Preparation method and application of engineered I-type photosensitizer

Abstract

The invention relates to the field of biological medicine and photodynamic therapy, in particular to a preparation method and application of an engineered I-type photosensitizer. The preparation method comprises the following steps: S1, synthesizing a core D-pi-A structural unit taking bithiophene as a framework and cyanoindanone as a receptor; s2, introducing a phenylcarbazole donor unit to a bithiophene aldehyde skeleton to form an intermediate 1; s3, connecting a strong receptor unit with the intermediate 1 to form a D-pi-A type photosensitizer; s4, introducing a phenylcarbazole donor unit to a bithiophene aldehyde skeleton to form an intermediate 2; s5, taking the intermediate 2 as a raw material to react with a strong receptor unit to obtain CzTTI; and S6, preparing the CzPhTTI nano particles. According to the invention, the treatment strategy of a plurality of core pathological pathways can be synchronously regulated and controlled, the problem of disjunction of anti-inflammation and repair of the existing therapy is solved, the bottleneck of strong oxygen dependence of the traditional II-type photosensitizer is overcome, and the targeting and safety of treatment are improved.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and photodynamic therapy, and in particular to an engineered method for preparing a type I photosensitizer and its application. Background Technology

[0002] Atopic dermatitis is a common chronic inflammatory skin disease, clinically manifested as dry skin, itching, erythema, and exudation, severely impacting patients' quality of life. Existing treatments, such as corticosteroids and calcineurin inhibitors, suffer from significant side effects and a high relapse rate. Photodynamic therapy, as a non-invasive treatment strategy, shows promising promise in the treatment of skin diseases. Type I photosensitizers generate reactive oxygen species under light irradiation via electron transfer pathways, exhibiting strong tissue penetration and potential for regulating oxidative stress.

[0003] Currently, existing treatment mechanisms only address the symptoms, not the root cause, and have significant side effects. Specifically, mainstream drugs (such as glucocorticoids and immunosuppressants) primarily work by broadly suppressing the immune response, but cannot fundamentally restore skin barrier function and immune balance. Long-term use can lead to local side effects such as skin atrophy, telangiectasia, and pigmentation, as well as systemic risks from systemic absorption (such as Cushing's syndrome, liver and kidney toxicity). While biologics offer stronger targeting, they pose risks such as conjunctivitis, high costs, and potential infection risks. Existing photosensitizers have limited mechanisms of action, resulting in insufficient penetration and safety. Specifically, type II photosensitizers used in traditional photodynamic therapy (PDT) (such as porphyrin derivatives) heavily rely on oxygen to generate singlet oxygen, leading to a sharp decline in efficacy in the hypoxic microenvironment often associated with atopic dermatitis. Furthermore, these photosensitizers are absorbed in the visible light spectrum (such as blue and green light), limiting their tissue penetration depth and making it difficult to effectively target immune cells in the dermis. In addition, they often cause persistent phototoxic reactions, requiring patients to avoid light for extended periods after treatment, severely impacting their quality of life. Furthermore, existing treatment strategies have high overall management costs and poor patient compliance. Specifically, AD treatment is a long-term process, and current protocols often result in high total treatment costs (especially for biologics), high frequency of visits, and lengthy treatment times. Frequent medication and follow-up visits place a heavy economic and emotional burden on patients and their families. The prolonged need to avoid light during photodynamic therapy also severely impacts patients' daily work and life, leading to decreased compliance. Summary of the Invention

[0004] The purpose of this invention is to address the problems of limited material mechanisms, insufficient penetration and safety of photosensitizers in the prior art, as well as the fact that existing treatments only treat the symptoms and not the root cause, and have high overall costs. This invention proposes an engineered method for preparing type I photosensitizers and their applications.

[0005] On the one hand, this invention proposes an engineered method for preparing a type I photosensitizer, comprising the following steps: S1. The core D-π-A structural unit with bithiophene as the backbone and cyanoindanone as the acceptor was synthesized by Knoevenagel condensation reaction, abbreviated as TTI; S2. Through the Suzuki-Miyaura coupling reaction, using boric acid as the organoboron reagent, the phenylcarbazole donor unit is introduced onto the bithiophene aldehyde skeleton to form intermediate 1. S3. The strong acceptor unit is linked to intermediate 1 through the Knoevenagel condensation reaction to form the target product D-π-A type photosensitizer, which is called the target product CzPhTTI. S4. Through the Suzuki-Miyaura coupling reaction, the organoboron reagent, using 9-phenyl-9H-carbazole-3-yl-3-boronic acid pinacol ester, introduces the phenylcarbazole donor unit onto the bithiophene aldehyde skeleton to form intermediate 2. S5. The target photosensitizer CzTTI was obtained by reacting intermediate 2 with a strong acceptor unit via Knoevenagel condensation reaction and purification. S6. Preparation of CzPhTTI nanoparticles.

