Current methods for attaching targeting ligands to liposomes and nanoparticles

MINIREVIEW Current Methods for Attaching Targeting Ligands to Liposomes and Nanoparticles LEILA NOBS,1 FRANZ BUCHEGGER,2 ROBERT GURNY,1 ERIC ALLE´MANN1 1

School of Pharmacy, University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland

2

Service of Nuclear Medicine, University Hospital of Geneva, Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland

Received 10 December 2003; revised 4 March 2004; accepted 4 March 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20098

ABSTRACT: Liposomes and nanoparticles have emerged as versatile carrier systems for delivering active molecules in the organism. These colloidal particles have demonstrated enhanced efficacy compared to conventional drugs. However, the design of liposomes and nanoparticles with a prolonged circulation time and ability to deliver active compounds specifically to target sites remains an ongoing research goal. One interesting way to achieve active targeting is to attach ligands, such as monoclonal antibodies or peptides, to the carrier. These surface-bound ligands recognize and bind specifically to target cells. To this end, various techniques have been described, including covalent and noncovalent approaches. Both in vitro and in vivo studies have proved the efficacy of the concept of active targeting. The present review summarizes the most common coupling techniques developed for binding homing moieties to the surface of liposomes and nanoparticles. Various coupling methods, covalent and noncovalent, will be reviewed, with emphasis on the major differences between the coupling reactions, on their advantages and drawbacks, on the coupling efficiency obtained, and on the importance of combining active targeting with long-circulating particles. ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1980–1992, 2004

Keywords: nanoparticle; liposome; active targeting; surface modification; antibody; drug targeting; carrier system; immunoliposome

INTRODUCTION In recent years, an increasing number of studies have been devoted to the development of drug delivery systems and drug targeting. First attempts in this direction were accomplished using monoclonal antibodies or antibody fragments Eric Alle´mann’s present address is Bracco Research SA, Route de la Galaise 31, 1228 Plan-les-Ouates, Geneva, Switzerland. Correspondence to: Robert Gurny (Telephone: þ 41 22 379 61 46; Fax: þ 41 22 379 65 67; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 93, 1980–1992 (2004) ß 2004 Wiley-Liss, Inc. and the American Pharmacists Association

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coupled with active compounds, such as antitumor drugs.1 One of the major problems facing the development and utilization of these drug– antibody conjugates was the preservation of both pharmacological and immunological activities. To maintain the binding activity of the antibody, only a relatively low amount of drug can be coupled to it, which is often insufficient to obtain the desired therapeutic effect. This shortcoming has encouraged research into other strategies, and one of the most challenging consists of the entrapment of drugs into liposomes or nanoparticles. These colloidal systems have proved to be versatile carriers for a wide variety of i.v. administered

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METHODS FOR ATTACHING TARGETING LIGANDS TO LIPOSOMES

active molecules.2 Their main advantage is that they offer a suitable means for delivering drugs combined with the potential of improving the therapeutic index while greatly reducing the side effects. However, these carrier systems initially presented two major drawbacks: the rapid uptake by phagocytic cells of the reticuloendothelial system (RES)3,4 and the lack of targeting specificity. To avoid the rapid clearance, ‘‘stealth’’ or ‘‘sterically stabilized’’ liposomes or nanoparticles have been developed. One of the major means to obtain stealth particles has been to coat them with amphipathic polyethylenglycol (PEG).5–9 Although capable of enhanced accumulation in tumor tissue10,11 compared to conventional particles, the interaction of the sterically stabilized carriers with tumor cells and tumor uptake appeared to be insufficient.12 Therefore, different strategies to enhance active targeting have been developed. Although in the field of liposomes many studies have been performed, only few studies have been carried out in the field of nanoparticles. Excellent review articles have been published dealing with liposomes and active targeting,12–16 particularly with tumor targeting.17–19 The aim of the present review is to describe and discuss the currently used coupling reactions for the attachment of ligands to colloidal carriers.

