CN112516311A - Experimental method for applying targeted graphene oxide compound to treatment of drug-resistant osteosarcoma - Google Patents
Experimental method for applying targeted graphene oxide compound to treatment of drug-resistant osteosarcoma Download PDFInfo
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Abstract
The invention discloses an experimental method for treating drug-resistant osteosarcoma by using a targeted graphene oxide compound, which comprises a basic integrated board, an input mechanism and an output mechanism, wherein a handle is fixedly connected to the basic integrated board, the input mechanism and the output mechanism are installed on the basic integrated board through a bracket, a tension pulley is installed between the input mechanism and the output mechanism, and a synchronous belt and the tension pulley synchronously rotate through a toothed belt. High efficiency, light weight, portability, durability and high reliability.
Description
Technical Field
The invention relates to the technical field of drug-resistant osteosarcoma treatment, in particular to an experimental method for treating drug-resistant osteosarcoma by using a targeted graphene oxide compound.
Background
Osteosarcoma (OS) is the most common aggressive primary malignant bone tumor, occurring mostly in children and young adults. Currently, the gold standard for treating osteosarcoma is: first with combination chemotherapy, followed by radical resection of the tumor (metastases may also be resected if the situation permits), and then again with combination chemotherapy. The 5-year survival rate for patients with non-metastatic osteosarcoma is 70%, whereas the 5-year survival rate for patients with metastatic or recurrent osteosarcoma is only about 20%. However, the effective proportion of this combination therapy strategy in osteosarcoma patients is still not more than 60%. Despite different treatment strategies, the prognosis of osteosarcoma has not improved significantly over the last decades. The main reason for the limited therapeutic effect at present is due to resistance to chemotherapeutic drugs. Causes of tumor resistance include: tumor cells overexpress an ATP-binding cassette transporter, such as P-glycoprotein (P-gP), which is an Adenosine Triphosphate (ATP) -dependent unidirectional efflux pump on the cell membrane and can expel anticancer drugs out of the cell; cell clones that develop the capacity to tolerate the corresponding chemotherapeutic drug appear after a long-term chemotherapeutic drug selection. Therefore, there is an urgent need to develop new therapies against new therapeutic targets to reverse tumor resistance.
In recent years, mitochondria have received much attention as an effective target for tumor therapy. Mitochondria, an indispensable subcellular organelle, is an energy factory of cells, hundreds to thousands of each, and plays a crucial role in various physiological processes of cells, including initiation of endogenous apoptotic pathways. Mitochondrial function changes are important markers for tumorigenesis, development, angiogenesis, and resistance to chemotherapeutic drugs. By inducing mitochondrial apoptosis, cytochrome c can be released, thereby activating caspase cascades. Therefore, induction of apoptosis of tumor cells by mitochondrial targeting has great potential in treating malignant tumors.
Synergistic phototherapy, represented by photodynamic therapy (PDT) combined with photothermal therapy (PTT), can be used to treat drug-resistant tumors, and the therapeutic effect is superior to chemotherapy due to the difference in the mechanism of action. Synergistic phototherapy is a non-invasive, highly selective, low systemic toxicity method for treating tumors. In phototherapy, light is absorbed by Photosensitizers (PS) or photothermal agents and converted to Reactive Oxygen Species (ROS) or local hyperthermia, which in turn leads to tumor cell death. By utilizing the photoinduced property, the tumor treatment can be synergistically promoted by reasonably combining PDT and PTT. However, if the excitation spectra of PDT and PTT do not match, two consecutive exposures may be required during the irradiation process, thus increasing the time and complexity of the treatment. In addition, some inherent drawbacks, such as the short lifetime of ROS (10-320 ns) and the relatively limited diffusion radius (10-55 nm), can reduce the therapeutic efficacy of PDT. Therefore, induction of ROS production in certain ROS-sensitive organelles using single-dose irradiation may be a promising therapeutic strategy.