[0006] Preferably, in S1, the synthesis of TTI includes the following steps: S11, Feeding: In a dry flask, weigh 5-bromo-2,2'-bithiophene-5'-carboxaldehyde (546.3 mg, 2.0 mmol) and 3-(dicyanomethylene)indan-1-one (388.4 mg, 2.0 mmol), add 15 mL of anhydrous ethanol as solvent, and stir to dissolve the solid; S12, Catalysis: Add 1 drop of piperidine as catalyst to the above mixture; S13, Reaction: Place the reaction system under nitrogen protection, heat to 90°C (oil bath temperature), and stir and reflux at this temperature for 12 hours; S14, Post-treatment: After the reaction is completed, cool the reaction mixture to room temperature, and then remove the solvent ethanol by rotary evaporation under reduced pressure to obtain the crude product; S15, Purification: Purify the crude product by silica gel column chromatography to obtain the target product TTI as a red solid.

[0007] Preferably, in S2, the formation process of intermediate 1 specifically includes the following steps: S21, Feeding: In a dry 50 mL Schlenk reaction tube, add sequentially: 9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)-9H-carbazole (1107.8 mg, 3.0 mmol), 5-bromo-2,2'-bithiophene-5'-carbaldehyde (546.3 mg, 2.0 mmol), dichloro(1,1'-bis(diphenylphosphine)ferrocene)palladium (Pd(dppf)Cl2) (73.2 mg, 0.1 mmol) and potassium carbonate (1105.6 mg, 8.0 mmol); S22, Solvent Deoxygenation: Add a mixed solvent of 1,4-dioxane (10 mL) and water (2.5 mL) to the reaction system. Then, perform a "vacuum-nitrogen purging" cycle three times. S23, Reaction: Heat the reaction system to 90°C under nitrogen protection and stir at this temperature for 12 hours. S24, Post-treatment: After the reaction is complete, cool the mixture to room temperature, pour the reaction solution into a separatory funnel, and extract with dichloromethane (30 mL each time, 3 extractions). Combine the organic phases and dry with anhydrous sodium sulfate. S25, Purification: Filter to remove the desiccant, concentrate the filtrate under reduced pressure to obtain the crude product, and purify the crude product by silica gel column chromatography to obtain a yellow solid intermediate 1.

[0008] Preferably, in step S3, the synthesis of the target product CzPhTTI includes the following steps: S31, Feeding: Intermediate 1 (867.2 mg, 2.0 mmol) and 3-(dicyanomethylene)indan-1-one (388.4 mg, 2.0 mmol) are placed in a dry flask, and 15 mL of anhydrous ethanol is added; S32, Catalysis and Reaction: 1 drop of piperidine is added to the mixture, and under nitrogen protection, the mixture is heated to 90 °C and stirred under reflux for 12 hours; S33, Post-treatment and Purification: After the reaction solution is cooled to room temperature, the solvent is removed by vacuum distillation, and the crude product is purified by silica gel column chromatography to obtain the purple solid target photosensitizer CzPhTTI.

[0009] Preferably, in S6, the raw materials for preparing CzPhTTI nanoparticles are CzTTI, 1,2-dimyristic-sn-glycerol-3-phosphocholine, cholesterol, and DSPEmPEG2000.

[0010] Preferably, the preparation of CzPhTTI nanoparticles includes the following steps: S61, dissolving 1 mg CzTTI, 8 mg 1,2-dimyristic-sn-glycerol-3-phosphocholine, 2 mg cholesterol and 0.5 mg DSPEmPEG2000 in chloroform, and then evaporating them into a thin film in a rotary evaporator; S62, hydrating the film with deionized water, and then sonicating it for 5 minutes with a micro-tip probe ultrasonic generator (Q125, QSonica, LLC) at 50% output power; S63, passing the resulting solution through a 0.22 μm polycarbonate filter membrane; S64, transferring the solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing with deionized water for 24 hours, and changing the water every 4 hours.

