Determinación de factores que influyen en el bajo nivel de

equilibration were analytically filtered. The solid content present in the liposome dispersions was separated by analytical filtration. The results o f these experiments are shown in Table 5.13.

Table 5.13 Effect of lowering the egg PL: cyA ratio in pro-liposomes converted in two stages on cyA association Eppendorf mixing of 0.640 g of cyA egg PL pro-liposome and 0.173 g o f deionised water Addition of hydrated pro-liposome to bulk deionised water after Eppendorf mixing Ease of filtration Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope

Immediate Immediate I-II 0.2 ± 0.0 Few spherical precipitates

Immediate Delayed for 24 hours II 0.2 ±0.1 Few spherical precipitates

Delayed for 24 hours Immediate I-II 1.3 ± 0.3 Considerable number of

spherical precipitates Delayed for 24 hours Delayed for 24 hours II 0.3 ±0.1 Few spherical precipitates

Immediate mixing of the egg PL pro-liposome with a small amount of deionised water for hydration generated a clear hydrated cyA pro-liposome. Moreover, if this hydrated cyA pro-liposome was left for 24 hours, the gel still remained optically clear. This clarity indicated that major precipitation had not developed during 24 hours of storage, which was confirmed by only slight increases in filter weight after analytical filtration of these cyA liposome dispersions. However, if the cyA pro-liposome and the deionised water for initial hydration were combined, but left for 24 hours before Eppendorf mixing, a hazy gel was generated. This haziness seemed to correlate with the formation of a considerable amount o f precipitation in the final cyA liposome dispersions. If this hazy gel was immediately dispersed in the bulk deionised water, over 1.0 mg of solid precipitate was detected after analytical filtration of this cyA liposome dispersion. This indicated that delaying the mixing by 24 hours followed by immediate dispersion in bulk deionised water reduced the loading o f cyA into the liposomes.

Chapter five-Solubilisation o f q/closporin A hy liposomes

Equilibrating the hydrated hazy gel described above by delaying the addition of the hydrated pro-liposome gel for a further 24 hours at room temperature, resulted in the disappearance of the haziness. Additionally, conversion of this clarified gel to a liposome dispersion, resulted in an average filter weight increase of about 0.3 mg. This equilibration period demonstrated that during 24 hours of storage a large proportion of the deposits re-dissolved in the hydrated cyA pro-liposome. Thus, if only a small amount of deionised water was added to this cyA pro-liposome, the surrounding media was sufficiently lipophilic to redissolve most of the cyA precipitate.

5.5.3.S Role of ethanol level for cyA association 5.5.3.5.1 High ethanol formulation

A high ethanol pro-liposome formulation, with an egg PL: ethanol: glycerol: cyA weight ratio of 60:48:26:2, was hydrated with three different levels o f deionised water. The resultant dispersions were analytically filtered to assess the amount of solid precipitate present (Table 5.14).

Table 5.14 Effect of mixing varying levels of deionised water for hydration with a pro-liposome with raised ethanol level

Amount o f high ethanol cyA egg PL

pro-liposome (g) Amount o f deionised water for hydration (g) Ease of filtration Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope

0.782 0.161 11 0.2 ±0.1 Darkened grey circles of fine

spherical precipitate

0.782 0.316 11 0.0 ± 0 .0 Clear

0.782 0.633 Il-III 1.3 ± 0 .2 Vast number o f spherical

precipitates

The addition of insufficient deionised water to the pro-liposome reduced the amount of cyA associated with the liposomes. If only 0.161 g of deionised water was added to 0.782 g of cyA pro-liposome in the first stage of hydration, the association of cyA with liposomes was incomplete. Under the light microscope, dark circles o f fine precipitate outlining the unsupported circular areas of the underlying filter support could be observed. The formation of precipitate in this case may have been attributed to the insufficient level of bilayer organisation prior to the dilution in bulk water. Therefore, the addition of insufficient amounts of water resembled a one stage dilution. However, if 0.316 g of deionised water for hydration was added and thoroughly mixed into the same amount of cyA pro-liposome, no precipitate was recovered from the final dispersions and the filter weight did not increase after filtering the dispersions. Hence, raising the ethanol level of the cyA pro-liposome enabled more deionised water to be

