Therapeutic applications of colloidal drug carriers

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Therapeutic applications of colloidal drug carriers Gillian M. Barratt Colloidal drug carriers such as liposomes and nanoparticles can be used to improve the therapeutic index of both established and new drugs by modifying their distribution, and thus increasing their efficacy and/or reducing their toxicity. This is because the drug distribution then follows that of the carrier, rather than depending on the physicochemical properties of the drug itself. If these delivery systems are carefully designed with respect to the target and the route of administration, they may provide one solution to some of the delivery problems posed by new classes of active molecules, such as peptides and proteins, genes and oligonucleotides. They may also offer alternative modes for more conventional drugs, such as highly hydrophobic

pheres) or of a reservoir system in which an oily core is surrounded by a thin polymeric wall (nanocapsules). Polymers suitable for the preparation of nanoparticles include poly(alkylcyanoacrylates), poly(methylidene malonate 2.1.2), and polyesters such as poly(lactic acid), poly(glycolic acid), poly (e-caprolactone) and their copolymers. Lipophilic drugs, which have some solubility in the polymer matrix or in the oily core of nanocapsules, are more readily incorporated than hydrophilic compounds, although the latter may be absorbed onto the particle surface. Methods for the preparation of nanoparticles can start from either a monomer or from a preformed polymer3,4.

small molecules. This review discusses the use of colloidal, particulate carrier systems (25 nm to 1 mm in diameter) in such applications.

▼ Liposomes consist of one or more phosphoGillian M. Barratt UMR CNRS 8612 Laboratoire de Pharmacie Galénique Faculté de Pharmacie Université Paris-Sud 5 rue J.B. Clément F-92296 Chatenay-Malabry France tel: 133 1 4683 5627 fax: 133 1 4661 9334 e-mail: Gillian.Barratt@ cep.u-psud.fr

lipid bilayers enclosing an aqueous phase. They were first proposed as carriers of biologically active substances in 1971 (Ref. 1), and have since been comprehensively studied. They can be classified as large multilamellar liposomes (MLV), small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV), depending on their size and the number of lipid bilayers. Water-soluble drugs can be included within the aqueous compartments, and lipophilic or amphiphilic compounds can be associated with the lipid bilayers. Methods for the preparation of liposomes are reviewed in Ref. 2. Several liposome-based pharmaceuticals are now on the market or in development (Table 1). Nanoparticles, based on biodegradable polymers and of a similar size to liposomes, show some advantages over the latter in terms of stability both during storage and in vivo3,4. They may consist of either a polymeric matrix (nanos-

Distribution and fate of colloidal drug carriers in vivo The possible therapeutic benefits of colloidal drug carriers depend on their distribution within the organism. After intravenous administration, these particles cannot extravasate except in tissues with a discontinuous capillary endothelium; that is, the liver, spleen and bone marrow. Even in these organs, the size of the gaps between endothelial cells (approximately 100 nm) means that only the smallest particles can penetrate into the tissue. Carriers may extravasate into solid tumours and into inflamed or infected sites, where the capillary endothelium is defective. However, for ‘conventional’ colloidal carriers, which have more or less hydrophobic surfaces, the usual fate is opsonization by plasma proteins, followed by uptake by phagocytic cells: either polymorphonuclear leukocytes in the blood or fixed macrophages, particularly the Küpffer cells in the liver5–7. Complement activation by the alternative pathway is an important component of carrier recognition and uptake8, but opsonization by other plasma proteins, for example nonspecific adsorption of IgG, also intervenes.

1461-5347/00/$ – see front matter ©2000 Elsevier Science Ltd. All rights reserved. PII: S1461-5347(00)00255-8

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Table 1. Liposome-based pharmaceuticals on the market or in clinical trials Use Cancer Daunorubicin Doxorubicin

Annamycin Cisplatin Tretinoin (retinoic acid) Edelfosine (ether lipid) Infections Amphotericin B

Nystatin Amikacin

ARDS* Prostaglandin E2

Status

Company

DaunoXome™ (available in 22 countries) Doxil™ (using Stealth® liposomes) Phase III (Evacet™), NDA filed Phase I/II Phase II (Platar) Phase I/II (Atragen®)

NeXstar (Gilead Sciences) Sequus (Alza) The Liposome Company Aronex Aronex Aronex

Phase I (TLC ELL-12)

The Liposome Company

AmBisome™ (available in 36 countries) Abelcet™ (approved in 22 countries) Amphocil™ (Amphotec™ in US) Phase III (Nyotran®) Phase II/III (MiKasome®)

NeXstar (Gilead Sciences) The Liposome Company Sequus (Alza) Aronex NeXstar (Gilead Sciences)

Available for partnership (Ventusª)

The Liposome Company

*Adult respiratory distress syndrome. Data obtained from the respective Web sites of the companies cited.

