Preparation method and application of photosensitizer molecular nano reagent for photodynamic response of singlet oxygen
Photodynamic response singlet oxygen nanoparticles prepared by DPPTPA compounds solve the biocompatibility and targeting of existing photosensitizers in photodynamic therapy, and achieve efficient tumor treatment and diagnostic effects, especially the treatment of osteosarcoma.
Patent Information
- Application Number
- CN202510752121.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-08-29
AI Technical Summary
The existing photosensitizers have problems such as poor biocompatibility, poor water solubility, light instability, poor tissue penetration and poor targeting in photodynamic therapy, which limits their wide application in cancer treatment, especially in the treatment effect of osteosarcoma and other parts.
DPPTPA compound is used to prepare photosensitizer nanoreagents that respond to singlet oxygen with photodynamic response. DPPTPA nanoparticles are formed through nanoparticle technology, which have excellent photodynamic and photothermal properties, and nanoparticles with good water-soluble and uniform particle size are prepared through a simple synthesis process.
It has achieved photodynamic treatment effects with strong tumor targeting, high biosafety and low toxic and side effects, and has good anti-tumor effects and in vivo fluorescence imaging capabilities, which are suitable for tumor diagnosis and treatment.
Smart Images
Figure CN120554362A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine and biomaterials, and in particular relates to a preparation method and application of a photosensitizer molecular nanoreagent that responds to singlet oxygen in a photodynamic manner. Background Art
[0002] With the development of pharmaceutical chemistry, laser pharmacy, and nanomaterial science, medical researchers have developed a variety of new methods for tumor diagnosis and treatment in order to more effectively treat cancer, especially malignant tumors such as osteosarcoma. Among them, light therapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has become a new direction and hope for the treatment of malignant tumors. Photodynamic therapy mainly relies on the highly selective absorption of photosensitizers by tumor tissues, and produces a series of reactions under the action of light to cause the denaturation of nucleic acids and proteins in tumor cells, thereby achieving the purpose of killing tumor cells. The main principle of photodynamic therapy is that under the stimulation of light of appropriate wavelength, photosensitizers (PS) can absorb light energy and produce reactive oxygen species (ROS, such as singlet oxygen) that are toxic to cells through a series of optical and chemical reactions in the cells. 1 O2), causing direct damage to cells and inducing cell apoptosis. In addition to producing ROS to directly kill tumors, PDT can also destroy the formation of microvessels in tumor tissues, cut off the nutrient delivery to the tumor, and play an immunomodulatory role, thereby indirectly killing tumor cells. Compared with traditional treatments, photodynamic therapy has the advantages of high tumor targeting, small tissue trauma, low toxicity and side effects, high biocompatibility, and almost no drug resistance. It is especially effective for tumors like osteosarcoma, which have few tissue vascular distributions, are difficult for drugs to reach, and are prone to residual and recurrence after surgery. It can replace traditional chemotherapy or radiotherapy and other treatments with large toxic and side effects, better cooperate with surgical treatment, remove residual lesions after surgery, and prevent tumor recurrence. It has a good application prospect in the treatment of osteosarcoma.
[0003] With its many advantages, photodynamic therapy has been increasingly studied and applied. However, the main reason limiting its widespread application lies in the photosensitizer itself. Currently, photosensitizers are almost all artificially synthesized compounds, and their safety for long-term use has not been verified and recognized, resulting in few clinical applications. Therefore, it is particularly important to find and develop natural compounds with photodynamic activity. Natural compounds that meet the requirements will have better clinical application potential as ideal photosensitizers. Summary of the Invention
[0004] The purpose of the present invention is to provide a method for preparing and applying a photosensitizer molecular nanoreagent that responds to singlet oxygen in a photodynamic manner. The nanoparticles not only have excellent photodynamic and photothermal properties, but also have a wide absorption spectrum and good biosafety. They can solve the defects of existing diagnostic and therapeutic reagents such as poor biocompatibility, poor water solubility, photostability, poor tissue penetration and poor targeting, and are expected to show good application prospects in anti-tumor diagnosis and precision treatment.
[0005] Another object of the present invention is to provide a method for preparing the photosensitizer molecular nanoreagent that responds to singlet oxygen in a photodynamic manner. The method is simple to operate, low in cost, and the obtained nanoparticles have good size uniformity.
[0006] The third object of the present invention is to provide the use of the photosensitizer nanoparticles in preparing in vivo fluorescent contrast agents and tumor phototherapy drugs.
[0007] The purpose of the present invention is achieved by adopting the following technical solutions: The photosensitizer molecular nanoreagent that responds to singlet oxygen in a photodynamic manner is expressed as DPPTPA, and its structure is:
[0008] The method for preparing the above-mentioned photodynamically responsive singlet oxygen photosensitizer molecular nanoreagent comprises the following steps: (1) Dissolve 10 mg of DPPTPA compound and 30 mg of DSPE-PEG in 5 mL of tetrahydrofuran and shake under ultrasonic conditions to fully dissolve them; (2) The mixed solution of DPPTPA and DSPE-PEG (5 mL) was slowly added dropwise to a mixed solution of 20 mL ultrapure water and 2 mL tetrahydrofuran under ultrasonic conditions; (3) After thorough shaking, stir in a ventilated place overnight to remove excess tetrahydrofuran; (4) DPPTPA nanoparticles were obtained after freeze-drying.
[0009] The photosensitizer nanoparticles that generate singlet oxygen in response to photodynamic stimulation can be used to prepare similar new anti-tumor drugs mediated by photodynamic and photothermal stimulation.
[0010] Compared with the prior art, the main advantages of the present invention include the following aspects: (1) the anti-tumor photosensitizer nanoparticles prepared by the present invention have a clear structure and a simple synthesis process; (2) the anti-tumor photosensitizer nanoparticles prepared by the present invention have good water solubility, uniform particle size, good tumor targeting, and excellent photodynamic and photothermal properties as well as in vivo fluorescence imaging capabilities; (3) the anti-tumor photosensitizer nanoparticles prepared by the present invention have excellent anti-tumor effects and biosafety, low toxic side effects, and have good application prospects as a new type of tumor phototherapy agent. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 :It is DPPTPA 1 The characteristic peaks of the H-NMR spectrum are consistent with the structure of DPPTPA.
[0012] Figure 2 : is the absorption spectrum of DPPTPA nanoparticles, indicating ultraviolet absorption at 600 nm.
[0013] Figure 3 : This is the TEM image of DPPTPA nanoparticles, indicating that the prepared nanoparticles are between 80-120nm.
[0014] Figure 4 : This is a cell viability diagram of human osteosarcoma 143B cells treated with DPPTPA nanoparticles.
[0015] Figure 5 : This is a diagram of the reactive oxygen species and cellular uptake of DPPTPA nanoparticles in human osteosarcoma 143B cells.
[0016] Figure 6 : This is a diagram of the treatment of human osteosarcoma tumors in nude mice with DPPTPA nanoparticles.
[0017] Figure 7 : Fluorescence imaging of DPPTPA nanoparticles in nude mice at different time points. DETAILED DESCRIPTION
[0018] Example 1 Preparation of DPPTPA A mixture of DPP (200.0 mg, 0.24 mmol), 4-bromotriphenylamine (194.4 mg, 0.60 mmol), platinum acetate (11.0 mg, 0.02 mmol), pivalic acid (20 mg, 0.20 mmol), and K2CO3 (83.0 mg, 0.60 mmol) was dissolved in 5 mL of N,N-dimethylacetamide (DMA), and then N2 was bubbled through the system to remove possible oxygen and water. Under nitrogen protection, the mixture was heated to 110°C for 12 h. The mixture was poured into saturated sodium chloride solution (150 mL), cooled to room temperature, and extracted three times with 100 mL of dichloromethane. The solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography using dichloromethane and hexane (1:2, v / v) as the developing solvent to obtain a dark blue solid (yield: 30%).
[0019] Example 2 Preparation of DPPTPA Nanoparticles 20 mg of DPPTPA prepared in Example 1 was weighed and dissolved in 10 ml of tetrahydrofuran. The solution was then slowly added dropwise (at a rate of approximately 10 drops / min) to 20 ml of rapidly stirred water (1500 rpm). Nitrogen was then added and stirred for 180 minutes. The tetrahydrofuran in the solution was removed, and the solution was centrifuged to obtain the photosensitizer nanoparticles DPPTPA NPs that generate singlet oxygen in response to photodynamic forces.
