Journal of Liposome Research, 2009; 19(2): 148–154
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
RESEARCH ARTICLE
Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes Arcan Güven1, Mayreli Ortiz1, Magdalena Constanti2, and Ciara K. O’Sullivan1,3 Nanobiotechnology and Bioanalysis Group, 2Department of Chemical Engineering, Universitat Rovira I Virgili, Tarragona, Spain, and 3Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain 1
Abstract Liposomes are colloidal structures formed by the self-assembly of lipid molecules in solution into spherical, self-closed structures through their amphiphilic properties. All liposome preparation protocols reported consist of several steps of preparation, homogenization, and purification, which are labor-intensive, arduous, and lengthy to execute. In this work, a new procedure has been developed to reduce the time of the postrehydration sizing of liposomes from multilamellar vesicles, while improving the uniformity of the resulting liposomes produced and achieving high encapsulation efficiencies. For the homogenization step, the typically used method of filter extrusion was substituted by centrifugation. Purification of liposomes to eliminate nonencapsulated molecules and lipids is routinely carried out via gel permeation chromatography, an extremely lengthy procedure, and in the method we report, this lengthy step was replaced by the use of molecular-weight cut-off filters. Using this novel method, large unilamellar vesicles were produced and the time required, postrehydration, was dramatically reduced from almost 48 to less than 2 hours, with a highly uniformly sized population of liposomes being produced—the homogeneity of the liposome population achieved using our method was 99%, as compared to 88% attained by using the traditional method of production. We have used this approach to encapsulate fluorescein isothiocyanate (FITC), and 160,000 FITC molecules were encapsulated and the liposomes were demonstrated to be stable for at least 10 weeks at 4°C.
Introduction Liposomes are colloidal structures formed by the selfassembly of lipid molecules in the solution into spherical, self-closed structures through their amphiphilic properties and consist of a lipid bilayer surrounding an interior aqueous region (Woodle et al., 1995). Liposome science and technology is one of the fastest growing scientific fields, contributing to areas such as drug delivery, cosmetics, waste water treatment, genetics, structure and function of biological membranes, and investigations of the origin of life, and they find a wide range of applications due to their biocompatible and controllable physicochemical properties and, most important, their ability to entrap molecules such as drugs, enzymes, fluorescent markers, and magnetic materials (Rongen et al., 1997; Edwards and Baumer, 2006; Ahn-Yoon et al.,
2003). This is due to several advantageous characteristics of liposomes, such as their ability to incorporate not only water, but also lipid-soluble agents, specific targeting to the required site in the body, and versatility in terms of fluidity, size, and charge (Mozafari, 2005). Liposomes can be prepared from a variety of lipids and lipid mixtures, with phospholipids being the most commonly used (Szoka and Papahadjopoulos,1980). Phospholipids have defined polar head groups and nonpolar hydrocarbon tails, and due to this, phospholipids organize themselves into ordered aggregates where the nonpolar regions orientate themselves toward the interior away from the aqueous phase, the polar regions being in contact with the aqueous phase (Lasic, 1988). Liposomes can be classified according to the method of their preparation or the number of bilayers present in the vesicle or by their size.
