Cell-targeting Aptamers Research Articles

Aptamer-Based Immune Drug Systems (AptIDCs) Potentiating Cancer Immunotherapy

Aptamers are artificial oligonucleotides with excellent molecule-targeting ability. Compared with monoclonal antibodies, aptamers have the advantages of low cost, no batch effect, and negligible immunogenicity, making them promising candidates for cancer immunotherapy. To date, a series of aptamer agonists/antagonists have been discovered and directly used to activate immune response, such as immune checkpoint blockade, immune costimulation, and cytokine regulation. By incorporating both tumor- and immune cell-targeting aptamers, multivalent bispecific aptamers were designed to pursue high tumor affinity and enhanced immune efficacy. More importantly, benefiting from feasible chemical modification and programmability, aptamers can be engineered with diverse nanomaterials (e.g., liposomes, hydrogels) and even living immune cells (e.g., NK cells, T cells). These aptamer-based assemblies exhibit powerful capabilities in targeted cargo delivery, regulation of cell–cell interactions, tumor immunogenicity activation, tumor microenvironment remodeling, etc., holding huge potential in boosting immunotherapeutic efficacy. In this review, we focus on the recent advances in aptamer-based immune drug systems (AptIDCs) and highlight their advantages in cancer immunotherapy. The current challenges and future prospects of this field are also pointed out in this paper.

Regulating the properties of XQ-2d for targeted delivery of therapeutic agents to pancreatic cancers.

Enhanced recognition ability, cell uptake capacity, and biostability are characteristics attributed to aptamer-based targeted anticancer agents, and are possibly associated with increased accumulation at the tumor site, improved therapeutic efficacy and reduced negative side effects. Herein, a phosphorothioate backbone modification strategy was applied to regulate the biomedical properties of pancreatic cancer cell-targeting aptamer for efficient in vivo drug delivery. Specifically, the CD71- targeting aptamer XQ-2d was modified into a fully thio-substituted aptamer S-XQ-2d, improving the plasma stability of S-XQ-2d and mitomycin C (MMC)-functionalized S-XQ-2d (MFSX), thus considerably prolonging their half-life in mice. Moreover, the binding and uptake capacities of S-XQ-2d were significantly enhanced. MFSX showed the same level of cytotoxicity as that of MMC against targeted cancer cells, but lower toxicity to non-targeted cells, highlighting its specificity and biosafety. Brief mechanistic studies demonstrated that XQ-2d and S-XQ-2d had different interaction modes and internalization pathways with the targeted cells.

Endogenous Stimuli-Responsive Autonomous Separation of Dual-Targeting DNA Guided Missile from Nanospacecraft for Intelligent Targeted Cancer Therapy.

Most conventional chemotherapeutics indiscriminately kill both cancerous and healthy cells and cause toxic side effects, limiting the maximum tolerated dose and thereby compromising therapeutic efficacy. To address this challenge, here dual-targeting intelligent DNA guided missile (GM)-integrated nanospacecraft (NSC) (abbreviated as GM-NSC) is demonstrated for staged chemotherapeutic drug delivery exclusively into cancer cells and then mitochondria (not into healthy cells). GM-NSC is essentially a core/shell nanocomposite composed of gold nanoparticles (AuNPs) surrounded by a high-density multilayer DNA crown that is self-assembled from DNA tetrahedral units (DNA Tetra) in a highly ordered manner. Each tetrahedral structural unit is equipped with three functional components: a cancer cell-targeting aptamer pointing toward the outside environment, a hidden mitochondria-targeting triphenylphosphonium (TPP), and an explosive bolt (E-bolt). GM-NSC can remain intact in fetal bovine serum solution over 12 h and has 53-fold improved systemic stability. Each GM-NSC accommodates 1250 anticancer doxorubicin (Dox), achieving a 48-63-fold improved drug payload capacity. When systemically administrated into a tumor-bearing xenograft murine model, Dox-loaded GM-NSC enters into tumor sites with 18-fold improved specificity followed by autonomous separation of GMs from the NSC core and specific mitochondrial accumulation due to the explosion of E-bolt upon stimuli of endogenous miRNAs. About 80% of Dox uptaken is transferred into mitochondria and induces mitochondria-mediated apoptosis. As a result, the growth of malignant tumor is almost 100% inhibited without detectable toxicity to healthy tissues. Due to the desirable systemic stability, good biocompatibility, high cargo loading capability, satisfactory in vivo biodistribution, and therapeutic efficacy without adverse effects, intelligible GM-NSC is expected to become an alternative drug delivery system for precision cancer therapy.

