Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of extrusion number on AChE encapsulation. The filter was made of polycarbonate under similar conditions as in figure 1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC113741&req=5

Figure 3: Effect of extrusion number on AChE encapsulation. The filter was made of polycarbonate under similar conditions as in figure 1.

Mentions: Vesicles formed by spontaneous swelling as described do have a rather polydisperse size distribution [13]. Narrow size distribution can be obtained by extruding the suspension through a filter of defined size. About 5 mg eggPC lipid films were solubilized with 1 ml of a 25 mM MOPS pH7 solution containing AChE. Then the tubes underwent ten freeze-thaw cycles and the liposome solutions were then subjected to different numbers of extrusions (from 0 to 20) through a disposable 200 nm cellulose acetate pore filter. Surprisingly we observed a strong decrease of encapsulation efficiency with the first extrusion and then a progressive recovery (Fig. 3). However, if the solution is passed through a new filter, no recovery was observed. This suggest the retension of much material inside the filter. Increasing the extrusion number did not result in an increase of encapsulation efficiency beyond the encapsulation observed following the freeze-thaw step.


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 extrusion number on AChE encapsulation. The filter was made of polycarbonate under similar conditions as in figure 1.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Effect of extrusion number on AChE encapsulation. The filter was made of polycarbonate under similar conditions as in figure 1.
Mentions: Vesicles formed by spontaneous swelling as described do have a rather polydisperse size distribution [13]. Narrow size distribution can be obtained by extruding the suspension through a filter of defined size. About 5 mg eggPC lipid films were solubilized with 1 ml of a 25 mM MOPS pH7 solution containing AChE. Then the tubes underwent ten freeze-thaw cycles and the liposome solutions were then subjected to different numbers of extrusions (from 0 to 20) through a disposable 200 nm cellulose acetate pore filter. Surprisingly we observed a strong decrease of encapsulation efficiency with the first extrusion and then a progressive recovery (Fig. 3). However, if the solution is passed through a new filter, no recovery was observed. This suggest the retension of much material inside the filter. Increasing the extrusion number did not result in an increase of encapsulation efficiency beyond the encapsulation observed following the freeze-thaw step.

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