Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes

advanced

drug delivery ELSEVIER

reviews Advanced Drug Delivery Reviews 14 (1994) 1 24

Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes Michel Monsigny, Annie-Claude Roche*, Patrick Midoux, Roger Mayer Laboratoire de Biochimie des GlycoconjuguOs et Leetines Endogdnes, Universit~ d'OrlOans et Centre de Biophysique Mol~eulaire, CNRS, Bdt. B, 1 rue Haute, 45071 Orleans Cedex 2, France (Received June 25, 1993; Accepted August 15, 1993)

Contents Abstract .............................................................................................................................................

2

1. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

2. M e m b r a n e lectins t h a t m e d i a t e e n d o c y t o s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4

3. N e o g l y c o p r o t e i n s o r glycosylated p o l y m e r s as carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. N e o g l y c o p r o t e i n s as carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. G l y c o s y l a t e d p o l y m e r s as carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. l m m u n o g e n i c i t y o f n e o g l y c o p r o t e i n s a n d glycosylated p o l y m e r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. D r u g - - c a r r i e r linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 8 8 9 10

4. D r u g t a r g e t i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. G l y c o p e p t i d e s as m a c r o p h a g e a c t i v a t o r s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. n e o g l y c o p r o t e i n b o r n e N - a c e t y l m u r a m y l d i p e p t i d e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.2. glycosylated p o l y m e r b o r n e N o a c e t y l m u r a m y l d i p e p t i d e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10 10 10 11

~ponding

author. Tel. (33) 38 51 55 37; Fax (33) 38 69 00 94.

Abbreviations: AGP - asialoglycoprotein or asialoorosomucoid; Ara-AMP = arabinofuranosyl adenine 5'-monophosphate; AZT 3'-azido 3'-deoxythymidine; BSA - bovine serum albumin; CD = calcium-dependent; CI = cation-independent; DAC = drug spacer arm conjugate; DALAC = drug acid-labile spacer arm conjugate; ddI = 2',3'-dideoxyribofuranosyl inosine; d4T = 2',3'-didehydro2',3'-dideoxythymidine; EC = endothelial cells; F = fluoresceinyl residue; FITC = fluoresceinylisothiocyanate; HIV = human immunodeficiency virus; HPP = 5'-phosphoribofuranosylhypoxanthine; HSV = herpes simplex virus; Lact = lactosyl residue = GalI34Glc-~-O-~p-NH-CS-; L D H = lactic dehydrogenase; LPS = lipopolysaccharide; Man6P = 6-phosphomannose; MDP = Nacetylmuramyldipeptide; ML = microsomal fraction; N K = natural killer; PC = parenchymal cells; PFU = plaque-forming unit; pl = isoelectric pH; pLK = poly-e-lysine; PMEA = phosphonomethoxyethyladenine; pSV2 luc = plasmid-containing luciferase gene under SV40 T antigen promotor; PTC = phenylthiocarbamyl residue; RES = reticuloendothelial system; RLU = relative light unit; SRBC = sheep red blood cells; TCA = trichloroacetic acid; T N F = tumor necrosing factor. 0169-409X/94/$27.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-409X(93)E0034-C

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1 ~ 4

4.1.3. antiviral activity ................................................................................................................ 4.2. Nucleoside analogues and derivatives as anti-infectious agents .......................................................... 4.2.1. Antiparasite agents ............................................................................................................ 4.2.2. Antiviral agents ................................................................................................................. 4.3. Oligonucleotides as drugs ............................................................................................................

11

12 12 12 13

5. Gene targeting ................................................................................................................................ 5.1. Transfection in the presence of chloroquine ................................................................................... 5.2. Transfection in the presence of fusogenic peptide ............................................................................ 5.3. Optimal conditions for transfection of HepG2 cells by pSV21uc/LactpLK complexes ............................ 5.4. Long term transfection ...............................................................................................................

16 17 20 20

6. Concluding remarks .........................................................................................................................

20

7. Acknowledgements ..........................................................................................................................

20

8. References ......................................................................................................................................

20

18

Abstract

Cell surface receptors are good candidates to selectively target drugs, oligonucleotides or even genes by making use of their specifc ligands. A large number of mammalian cells express cell surface sugar-binding proteins, also called "membrane lectins". Therefore, sugars may be used as specific recognition signals to specifically deliver biological active components. Tens of membrane lectins with different sugar specificities have been characterized; some of them actively carry their ligands to intracellular compartments, including endosomes, lysosomes and, in some cases, Golgi apparatus. In this review, we summarize the main properties of neoglycoproteins and glycosylated polymers; they have been developed to study the properties of endogenous lectins and to carry various drugs. Glycoconjugates have been successfully used to carry biological response modifiers such as N-acetylmuramyldipeptide. N-Acetylmuramyldipeptide is, in vitro, hundreds of times more efficient in rendering macrophages tumoricidal when it is bound to this type of carrier. In vivo, the N-acetylmuramyldipeptide bound to glycoconjugates containing mannose in a terminal nonreducing position, induces the eradication of lung metastases, occurring when treatment is started, in 70% of mice; free N-acetylmuramyldipeptide is strictly inactive. Similarly, N-acetylmuramyldipeptide bound to the same glycoconjugates induces an active antiviral effect. Glycoconjugates are also suitable for carrying antisense oligonucleotides specific for viral sequences. Antisense oligonucleotides protected at both ends and linked through a disulfide bridge to the glycoconjugates are 10 times more efficient than the corresponding free oligonucleotides. Poly-L-lysine containing about 190 lysine residues has been substituted by three components: sugars as recognition signal, antiviral (or antiparasite) agents as therapeutic elements and gluconoic acid as neutralizing and solubilizing agent. This type of neutral, highly water-soluble glycosylated polymer is a very efficient carrier to deliver drugs in infected cells according to the nature of the sugar borne on the polymer and to the specificity of the lectin present at the surface of the infected cells. Finally, poly-L-lysine (190 residues) partially substituted with sugars (60 units) is a polycationic glycosylated polymer which easily makes complexes with plasmids. These complexes are very efficient in transfecting cells in a sugardependent manner. The expression of reporter gene is greatly enhanced when cells are incubated with the plasmidglycosylated poly-L-lysine complex in the presence of either 100 IxM chloroquine or 10 I.tM fusogenic docosapeptide. Furthermore, this transfection method leads to a much larger number of stable transfectants than the classical method using calcium phosphate precipitate. The general properties of glycosylated proteins and of glycosylated polymers are presented and their efficiency in targeting genes in comparison with that of other available targeted transfection methods is discussed.

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

Key words: Allopurinol riboside; AZT; Cancer cells; Chloroquine; Drug carrier; Drug delivery; Endocytosis; Fusogenic peptides; Gene delivery; Gene targeting; Glycosylated poly-L-lysine; HIV; HSV; Leishmania; Macrophages; Membrane lectins; Neoglycoproteins; Oligonucleotides; PMEA; Transfection

I. Introduction

Site-specific drug delivery has been one of the main goals in therapeutics, since Paul Ehrlich's vision of targeted drugs as "magic bullets" for the eradication of diseases. The general aim in drug targeting by using macromolecular carriers is to increase the efficiency of a given drug by increasing its local concentration, and by preventing its excretion by renal filtration. Various carriers have been proposed such as: (a) resealed red blood cells or ghosts; (b) microspheres or nanoparticles; (c) neutral or negatively charged liposomes; (d) genuine proteins or enzymatically/chemically modified serum albumin, glycoproteins, hormones, antibodies etc.; (e) polymers which are not easily biodegradable by mam-

malian cells (such as polyacrylamide and derivatives or polyvinylpyrrolidone); and (f) biodegradable biopolymers (such as polyesters, poly-L-amino-acids or polysaccharides). Glycoconjugates (glycoproteins and glycolipids) are components of the plasma membrane of any mammalian cells. Sugar receptors are also plasma membrane components of many mammalian cells and are currently called lectins according to the definition given by Goldstein et al. [1]. The first membrane lectin was characterized on hepatocytes by Ashwell and Morell [for a review, see Ref. 2]. Endogenous lectins are found on many normal and malignant cells, and are involved in various biological functions, acting as specific receptors and/or mediating endocytosis of specific glycoconjugates [3-5].

