4 DRUG DELIVERY THROUGH DISPERSED COLLOIDAL SYSTEMS
4.2 Liposomes
4.2.1 Parenteral administration
For several decades, liposomes have been considered promising for drug delivery. There are many reasons for this, including the possibility to encapsulate both water-soluble, oil-soluble, and at least some surface-active substances, thereby, e.g. controlling the drug release rate, the drug degradation, and the drug bioavail-ability (44-50). Liposomes, similarly to other col-loidal drug carriers, may also have advantageous effects, e.g. for directed administration to tissues related to
the reticuloendothelial system (RES), e.g. liver, spleen and marrow, as adjuvants in vaccines formulations, etc. (see below). However, liposome-based formula-tions have also been found to have numerous weak-nesses and difficulties, e.g. related to complicated or at least expensive preparations, difficulties with steril-ization, poor storage stabilities, limitations concerning poor solubilization capacities for more hydrophobic drugs, difficulties in controlling the drug release rate, and limitations in how much the drug release can be sustained, etc. In parenteral administration, another problem with this type of formulation has been the rapid clearance from the bloodstream, thus resulting in poor drug bioavailability and local toxicity in RES-related tissues. However, during the last decade or so, the development of so-called Stealth® liposomes, i.e. liposomes which have been surface-modified by PEO derivatives, as well as other developments, have resolved at least some of these issues, and there has been an increased activity in this area. In fact, several liposome-based products have recently been commercialized (e.g. AmBisome™ (amphotericin B), DaunoXome™ (daunorubicine citrate) and Doxil™ (dox-orubicin)), while many more are currently being tested and documented.
On intravenous administration of liposomes and other colloidal drug carriers, these are accumulated in the RES, which leads to a short bloodstream circulation time and an uneven tissue distribution, with a pref-erential accumulation in RES-related tissues, such as the liver, spleen, and marrow (51-56). This, in turn, may cause poor drug bioavailability and accumulation-related toxicity effects. The RES uptake, as well as the drug circulation time and tissue distribution, depends on, e.g. the surface properties of the drug carrier.
This is related to the adsorption of serum proteins at the drug carrier surface, which induces biological responses related to complement activation, immune response, coagulation, etc. In fact, an inverse corre-lation has been found between the total amount of serum proteins adsorbed, on the one hand, and the bloodstream circulation time, on the other (Figure 1.5) (54, 55). In particular, through the use of PEO deriva-tives and surface modifications to induce steric stabi-lization, the adsorption of serum proteins at the drug carrier surface can be largely eliminated, which has been found to lead to an increased bloodstream circu-lation time and a more even tissue distribution (8, 44, 49-73).
An area where sterically stabilized liposomes are of particular interest is cancer therapy. Thus, by the
Circulation half-life (min)
Figure 1.5. Correlation between the total amount of pro-tein adsorbed and circulation time before plasma clear-ance of large unilamellar vesicles (LUVs) containing trace amounts of [3H]cholesteryl-hexadecyl ether administered intra-venously in CDl mice at a dose of about 20 umol of total lipid per 100 g of mouse weight. Results are shown for liposomes containing SM:PC:ganglioside GMl (72:18:10) (open square), PC:CH (55:45) (filled circle), PC:CH:plant PI (35:45:20) (filled square), SM:PC (4:1) (open triangle), PC:CH:dioleoylphosphatidic acid (DOPA) (35:45:20) (open diamond), and PC:CH:DPG (35:45:20) (open circle) (SM, sflngomyelin; PC, phosphatidyl choline; CH, cholesterol; PI, phosphatdylinositol; DPG, diphosphatidyl glycerol) (data from ref. (55))
use of such liposomes, enhanced antitumour capac-ity and reduced toxiccapac-ity of the encapsulated drug can be achieved for a variety of tumours, even those that do not respond to the free drug or the same drug encapsulated in conventional liposomes. Just to mention one example, Papahadjopoulos et al. investigated the use of PEO-modified liposomes consisting of distearoyl phosphatidylethanolamine-PEO1900, hydrogenated soy phosphatidylcholine and cholesterol, for the adminis-tration of doxorubicin to tumour-bearing mice (57). It was found that the liposomes have a longer bloodstream circulation time than liposomes composed of, e.g. egg phosphatidylcholine. Furthermore, the prolongation of the circulation time in blood was correlated to a decrease of accumulation in RES-related tissues such as liver and spleen, and a correspondingly increased accumulation in implanted tumours (Figure 1.6). These and other aspects of parenteral administration, e.g. in cancer therapy, have been extensively reviewed previously (8, 44, 47,49, 50).
