Anti-biological reduction protective agent applicable to phenothiazine photosensitive dye and application of anti-biological reduction protective agent in aspects of phototherapy and immunological enhancement

Abstract

The invention discloses an anti-biological reduction protective agent applicable to phenothiazine photosensitive dyes and application of the anti-biological reduction protective agent in the aspects of phototherapy and immunological enhancement. The protective agent is a biosafe electron acceptor (such as thymoquinone TQ, cytochrome c and ethidium dihydrogen), can effectively inhibit MB from being reduced into colorless LMB in a physiological simulation environment containing NADH / NADPH, accelerates reoxidation of LMB into active MB, and significantly improves the ROS yield (up to 4 times or more). The combined system not only greatly enhances the photodynamic effect of MB in tumor cells and bacteria, but also can strengthen the release of immunogenic death factors (CRT, HMGB1 and ATP), and provides a brand new strategy for developing high-performance phototherapy materials, in-vitro antibacterial systems and immune regulation tools.

Description

Technical Field

[0001] This invention relates to the fields of chemical and biotechnology, and in particular to a class of anti-bioreduction protectants suitable for phenothiazine photosensitive dyes and their application in phototherapy and immune enhancement. Background Technology

[0002] Phenothiazine structures, represented by methylene blue (MB), are a class of widely used optical functional dyes, primarily employed in bioimaging and phototherapy (antibacterial and antitumor), possessing significant commercial and research value. In antibacterial and antitumor phototherapy, MB can act as a photosensitizer to exert its phototherapy effect, reaching a triplet excited state upon red light excitation. 3 *Reactive oxygen species (ROS) generated by the reaction with oxygen in the environment damage biomolecules, thus achieving a therapeutic effect. In fact, MB also benefits from its prominent singlet oxygen (ROS). 1 O2 (a type of ROS) yield (56%) is a key focus in photodynamic therapy (PDT) and is being used in conjunction with other photosensitizers. 1 O2 production calibration. However, due to the tendency of heteroatoms in the structure to readily gain electrons, MB molecules are often reduced to the colorless LMB state in biological environments, losing their red light absorption and corresponding photodynamic therapy (PDT) capabilities. In particular, in the presence of coenzymes in the biological environment, such as nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), MB molecules are rapidly reduced upon photoexcitation, severely affecting their ROS generation capacity and thus blocking the PDT process. Therefore, addressing the issue of MB molecules being easily reduced to the LMB state in biological environments is a major challenge in improving this commercially available photosensitizer.

[0003] Currently, there are two main approaches to improving the reducibility of methanophores (MB): First, encapsulating NADH and other harmful substances in nanocarriers such as polyacrylamide and calcium phosphate to prevent MB reduction and inactivation in vivo. However, nano-encapsulation can shield MB's phototherapy performance and interfere with its ROS generation. Second, directly adding ionic salts such as iodide ions and isocyanate ions can also enhance the phototherapy effect of MB and reduce its self-degradation. However, these ionic additives are currently mainly believed to enhance therapeutic efficacy by converting low-toxicity superoxide radicals into high-toxicity free radicals, failing to address the significant limitation of the reduction of phenothiazine structures like MB under physiological conditions. Therefore, existing technologies and methods require further development. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to develop a class of "protective agents" to overcome the limitation that phenothiazine photosensitive dyes are easily reduced and lose their PDT activity under physiological conditions and in the presence of biological coenzymes, and to optimize their protective performance through formulation.

[0005] In a first aspect, the present invention provides a series of natural or safe anti-bioreduction protectants suitable for phenothiazine photosensitive dyes; said anti-bioreduction protectant is a biocompatible electron acceptor capable of inhibiting the reduction of said phenothiazine photosensitive dye to a colorless reduced state in a reducing environment, or promoting the re-oxidation of its colorless reduced state to a colored oxidized state, thereby maintaining its characteristic absorption at the therapeutic window; wherein: said phenothiazine photosensitive dye has the following general structural formula I (including but not limited to MB):

[0006] I Among them, R1, R2, R3, and R4 are independently selected from C1–C 10 At least one of the alkane chains, more preferably a C1–C6 alkyl group, more preferably a C1–C4 straight-chain alkyl group; X is selected from at least one of S, Se, and Te, and the parent structure is selected from phenothiazine or benzophenothiazine.

