achieving high encapsulation efficiency without subjecting the materials to be entrapped to
potentially damaging conditions such as sonication or the use o f organic solvents or
detergents. It is the method o f choice for sensitive biological m olecules such as proteins
and nucleic acids.
prepared as described above and converted to SU V by sonication or other techniques,
yielding a hom ogenous suspension, which, due to the high curvature o f SU V , w ill result in
up to 70% o f the total phospholipid present in the outer leaflet o f the lamella. On m ixing
the SU V with an aqueous solution o f the material to be entrapped, m ost o f the lipid is
directly exposed to the solute. Freeze-drying at this stage rem oves water, leaving an
intimate mixture o f the dry components (i.e. the drug and flattened lipid bilayers).
Rehydrating correctly is crucial: small, defined volum es o f water are slow ly added,
initiating fusion o f the vesicles, which form around adjacent solute m olecules, and
encapsulating a high proportion. Unentrapped material can be removed by
ultracentrifugation or dialysis. Dilution with isotonic buffer causes a large osm otic gradient
between the internal and external phases and this results in a redistribution o f solute within
the forming liposom es and with the external aqueous phase.
The procedure, described schematically in figure 1.8, leads to M LV containing up to 100%
o f the original amounts o f solute used. Entrapment values are usually high (60-100% ) for
macromolecules and lower (20-60% ) for smaller m olecules such as anticancer agents for
instance (Kirby and Gregoriadis, 1984; Kirby and Gregoriadis, 1999).
m
SUVi
MLVt
DRVs
Figure 1.7 A suspension o f MLV is sonicated to produce SUV. Materials to be entrapped
are are added and the SU V/solutes mixture is freeze-dried. Inset diagram shows intimate
contact o f flattened liposomes and drug molecules in a water-free environment. DRV are
generated as vesicles reform upon controlled rehydration, entrapping high amounts o f
solutes (Kirby and Gregoriadis, 1984).
1.3.4 Fate o f liposom es in vivo
Central to any further development o f liposomes as a delivery system is an understanding
o f their behaviour in vivo follow ing their administration. The body’s response to liposome
administration, particularly parenterally, is to mount a concerted defence leading to a
cascade o f events that effect gross and subtle changes to the liposom es them selves and their
Gregoriadis, 1974; Gregoriadis, 1976) with drug-containing M LV revealed a number o f
basic aspects. For instance, the rate o f clearance o f vesicles from the blood o f
intravenously injected rats was rapid, dose-dependent and biphasic. It became urgent to
elucidate pharmacokinetic parameters such as the rate o f clearance from the circulation, the
amount and location o f distribution in various tissues, the release profile o f encapsulated
contents and all the factors governing these events. It has been w ell established that
clearance o f liposom es o f large size and negative surface charge from the circulation is
rapid (Gregoriadis and Neerunjun, 1974; Juliano and Stamp, 1975). Within minutes o f
intravenous administration, such liposom es are found mainly in the fixed macrophages o f
the liver and spleen and, made appropriately, constitute the vehicle o f choice for the rapid
delivery o f drugs to these cells. It was also observed that neutral M LV and SU V exhibit a
longer residence time than negatively-charged vesicles and, more recently, positively
charged M LV and that SU V persisted for longest o f all. SU V also can cross from the
vascular compartment to reach tissues contiguous with extravascular space. The passage o f
small liposom es with diameters <0 . 1 pm, through the discontinuous capillaries o f the liver
has been w ell documented by Roerdink et al (1981) who found that, after penetrating the
discontinuous capillaries, SU V were taken up effectively by parenchymal cells. Although
the intercellular junctions o f discontinuous capillaries have a width ranging from 0 .1-
0.6pm , the mucopolysaccharide-rich interstitium in the spaces o f D isse, w hich surrounds
and lines the endothelium, restricts the passage o f macrom olecules or particles larger than
0.2pm (Roerdink et al, 1981; Poste et al, 1982; Scherphof et al, 1983). Similarly, the
intercellular junctions (2-6nm in width) o f the continuous capillaries, prevalent in skeletal,
cardiac and smooth m uscles, lung, skin, subcutaneous tissue and serous- and mucous-
membranes, preclude the transcapillary passage o f even the smallest liposom es (Poste et al.
1982). Liposom es, which were engulfed by the fixed macrophages o f the RES ended up in
intracellular lysosom al compartments and were degraded, presumably by the action o f
phospholipases present in the lysosom al milieu. Depending upon their molecular size and
ability to withstand the hostile environment o f the organelles, released drugs could then act
either locally e.g. hydrolysis o f stored sucrose by liposom al fructofuranosidase
(Gregoriadis and Buckland, 1973) or diffuse across the lysosom al membrane and operate in
other subcellular compartments, e.g. inhibition o f DNA-directed R N A synthesis by
liposom al actinom ycin D in partially hepatectomised rats (Black and Gregoriadis, 1974).
These early amassed data and understanding o f liposomal fate and behaviour in vivo
pointed the way to a number o f proposed applications, including the treatment o f certain
inherited metabolic disorders (Gregoriadis and Ryman, 1972a; Gregoriadis and Buckland,
1973; Belchetz et al, 1977), metal storage diseases (Rahman et al, 1973), intracellular
infections (Gregoriadis, 1973; N e w et al, 1978; A lving et al, 1978), cancer (Gregoriadis,
1973; Gregoriadis and Neerunjun, 1975), gene therapy (Gregoriadis and Ryman, 1972) and,
because o f antigen-presenting cell involvem ent in vesicle uptake, immunopotentiation and
vaccine delivery (A llison and Gregoriadis, 1974). Initial optim ism from findings that
liposom es coated with cell-specific ligands (e.g. antibodies and asialoglycoproteins) could
interact with cells (other than those o f the RES) expressing appropriate receptors, both in
vitro and in vivo (Gregoriadis and Neerunjun, 1975; Neerunjun et al, 1977), prompted the
concept o f v esicle targeting. At the same time, it was noted that the higher endocytic
activity o f som e tumour cells, combined with their augmented capillary permeation,
favoured preferential liposom al drug entry into the tumour mass, uptake o f locally released
drugs by vesicles, but also sufficiently extended residence time in order for circulating
loaded liposom es to encounter target tissues.
1.3.5 Retention o f drugs by liposom es
It had been observed that small water-soluble drugs such as 5-fluorouracil and penicillin G
leaked considerably from intravenously injected M LV (Gregoriadis, 1973), w hile large
entrapped solutes such as albumin and am yloglycosidase did not, at least to the same extent
(Gregoriadis and Ryman, 1972a; Gregoriadis and Neerunjun, 1974). It later became
apparent that such leakage was promoted by high-density lipoproteins (H D L), which
remove phospholipid m olecules from the vesicle bilayer (Scherphof et al, 1978) allowing
the formation o f pores on its surface and eventually progressing to disintegration. A s a
result, encapsulated solutes are released in the blood circulation at rates dependent on their
size (Kirby and Gregoriadis, 1980) and act as free drugs. This role o f HDL in the
destabilisation process was further established (Senior et al, 1983) when cholesterol-free