Reports indicate that mitochondria are most sensitive to ROS and high temperature mediated damage in all organelles, and can rapidly perturb mitochondrial function, alter mitochondrial membrane potential, reduce ATP production, and ultimately induce apoptosis in tumor cells. Therefore, selective mitochondrial generation of ROS and hyperthermia by phototherapy can significantly enhance their therapeutic effects. In addition, hyperthermia combined with ROS-mediated mitochondrial dysfunction, while inhibiting ATP production, should be an effective strategy to overcome tumor resistance. Therefore, mitochondrially targeted coordinated phototherapy may serve as an alternative strategy for drug-resistant osteosarcoma therapy.
Therefore, the photosensitizer and the photothermal agent are selectively and efficiently promoted to be gathered on mitochondria, the light beam is accurately focused on a tumor area, and the single near-infrared light is simultaneously excited to fully convert the light energy into heat energy and generate ROS. Therefore, during phototherapy, the tumor margins need to be delineated in real time to distinguish them from normal tissues, so that the light beams can be focused accurately on the tumor region. Studies have shown that Fluorescence (FL) imaging in the Near Infrared (NIR) region in the 700 nm-900 nm wavelength range can be used to monitor the dynamic distribution of photosensitizers and photothermal agents in vivo in real time, as well as to detect tumors and their margins. Furthermore, in order to improve the efficiency of treatment, reduce the number of procedures and the time required for treatment, and take full advantage of the synergistic effects of PDT and PTT, photosensitizers and photothermal agents must be delivered simultaneously to the mitochondria in the form of a complex structure and excited using a single dose of NIR. Thus, synergistic phototherapy relies on the overlapping absorption spectra of the photosensitizer and the photothermal agent. However, this special requirement and the complex synthesis process may limit its further applications. Therefore, there is a great need to develop a simple, efficient nanocomposite for the co-operative PDT and PTT.
Indocyanine green (ICG) is a near-infrared contrast agent approved by FDA in the united states, and not only can be used for near-infrared fluorescence imaging, but also can convert light energy into heat energy and generate ROS to realize synergistic phototherapy. Therefore, ICG is considered to be one of the ideal drugs for tumor therapy. However, the use of ICG in tumor therapy has several disadvantages: i) the half-life period in vivo is short; ii) poor cellular uptake due to its strong hydrophilicity; iii) is possibly discharged unidirectionally by P-gP; iv) lack of tumor specific aggregation; v) the efficacy of tumor phototherapy is low, and other therapeutic strategies need to be combined to improve the therapeutic outcome.
Disclosure of Invention
The invention aims to provide an experimental method for treating drug-resistant osteosarcoma by using a targeted graphene oxide compound, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
an experimental method for treating drug-resistant osteosarcoma by using a targeted graphene oxide compound and a mitochondrion targeted graphene oxide nano compound are used for synergistic phototherapy treatment of drug-resistant osteosarcoma under the guidance of fluorescence imaging, and the experimental method comprises the following steps: (1) performing xenografting on experimental cells, animals and tumors, implanting MG63/Dox cells under the back skin of a mouse, then performing living body imaging and phototherapy on the tumor-bearing mouse, (2) preparing PPG, TPP-PPG and TPP-PPG @ ICG, (3) characterizing the optical characteristics of the TPP-PPG, ICG and TPP-PPG @ ICG by adopting an ultraviolet visible near infrared spectrophotometer, (4) detecting singlet oxygen, detecting the generation of singlet oxygen by using a singlet oxygen green fluorescent probe, (5) performing a photothermal conversion experiment, irradiating by using near infrared light, recording the temperature change by using an infrared thermometer, (6) performing cell uptake and intracellular localization, incubating MG63/Dox cells, washing by PBS, then resuspending in a culture medium, detecting the average fluorescence intensity of a sample by using a flow cytometer, co-culturing MG63/Dox cells and TPP-PPG @ ICG, adding a mitochondrial fluorescent probe into PBS for incubation after washing cells, marking the