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1.
Methods Enzymol ; 700: 49-76, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38971612

RESUMO

High pressure is both an environmental challenge to which deep sea biology has to adapt, and a highly sensitive thermodynamic tool that can be used to trigger structural changes in biological molecules and assemblies. Lipid membranes are amongst the most pressure sensitive biological assemblies and pressure can have a large influence on their structure and properties. In this chapter, we will explore the use of high pressure small angle X-ray diffraction and high pressure microscopy to measure and quantify changes in the lateral structure of lipid membranes under both equilibrium high pressure conditions and in response to pressure jumps.


Assuntos
Pressão Hidrostática , Bicamadas Lipídicas , Difração de Raios X , Difração de Raios X/métodos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Espalhamento a Baixo Ângulo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Termodinâmica
2.
Proc Natl Acad Sci U S A ; 120(35): e2307772120, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37603747

RESUMO

Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.


Assuntos
Células Artificiais , Biomimética , Hidrogéis , Comunicação , Organelas
3.
Lab Chip ; 22(5): 972-985, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35107110

RESUMO

Simple diffusion of molecular entities through a phospholipid bilayer, is a phenomenon of great importance to the pharmaceutical and agricultural industries. Current model lipid systems to probe this typically only employ fluorescence as a readout, thus limiting the range of assessable chemical matter that can be studied. We report a new technology platform, the UV-DIB, which facilitates label free measurement of small molecule translocation rates. This is based upon the coupling of droplet interface bilayer technology with implemented fiber optics to facilitate analysis via ultraviolet spectroscopy, in custom designed PMMA wells. To improve on current DIB technology, the platform was designed to be reusable, with a high sampling rate and a limit of UV detection in the low µM regime. We demonstrate the use of our system to quantify passive diffusion in a reproducible and rapid manner where the system was validated by investigating multiple permeants of varying physicochemical properties across a range of lipid interfaces, each demonstrating differing kinetics. Our system permits the interrogation of structural dependence on the permeation rate of a given compound. We present this ability from two structural perspectives, that of the membrane, and the permeant. We observed a reduction in permeability between pure DOPC and DPhPC interfaces, concurring with literature and demonstrating our ability to study the effects of lipid composition on permeability. In relation to the effects of permeant structure, our device facilitated the rank ordering of various compounds from the xanthine class of compounds, where the structure of each permeant differed by a single group alteration. We found that DIBs were stable up to 5% DMSO, a molecule often used to aid solubilisation of pharmaceutical and agrochemical compounds. The ability of our device to rank-order compounds with such minor structural differences provides a level of precision that is rarely seen in current, industrially applied technologies.


Assuntos
Bicamadas Lipídicas , Fosfolipídeos , Difusão , Cinética , Bicamadas Lipídicas/química , Permeabilidade , Fosfolipídeos/química
4.
J R Soc Interface ; 18(185): 20210698, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34875877

RESUMO

The interactions between small molecules and keratins are poorly understood. In this paper, a nuclear magnetic resonance method is presented to measure changes in the 1H T1 relaxation times of small molecules in human hair keratin to quantify their interaction with the fibre. Two populations of small-molecule compounds were identified with distinct relaxation times, demonstrating the partitioning of the compounds into different keratin environments. The changes in relaxation time for solvent in hair compared with bulk solvent were shown to be related to the molecular weight (MW) and the partition coefficient, LogP, of the solvent investigated. Compounds with low MWs and high hydrophilicities had greater reductions in their T1 relaxation times and therefore experienced increased interactions with the hair fibre. The relative population sizes were also calculated. This is a significant step towards modelling the behaviour of small molecules in keratinous materials and other large insoluble fibrous proteins.


Assuntos
Cabelo , Queratinas , Humanos , Espectroscopia de Ressonância Magnética , Peso Molecular , Espectroscopia de Prótons por Ressonância Magnética
5.
Chem Sci ; 12(6): 2138-2145, 2021 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-34163978

RESUMO

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators. Here we report the activation of the bacterial mechanosensitive channel of large conductance (MscL) in a dioleoylphosphatidylcholine:dioleoylphosphatidylglycerol DIB by controlling membrane asymmetry. We show using electrical measurements that the incorporation of lysophosphatidylcholine (LPC) into one of the bilayer leaflets triggers MscL gating in a concentration-dependent manner, with partial and full activation observed at 10 and 15 mol% LPC respectively. Our findings could inspire the design of new minimal tissues where flux pathways are dynamically defined by lipid composition.

