Effective Encapsulation of Proteins into Size‐Controlled Phospholipid Vesicles Using Freeze‐Thawing and Extrusion

Abstract

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.

Hemoglobin encapsulation in vesicles retards NO and CO binding and O2release when perfused through narrow gas-permeable tubes

“…HbV was prepared as reported previously (33,40,45,47), with slight modifications. Human Hb solution was obtained through purification of outdated RBCs provided by the Japanese Red Cross Society (Tokyo, Japan).…”

“…The lipid bilayer comprised 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, cholesterol, 1,5-O-dihexadecyl-N-succinyl-Lglutamate (Nippon Fine Chemical, Osaka, Japan), and 1,2-distearoylsn-glycerol-3-phosphatidylethanolamine-N-PEG 5000 (NOF, Tokyo, Japan) at a molar composition of 5:5:1:0.033. The particle diameter was regulated using the extrusion method (45). The encapsulated HbCO was converted to oxyhemoglobin (HbO 2) by exposing the liquid membrane of HbV to visible light under an O2 atmosphere.…”

Encapsulation of Concentrated Hemoglobin Solution in Phospholipid Vesicles Retards the Reaction with NO, but Not CO, by Intracellular Diffusion Barrier

“…However, we use saturated phospholipids that essentially do not induce such reactions (39,40,42). The mean particle diameter was regulated by an extrusion method to 265-305 nm to study the influence of [Hb] in , and to 178, 265, and 452 nm at the same [Hb] in (equal to 35 g/dl) to study the influence of the particle diameter (32,34,43,44) (Table 1). After removing the unencapsulated Hb solution using ultracentrifugation, HbV was resuspended in the same PBS solution (pH 7.4) at [heme] ϭ 300 M. Therefore, the carbonylhemoglobin in HbV is converted to HbO 2 by photodissociation of CO by illuminating visible light under O 2 atmosphere.…”

Advanced Drug Carriers Targeting Bone Marrow

“…The liposomes were designed to have high entrapment capacity with the interfacial electrostatic interaction to form a unilamellar membrane (Sato et al, 2009;Sou et al, 2003;Sou 2011b). These characteristics are expected to facilitate the application of the bone marrow-targeted liposomes as pharmaceutical carriers to bone marrow.…”