difficulty in achieving batch-to-batch stability during scale-up production. In addition, some systems still have shortcomings in terms of stimulus responsiveness, photoactivation synergy, and fine-tuning of drug release, making it difficult to balance safety, controllability, and translational feasibility.

[0005] In view of this, there is an urgent need to provide a novel nanomedicine delivery material with a relatively simple structure and composition, a precisely defined drug loading ratio, both photoactivated and stimulus-responsive properties, and which is conducive to process scale-up and batch stability control, so as to achieve efficient delivery of immune adjuvants to tumor sites and further improve the efficacy of tumor immunotherapy. Summary of the Invention

[0006] This invention addresses the problems of existing tumor immunoadjuvants, such as systemic toxicity from free administration, difficulty in achieving effective enrichment and controlled release at the tumor site, and the complexity of existing nanomedicine delivery materials, difficulty in precisely defining drug loading ratios, and poor batch-to-batch consistency in preparation. It provides a photoactivated nanomedicine adjuvant drug delivery material, its preparation method, and its applications. This invention utilizes a photosensitizer monomer containing multiple azide groups, a crosslinking monomer containing dual DBCO groups, and a prodrug monomer containing stimulus-responsive breakable linkages and DBCO groups. Through copper-free click polymerization, precisely defined crosslinked nanoparticles are constructed, achieving efficient co-loading and precise proportioning of photosensitizers and immunoadjuvant prodrugs on the same nanoplatform. This allows for the synergistic triggering of photodynamic killing and controlled release of the immunoadjuvant at the tumor site via photoinduced reactive oxygen species generation and stimulus-responsive bond breaking, thereby enhancing the anti-tumor immune response. The photoactivated nanomedicine adjuvant drug delivery material of this invention is easy to design and control in structure and composition, possesses advantages such as high drug loading, good colloidal stability, and scalable preparation process, and is expected to provide a new and convertible nanomedicine delivery strategy for photodynamic-immunotherapy of malignant tumors.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a photoactivated nano-adjuvant drug delivery material. The present invention constructs a precursor polymerization unit for the photoactivated nano-adjuvant drug delivery material, specifically: the drug delivery material is a nanoparticle, and its cross-linked polymer backbone is composed of photosensitizer monomer A, cross-linking monomer B and prodrug monomer C through a copper-free click polymerization reaction.

[0009] The photosensitizer monomer A comprises a photochromophore P and at least three azide substituents attached to the photochromophore P and / or its linking arms. The photochromophore P generates reactive oxygen species upon light irradiation. The photochromophore P is selected from at least one of fluoroboron dipyrrole, porphyrin, phylloporin, and phthalocyanine chromophores; preferably, the photochromophore P is a fluoroboron dipyrrole chromophore. The structure of the photosensitizer monomer A is shown in formula (A):

[0010]

[0011] In equation (A), n is an integer from 2 to 10.

[0012] Furthermore, the photosensitizer monomer A has the following structure:

[0013]

[0014] Further, the crosslinking monomer B comprises two o-benzodiazepine octylene (DBCO) groups and a linking group connected between the two DBCO groups. Preferably, the linking group is a thioacetate linking group, the structure of which is shown in formula (B):

[0015]

[0016] In equation (B), x and y are integers from 1 to 9.

[0017] Furthermore, the crosslinking monomer B has the following structure:

[0018] .

[0019] Further, the prodrug monomer C comprises at least one DBCO group, a drug molecule group, and a ROS-responsive breakable linker group connecting the DBCO group and the drug molecule group. The ROS-responsive breakable chemical bond is at least one of a thioclase bond, an oxalate bond, and a phenylboronic acid ester bond. The drug molecule group is preferably the TLR7 / 8 agonist IMDQ or a pharmaceutically acceptable derivative thereof, the structure of which is shown in formula (C):

[0020]

[0021] In equation (C), p and q are integers from 1 to 9.

[0022] Furthermore, the prodrug monomer has the following structure:

[0023] .

