Phthalocyanine modified titanium dioxide loaded camptothecin nanoparticles and preparation method thereof
By using phthalocyanine-modified titanium dioxide loaded camptothecin nanoparticles, the stability and solubility problems of camptothecin in clinical applications were solved, effective drug delivery and rapid release in cancer treatment were achieved, and the anti-cancer effect was enhanced.
Patent Information
- Application Number
- CN202510994913.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-18
- Publication Date
- 2025-10-28
AI Technical Summary
The existing camptothecin has problems such as unstable lactone structure, poor solubility and high toxicity in clinical applications, which limits its effectiveness in cancer treatment.
Camptothecin nanoparticles were loaded with phthalocyanine-modified titanium dioxide. By encapsulating camptothecin in nanoparticles using nanotechnology, and combined with sonodynamic therapy, the stability and efficacy of camptothecin were enhanced.
Phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin rapidly release camptothecin in a simulated cancer cell environment, exhibiting superior therapeutic effects compared to camptothecin alone. Furthermore, they demonstrate good biocompatibility, show no significant organ damage, and enhance the anti-cancer effect.
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Figure CN120837639A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles and their preparation method. Background Technology
[0002] Sonodynamic therapy (SDT) utilizes ultrasound to activate a sonosensitive agent, triggering a sonochemical reaction that generates reactive oxygen species (ROS), cavitation effects, and thermal effects that kill tumor cells. Highly toxic ROS, such as singlet oxygen (SO2), are also present. 1 O2 and hydroxyl radicals (•OH) induce apoptosis or necrosis of cancer cells by oxidizing cell membranes, damaging DNA / proteins, and disrupting signaling function / gene transcription. Ultrasound can precisely focus on the tumor area, targeting and activating sonosensitive agents to selectively kill tumor cells while minimizing damage to adjacent normal organs and tissues, making it highly effective in treating deep malignant tumors. Therefore, sonodynamic therapy has become an important research direction in the treatment of many cancers.
[0003] Camptothecin (CPT) is a cytotoxic quinoline alkaloid extracted from the Camptotheca acuminata plant. It blocks the synthesis of topo isomerase I (topo I), an enzyme closely related to cell division. Blocking the production of this enzyme inhibits cancer cell growth, indicating that CPT targets topo I rather than topo isomerase II (topo II), which is CPT's unique anticancer mechanism. However, due to the instability of camptothecin's lactone structure, poor solubility, easy ring-opening decomposition, and high toxicity, its clinical application is limited. To overcome the shortcomings of camptothecin and enhance its antitumor effect, we can encapsulate camptothecin in nanomedicines using nanotechnology to form nano-formulations. These nano-formulations can then be combined with sonodynamics and immunotherapy to enhance the stability and efficacy of camptothecin.
[0004] There are currently no reports of combining these two technologies; therefore, this invention is proposed. Summary of the Invention
[0005] To address the technical problems existing in the prior art, researchers conducted extensive and in-depth research, ultimately leading to this invention. One objective of this invention is to provide phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin. Another objective is to provide a method for preparing the aforementioned phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin. A further objective is to provide the application of the aforementioned phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin in the preparation of anticancer drugs. To achieve the above objectives of this invention, the following technical solutions are adopted: According to a first aspect, the present invention provides phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles, said nanoparticles being made of titanium dioxide, phthalocyanine, and a pharmaceutically active ingredient.
[0006] Preferably, the active pharmaceutical ingredient is camptothecin and its derivatives or other active pharmaceutical ingredients with anticancer properties, wherein the camptothecin is natural camptothecin.
[0007] Experimental studies have shown that camptothecin selectively inhibits topoisomerase I (TopoI) and binds to the TopoI-DNA complex, stabilizing this complex and preventing the rejoining of broken DNA strands, thus inhibiting DNA replication and RNA synthesis and exhibiting an inhibitory effect on tumors. Preferably, synthesized phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin remain relatively stable in physiological saline, while rapidly releasing camptothecin in a simulated cancer cell growth environment, demonstrating superior therapeutic efficacy compared to using camptothecin alone.
[0008] Preferably, the phthalocyanine is one or more of cobalt phthalocyanine and phthalocyanine.
