title: 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.
authors.affiliation: Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Sou
authors.forename: Yoshiyasu
authors.initials: Y
authors.lastname: Naito
authors.forename: Taro
authors.initials: T
authors.lastname: Endo
authors.forename: Shinji
authors.initials: S
authors.lastname: Takeoka
authors.forename: Eishun
authors.initials: E
authors.lastname: Tsuchida
mesh_terms: D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis; D016503:Drug Delivery Systems / Q000379:methods*; D005615:Freezing; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008081:Liposomes / Q000138:chemical synthesis / Q000737:chemistry*; D008420:Manufactured Materials; D008563:Membrane Lipids / Q000737:chemistry*; D008567:Membranes, Artificial; D010316:Particle Size; D010743:Phospholipids / Q000737:chemistry*; D011312:Pressure; D014462:Ultrafiltration / Q000379:methods*
chemical_list: D020099:Coated Materials, Biocompatible; D006454:Hemoglobins; D008081:Liposomes; D008563:Membrane Lipids; D008567:Membranes, Artificial; D010743:Phospholipids