[0024] The drug delivery material of this invention exhibits good colloidal stability and high drug loading under physiological conditions. Upon near-infrared light irradiation, it generates reactive oxygen species (ROS), which cleave thioketone bonds, causing the nanoparticles to depolymerize and enabling photo-triggered, controllable release of the immunoadjuvant. Under photodynamic therapy, this drug delivery material efficiently generates reactive oxygen species, inducing immunogenic cell death in tumor cells. Simultaneously, the released immunoadjuvant activates the TLR7 / 8 pathway, promotes dendritic cell maturation, and enhances the systemic anti-tumor immune response, thereby inhibiting the growth of tumors in situ and at distant sites, reducing the risk of metastasis and recurrence. This provides a novel drug delivery strategy for constructing precisely defined photodynamic-immunotherapy nanomedicines. The average particle size of the nanoparticles in this invention is 20–200 nm, preferably 100–200 nm; the nanoparticles are in a colloidal dispersion system in an aqueous medium and have a narrow particle size distribution.

[0025] The preparation methods of photosensitizer monomer A, crosslinking monomer B, and prodrug monomer C are described below.

[0026] The preparation of the photosensitizer monomer A includes the following steps:

[0027] (1) According to the following reaction formula, p-hydroxyacetophenone and dibromoalkane are dissolved in acetone in the presence of potassium carbonate and etherified under heating conditions to obtain a para-substituted acetophenone intermediate with bromine substitution (A1).

[0028] (2) According to the following reaction formula, the para-substituted acetophenone intermediate (A1) with bromine substitution is dissolved in N,N-dimethylformamide to carry out a substitution reaction, and the para-substituted acetophenone compound (A2) with azid substitution is obtained.

[0029]

[0030] (3) According to the following reaction formula, p-hydroxybenzaldehyde and dibromoalkane are dissolved in acetone in the presence of potassium carbonate and etherified under heating conditions to obtain a para-substituted benzaldehyde intermediate with bromine substitution (A3).

[0031]

[0032] (4) According to the following reaction formula, the para-substituted benzaldehyde intermediate with bromine substitution (A3) is dissolved with sodium azide in N,N-dimethylformamide to carry out a substitution reaction to obtain the para-substituted benzaldehyde compound with azide substitution (A4).

[0033]

[0034] (5) According to the following reaction formula, the para-substituted acetophenone compound (A2) with azid substitution and the para-substituted benzaldehyde compound (A4) with azid substitution are dissolved in methanol in the presence of sodium hydroxide and condensed under reflux to obtain the α,β-unsaturated ketone intermediate shown in formula (A5).

[0035]

[0036] (6) According to the following reaction formula, compound (A5) and nitromethane are heated to 80°C in ethanol in the presence of potassium hydroxide, resulting in Michael addition and subsequent transformation, to obtain the nitro-substituted β-dicarbonyl compound shown in formula (A6).

[0037]

[0038] (7) According to the following reaction formula, compound (A6) is dissolved in n-butanol and reacted under reflux conditions. The pyrrole structural unit provided by ammonium acetate is used for condensation cyclization to obtain the compound shown in formula (A7).

[0039]

[0040] (8) According to the following reaction formula, compound (A7) was added to boron trifluoride diethyl ether complex BF3·Et2O and organic base triethylamine in dichloromethane solvent under nitrogen protection. The reaction was stirred at room temperature to coordinate dipyrrolemene with boron atoms and fluorinate it to obtain the compound shown in formula (A8).

[0041]

[0042] (9) According to the following reaction formula, the compound of formula (A8) is dissolved in dichloromethane, and the brominating agent N-bromosuccinimide (NBS) is added under nitrogen protection. The reaction is stirred at room temperature to introduce bromine atoms into the photosensitizer skeleton structure, thereby obtaining fluoroboron dipyrrole photosensitizer monomer A.

[0043]

[0044] The preparation of the crosslinking monomer B includes the following steps:

[0045] According to the following reaction formula, compounds B1 and B2 were dissolved in anhydrous dichloromethane, and the organic base triethylamine was added. The reaction was carried out under nitrogen protection and stirred at room temperature. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to prepare crosslinked monomer B with DBCO groups at both ends and a thioacetate linker in the center.

[0046]

[0047] The preparation of the prodrug monomer C includes the following steps:

[0048] (1) According to the following reaction formula, thiophene diol (compound C1) and p-nitrophenyl chloroformate were dissolved in anhydrous dichloromethane, and the organic base triethylamine was added. The mixture was slowly mixed under nitrogen protection and an ice bath, and then stirred at room temperature. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound C2, which has p-nitrophenyl carbonate groups at both ends and a thiophene linking group in the center.

