A supramolecularly assembled nanophotosensitizer, its preparation method and application

Abstract

This invention discloses a supramolecularly assembled nanophotosensitive agent, its preparation method, and its applications, belonging to the field of organic conjugated molecular assembly technology. Addressing the current technical challenge of single photosensitizers simultaneously achieving high-brightness NIR-II fluorescence, efficient type I ROS generation, and high photothermal conversion efficiency, this invention provides nanoparticles formed by supramolecular self-assembly of a type I photosensitizer and a multi-arm polyethylene glycol derivative, 8-arm-PEG-OH. The multi-arm structure of 8-arm-PEG-OH forms a tightly ordered nano-assembly with DBD molecules through hydrogen bonding, synergistically enhancing the photophysical properties of DBD. The resulting 8P@DBD nanoparticles exhibit a high fluorescence quantum yield of 2.97% in the near-infrared II (NIR-II) region, significantly improved type I reactive oxygen species (ROS) generation efficiency, and a photothermal conversion efficiency of 68.33%. This invention provides a universal strategy for developing high-performance NIR-II type I phototherapeutic reagents and has broad application prospects in biomedicine and other fields.

Description

Technical Field

[0001] This invention belongs to the field of organic conjugated molecular assembly technology, specifically relating to a universal method for constructing organic conjugated molecular nanoassemblies based on a multi-arm polyethylene glycol (PEG) supramolecular assembly strategy. Background Technology

[0002] Precision diagnosis and treatment of malignant tumors is a current research hotspot and challenge in the biomedical field. Phototherapy, due to its non-invasiveness, high precision, and low systemic toxicity, has become an important development direction in tumor treatment. Phototherapy mainly includes photodynamic therapy (PDT) and photothermal therapy (PTT). Organic conjugated molecules, as organic photosensitizers, have attracted much attention in the development of phototherapy agents due to their excellent biocompatibility and tunable optical properties. PDT utilizes photosensitizers (PS) to generate cytotoxic reactive oxygen species (ROS) under light irradiation to kill tumor cells, while PTT utilizes photosensitizers to convert light energy into heat energy, destroying tumors through thermal ablation. Furthermore, some photosensitizers can accumulate in tumors through the high permeability and retention (EPR) effect of solid tumors and serve as contrast agents for fluorescence imaging, enabling real-time guidance and monitoring of the treatment process. Near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging, due to its advantages such as deep tissue penetration, low scattering loss, and minimal autofluorescence interference, can achieve precise visualization of deep tumors. Traditional photosensitizers mostly rely on the type II photochemical pathway (energy transfer to molecular oxygen to produce singlet oxygen), and their efficacy is severely limited by the hypoxic microenvironment of tumor tissue. In contrast, type I photosensitizers generate superoxide anion radicals (O2) through an electron transfer pathway. •− ROS such as hydroxyl radicals (•OH) can effectively overcome the hypoxia problem.

[0003] Therefore, developing a "three-in-one" single-molecule photosensitizer integrating NIR-II fluorescence imaging, type I PDT, and PTT is considered an ideal strategy for overcoming deep, hypoxic, and drug-resistant solid tumors. However, progress in this field is limited by a key scientific challenge: how to simultaneously achieve high-brightness NIR-II fluorescence emission, efficient type I ROS generation, and excellent photothermal conversion efficiency (PCE) in a single molecule. These three functions have inherently conflicting requirements on the molecule's photophysical properties—strong luminescence requires high fluorescence quantum yield, efficient type I ROS generation requires promoting intersystem crossing to consume excited-state energy, and photothermal conversion requires enhancing nonradiative transitions. This trade-off makes maximizing all three properties simultaneously in a single molecule extremely challenging.

[0004] Limited by molecular design bottlenecks, most existing NIR-II imaging-guided PDT / PTT synergistic therapy systems have to adopt multi-component composite strategies, such as physically mixing or co-assembling fluorescent dyes, type I photosensitizers, and photothermal agents. This not only increases the complexity of the system and reduces preparation reproducibility but also poses a significant obstacle to its clinical translation. Although a few studies have reported "three-in-one" single-molecule photosensitizers, their overall performance still needs improvement, and a universally applicable design approach is lacking. Therefore, developing a universal strategy that can comprehensively improve the photophysical properties of water-insoluble photosensitizers and obtain a single phototherapeutic agent that integrates high-brightness NIR-II fluorescence, strong type I ROS generation, and high photothermal conversion efficiency is a pressing technical problem to be solved in this field. Summary of the Invention

