Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX.Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients’ blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses.Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression.Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes

Aptamers Increase Biocompatibility and Reduce the Toxicity of Magnetic Nanoparticles Used in Biomedicine

Aptamer-based approaches are very promising tools in nanomedicine. These small single-stranded DNA or RNA molecules are often used for the effective delivery and increasing biocompatibility of various therapeutic agents. Recently, magnetic nanoparticles (MNPs) have begun to be successfully applied in various fields of biomedicine. The use of MNPs is limited by their potential toxicity, which depends on their biocompatibility. The functionalization of MNPs by ligands increases biocompatibility by changing the charge and shape of MNPs, preventing opsonization, increasing the circulation time of MNPs in the blood, thus shielding iron ions and leading to the accumulation of MNPs only in the necessary organs. Among various ligands, aptamers, which are synthetic analogs of antibodies, turned out to be the most promising for the functionalization of MNPs. This review describes the factors that determine MNPs’ biocompatibility and affect their circulation time in the bloodstream, biodistribution in organs and tissues, and biodegradation. The work also covers the role of the aptamers in increasing MNPs’ biocompatibility and reducing toxicity

Aptamer-Based Viability Impedimetric Sensor for Bacteria

The development of an aptamer-based viability impedimetric sensor for bacteria (AptaVISens-B) is presented. Highly specific DNA aptamers to live <i>Salmonella typhimurium</i> were selected via the cell-systematic evolution of ligands by exponential enrichment (SELEX) technique. Twelve rounds of selection were performed; each comprises a positive selection step against viable <i>S. typhimurium</i> and a negative selection step against heat killed <i>S. typhimurium</i> and a mixture of related pathogens, including <i>Salmonella enteritidis</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i> to ensure the species specificity of the selected aptamers. The DNA sequence showing the highest binding affinity to the bacteria was further integrated into an impedimetric sensor via self-assembly onto a gold nanoparticle-modified screen-printed carbon electrode (GNP-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect <i>S. typhimurium</i> down to 600 CFU mL^–1 (equivalent to 18 live cells in 30 μL of assay volume) and distinguish it from other <i>Salmonella</i> species, including <i>S. enteritidis</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new venue for the aptamer-based viability sensing of a variety of microorganisms, particularly viable but nonculturable (VBNC) bacteria, using a rapid, economic, and label-free electrochemical platform

Aptamer-Based Impedimetric Sensor for Bacterial Typing

The development of an aptamer-based impedimetric sensor for typing of bacteria (AIST-B) is presented. Highly specific DNA aptamers to <i>Salmonella enteritidis</i> were selected via Cell-SELEX technique. Twelve rounds of selection were performed; each comprises a positive selection step against <i>S. enteritidis</i> and a negative selection step against a mixture of related pathogens, including <i>Salmonella typhimurium</i>, <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Citrobacter freundii</i>, to ensure the species-specificity of the selected aptamers. After sequencing of the pool showing the highest binding affinity to <i>S. enteritidis</i>, a DNA sequence of high affinity to the bacteria was integrated into an impedimetric sensor via self-assembly onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect <i>S. enteritidis</i> down to 600 CFU mL^–1 (equivalent to 18 CFU in 30 μL assay volume) in 10 min and distinguish it from other Salmonella species, including <i>S. typhimurium</i> and <i>S. choleraesuis</i>. This report is envisaged to open a new venue for the aptamer-based typing of a variety of microorganisms using a rapid, economic, and label-free electrochemical platform

Four steps for revealing and adjusting the 3D structure of aptamers in solution by small-angle X-ray scattering and computer simulation

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In Vivo Cancer Cells Elimination Guided by Aptamer-Functionalized Gold-Coated Magnetic Nanoparticles and Controlled with Low Frequency Alternating Magnetic Field