6.
Chem Commun (Camb) ; 56(92): 14499-14502, 2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33150883

RESUMO

Cholesterol is a crucial component of biological membranes and can interact with other membrane components through hydrogen bonding. NMR spectroscopy has been used previously to investigate this bonding, however this study represents the first 17O NMR spectroscopy study of isotopically enriched cholesterol. We demonstrate the 17O chemical shift is dependent on hydrogen bonding, providing a novel method for the study of cholesterol in bilayers.


Assuntos
Colesterol/química , Bicamadas Lipídicas/química , Isótopos de Oxigênio/química , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Solventes/química
7.
Langmuir ; 35(50): 16521-16527, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31702159

RESUMO

Dispersions of nonlamellar lipid membrane assemblies are gaining increasing interest for drug delivery and protein therapeutic application. A key bottleneck has been the lack of rational design rules for these systems linking different lipid species and conditions to defined lattice parameters and structures. We have developed robust methods to form cubosomes (nanoparticles with porous internal structures) with water channel diameters of up to 171 Å, which are over 4 times larger than archetypal cubosome structures. The water channel diameter can be tuned via the incorporation of cholesterol and the charged lipid DOPA, DOPG, or DOPS. We have found that large molecules can be incorporated into the porous cubosome structure and that these molecules can interact with the internal cubosome membrane. This offers huge potential for accessible encapsulation and protection of biomolecules and development of confined interfacial reaction environments.


Assuntos
Colesterol/química , Engenharia , Glicerofosfolipídeos/química
8.
Proc Natl Acad Sci U S A ; 116(34): 16711-16716, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31371493

RESUMO

To date, reconstitution of one of the fundamental methods of cell communication, the signaling pathway, has been unaddressed in the bottom-up construction of artificial cells (ACs). Such developments are needed to increase the functionality and biomimicry of ACs, accelerating their translation and application in biotechnology. Here, we report the construction of a de novo synthetic signaling pathway in microscale nested vesicles. Vesicle-cell models respond to external calcium signals through activation of an intracellular interaction between phospholipase A2 and a mechanosensitive channel present in the internal membranes, triggering content mixing between compartments and controlling cell fluorescence. Emulsion-based approaches to AC construction are therefore shown to be ideal for the quick design and testing of new signaling networks and can readily include synthetic molecules difficult to introduce to biological cells. This work represents a foundation for the engineering of multicompartment-spanning designer pathways that can be utilized to control downstream events inside an AC, leading to the assembly of micromachines capable of sensing and responding to changes in their local environment.


Assuntos
Células Artificiais , Compartimento Celular , Mecanotransdução Celular , Cálcio/metabolismo , Comunicação Celular/efeitos dos fármacos , Compartimento Celular/efeitos dos fármacos , Quelantes/farmacologia , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Fosfolipases A2/metabolismo
9.
J Colloid Interface Sci ; 538: 75-82, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30500469

RESUMO

The effect of glycerol with sodium chloride (NaCl) on the phase behaviour of sodium dodecyl sulfate (SDS) near the Krafft point was studied by surface tension analysis using the pendant drop method. The critical micelle concentration (CMC) and Krafft Temperature (TK) of SDS in water: glycerol mixtures, across the full composition range, and in NaCl solutions within 0.005-0.1 M were obtained. The pendant drop method successfully allowed us to determine the Krafft point of SDS in high glycerol systems where other traditional methods (e.g. conductivity) have been ineffective. Overall the addition of glycerol increases the CMC and the TK, thus shifting the Krafft point of SDS to higher temperatures (increasing crystallisation temperatures) and higher SDS content in the presence of glycerol, which is interpreted as a result of the reduction in solvent polarity which opposes micellization. The addition of NaCl to the SDS - water-glycerol systems brings the CMC back down, while having no significant effect on the TK. Our results establish a robust route for tuning the Krafft point of model surfactant SDS by adjusting solvent quality and salt content.

10.
Angew Chem Int Ed Engl ; 57(39): 12656-12660, 2018 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-30095209

RESUMO

Two-dimensional (2D) layered graphitic carbon nitride (gCN) nanosheets offer intriguing electronic and chemical properties. However, the exfoliation and functionalisation of gCN for specific applications remain challenging. We report a scalable one-pot reductive method to produce solutions of single- and few-layer 2D gCN nanosheets with excellent stability in a high mass yield (35 %) from polytriazine imide. High-resolution imaging confirmed the intact crystalline structure and identified an AB stacking for gCN layers. The charge allows deliberate organic functionalisation of dissolved gCN, providing a general route to adjust their properties.

