Pharmacotherapy by intracellular delivery of drugs using fusogenic liposomes: application to vaccine development

Advanced Drug Delivery Reviews 52 (2001) 177–186 www.elsevier.com / locate / drugdeliv

Pharmacotherapy by intracellular delivery of drugs using fusogenic liposomes: application to vaccine development Jun Kunisawa, Shinsaku Nakagawa, Tadanori Mayumi* Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1 -6 Yamadaoka, Suita, Osaka 565 -0871, Japan

Abstract We prepared fusogenic liposomes by fusing conventional liposomes with an ultra-violet inactivated Sendai virus. Fusogenic liposomes can deliver encapsulated contents into the cytoplasm directly in a Sendai virus fusion-dependent manner. Based on the high delivery rates into the cytoplasm, we originally planned to apply the fusogenic liposomes to cancer chemotherapy and gene therapy. We have recently also examined the use of fusogenic liposomes as an antigen delivery vehicle. In terms of vaccine development, cytoplasmic delivery is crucial for the induction of the cytotoxic T lymphocyte (CTL) responses that play a pivotal role against infectious diseases and cancer. In this context, our recent studies suggested that fusogenic liposomes could deliver encapsulated antigens into the cytoplasm and induce MHC class I-restricted, antigen-specific CTL responses. In addition, fusogenic liposomes are also effective as a mucosal vaccine carrier. In this review, we present the feasibility of fusogenic liposomes as a versatile and effective antigen delivery system.  2001 Elsevier Science B.V. All rights reserved. Keywords: Fusogenic liposome; Sendai virus; Tumor vaccine; Mucosal vaccine; Cancer chemotherapy; Gene therapy

Contents 1. Introduction ............................................................................................................................................................................ 2. Characteristics of fusogenic liposomes ...................................................................................................................................... 2.1. Historical background ...................................................................................................................................................... 2.2. Delivery of macromolecules (e.g., polysaccharides, proteins, DNA) into mammalian cells in vitro and in vivo........................ 3. Feasibility of fusogenic liposomes as an antigen delivery system ................................................................................................ 3.1. Cytotoxic T lymphocyte inducible type vaccine.................................................................................................................. 3.2. Mucosal vaccine .............................................................................................................................................................. 4. Future prospects ...................................................................................................................................................................... Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................

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1. Introduction *Corresponding author. Tel.: 1 81-6-6879-8175; fax: 1 81-66879-8179. E-mail address: [email protected] (T. Mayumi).

One way to enhance vaccine effects is to specifically deliver the antigen to target organs. Several

0169-409X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 01 )00214-9

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drug delivery systems including liposomes have been developed to achieve this goal and good results have sometimes been obtained, such as against bacterial toxins. However, vaccines against viruses and tumors have not been remarkable due to a lack of induction of cytotoxic T lymphocyte (CTL) responses against anomalous cells, such as virus-infected and tumor cells [1]. Exogenous antigens are usually taken up into cells by phagocytosis or endocytosis. After degradation by lysosomal enzymes, these exogenous antigens are presented in an MHC class II-restricted manner (Fig. 1). Such antigen presentation induces antigen-specific antibody production, but not CTL responses. In contrast, endogenous antigens (cytoplasmic antigens) are degraded by proteasomes in cytoplasm and presented with MHC class I molecules, eventually leading to the induction of CTL responses (Fig. 1). Thus, a key event for the induction of antigen-specific CTL responses is the delivery of antigens into cytoplasm. We developed a hybrid delivery system called fusogenic liposomes that are composed of conven-

tional liposomes and the Sendai virus. Our previous studies found that fusogenic liposomes delivered the encapsulated contents into cytoplasm efficiently and directly (Fig. 2) [2,3]. Thus, we confirmed the application of fusogenic liposomes as an efficient gene delivery vehicle [3–6]. Based on their characteristics, we supposed that fusogenic liposomes could be used for vaccine development, especially for CTL-inducible vaccines. Section 3 of this review describes our progress in developing CTL-inducible vaccines using fusogenic liposomes. Mucosal vaccines seem to represent a novel vaccination strategy because only immunization via the mucosal route (e.g., nasal or oral) can induce antigen-specific mucosal immune responses that provide a first barrier against pathogenic infection [7–9]. In general, mucosal immunization with antigens alone does not induce detectable antigenspecific immune responses since most antigens are not taken up into mucosal immune sites and are promptly excluded. Thus, a new system is needed to improve the efficiency of antigen delivery [10–12].

