RESUMO
In the field of nanomedicine there is a great demand for technologies that allow the creation of self-assembled structures of which the size and morphology can be accurately controlled. In the current study, we report a nanoparticle platform that is composed of a paramagnetic lipid and a fluorescently labeled lipopeptide. By judiciously controlling the ratio of the aforementioned amphiphilic molecules, a variety of well-defined nanosized supramolecular structures with different sizes and morphologies could be created. The hydrodynamic radii of the different structures were determined by dynamic light scattering. Cryo-TEM revealed the aggregate morphology to vary from small micellar structures to plate-like and even full grown ribbons of which the aspect ratios varied from a diameter of 5-8 nm to structures with a width of up to 25 nm and infinite length. Interestingly, nuclear magnetic resonance dispersion profiling revealed excellent properties for MRI and also showed that the relaxivity of the structures was tunable and morphology dependent. Finally, macrophage cells were treated with two selected nanoparticles and were shown to be avidly taken up. In conclusion we demonstrate a methodology to create structures that (1) are paramagnetic to enable their detection with MRI, (2) exhibit fluorescent properties, (3) can be tuned to defined sizes and shapes, and (4) are efficiently taken up by macrophage cells in vitro.
Assuntos
Apolipoproteínas E/química , Fluoresceína/química , Corantes Fluorescentes/química , Macrófagos/citologia , Imageamento por Ressonância Magnética/métodos , Nanoestruturas/química , Animais , Apolipoproteínas E/farmacocinética , Células Cultivadas , Fluoresceína/farmacocinética , Corantes Fluorescentes/farmacocinética , Gadolínio DTPA/análogos & derivados , Gadolínio DTPA/química , Gadolínio DTPA/farmacocinética , Luz , Bicamadas Lipídicas/química , Macrófagos/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Espalhamento de RadiaçãoRESUMO
The peculiar properties of osmotically shrunken liposomes acting as magnetic resonance imaging-chemical exchange saturation transfer (MRI-CEST) contrast agents have been investigated. Attention has been primarily devoted to assessing the contribution arising from encapsulated and incorporated paramagnetic lanthanide(III)-based shift reagents in determining the chemical shift of the intraliposomal water protons, which is a relevant factor for generating the CEST contrast. It is demonstrated that a highly shifted resonance for the encapsulated water can be attained by increasing the percentage of the amphiphilic shift reagent incorporated in the liposome bilayer. It is also demonstrated that the shift contribution arising from the bulk magnetic susceptibility can be optimized through the modulation of the osmotic shrinkage. In terms of sensitivity, it is shown that the saturation transfer efficiency can be significantly improved by increasing the size of the vesicle, thus allowing a high number of exchangeable protons to be saturated. In addition, the role played by the intensity of the saturating radiofrequency field has also been highlighted.
Assuntos
Meios de Contraste/química , Elementos da Série dos Lantanídeos/química , Lipossomos/química , Imageamento por Ressonância Magnética/métodos , Prótons , Magnetismo , Microscopia Eletrônica de Transmissão , Osmose , Tamanho da Partícula , Temperatura , Água/químicaRESUMO
One of the major challenges of MR imaging is the quantification of local concentrations of contrast agents. Cellular uptake strongly influences different parameters such as the water exchange rate and the pool of water protons, and results in alteration of the contrast agent's relaxivity, therefore making it difficult to determine contrast agent concentrations based on the MR signal only. Here, we propose a multimodal radiolabeled paramagnetic liposomal contrast agent that allows simultaneous imaging with SPECT and MRI. As SPECT-based quantification allows determination of the gadolinium concentration, the MRI signal can be deconvoluted to get an understanding of the cellular location of the contrast agent. The cell experiments indicated a reduction of the relaxivity from 2.7 ± 0.1 m m(-1) s(-1) to a net relaxivity of 1.7 ± 0.3 m m(-1) s(-1) upon cellular uptake for RGD targeted liposomes by means of the contrast agent concentration as determined by SPECT. This is not observed for nontargeted liposomes that serve as controls. We show that receptor targeted liposomes in comparison to nontargeted liposomes are taken up into cells faster and into subcellular structures of different sizes. We suggest that the presented multimodal contrast agent provides a functional readout of its response to the biological environment and is furthermore applicable in in vivo measurements. As this approach can be extended to several MRI-based contrast mechanisms, we foresee a broader use of multimodal SPECT/MRI nanoparticles to serve as in vivo sensors in biological or medical research.
