RESUMO
Aptamers are folded oligonucleotides that selectively recognize and bind a target and are consequently regarded as an emerging alternative to antibodies for sensing and therapeutic applications. The rational development of functional aptamers is strictly related to the accurate definition of molecular binding properties. Nevertheless, most of the methodologies employed to define binding affinities use bulk measurements. Here, we describe the use of fluorescence correlation spectroscopy (FCS) as a method with single-molecule sensitivity that quantitatively defines aptamer-protein binding. First, FCS was used to measure the equilibrium affinity between the CLN3 aptamer, conjugated with a dye, and its target, the c-Met protein. Equilibrium affinity was also determined for other functional aptamers targeting nucleolin and platelet-derived growth factors. Then, association and dissociation rates of CLN3 to/from the target protein were measured using FCS by monitoring the equilibration kinetics of the binding reaction in solution. Finally, FCS was exploited to investigate the behavior of CLN3 exposed to physiological concentrations of the most abundant serum proteins. Under these conditions, the aptamer showed negligible interactions with nontarget serum proteins while preserving its affinity for the c-Met. The presented results introduce FCS as an alternative or complementary analytical tool in aptamer research, particularly well-suited for the characterization of protein-targeting aptamers.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , Ligação Proteica , Proteínas Sanguíneas/metabolismo , Análise EspectralRESUMO
The p75 neurotrophin (NT) receptor (p75NTR) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75NTR and mutated (mut) p75NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75NTR , but not of mut p75NTR, to mediate immature NT (proNT)-induced apoptosis. Both p75NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.
Assuntos
Apoptose/fisiologia , Cones de Crescimento/fisiologia , Fatores de Crescimento Neural/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Camundongos , Camundongos Knockout , Sistema Nervoso/metabolismo , Fenômenos Fisiológicos do Sistema Nervoso/genética , Receptor de Fator de Crescimento Neural/genéticaRESUMO
Although molecular mechanisms driving tumor progression have been extensively studied, the biological nature of the various populations of circulating tumor cells (CTCs) within the blood is still not well understood. Tumor cell fusion with immune cells is a longstanding hypothesis that has caught more attention in recent times. Specifically, fusion of tumor cells with macrophages might lead to the development of metastasis by acquiring features such as genetic and epigenetic heterogeneity, chemotherapeutic resistance, and immune tolerance. In addition to the traditional FDA-approved definition of a CTC (CD45-, EpCAM+, cytokeratins 8+, 18+ or 19+, with a DAPI+ nucleus), an additional circulating cell population has been identified as being potential fusions cells, characterized by distinct, large, polymorphonuclear cancer-associated cells with a dual epithelial and macrophage/myeloid phenotype. Artificial fusion of tumor cells with macrophages leads to migratory, invasive, and metastatic phenotypes. Further studies might investigate whether these have a potential impact on the immune response towards the cancer. In this review, the background, evidence, and potential relevance of tumor cell fusions with macrophages is discussed, along with the potential role of intercellular connections in their formation. Such fusion cells could be a key component in cancer metastasis, and therefore, evolve as a diagnostic and therapeutic target in cancer precision medicine.
Assuntos
Biomarcadores Tumorais/sangue , Macrófagos/patologia , Metástase Neoplásica/patologia , Neoplasias/patologia , Animais , Humanos , Neoplasias/sangue , Células Neoplásicas Circulantes/patologiaRESUMO
Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Imagem Individual de Molécula/métodos , Aptâmeros de Nucleotídeos/química , Linhagem Celular Tumoral , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Microscopia de Fluorescência , Receptores da Transferrina/química , Receptores da Transferrina/metabolismoRESUMO
Peptide amphiphile micelles (PAMs) are attractive vehicles for the delivery of a variety of therapeutic and prophylactic peptides. However, a key limitation of PAMs is their lack of preferential targeting ability. In this paper, we describe our design of a PAM system that incorporates a DNA oligonucleotide amphiphile (antitail amphiphile-AA) to form A/PAMs. A cell-targeting DNA aptamer with a 3' extension sequence (tail) complementary to the AA is annealed to the surface to form aptamer-displaying PAMs (Aptamer~A/PAMs). Aptamer~A/PAMs are small, anionic, stable nanoparticles capable of delivering a large mass percentage peptide amphiphile (PA) compared to targeting DNA components. Aptamer~A/PAMs are stable for over 4 h in the presence of biological fluids. Additionally, the aptamer retains its cell-targeting properties when annealed to the A/PAM, thus leading to enhanced delivery to a specifically-targeted B-cell leukemia cell line. This exciting modular technology can be readily used with a library of different targeting aptamers and PAs, capable of improving the bioavailability and potency of the peptide cargo.