[0007] This invention primarily studies the effect of adding a protective agent on the anti-bioreduction properties of the above-mentioned phenothiazine structure, and does not involve new methods for synthesizing phenothiazine structures. Representative structures are selected as follows:

[0008] For the technical solution described above, a further preferred embodiment is that the anti-bioreduction protectant is selected from at least one of thymoquinone (TQ), cytochrome c, and dihydroethidium (DHE).

[0009] In a further preferred embodiment of the above-described technical solution, the standard reduction potential of the protective agent is higher than the redox couple between the phenothiazine photosensitive dye and its colorless reduced state, and it can undergo an electron transfer reaction with the colorless reduced state to promote its re-oxidation into a colored oxidized state.

[0010] This invention provides a photosensitizing composition comprising a phenothiazine-based photosensitizing dye as shown in general formula I and the aforementioned anti-bioreduction protectant, wherein the molar ratio of the protectant to the phenothiazine-based photosensitizing dye is 0.5–10:1. A preferred molar ratio is 1–3:1; the most preferred molar ratio is 2:1.

[0011] For the technical solution described above, a further preferred embodiment is that the photosensitizing composition is an aqueous system with a pH of 6.5–7.8. More preferably, the aqueous system includes, but is not limited to: phosphate-buffered saline (PBS, pH 7.4), Tris-HCl buffer (pH 7.0–8.0), HEPES buffer (pH 7.2–7.6), physiological saline, or other biocompatible aqueous solutions suitable for the dissolution and photochemical reaction of photosensitizing dyes. This system aims to provide a reproducible, controllable experimental platform with pH and ionic strength close to physiological conditions, for use only in non-therapeutic studies and material performance evaluation at the in vitro chemical, photophysical, or cellular level.

[0012] In addition, the present invention discloses a method for improving the ability of phenothiazine photosensitive dyes to generate reactive oxygen species (ROS) in a reducing system, which includes placing the aforementioned anti-bioreduction protectant and the phenothiazine photosensitive dye together in an aqueous system and applying light with a wavelength of 600–700 nm.

[0013] In this application, the term "reducing environment" (also referred to as "reducing system," "physiological environment," or "biological environment" in the specification) refers to an in vitro simulation system used to reproduce common reducing chemical conditions in vivo, and does not involve in vivo, animal, or human applications. This reducing environment is constructed by adding a reducing biological coenzyme to an aqueous system, typically 50–200 μM of nicotinamide adenine dinucleotide (NADH) and / or nicotinamide adenine dinucleotide phosphate (NADPH), where 100 μM NADH is the standard test concentration. This is widely used to evaluate the optical stability, reduction resistance, and reactive oxygen species generation properties of phenothiazine photosensitive dyes under such conditions.

[0014] Another aspect of the present invention protects the use of the photosensitizing composition for any of the following non-disease diagnostic or therapeutic purposes: (a) Used in the preparation of tumor phototherapy agents to maintain the photodynamic activity of phenothiazine photosensitive dyes in a reducing environment; (b) Used to prepare antibacterial photosensitizing agents, which inhibit or inactivate microorganisms under in vitro light irradiation conditions; (c) Used to prepare immune-enhancing agents to induce the release of immunogenic death-related factors from cells in vitro.

[0015] For the technical solution described above, a further preferred embodiment is that, in (a), the reducing environment contains NADH and / or NADPH, and the phototherapy-related photosensitive dye maintains its color state and reactive oxygen species generation capacity under 660 nm light irradiation.

[0016] In a further preferred embodiment of the above-described technical solution, in (b), the microorganisms include bacteria, fungi, or viruses, and the antibacterial photosensitizing agent generates reactive oxygen species through light activation to inhibit or inactivate the microorganisms.

[0017] For the technical solution described above, in a further preferred embodiment, in (c), the immunogenic death-related factor includes at least one of calreticulin (CRT), high-mobility group box 1 (HMGB1), and adenosine triphosphate (ATP).

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides the development, formulation, and phototherapy and immune-enhancing applications of an anti-bioreduction protectant suitable for methylene blue. Compared to existing technologies, this invention effectively solves the problem of easy reduction and inactivation of phenothiazine photosensitive dyes such as methylene blue in physiological reducing environments containing NADH / NADPH by introducing a biocompatible electron acceptor as an anti-bioreduction protectant. This protectant can inhibit the conversion of photosensitive dyes to a colorless reduced state or promote their rapid re-oxidation and restoration of activity within seconds, thereby maintaining their characteristic absorption at 660 nm and their efficient reactive oxygen species (ROS) generation capacity. Experiments show that in the presence of NADH, the addition of the protectant can increase the total ROS yield by more than four times.

