RESUMO
Lipospheres made from soy bean oil and a combination of the cationic lipid Metafectene and the helper lipid dioleoylphosphatidyl-ethanolamine were functionalized with magnetic nanoparticles (NPs) and small interfering RNA (siRNA). The resulting magnetic lipospheres loaded with siRNA are proven here as efficient nonviral vectors for gene silencing. Embedding magnetic NPs in the shell of lipospheres allows for magnetic force-assisted transfection (magnetofection) as well as magnetic targeting in both static and fluidic conditions mimicking the bloodstream.
Assuntos
Inativação Gênica , Lipídeos/química , Lipossomos/química , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Genes Reporter , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Magnetismo , Camundongos , Células NIH 3T3 , Fosfatidiletanolaminas/química , Óleos de Plantas/química , Glycine max/químicaRESUMO
A flow-cytometry-based assay is presented with which the uptake of polyelectrolyte capsules can be quantified. The cavity of the capsules is loaded with the pH-sensitive dye SNARF, which emits in the red and green in alkaline and acidic environments, respectively. By recording the fluorescence intensities in the red and green channels, the localization of capsules associated with cells can be determined. Capsules adherent to the outer cell membrane fluoresce in the red due to the alkaline pH of the cell medium, whereas capsules internalized by cells fluoresce in the green due to the acidic pH in the endosomal/lysosomal/phagosomal compartments in which incorporated capsules are located. Adding the SNARF readout to the scattering signal typically derived with flow cytometry analysis allows for a more detailed quantitative analysis of particle uptake, which can also distinguish between adherent and ingested particles.
Assuntos
Bioensaio/métodos , Cápsulas/metabolismo , Citometria de Fluxo/métodos , Polímeros/metabolismo , Animais , Linhagem Celular , Humanos , CamundongosRESUMO
We describe recent developments with multifunctional nanoengineered polymer capsules. In addition to their obvious use as a delivery system, multifunctional nanocontainers find wide application in enzymatic catalysis, controlled release, and directed drug delivery in medicine. The multifunctionality is provided by the following components: 1) Luminescent semiconductor nanocrystals (quantum dots) that facilitate imaging and identification of different capsules, 2) superparamagnetic nanoparticles that allow manipulation of the capsules in a magnetic field, 3) surface coatings, which target the capsules to desired cells, 4) metallic nanoparticles in the capsule wall that act as an absorbing antenna for electromagnetic fields and provide heat for controlled release, and 5) enzymes and pharmaceutical agents that allow specific reactions. The unique advantage of multifunctional microcapsules in comparison to other systems is that they can be simultaneously loaded/functionalized with the above components, allowing for the combination of their properties in a single object.
Assuntos
Sistemas de Liberação de Medicamentos , Nanocápsulas/administração & dosagem , Polímeros/administração & dosagem , Animais , Morte Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Corantes , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/administração & dosagem , Magnetismo , Permeabilidade , Polímeros/química , Pró-Fármacos , Ratos , Espectrometria de Fluorescência , Propriedades de Superfície , Células VeroRESUMO
We propose a combination of atomic force microscopy (AFM) and optical microscopy for the investigation of particle uptake by cells. Positively and negatively charged polymer microcapsules were chosen as model particles, because their interaction with cells had already been investigated in detail. AFM measurements allowed the recording of adhesion forces on a single-molecule level. Due to the micrometer size of the capsules, the number of ingested capsules could be counted by optical microscopy. The combination of both methods allowed combined measurement of the adhesion forces and the uptake rate for the same model particle. As a demonstration of this system, the correlation between the adhesion of positively or negatively charged polymer microcapsules onto cell surfaces and the uptake of these microcapsules by cells has been investigated for several cell lines. As is to be expected, we find a correlation between both processes, which is in agreement with adsorption-dependent uptake of the polymer microcapsules by cells.
Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Microesferas , Polímeros/química , Polímeros/farmacocinética , Adesividade , Movimento (Física)RESUMO
Cytotoxicity of CdSe and CdSe/ZnS nanoparticles has been investigated for different surface modifications such as coating with mercaptopropionic acid, silanization, and polymer coating. For all cases, quantitative values for the onset of cytotoxic effects in serum-free culture media are given. These values are correlated with microscope images in which the uptake of the particles by the cells has been investigated. Our data suggest that in addition to the release of toxic Cd(2+) ions from the particles also their surface chemistry, in particular their stability toward aggregation, plays an important role for cytotoxic effects. Additional patch clamp experiments investigate effects of the particles on currents through ion channels.