Assuntos
Aptâmeros de Nucleotídeos/química , Sistemas de Liberação de Medicamentos , Micelas , Peptídeos/química , Peptídeos/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Nanopartículas/química , Nanopartículas/ultraestruturaRESUMO
Stealth agents are extensively investigated as a means by which to prolong nanostructure residence time in the bloodstream by avoiding uptake by the reticuloendothelial system. Unfortunately, commonly used agents such as poly(ethylene glycol) can adversely impact targeting efficiency and promote immune reaction by the host organism. Therefore, there is an increasing interest in developing biocompatible, non-PEGylated organic nanostructures able to perform targeted delivery to increase the efficacy of liposomal technology. Here, a lipopeptide is presented that can be mixed with lipids commonly used in liposomal formulations in percentages ranging from 20% to 60% w/w. The resulting vesicles are thermally and chemically stable. The peptide coating limits serum-protein adsorption even upon prolonged incubation in pure serum in physiological conditions, outperforming PEGylated liposomes. This architecture can be easily modified to allow straightforward derivatization by standard bio-orthogonal conjugation. Upon derivatization with an anti-transferrin receptor aptamer, these vesicles show highly selective cellular internalization with minimal nonspecific uptake and pH-triggered doxorubicin release.
Assuntos
Antineoplásicos/administração & dosagem , Preparações de Ação Retardada/química , Doxorrubicina/administração & dosagem , Lipossomos/química , Nanopartículas/química , Peptídeos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Polietilenoglicóis/químicaRESUMO
Recent FDA approvals of mRNA vaccines, short-interfering RNAs, and antisense oligonucleotides highlight the success of oligonucleotides as therapeutics. Aptamers are excellent affinity reagents that can selectively label protein biomarkers, but their clinical application has lagged. When formulating a given aptamer for in vivo use, molecular design details can determine biostability and biodistribution; therefore, extensive postselection manipulation is often required for each new design to identify clinically useful reagents harboring improved pharmacokinetic properties. Few methods are available to comprehensively screen such aptamers, especially in vivo, constituting a significant bottleneck in the field. In this study, we introduce barcoded aptamer technology (BApT) for multiplexed screening of predefined aptamer formulations in vitro and in vivo. We demonstrate this technology by simultaneously investigating 20 aptamer formulations, each harboring different molecular designs, for targeting Non-Small Cell Lung Cancer cells and tumors. Screening in vitro identified a 45 kDa bispecific formulation as the best cancer cell targeting reagent, whereas screening in vivo identified a 30 kDa monomeric formulation as the best tumor-specific targeting reagent. The multiplexed analysis pipeline also identified biodistribution phenotypes shared among formulations with similar molecular architectures. The BApT approach we describe here has the potential for broad application to fields where oligonucleotide-based targeting reagents are desired.