COVALENT BINDING OF LIGANDS TO THE LIPOSOMAL SURFACE Two methods are used for attaching ligands to the liposomal surface: covalent and noncovalent

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coupling. In this first section, we will describe the frequently used covalent reactions (Table 1). Generally, ligands are bound to the surface of liposomes through hydrophobic anchors having functional groups. Long-chain fatty acid such as palmitic acid20–24 and phospholipids, such as phosphatidylethanolamine (PE)25 and phosphatidylinositol (PI),26 have been successfully used as anchors. These anchors are incorporated into the liposomal membrane during the formation of liposomes. There are essentially two approaches to covalently attach the ligand to the anchor. The first consists of carrying out the reaction between the ligand and the anchor and mixing the resulting ligand with the other constituents of the liposome.22 In the second case, the anchor is already included in the liposome bilayer and the coupling reaction occurs on the surface of preformed liposomes.27 Among the hydrophobic anchors, PE is frequently used because it can be easily derivatized to offer functional groups. Each covalent reaction used to attach ligands to phospholipid anchors will be described separately. More details on this topic can be found in a book chapter.28 Coupling of Ligands to the Surface of Liposomes Using Thioether Bonds The reaction between thiol functions and maleimide groups is a highly efficient reaction that gives a stable thioether bond (Fig. 1). Native thiol groups are present in some proteins, but in many others, thiol functions are either absent or present in insufficient amounts. Thus, they have to be

Table 1. Covalent Coupling Techniques Used to Bind Ligands to Liposomes Covalent Linkage

Ligand

Thioether

Fab0 fragments (
SH) SATA-modified ligand SPDP-modified ligand

Disulfide

Carboxamide Amide

PDP-modified ligand Fab0 fragments (
SH) SATA-modified ligand Ligand (
NH2) Ligand (
NH2)

Hydrazone

Oxidized ligand

Derivatized Anchor

Refs.

MPB–PE MP–PEG–DSPE MMC–PEG–DSPE MPB–DOPE PDP–DOPE PDP–PEG–DSPE PDP–PEG–PE PDP–PE

29–37 38,41 38,41 44 42 32,43 39 45–47

Ester (NHS) of the palmic acid DSPE–PEG–COOH NGPE Hz–PEG–DSPE

20–25 48–50 25,51–56 34,42 58,59

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Figure 1. Schematic diagram of the different coupling methods used. Reaction between maleimide and thiol functions (A), formation of a disulfide bond (B), reaction between carboxylic acid and primary amine group (C), reaction between hydrazide and aldehyde functions (D), crosslinking between two primary amine functions (E).

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added either via heterobifunctional crosslinking agents29,30 or be obtained by reducing existing disulfide bonds. Commonly, N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP) and succinimidyl-S-acetylthioacetate (SATA)29–31 are used as crosslinkers. Both offer one primary reactive amine residue for coupling with the ligand. In both cases, thiol functions are not directly available, and a deprotection of these functions is necessary before the reaction with liposomes. In the case of SPDP, dithiothreitol (DTT) or an alternative reducing reagent is used to reduce the disulfide bond to thiol functions. SATA offers one protected thiol function that can be made available by deacetylation with hydroxylamine. The deprotection is performed under milder conditions compared to those with a reducing reagent. Most frequently N-(4-(p-Maleimidophenyl) butyryl)phophatidylethanolamine (MPB-PE) has been used as a functionalized anchor.29–37 The extended spacer arm between the phospholipid head group and the maleimide moiety reduces the possibility of steric hindrance at the bilayer interface and thereby ensures favorable thiol reactivity. Once the liposomes with maleimide groups have been formed, the ligand, having thiol functions, can be coupled to the liposomes by a simple addition.30,32,35 Park et al.35 demonstrated that the internalization of immunoliposomes bearing anti-p185HER2 monoclonal antibodies, is possible and that the internalization is due to the specificity of the antibody coupled to the liposomes, because control liposomes lacking Fab0 showed no internalization by SK-BR-3 cells. Furthermore, immunoliposomes appeared to induce a high antiproliferative effect, which was superior to that of free monoclonal antibody. Another study29 with liposomes bearing AR-3 monoclonal antibodies showed that these liposomes were highly effective as antitumor agent and induced less systemic toxicity compared to the free drug. Immunoliposome technology can be combined with sterically stabilized liposomes technology to give long circulating vesicles capable of selectively delivering compounds to target cells. There are two ways of obtaining this type of liposome: (1) antibodies are bound to the surface of liposome in parallel with PEG,35,38 or (2) antibodies are linked to the distal end of PEG chains.38,39 For both approaches, PEG is incorporated into the bilayer membrane via an anchor such as distearoylphosphatidylethanolamine (DSPE).34,38,40–42 When antibodies were coupled to the termini of PEG,