mitochondria, using a laser scanning confocal microscope to scan and obtain images after washing the cells again by the PBS, (7) carrying out in vitro experiments, culturing MG63/Dox cell suspension, adding ICG, TPP-PPG and PPG @ ICG solutions with different concentrations, washing a non-irradiation group of PBS, culturing again after irradiating the irradiation group by NIR with 808nm, detecting cell viability and carrying out living cell/dead cell double staining simultaneously, adding MG63/Dox cells into PBS, TPP-PPG @ ICG, TPP-PPG, PPG @ ICG and TPP-PPG @ ICG, culturing, adding a double staining kit, observing by using near infrared fluorescence spectroscopy, (8) detecting cell apoptosis and intracellular ROS, culturing the NIR cells by MG63/Dox cells, centrifuging at a low speed to collect the cells after NIR irradiation, suspending and staining again in a buffer solution in a dark place, collecting cells by low-speed centrifugation, washing with PBS, diluting with a binding buffer solution, carrying out flow cytometry analysis, detecting the generation of ROS in the cells by a DCFH-DA kit, (9) detecting mitochondrial membrane potential, recording the change of the mitochondrial membrane potential by JC-1, collecting images by a laser confocal microscope, (10) detecting mitochondrial superoxide, detecting the generation of mitochondrial superoxide by a mitochondrial superoxide fluorescence probe, (11) detecting ATP, drawing an ATP concentration standard curve by the ATP detection kit, (12) imaging near-infrared fluorescence and thermal, injecting TPP-PPG @ ICG into the tail vein of MG63/Dox tumor-bearing mice, carrying out in-vitro near-infrared imaging by a near-infrared imaging system, carrying out real-time imaging on the temperature change of the tumor tissues of the mice by an infrared thermal imaging camera when NRI is used for irradiation, (13) carrying out in-vivo PDT/PTT combined treatment, tumor-bearing mice were randomly divided into 6 groups, PBS non-irradiated group (control group), TPP-PPG @ ICG non-irradiated group, ICG irradiated group, TPP-PPG irradiated group, PPG @ ICG irradiated group, and TPP-PPG @ ICG irradiated group, ICG was intravenously injected, irradiated group was irradiated at tumor site with 808nmNIR for 15 days, tumors and major organs were collected, and obtained viscera were stained with hematoxylin-eosin and observed under microscope.
As a further scheme of the invention: in the step (1), MG63/Dox cells are mixed in 100 mu L of matrix gel for transplantation, and when the tumor volume reaches 60mm3, living body imaging and phototherapy are performed.
As a further scheme of the invention: and (5) adopting near infrared light of 808nm as the near infrared light.
As a further scheme of the invention: in step (8), MG63/Dox cells were seeded overnight in 6-well plates, and then co-cultured for 24h with the addition of equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+).
As a further scheme of the invention: in the step (8), the DCFH-DA kit detects the generation of intracellular ROS, MG63/Dox cells are inoculated in a 24-well plate overnight, and equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) are added for co-culture for 24 h.
As a further scheme of the invention: in step (9), MG63/Dox cells were co-cultured with equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) for 24h, incubated for 24h after NIR irradiation, and the unirradiated group was incubated for 48h under the same conditions, followed by washing of the cells with PBS, co-culturing with 5mM JC-1 at 37 ℃ for 30min, washing of the cells with PBS and visualization with CLSM.
As a further scheme of the invention: in step (10), MG63/Dox cells were seeded overnight on a 35mm glass plate, and then an equivalent amount of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) were added for co-culture for 24h, after which the cells were washed twice with PBS, incubated with 5. mu.M mitochondrial superoxide fluorescence probe for 10min, washed twice with PBS and pictures were taken with CLSM.
As a further scheme of the invention: in the step (11), MG63/Dox cells are inoculated in a 96-well plate, 100 mu L DMEM is added to be cultured for 24 hours, then PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) with equivalent doses are added to be cultured for 24 hours together, the culture medium is removed and cell lysate is added, then an ATP detection kit is added, the fluorescence value is detected, the ATP concentration is calculated, each experiment is repeated three times, and the average value is calculated.