Assuntos
Meios de Contraste/administração & dosagem , Complexos de Coordenação/administração & dosagem , Lipossomos , Imageamento por Ressonância Magnética/métodos , Fosfatidiletanolaminas/administração & dosagem , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Meios de Contraste/farmacocinética , Complexos de Coordenação/análise , Sistemas de Liberação de Medicamentos , Células Endoteliais da Veia Umbilical Humana/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Lipossomos/administração & dosagem , Lipossomos/química , Lipossomos/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Oligopeptídeos/administração & dosagem , Fosfatidiletanolaminas/análise , PrótonsRESUMO
CryoTEM demonstrates that a CNA35-bearing liposomal MRI contrast agent selectively binds to poorly assembled collagen type I as opposed to well-assembled collagen fibrils, whereas monomeric CNA35 binds to all forms of collagen. It is shown that upon conjugation to liposomes and micelles CNA35 loses its ability to dissociate ordered collagen fibrils and thereby to create its own binding sites.
Assuntos
Colágeno/metabolismo , Meios de Contraste/metabolismo , Lipossomos/química , Imageamento por Ressonância Magnética/métodos , Animais , Sítios de Ligação , Colágeno/química , Colágeno/ultraestrutura , Meios de Contraste/química , Microscopia Crioeletrônica , Cavalos , Micelas , Microscopia Eletrônica de TransmissãoRESUMO
OBJECTIVES: The goal of this study was to use noninvasive conventional and molecular magnetic resonance imaging (MRI) to detect and characterize abdominal aortic aneurysms (AAAs) in vivo. BACKGROUND: Collagen is an essential constituent of aneurysms. Noninvasive MRI of collagen may represent an opportunity to help detect and better characterize AAAs and initiate intervention. METHODS: We used an AAA C57BL/6 mouse model in which a combination of angiotensin II infusion and transforming growth factor-ß neutralization results in AAA formation with incidence of aortic rupture. High-resolution, multisequence MRI was performed to characterize the temporal progression of an AAA. To allow molecular MRI of collagen, paramagnetic/fluorescent micellar nanoparticles functionalized with a collagen-binding protein (CNA-35) were intravenously administered. In vivo imaging results were corroborated with immunohistochemistry and confocal fluorescence microscopy. RESULTS: High-resolution, multisequence MRI allowed the visualization of the primary fibrotic response in the aortic wall. As the aneurysm progressed, the formation of a secondary channel or dissection was detected. Further analysis revealed a dramatic increase of the aortic diameter. Injection of CNA-35 micelles resulted in a significantly higher magnetic resonance signal enhancement in the aneurysmal wall compared with nonspecific micelles. Histological studies revealed the presence of collagen in regions of magnetic resonance signal enhancement, and confocal microscopy proved the precise co-localization of CNA-35 micelles with type I collagen. In addition, in a proof-of-concept experiment, we reported the potential of CNA-35 micelles to discriminate between stable AAA lesions and aneurysms that were likely to rapidly progress or rupture. CONCLUSIONS: High-resolution, multisequence MRI allowed longitudinal monitoring of AAA progression while the presence of collagen was visualized by nanoparticle-enhanced MRI.
Assuntos
Aneurisma da Aorta Abdominal/diagnóstico , Modelos Animais de Doenças , Imageamento por Ressonância Magnética , Animais , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Colágeno/análise , Meios de Contraste , Progressão da Doença , Aumento da Imagem , Imuno-Histoquímica , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
The preparation of particulate contrast agents for magnetic resonance imaging (MRI) based on biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanocarriers is reported. By spacer-aided covalent surface-grafting of the prominent chelating ligands diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), respectively, up to 236 µg gadolinium per mg PLGA can be immobilized in a stable manner. Due to the localisation at the particle surface, water protons may effectively interact with the gadolinium chelates and the modified particles exhibit high proton relaxivities as confirmed by T1 relaxivities of up to 17.5 mm(-1)s(-1) (25 °C, 1.41 T) in case of Gd-DOTA-functionalized carriers and also supported by NMRD profiles. The obtained values compare favorably with marketed low-molecular weight contrast agents and thus suggest suitability for in vivo use.
