RESUMO
Recent years have witnessed many breakthroughs in research on graphene (the first two-dimensional atomic crystal) as well as a significant advance in the mass production of this material. This one-atom-thick fabric of carbon uniquely combines extreme mechanical strength, exceptionally high electronic and thermal conductivities, impermeability to gases, as well as many other supreme properties, all of which make it highly attractive for numerous applications. Here we review recent progress in graphene research and in the development of production methods, and critically analyse the feasibility of various graphene applications.
RESUMO
The 9th-10th type III fibronectin domain pair (9-10FNIII) has found widespread use as a biomimetic surface for cell adhesion. However, the effect of mutations to 9-10FNIII on its surface adsorption characteristics have not been investigated. Here we address this issue using total internal reflection fluorescence (TIRF) and circular dichroism spectroscopy, comparing two conformationally stable 9-10FNIII mutants against the wild type. Desorption of the 9-10FNIII mutants from the silica surface was minimal in comparison to desorption of 9-10FNIII. The extent and rate of protein desorption from silica was empirically matched by loss of secondary structure upon adsorption, with only the spectrum for 9-10FNIII showing extensive loss of the beta-sandwich fold. For the proteins adsorbed to hydrophobic surfaces, only the CD spectra for the 9-10FNIII mutant constrained via an interdomain disulphide bridge showed similarity with the corresponding solution structure. Since the binding of 9-10FNIII to integrin alpha5beta1 is highly dependent on the relative spatial arrangement of the two domains, we suggest that the observed differences in cell adhesion and spreading on wild type 9-10FNIII and mutants may in part be attributed to the extent of protein desorption and unfolding at the surface.
Assuntos
Fibronectinas/química , Fibronectinas/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Conformação Proteica , Dobramento de Proteína , Adsorção , Substituição de Aminoácidos , Fibronectinas/genética , Humanos , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Dióxido de Silício , Propriedades de SuperfícieRESUMO
The 9th-10th type III fibronectin domain pair shows promise in tissue engineering and tumour vasculature targeting. Calorimetry and structure-function analysis were used to investigate the effects of solution formulation and lyophilisation of a mutant ((9-10)FNIII-P). A single endothermic transition for (9-10)FNIII-P in solution was observed at pH<8, irrespective of addition of sucrose or PEG. The temperature at the maximum heat capacity (T(m)) and enthalpy (deltaH) of the transition increased for increasing sucrose concentrations but decreased for increasing PEG concentrations. The transition was fitted to a single two-state unfolding mechanism (in contrast to unfolding in guanidine. x HCl) and was partially reversible only at pH 4, with increasing concentrations of sucrose causing a marked fall in deltaH between scans. Circular dichroism spectra for the thermal unfolding of (9-10)FNIII-P at pH 4 showed loss of native beta-sheet structure and loss of aromatic contributions to the peak centred around 226 nm yielding an intermediate conformation, which in the presence of sucrose was more disordered. Despite a glass transition (T(g)') for (9-10)FNIII-P(aq) of -70 degrees C, primary drying at -30 degrees C did not perturb its conformation upon reconstitution or its biological activity following lyophilisation; the addition of sucrose or PEG had no influence on structure or activity. The main consideration in the formulation of (9-10)FNIII-P was therefore pH.
Assuntos
Fibronectinas/química , Proteínas Recombinantes de Fusão/química , Animais , Calorimetria , Varredura Diferencial de Calorimetria , Adesão Celular , Dicroísmo Circular , Cricetinae , Temperatura Alta , Concentração de Íons de Hidrogênio , Polietilenoglicóis/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Sacarose/química , Engenharia TecidualRESUMO
Evidence is presented that a compartmentalised protein exists in its native state only within a particular size of aqueous cavity. This behaviour is shown to exist in AOT reverse micelles using fluorescence quenching and circular dichroism (CD) studies of human serum albumin (HSA). In particular, far ultraviolet CD measurements show that a reduction in quencher accessibility to the fluorophore is consistent with the protein being nearest to its native conformation at a waterpool size of around 80 A diameter. We also show that the biexponential fluorescence decay of N-acetyl-L-tryptophanamide (NATA) in AOT reverse micelles arises from the probe being located in two distinct sites within the interfacial region. The more viscous of these two sites is located on the waterpool side of the interface and the other is located on the oil side of the interface.