[0019] Unlike traditional nano-encapsulation or ionic additive strategies, this invention does not rely on physical isolation or free radical pathway modulation, but directly intervenes in the reduction process at the electron transfer level, avoiding shielding or interference with photodynamic properties. For example, without a protective agent, MB almost completely fades after 10 seconds of light exposure, while with the addition of thyquinone (TQ), its absorbance remains essentially unchanged, demonstrating excellent resistance to reduction. This method allows for the recycling and regeneration of the photosensitizer in an aqueous system, significantly reducing dependence on ambient oxygen.

[0020] Based on this, the photosensitizing composition of the present invention exhibits superior performance in non-therapeutic applications such as phototherapy materials, in vitro antibacterial agents, and induction of immune-related factors, providing a new approach for developing efficient and stable photosensitizing systems. Attached Figure Description

[0021] Figure 1 ac is the NADH decay absorption spectrum of the phenothiazine structure, the subject of this invention.

[0022] Figure 2 Absorption spectra of MB by different biological reducing agents, showing the absorption and decay interference.

[0023] Figure 3 ae represents the absorption spectra of the anti-bioreduction effect of MB in Examples 1-3 and Comparative Examples 1-2.

[0024] Figure 4 This is a graph showing the absorption recovery rate of LMB in Example 1.

[0025] Figure 5 The quantization diagrams of ROS generated by MB in a reducing environment under the protection of Examples 1-3 are shown.

[0026] Figure 6 ab shows the fluorescence and quantitative analysis of ROS generated by MB in cells under the protection of Example 1.

[0027] Figure 7 ac represents the evaluation of the antitumor PDT effect before and after the addition in Example 1.

[0028] Figure 8 ac represents the detection of immune enhancement and damage factor release induced by Example 1.

[0029] Figure 9 ac represents the evaluation of the antibacterial effect of PDT before and after its addition in Example 1. Detailed Implementation

[0030] This invention provides the development, formulation, and phototherapy and immune-enhancing applications of a class of methylene blue-based anti-bioreduction protective agents. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] The specific research examples and comparative examples of the anti-bioreduction "protective agents" used are as follows: Example 1 is a natural extract of thymequinone TQ selected from black cumin seeds, and its structure is as follows:

[0032] Example 2 is derived from cytochrome c, an essential substance in the mitochondrial respiratory chain, which is a protein structure.

[0033] Example 3 uses the cell-available ROS probe DHE, with the following structure:

[0034] Comparative Example 1 is selected from oxidized glutathione, i.e., GSSG, with the following structure:

[0035] Comparative Example 2 is selected from pyruvate, an oxidizing substance in the glycolysis pathway, i.e., Pyr, with the following structure:

[0036] In this embodiment of the invention, the chemical reagents were all from Anaiji Chemical Reagent Company or Bide Pharmaceuticals, the commercial dyes MTT, DHE, DCFH, and DCFH-DA were purchased from MCE Reagent Company, the mouse breast cancer cells 4T1 used were from the ATCC cell bank, and the E. coli bacterial strain was from Beina Biotechnology.

[0037] Verify the instability (decay) of a series of phenothiazine-structured photosensitizers in the presence of coenzyme NADH (in a bioreducing environment). This embodiment aims to quantitatively verify the technical problem mentioned in the background art and the problem that this invention aims to solve—namely, the phenomenon that phenothiazine photosensitizers (represented by MB) are easily inactivated under physiological reducing conditions.

[0038] 100 μM NADH was added to an aqueous environment to mimic the reducing environment under physiological conditions, and the reducing decay of photosensitizing agents such as MB (10 μM) under light irradiation was tested. Figure 1 As shown in Figures 1a-1c, the following data illustrates the effect of illumination for only 10 s (660 nm, 20 mW / cm²). 2 The absorbance of MB decreased from 0.8 to below 0.1, accompanied by the consumption of NADH, which is due to the electron transfer reaction between NADH and the excited state of MB. In contrast, NBSe also showed a more significant absorbance decay, from 0.5 to 0.3; while NBS, due to its low ROS production, showed a relatively weak absorbance decay. Overall, the phenothiazine structure, due to its unique mid-heteroatom structure, readily gains electrons in the excited state and is easily reduced to a colorless state, resulting in impaired photosensitivity. This demonstrates its poor photostability under physiological conditions and the difficulty in maintaining photodynamic activity.