METHODS FOR ATTACHING TARGETING LIGANDS TO LIPOSOMES

(maleimidomethyl)cyclohexanecarboxylate – PEG – DSPE (MCC–PEG–DSPE) and maleimido-phenylpropionate–PEG–DSPE (MP–PEG–DSPE) were used.38,41 Thiol groups of the Fab0 fragments38,40 or activated monoclonal antibodies (entire IgG) were then attached to the distal end of the PEG chains via the maleimide groups. When antibodies were coupled directly at the liposome surface, binding affinity for the target cells was reduced due to the steric hindrance caused by the high density of PEG coating.40,41 This was circumvented by attaching antibodies to the distal end of PEG chains.40,41 In this case, a linker with a long spacer arm is used that provides attachment of the antibody distant from the liposome bilayer. It is also possible to link maleimido antibodies to liposomes offering thiol functions.42,43 In the case of long-circulating liposomes, antibodies can be attached to the carrier either directly to the surface of the vesicles via PDP–dioleoylphosphoethanolamine (PDP–DOPE)42 or at the PEG terminus via PDP–PEG–DSPE42,43 or PDP– PEG–PE.39 Maleimido antibodies are obtained using a heterobifunctional crosslinker, succinimidyl 4-[p-maleimidophenyl]butyrate (SMPB),39,42,43 that is reactive with amino groups of antibodies and sulfhydryl groups (DTP-liposomes formed after reduction of the PDP). Hansen et al.42 showed that binding ligand at the PEG end chain yielded a coupling efficiency of approximately 60–70%. In contrast, when antibodies were directly bound to the surface of liposome via MPB–DOPE, the coupling efficiency was only 10%. In an other study,39 coupling efficiencies of nearly 100% were obtained by attaching antibodies to the terminus of PEG. PEG can also be directly linked to cholesterol (chol) instead of PE or DSPE.44 Pegylated Chol (PEG–Chol) derivatives are obtained more easily and the cost of production is lower. Despite these advantages, the amount of antibody coupled to liposomes is inferior than that with PEG–PE vesicles.44 Most probably, this is due to the fact that the functional groups are less accessible owing to the reduced mobility of PEG–Chol, which is located deeper in the liposome membrane. Attachment of Lligands to Liposomes via a Disulfide Linkage One of the most rapid and easy coupling chemistries involves the conjugation of two thiol functions to form a disulfide bond (Fig. 1). However,