Address for Correspondence: Mayreli Ortiz, and Ciara O’Sulivan, Nanobiotechnology and Bioanalysis Group, Universitat Rovira I Virgili, Avd. Països Catalans, 26, 43007 Tarragona, Spain. E-mail:
[email protected] and
[email protected] (Received 20 October 2008; accepted 08 December 2008) ISSN 0898-2104 print/ISSN 1532-2394 online © 2009 Informa UK Ltd DOI: 10.1080/08982100802674419
http://www.informapharmascience.com/lpr
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
Fluorescein encapsulating liposomes 149 Since their first report and definition by Bangham, lipid vesicles have been used extensively, first as models for biological membranes and more recently as carriers of various molecules, such as drugs, hormones, enzymes, genetic materials, or proteins. Liposomes are formed when thin lipid films are hydrated and stacks of liquid crystalline bilayers become fluid and swell (Zawada, 2004). The hydrated lipid films self-close to form multilamellar vesicles (MLVs) and large unilamellar vesicles (LUVs), which have a polydisperse size distribution (Barenholz, 2003). In order to subsequently produce homogenous populations of liposomes of reduced size, an input of energy, such as sonic energy (sonication) or mechanical energy (extrusion) is required (Deamer and Bangham, 1976). This sizing step is followed by purification to remove unencapsulated molecules and very small vesicles. An ideal method for producing liposomes would form unilamellar vesicles of uniform size, would be applicable to most lipid mixtures, and could efficiently trap ions, metabolites, or high-molecular-weight molecules. The diameter of the vesicles should be within the range of 0.1–1.0 m for versatility and to facilitate high trapping efficiency (Deamer and Bangham, 1976). Numerous procedures have been developed to prepare liposomes, but none of them, however, perfectly fulfill the two basic requirements of homogeneity and maximum encapsulation (Brandl et al., 1997). The most commonly used synthesis methodology is the lipid film hydration method coupled with the mechanical dispersion technique of filter extrusion used to obtain a more homogeneous distribution of defined mean size (Gregoriadis, 1984). The conventional method of liposome production involves three basic stages: drying down of lipids from organic solvents, dispersion of the lipids in aqueous media containing the buffer, salts, and any material desired for entrapment in the interior of the vesicles, homogenization, purification of the resultant liposomes, and subsequently, analysis of the final product (Mozafari, 2005). This method has the advantage of simplicity and relative stability, although it has a low encapsulation efficiency and is very irreproducible from batch to batch (Huang and Kennel, 1979). Sonication and extrusion are most often used for the dispersion step. Sonication generally produces small unilamellar vesicles (SUVs) 20–50 nm in diameter (Gregoriadis, 1984). In extrusion, the MLV solution is passed back and forth across a polycarbonate membrane of defined pore size, producing a vesicle population with a very homogeneous distribution and mean diameter proportional to the smallest pore size used (Chen et al., 1956). Extrusion is a simple and reproducible method for the preparation of liposomes with a narrow size distribution, but suffers from the drawback of low encapsulation in comparison with other methods (Szoka and Papahadjopoulos, 1980; Gregoriadis, 1984;
Huang and Kennel, 1979), and if an automated system is not used, requires intensive labor. These methods of homogenization and purification suffer from being arduous and lengthy to execute, requiring considerable “hands-on” time, typically 8 hours for homogenization (sizing) and a further 6 hours for purification (Nasseau et al., 2001; Olson et al., 1979). Considering the importance of liposomes, it is scientifically and industrially relevant to replace these laborious preparation steps with convenient and shorter procedures, which will result in a population of uniform liposomes capable of maximum encapsulation efficiency and demonstrating long-term stability under routine storage conditions. In this paper, we report on a new method, which dramatically reduces the time required for the preparation of stable monodisperse liposomes of maximal encapsulation efficiency for applications in drug delivery and biosensors, using careful tuning of centrifugal sizing and purification conditions for the production of a range of tailored monodisperse liposome populations, and is demonstrated using fluorescein isothiocyanate (FITC) as a “model” encapsulant.
Materials and methods 1,2-dipalmitoyl-sn-glycero-phosphocholine (DPPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (MPPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotynil) (B-DPPE) were obtained from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). Fluorescein-5-isothiocyanate (FITC), Sephadex G-100, and phosphorus standard solution were obtained from Sigma (St. Louis, Missouri, USA). Polycarbonate membranes and Microcon molecular-weight cut-off filters (centrifugal filter devices) were obtained from Millipore Milipore (Billerica, Massachusetts, USA). Ammonium molybdate (VI) tetrahydrate, sodium chloride, L-ascorbic acid, hydrogen peroxide, sulphuric acid, and solvents were obtained from Scharlau (Barcelona, Spain). HEPES was obtained from Acros Organics (Geel, Belgium). Thin lipid film formation and lipid film hydration was conducted in a Buchi Rotavapor R-200 (Büchi Labortechnik AG, Flawil, Switzerland). Extrusion was performed by using Avanti Lipid 1-mL syringes, an Avanti Lipid extrusion apparatus, and a Bibby heating block (Staffordshine, United kingdom). Centrifugation was performed by using an Eppendorf (Hamburg, Germany) Centrifuge 5417R. Hydrodynamic diameter of liposomes were determined by using dynamic light scattering (DLS) on a Malvern Instruments Zetasizer 3000-HS (Worcestershire, United Kingdom). Fluorescence measurements were performed with a Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, California, USA). Liposome images were taken