[0039] Interference of MB absorption and decay by different biological reducing agents To investigate the prevalence of photoinstability in micrografts (MB), this comparative study tested the effects of various common biological reducing agents. In fact, not only does NADH significantly interfere with the phototherapy performance of MB, but NADPH and vitamin C, two other reducing agents, also significantly inhibit MB activity. For example... Figure 2 As shown, under illumination (660 nm, 20 mW / cm²), 2 NADH, NADPH, and Vc, at the same concentration (100 μM), caused a similar decrease in absorbance of MB (10 μM), approximately 0.6. In contrast, the well-known reducing agent GSH had a weaker effect on MB. These results indicate that the photostability of MB is prevalent in various physiologically relevant reducing environments, further highlighting the need to develop a broad-spectrum and effective protective strategy.

[0040] Verification of the efficacy of MB anti-bioreduction protectant in Examples 1-3 and Comparative Examples 1-2 This embodiment is used to verify the ability of the selected substance as a protective agent to maintain the photostability of methylene blue (MB) in a reducing environment.

[0041] 1. Experimental grouping and solution preparation All experiments were conducted in air. The following solutions were prepared using phosphate-buffered saline (PBS, pH 7.4) as the solvent: Experimental groups 1-3: contained 10 μM MB, 100 μM NADH, and 50 μM thyquinone (TQ, Example 1) / cytochrome c (Cytc, Example 2) / dihydroethidium (DHE, Example 3), respectively.

[0042] Comparative Examples 1-2: Contained 10 μM MB, 100 μM NADH, and 50 μM oxidized glutathione (GSSG, Comparative Example 1) / pyruvate (Pyr, Comparative Example 2), respectively.

[0043] Positive control group: Contains only 10 μM MB and 100 μM NADH.

[0044] Negative control group: Contains only 10 μM MB (no NADH, no protectant).

[0045] All solutions were incubated at room temperature in the dark for 5 minutes to ensure thorough mixing of all components.

[0046] 2. Illumination Processing and Detection The solutions were irradiated for 10 seconds using a light source with a wavelength of 660 nm and an optical density of 20 mW / cm². The optical density (OD) of the solutions at a wavelength of 660 nm was measured immediately before and after irradiation using a UV-Vis spectrophotometer.

[0047] The above results demonstrate that the aforementioned series of electron acceptor substances aim to maintain the photosensitivity of the colorless LMB structure by plundering electrons to accelerate its recovery rate to the photosensitive state of MB, thereby acting as a "protective agent." TQ, Cytc, and DHE can serve as effective anti-bioreduction protectants for MB, while GSSG and pyruvate cannot; the results are as follows. Figure 3 As shown. Compared with the positive control group (MB+NADH), whose OD value dropped sharply after light exposure: The OD values ​​of experimental group 1 (MB+NADH+TQ) and experimental group 3 (MB+NADH+DHE) remained basically unchanged before and after illumination, indicating that TQ and DHE can almost completely inhibit the reduction of MB by NADH under the experimental conditions.

[0048] The OD value of experimental group 2 (MB+NADH+Cytc) decreased, but the decrease was significantly smaller than that of the positive control group, indicating that Cytc has a certain protective effect.

[0049] The OD value change curves of comparative group 1 (MB+NADH+GSSG) and comparative group 2 (MB+NADH+Pyr) basically overlapped with those of the positive control group, indicating that the addition of comparative group 1 (GSSG) and comparative group 2 (Pyr) did not change the fact that MB was rapidly reduced by NADH upon photoexcitation and could not act as a protective agent.

[0050] Although GSSG and pyruvate have certain oxidizing properties in a biochemical sense, neither of them effectively inhibited the reduction and inactivation of MB in the MB-NADH photoreduction system constructed in this invention. This indicates that not all oxidizing substances can be used as "anti-bioreduction protectants" in the sense of this invention—their effectiveness is highly dependent on their kinetic compatibility with the MB / NADH system and their electron transfer efficiency.

[0051] Example 4: Comparison of the recovery rates of TQ and oxygen This embodiment aims to investigate the rate at which the protective agent TQ restores the photoactivity of MB.

[0052] 1. Preparation of LMB Solution: A 10 μM MB solution was placed in a sealed reaction vessel filled with high-purity nitrogen (N2), and N2 was continuously introduced for at least 15 minutes to fully remove dissolved oxygen. Subsequently, 100 μM NADH was added under the N2 atmosphere, and the solution was irradiated with a 660 nm, 20 mW / cm² light source until the blue color of the solution completely disappeared (indicating that MB had been completely reduced to colorless LMB), thus obtaining the LMB solution. The prepared LMB solution was divided into two portions: Oxygen Experiment Group: An equal volume of fully aerated oxygen-saturated PBS solution (simulating an aerobic environment) was rapidly injected into a portion of LMB solution, and video monitoring was activated to track the process and real-time changes of LMB returning to blue MB after contact with oxygen.