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it has to be pointed out that disulfide bonds are relatively unstable under the reductive conditions in serum.45 For this reason the disulfide linkage has been progressively replaced by other more stable ones. As seen before, thiolated ligands can be generated either by reduction of disulfide bonds,45 or using SATA or SPDP.42,46,47 The thiolated ligands can then react with the pyridyldithio moiety of the anchor (PE-PDP) to form a disulfide linkage. Several studies have demonstrated that the coupling method resulted in efficient binding of antibody to the liposomes without denaturation of the ligand and that liposomes bearing Fab0 fragments recognized and bound selectively to target cells in vitro.45–47 Crosslinking between Carboxylic Acid Functions on the Surface of Liposomes and Primary Amines of the Ligand Ligands can be attached to the surface of liposomes by an amide bond (Fig. 1) using an anchor functionalized with carboxylic acid end groups. For this purpose, distearoyl-N-(3-carboxypropionoyl poly(ethylene glycol)succinyl)phosphatidylethanolamine (DSPE–PEG–COOH), which offers carboxylic acid groups at the distant end of surface-grafted PEG chains is commonly used.48,49 The coupling reaction is carried out in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and N-hydroxysulfosuccinimide (NHS) to form an acyl amino ester, which will subsequently react with the primary amine of the ligand, yielding an amide bond.48–50 The major advantage of this method is that no prior ligand modification is required, thus reducing the risk of denaturation and loss of its specific activity. Maruyama et al.49 prepared liposomes bearing monoclonal antibodies 273-32A (34A), specific for mouse pulmonary endothelial cells, and results indicated that the immunoliposomes recognized and bound specifically to target cells, in vivo. Ishida et al.48 have investigated the possibility of using transferrin (TF) as a ligand for active targeting to tumor cells. In vitro studies on mouse colon carcinoma cells (Colon 26) have indicated that TF-liposomes bound to these cells, and were efficiently internalized by receptormediated endocytosis. In vivo investigation on colon 26 tumor-bearing mice confirmed these results and showed that liposomes extravasated from the blood compartment into the solid tumor. PE can also be functionalized with glutaryl to give N-glutaryl-phosphatidylethanolamine JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

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(NGPE).25,51 –56 Torchilin et al.52 prepared liposomes bearing antimyosin antibody (AM) Fab0 and the measurement of the efficacy of the coupling reaction showed that between 65 and 75% of the Fab0 fragment were bound to the liposomes. Immunoreactivity of the modified Fab0 coupled to the liposome surface was measured and results revealed that unfortunately the immunoreactivity of the antibody decreased by 15 to 20 times compared to free antibody. This reduced immunoreactivity of a single Fab0 fragment was partially counterbalanced by the presence of a high number of antibodies per liposome. The efficacy of active targeting was shown in vivo, because liposomes bearing AM accumulated selectively in the infarcted myocardium, compared with native liposomes (without AM) that where present only in noninfarcted myocardium. Binding of Ligands to Liposomes via a Hydrazone Bond Antibodies can also be covalently bound through their carbohydrate moieties to hydrazide groups grafted onto the liposomal surface to form a hydrazide bond (Fig. 1). A mild oxidation of the carbohydrate groups on the constant region of the heavy chain of the immunoglobulin is required to produce aldehyde groups. These latter react with hydrazide groups of the anchor.34,57,58 The carbohydrate groups are oxidized either by galactose oxidase57 or by sodium periodate.34,42,57–60 It is obvious that the oxidation reaction must be performed under mild conditions to avoid any loss of antibody activity. Once the oxidized antibodies are formed, they can be either directly coupled to the lipid bilayer containing a hydrazide-hydrophobic anchor such as lauric acid hydrazide,57 or to the distal end of the PEG chains of sterically stabilized liposomes.34,42,58,59 In this latter case, a functionalized PEG–lipid hydrazide– PEG–DSPE (Hz–PEG–DSPE)61 is used. This coupling reaction theoretically offers the advantage that antibodies are correctly orientated once attached onto the surface of the liposomes, because only the Fc region is involved in the coupling reaction, leaving the antigen binding site available. However, a comparative study42 between different coupling methods revealed that this coupling method is not very efficient, because only a small percentage of antibodies (17%) were attached to the liposomal membranes. Nevertheless, positive results have been obtained in vitro by other research groups. Koning et al.34 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