150 Arcan Güven et al. by environmental scanning microscopy (ESEM) (Philips XL30; FEI Company, Hillsbore, Oregon, USA).
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
Liposome preparation and purification Before use, all glassware was washed with 2:1 chloroform:methanol and dried well. Lipid mixtures used in this study were composed of DPPC, MPPC, and B-DPPE, and a lipid molar ratio of 88:10:2 DPPC:MPPC:B-DPPE was employed. A phospholipid solution dissolved in a chloroform-methanol mixture was dried under vacuum to obtain a lipid film; this lipid film was subsequently hydrated at 45oC in a buffer solution of 10 mM of HEPES (pH 7.4), with 15 mM of NaCl, containing the FITC to be encapsulated—allowing 1 mL of hydration buffer per 12 mg (17 mol) of phospholipid mixture. The FITC concentrations used in this study were in the range from 50 M to 1 mM. The solvent was removed by evaporation under reduced pressure at room temperature until an even, thin, and transparent lipid film remained on the sides of the flask. The flasks were round-bottomed and of a volume greater than double the volume of solvent to be evaporated. The liposome suspension was divided in two batches: one of them was subjected to classical extrusion and purification by the Sephadex G-100 column (Bath A) and the second batch was centrifuged and purified by using Microcon centrifugal filters (Batch B). The details of both approaches are commented on as follows. Batch A Preparation The suspension obtained directly from the hydration procedure was subjected to classical extrusion with different polycarbonate membranes of 800, 600, 400, and 200 nm, sequentially at 45°C. It is important to remark that is only possible to extrude 1 mL each time and in order to obtain the best results; therefore, it is necessary to repeat this procedure 11 times with each membrane. This approach implicates a great effort and a large time (around 8 hours) of manipulation of the operator in order to extrude around 5 mL of liposome suspension. Purification The unencapsulated FITC was separated from liposomes by classical size-exclusion chromatography, using a 15 3 1 cm Sephadex G-100 gel permeation column equilibrated with the hydration buffer (HEPES). The purification process was monitored visually (due to the yellow color of the liposomes) and verified by fluorescence at 515 nm (exc = 495 nm). Clearly, the presence of the operator is advisable during the purification process and requires around 6 hours of manipulation.
Table 1. Estimation of FITC encapsulation in one 250-nm-sized liposome for different initial concentrations. Initial FITC Number of concentration encapsulated Encapsulation Encapsulation (mM) FITC efficiency (%) 0.05 20,000 0.01 10 0.1 40,000 0.02 40 0.2 60,000 0.04 60 0.4 80,000 0.05 40 0.6 100,000 0.06 40 0.8 120,000 0.07 40 1 150,000 0.10 30
Batch B Preparation The suspension obtained directly from the hydration procedure was centrifuged at different times and speeds at 4°C, depending on the required liposome diameter (see main article, Table 1). The principle of centrifugation for sizing purposes is “separation.” The larger sized liposomes precipitate. As the centrifugal force (speed) and duration are increased, smaller sized liposomes remain in the supernatant with low yields (Figure 4). Standard deviation for liposome size measurements was as follows: Area = Sum (Sum (I (i)) ; Mean = Sum (S (i)*I (i))/Area; StDev = sqrt (Sum (S (i)*S (i)*I (i))/Area 2 Mean*Mean, where “I” is the % intensity in size class I and S is the size class. Standard deviation depends on the quality of the sample (i.e., the number of different particle sizes present in the sample). Purification The free FITC was separated from liposomes by using Microcon centrifugal filters (YM-3, nominal weight cut-off: 3,000) at 10,500 rpm centrifugation speed for 12 minutes at 25°C. It is important to remark that not only the time of procedure is very short (around 2 hours), but also the manipulation time is only a few minutes. Size measurements. Size measurements were carried out by using dynamic light scattering, with a Zetasizer. The software was used to calculate the average size of the liposomes, percentage distributions of the liposome size, and standard error of this distribution. Total phosphorus content. Total phosphorus content was assayed by using the standard protocol. This assay is derived from the work of Chen et al., in which the phosphate is first digested by concentrated acid and is then complexed by ascorbic acid and ammonium molybdate to produce a colored product that can be detected spectrophotometrically (Chen et al., 1956).
