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In living organisms most chemical reactions take place within the confines of lipid-membrane bound compartments, while confinement within the bounds of a lipid membrane is thought to be a key step in abiogenesis. In previous work we demonstrated that confinement in the aqueous cavity of a lipid vesicle affords protection against hydrolysis, a phenomenon that we term here confinement effect (C e) and that we attributed to the interaction with the lipid membrane. Here, we show that both the size and the shape of the cavity of the vesicle modulate the C e. We link this observation to the packing of the lipid following changes in membrane curvature, and formulate a mathematical model that relates the C e to the radius of a spherical vesicle and the packing parameter of the lipids. These results suggest that the shape of the compartment where a molecule is located plays a major role in controlling the chemical reactivity of non-enzymatic reactions. Moreover, the mathematical treatment we propose offers a useful tool for the design of vesicles with predictable reaction rates of the confined molecules, e.g., drug delivery vesicles with confined prodrugs. The results also show that a crude form of signal transduction, devoid of complex biological machinery, can be achieved by any external stimuli that drastically changes the structure of the membrane, like the osmotic shocks used in the present work.
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With the aim of designing new metallosupramolecular architectures for drug delivery, research has focused on porous 3-dimensional (3D)-metallacages able to encapsulate cytotoxic agents protecting them from metabolism while targeting them to cancer sites. Here, two self-assembled [Pd2L4]4+ cages (CG1 and CG2) featuring 3,5-bis(3-ethynylpyridine)phenyl ligands (L) exo-functionalised with dipyrromethene (BODIPY) groups have been synthesised and characterised by different methods, including NMR spectroscopy and mass spectrometry. 1H NMR spectroscopy studies shows that the cages are able to encapsulate the anticancer drug cisplatin in their hydrophobic cavity, as evidenced by electrostatic potential (ESP) analysis based on XRD studies. The stability of the cages in an aqueous environment, and in the presence of the intracellular reducing agent glutathione, has been confirmed by UV-visible absorption spectroscopy. The luminescence properties of the cages enabled the investigation of their cellular uptake and intracellular localisation in human cancer cells by confocal laser scanning microscopy. In melanoma A375 cells, cage CG1 is taken up via active transport and endocytic trafficking studies show little evidence of transport through the early endosome while the cages accumulated in melanosomes rather than lysosomes. The antiproliferative activity of the lead cage was investigated in A375 together with two breast cancer cell lines, SK-BR-3 and MCF7. While the cage per se is non-cytotoxic, very different antiproliferative effects with respect to free cisplatin were evidenced for the [(cisplatin)2âCG1·BF4] complex in the various cell lines, which correlate with its different intracellular localisation profiles. The obtained preliminary results provide a new hypothesis on how the subcellular localisation of the cage affects the cisplatin intracellular release.
Asunto(s)
Cisplatino , Paladio , Compuestos de Boro , Línea Celular Tumoral , Cisplatino/química , Cisplatino/farmacología , Sistemas de Liberación de Medicamentos , Humanos , Paladio/químicaRESUMEN
In living cells, communication requires the action of membrane receptors that are activated following very small environmental changes. A binary all-or-nothing behavior follows, making the organism extremely efficient at responding to specific stimuli. Using a minimal system composed of lipid vesicles, chemical models of a membrane receptor and their ligands, we show that bio-mimetic ON/OFF assembly of high avidity, multivalent domains is triggered by small temperature changes. Moreover, the intensity of the ON signal at the onset of the switch is modulated by the presence of small, weakly binding divalent ligands, reminiscent of the action of primary messengers in biological systems. Based on the analysis of spectroscopic data, we develop a mathematical model that rigorously describes the temperature-dependent switching of the membrane receptor assembly and ligand binding. From this we derive an equation that predicts the intensity of the modulation of the ON signal by the ligand-messenger as a function of the pairwise binding parameters, the number of binding sites that it features and the concentration. The behavior of our system, and the model derived, highlight the usefulness of weakly binding ligands in the regulation of membrane receptors and the pitfalls inherent to their binding promiscuity, such as non-specific binding to the membrane. Our model, and the equations derived from it, offer a valuable tool for the study of membrane receptors in both biological and biomimetic settings. The latter can be exploited to program membrane receptor avidity on sensing vesicles, create hierarchical protocell tissues or develop highly specific drug delivery vehicles.
