Mitochondrial-targeted photodynamic therapy for keloids based on platinum complexes and its application

Abstract

This invention relates to the field of photodynamic therapy for keloids, and discloses a mitochondrial-targeted photodynamic keloid therapeutic agent based on platinum complexes and its application. The agent is prepared by coordinating the organic ligand TBQQ with an activated cisplatin derivative in a 1:1 molar ratio, followed by column chromatography purification to produce a high-purity TBQQPt powder with an active ingredient purity ≥98%. The prepared TBQQPt powder is then administered through a standardized drug formulation, intratumoral administration, and photo-activation process, combined with a multi-dimensional efficacy monitoring and safety evaluation system. Leveraging the D-A type planar square coordination structure of TBQQPt, which endows it with type I / II mixed photodynamic reaction characteristics, mitochondrial targeting, and photocatalytic oxidation of NADH, it synergistically exerts a dual effect of energy deprivation and genome disruption, effectively inducing mitochondrial dysfunction and apoptosis of keloid fibroblasts, and inhibiting their proliferation.

Description

Technical Field

[0001] This invention relates to the field of photodynamic therapy for keloids, specifically to a mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes and its application. Background Technology

[0002] Keloids are a pathological fibroproliferative disease characterized by excessive proliferation of fibroblasts and excessive deposition of extracellular matrix. Commonly used clinical treatments include surgical excision, corticosteroid injection, and radiotherapy, but these treatments generally have problems such as high recurrence rate, significant side effects, and limited long-term efficacy.

[0003] In recent years, photodynamic therapy (PDT) has shown promise in proliferative diseases due to its advantages such as spatial controllability, repeatability, and minimal invasiveness. Its core principle is that photosensitizers generate reactive oxygen species (ROS) under specific wavelengths of light, inducing cell death in the lesion. Currently, the photosensitizers used in PDT are mainly organic compounds such as porphyrins and phthalocyanines, whose mechanism of action is primarily an oxygen-dependent type II reaction (generating singlet oxygen). However, the hypoxic microenvironment often exists within keloid tissue, severely limiting the efficacy of these photosensitizers. Furthermore, traditional photosensitizers lack subcellular organelle specificity, easily leading to non-specific damage and having limited killing efficiency against apoptosis-resistant keloid fibroblasts.

[0004] In the prior art, some Pt(II) complexes have been reported for use in photodynamic therapy of tumors, but their use in the treatment of keloids, especially the design of multi-mechanism synergistic photosensitizers that combine mitochondrial targeting, type I / II mixed ROS generation, and direct interference with mitochondrial metabolic function, has not been reported. Furthermore, existing Pt(II) complex photosensitizers still have significant shortcomings in terms of hypoxia adaptation, diversity of mechanisms of action, and validation in fibrotic disease models, and cannot meet the clinical treatment needs of keloids. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a mitochondrial-targeted photodynamic therapy for keloids based on platinum complexes and its application. It has the advantages of strong mitochondrial targeting, excellent hypoxia tolerance, synergistic and efficient mechanism of action, and good safety. It solves the problems of poor targeting, limited efficacy in hypoxic environments, single mechanism of action that is difficult to overcome apoptosis resistance, insufficient safety and clinical translation value of existing photosensitizers in the treatment of keloids.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution: a mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes, wherein the treatment agent is prepared by a coordination reaction of an organic ligand TBQQ and an activated cisplatin derivative in a molar ratio of 1:1, and the chemical formula of the reaction is as follows:

[0009] In the formula, Combining cisplatin and The crude TBQQPt product obtained from the reaction was further purified by column chromatography, solvent removed by rotary evaporation, and vacuum dried to form TBQQPt powder therapeutic agent. The active ingredient with a purity ≥98% is the platinum(II) complex TBQQPt, whose chemical name is a platinum(II) coordination compound containing a triphenylamine-quinoline-benzothiadiazole structure, and its molecular formula is C. 35 H 30 N6PtS has a molecular weight of 796.1584.

[0010] Preferably, the therapeutic agent TBQQPt has a donor-acceptor (DA) type planar square coordination structure, wherein the triphenylamine group acts as an electron donor, the quinoline-benzothiadiazole skeleton acts as an electron acceptor, and the platinum (II) center binds to the ligand through a coordination bond to form a complex.

[0011] Preferably, the therapeutic agent has mitochondrial targeting properties and can specifically accumulate in the mitochondria of keloid fibroblasts. Its targeting mechanism depends on the lipid-soluble cationic properties of the complex and selective accumulation driven by mitochondrial membrane potential.

[0012] Preferably, the therapeutic agent has type I / II mixed photodynamic reaction characteristics, and under visible light irradiation with strong absorption in the 400-600nm visible light region, it can generate superoxide anions through the type I reaction pathway. It can also produce singlet oxygen through the type II reaction pathway¹ It also has the function of photocatalytic oxidation of NADH and metabolic interference.