[0005] To address the current technical challenge of simultaneously achieving high-brightness NIR-II fluorescence, efficient type I ROS generation, and high photothermal conversion efficiency with a single photosensitizer, this invention provides a novel phototherapy nanoparticle based on supramolecular assembly. This nanoparticle utilizes multi-arm PEG to assemble and regulate a single type I photosensitizer, synergistically enhancing its NIR-II fluorescence, type I ROS generation, and photothermal properties, thereby achieving highly efficient NIR-II imaging-guided phototherapy / phototherapy (PDT / PTT).

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a supramolecularly assembled phototherapy nanoparticle, wherein the nanoparticle is an 8P@DBD nanoparticle, which is formed by supramolecular self-assembly of a photosensitizer and a multi-arm polyethylene glycol (PEG) derivative; specifically, the photosensitizer is dissolved in an organic solvent, the multi-arm PEG is dissolved in water, the above are then mixed, assembled under ultrasound, and finally the organic solvent is removed to obtain an assembly of organic conjugated molecules.

[0008] The photosensitive molecule is an organic conjugated molecule with a conjugated structure;

[0009] The organic conjugated molecule is the compound DBD;

[0010] The structural formula of the compound DBD is as follows:

[0011] ;

[0012] The compound DBD has a donor-acceptor-donor (DAD) structure and is a type I photosensitizer.

[0013] The multi-arm polyethylene glycol (PEG) derivative is an 8-arm polyethylene glycol (8-arm-PEG-OH) with terminal hydroxyl-rich ends.

[0014] Furthermore, the molecular weight of the 8-arm polyethylene glycol (8-arm-PEG-OH) is 10 kDa to 40 kDa.

[0015] Furthermore, the mass ratio of the organic conjugated molecule to the terminal hydroxyl-rich 8-arm polyethylene glycol (8-arm-PEG-OH) is 1:5 to 1:50.

[0016] Furthermore, the nanoparticles exhibit a fluorescence quantum yield of 2.97% in the near-infrared II (NIR-II) region and a photothermal conversion efficiency of 68.33%.

[0017] The second aspect of the present invention provides a method for preparing supramolecularly assembled phototherapy nanoparticles, comprising the following steps: mixing a photosensitizer solvent with a multi-arm polyethylene glycol (PEG) derivative solution, subjecting the mixture to ultrasonic treatment, and then removing the organic solvent to obtain supramolecularly assembled phototherapy nanoparticles.

[0018] Furthermore, the photosensitizer is dissolved in an organic solvent, wherein the organic solvent is at least one of tetrahydrofuran, acetone or dimethyl sulfoxide, and is miscible with water.

[0019] Furthermore, methods for removing organic solvents include agitation evaporation, rotary evaporation, or dialysis.

[0020] Organic conjugated molecules DBD and 8-arm-PEG-OH interact through hydrogen bonding to form nanoassemblies with a dense and ordered stacked structure.

[0021] The third aspect of this invention provides an assembly mechanism for preparing organic conjugated molecular assemblies through a supramolecular assembly strategy, verified by molecular dynamics simulations. The specific mechanism is studied by optimizing the structure of 8-arm-PEG-OH and DBD molecules using density functional theory, and then exploring the assembly mechanism of 8-arm-PEG-OH and DBD through molecular dynamics simulations.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] This invention is the first to propose and verify a supramolecular assembly strategy using multi-arm PEG (such as 8-arm-PEG-OH) to confine a single photosensitizer (DBD) within a densely ordered nano-assembly. This structure significantly improves the NIR-II fluorescence quantum yield of the photosensitizer (up to 2.97% or more) while substantially enhancing the type I ROS (such as O2). •− It achieves high photothermal conversion efficiency and maintains extremely high photothermal conversion efficiency (up to 68.33% or more).

[0024] This invention can be prepared by one-step self-assembly of only a photosensitizer and a biocompatible multi-arm PEG, avoiding the complexity and uncontrollability of multi-component complex systems, and has good prospects for clinical translation.

[0025] The nanoparticles (8P@DBD nanoparticles) prepared in this invention exhibited excellent NIR-II fluorescence imaging capabilities in both in vivo and in vitro experiments, and could guide the synergistic treatment of type I PDT and PTT, ultimately achieving complete tumor ablation.