[0011] On the other hand, this invention proposes the application of engineered type I photosensitizers in phototherapy for atopic dermatitis.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects: 1. Mechanistic Objectives: Moving from "one-sided inhibition" to "synergistic regulation," this approach provides a treatment strategy that can simultaneously regulate multiple core pathological pathways. Under light exposure, it not only produces cytotoxic effects but also synergistically induces mitophagy, clearing dysfunctional mitochondria to alleviate oxidative stress. Simultaneously, it regulates the Nrf2 signaling pathway, enhancing the cell's antioxidant defense capabilities, thereby fundamentally improving the inflammatory microenvironment and barrier function of Alzheimer's disease (AD). This addresses the disconnect between "anti-inflammatory" and "repair" in existing therapies. Through the aforementioned synergistic regulatory mechanism, it effectively inhibits excessive immune responses while actively promoting the recovery of skin barrier function, reducing the disease recurrence rate. 2. Objectives at the Material and Performance Level: To overcome the bottleneck of "high oxygen dependence" in traditional type II photosensitizers, moving from "low efficiency and high toxicity" to "high efficiency and safety." This invention provides a type I photosensitizer that efficiently generates superoxide anions and other free radicals even in hypoxic environments via electron transfer pathways, ensuring stable and potent therapeutic efficacy within hypoxic lesion areas of Alzheimer's disease (AD); improving the targeting and safety of treatment. Through engineered design of the photosensitizer molecule, it is endowed with mitochondrial targeting capabilities, allowing it to accumulate at the target site, thereby improving efficacy and reducing the dosage. Simultaneously, through rational molecular design, its dark toxicity is reduced, and it is rapidly metabolized in vivo, significantly reducing the risk of long-term phototoxicity after treatment and improving patients' quality of life. Attached Figure Description

[0013] Figure 1 This is a schematic diagram illustrating the synthesis of the type I photosensitizer proposed in this invention; Figure 2 Schematic diagram of the physicochemical properties of TTI, CzPhTTI and CzTTI; Figure 3A schematic diagram illustrating the viability of HaCaT cells treated with CzPhTTI NPs. Figure 4 A schematic diagram showing the cell viability of endothelial cells and 293T cells treated with CzPhTTI NPs; Figure 5 A schematic diagram illustrating the mechanism of action of CzPhTTI NPs; Figure 6 A diagram illustrating how CzPhTTI NPs-mediated photodynamic therapy alleviates atopic dermatitis-like dermatitis in mice; Figure 7 A schematic diagram showing the body weight of mice in different groups; Figure 8 This is a schematic diagram of a hemolysis test; Figure 9 This is a schematic diagram of a pathological section of a major organ. Detailed Implementation

[0014] Example 1; as Figure 1 As shown, the present invention proposes an engineered method for preparing a type I photosensitizer, comprising the following steps: S1. The core D-π-A structural unit with bithiophene as the backbone and cyanoindanone as the acceptor was synthesized by Knoevenagel condensation reaction, abbreviated as TTI; S2. Through the Suzuki-Miyaura coupling reaction, using boric acid as the organoboron reagent, the phenylcarbazole donor unit is introduced onto the bithiophene aldehyde skeleton to form intermediate 1. S3. The strong acceptor unit is linked to intermediate 1 through the Knoevenagel condensation reaction to form the target product D-π-A type photosensitizer, which is called the target product CzPhTTI. S4, the same procedure as S2, was performed via the Suzuki-Miyaura coupling reaction, except that the organoboron reagent used was 9-phenyl-9H-carbazole-3-yl-3-boronic acid pinacol ester (1107.8 mg, 3.0 mmol), which introduced the phenylcarbazole donor unit onto the bithiophene aldehyde skeleton, forming an orange solid intermediate 2 with a yield of 574.9 mg and a yield of 66%. Its NMR data were similar to those of intermediate 1, confirming the successful connection of the bithiophene aldehyde structure. S5, the same procedure as S3, involves a Knoevenagel condensation reaction, except that intermediate 2 is used as the starting material to react with a strong acceptor unit. After purification, a dark blue solid target photosensitizer CzTTI is obtained, with a yield of 575.0 mg and a yield of 47%. S6. Prepare CzPhTTI nanoparticles, which are abbreviated as CzPhTTINPs.