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The biomedical application of discrete supramolecular metal-based structures, specifically self-assembled metallacages, is still an emergent field of study. Capitalizing on the knowledge gained in recent years on the development of 3-dimensional (3D) metallacages as novel drug delivery systems and theranostic agents, we explore here the possibility to target [Pd2L4]4+ cages (L = 3,5-bis(3-ethynylpyridine)phenyl ligand) to the brain. In detail, a new water-soluble homoleptic cage (CPepH3) tethered to a blood brain barrier (BBB)-translocating peptide was synthesized by a combination of solid-phase peptide synthesis (SPPS) and self-assembly procedures. The cage translocation efficacy was assessed by inductively coupled mass spectrometry (ICP-MS) in a BBB cellular model in vitro. Biodistribution studies of the radiolabeled cage [[99mTcO4]- â CPepH3] in the CD1 mice model demonstrate its brain penetration properties in vivo. Further DFT studies were conducted to model the structure of the [[99mTcO4]- â cage] complex. Moreover, the encapsulation capabilities and stability of the cage were investigated using the [ReO4]- anion, the "cold" analogue of [99mTcO4]-, by 1H NMR spectroscopy. Overall, our study constitutes another proof-of-concept of the unique potential of supramolecular coordination complexes for modifying the physiochemical and biodistribution properties of diagnostic species.
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Barrera Hematoencefálica , Paladio/química , Animales , Teoría Funcional de la Densidad , Sistemas de Liberación de Medicamentos/métodos , Técnicas In Vitro , Ligandos , Espectrometría de Masas/métodos , Ratones , Espectroscopía de Protones por Resonancia Magnética/métodos , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Cactus fibres have previously shown unusual mechanical properties in terms of bending and axial stiffness due to their hierarchical structural morphology. Bioinspiration from those cactus fibres could potentially generate architected materials with exciting properties. To that end we have built bioinspired artificial analogues of cactus fibres to evaluate their mechanical properties. We have generated 3D printed specimens from rendered models of the cactus structure using two different printing techniques to assess the reproducibility of the structural topology. Bioinspired additive manufactured materials with unusual mechanical properties constitute an ever-evolving field for applications ranging from novel wing designs to lightweight plant-inspired analogues. The cactus-inspired 3D printed specimens developed here demonstrate an unusually high bending to axial stiffness ratios regardless of the manufacturing method used. Moreover, when compared to their equivalent beam analogues the cactus specimens demonstrate a significant potential in terms of specific (weight averaged) flexural modulus. Imaging of the artificial cactus reinforcements has enabled the generation of a one-dimensional reduced order finite element model of the cactus structure, with a distribution of cross sections along the length that simulate the inertia and mechanical behaviour of the cactus topology. The novel bioinspired material structure shows an excellent reproducibility across different manufacturing methods and suggest that the tree-like topology of the cactus fibre could be very suited to applications where high bending to axial stiffness ratios are critical.
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Materiales Biomiméticos , Cactaceae , Impresión Tridimensional , Reproducibilidad de los ResultadosRESUMEN
Recently, 3-dimensional supramolecular coordination complexes of the metallacage type have been shown to hold promise as drug delivery systems for different cytotoxic agents, including the anticancer drug cisplatin. However, so far only limited information is available on their uptake and sub-cellular localisation in cancer cells. With the aim of understanding the fate of metallacages in cells by fluorescence microscopy, three fluorescent Pd2L4 metallacages were designed and synthesised by self-assembly of two types of bispyridyl ligands (L), exo-functionalised with boron dipyrromethene (BODIPY) moieties, with Pd(II) ions. The cages show high quantum yields and are moderately stable in the presence of physiologically relevant concentration of glutathione. Furthermore, the cages are able to encapsulate the anticancer drug cisplatin, as demonstrated by NMR spectroscopy. Preliminary cytotoxicity studies in a small panel of human cancer cells showed that the metallacages are scarcely toxic in vitro. The marked fluorescence due to BODIPY allowed us to visualise the cages' uptake and sub-cellular localisation inside melanoma cells using fluorescence microscopy, highlighting uptake via active transport mechanisms and accumulation in cytoplasmic vesicles.
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Antineoplásicos/química , Compuestos de Boro/química , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/química , Cisplatino/farmacología , Glutatión/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Microscopía Fluorescente , Paladio/química , Porfobilinógeno/análogos & derivados , Porfobilinógeno/químicaRESUMEN
Within the framework of designing new self-assembled metallosupramolecular architectures for drug delivery, seven [Pd2L4]4+ metallacages (L = 2,6-bis(pyridine-3-ylethynyl)pyridine) featuring different groups in exo-position, selected to enhance the cage solubility in aqueous environment, were synthesized. Thus, carboxylic acids, sugars, and PEG groups were tethered to the bispyridyl ligands directly or via disulfide bond formation, as well as via click chemistry. The ligands and respective cages were characterized by different methods, including NMR spectroscopy and high-resolution electrospray mass spectrometry (HR-ESI-MS). While the two ligands featuring carboxylic acid-functionalized groups showed improved solubility in water, the other ligands were soluble only in organic solvents. Unfortunately, all the respective self-assembled cages were also insoluble in water. Afterwards, the encapsulation properties of the anticancer drug cisplatin in selected [Pd2L4]X4 cages (X = NO 3 - , BF 4 - ) were studied by 1H, 1H DOSY, and 195Pt NMR spectroscopy. The effect of the counter ions as well as of the polarity of the solvent in the drug encapsulation process were also investigated, and provided useful information on the host-guest properties of these experimental drug delivery systems. Our results provide further experimental support for previous studies that suggest the desolvation of guests from surrounding solvent molecules and the resulting solvent rearrangement may actually be the primary driving force for determining guest binding affinities in metallacages, in the absence of specific functional group interactions.
