RESUMO
With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (RF ) group adjacent to the α-carbon. RF -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. RF -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (RF -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel RF -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs).
Assuntos
Antineoplásicos , Peptídeos Penetradores de Células , Fluorocarbonos , Transporte Biológico , Peptídeos Penetradores de Células/química , Aminoácidos/metabolismoRESUMO
Nanocapsules that collapse in response to guanosine triphosphate (GTP) have the potential as drug carriers for efficiently curing diseases caused by cancer and RNA viruses because GTP is present at high levels in such diseased cells and tissues. However, known GTP-responsive carriers also respond to adenosine triphosphate (ATP), which is abundant in normal cells as well. Here, we report the elaborate reconstitution of microtubule into a nanocapsule that selectively responds to GTP. When the tubulin monomer from microtubule is incubated at 37 °C with a mixture of GTP (17 mol%) and nonhydrolysable GTP* (83 mol%), a tubulin nanosheet forms. Upon addition of photoreactive molecular glue to the resulting dispersion, the nanosheet is transformed into a nanocapsule. Cell death results when a doxorubicin-containing nanocapsule, after photochemically crosslinked for properly stabilizing its shell, is taken up into cancer cells that overexpress GTP.
Assuntos
Nanocápsulas , Tubulina (Proteína) , Trifosfato de Adenosina/metabolismo , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
We developed a photoreactive molecular glue, BPGlue-N3, which can provide a universal strategy to enhance the efficacy of DNA aptamers by temporary-to-permanent stepwise stabilization of their conjugates with target proteins. As a proof-of-concept study, we applied BPGlue-N3 to the SL1 (DNA aptamer)/c-Met (target protein) conjugate system. BPGlue-N3 can adhere to and temporarily stabilize this aptamer/protein conjugate multivalently using its guanidinium ion (Gu+) pendants that form a salt bridge with oxyanionic moieties (e.g., carboxylate and phosphate) and benzophenone (BP) group that is highly affinitive to DNA duplexes. BPGlue-N3 is designed to carry a dual-mode photoreactivity; upon exposure to UV light, the temporarily stabilized aptamer/protein conjugate reacts with the photoexcited BP unit of adhering BPGlue-N3 and also a nitrene species, possibly generated by the BP-to-N3 energy transfer in BPGlue-N3. We confirmed that SL1, covalently conjugated with c-Met, hampered the binding of hepatocyte growth factor (HGF) onto c-Met, even when the SL1/c-Met conjugate was rinsed prior to the treatment with HGF, and suppressed cell migration caused by HGF-induced c-Met phosphorylation.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Aptâmeros de Nucleotídeos/química , Azidas/química , Benzofenonas/química , Linhagem Celular Tumoral , Movimento Celular , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Microscopia Confocal , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/química , Raios UltravioletaRESUMO
Transferrin (Tf) is known to induce transcytosis, which is a consecutive endocytosis/exocytosis event. We developed a Tf-appended nanocaplet (TfNCâsiRNA) for the purpose of realizing siRNA delivery into deep tissues and RNA interference (RNAi) subsequently. For obtaining TfNCâsiRNA, a macromonomer (AzGu) bearing multiple guanidinium (Gu+) ion units, azide (N3) groups, and trityl (Trt)-protected thiol groups in the main chain, side chains, and termini, respectively, was newly designed. Because of a multivalent Gu+-phosphate salt-bridge interaction, AzGu can adhere to siRNA along its strand. When I2 was added to a preincubated mixture of AzGu and siRNA, oxidative polymerization of AzGu took place along the siRNA strand, affording AzNCâsiRNA, the smallest siRNA-containing reactive nanocaplet so far reported. This conjugate was converted into Glue/BPNCâsiRNA by the click reaction with a Gu+-appended bioadhesive dendron (Glue) followed by a benzophenone derivative (BP). Then, Tf was covalently immobilized onto Glue/BPNCâsiRNA by Gu+-mediated adhesion followed by photochemical reaction with BP. With the help of Tf-induced transcytosis, TfNCâsiRNA permeated deeply into a cancer spheroid, a 3D tissue model, at a depth of up to nearly 70 µm, unprecedentedly.