[0020] The absorption spectrum of DPPTPA NPs was tested by UV-visible-near infrared absorption spectroscopy. Figure 2 As shown, DPPTPA nanoparticles exhibit a broad near-infrared absorption peak.
[0021] The morphology and size of DPPTPA nanoparticles were observed by transmission electron microscopy. Figure 3 It can be found that the nanoparticles are spherical and relatively uniform in size, with a particle size between 80 and 120 nm and a particle size less than 200 nm, indicating that DPPTPA nanoparticles can enter the tumor tissue through the strong permeability and long retention (EPR) effect.
[0022] Example 3 Inhibitory effect of DPPTPA nanoparticles on cell viability of human osteosarcoma 143B cells (1) Take 143B cells in the logarithmic growth phase, digest them with trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, add fresh culture medium and gently pipette to form a uniform cell suspension. Count the cells using a hemocytometer. Take the prepared cell suspension and inoculate it into a 96-well plate at a density of 5000-10000 cells / well, 100 μL per well, set up 6 replicate wells in parallel, and place it at 37 o C. Culture in an incubator with 5% CO2 for about 24-48 hours until the cell growth density reaches 70-80%.
[0023] (2) The DPPTPA NPs mother solution was diluted with low serum medium containing 2% FBS to a final concentration of 0, 1, 2, 5, 10, 20, 50, and 100 μg / mL. 100 μL of medium containing different concentrations of DPPTPA NPs was added to each well and the cells were placed back in the incubator for 12 h. The cells were divided into two groups, one 96-well plate was used as the light group, and the other 96-well plate was used as the dark group. The 96-well plate in the light group was illuminated with a 660 nm laser (0.2 W / cm 2 ) irradiated for 10 min, and then returned to the incubator for 12 h. The 96-well plates of the dark light group were kept in the incubator for culturing.
[0024] (3) Finally, the two groups of 96-well plates were taken out, 10 μL of MTT solution was added to each well, and the culture was continued for 3-4 h. The supernatant was aspirated and 100 μL of DMSO was added to each well. The plates were gently shaken to completely dissolve the crystals. The absorbance value (A) of each well was measured at a wavelength of 490 nm on a microplate reader. The inhibition rate of DPPTPA NPs on 143b cell viability was calculated based on the absorbance value, and the half-maximal inhibitory concentration (IC) was calculated. 50 values and draw a cell viability inhibition graph, such as Figure 4 shown.
[0025] Example 4: Reactive oxygen species and cellular uptake experiments of DPPTPA nanoparticles in 143B cells Take 1×10 4 143B cells were seeded in a 35 mm laser confocal microplate and cultured for 24 h. The culture medium was replaced with a culture medium containing DPPTPA NPs (20 mg / mL) and cultured for another 24 h. The old culture medium was discarded and the cells were washed with PBS buffer solution three times for 2 min each time. Then, 500 mL of DCFH-DA (10 mM) was added and incubated in the dark for 20 min. The cells were washed with PBS buffer solution three times for 2 min each time. Then, 1 mL of 4% paraformaldehyde solution was added for fixation for 20 min. After washing with PBS buffer solution, 1 mL of PBS buffer solution was added and the cells were illuminated with a xenon lamp (>600 nm, 20 mW / cm 2 ) for 5 min, and immediately take fluorescence images of the cells using a laser scanning confocal microscope. Figure 5 shown.
