A. Phase Transition Temperature
The phase transition temperature is defined as the temperature required to induce a change in the lipid physical state from the ordered gel phase, where the hydrocarbon chains are fully extended and closely packed, to the disordered liquid crystalline phase, where the hydrocarbon chains are randomly oriented and fluid.1 There are several factors which directly affect the phase transition temperature including hydrocarbon length, unsaturation, charge, and headgroup species. As the hydrocarbon length is increased, van der Waals interactions become stronger requiring more energy to disrupt the ordered packing, thus the phase transition temperature increases. Likewise, introducing a double bond into the acyl group puts a kink in the chain which requires much lower temperatures to induce an ordered packing arrangement.
When developing a new product, procedure, or method, controlling the transition temperature of the lipid could be useful. Choosing a high transition lipid where the lipid vesicle would always be in the gel phase would provide a non-leaky packaging system. Alternatively, a lipid with a transition temperature between the starting temperature and the ending temperature of the system would provide a means of releasing packaged material as the lipid passes through its phase transition temperature and the vesicle becomes leaky. Also, one should consider how the transition temperature of the lipid could impact the processing steps. Using a high transition lipid when filtration is necessary could present some technical problems.
The long term stability or shelf-life of a drug product containing lipids can be dramatically affected by the lipid species used in the formulation. Generally, the more unsaturated a compound, the easier the product is oxidized, and thus the shorter the shelf life of the product. Lipids from biological sources (e.g., egg, bovine, or soybean) typically contain significant levels of polyunsaturated fatty acids and therefore are inherently less stable than their synthetic counterparts. While saturated lipids offer the greatest stability in terms of oxidation, they also have much higher transition temperatures and thus present other difficulties in formulation. If unsaturation is a requirement, keep the degree of unsaturation as low as possible. In most cases, compounds containing oleic acid (18:1, cisD9) are sufficient to satisfy the need for unsaturation, and since they are monounsaturated, oleoyl-containing products are much more stable than polyunsaturated compounds.
Stability issues due to hydrolytic degradation is a general problem with lipid products. Aqueous formulations of drug products tend to be less stable since the presence of excess or bulk water leads to rapid hydrolytic degradation in lipid preparations.3,4,5 This hydrolysis is dependant on several factors including pH,3 temperature,3,5 buffer species,5 ionic strength, acyl chain length and headgroup,4 and the state of aggregation.4 A summary of the discussion on these factors can be found elsewhere.6 Others have shown that the cause of this hydrolysis is possibly due to the penetration of water into the membrane. Simon and McIntosh7 report water penetration depths in PE and PE:cholesterol membranes determined by X-ray diffraction and specific capacitance measurements. In PE membranes, water penetrates to near the deeper carbonyl group, while in PE membranes containing cholesterol, water only penetrates to the glycerol backbone. This indicates that cholesterol could play a role in stabilizing lipid membranes to hydrolysis.
Stabilizing membranes has been the subject of research for many years. The bulk of this research has been aimed at stabilizing intact liposomes in the dry powder form such that they retain their trapped internal contents upon reconstitution. Recently, lipid preparations have been stabilized using carbohydrates.8,9 The possible reason for the stabilizing effect carbohydrates have on lipid membranes is that the carbobohydrate could intercalate into the headgroup region near the membrane/water interface and displace water from that region. In dry lipid formulations, this would serve to maintain a “hydrated” lipid membrane and keep the liposome structure intact. If this is true, it stands to reason that in an aqueous environment, the carbohydrate could still enter this region and displace water. This would tend to stabilize the membrane to hydrolysis from the bulk water phase.
Many biological membranes carry a net negative charge on their surface. The charge is generally imparted by the presence of anionic phospholipid species in the membrane. The major naturally occuring anionic phospholipids are phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin. Some bacterial systems also contain phosphatidylglycerol. The charge may provide a special function for the membrane. Several steps of the blood coagulation cascade require a lipid membrane. The assembling of protein aggregates on the surface of platelets requires a negatively charged surface. For the conversion of prothrombin to thrombin, not only does it require a negative surface, the requirement is somewhat specific, limited to phosphatidylserine (PS) and phosphatidic acid (PA).10 Coagulation proteins bind as tightly to negatively charged surfaces containing phosphatidylglycerol and phosphatidylinositol as they do to PS or PA membranes, however, the activity is only a fraction of that obtained with PS or PA membrane. Therefore, in some systems, not only must the charge requirement be satisfied, the system specificity for a particular species must be satisfied.
D. Lipid Mixtures
In many cases, a single lipid species does not yield the exact physical properties needed for a particular system, or does not adequately mimic the natural system for which it is intended to replace or reproduce. For these issues, consider a complex lipid mixture composed of two or more individual lipid species, the composition designed to create or reproduce a particular charge ratio, unsaturation ratio, phase transition temperature, or biological function. To reproduce the function of native brain tissue extracts, a blend of synthetic lipids (dioleoyl acyl composition) in the ratio 5:3:2 (wt%), PE:PS:PC, has been found to be satisfactory.11 This represents the general phospholipid composition of most brain tissues. Also, many commercially available coagulation reagents which contained crude brain extracts in the past are being replaced by synthetic lipid blends. The advantages to this replacement system are the increased stability due to the lack of polyunsaturated fatty acids found in biological extracts, and the reproducibility of synthetic blends. Blending of multiple lipid species does not require much additional effort in sample preparation. If the quantity of lipid blend is sufficient, many times the lipid supplier will pre-blend to the users specifications and provide a ready-to-use product.
