Isolation and Identification of the High-Affinity DNA Aptamer Target to the Brain-Derived Neurotrophic Factor (BDNF).

Aptamers are functional oligonucleotide ligands used for the molecular recognition of various targets. The natural characteristics of aptamers make them an excellent alternative to antibodies in diagnostics, therapeutics, and biosensing. DNA aptamers are mainly single-stranded oligonucleotides (ssDNA) that possess a definite binding to targets. However, the application of aptamers to the fields of brain health and neurodegenerative diseases has been limited to date. Herein, a DNA aptamer against the brain-derived neurotrophic factor (BDNF) protein was obtained by in vitro selection. BDNF is a potential biomarker of brain health and neurodegenerative diseases and has functions in the synaptic plasticity and survival of neurons. We identified eight aptamers that have binding affinity for BDNF from a 50-nucleotide library. Among these aptamers, NV_B12 showed the highest sensitivity and selectivity for detecting BDNF. In an aptamer-linked immobilized sorbent assay (ALISA), the NV_B12 aptamer strongly bound to BDNF protein, in a dose-dependent manner. The dissociation constant (Kd) for NV_B12 was 0.5 nM (95% CI: 0.4-0.6 nM). These findings suggest that BDNF-specific aptamers could be used as an alternative to antibodies in diagnostic and detection assays for BDNF.

Unravelling the Glioblastoma Tumour Microenvironment: Can Aptamer Targeted Delivery Become Successful in Treating Brain Cancers?

The key challenges to treating glioblastoma multiforme (GBM) are the heterogeneous and complex nature of the GBM tumour microenvironment (TME) and difficulty of drug delivery across the blood-brain barrier (BBB). The TME is composed of various neuronal and immune cells, as well as non-cellular components, including metabolic products, cellular interactions, and chemical compositions, all of which play a critical role in GBM development and therapeutic resistance. In this review, we aim to unravel the complexity of the GBM TME, evaluate current therapeutics targeting this microenvironment, and lastly identify potential targets and therapeutic delivery vehicles for the treatment of GBM. Specifically, we explore the potential of aptamer-targeted delivery as a successful approach to treating brain cancers. Aptamers have emerged as promising therapeutic drug delivery vehicles with the potential to cross the BBB and deliver payloads to GBM and brain metastases. By targeting specific ligands within the TME, aptamers could potentially improve treatment outcomes and overcome the challenges associated with larger therapies such as antibodies.

The Diagnostic Potential of RNA Aptamers against the NS1 Protein of Dengue Virus Serotype 2.

Dengue infection, caused by the dengue virus, is a global threat which requires immediate attention and appropriate disease management. The current diagnosis of dengue infection is largely based on viral isolation, RT-PCR and serology-based detection, which are time-consuming and expensive, and require trained personnel. For early diagnosis of dengue, the direct detection of a dengue antigenic target is efficacious, and one such target is NS1. NS1-based detection is primarily antibody-centric and is beset by drawbacks pertaining to antibodies such as the high cost of synthesis and large batch-to-batch variation. Aptamers are potential surrogates of antibodies and are much cheaper, without exhibiting batch-to-batch variation. Given these advantages, we sought to isolate RNA aptamers against the NS1 protein of dengue virus serotype 2. A total of 11 cycles of SELEX were carried out, resulting in two potent aptamers, DENV-3 and DENV-6, with dissociation constant values estimated at 37.57 ± 10.34 nM and 41.40 ± 9.29 nM, respectively. These aptamers can be further miniaturized to TDENV-3 and TDENV-6a with an increased LOD upon their usage in direct ELASA. Moreover, these truncated aptamers are highly specific against the dengue NS1 while showing no cross-reactivity against the NS1 of the Zika virus, the E2 protein of the Chikungunya virus or the LipL32 protein of Leptospira, with target selectivity retained even in human serum. The usage of TDENV-3 as the capturing probe and TDENV-6a as the detection probe underpinned the development of an aptamer-based sandwich ELASA for the detection of dengue NS1. The sensitivity of the sandwich ELASA was further improved with the stabilization of the truncated aptamers and the repeated incubation strategy, which enabled a LOD of 2 nM when used with the target NS1 spiked in human serum diluted at 1:2000.

Cytotoxic effects of aptamer-doxorubicin conjugates in an ovarian cancer cell line.

