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Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

Colletier JP, Chaize B, Winterhalter M, Fournier D - BMC Biotechnol. (2002)

Bottom Line: Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application.To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion.We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologiques-Groupe de Biochimie des Protéines, Université Paul Sabatier, Toulouse, France. Colletie@ibs.fr

ABSTRACT

Background: We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.

Results: To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion. We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated.

Conclusion: We propose here an optimized procedure to encapsulate fragile enzymes into liposomes. Optimal encapsulation is achieved by induction of a specific interaction between the enzyme and the lipid surface.

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Effect of lipid concentration on AChE encapsulation. Two lipids compsitions were used: egg POPC (red) and POPS (blue).
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Figure 1: Effect of lipid concentration on AChE encapsulation. Two lipids compsitions were used: egg POPC (red) and POPS (blue).

Mentions: In a first series of experiments, we investigated the influence of the lipid concentration on the encapsulation. Various amounts of egg PC and POPS (from 0.5 to 10 mg in 0.1 to 1 ml buffer) were used and the encapsulation efficiency of AChE was recorded. Figure 1 clearly shows a linear relationship between the lipid concentration and the encapsulation efficiency. The liposome surface is proportional to the lipid concentration, in contrast the encapsulated inner volume is proportional to the lipid concentration to the power 3/2. Thus a double logarithmic plot of the encapsulated activity versus lipid concentration allows to discriminate between the relevant parameter. For eggPC and POPC lipids, values of the slope (0.86 +/- 0.01 and 1.16 +/- 0.04, respectively) were close to 1 suggesting that encapsulation is proportional to the number of lipids and thus proportional to the surface; and is significantly far from 1.5 relevant for encapsulation via internal volume.


Protein encapsulation in liposomes: efficiency depends on interactions between protein and phospholipid bilayer.

Colletier JP, Chaize B, Winterhalter M, Fournier D - BMC Biotechnol. (2002)

Effect of lipid concentration on AChE encapsulation. Two lipids compsitions were used: egg POPC (red) and POPS (blue).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC113741&req=5

Figure 1: Effect of lipid concentration on AChE encapsulation. Two lipids compsitions were used: egg POPC (red) and POPS (blue).
Mentions: In a first series of experiments, we investigated the influence of the lipid concentration on the encapsulation. Various amounts of egg PC and POPS (from 0.5 to 10 mg in 0.1 to 1 ml buffer) were used and the encapsulation efficiency of AChE was recorded. Figure 1 clearly shows a linear relationship between the lipid concentration and the encapsulation efficiency. The liposome surface is proportional to the lipid concentration, in contrast the encapsulated inner volume is proportional to the lipid concentration to the power 3/2. Thus a double logarithmic plot of the encapsulated activity versus lipid concentration allows to discriminate between the relevant parameter. For eggPC and POPC lipids, values of the slope (0.86 +/- 0.01 and 1.16 +/- 0.04, respectively) were close to 1 suggesting that encapsulation is proportional to the number of lipids and thus proportional to the surface; and is significantly far from 1.5 relevant for encapsulation via internal volume.

Bottom Line: Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application.To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion.We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire de Synthèse et Physicochimie des Molécules d'Intérêt Biologiques-Groupe de Biochimie des Protéines, Université Paul Sabatier, Toulouse, France. Colletie@ibs.fr

ABSTRACT

Background: We investigated the encapsulation mechanism of enzymes into liposomes. The existing protocols to achieve high encapsulation efficiencies are basically optimized for chemically stable molecules. Enzymes, however, are fragile and encapsulation requires in addition the preservation of their functionality. Using acetylcholinesterase as a model, we found that most protocols lead to a rapid denaturation of the enzyme with loss in the functionality and therefore inappropriate for such an application. The most appropriate method is based on lipid film hydration but had a very low efficiency.

Results: To improve it and to propose a standard procedure for enzyme encapsulation, we separate each step and we studied the effect of each parameter on encapsulation: lipid and buffer composition and effect of the different physical treatment as freeze-thaw cycle or liposomes extrusion. We found that by increasing the lipid concentration, increasing the number of freeze-thaw cycles and enhancing the interactions of the enzyme with the liposome lipid surface more than 40% of the initial total activity can be encapsulated.

Conclusion: We propose here an optimized procedure to encapsulate fragile enzymes into liposomes. Optimal encapsulation is achieved by induction of a specific interaction between the enzyme and the lipid surface.

Show MeSH
Related in: MedlinePlus