Table 1 Endocytosis of glycoproteins: oligosaccharides recognized by membrane lectins Liver parenchymal cells: asialoglycoprotein receptor

Galfl 4GlcNAcfl 2Mane - - 6 ~ M a n f l 4GlcNAcfl 4GlcNAcfl Galfl 4GlcNAcfl 4 ~ Galfl 4GlcNAcfl 2 j M a n e

3/

Kupffer and liver endothelial cells: ( S 0 3 ) 4GalNAc receptors

(SO3-) 4GalNAcfl 4GlcNAcfl 2Mane 6 \ .Manfl 4GlcNAcfl 4GIcNAc ~ (SO3-) 4GalNAcfl 4GIcNAcfl 2Mane 3j Macrophages and Bver endothelial cells: mannose receptor

Mane 2Mane 6 \ 3 / M a n e 6\ Mane ;Manfl 4GlcNAcfl 4GlcNAcfl ~ Mane 2Mane 2Mane - - 3/ Various cells: CI Man6P receptor

(HPO3-) 6~Man e 6 \ Mane - - 2

Mane 6 Mane 3/

(HPO3-)

6,

Mane

2/

/ M a n f l 4GlcNAcfl 4GIcNAcfl / X,Man~ 2Mane 3/

Adapted from Drickamer, 1991 [6].

M. Monsigny et al./Advanced Drug Defivery Reviews 14 (1994) 1-24

Therefore, glycoconjugates specifically recognized by membrane lectins can be used as carriers of metabolite inhibitors, toxic drugs, biological response modifiers and genetic material.

2. Membrane lectins that mediate endocytosis The endocytosis of modified plasma proteins takes place inside the liver in different types of cells: the parenchymal cells (PC), the Kupffer cells and the endothelial cells (EC). These cells have different receptors that mediate uptake of various ligands [6] (Table 1). Besides the wellknown receptor for galactoside-terminated glycoproteins present on and in PC, EC express scavenger and mannose receptors that rapidly internalize their ligands [7]. Membrane lectins of these cells can be efficiently targeted by glycosylated carriers [see Refs. 8 and 9] because they are in close contact with blood. Monocytic lineage cells play various important functions as effectors of the immune system and are also involved in protection against several infectious diseases. Monocytes and macrophages express a number of surface molecules that enable

them to interact with invading micro-organisms, host cells and macromolecules as part of the response to injury or infection [10]. Several of these surface molecules are membrane lectins, i.e. sugarbinding proteins. The expression of membrane lectins depends on the stage of differentiation and on the state of activation (Table 2). The macrophages are unique in that they have two carbohydrate recognition systems which mediate internalization of lysosomal enzymes, namely a mannose receptor [11] and a mannose-6-phosphate receptor [12]. The macrophage mannose receptor is a 175-kDa membrane glycoprotein; this lectin is however absent in monocytes and in many precursor cell lines [13]. The human mannose receptor has been cloned [14,15]. Its expression is precisely regulated. It is constitutively internalized and recycled through coated pits and early endosomes; internalized ligands are separated from the mannose receptor, allowing the unoccupied receptor to return to the cell surface. Two mannose-6-phosphate receptors are expressed in macrophages: a cation-independent (CI) receptor of 270 kDa also known as the "insulin-like growth factor (IGF II) receptor" [for a review, see Ref. 16] and a low-molecular-weight,

Table 2 Macrophage lectins that mediate endocytosis Receptor and Mr in kDa

Ligand

Expression

Function

Mannosyl fucosyl (175)

Manc~ 6 or GIcNAc~ 6 on high mannose core

Macrophage, hepatic endothelium

Man6P CI (270)

Man6P

Macrophages, monocytes and other cells

Endocytosis, i.e. phagocytosis and pinocytosis, secretion of glycoprotein Endocytosis

ASGP C

Biantennary glycans

Macrophages, leukocytes

Endocytosis

fl-Glucan

f13 D-glucan on heat-killed yeast, zymosan glucan particles

Peritoneal macrophages, human monocytes

Endocytosis

Fucosyl (88 77)

L-Fucosyl-glycoconjugates, GaI-BSA

Rat Kupffer ceils

Endocytosis

Galactosyl (30)

Gal particles

Rat Kupffer cells

Endocytosis

Adapted from [10] and [20].

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1 24

cation-dependent (CD) mannose-6-phosphate receptor (Mr 45 000) involved in lysosomal enzymetrafficking [17]. The CI receptor was demonstrated on freshly isolated blood monocytes on the basis of the uptake of phosphorylated mannose ligands [18]. Macrophages can be efficiently activated to a tumoricidal state by N-acetylmuramyl-dipeptide (MDP) carried on specific glycoconjugates, thanks to their numerous cell surface membrane lectins. Similarly, specific glycoconjugates have been shown to be efficient carriers of antiparasitic and antiviral drugs. Monocytes are putatively interesting target cells, as they are the first cells invaded by various pathogenic micro-organisms. We have recently shown that macrophages [19], monocytes as well as their precursors and also monocytic cell lines (U937, THP1) bind and internalize a variant (AGPC) of orosomucoid (~l-acid glycoprotein) [20]. This variant, isolated upon elution with a mannose solution from an affinity chromatography gel containing immobilized concanavalin A, bears two biantennary and three trior tetra-antennary glycans. The variant AGPA, which passes through the affinity column, bears five tri- or tetra-antennary glycans but lacks biantennary glycans and is not recognized by this receptor. Other lectins have been characterized: a lectin specific for L-fucose in Kupffer cells [21] and a liver macrophage membrane lectin that mediates phagocytosis of particles bearing galactose residues in a terminal non-reducing position [22]. Other lectins, such as sialoadhesin [23], are involved in cell recognition and do not seem to induce endocytosis; such lectins are not suitable to target drugs with a system that requires a transfer to endosomes and to the cytosol or to lysosomes. However, these surface receptors in a tumor have to be considered for targeting drug in systems based on the release of the drug at the cell surface. The presence of membrane lectins can be demonstrated by using glycoproteins or neoglycoproteins [24]. Neoglycoproteins are prepared by covalent attachment of sugars to proteins such as serum albumin by using one of the following methods: coupling O-phenyldiazoglycosides to a protein such as serum albumin [25,26] or ferritin

and peroxidase [27,28]; coupling ~-alkylimidate glycosides to serum albumin [29,30]; coupling Ophenylisothiocyanate glycosides to serum albumin [31,32]; coupling oligosaccharide to a protein by reducing the imine formed between protein amino groups and the sugar in reducing terminal position [33] with sodium cyanoborohydride [34]; coupling an oligosaccharide to p-aminoethylaniline by the Gray procedure and then transforming the aromatic amine into an isothiocyanate [35]; or coupling an oligosaccharide containing sugar lactone [36]. Neoglycoproteins bearing different sugars or oligosaccharides are suitable for detecting and determining the specificity of either soluble or membrane-bound lectins; their apparent binding constant is much higher than that of the related free sugars or glycosides [30,37-39]. Furthermore, neoglycoproteins can be easily labelled by either radioactive or fluorescent probes [31,32,40]. The binding of a neoglycoprotein to a lectin increases relatively with the number of neoglycoprotein-bound sugars, but the binding is more specific when the number of sugars bound to a protein molecule is close to 20 [32]. When the number of sugars on the serum albumin molecule is much higher, the interaction between a neoglycoprotein and a lectin is less dependent on the type of sugar. Neoglycoproteins obtained by reaction of glycosyl oxyphenyl isothiocyanate on the ~-amino group of protein lysine residues have a lower isoelectric point than the parent protein, and may interact with polyanion receptors such as a scavenger receptor as pointed out by Jansen et al. [41]. However, with many cells the interaction between such neoglycoproteins and cell surface components is primarily determined by recognition of the sugar moieties by membrane lectin. Fluorescent neoglycoproteins were used to visualize cell surface lectins and to study their binding, internalization and intracellular degradation. In order to select more specific neoglycoproteins able to bind membrane lectins, cells are incubated in the presence of various fluoresceinylated neoglycoproteins either at 4°C or at 37°C for a few minutes and up to 2 hours. After washing, the quantity of cell-associated neoglycoproteins is determined either by spectrofluorometry [31] or by