[0026] The supramolecular assembly strategy based on multi-arm PEG proposed in this invention can serve as a general platform to enhance the overall performance of other water-insoluble photosensitizers with similar structures, providing a new approach for developing high-performance NIR-II type I phototherapeutic agents. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a synthetic route diagram for DBD molecules.

[0029] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the DBD molecule.

[0030] Figure 3 Transmission electron microscopy characterization of 8P@DBD nanoparticles.

[0031] Figure 4 A statistical graph showing the total ROS generation of 8P@DBD nanoparticles.

[0032] Figure 5 This is a graph showing the photothermal conversion efficiency data for 8P@DBD nanoparticles.

[0033] Figure 6 This is a graph showing the statistical data of 4T1 cell killing experiments using 8P@DBD. Detailed Implementation

[0034] To gain a deeper understanding of this invention, we will provide a comprehensive and detailed description. However, this invention has various implementations and is not limited to the specific examples listed herein. These examples are presented to enhance a full understanding of the disclosure of this invention.

[0035] Example 1: Synthesis of photosensitizer DBD

[0036] Synthesize organic photosensitizing molecules DBD with DAD structure, such as Figure 1 As shown.

[0037] In a 50 mL pressure-resistant reaction flask, 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole (1.92 g, 5.0 mmol), tributyl(6-undecylthieno[3,2-b]thieno-2-yl)stanane (6.42 g, 11.0 mmol), tris(dibenzylacetone)dipalladium (30 mg, 0.03 mmol), and tris(2-methylphenyl)phosphine (50 mg, 0.16 mmol) were added sequentially. Under nitrogen protection, anhydrous toluene (25 mL) was added via syringe. The reaction mixture was stirred at 110 °C for 12 hours. After cooling to room temperature, the solvent was removed by rotary evaporation. The residue was dissolved in dichloromethane and washed with water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:3, v / v) to give 2.92 g of a red solid (compound 1), with a yield of 72%. The 1H NMR spectrum of compound 1 is as follows: 1 H NMR (400 MHz, CDCl3) δ 7.71 (s, 2H), 7.18 (s, 2H), 2.78 (t, J= 7.7 Hz, 4H), 1.79 (p, J = 7.5 Hz, 4H), 1.43-1.22 (m, 32H), 0.87 (t, J = 6.7Hz, 6H).

[0038] Compound 1 (2.3 g, 2.84 mmol) and triphenylphosphine (7.43 g, 28.4 mmol) were added to a three-necked round-bottom flask equipped with a reflux condenser. The system was evacuated and purged three times with nitrogen. Under nitrogen protection, o-dichlorobenzene (10 mL) was added via syringe. The reaction mixture was heated to 180 °C and stirred for 4 hours. After cooling to room temperature, the solvent was removed by vacuum distillation. The crude product was treated with methanol, precipitating a solid. The solid was collected by filtration to give compound 2 as an orange solid.

[0039] Compound 2 (2.84 mmol), potassium carbonate (7.85 g, 56.8 mmol), and potassium iodide (189 mg, 1.14 mmol) were added to a three-necked round-bottom flask. The flask was evacuated and purged three times with nitrogen. Under nitrogen protection, 1-bromo-2-hexyldecane (8.67 g, 28.4 mmol) and anhydrous N,N-dimethylformamide were added via syringe. The reaction mixture was heated to 110 °C and stirred for 12 hours. After cooling to room temperature, the reaction mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:6, v / v) to give compound 3 as an orange solid in 65% yield. The 1H NMR spectrum of compound 3 is shown below. 1 HNMR (400 MHz, CDCl3) δ 7.01 (s, 2H), 4.59 (d, J = 7.8 Hz, 4H), 2.82 (t, J =7.7 Hz, 4H), 2.07 (tt, J = 12.6, 6.0 Hz, 2H), 1.86 (p, J = 7.5 Hz, 4H), 1.50-0.62 (m, 98H).

