Anaplastic large cell lymphoma (ALCL) is the most common T-cell lymphoma in children. ALCL cells characteristically express surface CD30 molecules and carry the pathogenic ALK oncogene, both of which are diagnostic biomarkers and are also potential therapeutic targets. For precision therapy, we report herein a protamine nanomedicine incorporated with oligonucleotide aptamers to selectively target lymphoma cells, a dsDNA/drug payload to efficiently kill targeted cells, and an siRNA to specifically silence ALK oncogenes. The aptamer-equipped protamine nanomedicine was simply fabricated through a non-covalent charge-force reaction. The products had uniform structure morphology under an electron microscope and a peak diameter of 103 nm by dynamic light scattering measurement. Additionally, flow cytometry analysis demonstrated that under CD30 aptamer guidance, the protamine nanomedicine specifically bound to lymphoma cells, but did not react to off-target cells in control experiments. Moreover, specific cell targeting and intracellular delivery of the nanomedicine were also validated by electron and confocal microscopy. Finally, functional studies demonstrated that, through combined cell-selective chemotherapy using a drug payload and oncogene-specific gene therapy using an siRNA, the protamine nanomedicine effectively killed lymphoma cells with little toxicity to off-target cells, indicating its potential for precision therapy.

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.

Doxorubicin (DOX) is a common anti-tumor drug that binds to DNA or RNA via non-covalent intercalation between G-C sequences. As a therapeutic agent, DOX has been used to form aptamer–drug conjugates for targeted cancer therapy in vitro and in vivo. To improve the therapeutic potential of aptamer–DOX conjugates, we synthesized trifurcated Newkome-type monomer (TNM) structures with three DOX molecules bound through pH-sensitive hydrazone bonds to formulate TNM-DOX. The aptamer–TNM–DOX conjugate (Apt–TNM-DOX) was produced through a simple self-loading process. Chemical validation revealed that Apt–TNM-DOX stably carried high drug payloads of 15 DOX molecules per aptamer sequence. Functional characterization showed that DOX payload release from Apt–TNM-DOX was pH-dependent and occurred at pH 5.0, which reflects the microenvironment of tumor cell lysosomes. Further, Apt–TNM-DOX specifically targeted lymphoma cells without affecting off-target control cells. Aptamer-mediated cell binding resulted in the uptake of Apt–TNM-DOX into targeted cells and the release of DOX payload within cell lysosomes to inhibit growth of targeted lymphoma cells. The Apt–TNM-DOX provides a simple, non-toxic approach to develop aptamer-based targeted therapeutics and may reduce the non-specific side effects associated with traditional 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.

As a “chemical antibody”, oligonucleotide aptamers can specifically bind to their target molecules. However, clinical potential of aptamers in disease diagnosis is not yet fully explored. Using a tumor cell-based selection protocol, we developed single-stranded DNA aptamers for Hodgkin lymphoma (HL) tumor cells. The aptamers specifically bound to HL cells with a high affinity, reaching maximal cell binding at 10 nM final concentration. Importantly, the aptamers were able to selectively detect HL cells and did not react to other tumor or blood cells in mixed samples, indicating that the aptamers can be used as a specific probe for in vitro analysis of HL cells. Moreover, due to the inherent properties of DNA, the aptamers were stable in human serum, suggesting potential for in vivo detection of HL tumor cells.