[0053] TQ experimental group: An equal volume of PBS solution (containing 20 μM TQ, so that the final concentration is consistent with that in Example 3) was rapidly injected into another LMB solution, and the process and real-time changes of LMB returning to blue MB after contact with TQ were also monitored by video.

[0054] like Figure 4 As shown, the rate at which the protective agent TQ re-oxidizes LMB to active MB is much higher than the oxygen-dependent natural re-oxidation process in the physiological environment. This explains from a kinetic perspective why it can effectively maintain the photoactivity of MB.

[0055] Example 5: Effect of Protectant on ROS Generation in MB under Reducing Environment To investigate whether the addition of a protective agent improved the phototherapy performance of myocardium, the total ROS generation properties of myocardium were investigated in the presence of the reducing agent NADH (with DCFH as the ROS scavenger).

[0056] 1 Experimental Methods Prepare the following solutions in phosphate-buffered saline (PBS, pH 7.4), with the total volume being the same: Experimental groups 1-3: contain 10 μM MB, 100 μM NADH, 10 μM DCFH (ROS scavenger), and 10 μM TQ (Example 1) / Cytc (Example 2) / DHE (Example 3).

[0057] Positive control group: containing 10 μM MB, 100 μM NADH, and 10 μM DCFH.

[0058] Negative control group 1: Contains 10 μM MB and 10 μM DCFH (without NADH).

[0059] Each solution was placed in a cuvette, and fluorescence intensity was monitored in real time using a fluorescence spectrophotometer (excitation wavelength: 488 nm, emission wavelength: 525 nm). Irradiation was performed at 10-second intervals using a light source with a wavelength of 660 nm and an optical density of 20 mW / cm² for 40 seconds, and the fluorescence intensity was recorded over time. The increase in fluorescence intensity was directly proportional to the amount of ROS generated.

[0060] like Figure 5 As shown, in the positive control group (NADH+MB) without the addition of a protective agent, ROS production mainly depended on incompletely reduced MB during the 0-10 s period, showing a certain increase. However, ROS production remained almost unchanged during the subsequent 10-40 s period, indicating that the ROS generation capacity significantly decreased after rapid MB reduction. In contrast, the protective agents (TQ, Cytc, DHE) provided by this invention can effectively overcome the inhibition of MB photoactivity by the reducing environment and significantly improve its ROS yield. Among them, the addition of Examples 1 (MB+NADH+TQ), 2 (MB+NADH+Cytc), and 3 (MB+NADH+DHE) significantly improved the total ROS production of MB. In particular, the addition of TQ resulted in slightly higher ROS production than the negative control group, which may be due to the reduction of the impact of ROS on MB itself. The effect of Example 3 (DHE) being weaker than that of TQ is because the capture of ROS by this type of probe interferes with its actual yield, while the effect of Example 2 (Cytc) is because its protective effect is weaker than that of substances such as TQ.

[0061] Example 6 Analysis of changes in intracellular ROS production To investigate the effect of the addition of the protective agent on the phototherapy performance of MB under physiological conditions, the total ROS production before and after the addition of Example 1 in a cellular environment was tested (using DCFH-DA as a probe).

[0062] Experimental methods: 1. Cell Culture: Using murine breast cancer cells 4T1 as the research subject, the cells were cultured in DMEM medium (equipped with 1% penicillin antibiotics and 10% FBS) at 37°C, 5% CO2, and 95% air. For testing, the target cells were seeded into cell culture dishes and cultured in an incubator for 24 hours until the cell density reached approximately 60%.

[0063] 2. Drug Treatment and ROS Probe Imaging: The molecule DCFH-DA can be hydrolyzed by intracellular lipases to the structural DCFH, which reacts with reactive oxygen species (ROS) to dehydrogenate and convert into the conjugated fluorescent structural DCF. Intracellular ROS generation can be detected by CLSM imaging. In the experiment, the old culture medium was aspirated from the cell culture dish, and the prepared solutions for each group were added: Experimental group: containing 10 μM MB, 10 μM DCFH-DA (ROS scavenger), and 10 μM TQ (Example 1).