[0013] Preferably, the therapeutic agent has the ability to target mitochondrial DNA damage. Its planar aromatic structure and Pt(II) center endow it with a high affinity for mitochondrial DNA, causing oxidative damage to mtDNA under the synergistic effect of reactive oxygen species, resulting in downregulation of respiratory chain complex expression and mitochondrial dysfunction.

[0014] The application steps of a platinum-based mitochondrial-targeted photodynamic therapy for keloid scars are as follows:

[0015] Step 1, Drug Preparation: Dissolve TBQQPt powder in a solvent of a specified ratio to prepare a therapeutic solution;

[0016] Step 2, lesion drug administration: Apply the prepared TBQQPt solution topically to the keloid lesion site;

[0017] Step 3, photoactivation: Irradiate the lesion site after drug administration with a specific light source to activate the photodynamic activity of TBQQPt;

[0018] Step 4: Monitoring the efficacy of treatment: Regularly assess the growth of keloids and the treatment response during and after treatment;

[0019] Step 5: Safety evaluation: Monitor the subject's overall condition and major organ function during treatment to assess the safety of the treatment.

[0020] Preferably, in step one, the drug preparation involves dissolving TBQQPt powder in dimethyl sulfoxide to prepare an injection solution with a concentration of 200±0.05μM. The solution is then filtered through a 0.22μm microporous membrane to remove insoluble particles, followed by sterilization using a 0.22μm sterile membrane before use. This concentration is determined based on the effective concentration of 8±0.002μM observed in in vitro cell experiments and the conversion between the in vivo administration volume and the given formula:

[0021]

[0022] In the formula, Indicates the concentration of the solution for injection. Indicates the effective concentration in in vitro cell experiments. This represents the average volume of keloid tissue. This refers to the volume of a single intratumoral injection. This represents the drug retention coefficient in vivo.

[0023] Preferably, in step two, the lesion-specific drug delivery involves injecting the TBQQPt solution directly into the keloid tissue via intratumoral injection. The injection volume is adjusted according to the size of the keloid, controlling the single injection volume to be between 50-100 μL. The specific formula for calculating the single injection volume is as follows:

[0024]

[0025] In the formula, Indicates the volume of a single intratumoral injection. This represents the actual measured volume of the keloid tissue. The infiltration coefficient is 0.8 to 1.2, depending on the tissue density. Wait 30-60 minutes after administration.

[0026] Preferably, in step three, light irradiation activation involves using a white light source with a wavelength range of 400-700nm and a power density of 20mW / cm² to locally irradiate the lesion for 9-10 minutes.

[0027] Preferably, in step four, efficacy monitoring includes: measuring the volume of the keloid every 2-3 days and plotting a growth curve, observing changes in the color and texture of the keloid; and performing histological examination after treatment.

[0028] Safety evaluation in step five: During the entire treatment period, the subject's weight changes are monitored regularly. After the treatment, the heart, liver, spleen, lungs, kidneys and brain are collected for pathological histological examination to observe whether there is drug-related systemic toxicity or organ damage; at the same time, local reactions at the injection site are monitored.

[0029] Compared with existing technologies, this invention provides a mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes and its application, which has the following beneficial effects:

[0030] 1. This invention prepares high-purity TBQQPt powder by coordinating the organic ligand TBQQ with an activated cisplatin derivative in a 1:1 molar ratio, combined with column chromatography purification and other processes. This achieves the beneficial effects of improving the synthesis efficiency of the complex, ensuring the purity of the active ingredient ≥98%, avoiding impurities from interfering with the therapeutic effect, and reducing non-specific damage. At the same time, the clear chemical structure and molecular weight provide a precise basis for the quality control and dosage conversion of the therapeutic agent.

[0031] 2. This invention designs a DA-type planar square coordination structure to endow the therapeutic agent with mixed photodynamic response characteristics of type I / II and mitochondrial targeting, while also having the function of photocatalytic oxidation of NADH, so that it can adapt to the hypoxic microenvironment of keloids, achieve precise mitochondrial targeting, and achieve the dual effect of synergistic energy deprivation and genome destruction, ultimately effectively bypassing the apoptosis resistance mechanism of keloid fibroblasts.

[0032] 3. This invention ensures stable and repeatable treatment effects, reduces systemic toxicity, and improves treatment safety by standardizing drug preparation, intratumoral administration, and photo-activation procedures, combined with a multi-dimensional efficacy monitoring and safety evaluation system. It also provides reliable data support for clinical translation. Furthermore, its efficacy and safety have been verified by in vitro cell experiments and human keloid xenograft mouse models. Attached Figure Description

[0033] Figure 1 The chemical structural formula (A) of the platinum(II) complex TBQQPt and its control compound of the present invention, and its photophysical property characterization diagram (BE) and photochemical property characterization diagram (FN), including absorption spectrum, fluorescence spectrum, AIE characteristics, photostability, total ROS, singlet oxygen, superoxide anion generation and NADH photocatalytic oxidation curve.

