Efficient Preparation of Giant Vesicles as Biomimetic Compartment Systems with High Entrapment Yields for Biomacromolecules (original) (raw)
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Enzymes inside lipid vesicles: preparation, reactivity and applications
Biomolecular Engineering, 2001
There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior www.elsevier.com/locate/geneanabioeng Abbre6iations: Bz-Arg-pNA, benzoyl-L-Arg-p-nitroanilide; DDV, vesicles prepared by the detergent dialysis method; DMPC, 1,2prepared by the dehydration-rehydration method; DRV-MFV, vesicles prepared by microfluidization of DRV; DRV-VET 100 , vesicles prepared by using the dehydration-rehydration method first and then extruding through polycarbonate membranes with a mean pore diameter of 100 nm used in the last extrusion step; DSPE-PEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000 ]; EE, entrapment efficiency= (amount of enzyme entrapped in the vesicles)/(total amount of enzyme) × 100 (%); FAT-VET 200 , vesicles prepared by freeze-thaw cycles, followed by repeated extrusions through polycarbonate membranes with a mean pore diameter of 200 nm used in the last extrusion step; G M1 , monosialoganglioside; GUV, giant unilamellar vesicles; IFV, vesicles prepared by the so-called 'interdigitated-fusion method'; LUV, large unilamellar vesicles; lyso PC, lysophosphatidylcholine; lyso PE, lysophosphatidylethanolamine; lyso PI, lysophosphatidylinositol; MFV, vesicles prepared by using an homogenizer and the Microfluidizer™; MLS, multilamellar spherulites prepared by shearing a lamellar phase; MLV, multilamellar vesicles; MLV-FAT, vesicles prepared by repetitive freezing and thawing a MLV suspension; MLV-MFV, vesicles prepared from MLV by using the Microfluidizer™; MVVprepared by the reverse-phase evaporation method; REV-VET, REV that have been extruded through polycarbonate membranes of a defined pore size; SM, sphingomyelin; SOPC, 1-stearoyl-2-oleoyl-sn-glycero-3phosphocholine; Suc-Ala-Ala-Pro-Phe-pNA: succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide; SUV, small (sonicated) unilamellar vesicles as obtained by sonifying MLV; T m , main lamellar chain-melting phase transition temperature (also called 'lamellar gel-liquid crystalline phase transition temperature'); VEI, vesicles prepared by the ethanol injection method; VET 100 , vesicles prepared by the extrusion method (without freezing-thawing cycles) using for final extrusions polycarbonate membranes with mean pore diameters of 100 nm; VPL, vesicles prepared by the pro-liposome method; Z-Phe-Val-Arg-pNA, benzyloxycarbonyl-L-Phe-L-Val-L-Arg-p-nitroanilide.
Rapid preparation of giant unilamellar vesicles
Proceedings of the National Academy of Sciences, 1996
We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 ,um in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-a-phosphatidylcholine (lecithin), dipalmitoleoyl L-aphosphatidylcholine, and fJ-arachidonoyl y-palmitoyl L-aphosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cellsized proteoliposomes have been prepared in which y-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall. Liposomes consisting of single phospholipid membranes (unilamellar vesicles) are excellent model systems for studying the dynamics and structural features of many cellular processes, including viral infection, endocytosis, exocytosis, cell fusion, and transport phenomena. In addition to having importance for basic research in biological disciplines, liposomes are used as vehicles for drug application (1), for gene transfer in medical therapy and genetic engineering (2), and as microcapsules for proteolytic enzymes in the food industry (3). Vesicles also open many exciting possibilities for chemical reactions in small confined volumes, 10-12 to 10-21 liters. If
Preparation and characterization of polymer coated small unilamellar vesicles
Biochimica et Biophysica Acta (BBA) - General Subjects, 1991
Glucose oxidase was entrapped in small unilamellar vesicles composed of phosphatidylcholine, dicetyl phosphate and cholesterol. Prediction of the enzyme content of liposomes by calculations based on input concentrations of lipid and protein, dimensions of the lipids and the liposomes yielded one protein per vesicle. The entrapment efficiency was experimentally determined to be about 13%. On the other hand the entrapment efficiency for the small chromate ions was found to be significantly lower (0.1%). The liposomes were then coated with a polymer, poly(1,4-pyridinium diylethylene salt). It was possible to remove the lipoid material from underneath the polymer layer with various techniques. The effect of sonication and treatment with organic solvents (tested for this purpose) on enzyme activity were found to be very significant and Triton X-100 was chosen for this purpose. It was shown that the enzyme within the remaining net has 89% of its original activity.