Despite medical advances in treatment strategies over the past 30-years, epithelial ovarian cancer (EOC) continues to be defined by poor patient survival rates and aggressive, drug resistant relapse. Traditional approaches to cancer chemotherapy are typically limited by severe off-target effects on healthy tissue and aggressive drug-resistant recurrence. Recent shifts towards targeted therapies offer the possibility of circumventing the obstacles experienced by these treatments. While antibodies are the pioneering agents in such targeted therapies, several intrinsic characteristics of antibodies limits their clinical translation and efficacy. In contrast, oligonucleotide chemical antibodies, known as aptamers, are ideal for this application given their small size and lack of immunogenicity. This study explored the efficacy of a DNA aptamer, designed to target a well-established cancer biomarker, EpCAM, to deliver a chemotherapeutic drug. The results from this study support evidence that EpCAM aptamers can bind to epithelial ovarian cancer; and offers a valid alternative as a targeting ligand with tuneable specificity and sensitivity. It also supports the growing body of evidence that aptamers show great potential for application-specific, post-SELEX engineering through rational modifications. Through in vitro assays, these aptamers demonstrated cytotoxicity in both monolayer and tumoursphere assays, as well as in tumourigenic enriching assays. Further experimentation based on the results achieved in this project might aid in the development of novel cancer therapeutics and guide the novel designs of drugs for targeted drug delivery.

Natural Blockers of PD-1/PD-L1 Interaction for the Immunotherapy of Triple-Negative Breast Cancer-Brain Metastasis.

The limited treatment options for triple-negative breast cancer with brain metastasis (TNBC-BM) have left the door of further drug development for these patients wide open. Although immunotherapy via monoclonal antibodies has shown some promising results in several cancers including TNBC, it cannot be considered the most effective treatment for brain metastasis. This is due to the protective role of the blood-brain barrier (BBB) which limits the entrance of most drugs, especially the bulky ones such as antibodies, to the brain. For a drug to traverse the BBB via passive diffusion, various physicochemical properties should be considered. Since natural medicine has been a key inspiration for the development of the majority of current medicines, in this paper, we review several naturally-derived molecules which have the potential for immunotherapy via blocking the interaction of programmed cell death protein-1 (PD-1) and its ligand, PD-L1. The mechanism of action, physicochemical properties and pharmacokinetics of these molecules and their theoretical potential to be used for the treatment of TNBC-BM are discussed.

A Flow Cytometry-based Cell Surface Protein Binding Assay for Assessing Selectivity and Specificity of an Anticancer Aptamer.

A key challenge in developing an anticancer aptamer is to efficiently determine the selectivity and specificity of the developed aptamer to the target protein. Due to its several advantages over monoclonal antibodies, aptamer development has gained enormous popularity among cancer researchers. Systematic evolution of ligands by exponential enrichment (SELEX) is the most common method of developing aptamers specific for proteins of interest. Following SELEX, a quick and efficient binding assay accelerates the process of identification, confirming the selectivity and specificity of the aptamer. This paper explains a step-by-step flow cytometric-based binding assay of an aptamer specific for epithelial cellular adhesion molecule (EpCAM). The transmembrane glycoprotein EpCAM is overexpressed in most carcinomas and plays roles in cancer initiation, progression, and metastasis. Therefore, it is a valuable candidate for targeted drug delivery to tumors. To evaluate the selectivity and specificity of the aptamer to the membrane-bound EpCAM, EpCAM-positive and -negative cells are required. Additionally, a non-binding EpCAM aptamer with a similar length and 2-dimensional (2D) structure to the EpCAM-binding aptamer is required. The binding assay includes different buffers (blocking buffer, wash buffer, incubation buffer, and FACS buffer) and incubation steps. The aptamer is incubated with the cell lines. Following the incubation and washing steps, the cells will be evaluated using a sensitive flow cytometry assay. Analysis of the results shows the binding of the EpCAM-specific aptamer to EpCAM-positive cells and not the EpCAM-negative cells. In EpCAM-positive cells, this is depicted as a band shift in the binding of the EpCAM aptamer to the right compared to the non-binding aptamer control. In EpCAM-negative cells, the corresponding bands of EpCAM-binding and -non-binding aptamers overlap. This demonstrates the selectivity and specificity of the EpCAM aptamer. While this protocol is focused on the EpCAM aptamer, the protocol is applicable to other published aptamers.