have prepared liposomes bearing monoclonal antibodies against rat colon carcinoma CC531, and the results revealed that the anticancer prodrug, 5-fluoro-20 -deoxyuridine encapsulated in the bilayer of liposomes was selectively transferred to the surface of tumoral cells and internalized into the lysosomal compartment. Crosslinking between Primary Amines on the Surface of Liposomes and Primary Amines of the Ligand Among covalent coupling methods, direct amine– amine crosslinking has also been investigated (Fig. 1). Two homobifunctional crosslinkers have been used: glutaraldehyde62,63 and suberimidate.63 The advantage of this coupling approach is that no prior modification is required to add functional groups to the ligand. The coupling method consists of firstly activating the primary amine of PE on the liposome surface via the crosslinker and subsequently coupling the ligand. Results have demonstrated that almost 60% of the antibodies were coupled to the liposomes, and that they retained the binding capacity for the antigen.62 Despite the fact that the coupling reaction is efficient, the use of homobifunctional crosslinkers has been rarely exploited owing to the uncontrollable homopolymerization of ligand or liposomes during the crosslinking reaction.

Multistep Attachment Using the Avidin–Biotin Interaction The avidin–biotin strategy has become an extremely useful and versatile tool for active targeting, especially owing to the possibility of applying it in a multistep approach.64–66 The avidin–biotin technology has also been used in the field of liposomes.42,67 Furthermore, biotinylated antibodies can be easily obtained.68–70 Xiao et al.71 prepared liposomes for a three-step strategy by incorporating biotinyl amino-hexanoyl dihexadecanoyl PE into the liposomal membrane. In vitro trials were carried out on human ovarian cancer cells (OVCAR-3) to demonstrate the multistep targeting ability of the liposomes. Biotinylated antibodies were first incubated with the cells; subsequently, streptavidin was added followed by biotinylated liposomes. Results showed that the liposomes bound specifically to the antibody coated cell surface of OVCAR-3 cells but not to control cells, which do not express the specific antigen.

METHODS FOR ATTACHING TARGETING LIGANDS TO LIPOSOMES

It has to be kept in mind that the choice of the antibody is crucial for the multitargeting approach because the antibody should remain available and not be rapidly internalized after binding to the target antigen.

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approaches were not widely used, mainly due to the weak interaction between the carrier and the ligand and to the random distribution of the ligand on the surface of the liposomes.

LABELING POLYMERIC NANOPARTICLES WITH LIGANDS

NONCOVALENT METHODS USED TO BIND LIGANDS TO THE LIPOSOMAL SURFACE

As mentioned in the Introduction, nanoparticles have not yet been extensively investigated for active targeting of therapeutic agents, despite their improved stability in vivo and upon storage, a wide choice of materials (synthetic or natural polymers) and different techniques of preparation. They are also able to carry a high amount of active molecules. Nevertheless, there are still some improvements necessary to achieve selective targeting of nanoparticles to specific tissues or organs.79 Here, we will comment on the first attempts to bind ligands to the surface of nanoparticles (Table 2).

As stated previously, an alternative means to conjugate ligands to liposomes is the use of noncovalent techniques. These have the great advantage of being easy to be carried out without the need of aggressive reagents. A simple method is to merely add the ligand to the mixture of phospholipids during the preparation of the liposomes.72 However, the percentage of ligand attached to the carrier is relatively low (4–40%) and aggregation of the liposomes are frequently observed. Furthermore, the amount of ligand linked to the liposome is not easily controllable, and the correct orientation of the antibodies is not ensured. Finally, detachment of the antibody in vivo might occur. Other attempts have been described such as the attachment of heat-aggregated antibodies to the surface of liposomes,73,74 or the binding of antibodies via a hapten.75–78 But, overall, these

Adsorption of Ligands to the Surface of Polymeric Nanoparticles The most common approach described for the design of nanoparticles for active targeting is the adsorption of monoclonal antibodies to their surface.80–84 This noncovalent technique was widely investigated on cyanoacrylic nanoparticles. One

