Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet?

journal of

ELSEVIER

Journal of Controlled Release 41 (1996) 99-119

controlled release

Liposomes as a carrier for intracellular delivery of antisense oligonucleotides" a real or magic bullet? Olivier Zelphati, Francis C. Szoka Jr* University of Cal!fornia, School of Pharmacy, Department of Pharmacy and Pharmaceutical Chemist~', San Francisco, CA 94143-0446, USA

Received 20 May 1995; accepted 4 October 1995

Abstract Antisense oligonucleotides are specific inhibitors of gene expression. They represent a promising tool in fighting viral, malignant and inflammatory diseases. In many cases their activity is limited by their low cellular uptake and lack of target cell recognition. One approach to circumvent these problems is the use liposomes as an oligonucleotide carrier. The encapsulation of oligonucleotides in liposomes is useful for several reasons: (1) protection of oligonucleotides from nuclease degradation; (2) enhancement of cellular uptake in several cell types; (3) improvement of oligonucleotide potency, especially in vitro; (4) modification of their intracellular distribution (this is particularly true for cationic liposomes); (5) increased retention of the oligonucleotides in cells; (6) potential for slow release depots for modified oligonucleotides. However, encapsulation of oligonucleotides in liposomes may decrease the access of the oligonucleotide to tissues outside of the vascular system which may restrict the use of oligonucleotides encapsulated in liposomes or other particulate carriers to accessible cells or tissues. In this review results obtained in vitro and in vivo, using liposome encapsulated oligonucleotides are described and the benefits of liposomes as an oligonucleotide carrier are analyzed. Keywords: Antisense oligonucleotides; Liposomes; Drug delivery; Intracellular distribution; Cellular uptake

1. I n t r o d u c t i o n Most drugs used in clinical settings are specific at a molecular rather than a cellular level. Moreover, due to passive diffusion of the active drug in the body only a small fraction of the dose reaches the target; the remaining amount of drug acts on other tissues or is rapidly eliminated. Therefore, to obtain a therapeutic effect, a relatively high dose of drug

Corresponding author: Tel: +1-415-4763895 or +1-4154764676; Fax: +1-415-4760688; E-mail: [email protected] or [email protected].

must be administered; thus usual formulations are a balance between efficiency and toxicity. Antisense oligonucleotides, a new generation of antiviral and antitumoral agents, fall into this category. These single strands of DNA or RNA can hybridize to their complementary targets and can specifically inhibit, in principle, protein synthesis (Fig. 1). However, before they reach their intracellular target, antisense oligonucleotides must overcome several obstacles (Fig. 1) including their sensitivity to nucleases, their poor cellular uptake and their capacity to hybridize to their DNA or RNA target present in the cytoplasm a n d / o r in the nucleus of cells [1-4].

0168-3659/96/$15.00 © 1996 Elsevier ScienceIreland Ltd. All fights reserved PII S0168-3659(96)01361-2

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O. Zelphati, F.C. Szoka Jr/ Journal of Controlled Release 41 (1996) 99-119

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models compared to phosphodiester oligonucleotides [1-51. Antisense oligonucleotides have been reported to act by several different mechanisms; hence, each sequence may have its own pharmacology. They, and especially the phosphorothioate oligonucleotides, can have non-sequence-specific effects. These have been

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shown to be mainly due to non-specific interaction with some enzymes or cell surface molecules [1,4,6,7]. In most studies, however, antisense oligonucleotides have been shown to inhibit the protein synthesis by a sequence-specific mechanism (Fig. 3). Several such mechanisms have been reported [1,2,4,5,7]. Once hybridized to their target RNA, oligonucleotides can act by physical blockage of the site preventing the access or binding of various factors like ribosomes, spliceosomes or activation factors. In addition, the heteroduplex formed by oligonucleotide and its target can be recognized by RNase H which degrades only the targeted RNA. Finally a third process has been suggested, but never proved, where oligonucleotides can modify the secondary structure of the targeted mRNA and so improve the accessibility to ribonucleases which degrade the mRNA [1,2,4,6]. Oligonucleotides are relatively large hydrophilic

molecules (15-28-mers) with a molecular weight range of 5000-10 000 and therefore do not passively diffuse across cell membranes. Backbone-modifications have not resolved the problem of low cell membrane permeability. An alternative strategy to circumvent stability and permeability problems is to use liposomes as carriers for oligonucleotides. Indeed, liposomes are a potential way to deliver oligonucleotides since liposomes can protect various molecules from the external medium and can deliver molecules directly into cells [8]. Moreover, it is also possible to specifically target liposomes by coupling proteins or antibodies on their surface [9]. The purpose of this review is to describe the use of anionic, cationic, pH-sensitive, fusogenic and immunoliposomes to deliver the oligonucleotides intracellularly. We will discuss biological applications of various liposomes, particularly their protec-

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o. Zelphati, F.C. Szoka Jr/Journal of Controlled Release 41 (1996) 99-119

tive effects on oligonucleotides, their delivery efficiency and the resultant intracellular distribution of oligonucleotides. Finally, the potential of these oligonucleotide carrier systems for in vivo applications will be addressed in order to evaluate if lipid bilayer vesicles are a 'magic bullet' as dreamed Paul Ehrlich in 1906.

2. Cellular uptake and intracellular distribution of oligonucleotides.

103

uptake has not really been defined. Nevertheless, two models have been proposed. Passive diffusion across the celt membrane appears unlikely since this is a very slow process [22]. Most probably methylphosphonates are internalized by a non-specific fluid phase endocytosis [21,23]. In the case of other modified oligonucleotides their entry mechanisms into cells are not determined but seem charge and chemical modification dependent [1]. Oligonucleotides are also exocytosed from cells, but the nature and effects of the process are still unknown [4,12,20,24].

