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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 coencapsulation of stabilizers on AChE encapsulation. The measurements were done under similar conditions as in figure 1. (A) Encapsulation efficiency in absence (control) or with sugars (sucrose, mannose or trehalose). (B) Encapsulation efficiency as function of Polyethylene Glycol (PEG) concentration. (C) Encapsulation efficiency as function of Bovine Serum Albumin (BSA) concentration.
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Figure 6: Effect of coencapsulation of stabilizers on AChE encapsulation. The measurements were done under similar conditions as in figure 1. (A) Encapsulation efficiency in absence (control) or with sugars (sucrose, mannose or trehalose). (B) Encapsulation efficiency as function of Polyethylene Glycol (PEG) concentration. (C) Encapsulation efficiency as function of Bovine Serum Albumin (BSA) concentration.

Mentions: Most of proteins are denatured during freeze-thaw cycles. Since this step of the encapsulation process is a key step for the encapsulation efficiency, it cannot be skipped. One solution to avoid denaturation of proteins sensible to thermal fluctuations would be the coencapsulation of stabilizers. Indeed, addition of stabilizing additives in protein formulations is the most common tool to increasing the shelf life of the product. These compounds are often chosen on an empirical basis since the protective effect of solutes is variable, depending on protein characteristics. We therefore tried to coencapsulate AChE with different molecules (sugars, polymer or protein) usually employed to stabilize enzymes and we checked if these molecules affect encapsulation efficiencies. Here our enzyme was stable to resist the freeze-thaw cycle. We therefore investigated if the coencapsulation reduces the activity of the encapsulated enzyme. Mannose, sucrose and trehalose were coencapsulated at 100 mM with AChE as described in materials and methods. It appeared that those stabilizers did not affect encapsulation (data not shown), suggesting that proteins that would be stabilized by those sugars could be coencapsulated without any loss of efficiency. PEG (polyethylene glycol) and BSA (bovine serum albumin) protect the AChE from denaturation [14]. Enzyme buffered solutions containing various concentrations of PEG 3350 or BSA were used for encapsulation and efficiencies were compared. It turned out that PEG 3350 and BSA strongly disfavors encapsulation (Fig. 6) suggesting that PEG and BSA immobilize water molecules and compete with AChE for interaction with phospholipids.


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 coencapsulation of stabilizers on AChE encapsulation. The measurements were done under similar conditions as in figure 1. (A) Encapsulation efficiency in absence (control) or with sugars (sucrose, mannose or trehalose). (B) Encapsulation efficiency as function of Polyethylene Glycol (PEG) concentration. (C) Encapsulation efficiency as function of Bovine Serum Albumin (BSA) concentration.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Effect of coencapsulation of stabilizers on AChE encapsulation. The measurements were done under similar conditions as in figure 1. (A) Encapsulation efficiency in absence (control) or with sugars (sucrose, mannose or trehalose). (B) Encapsulation efficiency as function of Polyethylene Glycol (PEG) concentration. (C) Encapsulation efficiency as function of Bovine Serum Albumin (BSA) concentration.
Mentions: Most of proteins are denatured during freeze-thaw cycles. Since this step of the encapsulation process is a key step for the encapsulation efficiency, it cannot be skipped. One solution to avoid denaturation of proteins sensible to thermal fluctuations would be the coencapsulation of stabilizers. Indeed, addition of stabilizing additives in protein formulations is the most common tool to increasing the shelf life of the product. These compounds are often chosen on an empirical basis since the protective effect of solutes is variable, depending on protein characteristics. We therefore tried to coencapsulate AChE with different molecules (sugars, polymer or protein) usually employed to stabilize enzymes and we checked if these molecules affect encapsulation efficiencies. Here our enzyme was stable to resist the freeze-thaw cycle. We therefore investigated if the coencapsulation reduces the activity of the encapsulated enzyme. Mannose, sucrose and trehalose were coencapsulated at 100 mM with AChE as described in materials and methods. It appeared that those stabilizers did not affect encapsulation (data not shown), suggesting that proteins that would be stabilized by those sugars could be coencapsulated without any loss of efficiency. PEG (polyethylene glycol) and BSA (bovine serum albumin) protect the AChE from denaturation [14]. Enzyme buffered solutions containing various concentrations of PEG 3350 or BSA were used for encapsulation and efficiencies were compared. It turned out that PEG 3350 and BSA strongly disfavors encapsulation (Fig. 6) suggesting that PEG and BSA immobilize water molecules and compete with AChE for interaction with phospholipids.

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