Fluorescein encapsulating liposomes 151
Transmission electron microscopy (TEM) imaging was used to observe the size and lamellarity of liposomes after size reduction by homogenization. TEM images were recorded by using a Jeol (Tokio, Japan) Model 1011 transmission electron microscope operating at 100 kV. To prepare the TEM samples, 5 L of an aqueous solution of prepared and sized liposomes was dropped onto a carbon-coated copper grid directly from solution and dried prior to observation.
Results A lipid mixture of DPPC, MPPC, and B-DPPE in an 88:10:2 molar ratio was employed to form a thin lipid film, which was rehydrated in the presence of a range of concentrations of FITC in HEPES buffer at 45°C (Chen et al., 1956) for up to 20 hours. The liposomes were characterized by dynamic light scattering, fluorescence measurements, scanning electron microscopy, and total phosphorus content assay. As the driving force behind the formation of liposomes is hydration (Martorell et al., 1999; Sabin et al., 2006), in a first step to achieve maximum encapsulation while minimizing preparation time, a study of the required hydration time, using different FITC concentrations (from 50 M to 1 mM), was performed. As would be expected with increasing initial FITC concentrations, an increasing number of FITC molecules are encapsulated, with a maximum reached at 1 mM, the maximum solubility of FITC in HEPES (Figure 1). The number of molecules encapsulated in one liposome was estimated from three experimental quantities: the mean vesicle radius, the total phosphorus content, and the amount of encapsulated horseradish peroxidase Number of encapsulated FITC (×1012)
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
Transmission electron microscopy
500
1000 µM 800 µM
400 600 µM 300 200 100 0
400 µM 200 µM 100 µM 50 µM 0
5
10 15 Hydration time (h)
20
Figure 1. Dependence of encapsulation on rehydration time using 250-nm liposomes sized and purified by using the new procedure for liposome preparation reported here.
(HRP). First, the total number of lipid molecules per vesicle, Ntot, was estimated by using the data obtained from the literature as lipid bilayer thickness, polar head group area, and the vesicle-size data obtained from Zetasizer measurements (Hutchinson et al., 1989; Singh et al., 1995; Jones et al., 1993), as shown in Equation 1: N tot =
4τ Rv2 4 τ( Rv − t )2 + A A
(1)
where Rv is the unilamellar vesicle radius, t is the bilayer thickness [44.2 Å2 for DPPC (Lis et al., 1982), 64 Å2 for MPPC (Ramsammy Brockerhoff, 1982), and 41.9 Å2 for B-DPPE (Vaknin et al., 1993], and A is the average area per lipid molecule [52.3 Å2 for DPPC (Lis et al., 1982), 48 Å2 for MPPC (Heerklotz and Epand, 2001), and 120 Å2 for B-DPPE (Vaknin et al., 1993]. The next step was the estimation of the concentration of the liposome solution for a unimodal vesicle distribution, as shown in Equation 2: Cliposome =
C phosphate
(2)
N tot
To extend the calculation to arrive at encapsulated content per liposome, first, the total amount of encapsulated molecule is calculated based on a lysis assay, where the liposomes are lysed using surfactant, as shown in Equation 3:
( Fluorophore )inside =
(∆FI )inside (∆FI )due to 1 molecule
(3)
where FI is the change in fluorescein intensity. The number of particles encapsulated per liposome is then yielded by Equation 4: # of Fluorophores ( Fluorophore )inside = Liposome Cliposome ×Vassay
(4)
Independent of concentration, the time required to reach total saturation of encapsulation is 20 hours, although the rate of encapsulation is more rapid at higher FITC concentrations. This can be explained by the osmotic balance across the lipid membrane, which equilibrates the internal and external solute concentrations; the vesicle volume remains constant because of the osmotic balance, and the internal solute concentration increases in the same ratio that the external solute concentration does. It could be expected that the hydration time could be reduced with higher molecular weight encapsulants, such as enzymes. Although the time for rehydration could thus not be reduced without having an impact on the reproducibility of encapsulation, a maximum encapsulation efficiency for FITC was achieved and further experiments were carried
