Browsing by Author "Shen, HH"
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- ItemAnnexin V-containing cubosomes for targeted early detection of apoptosis in degenerative retinal tissue(Royal Society of Chemistry, 2018-10-26) Ding, Y; Chow, SH; Liu, GS; Wang, B; Lin, TW; Hsu, HY; Duff, AP; Le Brun, AP; Shen, HHNew drug delivery materials targeting damaged ocular tissues are of particular interest. In this work, we have formulated annexin/phosphatidylserine/phytantriol and annexin/phosphatidylserine/monoolein cubosomes based on incorporation of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (PS) lipid and annexin V (ANX) protein with phytantriol (Phy) and monoolein (MO) respectively. The incorporation of ANX is important because it can be used as a diagnostic tool for in vivo apoptosis detection due to its high affinity to phosphatidylserine in the presence of Ca2+. We have also prepared PS–Phy and PS–MO cubosomes without ANX as a comparison, and characterized them using dynamic light scattering, cryo-TEM images and small-angle X-ray scattering, showing that PS–Phy cubosomes have greater chemical stability, and that ANX–PS–Phy cubosomes have the potential for in vivo drug delivery. In addition, we have reconstituted an apoptotic biomimetic membrane on a surface to gain insights into cubosome–bilayer interactions using a quartz-crystal microbalance and neutron reflectometry. The neutron reflectivity data reveal that there is exchange of materials between the biomimetic apoptotic bilayer and ANX–PS–Phy cubosomes, with an accumulation of ANX between the membrane and cubosomes possibly being the reason for the reduced cytotoxicity of ANX–PS–Phy cubosomes. A rat model of laser-induced choroidal neovascularization showed that ANX–PS–Phy cubosomes specifically targeted apoptotic cells in vivo. We propose that ANX–PS–Phy cubosomes are a potential candidate for ocular drug delivery for eye diseases. © The Royal Society of Chemistry 2018
- ItemAn in vitro model to investigate the interactions between antimicrobial peptides and the outer membrane of gram-negative pathogens(Australian Institute of Nuclear Science and Engineering, 2016-11-29) Han, ML; Shen, HH; Zhu, Y; Le Brun, AP; Holt, SA; Roberts, K; Song, JN; Cooper, MA; Moskowitz, SM; Velkov, T; Li, JIncreasing antibiotic resistance in Gram-negative bacteria led to polymyxins as the last therapy. Polymyxins present their antimicrobial activity through an initial electronical interaction with lipid A in the outer membrane (OM) of GNB, and the most common mechanism of polymyxin resistance is through modifications of lipid A with positively charged groups, such as 4-amino-L-arabinose (L-Aar4N) or phosphoethanolamine (pEtN). However, it is notable that Gram-negative bacteria employ a combination of charge-charge repulsion mechanism and the modification to fatty acyl chains of lipid A to obtain high-level polymyxin resistance. Hence, we designed hydrophobic polymyxin-related lipopeptides in order to overcome modified lipid A to insert into the outer membrane of Gram-negative bacteria. In this study, we employed neutron reflectometry (NR) study to investigate the interactions between lipid A and polymyxins. Lipid A was extracted from polymyxin-susceptible and -resistant pseudomonas aeruginosa strains, and analysed using ESI-MS in the negative ion mode. The asymmetric lipid A: deuterated DPPC bilayers were deposited on SiO2 surfaces by combined Langmuir-Blodgett and Langmuir-Schaefer disposition methods, and characterised by neutron reflectometer. Our results showed L-Ara4N modified lipid A was observed in polymyxin-resistant PAKpmrB6 strain, but not in the wild-type PAK strain. The NR data obtained from unmodified lipid A: DPPC bilayer was fitted into a five-layer model. Whereas, a six-layer model containing an extra outer headgroup was established for L-Ara4N modified lipid A: d-DPPC bilayer. Our results showed a dense of PMB (volume fraction of >20%) bound to the surface of both unmodified and modified lipid A: DPPC bilayers. While it is notable that the significant changes in NR profiles obtained from H2O contrast indicated about 15.8% and 6.1% of PMB penetrated into the wild-type lipid A headgroup and fatty acyl chains, respectively, but without penetration into L-Ara4N-lipid A: d-DPPC bilayer. However, the employment of octpeptin A3 induced higher hydrophobic interactions with L-Ara4N-lipid A: d-DPPC bilayer. Our study provides an in vitro model to investigate the interactions of polymyxins with OM bilayers in GNB, and confirmed that lipid A modification with L-Ara4N was certainly to reduce the penetration of PMB into bacterial membranes. Remarkably, the higher binding affinity between octapeptin A3 and L-Ara4N modified lipid A indicated its potential to be the new generation antibiotics for the therapy of infections caused by multi-drug resistant Gram negative bacteria.