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

flow cytofluorometry [32,42,43]. In control experiments, in order to ascertain the sugar specificity of the fluoresceinylated neoglycoproteins, cells were systematically incubated with sugar-free fluoresceinylated serum albumin: the fluorescence associated with cells was always extremely low, roughly at the level of the cell autofluorescence. This result confirms the fact that fluoresceinylated neoglycoproteins prepared according to Roche et al. [31] and Monsigny et al. [32] are not recognized by the fluorescein moeities but are recognized by the sugar moieties. However, in some cases, neoglycoproteins substituted with fluorescein or with other compounds could also be recognized by those other substituents [44]. Membrane lectins usually recognize specific complex oligosaccharides with a high affinity and recognize single sugars with low affinity. For instance, the asialoglycoprotein receptor binds Nacetyl lactosamine GalI34GlcNAc[3-R type oligosaccharides or glycopeptides as well as glycoproteins bearing such oligosaccharides [2]. The binding constant of oligosaccharides containing a single N-acetyl lactosamine residue is about 104 1 x mol-1, that of oligosaccharides containing two antennae ended by N-acetyl lactosamine is about 105 1 x mol-1, and that of oligosaccharides containing three antennae (Table 1) is about 107 1 x m o l - I [45]. Similarly, synthetic compounds containing galactopyranosyl residues in a terminal non-reducing position are recognized with low affinity when they have one galactose residue per molecule, with medium affinity when they have two galactose residues per molecule, and with high affinity when they have three or more galactose residues per molecule [46]. Therefore, a cluster of sugar residues such as: Gal[3-1inker-O-CH2 ~ Gal[Minker-O-CH2 - - C - R Gal[3-1inker-O-CH

gar, for several reasons. Two sugars that have in common the chemical groups which are involved in the interaction with the binding site, in exactly the same position will have similar affinity: for instance, wheat germ agglutinin recognizes glycoconjugates with GlcNAc or Neu5Ac residues in a terminal non-reducing position [47]. The ring of both sugars has the same structure, the acetamido on C2 of GlcNAc and the acetamido on C5 of Neu5Ac are identically disposed, as well as the hydroxyl on C3 of GlcNAc and on C4 of Neu5Ac. Other lectins accommodate pairs of sugars with such similarities: for instance, Glc and Man are recognized by concanavalin A [48]. As pointed out previously [49], L-fucose (6-deoxy-Lgalactose) may accommodate a binding site for Dmannose, and L-rhamnose (deoxy-L-mannose) may accommodate a binding site for D-galactose, because of their steric similarities. Furthermore, a binding site which recognizes complex oligosaccharides may accommodate two or more sugars which do not share any significant similarities. For instance, LECAM 3, also known as "P selectin", is a lectin (expressed by endothelial cells under some circumstances) which binds with high affinity the oligosaccharide, Lewisx [50]. This compound contains Gal, Fuc and GlcNAc residues: Gal134(Fuc~3)GlcNAc13~R. Both Gal and Fuc residues are involved in the interaction with the binding site, and both neoglycoproteins containing Gal residues and neoglycoproteins containing Fuc residues are recognized by that lectin. Conversely, Lewis x, which contains a galactose residue in a terminal non-reducing position, is not a good ligand for other lectins which recognize oligosaccharides terminating with a galactose residue linked to otherwise non-substituted glucose or to N-acetylglucosamine: Gal[34Glc13--*R or GalI34GicNAc--*R.

2/

in a correct organization is a better ligand than isolated residues. The high affinity of neoglycoproteins containing single sugars is, to some extent, also related to the behavior of sugar clusters. Because lectins recognize complex oligosaccharides they recognize more than one type of su-

3. Neoglycoproteins or glycosylated polymers as carriers

A therapeutic agent, in order to be active, must reach its target cell and more precisely the correct compartment of the targeted cell. For a great majority of drugs, the required compartment is either

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

the cytosol or the nucleoplasm. A hydrophobic

endocytosed by liver parenchymal cells, by macro-

aru orno nlnomnmomrnnotn 0nt0r PhO!O ndInlinl olnr0n0

the cytosol, while a hydrophilic drug is not membrane-permeant and will possibly enter the cytosol from an intracellular organelle. On these bases, drug targeting may enhance the efficacy of a hydrophilic drug by carrying such a drug into the endocytotic compartment. The rationale of drug targeting using macromolecular glycoconjugates (see Table 3) includes the following considerations: (1) A low-molecular-weight hydrophilic drug is usually lost from the circulation in vivo within 30 minutes, while bound to a macromolecular carrier it may stay longer in the blood if the carrier is not rapidly endocytosed. When the carrier is actively

very fast. However, the drug is not excreted in the urine, but rather is concentrated in specialized organs. (2) A macromolecular carrier containing carbohydrate moieties as a recognition signal will be recognized by cell surface receptors (lectins) with a specificity depending on the nature of the carbohydrate moiety used and on the type of lectins present on the target cell. Therefore, the amount of drug at the level of the target cell will he much higher than that obtained when the drug is free. (3) A carbohydrate moiety used as a recognition signal allows the binding and the internalization of the drug-carrier conjugate because membrane lectins usually mediate endocytosis.

Table 3 Overview of glycoconjugate-based drug targeting

Carriers Glycoproteins (asialo-orosomucoids, asialofetuin, fl-glucuronidase etc.) Neoglycoproteins (lactosaminated albumin, mannosylated and other glycosylated albumins) Glycosylated hydrosoluble polymers Rationale Endogenous lectins (membrane sugar binding proteins) are expressed in various animal cells: (a) Their sugar specificity depends on the nature and on the differentiation state of the cells (b) They actively mediate endocytosis of their ligand Drug-carrier linkages Direct: Amide, ester.., bonds Indirect: Drug-arm-carrier (DAC): Peptide Drug-acid-labile-arm-carrier (DALAC): eis-Aconitic acid derivatives C-Ribofuranomaleic acid derivatives Phenylthiocarbamyl aspartic acid derivatives Hydrazone Through disulfide bridge Carried drugs Anticancer agents: Daunomycin, adriamycin, methotrexate, gelonin etc. Biological response modifiers: N-Acetylmuramyl dipeptide (MDP) and analogues Antiviral agents: Nucleoside and nucleotide analogues: fluoronucleotides, Ara-AMP, AZT, d4T, ddI, PMEA, ribavirin Antisense oligonucleotides Antiparasitic agents: Anti-malarial: Primaquine Anti-leishmania: Allopurinol riboside, methotrexate Genes Ara-AMP = arabinofuranosyl adenine-5'-monophosphate; AZT - 3'-azido-2',Y-dideoxythymidine; d4T - 2',Y-didehydrothymidine; ddl - 2',3'-dideoxyribofuranosylinosine; PMEA = phosphonomethoxyethyladenine.

M. Monsigny et al./Advaneed Drug Delivery Reviews 14 (1994) 1 24

(4) The drug can be attached to the carrier in such a way that it will be linked until it is released in a selected organelle inside the target cell. The endosomal compartment is a complex system starting at the level of plasma membrane as pits and vesicles; then endosomal vesicles migrate in the cytosol, and fuse with other vesicles and/or early endosomes. From early endosomes, other vesicles are detached and migrate to other compartments including Golgi apparatus, late endosomes, prelysosomes and putatively to endoplasmic reticulum and to the intermediate compartment between endoplasmic reticulum and Golgi apparatus. The various organelles including endoplasmic reticulum, the various compartments of the Golgi apparatus and of the endosomal system contain enzymes which are very precisely located in one of these compartments, such as cathepsin D in endosomes, specific glycosidases in endoplasmic reticulum, in cis-Golgi, and medium-Golgi, specific transferases in cis-, medium-, trans-Golgi and in trans-Golgi network, various hydrolases in lysosomes, etc. In addition, endosome compartments have two interesting properties: (1) their luminal pH goes from 7.4 (early vesicles) down to 6 or less in endosomes and late vesicles; (2) they contain reducing agents. In the light of those properties, it is clear that the drug may be specifically released from the carrier in a defined compartment according to the nature of the linkage between the drug and the carrier. 3.1. Neoglycoproteins as carriers