2.1. Cellular uptake 2.2. Intracellular distribution

Both backbone-modified and unmodified oligonucleotides have a poor capacity to cross cell membranes. Despite their relatively large molecular weight and ionic character (except for the methylphosphonate) these molecules enter cells to a limited extent [10]. Their internalization is dependent on temperature, energy, molecular size and concentration [10-13]. Oligonucleotide entry has been shown to be receptor-mediated, but the molecular characterization and the cellular function of these cell surface determinant(s) have not yet been completely elucidated. Some authors have reported the implication of cell surface protein(s) in the 80 kDa range [ 11,12,14], while others have demonstrated the association of oligonucleotides with 34 kDa [15] and 30 kDa cell surface proteins [16]. This may be attributable to different cell types and experimental conditions used in these studies. However, other studies have suggested also a non-receptor mediated uptake of oligonucleotides [17] or internalization by a pinocytotic mechanism [12,18]. Recently, Zamecnik and colleagues have suggested that internalization of oligonucleotides does not occur by an endocytotic process, but rather by a mechanism yet unclear that could be a caveolar, potocytotic mechanism [19]. In cultured cells, cellular uptake is inefficient and low ( < 4 % of total added oligonucleotides), although some authors have shown that, on certain cell types, the internalization is relatively rapid [13,20]. Due to their charge neutrality methylphosphonate oligonucleotides are picked up by cells by a distinct mechanism from that of phosphodiester or phosphorothioate oligonucleotides [21 ]. Their cellular

Fluorescent-microscopy studies have shown that oligonucleotides are localized into punctate cytoplasmic regions probably corresponding to endocytic compartments [5,11,13,23,25,26,56]. The mechanisms by which oligonucleotides are released from these compartments to reach their cytoplasmic or nuclear targets are still unknown. The inefficiency of cytoplasmic entry and the localization in acidic vesicles makes intracellular localization studies difficult. Nevertheless, microinjection experiments demonstrated rapid diffusion and accumulation of oligonucleotides in the nucleus in a temperature- and energy-independent manner [5,25,27-29]. Once inside cells, phosphodiester oligonucleotides are rapidly degraded [29], but phosphorothioates and a anomeric oligonucleotides persist at least 24 h [27,28]. In the nucleus oligonucleotides interact with small nuclear proteins [14,25,27,30]. Binding proteins are different depending on the structure of oligonucleotides. This may explain the different localization of oligonucleotides in the nucleus [25,30]. Also, other structural elements play important roles. For example, methylphosphonate oligonucleotides can interact with lipids and therefore their interaction with the membranes and organelles of the cell should affect their distribution [22]. The real significance of such interactions on the activity of oligonucleotides is not known, but too strong affinity towards some cellular components obviously may limit their availability to hybridize to their target. In this context, the intracellular distribution of oligonucleotides seems to favor nuclear action, but it

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O. Zelphati, F.C. Szoka Jr/ Journal of Controlled Release 41 (1996) 99-119

is likely that they act also in the cytoplasm. Indeed, antisense oligonucleotides have been shown to inhibit specifically the replication of the vesicular stomatitis virus (VSV) that replicates only in the cytoplasm [31,32]. Moreover, recent studies using fluorescent-resonance energy transfer (FRET), after microinjection of oligonucleotides, have shown the localization of these molecules in both cytoplasm and nucleus as well as the formation and dissociation of the duplexes formed by an oligonucleotide and its target [29]. Nevertheless, the microinjection experiments bypass the endocytosis steps and therefore do not give information on the internalization pathway of oligonucleotides. Intracellular localization studies of oligonucleotides, without microinjection, have been carried out, but the results are rather conflicting. Some studies have shown a preferential localization of phosphodiester and phosphorothioate oligonucleotides in the cytoplasm [5,11,23-25,33,34], while others have shown a preferential accumulation in the nucleus [14,19,34-36]. This divergence may be explained by the different cell types used (e.g. myeloid cell line, lymphocyte, fibroblast, epithelial cells, keratinocyte), chemical nature of oligonucleotides, and the experimental methods. Oligonucleotides have been, in part, also found in mitochondria [37]. Although backbone-modification of oligonucleotides has for the most part, resolved the problem of nuclease sensitivity, it has not really improved the low and slow cellular uptake. To solve this problem, some researches have used liposomes to increase the efficiency and the delivery of oligonucleotides in cell culture in vitro.

3. Anionic liposomes Anionic liposomes composed of phosphatidylserine have been used to deliver oligonucleotides into cells. In this case, liposomes were loaded with oligonucleotides in the presence of calcium in order to get a reasonable encapsulation efficiency [38]. This technique was necessary due to the repulsion of the negative charges of oligonucleotides and lipids to overcome the low encapsulation efficiency as shown with DNA and RNA. Phosphorothioate antisense oligonucleotides targeting the c-myc oncogene or the

