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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 freeze-thaw number on AChE encapsulation under similar conditions as in figure 1.
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Figure 2: Effect of freeze-thaw number on AChE encapsulation under similar conditions as in figure 1.

Mentions: Hydration of lipid films results in inhomogeneous multilamellar vesicles. Application of a series of freeze-thaw cycles breaks the multilamellar vesicles into unilamellar vesicles. As each freeze-thaw cycle usually causes denaturation of proteins, we investigated the effect number of freeze-thaw on encapsulation. About 5 mg eggPC lipid films were solubilized with 1 ml of a 25 mM MOPS pH 7 solution containing AChE. The tubes then underwent different numbers of freeze-thaw cycles from 0 to 20. The liposome solutions were then extruded. The encapsulation efficiencies were compared and are shown in figure 2. We observed an increase of encapsulation efficiency according to the number of freeze-thaw cycles without significant denaturation of the enzyme. This suggests that in the lipid hydration method, the encapsulation mainly occurs during the freeze thaw step. In the following experiments we fix the freeze-thaw cycles to 10.


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 freeze-thaw number on AChE encapsulation under similar conditions as in figure 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Effect of freeze-thaw number on AChE encapsulation under similar conditions as in figure 1.
Mentions: Hydration of lipid films results in inhomogeneous multilamellar vesicles. Application of a series of freeze-thaw cycles breaks the multilamellar vesicles into unilamellar vesicles. As each freeze-thaw cycle usually causes denaturation of proteins, we investigated the effect number of freeze-thaw on encapsulation. About 5 mg eggPC lipid films were solubilized with 1 ml of a 25 mM MOPS pH 7 solution containing AChE. The tubes then underwent different numbers of freeze-thaw cycles from 0 to 20. The liposome solutions were then extruded. The encapsulation efficiencies were compared and are shown in figure 2. We observed an increase of encapsulation efficiency according to the number of freeze-thaw cycles without significant denaturation of the enzyme. This suggests that in the lipid hydration method, the encapsulation mainly occurs during the freeze thaw step. In the following experiments we fix the freeze-thaw cycles to 10.

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