[0009] According to a second aspect, the present invention provides a method for preparing the above-mentioned phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin, the method comprising the following steps: (1) Anhydrous ethanol, polyvinylpyrrolidone aqueous solution, dilute hydrochloric acid and TiF4 aqueous solution were mixed evenly in a reaction vessel, and after standing at room temperature for 1 h, the temperature was raised to 180℃ and heated for 3 h. After cooling to room temperature, the mixture was centrifuged, washed three times with anhydrous ethanol and dried to obtain titanium dioxide nanoparticles. (2) Cobalt phthalocyanine was dissolved in N,N-dimethylformamide (cobalt phthalocyanine concentration 2 mg / mL), and titanium dioxide nanoparticles were also dissolved in N,N-dimethylformamide (titanium dioxide content 1 mg / mL). The two were mixed evenly, stirred at 60°C in the dark for 2 h, cooled to room temperature, centrifuged at 8000 rpm / min for 10 min, the supernatant was removed, and the mixture was rinsed with deionized water to obtain phthalocyanine-modified titanium dioxide. (3) The phthalocyanine-modified titanium dioxide was dissolved in deionized water and mixed with a bis(stearoylphosphatidylethanolamine)-methoxy polyethylene glycol 2000 (DSPE-PEG 2000) ethanol solution (DSPE-PEG-2000 concentration 100 mg / mL). The mixture was stirred at room temperature in the dark for 8-12 h to obtain phthalocyanine-modified titanium dioxide polyethylene glycol nanoparticles (PC-TiO2 NPs-PEG). (4) Dissolve the active pharmaceutical ingredient in a dimethyl sulfoxide organic solution (concentration of active pharmaceutical ingredient 5 mg / mL), then add PC-TiO2 NPs-PEG to it and mix evenly. Stir and react at room temperature in the dark for 8-12 h to obtain phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin.
[0010] More preferably, the prepared phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin have a particle size of 180-250 nm, and more preferably, a particle size of 170-240 nm.
[0011] According to another aspect, the application of the phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles described in this invention in the preparation of anticancer drugs is not particularly limited, as the diseases treatable by the drug are consistent with the diseases treated by the active pharmaceutical ingredient loaded in the nanoparticles. Especially suitable are various types of cancer cells, particularly oral squamous cell carcinoma.
[0012] (iii) Beneficial technical effects 1) The phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin exhibit good biocompatibility. At the molecular level, we tested the biochemical indicators of mice in the nanoparticle treatment group and the blank control group. The results showed no significant differences in liver function, cardiac function, and renal function indicators. At the mouse level, tissue sections of the major organs (heart, liver, spleen, lung, and kidney) of mice in each experimental group were stained with H&E, and the results showed no significant changes, indicating that the treatment with phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin does not cause damage to the liver, heart, kidneys, etc., indicating that the nanoparticles have good biocompatibility.
[0013] 2) The phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin of the present invention ingeniously utilize ultrasound to rapidly release the encapsulated camptothecin drug under conditions simulating tumor growth environment, thereby achieving targeted delivery to cancer sites to a certain extent.
[0014] The phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin of this invention also have the following advantages: both cobalt phthalocyanine and camptothecin have the effect of killing tumor cells in treating tumors. The phthalocyanine-modified titanium dioxide is not only a carrier but also a drug. The combination of these two drugs achieves better therapeutic effects than using camptothecin or cobalt phthalocyanine alone. The model of phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin and its therapeutic effect on oral squamous cell carcinoma mice will provide a feasible preliminary research basis for the application of this strategy in the treatment of oral squamous cell carcinoma.
[0015] 3) This invention utilizes a sonodynamic therapy approach involving the delivery of sonosensitizers to prepare a sonosensitizer-based drug delivery system using phthalocyanine-modified titanium dioxide, thus retaining the drug delivery advantages of nanoparticles. The process is relatively mature, allowing for rapid preparation of phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin through relatively simple heating without the need for large-scale equipment.