[0049]

[0050] (2) According to the following reaction formula, the DBCO derivative containing the terminal amino group (compound C3) and compound C2 are dissolved in anhydrous dichloromethane and stirred under nitrogen protection and room temperature in the presence of anhydrous triethylamine. The amino group on the DBCO derivative containing the terminal amino group undergoes amidation coupling with the active ester group in compound C2, and a DBCO group is introduced at one end of the molecule to obtain the compound shown in formula (C4).

[0051]

[0052] (3) According to the following reaction formula, the IMDQ drug molecule and the compound C4 are dissolved in anhydrous N,N-dimethylformamide, anhydrous triethylamine is added, and the reaction is continued to be stirred at room temperature so that the amino group on the IMDQ molecule condenses with the active ester group on the other side of the compound C4. After the reaction is completed, the solvent is removed, and the crude product is purified by silica gel column chromatography to obtain the target prodrug monomer C, that is, the prodrug molecule structure with DBCO at one end, IMDQ at the other end and connected by a thioketone bond in the middle.

[0053]

[0054] Secondly, the present invention provides a method for preparing the above-mentioned photoactivated nano-adjuvant drug delivery material. The method includes: dissolving photosensitizer monomer A, crosslinking monomer B, and prodrug monomer C in an organic solvent to obtain an organic phase solution; dispersing the organic phase solution in an aqueous phase containing a surfactant to form a nanoemulsion; and, under copper-free click reaction conditions, subjecting the azide substituents on the photosensitizer monomer A to click polymerization with the DBCO groups on the crosslinking monomer B and the prodrug monomer C, and removing the organic solvent to obtain nanoparticles with a crosslinked polymer backbone. The organic solvent is preferably selected from dichloromethane, chloroform, ethyl acetate, or mixtures thereof, and the surfactant is preferably selected from at least one of lecithin, polysorbate, or block copolymer surfactants. The copper-free click reaction is preferably carried out in the range of room temperature to 50°C. The resulting nanoparticles form a colloidal dispersion system in an aqueous medium, with an average particle size preferably of 20–200 nm, more preferably 100–200 nm.

[0055] Thirdly, this invention also provides the application of the above-mentioned photoactivated nano-adjuvant drug delivery material in the preparation of photodynamic-immunotherapy drugs for treating malignant tumors. The photoactivated nano-adjuvant drug delivery material of this invention can simultaneously achieve photodynamic killing of tumor cells and controlled release of immune adjuvants under near-infrared light irradiation at 600-800 nm, and is expected to be used to enhance tumor immune responses, inhibit the growth of primary and distant tumors, and reduce the risk of metastasis and recurrence.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] 1. This invention introduces a photosensitizer monomer A containing a hydrazine group, a crosslinking monomer B containing a double DBCO group, and a prodrug monomer C containing DBCO and a stimuli-responsive breakable linker bond into the same nanoplatform. By using copper-free click polymerization to construct crosslinked polymer nanoparticles within the confined space of a nanoemulsion, this invention achieves efficient co-loading and precisely defined molar ratio of photosensitizer and immune adjuvant prodrug in the same nanomaterial, overcoming the problems of complex composition and difficulty in precisely controlling the drug loading ratio of traditional nanocarriers.

[0058] 2. The photoactivated nano-adjuvant drug delivery material of the present invention uses photosensitizer monomer A as an endogenous luminescent and reactive oxygen species generating unit, cross-linking monomer B containing thiophene linkage group as ROS-responsive cross-linking network, and prodrug monomer C as an immune adjuvant prodrug that can be dissociated by stimulation-responsive linkage group. Under near-infrared light irradiation, it can simultaneously achieve photodynamic killing of tumor cells, cross-linking network depolymerization, and on-demand release of immune adjuvant, thereby forming a photodynamic-immune synergistic effect in the tumor site and effectively enhancing the anti-tumor immune response.

[0059] 3. The nanoparticles described in this invention are cross-linked polymer nanosystems with simple composition and designable structure. They do not rely on a large amount of pharmaceutically inert carriers during preparation, have high drug loading, good colloidal stability and controllable particle size distribution, are suitable for scale-up preparation and facilitate batch-to-batch quality consistency control, and have good potential for process scale-up and transformation.

