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In the treatment of hormone-dependent cancers, aromatase inhibitors (AI) are receiving increased attention due to some undesirable effects such as the risk of endometrial cancer and thromboembolism of SERMs (selective estrogen receptor modulators). Letrozole is the most active AI with 99% aromatase inhibition. Unfortunately, this compound also exhibits some adverse effects such as hot flashes and fibromyalgias. Therefore, there is an urgent need to explore new types of AIs that retain the same-or even increased-antitumor ability. Inspired by the letrozole structure, a set of new derivatives has been synthesized that include a ferrocenyl moiety and different heterocycles. The derivative that contains a benzimidazole ring, namely compound 6, exhibits a higher aromatase inhibitory activity than letrozole and it also shows potent cytostatic behavior when compared to other well-established aromatase inhibitors, as demonstrated by dose-response, cell cycle, apoptosis and time course experiments. Furthermore, 6 promotes the inhibition of cell growth in both an aromatase-dependent and -independent fashion, as indicated by the study of A549 and MCF7 cell lines. Molecular docking and molecular dynamics calculations on the interaction of 6 or letrozole with the aromatase binding site revealed that the ferrocene moiety increases the van der Waals and hydrophobic interactions, thus resulting in an increase in binding affinity. Furthermore, the iron atom of the ferrocene fragment can form a metal-acceptor interaction with a propionate fragment, and this results in a stronger coupling with the heme group-a possibility that is consistent with the strong aromatase inhibition of 6.
Assuntos
Neoplasias da Mama , Citostáticos , Humanos , Feminino , Letrozol/farmacologia , Inibidores da Aromatase/farmacologia , Inibidores da Aromatase/química , Aromatase/metabolismo , Metalocenos , Simulação de Acoplamento Molecular , Nitrilas/farmacologia , Triazóis/farmacologia , Células MCF-7RESUMO
Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomeric subunits that are non-covalently linked through exceptionally strong and kinetically inert interactions into a linear polymer chain. It has been shown that after its thermal depolymerisation into unfolded monomeric subunits, Caf1 is able to efficiently repolymerise in vitro to reform its polymeric structure. However, little is known about the nature of the repolymerisation process. An improved understanding of this process will lead to the development of methods to better control the lengths of the repolymerised species, and ultimately, to better design of the properties of Caf1-based materials. Here we utilize small-angle X-ray scattering to estimate the size of Caf1 polymers during the first 24 h of the re-polymerisation process. Analytical ultracentrifugation measurements were also used to investigate the process post-24 h, where the rate of repolymerisation becomes considerably slower. Results show that in vitro polymerisation proceeds in a linear manner with no evidence observed for the formation of a lateral polymer network or uncontrolled aggregates. The rate of Caf1 in vitro repolymerisation was found to be concentration-dependent. Importantly, the rate of polymer growth was found to be relatively fast over the first few hours, before continuing at a dramatically slower rate. This observation is not consistent with the previously proposed step-growth mechanism of in vitro polymerisation of Caf1, where a linear increase in polymer length would be expected with time. We speculate how our observations may support the idea that the polymerisation process may be occurring at the ends of the chains with monomers adding sequentially. Our findings will contribute towards the development of new biomaterials for 3D cell culture and bio-printing.
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Fímbrias Bacterianas , Materiais Biocompatíveis , Polímeros , Ultracentrifugação , Raios XRESUMO
Mesoporous carbonaceous materials (Starbons®) derived from low-value/waste bio-resources separate CO2 from CO2 /N2 mixtures. Compared to Norit activated charcoal (AC), Starbons® have much lower microporosities (8-32 % versus 73 %) yet adsorb up to 65 % more CO2 . The presence of interconnected micropores and mesopores is responsible for the enhanced CO2 adsorption. The Starbons® also showed three-four times higher selectivity for CO2 adsorption rather than N2 adsorption compared to AC.