Biotechnology Progress, 2003
We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-snglycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-L-glutamate-Nsuccinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 µm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL-1 and cooling rate of-140°C min-1. The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL-1) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 (20 nm.
Colloids and Surfaces B: Biointerfaces, 2009
Giant liposomes, or giant vesicles, are cell-size (∼5-100 m) compartments enclosed with phospholipid bilayers, and have often been used in biological research. They are usually generated using hydration methods, "electroformation" and "gentle hydration (or natural swelling)", in which dry lamellar films of phospholipids are hydrated with aqueous solutions. In gentle hydration, however, giant liposomes are difficult to prepare from an electrostatically neutral phospholipid because lipid lamellae cannot repel each other. In this study, we demonstrate the efficient formation of giant liposomes using the gentle hydration of neutral phospholipid (dioleoyl phosphatidylcholine, DOPC) dry films doped with nonelectrolytic monosaccharides (glucose, mannose, and fructose). A mixture of DOPC and such a sugar in an organic solvent (chloroform/methanol) was evaporated to form the films, which were then hydrated with distilled water or Tris buffers containing sodium chloride. Under these conditions, giant liposomes spontaneously formed rapidly and assumed a swollen cell-sized spherical shape with low lamellarity, whereas giant liposomes from pure DOPC films had multilamellar lipid layers, miscellaneous shapes and smaller sizes. This observation indicates that giant unilamellar vesicles (GUVs) of DOPC can be obtained efficiently through the gentle hydration of sugar-containing lipid dry films because repulsion between lipid lamellae is enhanced by the osmosis induced by dissolved sugar.
Colloids and Surfaces B: Biointerfaces, 2005
Giant unilamellar vesicles (diameter of a few tens of micrometers) are commonly produced by hydration of a dried lipidic film. After addition of the aqueous solution, two major protocols are used: (i) the gentle hydration method where the vesicles spontaneously form and (ii) the electroformation method where an ac electric field is applied. Electroformation is known to improve the rate of unilamellarity of the vesicles though it imposes more restricting conditions for the lipidic composition of the vesicles. Here we further characterize these methods by using fluorescence microscopy. It enables not only a sensitive detection of the defects but also an evaluation of the quantity of lipids in these defects. A classification of the defects is proposed and statistics of their relative importance in regard to both methods and lipid composition are presented: it shows for example that 80% of the vesicles obtained by electroformation from 98% 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine are devoid of significant defects against only 40% of the vesicles with the gentle hydration method. It is also shown that the presence of too many negatively charged lipids does not favor the formation of unilamellar vesicles with both methods. For the gentle hydration, we checked if the negatively charged lipids were inserted in the vesicles membrane in the same proportion as that of the lipid mixture from which they are formed. The constant incorporation of a negatively charged labeled lipid despite an increasing presence of negatively charged 1,2-Dioleoyl-sn-Glycero-3-[Phospho-l-Serine] tends to confirm that the composition of vesicles is indeed close to that of the initial mixture.