[0064] Negative control group 1: Contains 10 μM MB and 10 μM DCFH-DA.

[0065] Negative control group 2: Contained 10 μM MB.

[0066] Negative control group 3: containing 10 μM TQ.

[0067] After incubation in a constant temperature incubator in the dark for 2 hours, followed by 3 minutes of light exposure (660 nm, 20 mW / cm²), the samples were then subjected to light treatment. 2 The cell dish was imaged under a 60x oil immersion lens on a CLSM, with an excitation wavelength of 488 nm and an emission wavelength of 490-540 nm.

[0068] like Figure 6 As shown, the negative control groups 2-3, which were treated only with MB or TQ in a dark environment, produced almost no ROS in their cells, and their fluorescence intensity was negligible. The control group, however, showed green fluorescence under MB light, indicating that ROS could be produced in the cells at this time. In contrast, the addition of TQ further enhanced the fluorescence, with the fluorescence intensity increasing by approximately 1.8 times that of the negative control group 1.

[0069] Example 7 Evaluation of the anti-tumor PDT effect This embodiment aims to investigate and evaluate the enhancing effect of the protective agent TQ on photodynamic therapy of MB anti-tumor cells.

[0070] Experimental methods: 1. Cell Preparation: Using murine breast cancer cells 4T1 as the research subject, cells were cultured in DMEM medium (equipped with 1% penicillin antibiotics and 10% FBS) at 37 ℃, 5% CO2, and 95% air. Different cell types were cultured in 96-well cell culture plates at a density of 10,000 cells per well, and cultured in an incubator for 24 hours until the density reached 70% for use.

[0071] 2. MTT assay and cell viability calculation: Remove the old culture medium from the 96-well plate and add the pre-prepared series of drugs: Experimental light groups 1-2: DMEM culture medium solution containing 2.5 μM / 5 μM MB and 5 μM TQ.

[0072] Control groups 1-2: DMEM medium solution containing 2.5 μM / 5 μM MB.

[0073] Control groups 1-2 (no light): DMEM culture medium solution containing 2.5 μM / 5 μM MB.

[0074] Negative control group: DMEM medium solution without MB and TQ.

[0075] The sample was added to 96-well plates (5 parallel wells per group), incubated in an incubator for 2 hours, and then exposed to light for 10 minutes (660 nm, 20 mW / cm²). 2 After incubation in a dark environment overnight, the culture medium in the 96-well plate was removed, and 100 μL of medium containing 5 mg / mL MTT was added to each well. The plate was incubated for 4 hours, and after removing the medium, 100 μL of DMSO was added. The OD value (excitation: 490 nm) was measured using a microplate reader, and the cell viability was calculated using the following formula: Cell viability = ( - - ) × 100% In the formula, OD x The absorbance of the test group, ODbc The absorbance, OD represents the absorbance of the blank control group. c The absorbance represents the negative control group (pure cell group).

[0076] 3. Wound Healing Assay: The wound healing assay provides a more direct indication of drug treatment efficacy by showing the growth and recovery of cells in the missing area on a culture plate. Cells are seeded into cell culture dishes and cultured in an incubator for 48 hours until the cell density reaches approximately 90%. Different groups of drugs are added and incubated, followed by light or dark treatment. Experimental group: DMEM culture medium solution containing 2.5 μM MB and 5 μM TQ, light intensity (660 nm, 20 mW / cm²). 2 Processing time: 10 minutes.

[0077] Negative control group 1: DMEM medium solution containing 2.5 μM MB, light intensity (660 nm, 20 mW / cm²). 2 Processing time: 10 minutes.

[0078] Negative control group 2: DMEM medium solution containing 2.5 μM MB, treated in the dark.

[0079] Negative control group 2: DMEM medium solution containing 5 μM TQ, dark treatment.

[0080] Negative control group 2: DMEM culture medium solution, light (660 nm, 20 mW / cm²) 2 Processing time: 10 minutes.

[0081] After exposure to light, the initial cell wound gaps were photographed and recorded using a microscope. After incubation in an incubator for 24 hours, the cell wound gaps were photographed and recorded again. The ratio of the difference in wound distance to the initial distance is the wound recovery rate.