Neoglycopi'oteins, such as glycosylated serum albumin, can be further substituted with a few molecules of drugs. In fact, neoglycoproteins substituted with various drugs have been prepared and found to increase the efficiency of the carried drug in both in vitro and in vivo experiments [for reviews, see Refs. 4, 8 and 51-56]. One of the difficulties encountered during the preparation of drug-neoglycoprotein conjugates is that a neoglycoprotein is not soluble in organic solvents and the yield of the linking step is usually rather low. Furthermore, the use of natural proteins (other

than the recombinant human proteins) to make a therapeutic agent can be a source of trouble because of viral and bacterial contamination. In addition, drug-neoglycoprotein conjugates as well as neoglycoproteins may in principle induce the production of antibodies, and thus undesirable side effects may occur upon repeated injections. However, little immunogenicity was reported when homologous proteins such as lactosaminated murine albumin substituted with Ara-AMP and used in mice [57]. 3.2. Glycosylated polymers as carriers

Besides neoglycoproteins, many other macromolecular systems have been proposed as drug carriers. Various polymers are in use as drug vehicles [for reviews, see Refs. 58 and 59]. Among the desirable properties of such polymers, the basic ones are proper biocompatibility, sterilizability, water solubility, biodegradability and low immunogenicity. In experimental cancer therapy, the linkage of various toxic drugs to polycationic polypeptides has already been described [60]. However, the use of polycationic polypeptides leads to strong non-specific binding to cells because the cell surface contains a large number of electronegative charges. As a consequence, such polycationic polypeptides are also cytotoxic to non-tumor cells [61]. To improve the chemistry of the drug-glycosylated carrier conjugate and to make these carriers suitable for in vivo use, we developed a new glycosylated carrier, namely a poly-L-lysine derivative in which ~-amino groups are partially acylated by reaction with 6-gluconolactone, while the other amino groups are substituted by recognition signals (e.g. carbohydrate residues) and by drugs (Fig. 1) [62]. The poly-L-lysine based carrier is easily prepared with a high yield because the various chemical reactions can be conducted in organic solvent. The conjugate is highly water soluble because half of the lysine residues are substituted with a polyhydroxyalcanoyl moiety, such as a gluconoyl group. The main properties of glycosylated polymers are compared with those of previously developed neoglycoproteins in Table 4.

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1 24

9

0

C I"1=O H.40~. HO 0

H

OH.

o A/~--°-t,,o,,J _~ HO

°

HO-~co .

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H N I R

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V~N/~c0 H .

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Jl__

HN" ! R

Fig. 1. Chemical structure of a glycosylated poly-L-lysine based carrier of AZT. Recognition signal: ct-mannopyranosyloxyphenylacetyl-glycyl-glycyl. Water solubilizing and neutralizing substituent: gluconoyl. Antiviral drug: succinyl-5'-AZT.

3.3. Immunogenieity of neoglycoproteins and glycosylated polymers Homopolymers such as poly-L-lysine are known to be immunogenic in most animal species

[63,64] and their use as drug carriers may be limited. The antigenicity of neutral gluconoylated polymers (GlcA-pLK) bearing mannose residues as recognition signal, N-acetylmuramyldipeptide (MDP)

Table 4 Glycosylated macromolecular drug carriers

Starting material

Serum album&

Poly-L-lys&e

Available NH2 groups

-~ 57

-~ 190

Substituted material

Neoglycoprotein

Glycosylated polymer

Structure Form Size(nm) Mr (Da) Yield of linking (%) Solubility (mg/ml) Synthesis solvent Sugar derivative Sugars per molecule Drugs per molecule pl

globular spherical d = 6 -~ 68 000 ~ 50 -~ 100 aqueous osidyl PTC -~ 25 5-20 ~ 4

or-helix cylindrical d - 3; 1 = 30 -~ 80000 -~ 95 > 200 organic osidylphenylacetate -~ 50 10-80 neutral a

up to 100 partial _+ + + or _+b +

up to 100 total _ --

Biological efficiency Carried/free drug Biodegradability Unspecific cell binding lmmunogenicity Antigen presentation aAll remaining bHeterologous albumin is not d = diameter;

NH2 groups are acylated. serum albumin is highly immunogenic, i.e. glycosylated bovine serum albumin in mice, whereas homologous serum immunogenic, i.e. glycosylated mouse serum albumin in mice [57]. 1 = length; PTC = phenylthiocarbamyl.

10

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1 24

as macrophage activator and fluorescein residues was compared, as positive controls, with the antigenicity of neoglycoproteins, i.e. bovine serum albumin substituted with the same components, namely mannose, M D P and FITC (F). Conjugates were injected subcutaneously into mice and the production of antibodies was determined by ELISA (enzyme-linked immunoadsorbant assay) 1 week after the third injection. F4-, Man30-, MDP20-, GlcAIz0-pLK (polylysine substituted with mannose, MDP, FITC and gluconoyl residues) induced no antibody production while neoglycoproteins, derived from bovine serum albumin, induced a strong immune response in mice: antibodies reacting against fluorescein were detected in the serum of mice injected with F4-, Man20-, MDP10-BSA (bovine serum albumin sub0.7

3 06

0.5 \× \.'(

0.4 ob o.3

o.2

1

2:

o.1

\x \x \x \x \x \× \x \x \x \× \× \x

.--)~

o.o ABCD

ABCD

ABCD

wells Fig. 2. Detection of antibodies in serum from mice tentatively immunized with various glycosylated polymers. Mice received subcutaneously (300 lag in 100 ILl): (1) poly-L-lysine substituted with 4 fluorescein residues, 30 mannose residues and about 140 gluconoyl residues; (2) poly-L-lysine containing 4 fluorescein residues, 30 mannose residues, 20 N-acetylmuramyl dipeptide moieties and about 120 gluconoyl residues; (3) bovine serum albumin bearing 4 fluorescein residues, 20 mannose residues and 10 N-acetylmuramyl dipeptide moieties. Wells were coated with: (A) poly-L-lysine; (B) poly-L-lysine substituted with 30 mannose residues; (C) poly-L-lysine bearing 10 fluorescein residues; (D) poly-L-lysine bearing 10 fluorescein residues and 30 mannose residues. The ELISA was clearly positive with sera from mice immunized with bovine serum albumin conjugates used as controls; in 3C and 3D, sera contained antibodies against fluoresceinyl residues. These antibodies did not react with either mannose (3B) or poly-L-lysine itself (3A).

stituted with mannose, M D P and FITC) (Fig. 2). However, as above mentioned, neoglycoproteins, made with murine serum albumin, are hardly immunogenic in mice [57]. Therefore, by using either the neutral glycosylated poly-L-lysine derivatives or neoglycoproteins made from autologous serum albumin, the carriers are not immunogenic.

3.4. Drug-carrier linkage The main types of drug-carrier linkage which have been developed and the various drugs successfully carried by glycoconjugates are presented in Table 3. The drug-carrier linkage may be: (a) Cleavable in acidic medium (in the lumen of endosomes or lysosomes) as in the case of acidolabile heterobifunctional links: e.g., maleic acid derivatives such as cis-aconitic acid [65,66], C-ribofuranomaleic acid [4,67], phenylthiocarbamyl acidic s-amino acid derivatives leading to DALAC (drug acid-labile spacer arm conjugate) such as with p-benzylthiocarbamoyl-aspartic acid [68]; or (b) Cleavable under reducing conditions as in the case of disulfide bridges; gelonin linked through an - S S - bridge to glucosylated serum albumin is 100 times more active than free gelonin in killing Lewis lung carcinoma cells [31]; oligonucleotides bound to mannosylated serum albumin through an SS bridge are stable in the incubation medium but rapidly released inside the cells [69]; or (c) Cleavable through the action of an endosomal or lysosomal enzyme as in the case of peptidyl linkers as already described (DAC = drug spacer arm conjugate [70,71,72].