Assuntos
Meios de Contraste , Gadolínio DTPA/química , Compostos Heterocíclicos/química , Ácido Láctico/química , Imageamento por Ressonância Magnética , Nanosferas/química , Compostos Organometálicos/química , Ácido Poliglicólico/química , Nanosferas/ultraestrutura , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de SuperfícieRESUMO
PURPOSE: Atherosclerotic plaque macrophages express the peripheral cannabinoid receptor (CB2-R) and promote fibrous cap degradation by secretion of neutrophil gelatinase-associated lipocalin 2 (NGAL). In this study, we report the preparation, characterization, and in vitro and in vivo testing of double-labeled (MR and fluorescent) CB2-R- and NGAL-targeted micelles. PROCEDURES/RESULTS: Specific CB2-R agonists or antibodies directed to 24p3 (mouse homolog of NGAL) were incorporated into di-oleoyl-polyethylene glycol-phosphatidylethanolamine 1000 (DOPE-PEG1000) micelles or di-stearoyl-polyethylene glycol-phosphatidylethanolamine 2000 (DSPE-PEG2000) micelles. The hydrodynamic diameter, determined by dynamic light scattering, was 16.5 and 19.0 nm for CB2-R-targeted DOPE-PEG1000 and DSPE-PEG2000 micelles, respectively, and 23.0 nm for Ab-conjugated DSPE-PEG2000 micelles. In vitro and in vivo MRI and fluorescence microscopy showed specific binding of CB2-R-targeted and 24p3-targeted micelles to in vitro systems and to aortic plaque in apoE(-/-)/eNOS(-/-) mice, respectively. CONCLUSIONS: CB2-R- and NGAL-targeted micelles show promise as tools for in vivo characterization of vulnerable plaque.
Assuntos
Proteínas de Fase Aguda/metabolismo , Lipocalinas/metabolismo , Imageamento por Ressonância Magnética/métodos , Micelas , Imagem Molecular/métodos , Proteínas Oncogênicas/metabolismo , Placa Aterosclerótica/diagnóstico por imagem , Receptor CB2 de Canabinoide/metabolismo , Proteínas de Fase Aguda/antagonistas & inibidores , Animais , Células CHO , Cricetinae , Cricetulus , Estudos de Viabilidade , Lipocalina-2 , Lipocalinas/antagonistas & inibidores , Magnetismo/métodos , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Proteínas Oncogênicas/antagonistas & inibidores , Fosfatidiletanolaminas/química , Placa Aterosclerótica/metabolismo , Polietilenoglicóis/química , Radiografia , Receptor CB2 de Canabinoide/antagonistas & inibidoresRESUMO
CEST imaging is a recently introduced MRI contrast modality based on the use of endogenous or exogenous molecules whose exchangeable proton pools transfer saturated magnetization to bulk water, thus creating negative contrast. One of the critical issues for further development of these agents is represented by their limited sensitivity in vivo. The aim of this work is to improve the detection of CEST agents by exploring new approaches through which the saturation transfer (ST) effect can be enhanced. The performance of the proposed methods has been tested in vitro and in vivo using highly sensitive and highly shifted lipoCEST agents, and the results were compared with the standard ST evaluation mode. The acquired Z-spectra were interpolated locally and voxel-by-voxel by smoothing splines. Besides expressing the ST in the standard mode, we explore two methods, enhanced and integral ST, which better exploit all the information contained in the Z-spectrum. By combining different modes for ST assessment a significant improvement in the detection of the lipoCEST agents, both in vitro and in vivo, has been found. The results obtained from the application of the proposed methods outline the importance of post-processing analysis for highlighting the CEST-MRI contrast.
Assuntos
Imageamento por Ressonância Magnética/métodos , Animais , Bovinos , Linhagem Celular Tumoral , Simulação por Computador , Camundongos , Imagens de Fantasmas , Água/químicaRESUMO
The field of molecular imaging aims to visualize and quantify (patho)physiological processes at the cellular and molecular level. Sensitive and site-targeted contrast agents are employed to visualize molecular constituents of processes of interest. The principal aim of this study was to develop a magnetic resonance imaging (MRI) detectable liposome with high relaxivity and stability. To this end, Gd(III)DOTA-DSPE was synthesized and incorporated in a liposomal formulation. The resulting liposomes were extensively characterized in vitro in terms of contrast agent efficiency and structural properties. The liposomes were shown to have a high longitudinal relaxivity, which is crucial for the detection of low concentration molecular markers in molecular imaging studies. We also demonstrated that Gd(III)DOTA-DSPE exhibits no detectable transmetallation upon incubation with Zn(II). This is important as it significantly contributes to the biocompatibility of the contrast agent. The present liposome preparation will serve as versatile and well characterized platform for molecular imaging and targeted drug delivery studies.