Fig. 1. Antigen processing and presentation pathway. Exogenous antigens are usually taken up by endocytosis or phagocytosis and degraded by lysosomal enzymes. These degradation products are presented with MHC class II molecules. CD4 1 helper T cells recognize and are activated by these complexes on cell surfaces and assist antibody and cytotoxic T lymphocyte responses. In contrast, endogenous antigens are firstly degraded by proteasomes and transported into endoplasmic reticulum (ER) via transporters associated with antigen processing (TAP). At the ER compartments, degradation peptides bind to MHC class I molecules and are then transported to the cell surface. CD8 1 T cells bind to this complex and then exert a cytotoxic effect with the assistance of CD4 1 T cells against foreign cells expressing similar antigen–MHC class I complexes.

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Fig. 2. Features of fusogenic liposomes as efficient delivery vehicles into the cytoplasm. Fusogenic liposomes were prepared by fusing conventional liposomes with the Sendai virus at 378C and purified by discontinuous sucrose centrifugation. Fusogenic liposomes bind to the cell surface via HANA proteins and fuse with the cell membrane with F proteins, then directly deliver encapsulated molecules into the cytoplasm.

Because the parent Sendai virus naturally infects via mucosal sites [13,14], fusogenic liposomes may act as an effective delivery vehicle for mucosal vaccines. The function of fusogenic liposomes as a mucosal delivery vehicle of antigens in also examined in Section 3.

2. Characteristics of fusogenic liposomes

2.1. Historical background The Sendai virus belongs to paramyxoviridae and has a negative strand genomic RNA. On the surface of the virus membrane, two major proteins are involved in cellular infection. Hemagglutinating and neuraminidase (HANA) proteins are required to bind to a receptor (sialic acid) on the cell surface [15]. In addition, Fusion (F) protein interacts with the lipid layer of the cell membrane to induce cell fusion [16,17]. F protein is initially synthesized as a precursor form (F0), which is spliced to the active form (F1, F2) [18]. A novel HANA-dependent intermediate stage of Sendai virus-mediated membrane fusion has been identified [19]. However, the actual

process by which the Sendai virus infects cells remains unclear. In 1985, Okada et al. found that the Sendai virus can efficiently fuse with a conventional liposome as well as cell membranes at 378C [20] and a virus envelope protein is involved in this process [20,21]. Additionally, our recent study suggests that F protein, but not HANA protein, is required for the virus to fuse with conventional liposomes [22]. Furthermore, the fact that the Sendai virus receptor (sialic acid) does not exist on the liposomal membrane suggests that this receptor is not required for the fusion process. In addition to this unique fusion mechanism between the Sendai virus and liposomes, the Sendai virus–liposome fusion vehicle can fuse with mammalian cells [20]. This was the first report describing fusogenic liposomes. In collaboration with Nakanishi et al., we developed methods for the preparation of the fusogenic liposomes [2,3,6]. An ultra-violet Sendai virus and conventional liposomes readily fuse under optimal conditions (neutral pH, 378C) and fusogenic liposomes can be purified from the unreacted Sendai virus and conventional liposomes by discontinuous (12, 30, 50%) sucrose gradient centrifugation (77 000 3 g, 2 h, 48C). Thus, small uniform

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fusogenic liposomes can be prepared when unilamellar liposomes (d 5 300 nm) are fused with the purified Sendai virus (d 5 300 nm). The estimated diameter of fusogenic liposomes is 380 nm according to dynamic light scattering, suggesting that a single Sendai virus fuses with a single liposome. In addition, electron microphotography showed that fusogenic liposomes have a spike structure similar to that of the Sendai virus [3,6,23].