Modelling of mass transport and distribution of aptamer in blood-brain barrier for tumour therapy and cancer treatment.

The blood-brain barrier (BBB) is a strong barrier against the entrance of drugs, which has made brain cancer treatment a major challenge. We have previously shown that targeting transferrin receptors using aptamers increased brain drug delivery. To get a better understanding of this phenomenon, in the present article, a mathematical model based on the finite element method was developed accounting for the fluid flow and mass transport of the aptamer molecule inside an 8 µm capillary vessel across a 14 µm blood-brain barrier domain. The fluid flow and mass transport equations were coupled to calculate the blood velocity and aptamer concentration profiles across the BBB. It was identified that the thickness of the astrocyte and endothelial cell layers are key parameters affecting the concentration of the aptamer delivered to the last neuron dendrites in the BBB. The predicted efficacy of the drug delivery (Capt/Cin) of 10.9% to 13.8% was calculated at a porosity of 0.5 to 0.9, respectively, at a blood velocity of 0.38 mm/s, which was independent of the inlet concentration of the aptamer. This low efficacy was attributed to the mass transfer resistance across endothelial cells, astrocyte and pericyte layers, which decreased the concentration by 6.7%. It was also identified that the main mechanism of drug delivery is switched from convective mass transport in the capillary layer (with Peclet number > 50) to mixed convection mass transport (1 < Peclet number < 5) in the porous layers and to diffusion only once aptamer reached the brain parenchyma (Peclet number < 1).

Triple-negative breast cancer brain metastasis: An update on druggable targets, current clinical trials, and future treatment options.

The key challenges with the treatment of triple-negative breast cancer brain metastasis (TNBC-BM) are the lack of any targeted therapy and difficulties associated with drug delivery to the brain. These add to the high toxicity profile of existing treatments and the poor outcomes for patient. In this review, we introduce current drugs based on their molecular targets and look to improve brain drug delivery using more efficient and promising drug delivery systems. We describe ongoing clinical trials on druggable targets in TNBC-BM for a more targeted treatment and introduce the obstacles hindering drug delivery to the brain, bringing strategies and advancing knowledge for future steps in the treatment of patients with TNBC-BM.

Future of PD-1/PD-L1 axis modulation for the treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has the worst prognosis among the subtypes of breast cancer, with no targeted therapy available. Immunotherapy targeting programmed cell death protein-1 (PD-1) and its ligand (PD-L1) has resulted in some promising outcomes in cancer patients. The common treatments are monoclonal antibodies (mAbs). Despite novel methodologies in developing mAbs, there are several drawbacks with these medications. Immunological reactions, expensive and time-consuming production and requiring refrigeration are some of the challenging characteristics of mAbs that are addressed with using aptamers. Aptamers are nucleotide-based structures with high selectivity and specificity for target. Their small size helps aptamers penetrate the tissue better. In this review, we have discussed the nature of PD-1/PD-L1 interaction and summarised the available mAbs and aptamers specific for these two targets. This review highlights the role of aptamers as a future pathway for PD-1/PD-L1 modulation.

Diagnostics and Therapeutics in Targeting HER2 Breast Cancer: A Novel Approach.

Breast cancer is one of the most commonly occurring cancers in women globally and is the primary cause of cancer mortality in females. BC is highly heterogeneous with various phenotypic expressions. The overexpression of HER2 is responsible for 15-30% of all invasive BC and is strongly associated with malignant behaviours, poor prognosis and decline in overall survival. Molecular imaging offers advantages over conventional imaging modalities, as it provides more sensitive and specific detection of tumours, as these techniques measure the biological and physiological processes at the cellular level to visualise the disease. Early detection and diagnosis of BC is crucial to improving clinical outcomes and prognosis. While HER2-specific antibodies and nanobodies may improve the sensitivity and specificity of molecular imaging, the radioisotope conjugation process may interfere with and may compromise their binding functionalities. Aptamers are single-stranded oligonucleotides capable of targeting biomarkers with remarkable binding specificity and affinity. Aptamers can be functionalised with radioisotopes without compromising target specificity. The attachment of different radioisotopes can determine the aptamer's functionality in the treatment of HER2(+) BC. Several HER2 aptamers and investigations of them have been described and evaluated in this paper. We also provide recommendations for future studies with HER2 aptamers to target HER2(+) BC.