Assuntos
Micelas , Albumina Sérica/química , Triptofano/análogos & derivados , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Polarização de Fluorescência , Humanos , Fotoquímica , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
The micellar-like particle systems produced from poly-D,L-lactide-poly(ethylene glycol) (PLA-PEG) copolymers have been assessed using a range of physicochemical characterisation methods, followed by in vivo studies of their biodistribution after intravenous administration to the rat. The size of the PEG chain was kept constant at 5 or 2 kDa, while the PLA size increased within a series from 2 to 25 kDa. The results obtained reveal, that in an aqueous medium the copolymers assembled into micellar-like structures, with the PLA segments forming the core and the PEG segments the surrounding corona. The size of the PLA segments dominated the process of assembly of the molecules and the characteristics of the resultant micellar-like particles. The PLA-PEG micellar particles were found to be less dynamic than those obtained from conventional surfactants. Particles formed from the lower molecular weight PLA polymers allowed a level of chain mobility while the cores of the micellar particles formed from higher molecular weight PLA appeared to be solid-like in nature. The size of the micellar particles was dependent on the copolymer molecular weight and the z-average diameter increased from 25 to 76 nm as the molecular weight of the PLA moiety increased. This provides an ability to control the particle size by adjusting the molecular weight of the PLA moiety. Following intravenous administration to the rat model, micellar-like particles smaller than approximately 70 nm accumulated in the liver, despite the fact that the PEG corona provided an effective steric stabilization effect. Micellar-like particles with a diameter of more than approximately 70 nm exhibited prolonged systemic circulation and reduced liver uptake, although the steric stabilisation of these particles was shown to be less effective. These findings agree with recent observations from other research groups; that indicate a possibility that very small particulates can pass through the sinusoidal fenestrations in the liver and gain access to the parenchymal cells of the liver.
Assuntos
Lactatos/química , Lactatos/farmacocinética , Micelas , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacocinética , Coloides , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Lactatos/sangue , Fígado/metabolismo , Tamanho da Partícula , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
Magnetic Resonance Imaging experiments have been used to investigate the degradation of drug-loaded poly(glycolic lactic acid) (PGLA) 50:50 cylinders. Spatial variation in the rate of degradation throughout the polymer cross-section has been observed non-invasively using quantitative imaging of penetrant concentration, T(2) and self-diffusion coefficient. This spatial variation in the rate of degradation was attributed to the quicker degradation in the inner region of the sample due to autocatalysis by carboxyl end groups generated upon ester bond cleavage.
Assuntos
Imageamento por Ressonância Magnética/métodos , Poliésteres/química , Água/química , Catálise , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Estabilidade de Medicamentos , Glicolatos/química , Ácido Láctico/químicaRESUMO
Potent monomeric and dimeric cyclic peptide very late antigen-4 (VLA-4) inhibitors have been designed based on a tetrapeptide (Ile-Leu-Asp-Val) sequence present in a 25-amino acid peptide (CS-1) reported in the literature. The peptides, synthesized by the SPPS techniques, were evaluated in the in vitro cell adhesion assays and in the in vivo inflammation models. The N- to C-terminal cyclic peptides such as cyclo(Ile-Leu-Asp-Val-NH-(CH2)2-S-(CH2)2-CO) (28) and cyclo(MeIle-Leu-Asp-Val-D-Ala-D-Ala) (31), monomeric and dimeric peptides containing piperazine (Pip) or homopiperazine (hPip) residues as linking groups, e.g. cyclo(MeIle-Leu-Asp-Val-Pip-CH2CO-NH-(CH2)2-S-CH2-CO) (49) and cyclo(MeIle-Leu-Asp-Val hPip-CH2CO-MeIle-Leu-Asp-Val-hPip-CH2CO) (58) and cyclic peptides containing an amide bond between the side chain amino group of an amino acid such as Lys and the C-terminal Val carboxyl group, e.g. Ac-cyclo(D-Lys-D-Ile-Leu-Asp-Val) (62) and beta-Ala-cyclo(D-Lys-D-Leu-Leu-Asp-Val) (68) were more potent than CS-1 in inhibiting the adhesion of the VLA-4-expressing MOLT-4 cells to fibronectin. The more potent compounds were highly selective and did not affect U937 cell adhesion to fibronectin (VLA-5), PMA-differentiated U937 cell adhesion to intercellular cell adhesion molecule- 1-expressing Chinese hamster ovary cells (LFA-1) and ADP-induced platelet aggregation (GPIIb/IIIa). A number of the more potent compounds inhibited ovalbumin-induced delayed type hypersensitivity in mice and some were 100-300 times more potent (ED50 = 0.003-0.009 mg/kg/day, s.c.) than CS-1. Two peptides, Ac-cyclo(D-Lys D-Ile-Leu-Asp-Val) (62) and cyclo(CH2CO-Ile-Leu-Asp-Val-Pip-CH2CO-Ile-Leu-Asp-Val-Pip) (55), were formulated in poly(DL-lactide-co-glycolide) depots and the release profile was investigated in vitro over a 30-day period.