As a further scheme of the invention: the changes in tumor volume and mouse body weight in step (13) were monitored every 3 days for 15 days to evaluate the effect of the treatment.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, mitochondrion targeted TPP and ICG are combined with PEG and BPEI modified NGO, and are used for PDT/PTT cooperative therapy, the TPP-PPG @ ICG is gathered on tumor tissues due to EPR effect of the material, and the near-infrared imaging characteristic of the ICG can be used for near-infrared imaging, so that light beams are guided to focus on the tumor tissues, and the side effect of phototherapy can be reduced to the maximum extent; NGO is modified by PEG and BPEI, ICG dispersion and cell uptake are promoted, and mitochondrion targeting TPP enhances the PDT/PTT synergistic treatment effect of tumors; under the irradiation of single near infrared light, TPP-PPG @ ICG can rapidly disturb mitochondrial function, change mitochondrial membrane potential, reduce ATP generation and finally induce tumor cell apoptosis, and the strategy can effectively inhibit drug-resistant bone tumors.
Drawings
FIG. 1 is a schematic representation of TPP-PPG @ ICG targeting mitochondria for synergistic phototherapy.
FIG. 2 shows the TPP-PPG @ ICG characterization and biochemical features.
FIG. 3 shows cellular uptake and subcellular localization.
FIG. 4 is an in vitro synergistic phototherapy of TPP-PPG @ ICG on MG63/Dox cells.
FIG. 5 is a diagram showing the cellular mechanism of the synergistic phototherapy effect of TPP-PPG @ ICG.
FIG. 6 is a graph of the effect of TPP-PPG @ ICG in conjunction with phototherapy on mitochondrial function.
FIG. 7 is a measurement of the relative concentration of ATP in NIR-irradiated or non-irradiated cells.
FIG. 8 is TPP-PPG @ ICG in vivo co-phototherapy treatment of MG63/Dox tumor-bearing mice.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, in the embodiment of the present invention, an experimental method for treating drug-resistant osteosarcoma by using a targeted graphene oxide composite, and a mitochondrial targeted graphene oxide nanocomposite for synergistic phototherapy treatment of drug-resistant osteosarcoma under guidance of fluorescence imaging include the following steps: (1) experimental cells, animals and tumor xenografts. Human osteosarcoma cell line MG63 and doxorubicin-resistant osteosarcoma cell line MG63/Dox were cultured in RPMI-1640 medium (37 ℃ C., 5% CO2) containing 10% FBS and 1% penicillin/streptomycin double antibody. Nude mice (6 weeks old, 18 g-22 g in weight) were bred under SPF environment, and MG63/Dox cells (about 1X 107) were mixed in 100. mu.L of the matrix gel and implanted subcutaneously in the back of the mice. When the tumor volume reaches 60mm3In vivo imaging and phototherapy were performed with tumor-bearing mice.
(2) PPG, TPP-PPG and TPP-PPG @ ICG, and NGO is prepared according to an improved Hummer method, and the preparation method comprises the steps of firstly carrying out graphite sheet oxidation, then carrying out ultrasonic treatment, and then preparing the PG and the PPG. Grafting of TPP and PPG: 5mg of TPP was dissolved in 4ml of water and activated with EDC & HCl (15mg) and NHS (15mg) at room temperature for 15 min. Subsequently, 4mL of PPG (1.0mg/mL) solution was added to the reaction mixture and magnetically stirred at room temperature for 24 h. Finally, excess TPP was removed by filtration through a 10kDa dialysis ultrafiltration tube and washed repeatedly with double distilled water to obtain PPG-TPP (equivalent NGO-PEG concentration 0.5 mg/mL). Synthesis of TPP-PPG @ ICG: ICG (7.74mg) was dissolved in 1ml of anhydrous dimethyl sulfoxide as a stock solution (10mM) for further use. 200 μ l of ICG (10mM) and 1.8mL of TPP-PPG (0.5mg/mL) were mixed and stirred at room temperature for 24 hours. Then, the whole system was dialyzed in distilled water for 24 hours (molecular weight cut-off: 10 kDa). The final product (TPP-PPG @ ICG) was freeze-dried and stored below 4 ℃ until use. The concentration of ICG in TPP-PPG was determined from the standard curve and the peak absorbance at 781 nm.