[0015] Example 2: An engineered method for preparing type I photosensitizers proposed in this invention. Compared with Example 1, this example details the synthesis steps of TTI. In S1, the Knoevenagel condensation reaction is a process in which compounds containing active methylene groups (such as malonate esters and cyanoacetate esters) undergo a condensation reaction with aldehydes or ketones under weak base catalysis to generate olefinic compounds. This reaction is a powerful tool for synthesizing carbon-carbon double bonds (C=C), especially for extending carbon chains and preparing α,β-unsaturated carbonyl compounds. The synthesis of TTI includes the following steps: S11, Feeding: In a dry 100mL round-bottom flask, accurately weigh 5-bromo-2,2'-bithiophene-5'-carboxaldehyde (546.3mg, 2.0mmol) and 3-(dicyanomethylene)indan-1-one (388.4mg, 2.0mmol), add 15mL of anhydrous ethanol as a solvent, and stir to dissolve the solids as much as possible; S12, Catalysis: Add 1 drop of piperidine as a catalyst to the above mixture; S13, Reaction: The reaction mixture is... The mixture was heated to 90°C (oil bath temperature) under nitrogen protection and refluxed with vigorous stirring for 12 hours. The reaction process could be monitored by thin-layer chromatography (TLC), a rapid, trace, economical, and efficient separation and analysis technique, mainly used for: monitoring the progress of chemical reactions; identifying components in mixtures; determining the purity of compounds; and finding suitable separation conditions for column chromatography. S14. Post-processing: After the reaction, the reaction mixture was cooled to room temperature (25°C), and then the solvent ethanol was removed by rotary evaporation under reduced pressure to obtain the crude product. S15. Purification: The crude product was purified by silica gel column chromatography using a petroleum ether / dichloromethane mixed solvent with a volume ratio of 60:40. The red main band component was collected and concentrated by rotary evaporation to obtain the red solid target product TTI, with a yield of 518.2 mg and a yield of 70%.

[0016] Example 3: An engineered method for preparing type I photosensitizer proposed in this invention. Compared with Example 1, this example details the synthesis steps of intermediate 1. In S2, the Suzuki-Miyaura coupling reaction, often simply referred to as the "Suzuki reaction," is a reaction in which an organoboron reagent (such as boric acid) and an organohalide (or trifluoromethanesulfonate, etc.) undergo cross-coupling under the action of a palladium catalyst, thereby forming a new carbon-carbon bond. The formation process of intermediate 1 specifically includes the following steps: S21, Feeding: In a dry 50 mL Schlenk reaction tube, add sequentially: 9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)-9H-carbazole (1107.8 mg, 3.0 mmol), 5-bromo-2,2'-bithiophene-5'-carbaldehyde (546.3 mg, 2.0 mmol), dichloro(1,1'-bis(diphenylphosphine)ferrocene)palladium (Pd(dppf)Cl2) (73.2 mg, 0.1 mmol), and potassium carbonate (1105.6 mg, 8.0 mmol). S22, Solvent and Deoxygenation: Add a mixed solvent of 1,4-dioxane (10 mL) and water (2.5 mL) to the reaction system. Then, perform a "vacuum-nitrogen purging" cycle three times to ensure that the system is in an oxygen-free environment. S23, Reaction: Heat the reaction system to 90°C under nitrogen protection and stir at this temperature for 12 hours (overnight). S24, Post-treatment: After the reaction is completed, cool the mixture to room temperature, pour the reaction solution into a separatory funnel, and extract with dichloromethane (30 mL each time, 3 extractions). Combine the organic phases and dry with anhydrous sodium sulfate. S25, Purification: Filter to remove the desiccant, concentrate the filtrate under reduced pressure to obtain the crude product, and purify the crude product by silica gel column chromatography with petroleum ether / ethyl acetate mixed solvent at a volume ratio of 60:40. Collect the target component, concentrate it to obtain yellow solid intermediate 1 with a yield of 635.9 mg and a yield of 73%.

[0017] Example 4: An engineered method for preparing type I photosensitizers proposed in this invention. Compared with Example 1, this example details the synthesis steps of CzPhTTI. In S3, the synthesis of the target product CzPhTTI includes the following steps: S31, Feeding: Intermediate 1 (867.2 mg, 2.0 mmol) and 3-(dicyanomethylene)indan-1-one (388.4 mg, 2.0 mmol) are placed in a dry 100 mL round-bottom flask, and 15 mL of anhydrous ethanol is added; S32, Catalysis and Reaction: One drop of piperidine is added to the mixture, and under nitrogen protection, the mixture is heated to 90 °C and refluxed with stirring for 12 hours; S33, Post-treatment and Purification: After the reaction solution is cooled to room temperature, the solvent is removed by vacuum distillation. The crude product is purified by silica gel column chromatography using a petroleum ether / dichloromethane mixed solvent at a volume ratio of 40:60. The purple main band fraction is collected and concentrated to obtain the purple solid target photosensitizer CzPhTTI, with a yield of 513.9 mg and a yield of 42%.