[0016] The method for preparing phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin, as described in this invention, has the following advantages: no complex raw materials, no large-scale instruments or equipment required, mature synthesis technology, convenient and controllable concentration of camptothecin encapsulation, and minimal batch-to-batch biotoxicity and variability of the nanoparticles. More importantly, phthalocyanine-modified titanium dioxide, as a carrier material, achieves the encapsulation of the hydrophobic drug camptothecin and enables rapid release of camptothecin in tumor cells. Furthermore, phthalocyanine-modified titanium dioxide not only serves as a carrier but also functions as a tumor cell-killing agent, resulting in a synergistic effect. The nanoparticle preparation method is universal and enhances the bioavailability of camptothecin. Attached Figure Description
[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 This is a graph showing the diameter of phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin, prepared using a nanoparticle size and potentiometer as measured in Example 1 of this invention.
[0019] Figure 2 This is a graph showing the zeta potential changes of phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin in Example 1 of this invention.
[0020] Figure 3 This describes the uptake of nanomedicine by oral squamous cell carcinoma cells in Example 1 of this invention.
[0021] Figure 4 This describes the apoptosis of oral squamous cell carcinoma cells after drug treatment in Example 1 of this invention.
[0022] Figure 5 These are images of H&E staining, TUNEL staining, and Ki-67 immunohistochemical staining of tumor tissue in animal experiments in Example 1 of this invention.
[0023] Figure 6 In the animal experiment of Example 1 of this invention, the spleen and lymph nodes of mice in each group were analyzed by flow cytometry to determine the maturation of DC cells and the activation of cytotoxic T cells. Detailed Implementation
[0024] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments.
[0025] Unless otherwise expressly stated, numerical ranges throughout the application include any subranges therein and any numerical values incremented by the smallest subunit of a given value. Unless otherwise expressly stated, numerical values throughout the application represent approximate measures or limitations on the range of embodiments including minor deviations from a given value and having approximately the mentioned value as well as having the mentioned precise value. Except in the detailed description of the working embodiments provided at the end, all numerical values of parameters (e.g., quantities or conditions) in this application (including the appended claims) should in all cases be understood to be modified by the term “approximately,” regardless of whether “approximately” actually precedes the numerical value. “Approximately” indicates that the stated numerical value allows for slight inaccuracies (some close to precision at that value; approximately or reasonably close to the value; approximate). If the inaccuracy provided by “approximately” is not understood in this common sense in the art, then “approximately” as used herein at least indicates a variation that can be produced by common methods of measuring and using these parameters. For example, “approximately” can include variations less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%.
[0026] Titanium tetrafluoride was purchased from Sigma-Aldrich. Polyvinylpyrrolidone (Mw4000) was purchased from Aladdin Shanghai; Hydrochloric acid (analytical grade) was purchased from Tianjin Jiangtian Chemical Co., Ltd. Ethanol (analytical grade) was purchased from Tianjin Jiangtian Chemical Co., Ltd. Camptothecin was purchased from MCE; DSPE-PEG 2000 was purchased from MCE.
[0027] Raw material preparation: Weigh 24.7 mg of TiF4 using a balance to prepare 5.914 mL of a 40 mM TiF4 aqueous solution; Weigh 97.5 mg of polyvinyl pyrrolidone (Mw4000) (PVP) to prepare 10 mL of a 9.75 mg / mL PVP aqueous solution; Prepare 0.5 mL of 0.1 M dilute hydrochloric acid; Weigh 2 mg of cobalt phthalocyanine to prepare 1 mL of 2 mg / mL cobalt phthalocyanine N,N-dimethylformamide solution; Weigh 100 mg of DSPE-PEG-2000 to prepare 1 mL of 100 mg / mL anhydrous ethanol solution of DSPE-PEG-2000; Weigh 5 mg of camptothecin to prepare 1 mL of 5 mg / mL CPT dimethyl sulfoxide solution. All solutions are sonicated to ensure homogeneity.