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
152 Arcan Güven et al. out by using an initial concentration of 1 mM. From these results, the experimental maximum number of encapsulated FITC was 150,000 molecules for a 250-nm liposome. Following rehydration, sizing of liposomes was achieved by centrifugation at defined speed and duration at 4°C followed by purification, using commercial Microcon centrifugal molecular-weight cut-off filters, with a nominal cut-off of 3 kDa. The homogenity of the liposome population obtained was compared with those obtained by using a conventional subsequential extrusion of the same original rehydrated suspension through a series of polycarbonate membranes of defined size (800, 600, 400, and 200 nm) and purified by using gel permeation chromatography (Sephadex G-100). Using the developed method, homogenous populations of liposomes from 200 to 850 nm were produced by tailoring the centrifugation speed and duration at 4°C, demonstrating the flexibility of the approach (Table 2). For the comparison of the duration of the sizing step using the conventional method of extrusion and the use of the centrifuge, an extruder set-up of 1-mL volume was used as a model system. The capacity of extruder might be increased to have shorter processing times, Table 2. Comparison of length of sizing step and homogeneity of resulting liposome populations using the conventional extrusion technique and the new reported procedure. Extrusion Liposome Membrane pore size diameter Homogeneity (nm) Time (min) (nm) (%) 800 90 850/1,000 50 800 120 750 60 800 + 600 200 650 75 800 + 600 + 400 330 400 88 800 +600 +400 +200 480 250 88 Centrifugation Liposome diameter Homogeneity Speed (RPM) Time (min) (nm) (%) 7,000 15 850 85 7,000 30 870 90 10,500 15 700 90 7,000 + 10,500 30 (each) 550 90 7,000 + 10,500 60 (each) 450 95 7,000 +10,500 +14,000 30 (each) 400 99 7,000 +10,500 +14,000 60 (each) 120 90 14,000 15 450 95 14,000 30 300 90 14,000 45 290 95 14,000 60 250 99 Standard deviation for diameter measurements was calculated as ± 5%. For homogeneity calculations, different sizes in the range of standard deviation were accepted as the same size and the average of the sizes is presented here.
but then, one should consider the increase in the cost of the apparatus. Table 3 shows the encapsulated data obtained for different liposome sizes. In addition to drastically reducing the time required for the postrehydration sizing step from 8 to just 1 hour (with “hands-on” time being reduced from 8 hours to a few minutes), a much improved homogenity of the liposome population was achieved by using the new approach, as can be seen in Figure 2. TEM was used to characterize the prepared lipid vesicles and to observe the size and lamellarity of liposomes after sizing via centrifugation, demonstrating the prepared liposome to have a defined unilamellar morphology (Figure 3). By increasing centrifugal force and duration, smaller liposomes of more homogenous populations could be obtained; however, the yield after centrifugation A
B
C
Figure 2. (A) FITC-encapsulating liposomes after centrifugational separation (99% homogeneity). (B) FITC-encapsulating liposomes after extrusion (88% homogeneity). (C) Heterogeneous FITCencapsulating liposomes after spontaneous phospholipid swelling (rehydration) prior to sizing.
100 nm
Figure 3. TEM image of liposome prepared from DPPC-MPPCB-cap-DPPE, encapsulating FITC by using the thin-film hydration method and sized by using the centrifugation approach reported here. The size of the vesicle is 300 nm.