11.
Chem Sci ; 9(21): 4851-4858, 2018 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-29910937

RESUMO

Giant unilamellar vesicles (GUVs) are a well-established tool for the study of membrane biophysics and are increasingly used as artificial cell models and functional units in biotechnology. This trend is driven by the development of emulsion-based generation methods such as Emulsion Phase Transfer (EPT), which facilitates the encapsulation of almost any water-soluble compounds (including biomolecules) regardless of size or charge, is compatible with droplet microfluidics, and allows GUVs with asymmetric bilayers to be assembled. However, the ability to control the composition of membranes formed via EPT remains an open question; this is key as composition gives rise to an array of biophysical phenomena which can be used to add functionality to membranes. Here, we evaluate the use of GUVs constructed via this method as a platform for phase behaviour studies and take advantage of composition-dependent features to engineer thermally-responsive GUVs. For the first time, we generate ternary GUVs (DOPC/DPPC/cholesterol) using EPT, and by compensating for the lower cholesterol incorporation efficiencies, show that these possess the full range of phase behaviour displayed by electroformed GUVs. As a demonstration of the fine control afforded by this approach, we demonstrate release of dye and peptide cargo when ternary GUVs are heated through the immiscibility transition temperature, and show that release temperature can be tuned by changing vesicle composition. We show that GUVs can be individually addressed and release triggered using a laser beam. Our findings validate EPT as a suitable method for generating phase separated vesicles and provide a valuable proof-of-concept for engineering content release functionality into individually addressable vesicles, which could have a host of applications in the development of smart synthetic biosystems.

12.
Sci Rep ; 8(1): 4564, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540757

RESUMO

There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.


Assuntos
Células Artificiais/metabolismo , Organelas/metabolismo , Reatores Biológicos , Bicamadas Lipídicas , Técnicas Analíticas Microfluídicas/instrumentação , Modelos Biológicos
13.
Nat Commun ; 9(1): 1093, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29545566

RESUMO

Cell-sized vesicles have tremendous potential both as miniaturised pL reaction vessels and in bottom-up synthetic biology as chassis for artificial cells. In both these areas the introduction of light-responsive modules affords increased functionality, for example, to initiate enzymatic reactions in the vesicle interior with spatiotemporal control. Here we report a system composed of nested vesicles where the inner compartments act as phototransducers, responding to ultraviolet irradiation through diacetylene polymerisation-induced pore formation to initiate enzymatic reactions. The controlled release and hydrolysis of a fluorogenic ß-galactosidase substrate in the external compartment is demonstrated, where the rate of reaction can be modulated by varying ultraviolet exposure time. Such cell-like nested microreactor structures could be utilised in fields from biocatalysis through to drug delivery.


Assuntos
Raios Ultravioleta , Biocatálise , Hidrólise , beta-Galactosidase/metabolismo
14.
Int J Appl Glass Sci ; 8(4): 428-437, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29271977

RESUMO

This study aims to demonstrate that excellent bioactivity of glass can be achieved without the presence of an alkali metal component in glass composition. In vitro bioactivity of two sodium-free glasses based on the quaternary system SiO2-P2O5-CaO-CaF2 with 0 and 4.5 mol% CaF2 content was investigated and compared with the sodium containing glasses with equivalent amount of CaF2. The formation of apatite after immersion in Tris buffer was followed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), 31P and 19F solid state MAS-NMR. The dissolution study was completed by ion release measurements in Tris buffer. The results show that sodium free bioactive glasses formed apatite at 3 hours of immersion in Tris buffer, which is as fast as the corresponding sodium containing composition. This signifies that sodium is not an essential component in bioactive glasses and it is possible to make equally degradable bioactive glasses with or without sodium. The results presented here also emphasize the central role of the glass compositions design which is based on understanding of structural role of components and/or predicting the network connectivity of glasses.

