RESUMO
In vivo tumor targeting and drug delivery properties of small polymerized polydiacetylene (PDA) micelles (â¼10 nm) is investigated in a murine MDA-MB-231 xenograft model of breast cancer. Three micelles with different surface coatings are synthesized and tested for their ability to passively target tumor through the enhanced permeability and retention effect. After injection (24 h), fluorescence diffuse optical tomographic imaging indicates a tumor uptake of nearly 3% of the injected dose for the micelles with a 2 kDa poly(ethylene glycol) (PEG)-coating (PDA-PEG2000). The uptake of PDA micelles in tumors is confirmed by co-localization with [(18) F]-fluorodeoxyglucose (FDG) positron emission tomography. Although FDG has a higher diffusion rate in tumors, 40 ± 19% of the retained micelles is co-registered with the tumor volume visualized by FDG. Finally, PDA-PEG2000 micelles are loaded with the hydrophobic anticancer drug paclitaxel and used in vivo to inhibit tumor growth. These findings demonstrate the potential of PDA-PEG2000 micelles for both in vivo tumor imaging and drug delivery applications.
Assuntos
Diagnóstico por Imagem , Sistemas de Liberação de Medicamentos/métodos , Micelas , Neoplasias/metabolismo , Polímeros/química , Poli-Inos/química , Animais , Peso Corporal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Injeções Intravenosas , Camundongos , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Paclitaxel/farmacologia , Polímero Poliacetilênico , Polímeros/administração & dosagem , Polímeros/farmacocinética , Poli-Inos/administração & dosagem , Poli-Inos/farmacocinética , Tomografia por Emissão de Pósitrons , Espectroscopia de Luz Próxima ao Infravermelho , Distribuição Tecidual/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: 6-[(18)F]Fluoro-3,4-dihydroxy-L-phenylalanine (6-[(18)F]F-L-DOPA) is increasingly used for PET imaging of neuroendocrine tumours. In this study, we investigated the use of 6-[(18)F]F-L-DOPA to detect and to monitor the progression of medullary thyroid carcinoma (MTC) in a genetically engineered mouse model of multiple endocrine neoplasia type 2A (MEN2A). METHODS: Dynamic [(18)F]FDG and 6-[(18)F]F-L-DOPA small animal PET scans were acquired during 60 or 90 min in 8- to 20-month-old MEN2A transgenic mice. The kinetics of 6-[(18)F]F-L-DOPA, standardized uptake values (SUV) at 60 min and tumour volumes were recorded. The detection of MTCs using PET was confirmed by autopsy and histological analysis. RESULTS: 6-[(18)F]F-L-DOPA performs better than [(18)F]FDG for MTC detection in this transgenic mouse model. Uptake kinetics of 6-[(18)F]F-L-DOPA in MTCs are very different between mice but, in all cases, high contrast could be observed. Furthermore, 6-[(18)F]F-L-DOPA can detect tumours with sizes (1.8 mm(3)) that are near the resolution limit of PET, whereas they were undetectable by autopsy at the macroscopic level. CONCLUSION: 6-[(18)F]F-L-DOPA PET imaging can monitor the progression of MTCs in a genetically engineered mouse model.
Assuntos
Carcinoma Medular/diagnóstico por imagem , Di-Hidroxifenilalanina/análogos & derivados , Neoplasia Endócrina Múltipla Tipo 2a/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Animais , Camundongos , Camundongos Transgênicos , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The present study aims at investigating the mechanism by which membrane-type 4 matrix metalloproteinase (MT4-MMP), a membrane-anchored MMP expressed by human breast tumour cells promotes the metastatic dissemination into lung. We applied experimental (intravenous) and spontaneous (subcutaneous) models of lung metastasis using human breast adenocarcinoma MDA-MB-231 cells overexpressing or not MT4-MMP. We found that MT4-MMP does not affect lymph node colonization nor extravasation of cells from the bloodstream, but increases the intravasation step leading to metastasis. Ultrastructural and fluorescent microscopic observations coupled with automatic computer-assisted quantifications revealed that MT4-MMP expression induces blood vessel enlargement and promotes the detachment of mural cells from the vascular tree, thus causing an increased tumour vascular leak. On this basis, we propose that MT4-MMP promotes lung metastasis by disturbing the tumour vessel integrity and thereby facilitating tumour cell intravasation.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias Pulmonares/enzimologia , Metaloproteinase 17 da Matriz/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/secundário , Metástase Linfática , Neoplasias Mamárias Animais/irrigação sanguínea , Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Nus , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Transplante de Neoplasias , Neovascularização Patológica , Pericitos/metabolismoRESUMO
We have designed new nanoprobes applicable for both positron emission tomography (PET) and optical fluorescence in vivo imaging. Fluorine-18, which is commonly used for clinical imaging, has been coupled to phospholipid quantum dot (QD) micelles. This probe was injected in mice and we demonstrated that its dynamic quantitative whole body biodistribution and pharmacokinetics could be monitored using PET as well as the kinetics of their cellular uptake using in vivo fibered confocal fluorescence imaging. Phospholipid micelle encapsulation of QDs provides a highly versatile surface chemistry to conjugate multiple chemicals and biomolecules with controlled QD:molecule valency. Here, we show that, in contrast with several previous studies using other QD polymer coatings, these phospholipid QD micelles exhibit long circulation half-time in the bloodstream (on the order of 2 h) and slow uptake by reticulo-endothelial system.
