RESUMO
Sonoporation is a promising method to intracellularly deliver synthetic gene carriers that have lower endocytotic uptake than viral carriers. Here, we applied sonoporation to deliver genes via polyethylene glycol (PEG)-grafted polymeric carriers that specifically respond to hyperactivated protein kinase A (PKA). PEG-grafted polymeric carrier/DNA polyplexes were not efficiently delivered into cells via the endocytotic pathway because of the hydrophilic PEG layer surrounding the polyplexes. However, the delivery of polyplexes into cells was significantly increased by sonoporation. The delivered polyplexes exhibited PKA-responsive transgene expression in PKA-overexpressing cells, but not in cells with low PKA activation. These results show that the sonoporation-mediated delivery of PEG-modified PKA-responsive polyplexes is a promising approach for safely applying gene therapy to abnormal cells with hyperactivated PKA.
Assuntos
Doença/genética , Expressão Gênica , Técnicas de Transferência de Genes , Lipossomos/química , Sonicação/métodos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Terapia Genética/métodos , Células Hep G2 , Humanos , Microbolhas , Polietilenoglicóis/química , Polímeros/química , Transfecção/métodos , Transgenes/genéticaRESUMO
Here, we report the fluorometric detection of protein kinase Cα (PKCα) activity in a cancerous cell lysate using a polyion complex (PIC) composed of a quencher (BHQ3)-modified chondroitin sulfate [CS(X)] and a dendrimer modified with a cationic peptide substrate (FKKQGSFAKKK-NH(2)) and a near infrared (NIR) fluorophore (Cy5.5) (polymer 1). When polymer 1 formed the PIC with CS(X) through electrostatic interactions, the NIR fluorescence was quenched effectively via Förster resonance energy transfer (FRET) between Cy5.5 and BHQ3. However, this quenched fluorescence was recovered when the pendant peptides in polymer 1 were phosphorylated with PKCα due to the dissociation of the PIC. When PKCα was added to the PIC dispersion, a significant increase in fluorescence intensity was observed, whereas its fluorescence increase was inhibited with a PKCα inhibitor in a concentration-dependent manner. Furthermore, our PIC was robust enough to measure PKCα activity in a cancerous cellular lysate without purification. The PKCα-responsive PIC offers a simple, rapid, sensitive, and robust approach to detect PKCα activity in crude cellular lysates that would be suitable for drug screening formats and cancer diagnosis using crude cellular lysates.
Assuntos
Técnicas Biossensoriais/métodos , Sulfatos de Condroitina/química , Dendrímeros/química , Oligopeptídeos/síntese química , Proteína Quinase C-alfa/análise , Sequência de Aminoácidos , Carbocianinas , Extratos Celulares/química , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Fluorometria , Humanos , Dados de Sequência Molecular , Fosforilação , Espectroscopia de Luz Próxima ao Infravermelho , Eletricidade EstáticaRESUMO
We have reported a disease-cell specific gene expression system that is responsive to intracellular signaling proteins (e.g., protein kinases and proteases) hyperactivated in diseased cells. For this system, cationic peptide-grafted polymers were synthesized for polyplex formation with genes. Here, we modified poly(ethylene glycol) (PEG) to a protein kinase A (PKA)-responsive polymer to improve polyplex stability. PEG modification neutralized the surface charge of the polyplex and successfully increased polyplex stability at physiological conditions. However, PEG modification (PEG contents, 0.6 and 3.3 mol %) showed almost negligible effects on the reactivity of grafted peptides to PKA and the promotion of gene expression responding to PKA activity. Excessive modification of PEG (PEG contents, 6.8 mol %) inhibited polyplex formation. These results indicate that moderate modification of PEG to the enzyme-responsive polymer improves polyplex stability without inhibiting the reaction with enzymes.
