J. Guinea

INTERACTION OF THE GLYCOPROTEIN EXCRETED BY PSEUDOALTEROMONAS ANTARCTICA NF3 WITH PHOSPHATIDYLCHOLINE LIPOSOMES

Abstract The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative specie, Pseudoalteromonas antarctica NF 3 , to coat phosphatidylcholine (PC) unilamellar liposomes and to protect these bilayer structures against the solubilizing action of the non-ionic surfactant Triton X-100 (T X-100 ) has been investigated. TEM micrographs of freeze fractured liposome/GP aggregates reveal that the presence of GP in liposome suspensions resulted in the formation of a continuous thin film that coated tightly these bilayer structures. The complete coating of these vesicles was already achieved at a PC:GP weight ratio of about 9:1. Higher GP amounts (up to a PC:GP weight ratio of 1:1) led to a progressive growth of this film which exhibited at the highest GP proportion a multilayered structure. An increasing resistance of PC liposomes to be solubilized by T X-100 took place as the proportion of GP in the system rose, although this protective effect was more effective at low GP proportions (PC:GP weight ratios from 9:1 to 4:1). Thus, although in the range of PC:GP weight ratios from 9:1 to 6:4 a direct dependence was found between the growing of the covering structure and the resistance of the coated liposomes to be solubilized by T X-100 , the best protection effect occurred when this covering structure was a thin and continuous film with a thickness not higher than 15–20 nm (PC:GP weight ratios ranging from 9:1 to 4:1).

Assembly properties and applications of a new exopolymeric compound excreted by Pseudoalteromonas antarctica NF3

The self assembly properties and applications of an exopolymeric compound (EC) of a glycoprotein character excreted by a new Gram-negative species, Pseudoalteromonas antarctica NF3, have been reviewed. This compound exhibited surface-active properties in water, with a concentration of 0.20 mg ml-1 being the key value associated with its physicochemical properties. Unsonicated EC aqueous dispersions showed the coexistence of concentric multilamellar and small unilamellar aggregates by transmission electron microscopy (TEM). Sonication of these dispersions revealed that each lamellae of the initial multilamellar structures were made up of various subunits coiled coils. As for the ability of this exopolymeric biomaterial to coat phosphatidylcholine (PC) liposomes and to protect these vesicles against different surfactants, freeze-fracture TEM micrographs of liposome/EC aggregates revealed that the addition of the EC to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that coated very well the PC bilayers. The complete coating was already achieved at a PC : EC weight ratio of about 9 :1. An increasing resistance of PC liposomes to surfactants (in particular sodium dodecyl sulfate) occurred as the proportion of EC in the system rose, although this effect was more effective at low EC proportions (PC : EC weight ratios from 9 : 1 to 8 : 2). Although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactants, the best protection occurred when this structure was a thin film of about 20-25 nm formed by nine to ten layers of about 2-3 nm.

Ability of the exopolymer excreted by Pseudoalteromonas antarctica NF3, to coat liposomes and to protect these structures against octyl glucoside.

The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative specie Pseudoalteromonas antarctica NF3, to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant octyl glucoside (OG) has been investigated. TEM micrographs of freeze-fractured liposome/GP aggregates reveal that the addition of GP to liposomes led to the formation of a covering structure (polymer adsorbed onto the bilayers) that tightly coated PC bilayers. The complete coating was already achieved when the proportion of GP assembled with liposomes was approximately 10% (wt% vs total PC). Higher GP amounts resulted in a growth of this coating structure which exhibited at the highest GP proportion in the system (31% of assembled GP) a multilayered structure. An increasing resistance of PC liposomes to be affected by OG both at sublytic and lytic levels occurred as the proportion of GP in the system rose; this protective effect being more effective when the proportion of assembled GP was 10-20% in weight. Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by OG, the best protection occurred when the proportion of assembled GP was about 10 wt%.

