50 research outputs found
Visualization of membrane loss during the shrinkage of giant vesicles under electropulsation
We study the effect of permeabilizing electric fields applied to two
different types of giant unilamellar vesicles, the first formed from EggPC
lipids and the second formed from DOPC lipids. Experiments on vesicles of both
lipid types show a decrease in vesicle radius which is interpreted as being due
to lipid loss during the permeabilization process. We show that the decrease in
size can be qualitatively explained as a loss of lipid area which is
proportional to the area of the vesicle which is permeabilized. Three possible
mechanisms responsible for lipid loss were directly observed: pore formation,
vesicle formation and tubule formation.Comment: Final published versio
A practical guide to giant vesicles. Probing the membrane nanoregime via optical microscopy
Electrically addressable vesicles: Tools for dielectrophoresis metrology
Dielectrophoresis (DEP) has emerged as an important tool for the manipulation of bioparticles ranging from the submicron to the tens of microns in size. Here we show the use of phospholipid vesicle electroformation techniques to develop a new class of test particles with specifically engineered electrical propserties to enable identifiable dielectrophoretic responses in microfabricated systems. These electrically addressable vesicles (EAVs) enable the creation of electrically distinct populations of test particles for DEP. EAVs offer control of both their inner aqueous core and outer membrane properties; by encapsulating solutions of different electrolyte strength inside the vesicle and by incorporating functionalized phospholipids containing poly(ethylene glycol) (PEG) brushes attached to their hydrophilic headgroup in the vesicle membrane, we demonstrate control of the vesicles’ electrical polarizabilities. This combined with the ability to encode information about the properties of the vesicle in its fluorescence signature forms the first steps toward the development of EAV populations as metrology tools for any DEP-based microsystem.National Institutes of Health (U.S.) (Grant RR199652)National Institutes of Health (U.S.) (Grant EB005753)Merck/CSBi (Fellowship)Solomon Buchsbaum AT&T Research Fun
Melting Regime of the Anionic Phospholipid DMPG: New Lamellar Phase and Porous Bilayer Model
Extensive Bilayer Perforation Coupled with the Phase Transition Region of an Anionic Phospholipid
Studying the role of negatively charged membranes on the mode of action of Esc 1b (1-18)
FAPESPCNPqCAPESUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, BrazilFAPESPCNPqCAPESWeb of Scienc
A potential generic downstream process using Cibracon Blue resin at very high loading capacity produces a highly purified monoclonal antibody preparation from cell culture harvest
The use of a dye-ligand chromatography for the purification of monoclonal antibody (MAb) from cell culture and other feed streams has been largely overlooked in large scale production. Cibracon Blue dye (CB), a polycyclic anionic ligand, interacts with protein through a specific interaction between the dye, acting as a mimic of NAD+ and NADP+, or through non-specific electrostatic, hydrophobic, and other forces. In this paper, a CB resin was used to effectively and efficiently separate an IgG4 MAb from host and process impurities following the capture of the MAb on a Protein-A (PA) column. The CB unit operation, challenged at ≤180 g MAb/L of resin with the PA eluate, reduced BSA (1-2 log), host cell protein (HCP; 2-3 log), MAb oligomer (31-85%), fragment (from ∼0.8% to <0.1%), and other undesired MAb species. Purity, as measured by non-reducing (NR) SDS-PAGE, was improved 33-85%, to 92-99.5% overall (>99% by reducing SDS-PAGE). A facile three column scalable production scheme, employing CB as the second column in the process was used to generate highly purified MAb from cell culture harvest derived from two media of very different compositions. Free CB dye was ≤1 ng/mg in MAb preparations purified through the three column process and then concentrated and buffer exchanged into the appropriate buffer using tangential flow filtration (TFF). © 2006 Elsevier B.V. All rights reserved
Gene Transfection Mediated by Catiomers Requires Free Highly Charged Polymer Chains To Overcome Intracellular Barriers
The
prospective use of the block copolymers poly(ethylene oxide)<sub>113</sub>-<i>b</i>-poly[2-(diethylamino)ethyl methacrylate]<sub>50</sub> (PEO<sub>113</sub>-<i>b</i>-PDEA<sub>50</sub>)
and poly[oligo(ethylene glycol)methyl ether methacrylate]<sub>70</sub>-<i>b</i>-poly[oligo(ethylene glycol)methyl ether methacrylate<sub>10</sub>-<i>co</i>-2-(diethylamino)ethyl methacrylate<sub>47</sub>-<i>co</i>-2-(diisopropylamino)ethyl methacrylate<sub>47</sub>] (POEGMA<sub>70</sub>-<i>b</i>-P(OEGMA<sub>10</sub>-<i>co</i>-DEA<sub>47</sub>-<i>co</i>-DPA<sub>47</sub>)) as nonviral gene vectors was evaluated. The polymers are
able to properly condense DNA into nanosized particles (<i>R</i><sub>H</sub> ≈ 75 nm), which are marginally cytotoxic and
can be uptaken by cells. However, the green fluorescent protein (GFP)
expression assays evidenced that DNA delivery is essentially negligible
meaning that intracellular trafficking hampers efficient gene release.
Subsequently, we demonstrate that cellular uptake and particularly
the quantity of GFP-positive cells are substantially enhanced when
the block copolymer polyplexes are produced and further supplemented
by BPEI chains (branched polyethylenimine). The dynamic light scattering/electrophoretic
light scattering/isothermal titration calorimetry data suggest that
such a strategy allows the adsorption of BPEI onto the surface of
the polyplexes, and this phenomenon is responsible for increasing
the size and surface charge of the assemblies. Nevertheless, most
of the BPEI chains remain freely diffusing in the systems. The biological
assays confirmed that cellular uptake is enhanced in the presence
of BPEI and principally, the free highly charged polymer chains play
the central role in intracellular trafficking and gene transfection.
These investigations pointed out that the transfection efficiency
versus cytotoxicity issue can be balanced by a mixture of BPEI and
less cytotoxic agents such as for instance the proposed block copolymers