Liposomes are also destabilized by interactions with circulating lipoproteins, especially high-density lipoproteins8. Removal of phospholipids from the outer bilayer causes leakage of encapsulated drug and increased interaction with opsonins; this phenomenon depends on the phase transition temperature of the phospholipids and is reduced when the bilayer is stabilized by cholesterol5. When colloidal drug carriers are administered by other routes, such as by subcutaneous or intramuscular injection or topical application, they are generally retained at the site of administration for longer than free drug. When a liposomeassociated drug is applied to the skin, the amount penetrating into the superficial layers may be increased compared with free drug, while its passage to the systemic circulation may be reduced9. After subcutaneous or intraperitoneal administration, small liposomes and nanoparticles have been shown to be taken up by regional lymph nodes10,11. 164

The most convenient route of drug administration is the oral method. However, this route presents several barriers to the use of colloidal carriers, because the environment within the gastrointestinal (GI) tract can disrupt many of them. It has been shown that the concerted action of duodenal enzymes and bile salts destroys the lipid bilayers of most types of liposomes, thus releasing the drug12. Multilamellar liposomes prepared from phospholipids whose phase transition temperatures are above 378C and which contain cholesterol in their bilayers are the most resistant to degradation. Polymeric nanoparticles are more stable, although there is some evidence that polyesters can be degraded by pancreatic lipases13. Even if the carrier is stable, anatomical considerations mean that only a small proportion of the administered drug-carrier systems can be absorbed intact across the intestinal mucosa into the circulation or the lymphatics. Passage across enterocytes by diffusion is restricted to small, lipophilic molecules and transcytosis, which is rare, to particles of less than 200 nm in diameter. Passage by the paracellular pathway is impossible if the tight junctions are intact. Nevertheless, several studies have reported the appearance of particles in the circulation after oral dosing14.The current consensus is that uptake occurs via Peyer’s patches, which are specialized areas of the gut-associated immune system.Transcytosis of particles occurs across M cells and delivers them to the underlying lymphoid follicule. This antigen-sampling mechanism is important in developing a mucosal, and sometimes also a systemic, immune response15, but is probably not an important therapeutic route. Most particles are phagocytosed by antigen-presenting cells, although some nanoparticles may find their way into the portal circulation, and hence to the liver16. Possible therapeutic applications of colloidal drug carriers The therapeutic potential of drug carrier systems is influenced by their distribution. This section describes the possible spheres of application of ‘conventional’ liposomes and nanoparticles by different routes of administration. Structural modifications designed to modify their distribution will be discussed in the following section. Administration by the intravenous route The potential applications of colloidal drug carriers by the intravenous route can be summarized in terms of the concentration of drugs in accessible sites; the rerouting of drugs away from sites of toxicity; and increasing the circulation time of labile or rapidly eliminated drugs (such as peptides and proteins). Since colloidal drug carriers are naturally concentrated within macrophages, it is logical to use them to deliver drugs to these cells. A good example is the delivery of muramyldipeptide and chemically related compounds to stimulate

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100 Inhibition of metastases (%)