[0018] Example 5; This invention proposes an engineered method for preparing type I photosensitizers. Compared to Example 1, this example details the preparation process of CzPhTTI nanoparticles. In S6, the raw materials for preparing CzPhTTI nanoparticles are CzTTI, 1,2-distearyl-sn-glycerol-3-phosphocholine, cholesterol, and DSPEmPEG2000. DSPE (1,2-distearyl-sn-glycerol-3-phosphoethanolamine) is a phospholipid with a hydrophobic fatty acid chain (stearic acid) and a hydrophilic phosphoethanolamine head, commonly used to construct lipid bilayer structures (such as liposomes and nanoparticles). mPEG2000 (methoxy polyethylene glycol) is a hydrophilic polymer with good water solubility, biocompatibility, and "stealth" properties (reducing its recognition and clearance by the immune system). DSPEmPEG2000 is an amphiphilic conjugate formed by covalently linking DSPE and mPEG2000. Specifically, the preparation of CzPhTTI nanoparticles includes the following steps: S61, 1 mg CzTTI, 8 mg 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 2 mg cholesterol and 0.5 mg DSPEmPEG2000 are dissolved in chloroform and then evaporated into a thin film in a rotary evaporator; S62, the film is hydrated with deionized water and then sonicated for 5 minutes with a micro-tip probe ultrasonic generator (Q125, QSonica, LLC) at 50% output power; S63, the resulting solution is filtered through a 0.22 μm polycarbonate membrane; S64, the solution is transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500 Da, dialyzed with deionized water for 24 hours, with the water changed every 4 hours, and the resulting nanoparticles are stored in a refrigerator at 4°C for later use.

[0019] Example 6; as Figures 2-9 As shown, the engineered type I photosensitizer proposed in this invention is applied in phototherapy for atopic dermatitis. To verify the efficacy of the type I photosensitizer, the following verification was conducted: Total Reactive Oxygen Species (ROS) Detection: A fluorescent probe method (DCFH, DCFH-DA) was used to detect ROS generation from PS (Photosensitizer: a class of chemical molecules or materials that can convert light energy into chemical energy to generate reactive oxygen species and other reactive substances under white light excitation) in aqueous solution. DCFH and DCFH-DA are the most classic and widely used fluorescent probes for detecting intracellular ROS. DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate): is a precursor probe; it is not fluorescent itself but can penetrate the cell membrane to enter the cell. DCFH (2',7'-dichlorodihydrofluorescein): is the active form, present in the cell; it is not fluorescent itself but becomes a strongly fluorescent substance after reacting with ROS. DCFH was obtained by reacting DCFH-DA (0.5 mL, 1 mM ethanol solution) with 2 mL of NaOH aqueous solution (10 mM) at room temperature for 30 min. Hydrolysis products were neutralized with 7.5 mL of PBS buffer to obtain a 50 μM stock solution. PBS buffer (Phosphate-Buffered Saline, an aqueous solution commonly used in biological and medical experiments to simulate the fluid environment for cell survival in the human body; it is an "inert" liquid that provides a stable chemical environment without harming cells) containing 5 μM DCFH was mixed with different samples. The fluorescence of PS-sensitized DCFH was measured at different time intervals under red light irradiation (620–630 nm, 20 mW cm⁻²). PL spectra were measured at 488 nm, and emission was collected from 500 to 600 nm. The increase in DCFH fluorescence intensity (I / I₀) at 522 nm was used to indicate the total amount of ROS generated. Singlet oxygen (1O₂) detection: 1O₂ generation was detected using 9,10-anthratridiyl-bis(methylene)dimalonic acid (ABDA) as an indicator. PBS buffer containing 50 μM ABDA (stock solution: 5 mM, dissolved in DMSO) was mixed with different samples to a final concentration of 50 μM. The mixtures were then irradiated with red light (620-630 nm, 20 mW·cm−2), and the absorbance of ABDA at 378 nm was recorded. The decomposition rate (A0 / A) at 378 nm after irradiation was used to characterize the generation rate of 1O2.

[0020] Superoxide radical (O2•−) detection: The generation of O2•− was detected using dihydrorhodamine 123 (DHR123) as an indicator. Different samples were mixed with PBS buffer containing 5 μM DHR123 (stock solution: 2 mM, dissolved in DMSO) to a final concentration of 5 μM. The fluorescence of PS-sensitized DHR123 was measured at different time intervals under red light irradiation (620–630 nm, 20 mW cm−2). The PL spectrum was measured at 488 nm, and emission was collected from 500 to 600 nm. The increase in DHR123 fluorescence intensity at 526 nm (I / I0) indicated the total amount of ROS generated.

[0021] Hydroxyl radical (·OH) detection: The generation of ·OH was detected using terephthalic acid (TA) as an indicator. For TA testing, the fluorescence of PS (10 μM)-sensitized TA was measured at different time intervals under red light irradiation (620-630 nm, 20 mW cm−2). PL spectra were measured under 315 nm excitation.