Research progress of whole-cell-SELEX selection and the application of cell-targeting aptamer.

Aptamers refer to the artificially synthesized nucleic acid sequences (DNA/RNA) that can bind to a wide range of targets with high affinity and specificity, which are generally generated from systematic evolution of ligands by exponential enrichment (SELEX). As a novel method of aptamers screening, whole-cell-SELEX (WC-SELEX) has gained more and more attention in many fields such as biomedicine, analytical chemistry, and molecular diagnostics due to its ability to screen multiple potential aptamers without knowing the detailed structural information of target molecules. In recent years, with the deepening of research on application of aptamers, the traditional WC-SELEX cannot meet the practical application because of long experimental period, complicated operation process and low specificity, etc. Therefore, the development of more efficient methods for screening aptamer is always on the road. This paper summarizes the current research status of WC-SELEX for bacteria, parasites and animal cells, and reviews the latest advances of WC-SELEX techniques that are dependent on novel instruments, materials and microelectronics, including fluorescence-activated cell sorting-assisted SELEX, three-dimensional assisted WC-SELEX, and microfluidic chip system-assisted WC-SELEX. In addition, the application of aptamers targeting cells was discussed. Taken together, this review is aimed at providing a reference for WC-SELEX selection and application of aptamer targeting cells.

Integration of Palladium Nanoparticles with Surface Engineered Metal-Organic Frameworks for Cell-Selective Bioorthogonal Catalysis and Protein Activity Regulation.

Bioorthogonal catalysis provides a powerful tool to perform non-natural chemical reactions in living systems to dissect complex intracellular processes. Its potency to precisely regulate cellular function, however, is limited by the lack of bioorthogonal catalysts with cell selectivity. Herein, we report that palladium nanoparticles deposited on metal-organic frameworks, Pd@UiO-66, are highly efficient for intracellular bioorthogonal catalysis. In addition, introducing a cancer cell-targeting aptamer, AS1411, onto Pd@UiO-66 enables a threefold enhancement of catalysis efficiency in cancer cells. Moreover, AS1411@Pd@UiO-66 is effective in activating chemically caged 4-hydroxytamoxifen to regulate the activity of a protein destabilizing domain, ER50, and therefore protein function selectively in cancer cells. We show that the control over the activity of a bacterial effector, OspF, using AS1411@Pd@UiO-66 inactivates mitogen-activated protein kinase (MAPK) signaling of cancer cells to selectively prohibit tumor cell growth. We believe that the strategy developed herein for cell-selective bioorthogonal catalysis can expand the chemical biology toolbox for spatiotemporal control of protein function for advanced therapeutic applications.

Synthesis of a Single‐Stranded DNA Aptamer Modified Near‐infrared, Water‐Soluble Fluorophore for Lung Cancer Cell Imaging

AbstractIn recent years, aza‐boron dipyrromethene (aza‐BODIPY) derivatives have attracted considerable attention as fluorescent imaging agents. In this work, two polyethylene glycol functionalized water soluble aza‐BODIPY derivatives (a mono cycloaddition product 1PEG‐BOD and a double cycloaddition product 2PEG‐BOD) were synthesized through a cycloaddition click reaction between aza‐BODIPY and N3‐PEG2000‐COOH. The absorption and fluorescence profiles of these two compounds were in near‐infrared region. The fluorescence quantum yield of 2PEG‐BOD was 20 times higher than 1PEG‐BOD in water. The attachment of an A549 lung cancer cell targeting aptamer with 2PEG‐BOD afforded APT‐PEG‐BOD. The average fluorescence intensity of APT‐PEG‐BOD (5 μmol/L) in A549 lung cancer cells calculated from confocal microscopy images was 25–33 times stronger than that in other cell lines, such as MDA‐MB‐231, HT1080 and LO2. These results indicated that APT‐PEG‐BOD shows high affinity to A549 lung cancer cells.

Tumor-Specific Delivery of 5-Fluorouracil–Incorporated Epidermal Growth Factor Receptor–Targeted Aptamers as an Efficient Treatment in Pancreatic Ductal Adenocarcinoma Models

Fluoropyrimidine c (5-fluorouracil [5FU]) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor. We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)-selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy invitro and invivo. 5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre [KPC]), in an orthotopic cell line-derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up. Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell-targeting aptamers as the delivery platform.