[0060] 4. The photoactivated nano-adjuvant drug delivery material provided by this invention can be adapted to a variety of photosensitizer chromophores and various types of immune adjuvant drug molecular groups, and has good versatility and scalability. It is expected to provide a novel nano-drug delivery platform that can be modularly designed for photodynamic-immunotherapy of malignant tumors. Attached Figure Description

[0061] Figure 1 The results are the 1H NMR spectrum of photosensitizer monomer A.

[0062] Figure 2 The results are the 1H NMR spectrum of crosslinked monomer B.

[0063] Figure 3 The results are the 1H NMR spectrum of the prodrug monomer C.

[0064] Figure 4 Image showing the transmission electron microscope test results of a photoactivated nano-adjuvant drug delivery material;

[0065] Figure 5 A drug photostimulation-response release curve for a photoactivated nano-adjuvant drug delivery material;

[0066] Figure 6 This figure shows the cytotoxicity results of a photoactivated nano-adjuvant drug delivery material on 4T1 tumor cells. Detailed Implementation

[0067] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0068] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0069] Example 1: Preparation of photoactivated nano-adjuvant drug delivery materials

[0070] 1. Synthesis of photosensitizer monomer A

[0071] (1) As shown in the following reaction formula, p-hydroxyacetophenone (CAS No.: 99-93-4, 136.2 mg) and 1,3-dibromopropane (CAS No.: 109-64-8, 350 mg) were dissolved in acetone (50 mL), and potassium carbonate (250 mg) was added. The mixture was heated to reflux and stirred for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 5:1) to obtain the solid product, compound A1.

[0072]

[0073] (2) As shown in the following reaction formula, compound A1 (257 mg) and sodium azide (CAS No.: 26628-22-8, 100 mg) were dissolved in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reaction system was added to a water / dichloromethane mixture, and after separation and extraction, dichloromethane was removed by rotary evaporation to obtain the solid product, namely compound A2.

[0074]

[0075] (3) As shown in the following reaction formula, p-hydroxybenzaldehyde (CAS No.: 123-08-0, 122.1 mg) and 1,3-dibromopropane (CAS No.: 109-64-8, 350 mg) were dissolved in acetone (50 mL), and potassium carbonate (250 mg) was added. The mixture was heated to reflux and stirred for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to obtain the solid product, compound A3.

[0076]

[0077] (4) As shown in the following reaction formula, compound A3 (200 mg) and sodium azide (CAS No.: 26628-22-8, 100 mg) were dissolved in N,N-dimethylformamide (30 mL), and the mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reaction system was added to a water / dichloromethane mixture, and after separation and extraction, dichloromethane was removed by rotary evaporation to obtain the solid product, namely compound A4.

[0078]

[0079] (5) As shown in the following reaction formula, compound A2 (219.1 mg) and compound A4 (205.1 mg) were dissolved in methanol (30 mL), and sodium hydroxide (500 mg) was added. The mixture was heated to reflux and stirred for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to obtain the solid product, compound A5.

[0080]

[0081] (6) As shown in the following reaction formula, compound A5 (406.2 mg) and nitromethane (CAS No.: 75-52-5, 120 mg) were dissolved in ethanol (30 mL), and potassium hydroxide (300 mg) was added. The mixture was heated and stirred at 80 °C for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 5:1) to obtain the solid product, compound A6.

[0082]

[0083] (7) As shown in the following reaction formula, compound A6 (467.2 mg) and ammonium acetate (CAS No.: 631-61-8, 140 mg) were dissolved in n-butanol (50 mL), and the mixture was heated under reflux and stirred for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to obtain the solid product, namely compound A7.

[0084]

[0085] (8) As shown in the following reaction formula, compound A7 (845.4 mg) and boron trifluoride diethyl ether (CAS No.: 109-63-7, 120 mg) were dissolved in dichloromethane (50 mL), and triethylamine (30 mL) was added. The reaction was carried out under nitrogen atmosphere and stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: hexane / dichloromethane, volume ratio 1:2) to obtain the solid product, compound A8.

[0086]

[0087] (9) As shown in the following reaction formula, compound A8 (893.4 mg) and N-bromosuccinimide (CAS No.: 128-08-5, 360 mg) were dissolved in dichloromethane (50 mL) and reacted at room temperature in the dark for 24 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography (eluent: hexane / dichloromethane, volume ratio 1:5) to obtain the solid product, namely photosensitizer monomer A. The structure of the obtained photosensitizer monomer A was verified by 1H NMR spectroscopy, and the results are shown in the appendix. Figure 1 As shown.