[0034] Figure 2The figure shows the DFT / TD-DFT calculation results of TBQQPt in this invention (showing its frontier molecular orbital distribution and excited state characteristics).

[0035] Figure 3 The images show the simulated docking diagrams of molecules A, B, D, E, and G of TBQQPt interacting with DNA, and the experimental verification diagrams of C, F, and H (the images show its enhanced DNA binding ability).

[0036] Figure 4 This is a diagram illustrating the mitochondrial targeting and photoinduced apoptosis of TBQQPt in this invention.

[0037] Figure 5 This is a diagram illustrating mitochondrial DNA damage and dysfunction induced by TBQQPt-PDT of the present invention.

[0038] Figure 6 This is an in vivo therapeutic effect diagram of TBQQPt-PDT of the present invention in a mouse model of human keloid xenograft;

[0039] Figure 7 This is the molecular structure diagram of TBQQPt of the present invention. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Please see Figures 1-7 A mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes is prepared by a coordination reaction between the organic ligand TBQQ (a donor-acceptor type organic ligand containing a triphenylamine-quinoline-benzothiadiazole structure) and an activated cisplatin derivative in a 1:1 molar ratio. The chemical formula for the reaction is as follows:

[0042] In the formula, Combining cisplatin and The crude TBQQPt product obtained from the reaction was further purified by column chromatography, solvent removed by rotary evaporation, and vacuum dried to form TBQQPt powder therapeutic agent. The active ingredient with a purity ≥98% is the platinum(II) complex TBQQPt, whose chemical name is a platinum(II) coordination compound containing a triphenylamine-quinoline-benzothiadiazole structure, and its molecular formula is C. 35 H 30 N6PtS has a molecular weight of 796.1584.

[0043] By employing a ligand with an activated cisplatin derivative in a 1:1 molar ratio for coordination reaction, the reaction can be ensured to proceed fully, reducing raw material waste; through cisplatin and... The reaction preparation of activated cisplatin derivatives can effectively activate the coordination activity of platinum centers and improve the synthesis efficiency of complexes. The combined process of column chromatography purification, rotary evaporation to remove solvent and vacuum drying can effectively remove impurities, unreacted raw materials and solvent residues from the crude product, so that the purity of TBQQPt reaches more than 98%. The high purity of the active ingredient can avoid impurities from interfering with the therapeutic effect and reduce the risk of non-specific damage. At the same time, the clear molecular formula and molecular weight provide a precise chemical basis for the quality control and dosage conversion of the therapeutic agent.

[0044] Specifically, the therapeutic agent TBQQPt has a donor-acceptor (DA) type planar square coordination structure, in which the triphenylamine group acts as the electron donor, the quinoline-benzothiadiazole backbone acts as the electron acceptor, and the platinum (II) center binds to the ligand through a coordinate bond to form a complex. Its core structure is formed by the organic ligand TBQQ (a donor-acceptor molecule containing benzothiadiazole and quinoline) and the platinum (II) center through a coordinate bond. The specific structural formula is shown in [link to structural formula]. Figure 1 A and Scheme S1.

[0045] The advantages are: the DA-type planar square coordination structure is the core basis for TBQQPt to achieve photosensitivity. The combination design of triphenylamine donor and quinoline-benzothiadiazole receptor can achieve efficient intramolecular charge transfer (ILCT), giving the therapeutic agent strong absorption characteristics in the visible light region. The planar structure not only enhances the stability of the complex, but also provides structural support for its binding to DNA and mitochondrial targeting. The coordination binding of the platinum(II) center can promote intersystem crossing by utilizing the heavy atom effect, significantly improving the generation efficiency of reactive oxygen species (ROS), and solving the problems of low intersystem crossing efficiency and insufficient photosensitivity of traditional photosensitizers.

[0046] Specifically, the therapeutic agent possesses mitochondrial targeting properties, specifically accumulating in the mitochondria of keloid fibroblasts. Its targeting mechanism relies on the lipid-soluble cationic properties of the complex and selective accumulation driven by mitochondrial membrane potential. Confocal laser scanning microscopy (CLSM) confirmed that the red fluorescence of TBQQPt significantly overlapped with the mitochondrial green fluorescent probe (Mito-Tracker Green), while exhibiting poor co-localization with probes from other organelles such as lysosomes and cell membranes, confirming its highly efficient mitochondrial targeting specificity. Figure 4 B).

[0047] The advantages are: Mitochondria are the energy factories of fibroblasts in keloids and key organelles for their survival. The mitochondrial targeting properties of the therapeutic agent can concentrate the therapeutic effect on the mitochondria of the lesion cells, achieving precise targeting, reducing off-target damage to surrounding normal tissue cells, and reducing treatment side effects. At the same time, mitochondrial targeting can enrich TBQQPt in the core of the lesion, increase the local drug concentration, enhance the photodynamic therapy effect, and solve the defects of poor targeting and low treatment efficiency of traditional photosensitizers.