Assuntos
Aptâmeros de Nucleotídeos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Humanos , Animais , Camundongos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Oligonucleotídeos/química , Oligonucleotídeos/farmacocinética , Oligonucleotídeos/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Técnica de Seleção de Aptâmeros/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nanoparticles (NPs) are commonly functionalized using targeting ligands to drive their selective uptake in cells of interest. Typical target cell types are cancer cells, which often overexpress distinct surface receptors that can be exploited for NP therapeutics. However, these targeted receptors are also moderately expressed in healthy cells, leading to unwanted off-tumor toxicities. Multivalent interactions between NP ligands and cell receptors have been investigated to increase the targeting selectivity towards cancer cells due to their non-linear response to receptor density. However, to exploit the multivalent effect, multiple variables have to be considered such as NP valency, ligand affinity, and cell receptor density. Here, we synthesize a panel of aptamer-functionalized silica-supported lipid bilayers (SSLB) to study the effect of valency, aptamer affinity, and epidermal growth factor receptor (EGFR) density on targeting specificity and selectivity. We show that there is an evident interplay among those parameters that can be tuned to increase SSLB selectivity towards high-density EGFR cells and reduce accumulation at non-tumor tissues. Specifically, the combination of high-affinity aptamers and low valency SSLBs leads to increased high-EGFR cell selectivity. These insights provide a better understanding of the multivalent interactions of NPs with cells and bring the nanomedicine field a step closer to the rational design of cancer nanotherapeutics.
Assuntos
Nanopartículas , Neoplasias , Humanos , Receptores ErbB , Neoplasias/tratamento farmacológico , Linhagem Celular TumoralRESUMO
A significant fraction of non-small cell lung cancer (NSCLC) cases are due to oncogenic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR). Anti-EGFR antibodies have shown limited clinical benefit for NSCLC, whereas tyrosine kinase inhibitors (TKIs) are effective, but resistance ultimately occurs. The current landscape suggests that alternative ligands that target wild-type and mutant EGFRs are desirable for targeted therapy or drug delivery development. Here we evaluate NSCLC targeting using an anti-EGFR aptamer (MinE07). We demonstrate that interaction sites of MinE07 overlap with clinically relevant antibodies targeting extracellular domain III and that MinE07 retains binding to EGFR harboring the most common oncogenic and resistance mutations. When MinE07 was linked to an anti-c-Met aptamer, the EGFR/c-Met bispecific aptamer (bsApt) showed superior labeling of NSCLC cells in vitro relative to monospecific aptamers. However, dual targeting in vivo did not improve the recognition of NSCLC xenografts compared to MinE07. Interestingly, biodistribution of Cy7-labeled bsApt differed significantly from Alexa Fluor 750-labeled bsApt. Overall, our findings demonstrate that aptamer formulations containing MinE07 can target ectopic lung cancer without additional stabilization or PEGylation and highlights the potential of MinE07 as a targeting reagent for the recognition of NSCLC harboring clinically relevant EGFRs.
RESUMO
Evasion of immune destruction is a major hallmark of cancer. Recent US Food and Drug Administration (FDA) approvals of various immunomodulating therapies underline the important role that reprogramming the immune system can play in combating this disease. However, a wide range of side effects still limit the therapeutic potential of immunomodulators, suggesting a need for more precise reagents with negligible off-target and on-target/off-tumor effects. Aptamers are single-chained oligonucleotides that bind their targets with high specificity and affinity owing to their three-dimensional (3D) structures, and they are one potential way to address this need. In particular, bispecific aptamers (bsApts) have been shown to induce artificial immune synapses that promote T cell activation and subsequent tumor cell lysis in various in vitro and in vivo pre-clinical models. We discuss these advances here, along with gaps in bsApt biology at both the cellular and resident tissue levels that should be addressed to accelerate their translation into the clinic. The broad application, minimal production cost, and relative lack of immunogenicity of bsApts give them some ideal qualities for manipulating the immune system. Building upon lessons from other novel therapies, bsApts could soon provide clinicians with an immunomodulating toolbox that is not only potent and efficacious but exercises a wide therapeutic index.