4.2.2 Targeting of liposomes
An interesting use of liposomes related to their par-enteral administration concerns targeting of the drug
Time following injection (h)
Figure 1.6. Doxorubicin in tumour-bearing mice, either as the free drug (open symbols/dashed lines) or in liposomes consist-ing of distearoylphosphatidylethanolamine-PEO/hydrogenated soy phosphatidylcholine/cholesterol (0.2:2:1 mol/mol) (filled symbols/continuous lines) (data from ref. (57))
to a desired tissue or cell type. In particular, sterically stabilized liposomes and other types of PEO-modified colloidal drug carriers are of potential interest in this context, due to the long circulation times and rela-tively even tissue distributions of such systems after intravenous administration. If a biospecific molecule, e.g. a suitable antibody (fragment), a peptide sequence, oligosaccharide, etc., is covalently attached to such a carrier, the long circulation time reached ideally would improve the possibilities for targeting to a localized antigen. As an example of this, Khaw et al. investi-gated cytosceleton-specific immunoliposomes with the goal of either "sealing" hypotic cells or using them in the intracellular delivery of DNA (74, 75). Thus, by the use of antimyosin-immunoliposomes, a highly improved survival rate could be demonstrated for hypotic cells compared to those of the controls. Furthermore, by elec-tron microscopy, these investigators could infer that the liposomes act by "plugging" the microscopic cell lesions present in hypoxic cells. Furthermore, Holmberg et al.
investigated the binding of liposomes to mouse pul-monary artery endothelial cells (76). As can be seen in Figure 1.7, the amount of lipid bound to these cells was significant with two different relevant antibodies, and also displayed a strongly increasing binding with the liposome concentration, whereas the binding of both the bare liposomes and liposomes modified with an irrel-evant antibody was negligible. Positive results from the use of conjugated liposomes were also found, e.g. by Muruyama et al. (77), Ahmad et al. (78), Gregoriadis and Neerunjun (79), Torchilin and co-workers (80-82).
Bound protein (g protein/mol lipid) |ig Doxorubicin-equivalents per ml or g
Added liposome (|xg)
Figure 1.7. Binding to mouse pulmonary artery endothelial cells of two liposome preparations functionalized with rel-evant antibodies (34A and 201B) (filled and open circles, respectively), functionalized with an irrelevant antibody (open squares) or uncoated liposomes (filled squares) (data from ref. (76))
Naturally, liposomes as such are not unique in this context. Instead, the same approach can be used for other PEO-modified colloidal drug carriers, e.g.
copolymer micelles. For example, Kabanov et al. have demonstrated specific targeting of fluorescein isothio-cyanate solubilized in PEO-PPO-PEO block copoly-mer micelles conjugated with antibodies to the antigen of brain glial cells (o^-glycoprotein) (83, 84). Further-more, incorporation of haloperidol into such micelles was found to result in a drastically improved therapeu-tic effect in mice, as inferred from horizontal mobility and grooming frequency studies.
One should also note that although beneficial thera-peutic effects have been observed for both liposomes and micelles, the presence of the recognition moiety in the conjugated carrier may also have detrimental effects, e.g. causing the long circulation time in the absence of such entities to decrease drastically. As an example of this, Savva et al. conjugated a genetically modified recombinant tumour necrosis factor (TNF)-a to the termin(TNF)-al c(TNF)-arboxyl groups of liposome-gr(TNF)-afted PEO chains (85). However, although the liposomes in the absence of such conjugation displayed a long cir-culation in the bloodstream, incorporation of as lit-tle as 0.13% of the PEO chains resulted in a rapid elimination from the bloodstream. Clearly, the use of immunoliposomes for targeting may indeed be rather complex. The use of liposomes in parenteral drug deliv-ery has been extensively reviewed previously (44, 47, 49, 50).
