RESUMEN
Protein transduction domains comprised of basic amino acid-rich peptides, can efficiently deliver covalently fused macromolecules into cells. Quantum dots (QDs) are luminescent semiconductor nanocrystals that are finding increasing application in biological imaging. Previous studies showed that protein transduction domains mediate the internalization of covalently attached QDs. In this study, we demonstrate that arginine-rich intracellular delivery peptides (cell-penetrating peptides; CPPs), analogs of naturally-occuring protein transduction domains, deliver noncovalently associated QDs into living cells; CPPs dramatically increase the rate and efficiency of cellular uptake of QD probes. The optimal molecular ratio between arginine-rich CPPs and QD cargoes for cellular internalization is approximately 60:1. Upon entry into cells, the QDs are concentrated in the perinuclear region. There is no cytotoxicity following transport of QDs present at concentrations up to 200 nM. The mechanism for arginine-rich CPP/QD complexes to traverse cell membrane appears to involve a combination of internalization pathways. These results provide insight into the mechanism of arginine-rich CPP delivery of noncovalently attached cargoes, and may provide a powerful tool for imaging in vivo.
Asunto(s)
Péptidos de Penetración Celular/farmacocinética , Péptidos/farmacocinética , Puntos Cuánticos , Análisis de Varianza , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Ensayo de Cambio de Movilidad Electroforética , Endocitosis/efectos de los fármacos , Humanos , Tamaño de la Partícula , Péptidos/química , Transporte de Proteínas/efectos de los fármacos , Espectrometría de FluorescenciaRESUMEN
Protein transduction domains (PTDs) are small peptides with a high content of basic amino acids, and they are responsible for cellular uptake. Many PTDs, including arginine-rich intracellular delivery (AID) peptides, have been shown to transport macromolecules across membranes and into cells. In this study, we demonstrated for the first time that AID peptides could rapidly and efficiently deliver proteins into plant cells in both covalent and noncovalent protein transductions (CNPT) simultaneously. The optimal molecular ratio between an AID peptide carrier and cargo in CNPT was about 3:1. Fluorescence resonance energy transfer (FRET) analysis revealed protein-protein interactions between AID peptide carriers and cargos after CNPT in cells. The possible mechanisms of AID peptides-mediated cellular entry might involve a combination of multiple internalization pathways. Therefore, applications by AID peptide-mediated CNPT may provide a simple and direct transport strategy for delivering two proteins in agricultural systems.
Asunto(s)
Arginina/metabolismo , Péptidos/genética , Transporte Biológico , Transferencia Resonante de Energía de Fluorescencia , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Cinética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Microscopía Confocal/métodos , Péptidos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plásmidos , Prunus/genética , Prunus/metabolismo , Mapeo Restrictivo , Transducción Genética/métodosRESUMEN
Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of fluorescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.