4. Drug targeting

4.1. Glycopeptides as macrophage activators 4.1.1. Neoglycoprotein-borne N-acetylmuramyldipeptide (MDP) Reticuloendothelial cells, including free circulating monocytes, as well as macrophages present in various tissues, can be activated and rendered tumoricidal by targeting immunostimulating

M. Monsigny et al./AdvaneedDrug Delivery Reviews 14 (1994) 1-24 drugs such as N-acetylmuramyldipeptide (MDP). Free M D P upon intravenous injection is rapidly cleared from the circulation, mainly by renal filtration and consequently is inefficient in rendering macrophages tumoricidal in vivo. M D P covalently bound to serum albumin, a 68 000 Mr protein, stays longer in the blood than free MDP, but is still not efficient. M D P bound to mannosylated serum albumin is not cleared through the kidney and is more efficient than free M D P both in vitro and in vivo. The covalent attachment of M D P to BSA was performed by carbodiimide-mediated formation of an amide linkage between the glutamyl-y-carboxylic group of M D P and the c-amino group of lysine residues of the protein carrier [73]. The conjugate (MDP-BSA or MDP-, Man-BSA) was purified by gel chromatography. The number of M D P residues per carrier molecule was estimated according to the colorimetric method developed by Levvy and McAllan [74]. The absence of LPS contaminants (less than 0.1 ng/mg) was checked using a previously described method [75]. The in-vitro cytostatic activity of rat (or mouse) alveolar (or peritoneal) macrophages requires at least 100 times less neoglycoprotein-bound M D P than free M D P [73]. Furthermore, neoglycoprorein-bound M D P is much more efficient than free M D P in inducing the secretion of interleukin-1 and cytotoxic factors in in-vitro experiments [76]. In-vivo experiments gave even more interesting results [77]. In fact, upon injection of Lewis lung carcinoma cells in a rear leg of C57BL/6 mice, the mice develop a sizable tumor within 14 days. At that time, the lungs are already colonized and small metastatic fauci are easily detected. Upon excision of the primary tumor, 80% of mice die within 10 days if they receive either no treatment or a placebo, or a solution of free MDP, or a solution of the MDP-free carrier, mannosylated serum albumin. Conversely when mice, upon excision of the primary tumor, were treated by intravenous injections of MDP11-, Man25-BSA, the development of lung metastasis was reversed in 70% of the m i c e , 15% developed 2-5 lung metastases and 15% died after a few weeks. More than 70% of treated mice survived after 100 days. A similar result was obtained with beige mice which are deficient in N K (natural killer) cells, showing that

11

N K cells are not responsible for this metastatic reversion. 4.1.2. Glycosylated polymer-borne M D P 4.1.2.1. Cytotoxic activity Poly-L-lysine can be easily substituted both with biological response modifiers such as MDP, and with sugar derivatives such as mannoside or 6-phosphomannoside (Man6P) to allow the targeting of biological response modifiers to macrophages or monocytes. This targeting is cell-specific and can be used in vivo provided the number of residual positive charges is very low. M D P bound to poly-L-lysine substituted with mannosyl and gluconoyl residues was nearly as efficient as M D P bound to serum albumin substituted with mannosyl residues to activate macrophages to become cytotoxic [62]. The mannosylated conjugates with acetyl groups were less efficient than the mannosylated conjugates with gluconoyl groups in rendering macrophages cytotoxic. This result is consistent with a lower capacity of the former conjugate to inhibit the agglutination of red blood cells by concanavalin A. More surprisingly, the targeted M D P polylysine conjugate GlcA140-, Mans0-, MDPz0-pLK did not induce the release of soluble T N F as did the targeted MDP-BSA conjugate [76]. The cytotoxic activity induced by M D P bound to the glycosylated polymer is however related to a membrane-associated TNF, because anti-TNF antibodies reduced the cytotoxicity. 4.1.2.2. Stimulation of R E S functions Mice treated intravenously with 10 ~tg of M D P coupled to mannosylated and gluconoylated poly-L-lysine, 2 days before the intravenous inoculation of chromium-labeled sheep red blood cells (SRBC) were able to clear SRBC's from their circolation at a much higher rate than mice receiving carrier alone or free M D P (Fig. 3). 4.1.3. Antiviral activity Glycoconjugate-borne N-acetylmuramyldipeptide partially protects mice against death from hepatitis upon infection with HSV1 (MB strain). Virus, administered intravenously, induced within 2 or 3 days, both a viremia and a fulminant liver

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1 24

12

infection (Fig. 4). Conversely, intravenous administration of MDP bound to mannosylated serum albumin or to gluconoylated and mannosylated polylysine resulted in 40 and 60% survival, respectively. A similar protective effect was observed when mice infected with a HSV1 strain inducing pneumonitis were treated with the same conjugates. Control experiments including treatments with free MDP, with the carrier alone or with a mixture of both had no protective effect. 4.2. Nucleoside analogues and derivatives as antiinfectious agents

100

~X

75

50

25

'/ i

4

Glycosylated polymers can be substituted with various anti-infectious agents. 4.2.1. Anti-parasite agents Mannosylated polymers have been used to target allopurinol riboside to Leishamania-infected macrophages via the mannose-fucose specific membrane lectin. Briefly, O-p-phenyl-isothiocyanate 5'-phosphodiester allopurinol riboside was synthesized by reaction of allopurinol riboside

.

.

.

.

7

. . . . . . . . . .

8

•

12

I

I

16

20

24

DAYS POST INFECTION Fig. 4. Treatment of HSV-1 induced hepatitis with MDP conjugates. All mice received 1 LD80 of HSV-1, MB strain, intravenously on day 0. Two days prior to infection, on the day and 2 days following infection, mice received 10 lag of either free MDP ( i ) or conjugated MDP: MDPaa-, Man33-BSA (O); MDP47-, Man2s-, GlcAjls-GlyGIypLK (O). Ten mice per group were evaluated by Wilcoxon rank analysis. [Unpublished data, in collaboration with D. Gangemi, A. Ghaffar, E. Mayer, Columbia, SC., USA.]

with p-nitrophenyl phosphodichloridate to give Op-nitrophenyl-5'-phosphodiester allopurinol riboside, which was reduced and further reacted with thiophosgene to give the phenylisothiocyanate 5'phosphodiester derivative of allopurinol riboside. This compound, bound to a gluconoylated- and mannosylated poly-L-lysine was 80 times more active than the free drug in vitro [78].

L

Z o m [-. Z \\

--D \

o u

\ \\

\

O,

o

2m 2 10 0

I

I

i

I

I

2

4

6

8

10

TIMES AFTER INJECTION (minutes)

Fig. 3. Clearance of sheep red blood cells (SRBC). 10 lag of free MDP (O) or of MDP bound to 100 lag poly-L-lysine bearing 40 mannose residues ( 0 ) or the carrier alone (100 lag of poly-Llysine bearing 40 mannose residues) (D) were injected intravenously 2 days prior to inoculation of chromium-labeled sheep red blood cells. In all cases, the remaining amino groups were gluconoylated [unpublished data in collaboration with D. Gangemi, A. Ghaffar, E. Mayer, Columbia, SC, USA].

4.2.2. Antiviral agents Antiretroviral nucleoside analogues [for a review, see Ref. 79] have been coupled to glycosylated polymers. Fiume and co-workers showed that arabinopyranosyl adenine (Ara-A) and arabinopyranosyl adenine 5'-monophosphate (AraAMP) were efficient in inhibiting Ectromelia virus DNA synthesis in hepatocytes when these drugs are coupled either on asialofetuin [80] or lactosaminated serum albumin [81]; both carriers are recognized by the asialoglycoprotein receptor of the liver parenchymal cells. Ara-AMP conjugated with lactosaminated serum albumin also efficiently inhibits hepatitis B virus replication [8,81]. Succinyl-5'-azidothymidine (succinyl-AZT)

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

linked to the amino groups of glycylglycyl units used as spacers bound to mannosylated poly-Llysine was shown to be more efficient than free AZT or free succinyl-AZT against HIV multiplication in human macrophages (Fig. 5). To be active, AZT must be phosphorylated. In fact, the low efficiency of AZT, as well as of other 2',3'dideoxynucleotides, in inhibiting HIV replication in cultured human macrophages has been reported [83] to be associated with a low thymidine kinase activity in these cells; conversely, in lymphocytes and especially in activated lymphocytes, thymidine kinase activity is high. Thymidine kinase activity in monocyte-derived macrophages infected with HIV is even lower: 2.2 pmol/mg/min compared with 3.2 pmol/mg/min for uninfected macrophages, 66 pmol/mg/min for activated peripheral blood mononuclear cells infected with HIV, and 104 pmol/mg/min for activated periph50

40 co

I o 30

20

%)

10

-8

-7

-8

9

i0

log c o n c e n t r a t i o n (M) Fig. 5. Reverse transcriptase activity in macrophages infected with HIV-1 upon treatment with free or conjugated AZT. Human blood monocytes obtained by elutriation were cultured for 7 .days in AIM medium (106 cells/well in 24 well plate) and infected with a macrophage tropic HIV-1 strain (D117 III). After virus adsorption (5 h), the various conjugates were added. The cultures were maintained with the drugs during the whole experiment; drugs were replaced twice a week. AZT (11), succinyl-5'-AZT (1-]), succinyl-5'-AZT21-, Manss-, GlcAlla-pLK ([]), phosphoro-5'-AZT20-, Man60-, GlcAi10pLK ([~). Without any treatment, the reverse transcriptase activity was 30 000 cpm/ml. In this typical experiment, reverse transcriptase activity was measured in the supernatant after 10 days of culture. [Unpublished data, in collaboration with A.M. Aubertin and A. Kirn, Strasbourg, France.]