Protein Binding onto Surfactant-Based Synthetic Vesicles
Journal of Physical Chemistry B, 2007
Synthetic vesicles were prepared by mixing anionic and cationic surfactants, aqueous sodium dodecylsulfate with didodecyltrimethylammonium or cetyltrimethylammonium bromide. The overall surfactant content and the (anionic/cationic) mole ratios allow one to obtain negatively charged vesicles. In the phase diagram, the vesicular region is located between a solution phase, a lamellar liquid crystalline dispersion, and a precipitate area. Characterization of the vesicles was performed by electrophoretic mobility, NMR, TEM, and DLS and we determined their uni-lamellar character, size, stability, and charge density. Negatively charged vesicular dispersions, made of sodium dodecylsulfate/didodecyltrimethylammonium bromide or sodium dodecylsulfate/ cetyltrimethylammonium bromide, were mixed with lysozyme, to form lipoplexes. Depending on the protein/ vesicle charge ratio, binding, surface saturation, and lipoplexes flocculation, or precipitation, occurs. The free protein in excess remains in solution, after binding saturation. The systems were investigated by thermodynamic (surface tension and solution calorimetry), DLS, CD, TEM, 1 H NMR, transport properties, electrophoretic mobility, and dielectric relaxation. The latter two methods give information on the vesicle charge neutralization by adsorbed protein. Binding is concomitant to modifications in the double layer thickness of vesicles and in the surface charge density of the resulting lipoplexes. This is also confirmed by developing the electrophoretic mobility results in terms of a Langmuir-like adsorption isotherm. Charges in excess with respect to the amount required to neutralize the vesicle surface promote lipoplexes clustering and/or flocculation. Protein-vesicle interactions were observed by DLS, indicating changes in particle size (and in their distribution functions) upon addition of LYSO. According to CD, the bound protein retains its native conformation, at least in the SDS/CTAB vesicular system. In fact, changes in the R-helix and -sheet conformations are moderate, if any. Calorimetric methods indicate that the maximum heat effect for LYSO binding occurs at charge neutralization. They also indicate that enthalpic are by far the dominant contributions to the system stability. Accordingly, energy effects associated with charge neutralization and double-layer contributions are much higher than counterion exchange and dehydration terms.
Single-Molecule Enzymology of Chymotrypsin Using Water-in-Oil Emulsion
Biophysical Journal, 2005
Single-molecule studies allow the study of subtle activity differences due to local folding in proteins, but are time consuming and difficult because only a few molecules are observed in one experiment. We developed an assay where we can simultaneously measure the activity of hundreds of individual molecules. The assay utilizes a synthetic chymotrypsin substrate that is nonfluorescent before cleavage by chymotrypsin, but is intensely fluorescent afterward. We encapsulated the enzyme and substrate in micron-sized droplets of water surrounded by silicone oil where each microdroplet contains ,1 enzyme on average. A microscope and charge-coupled device camera are used to measure the fluorescence intensity of the same individual droplet over time. Based on these measurements, we conclude that enzymatic reactions could occur within this emulsion system, the statistical average activity of individual chymotrypsin molecules is similar to that measured in bulk, and the activity of individual chymotrypsin is heterogeneous.
Macromolecular Bioscience, 2017
Applications of enzymes are intensively studied, particularly for biomedical applications. However, encapsulation or immobilization of enzymes without deactivation and long-term use of enzymes are still at issue. This study focuses on the polymeric vesicles "PICsomes" for encapsulation of enzymes to develop a hecto-nanometer-scaled enzymeloaded reactor. The catalytic activity of a PICsome-based enzyme nanoreactor is carefully examined to clarify the effect of compartmentalization by PICsome. Encapsulation by PICsome provides a stability enhancement of enzymes after 24 h incubation at 37 °C, which is particularly helpful for maintaining the high effective concentration of β-galactosidase. Moreover, to control the microenvironment inside the nanoreactor, a large amount of dextran, a neutral macromolecule, is encapsulated together with β-galactosidase in the PICsome. The resulting dextran-coloaded nanoreactor contributes to the enhancement of enzyme stability, even after exposure to 24 h incubation at −20 °C, mainly due to the antifreezing effect.