[0048] Specifically, the therapeutic agent exhibits mixed type I / II photodynamic reaction characteristics. Under visible light irradiation with strong absorption in the 400-600nm visible light region, it can generate superoxide anions through the type I reaction pathway (…). It can also produce singlet oxygen through the type II reaction pathway (¹) It also possesses the function of photocatalytic oxidation of NADH, which interferes with metabolism. Experiments have verified that it works under white light (20mW cm⁻¹). -2 Under irradiation, significant total ROS can be detected using probes such as DCFH-DA, DHE, ABDA, and 3-CCA. ,¹ It generates and can efficiently catalyze the oxidation of NADH ( Figure 1 FL, Figure 1 N), while also possessing excellent photostability (N), Figure 1 E) and aggregation-induced emission (AIE) properties.

[0049] The advantages are: the mixed type I / II photodynamic response characteristics allow the therapeutic agent to adapt to the hypoxic microenvironment of keloids, wherein the type I response produces... With low oxygen dependence, it maintains high oxidative damage capacity even under hypoxic conditions, solving the problem of limited efficacy of traditional type II photosensitizers in the hypoxic environment of keloids; the photocatalytic oxidation of NADH can directly interfere with mitochondrial energy metabolism, depriving keloid fibroblasts of energy supply, and forming a synergistic effect with ROS oxidative damage to enhance cell killing efficiency; the strong absorption characteristics in the 400-600nm visible light region can avoid the problems of poor tissue penetration of short wavelength light and low excitation efficiency of long wavelength light, taking into account both tissue penetration and photosensitivity activation efficiency, and excellent photostability can ensure that the drug activity is not lost during treatment and prolong the duration of action.

[0050] Specifically, the therapeutic agent possesses the ability to target mitochondrial DNA damage. Its planar aromatic structure and Pt(II) center endow it with a high affinity for mitochondrial DNA, causing oxidative damage to mtDNA (8-OHdG marker) under the synergistic effect of reactive oxygen species, leading to downregulation of respiratory chain complex expression and mitochondrial dysfunction. Immunofluorescence staining verified that after TBQQPt-PDT treatment, the fluorescence signal of the mitochondrial DNA oxidative damage marker 8-OHdG perfectly colocalized with the mitochondrial outer membrane marker TOMM20, and the fluorescence intensity increased significantly. Figure 5 C, D); Real-time quantitative PCR (RT-qPCR) and Western blot experiments confirmed that mtDNA copy number, transcription level, and expression of key subunit IV of respiratory chain complex were all significantly decreased. Figure 5 FI).

[0051] The advantages are: Mitochondrial DNA is the core of maintaining mitochondrial function. Damage to it will directly lead to the collapse of the mitochondrial respiratory chain and loss of membrane potential, thereby triggering apoptosis. The targeted damage of mtDNA by the therapeutic agent can destroy the mitochondrial function of keloid fibroblasts from the root, achieving a double blow of genome destruction and energy deprivation. This effectively bypasses the apoptosis resistance mechanism of keloid fibroblasts, solving the problem of the single mechanism of action and difficulty in overcoming apoptosis resistance of traditional photosensitizers, and ultimately greatly improving the treatment effect.

[0052] The application steps of a platinum-based mitochondrial-targeted photodynamic therapy for keloid scars are as follows:

[0053] Step 1, Drug Preparation: Dissolve TBQQPt powder in a solvent of a specified ratio to prepare a therapeutic solution;

[0054] Step 2, lesion drug administration: Apply the prepared TBQQPt solution topically to the keloid lesion site;

[0055] Step 3, photoactivation: Irradiate the lesion site after drug administration with a specific light source to activate the photodynamic activity of TBQQPt;

[0056] Step 4: Monitoring the efficacy of treatment: Regularly assess the growth of keloids and the treatment response during and after treatment;

[0057] Step 5: Safety evaluation: Monitor the subject's overall condition and major organ function during treatment to assess the safety of the treatment.

[0058] Specifically, in step one, the drug preparation involves dissolving TBQQPt powder in dimethyl sulfoxide (DMSO) to prepare an injection solution with a concentration of 200 ± 0.05 μM. The solution is then filtered through a 0.22 μm microporous membrane to remove insoluble particles, followed by sterilization using a 0.22 μm sterile membrane. This concentration is determined based on the effective concentration of 8 ± 0.002 μM observed in in vitro cell experiments and the conversion between the in vivo administration volume and the given formula.

[0059]

[0060] In the formula, Indicates the concentration of the solution for injection. Indicates the effective concentration in in vitro cell experiments. This represents the average volume of keloid tissue (approximately 50 μL). This is the volume of a single intratumoral injection (20 μL). The in vivo drug retention coefficient (1.0 based on preliminary experimental measurements) ensures that the lesion site reaches the required drug concentration for treatment. The effective concentration of 8 μM in in vitro cell experiments was verified by cell viability and experiments, and showed no significant dark toxicity to human keloid fibroblasts (HKFs) under light-free conditions.