15.
Sci Rep ; 7(1): 12606, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28974701

RESUMO

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

16.
ACS Nano ; 11(7): 6549-6565, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28658575

RESUMO

Compartmentalization of biological content and function is a key architectural feature in biology, where membrane bound micro- and nanocompartments are used for performing a host of highly specialized and tightly regulated biological functions. The benefit of compartmentalization as a design principle is behind its ubiquity in cells and has led to it being a central engineering theme in construction of artificial cell-like systems. In this review, we discuss the attractions of designing compartmentalized membrane-bound constructs and review a range of biomimetic membrane architectures that span length scales, focusing on lipid-based structures but also addressing polymer-based and hybrid approaches. These include nested vesicles, multicompartment vesicles, large-scale vesicle networks, as well as droplet interface bilayers, and double-emulsion multiphase systems (multisomes). We outline key examples of how such structures have been functionalized with biological and synthetic machinery, for example, to manufacture and deliver drugs and metabolic compounds, to replicate intracellular signaling cascades, and to demonstrate collective behaviors as minimal tissue constructs. Particular emphasis is placed on the applications of these architectures and the state-of-the-art microfluidic engineering required to fabricate, functionalize, and precisely assemble them. Finally, we outline the future directions of these technologies and highlight how they could be applied to engineer the next generation of cell models, therapeutic agents, and microreactors, together with the diverse applications in the emerging field of bottom-up synthetic biology.

17.
Phys Chem Chem Phys ; 19(13): 9199-9209, 2017 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-28317964

RESUMO

Lanthanide salts have been studied for many years, primarily in Nuclear Magnetic Resonance (NMR) experiments of mixed lipid-protein systems and more recently to study lipid flip-flop in model membrane systems. It is well recognised that lanthanide salts can influence the behaviour of both lipid and protein systems, however a full molecular level description of lipid-lanthanide interactions is still outstanding. Here we present a study of lanthanide-bilayer interactions, using molecular dynamics computer simulations, fluorescence electrostatic potential experiments and nuclear magnetic resonance. Computer simulations reveal the microscopic structure of DMPC lipid bilayers in the presence of Yb3+, and a surprising ability of the membranes to adsorb significant concentrations of Yb3+ without disrupting the overall membrane structure. At concentrations commonly used in NMR experiments, Yb3+ ions bind strongly to 5 lipids, inducing a small decrease of the area per lipid and a slight increase of the ordering of the aliphatic chains and the bilayer thickness. The area compressibility modulus increases by a factor of two, with respect to the free-salt case, showing that Yb3+ ions make the bilayer more rigid. These modifications of the bilayer properties should be taken into account in the interpretation of NMR experiments.

18.
Lab Chip ; 16(23): 4621-4627, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27797387

RESUMO

In this study, we introduce an optofluidic method for the rapid construction of large-area cell-sized droplet assemblies with user-defined, re-writable, two-dimensional patterns of functional droplets. Light responsive water-in-oil droplets capable of releasing fluorescent dye molecules upon exposure were generated and self-assembled into arrays inside a microfluidic device. This biological architecture was exploited by the scanning laser of a confocal microscope to 'write' user defined patterns of differentiated (fluorescent) droplets in a network of originally undifferentiated (non-fluorescent) droplets. As a result, long lasting images were produced on a droplet fabric with droplets acting as pixels of a biological monitor, which can be erased and re-written on-demand. Regio-specific light-induced droplet differentiation within a large population of droplets provides a new paradigm for the rapid construction of bio-synthetic systems with potential as tissue mimics and biological display materials.


Assuntos
Dispositivos Lab-On-A-Chip , Lasers , Fenômenos Ópticos
19.
Soft Matter ; 12(37): 7731-7734, 2016 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-27722718

RESUMO

We report a new platform technology to systematically assemble droplet interface bilayer (DIB) networks in user-defined 3D architectures from cell-sized droplets using optical tweezers. Our OptiDIB platform is the first demonstration of optical trapping to precisely construct 3D DIB networks, paving the way for the development of a new generation of modular bio-systems.

20.
Chem Commun (Camb) ; 52(35): 5961-4, 2016 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-27056570

RESUMO

Compartmentalised structures based on droplet interface bilayers (DIBs), including multisomes and compartmentalised vesicles, are seen by many as the next generation of biomimetic soft matter devices. Herein, we outline a microfluidic approach for the construction of miniaturised multisomes of pL volumes in high-throughput and demonstrate their potential as vehicles for in situ chemical synthesis.


Assuntos
Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Gotículas Lipídicas/química , Aminas/química , Cápsulas , Compostos Heterocíclicos com 3 Anéis/química
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