Two stage conversion

Two stage conversion involved mixing a specified amount o f deionised water with the pro-liposome prior to the addition to the bulk deionised water at room temperature. This first stage involving the addition of deionised water to the pro-liposome will be referred to as the hydration stage. Mixing the deionised water for hydration with the pro­ liposome was achieved by repeatedly passing these two components between two Eppendorf syringes (5 ml) up to twenty times. This procedure is referred to in the text as Eppendorf mixing or mixing. This mixing usually resulted in the generation of a hydrated pro-liposome, which was transferred into a vial containing deionised water to give a final dispersion weight of 10 g. The hydrated pro-liposome was dispersed into the bulk aqueous phase by:

1) Syringing the pro-liposome in deionised water, or 2) Vigorously handshaking for 90 seconds, or

3) Leaving the pro-liposome to disperse in the deionised water without agitation.

This second stage involving the dispersion o f the hydrated pro-liposome in bulk deionised water will be referred to in the text as the dispersion stage.

3.4.4.1 Effect of Eppendorf mixing during hydration stage

Experiments were carried out at room temperature (20 °C-25 °C) to elucidate the optimal amount o f mixing required to form a homogeneous mix between the pro­ liposome and deionised water for hydration. In order to standardise the amount of mixing, 1.265 g (1.100 g +15% overage) of egg PL pro-liposome and 0.633 g (0.550 g +15% overage) o f deionised water for the first stage of hydration were passed between two Eppendorf syringes for a set number of times, ranging from 0 up to twenty times (section 3.4.4). Liposome dispersions were generated by adding and syringing 1.650 g o f hydrated pro-liposome ten times in 8.350 g of deionised water. The resultant dispersions were examined by light microscopy and sized by laser diffraction.

3.4.4.2 Effect of varying the amount of deionised water for hydration

Varying amounts of deionised water were added at room temperature to a fixed amount of the fluid egg PL pro-liposome. The amount of deionised water added to 1.265 g (1.100 g +15% overage) of pro-liposome ranged from 0.115 g (0.100 g +15% overage) up to 10.235 g (8.900 g +15% overage) of deionised water. These two components were intimately combined by Eppendorf mixing (twenty times) and the appearance of the

resultant mix was recorded. Eppendorf syringes with a volume of 5 ml were used if the total weight of pro-liposome and deionised water was below 4 g. However, for larger amounts of pro-liposome and deionised water 20 ml Eppendorf syringes were employed to mix the components. If required, the mixed pro-liposome and deionised water were added at room temperature to the remaining deionised water to give a final dispersion weight of 10 g. The liposome dispersion was converted by syringing the hydrated pro­ liposome in the bulk deionised water ten times (section 3.4.2). All samples were sized with light microscopy, laser diffraction and Coulter Counter (section 3.3.8). Freeze fracture replicas o f liposome dispersions generated from pro-liposomes (1.265 g) hydrated with half the weight of weight (0.633 g) and an equal weight of deionised water (1.265 g) were made and examined under EM (section 3.3.8.7).

3.4.4.3 Turbidity of diluted dispersions generated in two stages

Turbidity measurements investigating the effect of diluting egg PL dispersions, produced using the methodology described in section 3.4.4.1, were carried out. These studies involved diluting 100 pi of the dispersions in 10 ml o f 50 mg/g glucose and 10 ml o f deionised water. The turbidities of these diluted dispersions were monitored for 20 minutes at 550 nm as described in section 3.3.8.7.

3.4.4 4 Effect of agitation during dispersion stage

The two extremes of agitation during the dispersion stage of a hydrated pro-liposome on liposome size were investigated: vigorous handshaking for 90 seconds and leaving to hydrate without any agitation. The hydrated egg PL pro-liposome was formed by passing 0.633 g (0.550 g +15% overage) of deionised water and 1.265 g (1.100 g +15% overage) o f egg PL pro-liposome between two Eppendorf syringes twenty times, described in section 3.4.4. After Eppendorf mixing, 1.650 g of this gel was directly transferred into a glass vial containing 8.350 g of bulk deionised water. The deionised water and hydrated gel were immediately subjected to either:

Vigorous handshaking

The liposome dispersion was formed by vigorously handshaking the hydrated pro­ liposome with the bulk deionised water at room temperature. The intensity of handshaking was identical to the handshaking adopted for the single stage conversion.

Chapter three-P article size o f liposome dispersions

described in section 3.4.2.1. The duration of shaking required to disperse the hydrated pro-liposome was 90 seconds. Or,

No agitation

The hydrated pro-liposome was left to hydrate in the bulk deionised water without agitation at room temperature. The time taken for the hydrated pro-liposome to disperse without agitation was noted.

The two sets of dispersions were compared by sizing with light microscopy, laser diffraction and Coulter Counter (section 3.3.8).

3.4.4.5 Effect of lipid composition

3.4.4.5.1 Comparing the particle size of liposomes generated in two stages using pure soya PC and pure egg PC

The influence o f the difference in the degree of unsaturation between egg PC and soya PC on liposome particle size was investigated. This was achieved by comparing the size of liposomes generated from two pro-liposomes containing refined egg PC and refined soya PC. The pro-liposomes used to form these liposomes were produced at room temperature as described in section 3.3.2 by dissolving the requisite amount of either refined egg PC or refined soya PC in ethanol and subsequently adding glycerol. These two pro-liposomes were hydrated at room temperature by Eppendorf mixing 0.633 g (0.550 g +15% overage) of deionised water with 1.265 g (1.100 g +15% overage) of pro-liposome. After having Eppendorf mixed the two components twenty times, 1.650 g o f the resultant gels were dispersed at room temperature in 8.350 g o f bulk deionised water by vigorously handshaking the contents of the vial for 90 seconds. The resultant dispersions were sized by PCS, light microscopy and laser diffraction (section 3.3.8). To improve the reproducibility of the measurements, the isotonic media used for diluting these samples prior to sizing contained low amounts of Tween 80 (1 part by weight of Tween 80 to 100 parts by weight of lipid). The changes in turbidity were also qualitatively measured at 550 nm, described in section 3.3.8.7, after diluting 100 pi of the samples in 10 cm^ of 50 mg/g glucose, deionised water and 50 mg/g glucose with 0.006 mg/g Tween 80.

3.4.4.5 2 Effect of replacing soya PC with soya PL

The effect of blending soya PL with soya PC on the particle size of dispersions converted in two stages was examined. Six sets of liposome dispersions were generated

from pro-liposomes. The soya PC: soya PL weight ratios of the six pro-liposomes were 100:0, 93:7, 90:10, 75:25, 50:50 and 25:75. The pro-liposomes were made in the usual manner as described in section 3.3.2. The pro-liposomes were hydrated by Eppendorf mixing 1.265 g (1.100 g +15% overage) of each pro-liposome twenty times with 0.633 g (0.550 g +15% overage) of deionised water. The liposome dispersions were generated by vigorously handshaking 1.650 g of the hydrated pro-liposome in 8.350 g of deionised water for 90 seconds at room temperature. All dispersions were sized by light microscopy, PCS and laser diffraction, but only the number of particles in the dispersions with a soya PC: soya PL weight ratio of 90: 10 were counted using the Coulter Counter (section 3.3.8).