the antimicrobial and antitumoral activity of macrophages. Muramyldipeptide is a low molecular-weight, soluble synthetic compound derived from the peptidoglycan of mycobacteria, and, although it acts on intracellular receptors, it penetrates poorly into macrophages. Furthermore, muramyldipeptide is eliminated rapidly after intravenous administration.These problems can be overcome by encapsulation within liposomes or nanocapsules, especially when lipophilic derivatives such as muramyltripeptide-cholesterol17 or muramyltripeptide-phosphatidylethanolamine18 are used. Figure 1 shows the activity of muramyltripeptide-cholesterol within nanocapsules in a model of hepatic metastases in mice. Macrophages may also be sites for bacterial and parasitic infections. Liposomes or nanoparticles can be used to concentrate antibiotics at the site of infection, particularly when the microorganism is within the lysosomes19. For example, nanoparticles containing ampicillin were more effective than the free drug against both Salmonella typhimurium and Listeria monocytogenes. Colocalization of the particles and bacteria was seen in Salmonella-infected macrophages in vitro19.A liposomal formulation of amikacin (MiKasome®, Gilead Sciences, Foster City, CA, USA; Table 1) is currently in clinical trials against complicated bacterial infections, and is reputed to be better tolerated than the free antibiotic. The potential of liposomes as immunological adjuvants was recognized in 1974. In the case of protein antigens, encapsulation increases capture by antigen-presenting cells such as macrophages20. In an alternative strategy, immunogenic peptides have been coupled to the surface in order to directly activate B and T cell clones21. Liposomes have also been used as carriers in DNA vaccines22. Colloidal carriers can also be useful for diverting drugs from sites of toxicity after intravenous administration. For example, the anticancer drug doxorubicin (adriamycin) is active against a wide spectrum of tumours, but has dose-limiting cardiotoxicity. Encapsulation within liposomes or nanoparticles decreases this toxicity, by reducing the amount of drug that reaches the myocardium23,24. A corollary is that concentrations of doxorubicin in the liver increase considerably. In one study in mice, this was not associated with any gross toxicity24. However, another group reported a temporary depletion of Küpffer cells, and hence the ability to clear bacteria, in rats, which was less marked when long-circulating liposomes (see below) were used25. A systematic study using unloaded nanoparticles confirmed a reversible decline in the phagocytic capacity of the liver after prolonged dosing, as well as a slight inflammatory response 26. Thus, altered distribution may generate new types of toxicity and this must be considered when developing carrier systems. Accumulation of carrier-associated drug in the liver may also influence its elimination, because this organ is the site of

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50

25

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PIBCA

PVCA

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Figure 1. Antimetastatic activity of muramyltripeptide-cholesterol in nanocapsules. C57BL/6 mice were treated intravenously with 5 mg of drug in nanocapsules based on different polymers (black) or with an equivalent amount of unloaded nanocapsules (white) two days before and six days after receiving M5076 cells by the intravenous route. They were killed and liver metastases counted on day 14 (mean of seven mice per group). PLA, poly(D,L-lactic acid); PIBCA, poly(isobutylcyanoacrylate); PVCA, poly(vinyl chloride-co-vinyl acetate); MDP sol., solution of muramyldipeptide. Drawn from data in Ref. 17.

metabolism and of biliary excretion. It was shown that the biliary clearance of indomethacin was increased threefold by inclusion in nanocapsules27. Nanoparticle-associated doxorubicin also accumulates in bone marrow, leading to a myelosuppressive effect in one study28. However, this tropism of carriers can also be used to deliver myelostimulating compounds such as granulocyte colony stimulating factor29. The efficacy of doxorubicin in mice was considerably greater following treatment with doxorubicin-loaded poly(alkylcyanoacrylate) nanoparticles compared with free doxorubicin30. Tissue pharmacokinetic studies showed that the particles were initially concentrated within Küpffer cells, from which doxorubicin was progressively released, reaching the tumour cells in the hepatic parenchyme31.This can be considered as ‘indirect targeting’ to an accessible site which acts as a reservoir32. Interestingly, the association of doxorubicin with poly(alkylcyanoacrylate) nanoparticles also reversed the resistance to doxorubicin in a large number of multi-drug resistant cell lines32,33. The nanoparticle-associated drug was accumulated within the cells and appeared to avoid P-glycoprotein-dependent efflux. This reversal was only observed with poly(alkylcyanoacrylate) nanoparticles and was not the result of particle endocytosis. Rather, the formation of a complex between doxorubicin and polymer-degradation products seemed to favour 165