[0088] The specific 1H NMR spectrum results are as follows: 1 H NMR (500 MHz, Chloroform-d) δ 7.91-7.86 (m,4H), 7.77-7.71 (m, 4H), 6.97 (dd, J = 8.7, 6.4 Hz, 8H), 4.12 (dt, J = 19.3,5.8 Hz, 8H), 3.55 (dt, J = 15.4, 6.5 Hz, 8H), 2.09 (dt, J = 19.5, 6.3 Hz, 8H).

[0089] 2. Synthesis of crosslinking monomer B

[0090] As shown in the following reaction formula, compound B1 (DBCO-NHS ester, CAS No.: 1353016-71-3, 810 mg) and compound B2 (2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine, CAS No.: 22907-30-8, 194.1 mg) were dissolved in dichloromethane (50 mL), and triethylamine (100 mg) was added. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to obtain the solid product, namely crosslinking monomer B. The structure of the obtained crosslinking monomer B was verified by 1H NMR spectroscopy, and the results are attached. Figure 2 As shown.

[0091] The specific 1H NMR spectrum results are as follows: 1H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J =7.4 Hz, 2H), 7.50-7.27 (m, 12H), 7.24-7.19 (m, J = 7.1 Hz, 2H), 6.25 (d, J =22.8 Hz, 2H), 5.13 (d, J = 13.8 Hz, 2H), 3.69-3.60 (m, 2H), 3.40-3.20 (m,4H), 2.74 (dd, J = 16.2, 7.9 Hz, 2H), 2.66-2.58 (m, 4H), 2.35 (p, J = 8.5,7.5 Hz, 2H), 2.23-2.04 (m, 2H), 2.00-1.84 (m, 2H), 1.58 (s, 6H).

[0092]

[0093] 3. Synthesis of prodrug monomer C

[0094] (1) As shown in the following reaction formula, compound C1 (2,2'-(propane-2,2-diylbis(thionyl))diethanol, CAS No.: 1965253-60-4, 196.2 mg) was dissolved in anhydrous dichloromethane (25 mL), and p-nitrophenyl chloroformate (CAS No.: 7693-46-1, 405 mg) was dissolved in anhydrous dichloromethane (25 mL). The two were mixed under nitrogen protection and ice bath conditions, and then triethylamine (100 mg) was added. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 5:1) to obtain the solid product, namely compound C2, which has p-nitrophenyl carbonate groups at both ends and a thioacetate linker in the center.

[0095]

[0096] (2) According to the following reaction formula, DBCO-Amine (compound C3, CAS No.: 1255942-06-3) and compound C2 (263 mg) were dissolved in anhydrous dichloromethane (50 mL), followed by the addition of triethylamine (100 mg). The reaction was carried out under nitrogen protection and at room temperature with stirring for 24 hours. After the reaction was completed, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate, volume ratio 3:1) to obtain the solid product, compound C4.

[0097]

[0098] (3) As shown in the following reaction formula, compound C4 (663.2 mg) and the immunoadjuvant drug IMDQ (350.3 mg) were dissolved in N,N-dimethylformamide (30 mL), and triethylamine (100 mg) was added. The mixture was stirred at room temperature for 24 hours. After the reaction was completed, the reaction solution was poured into water and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using petroleum ether / ethyl acetate (5:1 v / v) as the eluent to obtain the prodrug monomer C. The structure of the obtained prodrug monomer C was verified by 1H NMR spectroscopy, and the results are shown in the appendix. Figure 3 As shown.

[0099]

[0100] The specific 1H NMR spectrum results are as follows: 1 H NMR (600 MHz, Chloroform-d): δ 7.64 (d, J =7.5 Hz, 2H), δ 7.60 (m, 2H), δ 7.50 (m, 2H), δ 7.45 (m, 4H), δ 7.50 (t, J =7.0 Hz, 2H), δ 7.35 (t, J = 6.8 Hz, 2H), δ 7.30 (d, J = 7.3 Hz, 2H), δ 6.95 (d, J = 5.3 Hz, 2H), δ 5.03 (d, J = 15.4 Hz, 2H), δ 3.97 (t, J = 6.0 Hz, 4H),δ 3.63 (d, J = 7.5 Hz, 2H), δ3.09 (m, 2H), δ 2.85 (m, 2H), δ 2.74 (t, J = 5.2Hz, 3H), δ 2.45 (m, 2H), δ 1.3 (m, 2H), δ 1.51 (t, 9H), δ 1.23 (s, 2H), δ1.07 (t, J = 7.0 Hz, 3H).