human or mRNA mouse IL-1 receptor were encapsulated in anionic liposomes [38,39]. All of these were efficient in cell culture. However, the delivery of the liposome contents requires fusion with cell membranes. In both studies, this has been achieved by polyethylene glycol treatment in serum-free medium. This limits their application. Anionic liposomes containing cardiolipin were used to carry oligonucleotides into cells. In order to improve the transport of oligonucleotides and to increase the encapsulation efficiency the authors have developed the minimal volume entrapment (MVE) technique [24,40]. This technique seems to be efficient in oligonucleotide encapsulation. Oligonucleotides were protected from degradation by nucleases present in serum-containing medium [24]. Cellular uptake mediated by these liposomes was increased by 7- to 18-fold in human T leukemia and ovarian carcinoma cells and the intracellular release of oligonucleotides was also facilitated. The oligonucleotides delivered by these liposomes were localized preferentially in the nucleus, whereas applied without liposomes they were localized mainly in the cytoplasm [24]. Moreover, it was demonstrated that liposomes enhanced also the retention of oligonucleotides in the cells. Biological effects have been demonstrated by antisense phosphorothioate oligonucleotides complementary to the 5' end of the coding region or to a loop-forming site in the multidrug resistance mdr-1 mRNA. P-glycoprotein synthesis and doxorubicin resistance were reduced for a multidrug resistance cell line after treatment by liposomes containing antisense oligonucleotides [40]. An anionic liposome-based vehicle for delivery of anti-HIV ribozymes to HIV-infected cells has also been reported [41]. Liposomes composed of 50% dipalmitoylphosphatidylglycerol (DPPG) increased ribozyme uptake. The reported results show that both lipid and ribozyme are taken up at the same rate and that this process reaches saturation after 24 h. Ribozymes were mainly localized in nucleus after their delivery by these liposomes. In order to determine whether the delivered ribozyme was functional, oligonucleotides containing gag target sequences and ribozymes designed to cleave the gag message of HIV-I in the region of the initiation codon were encapsulated into separate liposomes and incubated with human T lymphocytes. The results

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obtained demonstrate that some ribozymes can be delivered to an intracellular environment suitable for their cleavage activity [41]. The liposomal delivery of methylphosphonate antisense oligonucleotides complementary to specific regions of the b c r - a b l mRNA in chronic myelogenic leukemia has also been reported [42]. Efficient cellular uptake and augmentation of oligonucleotide activity have been shown, unfortunately, the liposome composition has not been reported. It has to be noted that these kinds of anionic liposomes were also used as model membranes to examine further the mechanisms by which oligonucleotides (modified or unmodified) pass through

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liposomes have been used [45,46]. Their principle of action is directly derived from enveloped viruses (vesicular stomatitis virus, influenza virus). They are endocytosed in coated pit vesicles and they merge with endosomal membranes at acidic pH to release their genome into the cytoplasm (Fig. 4) [47]. The lipidic composition of pH-sensitive liposomes, of which commonly used lipids is the dioleylphosphatidylethanolamine (DOPE), determines the stability at neutral pH and the capacity to be destabilize and/or merge at acid pH [45,46]. After destabilization and/or fusion with the endosomal membrane the liposome contents might be efficiently released into the cytoplasm. Several different molecules have been successfully delivered by such liposomes [45,46]. pH-sensitive liposomes interact non-specifically by electrostatic adsorption with cell membranes and are endocytosed. However, they can be specifically directed toward a cell surface molecule by coupling antibodies to their surface [45]. They are more efficient in cellular drug delivery than conventional liposomes. Nevertheless, only 10% of their contents have been shown to be actually released into the cytoplasm and the remainder is degraded in the lysosomes [48]. pH-sensitive liposomes have been used to deliver antisense oligonucleotides. These liposomes containing antisense oligonucleotides (15-mer) complementary to the AUG region of the e n v mRNA of Friend Retrovirus have inhibited viral spreading whereas the free oligonucleotide in solution did not display any effect [49]. The inhibition was sequence-specific. In that study, pH-sensitive and non-pH-sensitive liposomes were compared for their efficiency to deliver oligonucleotides. Antisense oligonucleotides encapsulated in pH-sensitive liposomes were the most efficient. These results are paralleled to those obtained in gene delivery [50,51]. This has led the authors to investigate the mechanism of delivery of oligonucleotides encapsulated in these liposomes into cells. Surprisingly, they have shown that in the absence of virus, liposomes remained mainly adsorbed on the mouse fibroblast surface without any internalization whereas in infected cells these liposomes were efficiently taken up [52]. This observation was also made with antisense oligonucleotides alone; indeed, it had been shown that the human T-cell leukemia virus type I t a x transformation is

associated with increased uptake of oligonucleotides in vitro and in vivo [16]. The fact that viral infection can increase the delivery of oligonucleotides by liposomes further encourages the approach of liposome-mediated antiviral oligonucleotide-delivery. However, the pH-sensitive liposomes, although efficient for intracellular delivery of DNA, are not very stable in plasma and serum [45,46]. The fragility of these liposomes leads to the leakage of their contents either by destabilization of the bilayer or by fusion or aggregation of liposomes. Nevertheless, the incorporation of cholesterol or ganglioside or 1,2-dipalmitoyl-sn-3-succinylglycerol or cholesterol hemisuccinate (CHEMS) can enhance the stability, but in exchange their capacity to merge in the acidic compartments is greatly diminished [45,46]. The real potential of the pH-sensitive liposomes to deliver oligonucleotides in vitro or in vivo requires further investigations.

