RESUMO
Nanoparticles are target-specific drug delivery agents that are increasingly used in cancer therapy to enhance bioavailability and to reduce off target toxicity of anti-cancer agents. Valrubicin is an anti-cancer drug, currently approved only for vesicular bladder cancer treatment because of its poor water solubility. On the other hand, valrubicin carrying reconstituted high density lipoprotein (rHDL) nanoparticles appear ideally suited for extended applications, including systemic cancer chemotherapy. We determined selected fluorescence properties of the free (unencapsulated) drug vs. valrubicin incorporated into rHDL nanoparticles. We have found that upon encapsulation into rHDL nanoparticles the quantum yield of valrubicin fluorescence increased six fold while its fluorescence lifetime increased about 2 fold. Accordingly, these and potassium iodide (KI) quenching data suggest that upon incorporation, valrubicin is localized deep in the interior of the nanoparticle, inside the lipid matrix. Fluorescence anisotropy of the rHDL valrubicin nanoparticles was also found to be high along with extended rotational correlation time. The fluorescence of valrubicin could also be utilized to assess its distribution upon delivery to prostate cancer (PC3) cells. Overall the fluorescence properties of the rHDL: valrubicin complex reveal valuable novel characteristics of this drug delivery vehicle that may be particularly applicable when used in systemic (intravenous) therapy.
Assuntos
Antineoplásicos/química , Meios de Contraste/química , Doxorrubicina/análogos & derivados , Lipoproteínas HDL/química , Nanopartículas/química , Antineoplásicos/metabolismo , Apolipoproteína A-I/química , Apolipoproteína A-I/metabolismo , Linhagem Celular Tumoral , Meios de Contraste/metabolismo , Doxorrubicina/química , Doxorrubicina/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Microscopia Confocal , Espectrometria de Fluorescência , TemperaturaRESUMO
We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype.
Assuntos
Cardiomiopatia Hipertrófica/genética , Mutação , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/genética , Músculos Papilares/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Diástole , Regulação da Expressão Gênica , Frequência Cardíaca , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Relaxamento Muscular , Contração Miocárdica , Miócitos Cardíacos/patologia , Cadeias Leves de Miosina/metabolismo , Músculos Papilares/diagnóstico por imagem , Músculos Papilares/patologia , Cultura Primária de Células , Transdução de Sinais , Técnicas de Cultura de Tecidos , Ultrassonografia Doppler de PulsoRESUMO
Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to â¼20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges.