formulations-for-freeze-drying-bacteria-their-influence-on-cell

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

The effect of the lipid polar headgroup on melittin-phospholipid interaction was investigated by cryo-TEM, fluorescence spectroscopy, ellipsometry, circular dichroism, electrophoresis and photon correlation spectroscopy. In particular, focus was placed on the effect of the lipid polar headgroup on peptide adsorption to, and penetration into, the lipid bilayer, as well as on resulting colloidal stability effects for large unilamellar liposomes. The effect of phospholipid headgroup properties on melittin-bilayer interaction was addressed by comparing liposomes containing phosphatidylcholine, -acid, and -inositol at varying ionic strength. Increasing the bilayer negative charge leads to an increased liposome tolerance toward melittin which is due to an electrostatic arrest of melittin at the membrane interface. Balancing the electrostatic attraction between the melittin positive charges and the phospholipid negative charges through a hydration repulsion, caused by inositol, reduced this surface arrest and increased liposome susceptibility to the disruptive actions of melittin. Furthermore, melittin was demonstrated to induce liposome structural destabilization on a colloidal scale which coincided with leakage induction for both anionic and zwitterionic systems. The latter findings thus clearly show that coalescence, aggregation, and fragmentation contribute to melittin-induced liposome leakage, and that detailed molecular analyses of melittin pore formation are incomplete without considering also these colloidal aspects.

Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers.

Comparison of melittin interaction with liposomes, bilayer disks and micelles showed that melittin binding to lipid aggregates is largely dictated by the amount of highly curved areas in the aggregates. The PEG-stabilised bilayer disks were characterised by a combination of small angle neutron scattering, cryo-transmission electron microscopy and dynamic light scattering. Importantly, the theoretically foreseen partial segregation of the lipid components, important for maintaining the structure of the bilayer disk, was confirmed. Steady state fluorescence spectroscopy indicated that melittin mainly resides at the rim of the bilayer disks. Results of the present study help increase the understanding of the mechanisms behind, and the physico-chemical factors affecting, melittin-lipid interaction. We suggest that bilayer disks, due to their stable structure, constitute interesting vehicles for transport of peptides that have high propensity to associate with lipid surfaces of high curvature.

Melittin-lipid interaction: a comparative study using liposomes, micelles and bilayer disks.

The membrane-destabilizing effect of the peptide melittin on phosphatidylcholine membranes is modulated by the presence of cholesterol. This investigation shows that inclusion of 40 mol % cholesterol in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes reduces melittin's affinity for the membrane. It is significant that the presence of cholesterol does not increase the amount of membrane-associated melittin needed to cause maximum leakage from, or major structural rearrangements of, the liposomes. Furthermore, comparison of microscopy and leakage data suggests that melittin-induced leakage occurs via different mechanisms in the cholesterol-free and cholesterol-supplemented systems. In the absence of cholesterol, leakage of carboxyfluorescein takes place from intact liposomes in a manner compatible with the presence of small melittin-induced pores. In the presence of cholesterol, on the other hand, adsorption of the peptide causes complete membrane disruption and the formation of long-lived open-bilayer structures. Moreover, in the case of cholesterol-supplemented systems, melittin induces pronounced liposome aggregation. Cryotransmission electron microscopy was used, together with ellipsometry, circular dichroism, turbidity, and leakage measurements, to investigate the effects of melittin on phosphatidylcholine membranes in the absence and presence of cholesterol. The melittin partitioning behavior in the membrane systems was estimated by means of steady-state fluorescence spectroscopy measurements.

Melittin-lipid bilayer interactions and the role of cholesterol.

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.

Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase.

Cryo-TEM and dynamic light scattering was used to investigate morphological changes induced by spray- and freeze-drying of liposomes and nanosized bilayer disks composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DSPE-PEG) from lactose solution. Particular focus was put on the identification of structural alterations that risk influencing the performance of liposomes and bilayer disks as carriers for protein and peptide drugs. Significant changes in the lipid aggregate structure and/or size was noted upon dehydration. Uni-lamellar liposomes tended to shrink in size and become bi-lamellar as a consequence of the drying process. The same transformation was observed upon deliberate establishment of a lactose gradient over the membranes of liposomes in solution. A mechanism based on an osmotically driven invagination of the liposomes is proposed to explain the change from uni- to bi-lamellar structures. PEGylation promoted formation of larger liposomes during spray-drying, and had a similar, but less pronounced, effect also during freeze-drying. The observed structural changes may have important consequences for the bioavailability of protein/peptide drugs bound to, or embedded in, the liposome membranes. The radius of bilayer disks increased upon both spray- and freeze-drying, but the drying procedure did not change the open single-bilayer structure of the disks.

Structural effects caused by spray- and freeze-drying of liposomes and bilayer disks.

Cryo-transmission electron microscopy was used in combination with turbidity and leakage measurements to explore and compare the membrane perturbing effects of melittin and alamethicin on POPC-based liposomes of varying composition. The results show that the two peptides, despite their differences in physico-chemical properties and proposed mode of action, induce similar structural effects on the liposomes. Importantly, whereas low peptide concentrations leave pure POPC liposomes intact and seemingly unperturbed, POPC liposomes supplemented with 40 mol.% cholesterol change their shape, rupture and fuse in response to the addition of both melittin and alamethicin. In the case of alamethicin, but not melittin, fusion is effectively prevented by inclusion of 10 mol.% POPG in the liposome membranes. By means of a competitive binding assay we furthermore show that alamethicin, in line with earlier findings for melittin, possess high affinity for positively curved lipid surfaces. Moreover, results from the present study show that magainin 2 has a similar preference for curved surfaces.

Effect of Î±-helical peptides on liposome structure: a comparative study of melittin and alamethicin.

Disaccharides are, in general, the first choice as formulation compounds when freeze-drying microorganisms. Although polysaccharides and other biopolymers are considered too large to stabilise and interact with cell components in the same beneficial way as disaccharides, polymers have been reported to support cell survival. In the present study we compare the efficiency of sucrose and the polymers Ficoll, hydroxyethylcellulose, hydroxypropylmethylcellulose and polyvinylalcohol to support the survival of three bacterial strains during freeze drying. The initial osmotic conditions were adjusted to be similar for all formulations. Formulation characterisation was used to interpret the impact that different compound properties had on cell survival.

Impact of matrix properties on the survival of freeze-dried bacteria.

Disaccharide sind in der Regel die erste Wahl als Formulierung Verbindungen beim Gefriertrocknen von Mikroorganismen. Obgleich Polysaccharide und andere Biopolymere zu gro&szlig; betrachtet sind, zu stabilisieren und die Interaktion mit dem Zellbestandteile vorteilhaft wie Disaccharide, wurde Polymere berichtet, Zelle &uuml;berleben zu unterst&uuml;tzen. In der vorliegenden Studie vergleichen wir die Effizienz der Saccharose und die Polymere Ficoll, Hydroxyethylcellulose, Hydroxypropylmethylcellulose und Polyvinylalcohol zu unterst&uuml;tzen, das &Uuml;berleben der drei Bakterienst&auml;mme w&auml;hrend der Trocknung Einfrieren. Osmotischen Anfangsbedingungen wurden angepasst, um f&uuml;r alle Formulierungen &auml;hnlich sein. Formulierung Charakterisierung wurde verwendet, um die Auswirkungen zu interpretieren, dass verschiedene zusammengesetzte Eigenschaften auf das &Uuml;berleben der Zelle hatte.

Auswirkungen der Matrix-Eigenschaften auf das &Uuml;berleben der gefriergetrockneten Bakterien.

Freeze-drying of bacterial cells with retained viability and activity after storage requires appropriate formulation, i.e. mixing of physiologically adapted cell populations with suitable protective agents, and control of the freeze-drying process. Product manufacturing may alter the clinical effects of probiotics and it is essential to identify and understand possible factor co-dependencies during manufacturing. The physical solid-state behavior of the formulation and the freeze-drying parameters are critical for bacterial survival and thus process optimization is important, independent of strain. However, the maximum yield achievable is also strain-specific and strain survival is governed by e.g. medium, cell type, physiological state, excipients used, and process. The use of preferred compatible solutes for cross-protection of Lactobacilli during industrial manufacturing may be a natural step to introduce robustness, but knowledge is lacking on how compatible solutes, such as betaine, influence formulation properties and cell survival. This study characterized betaine formulations, with and without sucrose, and tested these with the model lactic acid bacteria Lactobacillus coryniformis Si3. Betaine alone did not act as a lyo-protectant and thus betaine import prior to freeze-drying should be avoided. Differences in protective agents were analyzed by calorimetry, which proved to be a suitable tool for evaluating the characteristics of the freeze-dried end products.

A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying.

In this study we have compared the ability of the organic polymers Ficoll and hydroxyethylcellulose (HEC) and the disaccharides sucrose and trehalose to support cell survival during freeze-drying and subsequent storage of a gram-negative Sphingobium sp. In addition to determination of viability rates, cell integrity was evaluated using lipid peroxidation and RNA quality assays for the different storage conditions and formulation compositions. All formulations resulted in high initial cell survival rates after freeze-drying. However, the disaccharide formulations were superior to the polymer-based formulations in supporting cell survival during storage with the exception of Ficoll that upon storage under vacuum yielded bacterial survival rates equal to that of sucrose. Storage in the presence of both oxygen and moisture was detrimental for bacterial survival in all formulations tested, however, lipid peroxidation or RNA damages were not the controlling mechanisms for cell death in this system. The ability of Ficoll and HEC to support cell survival during freeze-drying show that organic polymers, expected to lack the water replacing capability of e.g. disaccharides, can successfully be used as lyoprotectants. For storage under vacuum conditions we suggest that the intracellular amount of sugars (i.e. trehalose), or other protective native cell components, is sufficient for a basic protection inside the bacteria cell and that the amorphous state is the most important aspect of the formulation excipient. However, when exposed to oxygen and moisture during storage this protection is not sufficient to prevent cell degeneration.

Article	Year
Effect of lipid headgroup composition on the interaction between melittin and lipid bilayers. Journal of colloid and interface science\| PubMed ID: 17383670	2007
Melittin-lipid interaction: a comparative study using liposomes, micelles and bilayer disks. Biochimica et biophysica acta\| PubMed ID: 18571494	2008
Melittin-lipid bilayer interactions and the role of cholesterol. Biophysical journal\| PubMed ID: 18658211	2008
Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase. The Journal of biological chemistry\| PubMed ID: 19767390	2009
Structural effects caused by spray- and freeze-drying of liposomes and bilayer disks. Journal of pharmaceutical sciences\| PubMed ID: 19894278	2010
Effect of Î±-helical peptides on liposome structure: a comparative study of melittin and alamethicin. Journal of colloid and interface science\| PubMed ID: 20226468	2010
Impact of matrix properties on the survival of freeze-dried bacteria. Journal of the science of food and agriculture\| PubMed ID: 21445855	2011
A case study on stress preconditioning of a Lactobacillus strain prior to freeze-drying. Cryobiology\| PubMed ID: 22266474	2012
Effects of di- and polysaccharide formulations and storage conditions on survival of freeze-dried Sphingobium sp. World journal of microbiology & biotechnology\| PubMed ID: 23475257	2013

Per Wessman

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