Table 2. Current Methods Used to Bind Ligands to the Surface of Polymeric Nanoparticles

Coupling Method Adsorption

Covalent Schiff’s base formation Periodate oxidation Carbodiimide reaction Polymer conjugate

Multistep approach

Composition of the Nanoparticles

Studies vitro

Poly(styrene) Bovine serum albumin

In — In — In In In

Poly(cyanoacrylic) Poly(cyanoacrylic) Poly(styrene) Gliadin Poly(lactic acid) Poly(lactic acid) Poly(styrene) Poly(lactic acid) Gelatin Human serum albumin Poly(lactic acid)/caprolactone

In In In In In — — — — — In

vitro vitro vitro/in vivo vitro vivo

Poly(cyanoacrylic)

vitro vivo vitro vitro/in vivo

vitro

Refs. 84 83 81 82 80 85 86 87 88 90,91 89 92 93 94 95 96 97 98

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of the first attempts was accomplished by Couvreur and Aubry84 with 17-1-A antibody adsorbed onto the surface of pre-formed nanoparticles. Large amounts of antibody were bound to the particles and the resulting nanoparticles were stable during storage. To optimize the coupling of the antibody to the nanoparticles, especially with respect to the orientation of the antibody, spacer molecules were used. Protein A (protein isolated from the cell wall of Staphyloccocus aureus) was chosen, which interacts specifically with the Fc fragment of IgG. After adsorption of Protein A on the surface of the nanoparticles, the antibody is simply added. Under these conditions, the IgG is correctly oriented with the Fab0 fragment accessible for binding to the specific antigen. Results demonstrated that almost 95% of the monoclonal antibody was coupled to the nanoparticles, which then interacted with target cells (human colorectal carcinomas HT-29 and HRT-18). However, in the absence of in vitro experiments in the presence of serum proteins, the competitive displacement of the adsorbed antibody was not studied. This investigation was subsequently carried out,81–83 but results were controversial. In fact, Manil et al.83 observed no significant desorption of the antibody (anti-a-fetoprotein antibody) when the nanoparticles were incubated with human serum protein. In contrast, other in vitro81,82 and in vivo80 studies provided evidence of competitive displacement of the adsorbed antibody by serum proteins. Furthermore, the in vivo results were disappointing, because the nanoparticles accumulated mostly in the liver and spleen. Blackwell et al.85 prepared polystyrene nanoparticles coated with humanized mAb HuEP5C7.g2 via Protein A, and the results indicated that these nanoparticles bound selectively to cellular expressed E-and P-selectin. Here again, further investigations must be carried out to confirm the efficacy of this approach. Covalent Binding of Ligands to the Surface of Nanoparticles To the best of our knowledge, very few studies dealing with the covalent coupling of antibodies to nanoparticles, and even fewer dealing with in vitro and in vivo experiments, have been published. In one of these studies,86 rabbit antihuman antibodies were attached to bovine serum albumin nanoparticles by formation of a Schiff’s base between the primary amine of the antibody JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