4.2.3 Topical administration
Another area where liposomes have been found use-ful is in topical and dermal drug delivery. Thus, the major problem concerning topical drug delivery is that the drug may not reach the site of action at a suf-ficient concentration to be efsuf-ficient, e.g. due to the barrier properties of the stratum corneum. To over-come this problem, topical formulations may contain so-called penetration enhancers, such as dimethyl sulfoxide, propylene glycol and Azone®. However, although these yield an improved transport of the drug, they typically also result in an increased systemic drug level, which is not always desired, and may cause irritative or even toxic effects (86-89). As discussed below, one way to achieve an increased drug penetration without the use of penetration enhancers is to use microemulsions.
Another approach for this, however, is to use liposomes or other types of lipid suspensions, e.g. so-called trans-fersomes (86). Although there are a large number of drugs which could be of interest in relation to liposomal transdermal drug delivery, perhaps of particular interest are local anaesthetics, retinoids and corticosteroids. For example, Gesztes and Mezei compared a formulation prepared by encapsulating tetracaine into a multilamel-lar liposome dispersion to a control cream formulation (Pontocaine™) and found the liposome formulation to be significantly more efficient (90). Positive results were found by Schafer-Korting et al. for tretinoin formula-tions for the treatment of acne vulgaris (91).
However, although liposomes have indeed been suc-cessfully used commercially (e.g. Pevaryl™ Lipogel, Ifenec™ Lipogel, Micotef™ Lipogel, Heparin Pur™ and Hepaplus Eugel™), other types of lipid dispersions are also interesting in this context. In particular, Cevc has convincingly argued for the advantages of so-called transfersomes, i.e. self-assembled lipid stuctures, which due to their highly deformable lipid bilayers have shown superior membrane penetration when compared to tra-ditional liposomes for a number of systems (86).
4.2.4 Liposomes in gene therapy
Yet another area where liposomes are of interest is gene therapy (48, 92-97). Thus, on mixing lipids with DNA, compact complexes may be obtained, particularly for cationic lipids. More specifically, most DNA conden-sation methods yield similar particles, i.e. torus-shaped with a 40-60 nm outer and 15-25 nm inner diameter, or rods of about 30 nm in diameter and a length of 200-300 nm, although this naturally depends on a num-ber of parameters, such as the lipid/DNA ratio (48). By
Bound lipid (ng)
the use of liposomes, an increased efficiency of DNA delivery has been observed. It has been found that the transfection efficiency depends on the net charge of the complex. However, this dependence is not straightfor-ward, and different cell lines require different complex charges for optimal expression (93). Although consid-erable work has been performed with both positively and negatively charged liposome complexes, as well as with titrating ones, cationic liposome complexes have received particular attention in gene theapy. However, there are several potential problems with the in vivo gene delivery through cationic charge-mediated uptake. For example, on intravenous administration the complex car-rier encounters net negatively charged serum proteins, lipoproteins and blood cells, with the risk of flocculation and emboli formation. Carriers administered through the airway, on the other hand, face problems related to the lung surfactants, etc. In both cases, there is a risk of the carriers not being able to maintain their positive charges until they reach their target, which will dete-riorate their performance. Furthermore, intravenously administered carriers are cleared from circulation rapidly by the RES. Despite these obstacles, however, DNA administered through cationic liposome complexes has been found to be more efficient than naked DNA deliv-ery (48).
Cationic lipid-based systems have also been found to be comparatively efficient for gene delivery to a range of tissues in vivo. These include, e.g. pulmonary epithe-lial cells, endotheepithe-lial cells after direct application to the endothelial surfaces or after intravenous administration, solid tumours after interstitial administration, metastases after intravenous delivery, etc. (93, and refs therein).
Furthermore, therapeutic cDNAs have been delivered by cationic liposomes in human gene therapy trials and no toxicity has been observed at the low doses adminis-tered. Naturally, the positive findings when using com-plexes between DNA and cationic liposomes and lipids are analogous to those of the enhanced gene deliv-ery efficiencies obtained for cationic (co)polymers or polymer complexes, as has been extensively reviewed recently (98). Comprehensive, recent reviews of the use of liposomes in gene therapy are also available (48, 92-94).