RESUMO
A facile, noncovalent solid-phase immobilization platform is described to assemble Janus-like supramolecular fusion proteins that are responsive to external stimuli. A chemically postmodified transporter protein, DHSA, is fused with (imino)biotinylated cargo proteins via an avidin adaptor with a high degree of spatial control. Notably, the derived heterofusion proteins are able to cross cellular membranes, dissociate at acidic pH due to the iminobiotin linker and preserve the enzymatic activity of the cargo proteins ß-galactosidase and the enzymatic subunit of Clostridium botulinum C2 toxin. The mix-and-match strategy described herein opens unique opportunities to access macromolecular architectures of high structural definition and biological activity, thus complementing protein ligation and recombinant protein expression techniques.
Assuntos
Toxinas Botulínicas/metabolismo , Albumina Sérica/metabolismo , beta-Galactosidase/metabolismo , Animais , Biotina/química , Biotina/metabolismo , Toxinas Botulínicas/química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares/química , Maleimidas/química , Maleimidas/metabolismo , Modelos Moleculares , Estrutura Molecular , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Albumina Sérica/química , beta-Galactosidase/químicaRESUMO
We describe the synthesis of a core-shell biohybrid consisting of a human serum albumin (HSA) core that serves as a reservoir for lipophilic molecules and a cationized shell region consisting of ethynyl-G2.0-PAMAM or ethynyl-G3.0-PAMAM dendrons. The binding capacity of lipophilic guests was quantified applying electron paramagnetic resonance (EPR) spectroscopy, and five to six out of seven pockets were still available compared with HSA. The attachment of ethynyl-G2.0-PAMAM dendrons to HSA yielded a nontoxic core-shell macromolecule that was clearly uptaken by A549 human epithelial cells due to the presence of the dendritic PAMAM shell. Significantly higher loading of doxorubicin was observed for dendronized G2-DHSA compared with the native protein due to the availability of binding pockets of the HSA core, and interaction with the dendritic shell. Dendronized G2-DHSA-doxorubicin displayed significant cytotoxicity resulting from high drug loading and high stability under different conditions, thus demonstrating its great potential as a transporter for drug molecules.
Assuntos
Dendrímeros/química , Dendrímeros/metabolismo , Portadores de Fármacos/farmacologia , Albumina Sérica/farmacologia , Carcinoma/tratamento farmacológico , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 7/efeitos dos fármacos , Caspase 7/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Doxorrubicina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Células Epiteliais/metabolismo , Humanos , Albumina Sérica/químicaRESUMO
Quantum dots (QDs) coated with an albumin-derived copolymer shell exhibit significant photoresponsiveness to DNA loading and have great potential for investigating gene delivery processes. The QDs reported herein are positively charged, have attractive optical properties, and are noncytotoxic and notably stable in live cells. Their complex formation with plasmid DNA leads to proportionally decreased photoluminescence and efficient gene transfection is observed. Therefore, they are suitable for live-cell bioimaging and mechanistic studies of nonviral gene delivery. Fluorescence correlation spectroscopy is applied for the first time to investigate individual QDs diffusing in large endosomes inside living cells, and serves as a valuable tool to study the physical properties of QDs inside live cells. The data obtained in this study strongly support the notable stability of these QDs, even in cell endosomes.
Assuntos
DNA/química , Técnicas de Transferência de Genes , Nanotecnologia/métodos , Pontos Quânticos , Albuminas/química , Calibragem , Cátions , Linhagem Celular Tumoral , Sobrevivência Celular , Diagnóstico por Imagem , Endossomos/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Polímeros/química , Sinais Direcionadores de Proteínas , Espectrometria de Fluorescência , TransfecçãoRESUMO
The phase separation of the polymer blend polystyrene/poly(methyl phenyl siloxane) (PS/PMPS) is studied in situ by laser scanning confocal microscopy (LSCM) and by fluorescence correlation spectroscopy (FCS) at macroscopic and microscopic length scales, respectively. It is shown for the first time that FCS when combined with LSCM can provide independent information on the local concentration within the phase-separated domains as well as the interfacial width.
Assuntos
Polímeros/química , Espectrometria de Fluorescência/métodos , Microscopia Confocal/métodos , Transição de FaseRESUMO
We report a new strategy for the preparation of well-defined and mechanically stable porous nanostructures with tunable porosity. Silica inverse opals, which are known as a model system for a porous periodic nanostructure, were grafted with brushes of the thermoresponsive poly(N-isopropylacrylamide) grown via atom transfer radical polymerization. By tuning the temperature, the swelling state of the brush layer is reversibly altered, and with this we were able to control the overall porosity of the system and, thus, the mobility of small penetrants. Fluorescence correlation spectroscopy, a method combining single molecule sensitivity with small probing volume (<1 µm3), was used to directly monitor and quantify in situ the changes in the penetrants' mobility.