Assuntos
Diagnóstico por Imagem/métodos , Radioisótopos de Flúor/química , Nanotecnologia/métodos , Fosfolipídeos/química , Pontos Quânticos , Imagem Corporal Total/métodos , Animais , Compostos de Cádmio/síntese química , Membrana Celular/metabolismo , Cinética , Camundongos , Camundongos Nus , Micelas , Microscopia de Fluorescência/métodos , Nanoestruturas/química , Tomografia por Emissão de Pósitrons/métodos , Compostos de Selênio/síntese químicaRESUMO
Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.
Assuntos
Proteínas Proto-Oncogênicas c-ret/genética , Técnica de Seleção de Aptâmeros/métodos , Células 3T3 , Substituição de Aminoácidos , Animais , Sequência de Bases , Chlorocebus aethiops , Inibidores Enzimáticos , Humanos , Camundongos , Dados de Sequência Molecular , Neoplasia Endócrina Múltipla Tipo 2b/genética , Células PC12 , Feocromocitoma , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Ratos , Transdução de Sinais , TransfecçãoRESUMO
Binding of aptamers is dependent on their target conformation, which in turn is conditioned by the target's environment. Therefore, selection of aptamers against the active forms of membrane proteins could require their correct membrane insertion in order to maintain their native conformation. Here, we compare different SELEX strategies to identify aptamers against the mutated form of the membrane receptor tyrosine kinase RET(C634Y). (1) selections S1 and S2 against living cells transformed to express the protein yielded a minority of RET-targeted aptamers while the bulk of aptamers recognized more abundant membrane proteins, suggesting that a high level of expression of the target protein is crucial to allow the isolation of aptamers at cell surface; (2) selection S3 against the purified extracellular moiety of RET yielded aptamers unable to recognize RET expressed at the cell membrane; (3) crossover selections S4 and S5 alternating cells and recombinant RET enhanced the enrichment of the aptamers directed against RET; however, these aptamers displayed a weaker affinity for Ret than those obtained with S1 and S2. In our case, using transformed cell lines as the partitioning matrix during SELEX appears to be essential in order to obtain aptamers able to recognize the RET receptor tyrosine kinase in its physiologic environment.
Assuntos
Aptâmeros de Nucleotídeos/isolamento & purificação , Aptâmeros de Nucleotídeos/metabolismo , Proteínas de Membrana/metabolismo , Técnica de Seleção de Aptâmeros/métodos , Substituição de Aminoácidos , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Reagentes de Ligações Cruzadas , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Células PC12 , Polimorfismo de Fragmento de Restrição , Ligação Proteica , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Fluorescence imaging techniques could be used in different ways to study the interaction of aptamers with biological systems from cell culture to animal models. Here, we present the methods developed in our laboratory for fluorescently labeled aptamers, study their internalization inside living cells using time-lapse microscopy, and monitor their biodistribution in mice bearing subcutaneous xenograft tumors using planar fluorescence imaging and fluorescence diffuse optical tomography (fDOT).
Assuntos
Aptâmeros de Nucleotídeos , Corantes Fluorescentes , Imagem Molecular/métodos , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Corantes Fluorescentes/química , Xenoenxertos , Humanos , Imageamento Tridimensional/métodos , Técnicas In Vitro , Camundongos , Camundongos Nus , Microscopia de Vídeo/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Coloração e Rotulagem , Imagem com Lapso de Tempo/métodos , Tomografia Óptica/métodosRESUMO
BACKGROUND: Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged. METHODOLOGY/PRINCIPAL FINDINGS: Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence. CONCLUSIONS/SIGNIFICANCE: Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application.
Assuntos
Adenocarcinoma/diagnóstico , Anexina A2/metabolismo , Aptâmeros de Nucleotídeos , Neoplasias da Mama/diagnóstico , Proteínas de Neoplasias/metabolismo , Adenocarcinoma/genética , Animais , Anexina A2/genética , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/isolamento & purificação , Neoplasias da Mama/genética , Células CHO , Cricetulus , Endocitose , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Achados Incidentais , Ligantes , Células MCF-7 , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Ligação Proteica , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Técnica de Seleção de AptâmerosRESUMO
Polydiacetylene (PDA) micelles coated with either carboxylate-, ammonium-, or methoxy-polyethyleneglycol (PEG) chains were assembled and loaded with a fluorescent dye (DiO). Their interaction with MCF-7 human breast tumor cells was investigated by epi-fluorescence microscopy and fluorescence-activated cell sorting (FACS) to determine their internalization pathway and intracellular fate. It was found that the ionic character of the micelles influenced their internalization kinetics through a caveolae-mediated pathway and that all micelle types behaved somewhat similarly inside cells.