[0061] The advantages are: by using DMSO as a solvent, the TBQQPt powder therapeutic agent can be fully dissolved, effectively avoiding uneven efficacy caused by drug insolubility; the dual treatment of 0.22μm microporous membrane filtration and sterile membrane filtration effectively removes insoluble particles and bacteria, reducing the risk of local infection and tissue irritation after injection; based on the precise conversion of the effective concentration in vitro, it can ensure that the concentration of the prepared solution can achieve the therapeutic effect while avoiding toxic reactions caused by excessive concentration, and the concentration design without dark toxicity further improves the safety of treatment.

[0062] Specifically, in step two, lesion-specific drug administration involves injecting TBQQPt solution directly into the keloid tissue via intratumoral injection. The injection volume is adjusted according to the size of the keloid, controlling the single injection volume to be between 50-100 μL. The specific formula for calculating the single injection volume is as follows:

[0063]

[0064] In the formula, Indicates the volume of a single intratumoral injection. This represents the actual measured volume of the keloid tissue. The infiltration coefficient is set at 0.8–1.2, depending on the tissue density (the lower limit is used for harder scar tissue, and the upper limit for softer tissue). This ensures that the drug fully infiltrates the entire lesion area without extravasation due to over-injection. After administration, wait 30–60 minutes to allow the drug to accumulate sufficiently within the mitochondria of the keloid fibroblasts. This time is based on… Figure 4 The cellular uptake kinetics of B were determined, ensuring that TBQQPt reached its peak mitochondrial enrichment before light exposure. Intratumoral injection allows the drug to act directly on the lesion core, avoiding concentration reduction due to systemic absorption and improving local drug utilization. Figure 6 B).

[0065] The advantages are: the above-mentioned intratumoral injection method has the advantages of strong targeting, high local drug concentration and low systemic exposure, which can reduce systemic toxicity; adjusting the injection volume according to the size and texture of the tumor can ensure that the drug fully infiltrates the entire lesion and avoid incomplete treatment due to uneven drug distribution; the 30-60 minute waiting time can ensure that TBQQPt is fully enriched in the mitochondria, so that the photodynamic effect can be maximized during light irradiation, improving treatment efficiency, while reducing the drug's residence in non-target sites.

[0066] Specifically, in step three, photo-activation involves using a white light source with a wavelength range of 400-700 nm and a power density of 20 mW / cm² to locally irradiate the lesion for 9-10 minutes. These light parameters are sufficient to stimulate TBQQPt to generate adequate reactive oxygen species and initiate the NADH oxidation catalytic reaction, while avoiding thermal damage to surrounding normal tissues. The parameters of the white light source have been verified through in vitro cell experiments and in vivo animal experiments; 20 mW / cm² is optimal. -2 The high power density and 9-10 minute irradiation time effectively activate the photodynamic activity of TBQQPt without producing a significant thermal effect. In in vitro experiments, a 5-minute irradiation time is sufficient to achieve a good cell-killing effect, while in vivo experiments extend the irradiation time to 9-10 minutes to ensure full activation of the lesions. Figure 4 C Figure 6 B).

[0067] The advantages are: the 400-700nm white light source perfectly matches the absorption peak of TBQQPt, which can maximize drug activity and improve ROS generation efficiency; the power density of 20mW / cm² and the irradiation time of 9-10 minutes ensure the therapeutic effect while effectively avoiding thermal damage to surrounding normal tissues caused by excessive light exposure, thus balancing efficacy and safety; the standardized design of light parameters provides a reliable basis for the reproducibility of treatment and clinical translation.

[0068] Specifically, in step four, efficacy monitoring involves measuring keloid volume and plotting growth curves every 2-3 days, observing changes in tumor color and texture; after treatment (e.g., on day 9), histological examination is performed, including H&E staining to observe changes in collagen arrangement and density, immunohistochemistry to detect the expression levels of cleaved caspase-3 (apoptosis marker) and PCNA (proliferation marker), and quantitative analysis of cell apoptosis rate and proliferation inhibition rate. In vivo experiments have verified that this monitoring method can clearly reflect the growth inhibition of keloids. H&E staining can observe the degree of collagen loosening, and immunohistochemistry can accurately quantify cell apoptosis and proliferation levels, providing objective and reliable experimental evidence for efficacy evaluation. Figure 6 CJ Figure 6 L).

[0069] The advantages are: by regularly measuring tumor volume and plotting growth curves, the treatment effect can be monitored in real time, and the treatment plan can be adjusted in a timely manner; histological examination and immunohistochemical detection can verify the treatment mechanism at the microscopic level, clarify the apoptosis and proliferation of fibroblasts in keloids, and ensure the scientificity and accuracy of efficacy evaluation; objective efficacy monitoring indicators provide data support for the optimization of treatment plans and clinical application.