Aptamers: Cutting edge of cancer therapies.

The development of an aptamer-based therapeutic has rapidly progressed following the first two reports in the 1990s, underscoring the advantages of aptamer drugs associated with their unique binding properties. In 2004, the US Food and Drug Administration (FDA) approved the first therapeutic aptamer for the treatment of neovascular age-related macular degeneration, Macugen developed by NeXstar. Since then, eleven aptamers have successfully entered clinical trials for various therapeutic indications. Despite some of the pre-clinical and clinical successes of aptamers as therapeutics, no aptamer has been approved by the FDA for the treatment of cancer. This review highlights the most recent and cutting-edge approaches in the development of aptamers for the treatment of cancer types most refractory to conventional therapies. Herein, we will review (1) the development of aptamers to enhance anti-cancer immunity and as delivery tools for inducing the expression of immunogenic neoantigens; (2) the development of the most promising therapeutic aptamers designed to target the hard-to-treat cancers such as brain tumors; and (3) the development of "carrier" aptamers able to target and penetrate tumors and metastasis, delivering RNA therapeutics to the cytosol and nucleus.

Profiling Cancer Cells by Cell-SELEX: Use of Aptamers for Discovery of Actionable Biomarkers and Therapeutic Applications Thereof.

The identification of tumor cell-specific surface markers is a key step towards personalized cancer medicine, allowing early assessment and accurate diagnosis, and development of efficacious targeted therapies. Despite significant efforts, currently the spectrum of cell membrane targets associated with approved treatments is still limited, causing an inability to treat a large number of cancers. What mainly limits the number of ideal clinical biomarkers is the high complexity and heterogeneity of several human cancers and still-limited methods for molecular profiling of specific cancer types. Thanks to the simplicity, versatility and effectiveness of its application, cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology is a valid complement to the present strategies for biomarkers' discovery. We and other researchers worldwide are attempting to apply cell-SELEX to the generation of oligonucleotide aptamers as tools for both identifying new cancer biomarkers and targeting them by innovative therapeutic strategies. In this review, we discuss the potential of cell-SELEX for increasing the currently limited repertoire of actionable cancer cell-surface biomarkers and focus on the use of the selected aptamers as components of innovative conjugates and nano-formulations for cancer therapy.

Novel Detection of Nasty Bugs, Prevention Is Better than Cure.

Hospital-acquired infections (HAIs) are a growing concern around the world. They contribute to increasing mortality and morbidity rates and are an economic threat. All hospital patients have the potential to contract an HAI, but those with weakened or inferior immune systems are at highest risk. Most hospital patients will contract at least one HAI, but many will contract multiple ones. Bacteria are the most common cause of HAIs and contribute to 80-90% of all HAIs, with Staphylococcus aureus, Clostridium difficile, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae accounting for the majority. Each of these bacteria are highly resistant to antibiotics and can produce a protective film, known as a biofilm, to further prevent their eradication. It has been shown that by detecting and eradicating bacteria in the environment, infection rates can be reduced. The current methods for detecting bacteria are time consuming, non-specific, and prone to false negatives or false positives. Aptamer-based biosensors have demonstrated specific, time-efficient and simple detection, highlighting the likelihood that they could be used in a similar way to detect HAI-causing bacteria.

Non-Invasive Delivery of Therapeutics into the Brain: The Potential of Aptamers for Targeted Delivery.

The blood-brain barrier (BBB) is a highly specialised network of blood vessels that effectively separates the brain environment from the circulatory system. While there are benefits, in terms of keeping pathogens from entering the brain, the BBB also complicates treatments of brain pathologies by preventing efficient delivery of macromolecular drugs to diseased brain tissue. Although current non-invasive strategies of therapeutics delivery into the brain, such as focused ultrasound and nanoparticle-mediated delivery have shown various levels of successes, they still come with risks and limitations. This review discusses the current approaches of therapeutic delivery into the brain, with a specific focus on non-invasive methods. It also discusses the potential for aptamers as alternative delivery systems and several reported aptamers with promising preliminary results.

Antibodies, Nanobodies, or Aptamers-Which Is Best for Deciphering the Proteomes of Non-Model Species?