Referring to fig. 2, an experimental method for treating drug-resistant osteosarcoma by using the targeted graphene oxide compound, (3) and characterization. And (3) acquiring a TPP-PPG @ ICG infrared spectrum, and confirming that the ICG is successfully loaded on the nano graphene oxide. And (3) detecting the size and the thickness of the TPP-PPG @ ICG nano material by using an atomic force microscope. TPP-PPG, ICG and TPP-PPG @ ICG optical characteristics are characterized by an ultraviolet visible near infrared spectrophotometer. The nanomaterial stability was tested as follows: TPP-PPG or TPP-PPG @ ICG was incubated with PBS, cell culture medium (RPMI-1640 medium) or serum, and the mixture was examined for the presence of precipitate. The release of the ICG from TPP-PPG @ ICG was studied by adding it to acidic (pH 5.0) and weakly basic (pH 7.4) Phosphate Buffered Saline (PBS) at 37 ℃ and 43 ℃, respectively, followed by centrifugation after a certain time, collecting PBS and measuring the amount of ICG released from TPP-PPG using uv-vis spectroscopy. The same volume of PBS was then added and the release assay was continued.
(4) And detecting singlet oxygen. The generation of singlet oxygen (1O2) was detected with a singlet oxygen green fluorescent probe (SOSG). SOSG was dissolved in a 2% aqueous methanol solution to a final concentration of 1mM, and a solution of ICG, TPP-PPG, and TPP-PPG @ ICG (ICG concentration 10 μ M) was added and mixed therewith. Then, the mixed solution was immediately irradiated with near infrared light at 808nm for 5min (energy density: 0.8w/cm 2). Then excited by 494nm light source, and 530nm SOSG emission peak is detected, thereby calculating the generation of singlet oxygen.
(5) And photothermal conversion experiments. 1ml of PBS, ICG, TPP-PPG and TPP-PPG @ ICG solutions were placed in a quartz cuvette and irradiated with 808nm near-infrared light (energy density: 0.8w/cm2) for 10min at intervals of 2.5 min. The temperature change was recorded using an infrared thermometer.
Referring to fig. 3, experimental methods for targeting graphene oxide complexes for the treatment of drug-resistant osteosarcoma, (6), cellular uptake and intracellular localization. MG63/Dox cells were seeded at a density of 1 × 105 cells/well in 6-well plates. After overnight incubation, 10. mu.M TPP-PPG @ ICG was added. After incubation for 1h, 4h, 8h, 12h and 24h, the cells were washed three times with PBS before trypsinization and resuspended in culture medium. Flow cytometry measures the mean fluorescence intensity of each sample. Subcellular localization: MG63/Dox cells were co-cultured with TPP-PPG @ ICG (ICG concentration 10. mu.M) in 35mm dishes for 24 h. The cells were washed three times with PBS, incubated for 10min with mitochondrial fluorescence probe, and mitochondria were labeled. The cells were washed three more times with PBS and scanned by laser scanning confocal microscope to obtain images.
Referring to fig. 4, an experimental method of targeting graphene oxide compound for treating drug-resistant osteosarcoma, (7), in vitro experiment. 100 μ L of MG63/Dox cell suspension (5X 103 cells/well) was seeded into 96-well plates and cultured for 24h, followed by the addition of different concentrations of ICG, TPP-PPG and PPG @ ICG solutions. The non-irradiated group was washed with PBS and then cultured for 24 hours again. The irradiated group was irradiated with NIR (energy density: 0.8w/cm2) at 808nm for 5min and then cultured again for 24 h. The CCK-8 method is used for detecting the cell viability. Meanwhile, live/dead cell double staining was performed for evaluation of TPP-PPG @ ICG synergistic phototherapy effect. MG63/Dox cells (1X 105 cells/well) were seeded in 6-well plates and cultured overnight. Equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+), and TPP-PPG @ ICG (+) (NGO 20. mu.g/mL; ICG 15. mu.M) were then added to co-incubate for 24h (-: no light, +: 0.8W/cm2NIR irradiation 5 min). And culturing for 24h, adding a Calcein AM/PI double-staining kit, incubating for 20min at 37 ℃, and observing by using near-infrared fluorescence spectrum.
