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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 addition of 1% DOGS-NTA-Ni to egg-PC lipids on AChE encapsulation efficiency in presence of 0.5 M NaCl.
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Figure 8: Effect of addition of 1% DOGS-NTA-Ni to egg-PC lipids on AChE encapsulation efficiency in presence of 0.5 M NaCl.

Mentions: The previous experiments suggest that electrostatic interactions between the enzyme and the lipidic surface favor encapsulation. However, increasing the unspecific electrostatic interactions with the lipid surface is rather limited as the ionic strength and the surface charge density of the protein can only be varied within a small range. In order to provide "universal method" for high efficient encapsulation we then test the possibility to use a specific interaction using the affinity of histidine tag for Nickel. The AChE we used has three histidine tag in a peptidic loop lying at the surface of the protein. This enzyme binds to NTA-Ni column chromatography confirming the functionality of the histidine tag. 5 mg of eggPC mixed with 0.12 mg DOGS-NTA-Ni lipids were used to formulate 1 ml lipid suspension. In order to observe the enhancement due to the specific interaction we minimize the electrostatic interaction by addition of 500 mM NaCl to the solution. It appeared (Fig. 8) that the addition of DOGS-NTA-Ni lipids in the composition of the liposomes enhanced the encapsulation efficiency of AChE.


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 addition of 1% DOGS-NTA-Ni to egg-PC lipids on AChE encapsulation efficiency in presence of 0.5 M NaCl.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Effect of addition of 1% DOGS-NTA-Ni to egg-PC lipids on AChE encapsulation efficiency in presence of 0.5 M NaCl.
Mentions: The previous experiments suggest that electrostatic interactions between the enzyme and the lipidic surface favor encapsulation. However, increasing the unspecific electrostatic interactions with the lipid surface is rather limited as the ionic strength and the surface charge density of the protein can only be varied within a small range. In order to provide "universal method" for high efficient encapsulation we then test the possibility to use a specific interaction using the affinity of histidine tag for Nickel. The AChE we used has three histidine tag in a peptidic loop lying at the surface of the protein. This enzyme binds to NTA-Ni column chromatography confirming the functionality of the histidine tag. 5 mg of eggPC mixed with 0.12 mg DOGS-NTA-Ni lipids were used to formulate 1 ml lipid suspension. In order to observe the enhancement due to the specific interaction we minimize the electrostatic interaction by addition of 500 mM NaCl to the solution. It appeared (Fig. 8) that the addition of DOGS-NTA-Ni lipids in the composition of the liposomes enhanced the encapsulation efficiency of AChE.

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