This planet is home to countless species, some more well-known than the others. While we have developed many techniques to be able to interrogate some of the "omics", proteomics is becoming recognized as a very important part of the puzzle, given how important the protein is as a functional part of the cell. Within human health, the proteome is fairly well-established, with numerous reagents being available to decipher cellular pathways. Recent research advancements have assisted in characterizing the proteomes of some model (non-human) species, however, in many other species, we are only just touching the surface. This review considers three main reagent classes-antibodies, aptamers, and nanobodies-as a means of continuing to investigate the proteomes of non-model species without the complications of understanding the full protein signature of a species. Considerations of ease of production, potential applications, and the necessity for producing a new reagent depending on homology are presented.

Bifunctional Aptamer-Doxorubicin Conjugate Crosses the Blood-Brain Barrier and Selectively Delivers Its Payload to EpCAM-Positive Tumor Cells.

The prognosis for breast cancer patients diagnosed with brain metastases is poor, with survival time measured merely in months. This can largely be attributed to the limited treatment options capable of reaching the tumor as a result of the highly restrictive blood-brain barrier (BBB). While methods of overcoming this barrier have been developed and employed with current treatment options, the majority are highly invasive and nonspecific, leading to severe neurotoxic side effects. A novel approach to address these issues is the development of therapeutics targeting receptor-mediated transport mechanisms on the BBB endothelial cell membranes. Using this approach, we intercalated doxorubicin (DOX) into a bifunctional aptamer targeting the transferrin receptor on the BBB and epithelial cell adhesion molecule (EpCAM) on metastatic cancer cells. The ability of the DOX-loaded aptamer to transcytose the BBB and selectively deliver the payload to EpCAM-positive tumors was evaluated in an in vitro model and confirmed for the first time in vivo using the MDA-MB-231 breast cancer metastasis model (MDA-MB-231Br). We show that colocalized aptamer and DOX are clearly detectable within the brain lesions 75 min postadministration. Collectively, results from this study demonstrate that through intercalation of a cytotoxic drug into the bifunctional aptamer, a therapeutic delivery vehicle can be developed for specific targeting of EpCAM-positive brain metastases.

Anything You Can Do, I Can Do Better: Can Aptamers Replace Antibodies in Clinical Diagnostic Applications?

The mainstay of clinical diagnostics is the use of specialised ligands that can recognise specific biomarkers relating to pathological changes. While protein antibodies have been utilised in these assays for the last 40 years, they have proven to be unreliable due to a number of reasons. The search for the 'perfect' targeting ligand or molecular probe has been slow, though the description of chemical antibodies, also known as aptamers, nearly 30 years ago suggested a replacement reagent. However, uptake has been slow to progress into the clinical environment. In this review, we discuss the issues associated with antibodies and describe some of the applications of aptamers that have relevancy to the clinical diagnostic environment.

Aptamer-Based Diagnostics and Therapeutics.

Aptamers were first described almost 30 years ago, with the publication of three separate research papers describing how a randomized library of RNA sequences could be incubated with a target to find a sequence that specifically binds via van der Waals forces, covalent and hydrogen bonding, and not Watson Crick base pairing [...].

Radiolabelled Aptamers for Theranostic Treatment of Cancer.

Cancer has a high incidence and mortality rate worldwide, which continues to grow as millions of people are diagnosed annually. Metastatic disease caused by cancer is largely responsible for the mortality rates, thus early detection of metastatic tumours can improve prognosis. However, a large number of patients will also present with micrometastasis tumours which are often missed, as conventional medical imaging modalities are unable to detect micrometastases due to the lack of specificity and sensitivity. Recent advances in radiochemistry and the development of nucleic acid based targeting molecules, have led to the development of novel agents for use in cancer diagnostics. Monoclonal antibodies may also be used, however, they have inherent issues, such as toxicity, cost, unspecified binding and their clinical use can be controversial. Aptamers are a class of single-stranded RNA or DNA ligands with high specificity, binding affinity and selectivity for a target, which makes them promising for molecular biomarker imaging. Aptamers are presented as being a superior choice over antibodies because of high binding affinity and pH stability, amongst other factors. A number of aptamers directed to cancer cell markers (breast, lung, colon, glioblastoma, melanoma) have been radiolabelled and characterised to date. Further work is ongoing to develop these for clinical applications.