Fluorescein encapsulating liposomes 153
900
C
B
A
10 um
10 um
10 um
Figure 5. Confocal images of FITC-encapsulating liposomes prior and following detergent addition: (A) prior to detergent addition (B); 15 minutes after detergent addition; and (C) 60 minutes after detergent addition.
800 700
dilution(lip) = 1:10
600
Diameter (nm)
Diameter (nm)
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
Table 3. Estimation of the number of encapsulated FITC obtained by using an initial concentration of 100 M. Theoretical Number of Size encapsulated maximum Encapsulation Encapsulation (× 109) efficiency (%) (nm) FITC (× 105) 750 3 2 0.02 37 650 2.5 1 0.02 57 400 1 0.3 0.02 47 250 0.4 0.1 0.02 40
500 400 300
40 20 0
200
dilution(lip) = 1:4
60
4
100 0
0
1
2
3 4 Time (hours)
5 Time (hours)
5
6
6
Figure 4. Hydrodynamic diameter of liposomes as a function of centrifugation time at 14,000 rpm and 4°C.
decreases as the centrifugal speed and duration increase. No experiment was performed to determine the yield after different centrifugational conditions, but the amount of liposomes obtained after any centrifugational condition was certainly quantitative and the yield is estimated to be no more than 10–15%, considering the size-distribution results. For example, at 14,000 rpm a range of liposome sizes was obtained at different times [Figure 4; with the most homogenous population being obtained after 60 minutes (with no further improvement at longer centrifugation times)]. After 6 hours of centrifugation, the yield of the liposomes remaining in the supernatant was not quantitative. It can be anticipated that rather than using a combination of centrifugation speeds that one speed for longer times will also result in a range of liposome sizes. Although the problem of irreproducibility associated with the film rehydration method is well established, when this method is coupled with centrifugation, the procedure is highly reproducible. The study was repeated three times, by two different operators, and in all cases, the same size and homogeneity of liposomes was produced under the same conditions. The liposomes prepared were stable for at least 10 weeks at 4°C in the dark, and no leakage of fluorophore molecules from lipid vesicles was observed during this time (data
not shown). Figure 5 shows the stored liposomes after 10 weeks of storage, and as seen in Figure 5A, the FITC molecules were well encapsulated within the liposome interior and were efficiently released upon lysis of the liposomes, making the reported method a promising approach for the preparation of homogenous liposomes encapsulating drugs, genetic material, cosmetic formulations, reporter molecules, etc.
Discussion and conclusion In this study, a new procedure has been demonstrated for the preparation of tailored, highly uniformly sized, and stable populations of liposomes. Maximal encapsulation was achieved by using the highest possible concentration of encapsulant in the rehydration step. The time required for postrehydration liposome sizing was reduced from 2 “hands-on” days to less than 2 hours, of which less than 10 minutes require operator input, thus making the procedure highly applicable to industrial scale-up and application. For scale-up purposes, centrifuge approach can be processed in parallel for the preparation of a much higher number of liposomes, whereas extrusion is limited to volume of syringes used and each set of extrusions has to be done sequentially, thus further demonstrating the flexibility of centrifuge approach and applicability to industrial scale-up. Extensions of this method to the encapsulation of other fluorescent and electrochemically active molecules as well as drugs are currently being studied.
Acknowledgments This work was carried out with financial support from the Commission of the European Communities specific RTD program, Smart Integrated Biodiagnostic Systems for Healthcare, SmartHEALTH, FP6-2004-IST-NMP-2016817, and as part of the INTERFIBIO: Grup de Recerca de la Interfície físico/biològica (project no.: 2005SGR00851). AG thanks the Generalitat de Catalunya for a FI
154 Arcan Güven et al. predoctoral scholarship. Financial support by the SAFE Network of Excellence (LSHB-CT-2004-503243) is also acknowledged.
Journal of Liposome Research Downloaded from informahealthcare.com by Memorial University of Newfoundland on 11/08/13 For personal use only.
