Dual effect of nano-polymer micelles mixed with doxorubicin and microRNA-34a on tumor cells
Foreword:
At present, with the development of new intelligent preparations, natural biomaterials are used to combine chemical drugs with RNA interference processes. It not only increases the sensitivity of biometrics, but also increases the activity of biopharmaceuticals. As a new type of drug carrier, polymer micelles can significantly increase the solubility of poorly soluble drugs, reduce toxic side effects, hydrophilic shell protects drugs from physiological environment damage; have active and passive targeting, change drugs The in vivo process improves the efficacy of the drug.
Matrix metalloproteinase 2 (MMP2) is a premise drug for doxorubicin, which can degrade various protein components in extracellular matrix, destroy the histological barrier of tumor cell invasion, and play a key role in tumor invasion and metastasis.
First, the drug is synthesized into a compound that can act on cells. This mixed nano-polymer micelle is mainly divided into three functional conjugated compounds. 1, doxorubicin (for metalloproteinase 2, MMP2) is covalently linked to the long chain of PEG2000, referred to as PEG2K-C LV-Dox/CLV-Dox. 2, miRNA-34a is disulfide-linked to the phospholipid molecule, abbreviated as miRNA-34a-SS-PE.3, osmotic polypeptide TATp (enhanced cell internalization, the drug is taken into the cell by endocytosis) The PEG1000 short chain binds and is linked to the phospholipid molecule, referred to as TAT-PEG1K-PE. Mixed polymer micelles can be delivered to the body in a variety of ways, including single conjugates, conjugates, and multiple conjugates.
Materials: CLV-Dox, TAT-PEG1K-PE/ CLV-Dox (TAT/CLV-Dox) micelles, TAT/CLV-Dox/miRNA-34a micelles, HT1080 (human fibroblasts)
The laser holographic imaging analysis system HoloMonitor M4 performs long-term tracking of cells, monitors the motility of cells in real time, and depicts the trajectories of cells. The laser holography technology can effectively distinguish between living cells and dead cells, and identify the G1, S, G2 and other periods in the cell cycle by using parameters such as the maximum optical thickness of the cells.
I. Cell migration research
CLV-Dox, TAT/CLV-Dox hybrid nano-polymer micelles were applied to HT1080 (human fiber tumor cells) for 12-hour time-lapse imaging using the Holomonitor M4 system, and 10 of them were tracked to observe the cells. Exercise and cell migration (Figure 1)
It can be clearly seen from the figure that the cell motility of the cells treated with CLV-Dox and TAT/CLV-Dox mixed nano-polymer micelles is significantly reduced, but the TAT/CLV-Dox mixed nano-polymer micelles are involved in cell migration. The treated HT1080 cells were significantly lower than the PBS control group and the CLV-Dox experimental group, and the images were processed by the Holomonitor M4 system. The correlation between the gray threshold and the cell volume and thickness was analyzed by 4D matrix analysis, indicating that the cells were treated with PBS. Continue to add value and split. However, the cell proliferation of CLV-Dox, TAT/CLV-Dox mixed nano-polymer micelles was stagnant.
Second, cell survival rate comparison
Compared with the survival rate of cells after different treatments, the survival rate of cells was significantly reduced by 40% under the dual action of TAT/CLV-Dox/ miRNA-34a, which was still lower than that treated by TAT/CLV-Dox alone. 15% showed that the tumor cell survival rate was effectively controlled at both RNA and cell levels under the dual action of doxorubicin and miRNA-34a.
Third, cell cycle research
Compared with the cell cycle distribution results in different drug treatments in Figure 3, the cells were arrested at the S phase when 0.5μM Dox and 10μM miRNA-34a were treated at a lower concentration, indicating that the drug replication was inhibited after the dual drug effect, and the drug was low. The amount of concentration used greatly reduces the toxic side effects after treatment.
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