Multicolor Two-Photon Nanosystem for Multiplexed Intracellular Imaging and Targeted Cancer Therapy.

The novel theranostic nanosystems based on two-photon fluorescence can achieve higher spatial resolution of deep tissue imaging for simultaneous diagnosis and therapy of a variety of cancers. Herein, we have designed and prepared FRET-based two-photon mesoporous silica nanoparticles (MTP-MSNs) for single-excitation multiplexed intracellular imaging and targeted cancer therapy for the first time. This nanosystem includes two constituents, containing (1) multicolor two-photon mesoporous silica nanoparticles and (2) cancer cell-targeting aptamers that act as gatekeepers for MTP-MSNs. After incubation with cancer cells, the Dox-loaded and aptamer-capped MTP-MSNs could be internalized into the cells, opening the pores and releasing the drug. Furthermore, using two-photon multicolor fluorescence, MTP-MSNs could serve as good contrast agents for multicolor two-photon intracellular imaging with increased imaging depth and improved spatial localization of tissue. In sum, these multicolor MTP-MSNs provide a promising system for traceable targeted cancer therapy with further applications in multiplex intracellular imaging and the screening of drug.

Tumor-Targeting Cholesterol-Decorated DNA Nanoflowers for Intracellular Ratiometric Aptasensing.

Probing endogenous molecular profiles is of fundamental importance to understand cellular function and processes. Despite the promise of programmable nucleic-acid-based aptasensors across the breadth of biomolecular detection, target-responsive aptasensors enabling intracellular detection are as of yet infrequently realized. Several challenges remain, including the difficulties in quantification/normalization of quencher-based intensiometric signals, stability issues of the probe architecture, and complex sensor operations often necessitating extensive structural modeling. Here, the biomimetic crystallization-empowered self-assembly of a tumor-targetable DNA-inorganic hybrid nanocomposite aptasensor is presented, which enables Förster resonance energy transfer (FRET)-based quantitative interpretation of changes in the cellular target abundance. Leveraging the design programmability and high-throughput fabrication of rolling circle amplification-driven DNA nanoarchitecture, this designer platform offers a method to self-assemble a robust nanosensor from a multifunctionality-encoded template that includes a cell-targeting aptamer, a ratiometric aptasensor, and a cholesterol-decorating element. Taking prostate cancer cells and intracellular adenosine triphosphate molecules as a model system, a synergistic effect in the targeted delivery by cholesterol and aptamers, and the feasibility of quantitative intracellular aptasensing are demonstrated. It is envisioned that this approach provides a highly generalizable strategy across wide-ranging target systems toward a biologically deliverable nanosensor that enables quantitative monitoring of the abundance of endogenous biomolecules.

Structural requirement of G-quadruplex/aptamer-combined DNA macromolecule serving as efficient drug carrier for cancer-targeted drug delivery

Photodynamic therapy (PDT) as a clinical cancer treatment method has been used to treat carcinomas in different organs, and G-quadruplex-based DNA nanocompartments serving as the carriers of cationic porphyrin photosensitizers, especially combined with cell-targeting aptamers, is considered to offer new opportunities for future cancer treatment. However, the structural features of G-quadruplex/aptamer complexes suitable for the capsulation of photosensitizers and target cell recognition is unexplored so far. In this study, unimolecular (UM), bimolecular (BM) and tetramolecular (TM) G-quadruplex structures were used as the drug loading compartments and grafted onto tumor cell-targeting aptamer Sgc8, constructing several targeting drug delivery vehicles (T-GMVs). Besides the binding affinity of resulting DNA architectures for target cells and cell recognition specificity were explored in a comparative fashion, the drug loading capability and cancer therapy efficacy were evaluated using TMPyP4 as the model porphyrin-based drug. The experimental results show that only TM G-quadruplex structure is suitable to combine with Sgc8 for the development of drug delivery vehicle and the as-prepared T-GMV- TMPyP4 complexes display the desirable cancer therapy efficacy, holding the potential application in the future cancer therapy. More importantly, T-GMV- TMPyP4 is expected to lay the scientific groundwork for the successful development of G-quadruplex-based photosensitizer drug delivery carriers for the targeted cancer therapy.