added without the formation of insoluble precipitate in the final cyA dispersions. The reason for this was probably due to the higher ethanol level increasing the lipophilic balance of the milieu surrounding the cyA pro-liposomes. However, even with this high ethanol formulation, over 1.0 mg of precipitate was formed if the amount of deionised water for hydration was raised to 0.633 g. These results again clearly illustrated the importance of adding the appropriate level of deionised water to the cyA pro-liposome in the first stage o f hydration to maximise cyA association.

5.5.3.S.2 Medium ethanol formulation

The medium ethanol formulation, which had an egg PL: ethanol: glycerol: cyA weight ratio of 60:36:26:2, was converted into a liposome dispersion in two stages and analytically filtered (Table 5.15).

Table 5.15 Effect of adding deionised water for hydration to an egg PL pro-liposome converted in two

Amount o f medium ethanol cyA egg PL pro-liposome

(g) Amount of deionised water for hydration (g) Ease of filtration Average increase in filter weight ± s.d. (mg)

Appearance o f filter under the light microscope

0.713 0.316 I-Il 0.2 ±0.1 Darkened grey circles of

fine spherical precipitate

After filtration o f the egg PL cyA liposome dispersion, traces o f a fine white powder could be seen by the naked eye on the upper surface of the filters. In addition, the filter weight increased by 0.2 mg, which confirmed that association of cyA was compromised if there was either insufficient ethanol in the cyA pro-liposome and/or excess deionised water for hydration was added to the cyA pro-liposome. This demonstrated that the amount of deionised water added to the first stage of hydration should not be considered in isolation, but should be viewed in conjunction with the ethanol level of the cyA pro­ liposome.

5.5.3 6 Medium ethanol formulation with soya PL blend

The same lipid: ethanol: glycerol: cyA weight ratio for the “medium” ethanol cyA formulation as used in section 5.5.3.5 was employed, but the egg PL was replaced with a soya PC: soya PL blend. The soya PC: soya PL: ethanol: glycerol: cyA weight ratio for the pro-liposome was 54:6:36:24:2. The analytical filtration results for this cyA dispersion are shown in Table 5.16.

Chapter five-Solubilisation of cyclosporin A by liposomes

Table 5.16 Effect o f adding deionised water for hydration to a soya PL blend pro-liposom e converted in ________ two stages on cyA association______________

Amount of cyA soya PL blend pro-liposome (g)

Amount of deionised water for hydration (g)

Ease of filtration Average increase in filter weight ± s.d. (mg) Appearance of filter under LM and SEM 0 . 7 1 3 0 . 3 1 6 0.0 ± 0.0 Clear

Replacing the egg PL with a soya PC/soya PL blend in a weight ratio of 90:10 (referred to in text as soya PL blend) improved the association of the cyA with the liposomes. This was shown by the fact that there were no increases in filter weight after analytical filtration of the soya PL blend dispersions containing cyA. Moreover, both light microscopy and SEM indicated that the polycarbonate filters were free from any form of precipitate after analytical filtration of the cyA dispersion (Plate 5.9). There are various possible explanations for the improved association of cyA with the soya PL blend liposomes. Firstly, the more unsaturated fatty acid chains of the soya PC may have been able to incorporate a higher cyA capacity than the more saturated fatty acids of egg PC. Secondly, the incorporation of cyA into the soya PC/soya PL blend bilayer may have been easier than the egg PL bilayer, as a result of the higher ethanol solubility of the soya lipids.

Due to the superior association of cyA with soya PL blend liposomes, this particular lipid blend was adopted for most of the subsequent association studies involving two stage conversion, unless otherwise stated.

Plate 5.9 Typical SEM of surface of polycarbonate filter used to filter a soya PL blend liposome dispersion containing cyA (Bar = 5 pm), described in section 5.5.3.6