13

eral blood mononuclear cells [84]. Furthermore, AZT is not catabolized in monocyte-derived macrophages and the slow decrease in 5'-monophosphate AZT cellular content is related to its excretion [84]. Instead of using succinylated AZT, phosphorylated AZT can be used. Two different phosphorylated derivatives have been synthesized; one contains an arylphosphodiester: R-CO-CH2-NH-CS-NH-q)-O-PO~ -5' AZT the other contains an alkylphosphodiester [85]: R-CO-CH2-NH2-CO-CH2-CH2-CO-NH-(CH2)3O-PO 2 -5' AZT where R is a glycine residue, or a glycyl residue bound to an e-amino group of poly-L-lysine. Conjugates with phosphoryl-AZT were more active than the conjugate bearing succinyl-AZT (Fig. 5). Independently, Meijer and co-workers prepared phosphorylated AZT-neoglycoprotein conjugates [86] by direct coupling of AZT monophosphate to the amino groups of the neoglycoprotein leading to a phosphoramide linkage [87]. This phosphorylated AZT-neoglycoprotein was shown to be efficient in protecting MT4 cells against the cytopathicity induced by HIV-1, in in-vitro experiments [56,86]. PMEA (phosphono-methoxy-ethyladenine) which inhibits herpes virus multiplication in macrophages is at least 50 times more active than free PMEA when it is linked to the mannosylated polymer [88]. Conversely, when drugs are linked to the recognition signal-free carrier, their activity is much lower than that of free drugs. These data show that even low molecular weight antiviral drugs linked to a carrier are more active than free drugs when the conjugate is efficiently taken up by cells, thanks to the surface receptors that bind the recognition signal borne by the carrier. The PMEA conjugate is also more active in vivo than free PMEA, as shown by treatment of HSV-l-infected mice (Fig. 6). 4.3. Oligonucleotides as drugs

Antisense oligonucleotides are putative antitu-

14

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24 3

2

(3

(3

/

control

free

conjugate

Fig. 6. Pulmonary HSV-1 titers in mice treated with free or conjugated PMEA; 3- to 4- week-old C3H/Hen mice were infected intranasally with 5000 Pfu (l LDg0) of HSV-1, VR3 strain of herpes virus. On the day of infection, mice received intravenous therapy with saline (control), or l0 mg/kg free PMEA or conjugated PMEA (PMEA-GIyGly:o-, ManGlyGly55-, AcGlyGly6-, GlcAllo-pLK (conjugate)). Mice were again treated on day 2 and day 4 post infection. The lungs were removed and 10% homogenates were prepared in RPMI. Viral infection was determined by standard plaque assay on Vero cells. PFU = plaque-forming unit. [Unpublished data, in collaboration with D. Gangemi, A. Ghaffar, E. Meyer, Columbia,

sc, USA.] mor and antiviral agents [for a review, see Ref. 89]. These agents are very sensitive to exonucleases, but can be derivatized at both 3'- and 5'ends in order to protect them. Currently, oligonucleotides specific for target sequences are substituted with a thiol group at one end and a fluorophore at the other. Oligonucleotides are polyanionic and highly hydrophilic. They are taken up by cells in vitro but only when they are used at relatively high concentration (usually from 1-20 tIM). In order to make them more efficient, oligonucleotide derivatives were linked to neoglycoproteins through a disulfide bridge, allowing the release of the nucleotide in the endosomic vesicles. By using radiolabeled or fluoresceinylated oligonucleotides bound to specific glycosylated carriers (Man-BSA or Man6P-BSA), we have shown that after 2 hours of incubation at 37°C the oligonucleotide concentration associated with cells was significantly increased in comparison with free oligonucleotides or oligonucleotides bound to unglycosylated BSA. The intracellular concentration of oligonucleotides carried by the neoglycoproteins was 4-

to 5-fold higher than the extracellular concentration whereas it was one fifth after incubation with free oligonucleotides [69]. Oligonucleotides bound to 6-phosphomannosylated serum albumin is internalized even 20-40 times more efficiently than free oligonucleotides. Alternatively, oligodeoxynucleotides substituted with biotin are efficiently carried by glycosylated streptavidin; this is an easy way to increase the intracellular concentration of oligonucleotides [90]. Oligonucleotides were modified at the 5'-end by coupling a fluoresceinyl group and at the 3'-end by coupling a biotinyl group via a disulfide bridge [F-5'-oligonucleotide-3'-SS-biotin]. The majority of the oligonucleotides were concentrated in vesicular compartments as shown by confocal microscopy (Fig. 7) and confirmed by subcellular cell fractionation (Fig. 8). Similar patterns were obtained with both ~-(T12) and [3-oligonucleotides (19-mer). 60% of the oligonucleotides targeted by a glycosylated carrier was recovered in the microsomal fraction (ML). However, 20-25% of the oligonucleotide was found in the cytosolic fraction; after 2 hours, oligonucleotides were released from the carrier inside the cells in an undegraded form. These results were obtained by electrophoresis analysis of radio-iodinated oligonucleotides [91]. Briefly, oligonucleotides were bound to the glycosylated carrier by their 3'-end through a disulfide bridge and labelled on their 5'-end by a radioactive iodine-substituted tyraminyl residue. Upon 4 hours incubation in the complete medium, the radioactivity was found at the level of the oligonucleotide-neoglycoprotein conjugate. Conversely, upon endocytosis, the major part of the radioactivity was found at the level of the free oligonucleotide, indicating that the oligonucleotide had been released from the carrier and had not yet been degraded [69]. In the cytosolic fraction, the radioactivity migrates at the level of the free oligonucleotide, the cytosolic fraction did not contain any significative amount of lysosomal enzymes as shown by the lack of acidic N-acetylglucosaminidase [91]. However, we cannot exclude the possibility that some oligonucleotides in the cytosolic fraction arise from partial breakdown of the endosomal fraction. The presence of oligonucleotides in the cytosolic fraction is important

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

1

15

2

3

4

Fig. 7. Intracellular localization of F-aTe2 and F-19mer oligonucleotides by confocal microscopy. J774E cells (mouse macrophage lines) were incubated for 24 hours at 37°C with 0.5 p.M free F-~TI2 (1), with 0.5 IxM F-~Tt2 bound to Man6PI6-BSA (2), with 5 p.M F19mer (3) or with 0.5 p.M F-19mer bound to Man6PI6-BSA (4). Scale bar = 10 p.m. Left: phase contrast. Right: fluorescence image.

since the targets of the oligonucleotides are in the n u c l e u s a n d / o r in t h e c y t o s o l . T o g e t b e t t e r effic i e n c y in t a r g e t i n g o l i g o n u c l e o t i d e s we m u s t f i n d a way to increase the cytosolic concentration. T h i s is b e i n g d e v e l o p e d in o u r l a b o r a t o r y b y using fusogenic peptides and peptides changing 90

o ~'~ CD

70

X Oq

6O 5O

E-~ ~3 I

40 .%O

E-* 2O

~:)

10 0

N

ML

P

S

Fig. 8. Subcellular distribution of oligonucleotides. Macrophage J774E cells, which express both the Man and the Man6P receptors, were incubated for 2 hours at 37°C with 0.2 pM radiolabeled free ~Ti2 oligonucleotide (11), 0.2 p.M radiolabeled ctTI2 bound to BSA (D), tO Man-BSA (k~), or to Man6PBSA ([]), (oligonucleotide/neoglycoprotein molar ratio = 2). After removal of the supernatant and extensive washing, cells were homogenized and submitted to differential centrifugation (800 x g) for 15 min to obtain the postriuclearfraction, and for 30 min to obtain the MLP fraction. This fraction~ after 1 hour centrifugation at 100 000 g, gave a fraction corresponding mainly to mitochondria and lysosomes (ML), a microsomal fraction (P) and a supernatant (S) corresponding to the cytosolic components. Fractions were characterized by their enzymes content: lactic dehydrogenase (LDH) in cytosol and N-acetylglucosaminidase in lysosomes. The radioactivity was monitored in each fraction after TCA precipitation in the presence of BSA (1 mg/ml). All the radioactivity was recovered in the precipitate as carrier-free oligonucleotides. *Tya = iodine-labeled tyraminyl moiety.