RESUMO
Neurodegeneration is a progressive deterioration of neural structures leading to cognitive or motor impairment of the affected patient. There is still no effective therapy for any of the most common neurodegenerative diseases (NDs) such as Alzheimer's or Parkinson's disease. Although NDs exhibit distinct clinical characteristics, many are characterized by the accumulation of misfolded proteins or peptide fragments in the brain and/or spinal cord. The presence of similar inclusion bodies in patients with diverse NDs provides a rationale for developing therapies directed at overlapping disease mechanisms. A novel targeting strategy involves the use of aptamers for therapeutic development. Aptamers are short nucleic acid ligands able to recognize molecular targets with high specificity and high affinity. Despite the fact that several academic groups have shown that aptamers have the potential to be used in therapeutic and diagnostic applications, their clinical translation is still limited. In this study, we describe aptamers that have been developed against proteins relevant to NDs, including prion protein and amyloid beta (Aß), cell surface receptors and other cytoplasmic proteins. This review also describes advances in the application of these aptamers in imaging, protein detection, and protein quantification, and it provides insights about their accelerated clinical use for disease diagnosis and therapy.
Assuntos
Aptâmeros de Nucleotídeos , Príons , Peptídeos beta-Amiloides/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/uso terapêutico , Humanos , Ligantes , Fragmentos de PeptídeosRESUMO
Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19-21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50-80 kDa, 175-250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.
Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Nanoestruturas/administração & dosagem , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Cães , Sistemas de Liberação de Medicamentos , Endocitose , Endossomos/metabolismo , Corantes Fluorescentes/química , Humanos , Leucemia de Células B/genética , Leucemia de Células B/metabolismo , Leucemia de Células B/terapia , Microscopia Confocal , Nanoestruturas/química , Nanotecnologia , Conformação de Ácido NucleicoRESUMO
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.
RESUMO
RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.
Assuntos
Aptâmeros de Nucleotídeos/genética , Corantes Fluorescentes , Genes Reporter/genética , Genes de Troca/genética , Microscopia de Fluorescência/métodos , RNA/genética , Técnicas Biossensoriais/instrumentação , Genes Sintéticos/genética , Imagem Molecular/métodosRESUMO
Aptamers able to bind efficiently cell-surface receptors differentially expressed in tumor and in healthy cells are emerging as powerful tools to perform targeted anticancer therapy. Here, we present a novel oligonucleotide chimera, composed by an RNA aptamer and a DNA decoy. Our assembly is able to (i) target tumor cells via an antitransferrin receptor RNA aptamer and (ii) perform selective codelivery of a chemotherapeutic drug (Doxorubicin) and of an inhibitor of a cell-survival factor, the nuclear factor κB decoy oligonucleotide. Both payloads are released under conditions found in endolysosomal compartments (low pH and reductive environment). Targeting and cytotoxicity of the oligonucleotidic chimera were assessed by confocal microscopy, cell viability, and Western blot analysis. These data indicated that the nuclear factor κB decoy does inhibit nuclear factor κB activity and ultimately leads to an increased therapeutic efficacy of Doxorubicin selectively in tumor cells.
RESUMO
Thanks to their ability to recognize biomolecular targets with high affinity and specificity, nucleic acid aptamers are increasingly investigated as diagnostic and therapeutic tools, particularly when their targets are cell-surface receptors. Here, we investigate the relationship between the folding of an anti-mouse transferrin receptor DNA aptamer and its interaction with the transferrin receptor both in vitro and in living cells. We identified and purified two aptamer conformers by means of chromatographic techniques. Fluorescence-anisotropy measurements showed that only one fold is able to bind mouse transferrin receptor. Besides displaying enhanced endocytosis in living mouse fibroblasts, the purified active fold is internalized also in human pancreatic cancer cells. Starting from these observations, we rationally designed variations of the parent sequence aimed at stabilizing the active fold, and consequently increase aptamer activity. A truncated version and full-length mutants with higher affinity than the parent sequence are shown.Molecular Therapy-Nucleic Acids (2014) 3, e144; doi:10.1038/mtna.2013.71; published online 28 January 2014.