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diffusion across the plasma membrane. However, these results have not yet been confirmed in vivo, because of the lack of a suitable animal model. Liposome-based formulations of doxorubicin and daunorubicin are already on the market (Table 1). Amphotericin B (AmB), an antifungal drug used in deep-seated systemic disease, is another drug with specific dose-limiting side effects, in this case to the kidneys. As early as 1984 it was noted that association with colloidal lipid systems could increase the maximum tolerated dose of AmB, and thereby enhance activity34. Many different colloidal delivery systems for AmB have since been developed: emulsions, nanoparticles, liposomes and lipid complexes. The reduced toxicity of these systems is probably the result of both an altered distribution and the physicochemical state of the AmB in the carrier systems. It has been shown that lipid or polymer association maintains AmB in its monomeric form, which is less disruptive to mammalian cell membranes than the self-associated form, while maintaining its activity towards fungal cell walls34. Colloidal preparations of AmB are also more effective than free drug against Leishmania infections35; this is an intracellular infection and the use of carriers increases the concentration of drug within macrophages. In contrast, lipid association also reduces some immunostimulating effects of AmB (such as nitric oxide and tumour necrosis factor-a production) compared with free AmB at the same dose, which may contribute to the reduced toxicity (M. Larabi, et al., unpublished). The circulating half-lives of cytokines such as gamma interferon and interleukin-2 (IL-2) (Ref. 36) have been shown to be prolonged by encapsulation within liposomes. Oral administration Colloidal drug carriers can be used to protect a labile drug from degradation in the GI tract; protect the GI tract from drug toxicity; and deliver antigens to the Peyer’s patches for oral immunization. Colloidal systems have been shown to protect insulin from enzymatic degradation in the GI tract by administration within such systems. In the 1970s and 1980s many studies with insulin encapsulated in liposomes gave controversial results, which may have been because of differences in liposome composition, affecting their stability in digestive fluids14. Later studies involved insulin encapsulated in poly(alkylcyanoacrylate) nanocapsules. Although these formulations were ineffective in reducing glycaemia in normal rats, they were effective in diabetic rats and in normal rats loaded with glucose37. A two-day lag was observed between administration and the fall in glucose levels, while the duration of the hypoglycaemia (up to 20 days), but not its intensity, was dose-dependent. It was shown that these nanocapsules could protect insulin from degradation by digestive enzymes in vitro38. The sustained effect was possibly because of the 166

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bioadhesive properties of the particles, which could be absorbed onto the intestinal mucosa and then slowly release the encapsulated insulin close to its site of absorption. Encapsulation within nanocapsules also improved and prolonged the therapeutic effect of a somatostatin analogue given by the oral route39. The bioadhesive properties of nanoparticles could potentially be used to improve the absorption of poorly water-soluble drugs. Incorporation within submicronic carriers can increase the surface area and thereby facilitate dissolution in GI fluids, while bioadhesion can increase the residence time in the GI tract.Targeting of particles to specific regions of the mucosa has also been envisaged40. Nanocapsules have been shown to be effective in protecting the GI mucosa of rats from the ulcerating effects of non-steroidal antiinflammatory drugs after oral administration41,42 (Fig. 2). The formulation did not affect drug absorption, because the plasma pharmacokinetics and systemic pharmacological effects of indomethacin were unchanged compared with the free drug41. Several groups are using particulate systems to elicit mucosal immune responses, for example, to increase resistance to microbial infection by this route43. This subject area is vast and thus merits a separate review. In general, the particles that are most readily captured by M cells are 5–10 mm in diameter and hydrophobic in nature15. Administration by other routes Colloidal drug carrier systems have been used to concentrate gamma-interferon in the skin for the treatment of cutaneous herpes. The cytokine accumulated in the stratum corneum, rather than remaining on the surface as occurred after administration of a simple solution44. The application of carrier formulations to the eye retards elimination of drug from the corneal surface. This has been demonstrated for beta-blockers45 and cyclosporin A46 within nanospheres and nanocapsules. Subcutaneous47 or intra-peritoneal48 administration of anticancer agents in liposomes has been shown to deliver the drug to lymphatic metastases. The use of nanocapsules has also been shown to reduce drug-related irritation, for example, after administration by the intramuscular route (S.S. Guterres et al., unpublished). Modification of the distribution of colloidal drug delivery systems Systems avoiding uptake by phagocytic cells Despite the promising results achieved with some ‘first-generation’ drug carrier systems, their value is limited by their distribution and, in particular, by their recognition by the mononuclear phagocyte system. As a result, a great deal of work has been focused on the development of so-called