5. Immunoliposomes Another means of bypassing problems of cellular membrane permeability, stability to nucleases (especially for phosphodiester oligonucleotides) and also to confer a target cell specificity to oligonucleotides is the use of antibody-targeted liposomes (immunoliposomes) as a transport system. The covalent coupling of an antibody enables liposomes to bind to target cells that express the cell surface molecule recognized by the antibody [53,54]. The quantity of liposomes bound is proportional to the density of targeted cell membrane molecules [55]. Immunoliposomes have been shown to enter into lymphoid cells and fibroblasts by an endocytic pathway and to release the encapsulated product intracellularly (Fig. 4). The efficiency of this process depends on the physiology of the targeted surface molecule, the type of cell, and the liposome size [56-58]. After internalization, immunoliposomes release their contents into the cytoplasm but the mechanism by which the encapsulated molecules reach the cytoplasm is unknown. Nevertheless, there is strong presumption that the acidification of endosome compartments plays a role in the release of liposome contents [59]. Immunoliposomes have been

O. Zelphati, F.C. Szoka Jr/Journal of Controlled Release 41 (1996) 99-119

studied as a transport system to deliver both membrane-permeable and impermeable molecules into cells [60-62]. Antibody-bearing liposomes containing antisense oligonucleotides have a double specificity: a particular cell selected by the targeting antibody on the liposome and a particular mRNA in the cell selected by sequence complementarity with the liposome-encapsulated oligonucleotide. Immunoliposomes have been used to transport antisense oligonucleotides into cells [62]. Encapsulation of phosphodiester oligonucleotides offers protection from extracellular degradation [63,64]. Encapsulation of antisense phosphodiester oligonucleotides complementary to several viral genes (5'-end region of the mRNA encoding the N protein of vesicular stomatitis virus or translation initiation regions of the t a t or r e v mRNA of HIV-I) has been shown to enhance delivery into cells and to specifically inhibit the expression of the targeted viral genes [7,63-65]. Phosphodiester antisense oligonucleotides, inactive in their free form, inhibited, in a sequencespecific manner, HIV replication in acutely infected cells after their encapsulation in immunoliposomes directed to HLA class I or CD7 molecules. However, the same encapsulated phosphodiester antisense oligonucleotides had no antiviral activity in chronically infected cells [7,65]. Antisense phosphorothioate oligonucleotides with specificity for the r e v gene of HIV-1 were also examined, either free in solution or encapsulated in immunoliposomes, for their antiviral activity. In acutely infected cells, phosphorothioate oligonucleotides free in solution inhibited the replication of H1V without sequence specificity and had slightly greater activity, also non-specifically, when encapsulated. The non-sequence-specific effects of these oligonucleotides are due to two mechanisms: (1) phosphorothioate oligonucleotides inhibit the activity of virus encoded reverse transcriptase; (2) they also inhibit the interaction between the virus gp 120 and the target CD4 molecules on human lymphocytes. However, they specifically blocked HIV replication in chronically infected cells with higher efficiency once encapsulated in targeted liposomes [7,65]. The use of immunoliposomes has permitted a direct comparison of the biological activity of a n t i - r e v phosphodiester and phosphorothioate oligonucleotides: the delivery of molecules encapsulated in liposomes of the same size and

107

targeted to the same molecule should be identical, whatever the encapsulated contents. In consequence, the parameters that govern membrane permeability and stability to extracellular nucleases are minimized and a direct comparison of their activity inside cells has been possible. Encapsulation in antibody-targeted liposomes permitted also intracellular delivery and distinction between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA. In this context and in order to identify the mechanisms of oligonucleotide action on HIV replication, phosphodiester and phosphorothioate oligonucleotides in a and /J-configurations directed against the initiation codon region of the HIV r e v gene were evaluated for their ability to inhibit HIV-1 replication in acutely and chronically infected cells. Results obtained have permitted four different mechanisms of antiviral activity for these antisense oligonucleotides to be distinguished [7]. One major limitation to this technology is the poor encapsulation efficiency of agents in liposomes sufficiently small to be taken up by target cells. To circumvent this problem, the use of cholesterol modified oligonucleotides for incorporation into liposomes was evaluated. Oligonucleotides covalently coupled to cholesterol via a potentially bioreversible disulfide linkage have improved incorporation of such oligonucleotides into liposomes [64,66]. The advantage of this modification is that it offers the potential of both increasing encapsulation of the conjugate in the lipid bilayer and permitting the release of the associated oligonucleotide in the reducing environment of endocytic vesicles. The biological activity of these cholesterol-S-S-oligonucleotides compounds complementary to the t a t mRNA of HIV-1 has also been reported [64]. However, in spite of their success in in vitro models, immunoliposomes have several drawbacks. They are internalized into the cells by an endocytotic pathway and are eventually destroyed in the lysosomes. The quantity of oligonucleotides escaping from this degradation and reaching their target in the cytoplasm or nucleus is unknown, but it is probably very low. It is likely that the poor escape from these acidic compartments limits the use of this delivery system. Recent studies have shown that immunoliposomes

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may bind to selected cells in vivo [67,68]. Since immunoliposomes may be immunogenic the resulting antibody response to the targeting antibody may reduce their half-life in the circulation as well as their ability to bind to the desired target cells [69]. Other types of targeted liposomes have also been reported to deliver oligonucleotides. Liposomes containing an ethylmaleimide derivative of distearoylphosphatidylethanolamine that are able to trigger endocytosis by human peripheral blood leukocytes in vitro were used to transport oligonucleotides [70]. Oligonucleotides specific for the 5' tat splice acceptor site of HIV were encapsulated in these liposomes. They have been shown to inhibit the replication of HIV by an as yet unknown mechanism since the sense but not the antisense configuration was active.

