Triple-negative breast cancer is an aggressive subtype of breast cancer that is primarily treated using systemic chemotherapy due to the lack of a specific cell surface marker for drug delivery. Cancer cell-specific aptamer-mediated drug delivery is a promising targeted chemotherapy for marker-unknown cancers. Using a poorly differentiated carcinoma cell-specific DNA aptamer (PDGC21T), we formed a self-assembling circinate DNA nanoparticle (Apt^21TNP) that binds triple-negative breast cancer cells. Using our previously designed pH-sensitive dendrimer-conjugated doxorubicin (DDOX) as the payload, we found that each nanoparticle loaded 30 doxorubicin molecules to form an Apt^21TNP-DDOX nanomedicine that is stable in human plasma. Upon cell binding, Apt^21TNP-DDOX is internalized by triple-negative breast cancer cells through the macropinocytosis pathway. Once inside cells, the low pH microenvironment in lysosomes induces doxorubicin drug payload release from Apt^21TNP-DDOX. Our in vitro studies demonstrate that Apt^21TNP-DDOX can preferentially bind triple-negative breast cancer cells to induce cell death. Furthermore, we show that Apt^21TNP-DDOX can accumulate in subcutaneous MDA-MB-231 tumors in mice following systemic administration to reduce tumor burden, minimize side effects, and improve animal survival. Together, our results demonstrate that Apt^21TNP-mediated doxorubicin delivery is a potent, targeted chemotherapy for triple-negative breast cancer that may alleviate side effects in patients.

Gemcitabine is a chemotherapeutic used clinically to treat a variety of cancers. However, because it lacks tumor cell specificity, gemcitabine may cause off-target cytotoxicity and adversely impact patients. To impart cancer cell specificity to gemcitabine and improve its therapeutic efficacy, we synthesized a unique aptamer–drug conjugate that carries a high gemcitabine payload (three molecules) via a dendrimer structure and enzymatically cleavable linkers for controlled intracellular drug release. First, linker–gemcitabinedendrimer–linker–gemcitabine products were produced, which had significantly lower cytotoxicity than an equimolar amount of free drug. Biochemical analysis revealed that lysosomal cathepsin B protease rapidly cleaved the dendritic linkers and released the conjugated gemcitabine as a free drug. Subsequently, the dendrimer–linker–gemcitabine was coupled with a cell-specific aptamer to form aptamer–gemcitabine conjugates. Functional assays confirmed that, under aptamer guidance, aptamer–gemcitabine conjugates were selectively bound to and then internalized by triple-negative breast cancer cells. Cellular therapy studies indicated that the aptamer–gemcitabine conjugates potentiated cytotoxic activity to targeted cancer cells but did not affect off-target control cells. Our study demonstrates a novel approach to aptamer-mediated targeted drug delivery that combines a high drug payload and an enzymatically controlled drug release switch to achieve higher therapeutic efficacy and fewer off-target effects relative to free-drug chemotherapy.

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

Although targeted cancer therapy can induce higher therapeutic efficacy and cause fewer side effects in patients, the lack of targetable biomarkers on triple-negative breast cancer (TNBC) cells limits the development of targeted therapies by antibody technology. Therefore, we investigated an alternative approach to target TNBC by using the PDGC21T aptamer, which selectively binds to poorly differentiated carcinoma cells and tumor tissues, although the cellular target is still unknown. We found that synthetic aptamer probes specifically bound cultured TNBC cells in vitro and selectively targeted TNBC xenografts in vivo. Subsequently, to identify the target molecule on TNBC cells, we performed aptamer-mediated immunoprecipitation in lysed cell membranes followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Sequencing analysis revealed a highly conserved peptide sequence consistent with the cell surface protein CD49c (integrin α3). For target validation, we stained cultured TNBC and non-TNBC cells with an aptamer probe or a CD49c antibody and found similar cell staining patterns. Finally, competition cell-binding assays using both aptamer and anti-CD49c antibody revealed that CD49c is the biomarker targeted by the PDGC21T aptamer on TNBC cells. Our findings provide a molecular foundation for the development of targeted TNBC therapy using the PDGC21T aptamer as a targeting ligand.