[0082] like Figure 7 As shown, the MTT assay revealed that with the addition of 5 μM TQ (at which TQ does not exhibit biotoxicity to cells), MB (2.5 μM) demonstrated stronger therapeutic performance than MB alone, with a 30% increase in cell killing effect. In contrast, the addition of 1:1 MB (5 μM MB) showed weaker protective ability and therapeutic improvement. The wound healing assay showed that the addition of TQ significantly improved the cell inhibition rate of MB, increasing it from 80% to 95%. These results demonstrate that Example 1 (TQ) can enhance the antitumor performance of MB and improve its PDT performance.

[0083] Example 8: Immune Enhancement Effect This embodiment aims to investigate the effect of the protective agent TQ on the immune response to MB-induced photodynamic therapy.

[0084] Experimental Methods: Photodynamic therapy can induce an immune response and enhance immunogenicity to a certain extent, mainly through the release of cell damage-related substances, such as cytokines like CRT, HMGB1, and ATP. Cells were seeded into cell culture dishes or 96-well plates according to the methods in Examples 6-7, and incubated overnight in an incubator until the cell density reached 70%. Cells were then treated with phototherapy (660 nm, 20 mW / cm²) in the experimental group (2.5 μM MB) and the control group (2.5 μM MB and 5 μM TQ, respectively). 2 After 10 minutes, incubate in the dark for 4 hours, and then stain according to the instructions of the corresponding detection kit. The fluorescence intensity of the experimental group and the control group is detected by CLSM and microplate reader, respectively.

[0085] like Figure 8 As shown, the immune response protein CRT induced by MB phototherapy was significantly enhanced upon the addition of TQ, with fluorescence enhancement of ~2-fold. Simultaneously, the extracellular concentrations of damage-related factors HMGB1 and ATP increased by 35% and 53%, respectively, with a significant increase in their release. These results demonstrate the beneficial effect of the protective agent on enhancing the PDT effect of MB and related immune-inducing effects.

[0086] Example 9 Evaluation of antibacterial PDT effect This embodiment aims to investigate the effect of the protective agent TQ on the photodynamic antibacterial efficiency induced by MB.

[0087] Experimental methods: 1. Bacterial smear test: Using E. coli (a negative bacterium) as the research object, the effect of TQ on the phototherapy antibacterial properties of MB was detected. Take 1 mL containing 10 8 CFU / mL E. coli bacterial culture was added to the wells of a 24-well plate (3 wells per group), and different drugs were added and the plates were then subjected to light or dark treatment. Experimental group: PBS solution containing 5 μM MB and 10 μM TQ, light irradiation (660 nm, 20 mW / cm²). 2 Processing time: 10 minutes.

[0088] Negative control group 1: PBS solution containing 5 μM MB, light irradiation (660 nm, 20 mW / cm²). 2 Processing time: 10 minutes.

[0089] Negative control group 2: PBS solution containing 5 μM MB, treated in the dark.

[0090] Negative control group 3: PBS solution containing 10 μM TQ, treated in the dark.

[0091] After treatment, the bacterial suspension was diluted 100-fold, and 100 μM was evenly spread onto LB agar plates. The plates were then inverted and incubated overnight at 37 ℃. Photos were taken and the colony counts for each group were recorded.

[0092] 2. Bacterial inhibition rate detection: The antibacterial phototherapy synergistic effect of TQ on different concentrations of MB was detected according to the MTT method in Example 7. Experimental groups 1-3: TQ concentration 10 μM, MB concentration 1.25 / 2.5 / 5 μM, irradiated for 10 minutes (660 nm, 20 mW / cm²). 2 )deal with.

[0093] Control groups 1-3: MB concentrations of 1.25 / 2.5 / 5 μM, subjected to 10 minutes of light irradiation (660 nm, 20 mW / cm²). 2 )deal with.

[0094] like Figure 9 As shown in the LB agar plate assay, compared to MB phototherapy, the addition of TQ in Example 1 significantly reduced the colony count, to only 50% of that in the MB phototherapy group, representing an improvement in its antibacterial phototherapy performance. Furthermore, the MTT assay showed that the addition of TQ (10 μM) had different effects on both treatments at different MB concentrations, indicating that the ratio affects the protective effect. Provided a safe dosage of TQ is maintained, a higher TQ to MB ratio results in better therapeutic improvement and a more significant enhancement.

