Spontaneous nanosized liposome formation from crude dried lecithin upon addition of glycerol

Abstract

Nanosized liposomal vesicles (NLV) were successfully prepared using natural sunflower lecithin without the use of high-pressure homogenization or filtration. Upon glycerol addition to dispersions of lecithin multilamellar vesicles (MLVs), these broke down spontaneously to liposomes with diameters in the range of 100-200 nm. Static light scattering demonstrated that glycerol addition above 30% (w/w) induced the complete transformation of MLVs into NLVs. Langmuir trough compression experiments showed a two-region compressional behavior. Upon 62% (w/w) glycerol addition, the compressional modulus of the liposomes decreased from 18.5 to 8.13 mN/m. Water activity and pulse NMR measurements also showed a divergence in behavior above 30% (w/w) glycerol. Liposomes were not birefringent in water but became strongly birefringent at and above 30% (w/w) glycerol, as determined by polarized light microscopy, and lost all birefringence above 80% (w/w). This was interpreted as the induction of stress-birefringence in the phospholipid bilayers above 30% (w/w) glycerol, and a relaxation of such stress above 80% (w/w) glycerol. We hypothesize that the mixture of phospholipids in the lecithin results in an effective non-zero intrinsic curvature for the molecular mixture, which lowers the bending energy of the bilayer, allowing for an easier break-up upon mixing. Secondly, glycerol addition decreases attractive van der Waals' interaction between lamellae in an MLV, thus weakening the multilamellar liposome walls. Glycerol also affects bilayer stability by strengthening the hydrogen bond network of water, which will affect phospholipid headgroup hydration. All these factors result in the spontaneous breakdown of MLVs into NLVs.

Keywords: Critical packing parameter; Glycerol; Lecithin; Liposomes; Nanosized; Spontaneous; Stress birefringence.

Fig. 1

Particle size distributions for 5% (w/w) Sunlec 25 lecithin liposomes prepared in glycerol-water mixtures, from 0 to 100% (w/w) glycerol, in 10% (w/w) increments. The different profiles correspond to experimental replicates.

Fig. 2

Particle size distributions for 5% (w/w) Sunlec 25 lecithin liposomes prepared in glycerol, propylene glycol and ethylene glycol and diluted 50% with water at room temperature without stirring. The different curves shown represent replicate experiments.

Fig. 3

Brightfield light micrographs of 5% (w/w) liposomal preparations of Sunlec25 lecithin in different water-glycerol mixtures, 0% (w/w) glycerol (a), 10% (w/w) glycerol (b), 20% (w/w) glycerol (c), 30% (w/w) glycerol (d), 40% (w/w) glycerol (e), 50% (w/w) glycerol (f), 60% (w/w) glycerol (g), 70% (w/w) glycerol (h), 80% (w/w) glycerol (i), and 100% (w/w) glycerol (j).

Fig. 4

Cryogenic transmission electron micrographs of 10% liposomal suspensions of Sunlec25 lecithin in 50% (w/w) glycerol in deionized water.

Fig. 5

Compression isotherms of 0.01% liposomal dispersions of Sunlec25 lecithin liposomal preparations. (a) Liposomes prepared and compressed in 62% (w/w) glycerol, (b) Liposomes prepared and compressed in deionized water.

Fig. 6

Polarized light micrographs of 5% (w/w) liposomal preparations of Sunlec25 lecithin in different water-glycerol mixtures, 0% (w/w) glycerol (a), 10% (w/w) glycerol (b), 20% (w/w) glycerol (c), 30% (w/w) glycerol (d), 40% (w/w) glycerol (e), 50% (w/w) glycerol (f), 60% (w/w) glycerol (g), 70% (w/w) glycerol (h), 80% (w/w) glycerol (i), and 100% (w/w) glycerol (j).

Fig. 7

Brightfield and polarized light micrographs of different 5% (w/w) Sunlec25 lecithin liposome preparations. (a) 10% (w/w) glycerol, (b) 10% (w/w) glycerol, (c) and (d) corresponding brightfield and polarized light micrographs of 40% (w/w) glycerol liposomes, (e) and (f) 50% (w/w) glycerol, (g) and (h) 62% (w/w) glycerol.

Fig. 8

Water activity as a function of glycerol content (w/w%) in for 5% Sunlec25 lecithin liposomes (red symbols). For comparison, data from Nakagama and Oyama for glycerol-water mixtures was adapted and plotted here (green symbols).

Fig. 9

(a) Changes in the proton spin-spin relaxation time (T₂) as a fuction of the glycerol-water mixtures containing 5% (w/W Sunlec lecithin 25 liposomes. (b) Experimentally-determined correlation between the water activity of water-glycerol solutions containing 5% (w/w) Sunlec25 lecithin liposomes, and the corresponding T₂.

Fig. 10

(a) Dynamic viscosity changes as a function of glycerol content (w/w %) for water-glycerol solutions with and without 5% Sunlec25 lecithin liposomes. (b) Small-angle oscillatory shear moduli for the water-glycerol solutions containing 5% (w/w) Sunlec25 lecithin liposomes. G’ = storage modulus, G” = loss modulus, tanδ = G”/G’.