2.2. Delivery of macromolecules (e.g., polysaccharides, proteins, DNA) into mammalian cells in vitro and in vivo One unique feature of fusogenic liposomes is that they can deliver encapsulated contents into the cytoplasm of a wide variety of mammalian cells. To

visually express this unique characteristic, we examined the delivery of a fluorescent tag by fusogenic liposomes by confocal microscopy. Fig. 3A shows that the cytoplasm was stained with FITC–dextran delivered by the fusogenic liposomes (Fig. 3A). In contrast, aggregated fluorescence, indicating endosomes, appeared in the cells cultured with FITC– dextran-encapsulated conventional liposomes (Fig. 3B). In addition, an endocytosis inhibitor (cytochalasin B) did not affect this delivery of fusogenic liposomes (Fig. 3C), while the fluorescence derived from conventional liposomes completely disappeared (Fig. 3D). These findings indicated that fusogenic liposomes delivered their contents into the cytoplasm via an endocytosis-independent pathway, whereas conventional liposomes were taken up by endocytosis. To confirm this feature of fusogenic lipo-

Fig. 3. Analysis of in vitro uptake by cultured cells. A murine intestinal epithelial cell line, MODE-K was exposed to fusogenic liposomes containing FITC–dextran (A) or with conventional liposomes containing FITC–dextran (B). After 1 h, the cells were washed and examined by confocal microscopy. The same experiment was repeated using an endocytosis inhibitor (cytochalasin B) (C and D).

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somes, a diphtheria toxin A subunit (DTA) was used as a probe. DTA can kill cells by deactivating elongation factor 2 (EF-2) in the cytoplasm [24]. However, DTA alone is absolutely non-toxic due to the absence of a receptor-binding hydrophobic domain (diphtheria toxin B subunit). Thus, DTA indicates delivery into the cytoplasm. Fusogenic liposomes carrying encapsulated DTA suppressed protein synthesis in cultured cells at 378C [2,3,6,23]. In contrast, conventional liposomes carrying encapsulated DTA were completely non-toxic. These findings confirmed that fusogenic liposomes could efficiently deliver DTA into the cytoplasm. Furthermore, fusogenic liposomes can deliver encapsulated DTA into a wide variety of mammalian cells (Table 1) [3,6]. However, primary human B and T lymphocytes (CD4 1 or CD8 1 ) and the human B cell line were not permissive to fusogenic liposome-mediated delivery [25]. In addition to being a marker of cytoplasmic delivery, DTA may become an effective anti-cancer drug if delivered into the cytoplasm of tumor cells. In this context, we demonstrated that an intraperiTable 1 Fusogenic liposomes can deliver the encapsulated molecules into wide variety cells Species

Cell (type)

Human

Hela HepG2 (hepatocyte) GOTO (neuroblastoma) Molt4 (T cell leukemia) Jurkat (T cell line) Primary fibroblast Primary endothelial cells

Monkey

LLCMK2 (kidney)

Hamster

CHO

Mouse

L EL4 (T thymoma) Sarcoma-180 B6 Melanoma MODE-K (intestinal epithelial cell) IC-21 (macrophage) DC2.4 (dendritic cell)

Rat

C6 glioma

Rabbit

Hepatocyte

181

toneal administration of DTA-encapsulated fusogenic liposomes induced the total disappearance of S-180 tumors from the abdominal cavity without any side effects [2]. In addition, an intratumor injection of fusogenic liposomes containing DTA significantly inhibited tumor growth [26]. These results suggest that fusogenic liposomes containing DTA is an effective anti-cancer chemotherapy. Over the past 20 years, gene therapy has been recognized as an effective approach to obtain clinical benefits from foreign therapeutic genes expressed in affected tissues. Since a therapeutic gene is easily and immediately split by lysosomal enzymes, development of a direct delivery system into the cytoplasm is essential. Fusogenic liposomes provide a vehicle for the delivery of the contents into the cytoplasm. We postulated that fusogenic liposomes might also be effective for gene therapy. To address this issue, we initially evaluated the efficiency of fusogenic liposomes as a gene transfer vector in vitro [3,5,6]. Gene transfer was swift in culture cells incubated with fusogenic liposomes compared with cationic liposomes. In addition, gene expression was also high even under high serum concentrations. In contrast, gene expression was not elevated when cationic liposomes were used as a vector [5]. Further analysis indicated that fusogenic liposomes could efficiently introduce encapsulated genes in vivo [4,5]. Namely, genes were expressed in ascites tumor cells following an intraperitoneal injection of fusogenic liposomes containing a model gene (luciferase)-expression plasmid [5]. In addition, tumors regressed when fusogenic liposomes containing the TNF-a expression gene were injected into an artery [4]. These findings indicated that fusogenic liposomes would constitute a novel system of gene delivery.