[0070] Specifically, in step five, safety evaluation involves regularly monitoring the subject's weight changes throughout the treatment period. After treatment, major organs such as the heart, liver, spleen, lungs, kidneys, and brain are collected for pathological histological examination (H&E staining) to observe for any drug-related systemic toxicity or organ damage. Simultaneously, local reactions at the injection site are monitored to ensure good biosafety at effective doses, providing safety data support for subsequent clinical applications. In vivo animal experiments verified that mice showed no significant weight abnormalities during treatment, no significant damage was observed in the pathological sections of major organs, and no significant inflammatory reaction was observed at the injection site, confirming that this treatment regimen has good in vivo safety. Figure 6 I, Figure S23A).

[0071] The advantages are: weight monitoring and pathological examination of major organs can comprehensively assess the systemic toxicity of the therapeutic agent; local reaction monitoring at the injection site can assess local safety; and multi-dimensional safety evaluation can ensure that the therapeutic agent has no obvious toxic side effects at effective therapeutic doses, reduce the risk of clinical application, and lay a safe foundation for the clinical translation of the therapeutic agent.

[0072] The TBQQPt powder therapeutic agent prepared above was applied in the following examples, specifically:

[0073] Example 1

[0074] The study aimed to verify the subcellular localization of TBQQPt in human keloid fibroblasts (HKFs) and its ability to induce apoptosis under light irradiation.

[0075] Method and steps:

[0076] T1. Cell culture: HKFs were seeded in confocal culture dishes and cultured to a suitable density;

[0077] T2, Mitochondrial Colocalization: HKFs were co-incubated with 8 μM TBQQPt at 37°C, then washed with PBS, and stained with Mito-Tracker Green (mitochondrial green fluorescent probe) and Hoechst 33342 (nuclear blue fluorescent probe). The colocalization of TBQQPt (red fluorescence) and Mito-Tracker Green (green fluorescence) was observed and analyzed using confocal laser scanning microscopy (CLSM).

[0078] T3. Apoptosis detection: HKFs were divided into four groups: control group (PBS), QPt group (8 μM QPt only), light group (L, white light irradiation 20 mW / cm², 5 min only), and QPt-PDT group (8 μM QPt incubation followed by the same light irradiation). After treatment, cells were collected and stained with Annexin V-FITC / PI apoptosis detection kit. The apoptosis rate was analyzed by flow cytometry. At the same time, cell proteins were extracted from each group, and the expression levels of apoptosis-related proteins Cleaved caspase-3 and Bcl-2 were detected by Western blotting.

[0079] Implementation results:

[0080] T1, mitochondrial colocalization: CLSM images showed that the red fluorescence of TBQQPt significantly overlapped with the green fluorescence of Mito-Tracker Green, while colocalization with other organelle probes (such as Lyso-tracker, DiO) was poor, confirming that TBQQPt can specifically accumulate in the mitochondria of HKFs. Figure 4 B);

[0081] T2, induction of apoptosis: Flow cytometry results showed that, compared with the control group, the QPt-only group, and the light-only group, the total apoptosis rate of HKFs (Annexin V positive) in the QPt-PDT group (L+QPt) was significantly increased. Figure 4 E,I), Western Blot results showed that the expression of the anti-apoptotic protein Bcl-2 was downregulated in the QPt-PDT group, while the expression level of the apoptosis-executing protein Cleaved caspase-3 was significantly upregulated. Figure 4 F,J,K).

[0082] Conclusion: TBQQPt can efficiently and specifically target the mitochondria of HKFs. Under light activation, TBQQPt can effectively induce mitochondrial-mediated endogenous apoptosis.

[0083] Example 2

[0084] TBQQPt-PDT induces mitochondrial DNA damage and dysfunction. The aim is to explore the molecular mechanism by which TBQQPt-PDT leads to the mitochondrial dysfunction of HKFs.

[0085] Method and steps:

[0086] T1. Cell treatment: HKFs were divided into control group (PBS), QPt group, light group (L) and QPt-PDT group, and the treatment method was the same as in Example 1.

[0087] T2. Mitochondrial membrane potential detection: After treatment, the cells were stained with MitoTracker Red CMXRos (a fluorescent dye that depends on mitochondrial membrane potential (ΔΨm)) and the changes in fluorescence intensity were observed by fluorescence microscopy to assess the loss of ΔΨm.

[0088] T3. Detection of mitochondrial DNA (mtDNA) oxidative damage: After treatment, the cells were fixed and permeabilized, and then co-incubated with anti-8-OHdG antibody (a marker of mitochondrial DNA oxidative damage) and anti-TOMM20 antibody (a marker of mitochondrial outer membrane). The colocalization and fluorescence intensity of 8-OHdG (red) and TOMM20 (green) were observed by immunofluorescence staining and CLSM.