6. Fusogenic liposomes As explained above, commonly encountered problem of liposomes is their internalization via endocytotic compartments into lysosomes. To directly introduce molecules into the cytoplasm, liposomes that merge with cell membranes have been developed. The principle mimics the way by which several viruses (HIV, Sendai virus) bind and merge with cell membranes at neutral pH and, thereafter, release their genome into the cytoplasm (Fig. 4) [47]. This kind of fusion between liposomes and cell membranes is not a spontaneous phenomenon and thus several methods have been used to facilitate this type of delivery. Fusion mediated by fusogenic agents like polyethylene glycol, glycerol and polyvinyl(alcohol) [71,72] or by reconstituted viral membranes have been used [73,74]. Liposomes prepared with fusion protein F of Sendai virus that is incorporated into the lipid bilayers, have acquired the capacity to merge with cell membranes [73,75]. Some molecules with very poor cellular membrane permeability (like the RNA duplex poly(rI)-poly(rC)) have been very efficiently delivered to cells [76]. Other viral proteins have been also incorporated to allow the fusion between membranes of liposomes and cells [74,75]. Surprisingly, these types of liposomes have been rarely reported to deliver antisense oligonucleotides

to cell culture in vitro. Nevertheless, Morishita et al. have encapsulated oligonucleotides into negatively charged liposomes complexed to the protein coat of an inactivated Sendai virus [77]. This method has been shown to result in a more rapid cellular uptake and in 10-fold higher transfection efficiency of oligonucleotides or plasmid DNA than lipofection (see next section) or passive uptake methods. The goal of this study was to develop an effective strategy to prevent the development of restinosis due to neointima formation after angioplasty injury. To achieve this aim, these authors have encapsulated antisense phosphorothioate oligonucleotides directed to the translation initiation sites of proliferating-cell nuclear antigen (PCNA) and cdc2 kinase mRNAs in their fusogenic liposomes. The two proteins have been identified as principal cell-cycle regulatory proteins of the final common pathway regulating cell proliferation. The effects of these oligonucleotides were first examined in vitro and their cotransfection inhibited serum-stimulated vascular smooth muscle cell growth [77]. Injection of these liposomes into balloon-injured arteries of rats led an inhibition of targeted mRNA expression. Moreover, the inhibitory effect of antisense oligonucleotides on neointima formation persisted for up to 8 weeks after a single transfection. However, the utilization of these fusogenic liposomes, also called virosomes, seems to be limited to a single injection due to their strong immunogenicity [78]. Moreover, the complexity of the preparative methods, their relatively poor cellular specificity and their poor stability in plasma and serum requires modifications of this approach.

7. Cationic lipids 7.1. Physicochemical characteristics

The transport of oligonucleotides by cationic lipids is based on the electrostatic interaction between negative charges of oligonucleotides and positive charges of lipids. Thus, in contrast to all other type of liposomes described above, cationic liposomes do not require any encapsulation step that limits the application of these carriers. In this case, oligonucleotides are directly mixed with preformed lipo-

o. Zelphati, F.C. Szoka Jr/Journal of Controlled Release 41 (1996) 99-119

somes. For oligonucleotides complexed with cationic lipids the charge ratio appears to be a critical parameter for their delivery efficiency. Despite the common use of cationic lipids for oligonucleotides transport, the physicochemical properties of oligonucleotide-cationic liposome interactions have been studied only recently. Aggregation, lipid fusion, and interactions with different lipid bilayers were examined [79]. Phosphorothioate oligonucleotides were associated with cationic liposomes composed of dimethyldioctadecylammonium bromide (DDAB) and dioleylphosphatidylethanolamine (DOPE) or of N-(1-( 2,3-dioleoyloxy )propyl )-N,N,N-trimethyl-

ammoniummethylsulfate (DOTAP) (Fig. 5). Oligonucleotides induced aggregation and fusion of cationic liposomes as a function of charge ratio [79]. Thus, one cannot really call these cationic structures 'liposomes' because after their complexation to oligonucleotides they do not necessarily retain the classic bilayer form surrounding an aqueous phase. In this context it seems preferable to call these complexes 'synthetic cationic amphiphile structures' as suggested by J.P. Behr [80]. Lipid fusion and interactions with different lipid bilayers indicated that either phosphatidylethanolamine or negative charges are required for fusion of cationic lipidsoligonucleotide complexes [79]. 7.2. Nuclease protection and cellular uptake

Because .oligonucleotides are complexed to cationic liposomes and thus unencapsulated, one would not necessarily expect protection against nucleases when using these carriers. However, it has been reported that degradation of phosphodiester oligonucleotides by nucleases was markedly prevented by DOTAP both in cell culture medium and in human serum [81]. This can be explained by a collapse or a 'coating' of the oligonucleotides after aggregation of the complexes, leading to structures where the oligonucleotides are completely covered by lipid bilayers. Nevertheless, antisense phosphodiester oligonucleotides associated with cationic lipids mainly failed to be active in cell culture in vitro [82-84]. Only one study has reported the activity of phosphodiester antisense oligonucleotides complexed to cationic lipids [85]. This points out the critical role of intracellular nucleases for oligonucleotide activity

109

and thus, the necessity to use backbone-modified oligonucleotides which are relatively resistant to both intracellular and extracellular nucleases. This is also suggested in a study with immunoliposomes in an HIV infection model [7]. Concerning the cellular uptake of oligonucleotides, many studies have shown that cationic lipids (Fig. 5) such as DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) with or without DOPE (Lipofectin®), dioctadecylamidoglycyl spermine (DOGS) and DDAB associated with DOPE markedly enhance the rate of oligonucleotide uptake in several different cell types [81,83,84,86]. Augmentation of cellular uptake was generally reported to be 15- to 25-fold relative to the uptake of oligonucleotides alone. In general, cationic lipids have enhanced oligonucleotide cell association in a time- and concentrationdependent manner [81,83,84]. However, in HL-60 and RBL-1 cells the oligonucleotide cellular uptake was unaffected by the presence of the cationic lipid DOTMA [83]. Although cationic liposomes have been used extensively in gene and oligonucleotide delivery, their mechanism of delivery is still controversial. It has been reported that the main mechanism of cellular delivery is the fusion of cationic lipids with negatively charged cell membranes thereby releasing directly their associated molecules into the cytoplasm [83,87]. However, we and others have found that cationic liposomes can deliver antisense oligonucleotides or plasmid DNA by an endocytotic process [51,88,89]. Thus, in order to better characterize the pathway of delivery by cationic liposomes we have used several inhibitors of endocytosis. CV-1 cells (kidney fibroblast from Monkey) were incubated with fluorescently labeled phosphorothioate oligonucleotides complexed to DOTAP in the presence of these inhibitors. The lysosomotropic agents Chloroquine, NH4CI , Monensin and Bafilomycin A which increase the pH of endocytotic vesicles, the microtubule depolymerizing-agent, Nocodazole and a drug interfering with membrane recycling between endoplasmic reticulum and cis golgi Brefeldin A, have no effect on the nuclear delivery of oligonucleotides. Thus, these results, in agreement with other results, suggest that the acidification of the endosomal vesicles and the later phases of endocytosis (since