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One approach to reduce the side effects of chemotherapy in cancer treatment is photodynamic therapy (PDT), which allows spatiotemporal control of the cytotoxicity. We have used the strategy of coordinating π-expansive ligands to increase the excited state lifetimes of Ir(III) half-sandwich complexes in order to facilitate the generation of 1O2. We have obtained derivatives of formulas [Cp*Ir(Câ§N)Cl] and [Cp*Ir(Câ§N)L]BF4 with different degrees of π-expansion in the Câ§N ligands. Complexes with the more π-expansive ligand are very effective photosensitizers with phototoxic indexes PI > 2000. Furthermore, PI values of 63 were achieved with red light. Time-dependent density functional theory (TD-DFT) calculations nicely explain the effect of the π-expansion. The complexes produce reactive oxygen species (ROS) at the cellular level, causing mitochondrial membrane depolarization, cleavage of DNA, nicotinamide adenine dinucleotide (NADH) oxidation, as well as lysosomal damage. Consequently, cell death by apoptosis and secondary necrosis is activated. Thus, we describe the first class of half-sandwich iridium cyclometalated complexes active in PDT.
Assuntos
Antineoplásicos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Antineoplásicos/farmacologia , Ligantes , Linhagem Celular Tumoral , Irídio/farmacologiaRESUMO
Ir(III) and Ru(II) polypyridyl complexes are promising photosensitizers (PSs) for photodynamic therapy (PDT) due to their outstanding photophysical properties. Herein, one series of cyclometallated Ir(III) complexes and two series of Ru(II) polypyridyl derivatives bearing three different thiazolyl-ß-carboline N^N' ligands have been synthesized, aiming to evaluate the impact of the different metal fragments ([Ir(C^N)2]+ or [Ru(N^N)2]2+) and N^N' ligands on the photophysical and biological properties. All the compounds exhibit remarkable photostability under blue-light irradiation and are emissive (605 < λem < 720 nm), with the Ru(II) derivatives displaying higher photoluminescence quantum yields and longer excited state lifetimes. The Ir PSs display pKa values between 5.9 and 7.9, whereas their Ru counterparts are less acidic (pKa > 9.3). The presence of the deprotonated form in the Ir-PSs favours the generation of reactive oxygen species (ROS) since, according to theoretical calculations, it features a low-lying ligand-centered triplet excited state (T1 = 3LC) with a long lifetime. All compounds have demonstrated anticancer activity. Ir(III) complexes 1-3 exhibit the highest cytotoxicity in dark conditions, comparable to cisplatin. Their activity is notably enhanced by blue-light irradiation, resulting in nanomolar IC50 values and phototoxicity indexes (PIs) between 70 and 201 in different cancer cell lines. The Ir(III) PSs are also activated by green (with PI between 16 and 19.2) and red light in the case of complex 3 (PI = 8.5). Their antitumor efficacy is confirmed by clonogenic assays and using spheroid models. The Ir(III) complexes rapidly enter cells, accumulating in mitochondria and lysosomes. Upon photoactivation, they generate ROS, leading to mitochondrial dysfunction and lysosomal damage and ultimately cell apoptosis. Additionally, they inhibit cancer cell migration, a crucial step in metastasis. In contrast, Ru(II) complex 6 exhibits moderate mitochondrial activity. Overall, Ir(III) complexes 1-3 show potential for selective light-controlled cancer treatment, providing an alternative mechanism to chemotherapy and the ability to inhibit lethal cancer cell dissemination.