Referring to fig. 5, experimental methods for targeting graphene oxide complexes for treatment of drug-resistant osteosarcoma, (8), apoptosis and intracellular ROS detection. MG63/Dox cells were seeded overnight in 6-well plates (2X 105 cells/well) and then co-cultured for 24h (—: no light irradiation, +: 0.8W/cm2NIR irradiation for 5min) with the addition of equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) (NGO 20. mu.g/mL; ICG 15. mu.M). After 6h NIR irradiation, cells were trypsinized, harvested by low speed centrifugation, and then washed twice with PBS. The collected cells were resuspended in 100. mu.L buffer and stained with 2. mu.L annexin V-FITC and 2. mu.L PI for 15min in the dark. After staining, cells were collected by low speed centrifugation, washed twice with PBS, and diluted with 400 μ L of binding buffer for flow cytometry analysis. DCFH-DA kit detected the production of intracellular ROS. MG63/Dox cells were seeded overnight in 24-well plates (1X 105 cells/well) and incubated for 24h with the addition of equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) (NGO 20. mu.g/mL; ICG 15. mu.M) (-) for 24h (-no light irradiation, +: 0.8W/cm2NIR irradiation for 5 min). Immediately after irradiation, cells were washed with PBS, incubated at 37 ℃ for 30min, and the ROS fluorescence signal was observed using a DMLRB inverted microscope.
Referring to fig. 6, an experimental method for treating drug-resistant osteosarcoma using the targeted graphene oxide compound, (9), mitochondrial membrane potential detection. Changes in mitochondrial membrane potential were recorded using JC-1 and images were taken using a laser confocal microscope (CLSM). MG63/Dox cells (1X 105 cells/well) were co-cultured with equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+), and TPP-PPG @ ICG (+) (NGO 20. mu.g/mL; ICG 15. mu.M) for 24h (-no light irradiation, +: 0.8W/cm2NIR irradiation for 5min) for 24 h. The NIR irradiation was followed by incubation for a further 24h and the unirradiated group was incubated under the same conditions for 48 h. Then, the cells were immediately washed with PBS and co-incubated at 37 ℃ for 30 minutes with the addition of 5mM JC-1. The cells were washed again with PBS and observed using CLSM.
(10) And detecting mitochondrial superoxide. Mitochondrial superoxide generation was detected using a mitochondrial superoxide fluorescence probe. MG63/Dox cells were seeded overnight on 35mm glass plates and then co-cultured for 24h (-no light, +: 0.8W/cm2NIR irradiation for 5min) with the addition of equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) (NGO 20. mu.g/mL; ICG 15. mu.M). After treatment, cells were washed twice with PBS, incubated for 10min with 5 μ M mitochondrial superoxide fluorescence probe, washed twice with PBS, and pictures were taken with CLSM observations.
Referring to fig. 7, an experimental method for treating drug-resistant osteosarcoma using targeted graphene oxide complex, (11), ATP detection. ATP concentration standard curves were drawn using the ATP detection kit. MG63/Dox cells were seeded in 96-well plates (1X 104 cells/well) and cultured in 100. mu.L DMEM for 24 hours. Equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) (NGO 20. mu.g/mL; ICG 15. mu.M) were then added and co-incubated for 24h (-: no light; 0.8W/cm2NIR irradiation 5 min). After 6 hours, the medium was removed and cell lysate was added. Then, an ATP detection kit is added to detect the fluorescence value, and the ATP concentration is calculated. Each experiment was repeated three times and the mean was calculated.