16

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1 24 30 25

@

0 "0

"'0 @

20 M

15

0 @

~0

5

0 09 09 co

>,

2

2

.~

09

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Fig. 9. Biodistribution of oligonucleotides, ctT12 oligonucleotides substituted on the 5'-end with a radioactive label (12sI) and on the 3'-end with a thiol function were injected intravenously into mice either in their free form (11) or coupled onto BSA ([--1), Man6P-BSA (k~) or M a n - B S A ([]), (oligonucleotide/neoglycoprotein molar ratio = 2). After 2 hours, mice were sacrificed and the radioactivity of each organ was determined (experiments in triplicate). [From Ref. 90.]

the intracellular traffic of internalized oligonucleotides. In vivo, the biodistribution of free oligonucleotides protected at their 3'- and 5'-end, and oligonucleotides bound to glycosylated carriers were also studied (Fig. 9); 2 hours after intravenous injection, the oligonucleotides were more abundant in liver and in spleen than when the same oligonucleotides were injected as free oligonucleotides. Mannosylated serum albumin is well recognized by macrophages and apparently directs oligonucleotides to liver and spleen which are organs rich in cells of the macrophage lineage. The highest concentration of oligonucleotides in those tissues was obtained in particular with mannosylated BSA as carrier.

5. Gene targeting Glycoconjugates have been shown to be efficient in targeting therapeutic drugs and oligonucleotides. On this basis, glycoconjugates could be also used to increase the efficiency of gene trans-

fer. In fact, a glycoconjugate that specifically binds to a membrane lectin allows a carried component to become concentrated inside endosomal compartments as shown above with the targeting of oligonucleotides. In the case of polynucleotides, it is easy to prepare stable conjugates simply by mixing a solution of a polynucleotide and a solution of a polycation such as polylysine; there is no need to make a covalent bridge between two polymers of opposite charges. Based on this property, various authors prepared polylysine-plasmid complexes and made use of recognition molecules such as transferrin [92,93], insulin [94] or asialo-orosomucoid [9,95] to help the polylysine-plasmid complex to enter into cells expressing the related receptor. Along that line, we made use of partially glycosylated polylysine to selectively transfer plasmid into cells expressing a membrane lectin able to recognize the sugar moieties attached to the glycosylated polylysine [96]. Poly-L-lysine (containing about 190 lysine residues) was substituted with 60 lactosyl residues (LactpLK) and used to form stable complexes with various plasmids (including pSV21uc which contains the luciferase gene under the control of the SV40 promotor) in order to target these plasmids to hepatocytes. Glycosylated polylysine is simply prepared by reacting galactopyranosyl 134-glucopyranosyl- 13-4-oxyphenylisothiocyanate with the p-toluene sulfonate salt of poly-L-lysine in dimethylsulfoxide in the presence of diisopropylethylamine. The glycosylated polymer is precipitated by adding 10 volumes of isopropanol, and the precipitate, dissolved in a buffer, is ready to use. The glycosylated polymer-plasmid complex is prepared by mixing a glycosylated polymer solution and a plasmid solution, there is no need for a chaotropic agent in contrast to the methods developed by Wu and Wu [95] and by Wagner et al. [92]. The number of sugar moieties on the poly-Llysine determines, at least in part, the conditions of preparation of the glycosylated poly-L-lysineplasmid complex, the efficacy of uptake by cells expressing a lectin able to bind these sugar moieties, and the expression of the related protein. With HepG2, the highest active enzyme (lucifer-

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

ase) expression was obtained when the glycosylated polymer contained 60_ 5 lactosyl residues. With a higher sugar content, the complex was not stable enough, and overall activity was lower [unpublished data]. The glycosylated poly-L-lysine/ plasmid ratio necessary to quantitatively form the complex increases from the sugar-free poly-L-lysine up to a glycosylated poly-L-lysine bearing 100 sugar moieties, in relation to the number of cationic charges left on the glycosylated polymer. The high efficacy (see below) of the glycosytated poly-L-lysine-plasmid complex with an optimal number of sugar moieties may also be related to the capacity of the complex to release the plasmid inside the targteted cells. This point is currently under investigation. In vitro, the hepatoma line HepG2, which expresses a membrane lectin able to bind and to mediate the uptake of 13-galactose-terrninated glycoproteins, efficiently takes up a DNA complexed with lactosylated polylysine but not a DNA complexed with a sugar-free polylysine. A fluoresceinylated lactosylated polylysine-plasmid complex incubated for 4 hours at 37°C with HepG2 cells is found in acidic compartments as evidenced by a 6-fold enhancement of cell fluorescence intensity upon post-incubation with monensin at 4°C (Fig.

17

10a). Monensin, a H+/Na ÷ ionophore, is known to neutralize all intracellular acidic compartments [97,98]; the fluorescein fluorescence is always quenched in acidic medium and reaches a maximum intensity at neutral pH [99,100]. When HepG2 cells were incubated for 4 hours at 37°C with a pSV21uc/LactpLK complex (3 nM DNA and 330 nM polymer), the luciferase activity measured 48 hours later was 7 x 103 RLU for 106 cells which is about 50 times higher than the value obtained with the sugar-free poly-L-lysine/pSV21uc complex, but not larger than when the plasmid was transfected by using the DEAE-dextran method. The relatively low gene expression upon transfection using LactpLK as carrier probably results from extensive degradation of the endocytosed complex into lysosomes which reduces the number of intact DNA molecules able to reach the nucleus. In order to inhibit the hydrolytic degradation, chloroquine may be used: chloroquine is a weak base known to neutralize the acidic pH of endocytotic vesicles and to inhibit various hydrolases contained in endosomes and/or lysosomes.

5.1. Transfection in the presence of chloroquine As shown by flow cytometry, the fluorescence

200

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Fig. 10. Flow cytometry analysis of plasmid uptake: (a) in the absence of chloroquine; (b) in the presence of chloroquine. Cellassociated fluorescence determinated by flow cytometry in HepG2 cells incubated with fluorescein labeled lactosylated poly-L-lysine/ pSV21uc complex for 4 hours at 37°C. (A) Control: cells incubated in the absence of the complex. (B) Cells incubated in the presence of the complex and then in the absence of monensin. (C) Cells incubated in the presence of the complex and then in the presence of 50 gM monensin at 4°C for 30 min. FL1 = fluorescence intensity of the channel 1, green fluorescence.

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

18

intensity of HepG2 cells incubated for 4 hours at 37°C with pSV21uc complexed with fluoresceinylated LactpLK in the presence of 100 taM chloroquine did not increase upon a post-incubation with monensin (Fig. 10b). Furthermore, in the presence of chloroquine, cell-associated fluorescence intensity after monensin post-treatment is much lower than when cells are incubated in the absence of chloroquine. Since the quantum yield of fluorescein linked to macromolecular structures is several times lower than that of free fluorescein or of fluorescein linked to small peptides [43], the relatively low fluorescence intensity is at least partially related to a lack of hydrolysis. When HepG2 cells were incubated with a pSV21uc/LactpLK complex for 4 hours at 37°C in the presence of chloroquine (100 ~tM), the luciferase activity measured 48 hours later was 60-fold higher (RLU: 7.9 x 105). Plasmid/polymer complexes present during the first 4 hours of incubation with or without 100 ~tM chloroquine present for up to 4 hours, did not induce any cytotoxic

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D

E

Fig. 11. Time course effect of chloroquine, pSV21uc (1.5 pmol) complexed with LactpLK (165 pmol) was incubated at 37°C with 3 x 105 HepG2 cells in 1 ml, either in the absence (A) or in the presence of 100 laM chloroquine (B to E). Chloroquine was added at t = 0 h (E), i.e. at the time when the pSV21uc/ LactpLK complex was added, 3 hours (B), 2 hours (C) or 1 hour (D) later. After 4 hours incubation, the medium was removed and the cells were further incubated in chloroquine-free and pSV21uc/LactpLK complex-free complete culture medium for 48 hours. Gene expression was determined by assaying the luciferase activity of three aliquots of cell lysates. The values of relative light units (RLU) shown represent the activity of 106 cells.

effect or cell detachment: the number of cells present in a well was identical to that of mock-treated cells. The effect of chloroquine on transfection efficiency appeared to be time-dependent: 100 I.tM chloroquine was added at t = 0 (E), i.e. at the time when pSV21uc/LactpLK was added, 1 or 2 or 3 hours later (Fig. 11). In control experiments, no chloroquine was added at any time. At t = 4 hours, in any case, cells were washed and cultured in the absence of chloroquine and of plasmid/ polymer complex for further 48 hours in complete medium. In the absence of chloroquine, the activity of luciferase was low. In the presence of chloroquine, a maximum luciferase activity was obtained when chloroquine was either added together with pSV21uc/LactpLK complex (t =0) or 1 hour later (t = 1 h). When chloroquine was present during a longer incubation period, no further increase in luciferase activity was detected.