[0028] Example 1 (1) Mix 2.5 mL of TiF4 aqueous solution, 4 mL of PVP aqueous solution, 250 μL of dilute hydrochloric acid and 27.6 mL of anhydrous ethanol and place them into a 100 mL reaction vessel. Mix evenly and let stand at room temperature for 1 h. Then heat to 180 °C for 3 h. After cooling to room temperature, centrifuge at 8000 rpm / min for 10 min. Wash three times with anhydrous ethanol and dry to obtain titanium dioxide nanoparticles. (2) Centrifuge the titanium dioxide nanoparticles, remove the supernatant, dissolve them with N,N-dimethylformamide until the titanium dioxide content is 1 mg / mL, add 86.3 μL of cobalt phthalocyanine N,N-dimethylformamide solution, mix them evenly, stir at 60℃ in the dark for 2 h, cool to room temperature, centrifuge at 8000 rpm / min for 10 min, remove the supernatant, rinse with deionized water to obtain phthalocyanine modified titanium dioxide; (3) Dissolve phthalocyanine-modified titanium dioxide in deionized water, control its concentration to 1 mg / mL, mix it with 120 μL LDSPE-PEG 2000 ethanol solution, stir and react at room temperature in the dark for 12 h to obtain PC-TiO2 NPs-PEG. (4) Add the above PC-TiO2 NPs-PEG to 600 μL camptothecin dimethyl sulfoxide solution, mix well, and stir at room temperature in the dark for 12 h to obtain phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin.
[0029] Experiment 1: 3 mL of phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin were diluted 1000 times in an aqueous solution and added to a plastic sample dish. The hydrated particle size and potential of the sample were measured using a nanoparticle size and potentiometer at 25 °C.
[0030] Depend on Figure 1 The results show that the particle size is between 170-240 nm; dynamic light scattering shows that the albumin-encapsulated capsaicin nanoparticles have an average particle size of 162.4 nm (n=3).
[0031] Depend on Figure 2 It can be seen that, compared with the potential of pure titanium dioxide of 14.6305 mV, the potential of titanium dioxide nanoparticles modified with cobalt phthalocyanine increases to 17.7918 mV.
[0032] Experiment 2: According to 1.0×10 5SCC7 cells were seeded in 24-well plates at a density of 1 cell / mL. 40 μL / mL of culture medium containing RhB-labeled phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin was added. After co-culturing for 10 h, the cells were sonicated for 6 minutes. Each well was washed three times with 500 μL PBS to remove particles that had not entered the cells. Hoechst 33342 dye was diluted 1:100 with DMEM medium, 500 μL per well. After incubation at 37°C in the dark for 15 min, the cells were washed three more times with PBS, and finally, 500 μL of DMEM medium was added. Cell uptake at 1, 3, 6, and 10 h after the addition of Rhdamine B-loaded nanoparticles was observed and photographed using a laser confocal microscope.
[0033] Depend on Figure 3 It can be seen that the red fluorescence of Rhodamine B in cells gradually increased within 10 hours, indicating that SCC7 cells took up more and more nanoparticles over time, proving that the drug can be taken up and has good performance.
[0034] Experiment 3: SCC7 cells were divided into groups of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 500 μL / well in 24-well plates and cultured for 24 hours before experiments. As shown in the figure, the experiment consisted of 6 groups: ctrl, ctrl+US, PC, PC+US, CPT, and CPT+US. After co-culturing the materials and cells for 10 hours, the sonication groups were sonicated for 6 minutes and then cultured for another 12 hours. Cells were collected, washed with PBS, and resuspended in Binding Buffer. 100 μL of cells from each group were added, and the mixture was incubated for 10 minutes at room temperature in the dark with 5 μL of Annexin V-FITC, followed by 5 μL of PI and incubation for 5 minutes at room temperature in the dark. Finally, 400 μL of PBS was added to each group to bring the volume to 500 μL, and the cells were analyzed using flow cytometry.
[0035] As shown in Figure 4, compared with the control group, the proportion of late apoptotic cells increased in the ctrl+US group, PC group, or PC+US group after treatment of SCC7 cells, but the changes were relatively small. The proportion of late apoptotic cells increased significantly in CPT and CPT+US, reaching 35.5% and 49.1%, respectively, which were significantly higher than the corresponding single treatment groups (P < 0.001). At the same time, the proportion of early apoptotic cells also increased significantly in the combined treatment group, indicating that the combination of ultrasound and nanomaterials can more effectively induce apoptosis in SCC7 cells.