3. Feasibility of fusogenic liposomes as an antigen delivery system

3.1. Cytotoxic T lymphocyte inducible type vaccine Cytotoxic T lymphocytes (CTLs) play a crucial role in protection against viral infections and malignancy [27–29]. Although much effort has been focused on the induction of CTL responses, inducing

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sufficient levels has been difficult. This is because standard vaccines are recognized as exogenous antigens and are taken up by endocytosis, and then presented with MHC class II molecules. This MHC class II-restricted presentation causes the induction of antigen-specific antibodies, not CTL responses (Fig. 1). The CTL responses are induced when antigens are processed in the cytoplasm, then presented with MHC class I molecules (Fig. 1). Thus, an imperative issue for the induction of CTL responses is the delivery of the antigens into the cytoplasm. Several delivery systems, including those involving liposomes, are aimed at delivering antigens into the cytoplasm to elicit CTL responses [30–32]. Although CTL responses were detectable with these systems, problems such as side effects and low induction efficacy remained. Since fusogenic liposomes could efficiently deliver encapsulated contents into the cytoplasm without any side effects, we supposed that fusogenic liposomes might be effective as a vehicle to deliver a CTL-inducible vaccine. In this context, we examined whether or not fusogenic liposomes could deliver antigens to the MHC class I-restricted antigen processing pathway [23]. Antigen presentation assays using a chicken egg ovalbumin (OVA)–MHC class I complex-specific T cell hybridoma revealed brisk MHC class I-restricted antigen presentation after exposure to OVA-encapsulated fusogenic liposomes. In contrast, such MHC class I-restricted antigen presentation was not determined in cells incubated with OVA-encapsulated conventional liposomes or fusogenic liposomes without any antigen. In addition, inactivation of membrane proteins (HANA and F proteins) of fusogenic liposomes by trypsin digestion did not induce MHC class Irestricted antigen presentation. Besides this inactivation of the virus protein, the inactivation of cytoplasmic degradation enzymes (proteasomes) inhibited MHC class I-restricted antigen presentation. These data indicated that fusogenic liposomes deliver encapsulated antigens into the cytoplasmic MHC class I-restricted antigen processing and presentation pathway in a manner that is dependent upon Sendai virus fusion. MHC class II as well as class I-restricted antigen presentation is important for inducing antigen-specific CTL and antibody responses. Thus, we examined whether or not fusogenic liposomes can deliver the encapsulated antigens to the MHC class

II-restricted antigen presentation pathway [33]. Antigen presentation assays using a hen egg lysozyme (HEL)–MHC class II complex-specific T cell hybridoma revealed brisk MHC class II-restricted antigen presentation in macrophages exposed to HEL-encapsulated fusogenic liposomes. In contrast, neither irrelevant antigen-encapsulated fusogenic liposomes nor empty fusogenic liposomes induced HEL-specific, MHC class II-restricted antigen presentation. In this MHC class II-restricted antigen presentation pathway, two antigen uptake pathways may be considered. In the first possibility, inactive fusogenic liposomes are taken up macrophages via phagocytosis and presented with MHC class II molecules through the conventional antigen processing pathway. In the second one, fusogenic liposomes deliver the encapsulated antigens into the cytoplasm and the antigens in the cytoplasm enter the endosome and then are processed in the conventional MHC class II-restricted antigen processing pathway. The second pathway is well documented [34,35]. Further analysis is needed to clarify which pathway contributes to this MHC class II-restricted antigen presentation induced by fusogenic liposomes. According to these antigen presentations both by MHC class I and class II molecules, subcutaneous immunization with fusogenic liposomes containing antigen-induced high levels of antigen-specific CTL responses compared with that induced by Freund’s complete adjuvant (CFA) (Table 2) [23]. In addition to the CTL responses, antigen-specific antibody production was also elevated in the sera following

Table 2 Efficient induction of OVA-specific CTL responses following subcutaneous immunization with OVA-encapsulated fusogenic liposomes Antigen

% Lysis (at E /T ratio550)

OVA alone OVA in conventional liposome OVA in fusogenic liposome OVA in CFA

11.261.6 10.861.4 68.367.4 42.563.2

Mice were subcutaneously immunized with OVA alone or OVA in conventional liposomes, fusogenic liposomes or Freund’s complete adjuvant (CFA). After 10 days, spleen cells were isolated and analyzed for these cytotoxic activity using 51Cr release assay. Data are presented as mean6S.E. for all mice in individual test groups (three mice per group).