[0095] The above specific embodiments demonstrate that: This invention utilizes protective agents, such as TQ, to intervene in and alter the reduction process of melanobacteria (MB) under light irradiation using biological coenzymes like NADH. This allows MB to maintain its blue dye state and exert its phototherapy effect even in the presence of NADH. In contrast, in air, 90% of the MB group without TQ is converted to a colorless LMB structure, with absorbance close to zero at the therapeutic wavelength of 660 nm. After adding TQ, the total ROS production of MB in a physiological environment with NADH is more than four times that of the group without TQ. Furthermore, unlike the slow recovery process of MB reduction dependent on ambient oxygen (>60 s), the addition of TQ can replace the role of oxygen, converting LMB to MB within 10 s, restoring its photosensitivity, and reducing the process's oxygen consumption and dependence. Through the improved anti-reduction ability of MB using protective agents such as TQ, the killing effect of MB on cancer cells can be increased by 30%, with its cell inhibition rate rising from 80% to 95%. Meanwhile, the enhanced phototherapy capabilities endow MB with stronger phototherapy-induced immune responses, with significantly improved release of its contents (a ~2-fold increase in pro-apoptotic calreticulin CRT; and a 35% and 53% increase in extracellular concentrations of high-mobility group box 1 (HMGB1) and adenosine triphosphate ATP, respectively), which can induce a stronger immune response. Furthermore, the addition of TQ can enhance the phototherapy-antibacterial effect of MB by 100%.

[0096] In summary, by introducing a specific type of biosafety electronic receptor as a "protective agent," this invention successfully solves the core problem of the easy inactivation of MB in physiological reducing environments. It not only restores and enhances its photodynamic activity, but also further expands its potential for clinical application in multiple dimensions such as tumor treatment, antibacterial disinfection, and immune regulation.

[0097] It should be understood that the above embodiments are only for illustrating the technical solutions of the present invention more clearly, and are not intended to limit the scope of protection thereof. Those skilled in the art can make various modifications and variations to the above embodiments without departing from the spirit and basic principles of the present invention, but these modifications and variations still fall within the protection scope of the claims of the present invention and their equivalents. The scope of protection of the present invention should be determined by the appended claims, and should not be limited to the specific embodiments described above.

Claims

1. A bioreduction-resistant protective agent for a phenothiazine photosensitizer, characterized in that: The structural formula of the phenothiazine photosensitizing dye is shown in general formula I: I wherein R1, R2, R3, R4are each independently selected from C1-C 10 at least one of an alkane chain; X is at least one selected from S, Se, and Te; the bioreduction-resistant protective agent is at least one selected from the group consisting of plumbagin, cytochrome c, and dihydroethidium.

2. The anti-bio-reduction protectant of claim 1, wherein: the phenothiazine photosensitizer is at least one selected from the group consisting of MB, NBS, and NBSe.

3. The anti-bio-reduction protectant of claim 1, wherein: the protective agent has a standard reduction potential higher than the redox potential between the phenothiazine photosensitizer and its colorless reduced state, and is capable of electron transfer reaction with the colorless reduced state to facilitate re-oxidation to the colored oxidized state.

4. A photosensitive composition characterized by: a photosensitive composition comprising a phenothiazine photosensitizer as shown in general formula I and a bioreduction-resistant protective agent as claimed in claim 1, wherein the molar ratio of the bioreduction-resistant protective agent to the phenothiazine photosensitizer is 0.5-10:

1.

5. The photosensitive composition of claim 4, wherein: the photosensitive composition is an aqueous system with a pH of 6.5-7.

8.

6. A method for improving the ability of a phenothiazine photosensitizing dye to generate active oxygen in a reductive system, characterized by: a method comprising co-locating the bioreduction-resistant protective agent as claimed in claim 1 with a phenothiazine photosensitizer in an aqueous system, and applying light with a wavelength of 600-700 nm.

7. Use of the photosensitive composition as claimed in claim 4 for any of the following non-disease diagnostic or therapeutic purposes: (a) for preparing a tumor phototherapy preparation to maintain the photodynamic activity of a phenothiazine photosensitizer in a reducing environment; (b) for preparing an antibacterial photosensitive preparation to inhibit or inactivate microorganisms under in vitro light exposure; (c) for preparing an immune-enhancing preparation to induce the release of immunogenic death-related factors from cells in vitro.

8. Use according to claim 7, characterized in that: In (a), the reducing environment contains NADH and / or NADPH, and the phototherapy-related photosensitizer maintains the color state and active oxygen generation ability of the phenothiazine photosensitizer under 660 nm light exposure.

9. The use according to claim 7, wherein: In (b), the microorganisms include at least one of bacteria, fungi, or viruses.

10. The use according to claim 7, characterized in that: In (c), the immunogenic death-related factors include at least one of calreticulin, high mobility group protein B1, and adenosine triphosphate.

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