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1.
Bioconjug Chem ; 31(12): 2737-2749, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33232126

RESUMO

Objective: Random formation of thrombi is classified as a pathological process that may result in partial or complete obstruction of blood flow and limited perfusion. Further complications include pulmonary embolism, thrombosis-induced myocardial infraction, ischemic stroke, and others. Location and full delineation of the blood clot are considered to be two clinically relevant aspects that could streamline proper diagnosis and treatment follow-up. In this work, we prepared two types of X-ray attenuating contrast formulations, using fibrinogen aptamer as the clot-seeking moiety. Methods: Two novel aptamer-targeted formulations were designed. Iodine-modified bases were directly incorporated into a fibrinogen aptamer (iodo-FA). Isothermal titration calorimetry was used to confirm that these modifications did not negatively impact target binding. Iodo-FA was tested for its ability to produce concentration-dependent contrast enhancement in a phantom CT. It was subsequently tested in vitro with clotted human and swine blood. This allowed for translation into ex vivo testing, using fluoroscopy. FA was also used to functionalize gold nanoparticles (FA-AuNPs), and contrast capabilities were confirmed. This formulation was tested in vitro using clotted human blood in a CT scan. Results: Unmodified FA and iodo-FA demonstrated a nearly identical affinity toward fibrin, confirming that base modifications did not impact target binding. Iodo-FA and FA-AuNPs both demonstrated excellent concentration-dependent contrast enhancement capabilities (40.5 HU mM-1 and 563.6 HU µM-1, respectively), which were superior to the clinically available agent, iopamidol. In vitro CT testing revealed that iodo-FA is able to penetrate into the blood clots, producing contrast enhancement throughout, while FA-AuNPs only accumulated on the surface of the clot. Iodo-FA was thereby translated to ex vivo testing, confirming target-binding associated accumulation of the contrast material at the location of the clot within the dilation of the external carotid artery. This resulted in a 34% enhancement of the clot. Conclusions: Both iodo-FA and FA-AuNPs were confirmed to be effective contrast formulations in CT. Targeting of fibrin, a major structural constituent of thrombi, with these novel contrast agents would allow for higher contrast enhancement and better clot delineation in CT and fluoroscopy.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Meios de Contraste/metabolismo , Fluoroscopia/métodos , Trombose/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Animais , Humanos , Suínos
2.
Chem Commun (Camb) ; 58(17): 2870-2873, 2022 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-35132974

RESUMO

Targeting of molecular constituents of thrombi with aptamer functionalized core-shell nanoparticles (CSN) allowed for high resolution clot delineation in T2-weighted magnetic resonance imaging. Meanwhile, the gold-coating demonstrated sufficient contrast capabilities in computed tomography (1697 HU µM-1). This targeted CSN formulation could allow for precise imaging of blood clots at low nanomolar concentrations.


Assuntos
Aptâmeros de Nucleotídeos/química , Fibrinogênio/química , Ouro/química , Nanopartículas Magnéticas de Óxido de Ferro/química , Imageamento por Ressonância Magnética , Trombose/diagnóstico por imagem , Humanos
3.
ACS Appl Mater Interfaces ; 13(8): 9412-9424, 2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33395250

RESUMO

Objective: An important clinical question in the determination of the extent of thrombosis-related vascular conditions is the identification of blood clot location. Fibrin is a major molecular constituent of blood clots and can, therefore, be utilized in molecular imaging. In this proof-of-concept study, we sought to prepare a fibrin-targeting magnetic resonance imaging contrast agent, using a Gd(III)-loaded fibrinogen aptamer (FA) chelate conjugate (Gd(III)-NOTA-FA) (NOTA = 1,4,7-triazacyclononane-1,4,7-triacetic acid), to endow the ability to specifically accumulate at the location of blood clots, thereby enhancing contrast capabilities. Methods: The binding affinity of FA for fibrin was confirmed by fluorescence microscopy and microscale thermophoresis. The preparation and effective loading of the chelate-aptamer conjugates were confirmed by mass spectrometry and a xylenol orange colorimetric test. Longitudinal and transverse relaxivities and the effects of target binding were assessed using T1- and T2-map sequences at 7 T. T1- and T2-weighted images were acquired after blood clots were treated with Gd(III)-NOTA-FA. Collagen was used as the protein control, while an unrelated aptamer sequence, FB139, was used as the aptamer control. Results: FA demonstrated a high affinity and selectivity toward the polymeric protein, with a Kd of 16.6 nM, confirming an avidity over fibrinogen. The longitudinal (r1) and transverse (r2) relaxivities of Gd(III)-NOTA-FA demonstrated that conjugation to the long aptamer strand shortened T1 relaxation times and increased T2 relaxation times (3.04 and 38.7 mM-1 s-1, respectively). These effects were amplified by binding to the fibrin target (1.73 and 46.5 mM-1 s-1, respectively). In vitro studies with thrombin-polymerized human blood (clots) in whole blood showed an unexpected enhancement of signal intensity (hyperintense) produced exclusively at the location of the clot during the T2-weighted scan, while the presence of fibrinogen within a whole blood pool resulted in T1 signal intensity enhancement throughout the pool. This is advantageous, as simply reversing the type of a scan from a typical T1-weighted to a T2-weighted would allow to selectively highlight the location of blood clots. Conclusions: Gd(III)-NOTA-FA can be used for molecular imaging of thrombi, through fibrin-targeted delivery of contrast to the location of blood clots in T2-weighted scans.