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subcutaneous immunization with antigen-encapsulated fusogenic liposomes [36]. These findings suggested that fusogenic liposomes are efficient delivery vehicles for the induction of CTL responses via the MHC class I-restricted antigen presentation. Based on these observations, we are currently investigating the application of fusogenic liposomes for use as a cancer vaccine.

3.2. Mucosal vaccine Parentally administered vaccines are generally ineffective for inducing antigen-specific mucosal immune responses that prevent microorganism transmission and dissemination to the regional lymph nodes or target organs. In contrast, mucosal (e.g., nasal and oral) immunization elicits both mucosal and systemic immune responses. Thus, intensive investigations are currently in progress for the development of mucosal vaccines [7,8,37]. In general, optimal antigen-specific immune responses are difficult to induce following mucosal immunization with a soluble protein antigen alone. Much effort has been directed towards improving the efficacy of soluble protein antigens, often via antigen delivery systems [10,12,38–40]. Among mucosal lymphoid tissues, nasopharyngeal-associated lymphoreticular tissue (NALT) is part of a mucosal inductive site that contacts inhaled antigens in the upper respiratory tract [7,11,41,42]. Murine and rat NALT consists of bilateral strips of non-encapsulated lymphoid tissue underlying the epithelium on the ventral aspect of the posterior nasal tract that assumes a bell-like shape in cross sections [43,44]. In humans, the pharynx is guarded by Waldeyer’s ring of tonsils and adenoids, which are considered to be NALT containing T and B cells, dendritic cells and epithelial cells including M cells, which are described below [45]. Due to recent understanding of the NALT features in terms of potential applications to developing mucosal vaccines [43,44,46,47], nasal immunization is recognized as an effective route for the induction of mucosal and systemic immune responses [42,48,49]. As we mentioned above, subcutaneous immunization with fusogenic liposomes carrying encapsulated antigen induced antigen-specific CTL and antibody responses in systemic lymphoid tissues in an MHC class I-dependent manner [23,36]. Since the Sendai

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virus naturally infects via the mucosal epithelium in the upper respiratory tract [13,14], fusogenic liposomes may effectively deliver antigens to the mucosal immune system and induce antigen-specific mucosal and systemic immune responses when immunized nasally. To address this issue, we examined the suitability of fusogenic liposomes as a nasal vaccine delivery vehicle [33]. Nasal administration of antigens using fusogenic liposomes delivered the antigens to NALT epithelial cells and macrophages more efficiently than conventional liposomes. M cells are scattered on the epithelial cell layer of NALT [50,51]. These M cells are morphologically different and specialized for the uptake and transcellular transport of particle antigens and microorganisms from the lumen to the lymphoid follicles [52,53]. Thus, M cells should constitute a suitable target for the efficient delivery of vaccine antigen into NALT. We, therefore, examined whether or not fusogenic liposomes fused and delivered the antigen to M cells. Confocal microscopy using M cell-specific lectin revealed that fusogenic liposomes delivered the antigen to M cells as well as to neighboring epithelial cells [33]. These findings demonstrated that fusogenic liposomes constitute an effective antigen delivery system for M cells, epithelial cells and Mac-1 1 cells in nasal immune compartments. Consistent with these high delivery activities of fusogenic liposomes, nasal immunization with fusogenic liposomes carrying encapsulated protein antigen induced high levels of antigen-specific helper T cell responses. Furthermore, antigen-specific CTL responses and antibody production were also elicited at both mucosal and systemic sites by nasal immunization with fusogenic liposomes carrying encapsulated antigen [33]. These results indicate that fusogenic liposomes comprise a versatile and effective system for stimulating antigen-specific immune responses in both mucosal and systemic compartments. Based on these findings, our present efforts are directed to applying fusogenic liposomes to mucosal HIV vaccines.

4. Future prospects This review presents the feasibility of fusogenic liposomes as an antigen delivery vehicle. We ex-

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amined the application of this system to the development of cancer and HIV vaccines. Our results will clarify whether or not fusogenic liposomes can induce sufficiently effective immune responses to have clinical applications. Our preliminary data indicate that fusogenic liposomes will be therapeutically effective. In addition to murine systems, we are also evaluating the effectiveness of fusogenic liposomes in a monkey system. We hope that clinical trials of fusogenic liposomes will start in the near future.

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Acknowledgements [13]

This research was supported, in part, by Grants-inAid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. J.K. is a Research Fellow of the Japan Society for the Promotion of Science.

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