Cholesterol is a membrane constituent widely found in biological systems which serves a unique purpose of modulating membrane fluidity, elasticity, and permeability. It literally fills in the gaps created by imperfect packing of other lipid species when proteins are embedded in the membrane. Cholesterol serves much the same purpose in model membranes. Unfortunately, cholesterol presents certain problems when used in human pharmaceuticals. High purity sources suitable for clinical applications are not widely available. Most cholesterol commercially available is derived from egg or wool grease (sheep derived). These animal sources are potentially not suitable for human pharmaceuticals due to the potential viral contamination. Also, cholesterol is readily oxidized creating a stability problem for lipid based drug products.12 Some of these oxidation by-products tend to be rather toxic in biological systems. The oxidation products 25-hydroxy cholesterol, 7-keto-cholesterol, 7a- and 7b-hydroxycholesterol, cholestane-3b,5a,6b-triol and the 5- and 7-hydroperoxides, were found in a concentrate which had activity causing aortic smooth muscle cells to die.13 This suggests that results from studies on atherosclerosis involving feeding experimental animals a diet containing cholesterol stored under adverse conditions (room temperature, open to air) could be ambiguous due to the potential presence of significant quantities of oxidized sterols.
There are two basic sources of phospholipids: synthetic and tissue-derived. Tissue-derived lipids are generally either egg-derived or bovine-derived. For clinical applications, either of these sources is not suitable due to stability problems and the possibility of viral or protein contamination. The U.S. Food and Drug Administration issued a letter restricting the source of bovine tissue used to isolate pharmaceutical products to countries and animals certified to be free of bovine spongiform encephalopathy (BSE). Cattle in the U.S. are not certified BSE-free and cannot be used to isolate pharmaceutical products. Egg sources are not currently restricted, however, additional testing for viral contamination may be required for pharmaceutical products. Regardless of the regulatory issues, animal-derived products do not offer any advantage to synthetic lipids. They are inherently less stable due to the polyunsaturated fatty acids, and in most cases the synthetic counterpart cost the same or less than the tissue-derived product.
Synthetic lipids from different sources are not necessarily equal either. Synthetic lipids can be prepared from glycerol or glycero-3-phosphocholine (GPC) derive from a plant or animal source. The latter is sometimes referred to as semi-synthetic lipids because a portion of the molecule is derived from a natural source. Lipids derived from glycerol require the chiral center be synthetically prepared which may lead to stereochemical impurities present in the final product. Lipids prepared using GPC obtained from an animal source may suffer from the same viral and protein contamination issues outlined above. The typical plant source for GPC is soybean lecithin.
- Small, D.M., Handbook of Lipid Research: The Physical Chemistry of Lipids, From Alkanes to Phospholipids, Vol. 4, Plenum Press, New York, 1986.
- Ellens, H., Bentz, J., and Szoka, F.C., Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature, Biochemistry, 25, 285, 1986.
- Frrkjaer, S., Hjorth, E.L., and Wrrts, O., Stability and storage of liposomes, in Optimization of Drug Delivery, Bundgaard, H., Bagger Hansen, A., and Kofod, H., Eds., Munksgaard, Copenhagen, 1982, 384.
- Kensil, C.R. and Dennis, E.A., Alkaline hydrolysis of phospholipids in model membranes and the dependence on their state of aggregation, Biochemistry, 20, 6079, 1981.
- Grit, M., de Smidt, J.H., Struijke, A., and Crommelin, D.J.A., Hydrolysis of phosphatidylcholine in aqueous liposome dispersions, Int. J. Pharm., 50, 1, 1989.
- Grit, M., Zuidam, N.J., and Crommelin, D.J.A, Analysis and hydrolysis kinetics of phospholipids in aqueous liposome dispersions, in Liposome Technology: Liposome Preparation and Related Techniques, Vol. 1, 2nd edn, Gregoriadis, G., Ed., CRC Press, Ann Arbor, 1993, 527.
- Simon, S.A. and McIntosh, T.J., Depth of water penetration into lipid bilayers, Meth. Enzymol., 127, 511, 1986.
- Crowe, J.H. and Crowe, L.M., Factors affecting the stability of dry liposomes, Biochim. Biophys. Acta, 939, 327, 1988.
- Crowe, J.H., Crowe, L.M., Carpenter, J.F., and Aurell Winstrom, C., Stabilization of dry phospholipid bilayers and proteins by sugars, Biochem. J., 242, 1 1987.
- Jones, M.E., Lentz, B.R., Dombrose, F.A., and Sandberg, H., Comparison of the abilities of synthetic and platelet-derived membranes to enhance thrombin formation, Thromb. Res., 39, 711, 1985.
- van den Besselaar, A.M.H.P., Neuteboom, J., and Bertina, R.M., Effect of synthetic phospholipids on the response of the activated partial thromboplastin time to heparin, Blood Coag. Fibrinol., 4, 895, 1993.
- Smith, L.L., Cholesterol Autoxidation, Plenum Press, New York, 1981.
- Taylor, C.B., Peng, S.K., Werthesen, N.T., Than, P., and Lee, K.T., Spontaneously occurring angiotoxic derivatives of cholesterol, Am. J. Clin. Nutri., 32, 40, 1979.
From Burgess, SW, Moore, JD, and Shaw, WA, Handbook of Nonmedical Applications of Liposome: From Design to Microreactors, Vol. 3, Y. Barenholz & D. Lasic, Eds., CRC Press, Ann Arbor, 1996, 5.