References Ahn-Yoon S, Decory TR, Baeumner AJ, Durst RA. Gangliosideliposome immunoassay for the ultrasensitive detection of cholera toxin. Anal Chem. 2003;75:2256–2261. Barenholz Y. Relevancy of drug loading to liposomal formulation therapeutic efficacy. J Lipos Res. 2003;13:1–8. Brandl M, Tardi C, Drechsler M, Bachmann D, Rezska R, Bauer KH, Schubert R. Three-dimensional liposome networks: freeze fracture electron microscopical evaluation of their structure and in vitro analysis of release of hydrophilic markers. Adv Drug Deliv Rev. 1997;24:161–164. Chen K, Toribara TY, Warner H. Microdetermination of phosphorus. Anal Chem. 1956;28:1756–1758. Deamer D, Bangham,AD. Large-volume liposomes by an ether vaporization method. Biochim Biophys Acta. 1976;443:629–634. Edwards KA, Baeumner AJ. Liposomes in analysis. Talanta. 2006; 68:1421–1431. Gregoriadis G. (Ed.). Liposome Technology. Vol. 1. Boca Raton, Florida, USA: CRC Press 1984. Heerklotz H, Epand RM. The enthalpy of acyl chain packing and the apparent water-accessible apolar surface area of phospholipids. Biophys J. 2001;80:271–279. Huang L, Kennel SJ. Binding of immunoglobulin G to phospholipid vesicles by sonication. Biochemistry. 1979;18:1702–1707. Hutchinson FJ, Francis SE, Lyle IG, Jones MN. The integrity of proteoliposomes adsorbed on a biosurface. Biochim Biophys Acta. 1989;987:212–216. Jones MA, Kilpatrick PK, Carbonell RG. Preparation and characterization of bifunctional unilamellar vesicles for enhanced immunosorbent assays. Biotechnol Prog. 1993;9:242–258.
Lasic DD. The mechanism of vesicle formation. Biochem J. 1988;256:1–11 Lis LJ, McAlister M, Fuller N, Rand RP. Interactions between neutral phospholipid bilayer membranes. Biophys J. 1982;37:657–666. Martorell D, Siebert STA, Durst RA. Liposome dehydration on nitrocellulose and its application in a biotin immunoassay. Anal Biochem. 1999;271:177–185. Mozafari MR. Liposomes: an overview of manufacturing techniques. Cell Mol Biol Lett. 2005;10:711–719. Nasseau M, Boublik Y, Meier W, Winterhalter M, Fournier D. Substrate-permeable encapsulation of enzymes maintains effective activity, stabilizes against denaturation, and protects against proteolytic degradation. Biotechnol Bioeng. 2001;75:615–618. Olson F, Hunt CA, Szoka FC, Vail WJ, Papahadjopoulos D. Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes. Biochim Biophys Acta. 1979;557: 9–23. Ramsammy LS, Brockerhoff H. Lysophosphatidylcholine-cholesterol complex. J Biol Chem. 1982;257:3570–3574. Rongen HAH, Bult A, van Bennekom WP. Liposomes and immunoassays. J Immunol Meth. 1997;204:105–133. Sabin J, Prieto G, Ruso JM, Hidalgo-Alvarez R, Sarmiento F. Size and stability of liposomes: a possible role of hydration and osmotic forces. Eur Phys J E. 2006;20:401–408. Singh KA, Kilpatrick PK, Carbonell RG. Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes. Biotechnol Prog. 1995;11:333–341. Szoka F, Papahadjopoulos D. Comparative properties and methods of preparation of lipid vesicles (liposomes). Ann Rev Biophys Bioeng. 1980;9:467–508. Vaknin D, Kjaer K, Ringsdorf H, Blankenburg R, Piepenstock M, Diederich A, Losche M. X-ray and neutron reflectivity studies of a protein monolayer adsorbed to a functionalized aqueous surface. Langmuir. 1993;9:1171–1174. Woodle MC. Sterically stabilized liposome therapeutics. Adv Drug Deliv Rev. 1995;16:249–265. Zawada ZH. A single-step method of liposome preparation. Cell Mol Biol Lett. 2004;9:603–615.