Assuntos
Antineoplásicos , Carbolinas , Complexos de Coordenação , Ensaios de Seleção de Medicamentos Antitumorais , Irídio , Fotoquimioterapia , Fármacos Fotossensibilizantes , Rutênio , Humanos , Irídio/química , Irídio/farmacologia , Rutênio/química , Rutênio/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ligantes , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/síntese química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/síntese química , Carbolinas/química , Carbolinas/farmacologia , Carbolinas/síntese química , Estrutura Molecular , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Piridinas/síntese química , Espécies Reativas de Oxigênio/metabolismo , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacosRESUMO
Fimbriae are long filamentous polymeric protein structures located upon the surface of bacteria. Often implicated in pathogenicity, the biosynthesis and function of fimbriae has been a productive topic of study for many decades. Evolutionary pressures have ensured that fimbriae possess unique structural and mechanical properties which are advantageous to bacteria. These properties are also difficult to engineer with well-known synthetic and natural fibres, and this has raised an intriguing question: can we exploit the unique properties of bacterial fimbriae in useful ways? Initial work has set out to explore this question by using Capsular antigen fragment 1 (Caf1), a fimbriae expressed naturally by Yersina pestis. These fibres have evolved to 'shield' the bacterium from the immune system of an infected host, and thus are rather bioinert in nature. Caf1 is, however, very amenable to structural mutagenesis which allows the incorporation of useful bioactive functions and the modulation of the fibre's mechanical properties. Its high-yielding recombinant synthesis also ensures plentiful quantities of polymer are available to drive development. These advantageous features make Caf1 an archetype for the development of new polymers and materials based upon bacterial fimbriae. Here, we cover recent advances in this new field, and look to future possibilities of this promising biopolymer.
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Antígenos de Bactérias , Yersinia pestis , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Fímbrias Bacterianas/metabolismo , Polímeros/química , Ciência dos Materiais , Yersinia pestis/química , Yersinia pestis/metabolismoRESUMO
An RNA-based coordination polymer is formed by the aqueous reaction of CuI ions with the thionucleoside enantiomer (-)6-thioguanosine, (6tGH). The resulting polymer, [CuI(µ3-S-thioG)]n1, has a one-dimensional structure based on a [Cu4-S4] core and undergoes extensive hierarchical self-assembly transforming from oligomeric chains â rod â cable â bundle through which a fibrous gel forms, that undergoes syneresis to form a self-supporting mass. The assembly involves the formation of helical cables/bundles and, in combination with the intrinsic photoemission of the polymer, results in the material exhibiting circularly polarised luminescence (CPL).
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There is a growing realization that 3D cell culture better mimics complex in vivo environments than 2D, lessening aberrant cellular behaviors and ultimately improving the outcomes of experiments. Chemically crosslinked hydrogels which imitate natural extracellular matrix (ECM) are proven cell culture platforms, but the encapsulation of cells within these hydrogel networks requires bioorthogonal crosslinking chemistries which can be cytotoxic, synthetically demanding, and costly. Capsular antigen fragment 1 (Caf1) is a bacterial, polymeric, fimbrial protein which can be genetically engineered to imitate ECM. Furthermore, it can, reversibly, thermally interconvert between its polymeric and monomeric forms even when chemically crosslinked within a hydrogel network. It is demonstrated that this meltable feature of Caf1 hydrogels can be utilized to encapsulate neonatal human dermal fibroblasts at a range of cell densities (2 × 105 -2 × 106 cells mL-1 of hydrogel) avoiding issues with chemical cytotoxicity. These hydrogels supported cell 3D culture for up to 21 d, successfully inducing cellular functions such as proliferation and migration. This work is significant because it further highlights the potential of simple, robust, Caf1-based hydrogels as a cell culture platform.
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Técnicas de Cultura de Células , Hidrogéis , Matriz Extracelular , Humanos , Hidrogéis/farmacologia , Recém-Nascido , PolímerosRESUMO
The aqueous equimolar reaction of Ag(i) ions with the thionucleoside enantiomer (-)6-thioguanosine, ((-)6tGH), yields a one-dimensional coordination polymer {Ag(-)tG} n , the self-assembly of which generates left-handed helical chains. The resulting helicity induces an enhanced chiro-optical response compared to the parent ligand. DFT calculations indicate that this enhancement is due to delocalisation of the excited state along the helical chains, with 7 units being required to converge the calculated CD spectra. At concentrations ≥15 mmol l-1 reactions form a sample-spanning hydrogel which shows self-repair capabilities with instantaneous recovery in which the dynamic reversibility of the coordination chains appears to play a role. The resulting gel exhibits circularly polarised luminescence (CPL) with a large dissymmetry factor of -0.07 ± 0.01 at 735 nm, a phenomenon not previously observed for this class of coordination polymer.