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Gold-based compounds are of great interest in the field of medicinal chemistry as novel therapeutic (anticancer) agents due to their peculiar reactivity and mechanisms of action with respect to organic drugs. Despite their promising pharmacological properties, the possible toxic effects of gold compounds need to be carefully evaluated in order to optimize their design and applicability. This study reports on the potential toxicity of three experimental gold-based anticancer compounds featuring lansoprazole ligands (1-3) studied in an ex vivo model, using rat precision cut kidney and liver slices (PCKS and PCLS, respectively). The results showed a different toxicity profile for the tested compounds, with the neutral complex 2 being the least toxic, even less toxic than cisplatin, followed by the cationic complex 1. The dinuclear cationic gold complex 3 was the most toxic in both liver and kidney slices. This result correlated with the metal uptake of the different compounds assessed by ICP-MS, where complex 3 showed the highest accumulation of gold in liver and kidney slices. Interestingly compound 1 showed the highest selectivity towards cancer cells compared to the healthy tissues. Histomorphology evaluation showed a similar pattern for all three Au(i) complexes, where the distal tubular cells suffered the most extensive damage, in contrast to the damage in the proximal tubules induced by cisplatin. The binding of representative gold compounds with the model ubiquitin was also studied by ESI-MS, showing that after 24 h incubation only 'naked' Au ions were bound to the protein following ligands' loss. The mRNA expression of stress response genes appeared to be similar for both evaluated organs, suggesting oxidative stress as the possible mechanism of toxicity. The obtained results open new perspectives towards the design and testing of bifunctional gold complexes with chemotherapeutic applications.
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Cisplatin occupies a crucial role in the treatment of various malignant tumors. However, its efficacy and applicability are heavily restricted by severe systemic toxicities and drug resistance. Our study exploits the active targeting of supramolecular metallacages to enhance the activity of cisplatin in cancer cells while reducing its toxicity. Thus, Pd2L4 cages (L = ligand) have been conjugated to four integrin ligands with different binding affinity and selectivity. Cage formation and encapsulation of cisplatin was proven by NMR spectroscopy. Upon encapsulation, cisplatin showed increased cytotoxicity in vitro, in melanoma A375 cells overexpressing αvß3 integrins. Moreover, ex vivo studies in tissue slices indicated reduced toxicity toward healthy liver and kidney tissues for cage-encapsulated cisplatin. Analysis of metal content by ICP-MS demonstrated that the encapsulated drug is less accumulated in these organs compared to the "free" cisplatin.
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Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Portadores de Fármacos/metabolismo , Integrina alfaVbeta3/metabolismo , Melanoma/tratamiento farmacológico , Estructuras Metalorgánicas/metabolismo , Paladio/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Humanos , Ligandos , Masculino , Melanoma/metabolismo , Estructuras Metalorgánicas/química , Paladio/química , Ratas WistarRESUMEN
The Armed Forces Health Surveillance Center's Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) supports and oversees surveillance for emerging infectious diseases, including respiratory diseases, of importance to the U.S. Department of Defense (DoD). AFHSC-GEIS accomplishes this mission by providing funding and oversight to a global network of partners for respiratory disease surveillance. This report details the system's surveillance activities during 2009, with a focus on efforts in responding to the novel H1N1 Influenza A (A/H1N1) pandemic and contributions to global public health. Active surveillance networks established by AFHSC-GEIS partners resulted in the initial detection of novel A/H1N1 influenza in the U.S. and several other countries, and viruses isolated from these activities were used as seed strains for the 2009 pandemic influenza vaccine. Partners also provided diagnostic laboratory training and capacity building to host nations to assist with the novel A/H1N1 pandemic global response, adapted a Food and Drug Administration-approved assay for use on a ruggedized polymerase chain reaction platform for diagnosing novel A/H1N1 in remote settings, and provided estimates of seasonal vaccine effectiveness against novel A/H1N1 illness. Regular reporting of the system's worldwide surveillance findings to the global public health community enabled leaders to make informed decisions on disease mitigation measures and controls for the 2009 A/H1N1 influenza pandemic. AFHSC-GEIS's support of a global network contributes to DoD's force health protection, while supporting global public health.