[0101] 4. Preparation of drug delivery materials

[0102] Photosensitizer monomer A, crosslinking monomer B, and prodrug monomer C were dissolved in dichloromethane to prepare stock solutions with concentrations of 2 mM, 4 mM, and 2 mM, respectively. The three stock solutions (photosensitizer monomer A (2 mM, 10 mL), crosslinking monomer B (4 mM, 9 mL), and prodrug monomer C (2 mM, 4 mL)) were mixed and then added to a polyvinyl alcohol (type 17-88) solution (2 wt%, 100 mL). The mixture was sheared at 16000 r / min for 3 min using a high-speed shear press. The resulting crude emulsion was then treated under a homogenizing pressure of 150 MPa to prepare a nanoemulsion. The nanoemulsion was placed in a beaker and stirred openly at room temperature for 24 h to allow the dichloromethane to evaporate. After centrifugation, washing with water, and freeze-drying, the photoactivated nano-adjuvant drug delivery material was obtained.

[0103] The final nanoparticle structure was characterized by transmission electron microscopy. The test results showed that the nanomaterial exhibited a uniform spherical nanoparticle morphology with an average particle size of about 180 nm.

[0104] Example 2: Photostimulation-Response Drug Release from Photoactivated Nanoparticles for Immunoadjuvant Drug Delivery

[0105] Weigh 2 mg of the photoactivated nano-adjuvant drug delivery material and add it to a dialysis bag with a molecular weight cutoff of 2 kDa. Place the bag in 30 mL of release medium (PBS solution containing 0.5% Tween 80 at pH 7.4) and pass it through a 660 nm laser (power 100 mW·cm⁻¹). -2 After irradiation for 3 min, the test system was incubated at 37°C and 100 r / min for 2 hours. Then, 1 mL of release medium was removed and an equal volume of blank release medium was added. This process was repeated 5 times. The IMDQ content in the collected samples was determined by high-performance liquid chromatography (HPLC), and the cumulative release rate was calculated. The chromatographic conditions were as follows: mobile phase: methanol-water (70:30); detection wavelength: 254 nm. The photoresponsive release kinetics of IMDQ are shown in the attached figure. Figure 5 As shown, without external light irradiation, the cumulative release rate of the photoactivated nano-adjuvant drug delivery material was less than 5% within 24 hours. However, under 660 nm light irradiation, the cumulative release rate exceeded 85% within 24 hours. This indicates that 660 nm light stimulation is a necessary prerequisite for the release of the adjuvant drug.

[0106] Example 3: Cytotoxicity experiment of photoactivated nano-adjuvant drug delivery material against 4T1 tumor cells

[0107] The cytotoxicity of the photoactivated nano-adjuvant drug delivery material to 4T1 cells under light- and light-free conditions was determined using a CCK8 assay: 4T1 cells were seeded at a density of 3000 cells per well in 96-well plates and cultured in a cell culture incubator for 24 hours. After cell attachment, the old culture medium was removed, and the medium was replaced with complete culture medium (100 μL) containing different concentrations of the drug. Cells were then cultured for another 48 hours. The old culture medium was then removed, and fresh medium containing 10% CCK8 (100 μL) was added, followed by 2 hours of culture. Finally, the absorbance of the 96-well plates at 450 nm was read using a microplate reader. In the light-treated group, cells were co-cultured with the drug for 12 hours and then irradiated with a 660 nm laser (100 mW / cm²). 2 (10 min). The results of the cytotoxicity test are attached. Figure 6 As shown, under 660 nm laser irradiation, the photoactivated nano-adjuvant drug delivery material exhibited cytotoxicity against 4T1 cells with increasing concentration. Conversely, without 660 nm light irradiation, the photoactivated nano-adjuvant drug delivery material showed negligible cytotoxicity against 4T1 cells.

