In vivo tumor suppression assay in animals
In vivo tumor suppression assay in animals
In vivo tumor suppression in animals can be applied to study the in vivo antitumor effects of drugs.
Operation method
bioluminescence labeling method
Principle
Principle of in vivo bioluminescence imaging: In small mammals, the luciferase protein produced by the expression of the reporter gene (luciferase gene) is utilized with its small-molecule substrate luciferin to consume ATP under the conditions of oxygen and Mg2+ presence to undergo a redox reaction, which converts part of the chemical energy into visible light energy release. Therefore luminescence occurs only in living cells. And the intensity of light is linearly correlated with the number of labeled cells.
Materials and Instruments
Nude Mouse Move I. Material Preparation 1. Animals: 9 nude mice, 6-8 weeks old, males; 24 C57 mice, 6-8 weeks old, males; all animals were fed with free water. 2. Cells: MDA-MB-231 human breast cancer cell line, labeled with luciferase gene. Methods: Human breast cancer cells were transfected with plasmid labeled with luciferase reporter gene, and co-transfected with selective marker gene to ensure the stability of the transfected cells, and form tumor cell lines expressing luciferase; B16 melanoma cells. Experimental steps 1. Preparation of mixed micelles of paclitaxel (1) Dissolve appropriate amount of paclitaxel in a small amount of anhydrous ethanol and propylene glycol. (2) Add the appropriate proportion of phospholipids and surfactant A and mix thoroughly to produce a stable paclitaxel mixed micelles precursor, with a drug loading concentration of 6 g/L. (3) When ready for use, press the dose of paclitaxel into a small amount of solvent. (3) Add saline to dilute and shake well according to the dosage at the time of use. The particle size and distribution characteristics of the preparation system were detected by dynamic laser light scattering particle size meter. 2. Establishment of MDA-MB-231 human breast cancer cells as a mouse model of tumorigenicity (1) Cultivate luciferase gene-labeled MDA-MB-231 human breast cancer cell line using DMEM medium (with 10% fetal bovine serum added) at 37 ℃ in a 5% CO2 incubator, and passaging once every 3 days. (2) When the cell density was 80%～90% and the total amount of cells reached the required requirement for the experiment, trypsin digestion was used and the cells were collected and resuspended in PBS solution. (3) If necessary, after the cells have been cultured for a certain period of time, the antibiotic Zeocin should be used for resistance screening to ensure that the expression of luciferase gene is sufficient. (4) Collect the cells, dilute them to a cell number of 1. 5×108/mL using PBS, and inoculate them in the axilla of nude mice, with an inoculum volume of 0.1 mL per nude mouse . 3. Animal in vivo imaging assay (1) After inoculation, the nude mice were randomly divided into 3 groups, namely, saline group, paclitaxel injection group, and paclitaxel mixed micellar preparation group, with 3 mice in each group. (2) The drug was administered at a dose of 15 mg/kg starting on the 8th day after tumor inoculation, and was administered by intraperitoneal injection once every 3 days. Starting from the 1st administration, biopsy was performed every 7 days. (3) Nude mice were anesthetized with pentobarbital at a dose of 60-80 mg/kg, and were in a comatose state after 10-15 min. (4) Fluorescein substrate (150 mg/kg) was injected intraperitoneally at a wavelength of 540-600 nm, and the highest fluorescence intensity was observed 15-35 min after the injection. 4. 4. Parameter setting of in vivo imager (1) Put 2~3 mice side by side in the dark room, the luminescence produced by the reaction between luciferase and the substrate does not need to be excited, and can be self-luminous, and only need to adjust the exposure time of the CCD camera to take pictures. (2) The exposure time of 3 min was selected, and the images were processed using the software Kodak MI In Vivo Fx Pro to increase the pseudo-colors. (3) Observe the image of the tumor area and measure the tumor size in the figure based on the in vivo imaging pictures for statistical analysis. 5. During the whole experiment, the weight of the nude mice was monitored while the drug was administered, the tumor size was measured by vernier calipers, the tumor volume was calculated by applying the formula V = π-(6ab2)-1, the tumor growth curve was plotted, and the tumor growth curve was compared with the results of the in vivo imaging, so as to examine the tumor inhibitory effect of the paclitaxel preparation as well as the feasibility and accuracy of the evaluation of tumor inhibition by the method of in vivo imaging. The results were compared with the results of in vivo imaging. 