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Demand continues to grow for biomimetic materials able to create well-defined environments for modulating the behaviour of living cells in culture. Here, we describe hydrogels based upon the polymeric bacterial fimbriae protein capsular antigen fragment 1 (Caf1) that presents tunable biological properties for enhanced tissue cell culture applications. We demonstrate how Caf1 hydrogels can regulate cellular functions such as spreading, proliferation and matrix deposition of human dermal fibroblast cells (hDFBs). Caf1 hydrogels exploring a range of mechanical properties were prepared using copolymers featuring controlled compositions of inert wild-type Caf1 subunits and a mutant subunit displaying the RGDS peptide motif. The hydrogels showed excellent cytocompatibility with hDFBs and the ability to modulate both cell morphology and matrix deposition. Interestingly, Caf1 hydrogels displaying faster stress relaxation were demonstrated to show the highest metabolic activities of growing cells in comparison with other Caf1 hydrogel formulations. The stiffest Caf1 hydrogel impacted cellular morphology, inducing alignment of the cells. This work is significant as it clearly indicates that Caf1-based hydrogels offer tuneable biochemical and mechanical substrates conditions suitable for cell culture applications.
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Materiais Biomiméticos , Hidrogéis , Técnicas de Cultura de Células , Fímbrias Bacterianas , Humanos , PolímerosRESUMO
The problems of resistance and side effects associated with cisplatin and other chemotherapeutic drugs have boosted research aimed at finding new compounds with improved properties. The use of platinum(IV) prodrugs is one alternative, although there is some controversy regarding the predictive ability of the peak reduction potentials. In the work described here a series of fourteen chloride Pt(II) and Pt(IV) compounds was synthesised and fully characterised. The compounds contain different bidentate arylazole heterocyclic ligands. Their cytotoxic properties against human lung carcinoma (A549), human breast carcinoma (MCF7) and human colon carcinoma (HCT116 and HT29) cell lines were studied. A clear relationship between the type of ligand and the anti-proliferative properties was found, with the best results obtained for the Pt(II) compound that contains an aniline fragment, (13), thus evidencing a positive effect of the NH2 group. Stability and aquation studies in DMSO, DMF and DMSO/water mixtures were carried out on the active complexes and an in-depth analysis of the two aquation processes, including DFT analysis, of 13 was undertaken. It was verified that DNA was the target and that cell death occurred by apoptosis in the case of 13. Furthermore, the cytotoxic derivatives did not exhibit haemolytic activity. The reduction of the Pt(IV) compounds whose Pt(II) congeners were active was studied by several techniques. It was concluded that the peak reduction potential was not useful to predict the ability for reduction. However, a correlation between the cytotoxic activity and the standard reduction potential was found.
Assuntos
Compostos de Anilina/química , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Compostos Organoplatínicos/farmacologia , Platina/química , Pró-Fármacos/farmacologia , Células A549 , Antineoplásicos/química , Apoptose , Proliferação de Células , Células HCT116 , Humanos , Células MCF-7 , Neoplasias/patologia , Compostos Organoplatínicos/química , Pró-Fármacos/químicaRESUMO
We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.