BIOPOLYMER EXCRETED BY PSEUDOALTEROMONAS ANTARCTICA NF3, AS A COATING AND PROTECTIVE AGENT OF LIPOSOMES AGAINST DODECYL MALTOSIDE

The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative species Pseudoalteromonas antarctica NF(3), to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant dodecyl maltoside was investigated. Transmission electron microscopy (TEM) micrographs of freeze fractured liposome/GP aggregates reveal that the addition of the glycoprotein to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that tightly coated PC bilayers. The complete coating was already achieved at a PC : GP weight ratio of about 9:1. Image analysis profiles of digitalized TEM micrographs (PC : GP weight ratio 8:2) show that this film was formed by a multilayer structure. The periods of the average distance of the pattern ordering in layer structures (9-10 layers) were of about 2-3 nm and the thickness of the complete film was of about 25 nm. Higher amounts of glycoprotein resulted in a growth of this film, which exhibited at the highest proportion of this compound (50% in weight) a multifilm structure. An increasing resistance of liposomes to be affected by dodecyl maltoside both at subsolubilizing and solubilizing levels occurred as the proportion of the glycoprotein in the system rose, although this protective effect was more effective at low proportions of this compound (PC : GP weight ratios from 9:1 to 8:2). Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactant, the more effective protection occurred when this structure was a thin film formed by the assembly of various layers of GP of about 2-3 nm. Copyright 1999 John Wiley & Sons, Inc.

Partitioning of SDS in liposomes coated by the exopolymer excreted by Pseudoalteromonas antarctica NF3 as a measure of vesicle protection against this surfactant.

The capacity of glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF3, to protect phosphatidylcholine (PC) liposomes against the action of the anionic surfactant sodium dodecyl sulfate (SDS) was studied in detail. To this end, changes in the surfactant partitioning between the lipid bilayer and the aqueous phase (partition coefficients, K) and in the effective surfactant to PC molar ratios (R e) were determined as a function of the amount of GP assembled with liposomes. The permeability of liposomes was determined by monitoring the changes in the fluorescence intensity of liposomes due to the release of the fluorescent dye 5(6)-carboxyfluorescein (CF) from the interior of vesicles to the bulk aqueous phase. Increasing GP amounts in the system resulted in the same interaction step as a rise in R e and a fall in the surfactant partitioning between the lipid bilayer and water. Hence, the higher the proportion of GP, the lower the surfactant ability to alter the permeability of liposomes and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in the same interaction step as a progressive increase of the free surfactant concentration (SW). The fact that the SW was always lower than the surfactant critical micelle concentration indicates that the interaction of SDS with coated liposomes was mainly ruled by the action of surfactant monomers in all cases.

Protective effect caused by the exopolymer excreted by Pseudoalteromonas antarctica NF3 on liposomes against the action of octyl glucoside

The capacity of the glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF(3), to protect phosphatidylcholine (PC) liposomes against the action of octyl glucoside (OG) was studied in detail. Increasing amounts of GP assembled with liposomes resulted for the same interaction step in a linear increase in the effective surfactant to PC molar ratios (Re) and in a linear fall in the surfactant partitioning between bilayer and the aqueous phase (partition coefficients K). Thus, the higher the proportion of GP assembled with liposomes the lower the surfactant ability to alter the permeability of vesicles and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in a progressive increase of the free surfactant concentration (S(W)) needed to produce the same alterations in liposomes. The fact that S(W) was always lower than the surfactant critical micelle concentration indicates that the interaction was mainly ruled by the action of surfactant monomers, regardless of the amount of assembled GP.

Protection of liposomes against triton X-100 by means of the new exopolymer excreted by Pseudoalteromonas antarctica NF3

Abstract The capacity of the glycoprotein (GP) excreted by Pseudoalteromonas antarctica NF 3 , to protect phosphatidylcholine (PC) liposomes against the action of Triton X-100 (T X-100 ) was studied in detail. Increasing GP amounts in the system led to a progressive adsorption of this compound on the surface of liposomes in equilibrium with the free GP in the aqueous phase. This fact resulted for the same interaction step in an increase in the effective surfactant to PC molar ratios (Re) and in a fall in the surfactant partitioning between lipid bilayer and the aqueous phase (partition coefficients K ). Thus, the higher the proportion of GP in the system the lower the surfactant ability to saturate and solubilize PC liposomes and the lower its affinity with these bilayer structures. In addition, increasing GP proportions resulted in a progressive increase of the free surfactant concentration ( S W ) needed to saturate or solubilize liposomes. The fact that S W at sublytic and lytic level was lower and similar (slightly higher) than the surfactant critical micelle concentration indicates that the interaction at these two levels was mainly ruled by the action of surfactant monomers and by the formation of complex mixed micelles surfactant/PC/GP.