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Mean lesional index

(a) 80 60 40 20

To ta l

Ile Ile

um

nu m Je

ju nu m

D

uo

de

St om

ac

nu m

h

0

(b) 140 Mean lesional index

105 70 35

D

al

um

To t

ju Je

uo

de

om

ac

nu m

h

0

St

‘Stealth™’ (Sequus Inc.) particles which are ‘invisible’ to macrophages. Early work49 had showed that small liposomes and those containing some negatively charged lipids (ganglioside GM1 or hydrogenated phosphatidylinositol) remained in the bloodstream for longer. Increased circulation time could also be achieved to some extent by administration of high doses, which saturated the phagocytic system. The major breakthrough, however, was the use of phospholipids substituted with poly(ethylene glycol) chains of molecular weight from 1000–5000, as 5–10% of the total lipid49. This provides a ‘cloud’ of hydrophilic chains at the particle surface which repels plasma proteins, as discussed theoretically by Jeon et al.50 Such ‘sterically stabilized’ liposomes have been shown to possess circulating half-lives of up to 45 h, as opposed to a few hours or even minutes for conventional liposomes. This prolongation is almost independent of the injected dose and of the particle diameter of between 50 and 300 nm. Thus they can function as reservoir systems and penetrate into accessible sites, such as tumours, other than mononuclear phagocytes51. The main application to date for long-circulating liposomes has undoubtedly been the treatment of cancers, either leukaemia or solid tumours. Cytosine arabinoside, vincristine, epirubicin and doxorubicin are among the drugs that have been formulated for this area51,52. A doxorubicin-containing formulation based on ‘Stealth™’ liposomes (Alza Corporation, Mountain View, CA, USA), Doxil™ (Alza Corp.), is commercially available for use in AIDS-related Kaposi’s sarcoma (Table 1). Although not containing PEG-substituted lipids, Daunosome™ (Gilead Sciences, Foster City, CA, USA) can be considered as a long-circulating formulation because of the small size of the liposomes (50 nm). As well as accumulating in solid tumours, long-circulating liposomes can extravasate into sites of inflammation and infection53. This provides possibilities for delivering antibiotic and anti-inflammatory agents to these sites, as well as the possibility of performing imaging using liposomes labelled with gammaemitters54. This type of liposome has also been shown to increase the circulating half-life of peptides and proteins, such as the cytokine interleukin-2 (Ref. 36). They may also be useful for prolonging the residence time of drugs administered locally by the subcutaneous55 and intraocular routes56. A similar strategy has been adopted for nanoparticles. Poly(ethylene glycol) can be introduced at the surface in two ways; either by adsorption of surfactants (for example, poloxamer 188), or by the use of block or branched co-polymers, usually based on polyesters, such as poly(lactic acid) (PLA). Although interesting results have been obtained with adsorbed surfactant, such as concentration of phthalocyanines in tumours using nanocapsules coated with poloxamer 407 (Ref.

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Figure 2. Protective effect of nanocapsules containing non-steroidal anti-inflammatory drugs by the oral route. Wistar rats were given 20 mg kg21 diclofenac (a) or 5 mg kg21 indomethacin (b), either as a solution (white) or within poly(D,L-lactic acid)-based nanocapsules (black), intragastrically on three consecutive days. The mean lesional index was determined after sacrifice on day four (mean of eight rats). Drawn from data in Ref. 42.

57), copolymers would seem to be a better choice as they are less easily desorbed from the surface. The surface characteristics (length and density of PEG chains) of nanospheres prepared from PLA–PEG have been optimized to reduce their interactions with plasma proteins and to increase their circulating half-life58,59. It was found that the average distance between two terminally attached chains had to be 2.2 nm or less for protein repulsion58, and that although PEG of 5000 Da was more effective than PEG of 2000 Da, further increases in the chain length did not confer any additional advantage at optimal surface density. Lipophilic drugs, such as lidocaine and cyclosporin A, as well as proteins, have been encapsulated and have been shown to be released in a controlled manner60. As well as their potential as circulating microreservoirs, 167

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Injected dose (%)

25 20 15 10 5 0 PLA/F68

PLA–PEG 5000 formulation

PLA–PEG 20000

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Figure 3. Tissue distribution of different nanocapsule formulations. CD1 mice were injected intravenously with different nanocapsule formulations (5 mg polymer/kg) containing 3H-labelled polymer. The bars show the radioactivity measured in blood (white), liver (black) and spleen (green) 90 min after injection as a percentage of the dose administered (mean of four animals). PLA/F68 poly(D,L-lactic acid) nanocapsules stabilized with adsorbed poloxamer 188. PLA–PEG 5000: poly(D,L-lactic acid) nanocapsules containing 10% (w/w of polymer) of poly(ethylene glycol) of 5000 Da covalently linked to the polymer; PLAPEG 20,000, poly(D,L-lactic acid) nanocapsules containing 10% (w/w of polymer) of poly(ethylene glycol) of 20,000 Da covalently linked to the polymer. Figure taken from V.C.F. Mosqueira et al., unpublished.