[0022] Overall ROS, mitochondrial ROS, and MMP detection: ROS detection was performed using commercially available kits according to the manufacturer's instructions. HaCaT cells (60% density) were cultured in 24-well plates for 24 hours. NPs were added to the NPs group and NPs+L group, and incubated for 24 hours; the NPs+L group and PBS+L group were incubated for 12 hours, irradiated with red light for 15 minutes, and then incubated for another 12 hours before ROS detection. MitoSOX and TMRE were used to quantitatively detect mitochondrial membrane potential (MMP), and fluorescence imaging was performed under an inverted PerkinElmer microscope at excitation wavelengths of 488nm, 510nm, or 576nm. The fluorescence microscopy product line is mainly aimed at high-end scientific research applications such as high-content cell imaging and in vivo animal imaging, ranging from basic cell morphology and fluorescence intensity to complex neural process growth and protein translocation within cells, and is widely used in fields such as tumor research, drug screening, and toxicology research.

[0023] Cell Culture and CCK-8 Assay: Adherent cells, including HaCaT cells, endothelial cells (EAhy926), and 293T cells, were used in this study. Cells were cultured in DMEM medium containing 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 µg / mL) at 37°C and 5% CO2. Cells were passaged when confluence reached 70%. Cell viability was assessed using a CCK-8 assay kit. After 24 hours of stable culture, the medium was replaced with 10% fetal bovine serum containing different concentrations of nanoparticles (0, 2.5, 5, 10, 15, 20, 25 μM), and cultured for 12 hours under either red light or non-red light irradiation, followed by another 12 hours of culture. CCK-8 solution was then added, and cells were incubated in the dark for 30 minutes. After incubation, absorbance was measured at 450 nm using a microplate reader. Results are expressed as cell viability (%).

[0024] Western blot analysis is a method for detecting and semi-quantitatively analyzing the presence, concentration, and molecular size of specific proteins in complex mixtures (such as cell or tissue extracts). Skin tissue or cells are lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein concentration is determined. Proteins are separated using 4E 20% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. β-tubulin (1:10000), Nrf (1:2500), Keap1 (1:5000), SOD3 (1:1000), P62 / SQSTM1 (1:10000), Pink1 (1:2000), and LC3A / B (1:1000) are incubated overnight at 4°C with primary antibodies. Sections are then incubated at room temperature for 1 hour in horseradish peroxidase-labeled goat anti-rabbit IgG (HÞL) (1:2000).

[0025] Immunofluorescence: HaCaT cells in confocal culture dishes were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and blocked with immunofluorescence rapid blocking solution for 15 minutes. Cells were incubated overnight at 4°C with Tomm20 (1:200) and LC3B (1:200). Next, cells were incubated with immunofluorescence secondary antibody at room temperature for 1 hour and counterstained with DAPI. Images were acquired using a STELLARIS5 laser confocal microscope (Leica, Germany). Mouse skin sections were fixed and blocked, and then incubated overnight at 4°C with SQSTM1 / P62 (1:1000) and Pink1 (1:1000). Subsequently, mouse skin samples were stained using a three-label four-color multiplex fluorescence staining kit. Finally, samples were stained with DAPI (a fluorescent dye widely used in life sciences and medical research, specifically for labeling cell nuclei), and images were acquired using the PerkinElmer quantitative pathological imaging system. Nrf, Keap1, and SOD3 form a crucial molecular combination in oxidative stress, antioxidant defense, and cell protection: Oxidative stress → Keap1 senses and releases Nrf2 → Nrf2 activation → Initiation of SOD3 and other gene expression → SOD3 enzyme synthesis and secretion extracellularly to scavenge superoxide → Reduced cellular oxidative stress levels, resulting in protection. P62 / SQSTM1, Pink1, LC3A / B, Tomm20, and LC3B are all essential protein markers in autophagy, particularly mitophagy, research. PINK1 is the core signal that initiates mitophagy; Tomm20 is used as an indicator of mitochondria themselves, and a decrease in its content is often considered evidence of mitophagy; P62 / SQSTM1 is an adaptor protein that recognizes Parkin-labeled ubiquitinated tags at one end and binds to LC3 at the other end; LC3-II is the gold standard marker for autophagosome formation, and its content is proportional to the number of autophagosomes. It is distributed on the autophagosome membrane and acts as a "hook" to bind to adaptor proteins such as P62, recruiting cargo into autophagosomes.