[0040] Under nitrogen protection, anhydrous N,N-dimethylformamide (0.5 mL) was added to a three-necked round-bottom flask. Phosphorus oxychloride (519 mg, 3.34 mmol) was added dropwise at 0 °C. After stirring for 30 minutes, a solution of compound 3 (400 mg, 0.33 mmol) in anhydrous 1,2-dichloroethane (12 mL) was added. The mixture was heated to 110 °C and stirred for 12 hours. After cooling to room temperature, the reaction mixture was poured into a saturated aqueous sodium bicarbonate solution and stirred for 6 hours. The mixture was extracted with dichloromethane, and the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification was achieved by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:2, v / v) to give compound 4 in 62% yield. The 1H NMR spectrum of compound 4 is as follows: 1 H NMR (400 MHz, CDCl3) δ 10.14 (s, 2H), 4.62 (d, J = 7.8 Hz, 4H), 3.20 (t, J = 7.7 Hz, 4H), 2.02 (dq, J = 12.2, 6.5 Hz, 2H), 1.92 (p, J =7.9 Hz, 4H), 1.53 – 0.64 (m, 98H).

[0041] 3-O-benzo[b]thiophene 1,1-dioxide (500 mg, 2.74 mmol), malononitrile (363 mg, 5.49 mmol), and sodium acetate (292 mg, 3.56 mmol) were added to anhydrous ethanol (10 mL), and the mixture was stirred overnight at 25 °C. Subsequently, the reaction mixture was diluted with water, and the pH was adjusted to 1–2 with 1 M hydrochloric acid. The precipitate was collected by vacuum filtration, washed thoroughly with water, and dried under vacuum to give compound 5 in 92% yield. The 1H NMR spectrum of compound 5 is as follows: 1 H NMR (600 MHz, CDCl3) δ 8.68 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.95 (t, J = 7.1 Hz, 1H), 7.90 (t, J = 7.5 Hz, 1H), 4.56 (s, 2H).

[0042] Compound 4 (156 mg, 0.13 mmol) and compound 5 (1.3 mmol) were added to acetic anhydride (8 mL), and the mixture was heated to 90 °C and stirred for 12 hours. After cooling to room temperature, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 1:1, v / v) to give DBD molecules in 63% yield. Figure 2 The following is the 1H NMR spectrum data of the DBD molecule: 1 H NMR (600 MHz, CDCl3) δ 9.22 (s, 2H), 8.92 (d,J = 7.7 Hz, 2H), 8.00 (d, J = 7.2 Hz, 2H), 7.91 – 7.83 (m, 4H), 4.70 (d, J =7.8 Hz, 4H), 3.20 (t, J = 7.6 Hz, 4H), 2.07 (s, 2H), 1.90 (p, J = 8.4 Hz, 4H), 1.43 – 0.65 (m, 98H).

[0043] Example 2: Preparation of 8P@DBD nanoparticles

[0044] The DBD photosensitizer synthesized in Example 1 was dissolved in tetrahydrofuran (THF) to prepare a solution with a concentration of 10 mg / mL. 8-arm-PEG-OH (molecular weight 40 kDa) was dissolved in ultrapure water to prepare a solution with a concentration of 20 mg / mL. 500 μL of the DBD THF solution was rapidly added to 10 mL of the 8-arm-PEG-OH aqueous solution under ultrasonic conditions. After continuing ultrasonication for 5 minutes, the mixture was placed in a fume hood and stirred overnight at room temperature to evaporate and remove the THF, thus obtaining an aqueous dispersion of 8P@DBD nanoparticles. The transmission electron microscopy characterization image of the 8P@DBD nanoparticles is shown below. Figure 3 As shown.

[0045] Example 3: Characterization of the assembly mechanism

[0046] Initial structural models of 8-arm-PEG-OH and DBD molecules were constructed using Chem3D software. Subsequently, the initial structures of both molecules were optimized in Gaussian 16 using the DFT B3LYP functional under the 6-311g(d) basis set. Force field parameter files for 8-arm-PEG-OH and DBD molecules were generated using the Sobtop program; all three molecules were described using GAFF force fields. The water molecule was modeled using the TIP3P model. A 18×18×18nm matrix was constructed using Gromacs 2023.3 software. 3 A box was constructed, and 45 8-arm-PEG-OH molecules and 15 DBD molecules were added to it, all initially randomly distributed within the box. Water molecules were then added to the box to solvate the system. All kinetic simulations were performed using Gromacs 2023.3, with 100 ns of kinetics simulations conducted after energy minimization and pre-equilibration with NVT and NPT systems. Simulation data showed strong hydrogen bonding interactions between the 8-arm-PEG-OH and DBD molecules, forming a stable and compact assembly.

