| Background:Breast cancer is, by far, the most frequently diagnosed cancer among women with an estimated1.38million new cancer cases in2008(23%of all cancers) and458,503deaths worldwide. In the treatment of breast cancer, doxorubicin is the preferred first-line drugs. It can be embedded in the DNA and inhibit the nuclear nucleic acid synthesis, producing a wide range of biochemical effects on the body, and also with strong cytotoxicity. Doxorubicin side effects are nausea, vomiting, gastrointestinal tract, decreasing in the number of peripheral white blood cells caused by bone marrow suppression, hair loss, and so many common side effects of chemotherapy. The most serious side effects of is cardiac toxicity, even can lead patients to death by heart failure. How to reduce the toxicity of doxorubicin and enhance the tolerance of the patients on the drug side effects, is one of the hot research areas. Aptamers are single-stranded oligonucleic acids or peptides that bind to specific targets with high affinity and specificity. Aptamers are derived from an iterative process called SELEX (Systematic Evolution of Ligands by Exponential enrichment). Compared with the antibody approach, aptamers have high affinity, excellent specificity and low toxicity. Moreover, they are stable and easy to synthesize, modify and manipulate. Aptamers’ target objects can be small organic molecules, metal ions, proteins, cells and even the organization.This experiment intended to detect the binding capacity of aptamers after generated from breast cancer cell-SELEX. Aptamer with excellent binding ability will be chose for following study. At first, aptamer can be specific internalized by target cells was studied, to confirm if they can used for drug carrier. And then by a simple chemical modification, making it for doxorubicin drug carriers, and determine whether it can be applied for the targeted therapy of breast cancer.Methods and Results:First, after sequencing, the binding ability of aptamers was tested by flow cytometry, using target cells and negative control cells. One aptamer, named KMF2-la, was confirmed that it have a good specific recognition of target cells, combined with stable and not affected by temperature and time. Further testing found that this aptamer could recognize the breast cancer cell lines (MCF-10AT1, MDA-MB-231), which not express estrogen receptor, progesterone receptor and HER-2. KMF2-1a is expected to become one aptamer that can specific recognize triple negative breast cancer.Secondly, flow cytometry and laser scanning confocal microscopy techniques were used for detecting if aptamer KMF2-1a could be specific internalized by target cells, MCF-10AT1. After KMF2-1a combined with cells, the fluorescence signal could be eliminated after cells were treated by trypsin. Take advantage of this phenomenon, the KMF2-1a and cell incubated at37℃, then treated by trypsin, after flow cytometry tested, there was still fluorescence signal which confirmed that KMF2-1a could be internalized by target cells. Confocal imaging further confirmed this conclusion, and KMF2-1a located in the endosome.Finally, the phenomenon of doxorubicin could be embedded in the principle of GC base complementary was used for target drug delivery. At one end of aptamer KMF2-1a, GC base complementary pair was added, used for a drug carrier. The impact of the targeted drugs on cell viability was tested by MTS assay, confirmed that this drug can reduce the killing of doxorubicin on normal cells, reduced the side effects of doxorubicin and achieve the purpose of targeting chemotherapy.Conclusion:The aptamer KMF2-1a has a good combination with the breast cancer cells, can be specifically internalized by target MCF-10AT1cells. It can be used for doxorubicin drug carriers, and reduce drug side effects to normal cells. |
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