- ItemThe interaction of cubosomes with supported phospholipid bilayers using neutron reflectometry and QCM-D(Royal Society of Chemistry, 2011-09-21) Shen, HH; Hartley, PG; James, M; Nelson, A; Defendi, H; McLean, KMWe present the results of a study of the interaction of lyotropic liquid crystalline dispersions with supported lipid bilayers based on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using a quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We investigated two types of phytantriol-based cubosome formulations, with subtly different internal nanostructures, with one formulation incorporating 2.5% of the biological lipid di-palmitoylphosphatidylserine (DPPS). The QCM-D data showed that cubosomes do not directly attach to the silica supporting surface but they can accumulate on the model membrane, confirming that there is an attractive interaction between POPC bilayers and the cubosome formulations. We have further used NR to quantify the amount of cubosomes adsorbed on the supported POPC bilayers and to examine the structural rearrangement of cubosomes on interaction with the supported lipid bilayer. The data show that the DPPS-containing cubosomes accumulate at the bilayer surface continuously for 15 hours. Pure phytantriol cubosomes accumulated over a longer time period (36 hours), but accumulated to a lesser degree overall. Furthermore, NR data revealed lipid exchange and structural rearrangements for both types of cubosomes, however, for the DPPS-containing cubosomes, these processes were greater in magnitude and faster. Confocal microscopy analysis of cubosome interactions with HeLa cells in vitro, showed increased membrane affinity for the DPPS-containing formulations, which were consistent with the NR and QCM-D observations. We interpret these observations as suggesting that membrane accumulation, cellular uptake and cytotoxicity of cubosome formulations are directly related to their DPPS content, and that this may be the result of increased propensity for liquid crystalline structural rearrangement, as postulated previously (H. H. Shen et al., Biomaterials, 2010, 31, 9473).© 2011, Royal Society of Chemistry
- ItemPolymyxin-induced lipid A deacylation in pseudomonas aeruginosa perturbs polymyxin penetration and confers high-level resistance(ACS Publications, 2017-11-28) Han, ML; Velkov, T; Zhu, Y; Roberts, KD; Le Brun, AP; Chow, SH; Gutu, AD; Moskowitz, SM; Shen, HH; Li, JPolymyxins are last-line antibiotics against life-threatening multidrug-resistant Gram-negative bacteria. Unfortunately, polymyxin resistance is increasingly reported, leaving a total lack of therapies. Using lipidomics and transcriptomics, we discovered that polymyxin B induced lipid A deacylation viapagL in both polymyxin-resistant and -susceptible Pseudomonas aeruginosa. Our results demonstrated that the deacylation of lipid A is an “innate immunity” response to polymyxins and a key compensatory mechanism to the aminoarabinose modification to confer high-level polymyxin resistance in P. aeruginosa. Furthermore, cutting-edge neutron reflectometry studies revealed that an assembled outer membrane (OM) with the less hydrophobic penta-acylated lipid A decreased polymyxin B penetration, compared to the hexa-acylated form. Polymyxin analogues with enhanced hydrophobicity displayed superior penetration into the tail regions of the penta-acylated lipid A OM. Our findings reveal a previously undiscovered mechanism of polymyxin resistance, wherein polymyxin-induced lipid A remodeling affects the OM packing and hydrophobicity, perturbs polymyxin penetration, and thereby confers high-level resistance. © 2017 American Chemical Society
- ItemA polytherapy based approach to combat antimicrobial resistance using cubosomes(Springer Nature, 2022-01-17) Lai, XF; Han, ML; Ding, Y; Chow, SH; Le Brun, AP; Wu, CM; Bergen, PJ; Jiang, JH; Hsu, HY; Muir, BW; White, J; Song, JN; Shen, HHA depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative ‘superbugs’ has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of “superbugs”. Open Access: This article is licensed under a Creative Commons Attribution 4.0 International Licence.
- ItemReconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes(Macmillan Publishers Limited., 2014-10-24) Shen, HH; Leyton, DL; Shiota, T; Belousoff, MJ; Noinaj, N; Lu, J; Holt, SA; Tan, K; Selkrig, J; Webb, CT; Buchanan, SK; Martin, LL; Lithgow, TIn biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines. © Macmillan Publishers Limited
- ItemTargeted detection of phosphatidylserine in biomimetic membranes and in vitro cell systems using annexin V-containing cubosomes(Elsevier, 2013-11-01) Shen, HH; Lake, V; Le Brun, AP; James, M; Duff, AP; Peng, Y; McLean, KM; Hartley, PGIn this work we have formulated Annexin V (ANX) decorated phosphatidylserine containing phytantriol (PSPhy) cubosomes to act as probes for the enhanced detection of apoptotic membranes in both model and in vitro cell systems. Small angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (Cryo-TEM) indicated that ANX-containing PSPhy (ANX-PSPhy) cubosomes retain the Pn3m cubic symmetry and cubic phase nanoparticle characteristics of PSPhy cubosomes. The interaction of ANX-PSPhy cubosomes with apoptotic model and cellular membranes was also investigated using both quartz crystal microbalance with dissipation and confocal microscopy which confirmed that ANX-PSPhy cubosomes can selectively bind to apoptotic cells and model membranes. Neutron reflectometry has also been used to show strong binding of ANX-PSPhy cubosomes to a model apoptotic membrane, and in addition reveals changes in both the bilayer structure and in the internal structure of the cubosome in a region adjacent to the membrane as a result of material exchange. This material exchange between cubosome and apoptotic model bilayer was further demonstrated using Cryo-TEM. We have demonstrated that lipid bound protein, in this case Annexin V, can be used to target cubosome systems to biological surfaces in vitro. © 2013, Elsevier Ltd.
- ItemAn x-ray and neutron reflectometry study of 'PEG-like' plasma polymer films(The Royal Society, 2012-05-07) Menzies, DJ; Nelson, A; Shen, HH; McLean, KM; Forsythe, JS; Gengenbach, TR; Fong, C; Muir, BWPlasma-enhanced chemical vapour-deposited films of di(ethylene glycol) dimethyl ether were analysed by a combination of X-ray photoelectron spectroscopy, atomic force microscopy, quartz crystal microbalance with dissipation monitoring (QCM-D), X-ray and neutron reflectometry (NR). The combination of these techniques enabled a systematic study of the impact of plasma deposition conditions upon resulting film chemistry (empirical formula), mass densities, structure and water solvation, which has been correlated with the films' efficacy against protein fouling. All films were shown to contain substantially less hydrogen than the original monomer and absorb a vast amount of water, which correlated with their mass density profiles. A proportion of the plasma polymer hydrogen atoms were shown to be exchangeable, while QCM-D measurements were inaccurate in detecting associated water in lower power films that contained loosely bound material. The higher protein resistance of the films deposited at a low load power was attributed to its greater chemical and structural similarity to that of poly(ethylene glycol) graft surfaces. These studies demonstrate the utility of using X-ray and NR analysis techniques in furthering the understanding of the chemistry of these films and their interaction with water and proteins. Copyright © The Royal Society 2012.