Biomedical applications of magnetic nanoparticles in a magnetic field have exceeded many expectations in cancer therapy. Magnetic nanoparticles are effective heat mediators, drug nanocarriers, and contrast agents; various strategies have been suggested to selectively target tumor cancer cells but not healthy cells. Our study presents magnetodynamic nanotherapy utilizing DNA aptamer-functionalized 50 nm gold-coated magnetic nanoparticles exposed to a low frequency alternating magnetic field for precise elimination of tumor cells in vivo. The cell specific DNA aptamer AS-14 binds to fibronectin protein in Ehrlich carcinoma and delivers gold-coated magnetic nanoparticles to a mouse tumor. An alternating magnetic field of 50 Hz causesthe nanoparticles to oscillate and pull fibronectin and integrins on the surface of the cell membrane resulting in massive cell apoptosis followed by necrosis without heating the tumor, adjacent healthy cells and tissues. The aptamer-guided nanoparticles and the low frequency alternating magnetic field demonstrates a unique technology of a non-invasive nanoscalpel for precise cancer surgery at a single cell level

Aptamer-Targeted Plasmonic Photothermal Therapy of Cancer

Novel nanoscale bioconjugates combining unique plasmonic photothermal properties of gold nanoparticles (AuNPs) with targeted delivery using cell-specific DNA aptamers have a tremendous potential for medical diagnostics and therapy of many cell-based diseases. In this study, we demonstrate the high anti-cancer activity of aptamer-conjugated, 37-nm spherical gold nanoparticles toward Ehrlich carcinoma in tumor-bearing mice after photothermal treatment. The synthetic anti-tumor aptamers bring the nanoparticles precisely to the desired cells and selectively eliminate cancer cells after the subsequent laser treatment. To prove tumor eradication, we used positron emission tomography (PET) utilizing radioactive glucose and computer tomography, followed by histological analysis of cancer tissue. Three injections of aptamer-conjugated AuNPs and 5 min of laser irradiations are enough to make the tumor undetectable by PET. Histological analysis proves PET results and shows lower damage of healthy tissue in addition to a higher treatment efficiency and selectivity of the gold nanoparticles functionalized with aptamers in comparison to control experiments using free unconjugated nanoparticles

In Vivo Cancer Cells Elimination Guided by Aptamer-Functionalized Gold-Coated Magnetic Nanoparticles and Controlled with Low Frequency Alternating Magnetic Field

Biomedical applications of magnetic nanoparticles in a magnetic field have exceeded many expectations in cancer therapy. Magnetic nanoparticles are effective heat mediators, drug nanocarriers, and contrast agents; various strategies have been suggested to selectively target tumor cancer cells but not healthy cells. Our study presents magnetodynamic nanotherapy utilizing DNA aptamer-functionalized 50 nm gold-coated magnetic nanoparticles exposed to a low frequency alternating magnetic field for precise elimination of tumor cells in vivo. The cell specific DNA aptamer AS-14 binds to fibronectin protein in Ehrlich carcinoma and delivers gold-coated magnetic nanoparticles to a mouse tumor. An alternating magnetic field of 50 Hz causesthe nanoparticles to oscillate and pull fibronectin and integrins on the surface of the cell membrane resulting in massive cell apoptosis followed by necrosis without heating the tumor, adjacent healthy cells and tissues. The aptamer-guided nanoparticles and the low frequency alternating magnetic field demonstrates a unique technology of a non-invasive nanoscalpel for precise cancer surgery at a single cell level

DNA Aptamers for the Characterization of Histological Structure of Lung Adenocarcinoma

Nucleic acid aptamers are becoming popular as molecular probes for identification and imaging pathology and, at the same time, as a convenient platform for targeted therapy. Recent studies have shown that aptamers may be effectively used for tumor characterization and as commercially available monoclonal antibodies. Here we present three DNA aptamers binding to whole transformed lung cancer tissues, including tumor cells, connective tissues, and blood vessels. Protein targets have been revealed using affinity purification followed by mass spectrometry analyses, and they have been validated using a panel of correspondent antibodies and 3D imaging of tumor tissues. Each of the proteins targeted by the aptamers is involved in cancer progression and most of them are crucial for lung adenocarcinoma. We propose the use of these aptamers in aptahistochemistry for the characterization of the histological structure of lung adenocarcinoma. The value of the presented aptamers is their application together or separately for indicating the spread of neoplastic transformation, for complex differential diagnostics, and for targeted therapy of the tumor itself as well as all transformed structures of the adjacent tissues. Moreover, it has been demonstrated that these aptamers could be used for intraoperative tumor visualization and margin assessment. Keywords: DNA-aptamer, lung adenocarcinoma, histological structur