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O. Zelphati, F.C. Szoka Jr/ Journal of Controlled Release 41 (1996) 99-119

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cose (energy depletion), N-ethyl maleimide (blocking NSF protein activity required in the fission of endosome membrane) and Cytochalasin B (blocking polymerization of microfilaments of actin which are

O. Zelphati, F.C. Szoka Jr/Journal of Controlled Release 41 (1996) 99-119

known to be implicated in the endocytic process of uncoated vesicles) inhibited DOTAP-mediated delivery of oligonucleotides to the nucleus [89]. These results led us to propose the following delivery mechanism (Fig. 6) which is in agreement with results obtained for plasmid DNA delivery by cationic lipids. Oligonucleotide-lipid complexes are mainly internalized via uncoated vesicles and oligonucleotides are released in the early steps of this

111

process. However, we cannot exclude the possibility that some cationic liposomes can destabilize or merge directly with the cell membrane (Fig. 6). Indeed, Monensin, an Na ÷ ionophore that disrupts Na ÷ and H ÷ gradients across biological membranes and blocks receptor-mediated endocytosis, has been shown to inhibit the nuclear accumulation of oligonucleotides mediated by DOTMA [83]. This result led the authors to conclude that monensin may

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112

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prevent fusion of DOTMA vesicles with the plasma membrane but this could mean blockage of endocytosis as well. Nevertheless, it appears that the fusion with plasma membrane is a rare event. 7.3. Intracellular distribution

Interestingly, it has to be noted that the enhancement of oligonucleotide cellular uptake (between 15and 25-fold) is not sufficient to explain the increase of oligonucleotide activity (at least 1000-fold with DOTMA) [82,83]. These observations have led to examine the question whether cationic lipids also modify the intracellular distribution of oligonucleotides. Moreover, the localization of oligonucleotides in the cells after their release by the carrier is a critical point since released free oligonucleotides have to reach their target in order to achieve a real antisense effect. Therefore, we and others have assessed the intracellular localization of fluorescently labeled oligonucleotides delivered with or without cationic lipids [5,83,86,89]. As mentioned earlier, the results obtained on the intracellular localization of oligonucleotides alone, without microinjection, are rather divergent. We and several other groups were unable to detect any 'naked' fluorescently labeled oligonucleotides in the nucleus. They were localized only in punctate cytoplasmic region consistent with endocytic vesicles [5,11,13,23,83,89]. Associated with DOTMA, Lipofectin® or DOTAP, however the cellular fluorescence markedly increased and the oligonucleotides were preferentially localized in the nucleus, although they can also seen in some diffuse structures in the cytoplasm [5,83,86,89]. The percentage of adherent cells (HUVEC, CV-1) exhibiting nuclear fluorescence fluctuated between 65 and 80% with DOTMA and DOTAP. Fluorescently labeled oligonucleotides were never visualized in the nucleoli and they seem to be preferentially concentrated in specific regions of the nucleus that could be particular domains of small nuclear proteins known to interact with oligonucleotides. Thus, cationic lipids largely modify the intracellular localization of oligonucleotides. Nuclear oligonucleotide delivery has been shown to be charge ratio, time and temperature dependent [89]. Another important parameter governs the efficiency of antisense oligonucleotides

is their ability to hybridize to their target RNA. In this context, if the cationic lipids are still complexed with oligonucleotides in the cells, they limit the availability of oligonucleotides to hybridize to their target. Thus, we have studied also the intracellular localization of fluorescein-labeled oligonucleotides associated with rhodamine-labeled phosphatidylethanolamine/DOTAP liposomes. The results obtained show that there is no co-localization of lipids and oligonucleotides in the cells. Oligonucleotides were mostly seen in the nucleus whereas lipids were entirely localized in the cytoplasm and particularly in some perinuclear regions [89]. Finally, cationic liposomes have increased the quantity of delivered oligonucleotides both in the cytoplasm and in the nucleus. The importance of the nuclear uptake of oligonucleotides on their efficiency is still unknown since oligonucleotides can also hybridize to their target in the cytoplasm. Nevertheless, for triple helix formation at least nuclear accumulation would be desirable. 7.4. In vitro models

Antisense oligonucleotides complexed to several different cationic liposomes have been widely used in cell culture in vitro. The most commonly used cationic lipid formulation is Lipofectin®: DOTMA/ DOPE. Lipofectin® increased the activity of antisense oligonucleotides complementary to the AUG translation initiation codon or to specific sequences in the 3'-untranslated region of the mRNA of human intracellular adhesion molecule- 1 (I-CAM- 1) [82,83,90,91]. Until now this cationic lipid is the most efficient oligonucleotide carrier system reported. It increased the potency of an antisense oligonucleotide at least 1000-Ibid. The activity was reported to be dependent on lipid concentration and time of incubation. DOTMA has been successfully used in several different biological models that demonstrate the potential of cationic lipids to deliver oligonucleotides to multiple cell types [82,85,9294]. In several systems, DOTMA or Lipofectin® was necessary to achieve any antisense effect [5,83,86,93-95]. This has been shown particularly with mouse NIH 3T3 or African Green monkey kidney CV-1 cells that were stably transfected with an internally deleted construct of the human gene of