[0036] Experiment 4: For example Figure 5As shown, BALB / c mice were randomly divided into six groups for treatment (same as the cell experiments above, n=3). After treatment, the mice were dislocated at the neck, and tumors were collected by dissection. H&E staining, TUNEL staining, and Ki-67 immunohistochemical staining were performed on the tumor tissues. The CPT+US group showed the most severe tumor necrosis, hemorrhage, and inflammatory cell infiltration, indicating a good anti-tumor effect. Then, H&E staining, TUNEL staining, and Ki-67 immunohistochemical staining were performed; the higher the index, the more severe the loss (where CPT is the abbreviation for phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin, and PC is the abbreviation for phthalocyanine-modified titanium dioxide nanoparticles).
[0037] Experiment 5: After treatment, tumors were collected from mice in each group. The tumors were cut into small pieces and ground into single-cell suspensions. These suspensions were then digested with a mixed enzyme solution at 37°C for half an hour and filtered through a 200-mesh filter. The suspensions were stained with fluorescently labeled antibodies (FITC anti-CD11c, PE anti-CD86, and APC anti-CD80) and analyzed by flow cytometry. For cytotoxic T lymphocytes, the treatment process was the same. Single-cell suspensions were stained with fluorescently labeled antibodies (FITC anti-CD3, PE anti-CD8a, Cy7 anti-CD45, and APC anti-CD4) and analyzed by flow cytometry. The CD86 and CD80 data in the figure show that the percentage of phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin increased from 10.90% to 14.10% after ultrasonic treatment, indicating that the treatment may have enhanced the activation state of dendritic cells. Furthermore, after treatment with phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin, combined with ultrasound therapy, the percentage of CD4+ T cells in the tumor area increased from 22.40% to 26.50%, and the CD8+ content increased from 24.00% to 26.10%. This indicates that ultrasound therapy with phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin can effectively increase the infiltration level of CD4+ and CD8+ T cells in the tumor area, thereby achieving anti-tumor immunity.
Claims
1. A phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticle, characterized in that, The nanoparticles are made of titanium dioxide, phthalocyanine, and pharmaceutical active ingredients.
2. The phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles according to claim 1, characterized in that, The active pharmaceutical ingredient is camptothecin or other active pharmaceutical ingredients with anticancer properties, and the camptothecin is natural camptothecin.
3. The phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles according to claim 1, characterized in that, The phthalocyanine is one or more of cobalt phthalocyanine and phthalocyanine.
4. A phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticle as described in any one of claims 1-3, characterized in that, The preparation method of the nanoparticles includes the following steps: (1) Anhydrous ethanol, polyvinylpyrrolidone aqueous solution, dilute hydrochloric acid and TiF4 aqueous solution were mixed evenly in a reaction vessel, and after standing at room temperature for 1 h, the temperature was raised to 180℃ and heated for 3 h. After cooling to room temperature, the mixture was centrifuged, washed three times with anhydrous ethanol and dried to obtain titanium dioxide nanoparticles. (2) Dissolve cobalt phthalocyanine in N,N-dimethylformamide and dissolve titanium dioxide nanoparticles in N,N-dimethylformamide. Mix the two evenly, stir at 60°C in the dark for 2 hours, cool to room temperature, centrifuge, and rinse with deionized water to obtain phthalocyanine-modified titanium dioxide. (3) The phthalocyanine-modified titanium dioxide was dissolved in deionized water and mixed with a bis(stearoylphosphatidylethanolamine)-methoxy polyethylene glycol 2000 (DSPE-PEG 2000) ethanol solution. The mixture was stirred at room temperature in the dark for 8-12 hours to obtain phthalocyanine-modified titanium dioxide polyethylene glycol nanoparticles (PC-TiO2 NPs-PEG). (4) Dissolve the active pharmaceutical ingredient in a dimethyl sulfoxide organic solution, then add PC-TiO2 NPs-PEG to it and mix evenly. Stir and react at room temperature in the dark for 8-12 hours to obtain phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin.
5. The phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles according to claim 1, characterized in that, The prepared phthalocyanine-modified titanium dioxide nanoparticles loaded with camptothecin had a particle size of 180-250 nm.
6. The application of phthalocyanine-modified titanium dioxide-supported camptothecin nanoparticles as described in any one of claims 1-5 in the preparation of anticancer drugs.