Abstract 5256: Toward biomarkers discovery: Profiling triple-negative breast cancer cells by cell-SELEX

Abstract Triple-negative breast cancer (TNBC) is a high heterogeneous group of tumors with a distinctly aggressive nature and high rates of relapse. So far, the lack of any known targetable proteins has not allowed a specific anti-tumor treatment, thus the identification of novel agents for specific TNBC recognition and treatment is desperately needed. Nucleic acids aptamers are emerging as the most promising candidates for targeting tumor cells. They are also termed "chemical antibodies" because their form unique tertiary structures, which allow specific binding to their target molecules. Recently, by cell-SELEX integrated to advanced Next Generation Sequencing and bioinformatics, we isolated a panel of six nuclease-resistant RNA aptamers able to bind at high affinity to TNBC cells, discriminating them from both normal cells and non-TNBC breast cancer cells, and interfere with their growth as mammospheres. Also, they diversify TNBC histological specimens in a tissue microarray without recognizing samples from both ER+, PR+, HER2+ breast cancers and normal adjacent tissues. Here, short versions of the aptamers have been generated, by truncating their length from 84 nt up to nearly 30 nt, which preserve efficacious targeting and anti-tumor properties as the parental moieties. In order to identify the targets of the above aptamers, which might be important surface cancer biomarkers crucial for the malignant phenotype, different biochemical approaches are harnessed that include aptamer-mediated affinity purification coupled to Mass Spectrometry and/or antibody array for human soluble receptors. Binding analyses on recombinant proteins, cells or tissues are used to verify the putative targets. Results obtained so far indicate that the target of one of the six aptamers is a secreted protein tightly and specifically associated to TNBC cell surface and with a poor prognosis. This protein identification and the functional role of its targeting by the aptamer will be shown here in detail. The proposed cell-targeting aptamer strategy could provide an important new avenue for selective TNBC therapy. Citation Format: Simona Camorani, Francesco Leonetti, Francesca Collina, Monica Cantile, Gerardo Botti, Antonella Zannetti, Monica Fedele, Laura Cerchia. Toward biomarkers discovery: Profiling triple-negative breast cancer cells by cell-SELEX [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5256.

Aptamer-Functionalized Drug Nanocarrier Improves Hepatocellular Carcinoma toward Normal by Targeting Neoplastic Hepatocytes

Site-specific delivery of chemotherapeutics specifically to neoplastic hepatocytes without affecting normal hepatocytes should be a focus for potential therapeutic management of hepatocellular carcinoma (HCC). The aptamer TLS 9a with phosphorothioate backbone modifications (L5) has not been explored so far for preferential delivery of therapeutics in neoplastic hepatocytes to induce apoptosis. Thus, the objective of the present investigation was to compare the therapeutic potential of L5-functionalized drug nanocarrier (PTX-NPL5) with those of the other experimental drug nanocarriers functionalized by previously reported HCC cell-targeting aptamers and non-aptamer ligands, such as galactosamine and apotransferrin. A myriad of well-defined investigations such as cell cycle analysis, TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) assay, and studies related to apoptosis, histopathology, and immunoblotting substantiated that PTX-NPL5 had the highest potency among the different ligand-attached experimental formulations in inducing selective apoptosis in neoplastic hepatocytes via a mitochondrial-dependent apoptotic pathway. PTX-NPL5 did not produce any notable toxic effects in healthy hepatocytes, thus unveiling a new and a safer option in targeted therapy for HCC. Molecular modeling study identified two cell-surface biomarker proteins (tumor-associated glycoprotein 72 [TAG-72] and heat shock protein 70 [HSP70]) responsible for ligand-receptor interaction of L5 and preferential internalization of PTX-NPL5 via clathrin-mediated endocytosis in neoplastic hepatocytes. The potential of PTX-NPL5 has provided enough impetus for its rapid translation from the pre-clinical to clinical domain to establish itself as a targeted therapeutic to significantly prolong survival in HCC.

Polymeric Engineering of Aptamer-Drug Conjugates for Targeted Cancer Therapy.