[0026] Transmission electron microscopy: NPs+L and PBS+L groups were incubated for 12 hours, followed by 15 minutes of light exposure, and then incubated for another 12 hours. All steps were performed in darkness. Additionally, TEM images of HaCaT cells after different treatments were collected by BIOSSCI (a company specializing in pathological sections and electron microscopy) to detect autophagosome aggregation and cell death patterns. Cells were collected after centrifugation. TEM fixative was added to tubes, and the precipitate was resuspended in the fixative. Samples were then fixed at 4°C for preservation and transport. Subsequent steps included agarose pre-embedding, post-fixation, room temperature dehydration, resin infiltration embedding, polymerization, ultrathin sectioning, staining, and observation under a transmission electron microscope (imaging magnification: 4,000x; accelerating voltage: 80kV).

[0027] The above experimental groups were divided into NC, AD + positive control (mometasone), AD + PBS (atopic dermatitis model + PBS), AD + PBS + L (atopic dermatitis model + PBS + light exposure), NPs (atopic dermatitis model + nanoparticles), and NPs + L (atopic dermatitis model + nanoparticles + light exposure). Atopic dermatitis modeling: sensitization with MC903 + OVA.

[0028] In summary, the present invention has the following technical effects: This invention represents a breakthrough in mechanism, moving from "unilateral inhibition" to "synergistic regulation": It successfully constructs a novel therapeutic system capable of synergistically regulating mitophagy and the Nrf2 signaling pathway. In vitro cell experiments confirmed that after treatment with the photosensitizer of this invention and subsequent light exposure, a significant increase in mitophagic flux (upregulation of the LC3-II / I ratio and a decrease in p62 protein levels), a decrease in mitochondrial membrane potential, and significant nuclear translocation of Nrf2, along with an increase in the white blood cell (WB) expression level of its downstream antioxidant gene SOD3, were observed. This dual regulatory mechanism, on the one hand, reduces ROS bursts at the source by clearing dysfunctional mitochondria, and on the other hand, activates endogenous antioxidant defenses, jointly reshaping the inflammatory microenvironment of AD skin, achieving simultaneous "anti-inflammatory" and "repair" processes. This invention overcomes the "oxygen dependence" bottleneck of traditional photodynamic therapy and improves penetration and safety: The photosensitizer of this invention is based on a strong D-π-A structure and is confirmed to be a type I photodynamic mechanism. Electron paramagnetic resonance spectroscopy directly detects that even under hypoxic conditions, it can still generate a large number of superoxide anion free radicals after light irradiation. In contrast, traditional type II photosensitizers (such as methylene blue) show a decrease in singlet oxygen production under the same hypoxic conditions. This allows the invention to maintain highly efficient therapeutic activity in hypoxic / inflammatory lesions common in Alzheimer's disease (AD). Excellent near-infrared absorption and deep tissue therapy capabilities are achieved: Through molecular structure engineering, the maximum absorption wavelengths of the photosensitizer CzPhTTI in this invention are red-shifted to 710 nm and 730 nm, respectively, located in the near-infrared I region window, and the molar extinction coefficients are both greater than 5.0 × 10⁻⁶. 4 M⁻¹cm⁻¹. At this wavelength, the laser penetration depth into mouse skin is approximately 2-3 times greater than that of 630nm light commonly used with traditional photosensitizers; Significantly reduced drug toxicity: After in vitro and in vivo validation, the cytotoxicity of CzPhTTINPs was assessed using the Cell Counting Kit-8 (CCK-8) assay. Under dark conditions, CzPhTTINPs exhibited extremely low cytotoxicity after 24 hours of co-incubation with HaCaT cells, endothelial cells, and 293T cells, maintaining over 80% cell viability even at high nanoparticle concentrations. To assess biocompatibility, body weight was monitored throughout the treatment process. Except for the control group, all groups experienced weight loss, while the treatment group showed a slight but significant recovery trend, indirectly reflecting the therapeutic effect. H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) revealed no significant inflammatory infiltration or structural damage, and hemolysis tests showed a hemolysis rate of less than 5%, confirming the excellent in vivo biocompatibility of the photosensitizer.

[0029] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. An engineered method for preparing a type I photosensitizer, characterized in that, Includes the following steps: S1. The core D-π-A structural unit with bithiophene as the backbone and cyanoindanone as the acceptor was synthesized by Knoevenagel condensation reaction, abbreviated as TTI; S2. Through the Suzuki-Miyaura coupling reaction, using boric acid as the organoboron reagent, the phenylcarbazole donor unit is introduced onto the bithiophene aldehyde skeleton to form intermediate 1. S3. The strong acceptor unit is linked to intermediate 1 through the Knoevenagel condensation reaction to form the target product D-π-A type photosensitizer, which is called the target product CzPhTTI. S4. Through the Suzuki-Miyaura coupling reaction, the organoboron reagent, using 9-phenyl-9H-carbazole-3-yl-3-boronic acid pinacol ester, introduces the phenylcarbazole donor unit onto the bithiophene aldehyde skeleton to form intermediate 2. S5. The target photosensitizer CzTTI was obtained by reacting intermediate 2 with a strong acceptor unit via Knoevenagel condensation reaction and purification. S6. Preparation of CzPhTTI nanoparticles.