Assuntos
Meios de Contraste , Lipossomos , Imageamento por Ressonância Magnética , Compostos Organometálicos , Fosfatidiletanolaminas , Espectroscopia de Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The cardiac muscle architecture lies at the basis of the mechanical and electrical properties of the heart, and dynamic alterations in fiber structure are known to be of prime importance in healing and remodeling after myocardial infarction. In this study, left ventricular remodeling was characterized using diffusion tensor imaging (DTI) in a mouse model of myocardial infarction. Myocardial infarction was induced in mice by permanent ligation of the left anterior descending coronary artery. Serial ex vivo DTI measurements were performed 7, 14, 28, and 60 days after ligation. Apparent diffusion coefficient, fractional anisotropy, the three eigenvalues of the diffusion tensor, and the myofiber disarray served as readout parameters. After myocardial infarction, the mouse hearts displayed extreme wall thinning in the infarcted area, which covered large parts of the apex and extended into the free wall up to the equator. Average heart mass increased by 70% 7-60 days after infarction. Histological analysis showed that the infarct at 7 days consisted of unstructured tissue with residual necrosis and infiltration of macrophages and myofibroblasts. At 14 days after infarction, the necrotic tissue had disappeared and collagen fibers were starting to appear. From 28 to 60 days, the infarct had fully developed into a mature scar. DTI parameters showed dynamic changes as a function of time after infarction. The apparent diffusion coefficient in the infarcted region was lower than in remote regions and increased as a function of time after infarction. The fractional anisotropy was higher in the infarcted region and was maximum at 28 days, which was attributed to the development of structured collagen fibers. Myofiber disarray, which was analyzed by considering the alignment of fibers in neighboring voxels, was significantly higher in infarcted regions. DTI provides a valuable non-destructive tool for characterizing structural remodeling in diseased myocardium.
Assuntos
Imagem de Difusão por Ressonância Magnética/métodos , Infarto do Miocárdio/complicações , Infarto do Miocárdio/diagnóstico , Disfunção Ventricular Esquerda/diagnóstico , Disfunção Ventricular Esquerda/etiologia , Remodelação Ventricular , Animais , Masculino , CamundongosRESUMO
Apoptosis plays an important role in the etiology of various diseases. Several studies have reported on the use of annexin A5-functionalized iron oxide particles for the detection of apoptosis with MRI, both in vitro and in vivo. The protein annexin A5 binds with high affinity to the phospholipid phosphatidylserine, which is exposed in the outer leaflet of the apoptotic cell membrane. When co-exposed to apoptotic stimuli, this protein was shown to internalize into endocytic vesicles. Therefore in the present study we investigated the possible internalization of commercially available annexin A5-functionalized iron oxide particles (r1 = 34.0 +/- 2.1 and r2 = 205.0 +/- 10.4 mm(-1) s(-1) at 20 MHz), and the effects of their spatial distribution on relaxation rates R2*, R2 and R1. Two different incubation procedures were performed, where (1) Jurkat cells were either incubated with the contrast agent after induction of apoptosis or (2) Jurkat cells were simultaneously incubated with the apoptotic stimulus and the contrast agent. Transmission electron microscopy images and relaxation rates showed that the first incubation strategy mainly resulted in binding of the annexin A5-iron oxide particles to the cell membrane, whereas the second procedure allowed extensive membrane-association as well as a small amount of internalization. Owing to the small extent of internalization, only minor differences were observed between the DeltaR2*/DeltaR2 and DeltaR2/DeltaR1 ratios of cell pellets with membrane-associated or internalized annexin A5 particles. Only the increase in R1 (DeltaR1) appeared to be diminished by the internalization. Internalization of annexin A5-iron oxide particles is also expected to occur in vivo, where the apoptotic stimulus and the contrast agent are simultaneously present. Where the extent of internalization in vivo is similar to that observed in the present study, both T2- and T2*-weighted MR sequences are considered suitable for the detection of these particles in vivo.