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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 composition on AChE encapsulation. Effect of eggPC, eggPC+5%POPE, POPC, POPS under similar conditions as in figure 1.
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Figure 7: Effect of lipid composition on AChE encapsulation. Effect of eggPC, eggPC+5%POPE, POPC, POPS under similar conditions as in figure 1.

Mentions: Different phospholipid compositions were used for the formulation of liposomes loaded with AChE, and encapsulation efficiencies were compared. Liposomes with the same polar head groups (eggPC and POPC) but with hydrocarbon chains of different length and saturation degrees had nearly the same encapsulation efficiencies (Fig. 7) suggesting that encapsulation efficiency does not depend on the hydrophobic component of the phospholipid. Encapsulation efficiency was better with POPC than with POPS (Fig. 1 and 7). Encapsulation was performed at pH 8.5, the enzyme peripheral surface was globally negatively charged. Surface of lipid membrane made with POPC was neutral and surface of lipid membrane with POPS was negatively charged. These data suggest that encapsulation depends on electrostatic interactions between the enzyme peripheral surface and the polar head group of phospholipids. With 5% of POPE, no significant encapsulation efficiency differences were observed.


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 composition on AChE encapsulation. Effect of eggPC, eggPC+5%POPE, POPC, POPS under similar conditions as in figure 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Effect of lipid composition on AChE encapsulation. Effect of eggPC, eggPC+5%POPE, POPC, POPS under similar conditions as in figure 1.
Mentions: Different phospholipid compositions were used for the formulation of liposomes loaded with AChE, and encapsulation efficiencies were compared. Liposomes with the same polar head groups (eggPC and POPC) but with hydrocarbon chains of different length and saturation degrees had nearly the same encapsulation efficiencies (Fig. 7) suggesting that encapsulation efficiency does not depend on the hydrophobic component of the phospholipid. Encapsulation efficiency was better with POPC than with POPS (Fig. 1 and 7). Encapsulation was performed at pH 8.5, the enzyme peripheral surface was globally negatively charged. Surface of lipid membrane made with POPC was neutral and surface of lipid membrane with POPS was negatively charged. These data suggest that encapsulation depends on electrostatic interactions between the enzyme peripheral surface and the polar head group of phospholipids. With 5% of POPE, no significant encapsulation efficiency differences were observed.

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