6. Determination of survival cycle (1) On the basis of in vivo imaging experiments, study the effects of different paclitaxel preparations on the survival of B16 melanoma C57 mice. (2) B16 melanoma cells were first cultured, and the cell number was adjusted to 5×106/mL with PBS. 0.1 mL per C57 mouse was used for subcutaneous injection in the right hind limb, i.e., the cell number was 5×105. (3) After inoculation, the C57 mice were fed with normal diet at room temperature. On the 6th day after tumor inoculation, when the tumor diameter reached about 0.2 cm, the C57 mice were randomly divided into 3 groups, namely, the saline group, the Taxol group, and the PMM group, with 8 mice in each group. (4) The drug was administered by intraperitoneal injection at a dose of 15 mg/kg every 3 days. (5) Observe the mice every day, record the time of death and plot the survival cycle curve in days, and calculate the average survival cycle. 7. Statistical methods All statistical experimental data were expressed as x ± s. SPSS software was used to perform t-test on the compared samples. Common Problems I. Discussion Bioluminescence-based principle of in vivo animal tumor imaging technology method can accurately label and obtain real-time in vivo tumor images with clear boundaries, which can effectively solve the shortcomings of dye fluorescence labeling technology, such as poor specificity, strong interfering signals, and inconspicuous localized signals. For more product details, please visit Aladdin Scientific website.
DMEM medium Fetal bovine serum Trypsin Fluorescein substrate Paclitaxel injection
Kodak Multimodal In Vivo Imaging System ZetePALS Type Laser Dynamic Light Scattering Particle Sizer
As an emerging development of real-time, in vivo and non-invasive monitoring technology, its safety, real-time, accuracy and other aspects have greater advantages, and can provide a more scientific and accurate basis for animal experimental research and evaluation of drug efficacy.
Of course, there are still many shortcomings and difficulties in the application of in vivo animal imaging technology, such as this paper, due to the limitation of the configuration of the instruments used, a two-dimensional in vivo imaging system is used, if a three-dimensional in vivo imaging system is used to obtain the three-dimensional image of in vivo tumors, then more information on the tumor morphology and size can be obtained.
In addition, there are fewer cell lines with bioluminescence characteristics, and the cells are usually self-made according to the needs, and the preparation technology process is complicated and costly, so there are difficulties when different tumor models need to be established, which limits the scope of application of this method.
In this paper, a hybrid micellar system made of phospholipids and a novel surfactant can effectively solve the solubility problem of paclitaxel and avoid the toxicity problem of Cremophor EL.
From the results of in vivo animal imaging after drug administration, it can be seen that both Taxol and PMM preparations can effectively inhibit tumor growth, but there is no significant difference between the two; however, from the changes in body weight of the nude mice and their status after drug administration, it can be seen that the nude mice in the mixed micelles group have normal feeding and activities, and their body weights are relatively stable, while the nude mice in the Taxol group are in depressed status, have less food, and their body weights have decreased.
These results indicated that the PMM formulation improved the compliance of the nude mice to the drug to some extent compared with Taxol, and the adverse effects were reduced; further results of the survival cycle experiments also showed that Taxol and PMM formulations improved the survival time of the C57 ruffed mice, and the C57 ruffed mice that were given PMM at the same time had a longer average survival cycle.
The reason for analyzing the tumor suppression effect of the PMM preparation and the improvement of survival time may be that the presence of phospholipids in PMM causes micelles to spontaneously form drug-lipid nanoparticles, which alters the in vivo distribution characteristics of the drug and enables it to better exert its efficacy; at the same time, the novel surfactant used can reduce histamine release, thus reducing the toxicity of the preparation and improving the safety of the drug.