5.2. Transfection in the presence of a fusogenic peptide The intracellular transmembrane passage of plasmid D N A is a critical process for its delivery into the cytosol and/or the nucleus where the gene should be expressed. Orthomyxoviruses possess an envelope glycoprotein containing a fusogenic peptide that, in an acidic endosomal medium, promotes fusion between the viral and the host plasma membranes allowing very efficient transfer of the viral genome into the cytosol. A peptide ( G L F E A I A E F I E G G W E G L I E G C A ) with a similar structure to the N-terminal moiety of influenza hemagglutinin (HA-2) was synthesized and used in order to increase the transmembrane passage of D N A molecules. This peptide is known to induce fusion of liposomes in acidic buffer [104-105]. When HepG2 cells were incubated for 4 hours at 37°C with a pSV21uc/LactpLK complex in the presence of 10 gM fusogenic peptide, luciferase activity measured 48 hours later was about 500-fold greater than with the complex in the absence of fusogenic peptide and 10-fold greater than that in the presence of chloroquine (100 gM) [96]. The effect of the fusogenic peptide on the transfection efficiency was also time-dependent (Fig. 12). Luci-

M. Monsigny et al./Advanced Drug Delivery Reviews 14 (1994) 1-24

Fig. 12. Time course effect of the fusogenic peptide. 1.5 nM pSV21uc was incubated at 37°C with HepG2 cells (A). 1.5 nM pSV21uc complexed with 165 nM LactpLK was incubated at 37°C with HepG2 cells, either in the absence (B) or in the presence of 10 laM fusogenic peptide (C to F). The peptide was added at t = 0 h (C), i.e. at the time when the pSV21uc/ LactpLK complex was added, 1 hour (D), 2 hours (E) or 3 hours (F) later. After 4 hours incubation, the medium was removed and the cells were further incubated in peptide-free and pSV21uc/LactpLK complex-free complete culture medium for 24 hours. Gene expression was determined by assaying the luciferase activity of three aliquots of cell lysates. The values of relative light units (RLU) shown represent the activity of 106 cells.

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ferase activity was maximal when the fusogenic p e p t i d e w a s a d d e d a t t i m e t = 0, i.e. t o g e t h e r w i t h the pSV21uc/LactplK complex, or 1 hour later

A

(t = 2 h), t h e t r a n s f e c t i o n e f f i c i e n c y w a s 5 - f o l d l o w er; 3 h o u r s l a t e r (t = 3 h), l u c i f e r a s e a c t i v i t y w a s as l o w as in c o n t r o l e x p e r i m e n t s , i.e. w i t h o u t p e p t i d e .

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DNA/pLK

10/50

10/25

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Fig. 13. Plasmid polylysine complex formation and gene transfer. (A) Electrophoretic analysis: Electrophoretic migration through a 0.6% agarose gel of plasmid DNA mixed with increasing amounts of LactpLK. Plasmid/LactpLK complexes were prepared by adding dropwise with constant mixing: 10 (a), 8 (b), 6 (c), 5 (d), 4 (e), 3 (t), 2 (g), 1 (h), 0.2 (i), 0 (j)/ag LactpLK in 60 lal DMEM to 2 lag pSV21uc plasmid in 140 lal DMEM. After incubation for 30 min at 20°C, 20 tll of each sample were analyzed by electrophoresis through a 0.6% agarose gel containing ethidium bromide to visualize DNA in a Tris borate EDTA buffer (95 mM Tris 89, mM boric acid and 2.5 mM EDTA), pH 8.6. (B) Gene expression: Complexes with 10 lag/ml pSV2Luc plasmid and 5, 15, 25 or 50 lag/ml LactpLK were formed in DMEM. HepG2 cells (5 x 105 cells per well) were incubated at 37°C for 4 hours in the presence of the indicated plasmid/LactpLK ratios and in the presence of 100 laM chloroquine. The medium was removed and cells were further incubated at 37°C for 48 hours in complete culture medium in the absence of chloroquine. Gene expression was determined by assaying, in triplicate, the luciferase activity of cell lysates. The values of relative light units (RLU) shown represent the activity of 106 cells.

20

M. Monsignyet al./AdvancedDrug DeliveryReviews14 (1994) 1 24

5.3. Optimal conditions for transfection of HepG2 cells by pSV2luc/LactpLK complexes Various ratios of LactpLK and pSV21uc plasmid were tested in order to determine the optimal conditions for D N A polymer complex formation and for gene delivery. Different complexes were made by adding dropwise, to a (10 gg/ml) plasmid solution, a solution containing 1-50 gg/ml polymer. Complexes were analyzed by electrophoresis through a 0.6% agarose gel (Fig. 13A). The LactpLK/plasmid complex did not migrate as a consequence of the neutralization of the anionic charges of D N A by the polylysine amino groups. When 10 lag/ml D N A was complexed with 25 gg/ml of LactpLK (polymer/plasmid molar ratio of 110:1, corresponding to a neutral complex), no migration of the D N A occurred. Luciferase activity was determined in cells transfected with complexes containing various plasmid/polymer ratios; maximum activity was observed with LactpLK/plasmid ratios corresponding to a complete lack of D N A migration (Fig. 13B).

cleotides and even genes. In the future, the selectivity of the glycoconjugates used can be further improved by using complex oligosaccharides instead of simple sugars, because a given membrane lectin accommodates, with rather fine specificity, a limited number of complex oligosaccharides. In addition, within the next few years, we may expect that ligands for several other membrane lectins will be defined and that it will be possible to use a large panel of glycosylated carriers to target drugs, oligonucleotides and genes to a number of well-defined cell types through their membrane lectins.

7. Acknowledgements

HOS cells (human osteosarcoma cells) possess a membrane lectin recognizing lactosyl residues. Upon transfection of HOS cells with a pSV2neo plasmid using either the calcium phosphate method or the pSV2neo/LactpLK complex in the presence of chloroquine, geneticin-resistant clones were detected. After 3 weeks in culture in the presence of geneticin, Petri dishes were stained with crystal violet. Transfection of HOS cells by pSV2neo/LactpLK complexes gave a greater number (about 30-fold) of resistant clones than transfection by the calcium phosphate method.

We thank Prof. Dirk K.F. Meijer for his kind advice, suggestions, and a critical reading of the manuscript. We thank Philippe Bouchard for the preparation of the sugar derivatives, Emmanuelle Martin for the synthesis of peptides, and MarieTh6r~se Bousser and Suzanne Nuques for their skill in using the cell biology techniques. This work was partly supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA), ARC (Association pour la Recherche sur le Cancer), W H O (World Health Organization) and CEC (Commission of the European Communities). P.M. and A.-C.R. are Research Directors of the Institut National de la Sant6 et de la Recherche M6dicale, R.M. is Research Director of the Centre National de la Recherche Scientifique and M.M. is Professor of Biochemistry at the University of Orleans and Head of the Department of Endogenous Lectins and Glycoconjugates, in the Centre de Biophysique Mol6culaire, Orleans.

6. Concluding remarks

8. References

5.4. Long-term transfection

Data accumulated during the past decade produced by us and others have clearly demonstrated that glycoconjugates are suitable to target drugs, oligonucleotides and even genes. This finding opens up interesting new ways to procedure cellspecific formulation of drugs, antisense oligonu-

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