such systems might be of value for the controlled release of antigens at mucosal surfaces. Poly(ethylene glycol) chains have also been covalently attached to poly(alkylcyanoacrylate) polymers by two different chemical strategies, and both types of particle have shown long-circulating properties in vivo61. Recently, we have prepared nanocapsules from PLA–PEG co-polymers, with the aim of creating circulating reservoirs with a high capacity for lipophilic drugs. Both PEG chain length and density were found to be important in reducing interactions with phagocytic cells in vitro62 and prolonging circulation time in vivo. We compared systems containing PLA–PEG with those stabilized by poloxamer 188. The latter showed some ‘Stealth™’ properties in vitro at low dilution, but these were lost at higher dilution and in vivo. Figure 3 shows the tissue distribution 90 min after administration. The nanocapsules containing PLA–PEG of 5000 Da, with a spacing of 4.3 nm, were better at avoiding capture by the liver than those containing PLA–PEG of 20,000 Da (spacing 7.8 nm), and both were better than nanocapsules with adsorbed poloxamer 188. However, when a denser coverage of PLA–PEG 20,000 was used, longer circulating times were obtained (V.C.F. Mosqueira, et al., unpublished). It should, however, be mentioned that the circulating half-lives that can be achieved with nanoparticulate systems are, for the moment, not as long as those obtained with liposomes; perhaps this is because the more rigid surface of the 168

polymeric systems is a more powerful complement activator. Another strategy for preparing long-circulating colloidal systems can be considered as biomimetic, in that it seeks to imitate cells or pathogens that avoid phagocytosis by reducing or inhibiting complement activation. One example is the development of liposomes with a membrane composition similar to that of erythrocytes; for example, liposomes containing GM1 (Ref. 49) or those coated with polysialic acids63. These systems may possess circulating half-lives as long as liposomes bearing PEG. Attempts to introduce sialic acid onto the surface of nanoparticles, either by adsorption of glycoproteins64 or by synthesis of a PLA–polysialic acid copolymer (P. Huve, et al., unpublished), were not successful; in the first case this was because of desorption in the presence of plasma proteins, and in the second because of either the water-solubility of the polymer or an inappropriate conformation of the polysaccharide. A more promising biomimetic approach is the use of heparin, as the hydrophilic part of amphiphilic copolymers capable of forming nanospheres. The hydrophobic segment was poly(methyl methacrylate), a non-biodegradable polymer, which was, however, suitable for validating the concept. Heparin is an anionic polysaccharide known to inhibit several steps of the complement cascade, and, indeed, these nanoparticles were not activators in an in vitro system. They avoided uptake by macrophages in vitro (N. Jaulin, et al., unpublished), and were longcirculating in vivo [half-life of 5 h or more compared with a few minutes for poly(methyl methacrylate) nanoparticles]65. It is interesting to note that nanoparticles prepared from a copolymer with dextran, intended as controls, had only low complement-activating potential and also showed long-circulating properties; thus, a steric hindrance effect caused by a brush-like arrangement of end-attached polysaccharide chains, similar to that obtained with PEG, may also determine the biological fate of these systems. Systems avoiding the lysosomal compartment It may be sufficient for a carrier system to concentrate the drug in the tissue of interest. However, in the case of hydrophilic molecules that experience difficulties in crossing the plasma membrane (such as nucleic acids66), intracellular delivery is required. If the carrier is taken up by endocytosis, its ultimate destination will be the lysosomes, in which hydrolytic enzymes will degrade both the carrier and its contents.Therefore, several liposome-based systems, have been developed to avoid the lysosomal compartment either by fusing directly with the plasma membrane with the help of fusogenic proteins or peptides67,68, or by destabilizing the endosome. Endosome disruption can be achieved by the use of pH-sensitive liposomes, in which the lipids undergo a phase change at acidic pH (Ref. 69) or by cationic liposomes70. In this way, the encapsulated material can be delivered to the cytoplasm. These systems are