O. Zelphati, F.C. Szoka Jr/Journal of Controlled Release 41 (1996) 99-119

the proce (I) chain of type I procollagen or two reporter genes (SV40 large T antigen and Escherichia coli/3-galactosidase). In these cases, phosphorothioate or C-5 propyne-substituted phosphorothioate antisense oligonucleotides were effective only after their association with Lipofectin® [5,94]. One other cationic liposome formulation (DDAB/ DOPE) has been successful in delivering antisense oligonucleotides targeted to the early genes of the human papillomavirus (HPV) in CaSki cells [84]. Interestingly, hammerhead ribozyme directed against tumor necrosis factor a was also delivered into human promyelocytic leukemia cells by cationic liposomes (Lipofectin®). These cationic liposomesdelivered ribozymes have been shown to be very efficient thereby proving that one of the major problems in the delivery of preformed inhibitory catalytic RNA to target cells can be bypassed by the use of these cationic agents [96]. However, the use of cationic liposomes is still greatly limited. Indeed, the amphiphilic compounds that bind to nucleic acids have detergent properties and they can be very cytotoxic [81,84,97-99]. The non-specific interactions of cationic lipids with some plasma or serum elements can also lead to an in vitro and in vivo cytotoxicity [100]. Nevertheless, concentrations of cationic lipids necessary to enhance oligonucleotides activity in cell culture in vitro are generally much less than those where cytotoxicity was observed. The most critical point in the use of cationic liposomes is the dramatic effect of serum on their delivery efficiency. All experiments described previously have used serum-free medium to incubate complexes oligonucleotide/cationic lipid on cells because serum is known to completely inhibit the efficiency of cationic liposomes [87,93]. There are at least two possible explanations to serum effects. First, non-specific interactions with plasma or serum components can neutralize the positive charge of complexes necessary for the electrostatic interaction (binding) with negative charges present on cell surfaces. Secondly, plasma or serum elements can destabilize the interaction between oligonucleotides and cationic agents. In this context, an in vivo approach is unleasable except for direct injection into targeted tissues [101]. However, in spite of these disadvantages, some in vivo studies have been carried out.

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7.5. In vivo studies

First preliminary in vivo study has been performed on human LOX ascites tumor in nude mice. Phosphorothioate antisense oligonucleotides complementary to a non-translated region at the 3' end of the p120 mRNA, the translation product of which is a human nucleolar antigen detected in most human malignant tumors, were injected intraperitoneally alone or in association with DOTMA. The tumor growth inhibitory effect of the antisense oligonucleotide complexed with DOTMA at a 0.65-6.5 m g / k g dose range (80-90% of inhibition) was significantly greater than that of oligonucleotide alone without any marked toxic effects [93]. To explain this activity despite to the inhibitory serum effect on cationic lipid efficiency, the authors suggested that different proteins present in ascites as compared to serum should interfere to a smaller extend with the ability to deliver oligonucleotides. However, the mechanism by which DOTMA increases the oligonucleotide activity in vivo is still unknown. In recent studies, phosphorothioate antisense oligonucleotides designed to hybridize to the translation initiation codon of murine protein kinase C-ce (PKC-ce) mRNA have been reported to inhibit the expression of PKC-cein vitro. In this case, the in vitro antisense oligonucleotide activity required cationic liposomes (Lipofectin)® [102]. These in vitro studies were extended to determine the ability of oligonucleotides to inhibit gene expression after systemic intraperitoneal administration in mice. Antisense oligonucleotides have reduced the P K C - a mRNA level in the liver in a dose-dependent and sequence-specific fashion (IC5o value: 30-50 mg/kg of body weight) [102]. However, the oligonucleotide activity in vivo did not require the presence of cationic liposomes or any other delivery system. There are at least three possible explanations for these results. First, in this in vivo model the serum largely blocks the delivery efficiency of cationic lipids in this organ. The second is related to the biodistribution and pharmacokinetics of oligonucleotides alone. Indeed, the liver is the major organ of oligonucleotide deposition [13,103-105] and these molecules have been reported to be relatively stable in the liver [103]. Thirdly, liposomes are naturally taken up by phagocytic cells and thus, macrophages

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present in the peritoneum can pick up lipid complexes. In the same way, the activity of phosphorothioate oligonucleotides associated with cationic liposomes for in vitro inhibition of ICAM-1 expression were greatly enhanced (see Section 7.4). Interestingly, the same phosphorothioate antisense oligonucleotides have been shown to block the heart allograft rejection alone or in combination with other immunosuppressive modalities without the need of any carrier [90]. Cationic liposomes appear very promising to deliver oligonucleotides. However, in spite of their numerous advantages in vitro, their real in vivo potential still needs to be established. Moreover, the relative success of some oligonucleotides in vivo raises the question of the real need for an oligonucleotide carrier system.