Nucleic acid aptamers, also known as "chemical antibodies", have been widely employed in targeted cancer therapy and diagnosis. For example, aptamer-drug conjugates (ApDCs), through covalent conjugation of cytotoxic warheads to aptamers, have demonstrated anticancer efficacy both in vitro and in vivo. However, a general strategy to endow ApDCs with enhanced biostability, prolonged circulation half-life, and high drug loading content remained elusive. Herein, we present a polymeric approach to engineer ApDCs via conjugation of cell-targeting aptamers with water-soluble polyprodrugs containing a reductive environmentally sensitive prodrug and biocompatible brush-like backbone. The resultant high-drug loading Aptamer-PolyproDrug Conjugates (ApPDCs) exhibited high nuclease resistance, extended in vivo circulation time, specific recognition, and cellular uptake to target cells, reduction-triggered and fluorescent-reporting drug release, and effective cytotoxicity. We could also further expand this design principle toward combination therapy by using two kinds of therapeutic drugs with distinct pharmacological mechanisms.

Aptamer/photosensitizer hybridized mesoporous MnO2 based tumor cell activated ROS regulator for precise photodynamic therapy of breast cancer

Herein, we report a turn-on strategy for selectively killing the tumor cell via combining the singlet-oxygen quenching MnO2 and tumor cell-targeting aptamer. The photosensitizers were in the quenching state when loaded in the mesoporous MnO2 (mMnO2) nanoparticles and sealed by the aptamer on the particle surface. The aptamer can selectively recognize the specific membrane protein on the tumor cell and release the photosensitizers, activating the photosensitizer and killing the tumor cells. The specific binding-induced “off-on” switching of singlet oxygen generation reduced the damage to the nearby healthy cells to a large extent. The high loading ability for photosensitizer and the GSH consumption property of mMnO2 endow the system with high local concentration of singlet-oxygen for killing the target tumor cell. The high selectivity and efficiency of the constructed singlet oxygen regulating system will pave a new way for utilizing PDT in cancer precise treatment.

Efficient Intracellular Delivery of RNase A Using DNA Origami Carriers

Delivery of proteins to carry out desired biological functions is a direct approach for disease treatment. However, protein therapy is still facing challenges due to low delivery efficiency, poor targeting during trafficking, insufficient therapeutic efficacy, and possible toxicity induced by carriers. Here, we present a novel delivery platform based on DNA origami nanostructure that enables tumor cell transportation of active proteins for cancer therapy. In our design, cytotoxic protein ribonuclease (RNase) A molecules are organized on the rectangular DNA origami nanosheets, which work as nanovehicles to deliver RNase A molecules into the cytoplasm and execute their cell-killing function inside the tumor cells. Cancer cell-targeting aptamers are also integrated onto the DNA origami-based nanoplatform to enhance its targeting effect. This DNA origami-protein coassembling strategy can be further developed to transport other functional proteins and therapeutic components simultaneously for synergistic effects and be adapted for integrated diagnostics and therapeutics.

Aptamer‐Decorated DNAsome for Targeted Cancer Therapy

AbstractDrug delivery using a nanocarrier is one promising approach for cancer therapy. However, the design of nanocarriers that allow the targeted delivery of drugs at the desired location upon demand remains a foremost challenge in cancer therapy. Herein, the design of a DNA based vesicle (DNAsome) as a nanocarrier from the self‐assembly of a DNA amphiphile is reported. The salient features of DNAsome include excellent biocompatibility, high drug encapsulation ability and DNA‐based surface addressability. The surface decoration of the DNAsome with highly information‐rich ssDNA is explored for the integration of cancer cell targeting aptamer through sequence specific DNA hybridization. Selective delivery of anticancer drug into a cancerous cell line is demonstrated using the aptamer decorated DNAsome in an efficient manner. Our results unequivocally show the potential of DNAsome for targeted cancer therapy, and hence could be a promising candidate as a nanocarrier for drug delivery applications.

Aptamer-Mediated Delivery and Cell-Targeting Aptamers: Room for Improvement.

Targeting cells with aptamers for the delivery of therapeutic cargoes, in particular oligonucleotides, represents one of the most exciting applications of the aptamer field. Perhaps nowhere has there been more excitement in the field than around the targeted delivery of siRNA or miRNA. However, when industry leaders in the field of siRNA delivery have tried to recapitulate aptamer-siRNA delivery results, they have failed. This problem stems from more than just the age-old problem of delivery to the cytoplasm, a challenge that has stymied the targeted delivery of therapeutic oligonucleotides since its inception. With aptamers, the problem is compounded further by the fact that many aptamers simply do not function as reported. This is distressing, as clearly, all published aptamers should be able to function as described. However, it is often challenging to recognize the details that might flag an unreliable aptamer from a viable one. As such, unreliable aptamers continue to be peer reviewed and published. We need to raise the bar and level of rigor in the field. Only then can we think about taking advantage of the unique attributes of these molecules and address the issues associated with their use as agents for targeted delivery.