[0089] T4. Mitochondrial function assessment: Total DNA was extracted from cells, and the relative copy number of the mtDNA encoding gene ND1 and the regulatory region D-loop was detected by real-time quantitative PCR (RT-qPCR). Total protein was extracted from cells, and the protein expression levels of the oxidative phosphorylation (OXPHOS) complex subunits (NDUFB8, SDHB, UQCRC1, MTCO2, ATPSA1) were detected by Western blotting.

[0090] Implementation results:

[0091] T1, membrane potential collapse: Compared with the control group, the MitoTracker Red CMXRos fluorescence intensity of cells in the QPt-PDT group was significantly reduced, indicating that the mitochondrial membrane potential (ΔΨm) underwent rapid and severe dissipation. Figure 5 Transmission electron microscopy (TEM) images (A, B) also confirmed that the treated group showed severe swelling and cristae damage in the mitochondria. Figure 5 E);

[0092] T2, Specific mtDNA damage: Immunofluorescence results showed that only in the QPt-PDT group was a strong 8-OHdG (red) fluorescent signal observed, and this signal was perfectly co-localized with TOMM20 (green) labeled mitochondria, proving that TBQQPt-PDT induced severe mtDNA oxidative damage in situ in mitochondria. Figure 5 C,D);

[0093] T3, respiratory chain breakdown: RT-qPCR results showed that the copy number of mtDNA and the transcription levels of ND1 and D-loop were significantly decreased in the QPt-PDT group. Figure 5 G,H), while Western blot results showed that the levels of key subunit proteins of mitochondrial respiratory chain complex IV were significantly reduced in all treatment groups (G,H). Figure 5 F,I).

[0094] Conclusion: TBQQPt-PDT specifically induces oxidative damage to mtDNA by generating ROS in situ in mitochondria. This disruption of genetic material leads to the inhibition of the synthesis of respiratory chain subunits encoded by mtDNA, which in turn triggers the disintegration of the mitochondrial respiratory chain complex, loss of membrane potential, and morphological and structural damage, ultimately resulting in the complete collapse of cellular bioenergy metabolism.

[0095] Example 3

[0096] The study aimed to evaluate the in vivo therapeutic effect of TBQQPt-PDT powder in a mouse model of human keloid xenograft, assessing its efficacy and safety in inhibiting human keloid growth at the in vivo level.

[0097] Method and steps:

[0098] T1. Model Establishment: Human keloid surgical excision tissue was trimmed into uniform small pieces of 4×4×5 mm³, and subcutaneously implanted into the backs of BALB / c nude mice to establish a human keloid xenograft model. Figure 6 A);

[0099] T2, Grouping and Treatment: Tumor-bearing mice were randomly divided into 4 groups (n=5): control group (G1, intratumoral injection of PBS), QPt alone group (G2, intratumoral injection of 200μM QPt only), light exposure alone group (G3, local white light irradiation of 20mW / cm² for 10min only), and combined treatment group (G4, intratumoral injection of 200μM QPt followed by the same light exposure). Figure 6 B) During treatment, mouse body weight and tumor volume were measured regularly;

[0100] T3. Evaluation of efficacy: After treatment, mice were euthanized, transplanted tumor tissue was dissected, and the tumor volume of each group was measured and compared. Hematoxylin and eosin (H&E) staining was performed on the tumor tissue sections to observe the tissue morphology, and immunohistochemical staining of Cleaved caspase-3 and PCNA was performed to assess cell apoptosis and proliferation in vivo.

[0101] The implementation results are as follows:

[0102] (1) Inhibition of tumor growth: Compared with the three control groups (G1, G2, G3), the combined treatment group (G4, QPt-PDT) showed the slowest growth in tumor volume during treatment, and the mean tumor volume at the treatment endpoint (day 9) was significantly smaller than that of the other groups. Figure 6 CH), during the entire treatment process, no significant abnormal changes were observed in the body weight of mice in any group ( Figure 6 I) No obvious pathological damage was observed in the H&E staining of the major organs (heart, liver, spleen, lung, kidney, and brain) (Figure S23A), indicating that the treatment regimen has good in vivo safety.

[0103] (2) Histological verification: H&E staining showed that the collagen arrangement of the tumor tissue in group G4 was looser than that in other groups. Figure 6 J). Immunohistochemical results showed that the number of Cleaved caspase-3 positive cells, a marker of apoptosis, was significantly increased in the G4 group tumors. Figure 6 K,L), while the expression level of the proliferation marker PCNA was significantly reduced (Figure S22G,H).

[0104] Conclusion: TBQQPt-PDT effectively inhibited keloid growth in a mouse model of human keloid xenograft, and its mechanism of action is related to inducing apoptosis and inhibiting the proliferation of scar fibroblasts in vivo. This treatment regimen did not exhibit significant systemic toxicity at effective doses and demonstrated good safety.