Assuntos
Adesão Celular/efeitos dos fármacos , Integrinas/antagonistas & inibidores , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Receptores de Retorno de Linfócitos/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CHO , Linhagem Celular , Cricetinae , Preparações de Ação Retardada , Dimerização , Estabilidade de Medicamentos , Humanos , Hipersensibilidade Tardia/prevenção & controle , Integrina alfa4beta1 , Ácido Láctico , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Peptídeos Cíclicos/química , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Células U937RESUMO
Additional structure-activity relationship studies on potent cyclic peptide inhibitors of very late antigen-4 (VLA-4) are reported. The new N- to C-terminal cyclic hexa-, hepta- and octapeptide inhibitors like cyclo(MeIle/MePhe-Leu-Asp-Val-X) (X = 2-4 amino acids containing hydrophobic and/or basic side chains) were synthesized using solid phase peptide synthesis methods. The peptides were evaluated in in vitro cell adhesion assays and in in vivo inflammation models. Many of the peptides like cyclo(MePhe-Leu-Asp-Val-D-Arg-D-Arg) (ZD7349) (17), cyclo(MeIle-Leu-Asp-Val-D-Arg-D-Arg-D-Phe) (20), cyclo(MeIle-Leu-Asp-Val-D-Arg-D-Arg-MePhe) (21) and cyclo(MePhe-Leu-Asp-Val-D-Arg-D-Arg-D-Ala-D-Ala) (23) were potent inhibitors of VLA-4-mediated cell adhesion and inhibited ovalbumin-induced delayed type hypersensitivity (DTH) response in mice. The more potent compounds were highly selective and did not affect U937 cell adhesion to fibronectin (VLA-5), phorbolmyristate acetate or PMA-differentiated U937 cell adhesion to intercellular cell adhesion molecule-1 (ICAM-1)-expressing Chinese hamster ovary cells (LFA-1) and adenosine diphosphate (ADP)-induced platelet aggregation (GPIIb/IIIa). In contrast to the inhibitors like Ac-cyclo(D-Lys-D-Ile-Leu-Asp-Val) and cyclo(CH2CO-Ile-Leu-Asp-Val-Pip-CH2CO-Ile-Leu-Asp-Val-Pip) described earlier, the new compounds were much more compatible with the depot formulations based on poly(DL-lactide-co-glycolide) polymers. The hexapeptide cyclo(MePhe-Leu-Asp-Val-D-Arg-D-Arg) (ZD7349) (17) inhibited MOLT-4 cell adhesion to fibronectin and vascular cell adhesion molecule-1 (VCAM-1) with IC50 values of 260 and 330 nM, respectively, and did not show any significant effect against other integrins (IC50 > 300 microM). ZD7349 inhibited ovalbumin-induced DTH response in mice when administered continuously using a mini-pump (ED50 0.01 mg/kg/day) or when given as an s.c. or i.v. bolus injection at a dose of 1-10 mg/kg. ZD7349 was also active in type II collagen-induced arthritis (CIA) and experimental autoimmune encephalomyelitis (EAE) tests at a dose of 3-10 mg/kg. The peptide was released from some formulations over a period of 10-20 days. ZD7349 is currently undergoing pre-clinical investigation.