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Materiais Biocompatíveis/química , Hidrogéis/química , Hidrogéis/metabolismo , Biossíntese de Proteínas/genética , Proteínas/genética , Alicerces Teciduais/química , Extratos Celulares , Linhagem Celular , DNA/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Polímeros/química , Polímeros/metabolismo , Sefarose/química , Sefarose/metabolismoRESUMO
The silver-nucleoside complex [Ag(i)-(N3-cytidine)2], 1, self-assembles to form a supramolecular metal-mediated base-pair array highly analogous to those seen in metallo-DNA. A combination of complementary hydrogen-bonding, hydrophobic and argentophilic interactions drive the formation of a double-helix with a continuous silver core. Electrical measurements on 1 show that despite having Ag···Ag distances within <5% of the metallic radii, the material is electrically insulating. This is due to the electronic structure which features a filled valence band, an empty conduction band dominated by the ligand, and a band gap of 2.5 eV. Hence, as-prepared, such Ag(i)-DNA systems should not be considered molecular nanowires but, at best, proto-wires. The structural features seen in 1 are essentially retained in the corresponding organogel which exhibits thixotropic self-healing that can be attributed to the reversible nature of the intermolecular interactions. Photo-reduced samples of the gel exhibit luminescence confirming that these poly-cytidine sequences appropriately pre-configure silver ions for the formation of quantum-confined metal clusters in line with contemporary views on DNA-templated clusters. Microscopy data reveals the resulting metal cluster/particles are approximately spherical and crystalline with lattice spacing (111) similar to bulk Ag.
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Capsular antigen fraction 1 (Caf1) is a robust polymeric protein forming a protective layer around the bacterium Yersinia pestis. Occurring as ≈1⯵m polymeric fibers, it shares its immunoglobulin-like fold with the majority of mammalian extracellular proteins such as fibronectin and this structural similarity suggests that this unusual polymer could form useful mimics of the extracellular matrix. Driven by the pressing need for reliable animal-free 3D cell culture environments, we showed previously that recombinant Caf1 produced in Escherichia coli can be engineered to include bioactive peptides, which influence cell behavior. Here, we demonstrate that through chemical crosslinking with a small palette of PEG-based crosslinkers, Caf1-based hydrogels can be prepared displaying a wide range of mechanical and morphological properties that were studied by rheology, compressive testing, SDS-PAGE and scanning electron microscopy. By varying the Caf1 protein concentration, viscoelasticity and stiffness (~11-2300â¯Pa) are reproducibly tunable to match natural and commercial 3D gels. Hydrogel porosity and swelling ratios were found to be defined by crosslinker architecture and concentration. Finally the hydrogels, which are 95-99% water, were shown to retain the high stability of the native Caf1 protein in a range of aqueous conditions, including extended immersion in cell culture media. The unusual Caf1 polymer thus offers the possibility of presenting bioactive protein subunits in a precisely tuneable hydrogel for use in cell culture and drug delivery applications.
Assuntos
Proteínas de Bactérias/química , Hidrogéis/química , Yersinia pestis/química , Elasticidade , Proteínas Recombinantes/química , ViscosidadeRESUMO
The ability to culture cells in three-dimensions has many applications, from drug discovery to wound healing. 3D cell culture methods often require appropriate scaffolds that mimic the cellular environments of different tissue types. The choice of material from which these scaffolds are made is of paramount importance, as its properties will define the manner in which cells interact with the scaffold. Caf1 is a protein polymer that is secreted from its host organism, Yersinia pestis, to enable escape from phagocytosis. In vitro, cells adhere poorly to the protein unless adhesion motifs are specifically introduced. Caf1 is a good candidate biomaterial due to its definable bioactivity, economical production and its ability to form hydrogels, through the use of cross-linkers. In this study, the thermostability of Caf1 was tested over a range of chemical conditions, and an initial characterisation of its rheological properties conducted in order to assess the suitability of Caf1 as a biomedical material. The results show that Caf1 retains its high thermostability even in harsh conditions such as extremes of pH, high salt concentrations and the presence of detergents. In solution, the concentrated polymer behaves as a complex viscous liquid. Due to these properties, Caf1 polymers are compatible with 3D bioprinting technologies and could be made to form a stimuli-responsive biomaterial that can alter its macrorheological properties in response to external factors. Caf1 biomaterials could therefore prove useful as 3D cell scaffolds for use in cell culture and wound repair.