and the free aldehyde groups of glutaraldehyde (bifunctional crosslinker) present on the surface of the pre-formed albumin nanoparticles. Results indicated that antibodies maintained their binding activity after the coupling reaction. Furthermore, in vitro and in vivo experiments demonstrated the specific targeting ability of the antibody coated nanoparticles, although with low affinity. The covalent binding of monoclonal antibodies to polymethacrylic nanoparticles was described by Rolland et al.87 In this study, an antiperipheral human T-lymphocyte antibody (CD3 monoclonal antibody) was linked to the surface of nanoparticles using the glutaraldehyde method. In vitro experiments showed that the specificity of the monoclonal antibody was maintained. A recent study88 on PEG-coated poly(cyanoacrylate) nanoparticles conjugated to transferrin (TF) revealed that this carrier system was useful for delivery of pDNA to target cells. TF was coupled to PEG coated nanoparticles by a periodate oxidation technique, and results revealed that 1–3% of the total PEG chains were linked to TF molecules. Interesting work has been accomplished with respect to the oral delivery of nanoparticles to increase their gastrointestinal uptake.89,90 Although a discussion of techniques to enhance the bioadhesion of orally administered nanoparticles is not the aim of this review, these studies described interesting coupling reactions that might be used for the coupling of ligand, such as antibodies, for active targeting. Various lectins have been successfully used, based on their primary amines available for reaction with carboxylic acid groups exposed on the surface of polystyrene90,91 or gliadin particles,89 and able to form an amide bond via a carbodiimide reaction. Another interesting approach is the synthesis of a conjugate of a polymer and a ligand, which is then directly used for the preparation of nanoparticles. Gautier et al.92 used this method for the preparation of biotinylated poly(acid lactic) (PLA) nanoparticles. A conjugate of biotin and poly (methyl methacrylate-co-methacrylic acid) was synthesized and coprecipitated with PLA into stable nanoparticles. For active targeting, such nanoparticles could be used for a multistep approach using the avidin–biotin interaction. Another promising strategy for active targeting was described by Maruyama et al.93 Here, nanoparticles exposing surface carbohydrates, which could bind to target cells via the carbohydratebinding proteins present on the surface of cells

METHODS FOR ATTACHING TARGETING LIGANDS TO LIPOSOMES

were prepared for the delivery of genetic material into cells. Various polysaccharides were investigated, and their binding to the nanoparticle was guaranteed by the presence of poly(L-lysine)grafted-polysaccharide in the PLA matrix of the nanoparticle. Another interesting study94 demonstrated the potential use of carbohydrate-conjugated nanoparticles as site-specific drug carrier. Serizawa et al.94 prepared poly(vinylamine)-grafted polystyrene nanospheres by the free radical polymerization of styrene and poly(N-vinylacetamide). The lactose was bound to the particles by an amide linkage. Results suggested that the use of lactoseconjugate nanoparticles could be a useful approach for site specific delivery of a drug carrier, because these particles recognized and bound specifically to lectin RCA120. As a multistep targeting approach, our own group is investigating the possibility of coating nanoparticles with homing moieties. The idea is to use the avidin–biotin interaction to attach specific antibodies on the surface of PLA nanoparticles. As a first step, functional thiol groups, were added to the surface of PLA nanoparticles; three different techniques have been investigated.95 The second step was to covalently attached NeutrAvidin1 to the nanoparticles, which will allow the subsequent binding of biotinylated antibodies to the carrier. A similar concept has already been used to prepare gelatine and human serum albumin nanoparticles96,97 as carriers for biotinylated compounds. More recently, Gref et al.98 have prepared nanoparticles composed of PLA and biotin-PEG-poly(e-caprolactone). Avidin was coupled to the surface of the nanoparticles followed by biotinylated WGA. In vitro studies revealed that the nanoparticles interacted specifically with target cells.

CONCLUSIONS In recent years, significant improvement in the field of colloidal carriers has been achieved. Nanoparticles and liposomes have emerged as versatile tools for delivering active compounds, such as antitumor drugs. The formulation of long circulating particles has given reliance in the usefulness of these carriers, and provide a solid basis for the development of particles for selective targeting. Many challenging questions arise when dealing with active targeting, such as those concerning the optimal size of the particles, the