[0108] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A photoactivated nano-adjuvant drug delivery material, characterized in that, The drug delivery material is a nanoparticle whose cross-linked polymer backbone is composed of photosensitizer monomer A, cross-linking monomer B and prodrug monomer C through a copper-free click polymerization reaction. The photosensitizer monomer A includes a photochromosome P and at least three azide substituents attached to the photochromosome P and / or its linking arm, wherein the photochromosome P generates reactive oxygen species when irradiated with light. The crosslinking monomer B comprises two o-benzodiazepine octylene (DBCO) groups and a linking group connected between the two DBCO groups; The prodrug monomer C includes at least one DBCO group, a drug molecule group, and a stimulus-responsive breakable linker group connecting the DBCO group and the drug molecule group; the photosensitizer monomer A crosslinks with the crosslinking monomer B and / or the DBCO group on the prodrug monomer C through a copper-free click polymerization reaction via its azide substituent to form the nanoparticles; The drug delivery material uses photosensitizer monomer A as an endogenous luminescent and reactive oxygen species generating unit, cross-linking monomer B containing thiophene linkage groups as a ROS-responsive cross-linking network, and prodrug monomer C as an immune adjuvant prodrug that can be dissociated by stimulation-responsive linkage groups. Under near-infrared light irradiation, it can simultaneously achieve photodynamic killing of tumor cells, depolymerization of the cross-linking network, and on-demand release of the immune adjuvant, thereby forming a photodynamic-immune synergistic effect in the tumor site and effectively enhancing the anti-tumor immune response.

2. The photo-activated nano-adjuvant drug delivery material according to claim 1, characterized in that, The photosensitive chromophore P is selected from at least one of fluoroboron dipyrrole, porphyrin, phytocyanin, and phthalocyanine chromophores.

3. The photo-activated nano-adjuvant drug delivery material according to claim 1, characterized in that, In crosslinking monomer B, the linking group is a thioketone linking group.

4. The photo-activated nano-adjuvant drug delivery material according to claim 1, characterized in that, The drug molecule group is an immune adjuvant, which is a TLR7 / 8 agonist IMDQ or a pharmaceutically acceptable derivative thereof.

5. The photo-activated nano-adjuvant drug delivery material according to claim 1, characterized in that, The stimulus-responsive breakable linker group is selected from at least one of the following: ROS-breakable thioketone bonds, oxalate bonds, and phenylboronic acid ester bonds.

6. The photoactivated nano-adjuvant drug delivery material according to any one of claims 1 to 5, characterized in that, The nanoparticles have an average particle size of 20–200 nm; the nanoparticles are a colloidal dispersion system in an aqueous medium and have a narrow particle size distribution.

7. A method for preparing the photoactivated nano-adjuvant drug delivery material according to any one of claims 1 to 6, characterized in that, The process includes the following steps: (1) dissolving photosensitizer monomer A, crosslinking monomer B and prodrug monomer C in an organic solvent to obtain an organic phase solution; (2) dispersing the organic phase solution in an aqueous phase containing a surfactant to form a nanoemulsion; (3) under copper-free click reaction conditions, causing the azide substituent on the photosensitizer monomer A to undergo a click polymerization reaction with the DBCO groups on the crosslinking monomer B and the prodrug monomer C, and removing the organic solvent to obtain a photoactivated nano-immunoadjuvant drug delivery material.

8. The preparation method according to claim 7, characterized in that, In step (1), the organic solvent is selected from dichloromethane, chloroform, tetrahydrofuran, ethyl acetate or mixtures thereof; in step (2), the surfactant is selected from at least one of lecithin, polysorbate or block copolymer surfactants; the copper-free click reaction is carried out in the range of room temperature to 50°C.

9. The use of the photoactivated nano-adjuvant drug delivery material according to any one of claims 1 to 6 in the preparation of a medicament for photodynamic-immunotherapy for treating malignant tumors, characterized in that, The malignant tumor includes at least one of breast cancer, colorectal cancer, and melanoma; the cumulative release rate of the photoactivated nano-adjuvant drug delivery material exceeds 85% within 24 hours under the condition of 600-800 nm light irradiation.

10. A prodrug monomer compound C, characterized in that, The prodrug monomer C includes at least one DBCO group, a drug molecule group, and a stimuli-responsive breakable linker group connecting the DBCO group and the drug molecule group; the drug molecule group is a TLR7 / 8 agonist IMDQ or a pharmaceutically acceptable derivative thereof.

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