8. Conclusions and perspectives Antisense oligonucleotides are promising drugs for antitumoral and antiviral therapeutic approaches. However, unmodified phosphodiester oligonucleotides have little pharmacological activity because of their sensitivity to nuclease degradation and their low cellular uptake [1,13]. One approach to circumvent the problem of nucleases degradation, has been the development of backbone-modified oligonucleotides [1]. Among these backbone-modified oligonucleotides, phosphorothioate oligonucleotides represent one of the most promising analogs since they have been shown to be very efficient in vitro and in several in vivo models [1,4,6]. Nevertheless, and in spite of some reported successful use of phosphorothioate oligonucleotides in several in vivo models [4,6], their use is still limited due to several persisting problems. Phosphorothioate and all other modified oligonucleotides have still not resolved the problem of their low cellular uptake. They are not concentrated on the cell surface and are diluted in the body. Other strategies have been pursued in order to improve the cellular uptake, the protection against nucleases, to direct intracellular localization, to reach the target sequence, to modify their biodistribution and pharmacokinetics. One strategy to improve oligonucleotide pharmacology is the use of lipo-

somes as oligonucleotide carrier systems. In all in vitro studies liposomes have improved the efficiency of oligonucleotides. The encapsulation of phosphodiester oligonucleotides in liposomes or their association with cationic lipids have protected them against nuclease degradation. Additionally, their isolation from the surrounding medium can also preclude some potential secondary effects of oligonucleotides. Liposomes have solved the problem of cell membrane permeability since they enhanced the cellular uptake of oligonucleotides in several cell types (e.g. fibroblast, macrophage, lymphocyte, keratinocyte, carcinoma, epithelial). Cationic liposomes also modify the intracellular distribution of oligonucleotides. Oligonucleotides are preferentially localized in the nucleus after delivery mediated by cationic lipids. It has also been reported that liposomes can increase the retention of oligonucleotides in cells and thus their effects can be prolonged. In the same way, some liposome formulations can be used as a slow release depot for modified oligonucleotides. Immunoliposomes confer a cellular specificity in addition to specific molecular recognition. Finally, each type of liposomes present their own advantages and inconveniences for the delivery of oligonucleotides. It is not yet possible to identify the best liposome formulation from the reported studies since no comparison studies have been carried out in the same biological model. Moreover, biodistribution and pharmacokinetics of liposomes are varying depending on several parameters including size and charge, composition and number of bilayers, nature of the transported molecules, the way of administration and interactions with external compounds [8,9]. Finally, due to their particular pharmacokinetics and biodistribution properties, liposomes can also modify and improve those of oligonucleotides. The in vivo behavior of oligonucleotides is well described [4,13,37,103,106-108]. But even when their pharmacokinetics and biodistribution seemed to be advantageous to target some tissues, their rapid clearance from blood or their inability to localize in lymph node or in brain limited their application. As of now the benefit of oligonucleotides carriers in in vivo applications is still unclear. In two recent studies, the oligonucleotide activity in vivo did not require the presence of cationic liposomes or any other delivery system [90,102]. Others have reported that liposomes increased the oligonucleotides ef-

O. Zelphati, F.C. Szoka Jr/ Journal of Controlled Release 41 (1996) 9 9 - 1 1 9

ficiency. T h e r e are several w a y s to explain these discrepancies. First, there are those related to the nature o f the p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s target (adhesion m o l e c u l e s , e n z y m e s , o n c o g e n e , viral gene) and thus, o f their particular localization in the body. F o r e x a m p l e , antisense o l i g o n u c l e o t i d e s c o m p l e m e n t a r y to the PKC-c~ m R N A w e r e efficient in the liver without any carrier; h o w e v e r , the liver represents the m a j o r organ o f o l i g o n u c l e o t i d e s deposition (see Section 7) [102]. In contrast, f u s o g e n i c l i p o s o m e s h a v e b e e n s h o w n to increase the activity o f o l i g o n u c l e o t i d e s to prevent the d e v e l o p m e n t of restinosis due to n e o i n t i m a f o r m a t i o n after angioplasty injury [77]. M o r e o v e r , the ' a n t i s e n s e ' effect o f p h o s p h o r o t h i o a t e o l i g o n u c l e o t i d e s needs further investigations because it is k n o w n that these m o l e c u l e s can act by non s e q u e n c e - s p e c i f i c m e c h a n i s m s especially on s o m e adhesion m o l e c u l e s and e n z y m e s [4]. Second, these d i v e r g e n c e s can be explain by the nature o f l i p o s o m e s used. Indeed, serum can dramatically inhibit the efficiency o f o l i g o n u c l e o t i d e s delivery by cationic l i p o s o m e s and thus greatly limit their advantages seen in vitro. M o r e o v e r , one i n c o n v e n i e n t feature o f l i p o s o m e s is their sizes that limit access o f the transported o l i g o n u c l e o t i d e s to s o m e target cells especially those behind e n d o t h e l i a with continuous b a s e m e n t m e m b r a n e s . In this context, the d e v e l o p ment o f smaller carrier systems seems to be necessary. A n y w a y , the real in v i v o potential o f l i p o s o m e s as carriers o f o l i g o n u c l e o t i d e s needs further studies that take into account the c h o i c e o f the o l i g o n u c l e o tide targets and the type o f liposomes. Finally, the ideal c o m b i n a t i o n s c o n c e r n i n g both m o d i f i e d o l i g o n u c l e o t i d e s or carrier systems are yet to be devised. H o w e v e r , the success o f s o m e lipos o m e formulations as drug carriers [109], suggests that future efforts in their use o f o l i g o n u c l e o t i d e carriers should be rewarding. In this context, the ' m a g i c bullet' d e s i g n e d by Paul Ehrlich to specifically transport drugs in the b o d y m i g h t be close at hand in l i p o s o m e encapsulated oligonucleotides.

Acknowledgments This w o r k was supported by N I H G M 3 0 1 6 3 , G l a x o Inc. and A s s o c i a t i o n p o u r le R e c h e r c h e contre le Cancer. We grateful a c k n o w l e d g e helpful corn-

ments f r o m Arto Urtti, Christian H e n d r e n and Chris L e a m o n .

115 Plank, W a y n e

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