[0105] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes, characterized in that, The therapeutic agent is prepared by a coordination reaction of the organic ligand TBQQ and an activated cisplatin derivative in a molar ratio of 1:

1. The chemical formula of the reaction is as follows: In the formula, Combining cisplatin and The crude TBQQPt product obtained from the reaction was further purified by column chromatography, solvent removed by rotary evaporation, and vacuum dried to form TBQQPt powder therapeutic agent. The active ingredient with a purity ≥98% is the platinum(II) complex TBQQPt, whose chemical name is a platinum(II) coordination compound containing a triphenylamine-quinoline-benzothiadiazole structure, and its molecular formula is C. 35 H 30 N6PtS has a molecular weight of 796.1584.

2. The mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes according to claim 1, characterized in that, The therapeutic agent TBQQPt has a donor-acceptor (DA) type planar square coordination structure, wherein the triphenylamine group acts as an electron donor, the quinoline-benzothiadiazole skeleton acts as an electron acceptor, and the platinum (II) center binds to the ligand through a coordination bond to form a complex.

3. The mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes according to claim 1, characterized in that, The therapeutic agent has mitochondrial targeting properties and can specifically accumulate in the mitochondria of keloid fibroblasts. Its targeting mechanism depends on the lipid-soluble cationic properties of the complex and selective accumulation driven by mitochondrial membrane potential.

4. The mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes according to claim 1, characterized in that, The therapeutic agent exhibits mixed type I / II photodynamic reaction characteristics. Under visible light irradiation with strong absorption in the 400-600nm visible light region, it can generate superoxide anions through the type I reaction pathway. It can also produce singlet oxygen through the type II reaction pathway¹ It also has the function of photocatalytic oxidation of NADH and metabolic interference.

5. The mitochondrial-targeted photodynamic keloid treatment agent based on platinum complexes according to claim 1, characterized in that, The therapeutic agent has the ability to target mitochondrial DNA damage. Its planar aromatic structure and Pt(II) center endow it with a high affinity for mitochondrial DNA. Under the synergistic effect of reactive oxygen species, it causes oxidative damage to mtDNA, resulting in downregulation of respiratory chain complex expression and mitochondrial dysfunction.

6. The application of a platinum complex-based mitochondrial targeted photodynamic keloid treatment agent, characterized in that, The application steps are as follows: Step 1, Drug Preparation: Dissolve TBQQPt powder in a solvent of a specified ratio to prepare a therapeutic solution; Step 2, lesion drug administration: Apply the prepared TBQQPt solution topically to the keloid lesion site; Step 3, photoactivation: Irradiate the lesion site after drug administration with a specific light source to activate the photodynamic activity of TBQQPt; Step 4: Monitoring the efficacy of treatment: Regularly assess the growth of keloids and the treatment response during and after treatment; Step 5: Safety evaluation: Monitor the subject's overall condition and major organ function during treatment to assess the safety of the treatment.

7. The application of the platinum complex-based mitochondrial targeted photodynamic keloid treatment agent according to claim 6, characterized in that, In step one, the drug preparation involves dissolving TBQQPt powder in dimethyl sulfoxide to prepare an injection solution with a concentration of 200 ± 0.05 μM. The solution is then filtered through a 0.22 μm microporous membrane to remove insoluble particles, followed by sterilization using a 0.22 μm sterile membrane. This concentration is determined based on the effective concentration of 8 ± 0.002 μM observed in in vitro cell experiments and the conversion between the in vivo administration volume and the given formula. In the formula, Indicates the concentration of the solution for injection. Indicates the effective concentration in in vitro cell experiments. This represents the average volume of keloid tissue. This refers to the volume of a single intratumoral injection. This represents the drug retention coefficient in vivo.

8. The application of the platinum complex-based mitochondrial targeted photodynamic keloid treatment agent according to claim 6, characterized in that, In step two, the lesion-specific drug delivery involves injecting TBQQPt solution directly into the keloid tissue via intratumoral injection. The injection volume is adjusted according to the size of the keloid, controlling the single injection volume to be between 50-100 μL. The specific formula for calculating the single injection volume is as follows: In the formula, Indicates the volume of a single intratumoral injection. This represents the actual measured volume of the keloid tissue. The infiltration coefficient is 0.8 to 1.2, depending on the tissue density. Wait 30-60 minutes after administration.

9. The application of the platinum complex-based mitochondrial targeted photodynamic keloid treatment agent according to claim 6, characterized in that, In step three, light irradiation activation involves using a white light source with a wavelength range of 400-700nm and a power density of 20mW / cm² to locally irradiate the lesion for 9-10 minutes.

10. The application of the platinum complex-based mitochondrial targeted photodynamic keloid treatment agent according to claim 6, characterized in that, In step four, efficacy monitoring involves measuring the volume of the keloid every 2-3 days and plotting its growth curve, observing changes in the color and texture of the tumor, and performing histological examination after treatment. Safety evaluation in step five: During the entire treatment period, the subject's weight changes are monitored regularly. After the treatment, the heart, liver, spleen, lungs, kidneys and brain are collected for pathological histological examination to observe whether there is drug-related systemic toxicity or organ damage. At the same time, local reactions at the injection site are monitored.

Images