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particularly appropriate for the delivery of genes and antisense oligonucleotides. For example, an antisense oligonucleotide against the Friend leukaemia virus was found to have a better antiviral effect in vitro when encapsulated in pH-sensitive liposomes than in conventional liposomes69. Systems targeted to specific cell populations An ideal drug carrier system would contain a specific ‘homing group’ capable of being recognized by the target cells. Much work has been devoted to coupling specific ligands to the surface of liposomes. Monoclonal antibodies or fragments thereof have often been used because of their specificity 71. Other targeting systems that have been investigated are sugar–lectin interactions, such as the mannose or fucose receptor of macrophages and the galactose receptor of hepatocytes, hormone and growth-factor receptors and receptors for cell nutrients such as transferrin and folic acid, which are over-expressed in some tumours. Impressive results have been obtained in vitro71 but, with the exception of targeting to the liver, these are not confirmed in vivo, because the use of specific ligands cannot overcome physiological constraints, as outlined below.. First, even targeted systems will be recognized by macrophages if their surfaces are not modified to avoid opsonization, although PEG chains can mask a ligand attached directly to the liposome surface. A solution might be to attach the targeting group to the end of the PEG chain, but to limit the degree of substitution so as to avoid recreating a surface on which opsonization can occur72. Second, the permeability of the vascular endothelium must continue to be taken into account.Third, if intracellular delivery is required, the targeting ligand must not simply be bound to the surface, but also internalized after binding, and the carrier system must be small enough to be taken up by receptor-mediated endocytosis in non-phagocytic cells, that is, 200 nm or less71. Furthermore, if this internalization occurs, it will lead to the lysosomal compartment unless an endosome-destabilizing element is present. In the light of such constraints and considerations, the most suitable targets for drug carriers would be cells in accessible sites such as liver metastases, circulating cells (such as leukaemia); cells in sites where the endothelium is leaky (tumours, inflammation, infection, including within the blood–brain barrier); and capillary endothelium, to concentrate the drug within a particular organ and allow it to diffuse from the carrier to the target tissue. An example of this last approach is the use of sterically stabilized liposomes containing AmB bearing an antibody specific for pulmonary endothelium at the end of the PEG chains73.This led to a greater accumulation of antibiotic in the lungs, as opposed to residence in the blood in the case of non-targeted PEG-bearing liposomes or accumulation in the liver in the case

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of conventional liposomes.This was accompanied by increased efficacy against experimental aspergillosis in mice. For the treatment of leukaemia, effective targeting of sterically stabilized liposomes containing doxorubicin and bearing anti-CD19 to malignant B cells has been observed in vitro and in vivo in mice74. However, antibody-targeted systems do not always show an advantage over simple sterically stabilized liposomes in solid tumours, probably because the larger antibodydecorated particles diffuse less easily within the tumours, where the hydrostatic pressure is increased because of the lack of lymphatic drainage52. However, PEG-liposomes bearing a smaller ligand, folic acid, have been shown to be effective carriers for intracellular delivery of nucleic acids and anti-cancer drugs to tumour cells in vitro75. Conclusion Colloidal drug delivery vehicles have been studied for almost 30 years, but have not yet become the ‘magic bullet’ foreseen by Paul Ehrlich. An improved knowledge of the physiological constraints governing the distribution and fate of these carriers has allowed for more rational design and the development of ‘second-generation’ systems. The formulations already on the market (Table 1) are mainly concerned with reducing the side effects of the encapsulated drugs. With the arrival of ‘Stealth™’ liposomes and nanoparticles that avoid rapid phagocytosis, the range of sites that can be reached has been extended. Even without specific targeting technologies, it has already been shown that sites of inflammation and infection and solid tumours can be reached, as well as intravascular sites. If specificity for a particular cell type is required, ligands such as monoclonal antibodies, sugars, lectins, or growth factors can be coupled to these long-circulating systems. Colloidal drug carriers are particularly useful for formulating new drugs derived from biotechnology (peptides, proteins, genes and oligonucleotides) because they can provide protection from degradation in biological fluids and promote their penetration into cells. They also have applications with respect to small hydrophobic molecules, because they can provide an ultradispersed form without the use of irritating solvents and allow rapid drug dissolution.Therefore, it is likely that colloidal systems able to improve the efficacy of both established drugs and new molecules will soon be available. Acknowledgements I wish to thank the Centre National de la Recherche Scientifique (Chatenay-Malabry, France) for financial support. I acknowledge all my colleagues, past and present, in the UMR CNRS 8612, and especially its founder and former director, Francis Puisieux, who provided a unique environment within which to develop this fascinating subject. 169

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