Referring to fig. 8, experimental methods for the use of targeted graphene oxide complexes for the treatment of drug-resistant osteosarcoma, (12), near-infrared fluorescence and thermal imaging. MG63/Dox tumor-bearing nude mice are used as experimental animals to evaluate the tumor targeting of the material, and TPP-PPG @ ICG is injected through tail vein according to the dose of 0.5 MG/kg. In vitro near infrared imaging was performed using a near infrared imaging system (Kodak). All settings and imaging conditions were performed as reported in the literature. 24h after injection of TPP-PPG @ ICG, mice were sacrificed and their internal organs were harvested. To assess in vivo PTT characteristics, mice were imaged in real time for tumor tissue temperature changes using an infrared thermographic camera while irradiated with NRI.
(13) And in vivo PDT/PTT combination therapy. When the tumor volume reached 60mm3, tumor-bearing mice were randomized into 6 groups of 5: PBS non-irradiated group (control group), TPP-PPG @ ICG non-irradiated group, ICG irradiated group, TPP-PPG irradiated group, PPG @ ICG irradiated group, and TPP-PPG @ ICG irradiated group. An equivalent dose of drug (ICG 750 μ M) was injected intravenously. The irradiation group was irradiated with 808nm (0.8w/cm2, 5min) NIR at the tumor site. Changes in tumor volume and mouse body weight were monitored every 3 days for 15 days to assess treatment efficacy. On day 15, animals were euthanized. Tumors and major organs (heart, liver, spleen, lung and kidney) were collected. The obtained viscera were stained with hematoxylin-eosin and observed under a microscope.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes in the embodiments and/or modifications of the invention can be made, and equivalents and modifications of some features of the invention can be made without departing from the spirit and scope of the invention.
Claims (9)
1. An experimental method for treating drug-resistant osteosarcoma by using a targeted graphene oxide compound, and a mitochondrion targeted graphene oxide nano compound for synergistic phototherapy treatment of drug-resistant osteosarcoma under guidance of fluorescence imaging, is characterized by comprising the following steps: (1) performing xenografting on experimental cells, animals and tumors, implanting MG63/Dox cells under the back skin of a mouse, then performing living body imaging and phototherapy on the tumor-bearing mouse, (2) preparing PPG, TPP-PPG and TPP-PPG @ ICG, (3) characterizing the optical characteristics of the TPP-PPG, ICG and TPP-PPG @ ICG by adopting an ultraviolet visible near infrared spectrophotometer, (4) detecting singlet oxygen, detecting the generation of singlet oxygen by using a singlet oxygen green fluorescent probe, (5) performing a photothermal conversion experiment, irradiating by using near infrared light, recording the temperature change by using an infrared thermometer, (6) performing cell uptake and intracellular localization, incubating MG63/Dox cells, washing by PBS, then resuspending in a culture medium, detecting the average fluorescence intensity of a sample by using a flow cytometer, co-culturing MG63/Dox cells and TPP-PPG @ ICG, adding a mitochondrial fluorescent probe into PBS for incubation after washing cells, marking the mitochondria, using a laser scanning confocal microscope to scan and obtain images after washing the cells again by the PBS, (7) carrying out in vitro experiments, culturing MG63/Dox cell suspension, adding ICG, TPP-PPG and PPG @ ICG solutions with different concentrations, washing a non-irradiation group of PBS, culturing again after irradiating the irradiation group by NIR with 808nm, detecting cell viability and carrying out living cell/dead cell double staining simultaneously, adding MG63/Dox cells into PBS, TPP-PPG @ ICG, TPP-PPG, PPG @ ICG and TPP-PPG @ ICG, culturing, adding a double staining kit, observing by using near infrared fluorescence spectroscopy, (8) detecting cell apoptosis and intracellular ROS, culturing the NIR cells by MG63/Dox cells, centrifuging at a low speed to collect the cells after NIR irradiation, suspending and staining again in a buffer solution in a dark place, collecting cells by low-speed centrifugation, washing with PBS, diluting with a binding buffer solution, carrying out flow cytometry analysis, detecting the generation of ROS in the cells by a DCFH-DA kit, (9) detecting mitochondrial membrane potential, recording