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choice and the density of the ligand, and the optimal coupling reaction. Various coupling methods have been described in this review, with their advantages and drawbacks. Covalent reactions appear to be an effective way to irreversibly fix ligands to the colloidal carriers, because the linkage formed is much more stable and reproducible when compared to noncovalent methods, such as adsorption. It has to be kept in mind that the coupling reaction must not affect the biological activity of the ligand. In the case of antibodies, all conjugation through primary amines will certainly involve a part of amine groups within the antigen binding region, enhancing thus the probability of affecting the antibody activity, especially if highly modified antibodies are prepared. The use of antibodies fragments having available thiol functions, is a good alternative, because no modification are required to add functional groups, preserving thus the antigen binding activity. Furthermore, a good orientation of the fragments on the liposomes surface is guaranteed. But, over all, there is not a optimal covalent coupling reaction, because the efficacy of the conjugation depends on various relevant parameters such as the type and the localization of the functional groups on the ligand, the number of available ligand attached to the carrier, and the type of crosslinker. The length of the crosslinker is also very important, because the accessibility of the ligand is directly related to the length of the crosslinker, and the use of an extended spacer arm can greatly reduce the steric hindrance. It also has to be considered that covalent reactions require the need of chemical reagents that can potentially interfere with subsequent reactions or damage the targeting device and/or the colloidal carrier. In addition, the use of some crosslinkers, such as glutaraldehyde, can induce undesired aggregation of ligand and/or carrier. Furthermore, the reactions and the purifications steps can lead to a decrease in the stability of the particle or the ligand. The ligand can be attached to the surface of the liposome either during their preparation of this, or on pre-formed vesicles. The major drawback of ligand incorporation by mixing with other components of the bilayer membrane is the possible presence of immobilized ligand on the inner surface of the liposomes and consequent unavailability for the binding activity. Furthermore, the number of ligands coupled to the liposome surface can be quite heterogeneous,20,21 thus impacting on the binding capacity to target cells. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 93, NO. 8, AUGUST 2004

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On the other hand, if the coupling reaction is achieved on pre-formed carrier, there is some risk of altering the structure of the particle and in some cases, the encapsulated compound. Therefore, it is essential to choose nonaggressive reagents and to work under mild, controlled conditions. One has to keep in mind that the ability to achieve active targeting in vivo depends directly on whether or not the target is accessible from the vasculature. In the first case, particles bearing homing moieties and having a long circulating half-life have a high probability of reaching the target. But when the target is extravascular, such as solid tumors, penetration of carriers to the bulk of the tumor cells is more difficult. The size of the carriers has to be well established for their extravazation and accumulation in solid tumors99 considering that the pore sizes of discontinuous tumor microvasculature varies between 100 to 780 nm.100,101 Although, as yet, these systems are far from perfect, we can conclude, as many authors have demonstrated, that the proof-of-concept for activetargeted drug delivery has been well established.

REFERENCES 1. Weiner LM. 2003. Monoclonal antibody therapy of cancer. Semin Oncol 26:43–51. 2. Martin FJ. 1998. Clinical pharmacology and antitumor efficacy of DOXIL (pegylated liposomal doxorubicin). In: Lasic DD, Papahadjopoulos D, editors. Medical applications of liposomes. New York: Elsevier Science B.V., pp 635–688. 3. Alle´mann E, Gurny R, Doelker E. 1993. Drugloaded nanoparticles-preparation methods and drug targeting issues. Eur J Pharm Biopharm 39:173–191. 4. Senior JH. 1987. Fate and behavior of liposomes in vivo: A review of controlling factors. Crit Rev Ther Drug Carrier Syst 3:123–193. 5. Stolnik S, Dunn SE, Garnett MC, Davies MC, Coombes AG, Taylor DC, Irving MP, Purkiss SC, Tadros TF, Davis SS. 1994. Surface modification of poly(lactide-co-glycolide) nanospheres by biodegradable poly(lactide)-poly(ethylene glycol) copolymers. Pharm Res 11:1800–1808. 6. Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R. 1994. Biodegradable long-circulating polymeric nanospheres. Science 263:1600–1603. 7. Allen TM, Hansen C, Martin F, Redemann C, Yau-Young A. 1991. Liposomes containing synthetic lipid derivatives of poly(ethylene glycol)

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