2. The engineered method for preparing type I photosensitizer according to claim 1, characterized in that, In S1, the synthesis of TTI includes the following steps: S11, Feeding: In a dry flask, weigh 5-bromo-2,2'-bithiophene-5'-carboxaldehyde (546.3 mg, 2.0 mmol) and 3-(dicyanomethylene)indan-1-one (388.4 mg, 2.0 mmol), add 15 mL of anhydrous ethanol as solvent, and stir to dissolve the solid; S12, Catalysis: Add 1 drop of piperidine as catalyst to the above mixture; S13, Reaction: Place the reaction system under nitrogen protection, heat to 90 °C (oil bath temperature), and stir under reflux for 12 hours; S14, Post-treatment: After the reaction is completed, cool the reaction mixture to room temperature, and then remove the solvent ethanol by rotary evaporation under reduced pressure to obtain the crude product; S15, Purification: Purify the crude product by silica gel column chromatography to obtain the target product TTI as a red solid.

3. The engineered method for preparing type I photosensitizer according to claim 1, characterized in that, In S2, the formation process of intermediate 1 specifically includes the following steps: S21, Feeding: In a dry 50 mL Schlenk reaction tube, add sequentially: 9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)phenyl)-9H-carbazole (1107.8 mg, 3.0 mmol), 5-bromo-2,2'-bithiophene-5'-carbaldehyde (546.3 mg, 2.0 mmol), dichloro(1,1'-bis(diphenylphosphine)ferrocene)palladium (Pd(dppf)Cl2) (73.2 mg, 0.1 mmol), and potassium carbonate (1105.6 mg, 8.0 mmol); S22, Solvent and Removal Oxygen: Add a mixed solvent of 1,4-dioxane (10 mL) and water (2.5 mL) to the reaction system. Then, perform a "vacuum-nitrogen purging" cycle three times. S23. Reaction: Heat the reaction system to 90°C under nitrogen protection and stir at this temperature for 12 hours. S24. Post-treatment: After the reaction is complete, cool the mixture to room temperature, pour the reaction solution into a separatory funnel, and extract with dichloromethane (30 mL each time, 3 extractions). Combine the organic phases and dry with anhydrous sodium sulfate. S25. Purification: Filter to remove the desiccant, concentrate the filtrate under reduced pressure to obtain the crude product, and purify the crude product by silica gel column chromatography to obtain a yellow solid intermediate 1.

4. The engineered method for preparing type I photosensitizer according to claim 1, characterized in that, In S3, the synthesis of the target product CzPhTTI includes the following steps: S31, Feeding: Intermediate 1 (867.2 mg, 2.0 mmol) and 3-(dicyanomethylene)indan-1-one (388.4 mg, 2.0 mmol) are placed in a dry flask, and 15 mL of anhydrous ethanol is added; S32, Catalysis and Reaction: 1 drop of piperidine is added to the mixture, and under nitrogen protection, the mixture is heated to 90 °C and stirred under reflux for 12 hours; S33, Post-treatment and Purification: After the reaction solution is cooled to room temperature, the solvent is removed by vacuum distillation, and the crude product is purified by silica gel column chromatography to obtain the purple solid target photosensitizer CzPhTTI.

5. The engineered method for preparing type I photosensitizer according to claim 1, characterized in that, In S6, the raw materials for preparing CzPhTTI nanoparticles are CzTTI, 1,2-dimyristic-sn-glycerol-3-phosphocholine, cholesterol, and DSPEmPEG2000.

6. The engineered method for preparing type I photosensitizer according to claim 5, characterized in that, The preparation of CzPhTTI nanoparticles includes the following steps: S61, 1 mg CzTTI, 8 mg 1,2-dimyristoyl-sn-glycerol-3-phosphocholine, 2 mg cholesterol and 0.5 mg DSPEmPEG2000 are dissolved in chloroform and then evaporated into a thin film in a rotary evaporator; S62, the film is hydrated with deionized water and then sonicated for 5 minutes with a micro-tip probe ultrasonic generator (Q125, QSonica, LLC) at 50% output power; S63, the resulting solution is filtered through a 0.22 μm polycarbonate membrane; S64, the solution is transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 24 hours, with the water changed every 4 hours.

7. The use of an engineered type I photosensitizer according to any one of claims 1-6 in phototherapy for atopic dermatitis.

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