the change of the mitochondrial membrane potential by JC-1, collecting images by a laser confocal microscope, (10) detecting mitochondrial superoxide, detecting the generation of mitochondrial superoxide by a mitochondrial superoxide fluorescence probe, (11) detecting ATP, drawing an ATP concentration standard curve by the ATP detection kit, (12) imaging near-infrared fluorescence and thermal, injecting TPP-PPG @ ICG into the tail vein of MG63/Dox tumor-bearing mice, carrying out in-vitro near-infrared imaging by a near-infrared imaging system, carrying out real-time imaging on the temperature change of the tumor tissues of the mice by an infrared thermal imaging camera when NRI is used for irradiation, (13) carrying out in-vivo PDT/PTT combined treatment, tumor-bearing mice were randomly divided into 6 groups, PBS non-irradiated group (control group), TPP-PPG @ ICG non-irradiated group, ICG irradiated group, TPP-PPG irradiated group, PPG @ ICG irradiated group, and TPP-PPG @ ICG irradiated group, ICG was intravenously injected, irradiated group was irradiated at tumor site with 808nmNIR for 15 days, tumors and major organs were collected, and obtained viscera were stained with hematoxylin-eosin and observed under microscope.
2. The experimental method for targeted graphene oxide compound for treating drug-resistant osteosarcoma according to claim 1, wherein in the step (1), MG63/Dox cells are mixed in 100 μ L of matrigel for transplantation, and the tumor volume reaches 60mm3In vivo imaging and phototherapy were performed.
3. The experimental method for treating drug-resistant osteosarcoma by using the targeted graphene oxide compound according to claim 1, wherein the near-infrared light in the step (5) is 808nm near-infrared light.
4. The experimental method for treating drug-resistant osteosarcoma by using the targeted graphene oxide complex according to claim 3, wherein in the step (8), MG63/Dox cells are inoculated in a 6-well plate overnight, and then equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) are added for co-culture for 24 h.
5. The experimental method for the targeted graphene oxide complex for treating drug-resistant osteosarcoma according to claim 3, wherein the DCFH-DA kit of step (8) detects the generation of intracellular ROS, MG63/Dox cells are inoculated into a 24-well plate overnight, and equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) are added for co-culture for 24 h.
6. The experimental method for treating drug-resistant osteosarcoma by using the targeted graphene oxide complex according to claim 5, wherein in step (9), MG63/Dox cells are co-cultured with PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) at equivalent doses for 24h, and then incubated for 24h after NIR irradiation, the unirradiated group is incubated for 48h under the same conditions, and then the cells are washed by PBS, and are co-cultured with 5mM JC-1 at 37 ℃ for 30min, and then washed by PBS and observed by CLSM.
7. The experimental method for targeted graphene oxide complex for treating drug-resistant osteosarcoma according to claim 1, wherein in step (10), MG63/Dox cells are inoculated in a 35mm glass culture dish overnight, and then equivalent doses of PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) are added for co-culture for 24h, after treatment, the cells are washed twice with PBS, incubated with 5 μ M mitochondrial superoxide fluorescence probe for 10min, washed twice with PBS, and the pictures are observed with CLSM.
8. The experimental method for treating drug-resistant osteosarcoma by using the targeted graphene oxide complex according to claim 7, wherein in the step (11), MG63/Dox cells are inoculated in a 96-well plate, 100 μ L of DMEM is added for culturing for 24 hours, then PBS (-), TPP-PPG @ ICG (-), ICG (+), TPP-PPG (+), PPG @ ICG (+) and TPP-PPG @ ICG (+) are added for co-culturing for 24 hours, the culture medium is removed, cell lysate is added, an ATP detection kit is added, the fluorescence value is detected, the ATP concentration is calculated, each experiment is repeated three times, and the average value is calculated.
9. The experimental method for the targeted graphene oxide complex for the treatment of drug-resistant osteosarcoma according to claim 7, wherein the change of tumor volume and mouse body weight is monitored every 3 days in the step (13) for 15 days to evaluate the treatment effect.
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