[0026] Example 5: Experiment on the inhibition of tumor growth in nude mice by DPPTPA nanoparticles 143B cells in the logarithmic growth phase were taken out and prepared into a medium concentration of about 1×10 7 / mL cell suspension was inoculated into the right axilla of nude mice, near the heart, with 1 mL of cell suspension per mouse. After inoculation, the mice were kept for 10-15 days to allow for tumor growth. The diameter and short diameter of the tumor were measured with a vernier caliper. The tumor volume was 100-150 mm. 3After 3 hours, the animals were randomly divided into groups and treatment began. The nude mice were randomly divided into 4 groups (5 in each group): (1) blank control group: normal saline solution was injected through the tail vein, and 6 hours after administration, 660 nm laser irradiation was performed; (2) DPPTPA dark light control group: normal saline solution with a concentration of 100 μg / mL DPPTPA NPs was injected through the tail vein, and the administration volume was calculated as 0.1 mL / 20 g. No laser irradiation was performed after administration; (3) DPPTPA light treatment group (0.2 W / cm2): normal saline solution with a concentration of 100 μg / mL DPPTPA NPs was injected through the tail vein, and the administration volume was calculated as 0.1 mL / 20 g. 6 hours after administration, 660 nm laser irradiation was performed; (4) DPPTPA light treatment group (1 W / cm2): normal saline solution with a concentration of 100 μg / mL DPPTPA NPs was injected through the tail vein, and the administration volume was calculated as 0.1 mL / 20 g. 6 hours after administration, 660 nm laser irradiation was performed. Figure 6 As shown, after the treatment, the tumor size in the light group was significantly reduced.
[0027] Example 6 Fluorescence imaging experiment of DPPTPA nanoparticles in nude mice at different time points Nude mice with established tumors were selected and injected with 100 μL of DPPTPANPs (100 μg / mL) in normal saline solution via tail vein. In vivo images were collected using the Tianneng X60 small animal in vivo imaging system before administration (0 h) and 2, 4, 6, 12, and 24 h after administration. Figure 7 As shown, the fluorescence intensity at the tumor site was the highest after 6 hours, and the tumor site still had fluorescence intensity after 24 hours, indicating that DPPTPANPs has good tumor targeting ability.
[0028] In the description of the present invention, it should be understood that the indicated orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present invention.
[0029] The above is only a specific implementation method of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with this technical field can easily think of changes or replacements within the technical scope disclosed in this application, which should be covered by the protection scope of the present application.
Claims
1. The photosensitizer molecule that responds to singlet oxygen in a photodynamic manner has the following chemical structure: 。 2. The method for preparing the photosensitizer molecular nanoreagent that responds to singlet oxygen in a photodynamic manner according to claim 1, characterized in that The steps include: ； Preparation of DPPTPA: A mixture of DPP (200.0 mg, 0.24 mmol), 4-bromotriphenylamine (194.4 mg, 0.60 mmol), platinum acetate (11.0 mg, 0.02 mmol), pivalic acid (20 mg, 0.20 mmol) and K2CO3 (83.0 mg, 0.60 mmol) was dissolved in 5 mL of N,N-dimethylacetamide (DMA), and then N2 was bubbled to drive out possible oxygen and water in the system. Under nitrogen protection, the mixture was heated to 110°C for 12 h. The mixture was poured into saturated sodium chloride solution (150 mL), cooled to room temperature, and extracted three times with 100 mL of dichloromethane. The solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography using dichloromethane and hexane (1:2, v / v) as the developing solvent to obtain a dark blue solid (yield: 30%). H NMR spectrum: δH 7.63-7.40 (2H, d), 7.28-7.20 (10H, m), 7.11-7.04 (10H, m), 7.03-6.96 (10H and m), 4.15-4.00 (4H, d), 1.89 (2H, s), 1.26-1.06 (63H, m) and 0.81-0.74 (13H, m).
3. The method for preparing the photosensitizer molecular nanoreagent for photodynamic response to singlet oxygen according to claim 2, characterized in that The solvent is tetrahydrofuran.
4. The method for preparing the photosensitizer molecular nanoreagent for photodynamic response to singlet oxygen according to claim 3, characterized in that 1-Bromoeicosane was added to the DPP reaction at a molar ratio of 1:
5.
5. The method for preparing the photosensitizer molecular nanoreagent for photodynamic response to singlet oxygen according to claim 2, characterized in that The reaction molar ratio of DPP to 4-bromotriphenylamine is 1:
3.
6. The method for preparing the photosensitizer molecular nanoreagent for photodynamic response to singlet oxygen according to claim 4, characterized in that The particle size of the nanoparticles is 80 to 120 nanometers.
7. Use of the photodynamically responsive singlet oxygen photosensitizer molecular nanoreagent according to claim 1 in the preparation of fluorescent imaging contrast agents and tumor photodynamic therapy drugs.