[0047] Example 5: Photophysical property characterization of nanoparticles

[0048] The performance of the nanoparticles prepared in Example 2 was tested.

[0049] (1) NIR-II fluorescence quantum yield test: The NIR-II fluorescence quantum yield of 8P@DBD was measured using a dye with a known quantum yield (IR26) as a reference. The results showed that the fluorescence quantum yield of 8P@DBD nanoparticles was significantly improved to 2.97%.

[0050] (2) Detection of reactive oxygen species (ROS): Specific fluorescent probes (such as DCFH-DA for total ROS detection, ABDA for singlet oxygen detection, and DHE for O2 detection) are used.•− The ability of 8P@DBD to generate ROS was detected by APF (Aspect-Protected Components) under laser irradiation, specifically by detecting •OH. Figure 4 As shown, the results indicate that the ROS generation efficiency of 8P@DBD nanoparticles is significantly improved, and under illumination, mainly type I ROS with •OH as the main component is generated.

[0051] (3) Photothermal Conversion Efficiency (PCE) Test: Aqueous dispersions of 8P@DBD nanoparticles at different concentrations were irradiated with an 808 nm laser, and the heating curves were recorded. The photothermal conversion efficiency was then calculated using the cooling curves. For example... Figure 5 The results showed that the photothermal conversion efficiency of 8P@DBD nanoparticles reached 68.33%, demonstrating excellent photothermal performance.

[0052] Example 6: In vitro cell experiments

[0053] The dark toxicity and phototoxicity of 8P@DBD nanoparticles to human breast cancer cells (e.g., 4T1 cells) were evaluated using the MTT assay and live / dead cell staining method. Cells were incubated with different concentrations of nanoparticles and then divided into a no-light group (for assessing dark toxicity) and a laser-irradiated group (for assessing phototoxicity). Figure 6 The results showed that 8P@DBD nanoparticles have low dark toxicity, but under laser irradiation, due to the synergistic effect of PDT and PTT, they can efficiently kill tumor cells.

[0054] Contents not described in detail in this specification are prior art known to those skilled in the art. Although illustrative specific embodiments of the invention have been described above to facilitate understanding by those skilled in the art, it should be understood that the invention is not limited to the scope of the specific embodiments. Various modifications are readily apparent to those skilled in the art as long as they fall within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions utilizing the concept of this invention are protected.

Claims

1. A supramolecularly assembled nanophotosensitizer, characterized in that: The photosensitizer is 8P@DBD nanoparticles, which are formed by supramolecular self-assembly of photosensitizing molecules and multi-arm polyethylene glycol derivatives. The photosensitive molecule is an organic conjugated molecule with a conjugated structure; The organic conjugated molecule is the compound DBD. The structural formula of the compound DBD is as follows: ； The multi-arm polyethylene glycol derivative is an 8-arm polyethylene glycol with terminal hydroxyl groups.

2. The supramolecularly assembled nanophotosensitizer according to claim 1, characterized in that: The molecular weight of the 8-arm polyethylene glycol is 10 kDa to 40 kDa.

3. The supramolecularly assembled nanophotosensitizer according to claim 1, characterized in that: The mass ratio of the organic conjugated molecule to the 8-arm polyethylene glycol with terminal hydroxyl groups is 1:5 to 1:

50.

4. The supramolecularly assembled nanophotosensitizer according to claim 1, characterized in that: The nanoparticles exhibit a fluorescence quantum yield of 2.97% in the near-infrared II region and a photothermal conversion efficiency of 68.33%.

5. A method for preparing a supramolecularly assembled nanophotosensitizer as described in claim 1, characterized in that: Includes the following steps: The photosensitizing molecule solution was mixed with a multi-arm polyethylene glycol derivative solution, subjected to ultrasonic treatment, and then the organic solvent was removed to obtain a supramolecularly assembled nanophotosensitizer.

6. The method for preparing a supramolecularly assembled nanophotosensitizer according to claim 5, characterized in that: The photosensitive molecule is dissolved in an organic solvent, wherein the organic solvent is at least one of tetrahydrofuran, acetone or dimethyl sulfoxide, and is miscible with water.

7. The method for preparing a supramolecularly assembled nanophotosensitizer according to claim 6, characterized in that: Methods for removing organic solvents include agitation evaporation, rotary evaporation, or dialysis.

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