Scanning Electron Microscopy (SEM) was performed on PC-based liposomes suspended in PBS buffer at pH 7.4, prepared according to the Morrisey protocol (see experimental section). Distribution of liposome diameters, after extrusion through a Ø450 nm porous filter, were found to be 102 ± 17 nm, excluding the presence of some aggregates with diameter > 400 nm, as shown in Figure 4.2.
Figure 4.2. SEM images of PC liposomes deposited on a Si substrate; white bars correspond to (a) 500 nm and (b) 50 nm.
88 Dynamic Light Scattering (DLS) experiments were performed to compare liposomes produced through two similar protocols: the Morrisey and Freeze-thaw protocols, both described in the experimental section.33-35 The hydrodynamic size distribution results of the obtained PC liposome suspensions measured via DLS can be observed in Figure 4.3. Figure 4.3a shows an example of a DLS measurement, at neutral pH, after extrusion through a Ø100 nm porous filter on a suspension of PC liposomes using the Freeze-thaw (blue) and Morrisey (red) protocols. For both preparation protocols, the presence of a singular peak at ~ 100 nm reveals that the liposome size is monodisperse, and no agglomeration/sedimentation of liposomes in solution is observed. Literature values on hydrodynamic size of liposomes show that liposomes with hydrodynamic diameter in the range of 50-200 nm are associated with Large Unilamellar Vesicles (LUV), i.e. one single bilayer of liposomes.6
Figure 4.3. (a) Example of a measurement on the PC liposome hydrodynamic size (d.nm) using the Freeze-thaw (blue) and Morrisey (red) protocols after extrusion through a Ø100 nm filter. (b) Hydrodynamic size of PC liposomes for Freeze-thaw protocol (red) and for Morrisey protocol (blue). Liposomes were extruded through a nylon membrane (450 nm pore diameter) followed by a second extrusion through another nylon membrane (100 nm pore diameter). The hydrodynamic size was measured immediately after extrusion, for t=30 min and for t=60 min.
The homogeneity of liposomes in suspension represents a key factor on liposomes efficiency as drug carriers, and as mimic lipid aggregates.36-37 Poly-Dispersive Index (PDI)
89 nanoparticles, and it is use to describe the variation on particles size: values close to 1 are correlated to high dispersity of particles size, while values close to 0 represent a population of particles that is monodisperse.36 The PDI for the PC liposomes hydrodynamic size was determined to be < 0.4 for both the Freeze-thaw and Morrisey protocols. PDI range obtained for PC liposomes prepared using both preparation protocols, is consistent with literature values for a suspension of monodisperse liposomes using natural phospholipids16-17, 36-38 For example, Jacquot et al.38studied the
morphology of natural-phospholipids based biomembranes through the analysis of their electrophoretic mobility. They reported PDI values of ~0.1 for synthetic-phospholipids based liposomes, which was further increased to PDI ~0.3 when a natural phospholipid was added to the liposome. Additionally, Tefas et al.36reported PDI values from 0.083 to 0.3 for increased % of natural PC phospholipids included on the liposome. Finally, Silva Malheiros et al.37 characterised the electrostatic stability of PC liposomes and reported
hydrodynamic sizes in the range of 150-200 nm with a correspondent PDI of 0.3-04. Figure 4.3b shows DLS measurements of liposomes suspended in a buffer at pH 7.4, prepared through two different methods. For both methods, liposome suspensions were extruded through Ø450 nm and Ø100 nm pore size nylon membranes (reported in the graph as 450 nm and 100 nm, respectively) after preparation of the liposome suspension in PBS. After the liposome suspension is extruded through the 450 nm pore size nylon membrane, the measured hydrodynamic mean diameter is d(450nm) = 235 ± 23 nm for the
Morrisey protocol and d(450nm) = 207 ± 9 nm for the Freeze-thaw protocol. A second
extrusion through 100 nm pore size nylon membranes, reduces the liposome mean diameter to d(100nm) = 124 ± 2 nm and d(100nm) = 124 ± 1 nm for the Morrisey and Freeze-
thaw methods, respectively. Therefore, considering the DLS diameter values obtained after the second extrusion, there were no significant differences in hydrodynamic size for liposomes obtained using both methods. These results are consistent with literature studies on liposomes size made of PC, using similar preparation protocols.37 For instance, Silva Malheiros et al.37 reported liposomes hydrodynamic diameter of ~ 150 nm for PC liposomes prepared through a film hydration protocol. Additionally, Jacquot
et al.38 used the vesicle fusion method for the preparation of PC-based liposomes and
90 The stability of liposomes in suspension is a key factor in the determination of the performance of liposomes in vivo.37 The liposome hydrodynamic size was measured as a function of time after filtered through the Ø100 nm nylon membrane; liposomes hydrodynamic size was measured at 3 different intervals: time = 0 min, when the liposome preparation is initiated, for time = 30 min, half-time after preparation and for time = 60 min, as shown in Figure 4.2b. With regards to the Morrisey protocol, the liposome hydrodynamic diameter was found to be d(t=30 min) = 126 ± 1 nm and d(t=60 min) =
126 ± 3 nm, whereas for the Freeze-thaw protocol, d(t=30 min) = 128 ± 1 nm and d(t=60 min) =
129 ± 1 nm. These results indicate that PC liposomes, produced either using the Freeze- thaw or the Morrisey protocols, show a great homogeneity and stability of their hydrodynamic sizes over 1 h in suspension. Silva Malheiros et al.37 studied the stability of PC liposomes in solution over a course of 24 days and reported a similar homogeneity on liposomes size and stable PDI for that time range. Given that no significant differences were observed on the liposomes size prepared using the two methods, the Morrissey protocol was chosen to be used from now on due to its simplicity when compared with the Freeze-thaw protocol.