Assuntos
Aptâmeros de Nucleotídeos/química , Meios de Contraste/química , Complexos de Coordenação/química , Trombose/diagnóstico por imagem , Aptâmeros de Nucleotídeos/metabolismo , Meios de Contraste/metabolismo , Complexos de Coordenação/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Gadolínio/química , Humanos , Imageamento por Ressonância Magnética , Estudo de Prova de Conceito , Trombose/metabolismo
4.
ACS Omega ; 5(36): 22691-22701, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32954116

RESUMO

Computed tomography (CT) and magnetic resonance imaging (MRI) are among the most well-established modalities in the field of noninvasive medical imaging. Despite being powerful tools, both suffer from a number of limitations and often fall short when it comes to full delineation of pathological tissues. Since its conception, molecular imaging has been commonly utilized to further the understanding of disease progression, as well as monitor treatment efficacy. This has naturally led to the advancement of the field of targeted imaging. Targeted imaging research is currently dominated by ligand-modified contrast media for applications in MRI and CT imaging. Although a plethora of targeting ligands exist, a fine balance between their size and target binding efficiency must be considered. This review will focus on aptamer- and peptide-modified contrast agents, outlining selected formulations developed in recent years while highlighting the advantages offered by these targeting ligands.

5.
ACS Chem Neurosci ; 10(1): 371-383, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30160936

RESUMO

The delivery of therapeutics across the blood-brain barrier remains a considerable challenge in investigating central nervous system related processes. In this work, a liposome vehicle was surface-modified with an aptamer that binds to the transferrin receptor and was loaded with two different dopamine-binding aptamer payloads. This system was effectively used to promote the delivery of the aptamer cargo from the peripheral injection site into the brain. The effect of these delivered aptamers on behavior was investigated in vivo in a locomotor task. The first dopamine binding aptamer assessed was a DNA aptamer, the binding of which had been previously validated through the aptamer-based biosensor development reported by several independent research groups. The second aptamer investigated was the result of a novel in vitro selection experiment described herein. Our data suggest that systemic administration of the modified liposomes led to delivery of the dopamine aptamers into the brain. Fluorescence microscopy revealed differential distribution of fluorescence based on the presence or absence of the transferrin receptor aptamer on the surface of fluorescently modified liposomes. In a behavioral experiment using cocaine administration to induce elevated concentrations of neural dopamine, systemic pretreatment with the dopamine aptamer-loaded liposomes reduced cocaine-induced hyperlocomotion. Multiple controls including a transferrin-negative liposome control and transferrin-positive liposomes loaded with either a nonbinding, base-substituted dopamine aptamer or a random oligonucleotide were investigated. None of these controls altered cocaine-induced hyperlocomotion. Chronic systemic administration of the modified liposomes produced no deleterious neurobehavioral or neural degenerative effects. Importantly, this work is one example of an application for this versatile multiaptamer payload/targeting system. Its general application is limited only by the availability of aptamers for specific neural targets.


Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/metabolismo , Encéfalo/metabolismo , Dopamina/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Locomoção/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Humanos , Locomoção/efeitos dos fármacos , Masculino , Camundongos
6.
Curr Protoc Chem Biol ; 9(4): 233-268, 2017 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-29241295

RESUMO

Aptamers, synthetic oligonucleotide-based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets. © 2017 by John Wiley & Sons, Inc.


Assuntos
Aptâmeros de Nucleotídeos/análise , Aptâmeros de Nucleotídeos/química , Técnicas de Química Combinatória , Bibliotecas de Moléculas Pequenas/química , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais , Sequenciamento de Nucleotídeos em Larga Escala
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