Toward the Selection of Cell Targeting Aptamers with Extended Biological Functionalities to Facilitate Endosomal Escape of Cargoes.

Over the past decades there have been exciting and rapid developments of highly specific molecules to bind cancer antigens that are overexpressed on the surfaces of malignant cells. Nanomedicine aims to exploit these ligands to generate nanoscale platforms for targeted cancer therapy, and to do so with negligible off-target effects. Aptamers are structured nucleic acids that bind to defined molecular targets ranging from small molecules and proteins to whole cells or viruses. They are selected through an iterative process of amplification and enrichment called SELEX (systematic evolution of ligands by exponential enrichment), in which a combinatorial oligonucleotide library is exposed to the target of interest for several repetitive rounds. Nucleic acid ligands able to bind and internalize into malignant cells have been extensively used as tools for targeted delivery of therapeutic payloads both in vitro and in vivo. However, current cell targeting aptamer platforms suffer from limitations that have slowed their translation to the clinic. This is especially true for applications in which the cargo must reach the cytosol to exert its biological activity, as only a small percentage of the endocytosed cargo is typically able to translocate into the cytosol. Innovative technologies and selection strategies are required to enhance cytoplasmic delivery. In this review, we describe current selection methods used to generate aptamers that target cancer cells, and we highlight some of the factors that affect productive endosomal escape of cargoes. We also give an overview of the most promising strategies utilized to improve and monitor endosomal escape of therapeutic cargoes. The methods we highlight exploit tools and technologies that can potentially be incorporated in the SELEX process. Innovative selection protocols may identify aptamers with extended biological functionalities that allow effective cytosolic translocation of therapeutics. This in turn may facilitate successful translation of these platforms into clinical applications.

Voltammetric aptamer based detection of HepG2 tumor cells by using an indium tin oxide electrode array and multifunctional nanoprobes

The authors describe a method for the detection and determination of human liver cancer cells in blood. The cytosensing system consists of a microfabricated chip-based electrochemical aptasensor that contains multifunctional hybrid electrochemical nanoprobes and an indium tin oxide (ITO) electrode array interface functionalized with cell-targeting aptamer and gold nanoparticles (AuNPs). The thiolated cell targeting aptamer (referred to as TLS11a) was immobilized on the ITO electrode/AuNPs for specific adhesion of hepatocellular carcinoma cells (HepG2). The hybrid nanoprobe system consists of hydroquinone (HQ) as an electrochemical probe, horseradish peroxidase (HRP), and an aptamer/hemin/G-quadruplex aggregate that was immobilized on gold/palladium-functionalized ZnO nanorods (ZnO@Au-Pd). The nanoprobes are capable of amplifying the voltammetric signal and capturing the target cells. Best operated at around −90 mV (vs Ag/AgCl), the electrode has a linear response that covers the 10^2 to 10^7 HepG2 cells per mL concentration range, with a 10 cell per mL detection limit. Captured cells may be released from the electrode via electrochemical desorption to break the Au-S bonds.

A novel 4-arm DNA/RNA Nanoconstruct triggering Rapid Apoptosis of Triple Negative Breast Cancer Cells within 24\u2009hours

Measuring at ~30 nm, a fully customizable holliday junction DNA nanoconstruct, was designed to simultaneously carry three unmodified SiRNA strands for apoptosis gene knockout in cancer cells without any assistance from commercial transfection kits. In brief, a holliday junction structure was intelligently designed to present one arm with a cell targeting aptamer (AS1411) while the remaining three arms to carry different SiRNA strands by means of DNA/RNA duplex for inducing apoptosis in cancer cells. By carrying the three SiRNA strands (AKT, MDM2 and Survivin) into triple negative breast MDA-MB-231 cancer cells, cell number had reduced by up to ~82% within 24 hours solely from one single administration of 32 picomoles. In the immunoblotting studies, up-elevation of phosphorylated p53 was observed for more than 8 hours while the three genes of interest were suppressed by nearly half by the 4-hour mark upon administration. Furthermore, we were able to demonstrate high cell selectivity of the nanoconstruct and did not exhibit usual morphological stress induced from liposomal-based transfection agents. To the best of the authors’ knowledge, this system represents the first of its kind in current literature utilizing a short and highly customizable holliday DNA junction to carry SiRNA for apoptosis studies.